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Chapter 2 Review of Literature
Department of Biotechnology, Jamia Hamdard 5
2. REVIEW OF LITERATURE
Our country is bestowed with a rich flora and a large area is covered with forests. These
forests have played key roles in the lives of people from the prehistoric age by providing
a diversity of valuable forest products for food, shelter and medicine (Kala, 2004). The
use of plants for medicines is, therefore, as old as our civilization. The first known
written record of curative plants was of Sumerian herbal of 2200 BC. In the 5th century
BC, the Greek doctor Hippocrates listed out 400 herbs in common use. Dioscorides, in
the 1st century AD mentioned 600 plants in one of his books which became the basis of
many later works. Although, traditional drugs of herbal, herbomineral and animal origin
have been used since the dawn of civilization to maintain and alleviate human suffering
from diseases, but the age-old traditional values attached with the medicinal plants have
gained tremendous importance in the present century (Stein, 2004; Kala , 2004). The
herbal medicines are, therefore, enjoying renaissance among the customers throughout
the world (Shinde et al., 2007) and according to an estimate of the world health
organization (WHO), about 80% of the world’s population uses herbs and other
traditional medicine for their primary health care needs (Khan et al., 2009). India and
China are two of the largest countries in Asia, which have the richest arrays of registered
and relatively well-known medicinal plants (Raven, 1998). In India, of the 17,000 species
of higher plants, 7500 are known for their medicinal uses (Shiva, 1996). The northern
part of India harbors a great diversity of medicinal plants because of the majestic
Himalayan range. So far about 8000 species of angiosperms, 44 species of gymnosperms
and 600 species of pteridophytes have been reported in the Indian Himalaya (Singh and
Hajra, 1996), of these 1748 species are known as medicinal plants (Samant et al., 1998).
The maximum medicinal plants (1717 species) have been reported around the 1800m
elevation range. On the regional scale, the maximum species of medicinal plants have
been reported from Uttaranchal (Kala, 2004) followed by Sikkim and North Bengal
(Samant et al., 1998). The trans-Himalaya sustains about 337 species of medicinal plants,
which is low compared to other areas of the Himalaya due to the distinct geography and
ecological marginal conditions (Kala, 2002). This proportion of medicinal plants is the
highest proportion of plants known for their medical purposes in any country of the world
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for the existing flora of that respective country. We, therefore, have an advantage of
making the traditional system of medicine, the basic source of healthcare.
These medicinal plants are also important source of other type of beneficial compounds
including the ingredients for functional foods. These functional foods promote better
health to prevent chronic illness. Some ingredients that make food functional are dietary
fibres, vitamins, minerals, antioxidants, oligosaccharides, essential fatty acids (omega-3),
lactic acid bacterial cultures and lignins. Many of these are present in medicinal plants.
Indian systems of medicine believe that complex diseases can be treated with complex
combination of botanicals unlike in the West, with single drugs. Whole foods are hence
used in India as functional foods rather than supplements. Some medicinal plants and
dietary constituents having functional attributes are spices such as onion, garlic, mustard,
red chilli, turmeric, clove, cinnamon, saffron, curry leaf, fenugreek and ginger. Some
herbs such as Bixa orellana and vegetables like amla, wheat grass, soybean and Garcinia
cambogia have antitumor effects (Dixit et al., 2005; Kulkarni et al., 2006). Other
medicinal plants with such functional properties include Aegle marmelos, Allium cepa,
Aloe vera, Andrographis paniculata, Azadirachta indica and Brassica juncea (Tilak and
Devasagayam, 2006).
Medicinal Plants also act as a rich source of antioxidants and radical scavengers
(Ravishankar and Shukla, 2007). In recent years, there is a tremendous interest in the
possible role of nutrition in prevention of diseases. In this context antioxidants, especially
derived from natural sources such as medicinal plants and herbal drugs derived from
them have gained special attention. Antioxidants neutralize the toxic and ‘volatile’ free
radicals. They have many potential applications, especially in relation to human health,
both in terms of prevention of diseases as well as therapy (Sies et al., 1996; Halliwell and
Gutteridge, 1997). In biological systems, oxygen gives rise to a large number of free
radicals and other reactive species, collectively known as ‘reactive oxygen species’
(ROS) and ‘reactive nitrogen species’ (RNS) (Kelly et al., 1998; Devasagayam et al.,
2004). In a normal healthy human, the generation of ROS and RNS are effectively kept
in check by the various levels of antioxidant defense. However, when the humans get
exposed to adverse physiochemical, environmental or pathological agents, this delicately
maintained balance is shifted in favour of pro-oxidants resulting in oxidative stress (Sies,
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1996). Cellular damage induced by oxidative stress has been implicated in the etiology of
a large number (>100) of human diseases as well as the process of ageing. Various
antioxidants may prevent and/or improve diseased states (Sies, 1996; Thomas and
Kalyanaraman, 1997; Devasagayam et al., 2004). These include the intracellular
antioxidant enzymes and the dietary or oral supplements in the form of vitamin C,
vitamin E, β-carotene, zinc and selenium (Knight et al., 2000; Fuente et al., 2000).
Antioxidants can also act at different levels of protection such as prevention, interception
and repair. The medicinal plants that show significant antioxidant activity include Acacia,
catechu, Achyranthes aspera, Aegle marmelos, Aglaia roxburghiana, Allium cepa, Allium
sativum, Aloe vera, Amomum subulatum, Andrographis paniculata, Asparagus
racemosus, Azadirachta indica, Bacopa monniera, Bauhinia purpurea, Brassica
campastris, Butea monosperma, Camellia sinensis, Capparis decidua, Capsicum annum,
Centella asiatica, Cinnamomum verum, Commiphora mukul, Crataeva nurvala, Crocus
sativus, Curcuma longa, Cymbopogan citrates, Emblica officinalis, Emilia sonchifolia,
Garcinia atroviridis, Garcinia kola, Glycyrrhiza glabra, Hemidesmus indicus,
Hypericum perforatum, Indigofera tinctoria, Melissa officinalis, Momordica charantia,
Morus alba, Murraya koenigii, Nigella sativa, Ocimum sanctum, Picrorrhiza kurroa,
Plumbago zeylanica, Premna tomentosa, Punica granatum, Rubia cordifolia, Sesamum
indicum, Sida cordifolia, Swertia decursata, Syzigium cumini, Terminalia arjuna,
Terminalia bellarica, Tinospora cordifolia, Trigonella foenum-graecum, Withania
somnifera and Zingiber officinalis (Devasagayam et al., 2001; Tilak et al., 2005; Tilak
and Devasagayam, 2006).
Apart from health care, medicinal plants are also the alternate income-generating source
for underprivileged communities (Myers, 1991). Strengthening this sector may, therefore,
benefit and improve the living standard of poor people, which is the basic need of any
developing country like India.
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Table 1. Some important medicinal plants and their therapeutic uses
Botanical name Family Parts used Therapeutic uses
Acorus calamus Linn Araceae Rhizome Nervine tonic, anti-
spasmodic (Satyavati et al .,
1976; Bose et al., 1960)
Aegle marmelos Linn Rutaceae Fruit Hypoglycemic;
chemopreventive
(Vyas et al., 1979; Dixit et
al., 2006)
Allium sativum Linn Alliaceae Bulbs Anti-inflammatory; anti-
hyperlipidemic, fibrinolytic
(Dixit et al., 2006)
Aloe barbadensis Mill.,
and Aloe vera Linn.
Alliaceae Gel Skin diseases- mild sunburn,
frostbite, scalds; wound
healing (Baliga, 2006)
Andrographis
paniculata
Acantahceae Whole plant Cold, flu, hepatoprotection
(Koul and Kapil-1994;
Sharma et al., 2002a)
Asparagus racemosus Alliaceae Roots Adaptogen, galactogogue
(Dahanukar et al.,
1997;Gupta and Mishra,
2006)
Bacopa monnieri Linn Scorphulariaceae Whole plant Anti-oxidant, memory
enhancer (Singh and
Dhawan, 1997)
Berberis aristata Berberidaceae Bark, fruit,
root, stem,
wood
Anti-protozoal,
hypoglycemic, anti-trachoma
(Dutta and Iyer, 1968;
Sharma et al., 2000a)
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Boerhavia diffusa Linn. Nyctaginaceae Roots Diuretic, anti-inflammatory
and anti-arthritic (Sharma et
al., 2000b; Harvey, 1966)
Boswellia serrata
Roxb.
Burseraceae Oleo resin Anti-rheumatic, anti-colitis,
anti-inflammatory, anti-
cancer (Sharma et al., 2000c)
Butea monosperma Fabaceae Bark,
leaves,
flowers,
seeds and
gum
Adaptogen; abortifacient,
anti-oestrogenic, anti-gout,
anti-ovulatory
(Sharma et al., 2000d)
Calotropis gigantean
Linn
Asclepiadaceae Flowers,
whole plant,
root, leaf
Anti-inflammatory,
spasmolytic, asthma
(Sharma et al., 2000e)
Callicarpa
macrophylla
Verbenaceae Leaves,
roots
Uterine disorders (Sood,
1995)
Cassia fistula Linn Leguminosae Resin Laxative, anti-pyretic, worm
infestation
(Joshi, 1998)
Celastrus paniculatus Celastraceae Whole plant Brain tonic; memory
enhancer; in the treatment of
depression (Joglekar and
Balwani, 1967;Tanuja, 1991)
Centella asiatica Linn Umbelliferae Whole plant Tranquilizer; memory
enhancer; wound healing-
(Suguna et al ., 1996;
Sharma et al., 2000 f)
Chlorophytum
boriavillianum
Alliaceae Roots Aphrodisiac (Farooqi et al.,
2001)
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Cissus quadrangularis
Linn.
(Vitaceae) Whole
plant, root,
stem and
leaf
Bone fracture; inflammation
(Udupa and Prasad, 1964b;
Deka et al., 1994)
Clerodendrum
serratum Linn.
Verbenaceae Root, leaf,
Stem
Malaria; anti-asthmatic, anti-
allergic
(Gupta and Gupta, 1967;
Sivarajan and Balachandran
1999a)
Commiphora mukul Burseraceae Resin Hypolipidemic; obesity,
rheumatoid arthritis
(Satyavati, 1991)
Crateva nurvala Capparidaceae Stem bark,
leaf
Urinary disorders including
stones (Anand et al., 1995)
Crocus sativus Linn. Iridaceae Stigma Aphrodisiac, anti-stress, anti-
oxidant (Billore et al.,
2004a)
Curculigo orchioides Amaryllidaceae Root stock Spermatogenesis enhancer
(Joshi, 2005)
Curcuma longa Linn. Zingiberaceae Rhizome Anti-inflammatory, wound
healing enhancer,
chemopreventive agent, anti-
oxidant, anti-cancer (Tripathi
et al., 1973), (Narasimhan et
al., 2006)
Desmodium
gangeticum Linn.
Papillionaceae Root Anti-oxidant, anti-
rheumatic- (Sharma et al.,
2001a) (Govindarajan and
Vijayakumar, 2006)
Eclipta alba Linn. Compositae Whole plant Hepatoprotecive , promotes
hair growth (Chandra et al.,
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1987)
Emblica officinalis Euphorbiaceae Fruit Adaptogen, anti-oxidant
(Rao and Siddiqui, 1964;
Vyas and Apte, 1977).
Eugenia jambolana
Linn.
Myrtaceae Seed, bark,
leaf
Hypoglycemic, anti-
inflammatory, anti-
diarrhoeal, anti-pyretic
(Sharma et al., 2001b)
Ficus religiosa Linn. Urticaeae Bark Anti-ulcer (gastric ulcer),
anti-inflammatory,
hypoglycemic agent-
(Ambike and Rao, 1967;
Sharma et al., 2001c)
Gymnema sylvestre Asclepiadaceae Roots and
leaves
Anti-diabetic, anti-
hyperglycemic
(Narasimhan et al., 2006)
Gloriosa superba Linn. Liliaceae Tuber Spasmolytic, oxytocic,
source plant for colchicine
(Sharma et al., 2002b)
Glycyrrhiza glabra
Linn.
leguminaceae Stem Expectorant, peptic ulcer
treatment
(Mitra and Rangesh, 2004a)
Hedychium spicatum Zingiberaceae Rhizome Soothening, expectorant,
anti-tussive
anti-asthmatic (Chaturvedi
and Sharma, 1975)
Hippophae rhamnoides
Linn.
Elaeagnaceae Fruits Extensively used in the
treatment of circulatory
disorders, wound healing
enhancer, duodenal ulcer etc.
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(Arora et al., 2006)
Holarrhena
antidysenterica Linn.
Apocynaceae Stem bark,
leaf, seed
Anti-spasmodic, anti-colitis,
hypoglycemic. (Mitra and
Rangesh, 2004b)
Inula racemosa Asteraceae;
Compositae
Roots Used in gastro intestinal
disorders, diuretic,
expectorant and allergic
disorders etc (Mishra, 2004a)
Leptadenia reticulata Asclepiadaceae Root, leaf,
fruit Galactogogue, vasodilator,
anabolic.
(Anjaria et al., 1975)
Momordica charantia
Linn.
Cucurbitaceae Root, leaf,
fruit, seed
Anti-diabetic (Ahmad et al.,
2001)
Mucuna pruriens Linn. Fabaceae;
Papilionaceae
Seeds, root,
leaf
Parkinson’s disorder, Male
sexual disorders. (Nath et al.,
1981; Satyavati et al., 1987c)
Myristica fragrans Myristicaceae Seed, aril,
oil
Aphrodisiac, hypolipidemic,
anti-inflammatory (Sharma
et al., 2002c)
Ocimum sanctum Linn. Lamiaceae Whole
plant, root,
leaf, seed
Adaptogen; anti-oxidant,
hypoglycemic,
immunomodulator, radio-
protector
(Uma Devi, 2006)
Oroxylum indicum
Linn.
Bignoniaceae Root, root
bark, leaf,
fruit, seed
Anti-inflammatory, Diuretic
(Gujral et al., 1955)
Phyllanthus amarus Euphorbiaceae Whole plant Hepatoprotective
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(Premalatha and
Govindarajan, 2004)
Picrorhiza kurroa Scorphulariaceae Tubers Hepatoprotective,
adaptogen. (Narasimhan et
al., 2006)
Piper longum Linn. Piperaceae Fruit, root Cough, asthma, fever
(Satyavati et al., 1987a;
Kohli and Aiman, 2006)
Piper nigrum Linn. Piperaceae Fruit Cough, asthma, fever
(Satyavati et al ., 1987a)
Plumbago zeylanica
Linn.
Plumbaginaceae Root, root
bark
Anti-pyretic, anti-cancer,
anti-coagulant, cytotoxic.
(Krishnaswamy and
Purushothaman, 1980;
Sharma et al., 2000g;)
Pterocarpus
marsupium
Fabaceae Bark,
leaves, gum,
flower
Hypoglycemic, anti-fungal.
(Pandey and Sharma, 1975;
Satyavati et al., 1987b)
Pueraria tuberosa Fabaceae Tuberous
root
Anti-implantation,
estrogenic, anti-
inflammatory,
dysmenorrhoea,
(Billore et al ., 2004b)
Rubia cordifolia Linn. Rubiaceae Root Anti-inflammatory, anti-
tumor, hypoglycemic
(Sharma et al., 2002d)
Rauvolfia serpentina Apocynaceae Root Hypertension, mental
disorders (Kohli and Aiman,
2006; Chauhan et al., 2006)
Saraca asoca Caesalpiniaceae Stem bark, Post menopausal syndrome
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flower, seed and Gynecological disorders
(Manjusha et al., 2004;
Narasimhan et al ., 2006)
Saussurea lappa Asteraceae Roots Analgesic, aphrodisiac,
asthma (Chaurasia, 2006)
Solanum xanthocarpum
Linn.
Solanaceae Whole plant Asthma and related
respiratory disorders (Sinha
et al., 2006)
Swertia chirata Gentianaceae Whole plant Anti-malarial,
hypoglycemic, febrifuge
(Gopal et al., 1981; Dixit et
al., 2006)
Symplocos racemosa Symplocaceae Bark Anti-diarrhoeal (Sharma et
al., 2002e)
Taxus baccata Linn. Taxaceae Source of
taxol
Used in the treatment of
metastatic breast cancer
(Chauhan et al., 2006)
Tecomella undulata Bignoniaceae Bark, seeds Anti-bacterial,
hypoglycemic,
hepatoprotective (Billore et
al., 2004c)
Terminalia arjuna Combretaceae Bark Heart diseases ( Gauthaman
and Mishra, 2004)
Terminalia chebula
Terminalia bellerica
Combretaceae Fruits Laxative, anti-oxidants
(Narasimhan et al ., 2006)
Terminalia arjuna Combretaceae Bark Heart diseases ( Gauthaman
and Mishra, 2004)
Tinospora cordifolia Menispermaceae Stem Adaptogen,
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immunomodulator. (Thatte
et al., 1994; Dahanukar et
al., 1997)
Tribulus terrestris
Linn.
Zygophyllaceae Whole plant Diuretic, anti-urolithiatic,
cytoprotective (Chakraborty
and Neogi 1978; Sangeetha
et al., 1993)
Vetiveria zizanioides
Linn.
Poaceae Root Vetiver oil for cosmetics
(Kumar et al., 1997)
Vitex negundo Linn. Verbenaceae Leaves,
root, bark,
flowers,
seed
Anti-inflammatory, anti-
arthritic, immunodmodulator
(Nair and Saraf, 1995)
Withania somnifera
Linn.
Solanaceae Root Adaptogen, anti-rheumatic
(Singh and Kumar, 1998) .
Zingiber officinale Zingiberaceae Rhizome Fever, cough, asthma, anti-
emetic
(Sharma et al, 2002f)
2.1. INDIGENOUS SYSTEMS OF MEDICINES
Our mother earth is blessed with a rich medicinal flora. On the basis of the therapeutic
properties of the medicinal plants, a large number of traditional systems of medicines are
widely practiced throughout the world. It is a well-known fact that the traditional systems
of medicines always played an important role in meeting the global health care needs.
They are continuing to do so at present and shall play major role in future also. The
systems of medicines, which are considered to be Indian in origin or the systems of
medicines, which have come to India from outside and got assimilated in to Indian
culture are known as Indian systems of Medicines (Prasad, 2002). More than 70% of
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India’s 1.1 billion population still uses these non-allopathic systems of medicines (Vaidya
and Devasagyam, 2007). India has the unique distinction of having six recognized
systems of medicine in this category. They are Ayurveda, Siddha, Unani, Yoga,
Naturopathy and Homoeopathy. Though Homoeopathy came to India in 18th
Century, it
completely assimilated in to the Indian culture and got enriched like any other traditional
system, hence, it is considered as part of Indian systems of medicines (Prasad, 2002).
These traditions have successfully set an example of natural resource use in curing many
complex diseases for more than 3,000 years. Millions of Indians use herbal drugs
regularly as spices, home-remedies, health foods as well as over-the-counter (OTC) as
self-medication or also as drugs prescribed in the non-allopathic systems (Gautam et al.,
2003). Many advantages of such eco-friendly traditions exist. The plants used for various
therapies are readily available, are easy to transport and have a relatively longer shelf life.
The most important advantage of herbal medicines is the minimal side effects and
relatively low cost compared to the synthetic medicines. A brief description of the
important Indian systems of medicine is given below.
2.1.1. Ayurvedic System of Medicine
Ayurveda literally means the Science of life. It is presumed that the fundamental and
applied principles of Ayurveda got organized and enunciated around 1500 BC.
Atharvaveda, the last of the four great bodies of knowledge known as Vedas, which
forms the backbone of Indian civilization, contains 114 hymns related to formulations for
the treatment of different diseases. In India, Ayurveda is considered not just as an ethno
medicine but also as a complete medical system that takes in to consideration physical,
psychological, philosophical, ethical and spiritual well being of the mankind. It lays great
importance on living in harmony with the universe and harmony of nature and science.
This universal and holistic approach makes it a unique and distinct medical system. This
system emphasizes the importance of maintenance of proper life style for keeping
positive health. This concept was in practice since two millennium and the practitioners
of modern medicine have now taken into consideration importance of this aspect. Not
surprisingly the WHO’s concept of health propounded in the modern era is in close
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approximation with the concept of health defined in Ayurveda (Kurup, 2004). Ayurveda
lays great emphasis on the diet regulation. The diagnosis is always done by considering
the patient as a whole object to be examined. The physician takes a careful note of the
patient’s internal physiological characteristics and mental disposition. Factors like the
affected bodily tissues, humors, the site at which the disease is located, patient’s
resistance and vitality, his daily routine, dietary habits, the gravity of clinical conditions,
condition of digestion and details of personal, social, economic and environmental
situation of the patient are also studied. The general examination is known as ten-fold
examination through which a physician examines ten different parameters in the patient
which include psychosomatic constitution, disease susceptibility, quality of tissues, body
build, anthropometry, adaptability, mental health, digestive power, exercise endurance
and age. In addition to this, examination of pulse, urine, stool, tongue, voice and speech,
skin, eyes and overall appearance is also carried out (Kurup, 2002).
The treatment lies in restoring the balance of disturbed humors (doshas) through
regulating diet, correcting life-routine and behavior, administration of drugs and
resorting to preventive non-drug therapies known as ‘Panchkarma’ (Five processes) and
‘Rasayana’ (rejuvenation) therapy. Before initiating the treatment many factors like the
status of tissue and end products, environment, vitality, time, digestion and metabolic
power, body constitution, age, psyche, body compatibility and type of food consumed are
taken in to consideration. The treatments are of different types which include Shodhana
therapy (purification treatment), Shamana therapy (palliative treatment), Pathya
Vyavastha (prescription of appropriate diet and activity), Nidan Parivarjan (avoidance of
causes and situations leading to disease or disease aggravation), Satvajaya
(psychotherapy) and Rasayan (adaptogens including immunomodulators, anti-stress and
rejuvenation drugs) therapy. Dipan (digestion) and Pachan (assimilation) enhancing
drugs are considered good for pacifying the vitiated doshas (humors). Shodhana therapy
provides purificatory effect through which therapeutic benefits can be derived. This type
of treatment is considered useful in neurological and musculo-skeletal disorders, certain
vascular or neuro-vascular states, respiratory diseases, and metabolic and degenerative
disorders. Shamana therapy involves restoring normalcy in the vitiated doshas (humors).
This is achieved without causing imbalance in other doshas. In this therapy use of
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appetizers, digestives, exercise and exposure to sun and fresh air are employed. In the
Pathya Vyavastha type of treatment, certain indications and contraindications are
suggested with respect to diet, activity, habits and emotional status. In Nidan Parivarjan
type of treatment, the emphasis is on avoiding known causes of the disease by the patient.
In Satvavajaya type of treatment, the emphasis is on restraining the mind from the desires
for unwholesome objects and Rasayana therapy deals with the promotion of strength and
vitality (http://www.indianmedicine.nac.in). According to Ayurvedic concepts food has
great influence over physical, temperamental and mental development of an individual.
The food is the basic material for the production of the body and life supporting vital
matter known as Rasa. The rasa is converted to body components and supports all types
of life activities.
The Ayurvedic system has eight major divisions which include Kayachikitsa (Internal
Medicine), Kaumar Bhritya (Pediatrics), Bhootavidya (Psychiatry), Shalakya
(Otorhinolaryngology and Ophthalmology), Shalya (Surgery), Agada Tantra
(Toxicology), Rasayana (Geriatrics) and Vajikarana (Aprhodisiacs and Eugenics).
Globalization of Ayurvedic practice has gained momentum in the past two decades.
Ayurvedic drugs are used as food supplements in USA, European Union and Japan.
Many physicians practice Ayurveda in many parts of the world. Facilities are available in
countries like USA, Argentina, Australia, Brazil, New Zealand, South Africa, Czech
Republic, Greece, Italy, Hungary, Netherlands, Russia, UK, Israel, Japan, Nepal and Sri
Lanka (Kurup, 2004) for imparting short and long-term training in Ayurveda.
2.1.2. Unani System of Medicine
Unani medicine has its origin in Greece. It is believed to have been established by the
great physician and philosopher, Hippocrates (460-377 BC). Galen (130-201 AD)
contributed for its further development. Aristotle (384-322 BC) laid down foundation of
anatomy and physiology. Dioscorides, the renowned physician of the 1st
Century AD has
made significant contribution to the development of pharmacology, especially of drugs of
plant origin. The next phase of development took place in Egypt and Persia (the present
day Iran). The Egyptians had well evolved pharmacy, which involved the preparation of
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different dosage forms like oils, powder, ointment, alcohol etc. (Ravishankar and Shukla,
2007). The Arabian scholars and physicians under the patronage of Islamic rulers of
many Arabian countries have played great roles in the development of this system. Many
disciplines like chemistry, pharmaceutical procedures like distillation, sublimation,
calcinations and fermentation were developed and refined by them (Ravishankar and
Shukla, 2007). According to the basic principles of Unani, the body is made up of four
basic elements i.e. earth, air, water and fire, which have different temperaments i.e. cold,
hot, wet and dry, respectively. They give rise through mixing and interaction with new
entities. The body is made up of simple and complex organs. They obtain their
nourishment from four humors namely blood, phlegm, black bile and yellow bile. These
humors also have their specific temperaments. In the healthy state of the body, there is
equilibrium among the humors and the body functions in normal manner as per its own
temperament and environment. Disease occurs whenever the balance of humors is
disturbed.
In this system also prime importance is given for the preservation of health. It is
conceptualized that six essentials are required for maintenance of healthy state. They are
air, food and drink, bodily movements and response, psychic movement and repose, sleep
and wakefulness and evacuation and retention (Khaleefathullah, 2002).
The human body is considered to be made up of seven components, which have
direct bearing on the health status of a person. They are Elements (Arkan), Temperament
(Mijaz). Humors (Aklat), Organs (Aaza), Faculties (Quwa) and Spirits (Arwah). These
components are taken in to consideration by the physician for diagnosis and also for
deciding the line of treatment (Khaleefathullah, 2002). Examination of the pulse occupies
a very important place in the disease diagnosis in Unani. In addition examination of the
urine and stool is also undertaken. The pulse is examined to record different features like
size, strength, speed, consistency, fullness, rate, temperature, constancy, regularity and
rhythm. Different attributes of urine are examined like odour, quantity, mature urine and
urine at different age groups. Stool is examined for colour, consistency, froth and time
required for passage etc.
Disease conditions are treated by employing four types of therapies which include
Regimental therapy, Dietotherapy, Pharmacotherapy and Surgery. Regimental therapy
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mainly consists of drug less therapy like exercise, massage, turkish bath, douches etc.
Dietotherapy is based on recommendation of patient‘s specific dietary regime.
Pharmacotherapy involves administration of drugs to correct the cause of the disease. The
drugs employed are mainly derived from plants, some are obtained from animals and
some are of mineral origin. Both single and compound preparations are used for the
treatment.
2.1.3. Siddha
Siddha system of medicine is practiced in some parts of South India, especially in the
state of Tamilnadu. It has close affinity to Ayurveda, yet it maintains a distinctive identity
of its own. This system occupies a close identification with Tamil civilization. The term
'Siddha' has come from 'Siddhi', which means achievement. Siddhars were the men who
achieved supreme knowledge in the field of medicine, yoga or tapa (meditation)
(Narayanaswamy, 1975). The materia medica of Siddha system of medicine depends to
large extent on drugs of metal and mineral origin in contrast to Ayurveda of earlier
period, which was mainly dependent upon drugs of vegetable origin. Similar to
Ayurveda, Siddha system also follows ashtanga concept with regards to treatment
procedures. However, the main emphasis is on the three branches namely Bala vahatam
(pediatrics), Nanjunool (toxicology) and Nayana vidhi (ophthalmology). The other
branches have not developed to the extent seen in Ayurveda. The surgical procedures,
which have been explained in great detail in Ayurvedic classics, do not find mention in
Siddha classics. The therapeutics in both the systems can be broadly categorized into
samana and sodhana therapies. The latter consists of well-known procedures categorized
under panchakarma therapy. This therapy is not that well developed in Siddha system and
only the vamana therapy has received attention of the Siddha physicians
(Narayanaswamy, 1975).
2.2 ALLOPATHIC MEDICINES AND NON-ALLOPATHIC MEDICINES
Traditional and modern systems of medicine were developed by different philosophies.
They look at health, diseases, and causes of diseases in different ways. These differences
bring different attitudes ranging from complete rejection of traditional medicines by
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modern medical practitioners and of modern medicine by traditional medical practitioners
to a parallel existence with little communication over patient care. Experience from many
countries, such as those in South East Asia, suggest that integration of traditional and
modern healthcare systems can solve much of the problems by providing the basic
healthcare services for people in developing countries, particularly the undeserved
majority. In these countries, both systems are equally developed and supplement each
other towards achieving optimal healthcare coverage (Getachew and Tadesse 2004)
Harmonization of traditional and modern medicine emphasizes the importance of
respectful coexistence. Within the model of harmonization, there is a requirement to
develop and hold a good understanding between traditional and modern medicine.
(Getachew and Tadesse, 2004). Many traditionally used medicinal plants contain
pharmacologically active compounds used in the preparation of both traditional and
modern medicines. Over 25% of the pharmaceutical preparations in the world and more
than 50% in the USA contain plant-derived active principles (Asfaw et al., 1999). At
present, most of the phytoconstituents and plant extracts find their way into modern
medicine to treat many critical diseases, and fill the gap between the contemporary
system and traditional system of medicine.
2.3. INDIGENOUS DRUGS SELECTED FOR STUDY
We have selected 10 crude indigenous drugs for our study, depending on their wide and
numerous medicinal uses. These drugs as well as their source medicinal plants are used
immensely in both Unani and Ayurvedic systems of medicine. The basic characteristic
features of their source plants and medicinal uses are given below:
2.3.1. Tukhm-e-kasoos (Seeds of Cuscuta reflexa)
Characteristic Features
Cuscuta reflexa belongs to the family convulvulacae and is one of the important herbs
used in the Indian system of medicine. It is commonly known as Akashbela, Aftimoon or
Kasoos and is distributed throughout Ceylon, India and Malaya. The seeds are termed as
Tukhm in the Unani system of medicine, therefore, the drug is known as Tukhm-e-
kasoos. Plant occurs for most part of the year, while fruits are found during February to
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April. It is a leafless parasitic annual plant with long stem. Mature fruits of the plant are
collected in the month of April and May. After drying, the seeds are separated from the
undesired plant parts. The seeds so obtained are winnowed, garbled and further dried.
Then these are packed in airtight containers and stored in cool and dry place.
Medicinal Uses
The drug Tukhm-e-kasoos is used for various therapeutic purposes. It is known to be a
good carminative and diuretic. The drug has anti-spasmodic, heamodynamic,
bradycardiac, anti-hypertensive, anti-inflammatory, anti-pyretic, laxative and anti-
steroidogenic properties (Gilani and Aftab, 1992; Gupta et al., 2003). In addition, it acts
as a good muscle relaxant and cardiotonic (Singh and Garg, 1973) and known to have
psycho-pharmacological (Pal et al., 2006), anti-viral and anti-convulsant (Gupta et al.,
2003) properties. Besides seeds, stems of C. reflexa are also used as drug (Anonymous,
1992). The methanol extract of C. reflexa exhibits antibacterial and free radical
scavenging activity (Gupta et al., 2003). The petroleum ether extract of C. reflexa and its
isolate are useful in treatment of androgen-induced alopecia by inhibiting the enzyme 5-
alpha-reductase (Uddin et al., 2007). It is also used to cure inflammation of liver and
stomach and treatment of patients suffering with diseases such as chronic fever,
constipation and jaundice (Anonymous, 1992).
2.3.2. Barg-e-sudab (Leaves of Ruta graveolens)
Characteristic Features
Ruta graveolens commonly known as Sudab belongs to the family Rutacea. The dried
leaves of Ruta graveolens are known as Barg-e-sudab, which is a crude indigenous drug
of high therapeutic value. Ruta graveolens is a strong scented, erect, glabrous herb
approximately 30-90 cm. in height. It is the native plant of the Mediterranean region and
sometimes cultivated in Indian gardens. The plant consists of 2-3 pinnate leaves and
segments oblong to speculate. The plant is covered with a bloom and strongly aromatic
small flowers, which have petals with dentate or wavy margins and small capsules with
lobes somewhat rounded (Anonymous, 1972).
Medicinal Uses
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The drug is prescribed to the patients suffering with gastric disorders and dizziness
(Conway and Slocumb, 1979). It is used as a sedative and antihelminthic (Skidmore
Roth, 2001). It is also used a remedy for deep headache and rheumatism (Miguel, 2003).
The drug also possess anti inflammatory, antiviral and antiplasmodic properties
(Yammamoto et al., 1989; Queener et al., 1991; Raghav et al., 2006). The herb contains
several alkaloids; stems and leaves contain skimmianine (C14H13O4N), graveolinine
(C17H13O3N) and kokusaginine (C14H13O4N), while the leaves contain dictamine and γ –
fagarine. The alkaloids graveoline and rutamine are also present in the herb (Anonymous,
1972). The plant acts as a good emolient, deobstruent, appetizer and diuretic. It decreases
the libido and thickens the sperm. It is also useful in the treatment of paralysis, tremors,
sciatica and arthralgia. It breaks the stones of kidney and urinary bladder. It is also
beneficial in the treatment of chest diseases and jaundice (Ghani, YNM).
2.3.3. Kurkum (Floral parts of Crocus sativus)
Characteristic Features
The dried stigmas of Crocus sativus are used as a drug possessing numerous medicinal
values in both Unani as well as Ayurvedic system of medicine. The drug is known as
Kurkum. The plant Crocus sativus belongs to the family Iridaceae and is also known as
Saffron, Zaafran or Kurkum. Its original habitat is doubtful but it is thought to be
indigenous to Greece, Asia minor and Persia. In these countries it grows wild. At present
time Spain produces bulk of European Saffron; small amounts come from France, Greece
and Persia (Wallis, 1985). Hay saffron forms a loosely matted mass of dark, reddish-
brown, flattened stigmas with a strong, characteristic odour and bitterish taste. When
fresh, it is unctuous to the touch and glossy, but after keeping it becomes dull and brittle
(Wallis, 1985).
Medicinal Uses
Since long it has been used as a medicine, spice and dye by Egyptians, Jews, Greeks and
Romans. It is known to be a brain tonic and stimulant (Anonymous, 1992). The drug also
possesses antispasmodic and acts as an appetizer (Anonymous, 1992). It is prescribed to
the patients suffering with small pox and impotency (Giacco, 1990). Its flowers contain
various chemical constituents (Abdullaev et al., 2002). Stigmas of the flower (saffron)
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contain crocin, anthocyanin, carotene and lycopene (Giaccio, 1990). These constituents
have various pharmacological effects on different illnesses, including anti-tumor effects
by inhibition of cell growth (Abdullaev, 2003). The styles and stigmas are anodyne,
antispasmodic, aphrodisiac, carminative, emmenagogue, expectorant and sedative
(Anonymous, 1992). They are used as a diaphoretic for children, to treat chronic
haemorrhages in the uterus of adults, to induce menstruation, treat period pains and calm
indigestion and colic. A dental analgesic is also obtained from the stigmas. It improves
complexion and hence is used for application on hyper pigmented lesions of the skin. Its
paste is applied on wounds. For weak eye sight, a mixture of rosewater and kesar is put in
the eyes. Its paste is also used in hepatitis. It is useful in nervous debility, migraine,
rheumatoid arthritis, pain caused by vata, loss of appetite, liver disorders, heart diseases,
blood disorders and dysuria. It is also useful in dysmenorrhoea, amenorrhoea and painful
labour (Giacco, 1990).
2.3.4. Senna (Leaves of Cassia acutifolia, Cassia angustifolia)
Characteristic Features
The drug is known as Senna which consists of the dried leaflets of C. acutifolia Delile
known in commerce as Alexandrian or Khartoum Senna and Cassia angustifolia Vahl,
known as Tinnevalley Senna (Trease and Evans, 2004). It belongs to the family
leguminoseae. The Senna plants are small shrubs, about 1 meter high with peripinnate
compound leaves. C. acutifolia (Senna) is indigenous to tropical Africa and is cultivated
in the Sudan. C. angustifolia is indigenous to Somaliland, Arabia, Sind and Punjab and is
cultivated in South India (Trease and Evans, 2004). The leaves and pods are the plant
parts used in various drug preparations. Alexandrian Senna is collected mainly in
September from both wild and cultivated plants and the whole leaves are usually sold for
the medicinal uses (Trease and Evans, 2004). Tinnevalley Senna is usually obtained from
cultivated plants of Cassia angustifolia grown in south India, N.W. Pakistan and Jammu.
It may grow either in dry land or in wetter conditions as a successor to rice. Being a
legume, it usually adds nitrogen to the soil (Trease and Evans, 2004).
Medicinal Uses
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Senna is a very good laxative and purgative. It stimulates the muscular coat of intestine
and produces purgation, which is not followed, as is commonly the case in constipation.
It is therefore, one of the most useful purgatives especially in cases of habitual
constipation (Ghani, YNM; Wallis, 1985). It is used in treatment of gout, migraine,
epilepsy, chronic headache, pleurisy, sciatica and rheumatoid arthritis (Ghani, YNM ;
Wallis, 1985).
2.3.5. Amla (Fruit of Emblica officinalis)
Characteristic Features
The fruit of Emblica officinalis or Amla is a popular drug and used in both fresh and
dried forms to treat various ailments. It is a medicinal plant described in Ayurveda as
well as Unani, the two major traditional medicinal systems of India (Gogate, 2000). It
belongs to the family euphorbiceae. Amla is a moderate sized deciduous tree and mainly
found in Deccan, the sea coast districts and Kashmir. The fruit is the most commonly
used part of this plant and is the richest natural source of vitamin C (Desouza et al.,
2005).
Medicinal Uses
The drug possesses enormous medicinal properties (Desouza et al., 2005; Saeed and
Tariq, 2006). It acts as carminative, antioxidant, stomachic, cardiotonic and enhances
memory (Parrota et al., 2001). It exhibits various properties like anti-inflammatory,
analgesic, antipyretic (Sharma et al., 2003), adaptogenic (Rege et al., 1999), diuretic
(Anon, 2006), antitumour (Jose et al., 2001), hepatoprotective (Jeena et al., 1999; Jose
and Kuttan, 2000), hypocholestrolemic (Kim et al., 2005), antioxidant (Bhattacharya et
al., 1999) and antiulcerogenic (Sairam et al., 2002). It is also useful in treatment of
haemmorage, diarrhea and dysentery (Parrota et al., 2001). The dried fruit has been
prescribed in Ayurveda for pancreas-related disorders (Garde, 1970; Gogate, 2000).
Leaves, roots, bark and flowers are also used sometimes. Fresh fruit acts as a good
refrigerant, diuretic and laxative. Green fruit is exceedingly acid and also acts as a
carminative and stomachic. Dried fruit is, however, sour and astringent. Flowers are
cooling and aperients. Bark is used as an astringent. Fresh fruit is used in inflammation of
lungs. The green fruits are made in to pickles and used to stimulate appetite (Nadkarni,
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1989). It is used for all Pitta diseases, all obstinate urinary conditions, anemia,
biliousness, bleeding, colitis, constipation, convalescence from fever, cough, diabetes,
gastritis, gout, hepatitis, hemorrhoids, liver weakness; relieve stress ,osteoporosis,
palpitation, spleen weakness, tissue deficiency, vertigo and rebuilds blood, bones, cells,
and tissues. It increases red blood cell count and regulates blood sugar; heart tonic,
cleanses mouth, stops gum bleeding, stops stomach and colon inflammation; cleanses
intestines, strengthens teeth, aids eyesight, removes acidity, cure eye and lung
inflammations, ulcerations, gastrointestinal disorders, painful urination, and internal
bleeding (Anonymous, 1992).
2.3.6. Zarishk (Bark of Berberis aristata)
Characteristic Features
The drug is known as Zarishk and its source plant is Berberis aristata. It belongs to the
family berberidaceae. It is distributed along Himalayas from chota Banghal to Nepal
ranging from 6000-10,500ft. It is a large deciduous shrub, usually 1.8-3.6m. high; but
attains a height of 4.5m. with stem. Twigs are whitish or pale yellowish in colour. The
bark is pale brown, deeply furrowed and rough in appearence.
Medicinal Uses
The drug Zarishk is known to exhibit numerous medicinal properties. The fruit, wood,
root, bark and extract of Indian Barberry have been used in Ayurvedic medicine from a
very remote period and its properties are said to be analogous to those of turmeric. It is
prescribed to patients in all types of inflammations (Gupta et al., 2008), ENT infections,
dysentery and indigestion (Saumya et al., 2009). It is useful in various uterine and
vaginal disorders and also used for healing of wounds (Fukuda et al., 1999). It also
possesses very high immunopotentiating property (Fukuda et al., 1999). It is used in the
treatment of opthalmia and eye diseases (Jain and Singh, 1994) and also for curing ulcers
and fever (Kirtikar and Basu, 2001). It is known to be a mild laxative and antihepatotoxic
(Gilani and Janbaz, 1992). It is used in skin diseases, menorrhagia and diarrhea (Kirtikar
and Basu, 2001). In painful micturition from bilius or acrid urine, a decoction of Indian
barberry and emblic myrobalan is given with honey. A decoction of the root bark is used
as a wash for unhealthy ulcers and is said to improve their appearance and promote
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cicatrization (Kirtikar and Basu, 2001). A decoction of root bark in 8 doses of one or two
ounces is given to patients for malarial fever and is found to be beneficial, although the
effect is quite slow. Roots of B.aristata contain an alkaloid berberine (BBR) with a long
history of medicinal use in China as a non-prescription drug to treat bacterial diarrhoea.
The chemical structure of BBR was first identified in 1910 and the total synthesis was
accomplished in 1969. BBR extracts and decoctions were shown to have significant anti-
microbial activities against a variety of organisms such as bacteria, viruses, fungi,
protozoans, helminths and chlamydia. Predominant clinical applications of BBR include
bacterial diarrhoea, intestinal parasitic infections and ocular trachoma infections (Birdsall
and Kelly, 1997). Various pharmacological properties of BBR have been recorded over
the years relating to inhibition of metabolism in certain micro-organisms (Ghosh et al.,
1985), bacterial enterotoxin formation, intestinal fluid accumulation and ion secretion
(Sack and Froelish, 1982), inflammation (Fukuda et al., 1999), cyclooxygenase-2 (COX-
2) transcription and N-acetyltransferase activity in colon and bladder cancer cell lines
(Lin et al., 1998), and the growth of mouse sarcoma cells in culture (Creasey, 1979).
2.3.7. Mulethi (Root of Glycyrrhiza glabra)
Characteristic Features
The drug Mulethi is a widely used drug in the Unani system of medicine. It is the dried
root of Glycyrrhiza glabra which belongs to the family leguminaceae. The plant is grown
in Spain, Italy, England, France, Germany and USA. It is also abundant in the wild state
in Galicia and central and southern Russia. It is a herbaceous perennial plant, growing up
to 1 m in height, with pinnate leaves about 7–15 centimetres (3–6 in) long, with 9–17
leaflets. The flowers are 0.8–1.2 cm (½–⅓ in) long, purple to pale whitish blue, produced
in a loose inflorescence. The fruit is an oblong pod, 2–3 centimetres (1 in) long,
containing several seeds (Huxley, 1991). It owes most of its sweet taste to glycyrrhizin,
the potassium and calcium salts of glycyrrhizinic acid (Trease and Evans, 1983). The
yellow color of liquorice is due to flavanoids. They include liquiritin, isoliquiretin, which
occur as glycosides and during drying, partly converted into liquiritin, liquiritigenin,
isoliquiritegenin and other compounds (Wallis, 1985). An examination of samples from
five countries has shown the flavanoid content to be geographically consistent, varying
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only in the relative proportions of constituents. Other active constituents of liquorice are
polysacchrides with a pronounced activity on the reticulo endothelial system. The roots
also contain about 1-2% of asparagines, 0.04-0.06% volatile compounds, β-sitosterol,
starch, protein, bitter principles glycyramin (Trease and Evans, 2002).
Medicinal Uses
This drug is used in the Hoxsey anti-cancer formula and is considered to be an adaptogen
which helps reregulate the hypothalamic-pituitary-adrenal axis. It can also be used for
auto-immune conditions including lupus, scleroderma, rheumatoid arthritis and animal
dander allergies (Winston et al., 2007). It exhibits antioxidant, antibacterial and anti
inflammatory properties (Vaya et al., 1997). Liquorice may be useful in conventional and
naturopathic medicine for both peptic ulcers and mouth ulcers (Das et al., 1989).
Liquorice is also a mild laxative and may be used as a topical antiviral agent for shingles,
ophthalmic, oral or genital herpes. It also acts as a carminative, diuretic, anti-
inflammatory, emmenogauge, laxative, expectorant and nervine tonic (Kabeeruddin,
YNM).
2.3.8. Filfil Siyah (Fruit of Piper nigrum)
Characteristic Features
The dried fruits of Piper nigrum possess numerous medicinal properties. Piper nigrum is
a perennial flowering vine in the family piperaceae, universally valued for its fruit, which
is usually dried and used as a spice and seasoning. While Piper nigrum is native to
southern Thailand and Malaysia, its most important habitat is the tropical regions of India,
particularly the Malabar Coast (Miller, 1969). Piper nigrum is cultivated in many areas,
including Sri Lanka, China and parts of Africa. Black pepper grows best in moist, well-
drained soil rich in organic matter. In Ayurveda, Piper nigrum is an important healing
spice, and along with long pepper and ginger, is a component of the preparation Trikatu,
renowned, for its ability to enhance the bioavailability and efficacy of other medicines
(Zutshi et al., 1985). Piper nigrum has been used as an ingredient in Indian cooking since
2000 BC.
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Medicinal Uses
In modern Ayurveda, Piper nigrum is used to treat digestive disorders, due to its anti-
inflammatory (Majumdar et al., 1990) and anti-microbial properties (Reddy et al., 2004).
It is useful in treatment of cholera, dyspensia, variety of gastric ailments and arthritic
disorders (Jung and Shin, 1998). It also possesses antioxidant (Kapoor et al., 2009) and
insecticidal properties (Kuchi et al., 1988; Park et al., 2002). When used externally, Piper
nigrum helps to alleviate neuralgia, scabies, piles and various skin disorders (Natakani et
al., 1986). It is used to relieve weakness following fevers, for vertigo, and as an
antiperiodic for malaria and arthritic disease. Piper nigrum also possesses cleansing and
antioxidant properties, enhancing oxygen flow to the brain, aiding digestion and
circulation, stimulating the appetite, and maintaining respiratory and joint health
(Nakatani et al., 1986). Ancient texts as early as the fifth century describe Piper nigrum
as an antidote for constipation, diarrhea, earache, gangrene, heart disease, hernia,
hoarseness, indigestion, insect bite, insomnia, joint pain, liver problems, lung disease,
oral abscess, sunburn, and toothache (Jack and Turner, 2004). In Chinese medicine, Piper
nigrum is used to improve the appetite and to treat cold, influenza, abdominal pain,
diarrhea and epilepsy. The spiciness of P. nigrum is, however, due to the alkaloid
piperine present in it.
2.3.9. Rewand chini (Root of Rheum emodi)
Characteristic Features
Rewand chini or the dried root of Rheum emodi is a widely used Unani drug. The plant
belongs to the family polygonaceae and is commonly known as Rhubarb. Other
synonyms are ravandehindi and ladakirevandachini. It is distributed around alpine and
subalpine Himalayas (11,000-12,000ft). The plant bears leafy stem and stout roots.
Medicinal Uses
Rewand chini has a sharp bitter taste and acts as a purgative, emmenagogue and diuretic.
According to the Unani system of medicine, the drug is known to be useful in
biliousness, sour eyes, piles, chronic bronchitis, asthma, corryza, pains and bruises. It is
known as antibacterial, antifungal and antidiabetic (Radhika et al., 2010). It also acts as
laxative, purgative and lowers serum cholesterol (Agarwal et al., 1976; Harvey and
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Waring, 1987; Cyong et al., 1987), and, thus, known to be a slimming agent. Its
stimulating effect, combined with aspirin properties, renders it especially useful in atonic
dyspepsia. It also has an astringent-like effect on the mucous membranes of the mouth
and nasal cavity. Other parts of the plant also hold several medicinal properties (Kirtikar
and Basu, 2001). The tuber is pungent, bitter, tonic and laxative. In Ayurvedic system of
medicine, it is useful in dysentery, loss of appetite and bad ulcers (Kirtikar and Basu,
2001). The root and stem of the plant are rich in anthraquinones, such as emodin and
rhein. These substances are cathartic and laxative. The root of the plant is also used in
traditional Chinese medicine. It was also used in the medieval Arabic and European
prescriptions. Rhubarb is used for making jams and sauces. It is also cooked with
strawberries or apples as a sweetener or with stem or root ginger, to make various types
of jams and sausages. The leaves of the plant are used to make an effective organic
insecticide for leaf-eating insects, such as cabbage caterpillars, aphids, peach and cherry
slug etc.
2.3.10. Unnab (Berries of Zizyphus jujube)
Characteristic Features
The berries of Zizyphus jujube are known as Unnab, which is a precious drug of the
Unani system of medicine. The plant belongs to the family moraceae. It is a sub
deciduous tree with 0.6m girth and 6m height. Bark is blackish to grey or brown.
Branches are usually armed with spines, usually in pairs, one straight and the other
carved. The plant is indigenous and naturalized throughout India, Burma, Ceylon (in the
outer Himalayas up to 4,500 ft), China, Afghanistan, Africa and Australia.
Medicinal Uses
Unnab is known to be a good blood purifier (Sharif et al., 2010) and used in skin
inflammations (Dinarello, 1997). It is also prescribed in bacterial sepsis and rheumatide
arthritis (Palladino et al., 2003). The root is known to cure biliousness and headache. The
bark cures boils, good in dysentery and diarrhea, acts as an antipyretic and reduces
obesity. The ripe fruit is aphrodisiac, tonic, laxative, invigorative, removes thirst and
vomiting and good in blood diseases. The dried fruit is a laxative, appetizer and removes
impurities from the blood. The leaves are antihelmintic, good in stomatitis and gum
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bleeding, heal wounds, good in ulcers, cures asthma and used in liver complaints. The
fruit is said to be mucilaginous, pectoral and styptic. The unripe fruit increases thirst,
lessens expectoration and biliousness. The ripe fruit is sweet and not good for digestion.
It also causes diarrhea in large doses and useful in fevers, wounds and ulcers. The seed is
astringent, tonic to heart and brain, aphrodisiac, cures eye diseases, cough, and asthma
and allays thirst. The berries are considered to purify blood and to assist digestion. The
root and bark acts as tonic. The bark is said to be a remedy in diarrhea and root is used as
decoction in fever, and as a powder it is applied to ulcers and old wounds (Kirtikar and
Basu, 2001).
2.4. ADULTERATION IN MEDICINAL PLANTS
A glance at the present scenario of the indigenous systems of medicines shows that the
traditional medicines are being successfully used to treat large number of ailments. The
ongoing growing recognition of traditional medicines is due to several reasons, including
escalating faith in herbal medicines. Allopathic medicine may cure a wide range of
diseases; however, its high prices and side-effects are causing many people to return to
the traditional medicines which have fewer side effects (Kala, 2005). Inspite of large
number of therapeutic uses of medicinal plants and their advantages over Allopathic
drugs, these plants suffer with lack of efficacy making the traditional medicinal systems
weak and less effective. The main reason behind it is the instant rising demand of plant-
based drugs, which is unfortunately creating heavy pressure on some selected high-value
medicinal plant populations in the wild due to over-harvesting. Several of these medicinal
plant species have slow growth rates, low population densities, and narrow geographic
ranges; therefore they are more prone to extinction (Jabloski, 2004). The deep insight in
to this problem concludes that the continuous increase in human population and over
exploitation of certain medicinal plants can be the major causes that lead to continuous
erosion of forests and the forest products. The World Health Organization (WHO) has
estimated that the present demand for medicinal plants is approximately US $14 billion
per year (Sharma, 2004). The demand for medicinal plant-based raw materials is growing
at the rate of 15 to 25% annually and according to an estimate of WHO, the demand for
medicinal plants is likely to increase more than US $5 trillion in 2050. In India, the
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medicinal plant-related trade is estimated to be approximately US $1 billion per year
(Joshi et al., 2004). The projected escalating demand of medicinal plants has led to the
over harvesting of many plants from wild which subsequently results in the loss of their
existing populations. For example, the large quantity of Himalayan yew (Taxus baccata)
has been gathered from the wild since its extract, taxol, is used in the treatment of ovarian
cancer. Aconitum heterophyllum, Nardostachys grandiflora, Dactylorhiza hatagirea,
Polygonatum verticillatum, Gloriosa superba, Arnebia benthamii and Megacarpoea
polyandra are other examples of north Indian medicinal plant species which have been
overexploited for therapeutic uses and have subsequently been placed today in rare and
endangered categories (Kala, 2006). Many medicinal plant species are used in curing
more than one disease (Kala, 2004; Kala, 2005) and as a result, these species are under
pressure due to over collection from wild. For example, Hemidesmus indicus is used to
cure 34 types of diseases, Aegle marmelos 31, Phyllanthus emblica 29, and Gloriosa
superba 28. Rising demands with shrinking habitats may lead to less accessibility to
many medicinal plant species, which results in deliberate adulteration by the suppliers of
these plant species. From the very beginning, herb authentication has presented a great
challenge for people using them for medical purposes. The authentication of medicinal
plants is a critical issue for the protection of consumers. Ideally, authentication should be
done from the harvesting of the plant material to the final product as herbal drugs are
normally processed parts of various plants, such as roots, stems, leaves, flowers, fruits,
seeds, etc. The pharmaceutical companies procure materials from traders, who are getting
these materials from untrained persons from rural and /or forest areas. This has given rise
to wide spread adulteration/substantiation (Meherotra and Rawat, 2000), leading to poor
quality of herbal formulations. Misidentification of herbs can be non-intentional
(processed plant parts are inherently difficult to distinguish) or intentional (profit-driven
merchants sometimes substitute expensive herbs with less-expensive look-alike ones), but
usage of wrong herb may be ineffective or worsen the conditions and may even cause
death (Khan et al., 2009). This comes out to be a matter of concern for public health
sector as adulteration not only decreases the therapeutic efficacy of these plants but
mixing of different plants can also have extreme consequences on the human body (Khan
et al., 2009). Adulteration is also one of the major reasons for unexpected toxicity which
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Department of Biotechnology, Jamia Hamdard 33
may be related to the mixtures of active compounds that they contain; their interactions
with other herbs and drugs, contaminants, adulterants; or their inherent toxicity. Plants
have complex mixtures of terpenes, alkaloids, saponins and other chemicals, increasing
the risk of adverse reactions to any one of them or to the additive or synergistic effects of
chemical interactions (Carson and Riley, 1995). Therefore, parallel with recent increasing
interest in alternative/herbal medicine for the prevention and treatment of various
illnesses, there is increasing concern about the safety and efficacy of medicinal plants
based drugs. Hence, correct identification and quality assurance is crucial to maintain the
efficacy of indigenous drugs.
2.5. METHODS OF AUTHENTICATION OF MEDICINAL PLANTS
Traditionally, people authenticated herbs by their appearance, smell and/or taste and
some of these methods are still skillful. During the early period of research, classical
strategies including comparative anatomy, physiology and embryology were employed in
genetic analysis to determine inter and intraspecies variability. Later on, herbs were
authenticated by inspection under microscopes, where the shape and content of various
plant cells are examined and analyzed. These methods, based on organoleptic markers or
anatomical characters, are sometimes imprecise. By and large analytical chromatography,
such as thin-layer chromatography (TLC), high-performance liquid chromatography
(HPLC), or liquid chromatography-mass-spectrometry (LC-MS) has been used for herb
authentication. Secondary metabolites, as markers have been extensively used in quality
control and standardization of herbal drugs, but these also suffer with few limitations.
During this decade, however, molecular markers also known as the DNA markers have
rapidly complemented the classical strategies (Weising, 1995). A Molecular marker is
generally referred to as landmark or DNA sequence in the genome of organism which is
readily detected and whose inheritance can be monitored. These Markers are unique,
conserved, stable, and ubiquitous to the plants. These DNA markers are not affected by
age, physiological condition as well as environmental factors (Chan, 2003). Different
types of DNA based markers viz., RAPD, RFLP, ISSR, AFLP, SCAR etc., are employed
for species discrimination of plants (Joshi et al., 2004).
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Department of Biotechnology, Jamia Hamdard 34
Markers can be categorized into three groups
1. Morphological markers
2. Biochemical Markers and Phytochemical Markers
3. Molecular markers
2.5.1. Morphological marker
Morphological markers have been routinely used to identify genetic diversity, but major
disadvantages associated with these markers are the limited number of morphological
characters available for analysis and these characters are also influenced by
environmental factors. Consequently different plant genotypes cannot be distinguished
(Staub and Meglic, 1993). The genetic basis of most morphological variations is
generally unknown and hence, these markers are incapable of providing desirable
information about the genome of the plants used in the study.
2.5.1.1. Characteristic of morphological markers:
The phenotype of most morphological markers can only be determined at the
whole plant level.
Allele frequency tends to be much lower with morphological loci.
It is generally associated with undesirable phenotypic effect.
Alleles at morphological loci interact in a dominant/recessive manner that limits
the identification of heterozygous genotypes.
More epistatic or pleiotropic effects are observed with morphological markers.
2.5.2. Biochemical marker and phytochemical marker
Isozymes (or isoenzymes) are different variants of the same enzyme which have identical
or similar functions and are present in the same individual. They are powerful tools to
study genetic variability within and between populations of plants and animals. Isozymes
have been used in taxonomic, genetic, evolutionary and ecological studies and
identification of cultivars and lines (Peirce and Brewbaker, 1973). Despite the use of
DNA markers such as RAPDs, AFLPs, RFLPs, isozymes etc; are still widely employed
in species delimitation and conservation (Booy and Van Raamsdonk, 1998; Chamberlain,
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Department of Biotechnology, Jamia Hamdard 35
1998), assessment of genetic variability in species and populations (Buso et al., 1998) and
gene flow studies (Gauthier et al., 1998). They are especially useful when several taxa,
accessions and individuals are to be compared, as the assumption of homology is more
accurate than with some DNA markers. Isozyme electrophoresis has been successfully
used to identify clones and to examine the clonal structure of plant populations (Mc
Neilly and Roose, 1984; Mc clintock and waterway, 1993; Johanson et al., 1996;
Lehman, 1997). Phytochemical markers which rely on differences in the amount of
secondary metabolites present in different species can also be used for identification of
species.
2.5.2.1. Limitations of Biochemical Markers
Much of the genome (including much of the most polymorphic portions of it that
are less subject to evolutionary restrictions) does not code for gene product and
hence, remains unanalyzed.
Different biochemical procedures are required to visualize allelic differences for
enzymes having different functions.
Many proteins undergo post transcriptional modifications and hence, can mask
variations present at DNA level (e.g., differences in tri-nucleotide sequences
coding for the same amino acid, introns sequences that are post transcriptionally
removed from the mRNA; all these factors contribute to reduced polymorphism
expression at the protein level compared to that at the DNA level).
2.5.3. Molecular markers (DNA Markers)
Molecular markers have been widely used to characterize diversity in different species of
plants. Molecular markers allow analysis of variation at the genomic level (Karp et al.,
1997) and permit detection of genetic variation at the molecular level (Rani et al., 2005).
These DNA-based markers have acted as versatile tools and have found their own
position in various fields like taxonomy, physiology, embryology, genetic engineering,
etc. They are no longer looked upon as simple DNA fingerprinting markers in variability
studies or as mere forensic tools. A variety of molecular assays could be used to assess
the genetic diversity and each method differs in principle, application, the amount of
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Department of Biotechnology, Jamia Hamdard 36
polymorphism detected, cost and time required. These DNA markers offer several
advantages over traditional phenotypic markers, as they provide data that can be analyzed
objectively. DNA fingerprinting in plants can be adapted to numerous applications and
uses, including characterizing individual plants to clarify errors in the identification of
accessions and cultivars (Aguirre et al., 1998; Saunders et al., 2001). Molecular linkage
maps are also being used successfully in many crop species for directed germplasm
improvement. Linkage maps facilitate the identification and localization of genes
controlling important traits, subsequently allowing marker assisted selection and
positional cloning of genes.
Molecular markers based on DNA sequences detect more polymorphism than
morphological and proteins based markers and constitute a new generation of genetic
markers (Tanksley et al., 1989). Polymorphism may be defined as simultaneous
occurrence within or between populations of multiple phenotypic forms of a trait
attributable to the alleles of a single gene or the homologes of a single chromosome
(Acquaah, 1992).
2.6. PROPERTIES DESIRABLE FOR IDEAL DNA MARKERS
Highly polymorphic in nature.
Co dominant inheritance (determination of homozygous and heterozygous
states of diploid organisms).
Frequent occurrence in genome.
Selective neutral behaviour (the DNA sequences of any organism are neutral
to environmental conditions or management practices).
Easy access (availability).
Easy and fast assay.
High reproducibility.
Easy exchange of data between laboratories.
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Department of Biotechnology, Jamia Hamdard 37
2.7. TYPES OF DNA BASED MARKERS
PCR based markers
Non PCR based markers
2.7.1. PCR based markers
PCR-based markers involve in vitro amplification of particular DNA sequences or loci,
with specific or arbitrarily chosen oligonucleotide sequences (primers) and a
thermostable DNA polymerase enzyme. The amplified products are separated
electrophoretically and banding patterns are detected by different methods such as
staining and autoradiography.
2.7.2. Non PCR based markers
Non PCR based markers work on the principle of DNA–DNA hybridization between
DNA/RNA probe and genomic DNA. The technique relies on differences between two or
more samples of homologous DNA molecules arising from differing locations of
restriction sites. The DNA sample is broken into pieces (digested) by restriction enzymes
and the resulting restriction fragments are separated according to their lengths by gel
electrophoresis.
Each marker, however, has its own advantage and disadvantage, but none is universally
ideal. The choice of technique is, therefore, often a compromise that depends upon the
nature of research pursued, the genetic resolution needed, financial constraints and the
technical expertise available.
In present study, we have attempted to use SCAR markers for authentication of
medicinal plants.
The most important DNA based markers used in revealing genetic diversity in plant
genome are given below:
2.8. RANDOMLY AMPLIFIED POLYMORPHIC DNA (RAPD)
These markers are very quick and easy to develop due to the arbitrary sequence of the
primers (Karp et al., 1997; Hansen et al., 1998). It is a PCR based technique and resolved
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Department of Biotechnology, Jamia Hamdard 38
most of the technical obstacle owing to its cost effective and easy to perform approach
(Welsh and McCleland, 1990; Williams et al., 1990). This efficient technique obviates
the need to work with radioisotope and gives satisfactory results even with crude DNA
preparations. RAPDs have, therefore, been extensively used in assessing genetic
relationship amongst various accessions of different plant species (Chalmers et al., 1992;
Adams et al., 1993; Castilione et al., 1993, Russel et al., 1993; Wachira et al., 1995).
RAPD has been used to identify medicinal plant tea (Camellia sinensis) (Wachira et al.,
1995), dried roots of P. ginseng, P. quinquefolius, P. notoginseng and their adulterants
(Shaw and But, 1995). The reproducibility of RAPDs is affected by DNA quality, primer
concentration, different thermal cyclers and the brand of DNA polymerase used (Meunier
and Grimont, 1993; Mac Pherson et al., 1993; Ellsworth et al., 1993). If the amplification
conditions (reagents and thermocycler parameters) are identical for all reactions, the
results are highly reproducible. Variation in the primer concentration is one of the main
sources of RAPD pattern variations (Hansen et al., 1998; Virk et al., 2000). Thus, the
lack of specificity and reproducibility are the major drawbacks of RAPD. The RAPD
amplicons have been reported to identify a large number of medicinal species from their
close relatives or adulterants including Panax species (Shaw and But,1995), Coptis
species (Cheng et al., 1997), Astragalus species (Cheng et al., 2000), Lycium barbarum
L. (Cheng et al., 2000), Panax ginseng species (Um et al., 2001), Echinacea species
(Neiri et al., 2003), turmeric (Sasikumar et al., 2004), Astragali radix (Na et al., 2004),
Dendrobium officinale L. (Ding et al., 2005), Typhonium species (Acharya et al., 2005),
Dendrobium species and its products (Zhang et al., 2005), Tinospora cordifolia (Rout,
2006), Mimosae tenuiflorae cortex (Rivera et al., 2007), Rahmannia glutinosa cultivars
and varieties (Qi et al., 2008), Desmodium species (Irshad et al., 2009), Glycyrrhiza
glabra (Khan et al., 2009), Piper nigrum (Khan et al., 2009) and Cuscuta reflexa ( Khan
et al., 2010).
2.9. SIMPLE SEQUENCE REPEAT (SSR)
Microsatellites or simple sequence repeat (SSR) markers are tandemly repeated DNA
sequences that occur throughout the eukaryotic genome. The length polymorphism arises
from variations in the number of repeated units, probably due to DNA polymerase
Chapter 2 Review of Literature
Department of Biotechnology, Jamia Hamdard 39
slippage during replication of the SSR (Levinson and Gutman, 1987; Eisen, 1999). The
frequency of SSRs in plant genome is estimated as one in every 6-7 Kb, based on the
information that can be found in public sequence database (Cardle et al., 2000). Thus,
these are abundant resources in the genome and have a high level of allelic diversity.
Consequently, they are frequently used as genetic markers in plant genetic studies
(Powell et al., 1996; McCouch et al., 1997). The codominant nature and allelic
polymorphism revealed by SSR markers have provided detailed information on genetic
structure (Bonnin et al., 2001) and gene flow (Konuma et al., 2000) in natural plant
populations. Construction of a genomic library or SSR enriched library has been the
principal means of discovering SSRs in eukaryotic genomes for which public DNA
sequence data is lacking. The SSR loci differ in the number of repetitive di, tri, or
tetranucleotide units present (Tautz et al., 1986; Tautz, 1989) and this length variation is
detected with the polymerase chain reaction (PCR) by utilizing pairs of primers flanking
each simple sequence repeats. These markers have been developed for plant species,
including Soyabean (Akkaya et al., 1992; Morgantae and Olivieri, 1993; Rongwen,
1995), and Rice (Wuk and Tankesley, 1993; Yang et al., 1994). Rapid evolution and the
properties of the replication slippage mechanism proposed for SSR polymorphism
generation, may not be suitable for estimating genetic similarities except in very closely
related taxa (Bowcock et al., 1994). Despite various advantages of SSR markers, their
development is time consuming.
2.10. INTER SIMPLE SEQUENCE REPEAT (ISSR)
Inter simple sequence repeat (ISSR) has been available since 1994 (Zietkiewicz et al.,
1994) and these are semiarbitrary marker(s) amplified by PCR in the presence of one
primer complementary to a target microsatellite. ISSR primers are derived from an
arbitrary nucleotide sequence of di and trinucleotide repeats with a 5’ or 3’ anchoring
sequence of a few nucleotides to prevent strand-slippage (14-22 bp). These nucleotide
repeats are based on the ubiquitous presence of simple sequence repeats that are
distributed throughout genomes. It is a powerful tool for investigating genetic variation
within species (Wolf and Liston, 1998; Gupta et al., 2008) especially when sequence
information about the study organism is limited. Amplification does not require genome
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Department of Biotechnology, Jamia Hamdard 40
sequence information and leads to multilocus and highly polymorphic patterns
(Zietkiewicz et al., 1994; Nagaoka et al., 1997). Each band corresponds to a DNA
sequence delimited by two-inverted microsatellites. Recent studies on genetic diversity of
clonal plant species have demonstrated the great discrimination power of ISSR markers
for genetic identification (Esselman et al., 1999; Camacho and Liston, 2001; Liston et al.,
2003; Wang et al., 2004). The plant Monimopetalum chinensis which is endangered
endemic species of Eastern China was assessed by ISSR marker (Xie et al., 2005). Like
RAPDs, ISSRs markers are quick and easy to handle, but they seem to have high
reproducibility like SSR markers because of the longer length of their primers.
2.11. RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP)
RFLP markers were the first to provide a means to directly detect variations present at
DNA level. RFLPs have been used to document genetic diversity in cultivated plants and
their wild relatives (Wang et al., 1992; Diers and Osborn, 1994). The polymorphism
detected by RFLP is mainly due to a variation at the restriction site, where as with AFLP,
an additional number of nucleotides, apart from restriction site are screened for
polymorphism (Becker et al., 1995). These markers were used for the first time in the
construction of genetic maps by Botstein et al (1980). Being a codominant marker, it can
detect coupling phase of DNA molecules as DNA fragments from all homologous
chromosomes are detected. They are very reliable markers in linkage analysis and
breeding study. This can easily determine the presence of any linked trait present in a
homozygous or heterozygous state in an individual and information is highly desirable
for recessive traits (Winter and Kahl, 1995). Although highly specific, performing RFLP
is quite tedious and expensive since it requires large amount of pure quality DNA and an
expertise in handling radioactivity.
2.12. SINGLE NUCLEOTIDE POLYMORPHISM (SNP)
DNA sequence variations that occur when a single nucleotide (A, T, C, or G) in the
genome sequence is altered, are the basis of SNP markersThe potentiality of single
nucleotide polymorphism (SNPs) to identify the genetic variability has been proved
(Hayashi et al., 2004). Because SNPs are highly abundant, occurs frequently throughout
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Department of Biotechnology, Jamia Hamdard 41
genome and tend to be genetically stable (Batley et al., 2003), their potential use as the
next generation of genetic marker in a species lacking polymorphism (e.g. peanut) should
be explored in future.
2.13. CLEAVED AMPLIFIED POLYMORPHIC SEQUENCE (CAPS)
Cleaved amplified polymorphic sequence (CAPS) is also known as PCR-RFLP marker,
based on STSs derived from ESTs. These markers have several advantages. First,
analysis of restriction fragment length polymorphism is based on PCR amplification and
is much easier and less time consuming, especially for species with large genome
(Wakamiya et al., 1993; Hizume et al., 2001) than analyzing alternative types of markers
that require southern hybridization. Second, the primers for CAPS markers based on
ESTs are more useful as genetic markers for comparative mapping study than those based
on anonymous, nonfunctional sequences such as microsatellite markers because the
coding regions of functional genes are generally well conserved, not only within but also
between species. Third, CAPS marker is inherited mainly in a codominant manner. If
STS and SCAR markers fail to reveal any polymorphism, then they can be easily
converted to CAPS by employing restriction enzyme digestion (Konieczny and Ausubel,
1993). Unlike RAPD marker, the CAPS marker is a PCR based codominant marker that
is reproducible and easier to manipulate in MAS (Caranta et al., 1999).
2.14. SELECTIVE AMPLIFICATION OF MICROSATELLITE POLYMORPHIC
LOCI (SAMPL)
A modification of the AFLP procedure called selective amplification of microsatellite
polymorphic loci (SAMPL) (Morgante and Vogel, 1994) combines the advantages of
AFLP with the analysis of highly variable microsatellite regions of eukaryotic genomes.
SAMPL has been reported to be more powerful than AFLPs in discriminating between
closely related individuals in several plant complexes (Paglia and Morgante, 1998 ; Roy
et al., 2002 ; Singh et al., 2002 ; Tosti and Negri, 2002) and in the rust pathogen,
Puccinia striiformis f. sp. tritici, which is strictly clonal (Stubbs, 1985 ; Keiper et al.,
2003). Keiper et al (2003) found SAMPL to be the most informative marker(s) system in
assessing genetic variation among isolates of five cereal rust pathogens. The SAMPLs,
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Department of Biotechnology, Jamia Hamdard 42
RAPDs and AFLPs were used for characterization of genetic variation among 11 cowpea
(Vigna unguiculata sub sp. unguiculata) land races and two commercial varieties showed
marginally greater diversity indeces for SAMPL than AFLP and RAPD (Tosti and Negri,
2002). Additionally, fewer SAMPL primer combinations were needed to obtain similar
discrimination when compared with AFLP and RAPD analyses. In another study,
SAMPL was found to be more efficient than AFLP in differentiating closely related
accessions of neem, Azadiracta indica (Singh et al., 2002).
2.15. SEQUENCE TAGGED SITE (STS)
RFLP probes specifically linked to a desired trait can be converted into PCR based STS
marker(s) based on nucleotide sequence of the probe giving polymorphic band pattern, to
obtain specific amplicon. Using this technique, tedious hybridization procedures involved
in RFLP analysis can be overcome. This approach is extremely useful for studying the
relationship between various species. When these markers are linked to some specific
traits; for example, powdery mildew resistance gene (Hartl et al., 1998) or stem rust
resistance gene in barley (Oh et al., 1994), they can be easily integrated into plant
breeding programmes for marker assisted selection of the trait of interest.
2.16. EXPRESSED SEQUENCE TAG (EST)
Adams et al. (1991) gave term ESTs for the markers obtained by partial sequencing of
random cDNA clones. Once generated, they are useful in cloning specific genes of
interest and synteny mapping of functional genes in various related organisms. ESTs are
popularly used in full genome sequencing and mapping programmes underway for a
number of organisms and for identifying active genes; thus, helping in identification of
diagnostic markers. Moreover, an EST that appears to be unique assists to isolate new
genes. EST markers are identified to a large extent for Rice, Arabidopsis, etc. wherein
thousands of functional cDNA clones are being converted into EST markers (Sasaki et
al., 1994).
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Department of Biotechnology, Jamia Hamdard 43
2.17. SINGLE STRAND CONFORMATION POLYMORPHISM (SSCP)
This is a powerful and rapid technique for gene analysis particularly for detection of point
mutations and typing of DNA polymorphism (Orita et al., 1989). It can identify
heterozygosity of DNA fragments of the same molecular weight and can even detect
changes of a few nucleotide bases as the mobility of the single stranded DNA changes
with change in its GC content due to its conformational change. To overcome problems
of reannealing and complex banding patterns, an improved technique called asymmetric-
PCR SSCP was developed (Ainsworth, 1991), wherein the denaturation step was
eliminated and a large-sized sample could be loaded for gel electrophoresis, making it a
potential tool for high throughput DNA polymorphism. It was found useful in the
detection of heritable human diseases. In plants, however, it is not well developed
although its application in discriminating progenies can be exploited, once suitable
primers are designed for agronomically important traits (Fukuoka, 1994).
2.18. AMPLIFIED FRAGMENT LENGTH POLYMORPHISM (AFLP)
Vos et al. (1995) developed AFLP (amplified fragment length polymorphism) technique
to detect polymorphism. This AFLP technique provides a novel and very powerful DNA
fingerprinting technique for DNAs of any origin or complexity. It has quickly become
one of the widely used methods of DNA fingerprinting for crops and wild plant species
(Mueller and Wolfenbarger, 1999; Ridout and Donini, 1999). In this technique, genomic
DNA is restricted with two different restriction endonucleases and a subset of the
resulting fragments is amplified using forward and reverse primers each with 1–4
(usually three) additional bases. The fragments are then visualized using radioactivity,
silver staining or fluorescent dyes. The resulting AFLP markers tend to show a strongly
asymmetric size distribution, with a much higher proportion of smaller fragments, and
this pattern does not appear to be affected by GC content or by genome size (Vekemans
et al., 2002).
2.19. SEQUENCE CHARACTERIZED AMPLIFIED REGION (SCAR)
SCAR markers (Sequence Characterized Amplified Region), initially developed for
downy mildew resistance genes in lettuce (Paran and Michelmore, 1993), are
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Department of Biotechnology, Jamia Hamdard 44
codominant, monolocus, and PCR-based markers that require the use of two specific
primers, designed from nucleotide sequence established in the cloned RAPD fragment
linked to a trait of interest. Hence, results with these markers are more reliable with
designed specific primers as compared to other DNA based molecular markers (Table 2).
Specific SCAR sequence primers for amplification may be located at any suitable
position within or flanking the unique RAPD amplicon and may be used to identify the
polymorphism in a population. Fig 1(a) shows a unique amplicon selected from the
RAPD profile and Fig 1(b) shows a fragment obtained with SCAR primers.
This PCR-based assay is fast, reliable, and easy to conduct in any laboratory. It can
be carried out in very short period using unknown genomic DNA from any
developmental stage and body part (Kethidi et al., 2003; Kiran et al., 2010).
Consequently, SCAR markers once developed, offer a practical method for screening
numerous samples, accurately at one time thus, adding to the cost efficiency of the
experiment (Kasai et al., 2004). SCARs allow for rapid marker development, even though
they are not highly polymorphic. These can be used as an allele-specific associated
primers (ASAPs) assay to detect the product eg. subspecies identification of plants (Gu et
al., 1995). Template DNA amplified using ASAPs at stringent annealing temperatures,
generate a single DNA fragment in individuals possessing the appropriate allele, thus
eliminating the need for electrophoresis to resolve the amplifications and increasing the
speedy analysis. The results are reliable, less sensitive to changes in the reaction
conditions, reproducible and are not affected by the physical forms and age of the sample
materials (Harnandez et al., 1999). Also SCAR markers are not affected by the presence
of introns that could eliminate the priming sites.
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Department of Biotechnology, Jamia Hamdard 45
Table 2: Comparison of DNA-based molecular markers
Parameters
DNA-based molecular marker
AFLP RAPD SSR RFLP SCAR
Quantity of
information
generated
High and
specific
High and
nonspecific
High and specific Low and
specific
Low and specific
Replicability High Variable High High High
Resolution of
genetic
differences
High Moderate High High Low
Ease of use
and
development
Moderate Easy Difficult Difficult Easy
Development
time
Short Short Long Long Short
Use of
radioactivity
Yes/No No Yes/No Yes No
Principle DNA
amplificatio
n
DNA
amplificatio
n
DNA amplification Restriction
digestion
DNA amplification
Recurring cost High Low High High Low
Reliability High Low High High High
Nature of
inheritance
Dominant Dominant Dominant/Codominan
t
Codominan
t
Dominant/Codominan
t
Single/multipl
e loci
Multiple Multiple Multiple/single Single Single
Parts of
genome
surveyed
Whole
genome
Whole
genome
Whole genome Generally
low copy
region
Part of genome
Skill required High Low High High Low
DNA quality
required
High Moderate to
high
High High Moderate to high
PCR-based Yes Yes Yes No Yes
Source : Kiran et al., 2010
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Department of Biotechnology, Jamia Hamdard 46
SCAR markers act as both dominant as well as codominant markers. In Asparagus, SCAR
markers were scored as a dominant marker. Amplification of locus M occurred in both
parents at 60°C annealing temperature, but when the annealing temperature was increased to
67°C, only one band was amplified in males and none in females (Jiang and Sink, 1997).
However, digesting the SCAR fragments produced at the 60°C annealing temperature with
four endonucleases (HaeIII, MboI, RsaI, AluI) failed to produce a small fragment length
polymorphism. This result differs from that of Paran and Michelmore (1993), where
codominant SCAR markers were obtained after digesting the monomorphic bands with
restriction enzymes.
2.19.2. Development of SCAR markers from other markers
The ease with which oligonucleotide from genomic sequence can be converted into a
simple, locus-specific marker varies according to the technique used to produce the
original multilocus profile. SCAR markers are developed by designing the
oligonucleotides (20-25 bp) that are used to amplify cloned RAPD amplicons (Tartarini
et al., 1999; Brisse et al., 2000; Cao et al., 2001) (Fig. 2). The designed SCAR primer
pair used to amplify genomic DNA from different species of a genus, including targeted
species, results in a single, distinct and bright band in the desired sample. SCAR primers
deduced from internal sequences are less polymorphic than those including initial RAPD
primer sequences, suggesting that the polymorphism is only present in the decamer
sequences derived from the RAPD primer sequence (Parasnis et al., 2000). The length
and GC content of the primer affects the specificity of SCAR marker (Vanichanon et al.,
2000). The specificity, temperature and time of annealing are partly dependent on primer
length and its GC content. In general, oligonucleotides between 18 and 24 bases are
extremely sequence specific.
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Department of Biotechnology, Jamia Hamdard 47
Fig. 2: Development of SCAR Marker from RAPD marker
Besides RAPD markers, SCAR markers can be developed from more reproducible
markers like AFLP (Vos et al., 1995), SSR (Litt and Luty, 1989; Tautz, 1989; Weber and
May, 1989) and ISSR (Zeitkeinicz et al., 1994) (Fig. 3). The development of SCAR
markers from these markers however, is very costly, difficult, time consuming and may
require whole genome sequence information.
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Department of Biotechnology, Jamia Hamdard 48
Fig 3: Development of SCAR Marker from other markers
2.19.3. Detection efficiency / sensitivity of SCAR markers
SCAR markers are robust and highly efficient. A nanogram or less of DNA sample is
sufficient. This method is more specific than other DNA fingerprinting methods using
arbitrarily chosen primers. These specific primers generate a sequence-characterized
amplified region, which can be particularly useful because they detect only a single locus,
their amplification is less sensitive to reaction conditions and they can potentially be
converted into allele-specific markers. To convert a selected unique RAPD, AFLP, ISSR
or SSR band to a SCAR marker, each unique band is isolated from agarose gel and the
isolated DNA is separated by electrophoresis. The nucleotide sequence is determined
using the automatic DNA sequencer and similarities of DNA sequence data is analyzed
using ncbi/BLAST. Further, the species-specific SCAR primers are synthesized.
A small amount (100 mg) of leaf powder of Echinacea species and Phyllanthus
emblica fruit powders (Amlacurna and Triphalacurna) were sufficient to develop a SCAR
marker (Dnyaneshwar et al., 2006; Adinolfi et al., 2007). The designed SCAR primers
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Department of Biotechnology, Jamia Hamdard 49
were used to amplify DNA which resulted in a sharp and reproducible band (343 bp)
from both (the commercial samples of P. emblica (Amlachurn and Triphalachurn)
formulations (Dnyaneshwar et al., 2006).
2.19.4. Authentication of medicinal herbs using SCAR markers
SCAR markers have been developed for authentication of large number of medicinal
plants, which are easily adulterated viz., Artemisia sp. (Lee et al., 2006), Phyllanthus sp.
(Dnyaneshwar et al., 2006), Panax sp. (Wang et al., 2001; Choi et al., 2008),
Atractylodes sp. (Huh et al., 2006), Echinacea sp. (Adinolfi et al., 2007), Phyllanthus
species (Theerakulpisut et al., 2008), Pueraria tuberose (Roxb. ex. Wild.) (Devaiah and
Subramanian, 2008), Angelica decursiva, Peucedanum praeruptorum, Anthricus
sylvestris (Choo et al., 2009), Jatropha curcas (Basha et al., 2009) and Curcuma
alismatifolia (Anuntalabhochai et al., 2007).
The Chinese plant, “Packchul”, (Atractylodes japonica) is a very important chinese
medicinal herb. The active components of A. japonica and another chinese plant A.
macrocephala, sold in the name of Pankchul are sesquiterpenoids such as atractylon,
atractylenolide III and 3β-acetoxyatractylon (volatile oils), but the content levels vary in
these two species (Sakamoto et al., 1996). Despite being considered less valuable than
Korean A. japonica with respect to its components and effects, A. macrocephala is
imported into Korea in huge amounts. These two species can be identified by leaf
morphology, flower color and size, and rhizome shape (Bang et al., 2003) but it is
impossible to distinguish between the two species when the rhizomes are sliced.
Therefore, in herbal markets A. macrocephala is illegally sold in the name of chinese
Packchul either without the correct label or by mixing it with Korean Packchul.
In a similar case, Hong Kong is a major entry port for roots of Panax ginseng (ginseng)
and Panax quinquefolius (American ginseng). P. quinquefolius from North America is 5
to 10 times more expensive than P. ginseng and substitution of the former by the latter is
found from time to time (Wang et al., 2001).
Artemisia species like A. princeps, A. argyi, A. capillaris, and A. iwayomogi are important
ingredients in traditional Asian medicinal formulations. Since the dried leaves of these
plants are used in medicinal applications, which are morphologically indistinguishable
Chapter 2 Review of Literature
Department of Biotechnology, Jamia Hamdard 50
from each other, a specific marker is required to efficiently discriminate different species.
Two primers Fb and R7, devised to amplify 254 bp fragment of genomic DNA extracted
from dried samples of A. princeps, and A. argyi were developed (Lee et al., 2006). This
reproducible SCAR marker (254 bp) efficiently discriminates A. princeps and A. argyi
from other Artemisia herbs, particularly from A. capillaries and A. iwayomogi (Table 3).
Adinolfi et al. (2007) developed a SCAR marker to differentiate Echinacea purpurea
from E. angustifolia and E. pallida. These different species, due to their difficult
identification, are commonly confused and probably used indifferently for the same
therapeutic purposes. A species-specific SCAR marker of size 330 bp was developed for
Echinacea purpurea from the 750 bp RAPD amplicon to distinguish it from related
species (E. angustifolia and E. pallida) (Adinolfi et al., 2007).
Plants belonging to Panax species are very similar in morphology but have diverse
physiological activities based on the different combinations and the quantity of various
ginsenosides found in each species (Kwan, 1995). Wang et al. (2001) converted a 420 bp
RAPD fragment of P. quinquefolius to a SCAR and used it successfully to authenticate
six Panax species by the presence of unique amplified band while two common
adulterants viz. Marabilis jalapa and Panax acinosa by absence of this unique band.
Moreover sequencing and alignment of the SCAR sequences from P. ginseng and P.
quinquefolius indicates the presence of a 25 bp insertion in P. quinquefolius.
Phyllanthus emblica L. (Indian gooseberry) fruit has applications in healthcare, food and
cosmetic industry. There is pool of material that can be used as adulterant for crude and
processed P. emblica fruits. The adulterant may be phyllogenetically close or distinct
(e.g. dried fruit pieces of pumpkin) from P. emblica (Dyneshwar et al., 2006). Dyneshwar
et al. (2006) developed a SCAR marker (343 bp) for correct genotype identification of P.
emblica from a species-specific amplicon developed by comparative analysis of RAPD
profiles of different cultivars of Phyllanthus. The marker was further used for
authentication of commercial samples of P. emblica fruit powders, a multi-component
formulation which contains fruit powders of P. emblica, Terminalia chebula Rotz and
Terminalia belerica Roxb.
SCAR markers were also used to provide a simple, cheap, and reliable procedure to
identify 22 geographically related olive-tree cultivars. The markers were designed by
Chapter 2 Review of Literature
Department of Biotechnology, Jamia Hamdard 51
cloning of prominant RAPD bands obtained in PCR performed on bulk DNA to retain the
genetic variability of each cultivar. Clones were partially or totally sequenced and new
primers derived from these sequences were used to obtain SCAR markers (Bautista et al.,
2003).
The species-specific SCAR marker was developed for Panax japonicas which
differentiated it from the other species of same genus (Choi et al., 2008). Amplified
fragment length polymorphism (AFLP) profile of P. japonicus, P. ginseng and P.
quinquefolius was compared and a unique AFLP marker for P. japonicus was generated.
JG14 Oligonucleotide primer (23 mer) was designed for amplifying 191 bp of the
sequence of JG14 (293 bp AFLP marker). PCR analysis revealed a clear amplified band
for P. japonicus but not in 3 other Panax species (P. ginseng, P. quinquefolius and P.
notoginseng).
Pueraria tuberosa, commonly known as Vidarikand in India, is an important plant used
in traditional medicine. However, there are at least three other botanical entities traded
under the same name, namely Ipomoea mauritiana, Adenia hondala and Cycas circinalis.
SCAR marker (320bp) was developed for identifying P. tuberosa, which is the authentic
vidari according to the Ayurvedic Pharmacopoeia of India (Devaiah and subramanian,
2008). The SCAR marker of 320 bp was found only in P. tuberosa and not in the other
species, thus aiding in distinguishing the authentic P. tuberosa from its commonly used
substitutes and adulterants.
One Curcuma variety called ‘patumma’ is of particular importance since it has strong
stalks, large symmetric inflorescence and is moderately resistant to fungal blight. It is
high yielding, and has been used to produce numerous high quality hybrids. ‘Patumma’ is
therefore, one of the key Curcuma varieties from which many hybrid crosses and induced
mutation varieties were developed. A SCAR marker of size 600 bp in length was
developed for differentiating ‘patumma’ variety (Anuntalabhochai et al., 2007). Since
new varieties of Curcuma are often dissimilar from their progenitors, this genomic
analysis allows a cost effective morphologically independent characterization of
Curcuma hybrid.
Astragalus radix is a well-known herbal material in traditional Chinese medicine.
Astragalus membranaceous and A. membranaceous var. mongholicus are known as the
Chapter 2 Review of Literature
Department of Biotechnology, Jamia Hamdard 52
authentic origins of Astragalus medicines whereas Hedysarum polybotrys is a common
adulterant of Astragalus radix in Taiwan. The single bands of 900 bp, 500 bp and 700 bp
were obtained in A. membranaceous var. mongholicus, A. membranaceous and H.
polybotrys, respectively as SCAR markers which were shorter than the original RAPD
fragments (Liu et al., 2008). SCAR markers were also used for genetic purity testing and
variety discrimination in Cantharanthus roseus (Menezes et al., 2002).
The Artemisia annua L. plants were screened using RAPD and SCAR techniques for high
artemisinin content (Zhang et al., 2006). The random primer could amplify a specific
band of approximately 1000 bp that was present in all high-artemisinin yielding strains,
but absent in all low-yielding strains in three independent replications. This specific band
was cloned and its sequence was analyzed for development of more stable SCAR
markers. Traditionally, Jatropha curcus seed and other plant parts have been used as oil,
soap and medicinal compounds. A SCAR marker was developed for authentication of
nontoxic Jatropha curcus from their toxic species (Basha et al., 2009). The development
of SCAR markers and their applications have been reviewed recently by us and can be
seen for more details (Kiran et al., 2010).
Table 3: Plant parts used and conditions required for developing SCAR markers in
some important medicinal plants.
S.N Medicinal plants Parameters
Part
used
Age of plant Annealing
temperature
Size of
SCAR
marker
Possible adulterants Development
of SCAR
marker
1. P. quinquefolius
(Wang et al.,
2001)
Root Post-
flowering
stage
420 bp M. jalapa and P.
acinosa
RAPD
2. Artemisia
(A. princeps and
A. argyii)
(Lee et al., 2006)
Leaf Pre-
flowering
stage
Not
Available
254 bp A. capillaries and
A. iwayomogi
RAPD
Chapter 2 Review of Literature
Department of Biotechnology, Jamia Hamdard 53
3. Phyllanthus
emblica
(Dnyaneshwar et
al., 2006)
Fruit Ripening
stage
55 ºC 343 bp Phyllanthus
distichus LINN.
Phyllanthus
reticulatus POIR.
Phyllanthus
urinaria LINN.
Phyllanthus simplex
RETZ.
Phyllanthus niruri
LINN.
Phyllanthus
indofischeri
BENNET.
RAPD
4. Panex
quinquiefolius
(Wang et al.,
2001)
Root Post-
flowering
stage
F1-56 ºC
R1
F1-60 ºC
R1
420 bp Panex ginseng RAPD
5. Echinacea
purpurea
(Adinolfi et al.,
2007)
Root Post-
flowering
stage
51.4 ºC 330 bp E. angustifolia and
E. pallida. RAPD
6. Atractylodes
japonica
A. macrocephala
(Huh et al., 2006)
Rhizom
e
Post-
flowering
stage
39 ºC Aj 1117
bp
Am 1325
bp
A. macrocephala RAPD
7. Panex japonicas
(Choi et al., 2008)
Root Post-
flowering
stage
62 ºC 191 bp P. ginseng,
P. quinquefolius
AFLP
8. Phyllanthus
amarus,
P. debilis Klein
ex
Willd
P. urinaria Klein
ex
Willd,
Whole
plant
Post-
flowering
stage
64ºC
62ºC
64ºC
408 bp
549 bp
321 bp
P. debilis and P.
urinaria
RAPD
Chapter 2 Review of Literature
Department of Biotechnology, Jamia Hamdard 54
(Theerakulpisu et
al., 2008)
9. Pueraria tuberose
(Roxb. ex. Wild.)
(Devaiah and
Venkatasubraman
ian, 2008)
Root Post-
flowering
stage
58 ºC 320 bp Ipomoea
mauritiana,
Adenia hondala
Cycas circinalis
RAPD
10. Angelica
decursiva,
Peucedanum
praeruptorum,
Anthricus
sylvestris
(Choo et al.,
2009)
Root Post-
flowering
stage
53 ºC 363 bp,
145 bp,
305 bp,
273 bp
Anthricus sylvestris RAPD
11. Jatropha curcas
L. (Basha et al.,
2009)
Whole
plant
Post-
flowering
stage
62 ºC, 54
ºC, 56 ºC
RSPJ1-F
RSPJ1-R
(961 bp)
RSPJ2-F
RSPJ2-R
(1077
bp)
ISPJ3-F
ISPJ3-R
(964 bp)
Toxic Jatropha
curcas
RAPD, ISSR
12. Curcuma
alismatifolia
(Anuntalabhochai
et al., 2007)
Dried
rhizom
e
Post-
flowering
stage
55 ºC 600 bp Curcuma species RAPD
13. Astragalus
membranaceous,
A.
membranaceous
var. mongholicus
(Liu et al., 2008)
Dried
root
Post-
flowering
stage
58 ºC,
64 ºC
500 bp,
900 bp
Hedysarum
polybotrys
Hand.-Mazz
RAPD
Chapter 2 Review of Literature
Department of Biotechnology, Jamia Hamdard 55
14. Artemisia annua
(Zhang et al.,
2006)
Young
leaf/
floret/
Branch
es
Pre-
flowering
stage
58 ºC 996 bp - RAPD
15. Cantharanthus
roseus (Menezes
et al., 2002)
Seeds Post-
flowering
stage
50 ºC - RAPD