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GENERAL INTRODUCTION

It is presumed that the science of Botany began with the passing of plant lore

from generation to generation since the time of Palaeolithic hunter-gatherers. They

passed on the information of different kinds of plants that they used for food, shelter,

poisons, medicines, ceremonies, rituals etc. The uses of plants by these pre-literate

societies influenced the way the plants were named and classified. The first written

records of plants were made in the Neolithic Revolution about 10,000 years ago, as

writing was developed in the settled agricultural communities, where plants and

animals were first domesticated. With these communities came the development of

the technology and skills needed for the domestication of plants and animals. The

emergence of the written word provided evidence for the passing of systematic

knowledge and culture from one generation to the next (Morton and Alan, 1981).

During the Neolithic Revolution plant knowledge increased most obviously

through the use of plants for food and medicine. All of today's staple foods were

domesticated in prehistoric times as a gradual process of selection of higher-yielding

varieties possibly unknowingly, over hundreds to thousands of years. Protobotany, the

first pre-scientific written record of plants, was born out of the medicinal literature

of Egypt, China, Mesopotamia and India (Reed and Howard, 1942).

Since time immemorial people have tried to find medications to alleviate pain

and cure different illnesses. In every period, the healing properties of certain

medicinal plants were identified, noted, and conveyed to the successive generations.

The benefits of one society were passed on to another, which upgraded the old

properties, discovered new ones, till present days. The continuous and perpetual

interest of the people in medicinal plants has brought about today's modern and

sophisticated fashion of their processing and usage.

In India the holy books Rigveda, Atharvaveda and Taittiriya Samhita

mentioned about the importance of a variety of plants and its medicinal properties,

which are abundant in the country. The importance of four medicinal plants are

mentioned in the epic ‘Ramayana’ which was used to rejuvenate the wounded

soldiers. The Atharvaveda contain prescriptions of herbs for various ailments. Many

other herbs and minerals used in Ayurveda were later described by ancient Indian

herbalists such as Charaka and Sushruta during the 1st millennium BC.

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The Sushruta Samhita (6th century BC) describes 700 medicinal plants, 64

preparations from mineral sources, and 57 preparations based on animal sources

(Girish and Shridhar, 2007).

The oldest written evidence of medicinal plants’ usage for preparation of

drugs has been found on a Sumerian clay slab from Nagpur, approximately 5000

years old. It comprised 12 recipes for drug preparation referring to over 250 various

plants, some of them alkaloid such as poppy, henbane, and mandrake (Kelly, 2009).

In ancient China lists of different plants and herb concoctions for

pharmaceutical purposes date back to 481 BC-221 BC. Many Chinese writers over the

centuries contributed to the written knowledge of herbal pharmaceuticals. There were

also the 11th century scientists and statesmen who compiled learned treatises on

natural history, emphasising herbal medicine (Needham et al., 1986)

The Chinese book on roots and grasses “Pen T’Sao,” written by Emperor Shen

Nung, 2500 BC, treats 365 drugs (dried parts of medicinal plants), many of which are

used even today such as the great yellow gentian, ginseng, jimson weed, cinnamon

bark, and Ephedra (Bottcher and Wiart,2006). The Ebers Papyrus(1550 BC),

represents a collection of 800 proscriptions referring to 700 plant species and drugs

used for therapy such as pomegranate, castor oil plant, aloe, senna, garlic, onion, fig,

willow, coriander, juniper, common centaury, etc.( Wikipaedia).

According to the data from the Bible and the holy Jewish book the Talmud,

aromatic plants like myrtle and incense were used during various rituals associated

with treatments (Dimitrova ,1999).There are also plants like Commiphera, Aloe vera,

Papaver somniferum, Asclepias acida, Cannabis indica and swallow wort which had

been used during crucifixion and anointing rituals of Jesus (Holger, 2001).

Theophrastus (371-287 BC) founded botanical science with his books “De

Causis Plantarium”— Plant Etiology and “De Historia Plantarium”—Plant History. In

the books, he generated a classification of more than 500 medicinal plants known at

the time (Beograd, 1958). The works of Hippocrates (459–370 BC) contain 300

medicinal plants classified by physiological action: wormwood and common centaury

were applied against fever; garlic against intestine parasites; opium, henbane, deadly

nightshade, and mandrake were used as narcotics; fragrant hellebore and haselwort as

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emetics; sea onion, celery, parsley, asparagus, and garlic as diuretics; oak and

pomegranate as astringents (Bojadzievski, 1992).

In his work “De re medica” the renowned medical writer Celsus (25 BC–50

AD) quoted approximately 250 medicinal plants such as aloe, henbane, flax, poppy,

pepper, cinnamon, the star gentian, cardamom, false hellebore, etc.(Tucakov,1948 ).

The work “De Materia Medica” offers plenty of data on the medicinal plants (50 and

70 AD by Pedanius). The work presents about 600 plants in all, along with some

animals and mineral substances, and around 1000 medicines made from these sources

(Krebs et al.,2003).

The Arabs introduced numerous new plants in pharmacotherapy, mostly from

India such as aloe, deadly nightshade, henbane, coffee, ginger, strychnos, saffron,

curcuma, pepper, cinnamon, rheum, senna etc;. Though in the Middle Ages people

used medicinal plants primarily as simple pharmaceutical forms like infusions,

decoctions and macerations, the demand for compound drugs was increasing. The

compound drugs comprised medicinal plants along with drugs of animal and plant

origin. (Toplak, 2005 and Bojadzievski, 1992)

In 18th century, in his work Species Plantarium (1753), Linnaeus (1707-1788)

provided a brief description and classification of the species described until then.

Early 19th century was a turning point in the knowledge and use of medicinal plants.

The discovery, substantiation, and isolation of alkaloids from poppy (1806),

Cephaelis ipecacuanha (1817), Strychnos (1817), quinine (1820), pomegranate

(1878) and other plants and the isolation of glycosides, marked the beginning of

scientific pharmacy. With the upgrading of the chemical methods, other active

substances from medicinal plants were also discovered such as tannins, saponosides,

etheric oils, vitamins, hormones, etc. (Dervendzi, 1992).

In late 19th and early 20th centuries, there was a great danger of elimination of

medicinal plants from therapy. Many authors wrote that drugs obtained from them

had many shortcomings due to the destructive action of enzymes, which cause

fundamental changes during the process of drying of medicinal plants, i.e. medicinal

plants’ healing action depends on the mode of drying. In 19th century, therapeutics,

alkaloids, and glycosides isolated in pure form were increasingly supplanting the

drugs from which they had been isolated. Many traditionally-used herbs have been

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put to the scientific test and have proven to possess remarkable curative powers. This

is one reason for the renewed interest in herbalism that we are seeing today. Herbs are

often proving to be effective and safe alternatives to dangerous and costly drugs.

Herbs are staging a comeback and herbal "renaissance" occurs all over the world.

According to the World Health Organization, 75% of the world's populations are

using herbs for basic healthcare needs. Up to now, the practice of herbal medicine

entails the use of more than 53,000 species, and a number of these are facing the

threat of extinction due to overexploitation. Herbalists today, believe to help

people build their good health with the help of natural sources. When herbs are taken,

the body starts to get cleansed. Unlike chemically synthesized, highly concentrated

drugs that may produce many side effects, herbs can effectively realign the

body's defences.

Most of the world’s medicinal plants were located in the tropical area, which

store about 2/3rd of all plant species, totaling about 2, 50,000 to 3, 00,000. India is a

botanical garden of the world and a gold mine of well practiced knowledge of herbal

medicine (Savithramma et al., 2011) and it has tremendous biodiversity, genetic

diversity as well as species and ecosystems. A list of over 20,000 medicinal plants has

been published (Deans and Svoboda, 1990) and with very much larger number of the

world's flowering plants.

Major pharmaceutical companies are currently conducting extensive research

on plant materials gathered from the rain forests and other places for their potential

medicinal value. Conservation of natural resources and its sustainable utility are

essential for the survival of human kind. Under the stress of over exploration and

habitat degradation a number of wild plants are essentially facing a constant threat of

extinction. Out of the 60,000 plant species that are listed as facing extinction, over

20,000 (or more) are from India alone. The botanical survey of India has prepared a

provisional list of threatened plants which includes a large number of wild (or) wild

relatives of food, horticultural, medicinal and aromatic plants. India is endowed with a

unique wealth of biota which includes a large number of medicinal and aromatic

plants. Many of these plants are rare and endemic and found only in wild sources.

Most of these wild medicinal and aromatic plants are highly habitat specific, found

only in forests and occupying highly specialized ecological niche with restricted

distribution.

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The conventional approaches to conservation include the in situ and ex situ

conservation strategies. In situ methods (gene banks, sanctuaries, national parks and

biosphere reservoirs etc.,) were traditional methods and were being widely followed

(Sudha, 1996). For many rare species, in situ preservation was not a viable option in

the face of increasing human disturbance. Species may decline and go extinct in the

wild due to genetic drift and inbreeding, demographic and environmental variation,

habitat loss, deteriorating habitat quality, competition from exotic species, disease or

over exploitation. Tissue culture, pollen bank and cryopreservation techniques used in

ex situ conservation were of recent origin and need highly sophisticated laboratory

facilities (Krogstrup et al., 1992 and Fay, 1994). Further there were number of

constraints for the propagation and conservation of many taxa through conventional

methods like vegetative and seed propagation.

So the urgency now is to conserve wild, rare, endangered and endemic flora

for future uses. At the same time, this also must ensure the mass cultivation of the

important medicinal plants which was being exploited from the wild by the

pharmaceutical industries (Akerele, 1991 and Thakur,1993). This has prompted

industries as well as scientists to take additional efforts that were inevitable to evolve

strategies to develop an appropriate mass propagation technique of several precious

medicinal plants and bring about the desired improvement for higher yield which was

warranted to meet the growing demand (Yeoman and Yeoman, 1996).

With a view to strengthen the medicinal plants sector all over the country as

well as to conserve the wild stock, the NMPB (National Medicinal Plants Board) was

set up by the Government of India in 2000. The prime objective of setting up the

board was to establish an agency which would be responsible for coordination of all

matters with respect to the medicinal plants sector, including drawing up policies and

strategies for in situ conservation, cultivation, harvesting, marketing, processing, drug

development, etc. (Kala and Sajwan 2007).

1.1 IN VITRO STUDIES

The galloping rate in the development of tissue culture is historically linked to

the discovery of cell and subsequently to the cell theory suggesting the totipotency of

cells. The knowledge on wound healing property and ‘polarity’ largely contributed to

the development of tissue culture techniques. The first successful culture to develop

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fully differentiated cells and the necessity of asepsis (Haberlandt,1902) formed the

basis for in vitro regeneration. Hanning (1904) successfully initiated the culture of

embryonic tissue on mineral salts and sugar solution which later became an important

area in in vitro techniques (Razdan, 2002). The credit for successful regeneration of

a bulky callus, buds and roots from poplar stem segments goes to Simon, 1908

(Razdan, 2002). This concept suggests that each plant cell has the ability to divide and

grow into a complete plant if suitable conditions of nutrition, light and temperature

are provided.

The development of culture media and the discovery of plant growth

regulators have paved way for understanding the theoretical and practical aspects of

plant tissue culture. Initially Knop’s mineral solution (Gautheret, 1939) was used for

plant callus cultures. Later there was gradual development of different media differing

essentially in mineral content. Media compositions were formulated considering

specific requirements. One of the earliest plant tissue culture medium was White’s

medium formulated for root culture. The MS medium (Murashige and Skoog, 1962)

and LS medium (Linsmaier and Skoog, 1965) were used successfully for the in vitro

propagation of dicot herbs and shrubs. B5 medium (Gamborg et al., 1968) was proved

valuable for protoplast culture. The Nitsch and Nitsch medium (Nitsch and Nitsch,

1969) has been developed for anther culture and it supports rapid embryogenesis in

protoplast culture. Woody Plant Medium was developed for propagation of

ornamentals, shrubs and trees (Lloyd and Mc Cown, 1980).

As the cells and tissues in culture medium lacks autotrophic ability external

carbon source for energy is required. The addition of external carbon source to the

medium enhances proliferation of cells and regeneration of green shoots

(Sathyanarayana and Dalia, 2007). Sucrose is the commonly used carbon source

followed by glucose, maltose and raffinose. Optimal shoot formation is favoured in

low concentration of sugar. Autoclaved sucrose is favoured for better growth as the

cells benefit from ready supply of glucose and fructose, brought about by the

hydrolysis of sucrose (Razdan, 2002).

Discovery of auxins and cytokinins in 1930s made it feasible for the

development of culture media that accelerated the growth, differentiation and

organogenesis of tissues. The requirement of these substances varies considerably

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with the tissue and their endogenous levels. A balance of auxin and cytokinin will

often produce an unorganized growth of cells, or callus, but the morphology of the

growth will depend on the plant species as well as the medium composition. An

excess of auxin will often result in a proliferation of roots, while an excess of

cytokinin may yield shoots. Media are usually gelled with agar to prevent tissue death

due to lack of oxygen. Agar, a polysaccharide extracted from sea weeds is an

excellent gelling agent as it do not react with media constituents and cannot be

digested by plant enzymes. Other substances that have successfully used are methacel,

alginate, phytagel and gelrite. The use of alternative gelling agents, like the Isubgol

product (Psyllium husk) and guar gum in the orchid species Dendrobium chrysotoxum

yielded good results (Jain and Babbar, 2002, 2011).Static liquid media gave good

results in the in vitro germination and plantlet growth in Doritaenopsis (Tsai and Chu,

2008), the micropropagation of Picrorhiza kurroa (Sood and Chauhan, 2009), Stevia

rebaudiana (Kalpana et al., 2009).

Prevention of contamination is an important and difficult aspect of successful

in vitro culture. Techniques like autoclaving and filter sterilisation is practised. Some

of the most commonly used surface sterilents are mercuric chloride, sodium

hypochlorite, ethyl alcohol, bromine water etc. Natrium hypochlorite as a sterilant in

culture media was tested positively in the species Ananas comosus (Teixeira et al.,

2006). The sterilization of culture media by using chlorine dioxide was tested, in

species Gerbera jamesonii (Cardoso and da Silva, 2012). Contamination was zero in

the multiplication stage and the treatment with 50 % chlorine dioxide gave optimal

results regarding multiplication rates.

In vitro culture is an efficient method for ex situ conservation of plant

biodiversity and multiplication of the endangered species. It enables the propagation

of the endangered species from minimum plant material that is available for

propagation. Single cells, plant cells without cell walls (protoplasts), pieces of leaves,

or (less commonly) roots can often be used to generate a new plant on culture media

given the required nutrients and plant hormones (Vidyasagar, 2006). Depending on

the species and culture conditions in vitro propagation can be achieved by four

different methods – enhanced axillary shoot proliferation, node culture, de novo

formation of adventitious shoots through organogenesis and somatic embryogenesis

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(Sathyanarayana and Dalia, 2007). The most commonly used method for commercial

production utilizes enhanced axillary shoot proliferation from cultured meristems.

The methods currently utilized for mass propagation of plants has its

advantages and limitations.

1. Somatic embryogenesis: This depends on stimulation of asexual embryos either

directly from cultured organ or indirectly from callus culture derived from cultured

organs (Takayama, 2002). A huge number of embryos can be regenerated from

various plant species. Such embryos can be further encapsulated with sodium alginate

and treated as synthetic seeds (Redenbaugh et al., 1991). However, wide expansion in

propagation by this method is usually hampered with the possible genetic

modifications that might appear, especially if intermediate callus phase is involved.

2. Adventitious bud formation: This method is based on the stimulation of organs

(stem, leaf, and root) on callus cultures through manipulation of growth regulators in

the medium (Akita and Takayama, 1994). High cytokinin/ auxin ratio in the medium

favours shoot initiation, while roots can be induced in the presence of high auxin/

cytokinin ratio. However, a balanced cytokinin/ auxin ratio leads to the regeneration

of complete plant. This method is characterized with relatively high number of

regenerated plants. Nevertheless, the chances of somaclonal or other variations might

be encountered in the developed plants.

3. Enhanced axillary branching in cultured shoot tips and lateral buds: This is

the most widely used method in the in vitro propagation programs (Hohnle and

Weber, 2007). The technique is based on the inhibition of the apical dominance by a

cytokinin, followed by stimulation of growth of bud primordia in the axils of leaves

within the cultured shoot tip or lateral bud. Shoots developed in this process are

severed and serially propagated, then individually rooted. This method is

characterized with moderate number of plant production with high genetic stability.

There is a limit to which shoot multiplication can be achieved in a single passage,

after which further axillary branching stops.

The credit for the development of micropropagation goes to Murashige who

showed that many plants could be propagated in vitro. Murashige (1974) described

the following basic stages in micropropagation.

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Development of aseptic cultures: This stage involves development of aseptic culture

of the selected plant material. The explants are surface sterilised and cultured in

selected media providing suitable conditions of light, temperature and relative

humidity. Explant quality and responsiveness is influenced by the physiological

condition of the donor plants. Tissue culture success mainly depends on the age, type

and position of explants because not all plant cells have the same ability to express

totipotency (Sasikumar et al., 2009). Pathogen free explants can be used if meristems

are selected or by maintaining disease free plants as stock.

Multiple shoot induction: In this stage the shoot propagules formed are cultured

again to increase their number. Sub cultures are maintained to produce large number

of shoots. Shoot production is encouraged by varying the medium with appropriate

amount of cytokinins and auxins. The possibility of number of subcultures from the

original culture depends on the type of species.

Rooting in vitro: This stage involves the in vitro rooting of the shoots prior to their

transfer to the soil. Special media are utilised to induce root formation. Ex vitro

rooting can also be induced for these shoots.

Acclimatization stage: The transfer of plants from in vitro to ex vitro environment is

extremely important. Plants developed through tissue culture are heterotrophic, lack

epicuticular wax (Adelberg et al., 2000), as well as having non-functional stomata

(Czynczyk and Takubowski, 2007). Such plants cannot survive the outside

unfavourable conditions. In acclimatization the plantlets are physiologically and

anatomically adjusted to the external environment. This is a slow process and may

take weeks. Success rate in hardening increases when elongated shoots with good

number of leaves and roots were used.

There were certain problems encountered while culturing. Microbial

contamination is the main problem faced in tissue culture. Microbes multiply and

compete with growing explant for nutrients, while releasing chemicals which can alter

culture environment like the pH and inhibit the growth of explants or cause death

(Leifert and Waites, 1992.). Autoclaving the media and equipments before the culture

process kills unwanted microbes. Other methods to reduce risk of contamination

include using a laminar flow hood with adequate air flow, keeping cell cultures in a

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room that is not commonly used, disinfect all fume hood surfaces that will be used

(Jamie Pighin, 2013).

Browning of tissues occurs in certain species. This is due to high

concentration of phenolic substances which are oxidised when cells are wounded.

Irreparable growth inhibition occurs when phenols are oxidised to highly active

quinine compounds. Browning can be minimized by adding antioxidants or phenol

absorbents for e.g. ascorbic acid, glutathione, activated charcoal and polyvinyl

pyrrolidone (Matkowski, 2000) or by transferring explants into new culture media on

regular intervals (Rout et al., 2000) or by inhibiting the action of phenol oxidase

enzymes by the addition of chelating agents (Sathyanarayana and Dalia, 2007).

Vitrification of tissues can be controlled by maximizing the air exchange

through the container closure or by the addition of phloroglucinol to the media. If the

callus formation is slow, its frequency can be enhanced by increasing the size of the

explants or the surface area of the wound site.

Apart from their use as a tool of research, plant tissue culture techniques in

recent years, have major importance in the area of plant propagation, disease

elimination, plant improvement and production of secondary metabolites.

Endangered, threatened and rare species have successfully been grown and conserved

by micro propagation because of high coefficient of multiplication. In addition, plant

tissue culture is considered to be the most efficient technology for crop improvement

by the production of somaclonal and gametoclonal variants. The micropropagation

technology has a vast potential to produce plants of superior quality, isolation of

useful variants in well-adapted high yielding genotypes with better disease resistance

and stress tolerance capacities (Franzon, 2004). Certain type of callus cultures give

rise to clones that have inheritable characteristics different from those of parent plants

due to the possibility of occurrence of somaclonal variability, which leads to the

development of commercially important improved varieties. It is a rapid propagation

processes that can lead to the production of virus free plants (Mitra, 2010). The

technique of in vitro conservation of germplasm is mainly used to conserve plant

which do not produce seeds or which have recalcitrant seeds which cannot be stored

under normal storage conditions in seed gene banks. Hence, vegetatively propagated

crops such as root and tubers, ornamentals, medicinal plants and many other tropical

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fruits can be conserved using in vitro methods (Akin-Idowu et al., 2009). Polyploids

has prospective in the development of agriculture and can be produced by treating the

cultured tissue with colchicine. Embryo rescue also is an application of tissue culture

technology. Micropropagation ensures a good regular supply of medicinal plants,

using minimum space and time (Prakash and Van Staden, 2007).

More recently, attention has turned to the mechanization and automation of

culture techniques to explore the possible beneficial effects of microorganisms in in

vitro plant cultures. Bioreactors helps to grow large and compact masses of shoot

meristem. The meristems are processed, sorted, distributed and allowed to develop

into rooted plantlets. The plantlets are automatically transplanted in the soil by

planting machine (Sathyanarayana and Dalia, 2007). The technique reduces labour

input and cost of production.

Mycorrhization in micropropagation, particularly the use of arbuscular

mycorrhizal fungi (AMF), is gaining momentum due to a demonstrated positive

impact on post transplant performance of in vitro grown plants (Rai, 2001). Improved

nutrient uptake, water relations, aeration, soil pH balance (Sylvia, 1998) and their

potential use as bioregulators have recently heightened research interest in AMF,

contributing to the development of effective AMF production methods,

mycorrhization of in vitro plants and screening for efficient AMF strains. The root

endophyte Piriformospora indica promotes explants hardening (Sahay and Varma,

1999); Psuedomonas sps. can reduce hyperhydricity (Bela et al., 1998) and Bacillus

pumilus, Alcaligenes faecalis and Psuedomonas spp. improve shoot multiplication

(Monier et al., 1998).

Cryobionomics is a new approach to study genetic stability in the

cryopreserved plant materials. The embryonic tissues can be cryopreserved for future

use or for germplasm conservation (Pasqual et al., 2008).

The encapsulation technology consists of the inclusion of some millimeter-

long plant portions in a nutritive and protective matrix. This technology represents a

further and promising tool for exchange of plant material between private and public

plant tissue culture laboratories, for short-term and medium-term storage of valuable

plant material and for use of in vitro derived or micropropagated propagules directly

in farm or in nurseries. After encapsulation, transport, storage and sowing in aseptic

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conditions, the enclosed explants (capsules) may evolve in shoots (regrowth) and be

employed for subsequent micropropagation or culture in vitro. When the encapsulated

explant evolves in plantlet (conversion) in in vitro or in vivo conditions, the product of

the encapsulation is defined as synthetic seed or artificial seed or synseed.

Encapsulated shoot buds can produce adventitious roots and germinate to produce

plants when planted in soil.

The development of genetic engineering and molecular biology techniques

allowed the appearance of improved and new agricultural products (Santos et

al.,2010) .These new approaches would have been impossible without the

development of tissue culture techniques, which provided the tools for the

introduction of genetic information into plant cells. One of the most promising

methods of producing proteins and other medicinal substances, such as antibodies and

vaccines, is the use of transgenic plants. Transgenic plants represent an economical

alternative to fermentation-based production systems. Plant-made vaccines or

antibodies are especially striking, as plants are free of human diseases, thus reducing

screening costs for viruses and bacterial toxins. Tissue culturing of medicinal plants is

widely used to produce active compounds for herbal and pharmaceutical industries.

Conservation of genetic material of many threatened medicinal plants also involves

culturing techniques.

1.2. PHYTOCHEMICAL STUDIES

The use of plants as medicines has been in use since the beginning of

civilization and passed on to subsequent generations. The continuous and perpetual

interest of the people in medicinal plants has brought about today's modern and

sophisticated fashion of their processing and usage. Nature has been a source of

medicinal agents for thousands of years and an impressive number of modern drugs

have been derived from natural sources, many of these isolations were based on the

uses of the agents in traditional medicine (Cragg and Newman 2001). India has a rich

cultural heritage of traditional medicines which chiefly comprised the two widely

flourishing systems of treatments i.e. Ayurvedic and Unani systems since ancient

times (Surana et al. 2008).

Indian herbal treatment dates back to 5000 BC and was recorded in Rig veda

and Atharva veda. The Charaka Samhita (900 B.C.) is the first recorded treatise fully

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devoted to the concepts and practice of Ayurveda. Ayurveda is considered as a

complete medical system that takes in to consideration physical, psychological,

philosophical, ethical and spiritual well being of mankind. The system involves

holistic approach to life and stresses the importance of living in harmony with nature.

The diagnostic and treatment procedures used are unique and based on its

foundational principles of panchamahabhutha (five basic elements of nature), tridosha

(three humours) and prakrithi (individual constitution) (Venkatasubramanian,

2007).Other texts that describe about the Indian traditional system of medicine are

Susruta Samhita (6th century BC), Astanga Hridayam (500 A.D) and Madhava Nidana

(800-900 A.D.)

India is one of the 12-mega biodiversity centres having about 10% of the

world’s biodiversity wealth, which is distributed across 16 agro-climatic zones (Shiva

1996). Medicinal plants are highly esteemed all over the world as a rich source of

therapeutic agents for the prevention of diseases and ailments (Sharma et al., 2008).

The search for eternal health and longevity and for remedies to relieve pain and

discomfort drove early man to explore his immediate natural surroundings and led to

the use of many plants, animal products, minerals etc. and the development of a

variety of therapeutic agents (Nair and Chanda, 2007). Integrative medicine is the

combination of traditional medicine with conventional or western medicine and

provides novel medicines for treatment of animals, human beings and their diseases

(Makkar et al., 2007). Recently, considerable attention has been paid to utilize eco-

friendly and bio-friendly based products for the prevention and cure of different

human diseases (Dubey et al., 2004). Therefore, such plants should be investigated to

better understand their properties, safety and efficiency. Plants have a limitless ability

to synthesize aromatic substances mainly secondary metabolites of which at least

12,000 have been isolated, a number estimated to be less than 10% of the total

(Mallikharjuna et al., 2007). More than 6000 plants in India including endemics are in

use in traditional folk and herbal medicine representing about 75% of medicinal needs

of the third world countries (Rajasekharan, 2002).

The rich knowledge of India and China in medicinal plants and health care has

led to the keen interest by pharmaceutical companies to use this knowledge as a

resource for research and development programs in the pursuit of discovering novel

drugs (Krishnaraju et al., 2005).

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There has been an explosive growth of herbal drug industry recently and data

analysis had shown that more and more people are consulting the herbal medicine

practitioners.

The use of traditional medicine and medicinal plants in most developing

countries, for the maintenance of good health, has been widely observed (UNESCO

1996). Furthermore, an increasing reliance on the use of medicinal plants in the

industrialized societies has been traced to the extraction and development of several

drugs and chemotherapeutics from these plants as well as from traditionally used rural

herbal remedies (UNESCO 1998).The Pharmaceutical Research and Development

Committee report of Ministry of Chemicals, Government of India also underscores

the importance of traditional knowledge (Mashelkar, 1999). The increasing use of

traditional therapies demands more scientifically sound evidence for the principles

behind such therapies and for effectiveness of medicines. Recent advances in the

analytical and biological sciences and with the innovations in genomics and

proteomics, validation of these therapies is possible. Western scientific community

views traditional medicines cautiously and stresses the concerns related to research,

development and quality (Patwardhan et al., 2003; Fabricant and Farnsworth

2001).Investigation of the chemical and biological activities of plants during the past

two centuries have yielded compounds for the development of modern synthetic

organic chemistry as a major route for discovery of novel and more effective

therapeutic agents (Nair et al., 2007).

The world is showing increasing attention to the importance of medicinal

plants and traditional health systems in solving the health care problems. Such a

revival in the traditional system is demanding the research on medicinal plants

phytochemically and pharmacognostically, often leading to the loss of natural habitats

and populations in the countries of origin. Most of the developing countries have

adopted traditional medical practice as an integral part of their culture. Historically,

all medicinal preparations are derived from plants, whether in the simple form of raw

plant materials or in the refined form of crude extracts, mixtures, etc (Krishnaraju et

al., 2005).

Worldwide trend towards the utilization of natural plant remedies has created

an enormous need for information about the properties and uses of medicinal plant as

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antitumor, anti analgesic and insecticides. Besides medicines, plants provides

thousand of novel compounds such as fragrance, flavorings, dyes, fibres, foods,

beverages, building materials etc. (Mungole and Chaturvedi, 2011). Several factors

are responsible for the recent curiosity in herbal remedies. Dissatisfaction with the

results from synthetic drugs and the belief that herbal medicines may be effective in

the treatment of certain diseases where conventional therapies and medicines have

proven to be inadequate. The high cost and side effects of most modern drugs was

another factor. The effectiveness of plant medicines and its source as renewable made

the people to be more inclined to herbal cure. There is also a commonly held belief

that herbal products are superior to manufactured products.

The rapid pace of research and development in herbal medicine has made it an

interdisciplinary science. The adverse effects of using antibiotics and other synthetic

compounds on human and animal health, and on product quality and safety have

regenerated interest in the fields of phytochemistry, phytopharmacology,

phytomedicine and phytotherapy during the last decade. Even though the use of

antibiotics is banned in the livestock health, its indiscriminate use has led to

pathogens becoming resistant to these chemicals. Hence it is fitting to research into

plant phytochemical constitution to use it as potential natural alternatives for

enhancing live stock productivity.

Knowledge of medicinal properties of herbs is growing as a result of research

and testing, which will make them an increasingly safe and preferred alternative to

allopathic medicine. There is a renewed interest in traditional medicine and an

increasing demand for more drugs from plant sources. This revival of interest in plant-

derived drugs is mainly due to the current widespread belief that “green medicine” is

safe and more dependable than the costly synthetic drugs, many of which have

adverse side effects (Parekh and Chanda, 2008).

1.2.1 Secondary metabolites

Plant cells and tissue cultures hold great promise for controlled production of

useful secondary metabolites on demand. Secondary metabolites are chemicals

produced by plants for which no role has yet been found in growth, photosynthesis,

reproduction, or other "primary" functions. These chemicals are extremely diverse;

many thousands have been identified in several major classes. Each plant family,

16

genus, and species produces a characteristic mix of these chemicals, and they can

sometimes be used as taxonomic characters in classifying plants. It has been estimated

that well over 3,00,000 secondary metabolites exist, and their primary function is to

increase likely hood of an organism’s survival repelling or attracting other organisms.

Secondary metabolites often play an important role in plant defense against herbivory

(Stamp and Nancy, 2003) and other interspecies defences (Samuni et al.,2012).

Secondary plant products are that occur usually only in special, differentiated

cells and are not necessary for the cells themselves but may be useful for the plant as

a whole. Humans use secondary metabolites as medicines, flavorings, and recreational

drugs. Apart from that, phenols, tannins and alkaloids are routinely used to give

antioxidant and antimicrobial activities. These are also used as antiseptics and

astringents (Usher et al., 2011). Secondary metabolites are chemicals produced by

means of secondary reactions resulting from primary carbohydrates, aminoacids and

lipids (Ting, 1982). Wahid and Ghazanfer (2004) and Wahid and Babu (2005)

reported that high level of secondary metabolites can enhance salt tolerance in

sugarcane and wheat respectively.

During the last 20 to 30 years, the analysis of secondary plant products has

progressed a lot. The use of modern analytical techniques like chromatography (in all

its variations), electrophoresis, isotope techniques and enzymology succeeded in the

elucidation of exact structural formulas and the most important biosynthetic

pathways. Many secondary compounds have signalling functions. Some plants, for

example, produce specific phytoalexines, in reponse to fungi infection, that inhibit the

spreading of the fungi mycelia within the plant. A number of substances is secreted

and influences the existence of other species. Many of them are antibiotic i.e. they

inhibit the existence of competing species in the surrounding of their producer thus

safeguarding its ecological niche. The mutual influence of plants by secretions is

called allelopathy. Allelopathic substances may damage the germination, growth and

development of other plants. Their influence is only rarely stimulating. Insects (and

other animals) have developed defence strategies against the insecticide effects of

some secondary plant products. Some species, need starting compounds for their

steroid synthesis that were originally meant to be a plant defence. It should be

mentioned that some plant products have psychopharmacological effects and

morphine or mescaline are even counted among the 'hard' drugs.

17

Secondary metabolites can be classified on the basis of chemical structure (for

example, having rings, containing a sugar), composition (containing nitrogen or not),

their solubility in various solvents, or the pathway by which they are synthesized

(e.g., phenylpropanoid, which produces tannins). A simple classification includes

three main groups: the terpenes (made from mevalonic acid, composed almost entirely

of carbon and hydrogen), phenolics (made from simple sugars, containing benzene

rings, hydrogen, and oxygen), and nitrogen-containing compounds (extremely

diverse, may also contain sulfur).

As secondary metabolites lack primary functions in the plant and have specific

negative impacts on other organisms such as herbivores and pathogens , it is

hypothesised that they have evolved because of their protective value. It is well

known now that their presence in different parts of the plant like root, leaves, bark etc.

deters feeding by slugs, snails, insects and vertebrates, as well as attacks by viruses,

bacteria and fungi (Winks and Schimmer, 1999). Many secondary metabolites are

toxic or repellant to herbivores and microbes and help defend plants producing them.

Production increases when a plant is attacked by herbivores or pathogens. Some

compounds are released into the air when plants are attacked by insects; these

compounds attract parasites and predators that kill the herbivores. Recent research is

identifying more and more primary roles for these chemicals in plants as

signals, antioxidants, and various other functions.

Consuming some secondary metabolites can have severe consequences.

Alkaloids can block ion channels, inhibit enzymes, or interfere with neuro-

transmission, producing hallucinations, loss of coordination, convulsions, vomiting,

and death. Some phenolics interfere with digestion, slow growth, block enzyme

activity and cell division, or just taste awful. Most herbivores and plant pathogens

possess mechanisms that ameliorate the impacts of plant metabolites, leading to

evolutionary associations between particular groups of pests and plants. Some

herbivores (for example, the monarch butterfly) can sequester plant toxins and gain

protection against their enemies. Secondary metabolites may also inhibit the growth

of competitor plants (allelopathy). Some metabolites such as terpenoids, carotenes,

phenolics, and flavonoids gives color to flowers and together with terpene and

phenolics release odours to attract pollinators.

18

Flavonoids, terpenes, phenols, alkaloids, sterols, waxes, fats, tannins, sugars,

gums, suberins, resin acids and carotenoids are among the many classes of

compounds known as secondary or special metabolites (Gottlieb, 1990).Most

pharmaceuticals are based on plant chemical structures, and secondary metabolites are

widely used for recreation and stimulation (the alkaloids nicotine and cocaine; the

terpene cannabinol). Psychoactive plant chemicals are central to some religions, and

flavors of secondary compounds shape our food preferences. The characteristic

flavors and aroma of cabbage and relatives are caused by nitrogen-and sulfur-

containing chemicals, glucosinolates, which protect these plants from many enemies.

The astringency of wine and chocolate derives from tannins. The use of spices and

other seasonings developed from their combined uses as preservatives (since they are

antibiotic) and flavorings.

A wide range of bioactive compounds have been studied and isolated from

medicinal plants. Eventhough plant produces these chemicals to protect itself, recent

research demonstrates that many phytochemicals can protect humans against diseases

(Kumar et al., 2009). Different phytochemicals have been found to possess a wide

range of activities, which may help in protection against chronic diseases. For

example phytochemicals such as saponins, terpenoids, flavonoids, tannins, steroids

and alkaloids have anti-inflammatory effects (Manch et al., 1996; Latha et al., 1998

and Akindele and Adeyemi, 2007). Glycosides, flavonoids, tannins and alkaloids have

hypoglycemic activities (Oliver, 1980; Cherian and Augstin, 1995). Rupasinghe et al.

(2003) have reported that saponins possess hypocholesterolemic and antidiabetic

properties. The terpenoids have been shown to decrease blood sugar level in animal

studies (Luo et al., 1999). Steroids and triterpenoids showed the analgesic properties

(Sayyah et al., 2004 and Malairajan et al., 2006). The steroids and saponins are

responsible for central nervous system activities (Argal and Pathak, 2006). Before the

discovery of modern pesticides, plant extracts containing nicotine and pyrethrin were

widely used in agriculture as insecticides.

Antioxidants are phytochemicals that help prevent the free radical damage.

Phytochemicals with antioxidant activity may reduce the risk of cancer and improves

heart health. They are also being investigated as possible treatments for

neurodegenerative disorders like Alzheimer’s disease, Parkinson’s disease etc.

19

Alkaloids are compounds containing basic nitrogen atoms. They can be

purified from crude extracts by acid-base extraction. Many alkaloids are toxic to other

organisms. They often have pharmacological effects and are used as medications,

as recreational drugs. Alkaloids are also known to regulate plant growth (Aniszewski

and Tadeusz, 2007). Alkaloids have a wide variety of chemical structures eg.

monocyclic, dicyclic, tricyclic, tetracyclic, and more complex cage structures, and are

classified according to the type of ring pyrrolidine, piperidine etc. and their

biosynthetic origin. Alkaloids are used as stimulant, adenosine receptor antagonist,

remedy for gout, sympathomimetic, vasodilator, antihypertensive, antipyretics,

antimalarial etc.

Phenols are a class of chemical compounds having a hydroxyl group (OH)

bonded directly to an aromatic hydrocarbon group. Some natural phenols can be used

as biopesticides. Some phenols are used as drugs like Crofelemer (USAN, trade name

Fulyzaq). This drug is under development for the treatment of diarrhoea associated

with anti-HIV drugs. Additionally, derivatives have been made of phenolic

compound, combretastatin A-4, an anticancer molecule, including nitrogen or

halogens atoms to increase the efficacy of the treatmen (Miriam et al., 2010).

Tannin is an astringent and bitter plant polyphenolic compounds that binds

and forms precipitates with proteins and various other organic compounds

including amino acids and alkaloids. The poliovirus, herpes simplex virus and various

enteric viruses are inactivated when incubated with red grape juice and red wines with

a high content of condensed tannins. (Bajaj, 1988). In tissue-cultured cell assays

tannins have shown antiviral (Lü L et al., 2004) antibacterial (Akiyama et al., 2001)

and antiparasitic effects (Kolodziej and Kiderlen, 2005) Tannins are mainly found in

bud and foliage tissues, seeds, bark, roots, sapwood and heartwood; but bark and

heartwood often contain the highest levels.

Saponins are a class of chemical compounds found in abundance in various

plant species and are amphipathic glycosides. The amphipathic nature of the class

gives them activity as surfactants that can be used to enhance penetration of

macromolecules such as proteins through cell membranes. Saponins have also been

used as adjuvants in vaccines.

20

Flavanoids have the general structure of a 15-carbon skeleton, which consists

of two phenyl rings and a heterocyclic ring. It inhibit coagulation, thrombus formation

or platelet aggregation, reduce risk of atherosclerosis, reduce arterial blood

pressure and risk of hypertension, reduce oxidative stress and related signalling

pathways in blood vessel cells. Dietary flavonoid intake is associated with reduced

gastric carcinoma risk in women (González et al., 2013) and reduced aero digestive

tract cancer risk in smokers (Woo and Kim, 2013). Of the 8000 known phenolic

compounds, around 4000 are flavonoids (Harborne, 2000). Flavonoids commonly

occur in foliage, bark, sapwood and heartwood in trees.

The discovery of new bioactive natural products is still a fascinating field in

organic chemistry as demonstrated by the recent paradigms of the anticancer drug

epothilon, the immunosuppressant rapamycin, or the proteasome inhibitor

salinosporamide, to name but a few of hundreds of possible examples. Finding new

secondary metabolites is a prerequisite for the development of novel pharmaceuticals,

and this is an especially urgent task in the case of antibiotics due to the rapid

spreading of bacterial resistances and the emergence of multiresistant pathogenic

strains, which poses severe clinical problems in the treatment of infectious diseases.

Years of testing are required before any new drug is approved for use in human

beings.

1.2.2 Screening of Secondary metabolites

The process of screening begins with extraction of compounds with various

solvents like water, methanol, ethanol, acetone etc. A beneficial compound may be

extracted with a toxic one that masks the benefits of the first. So it is necessary to

separate crude extracts into their various components and test each individually.

Previously the crude drugs were identified by comparison only with the standard

descriptions available, but recently due to advancement in the field of pharmacognosy

various techniques have been following for the standardization of crude drugs

(Savithramma et al., 2010).

The integration of herbal medicine into modern medical practises must take

into account the interrelated issues of quality, safety and efficacy. The lack of

pharmacological and clinical data on the majority of herbal medicinal products is a

major impediment to the integration of herbal medicines into conventional medical

21

practise. For valid integration pharmacological and clinical studies must be on those

plants lacking such data. It is very important that a system of standardization is

established for every plant medicine in the market because the scope for variation in

different batches of medicine is enormous (Ekka et al. 2008). A multidisciplinary

approach to drug development from medicinal plants used in traditional medicine was

tried out in several technical assistance programmes by UNIDO, if a successful plant

is identified, its large scale cultivation was to be implemented with FAO and clinical

assessment of the drug to be conducted with WHO participation.

The screening of plants for medicinal value has been carried out by number of

workers with the help of preliminary phytochemical analysis (Dan et al., 1978; Kumar

et al., 1990; Ram, 2001). Phytochemical screening is of paramount importance in

identifying new source of therapeutically and industrially valuable compound having

medicinal significance, to make the best and judicious use of available natural wealth.

Quantitative phytochemical determination is very important in identifying new

sources of therapeutically and industrially important compounds like alkaloids,

flavonoids, phenols, saponins, steroids, tannins, terpenoids etc (Akindele and

Adeyemi, 2007).

The chemical composition of herbal products varies depending on several

factors, such as botanical species, used chemotypes, the anatomical part of the plant

used (seed, flower, root, leaf, fruit rind, etc.), and the storage, humidity, type of

ground, time of harvest, geographic area etc. This variability can result in significant

differences in pharmacological activity, involving pharmacodynamics and

pharmacokinetics issues (Park, 2008). Strict guidelines have to be followed for the

successful production of a quality herbal drug. The medicinal plants should be

authentic and free from harmful materials like pesticides, heavy metals, microbial and

radioactive contamination. The source and quality of raw materials, good agricultural

practices and manufacturing processes are certainly essential steps for the quality

control of herbal medicines and play a pivotal role in guaranteeing the quality and

stability of herbal preparations. The herbal extract should be checked for biological

activity in experimental animal models. The bioactive extract should be standardized

on the basis of active compound. The bioactive extract should undergo limited safety

studies (De Smet 1997; Blumenthal et al., 1998; EMEA 2002; WHO 2004; Ahmad et

al., 2006; Samy and Gopalakrishnakone, 2007).

22

1.3 NUTRITIVE VALUE

Plants have great importance due to their nutritive value and continue to be a

major source of medicines as they have been found throughout human history

(Balick et al., 1996) 30 to 40% of today’s conventional drugs used in the medicinal &

curative properties of various plants are employed in herbal supplements,

nutraceuticals and drugs (Shulz et al., 2001). All human beings require number of

complex organic compounds as added (William, 1972) caloric requirements to meet

the need for their muscular activities, carbohydrates, fats and proteins, while minerals

and vitamins form comparatively a smaller part, plant materials form major portion of

the diet; their nutritive value is important (Indrayan AK et al., 2005).

A primary metabolite is a kind of metabolite that is directly involved in

normal growth, development, and reproduction. It usually performs a physiological

function in the organism (i.e. an intrinsic function). Primary metabolites comprise

many different types of organic compounds, including carbohydrates, lipids, proteins,

and nucleic acids. They are found universally in the plant kingdom because they are

the components or products of fundamental metabolic pathways or cycles such as

glycolysis, the Krebs cycle, and the Calvin cycle. Because of the importance of these

and other primary pathways in enabling a plant to synthesize, assimilate, and degrade

organic compounds, primary metabolites are essential. Examples of primary

metabolites include energy rich fuel molecules, such as sucrose and starch, structural

components such as cellulose, informational molecules such as DNA

(deoxyribonucleic acid) and RNA (ribonucleic acid), and pigments, such as

chlorophyll. In addition to having fundamental roles in plant growth and

development, some primary metabolites are precursors for the synthesis of secondary

metabolites.

A carbohydrate is a macromolecule of carbon (C), hydrogen (H), and oxygen

(O) atoms, usually with a hydrogen: oxygen atom ratio of 2:1 with the empirical

formula Cm (H2O) n. Carbohydrates perform numerous roles in living organisms.

Polysaccharides serve for the storage of energy (e.g., starch and glycogen), and as

structural components (e.g., cellulose in plants and chitin in arthropods). The 5-carbon

monosaccharide ribose is an important component of coenzymes (e.g., ATP, FAD,

and NAD) and the backbone of the genetic molecule known as RNA. The related

23

deoxyribose is a component of DNA. Saccharides and their derivatives include many

other important biomolecules that play key roles in the immune system, fertilization,

preventing pathogenesis, blood clotting, and development (Maton et al., 1993).

Proteins are large biological molecules, or macromolecules, consisting of one

or more long chains of amino acid residues. Proteins perform a vast array of functions

within living organisms, including catalyzing metabolic reactions, replicating DNA,

responding to stimuli, and transporting molecules from one location to another.

Proteins differ from one another primarily in their sequence of amino acids, which is

dictated by the nucleotide sequence of their genes, and which usually results

in folding of the protein into a specific three-dimensional structure that determines its

activity. The quality and quantity of proteins in the seeds are basic factors and

important for the selection of plants for nutritive value, systematic classification and

plant improvement programs (Nisar et al.,2009).

Lipids are a group of naturally occurring molecules that include fats, waxes,

sterols, fat-soluble vitamins, monoglycerides, diglycerides, triglycerides,

phospholipids, and others. The main biological functions of lipids include storing

energy, signalling, and acting as structural components of cell membranes (Fahy et

al., 2009 and Subramaniam et al., 2011). Lipids have applications in the cosmetic

and food industries as well as in nanotechnology (Mashaghi et al., 2013).

Nutritive importance of a plant can be analysed only if the toxicity of its

components are known. It was aptly quoted “In all things there is a poison, and there

is nothing without a poison. It depends on only upon the dose whether a poison is a

poison or not” (Paracelsus 1493-1541).

Humans consume a wide range of foods, drugs, and dietary supplements that

are derived from plants. It is presumed that ayurvedic drugs have lesser side effects as

compared to allopathic drugs. For the safety to use these plants and preparations (gel

and powder forms), the medicinal plants need to be evaluated for their toxicity. The

very defensive compounds that increase the reproductive fitness of plants by warding

off fungi, bacteria, and herbivores may also make them undesirable as food for

humans. Terpenes are toxins and feeding deterrents to many herbivorous insects and

mammals; thus they appear to play important defensive roles in the plant kingdom.

For example, monoterpene esters called pyrethroids, found in the leaves and flowers

24

of Chrysanthemum species, show striking insecticidal activity. Triterpenes that defend

plants against vertebrate herbivores include cardenolides and saponins.

Cardenolides are glycosides (compounds containing an attached sugar or

sugars) that taste bitter and are extremely toxic to higher animals. Isoflavonoids,

which are found mostly in legumes, have several different biological activities. Some,

such as rotenone, can be used effectively as insecticides, pesticides (e.g., as rat

poison), and piscicides (fish poisons). Other isoflavones have anti-estrogenic effects;

for example, sheep grazing on clover rich in isoflavonoids often suffer from

infertility. Tannins act as feeding repellents to a great variety of animals. Mammals

such as cattle, deer, and apes characteristically avoid plants or parts of plants with

high tannin contents. Unripe fruits frequently have very high tannin levels, which

deter feeding on the fruits until their seeds are mature enough for dispersal.

Cyanogenic glycosides release the well-known poisonous gas hydrogen cyanide

(HCN). The presence of cyanogenic glycosides deters feeding by insects and other

herbivores such as snails and slugs. The cyanogenic glycosides have aglycones

derived from amino acids. Several of these compounds can interfere with the iodine

utilisation and result in hypothyroidism. Some saponins induce photosensitisation and

jaundice.

However, plant-derived alkaloids, by function and chemical nature, are toxic

to mammals (Rattan RS, 2010). Most compounds responsible for the potency of arrow

and dart poisons belong to three plant chemical groups, namely the alkaloids (e.g.,

strychnine from Strychnos species), cardiac glycosides (e.g; ouabain from

Strophanthus species), and saponins (e.g., a monodesmoside glucoside from Clematis

species) (Bisset, 1989). Pyrrolizidine alkaloids are produced in Asteraceae

particularly in Senecio spp. and in Boraginaceae. Their adverse effect in man and

animals are hepatotoxicity after bioactivation. The pseudoalkaloids in Cicuta virosa

and Conium maculatum have effects on the central nervous system and taxine in yews

like T. baccata inhibits the ion transport of the heart.

The very potent little protein (lectin) ricin inhibits protein synthesis and induce

systemic effects in animals and humans, with gastrointestinal symptoms dominating.

Far less potent lectins are also present in seeds of several species of Fabaceae. Colic

25

and other gastrointestinal symptoms may occur if seeds are eaten without sufficient

heat treatment, which inactivates many lectins.

Plants, including most food and feed plants, produce a broad range of

bioactive chemical compounds via their so called secondary metabolism. These

compounds may elicit a long range of different effects in man and animals, depending

on plant species and the amount eaten. Plants with potent bioactive compounds are

often characterised as both poisonous and medicinal, and a beneficial or an adverse

result may depend on the amount eaten and the context of intake. For typical food and

feed plants with bioactive compounds with less pronounced effects, the intakes are

usually regarded as beneficial (Aksel, 2010).

1.4 SILVER NANOPARTICLES

Nanotechnology is the manipulation of matter on an atomic, molecular, and

supramolecular scale. The novel properties of nanoparticles have been exploited in a

wide range of potential applications in medicine, cosmetics, renewable energies,

environmental remediation and biomedical devices (De M, 2007; Ghosh and Paria S,

2012) Among them, silver nanoparticles (Ag-NPs or nanosilver) have attracted

increasing interest due to their unique physical, chemical and biological properties

compared to their macro-scaled counterparts (Sharma, 2009). Ag-NPs have distinctive

physico-chemical properties, including a high electrical and thermal conductivity,

surface-enhanced Raman scattering, chemical stability, catalytic activity and non

linear optical behaviour (Krutyakov et al., 2008). Ag-NPs exhibit broad spectrum

bactericidal and fungicidal activity (Ahamed et al.,2010). Besides silver exhibits low

toxicity (Jain et al.,2009); silver nanoparticles have diverse in vitro and in vivo

applications (Haes and Van Duyne,2002; McFarland and Van Duyne,2003). Although

there are many routes (Aymonier et al.,2002; Sun and Xia,2002) available for the

synthesis of silver nanoparticles, bioinspired synthesis using plant sources offers

several advantages such as cost-effectiveness, eco-friendliness, and the elimination of

high pressure, energy, temperature, and toxic chemicals necessary in the traditional

synthesis methods (Goodsell,2004 ).

Advances in nanotechnology have significantly impacted the field of

therapeutics delivery. This is evidenced by the increase in the number of nanoparticle

based therapeutic products in development over the last two decades. A 2006 global

26

survey conducted by the European Science and Technology Observatory (ESTO)

revealed that more than 150 companies are developing nanoscale therapeutics, and

twenty-four nanoparticle therapeutics are currently in clinical use (Wagner et al.,

2006). These drugs are being developed to treat a wide range of diseases, such as

fungal or bacterial infections, HIV infections, diabetes and cancers. There are several

advantages to using nanoparticles for therapeutics delivery. The use of materials on

the nanoscale level provides the unprecedented freedom to modify some of the most

fundamental properties of therapeutic carriers, such as solubility, diffusivity,

biodistribution, release characteristics and immunogenicity. Precise nanoparticle

engineering has yielded longer circulation half-life, superior bioavailability and lower

toxicity (Emerich and Thanos, 2007; Groneberg et al., 2006).

One strategy to further improve the therapeutic index of nanoparticle

therapeutics is to functionalize nanoparticles with targeting ligands. The addition of

targeting ligands allows the delivery of drug-encapsulated nanoparticles to uniquely

identified sites while having minimal undesired effects elsewhere. Since biologically

targeted nanoparticles have the potential to be the optimal drug delivery vehicle, there

has been tremendous amount of interest in developing novel targeted nanoparticles for

therapeutic applications.

Although acute toxicity of silver in the environment is dependent on the

availability of free silver ions, investigations have shown that these concentrations of

Ag+ ions are too low to lead toxicity (WHO, 2002). Metallic silver appears to pose

minimal risk to health, whereas soluble silver compounds are more readily absorbed

and have the potential to produce adverse effects (Pamela and Kyle, 2005). The wide

variety of uses of silver allows exposure through various routes of entry into the body.

Ingestion is the primary route for entry for silver compounds and colloidal silver

proteins. Dietary intake of silver is estimated at 70-90μg/day. Silver in any form is not

thought to be toxic to the immune, cardiovascular, nervous or reproductive system

and it is not considered to be carcinogenic. Therefore silver is relatively non-toxic

(Chen, 2008)

1.4.1 Green synthesis

In recent years, green synthesis of silver nanoparticles (AgNPs) has gained

much interest from chemists and researchers. In this concern, Indian flora has yet to

27

divulge innumerable sources of cost-effective non-hazardous reducing and stabilizing

compounds utilized in preparing AgNPs. Since noble metal nanoparticles are widely

applied to areas of human contact (Jae and Beom, 2009), there is a growing need to

develop environmentally friendly processes for nanoparticle synthesis that do not use

toxic chemicals. A quest for an environmentally sustainable synthesis process has led

to a few biomimetic approaches. Biomimetics refers to applying biological principles

in materials formation. One of the fundamental processes in biomimetic synthesis

involves bioreduction. Biological methods of nanoparticle synthesis using

microorganisms (Klaus et al., 1999; Nair and Pradeep, 2002; Konishi and Uruga,

2007), enzymes (Willner et al., 2006), fungus (Vigneshwaran et al., 2007), and plants

or plant extracts (Shankar et al., 2004; Chandran et al., 2006; Jae and Beom, 2009)

has been variously reported.

Nanobiotechnology is presently one of the most dynamic disciplines of

research in contemporary material science whereby plants and different plant products

are finding an imperative use in the synthesis of nanoparticles (NPs). In general,

particles with a size less than 100 nm are referred to as NPs. Entirely novel and

enhanced characteristics such as size, distribution and morphology have been revealed

by these particles in comparison to the larger particles of the mass material that they

have been prepared from (Van den Wildenberg, 2005). NPs of noble metals like gold,

silver and platinum are well recognized to have significant applications in electronics,

magnetic, optoelectronics and information storage (Gratzel, 2001; Okuda et al., 2005;

Dai and Bruening, 2002 and Murray et al., 2001).

This current emerging field of nanobiotechnology is at the primary stage of

development due to lack of implementation of innovative techniques in large

industrial scale and yet has to be improved with the modern technologies. Hence,

there is a need to design an economic, commercially feasible as well environmentally

sustainable route of synthesis of Ag NPs in order to meet its growing demand in

diverse sectors (Priya et al., 2014).

When Ag-NPs are produced by chemical synthesis, three main components

are needed: a silver salt (usually AgNO3), a reducing agent (i.e. ethylene glycol) and a

stabilizer or aping agent (i.e. PVP) to control the growth of the NPs and prevent them

from aggregating. In case of the biological synthesis of Ag-NPs, the reducing agent

28

and the stabilizer are replaced by molecules produced by living organisms. These

reducing and/or stabilizing compounds can be utilized from bacteria, fungi, yeasts,

algae or plants (Sintubin et al., 2012).

In summary, the biological method provides a wide range of resources for the

synthesis of Ag-NPs, and this method can be considered as an environmentally

friendly approach and also as a low cost technique. The rate of reduction of metal ions

using biological agents is found to be much faster and also at ambient temperature and

pressure conditions. In biological synthesis, the cell wall of the microorganisms pays

a major role in the intracellular synthesis of NPs. The negatively charged cell wall

interacts electrostatically with the positively charged metal ions and bioreduces the

metal ions to NPs (Thakkar et al., 2010). When microorganisms are incubated with

silver ions, extracellular Ag-NPs can be generated as an intrinsic defense mechanism

against the metal's toxicity. Other green syntheses of Ag-NPs using plant exacts as

reducing agents have been performed (Amaladhas et al., 2012 and Umadevi et al.,

2012). This defense mechanism can be exploited as a method of NPs synthesis and

has advantages over conventional chemical routes of synthesis. However, it is not

easy to have a large quantity of Ag-NPs using biological synthesis.

1.4.2. Antimicrobial activity

The outbreaks of re-emerging and emerging infectious diseases are a

significant burden on global economies and public health especially in the developing

countries. The outbreak of diarrhoea disease caused by an unusual serotype of Shiga-

toxin–producing Escherichia coli (O104:�H4) began in Germany with a large

number of cases of diarrhoea with 3167 without the hemolytic–uremic syndrome (16

deaths) and 908 with the hemolytic–uremic syndrome (34 deaths) (Rasko DA et al.,

2011). Transmission of infectious pathogens to the community has caused outbreaks

of diseases such as influenza (H5N1), diarrhoea (Escherichia coli), cholera (Vibrio

cholera), etc throughout the world. The growth of population and urbanization along

with poor water supply and environmental hygiene are the main reasons for the

increase in outbreak of infectious pathogens. These infectious diseases have not only

occurred in developing countries with low levels of hygiene and sanitation, but have

also been recognized in developed countries. Food and waterborne pathogens are the

main factors for the outbreak of these diseases, the transmission of these pathogens

29

endangering public health. Their emergence is thought to be driven largely by socio-

economic, environmental and ecological factors. To prevent further spread of the

infectious pathogens, disinfection methods should be done properly to eliminate these

pathogens from infected environmental areas, and effective treatments should also be

carried for patients in hospitals and in the community.

The increase of antibiotic resistance of microorganisms to conventional drugs

has generated a considerable interest in the search for new, efficient and cost effective

ways for the control of infectious diseases. There is a constant search for new organic

molecules with antibacterial activity, which could be cheap and readily available to

the local population as a means of improving primary health care. Two to three

antibiotics derived from microorganisms are launched each year (Cowan, 1999).

Scientists have realised that in order to cope with this slow pace, coupled with the fact

that previously discovered drugs are rendered obsolete by resistant bacterial strains,

plant based remedies would have to be considered as alternative sources of new drugs.

The comprehensive treatments of environments containing infectious

pathogens using advanced disinfectant nanomaterials have been proposed for

prevention of the outbreaks. Particularly, the noble metal Ag-NPs is drawing

increasing attention for potential prevention of bacterial/fungal and viral infections

due to their well-documented antimicrobial and disinfectant properties. The

generation of stable and efficient Ag-NPs forms offers an advanced perspective in the

field of environmental hygiene and sterilization. (Quang et al.,2013)

Plant based antibacterial have enormous therapeutic potential as they can serve

the purpose with lesser side effects that are often associated with synthetic

antibacterials. Biomolecules of plant origin appear to be one of the alternatives for the

control of these antibiotic resistant human pathogens (Kumaraswamy et. al., 2008).

Knowledge of the chemical constituents of plants is desirable because the medicinal

value of plant lies in the chemical substances that produce a definite therapeutic

action on the human body. Some of these important bioactive compounds are

alkaloids, flavonoids, tannins and phenolic compounds. In addition, the knowledge of

the chemical constituents of plants would further be valuable in the discovery of the

actual value of folkloric remedies. The phytochemical research based on

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ethnopharmacological information is generally considered an effective approach in

the discovery of new anti – infective agents from higher plants (Chhetri et al., 2008).

Furthermore, nanoparticles are alternatives to antibiotics allowing better action

against multidrug opposing bacteria and consequently plant-derived nanoparticles

have been proved better than other methods (Savithramma et al., 2011 and Song et

al., 2009). The method of the AgNPs antibacterial action has been efficiently

explained in conditions of their interaction with cell membranes of bacteria by

troubling its permeability and respiratory role (Vankar and Shukla, 2012 and Ghosh et

al., 2012).

Silver nanoparticles have the ability to anchor to the bacterial cell wall and

subsequently penetrate it, thereby causing structural changes in the cell membrane

like the permeability of the cell membrane and death of the cell. There is formation of

“pits” on the cell surface, and there is accumulation of the nanoparticles on the cell

surface (Sondi et al., 2004). The formation of free radicals by the silver nanoparticles

may be considered to be another mechanism by which the cells die. There have been

electron spin resonance spectroscopy studies that suggested that there is formation of

free radicals by the silver nanoparticles when in contact with the bacteria, and these

free radicals have the ability to damage the cell membrane and make it porous which

can ultimately lead to cell death (Danilcauk et al., 2006; Kim et al., 2007). It has also

been proposed that there can be release of silver ions by the nanoparticles (Feng et al.,

2008), and these ions can interact with the thiol groups of many vital enzymes and

inactivates them (Matsumura et al., 2003). The bacterial cells, when in contact with

silver takes up silver ions, which inhibit several functions in the cell and damage the

cells. The reactive oxygen formed through the inhibition of a respiratory enzyme by

silver ions becomes suicidal and attack the cell itself. Silver is a soft acid, and there is

a natural tendency of an acid to react with a base. Majority of cells are made up of

sulphur and phosphorus which are soft bases. The action of silver nanoparticles on the

cell can cause the reaction to take place and subsequently lead to cell death. Another

fact is that the DNA has sulphur and phosphorus as its major components; the

nanoparticles can act on these soft bases and destroy the DNA which would definitely

lead to cell death (Morones et al., 2005). It has also been found that the nanoparticles

can modulate the signal transduction in bacteria. The phosphorylation of protein

substrates in bacteria influences bacterial signal transduction. Dephosphorylation is

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noted only in the tyrosine residues of gram-negative bacteria. The phosphotyrosine

profile of bacterial peptides is altered by the nanoparticles. It was found that the

nanoparticles dephosphorylate the peptide substrates on tyrosine residues, which leads

to signal transduction inhibition and thus the stoppage of growth. It is however

necessary to understand that further research is required on the topic to establish the

claims (Hatchett et al., 1996.).

Silver nanoparticles are widely used for its unique properties in catalysis,

chemical sensing, biosensing, photonics, electronic and pharmaceuticals (Sarkar et al.,

2010) and in biomedicine especially as antibacterial agent (Rai et al., 2009) and

antiviral agent (Elechiguerra et al., 2005). Silver nanoparticles have a great potential

for use in biological including antimicrobial activity (Sap-Iam et al., 2010). Silver is

an effective antimicrobial agent that exhibits low toxicity (Farooqui et al., 2010). The

antibacterial activity of SNPs are well known since ancient times (Srivastava et al.,

2007). Biological synthesis of nanoparticles by plant extracts is at present under

exploitation as some researchers worked on it (Calvo et al., 2006) and tested for

antimicrobial activities (Saxena et al., 2010 and Khandelwal et al., 2010).

The antibacterial effect of nanoparticles can be attributed to their stability in

the medium as a colloid, which modulates the phosphotyrosine profile of the bacterial

proteins and arrests bacterial growth. Among the various inorganic metal

nanoparticles, silver nanoparticles have received substantial attention for various

reasons – silver is an effective antimicrobial agent, exhibits low toxicity (Jain et al.,

2009 and Sondi and Sondi, 2004). The antimicrobial effect of silver additives is

broadly used in various injection-moulded plastic products in textiles (Gao and

Cranston, 2008) and in coating based application including air ducts, counter tops and

food preparation areas (Galeano et al., 2003). Some important advantages of silver

based antimicrobials are their excellent thermal stability and their health and

environmental safety (Kumar and Munstedt, 2005).

The silver nanoparticles (SNPs) have various important applications.

Historically, silver has been known to have a disinfecting effect and has been found in

applications ranging from traditional medicines to culinary items (Chikramane, et al.,

2010) environmental and health (Singh et al., 2011). It has been reported that SNPs

are non-toxic to human and most effective against bacteria, virus and other eukaryotic

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microorganisms at low concentrations (Sharma et al., 2009). Several salts of silver

and their derivatives are commercially manufactured as antimicrobial agents

(Krutyakov et al., 2008). Antimicrobial capability of SNPs allows them to be suitably

employed in numerous household products such as textiles, food storage containers,

home appliances and in medical devices (Marambio and Hoek, 2010). The most

important application of SNPs is in medical industry such as tropical ointments to

prevent infection against burn and open wounds (Ip et al., 2006).

As herbal medicines are gaining much popularity, there is overexploitation of

plant species with medical values, which leads to threat to their existence in the

natural habitat. Moreover, development of road ways has paved enough opportunities

to reach remote places and forest areas to collect the material from wild. The species

growing in wild possess highly potential active principles when compared to

cultivated species. Due to this the plants are utilized in enormous quantity for

pharmaceutical preparations. As per IUCN data more than one plant species per

second is disappearing from globe.

Keeping this in view the present study was undertaken to fulfil the needs of

pharmaceutics and to meet its increasing demand, by developing an efficient protocol

through in vitro propagation using the various potential explants with the following

objectives.

OBJECTIVES

• To develop an efficient protocol for in vitro propagation of Clinacanthus

siamensis and Cissampelos pariera

• To screen the selected plants for qualitative and quantitative phytochemical

analysis.

• To determine the Nutritive values as the leaves are useful in the preparation of

medicine.

• To test the efficiency of the two taxa for biosynthesis of Silver nanoparticles

and their characterization.

• To evaluate and elucidate the potentiality of biologically synthesized Silver

nanoparticles against microbes.