Upload
others
View
0
Download
0
Embed Size (px)
Citation preview
1
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.
2
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
3
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
4
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.
5
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
6
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
7
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
8
(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.
9
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
10
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
11
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
12
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
13
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).
14
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
15
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
30
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
31
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
32
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.