In-Vitro Studies on the Protective Effect of Bacopa Monnieri on Oxidative Stress Induced Neurotoxici

International Journal of Bio-Technology and Research (IJBTR) ISSN (P): 2249–6858; ISSN (E): 2249–796X Vol. 10, Issue 1, Jun 2020, 87-96 © TJPRC Pvt. Ltd.

IN-VITRO STUDIES ON THE PROTECTIVE EFFECT OF BACOPA MONNIERI ON OXIDATIVE STRESS INDUCED NEUROTOXICITY SREELAKSHMI. R & LEKSHMI. R Scholar, Department of Bio-Technology, Milad-E-Sherif Memorial College, Kayamkulam, Kerala University, India ABSTRACT Neurodegenerative diseases can be defined as, ‘the disorders with selective neuron loss’. The disorders result mainly when neurons in brain and peripheral nervous system suffer a functional loss and progressive death. Most of such conditions have no cure. This may result in ‘ataxias’ or ‘dementias’. In some of the neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease; which are age related neurotoxicity arises mainly from protein aggregation. The attack of free radicals produced as a result of oxidative stress, on neurons also contributes to neurodegeneration. The free radicals which are toxic can cause protein and DNA degeneration, damage of tissues, inflammation and cell level apoptosis. Therefore, inhibiting aggregation of proteins and scavenging of free radicals will be potential therapeutic strategies for neurodegenerative disorders. It has been already proven that several phytochemicals like flavanoids can act as antioxidants scavenging stress induced free radicals in the brain. Here, we are

radicals. The study also aims to measure the neuroprotective activity of B.monnieri extracts. Phytochemical analysis was performed by qualitative assays which proved the presence of flavanoids and steroids. The plant extract was also found to have antioxidant potential which was confirmed by assay for reducing power. The anti apoptotic effect of B.monnieri extract were confirmed by FDA/EtBr staining. With potential anti-oxidant, free radical scavenging and anti apoptotic activity B.monnieri leaf extract may be used as an effective therapeutic agent in targeting neurodegenerative disorders.

Original Article

aiming to analyse the phytochemicals present in the leaf extract of Bacopamonnieri and how effectively it scavenges free

KEYWORDS: Neurodegeneration, Phytochemicals & Radical Scavenging

Received: Mar 01, 2020; Accepted: Mar 20, 2020; Published: May 20, 2020; Paper Id.: IJBTRJUN20208

1. INTRODUCTION Bacopa monnieri is a plant of great therapeutic importance. The use of B.monnieri in indigenous systems of medicine have been proven by several researchers. The alkaloid brahmine was isolated from B.monnieri in 1931.Several other alkaloids such as nicotine, herpestine, D-mannitol, saponin, hersaponin and potassium salts have also been isolated from B.monnieri extracts[1].Bacoside A and Bacoside B are two major saponins present in Bacopa extract which attributes to its nootropic activity[2].The plant extract also has memory enhancing activity, which is contributed by Bacoside A; chemically 3-(α-L-arabinopyranosyl)-O-ß-D glucopyranoside-10,20dihydroxy-16-keto-dammar-24-ene [3].Bacoside A and B are stereoisomers of which Bacoside A is levorotatory while the latter is dextrorotatory[4].

Other chemical constituents separated from the alcoholic extract of B.monnieri are triterpenoidsaponins like jujubogenin and pseudojujubogenin.Acid hydrolysis of Bacosides give aglycones bacogenin A1, A2, A3, A4 etc[5]. Phenylethnoid glycosides like monnierasides I, II and III and plantainoside B were isolated from the glycosidic fraction of B. monnieri [6]. Bacoside A is composed of four triglycosidicsaponins[7].Bacoside B has four diglycosidicsaponins[8].The plant aqueous extract has also given two dammarane glycosides[9], 20-O- α -Lwww.tjprc.org

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arabinopyranosyljujubogenin and 3-O-α-L-arabinopyranosyljujubogenin.Several other sterol glycosides like cucurbitacins, phenlethanoid glycosides etc have also been extracted from the plant extract. Studies have also shown the presence of phenylethanoid glycosides, flavonoids, amino acids alpha-alanine, aspartic acid, glutamic acid, and betulinic acid, stigmasterol, b-sitosterol and stigmastenol[10][11].

B.monnieri extract is used in Indian Ayurvedic medicinal system for enhancing memory and also intellect. It is also used in treating psyconeuro diseases and is used as a rejuvenator too. The presence of different triterpanoids have given the plant these therapeutic properties. Bacosides A and B in Bacopa, known as memory chemicals, increase the protein kinase activity in hippocampus, thereby help to repair the damaged brain neurons, enhance neuronal synthesis and nerve impulse transmission [12].

B.monnieri extract also have significant anti-oxidant properties. Its bacosides help to scavenge free radicals like peroxides, hydoxy radicals, superoxides etc. there by protecting several regions of brain, hippocampus, frontal cortex and striatum from oxidative damage[13].

Researchers have proven the anti-depressant activity of B.monnieri methanolic extract in mice[14].Also, it has improved the motor learning ability in rats. Bacosides A and B, Bacopasaponin I, II and Bacopasaponin present in Bacopa have well documented anti depressant activity[15].

The anti inflammatory effect of B.monnieri extract is also evident from various studies. The property is driven through inhibition of prostaglandin synthesis and by stabilisation of lysosomal membrane[16].When compared with the anti inflammatory drug indomethacin, Bacopa extract showed better results and also didn’t cause any gastric irritations[17][18].

Extracts of Bacopa monnieri in phenol, methanol and ethanol shows anti-microbial activity against several microbes like Streptococcus, Escherichia coli, Staphylococus aureus, Bacillus subtilis, Klebsiella pneumoniae, Pseudomonas aerogenosa [19].Betulinicacid, wogonin and oroxindin thedifferent phytochemicals isolated from the aerial part of B.monnieri showed significant antifungal activity against Alternaria alternata and Fusarium fusiformis[20]).

2. OBJECTIVES Bacopamonnieri, member of genus Bacopa and family Scrophulariaceae is reported to have significant influence on biological properties, especially free radical induced toxicity, therefore the aim of current study are; 

To analyze the phytochemicals present in the plant extract of Bacopamonnieri



To study the radical scavenging effect of plant extract of Bacopamonnieri by reducing power activity.



To measure the neuroprotection of Bacopamonnieri extracts by SRB assay and FDA staining.

3. EXPERIMENTS 3.1

Phytochemical analysis

Fresh leaves of Bacopamonnieri were collected and the leaves were shade dried in the laboratory. The shade dried leaves are powdered mechanically. 50 gm of the powdered sample was immersed in 70% ethyl acetate and incubated for 24 hours. Extract was taken and then filtered using a two layered muslin cloth, air dried, and then used for further studies.10 gm of powdered sample was subjected to cold extraction with ethanol and the extracts were dried. The presence of Impact Factor (JCC): 5.9084

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phytochemicals were analysed and determined. Flavanoids were analysed by adding a few drops of dilute HCl and a piece of Zinc chloride to alcoholic extract(0.5 ml) and boiled the solution for a few minutes.A reddish pink or dirty brown colour appeared which indicated positive flavanoid reaction. In order to test the presence of Phenols, Ferricchloride test was carried out. 1ml of alcoholic solution of plant extract was taken to which distilled water2ml) was added followed by a few drops of 10% aqueous ferric chloride solution. Blue colouration was not seen, indicating that phenols are absent in the plant extract. Presence of steroids was confirmed by Salkowskitest.1ml of concentrated H2SO4 was added to the plant extract in chloroform(2ml) along the side of test tube. Formation of a red colour in the chloroform layer showed the presence of steroids.

Figure 1: 50 Gm of Powdered Sample of Leaves was Immersed in 70%Ethyl Acetate.

Figure 2: Test for Flavanoid- Alchoholic Extract.

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Figure 3: Test for Phenol- Alcoholic Extract.

Figure 4: Test for Steroids- Chloroform Extract. a.

Reducing Power Determination

YEN and DUH method was used to determine the reducing power of plant extract. Various concentrations of the extract (12.5µl-200µl) were added. It was mixed with Phosphate buffer(2.5ml,200mM and pH 6.6)and Potassium Ferric cyanide(2.5ml) and boiled at 50⁰C for a period of 20minutes.Incubated and 10% Tri ChloroAcetic acid was added to the mixture, centrifugation is done at 650 xg and then kept for 10 minutes. 5 ml of supernatant was mixed with distilled water(5ml), and a millilitre of 0.1 % FeCl3 was added and the optical density was measured at 700nm.

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Cell Culture

SHSY5Y cell lines obtained from NCCS, Pune. The cell line was cultured in Dulbecco’s Modified Eagle’s Medium, FBS and streptomycin was added as supplements. The cells were grown to attain a confluency of 80-90% under humidified condition in a CO2incubator.The cells were sub cultured after trypsinisation with 0.25% in 0.5ml EDTA and were cultured at 37⁰Cunder CO2concentration of 5%. c.

Cell Fixation and Staining(Sulforhodamine staining)

Cold TCA(100µl,10%)was added into the well plates without discarding the supernatant and incubation was carried out at 4⁰C for an hour, the plates were then washed3-4times in slow running tap water. Excess water removed using paper towels.

The plates were dried properly using a blow dryer. SRB solution(100µl, 0.057%) was added to each well. Kept the stain for about 30 minutes, the plates were rinsed about 4 times in 1% acetic acid in order to remove unbound dye. Tris base solution (200µl, 10 mM, pH 10.5) was added to each well. The plates were kept in a shaker for about 5 minutes to solublize the dye bound to proteins. The Optical density was read in a microplate reader at 510 nm.

Percentage viability = Optical Density of test/Optical Density of control×100 d.

Determination of lipid peroxidation, reduced glutathione levels and apoptosis

For analysing lipid peroxidation 50µl tissue homogenate was added with alcohol(500µl,70%) and 1ml of Thio barbituric acid(TBA)1%. The tubes were kept in boiling water bath for about 20 minutes. After cooling to room temperature, acetone(50µl) was added to all the test tubes and the absorbance was read in spectrophotometer at 535 nm.

1 ml of tissue homogenised in the PBS was taken in a test tube, 0.5ml of phosphate buffer was added (0.2M pH 8).Distilled water (1.3ml) and DTNB(Dithiobis-2 nitrobenzoic acid,0.2ml,0.6 mM)were also added. The contents were mixed well and reading was taken on spectrophotometer at 420 nm [21]. GSH level was compared with a standard glutathione graph.

For determining apoptosis, two dyes were employed FDA and ethidium bromide. Used a permeable FDA dye that can easily pass the plasma membrane of healthy cells and stain the cytoplasm giving a green fluorescence. EtBr used is a nuclear dye which permease the cells whose membrane integrity is lost. EtBr intercalate with DNA and gives a red fluorescence to nucleus. Presence of red colour in nucleus is the indication of their damage. Staining solution consists of (10µg/ml FDA, 50µg/ml EtBr) was added to cell culture and incubated in dark for 15 minutes. After washing in PBS, the cells observed under fluorescent microscope using the blue filter. FDA (Sigma, St Lewis, Missouri, USA) was prepared by dissolving 100mg of FDA in 20ml of acetone which makes a final concentration of 5mg/ml. 1ml volumes of stock solution were distributed to 13*100mm screw caps test tubes and stored at 20⁰c; protected from light which hydrolyse FDA upon extended exposure.

4. RESULTS AND DISCUSSIONS e.

Phytochemical Analysis

Qualitative tests for flavonoids indicated a positive result with the appearance of a reddish pink colour with dilute HCl and zinc chloride.

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Ferric chloride test for phenols indicated its absence with no colour formation. Salkowski test for steroids gave a positive result(red colouration) showing the presence of steroids in Bacopa leaf extract. f.

Reducing Power Assessment

Figure 5 Reducing power of increasing concentrations of Bacopa leaf extract was determined by reducing power assay. A control was also taken. Concentrations of leaf extract taken were 12.5,25, 50,100 and 200Âľg/ml respectively. Optical densities determined at 700nm showed a remarkable increase from 0.204 to 1.181 with increasing concentrations of sample, which indicates the potential antioxidant activity of Bacopamonnieri leaf extract. g.

SRB Assay

Figure 6 SRB assay for determining the role of Bacopa leaf extract in enhancing cell viability was done. Leaf extracts were added to microwells in increasing concentrations 6.25,12.5,25 and 50Âľg/ml. Cell viability was found to increase respectively, indicated by decrease in absorbance from 0.1076 to 0.0882 from which percentage of cell viability is determined, which was increasing from78.34% to 81.97%.

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GSH Assay

Figure 7 GSH assay showed that glutathione level is maintained int act by the addition of Bacopa leaf extract, which was 0.00903µg/ml in control, with H2O2 it was reduced to 0.004214µg/ml. When Bacopa leaf extract was added to H2O2 GSH, the concentration was 0.008729µg/ml. This indicates its role in rendering protection against oxidative cell injuries. i.

Lipid Peroxidation Assay

Figure 8 As indicated in Figure 8, Bacopa leaf extract when added with H2O2was decreasing lipid peroxidation rate, which is indicated by the decrease in absorbance from0.264 to 0.106. j.

FDA/Ethidium Bromide Staining

Bacopa monnieri leaf extract when mixed with H2O2 was shown to keep the cells healthy with intact cell membrane rather than the cells, which were damaged when, treated withH2O2 only.

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4. CONCLUSIONS Incidence of neurodegenerative diseases is increasing in our modern society, and it is of great importance to develop effective strategies of treatment and management. Neurode generation occurs mainly by oxidative attack of free radicals on neurons. There are various studies indicating the role of oxidative stress on pathophysiology of neurodegenerative diseases.

These disorders can be prevented and treated by antioxidants. In this aspect, our objective is to determine the effectiveness of B.monnieri extract on neuro protection in H2O2 induced toxicity.

Herbal medicines are widely used by about 80% of people all over the world and is increasing in popularity across globe owing to its minimal side effects. The common Ayurvedic plant Bacopamonnieri act as an antioxidant in diet, with remarkable ways of action against neurodegenerative disorders and are used in experimental studies. The phytochemical analysis of Bacopa has shown the presence of both the steroids and flavanoids, which are potential scavengers of free radicals. Reducing power activity of Bacopa also confirmed the extract’s role as free radical scavenger.

The invitro studies of Bacopa used SH SY5Y cell lines for studying the neuroprotective effect and hydrogen peroxide was used as the toxicity inducer. In our study, we used SRB assay to measure the neuroprotective effect of Bacopa. The cell viability has been reduced to 59% on addition of hydrogen peroxide which was increased to 94% by Bacopa extract treatment. Further, the neuroprotective or anti apoptotic effect of Bacopa was determined by FDA/EtBr staining and the results showed the addition of extract retained most cells live. Hence, it can be concluded that the herb Bacopamonnieri has the potent ability for neuroprotection and more research can warrant isolation and identification of bioreactive principles from the extract.

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