South African Journal of Botany 72 (2006) 646–648 www.elsevier.com/locate/sajb

Short communication The in vitro antimicrobial activity of toothbrush sticks used in Ethiopia ⁎ S.F. van Vuuren a, , A.M. Viljoen b

a Department of Pharmacy and Pharmacology, Faculty of Health Sciences, University of the Witwatersrand, 7 York Road, Parktown 2193, Johannesburg, South Africa b School of Pharmacy, Tshwane University of Technology, Private Bag X680, Pretoria 0001, South Africa Received 25 November 2005; accepted 29 March 2006

Abstract

African toothbrush sticks have been used for centuries for maintaining oral hygiene. Seven different toothbrush sticks were acquired from Addis Ababa (Ethiopia); anisata, Dovyalis abyssinica, Maerua oblongifolia, Osyris quadripartite, Pittosporum viridiflorum, Sida cuneifolia and Stereospermum kunthianum. These have been screened for antimicrobial efficacy in an attempt to establish a scientific basis for their frequent use in maintaining oral hygiene. Minimal inhibitory concentrations (MIC) were determined on lipophilic and aqueous extracts against four Gram-positive bacteria (Bacillus cereus, Streptococcus mutans, Lactobacillus acidophilus and Staphylococcus aureus), two Gram- negative bacteria, (Escherichia coli and Klebsiella pneumoniae) and two yeasts ( and ). Results indicate moderate to good antimicrobial activity for most pathogens studied with S. cuneifolia having the greatest overall antimicrobial efficacy with MIC values mostly ranging between 0.4 and 6.0 mg mL− 1 for the solvent extracts. The aqueous extracts indicated similar or in selected samples better activities than the solvent extracts. © 2006 SAAB. Published by Elsevier B.V. All rights reserved.

Keywords: Clausena anisata; Dovyalis abyssinica; Maerua oblongifolia; Osyris quadripartite; Pittosporum viridiflorum; Sida cuneifolia; Stereospermum kunthianum; Toothbrush sticks; Antimicrobial

African toothbrush sticks have been used for centuries for the investigation of crude khat chewing sticks, which have not been maintenance of oral hygiene. The chewing sticks, termed as investigated previously. Kassu et al. (1999) assessed the most such due to the need for the user to chew the stick prior to common toothbrush sticks used by rural and urban Ethiopians. brushing, have been used throughout the Greek, Roman, Jewish Twenty species were identified of which; Maerua and Islamic Empires (Almas, 2001). Even though many people oblongifolia (‘Wawate’), Sida cuneifolia (‘Chifrig’), Osyris have abandoned the traditional toothbrush sticks and adapted to quadripartita (‘Keret’), Clausena anisata (‘Limich’), Stereos- the conventional tooth brushing method, some societies still permum kunthianum (‘Zana’) and Pittosporum viridiflorum make use of chewing sticks as a daily ritual to maintain oral (‘Ambilbaye’) together with Dovyalis abyssinica (‘Koshe ser’) hygiene. This is especially true in developing countries where acquired from Addis Ababa (Ethiopia) were selected for this economics, customs, religion and the availability of oral antimicrobial study. From the available literature, only one (C. hygiene tools play a role in their continued use (Kemoli et al., anisata) of the seven toothbrush stick species selected for this 2001). Often, chewing sticks are used by the majority of the study has previously been investigated (Kemoli et al., 2001). population, as is the case of Ethiopians (Fadulu, 1975; Olsson, The rationale of this study is to determine whether any of these 1978). Although the World Health Organization has promoted sticks exhibit in vitro antimicrobial properties and to establish a the use of toothbrush sticks (Wu et al., 2001) and has scientific basis for their frequent use. encouraged further research of their efficacy, few studies have Extracts were prepared by submerging the dried, macerated been undertaken on the potential antimicrobial properties of stems (with the exception of S. cuneifolia where the roots were chewing sticks. Al-hebshi et al. (2006) confirm this in their used) in dichloromethane and methanol (1:1). After 3 h at 40 °C, the extracts were filtered, evaporated and stored at 4 °C for ⁎ Corresponding author. further antimicrobial analysis. Aqueous extracts were prepared E-mail address: [email protected] (S.F. van Vuuren). where sufficient plant material was available. Macerated

0254-6299/$ - see front matter © 2006 SAAB. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.sajb.2006.03.009 S.F. van Vuuren, A.M. Viljoen / South African Journal of Botany 72 (2006) 646–648 647 ⁎⁎ 2 was

material was extracted in sterile distilled water, kept at ambient 3 temperature overnight, filtered and stored at −80 °C before ⁎ (clinical strain) L. acidophilus 2.5×10 lyophilization. 1

Antimicrobial activity was determined by the minimum S. mutans inhibitory concentration (MIC) assay (Eloff, 1998). The extracts ⁎⁎ 2 were investigated against four Gram-positive reference bacterial 3 strains, two Gram-negative strains, two reference yeasts and one ⁎ ATCC 90112 neoformans 1 clinical Gram-positive isolate (Lactobacillus acidophilus), cul- 2.5×10 tured from the oral mucosa (Table 1). Although most of the test organisms were not strictly oral pathogens, selection was based on ⁎⁎ 2 the role these organisms play in oral hygiene and potential for 3

causing dental diseases, periapical lesions, periodontal abscesses ⁎ ATCC 10231 1 1.3×10 =controls for all bacteria except and possible gingivitis (Lafl and Ababneh, 1995). Streptococcus c mutans and L. acidophilus have been associated with dental ⁎⁎ 2 caries and studies thereof are considered as a reliable means of 4 monitoring microbial levels in the oral cavity (Darout et al., ⁎ ATCC 8739 E. coli C. albicans C. 1 2002). Studies of these two oral pathogens differed from the 1.6×10 conventional MIC investigations by the addition of blood and/or

incubation under CO2. As results visualized with p-iodonitrote- ⁎⁎ 2 trazolium violet were not possible when culturing with blood, 3 aliquots for all wells were subcultured onto blood agar plates to ⁎ NCTC 9633 1 Micro-organism pneumoniae confirm inhibition profiles. Positive controls were included to 1.3×10 ensure sensitivity of the pathogens to conventional antimicrobial ⁎⁎

methods. Studies were done in duplicate or triplicate where 2 necessary. 3

Minimum inhibitory concentrations for the solvent extracts ⁎ ATCC 10449 1 (Table 1) indicate moderate to good antimicrobial activity for 0.3×10 most pathogens. Medicinal having MIC values below 8 mg −1 ⁎⁎ 2 mL have been reported as being effective for antimicrobial 3 therapy (Fabrey et al., 1998). Toothbrush stick extracts generally − 1 ⁎ 1 ATCC 29212 had MIC ranges between 0.25 and 8 mg mL for most patho- 0.8×10 gens. A few exceptions however were noted. With the oral −1

pathogen S. mutans, high MIC values (16 mg mL )were ⁎⁎ 2 obtained for O. quadripartita, P. viridiflorum, D. abyssinica and 3 S. kunthianum indicating the least sensitivity when compared ⁎ 1 ATCC 25923 with the other eight pathogens studied. Using the disc diffusion 6.3×10 assay, Kemoli et al. (2001) investigated the antimicrobial potential ⁎⁎ of C. anisata with seven other chewing stick extracts and no 2 3 activity was noted for S. mutans. In this study moderate sensitivity −1 (8 mg mL ) was observed. It is well known that results obtained ⁎ 1 ATCC 11778 3.0 b 4.0 2.0 8.0 4.0 8 4.0 3.0 2.0 6.0 3.0 4.0 3.0 2.0 3.0 8.0 b B. cereus S. aureus E. faecalis S. mutans K. 0.3×10 1.5 0.4 0.4 1.0 4.0 4.0 16 2.0 4.0 4.0 1.0 b 1.3 0.8 2.0 0.3 16 1.0 4.0 4.06.0 16 a 6.0 3.0 8.0 a 8.0 2.0 4.0 a 4.0 12 1.0 a 4.0 6.0 2.0 8.0 a 3.0 3.0 2.0 a 1.5 1.0 4.0 8.0 a b 1.5 a 0.4 a 4.0 4.0 3.0 4.0 8.0 8.0 16 a 8.0 2.0 6.0 4.0 6.0 4.0 2.0 0.5 6.0 a 4.0 a 6.0 a 1.0 8.0 16 a 8.0 a 4.0 4.0 3.0 4.0 2.0 1.0 8.0 a 3.0 1.0 1.5 1.0 4.0 b 16 4.0 6.0 b 6.0 4.0 0.8 0.5 0.3 0.2 16 1.0 with the disc diffusion method cannot always be correlated to ’ MIC data (Brantner and Grein, 1994; Janssen et al., 1986; Lafl ’ ’ ’ ’ ) for the Ethiopian toothbrush stick dichloromethane/methanol and aqueous extracts

and Ababneh, 1995; Njenga et al., 2005) and this could possibly 1 ’ ’ −

explain the discrepancy between the two studies. No antimicro- =water extracts, a=not determined due to insufficient sample; b=inactive at a starting concentration of 64 mg/mL Zana Wawate Chifrig Kosher ser Anbilbaye Keret Limich ⁎⁎ ‘ ‘ ‘ ‘ ‘ ‘ bial sensitivity trend was observed when comparing the overall ‘ activities of the toothbrush sticks i.e. S. kunthianum (Zana) having the highest efficacy against B. cereus, S. aureus and was penicillin; controls for yeasts were amphotericin B. E. coli, but also having one of the lowest efficacies against S. mutans and L. acidophilus. This could indicate that the extract

is particularly species specific. S. mutans and L. acidophilus S. mutans Bignoniaceae Capparidaceae Malvaceae Flacourtiaceae Pittosporaceae Santalaceae showed the least antimicrobial sensitivities against most extracts with S. cuneifolia (Chifrig) having the lowest MIC values (1.0 mg mL−1 and 0.4 mg mL−1 respectively). It is interesting to note that these two anaerobic organisms showed the highest sensitivity to

the only extract of root origin. Lafl and Ababneh (1995) reported c lower efficacies for chewing sticks used in Jordan against kunthianum oblongifolia viridiflorum quadripartita =methanol/dichloromethane (1:1) extracts, Table 1 Mean minimum inhibitory concentrations (mg mL ⁎ Stereospermum Chewing sticks Botanical name Family Local name Control ciprofloxacin; control for Maerua Sida cuneifolia Dovyalis abyssinica Pittosporum Osyris S. mutans and L. acidophilus when compared to S. aureus.Recent Clausena anisata 648 S.F. van Vuuren, A.M. Viljoen / South African Journal of Botany 72 (2006) 646–648 clinical studies by Almas and Al-Zeid (2004) on Miswak chewing sew and E. Dagne (University of Addis Ababa) are thanked for sticks indicate reduced microbial counts of S. mutans but no identifying plant material and confirming the local vernacular significant microbial reduction for L. acidophilus. S. cuneifolia names. (Chifrig) showed the highest efficacies (0.4–6mgmL−1)against all pathogens studied with the exception of S. aureus (16 mg References mL−1)havinglessactivity. As the traditional use of the sticks involves an aqueous Al-hebshi, N., Al-haroni, M., Skaug, N., 2006. In vitro antimicrobial and environment, extracts prepared with water were undertaken resistance-modifying activities of aqueous crude khat extracts against oral microorganisms. Archives of Oral Biology 51, 183–188. where sufficient plant material was available. Results indicate Almas, K., 2001. The antimicrobial effects of seven different types of Asian that for most samples activities were very similar to that found chewing sticks. Odonto-Stomatologie Tropicale 96, 17–20. with the dichloromethane and methanol extract, and even much Almas, K., Al-Zeid, Z., 2004. The immediate antimicrobial effect of a toothbrush better efficacies noted for some samples. M. oblongifolia water and Miswak on cariogenic bacteria: a clinical study. The Journal of – extracts tested against S. aureus and K. pneumoniae had a two Contemporary Dental Practice 5, 1 9. Brantner, A., Grein, E., 1994. Antibacterial activity of plant extracts used to three-fold increase in activity. D. abyssinica water extracts externally in traditional medicine. Journal of Ethnopharmacology 44, 35–40. tested against K. pneumoniae and C. neoformans had a four- Darout, I.A., Albandar, J.M., Skuug, N., Ali, R.W., 2002. Salivary microbiota fold increase in activity. The extracts of Osyris quadripartite levels in relation to periodontal status, experience of caries and miswak use and S. kunthianum tested against L. acidophilus showed the in Sudanese adults. Journal of Clinical Periodontology 29, 411–430. greatest variation with the water extracts having a sixteen times Eloff, J.N., 1998. A sensitive and quick microplate method to determine the minimal inhibitory concentration of plant extracts for bacteria. Planta greater efficacy. Increased efficacies were also noted for the Medica 64, 711–713. aqueous extracts of S. kunthianum against B. cereus, S. aureus, Fabrey, W., Okemo, P.O., Ansorg, R., 1998. Antibacterial activity of East S. mutans, C. albicans and C. neoformans. African medicinal plants. Journal of Ethnopharmacology 60, 79–84. Although one cannot always extrapolate in vitro results to Fadulu, S.O., 1975. The antibacterial properties of the buffer extracts of chewing – the in vivo situation, recent clinical studies on toothbrush sticks sticks used in Nigeria. Planta Medica 27, 122 126. Janssen, A.M., Scheffer, J.J.C., Baerheim Svendsen, A., 1986. Antimicrobial (Almas, 2001) have shown reduced oral microbial counts when activity of essential oils: a 1976–1986 literature review. Aspects of the test compared with conventional toothpaste. While the continuous methods. Planta Medica 53, 395–398. physical mechanism of tooth brushing will assist in plaque Kassu, A., Dagne, E., Abate, D., De Castro, A., van Wyk, B.-E., 1999. Ethno- removal and reduction in bacterial populations, the antimicro- medical aspects of the commonly used toothbrush sticks in Ethiopia. East – bial properties of the toothbrush sticks play an active role in oral African Medical Journal 11, 651 653. Kemoli, A.M., van Amerongen, W.E., de Soet, J.J., 2001. Antimicrobial and hygiene. At this stage we can only hypothesize to what natural buffer capacity of crude extracts of chewing sticks (Miswaki) from Kenya. compounds in the sticks are responsible for the observed Journal of Dentistry for Children 183–188 (May–June). antimicrobial activities seen in the toothbrush sticks studied. Lafl, T.A., Ababneh, H., 1995. The effect of the extract of the Miswaki (chewing Bioassay guided fractionation is recommended to identify sticks) used in Jordan and the Middle East on oral bacteria. International – the antimicrobial compounds found in the most active extract Dental Journal 45, 218 222. Njenga, E., van Vuuren, S.F., Viljoen, A.M., 2005. Antimicrobial activity of S. cuneifolia (Chifrig). Eriocephalus species. South African Journal of Botany 71, 81–87. Olsson, B., 1978. Efficiency of traditional chewing sticks in oral hygiene programs Acknowledgements among Ethiopian schoolchildren. Community Dental Oral Epidemiology 6, 105–109. The National Research Foundation (IKS), and Faculty of Wu, C.D., Darout, I.A., Skaug, N., 2001. Chewing sticks: timeless natural toothbrushes for oral cleansing. Journal of Periodontal Research 36, 275–284. Health Sciences Research Endowment Fund (WITS) are hereby acknowledged for the financial support. Professors S. Demis-

Edited by PJ Houghton