INTRODUCTION:

Free radicals are unstable and highly reactive atoms or molecules containing one or more unpaired electrons in its outermost shell which captures an electron from other stable molecules to gain stability triggering a chain reaction. They are of different types such as Reactive oxygen species (ROS) and Reactive Nitrogen Species (RNS). These are either produced within the body by different endogenous systems or induced by various physicochemical conditions and pathophysiological states leading to a number of diseases such as aging, cancer, Diabetes mellitus, arthritis and other diseases¹.

Antioxidants are molecules which can interact with free radicals and cease the chain reaction before the vital molecules get damaged. The free radical defense includes enzymatic antioxidants, non enzymatic antioxidants and antioxidants in food. Antioxidants may be synthetic or natural, like the secondary metabolites obtained from plants ^{2, 3}.

A significant amount of antioxidants such as flavonoids, phenolics and polyphenols (condensed and hydrolysable tannins) are produced by plants which help in preventing the oxidative stress caused by reactive oxygen species. Plants contain significant amount of the chief antioxidant nutrients such as vitamin A (as beta-carotene), C, E and selenium, which work synergistically to protect against oxidative damage. Potent antioxidant activity is exhibited by proanthocyanidins, belonging to a group of compounds known as bioflavonoids, which occurs naturally in fruits and vegetables. Green tea extract contains ample concentrations of a number of flavonoids and catechins, which have been well reviewed in relation to the prevention of oxidative stress ^{4, 5}.

Bacopa monnieri belongs to the family Scrophulariaceae is a water hyssop and locally known as brahmi in India. It is a small creeping herb with small oblong leaves bearing light purple or small white flowers with four or five petals. It is found in wet lands throughout the Indian subcontinent especially in damp, marshy or sandy areas near streams in tropical regions. The herb has been used for centuries in the Ayurveda, a holistic system of medicine originated from India.It was used as a brain tonic to enhance memory development, learning and to provide relief to patients with anxiety or epileptic disorders, digestive aid and to improve respiratory function in cases of broncho constriction. Specific uses include the treatment of asthma, insanity and epilepsy ⁶.

In a recent study, the neuroprotective role of Bacopa monnieri extract was investigated in hippocampus of temporal lobe of epileptic rats ⁷. Also studies were carried out on the anticholinergic effects of Bacopa monneiri which could be used as an alternative in treatment of Alzheimer’s disease ⁸.

The studies on antioxidant activity of B. monneiri were rather limited and hence this plant is selected and studied for the phytochemical composition and antioxidant properties.

MATERIALS AND METHODS:

Chemicals

Quercitin and 2, 2-diphenyl-1-picrylhydrazyl (DPPH) was purchased from Sigma-Aldrich Chemical Co. (Milwaukee, WI, USA). Sodium carbonate (Na₂CO₃), Sodium phosphate (NaH₂PO₄) was purchased from Himedia Laboratories Pvt. Ltd. (Mumbai, India). Methanol, Ferrozine, Ferrous chloride (FeCl₂), Ferric chloride (FeCl₃), Potassium Ferricyanide (K₃Fe (CN)₆), Trichloroacetic acid, Ascorbic acid, Folin- Ciocalteau reagent, Ethanol, Ascorbic acid, Gallic acid were purchased from SRL Pvt. Ltd. (Mumbai, India). Ammonium molybdate ((NH₄)₂MoO₄) and Aluminium chloride (AlCl₃) were purchased from SD Fine-Chem Chem. Ltd. (Mumbai, India). All other chemicals used were of analytical grade.

Plant material collection and Processing

Bacopa monnieri plant was collected from Alappuzha district (9.54°N 76.40°E) of Kerala, India, in the month of August 2012. The leaves of the plants were separated and the stem alone was taken. The stem was then washed in distilled water and allowed to shade dry at room temperature. The dried stem was then powdered uniformly in a mechanical grinder. About 10 grams of plant material was serially extracted in solvents like petroleum ether, chloroform, methanol and distilled water respectively for 24 hours in a rotary shaker. These extracts were then filtered through Whatman no.1 filter paper and then concentrated at 40°C under reduced pressure (72 mbar) using a rotary evaporator and dried with lyophilizer. Dried extracts were stored at 4°C for further use.

Phytochemical screening

The petroleum ether, chloroform, methanol and aqueous extracts of Bacopa monnieri stem was tested for tannins, flavanoids, saponins, carbohydrates, proteins, oils and fats using the standard protocols ⁹.

Antioxidant studies

DPPH radical scavenging activity

DPPH radical scavenging assay was performed for Petroleum ether, Chloroform, Methanol and Aqueous extracts in a dose dependent manner (20, 40, 60, 80 & 100 µg/ml) in triplicates. To 2ml of each dilution of the extract, 1ml of DPPH solution (0.2mM/ml in methanol) was added and mixed thoroughly and incubated at 20˚C for 40 minutes. The absorbance was measured at 517nm using UV- Visible spectrometer using methanol as the blank¹⁰.

The percentage DPPH radical scavenging activity was measured using the formula:

%DPPH radical scavenging = (Ac-At/Ac) X 100

Where, Ac is the absorbance of the control (DPPH) and At is the absorbance of test

Total antioxidant assay

A volume of 1 ml solution of the crude plant extract at different concentrations was mixed with the reaction mixture containing concentrated sulphuric acid, 0.335 g of sodium phosphate monobasic and 0.495 g of ammonium molybdate. The mixture was kept in water bath for one hour at 95º C. The solution containing 3 ml of reaction mixture and 1 ml of distilled water was used as blank. The absorbance was measured at 695 nm. Experiment was performed in triplicates at each concentration. Ascorbic acid is used as a standard¹¹.

Reducing power assay

The ferric reducing power assay¹² was used to determine the reducing power of the methanol and aqueous extracts of B.monnieri stem. A volume of 1 ml of the aqueous and methanol extract was taken in a dose dependent manner (125, 250, 500 and 1000 μg/ml). It was then mixed with phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and 2.5 ml of 1 % K₃Fe (CN)_6, after which it was incubated at 50˚C for 20 min. A volume of 2.5 ml of Trichloroacetic acid (10%) was added to the mixture, and was centrifuged at 3000 rpm for 10 min. Then, 2.5 ml of the supernatant was mixed with equal volume of distilled water and 0.5 ml FeCl₃ (0.1%). Absorbance was measured at 700 nm using a UV–Visible spectrophotometer. Ascorbic acid was used as positive control. The absorbance of the reaction mixture is directly proportional to the reductive potential of the extracts.

Antilipid Peroxidation (ALP) assay

Anti-lipid Peroxidation assay was performed according to the method of Mandal and Chatterjee, 1980 with few modifications¹³. Different concentrations of crude plant extract (125, 250, 500, 1000 µg/ml) was mixed with 2.8 ml of 10% goat liver homogenate and 0.1ml of 50mM FeSO₄ and incubated for 30 min at 37ºC. A volume of 2 ml of 10% TCA-0.67% TBA in acetic acid (50%)was added to 1ml solution of this extracts, was kept for boiling for 1 h at 100°C and subsequent centrifugation at 10,000 rpm for 5min. Absorbance of the test was recorded at 535 nm against the reagent blank. The control was without the extracts and FeSO₄ and induced without the extract. ALP percentage was calculated using the following formula.

% Antilipid peroxidation= [(Abs of Fe²⁺ induced peroxidation− Abs of sample) / (Abs of Fe²⁺ induced peroxidation − Absorbance of control)] × 100

Quantitative estimation of Total Phenolic content

Different concentrations of crude plant extracts (125, 250, 500, 1000 µg/ml) were prepared in triplicates. To 1ml of each dilution 2.5ml of Follins Ciocalteau reagent was added along with 2ml of 7.5%sodium carbonate. It was then incubated at 45˚C for 15 minutes. The absorbance was measured at 765nm using UV-Visible spectrometer. 4ml of 7.5 sodium carbonate in 5.2ml of distilled water was used as blank¹⁴.

Quantitative estimation of Total flavanoids content

The plant extracts were diluted to make concentrations of 125, 250, 500 and1000 µg/ml in triplicates. To 1ml of each dilution, 1 ml of aluminium chloride (2 grams of Aluminium chloride in 100ml ethanol) was added and incubated at room temperature for 1 hour. The absorbance was measured at 420nm using UV- Visible spectrometer with 3ml of aluminium chloride in 3ml distilled water as the blank¹⁵.

HPTLC Fingerprinting for the identification of polyphenols

HPTLC analysis was performed fallowing the method of Sasikumar et al, 2009¹⁶.To 50mg of aqueous extract of Bacopa monnieri stem, 1 ml of distilled water was added and centrifuged at 3000 rpm for 10 minutes. This solution was used as test sample for HPTLC analysis. About 2µl of test solution was loaded as 5mm band length in the 2 x 10 Silica gel 60F₂₅₄TLC plate using Hamilton syringe and Camag Linomat 5 instrument. For the spot development the plate loaded with samples was kept in TLC twin trough developing chamber (after saturated with Solvent vapor) with respective mobile phase (Polyphenol) and the plate was developed in the respective mobile phase up to 90mm.

For Photo-documentation the developed plate was dried by hot air to evaporate solvents from the plate.The plate was kept in Photo-documentation chamber (Camag Reprostar 3) and captured the images at Day light, UV 254nm and UV366nm. Then the developed plate was sprayed with respective spray reagent (Polyphenol) and dried at 100°C in Hot air oven. The plate was photo-documented in Day light and UV 366nm mode using Photo-documentation (Camag Reprostar 3) chamber.

Finally, before derivatization the plate was scanned at UV 254nm by fixing it on a scanner stage (Camag TLC Scanner 3). The Peak table, Peak display and Peak densitogram were noted using the software win CATS 1.3.4 version.

FT-IR analysis of plant sample

2 mg of the powdered Bacopa monnieri stem sample was mixed with 200 mg KBr (FT-IR grade) and pressed to make into a pellet. This sample pellet was then placed in the sample holder and FT-IR spectra were recorded in the range 4000-450 cm–1 in FT-IR spectroscopy^17,
18.

Statistical analysis

All the values were expressed as mean± standard deviation in triplicates and statistically analyzed using Graph pad prism v.5.00 (La Jolla, CA, USA) and Microsoft Excel 2007 (Roselle, IL, USA)

RESULTS AND DISCUSSION:

Ten grams of the dried stem powder of Bacopa monnieri was extracted serially using petroleum ether, chloroform, methanol and aqueous extracts by rotary shaker method. The yield of plant extract, which is the ratio of fresh mass to dry weight, was determined. The yield of methanol extract (1.5%) was higher compared to aqueous (1.4%), petroleum ether (1.2%) and chloroform extract (0.9%).

Phytochemical Screening

Phytochemical screening was performed to identify the presence of compounds within the plant extract which plays a vital role of various pharmacological activities of the plant. Phytochemical screening revealed the presence of major phytochemicals in the plant. Methanol extract showed the presence of flavonoids, saponins, carbohydrates, tannins, oils and fats. Aqueous extract contained flavanoids, saponins and carbohydrates. Petroleum ether and chloroform extract showed negative result for all the compounds. Results of phytochemical screening were mentioned in the table.1.

Table 1 Phytochemical screening of B. monnieri stem

Phytochemical Test	*B. monnieri* Stem
Phytochemical Test	Hexane	Chloroform	Methanol	Aqueous
Flavonoids	-ve	-ve	+ve	+ve
Saponins	-ve	-ve	+ve	+ve
Carbohydrates	-ve	-ve	+ve	+ve
Tannins	-ve	-ve	+ve	-ve
Oils and fats	-ve	-ve	+ve	-ve
Proteins	-ve	-ve	-ve	-ve

Here, +ve: positive, -ve: negative for the phytochemical tests performed

Antioxidant studies

In current scenario, humans in their busy life were used to junk foods that lack nutrition and were being exposed to pollute environment which are the major factors for free radicals production and cellular damage in the body that results in several diseases. Synthetic antioxidants were not preferred by the consumers because of their undesirable side effects. Hence demand on the usage of natural antioxidants has been increased and many plant products are being used in various disease conditions. In this perspective this study was undertaken to evaluate antioxidant activity of various stem extracts of B. monneiri against different types of free radicals by DPPH radical scavenging activity, Total antioxidant assay, Reducing power assay and Antilipid peroxidation assays. Extracts had shown elevated levels of antioxidant activities in a dose dependent manner.

DPPH radical scavenging assay

DPPH is a stable free radical .The phenolic compounds of the plant extract acts as a proton donor and donates a proton to the DPPH to make it stable, during which it loses its color from purple to yellow¹⁹.The inhibitory constant, IC₅₀was determined. It is the amount of sample required for showing 50% radical scavenging activity and is expressed in µg/ml. Lower the IC₅₀value, greater is the radical scavenging activity. IC 50 value of aqueous extract (5µg/ml) showed higher DPPH radical activity than methanol (49µg/ml), chloroform (85µg/ml) and petroleum ether (114µg/ml) extract. Percentage radical scavenging of the plant in different extracts was given in the figure.1. Aqueous and methanol extracts were showing highest radical scavenging activity in comparison with the previously reported medicinal plants^{20, 21}. Hence aqueous and methanol extracts were selected for further antioxidant studies.

Figure.1. Percentage radical scavenging of different extracts of B. monneiri in a dose dependent manner expressed as Mean± SD (Standard Deviation) in triplicates. PE: Petroleum Ether Extract; CE: Chloroform Extract; ME: Methanol Extract; AE: Aqueous Extract.

Total antioxidant assay

The ammonium molybdate method has been routinely used to evaluate the total antioxidant capacity of the extracts with slight modifications¹¹ .In the presence of the plant extract, the Mo (VI) is reduced to Mo (V) and forms a green colored ammonium molybdenum V complex. The absorbance is measured at 695nm using UV-Visible spectrometry. Aqueous extract showed higher antioxidant activity compared to methanol. Total antioxidant capacity of the extracts is shown in the figure.2.

Figure.2. Total antioxidant capacity of the methanol (ME) and aqueous extracts (AE) in a concentration dependent wise expressed as Mean ± SD in triplicates.

Reducing power assay

The reducing power is an indicator of antioxidant activity. In the experiment, ferric ions are reduced to ferrous ions and the color of the reaction mixture changes from yellow to bluish green. The plant extract acts as an electron donor. The aqueous extracts shows more reducing power activity than the methanol extract which was shown in the figure.3. Therefore aqueous extract was further tested for antilipid peroxidation ability.

Figure.3.Total reducing power assay of methanol (ME) and aqueous extracts (AE) of B. monneiri in a lower to higher concentration expressed as Mean± SD and n=3.

Anti-lipid peroxidation assay

The extracts of liver homogenate undergo rapid peroxidation when incubated with Feso₄and peroxide ²². Addition of Fe ²⁺ to the liver, cause increase lipid peroxidation. Aqueous extract showed inhibition of peroxidation of lipids at all the concentrations tested in a dose dependent manner which was shown in the Figure.4.

Figure.4.Anti-lipid Peroxidation assay of aqueous extract of B. monneiri stems expressed as Mean± SD in triplicates.

Total Phenolic content assay

Since methanol and aqueous extracts of B. monneiri has shown good antioxidant activities and previous reports proof on phenolic compounds in plants as antioxidants¹⁴, the amount of total phenols of aqueous and methanol

extracts were estimated in a dose dependent pattern. It was measured in Gallic acid equivalence. Aqueous extract (66.99mg/GAE/g of extract) showed higher phenol content than methanol extract (53mg/GAE/g) which was shown in the Figure.5.

Figure.5.Total phenolic content of methanol (ME) and aqueous extracts (AE) of B. monneiri stem in a dose dependent manner. Mean± SD; n=3.

Total Flavanoids

Flavonoids are a large class of naturally occurring polyphenolic compounds, with C6-C3-C6 backbone. It is responsible for antioxidant activity. The total flavonoids content of methanol extract and aqueous extract were calculated in terms of Quercitin equivalence. The aqueous extract (79.98mgQE/g of extract) shows higher flavanoids content than methanol extract (73.99mgQE/g of extract) has been given in the figure 6.

Figure.6. Total flavanoids content of methanol (ME) and aqueous extracts (AE) of B. monneiri stem in a dose dependent manner; Mean± SD; n=3.

HPTLC fingerprinting profile

HPTLC profile of aqueous extract of B. monneri has given bluish brown colored zones which were observed in the track by chromatogram after derivatization that indicates the presence of polyphenols in the sample. The peak heights of each polyphenols were given in the table.2.

Table 2. Peak table showing polyphenols in peaks 2, 3,6 respectively

Peak	Rf	Height	Area	Assigned substance
1	0.01	412.9	2983.4	Unknown
2	0.08	88.3	1305.8	Polyphenol 1
3	0.22	36.0	1249.2	Polyphenol 2
4	0.28	27.7	746.4	Unknown
5	0.36	14.0	354.6	Unknown
6	0.56	98.2	4617.9	Polyphenol 3
7	0.65	102.6	4254.8	Unknown
8	0.77	144.4	15731.2	Unknown
9	0.85	105.4	4077.3	Unknown
10	0.95	237.2	9932.3	Unknown

Figure 8: Peak densitogram display (Scanned at 254nm)

Figure 9: 3D display (Scanned at 254 nm)

The FT-IR analysis was carried out to predict the functional groups present in aqueous extract of B. monnieri stems. The resultant spectrum was reported in Figure 10. Absorption band at 3421.72 represent –OH group of phenolic compounds. It confirms the presence of phenolic compounds in the plant.

Figure 10. FT-IR analysis of the plant sample

CONCLUSION:

Antioxidants were a major cure for many of the disorders in the early days. Even after the discovery of many popular drugs the importance of antioxidants still remain. The quantity of total phenols, flavonoids, the intensity of DPPH radical scavenging in the petroleum ether, chloroform, methanol and aqueous extracts were determined. The presence of tannins, flavanoids, saponins, carbohydrates and oils and fats was detected in the methanol extract of the plant by the phytochemical screening. The phenolic content was also obtained by the phytochemical screening of aqueous extracts of Bacopa monnieri which is considered to be responsible for antioxidant activity. In comparision with the methanol extract, aqueous extract has shown the elevated levels of Phenolic (81.01 mg GAE/g extract) and Flavanoids (79.98 mg QE/g extract). The free radical scavenging and antioxidant properties of the extracts may be due to the presence of Polyphenols in the extracts. The investigations on Bacopa monnieri suggests that it posses significant antioxidant activity and hence it can be used as a good source of antioxidant against various diseases caused due to free radicals.

ACKNOWLEDGEMENTS:

The authors are grateful to the Management and Staff of VIT University, Vellore, TN, India for providing their support and encouragement to carry out this study.

REFERENCES:

1. Sivanandham V, Free radicals in health and diseases- A mini review. Pharmacologyonline Newsletter. 1; 2011: 1062-1077.

2. Singh A et al. Effect of Natural and Synthetic Antioxidants in a Mouse Model of Chronic Fatigue Syndrome.Journal of Medicinal Food.5 (4); 2002: 211-220.

3. Shahvar D, Rehman US and Raza AM. Acetyl cholinesterase inhibition potential and antioxidant activites of ferulic acid isolated from Impatiens bicolor Linn. Journal of Medicinal Plants Research. 4(3); 2010: 260-266.

4. Horzic D et al. The composition of polyphenols and methylxantine in teas and herbal infusions. Food Chemistry. 115; 2009: 441-448.

5. Cai YJ et al. Antioxidant effects of green tea polyphenols on free radical initiated peroxidation of rat liver microsomes. Chemistry and Physics of Lipids. 120(1-2); 2002: 109-117.

6. Patil et al. A Review on Bacopa monniera: Brahmi. International Journal of Pharmaceutical Sciences. 3(4); 2012: 3196-3211.

7. Khan R, Krishnakumar A and Paulose CS. Decreased glutamate receptor binding and NMDA R1 gene expression in hippocampus of pilocarpine-induced epileptic rats: neuroprotective role of Bacopa monnieri extract. Epilepsy Behaviour. 12(1); 2008: 54-60.

8. Saraf MK et al. Bacopa monnieri attenuates scopolamine-induced impairment of spatial memory in mice. Evidence. Based Complementary and Alternative Medicine. 7(8); 2011: 23-33.

9. Harborne JB. Phytochemical Methods. Chapman and Hall, London.1973.

10. Brand- Williams W, Cuvelier ME and Berset C. Use of a free radical method to evaluate antioxidant activity. Food Science Technology. 28; 1995: 25-30.

11. Prieto P, Pineda M and Aguilar M. Spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: specific application to the determination of vitamin E. Analytical Biochemistry. 7; 1999: 121-123.

12. Oyaizu M. Studies on products of browning reactions: Antioxidative activities of products of browning reaction prepared from glucosamine. Japanese Journal of Nutrition. 44; 1986: 307-315.

13. Mandal TK and Chatterjee SN. Ultraviolet- and Sunlight-induced Lipid Peroxidation in Liposomal Membrane. Radiation Research. 83; 1980: 290-302.

14. Hazra B, Biswas S and Mandal N. Antioxidant and free radical scavenging activity of Spondias pinnata. BMC Complement Alternative Medicine. 8(63); 2008: 63.

15. Adedapo AA et al. Antioxidant properties of the methanol extract of the leaves and stems of Celtis Africana. Records of Natural Products. 3(1); 2009: 23-31.

16. Sasikumar JM, Jinu U and Shamna R. Antioxidant activity and HPTLC analysis of Pandanus odoratissimus L. root. European Journal of Biological Sciences. 1 (2); 2009: 17-22.

17. Ramamurthy N and Kannan S. Fourier transform infrared spectroscopic analysis of a plant (Calotropis gigantea Linn) from an industrial village, Cuddalore dt, Tamilnadu, India. Romanian Journal of Biophysics. 17(4); 2007: 269-276.

18. Kumar G, Karthik L and Bhaskara Rao KV. Antimicrobial activity of latex of Calotropis gigantia against pathogenic microorganisms- An in vitro study. Pharmacologyonline. 3; 2010: 155-163.

19. Sanchez- Moreno C, Larrauri JA, Saura- Calixto F. Free radical scavenging capacity of selected red rose and white wine. Journal of the Science of Food and Agriculture.79 (10); 1999: 1301- 1304.

20. Kumar G, Karthik L and Bhaskara Rao KV. Phytochemical composition and in vitro antioxidant activity of aqueous extract of Aerva lanata (L.) Juss. ex Schult. Stem (Amaranthaceae). Asian Journal of Tropical Medicine. 6(3); 2013:180-187.

21. Priya et al., Antioxidant activity of Achyranthes aspera Linn stem extract. Pharmacologyonline. 2; 2010: 228-237.

22. Aruma OI. Characterization of drugs as antioxidant prophylactics. Free radical Biology and Medicine. 20 (7); 1996: 675-705.

Received on 05.06.2013 Modified on 30.06.2013

Research J. Pharm. and Tech. 6(9): September 2013; Page 1073-1078