PHYTOCHEMICAL SCREENING AND IN VITRO ANTIOXIDANT POTENTIALS OF EXTRACTS OF TEN MEDICINAL PLANTS USED FOR THE TREATMENT OF DIABETES MELLITUS IN SRI LANKA

Background: Over the last decade, extensive research work has focused on the potential health benefits of antioxidants while many medicinal plant extracts have been evaluated for their antioxidant profile. Medicinal plants selected for this study are widely used in traditional medicine for the treatment of diabetes mellitus in Sri Lanka some of which are recommended as dietary supplements to the existing therapies. The present study aimed at determining the total polyphenol contents and total antioxidant activities of aqueous extracts of 10 selected Sri Lankan medicinal plants by three in vitro methods; Materials and Methods: DPPH(2, 2-diphenyl-2-picrylhydrazyl), FRAP (ferric reducing power) and NO (nitric oxide) assays. The aqueous plant extracts were tested at the concentration of 0.05 g/mL. The total polyphenol content was determined according to the Folin-Ciocalteu method while the total antioxidant activity was evaluated by DPPH, FRAP and NO assays with L-ascorbic acid as reference compound. Results: The total polyphenol content of the plant extracts varied from 0.41 to 13.00 mg GAE (gallic acid equivalents) per gram dry weight. The antioxidant activities ranged in IC 50 of 36.89-101.27 µg/mL, IC 50 of 139.56-419.93 µg/mL, 0.12-8.98 µM for DPPH, NO, FRAP assays, respectively. A significant positive correlation was observed between total polyphenol content and antioxidant activities ( P < 0.05). Conclusion: Phytochemical analysis revealed the presence of polyphenolic compounds, alkaloids and flavonoids in the plant materials which also possessed in vitro antioxidant potentials. Polyphenolic compounds contribute significantly to the total antioxidant capacities of medicinal plant extracts.


Introduction
Over the years, research on antioxidants and medicinal plants has gained enormous popularity and proved as potential therapeutic avenue for preventing free radical-generated damage in the human body.Free radicals are well known for the pathogenesis of various degenerative diseases such as cardiovascular diseases, neurological disorder, diabetes mellitus, and also in aging (Shukla et al., 2009).Medicinal plants are being viewed as readily available, potent sources of natural antioxidants as they contain a mixture of different chemical compounds that may act individually or in synergy.The oxidative damage is a critical etiological factor implicated in several human diseases including diabetes mellitus (Pandey and Rizvi, 2009).The damage of lipids, proteins, and nucleic acids by free radicals with the simultaneous decline of antioxidant defense mechanisms leads to cell and tissue damage.It may be contributed to the secondary complications in diabetes (Bakirel et al., 2008).It has been postulated that most effects of oxidative stress in diabetes mellitus are diminished upon supplementation with certain plant antioxidants such as vitamins C, E and non-nutrient antioxidants as polyphenolic compounds (Sun et al., 2007).Thus, substantial effort is needed to ensure proper identification of safe natural antioxidants from plant species.Polyphenolic compounds virtually found in all families of plants are believed to possess preventive and curative effects on cellular oxidative damage.They are considered as affluent sources of free radical scavenging molecules which are rich in antioxidant activity (Rice-Evans et al., 1997).The evidence for their role in the inhibition of lipid peroxidation and scavenging reactive oxygen species such as hydroxyl, superoxide and peroxinitrite have been recognized in experimental diabetes (Kuttan and Sabu, 2009;Sharma et al., 2010).
As in many Asian countries in the world, Sri Lanka constitutes an apt example where medicinal plants are widely used in everyday life for culinary purposes and as medicinal remedies.The plants selected for this study are widely used in traditional medicine for the treatment of diabetes mellitus and some are recommended as dietary adjuncts to existing therapies (Ediriweera and Ratnasooriya, 2009).Till date, Sri Lankan medicinal plants included in the study have not been studied to assess their in vitro antioxidant potential and the relationship between their total polyphenol content and in vitro anti-oxidative activities.
Several reports indicate that the antioxidant potential of medicinal plants may be related to the concentration of the polyphenolic compounds (Anantharaman et al., 2010;Kiselova et al., 2006;Wong et al., 2006).The objectives of the study were to determine the total polyphenol content and in vitro antioxidant activities of aqueous extracts of ten selected Sri Lankan medicinal plants, in order to determine the relationship between their polyphenol content and antioxidant activities of plant extracts, and to examine the phytochemicals present in the plant extracts.

Preparation of extracts
Powdered plant material (5.0 g) was dissolved in 400.0 mL of distilled water and refluxed for 4 hr.The mixture was strained and the final volume was adjusted to 100.0 mL.The initial concentration of each plant extract was 0.05 g/mL.A series of extract dilutions were prepared (1-500 µg/mL) for DPPH and NO inhibition assays.All determinations were done in triplicates per plant species obtained from 3 different locations.
In the present study, we determined the total polyphenol content of selected medicinal plant extracts by classical Folin-Ciocalteu reagent method and total antioxidant activities by DPPH (2, 2-diphenyl-2-picrylhydrazyl hydrate) radical scavenging assay, FRAP (ferric reducing power)assay, and NO (nitric oxide) inhibition assay.

Total polyphenol content
Total polyphenol content was measured using Folin-Ciocalteu colorimetric method (Singleton et al., 1999).Plant extract (1.0mL) was mixed with 1.0 mL of 95% ethanol, 5.0 mL of distilled water and 0.5 mL of 50% Folin-Ciocalteu reagent.The mixture was allowed to react for 5 min and 1.0 mL of 5% Na 2 CO 3 was added.Thereafter, it was thoroughly mixed and placed in dark at room temperature (25°C) for 1 hr and the absorbance was measured spectrophotometrically at 725 nm.Quantification was done with respect to the standard curve of gallic acid (0-50 µg/mL).The results were expressed in gallic acid equivalents mgGAE/g of the dry weight.

DPPH radical scavenging activity
The total antioxidant activity was measured by the DPPH radical scavenging assay method (Brand Williams et al., 1995).The radical scavenging activity of plant extract against stable DPPH radical (DPPH*) was determined.When DPPH* reacts with an antioxidant compound in the plant extract, the antioxidant donates hydrogen and reduced.The change in color from deep violet to light yellow was measured spectrophotometrically at 517nm.Briefly, 0.004% DPPH solution was prepared with EtOH and the absorbance of the DPPH solution alone was measured (A control ) at 517 nm.EtOH was used as the sample blank.1.0 mL of each extract at different concentrations (1-500 µg/mL) was added to 3.0 mL of0.004% ethanol DPPH solution.The mixture was shaken vigorously, allowed to stand at 25°C in dark for 30 min.The decrease in absorbance of the resultant solution was measured spectrophotometrically at the same wave length (A sample ).L-Ascorbic acid was used as the reference compound.The antioxidant activity is expressed in terms of IC 50 (concentration of the extract /reference compound required to inhibit DPPH radical formation by 50%).% DPPH radical scavenging activity= (A control -A sample )/A control x100% Where, A control represents the absorbance of the control without the plant extract/reference compound, A sample represents the absorbance of the plant extract/reference compound.

Ferric reducing antioxidant potential (FRAP) assay
The FRAP assay was performed according to the method of Benzie and Strain (1999).The assay is based on the reducing power of a compound (antioxidant).A potential antioxidant reduces the ferric ion (Fe 3+ ) to ferrous (Fe 2+ ) ion; the latter forms a blue complex (Fe 2+ /TPTZ), which increases the absorption at 593 nm.The FRAP working reagent was prepared by mixing acetate buffer (300 mM, pH 3.6), a solution of 10mM TPTZ in 40 mM HCl and 20 mM FeCl 3 at 10:1:1 (v/v/v).The FRAP reagent (3.0 mL) and sample solution (100 µL) were mixed and the absorbance of the resulting solution was measured (t=0) at 593 nm spectrophotometrically (A sample t=o ) against a sample blank (FRAP working reagent).Thereafter the sample was placed in a water bath (37°C) and absorption was measured at the same wave length after 4 min.(A sample t=4 ).The ascorbic acid (100 µM) was used as the standard compound and preceded as in the same way.FRAP value of the plant extract (µM)=(A sample t=0-4 )/A standard t=0-4 ) x FRAP value of1000 µM ascorbic acid Where, (A sample t=0-4 ) represents the change in absorbance in a sample from 0 to 4 min, (A standard t=0-4 ) is the change in absorbance in the standard from 0-4min.FRAP value of ascorbic acid is 2.

Nitric oxide (NO) radical scavenging assay
Nitric oxide generated from SNP in aqueous solution at physiological pH, interacts with the Griess reagent and the absorbance of the chromophore formed was measured spectrophotometrically (Marcocci et al., 1972).Scavengers of nitric oxide compete with oxygen leads to reduce the production of nitric oxide.L-Ascorbic acid was used as the reference compound.1.0 mL of SNP (5 mM) was mixed with 4.0 mL of the plant extract at different concentrations (1-500 µg/mL) and incubated the resultant solution at 29°C for 2 hr.Incubated solution (2.0 mL) was mixed with 1.2mL of Griess reagent (1% sulfanilamide in 5% H 3 PO 4 and 0.1% N-(1-Naphthyl) ethylediamine dihydrochloride and measured the absorbance at 550 nm (A sample ) against a distilled water blank.The absorbance of the control without the plant extract/reference compound was also measured at the same wave length (A control ).The antioxidant activity is expressed in terms of IC 50 (micro-molar concentration required to inhibit NO radical formation by 50%).% NO radical scavenging activity= (A control -A sample )/A control x 100% Where, A control represents the absorbance of the control without the plant extract/reference compound, A sample is the absorbance of the plant extract/reference compound.

Phytochemical screening
Preliminary qualitative phytochemical screening for the presence of alkaloids, cardenolide glycosides, phenols, flavonoids, phytosterols, saponins, tannins, reducing sugars, and proteins was carried out by the reported protocol (Trease and Evans, 1978).

Statistical analysis
The replicates of each sample were used for statistical analysis and the values were expressed as mean ± standard deviation.Pearson's correlation and regression analysis was carried out using Minitab software to establish the relationship between total polyphenol content and antioxidant activities of selected medicinal plant extracts.The level of significance was set at P < 0.05.

Total polyphenol content
The total polyphenol content of the ten aqueous medicinal plant extracts was determined by regression equation of gallic acid calibration curve (y= -0.0203 + 0.0101x) and varied in a range of 0.41-13.00mgGAE/g of dry weight (Table 1).The highest and lowest polyphenol contents were in the extracts of G. arborea and K. zeylanica, respectively.Polyphenols possess ideal structural chemistry for free radical scavenging activity and are viewed as promising natural antioxidants.The antioxidant effect of polyphenolic compounds was mainly due to their redox properties and as a result of various possible mechanisms: free radical scavenging activity, metal chelating activity and/or singlet oxygen-quenching capacity (Corke et al., 2005).

Total antioxidant activities
The chemical approaches facilitate the study of the total antioxidant activity of medicinal plant extracts and the precise mechanisms of action of antioxidants.Thus far, numerous studies on antioxidant properties of many plant species have been conducted using different assay methods.The general recommendation is to employ at least three in vitro methods due to the presence of wide variety of oxidation systems (Kaul et al., 2010).
In the present study, antioxidant activity was evaluated by three spectrophotometric methods; DPPH, FRAP and NO assay.Water soluble antioxidant potentials are determined based on the traditional method in intake of plant extracts therapeutically.The IC 50 values of extracts were calculated for DPPH and NO inhibition assays to compare the antioxidant activities at different concentrations as described by many authors (Adesegun et al., 2009;Marwah et al., 2007).The results of three in vitro assays (DPPH, FRAP, NO) for antioxidant properties of the 10 plant extracts are given in Table 1.
All extracts and the standard compound exhibited concentration-dependent radical scavenging activities in DPPH and NO inhibition assays.The radical scavenging activities were expressed in IC 50 values in both assays to obtain a more precise single value over a range of concentration of plant extracts.The radical scavenging activity in DPPH assay was in the decreasing order of G. arborea, S. pinnata, C. grandis, S. dulcis, N. arbo-tristis, S. caryophyllatum, S. alnifolia, M. charantia, L. galanga, K. zeylanica followed by G. arborea, C. grandis, S. pinnata, S. caryophyllatum, M. charantia, N. arbo-tristis, S. dulcis, S. alnifolia, L. galanga, and K. zeylanicain NO assay.The high amount of polyphenol compounds leads to high radical scavenging activity (low IC 50 ) as shown in the extracts of G. arborea, C. grandis and S. pinnata in both assays.However the IC 50 of standard compound (L-Ascorbic acid) as a powerful antioxidant was more pronounced in DPPH and NO radical scavenging assays and values are comparable with previous studies (Zhu et al., 2004).The DPPH assay is reported to be a direct and reliable method for the determination of radical scavenging activity where the structure of electron donor (e.g.plant extract) is not known.DPPH assay method can afford data on reduction potential of the sample and hence can be helpful in comparing the reduction potential of unknown compounds.A key mediator released by activated macrophages that has been implicated in toxicity is nitric oxide.It has been pointed out that modulating nitric oxide production can modify tissue injury (Laskin et al., 1990).Thus, development of specific nitric oxide scavengers is considered important due to lack of endogenous enzymes responsible for nitric oxide in activation (Alisi and Onyeze, 2008).
The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activities (Meir et al., 1995).As shown in Table 1, the reducing power of extracts were between 0.12-8.98µMat the concentration of 0.05g/mL of plant extract and followed the decreasing order of G. arborea, C. grandis, S. pinnata, S. caryophyllatum, and N. arbo-tristis.The extract of K. zeylanica had the lowest ferric reducing power among selected plant extracts.The antioxidant activity of polyphenols determined using different free radical methods showed similar results to those obtained using the FRAP assay suggesting that the reducing ability of polyphenols seemed to be an important factor dictating free radical scavenging capacity of these compounds.FRAP assay has many advantages over many radical scavenging assays such as excellent reproducibility, linearity over a wide range and high sensitivity.In contrast, the FRAP assay measures the reducing capability by increased sample c FRAP( ferric reducing power).NA; not applicabl absorbance and the assay may not complete even several hours after the reaction starts, such that a single end point of the reaction cannot be determined (Prior et al., 2005).

Correlation between the total polyphenol content and antioxidant activity
The linear regression and correlation analysis was done to establish the correlation between total polyphenol content and antioxidant activities of medicinal plant extracts and to evaluate the suitability and reliability of the three in vitro assay methods.The correlation (R) is given in Table 2.A linear correlation between the total polyphenol content and antioxidant activity was established.This is consistent with previous findings for medicinal plant extracts (Sun et al., 2007).The total polyphenol content and antioxidant activity were positively correlated asR= 0.923, 0.978, 0.944 in DPPH, FRAP, and NO assays respectively (P< 0.05).This could be explained from the basic concept that polyphenolic antioxidants are reducing agents.All R values were positive at the P<0.05 significance level, indicating that the three antioxidant methods selected are suitable and reliable for assessing the total antioxidant potentials of plant extracts, although the differences in antioxidant capacities between assay methods were observed with samples.

Phytochemical screening
The phytochemical analysis conducted on all selected plant extracts revealed that the polyphenolic compounds, alkaloids and flavonoids are present in all plant species.Cardenolide glycosides were absent in the plant species of K. zeylanica, C. grandis, S. alnifolia and S. dulcis.In addition, saponins and sterols/tritepenoids were absent in the S. alnifolia and L. galanga.Anthracene glycosides and cyanogenic glycosides were not present in all of the selected plants.

Table 2:
The correlation between total polyphenol content and antioxidant activities of Sri Lankan medicinal plant extracts a a The correlation (R) is given between total polyphenol content (TPC) and 1/IC 50 in DPPH(2, 2-diphenyl-2-picrylhydrazyl hydrate), FRAP (ferric reducing power) value and IC 50 in NO (nitric oxide) inhibition assay.(IC 50 : Concentration of the extract required to inhibit DPPH/NO radical formation by 50%) The correlation is statistically significant at P < 0.05.

Conclusion
The data presented in this study demonstrate that almost all the plant species investigated possessed antioxidant activities by the virtue of the variety of chemical constituents present.The positive correlations obtained in this study suggest that polyphenolic compounds contribute significantly to the total antioxidant capacities of medicinal plant extracts.Results revealed that, bark extracts of G. arborea, S. pinnata and leaf extract of C. grandis were excellent free-radical scavengers containing potent natural polyphenolic compounds.Therefore, these extracts are most likely to exert health benefits in free radical pathologies in diabetes mellitus.

Table 1 :
Total polyphenol content and antioxidant activities of 10 selected Sri Lankan medicinal plant extracts All values were the mean of three measurements and expressed as mean ± SD.
a b,d Radical scavenging activity by DPPH (2, 2-diphenyl-2-picrylhydrazyl hydrate)assay and NO(Nitric oxide) inhibition assay was expressed in IC 50 (Concentration of the extract required to inhibit DPPH/NO radical formation by 50%) obtained by linear regression analysis.