Chapter 2 Review of Literature - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/13085/6/07-chapter 2.pdf · Ficus religiosa Linn. Urticaeae Bark Anti-ulcer (gastric ulcer),

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



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,



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.



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)



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)



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.,



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.



(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



(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



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,



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



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



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



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



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



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



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



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)


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



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)


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)



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)


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



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)


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,



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)


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



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)


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



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)


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.



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)


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



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)


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



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



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



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).



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,



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



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.



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



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



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



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



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,



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).



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



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.



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



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.



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.



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



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



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



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



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



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



(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



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

Documents

Chapter 2 Review of Literature - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/13085/6/07-chapter 2.pdf · Ficus religiosa Linn. Urticaeae Bark Anti-ulcer (gastric ulcer),