Journal of Ethnopharmacology 153 (2014) 386–391
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Antimycobacterial, anti-inflammatory and genotoxicity evaluation of plants used for the treatment of tuberculosis and related symptoms in South Africa B. Madikizela, A.R. Ndhlala, J.F. Finnie, J. Van Staden n Research Centre for Plant Growth and Development, School of Life Sciences, University of KwaZulu-Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa
art ic l e i nf o
a b s t r a c t
Article history: Received 7 November 2013 Received in revised form 17 January 2014 Accepted 16 February 2014 Available online 25 February 2014
Ethnopharmacological relevance: Emergence of drug-resistant tuberculosis strains and long duration of treatment has established an urgent need to search for new effective agents. The great floral diversity of South Africa has potential for producing new bioactive compounds, therefore pharmacological screening of plant extracts within this region offers much potential. To assess the in vitro antimycobacterial, antiinflammatory and genotoxicity activity of selected plants that are used for the treatment of TB and related symptoms in South Africa. Materials and methods: Ground plant materials from 10 plants were extracted sequentially with four solvents (petroleum ether, dichloromethane, 80% ethanol and water) and a total of 68 extracts were produced. A broth microdilution method was used to screen extracts against Mycobacterium tuberculosis H37Ra. The cyclooxygenase-2 (COX-2) enzyme was used to evaluate the anti-inflammatory activity of the extracts and the Salmonella microsome assay using two Salmonella typhimurium strains (TA98 and TA100) to establish genotoxicity. Results: Six out of 68 extracts showed good antimycobacterial activity. Three extracts showed good inhibition (470%) of COX-2 enzyme. All the extracts tested were non-genotoxic against the tested Salmonella strains. Conclusion: The results observed in this study indicate that some of the plants such as Abrus precatorius subsp. africanus, Ficus sur, Pentanisia prunelloides and Terminalia phanerophlebia could be investigated further against drug-resistant TB strains. & 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Ames test: anti-inflammatory Mycobacterium tuberculosis Terminalia phanerophlebia Pentanisia prunelloides
1. Introduction Tuberculosis (TB) is one of the oldest and most prevalent diseases, having infected about 8.7 million people worldwide in 2011 (Smith, 2003; Okunade et al., 2004; WHO, 2013). In the mid-20th century, the mortality rate of TB began to decrease and the decline is still evident in Europe and America with the incident rate continuing to be low (Daniel, 2006; WHO, 2013). In Africa, approximately half a million people died of TB in 2004. Africa remains the only continent
Abbreviations: 4-NQO, 4-nitroquinoline 1-oxide; BCG, Bacillus Calmette Guérin; CFU, colony-forming units; CO2, carbon dioxide; COX, Cyclooxygenase; DCM, dichloromethane; DMSO, dimethylsulfoxide; DPM, disintegrations per minute; EtOH, ethanol; HCL, hydrochloric acid; HIV, human immunodeficiency virus; INH, isoniazid; MDR, multi-drug resistant; MIC, minimum inhibition concentration; No., number; NRF, National Research Foundation; OADC, oleic acid-albumin-dextrosecatalase; PE, petroleum ether; REMA, resazurin microplate assay; TB, tuberculosis n Corresponding author. Tel.: þ 27 332605130; fax: þ 27 332605897. E-mail address: [email protected] (J. Van Staden). http://dx.doi.org/10.1016/j.jep.2014.02.034 0378-8741 & 2014 Elsevier Ireland Ltd. All rights reserved.
where TB rates are still increasing (Kamatou et al., 2007). TB symptoms are aggravated by the co-infection with Human Immunodeficiency Virus (HIV). Studies done have placed Mycobacterium tuberculosis as the most common human cause of TB worldwide (WHO, 2013), responsible for more human fatalities than any single bacterial species and infecting at least one-third of the world's population (Kamatou et al., 2007; Bansal et al., 2009; Knechel, 2009). Streptomyces-based antibiotics and other chemotherapeutic antimycobacterial agents often used in combination in the treatment of TB are no longer effective due to drug resistance exhibited by the organism (Lai et al., 2011). The treatments of TB take a long time resulting in poor compliance of patients which contributes to sustaining multi-drug resistant TB (MDR-TB) (Connolly et al., 2007). Neonates immunization with Bacillus Calmette Guérin (BCG) protects against mainly TB meningitis and miliary TB. However, the efficacy wanes 10–15 years post-vaccination, thus adults are not protected against pulmonary tuberculosis. Therefore, new vaccines and antitubercular agents with new modes of activity, shorter treatment duration as well as low toxicity are needed to reduce MDR-TB
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prevalence and stop the epidemic of TB. One of the TB symptoms is chest pain caused by inflammation of the membranes lining the lungs which leads to development of lung fibrosis. In the case of pulmonary TB, alveolar macrophages control persistant inflammatory responses in the lungs by producing chemical mediators that leads to granuloma formation (Shinohara et al., 2009). In response to infection of the host by Mycobacteria, granuloma formation represents protective immunity and inflammatory tissue destruction and repair (Shinohara et al., 2009). South Africa is endowed with a diverse flora which has potential for the discovery of metabolites that are active against Mycobacterium tuberculosis (Lall and Meyer, 1999; McGaw et al., 2008). Screening of plant extracts for antimycobacterial activity within South African medicinal plants with their great diversity offers much potential in the search for active new metabolites that may have activity against Mycobacterium tuberculosis and other opportunistic infections. Although medicinal plants have been used in therapy for many years, that does not mean that they are safe, as they may have side effects. Since medicinal plant use and prescription in South Africa is not standardized, the danger of misadministration especially if the plants are toxic is real (Fennell et al., 2004). It is important to determine if the antimicrobial activity exhibited by some plant extracts is not due to toxicity. In our previous study, we reported several plant extracts that exhibited good antibacterial activities against non-pathogenic Mycobacterium species and other strains associated with respiratory infections (Madikizela et al., 2013). As a result of research done in our laboratory on antimicrobial activity of plants used for treating TB and that most human fatalities caused by TB are caused by Mycobacterium tuberculosis, further investigations were carried out. The investigations included determining the antibacterial activity of selected plants used for treating TB and related symptoms against an Mycobacterium tuberculosis strain. The antiinflammatory (since inflammation is one of TB symptoms) activities of selected medicinal plants used in the previous study, as well as genotoxicity properties of the extracts that showed good antimicrobial activities in that previous study were carried out in the current study.
2. Materials and methods 2.1. Sample collection Plant materials were collected from the University of KwaZuluNatal botanical garden and Ukulinga research farm in Pietermaritzburg, South Africa. These plants have already been evaluated for their antimicrobial activity against strains of bacteria related to respiratory ailments in a previous study (Madikizela et al., 2013). The list of the plants used with family, species name, voucher specimen number, traditional uses and previously tested activities are outlined in Madikizela et al. (2013).
2.2. Plant extracts preparation Plant materials were oven-dried at 50 1C, ground into powder and 10 g was extracted sequentially with 200 ml of petroleum ether (PE), dichloromethane (DCM), 80% ethanol (EtOH) and water. The extracts were then filtered through Whatman No. 1 filter paper and concentrated under vacuum using a rotary evaporator for solvent extracts whereas the aqueous extracts were freezedried. The concentrated extracts were then dried under a stream of cold air and kept at 8 1C until required.
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2.3. Antimycobacterial activity using resazurin microplate assay The resazurin microplate assay (REMA) according to the method of Jadaun et al. (2007) was used as a antimycobacterial assay. Mycobacterium tuberculosis H37Ra (American type culture collection 25177) stored at 70 1C was thawed at room temperature for 10–15 min and subcultured in Middlebrook 7H9 broth supplemented with 0.2% glycerol and 10% oleic acid–albumin– dextrose–catalase (OADC). The strain was incubated at 37 1C for 4 weeks in 5% carbon dioxide (CO2) until logarithmic growth was reached. A turbidity equivalent to that of McFarland's no. 1 standard solution was achieved by mixing the culture with a sufficient volume of sterile supplemented Middlebrook 7H9 broth. The test inoculum was obtained by further dilution (1:20) of the suspension with the same culture medium to approximately 6 106 colony-forming units (CFU)/ml immediately prior to use. The extracts (50 mg/ml) were dissolved in 10% dimethylsulfoxide (DMSO) and maintained at room temperature for 1 h to assure their sterilization. Rifampicin and streptomycin were used as positive controls. The organic and aqueous extracts from each plant were assayed in duplicates. Each microplate was incubated for 5 days at 37 1C in a 5% CO2 atmosphere. After 5 days of incubation, 32 mL of freshly prepared resazurin solution was added to one growth control well. The microplates were incubated again at 37 1C in a 5% CO2 atmosphere for 24 h. When a color shift from blue to pink was observed in the growth control sample, 32 mL of resazurin solution was added to each of the remaining wells, and the microplate was further incubated for 24 h. A well-defined pink color was interpreted as positive bacterial growth, whereas a blue color indicated the absence of growth. 2.4. Anti-inflammatory activity The cyclooxygenase-2 inhibition assay was performed as described by Zschocke and Van Staden (2000). Cyclooxygenase-2 (COX-2) stock enzyme stored at 70 1C was activated with 1250 ml of co-factor solution and 200 ml of Tris buffer. Organic extracts were tested at a concentration of 250 μg/ml and aqueous extracts at 2 mg/ml. Three controls were used for the assay (background solvent blank and positive control). The negative controls were the background (enzyme inactivated with 4 N HCL before incubation) and the solvent blank (enzyme not deactivated). Indomethacin was used as a positive control at 200 mM. Arachidonic acid (20 ml ) was added to the eppendorfs to start the reaction before incubation at 37 1C in a water bath for 10 min. The following formula was used to calculate the percentage of inhibition for the test extracts: DPM sample DPM background COX inhibition ð%Þ ¼ 1 100 DPM blank DPM background
2.5. The Ames test A Salmonella microsome assay according to Maron and Ames (1983) modified by Mortelmans and Zeiger (2000) was used to evaluate genotoxicity potential of plant extracts that had good antimicrobial activity. The aliquots were prepared from previously dried plant extracts and dissolved in 10% DMSO to give three concentrations of 5000, 500 and 50 mg/ml. The Ames test was performed with two Salmonella typhimurium tester strains, TA98 and TA100 without metabolic activation. Bacterial stocks (100 ml) were incubated in 20 ml of Oxoid No. 2 nutrient broth at 37 1C on a rotary shaker for 16 h. The cultured bacteria (100 ml) were added to 100 ml of plant extract with 500 µl of phosphate buffer and 2 ml of top agar containing biotin–histidine (0.5 mM). The top agar mixture was then poured over the surface of a minimal agar plate and incubated at 37 1C for 48 h. The positive control used was
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4-nitroquinoline 1-oxide (4-NQO) at a concentration of 2 µg/ml whereas sterile distilled water was the negative control. All the samples tested were in triplicates and the results were expressed as the mean (7standard error) number of the revertant colonies per plate.
3. Results and discussion 3.1. Antimycobacterial assay The minimum inhibitory concentration (MIC) values of 68 extracts from 10 plants used in South Africa to treat TB and related symptoms are presented in Table 1. Analysis of the antimycobacterial efficacy of the plant samples was based on the following criteria: MIC values of less than 1.00 mg/ml were considered as having good activity (York et al., 2012). Out of 68 extracts obtained from different parts of 10 plant species screened for their inhibition of Mycobacterium tuberculosis H37Ra, six of them exhibited good antimycobacterial activity at MIC values ranging from 0.39– 0.78 mg/ml. The best activity was observed in the leaves (EtOH and water extracts) and twigs (water extracts) of Terminalia phanerophlebia Engl. & Diels having an MIC value of 0.39 mg/ml. Many reports often state that water extracts lack bioactivity, thus good antimycobacterial activity exhibited by the water extracts of Terminalia phanerophlebia (twigs and leaves) was noteworthy. The roots and twigs EtOH extracts of Terminalia phanerophlebia also showed good activity with an MIC value of 0.78 mg/ml. Terminalia plants were confirmed to have medicinal importance when Terminalia sericea Burch. ex DC. was included in the African herbal pharmacopeia representing the top 50 African medicinal plants (Eloff et al., 2008). The genus Terminalia has been reported to have antimycobacterial properties. This was evident in our previous study when the water extract of Terminalia phanerophlebia (leaves) Table 1 MIC values of plants used traditionally as remedies for the treatment of tuberculosis and related symptoms in South Africa against Mycobacterium tuberculosis H37Ra. Plant species
Abrus precatorius subsp. africanus Asparagus africanus Asparagus falcatus Brunsvigia grandiflora Ficus sur Indigofera arrecta Leonotis intermedia Pentanisia prunelloides Polygala fruticosa Terminalia phanerophlebia
Rifampicin Streptomycin
Plant part
Antimycobacterial (mg/ml)
Activity
MIC
PE
DCM
80% EtOH
Water
L
12.50
12.50
0.78
6.25
S L L Blb B R L R L Stm L R Wp L R T
12.50 12.50 12.50 12.50 12.50 12.50 12.50 12.50 12.50 12.50 12.50 12.50 12.50 12.50 12.50 12.50 0.024 0.048
12.50 12.50 12.50 12.50 12.50 12.50 12.50 12.50 12.50 12.50 12.50 12.50 12.50 12.50 12.50 12.50
12.50 6.25 6.25 12.50 6.25 0.78 6.25 12.50 3.125 6.25 12.50 0.78 6.25 0.39 0.78 0.78
6.25 12.50 3.13 6.25 1.56 6.25 6.25 12.50 3.13 12.50 3.13 3.13 3.13 0.39 12.50 0.39
L: leaves, R: roots, B: bark, Blb: bulb, S: seeds, Stm: stem, Wp: whole plant, T: twigs, MIC: minimum inhibitory concentration, DCM: dichloromethane, PE: petroleum ether, EtOH: ethanol. The values highlighted in bold are considered as very active. Rifampicin and Streptomycin are positive controls. The concentration of plant extracts was 50 mg/ml.
showed good antimycobacterial activity against Mycobacterium aurum A þ (Madikizela et al., 2013). Antimycobacterial properties of Terminalia were also evident in a study done by Kuete et al. (2010a) where compounds isolated from Terminalia superba Engl. & Diels exhibited inhibition of Mycobacterium species. The leaves and bark of Terminalia phanerophlebia were reported by Sibandze et al. (2009) to have a high flavonoid and phenolic content. Flavonoids are known to have antimicrobial, anti-inflammatory, anti-cancer and antiviral activity (Havsteen, 2002). Reports about antimycobacterial activities of flavonoids through inhibiting enzymes involved in biosynthesis of mycolic and fatty acid have been made (Kuete et al., 2010b). Additionally, flavonoids have been observed to possess modulatory properties of isoniazid (INH) and hence could be taken with anti-TB treatment for preventing or counteracting INH resistance (Macabeo et al., 2012). Good antimicrobial activity is sometimes associated with toxicity, however, the leafy extracts of Terminalia phanerophlebia demonstrated nontoxicity in a study done by Sibandze et al. (2009). The interaction or co-infection of TB microorganisms and malarial parasites is of importance. Of particular interest is a study by Sibandze et al. (2009) which confirmed the activity of Terminalia phanerophlebia as an antimalarial agent and this current study has demonstrated the same plant as an anti-TB agent. For Abrus precatorius subsp. africanus Verdc., only the EtOH extracts of the leaves exhibited good activity against Mycobacterium tuberculosis H37Ra with MIC value of 0.78 mg/ml and this extract exhibited good activity against Mycobacterium aurum in our previous study. The stem EtOH extracts of Abrus precatorius subps. africanus was found to have partial activity against Mycobacterium tuberculosis in a study done by Antoun et al. (2001). The roots EtOH extracts of Ficus sur Forssk. and Pentanisia prunelloides Schinz showed good antimycobacterial activity with both having MIC values of 0.78 mg/ ml. From a previous study good antibacterial activities were observed on tested extracts of the leaves, roots and bark of Ficus sur (Eldeen et al., 2005). The activities observed from the extracts of Abrus precatorius subsp. africanus, Ficus sur, Pentanisia prunelloides and Terminalia phanerophlebia advocates that these plants possibly have antimycobacterial compounds. Therefore further studies aimed at isolating bioactive compounds of these plants are necessary as the observed findings authenticate that they might comprise alternative future candidates for TB treatment. All the extracts of Asparagus africanus Lam., Asparagus falcatus (L.) Oberm., Brunsvigia grandiflora Lindl., Indigofera arrecta A. Rich.., Leonotis intermedia Lindl. and Polygala fruticosa P.J. Bergius did not display good antimycobacterial activity against the screened strain in our assay. However, in our previous research some of the extracts from these plants demonstrated antimycobacterial activity against Mycobacterium aurum Aþ with the exception of Brunsvigia grandiflora and Polygala fruticosa that did not display good antimycobacterial properties in both (our current and previous research) studies in spite of being reported to be used in the treatment of TB and related symptoms. 3.2. Anti-inflammatory results Pulmonary TB often leads to lung injury, pathological and inflammatory responses. Mycobacterium species are known to induce expression and induction of matrix metalloproteinase-9 which is suggested to be involved with COX-2 reliant signaling events (Bansal et al., 2009). COX-2 is an inducible enzyme with its expression activated by tissue damage and inflammation (Davies et al., 1997; Dey et al., 2003). Therefore, the development of selective COX-2 inhibitors could be an important step ahead in the therapeutic treatment of inflammatory pulmonary TB. Table 2 shows the results of COX-2 inhibitory activities exhibited by 68 extracts from 10 plants used for the treatment of TB and related
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Table 2 Anti-inflammatory activity (COX-2) of extracts from plants that are used for the treatment of TB and related symptoms in South Africa. Plant species
Plant part used
Abrus precatorius subsp. africanus
Percentage inhibition of Cyclooxygenase-2
L S L L Blb B R L R L St L R Wp L R T
Asparagus africanus Asparagus falcatus Brunsvigia grandiflora Ficus sur Indigofera arrecta Leonotis intermedia Pentanisia prunelloides Polygala fruticosa Terminalia phanerophlebia
PE
DCM
EtOH
Water
11.9 7 0 39.0 7 0 9.3 7 0 9.2 7 0 23.4 7 0 12.4 7 0 44.9 7 0.5 24.77 2.6 52.2 7 0 36.9 7 0 0.0 7 0 2.5 7 0 86.9 7 0 54.7 7 0 29.0 7 1.1 23.9 7 0 67.8 7 0.05
77.9 7 0 26.7 7 1.6 29.6 7 0 34.0 7 0 42.5 7 0 73.9 7 0 55.0 7 0 28.9 7 1.6 29.3 7 0 13.4 7 0 6.3 7 0.8 48.47 0 9.17 0.7 20.17 0 41.9 7 0 58.0 7 0 17.9 7 0
42.3 7 0 8.8 7 0 50.6 7 0 35.27 0 07 0 9.9 7 0 56.3 7 0 0.8 7 0 07 0 41.4 7 0 34.6 7 0 16.5 7 0 26.2 7 0 31.2 7 0 07 0 07 0 4.4 7 0
07 0 11.8 7 0 56.6 7 0 32.2 7 0 12.7 7 0.03 07 0 07 0 07 0 12.2 7 0 07 0 22.6 7 0 39.4 7 0 07 0 07 0 07 0 07 0 21.17 0
L: leaves, R: roots, B: bark, Blb: bulb, S: seeds, Stm: stem, Wp: whole plant, T: twigs, DCM: dichloromethane, PE: petroleum ether, EtOH: ethanol. Percentage inhibition by the positive control indomethacin was 79.2. n¼ 3.
Table 3 Number of revertant colonies of Salmonella typhimurium strains TA98 and TA100 induced by extracts of some plants used as remedies for the treatment of TB and related symptoms in South Africa. Plant species (part used)
Bio-active extract
Bacterial strains TA 98
TA 100
Extract concentration in mg/ml
Abrus precatorius subp. africanus (L) Abrus precatorius subp. africanus (S) Asparagus africanus (L) Asparagus falcatus (L) Ficus sur (B) Ficus sur (R) Indigofera arrecta (L) Leonotis intermedia (L) Pentanisia prunelloides (L) Pentanisia prunelloides (R) Terminalia phanerophlebia (L) Terminalia phanerophlebia (R) Terminalia phanerophlebia (T) Water 4NQO
DCM EtOH EtOH EtOH EtOH EtOH EtOH Water EtOH Water EtOH EtOH EtOH EtOH Water EtOH EtOH Water
5000
500
50
5000
500
50
24.337 0.33 26.337 0.88 28.337 3.33 26.677 0.89 20.337 2.60 20.007 1.00 22.007 1.00 22.677 0.33 21.007 2.31 21.677 1.20 26.007 1.15 20.337 1.76 26.337 0.33 28.007 0.57 25.677 2.72 23.007 1.15 28.677 0.89 23.677 0.89
25.007 0.58 22.677 0.33 20.337 2.40 27.677 1.45 20.337 0.33 28.007 1.52 22.677 0.88 21.337 0.67 22.337 1.33 21.677 0.33 26.677 2.33 22.007 1.15 27.677 0.87 27.007 0.58 26.677 1.20 22.677 0.67 25.007 0.58 23.337 2.19
19.3372.33 19.00 72.08 19.00 70.58 19.3370.33 20.3372.33 27.3371.20 20.6770.67 20.6770.89 20.6770.89 21.6771.76 26.00 71.53 22.3371.45 28.3373.2 25.6770.33 25.6770.67 22.3372.73 29.6771.86 23.6771.89
444.00 75.51 469.6774.33 446.67719.47 462.6776.67 430.3379.6 481.67740.91 427.3372.91 498 70.22 499.00 741.00 431.00 72.52 437.6772.84 406.33733.14 427.3372.91 439.6770.67 467.33714.88 421.33726.69 468.67730.14 437.6771.60
439.337 1.76 457.007 20.21 4407 16.37 467.677 6.23 465 7 9.61 455.677 10.17 427.677 1.45 496.007 1.54 484.007 2.31 431.677 0.89 429.677 1.20 452 7 15.28 427.677 1.45 430.007 4.58 472.007 7.81 440.677 13.38 409.337 4.58 452 7 1.05
438.677 1.76 427.677 19.23 432.677 18.99 474.007 4.16 477.007 5.20 456.337 24.17 426.007 2.00 487.007 0.50 498.677 19.54 433.677 1.86 428.337 0.88 458.007 21.07 426.007 2.00 425.007 2.52 461.677 5.17 481.007 9.84 422.007 13.32 440.677 1.20
217 1.05 1337 1.21
427.00 70.86 789.3371.45
L: leaves, R: roots, B: bark, S: seeds, T: twigs, MIC: minimum inhibitory concentration, DCM: dichloromethane, PE: petroleum ether, EtOH: ethanol, 4NQ: positive control.
symptoms in South Africa. For inhibition of the enzyme by extracts in our study, four different levels of activity were defined; inhibition below 20% was considered insignificant, between 20% and 40% as low, between 40% and 70% as moderate and between 70% and 100% as high (Tunón et al., 1995). The highest COX-2 inhibition was exhibited by petroleum ether extract from the roots of Pentanisia prunelloides at 86.9%. In studies done by Lindsey et al. (1999) and Yff et al. (2002) both the leaves and roots of Pentanisia prunelloides showed high inhibition of COX-2. The dichloromethane extracts of Abrus precatorius subsp. africanus (leaves) and Ficus sur (bark) exhibited high COX-2 inhibition with inhibition percentages of 77.9 and 73.9, respectively. Both the roots and
bark of Ficus sur are used traditionally for treating TB, these parts had high COX-1 inhibitory activity in a study by Eldeen et al. (2005) and low COX-2 inhibition. In our study a different solvent (DCM) from the one (ethyl acetate) used by Eldeen et al. (2005) showed the bark of Ficus sur to have high COX-2 inhibition. According to Jäger et al. (1996) a species with an antiinflammatory compound has a potential to be developed into an anti-inflammation product. COX-2 inhibition is beneficial in clinical conditions as it provides therapeutic effects and its high activity is desirable (Blobaum and Marnett, 2007). However, COX-2 inhibitors are currently seen as more critical, as prolonged use of COX-2 selective inhibitors can cause side effects in humans.
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Out of 68 extracts, only three exhibited high COX-2 inhibition, 14 moderate, 20 low and the remaining 31 showed insignificant inhibition. Generally the water extracts exhibited lower COX-2 activity when compared to organic extracts. Lipophilic compounds are more extractable by non-polar solvents, therefore low and insignificant activity toward COX-2 by water extracts could be due to the absence or small quantities of lipophilic compounds extracted (Tunón et al., 1995; Zschocke and Van Staden, 2000). When treating inflammation, targeting the desired area is not easy as the process has many mediators and pathways that can lead to many pathological changes. The results obtained in this study are dependent strongly on the test system used; more systems are needed to evaluate anti-inflammatory activity. 3.3. Genotoxicity results Considering the antimycobacterial results obtained in this study, one might suggest that the activity could probably be due to biological active or toxic compounds present. Therefore genotoxicity testing was done. The results of the genotoxic assay on plants selected based on MIC values being less than 1 mg/ml in our previous study are presented in Table 3. The Ames test without metabolic activation is designed only for direct mutagen detection. To designate a substance as a mutagen, a positive response in any single bacterial strain either with or without metabolic activation is sufficient (Zeiger, 2001). TA98 and TA100 bacterial strains are often used as they detect the great majority of mutagens (Verschaeve and Van Staden, 2008). Positive results from Salmonella typhimurium strain (TA98) detects frame-shift mutations based on spontaneous reversion of Salmonella typhimurium strain from His to His þ caused by crude plant extracts. While positive results caused by TA100 indicate base-pair substitution. The evaluated extracts must exhibit a dose-dependent increase in the number of revertants in order to be considered as genotoxic. Furthermore, the revertant colonies of the extracts must be equal to or greater than two times more of the negative control (Maron and Ames, 1983). However, in our study none of the tested extracts demonstrated a dose-dependent increase nor revertant colonies that are equal to or greater than twice the negative control. Therefore, the tested plant extracts lacked direct genotoxic compounds. In all cases, the values of the negative as well as the positive control were within normal limits and in accordance with literature (Elgorashi et al., 2003). Nongenotoxic activity demonstrated by the evaluated plant extracts does not confirm that they are safe for consumption as their metabolites could be genotoxic. It confirms that the substance is not genotoxic to the particular bacterial strain tested against and for the genetic endpoint tested. Non-genotoxicity on the tested extracts is a positive step forward in determining their safe use in treatment of TB and related symptoms. Therefore, more studies to evaluate genotoxicity and cytotoxicity of the extracts are required.
4. Conclusions The results indicate that some of the plants such as Abrus precatorius subsp. africanus, Ficus sur, Pentanisia prunelloides and Terminalia phanerophlebia have antimycobacterial compounds. Significant in vitro antimycobacterial activities have been demonstrated by extracts of South African plant species from different families and genera in this study. This provides supporting evidence that South Africa has diverse flora which has a potential for the discovery of metabolites that are active against Mycobacterium tuberculosis. The non-genotoxic results of the evaluated plant extracts might support safe usage of the plants in South African traditional medicine practice, but further confirmatory tests are necessary. Additionally, further phytochemical and pharmacological studies (isolation of active compounds) of these plants are evidently worthwhile as they
form a good preliminary basis for the selection of candidate plant species that might be an alternative to combat TB. Anti-inflammatory and antimycobacterial activity observed by some of the investigated plant extracts in this study indicate the presence of bioactive agents that warrant further investigation.
Acknowledgments The authors would like to thank the National Research Foundation (NRF), Pretoria, and Canon Collins GreenMatter, and Claude Leon Foundation for funding and providing fellowships for this project. We would also wish to thank Mrs. Alison Young (University of KwaZuluNatal-horticulturist) and Ms. Shanaz Ghuman for their assistance in plant collection and identification.
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Macabeo, A.P.G., Tudla, F.A., Krohn, K., Franzblau, S.G., 2012. Antitubercular activity of the semi-polar extractives of Uvaria rufa. Asian Pac. J. Trop. Med. 5, 777–780. Madikizela, B., Ndhlala, A.R., Finnie, J.F., Van Staden, J., 2013. In vitro Antimicrobial activity of extracts from plants used traditionally in South Africa to treat tuberculosis and related symptoms. Evid. Based Complement. Altern. Med. 2013, 1-8 (Article ID 840719). Maron, D.M., Ames, B.N., 1983. Revised methods for Salmonella mutagenicity test. Mutat. Res. 113, 173–215. McGaw, L.J., Lall, N., Meyer, J.J.M., Eloff, J.N., 2008. The potential of South African plants against Mycobacterium infections. J. Ethnopharmacol. 119, 482–500. Mortelmans, K., Zeiger, K., 2000. The Ames Salmonella/Microsome mutagenicity assay. Mutat. Res. 455, 29–60. Okunade, A.L., Elvin-Lewis, M.P.F., Lewis, W.H., 2004. Natural antimycobacterial metabolites: current status. Phytochemistry 65, 1017–1032. Shinohara, T., Pantuso, T., Shinohara, S., Kogiso, M., Myrvik, Q.N., Henriksen, R.A., Shibata, Y., 2009. Persistent inactivation of macrophage cyclooxygenase-2 in mycobacterial pulmonary inflammation. Am. J. Respir. Cell Mol. Biol. 41, 146–154. Sibandze, G.F., Van Zyl, R.L., Viljoen, A.N., 2009. Pharmacological Properties of Swazi Medicinal Plants. University of the Witswatersrand, Johannesburg (Thesis).
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Antimycobacterial, anti-inflammatory and genotoxicity evaluation of plants used for the treatment of tuberculosis and related symptoms in South Africa B. Madikizela, A.R. Ndhlala, J.F. Finnie, J. Van Staden n Research Centre for Plant Growth and Development, School of Life Sciences, University of KwaZulu-Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa
art ic l e i nf o
a b s t r a c t
Article history: Received 7 November 2013 Received in revised form 17 January 2014 Accepted 16 February 2014 Available online 25 February 2014
Ethnopharmacological relevance: Emergence of drug-resistant tuberculosis strains and long duration of treatment has established an urgent need to search for new effective agents. The great floral diversity of South Africa has potential for producing new bioactive compounds, therefore pharmacological screening of plant extracts within this region offers much potential. To assess the in vitro antimycobacterial, antiinflammatory and genotoxicity activity of selected plants that are used for the treatment of TB and related symptoms in South Africa. Materials and methods: Ground plant materials from 10 plants were extracted sequentially with four solvents (petroleum ether, dichloromethane, 80% ethanol and water) and a total of 68 extracts were produced. A broth microdilution method was used to screen extracts against Mycobacterium tuberculosis H37Ra. The cyclooxygenase-2 (COX-2) enzyme was used to evaluate the anti-inflammatory activity of the extracts and the Salmonella microsome assay using two Salmonella typhimurium strains (TA98 and TA100) to establish genotoxicity. Results: Six out of 68 extracts showed good antimycobacterial activity. Three extracts showed good inhibition (470%) of COX-2 enzyme. All the extracts tested were non-genotoxic against the tested Salmonella strains. Conclusion: The results observed in this study indicate that some of the plants such as Abrus precatorius subsp. africanus, Ficus sur, Pentanisia prunelloides and Terminalia phanerophlebia could be investigated further against drug-resistant TB strains. & 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Ames test: anti-inflammatory Mycobacterium tuberculosis Terminalia phanerophlebia Pentanisia prunelloides
1. Introduction Tuberculosis (TB) is one of the oldest and most prevalent diseases, having infected about 8.7 million people worldwide in 2011 (Smith, 2003; Okunade et al., 2004; WHO, 2013). In the mid-20th century, the mortality rate of TB began to decrease and the decline is still evident in Europe and America with the incident rate continuing to be low (Daniel, 2006; WHO, 2013). In Africa, approximately half a million people died of TB in 2004. Africa remains the only continent
Abbreviations: 4-NQO, 4-nitroquinoline 1-oxide; BCG, Bacillus Calmette Guérin; CFU, colony-forming units; CO2, carbon dioxide; COX, Cyclooxygenase; DCM, dichloromethane; DMSO, dimethylsulfoxide; DPM, disintegrations per minute; EtOH, ethanol; HCL, hydrochloric acid; HIV, human immunodeficiency virus; INH, isoniazid; MDR, multi-drug resistant; MIC, minimum inhibition concentration; No., number; NRF, National Research Foundation; OADC, oleic acid-albumin-dextrosecatalase; PE, petroleum ether; REMA, resazurin microplate assay; TB, tuberculosis n Corresponding author. Tel.: þ 27 332605130; fax: þ 27 332605897. E-mail address: [email protected] (J. Van Staden). http://dx.doi.org/10.1016/j.jep.2014.02.034 0378-8741 & 2014 Elsevier Ireland Ltd. All rights reserved.
where TB rates are still increasing (Kamatou et al., 2007). TB symptoms are aggravated by the co-infection with Human Immunodeficiency Virus (HIV). Studies done have placed Mycobacterium tuberculosis as the most common human cause of TB worldwide (WHO, 2013), responsible for more human fatalities than any single bacterial species and infecting at least one-third of the world's population (Kamatou et al., 2007; Bansal et al., 2009; Knechel, 2009). Streptomyces-based antibiotics and other chemotherapeutic antimycobacterial agents often used in combination in the treatment of TB are no longer effective due to drug resistance exhibited by the organism (Lai et al., 2011). The treatments of TB take a long time resulting in poor compliance of patients which contributes to sustaining multi-drug resistant TB (MDR-TB) (Connolly et al., 2007). Neonates immunization with Bacillus Calmette Guérin (BCG) protects against mainly TB meningitis and miliary TB. However, the efficacy wanes 10–15 years post-vaccination, thus adults are not protected against pulmonary tuberculosis. Therefore, new vaccines and antitubercular agents with new modes of activity, shorter treatment duration as well as low toxicity are needed to reduce MDR-TB
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prevalence and stop the epidemic of TB. One of the TB symptoms is chest pain caused by inflammation of the membranes lining the lungs which leads to development of lung fibrosis. In the case of pulmonary TB, alveolar macrophages control persistant inflammatory responses in the lungs by producing chemical mediators that leads to granuloma formation (Shinohara et al., 2009). In response to infection of the host by Mycobacteria, granuloma formation represents protective immunity and inflammatory tissue destruction and repair (Shinohara et al., 2009). South Africa is endowed with a diverse flora which has potential for the discovery of metabolites that are active against Mycobacterium tuberculosis (Lall and Meyer, 1999; McGaw et al., 2008). Screening of plant extracts for antimycobacterial activity within South African medicinal plants with their great diversity offers much potential in the search for active new metabolites that may have activity against Mycobacterium tuberculosis and other opportunistic infections. Although medicinal plants have been used in therapy for many years, that does not mean that they are safe, as they may have side effects. Since medicinal plant use and prescription in South Africa is not standardized, the danger of misadministration especially if the plants are toxic is real (Fennell et al., 2004). It is important to determine if the antimicrobial activity exhibited by some plant extracts is not due to toxicity. In our previous study, we reported several plant extracts that exhibited good antibacterial activities against non-pathogenic Mycobacterium species and other strains associated with respiratory infections (Madikizela et al., 2013). As a result of research done in our laboratory on antimicrobial activity of plants used for treating TB and that most human fatalities caused by TB are caused by Mycobacterium tuberculosis, further investigations were carried out. The investigations included determining the antibacterial activity of selected plants used for treating TB and related symptoms against an Mycobacterium tuberculosis strain. The antiinflammatory (since inflammation is one of TB symptoms) activities of selected medicinal plants used in the previous study, as well as genotoxicity properties of the extracts that showed good antimicrobial activities in that previous study were carried out in the current study.
2. Materials and methods 2.1. Sample collection Plant materials were collected from the University of KwaZuluNatal botanical garden and Ukulinga research farm in Pietermaritzburg, South Africa. These plants have already been evaluated for their antimicrobial activity against strains of bacteria related to respiratory ailments in a previous study (Madikizela et al., 2013). The list of the plants used with family, species name, voucher specimen number, traditional uses and previously tested activities are outlined in Madikizela et al. (2013).
2.2. Plant extracts preparation Plant materials were oven-dried at 50 1C, ground into powder and 10 g was extracted sequentially with 200 ml of petroleum ether (PE), dichloromethane (DCM), 80% ethanol (EtOH) and water. The extracts were then filtered through Whatman No. 1 filter paper and concentrated under vacuum using a rotary evaporator for solvent extracts whereas the aqueous extracts were freezedried. The concentrated extracts were then dried under a stream of cold air and kept at 8 1C until required.
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2.3. Antimycobacterial activity using resazurin microplate assay The resazurin microplate assay (REMA) according to the method of Jadaun et al. (2007) was used as a antimycobacterial assay. Mycobacterium tuberculosis H37Ra (American type culture collection 25177) stored at 70 1C was thawed at room temperature for 10–15 min and subcultured in Middlebrook 7H9 broth supplemented with 0.2% glycerol and 10% oleic acid–albumin– dextrose–catalase (OADC). The strain was incubated at 37 1C for 4 weeks in 5% carbon dioxide (CO2) until logarithmic growth was reached. A turbidity equivalent to that of McFarland's no. 1 standard solution was achieved by mixing the culture with a sufficient volume of sterile supplemented Middlebrook 7H9 broth. The test inoculum was obtained by further dilution (1:20) of the suspension with the same culture medium to approximately 6 106 colony-forming units (CFU)/ml immediately prior to use. The extracts (50 mg/ml) were dissolved in 10% dimethylsulfoxide (DMSO) and maintained at room temperature for 1 h to assure their sterilization. Rifampicin and streptomycin were used as positive controls. The organic and aqueous extracts from each plant were assayed in duplicates. Each microplate was incubated for 5 days at 37 1C in a 5% CO2 atmosphere. After 5 days of incubation, 32 mL of freshly prepared resazurin solution was added to one growth control well. The microplates were incubated again at 37 1C in a 5% CO2 atmosphere for 24 h. When a color shift from blue to pink was observed in the growth control sample, 32 mL of resazurin solution was added to each of the remaining wells, and the microplate was further incubated for 24 h. A well-defined pink color was interpreted as positive bacterial growth, whereas a blue color indicated the absence of growth. 2.4. Anti-inflammatory activity The cyclooxygenase-2 inhibition assay was performed as described by Zschocke and Van Staden (2000). Cyclooxygenase-2 (COX-2) stock enzyme stored at 70 1C was activated with 1250 ml of co-factor solution and 200 ml of Tris buffer. Organic extracts were tested at a concentration of 250 μg/ml and aqueous extracts at 2 mg/ml. Three controls were used for the assay (background solvent blank and positive control). The negative controls were the background (enzyme inactivated with 4 N HCL before incubation) and the solvent blank (enzyme not deactivated). Indomethacin was used as a positive control at 200 mM. Arachidonic acid (20 ml ) was added to the eppendorfs to start the reaction before incubation at 37 1C in a water bath for 10 min. The following formula was used to calculate the percentage of inhibition for the test extracts: DPM sample DPM background COX inhibition ð%Þ ¼ 1 100 DPM blank DPM background
2.5. The Ames test A Salmonella microsome assay according to Maron and Ames (1983) modified by Mortelmans and Zeiger (2000) was used to evaluate genotoxicity potential of plant extracts that had good antimicrobial activity. The aliquots were prepared from previously dried plant extracts and dissolved in 10% DMSO to give three concentrations of 5000, 500 and 50 mg/ml. The Ames test was performed with two Salmonella typhimurium tester strains, TA98 and TA100 without metabolic activation. Bacterial stocks (100 ml) were incubated in 20 ml of Oxoid No. 2 nutrient broth at 37 1C on a rotary shaker for 16 h. The cultured bacteria (100 ml) were added to 100 ml of plant extract with 500 µl of phosphate buffer and 2 ml of top agar containing biotin–histidine (0.5 mM). The top agar mixture was then poured over the surface of a minimal agar plate and incubated at 37 1C for 48 h. The positive control used was
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4-nitroquinoline 1-oxide (4-NQO) at a concentration of 2 µg/ml whereas sterile distilled water was the negative control. All the samples tested were in triplicates and the results were expressed as the mean (7standard error) number of the revertant colonies per plate.
3. Results and discussion 3.1. Antimycobacterial assay The minimum inhibitory concentration (MIC) values of 68 extracts from 10 plants used in South Africa to treat TB and related symptoms are presented in Table 1. Analysis of the antimycobacterial efficacy of the plant samples was based on the following criteria: MIC values of less than 1.00 mg/ml were considered as having good activity (York et al., 2012). Out of 68 extracts obtained from different parts of 10 plant species screened for their inhibition of Mycobacterium tuberculosis H37Ra, six of them exhibited good antimycobacterial activity at MIC values ranging from 0.39– 0.78 mg/ml. The best activity was observed in the leaves (EtOH and water extracts) and twigs (water extracts) of Terminalia phanerophlebia Engl. & Diels having an MIC value of 0.39 mg/ml. Many reports often state that water extracts lack bioactivity, thus good antimycobacterial activity exhibited by the water extracts of Terminalia phanerophlebia (twigs and leaves) was noteworthy. The roots and twigs EtOH extracts of Terminalia phanerophlebia also showed good activity with an MIC value of 0.78 mg/ml. Terminalia plants were confirmed to have medicinal importance when Terminalia sericea Burch. ex DC. was included in the African herbal pharmacopeia representing the top 50 African medicinal plants (Eloff et al., 2008). The genus Terminalia has been reported to have antimycobacterial properties. This was evident in our previous study when the water extract of Terminalia phanerophlebia (leaves) Table 1 MIC values of plants used traditionally as remedies for the treatment of tuberculosis and related symptoms in South Africa against Mycobacterium tuberculosis H37Ra. Plant species
Abrus precatorius subsp. africanus Asparagus africanus Asparagus falcatus Brunsvigia grandiflora Ficus sur Indigofera arrecta Leonotis intermedia Pentanisia prunelloides Polygala fruticosa Terminalia phanerophlebia
Rifampicin Streptomycin
Plant part
Antimycobacterial (mg/ml)
Activity
MIC
PE
DCM
80% EtOH
Water
L
12.50
12.50
0.78
6.25
S L L Blb B R L R L Stm L R Wp L R T
12.50 12.50 12.50 12.50 12.50 12.50 12.50 12.50 12.50 12.50 12.50 12.50 12.50 12.50 12.50 12.50 0.024 0.048
12.50 12.50 12.50 12.50 12.50 12.50 12.50 12.50 12.50 12.50 12.50 12.50 12.50 12.50 12.50 12.50
12.50 6.25 6.25 12.50 6.25 0.78 6.25 12.50 3.125 6.25 12.50 0.78 6.25 0.39 0.78 0.78
6.25 12.50 3.13 6.25 1.56 6.25 6.25 12.50 3.13 12.50 3.13 3.13 3.13 0.39 12.50 0.39
L: leaves, R: roots, B: bark, Blb: bulb, S: seeds, Stm: stem, Wp: whole plant, T: twigs, MIC: minimum inhibitory concentration, DCM: dichloromethane, PE: petroleum ether, EtOH: ethanol. The values highlighted in bold are considered as very active. Rifampicin and Streptomycin are positive controls. The concentration of plant extracts was 50 mg/ml.
showed good antimycobacterial activity against Mycobacterium aurum A þ (Madikizela et al., 2013). Antimycobacterial properties of Terminalia were also evident in a study done by Kuete et al. (2010a) where compounds isolated from Terminalia superba Engl. & Diels exhibited inhibition of Mycobacterium species. The leaves and bark of Terminalia phanerophlebia were reported by Sibandze et al. (2009) to have a high flavonoid and phenolic content. Flavonoids are known to have antimicrobial, anti-inflammatory, anti-cancer and antiviral activity (Havsteen, 2002). Reports about antimycobacterial activities of flavonoids through inhibiting enzymes involved in biosynthesis of mycolic and fatty acid have been made (Kuete et al., 2010b). Additionally, flavonoids have been observed to possess modulatory properties of isoniazid (INH) and hence could be taken with anti-TB treatment for preventing or counteracting INH resistance (Macabeo et al., 2012). Good antimicrobial activity is sometimes associated with toxicity, however, the leafy extracts of Terminalia phanerophlebia demonstrated nontoxicity in a study done by Sibandze et al. (2009). The interaction or co-infection of TB microorganisms and malarial parasites is of importance. Of particular interest is a study by Sibandze et al. (2009) which confirmed the activity of Terminalia phanerophlebia as an antimalarial agent and this current study has demonstrated the same plant as an anti-TB agent. For Abrus precatorius subsp. africanus Verdc., only the EtOH extracts of the leaves exhibited good activity against Mycobacterium tuberculosis H37Ra with MIC value of 0.78 mg/ml and this extract exhibited good activity against Mycobacterium aurum in our previous study. The stem EtOH extracts of Abrus precatorius subps. africanus was found to have partial activity against Mycobacterium tuberculosis in a study done by Antoun et al. (2001). The roots EtOH extracts of Ficus sur Forssk. and Pentanisia prunelloides Schinz showed good antimycobacterial activity with both having MIC values of 0.78 mg/ ml. From a previous study good antibacterial activities were observed on tested extracts of the leaves, roots and bark of Ficus sur (Eldeen et al., 2005). The activities observed from the extracts of Abrus precatorius subsp. africanus, Ficus sur, Pentanisia prunelloides and Terminalia phanerophlebia advocates that these plants possibly have antimycobacterial compounds. Therefore further studies aimed at isolating bioactive compounds of these plants are necessary as the observed findings authenticate that they might comprise alternative future candidates for TB treatment. All the extracts of Asparagus africanus Lam., Asparagus falcatus (L.) Oberm., Brunsvigia grandiflora Lindl., Indigofera arrecta A. Rich.., Leonotis intermedia Lindl. and Polygala fruticosa P.J. Bergius did not display good antimycobacterial activity against the screened strain in our assay. However, in our previous research some of the extracts from these plants demonstrated antimycobacterial activity against Mycobacterium aurum Aþ with the exception of Brunsvigia grandiflora and Polygala fruticosa that did not display good antimycobacterial properties in both (our current and previous research) studies in spite of being reported to be used in the treatment of TB and related symptoms. 3.2. Anti-inflammatory results Pulmonary TB often leads to lung injury, pathological and inflammatory responses. Mycobacterium species are known to induce expression and induction of matrix metalloproteinase-9 which is suggested to be involved with COX-2 reliant signaling events (Bansal et al., 2009). COX-2 is an inducible enzyme with its expression activated by tissue damage and inflammation (Davies et al., 1997; Dey et al., 2003). Therefore, the development of selective COX-2 inhibitors could be an important step ahead in the therapeutic treatment of inflammatory pulmonary TB. Table 2 shows the results of COX-2 inhibitory activities exhibited by 68 extracts from 10 plants used for the treatment of TB and related
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Table 2 Anti-inflammatory activity (COX-2) of extracts from plants that are used for the treatment of TB and related symptoms in South Africa. Plant species
Plant part used
Abrus precatorius subsp. africanus
Percentage inhibition of Cyclooxygenase-2
L S L L Blb B R L R L St L R Wp L R T
Asparagus africanus Asparagus falcatus Brunsvigia grandiflora Ficus sur Indigofera arrecta Leonotis intermedia Pentanisia prunelloides Polygala fruticosa Terminalia phanerophlebia
PE
DCM
EtOH
Water
11.9 7 0 39.0 7 0 9.3 7 0 9.2 7 0 23.4 7 0 12.4 7 0 44.9 7 0.5 24.77 2.6 52.2 7 0 36.9 7 0 0.0 7 0 2.5 7 0 86.9 7 0 54.7 7 0 29.0 7 1.1 23.9 7 0 67.8 7 0.05
77.9 7 0 26.7 7 1.6 29.6 7 0 34.0 7 0 42.5 7 0 73.9 7 0 55.0 7 0 28.9 7 1.6 29.3 7 0 13.4 7 0 6.3 7 0.8 48.47 0 9.17 0.7 20.17 0 41.9 7 0 58.0 7 0 17.9 7 0
42.3 7 0 8.8 7 0 50.6 7 0 35.27 0 07 0 9.9 7 0 56.3 7 0 0.8 7 0 07 0 41.4 7 0 34.6 7 0 16.5 7 0 26.2 7 0 31.2 7 0 07 0 07 0 4.4 7 0
07 0 11.8 7 0 56.6 7 0 32.2 7 0 12.7 7 0.03 07 0 07 0 07 0 12.2 7 0 07 0 22.6 7 0 39.4 7 0 07 0 07 0 07 0 07 0 21.17 0
L: leaves, R: roots, B: bark, Blb: bulb, S: seeds, Stm: stem, Wp: whole plant, T: twigs, DCM: dichloromethane, PE: petroleum ether, EtOH: ethanol. Percentage inhibition by the positive control indomethacin was 79.2. n¼ 3.
Table 3 Number of revertant colonies of Salmonella typhimurium strains TA98 and TA100 induced by extracts of some plants used as remedies for the treatment of TB and related symptoms in South Africa. Plant species (part used)
Bio-active extract
Bacterial strains TA 98
TA 100
Extract concentration in mg/ml
Abrus precatorius subp. africanus (L) Abrus precatorius subp. africanus (S) Asparagus africanus (L) Asparagus falcatus (L) Ficus sur (B) Ficus sur (R) Indigofera arrecta (L) Leonotis intermedia (L) Pentanisia prunelloides (L) Pentanisia prunelloides (R) Terminalia phanerophlebia (L) Terminalia phanerophlebia (R) Terminalia phanerophlebia (T) Water 4NQO
DCM EtOH EtOH EtOH EtOH EtOH EtOH Water EtOH Water EtOH EtOH EtOH EtOH Water EtOH EtOH Water
5000
500
50
5000
500
50
24.337 0.33 26.337 0.88 28.337 3.33 26.677 0.89 20.337 2.60 20.007 1.00 22.007 1.00 22.677 0.33 21.007 2.31 21.677 1.20 26.007 1.15 20.337 1.76 26.337 0.33 28.007 0.57 25.677 2.72 23.007 1.15 28.677 0.89 23.677 0.89
25.007 0.58 22.677 0.33 20.337 2.40 27.677 1.45 20.337 0.33 28.007 1.52 22.677 0.88 21.337 0.67 22.337 1.33 21.677 0.33 26.677 2.33 22.007 1.15 27.677 0.87 27.007 0.58 26.677 1.20 22.677 0.67 25.007 0.58 23.337 2.19
19.3372.33 19.00 72.08 19.00 70.58 19.3370.33 20.3372.33 27.3371.20 20.6770.67 20.6770.89 20.6770.89 21.6771.76 26.00 71.53 22.3371.45 28.3373.2 25.6770.33 25.6770.67 22.3372.73 29.6771.86 23.6771.89
444.00 75.51 469.6774.33 446.67719.47 462.6776.67 430.3379.6 481.67740.91 427.3372.91 498 70.22 499.00 741.00 431.00 72.52 437.6772.84 406.33733.14 427.3372.91 439.6770.67 467.33714.88 421.33726.69 468.67730.14 437.6771.60
439.337 1.76 457.007 20.21 4407 16.37 467.677 6.23 465 7 9.61 455.677 10.17 427.677 1.45 496.007 1.54 484.007 2.31 431.677 0.89 429.677 1.20 452 7 15.28 427.677 1.45 430.007 4.58 472.007 7.81 440.677 13.38 409.337 4.58 452 7 1.05
438.677 1.76 427.677 19.23 432.677 18.99 474.007 4.16 477.007 5.20 456.337 24.17 426.007 2.00 487.007 0.50 498.677 19.54 433.677 1.86 428.337 0.88 458.007 21.07 426.007 2.00 425.007 2.52 461.677 5.17 481.007 9.84 422.007 13.32 440.677 1.20
217 1.05 1337 1.21
427.00 70.86 789.3371.45
L: leaves, R: roots, B: bark, S: seeds, T: twigs, MIC: minimum inhibitory concentration, DCM: dichloromethane, PE: petroleum ether, EtOH: ethanol, 4NQ: positive control.
symptoms in South Africa. For inhibition of the enzyme by extracts in our study, four different levels of activity were defined; inhibition below 20% was considered insignificant, between 20% and 40% as low, between 40% and 70% as moderate and between 70% and 100% as high (Tunón et al., 1995). The highest COX-2 inhibition was exhibited by petroleum ether extract from the roots of Pentanisia prunelloides at 86.9%. In studies done by Lindsey et al. (1999) and Yff et al. (2002) both the leaves and roots of Pentanisia prunelloides showed high inhibition of COX-2. The dichloromethane extracts of Abrus precatorius subsp. africanus (leaves) and Ficus sur (bark) exhibited high COX-2 inhibition with inhibition percentages of 77.9 and 73.9, respectively. Both the roots and
bark of Ficus sur are used traditionally for treating TB, these parts had high COX-1 inhibitory activity in a study by Eldeen et al. (2005) and low COX-2 inhibition. In our study a different solvent (DCM) from the one (ethyl acetate) used by Eldeen et al. (2005) showed the bark of Ficus sur to have high COX-2 inhibition. According to Jäger et al. (1996) a species with an antiinflammatory compound has a potential to be developed into an anti-inflammation product. COX-2 inhibition is beneficial in clinical conditions as it provides therapeutic effects and its high activity is desirable (Blobaum and Marnett, 2007). However, COX-2 inhibitors are currently seen as more critical, as prolonged use of COX-2 selective inhibitors can cause side effects in humans.
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Out of 68 extracts, only three exhibited high COX-2 inhibition, 14 moderate, 20 low and the remaining 31 showed insignificant inhibition. Generally the water extracts exhibited lower COX-2 activity when compared to organic extracts. Lipophilic compounds are more extractable by non-polar solvents, therefore low and insignificant activity toward COX-2 by water extracts could be due to the absence or small quantities of lipophilic compounds extracted (Tunón et al., 1995; Zschocke and Van Staden, 2000). When treating inflammation, targeting the desired area is not easy as the process has many mediators and pathways that can lead to many pathological changes. The results obtained in this study are dependent strongly on the test system used; more systems are needed to evaluate anti-inflammatory activity. 3.3. Genotoxicity results Considering the antimycobacterial results obtained in this study, one might suggest that the activity could probably be due to biological active or toxic compounds present. Therefore genotoxicity testing was done. The results of the genotoxic assay on plants selected based on MIC values being less than 1 mg/ml in our previous study are presented in Table 3. The Ames test without metabolic activation is designed only for direct mutagen detection. To designate a substance as a mutagen, a positive response in any single bacterial strain either with or without metabolic activation is sufficient (Zeiger, 2001). TA98 and TA100 bacterial strains are often used as they detect the great majority of mutagens (Verschaeve and Van Staden, 2008). Positive results from Salmonella typhimurium strain (TA98) detects frame-shift mutations based on spontaneous reversion of Salmonella typhimurium strain from His to His þ caused by crude plant extracts. While positive results caused by TA100 indicate base-pair substitution. The evaluated extracts must exhibit a dose-dependent increase in the number of revertants in order to be considered as genotoxic. Furthermore, the revertant colonies of the extracts must be equal to or greater than two times more of the negative control (Maron and Ames, 1983). However, in our study none of the tested extracts demonstrated a dose-dependent increase nor revertant colonies that are equal to or greater than twice the negative control. Therefore, the tested plant extracts lacked direct genotoxic compounds. In all cases, the values of the negative as well as the positive control were within normal limits and in accordance with literature (Elgorashi et al., 2003). Nongenotoxic activity demonstrated by the evaluated plant extracts does not confirm that they are safe for consumption as their metabolites could be genotoxic. It confirms that the substance is not genotoxic to the particular bacterial strain tested against and for the genetic endpoint tested. Non-genotoxicity on the tested extracts is a positive step forward in determining their safe use in treatment of TB and related symptoms. Therefore, more studies to evaluate genotoxicity and cytotoxicity of the extracts are required.
4. Conclusions The results indicate that some of the plants such as Abrus precatorius subsp. africanus, Ficus sur, Pentanisia prunelloides and Terminalia phanerophlebia have antimycobacterial compounds. Significant in vitro antimycobacterial activities have been demonstrated by extracts of South African plant species from different families and genera in this study. This provides supporting evidence that South Africa has diverse flora which has a potential for the discovery of metabolites that are active against Mycobacterium tuberculosis. The non-genotoxic results of the evaluated plant extracts might support safe usage of the plants in South African traditional medicine practice, but further confirmatory tests are necessary. Additionally, further phytochemical and pharmacological studies (isolation of active compounds) of these plants are evidently worthwhile as they
form a good preliminary basis for the selection of candidate plant species that might be an alternative to combat TB. Anti-inflammatory and antimycobacterial activity observed by some of the investigated plant extracts in this study indicate the presence of bioactive agents that warrant further investigation.
Acknowledgments The authors would like to thank the National Research Foundation (NRF), Pretoria, and Canon Collins GreenMatter, and Claude Leon Foundation for funding and providing fellowships for this project. We would also wish to thank Mrs. Alison Young (University of KwaZuluNatal-horticulturist) and Ms. Shanaz Ghuman for their assistance in plant collection and identification.
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