Int J Pharm Pharm Sci, Vol 7, Issue 6, 225-230Original Article


QUANTIFICATION OF PHYTOCHEMICAL CONTENTS AND IN VITRO ANTIOXIDANT ACTIVITY OF EXACUM BICOLOR (ROXB.), AN ENDEMIC MEDICINAL PLANT

APPAJI M ASHWINI, MALA MAJUMDAR*

Department of Biotechnology, Centre for Post Graduate Studies, Jain University, # 18/3, 9th Main, Jayanagar, 3rdBlock, Bangalore, Karnataka, 560011, India
Email: malamajumdar51@gmail.com

Received: 16 Feb 2015 Revised and Accepted: 10 Mar 2015

ABSTRACT

Objective: To investigate different phytochemical contents and screening of antioxidant activity of sequentially extracted Exacum bicolor leaf with different solvents.

Methods: Leaf samples of E. bicolor were dried and subjected to sequential (hexane>chloroform>ethyl acetate>methanol>water) extraction using soxhlet apparatus. Total phenol contents (TPC) were estimated using Folin-ciocalteau method, flavonoids by aluminium chloride method, tannins by Folin-Denis reagent and alkaloids using bromocresol blue. In vitro antioxidant activities were estimated by using 1,1-Diphenyl-2-picrylhydrazyl (DPPH), 2,2’-azinobis-3-ethylbenzothiazoline-6-sulfonic acid ABTS·+and Nitric oxide (NO) scavenging assay.

Results: The total phenol, tannin and alkaloid contents in methanol extract were found to be 23.4±0.09 mg GAE/g, 23±0.57 mg TAE/g and 47.33±1.20 mg AE/g respectively. Hexane extract exhibited high flavonoid content (27.6±0.88 mg QE/g). Methanolic extracts exhibited dose dependent antioxidant activity against DPPH, ABTS·+and NO Scavenging methods; low IC50 value (14.84 µg/ml) was observed in DPPH scavenging activity. Findings showed positive and significant (P ≤ 0.05) correlation between TPC/DPPH (R2= 0.98).

Conclusions: The methanol extract showed higher antioxidative activity compared to other extracts. Positive correlation was obtained between TPC and DPPH activity signifying that the phenolic content may be responsible for the antioxidant activity of the plant. Further bioassay-guided purification of the bioactive compound is necessary. To the best of our knowledge, antioxidant activity and its correlation with TPC, is reported for the first time in E. bicolor.

Keywords: Exacum bicolor, DPPH, ABTS·+, NO scavenging assay, Correlation.

INTRODUCTION

Medicinal plants are the most important source of life saving drugs for the majority of the world’s population as they are having little or no side effects and also they are economical [1]. Plants are the important source for free radical scavenging molecules. Free radicals are highly reactive particles with an unpaired electron and are produced by radiation or as by-products of metabolic processes that lead to disintegration of cell membranes and cell compounds [2].

A serious imbalance between the production of free radicals and antioxidant defence system is responsible for oxidative stress. Oxidative stress is related to ageing process and some chronic diseases such as diabetes, cancer, cardiovascular diseases and inflammatory diseases [3]. Synthetic antioxidants, such as butylated hydroxy toluene (BHT) and butylated hydroxy anisole (BHA), have been widely used as antioxidants and may be responsible for side effects such as liver damage and carcinogenesis [4]. Therefore, the search for alternative source of natural antioxidant compound has gained importance nowadays.

During extraction, the solvent polarity plays a major role determining both quality and quantity of the extracted antioxidant compounds [5]. Higher the polarity, better the extraction of phenolic compounds which might be a major contributor for antioxidant activity [6]. Therefore, the investigation on phytochemicals and antioxidant activity from different non-polar and polar solvents will help to understand their importance and give information on rationale design or search for the new type of more potent bioactive compound helping in the treatment of oxidative stress related diseases.

E. bicolor Roxb. (Gentianaceae) is endemic to peninsular India [7] and now considered as endangered species [8]. They are extensively used locally in curing human ailments such as diabetes [9], antifungal [10], urinary, skin disorders [11], inflammation, purifies blood in treatment of malaria [12], paste is used against sores of leech bites [13], used as tonic for stomachic [14], febrifuge [15] also possess anthelmintic activity [16]. The plants possess secoiridoids, gentiopicroside and methylgrandifloroside [17]. As per literature survey, there are no reports of antioxidant activity of E. bicolor. Hence, E. bicolor has been selected in the present study for evaluating its antioxidant activity.

MATERIALS AND METHODS

Chemicals and reagents

Organic solvents (hexane, chloroform, ethyl acetate, methanol) SRL, Folin-Ciocalteu reagent (FC), 2,2-diphenyl-1-picrylhydrazyl radical (DPPH); 2,2’-azi-no-bis (3-ethylbenzthiazoline-6-sulphonic acid ABTS·+, Gallic acid, Ascorbic acid, Quercetin from Sigma–Aldrich (Sydney, Australia). Shimadzu 1800 UV Visible spectrophotometer was used for the photometric study.

Plant collection

Whole plant of E. bicolor was collected from Kumar Parvatha located near the famous shrine of Lord Subramanya at Kukke Subramanya in Sullia taluk, at 12 ° 33' 53'' N 75 ° 23' 11'' E of South Canara district, Karnataka, during the month of September, 2012. The plant specimen was identified and authenticated by Regional Research Institute Bangalore, India [Accession no: SMPU/MADRI/BNG/2010-11/557]. The voucher specimen was deposited in Biotechnology Department of Jain University, Bangalore.

Preparation of plant extract

Twenty grams of the dried and powdered leaf samples were subjected to sequential [Hexane (H)>Chloroform (C)>Ethyl acetate (EA)>Methanol (M)>Water (W)] extraction using the Soxhlet apparatus. The resulting extracts were then evaporated to dryness using the rotary evaporator and stored at 4 °C for further use.

Estimation of total phenolic content

The total phenolic content in different solvent extracts was estimated using Folin-Ciocalteu’s (FC) method with minor modifications [18]. Different concentrations of extracts were mixed with 0.5 ml FC reagent (diluted 1:10 v/v) and 5 min later 1 ml of sodium carbonate (20%) solution was added and allowed to stand in dark for 30 min at room temperature. Absorbance of sample was measured against the blank at 765 nm using UV-VIS spectrophotometer. Quantification was done on the basis of the standard curve of gallic acid. Total phenolic content was expressed as mg gallic acid equivalent (GAE) per gram of extract.

Estimation of flavonoids

Flavonoids were determined using aluminium chloride colorimetric method according to Chang et al. [19] with some modifications. An aliquot of 1 ml of leaf extracts were mixed with 0.5 ml of 5% sodium nitrite, after 5 min 0.5 ml of 10% aluminium chloride was added and the absorbance was measured at 420 nm. Flavonoid contents were determined from quercetin standard curve and were expressed as mg Quercetin equivalent (QE) per gram of extract.

Estimation of tannins

Amount of tannins in the leaf extracts were determined by Folin Denis method [20] extracts of various concentrations 1 ml mixed with 0.5 ml Folin-Denis reagent and after 5 min 1 ml of sodium carbonate solution was added. Absorbance of sample was measured against the blank at 700 nm. Tannic acid was used as standard the results were expressed as mg tannic acid equivalents (TAE) per gram of extract.

Estimation of alkaloids

Alkaloids were estimated [21] using 100 mg of extract dissolved in 2 N HCl and filtered. 1 ml of the filtered extract was taken and washed with chloroform in a separating funnel by adjusting the pH to 7.5 ml of BCG solution and 5 ml of phosphate buffer were also added. The mixture was shaken and complex extracted with chloroform by vigorous shaking, the extract was then collected in a 10 ml volumetric flask and diluted with chloroform. The absorbance of the complex in chloroform was measured at 470 nm in UV-Spectrophotometer against the blank. The results were expressed as mg Atropine equivalents (AE) per gram which was used as standard.

Antioxidant assay

1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity

Free radical scavenging activity of the different concentration of E. bicolor leaves extracts was determined by the method of Braca et al. [22] with little modifications. The reaction mixture (3 ml) consists of 1 ml of methanolic plant extract at different concentrations and 2 ml of 0.002% of methanolic solution of DPPH. Absorbance at 517 nm was determined after 30 min incubation in dark. Ascorbic acid was used as standard. The percentage inhibition activity was calculated by using the equation:

% scavenging activity= [(A0-A1)/A0] × 100

Where A0 is the absorbance of the control and A1 is the absorbance of the extract.

ABTS·+ [2, 2’-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)] radical cation scavenging method

ABTS radical cation (ABTS·+) decolorization assay was determined with minor modifications [23]. ABTS·+was produced by reacting ABTS solution (2 mM) with 17 mM potassium persulfate and the mixture was allowed to stand in dark at room temperature for 12-16 h before use. 2 ml of ABTS reagent was added to different concentration of extracts. Ascorbic acid was used as standard. The absorbance was read at 734 nm and %Inhibition was calculated using following formula.

% scavenging activity= [(A0-At)/A0] × 100

Where A0 is the absorbance of the control and At is the absorbance of the extracts.

NO scavenging activity

NO scavenging activity was performed according to Sreejayan and Rao [24] with little modification where, sodium nitroprusside (5 mM) in phosphate buffered saline was mixed with different concentrations of the E. bicolor extracts and incubated at 25 °C for 30 min. The incubated solutions were diluted with equal volume of Griess reagent (1% sulphanilamide, 2% phosphoric acid, and 0.1% N-(1-Naphthyl)ethylenediamine dihydrochloride). The absorbance of the chromophore formed were measured at 546 nm. The NO radical scavenging activity was expressed as the inhibition percentage (%I) and calculated as per the equation:

%I =[(Ablank-Asample)/Ablank ]×100

Where Ablank is the absorbance of the control reaction and Asample is the absorbance of the test compound with all reagents. Ascorbic acid was used as standard.

IC50 value of the extracts

The half maximal inhibitory concentration (IC50) value of each extract was determined, plotting the percentage of scavenging activity versus the concentration of extract using nonlinear regression analysis (curve fit). Lower the IC50 value, higher the radical scavenging effect.

Statistics

All analyses were performed in triplicate (n=3) using one way ANOVA and the data were presented as mean±SE. Graph Pad Prism for Windows version 7 (Graph Pad version 6 Software, San Diego, CA, USA) was used for all statistical analyses and IC50 determinations. P values ≤ 0.05 were considered to be significant.

RESULTS

Total Phenolic contents, Flavonoids, Tannins and Alkaloids

The amount of total phenols, flavonoids, tannins and alkaloids present in the sequential extracts of E. bicolor are shown in table 1. The methanolic extract recorded the highest amount of total phenols (23.4±0.09 mg GAE/g), tannins (23±0.57 mg TAE/g) and alkaloids (47.33±1.20 mg AE/g). Whereas the flavonoids were present in high amount in hexane (27.66±0.88 mg QE/g) extract.

Table 1: Total phenols, flavonoids and alkaloid content

Extracts

Total phenols

(mg GAE/g)

Flavonoids

(mg QE/g)

Tannins

(mg TAE/g)

Alkaloids

(mg AE/g)
H 3.6±0.04 27.66±0.88 2.33±1.52 8.66±0.88
C 7.5±0.12 21.33±0.88 12.33±1.52 15.0±0.57
EA 20.1±0.09 8.36±0.14 15.33±0.57 22.33±1.45
M 23.4±0.09 6.40±0.11 23.00±1.0 47.33±1.20
W 6.9±0.10 4.63±0.08 18.33±0.57 16.0±0.008

All values are expressed as mean±standard error (n=3). GAE=Gallic acid equivalents, QE=Quercetin equivalents, AE=Atropine equivalents H: Hexane, C: Chloroform, EA: Ethyl acetate, M: Methanol, W: Water extracts of E. bicolor.

DPPH radical scavenging activity

DPPH radical scavenging activity of hexane, chloroform, ethyl acetate, methanol and water extracts of E. bicolor are reported in fig. 1. All the extracts showed potent DPPH radical scavenging activity in concentration dependent manner. Among the solvent tested, the methanolic extract of E. bicolor leaves exhibited a maximum DPPH scavenging activity of (93.58±0.47%) at 100 µg/ml.

Fig. 1: DPPH radical scavenging assay
STD: Standard (Ascorbic acid), H: Hexane, C: Chloroform, EA: Ethyl acetate, M: Methanol, W: Water extracts of E. bicolor

ABTS ·+assay

ABTS scavenging assay, the E. bicolor leaves extract showed dose dependent effect. The %inhibition of methanolic extract was 82.68±1.14 µg/ml and was highest compared to all other extracts at 100 µg/ml.

Fig. 2: ABTS assay
STD: Standard (Ascorbic acid), H: Hexane, C: Chloroform, EA: Ethyl acetate, M: Methanol, W: Water extracts of E. bicolor

NO scavenging assay

NO scavenging assay exhibited concentration dependent increase in %inhibition among all the extract, where the maximum %inhibition of the extracts in methanolic extract of about 77.19±2.32% at 100 µg/ml compared to the other extracts.

IC50 values

IC50 values of hexane, chloroform, ethyl acetate, methanol and water extracts of E. bicolor for DPPH radical scavenging activity were found to be 192.5 µg/ml, 73.54 µg/ml, 38.13 µg/ml, 14.84 µg/ml and 28.45 µg/ml respectively. In ABTS assay the IC50 values of hexane, chloroform, ethyl acetate, methanol and water extracts of E. bicolor were 258.2 µg/ml, 197.4 µg/ml, 107.1 µg/ml, 34.33 µg/ml, and 66.91 µg/ml respectively. NO scavenging assay exhibited IC50 values for hexane, chloroform, ethyl acetate, methanol and water extracts such as 363.1 µg/ml, 289.0 µg/ml, 228.3 µg/ml, 34.03 µg/ml and 72.48 µg/ml respectively (table 2).

Fig. 3: Nitric oxide scavenging assay
STD: Standard (Ascorbic acid), H: Hexane, C: Chloroform, EA: Ethyl acetate, M: Methanol, W: Water extracts of E. bicolor


Table 2: IC50 values of different sequential extracts and standard (µg/ml)

Extracts DPPH ABTS NO
H 192.5 258.2 363.1
C 73.54 197.4 289.0
EA 38.13 107.1 228.3
M 14.84 34.33 34.03
W 28.45 66.91 72.48
STD 5.95 15.21 55.87

STD: Standard (Ascorbic acid), H: Hexane, C: Chloroform, EA: Ethyl acetate, M: Methanol, W: Water extracts of E. bicolor

Correlation of antioxidant activities with phenols, flavonoids and tannins contents

All the antioxidant activities showed positive and significant (P ≤ 0.05) correlations with total phenol content, flavonoids and tannins of methanolic extracts (fig. 4).

Fig. 4: The Correlation coefficient (R2) values of all the different concentration of extracts between Total phenol contents (TPC) and antioxidant activities are tabulated
A: DPPH-Phenols; B: DPPH-Flavonoids; C: DPPH-Tannins; D: ABTS·+-Phenols; E: ABTS·+-Flavonoids; F: ABTS·+-Tannins; G: NO-Phenols; H: NO-Flavonoids; I: NO-Tannins

DISCUSSION

Phenolic compounds are widely distributed in plants which have gained much attention because of their possible beneficial antioxidative effects on human health to cure various diseases such as diabetes, cancer and other degenerative conditions [25]. In the present study, the methanolic extracts of E. bicolor leaves revealed high phenols, tannins and alkaloids (23.4±0.09 mg GAE/g, 23±0.57 mg TAE/g and 47.33±1.20 mg AE/g) respectively. Maximum amount of flavonoids (27.6±0.88 mg QE/g) were observed in the hexane extract. Earlier findings revealed that in Swertia chirayita, the leaf extract possess total phenols and flavonoid content as 67.53±2.08 mg/g GAE and 60.30±2.46 mg/g QE respectively. Misra et al. [26] has reported that the secondary metabolites are responsible for the antioxidant activity of the plants.

DPPH is a stable free radical containing an odd electron having a characteristic absorption at 517 nm. The deep purple colour usually gets decolorized when exposed to antioxidant in the solution. Lower the absorption, higher the radical scavenging activity of the extract [27]. Current study revealed that E. bicolor exhibited dose-dependent inhibition of DPPH radical scavenging activity. It was observed that the methanolic extract showed significantly higher activity compared to other extracts. However, ascorbic acid which was used as positive control showed highest inhibition at the same concentration (100 µg/ml). IC50 values of hexane, chloroform, ethyl acetate, methanol and water extracts of E. bicolor for DPPH radical scavenging activity were found to be 192.5 µg/ml, 73.54 µg/ml, 38.13 µg/ml, 14.84 µg/ml and 28.45 µg/ml respectively (table 2).

The free radical scavenging ability of E. bicolor was also determined using ABTS•+radical cation. ABTS•+radical cation has been used for the evaluation of antioxidant activity of single compounds and complex mixtures. In this assay ABTS•+radical cation was generated directly in stable form using potassium per sulphate. Generation of radical before the antioxidants added prevents interference of compounds, which affect radical formation [28]. When stable absorbance is obtained the antioxidant sample is added to the reaction medium, and the antioxidant activity is measured in terms of decolourization. The IC50 values of hexane, chloroform, ethyl acetate, methanol and water extracts of E. bicolor were 258.2 µg/ml, 197.4 µg/ml, 107.1 µg/ml, 34.33 µg/ml and 66.91 µg/ml respectively.

NO is a reactive oxygen species which is associated with inflammation, cancer and other pathological conditions [29]. The ability of the extracts to scavenge NO may be considered advantageous for health as it can evade the ill effects of excessive NO generation. In E. bicolor the methanolic extract exhibited higher antioxidant activity than the other extracts with IC50 values for hexane, chloroform, ethyl acetate, methanol and water extracts were 363.1 µg/ml, 289.0 µg/ml, 228.3 µg/ml, 34.03 µg/ml and 72.48 µg/ml respectively.

The low EC50 value (27.70 µg/ml) of methanolic extract of Swertia chirayita exhibited effective antioxidant activity [30]. In Swertia longifolia the IC50 value of methanolic aerial part extracts showed 2.8 µg/ml [31]. In the present study, the methanolic leaf extract of E. bicolor showed low IC50 values for the three antioxidant assays exhibiting potent antioxidant activity compared to other extracts. Hence the methanolic extract of E. bicolor extract could be a good source of antioxidant.

The significant linear correlation (coefficient R2≥ 0.98and two-tailed p value ≤ 0.05) between the total phenolic content and DPPH activity of methanolic plant extracts concluded that the phenolic compounds might be responsible for the antioxidant activity [32].

CONCLUSION

In the present study the nonpolar to polar solvent systems showed difference in the total phenol content and antioxidant activity of E. bicolor. The polar solvents are more prominent than the nonpolar one in the present report, the former one can be a potential source of novel bioactive compounds for various stress related diseases. There was a significant positive correlation between total phenol content and DPPH activity, which signifies that the phenols may be the principal contributor of the antioxidant activities. To the best of our knowledge, antioxidant activity and its correlation with TPC, is reported for the first time in E. bicolor. Further investigations are required to purify the bioactive compounds and also to explore the pharmacologic and therapeutic potentials of the plant.

CONFLICT OF INTERESTS

We declare that we have no conflict of interest

ACKNOWLEDGEMENT

The authors gratefully acknowledge the authorities of Centre for Post Graduate Studies, Jain University, Bangalore and also the first author thank Jain University for providing the laboratory facilities and financial support.

CONFLICT OF INTERESTS

Declared None

REFERENCES

  1. Rangari VD. Pharmacognosy and phytochemistry. 1st ed. Nashik: Carrier Publication; 2002.
  2. Kaminidevi S, Thangavelu T, Lakshmanan A, Gunashekar D, Chirayil, Jamuna HT, et al. Preliminary phytochemical screening and antimicrobial activity of fresh plant extract of Indian folk medicinal plant, Gnaphalium polycaulon. Int J Phytomed 2014;6(1):82-6.
  3. Shanmugapriya K, Udayabhanu J, Thayumanavan T. Investigation of antioxidant activity in different solvents of Gnaphalium polycaulon. Int J Pharm Pharm Sci 2014;6(2):870-3.
  4. Grice HC. Safety evaluation of butylated hydroxyl toluene (BHT) in liver, lung and gastrointestinal tract. Food Chem Toxicol 1988;24:1127-30.
  5. Franco D, Sineiro J, Rubilar M, Sánchez M, Jerez M. Polyphenols from plant materials: extraction and antioxidant power. Electron. J Environ Agric Food Chem 2008;7(8):3210-6.
  6. Naczk M, Shahidi F. Phenolics in cereals, fruits and vegetables: Occurrence, extraction and analysis. J Pharm Biomed Anal 2006;41(5):1523-42.
  7. Brilliant R, Vincy MV, Joby P, Pradeepkumar AP. Vegetation analysis of Montane forest of Western Ghats with special emphasis on RET species. Int J Biodiversity and Conserv 2012;4(15):652-64.
  8. Sreelatha U, Baburaj TS, Kutty NC, Nazeem P, Bhaskar J. Cultivation prospects of Exacum bicolor Royle: an endangered, ornamental & anti-diabetic herb. Nat Prod Rad 2007;6(5):402-4.
  9. Marles RJ, Farnsworth NR. Antidiabetic plant and their active constituents. Phytomed 1995;2:137-9.
  10. Khare CP. Indian medicinal plants-an illustrated dictionary. Berlin: Springer; 2007. p. 77.
  11. Reddi STV, Naidu BVAR, Prasanthi S. In: Herbal remedies for diseases, Alikhan and A Khanum 1st ed. Hyderabad: Ukay Publications; 2005. p. 67-13.
  12. Megoneitso, Rao RR. Ethnobotanical studies in Nagaland medicinal plants used by the angami nagas. J Economic Taxonomic Botany 1983;4:167-72.
  13. Dey A, De JN. Traditional use of medicinal plants as febrifuge by the tribals of Purulia district, West Bengal, India. Asia Pac J Trop Dis 2012;2(2):800-3.
  14. Siddamallayya N, Yasmeen A, Gopakumar K. Medico-botanical survey of Kumara parvatha Kukke Subramanya. Indian J Traditional Knowledge 2010;9(1):96-9.
  15. Dey A, Gupta B, De JN. Traditional phytotherapy against skin diseases and in wound healing of the tribes of Purulia district, West Bengal, India. J Med Plant Res 2012;6(33):4825-31.
  16. Ashwini AM, Majumdar M. Qualitative phytochemical screening and in vitro anthelmintic activity of Exacum bicolor Roxb., an endemic medicinal plant from Western Ghats in India. Acta Biol Indian 2014;3(1):510-4.
  17. Das S, Barua RN, Sharma RP, Baruah JN, Kulanthaivela P, Herza W. Secoiridoids from Exacum tetragonum. Phytochem 1984;23:908-9.
  18. Singleton VL, Rossi JA. Colorimetry of phenols with phosphomolybdic–phospotungstic and reagents. Am J Enol Vitic 1965;37:144-8.
  19. Choi CW, Kim SC, Hwang SS, Choi BK, Hye J. Antioxidant activity and free radical scavenging capacity between Korean medicial plants and flavonoids by assay-guided comparison. Plant Sci 2002;163(6):1161-8.
  20. Polshettiwar SA, Ganjiwale RO. Spectrophotometric estimation of total tannins in some ayurvedic eye drops. Indian J Pharm Sci 2007;69(4):574-6.
  21. Shamsa F, Monsef H, Ghamooshi R, Verdian-rizi M. Spectrophotometric determination of total alkaloids in some Iranian medicinal plants. Thai J Pharm Sci 2008;32:17-20.
  22. Braca A, Politi M, Sanogo R, Sanou H, Morelli I, Pizza C, et al. Chemical composition and antioxidant activity of phenolic compounds from wild and cultivated Sclero caryabirrea (Anacardiaceae) leaves. J Agric Food Chem 2003;51:6689–95.
  23. Katalinic V, Milos M, Kulisic T, Jukic M. Screening of 70 medicinal plant extracts for antioxidant capacity and total phenols. Food Chem 2006;94(4):550–7.
  24. Sreejayan N, Rao MNA. Nitric oxide scavenging by curcuminoids. J Pharm Pharmacol 1997;49:105-7.
  25. Pandey KB, Rizvi SI. Plant polyphenols as dietary antioxidants in human health and disease. Oxid Med Cell Longevity 2009;2(5):270-8.
  26. Misra TK, Saha A. Soil sampling in tea plantation for fertility evaluation: A guideline. Assam Rev Tea News 2008;97:12-5.
  27. Ahmad N, Mukhtar H. Green tea polyphenols and cancer: biological mechanisms and practical implications. Nutr Rev 1999;57(3):78-83.
  28. Sanchez-Moreno C. Methods used to evaluate the free radical scavenging activity in foods and biological systems. Food Sci Tech Int 2002;8(3):121–37.
  29. Chen YH, Chang FR, Lin YJ, Hsieh PW. Identification of antioxidants from rhizome of Davallia solida. Food Chem 2008;107:684-91.
  30. Sharma N, Varshney VK, Kala RP, Bisht B, Sharma M. Antioxidant capacity and total phenolic content of Swertia chirayita (Roxb. ex Fleming) H Karst. In Uttarakhand. Int J Phar Sci Rev Res 2013;23(1):259-61.
  31. Hajimehdipoor H, Esmaeili S, Shekarchi M, Emrarian T, Naghibi F. Investigation of some biological activities of Swertia longifolia Boiss. Res Pharm Sci 2013;8(4):253-9.
  32. Jaishee N, Chakraborty. Evaluation of In vitro antioxidant activities of Pteris biaurita L. Int J Pharm Pharm Sci 2014;6(2):413-21.