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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 10  |  Issue : 1  |  Page : 60-65

In vitro Anti-Mycobacterium ulcerans and cytotoxic activities of some selected medicinal plants and an indoloquinoline alkaloid


1 Department of Pharmacognosy, Faculty of Pharmacy and Pharmaceutical Sciences, KNUST, Kumasi, Ghana
2 Department of Biochemistry, Faculty of Science, University of Bamenda, Bamenda, Cameroon
3 Department of Bacteriology, Noguchi Memorial Institute for Medical Research, College of Health Sciences, University of Ghana, Accra, Ghana
4 Department of Clinical Pathology, Noguchi Memorial Institute for Medical Research, College of Health Sciences, University of Ghana, Accra, Ghana

Date of Submission22-Dec-2020
Date of Acceptance13-Feb-2021
Date of Web Publication28-Feb-2021

Correspondence Address:
Patrick Valere Tsouh Fokou
University of Bamenda, 39 Bambili, Bamenda
Cameroon
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmy.ijmy_243_20

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  Abstract 


Background: Buruli ulcer (BU) is a neglected tropical disease caused by the Mycobacterium ulcerans. BU is an endemic disease in many communities in sub-Saharan Africa where population have long history of using medicinal plants for treatment. Indeed, several medicinal plants have been documented against BU and related conditions. The present study was undertaken to prove the efficacy of seven medicinal plants documented for the treatment of mycobacterial infections and related symptoms in Ghana. Method: Antimycobacterial activity of the stem bark extracts and reference control drugs were conducted using the resazurin microtiter assay (REMA) assay method in clear round bottom 96-well microtiter plates. The extracts that showed anti-mycobacterium ulcerans activity were assessed for cytotoxicity using the Alamar blue assay. Results: Overall, The Cryptolepis sanguinolenta root aqueous extract exhibited the highest antimycobacterial activity (MIC=64 μg/mL) followed by Cleistopholis patens (MIC=256 μg/mL). Based on the marked activity of the Cryptolepis sanguinolenta extracts, pure cryptolepine, its major metabolite recorded a MIC value of 32 μg/mL. These extracts with considerable antimycobacterial activity showed 50% cytotoxic concentration (CC50) ranging from 94 to 384 μg/mL. Conclusions: Thus, Cleistopholis patens and Cryptolepis sanguinolenta are primed for further studies and could afford novel drugs for the mitigation of buruli ulcer disease.

Keywords: Antimycobacterial activity, Cleistopholis patens, cryptolepine, Cryptolepis sanguinolenta, cytotoxicity, Mycobacterium ulcerans


How to cite this article:
Amponsah IK, Atchoglo PK, Ackah RY, Tsouh Fokou PV, Aboagye SY, Yeboah-Manu D, Appiah-Opong R, Mensah AY. In vitro Anti-Mycobacterium ulcerans and cytotoxic activities of some selected medicinal plants and an indoloquinoline alkaloid. Int J Mycobacteriol 2021;10:60-5

How to cite this URL:
Amponsah IK, Atchoglo PK, Ackah RY, Tsouh Fokou PV, Aboagye SY, Yeboah-Manu D, Appiah-Opong R, Mensah AY. In vitro Anti-Mycobacterium ulcerans and cytotoxic activities of some selected medicinal plants and an indoloquinoline alkaloid. Int J Mycobacteriol [serial online] 2021 [cited 2021 Apr 22];10:60-5. Available from: https://www.ijmyco.org/text.asp?2021/10/1/60/310514




  Introduction Top


Buruli ulcer (BU) is a neglected tropical disease caused by the M. ulcerans organism. It is a necrotizing infection of the skin and subcutaneous tissue[1],[2] that owes its virulence to a mycolactone that has cytotoxic and immunosuppressive properties.[3]

Large ulcers, resulting from the disease, often lead to scarring, contractual deformities, amputations, and irreversible disabilities especially in areas where treatment options are limited [Figure 1].
Figure 1: Clinical manifestation of Buruli ulcer

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The disease has been reported from over 33 countries from Africa, Asia, Latin America, and South Pacific with majority of the cases from the West African subregion.[4],[5] The magnitude of the disease burden remains largely unknown due to underreporting resulting from insufficient knowledge of the disease, especially among rural communities, ineffective functional health care, and monitoring systems.[6],[7] Côte d'Ivoire, Ghana, and Benin are the first three most endemic countries in the world.

Ghana, the second most endemic country, has cases reported from the Ashanti and Greater Accra regions with the former accounting for majority of the cases. BU, with a prevalence of 66 per 100,000, is the second most prevalent mycobacterial disease in Ghana after tuberculosis. Until 2004, surgical removal of the infected tissues from ulcers followed by skin grafting was the only approach for the management and treatment of the BU disease.[8] This treatment approach was really invasive and required long-term hospitalization and was expensive. This gave way to the WHO-approved antibiotic therapy regime of rifampicin (RIF) and streptomycin (STR) for 8 weeks (SR8) which was able to annihilate the M. ulcerans organism, arrested the skin ulcer progression, and promoted healing of the affected areas.[4] However, socioeconomic factors, beliefs, and practices have made patients more inclined to other treatment sources including the use of plant-based medicines.[9],[10]

Endemic communities in Ghana have been associated with a long history of medicinal plant parts used in BU and other related conditions. Several medicinal plants have been documented in literature, following an ethnobotanical study both in Ghana and abroad in this regard.[1],[11] Therefore, in continuation of research efforts by some scientists to scientifically validate the efficacy of these herbs used for the disease treatment,[1],[12] the present study was undertaken to prove the efficacy of seven medicinal plants documented for the treatment of mycobacterial infections and related symptoms[1],[13],[14] in Ghana. Their cytotoxic activities were also evaluated.


  Method Top


Plant collection and processing

Seven plants used in Ghana for treating and managing BU were selected solely based on their documented use for mycobacterial infections and related symptoms.[1],[13],[14] The plants were harvested between September and December 2016 and identified with the help of Mr Clifford Asare, an ethnobotanist, at the Herbal Medicine Department, KNUST, Kumasi, Ghana, where voucher specimens have been deposited at their herbarium. The fresh plant materials were cleaned and washed thoroughly with water. Stem barks and roots were diced and leaf samples air-dried under shade for 2–4 weeks and then pulverized with hammer mill. It was stored in an air-tight plastic bag for future use.

Nine medicinal plants belonging to Apocynaceae, Melastomataceae, Solanaceae, Bignoniaceae, Euphorbiaceae, and Annonaceae families were harvested for this study as suggested by their folkloric uses. Five of these had their stem barks harvested as the plant part of interest with three leaves, one root, and a whole plant completing the number of selected plants. Herbarium specimen numbers of the plants for this study were obtained from the Department of Herbal Medicine Department, KNUST, Kumasi, Ghana.

[Table 1] shows the profile of plants used for the antimycobacterial and cytotoxicity assays.
Table 1: Profile of plants used for the antimycobacterial and cytotoxicity assays

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Reagents and chemicals

All the chemicals, reagents, and consumables were of analytical grade. Milli-Q and distilled water was obtained from NMIMR, Legon, Ghana, ethanol: Mes laboratory reagents, Ghana, RIF and STR: Merck, Darmstadt, Germany, penicillin: Becton, Dickinson and Company, Sparks, USA, L-glutamine: Oxoid Ltd, Basingstoke, Hampshire, England, penicillin-STR: Sigma-Aldrich, Illinois, USA, 3 mm sterile glass beads: Sigma-Aldrich Steinheim, Germany, dimethyl sulfoxide (DMSO): Sigma-Aldrich, Steinheim, Germany, RPMI-1640: Merck, Darmstadt, Germany, fetal bovine serum: FBS-Merck, Darmstadt, Germany, trypsin-ethylenediaminetetraacetic acid: Thermo Fisher, Scientific, Massachusetts, USA, 96-well tissue culture microplates: Thermo Fisher, Scientific, Massachusetts, USA, phosphate-buffered saline: PBS-Sigma Chemicals, 7H9 GCOT: Difco, Sparks, MD, USA, and resazurin sodium salt: Thermo Fisher, Scientific, Massachusetts, USA. Chang liver cell line was obtained from the Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, and Nagasaki International University (Nagasaki, Japan). Pure cryptolepine was obtained from the Department of Pharmacology, KNUST.

Preparation of crude extract

Weighed samples were subjected to repeated hot extraction using a Soxhlet. Samples subjected to cold maceration were constantly shaken on a mechanical shaker for 72 h. Filtrates were concentrated using a rotary evaporator to remove solvents at reduced temperature and pressure. The ethyl acetate extracts were dried over a water bath, and aqueous and hydro-methanol (7:3) extracts were lyophilized.

In vitro antimycobacterial assays

The assay was performed by adapting the standard operating procedures from the Department of Bacteriology, Noguchi Memorial Institute for Medical Research (NMIMR). The assay was performed by adapting the standard operating procedures from the Department of Bacteriology, NMIMR and methods described by[1],[12] with slight modifications.

Culturing of mycobacterial strain and Preparation of inoculum

Stored standard clinical M. ulcerans isolate (NM 203 strain) was stored and maintained in the Department of Bacteriology, NMIMR, Legon, Accra, Ghana. NM 203 strain was thawed and subcultured on Lowenstein–Jensen (L-J slant) medium and incubated at temperatures of 31°C–32°C for 6–10 weeks. A loop full of the isolate NM 203 from an L-J medium was put into a cork-glass tube with 2–4 sterile glass beads and 2 mL of phosphate-buffered saline. The glass tube was then vortexed vigorously to obtain a uniform mixture with the turbidity checked and standardized to 1 McFarland standard. Particles were allowed to settle. A complete 7H9-GC medium (supplementation of 7H9 broth with glycerol, 0.2% (v/v) Sigma-Aldrich, Germany) was used to prepare 1 in 20 dilutions of the resulting mixture to get the inoculum ready for plating.

Stock solution preparations

Extracts for screening were weighed and reconstituted in distilled water (aqueous extract) and 100% DMSO (for the ethyl acetate and 70% ethanol extracts) to prepare 102.4 mg/mL and vortexed vigorously for the antimycobacterial assays. Reference controls were stocked at 2 mg/mL for RIF and 4 mg/mL for STR dissolved in absolute DMSO and sterile distilled water, respectively. The dried stem bark extracts and stock solutions were stored at 4°C and −20°C, respectively, until required for use.

Preparation of reference drug and extract working solutions

Solutions were prepared under aseptic conditions in a P3 facility at NMIMR. A 10-fold serial dilution from the RIF stock solution was obtained and diluted further (×25 dilution) with 7H9-GCOT medium to obtain the starting concentration of 8 μg/mL in the well of the reference drug control. The second reference drug, STR, was also prepared by performing a 20-fold dilution of the STR stock solution in sterile water. This concentration was further reduced at a ×25 dilutions in 7H9-GCOT to obtain the starting concentration of 16 μg/mL in the assay well plate. Similarly, a 1:100 dilution of the stock concentration of 102.4 mg/mL was done in 7H9-GCOT medium to achieve a concentration of 1024 μg/mL as stem bark extracts working concentration. All these working concentrations (RIF, STR, and extract) were aliquoted into their respective first wells for a 2-fold serial dilution to be done. These were further diluted to five lower concentrations. The final highest concentration of 4 μg/mL for RIF, 8 μg/mL for STR, and 512 μg/mL for the extracts were obtained after the addition of the bacteria culture suspension. Cryptolepine, the main metabolite of one of the plants being tested, was also prepared with a working concentration of 128 μg/mL.

Resazurin microtiter assay

Antimycobacterial activity of the stem bark extracts and reference control drugs was conducted using the resazurin microtiter assay (REMA) method in clear round bottom 96-well microtiter plates. In the REMA experiment, 100 μL of Middlebrook 7H9-GC broth containing 10% (v/v) (oleic acid, albumin, dextrose, and catalase) was dispensed in each well. In the test wells, 200 μL of the working solution of the extract (containing 1024 μg/mL in the complete medium) was added. Twofold serial dilutions were done to achieve concentrations of 1024, 512, 256, 128, 64, and 32 μg/mL. Finally, 100 μL of the M. ulcerans bacteria suspension (inoculum) was carefully added using the multichannel pipette to avoid cross-contamination. Initial concentrations of the extract were now halved and recorded as 512, 256, 128, 64, 32, and 16 μg/mL in the test wells. The reference controls, RIF and STR (with working concentrations of 8 and 16 μg/mL, respectively), resulted in final concentrations of 4 and 8 μg/mL after the addition of 100 μL of the bacteria inoculum, respectively. Similarly, the indoloquinoline alkaloid cryptolepine achieved concentrations of 128, 64, 32, 16, 8, and 4 μg/mL in the wells. The highest percentage concentration of the DMSO in reaction wells was <1.0%. Medium only was used as sterile control whereas medium with mycobacterium inoculum in wells was also used as the negative control. The plates were sealed in an airtight plastic bag and incubated at 31°C for 14 days. The test was conducted in duplicate. On the 14th day of incubation, the bacterial cell viability was determined by adding 20 μL of the resazurin dye (0.01% v/v) and further incubated at 31°C for a 24 h period. The minimum inhibitory concentration (MIC) values were determined by visual observation in comparison with the sterile, negative, and positive controls. Color change from blue to pink or red of resazurin dye indicated the reduction of the dye and hence bacterial growth. This would imply that the extract is inactive and should be discarded. However, the results were only accepted if the growth control turns pink while the sterility control containing only the culture medium remains unchanged within the incubation period. In the antimycobacterial experimental assay design, edging effect and evaporation was controlled by using sterile water in the entire perimeter well of the microtiter plate [Figure 2].
Figure 2: Assays plate design

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In vitro cytotoxic activity of active extracts

The extracts that showed anti-M. ulcerans activity were assessed for cytotoxicity using the Alamar blue assay described with slight modifications.[1] A complete RPMI 1640 medium containing 1% penicillin-STR-glutamine and 10% fetal bovine serum was used in maintaining cell culture preparations. Confluent liver cells in culture were prepared and counted to 1 × 105 cells/mL concentration for the assay. Cell concentrations were seeded (100 μL per) in triplicate in a 96-microtiter flat bottom well plate. The seeded plates were incubated at optimum cell culture conditions of 37°C with moderate humidity and 5% CO2 in the air. A minimum period of about 12 h (usually overnight) was allowed for the cells to adhere to the bottom of the plate. Each extract was tested by introducing 10 μL to the adhered cells. The assay plates were incubated again for 48 h. This procedure was repeated for curcumin used as the positive control. The concentration of extract in the assay wells was 1000–62.5 μg/mL. Similarly, cocentration of curcumin was 184.19–11.05 μg/mL and cryptolepine at 250–15.625 using RPMI 1640 as diluent. About 20 μL of 0.15 mg/mL Alamar blue solution was added to the wells after 48 h of incubation and re-incubation was done for another 4 h. With the aid of the spectrophotometer, infinite 200 PRO (Tecan, Grodig, Austria), the optical densities of the reaction mixtures in the wells were measured in the dark. Fluorescence was measured at an emission wavelength of 560 nm to the excitation wavelength of 590 nm. Normalization of data was done to percentage control activity (thus percentage cell viability) and subsequently, cytotoxic concentration (CC50) values representing the sample's concentration required to inhibit 50% of cell proliferation were calculated using GraphPad Prism 6.0 software (GraphPad Software Inc., San Diego, CA, USA) with data fitted by nonlinear regression.


  Results Top


Antimycobacterium ulcerans activity of selected plants

The in vitro antimycobacterial activity of plant extracts and reference drugs RIF and STR was evaluated against the M. ulcerans (NM 203) employing the REMA assay. Extract concentrations tested were between 16 μg/mL and 512 μg/mL in 2-fold serial dilutions. The different extracts exhibited varying degrees of antimycobacterial activities [Table 2].
Table 2: Anti-Mycobacterium ulcerans and cytotoxic activities plant extracts

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The Cryptolepis sanguinolenta root aqueous extract exhibited the highest antimycobacterial activity (MIC = 64 μg/mL) [Figure 3] followed by Cleistopholis patens (MIC = 256 μg/mL) [Table 2].
Figure 3: Cryptolepis sanguinolenta: leaves (a) and root (b)

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Based on the marked activity of the C. sanguinolenta extracts, pure cryptolepine, its major metabolite obtained from the Department of Pharmacology, KNUST, was tested against the M. ulcerans. This recorded a MIC value of 32 μg/mL [Table 2]. The activity of the extracts and cryptolepine was lower than that of the reference drugs STR and RIF which recorded MICs of 0.25 μg/mL and 0.125 μg/mL, respectively.

Cytotoxic activity

The plant extracts that showed considerable antimycobacterial activities were assessed for their cytotoxicity in vitro. Using the normal (noncancerous) human liver cells, some of the plant extracts showed cytotoxicity within the concentration range tested [Table 2]. All the active plant extracts with considerable antimycobacterial activity showed 50% CC50 ranging from 94 to 384 μg/mL compared to the reference agent curcumin (CC50 value of 6.9 μg/mL). However, cryptolepine, the main metabolite of the most active plant extract C. sanguinolenta, was the most cytotoxic [Table 2].

Phytochemical screening of active plants

Phytochemical analysis was performed on an active plant; plant extracts with MIC's of 256 μg/mL and below. This revealed the presence of secondary metabolites such as tannins, alkaloids, flavonoids, triterpenoids, and glycosides in the two active plants [Table 3].
Table 3: Qualitative phytochemical analysis of Cleistopholis patens and Cryptolepis sanguinolenta

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  Discussion Top


Extracts from nine selected medicinal plants were subjected to in vitro antimycobacterial assay using a clinical isolate of M. ulcerans (NM23). The standard clinical M. ulcerans clinical isolate (NM 203) was highly susceptible to the reference drugs [Table 2] which is expected as these are standard drugs for BU treatment. RIF and STR are used as combination therapy upon confirmation or detection of acid-fast bacilli in either early or late stages of the BU disease in Ghana. The clinical strain showed low susceptibility to three extracts (MIC's of 512 μg/mL) with some not showing any activity within the tested concentrations used. Two plant extracts showed some considerable activities [Table 2] of which one (C. sanguinolenta) exhibited marked activity (MIC = 64 μg/mL) against the M. ulcerans organism, even though the potency did not measure up to the reference drugs STR and RIF. The established criterion used in considering promising plant extract for further studies against M. ulcerans is a MIC value of <250 μg/mL.[12] Nevertheless, this does not rule out the fact that if MIC values are greater than this threshold, it cannot be considered. This convention was created to save time and resources when screening a large number of extracts against M. ulcerans. Thus, Cleistopholis patens and C. sanguinolenta are primed for further studies and could afford novel drugs for the mitigation of BU disease.

The most active compound from C. sanguinolenta, cryptolepine tested against M. ulcerans, recorded a MIC value of 32 μg/mL [Table 2] which is higher than its crude extract.

This again brings to bear the fact that purification of plant extracts could afford more active fractions or compounds. Thus, N. tobacum, S. campanulata, and T. crassa, with MIC's of 512 μg/m could be subjected to activity guided fractionation with the hope of obtaining more efficacious anti-M. ulcerans fractions or compounds. The activity of cryptolepine against M. ulcerans, in this study, showed a 4-fold increase in activity than other compounds isolated from H. floribunda and S. juglandifolia as reported by Yemoa et al.[12] and Tsouh Fokou et al.,[1] respectively, against the same organism. Four plant extracts did not show any activity thereby highlighting the need to validate traditional medicinal products used to treat diseases. The continuous use of plants with little or no efficacy against the M. ulcerans organism may lead to complications including amputations.

C. sanguinolenta (Periplocaceae) root decoction is commonly known and used as a strong antimicrobial and antimalarial agent in West Africa. In Ghana, the Centre for Plant Medicine Research formulated a product for therapeutic use as an antimalarial product called Nibima.[15] Antimycobacterial activity of cryptolepine in this study proves it as a promising anti-M. ulcerans agent or a scaffold for future BU drug development. Other reports indicated that this compound inhibited fast-growing mycobacteria with MICs ranging from 2 μg/mL to 32 μg/mL against six Mycobacterium species including Mycobacterium fortuitum, Mycobacterium abscessus, Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium aurum, and Mycobacterium bovis Bacille de Calmette et Guérin. According to the report, the MICs recorded were comparable to standard antibacterial drugs such as ethambutol, isoniazid, and STR sulfate.[16],[17] Despite the high cytotoxicity of cryptolepine when administered intraperitoneally in mice, its oral administration has been reported to be nontoxic.[15] This may be as a result of poor absorption and/or metabolism of an inactive metabolite. Studies have also shown that the liver enzyme aldehyde oxidase is a known oxidizing agent of cryptolepine which renders it inactive against malarial parasite P. falciparum in vitro. However, potent synthetic derivatives of cryptolepine have improved efficacy against malarial parasites in vitro (P. berghei) with no intercalation into DNA as well as DNA inhibition synthesis and of topoisomerase II. The derivative has less cytotoxicity even when injected intraperitoneally in mice. The 2,7-dibromocryptolepine, a synthetic derivative, also has been found to be active against Trypanosoma brucei.[15] Therefore, cryptolepine derivatives can also be considered in the development of agents against M. ulcerans, the causative organism for BU. This further supports the potential for cryptolepine and its derivatives to be used as a promising drug candidate for BU treatment. The leaf extracts of Periploca nigrescens Afzel (cited as Parquetina nigrescens) and Heterotis rotundifolia (Sm). Jacq.-Fel.(cited as Dissotis rotundifolia (Sm) Triana) confirm the same inactivity of the plant part used in this experiment.[1] However, Tsouh Fokou et al. reported activity against M. ulcerans at 250 μg/mL using root extracts of Spathodea campanulata and leaf extract of Alstonia boonei De Wild unlike in this study except for the stem bark extract of Spathodea campanulata which showed lower activity.[1] In vitro cytotoxicity studies performed on antimycobacterial extracts in this study revealed varying toxicity levels of the extracts though incomparable to the reference agent curcumin [Table 2]. The American National Cancer Institute considers CC50 values less than or equal to 30 μg/mL as the limit value to qualify a plant extract as cytotoxic.[18] The plant extracts and compound tested showed cytotoxic activity with CC50 values within the range 49.5-383.9 μg/mL. Cryptolepine showed cytotoxicity but not compared to the reference agent, curcumin.

Financial support and sponsorship

This work was partly supported by the the Bill and Melinda Gates Foundation through the postdoctoral fellowship training program in Infectious Diseases at the Noguchi Memorial Institute for Medical Research, Ghana under Global Health Grant [number OPP52155].

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Tsouh Fokou PV, Kissi-Twum AA, Yeboah-Manu D, Appiah-Opong R, Addo P, Tchokouaha Yamthe LR, et al. In vitro activity of selected West African medicinal plants against Mycobacterium ulcerans disease. Molecules 2016;21:445.  Back to cited text no. 1
    
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Yemoa A, Gbenou J, Affolabi D, Moudachirou M, Bigot A, Anagonou S, et al. Beninese medicinal plants as a source of antimycobacterial agents: Bioguided fractionation and in vitro activity of alkaloids isolated from Holarrhena floribunda used in traditional treatment of buruli ulcer. BioMed Res Int 2015;2015:835767.  Back to cited text no. 12
    
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Suffness M, Pezzuto J. Assays related to cancer drug discovery. In: KHostettmann, editor. Methods in Plant Biochemistry: Assays for Bioactivity. London, UK: Academic Press; 1990.  Back to cited text no. 18
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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