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الرحيم الرحمن الله بسم In the name of Allah, the Merciful, the Most Beneficent

‘A thousand mile journey begins with a single step’

-Lao Tzu

PHYTOCHEMICAL INVESTIGATION OF LAWSONIA INERMIS LINN AND ITS APPLICATIONS AS COLORANT

MAMOONA NIGHA M.Phil. Applied Chemistry2013-m.phil-app-chem-7UNIVERSITY OF ENGINEERING AND TECHNOLOGY, LAHORESUPERVISOR: DR. ABDUL GHAFFAR

CONTENTS• Abstract• Aims and Objectives• Background• Methods• Results• Conclusion• Future Plan

AbstractLawsonia inermis Linn leaves (Henna Plant) was investigated for: • Phytochemical components• Elemental analysis• Essential oil • Dyeing pigment (Lawsone) And then, their Proficiency of was evaluated by

antioxidant and antimicrobial/antibacterial action.

Moreover, Color combinations with

mordants, were made and their properties was tested on leather.

• To reveal antioxidant and antimicrobial/antibacterial potencies of essential oil of Lawsonia Inermis Linn leaves and their extracts.

• To estimate whether the metal content of Lawsonia Inermis Linn is within the recommendation ranges.

• Characterization of lawsone, and its applications on leather.

Aims and objectives

Background• The use of plants for the healing purposes

cited around 3000 B. C, in China, Egypt and subcontinent.

• In the subcontinent, the herbal medical system is very popular which dates about 800 B. C.

• The Ayurveda and Yunnan, which made use of extract of plants for the curing of diseases.

• In the background of the tremendous benefits of medicinal plants, many research institutes have been shown to carry out work on plants and reveal the miraculous powers present in them.

MethodsExtraction of Essential Oil• Powdered leaves were hydro

distilled in the modified Dean Stark apparatus.

• The percent yield was calculated based on the dried weight of the plant material and stored until analysis.

• The oil was then subjected for GC-MS analysis and for antioxidant, and antimicrobial activity.

MethodsCharacterization of Essential OilAntioxidant activity was determined using DPPH free radical scavenging assayAntimicrobial activity of essential oil was determined by paper disc method. Vancomycin was used as positive control. Test PathogensMenthicillin Resistant Styphylococcus Aureus (MRSA ATTC-43300) and Menthicillin Sensitive Styphylococcus Aureus (MSSA ATTC-25923)

MethodsPhytochemical Investigation of crude extracts of powdered leaves in hexane, toluene, THF, ethyl acetate, and ethanolQuantitative and qualitative test for AlkaloidsFlavonoidsSaponinsTanninsTerpenoidsPhenolic CompoundsSteroidsGlycosidesQuantitative test for CarbohydratesResinsAmino AcidFats

MethodsElemental analysis• Preparation of Ash• Digestion of Ash• Analysis through FAAS

Methods

Isolation and Characterization of Lawsone• Isolation was done

by maceration. • Identification of the

Lawsone was done by TLC and FT-IR.

MethodsAntioxidant and Antibacterial Activity• Antioxidant activity was evaluated through DPPH free

radical scavenging method• Antibacterial activity was determined by Paper Disc

method using Staphylococcus aureus ATCC 6538 and E. coil ATCC 10240

Results (GC-MS spectra)

Results (chemical constituents of EO, GC-MS)Sno# Compound Name Molecular Formula Molecular weight %composition Retention time

1 α- pinene C11H16 136 13.345 4.148

2 Eucalyptol C10H18O 154 18.141 5.453

3 Linalool C10H18O 154 10.527 7.118

4 Trans- pinocarveol C10H15O 151 0.811 7.919

5 α- terpineol C10H18O 154 1.205 8.417

6 2,6-octadien-1-ol C10H18O 154 3.195 9.784

7 Nerolic acid (2,6-octadienoic acid) C11H1802 182 1.408 10.683

8

2,6- octadien-1-ol, 3,7-dimethyl, acetate, (E) C12H20O2 196 5.636 11.678

9 α- caryophyllene C15H24 204 2.867 12.485

10 1,2-dimethoxy-4-[2-propenyl], benzene C11H14O2 178 2.129 12.909

11 1,6-octadien-3-ol, 3,7-dimethyl, formate C11H18O2 182 0.834 13.223

12 9-isopropyl-1-methyl-2-methylene-5-oxatricyclo [5,4,0,0 {3,8}] undecane

C15H24O 220 1.493 13.750

13 Durohydroquinone C10H14O2 166 7.856 14.465

Results (anti-oxidant activity of EO)

20 µL 40 µL 60 µL 80 µL 100 µL0

10

20

30

40

50

60

70

80

90

100

Essential oil BHT

10 µL 20 µL 30 µL 40 µL 50 µL0

10

20

30

40

50

60

70

80

90

Essential oil BHT

Results (anti-microbial activity of EO)

72737475767778798081

75

80Antimicrobial Activity

Microbial strains

%in

hibi

tion

MRSAMSSA

Organ

ism

Concentr

ation of

Vancomy

cin

Inhibition

zone Of

essential oil ±

SD for 10.9µL

pure oil

Inhibition zone

Of essential oil

± SD for 22.6µL

pure oil

Inhibition zone

Of Vancomycin

± SD

MRSA 20µL 22mm ± 5.0 25mm ± 5.0 20mm ± 0.0

MSSA 40µL 11mm ± 5.0 15mm ± 5.0 20mm ± 0.0

Orga

nism

Concentr

ation of

Vancom

ycin

MIC values in

µL/smL

For 10.9µL

MIC values in

µL/mL

For 22.6µL

Vancomycin

MRSA 20µL 3.3 ± 0.6 11.4± 0.6 -

MSSA 40µL 4.6 ± 0.6 14.9± 0.6 -

Results (FAAS)

Sodium Chromium Nickel Copper Potassium Cadmium Manganese Iron Zinc0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

Metals present in leaves of Lawsonia inermis Linn

Conc

entr

ation

in p

pm

Results (Phytochemical analysis)S.No Phytochemical

ConstituentsIdentification test Hexane Toluene THF Ethyl Acetate Ethanol

1 Alkaloids Wagner’s test _ + + + +2 Flavonoids Alkaline reagent

test_ _ + + +

3 Saponins Foam test _ + _ + +4 Triterpinoids - _ + + _ +5 Glycosides Killer Kilian’s test + - + + +

6 Steroids Salkowski test + _ + + +7 Tannins Ferric chloride test _ + + _ +

8 Carbohydrates Benedict test + + + + +9 Amino acids Biuret test _ _ _ _ _10 Fats Spot test + + + - +

Saponification test + + +

11 Resins Turbidity test _ _ _ _ _Ferric chloride test _ _ _ + _

Results (Phytochemical analysis)Compounds Hexane ± SD Toluene ± SD THF ± SD Ethyl Acetate ± SD Ethanol ± SD

Alkaloids - 18mg/L ± 2.02 22.42mg/L ± 0.28 24mg/L ± 1.44 22.16mg/L ± 0.28

Flavonoids - - 4.6mg/L ± 0.21 4.45mg/L ± 0.01 4.02mg/L ± 0.19

Saponins - 2.139mg/L ± 1.06 - 0.89mg/L ± 1.06

Triterpenoids - 0.1mg/L ± 0.08 - 0.272mg/L ± 0.05

Glycosides 0.158mg/L ± 0.19 - 2.365mg/L ± 0.36 2.818mg/L ± 0.62 1.65mg/L ± 0.05

Steroids 0.862mg/L ± 0.23 - 0.114mg/L ± 0.19 0.003mg/L ± 0.25 0.851mg/L ± 0.23

Tannins - 1.106mg/L ± 0.08 0.121mg/L ± 0.6 1.741mg/L ± 0.53

Total phenolic content 217mg/L 118mg/L 245mg/L 103mg/L 107mg/L

Results (FT-IR)

Results (FT-IR)

Results (TLC)Solvent system Rf value Reported Rf

valuesn – hexane: ethanol (6:4) 0.25 0.23

n – hexane: methanol (3:1) 0.85 0.79-0.85

ethanol: methanol: n – hexane (1:3:9) 0.38 0.39

toluene: ethyl acetate: acetic acid (8:1:1) 0.44 0.43

toluene: ethyl acetate: methanol: acetic acid (3:4:3:1) 0.71 0.73

chloroform: ethanol: acetic acid (4:5:1) 0.55 0.53

ethyl acetate: water: methanol (5:3:4) 0.23 0.23

Results (antioxidant activity of extracts)

o 2.4 4.4 4.7 5.20

10

20

30

40

50

60

70

80

90

100

Extracts in solvents of increasing polarities

Per

cent

age

inhi

bitio

n

20 µL 40 µL 60 µL 80 µL 100 µL0

10

20

30

40

50

60

70

80

90

100

BHT in solvents of increasing polarities

Perc

enta

ge in

hibi

tion

Results (antioxidant activity of extracts)

Calculations For hexane extract For toluene extract

For THF extract For ethyl acetate extract For ethanol extract

Weight of dry 25mL flask at room temperature 66.90gm 75.46gm 59.76gm 73.72gm 56.65gm

Weight of 25mLflask+sample+solvent 92.14gm 116gm 112.6gm 109gm

98.91gm

Weight of antioxidant in sample 25.24gm 40.54gm 52.84gm 35.28gm 42.26gm

Weight of 25mL flask solvent evaporated 73.22gm 91.42gm 108gm 88gm 86.11gm

Weight of residue+25mL flask 73.66gm 91.45gm 108.04gm 88.03gm 86.16gm

Dry weight of 25mL flask-weight of residue+25mL flask

6.76gm 15.99gm 48.28gm 14.31gm

29.51gm

%calculations 10% 21% 80% 19%

52%

Results (antibacterial activity of extracts)

Against E. coli Blank against E. coli Aganinst S. aureus Blank against S. aureus0

2

4

6

8

10

12

Hexane Toluene THF Ethyl Acetate Ethanol

Inhi

bitio

n zo

ne

Results (Formulation of Dye)formulations Lawsonia Inermis

Linn extractBeetroot

Extract

Oxalic acid Tartaric acid Citric acid Para- formaldehyde Ferrous sulphate Sodium picramate

L1 4 1 _ _ _ _ _ _L1a 5 1L1b 2 1L1c 1 2L2 4 1 _ 1 _ _ _ _L2a 5 1 1L2b 1 1 1L2c 2 2 1L3 4 1 1 _ _ _ _ _L3a 5 1 1L3b 1 1 1L3c 2 2 1L4 4 1 _ _ 1 _ _ _L4a 5 1 1L4b 1 1 1L4c 2 2 1L5 4 1 _ 1 _ 2 _ _L5a 5 1 1 2L5b 1 1 1 2L5c 2 2 1 2L6 4 _ _ _

 

 

_ _ _ 1L6a 4 2L6b 4 3L6c 4 4L6d 4 5L7 2 _ _ _ _ _ 2 1L7a 3 2 1L7b 1 2 1L7c 2 2 3L7d 4 2 2

Results (formulation of dyes, pH values)

formulations pH Formulations pH Formulations pH Formulations

pH

L1 5.90 L2 5.23 L3 5.80 L4 4.62

L1a 5.01 L2a 5.76 L3a 4.81 L4a 4.23

L1b 4.73 L2b 5.26 L3b 4.22 L4b 4.56

L1c 4.50 L2c 5.67 L3c 3.23 L4c 4.22

L5 4.68 L6 3.58 L7 3.98

L5a 4.51 L6a 3.23 L7a 3.64

L5b 4.50 L6b 3.80 L7b 3.30

L5c 4.48 L6c 2.94 L7c 2.95

L6d 2.81 L7d 2.81

Results (Fastness properties)Dye Mordant Light

fastnessWash fastness

Rub fastnessWet rubbing Dry rubbing

10% Without Mordant

3 4 4 320% 3 4 4 310% Ferrous

sulfate5 4-5 4 3-4

20% 5 5 4 410% Chrome 4 3-4 3-4 3-420% 4 4 4 410% Potassium

Permanganate4-5 4 3 4

20% 5 4 3 4

Results (fastness properties)

1 2 3 40

1

2

3

4

5

6

Wash fastness Dry rubbing Wet rubbing Light fastness

Fastness Properties

Fast

ness

raye

ConclusionThe study revealed that essential oil and leaves extract of Lawsonia inermis Linn consist of mainly terpenoids, flavonoids and phenolic compounds. Due to the presence of these compounds, it has high antioxidant and antimicrobial/antibacterial activity. The Lawsone from Lawsonia inermis Linn was applied on leather for its coloring effect.

Applications and Future path• The biological potencies of essential oil and lawsone

make them good ingredient to be use in the future drugs against various diseases probably skin infections. • Based on the dyeing method and selection of the

mordants variety of shades can be obtained.• Besides this dyeing processing cost is also low than

that of the synthetic dyes. • So the usage of natural dye is of great significance for

the future of commercial dyeing industry.

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