Reviews - European Journal of Applied Engineering and Scientific Research ( 2017) Volume 5, Issue 2
Bacopa monnieri, Bacoside, In vitro propagation, Conservation, Side effects
Bacopa monnieri is an important medicinal plant which rejuvenates memory and intellect.It has been used by Ayurveda in India for almost 3000 years [1]. It is belongs to ‘Scrophulariaceae’ family and a small perennial herb which grows in wetland and muddy shores habitat. The plant has a soft stem which is approximately 10-30 cm long, 1-2 mm in thickness. According to National Medicinal Plants Board (NMPB) it is also known as Gratiola monnieria (L.), Moniera cuneifoli, Herpestis monnieri (L.), Moniera cuneate andlocally known as brahmi, pan brahmi, water hyssop, jalbuti, jalnim, etc. Genus Bacopa includes more than 100 species of aquatic herbs. It is found in India, Nepal, Vietnam, Sri Lanka, China, Taiwan, Hawaii, Florida and some other Southern states of USA. In the list of important medicinal plants Bacopa monnieri is placed in second position. Whole plant of Bacopa monnieri is medicinally useful [2]. According to National Medicinal Plants Board the estimated market demand for Bacopa monnieri is around 1,000 tonnes per year. Bacopa monnieri also helps in repairing connective tissues and skin and smoothing out cellulite. It possesses different pharmacological activities due to presence of several compounds which include alkaloids, saponins, glycosides, flavonoids and stigmasterols. Saponins include hersaponin, monnierin, Bacoside A and Bacoside B [3]. Bacoside A is the major component that is responsible for the memory enhancement effect. Bacoside A differs from Bacoside B in optical rotation where Bacoside A is levorotatory and Bacoside B is dextrorotatory. The present review gives the detail study of in vitro propagation studies done in Bacopa monnieri and its conservation process. Further it also discusses the commercial production of bacosides and dosages and side effects
In vitro propagation
For conservation of B. monnieri tissue culture is one of the alternative methods. In short period of time clonal propagation through tissue culture can be achieved. Different in vitro propagation studies show that B. monnieri can be propagated through different parts of the plant which includes nodes, internodes, leaves, axillary bud and callus shown in Table 1. It was seen that generally Murashige and Skoog’s media used in most cases along with the growth hormones or elicitors or both for plant propagation [37-40].
| Explants | Media | Growth hormone | Shooting/Rooting/Callusing | References |
|---|---|---|---|---|
| Node | MS | BAP (1.0 mg/l)+IAA (0.5 mg/l) | Shooting | Dixit and Thakur [4] |
| Node | MS | BA (0.2 mg/l) | Shooting | Sharma et al. [5] |
| Node | MS | BA (0.2 mg/l) | Rooting | |
| Node | MS | BAP (3.0 mg/l) | Shooting | Shashikanta and Nibedita [6] |
| Leaf | MS | BAP (0.2 mg/l)+IAA (0.2 mg/l) | Shooting | Uma et al. [7] |
| Shoot tips, leaves, nodal and internodes | MS | BAP (2 mg/l)+Kinetin (1 mg/l)+ IAA (1 mg/l) | Shooting | Ayyappadas and Renugadevi [8] |
| 2,4-D (0.5 mg/l) | Callus induction | |||
| Nodal and leaf | MS | 2,4-D (2 mg/l) | Callus induction | Talukdar [9] |
| Nodal | MS | Kinetin (2 mg/l) | Shoot regeneration | Umesh [10] |
| Leaf | BAP (1 mg/l)+IAA (0.5 mg/l) | Callus induction | ||
| Leaf | MS | - | Shoot initiation | Koul et al. [11] |
| B5 | ||||
| Leaf, nodal and intermodal | MS | BAP (2.0 mg/l)+NAA (0.5 mg/l) | Callus induction | Priya [12] |
| Nodal | MS | BAP (1.5 mg/l) | Shoot multiplication | Pandey et al. [13] |
| IBA (1.0 mg/l) | Rooting | |||
| Nodal | MS | BAP (0.5 mg/l) | Shoot induction | Kumari et al. [14] |
| IBA (0.2 mg/l) | Rooting | |||
| Nodal | MS | BAP (1.5 mg/l)+IAA (0.5 mg/l) | Shoot multiplication | Kapil and Sharma [15] |
| IAA (1.0 mg/l) | Rooting | |||
| Nodal | MS | BAP (1.0 mg/l) | Shoot induction | Vollala et al. [16] |
| TDZ (1.0 mg/l) | ||||
| 2ip (4.92 mg/l) | ||||
| Nodal | MS | IAA (0.5 mg/l) | Axillary shoot proliferation | Mohanta and Sahoo [17] |
| NAA (0.5 mg/l) | ||||
| BAP (0.5 mg/l)+2,4 D (1 mg/l ) | Callus Initiation | |||
| BAP (1.0 mg/l)+IAA(1 mg/l) | Callus Regeneration | |||
| Leaf | B5 | 2,4-D (0.5 ml/l) | Callus induction | Jain et al. [18] |
| Nodal and Internodal | MS | BAP (1.0 mg/l)+NAA (0.5 mg/l) | Shoot initiation | Jain et al. [19] |
| Nodal | MS | BAP (1.0 mg/l) IBA (1.0 mg/l) |
Shoots multiplication | Kaur et al. [20] |
| Rooting | ||||
| Axillary buds | MS | BAP (4.44 µM) and NAA (0.53 µM) | Shoot regeneration | Sharath, et al. [21] |
| Leaf | MS | BAP (0.5 mg/l)+NAA (1.0, 2.0 mg/l), 2,4D (2.0 mg/l)+BAP (0.5, 1.0 mg/l) | Callus induction | Singh [22] |
| Nodal | MS | BAP (2.5 mg/l)+IAA (0.01 mg/l) | Shooting | Jain et al. [23] |
| Nodal | MS | BAP (1.5 mg/l) | Shoot induction | Pandiyan and Selvaraj [24] |
| IBA (1.0 mg/l)+NAA (0.5 mg/l) | Rooting | |||
| Nodal | MS | BAP (3 mg/l) | Shoot regeneration | Gurnani et al. [25] |
| BAP (1 mg/l)+IAA (3 mg/l) | Rooting | |||
| Auxiliary nodes and internodes | MS | IAA (0.5 mg/l)+Kn (0.5 mg/l) | Auxiliary shoot proliferation | Tanner et al. [26] |
| Leaves | NAA (0.5 mg/l)+TDZ (0.25 mg/l) | Callus induction | ||
| Nodal, Internodal and Leaf | MS | BAP (4.4 µM) | Shoot regenration | Yusuf et al. [27] |
| Leaf | MS | 2,4-D (0.5 mg/l) | Callus induction | Showkat et al. [28] |
| Leaf | MS | BAP (5 µM) | Shoots regeneration and multiplication | Parale et al. [29] |
| NAA (5 µM)+2,4-D (1 µM) | Callus induction | |||
| Leaf and node | MS | BAP (1 mg/l)+Kn (0.5 mg/l)+NAA (0.5 mg/l) | Shoot regeneration | Khan et al. [30] |
| IAA (0.5 mg/l) | Rooting | |||
| BAP (1 mg/l)+2,4D (0.5 mg/l) | Callus induction | |||
| Leaf | MS | BAP (6 µM) | Shoot regeneration | Pathak et al. [31] |
| IBA (2 µM) | Rooting | |||
| Nodal | MS | BAP (0.2 mg/l) | Shoot multiplication | Sharma et al. [32] |
| IBA (0.15 mg/l) | Rooting | |||
| Young shoot | MS | IBA (0.5 mg/l) | Rooting | Kumari et al. [33] |
| Internodal | MS | BAP (1.0 mg/l)+Kn (0.5 mg/l) | Shooting | Banerjee and Shrivastava [34] |
| Axillary nodes, young leaves and internodes | MS | BAP (1.1 µM)+IAA (0.2 µM) | Shoot regeneration | Yogesh et al. [35] |
| Leaf and nodal segments. | MS | BAP and NAA | Shoot regeneration | Mohapatra and Rath [36] |
| B5 | BAP and NAA |
Table 1: In vitro propagation of B. monnieri using different explants
Cryopreservation
In vitro shoot tips of B. monnieri was developed through Cryopreservation. Shoot tips (about 1 mm in length) were taken from 4 to 24 week old proliferating shoot cultures were pre-cultured on MS medium supplemented with various cryoprotectants before dehydration with PVS2 at 0°C. After rapid rewarming at 45°C the dehydrated shoot tips were directly immersed in liquid nitrogen, then they were quickly washed with 1.2 M sucrose solution and plated on solidified shoot culture medium [41-45]. By vitrification the shoot tips were successfully cryopreserved when they were pre-cultured on medium supplemented with 5% DMSO for two days before dehydration [46-51]. The survival rate and shoot regeneration rate of cryopreserved shoot tips is significantly improved by pre-culturing the shoot tips with sucrose at 25°C [52-56]. Regenerated plantlets were successfully transferred to soil [57,58]. A method was developed for slow growing, medium-term conservation of B. monneiri by using mineral oil. Nodes of B. monnieri were conserved using mineral oil for 24 months. Single node explants were inoculated in MS medium which contains 0.2 mg/l BA and were covered with mineral oil. There is significant enhancement of subculture duration from 6 to 24 months was observed on the above medium. Regeneration of plant from conserved culture was established in soil. Results show that mineral oil can be used for medium-term conservation of B. monnieri germplasm without any adverse genetical and biochemical effects [37].
Production of bacosides
In natural circumstances, the production of secondary metabolites is in fewer quantities so there is a requirement of large scale production. Secondary metabolite of B. monnieri from leaf, nodes and axillary buds was produced by using cheap alternative media [38]. In the same study MS medium used with 0.5 mg/l Indole Butyric Acid (IBA) and 3 mg/l Kinetin. For cost minimization, 70% strength MS medium with 20 g/l Jaggery, 10 g/l Sucrose and 5 g/l Agar were used [44-47]. Result shows that there were maximum multiple shoot regeneration from leaf up to number of shoots per explants- 7.25 ± 0.96, shoot length- 11.30 ± 0.26 cm, nodes number per shoots- 11.50 ± 0.58. Different compositions of 6-benzyl amino purine (BAP) were used for comparison instead of Kinetin and it was better than Kinetin [50-56]. Also, rooting response against NAA and IBA were compared and it was found that NAA gave better result than IBA and by this there was a 40% reduction in medium cost and 25% reduction in production cost [38]. The elevated production of saponins in short duration was measured using Gamboreg’s media with different combination of hormones [18]. Callus were formed and after 20 days viable callus were transferred to modified MS liquid medium which was supplemented with 1 mg/l naphthalene acetic acid, 0.5 mg/l kinetin, 1 g/l casein hydrolysate and 30 g/l sucrose [49]. After 40 days cell mass in suspension along with field grown natural plant system and plant parts were dried separately in dark for 4 days at 37°C. Total methanolic extraction of saponin was performed. Then spectrophotometric technique and TLC (Thin layer Chromatography) were performed and TLC plate exhibiting different bands of saponins obtained from Root system, Shoot system, Callus and Total plant system of B. monnieri [19]. Elicitors also help in the production of the secondary metabolites. It enhances the synthesis of secondary metabolites by the plants. [39] 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 [42]. The effect of 150 mg/l chitosan and 2 mg/ml yeast extract in whole plant cultures has showed an increased pseudojujubogenin glycoside production (40.83 ± 2.24 mg/g DW and 40.05 ± 2.37 mg/g DW, respectively) after 7 days. This production was 6 times higher than that of control cultures [40]. In a study when shoot cultures were co-cultivated with a novel symbiotic fungus Piriformospora indica, it showed an enhancement in the plant growth, biomass, bacoside contant, antioxidant activity and nuclear hypertrophy. Reported enhanced bacoside A production (4.4 mg/g) in in vitro shoot cultures treated for one week with 50 μM methyl jasmonate [59,60]. Increase in shoot growth, antioxidant defense and bacoside A content under the influence of NaCl has been reported [59] and recently, also observed enhanced bacoside A and bacoside I content in B. monnieri shoots when treated with a concentration of 10 μM Cadmium after a culture period of one week. In a study it was reported that rhizospheric microbes, i.e., Bacillus megaterium, Glomus intraradices, Trichoderma harzianum and their combinations used for the increase production of bacoside content and the production was 1.40-fold higher than control [42]. Effect of different abiotic elicitor’s, i.e., jasmonic acid, copper sulphate and salicyclic acid at varying concentrations increases the biomass and bacoside production. Shoot cultures treated with 45 mg/l of CuSO4 showed highest content of bacoside, i.e., 8.73 mg/g dry weight which was approximately 1.42-fold higher than in control cultures [40].
Dosage, side effects and toxicity
5-10 g of non-standardized powder of B. monnieri, 8-16 ml of infusion and 30 ml of Brahmi syrup are recommended for daily doses. For adults, 5-12 ml per day dosages of a 1:2 fluid extract and two and half to ml per day for children ages 6-12. For standardized to 20% Bacosides A and B, Bacopa monnieri extract the dosage is 200-400 mg daily for adults (in divided doses), and for children, 100-200 mg/day [43,48]. Therapeutic doses of B. monnieri are not associated with any known side effects. In a study it has been seen that LD50 of aqueous extracts of B. monnieri when administered to rats orally was 5 g/kg and the alcohol extract was 17 g/kg. None of the extract shows gross behavioural change at these concentrations [61]. In double-blind, placebo controlled clinical trial of healthy male volunteer’s safety of pharmacological doses of isolated bacosides over a four-week period was investigated. Daily concentrated bacosides given in form of single (20-30 mg) and multiple (100-200 mg) dose, and doses were well tolerated and without showing any adverse effects [62,63].
The present review shows that Bacopa monnieri contains various phytocompounds like alkaloids, saponins, glycosides, flavonoids, etc. and it has various pharmacological activities.
In vitro propagation studies showed that the plant has maximum growth in MS media with different concentration of auxins and cytokinins. Most commonly used cytokinin is BAP (0.2-3 mg/l) for the shooting and for rooting and callusing 2,4-D, IAA and NAA (0.2-2 mg/l). In future micropropagation and cryopreservation can be used for the conservation of the plant. Saponin is the major phytocompound. They are used to treat several kinds of diseases for this reason there is a requirement of large amount of saponin production. In cell suspension culture different types of elicitors and plant growth hormones were used to enhance the production of the saponin. In future, different concentrations of elicitors and growth hormones can be used for commercial production of saponin.
