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:
19
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
24
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.