Kothai S et al.

, IJSID, 2013, 3 (1), 43-50

ISSN:2249-5347

IJSID

International Journal of Science Innovations and Discoveries

Research Article

An International peer Review Journal for Science

Available online through www.ijsidonline.info

STREPTOCOCCUS PYOGENES

IN VITRO SYNERGISTIC EFFECT OF CHEWING STICKS (TOOTH BRUSH), CINNAMON AND HONEY AGAINST Meenakshi Agribusiness and Extension Service, No.69, III Cross, Indian Bank Colony, Tiruchirapalli, Tamil Nadu, India Kothai, S

Received: 14-01-2013 Accepted: 20-02-2013

*Corresponding Author

ABSTRACT Inhabitants. Chewing sticks chosen through ethno botanical studies were combined with used to assess the effect of different combinations of chewing sticks, cinnamon and honey. Chewing sticks and honey are some of the therapeutic agents used by Ethiopian

Cinnamon and honey brown/white and tested against Streptococcus pyogenes which is The results indicated that the antimicrobial activity of more than 50% of the chewing stick plants was enhanced by both brown and white honey. Cinnamon showed more

one of the causative organisms for strep-throat/tonsillitis. Agar well diffusion assay was antagonistic effect than synergistic effect with the chewing stick plants. 72% of the plants

Address: Name: Dr.S. Kothai Place: Tamil Nadu, India E-mail: kothaiseshathri@yahoo.co.in

showed synergistic effect with cinnamon and honey. Highest antibacterial activity was strep-throat caused by S.pyogenes.

showed by two combinations namely Justicia schimperiana with brown honey & J.schimperiana with cinnamon and white honey. These complex and unique combinations awaiting for further exploration into an effective, economical and affordable herbal cure to Justicia schimperiana, Cinnamon, honey

KEYWORDS: opportunistic infection, strep-throat, tonsillitis, Streptococcus pyogenes,

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Kothai S et al., IJSID, 2013, 3 (1), 43-50 caused by Streptococcus pyogenes is one of the opportunistic infection which deteriorates the meager health of the immunocompromised. Moreover, complexity, side effects associated with the allopathic drugs made both health care scientific research is needed to gain an understanding about the complexity of Ethiopian herbal plants and their mechanism of active ingredients of the plants used lies in the unique combination which produce synergistic effect i.e bactericidal to the synergistic activity, chewing sticks were combined with cinnamon and honey. Thus, the main focus of this research was to find the effect of combination of Ethiopian chewing sticks, cinnamon and honey against S.pyogenes. MATERIALS AND METHODS Honey Un-processed honey brown/white was purchased from Jimma Market. action against a particular microbe. Generally, traditional herbal medicines are combination of plants. Interaction between the practitioners and majority of the world population to turn towards alternative therapies, more likely towards herbal medicines [1]. Herbals have been used for thousands of years and undoubtedly have demonstrated health benefits. However, much Antibiotic therapy is incompetent in immune deficient patients due to several reasons particularly HIV. Strep-throat INTRODUCTION

microbe in question. Chewing sticks, cinnamon and honey are some of the therapeutic agents with ancient history and contemporary scientific proof. In spite of Spilanthes cure, tonsillitis remained as one of the frequently encountered oral

problem and chewing sticks showed less activity when compared to the standard antibiotics [2]. Therefore, to check the

Plant materials

schimperiana, Olea europaea, Phoenix reclinata, Rubus apetalus, Sida rhombifolia, Stereospermum kunthianum and Vernonia amygdalina [2] and cinnamon were used in this study. Extract preparation extracts were filtered through Whatman No.1 filter paper. The extracts were concentrated in a rotary evaporator at 35 0 C. A and cinnamon were prepared separately. Water was used in case of honey. Mixing of extracts was done in the following ratio for the antimicrobial study. a. c. b. Chewing stick extracts were mixed with honey brown/white at 1:1 ratio (v/v) Extract of chewing sticks and cinnamon were mixed at 1:1 ratio (v/v) and Chewing stick, Cinnamon and brown or white honey in the ratio of 1:1:1 (v/v) Plant materials were collected, dried, powdered and extracted with 70% of ethanol (Harbone 1963). The crude

Chewing stick plants viz., Clausena anisata, Clematis simensis, Clerodendrum myricoides Juniperus procera, Justicia

known quantity of the dried extract was reconstituted with DMSO and working concentration (100mg/ml) of chewing sticks

Test organism

Institute, Addis Ababa. The test organism at 0.5 McFarlands standard was prepared [3] in PPG1% (peptone water with red phenol supplemented with glucose 1% (w/v) and used for antimicrobial assay. Antimicrobial activity The expected values of a & b were calculated using the following formula [5]. the individual effect of chewing stick plants, cinnamon & honey and compared with the actual ZOI produced in combination. International Journal of Science Innovations and Discoveries, Volume 3, Issue 1, January-February 2013

The referral strain, Streptococcus pyogenes (ATCC19615) was obtained from Ethiopian Health and Nutrition Research Antimicrobial activity was determined by agar well diffusion [4] method. The expected values were calculated using

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If,

Calculated zone size = sum of zone size of 2 extracts/2 1. 2. 3.

Kothai S et al., IJSID, 2013, 3 (1), 43-50

100 to 0.78 mg/ml. PPG1% broth was used instead of Muller Hinton Broth, which indicates the MIC with persistence of red used as positive control. After incubation the tubes were observed for color change. The tubes which remained red indicated that there was inhibition of bacterial growth and tubes which turned yellow indicated microbial growth. Statistical analysis The results were analyzed using SPSS 16.0 version. RESULTS AND DISCUSSIONS

colour in the absence of microbes. 70% ethanol/DMSO and water were included as negative controls. Penicillin 10mg/ml was

The same formula was used to calculate the combination of three extracts (c).

Observed zone size = calculated zone size then the effect is additive

The MIC for the combination of extracts was determined by broth macro dilution assay with a concentration range of

Observed zone size > calculated zone size then the effect is synergistic

Observed zone size < calculated zone size then the effect is antagonistic

quality of life by having a serious impact on an individuals wellbeing and ability to fulfill desired socioeconomic functions [6]. Chewing sticks offer considerable and underexploited potential as sources of new antimicrobial backbones [7]. Kocry, (1983) [8] has elaborated the use of chewing sticks in preventive oral hygiene while Ogunbodede (1991) documented the role of traditional healers in dental care [9]. Ethno botanical studies in Jimma, Ethiopia revealed that 47% of the inhabitants use scientific advancement and non-availability of chewing sticks. Chewing sticks and honey irrevocably lost if quick efforts are not exerted to document this invaluable knowledge. Though the practice of using chewing chewing sticks to maintain oral hygiene [3, 4]. The use of chewing sticks to manage oral infections is in danger of being sticks is not new, their uses have been gradually disappeared in most of the developed and developing countries due to Effect of multi-plant extracts lies in the unique combination of compounds present in the plants to produce a desired effect. Ethno botanical survey on traditional medicinal plants used in Lake Victoria Region in Tanzania revealed that a maximum of chosen chewing sticks were combined with honey brown/white. The results of such study revealed that, eight plants viz., nutritional and health effects, including antibacterial, antioxidant, anti-inflammatory and prebiotic [12]. In the study area Jimma - Ethiopia, traditional healers use single or multi-plant extracts with or without additives [10]

Oral health is the total health of an individual to maintain personal & social well being. Oral diseases directly affect the

fifteen plant extracts were combined together to treat certain opportunistic infections [11]. Based on these concepts, the both brown and white honey (Fig. 1 & 2). In addition, honey is an established traditional medicine with a variety of putative

C.simensis, C. myricoides, J. procera, J. schimperiana, O. europaea, R. apetalus, S. kunthianum and V. amygdalina worked synergistically with brown honey as well as white honey. Among these plants J.schimperana produced highest activity with

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Kothai S et al., IJSID, 2013, 3 (1), 43-50

Figure 1 Effect of Combination of Chewing sticks and brown Honey

Chewing sticks and cinnamon

(http://www.wic.Herbal encyclopedia - on line). The results of the current study on the combination of chewing stick plants

extracts & cinnamon revealed that cinnamon was able to enhance the activity of C. anisata, C. simensis, J. schimperiana, O. europaea, and V. amygdalina and antagonistic with J. procera, P. reclinata, R. apetalus and Stereospermum kunthianum only (Fig. 3). Thus the results clearly indicated that cinnamon can enhance the activity of only certain plants with active ingredients which work synergistically with that of cinnamon and not other plants. The essential oil of Cinnamon [13], Clausenol & Clausenine in the stem bark of Clausenia [14], Alkaloids of J.schimperana [15] and other identified and unidentified compounds in other plants could have caused wide spectrum antibacterial activity against S.pyogenes in each unique combinations. International Journal of Science Innovations and Discoveries, Volume 3, Issue 1, January-February 2013

Generally, cinnamon is used as a stimulant to other herbs enabling the herbal remedies to work faster

Figure 2 Effect of Combination of Chewing sticks and white Honey

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Kothai S et al., IJSID, 2013, 3 (1), 43-50

Chewing sticks, cinnamon and brown/white honey

europaea, R. apetalus, S. kunthianum and V. amygdalina were increased by combination with cinnamon and brown honey at chewing stick plants mentioned above, cinnamon and honey have produced synergistic activity and a broad spectrum to produce the same synergistic effect with cinnamon and honey.

1:1:1 ratio (Fig. 4). Similar results were obtained for the combination of chewing stick plants, cinnamon and white honey (Fig.

5). Previous research on the individual effect of the same chewing stick plants against S.pyogenes has produced less effect [2] than the current combination study. Thus the results indicated that unique combination of the active ingredients of the eight

The antimicrobial activity of eight chewing stick plants namely, C. simensis, C. myricoides, J. procera, J. schimperiana, O.

Figure 3 Effect of Combination of Chewing sticks and Cinnamon

antimicrobial activity against S.pyogenes whereas three chewing stick plants (C.myricoides, P.reclinata and S.rhombifolia) failed

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Figure 4 Effect of Combination of Chewing sticks, Cinnamon and Brown honey

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Kothai S et al., IJSID, 2013, 3 (1), 43-50 Minimum Inhibitory Concentration (MIC) from 1 to 0.125mg/ml. The lowest MIC was recorded for J. schimperiana with white honey as well as its combination with cinnamon and brown honey. None of the combinations tested showed less than 0.125mg/ml of MIC. The data obtained through the determination of MIC for the combinations a, b & c revealed variability and ranged

Honey

http://www.dermnetnz.org. These are key body immune responses in the battle against infections. Fukuda, (2011) reported that oral intake of honey augmented antibody production in primary and secondary immune responses against thymusdependent and thymus independent antigens [16]. Current study showed that both brown and white honey on its own did not collected from Jimma and Saudi market. Synergistic effect a concentration ranging between 5 and 10 % honey and killed by a concentration ranging between 5 and15 % honey. The difference in antimicrobial activity of honey against S.pyogenes could be due to different floral sources present in the honey have any activity against S.pyogenes. In contrast to the current study, Halawani (2011) found that S. pyogenes was inhibited at

In addition to the antimicrobial properties, honey also appears to stimulate lymphocytic and phagocytic activity

A. Clausenia anisata B. Clematis simensis E. Justicia schimperiana F. Olea europea Sida rhombifolia J. Stereospermum kunthianum ZOI in mm = Mean Standard deviation ZOI Honey brown/white = 0.00mm ZOI

Figure 5 Effect of Combination of Chewing sticks, Cinnamon and White honey

C. Cleodendrum myricoides D. Juniperus procera G. Phoenix reclinata H. Rubus apitalus K. Vernonia amygdalina Positive control: Penicillin = 28.66 mm Cinnamon = 12.330.57 mm ZOI

results; additive, synergistic or antagonistic. Therefore, the effect differs with each combination. More number of chewing stick plants exhibited synergistic activity when combined with cinnamon and honey in this study whereas the same chewing stick plants as a single plant extract had less or no activity against S.pyogenes [ 2 ]. In addition to the synergistic effect produced by compounds in honey might have also contributed to the wide spectrum activity similar to traditional herbal formulation. Much International Journal of Science Innovations and Discoveries, Volume 3, Issue 1, January-February 2013 the secondary metabolites of chewing stick plants, cinnamon and honey, the hurdle effect of H 2O2, low pH and unidentified scientific exploration is needed to strengthen the above mentioned concept. Up to our knowledge such experiment on

From the result it is evident that the combination of each chewing sticks with cinnamon and honey produced variable

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combination of chewing sticks chosen in this study with cinnamon and honey was not reported before. The result of the study justifies the use of combination of plants and additives in traditional medicine. CONCLUSION (honey) has a potential in management of strep-throat caused by S.pyogenes. ACKNOWLEDGEMENT for availing the Library, Internet and Laboratory facilities. 1. 2. 3. 4. 5. 6. 7. 8. 9. rabbits. Acta Pharma Sci, 2009. 51: p. 33-38.

Kothai S et al., IJSID, 2013, 3 (1), 43-50

Herbal drugs are effective due to their complex combinations. Thus the multi-plant combination with an additive

Amharic and Oromo languages into English. Thanks to Jimma University College of Agriculture and Veterinary medicine Rafeeuddin, M., et al., Comparative efficacy of four Ayurvedic antidiabetic formulations in alloxaninduced diabetic REFERENCES pyogenes. Journal of phytology, 2011. 3(8): p. 34-37.

The author thanks everyone who helped her to conduct the survey, collection of chewing sticks and in translation of

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Samuel, T., Oral Health Issues in the African Region: Current Situation and Future Perspectives. J Dent Educ, 2006. 70(11): p. Ndukwe, K.C., Lamikanra, A., and Okeke, I.N, Antibacterial activity in plants used as chewing sticks in Africa. Drugs of the Kocry, T., The use of chewing sticks in preventive oral hygiene. Clinical Prev. Dent., 1983. 5: p. 11-14. Zone, Ethiopia. Ethiopian Journal of Health Science, 2005. 15(2): p. 89-106. for local healing in Tanzania. Afr. J. Trad. CAM, 2008. 5(2): p. 165 - 172. 103: p. 1023-1028 Ogunbodede, E.O., Dental Care: the role of traditional healer. Wld. Hlth. Forum, 1991. 12(4): p. 443-444.

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12. Alison, W., Sarah,E., Michelle,M., Harry,M., Andrew,McL., Maroussia,R., Jinny,W., Russell,S., and J.Sutherland, Demonstrating 13. Fabio, A., et al., Screening of the antibacterial effects of a variety of essential oils on microorganisms responsible for respiratory infections. Phytotherapy Research, 2007. 21(4): p. 374-377. anisata. Phytochem, 1995. 40(1): p. 295-8 the safety of manuka honey UMFw 201 in a human clinical trial with healthy individuals. British Journal of Nutrition, 2010.

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15. Zelalem, P., Eyasu,M and D.Asfaw, Tracheal relaxant effect of column chromatographic elutes of chloroform fraction of Adhatoda schimperiana leaves in Guinea pigs. Pharmacognosy magazine, 2009. 4: p. 86-91. honey enhances immune function and antitumor activity. Evid Based Complement Alternat Med, 2009. 2011: p. 1-8.

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16. Fukuda, M., Kobayashi, K.,Hirono, Y.,Miyagawa, M.,Ishida, T.,Ejiogu, E.C.,Sawai, M.,Pinkerton, K.E.and M.Takeuchi, Jungle

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