International Journal of Biotechnology and Bioengineering Research. ISSN 2231-1238, Volume 4, Number 7 (2013), pp. 687-696 © Research India Publications http://www.ripublication.com/ ijbbr.htm

Phytochemical Analysis, Free Radical Scavenging and

Antioxidant Profiling Using Chromatographic Techniques for Centella Asiatica

S.S. Desai*, Shruti K, Sana S, Sapna P, Zainab M, Swati H, and B.S. Hungund

Department of Biotechnology, B.V.B.Engineering and Technology, Vidyanagar, Hubli -580031, India.

Abstract Centella asiatica (Family:Apiceae) commonly known as mandukparni is a medicinally important herb. Almost all the parts of this plant are used in traditional medicinal system. It is one of the most important medicinal herb described by Charaka as antiaging plant. It is also called as “Brain food”. Hence this plant was choosen for present study.Various plant parts were extracted using solvents and it was analyzed. Phytochemical analysis of the methanolic extract revealed the presence of Reducing Sugar, Alkaloids, Flavanoids, Phenols, Saponins, Tannins, Anthraquinone, Steroids, Terpinoids and Cardiac glycosides. Thin Layer Chromatography (TLC) was performed using different solvent system for different molecules. Qualitative analysis by TLC for phenolic compounds revealed one band corresponding to Rf of 0.83 which is similar to that of standard gallic acid and for antioxidants one band was observed of Rf 0.63 which was similar to that of standard Vit.C. High Performance Liquid Chromatography (HPLC) fingerprinting for phenols in methanolic extract revealed 3 peaks of which one was similar to standard Gallic acid where as in hexane extract, 9 peaks was observed. Quantitative estimation of total phenol content was 500mg GAE (Gallic Acid Equivalents) /1 g of sample. Free radical scavenging activity as determined using 2,2-Diphenyl-1-picryl-hydrazyl radical (DPPH) indicated IC50 of sample to be 0.55 mg/ml as compared to 0.7 mg/ ml for standard Vit.C. This indicates that plant has high antioxidant properties and hence it has huge potential to be used in treatment of various ailments.

Keywords: Phytochemical, antioxidant, total phenol content, DPPH.

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1. Introduction Phytochemicals found in plants have beneficial effect on health or play active role in curing diseases. Plants have the ability to synthesize many secondary metabolites of which only 10% are identified. These metabolites serve remedy for various alimeties like antimicrobial, anti-inflammatory, antihypertensive anti diabetic ect.(Ayoola et al 2008,Mallikarjun et al 2007). Since these constituents influence human health in beneficial way, analysis of the constituents become important in view of pharmaceutical and health care products.

Certain chronic and degenerative diseases are induced by oxidative stress (Souri et al, 2008). Free radical, one of the factors to cause stress, are generated due to environmental pollutants, radiations, chemicals, toxins, metabolic process especially oxidation, make body cell to deteriorate and cause depletion of innate immunity, change in gene expression, if not used up. Antioxidants take up these free radicals and protect body against pathogenic conditions like anemia, arthritis, inflammation, neruo- degeneration, Parkinsons disease(Oke and Hamburger 2002, Odakoya et al 2006). Hence antioxidant property is important for study.

Antioxidant properties in medicinal plants are attributed to the wide range of amphipathic molecules broadly termed “Polyphenolic compounds”. Phenolic products act mainly due to its redox properties, which make them to act as hydrogen donor, reducing agents and singlet oxygen quencher. They also act as metal chelators (Rice-evans et al 2005) .In addition to pharmaceutical industry, in the food industry also, these phenolic compounds are of interest because they retard oxidative degradation of lipids and thereby improve the quality and nutritional value of food. Natural phenolic compounds are of interest to scientists, food manufacturers, and consumers due to its beneficial health effects (Huda-Foujan et al, 2009).

Due to depletion of Innate antioxidants, it becomes necessary to consume antioxidants as free radical scavenging. Synthetic antioxidants are being replaced by naturally occurring antioxidants, mainly due to low solubility and moderate antioxidant activity(Pourmoraf et al.2006). Hence, there is a demand to explore the antioxidant potential of plant species.Many plant species like Bridelia retusa, Gracilaria changii ect.,. have been investigated for antioxidant potential. Here, an attempt was made to screen the plant Centella asiatica for antioxidants properties. Centella asiatica (Family:Apiceae) commonly known as mandukparni is a medicinally important herb. Almost all the parts of this plant are used in traditional medicinal system.It is one of the most important medicinal herb described by Charaka as antiaging plant. It is also called as “Brain food”(Singh et al 2012). Hence, this plant was used for present investigation.

2. Methodology 2.1 Methods 2.1.1 Extract Preparation Various parts (stems, leaves, petiole) of Centella asiatica were collected randomly and brought to the laboratory in sterile bags. It was stored aseptically. The samples were

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cleaned thoroughly and sun dried for 8-10 days. The dried samples were macerated with methanol and allowed to stand for 48 hours (10 gm in 100 ml methanol) and filtered. The filtrate was then evaporated in hot air oven and extracted using methanol. The extracts were stored in refrigerator at 5°C and used for further analysis.

2.1.2 Phytochemical analysis Phytochemicals were analyzed using standard methods(Ayoola et al 2008, Odakoya et al 2006, Aiyelaagbe and Osamudiamenv 2009, Jack, I.R and Okorosaye-Odrubite 2008).

Reducing sugar Add 1 ml of Fehling’s solution to 100 µl of extract, followed by addition of 3 ml of distilled water and keep in boiling water bath. A brick red precipitate indicates the presence of reducing sugar. Saponin test Add 1 ml of distilled water to 100 µl of extract. Shake vigorously and observe for persistent froth. Mix 3 drops of olive oil. Formation of emulsion indicates the presence of saponins.

Alkaloid test 100 µl of extract was stirred with 3 ml of ethanol containing 3% tartaric acid. The filtrate was shared into 3 test tubes and tested for alkaloids as follows: Into the first test tube, Hagar’s reagent was added, into the second test tube, Mayer’s reagent was added and into the third test tube, Marquin’s reagent was added. Precipitation in any of the 3 test indicates the presence of alkaloids.

Flavonoid test Two gm of powdered sample was completely detanned with acetone. The residue was extracted in warm water after evaporating the acetone in a water bath. The mixture was filtered while, still hot. The filtrate was cooled and used. 5 ml of 20% sodium hydroxide was added to equal volume of the detanned water extract. A yellow solution indicates the presence of flavonoids.

Tannin test 100 µl of extract was added to 3 ml of freshly prepared potassium hydroxide solution and shaken to dissolve. Presence of dirty precipitate indicates the presence of tannin. Cardiac glycoside test 100 µl of extract was dissolved in I ml of glacial acetic acid containing 1 drop of ferric chloride solution. This was under layered with 1 µl of concentrated sulphuric acid. Presence of brown ring at the interface indicates the presence of cardiac glycosides.

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Steroid test About 100 µl of plant extract was dissolved in 2ml of chloroform. Sulphuric acid was carefully added to form a lower layer. A reddish brown color at the interface is indicative of the presence of steroidal ring.

2.2 Thin Layer Chromatograpy for phenols The powdered leaves of Centella was lixiviated in methanol on rotary evaporator for 24h. The condensed filtrate was used for chromatography. chloroform and methanol (27:0.3) solvent mixture was used for analysis of phenols. The colour and Rf values were recorded under visible light after spraying the plates with Folin-Ciocalteu’s reagent heating at 80oC/10min(Mallikarjun et al 2007). TLC for radical scavenging activity was performed using methanol: Chloroform (85:15 v/v) solvent mixture. The plates were developed in an unsaturated chamber. After chromatography, plates were dried and then sprayed by 0.04% DPPH solution for 5 seconds and images were observed under visible light at exactly 2 min after spraying. The area of bright yellow bands against the purple background determined radical scavenging activity (Sreenivasan et al 2007).

2.3 High Performance Liquid Chromatography (HPLC) 2.3.1HPLC instrumentation An isocratic HPLC (Aglient 1200 Infinite series) with quaternary pumps, variable wavelength programmable detector and a reverse phase Zorbex C18 column (250 mm x 4.6 mm) was used. The HPLC system was equipped with software chemstation. The mobile phase components acetonitrile: water (1:3) were filtered through 0.2 µm membrane filter before use and pumped from the solvent reservoir to the column at a flow rate 1 mL/min which yielded a column backpressure of 80-100 bar. The column temperature was maintained at 37oC. 20 µL of sample was injected using Rheodyne syringe (Model 7202, Hamilton). Peak area was determined(Mallikarjun et al 2007).Gallic acid (0.1%) was used as standard.

2.4 Total phenolic assay The amount of total phenolics in Centella extracts was determined by Folin-Ciocalteu reagent with some modifications. To 1 ml of each sample, 2.5 ml 1/10 dilution of Folin-Ciocalteau’s reagent and 2 ml of Na2CO3 (7.5%, w/v) were added and incubated at 45oC for 15 min. The absorbance of all samples was measured at 750 nm using a UV–Vis spectrophotometer (Elico India Ltd. ). Results were expressed as milligrams of gallic acid equivalent per gram of dry weight (mg GAE/1g) (Marinova et al 2005, Banerjee and Bonde2011).

2.5 Antioxidant activity by radical scavenging effect of DPPH (2,2-Diphenyl-1-Picryl Hyrazyl) The antioxidant activity of the plant extracts and the standard was assessed on the basis of the radical scavenging effect of the stable 1, 1-diphenyl-2-picrylhydrazyl (DPPH)-

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free radical activity (Sreenivasan et al 2007) 12. The reaction mixture contained 50 μl of test samples and 5 ml of a 0.04% (w/v) solution of DPPH in methanol. Ascorbic acid (1.0-25.0 mg/ml) was used as standard. These solution mixtures were incubated for 30 min and optical density was measured in spectrophotometer at 517 nm. Methanol (100µl) with DPPH solution was used as blank. The optical density was recorded and % inhibition was calculated using the formula given below:

Percent (%) inhibition of DPPH activity = Ao – A1 / Ao * 100 Where Ao was the absorbance of the control and A1 was the absorbance in the

presence of the sample. The actual decrease in absorption induced by the test compounds was compared with the positive controls. IC50 value was calculated using the dose inhibition curve. IC50 values denote the concentration of sample, which is required to scavenge 50% of DPPH free radicals.

3. Results and Discussion 3.1 Phytochemical screening of plant materials The phytochemical screening of methanolic extracts of Centella are shown in Table 1. The presence of some of the phtyochemicals has been reported earlier (Singh et al 2012, Arumugam et al 2011). However, it was observed by Arumugam et al 2011) that methanolic extract of leaf indicated presence of alkaloids, steroids, tannins flavanoids, reducing sugars and absence of saponins where as Sing et al 2012 observed the presence of saponins, alkaloids, glycoside, reducing sugar and absence of flavanoid and tannins. Leaf methanolic extracts of plant indicates presence of almost all phytochemicals except for sugars, tannins, flavones and coumarins(Samy et al 2011) . Some of our results are not in agreement with previous litratures which may be because of environmental conditions or the growth conditions of the plants in various areas.

Table 1: The phytochemical screening of the crude methanolic extracts of Centella.

Sl. No. Test Result

1 Alkaloids Positive 2 Flavonides Positive 3 Phenols Positive 4 Saponins Positive 5 Reducing sugars Positive 6 Steroids Positive 7 Tannins Positive 8 Terpenoid Positive 9 Anthraquinone Positive

10 Cardiac Glycosides Positive

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3.2 Thin Layer Chromatography and High Performance Liquid Chromatography analysis and phenol content estimation The thin layer chromatography for phenols using methanolic extract of centella in the solvent system gave a Retentinon factor (Rf) value of 0.83 which is similar to that of standard gallic acid. Also, Rf of 0.63 was obtained for vit.C which was similar to that of sample indicating the presence of antioxidants. Similar Rf value was observed by Sreenivasan et al (2007).

HPLC fingerprinting is best way for chemical characterization and therefore it was used for analysis using gallic acid as standard. The retention time for standard gallic acid (1mg/ml) was 0.891 with the peak area was 99.389% (Fig.1). HPLC analysis indicated 3 major peaks in methanolic extract of which one was similar to that of standard (Fig.2 Table 2) and 8 peaks in hexane extract (Fig.3 Table 3 ). Similar peak was observed for methanolic extract of plant indicating presence of gallic acid. This study of HPLC fingerprint indicates the presence of phenolic compounds that can act as antioxidant, antifungal, antibacterial, anti-inflammatory or other properties.

Figure 1: HPLC- chromatogram for gallic acid standard.

Figure 2: Chromatogram of Centella asiatica methanolic extract.

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Table 2: HPLC profile of Phenols (methanolic extract of Centella asiatica).

Peak No. Ret Time Area (%) 1 0.893 52.7784 2 1.307 14.4960 3 1.999 29.1864

Figure 3: HPLC Chromatogram for Hexane extract of Centella asiatica.

Table 3: HPLC profile of Phenols (hexane extract of Centella asiatica).

Peak No. Retention Time Area (%) 1 1.416 2.5840 2 1.526 7.1889 3 1.545 8.1648 4 1.645 8.4029 5 1.761 3.0897 6 1.870 5.4870 7 2.010 20.0808 8 2.195 10.4931 9 2.297 34.5087

Since, plant phenolics are a major compounds acting as primary antioxidants or

free radical scavengers(Demiray et al 2009). Therefore, it was reasonable to determine the total phenolic content in the plant extract. Total phenol content was 500 mg GAE/1 g of sample where as Hamid et a 2003 observed that the maximum phenol content was 117mg/g of leaf sample and in pegaga biscuits it was 620µg/g as observed by Rahmat et al 2011.

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3.3 Free radical scavenging activity Free radical scavenging activity as determined using 2,2-Diphenyl-1-picryl-hydrazyl radical (DPPH) indicated IC50 of sample to be 0.55 mg/ml as compared to for 0.7 mg/ ml for standard Vit C. Where as Barbosa et al 2009 observed that aqueous extract of Centella showed a high antioxidant activity, with an IC50 value of 31.25 μg/mL and for standards like ascorbic acid and butylated hydroxytoluene (BHT), IC50 values of 2.50 μg/mL and 7.58 μg/mL, respectively.

In the assay of DPPH, a molecule of DPPH is characterized as a stable fee radical by virtue of delocalization of the spare electron and hence gives a strong absorption band at 517 nm. When this electron becomes paired off, in the presence of free radical scavenger, the absorption reduces and solution is decolorized, the degree of reduction in absorbance measures the antioxidant power of the extract.

When a solution of DPPH is mixed with that of a substance that can donate a hydrogen atom, then this gives rise to the reduced form with the loss of this violet colour. Representing the DPPH radical by Z *and the donor molecule by AH, the reaction is

Z *+ AH = ZH + A* where ZH is the reduced form and A* is free radical produced in this first step.

This latter radical will then undergo further reactions which control the overall stoichiometry, that is, the number of molecules of DPPH reduced (decolorised) by one molecule of the reductant (Molyneux 2004).

The degree of decolorization by plant sample was recorded and IC50 was evaluated using curve(Fig. 4).IC50 value for the plant sample was less compared to that of standrard used (Vit.C) indicating that the plant has high antioxidant acitivity.

Figure 4: Dose inhibition curve and IC50 values of the crude extract of Centella asiatica.

Several studies show a very strong correlation between antioxidant activity and

phenolic compounds. Since Phenolic compounds are effective hydrogen donors, they are good antioxidants. Typical phenolics that possess antioxidant activity have been characterized as phenolic acids and flavonoids [Marinova et al 2005). Phenolic acids

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have repeatedly been implicated as natural antioxidants in fruits, vegetables, and other plants. Thus, therapeutic properties of the Centella asiatica can be attributed to the phenolic compounds present.

4. Conclusion In conclusion, the result of the present study suggest that Centella asiatica plant have various secondary metabolites and has good quantity of total phenols. Plant has potent free radical scavenging activity. HPLC analysis indicated presence of other metabolites also. However, we do not know what components in the plant extracts show these activities. Detailed studies on chemical composition, isolation of active constituents and pharmacological evaluation are essential to characterize them as biological antioxidants .The present findings of this study support the view that Centella asiatica is a promising source of potential antioxidant which can be used in treatment of various ailments.

5. Acknowledgement Authors would like to acknowledge Dr. Ashok Shettar, Principal, B.V.B.C.E.T, Hubli for his continuous support and encouragement. References

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