Chapter-01

INTRODUCTION, MATERIALS AND METHODS

(A) Introduction (B) Materials and methods

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(A) INTRODUCTION 1.1 General introduction:

Plants produce primary and secondary metabolites which encompass a wide array of

functions. Primary metabolites, which include amino acids, carbohydrates, nucleic acids and

lipids are compounds that are necessary for cellular processes. In addition to essential primary

metabolites, plants can synthesize a wide variety of compounds known as secondary metabolites

which have been subsequently exploited by humans for their beneficial role in a diverse role of

applications. Often, plant secondary metabolites may be referred to as natural products, in which

case they illicit effects on other organisms1

.

1.1.1 Secondary metabolites:

Secondary metabolites are organic compounds that do not have a recognized role in the

maintenance of fundamental life processes (normal growth, development and reproduction) in the

plants that synthesize them but they do have an important role in the interaction of the plant with

its environment. The production of these compounds is often low (less than 1% dry weight) and

depends greatly on the physiological and developmental stage of the plant. Unlike primary

metabolites, absence of secondary metabolites results not in immediate death, but in long-term

impairment of the organism's survivability/fecundity or aesthetics, or perhaps in no significant

change at all.

The function or importance of these compounds to the organism is usually of an

ecological nature as they are used as defenses against predators, parasites and diseases, for

interspecies competition and to facilitate the reproductive processes (coloring agents, attractive

smells, etc). Since these compounds are usually restricted to a much more limited group of

organisms, they have long been of prime importance in phytochemical research. Biomining is the

process of seeking organisms for the purpose of exploiting their natural products for drug or

other technological development directly, or as an inspiration for unnatural products. This will

concern secondary metabolites in plants, bacteria , fungi and many marine organisms (sponges,

tunicates, corals, snails).

Most of the secondary metabolites of interest to man are volatile oils, alkaloids,

flavonoids, steroids, glycosides, saponins, fatty acids, tannins and resins. These are the

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broad categories which classify secondary metabolites based on their biosynthetic origin. Since

secondary metabolites are often created by modified primary metabolite synthases, or "borrow"

substrates of primary metabolite origin, these categories should not be interpreted as saying that

all molecules in the category are secondary metabolites (for example the steroid category), but

rather that there are secondary metabolites in these categories2

.

1.1.2 Plant derived natural products:

Higher plants are rich source of bioactive constituents or phyto-pharmaceuticals used in

pharmaceutical industry. Some of the plant derived natural products include drugs such as

morphine, codeine, cocaine, quinine etc.; anticancer Catharanthus alkaloids, belladonna alkaloids,

colchicines, phytostigminine, reserpine and steroids like diosgenin, digoxin and digitoxin. Many

of these pharmaceuticals are still in use today and often no useful synthetic substituents have been

found that possess the same efficacy and the pharmacological specificity. Currently one-forth of

all prescribed pharmaceuticals in industrialized countries contain compounds that are directly or

indirectly, via semi-synthesis derived from plants. Furthermore, 11% of the 252 drugs considered

as basic and essential by WHO are exclusively derived from flowering plants1,2

. A significant

plant natural product to emerge as a new global drug is artemisinin, a sesquiterpene lactone from

a medicinal plant Artemisia annua. Artemisinin and its derivatives exhibited considerable activity

against cerebral malaria. Valerian from Valeriana species is known to exhibit CNS depressant

activity. Taxol from the bark of Taxus brevifolia is an anticancer agent. Campothecin, isolated

from Camptotheca acuminata is another potent anticancer agent. AIDS the most dreaded disease

has shown some hope of remedy now with the world’s scientific attention shifting towards

screening of plants for anti-HIV activity. Phytolacca americana, yielded an antiviral protein which

inhibited HIV-replication of picomolar concentration. Trichoxanthin, a protein produced

primarily in the tuberous roots of Trichosanthes kirilowii is known to selectively inhibit

replication of HIV virus in vitro by inhibiting ribosomal protein synthesis and cellular

reproduction3-10

.

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1.1.3 Himalayan biodiversity vis a vis the present study:

Focusing attention to the Himalaya one finds that this region possess luxuriant and varied

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vegetation, most of which is important from nutritional, aesthetic and medicinal view point.

Incidently, not much is known about the phytochemical aspect of the arboreous species of this

region and wherever such information is available, concerted efforts are called for verifying the

claims11

. Therefore, to initiate serious scientific efforts to observe the arboreous wealth of

Himalayan region, comprehensively study their constituents, find ways to explore them and

initiate their planned and systematic study, the author has surveyed the distribution of flora with

special reference to Lauraceae family. Research in the past few decades has established that

Laurels possess a wide range of flavonoids, mono and sesquiterpenoids and

furanosesquiterpenoids possessing varying pharmacognosical activities. Compared to the research

work in Lauraceae elsewhere in the world, the systematic chemical analysis of Himalayan

Lauraceae has not been attempted so far.

Therefore, a total of nine species of six genera viz. Neolitsea, Lindera, Dodecadenia,

Persea, Phoebe and Cinnamomum are being taken to study their terpenoid diversity,

chemotaxonomic and chemotypic studies along with some of their bioactive principles. Several

species of this family are known for their medicinal uses12,13

.

1.2 Lauraceae; an introduction:

1.2.1 Habitat and distribution:

The Lauraceae or Laurel family is a predominant arboreous family which comprises a group

of flowering plants included in the order Laurels. The family contains about 55 genera and more

than 2000 species worldwide, mostly in warm tropical regions and sometimes in temperate

regions. Most of them are evergreen trees and shrubs but Sassafras and one or two other genera

are deciduous and Cassytha is the only genus of parasitic vines13

. Trees of Laurel family

predominate in the world’s Laurel forests, which occur in a few humid subtropical and mild

temperate regions of the northern and southern hemispheres, including the Macaronesian islands,

southern Japan, Medagasker, southeast Asia, Brazil and central Chile. Six genera viz.

Cinnamomum, Lindera, Persea, Phoebe,

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Litsea and Dodecadenia are reported in the Himalayan forests varying from Kashmir to Bhutan

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up to 3000m13-16

. As far as the Kumaun and Garhwal regions concern, two species of

Cinnamomum (C. zeylanicum, C. tamala), two of Lindera (L. pulcherrima, L. lucida), three

species of Persea (P. duthiei P. odoratissima, P. gamblei), two species of Phoebe (P. lanceolata, P.

pallida), five species of Litsea (L. umbrosa, L. cuipala, L. elongata, L. monopetala, L. lanuginosa)

and only one of Dodecadenia (D. grandiflora) has been reported14-17

.

1.2.2 Medicinal importance:

As far as the family Lauraceae is concerned, the plant parts are used in traditional medicine,

spices, timber, wild edibles, oils etc13

. The leaves of Cinnamomum tamala (Tejpat) and Lindera

benjoin (Spicebush) are commonly used as spice. C. tamala holds in Indian cookery the same

status as that of ‘bay leaves’ (Laurus nobilis) in Europe. Besides flavoring it is also known for

hypoglycemic, stimulant and carminative used in traditional system of medicine in India. C.

camphora has religious importance while C. glanduliferum wood is odoriferous and have insect

repelling properties. Its durability has been widely used as a quality wood for building houses,

and making agricultural implements. The smoke of the dry leaf of C. impressinervium is used to

inhale in cold, cough and toothache. Laurus nobilis leaves and fruits are excite-aromatic which

are used as nerving against hysteria emmenagogue. In China the roots of Lindera strychnifolia are

used as a crude drug. The bark of Persea cordata, a Brazilian medicinal plant, is used by rural

communities for its inflammatory healing and antibacterial properties. In Congo, the stem bark of

Persea americana (Avocado) is taken to cure cough while its leaves are used for the treatment of

high blood pressure in Brazil and Jamaica. Ocotea bullata is one of the most frequently used

traditional medicine in southern parts of Africa and now it has become an endangered species17,18

.

1.2.3 Commercial uses:

Mankind has used the Lauraceae for their timber, as stinkwood (Ocotea bullata) from South

Africa, nan-mu (Persea nanmu) from China and ironwood (Eusideroxylon zwageri) from

Indonesia. Seeds used as seed fat (Litsea sebifera) from Indochina and

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laurel berry fat (Laurus nobilis) from Europe. Drugs are used from the bark of Aniba coto in

Bolivia and Ocotea rodiaei from British Guyana. The main economic uses of this family are due

to a high content of ethereal oils, which are important sources for spices and perfumes13

.

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Himalayan Lauraceae, therefore, offer a bright prospective in the field of phytochemical research,

where a lot is yet to be explored. Present investigation by the author is an attempt in this

direction.

1.3 Chemical markers; literature review:

The arylpropanoids, flavonoids, terpenoids and alkaloids are the principal secondary

metabolites found in Lauraceae and are valuable tools for studying their chemosystematics.

1.3.1 Arylpropanoids and flavonoids:

Arylpropanoids are a class of plant-derived organic compounds that are biosynthesized from

the amino acid phenylalanine. They have a wide variety of functions, including defense against

herbivores, microbial attack, or other sources of injury; as structural components of cell walls; as

protection from ultraviolet light; as pigments; and as signaling molecules. Arylpropanoids range

from cinnamoyl derivatives to the relatively very rare cinnamyl derivatives as well as from the

common allyl benzenes to a sole prophenyl benzene.

good chemotaxonomic marker of this family

19

. Another metabolite, whose origin clearly goes back

to phenylalanine, is 1-nitro-2-phenylethane which occurs together with allylbenzenes in Aniba

canenilla and in Ocotea pretiosa20

. In spite of its close association to phenylalanine, a ubiquitous

precursor; nitrophenylethane seems to be a fairly rare

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natural compound. So far only one additional source has been disclosed; the fruits of Dennettia

tripetala21

. Benzyl benzoate and benzyl salicylate though rather widespread in flowers, are

certainly exceptional as constituents of healthy wood of Aniba22

. This makes it even more

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surprising that these esters do occur in very substantial amounts in wood of most of the Aniba

species23,24

.

OO

safrole 1-nitro, 2-phenyl ethane benzyl benzoate benzyl salicylate

Aromatic derivatives of monocyclic 2-pyrones were reported from Aniba species25,26

. Lauric

acid was reported as the main constituent from the seeds of several species of Litsea,

Actinodaphne, Cinnamomum, Laurus, Lindera, Neolitsea, Sassafras and Umbellularia.

derivatives of 2-pyrones

methylnonylketone 2-methoxy undec-10-yne

The leaves of Litsea odorifera contains chiefly methylnonylketone while its bark was

found to be rich in 2-methoxyundec-10-yne. It is reasonable to consider the generation of both

these compounds linked to the biosynthesis of lauric acid27,28

. The presence of substantial amounts

of fatty oils in Lauraceae seeds is by no means a general character of the family. Many fruits

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accumulate oils in pericarp. In opposition to seed fats, the pericarp fats contain only the minor

proportion of lauric acid, while oleic acid predominates29,30

.

1.3.2 Terpenoids:

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1.3.2.1 Mono and Sesquiterpenoids:

The species belonging to this family have powerfully an odour of terpenes and have a large

amount of essential oils in their leaves, twigs and barks. The main components of the odour

consist of monoterpenes. E-Caryophyllene was found in all species examined while the remaining

sesquiterpenes were detected in limited species. C. camphora, grown for camphor which is

obtained from leaves and twigs. Other volatile constituents of the same species are 1,8-cineole,

limonene, linalool and terpinen-4-ol. C. tamala contains linalool, α-pinene, β-pinene, eugenol,

cinnamic aldehyde etc31-35

.

OH CHO

O O

camphor linalool 1, 4-cineole E-cinnamaldehyde

Persea americana is comprised of Z-nerolidol, β-caryophyllene and caryophyllene oxide as

major constituents while P. bombycina contains dodecenal, 11-dodecenal and decenal. On the

other hand, machikusanol, carrisone, γ-eudesmol and γ-selinene have been reported from P.

japonica. Bioactive ryanodane diterpenes are reported from P. indica36-40

.

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Lindera chunii is comprised of sesquiterpenoids. Lignans were reported from L. obtusiloba,

dihydrochalcone from L. lucida and E-nerolidol from L. benzoin41-45

. Camphene, limonene, α-

pinene and bornyl acetate were found to be major constituents of Ocotea comoriensis. Oleic acid

rich essential oil was reported from Phoebe attenuata

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seeds. P. porphyra contains 1,8-cineole, β-caryophyllene and spathulenol. Caparratriene, a

sesquiterpene hydrocarbon was isolated from Ocotea caparrapi46-50

.

Ryanodane diterpenes

The principal constituents of Brazilian Phoebe oil were carquejyl acetate, α-copaene, δ-

cadinene and β-eudesmol51,52

. Lauric acid and Linoleic acid are obtained from Litsea consimilis.

Citral was found predominantly followed by linalool, methyl heptanone and limonene from Litsea

cubeba53,54

. α-Pinene and 1,8-cineole were the main constituents of Laurus azorica55,56

. Umbellulone

was the chief constituent of Umbellularia while linalool was reported from the species of Aniba

and Cryptocarya57

.

OH

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O

β-caryophyllene caparratriene E-nerolidol umbellulone

Prerequisites to valid chemosystematic comments are a representative knowledge of the

distribution of individual compounds in the family and an assessment of the sequences of reaction

steps by which individual compounds arise from ubiquitous precursors. With respect to the

Lauraceae, all the biosynthetic schemes are based on comparative phytochemistry.

1.3.2.2 Furanosesquiterpenoids:

It is interesting that a large number of sesquiterpene furans have been isolated from a single

family possessing farnesane, germacrane, elemane, selinane and linderane

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skeletons. The occurrence of such compounds in this family is not wholly exceptional. The

presence of germacranolides, costunolides, parthenolides and aristolactones was noticed in

magnolidae31-33

. Germacranolides and eudesmanolides are typical constituents of the compositae

family which is also a source of furanosesquiterpenoids, even if these are not based on eudesmane,

but eremophyllane skeleton. The farnesane type sesquiterpene furans viz. sesquirosefuran and

longifolin are found in Actinodaphne longifolia.

sesquirosefuran

longifolin

Farnesane type furans

Germacrane type sesquiterpene furans have been isolated from Lindera strychnifolia, Neolitsea

aciculata, N. zeylanica and N. sericea. Elemane type furans, occurs in Lindera strychnifolia,

Neolitsea aciculata and N. sericea may be artifacts, produced from germacrane type sesquiterpene

furans.

OAc OAc

O

OO

O

O

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OC O

OC O

OC O

OC O

linderane litsealactone llitseaculane linderalactone

Germacrane type furans

Linderane type furans which were distributed in Neolitsea sericea and Lindera strychnifolia are

characteristic of the Lauraceae and may be derived from selinane type furans58-64

.

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Linderene type furans

The occurrence of the sesquiterpene furans is one of the characteristic chemosystematic

features of this family.

O O

epi-dihydroisolinderalactone isofuranogermacrene isosericenine isolinderalactone

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Elemane type furans 1.3.3 Alkaloids:

In Laurels, Ocotea syn. Phoebe is the genus which is well known for alkaloids. Oxo

aporphine alkaloids have been reported from the wood of Phoebe cinnamomifolia. Substituted

aporphine alkaloids have been reported from Phoebe molicella.

A pseudo alkaloid, anibene has been isolated from Aniba species65-67

. The occurrence of

simple phenylalanine derived alkaloids, such as benzyltetrahydroisoquinoline, aporphine and bis-

benzyltetrahydroisoquinoline are the other alkaloids limited to Lauraceae30

.

aprophine alkaloid anibene

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From the static viewpoint, Kosterman’s system of classification of Lauraceae genera seems

natural enough13

. Between two subfamilies, the Cassythoideae, represented by herbaceous,

parasitic vines; seem to be void of arylpropanoids. Within the arboreous Lauroidae,

arylpropanoids seem to concentrate in the tribe cinnamomeae. None have yet been found in the

Perseae, which are characterized by the presence of simple benzyltetrahydroisoquinoline

alkaloids.

The Litseae stand apart on account of their sesquiterpene chemistry and their surprisingly complex

flavonoids while the Cryptocaryeae distinguish themselves as producers of relatively more varied

gamut of alkaloidal type30

.

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1.4 Pharmacognosical importance:

The essential oils, extracts and their isolates from the members of family Lauraceae showed a

wide range of biological activities. Although the chemistry of furanosesquiterpenoids has been a

subject of numerous investigations, only a few reports have been made of the biological activities

which are mostly confined to the farnesane derivatives. Furanodienone is known to possess

insecticidal activity by inducing toxicity against larvae of the polyphagous pest insects.

Furanodienone, curzerenone and their structural analogues have also been shown to have

significant anti-inflammatory, antimicrobial and analgesic activities64-67

. Ryanodane diterpenes

from Persea indica were found to be antifeedant. The hypotensive effect of the constituents of the

leaves of Persea americana on arterial blood pressure was found in anaesththetized normotensive

rats. Methanol extract of the bark of Litsea glutinosa showed antibacterial activity. Further, the

essential oil of Laurus nobilis showed fumigant activity against insects. Anti-platelet and anti-

thrombotic activities were recorded in the essential oil of Ocotea quixos. Caparratriene, a

sesquiterpene hydrocarbon with significant growth inhibitory activity against CEM leukemia cells,

was isolated from the oil of O. caparrapi68-70

. Cinnamomum camphora, the camphor tree-bark was

found to contain in vitro anti-inflammatory and anti oxidative effects. Sesquiterpene lactones from

the water extract of the roots of Lindera strychnifolia were found to be cytotoxic against the

human small cell lung cancer

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cell, SBC-3. A diterpene was isolated from the fraction exhibiting antiallergenic activity obtained

from the bark of Cinnamomum cassia.

1.5 Biosynthetic pathways:

1.5.1 Mevalonate pathway:

The most important structural feature of nearly all the terpenoids is their derivation from

one monomer unit, isoprene C5H8. A fascinating area of research linking organic chemistry to

biology is the study of the biogenesis of natural products71

. The terpenoids have a diverse

functional role in plants as structural components of membranes, photosynthetic pigments,

electron carrier, hormones and are important flavoring and fragrant agents in foods, cosmetics and

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perfumes72,73

. Despite the remarkable diversity of plant isoprenoids, the various pathways that

direct the synthesis of these metabolites were thought to emerge from a single common

biosynthetic pathway-the mevalonate isoprenoid pathway, named after its known intermediate

mevalonic acid. The mevalonate isoprenoid pathway was first discovered in yeast and animals

through investigation of sterol biosynthesis.

This pathway involves first the synthesis of biological C5 isoprene unit,

isopentylpyrophosphate from three molecules of Acetyl-CoA via acetoacetyl-CoA and hydroxyl

methyl glutaryl-CoA (HMG-CoA). Hydroxyl methyl glutaryl-CoA (HMG-CoA) is reduced to

mevalonic acid which gets phosphorylated in two steps to form mevalonate pyrophosphate

(MVAPP) which subsequently decarboxylated to yield isopentyl pyrophosphate (IPP)74-86

. In the

second step isopentylpyrophosphate (IPP) isomerizes to dimethylallylpyrophosphate (DMAPP)

and these two isomers combine to yield geranyl pyrophosphate (GPP, C10) further condensation

with additional isopentylpyrophosphate (IPP) units form successively larger acyclic prenyl-

prenylpyrophosphate viz. farnesyl pyrophosphate (FPP, C15), geranylgeranylpyrophosphate

(GGPP, C20) etc. which undergo cyclization, coupling and rearrangement to produce the parent

carbon skeleton of each class. GPP (C10) and FPP (C15) yield monoterpenes and sesquiterpene

skeleton respectively. FPP (C15) can also dimerize in head to tail fashion to form squaline (C30),

the precursor of triterpene.

- 23 --

Similarly GGPP (C20) can dimerize to phytoene (C40), the precursor of tetraterpenoids

(Scheme 1.5.1).

OH

iii

iiiSCoA SCoA NADPH NADPH i

ii O

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OO COOH O COOH OH COOH

O SCoA acetyl-CoA acetoacetyl-CoA HMG-CoA

mevalonate mevalonic acid

OHOH v

vi PPO

vii PPO

ATP

ATP COOH

ATP COOH

PO

PPO MVA-5-phosphate MVA-5-diphosphate isopentyl pyrophosphate DMAPP

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i; Acetoacetyl-CoA-thiolase, ii; HMG-CoA synthase, iii;HMG-CoA reductase, iv; Mevalonate kinase, v; Phosphomevalonatekinase, vi; Mevalonate-5-diphosphatedecarboxylase, vii; IPP isomerase

Scheme-1.5.1; Mevalonic acid pathway

1.5.2 Deoxy-xylulose phosphate pathway:

Besides the ubiquitous MVA pathway, another completely different pathway that leads to the

formation of IPP and DMAPP, the main biological precursor of terpenoid biosynthesis77,78

. The non

mevalonate pathway is thought to be more or less similar to the valine biosynthetic route

involving glyceraldehyde 3-phosphate and pyruvate as the precursor for the C5 isoprene unit

which was also supported by 13

C labeling experiments in various plants79

. By comparison with

observed labeling pattern, it has been shown that the IPP/DMAPP units are biosynthesized via a

mevalonate independent pathway, which is called as triose-phosphate/pyruvate pathway or deoxy-

xylulose phosphate pathway (DOXP). The non-mevalonate DOXP pathway most likely involves a

free or

- 24 --

phosphorylated intermediate of 1-deoxy xylulose, resulting from condensation of pyruvate with

glyceraldehyde 3-phosphate. The role of 1-deoxyxylulose or its 5phosphate as a C5 precursor of

IPP was shown by successful incorporation of deuterium labeled 1-deoxyxylulose into isoprenoid

of various bacteria e.g. Escherichia coli and also in various higher plants. The 1-deoxy xylulose

phosphate was converted in multiple steps to IPP and DMAPP80

.

Recent literature search indicate that in higher plants, monoterpenes, diterpenes and phytol

chains of chlorophyll are formed via the DOXP and not by classical MVA pathway. Therefore,

one can presume that some terpenoids may be synthesized through the MVA pathway and other

by DOXP. Furthermore some terpenoids are also shown to have mixed origin by both pathways.

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Analysis of labeling patterns and quantitative 13

CNMR studies of sesquiterpene bisaboloxide-A

and chemazulen isolated from Matericaria recutita (chamomile) flowers showed that two of the

isoprene building blocks were predominantly formed by triose-phosphate/pyruvate pathway

whereas the third unit is mixed origin being derived from both MVA pathway and triose-

phosphate pyruvate pathway81

.

Although both pathways, MVA and DOXP, operate independently under normal

conditions, but interaction between them have been also reported with exchange of common IPP

and DMAPP units (Scheme-1.5.2).

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O OH

OH O ii

i OP+

OP

COOH OH

OP

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O OH OH OH

D-glyceraldehyde-D-1-deoxy-D-xylulose-2-methyl erythritol-

pyruvate

3-phosphate 5-phosphate 4-phosphate

NADPH _H Oiii/iv

2

NADPHOPP

OPP

OPP

OHO OHOH OH

_H2O

v OPP

OPP OPP OH

DMAPP isopentyl pyrophosphate

CH2OPP CH2OPP CH2OPP geranyl pyrophosphate farnesyl pyrophosphate polyisopropenyl pyrophosphate

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i; 1-deoxyxylulose-5-phosphate synthase, ii; 1-deoxyxylulose-5-phosphatereductoisomerase, iii; 4-diphosphocitydil-2-methyl-D-erythritol synthase, iv; 4-diphosphocytidyl-2-methyl-D-erythritol kinase, v; IPP isomerase

Scheme-1.5.2; Deoxy-xylulose phosphate pathway

1.5.3 Biosynthesis of Furano sesquiterpenes:

On the basis of the proposed biosynthetic pathway, chemosystematic considerations further

show that evolutionary lower species are only capable of carrying out a limited number of

biosynthetic steps during biosynthesis. On the other hand evolutionary more advanced species can

perform the complete biosynthetic sequence. Therefore, Actinodaphne longifolia containing

Farnesane type skeleton can be regarded as being evolutionary less advanced while Lindera

strychnifolia, having linderane type furans may be the most recent (scheme-1.5.3). The

sesquiterpene furans of Neolitsea aciculata greatly resemble those of Indian N. zeylanica. On the

other hand, the furans of L. strychnifolia and Neolitsea sericea are very similar to each other. The

furans with linear skeleton like those in Actinodaphne longifolia, however, clearly differ from

those of the other species68,69

.

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OPP

farnesyl pyrophosphate

O

O germacrane type farnesane type

O O

selinane type elemane type

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linderane type

Scheme-1.5.3; Biosynthesis of furanosesquiterpenoids

1.5.4 Biosynthesis of Phenylpropanoids:

Both eugenol and cinnamaldehyde belong to the group of compounds having a benzene ring

with a propane side branch C6-C3. The origin of the aromatic ring of the many natural

phenylpropanoids is regarded to be the cyclohexane derivative that arises by the cyclization of

sedoheptulose, a C7 sugar molecule. The key compound in the biosynthetic scheme is shikimic

acid. The key to this scheme was the discovery of a mutant strain of E. coli for five aromatic

compounds viz. phenylalanine, tyrosine, tryptophan, p-aminobenzoic acid and p-hydroxybenzoic

acid, could be completely satisfied by the single compound shikimic acid. Thus shikimic acid was

established as an obligate intermediate for the biosynthesis of aromatic rings in E. coli82

. Both

cinnamaldehyde and eugenol are formed through the shikimic acid pathway leading to lignin. It

would appear that cinnamaldehyde should be formed by a single step reduction

- 27 --

of cinnamic acid. A further step reduction will yield cinnamic alcohol, which could contribute

towards the formation of lignin. In species such as Cinnamomum, it may well be that a genetic

block occurs at the conversion of cinnamaic aldehyde to cinnamyl alcohol, and as a result there

can arise an accumulation of cinnamic aldehyde. The loss of ability to introduce p-oxygen to the

ring as a possible explanation for the cinnamaldehyde remaining as such; similarly a two step

reduction of the side chain of ferulic acid will yield coniferyl alcohol83

. Elimination of a terminal

hydroxy group in the side chain and rearrangement of the double bond will yield eugenol. In

Cinnamomum species there may be some enzyme responsible for such a transformation of

coniferyl alcohol. In view of the structural relationship within lignin, it is logical that cinnamic

aldehyde should be widespread in the plant kingdom. Among the commercially important

essential oils, cinnamic aldehyde is found in Cinnamomum, Cassia, Patchouli and Myrrh oils.

However, the tracer studies have shown interesting developments in cinnamic aldehyde and

eugenol biosynthesis84

. It is generally assumed that the allyl and propenyl groups attached to

phenolic nuclei in many plant constituents, such as in anethole, chavicol, estragol and eugenol,

originate from the cinnamic acid side chain through the reductive steps. The difference seems to

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lie in the position of the double bond in the side chain. In cinnamic aldehyde the double bond is

between C-1 and C-2. In such a situation the phenyl-propane skeleton of L-phenylalanine

incorporated into the molecule with retention of all carbon atoms. Decarboxylation of the side

chain took place at the ferulic acid stage and an 'extra' carbon atom was introduced to the side

chain, probably donated by S-adenosyl-methionine or an equivalent compound. Decarboxylation

at the ferulic acid stage was established as labeled ferulic acid incorporated into eugenol in

appreciable quantity. Thus it appears that the prophenyl and allyl side chains have independent

origins85,86

. Furthermore, as in case of eugenol, the allyl group only occurs when there is p-oxygen

attached to the ring. Thus the favored pathway for eugenol biosynthesis is accepted as L-phenyl-

alanine→ cinnamic acid→p-coumaric acid→ ferulic acid→eugenol (Scheme 1.5.4 & 1.5.5).

- 28 --

O O

phenylalanine cinnamic acid cinnamic aldehyde

OH

H3CO anithol HO

OCH3

eugenol cinnaamyl alcohol HO chavicol H3CO

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estragol Scheme-1.5.4

Formation of eugenol and related compounds from cinnamic acid

OO

OH

OH

OH HO

HO

HO

CHO CHO

OH caffeic acid

ferulic acid coniferyl alcohol

Scheme-1.5.5

Formation of eugenol from ferulic acid

1.6 References: 1. Zwenger, S., Basu, C., Biotechnology and Molecular Biology Reviews, 2008, 3, 01. 2 Namdeo, A.G., Pharmacognosy Reviews, 2007, 1, 69. 3 Thakurta, P., Bhowmic, P.M., Mukherjee, S., Hazra, T.K., Patra, A., Bag, P.K., Journal of Ethnopharmacology, 2007, 3, 607.

- 29 --

1 Tzenj, T.C., Lin, Y.L., Jong, T.T., Chang, C.M.T., Separation and Purification Technology, 2007, 56, 18. 2 Walsh, J.J., Coughlan, D., Heneghan, N., Gaynor, C., Bell, A., Bioorganic and Medicinal Chemistry Letters, 2007, 17, 3599. 3 Marder, M., Viola, H., Wasowski, C., Fernandez, S., Medina, J.H., Paladini, A.C., Pharmacology, Biochemistry and Behaviour, 2003, 75, 537. 4 Kingston, D.G.I., Phytochemistry, 2007, 68, 1844. 5 Pagnang, G., Sala, A., Neuroscience Letters, 2003, 336,163. 6 Kurinov, I.V., Uckun, F.M., Biochemical Pharmacology, 2003, 65, 1709. 7 Wang, S., Zheng, Z., Weng, Y., Yu, Y., Zhang, D., Fan, W., Dai, R., Hu, Z., Life Science, 2004, 74, 2468.

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8 Badoni, A.K., Journal of Himalayan Studies and Regional Development, 198788, 11 & 12,103. 9 Samuelsson, G., Farah, M.H., Claeon, P., Hagos, M., Thulin, M., Hedberg, O., Warfa, A.M., Hassan, A.O., Elmi, A.H., Abdurahman, A.D., Journal of Ethnopharmacology, 1992, 37, 93. 10 Kostermans, A.J.G.H., Reinwardtia, 1957, 4, 193. 11 Gupta, R.K., Flora Nainitalensis, A handbook of the flowering plants of Nainital, 1968, p.298. Navyug Traders, New Delhi. 12 Naithani, B.D., Flora of Chamoli, 1985, 2, p.550. Botanical survey of India. 13 Polunin, O., Stainton, A., Flowers of the Himalaya, 1984, p.351. Oxford University Press, Delhi. 14 Kubitzki, K., Rohwer, J.G., Bittrich, V., The families and genera of vascular plants, 1993, 2, 366. 15 Tallent, W.H., Horning, E.C., Journal of American Chemical Society, 1956, 78, 4467. 16 Birch, A.J., Chemical Plant Taxonomy (Edited by T. Swain), 1963, p. 143, Academic Press, London. 17 Gottlieb, O.R., Taviera, M., Journal of Organic Chemistry, 1959, 24, 1959.

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1 Okogun, J.I., Ekong, D.E.U., Chemistry and Industry, 1969, 1272. 2 Naves, Y.R., Mazuyer, G., Natural and Perfumery Material, 1947, p. 138, Reinhold, NewYork. 3 Naves, Y.R., Gottlieb, O.R., Taviera, M., Helvetica Chimica Acta, 1961, 44, 1121. 4 Hollands, R., Becher, D., Gaudemer, A., Polonsky, J., Ricroch, N., Tetrahedron, 1968, 24, 1633. 25. Mors, W.B., Taviera, M., Gottlieb, O.R., Chemizie Organic Natura, 1962, 20, 132. 5 Jewers, K., Private Communication to W.B. Mors, 1969, Tropical Products Institute, London. 6 Mathews, W.S., Pickering, G.B., Umoh, A.T., Chemistry and Industry, 1963, 122. 7 Bu'Lock, J.D., In Comparative Phytochemistry (Edited by T. Swain), 1966, p. 79, Academic Press, London. 8 Hilditch, T.P., Williams, P.N., The Chemical Constitution of Natural Fats, 4

th

Edn., 1964, p. 191, Chapman Hall London. 9 Gottlieb, O.R., Phytochemistry, 1972, 11, 1537. 10 Collera, O., Walls, F., Garcia, F. Flores, S.E., Herran, J., Chemical Abstract, 1964, 61, 9769. 11 Govindachari, T.R., Joshi, B.S., Kamat, V., Tetrahedron, 1965, 21, 1509. 12 Smith, M.M., Mayo, P.D., Smith, S.J., Stenlake, J.B., Williams, W.D., Tetrahedron Letters, 1964, 2391. 13 Herout, V., Sorm, F., Perspectives in Phytochemistry, 1969, 139. 14 Pelissier, Y., Marion, C., Prunac, S., Bessiere, J.M., Journal of Essential Oil Research, 1995, 7, 313. 15 Pino, J.A., Marbot, R., Rosado, A., Fuentes, V., Journal of Essential Oil Research, 2004, 16, 139. 16 Choudhary, S.N., Indian Journal of Chemistry, 2003, 42B, 641.

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1 Fraga, B.M., Terrero, D., Gutierrej, C., Gonzalez-Coloma, A., Phytochemistry, 2001, 56, 315. 2 Wang, C.C., Kuoh, C.S., Wu, T.S., Journal of Natural Products, 1996, 59, 409. 3 Gonzalez-Coloma, A., Terrero, D., Perales, A., Escoubas, P., Fraga, B. M., Journal of Agricultural and Food Chemistry, 1996, 44, 296. 4 Zhang, C.F., Nakamura, N., Tewtrakul, S., Hattori, M., Sun, Q.S. Wang, Z.T., Fuziwara, T., Chemical and Pharmaceutical Bulletin, 2002, 50, 1195. 5 Kwon, H.C., Choi, S.U., Lee, J.O., Bae, K.H., Zee, O.P., Lee, K.R., Archives of Pharmacal Research, 1999, 22, 417. 6 Leong, Y.W., Harrison, L.J., Bennet, G.J., Kadir, A.A., Connoly, J.D., Phytochemistry, 1998, 47, 891. 7 Brophy, J.J., Goldsack, R.J., Forster, P.I., Journal of Essential Oil Research, 1999, 11, 453. 8 Tucker, A.O., Macearello, M.J., Burbage, P.W., Sturtz, G., Economic Botany, 1994, 48, 333. 9 Ichino, K., Tanaka, H., Ito, K., Chemical and Pharmaceutical Bulletin, 1989, 37, 1426. 10 Menut, C., Bessiere, J.M., Said, H.M., Buchbauer, G., Schopper, B., Flavour and Fragrance Journal, 2002, 17, 459. 11 Kotoky, R., Kanjilal, P.B., Singh, R.S., Plant Archives, 2001, 1, 87. 12 Lopej, M.L., Zunino, M.P., Zygadlo, J.A., Lopej, A.G., Lucini, A.I., Faillasi, S.M., Journal of Essential Oil Research, 2004, 16, 129. 13 Palomino, E., Maldonado, C., Kempff, M.B., Ksebati, M.B., Journal of Natural Products, 1996, 59, 77. 14 Weyerstahl, P., Wahlburg, H.C., Splittgerber, U., Marschall, H., Flavour and Fragrance Journal, 1994, 9, 179. 15 Castro, C.O., Lopez, V.J., Vergara, G.A., Phytochemistry, 1985, 24, 203. � 53. Gaur, A., Bulletin of Pure and Applied Sciences, 2004, 23C, 77. � - 32 -- � 54. Nath, S.C., Hazarica, A.K., Singh, R.S., Ghosh, A.C., Indian Perfumer, 1994, 38, � 26. � 55. Uchiyama, N., Matsunaga, K., Kiuchi, F., Honda, G., Tsubouchi, A., Shimada, � J.N. Aoki, T., Chemical and Pharmaceutical Bulletin, 2002, 50, 1514. 16 Pedro, L.G., Santos, P.A.G., da Silva, J.A., Figueiredo, A.C., Barroso, J.G., Deans, S.G., Looman, A., Scheffer, J.C., Phytochemistry, 2001, 57, 245. 17 Govindachari, T.R., Joshi, B.S., Kamat, V., Tetrahedron, 1965, 21, 1509. 18 Takeda, K., Ikuta, M., Tetrahedron Letters, 1964, 6, 277. 19 Takeda, K., Minato, H., Horibe, H., Tetrahedron, 1963, 19, 2307. 20 Takeda, K., Minato, H., Ishikawa, M., Miyawaki, M., Tetrahedron, 1965, 20, 2655. 21 Takeda, K., Minato, H., Ishikawa, M., Journal of Chemical Society (Japan), 1964, 4578. 22 Takeda, K., Minato, H., Ishikawa, M., Miyawaki, M., Tetrahedron, 1964, 20, 2655. 23 Maradufu, A., Phytochemistry, 1982, 21, 677. 24 Hayashi, N., Sakao, T., Komae, H., ix

th

International congress of essential oils, (13-17 march, 1983), 1, 40, Singapore. 25 Hikino, H., Konno, C., Heterocycles, 1976, 4, 817. 26 Takeda, K., Horibe, I., Teraoka, M., Minato, H., Chemical Communications, 1968, 940. 27 Pandji, C., Grimm, C., Wray, V., Witte, L., Proksch, P., Phytochemistry, 1993, 34, 415.

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28 Dakebo, A., Dagne, E., Sterner, O., Fitoterapia, 2002, 73, 48. 29 Makabe, H., Maru, N., Kuwabara, A., Kamo, T., Hirota, M., Natural Product Research, 2006, 20, 680. 30 Tomita, Y., Uomori, A., Minato, H., Phytochemistry, 1969, 8, 2249. 31 Ohno, T., Nagatsu, A., Nakagawa, M., Inoue, M., Li, Y.m., Minatoguchi, S., Mizukami, H., Fujiwara, H., Tetrahedron Letters, 2005, 46, 8657.

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1 Ruzicka, L., Proceedings of Chemical Society, 1959, 341. 2 Turlings, T.C.J., Tumilinson, J.H., Lewis, W.J., Science, 1990, 250, 1251. 3 Mc.Garrey, D., Croteau, R., The Plant Cell, 1995, 7, 1015. 4 Quereshi, N., Porter, J.W., Biosynthesis of Isoprenoid Compounds, John Wiley, NewYork, 1981, 1, 17. 5 Gershenzon, J., Croteau, R., Lipid Metabolism in Plants, CRC Press, BocaRaton, 1993 p, 335. 6 Rohmer, M., Knani, M., Simmonin, P., Sutter, B., Sohm, H., Biochemistry Journal, 1993, 295, 517. 7 Takji, M., Kujuyama, T., Takashasi, S., Seta, H., Journal of Bacteriology, 2000, 182, 4153. 8 Rohmer, M., Seemann, M., Herbach, S., Bringer-Meyer, S., Sahm, H., Journal of American Chemical Society, 1996, 118, 2564. 9 Zeidler, J.G., Lichtenthaler, H.K. May, H.H., Lichtenthaler, F.W.Z., Zeitschrift für Naturforschung, 1997, 52, 1523. 10 Rohdich, F., Eisenreich, W., Wungsintweekul, J., Hechl, S., Schuhr, C.A., Bacher, A., European Journal of Biochemistry, 2001, 268, 3190. 11 Chappel, J., Wolf, F., Prolux, J., Cuellen, R., Saunders, C., Plant Physiology, 1995, 109, 1337. 12 Adam, K.P., Zapp, J., Phytochemistry, 1998, 48, 953. 13 Senanayake, U.M., Wills, R.B.H., Lee, T.H., Phytochemistry, 1978, 16, 2032. 14 Birch, A.J., Chemical Plant Taxonomy, T. swain edn., 1963, 141. 15 Manitto, P. Monti, O., Gramatica, P., Tetrahedron Letters, 1974, 17, 1587.

- 34 --

(B) MATERIALS AND METHODS

1. Plant materials:

Fresh leaves bark, flowers and fruits (according to availability) of individual plants were

collected from different regions of Kumaun and Garhwal Himalaya, India, (table 1.1-1.3). The

plants were identified at Botanical Survey of India (BSI), Dehradun and specimen herbaria of

samples were deposited in Phytochemistry Research Laboratory, Kumaun University, Nainital. A

total of nine species belonging to six genera (table1.1) were collected to investigate the terpenoid

diversity and in vitro antioxidant and antibacterial activity. Further, ten samples of Cinnamomum

tamala (table 1.2) and three samples of Cinnamomum camphora (table 1.3) were collected from

different regions of Uttarakhand to study their chemotypic behaviour.

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Table 1.1, Collection sites of Lauraceae species from Uttarakhand

(Voucher Numbers; Botanical Survey of India, Dehradun)

S. No. Species Voucher No. Collection site Altitude

1 Lindera pulcherrima BSD 101366 Barabey (Pithoragarh) 2000m

2 Neolitsea pallens BSD 3418 Khati (Bageshwar) 2210m

3 Dodecadenia BSD 108688 Cheena Peak (Nainital) 2500m grandiflora

4 Persea duthiei BSD 106489 Thalkedar (Pithoragarh) 1900m

5 Persea odoratissima BSD 71116 Mandal (Gopeshwer) 1800m

6 Persea gamblei BSD 91810 Didihat (Pithoragarh) 1700m

7 Phoebe lanceolata BSD 50760 Jeolikote (Nainital) 1350m

8 Cinnamomum tamala BSD 17433 Natural/Commercial1 -

9 Cinnamomum camphora BSD 20300 Natural/Commercial2 - 1,2

Natural/Commercial samples of C. tamala/ C. camphora were collected from different regions of Uttarakhand ranging an altitude of 1000m to 1800m are shown in table 1.2 and

1.3. Table 1.2, Collection of samples of Cinnamomum tamala (Voucher Numbers; Phytochemistry Research Laboratory,

- 35 --

Kumaun University, Nainital)

S. No. Voucher number Habitat Collection site*

1 No. Chem/DST/Ct-01 Natural/Fresh Pithoragarh

2 No. Chem/DST/Ct-02 Natural/Fresh Ranikhet

3 No. Chem/DST/Ct-03 Natural/Fresh Jeolikote

4 No. Chem/DST/Ct-04 Natural/Fresh Almora

5 No. Chem/DST/Ct-05 Commercial Ramnagar mkt.

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6 No. Chem/DST/Ct-06 Natural/Fresh Hedakhan

7 No. Chem/DST/Ct-07 Commercial Nainital mkt.

8 No. Chem/DST/Ct-08 Natural/Fresh Bageshwar

9 No. Chem/DST/Ct-09 Natural/Fresh Tanakpur

10 No. Chem/DST/Ct-10 Commercial Tanakpur mkt.

Table 1.3, Collection of samples of Cinnamomum camphora (Voucher Numbers; Phytochemistry Research Laboratory, Kumaun University, Nainital)

S. No. Voucher number Habitat Collection site*

1 No.Chem/DST/Cc-01 Cultivated/Fresh Ramnagar

2 No.Chem/DST/Cc-02 Natural/Fresh Bhimtal

3 No.Chem/DST/Cc-03 Natural/Fresh Dehradun

2. Extraction:

- 36 --

The fresh leaves, bark, flowers and fruits of individual plants were subjected to steam

distillation for 2h using a copper electric still, fitted with spiral glass condensers. The distillate

was saturated with NaCl and extracted with n-hexane and dichloromethane (2:1). The organic

phase was dried over anhydrous Na2SO4 and the solvent was distilled off in rotary vacuum

evaporator (Heidolph) at 30o

C to yield the essential oils. The yield was calculated in (v/w).

3. Gas Chromatography:

The oils were analyzed by using Nucon 5765 gas chromatograph fitted with Rtx-5 nonpolar

fused silica capillary column (30m × 0.32mm internal diameter). The column temperature was

programmed 60-2100

C @ 30

C/min using N2 as carrier gas at 4 Kg/cm2

. The injection temperature

was 2100

C, detector temperature 2100

C and the injection sizes 0.5μl using 10% solution of the oil

in n-hexane.

4. Gas Chromatography/Mass Spectrometry:

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GC-MS was done using Thermo quest Trace GC 2000 interfaced with Finnigan MAT

PolarisQ Ion Trap Mass Spectrometer fitted with Rtx-5 non polar fused silica capillary column

(30m × 0.25mm internal diameter). The column temperature was programmed 600

C-2100

C @

30

C/min using helium as carrier gas at 1.0ml/min. The injection temperature was 2100

C, ion source

temperature 2000

C, MS transfer line temperature 2750

C, injection size 0.1μl, split ratio 1:40. MS

were taken at 70 eV with mass range of m/z 40-450 amu.

5. Retention Indices:

Besides the spectral methods, the identification of essential oil was done by calculation of their

retention indices and comparison with those of the literature reports. Retention indices were

experimentally determined by the following formula:

RI= 100 × No + 100 [RTunknown – RTNo] / [RTN1–RTNo]

RTunknown= RI value of the compound to be identified.

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RTNo= n-alkane eluted before the unknown peak.

RTN1= n-alkane eluted after the unknown peak.

No= Carbon number from which the standardization is done.

6. Isolation of major constituents:

The essential oil (5-10ml) was fractionated using column chromatography (CC) on a

column (600 × 25mm) packed with silica gel (230-400 mesh, Merck) in hexane. The compounds

were eluted with hexane followed by hexane/ether mixture gradually increasing the concentrations

of the ether (5 to 25%). The fractions collected (10 to 20ml) were examined by TLC on silica gel-

G (Merck) plates using anisaldehyde-sulphuric acid-glacial acetic acid or vanillin-hydrochloric

acid as spraying reagents1

or observing under UV lamp. The fractions with identical compositions

were mixed finally giving some useful fractions among several others. The fractions were

concentrated and again examined by TLC followed by GC analysis.

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7. High performance liquid chromatography:

The impure fractions of column were further analyzed by using Thermoelectron quadrupole

High Performance Liquid Chromatograph fitted with Nucleosil non-polar column (25cm × 0.5mm

internal diameter). The separation was monitored on the basis of refractive index (RI-150) and

ultra violet (UV-1000) detector. The pressure programme was 0-3000 (Pump; P-4000) psi. Elution

was done by vacuum degassed hexane/ether (HPLC grade) as per requirement.

8. IR spectral analysis:

The IR spectra of essential oils and the isolated compounds were taken in the Perkin Elmer FT-

IR (Spectrum bx). Liquid samples were analyzed by salt plate method while diffuse reflectance

method was applied for solid samples.

9. 1

H-NMR and 13

C-NMR spectral analysis:

The NMR spectra of the pure isolates were taken in CDCl3 on a Bruker-Avance DRX 300 MHz

at 250

C using TMS as internal standard.

- 38 --

10. Identification of constituents:

The identification of the isolated compounds was done on the basis of linear retention index

(LRI), infra red spectral values (IR), mass spectral fragmentation pattern & library search (NIST

& WILEY) by comparing with the MS literature data2,3

and by 1

H-NMR and 13

C-NMR spectral

data. The percentage contents of constituents were determined on the basis of FID response on

GC. The known compounds were further confirmed by comparing with the authentic samples.

11. Statistical analysis:

The similarities and differences in the terpenoid compositions among the species of same

genus were determined by using BD-Pro software in order to discern chemotaxonomic

relationship and phylogenic studies. Bray-Curtius percentage was selected as the basis of cluster

analysis.

12. Antioxidant activity:

The in vitro antioxidant activity of the leaf essential oils of seven species viz. Lindera

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