CHAPTER 1-A

INTRODUCTION

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INTRODUCTION

Ayurveda in India, the origin of most forms of natural and

alternative medicine - has its mention in one of the oldest about

6,000 years philosophical texts of the world, the Rig Veda. Ayurveda

is the most sacred science of life, beneficial to humans both in this

world and the world beyond." The great sage- physician Charaka

authored Charaka Samhita revising and supplementing the text written

by Atreya, which has remained the most referred ayurvedic text on

internal medicine till date.

In the postmodern age, the popularity of this vibrant tradition

of ayurveda lies in its, subtle yet scientific, approach to heal a

person in its totality. It aims, not only at healing the body, but also

the mind and spirit, at one go. Its unique understanding of the

similarities of natural law and the working of human body, as well as

its holistic treatment methods, help it to strike a balance between the

two. This gives ayurveda an edge over other healing systems. Perhaps

that’s the reason behind ayurveda being the longest unbroken

medical tradition in the world, today.

The term "herb" refers to a plant used for medicinal purposes.

The use of medicinal plants for health reasons started thousands of

years ago and is still part of medical practice in China, Egypt, India

and other developing countries.

Medicinal plants have been the subjects of man's curiosity

since time immemorial (Constable, 1990). Almost every civilization has

a history of medicinal plant use (Ensminger et al., 1983).

Approximately 80% of the people in the world's developing countries

rely on traditional medicine for their primary health care needs, and

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about 85% of traditional medicine involves the use of plant extracts

(Vieira and Skorupa, 1993). Nature has been a source of medicines

for thousands of years and an impressive number of modern drugs

have been isolated from natural resources based on their use in

traditional medicine (Frombi, 2003). Interest in phytomedicine has

exploded in the last few years, and about 500 different plant species

are used as key ingredients, and many are still being collected from

the wild (Mendelsohnm and Balick, 1994).

Over the centuries, the use of medicinal herbs has become an

important part of daily life in the western world despite significant

progress in modern medical and pharmaceutical research. Since

World War II, the increasing availability of medicinal herbal products,

a desire for nutraceuticals of functional foods and alternative

medicine and concerns about the possible side effects of some

synthetic drugs have revived the use of medicinal herbs. Recently,

there has been a tremendous surge of interest in medicinal plants or

herbs and their products have become a multibillion dollar industry in

both North America and Europe.

Increasing knowledge of metabolic processes and the effects of

plants on human physiology have enlarged the range of application

of medicinal plants. Some lesser known plants have been found to

have significant, medicinal value. According to the report by the

World Bank in 1997 (Technical PaperNo.355), it is apparent that the

significance of plant-based medicines has been increasing all over the

world. Nearly 50 percent of medicines on the market are made of

natural basic materials. Interestingly, the market demand for medicinal

herbs likely to remain high because many of the active ingredients in

medicinal plants cannot yet be prepared synthetically. In spite of

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overwhelming influences and our dependence on modern medicine

and tremendous advances in synthetic drugs, a large segment of the

world population still prefer drugs from plants. In many developing

countries, the use of plant drugs is increasing because, modern life

saving drugs are beyond the reach of three quarters of the third

world's population although many such countries spend 40-50% of

their total wealth (Joy et al., 1998). Consequently, there is an urgent

need to identify plants of medicinal importance, which have made

their entry into the available literature as well as those, which are

popular between the tribal communities as ethnopharmacopia.

It is estimated that world market for plant derived drugs may

account for about Rs. 2,00,000 crores. It has been estimated that in

developed countries such as United States, plant drugs constitute as

much as 25% of the total drugs, while in fast developing countries

such as China and India, the contribution is as much as 80%. Of the

2,50,000 higher plant species on earth, more than 80,000 are

medicinal. India is one of the world's 12 biodiversity centres with the

presence of over 45,000 different plant species. India's diversity is

unmatched due to the presence of 16 different agro-climatic zones,

10 vegetation zones, 25 biotic provinces and 426 biomes (habitats of

specific species). Of these, about 15000-20000 plants have good

medicinal value. However, traditional communities use only 7000-7500

species for their medicinal values (Joy et al., 1998).

Keen observations, coupled with trial and error, have played an

important role in the genesis and evolution of plant-based medicines.

The casual observation, experimentation, standardization and

documentation are the four phases of development of any herbal

medicine. The knowledge of intermediate and long-term effects of

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eating particular roots, fruits and leaves have been accumulated over

the years. It has also been speculated that these effects must have

been observed either on humans or on animals. These observations

in terms of changes in the behavioural patterns after consumption of

a plant part would have helped in deciding whether the plant is

medicinal or harmful.

Medicinal substances found in plants are the products of

natural metabolic processes. However, each species has its own

genetic structure that governs the presence of chemical components

or bioactive molecules. In addition, the effects of environment and

differences among varieties or cultivars within each species create

variations in the quantity of compounds present. Thus, each plant

species or variety produces chemical compounds differently, and

some plants produce medicinally useful compounds, others do not or

do so in very small quantities (Thomas, 2002).

Recent research on medicinal plants and herbs has generated

a great deal of information about the biologically active chemical

components that are responsible for the claimed medicinal effects.

The level of active ingredients or chemical constituents has been

used as a standard or marker for the quality of raw plant materials

and value-added products. As modem medicine is often found to

reduce immunity and cause side effects, an increasing number of

people are turning to herbal cure. Today herbal treatments are

appreciated for their total health concepts (body as well as mind),

negligible side effects and enhancement of natural immunity. As a

result, today we find a number of plant-based modem medicines

around. There are around 21 indispensable mainstream plant based

drugs used in the treatment of cancer, heart diseases and chronic

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illnesses as in routine illness; aspirin, quinine, reserpine, digoxin,

diosgenin and taxol are just a few examples of such drugs. About

25% of the drugs prescribed worldwide come from plants, 121 such

active compounds being in current use. Of the 252 drugs considered

as basic and essential by the World Health Organisation, 11 % are

exclusively of plant origin and a significant number are synthetic

drugs obtained from natural precursors. Present day systematic

botany is a broad synthetic field sustained through the cooperative

endeavour of specialists in diverse fields of biology and biochemistry.

PHYTOCHEMISTRY

In recent years, phytotherapy is rapidly evolving throughout the

world. Phytochemicals are the naturally occurring bio-chemicals in the

plant that gives plant their colour, flavour, smell and texture. They

may help to prevent diseases like cancer and heart diseases besides

their role to inhibit the microorganisms causing many diseases in

human beings. More than 88000 secondary metabolites and every

year some 4000 new ones are being reported (Farnsworth, 1996).

Thus, there should be more than 1,00,000 secondary metabolites

known. This is only from the small percentage of all the species

studied so far.

In recent times, phytochemical studies have played a significant

role in aiding the solution of systematic problems on one hand and

in assisting in the search for additional raw material resources

industries on the other. Many of these indigenous medicinal plants

are used as spices and food plants. They are also sometimes added

to foods meant for pregnant and nursing mothers for medicinal

purposes (Okwu, 2001).

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The naturally occurring plant metabolites have been divided

into two groups. Primary metabolites involved directly in growth and

metabolism and secondary metabolites considered as end product

(by product) of primary metabolism and in general not involved in

metabolic activity. Secondary metabolites are compounds

biosynthetically derived from primary metabolites but more limited in

occurrence in the plant kingdom and may be restricted to a

particular taxonomic group. Secondary metabolites are mostly

accumulated by plant cell in smaller quantities than primary

metabolites.

These secondary metabolites are synthesized in specialized

cells at particular developmental stages, making their extraction and

purification difficult as compared to the primary products produced

by the whole plant or organ. Secondary metabolites exert in general

a profound physiological effect on the mammalian system and thus

are known as the active principle of plant. The physiological effects

of these active principles are used for curing ailments and therefore

these are drug of plant origin or natural drugs. These substances

serve to defend the plant against predation by microorganisms,

insects, and herbivorous animals. Certain terpenoids give plants their

odour. Quinones and tannins are responsible for plant pigments.

Terpenoids offers flavour to the plant. Some secondary metabolites

can be pharmacologically active and antimicrobial agents

(Hostettmann, 1999).

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Primary products are of prime importance and essentially

required for growth of plants e.g. amino acids, ascorbic acid,

carbohydrates, enzymes, lipids, nucleic acids and proteins etc. They

are of universal occurrence in plants. Amino acids are the building

blocks of proteins and many other secondary products. Chemically

ascorbic acid is more related to the monosaccharides as it is a

hexose derivative. It controls the cholesterol metabolism and helps in

the absorption and utilization of iron.

Phytochemical screening of bioactive plants extracts has

revealed the presence of alkaloids, tannins, flavonoids, sterols,

terpenes, carbohydrates, lactones, proteins, amino acids, glycosides

and saponins. These molecules have pharma and industrial values

including aromas, dyes, gums, resins, pulp, fibre etc. with high bearing

on health and commercial sectors. Most of the high value secondary

metabolites are produced in scarce amounts by plant, which are

poorly understood in totality. All the secondary metabolite pathways,

producing most of the natural products of use, originate as branch

points from primary metabolism with the origin at different places of

primary metabolic cycle.

Alkaloids

One of the largest groups of chemical compounds produced by

plants is alkaloid. The term alkaloid (alkali-like), was first used by W.

Meissner in 1891. Alkaloids are more or less toxic substances. They

act primarily on the central nervous system (CNS). They have a basic

character, containing heterocyclic nitrogen, and are synthesized in

plants from amino acids or their immediate derivatives. In most cases

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they are of limited distribution in the plant kingdom. Alkaloids are

very important in the medicinal world and are used as powerful drugs

mainly due to their sedative properties and powerful effect on the

nervous system. Many current drugs are derived from alkaloids. For

example the synthetic antimalarial drugs, local anaesthetist and the

antiparkinson drugs bromocryptine are derivatives of alkaloids

Quinine, cocaine and ergocryptine respectively. Alkaloids are produce

in actively growing tissue and rarely occur in dead tissue. Researches

on production of useful alkaloids by plant tissue culture have also

been carried out for more than 25 years.

Flavonoids

Flavonoids are another group of plant secondary metabolites

which are present almost universally in higher plants and contribute

to the flower and fruit colour. They impart mostly red, yellow, blue

and violet colour to plant organs. Chemically they are phenolic

compounds and most of them have flavone nucleus with two side

aromatic rings. Flavones occur as glycosides in plants. These

compounds appear to play vital role in defence against pathogens

and predators and contribute to physiological functions (Brenda,

1998). The distribution of flavonoids in plant kingdom is more or less

of taxonomic significance. Algae, fungi and bacteria lack any kind of

flavonoids, whereas mosses have a few types of them, ferns and

gymnosperms have many types of simple flavonoids whereas

angiosperms have a whole range of flavonoids. Soine (1964) have

pointed out the biological activities of flavonoids like anticoagulant,

estrogenic, antibacterial, molluscosidal, aithelmintic, sedative, analgesic

and hypothermal effects.

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Terpenoids

The terpenoids sometimes called ‘isoprenoids’ are a large and

diverse class of naturally-occurring organic chemicals similar to

terpenes, derived from five-carbon isoprene units assembled and

modified in thousands of ways. Most are multicyclic structures that

differ from one another not only in functional groups but also in

their basic carbon skeletons. These lipids can be found in all classes

of living things, and are the largest group of natural products. Plant

terpenoids are used extensively for their aromatic qualities. They play

a role in traditional herbal remedies and are under investigation for

antibacterial, antineoplastic and other pharmaceutical functions.

Terpenoids contribute to the scent of eucalyptus, the flavours of

cinnamon, cloves, and ginger, and the colour of yellow flowers. Well-

known terpenoids include citral, menthol, camphor, Salvinorin A in the

plant Salvia divinorum, and the cannabinoids found in Cannabis.

The steroids and sterols in animals are biologically produced

from terpenoid precursors. Sometimes terpenoids are added to

proteins, e.g., to enhance their attachment to the cell membrane; this

is known as isoprenylation. Terpenes are hydrocarbons resulting from

the combination of several isoprene units. Terpenoids can be thought

of as modified terpenes, wherein methyl groups have been moved or

removed, or oxygen atoms added. Just like terpenes, the terpenoids

can be classified according to the number of isoprene units used:

Hemiterpenoids, 1 isoprene unit; Monoterpenoids, 2 isoprene units;

Sesquiterpenoids, 3 isoprene units; Diterpenoids, 4 isoprene units;

Sesterterpenoids, 5 isoprene units; Triterpenoids, 6 isoprene units;

Tetraterpenoids, 8 isoprene units; Polyterpenoids with a larger number

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of isoprene units; Terpenoids can also be classified according to the

number of cyclic structures they contain.

Triterpenoids can be divided into at least four groups of

compounds: True triterpenes, Steroids, Saponins and Cardiac

glycosides (Harborne, 1973). Many triterpenes are known in plants

and new ones are regularly being discovered and characterized

(Kulshreshtha et al. 1972). At one time sterols were mainly considered

to be animal substances but such compounds have been detected in

plant tissues, occurrence of animal estrogen, estrone, in date palm

seed and pollen (Bennett and Heftmann, 1966), cholesterol occurs in

some plants of Malvaceae (Chauhan, 1984). Steroids are the

compounds known as terpenoids or isoprenoids. Terpenes are formed

by the polymerization of isoprene units and steroids are triterpenes

or triterpenoids. The term triterpenes refers to a group of natural

products containing 30 carbon atoms which rederived from six

isoprene (5 C) units. Most terpenes possess carbon content in

multiples of 5 C. Steroidal sapogenins are of economic importance as

main precursors of many medicinally useful steroidal hormones. The

sterols are most often discussed steroids in the plant literature. They

are crystalline steroids which contain an alcoholic group and may be

either saturated or unsaturated. Steroids have at least two functions.

As precursor in the formation of other steroids e.g. cholesterol and

sitosterol are precursors in the formation of saponins. Depending on

their origin, they are call zoosterol (from animals), phytosterols (from

plants), mycosterols (from fungi) and marine sterols (from marine

organisms e.g. sponges). Phytosterols have been isolated from large

number of plant species. Saponins are glycosides of both triterpenes

and sterols have been detected in over 70 families of plants (Basu et

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al., 1967). The expanding universe of the chemistry of natural

products is indicative of the organic chemist's interest in the plant

kingdom for finding new phyto-constituents of therapeutic value,

precursors for the synthesis of complex chemical substances, or new

sources of compounds of economic value. Wide phytochemical

surveys of alkaloids, saponins, tannins, etc., have been carried out in

different countries. In India, Badhwar (1944) surveyed 306 plants for

vegetable tannin material, Chakrabarthy (1961) examined 38 species

for saponins and Bhatnagar (1961) screened 175 plants belonging to

families alleged to possess medicinal properties in the Ayurvedic and

Unani systems of medicine.

The growing interest in secondary metabolites of plants has

directed attention to methods for their extraction. Natural products

are extracted by conventional methods such as Soxhlet and room

temperature solvent extraction, or by ultrasound, microwaves,

supercritical solvents or other methods. There are more than 4,20,000

distinct plant species, yet less than 10% of them have been fully

analyzed. The isolation and purification of these distinct species is a

major goal of the biotechnology and pharmaceutical industries with

screening procedures for phytochemical analysis. However, each of

the plant secondary metabolites has been successfully isolated and

purified using TLC. HPTLC is an excellent tool for the

qualitative/quantitative analysis of marker compounds in botanical

samples. Selecting a desired phytochemical is an appropriate method

of establishing a quantitative analysis for a marker compound. The

crystallization of alkaloids, both their bases and their salts from

different solvents has been phased out as a separation method for

isolating and purifying natural plant products.

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High Performance Thin Layer Chromatography (HPTLC)

Thin layer chromatography (TLC) and high performance thin

layer chromatography (HPTLC) – now also called planar

chromatography – is, like all chromatographic techniques, based on a

multistage distribution process. This process involves: a suitable

adsorbent (the stationary phase), solvents or solvent mixtures (the

mobile phase or eluent), and the sample molecules. For thin layer

chromatography the adsorbent is coated as a thin layer onto a

suitable support (e.g. glass plate, polyester or aluminium sheet). On

this layer the substance mixture is separated by elution with a

suitable solvent. The principle of TLC is known for more than 100

years now (Beyerinck, 1889). HPTLC has many advantages. It is fast,

flexible, cheap and highly reproducible. As the technology is highly

automated, the results are very reliable. Fractions from the generated

chemical pattern can be purified and used for further analysis.

Moreover, the choices of solvent system are innumerable and one

has the freedom of modifying the compositions. Many samples can

run parallel, which makes the technique fast. It has the possibility of

multiple evaluation of the plate with different parameters because all

fractions of the sample are stored on the plate.

HPTLC uses a stationary phase which is a thin layer (0.25-

2.0mm) of silica on a metal foil or a glass plate. The sample is

applied with the help of an automated applicator as a thin streak.

Sample is applied by spraying with the help of nitrogen gas. Since

the mass distribution is uniform over the full range of the bands,

densitometric estimation can be done by scanning. The plate is then

developed in a saturated chamber containing the developing solvents.

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The separation of compounds may be based on adsorption, partition,

chiral, ion exchange or molecular exclusion principles. The separation

rates of compounds are based on their distribution coefficients

between the mobile phase and the stationary phase. Once the mobile

phase reaches the front end of the plate, retardation factor (Rf) is

calculated as,

Densitometric estimation can be done by scanning in the

spectral range from 190-800nm.

A plant may produce innumerable bioactive compounds (Cowan,

1999) such as phenolics, terpenoids, alkaloids, saponins and lectins.

The Red Data Book of India has 427 entries of endangered

species of which 28 are considered extinct, 124 endangered, 81

vulnerable, 100 rare and 34 insufficiently known species (Thomas,

1997). Therefore, for retaining the quality of plant based products

either for commercial gains or for ethical reasons, a ceaseless supply

of plant material from perpetual source needs to be implemented, for

which in vitro cultivation of some of the medicinal plants becomes

vital.

The resurgence of public interest in plant based medicine

coupled with rapid expansion of pharmaceutical industries have

necessitated an increased demand for medicinal plants, leading to

over-exploitation that threatens the survival of many rare species.

Also, many medicinal plant species are disappearing at an alarming

rate due to rapid agricultural and urban development, uncontrolled

deforestation and indiscriminate collection of crude drugs.

Combinations of in vitro propagation techniques (Fay, 1992) and

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cryopreservation may help in conservation of biodiversity of locally

used medicinal plants. Cryopreservation is a reliable method for long-

term storage of the germplasm of endangered species (Bramwell,

1990). Several medicinal plant species have been successfully

cryopreserved (Bajaj, 1995; Naik, 1998). In vitro cell and tissue culture

methodology is envisaged as a mean for germplasm conservation to

ensure the survival of endangered plant species, rapid mass

propagation for large-scale revegetation, and for genetic manipulation

studies. Plants play a dominant role in the introduction of new

therapeutic agents, and also drugs from the higher plants continue to

occupy an important niche in modern medicine (Dev, 1997). Many

compounds used in today's medicine have a complex structure, and

synthesizing these bioactive compounds chemically at a low price is

not easy (Shimomura et al., 1997). With deforestation, medicinal

wealth is rapidly lost, such that many valuable plants are threatened

with extinction. Pharmaceutical companies depend largely upon

materials procured from wild resources that are being rapidly

depleted. Plant tissue culture is an alternative method of propagation

to keep these resources sustainable for future (George and

Sherrington, 1984) and is being used widely for the commercial

propagation of a large number of plant species, including many

medicinal plants (Rout et al., 2000).

The last two decades have witnessed the conversion of a large

amount of forest cover into agriculture land for human use.

Moreover, the increasing interest in medicinal plant–based treatments

has precipitated the over exploitation of these species from the wild.

The populations of medicinal plants are drastically degraded due to

anthropogenic interferences (Aguilar et al., 2008; Sarwat et al., 2008).

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The loss of one plant species means its depletion from the biological

Gene Bank (Hass, 2008). Thus, biodiversity is an irreplaceable

resource; once lost, it is lost forever.

Yield and quality of herbs collected from the wild are

unpredictable; both are significantly affected by the weather, pets and

other uncontrollable variables. Farming some of the popular herbs

would help reduce problems of inconsistent supply and would thus

regularize the trade. Furthermore, farmed products could be certified

as to source, identity and quality. However, cultivation of medicinal

plants is presently constrained by a lack of suitable technology,

which leads to low yield and products of poor quality. Field grown

plant material has generally been used but the quality of these

products may be affected by different environmental conditions that

can alter the medicinal value of plants (Murch et al., 2002).

Biotechnologists hope for a bypass to overcome this difficulty, by

introducing plant tissue culture technique and further multiplication of

important plants by micropropagation technique. The production of

useful metabolites from plant tissue culture has created a new

methodology for their commercialization.

Advanced biotechnological methods of culturing plant cells and

tissues provide a new means of conserving and rapidly propagating

valuable medicinal plants. Micropropagation methods are

advantageous, as we get true to type plants with lesser variation in

somatic embryo. Hairy roots established by transformation with

Agrobacterium rhizogenes are potentially applicable to medicinal

plants in which roots yield secondary metabolites. Combination of in

vitro propagation techniques (Fay, 1992) and cryopreservation will

help in conservation of biodiversity of locally used medicinal plants.

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In vitro cell and tissue culture methodology is envisaged as a means

for germplasm conservation to ensure the survival of endangered

plant species. Synthetic seed technology is one of the important

applications of somatic embryogenesis. In these synthetic seed

system, somatic embryo is encapsulated in a protective alginate

matrix, which provides mechanical support (Redenbaugh et al., 1986).

The artificial seeds can increase the efficiency in transportation and

delivery systems compared to other propagating material as it can be

packed in small vials thus, limiting space and ensuing viability.

Artificial seeds have many advantages over natural seeds since it

maintains genetic uniformity of plants and has potential for long term

storage without losing viability that helps to allow economical mass

propagation of elite plant varieties.

The continued commercial exploitation of medicinal plants has

resulted in receding the population of many species in their natural

habitat. Vacuum is likely to occur in the supply of raw plant materials

that are used extensively by the pharmaceutical industry as well as

the traditional practitioners. Consequently, cultivation of these plants

is urgently needed to ensure their availability to the industry as well

as to people associated with traditional system of medicine. If timely

steps are not taken for their conservation, cultivation and mass

propagation, they may be lost from the natural vegetation forever. In

situ conservation of these resources alone cannot meet the ever-

increasing demand of pharmaceutical industry. It is, therefore,

inevitable to develop cultural practices and propagate these plants in

suitable agro climatic regions. Commercial cultivation will put a check

on the continued exploitation from wild sources and serve as an

effective means to conserve the biodiversity and protect endangered

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species. The pharmaceutical industry, where the active medicinal

compound cannot be synthesized economically on large scale, the

product must be obtained from the cultivated plants. Systematic

conservation and large-scale cultivation of the important medicinal

plants are thus is the need of the hour. Efforts are required to

suggest appropriate cropping patterns for the incorporation of these

plants into the conventional agricultural and forestry cropping system.

The plant is threatened by increasing agriculture, cattle grazing

and ethnomedicinal usage. Information on how genetic variation is

distributed among the remaining population of an endangered species

can be used in designing recovery programmes. Moreover, opting for

conservation measures depends on their economic and medicinal

importance to individuals, a particular community or society at large.

Hence protecting an endangered species becomes an individual

conservation priority.

HPTLC is very good technique, but to identify compounds of

plant extract other techniques required, like GC-MS, NMR, etc.

Mass Spectrometry:

Mass spectrometry is a technique used by organic chemists to

characterize organic molecules in two principal ways:

1. To measure exact molecular weights, and from this, to measure

exact molecular formula

2. To indicate within a molecule the points at which it prefers to

fragment, and from this, the presence of certain structural units

in the organic compound can be recognized.

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Mass spectrometry is not a true spectroscopic technique

because absorption of electromagnetic radiation is not involved in

any manner.

The mass spectrometer bombards a neutral sample of a

volatile organic molecule (M) with a beam if high energy electrons,

which possess an energy (about 8-13 electron volts). Since this

energy is far in excess of the typical bond energies encountered in

organic compounds, the fragmentation is normally extensive. On

electron impact, the molecules are energised sufficiently to eject an

electron. This leads to the formation of a radical cation, symbolized

by (M+), which is called as Molecular Ion. This molecular ion

represents the intact molecule which has the same weight as the

original molecule (M). this is because the molecular weight depends

on the number of protons and neutrons, and thus it remains

unaffected even when an electron is ejected from the molecule,

leading to the formation of the molecular ion. The molecular ion

undergoes fragmentation, a process in which free radicals or neutral

molecules are lost from the molecular ion. The general tendency of

the molecular ion is to fragment into its most stable fragments. Post

fragmentation the radical products such as ᐧCH3 as well as neutral

molecules such as H2O, CO2 and H2C=C H2 does not appear in the

mass spectrum. Only cations bearing a positive charge can be

detected.

Each positive ion formed either directly on electron impact or

by fragmentation of the original molecule has a unique mass/charge

ratio called the m/z ratio, which is recorded on the graph paper. The

graphic representation of the mass spectrum of the compound is

constructed by plotting mass/charge ratio (m/z) versus relative

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abundance or percentage of the base peak, where the base peak is

the most intense in the spectrum. The base peak is assigned the

arbitrary value of 100, while all other peaks are given their

proportional value. The molecular weight of a compound is derived

from the mass spectrum of the compound. Although the molecular

ion formed by the initial electron bombardment usually undergoes

extensive fragmentation the m/z ratio of the ion is representative of

the molecular weight of the compound.

That an ion peak is indeed the molecular ion peak may be

known by inspecting the fragment ion peaks in the vicinity of that

peak in the mass spectrum. Losses of ᐧH (M-1 peak) and ᐧCH3 (M-15

peak) i.e., losses of 1 and 15 mass units respectively are commonly

observed for molecular ions of many organic compounds. Similarly

the presence of M-18 (loss of water) or an M-31 (loss of OCH3 from

methyl esters) can also give indications of the peak being the

molecular ion peak.

In addition to electron impact (EI) method, one can also

choose the chemical ionization (CI) method, which can also be used

for volatile compounds, in order to locate their molecular ion with

lesser fragmentation. In CI method the organic compound RH is

introduced along with a carrier gas such as methane. The methane

carrier gas, on impact of electron bombardment, forms primary ions

such as CH4+, CH3

+, etc. the primary ions react with more methane

molecules to form secondary ions such as CH5+ in an acid-base type

reaction.

CH+ᐧ+ CH4 —–———› CH5+ + ᐧCH3

A secondary ion like CH5+ is an energetic proton transfer

reagent which the organic compound RH to give RH2+. The neutral

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organic molecule M will now show a molecular ion (M+H)+ or simply

MH+ which has an m/z value one amu greater than that of the

molecular ion. Such an ion can now be termed as Quasimolecular

ion. The CI produced MH+ ions are generally prominent and undergo

less fragmentation, thus increasing the possibilities of locating the

molecular ion peak.

Mass spectra often contain peaks of significant intensity that

are attributed to the presence of isotopes. Two of these are called

the M +1 and M +2 peaks, where M is the mass of the parent ion

(M+ᐧ), while 1 and 2 represent the increase in mass value due to the

heavier isotopes of 1 or 2 mass units. Most elements occur naturally

in the form of several isotopes and generally the lightest of the

isotopes predominate while the heavier ones occur to a lesser extent.

Setting the mass of the lowest weight isotope to 100 percent and

computing the percentages of the other isotopes relative to it, the

following table can be constructed,

Isotopic composition of some common elements:

Element M+ M +1 M +2

Hydrogen 1H 100.0%

Carbon 12C 98.9% 13C 1.1%

Nitrogen 14N 99.6% 15N 0.4%

Oxygen 16O 99.8% 18O 0.2%

Sulphur 32S 95.0% 33S 0.8% 34S 4.2%

Chlorine 35Cl 75.5% 37Cl 24.5%

Bromine 79Br 50.5% 81Br 49.5%

Iodine 127I 100.0%

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Since about 1.1% of natural carbon is the 13C isotope, the

mass spectrum tends to show an additional peak having 1 amu

greater mass unit. For e.g. the mass spectrum of methane exhibits a

molecular ion peak at m/z 16. It also shows the presence of an

additional peak having m/z 17, whose intensity is 1.1% that if the

intensity of the m/z 16 peak.

The isotope of hydrogen, known as Deuterium, has just about

0.015% abundance and so its proportion is too small to be

considered and is normally ignored in the mass spectrum. For most

compounds the M +2 peak is small, however the compounds

containing chlorine, bromine or sulphur, the M +2 isotopic peak is

substantial. Fluorine and iodine are isotopically pure and so a single

mass value is observed for compounds containing either of them.

Compounds containing one bromine atom display their spectra in

their mass spectra pairs of peaks of roughly equal intensity and

separated by two mass units. This is due to an almost equal

abundance of the two isotopes, i.e., 1:1 ratio of 79Br and 81Br. Thus a

compound like methyl bromide CH3Br shows two very intense peaks

at m/z 94 and 96, which are the M+ and the M +2 peak respectively.

Similarly the spectra of monochloroalkanes show two molecular

ions, with a difference of two mass units due to the presence of

R35Cl and R37Cl, but here in a different ratio of 3:1. Similarly the

contribution of 34S isotope to the M +2 peak is of help in order to

detect the presence of sulphur in the molecule.

Iodine is recognized by the presence of the iodinium ion I+, at

m/z 127 in the mass spectrum. This clue is combined with a

characteristic 127 unit gap in the spectrum corresponding to the loss

of the iodine radical.

INTRODUCTION

22

In summary, a compound with no sulphur, chlorine or bromine

has a small M +1 and an even smaller M +2 peak.

If the ratio of intensities of the M+ᐧ and the M +2 peaks are

considered, it can reveal the presence of a number of possible

heteroatoms.

If the ratio M+ᐧ: M +2= 1:1 a bromine atom may be present

= 3:1 a chlorine atom may be present

If the M +2 peak is about 4.4% of the M+ᐧ then a sulphur atom

is likely to be present.

If the value M +2 peak is less than 1, this gives the indication

of the presence of an oxygen atom in the molecule.

The presence of a nitrogen atom is suggested by an odd m/z

value in the spectrum. However this holds true only if there are odd

number of nitrogen atoms in the molecule. The presence of an even

number of nitrogen atoms gives an even m/z value.

The intensity of the M +1 peak is can be used to know the

number of carbon as well as nitrogen atoms. In the absence of

nitrogen, the maximum number of carbon atoms present can be

calculated by dividing the relative intensity of the M +1 peak by 1.1

(which is the percentage of naturally occurring 13C atoms). For

example, a molecule having 12 carbon atoms will show the M +1

peak of 13.2%. in case nitrogen is present its contribution to the M

+1 peak will amount to 0.4 x number of nitrogen atoms. This quantity

must be subtracted from the measured relative intensity of the M +1

peak to know the number of carbon atoms (Kalsi, 2005).

INTRODUCTION

23

PLANT TISSUE CULTURE

(Barcelo et al., 2001; Fowler, 2000; Gamborg, 2002; Ramage and

Williams, 2002; Sugiyama, 1999)

Plant tissue culture now has direct commercial applications as

well as value in basic research into cell biology, genetics and

biochemistry. The techniques include culture of cells, anthers, ovules

and embryos on experimental to industrial scales, protoplast isolation

and fusion, cell selection and meristem and bud culture. Applications

include:

micropropagation using meristem and shoot culture to produce

large numbers of identical individuals

micropropagation can be used in to conserve rare or

endangered plant species

screening programmes of cells, rather than plants for

advantageous characters

large-scale growth of plant cells in liquid culture as a source

of secondary products

crossing distantly related species by protoplast fusion and

regeneration of the novel hybrid (somatic hybridization)

production of dihaploid plants from haploid cultures to achieve

homozygous lines more rapidly in breeding programmes

as a tissue for transformation, followed by either short-term

testing of genetic constructs or regeneration of transgenic

plants

removal of viruses by propagation from meristematic tissues

Practically any plant transformation experiment relies at some

point on tissue culture. There are some exceptions to this

generalisation, but the ability to regenerate plants from isolated cells

INTRODUCTION

24

or tissues in vitro underpins most plant transformation systems.

Micropropagation represents the optimum efficiency in terms of

vegetative plant propagation and allows large scale production in a

relatively shorter period of time under controlled conditions

throughout the year in a relatively small space. The rate of

micropropagation varies greatly form species to species, but it is

often possible to produce several million plants in the period of a

year starting with any explants.

Plasticity and totipotency

Two concepts, plasticity and totipotency, are central to

understanding plant cell culture and regeneration. Plants, due to their

sessile nature and long life span, have developed a greater ability to

endure extreme conditions and predation than have animals. These

cells are able to differentiate into a whole plant or a plant organ by

redifferentiation. These two phenomenons are inherent by the plant

cell and giving rise to a whole plant is described as cellular

totipotency. Since the plants cells are totipotent, all the necessary

genetic and physiological potential for natural product formation

should be present in an isolated cell (Zenk, 1978). According to this

theory cultured cells obtained from any part of a plant might be

expected to yield secondary metabolites similar to those of the plant

grown in vivo.

Many of the processes involved in plant growth and

development adapt to environmental conditions. This plasticity allows

plants to alter their metabolism; growth and development to best suit

their environment. Particularly important aspects of this adaptation, as

far as plant tissue culture and regeneration are concerned, are the

INTRODUCTION

25

abilities to initiate cell division from almost any tissue of the plant

and to regenerate lost organs or undergo different developmental

pathways in response to particular stimuli. When plant cells and

tissues are cultured in vitro they generally exhibit a very high degree

of plasticity, which allows one type of tissue or organ to be initiated

from another type. In this way, whole plants can be subsequently

regenerated.

This regeneration of whole organisms depends upon the

concept that all plant cells can, given the correct stimuli, express the

total genetic potential of the parent plant. This maintenance of

genetic potential is called ‘totipotency’. Plant cell culture and

regeneration do, in fact, provide the most compelling evidence for

totipotency. In practical terms though, identifying the culture

conditions and stimuli required to manifest this totipotency can be

extremely difficult and it is still a largely empirical process.

The culture environment

When cultured in vitro, all the needs, both chemical (see Table

1.1) and physical, of the plant cells have to met by the culture

vessel, the growth medium and the external environment (light,

temperature, etc.). The growth medium has to supply all the essential

mineral ions required for growth and development. In many cases, it

must also supply additional organic supplements such as amino acids

and vitamins. Many plant cell cultures, as they are not photosynthetic,

also require the addition of a fixed carbon source in the form of a

sugar. One other vital component that must also be supplied is

INTRODUCTION

26

water, the principal biological solvent. Physical factors, such as

temperature, pH, the gaseous environment, light (quality and duration)

and osmotic pressure, also have to be maintained within acceptable

limits.

Plant cell culture media

Culture media used for the in vitro cultivation of plant cells are

composed of three basic components:

(1) Essential elements, or mineral ions, supplied as a complex mixture

of salts;

(2) An organic supplement supplying vitamins and/or amino acids;

(3) A source of fixed carbon; usually supplied as the sugar sucrose.

Table No.1.1

Some of the elements important for plant nutrition and their

physiological function.

Element Function

Nitrogen Component of proteins, nucleic acids and

some coenzymes Element required in greatest

amount

Potassium Regulates osmotic potential, principal inorganic

cation

Calcium Cell wall synthesis, membrane function, cell

signalling

Magnesium Enzyme cofactor, component of chlorophyll

INTRODUCTION

27

Phosphorus Component of nucleic acids, energy transfer,

component of intermediates in respiration and

photosynthesis

Sulphur Component of some amino acids (methionine,

cysteine) and some cofactors

Chlorine Required for photosynthesis

Iron Electron transfer as a component of cytochromes

Manganese Enzyme cofactor

Cobalt Component of some vitamins

Copper Enzyme cofactor, electron-transfer reactions

Zinc Enzyme cofactor, chlorophyll biosynthesis

Molybdenum Enzyme cofactor, component of nitrate reductase

For practical purposes, the essential elements are further divided into

the following categories:

(1) Macroelements or Macronutrients;

(2) Microelements or Micronutrients;

(3) An iron source.

Media components

It is useful to briefly consider some of the individual

components of the stock solutions.

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28

Macroelements

As is implied by the name, the stock solution supplies those

elements required in large amounts for plant growth and

development. Nitrogen, phosphorus, potassium, magnesium, calcium

and sulphur (and carbon, which is added separately) are usually

regarded as macroelements. These elements usually comprise at least

0.1% of the dry weight of plants.

Nitrogen is most commonly supplied as a mixture of nitrate

ions (from the KNO3) and ammonium ions (from the NH4NO3).

Theoretically, there is an advantage in supplying nitrogen in the form

of ammonium ions, as nitrogen must be in the reduced form to be

incorporated into macromolecules. Nitrate ions therefore need to be

reduced before incorporation. However, at high concentrations,

ammonium ions can be toxic to plant cell cultures and uptake of

ammonium ions from the medium causes acidification of the medium.

In order to use ammonium ions as the sole nitrogen source, the

medium needs to be buffered. High concentrations of ammonium ions

can also cause culture problems by increasing the frequency of

vitrification (the culture appears pale and ‘glassy’ and is usually

unsuitable for further culture). Using a mixture of nitrate and

ammonium ions has the advantage of weakly buffering the medium

as the uptake of nitrate ions causes OH–ions to be excreted.

Phosphorus is usually supplied as the phosphate ion of

ammonium, sodium or potassium salts. High concentrations of

phosphate can lead to the precipitation of medium elements as

insoluble phosphates.

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Microelements

These elements are required in trace amounts for plant growth

and development, and have many and diverse roles. Manganese,

iodine, copper, cobalt, boron, molybdenum, iron and zinc usually

comprise the microelements, although other elements such as nickel

and aluminium are frequently found in some formulations. Iron is

usually added as iron sulphate, although iron citrate can also be

used. Ethylenediaminetetraacetic acid (EDTA) is usually used in

conjunction with the iron sulphate. The EDTA complexes with the iron

to allow the slow and continuous release of iron into the medium.

Uncomplexed iron can precipitate out of the medium as ferric oxide.

Organic supplements

Only two vitamins, thiamine (vitamin B1) and myoinositol

(considered a B vitamin) are considered essential for the culture of

plant cells in vitro. However, other vitamins are often added to plant

cell culture media for historical reasons. Amino acids are also

commonly included in the organic supplement. The most frequently

used is glycine (arginine, asparagine, aspartic acid, alanine, glutamic

acid, glutamine and proline are also used), but in many cases its

inclusion is not essential. Amino acids provide a source of reduced

nitrogen and, like ammonium ions; uptake causes acidification of the

medium. Casein hydrolysed can be used as a relatively cheap source

of a mix of amino acids.

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Carbon source

Sucrose is cheap, easily available, readily assimilated and

relatively stable and is therefore the most commonly used carbon

source. Other carbohydrates (such as glucose, maltose, galactose and

sorbitol) can also be used, and in specialised circumstances may

prove superior to sucrose.

Gelling agents

Media for in vitro plant cell culture can be used in either liquid

or ‘solid’ forms, depending on the type of culture being grown. For

any culture types that require the plant cells or tissues to be grown

on the surface of the medium, it must be solidified. Agar, produced

from seaweed, is the most common type of gelling agent. However,

because it is a natural product, the agar quality can vary from

supplier to supplier and from batch to batch. For more demanding

applications, a range of purer gelling agents are available. Purified

agar or agarose can be used, as can a variety of gellan gums.

These components are the basic necessities for plant cell

culture media. However, other additions are made in order to

manipulate the pattern of growth and development of the plant cell

culture.

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Plant growth regulators

Plant growth regulators are the critical media components in

determining the developmental pathway of the plant cells. The plant

growth regulators used most commonly are plant hormones or their

synthetic analogues.

Classes of plant growth regulators

There are five main classes of plant growth regulator used in

plant cell culture, namely:

(1) auxins; (2) cytokinins; (3) gibberellins; (4) abscisic acid; (5)

ethylene.

Auxins

Auxins promote both cell division and cell growth. The most

important naturally occurring auxin is IAA (indole-3-acetic acid), but

its use in plant cell culture media. Occasionally, amino acid

conjugates of IAA (such as indole-acetyl-L-alanine and indole-acetyl-L-

glycine), which are more stable, are used. 2, 4-Dichlorophenoxyacetic

acid (2, 4-D) is the most commonly used auxin and is extremely

effective in most circumstances. Other auxins are available (see Table

1.2), and some may be more effective or ‘potent’ than 2, 4-D in

some instances.

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Table 1.2

Commonly used Auxins, their abbreviation and chemical name

____________________________________________________________________

Abbreviation/name Chemical name

____________________________________________________________________

2, 4-D 2, 4-dichlorophenoxyacetic acid

2, 4, 5-T 2, 4, 5-trichlorophenoxyacetic acid

Dicamba 2-methoxy-3, 6-dichlorobenzoic acid

IAA Indole-3-acetic acid

IBA Indole-3-butyric acid

MCPA 2-methyl-4-chlorophenoxyacetic acid

NAA 1-naphthylacetic acid

NOA 2-naphthyloxyacetic acid

Picloram 4-amino-2, 5, 6-trichloropicolinic acid

Cytokinins

Cytokinins promote cell division. Naturally occurring cytokinins

are a large group of structurally related (they are purine derivatives)

compounds. Of the naturally occurring cytokinins, two have some use

in plant tissue culture media (see Table 1.3). These are Zeatin and

2iP (2-isopentyl adenine). Their use is not widespread as they are

expensive and relatively unstable. The synthetic analogues, kinetin and

BAP (benzylaminopurine), are therefore used more frequently. Non-

purine-based chemicals, such as substituted phenylureas, are also

used as cytokinins in plant cell culture media. These substituted

phenylureas can also substitute for auxin in some culture systems.

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Table 1.3

Commonly used Cytokinins, their abbreviation and chemical name

____________________________________________________________________

Abbreviation/name Chemical name

___________________________________________________________________

BAPa 6-benzylaminopurine

2iP (IPA) b [N6-(2-isopentyl) adenine]

Kinetina 6-furfurylaminopurine

Thidiazuronc 1-phenyl-3-(1, 2, 3-thiadiazol-5-yl) urea

Zeatinb 4-hydroxy-3-methyl-trans-2-

butenylaminopurine

____________________________________________________________________

a Synthetic analogues.

b Naturally occurring cytokinins.

c A substituted phenylurea-type cytokinin.

Gibberellins

There are numerous, naturally occurring, structurally related

compounds termed ‘gibberellins’. They are involved in regulating cell

elongation, and are agronomically important in determining plant

height and fruit-set. Only a few of the gibberellins are used in plant

tissue culture media, GA3 being the most common.

Abscisic acid

Abscisic acid (ABA) inhibits cell division. It is most commonly

used in plant tissue culture to promote distinct developmental

pathways such as somatic embryogenesis.

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Ethylene

Ethylene is a gaseous, naturally occurring, plant growth

regulator most commonly associated with controlling fruit ripening in

climacteric fruits. Some plant cell cultures produce ethylene, which, if

it builds up sufficiently, can inhibit the growth and development of

the culture. The type of culture vessel used and its means of closure

affect the gaseous exchange between the culture vessel and the

outside atmosphere and thus the levels of ethylene present in the

culture.

Plant growth regulators and tissue culture

Generalisations about plant growth regulators and their use in

plant cell culture media have been developed from initial observations

made in the 1950s. There is, however, some considerable difficulty in

predicting the effects of plant growth regulators; this is because of

the great differences in culture response between species, cultivars

and even plants of the same cultivar grown under different

conditions.

However, some principles do hold true and have become the

paradigm on which most plant tissue culture regimes are based.

Auxins and cytokinins are the most widely used plant growth

regulators in plant tissue culture and are usually used together, the

ratio of the auxin to the cytokinin determining the type of culture

established or regenerated. A high auxin to cytokinin ratio generally

favours root formation, whereas a high cytokinin to auxin ratio

favours shoot formation. An intermediate ratio favours callus

production.

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Culture types

Cultures are generally initiated from sterile pieces of a whole

plant. These pieces are termed ‘explants’, and may consist of pieces

of organs, such as leaves or roots, or may be specific cell types,

such as pollen or endosperm. Many features of the explant are

known to affect the efficiency of culture initiation. Generally, younger,

more rapidly growing tissue is most effective. Several different culture

types most commonly used in plant transformation studies.

Callus

Explants, when cultured on the appropriate medium, usually

with both an auxin and a cytokinin, can give rise to an unorganised,

growing and dividing mass of cells. It is thought that any plant tissue

can be used as an explant, if the correct conditions are found. In

culture, this proliferation can be maintained more or less indefinitely,

provided that the callus is subcultured on to fresh medium

periodically. During callus formation there is some degree of

dedifferentiation, both in morphology (callus is usually composed of

unspecialised parenchyma cells) and metabolism. One major

consequence of this dedifferentiation is that most plant cultures lose

the ability to photosynthesise. This has important consequences for

the culture of callus tissue, as the metabolic profile will probably not

match that of the donor plant. This necessitates the addition of other

components—such as vitamins and, most importantly, a carbon

source—to the culture medium, in addition to the usual mineral

nutrients. Callus culture is often performed in the dark as light can

INTRODUCTION

36

encourage differentiation of the callus. During long-term culture, the

culture may lose the requirement for Auxin and/or cytokinin. This

process, known as ‘habituation’, is common in callus cultures from

some plant species (such as sugar beet).

Callus cultures are extremely important in plant biotechnology.

Manipulation of the auxin to cytokinin ratio in the medium can lead

to the development of shoots, roots or somatic embryos from which

whole plants can subsequently be produced. Callus cultures can also

be used to initiate cell suspensions, which are used in a variety of

ways in plant transformation studies.

Cell-suspension cultures

Plant cell suspension cultures are mostly used for the

biochemical investigation of cell physiology, growth and metabolism

and for large scale production of secondary metabolites.

Callus cultures fall into one of two categories: compact or

friable. In compact callus the cells are densely aggregated, whereas

in friable culture types callus the cells are only loosely associated

with each other and the callus becomes soft and breaks apart easily.

Friable callus provides the inoculum to form cell-suspension cultures.

Explants from some plant species or particular cell types tend not to

form friable callus, making cell-suspension initiation a difficult task.

The friability of callus can sometimes be improved by manipulating

the medium components or by repeated subculturing. The friability of

the callus can also sometimes be improved by culturing it on ‘semi-

solid’ medium (medium with a low concentration of gelling agent).

When friable callus is placed into a liquid medium and then agitated,

INTRODUCTION

37

single cells and/or small clumps of cells are released into the

medium. Under the correct conditions, these released cells continue

to grow and divide, eventually producing a cell-suspension culture. A

relatively large inoculum should be used when initiating cell

suspensions so that the released cell numbers build up quickly. The

inoculum should not be too large though, as toxic products released

from damaged or stressed cells can build up to lethal levels. Large

cell clumps can be removed during subculture of the cell suspension.

Cell suspensions can be maintained relatively simply as batch

cultures in conical flasks. They are continually cultured by repeated

subculturing into fresh medium. This results in dilution of the

suspension and the initiation of another batch growth cycle. The

degree of dilution during subculture should be determined empirically

for each culture. Too great a degree of dilution will result in a

greatly extended lag period or, in extreme cases, death of the

transferred cells.

After subculture, the cells divide and the biomass of the

culture increases in a characteristic fashion, until nutrients in the

medium are exhausted and/or toxic by-products build up to inhibitory

levels—this is called the ‘stationary phase’. If cells are left in the

stationary phase for too long, they will die and the culture will be

lost. Therefore, cells should be transferred as they enter the

stationary phase. It is therefore important that the batch growth-cycle

parameters are determined for each cell-suspension culture.

INTRODUCTION

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Protoplasts

Protoplasts are plant cells with the cell wall removed.

Protoplasts are most commonly isolated from either leaf mesophyll

cells or cell suspensions, although other sources can be used to

advantage. Two general approaches to removing the cell wall can be

taken—mechanical or enzymatic isolation. Mechanical isolation,

although possible, often results in low yields, poor quality and poor

performance in culture due to substances released from damaged

cells. Enzymatic isolation is usually carried out in a simple salt

solution with a high osmoticum, plus the cell wall degrading enzymes.

It is usual to use a mix of both cellulase and pectinase enzymes,

which must be of high quality and purity. Protoplasts are fragile and

easily damaged, and therefore must be cultured carefully. Liquid

medium is not agitated and a high osmotic potential is maintained,

at least in the initial stages. The liquid medium must be shallow

enough to allow aeration in the absence of agitation. Protoplasts can

be plated out on to solid medium and callus produced. Whole plants

can be regenerated by organogenesis or somatic embryogenesis from

this callus. Protoplasts are ideal targets for transformation by a

variety of means.

Root cultures

Root cultures can be established in vitro from explants of the

root tip of either primary or lateral roots and can be cultured on

fairly simple media. The growth of roots in vitro is potentially

unlimited, as roots are indeterminate organs. Although the

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39

establishment of root cultures was one of the first achievements of

modern plant tissue culture, they are not widely used in plant

transformation studies.

Shoot tip and meristem culture

The tips of shoots can be cultured in vitro, producing clumps

of shoots from either axillary or adventitious buds. This method can

be used for clonal propagation. Shoot meristem cultures are potential

alternatives to the more commonly used methods for cereal

regeneration as they are less genotype-dependent and more efficient

(seedlings can be used as donor material).

Embryo culture

Embryos can be used as explants to generate callus cultures

or somatic embryos. Both immature and mature embryos can be

used as explants. Immature, embryo-derived embryogenic callus is the

most popular method of monocot plant regeneration.

Microspore culture

Haploid tissue can be cultured in vitro by using pollen or

anthers as an explant. Pollen contains the male gametophyte, which

is termed the ‘microspore’. Both callus and embryos can be produced

from pollen. Pollen-derived embryos are subsequently produced via

dehiscence of the mature anthers. The dehiscence of the anther

depends both on its isolation at the correct stage and on the correct

INTRODUCTION

40

culture conditions. Anthers can also be cultured in liquid medium,

and pollen released from the anthers can be induced to form

embryos, although the efficiency of plant regeneration is often very

low. Immature pollen can also be extracted from developing anthers

and cultured directly, although this is a very time-consuming process.

Some beneficial effects to the culture are observed when anthers are

used as the explant material. There is, however, the danger that

some of the embryos produced from anther culture will originate

from the somatic anther tissue rather than the haploid microspore

cells. If isolated pollen is used there is no danger of mixed embryo

formation, but the efficiency is low and the process is time-

consuming. Regeneration from microspore explants can be obtained

by direct embryogenesis, or via a callus stage and subsequent

embryogenesis.

Haploid tissue cultures can also be initiated from the female

gametophyte (the ovule). In some cases, this is a more efficient

method than using pollen or anthers. The ploidy of the plants

obtained from haploid cultures may not be haploid. This can be a

consequence of chromosome doubling during the culture period.

Chromosome doubling (which often has to be induced by treatment

with chemicals such as colchicine) may be an advantage, as in many

cases haploid plants are not the desired outcome of regeneration

from haploid tissues. Such plants are often referred to as ‘di-

haploids’, because they contain two copies of the same haploid

genome.

INTRODUCTION

41

Plant regeneration

Having looked at the main types of plant culture that can be

established in vitro, we can now look at how whole plants can be

regenerated from these cultures. In broad terms, two methods of

plant regeneration are widely used in plant transformation studies, i.e.

somatic embryogenesis and organogenesis.

Somatic embryogenesis

In somatic (asexual) embryogenesis, embryo-like structures,

which can develop into whole plants in a way analogous to zygotic

embryos, are formed from somatic tissues. These somatic embryos

can be produced either directly or indirectly. In direct somatic

embryogenesis, the embryo is formed directly from a cell or small

group of cells without the production of an intervening callus. Though

common from some tissues (usually reproductive tissues such as the

nucellus, styles or pollen), direct somatic embryogenesis is generally

rare in comparison with indirect somatic embryogenesis. In indirect

somatic embryogenesis, callus is first produced from the explant.

Embryos can then be produced from the callus tissue or from a cell

suspension produced from that callus. Somatic embryogenesis from

carrot is the classical example of indirect somatic embryogenesis.

Somatic embryogenesis usually proceeds in two distinct stages. In the

initial stage (embryo initiation), a high concentration of 2, 4-D is

used. In the second stage (embryo production) embryos are produced

in a medium with no or very low levels of 2, 4-D. Somatic embryos

may develop from single cells or from a small group of cells.

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42

Repeated cell divisions lead to the production of a group of cells

that develop into an organised structure known as a ‘globular-stage

embryo’. Further development results in heart and torpedostage

embryos, from which plants can be regenerated. Zygotic embryos

undergo a fundamentally similar development through the globular

(which is formed after the 16- cell stage), heart and torpedo stages.

Polarity is established early in embryo development. Signs of tissue

differentiation become apparent at the globular stage and apical

meristems are apparent in heart-stage embryos.

Furthermost to check genetic diversity between original plant

and cultured cells several DNA markers have been applied as a direct

approach. Among all the techniques RAPD technique requires less

time, low cost, small quantity of DNA for analysis and co-dominant

nature (Powell et al., 1996).

GENETIC STABILITY ANALYSIS

(RANDOM AMPLIFIED POLYMORPHIC DNA)

The distinguishing mark of every organism is its unique set of

DNA. Mutations occurring in the DNA sequences of an organism are

expressed as variations, which create new alleles in a population. This

phenomenon is termed as polymorphism (meaning, having many

forms). Characterizing such alterations in genetic structure is very

informative in understanding genetic polymorphism in a population. A

population is in a state of balanced polymorphism when non-identical

alleles for a trait are being maintained at frequencies greater than

1%. The extent of polymorphism within the gene pool of a species

determines not only the survival of a species but also its evolutionary

potential. In plants, about 15 to 30% of genes coding for enzymes

INTRODUCTION

43

are polymorphic. It means that genetic variation is very common in

natural populations of most plants. Such variations that distinguish

within species and among species can be called Molecular Markers.

DNA polymorphisms are important genetic markers in natural

populations. Knowledge about molecular markers based on

polymorphism have been exploited by plant biologists in the last two

decades for breeding, selection of high yielding varieties, identifying a

species, solving taxonomic issues, estimating phylogenetic

relationships, formulating conservation strategies, etc. Polymorphic

traits are influenced by environmental factors.

Until two decades ago, morphological and biochemical traits

like allozymes have been used to analyse genetic diversity within the

gene pool of a species. However, allozymes have proved insufficient

to assess genetic diversity within populations (Heun et al., 1994).

Therefore, the development of molecular based technologies such as

RAPD (Random Amplified Polymorphic DNA), ISSR (Inter Simple

Sequence Repeat), RFLP (Restricted Fragment Length Polymorphism)

have broadened the scope of such studies at the population level.

Among molecular markers, RAPDs have been extensively used in

genetic research owing to their speed and simplicity (Penner, 1996).

RAPD is a DNA polymorphism assay based on random amplification

of a DNA segment using arbitrary sequence primers. This method is

used in conservation, population and evolutionary biological studies

because of its technical swiftness, cost effectiveness and less labour

intensiveness. The technique does not require prior knowledge of DNA

sequences and generates DNA markers from very little tissue by

screening the entire genome. Visualisation of the result does not

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require the use of hazardous radioisotopes and can be directly

observed from the gel by ethidium bromide staining.

Information on genetic variation within and among populations

is crucial for conservation of endangered species. A species may be

considered as a group of individuals organized into populations that

share an amalgamation of indicative characters which are not found

outside the group. Survival chance of a species is indicated in

genetic diversity within the population (Balakrishna, 1999; Tsuda et

al., 2009). With greater variation in the genome, a species will be

able to adapt to new selection pressure caused by environmental

factors. Anthropogenic factors such as cattle grazing, agricultural

practices, unscrupulous exploitation for medicinal uses, and natural

calamities like flood and wild fire, have a detrimental effect on

preserving genetic diversity (Aguilar et al., 2008).

The gene pool, which comprises all the genes of the

population, usually occurs in two or more slightly different molecular

forms. Each of these forms is called an allele variation. This results

in the population as individuals inherit different combinations of these

alleles. A gene that exists as two or more alleles in a population is

called polymorphic, whereas a monomorphic gene exists as a single

allele in a population. By convention, a gene is considered

monomorphic when a single allele is found in at least 99% of all

instances of a gene. To be polymorphic, a gene has one or more

additional alleles that make up at least 1% of the alleles in a

population (Hartel and Jones, 1990).

The allele frequency will stay through the generation if there is

no mutation and the population is infinitely large and if it is isolated

from other populations of the same species. Additionally mating

INTRODUCTION

45

should be random regarding the alleles, and all individuals should

survive and reproduce equally (Hartel and Jones, 1990).

PCR based techniques

Polymerase chain reaction based techniques (Saiki et al., 1985)

are a better alternative to hybridization techniques for various

reasons. Primarily, PCR dependent techniques require less quantity of

DNA and sample purity is not a major concern. It is a quick method

without the requirement of radioactive labelling. Since primers are

employed for amplifications, these can be termed as 'sequence

tagged sites' (STS). Specificity and reproducibility of PCR depends on

a number of factors such as the concentration and quality of the

reaction ingredients, primer design, GC content of the primers,

number of cycles, temperature and duration. Many of these

disadvantages can be streamlined and an optimum condition can be

worked out (Westman and Kresovich, 1997).

Depending on the target sequence information, primers have to

be designed in PCR based marker assays. Arbitrary primers can be

designed if sequences are not known. In case of sequence tagged

sites (STSs) (Olson et al., 1989) presence or absences of the

fragment, or the difference in fragment length can be assessed to

generate data. This is vital in diploid organisms as the length of the

fragment can be assessed as co-dominant alleles.

Random Amplified Polymorphic DNA (RAPD)

Random Amplified Polymorphic DNA (RAPD) is based on the

random amplification of sequences using primers that are 10 bases

long (Williams et al., 1990). The primer anneals at several priming

INTRODUCTION

46

sites to the template at complementary sequences in both 'sense'

and 'antisense' orientation, and the regions between primers in

opposite orientation are amplified. However, geometric amplification

occurs only in those regions in which the 3' end of the annealed

primers face one another or opposite strands and not more than 3kb

apart. In other words, the primer annealing sites must be Inverted

repeats. Thus RAPD scans a genome for these small inverted repeats

and amplifies intervening DNA sequences of invariable length.

A primer generally produces 1-10 fragments. If there is

variation in the nucleotides of different sets of DNAs there will be

changes in the priming sites causing presence or absence of bands

which are generally polymorphic. The presence of a marker in more

than one individual means that the individuals share the same

sequence at the primer annealing sites and that these sites are

separate by the same number of base pairs. The absence of a

fragment in more than one individual does not indicate the same

level of homology (Dangi et al., 2004). The failure to amplify may be

due to changes in the annealing site, or increase levels of

competition from other amplified fragments. Therefore, the presence

of polymorphism is far more informative than its absence (Skroch et

al., 1993). However, this can lead to mis-scoring since the failure to

amplify is interpreted as the absence of an allele. As homozygous

bands cannot be distinguished from heterozygous, the presence of a

fragment is considered dominant over absence. RAPDs are dominant

markers, i.e., profiles are scored for the presence or absence of a

single allele. Dominant markers are not affected by the conditions

under which the plants are grown or the developmental stage of the

tissue from which the DNA is extracted. Though RAPD evaluates many

INTRODUCTION

47

loci simultaneously, it provides very little genetic information about

each locus.

One of the greatest concerns about RAPD is its reproducibility.

The reproducibility in RAPD using the same target DNA and primers

have been investigated in different laboratories. It was found that

most RAPD markers are reproducible, with difference between PCR

machines accounting for most of the variations (Penner et al., 1993).

This reproducibility problem is usually the case for bands with

lower intensity. This may arise from impurities in DNA preparation

(Micheli et al., 1994). Other steps such as consistent reaction

conditions, thermal profile during amplification and fixed DNA

template concentration can ensure reproducibility. Magnesium chloride

concentration above 2mM, primer to template concentration ratio,

fluctuation in the concentration of reagents in PCR reaction, Taq

polymerase above 2U/reaction also affects reproducibility. Annealing

temperature below 36°C gives altogether different banding pattern

(Penner, 1996). Pipeting error should be minimized. Moreover to

ensure the result, the assay should be carried out two or more times

and highest possible number of samples should be analysed to

eliminate artefacts. A modified method termed high annealing

temperature RAPD (HAT–RAPD) (Atienzar et al., 2000) relies on

increasing the temperature to 40-60oC and helps in generating

reproducible profile.

RAPD has various advantages. It is and easy technique and

does not require radioactive labelling as in RFLP, since the separation

of bands is done on an agarose gel and visualised by ethidium

bromide staining. It amplifies where no prior knowledge of sequence

exists. This is a much faster and cheaper method than RFLP.

INTRODUCTION

48

Moreover, it uses nanograms of genomic DNA which reduces the

damage done to a plant species. This is important if the plant

belongs to an endangered species (Winter and Kahl, 1995).

Applications of RAPD

RAPD has been employed in various areas due to its simplicity

and low cost. Some of the areas include biological control

programmes (Wang et al., 2008), genetic mapping (Kiss et al., 1993;

Hemmat et al., 1994; Sutherland et al., 2008), breeding programmes,

developing genetic markers linked to a trait of interest (Li et al.,

2008) without the necessity for mapping the entire genome. This has

been successful in identifying markers linked to disease resistance

genes in tomato (Martin et al., 1991), lettuce (Paran et al., 1991),

fungi (Ananga et al., 2008), barley (Jansen and Schaffrath, 2009),

testing purity in hybrid seeds (Singh et al., 2007) and genetic

diversity in a multitude of species (Ram et al., 2008; Sharma and

Chauhan, 2008; Jose et al., 2009; Orabi et al., 2009).

RAPD has also been used in a genome profiling strategy

termed Bulk Segregate Analysis (BSA) (Michelmore et al., 1991). In

this method bulked DNA samples from individuals that have the

target trait/gene are compared to bulked DNA samples of individuals

lacking the trait. Markers that are polymorphic between the pools will

be genetically linked to loci determining the trait used to construct

the pool. RAPD has been extensively used in population and

evolutionary genetics studies in spite of the need for large samples

of individuals from each population. This is essential if an accurate

estimate of allele and genotype frequencies is needed. Since RAPD is

a dominant marker, the estimated gene frequency is less accurate

INTRODUCTION

49

than those obtained with co-dominant markers such as Allozyme and

RFLP (Bardakci, 2001). Co-migrating bands derived from the same

primer is another problem with RAPD. But as the population diverges,

the co-migrating bands become less homologous. Therefore, RAPD

gives a better result between closely related species (Williams et al.,

1993; Megnegneau et al., 1993; Orabi et al., 2009). Species specific

(inter specific) markers unique to individuals from one species within

a genus can be estimated by RAPD (Arnold et al., 1991).

Applications of molecular markers

Looking at the history of the development of molecular markers

one can appreciate the advancement of markers in the last two

decades. It has evolved from an expensive, time consuming, elaborate

technique to an automated, accurate, reproducible and reliable

method. Markers also broadened their application from breeding

programmes to localising genes, marker assisted selection, evaluating

genetic diversity, solving taxonomic disputes, assessing genetic drift,

phylogenetic analysis and mapping (Condit and Hubbell, 1991; Gupta

et al., 1996; DeWalt and Hamrick, 2004; Varshney et al., 2005;

Sharma and Chauhan, 2008; Tsuda et al., 2009; Verma et al., 2009).

They have generated a vast array of information in the recent past

that helps in understanding and thus formulating policies for

establishing a balanced ecosystem.

Marker assisted selection

Identifying alleles of economic importance such as pathogen

resistance, stress tolerance and high yielding variety are vital in any

crop improvement programme. These markers have been used for

CHAPTER 1-B

OBJECTIVES AND INTRODUCTION

OF THE PLANT

OBJECTIVES AND INTRODUCTION TO THE TAXA

51

OBJECTIVES AND INTRODUCTION OF THE

PLANT

The present study was undertaken with taxa Argyreia nervosa

(Burm. f.) Bojer (Argyreia speciosa (L. f.) Sweet, medicinal plant with

high medicinal values.

The study was conducted with the following objectives:

A. Plant tissue culture

1. To standardize protocol for callus induction of different part

of the plant for large scale cell production and production

of secondary metabolites.

2. To standardize protocol for micropropagation of the taxa for

large scale propagation and conservation of the plant. To

standardize protocol for shoot induction of different part of

the plant.

3. To standardize protocol for cell suspension culture of

different part of the plant for large scale cell production

and production of secondary metabolites.

B. To study Pharmacognostic characters of plant: Macroscopical

and Microscopical (anatomy).

1. Leaf

a. T.S. of leaf with lamina

b. T.S. of petiole

OBJECTIVES AND INTRODUCTION TO THE TAXA

52

2. Stem

a. T.S. of Stem (primary growth)

b. T.S. of Stem (secondary growth)

3. Root

a. T.S. of Root (primary growth)

b. T.S. of Root (secondary growth)

4. Nodal anatomy

C. Qualitative and Quantitative analysis

1. Comparison of the phytochemical profile of the cultured

cell of the plant with the field grown plants by process

of High performance thin layer chromatography (HPTLC)

for alkaloids, Flavonoids, Tannin and Glycoside.

2. Qualitative analysis of secondary metabolites: Alkaloids,

Flavonoids, Tannins and Glycoside.

3. Protein, carbohydrate and lipid estimation(Quantitative) of

plant

4. Quantitative estimation of secondary metabolites:

Phenols, Tannins and Flavonoids.

D. Gas Chromatography – Mass Spectrometry/ NMR of plant

extract to identify active compounds of the plant

E. Antibacterial activity

a. Aqueous extract of leaf, stem and root

b. Methanolic extracts of leaf, stem and root

OBJECTIVES AND INTRODUCTION TO THE TAXA

53

F. Antifungal activity

a. Aqueous extract of leaf, stem and root

b. Methanolic extracts of leaf, stem and root

G. Genetic stability by Random Amplified Polymorphic DNA: To

know genetic variation between original plant and cultured cells

1. Screening the suitable primers for development of

diversity markers in Argyreia

2. To analyze the genetic diversity among the different

parts of cultured cells and original plant

3. Identification of DNA markers for best genotypes

OBJECTIVES AND INTRODUCTION TO THE TAXA

54

INTRODUCTION OF FAMILY

Convolvulaceae

Convolvulaceae is a large family, comprising approximately 50–

60 genera with some 1600–1700 species (Mabberley, 1987), exhibiting

a rich diversity of morphological characteristics and occupying a

broad range of ecological habitats. More than one-third of the species

are included in two major genera, Ipomoea and Convolvulus

(Cronquist, 1988). Convolvulaceae are distributed throughout the

world, but are primarily tropical, with many genera endemic to

individual continents. Although the family is best known in temperate

regions for its weedy representatives (e.g., Calystegia, Convolvulus),

many tropical species are valuable ornamentals, medicinal and food

crops. The sweet potato, Ipomoea batatas, is the world's second most

important root crop (>128 x 109 kg/yr; Simpson and Ogorzaly, 1995).

The record of microfossils attributed to the family is known as far

back as the Eocene ( 40–45 million years ago [mya]), but without

accompanying macrofossils.

Typical members of the family are annual or perennial vines,

with milky sap, internal (intraxylary) phloem, Leaves alternate, entire,

simple to lobed or pinnately divided to pectinate, exstipulate.

Inflorescence determinate, cymose, or flowers solitary, axillary, with

OBJECTIVES AND INTRODUCTION TO THE TAXA

55

jointed peduncles. Flowers actinomorphic, perfect, hypogynous, often

large and showy, ephemeral, usually with intrastaminal disc, generally

subtended by a pair of bracts (sometimes enlarged and forming an

involucre). Calyx of 5 sepals, distinct or sometimes basally connate,

sometimes unequal, imbricate, persistent. Corolla sympetalous, entire

to slightly 5-lobed, funnel form or salverform, plicate, brightly colored

(commonly red, violet, blue, or white), indup-licate-valvate and/or

convolute (twisted) in bud. Androecium of 5 stamens, epipetalous at

corolla base; filaments distinct, often unequal; anthers dorsifixed,

dehiscing longitudinally, usually introrse. Gynoecium of 1 pistil, 2-

carpellate; ovary superior, 2-locular or sometimes appearing 4-locular

due to false septa, sometimes with dense covering of hairs; ovules 2

in each locule, anatropous, sessile, placentation basal or basalaxile;

style simple and filiform or forked; stigma(s) 1 or 2, linear, lobed or

capitate. Fruit usually a 4-valved septifragal capsule; seeds smooth or

hairy; endosperm scanty, hard, cartilaginous; embryo large, straight or

curved, with folded or coiled, emarginated to bifid cotyledons,

surrounded by endosperm.

Distribution:

Primarily in the tropics and subtropics, with representatives

having ranges extending into north and south temperate regions;

particularly abundant in tropical America and tropical Asia. Major

genera: Ipomoea (500 spp.), Convolvulus (250 spp.), Cuscuta (145—

170 spp.), and Jacquemontia (120spp.).

The Convolvulaceae have been divided into three or four

subfamilies (sometimes segregated as distinct families) and/or three

to ten tribes. Although the relationships between these groups have

OBJECTIVES AND INTRODUCTION TO THE TAXA

56

been generally agreed upon, the taxonomic rank (family, subfamily, or

tribe) is a matter of controversy (Wilson, 1960). A notable segregate

group, the Cuscutoideae or Cuscutaceae (a monotypic taxon), has

been separated from the rest of the Convolvulaceae by some

botanists on the basis of the parasitic habit with related

specializations of the corolla and embryo (Momin, 1977).

Authors also disagree on the delimitation of the various genera within

the family, such as Ipomoea (Sengupta, 1972). The generic lines

depend upon characters of the bracts, sepals, corolla, pollen,

stigma(s), and fruit. For example, the sepals vary in size, shape, and

pubescence and the stigmas may be simple, lobed, or globose. In

addition, seed characters (e.g., type of pubescence) are important for

species delimitation.

Morning-glories are easy to spot in the field with their twining

habit and generally large, white or brightly colored, and funnel-shaped

corolla. The corollas are twisted clockwise in bud and strongly plicate

(Allard 1947). Usually a flower is open for only one day (for a few

hours); the corolla then incurves as it wilts. The corolla is

characteristically divided longitudinally by five obvious demarcations

that occur along the middle of the five lobes of the limb. These

markings taper toward the apex and usually twist in the clockwise

direction.

OBJECTIVES AND INTRODUCTION TO THE TAXA

57

INTRODUCTION OF PLANT

Classification: (Bentham and Hooker)

Kingdom Plant

Class Dicotyledones

Sub-class Gamopetalae

Series Bicarpellatae

Order Polemoniales

Family Convolvulaceae

Genus Argyreia

Species nervosa (Burm. f.) Bojer

OBJECTIVES AND INTRODUCTION TO THE TAXA

58

Other Latin names:

Argyreia speciosa (L. f.) Sweet

Convolvulus speciosus L.f.

Convolvulus nervosus Burm. f.

Vernacular Names:

Hindi: Samandar-ka-pat, Samudrasos, Samudra Shokha

Gujarati: Vardharo, Gha-vel, Chandpan

Kannada: Candrapada

Malayalam: Marikkunni, Marututari, samudrappacca

Sanskrit: Vrddhadarukah, Bastantri

Tamil: Samuttirappaccai, Samuttirappalai Kadarpalai

Telugu: Candrapada

Bengali: Bijarka

Nepalese: Samudra phool

Sinhalese: Vriddadaru

Unani: Samudar sokh

Indonesia: Areuy bohol keboh (Sundanese)

Philippines: Sedang-dahon (Tagalog)

OBJECTIVES AND INTRODUCTION TO THE TAXA

59

Thailand: Bai rabaat, Phak rabaat (central), Mueang mam (Bangkok)

English: Elephant creeper, elephant climber, elephant vine, Hawaiian

baby woodrose, silver morning glory, wood rose, woolly morning glory

French: Coup d'air, liane à minguet, liane d'argent, liane d'argentne à

minguet (Lavergne Christophe, 2006).

Mention of Argyreia nervosa in Veda: (Vaidyaratnam, 2005)

[“Bastantri visagandha vayojaradarika chagalantri

Visapatrikantravasta paryayairvrddhadarukam bhavati” (A.ma.)]

[“Vrddhadaruka avegi jungako dirghavalukah

Vrddhah kotarapuspi syadajantri chagalantryapi”(Dha.ni.)]

[“Vrddhadaruka avegi jongako jinabalakah

Antah kotarapuspi syat syama mahisavallari

Ajantri tu mahasyama vallari dirghabalakah” (Kai.ni.)]

OBJECTIVES AND INTRODUCTION TO THE TAXA

60

[“Vrddhadaruh kasayosnah katustikto rasayanah

Vrsyo vatamavatarsahsophamehakaphapranut

Suklayurbalamedhagnisvarakantikarah sarah" (Bha.pra.)]

[“Vrddhadaruh katustiktastathosnah kaphavatajit

Svayathukrmimehasravatodaraharah parah" (Dha.ni)]

[“Vrddhadaruh katustiktah kasayosno rasayanah

Suklayurbalamedhyagnisvarakantikarah sarah

Sophamavatavatasravatamehakaphapahah” (Kai. ni.)]

[“Sadharano vrddhadaruh katustiktah kasayakah

Rasiiyanosno madhuro medhyah svaryah sarognidah

Kantidhatukaro balyo rucyah pustikaro laghuh

Upadamasam panurogam kasayam kasam pramehakam

OBJECTIVES AND INTRODUCTION TO THE TAXA

61

Vataraktam camavatam. vatam sopham kapham jayet” (Ni.ra.)]

[“Vrddhadaro grahonmadapapalaksmi vinasanah

Apasmaramavataghnah sophasulapahognikrt

Balyah kanthyosthisamdhanakari vatarujapahah

Visucipratitunyadivyadhighati rasayanam” (So.ni.)]

[“Sramsini gulmahrdrogavisarocakanasini

Bastantri kapharogaghni mutrakrcchravinasini” (Ma.ni.)]

[“Vrddhadaruh kasayosah sarastikto rasayanam

Vrsyo vatamavatasrasophamehakaphan jayet” (Ma.vi.)]

Remarks: ‘Bastantrt’ of Syamadigana (Astangahrdayam) and

vrddhadaru of ‘Maharasnadikasayam’ (Sahasrayogam) are interpreted

as marikkunni in Malayalam by most of the commentators.

OBJECTIVES AND INTRODUCTION TO THE TAXA

62

In the ‘Arunodaya’ commentary on Astangahrdayam the

Malayalam name marututari is given for bastantri. In the

‘Osadhinighantu’ of Kumaran Krishnan, the Sanskrit-Malayalam

dictionary of Kanippayoor and ‘Ayurvedavisvakosam’ of Pandit K.K.

Panickar, chagalantri, vrddhadaruka. and vrddadaru are translated into

Malayalam as marikkunni. Bastantri is marututari or marukutari

according to ‘Ayurvedavisvakosam’. But there is no mention of this

word bastantri in ‘Osadhinighantu’ or Sanskrit-Malayalam dictionary.

Marikkunni and marututari are treated as two distinct raw drugs in

‘Osadhinighatu’ and ‘Ayurvedavisvakosam’. In the ‘Ayurvedic Formulary

of India’ also bastantri and vrddhadaru are treated as two distinct

ones, giving the Latin name as Argyreia speciosa and Ipomoea

petaloidea, respectively. In the ‘Glossary of Vegetable Drugs in

Brhaltrayi, Ipomoea pes-caprae is the Latin name given for chagalntri

and bastantri is treated as a synonym of chagaltintri. But in the

‘Pharmacognosy of Ayurvedic Drugs’ both Argyreia speciosa and

Ipomoea pes-caprae are regarded as vrddhadaruka, giving the

Malayalam names samudrapacca for Argyreia speciosa and aqampu

or cuvanna aqampu for Ipomoea pes-caprae. Dr S.N. Nesamani

considers Argyreia speciosa as samudrapacca in his book

‘Ausadhasasyannal.’ The commentators of ‘Bhavaprakasam’,

‘Kaiyadevanighantu’ etc., are of the opinion that the Latin name of

vrddhadaru is Argyreia speciosa.

As chagalantri is a synonym of bastantri and as marikkunni is

the Malayalam name given for chagalantri it amounts to regard

bastantri as marikkunni itself. Hence, the Malayalam name marikkunni

is applicable to bastantri as well as vrddhadaru.

OBJECTIVES AND INTRODUCTION TO THE TAXA

63

It is strange to note that all the Sanskrit synonyms of

vrddhadaru are given to Rourea santaloides W. & A. also, in ‘Indian

Medicinal Plants’. Being Highly poisonous is not advisable to use as

vrddhadaru.

Thus, it is clear that in Kerala Argyreia nervosa (A. speciosa) is

being used for marututari, marikkunni and samudrapacca.

Morphology:

Habitat/ecology:

A. nervosa is native to India, from Assam to Belgaum and

Mysore. It is common on the Bengal plain (Hooker, 1885). Specimens

have been recorded from Java, China and Mauritius, although it is

unclear whether these latter locations have included cultivated

specimens. This species exists in south and north-west India, as well

as Bengal (Stewart and Brandis 1874). A. nervosa is cultivated on the

Malay Peninsula (Hoogland 1952). A. nervosa is ‘originally in British

India, from Assam and Bengal to Belgaum and Mysore, cultivated in

other tropical countries; occasionally escaped from culture’

(VanOostroom 1943). A. nervosa has been erroneously listed as

native to Australia. The Queensland Herbarium has confirmed that it

considers that A. nervosa is a naturalized species in Australia

(Batianoff, pers. comm.). While most references state that A. nervosa

is native to India, Hawaiian and Polynesian people are reported to

have used this species as a drug over hundreds of years

(Anonymous, 2004a) and it could be speculated that other Indigenous

communities in Australia and elsewhere in the Pacific have used and

transported seeds from this plant for some time.

OBJECTIVES AND INTRODUCTION TO THE TAXA

64

Hawaiian Baby Woodrose’s been theorized that it was

introduced in Hawaii very early on and thrived in the tropical climate,

thus leading to Hawaii becoming known as its latter day “home” and

popular namesake. It is less popularly known as the Silver Morning

Glory (stemming from its origin in the Convolvulaceae Morning Glory

family), and the Monkey Rose, among other folk names. The plant is

also part of the indigenous flora of Australia and has been known to

grow wild in Africa. It is popular as an ornamental plant, as well as

an enthogenic intoxicant and legal inebriant, although the ingestion of

this plant in many parts of the world is now illegal, including the

United States.

A. nervosa prefers tropical and sub-tropical climates. References

that discuss the cultivation of A. nervosa state that the plant prefers

fertile, moist soil in a protected sunny position (Ellison, 1995).

Anecdotal information from Queensland National Parks and Wildlife

staff suggests that the plant can germinate quite readily in seemingly

undisturbed sites, including under rainforest canopies and among

dense grass cover in eucalypt woodland (P Williams, pers comm.

2004).

It is found in India throughout, up to an altitude of 300 meters

high, except in dry, western regions up to 1000 ft elevation, often

cultivated. The beautiful, woody, flowering trellis vine that is Hawaiian

Baby Woodrose flourishes in direct sunlight, in areas that promote

hot, humid climates. This plant has been used as a folk remedy in

India, and is valued for its aesthetics. It grows well in Hawaii,

California, Florida, and similar climates.

Description:

OBJECTIVES AND INTRODUCTION TO THE TAXA

65

"Clambering vine to several meters long; herbage velvety

pubescent, densely when young. It is a very large woody climber (Fig.

No. 1). Leaf blades 15-25 (30) cm long, 13-20 (30) cm wide, cordate,

acuminate-attenuate apically, cordate basally. The leaves are glabrous

above; persistently white tomentose beneath, petiole is long.; flowers

in cymes, on long, white-tomentose peduncles; sepals 13-15 mm long,

velvety like the herbage; pedicels to 15 cm long; flowers 5-7.5 cm

long, the corolla with a short tube and campanulate limb, lavender to

pink, the throat darker" (Welsh, 1998). Flowers trumpet-shaped (Fig.

No 2). Seeds are enclosed in a stone, pale yellow-brown globose,

apiculate, indehiscent berry 1.2 to 2 cm in diameter containing four

erect, curved embryos with corrugated cotyledons or two seeds

embedded in a meaty pulp (Fig. No.3.1). Seed pods dry into woody

"rosebuds," each one containing three to four seeds (Fig. No.3.2; Fig.

No.4). The seeds are known to be rich in psychoactive ergot alkaloids

and contain a naturally occurring tryptamine called LSA (Lysergic Acid

Amide). Root cylindrical, 1 to 1.5 cm thick; brown, smooth, round

wood is scant, flexible, and smooth, latex oozes at cuts. This vine

produces beautiful flowers and seeds of historic significance.

This plant is a rare example of a plant whose hallucinogenic

properties have only recently been discovered by non-Hawaiians.

While its cousins in the Convolvulaceae family, such as the Rivea

corymbosa (Ololiuhqui) and Ipomoea violacea (Morning Glory), were

used in shamanic rituals of Latin America for centuries, the Hawaiian

Baby Woodrose was not traditionally recognized as a hallucinogen. Its

properties were first brought to attention in the 1960s, despite the

fact that the chemical composition of its seeds is nearly identical to

those of the two species mentioned above, and the seeds contain

OBJECTIVES AND INTRODUCTION TO THE TAXA

66

the highest concentration of psychoactive compounds in the entire

family.

Seeds from Ghana are typically smooth and very light in

color. Seeds from the Ayurvedic strain from India are typically more

"pointed" and larger than most other Hawaiian Baby Woodrose

seeds. Ones from India typically have less LSA content, but look

roughly the same as the coveted strain from Hawaii, but don't have

much "fuzz" on them, and they are also typically slightly smaller in

size.

Propagation:

Berries dispersed by frugivorous birds. It may be propagated by

cuttings or seeds and in the spring by division. The seed may be

sprouted by making a small nick in the seed coat away from the

germ eye. Soak the seed until it swells. Plant 0.5 inch deep in loose

rich soil. After the cotyledons appear, water sparingly, letting the soil

surface dry out to a depth of 0.5 inch. Over-watering causes stem

and root rot. The plant grows slowly until it develops a half-dozen

leaves; after this it grows quickly. The next spring it will grow into a

very large vine and should produce flowers and seeds. The plant can

start growing flowers as early as its life cycle's second year. In India,

growing seasons are often accelerated, so one can often get seeds

within 18 months. For this to occur, there must be sufficient watering

and adequate room for the roots to grow; it can take up to 5 years

for the first signs of flowering to become visible.

The seeds will be found in the pods of the dried flowers.

These cannot be harvested until completely dried.

OBJECTIVES AND INTRODUCTION TO THE TAXA

67

Part Used:

Leaf, Stem, Root Seeds, Wood

Mythology:

The Hawaiian Baby Woodrose is a popular candidate for the

mythological Soma plant, of which the definitive botanical identity is

unknown today. “Soma” is the liquid potion derived from the Soma

plant, and the earthly counterpart of Ambrosia, the ancient, mythical

drink of immortality reserved for the gods. The name soma was

bestowed upon a deity (the moon; the god of plants), the plant itself

and the sacrificial drink that was prepared from the plant.

In the Hindu tradition, the moon (originally called soma), was

believed to be the ambrosia-filled drinking vessel of the gods. When

the moon was full, the vessel was full; by the time the new moon

appeared, the drinking vessel had been emptied. It filled up again as

the moon waxed.

To prepare this mystical concoction, the stems of the soma

plant were pressed to release the sap. The resulting juice, which was

believed to “dissolve all sins,” was then mixed with water and offered

to Indra, the Hindu God of Thunder. This soma ritual is thought to

have served as the prototype for the kava ceremony of the South

Pacific. It has also been widely speculated that the identity of the

Soma plant is actually Amanita muscaria.

It has been noted in various oral histories that the Huna

religion, the healing and spiritual shamanism of ancient Hawaii,

employed the seeds of the Hawaiian Baby Woodrose for their

OBJECTIVES AND INTRODUCTION TO THE TAXA

68

Shamanic rituals, using the seeds’ enthogenic and magical properties

to connect with the spirit world. The Huna Shaman prepared the

Hawaiian Baby Woodrose seeds by grinding them into a powder and

mixing the powder directly with water, resulting in a supernatural

libation that was then drunk by the Huna Shaman. This magical tonic

was used as a means for the Shaman to pass from this worldly

plane into the realm of the spirits.

Hawaiian Baby Woodrose seeds are perhaps one of the least

understood of modern-day entheogens and exotic botanicals. There

is much controversy in regards to its true place in Shamanic and

traditional history outside of its native culture and home; India.

Ayurvedic description: (Anonymous, 2004b)

Properties:

Rasa: Kuru, tikta, kasaya; Guna: Laghu, snigdha, sara; Virya: Usna.

Action: Vatakaphahara, sukravardhaka, vrsya, balya, rasayana. Medhya,

swarakantikara.

Therapeutic uses: (Anonymous, 2004b)

Klibato, daurbalya, amavata, vatarsa. Sotha.

Uses:

The leaves are antiphlogistic; they are applied over skin

diseases and wounds; the silky side of the leaf is applied over

tumors, boils, sores, and carbuncles; as an irritant to promote

maturation and suppuration. The leaves are also used for extracting

OBJECTIVES AND INTRODUCTION TO THE TAXA

69

guinea worms. A drop of the leaf juice is used in otitis. Dried leaves

are used for diabetes (Jain and Sharma, 1967). Traditionally leaves

used by Rajasthani tribes to prevent conception (Anonymous, 1988;

Nisteswar, 1988).

The roots are acrid, bitter, astringent, sweet, and emollient. It is

used in vitiated conditions kapha and vita, emaciation, wounds,

ulcers, anorexia, dyspepsia, flatulence. Roots are used in the

treatment of gonorrhea, rheumatism and diseases of nervous system.

It is also used in obesity, hoarseness, syphilis, anemia, diabetes,

tuberculosis and general debility. It is also used as a tonic (Prajapati

et al., 2003).

Powder of the root is given with "ghee" as an alternative; in

elephantiasis the powder is given with rice water. In inflammation of

the joints it is given with milk and a little castor oil. A paste of the

roots made with rice water is applied over rheumatic swelling and

rubbed over the body to reduce obesity.

LSD is the best-known synthetic hallucinogen and is

psychoactive at the microgram level. Although LSD does not occur in

nature, a close analogue, lysergic acid amide (LSA, ‘‘ergine’’) is found

in the seeds of Argyreia nervosa (Hawaiian baby woodrose) and

Ipomoea violacea (morning glory). Hallucinogenic activity of LSA

occurs with 2–5 mg, which provides a 4- to 8-hr intoxication that

reportedly has quantitative as well as qualitative differences from LSD

(Schultes and Hofmann, 1980). Seeds are crushed, germinated, eaten

whole, or an extract is drunk after the seeds are soaked in water.

Five to 10 seeds of Argyreia nervosa or 150–200 seeds (3–6 g) of

Ipomoea violacea yield average doses of LSA (Al Assmar, 1999;

OBJECTIVES AND INTRODUCTION TO THE TAXA

70

Borsutzky et al., 2002). The LSA content of Argyreia nervosa is 0.14%

of the dry weight of the seeds (Chao and Der Marderosian, 1973).

They are widely regarded as hallucinogens in today’s Australian

drug scene. The seeds and preparations made from them are utilized

and celebrated in Sex Magick rituals in certain underground

subcultures such as those associated with Aleister Crowley, the British

occultist.

The seeds are also used in a preparation known as Utopian

bliss balls, which consist of five Argyreia seeds, damiana herbage,

ginseng root, fo-ti-teng and bee pollen, and were very popular in the

sixties among the hippies and artists in California.

Suffice it to say that despite any controversy, there is enough

documented and anecdotal evidence to support the enthogenic

properties of the Hawaiian Baby Woodrose seeds. HBWR Seeds is

illegal in many parts of the world.

Three seeds considered sufficient to produce an LSD-like

experience, with psychonauts reporting colourful visions of a spiritual

nature, psychedelic patterns, all-over body sensations, a sense of

extreme relaxation, euphoria and deep spiritual awareness. Four to six

seeds are a standard dose, and there are reports of strong

hallucinogenic side effects after ingestion of 12 to 16 seeds. This

also depends on the age of the seeds, as some of the psychoactive

compounds found in Hawaiian Baby Woodrose seeds can break down

in as little to 6 to 9 months. The experience duration can range from

4 to 12 hours, with mild effects occasionally lasting about a day and

is usually accompanied with gastric discomfort, including severe

OBJECTIVES AND INTRODUCTION TO THE TAXA

71

nausea and flatulence. Other after effects of these seeds includes

sensitivity to light, and impaired motor skills. The removal of the

outer coating of the seeds is often recommended to reduce such

effects, although there appears to be no consensus as to this

practices efficacy and it has been suggested that it may not be true.

Ergot alkaloids, including LSD and LSA, can cause uterine

contractions, which may lead to miscarriage if the seeds are

consumed while pregnant. Contrary to popular belief, the seed's outer

coat does not contain cyanide or glycosides. The nausea associated

with consuming the seeds is mostly because of essential oils in the

seed itself.

The psychedelic properties of the seeds became known mainly

through their use in Hawaii, Haiti and Puerto Rico, where

impoverished members of the population would consume the seeds,

seeking a "cheap buzz" as an alternative to alcohol. A sample made

its way to Albert Hofmann, the creator of LSD, who confirmed the

effects and analyzed its chemical composition. It is still used by

some Hawaiians for a high.

Hawaiian Baby Woodrose seeds were traditionally used in

sacramental rituals of the Hawaiian and Polynesian islands. Traditional

use of the plant in India usually employed the leaves and roots of

the plants, which are not psychoactive, as antiseptic and anti-

inflammatory drugs.

The whole plant is reported to have antiseptic properties.

Principal Constituents:

OBJECTIVES AND INTRODUCTION TO THE TAXA

72

It contains many ergoline alkaloids. Argyreia nervosa seeds

contain 0.3-1 % ergot-alkaloids by weight. Ergine (d-lysergic acid

amide) (Figure No.: 5.1), isoergine (l-lysergic acid amide) (Figure No.:

5.2), ergometrine, lysergol (Figure No.: 5.4), isolysergol and

chanoclavine (Figure No.: 5.5) are present. 6, 7 Lysergol and

elymoclavine (Figure No.: 5.3) are reduction products of d-lysergic

acid. LSD which is a lysergic acid amide and although resembling the

natural lysergic acid amides both in structure and pharmacological

activity, it has not been found in nature. Except alkaloids many

secondary metabolites are also present in the plant. The plant

contains tannin and amber-coloured resin, soluble in ether, benzole;

partly soluble in alkalis; and fatty oil. Chemical analysis revealed the

presence of triterpenoids, flavonoids, sterol; saponins are also present

in the plant (Shrivastav and Shukla, 1998). It contains Argyroside, a

recently reported steroidal glycoside, (24R)-ergost-5-en-11-oxo-3beta-

ol-alpha-D-glucopyranoside (Rahman and Khan, 2003). A few of the

ergoline alkaloids reported in this plant are hallucinogenic. LSA (d-

lysergic acid amide) is Schedule III in the United States.

The petroleum ether extract of the leaves yielded 1-tricontanol,

epifriedelinol acetate, epifriedelinol and β-sitosterol (Sahu and

Chakravarti, 1971). The leaves were found rich in quercetin (Daniel,

1989). Extraction of the leaves with 90% methanol led to the

isolation of the flavonoids, quercetin and kaemperol together with the

latter’s glycoside kaemperol-3-o-l-rhamnopyranoside (Khan et al.,

1992). Two new flavone glycosides characterized as 7,8,3’,4’,5’-

pentahydroxyflavone5-o-α-lrhamnopyranoside and 7,8,3’,4’,5’-

pentahydroxyflavone5-o-α-l-glucopyranoside were also reported from

leaves (Ahmad et al., 1993).

OBJECTIVES AND INTRODUCTION TO THE TAXA

73

The hexane extract of the root yielded tetradecanyl palminate,

5,8-oxidotetracosan-10-one (Rani and Shukla, 1997) and two novel

aryl esters characterized as stigmasteryl p-hydroxycinnamate and

hexadecanyl phydroxycinnmate along with scopoletin (Shrivastava and

Shukla, 1998).

The seeds yielded fatty oil which found to contain the

glycerides of palminate, stearic, linoleic, linolenic and oleic acids

(Biswas et al., 1947; Batra and Mehta 1985). In another study, the

seed oil revealed the presence of myristoleic, myristic, palmitic,

linoleic, linolenic, oleic, stearic, nonadecanoic, eicosenoic,eicosanoic,

heneicosaoic and behenic acids identified as their corresponding

methyl esters through GLC (Kelkar et al., 1947). The ethanolic extract

of the seeds revealed the presence of a mixture of three alkaloids,

out of which only one was characterized as ergometrin. The other

constituents isolated were caffeic acid and ethyl caffeate (Agrawal

and Rastogi, 1974a), another study also revealed the presence of

ergoline alkaloids in the seeds (Nair et al., 1987). The ergolines were

indicated to be of clavine type (Nair et al., 1987). The free amino

acids reported in the seeds were glutamic acid, glycine, isoleucine,

leu-cine, lysine, phenylalanine, tyrosine, praline and α-amino butyric

acid (Jaiswal et al., 1984).

The fruits were reported to contain n-tricontanol, β-sitosterol,

p-hydroxycinnamoyloctadecanolate and caffeic acid (Purushothaman

et al., 1982).

OBJECTIVES AND INTRODUCTION TO THE TAXA

74

Pharmacology

Ergine, isoergine, ergometrine, elymoclavine and lysergol are

responsible for the psychedelic effects. The structurally similarity

between these alkaloids and the neurotransmitters dopamine,

noradrenaline and serotonine might explain the hallucinogenic activity

by mutual influence on the active sites of the central nervous system;

it appears that the psychoactive constituents are partial agonists on

the G-protein-linked a-adrenergic- and on various serotonergic-

receptors (the serotonergic receptor-subtype 5-HT2A appears to be

involved in hallucinogenic activity).

Ascorbic acid (vitamin C) doesn't change the intensity of the

experience, but it alters its quality. One can concentrate better,

develops less paranoia and is also less tired at the end of the

experience. MAO-inhibitors and sympathomimetic amines

(amphetamine, ephedrine etc.) have positive synergistic effects; they

prolong and intensify the experience.

Hashish or marihuana can also intensify the experience. Usually

produces positive feeling. Tricyclic-antidepressants antagonize the

effects.

Pharmacognostic parameters for the leaves of Argyreia nervosa

Burm were studied with the aim of drawing the pharmacopeial

standards for this species. Macroscopical and microscopical

characters, physio-chemical constants, quantitative microscopy

parameters, extractive values with different solvents, fluorescence

analysis of dry powder, its reaction after treatment with chemical

reagents under visible light and UV light at 254 nm and 366 nm.

Preliminary phyto-chemical screening on the leaves Argyreia nervosa

OBJECTIVES AND INTRODUCTION TO THE TAXA

75

Burm was studied by Krishnaveni and Santh, 2009.

PHARMACOLOGICAL STUDIES:

Although a lot of pharmacological investigations have been

carried out based on the ingredients resents but a lot more can still

be explored, exploited and utilized. A summary of the findings of

Argyreia nervosa is presented here. Aphrodisiac activity of the plant

studied by Subramonium et al., 2007; Immunomodulatory activity of

the plant studied by Gokhle et al., 2003; Hepatoprotective activity of

the plant studied by Habbu et al., (2008a); Central nervous system

activity of the plant studied by Galani and Patel, 2009; Hypoglycemic

of the plant studied by Hemet et al., 2008; Nootropic of the plant

studied by Joshi et al. 2007; Anti inflammatory activity of the plant

studied by Srivastava et al., 1998; Anticonvulsant activity of the plant

studied by Vyawahare and Bodhankar, (2009a); Analgesic activity of

the plant studied by Bachhav et al., 2009; Antibacterial activity of the

plant studied by (Kelkar et al., 1947; George and Pandalai, 1949;

Mishra and Chaturvedi, 1978; Habbu et al., (2008b); Modi et al., 2010a).

Antifungal activity of the plant was studied by Shukla et al., 1999;

Antiviral activity the plant was studied Babber et al., 1978;

Nematicidal activity of the plant was studied by Parveen et al., 1990;

Anti-diarrhoeal activity of the palnt was studied by Rao et al., 2004;

Physiological disposition of isoergine (d-isolysergamide, iso-LA)

obtained from the seeds of Argyreia nervosa (Burm. F.) Bojer was

determined in rat liver, brain and plasma was studied by Vogel et al.,

1971; Effect of Argyreia speciosa extract on learning and memory

paradigms in mice was studied by Vyawahare and Bodhankar,

OBJECTIVES AND INTRODUCTION TO THE TAXA

76

Argyreia nervosa

y = 0.0226x - 0.0198

R2 = 0.0568

y = 0.0226x - 0.0198

R2 = 0.0568

y = 0.0226x - 0.0198

R2 = 0.0568

y = 0.0226x - 0.0198

R2 = 0.0568

y = 0.0226x - 0.0198

R2 = 0.0568

y = 0.0226x - 0.0198

R2 = 0.0568

y = 0.0226x - 0.0198

R2 = 0.0568

y = 0.0226x - 0.0198

R2 = 0.0568

y = 0.0226x - 0.0198

R2 = 0.0568

y = 0.0226x - 0.0198

R2 = 0.0568

y = 0.0226x - 0.0198

R2 = 0.0568

y = 0.0226x - 0.0198

R2 = 0.0568

y = 0.0226x - 0.0198

R2 = 0.0568

y = 0.0226x - 0.0198

R2 = 0.0568

y = 0.0226x - 0.0198

R2 = 0.0568

y = 0.0226x - 0.0198

R2 = 0.0568

y = 0.011x - 0.1565

R2 = 0.1869

y = 0.011x - 0.1565

R2 = 0.1869

0

2

4

6

8

10

1926

1928

1930

1932

1934

1936

1938

1940

1942

1944

1946

1948

1950

1952

1954

1956

1958

1960

1962

1964

1966

1968

1970

1972

1974

1976

1978

1980

1982

1984

1986

1988

1990

1992

1994

1996

1998

2000

2002

2004

2006

Nu

mb

er

of

pap

ers

per

year

& t

ren

dlin

e

0

20

40

60

80

100

Pro

po

rtio

nal m

icro

in

dex &

tre

nd

lin

e.

(2009b).

Tissue culture work done on Argyreia nervosa:

Tissue culture work done on this plant by Dobberstein and

Staba (1968). General indole alkaloid was reported in their

investigation.

Popularity of Argyreia nervosa:

Argyreia nervosa has been very popular. Australian New Crops

Web Site has plotted graph of total papers mentioning Argyreia

nervosa per year from (1926-2006).

Popularity of Argyreia nervosa over time (Webmaster, Australian New Crops

Website)

[Plots of numbers of papers mentioning Argyreia nervosa (filled

column histogram and left hand axis scale) and line of best fit, 1926

OBJECTIVES AND INTRODUCTION TO THE TAXA

77

to 2006 (complete line, with equation and % variation accounted for,

in box on the left hand side); Plots of a proportional micro index,

derived from numbers of papers mentioning Argyreia nervosa as a

proportion (scaled by multiplying by one million) of the total number

of papers published for that year (broken line frequency polygon and

right hand scale) and line of best fit, 1926 to 2006 (broken line, with

equation and % variation accounted for, in broken line box on the

right hand side)]

Total Mentions (Biological Abstracts/Biosis Previews):

REFERENCES:

Harvey S E, Cumpston K L and Benson B E (2006): Serotonin toxicity

from the combination of Hawaiian Baby Woodrose and pro-

serotonergic pharmaceuticals. Clinical Toxicology 44. Contact: Harvey,

S. E.; New Mexico Poison and Drug Informat Ctr, Albuquerque, NM

USA.

Reddy K N and Subbaraju G V (2005): Ethnomedicine from

Maredumilli region of East Godavari District, Andhra Pradesh. Journal

of Economic and Taxonomic Botany 29, 476-81. Contact: Reddy, K.

N.; Laila Impex R and D Ctr, Taxon Div, Unit 1, Phase 3, Jawahar

Autonagar 520007, Vijayawada, India.

Bhatt D C, Patel N K, Mitaliya K D and Ant H M (2003): Herbal magic

by contact therapy among tribals and rurals of Gujarat. Journal of

Economic and Taxonomic Botany 27, 205-7. Contact: Bhatt, D. C.;

OBJECTIVES AND INTRODUCTION TO THE TAXA

78

Mitaliya, K. D.; Department of Marine Sciences, Bhavnagar University,

Bhavnagar, Gujarat, 364 002, India.

Borsutzky M, Passie T, Paetzold W, Emrich H M and Schneider U

(2002): Hawaiian baby woodrose: (Psycho-) Pharmacological effects of

the seeds of Argyreia nervosa. A case-orientated demonstration.

Nervenarzt 73, 892-6. Contact: Borsutzky, M.; Abteilung Klinische

Psychiatrie und Psychotherapie, Medizinische Hochschule Hannover,

30625, Hannover, Germany.

Bogenschutz M P (2001): Drugs on the internet. American Journal of

Psychiatry 158, 2094-5. Contact: Bogenschutz, Michael P.;

Albuquerque, NM, USA.

Halpern J H, Pope H G and Jr (2001): Drugs on the internet: Drs.

Halpern and Pope reply. American Journal of Psychiatry 158. Contact:

Halpern, John H. ; Pope, Harrison G., Jr.; Belmont, MA, USA.

Gupta V (2000): Structural changes in seed coat morphology during

dormancy breaking in some medicinal plants. Journal of Medicinal

and Aromatic Plant Sciences 22-23, 672-3. Contact: Gupta, Veena;

National Bureau of Plant Genetic Resources, Pusa Campus, New Delhi,

110 012, India.

Mann P, Tofern B, Kaloga M and Eich E (1999): Flavonoid sulfates

from the Convolvulaceae. Phytochemistry (Oxford) 50, 267-71.

Contact: Eich, Eckart; Inst. Pharm. II, Freie Univ. Berlin, Koenigin-Luise-

Strasse 2+4, 14195 Berlin, Germany.

OBJECTIVES AND INTRODUCTION TO THE TAXA

79

Tofern B, Kaloga M, Witte L, Hartmann T and Eich E (1999):

Occurrence of loline alkaloids in Argyreia mollis (Convolvulaceae).

Phytochemistry (Oxford) 51, 1177-80. Contact: Eich, Eckart; Institute

fuer Pharmazie II (Pharmazeutische Biologie), Freie Universitaet Berlin,

Koenigin-Luise-Strasse 2+4, D-14195, Berlin, Germany.

Murugesan S, Kumar S and Sundararaj R (1997): Blister beetles as a

threat to medicinal/ornamental plants of arid and semi-arid regions.

Indian Forester 123, 341-4. Contact: Div. Forest Protection, Arid Forest

Res. Inst., Jodhpur, India.

Srivastava S K, Mehrotra B N and Palvi S K (1992): Distributional

notes on some plants in Arunachal Pradesh. Journal of Economic and

Taxonomic Botany 16, 709-13. Contact: Srivastava, S. K.; Bot. Survey

India, Andaman and Nicobar Circle, Port Blair 744 102, India.

Furbee R B, Curry S C and Kunkle D B (1991): Ingestion of Argyreia

Nervosa Hawaiian Baby Woodrose Seeds. Veterinary and Human

Toxicology 33. Contact: FURBEE R B; SAMARITAN REGIONAL POISON

CENTER, PHOENIX, ARIZ 85006, USA.

Daniel M (1989): Polyphenols of Some Indian Vegetables. Current

Science (Bangalore) 58, 1332-4. Contact: DANIEL M; DEP BOTANY,

FACULTY SCIENCE, BARODA 390 002, INDIA.

OBJECTIVES AND INTRODUCTION TO THE TAXA

80

Nair G G, Daniel M and Sabnis S D (1988): Chemosystematics of

Convolvulaceae. Geobios (Jodhpur) 15, 241-4. Contact: NAIR G G;

PHYTOCHEM LAB, DEP OF BOTANY, MS UNIV OF BARODA, BARODA,

390 002, INDIA.

Srivastava R C (1983): A Taxonomic Study of the Genus Argyreia

Convolvulaceae in Madhya Pradesh India. Proceedings of the National

Academy of Sciences India Section B (Biological Sciences) 53, 37-42.

Contact: SRIVASTAVA R C; BOTANICAL SURVEY OF INDIA, CENTRAL

CIRCLE, ALLAHABAD-211 002, INDIA.

Nanda B K (1980): Histological Responses of Argyreia-Nervosa

Isolated Leaves to Hormones Applied across a Transverse Gradient.

Plant and Cell Physiology 21, 1133-42. Contact: NANDA B K; DEP

BOT, GM COLL, SAMBALPUR-768004, INDIA.

Pant D D and Bhatnagar S (1975): Morphological Studies in Argyreia

Convolvulaceae. Botanical Journal of the Linnean Society 70, 45-70.

Chao J M and Der Marderosian A H (1973): Ergoline Alkaloidal

Constituents of Hawaiian Baby Wood Rose Argyreia-Nervosa. Journal

of Pharmaceutical Sciences 62, 588-91.

Chao J M and Dermarderosian A H (1973): Identification of Ergoline

Alkaloids in the Genus Argyreia and Related Genera and Their Chemo

Taxonomic Implications in the Convolvulaceae. Phytochemistry (Oxford)

12, 2435-40.

OBJECTIVES AND INTRODUCTION TO THE TAXA

81

Vogel W H, Carapellotti R A, Evans B D and Der Marderosian A

(1972): Physiological Disposition of Isoergine from Argyreia-Nervosa-D

Convolvulaceae-D and Its Effect on the Conditioned Avoidance

Response in Rats. Psychopharmacologia 24, 238-42.

Crawford K W (1970): The Identification of Lysergic-Acid Amide in

Baby Hawaiian Woodrose-D by Mass Spectrometry. Journal of

Forensic Sciences 15, 588-94.

Der Marderosian A H and Chao J (1970): The Indole Alkaloid

Constituents of Argyreia-Nervosa-D Convolvulaceae-D. Lloydia

(Cincinnati) 33.

Miller M D (1970): Isolation and Identification of Lysergic-Acid Amide

and Iso Lysergic-Acid Amide as the Principal Ergoline Alkaloids in

Argyreia-Nervosa-D a Tropical Wood-Rose-D. Journal of the

Association of Official Analytical Chemists 53, 123-7.

Dobberstein R H and Staba E J (1969): Ipomoea-Violacea-D Rivea-

Corymbosa-D and Argyreia-Nervosa-D Tissue Cultures Influence of

Various Chemical Factors on Indole Alkaloid Production and Growth.

Lloydia (Cincinnati) 32, 141-7.

Hylin J W and Watson D P (1965): Ergoline alkaloids in tropical wood

roses. Science 148, 499-500. Contact: Hawaii Agr. Exp. Sta., Univ.

Hawaii, Honolulu, Hawaii, USA.

CHAPTER 1-B

OBJECTIVES AND INTRODUCTION

OF THE PLANT

OBJECTIVES AND INTRODUCTION TO THE TAXA

51

OBJECTIVES AND INTRODUCTION OF THE

PLANT

The present study was undertaken with taxa Argyreia nervosa

(Burm. f.) Bojer (Argyreia speciosa (L. f.) Sweet, medicinal plant with

high medicinal values.

The study was conducted with the following objectives:

A. Plant tissue culture

1. To standardize protocol for callus induction of different part

of the plant for large scale cell production and production

of secondary metabolites.

2. To standardize protocol for micropropagation of the taxa for

large scale propagation and conservation of the plant. To

standardize protocol for shoot induction of different part of

the plant.

3. To standardize protocol for cell suspension culture of

different part of the plant for large scale cell production

and production of secondary metabolites.

B. To study Pharmacognostic characters of plant: Macroscopical

and Microscopical (anatomy).

1. Leaf

a. T.S. of leaf with lamina

b. T.S. of petiole

OBJECTIVES AND INTRODUCTION TO THE TAXA

52

2. Stem

a. T.S. of Stem (primary growth)

b. T.S. of Stem (secondary growth)

3. Root

a. T.S. of Root (primary growth)

b. T.S. of Root (secondary growth)

4. Nodal anatomy

C. Qualitative and Quantitative analysis

1. Comparison of the phytochemical profile of the cultured

cell of the plant with the field grown plants by process

of High performance thin layer chromatography (HPTLC)

for alkaloids, Flavonoids, Tannin and Glycoside.

2. Qualitative analysis of secondary metabolites: Alkaloids,

Flavonoids, Tannins and Glycoside.

3. Protein, carbohydrate and lipid estimation(Quantitative) of

plant

4. Quantitative estimation of secondary metabolites:

Phenols, Tannins and Flavonoids.

D. Gas Chromatography – Mass Spectrometry/ NMR of plant

extract to identify active compounds of the plant

E. Antibacterial activity

a. Aqueous extract of leaf, stem and root

b. Methanolic extracts of leaf, stem and root

OBJECTIVES AND INTRODUCTION TO THE TAXA

53

F. Antifungal activity

a. Aqueous extract of leaf, stem and root

b. Methanolic extracts of leaf, stem and root

G. Genetic stability by Random Amplified Polymorphic DNA: To

know genetic variation between original plant and cultured cells

1. Screening the suitable primers for development of

diversity markers in Argyreia

2. To analyze the genetic diversity among the different

parts of cultured cells and original plant

3. Identification of DNA markers for best genotypes

OBJECTIVES AND INTRODUCTION TO THE TAXA

54

INTRODUCTION OF FAMILY

Convolvulaceae

Convolvulaceae is a large family, comprising approximately 50–

60 genera with some 1600–1700 species (Mabberley, 1987), exhibiting

a rich diversity of morphological characteristics and occupying a

broad range of ecological habitats. More than one-third of the species

are included in two major genera, Ipomoea and Convolvulus

(Cronquist, 1988). Convolvulaceae are distributed throughout the

world, but are primarily tropical, with many genera endemic to

individual continents. Although the family is best known in temperate

regions for its weedy representatives (e.g., Calystegia, Convolvulus),

many tropical species are valuable ornamentals, medicinal and food

crops. The sweet potato, Ipomoea batatas, is the world's second most

important root crop (>128 x 109 kg/yr; Simpson and Ogorzaly, 1995).

The record of microfossils attributed to the family is known as far

back as the Eocene ( 40–45 million years ago [mya]), but without

accompanying macrofossils.

Typical members of the family are annual or perennial vines,

with milky sap, internal (intraxylary) phloem, Leaves alternate, entire,

simple to lobed or pinnately divided to pectinate, exstipulate.

Inflorescence determinate, cymose, or flowers solitary, axillary, with

OBJECTIVES AND INTRODUCTION TO THE TAXA

55

jointed peduncles. Flowers actinomorphic, perfect, hypogynous, often

large and showy, ephemeral, usually with intrastaminal disc, generally

subtended by a pair of bracts (sometimes enlarged and forming an

involucre). Calyx of 5 sepals, distinct or sometimes basally connate,

sometimes unequal, imbricate, persistent. Corolla sympetalous, entire

to slightly 5-lobed, funnel form or salverform, plicate, brightly colored

(commonly red, violet, blue, or white), indup-licate-valvate and/or

convolute (twisted) in bud. Androecium of 5 stamens, epipetalous at

corolla base; filaments distinct, often unequal; anthers dorsifixed,

dehiscing longitudinally, usually introrse. Gynoecium of 1 pistil, 2-

carpellate; ovary superior, 2-locular or sometimes appearing 4-locular

due to false septa, sometimes with dense covering of hairs; ovules 2

in each locule, anatropous, sessile, placentation basal or basalaxile;

style simple and filiform or forked; stigma(s) 1 or 2, linear, lobed or

capitate. Fruit usually a 4-valved septifragal capsule; seeds smooth or

hairy; endosperm scanty, hard, cartilaginous; embryo large, straight or

curved, with folded or coiled, emarginated to bifid cotyledons,

surrounded by endosperm.

Distribution:

Primarily in the tropics and subtropics, with representatives

having ranges extending into north and south temperate regions;

particularly abundant in tropical America and tropical Asia. Major

genera: Ipomoea (500 spp.), Convolvulus (250 spp.), Cuscuta (145—

170 spp.), and Jacquemontia (120spp.).

The Convolvulaceae have been divided into three or four

subfamilies (sometimes segregated as distinct families) and/or three

to ten tribes. Although the relationships between these groups have

OBJECTIVES AND INTRODUCTION TO THE TAXA

56

been generally agreed upon, the taxonomic rank (family, subfamily, or

tribe) is a matter of controversy (Wilson, 1960). A notable segregate

group, the Cuscutoideae or Cuscutaceae (a monotypic taxon), has

been separated from the rest of the Convolvulaceae by some

botanists on the basis of the parasitic habit with related

specializations of the corolla and embryo (Momin, 1977).

Authors also disagree on the delimitation of the various genera within

the family, such as Ipomoea (Sengupta, 1972). The generic lines

depend upon characters of the bracts, sepals, corolla, pollen,

stigma(s), and fruit. For example, the sepals vary in size, shape, and

pubescence and the stigmas may be simple, lobed, or globose. In

addition, seed characters (e.g., type of pubescence) are important for

species delimitation.

Morning-glories are easy to spot in the field with their twining

habit and generally large, white or brightly colored, and funnel-shaped

corolla. The corollas are twisted clockwise in bud and strongly plicate

(Allard 1947). Usually a flower is open for only one day (for a few

hours); the corolla then incurves as it wilts. The corolla is

characteristically divided longitudinally by five obvious demarcations

that occur along the middle of the five lobes of the limb. These

markings taper toward the apex and usually twist in the clockwise

direction.

OBJECTIVES AND INTRODUCTION TO THE TAXA

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INTRODUCTION OF PLANT

Classification: (Bentham and Hooker)

Kingdom Plant

Class Dicotyledones

Sub-class Gamopetalae

Series Bicarpellatae

Order Polemoniales

Family Convolvulaceae

Genus Argyreia

Species nervosa (Burm. f.) Bojer

OBJECTIVES AND INTRODUCTION TO THE TAXA

58

Other Latin names:

Argyreia speciosa (L. f.) Sweet

Convolvulus speciosus L.f.

Convolvulus nervosus Burm. f.

Vernacular Names:

Hindi: Samandar-ka-pat, Samudrasos, Samudra Shokha

Gujarati: Vardharo, Gha-vel, Chandpan

Kannada: Candrapada

Malayalam: Marikkunni, Marututari, samudrappacca

Sanskrit: Vrddhadarukah, Bastantri

Tamil: Samuttirappaccai, Samuttirappalai Kadarpalai

Telugu: Candrapada

Bengali: Bijarka

Nepalese: Samudra phool

Sinhalese: Vriddadaru

Unani: Samudar sokh

Indonesia: Areuy bohol keboh (Sundanese)

Philippines: Sedang-dahon (Tagalog)

OBJECTIVES AND INTRODUCTION TO THE TAXA

59

Thailand: Bai rabaat, Phak rabaat (central), Mueang mam (Bangkok)

English: Elephant creeper, elephant climber, elephant vine, Hawaiian

baby woodrose, silver morning glory, wood rose, woolly morning glory

French: Coup d'air, liane à minguet, liane d'argent, liane d'argentne à

minguet (Lavergne Christophe, 2006).

Mention of Argyreia nervosa in Veda: (Vaidyaratnam, 2005)

[“Bastantri visagandha vayojaradarika chagalantri

Visapatrikantravasta paryayairvrddhadarukam bhavati” (A.ma.)]

[“Vrddhadaruka avegi jungako dirghavalukah

Vrddhah kotarapuspi syadajantri chagalantryapi”(Dha.ni.)]

[“Vrddhadaruka avegi jongako jinabalakah

Antah kotarapuspi syat syama mahisavallari

Ajantri tu mahasyama vallari dirghabalakah” (Kai.ni.)]

OBJECTIVES AND INTRODUCTION TO THE TAXA

60

[“Vrddhadaruh kasayosnah katustikto rasayanah

Vrsyo vatamavatarsahsophamehakaphapranut

Suklayurbalamedhagnisvarakantikarah sarah" (Bha.pra.)]

[“Vrddhadaruh katustiktastathosnah kaphavatajit

Svayathukrmimehasravatodaraharah parah" (Dha.ni)]

[“Vrddhadaruh katustiktah kasayosno rasayanah

Suklayurbalamedhyagnisvarakantikarah sarah

Sophamavatavatasravatamehakaphapahah” (Kai. ni.)]

[“Sadharano vrddhadaruh katustiktah kasayakah

Rasiiyanosno madhuro medhyah svaryah sarognidah

Kantidhatukaro balyo rucyah pustikaro laghuh

Upadamasam panurogam kasayam kasam pramehakam

OBJECTIVES AND INTRODUCTION TO THE TAXA

61

Vataraktam camavatam. vatam sopham kapham jayet” (Ni.ra.)]

[“Vrddhadaro grahonmadapapalaksmi vinasanah

Apasmaramavataghnah sophasulapahognikrt

Balyah kanthyosthisamdhanakari vatarujapahah

Visucipratitunyadivyadhighati rasayanam” (So.ni.)]

[“Sramsini gulmahrdrogavisarocakanasini

Bastantri kapharogaghni mutrakrcchravinasini” (Ma.ni.)]

[“Vrddhadaruh kasayosah sarastikto rasayanam

Vrsyo vatamavatasrasophamehakaphan jayet” (Ma.vi.)]

Remarks: ‘Bastantrt’ of Syamadigana (Astangahrdayam) and

vrddhadaru of ‘Maharasnadikasayam’ (Sahasrayogam) are interpreted

as marikkunni in Malayalam by most of the commentators.

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62

In the ‘Arunodaya’ commentary on Astangahrdayam the

Malayalam name marututari is given for bastantri. In the

‘Osadhinighantu’ of Kumaran Krishnan, the Sanskrit-Malayalam

dictionary of Kanippayoor and ‘Ayurvedavisvakosam’ of Pandit K.K.

Panickar, chagalantri, vrddhadaruka. and vrddadaru are translated into

Malayalam as marikkunni. Bastantri is marututari or marukutari

according to ‘Ayurvedavisvakosam’. But there is no mention of this

word bastantri in ‘Osadhinighantu’ or Sanskrit-Malayalam dictionary.

Marikkunni and marututari are treated as two distinct raw drugs in

‘Osadhinighatu’ and ‘Ayurvedavisvakosam’. In the ‘Ayurvedic Formulary

of India’ also bastantri and vrddhadaru are treated as two distinct

ones, giving the Latin name as Argyreia speciosa and Ipomoea

petaloidea, respectively. In the ‘Glossary of Vegetable Drugs in

Brhaltrayi, Ipomoea pes-caprae is the Latin name given for chagalntri

and bastantri is treated as a synonym of chagaltintri. But in the

‘Pharmacognosy of Ayurvedic Drugs’ both Argyreia speciosa and

Ipomoea pes-caprae are regarded as vrddhadaruka, giving the

Malayalam names samudrapacca for Argyreia speciosa and aqampu

or cuvanna aqampu for Ipomoea pes-caprae. Dr S.N. Nesamani

considers Argyreia speciosa as samudrapacca in his book

‘Ausadhasasyannal.’ The commentators of ‘Bhavaprakasam’,

‘Kaiyadevanighantu’ etc., are of the opinion that the Latin name of

vrddhadaru is Argyreia speciosa.

As chagalantri is a synonym of bastantri and as marikkunni is

the Malayalam name given for chagalantri it amounts to regard

bastantri as marikkunni itself. Hence, the Malayalam name marikkunni

is applicable to bastantri as well as vrddhadaru.

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63

It is strange to note that all the Sanskrit synonyms of

vrddhadaru are given to Rourea santaloides W. & A. also, in ‘Indian

Medicinal Plants’. Being Highly poisonous is not advisable to use as

vrddhadaru.

Thus, it is clear that in Kerala Argyreia nervosa (A. speciosa) is

being used for marututari, marikkunni and samudrapacca.

Morphology:

Habitat/ecology:

A. nervosa is native to India, from Assam to Belgaum and

Mysore. It is common on the Bengal plain (Hooker, 1885). Specimens

have been recorded from Java, China and Mauritius, although it is

unclear whether these latter locations have included cultivated

specimens. This species exists in south and north-west India, as well

as Bengal (Stewart and Brandis 1874). A. nervosa is cultivated on the

Malay Peninsula (Hoogland 1952). A. nervosa is ‘originally in British

India, from Assam and Bengal to Belgaum and Mysore, cultivated in

other tropical countries; occasionally escaped from culture’

(VanOostroom 1943). A. nervosa has been erroneously listed as

native to Australia. The Queensland Herbarium has confirmed that it

considers that A. nervosa is a naturalized species in Australia

(Batianoff, pers. comm.). While most references state that A. nervosa

is native to India, Hawaiian and Polynesian people are reported to

have used this species as a drug over hundreds of years

(Anonymous, 2004a) and it could be speculated that other Indigenous

communities in Australia and elsewhere in the Pacific have used and

transported seeds from this plant for some time.

OBJECTIVES AND INTRODUCTION TO THE TAXA

64

Hawaiian Baby Woodrose’s been theorized that it was

introduced in Hawaii very early on and thrived in the tropical climate,

thus leading to Hawaii becoming known as its latter day “home” and

popular namesake. It is less popularly known as the Silver Morning

Glory (stemming from its origin in the Convolvulaceae Morning Glory

family), and the Monkey Rose, among other folk names. The plant is

also part of the indigenous flora of Australia and has been known to

grow wild in Africa. It is popular as an ornamental plant, as well as

an enthogenic intoxicant and legal inebriant, although the ingestion of

this plant in many parts of the world is now illegal, including the

United States.

A. nervosa prefers tropical and sub-tropical climates. References

that discuss the cultivation of A. nervosa state that the plant prefers

fertile, moist soil in a protected sunny position (Ellison, 1995).

Anecdotal information from Queensland National Parks and Wildlife

staff suggests that the plant can germinate quite readily in seemingly

undisturbed sites, including under rainforest canopies and among

dense grass cover in eucalypt woodland (P Williams, pers comm.

2004).

It is found in India throughout, up to an altitude of 300 meters

high, except in dry, western regions up to 1000 ft elevation, often

cultivated. The beautiful, woody, flowering trellis vine that is Hawaiian

Baby Woodrose flourishes in direct sunlight, in areas that promote

hot, humid climates. This plant has been used as a folk remedy in

India, and is valued for its aesthetics. It grows well in Hawaii,

California, Florida, and similar climates.

Description:

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"Clambering vine to several meters long; herbage velvety

pubescent, densely when young. It is a very large woody climber (Fig.

No. 1). Leaf blades 15-25 (30) cm long, 13-20 (30) cm wide, cordate,

acuminate-attenuate apically, cordate basally. The leaves are glabrous

above; persistently white tomentose beneath, petiole is long.; flowers

in cymes, on long, white-tomentose peduncles; sepals 13-15 mm long,

velvety like the herbage; pedicels to 15 cm long; flowers 5-7.5 cm

long, the corolla with a short tube and campanulate limb, lavender to

pink, the throat darker" (Welsh, 1998). Flowers trumpet-shaped (Fig.

No 2). Seeds are enclosed in a stone, pale yellow-brown globose,

apiculate, indehiscent berry 1.2 to 2 cm in diameter containing four

erect, curved embryos with corrugated cotyledons or two seeds

embedded in a meaty pulp (Fig. No.3.1). Seed pods dry into woody

"rosebuds," each one containing three to four seeds (Fig. No.3.2; Fig.

No.4). The seeds are known to be rich in psychoactive ergot alkaloids

and contain a naturally occurring tryptamine called LSA (Lysergic Acid

Amide). Root cylindrical, 1 to 1.5 cm thick; brown, smooth, round

wood is scant, flexible, and smooth, latex oozes at cuts. This vine

produces beautiful flowers and seeds of historic significance.

This plant is a rare example of a plant whose hallucinogenic

properties have only recently been discovered by non-Hawaiians.

While its cousins in the Convolvulaceae family, such as the Rivea

corymbosa (Ololiuhqui) and Ipomoea violacea (Morning Glory), were

used in shamanic rituals of Latin America for centuries, the Hawaiian

Baby Woodrose was not traditionally recognized as a hallucinogen. Its

properties were first brought to attention in the 1960s, despite the

fact that the chemical composition of its seeds is nearly identical to

those of the two species mentioned above, and the seeds contain

OBJECTIVES AND INTRODUCTION TO THE TAXA

66

the highest concentration of psychoactive compounds in the entire

family.

Seeds from Ghana are typically smooth and very light in

color. Seeds from the Ayurvedic strain from India are typically more

"pointed" and larger than most other Hawaiian Baby Woodrose

seeds. Ones from India typically have less LSA content, but look

roughly the same as the coveted strain from Hawaii, but don't have

much "fuzz" on them, and they are also typically slightly smaller in

size.

Propagation:

Berries dispersed by frugivorous birds. It may be propagated by

cuttings or seeds and in the spring by division. The seed may be

sprouted by making a small nick in the seed coat away from the

germ eye. Soak the seed until it swells. Plant 0.5 inch deep in loose

rich soil. After the cotyledons appear, water sparingly, letting the soil

surface dry out to a depth of 0.5 inch. Over-watering causes stem

and root rot. The plant grows slowly until it develops a half-dozen

leaves; after this it grows quickly. The next spring it will grow into a

very large vine and should produce flowers and seeds. The plant can

start growing flowers as early as its life cycle's second year. In India,

growing seasons are often accelerated, so one can often get seeds

within 18 months. For this to occur, there must be sufficient watering

and adequate room for the roots to grow; it can take up to 5 years

for the first signs of flowering to become visible.

The seeds will be found in the pods of the dried flowers.

These cannot be harvested until completely dried.

OBJECTIVES AND INTRODUCTION TO THE TAXA

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Part Used:

Leaf, Stem, Root Seeds, Wood

Mythology:

The Hawaiian Baby Woodrose is a popular candidate for the

mythological Soma plant, of which the definitive botanical identity is

unknown today. “Soma” is the liquid potion derived from the Soma

plant, and the earthly counterpart of Ambrosia, the ancient, mythical

drink of immortality reserved for the gods. The name soma was

bestowed upon a deity (the moon; the god of plants), the plant itself

and the sacrificial drink that was prepared from the plant.

In the Hindu tradition, the moon (originally called soma), was

believed to be the ambrosia-filled drinking vessel of the gods. When

the moon was full, the vessel was full; by the time the new moon

appeared, the drinking vessel had been emptied. It filled up again as

the moon waxed.

To prepare this mystical concoction, the stems of the soma

plant were pressed to release the sap. The resulting juice, which was

believed to “dissolve all sins,” was then mixed with water and offered

to Indra, the Hindu God of Thunder. This soma ritual is thought to

have served as the prototype for the kava ceremony of the South

Pacific. It has also been widely speculated that the identity of the

Soma plant is actually Amanita muscaria.

It has been noted in various oral histories that the Huna

religion, the healing and spiritual shamanism of ancient Hawaii,

employed the seeds of the Hawaiian Baby Woodrose for their

OBJECTIVES AND INTRODUCTION TO THE TAXA

68

Shamanic rituals, using the seeds’ enthogenic and magical properties

to connect with the spirit world. The Huna Shaman prepared the

Hawaiian Baby Woodrose seeds by grinding them into a powder and

mixing the powder directly with water, resulting in a supernatural

libation that was then drunk by the Huna Shaman. This magical tonic

was used as a means for the Shaman to pass from this worldly

plane into the realm of the spirits.

Hawaiian Baby Woodrose seeds are perhaps one of the least

understood of modern-day entheogens and exotic botanicals. There

is much controversy in regards to its true place in Shamanic and

traditional history outside of its native culture and home; India.

Ayurvedic description: (Anonymous, 2004b)

Properties:

Rasa: Kuru, tikta, kasaya; Guna: Laghu, snigdha, sara; Virya: Usna.

Action: Vatakaphahara, sukravardhaka, vrsya, balya, rasayana. Medhya,

swarakantikara.

Therapeutic uses: (Anonymous, 2004b)

Klibato, daurbalya, amavata, vatarsa. Sotha.

Uses:

The leaves are antiphlogistic; they are applied over skin

diseases and wounds; the silky side of the leaf is applied over

tumors, boils, sores, and carbuncles; as an irritant to promote

maturation and suppuration. The leaves are also used for extracting

OBJECTIVES AND INTRODUCTION TO THE TAXA

69

guinea worms. A drop of the leaf juice is used in otitis. Dried leaves

are used for diabetes (Jain and Sharma, 1967). Traditionally leaves

used by Rajasthani tribes to prevent conception (Anonymous, 1988;

Nisteswar, 1988).

The roots are acrid, bitter, astringent, sweet, and emollient. It is

used in vitiated conditions kapha and vita, emaciation, wounds,

ulcers, anorexia, dyspepsia, flatulence. Roots are used in the

treatment of gonorrhea, rheumatism and diseases of nervous system.

It is also used in obesity, hoarseness, syphilis, anemia, diabetes,

tuberculosis and general debility. It is also used as a tonic (Prajapati

et al., 2003).

Powder of the root is given with "ghee" as an alternative; in

elephantiasis the powder is given with rice water. In inflammation of

the joints it is given with milk and a little castor oil. A paste of the

roots made with rice water is applied over rheumatic swelling and

rubbed over the body to reduce obesity.

LSD is the best-known synthetic hallucinogen and is

psychoactive at the microgram level. Although LSD does not occur in

nature, a close analogue, lysergic acid amide (LSA, ‘‘ergine’’) is found

in the seeds of Argyreia nervosa (Hawaiian baby woodrose) and

Ipomoea violacea (morning glory). Hallucinogenic activity of LSA

occurs with 2–5 mg, which provides a 4- to 8-hr intoxication that

reportedly has quantitative as well as qualitative differences from LSD

(Schultes and Hofmann, 1980). Seeds are crushed, germinated, eaten

whole, or an extract is drunk after the seeds are soaked in water.

Five to 10 seeds of Argyreia nervosa or 150–200 seeds (3–6 g) of

Ipomoea violacea yield average doses of LSA (Al Assmar, 1999;

OBJECTIVES AND INTRODUCTION TO THE TAXA

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Borsutzky et al., 2002). The LSA content of Argyreia nervosa is 0.14%

of the dry weight of the seeds (Chao and Der Marderosian, 1973).

They are widely regarded as hallucinogens in today’s Australian

drug scene. The seeds and preparations made from them are utilized

and celebrated in Sex Magick rituals in certain underground

subcultures such as those associated with Aleister Crowley, the British

occultist.

The seeds are also used in a preparation known as Utopian

bliss balls, which consist of five Argyreia seeds, damiana herbage,

ginseng root, fo-ti-teng and bee pollen, and were very popular in the

sixties among the hippies and artists in California.

Suffice it to say that despite any controversy, there is enough

documented and anecdotal evidence to support the enthogenic

properties of the Hawaiian Baby Woodrose seeds. HBWR Seeds is

illegal in many parts of the world.

Three seeds considered sufficient to produce an LSD-like

experience, with psychonauts reporting colourful visions of a spiritual

nature, psychedelic patterns, all-over body sensations, a sense of

extreme relaxation, euphoria and deep spiritual awareness. Four to six

seeds are a standard dose, and there are reports of strong

hallucinogenic side effects after ingestion of 12 to 16 seeds. This

also depends on the age of the seeds, as some of the psychoactive

compounds found in Hawaiian Baby Woodrose seeds can break down

in as little to 6 to 9 months. The experience duration can range from

4 to 12 hours, with mild effects occasionally lasting about a day and

is usually accompanied with gastric discomfort, including severe

OBJECTIVES AND INTRODUCTION TO THE TAXA

71

nausea and flatulence. Other after effects of these seeds includes

sensitivity to light, and impaired motor skills. The removal of the

outer coating of the seeds is often recommended to reduce such

effects, although there appears to be no consensus as to this

practices efficacy and it has been suggested that it may not be true.

Ergot alkaloids, including LSD and LSA, can cause uterine

contractions, which may lead to miscarriage if the seeds are

consumed while pregnant. Contrary to popular belief, the seed's outer

coat does not contain cyanide or glycosides. The nausea associated

with consuming the seeds is mostly because of essential oils in the

seed itself.

The psychedelic properties of the seeds became known mainly

through their use in Hawaii, Haiti and Puerto Rico, where

impoverished members of the population would consume the seeds,

seeking a "cheap buzz" as an alternative to alcohol. A sample made

its way to Albert Hofmann, the creator of LSD, who confirmed the

effects and analyzed its chemical composition. It is still used by

some Hawaiians for a high.

Hawaiian Baby Woodrose seeds were traditionally used in

sacramental rituals of the Hawaiian and Polynesian islands. Traditional

use of the plant in India usually employed the leaves and roots of

the plants, which are not psychoactive, as antiseptic and anti-

inflammatory drugs.

The whole plant is reported to have antiseptic properties.

Principal Constituents:

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72

It contains many ergoline alkaloids. Argyreia nervosa seeds

contain 0.3-1 % ergot-alkaloids by weight. Ergine (d-lysergic acid

amide) (Figure No.: 5.1), isoergine (l-lysergic acid amide) (Figure No.:

5.2), ergometrine, lysergol (Figure No.: 5.4), isolysergol and

chanoclavine (Figure No.: 5.5) are present. 6, 7 Lysergol and

elymoclavine (Figure No.: 5.3) are reduction products of d-lysergic

acid. LSD which is a lysergic acid amide and although resembling the

natural lysergic acid amides both in structure and pharmacological

activity, it has not been found in nature. Except alkaloids many

secondary metabolites are also present in the plant. The plant

contains tannin and amber-coloured resin, soluble in ether, benzole;

partly soluble in alkalis; and fatty oil. Chemical analysis revealed the

presence of triterpenoids, flavonoids, sterol; saponins are also present

in the plant (Shrivastav and Shukla, 1998). It contains Argyroside, a

recently reported steroidal glycoside, (24R)-ergost-5-en-11-oxo-3beta-

ol-alpha-D-glucopyranoside (Rahman and Khan, 2003). A few of the

ergoline alkaloids reported in this plant are hallucinogenic. LSA (d-

lysergic acid amide) is Schedule III in the United States.

The petroleum ether extract of the leaves yielded 1-tricontanol,

epifriedelinol acetate, epifriedelinol and β-sitosterol (Sahu and

Chakravarti, 1971). The leaves were found rich in quercetin (Daniel,

1989). Extraction of the leaves with 90% methanol led to the

isolation of the flavonoids, quercetin and kaemperol together with the

latter’s glycoside kaemperol-3-o-l-rhamnopyranoside (Khan et al.,

1992). Two new flavone glycosides characterized as 7,8,3’,4’,5’-

pentahydroxyflavone5-o-α-lrhamnopyranoside and 7,8,3’,4’,5’-

pentahydroxyflavone5-o-α-l-glucopyranoside were also reported from

leaves (Ahmad et al., 1993).

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The hexane extract of the root yielded tetradecanyl palminate,

5,8-oxidotetracosan-10-one (Rani and Shukla, 1997) and two novel

aryl esters characterized as stigmasteryl p-hydroxycinnamate and

hexadecanyl phydroxycinnmate along with scopoletin (Shrivastava and

Shukla, 1998).

The seeds yielded fatty oil which found to contain the

glycerides of palminate, stearic, linoleic, linolenic and oleic acids

(Biswas et al., 1947; Batra and Mehta 1985). In another study, the

seed oil revealed the presence of myristoleic, myristic, palmitic,

linoleic, linolenic, oleic, stearic, nonadecanoic, eicosenoic,eicosanoic,

heneicosaoic and behenic acids identified as their corresponding

methyl esters through GLC (Kelkar et al., 1947). The ethanolic extract

of the seeds revealed the presence of a mixture of three alkaloids,

out of which only one was characterized as ergometrin. The other

constituents isolated were caffeic acid and ethyl caffeate (Agrawal

and Rastogi, 1974a), another study also revealed the presence of

ergoline alkaloids in the seeds (Nair et al., 1987). The ergolines were

indicated to be of clavine type (Nair et al., 1987). The free amino

acids reported in the seeds were glutamic acid, glycine, isoleucine,

leu-cine, lysine, phenylalanine, tyrosine, praline and α-amino butyric

acid (Jaiswal et al., 1984).

The fruits were reported to contain n-tricontanol, β-sitosterol,

p-hydroxycinnamoyloctadecanolate and caffeic acid (Purushothaman

et al., 1982).

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Pharmacology

Ergine, isoergine, ergometrine, elymoclavine and lysergol are

responsible for the psychedelic effects. The structurally similarity

between these alkaloids and the neurotransmitters dopamine,

noradrenaline and serotonine might explain the hallucinogenic activity

by mutual influence on the active sites of the central nervous system;

it appears that the psychoactive constituents are partial agonists on

the G-protein-linked a-adrenergic- and on various serotonergic-

receptors (the serotonergic receptor-subtype 5-HT2A appears to be

involved in hallucinogenic activity).

Ascorbic acid (vitamin C) doesn't change the intensity of the

experience, but it alters its quality. One can concentrate better,

develops less paranoia and is also less tired at the end of the

experience. MAO-inhibitors and sympathomimetic amines

(amphetamine, ephedrine etc.) have positive synergistic effects; they

prolong and intensify the experience.

Hashish or marihuana can also intensify the experience. Usually

produces positive feeling. Tricyclic-antidepressants antagonize the

effects.

Pharmacognostic parameters for the leaves of Argyreia nervosa

Burm were studied with the aim of drawing the pharmacopeial

standards for this species. Macroscopical and microscopical

characters, physio-chemical constants, quantitative microscopy

parameters, extractive values with different solvents, fluorescence

analysis of dry powder, its reaction after treatment with chemical

reagents under visible light and UV light at 254 nm and 366 nm.

Preliminary phyto-chemical screening on the leaves Argyreia nervosa

OBJECTIVES AND INTRODUCTION TO THE TAXA

75

Burm was studied by Krishnaveni and Santh, 2009.

PHARMACOLOGICAL STUDIES:

Although a lot of pharmacological investigations have been

carried out based on the ingredients resents but a lot more can still

be explored, exploited and utilized. A summary of the findings of

Argyreia nervosa is presented here. Aphrodisiac activity of the plant

studied by Subramonium et al., 2007; Immunomodulatory activity of

the plant studied by Gokhle et al., 2003; Hepatoprotective activity of

the plant studied by Habbu et al., (2008a); Central nervous system

activity of the plant studied by Galani and Patel, 2009; Hypoglycemic

of the plant studied by Hemet et al., 2008; Nootropic of the plant

studied by Joshi et al. 2007; Anti inflammatory activity of the plant

studied by Srivastava et al., 1998; Anticonvulsant activity of the plant

studied by Vyawahare and Bodhankar, (2009a); Analgesic activity of

the plant studied by Bachhav et al., 2009; Antibacterial activity of the

plant studied by (Kelkar et al., 1947; George and Pandalai, 1949;

Mishra and Chaturvedi, 1978; Habbu et al., (2008b); Modi et al., 2010a).

Antifungal activity of the plant was studied by Shukla et al., 1999;

Antiviral activity the plant was studied Babber et al., 1978;

Nematicidal activity of the plant was studied by Parveen et al., 1990;

Anti-diarrhoeal activity of the palnt was studied by Rao et al., 2004;

Physiological disposition of isoergine (d-isolysergamide, iso-LA)

obtained from the seeds of Argyreia nervosa (Burm. F.) Bojer was

determined in rat liver, brain and plasma was studied by Vogel et al.,

1971; Effect of Argyreia speciosa extract on learning and memory

paradigms in mice was studied by Vyawahare and Bodhankar,

OBJECTIVES AND INTRODUCTION TO THE TAXA

76

Argyreia nervosa

y = 0.0226x - 0.0198

R2 = 0.0568

y = 0.0226x - 0.0198

R2 = 0.0568

y = 0.0226x - 0.0198

R2 = 0.0568

y = 0.0226x - 0.0198

R2 = 0.0568

y = 0.0226x - 0.0198

R2 = 0.0568

y = 0.0226x - 0.0198

R2 = 0.0568

y = 0.0226x - 0.0198

R2 = 0.0568

y = 0.0226x - 0.0198

R2 = 0.0568

y = 0.0226x - 0.0198

R2 = 0.0568

y = 0.0226x - 0.0198

R2 = 0.0568

y = 0.0226x - 0.0198

R2 = 0.0568

y = 0.0226x - 0.0198

R2 = 0.0568

y = 0.0226x - 0.0198

R2 = 0.0568

y = 0.0226x - 0.0198

R2 = 0.0568

y = 0.0226x - 0.0198

R2 = 0.0568

y = 0.0226x - 0.0198

R2 = 0.0568

y = 0.011x - 0.1565

R2 = 0.1869

y = 0.011x - 0.1565

R2 = 0.1869

0

2

4

6

8

10

1926

1928

1930

1932

1934

1936

1938

1940

1942

1944

1946

1948

1950

1952

1954

1956

1958

1960

1962

1964

1966

1968

1970

1972

1974

1976

1978

1980

1982

1984

1986

1988

1990

1992

1994

1996

1998

2000

2002

2004

2006

Nu

mb

er

of

pap

ers

per

year

& t

ren

dlin

e

0

20

40

60

80

100

Pro

po

rtio

nal m

icro

in

dex &

tre

nd

lin

e.

(2009b).

Tissue culture work done on Argyreia nervosa:

Tissue culture work done on this plant by Dobberstein and

Staba (1968). General indole alkaloid was reported in their

investigation.

Popularity of Argyreia nervosa:

Argyreia nervosa has been very popular. Australian New Crops

Web Site has plotted graph of total papers mentioning Argyreia

nervosa per year from (1926-2006).

Popularity of Argyreia nervosa over time (Webmaster, Australian New Crops

Website)

[Plots of numbers of papers mentioning Argyreia nervosa (filled

column histogram and left hand axis scale) and line of best fit, 1926

OBJECTIVES AND INTRODUCTION TO THE TAXA

77

to 2006 (complete line, with equation and % variation accounted for,

in box on the left hand side); Plots of a proportional micro index,

derived from numbers of papers mentioning Argyreia nervosa as a

proportion (scaled by multiplying by one million) of the total number

of papers published for that year (broken line frequency polygon and

right hand scale) and line of best fit, 1926 to 2006 (broken line, with

equation and % variation accounted for, in broken line box on the

right hand side)]

Total Mentions (Biological Abstracts/Biosis Previews):

REFERENCES:

Harvey S E, Cumpston K L and Benson B E (2006): Serotonin toxicity

from the combination of Hawaiian Baby Woodrose and pro-

serotonergic pharmaceuticals. Clinical Toxicology 44. Contact: Harvey,

S. E.; New Mexico Poison and Drug Informat Ctr, Albuquerque, NM

USA.

Reddy K N and Subbaraju G V (2005): Ethnomedicine from

Maredumilli region of East Godavari District, Andhra Pradesh. Journal

of Economic and Taxonomic Botany 29, 476-81. Contact: Reddy, K.

N.; Laila Impex R and D Ctr, Taxon Div, Unit 1, Phase 3, Jawahar

Autonagar 520007, Vijayawada, India.

Bhatt D C, Patel N K, Mitaliya K D and Ant H M (2003): Herbal magic

by contact therapy among tribals and rurals of Gujarat. Journal of

Economic and Taxonomic Botany 27, 205-7. Contact: Bhatt, D. C.;

OBJECTIVES AND INTRODUCTION TO THE TAXA

78

Mitaliya, K. D.; Department of Marine Sciences, Bhavnagar University,

Bhavnagar, Gujarat, 364 002, India.

Borsutzky M, Passie T, Paetzold W, Emrich H M and Schneider U

(2002): Hawaiian baby woodrose: (Psycho-) Pharmacological effects of

the seeds of Argyreia nervosa. A case-orientated demonstration.

Nervenarzt 73, 892-6. Contact: Borsutzky, M.; Abteilung Klinische

Psychiatrie und Psychotherapie, Medizinische Hochschule Hannover,

30625, Hannover, Germany.

Bogenschutz M P (2001): Drugs on the internet. American Journal of

Psychiatry 158, 2094-5. Contact: Bogenschutz, Michael P.;

Albuquerque, NM, USA.

Halpern J H, Pope H G and Jr (2001): Drugs on the internet: Drs.

Halpern and Pope reply. American Journal of Psychiatry 158. Contact:

Halpern, John H. ; Pope, Harrison G., Jr.; Belmont, MA, USA.

Gupta V (2000): Structural changes in seed coat morphology during

dormancy breaking in some medicinal plants. Journal of Medicinal

and Aromatic Plant Sciences 22-23, 672-3. Contact: Gupta, Veena;

National Bureau of Plant Genetic Resources, Pusa Campus, New Delhi,

110 012, India.

Mann P, Tofern B, Kaloga M and Eich E (1999): Flavonoid sulfates

from the Convolvulaceae. Phytochemistry (Oxford) 50, 267-71.

Contact: Eich, Eckart; Inst. Pharm. II, Freie Univ. Berlin, Koenigin-Luise-

Strasse 2+4, 14195 Berlin, Germany.

OBJECTIVES AND INTRODUCTION TO THE TAXA

79

Tofern B, Kaloga M, Witte L, Hartmann T and Eich E (1999):

Occurrence of loline alkaloids in Argyreia mollis (Convolvulaceae).

Phytochemistry (Oxford) 51, 1177-80. Contact: Eich, Eckart; Institute

fuer Pharmazie II (Pharmazeutische Biologie), Freie Universitaet Berlin,

Koenigin-Luise-Strasse 2+4, D-14195, Berlin, Germany.

Murugesan S, Kumar S and Sundararaj R (1997): Blister beetles as a

threat to medicinal/ornamental plants of arid and semi-arid regions.

Indian Forester 123, 341-4. Contact: Div. Forest Protection, Arid Forest

Res. Inst., Jodhpur, India.

Srivastava S K, Mehrotra B N and Palvi S K (1992): Distributional

notes on some plants in Arunachal Pradesh. Journal of Economic and

Taxonomic Botany 16, 709-13. Contact: Srivastava, S. K.; Bot. Survey

India, Andaman and Nicobar Circle, Port Blair 744 102, India.

Furbee R B, Curry S C and Kunkle D B (1991): Ingestion of Argyreia

Nervosa Hawaiian Baby Woodrose Seeds. Veterinary and Human

Toxicology 33. Contact: FURBEE R B; SAMARITAN REGIONAL POISON

CENTER, PHOENIX, ARIZ 85006, USA.

Daniel M (1989): Polyphenols of Some Indian Vegetables. Current

Science (Bangalore) 58, 1332-4. Contact: DANIEL M; DEP BOTANY,

FACULTY SCIENCE, BARODA 390 002, INDIA.

OBJECTIVES AND INTRODUCTION TO THE TAXA

80

Nair G G, Daniel M and Sabnis S D (1988): Chemosystematics of

Convolvulaceae. Geobios (Jodhpur) 15, 241-4. Contact: NAIR G G;

PHYTOCHEM LAB, DEP OF BOTANY, MS UNIV OF BARODA, BARODA,

390 002, INDIA.

Srivastava R C (1983): A Taxonomic Study of the Genus Argyreia

Convolvulaceae in Madhya Pradesh India. Proceedings of the National

Academy of Sciences India Section B (Biological Sciences) 53, 37-42.

Contact: SRIVASTAVA R C; BOTANICAL SURVEY OF INDIA, CENTRAL

CIRCLE, ALLAHABAD-211 002, INDIA.

Nanda B K (1980): Histological Responses of Argyreia-Nervosa

Isolated Leaves to Hormones Applied across a Transverse Gradient.

Plant and Cell Physiology 21, 1133-42. Contact: NANDA B K; DEP

BOT, GM COLL, SAMBALPUR-768004, INDIA.

Pant D D and Bhatnagar S (1975): Morphological Studies in Argyreia

Convolvulaceae. Botanical Journal of the Linnean Society 70, 45-70.

Chao J M and Der Marderosian A H (1973): Ergoline Alkaloidal

Constituents of Hawaiian Baby Wood Rose Argyreia-Nervosa. Journal

of Pharmaceutical Sciences 62, 588-91.

Chao J M and Dermarderosian A H (1973): Identification of Ergoline

Alkaloids in the Genus Argyreia and Related Genera and Their Chemo

Taxonomic Implications in the Convolvulaceae. Phytochemistry (Oxford)

12, 2435-40.

OBJECTIVES AND INTRODUCTION TO THE TAXA

81

Vogel W H, Carapellotti R A, Evans B D and Der Marderosian A

(1972): Physiological Disposition of Isoergine from Argyreia-Nervosa-D

Convolvulaceae-D and Its Effect on the Conditioned Avoidance

Response in Rats. Psychopharmacologia 24, 238-42.

Crawford K W (1970): The Identification of Lysergic-Acid Amide in

Baby Hawaiian Woodrose-D by Mass Spectrometry. Journal of

Forensic Sciences 15, 588-94.

Der Marderosian A H and Chao J (1970): The Indole Alkaloid

Constituents of Argyreia-Nervosa-D Convolvulaceae-D. Lloydia

(Cincinnati) 33.

Miller M D (1970): Isolation and Identification of Lysergic-Acid Amide

and Iso Lysergic-Acid Amide as the Principal Ergoline Alkaloids in

Argyreia-Nervosa-D a Tropical Wood-Rose-D. Journal of the

Association of Official Analytical Chemists 53, 123-7.

Dobberstein R H and Staba E J (1969): Ipomoea-Violacea-D Rivea-

Corymbosa-D and Argyreia-Nervosa-D Tissue Cultures Influence of

Various Chemical Factors on Indole Alkaloid Production and Growth.

Lloydia (Cincinnati) 32, 141-7.

Hylin J W and Watson D P (1965): Ergoline alkaloids in tropical wood

roses. Science 148, 499-500. Contact: Hawaii Agr. Exp. Sta., Univ.

Hawaii, Honolulu, Hawaii, USA.

CHAPTER 1-C

REVIEW OF LITERATURE

REVIEW OF LITERATURE

82

REVIEW OF LITERATURE

A large portion of the Indian population depends on the Indian

system of medicine “Ayurveda”. The well known treatises in Ayurveda

are Charaka Samhita and Sushruta Samhita. Sushruta arranged 760

herbs in 7 distinct sets based on some of their common properties.

Ayurvedic medicine stresses that psychic influences strongly affect the

body in health as well as disease, a fact which must also be taken

into account in modern therapeutics (Pizzorno and Murray, 1999;

Ahmad et al. 2006; Khare, 2007).

Many books and review articles are published till date which

synchronized the synonyms and medicinal properties of Indian

medicinal plants (Chopra et al. 1956; Kirtikar and Basu, 1975;

Anonymous, 1986; Sharma, 1991; Husain, 1992; Khare, 2007) and

also the number of other publications like, The Ayurvedic

Pharmacopoeia of India (Vol. I to IV); Medicinal Plants used in

Ayurveda (Rashtriya Ayurveda Vidyapeeth / National Academy of

Ayurveda, (1998); Natural Medicines Comprehensive Database, (2007).

Plants are the traditional source of many chemicals used as

pharmaceuticals. Most valuable phytochemicals are products of plant

secondary metabolism. Excellent reviews on the subject of secondary

metabolites and their production through cultures have been reported

(Bohm, 1980; Khanna, 1982, 1984; Vanishree et al. 2004; Christian

and Saxena, 2005; Sarin, 2005; Shinde et al.,, 2008). Product reviews

have been written very efficiently by Staba, 1963, Corduan, 1975 on

alkaloids biosynthesis and Stohs and Resenberg (1975) on steroids

and steroidal metabolism in plant tissue culture and numerous

reports are available describing the production of different secondary

REVIEW OF LITERATURE

83

metabolites, viz. alkaloids, saponins, steroidal alkaloids, coumarins and

several others (Zafar et al., 1992; Thorpe, 1994; Ramawat and

Merillon, 2000; Kumar, 2002; Mulabagal, 2004; Rathod, 2006). Scragg

et al. (1990) isolated serpentine from Catharanthus roseus.

Rasoanaivo et al. (1994) extracted alkaloids from plants of

Madagascar which were found to be resistant against Plasmodium

malaria.

PLANT TISSUE CULTURE:

The concept of plant cell and tissue culture was conceived by

Haberlandt in 1902, when he attempted to culture leaf of Lamium

pupureum on an artificial medium, with a view to develop tissue

cultures and eventually, regenerate a whole new plant. For about two

to three decades after Haberlandt's work very little was heard of

plant cell culture. The period of 1936 - 1956 was the period of

exploration and innovation in approach and techniques, which

provided model system for experimentation concerned with physiology

of nutrition, growth and morphogenesis. This gave rise to the

formulation of a number of artificial nutrient media. Since then

considerable progress had been made in the field of nutrient media.

Notable among them are White (1934, 1954); Nitsch (1951); Heller

(1953); Murashige and Skoog (1962); Gamborg et al., (1968); Chu

(1978) etc.

The studies of Camus (1949) led to important studies on

factors controlling vascular tissue differentiation (Wetmore and

Sorokin, 1955; Wetmore and Rier, 1963). The work of Miller and

Skoog (1953) on bud formation from cultured pith explants of

tobacco led to the discovery of kinetin. The first notable success in

REVIEW OF LITERATURE

84

the area of hormonal regulation of growth and differentiation came

from the work of Miller and Skoog (1957) on tobacco pith cultures

by manipulating auxin-cytokinin combinations in the nutrient media.

The regeneration of plants from explants of various origins has been

successfully accomplished in many taxa (Bhojwani and Razdan, 1983).

According to Thorpe (1993) plant tissue culture takes place by two

ways, organogenesis and embryogenesis. Numerous factors are

reported to influence the success of in vitro propagation of different

medicinal plants (Roy et al. 1994; Paek et al. 1995). It has been

observed that cytokinins are required, in optimal quantity, for shoot

proliferation in many genotypes but inclusion of low concentration of

auxins along with cytokinins triggers the rate of shoot proliferation

(Borthakur et al. 2002; Rai, 2002). Renowned researchers have

reviewed plant tissue culture successfully ensuring rapid clonal

propagation for conservation of germplasm of various plant species.

In 1941, it was demonstrated the coconut milk which normally

nourishes the developing coconut embryo, providing factors which

would encourage the growth of young, excised Datura embryos (Van

Overbeek et al. 1941).

Leaf culture:

Regeneration from the leaf callus cultures have been

successfully achieved by various investigators namely Nataraj and

Patil (1980) in Sida and Abutilon, Mroginski and Kartha (1981) in

Stylosanthes quianensis, Tanimoto and Harda (1982) in Rudbekia

bicolour, Webb and Emmanuel (1983) in Solanum tuberosum,

Srivastava et al., (1985) in Betula pendula, Rau and Forkmann (1986)

in Callistephus chinensis. Kantia and Kothari (2002) obtained plantlets

REVIEW OF LITERATURE

85

from leaf explant of Dianthus chinensis. Diaz and Alrvarez (2009)

obtained direct regeneration from Encyclia mariae. Gow et al., (2009)

obtained somatic embryos from leaf of Phalaenopsis orchids.

Regeneration from the leaf callus have been achieved by

Kothari and Chandra (1986) in Tagetes erecta, Katam and Padhya

(1990) in Azadirachta indica, Rout et al., (1992) in Rosa hybrida,

Dobos et al., (1994) in Sempervivum latorum, Rao et al., (1996) in

Paulonia species, Echeverrigaray et al., (2000) in Roman chanomile.

Saritha and Naidu (2008) obtained direct shoot regeneration from

leaf of Spilanthes acmella. An improved in vitro propagation system

for Spilanthes acmella using transverse thin cell layer culture system

was established by Shashikant et al., (2009).

Node/Internode culture:

Stem culture is useful for the clonal propagation in medicinal

plants and regeneration from it is achieved by various investigators

such as Lakshmiprasad and Shanthamma (1979) in Cenchrus glaucus.

Growth of multiple axillary shoots is stimulated when nodal explants

are cultured in medium with high cytokinin/auxin ratio as in

Eucalyptus (Mascarenhas, 1982). Multiple shoots were initiated from

nodal explant in Ochreinauclea missionis by Naomita and Ravishankar

(2001). Amin et al., (2002) reported axillary shoot formation on nodal

segments of lxora fulgens. In vitro regeneration was achieved in

Centella asiatica using nodal explants (Shashikala et al., (2005).

Gawde and Paratkar (2006) worked in Eclipta alba and obtained

multiple shoots from nodal explants. Neelofar jabeen et al., (2007)

obtained multiple shoots from node in lnula racemosa. Shravanan et

al., (2007) in node and internodes of Pedalium murex. Ricardo Daneil

REVIEW OF LITERATURE

86

et al., (2009) obtained multinodal segment culture of Habenaria

bractescens.

Axillary bud culture:

Several authors have worked on axillary bud like Mehra and

Cheema (1980) in Populus cilata. Axillary buds have been found to be

the most suitable for micropropagation in Morus niger as reported by

Yadav et al., (1990), Pattaniak et al., (1995) in mulberry, Karin and

Willi (1995) in Swarzia madagascariensis. Jagadishchandra et al.,

(1999) obtained in vitro culture of axillary buds from Pisonia alba,

Mousumi debnath (2008) has used axillary bud for regeneration in

Stevia rebaudiana.

Shoot tip culture:

Hussey, (1980) reported 6-Benzylaminopurine at high

concentration stimulates the development of the axillary meristems

and shoot tips of Atropa belladona. Barthe et al., (1987) produced

naringin and limonin in callus cultures and regenerated shoots from

Citrus sp. Gurel and Gulshan (1998) reported multiple shoot

proliferation from shoot tips of Amygdalus communis on MS medium

supplemented with the combination of 0.1 mg/l IBA and 1.0 mg/l

BAP. Multiple shoot formation were reported from shoot tips (1 - 2

cm) of field grown plants of Paederia foetida and Centella asiatica on

MS medium supplemented with BAP within 7 days of culture by (Singh

et al., 1999). Nasir et al., (2006) obtained multiple shoots from shoot

tip in an important medicinal plant called Artemisia scoparia. Plant

regeneration and in vitro flowering from shoot tip of Basilicum

polystachyon (L.) Moench done by Amutha et al., (2008).

REVIEW OF LITERATURE

87

Root culture:

Robbins (1922) was the first to develop a technique for the

culture of isolated roots. White in (1934) established continuous

growing root cultures of Lycopersion esculantum, Gautheret (1938) of

Salix purpurea, Populus nigra and others trees. During 1955-1960

period root cultures drew the attention to the role on vitamins in

tissue growth and the advanced the knowledge of the shoot-root

relationship (Street, 1959; 1966). Krikorian (1975) regenerated carrot

plantlet from secondary phloem cells of tap-root. Chang and Hsing

(1980) studied In vitro flowering of embryoids derived from mature

root callus of ginseng (Panax ginseng).

Seed culture:

Chaudhary et al., (1998) regenerated plantlets from explants of

hypocotyls and mature embryo of Phyllanthus amarus. Naomita and

Ravishankar (2000) induced in vitro shoots from hypocotyl, cotyledon

and cotyledonary node explants of Crotalaria lutescens. The effect of

growth regulators and their interactions on propagation of different

medicinal plants have been reproduced by Catapan et al., (2000).

Uddin et al., (2005) established a protocol for rapid multiplication of

shoots from cotyledonary node of Peltophorum. Kabir et al., (2008)

obtained in vitro plants of Abelmoschus esculentus using hypocotyl

explant. Mahendra and Bai (2009) were able to mass propagate

Satyrium nepalense via seed culture. Dutra et al., (2009) and Flores

et al., (2009) have propogated endangered plants like Cyrtopodium

punctatum and Oncidiun stramimeum respectively.

REVIEW OF LITERATURE

88

Anther culture:

Guha and Maheshwari (1964) reported the development of

haploid embryos in anther culture of Datura innoxia and subsequently

confirmed their origin from pollen grains.

Somatic embryogenesis:

The first report of somatic embryogenesis was from carrot root

tissue by Reinert (1958). Plant regenerabon via somatic

embryogenesis from single cells, that can be induced to produce an

embryo and then a complete plant, has been demonstrated in many

medicinal piant species. Arumugam and Bhojwani, (1990) noted the

development of somatic embryos from zygotic embryos of

Podophyllum hexandrum on MS medium containing 2 µM BA and 0.5

µM IAA. Embryogenic calluses and germination of somatic embryos in

nine varieties of Medicago sativa has been achieved by Fuentes

(1993). Asaka et al., (1993) worked on production of ginsenosides

saponins by culturing ginseng (Panax ginseng) embryogenic tissues in

bioreactors. Sagare et al., (2000) reported cytokinin-induced somatic

embryogenesis and plant regeneration in Corydalis yanhusuo

(Fumaraceae). Lee et al., (2001) studied formation of protoberberine-

type alkaloids by the tubers of somatic embryo-derived plants of

Corydalis yanhusuo. Sachdev et al., (2002) studied embryogenesis and

plantlet formation in callus culture of Gloriosa sllperba.

SECONDARY METABOLITES PRODUCTION BY PLANT TISSUE CULTURE:

Berlin et al., (1988) worked on the podophyllotoxins of root

cultures of Linum flavum. Ellis et al., 1996 studied taxol production in

nodule cultures of Taxus. Wongsamuth and Doran (1997) worked on

REVIEW OF LITERATURE

89

production of monoclonal antibodies by tobacco hairy roots. Rijhwani

and Shanks (1998) reported effects of elicitor dosage and exposure

time on biosynthesis of indole alkaloids by Catharanthus roseus hairy

root cultures. Zarate (1999) studied tropane alkaloid production by

Agrobacterium transformed hairy root culture of Atropa baetica. In

Bixa orellana, Sharon et al., (2000) regenerated plants from nodal

explants on MS medium supplemented with 2 isopentenyl adenine.

Alikaridis et al., (2000) produced flavonolignan from Silybum

marianum transformed and untransformed root cultures. Yu et al.,

(2000) worked on improvement of ginsenoside production by jasmonic

acid and some other elicitors in hairy root culture of ginseng (Panax

ginseng C.A. Mayer). Chen et al., (2001) cultured adventitious shoots

from internode explants of Adenophora triphylla an important

medicinal plant. Yu et al., (2002) studied Jasmonic acid improves

ginsenoside accumulation in adventitious root culture of Panax

ginseng C.A. Mayer. Shrishailappa et al. (2003) studied the antitumor

activity of total alkaloid fraction of Solanum pseudocapsic. Saifah et

al. (2004) isolated two new isoquioolone alkaloids named sauropine A

and sauropine B from Sauropus hirsutus and confirmed the presence

of isoquinoline alkaloids as the major constituents in a Sauropus

species.

Ammirato (1985) studied a large scale propagation of plants

through suspension culture. Woerdenbag et al., (1990) increased

podophyllotoxin production in Podophyllum hexandrum cell suspension

cultures after feeding coniferyl alcohol as a α-cyclodextrin complex.

The first time, in vitro taxol (a complex diterpene alkaloid) production

was carried out by the Christen et al. (1989), thereafter, similar

approached Sekh and Tsay, (1999). Wang et al., (1999) studied

REVIEW OF LITERATURE

90

significant improvement of taxane production in suspension cultures

of Taxus chinensis by sucrose feeding strategy. Choi et al., (2001)

enhanced production of paclitaxel by semi-continuous batch process

(SCBP) in suspension culture of Taxus chinensis. Chattopadhyay et al.,

(2002) worked on production of podophyllotoxin by plant cell cultures

of Podophyllum hexandrum in bioreactor.

Schiel et al., (1984) increased formation of cinnamoyl

putrescines by fedbatch fermentation of cell suspension cultures of

Nicotiana tabacum. Tabata and Fujita, (1985) produced shikonin by

plant cell cultures. Kim et al., (1990) worked on two stage culture for

the production of berberine in cell suspension culture of Thalictrum

rugosum. Suvarnalatha et al., (1993) computer-aided modeling and

optimization for capsaicinoid production by immobilized Capsicum

frutescens cells. Kobayashi et al., (1993) worked on large-scale

production of anthocyanin by Aralia cordata cell suspension cultures.

Jang et al., (1998) worked on production of a hepatoprotective

cerebroside from suspension cultures of Lycium chinense. Kitamura et

al., (1998) worked on induction of furanocoumarin biosynthesis in

Glehnia littoralis cell suspension cultures by elicitor treatment.

Ramachandra and Ravishankar (2000) worked on biotransformation of

protocatechuic aldehyde and caffeic acid to vanillin and capsaicin in

freely suspended and immobilized cell cultures of Capsicum

frutescens. Zhong et al., (2000) repoted high density cultivation of

Panax notoginseng cells in stirred bioreactors for the production of

ginseng biomass and ginseng saponin. Zhao et al., (2001) enhanced

catharanthine production in Catharanthus roseus cell cultures by

combined elicitor treatment in shake flasks and bioreactors. Ray and

Jha, (2001) reported production of withaferin in shoot cultures of

REVIEW OF LITERATURE

91

Withania somnifera. Orihara et al., (2002) reported abietane

diterpenoid from suspension cultured cells of Torreya nucifera var.

radicans. Phatak and Heble (2002) worked on organogenesis and

terpenoid synthesis in Mentha arvensis.

ANTIMICROBIAL ACTIVITY:

Mankind has always been under threat of diseases and

ailments. To cure such diseases, nature has gifted various plants to

humans. The knowledge of drugs has accumulated over thousands of

years as a result of man's inquisitive nature. In the past, almost all

the medicines used were from the plants, the plant being man's only

cure for ages even today. In 1985, the World Health Organization

(WHO) estimated that about 80% of the world's populations still rely

mainly on traditional remedies such as on herbs for their primary

health care needs (Farnsworth et al., 1985).

From the time immemorial the medicinal importance of plant is

well known to human beings. Antimicrobials are defined as those

secondary metabolites which are capable for inhibiting the growth of

other microorganisms (Kurzybski et al., 1967; Cochran and Hahn,

1975). A number of workers have investigated the occurrence of

antimicrobials active compounds from higher plants (Dhar et al., 1973;

Atal et al., 1978; Dhawan et al., 1977, 1980; Aswal et al., 1984).

The works on the antibacterial activity of medicinal plant have

been reviewed by a number of workers both in vivo (Gould and

Bowie, 1952; Dhar et al., 1968) and in vitro (Nickell, 1959, 1962;

Mathes, 1963, 1997; Misawa et al., 1974; Harsh and Nag 1984).

These studies have been reviewed by Skinner (1955) and Nickell

(1959) covering 174 plant species including 157 families for screening

of higher plants for biological activity.

REVIEW OF LITERATURE

92

A number of workers have investigated the occurrence of

antimicrobials active compounds from higher plants (Dhar et al., 1973;

Atal et al., 1978; Dhawan et al., 1977, 1980; Aswal et al., 1984). A

number of plant species have been known to possess antimicrobial

activity such as alkaloids of lndigofera microcarpa (De Morafs et al.,

1991). Mausumi debnath (2008) has developed a procedure for plant

regeneration and antimicrobial screening in Stevia rebaudiana.

REVIEW OF LITERATURE OF THE ARGYREIA NERVOSA:

Herbal drugs or medicinal plants, their extracts and their

isolated compound(s) have emonstrated spectrum of biological

activities. Such have been used and continued to be used as

medicine in folklore or food supplement for various disorders. Ethno-

pharmacological studies on such herbs/medicinally important plants

continue to interest investigators throughout the world. One such

plant, A. speciosa (Linn.f.) sweet, invites attention of the researchers

worldwide for its pharmacological activities ranging from aphordiasic

to nematicidal activistties (Subramonium et al., 2007; Gokhle et al.,

2003; Habbu et al., 2008a, 2008b; Galani and Patel, 2009; Hemet et

al., 2008; Joshi et al., 2007; Vyavhare and Bodhankar, 2009a, 2009b;

Bachhav et al., 2009; George and Pandalai, 1949; Mishra and

Chaturvedi, 1978; Shukla et al., 1999; Babber et al., 1978; Parveen et

al., 1990).

Traditionally the whole plant is used in stomach complaints,

sores on foot, small pox, syphilis, dysentery and diarrhea

(Anonymous, 2000; Guhabakshi et al., 1999). Leaf is used in

antiphlogistic, emollient, poultices of wounds, externally for skin

disease, gleet, gonorrhoea and chronic ulcers. Also used as a local

stimulant and rubefacient. Leaves of Argyreia nervosa are used by

REVIEW OF LITERATURE

93

Rajasthani tribes to prevent conception (Anonymous, 2004b; Kirtikar

and Basu, 1981). Externally used in the treatment of ringworm.

Eczema, itch and other skin diseases (Anomymous, 2004b). Root is

used in appetitiser, anaemia, aphrodisiac, anti–inflammatory, brain-

tonic, cardiotonic, cerebral disorders, diabetes, expectorant, obesity,

syphilius, tuberculosis, ulcers and wounds (Nadkarni 1995; Krishnaveni

and Thaakur, 2009). Seeds of A. nervosa found to possess

hypotension, spamolytic (Agarwal and Rastogi 1974b) and anti-

inflammatory activity (Gokhale et al., 2002). Chemical analysis

revealed the presence of triterpenoids, flavanoids, steroids and lipids

(Srivatasav et al., 1998). 24R-ergost-5- en-11-oxo-3 beta-ol alpha –D

glucopyranoside xylose was isolated from seeds of A. nervosa known

as Argyreioside (Rahman et al., 2003).

PHARMACOGNOSTIC STUDIES

The Macroscopical and microscopical features of the root, stem

and leaf have been studied.

Root

Macroscopical

The commercial samples of the root vary in size as well as in

thickness. The thin pieces of the root usually 2 – 4 mm in diameter

show somewhat smooth brownish exterior. When cut transversely such

pieces show a thin periderm and cambium appearing as a dark line

almost midway between the centre and the outer circumference

separating the outer phloem from inner central wood. The thicker

pieces of the root 5 - 25 mm in diameter or even more have a

rough exterior due to the presence of large number of lenticels. A

transversely cut surface of such root shows colourless tertiary phloem

REVIEW OF LITERATURE

94

and a pink coloured crescent shaped tertiary xylem (Singh, 1965;

Singh, 1972; Prasad and Chauhan, 1975).

Microscopical

Microscopically, the young root shows an epidermis composed

of small cubical parenchymatous cells, follo-wed by a wide cortex

consisting of mostly isodiametric or in some cases, slightly oval cells.

The primary vascular structure is tetrarch to pentarch. The mature

root possesses a narrow periderm of 6 - 8 layers of cork cells, a

single layer of phellogen and 10 - 12 layers of phelloderm cells, the

phelloderm cells close to the phellogen are somewhat tangentially

elongated and thin walled but become gradually polyhedral. Some of

them possess rosette crystals of calcium oxalate. The secondary

phloem is a wide zone, consisting of sieve tube elements with

companion cells and phloem parenchyma. Resin canals, small strands

of tertiary xylem and tertiary phloem a found scattered throughout

the region. The secondary xylem is composed of large xylem vessels,

tracheids, fibre tracheids and fibres. The vessels arc drum shaped,

having bordered pits on the walls. The tracheids are cylindrical and

possess bordered pits on the walls. The wood fibres are long and

tapering with pointed ends (Fig. No.6.1, 6.2, 6.3 6.4 and 6.5); (Singh,

1965; Singh, 1972; Prasad and Chauhan, 1975).

Stem

Macroscopical

The stem is white and tomentose in young stages. The older

stem (25 mm or so thick show vertical ridges and numerous lenticels,

REVIEW OF LITERATURE

95

which are mostly transversely elongated). (Singh, 1965; Singh, 1972;

Prasad and Chauhan, 1975).

Microscopical

The young stem microscopically, shows nonglandular hairs,

which are uniseriate, multicellular and usually 3- celled. Resin canals

are distributed throughout the cortex. Following the cortex is an

amphiphloic siphonostele. The mature stem shows the cork composed

of 10 - 15 layers of cells, which are stratified due to alternate

arrangement of 3 - 4 layers of large cells, followed by almost equal

number of shorter cells. The secondary phloem is wide and occupies

the greater portion. A tertiary cambium arises in the secondary

phloem and gives rise to tertiary phloem and tertiary xylem strands.

The xylem vessels are drum shaped with well marked perforation

rims. A few vessels are long and cylindrical. They have all bordered

pits on the walls. The tracheids are longer than the vessels. These

also have bordered pits on the walls and there are no end wall

opening. The xylem fibres are long with pointed tapering ends and

short lumen. They are however, shorter and narrower as compared to

the pericyclic fibres which have pointed ortruncated ends and show in

some cases peg like out-growths towards the tapering ends. The stem

is often substituted for the root and is also adulterated with the

stem cutting of cocculus hirsutus (Singh, 1965; Singh, 1972; Prasad

and Chauhan, 1975).

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96

Leaf

Macroscopical

The lower surface of the leaf is entirely covered with hair,

which gives the leaf a silvery soft wooly appearance; the upper

surface is green, glabrous and shows the markings of nerves by slight

depressions. The mature leaf is dorsiventral, unicostate with a strong

midnerve and several faint lateral nerves, alternate, petiole, acute at

the apex and cordate at the base. The margin is entire but slightly

wavy near the base. Lateral nerves 14 - 20 pairs arise alternatively

on the midrib; the single nerves bifurcate before reaching the edge,

the-anterior branch unites with the posterior one of the neighbouring

nerve; an arched nervule connects the two branches slightly above

the point of bifurcation. Neither the main secondary nerves nor their

branches reach the margin. Petiole stout and cylindrical, a little

shorter than the length of the blade is completely covered with wooly

tomentum (Singh, 1957; Shasikala et al., 1991).

Microscopical

The transverse section of the leaf near the apex shows a

prominent ridged midrib on the lower surface and a small groove on

the upper surface, while a section through the basal region presents

a small ridge on the upper side as well. The ventral cuticle is

stratified while the dorsal is thin and simple. The epidermal cells of

the upper side have synclinous walls with rubiaceous type of sunken

stomata. The openings of the latex canals are bound by 5 - 6 cells,

the epidermal cells or the under side differ from those of the upper

in possessing smaller cells and about twice the number of stomata

and openings of latex canals. The cells of the epidermis along the

REVIEW OF LITERATURE

97

veins on both sides of the leaf are roughly rectangular straight walled

and completely devoid of appendages. The spongy tissue is

composed of rounded cells enclosing air spaces and a few latex

canals. The palisade cells are nearly rectangular, roughly four times

longer than broad and are seen in the section usually in a single

row only and rarely in two rows. A few latex canals are sometimes

present in this zone as well. The vascular bundles are hexagonal in

transverse section and occur in characteristic, continuous single row

chains. The transverse section of the petiole at the base is grooved

along the ventral side while the groove becomes rather negligible at

the apex. Arrangement of the tissues in the petiole is as in the stem.

The vascular bundles are open, bicollateral and arranged in a ring.

The vasculature is represented by a shallow abaxial arc and a pair of

adaxial traces. Conjunctive parenchyma separates the xylem and the

phloem tissues distinctly. There are broad patches of phloem

parenchyma. Xylary tissues of the leaf and the petiole are identical.

Fresh vascular bundles are produced in the pith. The epidermal cells

are barrel shaped and most of them bear trichomes. Hypodermis or

any mechanical tissues are completely lucking. Hexagonal corticalcells

are smaller towards the periphery and the stele but are larger in the

central region. The corticle merge gradually with the phloem

parenchyma. The endodermis and pericycle are not made out even in

a very young petiole (Singh, 1957; Shasikala et al., 1991).

Pharmacognostical parameters for the leaves of Argyreia

nervosa Burm were studied with the aim of drawing the

pharmacopoeial standards for this species. Macroscopical and

microscopical characters, physio-chemical constants, quantitative

microscopy parameters, extractive values with different solvents,

REVIEW OF LITERATURE

98

fluorescence analysis of dry powder, its reaction after treatment with

chemical reagents under visible light and UVlight at 254 nm and 366

nm. Preliminary phyto-chemical screening on the leaves Argyreia

nervosa Burm was studied.

Powder analysis of Argyreia nervosa Burm.:

It is a pale green, fine, odourless powder with slight bitter

taste. The powder microscopy revealed the presence of glandular

&covering trichomes, xylem fibres, epidermal cells, cork cells, vessels

with bordered pits, xylem vessels with spiral thickenings were

recorded. The various qualitative chemical tests (Table 5) have shown

the presence of triterpenoids, saponins, sterols, flavanoids,

carbohydrates phenols, tannins and in large amount whereas aromatic

acids, gums and mucilage and volatile oils were totally absent in the

leaf extract of this plant.

Table 2.1

Ash Values of Argyreia nervosa

(Krishnaveni and Santh, 2009)

S. No. Ash type Percentage of Ash

1. Total ash 4.3% w/w

2. Acid insoluble ash 1.6% w/w

3. Water soluble ash 3.94%w/w

REVIEW OF LITERATURE

99

Table 2.2

Extractive Values of Argyreia nervosa

(Krishnaveni and Santh, 2009)

S. No. Solvent Percentage of

extractive

1. Petroleum ether 3.16% w/w

2. Chloroform 0.8% w/w

3. Ethyl acetate 1.4% w/w

4. Ethanol 0.2% w/w

5. Water 7.6% w/w

Table 2.3

Phyto constants of Argyreia nervosa

(Krishnaveni and Santh, 2009)

Leaf constants Report

Vein islet number 10.2/mm2

Vein termination number 12.6/mm2

Stomatal index (upper epidermis) 4.5/mm2

Stomatal index (lower epidermis) 16/mm2

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100

Similarly the fluorescence characteristic of the leaf powdered

leaf, when treated with various chemical reagents and its extracts

have also been extensively studied. The extractive values of the

powder with different solvent was determined and its result was

reported in table no: 2. The various qualitative chemical tests have

shown the presence of sterols, flavanoids, phenols, tannins and

saponins in large amount whereas aromatic acids, carbohydrates,

triterpenoids gums and mucilage and volatile oils were totally absent

in the leaf extract of this plant.

Powder as such:

Colour: Dark green; Taste: Slightly bitter; Odour: Characteristic.

Table No. 2.4

Behavioural characterstics of powdered leaves of Argyreia nervosa

with different chemical reagents.

(Krishnaveni and Santh, 2009)

Sr.N

o.

Particulars Under

Visible light

U.V. light

Short

Wavelengt

h

Long

wavelengt

h

1. Powder as such Dull green Dark

green

__

2. Powdered drug + Conc. HCl Dull green Pale

green

__

3. Powdered drug + Conc. H2SO4 Dull green Pale Green

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101

green

4. Powdered drug + Conc. HNO3 Yellow Dull

green

__

5. Powdered drug + Glacial Acetic

acid

Dull green Pale

green

Orange

6. Powdered drug + Aqueous NaOH Dark green Dark

green

__

7. Powdered drug + NaOH

(Alcoholic)

Dark green Dark

green

__

8. Powdered drug + 10% HCl Dull green Dull

brown

__

9. Powdered drug + 10% H2SO4 Dull brown __ __

10. Powdered drug + 10% HNO3 Dull green Dark

green

__

11. Powdered drug + 10% Glacial

Acetic acid

Dark green Dark

green

__

12. Powdered drug + Ferric chloride

(Aqueous)

Dark green Dark

green

__

13. Powdered drug + Ferric chloride

(Alcoholic)

Dark green Dark

green

__

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102

Table No. 2.5

Preliminary phytochemical screening of Argyreia nervosa

(Krishnaveni and Santh, 2009)

Sr.No. Tests Powder+ Water Ethanol extract Water extract

1. Alkaloids:

Dragendroff’s test

Mayer’s test

Hager’s test

Wagner’s test

+ ve

+ ve

+ ve

+ ve

+ ve

+ ve

+ ve

+ ve

+ ve

+ ve

+ ve

+ ve

2. Carbohydrates:

Fehling’s test

Molish test

+ ve

+ ve

+ ve

+ ve

+ ve

+ ve

3. Gums/Mucilage:

Water

Alcohol

-ve

-ve

-ve

-ve

- ve

- ve

4. Tannins:

Aq. FeCl3 Test

Alc. FeCl3 Test

+ ve

+ ve

+ ve

+ ve

+ ve

+ ve

5. Flavonoids:

Lead acetate test

Shinoda test

Mg/HCl

+ ve

+ ve

+ ve

- ve

- ve

- ve

+ ve

+ve

+ ve

6. Saponins:

Foam Test

Lead acetate test

+ ve

+ ve

+ ve

+ ve

- ve

+ ve

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103

7. Sterols:

Salowaski test

Libberman Burchad

test

+ ve

+ ve

+ ve

+ ve

+ ve

+ ve

PHARMACOLOGICAL STUDIES

Although a lot of pharmacological investigations have been

carried out based on the ingredients presents but a lot more can still

be explored, exploited and utilized. A summary of the findings of

these studies is presented below.

Aphrodisiac activity

The root, flower and to some extent, leaf (homogenate in 2%

gum acacia) of the plant showed aphrodisiac activity as evidenced by

an increase in mounting behavior of mice. When different extracts of

the root were tested, the activity was found in the alcohol extract

(200 mg/kg; p.o, single dose). The extract, 1 h after administration,

stimulated mounting behavior of male mice in a

concentrationdependent manner. The root- or flower-treated male

mice also exhibited a remarkable increase in mating performance.

Further, the number of males was found to be more among the pups

fathered by the herbal drug-treated mice compared to those by the

control mice. Thus, the plant has promising potential to be developed

into an effective medicine for stimulating male sexual activity with an

influence on sex ratio favoring males (Subramonium et al., 2007).

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104

Immunomodulatory activity

Oral administration of the ethanolic extract of A. speciosa root

(ASEE), at the doses of 50, 100 and 200 mg/kg in mice, dose-

dependently potentiated the delayed-type hypersensitivity reaction

induced both by sheep red blood cells (SRBC) and oxazolone. It

significantly enhanced the production of circulating antibody titre in

mice in response to SRBC. ASEE failed to show any effect on

macrophage phagocytosis. Chronic administration of ASEE significantly

ameliorated the total white blood cell count and also restored the

myelosuppressive effects induced by cyclophosphamide. The present

investigation reveals that ASEE possesses immunomodulatory activity

(Gokhle et al., 2003).

Hepatoprotective activity

The ethanolic extract and ethyl acetate extract (200 and 400

mg/kg) of A. speciosa roots decreased the elevated enzyme levels

induced by CCl4, thus protecting the structural integrity of hepatocyte

cell membrane or regeneration of damaged liver cells. These two

extracts are found to be capable of enhancing or maintaining the

activity of hepatic enzymes which are involved in combating Reactive

Oxygen Species. The hepatoprotective effect of A. speciosa roots was

evidenced by the amelioration of biochemical indicators of liver

damage and pathological disturbances caused by CCl4. From the

study we can conclude that root extracts of A. speciosa protects liver

from oxidative damage and could be used as an effective protector

in CCl4 induced damage (Habbu et al., 2008a).

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105

Central nervous system activity

The n-hexane (n-HF), chloroform (CF), ethyl acetate (EAF) and

water (WF) fractions of hydroalcoholic extract of roots of A. speciosa

were tested on the central nervous system. All the fractions (100,

200 and 500 mg/kg, p.o.) were evaluated for neuro-pharmacological

activity using spontaneous motor activity and pentobarbital-induced

sleeping time in mice. Chlorpromazine was used as a positive control.

Central nervous system depressant activity was observed with all the

fractions as indicated by the results in which they reduced

spontaneous motor activity and potentiated pentobarbital induced

hypnosis in mice (Galani and Patel, 2009).

Hypoglycemic

The hypoglycemic and antihyperglycemic activities of methanolic

extract of stem of A. speciosa sweet (A. speciosa and A. nervosa)

were done in normal and alloxan induced diabetic rats. The blood

glucose levels were measured at 0 h and 1, 2, 4, 6, 8, 12, 16 and

24 h after the treatment. Oral glucose tolerance test was performed

in normal, diabetic control, plant extract treated normal and diabetic

groups and tolbutamide also treated normal and diabetic groups. It

was found that alcoholic extract of A. speciosa showed significant (P

< 0.05) dose dependent percentage blood glucose reduction in

normal (26.42% at 250 mg/kg, 28.50% at 500 mg/kg and 34.25% at

750 mg/kg body weight) and in diabetic rats (24.72% at 250 mg/kg,

31.10% at 500 mg/kg and 40.47% at 750 mg/kg body weight)

respectively at 8 h. The hypoglycemic and antihyperglycemic effect of

A. speciosa was compared with the reference standard drug

tolbutamide (40 mg/kg) (Hemet et al., 2008).

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106

Nootropic

According to Joshi et al., (2007) effectiveness of aqueous

extract of AS on ageing, scopolamine and diazepam induced memory

deficits in mice was evaluated. Elevated plus maze and passive

avoidance paradigm were employed to assess short-term and long

term memory. In order to delineate the possible mechanism through

which AS elicits the anti-amnesic effects, the whole brain acetyl

cholinesterase (AChE) activity, was also assessed. Two doses (100

and 200 mg/kg, p.o.) of aqueous extract of AS were administered

orally for 6 successive days to both young and aged mice. AS

decreased transfer latencies and increased step down latencies in

both young and aged mice AS (100 and 200 mg/kg, p.o.)

successfully reversed amnesia induced by diazepam, scopolamine and

natural ageing.

Anti inflammatory activity

The alcoholic extract of the root exhibited statistically

significant anti-inflammatory activity against granuloma formation

technique in albino rats which comparable to acetylsalicylic acid. The

extract did not show much activity against formalin induced arthritis

in rats (Srivastava et al., 1972).

Anticonvulsant activity

The hydroalcoholic extract of A. speciosa at the dose of 200

and 400 mg/kg significantly delayed the latency to the onset of the

first clonus as well as onset of death in unprotected mice and

exhibited protection in 16.66 and 33.33% of pentylenetetrazole

REVIEW OF LITERATURE

107

treated mice respectively. Whereas in case of maximal electroshock

seizures, the dose of 200 and 400 mg/kg significantly reduced the

duration of hind limb extention and both the doses were statistically

found to be equipotent. The reference standards, clonazepam (0.1

mg/kg) and phenytoin (20 mg/kg) provided complete protection

(Vyavhare and Bodhankar, 2009a).

Analgesic activity

The methanolic extract of A. speciosa root was used in pain

and inflammation models. The analgesic activity of AS at the dose of

(30,100 and 300 mg/kg p.o) showed significant (P < 0.01) decreased

in acetic acid induced writhing, whereas ME of A. speciosa at the

dose of (30,100 and 300 mg/kg p.o) showed significant (P < 0.01)

increase in latency to tail flick in tail immersion method and elevated

mean basal reaction time in hot plate method (Bachhav et al., 2009).

Antibacterial activity

The alcoholic extract of the leaves revealed antibacterial

activity against staphylococcus aureas (George and Pandalai, 1949),

the seed oil was found to possess in vitro antibacterial activity

against Klebsiella sp., Escherichia coli, Pseudomonas aeruginosa and

Bacillus anthracis (Kelkar et al., 1947; Mishra and Chaturvedi, 1978).

Chansakaow et al., (2005) worked on five Thai medicinal plants,

Acanthus ilicifolius Linn. var. ilicifolius (leaves); Argyreia nervosa (Burm.

f.) Bojer (leaves); Punica granatum L. var. granatum (fruit rind);

Terminalia chebula Retz. var. chebula (fruits) and Zanthoxylum

myriacanthum Wall. ex Hook. F. (fruits) was extracted by several

extracting procedures with various solvents. Plant extracts tested for

REVIEW OF LITERATURE

108

their antibacterial activities against Staphylococcus aureus ATCC

25923 and Escherichia coli ATCC 25922 by well diffusion method.

The active extracts were found out for minimum inhibitory

concentration (MIC) and minimum bactericidal concentration (MBC) by

broth dilution method. The results revealed that the ethanolic extract

of Punica. Ethanolic extracts of leaves of Clerodendrum infortunatum

Linn, Argyreia nervosa and Vitex negundo were subjected to

preliminary screening for antimicrobial activity .All ethanolic extracts

exhibited significant anti-microbial activity comparable to the standard

drug tetracycline. Ethanolic extract of Clerodendrum infortunatum

shows more inhibition zone as compared to ethanolic extracts

Argyreia nervosa of and Vitex negundo. The mixture of all three

extracts together in equal concentration shows .When the three

extracts were mixed together in equal concentration(1:1:1), it shows

more inhibitory zone as compared to other individual extracts (Ashish

et al., 2010).

Antifungal activity

Hexadecanyl p-hydroxycinnamate and scopoletin isolated from

the root were tested for antifungal activity against Fusarium

fusiformis. F. semutectum and Alternaria alternate at a concentration

of 1000 ppm. It was found that both the compounds produced 100%

inhibition against A. alternate. The compounds also revealed

phytotoxicity in terms of root growth inhibition of germinating wheat

seeds (Shukla et al., 1999).

Antiviral activity

The extract of the plant and fruits had interferon-like antiviral

activity against vaccinia virus in CAM cultures, but was devoid of any

activity against Ranikhet disease virus (Babber et al., 1978).

REVIEW OF LITERATURE

109

Nematicidal activity

The effect of the aqueous and alcoholic extracts of the leaves

on the spontaneous movements of both the adult worm and a

nerve/muscle preparation of Setaria cervi, a filarial worm of cattle

and on the survival of microfilariae in vitro was studied. The aqueous

extract in a dose of 150mcg/ml caused a decreased in tone and

amplitude of spontaneous movements of the worm. A similar

response was produced by the alcoholic extract but a much lower

concentration of 75 mcg/ml. The aqueous extract produced complete

paralysis of the nerve/muscle preparation in a dose 25 mcg/ml

whereas with the alcoholic extract only 50 ng/ml was required

(Parveen et al., 1990).

CLINICAL STUDIES

A preparation made from this plant along with several other

ingredients is used for curing sexual disorders in males

(http://www.himalayahealthcare.com).

TOXICOLOGY

A few of the ergoline alkaloids reported in this plant are

hallucinogic

(http://www.himalayahealthcare.com).

INTRODUCTION

50

locating and modifying Quantitative Trait Loci (QTL) (Babu et al.,

2004; Noel et al., 2007) to introduce new characters in existing

germplasm. Markers associated with 40 traits of economic importance

have been reported in wheat (Gupta et al., 1999) which helps in

segregating a plant by a simple PCR early in its growth. Thus marker

assisted selection helps in eliminating unfavourable alleles in the early

stage of plant development. RAPD has been used to identify markers

linked to disease resistance genes in cereals (Adamblondon et al.,

1994) and other economically important crop species.

RAPD has been used in genome mapping of several plants

species including conifers. It is extremely difficult to map conifers due

to their large genome size (30-40 x l09nt) and high proportion of

repetitive DNA (Penner, 1996). RAPD has been used for mapping

where RFLP has not been able to detect polymorphism. RAPD has

often been converted to SCAR markers and used as diagnostic

markers.

Mapping helps in tagging traits such as yield, drought

resistance, stress tolerance, disease resistance, seed quality, etc.,

which are important for breeding. Mapped genes can also be used

for identifying other useful genes in the new genotype generated in a

hybrid programme.