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Antibacterial, antioxidant and fibroblast growth stimulation of aqueous extracts of Ficus asperifolia Miq. and Gossypium arboreum L., wound-healing plants of Ghana
Journal of Ethnopharmacology 119 (2008) 141–144
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Antibacterial, antioxidant and fibroblast growth stimulation of aqueous extracts of Ficus asperifolia Miq. and Gossypium arboreum L., wound-healing plants of Ghana Kofi Annan a , Peter J. Houghton b,∗ a b
Faculty of Pharmacy and Pharmaceutical Sciences, College of Health Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana Pharmacognosy Research Laboratories, Pharmaceutical Sciences Research Division, King’s College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, UK
a r t i c l e
i n f o
Article history: Received 7 February 2008 Received in revised form 27 May 2008 Accepted 18 June 2008 Available online 27 June 2008 Keywords: Ficus asperifolia Gossypium arboreum Antibacterial Antioxidant Fibroblast
a b s t r a c t Aim of the study: Use of in vitro tests to search for relevant activities in bark of Ficus asperifolia Miq. and leaves of Gossypium arboreum L., used in Ghana for wound healing. Materials and methods: Aqueous extracts of the relevant parts of the two species were tested for antibacterial activity against Staphylococcus aureus, Bacillus subtilis, Micrococcus flavus, Escherichia coli, Pseudomonas aeruginosa and resistant strains of Staphylococcus aureus SA1199B, RN4220 and XU212. The effects of the plants on fibroblast growth stimulation as well as antioxidant protective effect against hydrogen peroxide induced damage in the same cell line were also studied. Results: The extracts of Gossypium arboreum and Ficus asperifolia had weak antibacterial action against all bacteria tested. The extracts of the two plants had significant (p < 0.001) effects on the growth of human dermal fibroblast at 50 g/ml and lower. They also significantly protected fibroblast cells against oxidative damage at doses up to 50 g/ml. Gossypium arboreum leaf extract displayed the higher activity. Conclusion: The stimulatory effect on fibroblast growth and protection against peroxide-induced oxidative damage give some support to the traditional use of these two plants as wound-healing agents. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction More than 80% of the world’s population depends upon traditional medicines for various skin diseases (Steenkamp et al., 2004). Recently, the traditional use of plants for wound healing has received attention by the scientific community (Mensah et al., 2001; Subramoniam et al., 2001; Houghton et al., 2005). Wound healing is a complex process characterized by homeostasis, reepithelialisation, granulation tissue formation and remodeling of the extracellular matrix (Priya et al., 2002). Fibroblast cells play a very important role in all these processes (Bodeker and Hughes, 1998). Although the healing process progresses naturally, an infection can seriously delay this healing process by prolonging the inflammatory phase, disrupting the normal clotting mechanisms, promoting disordered leukocyte function and ultimately delaying
Abbreviations: CO2 , carbon dioxide; CFU, colony forming units; DPPH, 1,1diphenyl-2-picrylhydrazyl; FBS, foetal bovine serum; H2 O2 , hydrogen peroxide; HBSS, Hank’s balanced salt solution; IC50 , concentration that gives 50% inhibition; MIC, minimum inhibitory concentration; NCTCN, ational Collection of Type Culture; P, statistical significance. ∗ Corresponding author. E-mail address: [email protected] (P.J. Houghton). 0378-8741/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2008.06.017
angiogenesis (Subramoniam et al., 2001). Therefore, a variety of activities such as fibroblast growth stimulation, antioxidant and antimicrobial effects can explain the traditional use of a plant for helping wounds to heal (Houghton et al., 2005). Ficus asperifolia Miq. (Moraceae) and Gossypium arboreum L. (Malvaceae) were selected for testing, since they were frequently cited in the results of a survey conducted amongst 20 healers in the Ashanti region of Ghana for plants used topically for wound healing (Annan, 2007). The bark infusion of Ficus asperifolia is used for washing sores and ulcers and applied to circumcision wounds (Irvine, 1961). In Ghana, the rough leaves are used for scraping patches of ringworm before further treatment (Abbiw, 1990). The crushed leaf and seed kernel of Gossypium arboreum is applied to sores or used as poultice for bruises, wounds and swellings (Ghana Herbal Pharmacopoiea, 1992). In spite of their recorded uses, these two species have not yet been evaluated for their antibacterial and antioxidant activity and fibroblast growth-stimulant activities. Antioxidant activity was determined since it has been found to have positive effect on healing (Thiem and Grosslinka, 2003; Desneves et al., 2005) whereas fibroblasts play a crucial role in wound healing by initiating the proliferative phase of repair (Mensah et al., 2004).
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2. Experimental
Timbrell, 2006) to assess the effect of the extract on the growth of the cells.
2.1. Plant material 2.5. Antioxidant activity Plant materials were collected in the month of December 2003 and authenticated by Mr. Ofori Lartey, a senior research officer at the Centre for Scientific Research into Plant Medicine (CSRPM), Akwapim-Mampong, Ghana, where voucher specimen numbers 12/03/018 and 12/03/017 for Ficus asperifolia Miq. and Gossypium arboreum L., respectively, have been deposited. 2.2. Preparation of extracts 30 g of each powdered plant material was packed into a cellulose thimble (28 mm × 100 mm) and was soxhlet-extracted with 300 ml of distilled water over 48 h until the material was exhausted. The extraction was considered complete when the initial green colour of the percolate gradually changed to colourless. Each extract was allowed to cool and freeze-dried for 48 h using the LTE Scientific Freeze Drier to give a yield of 0.36% and 0.43% (w/w) for Ficus asperifolia and Gossypium arboreum, respectively. 2.3. Antibacterial assay The bacteria used for the tests were obtained from the National Culture Type Collection (NCTC), UK and included both Grampositive and Gram-negative bacteria. The Gram-positive bacteria used were Bacillus subtilis (NCTC 10073), Staphylococcus aureus (NCTC 4163), Streptococcus faecalis (NCTC 775), Micrococcus flavus (NCTC 7743), as well as resistant strains of Staphylococcus aureus SA1199B, RN4220 and XU212. Gram-negative bacteria used were Escherichia coli (NCTC 9002) and Pseudomonas aeruginosa (NCTC 10662). Inocula of the microorganisms were prepared from the 24 h Mueller–Hinton broth (Sigma) cultures and suspensions were adjusted to 105 CFU/ml. Aqueous extracts were reconstituted in distilled water. Minimal inhibition concentration (MIC) values of the extracts were determined based on a micro-well dilution method (Eloff, 1998). The 96-well sterile plates were prepared by dispensing 180 l of the inoculated broth plus a 20 l aliquot of the plant extract made up in broth or 20 l broth in the case of negative control in each well. Tetracycline (Sigma) was included as positive control. Plates were covered and incubated for 24 h at 37 ◦ C. Bacterial growth was determined after addition of 50 l p-iodonitrotetrazolium violet (0.2 mg/ml, Sigma). 2.4. In vitro test for fibroblast growth stimulation Confluent fibroblasts (142BR, Sigma) were trypsinised, centrifuged and resuspended in MEM/15% FBS/1% l-glutamine. The cells were counted using a haemocytometer and the suspension standardized at a concentration of 1 × 104 cells/ml in MEM/15% FBS/1% l-glutamine. Using a multi-channel pipette, the cells were seeded at a density of 1 × 103 cells/well in 96-well plate excluding the first row. The plates were maintained at 37 ◦ C in a humidified incubator of 5% CO2 :95% air atmosphere. The medium was replaced after 24 h with MEM containing 0.5% FBS and a range of concentrations of the extracts (1–50 g/ml), except for two columns which were maintained at MEM/0.5% FBS and MEM/15% FBS to serve as serve as starting and positive controls, respectively. The 0.5% FBS concentration is a maintenance dose needed for the production of healthy cells but does not significantly stimulate proliferation of cells. The cells were incubated and assayed after 5 days using the Neutral Red assay method (Weyermann et al., 2005; Fotakis and
2.5.1. DPPH radical scavenging activity The 2,2 -diphenylpicrylhydrazyl (DPPH) scavenging activity of the extracts was measured from the bleaching of a purple-coloured methanol solution of DPPH which was used as a reagent in a spectrophotometric assay (Yoshida et al., 1989; Gyamfi et al., 1999). 50 l of various concentrations of the extracts and compounds in methanol were added to 5 ml of a 0.004% methanol solution of DPPH. This was incubated at room temperature for 30 min after which absorbance was read against a blank at 517 nm on a Thermo Spectronic UV spectrophotometer. l-Ascorbic acid was used as positive control in these experiments. Inhibition of free radical DPPH, in percentage was calculated as
scavenging activity (%) =
A0 − A1 × 100 A0
where A0 is absorbance of blank at 517 nm. The IC50 value was obtained through extrapolation from linear analysis, using the Prism Software, and denoted the concentration of sample required to scavenge 50% of DPPH radicals. 2.5.2. Antioxidant activity of plant extracts on human skin fibroblasts The method used for the hydrogen peroxide assay was the one described by Yamasaki et al. (1994) and modified to evaluate the protective effect of the extracts on the cells against oxidant injury induced by hydrogen peroxide. Fibroblast cells were seeded at 5000 cells/well in a 96-well plate and incubated for 5 days until almost confluent. The growth medium was then discarded and the confluent cells subjected to three different types of experiment. In the first experiment, the cells were pre-treated with different concentrations of the extracts overnight after which they were exposed to 10−4 M hydrogen peroxide in the standard growth medium and incubated additionally for 3 h. In the second protocol, fibroblast cells were pre-incubated with the extracts of different concentrations overnight, before exposure to the same concentrations of the extracts together with 10−4 M hydrogen peroxide in the growth medium. In the third experiment, different concentrations of extracts were applied simultaneously with 10−4 M hydrogen peroxide in the growth medium and incubated for 3 h at 37 ◦ C. Catalase (250 units/ml), an antioxidant enzyme was used as positive control in all experiments. After the incubation period, the fibroblast cells were stained with Neutral Red and observed microscopically for cell damage, followed by the Neutral Red assay to quantify the degree of protection of fibroblast cells by extracts against hydrogen peroxide damage. 2.6. Statistical analysis One-way ANOVA was used for the comparison of the means. Results are expressed as mean ± S.D. (standard deviation) data, using the Prism Software. 3. Results and discussions The aqueous extracts of Ficus asperifolia and Gossypium arboreum presented MICs > 512 g/ml against all the selected bacteria, including the resistant strains of Staphylococcus aureus SA1199B, RN4220 and XU212. Micrococcus flavus was the most susceptible
K. Annan, P.J. Houghton / Journal of Ethnopharmacology 119 (2008) 141–144
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Table 1 Antibacterial activity of aqueous extracts of Ficus asperifolia and Gossypium arboreum expressed as minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) in g/ml Bacteria
Staphylococcus aureus Bacillus subtilis Micrococcus flavus Escherichia coli Pseudomonas aeruginosa SA1199B (Nor A) XU212 (Tet K) RN4220 (MsrA)
MIC (g/ml)
MBC (g/ml)
FA
GA
Tet
FA
GA
128 256 32 128 512 125 256 256
128 256 128 128 256 128 128 256
4 2 1 8 8 8 128 8
250 250 250 512 512 >1000 >1000 >1000
256 256 256 256 512 >1000 >1000 >1000
NI, no inhibition; FA, Ficus asperifolia stem bark; GA, Gossypium arboreum leaves; Tet, tetracycline (positive control); n = 3.
with MIC of 32 g/ml (Table 1). Tetracycline, a standard antibiotic that was used as a positive control, presented very low MICs of 1–8 g/ml except against Staphylococcus aureus XU212 (MIC 128 g/ml) which is resistant to the tetracyclines and overexpresses the Tet K efflux proteins. Therefore, neither of the extracts possessed appreciable antibacterial activity and it is unlikely that this activity contributes to any wound-healing properties. Both plant extracts had a significant dose-dependent effect (p < 0.01) on the growth of human dermal fibroblast (142BR) at a concentration of 50 g/ml (54% and 38% increase in growth for Gossypium arboreum and Ficus asperifolia, respectively), after which the extracts were toxic to the cells (Fig. 1). MEM/15% FBS alone, which was used as a positive control, recorded a growth of 88% compared to the control. The evidence of cytotoxicity of the extracts at doses above 50 g/ml implies that caution must be taken when using infusion of these plant species in treating wounds, since proliferation of new cells may be affected at high concentrations. The quantitative DPPH test on the two plant extracts revealed all of them having various degrees of antioxidant properties, with IC50 of 35.7 g/ml and 237 g/ml for Gossypium arboreum and Ficus asperifolia, respectively. l-Ascorbic acid, which was used as a positive control, recorded an IC50 value of 21.1 g/ml. This compared favorably with the literature value of 21.04 g/ml for l-ascorbic acid (Bizimenyera et al., 2007). Different protocols were used to assess the effects of the extracts on hydrogen peroxide induced damage on the fibroblast cells. In the first and second protocols, where the cells were pre-incubated with the extracts overnight before the application of the hydrogen peroxide, it was found by the Neutral Red assay that the cells
Fig. 1. Effect of aqueous plant extracts on 142BR cell proliferation (p < 0.01). FA, Ficus asperifolia; GA, Gossypium arboreum.
Fig. 2. Protection of fibroblast cells against hydrogen peroxide induced damage by simultaneous application of extracts and hydrogen peroxide (10−4 M; p < 0.01). GA, Gossypium arboreum; FA, Ficus asperifolia; control, catalase (250 units/ml).
were damaged and so were not protected. The cell damage however could be due to the inherent cytotoxicity of the extract rather than the effect of the hydrogen peroxide, due to the relatively high concentrations of the extracts used. In the third protocol, different concentrations of extracts were applied simultaneously with 10−4 M hydrogen peroxide in the growth medium and incubated for 3 h at 37 ◦ C. In this case, it was observed that fibroblast cells were protected against hydrogen peroxide damage by various degrees by the extracts. Gossypium arboreum offered the highest protection against hydrogen peroxide induced damage to cells with its activity (82% at 50 g/ml) almost comparable with that of catalase (control at 250 units/ml) (Fig. 2). Ficus asperifolia also showed 58% protection against oxidative damage to the fibroblast cells. The protective effect could therefore be due to the direct interaction of the extracts and the hydrogen peroxide rather than the extracts altering the cell membranes and limiting the damage induced by the hydrogen peroxide. The reported presence of biologically active flavonoids in Gossypium arboreum (Waage and Hedin, 1984) could be responsible for its observed effects on wound healing (Stipcevic et al., 2006). Acknowledgement We are grateful to Ghana Educational Trust Fund (GETFUND) for the financial support. References Abbiw, D., 1990. Useful Plants of Ghana. Intermediate Technology Publication Ltd., UK, pp. 182–205. Annan, K., 2007. Antibacterial and Wound Healing Properties of Some Indigenous Ghanaian Plants. Ph.D. Thesis. University of London, UK, pp. 30–32. Bizimenyera, E.S., Aderogba, M.A., Eloff, J.N., Swan, G.E., 2007. Potential of neuroprotective antioxidant-based therapeutics from Peltophorum africanum Sond. (Fabaceae). African Journal of Traditional, Complimentary and Alternative Medicines 4, 99–105. Bodeker, G., Hughes, M.A., 1998. Wound healing, traditional treatments and research policy. In: Prendergast, H.D.V., Etkin, N.L., Harris, D.R., Houghton, P.J. (Eds.), Plants for Food and Medicine. Royal Botanic Gardens, Kew, London. Desneves, K.J., Todorovic, B.E., Cassar, A., Crowe, T.C., 2005. Treatment with supplementary arginine, vitamin C and zinc in patients with pressure ulcers: a randomized controlled trial. Clinical Nutrition 24, 979–987. Eloff, J.N., 1998. A sensitive and quick microplate method to determine the minimal inhibitory concentration of plant extracts for bacteria. Planta Medica 64, 711–713. Fotakis, G., Timbrell, J.A., 2006. In vitro cytotoxicity assays: comparison of LDH, Neutral Red, MTT and protein assay in hepatoma cell lines following exposure to cadmium chloride. Toxicology Letters 160, 171–177. Ghana Herbal Pharmacopoiea, 1992. The Advent Press, Accra, pp. 24–36. Gyamfi, M.A., Yonamine, M., Aniya, Y., 1999. Free radical scavenging action of medicinal herbs from Ghana, Thonningia sanguinea on experimentally-induced liver injuries. General Pharmacology 32, 661–667.
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Houghton, P.J., Hylands, P.J., Mensah, A.Y., Hensel, A., Deters, A.M., 2005. In vitro tests and ethnopharmacological investigations: wound healing as an example. Journal of Ethnopharmacolgy 100, 100–107. Irvine, F.R., 1961. Woody plants of Ghana. Oxford University Press, London, UK, pp. 194, 208, 548. Mensah, A.Y., Sampson, J., Houghton, P.J., Hylands, P.J., Westbrook, J., Dunn, M., Hughes, M.A., Cherry, G.W., 2001. Effects of Buddleja globosa leaf and its constituents relevant to wound healing. Journal of Ethnopharmacology 77, 219–226. Mensah, A.Y., Houghton, P.J., Fleischer, T.C., Mensah, M.L.K., Agyare, C., Annan, K., Dickson, R., 2004. Antimicrobial and antioxidant aspects of Commelina diffusa, a Ghanaian plant used traditionally for wound healing. Journal of Pharmacy and Pharmacology 56, S-79. Priya, K.S., Gnanamani, A., Radhakrishnan, N., Babu, M., 2002. Healing potential of Datura alba on burn wounds in albino rats. Journal of Ethnopharmacology 83, 193–199. Steenkamp, V., Mathivha, E., Gouws, M.C., van Rensburg, C.E.J., 2004. Studies on antibacterial, antioxidant and fibroblast growth stimulation of wound healing remedies from South Africa. Journal of Ethnopharmacology 95, 353–357.
Stipcevic, T., Piljac, J., Berghe, D.V., 2006. Effect of different flavonoids on collagen synthesis in human fibroblasts. Plant Foods for Human Nutrition 61, 29–34. Subramoniam, A., Evans, D.A., Rajasekharan, S., Nair, G.S., 2001. Effect of Hemigraphis colorata (Blume) H.G. Hallier leaf on wound healing and inflammation in mice. Indian Journal of Pharmacology 33, 283–285. Thiem, B., Grosslinka, O., 2003. Antimicrobial activity of Rubus chamaemorus leaves. Fitoterapia 75, 93–95. Waage, S.K., Hedin, P.A., 1984. Biologically-active flavonoids from Gossypium arboreum. Phytochemistry 23, 2509–2511. Weyermann, J., Lockmann, D., Zimmer, A., 2005. A practical note on the use of cytotoxicity assays. International Journal of Pharmaceutics 288, 369–376. Yamasaki, T., Li, L., Lau, B.H.S., 1994. Garlic compounds protect vascular endothelial cells from hydrogen peroxide-induced oxidant injury. Phytotherapy Research 8, 408–412. Yoshida, T., Mori, K., Hatano, T., Okumura, T., Uehara, I., Komgoe, K., Fujita, Y., Okuda, T., 1989. Studies on inhibition mechanism of autooxidation by tannins and flavonoids. Radical scavenging effects of tannins and related polyphenols on 1,1-diphenyl-2-picrylhydrazyl radical. Chemical and Pharmaceutical Bulletin 37, 1919–1921.
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Antibacterial, antioxidant and fibroblast growth stimulation of aqueous extracts of Ficus asperifolia Miq. and Gossypium arboreum L., wound-healing plants of Ghana Kofi Annan a , Peter J. Houghton b,∗ a b
Faculty of Pharmacy and Pharmaceutical Sciences, College of Health Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana Pharmacognosy Research Laboratories, Pharmaceutical Sciences Research Division, King’s College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, UK
a r t i c l e
i n f o
Article history: Received 7 February 2008 Received in revised form 27 May 2008 Accepted 18 June 2008 Available online 27 June 2008 Keywords: Ficus asperifolia Gossypium arboreum Antibacterial Antioxidant Fibroblast
a b s t r a c t Aim of the study: Use of in vitro tests to search for relevant activities in bark of Ficus asperifolia Miq. and leaves of Gossypium arboreum L., used in Ghana for wound healing. Materials and methods: Aqueous extracts of the relevant parts of the two species were tested for antibacterial activity against Staphylococcus aureus, Bacillus subtilis, Micrococcus flavus, Escherichia coli, Pseudomonas aeruginosa and resistant strains of Staphylococcus aureus SA1199B, RN4220 and XU212. The effects of the plants on fibroblast growth stimulation as well as antioxidant protective effect against hydrogen peroxide induced damage in the same cell line were also studied. Results: The extracts of Gossypium arboreum and Ficus asperifolia had weak antibacterial action against all bacteria tested. The extracts of the two plants had significant (p < 0.001) effects on the growth of human dermal fibroblast at 50 g/ml and lower. They also significantly protected fibroblast cells against oxidative damage at doses up to 50 g/ml. Gossypium arboreum leaf extract displayed the higher activity. Conclusion: The stimulatory effect on fibroblast growth and protection against peroxide-induced oxidative damage give some support to the traditional use of these two plants as wound-healing agents. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction More than 80% of the world’s population depends upon traditional medicines for various skin diseases (Steenkamp et al., 2004). Recently, the traditional use of plants for wound healing has received attention by the scientific community (Mensah et al., 2001; Subramoniam et al., 2001; Houghton et al., 2005). Wound healing is a complex process characterized by homeostasis, reepithelialisation, granulation tissue formation and remodeling of the extracellular matrix (Priya et al., 2002). Fibroblast cells play a very important role in all these processes (Bodeker and Hughes, 1998). Although the healing process progresses naturally, an infection can seriously delay this healing process by prolonging the inflammatory phase, disrupting the normal clotting mechanisms, promoting disordered leukocyte function and ultimately delaying
Abbreviations: CO2 , carbon dioxide; CFU, colony forming units; DPPH, 1,1diphenyl-2-picrylhydrazyl; FBS, foetal bovine serum; H2 O2 , hydrogen peroxide; HBSS, Hank’s balanced salt solution; IC50 , concentration that gives 50% inhibition; MIC, minimum inhibitory concentration; NCTCN, ational Collection of Type Culture; P, statistical significance. ∗ Corresponding author. E-mail address: [email protected] (P.J. Houghton). 0378-8741/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2008.06.017
angiogenesis (Subramoniam et al., 2001). Therefore, a variety of activities such as fibroblast growth stimulation, antioxidant and antimicrobial effects can explain the traditional use of a plant for helping wounds to heal (Houghton et al., 2005). Ficus asperifolia Miq. (Moraceae) and Gossypium arboreum L. (Malvaceae) were selected for testing, since they were frequently cited in the results of a survey conducted amongst 20 healers in the Ashanti region of Ghana for plants used topically for wound healing (Annan, 2007). The bark infusion of Ficus asperifolia is used for washing sores and ulcers and applied to circumcision wounds (Irvine, 1961). In Ghana, the rough leaves are used for scraping patches of ringworm before further treatment (Abbiw, 1990). The crushed leaf and seed kernel of Gossypium arboreum is applied to sores or used as poultice for bruises, wounds and swellings (Ghana Herbal Pharmacopoiea, 1992). In spite of their recorded uses, these two species have not yet been evaluated for their antibacterial and antioxidant activity and fibroblast growth-stimulant activities. Antioxidant activity was determined since it has been found to have positive effect on healing (Thiem and Grosslinka, 2003; Desneves et al., 2005) whereas fibroblasts play a crucial role in wound healing by initiating the proliferative phase of repair (Mensah et al., 2004).
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2. Experimental
Timbrell, 2006) to assess the effect of the extract on the growth of the cells.
2.1. Plant material 2.5. Antioxidant activity Plant materials were collected in the month of December 2003 and authenticated by Mr. Ofori Lartey, a senior research officer at the Centre for Scientific Research into Plant Medicine (CSRPM), Akwapim-Mampong, Ghana, where voucher specimen numbers 12/03/018 and 12/03/017 for Ficus asperifolia Miq. and Gossypium arboreum L., respectively, have been deposited. 2.2. Preparation of extracts 30 g of each powdered plant material was packed into a cellulose thimble (28 mm × 100 mm) and was soxhlet-extracted with 300 ml of distilled water over 48 h until the material was exhausted. The extraction was considered complete when the initial green colour of the percolate gradually changed to colourless. Each extract was allowed to cool and freeze-dried for 48 h using the LTE Scientific Freeze Drier to give a yield of 0.36% and 0.43% (w/w) for Ficus asperifolia and Gossypium arboreum, respectively. 2.3. Antibacterial assay The bacteria used for the tests were obtained from the National Culture Type Collection (NCTC), UK and included both Grampositive and Gram-negative bacteria. The Gram-positive bacteria used were Bacillus subtilis (NCTC 10073), Staphylococcus aureus (NCTC 4163), Streptococcus faecalis (NCTC 775), Micrococcus flavus (NCTC 7743), as well as resistant strains of Staphylococcus aureus SA1199B, RN4220 and XU212. Gram-negative bacteria used were Escherichia coli (NCTC 9002) and Pseudomonas aeruginosa (NCTC 10662). Inocula of the microorganisms were prepared from the 24 h Mueller–Hinton broth (Sigma) cultures and suspensions were adjusted to 105 CFU/ml. Aqueous extracts were reconstituted in distilled water. Minimal inhibition concentration (MIC) values of the extracts were determined based on a micro-well dilution method (Eloff, 1998). The 96-well sterile plates were prepared by dispensing 180 l of the inoculated broth plus a 20 l aliquot of the plant extract made up in broth or 20 l broth in the case of negative control in each well. Tetracycline (Sigma) was included as positive control. Plates were covered and incubated for 24 h at 37 ◦ C. Bacterial growth was determined after addition of 50 l p-iodonitrotetrazolium violet (0.2 mg/ml, Sigma). 2.4. In vitro test for fibroblast growth stimulation Confluent fibroblasts (142BR, Sigma) were trypsinised, centrifuged and resuspended in MEM/15% FBS/1% l-glutamine. The cells were counted using a haemocytometer and the suspension standardized at a concentration of 1 × 104 cells/ml in MEM/15% FBS/1% l-glutamine. Using a multi-channel pipette, the cells were seeded at a density of 1 × 103 cells/well in 96-well plate excluding the first row. The plates were maintained at 37 ◦ C in a humidified incubator of 5% CO2 :95% air atmosphere. The medium was replaced after 24 h with MEM containing 0.5% FBS and a range of concentrations of the extracts (1–50 g/ml), except for two columns which were maintained at MEM/0.5% FBS and MEM/15% FBS to serve as serve as starting and positive controls, respectively. The 0.5% FBS concentration is a maintenance dose needed for the production of healthy cells but does not significantly stimulate proliferation of cells. The cells were incubated and assayed after 5 days using the Neutral Red assay method (Weyermann et al., 2005; Fotakis and
2.5.1. DPPH radical scavenging activity The 2,2 -diphenylpicrylhydrazyl (DPPH) scavenging activity of the extracts was measured from the bleaching of a purple-coloured methanol solution of DPPH which was used as a reagent in a spectrophotometric assay (Yoshida et al., 1989; Gyamfi et al., 1999). 50 l of various concentrations of the extracts and compounds in methanol were added to 5 ml of a 0.004% methanol solution of DPPH. This was incubated at room temperature for 30 min after which absorbance was read against a blank at 517 nm on a Thermo Spectronic UV spectrophotometer. l-Ascorbic acid was used as positive control in these experiments. Inhibition of free radical DPPH, in percentage was calculated as
scavenging activity (%) =
A0 − A1 × 100 A0
where A0 is absorbance of blank at 517 nm. The IC50 value was obtained through extrapolation from linear analysis, using the Prism Software, and denoted the concentration of sample required to scavenge 50% of DPPH radicals. 2.5.2. Antioxidant activity of plant extracts on human skin fibroblasts The method used for the hydrogen peroxide assay was the one described by Yamasaki et al. (1994) and modified to evaluate the protective effect of the extracts on the cells against oxidant injury induced by hydrogen peroxide. Fibroblast cells were seeded at 5000 cells/well in a 96-well plate and incubated for 5 days until almost confluent. The growth medium was then discarded and the confluent cells subjected to three different types of experiment. In the first experiment, the cells were pre-treated with different concentrations of the extracts overnight after which they were exposed to 10−4 M hydrogen peroxide in the standard growth medium and incubated additionally for 3 h. In the second protocol, fibroblast cells were pre-incubated with the extracts of different concentrations overnight, before exposure to the same concentrations of the extracts together with 10−4 M hydrogen peroxide in the growth medium. In the third experiment, different concentrations of extracts were applied simultaneously with 10−4 M hydrogen peroxide in the growth medium and incubated for 3 h at 37 ◦ C. Catalase (250 units/ml), an antioxidant enzyme was used as positive control in all experiments. After the incubation period, the fibroblast cells were stained with Neutral Red and observed microscopically for cell damage, followed by the Neutral Red assay to quantify the degree of protection of fibroblast cells by extracts against hydrogen peroxide damage. 2.6. Statistical analysis One-way ANOVA was used for the comparison of the means. Results are expressed as mean ± S.D. (standard deviation) data, using the Prism Software. 3. Results and discussions The aqueous extracts of Ficus asperifolia and Gossypium arboreum presented MICs > 512 g/ml against all the selected bacteria, including the resistant strains of Staphylococcus aureus SA1199B, RN4220 and XU212. Micrococcus flavus was the most susceptible
K. Annan, P.J. Houghton / Journal of Ethnopharmacology 119 (2008) 141–144
143
Table 1 Antibacterial activity of aqueous extracts of Ficus asperifolia and Gossypium arboreum expressed as minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) in g/ml Bacteria
Staphylococcus aureus Bacillus subtilis Micrococcus flavus Escherichia coli Pseudomonas aeruginosa SA1199B (Nor A) XU212 (Tet K) RN4220 (MsrA)
MIC (g/ml)
MBC (g/ml)
FA
GA
Tet
FA
GA
128 256 32 128 512 125 256 256
128 256 128 128 256 128 128 256
4 2 1 8 8 8 128 8
250 250 250 512 512 >1000 >1000 >1000
256 256 256 256 512 >1000 >1000 >1000
NI, no inhibition; FA, Ficus asperifolia stem bark; GA, Gossypium arboreum leaves; Tet, tetracycline (positive control); n = 3.
with MIC of 32 g/ml (Table 1). Tetracycline, a standard antibiotic that was used as a positive control, presented very low MICs of 1–8 g/ml except against Staphylococcus aureus XU212 (MIC 128 g/ml) which is resistant to the tetracyclines and overexpresses the Tet K efflux proteins. Therefore, neither of the extracts possessed appreciable antibacterial activity and it is unlikely that this activity contributes to any wound-healing properties. Both plant extracts had a significant dose-dependent effect (p < 0.01) on the growth of human dermal fibroblast (142BR) at a concentration of 50 g/ml (54% and 38% increase in growth for Gossypium arboreum and Ficus asperifolia, respectively), after which the extracts were toxic to the cells (Fig. 1). MEM/15% FBS alone, which was used as a positive control, recorded a growth of 88% compared to the control. The evidence of cytotoxicity of the extracts at doses above 50 g/ml implies that caution must be taken when using infusion of these plant species in treating wounds, since proliferation of new cells may be affected at high concentrations. The quantitative DPPH test on the two plant extracts revealed all of them having various degrees of antioxidant properties, with IC50 of 35.7 g/ml and 237 g/ml for Gossypium arboreum and Ficus asperifolia, respectively. l-Ascorbic acid, which was used as a positive control, recorded an IC50 value of 21.1 g/ml. This compared favorably with the literature value of 21.04 g/ml for l-ascorbic acid (Bizimenyera et al., 2007). Different protocols were used to assess the effects of the extracts on hydrogen peroxide induced damage on the fibroblast cells. In the first and second protocols, where the cells were pre-incubated with the extracts overnight before the application of the hydrogen peroxide, it was found by the Neutral Red assay that the cells
Fig. 1. Effect of aqueous plant extracts on 142BR cell proliferation (p < 0.01). FA, Ficus asperifolia; GA, Gossypium arboreum.
Fig. 2. Protection of fibroblast cells against hydrogen peroxide induced damage by simultaneous application of extracts and hydrogen peroxide (10−4 M; p < 0.01). GA, Gossypium arboreum; FA, Ficus asperifolia; control, catalase (250 units/ml).
were damaged and so were not protected. The cell damage however could be due to the inherent cytotoxicity of the extract rather than the effect of the hydrogen peroxide, due to the relatively high concentrations of the extracts used. In the third protocol, different concentrations of extracts were applied simultaneously with 10−4 M hydrogen peroxide in the growth medium and incubated for 3 h at 37 ◦ C. In this case, it was observed that fibroblast cells were protected against hydrogen peroxide damage by various degrees by the extracts. Gossypium arboreum offered the highest protection against hydrogen peroxide induced damage to cells with its activity (82% at 50 g/ml) almost comparable with that of catalase (control at 250 units/ml) (Fig. 2). Ficus asperifolia also showed 58% protection against oxidative damage to the fibroblast cells. The protective effect could therefore be due to the direct interaction of the extracts and the hydrogen peroxide rather than the extracts altering the cell membranes and limiting the damage induced by the hydrogen peroxide. The reported presence of biologically active flavonoids in Gossypium arboreum (Waage and Hedin, 1984) could be responsible for its observed effects on wound healing (Stipcevic et al., 2006). Acknowledgement We are grateful to Ghana Educational Trust Fund (GETFUND) for the financial support. References Abbiw, D., 1990. Useful Plants of Ghana. Intermediate Technology Publication Ltd., UK, pp. 182–205. Annan, K., 2007. Antibacterial and Wound Healing Properties of Some Indigenous Ghanaian Plants. Ph.D. Thesis. University of London, UK, pp. 30–32. Bizimenyera, E.S., Aderogba, M.A., Eloff, J.N., Swan, G.E., 2007. Potential of neuroprotective antioxidant-based therapeutics from Peltophorum africanum Sond. (Fabaceae). African Journal of Traditional, Complimentary and Alternative Medicines 4, 99–105. Bodeker, G., Hughes, M.A., 1998. Wound healing, traditional treatments and research policy. In: Prendergast, H.D.V., Etkin, N.L., Harris, D.R., Houghton, P.J. (Eds.), Plants for Food and Medicine. Royal Botanic Gardens, Kew, London. Desneves, K.J., Todorovic, B.E., Cassar, A., Crowe, T.C., 2005. Treatment with supplementary arginine, vitamin C and zinc in patients with pressure ulcers: a randomized controlled trial. Clinical Nutrition 24, 979–987. Eloff, J.N., 1998. A sensitive and quick microplate method to determine the minimal inhibitory concentration of plant extracts for bacteria. Planta Medica 64, 711–713. Fotakis, G., Timbrell, J.A., 2006. In vitro cytotoxicity assays: comparison of LDH, Neutral Red, MTT and protein assay in hepatoma cell lines following exposure to cadmium chloride. Toxicology Letters 160, 171–177. Ghana Herbal Pharmacopoiea, 1992. The Advent Press, Accra, pp. 24–36. Gyamfi, M.A., Yonamine, M., Aniya, Y., 1999. Free radical scavenging action of medicinal herbs from Ghana, Thonningia sanguinea on experimentally-induced liver injuries. General Pharmacology 32, 661–667.
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