Journal of Ethnopharmacology 157 (2014) 134–139
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Antimycobacterial activity of selected medicinal plants traditionally used in Sudan to treat infectious diseases Nadir Abuzeid a,b,c,1, Sadaf Kalsum c,1, Marie Larsson d, Mikaela Glader c, Henrik Andersson c, Johanna Raffetseder c, Elsje Pienaar c, Daniel Eklund c, Muddathir S. Alhassan a, Haidar A. AlGadir a, Waleed S. Koko a, Thomas Schön e, M. Ahmed Mesaik f,g, Omer M. Abdalla g, Asaad Khalid a, Maria Lerm c,n a
Medicinal and Aromatic Plants Research Institute, National Center for Research, P.O. Box: 2404, Khartoum 11111, Sudan Department of Clinical Microbiology, Faculty of Medical Laboratory Sciences, Omdurman Islamic University, P.O.Box 382, Omdurman,Sudan Division of Microbiology and Molecular Medicine, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linkoping University, 58185 Linkoping, Sweden d Department of Clinical Microbiology, Linköping University Hospital, 58185 Linkoping, Sweden e Department of Clinical Microbiology and Infectious diseases, Kalmar County Hospital, SE-391 85 Kalmar, Sweden f Faculty of Pharmacy, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, Kuala Lumpur 50300, Malaysia g Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan b c
art ic l e i nf o
a b s t r a c t
Article history: Received 19 June 2014 Received in revised form 9 September 2014 Accepted 15 September 2014 Available online 28 September 2014
Ethnopharmacological relevance: The emergence of multidrug-resistant strains of Mycobacterium tuberculosis underscores the need for continuous development of new and efficient methods to determine the susceptibility of isolates of Mycobacterium tuberculosis in the search for novel antimycobacterial agents. Natural products constitute an important source of new drugs, and design and implementation of antimycobacterial susceptibility testing methods are necessary to evaluate the different extracts and compounds. In this study we have explored the antimycobacterial properties of 50 ethanolic extracts from different parts of 46 selected medicinal plants traditionally used in Sudan to treat infectious diseases. Materials and methods: Plants were harvested and ethanolic extracts were prepared. For selected extracts, fractionation with hydrophilic and hydrophobic solvents was undertaken. A luminometrybased assay was used for determination of mycobacterial growth in broth cultures and inside primary human macrophages in the presence or absence of plant extracts and fractions of extracts. Cytotoxicity was also assessed for active fractions of plant extracts. Results: Of the tested extracts, three exhibited a significant inhibitory effect on an avirulent strain of Mycobacterium tubercluosis (H37Ra) at the initial screening doses (125 and 6.25 mg/ml). These were bark and leaf extracts of Khaya senegalensis and the leaf extract of Rosmarinus officinalis L. Further fractions of these plant extracts were prepared with n-hexane, chloroform, ethyl acetate, n-butanol, ethanol and water, and the activity of these extracts was retained in hydrophobic fractions. Cytotoxicity assays revealed that the chloroform fraction of Khaya senegalensis bark was non-toxic to human monocytederived macrophages and other cell types at the concentrations used and hence, further analysis, including assessment of IC50 and intracellular activity was done with this fraction. Conclusion: These results encourage further investigations to identify the active compound(s) within the chloroform fraction of Khaya senegalensis bark. & 2014 Published by Elsevier Ireland Ltd.
Keywords: Mycobacterium tuberculosis Sudanese medicinal plants Primary human macrophages Luminescence reporter assay Cytotoxicity assay
1. Introduction
n
Corresponding author. Tel.: þ 46 1010 32088; fax: þ 46 1010 3478. E-mail address: [email protected] (M. Lerm). 1 These authors contributed equally to the study.
http://dx.doi.org/10.1016/j.jep.2014.09.020 0378-8741/& 2014 Published by Elsevier Ireland Ltd.
Tuberculosis (TB) is a global public health threat and remains one of the major causes of death among infectious diseases. The patient incompliance associated with the long time required to reach sterilizing TB treatment ( Z6 months on 2–4 antibiotics) contributes to the emergence of multidrug- resistant TB (MDR-TB)
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and extensively drug-resistant TB (XDR-TB) (Jain and Dixit, 2008; Pinto and Menzies, 2011). Taking into consideration the low cure rates of MDR-TB and the high relative incidence, 28% of new TB cases in some areas of Eastern Europe, research promoting new antimycobacterial therapies is pivotal (WHO, 2010). In addition, the discovery of new classes of antimycobacterial drugs (Field et al., 2012) and possibly also virulence blockers (Keyser et al., 2008) and drugs stimulating immunity (Lam et al., 2012) may guide the development of a more rational and effective TB therapy that is several months shorter than the present regimen. A milestone of the development of the standard TB therapy was the implementation of short course chemotherapy in the 1970s (East African-British Medical Research Councils, 1972). The combination of four antibiotics allowed reduction of time of treatment to six months. However, this led to the conclusion that TB was finally defeated, but the emergence of MDR-TB in the United States rekindled the scientific interest in the disease (Frieden et al., 1993). Natural products can pave the way for new drug leads, since they provide an unlimited source of chemically diverse compounds. Today's first line drug to treat malaria, artemisinin, was found through the systematic search of anti-malarial compounds in herbs used in traditional Chinese medicine (Klayman, 1985). Several broad screening strategies have also been carried out with Sudanese medicinal plants for antibacterial, antifungal, antiviral, antimalarial and antihelminthic properties (Koko et al., 2008). In the present study, we screened 50 ethanolic extracts of 46 Sudanese plants with respect to antimycobacterial activity using a luminometry-based method (Eklund et al., 2010). Two hits were identified; Khaya senegalensis (bark and leaves) and Rosmarinus officinalis L. (leaves), and these extracts were subjected to fractionation with water, n-hexane, chloroform, ethanol, ethyl acetate and n-butane. The n-hexane, chloroform, ethyl acetate and nbutanol fractions were found to effectively inhibit the growth of Mycobacterium tuberculosis at a concentration of 6.25 mg/ml. The result was confirmed with other assays for mycobacterial growth.
2. Materials and methods
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calculated for each fraction (Table S2). The aqueous layer was finally freezed at 80 1C, stored at 40 1C and freeze-dried using a freeze dryer apparatus until completely dry and the yield was calculated. For experiments, 5 mg of dried plant extracts or fractions were dissolved in 50 ml of 100% DMSO to obtain stock solutions of 100 mg/ml. 2.3. pH measurement Litmus paper (MERCK) was dipped into the aliquot of plant extracts diluted to 125 and 6.25 mg/ml in broth supplemented with ADC and 0.05% Tween 80. The color obtained determined the pH of the extract as shown in Table S1. 2.4. Bacterial growth assay One ml of culture containing Mtb H37Ra-lux was centrifuged once at 5000g for 5 min at room temperature and the pellet was re-suspended in broth supplemented with ADC and 0.05% Tween 80. In order to remove bacterial aggregates, the suspension was passaged 10 times through a sterile syringe equipped with a 27gauge needle. The concentration was determined by optical density at 600 nm (OD600) as a function of colony-forming units (CFU) obtained from a standard curve derived from plated bacteria (not shown). For screening, 96-well plates were prepared with dilutions of the extracts at the final concentrations of 2.5 mg/ml, 125 mg/ml or 6.25 mg/ml. 2.5%, 0.125% and 0.00625% DMSO were used as solvent controls, respectively. A high dose of isoniazid (the first-line antibiotic, was selected to insure efficient inhibition) and broth only were included in all experiments as further controls. Before experiment, Mtb H37Ra-lux was added to the wells at a concentration of 105 CFU/ml and incubated at 37 1C for 5 days before analysis. As 2.5% DMSO had an inhibitory effect on the bacteria (Fig. S1), the highest concentration of extracts, 2.5 mg/ml was excluded from further investigation. Hits obtained in the screening procedure were re-confirmed by the same procedure in triplicates for the medium and lowest concentrations, and the isolated fractions were also tested with the same procedure in triplicates.
2.1. Mycobacterial culture 2.5. Analysis of bacterial growth by luminometry Mycobacterium tuberculosis strains H37Ra and H37Rv (American Type Culture Collection) harboring the pSMT1 plasmid (Eklund et al., 2010), which carry the gene for Vibrio harveyi Luciferase (Mtb H37Ra-lux/Mtb H37Rv-lux), were grown at 37 1C for one week before experiment in Middlebrook 7H9 broth (Becton Dickinson) supplemented with albumin dextrose catalase (ADC, Becton Dickinson), 0.05% Tween 80 and 100 mg/ml hygromycin as selection antibiotic.
To measure flash luminescence in the biosafety level (BSL) 2 facility, a Glomax Multiplus Reader (Promega) was used. The instrument is equipped with luminescence, fluorescence and absorbance functions and the presence of an injector module allowed the injection of the luciferase substrate n-decanal (Sigma-Aldrich, final concentration 1% decanal) in white, opaque 96-well plates (Eklund et al., 2010).
2.2. Plant extracts and fractionations
2.6. Determination of colony forming units (CFU)
Selected plant species were collected between January and April 2010 from different places in Sudan by botanists associated with the Medicinal and Aromatic Plant Institute in Khartoum, Sudan. Ethanolic extraction was carried out at the same institute, according to a method described by Harborne (Harborne, 1984; Rao et al., 2002). For fractionation of selected plant extracts (as shown in Table S1), a specific weight (listed in Table S2) was dissolved in 500 ml of distilled water and extracted three times by gentle shaking with 100 ml of n-hexane, each time using a separating funnel and the three samples were finally pooled. The aqueous fraction was re-extracted three times each with the following solvents: chloroform, ethyl acetate and n-butanol. Finally, the different fractions were evaporated under reduced pressure using a rotary evaporator apparatus and the yield percentage was
For confirmation of the results obtained with the luciferase assay, CFU plating of the 96-well plate cultures was done in parallel. To this end, dilutions of the cultures were streaked on 7H9 agar plates for determining CFU in the presence and absence of plant extracts. The plates were incubated for 3 weeks before evaluation of the number of colonies. All samples were analyzed in triplicates. 2.7. Separation and culture of Human monocyte-derived macrophages (hMDMs) Heparinized human whole blood was prepared from healthy donors (University Hospital in Linköping, Sweden) as described previously (Welin et al., 2011) and cultured in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 25 mM HEPES. The
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cells were allowed to adhere for 2 h at 37 1C in an incubator with 5% ambient CO2. After adhesion, non-adherent cells were washed away and monocytes were then covered with complete DMEM medium (containing 100 U/ml of penicillin and 100 mg/ml of streptomycin, 10% normal human serum (pooled from 5 donors) and 80 mM of L-glutamine and allowed to differentiate into macrophages for 5–8 days (Tanghe et al., 2001). 2.8. Calcein assay hMDMs were seeded at 100, 000 cells/well in black 96-well plates along with the extracts (at a concentration of 1 mg/ml or 0.1 mg/ml) and incubated for 5 days. After incubation for the indicated time period the cells were washed 3 times with PBS, and incubated with 4 mM calcein acetoxymethyl (calcein AM, Molecular Probes) for 30 min at room temperature. Viable cells easily take up the cell permeable non-fluorescent calcein AM and convert it to highly fluorescent calcein, which is non-permeable and thus trapped in living cells. Fluorescence thus proportional to the number of viable cells was measured in the Glomax Multiplus Reader using a 490 nm excitation filter. The assay was also performed on infected cells as indicated (Eklund et al., 2010). 2.9. Determination of IC50 value IC50 values were determined for the chloroform and ethyl acetate fractions of Khaya senegalensis bark in Mtb H37Ra-lux cultures using the luciferase assay. The test extracts were dissolved in 100% DMSO to obtain stock solutions of 100 mg/ml. Serial tenfold dilution of test solutions were prepared in 200 ml broth to give a series of 12 concentrations ranging from (10 mg/ml–1 10 7 mg/ml) in triplicate. Mycobacterial cultures were incubated with fractions at 37 1C for 5 days in 96-well plates. Luciferase substrate was added just before luminescence measurement and IC50 was calculated using linear fit log (graph pad prism version 5). 2.10. Infection of human macrophages with Mycobacterium tuberculosis One day before infection, hMDMs were re-seeded into 96-well plates in DMEM supplemented with 10% normal human serum and 80 mM of L-glutamine for infection with the virulent strain Mtb H37Rv-lux. In each well, 1 105 cells were seeded. On the day of infection, 10 ml of log-phase bacilli growing in broth were transferred to 10 ml tubes. The bacteria were centrifuged twice at 5000g for 5 min in PBS and 0.05% Tween 80. The supernatant was discarded and the pellet was re-suspended in 1 ml of DMEM with 25 mM HEPES and the suspension was passed 10 times through a 27-guage needle. The concentration of bacilli was determined by using optical density (OD600) as a function of CFU/ml and microscopy confirmed the efficient separation. The bacteria were added to hMDMs at a multiplicity of infection (MOI) of 10 in DMEM with 25 nM HEPES. The cells were incubated with the bacteria at 37 1C for 1 h. The medium was replaced with either DMEM supplemented with 10% normal human serum and 80 mM of L-glutamine or 0.1 mg/ml of fractions and incubated at 37 1C for 5 days (Tanghe et al., 2001; Eklund et al., 2010). 2.11. Cytotoxicity assay using cell lines An in vitro cytotoxicity assay was performed using the MDBK cell-line (American Type Culture Collection), and CC-1, a rat Wistar hepatocyte cell-line (European Collection of Cell Cultures). The CC-1 cells were grown in Minimum Essential Medium Eagle (MEM), supplemented with 10% FBS, 2 mM glutamine, 1% nonessential amino acids and, 20 mM HEPES, while the MDBK cells were cultured in RPMI-1640 complete media with 5% FBS. After
growth to 80% confluency, 100 ml of 6 104 cells/ml were added to 96 well-plates and incubated for 24 h at 37 1C and 5% CO2 environment. After the incubation and removal of media, cells were challenged with different concentrations (6.25, 12.5, 25, 50, 100 and 200 μg/ml) of A29 in DMSO (as vehicle) or DMSO alone (0.0625, 0.125, 0.25, 0.5, 1.0 and 2%). Then cells were incubated for 48 h at 37 1C in a CO2 incubator. Following exposure to the tested fractions, cell viability was assessed by using 0.5 mg/ml of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) for 4 h, followed by removal of supernatant and addition of DMSO to solubilize the formazan complex. Plates were read at 540 nm after one minute shaking (Mesaik et al., 2006). 2.12. Statistical analysis A one-way Anova (graph pad prism version 5) was performed to test whether there were any significant differences between means of the controls and tested extract fractions for various luminescence result. The differences were considered to be significant at levels of pr 0.05. 2.13. Ethics statement PBMCs were obtained from healthy human volunteers who gave written consent for research use of the donated blood in accordance with the Declaration of Helsinki. Since blood donation is classified as negligible risk to the donors and since only anonymized samples were delivered to the researchers, the research did not require ethical approval according to paragraph 4 of the Swedish law (2003:460) on Ethical Conduct in Human Research.
3. Results 3.1. Antimycobacterial screening of plant extracts against the avirulent strain Mtb H37Ra On the basis of ethnobotanical information, 46 plants traditionally used to treat infectious disease in Sudan were systematically collected and 50 ethanolic extracts were prepared from different parts (Table S1), evaporated and re-dissolved in DMSO. In order to screen the extract collection for possible antimycobacterial activity, broth cultures of H37Ra-lux were incubated for 5 days with two different concentrations of each extract, 125 mg/ml and 6.25 mg/ml before addition of luciferase substrate and measurement of luminescence as a correlate of bacterial growth. 12.5 mg/ml isoniazid (INH) was used as a positive control for growth inhibition and 0.125% and 0.00625% DMSO were used as negative solvent controls. With the lower concentration, three hits (defined as o20% of the growth observed in untreated controls) were identified as leaves of Rosmarinus officinalis L. (A22) and leaves (A28) and bark (A29) of Khaya senegalensis. The result of the screening is shown in Fig. S1 (B and C).We decided to prepare fractions of these three extracts in order to determine the fraction harboring the observed activity. To this end, the ethanolic extract was dissolved in water and subjected to sequential extraction using n-hexane, chloroform, ethyl acetate and n-butanol. The aqueous fraction and the solvent fractions were evaporated and re-dissolved in DMSO. The antimycobacterial activity of the fractions was assessed in the same way as the extracts, and the ethanolic extracts were used for reference (n¼5, Fig. 1). Five of the tested fractions were found to significantly inhibit mycobacterial growth at the concentration of 6.25 mg/ml. These include the n-hexane and chloroform fractions of Rosmarinus officinalis (A22) and the chloroform, ethyl acetate and n-butanol fractions of Khaya senegalensis bark (A29). Fractions of Khaya senegalensis leaves (A28) did not inhibit mycobacterial growth at
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the cut-off level. The results were confirmed with CFU plating to exclude possible artifacts resulting from the use of the luciferasebased inhibition assay.
150
Bacterial grow th in %
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100
3.2. Evaluation of cytotoxicity of fractions of plant extracts 50 *** ***
***
***
***
***
D
M SO IN A H 22 E A TO 22 H A n-H 22 X C H A Cl 22 3 A EA 22 B A UT 22 A H2 28 O E A TO 28 H A n-H 28 X C H A Cl 28 3 A EA 28 B A UT 28 A H2 29 O E A TO 29 H A n-H 29 X C H A Cl 29 3 A EA 29 B A UT 29 H 2O
0
Fig. 1. The antimycobacterial activity of Khaya senegalensis (A29) and Rosmarinus officinalis (A22) fractions (ETOH¼ ethanol, n-HX¼ n-hexane, CHCl3 ¼ Chloroform, EA¼ ethyl acetate, BUT¼butanol, H2O¼ water) were tested with an avirulent strain of Mtb H37Ra-lux (n¼ 5). The concentration of fractions used was 6.25 mg/ml while the controls used were isoniazide (INH, 12.5 mg/ml) and dimethylsulfoxide (DMSO, 0.00625%). Significant inhibition was obtained with the n-hexane and chloroform fractions of Rosmarinus officinalis leaves (A22), and the chloroform, ethyl acetate and n-butanol fractions Khaya senegalensis bark (A29). Statistical analysis using One-way Anova rendered ***pr0.001. Each bar and error bar represents mean7SEM.
The above initial screening with fractions of plant extracts showed that five fractions of two plant species with potential antimycobacterial activity. In order to rule out cytotoxicity, the viability of macrophages incubated with the fractions was assessed using a calcein-based assay. Due to limited materials and the high likelihood that the same compounds were localized to the hydrophobic solvents (chloroform vs. n-hexane/butanol), three fractions were selected (chloroform fractions of A22 and A29 and the ethyl acetate fraction of A29) and cell viability was analyzed after 3 days of treatment with the fractions at concentrations of 1 mg/ml and 0.1 mg/ml. All fractions were cytotoxic at the concentration of 1 mg/ml but the chloroform fraction of Rosmarinus officinalis leaves (A22) showed cytotoxic effect even at 0.1 mg/ml (Fig. 2). Cytotoxicity of the chloroform fraction of Khaya senegalensis bark (A29) was also tested on the MDBK and CC-1 cell line, which revealed no cytotoxicity at a concentration of 6.25 mg/ml (Fig. S2).
ns
3.3. Determination of IC50 values
***
Viability/Fluroscence arbitary units
***
Since the Rosmarinus officinalis fraction (A22) was cytotoxic also at 0.1 mg/ml, further experiments were performed only with the Khaya senegalensis fractions (A29). In order to determine the IC50 values, broth cultures of luciferase-expressing H37Ra were incubated for 5 days with 10 different concentrations of each extract before addition of luciferase substrate and measurement of luminescence (Fig. 3). The IC50 value for the chloroform fraction of Khaya senegalensis bark was 0.53 mg/ml (CI95 0.041–6.8 mg/ml) and for the ethyl acetate fraction 0.42 mg/ml (CI95 0.034–5.2 mg/ml).
***
80000
60000
40000
20000
3.4. Evaluation of intracellular killing of virulent strain H37 Rv 0 Cells
DMSO
A22 CHCl3 A29 CHCl3
A29 EA
ns *** Viability/Fluroscence arbitary units
ns ns
80000
To investigate whether the chloroform and ethyl acetate fractions of Khaya senegalensis bark (A29) were active against intracellular Mtb, hMDMs were infected with the virulent strain H37Rv-lux and treated with 0.1 mg/ml of the fractions for 5 days. Intracellular bacterial growth was assessed in the infected cells. As shown in Fig. 4, the chloroform extract of Khaya senegalensis bark did not significantly reduce the intracellular mycobacterial number, while the other fraction tested showed an increase in mycobacterial growth.
60000
3.5. Evaluation of specificity of the chloroform fraction of Khaya senegalensis bark
40000
20000
0 Cells
DMSO
A22 CHCl3 A29 CHCL3
A29 EA
Fig. 2. hMDMs were treated with the chloroform (CHCl3) fraction of Rosmarinus officinalis leaves (A22) and the chloroform and ethyl acetate (EA) fractions of Khaya senegalensis bark (A29) for 3 days at concentrations of 1 mg/ml (upper panel) and 0.1 mg/ml (lower panel) and cytotoxicity was assessed using a calcein-based assay. DMSO used as 1% (upper panel) and 0.1% (lower panel), Results shown are the mean 7 SEM of three independent experiments. One-way ANOVA was performed and statistical differences are indicated as ***p r 0.001 or ns as not significant.
Given the extremely long treatment for TB, any potential antimycobacterial drug should be as specific for mycobacteria as possible. Therefore, we assessed the growth of Escherichia coli and Staphylococcus aureus (as representatives of gram-negative and gram-positive bacteria, respectively) in shaking broth cultures in the presence of 125 mg/ml and 0.0625 mg/ml of the chloroform extract of Khaya senegalensis bark. The bacterial growth after 6 h was evaluated by CFU plating. The fraction did not cause any inhibition at the used concentrations in contrast to the positive control, which was a mixture of 100 U/ml of penicillin and 100 mg/ml of streptomycin (not shown).
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Bacterial number normalized against DMSO
Fig. 3. Dose response curves for Khaya senegalensis fractions. Mtb H37Ra-lux was incubated for 5 days with different concentrations of chloroform (CHCL3) and ethyl acetate fractions (EA) before addition of luciferase substrate and measurement of luminescence. IC50 values were calculated using non-linear fit log (inhibitor) vs. response. n¼ 3.
8
** 6
4
ns 2
0 DMSO
A29CHCl3
A29EA
Fig. 4. hMDMs were infected with H37Rv-lux and incubated for 5 days with the chloroform (CHCl3) and ethyl acetate (EA) fractions of Khaya senegalensis bark (A29). Intracellular bacterial growth was assessed at day 5 (n¼ 5). A statistically significant difference using One-way Anova is denoted by **Po 0.01 whereas ns means not significant. Bar graph shows mean 7SD.
4. Discussion The systematic search for pharmacological activity in traditionally used medicinal plants has many advantages, including the relatively high likelihood of finding active substances (as compared to extensive screening strategies involving non-selective collection of large numbers of plants) and the relatively low risk of extremely toxic hits. In addition, the strategy to use whole bacterial cells for screening has proven a method rendering the most promising hits, especially when it comes to mycobacteria, which have a highly impermeable cell wall constituting a challenging obstacle for drug development (Young et al., 2008). In the first part of this study, the 50 ethanolic plant extracts were investigated with respect to their pH before being screened for activity against an avirulent Mtb strain (H37Ra) (Mitscher et al., 1972), since a low pH could interfere with mycobacterial growth (Fischer et al., 2002; Deb et al., 2009). However, all extracts had a neutral pH around 6–7, ruling out effects related to acidity. Through the screening, we identified two interesting plants, Rosmarinus officinalis and Khaya senegalensis, that were subject for fractionation and then tested for activity against H37Ra-lux. We observed a potent antimycobacterial activity of the extracts and fractions of these plants with concentrations as low as 6.25 mg/ml, which, although still most likely a mixture of many compounds, is within the range of the physiological concentration of standard TB
drugs. The crude extract of Khaya senegalensis leaves (A28) showed antimycobacterial activity, however, the activity was lost in the fractionation procedure. This was possibly due to low initial concentration in the preparation. The fractionation of these plant extracts was carried out by sequential use of solvents from high to low polarity, which results in the separation of the high-polar from less-polar constituents. The fractionation of plant extracts always lead to some advantages or disadvantages which can also be observed in our study. The fractionation done in a variety of solvent allows a better discrimination between fractions that exhibit antimycobacterial activity or cytotoxicity (Vanden Berghe and Vlietinck, 1991). The low polarity fractions such as n-hexane, chloroform and ethyl acetate could possibly cross the lipid barrier of mycobacterial cell wall and appear as the most efficient mycobacterial inhibitors from this group (Jimenez-Arellanes et al., 2003; Eloff, 1998). The fractionation of plant extracts in a large variety of solvents also ensures the extraction of a large variety of the biological active compounds (Eloff, 1998). The extraction in organic solvents have been found to give more consistent antimicrobial activity compared to water extraction. The loss of antimycobacterial activity in some of the fractions obtained from ethanolic hits in further experiments agreed with the well-established facts that fractionation of some of the extracts frequently leads to a reduction or loss of the biological activity by compound break down or loss of additives or synergistic effects between analog constituents (Paul et al., 2006). The metabolite concentration also vary in the same plant tissues as in bark, heart wood, roots and branch bases and also vary among species from tree to tree and from season to season (Gottlieb et al., 2008). Some metabolic substances like acidic polysaccharides and tannins are generally found in almost every plant parts; bark, leaf, root, wood and fruit frequently produce a broad, non-selective activity against several microorganisms. The cytotoxicity analysis showed that the Khaya senegalensis fractions were not toxic to hMDMs at 0.1 mg/ml, and therefore, this concentration was used in a subsequent experiment, in which the activity against intracellularly growing Mycobacterium tuberculosis was tested. The chloroform fraction of Khaya senegalensis bark did not display any significant activity towards intracellular bacteria. Given the fact that the fraction is still a mixture of many compounds, the effective concentration of the active compound (s) is probably very low and further purification will allow testing and better investigation of activity at higher concentrations. The Khaya senegalensis fractions were not cytotoxic to MDBK and CC-1 cells either. Of the tested bacterial species, only Mycobacterium tuberculosis was sensitive to the Khaya senegalensis fractions, which
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is also promising, since broad-spectrum antibiotics are not suitable as TB therapy. Although not conclusive, further bioassay-guided fractionation need to be carried out to yield a potential candidate. Finding out the active lead will enable the addition of new compounds in the drug development pipeline. Acknowledgments This study was supported by the Ekhaga Foundation (# 2011-33) and N. Abuzeid by a scholarship from the Swedish Insitute. We express our gratitude to Dr. Wai´l S Abdalla and Yahia for identification of plants. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.jep.2014.09.020. References East African-British Medical Research Councils, 1972. Controlled clinical trial of short-course (6-month) regimens of chemotherapy for treatment of pulmonary tuberculosis. Lancet 1, 1079–1085. Deb, C., Lee, C.M., Dubey, V.S., Daniel, J., Abomoelak, B., Sirakova, T.D., Pawar, S., Rogers, L., Kolattukudy, P.E., 2009. A novel in vitro multiple-stress dormancy model for Mycobacterium tuberculosis generates a lipid-loaded, drug-tolerant, dormant pathogen. PLoS One 4, e6077. Eklund, D., Welin, A., Schon, T., Stendahl, O., Huygen, K., Lerm, M., 2010. Validation of a medium-throughput method for evaluation of intracellular growth of Mycobacterium tuberculosis. Clinical and Vaccine Immunology 17, 513–517. 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. Field, S.K., Fisher, D., Jarand, J.M., Cowie, R.L., 2012. New treatment options for multidrug-resistant tuberculosis. Therapeutic Advances in Respiratory Disease 6, 255–268. Fischer, M.A., Plikaytis, B.B., Shinnick, T.M., 2002. Microarray analysis of the Mycobacterium tuberculosis transcriptional response to the acidic conditions found in phagosomes. Journal of Bacteriology 184, 4025–4032. Frieden, T.R., Sterling, T., Pablos-Mendez, A., Kilburn, J.O., Cauthen, G.M., Dooley, S.W., 1993. The emergence of drug-resistant tuberculosis in New York City. The New England Journal of Medicine 328, 521–526. Gottlieb, T., Collignon, P.J., Robson, J.M., Pearson, J.C., Bell, J.M., 2008. Prevalence of antimicrobial resistances in Streptococcus pneumoniae in Australia, 2005:
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report from the Australian group on antimicrobial resistance. Communicable Diseases Intelligence Quarterly Report 32, 242–249. Harborne, J.B., 1984. In: Hall, C.A (Ed.), Phytochemical Methods a Guide to Modern Techniques of Plant Analysis, 22nd ed., Chapman and Hall, London New York. Jain, A., Dixit, P., 2008. Multidrug resistance to extensively drug resistance tubercluosis: what is next? Journal of Biosciences 33, 605–616. Jimenez-Arellanes, A., Meckes, M., Ramirez, R., Torres, J., Luna-Herrera, J., 2003. Activity against multidrug-resistant Mycobacterium tuberculosis in Mexican plants used to treat respiratory diseases. Phytotherapy Research 17, 903–908. Keyser, P., Elofsson, M., Rosell, S., Wolf-Watz, H., 2008. Virulence blockers as alternatives to antibiotics: type III secretion inhibitors against Gram-negative bacteria. Journal of Internal Medicine 264, 17–29. Klayman, D.L., 1985. Qinghaosu (artemisinin): an antimalarial drug from China. Science 228, 1049–1055. Koko, W.S., Mesaik, M.A., Yousaf, S., Galal, M., Choudhary, M.I., 2008. In vitro immunomodulating properties of selected Sudanese medicinal plants. The Journal of Ethnopharmacology 118, 26–34. Lam, K.K., Zheng, X., Forestieri, R., Balgi, A.D., Nodwell, M., Vollett, S., Anderson, H.J., Andersen, R.J., Av-Gay, Y., Roberge, M., 2012. Nitazoxanide stimulates autophagy and inhibits mTORC1 signaling and intracellular proliferation of Mycobacterium tuberculosis. PLoS Pathogens 8, e1002691. Mesaik, M.A., Zaheer, Ul.H., Murad, S., Ismail, Z., Abdullah, N.R., Gill, H.K., Atta, Ur.R., Yousaf, M., Siddiqui, R.A., Ahmad, A., Choudhary, M.I., 2006. Biological and molecular docking studies on coagulin-H: human IL-2 novel natural inhibitor. Molecular Immunology 43, 1855–1863. Mitscher, L.A., Leu, R.P., Bathala, M.S., Wu, W.N., Beal, J.L., 1972. Antimicrobial agents from higher plants. I. introduction, rationale, and methodology. Lloydia 35, 157–166. Paul, C., Anold, J., Dirk, V., Louis, M., 2006. Anti-infective potential of natural product: how to develop a stronger in vitro’proof-of-concept'. Ethnopharmacology 106, 290–302. Pinto, L., Menzies, D., 2011. Treatment of drug-resistant tuberculosis. Infection and Drug Resistance 4, 129–135. Rao, B.R., Kaul, P.N., Syamasundar, K.V., Ramesh, S., 2002. Water soluble fractions of rose-scented geranium (Pelargonium species) essential oil. Bioresource Technology 84, 243–246. Tanghe, A., D'souza, S., Rosseels, V., Denis, O., Ottenhoff, T.H., Dalemans, W., Wheeler, C., Huygen, K., 2001. Improved immunogenicity and protective efficacy of a tuberculosis DNA vaccine encoding Ag85 by protein boosting. Infection and Immunity 69, 3041–3047. Vanden Berghe, D.A., Vlietinck, A.J., 1991. Assays for Bioactivity (Methods in Plant Biochemistry). Academic Press, London (Journal of Ethnopharmacology). Welin, A., Raffetseder, J., Eklund, D., Stendahl, O., Lerm, M., 2011. Importance of phagosomal functionality for growth restriction of Mycobacterium tuberculosis in primary human macrophages. Journal of Innate Immunity 3, 508–518. WHO, 2010. Multidrug and extensively drug-resistant TB (M/XDR-TB) (Global Report on Surveillance and Response. WHO/HTM/TB/2010.3). World Health Organization, Geneva. Young, D.B., Perkins, M.D., Duncan, K., Barry, C.E., 2008. Confronting the scientific obstacles to global control of tuberculosis. Journal of Clinical Investigation 118, 1255–1265.
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Antimycobacterial activity of selected medicinal plants traditionally used in Sudan to treat infectious diseases Nadir Abuzeid a,b,c,1, Sadaf Kalsum c,1, Marie Larsson d, Mikaela Glader c, Henrik Andersson c, Johanna Raffetseder c, Elsje Pienaar c, Daniel Eklund c, Muddathir S. Alhassan a, Haidar A. AlGadir a, Waleed S. Koko a, Thomas Schön e, M. Ahmed Mesaik f,g, Omer M. Abdalla g, Asaad Khalid a, Maria Lerm c,n a
Medicinal and Aromatic Plants Research Institute, National Center for Research, P.O. Box: 2404, Khartoum 11111, Sudan Department of Clinical Microbiology, Faculty of Medical Laboratory Sciences, Omdurman Islamic University, P.O.Box 382, Omdurman,Sudan Division of Microbiology and Molecular Medicine, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linkoping University, 58185 Linkoping, Sweden d Department of Clinical Microbiology, Linköping University Hospital, 58185 Linkoping, Sweden e Department of Clinical Microbiology and Infectious diseases, Kalmar County Hospital, SE-391 85 Kalmar, Sweden f Faculty of Pharmacy, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, Kuala Lumpur 50300, Malaysia g Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan b c
art ic l e i nf o
a b s t r a c t
Article history: Received 19 June 2014 Received in revised form 9 September 2014 Accepted 15 September 2014 Available online 28 September 2014
Ethnopharmacological relevance: The emergence of multidrug-resistant strains of Mycobacterium tuberculosis underscores the need for continuous development of new and efficient methods to determine the susceptibility of isolates of Mycobacterium tuberculosis in the search for novel antimycobacterial agents. Natural products constitute an important source of new drugs, and design and implementation of antimycobacterial susceptibility testing methods are necessary to evaluate the different extracts and compounds. In this study we have explored the antimycobacterial properties of 50 ethanolic extracts from different parts of 46 selected medicinal plants traditionally used in Sudan to treat infectious diseases. Materials and methods: Plants were harvested and ethanolic extracts were prepared. For selected extracts, fractionation with hydrophilic and hydrophobic solvents was undertaken. A luminometrybased assay was used for determination of mycobacterial growth in broth cultures and inside primary human macrophages in the presence or absence of plant extracts and fractions of extracts. Cytotoxicity was also assessed for active fractions of plant extracts. Results: Of the tested extracts, three exhibited a significant inhibitory effect on an avirulent strain of Mycobacterium tubercluosis (H37Ra) at the initial screening doses (125 and 6.25 mg/ml). These were bark and leaf extracts of Khaya senegalensis and the leaf extract of Rosmarinus officinalis L. Further fractions of these plant extracts were prepared with n-hexane, chloroform, ethyl acetate, n-butanol, ethanol and water, and the activity of these extracts was retained in hydrophobic fractions. Cytotoxicity assays revealed that the chloroform fraction of Khaya senegalensis bark was non-toxic to human monocytederived macrophages and other cell types at the concentrations used and hence, further analysis, including assessment of IC50 and intracellular activity was done with this fraction. Conclusion: These results encourage further investigations to identify the active compound(s) within the chloroform fraction of Khaya senegalensis bark. & 2014 Published by Elsevier Ireland Ltd.
Keywords: Mycobacterium tuberculosis Sudanese medicinal plants Primary human macrophages Luminescence reporter assay Cytotoxicity assay
1. Introduction
n
Corresponding author. Tel.: þ 46 1010 32088; fax: þ 46 1010 3478. E-mail address: [email protected] (M. Lerm). 1 These authors contributed equally to the study.
http://dx.doi.org/10.1016/j.jep.2014.09.020 0378-8741/& 2014 Published by Elsevier Ireland Ltd.
Tuberculosis (TB) is a global public health threat and remains one of the major causes of death among infectious diseases. The patient incompliance associated with the long time required to reach sterilizing TB treatment ( Z6 months on 2–4 antibiotics) contributes to the emergence of multidrug- resistant TB (MDR-TB)
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and extensively drug-resistant TB (XDR-TB) (Jain and Dixit, 2008; Pinto and Menzies, 2011). Taking into consideration the low cure rates of MDR-TB and the high relative incidence, 28% of new TB cases in some areas of Eastern Europe, research promoting new antimycobacterial therapies is pivotal (WHO, 2010). In addition, the discovery of new classes of antimycobacterial drugs (Field et al., 2012) and possibly also virulence blockers (Keyser et al., 2008) and drugs stimulating immunity (Lam et al., 2012) may guide the development of a more rational and effective TB therapy that is several months shorter than the present regimen. A milestone of the development of the standard TB therapy was the implementation of short course chemotherapy in the 1970s (East African-British Medical Research Councils, 1972). The combination of four antibiotics allowed reduction of time of treatment to six months. However, this led to the conclusion that TB was finally defeated, but the emergence of MDR-TB in the United States rekindled the scientific interest in the disease (Frieden et al., 1993). Natural products can pave the way for new drug leads, since they provide an unlimited source of chemically diverse compounds. Today's first line drug to treat malaria, artemisinin, was found through the systematic search of anti-malarial compounds in herbs used in traditional Chinese medicine (Klayman, 1985). Several broad screening strategies have also been carried out with Sudanese medicinal plants for antibacterial, antifungal, antiviral, antimalarial and antihelminthic properties (Koko et al., 2008). In the present study, we screened 50 ethanolic extracts of 46 Sudanese plants with respect to antimycobacterial activity using a luminometry-based method (Eklund et al., 2010). Two hits were identified; Khaya senegalensis (bark and leaves) and Rosmarinus officinalis L. (leaves), and these extracts were subjected to fractionation with water, n-hexane, chloroform, ethanol, ethyl acetate and n-butane. The n-hexane, chloroform, ethyl acetate and nbutanol fractions were found to effectively inhibit the growth of Mycobacterium tuberculosis at a concentration of 6.25 mg/ml. The result was confirmed with other assays for mycobacterial growth.
2. Materials and methods
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calculated for each fraction (Table S2). The aqueous layer was finally freezed at 80 1C, stored at 40 1C and freeze-dried using a freeze dryer apparatus until completely dry and the yield was calculated. For experiments, 5 mg of dried plant extracts or fractions were dissolved in 50 ml of 100% DMSO to obtain stock solutions of 100 mg/ml. 2.3. pH measurement Litmus paper (MERCK) was dipped into the aliquot of plant extracts diluted to 125 and 6.25 mg/ml in broth supplemented with ADC and 0.05% Tween 80. The color obtained determined the pH of the extract as shown in Table S1. 2.4. Bacterial growth assay One ml of culture containing Mtb H37Ra-lux was centrifuged once at 5000g for 5 min at room temperature and the pellet was re-suspended in broth supplemented with ADC and 0.05% Tween 80. In order to remove bacterial aggregates, the suspension was passaged 10 times through a sterile syringe equipped with a 27gauge needle. The concentration was determined by optical density at 600 nm (OD600) as a function of colony-forming units (CFU) obtained from a standard curve derived from plated bacteria (not shown). For screening, 96-well plates were prepared with dilutions of the extracts at the final concentrations of 2.5 mg/ml, 125 mg/ml or 6.25 mg/ml. 2.5%, 0.125% and 0.00625% DMSO were used as solvent controls, respectively. A high dose of isoniazid (the first-line antibiotic, was selected to insure efficient inhibition) and broth only were included in all experiments as further controls. Before experiment, Mtb H37Ra-lux was added to the wells at a concentration of 105 CFU/ml and incubated at 37 1C for 5 days before analysis. As 2.5% DMSO had an inhibitory effect on the bacteria (Fig. S1), the highest concentration of extracts, 2.5 mg/ml was excluded from further investigation. Hits obtained in the screening procedure were re-confirmed by the same procedure in triplicates for the medium and lowest concentrations, and the isolated fractions were also tested with the same procedure in triplicates.
2.1. Mycobacterial culture 2.5. Analysis of bacterial growth by luminometry Mycobacterium tuberculosis strains H37Ra and H37Rv (American Type Culture Collection) harboring the pSMT1 plasmid (Eklund et al., 2010), which carry the gene for Vibrio harveyi Luciferase (Mtb H37Ra-lux/Mtb H37Rv-lux), were grown at 37 1C for one week before experiment in Middlebrook 7H9 broth (Becton Dickinson) supplemented with albumin dextrose catalase (ADC, Becton Dickinson), 0.05% Tween 80 and 100 mg/ml hygromycin as selection antibiotic.
To measure flash luminescence in the biosafety level (BSL) 2 facility, a Glomax Multiplus Reader (Promega) was used. The instrument is equipped with luminescence, fluorescence and absorbance functions and the presence of an injector module allowed the injection of the luciferase substrate n-decanal (Sigma-Aldrich, final concentration 1% decanal) in white, opaque 96-well plates (Eklund et al., 2010).
2.2. Plant extracts and fractionations
2.6. Determination of colony forming units (CFU)
Selected plant species were collected between January and April 2010 from different places in Sudan by botanists associated with the Medicinal and Aromatic Plant Institute in Khartoum, Sudan. Ethanolic extraction was carried out at the same institute, according to a method described by Harborne (Harborne, 1984; Rao et al., 2002). For fractionation of selected plant extracts (as shown in Table S1), a specific weight (listed in Table S2) was dissolved in 500 ml of distilled water and extracted three times by gentle shaking with 100 ml of n-hexane, each time using a separating funnel and the three samples were finally pooled. The aqueous fraction was re-extracted three times each with the following solvents: chloroform, ethyl acetate and n-butanol. Finally, the different fractions were evaporated under reduced pressure using a rotary evaporator apparatus and the yield percentage was
For confirmation of the results obtained with the luciferase assay, CFU plating of the 96-well plate cultures was done in parallel. To this end, dilutions of the cultures were streaked on 7H9 agar plates for determining CFU in the presence and absence of plant extracts. The plates were incubated for 3 weeks before evaluation of the number of colonies. All samples were analyzed in triplicates. 2.7. Separation and culture of Human monocyte-derived macrophages (hMDMs) Heparinized human whole blood was prepared from healthy donors (University Hospital in Linköping, Sweden) as described previously (Welin et al., 2011) and cultured in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 25 mM HEPES. The
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cells were allowed to adhere for 2 h at 37 1C in an incubator with 5% ambient CO2. After adhesion, non-adherent cells were washed away and monocytes were then covered with complete DMEM medium (containing 100 U/ml of penicillin and 100 mg/ml of streptomycin, 10% normal human serum (pooled from 5 donors) and 80 mM of L-glutamine and allowed to differentiate into macrophages for 5–8 days (Tanghe et al., 2001). 2.8. Calcein assay hMDMs were seeded at 100, 000 cells/well in black 96-well plates along with the extracts (at a concentration of 1 mg/ml or 0.1 mg/ml) and incubated for 5 days. After incubation for the indicated time period the cells were washed 3 times with PBS, and incubated with 4 mM calcein acetoxymethyl (calcein AM, Molecular Probes) for 30 min at room temperature. Viable cells easily take up the cell permeable non-fluorescent calcein AM and convert it to highly fluorescent calcein, which is non-permeable and thus trapped in living cells. Fluorescence thus proportional to the number of viable cells was measured in the Glomax Multiplus Reader using a 490 nm excitation filter. The assay was also performed on infected cells as indicated (Eklund et al., 2010). 2.9. Determination of IC50 value IC50 values were determined for the chloroform and ethyl acetate fractions of Khaya senegalensis bark in Mtb H37Ra-lux cultures using the luciferase assay. The test extracts were dissolved in 100% DMSO to obtain stock solutions of 100 mg/ml. Serial tenfold dilution of test solutions were prepared in 200 ml broth to give a series of 12 concentrations ranging from (10 mg/ml–1 10 7 mg/ml) in triplicate. Mycobacterial cultures were incubated with fractions at 37 1C for 5 days in 96-well plates. Luciferase substrate was added just before luminescence measurement and IC50 was calculated using linear fit log (graph pad prism version 5). 2.10. Infection of human macrophages with Mycobacterium tuberculosis One day before infection, hMDMs were re-seeded into 96-well plates in DMEM supplemented with 10% normal human serum and 80 mM of L-glutamine for infection with the virulent strain Mtb H37Rv-lux. In each well, 1 105 cells were seeded. On the day of infection, 10 ml of log-phase bacilli growing in broth were transferred to 10 ml tubes. The bacteria were centrifuged twice at 5000g for 5 min in PBS and 0.05% Tween 80. The supernatant was discarded and the pellet was re-suspended in 1 ml of DMEM with 25 mM HEPES and the suspension was passed 10 times through a 27-guage needle. The concentration of bacilli was determined by using optical density (OD600) as a function of CFU/ml and microscopy confirmed the efficient separation. The bacteria were added to hMDMs at a multiplicity of infection (MOI) of 10 in DMEM with 25 nM HEPES. The cells were incubated with the bacteria at 37 1C for 1 h. The medium was replaced with either DMEM supplemented with 10% normal human serum and 80 mM of L-glutamine or 0.1 mg/ml of fractions and incubated at 37 1C for 5 days (Tanghe et al., 2001; Eklund et al., 2010). 2.11. Cytotoxicity assay using cell lines An in vitro cytotoxicity assay was performed using the MDBK cell-line (American Type Culture Collection), and CC-1, a rat Wistar hepatocyte cell-line (European Collection of Cell Cultures). The CC-1 cells were grown in Minimum Essential Medium Eagle (MEM), supplemented with 10% FBS, 2 mM glutamine, 1% nonessential amino acids and, 20 mM HEPES, while the MDBK cells were cultured in RPMI-1640 complete media with 5% FBS. After
growth to 80% confluency, 100 ml of 6 104 cells/ml were added to 96 well-plates and incubated for 24 h at 37 1C and 5% CO2 environment. After the incubation and removal of media, cells were challenged with different concentrations (6.25, 12.5, 25, 50, 100 and 200 μg/ml) of A29 in DMSO (as vehicle) or DMSO alone (0.0625, 0.125, 0.25, 0.5, 1.0 and 2%). Then cells were incubated for 48 h at 37 1C in a CO2 incubator. Following exposure to the tested fractions, cell viability was assessed by using 0.5 mg/ml of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) for 4 h, followed by removal of supernatant and addition of DMSO to solubilize the formazan complex. Plates were read at 540 nm after one minute shaking (Mesaik et al., 2006). 2.12. Statistical analysis A one-way Anova (graph pad prism version 5) was performed to test whether there were any significant differences between means of the controls and tested extract fractions for various luminescence result. The differences were considered to be significant at levels of pr 0.05. 2.13. Ethics statement PBMCs were obtained from healthy human volunteers who gave written consent for research use of the donated blood in accordance with the Declaration of Helsinki. Since blood donation is classified as negligible risk to the donors and since only anonymized samples were delivered to the researchers, the research did not require ethical approval according to paragraph 4 of the Swedish law (2003:460) on Ethical Conduct in Human Research.
3. Results 3.1. Antimycobacterial screening of plant extracts against the avirulent strain Mtb H37Ra On the basis of ethnobotanical information, 46 plants traditionally used to treat infectious disease in Sudan were systematically collected and 50 ethanolic extracts were prepared from different parts (Table S1), evaporated and re-dissolved in DMSO. In order to screen the extract collection for possible antimycobacterial activity, broth cultures of H37Ra-lux were incubated for 5 days with two different concentrations of each extract, 125 mg/ml and 6.25 mg/ml before addition of luciferase substrate and measurement of luminescence as a correlate of bacterial growth. 12.5 mg/ml isoniazid (INH) was used as a positive control for growth inhibition and 0.125% and 0.00625% DMSO were used as negative solvent controls. With the lower concentration, three hits (defined as o20% of the growth observed in untreated controls) were identified as leaves of Rosmarinus officinalis L. (A22) and leaves (A28) and bark (A29) of Khaya senegalensis. The result of the screening is shown in Fig. S1 (B and C).We decided to prepare fractions of these three extracts in order to determine the fraction harboring the observed activity. To this end, the ethanolic extract was dissolved in water and subjected to sequential extraction using n-hexane, chloroform, ethyl acetate and n-butanol. The aqueous fraction and the solvent fractions were evaporated and re-dissolved in DMSO. The antimycobacterial activity of the fractions was assessed in the same way as the extracts, and the ethanolic extracts were used for reference (n¼5, Fig. 1). Five of the tested fractions were found to significantly inhibit mycobacterial growth at the concentration of 6.25 mg/ml. These include the n-hexane and chloroform fractions of Rosmarinus officinalis (A22) and the chloroform, ethyl acetate and n-butanol fractions of Khaya senegalensis bark (A29). Fractions of Khaya senegalensis leaves (A28) did not inhibit mycobacterial growth at
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the cut-off level. The results were confirmed with CFU plating to exclude possible artifacts resulting from the use of the luciferasebased inhibition assay.
150
Bacterial grow th in %
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100
3.2. Evaluation of cytotoxicity of fractions of plant extracts 50 *** ***
***
***
***
***
D
M SO IN A H 22 E A TO 22 H A n-H 22 X C H A Cl 22 3 A EA 22 B A UT 22 A H2 28 O E A TO 28 H A n-H 28 X C H A Cl 28 3 A EA 28 B A UT 28 A H2 29 O E A TO 29 H A n-H 29 X C H A Cl 29 3 A EA 29 B A UT 29 H 2O
0
Fig. 1. The antimycobacterial activity of Khaya senegalensis (A29) and Rosmarinus officinalis (A22) fractions (ETOH¼ ethanol, n-HX¼ n-hexane, CHCl3 ¼ Chloroform, EA¼ ethyl acetate, BUT¼butanol, H2O¼ water) were tested with an avirulent strain of Mtb H37Ra-lux (n¼ 5). The concentration of fractions used was 6.25 mg/ml while the controls used were isoniazide (INH, 12.5 mg/ml) and dimethylsulfoxide (DMSO, 0.00625%). Significant inhibition was obtained with the n-hexane and chloroform fractions of Rosmarinus officinalis leaves (A22), and the chloroform, ethyl acetate and n-butanol fractions Khaya senegalensis bark (A29). Statistical analysis using One-way Anova rendered ***pr0.001. Each bar and error bar represents mean7SEM.
The above initial screening with fractions of plant extracts showed that five fractions of two plant species with potential antimycobacterial activity. In order to rule out cytotoxicity, the viability of macrophages incubated with the fractions was assessed using a calcein-based assay. Due to limited materials and the high likelihood that the same compounds were localized to the hydrophobic solvents (chloroform vs. n-hexane/butanol), three fractions were selected (chloroform fractions of A22 and A29 and the ethyl acetate fraction of A29) and cell viability was analyzed after 3 days of treatment with the fractions at concentrations of 1 mg/ml and 0.1 mg/ml. All fractions were cytotoxic at the concentration of 1 mg/ml but the chloroform fraction of Rosmarinus officinalis leaves (A22) showed cytotoxic effect even at 0.1 mg/ml (Fig. 2). Cytotoxicity of the chloroform fraction of Khaya senegalensis bark (A29) was also tested on the MDBK and CC-1 cell line, which revealed no cytotoxicity at a concentration of 6.25 mg/ml (Fig. S2).
ns
3.3. Determination of IC50 values
***
Viability/Fluroscence arbitary units
***
Since the Rosmarinus officinalis fraction (A22) was cytotoxic also at 0.1 mg/ml, further experiments were performed only with the Khaya senegalensis fractions (A29). In order to determine the IC50 values, broth cultures of luciferase-expressing H37Ra were incubated for 5 days with 10 different concentrations of each extract before addition of luciferase substrate and measurement of luminescence (Fig. 3). The IC50 value for the chloroform fraction of Khaya senegalensis bark was 0.53 mg/ml (CI95 0.041–6.8 mg/ml) and for the ethyl acetate fraction 0.42 mg/ml (CI95 0.034–5.2 mg/ml).
***
80000
60000
40000
20000
3.4. Evaluation of intracellular killing of virulent strain H37 Rv 0 Cells
DMSO
A22 CHCl3 A29 CHCl3
A29 EA
ns *** Viability/Fluroscence arbitary units
ns ns
80000
To investigate whether the chloroform and ethyl acetate fractions of Khaya senegalensis bark (A29) were active against intracellular Mtb, hMDMs were infected with the virulent strain H37Rv-lux and treated with 0.1 mg/ml of the fractions for 5 days. Intracellular bacterial growth was assessed in the infected cells. As shown in Fig. 4, the chloroform extract of Khaya senegalensis bark did not significantly reduce the intracellular mycobacterial number, while the other fraction tested showed an increase in mycobacterial growth.
60000
3.5. Evaluation of specificity of the chloroform fraction of Khaya senegalensis bark
40000
20000
0 Cells
DMSO
A22 CHCl3 A29 CHCL3
A29 EA
Fig. 2. hMDMs were treated with the chloroform (CHCl3) fraction of Rosmarinus officinalis leaves (A22) and the chloroform and ethyl acetate (EA) fractions of Khaya senegalensis bark (A29) for 3 days at concentrations of 1 mg/ml (upper panel) and 0.1 mg/ml (lower panel) and cytotoxicity was assessed using a calcein-based assay. DMSO used as 1% (upper panel) and 0.1% (lower panel), Results shown are the mean 7 SEM of three independent experiments. One-way ANOVA was performed and statistical differences are indicated as ***p r 0.001 or ns as not significant.
Given the extremely long treatment for TB, any potential antimycobacterial drug should be as specific for mycobacteria as possible. Therefore, we assessed the growth of Escherichia coli and Staphylococcus aureus (as representatives of gram-negative and gram-positive bacteria, respectively) in shaking broth cultures in the presence of 125 mg/ml and 0.0625 mg/ml of the chloroform extract of Khaya senegalensis bark. The bacterial growth after 6 h was evaluated by CFU plating. The fraction did not cause any inhibition at the used concentrations in contrast to the positive control, which was a mixture of 100 U/ml of penicillin and 100 mg/ml of streptomycin (not shown).
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Bacterial number normalized against DMSO
Fig. 3. Dose response curves for Khaya senegalensis fractions. Mtb H37Ra-lux was incubated for 5 days with different concentrations of chloroform (CHCL3) and ethyl acetate fractions (EA) before addition of luciferase substrate and measurement of luminescence. IC50 values were calculated using non-linear fit log (inhibitor) vs. response. n¼ 3.
8
** 6
4
ns 2
0 DMSO
A29CHCl3
A29EA
Fig. 4. hMDMs were infected with H37Rv-lux and incubated for 5 days with the chloroform (CHCl3) and ethyl acetate (EA) fractions of Khaya senegalensis bark (A29). Intracellular bacterial growth was assessed at day 5 (n¼ 5). A statistically significant difference using One-way Anova is denoted by **Po 0.01 whereas ns means not significant. Bar graph shows mean 7SD.
4. Discussion The systematic search for pharmacological activity in traditionally used medicinal plants has many advantages, including the relatively high likelihood of finding active substances (as compared to extensive screening strategies involving non-selective collection of large numbers of plants) and the relatively low risk of extremely toxic hits. In addition, the strategy to use whole bacterial cells for screening has proven a method rendering the most promising hits, especially when it comes to mycobacteria, which have a highly impermeable cell wall constituting a challenging obstacle for drug development (Young et al., 2008). In the first part of this study, the 50 ethanolic plant extracts were investigated with respect to their pH before being screened for activity against an avirulent Mtb strain (H37Ra) (Mitscher et al., 1972), since a low pH could interfere with mycobacterial growth (Fischer et al., 2002; Deb et al., 2009). However, all extracts had a neutral pH around 6–7, ruling out effects related to acidity. Through the screening, we identified two interesting plants, Rosmarinus officinalis and Khaya senegalensis, that were subject for fractionation and then tested for activity against H37Ra-lux. We observed a potent antimycobacterial activity of the extracts and fractions of these plants with concentrations as low as 6.25 mg/ml, which, although still most likely a mixture of many compounds, is within the range of the physiological concentration of standard TB
drugs. The crude extract of Khaya senegalensis leaves (A28) showed antimycobacterial activity, however, the activity was lost in the fractionation procedure. This was possibly due to low initial concentration in the preparation. The fractionation of these plant extracts was carried out by sequential use of solvents from high to low polarity, which results in the separation of the high-polar from less-polar constituents. The fractionation of plant extracts always lead to some advantages or disadvantages which can also be observed in our study. The fractionation done in a variety of solvent allows a better discrimination between fractions that exhibit antimycobacterial activity or cytotoxicity (Vanden Berghe and Vlietinck, 1991). The low polarity fractions such as n-hexane, chloroform and ethyl acetate could possibly cross the lipid barrier of mycobacterial cell wall and appear as the most efficient mycobacterial inhibitors from this group (Jimenez-Arellanes et al., 2003; Eloff, 1998). The fractionation of plant extracts in a large variety of solvents also ensures the extraction of a large variety of the biological active compounds (Eloff, 1998). The extraction in organic solvents have been found to give more consistent antimicrobial activity compared to water extraction. The loss of antimycobacterial activity in some of the fractions obtained from ethanolic hits in further experiments agreed with the well-established facts that fractionation of some of the extracts frequently leads to a reduction or loss of the biological activity by compound break down or loss of additives or synergistic effects between analog constituents (Paul et al., 2006). The metabolite concentration also vary in the same plant tissues as in bark, heart wood, roots and branch bases and also vary among species from tree to tree and from season to season (Gottlieb et al., 2008). Some metabolic substances like acidic polysaccharides and tannins are generally found in almost every plant parts; bark, leaf, root, wood and fruit frequently produce a broad, non-selective activity against several microorganisms. The cytotoxicity analysis showed that the Khaya senegalensis fractions were not toxic to hMDMs at 0.1 mg/ml, and therefore, this concentration was used in a subsequent experiment, in which the activity against intracellularly growing Mycobacterium tuberculosis was tested. The chloroform fraction of Khaya senegalensis bark did not display any significant activity towards intracellular bacteria. Given the fact that the fraction is still a mixture of many compounds, the effective concentration of the active compound (s) is probably very low and further purification will allow testing and better investigation of activity at higher concentrations. The Khaya senegalensis fractions were not cytotoxic to MDBK and CC-1 cells either. Of the tested bacterial species, only Mycobacterium tuberculosis was sensitive to the Khaya senegalensis fractions, which
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