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CHAPTER 1

INTRODUCTION

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1.1 INTRODUCTION

The Himalayas have a great wealth of medicinal plants and traditional

medicinal knowledge. The Central Himalayan Region covering the new state of

India, provides excellent opportunities for studying the Traditional Knowledge

Systems1. The Western Himalayan region of Uttarakhand (India) is particularly

rich in medicinal and aromatic plants representing one of the twelve mega-

biodiversity centers of the world. This is due to topography and climatic

conditions that provide suitable environment for the growth of plants having

medicinal and economical values. The Western Himalayan region is considered

as a botanical treasure because of the presence of a large number of species in

this region, richness of flora and vegetation diversity 2. The rich plant diversity of

the Himalayan region has been utilized by the native communities in various

forms such as medicine, food, fodder, agricultural tools, fuels, building materials

etc. Over 4000 plants species are estimated to occur in Uttarakhand region.

The region supports 702 medicinal plants, 344 wild edibles, 279 common

fodder species, over 20 species of gymnosperms and 350 species of

pteridophytes. The Uttarakhand state has a large altitudinal range extending

from 300 m to 7,817 m and represents tropical, subtropical, temperate,

subalpine and alpine vegetation 3. The alpine and sub alpine zones are very

rich in medicinal and aromatic plants as compared to other zones 4.

The relationship existing between plants and humans is as old as

mankind, dating back to the origin of human civilization 5. Medicinal plants are

found and frequently used in China, India, Japan, Pakistan, Thailand and in

South Africa 6. Globally, the Indian Ayurvedic and Chinese are recognized as

the oldest and most developed traditional medicinal systems 7. Traditional

medicines also exist in southern America and Australia, but these are not as

developed as in Asia or Africa. Distinct traditional medicinal systems are found

universally in each geographical region, so that medicinal plant use is diverse

globally 8.

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Among ancient civilizations, the forests in India are the principal

repository of a number of medicinal and aromatic plants, which are largely

collected as raw materials for manufacturing drugs and perfumery products.

About 8,000 herbal remedies have been codified in Ayurveda. The Rigveda

(5000 BC) has recorded 67 medicinal plants, Yajurveda 81 species,

Atharvaveda (4500-2500 BC) 290 species. Charak Samhita (700 BC) and

Sushrut Samhita (200 BC) have described properties and uses of 1100 and

1270 species respectively, in compounding of drugs and still used in the

classical formulations, in the Ayurvedic system of medicine 9.

More than 200 economically important medicinal plants grow in

Uttarakhand (Kumaun and Garhwal region). Among these, several medicinal

herbs and essential oil bearing plants are used in Ayurvedic, Unani, Siddha and

Tibetan system of medicine in different forms 3, 10. Plants have been used by

man from prehistoric times for relieving suffering and curing ailments. The

indigenous system of medicine practiced in India is based mainly on the use of

plants. Charak Samhita records the use of 200 vegetable remedies. Ancient

medicine was not solely based on empiricism. This is evident from the fact that

some medicinal plants which were used in ancient times still have their place in

modern therapy. The plant sarpagandha (Rauwolfia serpentina) well known in

India as a remedy for insanity and hypertension, one of its constituent,

reserpine, is a wonderful drug today for curing mental ailments 11. Ephedra

plant is the world’s oldest medicine, have been used in traditional Chinese

medicinal system for over 5000 years. Traditional practitioners have used it for

a wide range of illnesses, from cold, asthma, and hay fever to various kidney

ailments. Over time, several pure amphetamine-like compounds called alkaloids

have been extracted from Ephedra. These include the strongly bioactive

isomeric molecules ephedrine and pseudoephedrine. Ephedrine was

recognized as a standard drug by the American Medical Association in 1927.

Recently Ephedra preparations have been marketed as stimulants and weight

loss agents 12. Quinine, obtained from the Cinchona tree is used as an

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important antimalarial drug of modern medicine 9. Some of the active molecule

cannot be synthesized economically; the product must be obtained from the

cultivation of plant material. About 121 (45 tropical and 76 subtropical) major

plant drugs have been identified for which no synthetic one is currently available

(Table 1.1). The scientific study of traditional medicines, derivation of drugs

through bioprospecting and systematic conservation of the concerned medicinal

plants are thus of great importance.

Table 1.1. Some major plant drugs for which no synthetic one is currently

available 13.

Drugs Plants Uses

Vinblastine Catharanthus roseus Anticancer

Ajmalacine Catharanthus roseus Anticancer, hypotensive

Rescinnamine Rauwolfia serpentina Tranquilizer

Reserpine Rauwolfia serpentina Tranquilizer

Quinine Cinchona species. Antimalarial,

amoebic dysentery

Pilocarpine Pilocarpus jaborandi Antiglucoma

Cocaine Erythroxylum coca Topical anaesthetic

Morphine Papaver somniferum Painkiller

Codeine Papaver somniferum Anticough

Atropine Atropa belladonna Spasmolytic, cold

Atropine Hyoscyamus niger Spasmolytic, cold

Cardiac glycosides Digitalis species For congestive heart

failure

Artemisinin Artemisia annua Antimalarial

Taxol Taxus baccata

T. brevifolia

Breast and ovary

cancer,

Antitumour

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Berberine Berberis For leishmaniasis

Pristimerin Celastrus paniculata Antimalarial

Quassinoids Ailanthusspecies Antiprotozoal

Plumbagin Plumbago indica Antibacterial, antifungal

Gossypol Gossypium species Antispermatogenic

Allicin Allium sativum Antifungal, amoebiasis

Emetine Cephaelis ipecacuanha Amoebiasis

Glycyrrhizin Glycyrrhiza glabra Antiulcer

Nimbidin Azadirachta indica Antiulcer

Catechin Acacia catechu Antiulcer

Sophoradin Sophora subprostrata Antiulcer

Magnolol Magnolia bark Peptic ulcer

Forskolin Coleus forskohlii Hypotensive, cardiotonic

Digitoxin, Digoxin Digitalis, Thevetia Cardio tonic

Thevenerin, Thevetia species Cardio tonic

Nerrifolin Thevetia species Cardio tonic

Podophyllin Podophyllum emodi Anticancer

Indicine N-oxide Heliotropium indicum Anticancer

Elipticine Ochrosia species Anticancer

Homoharringtonine Cephalotaxusspecies Anticancer

Camptothecine Camptotheca acuminate Anticancer

Medicinal and aromatic plants constitute a major segment of the flora

that provides raw materials for use in the pharmaceuticals, cosmetics, and drug

industries. The indigenous systems of medicines, developed in India for

centuries, make use of many medicinal herbs. These systems include

Ayurveda, Siddha, Unani, and many other indigenous practices. More than

9,000 native plants have established and recorded curative properties and

about 1500 species are known for their aroma and flavor. In one of the studies

by the World Health Organisation, it is estimated that 80 percent of the

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population of developing countries relies on traditional plant based medicines

for their health requirements 14. The basic composition of many modern

medicines are derived from medicinal plants and these have become

acceptable medicines due to easy availability, low prices, least side effects,

lasting curative property and environmental friendliness. India and China are

the two major producing countries, having 40 percent of the global biodiversity

and availability of rare species. These are well known as the home of medicinal

and aromatic crops that constitute a segment of the flora and provide raw

materials to the pharmaceutical, cosmetic, fragrance and flavour industries 15.

Phytochemistry has developed as a distinct discipline in natural product

organic chemistry and plant biochemistry and is closely related to both.

Phytochemistry or plant chemistry is concerned with the enormous variety of

organic substances that are elaborated and accumulated by plants and deals

with the chemical substances of these plants, their biosynthesis turnover and

metabolism, their distribution and biological function 16. Green plants are

producing amazingly diverse range of chemical products which can be

classified into primary and secondary metabolites. Primary metabolites are

those which are common to all species and can be sub divided in to

carbohydrate, proteins, fats and vitamins etc. Secondary metabolites are non

nutritional chemicals and can be sub divided on the basis of their biosynthetic

and biogenetic pathway into terpenoids, alkaloids, shikimates and polyketides.

These secondary plant metabolites play an important role and are known to

exhibit biological activity and drug formation 17, 18.

The therapeutic effect of medicinal plants for the treatment of various

diseases is based on the chemical compounds of these plants. The major

components are organic compounds, some of which have biological activity, but

none act independently and cannot replace the functions of the medicinal plant

as a whole. The active principles of many drugs were found in plants or

produced from one of the secondary metabolites. The remarkable contribution

of plants to the drug industries has been made possible because of the large

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number of phytochemical studies all over the world. These studies established

methods and techniques for extraction, separation, chemical identification and

biological testing of plants constituents. Many reagents have been developed

as diagnostic for different chemical principles and this progress resulted in

isolation of many active compounds19, 20.

A number of scientific investigations have highlighted the importance and

the contribution of many plant families i.e. Asteraceae, Liliaceae, Apocynaceae,

Solanaceae, Caesalpinaceae, Rutaceae, Piperaceae and Sopotaceae to

medicinal plants. Medicinal plants play a vital role for the development of new

drugs. Plant derived drugs are used to cure skin diseases, mental illness,

jaundice, hypertension, tuberculosis, diabetes and cancer. Vinblastine,

deserpidine, reseinnamine, vincristine and reserpine are higher plants derived

drugs which were introduced in the USA drug market during 1950-1970. From

1971 to 1990 new drugs such as ectoposide, (E)- and (Z)-guggulsterone,

teniposide, nabilone, plaunotol, lectinan, artemisinin and ginkgolides appeared

all over the world. Two percent of drugs were introduced from 1991 to 1995

including paciltaxel, toptecan, gomishin, irinotecan etc.21. Flavonoids are known

to exhibit several biological activities 22 including anti-HIV activity 23. The

dichloromethane and menthol extracts of Monotes africanus (Dipterocarpaceae)

have been isolated anti-HIV prenylated flavonoids as 6, 8-diprenylaromadendrin

and flavonol 6, 8-diprenylkaempferol along with lonchocarpol A. These

flavonoids exhibited HIV-inhibitory activity in the XTT-based, whole-cell screen.

Extract of various species of Valeriana are used in traditional medicine in many

parts of the world. The major traditional use is as tranquilliser or sedative,

hypotensive, antispasmodic, gastrointestinal, poison antidote and stomachic.

The essential oil from the root of Valeriana wallichii has been found to contain

α-santalene, α-curcumene, xanthorrhizol, patchouli alcohol and maaliol 24.

Phophyllotoxin is a constituent of Phodophyllum emodi used against testicular,

small cell lung cancer and lymphomas 21. Several alkaloids have been isolated

from the root bark of Rauwolfia serpentine including reserpine, ajmaline,

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ajmalicine, yohimbine etc. Reserpine is an indole alkaloid used for the treatment

of hypertension and lowering of blood pressure 25. Vincristine is used for acute

lymphocytic leukemia in childhood advanced stages of hodgkins,

lymophosarcoma, small cell lung, cervical and breast cancer. Vinblastine

isolated from Catharanthus rosesus is used for the treatment of hodgkins,

choriocarcinoma, non-hodgkins lymphomas, leukemia in children, testicular and

neck cancer 26.

1.2 CHEMOSYSTEMATC STUDY OF ESSENTIAL OILS

A chemotype is a chemically distinct entity in a plant and microorganism,

with difference in the composition of secondary metabolites. This variability in

the composition of secondary metabolites may be caused by the effects of

various climatic, genetic and environmental influences. In appearance, the

plants look identical, but their chemical compositions are different. These are

usually qualitative designations, making the concept of chemotypes somewhat

arbitrary, due to large amounts of variation in the chemistry of any particular

plant species. To address this ambiguity, researchers are increasingly using

multivariate statistical analysis of intra-specific variation in plant secondary

chemistry as a way to define chemotypes more precisely 27, 28.

The essential oils of four wild growing Origanum vulgare L. collected

from four locations in Kumaon region (Uttarakhand, India) representing the

presence of two chemotypes. The chemotype I shows p-cymene (6.7–9.8%), γ-

terpinene (12.4–14.0%), thymol (29.7–35.1%) and carvacrol (12.4–20.9%) as

major constituents while chemotype II shows linalool (6.7–9.7%), bornyl acetate

(12.6–16.8%), β-caryophyllene (10.5–13.8%) and germacrene D (6.3–11.3%)

as the major constituents 29 . Origanum vulgare L. growing in ten localities of

Vilnius district (Lithuania) had two chemotypes: ß-ocimene and germacrene D

type 30. D'antuono et al. (2000) 31 reported three chemotypes of O. vulgare:

carvacrol/thymol type, (E)-caryophyllene, γ-muurolene and high linalool type

and β-bourbonene, (E)-caryophyllene, γ-muurolene, germacrene D-4-ol and

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caryophyllene oxide type, collected from Northern Italy. Song and Chae (2004)

analyzed forty seven individual plants of Elsholtzia splendens collected from

eight regions in Korea and identified three chemotypes. The major oil

component of chemotype I was dihydrotagentone (75.0%) along with

naginataketone and elsholtziaketone. Chemotype II was naginataketone (NK)

type with more than 60% naginataketone. Chemotype III had more than 60% of

elsoltziaketone (EK) as a major volatile constituent 32. Many species in the

genus Thymus show evidence of polymorphic variation in monoterpene

production 33.

The essential oil from leaves of Clausena anisum-olens (Blanco) Merr.

Ver. anisum-olens (Rutaceae) was studied individually on 91 cultivated and wild

plants. Variation in the oil contents between individuals showed a distribution

pattern of apparent genetic origin, with three chemotypes: Chemotype I- pure

anethole oil, Chemotype II - pure methylchavicol oil and Chemotype III-anethole

and methylchavicol mixed oil 34. The study of citrus peel oil revealed the

presence of three chemotypes: limonene, limonene/gamma-terpinene and

linalyl acetate/limonene, while leaf oils showed three chemotypes; sabinene/

linalool, gamma-terpinene/ linalool and methyl N-methylanthranilate 35, 36. The

alkaloids, coumarins, flavanoids and lignans of the taxa Zanthoxylum and

Fagara were examined in detail and their chemotaxonomic markers have also

been documented in family Rutaceae 37.

Thymus vulgaris had six genetically distinct chemotypes that can be

distinguished on the basis of the dominant monoterpene produced in glandular

trichomes on the surface of the leaves 38. Each of the six chemotypes; geraniol

(G), α-terpineol (A), thuyanol-4 (U), linalool (L), carvacrol (C) and thymol (T), is

named according to its dominant monoterpene 39. Rosemary (Rosmarinus

officinalis L.) is a spice and medicinal herb showed mainly three chemotypes 1)

1,8-cineole chemotype from France, Greece, Italy and Tunisia 2) camphor-

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borneol chemotype from Spain and 3) α-pinene and verbenone chemotype from

Carsica and Algeria 40, 41, 42.

The Apiaceae (Umbelliferae) family comprises about 300 to 455 genera

and 3000 to 3750 species distributed in the Northen hemisphere 43. Ammi

visnaga (L.) Lam was found to exist as two chemotypes: one containing linalool,

isoamyl 2-methyl butyrate and isopentyl isovalerate as major constituents from

Tunisia 44 while the other has a high content of nerol and bisabolol from

Turkey45. There are 300 Umbellifer species which have been surveyed for their

leaf phenolics, using both fresh and herbarium tissue. The results showed that,

with few exceptions, species can be divided into two groups, those with flavone

(usually luteolin) and others with flavonol (kaempferol and/or quercetin) 46.

Chemosystematic comparison of essential oils from eight species of four

genera of the New Zealand Apiaceae family has been reported. The seed oil

from Gingidia and Scandia species contained high proportions of

phenylpropanoids while Anisotome haastii comprised of monoterpenoids

showing complete absence of phenylpropanoids. Out of the four Aciphylla

species, three contained neither terpenoids nor phenylpropanoids, but showed

the presence of fatty acids The oil from Aciphylla squarrosa was different as it

contained abundant phenylpropanoids. The composition of the leaf essential

oils of Scandia rosifolia and Gingidia montana differed significantly from their

corresponding seed oils. Outstandingly, the seed essential oil of G. montana

contained 62% estragole and that of Scandia geniculata contained 79% dill

apiole 47.

Cinnamon (Cinnamomum zeylanicum Blume, syn C. verum JS Presl,

family Lauraceae) is an important spice and aromatic tree cultivated in Sri

Lanka and India. On steam distillation, different parts of cinnamon yield volatile

oils of varying composition. The tender twig oil was rich in α-phellandrene

(3.4%), limonene (1.6%) and (E)-cinnamaldehyde (4.0%). The volatile oils from

pedicels were rich in neryl acetate (1.4–2.0%), (E)-cinnamyl acetate (58.1–

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64.5%) and β-caryophyllene (9.6–11.1%) 48. Higher amounts of (Z)-cinnamyl

acetate, α-humulene, δ-cadinene, humulene epoxide I, α-muurolol, α-cadinol

and α-bergamotene were observed in the oil of buds and flowers 49. The volatile

oils from leaves represented three chemotypes eugenol/ trans-β-caryophyllene,

eugenol, and eugenol/ linalool/ piperitone 50, 51, 52. The bark oil showed three

chemotypes: (E)-cinnamaldehyde /α-copane/O-methoxy cinnamaldehyde/(E)-

cinnamyl acetate (chemotype I), 1, 8-cineole/ (E)-cinnamaldehyde/ β-

caryophyllene (chemotype II) and cinnamaldehyde/ methyl cinnamate

(chemotype III) 51, 53, 54 . The fruit oil showed greater concentrations of α-pinene,

β-pinene and linalool 48. The above discussion showed that the whole plant of

cinnamon has different terpene distribution within the species and contained

different chemotypes.

1.3 TERPENE CHEMISTRY AND THEIR BIOSYNTHESIS

The Plants produce primary and secondary metabolites which

encompass a wide array of functions 55. Primary metabolites, including amino

acids, simple sugars, nucleic acids and lipids are the compounds necessary for

cellular processes. Secondary metabolites are produced by means of

secondary reactions resulting from primary carbohydrates, amino acids and

lipids 56. Plants can manufacture different types of secondary metabolites,

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

diverse array of applications. However, their ecological role 57 and particularly in

plant herbivore interaction 58, 59 and chemotaxonomy has been well established.

The secondary metabolites in plants include alkaloids, terpenoids, saponins,

steroids, tannins and organic acids.

Terpenoids are the important secondary plant metabolites, lipid soluble

and located in the cytoplasm of the plant cells. They also occur in special

glandular cells on the leaf surface 18. The term terpene, derived from turpentine,

appears to have originated in the writing of Kekule in 1866 for hydrocarbons

C10H16 to take place of such word as ‘terpene’; ‘camphene’ etc. 60, 61. The term

‘terpene’ later also included all compounds with distinct structural and chemical

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relation to simple C5H8 molecule. The term “Terpenoid” includes hydrocarbons,

alcohols, aldehydes, ketones etc. 62. The terpenes may be divided in to four

groups; (1) Acyclic terpenes (2) Monocyclic terpenes (3) Bicyclic terpenes (4)

Tricyclic terpenes 63.

All the terpenoids are derived from the same monomeric unit, isoprene

C5H8 64, 65. Isoprene is 2-methyl-1, 3-butadiene and can be represented as

given below:

or

C C

C

H

HH

C

H

H

H

The isoprene units are linked together in a head to tail manner forming

open chain or cyclic compounds. The terpenoids are classified on the basis of

the number of isoprene units which they contain. As early as 1887, Von Wallach

(1887) suggested that isoprene (2-methylbutadiene) is the building block of

terpenes and he earned the Nobel Prize in chemistry in 1910 for this concept.

The isoprene units are ‘linked in a special way according to the isoprene rule 66,

64. Hence, the tail of one unit is linked to the head of the next one. The terpenes

are classified based on the number of isoprene-units linked (Table 1.2). More

than 6000 compounds with mono-, sesqui-, and diterpene structures have been

detected and their structures determined. The vast majority of these are

hydrocarbons or oxygen containing terpenes such as alcohols. There are

compounds with the isoprenoid skeleton, either lacking one, or with one extra

carbon atom, named nor-terpenes and homo-terpenes respectively.

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Table 1.2. Classification of terpenes based on Sjöström (1981) 68.

Prefix Number of carbon

atoms

Number of Isoprene

(C5H8) units

Hemiterpenoids 5 1

Monoterpenoids 10 2

Sesquiterpenoids 15 3

Diterpenoids 20 (e.g. resin acids) 4

Sesterterpenoids 25 5

Triterpenoids 30 (e.g. steroids) 6

Tetraterpenoids 40 (e.g. carotenoids) 8

Polyterpenoids >40 (e.g. rubber) >8

Hemiterpenoids do not occur in nature. The mono- and sesquiterpenoids

are steam volatile constituents and are responsible for the characteristic odour

of plants. The biosynthesis of terpenoids has been the subject of much

research. Isotopic studies on plants, animals and microorganisms have

revealed a great deal on the biosynthesis of terpenes 69, 70. There are some

different types of biosynthetic precursors which are responsible for the

synthesis of terpenes and terpenoids by cyclization and rearrangements. These

biosynthetic precursors are Isopentenyl pyrophosphate (IPP) for

hemiterpenoids (C5H8), Geranyl pyrophosphate (GPP) for monoterpenoids

(C10H16), Farnesyl pyrophosphate (FPP) for sesquiterpenes (C15H24), Geranyl

geranyl pyrophosphate for diterpenoids (C20H32), Geranyl farnesyl

pyrophosphate for sesterterpenoids (C25H40), squalene for triterpenoids

(C30H48), Phytoene for tetraterpenoids (C40H64) and Geranyl geranyl

pyrophosphate for polyterpenoids (C5H8)n 71. Biosynthesis of terpenoids is given

in Figure 1.1. In the first step of biosynthetic pathway, acetyl-CoA (C2) and

acetoacetyl-CoA (C4) condence to form β-Hydroxy-β-methylglutaryl-CoA (C6).

This step is followed by an enzymatic reduction of the thiol ester group of β-

Hydroxy-β-methylglutaryl-CoA to the primary alcohol of mevalonic acid. The

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enzyme that catalyzes this step is called HMG-CoA reductase (HMG is β-

Hydroxy-β-methylglutaryl). In this pathway, Mevalonic acid is an intermediate

and this C6 compound could be transformed into 3-methyl-3-butyenyl

pyrophosphate by successive phosphorylation and decarboxylation reactions 72.

3-Methyl-3-butyenyl pyrophosphate (Syn. IPP Isopentenyl pyrophosphate) is

the precursor of Hemiterpenods. 3-Methyl-2-butyenyl pyrophosphate is formed

by the isomerisation of 3-methyl-3-butyenyl pyrophosphate. The combination of

3-methyl-2-butyenyl pyrophosphate and 3-methyl-3-butyenyl pyrophosphate

involves enzymatic formation of an allylic cation. The two compounds condense

to form geranyl and neryl pyrophosphate, a C10 compound. Geranyl

pyrophosphate is the precursor of the mono terpenes. Geranyl pyrophosphate

subsequently condenses with another molecule of 3-methyl-3-butyenyl

pyrophosphate to form the C15, precursor for sesquiterpenes, farnesyl

pyrophosphate and so on 73, 74.

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Figure 1.1 Biosynthetic pathway of terpenoids

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1.4 ROLE OF TERPENES IN NATURE

Terpenes are constituents of essential oils and prevalent in many plants.

In general, terpenes represent a heterogeneous group of chemical substances

constructed from hydrocarbons. Variation in additional chemical compounds,

such as alcohols, aldehydes or ketones (terpenoids), defines the variations of

the different terpene compositions. Conservative estimates suggest that at least

40,000 different terpenoids (isoprenoids) exist in nature, many of which are of

plant origin 75. Many terpenoids are essential for plant growth, development and

general metabolism 76. These terpenoids are found in almost all plant species.

The distribution of terpenes in nature has been studied extensively. To

better understand terpene and other volatile organic compound emissions, from

loblolly pine (Pinus taeda), Thompson et al. (2006) 77 analyzed tree core

samples. They found the highest concentrations of terpenes in heartwood,

lowest in outer sapwood and moderate levels in the inner sapwood. In a less

invasive study by Martin et al. (2003) 78 methyl jasmonate was applied onto

foliage of Norway spruce (Picea abies) trees which led to a two fold increase of

terpenes within the needles. In another investigation, the amounts of different

terpenes in Scots pine (Pinus sylvestris) needles varied across Finnish and

Turkish regions, showing that diversity of terpene distribution can vary within a

species 79 .

Terpenoids are secondary metabolic products but they have specific

function within the plants. The most important physical characteristic of the

monoterpenes is their odour; insects as well as animals are sensitive to this.

Terpene emissions and attracting mechanisms play an important indirect role in

plant defence mechanisms. Terpenoids have been shown to attract beneficial

mites, which feed on the herbivorous insects 80. Nepeta cataria has a particular

attraction for the domestic cats because of monoterpene lactone, nepeta

lactone 18 which also exhibits insect repelling activity 81. Herbivorous insects can

induce terpene release from a plant and also cause the plant to release signals

which attracts predatory species 82. Arabidopsis emits different volatiles

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including terpenes which may play an important role as insect attractant in

reproduction 83. Some monoterpene derivatives have been implicated as sex

and food attractant for certain insects 84. Farnesol, interestingly plays an

important role among the insect both as a juvenile hormone and in other cases

as a male sex attractant 85. Plant terpenes have also been used as models for

phylogenetic studies 86.

Ecological roles of terpenes extend beyond plant insect co evolution.

Terpenes may also act as chemical messengers that influence the expression

of genes involved in plant defense mechanisms or even influence gene

expression of neighboring plants 87. Terpenes also showed allelopathic toxic

activity, acting as phytotoxins. It has been shown that Salvia leucophylla

liberates 1, 8-cineole, camphor and related compounds through its leaves in to

the surrounding atmosphere which are absorbed by the dry soil. These

compounds inhibit the germination and growth of grassland herbs which would

otherwise have taken place with the arrival of rain water 88. Plants containing

secondary metabolites particularly alkaloids, saponins and tannins are generally

avoided by grazing animals and leaf feeding insects. Their presence in plants

and intake at high level reduces the nutrient utilization, feed efficiency, animal

productivity and in some cases death of animals 89. Recently, Wahid and

Ghazanfar (2004) 90 and Wahid and Babu (2005) 91 reported that high level of

secondary metabolites can enhance salt tolerance in sugarcane and wheat,

respectively.

The growth regulating properties of terpenoids are well established.

Sesquiterpenoids, abscisic acid, xanthinin and diterpenoid-based gibberellins

are the major classes of growth regulators. Abscisic acid is best known as the

principal hormone controlling dormancy in seeds of herbaceous plants and in

the buds of the plants 18. Xanthinin acts as an auxin-antagonist in plant

physiology 92. Ipomea marone is a sesquiterpene produced by sweet potato

roots in response to infection with black rot fungus Ceratocyctis Fimbrista.

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Ipmoea marone also exhibits potent antifungal action against the pathogenic

fungus 93.

Many plants contribute to the earth’s atmospheric composition by releasing

volatile organic compounds, which includes terpenes. It has been estimated

that the annual global emission of isoprenes is 500 teragrams 94. Volatile

organic compounds (VOCs) including terpenoids are emitted into the

atmosphere from both stressed and non-stressed vegetation. In European

boreal zone, the natural VOC sources are known to surpass the anthropogenic

ones. Mechanical stress and damage of plants often strongly increases their

monoterpene emissions. As the forests in European boreal zone are under

intense economic use, forest management operations can be a significant

source of terpenes into the atmosphere of this area 95. Appreciable quantities of

volatile terpenes are emitted into the atmosphere from plants such as conifers

and oaks 96, 97. Since they potentially serve as photochemical oxidant

precursors, their global emission rate 98, 97, their atmospheric reactivity 99 and

their ambient concentration 100, 101 have been studied to determine their

contribution to air pollution. To estimate the contribution of monoterpenes to air

pollution, it is necessary to establish a clear relationship between monoterpene

emission rates and environmental conditions. Monoterpene emission rate

increases exponentially with temperature and is also influenced by light 102.

Terpene emission depends on the vapor pressure of terpenes, the humidity of

the air surrounding the leaf and the surface area of essential oil present on the

leaves 103.

1.5 ESSENTIAL OILS AND THEIR BIOLOGICAL ACTIVITY

Essential oil is the essence of a plant and is produced in various parts of the

plant. Cooksley described them as “Tiny droplets” contained in glands,

glandular hairs, sacs or veins of different plants parts: leaves, stems, bark,

flowers, roots and fruits. Two common essential oils are obtained from sandal-

wood and cedar tree. Rose oil comes from petals, peppermint oil from the

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glands on the leaves while it is present in the rind, leaves, fruits and flowers of

orange 104. It can be extracted using a variety of methods such as steam

distillation and solvent extraction. It is widely used in perfumery,

aromatherapy, cosmetics, incense, medicine, household cleaning product as

well as flavoring food and drink industries. Essential oils are also known as

volatile oils and ethereal oils. Sometimes, it may also be referred to as oil of the

raw plant materials, from which it was extracted, for example oils of clove 105. It

is important to understand the effect that the oils have and the way it works

before using the essential oils as a part of treatment. The active

constituents of the essential oils are terpene hydrocarbons and their

oxygenated derivatives. Main components of the essential oils and their

therapeutic properties have been discussed below in brief.

1.5.1 Terpenes

1.5.1.1 Monoterpene hydrocarbons- The essential oils from certain

umbellifers, pine, eucalyptus, cumin, carrot, rosemary, lemon,

coriander and tea tree are rich in terpene hydrocarbons like pinene,

camphene, limonene, myrcene, phellandrene etc. They act as

strengthening agents, stimulants and have antiseptic properties.

Certain conifers and their resin oils have stimulating effect on the

connection between the pituitary gland and the suprarenal gland

cortex 106. Juniper species and berry oils rich in monoterpene

hydrocarbons, showed cytotoxic and antimicrobial activity 107.

Limonene is used as an insecticide and marketed to relieve

gastroesophagal reflux disease and heart burn 108.

1.5.1.2 Sesquiterpene hydrocarbons- Sesquiterpene hydrocarbons like

cadinene, selinene, β-caryophyllene, bisabolene, humulene,

farnesene, germacrene D and guainene have been present as major

constituents in the essential oil of Origanum, celery, clove juniper,

cedar and showed potent larvacidal activity 109, antibacterial and

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antifungal activities 110, 111. It has been reported that essential oils

containing germacrene D from members of Asteraceae and Rutaceae

showed activity like sex hormone 112. β-Caryophyllene have anti

inflammatory properties 113. While bisabolane compounds possess

antitumor, antibacterial and antifeedent activities 114, 115, 116.

1.5.2 Terpenoids

1.5.2.1 Phenols- Phenolic terpenes such as carvacrol, thymol, eugenol and

guaiacol commonly available in high amount in Origanum, Ocimum,

Thyme , cloves, cinnamon essential oils. Eugenol and methyl eugenol

have been reported as attractants117. Phenolic terpene rich

Origanum, Ocimum and Thymus are reported to have insecticidal,

antibacterial and antifungal activities118, 119, 120. Aromatic phenols,

stimulate the immune system (greatly increase the α-globulene),

evoke general activity of a “hyper” nature (hyperthermic hypertone),

generally stimulating for the nervous system, circulation, liver and

digestion 106.

1.5.2.2 Alcohols- The essential oils from thyme, orange, lemon, lemongrass,

marjoram, lavender, rosemary and mint were found to be rich in

alcoholic terpenes. Some of them are citronellol, borneol, menthol,

nerol, geraneol, carotol, santalol, viridiflorol etc. Essential oils from

some herbs and spices possess cancer chemo preventive activities.

Zu et al. (2010) 121 reported the anticancer activity of mint, lemon,

lavender, chamomile, thyme, rose and Cinnamomum essential oils

against PC-3, A-549 and MCF-7 cancer cells and found that thyme

essential oil has strongest cytotoxic activity against cancerous cells.

α-Bisabolol possesses anti-inflammatory activity 122 .

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1.5.2.3 Oxides- Terpene oxides such as 1, 8-cineole, linalool oxide,

ascaridol, bisabobole oxide, safrol, piperiton oxide and piperonal

butoxide obtained from eucalyptus, mint, cardamom, rosemary,

lavender and Cinnamomum. Piperonal butoxide, a modified

compound of safrol oil has been reported as the most potent

synergist 123. 1,8-cineole isolated from Salvia fruiticosa showed

antimicrobial activity against Escherichia coli, Pseudomonas

aeruginosa, Staphylococcus aureus, Rhizobium leguminosarum and

Bacillus subtillis and also exhibits cytotoxic activity against African

green monkey kidney cells and high level of virucidal activity agaist

herpes simplex virus, a ubiquitous human virus 124.

1.5.2.4 Ethers- Methyl chavicol, methyl salicylate, methyl cinnamate, methyl

eugenol, trans-anethole, methyl ethers (thymol, carvacrol) are known

as aromatic ethers. Some plants that are rich in these components

are bay laurel, wintergreen, anise and clove. Anethole is an effective

insect repellent against mosquitos 125. Anethole has potent

antibacterial 126, antifungal 127, antihelmintic 128 and nematicidal

activity 129. These are used for conditions of sedative, anxiety and

depression106.

1.5.2.5 Ester- Lavender, bergamot, rosemary, pine, cinnamon, eucalyptus,

peppermint and cloves are rich sources of terpene esters. These are

linalyl acetate, bornyl acetate, cinnamic acetate, terpenyl acetate,

neryl acetate, menthyl acetate, eugenyl acetate, myrtenyl acetate etc.

Terpene esters showed antifungal, anti inflammatory, antispasmodic,

calming, tonic to nervous system and effective for skin rashes 130.

1.5.2.6 Aldehydes- Terpene aldehydes such as benzaldehyde,

cinnamaldehyde, anisaldehyde, cuminaldehyde, citrol, citronellol,

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citral, neral, vanillin and myrtenol possess vasodilator, hypotensive

and antipyretic properties130. Benzaldehyde and cinnamaldehyde,

reported from Pogostemon parviflorus have tendency to produce 50%

mortality in adult insects 131.

1.5.2.7 Ketones- Carvone thujon, camphon, verbenon, cryptone, borneone,

fanchone are ketonic terpenes. Thymoquinone, the major constituent

of Nigela sativa essential oils shows promising in vitro and in vivo

antineoplastic growth inhibition against various tumour cell lines. This

activity may be attributed to its inhibitory affects on cancer cell growth

and its capability for inducing apoptosis 132. Aromatic ketones also

showed analgesic, digestive, wound healing, calming and sedative

properties130.

Due to the bactericidal and fungicidal properties of essential oils,

their use in pharmaceutical and food industry is becoming more

important as alternatives to synthetic chemical products to maintain the

ecological equilibrium. Currently, about 300 essential oils, out of

approximately 3,000 are commercially important especially for the

pharmaceutical, agronomic, food, sanitary, cosmetic and perfume

industries 133. Some of the essential oils and their bioactive components

such as limonene, geranyl acetate or carvone are also used as an

important ingredient in tooth pastes and hygiene products. Essential oils

play a very important role in aromatherapy as they are believed to exhibit

certain medicinal benefits for curing organ dysfunction or systematic

disorder134. Many of the essential oil components like menthol citral,

eudesmol, piperiton etc are used for flavours, cosmetics, perfumes and

manufacture of vitamin A 135.

Besides the pharmaceutical and medicinal uses, the essential oils are

employed as flavours for food, in spice, perfumes and cosmetic trades.

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Many of them are used as raw material for chemical modification to

synthetic valuable flavor and perfume materials. About one third of

pharmaceuticals are derived from plants and most of the pharmaceutical

preparations are plant based. Side effects of the drugs from plants are

less than those of synthetic one 136.

1.6 TOXICITY OF HERBAL DRUGS

Herbal medicines have been the main source of primary health care in

many developing nations. It is well known that some plants must be used with

caution because they may be toxic for liver (pyrrolizidine alkaloids, apiole,

safrole, lignans etc.), kidney (terpenes, saponins), skin (sesquiterpene lactone,

furano coumarins, etc) and other tissues 137,138, 139, 140. Mainly widely used

medicinal plants have been implicated as possible causes of long term

diseases manifestations such as liver and kidney diseases. The wide spread

use of Scenecio, Crotalaria and Cynoglossum has been implicated in the

occurrence of liver lesions and tumors, lung and kidney diseases in certain area

of Ethiopia 141. Lobelia and Euonymus species that has powerful actions, often

causing nausea or vomiting, although they are safe under appropriate

conditions. There is also an indiosyncratic grouping of herbs that have been

allerged with some scientific support, to exhibit specific kind of toxicity. The best

known example is the hepatotoxicity of pyrrolizidine alkaloid-containing plants

such as Symphytum, Dryopteris, Viscum and Corynanthe 142. Valerian roots,

used in herbal trees contain alkylating agents that inhibit thymidine

incorporation into DNA, leading to impaired mitochondrial function 143. Various

phytotoxic furanocoumarins have been reported from apiaceae and rutaceae

144. Some herbs like Abrus precatorius, Adhatoda vasaka, Riccinus communis

etc. are tetragenic in nature (harmful to foetus during pregnancy) and thus lead

to birth defects or aborption145. Psoralea corylifolia Linn. is used for treating

conditions like psoriasis, leucoderma and non healing ulcers and wounds is

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known to cause hepatosplenomegaly in experimental animals 146. Kawa-Kawa,

a well-established hypnotic drug, was reported to show hepatotoxicity and had

to be eventually baned in most countries worldwide 147.

The safety and quality of raw medicinal plant materials and finished

products depends on intrinsic or extrinsic factors like environment, collection,

cultivation, transport, storage etc. Plants collected from wild sources may be

contaminated by other species or accidental contamination. Toxic effects can

be also attributed to several factors including toxicity of constituents,

contamination by pesticides, microorganisms, heavy metals etc. 148, 149.

1.7 AROMATIC AND MEDICINAL FLORA OF UTTARAKHAND

In the Indian Himalayan region (IHR), medicinal and aromatic plants form

one of the important components for the socio-economic development of native

communities. Due to this particular feature, The IHR has been identified one of

the biodiversity hot spot. This rich biodiversity is being utilized by the

inhabitance of the region for medicines, wild edibles, fodder, fuel, timber, in

making agriculture implements, religious and various other purposes 2. A large

number of medicinal and aromatic plants are used in Indian system of medicine,

pharmaceutical and oil industries.

Uttarakhand Himalaya is located in the eastern most part of the west

Himalaya of India and is very rich in flora and fauna and stretches from 300-

4500 m altitudes. The diversified topography of Uttarakhand with a range of

elevation from almost sea level to alpine height possess a wide variety in its

natural vegetation including medicinal and aromatic flora with immense

potential for providing new drugs , flavor chemicals and other valuable

bioactive substances. Some of the essential oil bearing aromatic and medicinal

plants of this region are: Cedrus deodara,Aegle marmelos (L.) Correa Pinus

longifolia, Artemisia vulgaris, Nepeta elliptica, Nepeta leucophylla, Origanum

vulgare, Valeriana wallichii, Salvia leucantha, Erigeron multiradiatus, Murraya

koengii, Aster tricephalus, Boenninghausenia albiflora, Cymbopogon distans,

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Brunella vulgaris, Carum anethfolium, Elsholzia strobilifera, Hedychium

spicatum, Heracleum candicans, Inula cuspidata, Lantana indica, Litsea

umbrosa, Mentha longifolia, Selinum tenuifolium, Picea morinda, Rosmarinus

officinalis, Zanthoxylum alatum 2, 150.

1.8 AROMATIC AND MEDICINAL PLANTS UNDER

INVESTIGATION

The kumaon region in Himalaya possesses great diversity of flora. Most

of these are used in herbal medicines, essential oils and dyes. The author has

selected the aromatic and medicinal plants mainly belonging to Lauraceae

Apiaceae and Rutaceae family of kumaon Himalayas. The author has

undertaken the study of essential oils bearing medicinal plants viz

Cinnamomum glanduliferum Meissn., Feronia elephantum Correa, Bupleurum

species and Cyclospermum leptophyllum (Pers.) Sprague ex Britton & P.

Wilson to determine their essential oil composition, chemosystematics and

antibacterial activity against different microorganisms.

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