Short Communication DOI: http://dx.doi.org/10.18320/JIMD/201502.02104 Journal of International Medic ine and Dentistry 201 5; 2(2): 104-110 JOURNAL OF INTERNATIONAL MEDICINE AND DENTISTRY To search……………..to know………..…….to share ISSN.No. 2350-045X Anticandidal effect of extract of Bridelia s tipularis Sachidananda Mallya P 1, Sudeendra Prabhu 2, Maji Jose 3, Shrikara Mallya P 4 Abstract: Medicinal and aromatic plants are gift of nature and are being used against various infections and diseases in the world since ages. Species of the genus Bridelia stipularis var scandens is reported to be used traditionally for treating various oral diseases . However, the antimicrobial effect of t hese plant materials against oral pathogens is not proved. Therefore, we have done the present study. Aim is to find out the anticandidal effect of water extract of Bridelia stipularis against four common oral candidal p athogens. The leaves after identif ication and authentication by a b otanist were collected, air dried, pulverized to fine powder using household blender. The water extract was prepared using cold percolation method. The standard Candida species, Candida albicans , Candida parapsilosis , Candida glabrata and Candida tropicalis obtained from P ost Graduate Institute (PGI) , Chandigarh was procured . Antifungal activity was determined by Kirby B auer well diffusion method and Time kill assay. All four species of Candida showed variable results w ith diameter of zone of inhibition ranging from 12mm to 21mm on Sabouraud’s dextrose agar with both 6 hour and 24 hour peptone water subculture. Time kill assay showed inconsistent result s even after 24 hours of exposure with the crude extract of Bridelia stipularis . All four species of Candida causing oral infections are moderately sensitive to crude water extract of dried leaves of Bridelia stipularis. Key words: Bridelia stipularis , Candida species, antifungal activity Introduction: Use of medicinal plants for routine Herbal medicine has been known to man for cleaning of teeth and treating various oral centuries. The therapeutic efficacies of many diseases has long been a part of Indian indigenous plants for several disorders have culture, and Karnataka has its own been described by the practi tioners of tradition. 1,2 Species of the genus Bridelia traditional medicine. Natural products belonging to th e family Euphorbiaceae, are currently are the leading source in the searc h commonly seen in various parts of Indian for new biologically active compounds .It has sub continent and is reported to be used been well documented that traditional traditionally for treating dental decay and medicinal plants confer considerable related pain by people of Dakshina antibacterial activity against various Kannada. 3 Previous studies have revealed microorganisms. Many plants were reported to that this plant has many pr operties such as inhibit the growth of many oral bacteria antibacterial effects against various gram particularly Streptococcus mutans and control positive and gram negative organisms. 4,5,6 plaque and thus prevent caries. Use of plant However, the beneficial effects of this based alternatives for oral health has been plant materials against oral pathogens is successfully promoted; for example, the use of not e valuated. Therefore , we have done the antibacterial chewsticks (meswak) have been present investigation to exa mine the widely advocated by health agencies where antimicrobial activities of extract of leaves their use is culturally acceptable. Mallya P S et al: Anticandidal effect of extract of Bridelia stipularis www.jimd.in of Bridelia stipularis on various Candida glabrata (NCCPF 100018) and Candida species. parapsilosis (ATCC 22019) were tested. Materials & Methods: 6 hours and 24 hours peptone water culture of Candida were used. Selection of plant The leaves were collected from the local wild plants and were ide ntified and authenticated by a botanist ( Fig ure I). Figure II: Powder of dried l eaves of Bridelia stipularis Fig ure I: Leaves of Bridelia s tipularis Preparation of extract The collected leaves were washed, air dried at room temperature for two weeks and pulverized to fine powder usin g house hold blender and were stored in air tight bottles till further use ( Fig ure II ). Fig ure III: Extract of leaves prepared The aqueous extract was prepared by cold by c old percolation method percolation method ( keeping the bottle for 48 hours at room temperature with In-vitro studies on antimicrobial activity frequent shaking (Figure III ). of plant extracts Two concentrations were prepared -5gm of Antimicrobial activity was determined by powder suspende d in 50 ml of distilled Kirby Bauer a gar well diffusion water ( 100 mg/ml), and 2.5 gm of powde r technique. 7 The inoculum s ize of candidal in 50 ml of distilled water (50 mg /ml ), culture was standardized according to the filtered, centrifuged at 2000rpm for 10 National Committee for Clinical minutes. Extracts were stored in bijou Laboratory Standards. 8 The peptone water bottle at 4˚C. culture incubated for 6 hours as well as 24 Four standard species of Candida hours were swabbed on separate agar (obtained from PGI Chandigarh) – plates. Wells we re charged with extracts Candida albicans (ATCC 90028), (100mg/ml and 50mg/m l).The plates with Candida tropicalis (ATCC 750), Candida fungal growth were incubated at 37 oC for 48 hours. The antimicrobial activity was 105 Journal of International Medic ine and Dentistry 2015; 2 (2): 104 -110 Mallya P S et al: Anticandidal effect of extract of Bridelia stipularis www.jimd.in evaluated by measuring the diameter of MIC determination was performed by zone of inhibition expressed in millimeters broth microdilution method in microtiter against the test pathogens (Figure IV and plates which were inoculated with V). Chlorhexidine disc was used as 0.5x10 3/ml of Candida isolates and positive control and blank disc incubated at 30 oC. Cell density per plate (impregnated with solvent) as negative was measured after 48 hours of incubation. control. MIC was defined as the lowest The effective plant extract was further concentration of extract that inhibits the subjected for determination of MIC by growth. 7 broth dilution method. 7,8 The inhibitory effect of the extract was studied by Time Results: kill assay 9 and determination of Minimum All four Candida species showed varied Inhibitory Concentration (MIC). In Time results in their susceptibility with diameter kill assay 8, the inoculum containing of zone of inhibition ranging from 12- approximately 5x10 cfu /ml was 21mm with both the concentrations of introduced into the Mueller Hinton broth extract as well as in different period of containing various extracts and incubated incubation (Table I and II ). Candida at 37 ˚ C. 500 μl sample was removed from albicans showed variable results on plates culture at 6, 12, 18 and 24h, diluted swabed with 6 hour peptone water culture serially and 100 μl of the diluted samples in both the concentration of extract. were inoculated on Mueller Hinton agar Candida parapsilosis showed no growth. and Sabouraud’s dextrose agar plate, There was not much difference in zone incubated at 37 oC for 24hours. Control size of all Candida species with both the included extract free Mueller Hinton broth concentration of extract. Determination of seeded with the test inoculum. Viable Minimum Inhibitory Concentration ( MIC) counts were calculated to give cfu/ml. and Time kill assay (Figure VI ) even after There was not much difference in test as 24 hours of incubation showed well as control plate. inconsistent results. Table I: Antimicrobial activity of extract (100mg/ml) against Candida species Candida Species Zone of Inhibition ( 100mgm/ml) (5gm+50 ml) 6 hours 24 hours of incubation Candida albicans Variable 20 mm Candida tropicalis 18 mm 20 mm Candida glabrata 12 mm 15 mm Candida parapsilosis No growth 21 mm 106 Journal of International Medicine and Dentistry 2015; 2 (2): 104-110 Mallya P S et al: Anticandidal effect of extract of Bridelia stipularis www.jimd.in Figure IV: Zone of i nhibition with 100mg /m l of extract after 6 hours of i ncubation Figure V: Zone of inhibition with 100mg/ml of extract after 24 hours of incubation 107 Journal of International Medic ine and Dentistry 2015; 2 (2): 104 -110 Mallya P S et al: Anticandidal effect of extract of Bridelia stipularis www.jimd.in Table II: Antimicrobial activity of extract(50mg/ml) against Candida species Candida species Zone of Inhibition ( 50 mgm/ml ) ( 2.5 gm + 50 ml) 6 hours 24 hours of incubation Candida albicans Variable 20 mm Candida tropicalis 18 mm 18 mm Candida glabrata 16 mm 18 mm Candida parapsilosis No growth 19 mm Figure VI: Time Kill Assay Discussion: types of oral ailments like toothache, plaque and caries, pyorrhea and aphthae. The antibacterial activity of Bridelia Sixteen of these plants were new for the stipularis var scandens has been reported treatment of oral ailme nts and not by many workers. previously reported. Basella alba, Hebbar et al covered eth nomedicine Blepharis repens, Capparis sepiaria, Oxalis survey of plants used in oral care in corniculata and Ricinus communis are Dharwad district , Karnataka, India . It was used for the treatment of aphthae; Azima revealed that 35 plants belon ging to 26 tetracantha, Caesalpinia coriaria,Cleome families are being used to treat different gynandra, Gossypium herbacium, Leucas aspera, Merremia chryseides, Pergularia 108 Journal of International Medic ine and Dentistry 2015; 2 (2): 104 -110 Mallya P S et al: Anticandidal effect of extract of Bridelia stipularis www.jimd.in daemia, Prosopis juliflora and Solanum Adetutu et al concluded that the nigrum are used to treat tooth ache and antimicrobial and relatively strong Cassia hirsuta and Cassia tora are used in antioxidant activity lend some support to the treatment of plaque and caries.1 the topical use of Bridelia ferruginea leaf V H Harsha et al used 45 species of plants for wound healing in the traditional used by Kunabi community people based medicine.