ANTIOXIDANT AND ANTIMICROBIAL ACTIVITIES OF ... - wjpps

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES Kaladhar et al.

World Journal of Pharmacy and Pharmaceutical Sciences

Volume 3, Issue 5, 1180-1189.

Research Article

ISSN 2278 – 4357

ANTIOXIDANT AND ANTIMICROBIAL ACTIVITIES OF RHIZOME EXTRACTS OF Kaempferia galanga Narasinga Rao V, DSVGK Kaladhar* Dept. of Biochemistry, GIS, GITAM University, Vishakhapatnam, AP, India

Article Received on 06 March 2014, Revised on 20 March 2014, Accepted on 24 April 2014

ABSTRACT Several Zingiberaceae family members show active principles in treatment of various human ailments. An experiment on antioxidant and antimicrobial activity in rhizome extracts of Kaempferia galanga has been conducted. The Kaempferia galanga rhizome extracts shown

*Correspondence for Author Dr. Kaladhar Dowluru Dept. of Biochemistry, GIS,

antioxidant activity of the tested plant extracts (IC50: 490µg/ml to 720µg/ml) is less compared to the ascorbic acid (IC50: 230µg/ml).

GITAM University,

Methanol, ethylacetate, ethanol and aqueous rhizome extracts has

Vishakhapatnam, AP, India

shown good antimicrobial activity against tested microbes (9.5 to 22mm zone diameter). The extracts was shown good antimicrobial

activity against fungi (11 to 22mm) when compared with the gram negative bacteria (9.5 to 15mm) and gram positive bacteria (9.5 to 13). Hence Kaempferia galanga rhizome extracts was shown good antioxidant and antimicrobial activities. Key words: Kaempferia galanga, solvent extraction, antioxidant and antimicrobial studies INTRODUCTION Plants have a precious medicinal source of natural products for maintain high-quality of human health with added intensive studies for natural therapies [1]. The use of plant metabolites for pharmaceutical implements has progressively increased in India [2]. Microorganisms comprise the genetic capability to transmit and attain resist to antibiotics, which are used as therapeutic agents [3]. In the present years, microbial resistance is developing and applying of antimicrobial agents in the prospect is still uncertain [4]. Thus, the therapeutic actions must be taken to diminish these problems and to supervise the use of antibiotics. Highly developed research strategies can make available improved understanding in the genetic mechanisms of resistance in microbes. The crucial aim is to produce a suitable

www.wjpps.com

Vol 3, Issue 5, 2014.

1180

Kaladhar et al.


and efficient antimicrobial drug to the patients [5]. About 75% of individuals use traditional medicine, which are resultant from medicinal plants in a variety of developed countries [6]. Hence, the search of different phyto-components from medicinal plants should be to recognize the properties and efficiency with possible treatment against diseases [7]. The family Zingiberaceae has enormous number of medicinal plants and is well known for use in ethnomedicine. The wisdom of Zingiberaceae members indicates systematically analyzes compounds for the management and prevention of various human ailments from India. The phytochemical assessments from the plants complete humans for studies in biodiversity and its protection for future pharmacological studies [8]. A number of medicinal plants have good sources with nutrient and non nutrient molecules, lots of which have anti-fungal, anti-bacterial, anti-oxidant and anti-viral properties that can guard the human system against cellular oxidation reactions and pathogens [9]. Essential oils have good anti-fungal, anti-viral, insecticidal, anti-bacterial and antioxidant properties. Several oils are used in various skin disorders [10]. Essential oils are also having industrial applications like fragrance, food preservation and aromatotherapy [11]. The essential oils of aromatic plant extracts are rich sources of biologically vigorous compounds which are enhanced interest in looking at anti-microbial properties [12]. So, it is sensible to anticipate a diversity of plant compounds in these oils with the appropriate general antibiotic potential and antimicrobial activity [13]. The severe infections caused by various disease causing microorganisms enhanced and an important cause of morbidity and mortality patients in worldwide [14]. Nearly three quarters of the population in developing countries used in their traditional medicinal system depends upon plant based on measures and as the basic requirements for human primary health care [15]. Hence, several medicinal plants need to be necessary possible antimicrobial activity evaluation and to get admirable remedy for a microbial origin from a variety of ailments [16]. The living organisms protected from several antioxidants and prevent scratches caused by uninhibited formation of reactive oxygen species; concurrent lipid per oxidation, protein damage and DNA strand breakage [17]. Antioxidant that can decrease free radicals activity can prevent the oxidation of various molecules and may possibly have health promoting special effects in the interference of degenerative diseases [18]. In accumulation there is a contrary relationship between the frequency of human diseases and dietary intake of

www.wjpps.com

Vol 3, Issue 5, 2014.

1181

Kaladhar et al.


antioxidant rich food [19]. The natural antioxidants had shown a extensive range of biochemical activities, like inhibition of Reactive Oxygen Species (ROS) making, through direct or indirect scavenge of free radicals, and differences of intra cellular redox potential [20]. The ROS are free radicals such as superoxide anion radicals (O2-), hydroxyl radicals (OH-) and other non-free-radical species such as H2O2 and singlet oxygen (1O2) are several forms of activated oxygen [21]. The importance of free radicals provided increasing attention over the decades in understanding antioxidant activity. The cellular injury and in ageing process these molecules are exacerbating factors [22]. ROS have important interest between scientist and their extensive range of property on biological and medicinal systems studied in many experimental investigations [23]. In living organisms, several ROS can form signaling networks in knowing different components used in different traditions. Normal aerobic respiration stimulates macrophages, peroxisomes and polymorph nuclear leukocytes that emerge to be the chief endogenous sources of mainly the oxidants formed by cells. The exogenous or external sources of ROS include certain pesticides, tobacco smoke, pollutants and organic solvents [24]. Ageing is one of the unique features in all living organisms. The impaired functions of many systems characterize aging. The impairments occur in the brain causes susceptibility to neurodegenerative diseases that amplifies significantly. The free radical presumption of aging posits the functional impairments in brains that are due to the attack on serious cellular mechanism by reactive nitrogen species, free radicals and ROS produced during normal metabolism [25]. MATERIALS AND METHODS Collection of Plant Material Fresh plants consisting of rhizomes of Kaempferia galanga (K. galanga or KG) were collected from Visakhapatnam District, Andhra Pradesh and some regions of Kerala during July and August 2011. The plant is authenticated by Dr. P.V. Arjun Rao, Ethanobotanist, Dept. of Botany, Phytopharma Technology Laboratory, Visakhapatnam (No. Res/1 dated 2109-2010). K. galanga is confirmed based on details given in “Flora of the Presidency of Madras” by J.S. Gamble, Vol. iii, page numbers 1483-1484, Bishen Singh Mahendra Pal Singh Publishers, India (2004). Fully matured rhizomes were washed systematically and

www.wjpps.com

Vol 3, Issue 5, 2014.

1182

Kaladhar et al.


dried out in sunlight. The desiccated plant rhizomes were grinded to powder and about 150gms of the dehydrated plant material were used for the extraction. Extraction Process Nearly 30g of air dried powder were taken in 200ml of ethyl acetate, methanol, ethanol and aqueous separately, plugged with cotton wool and then kept on orbital shaker for 48 hours with 150rpm at room temperature. The extracts were filtered with whatmann no 1 filter paper and collect the supernatant. Then solvent evaporated through rotavapour and make the final volume one-fourth of the original volume and stored at the 4oC in air tight containers. Antioxidant Activity with DPPH Chemicals & Reagents Standard: Ascorbic acid a. 100µM of DPPH (1,1-diphenyl-2-picrylhydrazyl): Nearly 3.9432 mg of DPPH was dissolved in 3ml of methanol and made up to 100 ml to obtain a final concentration of 100µM. b.Stock solutions of test items: 3mg/ml stock solutions were prepared in DMSO. c. Test items preparation: Appropriate dilutions (0 to 1000µg/ml) of test items were prepared. The reaction mixture was made by addition of 20µl of test items and 280µl of DPPH reagent to reach a final volume of 300µl and kept incubation in dark for 50 minutes and then absorbance was read at 517 nm using spectrophotometer. An IC50 value was determined as the concentration that elicits the half maximal response. Antimicrobial Activity The antimicrobial activity was conducted based on zone method. Microorganisms Microbes from ATCC (American Type Culture Collection), USA have been used in the present study. Several bacteria used in the current research work are Bacillus subtilis (ATCC 6051) and Klebsiella pneumonia (ATCC 13883) belongs to gram positive bacteria, and Serratia marcescens (ATCC 14756), Pseudomonas aeruginosa (ATCC 15442), Enterobacter aerogenes (ATCC 13048) and Escherichia coli (ATCC 25922) belong to gram negative bacteria. Fungi used in the work are Aspergillus niger (ATCC 6275) and Candida albicans (ATCC 10231).

www.wjpps.com

Vol 3, Issue 5, 2014.

1183

Kaladhar et al.


Antimicrobial Activity Using Zone Method Antimicrobial activity has been conducted based on zone method. The medium used in this experimentation are Muller-Hinton Agar (Hi Media Pvt. Ltd., Mumbai., India) for bacteria grown at 370C for 24 hours and fungi in Sabourand Dextrose Agar (Hi Media Pvt. Ltd., Mumbai., India) at 250C for 48 hours. The freshly prepared nutrient broth has been prepared and inoculated the broth cultures. The broth was inoculated into growth medium. The growth medium has prepared in the petriplates. Wells of 8mm size were made in the growth media with sterile borer. Nearly 50µl of extracts were added to the wells of growth media. The MHA (Muller-Hinton Agar) plates of bacteria were incubated at 370C for 24hrs. The Sabourand Dextrose Agar plates were incubated using micro pipette at 250C for 72 hours for fungi. After incubation diameter of zones of inhibition was measured using Hi Media zone reader in mm. RESULTS AND DISCUSSION India has rich sources of medicinal plants shows important part of life from ancient times. An important part of this indigenous knowledge was consistent from the past into the organized systems of medicines such as Sidha, Yunani and Ayurveda or other systems (Chetna and Anoop, 2009). Caffeic acid (3, 4-dihydroxycinnamic acid) is a powerful antioxidant isolated from plants (Gülçin, 2006). One such plant Kaempferia galanga belongs to Zingiberaceae family revealed to be having antioxidant and antimicrobial activities by present investigation. K. galanga is a precious medicinal plant, which is rarely available in few countries and becoming extinct due to change of climatic conditions and human factors. These plant rhizomes shown good secondary metabolite products and medicinal activities have not been widely recognized. Currently the entire plant K. galanga are used for antifungal and antibacterial properties for extracting plant metabolites against fungi and bacteria using agar diffusion and the zones of inhibition methods. The antioxidant activity can be calculated using DPPH method. The K. galanga rhizome extracts shown antioxidant activity with IC50 value for ethanol is 490µg/ml, 590µg/ml for methanol, ethyl acetate is 640µg/ml and aqueous extract shown is 720µg/ml. The IC50 value for standard (ascorbic acid) obtained in the experimentation is 230µg/ml (Figure 1, Table 1). Though the antioxidant activity of the tested plant extract is less compared to the standard, the plant has shown good antioxidant activity

www.wjpps.com

Vol 3, Issue 5, 2014.

1184

Kaladhar et al.


Table 1: Antioxidant activity (IC50) for crude rhizome extracts of K. galanga Table 1: Percentage of Inhibition of DPPH radical for antioxidant activities of K. galanga Log conc. Ethanol (µg/ml) 0 31.25 62.5 125 250 500 1000

0 8.39 16.33 27.45 38.66 50.45 89.54

Methanol

0 6.45 12.23 23.44 35.24 44.44 76.33

Ethyl acetate 0 4.44 11.43 22.75 30.33 42.33 72.33

Aqueous

0 4.02 8.02 17.34 27.34 39.43 65.43

Standard (Ascorbic acid) 0 23.13 34.33 41.67 52.73 66.23 92.78

The plant rhizome extracts were analyzed for antimicrobial activity against various test microorganisms. All the prepared extracts were shown fine antimicrobial activity (Table 2). Ethyl acetate, Ethanol, Methanol and aqueous rhizome extracts of K. galanga was revealed good antimicrobial activity against the tested bacteria (9.5 to 15 mm). The extracts have shown good activity against fungi (11 to 22) while compared with gram negative bacteria (9.5 to 13) and gram positive bacteria (10 to 15mm). The zone of inhibition for the bacterial standard, tetracycline has shown inhibition zone from 10 to 44 mm at 50µg/ml. The zone of inhibition for fungal standard, Fluconazole has shown inhibition zone for 13 and 16 mm for Aspergillus niger and Candida albicans respectively at 50µg/ml. Antifungal medicinal plants belong to the Zingiberaceae family has also been previously reported in plants like Curcuma longa L., Curcuma zedoaria Rosc., Curcuma malabarica Vel., and A. galanga supports the use of their rhizomes in traditional medicine for the treatment of fungal and bacterial infections [26].

www.wjpps.com

Vol 3, Issue 5, 2014.

1185

Kaladhar et al.


Table 2: Antimicrobial activity for crude rhizome extracts of K. galanga Note: Standard antibiotic for bacteria is Tetracyclin and for fungi is Fluconazole Microorganism

Zone of inhibition in mm (including well size of 8mm) at 50 µg/ml Methanol Ethanol Ethyl Aqueous Antibiotic Control extract extract acetate extract (DMSO) extract

Bacteria Bacillus subtilis Klebsiella pneumoniae Serratia marcescens Pseudomonas aeruginosa Enterobacter aerogenes Escherichia coli Fungi Aspergillus niger Candida albicans

11 12 13 9.5 11

12.5 11.5 13 11 12

15 15 12 9.5 11

11 10 10.5 10 9.5

26 31 11 38 44

Nil Nil Nil Nil Nil

10.5

12

11

10

10

Nil

18 20

20 21

22 21

13 11

13 16

Nil Nil

K. galanga belongs to Zingiberaceae family used in the treatment of several diseases. Most of the medicinal plants are widely used for the treatment of several diseases in India and China [27]. The practice of plants like turmeric may decrease the risk of different kinds of cancers and provide other protective biological effects in humans. These biological effects are due to constituent curcumin that widely studied for its anti-oxidant, wound healing, anti-cancer and anti-inflammatory effects [28]. The possible compounds for developing antimicrobials from plants appear valuable, as it will lead to the enhancement of a phytomedicine to act against microbes. The Plant based antimicrobials have immense therapeutic potential as they can provide the purpose with slighter side effects that are frequently associated with synthetic antimicrobials [29]. CONCLUSION The crude rhizome extracts of K. galanga has shown antioxidant activity. The K.galanga has shown extensive antimicrobial activity and hence the naturally available crude rhizome extracts of K. galanga could be potential alternative against microbes. Hence the crude rhizome extracts of K. galanga shows antioxidant and antimicrobial components. ACKNOWLEDGEMENT The author would like to thank GITAM University for providing lab facility and access to ejournals to carry out the entire research work.

www.wjpps.com

Vol 3, Issue 5, 2014.

1186

Kaladhar et al.


REFERENCES 1. Grabley S. and Isabel S. Natural products for lead identification: nature is a valuable resource for providing tools. Modern Methods of Drug Discovery. Birkhäuser Basel. 2003; pp. 87-107. 2. Smetanska I. Production of secondary metabolites using plant cell cultures. In Food Biotechnology .2008; pp. 187-228. Springer Berlin Heidelberg. 3. Chopra I, Roberts M. Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiology and Molecular Biology Reviews. 2001; 65(2), 232-260. 4. Talbot G H, Bradley J, Edwards J E., Gilbert D, Scheld, Bartlett J G. Bad bugs need drugs: an update on the development pipeline from the Antimicrobial Availability Task Force of the Infectious Diseases Society of America. Clinical infectious diseases. 2006; 42(5), 657-668. 5. Dellit T H, Owens R C, McGowan J E, Gerding D N, Weinstein R A, Burke J P , Huskins, WC, Paterson DL, Fishman NO, Carpenter CF,Brennan PJ, Billeter M. and Hooton TM. Infectious Diseases Society of America and the Society for Healthcare Epidemiology of America guidelines for developing an institutional program to enhance antimicrobial stewardship. Clinical Infectious Diseases. 2007; 44(2), 159-177. 6. Fabricant D S, Farnsworth N R. The value of plants used in traditional medicine for drug discovery. Environmental health perspectives .2001; 109(Suppl 1), 69. 7. Nascimento GG, Locatelli J, Freitas PC, Silva GL. (Antibacterial activity of plant extracts and phytochemicals on antibiotic-resistant bacteria). Brazilian

journal of 72

microbiology, 2000; 31(4):247-56. 8. Basak S, Sarma GC , Rangan

L. Ethnomedical uses of Zingiberaceous plants of

Northeast India. Journal of ethnopharmacology. 2010;132(1), 286-96, 9. Sengul M, Yildiz H, Gungor N, Cetin B, Eser Z, Ercisli S. (Total phenolic content, antioxidant and antimicrobial activities of some medicinal plants). Pak. J. Pharm. Sci, 2009; 22(1):102-6. 10. Megraj, K V K, Koneri Raju R, Meenakshisundaram K. Biological Activities of Some Indian medicinal plants. Journal of Advanced Pharmacy Education & Research. 2011; 1, 12-44. 11. Christaki E, Bonos E, Giannenas I, Florou-Paneri P. Aromatic Plants as a Source of Bioactive Compounds. Agriculture. 2012; 2(3), 228-243.

www.wjpps.com

Vol 3, Issue 5, 2014.

1187

Kaladhar et al.


12. Prabuseenivasan S, Jayakumar M, Ignacimuthu S. In vitro antibacterial activity of some plant essential oils. BMC Complementary and Alternative Medicine. 2006; 6(1), 39. 13. Darokar MP, Mathur A, Dwivedi S, Bhalla R, Khanuja SPS, Kumar S (Detection of antibacterial activity in the floral petals of some higher plants). Curr Sci, 1998; 75:187. 14. Al-Bari

MAA, Sayeed MA. and Rahman MS. Characterization and antimicrobial

activities of a phenolic acid derivative produced by Streptomyces bangladeshiensis: A novel species collected in Bangladesh. Res J Med Sci., 2006; Vol. 1, pp. 77–81, 15. Mthethwa N S. Antimicrobial activity testing of traditionally used plants for treating wounds and sores at Ongoye area KwaZulu-Natal, South Africa (Doctoral dissertation, University of Zululand). 2009. 16. Ubramani SP, Goraya GS.( Some folklore medicinal plants of Kolli hills: Record of a Watti vaidyas Sammelan). J Econ Taxon Bot. 2003;27: 665–78. 17. Gill SS, Tuteja N. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiology and Biochemistry. 2010; 48(12), 909-930. 18. Young I S, Woodside JV. Antioxidants in health and disease. Journal of clinical pathology. 2001; 54(3), 176-186. 19. Leonard SS, Cutler D, Ding M, Vallyathan V, Castranova V, Shi X.( Antioxidant properties of fruit and vegetable juices: More to the story than ascorbic acid). Ann Clin Lab Sci. 2002; 32:193–200. 20. Ju E M, Lee S E, Hwang H J, Kim J H. Antioxidant and anticancer activity of extract from< i> Betula platyphylla var.< i> japonica. Life sciences. 2004; 74(8), 10131026. 21. Gülçinİ.(Antioxidantactivityofcaffeicacid(3,4dihydroxycinnamicacid). Toxicology. 2006;217(2):213-20. 22. Lai L S, Chou S T, Chao WW. Studies on the antioxidative activities of Hsian-tsao (Mesona procumbens Hemsl) leaf gum. Journal of Agricultural and Food Chemistry. 2001; 49(2), 963-968. 23. Oktay M, Gülçin I, Küfrevioğlu Öİ. Determination of in vitro antioxidant activity of fennel (< i> Foeniculum vulgare) seed extracts. LWT-Food Science and Technology. 2003; 36(2), 263-271. 24. Kumar RS, Sivakumar T, Sunderam RS, Gupta M, Mazumdar UK, Gomathi P, Kumar KA. (Antioxidant and antimicrobial activities of Bauhinia racemosa L. stem bark). Brazilian journal of medical and biological research, 2005; 38(7):1015-24.

www.wjpps.com

Vol 3, Issue 5, 2014.

1188

Kaladhar et al.


25. Poon HF, Calabrese V, Scapagnini G, Butterfield D A (Free radicals: key to brain aging and heme oxygenase as a cellular response to oxidative stress). The Journals of Gerontology Series A: Biological Sciences and Medical Sciences,2004;59(5):M478M493 26. Maria JA, María A, and Paulina B. (Active antifungal substances from natural sources). ARKIVOC 2007; (vii): 116-45. 27. Kaladhar DSVGK, Harasreeramulu S, Duddukuri GR, et al. In Vitro Regeneration of the Medicinal Herb, Evolvulus Nummularius L. From Shoot Tip and Flower Explants. Indian Journal of Fundamental and Applied Life Sciences. 2011; 1 (2): 81-89. 28. Maheshwari RK, Singh AK, Gaddipati J, et al. Multiple biological activities of curcumin: a short review. Life sciences. 2006; 78(18): 2081-2087. 29. Martins AP, Salgueiro L Goncalves MJ. (Essential oil composition and antimicrobial activity of three Zingiberaceae from S.Tomee Principle). Planta med. 2001; 67:580-84.

www.wjpps.com

Vol 3, Issue 5, 2014.

1189