Journal of Ethnopharmacology 155 (2014) 852–855
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Ethnopharmacological communication
Antibacterial activities of extracts from Ugandan medicinal plants used for oral care Francis Ocheng a,b,c,n, Freddie Bwanga b, Moses Joloba b, Ann-Karin Borg-Karlson d, Anders Gustafsson c, Celestino Obua e a
Department of Dentistry, School of Health Sciences, College of Health Sciences, Makerere University, P.O. Box 7072, Kampala, Uganda Department of Medical Microbiology, School of Biomedical Sciences College of Health Sciences, Makerere University, P.O. Box 7072 Kampala, Uganda c Unit of Periodontology, Department of Dental Medicine, Karolinska Institutet, P.O. Box, 4064, SE-141 04, Huddinge, Sweden d Ecological Chemistry Group, Department of Chemistry, School of Chemical Science and Engineering, Royal Institute of Technology, SE-100 44 Stockholm, Sweden e Department of Pharmacology and Therapeutics, School of Biomedical Sciences College of Health Sciences, Makerere University, P.O. Box 7072, Kampala, Uganda b
art ic l e i nf o
a b s t r a c t
Article history: Received 16 September 2013 Received in revised form 6 May 2014 Accepted 6 June 2014 Available online 16 June 2014
Ethnopharmacological relevance: Medicinal plants are widely used for treatment of oral/dental diseases in Uganda. Aim of the study: To investigate antibacterial activities of 16 commonly used medicinal plants on microorganisms associated with periodontal diseases (PD) and dental caries (DC). Materials and methods: Pulp juice and solvent extracts (hexane, methanol and water) from the plants were tested against Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia associated with PD and Streptococcus mutans, Streptococcus sobrinus, Lactobacillus acidophilus associated with DC. Tests were done using agar well-diffusion (pulp juice) and agar-dilution (Solvent extracts) assays. Results: Pulp juice from Zanthoxylum chalybeum and Euclea latidens showed activity against all the bacteria, Zanthoxylum chalybeum being most active. Hexane extract from aerial part of Helichrysum odoratissimum was most active (MIC: 0.125–0.5 mg/ml). Methanol extract from leaves of Lantana trifolia showed activity against all bacteria (MIC: 0.25–1 mg/ml). Conclusion: Several of the tested plants showed antibacterial activities against bacteria associated with PD and DC, meriting further investigations. & 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Periodontal diseases (PD) and dental caries (DC) are common oral conditions, caused by bacterial plaque (Marcenes et al., 2013). In PD, presence of bacteria in the gingival crevice lead to inflammations that cause destruction of tissues attaching the teeth to the jawbone. Gram-negative bacteria such as Porphyromonas gingivalis, Tannerella forsythia and Aggregatibacter actinomycetemcomitans are associated with PD (Ximénez-Fyvie et al. 2000). DC is a destruction of dental hard tissues involving acidogenic bacteria that include Streptococcus mutans, Streptococcus sobrinus and Lactobacillus spp. (Takahashi and Nyvad, 2011). The use of plants as alternative tools for oral care is an ancient custom, which remains widespread across many parts of the world
n
Correspondence to: Tel.: þ 256 41 4532803; fax: þ256 41 4272861. E-mail addresses: [email protected], [email protected], [email protected] (F. Ocheng). http://dx.doi.org/10.1016/j.jep.2014.06.027 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.
(Hyson, 2003). Extracts from some plants have been found to be effective against oral bacteria (More et al., 2008). The WHO (1987) has recommended and encouraged the use of plants as tools for oral hygiene in areas where this is customary. In Uganda, the practice of medicinal plants for oral care is well established, but there is limited data on their antibacterial activities. The aim of the present study was to investigate the in vitro antibacterial activities of fresh pulp juice and solvent extracts from 16 Ugandan medicinal plants used for oral care.
2. Material and methods 2.1. Plant materials Plants used for traditional treatment of oral/dental diseases were selected (Table 1) and collected from different parts of Uganda during the second half of 2011. Plant parts to be used for extraction
F. Ocheng et al. / Journal of Ethnopharmacology 155 (2014) 852–855
853
Table 1 Ugandan medicinal plants used for treatment of dental/oral diseases. Family and botanical name (Voucher number)a APOCYNACEAE Carissa edulis.Vahl (FO-001) ASTERACEAE Bidens pilosa (FO-002) Crassocephalum vitellinum (FO-003) Helichrysum odoratissimum (FO-004) Vernonia amygdalina (FO-005) EBENACEAE Euclea latidens. Stapf (FO-006) CUCURBITACEAE Momordica foetida K.Schumach (FO007) LAMIACEACE Hoslundia opposite (FO-008) Ocimum gratissimum (FO-009) POACEAE Cymbopogon citrates (FO-010) Cymbopogon nardus (FO-011) RANUNCULACEAE Clematis hirsuta. Guill. & Perr. (FO-012) RUTACEAE Teclea nobilis. Delile (FO-013) Zanthoxylum chalybeum (FO-014) SOLANACEAE Solanum nigrum (FO-015) VERBENACEAE Lantana trifolia (FO-016)
Local name/tribe
Plant part: Ethno-medical use(s) (references)
Dental/oral disease(s) treated
Achuga/Langi
LT: pound, add water and squeeze few drops on gumb
Teething syndrome
b
Enyabarashana/Rukiga Paroti / Kupsabiny Chebushe / Kupsabiny Okello-okello/Langi
LT: chew fresh LT: Dry, burn and rub ash on false teethb LT: Dry, burn and rub ash on false teethb ST: Brush teeth, twigs chewed freshb,c
Amuru-dyek/Langi
RT: Use to brush teethb
Toothache Teething syndrome Teething syndrome Dental caries Dental caries b
Bomo/Langi
LT: Pound, squeeze juice onto the wound.
Kamunye/Baganda Omujaja/Rukiga
LT: Chew freshd,e LT: Chew freshb
Mouth wounds Toothache
Kasubi/Baganda Ettette/Rukiga Bad breath, dental caries.
LT: taken as tea; chewed freshc Root: chewed; LT: Young part chewed and used for cleaningb
Bad breath, toothache
Adwe/Langi
LT: pound and apply paste on painful tooth;Infusion drunkb,e
Toothache Sore throat
Nzo/Baganda Owucu/Langi Songowowo/ Pokot
ST: Use to brush teethd RT: Use to brush;ST: Bark chewede
Dental caries Dental caries Toothache
Osuga/Langi
LT, FT: crush into paste and rub on the gume
Teething syndrome
Kayuki-yuki/Baganda
ST: Use as toothbrush to clean and freshen; LT: infusion swallowedd,f Oral Hygiene Tonsillitis
Teething syndrome
a
Voucher specimen number at Herbarium, Makerere University; Mubiru et al., 1994; Hirt and M'Pia, 2008; d Hamill et al., 2003; e Kokwaro, 1993; f Odongo et al., 2011; FT ¼Fruit; LT ¼Leave; RT ¼ Root; ST¼Stem. b c
of fresh pulp juice were transported in an ice cooled box and stored at 80 1C until use. Identities of the plants were confirmed by botanists at the Herbarium, Department of Botany, Makerere University, Uganda, where voucher specimens were kept. 2.2. Preparation of fresh pulp juice Fresh pulp juice was expressed from pieces of fresh plant parts cited in Table 1 using a manual fruit juicer and tested immediately at full strength and at 50 and 25%. Dilutions were made using brain heart infusion (BHI) broth. 2.3. Preparation of solvent extracts Preparation of solvent extracts was adapted from Matu and van Staden (2003) with modifications. Three solvents (hexane, methanol and water) were used. Hexane extract (HE) and methanol extracts (ME) were obtained by macerating 300–500 g of powdered plant material in 600–2000 mls of hexane or methanol, for 72 h and filtered using Whatman filter paper. The filtrate was concentrated using a rotary vacuum evaporator to approximately 50 mls and the resultant concentrate dried in an oven at 40–50 1C. The extract was stored at 4 1C until use. For water extract (WE), 10–35 g of powdered plant material was heated in 200–500 mls of distilled water to boiling point for about 20 min and the extract filtered using Whatman filter paper and concentrated using a freeze dryer. The residue was stored at 4 1C.
2.4. Bacterial strains and culture conditions The bacterial strains from Culture Collection of the University of Gothenburg (CCUG) were used: Streptococcus mutans (CCUG 6519 T), Streptococcus sobrinus (CCUG 25735 T), Lactobacillus acidophilus (CCUG 5917 T), Porphyromonas gingivalis (CCUG 25226), Aggregatibacter actinomycetemcomitans (CCUG 56173), Tannerella forsythia (CCUG 21028 AT). The strains were grown and maintained as described by Sofrata et al. (2008), with the following modifications: 5% sheep blood was used instead of citrated horse blood; Colombia base agar for growing Streptococcus mutans and Lactobacillus acidophilus was not supplemented with tryptophan; Tannerella forsythia was grown anaerobically in chocolate agar supplemented with hemin (0.05 mg/ml) and vitamin K (0.01 mg/ml). 2.5. Antibacterial activity testing 2.5.1. Fresh pulp juice Agar well-diffusion assay was used to test for antibacterial activities of pulp juice. Streptococcus mutans, Streptococcus sobrinus, Lactobacillus acidophilus and Aggregatibacter actinomycetemcomitans were suspended in phosphate buffered saline (PBS). Porphyromonas gingivalis and Tannerella forsythia were suspended in peptone yeast glucose. The density of each bacterial suspension was adjusted to equal that of 0.5 McFarland standards. Each bacterial suspension was swabbed over agar plate (see Section 2.4) and a 7 mm core was removed from three positions on each plate. The wells were aseptically filled as follows: i) 0.1 mls of fresh pulp juice or dilutions; ii) 0.1 mls of doxycycline (30 mg) solution in normal saline to act as positive control; iii) 0.1 mls
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F. Ocheng et al. / Journal of Ethnopharmacology 155 (2014) 852–855
of BHI broth to act as negative control. The plates were incubated under the conditions described by Softrata et al. (2008). The experiments were repeated three times and the antibacterial activity expressed as the ratio of the inhibition zone produced by the pulp juice and the inhibition caused by the doxycycline in the plate. 2.5.2. Solvent extracts Agar-dilution assay was used to screen for antibacterial activities of 66 solvent extracts in the special media prepared for each bacteria in Section 2.4. The extracts were first screened at a concentration of 1 mg/ml, and if active, the minimum inhibitory concentrations (MIC) determined following the European Committee for Antimicrobial Susceptibility Testing (EUCAST) protocol for determination of MIC by agar dilution (EUCAST, 2000), with modifications. For testing, 0.5 mls of extract at a concentration of 20 mg/ml in 10% dimethyl sulfoxide (DMSO) or PBS (for WE) was added to 9.5 mls of melted agar in a falcon tube, shaken and poured in petri-dish. The bacteria were suspended as described in Section 2.5.1 and each suspension adjusted to give 107 CFU/ml. 1 ml of the suspension was spotted onto the surface of agar plate. A plate with 1% DMSO and another incorporated with 10 mg doxycycline were also inoculated to serve as solvent and positive controls respectively. The plates were incubated anaerobically at 37 1C for 24–48 h. The extract was declared active if it completely inhibited visible growth of bacteria as judged by the naked eye. For MIC determination, two-fold dilutions of the extracts in DMSO or PBS (for WE) were prepared at final in-test concentrations ranging from 0.0313–1 mg/ml, and the bacteria prepared and spotted as above. The MIC of doxycycline for each bacterium was also run to validate the procedures. The final in-test concentrations of doxycycline ranged from 7.8 10 5 to 2.5 10 3 mg/ml. All tests were performed in duplicates
3. Results 3.1. Fresh pulp juice Fresh pulp juice prepared from 16 plants species were screened. Only pulp juice from Zanthoxylum chalybeum and Euclea
latidens showed activity against all the bacteria tested and at all strengths. The most active pulp juice was from Zanthoxylum chalybeum. It exhibited best activity against Lactobacillus acidophilus, producing 63, 52 and 45% zones of inhibition with the undiluted, 50 and 25% strengths respectively compared to the doxycycline control. Pulp juice from Ocimum gratissimum exhibited mild antibacterial activities on two bacterial species, Aggregatibacter actinomycetemcomitans and Tannerella forsythia, at undiluted and 50% strength.
4. Solvent extracts Extracts from 15 plants species were screened, and MIC of active extracts are summarized in Table 2. One plant species, Carissa edulis could not be obtained for solvent extraction. HE from the aerial part of Helichrysum odoratissimum exhibited the best activity with MIC ranging from 0.0125 to 0.5 mg/ml on all the bacteria tested except Aggregatibacter actinomycetemcomitans. ME from Lantana trifolia was active on all bacteria tested with MIC ranging 0.25–1 mg/ml. Lactobacillus acidophilus was sensitive to plant extracts with all three solvents.
5. Discussion and conclusion Pulp juice from Zanthoxylum chalybeum exhibited the strongest inhibitory activity. Most studies on Zanthoxylum chalybeum (Olila et al., 2001; Matu and van Staden, 2003) used solvent extracts from dried plant materials or different bacterial species, making direct comparisons difficult. We showed that pulp juice from Zanthoxylum chalybeum and Euclea latidens were active at 50 and 25% strength, confirming their use as chewing sticks for brushing teeth, where during normal use, pulp juice leach into the mouth and is diluted by saliva. The 50 and 25% strengths of pulp juice thus mimic the conditions in the mouth. To the best of our knowledge, this is the first time antibacterial activity of Euclea latidens is being shown.
Table 2 Minimum inhibitory concentrations of solvent extracts with antibacterial activity. Plant species
Plant part/Ea
MIC (mg/ml) A.ab
Crassocephalum vitellinum Helichrysum odoratissimum Helichrysum odoratissimum Vernonia amygdalina Euclea latidens Momordica foetida Momordica foetida Hoslundia opposita Hoslundia opposita Cymbopogon citratus Cymbopogon nardus Teclea nobilis Zanthoxylum chalybeum Zanthoxylum chalybeum Zanthoxylum chalybeum Zanthoxylum chalybeum Zanthoxylum chalybeum Lantana trifolia Lantana trifolia Lantana trifolia Doxycycline DMSO 1% (Solvent control) a
AE/H AE/H AE/M LF/M SB/M RT/M RT/W LF/M ST/W AE/W AE/M LF/H LF/W SB/H SB/W RB/H RB/M LF/H LF/M LF/W
P.g
T.f
S.m
S.s
L.a
1 0.125 0.5
0.5
0.25 1
0.125 1
0.125 0.5
1
0.5
1 1
1
0.5 1 0.5 1 1 1 0.5
1 1
1 1 1 1 1
0.5 1 0.5 0.5 0.25 1 1.56 10 4
0.5 0.5 1 3.13 10
1 1 1
0.5 4
6.25 10
4
1.56 10
1 4
3.13 10
1 4
12.5 10 4
Active plant part/E: AE ¼ Aerial part; LF ¼Leaves; SB ¼ Stem bark; ST¼Stem; RT ¼Root; RB ¼Root bark; H ¼ Hexane; M ¼Methanol; W ¼Water. Microorganisms: A.a ¼Aggregatibacter actinomycetemcomitans; P.g¼ Porphyromonas gingivalis; T.f ¼ Tannerella forsythia; S.m ¼ Streptococcus mutans; S.s ¼ Streptococcus sobrinus, L.a ¼ Lactobacillus acidophilus. b
F. Ocheng et al. / Journal of Ethnopharmacology 155 (2014) 852–855
ME from the leaves of Lantana trifolia was active against all bacteria tested, which is in agreement with earlier studies (Rwangabo et al., 1988). The activities in Lantana trifolia seemed to be concentrated in the leaves. This is of importance because harvesting plant leaves is more sustainable compared to harvesting of stems or roots. Solvent extracts from Bidens pilosa, Ocimum gratissimum, Clematis hirsuta, and Solanum nigrum did not exhibit any activity, although antimicrobial activities have previously been reported in Bidens pilosa (Rabe and van Staden, 1997) and Clematis hirsuta, (Cos et al., 2002). Active components in plant can be affected by the locality of the plant, time of collection, storage conditions, study design, and this could explain the contrasting results (Samuelsson and Bohlin, 2010). In conclusion, pulp juice prepared from fresh root of Zanthoxylum chalybeum and Euclea latidens have antibacterial activities against bacteria associated with PD and DC. Antibacterial activities were also demonstrated in solvent extracts from Helichrysum odoratissimum, Zanthoxylum chalybeum, Lantana trifolia, Cymbopogon nardus, thus providing potential for further development. Acknowledgment This work was supported by Swedish International Development Agency (grant no. 75007369). References Cos, P., Hermans, N., De-Bruyne, T., Apers, S., Sindambiwe, J.B., Vanden Berghe, D., Pieters, L., Vlietinck, A.J., 2002. Further evaluation of Rwandan medicinal plant extracts for their antimicrobial and antiviral activities. Journal of Ethnopharmacology 79, 155–163. EUCAST, 2000. Determination of minimum inhibitory concentrations (MICs) of antibacterial agents by agar dilution. Clinical Microbiolgy and Infection 6, 509–515.
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Hamill, F.A., Apio, S., Mubiru, N.K., Bukenya-Ziraba, R., Mosango, M., Maganyi, O.W., Soejarto, D.D., 2003. Traditional herbal drugs of Southern Uganda, II: literature analysis and antimicrobial assays. Journal of Ethnopharmacology 84, 57–78. Hirt, H.M., M'Pia, B., 2008. Natural Medicine in the Tropics 1: Foundation, third ed. Marianum Press, Kisubi. Hyson Jr., J.M., 2003. History of the toothbrush. Journal of the History of Dentistry 51, 73–80. Kokwaro, J.O., 1993. Medicinal Plants of East Africa, second ed. Kenya Literature Bureau, Nairobi. Marcenes, W., Kassebaum, N.J., Bernabe, E., Flaxman, A., Naghavi, M., Lopez, A., Murray, C.J., 2013. Global burden of oral conditions in 1990-2010: a systematic analysis. Journal of Dental Research 92, 592–597. Matu, E.N., van Staden, J., 2003. Antibacterial and anti-inflammatory activities of some plants used for medicinal purposes in Kenya. Journal of Ethnopharmacology 87, 35–41. More, G., Tshikalange, T.E., Lall, N., Botha, F., Meyer, J.J., 2008. Antimicrobial activity of medicinal plants against oral microorganisms. Journal of Ethnopharmacology 119, 473–477. Mubiru, N.K., Kakoooko, A.B., Mutyaba, J.B., Amai, C.A., Apio, S.K., A.M., A., Ndugga, J. Z.B.S., Ndugga, E.S., 1994. Ethnomedicine in Uganda. National Chemotherapeutic Research Laboratory (NCRL), Ministy of Health, Uganda. Odongo, C.O., Musisi, N.L., Waako, P., Obua, C., 2011. Chewing-stick practices using plants with anti-streptococcal activity in a Ugandan rural community. Frontiers in Pharmacology 2, 1–5. Olila, D., Olwa-Odyek, Opuda-Asibo, J., 2001. Antibacterial and antifungal activities of extracts of Zanthoxylum chalybeum and Warburgia ugandensis, Ugandan medicinal plants. African Health Sciences 1, 66–72. Rabe, T., van Staden, J., 1997. Antibacterial activity of South African plants used for medicinal purposes. Journal of Ethnopharmacology 56, 81–87. Rwangabo, P.C., Claeys, M., Pieters, L., Corthout, J., Vanden Berghe, D.A., Vlietinck, A. J., 1988. Umuhengerin, a new antimicrobially active flavonoid from Lantana trifolia. Journal of Natural Products 51, 966–968. Samuelsson, G., Bohlin, L., 2010. Drugs of Natural Origin: A Treatise of Pharmacognosy, sixth ed. Swedish Pharmaceutical Press, Stockholm. Sofrata, A.H., Claesson, R.L., Lingstrom, P.K., Gustafsson, A.K., 2008. Strong antibacterial effect of miswak against oral microorganisms associated with periodontitis and caries. Journal of Periodontology 79, 1474–1479. Takahashi, N., Nyvad, B., 2011. The role of bacteria in the caries process: ecological perspectives. Journal of Dental Research 90, 294–303. WHO, 1987. Prevention of Oral Diseases. World Health Organization, Geneva p. 83 (offset publication No. 103). Ximénez-Fyvie, L.A., Haffajee, A.D., Socransky, S.S., 2000. Microbial composition of supra- and subgingival plaque in subjects with adult periodontitis. Journal of Clinical Periodontology 27, 722–732.
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Ethnopharmacological communication
Antibacterial activities of extracts from Ugandan medicinal plants used for oral care Francis Ocheng a,b,c,n, Freddie Bwanga b, Moses Joloba b, Ann-Karin Borg-Karlson d, Anders Gustafsson c, Celestino Obua e a
Department of Dentistry, School of Health Sciences, College of Health Sciences, Makerere University, P.O. Box 7072, Kampala, Uganda Department of Medical Microbiology, School of Biomedical Sciences College of Health Sciences, Makerere University, P.O. Box 7072 Kampala, Uganda c Unit of Periodontology, Department of Dental Medicine, Karolinska Institutet, P.O. Box, 4064, SE-141 04, Huddinge, Sweden d Ecological Chemistry Group, Department of Chemistry, School of Chemical Science and Engineering, Royal Institute of Technology, SE-100 44 Stockholm, Sweden e Department of Pharmacology and Therapeutics, School of Biomedical Sciences College of Health Sciences, Makerere University, P.O. Box 7072, Kampala, Uganda b
art ic l e i nf o
a b s t r a c t
Article history: Received 16 September 2013 Received in revised form 6 May 2014 Accepted 6 June 2014 Available online 16 June 2014
Ethnopharmacological relevance: Medicinal plants are widely used for treatment of oral/dental diseases in Uganda. Aim of the study: To investigate antibacterial activities of 16 commonly used medicinal plants on microorganisms associated with periodontal diseases (PD) and dental caries (DC). Materials and methods: Pulp juice and solvent extracts (hexane, methanol and water) from the plants were tested against Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia associated with PD and Streptococcus mutans, Streptococcus sobrinus, Lactobacillus acidophilus associated with DC. Tests were done using agar well-diffusion (pulp juice) and agar-dilution (Solvent extracts) assays. Results: Pulp juice from Zanthoxylum chalybeum and Euclea latidens showed activity against all the bacteria, Zanthoxylum chalybeum being most active. Hexane extract from aerial part of Helichrysum odoratissimum was most active (MIC: 0.125–0.5 mg/ml). Methanol extract from leaves of Lantana trifolia showed activity against all bacteria (MIC: 0.25–1 mg/ml). Conclusion: Several of the tested plants showed antibacterial activities against bacteria associated with PD and DC, meriting further investigations. & 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Periodontal diseases (PD) and dental caries (DC) are common oral conditions, caused by bacterial plaque (Marcenes et al., 2013). In PD, presence of bacteria in the gingival crevice lead to inflammations that cause destruction of tissues attaching the teeth to the jawbone. Gram-negative bacteria such as Porphyromonas gingivalis, Tannerella forsythia and Aggregatibacter actinomycetemcomitans are associated with PD (Ximénez-Fyvie et al. 2000). DC is a destruction of dental hard tissues involving acidogenic bacteria that include Streptococcus mutans, Streptococcus sobrinus and Lactobacillus spp. (Takahashi and Nyvad, 2011). The use of plants as alternative tools for oral care is an ancient custom, which remains widespread across many parts of the world
n
Correspondence to: Tel.: þ 256 41 4532803; fax: þ256 41 4272861. E-mail addresses: [email protected], [email protected], [email protected] (F. Ocheng). http://dx.doi.org/10.1016/j.jep.2014.06.027 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.
(Hyson, 2003). Extracts from some plants have been found to be effective against oral bacteria (More et al., 2008). The WHO (1987) has recommended and encouraged the use of plants as tools for oral hygiene in areas where this is customary. In Uganda, the practice of medicinal plants for oral care is well established, but there is limited data on their antibacterial activities. The aim of the present study was to investigate the in vitro antibacterial activities of fresh pulp juice and solvent extracts from 16 Ugandan medicinal plants used for oral care.
2. Material and methods 2.1. Plant materials Plants used for traditional treatment of oral/dental diseases were selected (Table 1) and collected from different parts of Uganda during the second half of 2011. Plant parts to be used for extraction
F. Ocheng et al. / Journal of Ethnopharmacology 155 (2014) 852–855
853
Table 1 Ugandan medicinal plants used for treatment of dental/oral diseases. Family and botanical name (Voucher number)a APOCYNACEAE Carissa edulis.Vahl (FO-001) ASTERACEAE Bidens pilosa (FO-002) Crassocephalum vitellinum (FO-003) Helichrysum odoratissimum (FO-004) Vernonia amygdalina (FO-005) EBENACEAE Euclea latidens. Stapf (FO-006) CUCURBITACEAE Momordica foetida K.Schumach (FO007) LAMIACEACE Hoslundia opposite (FO-008) Ocimum gratissimum (FO-009) POACEAE Cymbopogon citrates (FO-010) Cymbopogon nardus (FO-011) RANUNCULACEAE Clematis hirsuta. Guill. & Perr. (FO-012) RUTACEAE Teclea nobilis. Delile (FO-013) Zanthoxylum chalybeum (FO-014) SOLANACEAE Solanum nigrum (FO-015) VERBENACEAE Lantana trifolia (FO-016)
Local name/tribe
Plant part: Ethno-medical use(s) (references)
Dental/oral disease(s) treated
Achuga/Langi
LT: pound, add water and squeeze few drops on gumb
Teething syndrome
b
Enyabarashana/Rukiga Paroti / Kupsabiny Chebushe / Kupsabiny Okello-okello/Langi
LT: chew fresh LT: Dry, burn and rub ash on false teethb LT: Dry, burn and rub ash on false teethb ST: Brush teeth, twigs chewed freshb,c
Amuru-dyek/Langi
RT: Use to brush teethb
Toothache Teething syndrome Teething syndrome Dental caries Dental caries b
Bomo/Langi
LT: Pound, squeeze juice onto the wound.
Kamunye/Baganda Omujaja/Rukiga
LT: Chew freshd,e LT: Chew freshb
Mouth wounds Toothache
Kasubi/Baganda Ettette/Rukiga Bad breath, dental caries.
LT: taken as tea; chewed freshc Root: chewed; LT: Young part chewed and used for cleaningb
Bad breath, toothache
Adwe/Langi
LT: pound and apply paste on painful tooth;Infusion drunkb,e
Toothache Sore throat
Nzo/Baganda Owucu/Langi Songowowo/ Pokot
ST: Use to brush teethd RT: Use to brush;ST: Bark chewede
Dental caries Dental caries Toothache
Osuga/Langi
LT, FT: crush into paste and rub on the gume
Teething syndrome
Kayuki-yuki/Baganda
ST: Use as toothbrush to clean and freshen; LT: infusion swallowedd,f Oral Hygiene Tonsillitis
Teething syndrome
a
Voucher specimen number at Herbarium, Makerere University; Mubiru et al., 1994; Hirt and M'Pia, 2008; d Hamill et al., 2003; e Kokwaro, 1993; f Odongo et al., 2011; FT ¼Fruit; LT ¼Leave; RT ¼ Root; ST¼Stem. b c
of fresh pulp juice were transported in an ice cooled box and stored at 80 1C until use. Identities of the plants were confirmed by botanists at the Herbarium, Department of Botany, Makerere University, Uganda, where voucher specimens were kept. 2.2. Preparation of fresh pulp juice Fresh pulp juice was expressed from pieces of fresh plant parts cited in Table 1 using a manual fruit juicer and tested immediately at full strength and at 50 and 25%. Dilutions were made using brain heart infusion (BHI) broth. 2.3. Preparation of solvent extracts Preparation of solvent extracts was adapted from Matu and van Staden (2003) with modifications. Three solvents (hexane, methanol and water) were used. Hexane extract (HE) and methanol extracts (ME) were obtained by macerating 300–500 g of powdered plant material in 600–2000 mls of hexane or methanol, for 72 h and filtered using Whatman filter paper. The filtrate was concentrated using a rotary vacuum evaporator to approximately 50 mls and the resultant concentrate dried in an oven at 40–50 1C. The extract was stored at 4 1C until use. For water extract (WE), 10–35 g of powdered plant material was heated in 200–500 mls of distilled water to boiling point for about 20 min and the extract filtered using Whatman filter paper and concentrated using a freeze dryer. The residue was stored at 4 1C.
2.4. Bacterial strains and culture conditions The bacterial strains from Culture Collection of the University of Gothenburg (CCUG) were used: Streptococcus mutans (CCUG 6519 T), Streptococcus sobrinus (CCUG 25735 T), Lactobacillus acidophilus (CCUG 5917 T), Porphyromonas gingivalis (CCUG 25226), Aggregatibacter actinomycetemcomitans (CCUG 56173), Tannerella forsythia (CCUG 21028 AT). The strains were grown and maintained as described by Sofrata et al. (2008), with the following modifications: 5% sheep blood was used instead of citrated horse blood; Colombia base agar for growing Streptococcus mutans and Lactobacillus acidophilus was not supplemented with tryptophan; Tannerella forsythia was grown anaerobically in chocolate agar supplemented with hemin (0.05 mg/ml) and vitamin K (0.01 mg/ml). 2.5. Antibacterial activity testing 2.5.1. Fresh pulp juice Agar well-diffusion assay was used to test for antibacterial activities of pulp juice. Streptococcus mutans, Streptococcus sobrinus, Lactobacillus acidophilus and Aggregatibacter actinomycetemcomitans were suspended in phosphate buffered saline (PBS). Porphyromonas gingivalis and Tannerella forsythia were suspended in peptone yeast glucose. The density of each bacterial suspension was adjusted to equal that of 0.5 McFarland standards. Each bacterial suspension was swabbed over agar plate (see Section 2.4) and a 7 mm core was removed from three positions on each plate. The wells were aseptically filled as follows: i) 0.1 mls of fresh pulp juice or dilutions; ii) 0.1 mls of doxycycline (30 mg) solution in normal saline to act as positive control; iii) 0.1 mls
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F. Ocheng et al. / Journal of Ethnopharmacology 155 (2014) 852–855
of BHI broth to act as negative control. The plates were incubated under the conditions described by Softrata et al. (2008). The experiments were repeated three times and the antibacterial activity expressed as the ratio of the inhibition zone produced by the pulp juice and the inhibition caused by the doxycycline in the plate. 2.5.2. Solvent extracts Agar-dilution assay was used to screen for antibacterial activities of 66 solvent extracts in the special media prepared for each bacteria in Section 2.4. The extracts were first screened at a concentration of 1 mg/ml, and if active, the minimum inhibitory concentrations (MIC) determined following the European Committee for Antimicrobial Susceptibility Testing (EUCAST) protocol for determination of MIC by agar dilution (EUCAST, 2000), with modifications. For testing, 0.5 mls of extract at a concentration of 20 mg/ml in 10% dimethyl sulfoxide (DMSO) or PBS (for WE) was added to 9.5 mls of melted agar in a falcon tube, shaken and poured in petri-dish. The bacteria were suspended as described in Section 2.5.1 and each suspension adjusted to give 107 CFU/ml. 1 ml of the suspension was spotted onto the surface of agar plate. A plate with 1% DMSO and another incorporated with 10 mg doxycycline were also inoculated to serve as solvent and positive controls respectively. The plates were incubated anaerobically at 37 1C for 24–48 h. The extract was declared active if it completely inhibited visible growth of bacteria as judged by the naked eye. For MIC determination, two-fold dilutions of the extracts in DMSO or PBS (for WE) were prepared at final in-test concentrations ranging from 0.0313–1 mg/ml, and the bacteria prepared and spotted as above. The MIC of doxycycline for each bacterium was also run to validate the procedures. The final in-test concentrations of doxycycline ranged from 7.8 10 5 to 2.5 10 3 mg/ml. All tests were performed in duplicates
3. Results 3.1. Fresh pulp juice Fresh pulp juice prepared from 16 plants species were screened. Only pulp juice from Zanthoxylum chalybeum and Euclea
latidens showed activity against all the bacteria tested and at all strengths. The most active pulp juice was from Zanthoxylum chalybeum. It exhibited best activity against Lactobacillus acidophilus, producing 63, 52 and 45% zones of inhibition with the undiluted, 50 and 25% strengths respectively compared to the doxycycline control. Pulp juice from Ocimum gratissimum exhibited mild antibacterial activities on two bacterial species, Aggregatibacter actinomycetemcomitans and Tannerella forsythia, at undiluted and 50% strength.
4. Solvent extracts Extracts from 15 plants species were screened, and MIC of active extracts are summarized in Table 2. One plant species, Carissa edulis could not be obtained for solvent extraction. HE from the aerial part of Helichrysum odoratissimum exhibited the best activity with MIC ranging from 0.0125 to 0.5 mg/ml on all the bacteria tested except Aggregatibacter actinomycetemcomitans. ME from Lantana trifolia was active on all bacteria tested with MIC ranging 0.25–1 mg/ml. Lactobacillus acidophilus was sensitive to plant extracts with all three solvents.
5. Discussion and conclusion Pulp juice from Zanthoxylum chalybeum exhibited the strongest inhibitory activity. Most studies on Zanthoxylum chalybeum (Olila et al., 2001; Matu and van Staden, 2003) used solvent extracts from dried plant materials or different bacterial species, making direct comparisons difficult. We showed that pulp juice from Zanthoxylum chalybeum and Euclea latidens were active at 50 and 25% strength, confirming their use as chewing sticks for brushing teeth, where during normal use, pulp juice leach into the mouth and is diluted by saliva. The 50 and 25% strengths of pulp juice thus mimic the conditions in the mouth. To the best of our knowledge, this is the first time antibacterial activity of Euclea latidens is being shown.
Table 2 Minimum inhibitory concentrations of solvent extracts with antibacterial activity. Plant species
Plant part/Ea
MIC (mg/ml) A.ab
Crassocephalum vitellinum Helichrysum odoratissimum Helichrysum odoratissimum Vernonia amygdalina Euclea latidens Momordica foetida Momordica foetida Hoslundia opposita Hoslundia opposita Cymbopogon citratus Cymbopogon nardus Teclea nobilis Zanthoxylum chalybeum Zanthoxylum chalybeum Zanthoxylum chalybeum Zanthoxylum chalybeum Zanthoxylum chalybeum Lantana trifolia Lantana trifolia Lantana trifolia Doxycycline DMSO 1% (Solvent control) a
AE/H AE/H AE/M LF/M SB/M RT/M RT/W LF/M ST/W AE/W AE/M LF/H LF/W SB/H SB/W RB/H RB/M LF/H LF/M LF/W
P.g
T.f
S.m
S.s
L.a
1 0.125 0.5
0.5
0.25 1
0.125 1
0.125 0.5
1
0.5
1 1
1
0.5 1 0.5 1 1 1 0.5
1 1
1 1 1 1 1
0.5 1 0.5 0.5 0.25 1 1.56 10 4
0.5 0.5 1 3.13 10
1 1 1
0.5 4
6.25 10
4
1.56 10
1 4
3.13 10
1 4
12.5 10 4
Active plant part/E: AE ¼ Aerial part; LF ¼Leaves; SB ¼ Stem bark; ST¼Stem; RT ¼Root; RB ¼Root bark; H ¼ Hexane; M ¼Methanol; W ¼Water. Microorganisms: A.a ¼Aggregatibacter actinomycetemcomitans; P.g¼ Porphyromonas gingivalis; T.f ¼ Tannerella forsythia; S.m ¼ Streptococcus mutans; S.s ¼ Streptococcus sobrinus, L.a ¼ Lactobacillus acidophilus. b
F. Ocheng et al. / Journal of Ethnopharmacology 155 (2014) 852–855
ME from the leaves of Lantana trifolia was active against all bacteria tested, which is in agreement with earlier studies (Rwangabo et al., 1988). The activities in Lantana trifolia seemed to be concentrated in the leaves. This is of importance because harvesting plant leaves is more sustainable compared to harvesting of stems or roots. Solvent extracts from Bidens pilosa, Ocimum gratissimum, Clematis hirsuta, and Solanum nigrum did not exhibit any activity, although antimicrobial activities have previously been reported in Bidens pilosa (Rabe and van Staden, 1997) and Clematis hirsuta, (Cos et al., 2002). Active components in plant can be affected by the locality of the plant, time of collection, storage conditions, study design, and this could explain the contrasting results (Samuelsson and Bohlin, 2010). In conclusion, pulp juice prepared from fresh root of Zanthoxylum chalybeum and Euclea latidens have antibacterial activities against bacteria associated with PD and DC. Antibacterial activities were also demonstrated in solvent extracts from Helichrysum odoratissimum, Zanthoxylum chalybeum, Lantana trifolia, Cymbopogon nardus, thus providing potential for further development. Acknowledgment This work was supported by Swedish International Development Agency (grant no. 75007369). References Cos, P., Hermans, N., De-Bruyne, T., Apers, S., Sindambiwe, J.B., Vanden Berghe, D., Pieters, L., Vlietinck, A.J., 2002. Further evaluation of Rwandan medicinal plant extracts for their antimicrobial and antiviral activities. Journal of Ethnopharmacology 79, 155–163. EUCAST, 2000. Determination of minimum inhibitory concentrations (MICs) of antibacterial agents by agar dilution. Clinical Microbiolgy and Infection 6, 509–515.
855
Hamill, F.A., Apio, S., Mubiru, N.K., Bukenya-Ziraba, R., Mosango, M., Maganyi, O.W., Soejarto, D.D., 2003. Traditional herbal drugs of Southern Uganda, II: literature analysis and antimicrobial assays. Journal of Ethnopharmacology 84, 57–78. Hirt, H.M., M'Pia, B., 2008. Natural Medicine in the Tropics 1: Foundation, third ed. Marianum Press, Kisubi. Hyson Jr., J.M., 2003. History of the toothbrush. Journal of the History of Dentistry 51, 73–80. Kokwaro, J.O., 1993. Medicinal Plants of East Africa, second ed. Kenya Literature Bureau, Nairobi. Marcenes, W., Kassebaum, N.J., Bernabe, E., Flaxman, A., Naghavi, M., Lopez, A., Murray, C.J., 2013. Global burden of oral conditions in 1990-2010: a systematic analysis. Journal of Dental Research 92, 592–597. Matu, E.N., van Staden, J., 2003. Antibacterial and anti-inflammatory activities of some plants used for medicinal purposes in Kenya. Journal of Ethnopharmacology 87, 35–41. More, G., Tshikalange, T.E., Lall, N., Botha, F., Meyer, J.J., 2008. Antimicrobial activity of medicinal plants against oral microorganisms. Journal of Ethnopharmacology 119, 473–477. Mubiru, N.K., Kakoooko, A.B., Mutyaba, J.B., Amai, C.A., Apio, S.K., A.M., A., Ndugga, J. Z.B.S., Ndugga, E.S., 1994. Ethnomedicine in Uganda. National Chemotherapeutic Research Laboratory (NCRL), Ministy of Health, Uganda. Odongo, C.O., Musisi, N.L., Waako, P., Obua, C., 2011. Chewing-stick practices using plants with anti-streptococcal activity in a Ugandan rural community. Frontiers in Pharmacology 2, 1–5. Olila, D., Olwa-Odyek, Opuda-Asibo, J., 2001. Antibacterial and antifungal activities of extracts of Zanthoxylum chalybeum and Warburgia ugandensis, Ugandan medicinal plants. African Health Sciences 1, 66–72. Rabe, T., van Staden, J., 1997. Antibacterial activity of South African plants used for medicinal purposes. Journal of Ethnopharmacology 56, 81–87. Rwangabo, P.C., Claeys, M., Pieters, L., Corthout, J., Vanden Berghe, D.A., Vlietinck, A. J., 1988. Umuhengerin, a new antimicrobially active flavonoid from Lantana trifolia. Journal of Natural Products 51, 966–968. Samuelsson, G., Bohlin, L., 2010. Drugs of Natural Origin: A Treatise of Pharmacognosy, sixth ed. Swedish Pharmaceutical Press, Stockholm. Sofrata, A.H., Claesson, R.L., Lingstrom, P.K., Gustafsson, A.K., 2008. Strong antibacterial effect of miswak against oral microorganisms associated with periodontitis and caries. Journal of Periodontology 79, 1474–1479. Takahashi, N., Nyvad, B., 2011. The role of bacteria in the caries process: ecological perspectives. Journal of Dental Research 90, 294–303. WHO, 1987. Prevention of Oral Diseases. World Health Organization, Geneva p. 83 (offset publication No. 103). Ximénez-Fyvie, L.A., Haffajee, A.D., Socransky, S.S., 2000. Microbial composition of supra- and subgingival plaque in subjects with adult periodontitis. Journal of Clinical Periodontology 27, 722–732.
