Research Journal of Pharmacognosy and Phytochemistry. 6(4): October-December, 2014, 151-155 ISSN 0975-2331 (Print) www.anvpublication.org 0975-4385 (Online) RESEARCH ARTICLE Antioxidant Profiling and Antibacterial Activity of the Vaccinium sikkimense from the Lachen Valley of the Sikkim Himalayas Smrita Pradhan*, Sushen Pradhan, K.B. Subba, Bharat Basistha State Council of Science and Technology, Bigyan Bhawan, Deorali, Gangtok East Sikkim *Corresponding Author E-mail: [email protected] ABSTRACT: Vaccinium sikkimense under the family of Ericaceae is a low bush blueberry commonly found in Northern area of Sikkim Himalayas. The berry, leaves and the twigs of the plant were collected shade dried and grounded. The solvent extraction of the dried powder of the leaves, berry and the twigs and the fresh berry was carried out in different solvents to get a semi solid extract which was used for further studies like antibacterial activity against E.coli, P.aeruginosa, Klebsiella pneumoniae and S.aureus by well diffusion method where percentage inhibition was calculated using the formula and the graph was plotted. studies of the extract was carried out by DPPH method using UV/Vis spectrophotometer and the percentage scavenging activity of the reading was calculated using the formula and IC50 value was calculated. KEYWORDS: V. sikkimense; Antibacterial activity; Antioxidant profiling; DPPH INTRODUCTION: METHODOLOGY: Vaccinium sikkimense C. B. Clarke also known as ‘Sikkim Collection of plant Material: Blueberry’ is native to eastern Himalayas[11][13] and is found The plant material mainly the berry ,leaves and the stem of in high altitude areas of the north Sikkim mainly in Lachen the plant were collected from the Lachen valley of North and Lachung valley. It was first collected and reported by Sikkim and refrigerated for further processing. C.B. Clarke and J. D. Hooker from Lachen, North Sikkim which has the altitude around 13000 ft and published in The Flora of British India 3(9): 451. 1882. (Fl. Brit. India.[4] It is a evergreen small and rigid shrub around 30-60cm tall placed under the family Ericaceae. It is multi stemmed , twigs are angled, leaves are oblong or obovate, acute serrulate, racemes short solitary or clustered, calyx-teeth very short obtuse nearly glabrous, margin recurred. Flowers are pinkish and fruits are in the form of dark blue berry, 10- pseudoloculed, 5–9 mm in diameter which are edible.[4]Many Vaccinium sp. like blue berry, bilberry, cranberry are reported to have a potential antioxidant property and antimicrobial activity.[1]. Similarly Vaccinium sikkimense a native to the Sikkim Himalayas has a potential anti-bacterial and antioxidant properties. Received on 10.07.2014 Modified on 20.07.2014 Accepted on 27.08.2014 ©A&V Publications All right reserved Fig. No.1: a) Leaves b) Berries c) and Twigs of V. sikkimense Res. J. Pharmacognosy & Phytochem. 6(4):Oct. - Dec.2014; Page 151-155 151 Research Journal of Pharmacognosy and Phytochemistry. 6(4): October-December, 2014, 151-155 Drying and Grinding: Media Preparation: The berry, leaves and the stem of the plant were shade dried The nutrient agar media (Himedia) was prepared, for few days till the 60% of the moisture content was lost. It autoclaved and 20 ml each of the media was poured in was then grounded to fine powder and stored for extraction. petriplates inside Laminar air flow and allowed to solidify. The sterilised cork borer of 6mm diameter was used to Extraction and Filtration: make a well in the centre of the petriplates. 15% w/v of the dried powder of leaves, twigs and berry was weighed and added to 100ml ethanol for leaves and Sample preparation: twigs and 100ml methanol for the dried berries powder. 10 mg/ml concentration of the extract was prepared using Similarly, the refrigerated fresh berries were taken and 10% DMSO (di methyl sulphyl oxide) for the ethanol crushed to make fine paste with the help of mortar and extract of dried berry, leaves and stem ,methanol extract of pestle .15% w/v of the berry paste were weighed and added berry and ethanol extract of fresh berry. to 100ml of ethanol for solvent extraction. Extraction procedure was carried out using Soxhlet apparatus at 80oC Inoculation & Incubation: for 8-10 hours. 100 µl of each sample prepared were added to the well using micropipette in four petriplates for four different After extraction the filtrate was separated from the residue bacteria respectively to give around 1mg concentration. using No.1 Whatman filter paper. The filtrate was Similarly the same procedure was carried out for other concentrated and solvent were evaporated in rota- samples. Sterilized cotton was wrapped around the stick to evaporator of Buchi, (Heating Bath B-491) at 30oC to form prepare a cotton swab which was used to inoculate and semi-solid crude extract which was dried in hot air oven at evenly spread the known concentration of the bacterial 35oC. The extract was weighed and stored for further culture suspension in each petriplates. 100 µl of sterilized studies.[4] distilled water and the DMSO were used as negative control. 50 µg of tetracycline solution was used as positive Crude extract sample: control. Petriplates were incubated in the B.O.D incubator DLEE:-Dried leaves ethanol extract; DTEE= Dried twigs at 32oC for 24 - 48 hours. ethanol extract; WBEE= Wet berry ethanol extract; DBME = Dry berry methanol extract This experiment was conducted in triplicates. Antibacterial activity: Zone of Inhibition:- Antibacterial activity was conducted by agar well diffusion The effectiveness of extracts as antibacterial against method. [5][8][10][15] different bacteria was observed by measuring the Zone of Inhibition in diameter(mm) which was compared with the Culture procurement: Zone of Inhibition of positive control and mean and The pure culture slant of bacterial culture mainly E.coli, standard deviation (n=3 )was calculated.[1] Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella sp. was obtained from the Sikkim Manipal University. Table No. 1:-Zone of Inhibition (mm) test . Test Organism DLEE DSEE WBEE DBME PC S. aureus 22.3mm + 2 8.5 mm + 0.8 14.3mm + 0.6 10.7mm + 2 25.3 + 0.58 E. coli 13.3mm + 1.2 13.7mm + 2.5 10mm + 1 13.8mm + 0.2 9.3 + 1.5 P. aeruginosa 7mm + 1 1.7mm + 0.5 6.5m + 1.3 7.5mm + 0.5 17 + 1 K. pneumonia 15.7mm + 2 4.2mm + 1 18mm + 2 18.3mm + 1.5 21.7 + 1.2 Fig. No.2: Zone of Inhibition graph 152 Research Journal of Pharmacognosy and Phytochemistry. 6(4): October-December, 2014, 151-155 Fig No. 3: Percentage inhibition of the crude extracts against bacteria Percent Inhibition:- Relative percent Inhibition:- Relative percentage Inhibition of the test extract was measured using the formula. [6] Relative % Inhibition = 100 x (a - b) (c - b) Relative percentage inhibition of the test extract = Where, a : total area of inhibition of the test extract b : total area of inhibition of the solvent c : total area of inhibition of the standard drug The total area of the inhibition was calculated by using area = πr2; where, r = radius of zone of Inhibition Table 2 Sl. Microorganism Percent Inhibition No. DLEE DTEE WBEE DBME 1 Staphylococcus 77.8 11.3 32.01 17.72 aureus 2 Escherichia coli 47.6 49.9 26.8 51.2 3 Pseudomonas 16.9 0.96 14.6 19 aeruginosa 4 Klebsiella 52.2 3.7 69 71.6 pneumoniae Antioxidant Profiling:- Free Radical scavenging activity by DPPH method:- The antioxidant assay of the extracts was done by DPPH method with slight modification. [2][7] 0.006 mM DPPH solution was freshly prepared using methanol as solvent and stored in dark for further use. The µg/µl stock solution of each extracts viz; DLEE, DTEE, WBME, DBEE and standard as Ascorbic acid and BHT were prepared in methanol and these samples and the standards were used for free radical scavenging activity by DPPH method. 153 Research Journal of Pharmacognosy and Phytochemistry. 6(4): October-December, 2014, 151-155 Table No. 3:- IC50 value (µg/µl) using Graphpad prism software (version 6.04) Sl. No Sample IC50 (µg/µl) 1 DLEE 481.8 2 DTEE 497.1 3 WBEE 442.9 4 DBME 417.8 RESULT AND OBSERVATION:- The maximum zone of inhibition was seen against S.aureas by dried twig ethanol extract which was 22.3mm + 2 and the minimum zone of inhibition was seen against P.aeruginosa by dried leaves ethanol extract which was 1.7mm + 0.5. Maximum relative percentage inhibition was shown by dried leaves ethanol extract(DLEE) against S.aureas which was around 77.8% and the minimum relative inhibition was shown against P. aeruginosa which Fig. No. 4: Highest zone of Inhibition observed for four different was around 0.9% by dried twigs ethanol extract.(DTEE) crude extract against bacteria, 1) DLEE = S. aureus, 2) DSEE=E. coli, 3) WBEE=Klebsiella sp., 4)DBME= E. coli Antioxidant activity of the extracts was determined by calculating the percentage inhibition. Percentage inhibition Different concentration of aliquots of solution ranging of 100 µg/µl conc. of the DLEE which was the lowest from 100µl,200 µl,300 µl,400 µl,500 µl,600 µl,700 µl,800 concentration is 64.7 + 0.02 and that of 800 µg/µl which µl and 900 µl was prepared in test tubes and finally was was the highest concentration is 72.3 + 0.04 .In case of made it up to 1ml by adding methanol.2ml of freshly DTEE, the percentage inhibition of the lowest prepared DPPH was added to each test tubes and was concentration 100 µg/µl was 61 + 0.01 and that of highest incubated in dark at room temperature for 30 minutes.