Ethnobotanical Leaflets 14: 402-412, 2010.

In Vitro Antimicrobial Activity of Sapindus mukorossi and Emblica officinalis Against Dental Caries Pathogens

Kamal Rai Aneja, Radhika Joshi* and Chetan Sharma

Department of Microbiology, Kurukshetra University, Kurukshetra -136119. India

Corresponding author Email:

Issued: April 01, 2010

Abstract

The in vitro antimicrobial activity of Sapindus mukorossi and Emblica officinalis fruit extracts were studied against Streptococcus mutans, Staphylococcus aureus, Lactobacillus acidophilus, Candida albicans and Saccharomyces cerevisiae. The acetone, ethanol, methanol, hot water and cold water extracts of S.mukorossi exhibited antimicrobial activity against one of the tested microorganisms i.e. S.cerevisiae. All the five extracts of E.officinalis showed inhibitory activity against S.mutans while the acetonic, hot and cold aqueous extracts showed inhibitory activity against S.aureus also. The largest zone of inhibition was obtained with the acetonic extract of S.mukorossi against S.cerevisiae (29.65mm) and hot water extract of E.officinalis against S.aureus (40.32mm). Minimum inhibitory concentrations (MIC) of the extracts were also determined against the selected microorganisms showing zones of inhibition 8mm. This study depicts that the fruits of Sapindus mukorossi and Emblica officinalis possess very good antifungal and antibacterial activities respectively and can be used as a potential source of novel antimicrobial agents used to cure dental caries.

Key words : Dental caries, Sapindus mukorossi, Emblica officinalis, antimicrobial activity, zone of inhibition, minimum inhibitory concentration.

Introduction

Dental caries is a very common problem that affects all age groups. It is a process in which the enamel and the dentine are demineralised by acids produced by bacterial fermentation of carbohydrates (de Soet and de Graff, 1998). In real life, it is the most common infectious disease affecting human beings (Balakrishnan et al., 2000). Medicinal plants since ancient times have been employed for prophylactic and curative purposes (Joshi and Joshi, 2005; Amadi et al., 2007). The present study reports the antimicrobial activity of five different solvent extracts of Sapindus mukorossi and Emblica officinalis on dental caries causing microorganisms.

Sapindus mukorossi Gaertn., a member of the family Sapindaceae, is commonly known by several names such as soapnut, soapberry, washnut, ritha, reetha, aritha, dodan and doadni. It is a deciduous tree widely grown in upper reaches of Indo-Gangetic plains, Shivaliks and sub Himalayan tracts at altitudes from 200m to 1500m. It is one of the most important trees of tropical and sub-tropical regions of Asia, usually with straight trunk, nearly 4-5m in height, pinnately compound leaves and lanceolate leaflets. Flowers are small, terminal and polygamous. The fruit is valued for the saponins (10.1 %) present in the pericarp and constitutes up to 56.5 per cent of the drupe known for inhibiting tumor cell growth. The fruits are expectorant, emetic, contraceptive, for treatment of excessive salivation, epilepsy, to treat chlorosis, head lice, migranes, eczema, psoriasis and for removing freckles (Kirtikar and Basu, 1991). The powdered seeds are employed in the treatment of dental caries, arthritis, common cold, constipation and nausea (Dhar et al., 1989). It cleanses the skin of oily secretion and is even used as a cleanser for washing hair and a hair tonic, and forms a rich, natural lather.

Emblica officinalis Gaertn., a member of the family Euphorbiaceae, is commonly called by several names such as amalaka, aavalaa, amla, amlaki and Indian gooseberry. It is found throughout India, Pakistan, Bangladesh, China, Sri Lanka and the Malayan Peninsula. It is a deciduous tree, 8-15 m tall, with alternate, subsessile leaves and greenish to creamy-yellow, unisexual, actinomorphic, trimerous flowers (Warrier et al., 1995). The fruit is a very rich source of Vitamin C (600mg/100g). The leaves, bark, fruit and root bark are used to cure many diseases (Dey, 1980). The fruit has anti-viral, antibacterial, anti-cancer, anti-allergy, and anti-mutagenic properties. It is rich in phenols, tannins, polyphenols, flavonoids, kaempferol, ellagic acid and gallic acid (Nair and Chanda, 2007).

Materials and Methods

Sapindus mukorossi and Emblica officinalis fruits were obtained from the local market of Delhi, India. Dr. B.D.Vashistha, Botany Department, Kurukshetra University, Kurukshetra confirmed the identification of the samples.

Extraction of plant material

The samples were carefully washed under running tap water followed by sterile distilled water. These were air dried at room temperature (40^OC) for five days and pulverized to a fine powder using a sterilized mixer grinder and stored in air-tight bottles. Four different solvents namely ethanol, methanol, acetone and aqueous (hot and cold) were used for extraction. A 10g amount of pulverized fruits was separately soaked in 100ml of acetone, ethanol, methanol (100% each) and cold sterile distilled water for 24h. Also the same amount (i.e. 10g) of pulverized fruits was immersed in 100ml of hot sterile distilled water and allowed to stand for 30min on a waterbath with occasional shaking and kept undisturbed for 24h. Each preparation was filtered through a sterilized Whatman No.1 filter paper (Ogundiya et al., 2006) and the filtered extract was concentrated under vacuum below 40^oC using Heidolph, VE-11 rotaevaporator (Bag et al., 2009). The dried extract thus obtained was exposed to UV rays for 24hrs and checked for sterility on nutrient agar plates and stored in labelled sterile bottles in a freezer at 4^oC until further use (Nkere and Iroegbu, 2005).

Test Microorganisms

Three dental caries causing bacteria Streptococcus mutans (MTCC*497), Staphylococcus aureus (MTCC 740), Lactobacillus acidophilus (MTCC *447) and two yeasts Candida albicans (MTCC 227) and Saccharomyces cerevisiae (MTCC 170) were procured from Microbial Type Culture Collection, IMTECH, Chandigarh. The microorganisms were subcultured on the specific media recommended for different microorganisms such as Brain heart infusion agar (S.mutans), Nutrient agar (S.aureus), Lactobacillus MRS agar (L.acidophilus), Malt yeast agar (C.albicans and S.cerevisiae) and incubated aerobically at 37^OC. The media were procured from HiMedia Laboratory Pvt. Ltd., Bombay, India. Identification of all the strains was confirmed by standard biochemical and staining methods (Aneja, 2003; Benson, 2004; Cappuccino and Sherman, 1995).

Antimicrobial Assay

The acetone, methanol, ethanol, cold and hot water extracts were used for the screening. Antimicrobial activity of various extracts was determined by the agar well diffusion method (Okeke et al., 2001). In this method, pure isolate of each microbe was subcultured on the recommended specific media for each microorganism at 37^OC for 24hrs. A plate of each microorganism was taken and a minimum of four colonies were touched with a sterile loop and transferred into normal saline (0.85%) under aseptic conditions. Density of each microbial suspension was adjusted equal to that of 10⁶ cfu/ml (standardized by 0.5McFarland standard) and used as the inoculum for performing agar well diffusion assay (Aneja et al., 2010). One hundred microlitre (100l) of inoculum of each test organism was spread onto the specific media plates so as to achieve a confluent growth. The agar plates were allowed to dry and wells or cups of 8mm were made with a sterile borer in the inoculated agar plates and the lower portion of each well was sealed with a little specific molten agar medium. The extracts were reconstituted in 20% DMSO for the bioassay analysis (Rajasekaran et al., 2008). A 100l volume of each extract was propelled directly into the wells (in triplicates) of the inoculated specific media agar plates for each test organism. The plates were allowed to stand for 10 minutes for diffusion of the extract to take place and incubated at 37^OC for 24h (Aneja et al., 2009; Khokra et al., 2008; Rios et al., 1980). Sterile DMSO (20%) served as the negative control and ciprofloxacin (for bacteria) and amphotericin-B (for fungi) served as the positive control. The antimicrobial activity, indicated by an inhibition zone surrounding the well containing the extract, was recorded if the zone of inhibition was greater than 8mm (Hammer et al., 1999). The experiments were performed in triplicates and the mean values of the diameter of inhibition zones with standard deviation were calculated (Aneja and Joshi, 2009a, b).

Determination of Minimum Inhibitory Concentration (MIC)

MIC is defined as the lowest concentration of a compound/extract/drug that completely inhibits the growth of the microorganism in 24h (Thongson et al., 2004). The MIC was determined by following the modified agar well diffusion method (Rajasekaran et al., 2008). A twofold serial dilution of each extract was prepared by first reconstituting the powder (100:1mg/ml) in 20% dimethylsulphoxide (DMSO) followed by dilution in sterile distilled water (1:1) to achieve a decreasing concentration range of 50mg/ml to 0.39mg/ml. A 100 l volume of each dilution was introduced into wells (triplicate) in the specific media agar plates already seeded with 100l of microbial inoculum adjusted with sterile distilled water equal to that of 10⁶ cfu/ml (standardized by 0.5McFarland standard) of the test microbial strain. All test plates were incubated aerobically at 37^oC for 24 hrs and observed for the inhibition zones. The lowest concentration of each extract showing a clear zone of inhibition (>8mm), considered as the MIC, was recorded for each test organism (Nkere and Iroegbu, 2005; Aneja et al., 2009).

Results and Discussion

The results of antimicrobial activity of the five extracts of S.mukorossi and E.officinalis by agar well diffusion method have been shown in Table 1 and Table 2 respectively. From the data presented in the table 1, it is evident that all the five extracts of S.mukorossi i.e. hot aqueous, cold aqueous, acetonic, methanolic and ethanolic showed antimicrobial inhibitory activity against only one of the five tested dental caries causing microorganism S.cerevisiae, with the mean diameter of the highest zone of inhibition being 29.65mm. S.cerevisiae survived upto 12.5mg/ml in the acetonic extract, thus having a MIC of 25mg/ml (Table 3). Although no inhibitory activity of S.mukorossi was shown against S.mutans, S.aureus, L.acidophilus and C.albicans, the antifungal activity was much higher than the control (amphotericin-B, with the mean diameter being 11.94mm), thus S.mukorossi possesses very good antifungal properties against S.cerevisiae. The major active ingredient responsible for antifungal activity of S.mukorossi might be saponin. Saponins are generally known as nonvolatile, surfaceactive compounds that are widely distributed in nature, occurring primarily in the plant kingdom (Oleszek, 2002). Saponins have a diverse range of properties, including foaming and emulsifying (Price et al., 1987), pharmacological and medicinal properties (Attele et al., 1999), haemolytic properties (Sparg et al., 2004), as well as antimicrobial, insecticidal, spermicidal and molluscicidal activities (Saxena et al., 2004; Shiau et al., 2009).

All the five tested extracts of E.officinalis as presented in table 2 showed antimicrobial activity against S.mutans with the mean diameter of the highest zone of inhibition being 18.96mm and an MIC of 50mg/ml (table 4), as it survived upto 25mg/ml, while the acetonic, hot and cold aqueous extracts of E.officinalis showed excellent inhibitory activity against S.aureus with the mean diameter of highest zone of inhibition being 40.32mm produced by hot aqueous extract (much higher than the positive control ciprofloxacin 34.66mm) and an MIC of 12.5mg/ml, as it survived upto 6.25mg/ml. L.acidophilus, C.albicans and S.cerevisiae did not show any inhibitory activity when assayed against E.officinalis fruit extracts. Thus E.officinalis possesses good antibacterial activity but no antifungal activity. The excellent activity of E.officinalis against S.aureus shows a very good potential of E.officinalis to treat the diseases caused by S.aureus. The possible reason for the antibacterial activity of E.officinalis might be due to the tannins present in its fruits. The fruits have 28% of the total tannins distributed in the whole plant. The fruit contains two hydrolysable tannins Emblicanin A and B, which has antioxidant properties, one on hydrolysis gives gallic acid, ellagic acid and glucose wherein the other gives ellagic acid and glucose. The fruit also contains Phyllemblin (Wealth of Asia, 1998; Ghosal, 1996; Dictionary of Indian Medicinal Plants, 1988).

Table 1: Antimicrobial activity of fruit extracts of Sapindus mukorossi on dental caries causing microorganisms determined by agar well diffusion method on specific media for each test microorganism.

(-) = no activity, nt = not tested, S.m. = Streptococcus mutans, S.a.= Staphylococcus aureus, L.a.= Lactobacillus acidophilus, C.a.= Candida albicans, S.c.= Saccharomyces cerevisiae, ^* Values, including diameter of the well (8 mm), are means of three replicates, Standard deviation.

Table 2: Antimicrobial activity of fruit extracts of Emblica officinalis on dental caries causing microorganisms determined by agar well diffusion method on specific media for each test microorganism.

(-) = no activity, nt = not tested, S.m. = Streptococcus mutans, S.a.= Staphylococcus aureus, L.a.= Lactobacillus acidophilus, C.a.= Candida albicans, S.c.= Saccharomyces cerevisiae, ^* Values, including diameter of the well (8 mm), are means of three replicates, Standard deviation.

Table 3: MIC of fruit extracts of Sapindus mukorossi against dental caries causing microorganisms on specific media for each microorganism, determined by modified agar well diffusion method.

Table 4: MIC of fruit extracts of Emblica officinalis against dental caries causing microorganisms on specific media for each microorganism, determined by modified agar well diffusion method.

Conclusion

Since the acetonic and hot water extracts of fruits of Sapindus mukorossi and Emblica officinalis were most effective against the tested dental caries causing microorganisms, purification and toxicological studies of the plants and in vivo trials should be carried out so that it can be used as a potential source for the development of a phytomedicine to act against dental caries causing microbes. The antimicrobial activities can be enhanced if the phytoactive components are purified and adequate dosage determined for proper administration.

Acknowledgement

We would like to thank Dr. B.D. Vashishta, Department of Botany, Kurukshetra University, Kurukshetra, for rending help for the confirmation of identification of the plants, Dr. Tapan Chakrabarti, Institute of Microbial Technology, Chandigarh, for providing the microbial cultures and the Chairperson, Department of Microbiology, Kurukshetra University, Kurukshetra for providing the lab facilities.

References

Amadi, E.S., Oyeka, C.A., Onyeagba, R.A., Ogbogu, O.C. and Okoli, I 2007. Antimicrobial screening of Breynia nivosus and Ageratum conyzoides against dental caries causing organisms, J. Biological Sciences 7(2): 354-358.

Aneja, K.R 2003. Experiments in Microbiology Plant Pathology and Biotechnology, 4^th ed., New Age International Publishers, New Delhi.

Aneja, K.R. and Joshi, R 2009a. Antimicrobial activity of Amomum subulatum and Elettaria cardamomum against dental caries causing pathogens, Ethnobotanical Leaflets 13: 840-849.

Aneja, K.R. and Joshi, R 2009b. Evaluation of antimicrobial properties of fruit extracts of Terminalia bellerica against dental caries pathogens, Jundishapur J. Microbiology 2(3): 105-111.

Aneja, K.R., Joshi, R. and Sharma, C 2009. Antimicobial activity of dalchini (Cinnamomum zeylanicum bark) extracts on some dental caries pathogens, J. Pharmacy Research 2(9): 1387-1390.

Aneja, K.R., Joshi, R. and Sharma, C 2010. The antimicrobial potential of ten often used mouthwashes against dental caries pathogens, Jundishapur J. Microbiology 3(1): 15-27.

Attele, A.S., Wu, J.A. and Yuan, C.S 1999. Ginseng pharmacology. Multiple constituents and multiple actions, Biochemical Pharmacology 58: 16851693.

Bag, A., Bhattacharya, S.K., Bharati, P., Pal, N.K. and Chattopadhyay, R.R 2009. Evaluation of antibacterial properties of Chebulic myrobalan (fruit of Terminalia chebula Retz.) extracts against methicillin resistant Staphylococcus aureus and trimethoprim-suphamethoxazole resistant uropathogenic Escherichia coli, African J. Plant Sciences 3(2): 25-29.

Balakrishnan, M., Simmonds, R.S. and Tagg, J.R 2000. Dental Caries is a preventable infectious disease, Australian Dental J. 45: 235-245.

Benson, H.J 2004. Microbiological Applications: Laboratory Manual in General Microbiology, McGraw Hill Publication, USA.

Cappuccino, J.G. and Sherman, N 1995. Microbiology Lab Manual, Benjamin-Cummings Publishing Company, USA.

de Soet, J.J. and de Graaff, J 1998. Microbiology of carious lesions, Dental Update 25: 319-324.

Dey, A.C 1980. Indian Medicinal Plants Used in Ayurvedic Preparations. Bishen Singh, Mahendra Pal Singh, Dehra Dun.

Dhar, J.P., Bajpai, V.K., Setty, B.S. and Kamboj, V.P 1989. Morphological changes in human spermatozoa as examined under scanning electron microscope after in vitro exposure to saponins isolated from S.mukorossi, Contraception 39(5): 563-568.

Dictionary of Indian Medicinal plants, 1988, CIMAP, Lucknow.

Ghosal, S 1996. Active constituents of Emblica officinalis: Part 1 - The chemistry and antioxidative effects of two new hydrolysable tannins, emblicanin A and B, Indian J. Chemistry 35B: 941-948.

Hammer, K.A., Carson, C.F. and Riley, T.V 1999. Antimicrobial activity of essential oils and other plant extracts, J. Applied Microbiology 86: 985-990.

Joshi, A.R. and Joshi, K 2005. Ethnobotany and Conservation of Plant Diversity in Nepal, Rub Rick, Kathmandu, Nepal.

Khokra, S.L., Prakash, O., Jain, S., Aneja, K.R. and Dhingra, Y 2008. Essential oil composition and antibacterial studies of Vitex negundo Linn. extracts, Indian J. Pharmaceutical Sciences 70(4): 522 526.

Kirtikar, K.R. and Basu, B.D 1991. Indian Medicinal Plants, B.L.M. Publication, Allahabad.

Nair, R. and Chanda, S.V 2007. Antibacterial activities of some medicinal plants of the Western Region of India, Turkish J. Biology 31: 231-236.

Nkere, C.K. and Iroegbu, C.U 2005. Antibacterial screening of the root, seed and stem bark extracts of Picralima nitida, African J. Biotechnology 4(6): 522-526.

Ogundiya, M.O., Okunade, M.B. and Kolapo, A.L 2006. Antimicrobial activities of some Nigerian chewing sticks, Ethnobotanical Leaflets 10: 265-271.

Okeke, M.I., Iroegbu, C.U., Eze, E.N., Okoli, A.S. and Esimone, C.O 2001. Evaluation of extracts of the root of Landolphia owerrience for antibacterial activity, J. Ethnopharmacology 78: 119-127.

Oleszek, W.A 2002. Chromatographic determination of plant saponins, J. Chromatography 967: 147162.

Price, K.R., Johnson, I.T. and Fenwick, G.R 1987. The chemistry and biological significance of saponins in foods and feedstuffs, CRC Critical Reviews Food Science Nutrition 26: 27135.

Rajasekaran, C., Meignanam, E., Vijayakumar, V., Kalaivani, T., Ramya, S., Premkumar, N., Siva, R. and Jayakumararaj, R 2008. Investigations on antibacterial activity of leaf extracts of Azadirachta indica A. Juss (Meliaceae): a traditional medicinal plant of India, Ethnobotanical Leaflets 12: 1213-1217.

Rios, J.L., Recio, M.C. and Villar, A 1980. Screening methods for natural products with antimicrobial activity: a review of the literature, J. Ethnopharmacology 23: 127-149.

Saxena, D., Pal, R., Dwivedi, A.K. and Singh, S 2004. Characterisation of sapindosides in Sapindus mukorosii saponin (reetha saponin) and quantitative determination of Sapindoside B, J. Scientific Industrial Research 63(2): 181186.

Shiau, I.L., Shih, T.L., Wang, Y.N., Chen, H.T., Lan, H.F., Lin, H.C., Yang, B.Y., Ko, C.H. and Murase, Y 2009. Quantification for saponin from a soapberry (Sapindus mukorossi Gaertn) in cleaning products by a chromatographic and two colorimetric assays, J. Faculty Agriculture Kyushu University 54 (1): 215221.

Sparg, S.G., Light, M.E. and van Staden, J 2004. Biological activities and distribution of plant saponins, J. Ethnopharmacology 94: 219243.

Thongson, C., Davidson, P.M., Mahakarrchanakul, W. and Weiss, J 2004. Antimicrobial activity of ultrasound-assisted solvent-extracted spices, Letters Applied Microbiology 39: 401-406.

Warrier, P.K., Nambiar, V.P.K. and Ramankutty, C 1995. Indian Medicinal Plants, Orient Longman Ltd., Madras.

Wealth of Asia, 1998, CD-ROM, NISCOM, New Delhi.