Upload
truongxuyen
View
220
Download
0
Embed Size (px)
Citation preview
Chapter-01
INTRODUCTION, MATERIALS AND METHODS
(A) Introduction (B) Materials and methods
Estelar
(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
- 12 --
Estelar
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
.
- 13 --
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
Estelar
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,
- 14 --
Litsea and Dodecadenia are reported in the Himalayan forests varying from Kashmir to Bhutan
Estelar
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
- 15 --
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
.
Estelar
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
- 16 --
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
Estelar
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
Estelar
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:
- 17 --
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
.
Estelar
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
- 18 --
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
Estelar
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
Este
lar
- 19 --
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
Estelar
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
.
- 20 --
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
Estelar
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
- 21 --
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
.
Estelar
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
- 22 --
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
Estelar
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
Estelar
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
Estelar
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.
Estelar
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).
- 25 --
O OH
OH O ii
i OP+
OP
COOH OH
OP
Estelar
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
Estelar
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
.
Este
lar
- 26 --
OPP
farnesyl pyrophosphate
O
O germacrane type farnesane type
O O
selinane type elemane type
Estelar
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
Estelar
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
Estelar
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.
Estelar
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.
- 30 --
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.
Estelar
- 31 --
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.
Estelar
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.
- 33 --
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.
Estelar
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.
Estelar
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:
Estelar
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.
- 37 --
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.
Estelar
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
Estelar