Ethnobotanical Leaflets 10: 24-40. 2006.

Antifungal Activities of Ethanol and Aqueous Crude Extracts of Four Nigerian Chewing Sticks

*A. A ADEKUNLE AND K. A. ODUKOYA

Department of Botany and Microbiology

University of Lagos, Akoka Yaba

Lagos, Nigeria

Email:

*Corresponding author

Issued 6 January 2006

ABSTRACT

The antifungal activities of the ethanol and aqueous crude extracts of four Nigerian chewing sticks were investigated. Also a preliminary phytochemical analysis of the plants was done. The chewing sticks include Anogeissus schimperi, Distemonanthus benthmianus, Vernonia amagdalina and Xanthoxylum zanthoxyloides. All the plants tested, except Anogeissus Schimperi, displayed antifungal activities, zone of inhibition above the 10mm standard mark.  The ethanol crude extracts of the chewing sticks had a greater zone of inhibition in comparison with the aqueous extract.  Among the individual plant extracts, D. benthmianus had the highest antifungal activity against Candida albicans, Aspergillus flavus and Microsporium gypseum and Trichophyton metagrophytes. The chewing sticks contain antifungal agents, though the concentration and composition of the bioactive substances may differ amongst the plants.  Distemonath benthmianus exhibited a better antifungal activity and thus made it more suitable for better dental care.

            Flavonoid was present in all the plant extracts.  Tannin was present in all the plant extract, except that of Anogeissus schimperi.  Alkaloids were absent in all the plant extract.  The ethanolic extracts had more phytochemical compounds than the aqueous extracts.

KEYWORDS: Chewing Sticks; antifungal activity; Ethanol and aqueous extract; flavonoid; Tannin

INTRODUCTION

In many African houses, teeth are cleaned in the morning by chewing the root or skin stem of certain plants, until they acquire brush like ends (EL-Said et al., 1971).  The fibrous end is then cleaned and it brushes the teeth thoroughly.  In certain parts of West Africa, e.g. Ghana, Nigeria, Senegal, Chewing sticks are used frequently during the day.  These chewing sticks impact varying taste sensation; a tingling peppery taste, a bitter taste and numbness is provided (Buada and Boakye-Yiadom, 1973).

Investigations carried out on some of these chewing sticks, shows that they all posses antimicrobial activity against oral microbial flora, but to varying degrees when tested by cup-plate agar method (Enwonwu, 1997).  This could indicate therefore that the chewing sticks, in addition to providing mechanical stimulation of the gums, also destroy microbes, presenting the mouth a feature, which is absent in the common toothpaste and brush method (Enwonwu, 1997). The advantage of the chewing sticks over the conventional toothpaste and brush could explain why many Africans have strong teeth (Ugoji et al., 2000).

Some of the plants employed as chewing sticks for dental care include Anogeissus schiemperi (Hochst); Distemonathus benthmianus (Baill); vernonian amygdalina (Del.) and Xanthoxyllum zanthoxyloides (Lam.) “formerly Fagara zanthoxyloides” ( Burkill,1997).  A. schimperi is in the family combretaceae found in southern Nigeria, Senegal, Ivory Coast and Togo, and commonly called “Pako dudu” in Yoruba.  Distemonanthus benthmianus known as “Ayan” in Yoruba is in the family caesalpinaceae. Xanthoxyllum zanthozyloides “orin ata” is in the family Rutaceae while Vernonia amagdalina commonly called bitter leaf is in the family compositae.  Ugoji et al., (2000) determined the antibacterial activities of the aqueous extracts of these chewing sticks, except Distemonanthus benthmianus, on oral pathogens and found that they displayed broad spectrum activity on aerobic and anaerobic bacteria.  The antifungal activities of these chewing sticks were not reported, although some of them have documented reports of the antifungal properties of their leaves or bark Sofowora (1982).

The aim of this paper is to report the antifungal activities of ethanol and aqueous extracts of Anogeissus schimperi, Distemonanthus benthmianus, Xanthoxyllum Zanthoxyloides and Vernonia amagdalina on oral pathogens and some dermatophytes using the paper disc method.  Also the preliminary phytochemistry of the extracts will be examined.

MATERIALS AND METHODS

Source of plant materials:

The stem or root of the plant used as chewing stick were purchased from Ketu and Tejuosho markets of Lagos State, Nigeria.  The plant species parts were authenticated by a botanist at the Department of Botany and microbiology University of Lagos as well as the Forests research Institute, Ibadan, Nigeria.

Source of microorganisms:

Aspergillus flavus, Candida albicans, Microsporum gypseum, Trichophyton metagrophytes were colleted from patients at the Department medical microbiology, college of medicine, Idi-Araba, University of Lagos, Nigeria.  The fungi were storted on Sabouraud dextrose agar (SDA) slants in the refrigerator, 4⁰C, prior to use.

Extract Preparation:

The ethanol and aqueous extracts were carried out using modified methods of Saxena and Mathela (1996).  The stem or roots of the different plants (chewing sticks) were chopped into tiny bits.  They were then pounded with local pistil and mortar before blending with an electric blender.  Twenty grammes of the ground plant part was soaked separately in 100ml, 70% aqueous ethanol, and another twenty grammes in sterilised distilled water (100ml) for 24 hours.  The fluids were then filtered using Whatman No. 1 filter paper.  The ethanol and water extract were concentrated using a Rotatory evaporator at 40⁰C.  The concentrated extracts were the ethanol and aqueous extract of the plants.  They were kept in the fridge prior to use.

Antifungal tests:

The paper disc diffusion method if Irobi and Daramola (1994) was modified here.  Spore of conidia suspension of 10⁵ – 10⁷ cells, counted with haemocytometre were made.  About 10ml Sabouraud dextrose agar, (SDA) water poured into Petri dishes and allowed to solidify.  A micropipette was used to introduce 0.1ml of the spore or conidia suspensions on to the agar plate, and spread with glass spreading rod under sterile conditions.  Sterilised disc (6mm, Whatman No AA2017006) were soaked in each of the extracts (100mg/ml) being assayed for 6 hours.  Four of these soaked discs were spread on a fungal spore or conidia seeded plate with the help of sterile forceps.  There was a control which contains the SDA and fungal inoculums but the discs were soaked in sterilised distilled water only without extracts.  Three replicates were produced for each fungus.  All the plates containing the discs were then incubated at 28 – 31⁰C.  Zone of inhibition was measured after 48 – 72 hours.  Results were statistically analysed using standard deviation (S.D), analysis of variance (ANOVA, F – Test) and Duncan multiple range test (Parker, 1979).

Preliminary Phytochemical Studies:

Supporting phytochemical studies were carried out using methods described by Fadeyi et al., (1987) and Harbone, (1998).  Basic phytochemical screening were performed on the chewing stick extracts.  They were screened for the presence of anthocyanin, anthraquinone, alkaloids, flavonoids, phlobatarnnin, saponin, steroid and tannin.

RESULTS

The ethanolic, aquesous extract of each of the chewing sticks as well as the missed extract inhibited the growth of all the fungi tested, except the extract of Anogeissus schimperi which did not inhibit the growth of C. albicans (Tables 1 – 2).  Only the extracts of A. schimperi had zone of the inhibition less than 10mm (Tables 1 and 2). The ethanol crude extracts of the chewing sticks had a greater zone of inhibition in comparison with the aqueous extract.   Amongst the plant extracts D. benthmianus had the highest antifungal activity against C. albicans and A. flavus.  The mixed extracts had the highest zone of inhibition on M. gypseum and T. mentagrophytes. There were significant differences between the zones of inhibition among the extracts of some of the plant part tested.  The control, which was with sterilised distilled water, yielded no zone of inhibition.

Table 3 shows the phychemical compounds present in the aqueous and ethanol crude extracts of the four plants.  Flavonoids was present in all four plants extracts.  Tannin was present in all the extract except in the aqueous and ethanol extract of Anogeissus schimperi.  Alkaloid was not present in any of the plant extract.  The ethanolic extracts had more phytochemical compounds than the aqueous extracts.  The mixed extracts (ethanolic) and ethanolic extracts of Vernonia amagdalina had the highest range of phytochemical compounds (Table 3).

DISCUSSION

The result presented here shows the presence of antifungal substances in all the ethanol chewing stick extracts.  The aqueous extracts had lower fungal inhibition which might be because the bioactive components were more soluble in the ethanol.  This is in agreement with findings of Barnabas and Nagarajan (1988) on other plants.  Of all the plant extract tested, D. benthmianus was the most active against the fungi tested, since it possessed the highest antifungal inhibition.  A. schimperi extracts do not contain much phytochemical compound, especially tannin, which probably might be attributed to its low activity.  Burapadaja and Bunchoo (1995) reported the presence of tannins in Terminalia citrina extracts and explained that the tannins inhibited cell wall formation in fungi leading to the death of the micro-organism.  A. schimperi extracts might not contain potent antifungal substances even though Ugoji et al., (2000) reported that the aqueous extract contain antibacterial substances.  This observation supported Valenciennes et al., (1999), who reported that antibacterial agents might not necessarily be antifungal.  It is of significance to note the high sensitivity of some of the fungi to the D. benthmianus extracts, because it is claimed locally that the use D. benthmianus might reduce or completely stop mouth odour.  The mixed extracts (ethanol or aqueous) of all the chewing sticks proved to inhibit the fungi better than the extracts of most be due to the presence of more phytochemical compounds or bioactive components in the mixed extracts as observed in the result of the phytochemical study.

Conclusively, this study has shown the presence of antifungal substances in the chewing sticks.  The concentration and composition of the bioactive substances may differ from one plant to another as indicated in the degree of potency and presence of phytochemical compounds.  The regular use of the Nigerian chewing sticks may decrease the incidence of dental disease caused by microbes.  The chewing stick of Distemonathus benthmianus because of its better antifungal properties might be recommended for good oral health.

REFERENCES

Barnabas, C. G. and Nagarajan, S. (1988). Antimicrobial activity of flavonoids of some medicinal plants.  Fitoterapia 3; 508-510.

Buada, C. V. and Boakye – Yiadom, K. (1973). The antibacterial activity of some Ghanian chewing sticks.  Ghana pharmaceutical Journal 1: 150 – 151.

Burapadja, S. and Bunchoo, A. (1995). Antimicrobial activity of Tannins from Terminalia citrina Planta medica 61(4): 365 – 366.

Burkhill, H. M. (1997). Usefull plants of Tropical West Africa. Vol. 4. Royal botanic gardens, kew. 969pp.

EL-Said, F; Fadulu, S. O.; Kuye. J. O.; and Sofowora, E. A. (1971). Native cures in Nigeria Part II: The antimicrobial properties of the buffer extract of chewing sticks. Lioydia 34: 172 – 174

Enwonwu, C. O. (1974). Socio – economic factors in the dental caries prevalence and frequency.  Nigerian carries res. 8; 155 – 177.

Fadeyi, M. O.; Adeoye, A. O. and Olowokudejo, I. D. (1987). Epidermal and Phytochemical Studies in the genus Bohervia (Nyctaginaceae) in Nigeria.  International Journal of Crude drug research 27:178-184.

Harbone, J. B. (1998). Phytochemical methods: A guide to modern techniques of plant analysis.  3^rd Edition.  Chapman and Hill, London. 279pp.

Irobi, O. N. and Daramola, S. O. (1994). Antifungal activities of crude extracts of Mitracarpus villosus (Rubiaceae).  Journal of Ethnopharmacology 38: 604 – 610.

Parker, R. E. (1979). Introductory Statistics for biology.  2^nd Edition.  Edwards Arnold, London. 112pp.

Saxena, J. and Methela, C. S. (1996). Antifungal activity of New compound from Nepeta leucophylla and Nepeta clarkeii.  Applied and Environmental microbiology 62(2): 702 – 704.

Sofowora, E. A. (1982). Medicinal plants and traditional medicine in Africa.  2^nd Edition. John Wiley and Sons Limited, Chitchestes.  404pp.

Ugoji, E; Egwari, L. O.; and Obisesan, B. (2000). Antibacterial activities of aqueous extracts of ten African chewing sticks on oral pathogens. Nig. Journal of Internal medicine 3(1) : 7-11.

Valenciennes, E; Smadja, J. and Conan, J. Y. (1999). Screening for biological activity and chemical composition of Euodia bordonica var. borbonica (Rutaceae), a medicinal plant in Reunion Island. Journal of Ethnopharmacology 43: 283 – 288.

Table 1. Antifungal Activity of the Ethanol Crude Extracts of Four Nigerian Chewing Sticks.

*          Zone of inhibition with similar alphabets shows no significant difference p = 0.05

*          Zone of inhibition with different alphabets shows significant difference p = 0.05

Table 2. Antifungal Activity of the Aqueous Crude Extracts of Four Nigerian Chewing Sticks.

*          Zone of inhibition with similar alphabets shows no significant difference p = 0.05

*          Zone of inhibition with different alphabets shows significant difference p = 0.05

Table 3. Phytochemical Compounds in the Crude Extracts (Aqueous and 70% Aqueous Ethanol) of Four Nigerian Chewing Sticks.

(+)           Presence of the phytochemical compound

(–)           Absence of the phytochemical compound