Ethnobotanical Leaflets 13: 840-49, 2009.

 

�� Antimicrobial Activity of Amomum subulatum and Elettaria cardamomum Against Dental Caries Causing Microorganisms

 

K.R.Aneja and Radhika Joshi*

 

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

�����������������

���������������� *Corresponding Author: joshi_radhika31282@yahoo.com

�������������������������������������������������������������

Issued July 01, 2009

 

Abstract

The in vitro antimicrobial activity of Amomum subulatum and Elettaria cardamomum fruit extracts were studied against Streptococcus mutans, Staphylococcus aureus, Lactobacillus acidophilus, Candida albicans and Saccharomyces cerevisiae. The acetone, ethanol and methanol extracts of the selected plants exhibited antimicrobial activity against all tested microorganism except L. acidophilus. The most susceptible microorganism was S.aureus followed by S.mutans, S.cerevisiae and C.albicans in case of Amomum subulatum while in the case of Elettaria cardamomum; S.aureus was followed by C.albicans, S.cerevisiae and S.mutans. The largest mean zone of inhibition was obtained with the ethanolic extract of A.subulatum and acetonic extract of E.cardamomum against Staphylococcus aureus (16.32mm and 20.96mm respectively). Minimum inhibitory concentrations (MIC) of the extracts were also determined against the four selected microorganisms showing zones of inhibition 10mm. This study depicts that ethanol and acetone extracts of fruits of Amomum subulatum and Elettaria cardamomum can be used as a potential source of novel antimicrobial agents used to cure dental caries.

Keywords: Dental caries, Amomum subulatum, Elettaria cardamomum, 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 Graaff, 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 (Amadi et al., 2007). This study reports the antimicrobial effects of Amomum subulatum (Badi elaichi) and Elettaria cardamomum (Chhoti elaichi) on dental caries causing microorganisms.

������� The large cardamom (Amomum subulatum Roxb., family Zingiberaceae) is one of the major cash crops cultivated between elevations of 600 and 2000 m in tropical wet evergreen forests of Eastern Himalayas in India (Sikkim and Darjeeling areas), Nepal and Bhutan. The fruit is a trilocular, reddish brown to dark pink, many seeded capsule and seeds contain 2�3% essential oils, which possess medicinal properties and are used as adjuncts to various medicinal preparations (Gupta et al., 1984; Sinu and Shivanna, 2007; Hussain et al., 2009). The seeds have properties similar to those of true cardamom (Elettaria cardamomum) but are much larger in size and are used as a condiment for culinary and other preparations. Amomum seeds are used as spices and their plant parts are used in traditional medicine for curing toothache, dysentery, diarrhoea, rheumatism, vomiting, dyspepsia and lung diseases (Dutta et al., 2000; Sabulal et al., 2006).

The small cardamom Elettaria cardamomum also belongs to family Zingiberaceae and is historically known as the �Queen of all Spices�. It grows in the understory of tropical rain forests at elevations of 762-1524m, where it rains about 381cm per year. It is cultivated commercially in India, Sri Lanka, Guatemala and Tanzania. The fruits are thin walled, smooth skinned, oblong, green capsules containing 15-20 aromatic reddish brown seeds. The seeds contain a volatile oil, used for flavouring cakes, curries, bread and other culinary purposes, like flavouring coffee and confectionery. It was traditionally used in various gastrointestinal, cardiovascular and neural disorders (Arora and Kaur, 2007; Dhulap et al., 2008). Studies have revealed its use as an effective skin penetration enhancer for certain drugs, anticarcinogenetic agent, anti ulcerogenic agent and anti microbial and anti convulsant agent (Dhulap et al., 2008).

Materials and Methods

������ Amomum subulatum and Elettaria cardamomum fruits were obtained from the local market of Delhi, India (February, 2009). The plants were identified and authenticated taxonomically by a botanist of the Department of Botany, Kurukshetra University, Kurukshetra. The samples were carefully washed under running tap water followed by sterile distilled water and then air dried for two days, pounded using a mixer grinder and stored in airtight bottles.

Extraction of plant material

Four different solvents namely ethanol, methanol, acetone and water (at two different temperatures hot and cold) were used for extraction.

Cold aqueous extraction: Ten grams of fruit powder was soaked in 100ml cold sterile distilled water in a conical flask and left undisturbed for 24h, then filtered off using a sterile Whatman filter paper no1 (Ogundiya et al., 2006). The filtered extract was concentrated under vacuum below 40oC using a rotaevaporator (Heidolph, VE-11). The weight of the solid residue was recorded and taken as the yield of crude extract (Bag et al., 2009).

Hot aqueous extraction: Hot aqueous extract was prepared by boiling 10g of fruit powder in 100ml of sterile distilled water for 30min and kept undisturbed in a conical flask for 24h. The other steps were same as followed in the case of cold aqueous extract.

Organic solvent extraction: Ten grams of fruit powder was kept in 70% methanol, ethanol and acetone for 3 consecutive days at room temperature and then filtered followed by concentrating under vacuum using the rotaevaporator (Bag et al., 2009).

The extracts thus obtained were stored in labelled sterile bottles and kept in the freezer at 4oC until further use for the screening of antimicrobial activity. The extracts were reconstituted in 20% DMSO for the bioassay analysis (Rajasekaran et al., 2008).

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 cervisiae (MTCC 170) were procured from Microbial Type Culture Collection, IMTECH, Chandigarh. The microorganisms were subcultured on different media such as S.mutans on Brain heart infusion agar, S.aureus on Nutrient agar, L.acidophilus on Lactobacillus MRS agar, C.albicans and S.cerevisiae on Malt yeast agar (Himedia Laboratory Pvt. Ltd., Bombay) and incubated aerobically at 37OC.

Antimicrobial assay

The antimicrobial activity was tested using agar well diffusion method. Agar plates were swabbed with 100�l of the respective broth cultures (1.5�108CFU/ml, standardized by 0.5 Mac Farland) and were kept at room temperature for 15 min for absorption to take place. Wells of 8mm size were made with sterile borer in the inoculated agar plates and loaded with 100�l of plant extracts. DMSO was used as a negative control whereas Ciprofloxacin (bacteria) & Amphotericin-B (yeast) were used as positive control. Prior to incubation at 37oC for 24 hrs, the Petri dishes were kept at room temperature for 15min in order to promote diffusion of the extracts into the agar (Khokra et al., 2008; Rios et al., 1988). All the tests were made in triplicate and the mean diameter of the inhibition zones in millimetre and the standard deviation was calculated.

Determination of Minimum Inhibitory Concentration (MIC)

The MIC was determined only for the three organic solvent extracts which showed positive antimicrobial activity against four tested microorganisms by modified agar well diffusion method (Okeke et al., 2001).In this technique, a twofold serial dilution of the extracts was prepared by first reconstituting in 20% dimethylsulphoxide (DMSO). They were diluted in sterile distilled water to achieve a decreasing concentration range of 10mg/ml to 0.04mg/ml. A 100 �l volume of each dilution was introduced in wells in the respective agar plates already seeded with the standardized inoculum (1.5 �108 cfu/ml) of the test microbial strain. All the test plates were incubated at 37oC for 24 hrs. MIC was considered the lowest concentration of the A.subulatum and E.cardamomum fruit extracts that showed visible zone of inhibition (Nkere and Iroegbu, 2005).

Results and Discussion

������� We chose Streptococcus mutans, Lactobacillus acidophilus and Candida albicans as test microorganisms for our study because they have been implicated in dental caries (Lee et al., 2004; Joshi and Joshi, 2005). C.albicans is the most common yeast isolated from the oral cavity, and is associated with fungal oral infections, endocarditis and septicemia (Bagg, 1999). Staphylococcus aureus a major human pathogen responsible for a number of hospital � acquired infections propagates mainly in mouth and hands in the hospital environment (Knighton, 1960; Lowy, 1998; Piochi and Zelante, 1975). We also chose Saccharomyces cerevisiae as a test organism for our study. While it is considered to be an opportunistic pathogen in the oral cavity, it may induce significant oral risks by acting as a tertiary colonizer in dental caries.

The results of antimicrobial activity of the five extracts of Amomum subulatum and Elettaria cardamomum by agar well diffusion method have been shown in Table 1 and Table 2 respectively. From the present data it is evident that the acetonic, methanolic and ethanolic extracts of A.subulatum showed antimicrobial inhibitory activity against two bacteria S.mutans and S.aureus and two fungi C.albicans and S.cerevisiae, with the mean of the highest zone of inhibition being 16.32mm and MIC of 2.5mg/ml (Table 3), shown by ethanolic extract of A.subulatum against S.aureus (Figure 1). The hot and cold water extracts of A.subulatum did not show any inhibitory effect against any of the five microorganisms tested. Similarly the acetonic, methanolic and ethanolic extracts of E.cardamomum showed inhibitory activity against S.mutans, S.aureus, C.albicans and S.cerevisiae with the mean of the highest zone of inhibition being 20.96mm and MIC of 1.25mg/ml (Table 4), shown by acetonic extract of E.cardamomum against S.aureus. The cold water extract of E.cardomomum showed antimicrobial activity against S.mutans and S.aureus while the hot water extract showed no activity at all.

The ethanol and acetone extracts of fruits of Amomum subulatum and Elettaria cardamomum showed greater antimicrobial activity than the corresponding water and methanolic extracts. This finding is interesting, because in the traditional method of treating a microbial infection, decoction of the plant parts or boiling the plant in water was employed. Whereas, according to the present study, preparing an extract with an organic solvent (acetone and ethanol) shows a better antimicrobial activity, in accordance with the results obtained by Nair et al. (2005).

Conclusion

���������� Since the ethanol and acetone extracts of fruits of Amomum subulatum and Elettaria cardamomum were effective against the tested dental caries causing microorganisms, purification and toxicological studies of the plant 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.

Acknowledgements

������ We acknowledge the Vice chancellor and the Chairperson of Microbiology Department for providing us research facilities in the Department of Microbiology, Kurukshetra University, Kurukshetra. We would like to thank Dr. B.D. Vashishta, Department of Botany, Kurukshetra University, Kurukshetra, for helping with the identification of the plants. We are grateful to Dr. Tapan Chakrabarti, Institute of Microbial Technology, Chandigarh, for providing the microbial cultures.

Table 1: Antimicrobial activity of fruit extracts of Amomum subulatum on the test organisms.

Amomum subulatum

extracts (mg/ml)

Diameter of growth of inhibition zones ( mm )

Streptococcus mutans

Staphylococcus aureus

Lactobacillus acidophilus

Candida albicans

Saccharomyces cerevisiae

Acetone

12.31a�0.57b

16�0

-

29.30�1.15

16.65�0.57

Methanol

12.94�1

14.31�0.57

-

18.96�1

17.65�0.57

Ethanol

14.95�1

14.65�0.57

-

11.64�0.57

18.32�0.57

Hot water

10.64�0.57

14�0

-

-

-

Cold water

10�0

10.31�0.57

-

-

-

Ciprofloxacin

(5 �g/ml)

27.32�0.57

34.66�0.57

25.65�0.57

nt

nt

Amphotericin B (100 units/ ml)

nt

nt

nt

13�0

11.94�1

DMSO

-

-

-

-

-

(-) = no activity, nt = not tested

aValues, including diameter of the well (8 mm), are means of three replicates

�� b � Standard deviation

Table 2: Antimicrobial activity of fruit extracts of Elettaria cardamomum on the test organisms.

Elettaria cardamomum extracts (mg/ml)

Diameter of growth of inhibition zones ( mm )

Streptococcus mutans

Staphylococcus aureus

Lactobacillus acidophilus

Candida albicans

Saccharomyces cerevisiae

Acetone

12.94a�1b

20.96�1

-

13.31�0.57

11.94�1

Methanol

10.93�1

13.31�0.57

-

14.31�1

13.95�1

Ethanol

11.94�1

18.65�0.57

-

15�0

12.94�1

Hot water

-

��������� -

-

-

-

Cold water

10.93�1

10.58�1.15

-

-

-

Ciprofloxacin

(5 �g/ml)

27.32�0.57

34.66�0.57

25.65�0.57

nt

nt

Amphotericin B (100 units/ ml)

nt

nt

nt

13�0

11.94�1

DMSO

-

-

-

-

-

(-) = no activity, nt = not tested

aValues, including diameter of the well (8 mm), are means of three replicates

�� b � Standard deviation

Table 3: MIC of fruit extracts of Amomum subulatum.

Amomum subulatum fruit extracts

MIC (mg/ml)

Streptococcus mutans

Staphylococcus aureus

Lactobacillus acidophilus

Candida albicans

Saccharomyces cerevisiae

Acetone

5

2.5

nt

5

5

Methanol

5

2.5

nt

5

5

Ethanol

5

2.5

nt

5

5

(-) = no activity, nt = not tested

Table 4: MIC of fruit extracts of Elettaria cardamomum.

Elettaria cardamomum fruit extracts

MIC (mg/ml)

Streptococcus mutans

Staphylococcus aureus

Lactobacillus acidophilus

Candida albicans

Saccharomyces cerevisiae

Acetone

5

1.25

nt

2.5

5

Methanol

5

1.25

nt

2.5

5

Ethanol

5

1.25

nt

2.5

5

(-) = no activity, nt = not tested

 


Figure 1: Zone of inhibition shown Elettaria cardamomum fruit acetone extract against Staphylococcus aureus and negative control (DMSO).

 

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. Biol. Sci. 7(2): 354-358.

Arora, D.S. and Kaur, G.J. 2007. Antibacterial activity of some Indian medicinal plants, J. Nat. Med. 61: 313-317.

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, Afr. J. Plant Sciences 3(2): 25-29.

Bagg J. 1999. Essentials of Microbiology for Dental Students, Oxford University Press, New York.

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

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

Dhulap, S., Anita, M. and Hirwani, R.R. 2008. Phyto-pharmacology of Elettaria cardamom, Phcog. Rev. 2(4): 27-35.

Dutta, S., Ahmed, R. and Pathak, M.G. 2000. Essential oil composition of Amomum linguiforme Benth. from north-east India, Indian Perfum. 44: 11�13.

 

Gupta, P. N., Naqvi, A. N., Mishra, L. N., Sen, T. and Nigam, M. C. 1984. Gas chromatographic evaluation of the essential oils of different strains of Amomum subulatum Roxb. growing wild in Sikkim, Sonderdruck aus Parfumeric kodmetik 65, 528�529.

 

Hussain, J., Khan, A.L., Rehman, N., Zainullah, Khan, F., Hussain, S.T. and Shinwari, Z.K. 2009. Proximate and nutrient investigations on selected medicinal plant species of Pakistan, Pak. J. Nutr. 8(5): 620-624.

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, Ind. J. Phar. Sci. 70(4): 522 � 526.

Knighton, H.T. 1960. Study of bacteriophage types and antibiotic resistance of Staphylococci isolated from dental students and faculty members, J. Dent. Res. 39: 906-911.

Lee, S.S., Zhang, W. and Li, Y. 2004. The antimicrobial potential of 14 natural herbal dentifrices: Results of an in vitro diffusion method study, J. Am. Dent. Assoc. 135: 1133-1141.

Lowy, F.D. 1998. Staphylococcus aureus infections, N. Engl. J. Med. 339(8):520-532.

Nair, R., Kalariya, T., Sumitra, C. 2005. Antibacterial activity of some selected Indian medicinal flora, Turk .J. Biol. 29: 41-47.

Nkere, C.K. and Iroegbu, C.U. 2005. Antibacterial screening of the root, seed and stem bark extracts of Picralima nitida, Afr. J. Biotech. 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.Evluation of extracts of the root of Landolphia owerrience for antibacterial activity, J. Ethnopharmacology 78: 119-127.

Piochi, B.J. and Zelante, F. 1975. Contribution to the study of Staphylococcus isolated in the mouth.III. Staphylococcus isolated from dental plaque, Rev. Fac. Odontol. Sao. Paulo.13 (1): 91-97.

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.

Sabulal, B., Dan, M., Pradeep, N.S., Valsamma, R.K., George, V. 2006. Composition and antimicrobial activity of essential oil from the fruits of Amomum cannicarpum, Acta. Pharm. 56: 473-480.

Sinu, P.A. and Shivanna, K.R. 2007. Pollination biology of large cardamom (Amomum subulatum), Current Science 93(4): 548-552.

 

 

 

 

 

 

 

 

 

���������������������������������������������������������������������������������������������������������������������������������