Javid Ahmad Parray et al. / Journal of Pharmacy Research 2011,4(7),2170-2174 Research Article Available online through ISSN: 0974-6943 http://jprsolutions.info Phytochemical screening,antifungal and antioxidant activity of Euryale ferox- SALISB. a threatened aquatic of Kashmir Himalaya. Javid Ahmad Parray1, Azra N. Kamili1, Rehana Hamid2, Bashir A Ganai3, Khalid Ghazanfar Mustafa3 and Raies A. Qadri4 1 Department of Environmental Science, University of Kashmir, Srinagar-190006, J and K, . 2 Department of Botany, Jamia Hamdard, New Delhi, 1100621, India. 3 Department of Biochemistry, University of Kashmir, Srinagar-190 006 J and K, India 4 Department of Biotechnology, University of Kashmir, Srinagar-190006, J and K, India. Received on: 12-04-2011; Revised on: 18-05-2011; Accepted on:21-06-2011 ABSTRACT Multiple drug resistance in human pathogenic microorganisms has developed due to indiscriminate use of modern antimicrobial drugs generally used in the management of infectious diseases. This increases the importance of exploiting the natural sources instead of modern drugs. The present study was carried out to evaluate the possible medicinal value of Euryale ferox extracts, a threatened aquatic plant of Kashmir valley, by screening it for antifungal activity, antioxidant activity and phyto-chemical screening. The and extracts exhibited its scavenging effect in concentration dependent manner on superoxide anion radicals, hydroxyl radicals and exhibited remarkable antifungal activity which is due to the presence of various classes of secondary metabolites as estimated by the phyto-chemical screening. The extracts were able to exhibit maximum inhibitory activities against tested fungal strains. The five out of the nine fungal strains tested were susceptible to both extracts with Candida albicans and Pencillium notatum exhibited highest zone of inhibition. Both extracts were also investigated for antioxidant activities through three in vitro assay systems, i.e. DPPH, Deoxyribose assay and superoxide radical scavenging method. The percentage inhibition for seed and leaf extract was 89.50 and 83.06 respectively as compared to BHT (97.00 %) at concentration of 500 µg/ml. DPPH assay showed about 88.21 and 74.89 percentage inhibitions at a concentration of 500 µg/ml compared to control (92.03 %) inhibition and superoxide radical scavenging assay exhibited 80.32 % and 66.82 % inhibitory activity while as control showed 89.05 % inhibition. The results provided evidence that the studied plant might indeed be potential sources of natural antioxidant and antimicrobial agents.

Key words:Antioxidant activity, antifungal activity, Euryale ferox, methanol.

INTRODUCTION Providing effective health care is a challenge under the best of economic cir- Antioxidant activities of E. ferox and its glycosides composition in vitro reveal cumstances in the world’s poorest countries where infectious diseases are rife that the plant extracts have potent reactive oxygen species scavenging activity and resources limited this challenge can assume overwhelming proportions hence suggesting that seed of this plant have cardio protective properties which may the resurgence in the use of herbal preparations to treat diseases. So medicinal link with ability of makhana to induce TRP32 and TrX-1 proteins and to plant extracts can be used directly or indirectly for the treatment of different scavenge ROS (13).Very recent studies have also revealed that E. ferox extracts ailments. Therefore, the use of traditional medicine and medicinal in exhibit significant antibacterial activity against various clinically isolated bacte- most developing countries, as a basis for the maintenance of good health, has rial strains (14). been widely observed (1). Scientists throughout the world are trying to explore the precious assets of medicinal plants to help the suffering humanity. Further- It is in this context, the present study was carried out to determine the medicinal more, in the world more than 30% of the pharmaceutical preparations are based value of the E. ferox, a threatened aquatic plant which was present in almost all on plants (2). the valley lakes in early 1990’s but now is present in Manasbal Lake of Kashmir Valley, J&K, India. The goals of this study was to obtain methanolic extracts The screening of plant extracts for antimicrobial and antioxidant activity re- from E. ferox, to emphasize the anti fungal activity, phytochemical screening vealed the potential of higher plants as a source of new agents to serve the and scavenging activity of these extracts against reactive oxygen species i.e. processing of natural products (6). Reactive oxygen species such as superoxide superoxide anion and 1,1-diphenyl-2-picrylhydrazyl (DPPH) and total antioxi- anion radical, hydroxyl radicals, non free radicals (H O and ¹O ) are involved in 2 2 2 dant activity by DNA Damage assay. various physiochemical processes and diseases such as ageing, cancer and activ- ity atherosclerosis. Secondary metabolites isolated from plants are important in MATERIAL AND METHODS providing protection against oxidative damage from free radicals in addition to cell’s own defense system. E.ferox has been widely used in traditional oriental Plant material medicine to treat a variety of illness; however very little is known about the E. ferox is an annual plant and its and were collected from Manasbal cellular actions by which this plant mediates its therapeutic effects. The dry Lake of Kashmir Himalaya, J&K, India in September 2008. Sampling was car- seeds of E. ferox of the plant are being used as immuno-stimulant for mothers ried out immediately after seed formation and plants were collected manually at after child birth and invalids with relatively poor immune status (7). The plant the age of 7 months in bulk from the Lake. All the two parts i;e leaves and seeds has been used throughout the world to cure many diseases including chronic were separated, washed and dried under shade for few days and then cut into small diarrhhoea, kidney problem, leucorrehea and spleen hypofunction (8) and is pieces. These small pieces were then made in powdered form in a wood grinder internally taken to treat vaginal discharge, impotency, premature and involun- under sterilized conditions. Plant was identified in Kashmir University Her- tary ejaculation and nocturnal emission (8). The plant as a whole is used as an barium (KASH), Centre of Plant , Department of Botany, University analgesic, aphrodisiac, astringent, deobstruent, oxytonic and tonic in China (9) of Kashmir, Srinagar. The herbarium of the same was submitted in the above while leaves are being used in the case of difficult parturition (10). Various centre under voucher no.1015. Fifty grams of dried powder of leaves and seeds aspects of significant antioxidant activity have been evaluated in total extracts were separately loaded in Soxhlet extractor and which then was defatted with and fractions derived from the plant. E. ferox also has been shown to enhance methanol in Soxhlet extractor (for 6 hrs at 800C). The methanolic seed and leaf the activities of superoxide dismutase, catalase and glutathione peroxidase in extracts collected were later dried and weighed and kept for further usage in V79-4 cells (11). sterilized caped vials at 40C.

*Corresponding author. Chemicals: Dr. Raies A Qadri, All the solvents used were of analytical grade. Ammonium thiocyanate (Merck), Department of Biotechnology, 1,1-diphenyl-2-picrylhydrazyl (DPPH), Trichloroacetic acid, Ascorbic acid, Nitro Blue tetrazolium (NBT), NADH, Butylated Hydroxy Toleune (BHT) and University of Kashmir, Srinagar, Phenazine methosulphate (PMS) were purchased from Sigma Chemicals Co (St, JK India-190006 Louis, MO, USA). All other chemicals i.e. sodium hydroxide, sodium nitrite, ferrous chloride, ammonium thiocyanate, potassium dihydrogen phosphate,

Journal of Pharmacy Research Vol.4.Issue 7. July 2011 2170-2174 Javid Ahmad Parray et al. / Journal of Pharmacy Research 2011,4(7),2170-2174 Sabourand dextrose agar (SDA), EDTA, Dipotassium hydrogen phosphate, used Superoxide anion radical scavenging activity method in this study were purchased from Hi media (Mumbai Pvt. Ltd.) Super oxide radicals are generated in PMS-NADH systems by oxidation of NADH and assayed by the reduction of NBT (17). In this experiment, the super Preliminary Phyto-chemical screening tests oxide radicals were generated in 3.0ml of Tris-HCl buffer (16mM, pH 8.0) To detect the presence of possible phyto-chemicals in methanolic seed and leaf containing 1.0ml of NBT (50 µM) solution, 1.0 ml NADH (78 µM) solution and extracts, preliminary phyto-chemical testing was performed. (i) Test for alka- sample solution of the extracts (50-300 µg/ml) in water. The reaction started by loids: (Dragendroff’s test) Dissolve few mg of alcoholic or aqueous extract in 5 adding 1.0 ml of phenazine methosulphate (PMS) solution (10 µM) to the ml of distilled water, add 2 M HCl until an acidic reaction occurs, then add 1ml mixture. The reaction mixture was incubated at 25 °C for 5 min, and the of Dragendroff’s reagent, an orange or orange red ppt. produced immediately absorbance at 560 nm was measured against blank samples. L-Ascorbic acid was confirms the presence of alkaloids (19). (ii) Steroids: (Libermann-Burchard’s used as a control. Decreased absorbance of the reaction mixture indicates in- test), Add 2ml of acetic anhydride solution to 1ml of petroleum ether of the creased super oxide anion scavenging activity. The % inhibition of super oxide extract in chloroform followed by 1ml of conc. Sulphuric acid, a greenish color anion generation was calculated using the following formula: is developed which turns to blue (19). (iii) Tannins: (Ferric chloride test) To 1- SOD scavenged (%) = 100(Ac-As)/Ac 2ml of aqueous extract add few drops of 5% aq. Ferric chloride solution, a bluish Where Ac is the absorbance of the controlled reaction and As is absorbance of black color is produced which disappears on addition of a few ml of dil. Sulphuric reaction with sample extracts. The test was repeated thrice. acid solution followed by the formation of a yellowish brown precipitation indicated the presence of Tannins (19) (iv) Flavonoids: In a test tube containing Hydroxyl scavenging activity-Deoxyribose assay 0.5ml of alcoholic extract, add 5-10 drops of dil. HCl followed by a piece of The highly reactive radical i;e. hydroxyl radical is generated by both reaction of Zinc/Magnesium. Boil the solution for few minutes, in the presence of fla- +3 H2O2 by Fe2O3. The hydroxyl radical is generated by using ferric nitrate (Fe ), vonoids a pink or reddish pink or brown color produced indicated the presence ascorbic acid and 30 mM H2O2 and extract of different concentrations was added of Flavnoids (20). (v) Phenols: (Ferric chloride test) Dissolve a small quantity to find its protective effect on DNA damage. In this assay (18), 500 µl of DNA of alcoholic or aqueous extract in 2ml of D. Water, add a few drops of 10% was added to 100 µl of different concentration of test samples (leaf and seed aqueous ferric chloride solution, a blue or green color is produced (20). (vi) extract) and 100 µl of ascorbic acid (500 mM), 100 µl ferric nitrate (20 mM), 30

Saponins: Small quantity of alcoholic extract is mixed some drops of sodium bi- µl of H2O2 and final volume was made to 1 ml by tris HCl buffer (0.01M, pH 7.5). corbnate and leave for 5 minutes. Heavy comb like froth is formed, confirms The mixture was than incubated at 30 0C for 20 hrs. The reaction was termi- the presence of saponins (21). (vi) Glycosides: Dissolve a small quantity of nated by adding 1 ml TCA (25 %) and if there was any precipitation, it was than alcoholic extract after drying in 1 ml of water and add NaOH solution, a yellow centrifuged at 3000 rpm. To supernatant, 1 ml of TBA (1.68%) was added. The color indicates the presence of glycosides (22). deoxyribose degradation was assessed by estimation of Thiobarbuturic acid reac- tive species (TBARS) formation after boiling in water bath for 10 minutes and Anti-fungal Activity tubes were than cooled in an ice bath followed by centrifugation at 10000 rpm. Test Microorganisms: TBARS formation was estimated at 535 nm by spectrophotometer. For the evaluation of Antifungal activity of E. ferox extracts, all the pure fungal % inhibition = 100(A -A )/A strains viz, Aspergillus fumigates, candida albicans, Candida stealoidea, Can- c s c where A is the absorbance of the controlled reaction and As is absorbance of dida tropicals, Candida kruesei, Candida parapsilosa, Cryptococcus neoformans, c reaction with sample extracts. The test was repeated thrice. BHT was used as Rhizopus spp. and Pencillum notatum were provided by Mycology section, control. Deptt. Of Microbiology, SKIMS, Soura, Srinagar J&K, India. Statistical Analysis Antifungal Susceptibility Tests Each sample was analyzed individually in triplicates and data reported as mean The disc diffusion method (15) was used to determine the antifungal activity of ± SEM. Data was analyzed by using one-way analysis of variance (ANOVA) with leaf and seed extract of E. ferox using Sabourand Dextrose Agar (SDA) (Hi- multiple comparisons by Dennett’s test. Data was considered significant for Media). The SDA plates were prepared by pouring 15ml of SDA into sterile Petri P<0.05. plates. The plates were allowed to solidify for 15 minutes and 0.1 ml (0.5 Mac Farland) inoculum suspension was swabbed uniformly and the plates were al- RESULTS AND DISCUSSIONS lowed to dry for 5 -10 minutes. The concentrations of extracts (200 µg and 400 Phytochemical Screening of crude extracts of E. ferox revealed that methanolic µg /disc) were loaded on 6 mm sterile discs (Hi Media). The loaded discs were active fractions of seed and leaf contain alkaloids, phenols, tannins, flavnoids placed on the surface of the medium and the extracts were allowed to diffuse for and steroids. However, glycosides and saponins were only present in seed extract 5 minutes and the plates were kept for incubation. Inhibition zones formed (Table 1). MSLE were tested against different fungal strains and showed broad around the discs were measured with transparent ruler (in mm). Antibiotic disc antifungal activity at different concentrations (Table 2). Among the fungal (Nystatin) and solvent (Methanol) were taken as positive and negative control strains tested, C. neoformans, A. fumigates, Rhizopus spp and C. parapsilosis respectively. were found resistant towards both extracts (Fig. 1a, b; 2a, b). However C. albicans, C. stealoidea and C. kruesie were more susceptible towards both Anti-oxidant activity: extracts followed by P. notatum. (Fig. 3a, b; 4a, b) However C. tropical was For evaluation of antioxidant activity of methanolic seed and leaf extracts susceptible to only higher concentrations of extracts (Fig. 5). It was also found (MSLE), different methods followed were as: that seed extracts exhibited higher antifungal activity than leaf extracts. Nysta- tin and methanol were used as positive and negative control respectively and DPPH assay methanol was found resistant in all strains. The overall results revealed that The radical scavenging activity of MSLE were measured with 1,1 diphenyl-2- inhibitory activity of microorganisms is concentration dependent i.e. that the picryl hydrazyl radical (DPPH) spectrophotometricaly at 517 nm (16). The inhibition zone increases with increase in concentration of extracts. The major- stock solution of crude seed and leaf extracts (5 mg/ml) was prepared by dissolv- ity of plant extracts or antifungal agents have an inhibitory action on the ing a known amount of dry extract in 98% methanol. The working solutions (50,100,150, 200, 250, 300, 350, 400 and 500 µg/ml) of the extracts were Table 1. Qualitative analysis for various secondary metabolites in ex- prepared from the stock solution using suitable dilution. 0.005% DPPH was also tracts of Euryale ferox. prepared in methanol. About 50 µl of different concentrations was added to 2.950 ml of DPPH solution. The scavenging activity was observed by bleaching S. No. Compound Leaf extract Seed extract of DPPH solution from violet color to light yellow. Ascorbic acid was used as 01 Alkaloids + + control. 02 Glycoside - + % inhibition = 100(A -A )/A 03 Phenols + + c s c 04 Tannins + + Where Ac is the absorbance of the controlled reaction and As is absorbance of 05 Saponins _ + reaction with sample extracts. The test was repeated thrice. 06 Flavanoids + + 07 Steroids + + += Present; - = absent Journal of Pharmacy Research Vol.4.Issue 7. July 2011 2170-2174 Javid Ahmad Parray et al. / Journal of Pharmacy Research 2011,4(7),2170-2174 Table 2: Antifungal activity of methanolic extract of leaf and Seed extracts of Euryale ferox gosterol in fungal cell membranes has also been found to be the basis for the selec- S. No. Fungal Strains Leaf extract Seed extract 200 µg 400 µg 200 µg 400 µg tion action of most of the fungicides cur- rently used in clinical practice. The anti- 01 Crytococcus neoformans NA NA NA NA 02 Aspergilus fumigates NA NA NA NA fungal agents inhibit fungal growth by bind- 03 Candida albicans 9± 0.81 16± 0.70 7±0.3 12± 0.81 ing the ergosterol in the plasma mem- 04 C. kruesie 8± 0.48 10±0.28 7±0.3 9± 0.00 05 C.Stealoidea 10± 0.62 15±0.34 8±0.2 11± 0.5 branes of fungi and forming channels that 06 C. tropicals NA 15±0.34 NA 18± 0.14 lead to the loss of cell integrity (25). Mi- 07 C. parapsilosis NA NA NA NA 08 Rhizopus spp NA NA NA NA crotubules play a key role in a variety of 09 Pencillum notatum 10± 0.81 15±0.60 8±0.21 9± 0.84 processes including cell division, motility, Fig. 5: C. tropicals cytoplasmic transport, secretion and mor- *Results are mean diameter of zone of inhibition of three replicates followed by standard phogenesis. Alkaloids like colchicines, SI=200 µg, S2=400 µg = Seed metha- deviation; Nystatin was used as positive control and Methanol as negative control in all vinblastine, taxol have been reported to nol extract. L1=200 µg, L2=400 µg strains, NA=No activity inhibit the assembly of microtubules by = Leaf methanol extract, Ab= Anti- Antifungal activity of methanolic seed and leaf extract of E. ferox against binding to tubulin dimmers and inhibit fun- biotic; M=methanol Fungal Strains: gal mitosis (26). The seed extract exhibited significant inhibitory activity against Candida sps as was also studied by Mashhadian and Rakhshandeh27 for methanolic extract of Nigella sativa seeds. Candida sps have become common opportunis- tic pathogens in compromised hosts. The predominant clinical isolate C. albicans is the fifth most common cause of nosocomial blood stream infection (28) and with the advent of antimicrobial activity of E. ferox extracts particularly against Candida sps drugs formulated from this plant would be effective against yeast infections. Likewise many plants have been reported to have good antifungal activity against Candida sps i.e. Euphorbia characias (29), Centella asiastica (30). The fungal sps like A. Fumigates, C. neoformans were found more resis- tant to the extracts of E. ferox in present study and our finding coincides with the results of Radiusiene et al 31. The results revealed that both extracts exhibited good antifungal activity and our findings are corroborated by Singh and Singh32. Fig. 1a: Cryptoccocus neoformans Fig. 1b: Aspergillus fumigates Antioxidant activity of MSLE carried out using DPPH radical assay, Deoxy- ribose assay and Superoxide anion radical scavenging activity method revealed Table. 3: De-oxyribose assay.

Concentration *Inhibitory activity (%) (µg/ml) Seed extract Leaf extract Control

50 40.35±0.23 33.20±0.12 50.00±0.43 100 47.23±0.54 40.67±0.47 58.78±0.67 150 55.16±0.63 49.24±0.69 65.09±0.71 200 61.72±0.04 54.13±0.08 74.32±0.03 250 69.12±0.65 62.32±0.43 78.43±0.39 300 74.45±0.73 66.17±0.67 83.91±0.86 350 77.58±0.20 74.56±0.38 86.26±0.47 400 79.63±0.72 79.38±0.56 91.09±0.20 450 86.29±0.04 80.00±0.06 93.42±0.10 Fig. 2a: C. parapsilosis Fig. 2b: Rhizopus spp 500 89.50±0.74 83.06±0.43 96.90±0.75

*Values are mean±SD of three replicates. Table. 4: Effect of MSLE on Superoxide radical inhibition. Concentration(µg/ml) *Inhibitory activity (%) Seed extract Leaf extract Control

50 43.53±0.53 30.50±0.83 42.08±0.43 100 52.20±0.49 39.25±0.72 56.94±0.67 150 59.74±0.58 46.50±0.41 64.23±0.71 200 68.18±0.15 55.23±0.28 75.62±0.03 250 75.27±0.05 61.23±0.03 80.43±0.09 300 80.32±0.61 66.82±0.77 89.05±0.90

Fig. 3a: C. albicans Fig. 3b: C. Kruesie *Values are mean±SD of three replicates.

Table 5: Effect of MSLE on DPPH radical inhibition

Concentration(µg/ml) *Inhibitory activity (%) Seed extract Leaf extract Control

100 50.83±0.26 45.23±0.43 60.00±0.61 200 62.36±0.44 50.73±0.30 66.25±0.67 300 71.56±0.63 63.27±0.49 78.38±0.81 400 80.30±0.24 68.40±0.18 83.23±0.33 500 88.21±0.75 74.89±0.83 92.03±0.59

*Values are mean±SD of three replicates.

that inhibitory activity is concentration dependent. The % inhibition for con- Fig. 4a: P. notatum Fig. 4b: C. stealoidea trols (ascorbic acid and BHT) used were comparatively found higher at all synthesis of cell wall components that are unique to fungi. The cell walls of fungi concentrations used. The general free radical scavenging assay (table 3) illus- such as Candida sps are composed of many components including manoproteins, trates the antioxidant activity of both active fractions of seed and leaf i.e. 88.21 chitin and ß- glucans (23, 24). Many antifungal agents of crude plant extracts and 74.89 percentage inhibitions respectively at a concentration of 500 µg/ml exhibit inhibitory effects against ß- glucans biosynthesis. The presence of er- compared to ascorbic acid (92.03 %) inhibition thereby exhibited significant Journal of Pharmacy Research Vol.4.Issue 7. July 2011 2170-2174 Javid Ahmad Parray et al. / Journal of Pharmacy Research 2011,4(7),2170-2174 activity almost equal to control used. The scavenging of the hydroxyl radicals vis a vis its habitat because of its threatened status as it is found nowhere in this generated by Fenton’s reaction by extracts of E. ferox is shown in (table 4) and state of Jammu and Kashmir except in Manasbal lake of Kashmir Valley J&K percentage inhibition recorded was 89.50 and 83.06 for seed and leaf extract as India. compared to BHT (97.00 %) used as control at concentration of 500 µg/ml. It shows that methanolic fraction of seed extract exhibits maximum activity as ABBREVIATIONS: compared with leaf extract. The scavenging activity of superoxide radicals by BHT: butylated hydroxy toleune; DPPH: 1,1-diphenyl-2-picrylhydrazyl; MSLE: MSLE (table 5). Both extracts exhibit scavenging activity with percentage methanolic seed and leaf extract, NBT; nitroblue tetrazolium, SDA: Sabourand inhibition of 80.32 and 66.82 for seed and leaf extracts respectively compared Dextrose Agar. to the ascorbic acid which showed 89.05% inhibition. MSLE shows significant inhibitory activities towards all assay systems as compared with respective ACKNOWLEDGMENT: controls at P<0.05. The authors are highly thankful to RRIUM Hazratbal Srinagar and SKIMS Soura, Sinagar J&K India for providing Laboratory facilities to complete the Antioxidant compounds act by several mechanisms such as, inhibition of gen- study . eration and scavenging activity against reactive oxygen species (ROS), reducing power, metal chelation activity as antioxidative enzymes, inhibition of oxida- REFERENCES tive enzymes. Oxidative damage caused by ROS causes DNA lesions, loss of 1. Edward A. 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