15

CHAPTER 2

REVIEW OF LITERATURE

2.1 Bacopa monnieri (L.) Wettst

Bacopa monnieri (L.) Wettst. [syn. Bacopa monniera (L.) Pennell.; Gratiola

monnieri (L.); Monniera cuneifolia Michaux; Herpestis monniera (L.) Kunth],

commonly known as Water hyssop, Brahmi, Jal Brahmi, Nira-brahmi and Saraswati,

belongs to the family Scrophulariaceae which has 220 genera and 4500 species.

2.1.1 Classification, Distribution and Habitat

The plant belongs to Kingdom – Plantae, Division – Angiospermae, Class –

Dicotyledonae, Subclass – Gamopetalae, Series – Bicarpellatae, Order – Personale,

Family – Scrophulariaceae, Genus – Bacopa and Species – monnieri.

Genus Bacopa comprises of 146 species of aquatic herbs distributed throughout the

warmer regions of the world. Apart from India, Nepal, Sri Lanka, China, Taiwan and

Vietnam, it is also found in Florida and other southern states of USA. In United

States, the herbs are recognized as weeds in rice fields and found growing abundantly

in marshes and wetlands of warmer regions (Barrett and Strother, 1978). In India, it

grows in damp, marshy places and on the banks of slow flowing rivers and lakes,

ascending up to an attitude of 1,320 m (Russo and Borrelli, 2005).

2.1.2 Botanical Features B. monnieri is a small creeping, spreading, succulent herb with numerous branches

and small fleshy, oblong leaves. Flowers and fruits appear in summer and the whole

plant is medicinally important (Chopra et al., 1956). The salient botanical features

are: Stem - prostrate, (sub) succulent, herbaceous; Leaves - decussate, simple, oblong,

1 × 0.4 cm, succulent, punctate, penninerved, margin entire, apex obtuse, sessile;

Flower(s) - axillary, solitary, bracteate, linear, purple, pink or white in colour; Calyx

- 5 lobes (unequal); outer 2 lobes larger, oval, 7 × 3.5 mm; inner 2 lobes linear, 5.5 ×

0.7 mm; median 1 lobe oblong, 5.5 × 2 mm, imbricate, (sub) succulent, punctuate,

obtuse, acute; Corolla - white with violet and green bands inside the throat, 0.8 cm

across, 5 mm tube; 5 lobes, obscurely 2-lipped, obtuse or emarginated; Stamens - 4,

16

didynamous; filament pairs 1 and 2.5 mm anthers oblong, contiguous, 1.5 mm; Ovary

- oblong-globose, 2 mm; style slightly deflexed, 5.5 mm; Stigma - flat capsule,

oblong-globose, 5×2.5 cm septicidal or locilicidal or 4 valved; Seed - oblong, testa

striate; Fruit - small, capsule form, less than 0.5 inch in length (Rao et al., 2011).

2.1.3 Propagation and Cultivars

Warm (30-40 °C) and humid (65-80 %) climatic conditions with plenty of sunshine

and ample rainfall are ideal for growth of Bacopa monnieri and therefore it is

propagated by stem cuttings as a summer-rainy season (March-June) crop in north

India (CIMAP, 2007). So far, three cultivars Pragyashakti, Subodhak and Cim-Jagriti

have been released by Central Institute of Medicinal and Aromatic Plants (CIMAP),

Lucknow which can be grown as perennials with at least two harvests per year.

Pragyashakti is a selection from Orissa with dry herb yield of 65

quintals/hectare/harvest and 1.8 % bacoside A, whereas Subodhak is a selection from

wild collections having a dry herb yield of 47 quintals/hectare/harvest and 1.6 %

bacoside A (Mathur et al., 1999). Cim-Jagriti has a potential of producing dry herb

yield of 40 quintal/hectare with 2 % bacoside A under Lucknow conditions (Darokar

et al., 2007). Indian Institute of Integrative Medicine (IIIM), Jammu has also

developed an improved accession RRL-01BM containing 1.8-2.2 % Bacoside A

(Gupta, 2000).

2.1.4 Phytochemistry

In view of the therapeutic importance of B. monnieri in indigenous systems of

medicine, systematic chemical examinations of the plant have been carried out by

several groups of researchers. Detailed investigations were first documented as early

as 1931, when Bose and Bose reported the isolation of the ‘brahmine’ (alkaloid) from

B. monnieri followed by identification of other alkaloids like nicotine and herpestine

(Chopra et al., 1956), D-mannitol, saponin, hersaponin and potassium salts by Sastri

et al. (1959).

The nootropic activity of the extract has been attributed to the presence of two major

saponins bacoside A and B (Singh et al., 1988; Singh and Dhawan, 1997). However,

major chemical entity shown to be responsible for the memory facilitating action of B.

17

monnieri is Bacoside A, assigned as 3-(α-L-arabinopyranosyl)-O-ß-D-

glucopyranoside-10,20-dihydroxy-16-keto-dammar-24-ene (Chatterji et al., 1965).

During the isolation of Bacoside A, an artefact Bacoside B usually co-occurs with

bacoside A. Bacoside A was found to be levo-rotatory and bacoside B dextro-rotatory

due to differences in their carbohydrate chain configuration (Figure 1).

The major chemical constituents isolated and characterized using various major

spectral, 2D NMR and chemical studies by various research groups from the alcoholic

extract of the herb are dammarane type of triterpenoid saponins with jujubogenin and

pseudojujubogenin as aglycones (Table 2 ).

The chemical composition of bacosides contained in the polar fraction has also been

established on the basis of chemical and physical degradation studies. On acid

hydrolysis, bacosides yield a mixture of aglycones, bacogenin A1, A2, A3, A4

(Kulshreshtha and Rastogi, 1973, 1974; Chandel et al., 1977; Rastogi et al., 1994)

among which the major component was ebelin lactone pseudojujubogenin (bacogenin

A4). In view of the increasing interest for this herbal drug, Chakravarty et al. (2002)

isolated three phenylethnoid glycosides, viz. monnierasides I-III along with the

known analogue plantainoside B from the glycosidic fraction of B. monnieri. The

composition of bacoside A has been established as a mixture of four triglycosidic

saponins i.e; Bacoside A3, Bacopaside II, 3-O-[α-L-arabinofuranosyl-(1→2)-{ß-

Dglucopyranosyl-(1→3)-}-α-L-arabinopyranosyl] jujubogenin and Bacopasaponin C

(Deepak et al., 2005). Bacoside B has also been reported as a mixture of four

diglycosidic saponins i.e; Bacopaside N1, Bacopaside N2, Bacopaside-IV and

Bacopaside-V (Sivaramakrishna et al., 2005) and its identity needs further

establishment (Mundkinajeddu and Agarwal, 2013). Pawar and Bhutani (2006)

isolated two dammarane glycosides from aqueous extracts of the plant. Chemical

structure of these compounds have been established as 20-O- α -L-arabinopyranosyl

jujubogenin and 3-O-α-L-arabinopyranosyl jujubogenin on the basis of LC-MS, IR,

1D- and 2D-NMR studies.

A new sterol glycoside, bacosterol-3-O-ß-D-glucopyranoside along with

bacopasaponin-C, bacopaside-I, bacopaside-II, bacosterol, bacosine, luteolin-7-O-ß -

glucopyranoside and four cucurbitacins, bacobitacin A (I)-D, a known cytotoxic,

cucurbitacin E, together with three known phenylethanoid glycosides, monnieraside I,

18

III and plantioside B were also isolated from B. monnieri (Bhandari et al., 2006,

2007). Zhou et al. (2007) isolated three new triterpene glycosides, bacopasides VI-

VIII (1-3), together with 3 known analogs, bacopaside I (4), bacopaside II (5), and

bacopasaponin C (6) from the whole plant. Suresh et al. (2010) also extracted using

ethyl acetate a chalcone type compound 2,4,6-trihydroxy-5-(3,3-di-Me propenyl)-3-

(4-hydroxyphenyl) propiophenone from B. monnieri.

Other major compounds reported in this plant include; phenylethanoid glycosides,

flavonoids, amino acids such as alpha-alanine, aspartic acid, glutamic acid, and

betulinic acid, stigmasterol, b-sitosterol and stigmastenol (Chatterji et al., 1963; Jain

and Kulshreshtha, 1993; Russo and Borrelli, 2005).

2.1.5 Therapeutic Applications Ayurvedic medicine classifies Bacopa as belonging to a group of plant medicines

known as medhya rasayana - that improve mental health, intellect and memory

(medhya) and promote longevity and rejuvenation (rasayana) (Singh and Singh,

1980). The Sanskrit name Brahmi stems from Brahma - the creative aspect of God

and since the brain is seen as the creative centre of humans, it is so named (Russo and

Borrelli, 2005). B. monnieri has been used for centuries as a brain tonic, memory

enhancer, revitaliser of sensory organs, anti-anxiety, cardio-tonic, diuretic,

antidepressant and anticonvulsant agent (Chopra et al., 1969; Monograph, 2004). In

India and Pakistan, the plant is also used for all sorts of skin problems - eczema,

psoriasis, abscess, ulcerations, leprosy, for chronic rheumatism as an ointment,

asthma and hoarseness of the voice (Shakoor et al., 1994).

It has a very important role in Ayurvedic therapies for the treatment of cognitive

disorders of aging (Russo and Borrelli, 2005; Ernst 2006) and also possesses anti-

inflammatory, analgesic, antipyretic, epilepsy, anticancer and antioxidant activities

(Tripathi et al., 1996; Sinha and Saxena, 2006).

2.1.6 Pharmacological studies The mechanism of action of B. monnieri is evident from several studies, as detailed

below:

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Cognition and Neuropharmacological properties The cognition facilitating activity of B. monnieri extract is due to the presence of

different tetracyclic triterpenoid saponins which have been used to promote memory

and intellect to treat psychoneuro disorders and as a rejuvenator from Ayurvedic era

(Aithal and Sirsi, 1961; Prakash and Sirsi, 1962; Gold et al., 1998). The plant extract

has been extensively investigated in several labs for their neuropharmacological

effects and a number of reports are available confirming their nootropic action.

According to scientists at the Central Drug Research Institute, Lucknow, India;

certain "memory chemicals" in Bacopa, called bacosides A and B, help repair

damaged neurons by enhancing protein kinase activity in the hippocampus which aids

in repair of damaged neurons by enhancing kinase activity, neuronal synthesis and

restoration of synaptic activity and ultimately nerve impulse transmission (Rastogi et

al., 1994). Treatment with the ethanolic extract of B. monnieri plant enhanced

learning ability in rats due to two active saponins, bacosides A and B (Singh and

Dhawan, 1997). It has been suggested that bacosides induce membrane

dephosphorylation, with a concomitant increase in protein and RNA turnover in

specific brain areas (Singh et al., 1990) and they have also been shown to enhance

protein kinase activity in the hippocampus which could contribute to its nootropic

action (Singh and Dhawan, 1997).

Effect of B. monnieri extract on cognition performance during ageing has been

studied (Roodenrys et al., 2002; Bhaskar and Jagtap, 2011). Studies on animal models

indicate the effectiveness of Bacopa extracts for increasing memory capacity and

neuroprotectant activity against Alzheimer’s disease (Uabundit et al., 2010; Sudharani

et al., 2011).

Antioxidant properties

Antioxidants have been reported to prevent oxidative damage by free radicals that are

responsible for number of human disorders such as artherosclerosis, hypertension,

arthritis, gastritis, ischemia, Alzheimer’s disease, diabetes mellitus and AIDS

(Kikusaki and Nakatani, 1993; Deepak et al., 2003). Bacosides are reported to

scavenge free radicals such as peroxides, superoxides and hydroxyl radicals (Cook

and Samman, 1996; Bafna and Balaraman, 2005; Singh et al., 2006; Shah et al.,

2012).

20

Antioxidant activity of alcoholic and hexane extract of B. monnieri on lipid

peroxidation by ferrous sulphate and cumene hydroperoxide in rat liver homogenate is

documented (Tripathi et al., 1996). Based on animal studies, bacosides were shown to

have antioxidant activity in the hippocampus, frontal cortex and striatum

(Bhattacharya et al., 2000) and found to modulate the expression of certain enzymes

involved in generation and scavenging of reactive oxygen species in the brain

(Govindarajan et al., 2005). Pawar et al. (2001) demonstrated that Bacoside A3 in the

hydroalcoholic extract of the whole plant exhibited an inhibitory effect on

superoxides released from polymorphonuclear cells in a nitroblue tetrazolium assay.

Sumathy et al. (2001) investigated the hepatoprotective activity of its alcoholic

extract, administered orally, on the liver antioxidant status of morphine-treated rats.

The same research group (Sumathy et al., 2002), reported the protective effect of the

plant extract on morphine-decreased brain mitochondrial enzyme activity in rats.

Russo et al. (2003) showed the protective role of methanolic extract against the

toxicity induced by the NO donor (S-nitroso-N-acetyl-penicillamine, SNAP) in

culture of rat astrocytes, consequently preventing DNA damage. The neuroprotective

effect of the herb against aluminium induced oxidative stress in the hippocampus of

rat brain has also been proved (Janani et al., 2008). Sharan et al. (2011) reported the

free radical-scavenging activity of the methanolic extract of the plant provided

protection against DNA damage in human non-immortalized fibroblasts.

Anti - depressant and Sedative properties Mental depression, a chronic illness affecting person’s mood, physical health and

behavior are common problems in today’s stressful and competitive world (Alan et

al., 2011). Patients with depression have symptoms that reflect decrease in brain

monoamine neurotransmitters, specifically norepinephrine, serotonin and dopamine.

Methanolic extract of B. monnieri (20-40 mg kg-1) given once daily for 5 days showed

significant antidepressant activity (Singh and Dhawan, 1997). Performance of rats in

motor learning was improved by administration of alcoholic extract of the herb and

chlorpromazine (Prakash and Sirsi, 1962). It has been reported that Bacosides A and

B, Bacopasaponins I and II and Bacopasaponin C exhibit antidepressant activity

(Zhou et al., 2007). Methanolic extract given in the dose of 20 and 40 mg kg-1 to

rodent models of depression, orally once daily for 5 days was compared with standard

antidepressant drug imipramine (15 mg kg-1 ip) which proved its beneficial effect in

21

learning (Sairam et al., 2002). Hersaponin, a glycoside isolated from B. monnieri has

been analyzed for its sedative effect (Malhotra and Das, 1959).

Anti-epileptic properties

Extracts of B. monnieri provide relief to patients suffering with anxiety or epileptic

disorders (Das et al., 2002, Achaliya et al., 2005 and Saba et al., 2012). Clinical

studies reported the effectiveness of alcoholic extract of Bacopa in decreasing

symptoms of epileptic seizures (Dhanasekaran et al., 2007). Paulose et al. (2008)

reported the neuroprotective activity of extract of the plant in glutamate mediated

excitotoxicity during seizures and cognitive damage in Pilocarpine induced epilepsy.

Brahmigrith, a medicated ghee prepared from B. monnieri is beneficial for treatment

of epilepsy and hysteria (Limpeanchob et al., 2008).

Table 2: Saponins characterized using spectroscopic, 2D NMR and chemical transformation methods reported from Bacopa monnieri

Name Derivative Reference

Jujubogenin derivatives Bacoside A1 3-O-[α-L-arabinofuranosyl(1→3)]-α-L-arabinopyranoside Jain and Kulshreshtha, 1993

Bacoside A3 3-O-α-L-arabinofuranosyl-(1→2)-[β-D-glucopyranosyl-(1→3)]-β-D-glucopyranoside Rastogi et al., 1994

Bacopasaponin A 3,20-di-O-α-L-arabinopyranoside Garai et al., 1996a

Bacopasaponin E 3-O-α-L-arabinofuranosyl-(1→2)-[β-D-glucopyranosyl-(1→3)]-α-L-arabinopyranoside, 20-O-α-L-arabinopyranoside

Mahato et al., 2000

Bacopasaponin F 3-O-α-L-arabinofuranosyl-(1→2)-[β-D-glucopyranosyl-(1→3)]-β-D-glucopyranoside,20-O-α-L-arabinopyranoside

Bacopasaponin G 3-O-[α-L-arabinofuranosyl-(1→2)]-α-L-arabinopyranoside Hou et al., 2002

Bacopaside III 3-O-α-L-arabinofuranosyl-(1→2)-ß-D-glucopyranosyl Chakravarty et al., 2003 Bacopaside IV 3-O-ß-D-glucopyranosyl-(1→3)-α-L-arabinopyranosyl

Bacopaside IX 3-O-{ß-D-glucopyranosyl(1→4)[α-L-arabinofuranosyl -(1→2)]-ß-D-glucopyranosyl}-20-O-α-L-arabinopyranosyl

Zhou et al., 2009

Pseudojujubogenin derivatives Bacopasaponin B 3-O-[α-L-arabinofuranosyl-(1→2)]-α-L-arabinopyranoside Garai et al., 1996a,b

Bacopasaponin C 3-O-α-L-arabinofuranosyl-(1→2)-[β-D-glucopyranosyl-(1→3)]-α-L-arabinopyranoside

Bacopasaponin D 3-O-[α-L-arabinofuranosyl-(1→2)]-β-D-glucopyranoside

Bacoside A2

3-O-α-L-arabinopyranosyl-(1→5)-[α-L-arabinofuranosyl-(1→6)]-α-D-glucofuranoside Rastogi and Kulshreshtha, 1999

Bacopaside III 3-O-[6-O-sulfonyl-β-D-glucopyranosyl-(1→3)]-α-L-arabinopyranoside Hou et al., 2002

Bacopaside I , Bacopaside V

3-O-α-L-arabinofuranosyl-(1→2)-[6-O-sulfonyl-β-D-glucopyranosyl-(1→3)]-α-L-arabinopyranoside, 3-O-ß-D-glucopyranosyl-(1→3)-α-L-arabinofuranosyl

Chakravarty et al., 2001, 2003

Bacopaside II 3-O-α-L-arabinofuranosyl-(1→2)-[β-D-glucopyranosyl-(1→3)]-β-D-glucopyranoside

Bacopasaponin H 3-O-[α-L-arabinopyranosyl] Mandal and Mukhopadhyay, 2004

Bacopaside XI 3-O-[ß-D-arabinofuranosyl (1→3)]-6-O-sulfonyl-ß-D-glucopyranosyl Bhandari et al., 2009

Bacopaside XII 3-O-{ß-D-glucopyranosyl(1→3)[ß-D-arabinofuranosyl(1→2)]-ß-D-glucopyranosyl}-20-O-ß-D-arabinopyranosyl

22

23

Figure 1: Chemical structure of bacosides isolated from B. monnieri.

Bacoside A2 Bacoside A3

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Anti-ulcerative properties

Stress, improper dietary habits, ingestion of non-steroidal anti-inflammatory agents

and infection by Helicobacter pylori may be responsible for the development of ulcer

(Jain et al., 1994). Goel et al. (2003) reported anti-Helicobacter pylori activity of

standardized B. monnieri extract in vitro at a dose of 1000 µg ml-1. Plant extract

possesses ulcer healing activities due to its effects on various mucosal offensive and

defensive factors (Dharmani and Palit, 2006; Subhan et al., 2010). Fresh juice of the

plant has been reported to have significant antiulcerogenic activity (Rao et al., 2000;

Sairam et al. 2001; Dorababu et al., 2004).

Anti-cancerous properties

The anti-cancer nature of the ethanolic extract of B. monnieri was reported in Walker

carcinosarcoma 256 in rat (Bhakuni et al., 1969) and sarcoma-180 cell culture

(Elangovan et al., 1995). In addition, bacoside A fraction and its individual

components were found to be more active than the bacoside B fraction

(Sivaramakrishna et al., 2005). Bacosides A and B, bacopasides I, II, X and

bacopasaponin C showed improved activity in a brine shrimp lethality assay (an assay

that is predictive of potential anticancer) activity (McLaughlin et al., 1998; D’Souza

et al., 2002).

Anti-diabetic properties

Animal studies demonstrated that ethanolic extract of B. monnieri has a positive effect

on haemoglobin glycosylation in vivo, anti-oxidant potential and in vitro peripheral

glucose utilization (Ghosh et al., 2011). Bacosine, a triterpenoid isolated from the

plant extract has been found to increase glycogen content in the liver of diabetic rats

and peripheral glucose utilization in the diaphragm (Ghosh et al., 2011).

Attention-deficit disorder

Alcoholic extract of B. monnieri has also studied for its attention-deficit hyperactivity

disorder (ADHD) in children with significant improvement in the areas of sentence

repetition, logical memory, and pair associative learning in ADHD children (Mishra,

1998).

25

Anxiety and Blood pressure

Singh and Singh (1980) observed 20% lower anxiety levels in patients suffering from

anxiety neurosis. Ethanolic extract of B. monnieri has shown broncho-vasodilatory

activity with concurrent involvement of Ca++ channels, β adrenoreceptors and

prostaglandins (Dar and Channa, 1997, 1999). Animal trials reveal relaxant effect of

the plant extract on chemically induced broncho-constriction and effect via inhibition

of Ca++ influx into cell membrane and has a similarity in activity with quinidine

(cardiac depressive drug) (Rashid et al., 1990).

Anti-inflammatory properties

B. monnieri extract showed anti-inflammatory activity via inhibition of prostaglandin

synthesis and lysosomal stabilization (Aloe et al., 2002; Sharma, 2006). Effect of

plant extract compared with that of indomethacin (anti-inflammatory drug) showed its

effective suppression in experimentally induced inflammatory reactions, by inhibiting

prostaglandin synthesis and partly by stabilizing lysosomal membranes, and did not

cause gastric irritation at anti-inflammatory doses (Jain et al., 1994; Holocomb et al.,

2006).

Anti-microbial properties

Propanoic, methanolic and ethanolic extracts of B. monnieri exhibited anti-microbial

activity against many bacterial etiological agents e.g. Streptococcus, Escherichia

coli, Staphylococus aureus, Bacillus subtilis, Klebsiella pneumoniae, Pseudomonas

aerogenosa (Sairam et al., 2001; Rakesh et al., 2009; Hema et al., 2013). The

phytochemicals such as betulinic acid, wogonin and oroxindin isolated from the

aerial parts of B. monnieri showed significant antifungal activity against Alternaria

alternata and Fusarium fusiformis (Chaudhuri et al., 2004).

2.1.7 Substituents and Adulterants

Bacopa monnieri is often substituted and confused with Centella asiatica since both

the plants are considered as ‘Medhya rasayanas’ (brain tonic) in Ayurveda and

possess the same vernacular name Brahmi (Singh et al., 2008). However, these plants

differ in their therapeutic properties and chemical constituents. In ancient Sanskrit

writings, B. monnieri was known as Brahmi, Jala-brahmi or water-brahmi whereas the

26

name Mandukaparni was assigned to C. asiatica (AYUSH, 2004). Brahmi is used to

treat specific mental disorders such as insanity and epilepsy (Gohil and Patel, 2010),

while Mandukaparni is a general rejuvenative tonic which improves mental health

(Raghavendra et al., 2009).

The Charak Samhita considers them as promoters of cognitive functions, but it

suggests that Brahmi is superior to Mandukaparni (Nadkarni, 1954). Chemically both

species are rich in saponins, Bacoside A and B from B. monnieri and Madecassoside

and Asiaticoside from C. asiatica (Sukhdev, 2006). The Sushruta Samhita also

defines the properties of the herbs wherein Brahmi belongs to tikta rasa (bitter), while

Mandukaparni belongs to kasaya rasa (astringent) (Bhishagratna, 1991).

Mandukaparni is cooling, making it better for pitta whereas Brahmi is warming,

indicated in kapha/vata. Mandukaparni is also indicated in skin issues and for wound-

healing, whereas Brahmi has additional properties for helping throat and lung

infections (CSIR, 1988, 1992).

2.1.8 Ayurvedic preparations

B. monnieri forms the basis for many commercial Ayurvedic preparations like

Brahmighrita (in clarified butter), Sarasvatarishta (a decoction used as brain tonic),

Brahmirasayana (a rejuvenating formulation), Brahmitaila (medicated oil) and Brahmi

Sarbat/Brahmi Panaka (a cooling drink usually used in summer) available in Indian

markets due to its therapeutic values (Pravina et al., 2007; Prasad et al., 2008). Other

commercial formulations containing extracts of the herb include Brahmi Vati (tablet

containing powders of Brahmi, other herbs and minerals), Brahmi Capsules,

Memokriti Capsules, Brain-Act Capsules, BacoMind, Brain-Fit (Herbal Fit for Brain),

Memory Booster, Mind Power, More Memory Capsules, etc.

2.1.9 Field Performance studies

Domestication of different accessions of B. monnieri under field conditions has been

extensively studied during different seasons (Mathur et al., 2000, 2002; Ganjewala et

al., 2001). Highest bacoside content was found during the monsoon season in

comparison to other seasons which in general increased with increase in leaf dry

weight. Favourable effects of monsoon season on vegetative growth, bacoside A and

saponin production were studied (Phrompittayarat et al., 2011; Sharma et al., 2013).

27

Recently, Naik et al. (2012) analyzed by HPLC the bacoside A content in 22

accessions of B. monnieri collected from Karnataka, India and reported the highest

bacoside A concentration (18.36 mg g-1 DW) in accession Bm2 collected from

Belgaum. Evaluation of bacoside A content in various plant parts revealed the highest

content in stolons (9.54 mg g-1 DW) followed by that in leaves, roots, whole plant,

internodes, and nodes (4.73, 4.48, 3.62, 3.35, 3.02 mg g-1 DW, respectively).

2.2 Tissue Culture Studies A number of studies have been conducted utilizing tissue cultures and other

biotechniques for plantlet regeneration and bacoside production in agarified and liquid

culture medium. This review attempts to present a synopsis on such studies in the

herb including micropropagation leading to plantlet generation, genetic

transformation, production of bacosides from in vitro cultures, elicitation and use of

molecular markers.

2.2.1 Micropropagation leading to plant regeneration Micropropagation, regeneration protocols, morphogenetic potential of leaf, internodal

and nodal segments for B. monnieri have been reported by various scientific and

research groups (Table 3). Plantlets have been regenerated from leaf explants

(Mohapatra and Rath, 2005; Papori and Sharma, 2007; Vijayakumar et al., 2010;

Joshi et al., 2010; Showkat et al., 2010; Sharma et al., 2013), nodal explants (Tiwari

et al., 2001, 2006; Sharma et al., 2007; Ramesh et al., 2009; Patil et al., 2009; Prabha

et al., 2010; Vijayakumar et al., 2010; Sharma and Khan, 2011; Chandra et al., 2012;

Mehta et al., 2012; Pandiyan and Selvaraj, 2012; Asha et al., 2013), internodal

explants (Banerjee and Shrivastava, 2008; Subashri and Koilpillai, 2013) and

leaf/internodal explants (Tiwari et al, 2001; Joshee et al., 2007; Ceasar et al., 2010;

Tiwari and Singh, 2010; Sharma and Khan, 2011; Karatas et al., 2013; Bhusari et al.,

2013).

Shoot regeneration of B. monnieri has been reported from leaf, nodal and internodal

segments cultured on growth regulator free medium (Thakur and Ganpathy, 1978;

Mathur and Kumar, 1998), while, multiple shoot bud induction on MS medium in the

presence of BAP/KN without callus formation was studied by Tiwari et al. (1998).

Shrivastava and Rajani (1999) described the processes for adventitious shoot bud

28

induction from leaf explants of shoot cultures grown on MS medium supplemented

with BAP (2μM) and gelled with gelrite (0.2%). Shoot tip and nodal explants induced

multiple shoots on MS medium supplemented with KN/2-ip (0.1 mg l-1 each) and KN

(1.0 mg l-1) respectively (Tejavathi et al., 2001) has also been well documented.

Tiwari et al. (2001) used different cytokinins (BAP, TDZ, KN, 2-ip) for multiple

shoot induction and found that TDZ (6.8 μM) and BAP (8.9 μM) gave better response

as compared to other treatments. Tiwari et al. (2006) established contamination free

nodal cultures of B. monniera using growth regulator free MS medium containing

antibiotic trimethoprim (50 mg l-1) and fungicide bavistin (150 mg l-1). George et al.

(2004) developed an effective protocol for the storage of shoot cultures with 90%

viability under slow-growth conditions for up to 20 months in half-strength MS

medium with sucrose (20 g l-1) in polypropylene-capped culture bottles. Mohapatra

and Rath (2005) compared the micropropagating efficiency of MS and GB media and

reported that MS medium supplemented with BAP (2.0 mg l-1) was better suited for

regeneration of leaf and nodal explants.

Shoot bud proliferation from nodal segments, young leaves, internodes and shoot tip

(Binita et al., 2005) on MS medium supplemented with low concentration of BAP

(1.0 mg l-1) and KN (0.5 mg l-1) (Banerjee and Shrivastava, 2008) and BAP (1.5 mg l-

1), KN (1.5 mg l-1) and adenine sulphate (15 mg l-1) at pH 5.8 (Modi and Banerjee,

2009 ) was achieved. Successful plantlet regeneration on MS medium with KN (0.1µg

ml-1), NAA (0.5 µg ml-1), IBA (l.0 µg ml-1) and BAP (0.5 mg l-1) + 2,4-D (0.5 mg l-1)

using leaf explants has been reported (Joshee et al., 2007; Papori and Sharma, 2007).

Sharma et al. (2007) reported active shoot proliferation (22.2 shoots/explants) and

rooting on MS medium having BAP (0.2 mg l-1) without callus formation after a

culture period of 8 wks. Maximum number of proliferative shoots on MS medium

having BAP (1.0 mg l-1) (Prabha et al., 2010), TDZ (1.5 mg l-1) and NAA (0.5 mg l-1)

(Ceasar et al., 2010) and BAP (1.0 mg l-1) + KN (0.4 mg l-1) + NAA (0.4 mg l-1)

(Vijayakumar et al., 2010) has been documented. MS medium supplemented with

different concentrations of BAP (2 -12 μM) were used to establish in vitro cultures of

B. monnieri (Joshi et al., 2010).

Multiple shoot regeneration and rooting was achieved on MS medium supplemented

with BAP (3.0 mg l-1) and BAP (1.0 mg l-1) + IAA (3.0 mg l-1) respectively (Chandra

et al., 2012). Shoot regeneration and callus induction from nodal segments in MS

29

medium having BAP (1.0 mg l-1) and rooting on medium supplemented with IBA (2.0

mg l-1 ) was also studied (Mehta et al., 2012). Pandiyan and Selvaraj, 2012 observed

maximum shoots on BAP + KN + NAA (0.5 to 2.0 mg l-1) supplemented medium

which were rooted with NAA (0.5 mg l-1) + IBA (1.0 mg l-1) and transferred to field

conditions.

Sharma et al., 2013 observed higher number of adventitious shoots induced from leaf

explants in agar solidified (85±0.5 shoots/explant) and liquid MS medium (200±0.5

shoots/explant) containing KN (10 μM). Subashri and Koilpillai (2013) reported shoot

induction from callus on MS medium containing KN (0.1 mg l-1) and 2,4-D (1.0 mg l-

1). Half strength semi solid MS medium containing KN (3.0 mg l-1) and IBA (0.5 mg

l-1) produced maximum number of shoots (7.25±0.96 shoots/explants) from leaf

explants (Bhusari et al., 2013). Multiple shoots were reported recently, from nodal

explants in MS medium containing BAP (2.0 mg l-1) (Asha et al., 2013) and BAP

(0.25 mg l-1) + NAA (0.25 mg l-1) (Karatas et al., 2013)

2.2.2 Studies on micropropagation/regeneration in Liquid medium

In vitro shoots regenerated from nodal explants of B. monnieri have been successfully

propagated using MS liquid medium (Tiwari et al., 2000). GR free liquid medium

induced 4 to 5 shoots/nodal explants after 4 wks which was 4.8 times higher than that

produced in GR free agarified medium. However, addition of BAP (0.01-0.1 mg l-1)

in the media resulted in 6 to 7 shoots/explants in agarified medium which was higher

to than that cultured in liquid medium. A cost effective and efficient in vitro culture

technique for rapid production of shoot cultures using Growtek vessel has been

patented by Mandal and Sheeja (2006). Jain et al. (2012) attempted shoot biomass

production in different vessels using MS liquid medium containing BAP (2.5 mg l-1)

and IAA (0.01 mg l-1) with 3% sucrose and found Growtek bioreactor as an effective

system for biomass production without the loss of antioxidant properties (Table 4).

Table 3: Studies on in vitro manipulations for micropropagation/plantlet regeneration in Bacopa monnieri

S.

No

Explants used Medium + PGR (Conc)* Regeneration

pathway

Ca

llu

s

Ro

oti

ng

Pla

ntl

et

reg

ener

ati

on

No. of shoots/explants References

1 Node, Internode, Leaf

MS+BAP (1.5 or 2.0 mg l-1) Callus, root, shoot

37/leaf explant (4 wks) Tiwari et al., 1998

2 Stem, leaf MS+ BAP (2 µM)+gelrite (0.2 %) Shoot buds x 100 shoot buds/leaf explant (4 wks)

Shrivastava and Rajani, 1999

3 Shoot tip, Node MS+KN, 2 iP (0.1 -1.0 mg l-1) Multiple shoots x - Tejavathi et al., 2001

4 Node , Internode, Leaf

MS+TDZ (6.8 μM), BAP (8.9 μM)

Shoot buds x 93 shoot buds/leaf explant (7 wks) Tiwari et al., 2001

5 Leaf MS+0.1 mg l-1NAA+1.5 mg l-1

BAP + 0.1 mg l-1 GA3 Agrobacterium tumefaciens strain EHA105, vector pBE2113

Multiple shoots x x - Nisha et al., 2003

6 Shoot tip , Node MS+BAP 0.5 mg l-1 Multiple shoots x Slow growth @ 28 months George et al., 2004

7 Node MS+IAA 0.2 mg l-1 + BAP 1.5 mg l-1+ AdSO4 60 mg l-1

Multiple shoots x 18 (4 wks) Ramesh et al., 2005

8 Node, Internode, leaf

MS+300 mg l-1 TMP or BVN Shoot x 22.5 shoots/internode explants (4 wks)(TMP); 98.6 shoots/internode explants (4 wks )(BVN)

Tiwari et al., 2006

9 Node MS+0.25 mg l-1 BAP 0.001 and 0.01% colchicine

Shoot x 18.37 shoots/explant Escandón et al., 2006

10 Leaf MS+0.1 µg ml-1 kn+ 0.5 µg ml-1 NAA+ l.0 µg ml-1 IBA

shoot x - Papori and Sharma, 2006

11 leaf , Internode

MS+0.5 mg l-1 BAP + 0.5 mg l-1

2,4-D Multiple shoots x - Joshee et al., 2007

12 Node MS+0.2 mg l-1 BAP Multiple shoots x 22.2 (8 wks) Sharma et al ., 2007

13 Node MS+5 mg l-1 BAP+ 0.2% NaCl MS+5mg l-1BAP+ 8% mannitol

Callus, shoot

x

20 (3 wks) Debnath, 2008

30

14 Leaf

MS+15 mg l-1kanamycin MS+10 mg l-1hygromycin MS+60 mg l-1adriamycin MS+ 20 mg l-1chloramphenicol

Shoot x - Wu and Wang, 2008

15 Internode MS+BAP 1.0 mg l-1 + KN 0.5 mg l-1

Shoot, Root x 18 (4 wks) Banerjee and Shrivastava, 2008

16 Leaf, node MS+1 mg l-1 2,4-D+ 1 mg l-1 KN MS+2 mg l-1 IBA

Callus Root

- Singh et al., 2009

17 Node MS+ 1.5 mg l-1 BAP and 1.0 mg l-

1 NAA 3.0 % Na alginate and 80 mM CaCl2 .2 H2O+3.0 mg l-1 bavistin

Shoot, Root x 45.6 (7 wks) Ramesh et al., 2007

18 Node MS+ BAP 0.5μM + NAA 0.5μM Callus - Patil et al., 2009

19 Internode MS+ BAP 1.5 mg l-1, KN 1.5 mg l-1& AdSO4 15 mg l-1

Shoot x - Modi and Banerjee, 2009

20 Node MS (40ml)+1.0 mg l-1KN + Aulosira extract (60 ml)

Shoot, Root x 56 (4 wks) Banerjee and Modi, 2010

21 Leaf MS+1.0 mg l-1IAA+ 1.0mg/l IBA; MS+ 0.5 mg l-1 2,4‐D

Shoot, Callus

23-25 (2 wks) Showkat et al., 2010

22 Node MS+ BAP 0.2 mg l-1 Shoot x - Sharma et al., 2010

23 Leaf MS+ 6µM BAP 1/2MS+2µM IBA + 1 % sucrose(liquid culture)

Shoot, Root

x 30 shoots (4 wks)

Joshi et al., 2010

24 Leaf. Internode MS+1.5 mg l-1 TDZ + 0.5 mg l-1

NAA MS+0.5 mg l-1 BAP ½ MS+1.0 mg l-1 IBA + 0.5 mg l-

1phloroglucinol

Shoot Shoot Root

x x x

x x

x x

135 shoots

Ceasar et al., 2010

25 Node MS+1.0 mg l-1 BAP MS+0.5 mg l-1IBA

Shoot Root

x

65 (4 wks) Prabha et al., 2010

26 Node MS+0.1 mg l-1 NAA, 1.0 mg l-1

BAP + 0.1 mg l-1 GA3 Agrobacterium EHA 105 strain vector pCAMBIA 1301

Callus - Ramesh et al., 2011

27 Shoots MS basal Agrobacterium

rhizogenes strains LBA 9402 and A4

Callus 2 fold increase transformed plant (12 wks)

Majumdar et al., 2011

31

28 Leaf, Internode MS+ 0.1 mg l-1 KN + 0.1 mg l-1

2,4-D MS+ 0.5 mg l-1 KN + 0.5 mg l-1

IAA; 0.1% colchicine (1 & 3 hr)

Callus, Shoot Mean 11.5 shoots/leaf explants (90 days) 63.5 shoots/leaf explants (3 hrs)

Sangeeta and Ganesh, 2011

29 Leaf, Node, Internode

MS + KN (3 mg l-1 ) Shoot x x x 20.9% flowering/node Tejovathi et al.,2011

30 Node MS+ 3 mg l-1 BAP MS+1 mg l-1 BAP + 3 mg l-1 IAA

Shoot Root

x 90 % shoot regeneration (4 wks) Chandra et al., 2012

31 Node, shoot tip MS+ 1.5 mg l-1 BAP MS+ 1 mg l-1 IBA + 0.5 mg l-1

NAA

Shoot Root

x Mean 18.4 shoots/node (4 wks)

Pandiyan and Selvaraj, 2012

32 Node MS+ 1 mg l-1 BAP MS+ 2 mg l-1 IBA MS+0.25 mg l-1 2, 4-D+ 0.5 mg l-1

KN

Shoot Root Callus

Mean 3.42 (3 wks) Mehta et al., 2012

33 Node, Internode, Leaf

MS+TMP+BVN (200mg dm-3) Shoot 135.2 shoot buds/Internode (4 wks)

Tiwari et al., 2012

34 Internode, Leaf MS+BAP+NAA (0.25 mg l-1

each) Shoot Mean 23.11 shoots/internode

(4 wks) Karatas et al., 2013

35 Leaf MS+KN (10µM) MS+NAA (10µM) MS+IBA (10µM)

Shoot 85(8 wks)in semisolid medium; 200 (8 wks) in liquid medium; 95% callus induction; Mean 17 roots

Sharma et al., 2013

36 Internode MS+KN+2,4-D (7µM) MS+KN(0.1 mg l-1 )+2,4-D (1 mg l-1 )

Callus, Shoot Callus (2 wks) 78% shoot response (5 wks)

Subashri et al., 2013

37 Node MS+BAP (2 mg l-1 ) Shoot x Mean 17 shoots/node (5 wks) Asha et al., 2013

38 Shoot tip, Node, Leaf

½ MS+KN(3 mg l-1 )+IBA(0.5 mg l-1)

Shoot x x Mean 7.25 shoots/leaf explants (4 wks) 100% rooting

Bhusari et al., 2013

32

33

2.2.3 Production of bacosides from in vitro cultures

In vitro organ cultures of plants provide an excellent experimental system to study

production, regulation and enhancement of secondary metabolites under complete

controlled conditions. Fragmented studies are available (Table 4 and 5) with respect

to production of bacosides from in vitro cultures of B. monnieri (Rahman et al., 2002;

Ahuja et al., 2005; Praveen et al., 2009; Naik et al., 2010, 2011; Parale et al., 2010;

Mendhulkar et al., 2011).

The potential of cell suspension cultures of B. monnieri for bacoside (A and B)

production was reported by Rahman et al. (2002). Ahuja et al. (2005) reported total

bacoside content to vary between 1.76-2.70% on dry wt. basis from in vitro shoot

cultures, acclimatized plants in Green house and in field beds. Praveen et al. (2009)

reported higher bacoside A content in shoots regenerated in liquid medium (11.92 mg

g-1 DW) which was 2.2-fold higher as compared to shoots grown on semisolid

cultures. Medium supplemented with 2% sucrose and pH 4.5 was found most suitable

for shoot regeneration (151 shoots/explant) from leaf explants and bacoside A

production (13.09 mg g-1 DW) after 8 wks of culture (Naik et al., 2010). The effects

of different strengths of macro elements (NH4NO3, KNO3, CaCl2, MgSO4 and

KH2PO4) and nitrogen source (NH4+/NO3

-) of MS medium on biomass and bacoside

production indicated that an optimum number of adventitious shoots (99.33

shoots/explant), fresh weight (1.841g), dry weight (0.150 g) and bacoside A

production (17.935 mg g-1 DW) were obtained in medium with 2.0X strength of

NH4NO3 (Naik et al., 2011). Maximum number of shoots (70.0 shoots/ explants),

biomass (1.137g FW and 0.080g DW) and bacoside A content 27.106 mg g-1 dry

weight was obtained at NH4+/NO3

- ratio of 14.38/37.60 mM. Influence of organic

supplements glycine, ferulic acid, phenylalanine, α-ketoglutaric acid and pyruvic acid

on production of bacoside A in shoots and callus showed a 4.0 and 3.8 times

respective increase in bacoside A content in MS medium containing 100 µM pyruvic

acid (Parale et al., 2010). Maximum bacoside A content (4.6±0.02 μg mg-1) in

suspension cultures treated with 1% DMSO for 3 and 6 hrs as compared to the control

(2.1±0.05 and 3.2±0.0102 μg mg-1, respectively) has been reported by Mendhulkar et

al. (2011).

Table 4: Studies on use of liquid culture media for in vitro regeneration and bacoside production in Bacopa monnieri

S.

No.

Type of vessel Medium + PGR Culture

type*

No. of shoots/Fold increase/GI

(DW)

Bacoside

content

Reference

1 Flask (250 ml) MS basal S Mean 4.3 shoots/agarified medium 4.4 shoots/liquid medium

- Tiwari et al., 2000

2 Flask (250 ml) MS+NAA +Kn 0.5 mg l-1+ casein hydrolysate1 1 g l-1

C 5 – 6 fold @40 days Bacoside A (1.05%) Bacoside B (0.37%)

Rahman et al., 2002

3 Flask (250 ml) agar and liquid medium MS+BA (1.1 μM) and IAA (0.2 μM).

S+R 28 shoots/internode explants, 110 shoots/leaf explants @ 3 wks

- Binita et al., 2005

4 Growtek (1L) - S 610 viable plantlets @ 8 wks - Mandal and Sheeja, 2006

5 Flask (250 ml) ) agar and liquid medium

MS+KN 2.0 mg l-1 S 64.4 shoots/agarified medium 155.6 shoots/liquid medium @ 8 wks

11.92 mg g-

1DW Parveen et al., 2009

6 Flask (100,250 ml), Majenta Box(400 ml),Glass jar(500 ml),Growtek (1L) with aeration

MS+ BAP 2.5 mg l-1 +IAA 0.01 mg l-1

S 10.0 (GI,DW) 1,979 shoots/L

- Jain et al., 2012

*C-Callus, S-Shoot, R-Roots

34

Table 5: Studies on in vitro manipulations in static and liquid culture media for bacoside production

S.

No.

Explant

used

Medium + PGR Culture

type*

Elicitor/organic

suppliments

No. of shoots/Fold

increase/GI (DW)

Bacoside content References

1 Node MS+ 1 mg l-1 BAP+ 1 mg l-1 IBA

S,P - 18.35 shoots (4 wks) 1.76-2.70% DW Ahuja et al., 2005

2 Stem MS+0.1/0.5 mg l-1 TDZ

P - 117 shoots/explant pseudojujubogenin glycosides 30.62 mg g-1 DW

Kamonwannasit et

al., 2008

3 Shoots MS+BAP 1 mg l-

1+IBA 1 mg l-1

S Salicyclic acid (1 mg l-1l) , MeJ (0.5 µm l-1) Bacterial culture

321.57(GI,DW) bacterial elicitor 17 shoots/flask ( 4 wks)

2.53% DW Potukuchi et al., 2009

4 Leaf MS+KN 2.0 mg l-1 + 2% sucrose, pH 4.5

S - 150.50 shoots/explants (8 wks)

13.09 mg g-1DW Naik et al., 2010

5 Leaf MS+KN 2.0 mg l-1 2.0X NH4NO3

S - 99.33 shoots/explants (8 wks)

17.93 09 mg g-1DW Naik et al., 2011

6 Leaf MS+BAP 5µM MS+2,4D 1µM +NAA5µM

S C

Glycine, Ferulic Acid, Phenylalanine, α-ketoglutaric acid, pyruvic acid (100 µM)

- 35.2 09 mg g-1DW 12. 09 mg g-1 DW

Parale et al., 2010

7 Node MS+BAP 1 mg l-1

S NaCl (100 mM) - 1.32 mg g-1 DW Ahire et al., 2013

*C-Callus, S-Shoot, R-Roots

35

36

2.2.4 Genetic transformations studies

Attempts have also been made to induce Ti and Ri based genetic transformations in B.

monnieri. Nisha et al. (2003), Ramesh et al. (2011) attempted transformation using

Agrobacterium tumefaciens strain EHA105 that harbored the binary vector pBE2113

and pCAMBIA 1301 containing hpt and gus genes respectively. The final

transformation frequency reported by the later in nodal segments using hygromycin

phosphotransferase as selection marker was higher than that reported by the former

with a final transformation frequency of 63.50% using neomycin phosphotransferase

as selection marker. Agrobacterium rhizogenes strains LBA 9402 and A4 have been

used to study the effect of Ri T-DNA on biomass and saponin accumulation in

transformed shoots and roots culture after a culture period of 12 wks which was found

to be significantly higher i.e. shoots (twofold more) and roots (4.3-fold more) than in

the non transformed plants of similar age (Majumdar et al., 2011). Recently,

Aggarwal et al., (2013) reported stable protocols for T-DNA delivery and subsequent

transformed rooted shoots using Agrobacterium tumefaciens.

2.2.5 Elicitation studies

Few reports are available for bacoside production in B. monnieri using biotic and

abiotic elicitors (Debnath, 2008; Kamonwannasit et al., 2008; Prasad et al., 2008;

Potukuchi et al., 2009; Banerjee and Modi, 2010; Sharma et al., 2013; Ahire et al.,

2013; Gupta et al., 2014).

Debnath (2008) investigated the effect of abiotic stress (salinity and drought) on

proline and protein content in in vitro shoot culture for a period of 5, 15 and 30 days

and showed an increase in proline but decease in protein content with an increase in

both mannitol and NaCl concentration as well with culture age. The effect of chitosan

at 150 mg l-1 and yeast extract at 2 mg ml-1 in whole plant cultures showed an

increased pseudojujubogenin glycoside production (40.83± 2.24 mg g-1 DW and

40.05±2.37 mg g-1DW, respectively) after 7 days, which was 6-fold higher than that

in the control cultures (Kamonwannasit et al., 2008). Shoot cultures when co-

cultivated with a novel symbiotic fungus Piriformospora indica showed an

enhancement in plant growth, biomass, bacoside contant, antioxidant activity and

nuclear hypertrophy (Prasad et al., 2008, 2013). Multiple shoot cultures of B.

monnieri cultured on MS medium supplemented with BAP + IBA (1.0 mg l-1 each)

37

and when treated with an abiotic elicitor methyl jasmonate (MeJ) and a biotic

elicitor,recorded higher bacoside content of 2.53% in bacterial elicitor treated cultures

(Potukuchi et al., 2009). Banerjee and Modi (2010) studied the effect of

cyanobacterial extract of Aulosira fertilisima on shoot induction and plantlet

regeneration. Sharma et al. (2013) reported enhanced bacoside A production (4.4 mg

g-1) in in vitro shoot cultures treated for one wk with 50 μM methyl jasmonate (MeJ).

Increase in shoot growth, antioxidant defense and bacoside A content under the

influence of NaCl has been reported (Ahire et al., 2013) and recently, Gupta et al.,

(2014) also observed enhanced bacoside A and bacopaside I content in B. monnieri

shoots when treated with a concentration of 10 µM Cadmium after a culture period of

one wk.

2.2.6 Molecular studies

Darokar et al. (2001) detected low level of genetic diversity in 24 geographically

distinct accessions of Bacopa monnieri by RAPD analysis. However, Manikandan et

al. (2010) reported low to moderate level of genetic variation (21.5%) between

randomly collected micropropagated, synthetic seed derived and hardened plants of B.

monnieri.

Highly efficient shoot regeneration using a two-stage culture procedure wherein MS

medium containing BAP (0.5 mg l-1) produced 135 shoots having a length of 7.8 cm

with more nodes (6) and assessment of genetic integrity of micropropagated B.

monnieri plants using RAPD analysis was attempted (Ceasar et al., 2010). A RAPD-

based SCAR marker system has been developed to identify B. monnieri from its

adulterant candidates namely Centella asiatica, Eclipta alba and Malva rotundifolia

(Yadav et al., 2012). Genetic variation among different accessions of B. monnieri

collected from different locations of Southern India (Karthikeyan et al., 2011) and

Central India (Tripathi et al., 2012) has been evaluated using RAPD and inter simple

sequence repeats (ISSR) marker systems individually or combined. Bansal et al.,

(2014) investigated the morphogenetic potential of 14 accessions collected from

different locations of India and analysed their biochemical and molecular diversity

using RAPD and ISSR markers.