prr.hec.gov.pkprr.hec.gov.pk/jspui/bitstream/123456789/9239/1/T... · vi ACKNOWLEDGEMENTS In the name of ALLAH, the merciful, the beneficent If oceans turn into ink and all of the

i

ii

BIOACTIVITY GUIDED ISOLATION OF ANTICANCER

COMPOUNDS FROM OLEORESIN OF BOSWELLIA AND

PINUS SPECIES

By

MUHAMMAD ADNAN AYUB

M. Phil (GCUF)

2007-ag-1396

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE

REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

IN

CHEMISTRY

DEPARTMENT OF CHEMISTRY

FACULTY OF SCIENCES

UNIVERSITY OF AGRICULTURE,

FAISALABAD

2018

iii

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v

I want to consecrate this humble effort to the gleaming tower of knowledge

Hazrat Muhammad

(May Peace and Blessings of Allah be upon Him)

&

My Affectionate Parents

&

To my beloved brother & sisters

Who‘s esteemed love enabled me to get the success and whose hearts are

always beating to wish for me maximum felicity in life.

vi

ACKNOWLEDGEMENTS

In the name of ALLAH, the merciful, the beneficent

If oceans turn into ink and all of the woods become pens, even then the praises of ―ALLAH‖

Almighty cannot be expressed. He who created the universe and knows whatever is there in it,

hidden or evident and who bestowed upon me the intellectual ability and wisdom to search for its

secrets. All praises and respects to His Holy Prophet, ―MOHAMMAD‖ (Peace Be Upon Him). The

most perfect and exalted among and of ever born on the surface of earth, which is forever a torch of

guidance and knowledge for humanity as a whole.

All praises to Almighty Allah, the most Merciful and the most Beneficent, Who guides us in

darkness and helps in every difficult moment. Lord is most Bounteous. Who taught the man by the

pen ought what he knew not. I offer my humble thanks to Almighty Allah for Bestowing upon me

the potential and ability for successful accomplishment of this research work.

About all I am indebted to Almighty Allah, Lord of my life and everything in the universe

and His Holy Prophet Hazrat Muhammad (P.B.U.H) Who is forever model of guidance and

knowledge for humanity and Whose blessings enabled me to perceive and suit higher ideas of life.

It is a matter of gratification to express my deep sense of devotion to my worthy supervisor

Dr. M. Asif Hanif, Department of Chemistry, University of Agriculture, Faisalabad, under whose

kind supervision and constructive criticism, the present research was completed.

Appreciation is also extended to my supervisory committee, Dr Raja Adil Sarfraz, Assistant

professor Department of Chemistry, University of Agriculture, Faisalabad; and Dr. Muhammad

Shahid, Associate professor, Department of Biochemistry, University of Agriculture, Faisalabad, for

providing me valuable help.

The whole work will remain incomplete if I do not record my indebted and immense gratitude

and appreciation to my affectionate parents and my loving brothers Dr. Muhammad Yousaf

Ansari and Muhammad Younas Ansari and sisters, whatever I have got, so far, in the fields of

education and for their unforgettable sacrifices, moral and financial support and countless prays

throughout my education career. Words are lacking to express my humble obligations to my

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List of Tables:

Tab. Title Page No

3.1 List of instruments 28

4.1 Essential oil yield of Pinus roxburghii oleoresin by different

extraction methods 45

4.2 Essential oil yield of Boswellia serrata oleoresin by different

extraction methods 47

4.3 Fractionation of hydro distilled essential oils of Pinus roxburghii

oleoresin. 49-50

4.4 Fractionation of steam distilled essential oils of Pinus roxburghii

oleoresin. 51-52

4.5 Fractionation of hydro distilled essential oils of Boswellia serrata

oleoresin. 53-54

4.6 Fractionation of steam distilled essential oils of Boswellia serrata

oleoresin. 55

4.7 Concentration of phenolic acids in Boswellia serrata and Pinus

roxburghii oleoresin (mg/L) 148

4.8

Chemical composition of Pinus roxburghii oleoresin essential oils

isolated through different extraction methods and most active sub

fractions

152-154

4.9

Chemical constituents of Pinus roxburghii oleoresin essential oils


fractions.

159

4.10

Chemical composition of Boswellia serrata oleoresin essential oils


fractions

163-164

4.11 Chemical constituents of Boswellia serrata oleoresin essential oils

isolated through different extraction methods and most active sub 169

xiv

fractions.

List of Figures:

Sr. No Title Page No

2.1 Typical image of Pinus roxburgii tree 11

2.2 Typical image of Boswellia serrata tree

20

3.1 Collection of plant materials 31

3.2

Schematic diagram of extraction of essential oils from oleoresins

Boswellia serrata and Pinus roxburghii through hydro distillation,

steam distillation and supercritical fluid extraction methods.

33

3.3 Schematic diagram of Boswellia serrata and Pinus roxburghii

oleoresin essential oils fractionation.

34

3.4 Calibration curve for total phenolic contents 36

3.5 Calibration curve for total flavonoid contents 37

3.6 Calibration curve for Total antioxidant contents 38

3.7 A typical agar plate showing the inhibition zones exhibited by essential

oils (1-9)

40

3.8 A typical plate in resazurin microtitre-plate assay showing the color

change due to antibacterial effect of essential oils

42

3.9 Schematic diagram of Boswellia serrata and Pinus roxburghii oleoresin

essential oils fractionation. 43

4.1 Total phenolic contents of Pinus roxburghii oleoresin 57

4.2 Total phenolic contents of Boswellia serrata oleoresin 60

4.3 Total flavonoid contents of Pinus roxburghii oleoresin 62

4.4 Total flavonoid contents of Boswellia serrata oleoresin. 64

xv

4.5 DPPH free radical scavenging activity of Pinus roxburghii oleoresin. 66

4.6 DPPH free radical scavenging activity of Boswellia serrata oleoresin. 68

4.7 Percent inhibition of linoleic acid oxidation of Pinus roxburghii

oleoresin. 72

4.8 Percent inhibition of linoleic acid oxidation of Boswellia serrata

oleoresin. 73

4.9 Hydrogen peroxide scavenging activity of Pinus roxburghii oleoresin 75

4.10 Hydrogen peroxide scavenging activity of Boswellia serrata oleoresin 77

4.11 Total antioxidant contents/ FRAP assay of Pinus roxburghii oleoresin 80

4.12 Total antioxidant contents/ FRAP assay of Boswellia serrata oleoresin 82

4.13 Antibacterial activity of Pinus roxburghii oleoresin against E. coli 85

4.14 Antibacterial activity of Pinus roxburghii oleoresin against S. aureus 86

4.15 Antibacterial activity of Pinus roxburghii oleoresin against P.

multocida 87

4.16 Antibacterial activity of Pinus roxburghii oleoresin against B. subtilis 88

4.17 Antibacterial activity of Boswellia serrata oleoresin against E.coli 91

4.18 Antibacterial activity of Boswellia serrata oleoresin against S. aureus 92

4.19 Antibacterial activity of Boswellia serrata oleoresin against P.

multocida 93

xvi

4.20 Antibacterial activity of Boswellia serrata oleoresin against B. subtilis 94

4.21 Antibacterial activity of Pinus roxburghii and Boswellia serrata

oleoresin essential oils, fractions and sub-fractions. 97

4.22 Minimum inhibitory concentration of Pinus roxburghii oleoresin

against E. coli 99


against S. aureus 100


against P. multocida 101


against B. subtilis 102

4.26 Minimum inhibitory concentration of Boswellia serrata oleoresin

against E.coli 106


against S. aureus 107


against P. multocida 108


against B. subtilis 109

4.30

Represents the minimum inhibitory concentration of Boswellia serrata

and Pinus roxburghii oleoresin essential oils, fractions and sub-

fractions.

110

4.31 Antifungal activity of Pinus roxburghii oleoresin for F. solani. 113

xvii

4.32 Antifungal activity of Pinus roxburghii oleoresin for A. niger 114

4.33 Antifungal activity of Pinus roxburghii oleoresin against A. alternata 115

4.34 Antifungal activity of Pinus roxburghii oleoresin against A. flavus 116

4.35 Antifungal activity of Boswellia serrata oleoresin against F. solani. 119

4.36 Antifungal activity of Boswellia serrata oleoresin against A. niger 120

4.37 Antifungal activity of Boswellia serrata oleoresin against A. alternata 121

4.38 Figure 4.36: Antifungal activity of Boswellia serrata oleoresin against

A. flavus 122

4.39 Represents the antifungal activity of Pinus roxburghii and Boswellia

serrata oleoresin essential oils, fractions and sub-fractions. 124


against F. solani 127


against A. niger 128


against A. alternata 129


against A. flavus 130


against F. solani 133


against A. niger 134


against A. alternata 135


against A. flavus 136

xviii

4.48 Crown gall antitumor activity of Pinus roxburghii oleoresin. 139

4.49 Crown gall antitumor activity of Boswellia serrata oleoresin 142

4.50 Hemolytic activity of Pinus roxburghii oleoresin 144

4.51 Hemolytic activity of Boswellia serrata oleoresin 146

4.52 HPLC chromatogram of F2 c sub-fraction of Pinus roxburghii

oleoresin hydro distilled essential oil of 160 oC.

149

4.53 HPLC chromatogram of F3 fraction of Boswellia serrata oleoresin

hydro distilled essential oil of 140 oC.

149

4.54 Variations in major components of Pinus roxburghii oleoresin essential

oils extracted through different extraction methods. 154

4.55 GC-MS chromatogram of hydro distilled essential oil isolated at 160 ⁰C

from Pinus roxburghii oleoresin. 155

4.56 GC-MS chromatogram of steam distilled essential oil isolated at 160 ⁰C

from Pinus roxburghii oleoresin. 155

4.57 Chromatogram of super critical fluid extracted essential oil isolated at

40 ⁰C, 80 bar pressure from Pinus roxburghii oleoresin. 156

4.58 Chromatogram of sub fraction F2 c of hydro distilled essential oil

isolated at 160 ⁰C from Pinus roxburghii oleoresin. 156

4.59 Chromatogram of sub fraction F2 a of steam distilled essential oil

isolated at 160 ⁰C from Pinus roxburghii oleoresin 157

4.60 Variations in major components of Boswellia serrata oleoresin

essential oils extracted through different extraction methods. 164

4.61 Chromatogram of hydro distilled essential oil isolated at 140 ⁰C from

Boswellia serrata oleoresin. 165

4.62 Chromatogram of steam distilled essential oil isolated at 120 ⁰C from

Boswellia serrata oleoresin. 165

4.63 Chromatogram of super critical fluid extracted essential oil isolated at

40 ⁰C, 80 bar pressure from Boswellia serrata oleoresin. 166

xix

4.64 Chemical composition of sub fraction F1 c of hydro distilled essential

oil isolated at 140 ⁰C from Boswellia serrata oleoresin. 167

4.65 Chromatogram of sub fraction F1 c of steam distilled essential oil

isolated at 120 ⁰C from Boswellia serrata oleoresin. 168

xx

Abstract

Oleoresins are secondary metabolites of resinous plants, mainly composed of essential oil and

resin acids. The present research work was conducted for the development of methodology for

isolation of anticancer compounds from oleoresins of Boswellia and Pinus species. Conventional

and advanced extraction techniques such as Hydro-distillation, steam distillation and

supercritical fluid extraction (SCFE) method were used for extraction of essential oil from

oleoresins. For optimum extraction yield of essential oil, hydro-distillation and steam distillation

methods were performed under different temperature conditions. It was observed that essential

oil yield in both plants oleoresin was increased on increasing extraction temperatures. These

essential oils were further separated in to different fractions and sub fractions on the basis of

their boiling points by vacuum fractional distillation method. Moreover, the biological activity of

essential oils, fractions and sub fractions were performed under bioactivity guided assays.

Antioxidant potential of essential oils, fractions and sub fractions was determined through

different antioxidant assays. It was observed that most the essential oils, fractions and sub

fractions revealed moderate level of antioxidant activity. Moreover, it was found that all the

essential oils, fractions and sub fractions showed excellent antimicrobial and antitumor activity.

Cytotoxicity was determined through haemolytic assay and their results expressed that all the

tested essential oils, fractions and sub fractions exhibit weak cytotoxic activity. The chemical

composition of essential oils was determined through GC-MS. The results showed that α-pinene,

β-pinene, 3-carene and longifolene were the major chemical components of Pinus roxburghii

oleoresin essential oils. Similarly, the chemical composition of Boswellia serrata oleoresin

essential oils showed that α-pinene, β-pinene, pinocarveol and verbenol were the main chemical

compounds and these chemical compounds might be responsible for their biological activity.

Moreover, it was observed that the chemical composition of essential oils significantly vary with

extraction methods.

1

Chapter 1

Introduction

Plants have played an important role in the development of various sophisticated

traditional systems of medicines like Chinese, African, Unani, Australian and Ayurveda.

According to World Health Organization (WHO) about 65% of world‘s population follow these

traditional systems of medicines for their primary health care (Cragg et al., 2009). More than 122

pure compounds have been isolated from 94 plant species and use as drugs against various

diseases (Gurib-Fakim, 2006). Plants synthesize various secondary metabolites such as

phenolics, terpenoids, oleoresins, cyanogens, carotenoids, flavonoids, alkaloids, anthocyanins

and glucosinolates (Wink, 2015). Previously, it was considered that these secondary metabolites

are the plant waste products. Now, it has been widely accepted that these secondary metabolites

are rich source of bioactive compounds and major source of new drugs. Oleoresins are the

secondary metabolites of the resinous plants and contain significant amount of essential oil

naturally synthesized by the trees. Oleoresin ducts contain plastid-enriched epithelial cells lines

that produce terpenoid rich oleoresin and secrete them into an extracellular lumen where they

store under pressure. Whenever plant undergoes physical injury by wounding or an invading

organism, oleoresin is secreted and remove the organism out of the bark or capture the organism

in sticky oleoresin and toxic components of oleoresin kill invader (Christiansen and Kucera,

1999; Nagy et al., 2000). With the passage of time volatile components (essential oil) of

oleoresin evaporates and nonvolatile components (rosin) sterilize and cover the wounded region

of plant. Oleoresins are considered to be a defense against bark-boring insects and microbes

(Hudgins and Franceschi, 2004). It has been reported that chemical composition of oleoresin

significantly changed with season and depth of the draining (drilling) holes in the tree. As the

temperature change from winter to summer, the chemical composition of volatile compounds

gradually decrease and more depth drilling gives more volatile compounds (Mita et al., 2002).

Chemically, oleoresins are mixture of terpenes and rosin (Trapp and Croteau, 2001; Figueiredo

et al., 2008). Terpenes are complex mixture of monoterpenes, sesquiterpenes, diterpenes,

oxygenated monoterpenes, oxygenated diterpenes and oxygenated sesquiterpenes While, rosin is

complex mixture of resin acids (da Silva Rodrigues-Corrêa et al., 2013).

2

Since the ancient times, oleoresin has been used for the treatment of various diseases in

traditional medicinal systems such as dragon‘s blood resin has been used as an antidiarrhetic and

hemostatic drug. Frankincense is an oleoresin excreted from bark of Boswellia species, essential

oils and extracts, isolated from these species has been used against asthma, ulcerative colitis,

rheumatic and cough. Moreover, Frankincense oleoresin contained α and β-boswellic acids that

showed Immunomodulatory and anti-inflammatory activities.

Monoterpenes

H

H

CH3

CH3

H3C

3-Carene

CH3

H3C

H3C

α-pinene

CH3

CH3

CH2

β-pinene

CH3

H3C CH3

α-phellandrene

CH3H2C

CH3

1,5,8-p-methatriene

CH2

CH2

H3C CH3

β-myrcene

Sesquiterpenes

H3C

CH3

H3C CH3

α-cubebene

H3C

H2C

CH3CH3

1, 4- Methenoazulene

H

H

HH3C

CH3

α- patchoulene

HH

CH3

CH3

H2C

CH3

β-caryophyllene

H3C

CH3

H3C CH3

α-bisabolene

H3C

HH3C CH3

CH3

α-cedran

3

Diterpenes

CH3

CH3

H3C

CH3

CH3

8,13-Abietadiene

HH

CH3

CH3

H3C

CH3

H

α-Ylangene

Oxygenated

monoterpenes

CH3

H3C

CH3

O

Verbenene

CH3

H3C CH3

O

α-Campholene

H

H

H3C

CH3

O

CH3

Filifolone

CH3

O

CH3

CH3

Camphanone

oxygenated

sesquiterpenes

H3C CH3

CH2

H2C

OH

CH3

Nerolidol

HH

CH3H3C

H

H

H3C

o

Aromadendreneoxide

Oxygenated diterpenes

HH3C

CH3HO

CH3

OH

H2C

Isopimaradiene-diol

Resin acids

CH3

H2C

CH3

HO

O

CH3

Pimaric acid

CH3

O

HO

CH3H

H3C

CH2

CH2

Communic acid

O

HOCH3

H

CH3

H

H3C

CH3

Dehydroabietic acid

4

OH

H3C

HCH3

CH3H

HHO

CH3

O

H3C

CH3

OH

H3C

HCH3

CH3H

HHO

CH3

O

H3C

CH3

O

O

HOCH3

H

CH3CH3

H

H3C

CH3

β-Boswellic acid Acetyl-keto- β-boswellic acid Palustric/Levopimaric

acids

CH3H3C

OH

H3C

HCH3

CH3H

HHO

CH3

CH3

O

CH3

CH3

H

CH3

CH3H

OHO

H3C COOH

CH3CH2

O

α-Boswellic acid Abietic acid Lambertianic acid

It is very difficult to isolate pure bioactive compounds from oleoresins as it may contain

thousands of compounds. For isolation of pure bioactive compounds, various separation

techniques are used. The selection of suitable separation techniques depend on stability,

solubility and volatility of the compounds to be separated. Extraction is the first step in isolation

of bioactive compounds from oleoresins. Maceration, soaking, soxhlet extraction, hydro, steam

distillation and sonication are the traditional methods used for the extraction of essential oils and

extracts. Hydro and steam distillation methods are frequently used for extraction of essential oil

from oleoresins, while few reports are available on supercritical fluid extraction.

Steam distillation is one of official and earliest approved method used for isolation of

pure essential oil from oleoresins. The oleoresin is packed in alembic and high temperature

steam is passed from bottom of alembic. The high temperature steam evaporates essential oil

from oleoresin, pass through condenser and collects in separation funnel. Steam distillation have

major advantages our hydro distillation with least chance of degradation of heat sensitive

compounds and less consumption of fuel (Sadgrove and Jones, 2015).

5

In hydro-distillation, oleoresin is dipped in water and whole mixture is then heated in an

alembic above its boiling point. During hydro distillation, an azeotropic mixture of volatile oil

and water is formed that can evaporate and pass through condenser. Due to low temperature in

condenser, essential oil and water vapors are condensed and collect in separation funnel, where

essential oil and water is easily separated (Sadgrove and Jones, 2015). Hydro distillation has

major advantage over steam distillation that it requires least amount of initial investment of

equipment than steam distillation (Wang et al., 2010; Moradalizadeh et al., 2013). Similarly,

extraction through organic solvents is another choice, where oleoresins are macerated in organic

solvent for specific time period and after that the solvent is removed under reduced pressure. The

extracts obtained through this method contained trace amount of organic solvents. Therefore,

they cannot use in fragrance and food products (Sadgrove and Jones, 2015). These conventional

methods have some major drawbacks, including potential degradation of labile compounds,

unsatisfactory extraction efficiency, large consumption of expensive, environmental unfriendly

organic solvents and long extraction periods. In last few years advance extraction systems have

been developed that overcome the problems of traditional extraction methods (Sticher, 2008).

Among these advance extraction techniques, microwave-assisted extraction, supercritical fluid

extraction and pressurized liquid extraction are frequently used for the extraction due to better

extraction along with maximum analyte recovery, reduce the extraction time and minimum

solvent consumption (Wang and Weller, 2006; Wijngaard et al., 2012; Gañán and Brignole,

2013).

Supercritical fluid extraction (SFE) is interesting alternative technique as compare to

conventional solid–liquid extraction with lower working temperature, environmental friendly and

lower solvent consumption. It is type of liquid extraction where supercritical fluid used as

extractor instead of the usual liquid solvent. Wide ranges of supercritical fluids are available but

carbon dioxide is extensively used due to its comparatively lower critical temperature and

pressure. CO2 as supercritical fluid give better extraction for non-polar and moderate polar

compounds and polar organic solvent added as modifiers for the polar compounds extraction

(Gañán and Brignole, 2013). Online-SFE is coupled with different chromatographic instruments

like GC-MS, HPLC, HPLC-MS or supercritical fluid chromatograph (SFC) that determines

online chemical composition of extracts obtained through SFE. Pressurized liquid extraction

derived from SFE with advantage of suitable for temperature sensitive compounds, lower solvent

6

consumption, and higher extraction efficiency as compare to conventional extraction techniques.

It works under elevated temperature form 50-200 0C and high pressure (between 10–15 MPa).

Purpose of the higher pressure is to keep the solvent in liquid phase above its boiling

temperature, helps the solvent to rapidly penetrate in to matrix pores and rapid filling of

extraction chamber. Elevated temperature increases the diffusion rate, solubility and mass

transfer. SFE is better choice for extraction of thermo-labile compounds as compare to PLE

(Sticher, 2008). After extraction the next step is separation of pure compounds from these

extracts and essential oils. Various separation techniques such as preparative thin layer

chromatography (PTLC), vacuum fractional distillation (VFD), flash chromatography (FC),

column chromatography (CC), Preparative HPLC, Preparative gas chromatography (PGC) and

high speed counter current chromatography (HSCCC) have been used for isolation of pure

bioactive compounds from crude extracts and essential oils. PTLC is frequently used for

separation of extracts and essential oils. It is easily available, low in cost and gram to milligram

material can be separated. PTLC has few drawbacks such as long separation time, removal of

separated compounds from plate, impurities in final separated compounds and non-

reproducibility of results (Liu et al., 2013).

VFD is a separation technique used for fractionation and purification of essential oils on

the basis of their boiling points (Olmedo et al., 2014). This technique works under low

temperature and high vacuum, therefore, it has minimum chance of thermal decomposition of

heat sensitive compounds present in essential oils (Tovar et al., 2010). Flash chromatography

(FC) is modified form of column chromatography (CC) in which pressurize gas such as nitrogen

or air is passed through glass column filled with an adsorbent such as silica gel with particle size

40–63 mm or 35–70 mm. Mostly, It is used for fractionation of crude extract and essential oil

that is further purified through PTLC and preparative HPLC. FC gives better separation than

open column chromatography, with better peak resolution and shorter separation time. Moreover,

separated compounds and fractions in FC can be detected by coupling with online UV detector

(Bucar et al., 2013).

Preparative HPLC is a rapid, versatile and robust technique used for isolation of pure

compounds from complex mixtures. Preparative HPLC is different from other separation

techniques due to its reproducibility, high pressure of mobile phase and smaller particle size of

7

stationary phase. Normally, 3-10 µm particle size stationary phases are used in Preparative

HPLC, such a small particle size increases surface area to interact the solutes with stationary

phase and better separation of complex mixtures is possible. However, HPLC strictly requires

complex pretreatments to elimination of solid particles from samples (Tsai, 2001). Additionally,

some highly viscos sample easily absorb on to the solid matrix cannot be separated by

preparative HPLC. PGC is another alternative technique used for separation of volatile

compounds from essential oils. Usually large diameter columns up to 40 mm are used for

isolation of large amount of essential oil. There are few draw backs of this technique such as,

less resolution, requires large amount of carrier gas and denaturation of heat stable compounds

(Zuo et al., 2013).

Recently, the scientific interest in HSCCC is increased. It is an efficient preparative scale

separation technique that separates complex mixture of multi-gram samples of essential oils and

crude extracts with in few hours. It is liquid-liquid separation technique and has more advantages

than liquid-solid separation techniques such as no irreversible adsorption of the sample on

stationary phase, no need of expensive columns, minimum sample denaturation, maximum

sample recovery, minimum mobile phase consumption, minimum peak tailing (Sticher, 2008).

Nevertheless, it requires specific technical knowledge for selection of experimental conditions

(Ito, 2005). Interaction volume of sample with stationary phase in HSCCC is much higher than

Preservative HPLC. Therefore, it shows better separation of compounds than preparative HPLC.

Moreover, in HSCCC flow direction of mobile phase can reverse any time during operation

(Marston and Hostettmann, 2006).

The genus Pinus consists of 110 to 120 species that are distributed throughout boreal,

temperate, and tropical regions of the world, from sea level to more than 10,000 feet (da Silva

Rodrigues-Corrêa et al., 2013; Kaushik et al., 2013). Its ability to successfully grow in

inhospitable habitats (such as semiarid and subalpine/subartic regions) and its low technical

requirements for planting make Pinus one of the most suitable woody species for cultivating and

recovering of uninhibited and degraded agricultural lands, as well as nonagricultural or marginal

areas (Keeling and Bohlmann, 2006; Fox et al., 2007). Within different species of Pinus, Pinus

roxburgii is an important oleoresin producing plant that found in subtropical region of Pakistan,

North India, Kashmir, Nepal, Sikkim and the southern part of Tibet (Satyal et al., 2013). It is

commercially important tree due to its high quality timber, paper pulp, turpentine and oleoresin

8

(Tewari, 1994; Khare, 2008). Moreover, all parts of the plant are believed to possess medicinal

qualities in Ayurvedic and Unani systems of medicine (Siddiqui et al., 2009). Its wood is

aromatic, deodorant, haemostatic, stimulant, anthelmintic, digestive, liver tonic, diaphoretic, and

diuretic. It is useful in eye, ear, and pharynx diseases, foul ulcers, haemorrhages, haemoptysis,

worn infections, flatulence, liver diseases, bronchitis, inflammations, skin diseases, pruritus, and

giddiness (Kaushik et al., 2012). The plant aerial parts, barks and needles are rich in various

classes of bioactive compounds such as essential oil, resin acids, phenolic compounds,

flavonoids, tannins, alkaloids. During insect attack or physical injury, Pinus roxburghii exudes

oleoresin from trunk, branches and sometimes from cone (Kala, 2004). Pinus roxburghii

oleoresin is fractionated into monoterpene rich turpentine and diterpenoid resin acids or rosin.

These monoterpenes and diterpenoids are feedstocks for renewable chemicals widely used in

products that compete with petroleum derived feedstocks such as solvents, pesticides,

pharmaceuticals, flavors, cosmetics, household cleaners, printing inks, adhesives, coatings and

paper-sizing (Susaeta et al., 2014). Moreover, phytochemical studies have shown that these types

of oleoresins consist mainly of tricyclic acid diterpenes of the pimarane and abietane class as

well as bicyclic diterpenes, especially labdanes (Langenheim, 2003). Among the various classes

of plant terpenes, diterpenes stand out: they present several well-known biological activities such

as antiparasite (Tóro et al., 2003), anti-inflammatory, antifungal, and vascular smooth muscle

relaxant actions (Boeck et al., 2005; Rios and Recio, 2005; Ambrosio et al., 2006; Kaushik et

al., 2012).

The genus Boswellia contains nearly 43 different species that are distributed throughout

Arabian Peninsula, Pakistan, North Africa and India. Boswellia serrata is a deciduous middle-

sized tree. It is grown in tropical regions of Nigeria, India and Pakistan (Aman et al., 2010).

Boswellia serrata oleo gum resin is used to cure asthma, stomach, intestine, cough, diarrhea,

hemorrhoids, dysentery and pulmonary diseases in Unani and Ayurvedic medicinal systems

(Aman and Balu, 2009). Moreover, it was reported that different parts of this plant showed

antibacterial, anti-inflammatory, antifungal and anticancer activities (Aman et al., 2010; Sharma

et al., 2010). Phytochemical studies showed that monoterpene hydrocarbons and oxygenated

monoterpenes are major class of chemical compounds present in essential oil isolated from oleo

gum resin. Moreover, tetrahydro-linalool,α-thujene, carene-3, benzyl tiglate, methyl isoeugenol,

9

p-cymene, α- terpineol, epi-cubenol and α-pinene were the major components of its essential oil

(Singh et al., 2007).

Aims and objectives

The extraction techniques, chemical composition and anticancer activity of oleoresins are

still not well defined. Therefore, the present research project was designed with following

objectives.

1. To extract essential oil from oleoresins of Boswellia and Pinus species.

2. To isolate and purify major bioactive compounds using seperation techniquies.

3. To characterization the most active essential oils and bioactive isolates.

4. To evaluate anticancer activity of essential oils, fractions and sub fractions using in vitro

anticancer assays.

10

Chapter 2

Review of literature

2.1 Pinus roxburghii

2.1.1 Introduction

Pinus roxburghii is a tree belongs to the Pinaceae family. It is commonly known as long-

leaved pine and Chir pine and distributed in subtropical region of Pakistan, India, Sikkim, Azad

Jammu Kashmir, Nepal and Tibet at 450-2300 m in the outer ranges of Himalaya where the full

force of the monsoon is felt (Satyal et al., 2013). It is commercially important tree due to its high

quality timber, paper pulp, turpentine and oleoresin (Tewari, 1994; Khare, 2008). Moreover,

nearly all parts of this plant like stem, leaves, oleoresin, cones and roots reveals medicinal values

in Unani and Ayurvedic systems of medicine (Siddiqui et al., 2009). Wood is effective against

liver, stomach, eye, skin, pharynx and ear diseases (Kaushik et al., 2012). The plant aerial parts,

barks and needles are rich in various classes of active compounds such as essential oil, resin

acids, phenolic compounds, flavonoids, tannins, alkaloids. During insect attack and physical

injury, Pinus roxburghii exudes oleoresin from trunk, branches and cones (Kala, 2004), that is

rich source of monoterpenes, bicyclic and tricyclic acid especially abietane, labdanes and

pimarane (Langenheim, 2003). Essential oil isolated from leaves, stem and oleoresin is

renewable chemical source that compete with petroleum. It is also used in cosmetics, pesticides

and pharmaceutical industry (Susaeta et al., 2014). Among the different classes bioactive

compounds terpenes, diterpenes, resin acids, phenolics, flavonoids and tannins exhibited

significant biological activities like antioxidant, antibacterial, antiparasite (Tóro et al., 2003),

anti-inflammatory, antifungal and anticancer activities (Boeck et al., 2005; Rios and Recio,

2005; Ambrosio et al., 2006; Kaushik et al., 2012).

The generic name, Pinus, comes from Latin meaning pit (resin) (Little and Critchfield,

1969). In early 19th

century, it was known as fir, this name comes from Old Norse fyrre that is

still used in European countries (Richardson, 2000). In Sanskrit it is known as Bhadradaru,

Manojna; in Hindi, Chir, Chil, Salla; in Bengali Saralgachha; Saraladeodara in Gujarati;

Simaidevadari in Tamil, Chir in urdu; Salla, Charalam in Malayalam; and Devadaru in Telugu

(Kirtikar and Basu, 1918). Pinus roxburghii is distributed in Indian occupied Kashmir,

Uttarakhand, Himachal Pradesh, Bhutan, Nepal, Sikkim, Afghanistan and the southern part of

https://en.wikipedia.org/wiki/Pinaceae

https://en.wikipedia.org/wiki/Old_Norse_language

11

Tibet at 450-2300 m in the outer ranges of Himalaya where the full force of the monsoon is felt

(Satyal et al., 2013; Shuaib et al., 2013). In Pakistan, it is growing in, Punjab, lower parts of

NWFP and Azad Kashmir (Siddiqui et al., 2009).

Figure 2.1: Typical image of Pinus roxburgii tree

Pinus roxburghii is a tall tree with length of 55 to 100 meter and diameter up to 100

centimeter (Siddiqui et al., 2009). Its bark is thick, non-resinous, dark red-brown in color, scaly

and longitudinally fissured; winter buds are ovoid, small and brown in color. Each bundle

contained three needles like leaves; differentiate it from other Pinus species. Its cones are shortly

pediculate, ovoid, 10 to15 × 6 to 9 centimeters. Seeds are 8 to 12 millimeter in length with a 2.5

centimeter long wing, at maturation they attain height of 45-54 meter and start bearing cones

from 15-40 years of age (Shuaib et al., 2013).

2.1.2 Chemical composition

Pinus roxburghii is an aromatic and ornamental plant. Within different species of Pinus,

present specie is famous with high quality timber, terpene and its oleoresin. Various part of this

plant contains essential oil including cones, aerial parts, shoots, truck and oleoresin (Rawat et al.,

2006). α-pinene, longifolene, β-pinene and car-3-ene are the major compounds present in its

essential oil (Kaushik et al., 2012). Pinusoic acid, quinoroxburghianoic acid, dehydroabietic acid

and 12- hydroxydehydroabietic acid are the major resin acids present in oleoresin (Shuaib et al.,

12

2013). Pinus roxburghii is a rich source of essential oil, oleoresin, flavonoids, alkaloids, phenolic

compounds, xanthones, vitamin C, resin acids, tannins and other compounds (Hassan and Amjid,

2009). Its seeds are edible, contained sufficient amount of edible oil with high polyunsaturated

fatty acids and linoleic acid (Ahmad et al., 1990). Fresh bark contained 4.9-6.8% total sugars

with 1.25- 2.49 % glucose, 1.2-2.9 % fructose and 1.17-1.87 % arabinose (Ahmad et al., 1990).

Distillation of oleoresin yields essential oil and rosin used in chewing gums (Wiyono et al.,

2006). Numerous chemical compounds isolated from different parts of Pinus roxburgii.

Compounds isolated from bark are hexacosylferulate (Hussain, 1987), 1,5-diliydroxy-3,6,7-

triniethoxy-8-allyloxyxanthone, taxifolin, rhamnetin,1-hydroxy-3,6-diinethoxy-2-β-D-

glucopyranoxanthone (Kirtikar and Basu, 1918), friedelin, ceryl alcohol, β-sitosterol (Arshad and

Ahmad, 2004), quercetin, 3,4-dihydroxybenzoic acid, catechin, 3,4-dihydroxycinnamic acid,

kaempferol, pinosylvin, resin acid, pinoresinol, sterols, gallocatechin (Uniyal et al., 2006). The

essential oil isolated from leaves and stems contained caryophyllene, camphene, α-terpeniol,

fenchene, 3-carene, α-pinene, longifolene, α-humulne, dipentene, β-pinene and polymeric

terpenes (Kala, 2004). Chemical composition of monoterpenes present in essential oil

significantly varies with geological position and origin of seed (Kaushik et al., 2012).

Pinus roxburghii is commercially important due to its high quality oleoresin which

contained α-terpinene, 3-carene, α-terpineol, pinene, dl-camphor, longifolene (Rawat et al.,

2006), limonene, d-borneol and camphene (Willför et al., 2009). Some novel resin acids isolated

from oleoresin are pinusoic acid, quinoroxburghianoic acid, dehydroabietic acid and 12-

hydroxydehydroabietic acid (Shuaib et al., 2013). Essential oil isolated from Pinus roxburgii

needles contained numerous bioactive compounds including α-pinene, 3-carene, caryophyllene

as the major compounds followed by some minor compounds including α-terpineol, terpinyl

acetate, α-longipinene, caryophyllene oxide, borneol acetate and β-myrecene (Iqbal et al., 2011).

Similarly, (Hassan and Amjid, 2009) isolated essential oil from stems of Pinus roxburghii

through hydro-distillation method and chemical constituent was determined by GC-MS. The

results of GC-MS analysis showed α-pinene, 3-carene and caryophyllene was the major

compounds followed by minor compounds including p-cymene, 1-terpinen-4-ol, o-cymene,

terpinenol, phallenderene, limonene, borneol acetate, camphene, caryophyllene oxide, farnesene,

Ɣ-terpinene, tepinyl acetate, butanoic acid, 3-methyl- 2-phenylethyl ester and farnesyle acetate.

13

Essential oil of fresh fruit of Pinus roxburghii contained α-pinene, β-pinene, camphene, l-β-

pinene, β-caryophyllene, limonene and α-terpinol (Shah et al., 2014).

Needle and bark contained different phenolic acids and flavonoids such as 3,4-

dihydroxybenzoic acid, quecetin, kampherol, pinosylvin, rhamnetin, catechin, pinoresinol,

isorhamnetin, 3,4-dihydroxycinnamic acid, myrcetin, dihydro-monomethyl, taxifolin and

secoisolariresinol were major phenolic for flavonoids (Naeem et al. 2010).

H

H

CH2

CH2

H3C CH3

α-pinene 3-carene β-myrecene

HH

CH3

CH3

H2C

CH3

HO

OCH3

CH3CH3

H2C

CH3

CH3

H

CH3

CH3H

OHO

β-caryophyllene Isopimaric acid Neoabietic acid

2.1.3 Uses

2.1.3.1 Common uses

Timber of Pinus roxburghii is used for shelterbelts and as fuel for cooking (Siddiqui et

al., 2009). Its seeds are edible, contain sufficient amount of edible oil (Puri et al., 2011).

Different parts of Pinus roxburghii were used in folk medicine in Indo-Pak region including

cones, oleoresin, trunk, essential oil, wood, bark, needles and leaves (Siddiqui et al., 2009).

Aerial parts especially leaves are used to prevent contamination of food and life stock during

rainy season (Siddiqui et al., 2009). Barks are used for melting metals and designing of various

house hold metallic outfits. Oleoresin and wood are used for lighting. Cones are used for edible

oil, decoration, lighting and replantation. Twigs are used for the treatment of asthma (Siddiqui et

al., 2009). Oleoresin is used for repair earthenware (Abbasi et al., 2010). In Nepal, equal

concentration of oleoresin and common salt boils with 100 ml of water and used against common

14

coughs. Oleoresin is used as plaster for bone fracture, boils, heel cracks and soften the scar tissue

(Smaleh et al., 1976). The bark contend significant amount of tannins that is used for coloring

the leather industry (Maimoona et al., 2011).

2.1.3.2 Traditional uses

Pinus roxburghii is traditional used for the treatment of liver, stomach, eye, blood, skin,

pharynx and ear diseases (Kaushik et al., 2012). Its oleoresin is used to heel boils and external

injury (Naeem et al., 2010). In Nepal, Pinus roxburghii oleoresin is used to cure cough, gastric

problem and tuberculosis (Joshi et al., 2011; Satyal et al., 2013). oleoresin is also used to remove

hair from body (Beri, 1970). In India and Pakistan, Pinus roxburghii oleoresin is locally known

as ganda baroja and baroja respectively that is used in treatment of skin diseases, snake bite,

ulcer and scorpion stings (Zafar et al., 2010). Ash of resinous wood mixed with mustard oil and

used inside and lower eyelids of the eyes to keep it attractive and clean (Naeem et al., 2010). The

wood of the Pinus roxburghii wood is stimulant and diaphoretic in nature and used for the

treatment of ulceration and cough, fainting (Swales and Dev, 1979). The needles are used as

diuretic (Ahmad et al., 1990). Essential oil isolated from stem, wood and leaves are used as

nerve tonic, hemostatic and also used for the treatment of chronic bronchitis, typhoid. Inhaling

the essential oil vapors is effective for relief of bronchitis. Bark is used for skin diseases, burns,

and cracks (Maimoona et al., 2011). The bark from the roots and stems of is also used as an

antidiabetic (Khan et al., 2012).

2.1.3.3 Pharmacological uses

Pinus roxburghii is known to be a rich source of essential oil, flavonoids, tannins,

alkaloids, phenolic compounds, xanthones, vitamin C, resin acids and other compounds (Hassan

and Amjid, 2009). Almost all the parts of plant are found to contain biologically active

compounds. Pinus roxburghii oil is known to have strong antimicrobial property. Literature

survey has shown all parts of plant contain potent antimicrobial, anticancer, Antidyslipidemic,

analgesic, anti-inflammatory, anti-asthmatic, anti-oxidant, hepatoprotective, anthelmintic and

anti- insecticidal activities.

2.1.3.3.1 Hepatoprotective activity

Essential oil isolated from Pinus roxburghii wood was evaluated for their

hepatoprotective activity at different concentration. The authors observed significant increase in

serum levels of alkaline phosphatase, total bilirubin, malondialdehyde, aspartate

15

aminotransferase, alanine aminotransferase as well as decreased in total protein induced and

level of reduced glutathione (GSH). Furthermore, they observed that at concentration of 200 and

300 mg/kg hepatotoxins were suggestively restored towards the normal levels that might be due

to the triterpenes and steroids in essential oil that inhibit free radical formation and reduced lipid

peroxidation (Khan et al., 2012).

2.1.3.3.2 Antibacterial and antifungal activities

All part of Pinus roxburgii including stems, needles, leaves, male and female cones, fresh

fruit, wood and oleoresin extracts/ essential oils showed significant antibacterial and antifungal

activity against plant and human pathogenic microbes. Essential oil isolated from of the needles

of Pinus roxburghii inhibited the growth of gram positive bacteria such as Bacillus subtilis and

Staphylococcus aureus while showed inhibition against gram negative bacteria such as

Escherichia coli, Salmonella typhi and Enterobacter aerogenes (Murad et al., 2011). Similar

finding was observed in another study where stem essential oil showed antibacterial activity

against gram positive bacterial strains including Bacillus subtilis and Staphylococcus aureus,

while no antibacterial activity was detected against gram negative bacteria including

Enterobacter aerogenes and Escherichia coli (Kaushik et al., 2013). In case of antifungal

activity, stem essential oil showed significant antifungal activity and dose-dependent inhibition

the fungal growth against different fungi (Melkania et al., 1982). Similarly in another report,

stem essential oil showed antifungal activity against Trichoderma viride, Aspergillus flavus,

Aspergillus vessicolor, Aspergillus candidus, Aspergillus terrus, and Aspergillus niger (Kaushik

et al., 2013).

Alcoholic and aqueous extracts of male, female cone, bark, leaves and stem were tested

against one plant pathogenic bacteria, Agrobacterium tumefaciens and four human pathogenic

bacteria, Salmonella arizonae, Staphylococcus aureus, Salmonella typhi and Escherichia coli.

Alcoholic and aqueous extracts inhibited the bacterial growth against Agrobacterium

tumefaciens, while only stem extracts showed antibacterial activity against Escherichia coli

(Kaushik et al., 2012). In another report, antibacterial and antifungal activity of Pinus roxburghii

wood essential oil, methanol and chloroform extracts were evaluated through disc diffusion

assay against Aspergillus clavatus, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus

subtilis, Aspergillus niger, Escherichia coli, Candida albicans and Streptococcus pyogenes

strains. Essential oil and chloroform extract exhibited good antibacterial and antifungal activity

16

against all tested strains, while methanol extract showed least antibacterial and antifungal

activities. Essential oil isolated form bark, needles and wood were active against pathogenic

bacteria except Erwinia amylovora strain in disc diffusion assay (Salem et al., 2014).

Furthermore, wood essential of showed highest MIC 64 µL/mL and 250 µL/mL against Baccilus

subtilis and Staphylococcus aureus, respectively. Alcohol and water extracts of bark, leaf, stem,

male and female cone of Pinus roxburghii inhibited the bacterial growth of Salmonella arizonae,

S. typhi and Staphylococcus aureus, Agrobacterium tumefaciens except in Escherichia coli

(Parihar et al., 2006). Antibacterial activity of methanol extract of oleoresin against Gram

positive and Gram negative bacteria (Enterococcus faecalis, Shigella dysenteriae,

Staphylococcus aureus, Escherichia coli, Micrococcus luteus, Bacillus subtilis, Pseudomonas

aeruginosa and Salmonella typhi) was evaluated via agar-well diffusion assay (Shuaib et al.,

2013). It was observed that methanol extracts showed much better activity against Gram positive

bacterial activity as compare to Gram negative bacteria. Moreover, methanol extract was not

active against Pseudomonas aeruginosa as well as Salmonella typhi strain, at 100 µg/ml

concentration, while showed variable inhibition against all other strains.

Antifungal activity of chir pine oleoresin and its fractions were evaluated through micro

dilution broth method against Macrophomina phaseolina, Fusarium solani, Lasiodiplodia

theobromae, Colletotrichum gloeosporioides, Phytophthora infestans, Sclerotium rolfisii and

Fusarium oxysporum. Oleoresin and its fractions showed significant antifungal activity with

MIC in range of 1.95 to 1000 μg/mL (Andrade et al., 2014). Essential oil isolated from needles

evaluated for their antibacterial and antifungal activity through disc diffusion assay at different

concentrations. Antibacterial activity of essential oil of the needles indicated that this oil showed

maximum activity against Staphylococcus aureus and Bacillus subtilis with inhibition zone of

12.12-36.91 mm, while no activity was observed against Escherichia coli, Salmonella typhi and

Enterobacter aerogenes (Zafar et al., 2010). In antifungal activity, volatile oil inhibited the

fungal growth of Aspergillus terrus, Aspergillus flavus and Trichoderma viride with inhibition

zone in range of 11.5-40.5 mm. In another report aqueous and ethanol leaf extracts inhibited the

growth of Salmonella typhi and Escherichia coli (Parihar et al., 2006). Moreover, isolated

essential oil through steam distillation method did not showed the antibacterial activity against

Trichoderma viride at 5 µl, but at this low concentration of essential oil showed antibacterial

activity against Aspergillus flavus and Aspergillus terrus with inhibition zone 13 and 20 mm,

17

respectively (Motiejūnaite and Peciulyte, 2003). Essential oil isolated from stems was evaluated

for the antimicrobial activity against numbers pathogenic microorganism through agar disc

diffusion assay. Authors observed Escherichia coli and Enterobacter aerogenes showed high

resistance against all concentrations of essential oil, while showed inhibition against Bacillus

subtilis, Salmonella typhi and Staphylococcus aureus with inhibition zone in range of 8.5 -33

mm. In antifungal activity essential oils at different concentration inhibited the fungal growth

against Aspergillus terrus, Aspergillus flavus and Trichoderma with inhibition zone 8.5- 38.5

mm, while no activity was observed against Aspergillus candidus, Aspergillus vessicolor and

Staphylococcus niger up to 20 µl of pure essential oil (Hassan and Amjid, 2009). Essential oil

isolated from fresh fruit exhibited bacterial growth against Bacillus subtilis, Staphylococcus

aureus, K. pneumonia, Escherichia coli, Pseudomonas aeruginosa and Poteus vulgaris (MTCC-

1771) in agar well diffusion assay. In agar well diffusion assay, highest activity was observed

against P. vulgaris 32 mm zone of inhibition and MIC 12.8 mg/mL, while positive control

Streptomycin sulphate showed inhibition zone of 30 mm (Shah et al., 2014).

2.1.3.3.3 Antioxidant activity

Antioxidant activity of needles extracts including ethanol and its fractions were evaluated

by Trolox equivalent antioxidant capacity assay (Puri et al., 2011). The author reported that

ethanol extract and their fractions (n-hexane, chloroform and n-butanol) exhibited significant

antioxidant activity. In another report antioxidant activity of needle and bark extracts was

assessed by free radical scavenging assay (Maimoona et al., 2011) and reported that polar

fraction of needle and bark extracts showed substantial antioxidant activity. DPPH free radical

scavenging potential of essential oil isolated from fresh fruit showed 10% free radical

scavenging at concentration of 100 mg/mL of the oil (Shah et al., 2014)

2.1.3.3.4 Anti-dyslipidemic activity

Ethanol extract and their fractions (n-hexane, chloroform and n-butanol) of Pinus

roxburghii needles were investigated for anti-dyslipidemic activity in hyperlipidemic golden

Syrian hamsters (Puri et al., 2011). All the extracts exhibited significant potential to lower

plasma lipid level. Moreover, all extracts showed good effect on high density lipoprotein and its

ratio with total cholesterol level in dyslipidemic hamster model.

2.1.3.3.5 Analgesic and anti-inflammatory activity

18

The ethanol extract of bark exhibited high anti-inflammatory as well as analgesic activity

in Wister rat and albino mice assays, respectively (Kaushik et al., 2012). The authors proposed

that flavonoids were responsible of anti-inflammatory and analgesic activity.

2.1.3.3.6 Anti-asthmatic activity

Anti-asthmatic activity of alcoholic extract of Pinus roxburghii was assessed through

different in-vitro and in-vivo assays. There results showed that Pinus roxburghii alcoholic extract

showed appreciable anti-asthmatic activity against different in-vitro and in-vivo assays (Kaushik

et al., 2013).

2.1.3.3.7 Anticancer activity

Essential oil isolated from fresh fruits showed excellent anticancer activity against

different human cell lines including A549 (lung), C6 (glioma), T47D (breast), MCF (breast) and

TH-1(colon) cell lines in MTT assay. Essential oil showed excellent anticancer activity against

all cell line and MIC values are nearly equal to positive control Mitomycin C. Highest MIC

value was observed against lungs cancer cell lines with MIC 90 ug/mL, while positive control

Mitomycin C showed MIC 92 ug/mL. Moreover, it was reported that complex mixture of mono

and sesquiterpenes might be responsible of their anticancer activity (Shah et al., 2014). In

another report cone essential oil showed remarkable anticancer activity with 100% killing of

MCF-7 cells at 100 ug/ml (Satyal et al., 2013).

2.1.3.3.8 Anthelmintic, anti-insecticidal activity and other biological activities

Traditionally, Pinus roxburghii used for the treatment of intestinal worm infection. There

are numerous reports are available on their anthelmintic activity. Essential oil isolated from

needles was evaluated for their anthelmintic activity in adult earthworms Pheretima posthuma at

different concentrations. The authors reported that higher concentration of essential oil cause to

happen early paralytic effect and death of worms. Moreover, it was reported that essential oil or

its active compounds may bind with free proteins in the gastrointestinal tract of the host animal

or may be bind with to glycoprotein on the cuticle surface of the parasite and cause death

(Langley and Mort, 2012). Most of the commercially available chemical insecticides are harmful

for living beings as well as environment. Oleoresin and its essential oil have insecticidal activity

and at low concentration are non-toxic and environmental friendly. Previously, It was reported

that essential oil and its isolated compounds, showed high repellent activity against cockroaches

and house flies (Singh et al., 1990). Alpha-pinene, camphor and limonene are the main

19

components of Pinus roxburgii essential oil and reported as an insect repellents (Nerio et al.,

2010). Moreover, geraniol is another component of its essential oil and effective against

mosquito repellent (Barnard and Xue, 2004). Anti-parasitic activity of oleoresin, chloroform

fraction, steam oil, α and β-pinenes at the concentration of 0.5 and 1 ppm were examined against

Lernaea cyprinacea (Tóro et al., 2003). The results indicated that chloroform fractions showed

higher activity than pure compounds α and β-pinenes. Essential oil isolated from aerial parts was

not activity against Culex quinquefasciatus and Aedes aegypti mosquito (Makhaik et al., 2005).

2.2 Boswellia serrata

2.2.1 Introduction

Boswellia serrata Roxb. commonly known as kundar, is an annual tree belonging to

Burseraceae family that is found in India, Nigeria, Yemen, Arabia, Oman and Pakistan

(Maupetit, 1984; Singh et al., 2008; Khan and Abourashed, 2011). It is a commercially important

plant due to its highly valuable oleoresin, used in food, flavor and perfume industries. Moreover,

different parts of this plant have been used for treatment of various health problems in Unani and

Ayurvedic medicinal systems (Nigrami, 1995; Lubhaya, 1979). It is a moderate to large sized

branching tree, height up to 18 m and girth 2.4 m. Barks are thin, yellow, greenish grey, and

yellow in color (Alam et al., 2012). The Burseraceae family contains 17 genera and 600 species

wide-spread in all tropical regions. The genus Boswellia contains nearly 43 different species that

are distributed throughout Arabian Peninsula, Pakistan, North Africa and India (Aman et al.,

2010).

Boswellia serrata oleoresin is complex mixture of essential oil, resin acids and

polysaccharides. The presence of significant amount of essential oil gives it pleasant smell and

high commercial values. Hydro and steam distillation methods are used to isolate the essential oil

from oleoresins that can used in aromatherapy, paints, beauty products, perfumes and varnishes.

Tapping is a most common method used to extract golden yellow oleoresin from Boswellia

serrata. Initially, oleoresin is transparent liquid, with the passage of time, it becomes hard and

color changes to golden yellow (Alam et al., 2012; Ismail et al., 2014). The trees have been

wounded in April or March and oleoresin is collected throughout summer and autumn seasons.

One tree gives good quality of oleoresin continuously up to three years, after that period the

quality of oleoresin is gradually decreased. Therefore, it is important to rest the tree for next

20

three years to get better quality of oleoresin in future (Siddiqui, 2011). In last several decays,

Boswellia serrata oleoresin has been used to cure various aliment especially rheumatism, skin

diseases dysentery, dyspepsia, lung diseases, haemorrhoids, rheumatism, urinary disorders and

corneal ulcer in Unani and Ayurvedic system of medicines. Moreover, it is an important

ingredient of different compounds formulations that is used for the treatment of rental disorder

(Nigrami, 1995; Lubhaya, 1979). Furthermore, It possesses anti-carcinogenic (Huang et al.,

2000), anti-fungal (Garg, 1974), antimicrobial, anti-complementary (Kapil and Moza, 1992)

Juvenomimetic (Dennis et al., 1999) anti-inflammatory, anti-arthritic and analgestic activity

(Kimmatkar et al., 2003). It showed beneficial effects in Immunomodulation (Sharma et al.,

1996), Bronchial asthma (Gupta et al., 1998), Polyarthritis (Sander et al., 1998), Hepatitis C-

virus (Hussein et al., 2000), Colitis (Gupta et al., 2001) and Crohn's disease (Gerhardt et al.,

2001). In English, Boswellia serrata oleoresin is known as Indian olibanum and Indian

frankincense, in Hindi, Urdu and Gujarati as Kundar and Salai, in Malayalam, Tamil and Telugu

as Parangi, Saambraani, in Sanskrit as Ashvamutri, Kundara and Shalliki (Siddiqui, 2011).

Boswellia serrata is one of the most ancient herb, native to Indo-Pak region and found other

regions of earth including northern Africa and the middle East (Maupetit, 1984; Khan and

Abourashed, 2011). This plant is commercial grown for its high quality oleoresin in Madhya

Pradesh, Chhattisgarh, Andhra Pradesh, Jharkhand and Gujarat regions of India (Siddiqui, 2011).

Moreover, it is found in Northern Africa, Pakistan and few Middle East counties (Maupetit,

1984; Khan and Abourashed, 2011; Ismail et al., 2014).

Figure 2.2: Typical image of Boswellia serrata tree

21

Boswellia serrata is a deciduous, medium to large sized tree that can attain height up to

18-20 m and grid up to 2.0-2.4 m, bark is resinous, thin and ashy, greyish green color (Ismail et

al., 2014) The leaves are crowded at the end branch, ex-stipulate, alternate and 30-45 cm long.

The flowers are white in color, bisexual, shorter than leaves and crowded at the end. The leaflets

are 8-15 in number and 2.5- 6.3 cm long. The petals are 0.4-0.8 cm long, erect, ovate shaped

with basal disk. The fruits are 1.25-1.30 cm in length, cotyledous, trigonous that splits in to three

valves (Siddiqui, 2011).

2.2.2 Chemical composition

Boswellia serrata oleoresin is composed of resin acids, essential oil and polysaccharides

(Shama and Varma, 1980; Gangwal and Vardhan, 1995). Essential oil from oleoresin can be

separated by hydro and steam distillation and their chemical composition may vary with

extraction method, part of plant, geological location and season (Hussain et al., 2013). GC-MS

analysis of steam distilled essential oil showed that n-octyl acetate, n-octanol, limonene, α-

pinene, verticilla-4(20),7,11-triene, incensole acetate are the major compounds followed by α-

thujene, camphene, myrcene, α-terpinene, p-cymene, Z-ocimene, E-ocimene, γ-terpinene,

eucalyptol, linalool, terpinen-4-ol, α-terpineol, carvone, α-copaene, thunbergene, neocembrene

A, 1-methoxy-2-methyl-, 3,5-dimethoxytoluene, n-octyl formiate, citronellyl acetate, neryl

acetate, geranyl acetate, n-decyl acetate as minor compounds (Camarda et al., 2007). Similarly,

(Ahmed et al., 2014) reported the chemical composition of hydro distilled essential oil of

Boswellia serrata oleoresin with terpinyl acetate, sabinene and terpinen-4-ol as major

compounds. In another study (Hamm et al., 2005) determined the chemical composition of

Boswellia serrata oleoresin essential oil through SPME–GC/MS. They reported that α-thujene,

β-myrcene, p-cymene, methylchavicol, β-bourbonene and methyl-eugenol were the most

prominent components of Boswellia serrata oleoresin essential oil. (Verghese et al., 1987)

reported that α-thujene was the major component of Indian Boswellia serrata oleoresin essential

oil, followed by α-pinene, estragol, α-phellandrene, linalool, β-pinene, limonene, α–terpineol.

(Gupta et al., 2016) studied the effect of geographical variation on chemical composition of

Boswellia serrata oleoresin essential oil. They observed that oleoresin collected from different

locations of India revealed significant variation in their chemical composition and these

variations directly effects on biological activity. They found that essential oil isolated from

22

Madhya Pradesh, India contained higher percentage of sesquiterpenes and oxygenated

monoterpenoids than other essential oils. (Kasali et al., 2002) determined the chemical

composition of Boswellia serrata bark essential oil. The results showed that α–pinene as a major

component of essential oil, followed by β-pinene, p-cymene, trans–pinocarveol, thuja-2,4(10)-

diene, cis-verbenol, limonene, borneol, verbenone and myrcene. In sesquiterpenes, α–copaene

was the only compound present in essential oil. (Krohn et al., 2001) isolated four boswellic

acids, O-acetyl-11-keto-β-boswellic acid, β-boswellic acid, 11-keto-β-boswellic acid and 3-O-

acetyl-β-boswellic acid from oleoresin of Boswellia serrata. Similarly, (Ganzera and Khan,

2001) identified six boswellic acids including 3-acetyl- β-boswelic acid, 11-keto-β-boswellic

acid, β-boswellic acid, 3-α-acetyl-11-keto-β-boswellic acid , 3-acetyl-α-boswelic acid and α-

boswelic acid in Boswellia serrata.

2.2.3 Pharmacological uses

Boswellia serrata is known to be a rich source of essential oil, flavonoids, tannins,

alkaloids, phenolic compounds, xanthones, vitamin C, boswellic acids and other compounds.

Almost all the parts of plant are found to contain biologically active compounds. Literature

survey has shown all parts of plant contain potent antimicrobial, anticancer, immunomodulatory,

anti-hyperlipidmic, antibiofilm, anti-convulsant, antidepressant, hepatoprotective, antiulcer and

antiimflammetry activities.

2.2.3.1 Antimicrobial activity

Antimicrobial activity of methanol extract of Boswellia serrata leaves and flowers were

determined against various bacterial strains by disc diffusion assay (Aman et al., 2010). They

found that 1.25 mg/disc concentration of both extracts showed significant antibacterial activity

against all the bacterial strains except Bacillus cereus and Micrococcus. Similarly, (Kasali et al.,

2002) reported that essential oil isolated from Boswellia serrata bark show good antibacterial

activity against Proteus Mirabilis, Staphylococcus aureus and Escherichia coli strains. In

another report (Patil et al., 2010) observed significant antibacterial activity of bark and stem

extracts of Boswellia serrata against Bacillus subtilis, Klebsiella pneumoniae and Escherichia

coli. Moreover, (Padhi and Mahapatra) evaluated the antibacterial activity of petroleum ether,

methanol, ethanol and chloroform extracts of Boswellia serrata leaves against three gram

positive (Staphylococcus epidermidis, Streptococcus pneumoniae, Staphylococcus aureus) and

three gram negative (Proteus vulgaris ,Escherichia coli and Pseudomonas aeruginosa) bacterial

https://en.wikipedia.org/wiki/Staphylococcus_aureus

https://en.wikipedia.org/wiki/Escherichia_coli

23

strains by Cup plate assay. They found that all the extracts showed activity against bacterial

strains. Methanol extract exhibited highest activity with higher zone of inhibitions than other

extracts. Moreover, they reported that antibacterial activity of these extracts may due to resin

acids, phenolics and terpenes. (Avasthi and Purkayastha, 2013) reported antibacterial activity of

methanol extract and its fractions of Boswellia serrata oleo gum resin against multi-drug

resistant clinically isolated bacterial strains. They observed that all the extracts and fractions

showed resistance against bacteria but the dichloromethane fraction showed maximum resistance

against tested bacterial stains. These results were further confirmed by direct bio-autography

assay that showed same results as in disc diffusion assay. Furthermore, they reported that

antibacterial activity of extract and fractions might be attributed due to triterpenoids, alkaloids,

steroids and flavonoids present in oleo gum resin.

Steam distilled essential oil of Boswellia serrata oleo gum resin was evaluated against

normal and methicillin-drug resistant gram positive and gram negative bacterial and human

pathogenic fungal strains (Camarda et al., 2007). Overall, steam distilled resin oil showed higher

antibacterial activity with low MIC values in range of 12.86 - 107.18 ug/ml against gram

negative bacterial strains than gram positive strains 89.17 - 107.20 ug/ml. In both fungal strains,

Candida albicans and Candida tropicalis stream distilled resin oil exhibited same antifungal

activity with MIC values 12.86 ug/ml. Moreover, they reported that antimicrobial activity of

steam distilled resin oil may attribute due to synergistic effect of several components.

Antibacterial activity of silver nano particles synthesized by reduction of silver nitrate with

Boswellia serrata flower extract was determined through disc diffusion assay (Kudle et al.,

2013). They observed that 5ul extract nano particles significantly inhibited the bacterial growth

against both gram positive and negative strains.

Acetyl-11-keto-β-boswellic acid, β-boswellic acid and 11-keto-β-boswellic acids are the

pentacyclic triterpenes boswellic acids and reported to have antibacterial activity against

different bacterial strains. (Raja et al., 2011) reported that acetyl-11-keto-β-boswellic acid

exhibited higher antibacterial activity with low MIC range of 2 – 8 ug/ml against gram positive

strains than other two compounds. Acetyl-11-keto-β-boswellic acid killed Staphylococcus aureus

ATCC 29213 in concentration dependent manner up to 8 × MIC. Moreover, this compound

inhibited the biofilms generated by Staphylococcus epidermidis and Staphylococcus aureus.

2.2.3.2 Analgesic and Psychopharmacological activity


24

The Boswellia serrata oleoresin showed significant analgesic effect in an animal

experimental model. It was observed that Boswellia serrata oleoresin significantly reduced the

natural activity and induces ptosis in rats (Menon and Kar, 1971).

2.2.3.3 Anti-hyperlipidmic activity

In an animal experimental model, the anti-hyperlipidmic activity of Boswellia serrata

oleoresin was studied on rabbits. The authors observed that animals treated with different

concentration of alcoholic extract of Boswellia serrata oleoresin show significant reduction in

triglycerides and serum cholesterol level. The daily dose of 25-50 mg/Kg of extract reduced the

cholesterol level (30-50 %) and triglycerides level (20-60 %) (Zutshi et al., 1986).

2.2.3.4 Anti-convulsant activity

In vivo anticonvulsant activity of Boswellia serrata oleoresin was performed on group of

mice. It was observed that the treatment with Boswellia serrata in range of (10 and 200 mg/kg)

postponed the start of convulsion along with duration of tonic-clonic convulsions as well as it

significantly reduced PTZ and STR-induced mortality in mice. It also considerably reduced

severity of electrically kindled seizures in rats and total number of rats seizure per group. Mice

treated with Boswellia serrata (10 and 200 mg/kg) significantly amplified level of brain GABA,

while it considerably reduced the elevated level of brain NO and XO. As a result, the discoveries

of this study deliver pharmacological acceptance to anticonvulsant potential of Boswellia

serrata. The defense against the convulsions and reestablishment of enzyme level give an

inference to its possible procedure of action which may be arbitrated to the GABA ergic pathway

and inhibition of oxidative injury (Abdel Wahab et al., 1987).

2.2.3.5 Anti-depressant activity

A polyherbal formulation (Trans-01) was investigated for its antidepressant properties.

Trans-01 has the following composition: Valeriana wallichii (45%), Convolvulus microphyllus

(30%), Plumbago zeylanica (7.5%), Boswellia serrata (15%), and Acorus calamus (3.5%). The

effect of different doses of Trans-01 (25, 50, 75 and 100 mg/kg; PO) were studied and it was

found to safe up to a dose of 5000 mg/kg as no mortality was observed within 48 h of

administration. Trans-01 showed a dose-dependent decrease in immobility time in TST, which is

an indication of its antidepressant effect; this finding was further reinforced in the FST, where a

significant effect on immobility was witnessed. Trans-01 significantly attenuated the elevated

corticosteroid levels. To ascertain whether the antidepressant effect of Trans-01 included general

25

body stimulation, locomotor activity test was also done. These results indicate that Trans-01 can

be a potential candidate for managing depression. However, further studies are required to

substantiate the same (Sultana et al., 2013).

2.2.3.6 Hepatoprotective activity

The hepatoprotective effect of hexane extract of Boswellia serrata oleoresin on liver

injury induced by carbon tetrachloride, paracetamol or thioacetamide was evaluated. This was

given in two different doses (87.5mg/kg p.o. and 175mg/kg p.o.) and standard Silymarin was

used. The least dose of this extract (87.5mg/kg p.o.) considerably decreased the raised levels of

serum marker enzymes and prevented the increase in liver weight in all three models of liver

injury, while the higher dose showed mild hepatoprotective activity. It was concluded that

hexane extract of Boswellia serrata oleoresin in lower doses possess hepatoprotective activity,

which was supported by changes in histopathology (Jyothi et al., 2006).

2.2.3.7 Hypoglycemic activity

Herbal extract of Boswellia serrata oleoresin as one of the constituents has been reported

to produce important anti-diabetic activity on non-insulin dependent diabetes mellitus in

streptozocin induced diabetic rat model. The results showed that herbal extract of Boswellia

serrata oleoresin reduced the blood-glucose level comparable to that of (standard) phenformin

(al-Awadi et al., 1991).

2.2.3.8 Anti-ulcer activity

Antiulcer potential of boswellic acids, β-boswellic acid, α-boswellic acid, acetyl-β-

boswellic acid, acetyl-α-boswellic acid, 11-keto-β-boswellic acid and acetyl-11-keto-β boswellic

acid isolated from Boswellia serrata oleoresin was determined in male Wister rats. They

observed that all the boswellic acids showed significant antiulcer activity in dose dependent

manner against all assay. Moreover, they proposed that these resin acids may increase synthesis

of cytoprotective prostaglandins and inhibit leukotriene synthesis (Singh et al., 2008).

2.2.3.9 Osteoarthritis activity

Osteoarthritis is a common, chronic, progressive, skeletal, degenerative disorder, which

commonly affects the knee joint. Boswellia serrata oleoresin is very effective against

osteoarthritis and reduced total WBC count in the joint fluid, restoring the integrity of blood

vessels obliterated by spasm or internal damage. Boswellia serrata gum extract reduced the knee

pain in 30 patients of Osteoarthritis. Moreover, consistant use of 333mg of gum extract for 8

26

weeks increased walking distance, knee flexion and decreased the knee joint swelling

(Kimmatkar et al., 2003).

2.2.3.10 Anti-inflammetry activity

In vitro studies and animal models show that boswellic acids were found to inhibit the

synthesis of pro-inflammatory enzyme, 5-lipoxygenase (5-LO) including 5-

hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B4 (LTB-4), which cause

bronchoconstriction, chemotaxis, and increased vascular permeability (Ammon et al., 1991;

Etzel, 1996). Other antiinflammatory plant constituents, such as quercetin, also block this

enzyme, but they do so in a more general fashion, as an antioxidant, whereas boswellic acids

seem to be specific inhibitor of 5-LO (Ammon, 1996). 5-LO generates inflammatory

leukotrienes, which cause inflammation by promoting free radical damage, calcium dislocation,

cell-adhesion and migration of inflammation-producing cells to the inflamed body area. In

contrast to non-steroidal antiinflammatory drugs (NSAIDS), which are well known to disrupt

glycosaminoglycan synthesis, thus accelerating articular damage in arthritic conditions,

boswellic acids have been shown to significantly reduce glycosaminoglycan degradation (Lee

and Spencer, 1969; Palmoski and Brandt, 1979; Dekel et al., 1980; Brandt and Palmoski, 1984).

2.2.3.11 Anticancer activity

In vitro anticancer activity of Boswellia serrata oleoresin and 3-O-acetyl-11-keto-β-

boswellic acid (AKBA) was determined against HFFF2, HaCaT and NCTC human cell lines

through DNA, MTT and neutral red uptake (NRU) assays (Burlando et al., 2008). Both the

extract and 3-O-acetyl-11-keto-β-boswellic acid exhibited moderate level of cytotoxicity against

all the cell lines while in DNA assay extract showed low toxicity against HFFF2 than HaCaT and

NCTC cell lines. (Khan et al., 2014) studied the in vivo and in vitro anticancer activity of

Boswellia serrata extract against Wistar rats and hepatocellular carcinoma cell lines. In vitro

study was carried out through MTT assay that showed IC50 values 21.21 and 18.65 𝜇g/mL

against Hep3B and HepG2 respectively, while positive control doxorubicin showed IC50 values

1.92 and 1.06 ug/ml respectively. Synergistic effect of doxorubicin and Boswellia serrata extract

was evaluated by isobolographic analysis method that showed significant synergistic effect with

combination index values range from 0.53 - 0.76 for 50 % cell kill. Moreover, Boswellia serrata

extract revealed dose dependent enhancement in IL-6 level, TNF-𝛼 level and caspase-3 activity

27

that was higher than positive control doxorubicin and combination of Boswellia serrata extract

and doxorubicin.

2.2.3.12 Antioxidant activity

Antioxidant activity of Boswellia serrata oleo gum resin essential oil isolated from

different locations of India revealed free radical scavenging activity in range of 47.5 – 79.8 %

(Gupta et al., 2016). (Upaganlawar and Ghule, 2009) assessed essential oils of Boswellia spp. for

antioxidant activity. The essential oils revealed significant antioxidant activity with IC50 values

of 121.40 μg/ mL, 211.2 μg/mL, and 175.2 μg/mL for B. socotrana, B. elongate, and B. ameero,

respectively in DPPH free radical scavenging assay. Similarly, (Mothana et al., 2011) reported

the DPPH free radical scavenging activity of Boswellia species essential oils. They reported that

Boswellia species essential oils showed weak antioxidant activity in DPPH free radical

scavenging assay.

28

Chapter 3

MATERIALS AND METHODS

The research work presented in this dissertation was conducted in the laboratories of the

Department of Chemistry; Department of Biochemistry, University of Agriculture, Faisalabad,

Pakistan and School of Chemistry and Molecular Bioscinces, The University of Queensland,

Australia.

3.1 Materials

3.1.1 Chemicals and standard compounds

Linoleic acid, 2, 2,-diphenyl-1-picrylhydrazyl, gallic acid, Folin-Ciocalteu reagent,

ascorbic acid, trichloroacetic acid, sodium nitrite, aluminum chloride, ammonium thiocyanate,

ferrous chloride, ferric chloride, potassium ferricyanide, butylated hydroxytoluene (99.0 %),

dimethyl sulfoxide, homologous series of C9-C24 n-alkanes and various reference chemicals (α-

pinene, camphene, β- pinene, β-myrcene, limonene, p-cymene, γ-terpinene, estragole,

caryophyllene, and caryophyllene oxide etc.) used to identify the constituents were obtained

from Sigma Chemical Co. (St Louis, MO, USA). Sterile resazurin tablets were obtained from

BDH Laboratory Supplies. All other chemicals (analytical grade) i.e. anhydrous sodium

carbonate, ferrous chloride, ammonium thiocyanate, chloroform, ethanol and methanol used in

this study were purchased from Merck (Darmstadt, Germany), unless stated otherwise. All

culture media and standard antibiotic discs were purchased from Oxoid Ltd., (Hampshire, UK).

3.1.2. Instruments

The instruments used for different processes during the study with their company are as

follows

Table: 3.1 List of instruments

Sr. No. Name of Instrument Manufacturing Company

29

1 Autoclave Omron, Japan

3 Laminar air flow Dalton, Japan

4 Incubator Sanyo, Germany

5 Electric Balance MP-300 Ohyo, Japan

6 Micropipettes Gilson, USA

7 Orbital shaker Gallenkamp, UK

8 Oven IM-30, Irmec, Germany

9 Centrifuge H-200 NR Kokusan, Japan

10 Refrigerator Dawlance, Pakistan

11 pH meter PSH-3BW, China

12 Glass slides &Coverslips Bolton, UK

14 Vortex Velp Scientifica, Italy

15 Micro-centrifuge 235C, Fischer Scientific, USA.

17 Micro Quant Elisa Plate Reader Bio Tek, U.S.A

19 Haemacytometer Fisher ultra plane, Japan

20 Water bath Memmert, Japan

21 Spectrophotometer U-2001 Hitachi, Japan

22 GC/MS (6890N) Agilent-Technologies, California, USA.

30

3.1.3. Collection of plant materials

Oleoresins of Boswellia serrata and Pinus roxburghii were collected from hilly area of

Zhob district of Balochistan and Mansehra district of Khyber Pakhtunkhwa, respectively. The

plant materials were further identified and authenticated by a Taxonomist, Dr. Mansoor Hameed,

Assistant Professor, Department of Botany, University of Agriculture, Faisalabad, Pakistan.

Figure 3.1: Collection of plant materials

31

3.1.4. Microbial strains utilized to evaluate the antimicrobial and antitumor activity of

essential oils, fractions and sub fractions.

Bacterial strains

I. Escherichia coli (E. coli) (ATCC 25922)

II. Staphylococcus aureus (S. aureus ) (ATCC 25923)

III. Bacillus subtilis (B. subtilis ) (ATCC 10707)

IV. Pasteurella multocida (P. multocida) (ATCC 43137)

Fungal strains

I. Aspergillus niger (A. niger) (ATCC 10575)

II. Fusarium solani (F. solani) (ATCC 36031)

III. Aspergillus flavus (A. flavus) (ATCC 20046)

IV. Alternaria alternata (A. alternate) (ATCC 20084)

The antimicrobial and antitumor activity of the Boswellia serrata and Pinus roxburghii

oleoresins essential oils, fractions and sub-fractions was evaluated against four bacterial, four

fungal and one tumor strain obtained from American Type Culture Collection, Biological

Division of the Nuclear Institute for Agriculture and Biology (NIAB), Faisalabad, Pakistan. The

purity and identity were further confirmed from Institute of Microbiology, University of

Agriculture Faisalabad, Pakistan. Fungal strains were grown on potato dextrose agar (PDA) at 25

⁰C for 72 hours, bacterial strains were grown overnight on nutrient agar (NA) at 37 ⁰C and

Agrobacterium tumefaciens (At10) was grown for 48 hours in Lauria broth. Microbial cell

suspension in 0.9% NaCl solution was adjusted at 0.5 McFarland to get approximately 106

cfu/ml.

3.2. Isolation of essential oils

Hydro-distillation, steam distillation and supercritical fluid extraction methods were used

for isolation of essential oil from oleoresin of Boswellia serrata and Pinus roxburghii.

3.2.1. Hydro and steam distillation method

32

The air-dried and finely ground (80 mesh) oleoresin (300 g) was subjected to hydro-

distillation and steam distillation at 120 ⁰C, 140 ⁰C, 160 ⁰C and 180 ⁰C for 3 h. Distillates of

essential oils were dried over anhydrous sodium sulfate, filtered and stored at +4 ⁰C until further

analysis. The process of extraction of oil was repeated five times to ensure the reproducibility.

Figure 3.2: Schematic diagram of extraction of essential oils from oleoresins of Boswellia

serrata and Pinus roxburghii through hydro distillation, steam distillation and supercritical

fluid extraction methods.

3.2.2. Supercritical CO2 extraction

Extraction of essential oil from Pinus roxburghii and Boswellia serrata oleoresin was

experimentally performed through Speed SFE instrument (Applied Separations Inc., Allenton,

PA, USA). Liquid carbon dioxide was pressurized with a high-pressure pump and then charged

into the extraction column to desired pressure. The pressure was controlled to an accuracy of

about 1% over the measuring range. The extraction column was 32 ml with 14.40 mm inner

diameter and 195 mm length, being packed with raw materials and glass beads. The extraction

column was heated with an oven and its temperature was indicated and controlled by a

thermocouple to within ±1 ⁰C. The supercritical CO2 with dissolved compounds passed through

33

a heated micrometer valve, and was subsequently expanded to ambient pressure. The extract was

precipitated in a collect vial at ambient pressure and temperature. A calibrated wet-test meter at

known temperature and pressure measured the total amount of CO2. For each extraction test, the

extractor was charged with about 50 g of oleoresin. CO2 flow rates ranging about 2 L/min were

used. The oil weight was measured by precision balance until no oil was extracted out from

oleoresin (Guan et al., 2007).

3.3. Fractionation of essential oils

Boswellia serrata and Pinus roxburghii oleoresin essential oils obtained by hydo-

distillation and steam distillation methods at different temperatures were seperated into different

fractions and sub-fractions by short path molecular vaccum distillation as previosuly described

by (Olmedo et al., 2014) with minor modifications. The 100 ml of essential oil was taken in the

reception flask and different fractions were obtained by modifying the temperature while

pressere and flow rate were kept constant. Pinus roxburghii oleoresin hydro-distilled essential oil

of 120 0C gives five different fractions (F1, F2, F3, F4 and F5) at various temperatures. Among

these five fractions, F1, F2 and F3 fraction was further separated in to sub-fractions (F1 a, F1 b,

F1 c and F1 d), (F2 a, F2 b and F2 c) and (F3 a, F3 b, F3 c and F3 d), respectively, on the basis

of their boiling points. Similar process was carried out with Boswellia serrata and Pinus

roxburghii oleoresin essential oils extracted at various temperatures with different extraction

methods. Fractions and sub-fractions of approximate volumes were collected during the

fractional distillation and analyzed by GC-MS.

3.4. Chromatographic analysis

3.4.1. Gas chromatography/mass spectrometry (GC-MS) analysis

GC–MS analysis of the essential oils was performed using an Agilent-Technologies

(Little Falls, California, USA) 6890N Network gas chromatographic (GC) system, equipped with

an Agilent-Technologies 5975 inert XL Mass selective detector and Agilent-Technologies 7683B

series auto injector. Compounds were separated on HP-5 MS capillary column (30 m x 0.25 mm,

film thickness 0.25 μm; Little Falls, CA, USA). A sample of 5.0 μL was injected using the split

mode (split ratio 1:100). For GC/MS detection, an electron ionization system, with ionization

energy of 70 eV, was used. Column oven temperature was programmed from 80 oC to 220

oC at

34

the rate of 4 oC min

-1; initial and final temperatures were held for 3 and 10 minutes, respectively.

Helium was used as a carrier gas at a flow rate of 1.5 mL min-1

. Mass scanning range was 50 –

550 m/z while injector and MS transfer line temperatures were set at 220 and 290 ºC,

respectively (Hussain et al., 2013; Hanif et al., 2017).

3.4.2. Compounds identification

The identification of the components was based on comparison of their mass spectra with

those of NIST mass spectral library and with the help of a built-in data-handling program as

advised by the manufacturer (Adams, 2007; Hussain et al., 2013; Hanif et al., 2017).

Figure 3.3: Schematic diagram of Boswellia serrata and Pinus roxburghii oleoresin essential

oils fractionation.

3.5. Biological activities

3.5.1. Antioxidant activity

35

Antioxidant potential of Boswella serrata and Pinus roxburghii oleoresin essential oils,

fractions and sub-fractions was determined through following assays.

3.5.1.1. Total phenolic contents

To 1.0 mL of each essential oil, fractions and sub-fractions of Pinus roxburghii and

Boswellia serrata oleoresin, Gallic acid standard solution (20, 40, 60, 80 and 100 mg/L), 5 mL of

Folin-Ciocalteu and 4 mL of sodium carbonate (7% w/v) were added and samples were shaken

to mix the components completely. After keeping all the samples in dark for 30 min, absorbance

was measured at 765 nm using a spectrophotometer (model 721D). Reagent solution was

expressed as Gallic acid equivalent (GAE) in milligram per liter of dry weight basis (Khan et al.,

2012). The Calibration curve of Gallic acid (Figure 3.4) is shown in below.

Figure 3.4: Calibration curve for total phenolic contents

3.5.1.2. Total flavonoid contents

The total flavonoid contents in essential oils, fractions and sub-fractions were determined

using aluminum trichloride colorimetric assay as describe by (Zhishen et al., 1999) with minor

modifications. To 1.0 mL of essential oils isolated through different extraction methods, most

active fractions, sub fractions or catechin standard solution (20, 40, 60, 80 and 100 mg/L) was

mixed with 4.0 mL of water in 10 mL volumetric flask followed by addition of 0.3 mL of 5 %

NaNO2. After 5 min, 0.3 mL of 10% AlCl3 was added and after waiting for one more min, 2 mL

of 1 M NaOH was added and total volume was made up to 10 mL using deionized distilled water

y = 0.0088x + 0.0422 R² = 0.99

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 20 40 60 80 100 120

Ab

sorb

an

ce

Concentration (mg/L)

36

(DDW). After mixing solution properly, the absorbance reading was measured at 510 nm using

reagent blank. The amount of total flavonoids was expressed as catechin equivalent in milligram

per liter of dry plant materials.

Figure 3.5: Calibration curve for total flavonoid contents

3.5.1.3. DPPH free radical scavenging activity

The DPPH assay was performed as described by (Bozin et al., 2006) with minor

modifications. To 2.5 mL of essential oils isolated through different extractions methods, most

active fractions and sub fractions were mixed with 1 mL of 0.09 mM DPPH solution and filled

up with 95% MeOH, to a final volume of 4 mL. The absorbance of the resulting solutions and

the blank were recorded after 1 h at room temperature in dark at 515 nm using a

spectrophotometer. Butylated hydroxyl toluene (100 ppm) was used as a positive control.

Inhibition of free radical by DPPH in percent (%) was calculated in the following way:

I (%) = 100 × (Ablank − Asample/Ablank)

Where Ablank is the absorbance of the control reaction mixture and Asample is the absorbance of the

test compounds.

3.5.1.4. Total antioxidant contents (FRAP assay)

Total antioxidant contents were estimated using ferric reducing antioxidant power

(FRAP) assay as describe (Chan et al., 2007) by with minor modifications. One milliliter of

y = 0.0009x - 0.0011 R² = 0.9866

0

0.02

0.04

0.06

0.08

0.1

0 20 40 60 80 100 120

Ab

sorb

an

ce a

t 5

10

nm


37

essential oils isolated through different extraction methods, most active fractions and sub

fractions was mixed with 2.5 mL of phosphate buffer (0.2 M; pH 6.6) and 2.5 mL of potassium

ferricyanide (1% W/V). The test tubes were incubated in water both at 50 ⁰C for 25 min. Then

2.5 mL of trichloro-acetic acid solution (10% W/V) was mixed to each reaction mixture. Then

2.5 mL of each reaction mixture was taken in separate test tubes and diluted with 2.5 mL of

distilled water, followed by addition of 500 µL of ferric chloride (0.1% W/V) solution. The test

tubes were incubated for 30 min at room temperature. The absorbance of each reaction mixture

was measured at 700 nm using UV–vis spectrophotometer. Total antioxidant activity was

calculated using Fig gallic acid calibration curve (0-100 mg/mL) and total antioxidant contents

were expressed as mg/mL of gallic acid equivalents.

Figure 3.6: Calibration curve for Total antioxidant contents

3.5.1.5. Percentage inhibition in linoleic acid system

The antioxidant activity of essential oils isolated through different extraction methods,

most active fractions and sub fractions was determined by the method as described by (Singh et

al., 2006) with minor modifications. The essential oils isolated through different extraction

methods, most active fractions and sub-fractions (50 µL) were dissolved to a 1 mL of ethanol,

mixed with linoleic acid (2.5%, v/v), 99.5% ethanol (4 mL) and 4 mL of 0.05 M sodium

phosphate buffer (pH 7). The solution was incubated at 40 ⁰C for 175 h. The extent of oxidation

y = 0.021x + 0.0151 R² = 0.99

0

0.5

1

1.5

2

2.5

0 20 40 60 80 100 120

Ab

sorb

an

ce


38

was measured by peroxide value using the colorimetric method described by (Yen et al., 2000).

To 0.2 mL sample solution, 10 mL of ethanol (75%), 0.2 mL of an aqueous solution of

ammonium thiocyanate (30%) and 0.2 mL of ferrous chloride solution (20 mM in 3.5% HCl)

were added sequentially. After 3 min of stirring, the absorbance was measured at 500 nm, using

a spectrophotometer. A control was performed with linoleic acid without essential oils. Butylated

hydroxytoluene (BHT) was used as positive control. Inhibition of linoleic acid oxidation

expressed as percent was calculated as follows:

% inhibition of linoleic acid oxidation = 100 − [(Abs. Increase of sample at 175 h/Abs. Increase

of control at 175 h) × 100]

3.5.1.6. Hydrogen peroxide scavenging activity

The ability of the essential oils isolated through different extraction methods, most active

fractions and sub fractions to scavenge hydrogen peroxide was determined spectro-

photometrically as described previously (Bakkali et al., 2008) with minor modifications. Briefly,

a solution of hydrogen peroxide (2 mM) was prepared in 0.17 M phosphate buffer (pH = 7.4).

600 µL essential oil, fractions, sub fractions and Ascorbic acid (0–10 mg/mL) were added to the

reaction mixture containing 600 µL, 2 mM hydrogen peroxide. After 10 min of incubation at

room temperature, the absorbance was read against a blank at 230 nm. The percentage hydrogen

peroxide scavenging activity of samples was calculated as follows:

Percentage hydrogen peroxide scavenging = (A0− A1/A0) × 100

Here, A0 was the absorbance of the control and A1 was the absorbance of the presence of

samples.

3.6.2. Evaluation of antimicrobial activities of essential oils

The essential oils isolated through different extraction methods, most active fractions and

sub-fractions of Pinus roxburghii and Boswellia serrata oleoresins were individually tested

against a panel of selected microorganisms. Bacterial strains were cultured overnight at 37 ºC in

nutrient agar (NA) while the fungal strains were cultured overnight at 30 ºC using potato

dextrose agar (PDA). Following antimicrobial assays were employed for the determination of

antimicrobial potential of essential oils isolated through different extraction methods, most active

fractions and sub-fractions of Pinus roxburghii and Boswellia serrata oleoresins.

39

3.6.2.1. Agar well diffusion method

The antibacterial and antifungal activity of Boswellia serrata and Pinus roxburghii

oleoresins essential oils isolated through different extraction methods, most active fractions and

sub fractions were performed by agar well diffusion method as previously describe by (Rashid et

al., 2013) with minor modification. The overnight cultures of the indicator strains of bacteria and

72 hour cultures of fungi were transferred to 25 ml of liquid nutrient agar and potato dextrose

agar, respectively. The contents of the flasks were transferred to medium size petri plates and

wait for its solidification at room temperature. Sterilize cork borer was used for well formation.

These wells were filled with 10 µl and 20 µl of essential oils, fractions, sub fractions and

standard drugs for antibacterial and antifungal activity, respectively. The perti plates were

incubated at 37 ⁰C for 24 h and 28 ⁰C for 72 h for bacteria and fungi, respectively. After the

incubation period, antimicrobial activity was evaluated by measuring the width of the zones of

inhibition (mm). Ampicillin (1 mg/mL) were used as positive controls for bacteria, while as

Terbinafine (1 mg/mL) was used as standard for antifungal activity.

Figure 3.7: A typical agar plate showing the inhibition zones exhibited by essential oils (1-9)

3.6.2.2. Resazurin microtitre-plate assay

For the measurement of minimum inhibitory concentration (MIC) of essential oils

isolated through different extraction methods, most active fractions and sub fractions, a modified

resazurin microtitre-plate assay was used, as reported by (Sarker et al., 2007). Dimethyl

40

sulfoxide (DMSO) was diluted with distilled water (10 mL/100 mL) for preparing the working

solutions of essential oils, fractions, sub fractions and antibiotic. A volume of 10 µL essential

oils, fractions and sub fractions solutions (8.4 mg/mL, w/v in DMSO) and standard antibiotic

(1.0 mg/mL in DMSO) was pipetted into the first row of the 96 well plates. To all other wells 50

µL of nutrient broth was added. Two fold serial dilutions were performed using a multichannel

pipette such that each well had 50 µL of the test material in serially descending concentrations. A

volume of 30 µL of 3.3× strength isosensitised broth and 10 µL of resazurin indicator solution

(prepared by dissolving 270 mg tablet in 40 mL of sterile distilled water) were added in each

well. Finally, 10 µL of bacterial suspension was added to each well to achieve a concentration of

approx 5 × 105 cfu/ml. Each plate was wrapped loosely with cling film to ensure that bacteria did

not become dehydrated. Each plate had a set of controls: a column with a positive control, a

column with all solutions with the exception of the test compound, a column with all solutions

with the exception of the bacterial solution adding 10 µL of nutrient broth instead and a column

with DMSO solution as a negative control. The plates were prepared in triplicate, and incubated

at 37 0C for 24 h. The change in the colour of resazurin indicator was then assessed visually. The

growth was indicated by colour changes from purple to pink or colourless. The lowest

concentration at which colour change occurred was taken as the MIC value.

3.6.2.3. Micro-dilution broth susceptibility assay

For minimum inhibitory concentration (MIC), a micro-dilution broth susceptibility assay

was used, as reported in (Wayne, 2008). Essential oils, fractions and sub fractions were

solubilized in dimethylsulfoxide (10% DMSO) then diluted in culture media for use. Dilutions

series (0.66 to 1351.08 µg/mL) of the essential oils, fractions and sub fractions in a 96-well

microtitre plate, including one growth control, solvent control and one sterility control were

prepared. 160 μL of sabouraud dextrose broth were added onto microplates and 20 μL of test

solution. Then, 20 μl of 5 x 105 cfu/mL (confirmed by viable count) of standard\microorganism

suspension were inoculated onto microplates. Plates were incubated at 30 ºC for 48 h for fungi.

Fluconazole (1.0 mg/mL in 10% DMSO) were used as positive control. The growth was

indicated by the presence of a white ‗‗pellet‘‘ on the well bottom. The MIC was calculated as the

highest dilution showing complete inhibition of the test strains.

41

Figure 3.8: A typical plate in resazurin microtitre-plate assay showing the color change due

to antibacterial effect of essential oils

3.6.3. Antitumor activity

3.6.3.1. Crown gall antitumor assay

Antitumor activity of essential oils isolated through different extraction methods, most

active fractions and sub fractions were determined by crown gall antitumor assay (potato disc

assay) as describe by (Lellau and Liebezeit, 2003; Hussain et al., 2007) with minor

modifications. Vincristine sulfate was used as positive and 10 % DMSO as negative control. A.

tumefaciens from storage culture was inoculated into sterilized growth medium using aseptic

techniques. The culture was vigorously shaken and then placed in a shaker for 48 hours at 28 ⁰C.

Red skinned potatoes were surface sterilized in 10 % sodium hypochlorite for 30 minutes and

extensively washed with autoclaved distilled water and cut into 4 mm thick discs of 8 mm

diameter using cork borer and surgical blades sterilized by γ-irradiation (2.5 MRads). Nutrient

agar solution was prepared by dissolving nutrient agar powder in autoclaved distilled water.

Nutrient agar was poured into sterilized petri plates and allowed to solidify in laminar air flow.

Eight potato discs were placed on each agar plate using sterilized forceps. A 30 µL sample of

inoculum was placed on the surface of each disc. Plates were wrapped with parafilm and

incubated at 27 ⁰C for 21 days. After 21 days, discs were stained with Lugol solution (10 % KI,

5% I2) for 20 minutes and tumors were counted on each disc using a dissecting microscope.

Percent inhibition was calculated using the formula.

42

% Inhibition= [1 - no of tumours in sample/no of tumours in negative control] × 100

Figure 3.9: Schematic diagram of Boswellia serrata and Pinus roxburghii oleoresin essential

oils fractionation.

3.6.4. Cytotoxicity

3.6.4.1. Hemolytic activity

The cytotoxicity of essential oils isolated through different extraction methods, fractions

and sub fractions were analyzed by hemolytic activity as describe (Yang et al., 2005; Kumar et

al., 2016). 3 mL of freshly obtained blood was added in heparinized tubes to avoid coagulation

and gently mixed, poured into a sterile 15 mL falcon tube and centrifuged for 5 min at 850×g.

The supernatant was poured off and RBCs were washed three times with 5 mL of chilled (4oC)

sterile isotonic phosphate buffer saline (PBS) solution, adjusted to pH 7.4. The washed RBCs

were counted on heamacytometer. The RBCS count was maintained to 7.068 x 108 cells per ml

for each assay. The 20 µL of essential oils, fractions and sub fractions were taken in 2 mL

eppendorf tubes, then added 180 µL diluted blood cell suspension. These samples were

incubated for 35 minutes at 37oC. Agitated it after 10 minutes and after incubation, the tubes

placed it on ice for 5 minutes and centrifuged for 5 minutes at 1310 x g. After centrifugation, 100

43

µL supernatant was transferred to another eppendorf tube and diluted with 900 µL chilled PBS.

All eppendorfs were maintained on ice after dilution. After this 200 µL mixture from each

eppendorfs was added into 96 well plates. For each assay, 0.1% triton X- 100 was taken as a

positive control and phosphate buffer saline (PBS) was taken for each assay as a negative

control. The absorbance was noted at 576 nm with a Bio TeK, µ Quant (BioTek, Winooski, VT,

USA). Triton-X 100 (0.1 %) was used as positive control for 100% lyses and PBS buffer as

Negative control 0% lyses. The experiment was performed in triplicate and results were average.

% Hemolytic inhibition was calculated with the help of following formula:

Lysis of RBCs (%) = (Absorbance of sample –Absorbance of Negative control/Absorbance of

Positive control) ×100.

3.7. High performance liquid chromatography (HPLC) analysis

HPLC is the selected method for identification and quantification of phenolic compounds

from Boswellia serrata and Pinus roxburghii oleoresin essential oils. HPLC analysis of phenolic

compounds was performed using Shimadzu LC-20AC pumps (Shimadzu Co., Kyoto, Japan),

SPD-M20A with a UV/Visible detector, and chromatographic separations were performed on a

shim-pack CLC C-18 column (4.6 mm× 25 cm, i.d. 5 µm). The composition of solvents and the

gradient elution conditions used were as described previously (Butsat et al., 2009) with some

modifications. The mobile phase consisted of purified water with acetic acid 94:6 (pH 2.27)

(solvent A) and acetonitrile 100 % (solvent B) at a flow rate of 1 mL/min. Gradient elution was

performed as follows: from 0 to 15 min, linear gradient from 15% solvent B; from 15 to 30 min,

45% solvent B; from 30 to 45 min, linear gradient of 100% solvent B. Operating conditions were

as follows: column temperature, 38 0C, injection volume 20 µL, and UV-visible detection at 280

nm at a flow-rate of 1 mL/min. Phenolic compounds in the samples were identified by

comparing their relative retention times and UV spectra with those of standard compounds and

were detected using an external standard method.

3.8. Statistical analysis

All the experiments were conducted in triplicate and statistical analysis of the data were

performed by analysis of variance (ANOVA) using STATISTICA 5.5 (Stat Soft Inc., Tulsa, OK,

USA) software. A probability value at p ≤ 0.05 was considered statistically significant. Data are

presented as mean values ± standard deviation calculated from triplicate determinations.

44

CHAPTER 4

Results and Discussions

4.1. Essential oil yield

Essential oil yield of Pinus roxburghii and Boswellia serrata oleoresin was calculated as

the gram weight of essential oil divided by the gram weight of dry oleoresin used for extraction.

The temperature used for extraction studies was the temperature of heating source which affected

the rate of vapors production.

4.1.1. Essential oil yield of Pinus roxburghii oleoresin

Essential oil yield of Pinus roxburghii oleoresin isolated through hydrodistillation, steam

distillation and supercritical fluid extraction method is given in Table 4.1. It was observed that

essential oil yield of Pinus roxburghii oleoresin increased with increasing the extraction

temperature. In hydrodistillation method, maximum essential oil yield (19.18 ± 0.31 %) was

obtained at 180 ⁰C, while minimum essential oil yield (12.46 ± 0.18 %) was found at 120 ⁰C.

Similar, trend was found in steam distillation method, where essential oil isolated at 180 ⁰C

showed highest essential oil yield (19.91 ± 0.22 %), while essential oil obtained at 120 ⁰C

exhibited lowest essential oil yield (16.01 ± 0.21 %). Essential oil yield of Pinus roxburghii

oleoresin found in the present study is lower than a previous study (Coppen et al., 1988) which

reported that Pinus roxburghii oleoresin contained 26.7 % essential oil. Moreover, literature

reports confirmed that essential oil isolated from other parts of Pinus roxburghii revealed the

smaller amount of essential oil than oleoresin. It was reported that wood, bark, and needles of

Pinus roxburghii contained 0.30, 0.75 and 1.25 % of essential oil, respectively (Salem et al.,

2014). Similarly, it was found that stems and needles of Pinus roxburghii exhibited 0.24% and

0.11 % essential oil, respectively (Hassan and Amjid, 2009). In another study, leaves of Pinus

roxburghii contained 1.83 % essential oil (Kaushik et al., 2013). Such variations in essential oil

yield may be due to the difference in geological, seasonal and agro-climatic conditions. It has

been reported that yield and chemical composition of oleoresin essential oil significantly

changed with season, genotype, extraction condition, depth of the draining (drilling) holes in the

45

tree, plant nutrition, location of plant, parts of plant, environment stress, extraction method and

storing conditions (Mita et al., 2002; Hussain et al., 2008; Raut and Karuppayil, 2014).

The comparison of essential oil yield with different extraction methods showed that

steam distilled essential oils contained higher yield (16.01 ± 0.21 - 19.91 ± 0.22 %), followed by

hydro distilled essential oils (12.46 ± 0.18 - 19.18 ± 0.31 %) and supercritical fluid extracted

essential oil (1.04 ± 0.13 %). The variations in essential oil yields of hydro-distillation, steam

distillation and supercritical fluid extraction method may be due to the polarity of CO2 as it is

non-polar solvent with different extracting power to those of water as a polar solvent. Moreover,

it may possible that some of the volatile compounds escape with CO2 from the container. These

experimental results coincide with previous studies of (Bagheri et al., 2014) and (Ferreira et al.,

1999) how reported that hydro and steam distillation methods gave higher essential oil yield than

supercritical fluid extraction.

Table 4.1: Essential oil yield of Pinus roxburghii oleoresin by different extraction methods

Extraction technique Temperature

(⁰C)

Sample

weight

(g)

Hydrosol

volume (ml)

Extraction

time (hr)

Oil

(g)

Percent

yield

Hydro distillation

120 200 400 3 24.92 12.46±0.18f

140 200 610 3 31.02 15.51±0.29e

160 200 1170 3 34.4 17.20±0.24c

180 200 1600 3 38.37 19.18±0.31b

Steam distillation

120 200 184 3 32.02 16.01±0.21d

140 200 330 3 34.38 17.19±0.16c

160 200 650 3 37.8 18.90±0.25b

180 200 1050 3 39.81 19.91±0.22a

Supercritical fluid

extraction

40 ⁰C, 80

Bar 5000 Nil 3 52.2 1.04±0.13

g

Values are mean ± standard deviation of three separate determinations. Different letters in

superscripts represent significant difference with P ≤ 0.05 among Pinus roxburghii oleoresin

46

essential oils isolated at different temperatures with hydro-distillation, steam distillation and


4.1.2. Essential oil yield of Boswellia serrata oleoresin

Essential oil yield of Boswellia serrata oleoresin isolated by hydro-distillation, steam

distillation and supercritical fluid extraction method is given in Table 4.2. It was observed that

essential oil yield from Boswellia serrata oleoresin increased with increasing the extraction

temperature. In hydro-distillation method, maximum essential oil yield (9.37 ± 0.12 %) was

obtained at 180 ⁰C, while lowest essential oil yield (6.71 ± 0.10 %) was obtained at 120 ⁰C.

Similar, trend was seen in steam distillation method, where essential oil isolated at 180 ⁰C

contained highest essential oil yield (4.90 ± 0.12 %), while essential oil obtained at 120 ⁰C

showed lowest essential oil yield (3.30 ± 0.09 %). These experimental results are in good

agreement with a previous study (Singh et al., 2007) which reported that Boswellia serrata

oleoresin collected from different locations of India, contained (5.0 – 9.0 %) essential oil. The

comparison of oleoresin essential oil yield between Boswellia serrata and other Boswellia

species shows that Boswellia serrata oleoresin contains higher amount of essential oil (3.30 -

9.37 %) than Boswellia elongate (2.3%), Boswellia socotrana (1.2%), Boswellia sacra (5.5%)

and Boswellia ameero (1.8%) (Al-Harrasi and Al-Saidi, 2008; Ali et al., 2008). Moreover,

literature studies showed that essential oil isolated from barks of Boswellia species exhibited less

amount of essential oil than oleoresin. It was reported that barks of Boswellia socotrana,

Boswellia dioscorides and Boswellia elongate contained 0.23%, 0.28% and 0.44% essential oil,

respectively (Mothana et al., 2011). In another study, barks of Boswellia carterii contained 0.42

% essential oil (Prakash et al., 2014). Such variation in essential oil yield may be due to the

difference in geological, seasonal and agro-climatic conditions. Furthermore, it has been reported

that yield and chemical composition of oleoresin essential oil significantly changed with season,

genotype, extraction condition, depth of the draining (drilling) holes in the tree, plant nutrition,

location of plant, parts of plant, environment stress, extraction method and storing conditions

(Mita et al., 2002; Hussain et al., 2008; Raut and Karuppayil, 2014). The comparison of essential

oil yield with extraction methods showed that essential oils extracted by hydro-distillation

method has highest essential oil yield (6.71 ± 0.10 - 9.37 ± 0.12 %), followed by essential oils

obtained by steam distillation method (3.30 ± 0.09 - 4.96 ± 0.12 %) and essential oil collected

47

by supercritical fluid extraction method (0.31 ± 0.03 %). These results are supported by previous

studies of (Bagheri et al., 2014) and (Ferreira et al., 1999) which reported that hydro and steam

distillation methods gave higher essential oil yield than supercritical fluid extraction. The

variation in essential oil yield of hydro-distillation, steam distillation and supercritical fluid

extraction method may be due to the polarity of CO2 as it is non-polar solvent with different

isolating power to those of water as polar solvent. Moreover, it may possible that some of the

volatile compounds escape with CO2 from the container. Previously, it was reported that essential

oil yield of clove buds varied with extraction condition and extraction method. Moreover, yield

and chemical composition of essential oil significantly affected by extraction temperature (Guan

et al., 2007). In another study, it was reported that Carum copticum essential oils extracted

through supercritical fluid CO2 extraction method showed higher essential oil yield (1.0–5.8%)

than essential oil obtained by hydro-distillation method (2.8 %) (Khajeh et al., 2004). The

variations in essential oil yield of our results and previously literature reports may be due to the

less penetrating power of supercritical fluid CO2 through Boswellia serrata oleoresin.

Table 4.2: Essential oil yield of Boswellia serrata oleoresin by different extraction methods

Extraction

technique

Temperature

(⁰C)

Sample

weight (g)

Hydrosol

volume (ml)

Extraction

time (hr)

Oil

(g)

Percentage

yield

Hydro-

distillation

120 200 380 3 13.42 6.71±0.10d

140 200 605 3 16.35 8.18±0.15c

160 200 1200 3 17.51 8.76±0.13b

180 200 1610 3 18.74 9.37±0.12a

Steam

distillation

120 200 200 3 6.6 3.30±0.09g

140 200 350 3 7.04 3.52±0.16f

160 200 670 3 9.52 4.76±0.03e

180 200 1110 3 9.8 4.90±0.12e

Supercritical

fluid extraction

40 ⁰C, 80

Bar 7000 Nil 3 24.3 0.31±0.03

h


superscripts represent significant difference with P ≤ 0.05 among Boswellia serrata oleoresin

48

essential oils isolated at different temperatures with hydro distillation, steam distillation and


4.2. Fractionation of essential oils

4.2.1. Fractionation of Pinus roxburghii oleoresin essential oils

Essential oils are the complex mixture of hundreds of molecules that have strong

synergism between them (Bakkali et al., 2008). Therefore, the concentrations of certain

molecules are more important than pure compound. A short path vacuum fractional distillation is

efficient technique for separation of essential oil components. It works under low temperature

conditions to avoid the degradation of thermo labile compounds. Moreover, the essential oil

components are separated on the basis of their boiling points under constant reduce pressure. The

results of Pinus roxburghii oleoresin essential oils, fractions and sub-fractions along with their

boiling points and yields are given in Table 4.3. The hydro-distilled essential oil obtained at 120

⁰C was separated into five different fractions (F1, F2, F3, F4 and F5) on the basis of their boiling

points (Table 4.3). It was observed that F3 fraction contained highest yield (20 mL), while F5

fraction showed lowest yield (3.5 mL). Among these five fractions, F1, F2 and F3 fraction was

further separated in to sub-fractions (F1 a, F1 b, F1 c and F1 d), (F2 a, F2 b and F2 c) and (F3 a,

F3 b, F3 c and F3 d) with yield of (4.75, 8.25, 5 and 0.5 mL), (5, 3 and 1.5 mL) and (5, 6, 4.5 and

2.8 mL), respectively. Similarly, the hydro distilled essential oil isolated at 140 ⁰C was converted

into fractions (F1, F2, F3 and F4) on the basis of their boiling points (Table 4.3). It was observed

that F3 fraction contained highest yield (22.5 mL), while F4 fraction showed least yield (9 mL).

All the four fractions were further separated in to sub-fractions (F1 a, F1 b and F1 c), (F2 a, F2 b,

F2 c and F2 d), (F3 a, F3 b and F3 c) and (F4 a and F4 b) with yields of (3.3, 6.2 and 1.3 mL),

(9.5, 5.8, 3.5 and 1 mL), (8.2, 5.5 and 6.3 mL) and (2.3 and 5.7 mL), respectively. In the similar

way hydro-distilled essential oil isolated at 160 ⁰C was separated into four fractions (F1, F2, F3

and F4) on the basis of their boiling points (Table 4.3). All the four fractions were further

separated in to sub-fractions (F1 a and F1 b), (F2 a, F2 b, F2 c and F2 d), (F3 a, F3 b, F3 c and

F3 d) and (F4 a and F4 b) with yield of (3.5 and 3.5 mL), (4.5, 3, 8.5 and 6 mL), (2.5, 5.5, 7 and

9 mL) and (2.5 and 6.5 mL) respectively. Finally, the hydro-distilled essential oil isolated at 180

⁰C was separated into four fractions F1, F2, F3and F4 on the basis of their boiling points (Table

49

4.3). It was found that F2 fraction contained highest yield (25 mL), while F4 fraction showed

least yield (3.5 mL). Among these four fractions, F1, F2 and F3 fraction was further separated in

to sub-fractions (F1 a, F1 b, F1 c and F1 d), (F2 a, F2 b, F2 c and F2 d) and (F3 a and F3 b) with

yield of (7, 10, 3 and 3 mL), (6.25, 10, 5 and 2.5 mL) and (1 and 7 mL) respectively.

Similar process was used for essential oils isolated by steam distillation method, where

essential oil of 120 ⁰C was separated into four fractions F1, F2, F3 and F4 on the basis of their

boiling points with yields of 25, 32.5, 4.5 and 3.5, respectively (Table 4.4). Among these

fractions, F1 and F2 fraction was further separated into sub-fractions F1 a, F1 b, F1 c and F2 a,

F2 b, F2 c, F2 d that gave yields of 8.25, 6.25, 8.5 and 6.25, 8.25, 6.25 and 8 mL, respectively.

Table 4.3.: Fractionation of hydro distilled essential oils of Pinus roxburghii oleoresin.

Fractions and sub-fractions of Pinus roxburghii oleoresin essential oil through hydro-

distillation at various temperatures

120 ⁰C HD 140 ⁰C HD

No BP (⁰C) Yield (mL) No BP (⁰C) Yield (mL)

F1 63.9-67.1 18.25 ± 0.22b F1 44.4-66.6 13.00 ± 0.19

c

F2 67.1-68.1 10.25 ± 0.12c F2 66.6-69 21.50 ± 0.32

b

F3 68.1-74.8 20.00 ± 0.24a F3 69-74.2 22.50 ± 0.34

a

F4 74.8-77.4 4.50 ± 0.05h F4 > 74.2 9.00 ± 0.13

e

F5 > 77.4 3.50 ± 0.04i F1 a 51-64.4 3.30 ± 0.05

l

F1 a 51.3-67.2 4.75 ± 0.05g F1 b 64.4-67.5 6.20 ± 0.09

g

F1 b 67.2-69.1 8.25 ± 0.09d F1 c > 67.5 1.30 ± 0.01

n

F1 c 69.1-73.8 5.00 ± 0.06f F2 a 57.4-58.9 9.50 ± 0.14

d

F1 d > 73.8 0.50 ± 0.00m

F2 b 61.1 5.80 ±0.08h

F2 a 63.1-67.2 5.00 ± 0.04f F2 c 64.8 3.50 ± 0.05

k

F2 b 67.2-69.7 3.00 ± 0.03j F2 d > 64.8 1.00 ± 0.01

o

F2 c > 69.7 1.50 ± 0.01l F3 a 15.3-65 8.20 ± 0.12

f

F3 a 62.2-68.6 5.00 ± 0.04f F3 b 65-68.2 5.50 ± 0.06

j

F3 b 68.6-70.2 6.00 ± 0.07e F3 c > 68.2 6.30 ± 0.05

g

F3 c 70.2-73.2 4.50 ± 0.05h F4 a 73.5-76.5 2.30 ± 0.02

m

50

F3 d > 73.2 2.80 ± 0.03k F4 b > 76.5 5.70 ± 0.08

i

160 ⁰C HD 180 ⁰C HD


F1 64.2-67.5 10.00 ± 0.21d F1 61.4-67.5 24.50 ± 0.61

a

F2 67.5-69 25.00 ± 0.52b F2 67.5-70.2 25.00 ± 0.53

a

F3 69-71.2 27.00 ± 0.56a F3 70.2-71.4 12.00 ± 0.33

b

F4 > 71.2 12.50 ± 0.26c F4 > 71.4 3.50 ± 0.08

i

F1 a 25.9-43 3.50 ± 0.07l F1 a 68-71 7.00 ± 0.14

d

F1 b > 43 3.50 ± 0.05l F1 b 71-74 10.00 ± 0.24

c

F2 a 34.9 4.50 ± 0.09k F1 c 66-68 3.00 ± 0.07

j

F2 b 34.9-42.1 3.00 ± 0.03m

F1 d > 68 3.00 ± 0.05j

F2 c 42.1-44.5 8.50 ± 0.18f F2 a 68-70 6.25 ± 0.16

e

F2 d > 44.5 6.00 ± 0.13i F2 b 70-74 10.00 ± 0.22

c

F3 a 50.3-54.1 2.50 ± 0.05n F2 c 75-78 5.00 ± 0.12

f

F3 b 57.9 5.50 ± 0.11j F2 d > 78 2.50 ± 0.05

k

F3 c 63.8 7.00 ± 0.15g F3 a 42-72 1.00 ± 0.01

l

F3 d > 63.8 9.00 ± 0.19e F3 b > 72 7.00 ± 0.13

d

F4 a 61.9-70.5 2.50 ± 0.05n --- --- ---

F4 b > 70.5 6.50 ± 0.14h --- --- ---


superscripts represent significant difference with P ≤ 0.05 among fractions and sub-fractions of

Pinus roxburghii oleoresin essential oils isolated at different temperatures with hydro-distillation

method.

Similarly, steam distilled essential oil of 140 ⁰C was separated into four fractions F1, F2,

F3 and F4 on the basis of their boiling points with yields of 21, 12.5, 14.5 and 8.5 mL

respectively. Among these fractions, F1, F2 and F3 fraction was further separated into sub-

fractions F1 a, F1 b, F1 c and F2 a, F2 b and F3 a, F3 b, F3 c with yield 4.5, 10, 4.5 and 3, 7.5,

and 3.5, 2, 6 mL, respectively. In a similar way, essential oil isolated at 160 ⁰C was separated

into four fractions F1, F2, F3 and F4 on the basis of their boiling points with yields of 24, 22.5,

7.5 and 12, respectively. F1 and F2 fraction was further separated into sub-fractions F1 a, F1 b,

51

F1 c, F1 d and F2 a, F2 b, F2 c with yields of 3.5, 3, 5, 8.5 and 5, 7, 8.5 mL, respectively.

Finally, steam distilled essential oil isolated at 180 ⁰C was separated into four fractions F1, F2,

F3 and F4 with yields of 11, 24.5, 16 and 15 mL, respectively. Among these fractions F1, F2 and

F3 fraction was further separated into sub-fractions. The F1 fraction was separated into F1 a, F1

b and F1 c with yield of 2, 2.5 and 4.25, respectively. Similarly, F2 fraction was converted into

four sub-fractions F2 a, F2 b, F2 c and F2 d with yields of 4.5, 7.5, 7.25 and 2.5, respectively.

Moreover, F3 fraction was separated into three sub-fractions F3 a, F3 b and F3 c with yields of

5.25, 5.25 and 3 mL, respectively.

Table 4.4: Fractionation of steam distilled essential oils of Pinus roxburghii oleoresin.

Fractions and sub-fractions of Pinus roxburghii oleoresin essential oil through steam


120 ⁰C SD 140 ⁰C SD


F1 57-61.1 25.00 ± 0.31b F1 61.5-67.2 21.00 ± 0.27

a

F2 61.1-64.7 32.50 ± 0.40a F2 67.2-70.8 12.50 ± 0.16

c

F3 64.7-68.2 4.50 ± 0.05g F3 70.8-74.8 14.50 ± 0.19

b

F4 > 68.2 3.50 ± 0.04h F4 > 74.8 8.50 ± 0.11

e

F1 a 64-69 8.25 ± 0.11d F1 a 66-68 4.50 ± 0.05

h

F1 b 69-72 6.25 ± 0.07f F1 b 68-72 10.00 ± 0.13

d

F1 c > 72 8.50 ± 0.12c F1 c > 72 4.50 ± 0.05

h

F2 a 68-70 6.25 ± 0.07f F2 a 66-67 3.00 ± 0.03

j

F2 b 70-72 8.25 ± 0.10d F2 b > 67 7.50 ± 0.10

f

F2 c 72-75 6.25 ± 0.05f F3 a 68-70 3.50 ± 0.06

i

F2 d >75 8.00 ± 0.13e F3 b 67-68 2.00 ± 0.01

k

--- --- --- F3 c > 68 6.00 ± 0.07g

160 ⁰C SD 180 ⁰C SD


F1 64-69.5 24.00 ± 0.39a F1 52.4-71.5 11.00 ± 0.15

d

F2 69.5-73.9 22.50 ± 0.36b F2 71.5-75 24.50 ± 0.33

a

F3 73.9-74.8 7.50 ± 0.13e F3 75-78.9 16.00 ± 0.22

b

52

F4 > 74.8 12.00 ± 0.19c F4 > 78.9 15.00 ± 0.20

c

F1 a 60-68 3.50 ± 0.06h F1 a 68-70 2.00 ± 0.03

m

F1 b 68-70 3.00 ± 0.04i F1 b 70-72 2.50 ± 0.04

l

F1 c 70-73 5.00 ± 0.09g F1 c > 72 4.25 ± 0.07

j

F1 d > 73 8.50 ± 0.11d F2 a 70-71 4.50 ± 0.05

i

F2 a 70-74 5.00 ± 0.06g F2 b 71-74 7.50 ± 0.10

f

F2 b 74-76 7.00 ± 0.11f F2 c 74-76 7.25 ± 0.12

g

F2 c > 76 8.50 ± 0.14d F2 d > 76 2.50 ± 0.01

l

--- --- --- F3 a 71-75 5.25 ± 0.04h

--- --- --- F3 b 75-78 5.25 ± 0.07h

--- --- --- F3 c > 78 3.00 ± 0.03k



Pinus roxburghii oleoresin essential oils isolated at different temperatures with steam distillation

method.

4.2.2. Fractionation of Boswellia serrata oleoresin essential oils

The essential oils isolated by hydro and steam distillation methods were separated into

different fractions and sub-fractions on the basis of their boiling points and their results given in

Table 4.5. The hydro-distilled essential oil of 120 ⁰C was separated into three fractions F1, F2

and F3, on the basis of their boiling points, with yield of 23, 10.25 and 11.5 mL, respectively

(Table 4.5). Among these three fractions, F1 and F2 fraction was further separated in to sub-

fractions F1 a, F1 b, F1 c, F1 d, F1 e and F2 a, F2 b with yield of 4.2, 2.5, 2.8, 5, 5 mL and 5, 3.5

mL, respectively. Similarly, the hydro distilled essential oil of 140 ⁰C was separated into four

fractions F1, F2, F3 and F4, on the basis of their boiling points, with yields of 7.5, 11, 11.5 and

14.5 mL, respectively. Among these four fractions, F2 and F3 fractions were further separated in

to sub-fractions F2 a, F2 b, F2 c and F3 a, F3 b, F3 c with yields of 5.2, 2.5, 1.25 mL and 4.75,

2.25 and 2.5 mL, respectively. In the similar way hydro distilled essential oil of 160 ⁰C was

separated into three fractions F1, F2 and F3 with yields of 10.5, 26.5 and 15 mL, respectively. F1

and F2 fraction was further separated into sub-fractions F1 a, F1 b, F1 c and F2 a, F2 b, F2 c, F2

d with yields of 4.5, 3, 1 mL and 12, 7.5, 2.5, 2 mL, respectively. Finally, the hydro distilled

53

essential oil of 180 ⁰C was separated into four fractions F1, F2, F3, F4 that gave yields of 8,

10.5, 19.5 and 16.5 mL, respectively. It was found that F3 fraction contained highest yield 19.5

mL, while F1 fraction showed least yield 8 mL. All of these four fractions F1, F2, F3 and F4

were further separated into sub-fractions (F1 a, F1 b, F1 c), (F2 a, F2 b, F2 c), (F3 a, F3 b, F3 c)

and (F4 a, F4 b, F4 c) with yields (3.5, 2, 1.5 mL), (5.5, 2, 1 mL), (5.25, 10.4, 2 mL) and (3.2,

4.3 and 2.00 mL), respectively.

In the similar way, steam distilled essential oil of 120 ⁰C was separated into three

fractions F1, F2 and F3, on the basis of their boiling points, with yields of 14, 25 and 11.5 mL,

respectively as shown in Table 4.6 . Among these three fractions, F1 and F2 fractions were

further separated into sub-fractions F1 a, F1 b, F1 c and F2 a, F2 b, F2 c with yields of 5.5, 2,

4.25 and 8, 9.25and 4.25 mL, respectively. Similarly, steam distilled essential oil of 140 ⁰C was

separated into three fractions F1, F2 and F3 on the basis of their boiling points with yields of

15.5, 12.5 and 18.5 mL, respectively. Among these fractions, F1 and F2 fractions were further

separated into sub-fractions F1 a, F1 b, F1 c and F2 a, F2 b, F2 c with yields of 4, 9.5, 1 mL and

3.25, 3.25 and 1.5 mL, respectively. In the similar way, essential oil isolated at 160 ⁰C was

separated into three fractions F1, F2 and F3 on the basis of their boiling points with yields of 5,

14.25 and 15.25 mL, respectively. F2 and F3 fraction was further separated into sub-fractions F2

a, F2 b, F2 c and F3 a, F3 b, F3 c with yields of 4.25, 3.8, 3.5 mL and 3.75, 6, 3.5 mL,

respectively. Finally, steam distilled essential oil isolated at 180 ⁰C was separated in to three

fractions F1, F2and F3 with yields of 16, 10 and 12 mL, respectively.

Table 4.5: Fractionation of hydro distilled essential oils of Boswellia serrata oleoresin.

Fractions and sub-fractions of Boswellia serrata oleoresin essential oil through hydro-


120 ⁰C HD 140 ⁰C HD


F1 66-70 23.00 ± 0.42a F1 52-57.5 7.50 ± 0.09

d

F2 70-71 10.25 ± 0.22c F2 57.5-60 11.00 ± 0.15

c

F3 > 71 11.50 ± 0.31b F3 60-61 11.50 ±0.14

b

F1 a 54-60 4.20 ±0.21e F4 > 61 14.50 ± 0.30

a

54

F1 b 60-62.5 2.50 ± 0.01h F2 a 61-63 5.20 ± 0.08

e

F1 c 62.5-63.3 2.80 ± 0.02g F2 b 63-64 2.50 ± 0.02

g

F1 d 63.5-64 5.00 ±0.04d F2 c > 64 1.25 ± 0.03

i

F1 e > 64 5.00 ± 0.03d F3 a 61-61.5 4.75 ± 0.10

f

F2 a 60-64.5 5.00 ±0.06d F3 b 61.5-63 2.25 ± 0.04

h

F2 b > 64.5 3.50 ± 0.01f F3 c > 63 2.25 ± 0.03

h

160 ⁰C HD 180 ⁰C HD


F1 57-61 10.50 ± 0.12d F1 54-62 8.00 ± 0.09

d

F2 61-62.5 26.50 ± 0.15a F2 62-64 10.50 ± 0.16

c

F3 > 62.5 15.00 ± 0.19b F3 64-66 19.50 ±0.25

a

F1 a 59-60 4.25 ± 0.08f F4 > 66 16.50 ± 0.35

b

F1 b 60-61 3.00 ± 0.05g F1 a 60-64 3.50 ± 0.05

h

F1 c > 61 1.00 ± 0.01j F1 b 64-65 2.00 ± 0.02

j

F2 a 57-63 12.00 ± 0.27c F1 c > 65 1.50 ± 0.03

k

F2 b 63-64 7.50 ± 0.14e F2 a 61-63 5.50 ± 0.11

e

F2 c 64-65 2.50 ± 0.05h F2 b 63-63.5 2.00 ± 0.04

j

F2 d > 65 2.00 ± 0.06i F2 c > 63.5 1.00 ± 0.03

l

--- --- --- F3 a 61-63 5.25 ± 0.06f

--- --- --- F3 b 63-66 10.40 ± 0.24c

--- --- --- F3 c > 66 2.00 ± 0.03j

--- --- --- F4 a 59-63.5 3.20 ± 0.05i

--- --- F4 b 63.5-64.5 4.30 ± 0.02g

--- --- F4 c > 64.5 2.00 ± 0.01j



Boswellia serrata oleoresin essential oils isolated at different temperatures with hydro-

distillation method.

F1 fraction was separated into F1 a, F1 b and F1 c sub-fractions with yields of 3.25, 6.25

and 5 mL, respectively and F2 fraction was converted into three sub-fractions F2 a, F2 b and F2

c, on the basis of their boiling points, with yields of 4.5, 1.5 and 1 mL, respectively.

55

Table 4.6: Fractionation of steam distilled essential oils of Boswellia serrata oleoresin.

Fractions and sub-fractions of Boswellia serrata oleoresin essential oils through steam


120 ⁰C SD 140 ⁰C SD


F1 61-66 14.00 ± 0.17b F1 59-65 15.50 ± 0.18

b

F2 66-67 25.00 ± 0.38a F2 65-66.5 12.50 ± 0.19

c

F3 > 67 11.50 ± 0.15c F3 > 66.5 18.50 ± 0.24

a

F1 a 60-62.5 5.50 ± 0.12f F1 a 64-64.5 4.00 ± 0.08

e

F1 b 62.5-63 2.00 ± 0.03h F1 b 64.5-65.5 9.50 ± 0.16

d

F1 c > 63 4.25 ± 0.04g F1 c > 65.5 1.00 ± 0.01

h

F2 a 63-65 8.00 ± 0.18e F2 a 61.5-63 3.25 ± 0.08

f

F2 b 65-66.5 9.25 ± 0.17d F2 b 63-64 3.25 ± 0.06

f

F2 c > 66.5 4.25 ± 0.08g F2 c > 64 1.50 ± 0.03

g

160 ⁰C SD 180 ⁰C SD


F1 60-64 5.00 ± 0.06d F1 56-64 16.00 ± 0.19

b

F2 64-66.5 14.25 ± 0.21b F2 64-65 10.00 ± 0.15

c

F3 > 66.5 15.25 ± 0.20a F3 > 65 12.00 ± 0.16

a

F2 a 62-65 4.25 ± 0.09e F1 a 62-64 3.25 ± 0.07

e

F2 b 65-66 3.80 ± 0.06f F1 b 64-65 6.25 ± 0.10

d

F2 c > 66 3.50 ± 0.03g F1 c > 65 5.00 ± 0.05

h

F3 a 60-61.5 3.75 ± 0.08f F2 a 63-65.5 4.50 ± 0.10

f

F3 b 63-65.5 6.00 ± 0.11c F2 b 65.5-68 1.50 ± 0.03

f

F3 c > 65.5 3.50 ± 0.07g F2 c > 68 1.00 ± 0.02

g



Boswellia serrata oleoresin essential oils isolated at different temperatures with steam distillation

method.

56

4.3. Antioxidant activity

4.3.1. Total phenolic contents of Pinus roxburghii oleoresin

Phenolic compounds are most important plant secondary metabolites and act as

antioxidants due to their hydroxyl groups (Jung et al., 2003). For determination of total phenolic

compounds, Folin–Ciocalteu reagent is frequently used that measure a samples reducing capacity

(Huang et al., 2005). The total phenolic contents in essential oils extracted through different

extraction methods, fractions and sub-fractions of most active essential oils were determined

from regression equation of a calibration curve (y = 0.0088x + 0.0422, R² = 0.99) and expressed

as mg/L of Gallic acid equivalents (GAE) given in Figure 4.1. Total phenolic contents in

essential oils extracted through different extraction methods, fractions and sub-fractions of most

active essential oils, varied from 32.24 ± 4.81 to 2234.89 ± 15.87 mg/L of Gallic acid

equivalents. In essential oils isolated through hydro-distillation method, essential oil of 180 ⁰C

revealed maximum amount of total phenolic contents (2234.89 ± 15.87 mg/L of Gallic acid

equivalents). Therefore, fractions and sub-fractions of 180 ⁰C essential oil were further evaluated

for total phenolic contents, in order to find the most active fraction and sub-fraction. It was

observed that total phenolic contents in pure essential oils increased on increasing the extraction

temperature (Figure 4.1). In fractions of hydro distilled essential oil extracted at 180 ⁰C, F1

fraction contained highest amount of total phenolic contents (1225.23 ± 12.64 mg/L of Gallic

acid equivalents), while F4 fraction showed lowest amount of total phenolic contents (205.76 ±

5.14 mg/L of Gallic acid equivalents). Similarly, in sub-fractions of hydro distilled essential oil

extracted at 180 ⁰C, F2 b sub-fraction showed maximum amount of total phenolic contents

(2234.89 ± 15.87 mg/L of Gallic acid equivalents), while F2 a sub-fraction showed minimum

amount of total phenolic contents (398.59 ± 10.96 mg/L of Gallic acid equivalents). Overall, it

was observed that sub-fractions showed highest total phenolic contents, followed by fractions

and pure essential oils extracted at different extraction temperatures (Figure 4.1). A much similar

trend was observed in essential oils isolated through steam distillation method, in which essential

oil of 180 ⁰C showed highest total phenolic contents (660.65 ± 14.52 mg/L of Gallic acid

equivalents). Moreover, amount of total phenolic contents was increased with increasing the

extraction temperatures. In fractions of steam distilled essential oil extracted at 180 ⁰C, F4

fraction contained maximum amount of total phenolic contents (287.73 ± 11.19 mg/L of Gallic

57

Figure 4.1: Total phenolic contents of Pinus roxburghii oleoresin.

33.78

152.67 165.63

382.29

1225.23

1132.34

1044.15

205.76

1368.22

1490.07

946.37

610.82

398.59

2234.89

1087.85

1530.82 1425.62

1199.71

183.71

372.24

637.21

660.65

115.78

109.68

133.71

287.73

32.24 60.29

361.64

103.49 114.06 208.4

500.42

128.46

144.68

412.97 346.29

0

250

500

750

1000

1250

1500

1750

2000

2250

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F1 c

F1 d

F2 a

F2 b

F2 c

F2 d

F3 a

F3 b

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F1 c

F2 a

F2 b

F2 c

F2 d

F3 a

F3 b

F3 c

40

⁰C

HD SD SCF

To

tal

ph

eno

lic

con

ten

ts

(mg

/L o

f G

AE

)

Pinus roxburghii oleoresin essential oils, fractions and sub-fractions

58

acid equivalents), whereas in sub-fractions of 180 ⁰C steam distilled essential oil, F3 c sub-

fraction showed highest amount of total phenolic contents (412.97 ± 10.33 mg/L of Gallic acid

equivalents). Overall, it was found that pure essential oils extracted at different extraction

temperatures showed maximum total phenolic contents followed by sub-fractions and fractions

(Figure 4.1). The comparison of total phenolic contents in pure essential oils with different

extraction methods showed that steam distilled essential oils contained highest amount of total

phenolic contents, varied from 183.71 ± 8.59 to 660.65 ± 14.52 mg/L of Gallic acid equivalents,

followed by hydro-distilled essential oils (33.78 ± 0.84 – 382.29 ± 8.55 mg/L of GAE) and

supercritical fluid extracted essential oil (346.29 ± 9.44 mg/L of GAE) respectively. Previously,

it has been reported that total phenolic contents may vary with extraction conditions and

extraction methods (Guan et al., 2007; Meullemiestre et al., 2014). The literature study shows

that there is no early report is published on total phenolic contents of Pinus roxburghii oleoresin

essential oil, fractions and sub-fraction to compare the results of present study. While few reports

are available on total phenolic contents of Pinus species extracts. (Maimoona et al., 2011)

reported that bark extracts of Pinus roxburghii contained (810 ± 0.31 – 1331 ± 0.24 mg/100 g of

Gallic acid equivalents) total phenolic contents, while needle extracts revealed (394 ± 0.03 –

1008 ± 0.06 mg/100 g of Gallic acid equivalents) total phenolic contents. Similarly, (Dudonné et

al., 2009) found that aqueous extract of Pinus maritima contained (360.76 mg/g of Gallic acid

equivalents) total phenolic contents.

4.3.2. Total phenolic contents of Boswellia serrata oleoresin

Phenolic compounds are most important plant secondary metabolites and act as

antioxidants due to their hydroxyl groups (Jung et al., 2003). For determination of total phenolic

compounds, Folin–Ciocaldteu reagent is frequently used that measures a sample reducing

capacity (Huang et al., 2005). The total phenolic contents in Boswellia serrata oleoresin essential

oils extracted through different extraction methods, fractions and sub-fractions of most active

essential oils were determined from regression equation of a calibration curve (y = 0.0088x +

0.0422, R² = 0.99) and expressed as mg/L of Gallic acid equivalents (GAE) shown in Figure 4.2.

Total phenolic contents in Boswellia serrata oleoresin essential oils extracted through different

extraction methods, fractions and sub-fractions of most active essential oils, varied from 89.33 ±

6.23 to 768.22 ± 12.21 mg/L of Gallic acid equivalents. In essential oils extracted by hydro

59

distillation method, essential oil of 180 ⁰C showed maximum amount of total phenolic contents

(749.44 ± 16.74 mg/L of Gallic acid equivalents). Therefore, fractions and sub-fractions of 180

⁰C essential oil were further evaluated for total phenolic contents. It was observed that total

phenolic contents increased on increasing the extraction temperature (Figure 4.2). In fractions of

hydro distilled essential oil extracted at 180 ⁰C, F4 fraction showed highest amount of total

phenolic contents (452.98 ± 11.32 mg/L of Gallic acid equivalents), while F1 fraction showed

lowest amount total phenolic contents (95.66 ± 2.39 mg/L of Gallic acid equivalents). In sub-

fractions, F4 c showed maximum total phenolic contents (768.22 ± 12.21 mg/L of Gallic acid

equivalents), while F1 a showed minimum amount of total phenolic contents (147.85 ± 3.69

mg/L of Gallic acid equivalents). It was observed that sub-fractions showed highest total

phenolic contents, followed by pure oils than fractions, respectively (Figure 4.2). A much similar

trend was observed in steam distillation method, in which essential oil extracted at 180 ⁰C

showed highest total phenolic contents (543.71 ± 13.59 mg/L of Gallic acid equivalents) and

amount of total phenolic contents increased on increasing the extraction temperature. In fractions

of steam distilled essential oil extracted at 180 ⁰C, F1 fractions showed maximum total phenolic

contents (345.12 ± 8.63 mg/L of Gallic acid equivalents), while in sub-fractions of 180 ⁰C steam

distilled essential oil, F2 b sub-fraction contained highest total phenolic contents (371.56 ± 9.29

mg/L of Gallic acid equivalents). Overall, it was found that steam distilled essential oils showed

maximum total phenolic contents followed by sub-fractions and fractions (Figure 4.2). The

comparison of total phenolic contents in pure essential oils extracted through different extraction

methods showed that hydro-distilled essential oils contained higher amount of total phenolic

contents varied from 374.32 ± 9.36 to 749.44 ± 16.74 mg/L of Gallic acid equivalents, followed

by supercritical fluid extracted essential oil (598.24 ±14.15 mg/L of Gallic acid equivalents) and

steam distilled essential oils (400.42 ± 10.01 – 543.71 ± 13.59 mg/L of Gallic acid equivalents),

respectively. The variations of total phenolic contents in these essential oils may be due to

different extraction methods and extraction conditions. Previously, it has been reported that total

phenolic contents vary with extraction conditions and extraction methods (Guan et al., 2007;

Meullemiestre et al., 2014). It was reported that essential oil extracted by solvent free microwave

extraction methods showed higher total phenolic contents than essential oil extracted by hydro

distillation method (Berka-Zougali et al., 2012). The literature studies show that there is no early

report published on total phenolic contents of Boswellia serrata oleoresin essential oil, its

60

Figure 4.2: Total phenolic contents of Boswellia serrata oleoresin

.

374.32

581.88

696.26

749.44

95.66 112.37

122.24

452.98

147.85

252.67

345.29

271.56 243.78

268.09

171.56

296.03 289.56

318.22 338.22

768.22

400.42

443.46

426.15

543.71

345.12

298.34

121.76

207.11 179.33

264.89

336.13

371.56

89.33

598.24

0

100

200

300

400

500

600

700

8001

20

⁰C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F1 c

F2 a

F2 b

F2 c

F3 a

F3 b

F3 c

F4 a

F4 b

F4 c

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3

F1 a

F1 b

F1 c

F2 a

F2 b

F2 c

40

⁰C

HD SD SCF

To

tal

ph

eno

lic

con

ten

ts

(mg

/L o

f G

AE

)

Boswellia serrata oleoresin essential oils , fractions and sub-fractions

61

fractions and sub-fractions to compare the results of the present study. While few reports are

available on total phenolic contents of other Boswellia species essential oil and extracts. Our

results of total phenolic contents in essential oils extracted through different extraction methods

was higher than (Prakash et al., 2014) how found total phenolic contents (35.83 mg/g of Gallic

acid equivalents) in Boswellia carterii oleoresin essential oil. Similarly, it was reported that

methanol extracts of Boswellia serrata flowers and leaves contained (56.37 and 60.5 mg/g of

Gallic acid equivalents, respectively) total phenolic contents (Aman et al., 2010). In another

study, it was reported that ethanol extract of Boswellia serrata oleoresin contained 0.021 mg/g of

Gallic acid equivalents total phenolic contents (Bapat et al.). (Singh et al.) reported that aqueous

and ethanol extracts of Boswellia serrata barks showed total phenolic contents 28.46 and 12.73

mg/g of Gallic acid equivalents, respectively. (DEVI et al., 2014) reported that methanol

extractof Boswellia serrata leaves contained 277.0 ± 4.36 mg/ g of Gallic acid equivalents total

phenolic contents.

4.3.3. Total flavonoid contnents of Pinus roxburghii oleoresin

The total flavonoid contents in essential oils extracted through different extraction

methods, fractions and sub-fractions of most active essential oils were determined from

regression equation of a calibration curve (y = 0.0009x - 0.0011, R² = 0.9866) and expressed as

mg/L of catechin equivalents are shown in Figure 4.3. The total flavonoid contents in essential

oils extracted through different extraction methods, fractions and sub-fractions of most active

essential oils varied from 13.78 ± 1.40 – 446.98 ± 8.17 mg/L of catechin equivalents. In essential

oils extracted through hydro-distillation method, the essential oil of 180 ⁰C revealed maximum

amount of total flavonoid contents (30.90 ± 1.77 mg/L of catechin equivalents). Therefore,

fractions and sub-fractions of 180 ⁰C essential oil were further evaluated for total flavonoid

contents. It was observed that total flavonoid contents increased on increasing the extraction

temperature (Figure 4.3). In fractions of hydro-distilled essential oil extracted at 180 ⁰C, F1

fraction showed highest total flavonoid contents (99.31 ± 2.48 mg/L of catechin equivalents),

while F4 fraction contained lowest total flavonoid contents (19.01 ± 2.07 mg/L of catechin

equivalents). In sub-fractions, F2 b contained maximum total flavonoid contents (446.98 ± 8.17

mg/L of catechin), while F1 d showed minimum total flavonoid contents (37.40 ± 1.93 mg/L of

catechin). It was observed that sub-fractions showed highest total flavonoid contents, followed

by fractions and pure essential oils (Figure 4.3). In essential oils extracted through steam

62

Figure 4.3: Total flavonoid contents of Pinus roxburghii oleoresin

18.84

23.28

24.71 30.9

99.31

86.61 76.92

19.01

195.35

304.1

57.94 37.4

49.83

446.98

108.78

76.54 59.4

349.91

13.78

88.22

170.44

269.33

23.13

42.32 31.33

82.26

18.72

156.89

250.52

18.09

56.14 50.54

87.51 110.27

203.51

61.5 84.24

0

50

100

150

200

250

300

350

400

4501

20

⁰C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F1 c

F1 d

F2 a

F2 b

F2 c

F2 d

F3 a

F3 b

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F1 c

F2 a

F2 b

F2 c

F2 d

F3 a

F3 b

F3 c

40

⁰C

HD SD SCF

To

tal

fla

vo

no

ids

con

ten

ts

(mg

/L o

f C

E)

Pinus roxburghii oleoresin essential oils , fractions and sub-fractions

63

distillation method, the essential of 180 ⁰C showed higher total flavonoid contents (269.33 ±

6.73 mg/L of catechin equivalents) than other steam distilled essential oils. It was observed in

steam distilled essential oils that total flavonoid contents were increased on increasing the

extraction temperature. In fractions of 180 ⁰C steam distilled essential oil, F4 fractions showed

maximum total flavonoid contents (82.26 ± 2.05 mg/L of catechin equivalents), whereas in sub-

fractions, F1 c sub-fraction showed maximum total flavonoid contents (250.52 ± 6.26 mg/L of

catechin equivalents). Overall, it was found that essential oils contained higher total phenolic

contents followed by sub-fractions and fractions (Figure 4.3). The comparison of total flavonoid

contents in essential oils extracted through different extraction methods, steam distilled essential

oils showed higher total flavonoid contents (13.78 ± 1.40 – 269.33 ± 6.73 mg/L of catechin

equivalents), followed by supercritical fluid extracted essential oil (84.24 ± 3.74 mg/L of

catechin equivalents) and hydro distilled essential oils (18.84 ± 2.20 – 30.09 ± 1.77 mg/L of

catechin equivalents). Previously, it was reported that bark extracts of Pinus roxburghii

contained total flavonoid contents in range of 97.4 ± 5.12 – 740 ± 2.09 mg/ 100 g of Quercetin

equivalents, whereas the needle extracts revealed total flavonoid contents in range of 108 ± 3.12

– 428 ± 2.17 mg/ g of Quercetin equivalents (Maimoona et al., 2011).

4.3.4. Total flavonoid contnents of Boswellia serrata oleoresin

The total flavonoid contents in Boswellia serrata oleoresin essential oils extracted

through different extraction methods, fractions and sub-fractions of most active essential oils

were determined from regression equation of a calibration curve (y = 0.0009x - 0.0011, R² =

0.9866) and expressed as mg/L of catechin equivalents given in Figure 4.4. The total flavonoid

contents in Boswellia serrata oleoresin essential oils extracted through different extraction

methods, fractions and sub-fractions of most active essential oils, varied from 7.02 ± 0.02 –

444.89 ± 10.14 mg/L of catechin equivalents. In essential oils extracted through hydro

distillation method, essential oil of 180 ⁰C showed maximum amount of total flavonoid contents

(444.89 ± 10.14 mg/L of catechin equivalents). Therefore, fractions and sub-fractions of 180 ⁰C

essential oil were further evaluated for total flavonoid contents. It was observed that total

flavonoid contents were increased on increasing the extraction temperature (Figure 4.4).

Moreover, it was observed that every last fraction and sub-fraction showed higher total flavonoid

contents than previous fractions and sub-fractions (Figure 4.4). In fractions of 180 ⁰C hydro

distilled essential oil, F4 fraction showed highest amount of total flavonoid contents (68.57 ±

https://www.google.com.pk/search?espv=2&biw=1352&bih=635&q=quercetin&spell=1&sa=X&ved=0ahUKEwiwhaf9wL3QAhUHNhoKHQXxDM8QvwUIFSgA

https://www.google.com.pk/search?espv=2&biw=1352&bih=635&q=quercetin&spell=1&sa=X&ved=0ahUKEwiwhaf9wL3QAhUHNhoKHQXxDM8QvwUIFSgA

64

Figure 4.4: Total flavonoid contents of Boswellia serrata oleoresin

207.11

328.22

324.89

444.89

7.02 10.82 21.87

68.57

132.67

56.76

297.11

100.17

53.46

257.11

32.12 31.76

198.22

90.78 96.34

112.68

202.66

208.22

299.33

327.11

70.17 67.36

29.44 33.21

35.54

178.09

32.12

59.07

163.58

376.18

-40

10

60

110

160

210

260

310

360

410

4601

20

⁰C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F1 c

F2 a

F2 b

F2 c

F3 a

F3 b

F3 c

F4 a

F4 b

F4 c

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3

F1 a

F1 b

F1 c

F2 a

F2 b

F2 c

40

⁰C

HD SD SCF

To

tal

fla

vo

no

ids

con

ten

ts

(mg

/L o

f C

E)

Boswellia serrata oleoresin essential oils, fractions and sub-fractions

65

2.72 mg/L of catechin equivalents), while F1 fraction contained lowest amount of total flavonoid

contents (7.02 ± 0.02 mg/L of catechin equivalents). In sub-fractions of hydro distilled essential

oil at 180 ⁰C, F1 c contained maximum total flavonoid contents (297.11 ± 7.42 mg/L of catechin

equivalents), while F3 b sub-fraction showed minimum amount of total flavonoid contents

(31.76 ± 2.79 mg/L of catechin equivalents). Moreover, it was found that essential oils showed

higher total flavonoid contents, followed by sub-fractions and fractions (Figure 4.4). In essential

oils extracted through steam distillation method, the essential oil of 180 ⁰C showed higher

amount of total flavonoid contents (327.11 ± 6.17 mg/L of catechin equivalents) than other steam

distilled essential oils. It was observed that total flavonoid contents were increased on increasing

the extraction temperature. In fractions of steam distilled essential oil extracted at 180 ⁰C, F1

fraction showed maximum amount of total flavonoid contents (70.17 ± 1.75 mg/L of catechin

equivalents), while F3 fraction showed lowest amount of total flavonoid contents (29.44 ± 2.74

mg/L of catechin equivalents). In sub-fractions, F1 c exhibited maximum amount of total

flavonoid contents (178.09 ± 5.45 mg/L of catechin equivalents), while F2 a showed minimum

amount of total flavonoid contents (32.12 ± 3.93 mg/L of catechin equivalents). It was observed

that pure essential oils showed higher amount of total flavonoid contents, followed by sub-

fractions and fractions (Figure 4.4). Moreover, it was observed that every last sub-fraction

contained higher amount of total flavonoid contents than previous sub-fractions (Figure 4.4). In

comparison with different extraction methods, it was found that hydro-distilled essential oils

contained higher total flavonoid contents (207.11 ± 5.17 – 444.89 ± 10.14 mg/L of catechin

equivalents), followed by supercritical fluid extracted essential oil (376.18 ± 7.42 mg/L of

catechin equivalents) and hydro-distilled essential oils (202.66 ± 5.06 – 327.11 ± 6.17 mg/L of

catechin equivalents). Previously, it has been reported that total flavonoid contents vary with

extraction conditions and extraction methods. Our findings are higher than previously reported

data that showed the methanol extract of Boswellia serrata leaves contained 150.0 ± 2.89 mg/ g

of catechin equivalents total flavonoid contents (DEVI et al., 2014). Similarly in another study, it

was reported that ethanol extract of Boswellia serrata oleoresin exhibited 0.286 mg/ g of rutin

equivalents total flavonoid contents (Bapat et al.).

4.3.5. DPPH free radical scavanging activity of Pinus roxburghii oleoresin

Free radical scavenging activity of essential oils extracted through different extraction

methods, fractions and sub-fractions of most active essential oils was determined by DPPH assay

66

Figure 4.5: DPPH free radical scavenging activity of Pinus roxburghii oleoresin.

63.9 59.04

66.91

73.77

38.01

48.09

73.2

49.59 54.1

59.68 56.25

61.04

52.24

57.89

58.82

59.75 54.74

80.5

62.4

54.67

60.68 63.33

93.94

48.74

96.97

58.11

80.51 75.92

80.72

73.99 74.71 76.21 80.28 78.28

96.74

83.65

49.53

97.92

0

10

20

30

40

50

60

70

80

90

100

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F1 c

F1 d

F2 a

F2 b

F2 c

F2 d

F3 a

F3 b

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F1 c

F2 a

F2 b

F2 c

F2 d

F3 a

F3 b

F3 c

40

⁰C

BH

T

HD SD SCFControl

% F

ree

rad

ica

l sc

av

an

gin

g


67

and their results are shown in Figure 4.5. It was observed that all the essential oils, fractions and

sub-fractions exhibited significant free radical scavenging capacity, varied from 38.01 ± 0.95 –

96.97 ± 1.53 %, whereas the standard BHT showed maximum free radical scavenging 97.92 ±

1.92 %. In essential oils extracted by hydro-distillation method, essential oil of 180 ⁰C showed

maximum free radical scavenging (73.77 ± 1.12 %). Therefore, fractions and sub-fractions of

essential oil extracted at 180 ⁰C were tested for free radical scavenging activity to find the most

active fraction and sub-fraction. In fractions, F3 fraction showed highest free radical scavenging

(73.20 ± 0.96 %), while in sub-fractions, F3 b sub-fraction showed maximum free radical

scavenging (80.50 ± 1.12 %). It was found that every last sub-fraction showed more free radical

scavenging capacity than previous sub-fractions. Overall, essential oils extracted by hydro-

distillation method at different temperatures showed higher free radical scavenging activity than

fractions and sub-fractions. Similar trend was found in essential oils extracted by steam

distillation method, where the essential oil of 180 ⁰C showed highest free radical scavenging

(63.33 ± 0.87 %). In fractions of steam distilled essential oil at 180 ⁰C, F3 fraction exhibited

highest free radical scavenging (96.97 ± 1.53 %), while F2 fraction showed lowest DPPH free

radical scavenging potential (48.74 ± 0.62 %). In sub-fractions, F3 b sub-fraction exhibited

maximum DPPH free radical scavenging (96.74 ± 1.56 %). In comparison with different

extraction methods, it was found that hydro-distilled essential oils showed higher DPPH free

radical scavenging capacity, varied from 59.04 ± 0.71 - 73.77 ± 1.12 %, followed by hydro-

distilled essential oils (54.67 ± 0.68 - 63.33 ± 0.87 %) and supercritical fluid extracted essential

oil (49.53 ± 0.31 %). Previously, it has been reported that free radical scavenging activity of

essential oils vary with extraction conditions and extraction methods (Danh et al., 2013; Bagheri

et al., 2014). The DPPH free radical scavenging activity of hydro-distilled essential oils are less

than (Salem et al., 2014) how reported that needle, wood and bark essential oils of Pinus

roxburghii revealed 50, 82 and 85 % DPPH free radical scavenging activity, respectively. In

another study it was reported that aqueous extract of Pinus maritima showed 94.51 % DPPH free

radical scavenging activity (Dudonné et al., 2009). (Shah et al., 2014) reported that 100 mg/mL

of Pinus roxburghii essential oil revealed 10 % DPPH free radical scavenging.

4.3.6. DPPH free radical scavanging activity of Boswellia serrata oleoresin

Free radical scavenging capacity of Boswellia serrata oleoresin essential oils extracted

through different extraction methods, fractions and sub-fractions of most active essential oils was

68

Figure 4.6: DPPH free radical scavenging activity of Boswellia serrata oleoresin

58.25 53.39

56.04 59.33

59.61

58.76

58.47

72.2

65.76 63.47

64.05

62.97 66.26 68.27

58.68 58.61

66.76

55.32

35.44

57.39

59.39

58.12 55.75

71.77

49.88

49.45

46.52

67.63

42.73

94.63

50.67

95.77 92.73

96.16 97.92

0

10

20

30

40

50

60

70

80

90

1001

20

⁰C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F1 c

F2 a

F2 b

F2 c

F3 a

F3 b

F3 c

F4 a

F4 b

F4 c

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3

F1 a

F1 b

F1 c

F2 a

F2 b

F2 c

40

⁰C

BH

T

HD SD SCFControl

% F

ree

rad

ica

l sc

av

an

gin

g


69

determined by DPPH free radical scavenging assay and their results are shown in Figure 4.6. It

was observed that all the essential oils, fractions and sub-fractions revealed significant free

radical scavenging capacity, varied from 35.44 ± 0.42 – 96.16 ± 1.57 %, while the standard BHT

showed maximum free radical scavenging (97.92 ± 1.92 %). In essential oils extracted by hydro

distillation method, essential oil of 180 ⁰C exhibited maximum free radical scavenging capacity

(59.33 ± 0.78 %). Therefore, fractions and sub-fractions of 180 ⁰C essential oil were tested for

free radical scavenging activity to find the most active fraction and sub-fraction. In fractions, F4

fraction showed highest free radical scavenging capacity (72.20 ± 1.18 %), while F2 c sub-

fraction exhibited maximum free radical scavenging (68.27 ± 0.96 %). It was found that every

last sub-fraction showed higher free radical scavenging capacity than previous sub fractions

except in sub-fractions of F1 fraction. Similar trend was found in essential oils isolated by steam

distillation method, where essential oil of 180 ⁰C showed highest free radical scavenging (71.77

± 1.16 %). In fractions of steam distilled essential oil at 180 ⁰C, F1 fraction showed highest free

radical scavenging (49.88 ± 0.54 %), while F3 fraction showed lowest DPPH free radical

scavenging capacity (46.52 ± 0.32 %). In sub-fractions, F2 b sub-fraction showed maximum

DPPH free radical scavenging capacity (95.77 ± 1.35 %). The comparison of DPPH free radical

scavenging activity with different extraction methods, it was observed that essential oil extracted

through supercritical fluid method showed higher DPPH free radical scavenging capacity (96.16

± 1.57 %), followed by steam distilled essential oils (55.75 ± 0.66 - 71.77 ± 1.16 %) and hydro

distilled essential oils (53.39 ± 0.43- 59.33 ± 0.78 %). Previously, it was reported that free

radical scavenging activity of essential oils vary with extraction conditions and extraction

methods (Danh et al., 2013; Bagheri et al., 2014). It was reported that Myrtus communis essential

oil isolated by solvent free microwave extraction method showed higher DPPH free radical

scavenging capacity than essential oil isolated through hydro-distillation method. Moreover, the

variations in DPPH free radical scavenging activity may be due to difference in chemical

composition of these essential oils isolated through different extraction methods (Berka-Zougali

et al., 2012). In another study, it was reported that chemical composition and DPPH free radical

scavenging activity of Boswellia serrata essential oils vary with geographical location (Gupta et

al., 2016). (Afsar et al., 2012) reported that methanol extract of Boswellia serrata leaves

contained significant DPPH free radical scavenging activity (IC 50 26.02 μg/ml). (Bapat et al.)

found that 1 mg/ ml of Boswellia serrata oleoresin essential oil showed (8.28 ± 0.14 %) free

70

radical scavenging. (Prakash et al., 2014) reported that Boswellia carterii oleoresin essential oil

showed IC 50 0.64 µl/ml. (Singh et al.) found DPPH free radical scavenging activity of aqueous

and alcoholic extract of Boswellia serrata barks (23.53 and 91.97μg/ml of IC 50 respectively).

(Mothana et al., 2011) determined DPPH free radical scavenging activity (22, 21 and 28 %,

respectively) of Boswellia dioscorides, Boswellia elongata and Boswellia socotrana oleoresins

essential oils. (Alizadeh et al., 2010) determined the DPPH free radical scavenging activity (IC50

of 8.45 μg/ml) in Satureja hortensis L. essential oil.

71

4.3.7. Percent inhibition of linoleic acid oxidation of Pinus roxburghii oleoresin

The percent inhibition of linoleic acid oxidation as revealed the Pinus roxburghii

oleoresin essential oils extracted through different extraction methods, fractions and sub-

fractions of most active essential oils is given in Figure 4.7. In linoleic acid assay, greater the

number of peroxides formed during the reaction, higher will be the absorbance which ultimately

shows the lowest antioxidant activity. Pinus roxburghii oleoresin essential oils extracted through

different extraction methods, fractions and sub-fractions of most active essential oils under

observation suppressed the oxidation of linoleic acid by 31.71 ± 0.73 - 98.92 ± 1.85 %. It was

found that most of essential oils, fractions and sub-fractions showed good antioxidant activity

with lowest amount of linoleic acid peroxide formation. In essential oils extracted by hydro-

distillation method, the essential oil of 180 ⁰C showed maximum ability to inhibit the linoleic

acid peroxide formation (97.84 ± 1.73 %). Therefore, fractions and sub-fractions of this essential

oil were further tested. In fractions, F1 fraction showed the maximum ability to inhibit the the

linoleic acid peroxide formation (97.60 ± 1.83 %). In sub-fractions, F3 b showed maximum

ability to inhibit linoleic acid peroxide formation (98.04 ± 1.68 %). Moreover, it was observed

that every last sub-fraction showed more inhibition of linoleic acid peroxidation than previous

sub-fractions (Figure 4.7). A much similar trend was found in essential oils extracted by steam

distillation method, where essential oil extracted at 180 ⁰C showed maxmimum inhibition of

linoleic acid peroxide formation (96.55 ± 1.71 %). In fractions, F4 fraction showed maximum

inhibition of linoleic acid peroxidation formation (98.92 ± 1.85 %). In sub-fractions, F3 a sub-

fraction showed highest inhibition of linoleic acid peroxidation formation (98.72 ± 1.74 %). In

comparison with different extraction methods, hydro-distilled essential oils showed maximum

inhibition of linoleic acid peroxide formation (82.23 ± 1.46 – 97.84 ± 1.73 %), followed by

steam distilled essential oils (80.46 ± 1.42 – 96.55 ± 1.71%) and supercritical fluid extracted

essential oil (88.37 ± 1.92 %). The variaitions in inhibition of linoleic acid peroxide formation

may be due to difference in chemical composition of essential oils.

4.3.8. Percent inhibition of linoleic acid oxidation of Boswellia serrata oleoresin

The percentage inhibition of linoleic acid oxidation as revealed the Boswellia serrata

oleoresin essential oils extracted through different extraction methods, fractions and sub-

fractions of most active essential oils is given in Figure 4.8. In linoleic acid assay, greater the

72

Figure 4.7: Percent inhibition of linoleic acid oxidation of Pinus roxburghii oleoresin.

82.23 87.35

95.45

97.84 97.6

92.21 86.31

83.02

38.98

57.29

80.21 85.87

47.01

79.88 79.43 82.13

79.14

98.04

80.46

92.18 93.16

96.55 96.79

40.34

97.03 98.92

81.04

61.93

97.42

73.84

80.92

98.2 97.75 98.72

31.71

97.23

88.37

98.85

0

20

40

60

80

100

1201

20

⁰C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F1 c

F1 d

F2 a

F2 b

F2 c

F2 d

F3 a

F3 b

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F1 c

F2 a

F2 b

F2 c

F2 d

F3 a

F3 b

F3 c

40

⁰C

BH

T

HD SD SCF

% i

nh

ibit

ion

of

lin

ole

ic a

cid

oxid

ati

on


73

Figure 4.8: Percent inhibition of linoleic acid oxidation of Boswellia serrata oleoresin.

43.53

68.56 70.53

93.28

96.1 95.53 95.2

98.02 96.04

94.28 94.49

92.97 96.02

98.02

92.78 93.56

96.88

90.75

26.37

92.62

71.37

79.58

42.75

82.28

62.15 60.47

88.04

58.73

26.51

96.2

64.12

98.05

92.32 94.18

98.85

0

20

40

60

80

100

1201

20

⁰C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F1 c

F2 a

F2 b

F2 c

F3 a

F3 b

F3 c

F4 a

F4 b

F4 c

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3

F1 a

F1 b

F1 c

F2 a

F2 b

F2 c

40

⁰C

BH

T

HD SD SCF

% i

nh

ibit

ion

of

lin

ole

ic a

cid

oxid

ati

on


74

number of peroxides formed during the reaction, higher will be the absorbance which ultimately

shows the lowest antioxidant activity. Boswellia serrata oleoresin essential oils extracted through

different extraction methods, fractions and sub fractions of most active essential oils under

observation suppressed the oxidation of linoleic acid by (26.37 ± 0.53 - 98.05 ± 1.76 %). It was

found that most of essential oils, fractions and sub-fractions showed good antioxidant activity

with least amount of linoleic acid peroxide formation. In essential oils extracted by hydro-

distillation method, the essential oil of 180 ⁰C showed maximum ability to inhibit the linoleic

acid peroxide formation (93.28 ± 1.53 %). Therefore, fractions and sub-fractions of this essential

oil were further tested. In fractions, F4 fraction exhibited the maximum ability to inhibit the the

linoleic acid peroxide formation (98.02 ± 1.04 %). In sub-fractions, F2 c showed maximum

ability to inhibit linoleic acid peroxide formation (98.02 ± 1.96 %). Similar trend was found in

essential oils extracted by steam distillation method, where essential oil extracted at 180 ⁰C

showed maxmimum inhibition of linoleic acid peroxide formation (82.28 ± 1.27 %). In fractions,

F3 fraction showed maximum inhibition of linoleic acid peroxidation formation (88.04 ± 1.46

%). F2 b sub-fraction revealed highest inhibition of linoleic acid peroxidation formation (98.05 ±

1.76 %) in sub-fractions. In comparison with different extraction methods, supercritical fluid

extracted essential oil showed maximum inhibition of linoleic acid peroxide formation (94.18 ±

1.47 %), followed by hydro-distilled essential oils (43.53 ± 0.87 – 93.28 ± 1.53 %) and steam

distilled essential oil (42.75 ± 0.78 – 82.28 ± 1.27 %). The variaitions in inhibition of linoleic

acid peroxide formation may due to difference in chemical composition of essential oils.

4.3.9. Hydrogen peroxide scavenging activity of Pinus roxburghii oleoresin

The ability of essential oils isolated by different extraction methods, fractions and sub-

fractions of most active essential oils to scavenge hydrogen peroxide was determined by

hydrogen peroxide scavenging assay and their results are given in Figure 4.9. The hydrogen

peroxide radical scavenging capacity of essential oils isolated by different extraction methods,

fractions and sub-fractions of most active essential oils was found in range of 48.22 ± 0.52 –

69.31 ± 0.87 %, while the standard (Ascorbic acid 100 ppm) showed 85.57 ± 0.46 % hydrogen

peroxide radical scavenging capacity. Overall, essential oils obtained by different extraction

methods, fractions and sub-fractions of most active essential oils showed moderate level of

hydrogen peroxide radical scavenging activity. In essential oils isolated by hydro-distillation

75

Figure 4.9: Hydrogen peroxide scavenging activity of Pinus roxburghii oleoresin

50.12 54.77

56.63 58.96

56.72

48.22

64.23

52.09

59.1 64.18

59.51 60.04

57.95 57.93

58.11 57.75

60.38

66.29

55.32 53.76

50.43

59.42

65.54

48.23

67.23 69.31

55.88 50.06

50.45 49.59

48.48

56.12 52.21

52.68 59.56

48.26 52.49

85.57

0

10

20

30

40

50

60

70

80

90

1001

20

⁰C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F1 c

F1 d

F2 a

F2 b

F2 c

F2 d

F3 a

F3 b

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F1 c

F2 a

F2 b

F2 c

F2 d

F3 a

F3 b

F3 c

40

⁰C

Asc

orb

ic a

cid

HD SD SCF

% H

yd

rog

en p

ero

xid

e sc

av

eng

ing

act

ivit

y

Pinus roxhburgii oleoresin essential oils, fractions and sub-fractions

76

methods, essential oil of 180 ⁰C exhibited the highest hydrogen peroxide radical scavenging

capacity (58.96 ± 0.82 %). Therefore, hydrogen peroxide radical scavenging capacity of this oil

fractions and sub-fractions was further determined. In fractions, F3 fraction showed highest

hydrogen peroxide radical scavenging (64.23 ± 0.86 %), while F2 fraction showed lowest

hydrogen peroxide radical scavenging (48.22 ± 0.52 %). In sub-fractions, F3 b sub-fraction

exhibited highest hydrogen peroxide radical scavenging (66.29 ± 0.85 %), while, F2 d sub-

fraction showed lowest hydrogen peroxide radical scavenging capacity (57.75 ± 0.44 %).

Overall, F3 b sub-fraction exhibited highest hydrogen peroxide radical scavenging (66.29 ± 0.85

%) in all essential oils isolated by different extraction methods, fractions and sub-fractions of

most active essential oils. Similarly, in essential oils obtained by steam distillation method,

essential oil of 180 ⁰C exhibited the highest hydrogen peroxide radical scavenging (59.42 ± 0.52

%). Therefore, hydrogen peroxide radical scavenging capacity of this oil fractions and sub-

fractions was further determined. In fractions, F4 fraction showed highest hydrogen peroxide

radical scavenging (69.31 ± 0.87 %), while F2 fraction showed lowest hydrogen peroxide radical

scavenging (48.23 ± 0.32 %). In sub-fractions, F3 b sub-fraction exhibited highest hydrogen

peroxide radical scavenging (59.56 ± 0.89 %), while, F3 c sub-fraction showed lowest hydrogen

peroxide radical scavenging (48.26 ± 0.39 %). Overall, F4 fraction exhibited highest hydrogen

peroxide radical scavenging (69.31 ± 0.87 %), in essential oils isolated by different extraction

methods, fractions and sub-fractions of most active essential oil.

In comparison with different extraction methods, essential oils obtained by hydro

distillation method showed highest hydrogen peroxide radical scavenging capacity, varied from

50.12 ± 0.45 – 58.96 ± 0.82 %. Moreover, it was observed that hydrogen peroxide radical

scavenging capacity increased on increasing the extraction temperatures (Figure 4.9). Essential

oil obtained by supercritical fluid extraction method showed lowest hydrogen peroxide radical

scavenging capacity (52.49 ± 0.46 %). The variations in hydrogen peroxide radical scavenging

capacity of essential oil obtained by different extraction methods may be due to variations in

chemical composition of these essential oils.

4.3.10. Hydrogen peroxide scavenging activity of Boswellia serrata oleoresin

The ability of essential oils isolated by different extraction methods, fractions and sub

fractions of most active essential oils to scavenge hydrogen peroxide was determined by

77

Figure 4.10: Hydrogen peroxide scavenging activity of Boswellia serrata oleoresin.

54.46

56.71

51.05

59.19 58.37

55.23 54.75

68.06

54.62

50.84

54.25 53.58

49.98

66.02

52.16 54.75

58.34 54.91

65.71 64.98

50.74 49.73

53.84 56.79

68.22 67.13

58.57

66.67

67.39 69.35

68.4 69.54

67.78 68.25

85.57

0

10

20

30

40

50

60

70

80

90

100

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F1 c

F2 a

F2 b

F2 c

F3 a

F3 b

F3 c

F4 a

F4 b

F4 c

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3

F1 a

F1 b

F1 c

F2 a

F2 b

F2 c

40

⁰C

Asc

orb

ic a

cid

HD SD SCF

% H

yd

rog

en p

ero

xid

e sc

av

eng

ing

act

ivit

y


78

hydrogen peroxide scavenging assay and their results are given in Figure 4.10. The hydrogen

peroxide radical scavenging capacity of essential oils isolated by different extraction methods,

fractions and sub-fractions of most active essential oils, varied from 49.73 ± 0.59 – 69.54 ± 1.04

%, while the hydrogen peroxide radical scavenging capacity for standard was 85.57 ± 0.46 %.

Overall, essential oils obtained by different extraction methods, fractions and sub-fractions of

most active essential oils showed moderate level of hydrogen peroxide radical scavenging

activity. In essential oils extracted by hydro-distillation methods, essential oil of 180 ⁰C

exhibited the highest hydrogen peroxide radical scavenging (59.19 ± 0.72 %). Therefore,

hydrogen peroxide radical scavenging capacity of this oil fractions and sub-fractions was further

determined. In fractions, F4 fraction showed highest hydrogen peroxide radical scavenging

(68.06 ± 0.92 %), while F3 fraction showed lowest hydrogen peroxide radical scavenging (54.75

± 0.89 %). In sub-fractions, F2 c sub-fraction exhibited highest hydrogen peroxide radical

scavenging (66.02 ± 0.78 %), while, F2 b sub-fraction showed lowest hydrogen peroxide radical

scavenging (49.98 ± 0.58 %). Overall, F4 fraction exhibited highest hydrogen peroxide radical

scavenging (66.02 ± 0.78 %) in all essential oils obtained by different extraction methods,

fractions and sub-fractions of most active essential oils. Similarly, in essential oils obtained by

steam distillation method, essential oil of 180 ⁰C exhibited the highest hydrogen peroxide radical

scavenging capacity (56.79 ± 0.73 %). Therefore, hydrogen peroxide radical scavenging capacity

of this oil fractions and sub-fractions was further determined. In fractions, F1 fraction showed

highest hydrogen peroxide radical scavenging (68.22 ± 0.79 %), while F3 fraction showed lowest

hydrogen peroxide radical scavenging capacity (58.57 ± 0.68 %). In sub-fractions, F2 b sub-

fraction exhibited highest hydrogen peroxide radical scavenging capacity (69.54 ± 1.04 %),

while, F1 a sub-fraction showed lowest hydrogen peroxide radical scavenging capacity (66.67 ±

0.73 %). Overall, F2 b sub-fraction exhibited highest hydrogen peroxide radical scavenging

(69.54 ± 1.04 %), in essential oils isolated by different extraction methods, fractions and sub-

fractions of most active essential oil. In comparison with different extraction methods, essential

oil obtained by supercritical fluid extraction method showed highest hydrogen peroxide radical

scavenging capacity (68.25 ± 1.02 %), followed by hydro-distillated essential oils (51.05 ± 0.65

– 59.19 ± 0.72 %) and steam distilled essential oils (49.73 ± 0.59 – 56.79 ± 0.73 %). The

variation in hydrogen peroxide radical scavenging capacity of essential oil isolated by different

extraction methods may due to variation in chemical composition of these essential oils.

79

4.3.11. Total antioxidant contents/ FRAP assay of Pinus roxburghii oleoresin

Total antioxidant contents of essential oils isolated from Pinus roxburghii oleoresin by

different extraction methods, fractions and sub fractions of most active essential oils were

determined from regression equation of a calibration curve (y = 0.021x - 0.0151, R² = 0.99) and

expressed as mg/L of Gallic acid equivalents (GAE), shown in Figure 4.11. The total antioxidant

contents in essential oils isolated by different extraction methods, fractions and sub fractions of

most active essential oils were varied from 13.56 ± 0.47 – 164.9 ± 2.45 mg/L of Gallic acid

equivalents. It was observed that most of the sub fractions showed higher total antioxidant

contents than essential oils and fractions. In essential oils isolated by hydro distillation method at

different temperatures, essential oil isolated at 180 ⁰C showed highest total antioxidant contents

(111.73 ± 1.16 mg/L of gallic acid equivalent). Therefore, fractions and sub fractions of this oil

were fruther evalated for total antioxidant contents to find most active fraction and sub fraction.

In fractions, F4 fraction showed highest total antioxidant contents (68.77 ± 1.32 mg/L of gallic

acid equivalent), while F3 fraction showed lowest amount of total antioxidant contents (47.41 ±

0.85 mg/L of gallic acid equivalent). Similarly, in sub fractions F3 b exhibited maximum amount

of total antioxidant contents (164.90 ± 2.45 mg/L of gallic acid equivalent), while F1 c sub

fraction exhibited minumum amount of total antioxidant contents (13.56 ± 0.47 mg/L of gallic

acid equivalent). Overall, F3 b sub fraction contained highest amount of total antioxidant

contents (164.90 ± 2.45 mg/L of gallic acid equivalent) in essential oils isolated by hydro

distillation method, fractions and sub fractions of most active essential oil. In essential oils

extracted by steam distillation method at different temperatures, essential oil of 180 ⁰C showed

highest total antioxidant contents (134.49 ± 1.49 mg/L of gallic acid equivalent). Therefore,

fractions and sub fractions of this oil were fruther evalated for total antioxidant contents to find

most active fraction and sub fraction. In fractions, F4 fraction showed highest total antioxidant

contents (78.42 ± 1.02 mg/L of gallic acid equivalent) and F1 fraction showed lowest amount of

total antioxidant contents (54.14 ± 0.96 mg/L of gallic acid equivalent). Similarly, in sub

fractions F3 c showed maximum amount of total antioxidant contents (124.33 ± 1.25 mg/L of

gallic acid equivalent), while F2 b sub fraction exhibited minumum amount of total antioxidant

contents (34.42 ± 0.57 mg/L of gallic acid equivalent). Overall, essential oil isolated at 180 ⁰C

contained highest amount of total antioxidant contents (134.49 ± 1.49 mg/L of gallic acid

equivalent).

80

Figure 4.11: Total antioxidant contents/ FRAP assay of Pinus roxburghii oleoresin.

109.09

87.63 95.28

111.73

55.47 52.3

47.41

68.77

34.42 25.56

13.56

118.42

38.77

25.98

93.38

15.85

145.56

164.9

67.44

102.07

127.12 134.49

54.14

56.29 56.9

78.42

52.13

82.77

97.28

40.14 34.42

90.23

114.23

38.77

52.99

124.33

99.92

0

20

40

60

80

100

120

140

160

180

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F1 c

F1 d

F2 a

F2 b

F2 c

F2 d

F3 a

F3 b

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F1 c

F2 a

F2 b

F2 c

F2 d

F3 a

F3 b

F3 c

40

⁰C

HD SD SCF

To

tal

an

tio

xid

an

t co

nte

nts

/ F

RA

P

(mg

/l o

f G

AE

)

Pinus roxburghii oleoresin essential oils , fractions and sub fractions

81

In comparison with different extraction methods, steam distillated essential oils showed

maximum amount of total antioxidant contents, varied from 67.44 ± 0.95 – 134.49 ± 1.34 mg/L

of Gallic acid equivalent, followed by hydro-distillated essential oils 87.63 ± 0.94 – 111.73 ±

1.16 mg/L of Gallic acid equivalent and supercritical fluid extracted essential oil 99.92 ± 1.06

mg/L of Gallic acid equivalent. Such variations in hydrogen peroxide radical scavenging

capacity of essential oil isolated by different extraction methods may be due to difference in

chemical composition of these essential oils.

4.3.12. Total antioxidant contents/ FRAP assay of Boswellia serrata oleoresin

Total antioxidant contents of essential oils isolated from boswellia serrata oleoresin by

different extraction methods, fractions and sub-fractions of most active essential oils were

determined from regression equation of a calibration curve (y = 0.021x - 0.0151, R² = 0.99) and

expressed as mg/L of gallic acid equivalents (GAE), given in Figure 4.12. The total antioxidant

contents in essential oils isolated by different extraction methods, fractions and sub-fractions of

most active essential oils, varied from 24.65 ± 0.65 – 134.33 ± 1.47 mg/L of gallic acid

equivalents. In essential oils isolated by hydro-distillation method at different temperatures,

essential oil of 180 ⁰C showed highest total antioxidant contents (124.65 ± 1.49 mg/L of gallic

acid equivalent). Therefore, the total antioxidant contents of fractions and sub-fractions of this

oil was determined. In fractions, F3 fraction showed highest total antioxidant contents (109.03 ±

1.18 mg/L of gallic acid equivalent), while F1 fraction showed lowest amount of total

antioxidant contents (75.12 ± 0.84 mg/L of Gallic acid equivalent). Similarly, in sub-fractions F3

c showed maximum amount of total antioxidant contents (108.90 ± 1.25 mg/L of gallic acid

equivalent), while F4 c sub-fraction exhibited minumum amount of total antioxidant contents

(24.65 ± 0.65 mg/L of Gallic acid equivalent). Overall, essential oil isolated at 180 ⁰C contained

highest amount of total antioxidant contents (124.65 ± 1.49 mg/L of Gallic acid equivalent) in

essential oils isolated by hydro-distillation method, fractions and sub-fractions of most active

essential oil. In essential oils isolated by steam distillation method at different temperatures,

essential oil isolated at 180 ⁰C showed highest total antioxidant contents (126.39 ± 1.56 mg/L of

Gallic acid equivalent). In fractions, F1 fraction showed highest total antioxidant contents (98.58

± 1.14 mg/L of Gallic acid equivalent) and F2 fraction showed lowest amount of total

antioxidant contents (70.65 ± 0.89 mg/L of Gallic acid equivalent). Similarly, in sub fractions F1

c showed maximum amount of total antioxidant contents (68.58 ± 0.89 mg/L of Gallic acid

82

Figure 4.12: Total antioxidant contents/ FRAP assay of Boswellia serrata oleoresin.

121.4

91.66

106.81

124.65

75.12

90.62

109.03

89.98

74.24

92.86

95.05

55.92 49.76

97.83

59.95

47.76

108.9

52.68 56.11

24.65

94.39

115.94

81.44

126.39

98.58

70.65

79.38

59.73

43.51

68.58

38.99 37.06

56.74

134.33

0

20

40

60

80

100

120

140

160

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F1 c

F2 a

F2 b

F2 c

F3 a

F3 b

F3 c

F4 a

F4 b

F4 c

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3

F1 a

F1 b

F1 c

F2 a

F2 b

F2 c

40

⁰C

HD SD SCF

To

tal

an

tio

xid

an

t a

ctiv

ity

/FR

AP

(mg

/L G

AE

)

Boswellia serrata oleoresin essential oils , fractions and sub-fractions

83

equivalent), while F2 b sub-fraction exhibited minumum amount of total antioxidant contents

(37.06 ± 0.57 mg/L of Gallic acid equivalent). Overall, steam distilled essential oil of 180 ⁰C

contained highest amount of total antioxidant contents (126.39 ± 1.56 mg/L of Gallic acid

equivalent).In comparison with different extraction methods, supercritical fluid extracted

essential oil showed maximum amount of total antioxidant contents (134.33 ± 1.47 mg/L of

Gallic acid equivalents), and followed by steam distillated essential oils (81.44 ± 0.97 – 126.39 ±

1.56 mg/L of Gallic acid equivalents) and hydro-distilled essential oils (91.66 ± 1.04 – 124.65 ±

1.49 mg/L of Gallic acid equivalents). The variation in hydrogen peroxide radical scavenging

capacity of essential oil isolated by different extraction methods may due to variation in chemical

composition of these essential oils. Previously, it was reported that aqueous and alcoholic

extracts of Boswellia serrata showed significant amount of total antioxidant contents (Azemi et

al., 2012).

4.4. Antimicrobial activity

4.4.1. Antibacterial activity of Pinus roxburghii oleoresin by Well diffusion method

Antibacterial activity of Pinus roxburghii oleoresin essential oils isolated by different

extraction methods, most active fractions and sub-fractions was determined by well diffusion

method and their results are given in figure 4.13 to 4.16. The values of inhibition zones for

essential oils isolated by different extraction methods, most active fractions and sub-fractions

were 8.31 – 31.80 mm, while the data acquired for positive control (Amoxicillin) was 21.11-

28.53 mm. It was found that most of essential oils, fractions and sub-fractions showed excellent

antibacterial activity. Moreover, it was observed that sub-fractions showed higher antibacterial

activity than its essential oil and fractions. The values of inhibition zones showed that P.

multocida and S. aureus were the most sensitive bacterial strains presenting larger inhibition

zones 10.02 - 27.32 mm and 13.58 – 31.80 mm respectively, while E. coli and B. subtilis were

the least sensitive bacterial strains presenting small inhibition zones 8.31 – 25.82 mm and 10.44

– 24.52 mm, respectively. The antibacterial activity of Pinus roxburghii oleoresin essential oils

may due to presence of high concentration of α–pinene. Previously, it was reported that α–pinene

showed significant antibacterial activity against S. aureus, P. multocida and B. subtilis (Hassan

and Amjid, 2009; Zafar et al., 2010).

84

In essential oils obtained through hydro distillation method at different temperature

conditions, the essential oil of 160 ⁰C showed the highest antibacterial activity with larger

inhibition zones (16.24 - 26.00 mm) against all test bacterial strains. Therefore, the fractions and

sub-fractions of this essential oil were tested for antibacterial activity to find most active fraction

and sub-fraction. In fractions of 160 ⁰C essential oil, F1 fraction showed highest antibacterial

activity against E. coli and S. aureus with inhibition zones of 19.02 and 27.10 mm respectively,

while F3 fraction exhibited highest antibacterial activity against P. multocida and B. subtilis with

zone of inhibitions 20.78 - 20.40 mm, respectively. In sub-fractions, F2 c sub-fraction showed

highest activity against E. coli and B. subtilis with zone of inhibition 22.52 and 24.52 mm

respectively, while F2 b and F3 a sub-fractions revealed maximum activity against P. multocida

and S. aureus with inhibition zones 26.13 mm and 31.18 mm, respectively. A much similar trend

was observed in essential oils isolated at different temperature conditions by steam distillation

method, where essential oil isolated of 160 ⁰C showed maximum antibacterial activity against all

bacterial strains with inhibition zones in range of 14.45 - 24.74 mm. The essential oils showed

highest activity against S. aureus with inhibition zones 14.22 – 24.74 mm, while least activity

was observed against E. coli with inhibition zones 12.86 – 14.45 mm. In fractions of 160 ⁰C

essential oil, F4 fraction showed maximum antibacterial activity with larger inhibition zones

against all bacterial strains. In sub-fractions, F2 c sub-fraction showed highest activity against E.

coli and S. aureus with inhibition zones 17.74 mm and 27.94 mm, respectively, while F2 a sub-

fraction exhibited highest bacterial activity with inhibition zones of 20.01 mm and 23.38 mm

against P. multocida and B. subtilis, respectively.

In comparison of hydro, steam and supercritical fluid extraction methods, essential oils

obtained through hydro-distillation method showed higher antibacterial activity with larger

inhibition zones in range of 16.24 - 26.00 mm for all the test bacterial strains. While, essential oil

obtained through supercritical fluid extraction method showed lowest antibacterial activity with

smaller inhibition zones 10.14 – 16.30 mm. Such variations in antibacterial activity of essential

oils may due to difference in chemical composition of essential oils. Previous literature studies

confirmed that chemical compositions of essential oils vary with extraction methods and such

variation directly affects the antibacterial activity (Kokoska et al., 2008; Okoh et al., 2010). It

was observed that all essential oils isolated through different extraction methods showed

excellent antibacterial activity against all test bacterial strains. The values of inhibition zones

85

Figure 4.13: Antibacterial activity of Pinus roxburghii oleoresin against E. coli

14.02

13.65

16.24

13.21

19.02 18.58

18.31

11.32 12.43

14.8

18.42

20.26

22.52

16.13

20.11 19.92

18.33

13.32

18.9

8.31

13.9 12.86

14.45 13.12

13.27

10.23

18.42

25.82

13.4

9.54

15.56 14.96

17.2 16.24

17.74

14.77

21.11

0

5

10

15

20

25

30

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F2 a

F2 b

F2 c

F2 d

F3 a

F3 b

F3 c

F3 d

F4 a

F4 b

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F1 c

F1 d

F2 a

F2 b

F2 c

40

⁰C

Am

oxi

cilli

n

HD SD SCF

Zo

ne

of

inh

ibit

ion

(m

m)

Pinus roxburghii oleoresin essential oils, fractions and sub fractions against E. coli

86

Figure 4.14: Antibacterial activity of Pinus roxburghii oleoresin against S. aureus

24.61 23.42

26

21.23

27.1

24.61 25.2

17.45

24.74 25.66

28.01

31.6 30.32

26.21

31.8

29.49 28.12

23.08

28.95

13.58

21.12

14.22

24.74

22

28.12

24.49

28.3 30.32

26.5 26.5 27.35 26.62 27.45

23.5

27.94

16.3

24.38

0

5

10

15

20

25

30

351

20

⁰C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F2 a

F2 b

F2 c

F2 d

F3 a

F3 b

F3 c

F3 d

F4 a

F4 b

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F1 c

F1 d

F2 a

F2 b

F2 c

40

⁰C

Am

oxi

cilli

n

HD SD SCF

Zo

ne

of

inh

ibit

ion

(m

m)

Pinus roxburghii oleoresin essential oils, fractions and sub fractions against S. aureus

87

Figure 4.15: Antibacterial activity of Pinus roxburghii oleoresin against P. multocida

17.22 16.21

17.45 16.78

17.77

20.06 20.78

13.66

19.36 20.31

23.17

26.13 24.91

18.92

24.57

22.09 21.43

15.6

23.54

10.08

16.12

10.02

16.8 15.9

15.23 14.85

20.63

27.32

16.5 15.74

19.3 19.12

20.01

18.23

19.34

10.14

28.53

0

5

10

15

20

25

301

20

⁰C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F2 a

F2 b

F2 c

F2 d

F3 a

F3 b

F3 c

F3 d

F4 a

F4 b

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F1 c

F1 d

F2 a

F2 b

F2 c

40

⁰C

Am

oxi

cilli

n

HD SD SCF

Zo

ne

of

inib

itio

n (

mm

)

Pinus roxburghii oleoresin essential oils, fractions and sub fractions against P. multocida

88

Figure 4.16: Antibacterial activity of Pinus roxburghii oleoresin against B. subtilis.

15.32

14.18

17.07

14.9

20.36

18.67

20.4

14.72

16.9

18.4

21.57

24.23 24.52

19.03

23.54

19.94

18.34

13.87

17.71

12.87

14.7

13.2

15.45 14.15

20.61

16.8

21.3

24.13

21.73

20.09 21.45

20.66

23.28 22.26

20.45

10.44

22.32

0

5

10

15

20

25

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F2 a

F2 b

F2 c

F2 d

F3 a

F3 b

F3 c

F3 d

F4 a

F4 b

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F1 c

F1 d

F2 a

F2 b

F2 c

40

⁰C

Am

oxi

cilli

n

SD SCF

Zo

ne

of

inh

ibit

ion

(m

m)

Pinus roxburghii oleoresin essential oils, fractions and sub fractions against B. subtilis

89

showed that S. aureus and P. multocida were the most sensitive bacterial strains presenting larger

inhibition zones 14.22 – 26.00 mm and 10.02 – 17.45 mm, respectively, while E. coli and B.

subtilis showed least sensitivity with smaller inhibition zones 12.86 -16.24 and 10.44 – 17.07

mm, respectively. These experimental results are similar to (Salem et al., 2014) how reported

that essential oil isolated from bark, needle and wood of Pinus roxburghii showed good

antibacterial activity. In another study, it was reported that Pinus roxburghii needles essential oil

revealed antibacterial potential against S. aureus and B. subtilis, while no antibacterial activity

was observed against E. coli, E. aerogenes and Salmonella typhi (Zafar et al., 2010). In another

report (Parihar et al., 2006) found that leaf extract of Pinus roxburghii showed antibacterial

activity against E. coli. (Shah et al., 2014) demonstrated that Pinus roxburghii fruit essential oil

exhibited stronger antibacterial activity against Bacillus subtilis, Pseudomonas aeruginosa,

Staphylococcus aureus, K. pneumonia, Escherichia coli and P. vulgaris. (Mahajan and Sharma,

2011) reported that essential oil and chloroform extract of Pinus roxburghii revealed good

antibacterial activity against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus,

Streptococcus pyogenes, Bacillus subtilis. (Shuaib et al., 2013) reported that methanol extract of

Pinus roxburghii oleoresin exhibited moderate antibacterial activity against four Gram positive

and four Gram-negative bacterial strains.

Overall, essential oils obtained by different extraction methods, fractions and sub-

fractions showed higher antibacterial activity with larger inhibition zones against Gram positive

bacterial strains than Gram negative bacterial strains. In comparison of antibacterial activity of

essential oils obtained through different extractions methods, hydro-distilled essential oil showed

highest antibacterial activity against all tested bacterial strains. In hydro-distilled essential oils,

fractions and sub-fractions F2 c sub fraction showed highest activity against E. coli and B.

subtilis, while F2 b and F3 a sub-fractions revealed maximum activity against P. multocida and

S. aureus. In steam distilled essential oils, fractions and sub-fractions F4 fraction exhibited

highest antibacterial activity against all tested bacterial strains.

4.4.2. Antibacterial activity of Boswellia serrata oleoresin by Well diffusion method

Antibacterial activity of Boswellia serrata oleoresin essential oils isolated through

different extraction methods, most active fractions and sub-fractions was determined by well

diffusion method and their results are given in Figure 4.17 to 4.20. The values of inhibition zones

90

for essential oils isolated through different extraction methods, most active fractions and sub-

fractions against E. coli, S. aureus, P. multocida and B. subtilis were in the range of 7.57 – 30.23,

8.00 - 17.65, 10.79 - 38.28 and 8.47 - 21.05 mm, respectively, while the data acquired for

positive control (Amoxicillin) was 21.11-28.53 mm. The GC-MS analysis of pure essential oils

showed that α-pinene, β-pinene, verbenol and pinocarveol were the major components of Boswellia

serrata oleoresin essential oils and these compounds may be responsible for their antibacterial

activity. Previously, it was reported that the antibacterial activity of Boswellia serrata oleoresin

essential oil is not attributed due to single compound, but to the synergistic effect of several

compounds (Camarda et al., 2007). Our finding are opposite to (Shao et al., 1998) how reported

that limonene is the major compound present in Boswellia serrata oleoresin essential oil and

responsible for antibacterial activity. It was observed that most of essential oils, fractions and

sub-fractions showed excellent antibacterial activity for all test bacteria. Moreover, it was found

that most of sub-fractions showed higher antibacterial activity than essential oils and fractions.

The values of inhibition zones showed that P. multocida and E. coli were the most sensitive

bacteria presenting larger inhibition zones 10.79 - 38.28 and 7.57 – 30.23 mm, respectively,

while S. aureus and B. subtilis were the least sensitive bacteria presenting small inhibition zones

8.00 – 17.65 mm and 8.47 – 21.05 mm, respectively. Overall, values of inhibition zones for

essential oils isolated through different extraction methods, most active fractions and sub-

fractions were in range of 7.57 – 38.28 mm.

Antibacterial activity of essential oils isolated through hydro-distillation method at

different temperature conditions showed that essential oil isolated at 140 ⁰C showed higher

antibacterial activity with larger inhibition zones in range of 16.24 - 26.00 mm against all the test

bacteria. Therefore, antibacterial activity of fractions and sub-fractions of 140 ⁰C essential oil

was tested to find the most active fraction and sub-fraction. In fractions of hydro distilled

essential oil of 140 ⁰C, F4 fractions showed higher sensitivity with larger inhibition zones for E.

coli, S. aureus, P. multocida and B. subtilis with inhibition zones 24.33, 14.67, 30.16 and 16.40

mm, respectively. In sub-fractions, F3 c sub-fraction showed higher sensitivity for E. coli, S.

aureus, P. multocida and B. subtilis with inhibition zones 22.81, 13.88, 28.32 and 15.88 mm,

respectively. Overall, F4 fraction showed higher antibacterial activity than essential oils isolated

by hydro-distillation method, its fractions and sub-fractions.

91

Figure 4.17: Antibacterial activity of Boswellia serrata oleoresin against E.coli

14.55 15.1 14.73 13.43

18.02

0 0

24.33

10.2

18.05 19.25

0 0

22.81

16.8 18.7

12.27 11.47

0 0

25.58

0 0

30.23

26 27.26

22.06

7.57

21.11

0

5

10

15

20

25

30

35

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F2 a

F2 b

F2 c

F3 a

F3 b

F3 c

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3

F1 a

F1 b

F1 c

F2 a

F2 b

F2c

40

⁰C

Am

oxi

cilli

n

H.D S.D SCF

Zo

ne

of

inh

ibit

ion

(m

m)

Boswellia serrata oleoresin essential oils ,fractions and sub fractions against E.coli

92

Figure 4.18: Antibacterial activity of Boswellia serrata oleoresin against S. aureus

10.8

12.9

10.4 9.89

12.48

0 0

14.67

8

10

12.66

0 0

13.88 14.15 14.35 13.24

12.45

0 0

14.11

0 0

17.65

10.65

16.42

9.12 9.87

24.38

0

5

10

15

20

25

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F2 a

F2 b

F2 c

F3 a

F3 b

F3 c

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3

F1 a

F1 b

F1 c

F2 a

F2 b

F2c

40

⁰C

Am

oxi

cilli

n

H.D S.D SCF

Zo

ne

of

inh

ibit

ion

(m

m)

Boswellia serrata oleoresin essential oils, fractions and sub-fractions against S. aureus

93

Figure 4.19: Antibacterial activity of Boswellia serrata oleoresin against P. multocida

22.92

24.21

23.11 22.27

25.15

30.16

18.32

24.04

26.24

0 0

28.32

21.07 21.54 19.87 20.27

31.13

0 0

38.28

22.74

26.12

20.23

10.79

28.53

0

5

10

15

20

25

30

35

40

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F2 a

F2 b

F2 c

F3 a

F3 b

F3 c

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3

F1 a

F1 b

F1 c

F2 a

F2 b

F2c

40

⁰C

Am

oxi

cilli

n

H.D S.D SCF

Zo

ne

of

inh

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ion

(m

m)

Boswellia serrata oleoresin essential oils , fractions and sub-fractions against P. multocida

94

Figure 4.20: Antibacterial activity of Boswellia serrata oleoresin against B. subtilis.

12.67

14.22

12.22

10.23

13.6

16.4

9.44

12.01 13.23

0 0

15.88

14.47 13.82

11.25 12.16

18.03

0 0

21.05

15.58

17.28

14.32

8.47

22.32

0

5

10

15

20

251

20

⁰C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F2 a

F2 b

F2 c

F3 a

F3 b

F3 c

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3

F1 a

F1 b

F1 c

F2 a

F2 b

F2c

40

⁰C

Am

oxi

cilli

n

H.D S.D SCF

Zo

ne

of

inh

ibit

ion

(m

m)

Boswellia serrata oleoresin essential oils, fractions and sub-fractions against B. subtilis

95

Similar trend was observed in essential oils obtained at different temperature conditions

through steam distillation method, where essential oil of 140 ⁰C showed maximum antibacterial

activity for all test bacteria with inhibition zones in range of 13.82 - 21.54 mm. The isolated

essential oils showed highest antibacterial activity against P. multocida with inhibition zones

19.87 – 21.54 mm, while lowest activity was observed against S. aureus with inhibition zones

12.45 – 14.45 mm. In fractions of 140 ⁰C essential oil, F3 fraction showed maximum growth

inhibition for E. coli, S. aureus, P. multocida and B. subtilis with diameter of inhibition zones

25.58, 14.11, 21.54 and 13.82 mm, respectively. In sub-fractions, F1 c sub-fraction showed

highest growth inhibition against E. coli, S. aureus, P. multocida and B. subtilis with diameter of

inhibition zones 30.23, 17.65, 38.38 and 21.05 mm, respectively, Overall, in steam distilled

essential oils, its fractions and sub-fractions F1 c exhibited highest antibacterial activity with

larger inhibition zones for all test bacteria.

In comparison of hydro, steam and supercritical fluid extraction methods, essential oils

obtained through steam distillation method showed higher antibacterial activity with larger

inhibition zones in range of 13.82 - 21.54 mm against all test bacteria. While, essential oil

obtained through supercritical fluid extraction method showed lowest antibacterial activity with

smaller inhibition zones (7.57 – 10.79 mm). Such variations in antibacterial activity of essential

oils may be due to difference in chemical composition of essential oils. It was observed that all

essential oils isolated through different extraction methods showed good antibacterial activity

against all test bacteria except supercritical fluid extracted essential oil that showed weak

antibacterial activity. The values of inhibition zones for essential oils isolated through different

extraction methods showed that P. multocida and E. coli were the most sensitive bacterial strains

presenting larger inhibition zones 10.79 – 24.21 mm and 7.57 – 18.70 mm, respectively, while S.

aureus and B. subtilis were the least sensitivity bacterial strains presenting smaller inhibition

zones 9.87 -14.35 and 8.47 – 14.47 mm, respectively. These experimental results are in good

agreement with (Camarda et al., 2007) which reported that Boswellia serrata oleoresin essential

oil exhibited good antibacterial activity against Gram positive than Gram negative bacterial

strains. Similarly, it was reported that Boswellia serrata bark essential oil showed good

antibacterial activity against Proteus Mirabilis, Staphylococcus aureus and Escherichia coli

(Kasali et al., 2002). In another study, it was demonstrated that methanol extract and its fractions

of Boswellia serrata oleoresin showed significant antibacterial activity against multi-drug


https://en.wikipedia.org/wiki/Escherichia_coli

96

resistant clinically isolated bacterial strains. Furthermore, they reported that antibacterial activity

of extract and fractions might be attributed due to triterpenoids, alkaloids, steroids and

flavonoids present in oleoresin (Avasthi and Purkayastha, 2013). In another study, (Patil et al.,

2010) reported that bark and stem extracts of Boswellia serrata inhibited the bacterial growth of

Bacillus subtilis, Klebsiella pneumoniae and Escherichia coli. (Padhi and Mahapatra) reported

that different extracts of Boswellia serrata leaves inhibited the bacterial growth of Gram positive

and Gram negative bacteria. Moreover, the antibacterial activity of these extracts might be due to

presence of resin acids, phenolics and terpenes.

Overall, essential oils obtained by different extraction methods, fractions and sub-

fractions showed higher antibacterial activity with larger inhibition zones against Gram negative

bacterial strains than Gram positive bacterial strains. In comparison of antibacterial activity of

essential oils obtained through different extractions methods, steam distilled essential oils

showed highest antibacterial activity against all tested bacterial strains. In hydro-distilled

essential oils, fractions and sub-fractions F4 fraction showed highest activity against all bacterial

bacteria strains. In steam distilled essential oils, fractions and sub-fractions, F1 c sub-fraction

exhibited highest antibacterial activity against all tested bacterial strains.

Figure (a)

97

Figure (b)

Figure (c)

Figure 4.21 (a), (b) and (c): Represents the antibacterial activity of Pinus roxburghii and

Boswellia serrata oleoresin essential oils, fractions and sub-fractions.

98

4.4.3. Minimum inhibitory concentration of Pinus roxburghii oleoresin by Resazurin

microtiter-plate assay

The minimum inhibitory concentration (MIC) of Pinus roxburghii oleoresin essential oils

isolated by different extraction methods, most active fractions and sub-fractions against two

Gram positive and two Gram negative bacterial strains was determined by Resazurin microtiter

plate assay and their results are given in Figure 4.22 to 4.25. The MIC values for essential oils

isolated by different extraction methods, most active fractions and sub-fractions were 2.62 –

337.80 µg/mL, while the data acquired for positive control (Amoxicillin) was 2.92-13.33 µg/mL.

It was found that most of essential oils, fractions and sub fractions showed excellent antibacterial

activity against all bacterial strains. Moreover, it was observed that sub fractions showed higher

antibacterial activity with smaller MIC values than its essential oil and fractions. The MIC values

showed that P. multocida and S. aureus were the most sensitive bacterial strains presenting

smaller MIC values 12.31 - 281.47 µg/mL and 2.68 – 168.88 µg/mL, respectively, while E. coli

and B. subtilis were the least sensitive bacterial strains presenting small inhibition zones 12.31 –

337.77 µg/mL and 24.62 – 281.47 µg/mL, respectively. In essential oils isolated through hydro

distillation method at different temperature conditions, the essential oil of 160 ⁰C showed highest

antibacterial activity with smaller MIC values in range of 8.79 - 98.51 µg/mL against all the

bacterial strains. Therefore, the fractions and sub fractions of this oil was evaluated for

antibacterial activity to find most active fraction and sub fractions. In 160 ⁰C essential oil

fractions, F1 fractions showed highest activity against E. coli and S. aureus with MIC values

70.36 and 28.15 µg/mL, respectively. While F3 fraction exhibited highest activity against P.

multocida and B. subtilis with MIC values 56.29 – 70.36 µg/mL, respectively. In sub fractions,

F2 c sub fraction showed highest activity against E. coli and B. subtilis with MIC values 56.29

and 42.22 µg/mL respectively, while F2 b and F3 a sub fractions showed highest antibacterial

activity against P. multocida and S. aureus with MIC values 35.18 and 2.68 µg/mL, respectively.

Similar trend was observed in essential oils isolated at different temperature conditions

by steam distillation method, where essential oil isolated at 160 ⁰C showed highest antibacterial

activity against all bacterial strains with MIC values in range of 42.22 – 112.60 µg/mL. It was

observed that essential oils showed highest activity against S. aureus with MIC values in range

of 42.22 – 168.88 µg/mL, while least activity was found against E. coli with MIC values in range

99

Figure 4.22: Minimum inhibitory concentration of Pinus roxburghii oleoresin against E. coli

112.58 126.61

98.51

140.73

70.36 76.36 84.44

168.88

140.73

112.58

84.44 68.36

56.29

112.58

70.36

168.88

84.44

140.73

84.44

337.77

112.58

168.88

112.58

140.73 144.8

281.47

70.36

12.31

140.73

225.18

119.61 112.58

84.44 98.51

84.44

112.58

5.83

0

50

100

150

200

250

300

350

12

0 ⁰

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14

0 ⁰

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16

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C

18

0 ⁰

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F1 F2 F3 F4

F1 a

F1 b

F2 a

F2 b

F2 c

F2 d

F3 a

F3 b

F3 c

F3 d

F4 a

F4 b

12

0 ⁰

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14

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C

16

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C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F1 c

F1 d

F2 a

F2 b

F2 c

40

⁰C

Am

oxi

cilli

n

HD SD SCF

MIC

(µ

g/m

l)

Pinus roxburghii oleoresin essntial oils, fractions and sub-fractions against E. coli

100

Figure 4.23: Minimum inhibitory concentration of Pinus roxburghii oleoresin against S. aureus

24.62

35.18

8.79

56.29

28.14

49.25

35.18

112.58

49.25

35.18

12.31 3.51 4.39

28.14

3.51 5.27 5.27

56.29

4.39

112.58

56.29

168.88

42.22

56.29

5.27

35.18

5.27 3.51 5.27

28.14

12.31 17.59

12.31

35.18

4.39

17.59

2.916

0

20

40

60

80

100

120

140

160

180

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

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F1 F2 F3 F4

F1 a

F1 b

F2 a

F2 b

F2 c

F2 d

F3 a

F3 b

F3 c

F3 d

F4 a

F4 b

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

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F1 F2 F3 F4

F1 a

F1 b

F1 c

F1 d

F2 a

F2 b

F2 c

40

⁰C

Am

oxi

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n

HD SD SCF

Min

imu

m I

nh

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ory

Co

nce

ntr

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on

(µ

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l)

Pinus roxburghii oleoresin essential oils, fractions and sub-fractions against S. aureus

101

Figure 4.24: Minimum inhibitory concentration of Pinus roxburghii oleoresin against P. multocida

24.62

49.25

24.62 35.18

84.44 70.36

56.29

140.73

70.36 56.29

49.25 35.18

42.22

70.36

42.22 56.29

70.36

140.73

49.25

281.47

140.73

281.47

112.58

168.88

112.58

140.74

49.25

12.31

112.58

130.64

70.36 84.44

49.25

84.44 70.36

281.47

8.33

0

50

100

150

200

250

300

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F2 a

F2 b

F2 c

F2 d

F3 a

F3 b

F3 c

F3 d

F4 a

F4 b

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F1 c

F1 d

F2 a

F2 b

F2 c

40

⁰C

Am

oxi

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n

HD SD SCF

Min

imu

m I

nh

ibit

ory

Co

nce

ntr

ati

on

(µ

g/m

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Pinus roxburghii oleoresin essential oils , fractions and sub-fractions against P. multocida

102

Figure 4.25: Minimum inhibitory concentration of Pinus roxburghii oleoresin against B. subtilis

70.36

98.51

49.25

70.3 70.36 84.44

70.36

140.73

112.5866667

84.44

56.29 46.22 42.22

70.36

49.25

70.36 84.44

140.73

84.44

225.17

140.73

281.47

56.29 70.36

49.25

112.58

42.22

24.62

49.25 56.29

40.25 49.25

35.18 42.22

49.25

168.88

13.33

0

50

100

150

200

250

300

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F2 a

F2 b

F2 c

F2 d

F3 a

F3 b

F3 c

F3 d

F4 a

F4 b

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F1 c

F1 d

F2 a

F2 b

F2 c

40

⁰C

Am

oxi

cilli

n

HD SD SCF

Min

imu

m I

nh

ibit

ory

Co

nce

ntr

ati

on

(µ

g/m

l)

Pinus roxburghii oleoresin essential oils, fractions and sub-fractions against B. Subtilis

103

of 112.58 – 168.88 µg/mL. In fractions of 160 ⁰C essential oil, F4 fraction showed highest

antibacterial activity with smaller MIC values against all bacterial strains in range of 3.51 –

24.62 µg/mL. In sub-fractions, F2 c sub-fraction showed highest activity against E. coli and S.

aureus with MIC values 84.44 and 4.39 µg/mL, respectively, while F2 a sub-fraction exhibited

highest activity with MIC values 49.25 and 35.18 µg/mL against P. multocida and B. subtilis

respectively. In comparison of hydro, steam and supercritical fluid extraction methods, essential

oils obtained through hydro-distillation method showed highest antibacterial activity with

smaller MIC values in range of 8.79 - 140.73 µg/mL against all the bacterial strains. While,

essential oil isolated through supercritical fluid extraction method showed lowest antibacterial

activity with higher MIC values in range of 112.58 – 281.47 µg/mL. Such variations in

antibacterial activity of essential oils may be due to difference in chemical composition of

essential oils. Previous literature studies confirmed that the chemical compositions of essential

oils vary with extraction methods and such variations are directly affected the antibacterial

activity (Kokoska et al., 2008; Okoh et al., 2010). It was observed that all essential oils isolated

through different extraction methods showed excellent antibacterial activity against all bacteria.

The MIC values for essential oils showed that S. aureus and P. multocida were the most sensitive

bacterial strains presenting smaller MIC values 8.79 – 168.8 and 24.62 – 281.47 µg/mL,

respectively, while E. coli and B. subtilis showed least sensitivity with larger MIC values 98.51 –

168.88 and 49.25 – 281.47 µg/mL, respectively. These experimental results coincides with (Shah

et al., 2014) and (Salem et al., 2014) how reported that essential oils isolated from different parts

of Pinus roxburghii showed good antibacterial activity against Gram positive and Gram negative

bacteria. In another study, it was reported that alcohol and water extracts of bark, leaf, stem,

male and female cone of Pinus roxburghii inhibited the bacterial growth of Salmonella arizonae,

S. typhi and Staphylococcus aureus, Agrobacterium tumefaciens except in Escherichia coli

(Parihar et al., 2006). Overall, essential oils obtained through different extraction methods,

fractions and sub-fractions showed higher antibacterial activity with smaller MIC values against

Gram positive bacterial strains than Gram negative bacterial strains. In comparison of

antibacterial activity of essential oils isolated through different extractions methods, hydro

distilled essential oil showed highest antibacterial activity against all tested bacterial strains. In

hydro-distilled essential oils, fractions and sub-fractions F2 c sub-fraction showed highest

activity against E. coli and B. subtilis, while F2 b and F3 a sub-fractions showed maximum

104

antibacterial activity against P. multocida and S. aureus. In steam distilled essential oils,

fractions and sub-fractions F4 fraction showed highest antibacterial activity against all tested

bacterial strains.

4.4.4. Minimum inhibitory concentration of Boswellia serrata oleoresin by Resazurin

microtiter-plate assay

The minimum inhibitory concentration (MIC) of Boswellia serrata oleoresin essential oils

isolated through different extraction methods, most active fractions and sub-fractions against two

Gram positive and two Gram negative bacterial strains was determined by Resazurin microtiter

plate assay and their results are given in Figure 4.26 to 4.29. The MIC values for essential oils

isolated by different extraction methods, most active fractions and sub-fractions against E. coli,

S. aureus, P. multocida and B. subtilis were in range of 4.39 – 337.77, 70.36 – 337.77, 2.20 –

281.47 and 56.29 – 281.47 µg/mL, respectively, while the data acquired for positive control

(Amoxicillin) was 2.92 – 13.33 µg/mL. It was found that most of essential oils, fractions and

sub-fractions showed excellent antibacterial activity against all tested bacteria. Moreover, it was

observed that most of sub-fractions showed higher antibacterial activity than essential oils and

fractions. The MIC values showed that P. multocida and E. coli were the most sensitive bacterial

strains presenting smaller MIC values in range of 2.20 – 281.47 µg/mL and 4.39 – 337.77 µg/mL

respectively, while S. aureus and B. subtilis were the least sensitive bacterial strains presenting

small inhibition zones 70.36 – 337.77 µg/mL and 56.29 – 281.47 µg/mL, respectively. Overall,

MIC values for essential oils isolated by different extraction methods, most active fractions and

sub-fractions were in range of 2.20 – 337.77 µg/mL.

In essential oils isolated by hydro-distillation method at different temperature conditions,

the essential oil of 140 ⁰C showed highest antibacterial activity with smaller MIC values in range

of 35.18 – 112.58 µg/mL against all the bacterial strains. Therefore, the fractions and sub-

fractions of this oil were evaluated for antibacterial activity to find most active fraction and sub-

fraction. In fractions of 140 ⁰C essential oil, F4 fractions revealed highest activity against E. coli,

S. aureus, P. multocida and B. subtilis with inhibition zones 31.66, 70.36, 4.39 and 70.36 µg/mL,

respectively. In sub-fractions, F3 c sub-fraction showed highest activity against E. coli, S.

aureus, P. multocida and B. subtilis with MIC values 49.25, 98.51, 12.31 and 105.52 µg/mL,

105

respectively. Overall, F4 fraction showed highest antibacterial activity among essential oils

isolated through hydro-distillation method, its fractions and sub-fractions.

Similar trend was observed in essential oils obtained at different temperature conditions

by steam distillation method, where essential oil obtained at 140 ⁰C showed maximum activity

against all bacterial strains with MIC values in range of 2.20 – 337.77 µg/mL. The isolated

essential oils showed highest activity against P. multocida with inhibition zones 2.20 – 281.47

µg/mL, while least activity was observed against S. aureus with MIC values 84.44 – 337.77

µg/mL. In fractions of 140 ⁰C essential oil, F3 fraction revealed maximum activity against E.

coli, S. aureus, P. multocida and B. subtilis with MIC values 33.42, 112.59, 3.52 and 70.36 µg/

mL, respectively. In sub-fractions, F1 c sub-fraction showed highest activity against E. coli, S.

aureus, P. multocida and B. subtilis with inhibition zones 4.39, 84.44, 2.20 and 56.29 µg/ mL,

respectively. Overall, in steam distilled essential oils, its fractions and sub-fractions F1 c

exhibited highest activity against all bacterial strains. In comparison of hydro, steam and

supercritical fluid extraction methods, essential oils obtained by steam distillation method

showed highest antimicrobial activity with larger inhibition zones in range of 63.33 – 168.88

µg/mL against all the bacterial strains. While, essential oil obtained through supercritical fluid

extraction method showed lowest activity with larger MIC values 168.88 – 337.77 µg/mL. Such

variations in antibacterial activity of essential oils may be due to the difference in chemical

composition of essential oils. Previous literature reports confirmed that chemical compositions of

essential oils vary with extraction methods and such variation in chemical composition of

essential oil directly affected their antibacterial activity (Kokoska et al., 2008; Okoh et al.,

2010). It was observed that all essential oils obtained through different extraction methods

showed good antibacterial activity against all bacteria except supercritical fluid extracted

essential oil that show weak antibacterial activity. The MIC values for essential oils obtained by

different extraction methods showed that P. multocida and E. coli were the most sensitive

bacterial strains presenting smaller MIC values 35.18 - 281.47 µg/mL and 70.36 – 337.77

µg/mL, respectively. The GC-MS analysis of pure essential oils showed that α-pinene, β-pinene,

verbenol and pinocarveol were the major components of Boswellia serrata oleoresin essential oils

and these compounds might be responsible for their antibacterial activity. Previously, it was

reported that antibacterial activity of Boswellia serrata oleoresin essential oil is not attributed

106

Figure 4.26: Minimum inhibitory concentration of Boswellia serrata oleoresin against E.coli

98.51

70.36

98.51

140.73

84.44

31.66

337.77

140.73

70.36

0 0

49.25

98.51 84.44

197.03

281.47

33.42

0 0 4.39

28.14 24.62

49.25

337.77

5.83

0

50

100

150

200

250

300

350

4001

20

⁰C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F2 a

F2 b

F2 c

F3 a

F3 b

F3 c

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3

F1 a

F1 b

F1 c

F2 a

F2 b

F2c

40

⁰C

Am

oxi

cilli

n

H.D S.D SCF

Min

imu

m I

nh

ibit

ory

Co

nce

ntr

ati

on

(µ

g/m

l)

Boswellia serrata oleoresin essential oils, fractions and sub-fractions against E.coli

107

Figure 4.27: Minimum inhibitory concentration of Boswellia serrata oleoresin against S. aureus

225.17

112.58

190.64 168.88

140.73

70.36

225.18

337.77

197.03

0 0

98.51

112.58

105.14

217.82

225.17

112.59

0 0

84.44

337.77

98.52

225.17

168.88

2.92 0

50

100

150

200

250

300

3501

20

⁰C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F2 a

F2 b

F2 c

F3 a

F3 b

F3 c

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3

F1 a

F1 b

F1 c

F2 a

F2 b

F2c

40

⁰C

Am

oxi

cilli

n

H.D S.D SCF

Min

imu

m I

nh

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ory

Co

nce

ntr

ati

on

(µ

g/m

l)

Boswellia serrata oleoresin essential oils, fractions and sub-fractions against S. aureus

108

Figure 4.28: Minimum inhibitory concentration of Boswellia serrata oleoresin against P. multocida

70.36

35.18 49.25

56.29 45.73

4.39

98.51

49.25

24.62

0 0 12.31

70.36 63.33

84.44 77.23

3.52 0 0 2.2

49.25 35.18

70.5

281.47

8.33

0

50

100

150

200

250

3001

20

⁰C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F2 a

F2 b

F2 c

F3 a

F3 b

F3 c

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3

F1 a

F1 b

F1 c

F2 a

F2 b

F2c

40

⁰C

Am

oxi

cilli

n

H.D S.D SCF

Min

imu

m I

nh

ibit

ory

Co

nce

ntr

ati

on

(µ

g/m

l)

Boswellia serrata oleoresin essential oils, fractions and sub-fractions against P. multocida

109

Figure 4.29: Minimum inhibitory concentration of Boswellia serrata oleoresin against B. subtilis

112.58 98.52

140.73

168.88

90.51

70.36

281.47

140.73

112.58

0 0

105.52

112.58

168.88

281.47

225.17

70.36

0 0

56.29

112.58

84.44

140.73

281.47

13.33

0

50

100

150

200

250

3001

20

⁰C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F2 a

F2 b

F2 c

F3 a

F3 b

F3 c

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3

F1 a

F1 b

F1 c

F2 a

F2 b

F2c

40

⁰C

Am

oxi

cilli

n

H.D S.D SCF

Min

imu

m I

nh

ibit

ory

Co

nce

ntr

ati

on

(µ

g/m

l)

Boswellia serrata oleoresin essential oils, fractions and sub-fractions against B. subtilis

110

Figure (a)

Figure (b)

Figure 4.30 (a) and (b): Represents the minimum inhibitory concentration of Boswellia

serrata and Pinus roxburghii oleoresin essential oils, fractions and sub-fractions.

111

due to single compound, but to the synergistic effect of several compounds (Camarda et al.,

2007).

Overall, it was concluded that all essential oils isolated by different extraction methods,

fractions and sub fractions showed higher antibacterial activity with smaller MIC values against

Gram negative bacterial strains than Gram positive bacterial strains. In comparison of

antibacterial activity of essential oils isolated by different extractions methods, steam distilled

essential oils showed highest antibacterial activity against all tested bacterial strains. In hydro

distilled essential oils, fractions and sub fractions F4 fraction showed highest activity against all

bacterial bacteria strains. In steam distilled essential oils, fractions and sub fractions F1 c sub

fraction exhibited highest antibacterial activity against all tested bacterial strains.

4.4.5. Antifungal activity of Pinus roxburghii oleoresin by Well diffusion assay

Antifungal activity of Pinus roxburghii oleoresin essential oils isolated by different

extraction methods, most active fractions and sub-fractions against F. solani, A. niger, A.

alternate and A. flavus was determined by well diffusion assay and their results are given in

Figure 4.31 to 4.34. The values of inhibition zones for essential oils obtained by different

extraction methods, most active fractions and sub-fractions against all fungal strains were 7.00 –

28.20 mm, while the data acquired for positive control (Terbinafine) was 22.61 - 28.82 mm. It

was observed that most of essential oils, fractions and sub-fractions showed excellent antifungal

activity against all test fungal strains. The inhibition zone values of essential oils isolated through

different extraction methods, fractions and sub-fractions against F. solani, A. niger, A. alternate

and A. flavus were 9.00 – 26.63 mm, 7.00 – 21.16 mm, 10.00 - 28.20 mm and 8.06 – 22.31 mm,

respectively, while the data acquired for positive control was 24.36, 22.61, 24.82 and 23.32 mm,

respectively. These results indicated that most of essential oils, fractions and sub-fractions

revealed excellent antifungal activity against all test fungal strains. Moreover, it was observed

that sub-fractions showed higher antifungal activity than its essential oil and fractions except in

essential oils isolated by steam distillation method and its fractions and sub-fractions where

essential isolated at 140 ⁰C revealed highest antifungal activity with larger inhibition zones.


the essential oil of 140 ⁰C showed highest antifungal activity with larger inhibition zones 20.75,

112

14.27, 21.12 and 15.15 mm against F. solani, A. niger, A. alternate and A. flavus, respectively.

Therefore, the fractions and sub-fractions of this 140 ⁰C oil was further evaluated for antifungal

activity to find most active fraction and sub-fraction. In fractions of 140 ⁰C essential oil, F1

fractions showed highest activity against F. solani, A. niger, A. alternate and A. flavus with

inhibition zones 18.46, 14.10, 19.56 and 17.22 mm, respectively, while F3 fraction exhibited

lowest activity against all test strains. In sub-fractions, F1 c sub-fraction showed highest activity

with larger inhibition zones 26.63, 21.16, 28.20 and 22.31 mm against F. solani, A. niger, A.

alternate and A. flavus, respectively. It was observed that most of sub-fractions showed higher

antifungal activity with larger inhibition zones than pure essential oils and fractions.

A much similar trend was observed in essential oils obtained at different temperature

conditions by steam distillation method, where essential oil of 140 ⁰C showed higher antifungal

activity with inhibition zones 24.03, 17.84, 22.00 and 19.80 mm against F. solani, A. niger, A.

alternate and A. flavus, respectively. In fractions of 140 ⁰C oil, F3 fraction showed highest

antifungal activity with larger inhibition zones 20.96, 14.45, 22.15 and 15.64 mm against F.

solani, A. niger, A. alternate and A. flavus, respectively, while F4 fraction showed no activity

against all test fungal strains. In sub-fractions, F3 c sub fraction showed highest activity with

inhibition zones 22.72, 15.00, 23. 48 and 16.00 mm against F. solani, A. niger, A. alternate and

A. flavus respectively, while F3 c sub fraction exhibited no activity against all test strains.

Opposite trend was observed in essential oil isolated by steam distillation method, its fractions

and sub-fractions, where pure oil at 140 ⁰C showed maximum antifungal activity than its

fractions and sub-fractions. Such variation in inhibition zones of essential oil, fraction and sub

fractions indicated the synergistic behavior of components present in essential oil.

In comparison with hydro, steam and supercritical fluid extraction methods, essential oils

isolated through steam distillation method showed highest antifungal activity with larger

inhibition zones in range of 17.84 – 24.03 mm against all test fungal strains. While, essential oil

isolated through supercritical fluid extraction method showed lowest antifungal activity with

smaller inhibition zones in range of 10.58 – 15.02 mm. The variations in antifungal activity of

essential oils isolated through different extraction methods may be due to the difference in

chemical composition of essential oils. It has been reported that chemical compositions of

essential oils vary with extraction methods and such variations directly affects the antifungal

113

Figure 4.31: Antifungal activity of Pinus roxburghii oleoresin for F. solani

13.1

20.75

10.02

15.13

18.46 17.34

15.96 16.32

17.74

12.85

26.63

13.44

0

19.46 20.46

0

16.22 15.96

22.76

9.82 10.5

24.03

12.13

9

17.32 16.82

20.96

0

20.6

15.21 15.06

17.12 16.42

20.46

22.73

0

12.42

24.36

0

5

10

15

20

25

30

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F1 c

F2 a

F2 b

F2 c

F2 d

F3 a

F3b

F3 c

F4 a

F4 b

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F 4

F1 a

F1 b

F1 c

F2 a

F2 b

F3 a

F3 b

F3 c

40

⁰C

Terb

inaf

ine

HD SD SCF

Zo

ne

of

inh

ibit

ion

(m

m)

Pinus roxburghii oleoresin essential oils, fractions and sub-fractions for F. solani

114

Figure 4.32: Antifungal activity of Pinus roxburghii oleoresin for A. niger

10.29

14.27

7.63

11.33

14.1

10.32

8.82

10.12

13.7

11.17

21.16

10.12

0

11.62

10.02

0

9.74 8.82

15.05

8.5 9

17.84

10.02

7

12.42

14.18 14.45

0

14

12 11.15

12.3

13.5 13

15

0

10.58

22.61

0

5

10

15

20

251

20

⁰C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F1 c

F2 a

F2 b

F2 c

F2 d

F3 a

F3b

F3 c

F4 a

F4 b

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F 4

F1 a

F1 b

F1 c

F2 a

F2 b

F3 a

F3 b

F3 c

40

⁰C

Terb

inaf

ine

HD SD SCF

Zo

ne

of

inh

ibit

ion

(m

m)

Pinus roxburghii oleoresin essential oils, fractions and sub-fractions against A. niger

115

Figure 4.33: Antifungal activity of Pinus roxburghii oleoresin against A. alternata

13

21.12

10.75

17.83

19.56

12.56

17.05

11.25

18.34

16.22

28.2

14.54

0

18

20.32

0

11.04

17.05

23.04

10 11

22

14.5

10

22.15

18.22

21.68

0

21.22

17.06

16.89

20.21

17.22

22.4 23.48

0

15.02

24.82

0

5

10

15

20

25

301

20

⁰C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F1 c

F2 a

F2 b

F2 c

F2 d

F3 a

F3b

F3 c

F4 a

F4 b

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F 4

F1 a

F1 b

F1 c

F2 a

F2 b

F3 a

F3 b

F3 c

40

⁰C

Terb

inaf

ine

HD SD SCF

Zo

ne

of

inh

ibit

ion

(m

m)

Pinus roxburghii oleoresin essential oils, fractions and sub-fractions against A. alternata

116

Figure 4.34: Antifungal activity of Pinus roxburghii oleoresin against A. flavus

9.73

15.15

9

13

17.22

11.23

9.11

10.92

16.5

11.5

22.31

11.23

0

12.44 11.39

0

10.5

9.11

15.5

8.9 8.06

19.8

11

8.97

13.45 12.66

15.64

0

15.1

13.5

12.01 12.5

12

14

16

0

11.46

23.32

0

5

10

15

20

251

20

⁰C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F1 c

F2 a

F2 b

F2 c

F2 d

F3 a

F3b

F3 c

F4 a

F4 b

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F 4

F1 a

F1 b

F1 c

F2 a

F2 b

F3 a

F3 b

F3 c

40

⁰C

Terb

inaf

ine

HD SD SCF

Zo

ne

of

inh

ibit

ion

(m

m)

Pinus roxburghii oleoresin essential oils, fractions and sub-fractions against A. flavus

117

activity (Wenqiang et al., 2006; Danh et al., 2013). It was observed that essential oils isolated

through different extraction methods showed good antifungal activity against all test fungi. The

values of inhibition zones for essential oils indicated that F. solani and A. alternate were the

most sensitive fungal strains presenting larger inhibition zones in range of 9.00 – 24.03 mm and

10.00 – 22.00 mm respectively, while A. niger and A. flavus were the least sensitivity fungal

strains with smaller inhibition zones 7.00 - 17.84 and 10.00 – 22.00 mm, respectively. These

experimental results are in good agreement with (Zafar et al., 2010) how described that Pinus

roxburghii essential oil showed good antifungal activity against Aspergillus flavus. Similarly in

another study, it was reported that essential oil and chloroform extract of Pinus roxburghii

revealed good antifungal activity against Candida albicans, Aspergillus niger, and Aspergillus

clavatus (Mahajan and Sharma, 2011). Moreover, it was reported that Pinus roxburghii essential

oil showed antifungal activity in concentration dependent manner (Hassan and Amjid, 2009). In

another study (Motiejūnaitė and Dalia Pečiulytė, 2004) noticed that steam distilled essential oils

inhibited the fungal growth of Aspergillus flavus and Aspergillus terrus.

Overall, it was concluded that essential oils obtained by different extraction methods,

most active essential oils fractions and sub fractions showed good antifungal activity with

different inhibition zones except sub fraction F2 b, F3 a of hydro distilled essential oil of 140 ⁰C

and fraction F4, sub fraction F3 c of steam distilled essential oil 140 ⁰C that showed no activity

against all test fungal strains. In comparison of essential oils isolated by different extractions

methods, steam distilled essential oil showed highest antifungal activity against all test fungal

strains. In hydro distilled essential oils, fractions and sub-fractions, F1 c sub-fraction showed

highest activity against all test fungal strains. In steam distilled essential oils, fractions and sub-

fractions, 140 ⁰C essential oil exhibited highest antifungal activity against all test fungal strains.

4.4.6. Antifungal activity of Boswellia serrata oleoresin by Well diffusion assay

Antifungal activity of Boswellia serrata oleoresin essential oils isolated by different

extraction methods, most active fractions and sub-fractions against F. solani, A. niger, A.

alternate and A. flavus was determined by well diffusion assay and their results are given in

Figure 4.35 to 4.38. The values of inhibition zones for essential oils isolated by different

extraction methods, most active fractions and sub-fractions against all fungal strains were 8.80 –

28.80 mm, while the data acquired for positive control (Terbinafine) was 22.61 - 28.82 mm. It

118

was found that all the pure essential oils showed significant antifungal activity against all test

fungal strains. In fractions and sub fractions, F3 fraction of 120 ⁰C hydro distilled essential oil

showed antifungal activity, while all the other fractions and sub fractions showed no activity

against all test fungal strains. The values of inhibition zones of essential oils isolated by different

extraction methods, fractions and sub fractions against F. solani, A. niger, A. alternate and A.

flavus were 10.12 - 28.80 mm, 8.80 – 17.80 mm, 16.15 - 22.34 mm and 11.62 – 17.95 mm

respectively, while the data acquired for positive control was 24.36, 22.61, 24.82 and 23.32 mm

respectively.

In essential oils isolated by hydro distillation method at different temperature conditions,

the essential oil of 120 ⁰C showed highest antifungal activity with larger inhibition zones 16.06,

15.81, 21.64 and 16.78 mm against F. solani, A. niger, A. alternate and A. flavus, respectively.

Therefore, the fractions and sub fractions of 120 ⁰C essential oil was further evaluated for

antifungal activity to find most active fraction and sub fraction. In fractions of 120 ⁰C essential

oil, only F3 fractions showed antifungal activity against F. solani, A. niger, A. alternate and A.

flavus with inhibition zones 14.80, 12.34, 18.42 and 16.00 mm, respectively, while in sub

fractions no activity was observed.

In essential oils isolated at different temperature conditions by steam distillation method,

essential oil isolated at 160 ⁰C showed higher antifungal activity with inhibition zones 28.80,

17.80, 22.34 and 15.64 mm against F. solani, A. niger, A. alternate and A. flavus, respectively. It

was found that all fractions and sub fractions showed no activity against all test fungal strains.

These results of steam distilled essential oil of 160 ⁰C, its fractions and sub fractions indicated

the synergistic effect or combine effect of chemical components present in essential oil. In

comparison of antifungal activity of essential oils obtained by hydro, steam and supercritical

fluid extraction methods, the essential oils isolated through steam distillation method showed

highest antifungal activity with larger inhibition zones in range of 12.08 – 28.08 mm against all

test fungal strains. While, essential oil isolated through supercritical fluid extraction method

showed lowest antifungal activity with smaller inhibition zones in range of 8.80 – 16.24 mm.

The variation in antifungal activity of essential oils isolated through different extraction methods

might be due to difference in chemical profiles. Literature studies showed that chemical profile

of essential oils varies with extraction methods and such variation directly affects the antifungal

119

Figure 4.35: Antifungal activity of Boswellia serrata oleoresin against F. solani

14.07

12.37

14.02

16.06

0 0

14.8

0 0 0 0 0 0 0

23

25.81

28.8

20

0 0 0 0 0 0 0 0 0

10.12

24.36

0

5

10

15

20

25

30

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3

F1 a

F1 b

F1 c

F1 d

F1 e

F2 a

F2 b

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3

F2 a

F2 b

F2 c

F3 a

F3 b

F3 c

40

⁰C

Terb

inaf

ine

HD SD SCF

Zo

ne

of

inh

ibit

ion

(m

m)

Boswellia serrata oleoresin essential oils, fractions and sub-fractions against F. solani

120

Figure 4.36: Antifungal activity of Boswellia serrata oleoresin against A. niger

15.81

10.7

13.65 13.87

0 0

12.34

0 0 0 0 0 0 0

12.08 12.58

17.8

12.6

0 0 0 0 0 0 0 0 0

8.8

22.61

0

5

10

15

20

251

20

⁰C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3

F1 a

F1 b

F1 c

F1 d

F1 e

F2 a

F2 b

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3

F2 a

F2 b

F2 c

F3 a

F3 b

F3 c

40

⁰C

Terb

inaf

ine

HD SD SCF

Zo

ne

of

inh

ibit

ion

(m

m)

Boswellia serrata oleoresin essential oils, fractions and sub-fractions against A. niger

121

Figure 4.37: Antifungal activity of Boswellia serrata oleoresin against A. alternata

20.12

21.64

19 20.1

0 0

18.42

0 0 0 0 0 0 0

16.73 17.57

22.34

16.15

0 0 0 0 0 0 0 0 0

16.24

24.82

0

5

10

15

20

251

20

⁰C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3

F1 a

F1 b

F1 c

F1 d

F1 e

F2 a

F2 b

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3

F2 a

F2 b

F2 c

F3 a

F3 b

F3 c

40

⁰C

Terb

inaf

ine

HD SD SCFControl

zon

e o

f in

hib

itio

n (

mm

)

Boswellia serrata oleoresin essential oils, fractions and sub-fractions agianst A. alternata

122

Figure 4.38: Antifungal activity of Boswellia serrata oleoresin against A. flavus

16.78

13.64

11.95

16.1

0 0

16

0 0 0 0 0 0 0

12.71

15.09 15.04

17.95

0 0 0 0 0 0 0 0 0

11.62

23.32

0

5

10

15

20

251

20

⁰C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3

F1 a

F1 b

F1 c

F1 d

F1 e

F2 a

F2 b

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3

F2 a

F2 b

F2 c

F3 a

F3 b

F3 c

40

⁰C

Terb

inaf

ine

HD SD SCF

Zo

ne

of

inh

ibit

ion

(m

m)

Boswellia serrata oleoresin essential oils, fractions and sub-fractions against A. flavus

123

Figure (a)

Figure (b)

124

Figure (c)

Figure 4.39 (a), (b) and (c): Represents the antifungal activity of Pinus roxburghii and

Boswellia serrata oleoresin essential oils, fractions and sub-fractions.

activity (Wenqiang et al., 2006; Danh et al., 2013). It was observed that essential oils isolated

through different extraction methods showed good antifungal activity against all test strains. The

values of inhibition zones for essential oils indicated that F. solani and A. alternate were the

most sensitive fungal strains presenting larger inhibition zones in range of 10.12 – 28.80 mm and

16.15 – 22.34 mm, respectively, while A. niger and A. flavus were the least sensitivity fungal

strains with smaller inhibition zones 8.80 - 17.80 and 11.62 – 17.95 mm respectively. The

chemical composition of pure essential oils showed that α-pinene, β-pinene, verbenol and

pinocarveol were the major components of Boswellia serrata oleoresin essential oils and these

compounds might be responsible for their antibacterial activity. Prevously, it was reported that

antimicrobial activity of Boswellia serrata oleoresin essential oil is not attributed due to single

compound, but to the synergistic effect of several compounds (Camarda et al., 2007). Similarly,

in another study, it was reported that antifungal activity of Boswellia serrata oleoresin essential

oil might be attributed to high concentration of monoterpene hydrocarbons such as α-thujene, δ-

3-carene and α-pinene (Gupta et al., 2016).

125

Overall, it was concluded that essential oils obtained by different extraction methods

showed good antifungal activity with different inhibition zones, while in all fractions and sub

fractions F3 fraction of hydro distilled oil showed antifungal activity. In comparison with

essential oils isolated by different extractions methods, steam distilled essential oil showed

highest antifungal activity against all test fungal strains.

4.4.7. Minimum inhibitory concentration of Pinus roxburghii oleoresin by micro-dilution

broth susceptibility assay

The minimum inhibitor concentration (MIC) is the concentration that completely inhibits

the growth of microorganism. The MIC of Pinus roxburghii oleoresin essential oils isolated by

different extraction methods, most active fractions and sub-fractions against F. solani, A. niger,

A. alternate and A. flavus was determined by micro dilution broth assay and their results are

given in figure 3.40 to 3.43. The MIC values of essential oils isolated by different extraction

methods, most active fractions and sub-fractions against all fungal strains were 14.06 – 675.54

µg/mL, while the data acquired for positive control (Terbinafine) was 5.00 – 8.42 µg/mL. The

MIC values of essential oils isolated by different extraction methods, fractions and sub-fractions

against F. solani, A. niger, A. alternate and A. flavus were 31.66 – 506.65 µg/mL, 84.44 – 675.54

µg/mL, 14.06 – 337.76 µg/mL and 56.29 – 562.94 µg/mL, respectively, while the data acquired

for positive control was 5.88, 8.42, 5.00 and 6.66 µg/mL, respectively. These MIC values

indicated that most of essential oils, fractions and sub-fractions showed good antifungal activity

against all test fungal strains. Moreover, it was observed that sub fractions showed higher

antifungal activity with smaller MIC values than its essential oil and fractions except in essential

oils isolated by steam distillation method and its fractions and sub-fractions where essential

isolated at 140 ⁰C revealed highest antifungal activity with smaller MIC values.

In essential oils isolated by hydro-distillation method at different temperatures, the

essential oil of 140 ⁰C showed highest antifungal activity with smaller MIC values 84.44,

197.02, 70.36 and 168.88 µg/mL against F. solani, A. niger, A. alternate and A. flavus,

respectively. Therefore, the fractions and sub-fractions of this 140 ⁰C oil were further tested for

antifungal activity to find most active fraction and sub fraction. In fractions of 140 ⁰C essential

oil, F1 fractions revealed highest activity against F. solani, A. niger, A. alternate and A. flavus

with inhibition zones 116.55, 217.06, 104.47 and 132.62 µg/mL, respectively, while F3 fraction

126

exhibited least activity against all test strains. In sub-fractions, F1 c sub-fraction showed highest

activity with MIC values 31.66, 84.44, 14.06 and 56.29 µg/ml against F. solani, A. niger, A.

alternate and A. flavus, respectively. It was observed that most of sub-fractions revealed higher

antifungal activity with smaller MIC values than pure essential oils and fractions.

Similar trend was observed in essential oils isolated at different temperature conditions

by steam distillation method, where essential oil isolated at 140 ⁰C showed higher antifungal

activity with MIC values 42.22, 112.58, 70.36 and 84.44 µg/mL against F. solani, A. niger, A.

alternate and A. flavus, respectively. In fractions of 140 ⁰C oil, F3 fraction showed highest

antifungal activity with MIC values 78.33, 210.10, 50.33, 160.77 µg/mL against F. solani, A.

niger, A. alternate and A. flavus, respectively, while F4 fraction showed no activity against all

test fungal strains. In sub-fractions, F3 b sub-fraction showed highest activity with MIC values

42.22, 168.88, 56.29 and 140.73 µg/mL against F. solani, A. niger, A. alternate and A. flavus

respectively, while F3 c sub-fraction exhibited no activity against all test strains. Opposite trend

was observed in essential oil isolated by steam distillation method, its fractions and sub-

fractions, where pure oil at 140 ⁰C showed maximum antifungal activity than its fractions and

sub-fractions. Such variation in MIC values of essential oil, fraction and sub-fractions indicated

the synergistic behavior of components present in essential oil.

In comparison with hydro, steam and supercritical fluid extraction methods, essential oils

extracted through steam distillation method showed highest antifungal activity with smaller MIC

values in range of 42.22 – 675.54 µg/mL against all test fungal strains. While, essential oil

extracted through supercritical fluid extraction method showed lowest antifungal activity with

larger MIC values in range of 160.77 – 329.66 µg/mL. The variations in antifungal activity of

essential oils extracted through different extraction methods may be due to difference in

chemical profiles of essential oils. It has been reported that chemical compositions of essential

oils vary with extraction methods and such variation in chemical profile directly affects the

antifungal activity (Wenqiang et al., 2006; Danh et al., 2013). It was observed that essential oils

extracted through different extraction methods showed good antifungal activity against all test

strains. The MIC values of essential oils indicated that F. solani and A. alternate were the most

sensitive fungal strains presenting smaller MIC values 84.44 – 506.6 µg/mL and 70.36 – 337.76

µg/mL, respectively, while A. niger and A. flavus were the least sensitivity fungal strains with

127

Figure 4.40: Minimum inhibitory concentration of Pinus roxburghii oleoresin against F. solani

253.32

84.44

337.76

168.88

116.55 126.66

168.88

126.66 126.66

168.88

31.66

142.88

0

126.66

84.44

0

126.66

168.88

63.33

337.76 337.76

42.22

253.32

506.65

126.66

168.88

78.33

0

84.44

168.88

168.88

126.66

168.88

84.44

42.22

0

245.22

5.88

0

100

200

300

400

500

600

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F1 c

F2 a

F2 b

F2 c

F2 d

F3 a

F3b

F3 c

F4 a

F4 b

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F 4

F1 a

F1 b

F1 c

F2 a

F2 b

F3 a

F3 b

F3 c

40

⁰C

Terb

inaf

ine

HD SD SCF

Min

imu

m I

nh

ibit

ory

Co

nce

ntr

ati

on

(µ

g/m

l)

Pinus roxburghii oleoresin essential oils and fractions against F. solani

128

Figure 4.41: Minimum inhibitory concentration of Pinus roxburghii oleoresin against A. niger

323.69

197.02

675.54

281.46

217.06

337.76

478.49

337.76

225.17

281.46

84.44

337.76

0

267.39

337.76

0

436.28

478.49

168.88

450.35 478.49

112.58

337.76

675.54

253.32

218.1 210.1

0

218.1

281.46

295.53

253.32 225.17

232.24

168.88

0

329.66

8.42

0

100

200

300

400

500

600

7001

20

⁰C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F1 c

F2 a

F2 b

F2 c

F2 d

F3 a

F3b

F3 c

F4 a

F4 b

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F 4

F1 a

F1 b

F1 c

F2 a

F2 b

F3 a

F3 b

F3 c

40

⁰C

Terb

inaf

ine

HD SD SCF

Min

imu

m I

nh

ibit

ory

Co

nce

ntr

ati

on

(µ

g/m

l)

Pinus roxburghii oleoresin essential oil, fractions and sub fractions against A. niger

129

Figure 4.42: Minimum inhibitory concentration of Pinus roxburghii oleoresin against A. alternata

281.46

70.36

281.46

126.68

104.47

225.17

137.68

281.46

112.58

140.73

14.06

168.88

0

112.58

84.44

0

281.46

137.68

28.14

337.76

281.46

70.36

168.88

337.76

50.33

126.55

62.22

0

70.36

137.68 140.73

84.44

137.68

70.36 56.29

0

160.77

5

0

50

100

150

200

250

300

3501

20

⁰C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F1 c

F2 a

F2 b

F2 c

F2 d

F3 a

F3b

F3 c

F4 a

F4 b

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F 4

F1 a

F1 b

F1 c

F2 a

F2 b

F3 a

F3 b

F3 c

40

⁰C

Terb

inaf

ine

HD SD SCF

Min

imu

m I

nh

ibit

ory

Co

nce

ntr

ati

on

(µ

g/m

l)

Pinus roxburghii oleoresin essential oils, fractions and sub-fractions against A. Alternata

130

Figure 4.43: Minimum inhibitory concentration of Pinus roxburghii oleoresin against A. flavus

450.35

168.88

562.94

225.17

132.62

281.46

422.8

289.57

140.73

267.39

56.29

281.46

0

225.17

281.46

0

337.76

422.8

168.88

450.35

562.94

84.44

281.46

422.8

225.17

225.17

160.77

0

168.88 197.05

281.46

225.17

337.76

189.98

140.73

273.33

6.66

0

100

200

300

400

500

6001

20

⁰C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F1 c

F2 a

F2 b

F2 c

F2 d

F3 a

F3b

F3 c

F4 a

F4 b

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F 4

F1 a

F1 b

F1 c

F2 a

F2 b

F3 a

F3 b

F3 c

40

⁰C

Terb

inaf

ine

HD SD SCF

Min

imu

m I

nh

ibit

ory

Co

nce

ntr

ati

on

(µ

g/m

l)

Pinus roxburghii oleoresin essential oils and fractions against A. flavus

131

larger MIC values 112.58 – 478.49 and 84.44 – 562.94 µg/mL, respectively. These experimental

results coincides with (Andrade et al., 2014) how reported that Pinus roxburghii oleoresin and its

fractions showed good antifungal activity with MIC values in range of 1.95 to 1000 μg/mL

against different fungal strains.

Overall, conclusion is that essential oils extracted by different extraction methods, most

active essential oils fractions and sub-fractions showed good antifungal activity with different

MIC values except sub fraction F2 b, F3 a of hydro distilled essential oil of 140 ⁰C and fraction

F4, sub-fraction F3 c of steam distilled essential oil of 140 ⁰C that showed no activity against all

test fungal strains. In comparison of essential oils isolated by different extractions methods,

steam distilled essential oil showed highest antifungal activity against all test fungal strains. In

hydro distilled essential oils, fractions and sub-fractions, F1 c sub-fraction showed highest

activity against all test fungal strains. In steam distilled essential oils, fractions and sub-fractions,

140 ⁰C essential oil exhibited highest antifungal activity against all test fungal strains.

4.4.8. Minimum inhibitory concentration of Boswellia serrata oleoresin by micro-dilution

broth susceptibility assay

The minimum inhibitory concentration of Boswellia serrata oleoresin essential oils

isolated by different extraction methods, most active fractions and sub-fractions against F.

solani, A. niger, A. alternate and A. flavus was determined by micro-dilution broth assay and

their results are given in figure 3.44 to 3.47. The MIC values of essential oils isolated by

different extraction methods, most active fractions and sub-fractions against all fungal strains

were 28.14 – 562.94 µg/mL, while the data acquired for positive control (Terbinafine) was 5.88 -

8.42 µg/mL. It was found that all the pure essential oils showed significant antifungal activity

against all test fungal strains. In fractions and sub-fractions, F3 fraction of 120 ⁰C hydro distilled

essential oil showed antifungal activity, while all the other fractions and sub fractions showed no

activity against all test fungal strains. The MIC values of essential oils isolated by different

extraction methods, fractions and sub fractions against F. solani, A. niger, A. alternate and A.

flavus were 28.14 – 337.76 µg/mL, 112.58 – 562.94 µg/mL, 70.36 – 168.88 µg/mL and 112.58 –

337.76 µg/mL, respectively, while the data acquired for positive control was 5.88, 8.42, 5.00 and

6.66 µg/mL, respectively.

132


the essential oil of 120 ⁰C showed highest antifungal activity with smaller MIC values 140.73,

140.73, 70.36 and 133.18 µg/mL against F. solani, A. niger, A. alternate and A. flavus,

respectively. Therefore, the fractions and sub-fractions of this 120 ⁰C oil were further tested for

antifungal activity to find most active fraction and sub-fraction. In fractions of 120 ⁰C essential

oil, only F3 fractions revealed antifungal activity against F. solani, A. niger, A. alternate and A.

flavus with MIC values 168.88, 281.46, 126.65 and 140.73 µg/mL, respectively, while in sub-

fractions no activity was observed. In essential oils isolated at different temperature conditions

by steam distillation method, essential oil of 160 ⁰C showed higher antifungal activity with MIC

values 28.14, 112.58, 70.36 and 112.58 µg/mL against F. solani, A. niger, A. alternate and A.

flavus, respectively. It was found that all fractions and sub-fractions showed no activity against

all test fungal strains. These results of steam distilled essential oil of 160 ⁰C, its fractions and

sub-fractions indicated the synergistic effect or combine effect of chemical components present

in essential oil. In comparison of antifungal activity of essential oils isolated by hydro, steam and

supercritical fluid extraction methods, the essential oils isolated through steam distillation

method showed highest antifungal activity with smaller MIC values in range of 28.14 – 281.46

µg/mL against all test fungal strains. While, essential oil isolated through supercritical fluid

extraction method showed lowest antifungal activity with larger MIC values in range of 140.73 –

478.49 µg/mL. The variations in antifungal activity of essential oils isolated through different

extraction methods might be due to difference in chemical composition of essential oils. Previous

literature reports confirmed that chemical compositions of essential oils vary with extraction

methods and the variation in chemical composition of essential oils directly affect their

antifungal activity (Prakash et al., 2014). It was observed that essential oils isolated through

different extraction methods showed significant antifungal activity against all test strains. The

MIC values of essential oils indicated that F. solani and A. alternate were the most sensitive

fungal strains presenting smaller MIC values in range of 28.14 – 337.76 and 70.36 – 168.88

µg/mL, respectively, while A. niger and A. flavus were the least sensitivity fungal strains with

larger MIC values 112.58 – 562.94 and 112.58 – 337.76 µg/mL, respectively. Our findings are in

good agreement with (Gupta et al., 2016) how reported that essential oils isolated from different

locations of India showed good antifungal activity. Moreover, the antifungal activity of

Boswellia serrata oleoresin essential oil might be attributed to δ-3-carene.

133

Figure 4.44: Minimum inhibitory concentration of Boswellia serrata oleoresin against F. solani

225.17

281.46

225.18

140.73

0 0

168.88

0 0 0 0 0 0 0

56.29

35.18 28.14

84.44

0 0 0 0 0 0 0 0 0

337.76

5.88

0

50

100

150

200

250

300

350

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3

F1 a

F1 b

F1 c

F1 d

F1 e

F2 a

F2 b

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3

F2 a

F2 b

F2 c

F3 a

F3 b

F3 c

40

⁰C

Terb

inaf

ine

HD SD SCF

MIC

(u

g/m

l)

Boswellia serrata oleoresin essential oils , fractions and sub-fractions against F. solani

134

Figure 4.45: Minimum inhibitory concentration of Boswellia serrata oleoresin against A. niger

140.73

562.94

225.17 225.17

0 0

281.46

0 0 0 0 0 0 0

281.46

225.17

112.58

225.17

0 0 0 0 0 0 0 0 0

478.49

8.42

0

100

200

300

400

500

600

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3

F1 a

F1 b

F1 c

F1 d

F1 e

F2 a

F2 b

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3

F2 a

F2 b

F2 c

F3 a

F3 b

F3 c

40

⁰C

Terb

inaf

ine

HD SD SCF

MIC

(u

g/m

l)

Boswellia serrata oleoresin essential oils, fractions and sub-fractions against A. niger

135

Figure 4.46: Minimum inhibitory concentration of Boswellia serrata oleoresin against A. alternata

84.44

70.36

112.58

84.44

0 0

126.65

0 0 0 0 0 0 0

140.73

112.58

70.36

168.88

0 0 0 0 0 0 0 0 0

140.73

5

0

20

40

60

80

100

120

140

160

180

200

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3

F1 a

F1 b

F1 c

F1 d

F1 e

F2 a

F2 b

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3

F2 a

F2 b

F2 c

F3 a

F3 b

F3 c

40

⁰C

Terb

inaf

ine

HD SD SCF

MIC

(u

g/m

l)

Boswellia serrata oleoresin essential oils, fractions and sub-fractions against A. alternata

136

Figure 4.47: Minimum inhibitory concentration of Boswellia serrata oleoresin against A. flavus

133.18

281.46

337.76

140.73

0 0

140.73

0 0 0 0 0 0 0

225.17

168.88 168.88

112.58

0 0 0 0 0 0 0 0 0

267.39

6.66

0

50

100

150

200

250

300

3501

20

⁰C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3

F1 a

F1 b

F1 c

F1 d

F1 e

F2 a

F2 b

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3

F2 a

F2 b

F2 c

F3 a

F3 b

F3 c

40

⁰C

Terb

inaf

ine

HD SD SCF

MIC

(u

g/m

l)

Boswellia serrata oleoresin essential oils, fractions and sub-fractions against A. flavus

137

Similarly, (Prakash et al., 2014) reported that Boswellia carterii oleoresin essential oil showed

good antifungal activity with MIC value (1.75 µL/mL) against A. flavus. In another study

(Camarda et al., 2007) reported that steam distilled essential oil of Boswellia serrata oleoresin

contained similar MIC values (12.86 µg/mL) against Candida albicans and Candida tropicalis.

Moreover, they reported that antimicrobial activity of steam distilled resin oil may attribute due

to synergistic effect of several components. The chemical composition of pure essential oils

showed that α-pinene, β-pinene, verbenol and pinocarveol were the major components of

Boswellia serrata oleoresin essential oils and these compounds may responsible for their

antifungal activity. (Shao et al., 1998) reported that limonene is the major compound present in

Boswellia serrata oleoresin essential oil and responsible for antifungal activity. In another study

(Gupta et al., 2016) reported that antifungal activity of Boswellia serrata oleoresin essential oil

might be attributed to high concentration of monoterpene hydrocarbons such as α-thujene, δ-3-

carene and α-pinene.

Overall, conclusion is that essential oils isolated by different extraction methods showed

good antifungal activity with different MIC values, while in all fractions and sub-fractions F3

fraction of hydro distilled oil showed antifungal activity. In comparison with essential oils

isolated by different extractions methods, steam distilled essential oil showed highest antifungal

activity against all test fungal strains.

4.5. Anticancer activity

Agrobacterium tumefaciens is a rod shape, Gram negative bacteria that indices tumor

(crown gall) in various plant species. The tumor produced by Agrobacterium tumefaciens is

histologically similar to human and animal tumor. The Ti-plasmid induces tumor that enhance

cell proliferation and block apoptosis in the same as in human and animal cancer cells (Agrios,

1997). Therefore, Agrobacterium tumefaciens is used in potato disc assay to induce crown gall

on potato discs. Potato disc assay is extensively utilized for primary screening of antitumor

compounds isolated from essential oils and plant extracts (Trigui et al., 2013). It has been

reported that the tumorogenic mechanism initiated by Agrobacterium tumefaciens in plants are

similar to Bartonella henselae that cause tumor in human (Srirama et al., 2008). Similarly,

(Kempf et al., 2002) observed that Bartonella henselae followed the same pathogenicity strategy

as the Agrobacterium tumefaciens followed in plants. Various studies indicated that both

138

pathogenic bacteria have significant similarity in their mechanism of action such as use of

common toxins, regulation, secretion system, invasion and adhesion mechanism (Guttman,

2004). Moreover, it have been confirmed from different studies that there is significant

correlation between inhibition of grown gall formation and in vitro and in vivo anti-leukemic

activity of different compounds and plants extracts (Galsky et al., 1980; Ferrigni et al., 1982;

Coker et al., 2003; Islam et al., 2013). Furthermore, various studies have confirmed that

anticancer compounds such as Vinblastine, Podophyllin, Vincristine, Camptothecin and Taxol

have revealed good antitumor activity against 3PS leukemic mouse assay. Similarly, these

compounds have shown good inhibition of tumor formation in potato disc assay (Galsky et al.,

1980; Kahl, 1982; Spjut, 1985; Gordaliza et al., 1994; Islam et al., 2011).

4.5.1. Crown gall antitumor activity of Pinus roxburghii oleoresin

The antitumor activity of Pinus roxburghii oleoresin essential oils isolated by different

extraction methods, most active essential oils fractions and sub-fractions was determined by

crown gall antitumor assay and their results are given in Figure 3.48. The percent tumor

inhibition of essential oils isolated by different extraction methods, most active essential oils

fractions and sub-fractions were in range of 22.23 - 90.04 %, while the data acquired for positive

control (Vincristine sulphate) was 92.13 %. It was observed that all the essential oils, fractions

and sub-fractions showed antitumor activity. In essential oils extracted by hydro-distillation

method at different temperature conditions, the essential oil of 160 ⁰C showed maximum percent

tumor inhibition 85.29 %. Therefore, the fractions and sub-fractions of this oil were evaluated for

antitumor activity to find most active fractions and sub-fractions. In fractions of 160 ⁰C essential

oil, F3 fraction showed highest antitumor activity with percent tumor inhibition 55.56 %. In sub-

fractions, F2 c exhibited maximum antitumor activity with percent tumor inhibition 90.04 %. It

was observed that sub-fractions showed higher antitumor activity than pure oils and fractions.

In essential oils extracted by steam distillation method at different temperature

conditions, the essential oil of 160 ⁰C showed maximum percent tumor inhibition 88.24 %. In

fractions of 160 ⁰C, F4 fractions showed maximum antitumor activity with 81.82 percent tumor

inhibition. In sub-fractions, F2 a exhibited highest antitumor activity with 72.78 percent tumor

inhibition. It was observed that pure essential oils revealed highest antitumor activity followed

by fractions and sub-fractions. In comparison with hydro, steam and supercritical fluid extraction

139

Figure 4.48: Crown gall antitumor activity of Pinus roxburghii oleoresin.

58.83

47.05

85.29

64.71

55.3

48.42

55.56

44.45

83.33

22.32

77.78

66.85

90.04

38.9

72.94 72.22

61.12

22.23

55.36

61.01

73.53 76.47

88.24

80.23

70.32

61.11

45.46

71.82 68.19

70.72

36.36 40.48

72.78

45.46

36.3

75.56

92.13

0

10

20

30

40

50

60

70

80

90

1001

20

⁰C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F2 a

F2 b

F2 c

F2 d

F3 a

F3 b

F3 c

F3 d

F4 a

F4 b

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F1 c

F1 d

F2 a

F2 b

F2 c

40

⁰C

Vin

cris

tin

e s

ulp

hat

e

HD SD SCF

Per

cen

t tu

mo

r in

hib

itio

n

Pinus roxburghii oleoresin essential oils, fractions and sub fractions against A. tumefaciens

140

methods, steam distilled essential oils showed higher antitumor activity with percent tumor

inhibition 73.53- 88.24 % followed by hydro-distillation 47.05 – 85.29 % and supercritical fluid

extraction methods 75.56 %. It was observed that all the essential oils isolated through different

extraction methods showed good antitumor activity. No early literature reports are available on

antitumor activity of Pinus roxburghii oleoresin essential oil determined through crown gall

assay to compare our results. But few literatures reports are available on anticancer activity of

Pinus roxburghii essential oils and extracts determined through MTT assay. (Shah et al., 2014)

reported that Pinus roxburghii fruits essential oil showed excellent anticancer activity through

MTT assay against different human cell lines. Moreover, they reported that complex mixture of

mono and sesquiterpenes might be responsible of their anticancer activity. In another report cone

essential oil showed remarkable anticancer activity with 100% killing of MCF-7 cells at 100

µg/ml (Satyal et al., 2013). Similarly, 15-ethyl-18-methyl pinifolate isolated from Pinus

sylvestris exhibited significant cytotoxicity against human carcinoma cell lines (Wang et al.,

2008). Dehydroabietic acid, podocarpic acid and ortho-methylpodocarpic acid isolated from

Pinus merkusii oleoresin exhibited cytotoxic activity on human epithelial and fibroblast cells

lines. Furthermore, author observed higher concentration of resin acids and longer exposure time

showed significantly higher activity (Kiliç and Koçak, 2014). Anticancer activity of five novel

diterpenoids and fourteen known diterpenoids from Pinus massoniana oleoresin against A431

and A549 cells (Yang et al., 2010). The results showed compounds with less polarity (19-

acetoxy-8(14),12E,15-labdatrien, elliotinol, 7α-hydroxy-dehydroabietic acid) exhibited

significant anticancer activity against A431 and A549 cancer cells, whereas compounds with

high polarity was not active against cancer cell lines. (Tanaka et al., 2008) reported strong in

vitro cytotoxic activity of resin acids isolates including isopimaric acid, neoabietic acid,

dehydroabietic acid and their derivatives 13a-H-D8 dihydroabietic acid, fumaropimaric acid

from Pinus massonia species against skin cancer cell lines. Among these isolates, isopimaric

acid, dehydroabietic acid and 13a-H-D8 dihydroabietic acid were further evaluated for in vivo

skin carcinogenesis. Dehydroabietic acid and 13a-H-D8 dihydroabietic acid exhibited higher

activity with less papilloma formation at 85 nmol concentration of respective isolates. In vitro

cytotoxic of dehydroabietic acid, podocarpic acid and O-methylpodocarpic acid isolated from

Pinus oleoresin were studied on human epithelial and fibroblast cells (Söderberg et al., 1996).

141

All the compounds exhibited cytotoxic activity, longer exposure time and higher concentration

of compounds showed significant increase in cell death.

4.5.2. Crown gall antitumor activity of Boswellia serrata oleoresin

The antitumor activity of Boswellia serrata oleoresin essential oils isolated by different

extraction methods, most active essential oils fractions and sub-fractions was determined by

crown gall antitumor assay and their results are given in Figure 3.49. The percent tumor

inhibition of essential oils isolated by different extraction methods, most active essential oils

fractions and sub-fractions were in range of 11.14 – 88.58 %, while the data acquired for positive

control (Vincristine sulphate) was 92.13 %. It was observed that all the essential oils, fractions

and sub-fractions showed antitumor activity. In essential oils isolated by hydro-distillation

method at different temperature conditions, the essential oil of 140 ⁰C showed maximum percent

tumor inhibition 85.29 %. Therefore, the fractions and sub-fractions of this oil were evaluated for

antitumor activity to find most active fraction and sub-fraction. In fractions of 140 ⁰C essential

oil, F3 fraction showed highest antitumor activity with percent tumor inhibition 88.58 %. In sub-

fractions, F1 c exhibited maximum antitumor activity with percent tumor inhibition 52.78 %. It

was observed that fractions showed higher antitumor activity than pure oil and sub-fractions.

In essential oils isolated by steam distillation method at different temperature conditions,

the essential oil of 120 ⁰C showed maximum percent tumor inhibition 83.14 %. In fractions of

120 ⁰C, F2 fractions showed maximum antitumor activity with 68.12 percent tumor inhibition. In

sub-fractions, F2 c exhibited highest antitumor activity with 60.12 percent tumor inhibition. It

was observed that pure essential oil showed highest antitumor activity followed by fractions and

sub-fractions. In comparison with hydro, steam and supercritical fluid extraction methods, hydro

distilled essential oils showed higher antitumor activity with percent tumor inhibition in range of

58.82 – 85.29 % followed by steam distilled essential oils 64.71 – 83.14 % and supercritical fluid

extraction methods 78.90 %. It was observed that all the essential oils extracted by different

extraction methods showed good antitumor activity. No early literature reports are available on

antitumor activity of Boswellia serrata oleoresin essential oil determined through crown gall

assay to compare our results. But few literatures reports are available on anticancer activity of

Boswellia serrata oleoresin, its essential oil and extracts determined through MTT assay.

142

Figure 4.49: Crown gall antitumor activity of Boswellia serrata oleoresin.

58.82

85.29 82.35 80.35

11.14

27.78

88.58

66.76

25

47.24

52.78

33.54

22.24

30.56

83.14 78.23

70.19

64.71 66.35 68.12

33.35 36.43

44.32

55.87 54.25

38.72

60.12

78.9

92.13

0

10

20

30

40

50

60

70

80

90

100

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F1 c

F2 a

F2 b

F2c

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3

F1 a

F1 b

F1 c

F2 a

F2 b

F2c

40

⁰C

Vin

cris

tin

e s

ulp

hat

e

H.D S.D SCF

Per

cen

t T

um

or

Inh

ibit

ion

Boswellia serrata oleoresin essential oils, fractions and sub-fractions against A. tumefaciens

143

(Burlando et al., 2008) reported that Boswellia serrata oleoresin and its pure compound 3-O-

acetyl-11-keto-β-boswellic acid (AKBA) showed moderate level of anticancer activity against

HFFF2, HaCaT and NCTC human cell lines. Similarly, (Khan et al., 2014) observed that

Boswellia serrata oleoresin exhibited good anticancer activity against Wistar rats and

hepatocellular carcinoma cell lines. (Ahmed et al., 2014) reported that Boswellia serrata oleo

gum resin essential oil, petroleum ether, methanol and its fraction revealed good anticancer

activity against HCT 116 and HepG2 cell lines.

4.6. Hemolytic activity

4.6.1. Hemolytic activity of Pinus roxburghii oleoresin

The cytotoxicity of Pinus roxburghii oleoresin essential oils extracted by different

extraction methods, fractions and sub-fractions of most active essential oils were determined by

hemolytic assay and their results are given in Figure 4.50. It was found that all the essential oils,

fractions and sub-fractions showed low hemolytic activity with percent hemolysis in range of

29.89 - 0.56 %. Moreover, it was observed that essential oils extracted by different extraction

methods showed higher hemolytic activity with percent hemolysis in range of 2.58 – 29.89 %,

followed by sub-fractions 1.07 – 13.96 and fractions 0.56 – 13.13 %. In essential oils extracted

by hydro-distillation method under different temperature conditions, essential oil of 140 ⁰C

showed maximum percent hemolysis 29.89 %. Therefore, fractions and sub-fractions of 140 ⁰C

essential oil were further tested for hemolytic activity. The hemolytic activity of 140 ⁰C essential

oil fractions were found in range of 0.56 – 2.51 %. In fractions, F4 fraction showed highest

hemolytic activity with percent hemolysis 2.51 %, while F2 fraction exhibited lowest percent

hemolysis 0.56 %. In sub-fractions, F4 a showed highest hemolytic activity with percent

hemolysis 12.01 while F2 a sub fraction revealed least hemolytic activity 1.07 %.

In essential oils extracted by steam distillation method under different temperature

conditions, essential oil of 120 ⁰C showed highest hemolytic activity with percent hemolysis

24.58 %. Therefore, fractions and sub-fractions of 120 ⁰C essential oil were tested for hemolytic

activity. The hemolytic activity of 120 ⁰C essential oil fractions was found in range of 13.13 –

1.96 %. In fractions, F1 fraction showed highest hemolytic activity with percent hemolysis 13.13

% and F2 fraction revealed lowest hemolytic activity with percent hemolysis 1.96%. In sub-

144

Figure 4.50: Hemolytic activity of Pinus roxburghii oleoresin

10.62

29.89

12.29

10.62

1.68 0.56

1.4 2.51

5.31 4.31

1.12 1.07

3.07 3.73

5.03 4.19

2.79 3.35

12.01

1.12

24.58

7.54

19.27

5.58

13.13

1.96

3.91

5.59

13.96

11.73

13.42

8.38

2.23

9.22

7.26

2.84

0

5

10

15

20

25

30

35

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F1 c

F2 a

F2 b

F2 c

F2 d

F3 a

F3 b

F3 c

F4 a

F4 b

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F1 c

F2 a

F2 b

F2 c

F2 d

40

⁰C

HD SD SCF

Per

cen

t h

emo

lysi

s

Pinus roxburghii oleoresin essnetial oils, fractions and sub fractions

145

fractions, F1 a showed highest hemolytic activity with percent hemolysis 13.96 %, while, F2 b

sub-fraction exhibited lowest hemolytic activity with percent hemolysis 2.23 %. Such variation

of hemolytic activity in essential oils, fractions and sub-fractions may be due to difference in

chemical composition of essential oils, its fractions and sub-fractions. In comparison with hydro,

steam and supercritical fluid extraction methods, hydro distilled essential oils showed higher

hemolytic activity with percent hemolysis 29.89 % followed by steam distilled essential oils 5.58

– 24.58 % and supercritical fluid extracted essential oil 2.84 ± 0.17 %.

4.6.2. Hemolytic activity of Boswellia serrata oleoresin

The cytotoxicity of Boswellia serrata oleoresin essential oils isolated by different

extraction methods, fractions and sub-fractions of most active essential oils were determined by

hemolytic assay and their results are given in Figure 4.51. It was observed that all the essential

oils, fractions and sub-fractions showed low hemolytic activity with percent hemolysis in range

of 0.40 – 15.08 %. Moreover, it was found that essential oils extracted by different extraction

methods showed higher hemolytic activity with percent hemolysis in range of 3.40 – 15.08 %

followed by sub-fractions 0.40 - 11.17 % and fractions 0.56 – 7.82 %. In essential oils isolated

by hydro-distillation method under different temperature conditions, essential oil of 180 ⁰C

revealed maximum percent hemolysis 15.08 %. Therefore, fractions and sub-fractions of 140 ⁰C

essential oil were further tested for hemolytic activity. The hemolytic activity of 180 ⁰C essential

oil fractions were found in range of 0.56 – 2.51 %. In fractions, F1 fraction showed highest

hemolytic activity with percent hemolysis 7.82 %, while F4 fraction exhibited lowest percent

hemolysis 1.68 %. In sub-fractions, F1 c showed highest hemolytic activity with percent

hemolysis 11.17 %, while F4 b sub-fraction revealed lowest hemolytic activity 0.84 %

hemolysis.

In essential oils extracted by steam distillation method under different temperature

conditions, essential oil of 160 ⁰C revealed maximum hemolysis 10.34 %. Therefore, fractions

and sub-fractions of 160 ⁰C essential oil were further tested for hemolytic activity. The

hemolytic activity of 160 ⁰C essential oil fractions was found in range of 10.89 – 0.40 %. In

fractions of 160 ⁰C essential oil, F2 fraction showed highest hemolytic activity with percent

hemolysis 6.42 % and F3 fraction revealed lowest hemolytic activity with percent hemolysis

0.56 %. In sub-fractions, F1 c showed highest hemolytic activity with percent hemolysis 10.89

146

Figure 4.51: Hemolytic activity of Boswellia serrata oleoresin

7.54

9.77

13.68

15.08

7.82

6.15

2.51

1.68

5.59

6.42

11.17

6.15 5.59

1.96

5.03

10.89

8.94

2.23

0.84

4.47

9.77

6.15

10.34

5.59

2.23

6.42

0.56

2.79

6.7

10.89

1.96

1.12 0.4

3.4

0

2

4

6

8

10

12

14

16

18

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3 F4

F1 a

F1 b

F1 c

F2 a

F2 b

F2 c

F3 a

F3 b

F3 c

F4 a

F4 b

F3 c

12

0 ⁰

C

14

0 ⁰

C

16

0 ⁰

C

18

0 ⁰

C

F1 F2 F3

F1 a

F1 b

F1 c

F2 a

F2 b

F2 c

40

⁰C

HD SD SCF

Per

cen

t h

emo

lysi

s

Boswellia serrata oleoresin essential oils , fractions and sub fractions

147

%, while F2 c exhibited lowest hemolytic activity with percent hemolysis 0.4 %. Such variation

of hemolytic activity in essential oils, fractions and sub-fractions may due to difference in

chemical composition of essential oils, its fractions and sub-fractions. In comparison with hydro,

steam and supercritical fluid extraction methods, hydro distilled essential oils showed higher

hemolytic activity with percent hemolysis 7.54 - 15.08 % followed by steam distilled essential

oils 5.59 – 10.34 % and supercritical fluid extracted essential oil 3.4 %.

4.7. High performance liquid chromatography of Boswellia serrata and Pinus roxburghii

oleoresin

High performance liquid chromatography (HPLC) is not frequently used for detection of

chemical composition of volatile components in essential oils. Mostly, it has been used for the

fractionation and determination of non-volatile compounds (e.g phenolic compounds) from

essential oils and extracts (Lockwood, 2001; Frérot and Decorzant, 2004). The phenolic

compounds in most active antitumor essential oil, fraction or sub-fraction of Boswellia serrata

and Pinus roxburghii oleoresins were determined through HPLC and their results are given

Table 4.7. The HPLC results showed that vanillic acid, p-coumaric acid and m-coumaric acid

were the phenolic acids present in F2 c sub-fraction of Pinus roxburghii oleoresin hydro distilled

essential oil of 160 oC. Vanillic acid is the major phenolic acid present in Pinus roxburghii

oleoresin essential oil (9.72 mg/L), followed by m-coumaric acid (3.75 mg/L) and p-coumaric

acid (1.28 mg/L), respectively. Similarly, the HPLC results of F3 fraction of Boswellia serrata

oleoresin hydro distilled essential oil of 140 oC showed that vanillic acid, caffeic acid,

chlorogenic acid and cinnamic acid were the phenolic acids. Chlorogenic acid is the major

phenolic acid present in Boswellia serrata oleoresin essential oil (20.79 mg/L), followed by

cinnamic acid (10.67 mg/L), caffeic acid (5.22 mg/L)and vanillic acid (5.21 mg/L), respectively.

Previously, it has been reported that most of the phenolic compounds exhibited the anticancer

activity against different cancer cell lines (Rosa et al., 2016). The antitumor activity of both

oleoresin essential oils may be due to vanillic acid, caffeic acid, p-coumaric acid, cinnamic acid

and chlorogenic acid. Literature reports have been shown that vanillic acid, p-coumaric acid and

chlorogenic acid exhibited significant antitumor activity. Vanillic acid and chlorogenic acid

isolated from methanol-water extract of Polygonum Bistorta L showed good antitumor activity

against human hepatocellular carcinoma cell line (Intisar et al., 2012). In another study, p-

148

coumaric acid isolated Cinnamon tamala leaf exhibited good anticancer activity against human

ovarian cancer cell lines (Shahwar et al., 2015). Similarly, it has been reported that p-coumaric

acid exhibited anticancer activity against human colorectal carcinoma cell lines (Jaganathan et

al., 2013). (Chang and Shen, 2014) reported that p-coumaric acid showed good inhibitory effects

against liver, breast and colorectal cancer cells. Similarly, caffeic acid and coumaric acid showed

significant anticancer activity against colon adenocarcinoma (HT29-D4) cancer cell lines

(Bouzaiene et al., 2015).

Table 4.7: Concentration of phenolic acids in Boswellia serrata and Pinus roxburghii

oleoresin (mg/L)

Compounds

Pinus roxburghii

(HD 160 oC)

Boswellia serrata

(HD 140 oC )

F2 c F3

Vanillic acid 9.72±0.21 5.18±0.17

p-Coumaric acid 1.28±0.09 ---

m-Coumaric acid 3.75±0.12 ---

Caffeic acid --- 5.22±0.23

Chlorogenic acid --- 20.79± 0.37

Cinnamic acid --- 10.67±0.29

Values are mean ± Standard Deviations of three separate experiments.

149

Figure 4.52: HPLC chromatogram of F2 c sub-fraction of Pinus roxburghii oleoresin hydro

distilled essential oil of 160 oC.

Figure 4.53: HPLC chromatogram of F3 fraction of Boswellia serrata oleoresin hydro

distilled essential oil of 140 oC.

150

4.8.1. GC-MS analysis of Pinus roxburghii oleoresin.

The chemical composition of essential oils and sub-fractions with highest antitumor

activity was determined by GC-MS and their results are shown in Table 4.8. GC-MS results of

hydro-distilled essential oil of 180 ⁰C showed that essential oil contained 17 compounds with α-

pinene (33.89 %) as a major compound, followed by longifolene (20.91 %), β-pinene (20.90 %),

and 3-carene (13.27 %). These results are in good agreement with (Kaushik et al., 2013) how

reported that α-pinene, β-pinene and 3-carene were the major compounds of Pinus roxburghii

essential oil grown in India. Similarly, (Hassan and Amjid, 2009) and (Zafar et al., 2010) found

alpha-pinene and 3-carene as major compounds in barks and needles essential oils of Pinus

roxburghii from Pakistani flora. In another study (Smaleh et al., 1976) reported that alpha-pinene

and 3-carene were the major compounds of Pinus roxburghii oleoresin essential oil. (Shah et al.,

2014) found that α-pinene and β-pinene was the major compound of Pinus roxburghii essential

oil. The variation in chemical composition of essential oil may due to difference in agro-climatic,

genetic and seasonal conditions. Analyzed essential oil showed that essential oil mainly

consisted of monoterpenes (74.11 %), followed by sesquiterpenes hydrocarbons (23.84 %),

oxygenated sesquiterpenes (1.34 %) and oxygenated monoterpenes (0.70 %) (Table 4.9). Our

findings are opposite to (Satyal et al., 2013) how reported that sesquiterpenes were the major

class of Pinus roxburghii bark essential oil. In another study (Hassan and Amjid, 2009) observed

that monoterpenes were the major class of Pinus roxburghii stems essential oil. α-pinene, β-

pinene and 3-carene were the major monoterpenes. In sesquiterpene hydrocarbon, longifolene

and α-longipinene were main compounds. In oxygenated monoterpenes myrtenol and trans-2-

caren-4-ol were compounds, while caryophyllene oxide was the only oxygenated sesquiterpene

present in essential oil.

The chemical composition of steam distilled essential oil of 160 ⁰C showed that essential

oil contained 13 compounds with 3-carene (42.65 %) as major compound, followed by α-pinene

(25.97 %), β-pinene (15.44 %), and longifolene (9.43 %) (Table 4.8). These results coincides

with (Kaushik et al., 2013) how reported that Pinus roxburghii oleoresin contained α-pinene, β-

pinene, car-3-ene and longifolene hydrocarbons as major compounds. Similarly, (Rawat et al.,

2006) found car-3-ene, pinene, longifolene as major compounds of Pinus roxburghii essential

oil. Furthermore, they reported that chemical composition of monoterpenes especially β-pinene

β-phellandrene, myrcene and limonene vary with geological pattern and origin of seed. Analyzed

151

essential oil showed that essential oil mainly consisted of monoterpenes (87.81 %), followed by

sesquiterpenes hydrocarbons (12.12 %) and oxygenated monoterpenes (0.06 %) (Table 4.9). 3-

carene, α-pinene, β-pinene and α-terpinolene were the major monoterpenes. In sesquiterpene

hydrocarbon, longifolene and caryophyllene were main compounds, while estragole was the only

oxygenated monoterpenes present in essential oil. The GC-MS results of F2 c sub fraction

showed that sub fraction contained 8 compounds with α-pinene (58.15 %) as major compound,

followed by β-pinene (38.66 %), and camphene (0.92 %) (Table 4.8). It was found that

monoterpene hydrocarbons (98.04 %) were the major class of compounds, followed by

oxygenated monoterpenes (0.81 %). α-pinene, β-pinene, camphene and 2-ethenyl-1,3,3-

trimethylcyclohexene were the monoterpene hydrocarbons. α-pinene oxide, myrtenol, verbenol

and verbenone were the oxygenated monoterpenes. Similarly, F2 a sub-fraction contained 8

compounds with α-pinene (53.11 %), β-pinene (20.38 %) and 3-carene (24.26 %) as major

compounds. The GC-MS analysis of essential oil isolated through supercritical critical fluid

extraction method showed that essential oil contained 16 compounds with 3-carene (38.67 %) as

major compound, followed by α-pinene (32.75 %), β-pinene (14.37 %), and longifolene (6.62

%). Moreover, it was found that monoterpene hydrocarbon (89.93 %) was the major class

compounds contained 8 compounds, followed by sesquiterpenes hydrocarbons (8.92 %) revealed

6 compounds, oxygenated monoterpenes (0.97 %) and oxygenated sesquiterpenes (0.17 %)

contained two compounds. Alpha-pinene, β-pinene and 3-carene were the major compounds of

monoterpenes. In sesquiterpene hydrocarbons, longifolene and caryophyllene were main

compounds, while oxygenated monoterpenes and oxygenated sesquiterpenes contained

citronellal and caryophyllene oxide, respectively. It was observed that chemical composition of

essential oils significantly varied (p ≤ 0.05) with extraction methods (Figure 4.54). The chemical

components that varied with extraction methods were α-pinene, camphene, β-pinene, α-

longipinene, longicyclene, sativene, longifolene and α-himachalene (highest amount in hydro-

distilled essential oil: 33.89 %, 0.39 %, 20.90 %, 1.40 %, 0.96 %, 0.34 %, 20.91 % and 0.24 %

respectively), 3-carene, γ-terpinene and α-terpinolene (maximum contribution in steam distilled

essential oil: 13.27 %, 0.25 % and 3.17 % respectively).

152

Table 4.8: Chemical composition of Pinus roxburghii oleoresin essential oils isolated through different extraction methods,

fractions and most active sub fractions

Components RI

% Composition Method of

identification HD F1 F2 F3 F4 F2 a F2 b F2 c F2 d F3 a F3 b F3 c F3 d F4 a F4 b

Monoterpene hydrocarbon

α-Thujene 923 0.28 0.47 0.37 0.27 ---- 0.48 0.39 0.28 0.12 0.34 0.28 0.22 0.04 0.07 0.54 a, b

α-Pinene 933 27.51 54.33 48.83 32.87 4.68 70.24 67.29 54.82 20.26 59.13 47.87 36.5 10.37 13.78 ---- a, b

Camphene 952 0.3 0.59 0.53 0.39 0.08 0.54 0.51 0.48 0.25 0.52 0.48 0.4 0.18 0.21 ---- a, b

Verbenene 972 0.53 0.97 0.71 0.6 0.52 0.45 0.42 0.64 0.49 0.6 0.56 0.48 0.28 0.68 0.25 a, b

β-Pinene 988 13.27 20.02 21.67 23.53 10.49 16.8 18.23 22.14 22.31 2055 23.42 25.34 20.21 23.15 4.46 a, b

1,5,8-p-menthatriene 1004 ---- 0.14 0.09 0.05 0.06 ---- ---- 0.07 0.03 ---- 0.02 ---- ---- 0.07 ----

a, b

3- Carene 1011 44.34 16.38 24.61 39.7 39.77 10.12 12.77 18.26 53.14 16.67 26.44 36.05 63.54 58.77 31.9 a, b

Trans-3-caren-2-ol 1021 ---- 0.13 0.09 0.05 0.07 ---- ---- 0.08 ---- ---- ---- ---- 0.09 0.08 0.04 a, b

m-Cymene 1023 0.35 0.24 0.21 0.31 0.74 ---- ---- 0.11 0.34 0.09 0.1 0.15 0.32 0.59 0.64 a, b

p-Cymene 1027 0.52 0.47 0.51 0.78 1.25 0.11 0.11 0.33 0.95 0.26 0.34 0.51 1.36 1.3 1.66

Limonene 1031 0.26 ---- 0.09 0.35 0.61 ---- ---- ---- 0.33 ---- 0.08 0.16 1 0.16 0.53 a, b

α-Terpinolene 1088 1.04 ---- 0.08 0.54 2.09 ---- ---- ---- 0.56 ---- ---- 0.15 ---- 0.23 ---- a, b

Oxygenated monoterpene

Linalool 1098 ---- 0.95 0.55 0.22 0.18 0.66 0.28 0.61 ---- 0.86 0.34 0.04 ---- 0.08 4.22 a, b

Thujol 1109 ---- 0.21 0.11 ---- ---- ---- ---- 0.11 ---- 0.11 ---- 2.28 0.11 0.05 a, b

Cis-limonene

oxide 1138 0.12 0.87 0.46 0.12 0.26 0.23 ---- 0.29 0.07 0.39 0.07 ---- ---- 0.36 0.04 a, b

Trans-limonene

oxide 1139 0.05 0.52 0.21 ---- 0.82 0.12 ---- ---- ---- 0.24 ---- ---- ---- 0.39 0.59 a, b

Trans-verbenol 1144 0.23 0.27 0.07 ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- 0.09 a, b

Pinocarvone 1162 ---- 0.77 0.24 0.06 0.15 ---- 0.32 ---- 0.12 ---- ---- ---- ---- 0.23 a, b

Myrtenal 1193 ---- 0.37 0.18 ---- 1.45 0.1 ---- 0.23 ---- 0.12 ---- ---- ---- ---- 1.44 a, b

UI ---- ---- 0.33 0.16 ---- ---- ---- 0.25 ---- ---- ---- ---- ---- ---- ---- a, b

Verbenone 1204 ---- 0.3 0.11 ---- 0.26 ---- ---- 0.18 ---- ---- ---- ---- ---- ---- ---- a, b

Eucarvone ---- ---- 0.53 0.12 ---- ---- ---- 0.8 ---- ---- ---- ---- ---- ---- ---- a, b

Bornyl acetate 1285 0.05 ---- ---- ---- 0.79 ---- ---- ---- ---- ---- ---- ---- 18.628 1.15 ---- a, b

153

Sesquiterpene hydrocarbons

α-longipinene 1351 0.59 1.04 ---- ---- 2.64 ---- ---- ---- ---- ---- ---- ---- ---- ---- 3.73 a, b

α-Ylangene 1367 0.08 ---- ---- ---- 0.34 ---- ---- ---- ---- ---- ---- ---- ---- ---- 0.48 a, b

Longicyclene 1373 0.26 ---- ---- ---- 1.2 ---- ---- ---- ---- ---- ---- ---- ---- ---- 1.41 a, b

Sativene 1393 0.17 0.1 ---- ---- 0.55 ---- ---- ---- ---- ---- ---- ---- ---- ---- 0.85 a, b

Junipene 1408 8.84 ---- ---- 0.16 28.7 ---- ---- ---- 0.15 ---- ---- ---- 0.33 0.15 41.41 a, b

Trans-caryophyllene 1418 0.63 ---- ---- ---- 2.1 ---- ---- ---- ---- ---- ---- ---- ---- ---- 3.34

a, b

Oxygenated sesquiterpene hydrocarbons

Caryophyllene

oxide 1581 0.56 ---- ---- ---- 0.8 ---- ---- ---- ---- ---- ---- ---- ---- ---- 0.94 a, b

Components RI


identification SD F1 F2 F3 F1 a F1 b F1 c F1 d F2 a F2 b

α-Thujene 923 0.25 0.31 0.25 0.05 0.48 0.47 0.33 0.16 0.37 0.25 a, b

α-Pinene 933 25.78 52.92 40.26 16.59 76.46 73.03 63.06 26.36 64.2 46.1 a, b

Camphene 952 0.27 0.45 0.42 0.23 0.54 0.52 0.5 0.32 0.54 0.49 a, b

Verbenene 972 0.31 0.55 0.61 0.58 0.39 0.33 0.43 0.66 0.51 0.66 a, b

β-Pinene 988 12.56 16.26 17.56 17.14 11.58 12.95 16.23 18.66 15.82 19.68 a, b

1,5,8-p-menthatriene 1004 --- 0.06 0.08 --- --- --- --- 0.07 --- 0.07 a, b

3- Carene 1011 51.05 28.23 38.37 62.99 9.43 12.43 19.06 51.52 17.81 30.41 a, b

Trans-3-caren-2-ol 1021 0.05 --- 0.08 --- --- --- --- 0.06 --- 0.09 a, b

m-Cymene 1023 0.19 0.16 0.24 0.44 --- --- --- 0.31 0.02 0.13 a, b

p-Cymene 1027 0.55 0.37 0.53 0.99 0.07 0.13 0.11 0.73 0.11 0.34 a, b

Limonene 1031 0.34 0.09 0.13 0.22 --- --- --- 0.15 --- 0.06 a, b

α-Terpinolene 1088 0.16 --- --- --- --- --- --- --- --- a, b

Linalool 1098 0.11 --- --- --- --- --- --- --- --- 0.41 a, b

Thujol 1109 1.79 --- 0.09 0.37

0.21 --- 0.12 a, b

Cis-limonene oxide 1138 --- 0.42 0.51 --- 0.78 0.14 0.28 0.34 0.5 0.2 a, b

Trans-limonene oxide 1139 --- 0.08 0.13 0.09 --- --- --- 0.12 --- --- a, b

Trans-verbenol 1144 --- --- 0.14

--- --- --- 0.05 --- --- a, b

Pinocarvone 1162 --- --- 0.18 0.15 --- --- --- 0.18 --- 0.13 a, b

Myrtenal 1193 --- 0.1 0.11 --- 0.16 --- --- --- 0.12 0.43 a, b

UI ---- --- --- 0.09 --- 0.11 --- --- --- --- --- a, b

Verbenone 1204 --- --- 0.16 --- --- --- --- --- --- --- a, b

Eucarvone ---- 0.21 --- --- --- --- --- --- --- --- --- a, b

154

Bornyl acetate 1285 0.03 --- --- --- --- --- --- --- --- a, b

α-longipinene 1351 0.16 --- --- --- --- --- --- --- --- 0.43 a, b

α-Ylangene ---- 0.08 --- --- --- --- --- --- --- --- --- a, b

Longicyclene 1373 5.44 --- 0.06 0.16 --- --- --- 0.1 --- --- a, b

Sativene 1393 0.72 --- --- --- --- --- --- --- --- --- a, b

Values are mean ± Standard Deviations of three separate determinations.

Different letter in superscripts represent significant difference among Pinus roxburghii oleoresin essential oils isolated by different extraction methods and most

active antitumor sub-fractions.

a = Identification based on retention index.

b = identification based on comparison of mass spectra. A Compound listed in order of elution from a HP-5MS column.

BRetention indices on the HP-5MS column.

155

Figure 4.54: Variations in major components of Pinus roxburghii oleoresin essential oils

extracted through different extraction methods.

0

10

20

30

40

50

HD EO SD EO SCF EO

%

α-Pinene β-Pinene 3-Carene Longifolene

0

0.5

1

1.5

2

2.5

3

3.5

HD EO SD EO SCF EO

%

α-Terpinolene α-Longipinene

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

HD EO SD EO SCF EO

%

Camphene γ-Terpinene Longicyclene Sativene α-Himachalene

156

Figure 4.55: GC-MS chromatogram of hydro distilled essential oil isolated at 160 ⁰C from

Pinus roxburghii oleoresin.

Figure 4.56: GC-MS chromatogram of steam distilled essential oil isolated at 160 ⁰C from

Pinus roxburghii oleoresin.

157

Figure 4.57: Chromatogram of super critical fluid extracted essential oil isolated at 40 ⁰C,

80 bar pressure from Pinus roxburghii oleoresin.

Figure 4.58: Chromatogram of sub fraction F2 c of hydro distilled essential oil isolated at

160 ⁰C from Pinus roxburghii oleoresin.

158

Figure 4.59: Chromatogram of sub fraction F2 a of steam distilled essential oil isolated at

160 ⁰C from Pinus roxburghii oleoresin.

159

Table 4.9: Chemical constituents of Pinus roxburghii oleoresin essential oils isolated through different extraction methods and

most active sub fractions.

Class of Compounds HD 160 ⁰C SD 160 ⁰C SCF F2 c F2 a

No of

Comp % age

No of

Comp % age

No of

Comp % age

No of

Comp % age

No of

Comp

%

age

Monoterpene hydrocarbon 8 74.11 6 87.81 8 89.93 4 98.04 6 99.43

Oxygenated monoterpenes 2 0.7 1 0.06 1 0.97 4 0.81 1 0.23

Sesquiterpene hydrocarbon 5 23.84 6 12.12 6 8.92 0 0 1 0.32

Oxygenated Sesquiterpenes 1 1.34 0 0 1 0.17 0 0 0 0

Total 16 99.99 13 99.99 16 99.99 8 98.85 8 99.98

http://webbook.nist.gov/cgi/cbook.cgi?ID=C125122&Mask=200


160

4.8.2. GC-MS analysis of Boswellia serrata oleoresin

The chemical composition of Boswellia serrata oleoresin essential oils isolated through

different extraction methods and sub-fractions with highest antitumor activity was determined by

GC-MS and their results are shown in Table. 4.10. GC-MS results of hydro-distilled essential oil

of 140 ⁰C showed that essential oil contained 16 compounds with α-pinene (75.07 %) as major

compound, followed by β-pinene (4.61 %), verbenol (4.58 %) and pinocarveol (3.75 %). A much

similar chemical composition with α-pinene as major compound from Boswellia serrata

oleoresin essential oil was reported by (Verghese et al., 1987). Analyzed essential oil showed

that essential oil mainly consisted of monoterpenes (86.53 %), followed by oxygenated

monoterpenes (12.74 %), oxygenated triterpenes (0.45 %) and oxygenated diterpenes (0.27 %)

(Table.4.11). α-pinene, β-pinene and m-mentha-1,8-diene were the major monoterpenes. In

oxygenated monoterpenes, pinocarveol and verbenol were main compounds, while verticillol and

2,3-oxidosqualenen were the only oxygenated diterpene and oxygenated triterpene present in

essential oil. These results coincides with (Kasali et al., 2002) how found that Boswellia serrata

bark essential oil contained α-pinene as major compound, followed by β-pinene, verbenol and

pinocarveol. Moreover, they reported that monoterpene hydrocarbons were the major class of

compounds present in Boswellia serrata essential oil. Similarly, (Gupta et al., 2016) reported

that monoterpene hydrocarbons were the dominant class of compounds present in Indian

Boswellia serrata oleoresin.

The chemical composition of steam distilled essential oil of 120 ⁰C showed that essential

oil contained 9 compounds with α-pinene (86.21 %) as major compound, followed by, β-pinene

(2.81 %), verbenol (2.17 %) and pinocarveol (2.06 %) (Table 4.10). Analyzed essential oil

showed that essential oil mainly consisted of monoterpenes (91.04 %), followed by oxygenated

monoterpenes (6.48 %) (Table 4.10). α-pinene, β-pinene and o-cymene were the major

monoterpenes. In oxygenated monoterpenes, pinocarveol, verbenone and verbenol were main

compounds. Similarly, the GC-MS analysis of essential oil isolated through supercritical critical

fluid extraction method showed that essential oil contained 12 compounds with α-pinene (46.49

%) and citronellal (45.74 %) as major comopounds. Moreover, it was found that monoterpene

hydrocarbon (51.19 %) was the dominant class of compounds contained 6 compounds, followed

by oxygenated monoterpenes (47.82 %) revealed 4 compounds, sesquiterpene hydrocarbons

(0.43 %) and oxygenated triterpenes (0.53 %) contained two compounds. In monoterpene

161

hydrocarbons, α-pinene, β-pinene and p-menthene were dominant compounds. In oxygenated

monotepenes, citronellal and verbenone were main compounds, while caryophyllene and 2,3-

oxidosqualene were the only sesquiterpene hydrocarbon and oxygenated triterpene, respectively.

The GC-MS results of F1c sub fraction of steam distilled essential oil of 120 ⁰C showed

that sub fraction contained 10 compounds with α-pinene (77.18 %) as major compound, followed

by m-mentha-1,8-diene (6.52 %), and β-pinene (4.99 %) (Table 4.10). It was found that

monoterpene hydrocarbons (96.31 %) were the major class of compounds, followed by

oxygenated monoterpenes (2.71 %). α-pinene, β-pinene, o-cymene and m-mentha-1,8-diene were

the major monoterpene hydrocarbons. Pinocarveol and verbenol were the oxygenated

monoterpenes. Similarly, F1 c sub fraction of hydro distilled essential oil of 140 ⁰C contained 16

compounds with α-pinene (72.37 %) as major compound. Moreover, sub fraction contained

monoterpene hydrocarbons (88.88 %) and oxygenated monoterpenes (11.11 %). In monoterpene

hydrocarbons, α-pinene, β-pinene, m-mentha-1,8-diene and o-cymene were the main

compounds, while the contraction of camphene, β-thujene, Bicyclo[4.2.0]oct-1-ene, 7-endo-

ethenyl- and β-myrcene were very small. In oxygenated monoterpenes, pinocarveol and verbenol

were the major compounds. It was observed that chemical composition of essential oils

significantly varied (p ≤ 0.05) with extraction methods (Figure 4.60). The chemical components

that varied with extraction methods were α-pinene (revealed maximum amount 86.21 % in steam

distilled essential oil), camphene, β-pinene and o-cymene (highest amount in hydro-distilled

essential oil: 1.11 %, 4.61 % and 1.64 % respectively), α-campholenal and verbenone (maximum

contribution in supercritical fluid extracted essential oil: 0.83 % and 1.72 % respectively).

Previous literature reports confirmed that chemical composition of essential oils vary with

extraction methods. (Guan et al., 2007) reported that essential oil isolated from clove buds

through hydro, steam distillation, soxhlet extraction and supercritical fluid extraction method

showed significant variation in concentration of chemical components present in these essential

oil. Similarly, (Khajeh et al., 2004) found that there is quantitative difference in chemical

composition of Carum copticum essential oils isolated through supercritical fluid CO2 extraction

method and hydro distillation method.

The literature reports showed that Boswellia serrata oleoresin contained α-pinene as a

major compound, followed by copaene, cis-verbenol, thuja-2,4(10)-diene, trans –pinocarveol,

162

borneol, myrcene, p-cymene, verbenone and limonene (Kasali et al., 2002). Moreover, they

reported that monoterpene hydrocarbon was the main class of compounds present in essential oil.

Similarly, (Verghese et al., 1987) found steam distilled essential oil of Boswellia serrata

oleoresin contained α-pinene as a major compound. (Hamm et al., 2005) reported that α-pinene,

limonene and β-myrcene are the main monoterpene hydrocarbons. Caryophyllene oxide, β–

caryophyllene, α-copaene and α-humulene were the dominant sesquiterpene. Opposite results

was reported by (Singh et al., 2007) how found α–thujene as major compound from oleoresin

essential oil of Boswellia serrata collected from different locations of India. They found that

monoterpene hydrocarbons were major compounds in essential oils. Moreover, they reported that

benzyl tiglate, α -pinene, p-cymene, tetrahydro-linalool, linalool acetate, terpineol, carene-3,

Menthone, methyl isoeugenol and epi-cubenol were the compounds present in all commercial

and natural habitat essential oil. Similarly, (Gupta et al., 2016) reported that commercial

oleoresin essential contained high concentration of monoterpene hydrocarbons with α-thujene, α-

pinene, sabinene, δ-3-carene, p-cymene and limonene as most dominant compounds.

Furthermore, they reported that commercial oleoresin essential oils contained higher amount of

sesquiterpenes than natural habitat oleoresin essential oil.

163

Table 4.10: Chemical composition of Boswellia serrata oleoresin essential oils isolated through different extraction methods

fractions and most active sub-fractions

Components Retention

index


identification SD F1 F2 F3 F1 a F1 b F1 c F2 a F2 b F2 c


α-Thujene 923 3.56 5.6 5.82 2.43 6.23 6.01 6.43 8.02 7.36 4.54 a, b

α-Pinene 933 82.21 86.53 86.26 33.05 86.33 82.68 79.97 87.46 87.11 74.93 a, b

Camphene 952 1.21 1.14 1.45 1.07 0.84 1.13 1.91 1.84 1.94 1.61 a, b

Verbenene 972 0.37 0.37 0.53 0.84 0.19 0.34 0.73 0.35 0.49 1.03 a, b

Sabinene 976 1.38 1.02 1.51 2.41 0.43 0.66 1.86 0.93 1.35 2.91 a, b

β-Pinene 988 0.85 0.24 0.59 1.8 0.42 0.06 0.68 0.28 0.48 1.35 a, b

m-Cymene 1023 1.44 0.58 1.18 6.15 0.1 0.31 1.75 0.32 0.48 3.93 a, b

p-Cymene 1027 1.5 0.86 1.63 8.64 0.26 0.34 2.04 0.32 0.55 4.87 a, b

Oxygenated monoterpene

1,8-Cineole 1033 0.16 --- 0.11 1.04 0.59 0.21 0.15 --- --- 0.3 a, b

Linalool 1098 0.5 0.99 0.26 2.31 1.24 2.08 1.04 0.24 0.13 0.85 a, b

α-Thujone 1116 0.48 1.05 --- 2.03 --- 2.35 1.2 --- --- 0.84 a, b

α-campholenal 1125 0.33 --- --- 2 --- --- --- --- --- 0.14 a, b

β-thujone 1139 1.82 0.35 0.19 10.21 0.13 0.42 0.61 --- --- 0.98 a, b

Trans verbenol 1144 1.91 0.81 0.26 11.63 0.47 1.42 1.01 --- --- 1.07 a, b

Isopinocamphone 1173 0.27 0.09 --- 2.84 0.5 --- --- --- --- --- a, b

Terpineol-4 1179 0.2 --- --- 0.92 --- --- --- --- --- --- a, b

P-Cymen-8-ol 1183 --- --- --- 1.33 --- 0.29 --- --- --- --- a, b

Myrtenol 1194 0.58 --- --- 3.18 --- 1.07 --- --- --- 0.12 a, b

d-Verbenone 1205 0.84 0.37 --- 4.8 0.55 --- 0.48 --- --- 0.46 a, b


Different letter in superscripts represent significant difference among Boswellia serrata oleoresin essential oils isolated by different extraction methods and most

active fractions and sub-fractions.




164

Components Retention

index


identification HD F1 F2 F3 Residue F2a F2 b F3 a F3 b F3 c


α-Thujene 923 4.88 5.71 5.92 6.37 2.99 7.37 6.87 7.32 6.05 4.74 a, b

α-Pinene 933 80.69 90.36 91.67 88.07 47.1 90.01 89.22 89.32 90.36 73.83 a, b

Camphene 952 1.4 0.82 1.28 1.39 1.18 1.28 1.38 1.5 1.44 1.47 a, b

Verbenene 972 0.43 0.24 0.23 0.45 0.61 0.23 0.38 0.31 0.34 1.06 a, b

Sabinene 976 1.27 0.64 0.57 1.19 1.69 0.58 0.92 0.8 0.94 2.67 a, b

β-Pinene 988 1.03 0.11 0.12 0.51 1.86 0.11 0.34 0.3 0.27 1.67 a, b

m-Cymene 1023 1.05 0.26 0.11 0.79 3.64 0.14 0.27 0.15 0.19 3.9 a, b

p-Cymene 1027 1.98 0.46 0.18 1.23 6.98 0.16 0.47 0.3 0.41 5.53 a, b

Oxygenated Monoterpene

1,8-Cineole 1033 0.14 --- --- --- 0.78 --- --- --- --- 0.26 a, b

Linalool 1098 0.24 0.58 0.1 --- 0.47 --- 0.15 --- --- 0.74 a, b

Thujol 1109 0.1 0.45 --- --- 0.21 --- --- --- --- 0.61 a, b

α-Thujone 1116 0.32 --- --- --- 1.26 --- --- --- --- 0.39 a, b

α-campholenal 1125 0.32 --- --- --- 1.47 --- --- --- --- 0.23 a, b

β-thujone 1139 2.14 0.05 --- --- 7.9 --- --- --- --- 0.95 a, b

Trans verbenol 1144 2.01 0.29 --- --- 8.15 --- --- --- --- 0.93 a, b

Isopinocamphone 1173 0.24 --- --- --- 3.67 --- --- --- --- 0.23 a, b

Terpineol-4 1179 0.25 --- --- --- 1.07 --- --- --- --- --- a, b

P-Cymen-8-ol 1183 0.24 --- --- --- 1.14 --- --- --- --- --- a, b

Myrtenol 1194 0.44 --- --- --- 2.8 --- --- --- --- 0.24 a, b

d-Verbenone 1205 0.67 --- --- --- 3.54 --- --- --- --- 0.55 a, b

Bornyl acetate 1285 0.16 --- --- --- 0.67 --- --- --- --- --- a, b


Different letter in superscripts represent significant difference among Boswellia serrata oleoresin essential oils isolated by different extraction methods and most

active fractions and sub-fractions.




165

Figure 4.60: Variations in major components of Boswellia serrata oleoresin essential oils

extracted through different extraction methods.

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

HD EO SD EO SCF EO

Pe

rce

nta

ge

Camphene β-Pinene o-Cymene

-0.2

0.3

0.8

1.3

1.8

HD EO SD EO SCF EO

Pe

rce

nta

ge

α-Campholenal Verbenone

0

20

40

60

80

100

HD EO SD EO SCF EO

Pe

rce

nta

ge

α-Pinene Monoterpene hydrocarbon Oxygenated monoterpenes

166

Figure 4.61: Chromatogram of hydro distilled essential oil isolated at 140 ⁰C from


Figure 4.62: Chromatogram of steam distilled essential oil isolated at 120 ⁰C from


167

Figure 4.63: Chromatogram of super critical fluid extracted essential oil isolated at 40 ⁰C,

80 bar pressure from Boswellia serrata oleoresin.

Figure 4.64: Chemical composition of sub fraction F1 c of hydro distilled essential oil

isolated at 140 ⁰C from Boswellia serrata oleoresin.

168

Figure 4.65: Chromatogram of sub fraction F1 c of steam distilled essential oil isolated at

120 ⁰C from Boswellia serrata oleoresin.

169

Table 4.11: Chemical constituents of Boswellia serrata oleoresin essential oils isolated through different extraction methods

and most active sub-fractions.

Class of Compounds HD 140 ⁰C SD 120 ⁰C SCF F1 c F1 c

No of

Comp % age

No of

Comp % age

No of

Comp % age

No of

Comp % age

No of

Comp % age

Monoterpene hydrocarbon 6 86.53 4 91.04 6 51.19 6 96.31 8 88.88

Oxygenated monoterpenes 8 12.74 5 6.48 4 47.82 4 2.71 8 11.11

Sesquiterpene hydrocarbon 0 0 0 0 1 0.43 0 0 0 0

Oxygenated diterpene 1 0.27 0 0 0 0 0 0 0 0

Oxygenated triterpene 1 0.45 0 0 1 0.53 0 0 0 0

Total 16 99.99 9 97.52 12 99.97 10 99.02 16 99.99


170

Chapter 5

Summary

The research work presented in this dissertation was conducted in the laboratories of the

Department of Chemistry; Department of Biochemistry, University of Agriculture, Faisalabad,

Pakistan and Department of Chemistry, Forman Christian College, Lahore, Pakistan. Boswellia

serrata and Pinus roxburghii oleoresin was collected form Zhob district of the north west

of Balochistan and Neelum Valley, north-east of Muzaffarabad, Azad Kashmir, Pakistan,

respectively. Experiments were performed to evaluate the percentage yield, fractionation,

antioxidant, antibacterial, antifungal, cytotoxic and antitumor activity of essential oils, fractions

and sub fractions. Essential oils were extracted through hydro distillation, steam distillation and

supercritical fluid extraction methods under different temperature conditions. Among all the

Pinus roxburghii oleoresin essential oils, the essential oil obtained through steam distillation

method at 180 0C showed highest essential oil yield (19.91 ± 0.22%), while supercritical fluid

extracted essential oil showed lowest essential oils yield (1.04 ± 0.13 %). A much similar trend

was observed in Boswellia serrata oleoresin essential oils, where essential oil obtained by hydro-

distillation method at 180 0C showed highest essential oil yield (9.37 ± 0.12 %) and supercritical

fluid extracted essential oil had lowest essential oils yield (1.04 ± 0.13 %). All the essential oils

were separated into different fractions and sub fractions on the basis of their boiling points and

their biological activities were determined under bioactivity guided assays. Antioxidant activity

was determined through DPPH assay, H2O2 assay, percentage inhibition in linoleic acid system and

total antioxidant contents /FRAP assay. All the essential oils, most active fractions and sub

fractions showed moderate level of antioxidant activity. In Pinus roxburghii oleoresin essential

oils extracted at different temperatures, the 180 0C showed highest antioxidant activity in all

tested assays. While in fractions and sub fractions, F3 b sub fraction showed highest antioxidant

activity. Similarly in steam distilled essential oils, 180 0C essential oils showed highest

antioxidant activity in all tested assays and in fractions and sub fractions, F4 fraction showed

highest antioxidant activity. In Boswellia serrata oleoresin hydro distilled essential oils, essential

oil of 180 0C exhibited highest antioxidant activity, while in fractions (F4) and sub fractions (F2

c) showed highest antioxidant activity. In steam distilled essential oils, the essential oil of 180 0C

171

showed highest antioxidant activity while in fractions (F1) and sub fractions (F2 c) exhibited

optimum antioxidant activity.

All the essential oils, fractions and sub fractions showed good antimicrobial activity

against all the test microbial strains. In hydro distilled essential oils of Pinus roxburghii, the

essential oils of 160 0C showed highest antibacterial activity, while in fractions F1 and F3 and in

sub fractions F2 c exhibited highest antibacterial activity. Similar trend was observed in steam

distilled essential oils, where essential oil of 160 0C showed highest antibacterial activity. While

in fractions (F4) and in sub fractions (F2 c) showed highest antibacterial activity. In hydro

distilled essential oils of Boswellia serrata oleoresin, the essential oils of 140 0C showed highest

antibacterial activity, while in fractions (F4) and in sub fractions (F2 c) showed highest

antibacterial activity against all the tested bacterial strains. In steam distilled essential oils, 140

0C essential oil showed highest antibacterial activity, in fractions (F3) and in sub fractions (F1 c)

showed highest antibacterial activity. Antifungal activity of essential oils, most active fractions

and sub fractions was determined through disc diffusion and micro broth susceptibility assays. In

Pinus roxburghii oleoresin hydro distilled essential oils, fractions and sub fractions, F1 c sub

fraction showed highest inhibition zones (21.16 - 28.20 mm) and lowest MIC values (14.06 -

84.44 µg/mL) against all the tested fungal strains. In Pinus roxburghii oleoresin steam distilled

essential oils, fractions and sub fractions, 140 0C essential oils showed highest inhibition zones

(17.84 - 24.03 mm) and lowest MIC values (42.22 - 112.58 µg/mL) against all the tested fungal

strains. In Boswellia serrata hydro distilled essential oils, its most active fractions and sub

fractions, 120 0C essential oil showed the highest inhibition zones (15.81- 16.78 mm) and lowest

MIC values (70.36 – 140.73 µg/mL), while in steam distilled essential oils, most active fractions

and sub fractions, 160 0C essential oil showed highest antifungal activity with inhibition zones

(17.80 – 28.08 mm) and MIC values (28.14 – 112.58 µg/mL). Cytotoxicity of essential oils,

fractions and sub fraction was determined through hemolytic assay. The results of hemolytic

assay showed that all the essential oils, fractions and sub fractions showed weak cytotoxicity.

Pinus roxburghii hydro distilled essential oil of 140 0C showed highest percent hemolysis (29.89

%), while in steam distilled essential oils, fractions and sub fractions, essential oil of 120 0C

showed highest percent hemolysis (24.58 %). In Boswellia serrata oleoresin essential oils,

fractions and sub fractions, hydro distilled essential oil of 180 0C showed optimum hemolysis

(15.08 %). Antitumor activity of essential oils, fractions and sub fractions was determined

172

through crown gall antitumor assay. The results showed that F2 c sub fraction exhibited highest

tumor inhibition potential (90.04 %) in Pinus roxburghii hydro distilled essential oils, steam

distilled essential oils, most active fractions and in sub fractions. In hydro distilled essential oils,

steam distilled essential oils, most active fractions and sub fractions of Boswellia serrata

oleoresin, F3 fraction of hydro distilled essential oil of 140 0C showed maximum percentage

tumor inhibition (88.58 %). The chemical composition of essential oils and sub fractions with

highest antitumor activity was determined through GC-MS. It was observed that α-pinene, β-

pinene, 3-carene and longifolene were the major chemical components of Pinus roxburghii

oleoresin essential oils and most active sub fractions and these compounds might be responsible

for their biological activity. Similarly, the chemical composition of Pinus roxburghii oleoresin

essential oils and most active sub fractions showed that α-pinene, β-pinene, pinocarveol and

verbenol were the main chemical compounds. Moreover, it was observed that the chemical

composition of essential oils significantly vary with extraction methods.

173

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prr.hec.gov.pkprr.hec.gov.pk/jspui/bitstream/123456789/9239/1/T... · vi ACKNOWLEDGEMENTS In the name of ALLAH, the merciful, the beneficent If oceans turn into ink and all of the