review of literature - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/21241/8/08_chapter...

REVIEW OF LITERATURE

Traditional health care has been flourishing in India for many centuries

(Subromoniam, 2001). Ayurveda and Unani, which has survived through more

than 3000 years, mainly use plant based drugs in their medical system (Kamboj et

al., 2000; Mukherjee et al., 2003). The ancient texts like Rig veda and Athar veda

(2000 BC) mention the use of several plants as medicine. The books on ayurvedic

medicine such as ‘Charak samhita’ (300 BC) and ‘Susruta samhita’ (1300 BC) refer

to the use of more than 700 herbs (Jain, 1968). Indian systems of medicine use

around 8,000 species of plants, which include trees (33%), herbs (32%), shrubs

(20%), climbers (12%) and a variety of epiphytes, grasses, lichens, ferns and algae

(3%). Of the 2,000 drugs being used in curing human ailments in India, only 200 are

of animal origin and the rest 1500 drugs are extracted from plants (Dubey et al.,

2004; Mitra et al., 2007). About 960 plant species are used by the Indian herbal

industry, of which 178 are of high volume exceeding 100 metric tonnes a year.

There are about 300 natural products used as raw material in flavor and fragrance

industry in the form of essential oil, extracts, oleoresin, concretes, absolutes,

resinoids and tinctures. Modern pharmacopoeia still contains at least 25% drugs

derived from plants and many others, which are synthetic analogues, built on

prototype compounds isolated from plants (Rates, 2001). According to World

Health Organisation, medicinal plants should be investigated to better understand

their properties, safety, efficacy (Nascimento et al., 2000, Akerele, 1993). Global

trend leading to increased demands of medicinal plants for pharmaceuticals,

phytochemicals, neutraceuticals, cosmetics and other products is an opportunity

sector for Indian trade and commerce. Increased demand of medicinal plants

causes the adulteration/substitution. Adulterated material can be harmful, and

sometimes it is dangerous for using same preparation of end products. Substitutes

have become so popular in many cases that the manufacturers have forgotten about

the original plant. These substitutes do not contain the active ingredients available

through the genuine plants which are substituted nor the effects of the end

product. Some of these plants are high-valued medicinal plants and are also in

CHAPTER –TWO REVIEW OF LITERATURE

Department of Botany, Jamia Hamdard, New Delhi 5

endangered state. Increasing demand and use of herbal medicines by the public

require evaluation of health claim and development of standards of quality and

manufacture (Marwick, 1995; Barnes et al., 2003).

One of the great issues with the herbal medicines is the authentic

identification of genuine plant because of wide spread adulteration/substitution in

the crude drug market, leading to poor quality of medicinal formulations.

Authentication of the plant material at various stages from plant harvest to the final

product is a need of the hour. Correct identification and quality assurance of the

plant material is also needed to ensure reproducible quality of herbal medicine,

which contributes to its safety and efficacy (Sagar et al., 2004; Joshi et al., 2004).

Most of the regulatory guidelines and pharmacopoeias suggest pharmacognostic

techniques like macroscopic and microscopic evaluation and chemical profiling of

botanical materials for quality control and standardization (anonymous WHO,

1996; Patra et al., 2010). However, these parameters are judged subjectively.

Substitutes and adulterants may closely resemble the genuine material. Chemical

profiling establishes a characteristic chemical pattern for a plant material, its

fraction or extracts. Metabolic profiling (TLC/DLC) and other analytical techniques

like volumetric analysis, gravimetric analysis, gas chromatography and

spectrophotometers methods are frequently used for quality control but they have

several limitations because of their variable sources and complexity. Molecular

biology offers various modern techniques that can be applied for plant

identification. Genetic polymorphism in controversial medicinal plants has been

widely studied which helps in distinguishing plants at inter/intra species level.

Molecular markers provide a useful tool for safe identification and assessment of

the genetic variability of the medicinal plants. DNA-based markers have been used

for a wide range of applications in food crops and horticultural crops but it has

been extensively used now-a-days for authentic identification of medicinal plant

materials. In the present chapter, reasons for adulteration/substitution are

provided and further strategies involved in the identification and characterization

of medicinal plants are given.



2.1 PROBLEMS ASSOCIATED WITH THE EFFICACY OF MEDICINAL PLANTS

Despite considerable increase in the popularity of herbal drugs, increased concern

is being voiced regarding the efficacy and safety of medicinal herbal drug.

Maintenance of the efficacy and safety of medicinal herbal drug is facing several

problems. Some of the most common problems are given below:

1. Problem of intentional and un-intentional adulteration of plant source

2. Substitution of substandard variety

3. Variation in the content of active principle

4. Herb-drug interaction

2.1.1 Problem of adulteration in medicinal plants

Now-a-days, drugs are adulterated with substandard commercial varieties, inferior

drug or artificially manufactured commodities. Adulterants are the herbs which

look physically similar to that of the original plant with the result they are

adulterated in such a way that for an ordinary person it becomes very difficult to

make the difference between the real and the part adulterated with the same.

Sometimes the adulterated product has no resemblance with the genuine article,

may or may not have any therapeutic or chemical component desired. For example,

leaves of Ailanthus species are adulterated with the leaves of Belladonna, Cassia etc.

Leaves of Phytolacca and Scopalia are adulterated with the leaves of Belladonna.

Similarly, leaves of Xanthium are adulterated with the leaves of Stramonium.

Sometimes, they are adulterated even with synthetic principles like addition of

citral to oil of lemon and benzyl benzoate to balsam of Peru. There are medicinal

plants which have been added with adulterants either to increase its concentration

or due to its less availability (Table 1). Black pepper which is often called ‘Kings of

spices’ is commonly adulterated with dried papaya seeds which closely resemble

them in color, size and shape. It is often used to increase the bulk of the sample but

has deleterious effects (Tremlova et al., 2001; Bhalla et al., 1975). Another good

example of adulteration is Chilli, which is used in all types of curries in India.

Artificial colors such as coal tar red, sudan red, para-red etc., synthetic pungent



compounds, brick powder are the non-plant based adulterants reported in chilli

powder (Mitra et al., 1961; Mazzetti et al., 2004). Turmeric powder, prepared from

the processed rhizome, is frequently adulterated with rhizomes of cheaply

available related species, especially with those containing the colouring pigment

curcumin. The related curcuma species, which have been used as adulterants are C.

zedoaria Rosc or “yellow shotti” syn. C. xanthorrhiza Roxb or “Manjakua” and C.

malabarica, “shotti” (Sen et al., 1974; Tripathi et al., 2007).

Table 1. Adulterants found in some of the major traded medicinal products

Commodity Adulterants

Chemical/

earthy material

Biological material

Ginger (Zingiber officinale)

Lime, capsaicin Exhausted ginger (volatile oil extracted).

Ginger powder Lime Capsicum, Grains of paradise; turmeric; exhausted ginger fortified with flavours; Japanese ginger (Zingiber mioga).

Black pepper berries (Piper nigrum)

Mineral oil Dried papaya seed ( Carica papaya); wild Piper Spp. (P. attenuatum and P. galeatum); fruits of Lantana camara and Embelia ribes; seeds of Mirabilis jalapa; berries of Schinus molle; exhausted black pepper: light berries, stems and chaff of black pepper

Black pepper powder

Dye Powdered papaya seed; wild Piper berries; Lantana camara; Embelia ribes; Mirabilis jalapa seeds; Schinus molle berries; exhausted black pepper and light berries; starch from cheaper source.

Chilli fruits (Capsicum annum)

Dyes, mineral oil -

Chilli powder Dye-coal tar red, sudan red, para red; vanilyl-n-nonamide; Mineral oil; talc powder; brick powder; salt powder

Powder fruits of ‘Choti ber’ (Ziziphus nummularia); red beet pulp; almond shell dust; extra amounts of bleached pericarp, seeds, calyx and peduncle of chilli; starch of cheap origin; tomato wastes.

Turmeric powder (Curcuma longa)

Dye Mentanil Yellow, Orange II

Wild Curcuma spp, C zeodaria Rose or ‘yellow shotti’ syn. C. malabrica; starch



lead chromate; chalk powder; brick powder

from cheaper source; saw dust.

Cinnamon bark - Cassia (Cinnamomum cassia)

Cinnamon powder

Eugenol, cylon oil, yellow brown dye

Aromatized and powdered beechnut husk; hazel nut; almond shell dust.

Cardamom fruits (Elettaria cardamomum)

Small pebbles Orange seeds; unroasted coffee seeds.

Cardamom seeds - Seeds of Amomum aromaticum, A. subalatum and A. cardamomum

Cardamom seed powder

- Powdered cardamom bulls

Nutmeg (Myristica fragrans)

Pieces of clay for repairing broken nutmeg

Wild species- Macassar (Myristica argentea), Bombay nutmeg (M. Malabarica) and M. otoba

Mace (Myristica fragrans)

- Bomabay mace (Myristica malabarica); Macassar mace (M. argentea)

Clove Magnesium salt, sand, earth

Exhausted above (volatile oil extracted); stem and fruits of clove.

Mustard seed - Argemone seeds (Argemone mexicana); rape seed; ragi

Mustard seed powder

- Added starch; turmeric

Cassia bark (Cinnamomum cassia)

- Bark of Cinnamomum japonicum, C. mairei, C. Burmanii.

Allspice powder (Pimento dioica)

- Powdered clove stem; berries of Myrtus tobasco and Lindera benzoin

Aniseed Fine earth metals Hemlock fruit; parsley; dill fruit

Aniseed powder - Fennel

Nigella seeds (Nigella sativa)

- Onion seeds

Caraway (Caravum carvi)

- Cumin; Carum bulbocastanum

Fennel - Exhausted or partially exhausted fennel fruits; stem tissue and stalks of fennel; umbelliferous seeds.

Poppy seed (Papaver sommniferum)

- Rajeera seeds (Amaranthus paniculatus)

European dill Terpenes Indian dill

Ajowain Earthy materials Exhausted ajowain seeds; excess stem and chaff

Mediterranean - Origanum majorana; O. syriacum; O.



oregano vulgare; Satureja montana.

Asafoetida Coal tar dyes; gypsum; red clay; chalk

Foreign resin-Gum Arabic, gum resin colophony, galbanum, moriacum, resin, rosin; Barley; wheat or rice flour; slices of potato

Saffron (Crocus sativus)

Synthetic dyes-tartrazine, ponceau 2R, sunset yellow, amaranth, orange GG, methyl orange, eosin and Erythrosine; oil; honey; glycerine; solutions of potassium or ammoniumnitrate; sodium sulphate; magnesium sulphate; barium sulphate; borax

Different parts of the saffron flower itself (styles, stamen, strips of the corolla); dried petals of safflower and Scotch marigold; calendula; poppy; arnica; onion skins; turmeric; annatto; stigmata from other species of Crocus, pomegranate, Spanish oyster and maize; dyed corn silk; meat fibre; red sandal wood; turmeric powder; paprika powder.

Vanilla beans - Tonka beans (Dipteryx odorata); Dipteryx oppositifolia; vanillon (Vanilla pompona); little vanilla (Selenipedium chica); leaves of orchid Angreacum fragrans and Orchis fusca, spirnathes cernua, Trilisa odoratissima, Nigritella angustifolia and Melilotus spp.

Vanilla extract Synthetic vanillin, ethyl vanillin, vertraldehyde, piperonal, vanitrope and coumarin

-

2.1.2 Substitution in medicinal plants

Substitution involves intentional replacement with another plant species or

intentional addition of a foreign substance to increase the weight or potency of the

product or to decrease its cost. Over-exploitation, unsustainable harvesting, habitat

degradation due to increased human activities, illegal trade and virtual decimation

of several valuable, rare and endangered medicinal plants are the main factors

leading to the current rate of extinction (Nautiyal et al., 2002; Jablonski., 2004).



Highly priced market value plants which are commonly substituted are

Chlorophytum borivilianum with Asparagus racemosus, Swertia chirayita with

Andrographis paniculata, American ginseng (Radix quinquefolius) with ginseng (Radix

ginseng). Due to limited supply, Hypericum perforatum is replaced by H. patulum and

Podophyllum hexandrum with Valeriana jatamansi in India. Picrorhiza kurruoa, a liver

stimulating drug plant which dwells in Himalayas, is often substituted with Lagotis

cashmeriana or Holoptelia integrifolia. There are many medicinal plants which have

controversial status, as they have same common name but belong to different

genus/species and these are the main common alternatives used in pharmaceutical

industries like Hemidesmus indicus/Tylophora indica (Anantmool), Pluchea

lanceolata/Vanda roxburghii (Rasana), Onosma echinoides/Jatropha curcas (ratanjot).

Confusion also exists in the identification of plant materials where the origin of

particular drug is assigned to more than one plant, sometimes having vastly

different morphological and taxonomical characters. There are few others, where

the identity of plant sources is doubtful or still unknown.

There can be ignorant or intentional substitution with cheaper plant

material. Mistaken identity due to multiple synonyms and local names often leads

to wrong plant source being used in preparations. Anacyclus pyrethrum and

Spilanthes acmella are both commonly known as ‘Akarkara’ which is a constituent of

‘Chywanprash’. Spilanthes acmella is indigenous to India and Anacyclus pyrethrum is

not indigenous to India. Therefore, the former is easily substituted by the later one.

Several pharmaceutical companies are using Spilanthus acmella in place of Anacyclus

pyrethrum. Another classical example is that of Shankhpuspi. Canscora decussata,

Convolvulus pluricalis, Clitorea ternatea and Evolvulus alsinoides. Convolvulus pluricaris

has been described as the genuine plant material and the other as its substituents.

The true source of the crude drug in such case can be located only after detailed

chemical and pharmacological studies. For example, detailed chemical

investigation on Bacopa monnieri and Centella asiatica, described by the same

vernacular name ‘Brahmi’ and easily substituted with one another, have revealed

entirely different phytochemical composition. The former contains alkaloid

Brahmine, Herpestrine and Bacoside A & B which have been found to have



important action on brain function, while Centella asiatica contains asiaticoside,

hydrocotyline etc. which have hardly common relationship with the properties

ascribed to the drug ‘Brahmi’ in the text. Moreover, there is a single plant that has

different vernacular name and many herbs that have single vernacular name in

different parts of India (Table 2).

Table 2. Substituents used in various traded medicinal plants.

Vernacular name/ Trade name

Genuine plant Substituents

Sankhpuspi Evolvulus alsinoides

Clitorea ternatea Ephedra gerardiana Convolvulus microphyllus

Jeevaka Malaxis acuminate Tinospora cordifolia

Ratanjot Onosma echinoides

Clausena pentaphylla Canscora decussate

Brahmi Bacopa monnieri Centella asiatica

Chiraita Swertia chirayita Andrographis paniculata

Ativisha Aconitum heterophyllum Cyperus rotundus

Rishabaka Microcystis wallichii Bambusa arundinacea

Chitraka Plumbago zeylanica Baliospermum montanum

Meda Polygonatum cirrihifolium Withania somnifera

Lakshmana Ipomea sepiaria Elephantopus scaber

Mahameda Polygonum verticillatum Hemidesmus indicus

Pushkarmula Iris germanica Saussurea lappa

Kakoli dwaya Roscaea procera Asparagus racemosus

Murva Marsedenia tenacissima Rubia cordifolia

Nagakesara Messua ferrea Callophyllum inophyllum

Senna Cassia senna Cassia angustifolia

Kateli Solanum xanthocarpam Clerodendron indicum

Gokhru Tribulus terrestris Pedalium murg

Agnimantha Clerodendron phlomoides Premna oblusifolia

Kutki Gentiana kuroo Picrorhiza kurroa

Rasna Pluchea lanceolata Alpinia officinarum Vanda roxburghii Geranium wallichianum

Somlata Sarcostemma acidum Abies webbiana

Parpatta Fumaria parviflora Mollungo pentaphylla

Punarva Boerhavia diffusa

Trianthema portulacasstrum



2.1.3 Variation in active principles

Geographical distribution of medicinal plants seems to be an important factor

related to variation in the active compounds on medicinal plants (Gairola et al.,

2010). Variations in artemisin content of Artemisia annua has been found in different

regions of the world. The artemisin content has been found to differ markedly with

geographical conditions, being 0.01 to 0.09 % in different parts of India (Singh et al.,

1986), 0.01 to 0.5 % in China (Luo et al., 1980) and 0.06 to 0.1% in Argentina (Liersch

et al., 1986) and 0.06 to 0.22 percent in the USA (Ferreira et al., 1995). The local

ecology has been found to effect markedly the alkaloid production in Aconitum

napellus and A. heterophyllum (Beigh et al., 2008). Effect of altitude was apparent in

the production of Aconitum alkaloids (Beigh et al., 2008). It was seen that the

alkaloid production increased with the altitude. A. heterophyllum grown at a lower

altitude has shown a marked decrease in the morphological, physiological and

biochemical responses of plant species to environment. Similar variation was

observed for podophyllotoxin content in Podophyllum hexandrum (Iqbal et al., 2004).

The increase in the alkaloid production with increasing altitude may be related to

the biochemical adaptation to the species in a particular, ecological, edaphic or

climatic niche. It has been known that beyond their ecological niche or under stress

condition medicinal plants behave differently, they either show increased or

decreased level of bioactive compounds. This is the reason why the A. heterophyllum

grown at lower altitude has shown a marked decrease in the morphological,

physiological traits. The biosynthesis of alkaloids in the above given species

however are genetically controlled but they are greatly influenced by the biotic or

abiotic environmental factors of the given habitat. From time to time it has been

seen that there has been variations in the biochemical constituents of some major

important medicinal plants like withafarin content varied in different

concentrations in Ashwagandha (Sangwan et al., 2004), Bacoside A content varied in

different accessions of Bacopa monnieri (Mathur et al., 2002). Vascicine contents

varied in different accessions of Adhatoda zelaynica (Bagachi et al., 2003).



Several studies have examined the effects of increased temperatures on

secondary metabolite production of plants (Jochum et al., 2007). Researchers have

postulated that climate change could affect the chemical composition and

ultimately the survival of some medicinal plants in high altitude region.

Temperature stress affects the secondary metabolites and other compounds

(Zobayed et al., 2005).

Effect of seasonal variation on the active principles of different plants of

Mentha longifolia (Ahmad et al., 2011), Hypericum perforatum (Southwell et al., 2001)

revealed that medicinally active ingredients are in increased concentration in

specific season and their collection is better in this time. Different medicinal plant

species show a marked variation in active ingredients during different seasons,

these have been widely attributed to variations in environmental variables such as

temperature and rainfall (Ghimire et al., 2006). Calotropis procera samples were

collected in three seasons i.e. winter, summer and monsoon from Jodhpur, samples

were processed and secondary metabolites viz. alkaloids, flavonoids and sterols

were extracted. Results showed that optimum yield of total extractives in flowers

were obtained in monsoon (Rathore et al., 2010). Extracts of the young leaves of

Mikania glomerulata contained highest coumarin level during the early evening of

December and July (Pereira et al., 2000). Content of the four iridoids in cultivars of

Antirrhinum majus (antirrhinoside, antirrhide, 5-glucosyl-antirrhinoside and

linarioside) showed a marked bimodal distribution with high total values in early

and late season and a very low content of all iridoids with the onset of flowering

(Hogedal et al., 2000). Seasonal variation in content of bioactive alkaloids including

aconitine in different plant parts of two species of Aconitum, A. nagarum and A.

elwesii, showed that alkaloids in root was found to be highest in the month of

November in both the species of Aconitum. The alkaloid content of leaves was

highest in the pre-flowering season in both these species. However, aconitine

content decreased significantly (at 5% level of significance) during October–

November in A. nagarum leaves (Sinam et al., 2011).

Genotypic changes due to regional factors also influence the variations in

secondary metabolites found in the different stages of vegetative growth. The



experiment to prove the genetic variations was performed in Hypericum perforatum

and was found that hypericin contents, total phenols, and flavonoids varied in the

above plants (Ghavamaldin et al., 2012). The presence of significant genotype by

environment interaction effects (GxE) suggests that genotypes are specialized when

they exploit environments differentially, while in the absence GxE effects suggest

that genotypes are generalized equally responsive to changes in environmental

conditions. Studies simultaneously examining the effects of genotype and

environment are rare (Gauch and Zobel, 1996).

Most of the botanicals keep confined the bioactive compounds in specialized

structure or pockets. The knowledge of location of such compounds in plants is of

paramount importance to ensure proper harvesting and collection of the botanical.

In some plants, maximum amount of metabolites are present in particular stage

and position of leaves (Digitalis, Mentha). In Costus speciosus, reproductive stage

registers maximum diosgenin content. So it is better to collect the medicinal plant

in that stage when there is maximum amount of secondary metabolite. The

psoralen content in Psoralea corylifolia was highest in seeds, followed by leaves, and

stem and roots (Ali et al., 2008). In Aconitum heterophyllum, it was found that tubers

had the highest alkaloid content while the aerial stem contained the least. Within

the plant, the alkaloid content decreased in the order Tubers > Leaves > Flowers >

Stem. (Beigh et al., 2008).

2.1.4 Consequences of adulteration/substitution on human health

Herbal products are not completely free from side-effects. Adulteration in herbal

drugs can cause damage to human body (Jordan et al., 2010). The WHO database

has over 16,000 suspected herbal case reports. The common adverse effects by

adulterated herbal products are hypertension, hepatitis, face, oedema, angiodema,

convulsions, thrombocytopenia, dermatitis and death. There are various side

effects such as, cardiovascular problem with the use of Ephedra, hepatoxic effect

due to Kava-Kava consumption, anticholinergic effect due to Datura betel etc.

(Cuzzolin et al., 2006). Sometimes an incorrect botanical quality with respect to the

labeling can create a quite fuss. For example in 1990’s a South American product



labelled as ‘Paraguay Tea’ was responsible for a major outbreak of anticholinergic

poisoning in New York. After a chemical analysis of the Labelled ‘Tea’, it was

found that metabolites found in the above were different from those present in

‘Paraguay Tea’ (Hsu et al., 1995). ‘Tung Shueh pills’, used for pain relief sampled

from Singapore market, was found to contain four undeclared drugs caffeine,

diazepam, indomethacin and prednisolone which have the potential to cause

mental depression, bone loss, spontaneous fractures and coma. ‘Wonder drug’ used

for reducing fat from the body was found to contain Phenoformin, a drug which is

banned in Singapore (Yee et al., 2005). The replacement of Stephania tetrandra

(fangji) with the root of Aristolochia fangchi (guangfagji) in a slimming treatment

that included conventional medicines had resulted in numerous cases of

progressive renal interstitial fibrosis, complicated in some persons by urothelial

carcinoma (Nortier, 2000). Such kind of misunderstanding may lead to serious

adverse conditions. With the growing need for safer drugs, attention has been

drawn to their quality, safety and efficacy and standards of the traditional Indian

medicine formulations. Therefore, quality control standards of various medicinal

plants used in indigenous system of medicine are becoming more relevant today in

view of commercialization of formulations based on medicinal plant.

2.1.5 Herb-drug interactions

Due to growing use of herbals and other dietary supplements healthcare providers

and consumers need to know whether problems might arise from using this

preparation in combination with conventional drugs. However, the evidence of

interactions between natural products and drugs is based on known or suspected

pharmacologic activity, data derived from in vitro or animal studies, or isolated

case reports that frequently lack pertinent information. The usefulness of such

information is questionable. More recently an increasing number of documented

case reports, in vivo studies, and clinical trials evaluated herbal-drug reactions.

Herb-drug interactions may have little or no effect in patients. For those herb-drug

reactions which are potentially harmful, the effects may either be in a small

proportion of patients or serious interactions may occur and these may be life



threatening. There is only limited information available in the literature concerning

the interaction of herbal medicines and conventional medicines. Despite the

common use of Herbal drugs it is quite unknown that the herbal drugs have the

tendency to alter the efficacy of the co-administered prescription drugs.

Interactions between herbs and drugs may increase or decrease the

pharmacological or toxicological effects of either component. Synergistic

therapeutic effects may complicate the dosing of long-term medications, e.g. herbs

traditionally used to decrease glucose concentrations in diabetes could theoretically

precipitate hypoglycemia if taken in combination with conventional drugs (Tirona

and Bailey, 2006). Herbal medicines are mixtures of more than one active

ingredient. The multitude of pharmacologically active compounds obviously

increases the likelihood of interactions taking place. Hence, the likelihood of herb–

drug interactions is theoretically higher than drug–drug interactions, if only

because synthetic drugs usually contain single chemical entities. There is evidence

that taking herbal preparations can result in pharmacokinetic or pharmacodynamic

interactions that represent a potential risk to patients taking conventional

medicines. Natural products, unlike conventional drugs, provide a complex

mixture of bioactive entities, which may or may not provide therapeutic activity.

Interactions may occur when constituents of herbal products have either synergistic

or antagonistic activity in relation to a conventional drug. Experimental data in the

field of herb-drug interaction are limited, case reports are less and case series are

also very less reported. A case study of 1000 elderly people admitted to hospital

from the emergency department found that 538 patients were exposed to 1087

drug-drug interactions, 30 patients’ experienced adverse effects as a consequence of

these interactions (Doucet et al., 2002). Different types of herbal-drug interactions

have been shown in Table 3.



Table 3. Herb-Drug Interactions

Herbal product Drug Induced genes Result of interaction

Hypericum perforatum

Cyclosporin, indinavir, ritonavir, omeprazole, digoxin, imatinib, oral contraceptives, Alprazolam, warfarin

CYP3A4, P-glycoprotein, CYP2C9, CYP2C19, CYP1A2, CYP2E1

Decreased plasma concentration, digoxin concentration, Hypomania, decreased anti-coagulant effect,

Echinacea purpurea

Midazolam CYP3A4 Increased or decreased clearance

Gingkgo biloba Omeprazole, Aspirin, paracetamol, Trazodone, warfarin

AhR activation Spontaneous hyphema, serious bleeding disorder, coma

Panax sp Warfarin, AhR activation Over-anticoagulation, insomnia, excitation, decreased INR in rats

Areca catechu Flupenthixol, procyclidine, prednisone,

Rigidity, inadequate control of asthma, bradykinesia, stiffness,

Angelica sinensis

Warfarin Dong quai contains coumarins, increased INR and widespread bruising

Glycirrhiza glabra

Prednisolone, oral contraceptives

AhR activation Decreases plasma clearance orally, increases AUC hypertension, causes insomnia, oedema,

Ephedra Guanethidine Enhances sympathomimetic effect

Shankhpuspi Phenytoin Decreased phenytoin concentrations

Allium sativum Warfarin, Saquinavir MDR1 Increased INR post-operative bleeding and spontaneous spinal epidural haematoma



2.1.6 Regulatory norms for standardization

Some of the parameters that help in understanding the development of herbal drug

regulation in a given nation are general policy structure, drug registration system,

development of pharmacopoeia, natural monographs, inclusion in essential

medicine list and drug type. Herbal drug regulation in different countries has been

recorded in Table 4. Of the eighteen countries studied, except Bhutan, Sri Lanka

and Maldives, all countries have herbal drug regulation and registration system.

Nine countries (Korea, Indonesia, India, Myanmar, Sri Lanka, Thailand, China,

Malaysia, and Vietnam) have their National monographs for herbal drugs. In

Bhutan, Nepal and Philippines the development of monographs for herbal drugs

are in progress. Pharmacopoeias for the herbal medicines are developed only in

fifteen countries (except for Maldives, Malaysia and Singapore). In some countries

the herbal drugs come under essential medicines list. Philippines have the highest

number included in the list with 2000 herbal drugs followed by China with 1242

herbal drugs. India has separate essential medicine lists for the traditional herbal

drugs which are available as OTC drugs.

Table 4. Herbal drugs regulation in selected countries.

Country Regulation on

herbal drug (yr)

National monograph Pharmacopoeia Drug type

Bangladesh 1992 No Bangladesh national formularies

on Unani and Ayurvedic medicine

Prescription and

OTC

Bhutan No In development In development Prescription

India 1940 Yes Ayurvedic Pharmacopoeias of

India and Unani, Pharmacopoeia of

India

Prescription and

OTC

Indonesia 1993 Materia medica Indonesia Farmakope Indonesia OTC

Myanmar 1996 Monograph of Myanmar

medicinal plants

In development OTC

Nepal 1978 In development In development Prescription and

OTC

Sri Lanka No Compendium of

medicinal plants

Ayurvedic Pharmacopoeia Prescription and

OTC

Australia 1989 No British Pharmacopoeia OTC

China 1963 Yes Chinese Pharmacopoeia Prescription and

OTC

Japan 1960 No Japanese Pharmacopoeia Prescription and

OTC

Malaysia 1984 Malaysian herbal

monograph

No OTC

Philippines 1984 In development In development OTC

Singapore 1998 No No OTC

Korea 1999 Korean Herbal medicine

monographs

Pharmacopoeia of the Democratic

Democratic People’s Republic of

Korea

Prescription and

OTC



2.2 STRATEGIES FOR THE IDENTIFICATION OF GENUINE MEDICINAL PLANTS In order to overcome this perplexing situation of misidentification, intentional

adulteration and substitution of sub-standard medicinal plant, different methods

have been used for the authentication of different medicinal plant. Several markers

such as taxonomic, chemical, genomic and proteomic help in identification of

herbal drug components. Such methods encompass morphological (macroscopic),

and anatomical (microscopic) study, chemical analysis such as HPLC/MS, protein

analysis and the use of molecular markers (Bhutani et al., 2003; Mosihuzzamam et

al., 2008; Patwardhan et al., 2005). The chemical markers are defined as constituents

of herbal medicinal products which are of interest of quality control purposes. They

are helpful for the identification of adulterants, differentiation of herbal medicines

with different sources, stability testing of proprietary product etc (Lazarowych et

al., 1998). As the genetic composition is unique for each species and is not affected

by age, physiological conditions and environmental factors. DNA based markers

are also used in identification of medicinal plant (Shikha et al., 2009). Some of the

methods are described here.

2.2.1 Organoleptic evaluation

Organoleptic evaluation of drugs refers to the evaluation of drug by colour, odour,

size, shape, taste and special features including touch, texture etc. Since the

majority of information on the identity, purity, and quality of the material can be

drawn from these observations, they are of primary importance before any further

testing can be carried out. Organoleptic evaluation can be done by means of organs

of sense which includes the above parameters and thereby define some specific

characteristics of the material which can be considered as a first step towards

establishment of identity and degree of purity. For this purpose authentic specimen

of the material under study and samples of pharmacopoeial quality should be

available to serve as a reference. This evaluation process provides the simplest,

quickest means to establish the identity and purity and thereby ensure quality of a

particular sample. However, judgement based on the sensory characteristics like



odour, taste etc may vary from person to person and time to time based on

individuals nature. These features are useful in judging the material in its entirety.

No preliminary treatment is necessary for evaluating the sample in this manner.

Uptill now, traditional morphological inspection is still widely used to distinguish

the herbs. For example, in the traditional authentication of Radix Codonopsis, the

following criteria are used: the root is cylindrical, slightly curved, 10-35 cm long

and 0.4-2.0 cm in diameter and the odour is characteristic, aromatic and tastes

sweet. The morphological inspection to authenticate herbal is simple and direct but

its accuracy depends heavily on the examiners, which are sometimes subjective

(Indian Herbal Pharmacopoeia, 2002; British Herbal Pharmacopoeia, 1996).

2.2.2 Macroscopic and microscopic evaluation

Histological techniques based on microscopic examinations are used to reveal the

characteristics of tissue structure and arrangement in cork cell, cortex, sieve tubes,

xylem vessels and cell components or content of a manufactured product. The

thickness of the exodermal cell walls, diameter of exodermis, number of transfusion

cells, and number of vascular bundles were used as markers for identifying the

plant source of Herba dendrobii (Li et al., 1995) and Radix Codonopsis is identified by

its cortex and cork cells (Namba et al., 1981). Histological identification is not

applicable to modern herbal drugs, for example, herbal capsules etc. Related

species may share similar histological characteristics, making this approach not so

accurate. Prior to processing of any plant into a finished botanical product, an

accurate identification of the plant species by a trained individual is needed to

assure that the correct source material has been used. GAP guidelines dictate that

the authenticity and quality of the final botanical product is directly related to the

authenticity and proper identification of the source material (Anonymous, WHO,

1996). Generally plants may be accurately identified up to species, whether wild or

cultivated, in their whole form. Voucher specimens, consisting plant or portions of

the plant that illustrate relevant taxonomic features needed for identification,

should really be retained from each processing batch for future verification

purposes. Botanical identification is accomplished via examination of the intact,



whole plant material, most readily during the time of collection or harvest, while

macroscopic examination is done by studying the morphological or organoleptic

characteristics of relevant plant parts, as a whole or cut into chunks and or

fragments. A trained individual is needed for identification, having easy access to

working herbaria at which comparisons of the voucher specimens against others of

known identity may be made. Voucher specimens and macroscopic samples need

to be stored in ideal conditions with space-consuming storage facilities.

Macroscopic techniques are recognized by subtle gradations within plant species,

such as chemotypes or ecotypes, and in some cases may not allow identification

even to the species level.

Microscopic method allows for identification of botanicals through an

assessment of whole, fragmented, or powdered crude plant material. Plant parts

examined by microscope include trichomes, stomata, cell types, fibres, granular

objects (e.g starch grains, calcium oxalate crystals) as well as minute floral and fruit

characteristics. This technique is relatively rapid and repeatable. Identification of

herbal medicine in all pharmacopoeias is currently based on primarily on the

microscopic characteristics (Joshi et al., 2006).

2.2.3 Phytochemical methods for the identification of plants

The traditional methods of pharmacognostic analysis are mainly based on

differences in morphological characters and analysis of compounds. Chemical

profiling establishes a characteristic chemical pattern for a plant material, its

fractions or extracts. Thin layer chromatography (TLC) and high performance thin

layer chromatography (HPTLC) are routinely used as valuable tools for qualitative

determination of small amounts of impurities. HPTLC and HPLC fingerprints have

been used to distinguish genuine plant from adulterants (Wang et al., 2005). The

accuracy of authentication has limitations because of the amounts of samples, the

stability of chemical constituents, the variable sources and the chemical complexity.

In addition, many analytical techniques such as volumetric analysis, gravimetric

determination, gas chromatography and column chromatography, high

performance liquid chromatography and spectroscopic methods are also used



frequently for quality control and standardization. In order to ensure efficacy,

selection of a correct chemotype of the plant is necessary. But it is difficult to select

the right chemotype, to which the clinical effects are attributed, even there are

many known chemotypes of a plant species. Assaying for those herbal ingredients

with known active principles is another method of ensuring products identity and

purity. Purity is closely linked with the safe use of drugs and deals with factors

such as ash values, contaminants, heavy metals and other adulterant species.

Analytical methods such as TLC, HPLC and Gas Chromatography can be

employed in order to establish the constant composition of herbal preparations.

TLC, HPLC, GC, quantitative TLC (Q-TLC), and high-performance TLC (HPTLC)

can determine the homogeneity of a plant extract. Over-pressured layer

chromatography (OPLC), infrared and UV-visible spectrometry, GC-MS (Gas

chromatography-mass spectrometry), LC-MS (Liquid chromatography-mass

spectrometry) used alone, or in combinations, are powerful tools for

standardization and to control the quality of both the raw material and the finished

product. The results from these sophisticated techniques provide a chemical

fingerprint as to the nature of chemicals or impurities present in the plant or

extract. The standardization of various marketed herbal and polyherbal

formulation Madhumehari Churna (Baidynath) containing the mixture of eight

herbal antidiabetic drugs Momordica charantia (seeds), Syzigium cuminum (seeds),

Trigonella foenum (seeds), Azadirachta indica (leaves), Emblica officinalis (fruits),

Curcuma longa (rhizomes), Gymnema sylvestre (leaves), Pterocarpus marsupium (heart-

wood) (Chandel et al., 2011), “Pancasama Churna” known to be effective in

gastrointestinal disorder (Meena et al., 2010), “Dashmularishta”, a traditional

formulation, used in the normalization of physiological processes after child birth

(Sanjay et al., 2009), “Gokshuradi Churna” (Kumar et al., 2011), Jawarish-e-

Darchini (Meena et al., 2010) have been reported.



Thin layer chromatography / High pressure thin layer chromatography (TLC &

HPTLC).

TLC is the common method of choice for herbal analysis because it provides first

characteristic fingerprints of herbs before instrumental chromatography methods

like GC and HPLC. TLC is used as an easier method of initial screening with semi-

quantitative evaluation together with other chromatographic techniques, as there is

relatively less change in the simple TLC separation of herbal medicines than with

other instrumental chromatography. HPTLC has the advantages of many-fold

possibilities of detection in analyzing herbal medicines. HPTLC has been

investigated for simultaneous assay of several components in a multi-component

formulation. It has been widely employed in pharmaceutical industry in process

development, identification and detection of adulterants in herbal product and

helps in quality control of herbs and health foods. Some of the examples of the use

of TLC and HPTLC are - quantification of bergenin, catechin, gallic acid from

Berginia lingulata (Dhalwal et al., 2008), capsaicin and piperine by Milangi thailam

(Manimaran et al., 2005), genistein and daidzen in Glycine max (Suthar et al., 2002),

berginin in Caesalpinia digyna (Mahadevan et al., 2005), luteolin in Thymus vulgaris

(Bazylko et al., 2007), D-rhamnoside in Euphorbia hirta ( Mallavadhani et al., 2002),

picroside-I and picroside II in Picrorhiza Kurruoa (Singh et al., 2005), curcumin in

herbal formulation (Ansari et al., 2005), beta-sitosterol-d-glucoside & withaferin A

in Withania somnifera (Mahadevan et al., 2003), bacosisde-A in Bacopa monnieri

(Shahare et al., 2010), hepatoprotective diterpenoids from Andrographis paniculata

(Saxena et al., 2000). It has also been used to detect the presence of ground papaya

seed in ground black pepper (Paradkar, 2001) and for identification of Fructus

Xanthii (Yin et al., 2005). It has also been used to detect swertiamarin in marketed

polyherbal antidiabetic formulation (Patel et al., 2007).

High Performance liquid chromatography (HPLC)

HPLC is popularly used for the analysis of herbal medicines because it is easy to

perform and its use is not limited by the volatility and stability of the sample

compound. Thus it has been extensively used for the application and analysis of



herbal medicines. Reverse phase (RP) columns have gained much importance now-

a-days for the separation of different herbal medicines. It is seen that the optimal

separation for the HPLC involves many factors like different composition of mobile

phases, pH adjustment and pump pressures etc. It has also varied applications.

One of the main advantages of HPLC is that it can be coupled with UV, DAD,

ELSD, FLD, RID, MS, and NMR which supplies different possibilities for detecting

different constituent types. Caulometric electrode array detection (HPLC-CEAD)

and charged aerosol detection CAD has also been introduced for analysis of herbal

formulations. HPLC method with various detectors has been developed for

qualitative and quantitative analysis in Piper nigrum fruits (Jain et al., 2007, Ertas et

al., 2007), constituents such as chiconic acidin in Poisidonia oceanica, (HPLC-UV

detector) (Haznedaroglu et al., 2007), simultaneous determination of puerarin,

daidzin, paeoniflorin, liquiritin, cinnamic acid, cinnamaldehyde and glycyrrhizin in

Kampo medicines (Okamura et al., 1999), phenolic compounds in Achillea

millefolium (Benetis et al., 2008), chlorogenic acid in tobacco leaves (HPLC-UV)

(Chen et al., 2007), Angelia dahurica (HPLD-DAD) (Kang et al., 2008), phenolic

compound in Chyawanprash by RP column with HPLC UV-diode array detection

(Govindarajan et al., 2007), ‘Qi-shen-yi-qi preparation by LC-UV and LC-ELSD (Li

et al., 2008), determination of flavonoids in Hypericum japonicum by HPLC-ESI-MS

(Su et al., 2008). RP-HPLC has been used for detection of adulteration in olive oil

(El et al., 1995). Now-a-days, UPLC, RRLC and RSLC have been used for analysis of

various compounds in plants, like Panax notoginseng (Chan et al., 2008), Epimedium

(Chen et al., 2008), Carthamus tinctorius (Jin et al., 2008). It has also been used for

standardization of ‘Triphala’ (Singh et al., 2007) and Du-shen-Tang (Li et al., 2010).

Standardization of ‘Danning tablets’ (Liang et al., 2007) and ‘Longdan-Xiegan

decoction’ (Wang et al., 2007) by HPLC fingerprints are other good examples.

Gas chromatography (GC)

Gas chromatography has been effectively used for analyzing essential oils. GC

coupled with MS has been fruitful for the analysis of volatile constituents of herbal

medicines due to sensitivity, stability and high efficiency. Reliable information has



been achieved for the quantitative analysis of the complex constituents when GC

hyphenated procedures are used. GC combined with MS has been used as a

powerful separation method. It has been used for identification and quantification

of chemical constituents in MEGNI which consists of 9 different constituents

namely Myristica fragrans, Eucalyptus globules, Glautheria procumbens and Mentha

piperita (Kasthuri et al., 2010). HS-SPME is a modern procedure which has been

used as it is fast, simple, solvent–free extraction can be done. It has been applied for

the analysis of Ligusticum chuanxiong (Deng et al., 2005), Chrysanthemum (Shen et al.,

2004; Dong et al., 2007)) and Artemisia argyi (Li et al., 2008). It has been applied for

the analysis of Rhioxma Curcumae aeruginosae (Shaa et al., 2004). Further

modification of the above procedure is HS-SDME. In this technique, extraction of

the analyte is done using single microdrop of solvent such as benzyl alcohol or

octanol. It has been applied in Artemisia haussknechtii (Heravi et al., 2007), Angelica

sinensis (Deng et al., 2005), Atractylodes (Guo et al., 2006), Cloves (Myinff et al.,

1996).

Electrophoretic methods

Capillary electrophoresis technique is popularly used nowadays for natural

products analysis. Alkaloids and flavonoids have been extensively studied by this

method. Only evaluation of single herb is done at a time and it has proved to be

highly specific and sensitive. Capillary electrophoresis method was two-times

shorter than HPLC method. It has been applied on Flos carthami (Sun et al., 2003)

and Radix scutellariae (Wang et al., 2005). The introduction of capillary

electrophoresis has to a great extent paralleled that of liquid chromatography.

Moreover nowadays the techniques used are Capillary Zone Electrophoresis (CZE),

Capillary Gel Electrophoresis (CGE) and Capillary Isoelectric Focusing (CIEF).

Recently, several studies dealing with herbal medicines have been reported and

two kinds of medicinal compounds, i.e. alkaloids and flavonoids have been studied

extensively. In general, CE is a versatile and powerful separation tool with a high

separation efficiency and selectivity when analyzing mixtures of low-molecular-

mass components. It has been used to ascertain the botanical identity and quality of



Ephedra herbae (Liu et al., 1993), Coptidis rhizome (Liu et al., 1994), Ginseng radix

(Chuang et al., 1995).

Capillary electrochromatography is considered to be combination of both

HPLC and CE which takes the benefits of both i.e low sample/solvent

consumption and being highly efficient. It has been used to analyse different

coumarins like xanthotoxol, osthenol, oxypeucedanin hydrate and byankangelicin

in Angelica dahurica commonly called ‘Bai Zi’ in chinese (Chen et al., 2006).

Spectroscopic Method

Because of their uniqueness and beneficial advantages they are preferred over

chromatography methods. They combine speed and non-destructive character with

the need for no or only little sample preparation. This is one of the important

reasons that NIR and NMR spectroscopy is popular for purity and identity

evaluation in industry (Singh et al., 2010; Krishnan et al., 2005). Wu and colleagues

(2008) described the use of MIR and NIR spectroscopy for the quantitative

determination of α-pinene, methyl salicylate and eugenol in ‘Honghua oil’. This

preparation is composed of several essential oils like (wintergreen, turpentine,

clove and Cassia leaf) and is utilized in the treatment of rheumatism and joint

problems. It has been used for the analysis of Ephedra species (Kim et al., 2005),

Herba Epimedii (Pie et al., 2008) and Ginseng (Shin et al., 2007). For the compounds

of interest, characteristic absorption bands in the IR spectra was assigned, and

based on the quantitative GC results, the areas of these bands were correlated with

the respective quantities.

Limitations of chemical fingerprinting

Because of variable sources and chemical complexity the use of chromatographic

techniques and marker compounds has limitations in the standardization of

botanical preparations. Intrinsic factors such as genetics and extrinsic factors, such

as cultivation, harvesting, drying and storage conditions are few examples that can

affect the chemical profile of any herb. Chemotaxonomic study of herbs provides

only a quantitative account of secondary metabolites. As compared to the

conventional methods this approach has several advantages. The conventional



methods usually determine the individual compounds present in the herbal drugs.

We cannot determine whether they are active or specific or not. For quantitative

studies, use of specific markers to distinguish between varieties, has been useful.

These metabolites, used as markers, may or may not be therapeutically active, but

should ideally be neutral to environmental effects and management practices (Joshi

et al., 1999). Problem may also arise due to different chemotypes, e.g., Withania

somnifera is reported to have three chemotypes depending upon the steroidal

lactones. Withaferin A and other biologically active compounds may similarly vary

depending upon the environment, genotype and time of collection of plant

material. Thus, selection of right chemotype is very necessary to ensure the efficacy.

Moreover the problem is further added while selecting correct plant material to

establish the identity of certain species that is known by different binomial

botanical names in different regions e.g. Shankhpuspi is equated with any one of

the following plants in India ‘Canscora decussata, Evolvulus alsinoides and Clitorea

ternatea.

2.2.4 Molecular methods for the identification of plants

Genetic markers have proved their utility in fields like taxonomy, physiology,

embryology, genetics etc. As the science of plant genetics progressed, researchers

have tried to explore these molecular markers techniques for their applications in

commercially important plants such as food crops, horticultural plants, etc. and

recently in pharamacognostic characterization of herbal plants (Shaw et al., 2002;

Lum et al., 2006; Yip et al., 2007). Molecular markers are generally referred to as

biochemical constituents, including primary and secondary metabolites in plants

and macromolecules, viz., proteins and deoxyribonucleic acid (DNA). Secondary

metabolites as markers have been extensively used in quality control and

standardization of herbal drugs, but these also suffer with few limitations (Joshi et

al., 2004). The main focus is now on the development of markers based on genetic

composition and, hence, is unique, stable, and ubiquitous to the plants. The other

one, genetic markers have assisted in genomic characterization of various crop

species (Rafalski and Tingey, 1993). Although other genetic marker systems are in



use, most relevant marker is based on DNA sequence polymorphisms. Each system

has unique advantages and disadvantages based on cost, experimental objectives,

and type of information desired. Regardless of its inherent methodology, the

effective deployment of any marker system for genetic analysis requires that it

should be reliable and consistent. Various DNA-based markers, viz., RAPD, RFLP,

SSR, AFLP and SSR, are employed for plant species discrimination coupled with

methods of plant identification involving taxonomy, physiology and embryology

(Zhang et al., 2007; Sucher and Carles, 2008). Genome analysis based on molecular

markers has generated a vast amount of information and a number of data bases

are being generated to preserve and popularize it.

Randomly Amplified Polymorphic DNA (RAPD)

Randomly amplified polymorphic DNA is based on PCR amplification of genomic

DNA by random primer. In this reaction, a single species of primer (8-10 bp)

anneals to the genomic DNA at different sites on complementary strands of DNA

template. If these priming sites are within amplifiable range of each other, a

discrete DNA product is formed through thermo cyclic amplification. On an

average each primer directs amplification of several discrete loci in the genome

making the assay useful for efficient screening of nucleotide sequence

polymorphism between individuals (William at al., 1990; Welsh et al., 1990). This

approach requires no prior genetic information.

RAPD–based molecular markers have been found to be useful in

differentiating different accessions of Glycirrhiza species (Yamasaki et al., 1994),

Atractylodes plants (Kohjyouma et al., 1997), Piper nigrum (Khan et al., 2010),

Andrographis paniculata (Fu et al., 2003). Dried fruit samples of Lycium barabarum

were differentiated from its related species (Zhang et al., 2001). RAPD has been

used identify eight types of dried Coptis rhizomes (Cheng et al., 1997) and

Picrorrhiza rhizome (Um et al., 2001), Ammomum villosum and its adulterants (Wang

et al., 2000), Panax sp. (Shaw et al., 1995; Shim et al., 2003). The RAPD has been used

for the characterization of three different species of Scutellaria (Hosokawa et al.,

2000), three sub-species of Melissa officinalis (Wolf et al., 1999), identification of



Cuscuta reflexa from its adulterant, Cuscuta chinenesis (Khan et al., 2010 and

Echinacea species from its adulterants (Wolf et al., 1999; Nieri et al., 2003). DNA

profiling was also done to identify Chinese formulation Ping-Feng San (Cheng et

al., 1998).

AP-PCR, Arbitrary polymerase chain reaction is a special case of RAPD

where single primer of 10-50 bp is used. In AP-PCR the three steps are required, the

first two are non-stringent and the last one is stringent. AP-PCR has been applied

for analysis of species and genetic variation in plants. AP-PCR has been used to

authenticate Chinese drug Ku-Di-Dan (Herba elephantopi) from its substitutes (Cao

et al., 1996) and ‘Herba Taraxaci’ and its adulterants of six species of Compositae

(Cao et al., 1997).

DAF-DNA Amplification fingerprinting is a variant and independently

developed method. Short primers of 5-8 nucleotides are used that produces a

complex banding pattern. Only two temperature cycles are used. The products are

then separated by polyacrylamide gels and detected by silver staining. It has been

used to identify Magnoliae officinalis (Wang et al., 2001).

Both DAF and AP-PCR are different with respect to the length of random

primers, amplification conditions and visualization methods. Both do not require

prior genetic information and a single random primer is required.

Restriction Fragment Length Polymorphism (RFLP)

In RFLP, the genome of the plant is digested with suitable restriction endonuclease

enzyme and fragments of genomic DNA are run on gel electrophoresis. Thereafter,

the fragment of DNA is transferred to a membrane for Southern blotting.

Hybridization of the membrane to a labeled DNA probe determines the length of

the fragments which are complementary to the probe. A RFLP occurs when the

length of the detected fragment varies between individuals. Each fragment length

is considered an allele, and can be used in genetic analysis. The polymorphism is

detected by the presence or absence of bands. Analysis of RFLP variation was an

important tool in genome mapping, localization of genetic disease genes, genetic

fingerprinting etc. Such a polymorphism can be used to distinguish plant species,



genotypes and in some cases, individual plants. This technique is time consuming,

labour intensive and requires a large quantity of good quality and undegraded

DNA.

Different medicinal plants have been evaluated using RFLP technique. RFLP

was used to identify different Fritillaria species (Tsoi et al., 2003), Rheum species

(Yang et al., 2004), differentiation of Panax ginseng and Panax quinquefolius (Ngan et

al., 1999). RFLP has been used differentiate Codonopsis from its adulterants (Fu et

al., 1999), Lupinus (Yumazaki et al., 1993), Hedysarum species (Trifi-Farah et al.,

2001), Dendrobium species (Li et al., 2005) and Bulbus Fritillaria (Wang et al., 2005).

Amplified Fragment Length Polymorphism (AFLP)

The AFLP technique is a powerful DNA fingerprinting technology applicable to

any organism without the need for prior sequence knowledge. In this technique

restriction fragment analysis along with selective PCR amplification is carried out

for getting DNA fingerprinting pattern. In the first step, the whole genomic DNA is

digested with two or more restriction endonuclease enzyme with sticky ends. The

fragments are ligated to specific adaptor sequence which is then, used for

amplification of DNA fragments which are obtained through restriction digestion.

PCR amplified products are separated on a polyacrylamide gel. AFLP fragments

correspond to unique position on the genome and hence can be exploited as

landmarks in genetic and physical mapping. This technique is used to distinguish

closely related individuals at the sub-species level and can also map genes.

Identification of large number of medicinal plants was carried out by AFLP.

Some examples are - Panax japonicus (Choi et al., 2008), Panax ginseng from Panax

quinquefolius (Ha et al., 2002), used to distinguish between different Plectranthus

species, (Passinho-Soares et al., 2006). An AFLP map has also been developed for

Actaea racemosa (Zerega et al., 2002), Cannabis (Datwyler et al., 2006), and Rehmannia

(Qi et al., 2008). Species-specific markers were developed to identify different

Swertia sp, S. chirayita, S. angustifolia, S. bimaculata, S.ciliata, S.cordata and S.alata

(Misra et al., 2010)



Inter Simple Sequence Repeats (ISSR)

ISSR involves amplification of DNA segments present at an amplifiable distance in

between two identical microsatellite repeat regions oriented in opposite direction.

The technique uses microsatellite as primers in a single primer PCR reaction,

targeting multiple genomic loci to amplify mainly inter simple sequence repeats

(ISSR) of different sizes. ISSR analysis represents a compromise between AFLP and

RAPD in that the method is inexpensive and easy to perform. ISSR primers are not

totally arbitrary, but are instead composed of repeated di-, tri-, tetra- or penta-

nucleotide motifs. Such simple sequence repeats are known to be scattered

throughout the genome of eukaryotes. ISSR exhibits specificity of microsatellite

markers but needs no sequence information for primers synthesis enjoying the

advantage of random markers. This technique is quick and simple and the use of

radioactivity is not essential. ISSR markers are more in demand being abundant,

reproducible, highly polymorphic, informative and quick to use. It has been used to

identify several medicinal plants like, Cistanche (Shi et al., 2009), Fritillaria (Li et al.,

2009), Salvia (Song et al., 2010), Vitex (Hu et al., 2007), Cannabis (Kojoma et al., 2002).

It has been used for the authentication of Dendrobium officinale (Shen at al., 2006).

Microsatellite-based marker

Hyper-variable repetitive DNA sequences such as microsatellites, minisatellites or

midisatellites are helpful in assessing high level polymorphism. Microsatellite or

short tandem repeats or simple sequences repeats are monotonous repetitions of

very short (2-6) nucleotide motifs, which occur as interspersed elements in the

eukaryotic genomes. These are also known as simple sequence repeats (SSRs), short

tandem repeats (STRs) or simple sequence length polymorphism (SSLPs), which

are the smallest class of simple repetitive DNA sequences. It is widely used for

DNA fingerprinting, paternity testing, linkage map construction and popular

genetic studies but are of less importance for species identification. Variation in the

number of tandemly repeated units is mainly due to strand slippage during DNA

replication where the repeats allow matching via excision or addition of repeats.



Microsatellite assays show extensive inter-individual length polymorphism during

PCR analysis of unique loci using discriminatory primer sets. The reproducibility

of microsatellites is such that, they can be used efficiently by different research

laboratories to produce consistent data. This technique has been used to evaluate

Panax ginseng (Kim et al., 2007), Acanthopanax senticosus (Kim et al., 2007),

Dendrobium fimbriatum (Fan et al., 2009), Cymbopogon species (Kumar et al., 2007),

Bupleurum (Chun et al., 2009) and Schisandra (Boqian et al., 2009).

Cleaved Amplified Polymorphic Sequence (CAPS)

The CAPS technique provides a way to utilize the DNA sequences of mapped

RFLP markers to develop PCR-based markers, thereby eliminating the tedious

DNA blotting. Therefore, CAPS are also known as PCR-RFLP markers. The CAPS

deciphers the restriction fragment length polymorphisms caused by single base

changes like SNPs insertions/deletions, which modify restriction endonuclease

recognition sites in PCR amplicons. The CAPS assay is performed by digesting

locus-specific PCR amplicons with one or more restriction enzymes, followed by

seperation of the digested DNA on agarose or polyacrylamide gels. Primers are

synthesized on the sequence information available in data bank of genomic or

cDNA sequences or cloned RAPD bands. The CAPS analysis is versatile and can be

combined with SSCP, SCAR, AFLP, RAPD analysis to increase the possibility of

finding DNA polymorphisms. It has been used for authentication of Alisma (Li et

al., 2007), Angelica (Watanabe et al., 1998), Ephedra (Guo et al., 2006), Fritillaria

(Wang et al., 2007), Artemisia (Lee et al., 2009), Panax (Diao et al., 2009), Astragalus

(Lu et al., 2009), Dendrobium (Zhang et al., 2005) and Codonopsis (Fu et al., 1999).

Direct Amplification of Length Polymorphisms (DALP)

This method uses an arbitrarily primed PCR to produce genomic fingerprints and

to enable sequencing of DNA polymorphisms in virtually any species. The

uniqueness of DALP relies upon the specific design of primer pairs. It uses a

selective forward primer containing a 5’ core sequence of the universal M13

sequencing primer plus additional bases at the 3’ end, and a common reverse M13



primer. Any of the bands generated by PCR can be excised from the gel and

sequenced directly using forward and reverse primers. After sequencing the

polymorphic bands among the samples, species specific primers can be designed. It

has been used to authenticate Stephania yunnanenis (Ma et al., 2008), Panax ginseng

and Panax quinquefolius (Ha et al., 2001).

Amplification refractory mutation system (ARMS)

ARMS, also known as allelic specific polymerase chain reaction (ASPCR). It is a

simple, time-saving, and effective method for detecting any mutation involving

single base changes. The basis of ARMS is that oligonucleotides with a mismatched

3’ residue will not function as primers in the PCR. ARMS allow amplification of test

DNA only when the target allele is contained within the sample and will not

amplify the non-target allele. Following an ARMS reaction, the presence and

absence of a PCR product is diagnosed for the presence of the target allele. The

main advantage of ARMS is that amplification step and authentication step are

combined in the presence or absence of the target allele. This technique has been

applied in the authentication of Alisma orientale (Li et al., 2007), Panax ginseng (Diao

et al., 2009), Rheum species (Yang et al., 2004), Dendrobium officinale (Ding et al.,

2008).

Sequence Characterized Amplified Region (SCAR)

The SCAR is a PCR based marker that represents single genetically defined locus

identified by amplification of genomic DNA with a pair of specific oligonucleotide

primers. These primers are designed from polymorphic RAPD fragments. The

polymorphic region from RAPD is selected among amplified fingerprints. After

cloning and sequencing for a selected polymorphic region, a pair of internal

primers is designed to amplify a unique and specific sequence. This sequence is

designated as SCAR marker. PCR results in a positive or negative amplification in

target containing and non-target containing samples, respectively. SCAR sequence

primers used 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. A SCAR marker, generated from polymorphic

regions that differ in size between species, permits sample authentication based on

SCAR size shifts. Different DNA-based markers viz., AFLP, SSR, ISSR and RAPD

can also be used to generate these markers. It is fast, reliable, less sensitive to

reaction conditions and easy to conduct in any laboratory. It can be carried out

using unknown genomic DNA from any developmental stage and body part.

SCAR method offers a practical application for screening numerous samples,

accurately at one time, thus adding to the cost efficiency of the experiment. SCAR

does not face the problem of low reproducibility, as generally encountered with

RAPD. The arbitrary marker techniques are sensitive to changes in the reaction

condition.

Significant advantage of SCAR markers versus RAPD markers is the

production of unique (single band) PCR products that are less influenced by

reaction conditions (Paran et al., 1993, Penner et al., 1993). Some well-documented

conditions known to influence PCR performance include MgCl2 concentration,

template DNA (purity, concentration and source), Taq polymerase, and

thermocycler. SCAR markers are robust and highly efficient. A nanogram or less

DNA sample is efficient to develop the marker. The DNA can be extracted from

fresh as well as dried samples. Samples can be taken from any part of the plant

stem, leaves, fruits, roots, capsules etc. SCAR markers have high detection

efficiency as the SCAR primers are able to retrieve a single clear band from the

genuine sample, differentiating it from the adulterants.

Examples of application of SCAR marker in the identification of medicinal

plants is described here. In China, Atractylodes is sold in the name of Atractylodes

macrocephala ‘Packhul’ and Atractylodes japonica ‘Sabju’ in korea. Atractylodes

japonica and Atractylodes macrocephala are frequently adulterated with each other.

However, Chinese packchul is less valuable than Korean A. japonica with respect to

their components and effects. SCAR Marker has been successfully applied in the

differentiation of A. japonica and A. macrocephala. For this purpose, AjR1 and AmR1

(forward and reverse) were used (Huh et al., 2006).



Pueraria tuberosa, commonly sold in the Indian market as ‘vidari’, is used as

aphrodisiac and galactagogue and used in preparation of ‘chywanaprash’. It is

commonly adulterated with Ipomea mauritiana, Adenia hondala and Cycas circinalis. A

320 bp SCAR marker was developed for the identification of P. tuberosa. The

designed SCAR primers were able to identify the authentic Vidari from its

adulterants and substitutes. In another case, Ipomea mauritiana, was distinguished

from Pueraria tuberosa, Adenia hondala and Cycas circinalis by designing IM1F and

IM1R SCAR primers, which produced a specific 323 bp band in Ipomea circinalis

(Devaiah et al., 2010.).

Embelia ribes, which has been constituent of different Ayurvedic drugs in

India, has also been substituted with Embelia tsjeriam-cottam, Myrsine Africana and

Maesa indica . ER1 and ER2 (forward and reverse) SCAR primers were designed to

get 594 bp band in E. ribes only (Devaiah et al., 2008). Similarly, Astragalus radix

commonly called as ‘Huang Qi’, is a well known basic drug in traditional Chinese

medicine. Astragalus membranaceous was discriminated from Astragalus radix and H.

polybotrys by SCAR analysis. Identification was done on the basis of A1, A2 and H1

primers (Liu et al., 2008).

Phyllanthus amarus, P. debilis and P. urinaria are morphologically very

similar. These are grown in overlapping populations in the same habitat and

collectively called ‘Luk-tai-bai’ or Ya-tai-bai. Authentication of these medicinal

species of Phyllanthus, P. amarus, P. debilis, P. urinaria was done on the basis of PA-

B7, PA-B17, PU-J17 and PU-J19 SCAR primers. Another identification method for

discrimination of P. amarus, P. fraternus, P. debilis and P. urinaria was done on the

basis of GRB/EA1150, GRB/EF317, GRB/BD980, and GRB/BU550 primer

(Theerakulpisut et al., 2008). Three species of Echinacea, E. angustifolia, E. pallida and

E. purpurea was discriminated by SCAR marker. A 330 bp SCAR marker was

developed to authenticate E. purpurea from its adulterant (Adinolfi et al., 2007).

SCAR markers have been used for the discrimination of three species of medicinal

plants, Angelica decursivum, Pseucedanum praeruptum and Anthricus sylvestris have

been successfully used (Choo et al., 2009).



Likewise, development of SCAR markers have been done for other

medicinal plants, Panax (Wang et al., 2001), Artemisia (Lee et al, 2006; Zhang et al.,

2006), Zingiber (Chavan et al., 2008), Cynanchum (Moon et al., 2010).

review of literature - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/21241/8/08_chapter...

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