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Isolation, characterization and quantification of bioactive molecules…… Chapter 1

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Isolation, characterization and quantification of bioactive molecules from Cedrus

deodara, Albizzia chinensis and Podophyllum hexandrum

1.1 Isolation, characterization and quantification of bioactive molecules from

Cedrus deodara (Roxb.) Loud.

1.1.1 Introduction

The genus of true cedars, Cedrus consists of

four closely related species with

geographically separated distributions in

Mediterranean and western Himalayas

[Farjoń (1990, 2001)], i.e. C. deodara

(Roxb.) Loud. in the Hindu Kush, Karakoram

and Indian Himalayas, C. libani A. Rich. in

Turkey, Lebanon and Syria, C. brevifolia

(Hook. f.) Henry in Cyprus, and C. atlantica

(Endl.) Manetti ex Carrie´re in North Africa (Algeria, Morocco) (Table 1.1.1).

The Himalayan cedarwood, Cedrus deodara (Roxb.) Loud., grows extensively on the

slopes of the Himalayas in northern India, Pakistan and Afghanistan and is often the most

important conifer at the elevations of 1650-2400 m. In India, deodar forests are naturally

distributed in an average estimated area of about 203263 hectares comprising of 69872,

20391, and 113000 hectares in Himachal Pradesh, Uttar Pradesh and Jammu & Kashmir.

respectively, yielding 0.75 million m3 annual production of wood [Anonymous (1950)].

Sizeable quantities of wood are employed for distillation of essential oils, used worldwide

in the soap industry as an inexpensive source of perfume. The oil is distilled from roots and

stumps of the plant left after cutting of trees for timber extraction. Himalayan cedarwood

oil is relatively a recent addition to the list of cedarwood oils produced commercially.

Production began in India in late 1950s and is estimated to be around 150 tonnes per

annum out of world’s production of cedarwood oil of 3000 tonnes per annum [Coppen

(1995)].

The plant leaves (called needles) are widely used for flavoring foods, beverages, (powders,

wine, and tea) [Kim and Chung (2000)] and for the treatment of rheumatism, diabetes,

obesity, gonorrhea, chronic bronchitis, cancer, stomach and cardiovascular diseases [Zhang

et al. (2011); Atwal et al. (1976)]. Their application lessen the inflammation in tuberculous

glands and have mild terebinthinate properties [Krishnappa and Dev (1978); Bhan et al.


2

(1984); Mukherjee (2001)]. It is recorded in the dictionary of Chinese Crude Drugs as an

effective herbal drug for expelling wind, removing dampness, destroying parasites,

termites, moths, beetles and relieving itching. The wood has been used since ancient time

in Indian medical practice for the treatment of inflammations and rheumatoid arthritis

[Kirtikar and Basu (1933); Agarwal et al. (1980)]. The wood and bark extracts are

carminative, diaphoretic and useful in fever, piles, kidney stones, flatulence, pulmonary,

urinary, diarrhoea, dysentery problems [Parveen et al. (2010); Sharma et al. (1997);

Bhushan et al. (2006)]. The root oil is used as anti-ulcer drug by Hakims. Previous

chemical investigations indicated the presence of terpenes, lignans [Agarwal et al. (1982)],

and flavonoids [Agarwal et al. (1980)]. The lignans have been known for a number of

pharmacological activities such as antioxidant, antimitotic, antiviral, antitumor [Mercer and

Towers (1984)].

Table 1.1.1: List of Cedar trees with botanical/common names

Botanical Name Common Name

Family Pinaceae

Cedrus deodara Himalayan cedarwood, deodar

Cedrus libani Lebanon cedar or Cedar of Lebanon

Cedrus brevifolia Cyprus cedar

Cedrus atlantica Atlas cedar

Family Cupressaceae

Cupressus funebris Endl. Chinese cedarwood

Juniperus virginiana L. Virginia cedarwood, Eastern red cedar

J. mexicana Schiede Texas cedarwood

J. procera Hochst East African cedarwood

Widdringtonia whytei Rendle Mulanje cedarwood

1.1.2 Chemical constituents

Phytochemical research carried out on C. deodara has led to the isolation of

sesquiterpenes, flavonoids, alkaloids, tannins, saponins, lignans, organic acids and few

other classes of chemical constituents from its different parts. Sesquiterpenes are present in

almost all parts of C. deodara, in fact, they are mainly responsible for the pharmacological

activities of the plant.

1.1.2.1 Constituents of wood essential oil

The cedarwood oil is used as perfume fixative in cosmetic, soap and essence for household

or industrial use. The oil enriched with himachalenes is known as ‘Supper Rectified Oil’


3

and that in atlantones is called ‘Perfumery Grade’. The therapeutic actions of Cedrus

species have partially been ascribed to sesquiterpenes. Generally, the Cedrus oils were

characterized by high percentage of himachalenes [Bhushan et al. (2006)]. In the essential

oil of C. deodara wood, a number of sesquiterpenes including the himachalenes (�, � and

�) (1-3), isocentdarol (4), himachalol (5), allohimachalol (6) [Bisarya and Dev (1968)],

aryl himachalene (7), (E)-�-atlantone (8), (E)-γ-atlantone (9), (Z)-�-atlantone (10), (Z)-γ-

atlantone (11) [Pande et al. (1971)], deodarone (12), [Shankaranarayan et al. (1973);

Gopichand and Chakravarti (1974)], oxidohimachalene (13), �-himachalene monoepoxide

(14), atlantolone (15) [Shankaranarayan et al. (1977)], deodardione (16), diosphenol (17),

limonene carboxylic acid (18) were reported [Krishnappa and Dev (1978)]. Nigam et al.

(1990) described the presence of twenty three sesquiterpenic compounds in the essential

oil.

H

H

HO

OH OH

HO

H

(1) (2) (3) (4) (5) (6)

O

O

O O

(7) (8) (9) (10) (11)

O

O

O

O

O

HO

O

OH

O

(12) (13) (14) (15) (16)

OH

O

COOHH

(17) (18)

1.1.2.2 Constituents of stem bark and wood extract

Flavonoids [taxifolin (19), taxifolin-3'-glucoside (20), deodarin (21), cedeodarin (22),

cedrin (23), cedrinoside (24), quercetin (25)] [Raghunathan et al. (1971)], neolignans

[dihydrodehydroconiferyl alcohol (26), cedrusin (27), cedrusinin (28), triacetyl cedrusinin

(29)], lignans [lariciresinol (30), isolarciresinol (31), secoisolaricirosinol (32), meso-

secoisolariciresinol (33), (-)-wikstromal (34), (-)-matairesinol (35), dibenzylbutyrolactol

(36), (-)-nortrachelogenin (37)], sesquiterpenoids [himachalol (5), centdarol (38)], phenolic


4

sesquiterpene [himasecolone (39)] [Agarwal and Rastogi (1981)], diterpene [isopimaric

acid (40)], fatty acid ester [ethyl-23-methyl pentacosanoate (41)] [Khan and Naheed

(1988)] and acids [� 10-dehydroepitodomatuic acid (42), �7-dehydrotodomatuic acid (43) and

7-hydroxytodomatuic acid (44)] [Agarwal et al. (1982); Bhan et al. (1984)] were identified

from the extracts of wood and stem bark.

O

OH

HO

O

OR4

R3

OH

OH

R1

R2

O

OH

HO

O

OH

OH

OH

R

R1 R2 R3 R4 R

H H H H (19) H (25)

H H H glu (20) OH (45)

H Me H H (21)

Me H H H (22)

Me H OH H (23) Me H OH glu (24)

H H OH H (46)

OROH2C

OR1

OR2

OMe

R3

O

HO

H H

OMe

OH

MeO

HO

R R1 R2 R3 (30)

H H H OMe (26) H H H OH (27)

H H H H (28) Ac Ac Ac H (29)

MeO

HO

OH

OMe

CH2OH

CH2OH

HO

MeO

OH

OMe

CH2OH

CH2OH

HO

MeO

OH

OMe

CH2OH

CH2OH

(31) (32) (33)

1.1.2.3 Constituents of needles and roots

The compounds obtained from 95% ethanolic extract of C. deodara needles were

characterized as taxifolin (19), quercetin (25), myricetin (45), 2R,3R-dihydromyricetin


5

(46), cedrusone A (47), �-sitosterol (48), 1-[3-(4-hydroxyphenyl)-2-propenoate]-�-D-

glucopyranoside (49), 10-nonacosanol (50), dibutyl phthalate (51), phthalic acid bis-(2-

ethylhexyl)ester (52), protocatechuic acid (53), shikimic acid (54) and 5p-trans-

coumaroylquinic acid (55) [Zhang et al. (2011); Liu et al. (2011)]. A diterpene acid,

centdaroic acid (56) has been reported from roots [Srivastava et al. (2001)].

O

MeO

HO

O

OH

OMe

O

MeO

HO

O

OH

OMe

HO

H

O

MeO

HO

OH

OMe

OH

(34) (35) (36)

O

O

MeO

HO

OH

OMe

H

H

OH

OH

OH

O

Me

Me

HOOCH COOC2H520

(37) (38) (39) (40) (41)

HHO

HOOC

HO

HOOC

HO

HOOC

OH

(42) (43) (44)

O

OMe

OH

OMe

O

OOH

HO

O

O

O

HO

OH

OH

H

HH

HO

H

O

O

O

HOHO

OH

OH

OH

(47) (48) (49)

OH

18

O

O

O

O

O

O

O

O

(50) (51) (52)


6

O OH

OH

OH

O OH

OHHO

OH OH

OH

HOOC

HO

O

O

OH

Me

Me

Me

Me COOH

(53) (54) (55) (56)

1.1.3 Pharmacological and biological activities

The use of C. deodara (Roxb.) Loud. is recommended in the Ayurvedic system of

medicine for treatment of various ailments [Nayar and Chopra (1956)]. The alcoholic

extract of C. deodara is known to possess a variety of biological effects such as anticancer,

anti-inflammatory, diuretic and spasmolytic activities [Kulshreshtha and Rastogi (1976);

Agarwal et al. (1980)]. Major pharmacological activities included insecticidal,

molluscicidal, antifungal and anti-inflammatory activities [Zhang et al. (2009)].

1.1.3.1 Insecticidal activity

Cedarwood oil, being non-pollutant, has been used as an alternative for conventional

pesticides against different insect-pests. Chromatography fractions of the oil showed

insecticidal activity against Callosobruchus analis and Musca domestica; himachalol (5)

and �-himachalene (2) being active against C. analis [Singh and Agarwal (1988)]. The oil

showed Knock Down (KD) property against adult Anopheles stephensi, at low

concentrations (KD50 0.4452% in acetone) [Singh et al. (1984)]. The commercial products of C.

deodara such as Himax and Pestoban were found effective against skin diseases of goats,

sheep and dogs [Hazarika et al. (1995); Sharma et al. (1997); Dimri and Sharma (2004b)].

Oils obtained from A. indica, C. deodara and their combination (1:1) exhibited fumigant

potential against adults of Callosobruchus chinensis L. [Raguraman and Singh (1997)].

Essential oil of C. deodara, in combination with other oils showed low activity against the

adults of Aedes agypti (LC50 2.48%) [Makhaik (2005)].

1.1.3.2 Molluscicidal activity

Different combinations of the essential oils of cedar and neem tree in combination with

powder from bulbs of Allium sativum Linn., oleoresin extracted from rhizomes of Zingiber

officinale Rosc., custard apple seed powder, fruit powder of Embelia ribes showed toxicity

against the snail Lymnaea acuminata L. [Singh and Singh (1998); Singh and Singh (2001);

Rao and Singh (2001); Singh and Singh (2004)]. C. deodara along with other plant-derived

molluscicides exhibited dose and time dependent toxicity against Achatina fulica [Rao and

Singh (2000)]. Sublethal in vivo 24 h exposure of oil of Azadirachta indica, C. deodara,

bulb powder of Allium sativum and bark powder of Nerium indicum (LC50 40% and 80%)


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significantly reduced the activity of A. fulica by altering acetylcholinesterase (AChE),

lactic dehydrogenase (LDH) and acid/alkaline phosphatase enzymes activity [Rao et al.

(2003)].

1.1.3.3 Antifungal activity

The oil proved to be effective against Absidia sp., Alternaria alternata, A. porri,

Aspergillus flavus, A. fumigatus, A. niger, A. ruber, A. versicolor, Cladosporium

cladosporioides, Curvularia lunata, Paecilomyces variotii, Fusarium oxysporum and

Rhizopus spp. [Dikshit et al. (1983); Pawar and Thaker (2007)]. Himachalol (5) exhibited

a significant protection and reduced colony forming units against A. fumigatus (minimum

inhibitory concentration, MIC, 46.4 �g/ml) [Khan and Jain (2000); Chowdhry et al. (1996);

Parveen et al. (2010)] and showed protection against invasive Aspergilli [Chowdhry et al.

(1997)]. The essential oil exhibited absolute toxicity, inhibiting the mycelial growth of A.

niger and Curvularia ovoidea, the two storage fungi found in blackgram, Vigna mungo L.

showing MIC of 1000 ppm [Singh and Tripathy (1999)]. The oil showed a broad

fungitoxic spectrum inhibiting the mycelial growth of fungi (Achaetomium strumarium

Guarro, Acremonium album Cattaneo, Alternaria alternata (Fr.) Keisaler, Aspergillus

aculeatus Iizuka, A. flavus Link ex Fries, A. japonicus Saito, A. niger van Teighem, A.

tamarri Kita, A. terreus Thom., Curvularia ovoidea (Hiroe & Watanabe) Muntanola,

Fusarium moniliforme Scheldon, F. oxysporum Schl., Penicillium chrysogenum Thom. and

P. funiculosum Thom., Rhizopus arrhizus Fischer [Singh et al. (1999)]. Eleven �-

himachalene derivatives displayed moderate to excellent activity against Botrytis cinerea

after 6 days; dihydroxyhimachalene derivative was found most promising [Daoubi et al.

(2005)]. Ethanolic extract of fresh plants of C. deodara caused complete inhibition of

mycelial growth of Sclerotium rolfsii Sacc. [Devi et al. (2007)].

1.1.3.4 Anti-inflammatory

The wood of C. deodara has been used since ancient days in Ayurvedic medical practice

for the treatment of inflammations and arthritis [Kirtikar and Basu (1933)]. The activity of

the oil could be attributed to its mast cell stabilizing activity and the inhibition of

leukotriene synthesis [Shinde et al. (1999a)]. The essential oil reduced the number of

neutrophils at the site of inflammation and the release of inflammatory mediators. It

produced significant inhibition of carrageenan-induced rat paw edema and of both

exudative-proliferative and chronic phases of inflammation in adjuvant arthritic rats at

doses of 50 and 100 mg/kg body weight [Shinde et al. (1999b); Tandan et al. (1998);

Baylac and Racine (2004)].


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1.1.3.5 Other activities

The spasmolytic activity was shown by 50% ethanolic extract of the wood of C. deodora

and himachalol (5) [Dhar et al. (1968); Kar et al. (1975)]. Antibacterial action of the

ethanolic extract was found against Staphylococcus aureus, Enterococcus faecalis, Bacillus

cereus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli [Zeng et al.

(2011)]. A standardized lignan composition (AP9-cd) from C. deodara consisting of (-)-

wikstromal (34), (-)-matairesinol (35) and dibenzylbutyrolactol (36) showed cytotoxicity in

several human cancer cell lines [Bhushan et al. (2006)]. Higher doses (100 & 200 mg/kg)

of alcoholic extract of heart wood of the plant showed anxiolytic, anticonvulsant activities

and significant CNS depression by reducing locomotor activity in mice through modulation

of GABA levels in brain [Dhayabaran et al. (2010)]. The mortality was found against

larvae of Culex quinquefasciatus by hot water, acetone, and methanolic extracts of stem

bark with LC50 values of 133.85, 141.60, 95.19 ppm and LC90 values of 583.14, 624.19 and

639.99 ppm, respectively [Rahuman (2009)]. Study showed that petroleum ether extract of

the heart wood exhibited protection against sodium oxalate induced nephrolithiasis, thus

showing diuretic and anti-urolithiatic activities [Ramesh et al. (2010)].

There has been a tremendous interest in this plant as evidenced by the voluminous work

carried out by different researchers in the identification of chemical constituents of

essential oil. Yet very less work on the extracts from wood of this species has so far been

reported. With the efforts to search for the novel bioactive constituents from natural source,

we aimed to work on isolation and identification of bioactive constituents of C. deodara

and their quantification by different analytical techniques. In the following pages results

and discussion section followed by experimental sections are mentioned.

1.1.4 Results and discussion

1.1.4.1 Phytochemical studies

The sawdust of C. deodara stump was successively extracted with n-hexane (hexane),

followed by chloroform (CHCl3), methanol (MeOH) and water (H2O) using percolation

extraction. From hexane extract one known sesquiterpene, (E)-�-atlantone (8) and from

chloroform extract, two new sesquiterpenes, (E)-(2S, 3S, 6R)-atlantone-2,3-diol (57), (E)-

(2S, 3S, 6S)-atlantone-2,3,6-triol (58) and one known sesquiterpene atlantolone (15) were

isolated by repeated column chromatography over silica gel.

The ethanolic extract of the needles was fractionated into petroleum ether, ethyl acetate

(EtOAc), n-butanol (n-BuOH) and water fractions. From petroleum ether fraction


9

protocatechuic acid (53) and from ethyl acetate fraction, taxifolin (19) and myricetin (45)

were isolated and characterized.

The chemical composition of the essential oil obtained by hydrodistillation of woodchips

of C. deodara by GC-MS was investigated for their secondary metabolites. A mixture of

three himachalenes: �-himachalene (1), �-himachalene (2), γ-himachalene (3) were isolated

and characterized from essential oil.

1.1.4.1.1 (E)-�-Atlantone (8)

Compound 8 was isolated as a yellowish gum. Its positive ESI-QTOF-MS showed

molecular ion peak at m/z 219.3411 [M+H]+ (calcd. 219.3425) corresponding to the

molecular formula C15H23O. HPLC chromatogram showed 88.1% purity (tR 8.03 min)

using water:methanol (20:80, v/v) as mobile phase (Figure 1.1.1). UV spectrum showed

absorption maxima at 268 nm. FT-IR spectrum indicated absorption maxima for �,�-

unsaturated ketone (1665, 1614 cm-1

) and olefinic groups (3139, 2979 cm-1

). 1H NMR

spectrum (Table 1.1.2) showed three methine signals at � 5.32 (1H, br s), 5.98 (2H, s)

assignable to H-2, H-8 and H-10 respectively. Six methylene protons resonated at � 1.87-

1.97 (4H, m) and 1.68-1.72 (2H, m) corresponding to carbons at C-1, C-4 and C-5

positions whereas four methyls were observed at � 2.08 (6H), 1.80 (3H) and 1.52 (3H).

OH

31

114

2

79

0.0 2.5 5.0 7.5 10.0 12.5 min

0

500

1000

1500

2000

2500

3000mAU

Figure 1.1.1: Chemical structure of 8 and its HPLC chromatogram

HMBC

31

114

2

79

OH

Figure 1.1.2: Selected HMBC correlations of 8

13C NMR spectrum displayed fifteen signals including one �,�-unsaturated carbonyl at �

191.9 (C-9) and three quaternary carbons at � 153.9 (C-11), 161.6 (C-7) and 133.5 (C-3).

Three tertiary carbon signals at � 120.1, 125.5 and 124.2 were assigned to C-2, C-8 and C-

10 respectively. The long-range interactions were observed in HMBC spectrum between

the protons at �H 5.98 with the carbons resonating at � 191.9 (C-9), 153.9 (C-11) and 161.6

(C-7) suggesting that these carbons are present in the same chain (Figure 1.1.2). The


10

methyl protons resonating at �H 1.52 exhibited long-range interactions with the carbons at �

133.5 (C-3), 30.4 (C-1) and 30.3 (C-4). The HMBC correlations displayed interaction of C7

methyl (�H 2.08) and C-8 methylene (�H 5.98) protons with C-6 carbon (� 44.8). Thus, on

the basis of above spectral data and comparison with earlier reported spectral values

[Shankaranarayan et al. (1977); Pande et al. (1971); Crawford et al. (1972)], the structure

of compound 8 was assigned as (E)-�-atlantone (Figure 1.1.1).

Table 1.1.2: 1H (300 MHz) and 13C NMR (75.4 MHz) data of 8 in CDCl3 Position �C (ppm) �H (ppm) m (J Hz) Position �C �H (ppm)m (J Hz)

1 30.4 1.87-1.97 m 9 191.9 -

2 120.1 5.32 br s 10 124.2 5.98 s

3 133.5 - 11 153.9 -

4 30.3 1.87-1.97 m 11-trans-CH3 27.4 1.80 s

5 27.3 1.68-1.72 m 3-CH3 23.4 1.52 s

6 44.5 2.25-2.28 m 7-CH3 17.4 2.08 s

7 161.6 - 11-cis-CH3 20.8 2.08 s 8 125.5 5.98 s

1.1.4.1.2 (E)-(2S, 3S, 6R)-atlantone-2,3-diol (57)

Compound 57 was obtained as a light brownish gum and displayed a molecular ion peak at

m/z 253.3562 [M+H]+ (calcd. 253.3572) in its HRESI-QTOF-MS, corresponding to the

formula C15H25O3 suggesting 4 degrees of unsaturation. FT-IR spectrum indicated

absorption maxima for hydroxyl (3421 cm-1) and �,�-unsaturated ketone (1660, 1617 cm-1)

functional groups. The UV spectrum showed absorption maxima at �max 296 nm. The

compound showed 92.3% purity (tR 4.83 min) determined by HPLC using

water:acetonitrile (30:70, v/v) as mobile phase (Figure 1.1.3). 1H,

13C and DEPT NMR

spectra revealed 15 carbon signals constituting four methyls, three methylenes, four

methines and four quaternary carbons. The four methyl singlets were found at � 1.24, 1.93,

2.14 and 2.15 corresponding to carbons at � 27.8 (C3-CH3), 27.8 (C11-trans-CH3), 18.1 (C7-

CH3) and 20.9 (C11-cis-CH3), respectively, two olefinic protons at � 6.16 (C-8, C-10) and

one hydroxymethine signal at � 3.59 (C-1) (Table 1.1.3). One carbonyl moiety at � 194.3

was assignable to C-9, the characteristic signal of an �,�-unsaturated ketone moiety. Four

olefinic signals at � 164.1, 125.4, 127.5 and 156.0 revealed the position of two double

bonds at C-7 and C-8; C-10 and C-11. Since the 13C NMR spectral data of 57 was similar

to (E)-�-atlantone, it was assumed to be an atlantone type of sesquiterpene.

Analysis of its COSY data disclosed two proton-proton networks corresponding to H-2, H-

1, H-5, H-6, H-7, H3-7, H-8 and H-10, H-11 and H3-11 (Figure 1.1.4). Long-range proton-

carbon correlations observed in the HMBC spectrum (Figure 1.1.4) provided corroborative

evidence to support these subunits deduced from COSY data. The HMBC spectrum


11

showed correlations of H2-1 (�H 1.99-2.08, 1.61-1.69)/C-2, H3-3 (�H 1.24)/C-2, H2-4 (�

1.73-1.78, 1.46-1.55)/C-2, H1-2 (�H 3.59)/C-3, CH3-3 (�H 1.24)/C-3, and H2-4 (�H 1.73-

1.78, 1.46-1.55)/C-3. The NOESY correlations H-2 (�H 3.59)/H-1 (�H 1.99-2.08); H3-3 (�H

1.24)/H-1 (�H 1.99-2.08); H-5 (�H 1.73-1.78)/H-6 (�H 2.39-2.46); H-1 (�H 1.61-1.69)/H-6

(�H 2.39-2.46) supported a trans configuration between C-2 and C-3 hydroxyl groups

[Werf et al. (1999); Demyttenaere et al. (2001)]. MS2 spectra generated the fragments at

m/z 275 [M+Na]+ due to the sodiated molecular ion peak and the fragments at m/z 235 [M-

H2O]+, 217 [M-2H2O]+ were due to the sequential loss of two 18 (H2O) mass units

confirming presence of two free hydroxyl groups. Thus, on the basis of above evidences

compound 57 was unambiguously characterized as (E)-(2S, 3S, 6R)-atlantone-2,3-diol

(Figure 1.1.3).

O

OHOH

31

8

11 65

4

279

10

0.0 2.5 5.0 7.5 10.0 12.5 min

0

25

50

75

100

mAU

Figure 1.1.3: Structure of compound 57 and its HPLC chromatogram

H-H COSY

HMBC

O

OHOH

Figure 1.1.4: Selected HMBC and COSY correlations of 57

Table 1.1.3: 1H NMR (300 MHz) and 13C NMR (75.4 MHz) data of 57 in CD3OD Position �C (ppm) �H (ppm) m (J Hz) Position �C (ppm) �H (ppm) m (J Hz)

1 34.8 1.99-2.08 m; 1.61-1.69 m 9 194.3 -

2 74.2 3.59 br s 10 127.5 6.16 s

3 71.5 - 11 156.0 -

4 34.2 1.73-1.78 m; 1.46-1.55 m 11-trans-CH3 27.8 1.93 s

5 27.0 1.73-1.78 m; 1.46-1.55 m 3-CH3 27.8 1.24 s 6 42.4 2.39-2.46 m 7-CH3 18.1 2.14 s

7 164.1 - 11-cis-CH3 20.9 2.15 s

8 125.4 6.16 s

1.1.4.1.3 (E)-(2S, 3S, 6S)-atlantone-2,3,6-triol (58)

Compound 58 was obtained as a brownish gum. Its positive HR-ESI-QTOF-MS showed a

molecular ion peak at m/z 269.3581 [M+H]+ (calcd. 269.3566) correspond to the molecular

formula C15H25O4. FT-IR spectrum indicated absorption maxima for a hydroxyl (3404 cm-

1) and �,�-unsaturated ketone (1650, 1615 cm

-1). UV spectrum showed absorption maxima

at �max 293 nm. The purity (95.6%) of the compound (tR 3.84 min) was determined by


12

HPLC using water:ACN (30:70, v/v) as mobile phase (Figure 1.1.5). It possessed

spectroscopic data closely comparable to those of 57 except that proton (� 2.39) at C-6

position in 57 replaced with a hydroxyl group as evident from its 1H NMR data (Table

1.1.4). The presence of OH functional group at C-6 (� 77.0) was further confirmed by the

HMBC correlations H3-7 (�H 2.10)/C-6 and H-8 (�H 6.50)/C-6 (Figure 1.1.6). The NOESY

showed correlations of H-1 (�H 1.55-1.60)/H-5 (�H 1.41-1.49); H3-3 (�H 1.28)/H-1 (�H 1.55-

1.60) and H-5 (�H 2.17-2.18)/H-4 (�H 1.98-2.02).

31

8

11

6

54

279

10

O

OHOH

OH

0.0 2.5 5.0 7.5 10.0 12.5 min

0

250

500

750mAU

Figure 1.1.5: Structure of compound 58 and its HPLC chromatogram

MS2 spectra generated the fragments at m/z 291 [M+Na]+ due to the sodiated molecular

ion peak and the fragments at m/z 251, 233, 215 were due to the sequential loss of three 18

(H2O) mass units confirming presence of three free hydroxyl groups. On the basis of 1H-1H

COSY, HMBC and NOESY correlations the structure of 58 was deduced as (E)-(2S, 3S,

6S)-atlantone-2,3,6-triol (Figure 1.1.5) [Thappa et al. (1976); Kozma et al. (2004)].

H-H COSY

HMBC

O

HOOH

OH

Figure 1.1.6: Selected HMBC and COSY correlations of 58


1 36.4 2.25-2.31 m; 1.55-1.60 m 9 195.0 -

2 75.2 3.56 br s 10 127.5 6.20 s

3 71.6 - 11 156.7 -

4 30.1 1.98-2.02 m; 1.41-1.49 m 11-trans-CH3 27.8 1.93 s

5 31.6 2.17-2.18 m; 1.41-1.49 m 3-CH3 27.3 1.28 s 6 77.0 - 7-CH3 15.6 2.10 s

7 162.3 - 11-cis-CH3 20.9 2.15 s

8 124.7 6.50 s

1.1.4.1.4 Atlantolone (15)

Compound 15 was isolated as brownish oil. Its positive ESI-QTOF-MS showed molecular

ion peak at m/z 237.3563 [M+H]+ (calcd. 237.3578) corresponding to the molecular

formula C15H25O2. UV spectrum showed absorption maxima at 241 nm. FT-IR spectrum

indicated absorption maxima for hydroxyl (3435 cm-1

) and �,�-unsaturated ketone (1664,


13

1604 cm-1

) and olefinic (2914 cm-1

) functional groups. The purity (85.6%) of the

compound (tR 3.37 min) was determined by HPLC using water:ACN (20:80, v/v) as mobile

phase (Figure 1.1.7). 1H and 13C NMR spectra revealed fifteen carbon signals and

constituted four methyls, four methylenes, three methines and four quaternary carbons as

evident from DEPT spectra. The four methyl singlets were found to be at � 2.14, 1.64, 1.26

corresponding to carbons at � 18.1 (C7-CH3), 23.5 (C3-CH3), 29.5 (C11-trans-CH3, C11-cis-

CH3), respectively, two olefinic protons at � 5.39 (C-2, 1H, br s) and � 6.01 (C-8, 1H, s)

(Table 1.1.5).

OHO H

31

811 6

54

2

7910

0.0 2.5 5.0 7.5 10.0 12.5 min

0

25

50

75

mAU

Figure 1.1.7: Chemical structure of compound 15 and its HPLC chromatogram

HMBC

OHO

31

11 5

2

79

Figure 1.1.8: Selected HMBC correlations of 15

Table 1.1.5: 1H NMR (300 MHz) and

13C NMR (75.4 MHz) data of atlantolone in CDCl3

Position �C (ppm) �H (ppm) m (J Hz) Position �C (ppm) �H (ppm) m (J Hz)

1 30.4 1.98-2.04 m 9 202.9 -

2 120.0 5.39 br s 10 54.4 2.58-2.60 m

3 134.0 - 11 70.1 -

4 30.3 1.98-2.04 m 11-trans-CH3 29.5 1.26 s

5 27.5 1.75-1.84 m 3-CH3 23.5 1.64 s

6 44.7 2.19-2.23 m 7-CH3 18.1 2.14 s

7 164.8 - 11-cis-CH3 29.5 1.26 s

8 122.7 6.01 s

One carbonyl moiety at � 202.9 was assignable to C-9, the characteristic signal of an �,�-

unsaturated ketone moiety. Four olefinic signals at � 120.0, 134.0, 164.8 and 122.7

revealed the position of two double bonds at C-2 and C-3; C-7 and C-8. The presence of

OH functional group at C-11 (� 70.1) was confirmed by the HMBC correlations H3-3 (�H

1.26)/C-11, H2-10 (�H 2.58-2.60)/C-11 (Figure 1.1.8). The HMBC spectrum showed

correlations of H3-3 (�H 1.64) to C-1, C-2, C-3; H3-7 (�H 2.14) to C-6, C-8 and H2-5 (�H

1.75-1.84) to C-1, C-4, C-7. MS2 spectra generated the fragments at m/z 259 [M+Na]

+ due


14

to the sodiated molecular ion peak and the fragment at m/z 219 was due to the loss of one

18 (H2O) mass units confirming presence of one free hydroxyl groups. Thus, on the basis

of above spectral data and comparison with previously known spectral values

[Shankaranarayan et al. (1977)], the structure of compound 15 was assigned as atlantolone

(Figure 1.1.7).

1.1.4.1.5 Protocatechuic acid (53)

COOH

OH

OH

126

4

O

OH

HO

OH

O

OH

OH

O

OH

HO

OH

OH

O

OH5

8

4a

4'

OH

2

53 19 45

Figure 1.1.9: Chemical structures of compounds 53, 19 and 45

Compound 53 was isolated as a white amorphous powder. Its positive ESI-QTOF-MS

showed molecular ion peak at m/z 319.2411 [M+H]+ (calcd. 319.2430) corresponding to

the molecular formula C7H7O4. The FT-IR spectrum showed the absorption bands at 3456,

1680, 1544, 1167 cm-1

suggesting the existence of hydroxyl, carbonyl, C=C and ether

functionalities, respectively.

Table 1.1.6: 1H NMR (300 MHz) and 13C NMR (75.4 MHz) data of 53 in CD3OD Position �C (ppm) �H (ppm) m (J Hz)

1 122.0 -

2 116.7 7.46 s

3 145.0 -

4 150.5 -

5 114.7 6.81 d (8.6)

6 122.9 7.43 d (8.1)

COOH 169.2 -

1H NMR spectrum of this compound (Table 1.1.6) showed signals at � 7.46 (1H, s), 7.43

(1H, d, J = 8.1 Hz), and 6.81 (1H, d, J = 8.6 Hz) indicating the presence of a 1,3,4-

trisubstituted benzene ring. The 13C NMR spectrum showed the presence of seven carbons

with a signal at � 169.2 assignable to carboxyl carbon (COOH). The four signals at � 122.9,

122.0, 116.7, and 114.7 were assigned to C-6, C-1, C-2, and C-5 carbons respectively, by

HMQC and HMBC correlations. The signals at � 145.0 and 150.5 were assigned to C-3 and

C-4 aromatic carbons bearing hydroxyl groups. MS2 spectra generated the fragments at m/z

155 [M+H]+ due to the protonated molecular ion peak and the fragments at m/z 137

[M+H-H2O]+ and 111 [M+H-CO2]+, showed presence of hydroxyl and acidic groups. Thus,

compound 53 was identified as 3,4-dihydroxybenzoic acid (protocatechuic acid) (Figure

1.1.9) on the basis of spectral data that matched with the reported values [He et al. (2009)].


15

1.1.4.1.6 Taxifolin (19)

Compound 19 was isolated as a yellow amorphous powder. Its positive ESI-QTOF-MS

showed molecular ion at m/z 305.2579 [M+H]+ (calcd. 305.2595) corresponding to the

molecular formula C15H13O7. FT-IR spectrum showed the absorption bands at 3566, 1663,

1588, 1128 cm-1

suggesting the existence of hydroxyl, carbonyl, C=C and ether

functionalities, respectively. The 1H NMR spectrum gave signals (Table 1.1.7) due to C-2

and C-3 protons at � 4.94 (1H, m), 4.52 (1H, d, J = 11.2 Hz). The coupling constant and �

value indicated that C-2 and C-3 protons were of trans-type and of (2R, 3R) configuration

[Sakushima et al. (2002)]. The signals at � 6.98 (1H, br s), 6.83 (1H, m), and 6.80 (1H, m)

indicated the presence of a 1,3,4-trisubstituted benzene ring. 13

C NMR spectrum displayed

fifteen signals including one carbonyl at � 198.0 (C-4) and eight oxygen bearing quaternary

carbons at � 169.1 (C-5), 165.7 (C-7, C-8a), 147.5 (C-3'), 146.7 (C-4'), 85.5 (C-2), 74.1 (C-

3). Five methine signals at � 121.3, 116.5, 116.2, 97.7 and 96.7 were assigned to C-6', C-5'

and C-2', C-6 and C-8 carbons respectively. Thus, 19 was identified as (2R, 3R)-Taxifolin

(Figure 1.1.9) and spectral data of 19 matched with the previous literature [Han et al.

(2007)].

Table 1.1.7: 1H NMR (300 MHz) and

13C NMR (75.4 MHz) data of 19 in CD3OD


1 - 8a 165.7 -

2 85.5 4.94 m 4a 102.3 -

3 74.1 4.52 d (11.2) 1' 130.3 -

4 198.0 - 2' 116.2 6.98 br s

5 169.1 - 3' 147.5

6 97.7 5.94 br s 4' 146.7

7 165.7 - 5' 116.5 6.80 m

8 96.7 5.90 br s 6' 121.3 6.83 m

1.1.4.1.7 Myricetin (45)

Compound 45 was isolated as a yellow amorphous powder. Its positive ESI-QTOF-MS

showed molecular ion at m/z 319.2411 [M+H]+ (calcd. 319.2430) corresponding to the

molecular formula C15H10O8. UV spectrum showed absorption maxima at 296 and 375 nm

characteristic of flavonols. FT-IR spectrum showed the absorption bands at 3556, 1675 cm-

1 suggesting the existence of hydroxyl and carbonyl groups, respectively. The IR spectrum

also exhibited absorptions at 1590 and 1025 cm-1 representing C=C and ether

functionalities, respectively. 1H NMR spectrum (Table 1.1.8) showed aromatic signals at �


16

7.35 (2H, br s), 6.39 (1H, br s), 6.19 (1H, br s) assignable to H-2', H-6', H-8, and H-6

respectively. 13C NMR spectrum displayed fifteen signals including one flavonol carbonyl

at � 177.4 (C-4) and eight oxygen bearing quaternary carbons at � 165.7 (C-7), 162.6 (C-5),

158.3 (C-8a), 148.1 (C-2), 146.8 (C-3', C-5'), 137.5 (C-4'), 137.1 (C-3). Four methine

signals at � 108.7, 99.3 and 94.5 were assigned to C-2', C-6', C-6 and C-8 respectively.

Thus, on the basis of above spectral data and comparison with previously known spectral

values [He et al. (2009)], the structure of compound 45 was assigned as myricetin (Figure

1.1.9).


1 - - 8a 158.3 -

2 148.1 - 4a 104.6 -

3 137.1 - 1' 123.2 -

4 177.4 - 2' 108.7 7.35 s

5 162.6 - 3' 146.8 -

6 99.3 6.19 br s 4' 137.5 -

7 165.7 - 5' 146.8 -

8 94.5 6.39 brs s 6' 108.7 7.35 s

1.1.4.1.8 �-, �- and �-Himachalene (1-3)

1 2 3

Figure 1.1.10: Chemical structures of mixture of three himachalenes (1-3)

Compound (1-3) was isolated as colorless oily liquid. The GC-MS showed three peaks at

m/z 204 corresponding to the molecular formula C15H24. 1H,

13C NMR, IR and GC-MS

showed a mixture of three compounds identified as �- (1), �- (2) and �-himachalene (3)

(Figure 1.1.10). 1H,

13C NMR and MS values are detailed in section 1.4.1.1.3.3 and were

compared with literature values [Bisarya and Dev (1968); Daoubi et al. (2005)].

1.1.4.2 Chemical composition of hydrodistilled and solvent volatiles extracted from

woodchips of C. deodara

The yields of wood essential oils and extracts from the hydrodistillation and percolation of

woodchips of C. deodara were 1.0% and 14.5% on dry weight basis, respectively. GC and

GC-MS analyses resulted in the identification of thirty four and twenty six constituents in

oil and extract of woodchips of C. deodara, respectively. Table 1.1.9 indicated the


17

constituents identified, their percentage composition and Kovats index (KI) values listed in

order of elution from the BP-20 capillary column. The identified constituents identified

were 26.7-79.5% sesquiterpene hydrocarbons and 18.9-67.9% oxygenated sesquiterpenes.

A total of twenty sesquiterpene hydrocarbons and nineteen oxygenated sesquiterpenes were

observed in the essential oil and extract; major constituents being himachalenes (23.5-

68.5%) and atlantones (15.0-61.6%). The other constituents identified were himachalene

oxide, himachalol, oxidohimachalene, dehydro-ar-himachalene and cis-�-bisabolene. C.

libani wood extract was reported to be rich in himachalenes (~42%) and needle oil in �-

pinene (~24%) and carophyllene (~7%) [Fleisher (2000)]. Himachalenes (68.5%),

atlantones (15.0%), himachalol (1.0%) present in the oil were reported in various

publications variation in their percentage composition [Nigam et al. (1990); Fleisher

(2000)]. A significantly higher percentage of atlantones (~67%) was observed in the C.

deodara extract as compared to other species whereas these constituted less than 10% of

the total oil. It may be pointed that the essential oil or extract content may differ greatly in

the same genus or different organs of same species [Salido et al. (2002); Cavaleiro et al.

(2002); Duquesnoy et al. (2006)]. Some earlier reported major constituents (cedrene and

cedrol) were not detected in this study [Nigam et al. (1990)].

Table 1.1.9: Chemical composition C. deodara woodchips essential oil and extract

Compounds KI Oil % Extract % Compounds KI Oil % Extract %

Longifolene 1517 0.6 - �-Dehydro-ar-himachalene 1849 0.7 0.3

Aromadendrene 1560 0.6 - trans-�-Bergamotene 1873 - 0.6

allo-Aromadendrene 1579 0.3 - Vestitenone 1883 0.4 1.6

� –Himachalene 1593 17.1 4.7 cis-�-Bergamotene 1890 - 0.6

� –Humulene 1633 0.5 - Oxidohimachalene 1931 0.3 0.2

Z-�-Farnesene 1637 0.4 - �-Himachaleneoxide 1965 0.3 -

�-Himachalene 1646 12.6 3.3 Caryophyllene oxide 1993 0.4 0.5

Cubinene 1649 2.3 0.6 �-Bisabolol 2036 - 0.2

�-Himachalene 1666 38.8 15.5 Longiborneol 2099 0.2 0.2

�-Cadinene 1672 0.4 - �-Atlantone 2127 0.4 0.8

8-Cedren-13-ol-acetate 1675 0.4 0.2 (Z)-�-Atlantone 2152 2.3 7.8

�-Cadinene 1677 0.7 0.2 Himachalol 2158 1.0 1.5

(E),(E)-Farnesol 1680 0.2 - (E)-�-Atlantone 2173 2.4 10.2

Albicanol 1685 0.2 - Deodarone 2181 0.3 0.8

4,5-Dehydroisolongifolene 1690 0.2 - Deodarone isomer 2185 0.3 0.9

�-Vativenene 1694 0.2 - (Z)-�-Atlantone 2200 1.4 4.3

cis-�-Bisabolene 1734 2.2 0.7 Aristolone 2265 - 0.1

ar-Curcumene 1773 0.9 - (E)-�-Atlantone 2278 8.6 38.5

�-Dehydro-ar-himachalene 1811 0.7 0.2 14-Oxy-�-muurolene 2292 - 0.2

9,10-Dehydroisolongifolene 1818 0.3 - Total identification 98.3 94.6


18

1.1.4.3 Chemical constituents of essential oil fractions

Total forty compounds were identified using GC and GC-MS analyses from n-pentane and

acetonitrile fractions. The identified constituents, percentage composition and their Kovats

index (KI) values are shown in Table 1.1.10. In the n-pentane fraction, twenty seven

compounds were identified representing 90.89% of the total constituents detected. A total

of eleven sesquiterpene hydrocarbons and sixteen oxygenated sesquiterpenes were

identified constituting 59.68 and 31.21%, respectively.

Table 1.1.10: Chemical constituents of fractions of C. deodara essential oil Compounds KI A5 (%) A6 (%) A4 (%) A3 (%)

4-Acetyl-1-methylcyclohexene 1499 - 2.10 - -

Longifolene 1503 0.91 - - -

Aromadendrene 1545 0.25 - - -

�-Himachalene 1579 13.29 2.56 20.33 -

�-Humulene 1617 0.48 - - - �-Himachalene 1627 11.28 2.29 17.95 -

�-Curcumene 1629 - 0.40 - -

�-Himachalene 1651 27.78 6.85 52.61 -

3-Cyclohexene-1-methanol 1660 - 0.33 - -

8-Cedren-13-ol-acetate 1677 0.21 - - -

�-Vativenene 1702 0.50 - - -

cis-�-Bisabolene 1715 1.58 0.36 - -

3-Methylacetophenone 1716 - 1.30 - -

4,5-Dehydroisolongifolene 1755 0.23 - - -

�-Dehydro-ar-himachalene 1791 1.94 0.96 - -

�-Dehydro-ar-himachalene 1826 1.44 1.02 - - Vestitenone 1860 0.55 3.00 - -

�-Himachalene oxide 1923 9.12 1.17 - -

Calarene epoxide 1969 0.83 1.74 - -

Nerolidol 1977 - 0.55 - -

Carophyllene oxide 2002 0.25 0.50 - -

(+)-8(15)-Cedren-9-ol 2006 - 0.47 - -

Aromadendrene oxide 2009 0.96 - - -

Longiborneol 2070 0.58 2.01 - -

�-Bisabolol 2077 0.22 0.48 - -

�-Atlantone 2097 0.94 2.71 - 1.95

(Z)-�-Atlantone 2122 0.79 8.63 - 11.38 Himachalol 2129 2.04 4.51 - -

m-Tolyldimethylactealdehyde 2137 - 0.45 - -

(E)-�-Atlantone 2143 0.87 8.83 - 15.70

Deodarone 2152 0.53 4.18 - -

Deodarone isomer 2156 - 3.70 - -

Humulane-1,6- dien-3-ol 2161 - 4.37 - -

(Z)-�-Atlantone 2172 3.40 5.23 - 4.99

(E)-�-Atlantone 2248 9.45 16.00 - 61.82

Longifolenaldehyde 2303 0.47 - - -

2-Butyl-1-methyl-1,2,3,4-tetrahydro-naphthalen-

1-ol

2345 - 1.09 - -

8-�-Acetoxyelemol 2349 - 0.47 - - 7�,3�-Dihydroxy-1�-2,6-cyclohimachalane 2401 - 0.36 - -

14-Hydroxy-9-epi-(E)-caryophyllene 2412 - 0.62 - -

Total identification 90.89 89.24 90.89 95.74 A5: n-pentane fraction; A4: himachalenes; A3: atlantones; A6: acetonitrile fraction; A2: crude oil

In the acetonitrile fraction, thirty one compounds were identified representing 89.24% of

the constituents of oxygenated monoterpene (3.73%), sesquiterpene hydrocarbon (14.44%),


19

and oxygenated sesquiterpene (71.07%) types. The major constituents in n-pentane fraction

were himachalenes (52.35%) while in acetonitrile fraction were atlantones (41.40%). The

other constituents identified in these fractions were himachalene oxide (1.17-9.12%),

himachalol (2.04-4.51%), �-dehydro-ar-himachalene (0.96-1.94%), cis-�-bisabolene (0.36-

1.58%) and �-dehydro-ar-himachalene (1.02-1.44%). Further chromatography of n-pentane

and acetonitrile fractions led to the increase in percentage of himachalenes and atlantones

to 90.89 and 95.74%, respectively. The results of the present study are indicative of the

utilization of oil, major constituents and extract of C. deodara for further modifications.

1.1.4.4 Determination of major flavonoids by UPLC-MS in C. deodara needles extract

A simple, sensitive, selective, precise and robust ultra-performance liquid

chromatography–tandem mass spectrometry (UPLC-MS) method was developed and

validated for determination of four flavonoids, taxifolin, quercitrin, myricetin, quercetin in

needles of C. deodara. All the four flavonoids were detected and determined in the needles

extracts. The chromatographic separation of four flavonoids was achieved in less than 8

min by UPLC® BEH C18 column (100 × 2.1 mm i.d., 1.7 �m) using linear gradient elution

of water (0.05 % formic acid) and acetonitrile with flow rate of 0.3 ml/min at � 254 nm.

The different extraction techniques such as soxhlet, maceration, ultrasound-assisted

extraction (UAE) and microwave-assisted extraction (MAE) were applied and compared to

bring out the best extraction procedure for flavonoids with methanol. In soxhlet extraction,

maximum yield of flavonoids was obtained. Soxhlet and microwave were found to show

comparable results, but taking into consideration the solvent and time consumption for

extraction, MAE was found to be the best approach for the rapid and efficient extraction of

flavonoids.

R1 R2 R3

OH OH H Myricetin

OH H rha Quercitrin

OH H H Quercetin

Taxifolin

Figure 1.2.14: Chemical structures of myricetin, quercitrin, quercetin and taxifolin

In UPLC analysis, various mobile phase compositions and chromatographic conditions

were tried to find the optimal chromatographic conditions. For optimization of

chromatographic conditions, good resolution was recorded with acetonitrile-water-formic

acid system for all active components than with methanol-water or acetonitrile-water

system. Water with formic acid (0.05%) (solvent A) and ACN (solvent B) was used as

O

OH

HO

OH

OR3

O

R1

R2 O

OH

HO

OH

O

OH

OH


20

mobile phase for the analysis with a linear gradient elution as follows: 0-2.5 min, 15% B;

2.5-4.0 min, 15-30% B; 4.0-5.5 min, 30% B, 5.5-6.0 min, 30-15% B, 6.0-8.0 min, 15% B.

The peak resolution was also recorded with variation in the column temperature. Column

temperature was optimized systematically from 25 to 40°C, and it was observed that all the

components achieved a baseline resolution at 35°C. All parameters like optimal separation,

high sensitivity and good peak shape without tailing of the analytes were standardized.

Optimal chromatographic conditions were obtained after running different mobile phases

with a reversed-phase C18 column (BEH C180, 100 × 2.1 mm i.d., 1.7 �m). Four

flavonoids, viz. taxifolin (RT: 1.36 min), quercitrin (RT: 4.79 min), myricetin (RT: 5.11

min), and quercetin (RT: 6.14 min) were well resolved. The representative chromatograms

of the standard mixture and sample of C. deodara have been shown in Figures 1.1.11 and

1.1.12. The chromatographic peaks were identified and confirmed by comparing their

retention times with reference compounds, overlaying of UV spectra with those of

reference compounds and spiking of samples with the reference compounds. The results

indicated that flavonoids were well resolved and their quantitative determination in C.

deodara was possible. Taxifolin (1.16-1.92%) and myricetin (0.54-1.25%) were detected in

major amount in the extract as compared to quercitrin (0.19-0.29%) and quercetin (0.17-

0.27%) (Table 1.1.14).

The calibration curves were linear in the range of 1.56-100 �g/ml for taxifolin, quercitrin,

myricetin, and quercetin. Regression equation and coefficient of correlation (r2 = 0.9952-

0.9962) revealed a good linearity response for developed method and are presented in

Table 10. The LODs for taxifolin, quercitrin, myricetin, and quercetin were 0.20, 0.10,

0.78, 0.39 and LOQs for same analytes were found to be 0.66, 0.32, 2.34, and 1.25 �g/ml,

respectively (Table 1.1.11). This indicated that the proposed method exhibited a good

sensitivity for the quantification of flavonoids. The intra- and inter-day precisions

(expressed in terms of %RSD) were observed in the range of 0.21-0.70% and 0.42-0.51%

respectively, demonstrating the good precision of the proposed method (Table 1.1.12).

Accuracy of the proposed method was expressed as the recovery of standard compounds

added to the pre-analyzed sample. Samples spiked with 150, 75, 37.5 �g/ml of taxifoiln,

50, 25, 12.5 �g/ml of quercitrin, quercetin and 100, 50, 25 �g/ml of myricetin, were used in

triplicate to assess accuracy. The amount of compounds was calculated from related linear

regression equation. The percentage recovery ranged from 95.99 to 99.86% with RSD

values in the range 0.11-1.53%, for the detected compounds (Table 1.1.13).


21

a

b

c

d

Figure 1.1.11: UPLC chromatogram of standard mixture of flavonoids; a = taxifoiln; b =

quercitrin; c = myricetin; d = quercetin

a

dbc

Figure 1.1.12: UPLC chromatogram of extract C. deodara needles; a = taxifoiln; b =

quercitrin; c = myricetin; d = quercetin

Table 1.1.11: Method validation data of four compounds in C. deodara needles

Analytes rt Regression

equation Linearity range

(�g/ml) r

2

LOD (�g/ml)

LOQ (�g/ml)

Taxifoiln 1.36 121.15x+181.84 1.56-100 0.9953 0.20 0.66

Quercitrin 4.79 177.47x+244.36 1.56-100 0.9952 0.10 0.32

Myricetin 5.11 119.23x+4.646 1.56-100 0.9957 0.78 2.34

Quercetin 6.14 138.82x+142.9 1.56-100 0.9962 0.39 1.25

The UV spectra suggested that the detected flavonoids present in the extracts were

flavanols and dihydroflavonols. The peaks showed characteristic absorption bands at 346-

371 nm (band A) and 251-265 nm (band B), indicating that the flvonols in extracts were

substituted in the 3-OH position. In dihydroflavonols, band A was reduced to little more

than a shoulder at 327 nm and band B, at 288 nm, detected as main peak.

Table 1.1.12: Inter-, intra-day precision of detected compounds in needles of C. deodara

Analytes Intra-day Precision (n=3) Inter-day Precision (n=9)

Day 1 Day 2 Day 3

RT* PA** RT* PA** RT* PA** RT* PA**

Taxifoiln 0.42 0.58 0.43 0.70 0.86 0.40 0.75 0.51

Quercitrin 0.12 0.50 0.12 0.55 0.12 0.44 0.13 0.49

Myricetin 0.11 0.59 0.11 0.40 0.11 0.45 0.13 0.42

Quercetin 0.09 0.50 0.09 0.21 0.09 0.21 0.09 0.43

RT*, %RSD of retention time. PA**, %RSD of peak area.

ESI-QTOF-MS/MS (in positive ion mode) of the constituents showed characteristic

distribution of fragment ions as illustrated in Figure 1.1.13. The fragmentation patterns

observed in the mass spectrum were useful in characterization of the compounds.


22

Dihydroflavonols (taxifolin) exhibited an initial dehydration of [M+H]+

to [M+H-H2O]+

(m/z 287) and sequential losses of two carbonyl groups to form [M+H-H2O-CO]+ (m/z 259)

and [M+H-H2O-2CO]+ (m/z 231) (Table 1.1.15). Additional fragments were the 1,3A+ (m/z

153), [M+H-B ring]+

(m/z 159), 0,2

A+ (m/z 165) and

1,4B

+ (m/z 179) [Tsimogiannis et al.

(2007)]. Flavonol (quercetin) [M+H]+ product ions underwent dehydration, followed by

two sequential losses of CO: [M+H-H2O-CO]+ (m/z 257) and [M+H-H2O-2CO]+ (m/z

229). Furthermore the C-ring cleavage at bonds 0,2 and 1,3 produced the respective RDA

fragments 0,2B+ (m/z 137) and 1,3A+ (m/z 153) of the protonated molecule.

Table 1.1.13: Accuracy for the quantitative determination of four compounds C. deodara

needles

Added

amount (�g/ml)

Average

Recovery (%) RSD (%)

Taxifoiln 150 99.22 1.27

75 97.92 0.55

37.5 97.41 0.93

Quercitrin 50 99.65 0.66

25 96.34 0.11

12.5 98.76 0.61

Myricetin 100 99.86 0.05

50 99.22 0.97

25 98.86 1.53

Quercetin 50 99.22 0.87

25 97.35 0.13

12.5 95.99 0.13

Table 1.1.14: Quantitative determination of four compounds in the extract of C. deodara

by different extraction techniques

rt

Ultrasonication

(%w/w)

Microwave

(%w/w)

Soxhlet

(%w/w)

Maceration

(%w/w)

Taxifoiln 1.36 1.52 1.80 1.92 1.16

Quercitrin 4.79 0.24 0.26 0.29 0.19

Myricetin 5.11 0.87 0.93 1.25 0.54

Quercetin 6.14 0.21 0.23 0.27 0.17

Table 1.1.15: Identification of major constituents in methanolic extracts of C. deodara

needles

Peak tR

(min)

UV

spectra

Calcd.

MW

(+) ion mode Detected

Compounds [M+H]+ MS/MS (m/z)

a 1.34 282,

327sh 304 305

305, 287, 259, 231, 195, 179, 165, 153, 123, 108

Taxifolin

b 4.88 265, 346 448 449

303, 285, 257, 247, 229,

219, 201, 183, 165, 153,

137, 109, 81, 69

Quercitrin

c 5.11 251, 371 318 319 290, 245, 179, 153, 123,

79 Myricetin

d 6.15 253, 368 302 303 257, 229, 201, 183, 153,

137, 111, 95, 69 Quercetin


23

O

OH

HO

OH

O

OH

OH

O

OH

HO

O

O

[M+H-B ring]+ = 195

O

O

O

OH

1,4B+ = 179

O

OH

HO

O

1,3A+ = 153

OH

HO O

O

0,2A+ = 165

[M+H]+ = 305

0

3

2

4

A

B

C

I

O

OH

HO

OH

O

OH

OH

0,2B+ = 137

1,3A+ = 153

0

3

2

4

[M+H]+ = 303

A C

B

II

Figure 1.1.13: Fragmentation pattern of (I) taxifolin and (II) quercetin


Albizzia chinensis (Osbek) Merril

1.2.1 Introduction

Albizia or Albizzia is a pantropical genus

(subfamily Mimosoideae) with about 150

species of which 35 occur in Asia, 48 in

Africa and 35 in tropical and subtropical

America [Nielson (1981)]. Most species of

the genus have high biomass production, a

spreading crown and light feathery foliage

in addition to nodulation and nitrogen

fixing capabilities [Allen and Allen (1981)] and consequently are good shade trees for tea

and coffee plantations. These are deciduous woody trees and shrubs. They are easily

identified by their bipinnately compound leaves. Several Albizzia species are planted as

ornamentals or as a source of tannin extracts and are socially significant for producing high

quality timber and as a valuable resource for gum yield. A. julibrissin, A. lebbeck, A.

procera and A. amara are important in ayruvedic medicine. Seeds are regarded as

astringent, and used in the treatment of piles, diarrhea and gonorrhea [Anonymous (1989)].

Many Albizia species such as A. chinensis, A. amara, A. lebbeck, A. odoratissima, A.


24

procera, A. stipulata, A. thomsonii, A. kalkora, A. lucida and A. orissensis are endemic to

Indian subcontinent.

Albizzia chinensis (Osbek) Merr., belonging to the family Leguminosae, is a large

deciduous tree with feathery foliage and large stipules. It occurs naturally in India,

Myanmar, Thailand, Indo-China, China, Java and the Lesser Sunda Islands (Bali and Nusa

Tenggara). It is a native of mixed deciduous forest in humid tropical and subtropical

monsoon climates with annual rainfall varying from 1000-5000 mm. In India, it occurs

chiefly in moist localities throughout the sub Himalayan tract and valleys up to an

elevation of about 1200 m from Himachal Pradesh eastwards, through Uttaranchal and also

in West Bengal, Assam, Andaman and Nicobar islands. It is excellent for restoration of

degraded lands, produces fuel and small timber, its leaves form excellent fodder for cattle,

and it also serves as the suitable host for Lac insect. The tree is extensively cultivated in tea

gardens for shade and improving the fertility of soil. It is also used for box making

especially tea boxes and heavy packing cases. The tree exudes a gum from the stem, which

is used sometimes for sizing hand made paper. Due to multifarious nature of this species, it

has been overexploited throughout the hills for fuel, fodder and timber requirements

[Dhanari et al. (2003)]. The anticancer and antioxidant activities of the plant were

attributed due to the presnebce of saponinas and flavonoids [Liu et al. (2009)].


Many important classes of compounds i.e. saponins, alkaloids and polyphenolic

compounds were reported from Albizzia species. Most of the biological activities of this

plant have been attributed to the presence of saponins, alkaloids and flavonoids. However,

only few saponins and flavonoids were identified from the less explored species i.e. A.

chinensis.

1.2.2.1 Saponins

Five oleanane-type triterpene saponins, albizosides A-E (59-63) were isolated from the

stem bark of A. chinensis [Liu et al. (2009a); Liu et al. (2010)]. The saponins of julibroside

type were isolated from stem bark of other Albizzia sps. and were identified as julibroside

J26 (64), julibroside J1 (65) [Zou et al. (1999)], julibroside J5 (66), julibroside J8 (67),

julibroside J12 (68), julibroside J13 (69) [Zou et al. (2005)], julibroside J28 (70), julibroside

III (71), julibroside J14 (72) [Liang et al. (2005)], julibroside J29 (73), julibroside J30 (74),

julibroside J31 (75) [Zheng et al. (2006)], julibroside II (76) julibroside J16 (77), julibroside

J21 (78) and julibroside J17 (79) [Zou et al. (2010)].


25

OOH

OH

R1

OO

OH

O

OR2

OHO

OH

O

OH

OH

OH

O

O

OH

O

O

OH

HO

OO

OHO

Me

OH

O

HO

OH

O

OH

OH

OH

HO

O

O

O

OH

OH

Me

O

O

H

OO

O

OHOR3

OH

Me

O

O

O

OHOH

OH

Me

OH

=S2

R1 R2 R3

Me H S2 (59)

Me Glc2 S2 (60)

H Glc2 H (61)

OOH

OH

R1

OO

OH

O

OR2

OHO

OH

O

OH

OH

OH

O

O

OH

O

O

OH

HO

OO

OHO

Me

OH

O

HO

OH

O

OH

OH

OH

HO

O

O

OH

OH

Me

O

O

H

OO

O

OHOR3

OH

Me

O

O

O

O

OHOH

OH

Me

OH

MT-Q

R1 R2 R3 Me Glc2 H (62)

H H MT-Q (63)


26

OOH

OH

OO

OH

O

OH

OHO

OH

O

OH

OH

R1

O

O

O

OH

O

O

OH

HO

OO

OHO

Me

OH

O

HO

OH

O

OH

OH

OH

HO

O

OR2

O

OH

OH

Me

OH

O

O

OH

O

OH

OH

Me

=S

R1 R2

H H (64)

OH S (65)

OOH

OH

Me

OO

OH

O

R

OH

O

OH

O

OH

OH

OH

O

O

O

OH

O

O

OH

HO

OO

OHO

Me

OH

O

HO

OH

O

OH

OH

OH

HO

O

O

O

OH

OH

Me

O

O

OH

O

OH

OH

Me

OH

6

R C(6)

OH (R) (66)

OH (S) (67)

NHAc (R) (68)

NHAc (S) (69)


27

OOH

OH

R1

OO

OH

O

R2

OH

O

OH

O

OH

OH

OH

O

O

O

OH

O

O

OH

HO

OO

OHO

Me

OH

O

HO

OH

O

OH

OH

OH

HO

O

O

O

OH

OH

Me

O

O

OH

O

OH

OH

Me

6

R1 R2 C(6) CH3 NHAc (R) (70)

CH3 NHAc (S) (71) H OH (R) (72)

.

OOH

OH

Me

OO

OH

O

R1

OH

O

OH

O

OH

OH

OH

O

O

O

OH

O

O

OH

HO

OO

OHO

Me

OH

O

HO

OH

O

OH

OH

OH

HO

O

OHOH

OH

R2

O

H

R3

R1 R2

R3

NHAc Me OH (73) NHAc H OH (74)

O-glc Me OH (75)


28

OH

OHO

HO OH

O

O

OH

HOO

H

H

R1

OHO

HOOH

OO

HOOH

O

O

R2

O

O

OOHO

HO OH

R3

O

O

O

OH

OHO

HO

O

O

OH

OO

HOHO

OH

OH

OH

OHO

HO

OH

6

R1 R

2 R

3 C(6)

Me Me Me (S) (76)

Me Me Me (R) (77)

H HO-CH2 H (R) (78)

OH

OHO

HO OH

O

O

OH

HOO

H

H

OHO

HOOH

OHOO

OH

O

O

O

O

OOHO

HO OH

O

O

O

OH

OHO

HO

O

O

OH

OO

HOHO

OH

OH

OH

OHO

HO

OH

OH

(79)

1.2.2.2 Polyphenols

Recently, eight compounds were isolated from 95% ethanol and methanolic extracts of A.

chinensis leaves and their structures were elucidated as quercetin (25), kaempferol (80),

luteolin (81), quercetin-di-glycoside (82), quercetin-3-O-�-L-rhamnopyranoside (83), rutin

(84), luteolin-7-O-�-D-glucopyranoside (85), kaempferol-3,7-di-O-�-D-glucopyranoside

(86), kaempferol-3-O-�-L-rhamnopyranoside (87), (+)-lyoniresinol-3�-O-�-D-

glucopyranoside (88), (-)-lyoniresinol-3�-O-�-D-glucopyranoside (89) [Liu et al. (2009b);

Ghaly et al. (2010)]. The polyphenols identified from other Albizzia sps. were named as

isoquercitrin (90), sulfuretin (91), hyperoside (92) [Kang et al. (2000); Jung et al. (2003)],

albibrissinosides A (93), and B (94) [Jung et al. (2004)].


29

O

O

HO

OH

OH

OH

O

O

HO

OH

OH

OH

(80) (81)

O

OH

HO

O

OH

O

O

O

OH

HO

HO

OHO OH

OH

O

O

OH

HO

OH

HO

O

OH

HO

OH

OH

O

O

O

OH

OH

OH

(82) (83)

O

OH

HO

OH

OH

O

O

O

OH

HO

HO

OHO OH

OH

O

O

OH

O

OH

O

O

HO

OH

HO

OH

OH

(84) (85)

O

OH

O

OH

O

O

OOHHO

HO

OH

O

HO

OH

HO

OH

O

OH

HO

OH

O

O

O

OH

OH

OH

(86) (87)

O

OMe

MeO

MeO

OH

OMe

OH

OGlcHO

O

OMe

MeO

MeO

OH

OMe

OH

OGlcHO

O

OH

HO

OH

O

O

OOHHO

HO

CH2OH

(88) (89) (90)

1.2.2.3 Alkaloids

The CH2Cl2 extract of A. gummifera stem bark yielded four alkaloids, budmunchiamine G

(95), budmunchiamine K (96), 6'�-hydroxybudmunchiamine K (97), and 9-

normethylbudmunchiamine K (98) [Rukunga and Waterman (1996a)] and other

budmunchiamines were characterized from stem bark of A. schimperana [budmunchiamine


30

A (99), 6'�-hydroxybudmunchiamine C (100), 5-normethylbudmunchiamine K (101), 6'�-

hydroxy-5-normethylbudmunchiamine K (102) 14-normethylbudmunchiamine K (9)]

[Rukunga and Waterman (1996b)]. The macrocyclic alkaloids, named as budmunchiamines

L4 (104), L5 (105) and L6 (106), were identified from methanolic extract of the seeds of A.

lebbek [Dixit and Misra (1997); Ovenden et al. (1997)].

Syringin (107) was also isoleted from A. chinensis leaves [Liu et al (2009)]

HO O

O

HO OH

O

OH

HO

OH

OH

O

O

O

HO

OHOH

OH

O

O

OH OH

O

OMe

HO

MeO

OHO

HOO

O

OH OMe

R1

R2

(91) (92) R1 R2

OMe OMe (93)

OH H (94)

N

N

N

N

R3 R1

OHR2

(CH2)5 (CH2)n

R

CH3

12

13

11

15

7

9 51

6'

n R R1 R2 R3

6 H H Me Me (95)

8 H Me Me Me (96) 8 OH Me Me Me (97)

8 H Me Me H (98)

4 H Me Me Me (99)

6 OH Me Me Me (100)

8 H H Me Me (101)

8 OH H Me Me (102)

8 H Me H Me (103)

NH HN

HNNH

O( )n

R

O

O

OO

OH

HO

HO

OHH

HO

n R (107) 5 CH2CH(OH)CH2CH2CH3 (104)

9 CH=CHCH2CH2CH3 (105) 7 CH=CHCH2CH2CH3 (106)

1.2.3 Pharmacology and biological activities

1.2.3.1 Cytotoxicity

Two oleanane-type triterpene saponins albizosides D (62) and E (63) from A. chinensis

were evaluated for cytotoxic activities against five human tumor cell lines i.e. HCT-8


31

(human colon cancer), Bel-7402 (human hepatoma cancer), BGC-823 (human gastric

cancer), A549 (human lung epithelial cancer), and A2780 (human ovarian cancer) using

the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay

(paclitaxel as positive control) and these showed moderate cytotoxicity with IC50 values of

7.70 ± 0.15, 0.70 ± 0.08, 0.08 ± 0.02, 0.30 ± 0.07, 0.90 ± 0.05 �M for albizosides D and

>10, 0.60 ± 0.03, 0.30 ± 0.04, 1.20 ± 0.14, 0.30 ± 0.09 �M for albizosides E, respectively

[Liu et al. (2010)]. Three saponins, albizosides A-C (59-61) from A. chinensis exhibited

cytotoxic activity against above mentioned human tumor cell lines with IC50 value <6 �M

using camptothecin as positive control [Liu et al. (2009a)]. The saponins from A.

adianthifolia i.e., adianthifoliosides A, B and D were found to exhibit a cytotoxic effect on

human leukemia T-cells (Jurkat cells), whereas, the prosapogenins (Pro1 and Pro2) were

found to exert a lymphoproliferative effect on this cell type. These compounds were found

to exert a synergistic lymphoproliferative activity and induced apoptosis and a disrupted

mitochondrial membrane potential in Jurkat cells [Haddad et al. (2004)]. Julibroside J28

(70) displayed significant antitumor activity in vitro against PC-3M-1E8 (prostrate cancer),

Bel-7402, and HeLa cancer (cervical cancer) cell lines using by sulforhodamine B (SRB)

assay; the inhibitory rates were 80.47, 70.26, and 58.53%, respectively, at 10.0 �M dose

[Liang et al. (2005)]. The diastereoisomeric saponins julibroside J8 (67) and J13 (69) from

A. julibrissin showed marked cytotoxic activities against Bel-7402 cancer cell line with

86.66 and 93.33% inhibition respectively at 100 �g/ml concentration using SRB method

[Zou et al. (2005)]. Julibroside J29 (73), julibroside J30 (74), and julibroside J31 (75)

displayed significant in vitro antitumor activities against PC-3M-1E8, HeLa, and

MDAMB-435 (breast cancer) cancer cell lines at 10 �M concentration assayed by SRB and

MTT methods [Zheng et al. (2006b)]. Julibroside J8 (67) significantly inhibited growth in

BGC-823 (gastric cancer), Bel-7402, HeLa cell lines in vitro by MTT and SRB methods at

100 �g/ml dose; with typical apoptotic changes in morphology, nuclear damage and DNA

fragmentation of HeLa cells was observed. The treatment of HeLa cells with 20 �mol/L of

julibroside J8 (67) for 12 h and 24 h induced approximately 72% and 87% of HeLa cell

death [Zheng et al. (2006a)]. The saponins 3-O-[�-D-xylopyranosyl-(1�2)-�-L-

arabinopyranosyl-(1�6)-2-acetamido-2-deoxy-�-D-glucopyranosyl] echinocystic acid, 3-

O-[�-L-arabinopyranosyl-(1�2)-�-L-arabinopyranosyl-(1�6)-2-acetamido-2-deoxy-�-D-

glucopyranosyl] echinocystic acid exhibited cytotoxic effect with IC50 9.13 �g/ml and 10.0

�g/ml, respectively. The presence of the lactone ring in the triterpene and arabinose moiety

induced the inhibition of cytotoxic activity [Melek et al. 2007]. Julibroside II (76),


32

julibroside J16 (77), julibroside J21 (78) displayed good in vitro inhibitory activities against

Bel 7402 human cancer cell line at 100 �g/ml concentration with 91.9, 72.4 and 76.6%

cytotoxic activity [Zou et al. (2010)].

The potential cytotoxicity of the nine alkaloids [budmunchiamine G (95), budmunchiamine

K (96), 6'�-hydroxybudmunchiamine K (97), 9-normethylbudmunchiamine K (98),

budmunchiamine A (99), 6'�-hydroxybudmunchiamine C (100), 5-

normethylbudmunchiamine K (101), 6'�-hydroxy-5-normethylbudmunchiamine K (102),

14-normethylbudmunchiamine K (103)] were evaluated using the brine shrimp cytotoxicity

assay (BSCA). The fully N-methylated budmunchiamines, 96 and 99 without side chain

hydroxylation were strongly cytotoxic (<10 �g/ml dose). Two to three times higher

concentrations were required to obtain the same level of activity from the four alkaloids

(95, 96, 101, and 103) where one of the N-methyl groups was lost. The three 6'�-

hydroxylated compounds (97, 100, and 102) showed reduced toxicity by an order of

magnitude [Rukunga et al. (1996a)]. The toxicity of the n-hexane (HX), carbon

tetrachloride (CT), chloroform (CF) and aqueous soluble (AQ) fractions of the methanolic

extract of A. lebbek against brine shrimp was evaluated after 24 h (positive control,

vincristine sulphate, VS) and the LC50 were found to be 2.14, 2.15, 3.14, 6.99 and 0.30

�g/ml for HX, CT, CF, AQ and VS, respectively; n-hexane and carbon tetrachloride

soluble fractions being significantly toxic [Hussain et al. (2008)].

1.2.3.2 Antimicrobial activity

The antibacterial activity of kaempferol-3-O-�-L-rhamnopyranoside (87), quercetin-3-O-�-

L-rhamnopyranoside (83) and luteolin (81) were carried out against gram +ve bacteria,

Bacillus subtilis NRRL B-543 and Staphylococcus aureus NRRL B-313, as well as gram -

ve bacteria, Escherichia coli NRRL B-210 using nutrient agar medium. The results

revealed that these compounds exhibited moderate inhibiting activity against gram +ve and

gram -ve bacteria with zone of inhibition in the range of 16-17.5 mm [Ghaly et al. (2010)].

The nine budmunchiamines alkaloids were tested against gram +ve (B. subtilis, S. aureus)

and gram -ve (Escherichia coli, Pseudomonas aeruginosa) bacteria using the simple disk

diffusion assay. They exhibited zones of inhibition of at least 7 mm diameter against all the

bacteria at a loading of 50 �g/disk; yet found not active as the standard chloramphenicol

[Rukunga et al. (1996a)]. Antibacterial activities of ethyl acetate, ethanol and aqueous

extracts of A. adiantifolia bark and roots showed MIC against B. subtilis, E. coli, Klebsiella

pneumoniae, S. aureus and Micrococcus luteus in the range of 6.250-12.5 mg/ml [Eldeen

et al. (2005)]. Aqueous, ethanol and ethyl acetate extracts of A. gummifera bark showed


33

MIC against B. subtilis, E. coli, K. pneumoniae, M. luteus in the range of 0.39-12.5 mg/ml

[Buwa and van Staden (2006)].

n-Hexane and methanolic extracts of stem bark of A. procera showed >12 mm zone of

inhibition against B. subtilis, S. aureus, S. epidermidis, Enterococcus faecalis at

concentration of 5 mg/disc [Duraipandiyan et al. (2006)]. Antibacterial activities of A.

harveii bark against methicillin-sensitive S. aureus were observed at 2 mg/ml MIC

[Heyman et al. (2009)]. The methanol extracts of A. lebbek bark was screened for

antibacterial activity against eight strains of enteropathogenic bacteria, including multi-

drug resistant Vibrio cholerae (SG 24, NB2, PC4 and PC 65), Aeromonas hydrophila and

B. subtilis using broth microdilution method gave minimum inhibitory concentration

(MIC) (16-24 mg/ml) and minimum bactericidal concentration (MBC) (16-24 mg/ml)

[Acharyya et al. (2009)]. The crude hydro-alcoholic extract of stem and leaves exhibited

complete growth inhibition of N. gonorrhoeae strains at 500 �g/ml concentration.

Antimicrobial efficiency of crude leaf extracts of water, benzene and acetone of A. lebbeck

were tested against E. coli, S. aureus, K. pneumoniae, B.cereus, V. cholerae and Candida

albicans. The extracts displayed profound antimicrobial activity (>11 mm inhibition zone),

with 0.40-0.60 mg/ml and 0.50-0.80 mg/ml values of MIC and minimum bactericidal

concentration (MBC) [Maji et al. (2010)]. The carbon tetrachloride, chloroform and

aqueous fractions of methanolic extract of A. lebbek roots exhibited moderate inhibitory

activity against gram+ve bacteria (B. cereus, B. megaterium, B. subtilis, S. aureus, Sarcina

lutea) and gram-ve bacteria (E. coli, P. aeruginosa, Salmonella paratyphi, S. typhi,

Shigella boydii, S. dysenteriae, V. mimicus, V. Parahemolyticus) and fungal strains (C.

albicans, Aspergillus niger, Sacharomyces cerevacae) with the zone of inhibition of 9-17

mm, 9-18 mm and 9-16 mm, respectively at 400 �g/disc concentration. The crude

methanolic extract showed very weak inhibitory activity and the hexane partitionate

remained insensitive to the microorganisms [Hussain et al. (2008)]. The seed pods of A.

lebbeck showed antibactaerial activity with zone of inhibition 7 mm against S. aureus. The

in vitro antibacterial activities of 80% ethanolic and aqueous fractions from A. gummifera

seeds were tested for inhibitory activity against the clinical isolates of six Streptococcus

pneumonae and twenty two S. pyogenes using agar dilution method and it was found to

have antibacterial effects to all assayed bacteria while aqueous fractions did not exhibit any

effect. MIC of 80% ethanolic fractions was ranged from 500 to 1000 �g/ml depicting that

the extracts may contain bioactive compounds of therapeutic interest [Unasho et al.

(2009)].


34

1.2.3.3 Antioxidant activity

The antioxidant activity of methanol extract of A. julibrissin stem bark was evaluated for

its potential to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, authentic

peroxynitrites (ONOO-) and to inhibit the generation of the hydroxyl radical (.OH), total

reactive oxygen species (ROS). The antioxidant activity of its further fractions were

observed in the order of EtOAc>n-BuOH>CH2CI2> and H2O Sulfuretin (91) from EtOAc

fraction exhibited good activity in all tested model systems and exhibited five times more

inhibitory activity on the total ROS than Trolox [Jung et al. (2003)]. The methanol extract

of A. chevalieri leaves showed promising DPPH scavenging activity of 59.58, 68.48,

77.24, 85.93 and 94.73% of at 10, 25, 50, 125 and 250 �g/ml concentrations, respectively.

No significant difference (p<0.05) in the antioxidant activity was found between the extract

and standards (ascorbic and gallic acids) at 50, 125 and 250 �g/ml concentrations. The

reducing power of the extract (0.113 ± 0.056 nm) was found to be higher than the gallic

acid standard (0.096 ± 0.035 nm) [Aliyu et al. (2009)].

1.2.3.4 Antimalarial activity

Crude MeOH extract from A. adinocephala stem bark and leaves inhibited the activity of

malarial enzyme plasmepsin II which has profound effect on Plasmodium falciparum

parasite multiplication in vitro. Budmunchiamines L4 (104) and L5 (105) displayed mild

activity against plasmepsin II, with IC50 values of 14 and 15 mM, respectively [Ovenden et

al. (2002)]. The fractions of methanolic extract of A. gummifera showed low activity

against chloroquine sensitive (NF54) and resistant (ENT30) strains of P. falciparum with

IC50 above 3 �g/ml. The alkaloidal fraction exhibited strong activity against NF54 and

ENT30 with IC50 of 0.16 ± 0.05 and 0.99 ± 0.06 �g/ml, respectively. Five spermine

alkaloids [budmunchiamine K (96), 6'�-hydroxybudmunchiamine K (97), 5-

normethylbudmunchiamine K (101), 6'�-hydroxy-5-normethylbudmunchiamine K (102), 9-

normethylbudmunchiamine K (98)] exhibited activities against NF54 and ENT30 strains

with IC50 ranging from 0.09 ± 0.02 to 0.91 ± 0.10 �g/ml. The alkaloids showed percentage

chemosuppression of parasitaemia in mice ranging from 43 to 72% [Rukunga et al.

(2007)]. The methanolic extract of A. zygia stem bark exhibited antiprotozoal activity

against P. falciparum K1 strain with IC50 values of 1.0 �g/ml [Lenta et al. (2007)]. The

larval toxicity and smoke repellent potential of A. amara was evaluated at different

concentrations (2, 4, 6, 8 and 10%) against the different instar (I, II, III and IV) larvae and

pupae of Aedes aegypti (dengue vector). The (Lethal concentration) LC50 values of 5.41,

6.48, 7.11 and 7.52% were observed for I, II, III and IV instar larvae respectively, while,


35

the LC50 and LC90 values of 6.79% and 16.93% were found for pupae [Murugan et al.

(2007)].

1.2.3.5 Anti-inflammatory activity

The chloroform extract of A. chinensis bark at 200 mg/kg and 400 mg/kg dose levels

significantly decreased the carrageenan induced paw oedema paw volume in rats (P<0.001)

as compared to control (carboxy methyl cellulose) and positive control (diclofenac sodium)

at the end of 3 h [Perumal et al. (2010)]. A botanical formulation, Aller-7/NR-A2, was

developed for the treatment of allergic rhinitis which is a combination of medicinal plant

extracts from Phyllanthus emblica, Terminalia chebula, T. bellerica, A. lebbeck, Piper

nigrum, Zingiber officinale and P. longum. This formulation demonstrated potent

antihistaminic, anti-inflammatory, antispasmodic, and mast-cell-stabilization activities

[Amit et al. (2003)]. In carrageenan induced rat paw edema model, the equal mixture of

petroleum ether, ethyl acetate and methanol cold extracts at the 200 mg/kg and 400 mg/kg

dose level showed 36.68% and 27.51% inhibition of oedema volume at the end of 4 h

[Saha et al. (2009)]


The chloroform extract of A. chinensis bark at 200 and 400 mg/kg dose levels showed

significantly reduction in the ulcer index and increase in the ulcer protective effect as

compared to control and positive control [Perumal et al. (2010)]. Albizosides A-C (59-91),

from A. chinensis exhibited hemolytic activity against rabbit erythrocytes [Liu et al.

(2009)]. The sedative activity of flavonol glycosides, quercitrin (83) and isoquercitrin (90)

was evaluated and both compounds increased pentobarbital-induced sleeping time in dose-

dependent manner when administered intraperitoneally (i.p.) to mice. These compounds

were found devoid of any lethal effect in mice and no apparent behavioural change was

observed [Kang et al. (2000)].

However, numbers of reports are available on chemical investigations and biological

activities of different Albizzia species, yet, only few reports are available on chemical

investigations of A. chinensis growing in India. Thus, the present study has been envisaged

to systematically investigate A. chinensis growing in Himachal Pradesh, India for isolation

and characterization of bioactive molecules and development of new analytical methods for

quality assessment. The following pages describe the chemical investigation and biological

activity of A. chinensis performed in the present work. The results and discussion is

followed by experimental section and references are included at the end of the chapter.


36



For the isolation of bioactive constituents from flowers of A. chinensis, the dried powdered

plant material was extracted with 90% ethanol using percolation. The crude extract was

fractionated with n-hexane, ethyl acetate, n-butanol and water. Different fractions obtained

were chromatographed for the isolation of bioactive constituents. Repeated column

chromatography of ethyl acetate and n-butanol fractions led to the isolation of nine

flavonoids, namely, quercetin (25), kaempferol (80), quercetin-3-O-�-L-rhamnopyranoside

(quercitrin) (83), rutin (84), quercetin-3-O-�-L-galactopyranoside (92), quercetin-3-O-�-L-

arabinofuranoside (108), kaempferol-3-O-�-L-arabinofuranoside (109), myricetin (45) and

myricetin-3-O-�-L-rhamnopyranoside (myricitrin) (110).

The methanolic extract of bark on repeated column chromatography (normal and reverse

phase) led to isolation and characterization of five constituents, namely, catechin (111),

ferulic acid (112), caffeic acid (113) and �-sitosterol (48).

1.2.4.1.1 Quercitrin (83)

36

8

4a

3'

4'

1''

2''

6''

O

OH

HO

OH

OH

O

O

O

OH

OH

OH

Figure 1.2.1: Chemical structure of 83

Compound 83 was isolated as a yellow amorphous powder. The positive HRESI-QTOF-

MS showed a molecular ion peak at m/z 449.3824 [M+H]+ (calcd. 449.3848) indicated the

molecular formula as C21H21O11. UV spectrum showed absorption maxima at 255 and 350

nm, characteristic of flavonols. 1H NMR spectrum showed five aromatic signals at � 7.26

(1H, br s), 7.22 (1H, d, J = 8.7), 6.83 (1H, d, J = 8.1 Hz), 6.26 (1H, br s) and 6.10 (1H, br

s) assignable to H-2', H-6', H-5', H-8 and H-6 protons respectively (Table 1.2.1). 13

C NMR

and HMBC spectra displayed seven oxygenated carbons at � 164.3, 161.7, 157.9, 157.0,

148.3, 144.9, 134.8 and assignable to C-3, C-2, C-8a, C-3', C-4', C-5 and C-7 respectively.

The signal at � 178.2 assigned to C-4 further suggested the presence of flavonol type of

skeleton.

The sugar was identified as �-L-rhamnopyranosyl on the basis of NMR spectral signals at �

5.27, 4.16 3.68, 3.24-3.35, and 0.87 which are characteristic for �-L-rhamnopyranosyl

sugar. The attachment of sugar at C-3 position was deduced on the basis of above spectral


37

data and comparison with previously known spectral values. Thus, on the basis of above

spectral data and comparison with previously known spectral values [Manguro et al.

(2004); Yoshioka et al. (2004)], the structure of compound 83 was assigned as quercetin-3-

O-�-L-rhamnopyranoside (Figure 1.2.1).

Table 1.2.1: 1H (300 MHz) and


Position �C �H m (J Hz) Position �C �H m (J Hz)

1 - - 2' 115.0 7.26 br s

2 157.9 - 3' 144.9 -

3 134.8 - 4' 148.3 -

4 178.2 - 5' 115.6 6.83 d (8.1)

5 161.7 - 6' 121.5 7.22 d (8.7)

6 98.4 6.10 br s 1'' 102.1 5.27 br s

7 164.3 - 2'' 70.7 3.68 m

8 93.3 6.26 br s 3'' 70.6 3.24-3.35 m

8a 157.0 - 4'' 71.9 3.24-3.35 m

4a 104.5 - 5'' 70.5 4.16 s

1' 121.5 - 6'' 16.3 0.87 d (5.4)

1.2.4.1.2 Quercetin (25)

O

OH

HO

OH

O

OH

OH


Compound 25 was isolated as a yellow amorphous powder. Its positive HRESI-QTOF-MS

showed a molecular ion peak at m/z 303.0518 [M+H]+ (calcd. 303.2436) corresponding to

molecular formula C15H11O7. In UV spectrum, the absorption maxima were observed at

256, 372 nm. The UV absorptions were consistent with the presence of a 3,5,7,3',4'-

pentahydroxyflavone structure. 1H and

13C NMR spectra exhibited resonances due to

aromatic systems. In 1H NMR spectrum, the aromatic region showed signals at � 8.66 (1H,

s), 8.14 (1H, d, J = 8.1 Hz), 7.43 (1H, d, J = 8.1 Hz), and 6.43 (1H, br s) and 6.39 (1H, br

s) assignable to H-2', H-6', H-5', H-6 and H-8 protons (Table 1.2.2).

13C and HMBC NMR spectra showed the presence of fifteen aromatic carbon signals

corresponding to ten quaternary and five methine carbons. 13

C NMR signals were assigned

with the help of an HMQC data and it included one flavone carbonyl at � 178.1 (C-4) and

seven oxygen bearing quaternary carbons at � 166.3 (C-7), 163.2 (C-5), 158.3 (C-8a),


38

148.5 (C-4'), 147.9 (C-2), 147.9 (C-3') and 138.7 (C-3). Five signals at � 121.9, 117.5,

117.5, 100.0 and 95.1 were assigned to C-6', C-5', C-2', C-6 and C-8 respectively. Thus, on

the basis of above spectral data and comparison with previously known spectral values

[Xiao et al. (2006)], the structure of compound 25 was assigned as quercetin (Figure 1.2.2).


13C NMR (75.4 MHz) data of 25 in C5D5N


1 - - 8a 158.3 -

2 147.9 - 4a 105.3 -

3 138.7 - 1' 123.7 -

4 178.1 - 2' 117.5 8.66 br s

5 163.2 - 3' 147.9 -

6 100.0 6.43 br s 4' 148.5 -

7 166.3 - 5' 117.5 7.43 d (8.1)

8 95.1 6.39 br s 6' 121.9 8.14 d (8.1)

1.2.4.1.3 Rutin (84)

O

OH

HO

OH

OH

O

O

O

OH

HO

HO

OHO OH

OH

O

4

8

4a

8a

3'

4'

1'''6'''1'' 6''


Compound 84 was isolated as a yellow amorphous powder. Its positive HRESI-QTOF-MS


the molecular formula C27H31O16. The UV spectrum, absorption maxima were observed at

257, 353 nm. In 1H NMR (Table 1.2.3), the aromatic protons exhibited one ABX coupling

system at � 7.68 (1H, d, J = 2.1 Hz, H-2'), 7.64 (1H, dd, J = 2.1, 8.4 Hz, H-6') and 6.89

(1H, d, J = 8.4 Hz, H-5'). The other AX coupling system at � 6.40 (1H, br s) and 6.22 (1H,

br s) was assigned to H-8 and H-6 protons, respectively. 1H NMR spectrum supported the

presence of one rhamnose and one glucose moieties with the � values are similar to that of

rhamnose and glucose proton signals. 13C NMR spectrum displayed twenty seven carbon

signals including fifteen carbon signals due to the flavonol skeleton. By comparison with

the 13

C NMR spectral data of quercetin (25), we found that C-3 (� 135.7) was upfield

shifted, demonstrating glycosylation at C-3 position. The signals due to one flavonol

carbonyl at � 179.5 (C-4) and seven oxygen bearing quaternary carbons at � 166.1 (C-7),

163.0 (C-5), 158.6 (C-8a), 149.8 (C-4'), 159.4 (C-2), 145.9 (C-3') and 135.7 (C-3) were


39

observed. Five signals at � 123.6, 117.2, 116.1, 100.0 and 94.9 were assigned to C-6', C-2',

C-5', C-6 and C-8 respectively. The HMBC spectrum was utilized to identify the linkage

between the aglycone and sugar units. Accordingly, the structure of compound 84 was

established as rutin (Figure 1.2.3) on the basis of above spectral data and comparison with

previous reports [Wu et al. (2007)]




1 - - 5' 116.1 6.89 d (8.4)

2 159.4 - 6' 123.6 7.64 dd (8.4, 2.1)

3 135.7 - 1'' 104.8 5.12 d (7.4)

4 179.5 - 2'' 75.8 3.43-3.49 m

5 163.0 - 3'' 78.2 3.43-3.49 m

6 100.0 6.22 br s 4'' 71.5 3.25-3.33 m

7 166.1 - 5'' 77.3 3.25-3.33 m

8 94.9 6.40 br s 6'' 68.6 3.82 d (10.4);

3.36-3.38 m

8a 158.6 - 1''' 102.5 4.53 br s

4a 105.7 - 2''' 72.1 3.64-3.66 m

1' 123.2 - 3''' 72.3 3.52-3.57 m

2' 117.2 7.68 d (2.1) 4''' 74.0 3.25-3.33m

3' 145.9 - 5''' 69.8 3.25-3.33 m

4' 149.8 - 6''' 17.9 1.14 d (6.2)

1.2.4.1.4 Quercetin-3-O-�-L-arabinofuranoside (108)

HO

OH

O

O

OH

O

O H

OH

H

H

CH2OH

H

OH

1

3

5

7 8a

4a

3'4'

1''

3''

5''

OH


Compound 108 was isolated as a yellow amorphous powder. The negative ESI-QTOF-MS

showed a molecular ion peak at m/z 435 [M+H]+ indicating the molecular formula as

C20H19O11. UV spectrum showed absorption maxima at 255 and 356 nm characteristic of

flavonols. 1H NMR spectrum showed aromatic signals at � 7.45 (1H, br s), 7.41 (1H, br s),

6.84 (1H, d, J = 8.1 Hz), 6.33 (1H, br s) and 6.14 (1H, br s) assignable to H-6', H-2', H-5',

H-8 and H-6 respectively (Table 1.2.4). 13

C and HBMC NMR spectra displayed showed


40

seven oxygenated carbons at � 164.7, 161.7, 157.9, 148.4, 157.2, 144.9, 133.5, and

assignable to C-7, C-5, C-3, C-2, C-8a, C-3', and C-4' carbons respectively. The signal at �

178.6 was assigned to C-4 that further suggested the presence of flavonol type of skeleton.

The presence of five signals for sugar included a downfield anomeric signal at � 108.1 (C-

1'') and the sugar was identified as �-L-arbinofuranosyl on the basis of NMR spectral

values at � 5.40, 4.26, 3.83, 3.78-3.80, 3.42-3.43 that are characteristic for �-L-

arbinofuranosyl sugar. The attachment of sugar at C-3 position was deduced on the basis of

above spectral data and comparison with previously known spectral values [Shen et al.

(2009)], compound 108 was assigned as quercetin-3-O-�-L-arabinofuranoside (Figure

1.2.4).




1 - - 2' 115.0 7.41 br s

2 157.9 - 3' 144.9 -

3 133.5 - 4' 148.4 -

4 178.6 - 5' 115.4 6.84 d (8.1)

5 161.7 - 6' 121.5 7.45 br s

6 98.5 6.14 br s 1'' 108.1 5.40 br s

7 164.7 - 2'' 81.9 4.26 br s

8 93.4 6.33 br s 3'' 77.3 3.83 m

8a 157.2 - 4'' 86.6 3.78-3.80 m

14a 105.0 - 5'' 61.1 3.42-3.43 m

1' 121.5 -

1.2.4.1.5 Quercetin-3-O-�-L-galactopyranoside (92)

O

OH

HO

OH

OH

O

O

O

HO

OHOH

OH

1

3

5

8

3'

4'

1''

3''

6''


Compound 92 was isolated as a yellow amorphous powder. The positive ESI-QTOF-MS

showed a molecular ion peak at m/z 465 [M+H]+ indicated the molecular formula as


flavonols. 1H NMR spectrum showed five aromatic signals at � 8.45 (1H, br s), 8.12 (1H,

d, J = 8.4), 7.26 (1H, d, J = 8.1 Hz), 6.69 (1H, br s) and 6.63 (1H, br s) and these were

assignable to H-2', H-6', H-5', H-8 and H-6 protons respectively (Table 1.2.5). 13

C and


41

HMBC NMR spectra displayed seven oxygenated carbons at � 166.3, 163.0, 158.2, 157.9,

151.1, 147.1, and 135.5 assignable to C-7, C-5, C-2 C-8a, C-4', C-3' and C-3 carbons

respectively. The signal at � 179.2 assigned to C-4 further suggested the presence of

flavonol type of skeleton. The presence of five signals for sugar at � 6.07, 4.78-4.84, 4.60,

4.27-4.43, 4.15-4.18 including a anomeric signal at � 105.5 (C-1'') are characteristic for �-

L-galactopyranosyl. The attachment of sugar at C-3 position was deduced on the basis of

above spectral data and comparison with previously known spectral values [Xiao et al.

(2006); He et al. (2010)], the compound 92 was assigned as quercetin-3-O-�-L-

galactopyranoside (Figure 1.2.5).



1.2.4.1.6 Kaempferol-3-O-�-L-arabinofuranoside (109)

HO

OH

O

O

OH

O

O H

OH

H

H

CH2OH

H

OH

1

36

8 4'

1''

3''

5''


Compound 109 was isolated as a yellow amorphous powder. The negative ESI-QTOF-MS

showed a molecular ion peak at m/z 419 [M+H]+ indicated the molecular formula as


flavonols. 1H NMR spectrum revealed two sets of meta-coupled broad singlets at � 6.33

(1H, br s) and 6.14 (1H, br s) assigning to H-8 and H-6 protons respectively. The presence

of a set of A2B2 doublets at � 7.89 (1H d, J = 8.4 Hz) and 6.85 (2H, J = 8.4 Hz) each


1 - - 2' 116.6 8.45 br s

2 158.2 - 3' 147.1 -

3 135.5 - 4' 151.1 -

4 179.2 - 5' 118.2 7.26 d (8.5)

5 163.0 - 6' 123.1 8.12 d (8.4)

6 100.1 6.63 s 1'' 105.5 6.07 d (8.0)

7 166.3 - 2'' 73.7 4.78-4.84 m

8 94.9 6.69 s 3'' 75.8 4.60 m

8a 157.9 - 4'' 70.1 4.15-4.18 m

4a 105.8 - 5'' 78.0 4.78-4.84 m

1' 122.6 - 6'' 62.3 4.27-4.43 m


42

integrating for two protons have been assigned to H-2', H-6' and H-3', H-5' respectively

(Table 1.2.6). 13C and HMBC NMR spectra showed six oxygenated carbons at � 164.6,

161.7, 136.6, 160.1, 158.0, and 157.2 assignable to C-7, C-5, C-4', C-8a, C-2, and C-3,

respectively. The signal at � 178.5 assigned to C-4 further suggested the presence of

flavonol type of skeleton. The presence of five signals for sugar (� 5.41, 4.25, 3.83, 3.71-

3.73, 3.40-3.41) including a downfield anomeric signal at � 108.2 (C-1''), clearly suggested

pentose sugar in furanose form. The sugar was identified as �-L-arbinofuranosyl on the

basis of NMR spectral data. The attachment of sugar at C-3 position was deduced on the

basis of above spectral data and comparison with previously known spectral values [Xiao

et al. (2006)], compound 109 was assigned as kaempferol 3-O-�-L-arabinofuranoside

(Figure 1.2.6).




1 - - 2' 130.6 7.89 d (8.4)

2 157.2 - 3' 115.1 6.85 d (8.4)

3 136.6 - 4' 160.1 -

4 178.5 - 5' 115.1 6.85 d (8.4)

5 161.7 - 6' 130.6 7.89 d (8.4)

6 98.5 6.14 br s 1'' 108.2 5.41 br s

7 164.6 - 2'' 81.9 4.25 br s

8 93.4 6.33 br s 3'' 77.2 3.83 m

8a 158.0 - 4'' 86.6 3.71-3.73 m

4a 104.0 - 5'' 61.1 3.40-3.41 m

1' 121.4 -

1.2.4.1.7 Myricetin (45)

O

OH

HO

OH

OH

O

OH5

4a

8a

3'

4'

OH


Compound 45 was isolated as a yellow amorphous powder. Its positive ESI-MS showed a


molecular formula C15H11O8. In UV spectrum, absorption maxima were observed at 251,

371 nm. 1H and 13C NMR spectra displayed resonances due to aromatic systems. 13C NMR

signals were assigned with the help of HMQC and HMBC data. In 1H NMR spectrum, the

aromatic region showed signals at � 7.35 (2H, br s), 6.38 (1H, br s) and 6.20 (1H, br s)


43

assignable to H-2', H-6', H-8 and H-6 protons (Table 1.2.7). 13

C NMR spectrum showed

the presence of fifteen aromatic carbon signals corresponding to eleven quaternary and four

methine carbons; including one flavones carbonyl at � 178.0 (C-4) and eight oxygen

bearing quaternary carbons at � 164.3 (C-7), 161.6 (C-5), 157.2 (C-8a), 145.5 (C-3', 5'),

136.4 (C-4'), 157.2 (C-2), 136.0 (C-3). Four signals at � 107.7, 107.7, 98.4 and 93.7 were

assigned to C-2', C-6', C-6 and C-8 carbons respectively. Based on the above spectral data

and comparison of the data with the literature [He et al. (2009)], the structure of compound

45 was identified as myricetin (Figure 1.2.7).

Table 1.2.7: 1H (300 MHz) and 13C NMR (75.4 MHz) data of 45 in CD3OD Position �C �H m (J Hz) Position �C �H m (J Hz)

1 - - 8a 157.2 -

2 157.2 - 4a 104.5 -

3 136.0 - 1' 122.2 -

4 178.0 - 2' 107.7 7.35 br s

5 161.6 - 3' 145.5 -

6 98.4 6.20 br s 4' 136.4 -

7 164.3 - 5' 145.5 -

8 93.7 6.38 br s 6' 107.7 7.35 br s

1.2.4.1.8 Myricetin-3-O-�-L-rhamnopyranoside (110)

36

8

4a

3'

4'

1''

2''

6''

O

OH

HO

OH

OH

O

O

O

OH

OH

OH

OH


Compound 110 was isolated as a yellow amorphous powder. The positive ESI-MS showed

a molecular ion peak at m/z 465 [M+H]+ indicated the molecular formula as C21H21O12. UV

spectrum showed absorption maxima at 258 and 352 nm characteristic of flavonols. 1H

NMR spectrum showed four aromatic signals at � 6.89 (2H, br s), 6.26 (1H, br s), and 6.10

(1H, br s) assignable to H-2', H-6', H-8 and H-6 protons respectively (Table 1.2.8). The

detailed analyses of 13

C and HMBC NMR spectra indicated the presence of eight

oxygenated quaternary carbons at � 164.3, 161.6, 158.0, 157.0, 145.0, 145.0, 136.4, and

134.9 assignable to C-7 C-5 C-2, C-8a, C-3', C-5', C-4', and C-3 carbons respectively. The

signal at � 178.2 assigned to C-4 further suggested the presence of flavonol type of

skeleton. The presence of six signals (� 5.25, 4.19, 3.75-3.77, 3.43-3.57) including a signal


44

at � 102.1 (C-1''), suggesting the sugar in pyranose form. The sugar was identified as �-L-

rhamnopyranosyl on the basis of NMR spectral data. The attachment of sugar at C-3

position was deduced on the basis of above spectral data and comparison with previously

known spectral values [Adebayo et al. (2011)], the compound 110 was assigned as

myricetin-3-O-�-L-rhamnopyranoside or myricitrin (Figure 1.2.8).




1 - - 2' 107.9 6.89 br s

2 158.0 - 3' 145.0 -

3 134.9 - 4' 136.4 -

4 178.2 - 5' 145.0 -

5 161.6 - 6' 108.3 6.89 br s

6 98.5 6.10 br s 1'' 102.1 5.25 br s

7 164.3 - 2'' 70.7 3.75-3.77 m

8 93.3 6.26 br s 3'' 70.5 3.43-3.57 m

8a 157.0 - 4'' 71.9 3.43-3.57 m

4a 104.5 - 5'' 70.1 4.19 s

1' 121.6 - 6'' 16.3 0.90 d (6.0)

1.2.4.1.9 Catechin (111)

O

OH

HO

OH

OH

OH

4

2

6

8

2'

5'6'


Compound 111 was isolated as a white amorphous powder. It showed a molecular ion peak

at m/z 291.2736 [M+H]+ (calcd. 291.2760) in its positive ion HR-ESI-QTOF-MS spectrum,

in accordance with the formula C15H15O6. UV spectra showed �max at 220 and 280 nm. 1H

NMR spectra indicated five aromatic protons at � 6.85 (1H, br s, H-2'), 6.75 (2H, d, J = 8.8

Hz, H-5', H-6'), 5.94 (1H, br s, H-8) and 5.87 (1H, br s, H-6). Four aliphatic protons were

found at � 4.56-4.59 (1H, m, H-2), 3.97-4.00 (1H, m, H-3), 2.82-2.89 (1H, m, H-4) and

2.47-2.55 (1H, m, H-4) (Table 1.2.9). In 13C and HMBC NMR spectra, fifteen carbon

signals including seven oxygen bearing carbons at � 157.9, 157.7, 157.0, 146.4, 146.4,

83.0, and 68.9 due to C-5, C-7, C-8a, C-3', C-4', C-3, C-2 carbons respectively and five

aromatic methine carbons showed peaks at � 120.2, 116.2, 115.4, 96.5, 95.7 assignable to

C-6', C-5', C-2', C-6 and C-8 carbons respectively. Based on the NMR, mass, optical


45

rotation data and previous literature [Hye et al. (2009); Cren-Olive et al. (2002)], the

structure of compound 111 was identified as catechin (Figure 1.2.9).

Table 1.2.9: 1H (300 MHz) and 13C NMR (75.4 MHz) data of 111 in CD3OD Position �C �H m (J Hz) Position �C �H m (J Hz)

1 - - 8a 157.0 -

2 83.0 4.56-4.59 m 4a 101.0 -

3 68.9 3.97-4.00 m 1' 132.4 -

4 28.6 2.82-2.89 m; 2.47-2.55 m 2' 115.4 6.85 br s

5 157.9 - 3' 146.4 -

6 96.5 5.87 br s 4' 146.4 -

7 157.7 - 5' 116.2 6.75 d (8.8)

8 95.7 5.94 br s 6' 120.2 6.75 d (8.8)

1.2.4.1.10 Ferulic acid (112)

HO

H3COOH

O

3

5

7

8


Table 1.2.10: 1H (300 MHz) and 13C NMR (75.4 MHz) data of 112 in CD3OD Position �C �H m (J Hz)

9 171.0 -

8 116.0 6.31 d (15.8)

7 146.9 7.60 d (15.8)

1 127.9 -

6 124.0 7.06 d (8.1)

5 116.5 6.81 d (8.1)

4 149.4 -

3 150.5 -

2 111.8 7.17 s

3-OCH3 56.3 3.89 s

Compound 112 was isolated as a white amorphous powder. Its positive ESI-QTOF-MS


the molecular formula C10H11O4. 1H and

13C NMR spectra exhibited resonances due to

aromatic system. 13

C NMR signals were assigned with the help of an HMQC and HMBC

data. In 1H NMR spectrum, the aromatic region showed signals at � 7.60 (1H, d, J = 15.8

Hz,), 7.17 (s, 1H), 7.06 (1H, d, J =8.1 Hz,), 6.81 (1H, d, J =8.1 Hz,), 6.31 (1H, d, J = 15.8

Hz,) assignable to H-7, H-2, H-6, H-5 and H-8 protons (Table 1.2.10). 13C NMR spectrum


46

showed the presence of ten carbon signals including six aromatic carbons at � 150.5 (C-3),

149.4 (C-4), 124.0 (C-6), 116.5 (C-5), 111.8 (C-2) and one carboxylic carbon at � 171.0

(C-9). Three signals of two methines at � 146.9, 116.0 and one methoxy carbon at � 56.3,

were assigned to C-7, C-8 and C3-OCH3 carbons respectively. Based on the above spectral

data and comparison of the data given in the literature [Bunzel et al. (2005); Yoshioka et

al. (2004)], the structure of compound 112 was identified as ferulic acid (Figure 1.2.10).

1.2.4.1.11 Caffeic acid (113)

HO

HOOH

O

1

4

39

7


Table 1.2.11: 1H (300 MHz) and 13C NMR (75.4 MHz) data of 113 in CD3OD

Position �C �H m (J Hz)

1 127.9 -

2 115.3 7.04 s

3 149.5 -

4 146.4 -

5 116.6 6.78 d (8.2)

6 123.0 6.93 d (8.2)

7 147.2 7.54 d (15.9)

8 115.6 6.23 d (15.9)

9 171.2 -

Compound 113 was isolated as a white amorphous powder. Its positive ESI-MS showed a


molecular formula C9H9O4. 1H NMR spectrum exhibited signals for three aromatic protons

in a 2,5,6 substitution pattern at � 7.04 (s, 1H, H-2), 6.93 (d, 1H, J = 8.2 Hz, H-6), 6.78 (d,

1H, J = 8.2 Hz, H-5) The two protons of a trans-double bond at � 7.54 (1H, d, J = 15.9 Hz)

and 6.23 (1H, d, J = 15.9 Hz) indicated the presence of a 3,4-dihydroxy-trans-cinnamate

moiety (Table 1.2.11). The 13

C and HBMC NMR spectra indicated the presence of

resonances attributable to a carbonyl group at � 171.2 (C-9), two deshielded oxygen

bearing quaternary carbons at � 149.5 (C-3), 146.4 (C-4), three aromatic methines at �

123.0 (C-6), 116.6 (C-5) and 115.3 (C-2) and two trans doubly bonded carbons at � 147.2

(C-7), 115.6 (C-8). The structure of compound 113 was identified as caffeic acid (Figure

1.2.12) on comparison of the above spectral data and literature values [He et al. (2009);

Hoeneisen et al. (2003)].


47

1.2.4.1.12 �-Sitosterol (48)

HO

1

4 6

19

12

14

17

18

2122

24

29

26

27



13C NMR (75.4 MHz) data of 113 in CDCl3


1 37.2 1.05-1.08 m 16 28.4 1.75 m

2 31.8 1.40-1.45 m 17 56.2 1.92 m

3 71.9 3.41-3.46 m 18 12.0 0.62 s

4 38.8 2.19 m 19 19.5 0.83-0.95 m

5 140.9 - 20 36.3 2.10 m

6 121.8 5.28 br s 21 18.9 0.83-0.95 m

7 32.0 2.10 m 22 34.2 1.63 m

8 32.0 1.40-1.45 m 23 26.5 1.63 m

9 50.3 0.83-0.95 m 24 46.0 1.40-1.45 m

10 36.9 - 25 29.3 1.40-1.45 m

11 21.2 1.40-1.45 m 26 19.9 0.71-0.75 m

12 39.9 1.92 m 27 19.2 0.71-0.75 m

13 42.4 - 28 23.2 1.40-1.45 m

14 56.9 1.05-1.18 m 29 12.1 0.71-0.75 m

15 24.4 1.40-1.45 m

Compound 48 was isolated as colorless needles. The 1H NMR showed the proton of H3

appeared as a multiplet at � 3.41-3.46 (m, 1H) and revealed the existence of signals for

olefinic proton at � 5.28 (1H, br s) (Table 1.2.12). Angular methyl proton at � 0.62 (s),

0.83-0.85 (m) correspond to H-18 and H-19 protons respectively. 13C NMR displayed

signals at � 140.9 and 121.8, which are assigned C-5 and C-6 double bonded carbons

respectively. The signal at � 19.5 was assignable to angular carbon C-19. The spectra

depicted twenty nine carbon signal including six methyls, eleven methylenes, nine methine

and three quaternary carbons. On the basis of NMR, mass, optical rotation data and

comparison with previously known spectral values [Saxena and Albert (2005); Kamboj and

Saluja (2011)], compound 48 was assigned as �-sitosterol (Figure 1.2.12).


48

1.2.4.2 Identification of phenolic compounds by UPLC-DAD-ESI-QTOF-MS in A.

chinensis

Out of varios moblile phase compositions, water (0.05% formic acid) (A) and methanol (B)

was chosen as the best mobile phase based on the chromatographic peak shape. Increasing

flow rates shorten the run time, but have detrimental effects on resolution. A flow rate of

0.275 ml/min was finally chosen for faster separation and better resolution. The column

temperature was set to 28ºC in order to obtain better resolution and appropriate column

pressure. By UPLC-MS analysis the presence of phenolic constituents (flavonols, their

glycosides, procyanidins and galloyl tannins) was detected consisting of fifteen known and

unknown compounds (Figure 1.2.14). The flavonols were found to be glycosides of

quercetin, kaempferol and myricetin. UV spectra showed characteristic absorption bands at

350-367 and 254-266 nm, indicating the presence of flavonols in the extracts. Flavonols

and their glycosides have been identified in other Albizzia species [Kumar et al. (2007);

Lau et al. (2007)]. Four flavonoids were identified by comparison of their retention time

(tR) and UV spectra as myricetin-3-O-rhamnoside, quercetin-3-O-arabinofuranoside,

quercetin-3-O-rhamnoside and quercetin (Figure 1.2.13). The flavonol glycosides (peak 5-

15) were eluted in the order of myricetin, quercetin and kaempferol glycosides,

respectively. The peaks (1-4) show spectral characteristics close to those of procyanidins as

their UV �max values (210 and 262-283 nm) were close to those of catechin [Rohr et al.

(2000); Harris et al. (2007)].

R1 R2 R3

OH OH rha Myricetin-3-O-rhamonside

OH H ara Quercetin-3-O-arabinoside

OH H rha Quercetin-3-O-rhamonside

OH H H Quercetin

Figure 1.2.13: Chemical structures of myricetin-3-O-rhamonside, quercetin-3-O-

arabinoside, quercetin-3-O-rhamonside and quercetin

The phenolics and their glycosides were further characterized by UPLC-ESIMS/MS

analysis in the positive ion mode after tentative identification from UV data allowed the

partial determination of structure. Positive ion UPLC-ESI-MS of ethanolic extracts of A.

chinensis showed protonated [M+H]+ molecules.

O

OH

HO

OH

OR3

O

R1

R2


49

Figure 1.2.15: UPLC-DAD chromatograms of (A) standard mixture, (B) flowers, (C)

pods, (D) leaves and (E) bark of A. chinensis methanolic extracts

A

6

11

15

10

2 3

4 20 11

19

D 15

18

8 7

1

10

18

8 7

1

10

E

1

6 7 8

9

10

13

15

14

11 B

2

9

11 12

13

14

15

20

19 3 6

17 16

C


50

A difference of 162, 146 and 132 mass units indicated the presence of a hexose (possibly

glucose or galactose), rhamnose, and a pentose (possibly xylose or arabinose), which were

calculated from the difference in mass of molecular ion and fragment ion peaks.

Comparison of retention times and MS data with those of standard compounds revealed the

presence of phenolic compounds; corroborate previous findings [David et al. (1996);

Schiebera et al. (2002)]. ESI-QTOF-MS/MS (in positive mode) of the majority of these

ions showed the characteristic distribution of fragment ions in the A. chinensis extracts

(Figure 1.2.15). Out of eleven flavonoids, six quercetin (peak 7-11, 15), three kaempferol

(peak 12-14), and two myricetin (peak 5-6) derivatives were identified. Other peaks (peak

2-5) were expected to be procyanidins on the basis of UV spectra; however, corresponding

mass signal peaks were not observed for these peaks.

Table 1.2.14: Identification of phenolic compounds in methanolic extracts of different

parts of A. chinensis Peak tR

min)

UV

Spectra

Calculated

MW

Positive ion mode Phenolic compounds

MS MS-MS

1 0.92 210, 272 380 381 219, 201 Procyanidinc

2 1.30 210, 262 382 383 367, 247, 217, 166 Procyanidinc

3 1.50 210, 283 - - - Procyanidinc

4 1.90 210, 262 408 409 300, 247, 185 Procyanidinc

5 3.31 258, 352 486 487 365, 267, 215, 319,

267, 205, 175

Myricetin-3-O-glycoside

6 3.64 259, 352 464 465 361, 341, 319, 205,

175

Myricetin-3-O-

rhamnosidea

7 4.08 256, 354 464 465 383, 367, 303, 229,

205, 175, 121, 109

Quercetin-3-O-galactose

8 4.40 256, 355 434 435 303, 233, 205, 175 Quercetin-3-O-pentosideb

9 4.51 255, 356 434 435 349, 303, 213, 139 Quercetin-3-O-pentosideb

10 4.94 256, 353 434 435 303, 245, 209, 102 Quercetin-3-O-

arabinofuranosidea

11 5.16 256, 350 448 449 303, 147, 129 Quercetin-3-O-

rhamnosidea

12 5.70 266, 356 498 499 287, 175 Kaempferol-3-O-

glycosidec

13 5.99 266, 356 418 419 404, 349, 326, 287 Kaempferol-3-O-

arabinofuranosideb

14 6.27 264, 356 432 433 407, 389, 350, 287,

148, 103, 85, 71

Kaempferol-3-O-

rhamnosideb

15 6.73 256, 367 302 303 285, 257, 247, 229,

201, 183, 153, 137,

111, 95, 69

Quercetina

aCompounds conclusively identified by comparison with authentic standard.

bCompounds tentatively

identified by UV and mass spectral data. cSamples tentatively identified by UV spectral data

Flavonol (quercetin) [M+H]+ molecular ions dehydrated to [M+H-H2O]+ (m/z 285),

followed by two sequential losses of CO: [M+H-H2O-CO]+ (m/z 257) and [M+H-H2O-

2CO]+ (m/z 229). These losses of carbon monoxide were also observed directly from the


51

protonated flavonoid: [M+H-CO]+ and [M+H-2CO]

+ (m/z 247). Furthermore the C-ring

was cleaved at bonds 0,2 and 1,3 and produced the respective RDA fragments (0,2A+, 0,2B+

and 1,3A+, 1,3B+) of the protonated molecule (Figure 1.2.15).

0,4A+

O

OH

HO

OH

O

OH

OH

1,4B+ = 179

1,3A+ = 153

0,2A+ = 165

0

3

2

4

A

B

H+

1,3B+

0,2B+ = 137

0,4B+

C

Figure 1.2.15: Fragmentation pattern of quercetin

The plant isolated antioxidant quercitrin has been encapsulated on poly-D,L-lactide (PLA)

nanoparticles by solvent evaporation method to improve the solubility, permeability and

stability of this molecule. The size of quercitrin-PLA nanoparticles was found to be

250±68nm whereas that of PLA nanoparticles was 195±55 nm. The encapsulation

efficiency of nanoencapsulated quercitrin was evaluated by HPLC. The presence of

quercitrin specific peaks on FTIR of quercitrin loaded PLA nanoparticles provides an extra

evidence for the encapsulation of quercitrin into PLA nanoparticles. These properties of

quercitrin nanomedicine provide a new potential for the use of such less useful highly

active antioxidant molecule towards the development of better therapeutic for intestinal

anti-inflammatory effect and nutraceutical compounds.

1.2.4.3 Determination of total flavonoid and phenolic contents of aerial parts of A.

chinensis

Table 1.2.15: Total phenolic and flavonoid contents of different parts of A. chinensis

Plant Part TFCa (mg/g) TPC

b (mg/g)

Flower 8.4 ± 0.09 24.1 ± 0.6

Pods 5.1 ± 0.05 23.1 ± 0.7 Leaf 11.8 ± 0.4 24.6 ± 0.4

Bark 19.6 ± 0.9 26.3 ± 2.0 aTotal flavonoid content; bTotal phenolic content

Various authors have identified and quantified the polyphenols in Albizzia species [Lau et

al. (2007)]. In this study, the total phenol contents (TPC) of different parts of A. chinensis

were measured by the Folin Ciocalteu reagent in terms of gallic acid equivalents (standard

curve equation: y= 0.1194x-0.0438, r2 = 0.9991) (Table 1.2.15). The total phenol content

varied from 23.3 ± 0.7 to 26.3 ± 2.0 mg/g in the extracts in terms of gallic acid equivlents.


52

Total flavonoid contents (Table 1.2.15) of extracts of aerial parts (flowers, pods, leaves,

bark) of A. chinensis were determined. The flavonoids content of the extracts in terms of

catechin equivalents (standard curve equation: y=0.0131x-0.0149, r2 = 0.9873) were found

between 5.1 ± 0.05 and 19.6 ± 0.9 mg/g; the highest amount was present in the bark extract

(19.6 ± 0.9 mg/g).


Podophyllum hexandrum Royle

1.3.1 Introduction

The name Podophyllum was derived

from the Greek words podos and

phyllon meaning foot shaped leaves.

The genus Podophyllum

(Podophyllaceae) has four well known

species, Himalayan, P. hexandrum,

commonly distributed in the Himalayan

regions of Asian continent popularly

known as Himalayan Mayapple,

American, P. peltatum, commonly distributed in Atlantic North America popularly called

as American Mayapple [Chaurasia et al. (2012)] and two of Chinese origin, P. versipelle

and P. aurantiocaule. Several other less-known Chinese species (P. mairei, P. delavayi, P.

difforme and P. pleianthum) have also been identified and chemically investigated

[Rahman et al. (1995)]. They are woodland plants, typically growing in colonies derived

from a single root. Podophyllum rhizomes have a long medicinal history among native

North American tribes who used a rhizome powder as a laxative or an agent that expels

worms (anthelmintic). The dried roots and rhizomes are called podophyllum, which is

enriched in lignans and its first modern botanical name was given by Linnaeus in 1753 [Liu

et al. (2007)]. The natives of the Himalayas as well as the American Indians independently

discovered that the rhizomes extract possessed a cathartic action. The Indians introduced

podophyllin, a resin obtained by ethanolic extraction of the roots and rhizomes, to colonists

for the use as a catharic, an anthelmentic and misuse as a lethal poison. The colonists also

used this resin as an emetic and cholagogue. Podophyllin was included in the first U.S.

Pharmacopoeia in 1820 and the use of this resin was prescribed for the treatment of

venereal warts and as a cathartic. Its first serious chemical investigation was carried out by


53

Podwyssotzki [Podwyssotzki (1880)]. Because of its severe toxicity, the drug was removed

from the 12th edition of Pharmacopoeia in 1942 [Ayres and Loike (1990); Horwitz and

Loike (1977)]. In the same year, however, it was reported that venereal warts (Condyloma

acuminata) could be selectively destroyed by the topical application of podophyllin.

Renewed interest in the Podophyllum plant was generated in the 1940s when Kaplan

demonstrated the curative effect of podophyllin, an alcoholic extract of the Podophyllum

rhizomes in C. acuminata.

Podophyllum hexandrum Royle (syn. P. emodi Wall.), Podophyllaceae, is an important

high altitude medicinal plant, commonly known as ‘Indian mayapple’ because its fruits

ripen in spring. It is found in Alpine Himalayas (3000-4000 msl), Jammu and Kashmir,

Himachal Pradesh, Sikkim and Arunachal Pradesh. The plant has been used extensively for

its antitumour, antifungal and immunostimulatory properties [Kamil and Dewick (1986);

Foster (1993); Canel et al. (2000)]. Traditionally, dried rhizomes of the plant were mixed

with liquid and taken as a laxative or to get rid of intestinal worms as a powerful purgative.

Powder of the rhizome was used as a poultice to treat warts and tumorous growth on skin.

Physicians in Missouri, Mississpi and Lousiana used the resin for the treatment of genital

warts. The resin was applied to treat cancerous tumors, polyps and granulations in

traditional medicines. Extracts of Podophyllum species were used as antidotes against

poison, treatments for skin disorders or as purgative, antihelminthic, vesicant, and suicide

agents. P. hexandrum was extensively exploited in Ayurvedic system of medicine for

treating ailments like constipation, cold, biliary fever, septic wounds, inflammation,

burning sensation, mental disorder, genital warts, monocytoid leukemia, Hodgkin’s and

non Hodgkin’s lymphoma [Singh and Shah (1994)].

Two species of the genus Podophyllum, P. hexandrum and P. peltatum are the

commercially most exploited species for the production of podophyllotoxin [Stahelin and

Von Wartburg (1991); Imbert (1998)], that acts as starting material for the preparation of

semisynthetic compounds used in the treatment of lung cancer, a variety of leukemias and

other solid tumours [Van Uden et al. (1989); Canel et al. (2000); Canel et al. (2001)].

Podophyllotoxin is the most abundant lignan isolated from podophyllin. P. hexandrum has

been reported to contain approximately higher podophyllotoxin content (4.3%) in

comparison to the American species, P. peltatum (0.25%) [Jackson and Dewick (1984)].

Podophyllotoxin was also produced in relatively minute quantities by other plant species,

viz., P. aurantiocaule, P. delavayi, P. pleianthum P. peltatum, P. versipelle, Linum flavum,

L. album and Juniperous chinensis.


54


Extensive chemical investigation of roots, rhizomes and leaves of P. hexandrum and P.

peltatum revealed the presence of lignans and their glycosides such as podophyllotoxin

(114) [Podwyssotzki (1880), Liu and Jiao (2006)], �-peltatin (115) [Hartwell (1947)], �-

peltatin (116) [Hartwell and Detty (1948)], 4'-O-demethylpodophyllotoxin (117) [Nadkarni

et al. (1952); Liu et al. (2006)], 4-O-(�-D-glucopyranosy1)-picropodophyllin or

picropodophyllin glucoside (118) [Nadkarni et al. (1953); Liu and Jiao (2006)],

dehydropodophyllotoxin (119) [Kofod and Jorgensen (1954); Liu and Jiao (2006)],

deoxypodophyllotoxin (120) [Kofod and Jorgensen (1955); Dewick and Jackson (1981);

Liu and Jiao (2006)], podophyllotoxone (121) [Dewick and Jackson (1981); Liu and Jiao

(2006)], isopicropodophyllone (122), 4'-O-demethyldeoxypodophyllotoxin (123), 4'-O-

demethylpodophyllotoxone (124), 4'-O-demethylisopicropodophyllone (125) [Dewick and

Jackson (1981); Jackson and Dewick (1984)], picropodophyllotoxin (126) [Chakravarti and

Chakraborty (1954); Liu and Jiao (2006)], 4'-O-demethylpodophyllotoxin-4-O-�-D

glucoside (127), picropodophyllotoxone or picropodophyllone (128) [Singh and Shah

(1994); Chaudhary et al. (2011)], epipodophyllotoxin (129) [Rahman et al. (1995); Liu and

Jiao (2006)], 4'-O-demethyldehydropodophyllotoxin (130) [Rahman et al. (1995)],

podophyllotoxin-4-O-�-D-glucoside (131) and podophyllotoxin-4-O-�-D-glucoside (132)

[Sultan et al. (2010a)].

O

O

O

O

OCH3

OR3

H3CO

R4 R1 R2

O

O

O

O

OCH3

OCH3

H3CO

O

OHO

OHOH

OH

O

O

O

O

OCH3

OR

H3CO

OH

R1 R2 R3 R4 (118) R

OH H Me H (114) CH3 (119)

H H H OH (115) H (130)

H H Me OH (116)

OH H H H (117)

H H Me H (120)

H H H H (123)

H OH Me H (129)


55

O

O

O

O

OCH3

OR

H3CO

O

O

O

O

O

OCH3

OR

H3CO

O

O

O

O

O

OCH3

OCH3

H3CO

OH

R R (126) CH3 (121) CH3 (122)

H (124) H (125)

O

O

O

O

OCH3

OCH3

H3CO

O

O

O

O

O

OCH3

OR

H3CO

O

OHO

OHOH

OH

O

O

O

O

OCH3

OCH3

H3CO

O

OHO

OHOH

OH

(128) R (132) CH3 (131)

H (127)

O

OH

HO

OH

O

O

OOHHO

HO

OH

O

OH

HO

OH

O

O

O

OH

HO

HO

OHO OH

OH

O

O

OH

HO

O

OH

OH

(133) (134) (135)

R

O OH

O

H

R

CH3(CH2)10- (136)

CH2=CH-(CH2)10- (137)

CH3(CH2)12- (138)

A few flavonoids such as quercetin (25) [Singh and Shah, (1994)], kaempferol (80) [Shen

and Tian (2006)], quercetin-3-O-�-D-glycoside [Sultan (2010a)], kaempferol-3-O-�-D-

glucopyranoside (133) [Chaudhary et al. (2011)], rutin (84), kaempferol-3-O-rutinoside

(134) [Zhao et al. (2001)] and 8-prenylkaempferol (135) [Shang et al. (2000)] were

reported from roots, rhizomes and fruits. A mixture of three 3-acyl-4-hydroxy-5,6-

dihydropyrones [podoblastin A (136), B (137) and C (138)] were characterized from

chloroform extract of aerial parts of P. peltatum [Miyakado et al. (1982)].


56

1.3.3 Chromatographic studies

The most frequently used analytical techniques for determination of lignans and flavonoids

in Podophyllum species so far were high performance liquid chromatography (HPLC), high

performance thin layer chromatography (HPTLC) and micellar electrokinetic

chromatography (MEKC). The majority separations were carried out in the reversed-phase

HPLC mode, however, analysis on conventional silica-gel columns were also reported.

A HPLC method was developed for separation of three lignans namely �- peltatin (115), �-

peltatin (116) and podophyllotoxin (114) in P. peltatum resin using 1.8% ethanol in

chloroform as mobile phase on Perkin-Elmer silica A column at 254 nm with a flow rate of

0.8 ml/min [Treppendahl and Jakobsen (1980)]. Seven diastereoisomers of

podophyllotoxin (114) were successfully separated on silica (Hypersil) and reverse phase

(ODS-Hypersil) column using n-heptane-dichloromethane-methanol (90:10:4) and

methanol-water (45:55) as mobile phase, respectively [Lim and Ayres (1983)].

Furthermore, a method was developed using a Taxsil B column for determination of eight

lignans such as podophyllotoxin (114), epipodophyllotoxin (129), �-peltatin (115), �-

peltatin (116), 4'-O-demethylpodophyllotoxin (117), podophyllotoxin-4-O-�-D-

glucopyranoside (131), epipodophyllotoxin-4-O-�-D-glucopyranoside (139) and 1,2,3,4-

dehydrodesoxypodophyllotoxin (140) by solvent system of (A) reagent alcohol [90.6%

ethanol, 4.5% CH3OH, 4.9% isopropanol)-tetrahydrofuran-methyl-t-butyl ether

(40:30:30)], (B) methanol:water:acetic acid (15:84:1) containing 0.1% ammonium acetate

and (C) acetonitrile in gradient elution in P. peltatum and P. emodi [Bastos et al. (1995);

Bastos et al. (1996)]. In another study, eight lignans and their glucosides [podophyllotoxin

(114), 4'-O-demethylpodophyllotoxin (117), podophyllotoxin-4-O-�-D-glucoside (131), 4'-

O-demethylpodophyllotoxin-�-D-glucoside (127), �-peltatin (115), �-peltatin (116) and

their glucosides] were separated on RP-HPLC (ODS-Hypersil) column with CH3OH-H2O,

CH3CN-H2O, CH3OH-CH3COONH4 or CH3CN-CH3COONH4 as mobile phase at 280 nm

[Lim (1996)].

A microanalytical technique was developed for determination of podophyllotoxin content

(1 to 2 mg) in P. hexandrum resin by RP-HPLC and RP-HPTLC at 230 and 217 nm

respectively using (A) acetonitrile and (B) water as mobile phase [Mishra et al. (2005)].

Another HPLC method was developed for simultaneous determination of nine lignans

[picropodophyllin glucoside (118), 4'-O-demethylpodophyllotoxin (117),

epipodophyllotoxin (129), picropodophyllin (126), picropodophyllone (128),

podophyllotoxin (114), podophyllotoxone (121), deoxypodophyllotoxin (120),


57

dehydropodophyllotoxin (119)] and two flavonoids [quercetin (25), kaempferol (80)] in P.

emodi with a mobile phase (A) 25 mM phosphate buffer, pH 2.5 and (B) methanol using

gradient elution [Liu and Jiao (2006)]. Later on, a quantitative LC/MS/MS method was

developed to analyze podophyllotoxin (114), quercetin (25) and kaempferol (80) in

podophyllin. Chromatographic separation was performed on a reversed-phase C18 column

using a gradient of mobile phase of 0.25% formic acid and methanol [Lin et al. (2008)].

Five lignans [podophyllotoxin (114), 4'-O-demethylpodophyllotoxin (117),

podophyllotoxin-4-O-�-D-glycoside (132), podophyllotoxin-4-O-�-D-glycoside (131) and

quercetin-3-O-�-D-glycoside] and two unknown compounds were analyzed in twelve

different accessions of Podophyllum using HPLC-MS on RP-18 column using methanol

and water (60:40) at 290 nm [Sultan et al. (2010a)].

O

O

O

O

OCH3

OCH3

H3CO

O

OHO

OHOH

OH

O

O

O

O

OCH3

OCH3

H3CO

O

O

O

O

OCH3

OR2

H3CO

O

OO

OR1

HOOH

(139) (140) R1 R2

CH3 H (141)

S H (142) Recently, an UPLC-UV-MS was developed for rapid analysis of four lignans [4-O-

demethylpodophyllotoxin (117), podophyllotoxin (114), �-peltatin (115) and �-peltatin

(116)] in P. peltatum leaves on a reversed-phase C18 column using (A) water and (B)

acetonitrile, both containing 0.05% formic acid within 3 min [Avula et al. (2011)]. A

micellar electrokinetic chromatography (MEKC) method was reported for quantitative

analysis of seven lignans [4'-O-demethylpodophyllotoxin (117), epipodophyllotoxin (129),

picropodophyllin (126), podophyllotoxin (114), picropodophyllone (128),

podophyllotoxone (121), deoxypodophyllotoxin (120)] in P. emodi using 10 mM

NaH2PO4-5 mM borate-100 mM sodium dodecylsulfate- 30% isopropanol [Liu et al.

(2001)]. Later, seven pairs of diastereoisomeric (at C-2 and C-4 position) Podophyllum

lignans were separated by MEKC within 35 and 20 min, respectively [Liu et al. (2002a);

Liu et al. (2002b)].


58

1.3.4 Pharmacological and biological activities

The plant is used extensively for its medicinal antitumour, antifungal and

immunostimulatory properties of the rhizomes [Kamil and Dewick (1986); Foster (1993);

Canel et al. (2000)]. In the modern allopathic system of medicine, it has been utilized for

the treatment of various metabolic disorders, cancer, bacterial and viral infections [Gowdey

et al. (1995); Cobb (1990)], venereal warts [Beutner and Krogh (1990)], rheumatoid

artharalgia associated with limb numbness and infections of skin tissue [Wong et al.

(2000)], AIDS-associated Kaposis sarcoma and different cancers of brain, lung and bladder

[Blasko and Cordell (1998)]. P. hexandrum extracts has been found to offer radioprotection

by modulating free radical flux involving the role of lignans presents [Chawla et al.

(2006)]. A number of lignans isolated from Podophyllum species have shown a wide range

of biological activities such as antitumor, antimitotic and antiviral. Some of them exhibited

toxicity to fungi, insects and vertebrates.

1.3.4.1 Antitumor activity

The semisynthetic derivatives [etoposide (141) and teniposide (142)] of podophyllin

lignans (podophyllotoxin) were successfully applied as antitumor agents [Farkya et al.

(2004)]. The glucopyranoside derivatives of podophyllotoxin (114) and

deoxypodophyllotoxin (120) were recognized for antihyperlipidimic and antiproliferative

properties [Kusari et al. (2011)]. The ethanol and water extracts of root and rhizome,

podophyllotoxin (114), deoxypodophyllotoxin (120) and 4'-O-

demethyldeoxypodophyllotoxin (123) demonstrated cytotoxicity and inhibitory effects

towards cultured human and mice leukemia, breast, cervical and neuroblastoma cancer cell

lines (K562, MDA468, MCF7, L1210 and L7712, HeLa, SH-SY5Y) [Goel et al. (1998);

Chattopadhyay et al. (2004); Wang et al. (1997); Kumar et al. (2003); Zhang et al. (2005)].

Sequential doses of aqueous extract of P. hexandrum (a daily dose of 34.5 mg/kg b. w. for

15 days) enhanced tumour doubling time (TDT) from 1.94 +/- 0.26 days to 19.1 +/- 2.5

days [Goel et al. (1998)].

1.3.4.2 Radioprotective and antioxidant activities

P. hexandrum was investigated for its radioprotective capabilities, including free radical

scavenging, inhibition of apoptosis and cell cycle arrest-related activities both in vitro and

in vivo models [Arora et al. (2006)]. Methanolic, hydro-alcoholic and chloroform extracts

of P. hexandrum were reported to protect the mice when administered 1-2 h before lethal

whole-body 10 Gy and 20 Gy radiations [Goel et al. (2002); Goel et al. (2007)]. The

radioprotective properties were comparable to synthetic radioprotectors like diltiazem etc


59

[Goel et al. (1998)]. Various polyphenols and flavonoids in chloroform extract (REC-2006)

contributed to scavenge the radiation-induced radicals, inhibited superoxide anions,

prevented DNA damage and stimulated DNA repair [Chaudhary et al. (2011)].

Administration of herbal water extract reduced the apoptosis incidence of jejunum villi

cells that underwent radiations [Salin et al. (2001)], protect spermatogenesis disorder

[Samanta and Goel (2002)], physiological markers change [Goel et al. (2002)] and neuron

injury [Sajikumar and Goel (2003)] induced by radiation. Quercetin-3-O-�-D-glycoside

isolated from the hydroalcoholic extract exhibited protective effects on supra-lethal �-

radiation-induced lipids and proteins damage in renal and neuronal systems [Chawla et al.

(2005a); Chawla et al. (2005b)].

Podophyllotoxin (114) also showed protective effect on Co-�-radiation induced damage in

Saccharomyces cerevisiae yeast cells [Bala and Goel (2004)]. The water extracts produced

increased superoxide dismutase activity [Mittal et al. (2001)], decreased reactive oxygen

species and NO production [Gupta et al. (2003)], inhibited radiation-induced decrease in

mitochondrial membrane potential [Gupta et al. (2004)], decreased lipid peroxidation and

increased thiol content [Samanta et al. 2004)], and regulated the protein expressions related

to cell death [Kumar et al. (2005)]. The EtOAc extract showed concentration dependent

superoxide radical scavenging activity, hydrogen peroxide radical scavenging activity

[Ganie et al. (2011)]. The polar fraction (alcoholic) extract exhibited potent antioxidant

activity in terms of reducing power assay [Chawla et al. (2005a)]. The methanolic extract

showed potential antioxidant effects against free radical mediated damages; monitored by

assaying the activities of different antioxidant enzymes viz., superoxide dismutase (SOD),

glutathione peroxidase (GPx), glutathiaone reductase (GR) and glutathiaone S-transferase

(GST) [Ganie et al. (2010)].

1.3.4.3 Hepatoprotective activity

Oral administration of ethanol extracts of the root, rhizome and fruit, as well as

podophyllotoxin (114), inhibited increases in liver index, serum glutamic-pyruvic

transaminase and serum glutamic-oxaloacetic transaminase in CCl4-treated mice, exhibited

hepatoprotective effects. The EtOAc extract of P. hexandrum depicted a liver-protective

effect against CCl4-induced hepatotoxicity in male Wistar rats. Rats pretreated with EtOAc

extract at 20, 30, and 50 mg/kg dose prior to CCl4 administration (1 ml/kg, 1:1 in olive oil)

showed remarkably reduced CCl4-induced toxicity, particularly hepatotoxicity, by

inhibiting lipid peroxidation, suppressing alanine aminotransferase (ALT), aspartate

aminotransferase (AST), and lactate dehydrogenase (LDH) activities [Ganie et al. (2011)].


60


The water extract of P. hexandrum inhibited lipopolysaccharide-induced nitrite production,

interferon-�, interleukin-6 and tumor necrosis factor-� secretion in peritoneal macrophages,

and exhibited anti-inflammatory activities, in vitro [Prakash et al. (2005)].

Deoxypodophyllotoxin (120) demonstrated remarkable potential against Herpes simplex

virus, exhibited antiproliferative, antiplatelet aggregation, in vivo antiasthmatic, insecticidal

and antiallergic activities. Podophyllotoxin (114), deoxypodophyllotoxin (120), 4'-O-

demethyldehydropodophyllotoxin (130) and picropodophyllone (128) demonstrated

insecticidal, antimicrobial activities [Miyazawa et al. (1999); Rahman et al. (1995); Gao et

al. (2004); Kusari et al. (2011)]. The potent immunosuppressive activity of 4'-

demethyldeoxypodophyllotoxin (123) was shown by use of a T-cell-mediated immune

response with respect to their suppression of activated splenocytes [Gordaliza et al.

(1996)].

Numbers of reports are available on biological activities and chemical investigations, still

there is gap of systematic analytical procedures for detailed chemical investigations of the

plant growing in the subtropical regions of Himachal Pradesh. Thus, keeping in view the

importance of P. hexandrum in traditional system of medicine, the present work was

directed towards the phytochemical studies and development of new analytical methods for

quality assessment in P. hexandrum. In the following pages results and discussion followed

by experimental sections are described.



The rhizomes of P. hexandrum were sequentially extracted at room temperature using n-

hexane, chloroform, methanol and water. From chloroform extract, six known lignans

podophyllotoxin (114), 4'-O-demethylpodophyllotoxin (117), deoxypodophyllotoxin (120),

�-peltatin (116), podophyllotoxone (121), and isopicropodophyllone (122) and from

methanolic extract one lignan, podophyllotoxin-4-O-�-D-glucopyranoside (131) and four

flavonoids- kaempferol (80), quercetin (25), quercitrin (83) and rutin (84) were

respectively isolated and characterized.

1.3.5.1.1 Podophyllotoxin (114)

Compound 114 was obtained as colorless crystals. Its positive HRESI-QTOF-MS showed a


molecular formula C22H23O8 indicating 13 degrees of unsaturation. FT-IR spectrum of 114

showed the absorption bands at 3525 (-OH), 3490 (-OH) and 1765 (C=O) cm-1

. The


61

absorptions at 2838, 1580 and 1005 cm-1

represented C-H, C=C and C-O functionalities,

respectively. UV spectrum showed absorption maxima at �max 243 and 291 nm.

4a6 3

2'6'

12a

8

O

O

O

O

OCH3

OCH3

H3CO

OH

4a6

3

2'6'

12a

8

O

O

O

O

OCH3

OCH3

H3CO

OH

HMBC

Figure 1.3.1: Chemical structure and selected HMBC correlations of 114



Position �C (ppm) �H (ppm) m (J Hz) �C (ppm) �H (ppm) m (J Hz) �C (ppm)

1 44.2 4.48-4.53 m 8a 133.5 -

2 45.5 2.66-2.68 m 1' 135.6 -

2a 174.6 - 2' 108.6 6.34 s

3 40.9 2.66-2.68 m 3' 152.7 -

3a 71.5 3.96-4.00 m; 4.48-4.53 m 4' 137.4 -

4 72.9 4.66-4.69 d (9.0) 5' 152.7 -

4a 131.3 - 6' 108.6 6.34 s

5 106.4 7.10 s -OCH2O- 101.6 5.93 d (1.3), 5.92 d (1.3)

6 147.9 - C-3'-OCH3 56.4 3.64 s

7 147.8 - C-4'-OCH3 60.9 3.70 s

8 109.9 6.47 s C-5'-OCH3 56.4 3.64 s

1H NMR spectrum in CDCl3 indicated the presence of three methoxy groups at � 3.70 (3H,

s), 3.64 (6H, s), two methylene dioxy protons at � 5.93 (1H, d, J = 1.3 Hz), 5.92 (1H, d, J =

1.3 Hz) and three singlets of four aromatic protons at � 7.10 (1H, s), 6.47 (1H, s), 6.34 (2H,

s) (Table 1.3.1). Two singlets resonating at � 7.10 and 6.47 were assigned to the protons of

aromatic ring, placed para to each other. A 2H singlet at � 6.34 was assigned to the

aromatic protons (H-2' and H-6') of the phenyl substituent. The upfield shift of H-2' and H-

6' indicated that this benzene ring bears some electron donating substituent. The signals at

� 4.66-4.69 (1H, d, J = 9.0 Hz), 4.48-4.53 (1H, m), 2.66-2.68 (2H, m) were assigned to H-

4, H-1, H-2 and H-3 protons. Two multiplets at � 3.96-4.00 (1H, m) and 4.48-4.53 (1H, m)

were attributed to the H-3a � and � methylene protons sandwiched between a methine and

oxygen. 13

C NMR and DEPT spectral data revealed the existence of nine quaternary

carbons, eight methine carbons, two methylene carbons and three methoxy carbons. An

ester carbonyl carbon resonated at � 174.6 (C-2a), a methylenedioxy carbon appeared at �

101.6 and a downfield methylene carbon resonated at � 71.5 (C-3a). HMQC spectrum


62

showed interactions of H1-5 (� 7.10)/C-5; H1-8 (� 6.47)/C-8; H1-2' (� 6.34)/C-2', H1-6' (�

6.34)/C-6', H1-3a (� 3.96-4.00)/C-3a and H1-3a (� 4.48-4.53)/C-3a. HMBC correlation

displayed interactions of H1-2' (� 6.34)/C-3'; H1-6' (� 6.34)/C-5'; H1-5 (� 7.10)/C-7, C-4a;

H1-3a (� 3.96-4.00)/C-3 and H1-8 (� 6.47)/C-7, C-8a, C-1. Thus, on the basis of above

spectral data and comparison with previously known spectral values [Berkowitz et al.

(2000)], the structure of 114 was assigned as podophyllotoxin (Figure 1.3.1).

1.3.5.1.2 4'-O-Demethylpodophyllotoxin (117)

Compound 117 was isolated as colorless crystals. The molecular formula was determined

as C21H21O8 by HRESI-QTOF-MS with molecular ion peak at m/z 401.3843 [M+H]+

(calcd. 401.3866). UV spectrum showed absorptiom bands at �max 242, 291 and 296 (sh)

nm. IR bands at 3620, 3555, 1774 cm-1

were observed due to the presence of hydroxyl and

carbonyl groups, respectively. 1H NMR spectrum in CD3OD revealed proton signals of two

methoxyl groups at � 3.56 (6H, s) and four aromatic protons at � 6.99 (1H, s), 6.29 (1H, s),

6.28 (2H, s). Broad proton signals of -CH2- at � 5.76 (2H, br s) and the aliphatic proton

signals at � 4.54-4.57 (1H, d, J = 8.4 Hz), 4.39-4.41 (1H, m), 4.30-4.31 (1H, m), 3.90-3.96

(1H, m) and 2.58-2.68 (2H, m) were observed (Table 1.3.2). 13

C NMR spectrum contained

seventeen signals constituting twenty one carbons. Seven of the 13

C signals appeared to be

from saturated carbons, while ten signals appeared to be from unsaturated carbons as

evident from DEPT spectrum. One of the saturated carbons at � 102.7 was exceptionally

deshielded, indicating that it was probably bonded to two oxygens indicating a methylene

oxide group. One of the unsaturated carbon signals at � 177.2 (C-2a) indicated the presence

of a carbonyl group, possibly from an ester or an acid and while signal at � 72.9

represented a downfield methylene carbon. The aromatic ring substituted at C-1 contained

two pairs of identical carbons (the two methoxy carbons appearing at � 56.8, the two

carbons at which the -OCH3 groups are attached resonating at � 148.5 and the two carbons

ortho to the methoxy group resonating at � 109.6). The signals for the C-1' and C-4'

quaternary carbons were appeared at � 135.6 and 132.7, respectively.

4a6 3

2'6'

12a

8

O

O

O

O

OCH3

OH

H3CO

OH

O

O

O

O

OCH3

OH

H3CO

OH

H-H COSY

HMBC

Figure 1.3.2: Chemical structure and selected HMBC and COSY correlations of 117


63

HMBC interactions were observed for H1-5 (� 6.99)/C-7; H1-3a (� 3.90-3.96, 4.39-4.41)/C-

2a; H1-8 (� 6.29)/C-1, C-7, C-8a (Figure 1.3.2). Analysis of COSY data supported the

proton-proton network between H-1, H-2, H-3, H-3a and H-4 protons (Figure 1.3.2). Thus,

on the basis of above spectral data and comparison with previously known spectral values

[Xu et al. (2011)], the structure of compound 117 was assigned as 4'-O-

demethylpodophyllotoxin (Figure 1.3.2)

Table 1.3.2: 1H (300 MHz) and 13C NMR (75.4 MHz) data of 117 in CD3OD Position �C (ppm) �H (ppm) m (J Hz) Position �C (ppm) �H (ppm) m (J Hz)

1 45.2 4.30-4.31 m 8a 135.4 -

2 46.2 2.58-2.68 m 1' 135.6 -

2a 177.2 - 2' 109.6 6.28 s

3 41.8 2.58-2.68 m 3' 148.5 -

3a 72.9 3.90-3.96 m; 4.39-4.41 m 4' 132.7 -

4 73.0 4.54-4.57 d (8.4) 5' 148.5 -

4a 132.8 - 6' 109.6 6.28 s

5 107.5 6.99 s -OCH2O- 102.7 5.76 br s

6 148.7 - C-3'-OCH3 56.8 3.56 s

7 148.7 - C-5'-OCH3 56.8 3.56 s

8 110.6 6.29 s

1.3.5.1.3 Deoxypodophyllotoxin (120)

4a6 3

2'6'

12a

8

O

O

O

O

OCH3

OCH3

H3CO


Compound 120 was obtained as a white amorphous solid. Its positive HRESI-QTOF-MS

showed a molecular ion peak at m/z 399.4122 [M+H]+ (calculated 399.4138) corresponding

to the molecular formula C22

H23

O7. FT-IR spectrum indicated absorption maxima for

lactone carbonyl (1774 cm-1

) and C=C moieties (1590 cm-1

). UV spectrum showed

absorption maxima at �max 210, 240 (sh) and 290 nm. 1H NMR spectrum in CDCl3

indicated the presence of three methoxy groups at � 3.78 (3H, s), 3.73 (6H, s) two of which

were equivalent; two methylene dioxy protons at � 5.91 (2H, br s) and three singlets of four

aromatic protons at � 6.64 (1H, s), 6.50 (1H, s) and 6.34 (2H, s) (Table 1.3.3). Two singlets

resonating at � 6.64 (1H, s) and 6.50 (1H, s) were assigned to protons of the aromatic ring,


64

disposed para to each other. A 2H singlet at � 6.34 (1H, s) was assigned to the aromatic

protons (H-2' and H-6') of the phenyl substituent. The multiples at � 4.57 (1H, m) and 3.03-

3.07 (1H, m) were ascribed to H-1 and H-4 respectively. Overlapping multiples at � 2.71

(3H, m) were assigned to H-2, H-3 and H-4. Two multiplets at � 3.88 (1H, m) and 4.42

(1H, m) were attributed to the H-3a � and � methylene protons sandwiched between a

methine and oxygen.

Table 1.3.3: 1H (300 MHz) and 13C NMR (75.4 MHz) data of 120 in CDCl3 Position �C (ppm) �H (ppm) m (J Hz) Position �C (ppm) �H (ppm) m (J Hz)

1 43.9 4.57 m 8a 130.9 -

2 47.6 2.71 m 1' 136.4 -

2a 175.0 - 2' 108.7 6.34 s

3 32.9 2.71 m 3' 152.7 -

3a 72.1 3.88 m; 4.42 m 4' 137.5 -

4 33.2 2.71 m;

3.03-3.07 m 5' 152.7 -

4a 128.5 - 6' 108.7 6.34 s

5 108.7 6.64 s -OCH2O- 101.3 5.91 br s

6 147.2 - C-3'-OCH3 56.4 3.73 s

7 146.9 - C-4'-OCH3 60.8 3.78 s

8 110.6 6.50 s C-5'-OCH3 56.4 3.73 s

13C NMR spectrum contained eighteen signals constituting twenty two carbons. Eight of

the 13C signals appeared to be from saturated carbons, while ten signals appeared to be

from unsaturated carbons as evident from DEPT spectrum. The compound (120) possessed

spectroscopic data closely comparable to that of podophyllotoxin (114) except that there is

absence of hydroxyl group at C-4 position as evident from its NMR data (Table 1.3.3).

This was further confirmed by loss of 16 units in mass spectral data (m/z 399). On the basis

of spectral data and comparison with literature data [Hendrawati et al. (2011)], the

structure of compound 120 was assigned as deoxypodophyllotoxin (Figure 1.3.3).

1.3.5.1.4 �-Peltatin (116)



to the molecular formula C22H23O8. FT-IR spectrum indicated absorption maxima for

hydroxyl (3615 cm-1) and lactone carbonyl groups (1765 cm-1). UV spectrum showed

absorption maxima at �max 271 nm.


65

4a6 3

2'6'

12a

8

O

O

O

O

OCH3

OCH3

H3CO

OH

O

O

O

O

OCH3

OCH3

H3CO

OH

H-H COSY

HMBC





1 44.2 4.60 m 8a 137.3 -

2 33.1 2.70 m 1' 136.7 -

2a 175.7 - 2' 108.6 6.37 s

3 47.7 2.70 m 3' 152.8 -

3a 72.8 3.90-3.94 m; 4.49-4.51 m 4' 137.3 -

4 27.3 2.49-2.54 m; 3.19-3.24 m 5' 152.8 -

4a 118.6 - 6' 108.6 6.37 s

5 132.0 - -OCH2O- 101.9 5.92 br s

6 133.3 - C-3'-OCH3 56.5 3.75 s

7 147.6 - C-4'-OCH3 61.1 3.81 s

8 103.7 6.22 s C-5'-OCH3 56.5 3.75 s

1H,

13C NMR and DEPT spectral data revealed the existence of ten quaternary carbons, six

methine carbons, three methylene carbons and three methoxyl carbons. Both 1H and

13C

NMR spectra (Table 1.3.4) revealed close correspondence with those of podophyllotoxin

(114), however, one of the aromatic proton at C-5 carbon was replaced by an aromatic

hydroxyl group. In addition, there is absence of hydroxyl group at C-4 position as in

deoxypodophyllotoxin (120). HMBC interactions showed correlations of methylenedioxy

protons at �H 5.92 (2H, br s) to C-6 and C-7 carbons. The methylene protons at �H 2.49-

2.54 (1H, m), 3.19-3.24 (1H, m) showed correlations with hydroxyl bearing carbon (C-5).

Analysis of COSY data supported the proton-proton network between to H-1, H-2, H-3, H-

3a and H-4 (Figure 1.3.4). Thus, on the basis of above evidences and literature data

[Jackson and Dewick (1984); Schmidt et al. (2006)], the compound 116 was

unambiguously characterized as �-peltatin (Figure 1.3.4).

1.3.5.1.5 Podophyllotoxone (121)

Compound 121 was obtained as a white amorphous solid. The exact mass was obtained by

HRESI-QTOF-MS and was found to be m/z 413.3950 (calculated 413.3973), and


66

correspond to the molecular formula C22H21O8. UV spectrum displayed absorption bands at

�max 205, 234, 279 and 315 nm characteristic of a podophyllone skeleton [Gensler et al.

(1960)]. IR spectrum showed strong absorption bands at 1775 and 1665 cm-1, characteristic

of a lactone carbonyl and a ketone moiety conjugated with an aromatic ring, respectively.

The absorptions at 2842, 1576 and 1132 cm-1

represented C-H, C=C and C=O

functionalities, respectively. 1H NMR spectrum displayed signals representing four

aromatic protons, nine protons for three methoxy group and two pairs of methylene protons

(Table 1.3.5). The proton signals of two methoxy group at � 3.79 (3H, s) and 3.73 (6H, s)

four aromatic protons at � 7.52 (1H, s), 6.68 (1H, s) and 6.36 (2H, s), two methylene dioxy

protons at � 6.08 (1H, br s) and 6.06 (1H, br s) were observed. The downfield chemical

shift of H-5 (� 7.52) could be due to the electron withdrawing and magnetic anisotropy

effects of the carbonyl group at C-5. A set of geminally coupled diastereotopic protons

resonating at � 4.51-4.56 (1H, m) and 4.33 (1H, t, J = 9.8 Hz) represented a methylene

group (H-3a) sandwiched between an oxygen and a methine. The signals at � 3.45-3.52

(1H, m) and a doublet of doublet at � 3.27 (1H, dd, J = 15.5, 4.0 Hz) could be assigned to

H-2 and H-3 methine protons. A doublet at � 4.83 (1H, d, J = 3.7 Hz) was assigned to the

H-1 methine, appeared downfield due to the deshielding effect of the carbonyl group. 13

C

NMR and DEPT spectra showed nineteen signals representing twenty two carbons

containing ten quaternary, seven methine, two methylene and three methoxy methyl

carbons. HMBC spectrum showed correlations of H1-3 (�H 3.45-3.52)/C-5 and H1-5 (�H

7.52)/C-5. The other HMBC correlations were found to be similar to that of

podophyllotoxin (114). COSY spectrum displayed crosspeaks between signals at �H 4.51-

4.56 and 4.33 due to their geminal disposition, while the cross-peaks between �H 4.51-4.56,

and 4.33 represented vicinal coupling of methylenic protons with the neighbouring methine

proton at C-3 position (�H 3.45-3.52). Thus, on the basis of above evidences and literature

data [Rahman et al. (1995)], compound 121 was unambiguously characterized as

podophyllotoxone (Figure 1.3.5).

4a6 3

2'6'

12a

8

OO

O

OCH3

OCH3

H3CO

O

O

O

O

O

O

OCH3

OCH3

H3CO

O

H-H COSY

HMBC



67




1 46.8 4.83 d (3.7) 8a 128.4 -

2 44.8 3.27 dd (15.5, 4.0) 1' 132.3 -

2a 173.2 - 2' 107.8 6.36 s

3 43.6 3.45-3.52 m 3' 153.2 -

3a 67.1 4.33 t (9.8);

4.51-4.56 m

4' 137.8 -

4 192.6 - 5' 153.2 -

4a 141.7 - 6' 107.8 6.36 s

5 106.2 7.52 s -OCH2O- 102.6 6.06 br s;

6.08 br s

6 153.3 - C-3'-OCH3 56.4 3.73 s

7 148.3 - C-4'-OCH3 60.9 3.79 s

8 109.8 6.68 s C-5'-OCH3 56.4 3.73 s

1.3.5.1.6 Isopicropodophyllone (122)



to the molecular formula C22H21O8. In UV spectrum, absorption maxima were observed at

�max 205, 235, 270 and 321 nm, typical of a podophyllotone skeleton. IR spectrum showed

the presence of lactone and conjugated ketone carbonyls through the band at 1765 and

1669 cm-1

, respectively. 1H NMR spectrum showed the presence of four aromatic protons

at � 7.40 (1H, s), 6.67 (1H, s), 6.27 (2H, s) and three methoxy methyls at � 3.78 (3H, s) and

3.70 (6H, s) (Table 1.3.6). Two broad singlets at � 6.06 (1H, br s) and 6.04 (1H, br s) were

assigned to the methylene dioxy protons. A proton resonating at � 4.55 (1H, d, J = 5.3 Hz)

was ascribed to H-1. Overlapping multiplets at � 3.54-3.59 (2H, m) were assigned to H-2

and H-3 protons. Two multiplets at � 3.80-3.86 (1H, m) and 4.47-4.53 (1H, m) were

attributed to the H-3a � and � methylene protons sandwiched between a methine and

oxygen. 13

C NMR spectrum showed the presence of three methoxy, two methylenes, seven

methines and nine quaternary carbons.

OO

O

OCH3

OCH3

H3CO

O

5

8 1

3

3a

2a

2'

3'

6'

O

8a

Figure 1.3.6: Chemical structure of compound 122


68

Further investigations by 2D experiments (HMQC and HMBC) led to the conclusion that

the compound (122) has same structure as reported earlier for isopicropodophyllone.

Owing to the deficiency of a hydroxyl group at C-4 position, its protonated molecular ion

(m/z 413) could not produce the ion peak at m/z 397 by elimination of a water molecule.

However, by elimination of a trimethoxybenzene molecule, fragment ion at m/z 245 was

observed in their MS/MS spectra. This ion further produced the ion at m/z 201 after

elimination of a carbon dioxide molecule. Thus, on the basis of above evidences and

literature data [Jackson and Dewick (1984)], compound 122 was unambiguously

characterized as isopicropodophyllone (Figure 1.3.6).




1 44.2 4.55 d (5.3) 8a 128.7 -

2 45.0 3.54-3.59 m 1' 133.9 -

2a 175.4 - 2' 106.5 6.27 s

3 44.7 3.54-3.59 m 3' 153.3 -

3a 69.5 3.80-3.86 m; 4.47-4.53 m 4' 137.6 -

4 194.2 - 5' 153.3 -

4a 139.0 - 6' 106.5 6.27 s

5 106.0 7.40 s -OCH2O- 102.3 6.04, 6.06 brs

6 153.4 - C-3'-OCH3 56.1 3.70 s

7 148.3 - C-4'-OCH3 60.9 3.78 s

8 108.5 6.67 s C-5'-OCH3 56.1 3.70 s

1.3.5.1.7 Podophyllotoxin-4-O-�-D-glucopyranoside (17)

Compound 131 was obtained as white powder. Its molecular formula was determined as

C28H33O13 by HRESI-TOF-MS 599.5329 (calculated 599.5357). UV spectrum showed the

absorption bands at �max 210 and 285 nm, suggesting the existence of the benzene rings. IR

spectrum displayed the absorption bands at 3545, 3355 and 1789 cm-1

, suggesting the

existence of hydroxyl and carbonyl groups, respectively. 1H NMR spectrum revealed

proton signals of three methoxyl groups at � 3.85 (3H, s), 3.62 (6H, s), and four aromatic

protons at � 7.03 (1H, s), 6.84 (2H, s), 6.74 (1H, s), methylenedioxy protons at � 6.02 (2H,

br s), and six aliphatic proton signals at � 5.47 (1H, m), 5.06 (1H, m), 4.84 (2H, m) and

3.30 (2H, m) (Table 1.3.7). 13

C NMR and DEPT spectral data revealed the existence of

nine quaternary carbons, thriteen methine carbons, three methylene carbons and three

methoxyl carbons.


69

4a63

2'6'

12a

8

1''

6''

O

O

O

O

OCH3

OCH3

H3CO

O

OHO

OHOH

OH





1 40.6 3.30 m 3' 153.9 -

2 46.2 3.30 m 4' 138.6 -

2a 175.5 - 5' 153.9 -

3 45.1 4.84 m 6' 109.8 6.84 s

3a 72.7 5.06 m -OCH2O- 102.5 6.02 br s

4 79.7 5.47 m C-3'-OCH3 56.7 3.62 s

4a 132.9 - C-4'-OCH3 61.1 3.85 s

5 110.0 7.93 s C-5'-OCH3 56.7 3.62 s

6 148.7 - 1'' 104.1 5.06 m

7 148.4 - 2'' 75.5 4.05 m

8 110.4 6.74 s 3'' 79.4 4.23 m

8a 133.2 - 4'' 72.2 4.23 m

1' 137.4 - 5'' 79.1 4.23 m

2' 109.8 6.84 s 6'' 63.3 4.54-4.58 m

The carbon chemical shifts of the aglycone were very similar to those of podophyllotoxin

(114), in addition, there were signals present due to a sugar moiety. 1H and 13C NMR

spectral data showed one anomeric signal of proton at � 5.06 (1H, m) and a carbon at �

104.1, respectively, indicating it to be a monoglycoside. The sugar was identified as �-D-

glucopyranosyl on the basis of NMR spectral data. The C-1 of glucose was attached to the

4-OH of the aglycone part, as indicated by downfield C-4 chemical shift (� 79.7), and the

correlation of H-1 of glucose and C-4 of the aglycone in HMBC. Based on these findings

and literature data [Sultan et al. (2010); Canel et al. (2000)], the compound 131 was

identified as podophyllotoxin-4-O-�-D-glucopyranoside (Figure 1.3.7).


70

1.3.5.1.8 Kaempferol (80)

O

OH

HO

O

OH

OH


Compound 80 was isolated as a yellow amorphous powder. The positive ESI-QTOF-MS

showed a molecular ion peak at m/z 287.2449 [M+H]+ (calculated 287.2442) indicated the

molecular formula as C15H11O6. UV spectrum showed absorption maxima at �max 265 and

365 nm characteristic of flavonols. 1H NMR spectrum revealed a set of broad singlets at �

6.38 (1H, br s) and 6.17 (1H, br s) assigning to H-8 and H-6 protons respectively. The

presence of a set of A2B2 doublets at � 8.07 (2H, J = 8.5 Hz) and 6.89 (2H, J = 8.6 Hz)

each integrating for two protons were assigned to H-2', H-6' and H-3', H-5' respectively

(Table 1.3.8).




1 - - 8 94.5 6.38 s

2 148.1 - 8a 158.3 -

3 126.8 - 1' 128.8 -

4 177.4 - 2' 130.7 8.07 d (8.5)

4a 104.6 - 3' 116.4 6.89 d (8.6)

5 162.5 - 4' 160.6 -

6 99.3 6.17 br s 5' 116.4 6.89 d (8.6)

7 165.6 - 6' 130.7 8.07 d (8.5)

13C NMR spectrum showed six oxygenated carbons at � 165.6, 162.5, 160.6, 158.3, 148.1,

and 126.8 assignable to C-7, C-5 C-4', C-8a, C-2, and C-3, respectively. The signal at �

177.4 assigned to C-4 further suggested the presence of flavonol type of skeleton. Thus on

the basis of above spectral data and comparison with previously known spectral values

[Xiao et al. (2006)], the structure of compound 80 was assigned as kaempferol (Figure

1.3.8).

Other compounds were identified as quercetin (25), quercitrin (83) and rutin (84) have

been already discussed in the results and discussion part of Albizzia chinensis


71

1.3.5.2 Determination of major lignans and flavonoid by HPTLC

The lignans are interesting group of molecules because of their potent biological activities

[Gordaliza et al. (2000); Gordaliza et al. (2004)]. In recent years, polyphenols have also

become of prominent interest. High intake of flavonoids is associated with a variety of

human health benefits, including prevention of cancer, cardiovascular diseases, and

osteoporosis [Aalvarez et al. (2010); Kimata et al. (2000)].

The lignans and flavonoids were quantified simultaneously by HPLC [Liu and Jiao (2006);

Lin et al. (2008)] and separately by both HPLC [Kumar et al. (2008); Willfor et al. (2006)]

and HPTLC [Bhandari et al. (2007); Mishra et al. (2005)]. Yet no report has been

published till date on simultaneous quantification of lignans and flavonoids in P.

hexandrum by HPTLC that is simple and cost effective and an important tool for the

qualitative, semi-quantitative and quantitative phytochemical analysis of medicinal plants

for the development of TLC fingerprint profiles and estimation of chemical markers and

biomarkers [Bagul et al. (2005)]. Owing to the recent interest and in continuation to our

work on the development of rapid and simple methods for quality assessment of various

medicinal plants of commercial utility [Kaur et al. (2009)], we have optimized, developed

and validated a rapid, sensitive and accurate HPTLC method for the simultaneous

determination of lignans and flavonoid [4'-O-demethylpodophyllotoxin (117),

podophyllotoxin (114), kaempferol (80), podophyllotoxone (121) and

deoxypodophyllotoxin (120)] in P. hexandrum rhizomes.

1.3.5.2.1 Optimization of chromatographic conditions

Five different mobile phases such as acetonitrile:water, ethyl acetate:hexane,

chloroform:hexane and ethyl acetate:methanol:formic acid:water, toluene:ethyl

acetate:acetic acid in different proportions were tested for the separation of lignans and

flavonoids, using silica gel HPTLC plates. The best resolution was achieved with test

mixture of toluene:ethyl acetate:acetic acid (15:7.5:0.5, v/v). The resolution achieved by

the use of these solvent systems provided good separation of the lignans and flavonoid and

offered an advantage over the earlier reported methods [Mishra et al. (2005); Ahmad et al.

(2007)] that described the determination of only one compound i.e. podophyllotoxin in P.

hexandrum by RP-HPTLC using acetonitrile:H2O as mobile phase. In our method, on

applying toluene:ethyl acetate:acetic acid (15:7.5:0.5, v/v) as solvent system, we were able

to quantify four lignans and one flavonoid [4'-O-demethylpodophyllotoxin (117),

podophyllotoxin (114), kaempferol (80), podophyllotoxone (121) and

deoxypodophyllotoxin (120)] simultaneously. Thereafter, the effect of extracting solvents


72

was studied with respect to the content of major constituents. Various extracts viz.

chloroform, ethyl acetate, methanol, acetone, methanol:water and water were used to

evaluate the extraction efficiency. The five compounds i.e. 4'-O-demethylpodophyllotoxin

(117), podophyllotoxin (114), kaempferol (80), podophyllotoxone (121) and

deoxypodophyllotoxin (120) were quantified by TLC densitometric methods. The identity

of bands of compounds in the different samples was confirmed by the Rf values and by

overlaying the UV-Vis absorption spectrum with that of standards using the CAMAG TLC

Scanner 3.

1.3.5.2.2 Method validation

Linearity

Working stock solutions containing reference compounds of 4'-O-

demethylpodophyllotoxin (117), podophyllotoxin (114), kaempferol (80),

podophyllotoxone (121) and deoxypodophyllotoxin (120) were prepared in different

dilutions and applied on HPTLC plate for preparing five points linear calibration curves.

The solutions prepared for 4'-O-demethylpodophyllotoxin (117), podophyllotoxin (114),

podophyllotoxone (121) were applied at 1.0, 2.0, 4.0, 6.0 and 8.0 �l, while that of

kaempferol (80) and deoxypodophyllotoxin (120) were applied at 2.0, 4.0, 6.0, 8.0 and 10.0

�l. Sample solutions (5 �l) were applied on TLC plate with similar band pattern. The

calibration curves in this study were plotted between the amounts of analyte versus the

average response (peak area). A linear relationship was found in the calibration range of 1-

8 �g/band for 4'-O-demethylpodophyllotoxin (117), podophyllotoxin (114) and

podophyllotoxone (121) and 2-10 �g/band for kaempferol (80) and deoxypodophyllotoxin

(120). The regression data obtained showed a good linear relationship (r2 = 0.9903-0.9976)

(Table 1.3.9).

Specificity

The specificity of the method was ascertained by analyzing standards and sample. The

bands for four tested lignans [4'-O-demethylpodophyllotoxin (117), podophyllotoxin (114),

podophyllotoxone (121), deoxypodophyllotoxin (120)] and one flavonoid [kaempferol

(80)] in samples were confirmed by comparing the Rf and UV spectra of the spot with that

of standard. The peak purity of individual compounds was assessed by comparing the

spectra at peak start, peak apex, and peak end positions of the band.

Precision

For checking the instrumental precision, five bands of 2 �l for each of five compounds

were applied and analyzed according to the proposed method. The percentage relative


73

standard deviation (%RSD) for the instrumental precession were found to be 0.86, 2.85,

1.04, 1.71 and 1.34 for 4'-O-demethylpodophyllotoxin (117), podophyllotoxin (114),

kaempferol (80), podophyllotoxone (121), deoxypodophyllotoxin (120), respectively

(Table 1.3.9). Intraday precision were determined by applying different concentration

levels of five reference compounds five times within 1 day and over a period of 5 days for

interday precision. The intra- and inter-assay precisions were found in the range of 0.97-

2.92% and 0.94-2.87%, respectively in terms of % RSD.

Accuracy

The accuracy was tested by determination of recovery of the compounds in the sample. The

preanalyzed sample, i.e. methanol:water extract, was spiked with three different

concentrations (25, 50, 100%) of compounds, extracted in triplicate and then analyzed by

proposed HPTLC method. Results from measurement of recovery were in the range of

96.38 ± 1.92 to 101.84 ± 1.05% (Table 1.3.10).

Table 1.3.9: Method validation data for five detected compounds in the extract of P.

hexandrum rhizomes 117 114 80 121 120

Rf (± 0.02)a) 0.22 0.37 0.49 0.55 0.7

Regression Equation 2605.3+748.78x 1813.9+589.55x 4464.4+1436x 3327.1+1215.9x 1685.4+463.22x

r2 0.9918 0.9903 0.9976 0.9961 0.9927

Instrument Precisionb)

0.86 2.85 1.04 1.71 1.34

LOD (ng/band) 263 250 617 259 371

LOQ (ng/band) 868 875 1974 856 1188

Robustnessb)

Mobile phase

composition 2.34 1.87 2.35 2.32 2.88

Mobile phase volume 2.98 1.58 1.76 1.85 2.58

Duration of saturation 2.78 1.61 2.35 1.63 1.59

Developing distance 1.26 1.73 2.94 1.72 1.84

Time from spotting to

chromatography 1.22 2.44 2.45 2.04 2.30

Time from

chromatography to

scan

1.58 2.83 1.23 1.63 2.90

a)SD, b)%RSD (n = 5), 117= 4'-O-demethylpodophyllotoxin, 114= podophyllotoxin, 80= kaempferol, 121=

podophyllotoxone, 120= deoxypodophyllotoxin

Robustness

Robustness is a measure of the capacity of a method to remain unaffected by small but

deliberate variations in the method conditions, and is an indication of the reliability of the

method. The robustness was studied by determining the effects of small variation of mobile

phase composition (± 0.1 ml for each component), mobile phase volume (± 5%), chamber

saturation period (20, 30, 40 min) and development distance (80, 85, and 90 cm), time from

spotting to chromatography (5, 10 and 20 min), time from chromatography to scan (5, 10


74

and 20 min). The % RSD of peak areas was calculated for each parameter. The overall low

values of %RSD (1.22-2.98%) indicated the robustness of the method (Table 1.3.9).

Quantitation was not significantly effected by changing scanning wavelength by ± 5 nm.

Limits of detection (LOD) and quantification (LOQ)

The limits of detection (LOD) and quantification (LOQ) were determined by injecting a

series of dilute solutions of known concentration. LOD was determined based on the

lowest concentration detected by the instrument from the standard while the LOQ was

found based on the lowest concentration quantified in the sample. In order to estimate the

LOD and LOQ, blank methanol was spotted six times following the same method as

explained. LOD was determined at S/N of 3:1 and LOQ at S/N of 10:1. LOD and LOQ

were in the range of 250-617 and 856-1974 ng/band indicating a high sensitivity for the

investigated compounds (Table 1.3.9).

Table 1.3.10: Results from the recovery analysis to assess accuracy of the method

Amount present

[�g/mg]

Amount added

[�g/mg]

Amount

recovered [%]a)

117 3.99 1.00 96.46 ± 2.60

3.99 2.00 98.78 ± 1.69

3.99 4.00 101.84 ± 1.05

114 8.05 2.00 98.87 ± 1.05

8.05 4.00 97.04 ± 2.31

8.05 8.00 101.37 ± 1.58

80 3.08 0.75 97.04 ± 2.02

3.08 1.50 98.75 ± 0.67

3.08 3.00 96.38 ± 1.92

121 0.94 0.25 96.92 ± 2.18

0.94 0.50 97.45 ± 1.09

0.94 1.00 98.80 ± 1.98

20 1.47 0.35 97.80 ± 2.45

1.47 0.70 98.62 ± 1.24

1.47 1.40 97.79 ± 2.88 a)

Average ± %RSD (n = 3), 117= 4'-O-demethylpodophyllotoxin, 114= podophyllotoxin, 80= kaempferol,

121= podophyllotoxone, 120= deoxypodophyllotoxin

1.3.5.2.3 Quantitative evaluation of extracts

Thin layer chromatographic method was used for quantification of lignans and flavonoids

to resolve the compounds present in the extract. The mobile phase of toluene:ethyl

acetate:acetic acid (15:7.5:0.5, v/v) showed highest selectivity for resolution of lignans and

flavonoids. Five well-separated bands of 4'-O-demethylpodophyllotoxin (Rf 0.22),

podophyllotoxin (Rf 0.37), kaempferol (Rf 0.49), podophyllotoxone (Rf 0.55),

deoxypodophyllotoxin (Rf 0.70) were observed on the F254 TLC plate (Figure 1.3.9) and in

the densitogram (Figure 1.3.10).


75

Table 1.3.11: Amounts of detected compounds in P. hexandrum

117 a) 114 80 121 120

Ethyl acetate 3.96 ± 1.54b)

9.30 ± 1.33 3.58 ± 1.18 1.81 ± 0.70 2.51 ± 2.86

Chloroform 4.42 ± 1.06 9.92 ± 1.08 3.37 ± 1.11 1.86 ± 1.27 2.81 ± 1.35

Methanol 3.86 ± 1.91 9.14 ± 0.35 9.02 ± 0.85 1.45 ± 0.55 2.37 ± 1.08

Water 3.69 ± 0.77 4.26 ± 0.53 8.87 ± 0.79 n.d.c)

n.d.

Acetone 4.47 ± 2.12 9.81 ± 0.49 3.16 ± 2.03 1.86 ± 0.74 2.68 ± 1.73

Methanol:water (1:1) 3.99 ± 2.22 8.05 ± 0.90 3.08 ± 1.13 0.94 ± 0.47 1.47 ± 0.80 a)�g/mg of the dry weight of the plant sample, b)mean value (n = 3) ± %RSD, c)not detected, 117= 4'-O-

demethylpodophyllotoxin, 114= podophyllotoxin, 80= kaempferol, 121= podophyllotoxone, 120=

deoxypodophyllotoxin

Figure 1.3.9: CCD image of TLC plate of P. hexandrum at 254 nm. Lines: 1–5: standard

tracks, 6–11: sample tracks of ethyl acetate, chloroform, methanol, water, acetone and

methanol:water (1:1) extracts.

Figure 1.3.10: TLC densitogram of standards 4'-O-demethylpodophyllotoxin,

podophyllotoxin, kaempferol, podophyllotoxone and deoxypodophyllotoxin at 254 nm

All the five compounds were detected in different extracts in the range of 0.94-9.92 �g/mg

(Table 1.3.11) except in water extract in which low polar lignans (podophyllotoxone,

deoxypodophyllotoxin) were not detected. As evident from the table, chloroform was best

extracting solvent for lignans, but as far as extraction of flavonoids was concerned,

podophyllotoxone

podophyllotoxin

kaempferol

4'-demethylpodophyllotoxin

deoxypodophyllotoxin


76

methanol was found to be better extracting maximum content of kaempferol (9.02 �g/mg

of plant material). Podophyllotoxin was found in highest (9.92 �g/mg) and

podophyllotoxone in lowest content (0.94 �g/mg) in chloroform and methanol:water

extracts respectively.

1.3.5.3 Simultaneous determination of lignans and flavonoids by UPLC-MS

A simple, sensitive, selective, precise and robust ultra-performance liquid chromatography-

tandem mass spectrometry (UPLC-MS) method was developed and validated for

determination of seven compounds including three lignans [podophyllotoxin (114), 4'-O-

demethylpodophyllotoxin (117), podophyllotoxin-4-O-�-D-glucoside (131)] and four

flavonoids [rutin (84), quercitrin (83), quercetin (25), kaempferol (80)] in the extract of P.

hexandrum rhizomes. All the seven compounds were detected and quantified in the extract.

The chromatographic separation of compounds was achieved in less than 8 min by RP-

UPLC (BEH C18 column, 100 × 2.1 mm i.d., 1.7 �m) using linear gradient elution of water

(0.1% formic acid) and methanol:acetonitrile (25:75, v/v) with flow rate of 0.3 ml/min at

�max 290 nm.

1.3.5.3.1 Optimization of chromatographic conditions

To achieve better resolution in short period for seven compounds, the mobile phase was

standardized after several trials with ACN, methanol and water in various proportions. A

mobile phase consisting of water with 0.1% formic acid (solvent A) methanol:acetonitrile

(25:75, v/v) (solvent B) with a linear gradient elution as follows: 0-1 min, 28% B; 1.0-1.4

min, 28-30% B; 1.4-3.5 min, 30% B, 3.5-4.0 min, 30-50% B, 4.0-5.0 min, 50% B, 5.0-5.5

min, 50-28% B, 5.5-8.0 min, 28% B was finally selected in order to achieve optimal

separation, high sensitivity, and good peak shape. The peak resolution was also recorded

with variation in the column temperature. Column temperature was optimized

systematically from 25 to 40°C, and it was observed that all the components achieved a

baseline resolution at 35°C. Optimal chromatographic conditions were obtained after

running different mobile phases with a reversed-phase C18 column (BEH C18 column, 100

× 2.1 mm i.d., 1.7 �m). Seven compounds viz., rutin (RT: 1.23 min), quercitrin (RT: 1.77

min), quercetin (RT: 3.92 min), 4'-O-demethylpodophyllotoxin (RT: 4.26 min),

podophyllotoxin-4-O-�-D-glucoside (RT: 4.73 min), kaempferol (RT: 5.09 min) and

podophyllotoxin (RT: 5.31 min) were well resolved. The representative chromatograms of

the standard mixture and sample of P. hexandrum rhizome extract have been shown in

Figures 1.3.11 and 1.3.12, respectively. The chromatographic peaks were identified by

comparing their retention times with reference compounds and spiking of samples with the


77

reference compounds. The results indicated that compounds were well resolved and their

quantitative determination in P. hexandrum was possible.

Figure 1.3.11: UPLC chromatogram of standard mixture of lignans and flavonoids; peaks

5 = rutin, 8 = quercitrin, 12 = quercetin, 13 = 4'-O-demethylpodophyllotoxin, 14 =

podophyllotoxin-4-O-�-D-glucoside, 16 = kaempferol, 17 = podophyllotoxin

Rhizome extract

Figure 1.3.12: UPLC chromatogram of extract P. hexandrum rhizomes

1.3.5.3.2 Validation parameters

The standard solutions were injected in minimum of nine different concentrations and

linearity was observed with the regression coefficient (r2) > 0.99 presented in Table 1.3.12.

The calibration curves were linear in the range of 6.25-100 �g/ml for rutin, quercitrin,

quercetin, 12.5-200 �g/ml for podophyllotoxin-4-O-�-D-glucoside, 4'-O-

demethylpodophyllotoxin, podophyllotoxin, and 3.13-50 �g/ml for kaempferol (Table

1.3.12).

The LODs for rutin, quercitrin, quercetin, 4'-O-demethylpodophyllotoxin,

podophyllotoxin-4-O-�-D-glucoside, podophyllotoxin and kaempferol were 0.39, 0.39,

0.20, 0.78, 1.56, 0.10 and 0.39 �g/ml and the LOQs for same analytes were found to be

1.25, 1.29, 0.64, 2.03, 4.69, 0.32 and 1.13 �g/ml, respectively (Table 1.3.12). This

indicated that the proposed method exhibited a good sensitivity for the quantification of


78

lignans and flavonoids. The intra- and inter-day precisions (expressed in terms of %RSD)

were observed in the range of 0.33-1.10% and 0.78-2.80%, respectively, demonstrating

good precision of the proposed method.

The accuracy of the proposed method was expressed as the recovery of standard

compounds added to the pre-analyzed sample. Samples spiked with 50, 25, 12.5 �g/ml of

rutin, quercitrin, quercetin, 4'-O-demethylpodophyllotoxin, kaempferol and 200, 100, 50

�g/ml of podophyllotoxin-4-O-�-D-glucoside, podophyllotoxin were used in triplicate to

assess accuracy. The amount of compunds was calculated from related linear regression

equation. The percentage recovery ranged from 95.89 to 100.03% with %RSD values in

the range 0.46-2.93%, for the detected compounds (Table 1.3.13).

1.3.5.3.3 Quantification of major constitutes in the extract

The presence of lignans and flavonoids in the extract was confirmed by comparison of their

retention times and overlaying of UV spectra with those of standard compounds. The

methanol extract of P. hexandrum rhizomes showed the presence of twenty compounds out

of which seven compounds were identified and quantified by comparison of their tR and

UV spectra with those of reference standards analyzed under identical chromatographic

conditions (Table 1.3.12). The lignans content in the extract were found to be in higher

amount (5.05%) as compared to flavonoid content (0.67%). Podophyllotoxin was detected

in major amount (2.70%) followed by podophyllotoxin-4-O-�-D-glucoside (1.77%), and 4'-

O-demethylpodophyllotoxin (0.58%). Amongst flavonoids, quercitrin was found in higher

amount (0.24%) and quercetin in very low amount (0.15%).

Table 1.3.12: Method validation data for seven detected compounds in the extract of P.

hexandrum rhizomes

Analytes tR Regression

equation

Linearity

(�g/ml)

r2

LOD

(�g/ml)

LOQ

(�g/ml)

Intraday Precision

(n=5)a

Interday Precision

(n=5) a

%

(w/w)

84 1.23 y = 61.271x - 101.28 6.25-100 0.9987 0.39 1.25 1.05 1.01 0.18

83 1.77 y = 51.457x - 132.26 6.25-100 0.9969 0.39 1.29 0.72 2.68 0.24

25 3.92 y = 156.88x - 44.698 6.25-100 0.9999 0.20 0.64 0.59 2.71 0.10

117 4.26 y = 41.773x - 43.909 12.5-200 0.9996 0.78 2.03 0.65 1.46 0.58

131 4.73 y = 20.964x + 7.2129 12.5-200 0.9991 1.56 4.69 1.10 2.80 1.77

80 5.09 y = 129.16x + 25.646 3.13-50 0.9999 0.10 0.32 0.33 0.78 0.15

114 5.31 y = 36.065x - 35.072 12.5-200 1.0000 0.39 1.13 0.50 1.35 2.70

a%RSD, 84= rutin, 83= quercitrin, 25= quercetin, 117= 4'-O-demethylpodophyllotoxin, 131=

Podophyllotoxin-4-O-�-D-glucoside, 80= kaempferol, 114= deoxypodophyllotoxin


79

Table 1.3.13: Accuracy for the quantitative determination of seven compounds in the

extract of P. hexandrum rhizomes

Analytes Added

amount (�g/ml)

Average

Recovery (%) RSD (%)

84 50 97.23 0.46

25 95.89 0.86

12.5 99.17 0.69

83 50 97.56 2.93

25 100.03 2.27

12.5 96.37 1.20

25 50 98.33 1.51

25 97.67 2.12

12.5 97.86 2.19

117 50 97.24 1.17

25 97.36 0.46

12.5 98.31 1.88

131 200 99.61 0.68

100 99.04 0.51

50 98.90 2.03

80 50 98.90 1.41

25 96.52 2.14

12.5 97.52 1.94

114 200 99.31 2.21

100 96.98 2.43

50 99.28 0.85

84= rutin, 83 quercitrin, 25= quercetin, 117= 4'-O-demethylpodophyllotoxin, 131=

podophyllotoxin-4-O-�-D-glucoside, 80= kaempferol, 114= deoxypodophyllotoxin

1.3.5.3.4 Identification of constituents by UPLC-ESI-TOF-MS

Ultra-performance liquid chromatography coupled with electrospray ionization-quadrupole

time of flight mass spectrometry (UPLC-ESI-TOF-MS) was used to study phenolic

composition in the methanolic extract of P. hexandrum rhizomes. The phenolic

constituents were further investigated by ESI-TOF-MS/MS in a positive ion mode. Peaks

5, 8, 12-14, 16, and 17 were identified based on comparison with pure reference

compounds using UPLC, molecular weights and fragment ions by LC–MS/MS and UV

spectra. Peaks 6, 7, 9-11, 15 and 18-20 were identified based on molecular weights,

fragment ions and UV spectra. The molecular weights and the fragment ions were

summarized in Table 1.3.14.

By UPLC-MS analysis presence of twenty known and unknown compounds (lignans,

flavonoids, their glycosides, procyanidins) were detected. The lignans and flavonoids were

found to be glycosides of podophyllotoxin, 4'-O-demethylpodophyllotoxin, quercetin and

kaempferol. The lignans peaks were assigned unambiguously from their characteristic UV

spectra, displaying two absorption bands at �max 240 and 290 nm. The peaks (10, 13-15, 17)

showed characteristic absorption bands at �max 285-290 and 227-243 nm, indicating that the


80

presence of podophyllotoxin skeleton type lignans in the extract [Jackson and Dewick

(1984); Bastos et al. (1995)]. The absorption bands at 205, 234, 279, and 315 were found

as the characteristic of podophyllone type skeleton (peaks 18, 20). The UV spectrum

exhibited absorption bands at �max 232, 267, 298 and 361 nm, characteristic of a

tetradehydropodophyllotoxin lignan nucleus (peak 19) [Rahman et al. (1995)]. The UV

spectra suggested that the major flavonoids present in the extracts were flavan-3-ols. The

peaks showed characteristic absorption bands at �max 350-367 and 254-266 nm, indicating

that the flvonols in extracts were substituted in the 3-OH position [Lin et al. (2008); Liu

and Jiao (2006)].

Table 1.3.14: Identification of chemical constituents in methanolic extracts of rhizomes of

P. hexandrum

Peak tR

(min)

UV

spectra

Calcd

MW

(+) ion mode

Detected Compounds [M+H]+/

[M+Na]+

ms/ms (m/z)

1 0.78 210, 262 - - - Procyanidinc

2 0.94 210, 276 - - - Procyanidinc

3 1.02 210, 261 - - - Procyanidinc

4 1.18 210, 274 - - - Procyanidinc

5 1.23 257, 353 610 611 465, 303 Rutina

6 1.39 255, 254 464 465

303, 285, 213, 166,

145, 257, 229, 153,

111

Quercetin-3-O-

glycosideb

7 1.51 254, 352 464 465 303 Quercetin-3-O-

glycosideb

8 1.77 255, 347 448 449 303 Quercitrina

9 2.10 266, 345 449 471 287, 213, 153, 121 Kaempferol-3-O-

glycosideb

10 2.51 243, 285 562 585 383, 229, 333, 299,

267, 85

4'-O-demethyl podo

phyllotoxin glucosideb

11 2.73 265, 347 Kaempferol-3-O-

glycosideb

12 3.92 254, 370 302 303 257, 229, 201, 165,

153, 137, 109, 111, 69 Quercetina

13 4.26 242, 287 400 423 383, 333, 299, 247,

239, 229, 185, 147, 85

4'-O-Demethyl

podophyllotoxina

14 4.73 227, 290 576 599 397, 229, 313, 282, Podophyllotoxin-4-O-

�-glucosidea

15 4.90

235,

268sh,

292

415 397, 247, 229 Epipodophyllotoxinb

16 5.09 265, 365 286 287 287, 258, 213, 153 Kaempferola

17 5.31 235, 291 414 415

397, 351, 313, 282,

247, 229, 195, 169,

147, 85

Podophyllotoxina

18 5.67 236, 287,

324 412 413 245, 201, 353, Picropodophyllone

b

19 5.74 232, 267,

298, 361 410 397 353

4'-O-demethyldehydro

podophyllotoxinb

20 6.14 240, 263, 286, 327

412 413 245, 217, 201, 189,

169, 143, 115 Podophyllotoxone

b

aCompounds conclusively identified by comparison with authentic standard

bCompounds tentatively identified by UV and mass spectral data cSamples tentatively identified by UV spectral data


81

Four flavonoids were identified by comparison of their tR and UV spectra with those of

standard flavonoids analyzed under identical chromatographic conditions, namely, rutin

(peak 5), quercetin-3-O-rhamnoside (peak 8), quercetin (peak 12) and kaempferol (peak

16). On the basis of presence of polarity of phenolic groups, these flavonol glycosides were

eluted in the order of quercetin and kaempferol glycosides respectively. The remaining

peaks (peaks 1-4) show spectral characteristics close to those of procyanidins, their UV

�max values were close to with catechin-matching absorption spectra, i.e. �max 210 and 261-

274 nm.

1.3.5.3.5 ESI (+)-MS and ESI (+)-MS/MS analysis

Whilst UV data allowed the partial determination of structure, conclusive structural

information could be obtained from the LC-MS/MS analysis. Therefore, the phenols and

their glycosides were further characterized by UPLC-ESI-MS/MS analysis in positive ion

mode. The fragmentation patterns observed in the mass spectrum were useful in

characterization of lignans and flavonoids. The podophyllotoxin type aryltetralin lignans

(podophyllotoxin, 4'-O-demethylpodophyllotoxin and their glycosides) exhibited a

characteristic fragment ion [A+H]+ formed by loss of the C-4 oxygen substituent along

with the entire lactone ring followed by retro-Diels-Alder rearrangement leading to a

stabilised anthracene derivative (Figure 1.3.13) [Schmidt et al. (2006)].

OO

O

OH

X

O

X

H

OO

O

OH

O

X

H

H

[B + H]+

OO

O

X

O

-H2O

[M + H-H2O]+

O

OX

[A + H]+

H

H

OO

O

H O

H

-H2O

[B + H-H2O]+

X

H

Figure 1.3.13: ESI/MS fragmentation of P. hexandrum lignans

Secondly, the fragments were produced by loss of the pendant phenyl substituent [B+H]+

and subsequent loss of H2O from the lactone showed the dehydration product [B-H2O+H]+

with higher intensity. In case of podophyllone type skeleton (podophyllotoxone,

picropodophyllone), the protonated molecule [M+H]+ could not produce the fragment ion


82

by elimination of a water molecule. However, by elimination of a trimethoxybenzene

molecule, fragment ion [B+H]+ was observed in their MS/MS spectra. This ion further

produced the ion [B+H-CO2]+ after elimination of a carbon dioxide molecule. Twenty

compounds were detected in the P. hexandrum extract including five quercetin derivatives

(peaks 5-8, 12), three kaempferol derivatives (peaks 9, 11 and 16), six podophyllotoxin

derivatives (peaks 10, 13-15, 17, 19) and two podophyllone derivatives (peak 18 and 20).

Peaks 1-4 were expected to be procyanidins on the basis of UV spectra; however,

corresponding mass signal peaks were not observed for these peaks.

1.4 Experimental

1.4.1 Isolation, characterization and quantification of bioactive molecules from

Cedrus deodara (Roxb.) Loud.


1.4.1.1.1 Instrumentation and conditions

Optical rotation were determined on Horiba Sepa-300 Polarimeter, UV spectra performed

on a Shimadzu UV-2450 instrument (Japan) and IR spectra were obtained on a Nicolet

5700 FTIR (Thermo, USA) spectrophotometer in the region 4000-400 cm-1

using KBr

discs. Mass spectra were recorded on a Waters QTOF-MS with ESI using Waters

MassLynx 4.1 software. 1H and

13C NMR spectra were recorded in Bruker Avance-300.

HPLC analysis was performed on a Shimadzu Prominence HPLC system, equipped with

LC-20AT quaternary gradient pump, SPD-M20A diode array detector (DAD), CBM-20A

communication bus module, CTO-10AS VP column oven, Rheodyne injector and

Shimadzu LC solution (ver. 1.21 SP1) software. Peak purity of compounds was determined

on a LiChroCART® 250-4 LiChrospher

® 100 RP-18 (5 µm) column from Merck

(Darmstadt, Germany). Temperature of the column was set at 30ºC. The mobile phase was

isocratic and composed of water (A) and methanol (B) with a flow-rate of 1.0 ml/min.

Analysis wavelength was set at 290 nm. Column chromatography was carried out with

Merck silica gel 60-120, 230-400 mesh and RP-C18. TLC was run on Merck aluminium

pre-coated silica gel 60 F254 and RP C-18 plates. All the chemicals were purchased from

Merck India Ltd. GC analysis of the oil samples was performed on Shimazdu Gas

Chromatograph (GC 2010) using nitrogen as a carrier gas with a flow rate 1.0 ml/min,

equipped with FID detector and carbowax phase, BP-20 capillary column (30 m × 0.25 mm

i.d. with film thickness 0.25 �m). The injector temperature was programmed from 40-

220ºC @ 4ºC/min rise with 4 min hold at 40ºC and 15 min hold at 220ºC. Injector and

interface temperatures were 250ºC for both. Ion source temperature was 200ºC. Sample (20


83

�L) was dissolved in 2 ml GC grade dichloromethane (CH2CI2) and sample injection

volume was 2 �L. Relative percentages of constituents were calculated from the FID, the

automated integrator. GC–MS analysis was conducted on a Shimadzu QP2010 GC–MS

system with 2010 GC. A carbowax phase, BP-20 capillary column (30 m × 0.25 mm i.d.

with film thickness 0.25 �m) was used with helium as a carrier gas at a flow rate of 1.1

ml/min on split mode (1:50) using the same conditions as in GC-FID.

1.4.1.1.2 Plant material

Samples of C. deodara sawdust, woodchips and needles were deposited (PLP 5969) in the

Herbarium of Institute of Himalayan Bioresource Technology (CSIR), Palampur, India.

The plant material was air dried in shade, subjected to grinding to a coarse particle size

powder and stored at ambient temperature before extraction and analysis.

1.4.1.1.3 Isolation and characterization of chemical constituents

1.4.1.1.3.1 Extraction and isolation from sawdust

The sawdust of C. deodara (1.5 kg) was extracted with hexane (4×1L), chloroform (4×1L),

methanol (4×1L) and water (4×1L) at room temperature and filtered. The filtrates were

evaporated under reduced pressure to yield the respective extracts.

The hexane extract (8 g) was chromatographed on a silica gel column, eluting with a

gradient of hexane-EtOAc (1:0�0:1) to give ten fractions (A1-A10). Fraction A3 (1.5 g)

was subjected to silica gel CC with hexane-EtOAc (9.5:0.5�9:1) resulted in the isolation

of (E)-�-atlantone (8) (50.1 mg). The CHCl3 extract (30 g) was loaded on silica gel CC

eluting with a 100% CHCl3 to give eight fractions (B1-B8). The separation of fraction B2

(550 mg) on silica gel CC eluted with isocratic hexane: EtOAc (3:2) to yield fractions B2-1

to B2-7. Fraction B2-1 (230 mg) was further subjected to reverse phase C-18 CC using a

gradient of MeOH: H2O (1:3�1:1) to afford (E)-(2S, 3S, 6R)-atlantone-2,3-diol (57) (61.9

mg). The separation of subfraction B3 (1.91 g) over silica gel column eluting with isocratic

hexane: EtOAc (3:7) to give fractions B3-2 to B3-8. Fraction B3-2 (350 mg) was further

purified by reverse phase C-18 CC using MeOH: H2O (1:5�1:1) to give compound (E)-

(2S, 3S, 6S)-atlantone-2,3,6-triol (58) (57.5 mg). The fraction B8 (250 mg) was

chromatographed over reverse phase C-18 column using MeOH:H2O (0.5:9.5�1:1) to

afford atlantolone (15) (30.6 mg).

The air-dried powdered needles of C. deodara (3.0 kg) were extracted with ethanol (3×1L)

to afford crude extract (350.9 g). The crude extract was suspended in water, and

fractionated with petroleum ether, ethyl acetate, and n-butanol, separately. The petroleum

ether fraction (45.7 g) was chromatographed on a silica gel column by a gradient elution


84

with hexane–ethyl acetate (9:1�1:9) to yield protocatechuic acid (53) (20.2 mg). The ethyl

acetate extract (50.1 g) was subjected to silica gel column chromatography and eluted with

a gradient elution of methylene chloride- methanol (36:1�0:100) to give 20 fractions.

Fraction 6 (2.05 g) was applied on silica gel column and Sephadex LH-20 column to give

taxifolin (19) (29.7 mg). Similarly, fraction 10 (2.9 g) was then applied on silica gel

column and Sephadex LH-20 column to give myricetin (45) (31.6 mg).

1.4.1.1.3.2 Essential oil extraction and fractionation

Woodchips (1.5 kg) of C. deodara were subjected to hydrodistillation using a Clevenger-

type apparatus for 6 h. The oil was dried over anhyd. sodium sulphate and stored in airtight

containers at an ambient temperature until analysed. Woodchips (600 g) were extracted by

percolation technique with hexane (3 L) and this process was repeated thrice. After

filtration, the filtrate was evaporated to dryness at 40ºC under reduced pressure. The

essential oil (50 ml) was fractionated between n-pentane and acetonitrile (3×50 ml) (Figure

1.4.1). All the samples were evaporated to dryness at 40ºC under reduced pressure and

stored in refrigerator at 4�C prior to analysis by gas chromatography flame ionization

detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS).

Figure 1.4.1: Fractionation protocol for essential oil of C. deodara

1.4.1.1.3.3 Chromatography and characterization of essential oil fractions

Aliquots of n-pentane and acetonitrile fractions were subjected to chromatography over

silica gel and eluted sequentially with hexane/EtOAc gradients and finally with EtOAc.

Fractions were monitored by TLC and compounds with similar Rf values were pooled

together to give sub fractions. From n-pentane and acetonitrile fractions, mixture of

himachalenes and atlantones were isolated, respectively, and were identified by GC and

GC-MS analyses. Different fractions of essential oil were designated as A2-A6 (A2: crude

oil; A3: atlantones; A4: himachalenes A5: n-pentane fraction and A6: acetonitrile fraction).

(E)-�-Atlantone (8)

Cedrus deodara essential oil

Fractionation

Acetonitrile fraction

Himachalenes mixture Atlantones mixture

Column chromatography Column chromatography

n-Pentane fraction


85

Yellowish gum; [�]D25

+21° (c 0.50, MeOH); UV (MeOH): �max 268 nm; IR (KBr, cm-1

):

3139, 2979, 1665, 1614, 1216, 768; 1H NMR (CDCl3, 300 MHz): see Table 1.1.2; 13C

NMR (CDCl3, 75.4 MHz): see Table 1.1.2; HRMS-ESI: m/z [M+H]+ for C15H23O,

calculated 219.3425, observed 219.3411; MS-MS-ESI: m/z 219, 201, 161, 145, 125, 107,

83.

(E)-(2S, 3S, 6R)-atlantone-2,3-diol (57)

Light brownish gum; [�]D25 +10.0° (c 0.50, MeOH); UV (MeOH): �max 296 nm; IR (KBr,

cm-1): 3421, 2923, 1660, 1617, 1440, 1375, 1217, 1048, 769; 1H NMR (CD3OD, 300

MHz): see Table 1.1.3; 13C NMR (CD3OD, 75.4 MHz): see Table 1.1.3; HRMS-ESI: m/z

[M+H]+ for C15H25O3, calculated 253.3572, observed 253.3562; MS-MS-ESI: m/z 275,

253, 235, 217, 199, 189, 135, 119, 83, 59.

(E)-(2S, 3S, 6S)-atlantone-2,3,6-triol (58)

Brownish gum; [�]D25 +19.0° (c 0.50, MeOH); UV (MeOH): �max 293 nm; IR (KBr, cm-1):

3404, 2917, 1650, 1615, 1431, 1369, 1215, 1046, 770; 1H NMR (CD3OD, 300 MHz): see

Table 1.1.4; 13

C NMR (CD3OD, 75.4 MHz): see Table 1.1.4; HRMS-ESI: m/z [M+H]+ for

C15H25O4, calculated, 269.3566, observed 269.3581; MS-MS-ESI: m/z 291, 269, 251, 233,

215, 195, 177, 159, 135, 107, 95, 83.

Atlantolone (15)

Brownish oil; [�]D25 +9° (c 0.50, MeOH); UV (MeOH): �max 241 nm; IR (KBr, cm-1):

3435, 2914, 1664, 1604, 1432, 1371, 1217, 771; 1H NMR (CDCl3, 300 MHz): see Table

1.1.5; 13

C NMR (CDCl3, 75.4 MHz): see Table 1.1.5; HRMS-ESI: m/z [M+H]+ for

C15H25O2, calculated 237.3578, observed 237.3563; MS-MS-ESI: m/z 237, 219, 179, 163,

135, 107, 69.

Protocatechuic acid (53)

White amorphous powder; mp 198-201°C; IR (KBr, cm-1): 3456, 3223, 1680, 1615, 1544,

1254, 1184, 1167; 1H NMR (CD3OD, 300 MHz): see Table 1.1.6; 13C NMR (CD3OD, 75.4

MHz): see Table 1.1.6; HRMS-ESI: m/z [M+H]+ for C7H7O4, calculated 155.1281,

observed 155.1258; MS-MS-ESI: m/z 155, 137, 111.

Taxifolin (19)

Yellow amorphous powder; mp 241-243°C; [�]D25

+50° (c 0.50, MeOH); IR (KBr, cm-1

):

3566, 1663, 1588, 1485, 1443, 1128, 1028; 1H NMR (CD3OD, 300 MHz): see Table 1.1.7;

13C NMR (CD3OD, 75.4 MHz): see Table 1.1.7; HRMS-ESI: m/z [M+H]+ for C15H13O7,


153, 123, 108.


86

Myricetin (45)

Yellow amorphous powder; mp 230-231°C; UV (EtOH): �max 296, 355 nm; IR (KBr, cm-

1): 3556, 1675, 1590, 1535, 1490, 1455, 1130, 1025; 1H NMR (CD3OD, 300 MHz): see

Table 1.1.8; 13


C15H11O8, calculated 319.2430, observed 319.2411; MS-MS-ESI: m/z 319, 273, 245, 217,

179, 165, 153, 137, 111.

Himachalenes (1-3)

Colorless oil; IR (KBr, cm-1): 3056, 1606, 1643, 1362, 720, 945; 1H NMR (CDCl3, 300

MHz): � 5.49, 5.42, 5.36, 4.71, 4.66, 2.83, 2.77, 2.58, 2.56, 2.32, 2.08, 1.80, 1.79, 1.68,

1.65, 1.61, 1.50, 1.38, 1.37, 1.32, 0.94, 0.92, 0.91; 13

C NMR (CDCl3, 75.4 MHz): � 157.7,

137.9, 134.6, 134.0, 133.8, 131.2, 129.0, 126.5, 125.4, 124.5, 122.5, 111.2, 47.7, 47.3,

46.0, 45.0, 42.8, 39.9, 39.1, 38.2, 36.5, 34.7, 34.5, 34.0, 32.0, 31.5, 30.1, 29.4, 26.5, 26.0,

25.0, 24.0, 23.6, 22.6, 20.1; GC-MS: m/z 204, 189, 175, 161, 147, 134, 119, 105, 93, 79,

69, 55.

1.4.1.1.4 Analysis of extract, essential oil and its fractions

Kovats indices (KI) of the compounds relative to a mixture of n-alkanes (C8-C23) were

calculated. Identification of compounds was first attempted using mass spectral libraries

Wiley 7 and NIST 02 [McLafferty (1989); Stein (1990)]. Corroboration of the

identification was conducted by matching the mass spectra of compounds with those

present in our own library and in the literature [Adams (1995); Jennings and Shibamoto

(1980)] and finally by matching the KI of the compounds reported on column having

equivalent binding phase.

1.4.1.1.5 Determination of flavonoids by UPLC-MS in C. deodara needles extract

1.4.1.1.5.1 Chemicals

Standard compounds myretin, quercitrin, quercetin were purchased from Sigma-Aldrich.

Taxifolin was isolated by chromatographic methods, and identified by spectral data (IR,

1D- and 2D-NMR, ESI-MS) that was compared with published spectral data [Sakushima et

al. (2002); Kuspradini and Ohashi (2009)]. Acetonitrile, water and formic acid were of

HPLC grade, purchased from J. T. Baker (USA).

1.4.1.1.5.2 Preparation of standard solutions

An individual stock solution of standard compounds containing taxifolin, quercitrin,

myricetin and quercetin was prepared by dissolving approximately 5 mg each, accurately

weighed, in 50 ml methanol in volumetric flasks.

1.4.1.1.5.3 Sample Preparation


87

Comparison of extraction techniques

Comparison of four extraction techniques, i.e., UAE, percolation, soxhlet and maceration

were carried out using single optimized extraction solvent mixture. All the extracts were

analyzed by UPLC for the extracted content of individual flavonoid.

Ultrasound assisted extraction (UAE)

Powdered plant material (200 mg) was sonicated for 20 min at a controlled temperature (40

± 5°C) with 20 ml of methanol in an ultrasonicator bath. The extracts were filtered and

concentrated to dryness under vacuum (temperature, 40-45°C) and then subjected to

lyophilization until a constant weight was obtained.

Microwave-assisted extraction (MAE)

Powdered plant material (200 mg) was extracted with 20 ml of methanol in a domestic

microwave for 20 min at 100 W microwave power. The extracts were filtered and

concentrated to dryness under vacuum (temperature, 40-45°C) and then subjected to

lyophilization until a constant weight was obtained.

Soxhlet Extraction

Powdered plant material (200 mg) was extracted with 50 ml of methanol for 8 h in a

Soxhlet apparatus. The extracts were filtered and concentrated to dryness under vacuum

(temperature, 40-45°C) and then subjected to lyophilization until a constant weight was

obtained.

Maceration

Powdered plant material (200 mg) was packed in a percolator and soaked with 20 ml of

methanol through the powder packing and collected. The collected percolations were

filtered and residues were washed with methanol. The extracts were concentrated to

dryness under vacuum (temperature, 40-45°C) and then subjected to lyophilization until a

constant weight was obtained.

All of the extractions were performed in triplicate. All samples were kept in a nitrogen

atmosphere and at -20°C until further use. For the quantitative determination of compounds

by UPLC, concentrated extracts were dissolved in 2 ml of methanol (analytical grade). The

extracts were filtered through a 0.22 �m membrane filters prior to use.

1.4.1.1.5.4 Instrumentation and chromatographic conditions

The analysis was performed using a Waters ACQUITY™ UPLC system (Waters, Milford,

MA, USA). An ACQUITY UPLC® BEH C18 column (100 × 2.1 mm i.d., 1.7 �m), also

from Waters, was used for achieving separation. The column and sample temperature were

maintained at 35°C and 15°C, respectively. The mobile phase consisted of (A) 0.05%


88

formic acid in water and (B) acetonitrile at a flow rate of 0.3 ml/min. Gradient elution was

employed starting at 15% B for 2.5 min, then shifted linearly B to 30% for 1.5 min, held at

30% for 1.5 min, shifted B to 15% for 0.5 min and re-equilibrated for 2.0 min, giving a

total cycle time of 8.0 min. The injection volume was 1 �L with partial loop injection using

needle overfill mode. The peaks were detected at 254 nm. Strong needle wash solution

(90:5, acetonitrile-water) and weak needle wash solution (10:90, acetonitrile-water) were

used. All the solutions mentioned were filtered via 0.22 �m membranes under vacuum and

degassed before their usage. A time-of-flight mass spectrometer with electrospray

ionization (ESI-MS) inter face was used for fingerprinting (Micromass, Manchester, UK).

For UPLC analysis, data acquisition was performed using positive ion mode over a mass

range of m/z 50-1000. The general conditions were: source temperature 80ºC, capillary

voltage 3.1 kV and cone voltage 23 V. Positive ion ESI-MS analysis was performed by

direct infusion with a flow rate of 10 �L/min using a syringe pump. Mass spectra were

acquired and accumulated over 60 sec and spectra were scanned in the range between 50

and 1000 m/z. MassLynx 4.1 (Waters, Manchester, UK) was used for data analysis.

Tandem mass spectrometry for structural analysis of single molecular ion in the mass

spectra from needles extracts was performed by mass-selecting the ion of interest, and in

turn submitted to 15-35 eV collisions with argon in the collision quadrupole.

1.4.1.1.5.5 Method validation

For validation of the developed method, various parameters- linearity, analytical limits,

repeatability, accuracy and recovery were examined using ICH guidelines.

Calibration curves

Stock solution containing four analytes were prepared and diluted to appropriate

concentration in the range of from 0.39-100 �g/ml for establishing calibration curves. For

quantitative analysis, seven different concentrations of four analytes were injected in

triplicate. The calibration curves were constructed by plotting the peak areas versus the

concentration of each analyte.

Selectivity

The selectivity of the method was determined by analysis of standard compounds and

samples. The peaks of compounds were identified by comparing their retention times and

UV spectra with those of the standards.


89

Limit of detection (LOD) and quantification (LOQ)

In order to estimate the LOD and LOQ, blank methanol was spotted six times following the

same method as explained. Limit of detection was determined at a signal to noise ratio of

3:1 and limit of quantitation at a signal-to-noise ratio of 10:1.

Precision and accuracy

The measurements of intra-day and inter-day variability were utilized to assess the

repeatability and reproducibility of the developed method. Intra- and inter-day precisions

(expressed as %RSD) for the four compounds were determined by applying different

concentration levels of five reference compounds five times within 1 day and over a period

of 5 days for interday precision.

Accuracy was evaluated by means of recovery assays carried out by adding known

amounts of the reference compounds to the sample solutions. The amounts of analytes

added correspond to 25, 50, and 100% of compounds concentrations in samples. The

spiked samples were extracted in triplicate and analyzed under the above-mentioned

conditions. The percent recovery and average percent recoveries were calculated.

1.4.2 Isolation, characterization and quantification of bioactive molecules from

Albizzia chinensis (Osbek) Merril



Optical rotation were determined on Horiba Sepa-300 Polarimeter, UV spectra performed

on a Shimadzu UV-2450 instrument (Japan). Mass spectra were recorded on a Waters

QTOF-MS with ESI using Waters MassLynx 4.1 software (Micromass, Manchester, UK).

1H and

13C NMR spectra were recorded in Bruker Avance-300. Column chromatography

was carried out with Merck silica gel 60-120, 230-400 mesh and RP-C18. TLC was run on

Merck aluminium pre-coated silica gel 60 F254 and RP-18 plates. TLC plates were

visualized by the UV irradiation (254 and 365 nm), iodine spray and visualising agents. All

the chemicals were purchased from Merck India Ltd.


The plant material was collected from Palampur region of Himachal Pradesh. The samples

were authenticated (PLP 11352) by biodiversity department (CSIR-IHBT) and voucher

specimens were deposited in our herbarium section. The flowers were air dried in shade,

subjected to grinding to a coarse particle size powder and stored at ambient temperature.


90


The air dried powder of A. chiensis flowers (1.5 kg) was extracted with 90% ethanol (1L ×

3) to afford crude extract. The crude extract (200.7 g) was then suspended in H2O (1L × 3),

extracted with n-hexane (1L × 3), EtOAc (1L × 3) and n-BuOH (1 L× 3), successively. The

extracts were concentrated in vacuo to yield semisolid mass. Ethyl acetate soluble part

(45.3 g) was subjected to dry column chromatography on silica gel H. Elution was carried

out in varying percentage of EtOAc:hexane to give fractions A-G. Fractions A-C (01:99,

05:95 and 15:85, v/v, EtOAc:hexane) were pooled and on column chromatography over

silica gel (230-400 mesh) by elution with EtOAc/hexane (05:95, 08:92, 10:90) afforded

three fractions H, I and J. Fraction H was crystallized to yield kaempferol (80) (29.1 mg).

The fractions I and J on silica gel (60-120 mesh) column chromatography on elution with

MeOH:CHCl3 (08:92�12:88, v/v) yielded quercetin (25) (35 mg) and myricetin (45) (33.4

mg).

Fractions D and E on silica gel (230-400 mesh) column chromatography led to fractions K,

L and M (15:85�40:60, v/v, EtOAc/hexane). Fractions K and L resulted in the isolation of

kaempferol-3-O-�-L-arabinofuranoside (108) (17.3 mg) and quercitrin (83) (116.4 mg) and

quercetin-3-O-�-L-arabinofuranoside (107) (22.8 mg) on silica gel (60-120 mesh) column

chromatography on elution with MeOH/CHCl3 (05:95�30:70, v/v). n-Butanol soluble part

(55.7 g) was subjected to dry column chromatography on silica gel H. Elution was carried

out in varying percentage of MeOH:CHCl3 (10:90�70:30, v/v) give fractions N1-N5.

Fractions N1-N3 were pooled and on column chromatography over silica gel (60-120

mesh) by elution with EtOAc/hexane (15:85�40:60, v/v) afforded quercetin-3-O-�-L-

galactopyranoside (92) (16.6 mg), myricetin-3-O-�-L-rhamnopyranoside (myricitrin) (109)

(45 mg) and rutin (84) (27.1 mg).

Dried bark powder of A. chinensis (1.5 kg) were extracted with MeOH (1L � 3) at room

temperature and the solvent was removed under reduced pressure to afford a brown

semisolid mass. One portion (100.5 g) of this mass was suspended in water, and extracted

with petroleum ether, ethyl acetate, and n-butanol, separately. The petroleum ether residue

(35 g) was chromatographed on a silica gel column gradiently eluted with petroleum

ether:EtOAc (9:1�1:9) to yield compounds �-sitosterol (48) (25 mg). The EtOAc fraction

was chromatographed on a silica gel column gradiently eluted with MeOH:CHCl3

(1:10�1:0) to afford catechin (110) (29.7 mg). Second portion of the methanolic extarct

was redissolved in water and adjusted to pH 2.0 with 2 N HCl before being extracted twice

with EtOAc. The EtOAc-soluble acidic phases were combined and partitioned twice


91

between a 5% sodium hydrogen carbonate solution. The EtOAc-soluble neutral phases

were combined and concentrated in vacuo. The resulting residue (18.9 g) was first

fractionated by column chromatography on silica gel (EtOAc/hexane 1:5�1:1) to give five

fractions P1-P5. Fraction P5, obtained by elution with 40% EtOAc, was chromatographed

on a silica gel column (CHCl3/MeOH). Fraction P51, obtained by elution with 2%

MeOH/CHCl3, was further chromatographed on a silica gel column (EtOAc/n-hexane

1:3�2:3) to afford ferulic acid (112) (16.3 mg) and caffeic acid (113) (22.3 mg).

Structures of compounds were elucidated by NMR and ESI-MS-MS spectral data and

further confirmed by comparing the spectral data with literature values [Manguro et al.

(2004); Yoshioka et al. 2004; Xiao et al. (2006); He et al. (2009)].

Quercetin-3-O-�-L-rhamnoside (83)

Yellow powder; UV (MeOH): �max 255, 350 nm; 1H NMR (CD3OD, 300 MHz): see Table

1.2.1; 13C NMR (CD3OD, 75.4 MHz): see Table 1.2.1; HRMS-ESI: m/z [M+H]+ for

C21H21O11, calculated 449.3848, observed 449.3824; MS-ESI: m/z 449, 303, 147.

Quercetin (25)

Yellow powder; UV (MeOH): �max 256, 372 nm; 1H NMR (C5D5N, 300 MHz): see Table

1.2.2; 13

C NMR (C5D5N, 75.4 MHz): see Table 1.2.2; HRMS-ESI: m/z [M+H]+ for

C15H11O7, calculated 303.2436, observed 303.0518; MS-ESI: m/z 303, 285, 257, 247, 229,

219, 201, 183, 165, 153, 137, 109, 81, 69.

Rutin (84)


1.2.3; 13


C27H30O16, calculated 611.5254, observed 611.5237; MS-ESI: m/z 611, 465, 303, 147.

Quercetin-3-O-�-L-arabinofuranoside (108)

Yellow powder; m. p. 220-224°C; UV (MeOH): �max 255, 356 nm; 1H NMR (CD3OD, 300

MHz): see Table 1.2.4; 13C NMR (CD3OD, 75.4 MHz): see Table 1.2.4; MS-ESI: m/z 303

[M+H] for C20H19O11, 245, 209, 102.

Quercetin-3-O-�-L-galactopyranoside (92)

Yellow powder; m. p. 234-236 °C; UV (MeOH): �max 256, 354 nm; 1H NMR (CD3OD, 300

MHz): see Table 1.2.5; 13

C NMR (CD3OD, 75.4 MHz): see Table 1.2.5; MS-ESI: m/z 465

[M+H] for C21H20O12, 383, 367, 303, 229, 205, 175, 121, 109

Kaempferol-3-O-�-L-arabinofuranoside (109)


92

Yellow powder; m. p. 229-232 °C; [�]D25

-124° (c 0.50, MeOH); UV (MeOH): �max 265,

356 nm; 1H NMR (CD3OD, 300 MHz): see Table 1.2.6; 13C NMR (CD3OD, 75.4 MHz):

see Table 1.2.6; MS-ESI: m/z 419 [M+H]+ for C20H19O10, 404, 349, 326, 287.

Myricetin (45)


1.2.7; 13


C15H11O8, calculated 319.2430, observed 319.2411; ESI-MS: m/z 319, 290, 273, 245, 217,

195, 189.

Myricetin-3-O-�-L-rhamnopyranoside (110)

Yellow powder; m. p. 195-197 °C; UV (MeOH): �max 258, 352 nm; 1H NMR (CD3OD, 300


C NMR (CD3OD, 75.4 MHz): see Table 1.2.8; HRMS-ESI:,

calculated 319.2430, observed 319.2411; ESI-MS: m/z 465 [M+H]+ for C21H21O12, 361,

341, 319, 205, 175.

Catechin (111)

White powder; m. p. 172-175 °C; [�]D25

+25° (c 0.50, MeOH); UV (MeOH): �max 220, 280

nm; 1H NMR (CD3OD, 300 MHz): see Table 1.2.9;

13C NMR (CD3OD, 75.4 MHz): see

Table 1.2.9; HRMS-ESI: m/z [M+H]+ for C15H15O6, calculated 291.2760, observed

291.2736. ESI-MS: m/z 291, 273, 165, 139, 123.

Ferulic acid(112)

White powder; 1H NMR (CD3OD, 300 MHz): see Table 1.2.11; 13C NMR (CD3OD, 75.4


observed 195.1937; ESI-MS: m/z 195, 177, 145.

Caffeic acid (113)

White powder; 1H NMR (CD3OD, 300 MHz): see Table 1.2.12;

13C NMR (CD3OD, 75.4


observed 181.1626; ESI-MS: m/z 181, 163, 145.

�-Sitosterol (48)

Colorless needles; m.p. 137-140°C; 1H NMR (CD3OD, 300 MHz): see Table 1.2.13;

13C

NMR (CD3OD, 75.4 MHz): see Table 1.2.13.

1.4.2.2 Identification of phenolic compounds by UPLC-DAD-ESI-QTOF-MS in A.

chinensis


The aerial parts (flower, leaves, pods and bark) of A. chinensis were collected from around

the campus of the IHBT (CSIR), Palampur, India, in May 2007. Voucher specimens were


93

deposited (PLP 11352) in the Herbarium of IHBT. After harvest, each of the plant

materials was oven dried at a temperature of 40ºC, pulverized and stored at ambient

temperature (27ºC) before analysis.

1.4.2.2.2 Sample preparation

The powdered plant material (1 g) was sonicated with 25 ml of ethanol in an ultrasonicator

bath (Elma Ultrasonic, Germany) at 35 ± 5ºC for 30-60 min. An extraction time of 40 min

was taken as optimum on a mass yield basis. The extracts were filtered, concentrated to

dryness under vacuum at 40-45ºC, and lyophilized to constant weight.

1.4.2.2.3 Instrumentation and separation conditions

UPLC was performed using a Waters ACQUITY UPLC System (Waters, Milford MA,

USA). Separation was achieved using an ACQUITY UPLC® BEH C18 column (100 mm,

2.1 mm i.d., 1.7 �m particle size; Waters) maintained at 28ºC, with a mobile phase flow

rate of 0.275 ml/min. The system operating pressure was 11000 psi at initial gradient

conditions. The mobile phase contained water, 0.05% formic acid (A) and methanol (B).

Gradient elution was employed starting at 35% B, held for 1.0 min, then shifting linearly B

to 40% over 3.0 min, 50% over 5.0 min, then decreasing to 40% over 6.5 min, 35% over

7.0 min and re- equilibrated for 1.0 min, giving a total cycle time of 8.0 min. The injection

volume was 1 �L with partial loop injection using needle over fill mode. The peaks were

detected at 254 and 360 nm.

A time-of-flight mass spectrometer with electrospray ionization (ESI-MS) inter face was

used for fingerprinting (Micromass, Manchester, UK). For UPLC analysis, data acquisition

was performed using positive ion mode over a mass range of m/z 50-1000. The general

conditions were: source temperature of 80ºC, capillary voltage of 3.1 kV and cone voltage

of 23 V. Positive ion ESI-MS analysis was performed by direct infusion with flow rate of

10 �l min-1 using a syringe pump. Mass spectra were acquired and accumulated over 60 s

and spectra were scanned in the range between 50 and 1000 m/z. Mass Lynx 4.1 (Waters,

Manchester, UK) was used for data analysis. Structural analysis of single molecular ion in

the mass spectra from flower, leaf, bark and pods extracts was performed by mass-selecting

the ion of interest, which was in turn submitted to 15-35 eV collisions with argon in the

collision quadrupole.

1.4.2.3 Determination of total phenolic content

The total phenolic content was measured using Folin-Ciocalteu’s method [Swain and Hillis

(1959)]. For preparation of a calibration curve 20, 40, 60, 80, and 100 �L aliquots of

aqueous gallic acid were mixed with 0.5 ml of 1 N Folin-Ciocalteu’s phenol reagent and


94

1.0 ml of 35% Na2CO3 (w/v) in a 25 ml volumetric flask and the solution was made to 25

ml in distilled water. After 35 min of incubation at ambient temperature the absorbance

relative to that of the blank was measured using a XP-2001 Explorer UV

spectrophotometer at 730 nm. The total phenolic content of the sample is expressed as mg

of gallic acid equivalent (GAE)/g of plant material. A total of 50 �L of ethanolic extract of

samples were mixed with the same reagent as described above, and after 35 min of

incubation, the absorption was measured at 730 nm for determination of total phenolics.

All determinations were performed in triplicate.

1.4.2.4 Determination of total flavonoid content

Total flavonoid content (TFC) in the extracts was measured using a modified colorimetric

method [Zhishen et al. (1999)]. For preparation of calibration curve 20, 40, 60, 80,100 �L

of catechin was mixed with 0.3 ml 5% NaNO2 (w/v). After 5 min, 0.3 ml of 10% AlCl3

(w/v) and at 6th min, 2 ml 1M NaOH (w/v) were added in 10 ml volumetric flask. The

absorbance of the reaction mixture relative to blank was measured using spectrophotometer

at 510 nm. The total flavonoid contents of extracts were expressed as mg of catechin

equivalent/g of dry plant material. Aliquots (0.5 ml) of extracts were mixed with the same

reagent as described above, and after 30 min, the absorption at 510 nm was measured for

determination of total flavonoids. All measurements were carried out in triplicate.

1.4.2 Isolation, characterization and quantification of bioactive molecules

from Podophyllum hexandrum Royle



UV spectra performed on a Shimadzu UV-2450 instrument (Japan) and IR spectrum was

recorded FTIR (Perkin Elmer). A time-of-flight mass spectrometer with electrospray

ionization (ESI-MS) inter face was used for recording mass spectra (Micromass,

Manchester, UK) using Waters MassLynx 4.1 software. 1H and 13C NMR spectra were

recorded in Bruker Avance-300. Column chromatography was carried out with Merck

silica gel 60-120, 230-400 mesh and RP-C18. TLC was run on Merck aluminium pre-

coated silica gel 60 F254 and RP C-18 plates. All the chemicals were purchased from Merck

India Ltd.

1.4.2.1.2 Plant material and chemicals

The plant material of P. hexandrum was collected from Chamba district, Himachal

Pradesh, in the month of July, 2009. The samples were authenticated (PLP 9763) by

biodiversity department (CSIR-IHBT) and voucher specimens were deposited in the


95

herbarium section. The rhizomes of P. hexandrum were air dried in shade, subjected to

grinding to a coarse particle size powder and stored at ambient temperature.


The dried powdered plant material (1 kg) was sequentially extracted at room temperature

using n-hexane, chloroform, methanol and water by percolation method and the extracts

were concentrated in vacuo to yield semisolid mass. Chloroform soluble part (59.6 g) was

subjected to dry column chromatography on silica gel H. Elution was carried out in varying

percentage of chloroform in n-hexane to give fractions A-F. Fractions A-C (10:90, 20:80

and 40:60 chloroform:hexane) were pooled and on column chromatography over silica gel

(230-400 mesh) by elution with ethyl acetate/n-hexane (30:70, 40:60, 50:50) afforded three

fractions G, H and I. Fraction G was crystallized to yield deoxypodophyllotoxin (120)

(43.2 mg). The fractions H and I on RP-18 column chromatography on elution with

methanol:water (60:40) yielded isopicropodophyllone (122) (30 mg) and �-peltatin (116)

(19.4 mg) and methanol:water (70:30) afforded podophyllotoxone (121) (33.6 mg).

Fractions D and E on silica gel (230-400 mesh) column chromatography led to fractions J

(50:50 ethyl acetate/n-hexane) and K (55:45 ethyl acetate/n-hexane) and resulted in the

isolation of podophyllotoxin (114) (100 mg) and 4'-O-demethylpodophyllotoxin (117)

(113.8 mg) respectively. The methanolic extract (15 g) was chromatographed over silica

gel (230-400 mesh) by ethyl acetate/n-hexane (60:40, 50:50, 70:30 and 100:0) gave four

fractions L M, N and O. Fraction L yielded podophyllotoxin-4-O- �-D-glucoside (131)

(36.1 mg) and other three fractions (M to O) led to the isolation of kaempferol (80) (18.9

mg), quercetin (25) (32.1 mg), quercitrin (83) (18.2 mg) and rutin (84) (22.7 mg) on

purification by chloroform-methanol mixture (05:95�30:70). Structures of compounds

were elucidated by NMR and ESI-MS-MS spectral data and further confirmed by

comparing the spectral data with literature values [Li et al. (2001); Hadimani et al. (1996);

Rahman et al. (1995)].

Podophyllotoxin (114)

Colorless crystals; mp 180-182°C; [�]D25

-141.3° (c 0.50, CHCl3); UV (EtOH): �max 243,

291, 295 (sh) nm; IR (KBr, cm-1

): 3525, 3490, 2838, 1765, 1580, 1515, 1225, 1005; 1H

NMR (CDCl3, 300 MHz): see Table 1.3.1; 13

C NMR (CDCl3, 75.4 MHz): see Table 1.3.1;

HRMS-ESI: m/z [M+H]+ for C22H23O8 calculated 415.4132, observed 415.4124; MS-MS-

ESI: m/z 415, 397, 313, 247, 229, 169.


96

4'-O-Demethylpodophyllotoxin (117)

Colorless crystals; mp 248-250°C; [�]D25 -122.3° (c 0.50, CHCl3); UV (EtOH): �max 242,

291, 296 (sh) nm; IR (KBr, cm-1): 3620, 3555, 1774, 1635, 1543; 1H NMR (CD3OD, 300


C NMR (CD3OD, 75.4 MHz): see Table 1.3.2; HRMS-ESI: m/z

[M+H]+ for C21H21O8 calculated 401.3866, observed 401.3843; MS-MS-ESI: m/z 401, 383,

339, 299, 247, 229, 185, 155.

Deoxypodophyllotoxin (120)

White powder; C22H22O7; mp 160-163°C; [�]D25 -108.9° (c 0.50, CHCl3); UV (EtOH): �max

210, 240 (sh), 290 nm; IR (KBr, cm-1): 1774, 1590; 1H NMR (CDCl3, 300 MHz): see Table

1.3.3; 13

C NMR (CDCl3, 75.4 MHz) see Table 1.3.3; HRMS-ESI: m/z [M+H]+ for

C22H23O8 calculated 399.4138, observed 399.4122; MS-MS-ESI: m/z 399, 231, 187, 169,

129.

�-Peltatin (116)

White powder; mp 235-238°C; UV (EtOH): �max (log �) 271 nm; IR (KBr, cm-1): 3615,

1765, 1638, 1590, 1512; 1H NMR (CDCl3, 300 MHz): � see Table 1.3.4;

13C NMR (CDCl3,

75.4 MHz): see Table 1.3.4; HRMS-ESI: m/z [M+H]+ for C22H23O8 calculated 415.4132,

observed 415.4118; MS-MS-ESI: m/z 415, 247.

Podophyllotoxone (121)

White powder; mp 184-186°C; mp 178-182°C; UV (EtOH): �max 205, 234, 279, 315 nm; IR

(KBr, cm-1): 2842, 1576, 1665, 1775, 1132; 1H NMR (CDCl3, 300 MHz): see Table 1.3.5;

13C NMR (CDCl3, 75.4 MHz): see Table 1.3.5; HRMS-ESI: m/z [M+H]

+ for C22H21O8


169, 143.

Isopicropodophyllone (122)

White powder; UV (EtOH): �max 205, 235, 270, 321 nm; IR (KBr, cm-1): 2842, 1765, 1669,

1585, 1126, 1027; 1H NMR (CDCl3, 300 MHz): see Table 1.3.6; 13C NMR (CDCl3, 75.4

MHz): see Table 1.3.6; HRMS-ESI: m/z [M+H]+ for C22H21O8 calculated 413.3973,

observed 413.3950; MS-MS-ESI: m/z 413, 395, 245, 217, 201, 169, 138.

Podophyllotoxin-4-O-�-D-glucopyranoside (131)

White powder; UV (EtOH): �max 210, 285 nm; IR (KBr, cm-1

): 3545, 3355, 1789; 1H NMR

(C5D5N, 300 MHz): see Table 1.3.7; 13C NMR (C5D5N, 75.4 MHz): see Table 1.3.7;

HRMS-ESI: m/z [M+H]+ for C28H33O13 calculated 599.5357, observed 599.5329; MS-MS-

ESI: m/z 599, 397, 353, 313, 229, 203.


97

Kaempferol (83)

Yellow powder; mp 270-272°C; UV (MeOH): �max 265, 365 nm; 1H NMR (CD3OD, 300

MHz): see Table 1.3.8; 13C NMR (CD3OD, 75.4 MHz): see Table 1.3.8; HRMS-ESI: m/z

[M+H]+ for C15H10O6 calculated 287.2442, observed 287.2449; MS-MS-ESI: m/z 287, 257,

240, 21, 185, 164, 152, 137, 121.

1.4.2.2 Determination of major lignans and flavonoid by HPTLC


The plant material of P. hexandrum was collected from Chamba district, Himachal

Pradesh, in the month of July 2009. The rhizomes of P. hexandrum were air dried in shade,

subjected to grinding to a coarse particle size powder and stored at ambient temperature.

1.4.2.2.2 Preparation of standard solutions

Stock solutions of 4'-demethylpodophyllotoxin, podophyllotoxin, kaempferol,

podophyllotoxone and deoxypodophyllotoxin were prepared by dissolving approximately 5

mg of each in 5 ml methanol in volumetric flasks. These standard solutions were spotted to

HPTLC plates to obtain 4'-O-demethylpodophyllotoxin, podophyllotoxin,

podophyllotoxone in the range of 1.0-8.0 �g/band and kaempferol, deoxypodophyllotoxin

in the range of 2.0-10.0 �g/band.

1.4.2.2.3 Preparation of sample solutions

Powdered rhizomes (500 mg) were extracted with five different solvents- ethyl acetate,

chloroform, methanol, water, acetone and methanol:water (1:1, v/v) (25 ml each) for 20

min at 25°C in ultrasonicator bath (Elma, Germany). All the extractions were performed in

triplicate. The extracts were concentrated under vacuum to remove solvents. Concentrated

extracts were dissolved in methanol (2 ml) and filtered through 0.45 µm membrane.

1.4.2.2.4 Chromatography

A Camag HPTLC system equipped with an automatic TLC sampler ATS4, TLC scanner 3

and an integrated software Win-CATS version 1.2.3 was used for the analysis. HPTLC was

performed on pre-coated silica gel HPTLC 60 F254 (20 cm × 10 cm) plate of 0.2 mm layer

thickness. Samples and standards were applied on precoated plates, as 6 mm bands, with a

Camag automatic TLC applicator (ATS4), ATS4 equipped with 25 �L syringe under N2

gas flow, 10 mm from the bottom and 10 mm from the side and the space between two

bands was 15.4 mm of the plate. Ascending development of the plate, migration distance

90 mm, was performed at 25 ± 2°C with single run using toluene:ethyl acetate:acetic acid

(15:7.5:0.5, v/v) as mobile phase in a Camag twin-trough chamber saturated with mobile

phase vapour. After development, the plate was removed, dried and spots were visualized


98

under UV light at 254 nm with Wincats Software, using the deuterium light source, slit

width 6 × 0.45 mm, scanning speed 20 mm/s, and data resolution 100 �m/step.

1.4.2.3 Determination of polyphenols by UPLC-MS

1.4.2.3.1 Plant material and chemicals

The plant material of P. hexandrum rhizomes was collected in the month of July, 2010.

The rhizomes of P. hexandrum were washed, air dried in shade, subjected to grinding to a

coarse particle size powder and stored at ambient temperature. Standard compounds rutin,

kaempferol, podophyllotoxin, quercitrin and quercetin were purchased from Sigma

Aldrich. 4'-O-demethylpodophyllotoxin, podophyllotoxin-4-O-�-D-glucoside were isolated

and identified by spectral data (IR, 1D-, 2D-NMR and ESI-MS) in comparison with

published spectral data [Rahman et al. (1995); Jackson and Dewick (1984)]. Analytical

grade acetonitrile, methanol, water and formic acid were purchased from J. T. Baker

(USA).

1.4.2.3.2 Preparation of standard solutions

An individual stock solution of standard compounds containing rutin, quercitrin, quercetin

(0.1 mg/ml), 4'-O-demethylpodophyllotoxin, podophyllotoxin-4-O- �-D-glucoside,

podophyllotoxin (0.2 mg/ml) and kaempferol (0.05 mg/ml) was prepared in methanol.

1.4.2.3.3 Preparation of sample solution

About 200 mg of powdered plant material was extracted with 20 ml of methanol in an

ultrasonicator bath (Elma Ultrasonic, Germany) at a controlled temperature (40°C) for 20-

40 min. An extraction time of 30 min was taken as optimum on mass yield basis. The

extracts were filtered and concentrated to dryness under vacuum. Concentrated extracts

were redissolved in 2.0 ml methanol (HPLC grade). As the UPLC analysis, the more

sensitive method, the samples were diluted to get a final concentration of 10 mg/ml. The

injection volume was 1 �L.

1.4.2.3.4 Instrumentation and chromatographic conditions

The analysis was performed using a Waters ACQUITY™ UPLC system (Waters, Milford,

MA, USA). An ACQUITY UPLC® BEH C18 column (100 × 2.1 mm i.d., 1.7 �m), also

from Waters, was used for achieving separation. The column and sample temperature were

maintained at 35 and 15°C, respectively. The mobile phase consisted of (A) 0.1% formic

acid and (B) methanol:acetonitrile (25:75, v/v) at a flow rate of 0.3 ml/min. Gradient

elution was employed starting at 28% B for 1.0 min, then shifted linearly B to 30% for 0.4

min, held at 30% for 2.1 min, shifted again to 50% for 0.5 min, held at 50 % for 1.0 min,

shifted B to 28% for 0.5 min and re-equilibrated for 2.5 min, giving a total cycle time of


99

8.0 min. The injection volume was 1 �L with partial loop injection using needle overfill

mode. The peaks were detected at 290 nm. The peaks were assigned with respect to the UV

spectra of the compounds and comparison of the retention times.

Strong needle wash solution (90:5, acetonitrile-water) and weak needle wash solution

(10:90, acetonitrile-water) were used. All of the solutions mentioned were filtered via 0.22

�m membranes under vacuum and degassed before their usage.

A time-of-flight mass spectrometer with electrospray ionization (ESI-MS) inter face was

used for fingerprinting (Micromass, Manchester, UK). For UPLC analysis, data acquisition

was performed using positive ion mode over a mass range of m/z 50-1000. The general

conditions were: source temperature 80ºC, capillary voltage 3.1 kV and cone voltage 23 V.

Positive ion ESI-MS analysis was performed by direct infusion with a flow rate of 10

�L/min using a syringe pump. Mass spectra were acquired and accumulated over 60 s and

spectra were scanned in the range between 50 and 1000 m/z. Mass Lynx 4.1 (Waters,

Manchester, UK) was used for data analysis. Tandem mass spectrometry for structural

analysis of single molecular ion in the mass spectra from rhizomes extracts was performed

by mass-selecting the ion of interest, and was in turn submitted to 15-35 eV collisions with

argon in the collision quadrupole.

1.4.2.3.5 Method validation

Calibration curves

Stock solution containing seven analytes were prepared and diluted to appropriate

concentration in the range of 0.39 to 100 �g/ml for rutin, quercitrin, quercetin, 0.78 to 200

�g/ml for 4'-O-demethylpodophyllotoxin, podophyllotoxin-4-O-�-D-glucoside,

podophyllotoxin and 0.20 to 50 �g/ml for kaempferol for establishing calibration curves.

For quantitative analysis, nine different concentrations of seven analytes were injected in

triplicate. The calibration curves were constructed by plotting the peak areas versus the

concentration of each analyte.

Selectivity

The selectivity of the method was determined by analysis of standard compounds and

samples. The peaks of rutin, quercitrin, quercetin, 4'-demethylpodophyllotoxin,

podophyllotoxin-4-O-�-D-glucoside, kaempferol and podophyllotoxin were identified by

comparing their retention times and UV spectra with those of the standards.


100

Limit of detection (LOD) and quantification (LOQ)

In order to estimate the LOD and LOQ, blank methanol was spotted six times following the

same method as explained. Limit of detection was determined at a signal to noise ration of

3:1 and limit of quantitation at a signal-to-noise ratio of 10:1.

Precision and accuracy

The measurements of intra-day and inter-day variability were utilized to assess the

repeatability and reproducibility of the developed method. Intra- and inter-day precisions

(expressed as %RSD) for the seven compounds were determined by applying different

concentration levels of reference compounds five times within 1 day and over a period of 5

days for interday precision.

Accuracy was evaluated by means of recovery assays carried out by adding known

amounts of the reference compounds to the sample solutions. The amounts of analytes

added correspond to 25, 50, and 100% of compounds concentrations in samples. The

spiked samples were extracted in triplicate and analyzed under the above-mentioned

conditions. The percent recovery and average percent recoveries were calculated.

1.5 Conclusion

For exploration of secondary metabolites from western Himalayan region, three

medicinally important plants were studied for the isolation, characterization and

development of new analytical methods using modern hyphenated techniques. From

Cedrus deodara ten compounds (including two novel) were isolated and characterized. For

the first time, two new sesquiterpenes, (E)-(2S, 3S, 6R)-atlantone-2,3-diol and (2S, 3S, 6S)-

atlantone-2,3,6-triol were identified and their structures were elucidated using 1D and 2D

NMR and mass spectral data. Analyses of essential oil and extracts demonstrated highest

content of himachalenes and atlantones in the extract of C. deodara. Furthermore, a newly

developed UPLC method was applied for identification and quantification of four

flavonoids in Cedrus needles. Analysis of P. hexandrum rhizomes led to isolation and

characterization of seven lignans and four flavonoids. In addition, simple and rapid

analytical procedures (HPTLC, UPLC) have been developed for simultaneous

determination and quantification of lignans and flavonoids in P. hexandrum. From flowers

and bark of A. chinensis fourteen compounds including phenolics and terpenes were

isolated and characterized. This is the first report on complete chemical fingerprinting of

different parts of A. chinensis for identification of secondary metabolites conducted by

UPLC-ESI-QTOF-MS.


101

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121

Spectral data of some compounds

1H NMR (in CDCl3) spectrum of (E)-(2S, 3S, 6R)-atlantone-2,3-diol (57)

220 200 180 160 140 120 100 80 60 40 20 ppm 13

C NMR (in CDCl3) spectrum of (E)-(2S, 3S, 6R)-atlantone-2,3-diol (57)

220 200 180 160 140 120 100 80 60 40 20 ppm DEPT spectrum of (E)-(2S, 3S, 6R)-atlantone-2,3-diol (57)

9 8 7 6 5 4 3 2 1 ppm

O

OHOH


122

HMBC spectra of (E)-(2S, 3S, 6R)-atlantone-2,3-diol (57)

HMQC spectra of (E)-(2S, 3S, 6R)-atlantone-2,3-diol (57)

HRESI-QTOF-MS of (E)-(2S, 3S, 6R)-atlantone-2,3-diol (57)


123

1H NMR (in CDCl3) spectrum of (E)-(2S, 3S, 6R)-atlantone-2,3-diol (58)

220 200 180 160 140 120 100 80 60 40 20 ppm 13C NMR (in CDCl3) spectrum of (E)-(2S, 3S, 6R)-atlantone-2,3-diol (58)

220 200 180 160 140 120 100 80 60 40 20 ppm DEPT spectrum of (E)-(2S, 3S, 6R)-atlantone-2,3-diol (58)

9 8 7 6 5 4 3 2 1 ppm

O

OHOH

OH


124

HMBC spectra of (E)-(2S, 3S, 6R)-atlantone-2,3-diol (58)

HMQC spectra of (E)-(2S, 3S, 6R)-atlantone-2,3-diol (58)

HRESI-QTOF-MS of (E)-(2S, 3S, 6R)-atlantone-2,3-diol (58)

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