Anticancer activity of Bacopa monnieri through apoptosis induction and mitophagy-dependent NLRP3 inflammasome inhibition in oral squamous cell carcinoma.

BACKGROUND: Bacopa monnieri (BM) is traditionally used in human diseases for its antioxidant, anti-inflammatory and neuroprotective effects. However, its anticancer potential has been poorly understood. AIM: The aim of this study was to explore the detailed anticancer mechanism of BM against oral cancer and to identify the bioactive BM fraction for possible cancer therapeutics. RESULTS: We performed bioactivity-guided fractionation and identified that the aqueous fraction of the ethanolic extract of BM (BM-AF) had a potent anticancer potential in both in vitro and in vivo oral cancer models. BM-AF inhibited cell viability, colony formation, cell migration and induced apoptotic cell death in Cal33 and FaDu cells. BM-AF at low doses promoted mitophagy and BM-AF mediated mitophagy was PARKIN dependent. In addition, BM-AF inhibited arecoline induced reactive oxygen species production in Cal33 cells. Moreover, BM-AF supressed arecoline-induced NLR family pyrin domain containing 3 (NLRP3) inflammasome activation through mitophagy in Cal33 cells. The in vivo antitumor effect of BM-AF was further validated in C57BL/6J mice through a 4-nitroquinolin-1-oxide and arecoline-induced oral cancer model. The tumor incidence was significantly reduced in the BM-AF treated group. Further, data obtained from western blot and immunohistochemistry analysis showed increased expression of apoptotic markers and decreased expression of inflammasome markers in the tongue tissue obtained from BM-AF treated mice in comparison with the non-treated tumor bearing mice. CONCLUSION: In conclusion, BM-AF exhibited potent anticancer activity through apoptosis induction and mitophagy-dependent inhibition of NLRP3 inflammasome activation in both in vitro and in vivo oral cancer models. Moreover, we have investigated apoptosis and mitophagy-inducing compounds from this plant extract having anticancer activity against oral cancer cells.

Bacopa monnieri reduces Tau aggregation and Tau-mediated toxicity in cells.

Alzheimer's disease is a neurodegenerative disease characterized by progressive memory loss and behavioral impairments. In the present study, the ethanolic extract of Bacopa monnieri was studied for its potency to inhibit Tau aggregation and rescuing of the viability of Tau-stressed cells. Bacopa monnieri was observed to inhibit the Tau aggregation in vitro. The cells exposed to Bacopa monnieri were also observed to have a low level of ROS and caspase-3 activity. The immunoblot and immunofluorescence analysis showed that Bacopa monnieri acts as an antioxidant and restored the Nrf2 levels in Neuro2a cells. Bacopa monnieri treatment to Neuro2a cells was observed to reduce the phospho-Tau load in formaldehyde-stressed cells. Furthermore, the treatment of Bacopa monnieri reduced the phosphorylation of GSK-3ß in formaldehyde-stressed cells. Ran and NUP358 are the key proteins involved in nuclear transport. It was observed that formaldehyde treatment impaired the nuclear transport by missorting the NUP358 arrangement in Neuro2a cells. On the contrary, Bacopa monnieri treatment restored the NUP358 arrangement in cells. The overall results of the present study suggested that Bacopa monnieri could be considered a potent herb against Tau phosphorylation and Tau aggregation, which projects it as a promising formulation for Alzheimer's disease.

Computational Analysis of Bacopa monnieri (L.) Wettst. Compounds for Drug Development against Neurodegenerative Disorders.

AIM: With several experimental studies establishing the role of Bacopa monnieri as an effective neurological medication, less focus has been employed to explore how effectively Bacopa monnieri brings about this property. The current work focuses on understanding the molecular interaction of the phytochemicals of the plant against different neurotrophic factors to explore their role and potential as potent anti-neurodegenerative drugs. BACKGROUND: Neurotrophins play a crucial role in the development and regulation of neurons. Alterations in the functioning of these Neurotrophins lead to several Neurodegenerative Disorders. Albeit engineered medications are accessible for the treatment of Neurodegenerative Disorders, due to their numerous side effects, it becomes imperative to formulate and synthesize novel drug candidates. OBJECTIVE: This study aims to investigate the potential of Bacopa monnieri phytochemicals as potent antineurodegenerative drugs by inspecting the interactions between Neurotrophins and target proteins. METHODS: The current study employs molecular docking and molecular dynamic simulation studies to examine the molecular interactions of phytochemicals with respective Neurotrophins. Further inspection of the screened phytochemicals was performed to analyze the ADME-Tox properties in order to classify the screened phytochemicals as potent drug candidates. RESULTS: The phytochemicals of Bacopa monnieri were subjected to in-silico docking with the respective Neurotrophins. Vitamin E, Benzene propanoic acid, 3,5-bis (1,1- dimethylethyl)- 4hydroxy-, methyl ester (BPA), Stigmasterol, and Nonacosane showed an excellent binding affinity with their respective Neurotrophins (BDNF, NT3, NT4, NGF). Moreover, the molecular dynamic simulation studies revealed that BPA and Stigmasterol show a very stable interaction with NT3 and NT4, respectively, suggesting their potential role as a drug candidate. Nonacosane exhibited a fluctuating binding behavior with NGF which can be accounted for by its long linear structure. ADME-Tox studies further confirmed the potency of these phytochemicals as BPA violated no factors and Vitamin E, Stigmasterol and Nonacosane violated 1 factor for Lipinski's rule. Moreover, their high human intestinal absorption and bioavailability score along with their classification as non-mutagen in the Ames test makes these compounds more reliable as potent antineurodegenerative drugs. CONCLUSION: Our study provides an in-silico approach toward understanding the anti-neurodegenerative property of Bacopa monnieri phytochemicals and establishes the role of four major phytochemicals which can be utilized as a replacement for synthetic drugs against several neurodegenerative disorders.

TiO2 nanoparticle synthesis, characterization and application to shoot regeneration of water hyssop (Bacopa monnieri L. Pennel) in vitro.

Water hyssop (Bacopa monnieri L. Pennel) is a medicinal aquatic herb used to treat diseases in South Asia. Various regeneration protocols have been developed or modified in vitro to ensure the availability of biomass and secondary metabolites of Bacopa. We applied hydrothermally treated titanium dioxide (TiO2) nanoparticles (NPs) (TiO2-NPs) at different concentrations. Three explants, distal portion of half leaf (DPHL), proximal portion of half leaf (PPHL) and full leaf (FL), were used to evaluate response to TiO2. Regeneration from the three explants in vitro was similar except for shoot length. Application of TiO2-NPs exerted significant, but variable, effects on all parameters except percentage of shoot formation, which was 100%. Interactive effects of explant and TiO2-NPs exhibited significant, but variable, effects on fresh weight and percentage of callus formation. All explants produced more shoots using TiO2-NPs compared to control treatments. DPHL explants with application of 8 mg/l TiO2 produced more shoots than controls. Similarly, FL explant treated with 2 mg/l TiO2-NPs produced more shoots/explant than controls. All concentrations of TiO2-NPs produced significantly longer shoots compared to controls. Increased elongation of shoots justifies use of TiO2-NPs for propagation of plants in vitro during acclimatization. Use of TiO2-NPs for rapid elongation of shoots ultimately fosters survival of plants.

A comprehensive review on tissue culture studies and secondary metabolite production in Bacopa monnieri L. Pennell: a nootropic plant.

Bacopa monnieri L. Pennell, commonly known as Brahmi, is an important medicinal plant that belongs to the family Plantaginaceae. Brahmi is rich in innumerable bioactive secondary metabolites, especially bacosides that can be employed to reduce many health issues. This plant is used as a neuro-tonic and treatment for mental health, depression, and cognitive performance. Brahmi is also known for its antioxidant, anti-inflammatory, and anti-hepatotoxic activities. There is a huge demand for its raw materials, particularly for the extraction of bioactive molecules. The conventional mode of propagation could not meet the required commercial demand. To overcome this, biotechnological approaches, such as plant tissue culture techniques have been established for the production of important secondary metabolites through various culture techniques, such as callus and cell suspension cultures and organ cultures, to allow for rapid propagation and conservation of medicinally important plants with increased production of bioactive compounds. It has been found that a bioreactor-based technology can also enhance the multiplication rate of cell and organ cultures for commercial propagation of medicinally important bioactive molecules. The present review focuses on the propagation and production of bacoside A by cell and organ cultures of Bacopa monnieri, a nootropic plant. The review also focuses on the biosynthesis of bacoside A, different elicitation strategies, and the over-expression of genes for the production of bacoside-A. It also identifies research gaps that need to be addressed in future studies for the sustainable production of bioactive molecules from B. monnieri.

Neuroprotective Potential of Bacopa monnieri: Modulation of Inflammatory Signals.

BACKGROUND: To date, much evidence has shown the increased interest in natural molecules and traditional herbal medicine as alternative bioactive compounds to fight many inflammatory conditions, both in relation to immunomodulation and in terms of their wound healing potential. Bacopa monnieri is a herb that is used in the Ayurvedic medicine tradition for its anti-inflammatory activity. OBJECTIVE: In this study, we evaluate the anti-inflammatory and regenerative properties of the Bacopa monnieri extract (BME) in vitro model of neuroinflammation. METHODS: Neuronal SH-SY5Y cells were stimulated with TNFα and IFNγ and used to evaluate the effect of BME on cell viability, cytotoxicity, cytokine gene expression, and healing rate. RESULTS: Our results showed that BME protects against the Okadaic acid-induced cytotoxicity in SH-SY5Y cells. Moreover, in TNFα and IFNγ primed cells, BME reduces IL-1ß, IL-6, COX-2, and iNOS, mitigates the mechanical trauma injury-induced damage, and accelerates the healing of wounds. CONCLUSION: This study indicates that BME might become a promising candidate for the treatment of neuroinflammation.

Biotechnological production of bacosides from cell and organ cultures of Bacopa monnieri.

Bacopa monnieri (L.) Wettst. (BM), also known as 'Brahmi' or 'Water Hyssop', has been utilized as a brain tonic, memory enhancer, sensory organ revitalizer, cardiotonic, anti-anxiety, antidepressant and anticonvulsant agent in the Indian system of medicine Ayurveda for centuries. BM is beneficial in the treatment of Parkinson's disease, Alzheimer's disease, epileptic seizures and schizophrenia in recent pharmacological research. Dammarane-type triterpenoid saponins containing jujubogenin and pseudojujubogenin as aglycones, also known as bacosides, are the principal chemical ingredients identified and described from BM. Bacosides have been shown to have anti-ageing, anticancer, anticonvulsant, antidepressant, anti-emetic, anti-inflammatory and antibacterial properties in a variety of pre-clinical and clinical studies. The pharmaceutical industry's raw material comes from wild sources; nevertheless, the concentration of bacosides varies in different regions of the plants, as well as seasonal and genotypic variation. Cell and tissue cultures are appealing alternatives for the long-term manufacture of bioactive chemicals, and attempts to produce bacosides using in vitro cultures have been made. This review discusses the biotechnological approaches used to produce bacosides, as well as the limitations and future potential. KEY POINTS: • Bacosides extracted from Bacopa monnieri are important pharmaceutical compounds. • The current review provides insight into biotechnological interventions for the production of bacosides using in vitro cultures. • Highlights the prospects improvement of bacoside production through metabolic engineering.

Biotechnology for propagation and secondary metabolite production in Bacopa monnieri.

Bacopa monnieri (L.) Wettst. or water hyssop commonly known as "Brahmi" is a small, creeping, succulent herb from the Plantaginaceae family. It is popularly employed in Ayurvedic medicine as a nerve tonic to improve memory and cognition. Of late, this plant has been reported extensively for its pharmacologically active phyto-constituents. The main phytochemicals are brahmine, alkaloids, herpestine, and saponins. The saponins include bacoside A, bacoside B, and betulic acid. Investigation into the pharmacological effect of this plant has thrived lately, encouraging its neuroprotective and memory supporting capacity among others. Besides, it possesses many other therapeutic activities like antimicrobial, antioxidant, anti-inflammatory, gastroprotective properties, etc. Because of its multipurpose therapeutic potential, it is overexploited owing to the prioritization of natural remedies over conventional ones, which compels us to conserve them. B. monnieri is confronting the danger of extinction from its natural habitat as it is a major cultivated medico-botanical and seed propagation is restricted due to less seed availability and viability. The ever-increasing demand for the plant can be dealt with mass propagation through plant tissue culture strategy. Micropropagation utilizing axillary meristems as well as de novo organogenesis have been widely investigated in this plant which has also been explored for its conservation and production of different types of secondary metabolites. Diverse in vitro methods such as organogenesis, cell suspension, and callus cultures have been accounted for with the aim of production and/or enhancement of bacosides. Direct shoot-organogenesis was initiated in excised leaf and internodal explants without any exogenous plant growth regulator(s) (PGRs), and the induction rate was improved when exogenous cytokinins and other supplements were used. Moreover, biotechnological toolkits like Agrobacterium-mediated transformation and the use of mutagens have been reported. Besides, the molecular marker-based studies demonstrated the clonal fidelity among the natural and in vitro generated plantlets also elucidating the inherent diversity among the natural populations. Agrobacterium-mediated transformation system was mostly employed to optimize bacoside biosynthesis and heterologous expression of other genes. The present review aims at depicting the recent research outcomes of in vitro studies performed on B. monnieri which include root and shoot organogenesis, callus induction, somatic embryogenesis, production of secondary metabolites by in vitro propagation, acclimatization of the in vitro raised plantlets, genetic transformation, and molecular marker-based studies of clonal fidelity. KEY POINTS: • Critical and up to date records on in vitro propagation of Bacopa monnieri • In vitro propagation and elicitation of secondary metabolites from B. monnieri • Molecular markers and transgenic studies in B. monnieri.

The Evolving Roles of Bacopa monnieri as Potential Anti-Cancer Agent: A Review.

The intermingled interrelationship of Bacopa monnieri and human health dates backs to the ancient times in the history of ayurveda making the plant an enriched source of alternative drug development in a nontoxic manner. In recent years, research on the biological effects of Bacopa monnieri has flourished as promising neuroprotective, memory boosting and more importantly as both chemopreventive and anti-neoplastic agent. Each naturally synthesized chemical constituent identified from Bacopa monnieri leaf extract with different solvents, has significant anti-metastatic, anti-angiogenic and anti-proliferative activity on different type of cancer cells. In this context, a substantial literature survey allows a deep understanding of the involvement of specific bioactive molecules along with the whole plant extract of Bacopa monnieri with their divergent effective molecular pathways. This comprehensive review covers literature up to the year 2020 highlighting all the anticancer efficacy along with signaling pathways activated by secondary metabolites found in bacopa plant.

Transcriptome-wide miRNA identification of Bacopa monnieri: a cross-kingdom approach.

Bacopa monnieri known as 'Brahmi' is a well-known medicinal plant belonging to Scrophulariaceae family for its nootropic properties. To the best of our knowledge, no characterization data is available on the potential role of micro RNAs (miRNAs) from this plant till date. We present here the first report of computational characterizations of miRNAs from B. monnieri. Owing to the high conservation of miRNAs in nature, new and potential miRNAs can be identified in plants using in silico techniques. Using the plant miRNA sequences present in the miRBase repository, a total of 12 miRNAs were identified from B. monnieri which pertained to 11 miRNA families from the shoot and root transcriptome data. Furthermore, gene ontology analysis of the identified 68 human target genes exhibited significance in various biological processes. These human target genes were associated with signaling pathways like NF-kB and MAPK with TRAF2, CBX1, IL1B, ITGA4 and ITGB1BP1 as the top five hub nodes. This cross-kingdom study provides initial insights about the potential of miRNA-mediated cross-kingdom regulation and unravels the essential target genes of human with implications in numerous human diseases including cancer.

Bacopa monnieri prevents colchicine-induced dementia by anti-inflammatory action.

Inflammation is considered as an early event in the development of Alzheimer's disease (AD) that precedes the formation of Aß plaques and neurofibrillary tangles. Therefore, strategies aimed at attenuating inflammation by phytochemicals may be a potential therapeutic intervention against AD. The present study was designed to evaluate if colchicine-induced inflammation and Aß production could be prevented by Bacopa monnieri (BM) supplementation. Dementia was induced by a single intracerebroventicular injection of colchicine (15 µg/5 µl), whereas, BM extract was administered orally (50 mg/kg body weight, daily) for 15 days. Assessment of cognitive functions using Morris water maze revealed deficits in colchicine administered animals. This was accompanied by significant increase in oxidative stress in terms of accentuated ROS and NO production. Expression of pro-inflammatory cytokines (IL-6, TNF-α) and chemokine (MCP-1) increased in the brain regions. Furthermore, COX-2 and iNOS expression also increased significantly in the brain regions of colchicine-administered animals. In addition, BACE-1 activity increased in the colchicine treated animals, which was accompanied by enhanced Aß production. On the other hand, BM supplementation was able to improve cognitive functions, suppress Aß formation by reducing BACE-1 activity. Inflammatory and oxidative stress markers were attenuated in the brain regions of BM supplemented animals. Taken together, the findings reveal that BM reverses colchicine-induced dementia by its anti-inflammatory and anti-oxidant action suggesting that it may be an effective therapeutic intervention to ameliorate progression of AD.

Zinc oxide nanoparticles (ZnO NP) mediated regulation of bacosides biosynthesis and transcriptional correlation of HMG-CoA reductase gene in suspension culture of Bacopa monnieri.

Bacopa monnieri (L.) Wettst. is a well documented nootropic plant, extensive known for alleviating symptoms of neurological disorder, along with other symptomatic relief. This property is attributed to the active phytocompounds, saponins (bacoside A) present in the plant. However, lack of stringent validation guidelines in most of the countries bring to the market, formulations differing in phytocompounds yield, thereby suggesting possible variation in therapeutic efficacy. The in-vitro suspension cultures of the Bacopa monnieri, provide an ease of scale-up, but regulating saponin yield is a stringent task. The aim of the study is to explore the effects of different concentrations (0, 0.25, 0.50, 0.75 and 1.0 ppm) of zinc oxide nanoparticles (ZnO NP) (24 nm in size), in regulating growth rate, bacoside yield and transcriptional profile of HMG CoA reductasegene in the suspension cells of Bacopa monnieri. Results showed a linear correlation between Bacoside A yield and ZnO NP concentrations with around 2 fold increase in total bacoside A concentration at 1 ppm. Also, ZnO NP supplemented suspension cells showed variation in the specific growth rate. Neuroprotective properties, analyzed using methanolic extracts of suspension cells again obtrude the extract of ZnO NP supplemented (0.75 ppm and 1 ppm) culture for better response in alleviating oxidative stress mediated damage to neuronal cells. ZnO NP supplemented system showed lower expression of HMG CoA reductasegene (the rate limiting step in bacoside A biosynthesis) but higher concentration of bacoside A, suggesting possible role of ZnO NP in isoprenoid pathway than MVA pathways.

Role of ethanolic extract of Bacopa monnieri against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced mice model via inhibition of apoptotic pathways of dopaminergic neurons.

Parkinson's disease (PD) is a neurodegenerative disease which causes rigidity, resting tremor and postural instability. The neuroprotective effects of an ethanolic extract of Bacopa monnieri (BM) were evaluated in a Parkinsonian mice model induced by the MPTP. The present study investigates the mechanisms of neuroprotection elicited by BM, an herb traditionally recognized by the Indian system of medicine, Ayurveda. An ethanolic extract of BM was co-treated with the MPTP induced mouse model of PD and was shown to significantly rescue the motor behaviour (Rotarod, Grip Strength and Foot Printing test). Furthermore, on biochemical parameters too BM significantly showed protective effect as Catalase, LPO, Nitrite, SOD, GR, GPx parameters showed marked improvement and levels of Dopamine, DOPAC and HVA were enhanced significantly. There was a significant reduction in tyrosine hydroxylase (TH) immunoreactivity in the substantia nigra (SN) in MPTP treated group, which was considerably restored by the use of BM extract. BM also facilitated neuroprotection by creating an anti-apoptotic environment indicated by reduced apoptotic (Bax and caspase-3) and increased levels of anti-apoptotic (Bcl2) protein expression, respectively. Altogether, the present study suggests that BM treatment provides nigrostriatal dopaminergic neuroprotection against MPTP induced Parkinsonism by the modulation of oxidative stress and apoptotic machinery possibly accounting for the behavioural effects.

Microbial interference mitigates Meloidogyne incognita mediated oxidative stress and augments bacoside content in Bacopa monnieri L.

Microbial interference plays an imperative role in plant development and response to various stresses. However, its involvement in mitigation of oxidative stress generated by plant parasitic nematode in plants remains elusive. In the present investigation, the efficacy of microbe's viz., Chitiniphilus sp. MTN22 and Streptomyces sp. MTN14 single and in combinations was examined to mitigate oxidative stress generated by M. incognita in medicinal plant, Bacopa monnieri. Microbial combination with and without pathogen also enhanced the growth parameters along with secondary metabolites (bacoside) of B. monnieri than the pathogen inoculated control. The study showed that initially the production of hydrogen peroxide (H2O2) was higher in dual microbes infected with pathogen which further declined over M. incognita inoculated control plants. Superoxide dismutase and free radical scavenging activity were also highest in the same treatment which was linearly related with least lipid peroxidation and root gall formation in B. monnieri under the biotic stress. Microscopic visualization of total reactive oxygen species (ROS), H2O2, superoxide radical and programmed cell death in host plant further extended our knowledge and corroborated well with the above findings. Furthermore, scanning electron microscopy confirmed good microbial colonization on the host root surface around nematode penetration sites in plants treated with dual microbes under pathogenic stress. The findings offer novel insight into the mechanism adopted by the synergistic microbial strains in mitigating oxidative stress and simultaneously stimulating bacoside production under pathogenic stress.

In vitro cultures of Bacopa monnieri and an analysis of selected groups of biologically active metabolites in their biomass.

CONTEXT: Bacopa monnieri L. Pennell (Scrophulariaceae) is one of the most important plants in the system of Indian medicine (Ayurveda). OBJECTIVE: This paper studies the optimal growth of B. monnieri for effective accumulation of metabolites. Biomass growth of this plant could be accomplished in liquid cultures on Murashige & Skoog medium. MATERIALS AND METHODS: Powdered shoots of in vitro cultures of B. monnieri were extracted by methanol for indole compounds, phenolic compounds and bacosides for RP-HPLC analysis. Fatty acid analysis was performed via gas chromatography. Anti-inflammatory effect of B. monnieri extracts was evaluated in the A549 cells. COX-2 and cPGES expression was analyzed using Western blots. RESULTS: l-Tryptophan and serotonin were found in biomass from in vitro cultures of B. monnieri on MS medium and in biomass from the MS mediums enriched with the different additions such as of 0.1 g/L magnesium sulphate, 0.1 g/L zinc hydroaspartate, 0.1 g/L l-tryptophan, 0.25 g/L serine, 0.5 g/L serine and 0.5 mg/L anthranilic acid. The content of l-tryptophan and serotonin compounds was significant in biomass from medium with the addition of 0.1 g/L zinc hydroaspartate (0.72 mg/g dry weight and 1.19, respectively). Phenolic compounds identified in biomass from the same variants of MS medium were chlorogenic acid (ranging from 0.20 to 0.70 mg/g dry weight), neochlorogenic acid (ranging from 0.11 to 0.40 mg/g dry weight) and caffeic acid (ranging from 0.01 to 0.04 mg/g dry weight). The main group of fatty acids in biomass was saturated fatty acids (53.4%). The predominant fatty acid was palmitic acid. A significant decrease of COX-2 and cPGES expression was observed in the A549 cells activated with LPS and treated with B. monnieri extracts. DISCUSSION AND CONCLUSIONS: As far as we know, this is the first analysis of indole compounds and phenolic acids in this plant. The multi-therapeutic effect of B. monnieri is expressed by the activity of bacosides. Information about the presence of indole and phenolic compounds, and fatty acids in this plant is limited, but the content of these compounds might participate in the physiological activity of B. monnieri.

Trace metals accumulation in Bacopa monnieri and their bioaccessibility.

Bacopa monnieri is commonly known as "Brahmi" or "Water hyssop" and is a source of nootropic drugs. Aboveground parts of plant samples collected from peri-urban Indian areas were analysed for total trace metal concentrations. Subsequently, three samples with high concentrations of Cd and Pb were subjected to in vitro gastrointestinal digestion to assess the bioaccessibility of the trace metals in these plants. The total concentrations of trace metals on a dry weight basis were 1.3 to 6.7 mg·kg⁻¹ Cd, 1.5 to 22 mg·kg⁻¹ Pb, 36 to 237 mg·kg⁻¹ Cu, and 78 to 186 mg·kg⁻¹ Zn. The majority of Bacopa monnieri samples exceeded threshold limits of Cd, Pb, Cu, and Zn for use as raw medicinal plant material or direct consumption. Therefore, it is necessary to evaluate Bacopa monnieri collected in nature for their trace metal content prior to human consumption and preparation of herbal formulations.

Exiguobacterium oxidotolerans, a halotolerant plant growth promoting rhizobacteria, improves yield and content of secondary metabolites in Bacopa monnieri (L.) Pennell under primary and secondary salt stress.

Brahmi (Bacopa monnieri), an integral component of Indian Ayurvedic medicine system, is facing a threat of extinction owing to the depletion of its natural populations. The present study investigates the prospective of exploitation of halotolerant plant growth promoting rhizobacteria (PGPR) in utilising the salt stressed soils for cultivation of B. monnieri. The effects of two salt tolerant PGPR, Bacillus pumilus (STR2) and Exiguobacterium oxidotolerans (STR36) on the growth and content of bacoside-A, an important pharmaceutical compound in B. monnieri, were investigated under primary and secondary salinity conditions. The herb yields of un-inoculated plants decreased by 48 % under secondary salinization and 60 % under primary salinization than the non salinised plants. Among the rhizobacteria treated plants, E. oxidotolerans recorded 109 and 138 %, higher herb yield than non-inoculated plants subjected to primary and secondary salinity respectively. E. oxidotolerans inoculated plants recorded 36 and 76 % higher bacoside-A content under primary and secondary salinity respectively. Higher levels of proline content and considerably lower levels of lipid peroxidation were noticed when the plants were inoculated with PGPR under all salinity regimes. From the results of this investigation, it can be concluded that, the treatments with salt tolerant PGPR can be a useful strategy in the enhancement of biomass yield and saponin contents in B. monnieri, as besides being an eco-friendly approach; it can also be instrumental in cultivation of B. monnieri in salt stressed environments.

Investigation of biochemical responses of Bacopa monnieri L. upon exposure to arsenate.

Widespread contamination of arsenic (As) is recognized as a global problem due to its well-known accumulation by edible and medicinal plants and associated health risks for the humans. In this study, phytotoxicity imposed upon exposure to arsenate [As(V); 0-250 µM for 1-7 days] and ensuing biochemical responses were investigated in a medicinal herb Bacopa monnieri L. vis-à-vis As accumulation. Plants accumulated substantial amount of As (total 768 µg g(-1) dw at 250 µM As(V) after 7 days) with the maximum As retention being in roots (60%) followed by stem (23%) and leaves (17%). The level of cysteine and total nonprotein thiols (NP-SH) increased significantly at all exposure concentrations and durations. Besides, the level of metalloid binding ligands viz., glutathione (GSH) and phytochelatins (PCs) increased significantly at the studied concentrations [50 and 250 µM As(V)] in both roots and leaves. The activities of various enzymes viz., arsenate reductase (AR), glutathione reductase (GR), superoxide dismutase (SOD), guaiacol peroxidase (GPX), ascorbate peroxidase (APX), and catalase (CAT) showed differential but coordinated stimulation in leaves and roots to help plants combat As toxicity up to moderate exposure concentrations (50 µM). However, beyond 50 µM, biomass production was found to decrease along with photosynthetic pigments and total soluble proteins, whereas lipid peroxidation increased. In conclusion, As accumulation potential of Bacopa may warrant its use as a phytoremediator but if Bacopa growing in contaminated areas is consumed by humans, it may prove to be toxic for health.

Use of the cryptogein gene to stimulate the accumulation of Bacopa saponins in transgenic Bacopa monnieri plants.

Genetic transformation of the Indian medicinal plant, Bacopa monnieri, using a gene encoding cryptogein, a proteinaceous elicitor, via Ri and Ti plasmids, were established and induced bioproduction of bacopa saponins in crypt-transgenic plants were obtained. Transformed roots obtained with A. rhizogenes strain LBA 9402 crypt on selection medium containing kanamycin (100 mg l(-1)) dedifferentiated forming callus and redifferentiated to roots which, spontaneously showed shoot bud induction. Ri crypt-transformed plants thus obtained showed integration and expression of rol genes as well as crypt gene. Ti crypt-transformed B. monnieri plants were established following transformation with disarmed A. tumefaciens strain harboring crypt. Transgenic plants showed significant enhancement in growth and bacopa saponin content. Bacopasaponin D (1.4-1.69 %) was maximally enhanced in transgenic plants containing crypt. In comparison to Ri-transformed plants, Ri crypt-transformed plants showed significantly (p ≤ 0.05) enhanced accumulation of bacoside A(3), bacopasaponin D, bacopaside II, bacopaside III and bacopaside V. Produced transgenic lines can be used for further research on elicitation in crypt-transgenic plants as well as for large scale production of saponins. Key message The cryptogein gene, which encodes a proteinaceous elicitor is associated with increase in secondary metabolite accumulation-either alone or in addition to the increases associated with transformation by A. rhizogenes.

Genetic transformation of Bacopa monnieri by wild type strains of Agrobacterium rhizogenes stimulates production of bacopa saponins in transformed calli and plants.

We have developed an efficient transformation system for Bacopa monnieri, an important Indian medicinal plant, using Agrobacterium rhizogenes strains LBA 9402 and A4. Transformed roots induced by strain LBA 9402 spontaneously dedifferentiated to callus while excised roots induced by strain A4 spontaneously showed induction of shoot buds within 10 days. PCR and RT-PCR analysis revealed the presence and expression of the rolAB and rolC genes at the transcription level in pRi A4 transformed cultures indicating that the TL-DNA was integrated retained and expressed in the A4-Ri transformed shoots. Transformed calli showed the presence of rolAB or rol A, TR and ags genes. Transformed plants showed morphological features typically seen in transgenic plants produced by A. rhizogenes. Growth and biomass accumulation was significantly higher in the transformed shoots (twofold) and roots (fourfold) than in the non-transformed (WT) plants. In pRi A4-transformed plants, the content of bacopasaponin D, bacopasaponin F, bacopaside II and bacopaside V was enhanced significantly as compared to WT plants of similar age while bacoside A3 and bacopasaponin C content was comparable with that of WT plants. Significant increase in content of five bacopa saponins could be detected in pRi 9402-transformed callus cultures. There is an overall stimulatory effect on accumulation of bacopa saponins in transformed plants and cells of B. monnieri establishing the role of endogenous elicitation by Ri T-DNA of A. rhizogenes.