Update on Medicinal Plants with Potency on Mycobacterium ulcerans

Mycobacterium ulcerans disease has been a serious threat for people living in rural remote areas. Due to poverty or availability of traditional medicine these populations rely on herbal remedies. Currently, data on the anti-Mycobacterium ulcerans activity of plants, so far considered community-based knowledge, have been scientifically confirmed, concomitantly with some medicinal plants used to treat infectious diseases in general. Products derived from plants usually responsible for the biological properties may potentially control Mycobacterium ulcerans disease; numerous studies have aimed to describe the chemical composition of these plant antimicrobials. Thus, the present work provides the first compilation of medicinal plants that demonstrated inhibitory potential on Mycobacterium ulcerans. This work shows that the natural products represent potential alternatives to standard therapies for use as curative medicine for Mycobacterium ulcerans disease.


Background
Mycobacterium ulcerans disease has been a serious threat for people living in rural remote areas. Due to cultural belief, convenience or inaccessibility of modern therapy, in many rural folk, usually relies on traditional medicine for ulcer management [1]. Besides, many people with M. ulcerans disease suffer mutilation and amputation because modern treatment seems to be expensive and also associated with side effects [2]. These traditional medicines are usually sold in markets and public places or administered by traditional healers in their clinics. Most remedies are mixtures of two or more plants species and solvents used including water, palm wine, or oils. Health problems are often treated through self-medication, first with the popular pharmacopoeia [3,4] or specialized pharmacopoeia from traditional healers for difficult health problems.
Traditional treatment for Mycobacterium ulcerans disease is done in four steps that involve diagnosis, necrosis ablation, wound curing, and exorcism [1]. The use of medicinal plants takes place in the second and the third stages of the treatment process [1]. Although medicinal plants may play a significant role in Buruli ulcer case management and control of the disease at an affordable cost for the local population, no attempt has been made to document potent medicinal plants against Buruli ulcer. We previously compiled ethnopharmacological data on plants used traditionally to treat Buruli ulcer which addressed only the activities of ethnopharmacolocally used medicinal plants excluding plants for which there was no report on their use against Buruli ulcer. In this review we

An Overview of the Assay Methods
Used to Estimate Antimycobacteria Activity 2.1. Antimicrobial Assays. Several M. ulcerans strains and isolates have been successfully adapted to in vitro culture. They were employed in the activity screening described. Two different assays were used to determine the antimicrobial activity of the extracts, fractions, and pure compounds and where possible were compared to commercially available antimicrobial agents, such as rifampicin (MIC = 2 g/mL) [20].

The Proportion
Method. The proportion method, which is still the main method used for drug or products screening of Mycobacterium ulcerans by many researchers, applied Löwenstein-Jensen medium. This involved the use of a drug containing media associated with or without dilution of the drug and drug-free media. A standardized inoculum was applied to the drug containing and drug-free media and incubated. After numbering the colonies formed on drug containing medium and on drug-free medium, the MIC was defined as the minimum concentration that inhibited at least 99% mycobacteria [53]. From the studies reviewed, serial dilution of the plant extracts at concentration ranging from 0.4 g/mL to 50 g/mL (25% vol/vol to 0.20% vol/vol) [5] or 0.5 to 50 mg/mL [33,43] was incorporated into Löwenstein-Jensen media. Thereafter, inocula size of 10 4 to 10 5 mycobacteria/mL was applied and incubated for 8 to 12 weeks at 30-33 ∘ C [5,33,43].

The Resazurin Microtiter Assays (REMA).
Resazurin or alamar Blue-based assays are sensitive and powerful tools used to detect growth inhibition of a given substances in various concentration ranges on mammalian cell lines and microorganism including mycobacteria [66]. The resazurin microtiter assay was first proposed as a simple, rapid, and cheap alternative for Mycobacterium tuberculosis susceptibility testing by Martin et al. [67] and was then adapted by Yemoa et al. in 2011 [21] for Mycobacterium ulcerans susceptibility testing. This method is performed usually in the 96well microplates and can be read with a spectrophotometer or spectrofluorometer or visually without any equipment. In the latter case, which is the one used in the review studies, the minimum inhibitory concentration (MIC) is defined as the lowest concentration of extract that prevents a color change of resazurin from blue to pink. In fact, viable mycobacteria will reduce the blue resazurin into the pink-coloured resorufin. In all cases, extracts or compounds were serially diluted in a 96-well plate and incubate with the inoculum of mycobacteria 10 5 cfu/mL in Middlebrook 7H9 broth supplemented with oleic acid-albumin-dextrose-catalase with concentration ranging from 7.81 to 12,500 g/mL. After 15 days of incubation, resazurin was added and a colour change was visible after additional 48 hours of incubation [11,21].

Medicinal Species with Anti-Mycobacterium ulcerans
Activities. Several authors have reported medicinal species with biological activity against M. ulcerans in vitro. In this work, we have reviewed 33 species either alone or in combination for their potential on M. ulcerans in vitro (Table 1).
vera is an evergreen perennial medicinal plant known for centuries for its beauty and medicinal and skin care properties [51]. Classically, A. vera has been used for its wound healing potential [51]. Recently Seefeld et al. [68] reported its use in the traditional treatment of Buruli ulcer while Addo et al. [5] have demonstrated its in vitro activities on 7 different M. ulcerans strains and isolates with MIC mean value of 40 g/mL (Table 1). Phenolic compounds such as chromone, anthrone, anthraquinone [52], aloin, and emodin have been identified and are known for their antibacterials effect [51]. Also, its anti-inflammatory action through the inhibition of the cyclooxygenase pathway and reduction of prostaglandin E2 production from arachidonic acid was reported. Recently, the novel antiinflammatory compound called C-glucosyl chromone was isolated from gel extracts [51] of A. vera. The anti-inflammatory activity combined with the antibacterial effect and the wound healing activity of A. vera support its use in Buruli ulcer case management. In fact, its high water content can keep the wound moist and increase epithelial cell migration.
In addition, its content of glucomannan, a mannose-rich polysaccharide, and gibberellin, a growth hormone, interacts with growth factor receptors on fibroblasts and macrophages, thereby significantly increasing collagen and proteoglycan synthesis, and the degree of collagen cross linking after topical and oral administration resulting in accelerated tissue repair [51,52]. Furthermore, acemannan, saponin, and genin isolated from Aloe leaves have been shown to accelerate wound healing [52,69,70].

Alstonia boonei De
Wild. Alstonia boonei is a large deciduous evergreen tree, usually up to 45 m tall and 1.2 m in diameter, and is also called devil tree in tropical and subtropical Africa, Central America, and Australia [71]. The plant is used to treat various diseases across Africa and the stem bark has been listed as an agent useful for treatment of ulcer or wounds [72]. Addo et al. [5] reported in vitro activity of infusion of leaves on 7 M. ulcerans strains and isolates with MIC mean value of 40 g/mL (Table 1). A wide array of chemical compounds, including alkaloids (echitamine, echitamidine, voacangine, akuammidine, N-formylechitamidine, N -formyl-12-methoxyechitamidine), tannins, iridoids (boonein, loganin), steroids, saponins, glycosides, flavonoids, and terpenoids and triterpenoids (lupeol, ursolic acid, and -amyrin) [19,[71][72][73], have been isolated from the plant that might support its reported Mycobacterium ulcerans inhibitory effects.

Pupalia lappacea Juss.
Pupalia lappacea is a widespread medicinal plant found in savannah and woodland localities        [5,32,[59][60][61][62][63][64][65] w/v = 20% w/v (200 g/mL) of herbal preparations (infusions, decoctions and juices) were each incorporated at 1 : 5 dilution into L-J medium corresponding to a final concentration of 40 g/mL. V/V = The herbal preparations (infusions, decoctions and juices) at 20% w/v were incorporated into L-J media at final concentrations ranging from 25% vol/vol to 0.20% vol/vol corresponding to 50 g/mL to 0.4 g/mL. 8 BioMed Research International as well as forests in tropical Africa and in Asia [74]. It has been used in the management of wound [5,6] and its leaves extracts have shown inhibitory activity on 7 M. ulcerans strains and isolates with MIC mean value of 40 g/mL (cited as 1 : 5 (20% w/v)) ( Table 1) [1]. Chemical investigations show that P. lappacea leaf extract contains alkaloids, glycosides, flavonoids, saponins, tannins, coumarins, terpenoids, and steroids such as 1-docosanol, stearic acid, stigmasterol, sitosterol, N-benzoyl-L-phenylalaninol acetate, setosterol-3-O-Dglucopyranoside, stigmasterol-3-O-D-glucopyranoside, and 20-hydroxyl ecdysone. These compounds have been shown to promote the wound healing process in animals and humans. Stigmasterol found in the plant has been shown to exhibit haemostatic and anti-inflammatory activities. Similarly, 20hydroxyl ecdysyne also present in the plant promotes protein synthesis and wound healing in animals and humans. Since some of the compounds contained in P. lappacea leaves extract have antibacterial activities and also promote the wound healing process, the plant extract may exhibit wound healing activities as claimed by the traditional practitioners [6][7][8].

Lannea nigritana (Sc. Elliot) Keay. Lannea nigritana
is a small tree of 3-6 m of height found in the tropical rain forest [14]. It is used in traditional medicine for the treatment of various infectious diseases [14] including wounds [5]. 2.2.7. Cleome viscosa L. Cleome viscosa is commonly known as tickweed, wild mustard, or spider plant that occurs in woodland and grassland. It is a weed found in fallow land, fields, roadsides, and wasteland. It often grows on sandy soils but sometimes grows on calcareous and rocky soils. It is a widely distributed herb with yellow flowers and long slender pods containing seeds [77]. The leaves and whole plant of C. viscosa are used as a folk remedy to cure wounds, ulcers, inflammations, and skin infections [27]. Its leaves have shown MIC mean value of 40 g/mL (cited as 1 : 5 (20% w/v)) on 7 M. ulcerans isolates (Table 1) [5]. Methanolic extract of the aerial parts of the plant is also reported to have significant wound healing properties on experimentally induced excision and incision wound models in rats in addition to its previously reported analgesic, antimicrobial, and antiulcer activities [77]. All of this supports the traditional use of the plant. Its content of alkaloids, tannins, saponins, flavonoids, terpenes, and carbohydrates [27,28] may be probably responsible for the observed wound healing activity.

Phyllanthus fraternus G. L. Webster. Phyllanthus fraternus
commonly is a small, erect, annual herb that grows 30-40 cm in height [78] indigenous to the rainforests of the Amazon and other tropical areas throughout the world [79]. The plant has numerous uses by indigenous peoples to treat blennorrhagia, colic, diabetes, dysentery, fever, flu, tumors, jaundice, vaginitis, dyspepsia, and pain [80]. P. fraternus has previously demonstrated an MIC mean value of 40 g/mL (cited as 1 : 5 (20% w/v)) on 7 M. ulcerans (Table 1) [5] isolates and strains. It has been shown to contain alkaloids, steroids, glycosides, tannins, saponin, flavonoids, other compounds (estradiol, corilagin, ellagic acid, gallic acid, rutin, gernanine, rutinoside, lupa, lupeol, and methyl salicylate), and saponins (triacontanal, triacontanol) [45,79]. The reported antimicrobial activity may be attributed to the presence of some of the reported secondary metabolites. However, the active compound(s) known to give this observed activity need to be identified.

Bridelia ferruginea Benth. Bridelia ferruginea
Benth. is a gnarled shrub which can reach the sizes of a tree in suitable condition [30]. B. ferruginea bark is used for treatment of bacterial infections on wound [81] and has been shown to inhibit the growth of 7 M. ulcerans strains and isolates with an MIC mean value of 40 g/mL (cited as 1 : 5 (20% w/v)) ( Table 1) [5]. The presence of phytochemical such as polyphenols, steroids, saponins, tannins, terpenoids, and alkaloids [30,31] may support the demonstrated antimicrobial activity against mycobacteria.

2.2.11
. Psidium guajava L. Psidium guajava, commonly known as guava, is a native plant of tropical America and has been used in indigenous system of medicine for the treatment of various human ailments such as wounds, ulcers, bowels, and cholera [40,83]. In Central and West Africa, its decoctions are used externally for skin ulcers [84]. P. guajava also possesses antibacterial and anti-inflammatory properties and also inhibited the growth of 7 M. ulcerans strains and isolates with MIC mean value of 40 g/mL (cited as 1 : 5 (20% w/v)) ( Table 1) [5]. A number of secondary metabolites in good yield have been isolated and some, which are mainly phenolic, flavonoid, carotenoid, volatile oil, tannins, terpenoid, and triterpene, have been shown to possess useful biological activities [40].

Capsicum annum L.
Capsicum annum is a perennial shrub, with woody trunk, which bears green fruits that ripen to red. The active ingredient in the plant is capsaicin that is used for the management of various medical conditions [48]. This plant possesses antibacterial and wound healing properties. It is used often in combination with Pothos scandens L. and Allium sativum to heal wounds [85]. The maceration of fruit, used to treat Buruli ulcer, has showed MIC mean value of 40 g/mL (cited as 1 : 5 (20% w/v)) on 7 M. ulcerans strains and isolates ( diseases, respiratory ailments, and genital-urinary system disorders [24] as well as relief for skin conditions, swollen cheeks, and body rashes and Buruli ulcer. The leaves and stem bark paste are used to bandage ulcers while infusions of the leaves, root, and bark are also used to clean ulcers [4,5,20,88,89]. The decoction of the root showed inhibitory activity on 7 M. Ulcerans strains isolates with MIC mean value of 25 g/mL (cited as 12.50% (V/V)) ( Table 1) [5]. Biologically active phytochemicals have been identified such as alkaloids, tannins, saponins, glycosides, anthraquinone glycosides, steroids, flavonoids, tannins, and glycosides [24][25][26].

2.2.15
. Allium sativum L. Allium sativum has been widely recognized as a valuable spice and a popular remedy for various ailments and physiological disorders. The plant appears to have originated from Central Asia and then spread to China, the Near East, and the Mediterranean region before moving west to Central and Southern Europe, Northern Africa, and Mexico [90]. The juice of A. sativum cloves is used to treat Buruli ulcer conditions [5]. It has been shown experimentally to accelerate the wound healing process in mice [91]. Besides, aqueous extract of A. sativum in combination with honey has shown acceleration of wound healing in rats [92]. It has shown growth inhibition on 7 M. ulcerans strains and isolates with MIC mean values of 0.78-6.25 g/mL (cited as 0.39-3.13% (V/V)) ( Table 1) [5]. Active constituents such as alliin, allicin and -glutamylcysteine, ajoenes, and vinyldithiins have been identified [9,10]. Allicin has antimicrobial effects against many bacteria and fungi [90] usually found in ulcers. Its activity on M. ulcerans combined with its antibacterial, antifungal and anti-inflammatory, antioxidant, [90,93], and wound healing potency supports its traditional use in Buruli ulcer treatment.

Syzygium aromaticum (L.) Merr. & L. M.
Perry. Syzygium aromaticum is an evergreen tree found worldwide [41]. It is a natural analgesic and antiseptic used primarily in dentistry because of its main ingredient eugenol. It has been traditionally used externally or locally for the treatment of minor infections of the mouth and skin, dressing of minor wounds, and Buruli ulcer [5]. Its seeds have showed MIC mean value of 25 g/mL (cited as 12.50% (V/V)) on 7 M. ulcerans isolates (Table 1) [5]. Many active ingredients have been identified from S. aromaticum including essential oils, tannins, gallotannic acid, methyl salicylate, flavonoids eugenin, and triterpenoids like oleanolic acid [41].

Hydrastis canadensis L.
Hydrastis canadensis is an herbaceous perennial growing short yellowish rhizome [94]. H. canadensis is widely used to treat many ailments, including arrow wounds [46]. It inhibited the growth of 7 M. ulcerans strains and isolates with MIC values of 0.39-6.25 g/mL (cited as 0.20-3.13% (V/V)) on (Table 1) [5]. In an antimicrobial screening program, H. canadensis extract also exhibited significant activity against multiple drug resistant strains of M. tuberculosis and other Mycobacterium species as well as other human pathogens [95]. H. canadensis is found to contain alkaloids, flavonoids, organic acids, sterols (sitosterol 3-O--D-glucoside), volatile oil, resin, and fatty acids [46]. Bioassay-guided fractionation revealed berberine to be the active constituent on mycobacteria [95]. This in combination with its wound healing properties can explain the higher activity observed against M. ulcerans and support the indigenous treatment of Buruli ulcer.
The presence of a diversity of essential oils, alkaloids, and several aliphatic and aromatic amides [47] strengthens claims of effectiveness of this plant in its traditional use for treatment of Buruli ulcer.

Gratiola officinalis L. Gratiola officinalis
Linn. is a glabrous perennial herb and is native to the south of Europe, and its favourable habitat is damp grounds. Gratiola officinalis L., commonly known as common Hedgehyssop or "Herb of Grace" is well known for its pharmacological properties. Various parts of this plant (root and herb) are used in phytomedicines to treat skin diseases [42]. This may explain the observed good inhibitory activity on 7 M. ulcerans strains and isolates with MIC values 1.56-25-g/mL (cited as 0.78-12.5% (V/V)) ( Table 1) [5]. The active constituents found in G. officinalis include gratiogenin, 16-hydroxygratiogenin, cucurbitacins E and I, glycosides gratiogenin-3beta-D-glucoside, gratioside, elaterinide, flavonoids, alkaloids, lignans, coumarin, and saponins which have many biological properties [42].

2.2.20
. Jatropha curcas L. Jatropha curcas Linn belonging to the family Euphorbiaceae is a drought-resistant shrub originating in Central and South America but now thrives in many parts of the tropics and subtropics in Africa and Asia [97]. J. curcas has been used as traditional medicine to cure Buruli ulcer [4]. Its leaf ethanolic extract inhibited the growth of M. ulcerans ATCC 19423 with MIC value of 250 g/mL and the crude bark extract of Jatropha curcas has been shown to be very effective in accelerating wound healing process in rat [98]. Researchers have isolated and characterized a number of biologically active constituents such as flavonoids, apigenin, vitexin, isovitexin, stigmasterol, -D-sitosterol, -D-glucoside, sapogenins, alkaloids, triterpene alcohol, and 1triacontanols from all parts of this plant [32].

Holarrhena floribunda (G. Don) T.
Durand. Holarrhena floribunda grows as a shrub or tree up to 25 m tall, with a stem diameter of up to 30 cm [4,21]. Bark is used as an enema or in baths to treat skin infections and the leaf sap is sprinkled on wounds as a haemostatic. A sap extracted from its leaves is sprinkled onto wounds to act as a haemostatic [22]. Yemoa et al. [4] report its use in the traditional treatment of Buruli ulcer and hydroethanolic extract and CH 2 Cl 2 fraction of root inhibited the growth of M. ulcerans ATCC 19423 with MIC value of 125 g/mL [20] while that of the alkaloid enriched fraction had an MIC of 62.5 g/mL. These give some support to the use of this plant in traditional medicine (Table 1) [21]. It contains active compounds such as saponins, polar steroidal glycosides, steroidal glycosides, and alkaloids including holaphylline, holaphyllamine, holamine, holaphyllinol, holaphyllidine, holadysamine, holarrhesine, conessine, and progesterone [21,22].

Sorindeia juglandifolia (A. Rich.) Planch. ex Oliv.
Sorindeia juglandifolia is a shrub or small tree that grows to 23 m tall and 40 cm in diameter. It is widespread in the West and Central Africa subregion. This plant is found on the edges of dry deciduous forest and regrowth in humid forest and in the galleried Sudanian forest of Senegal to Dahomey and also in Ubangi-Shari, Angola, and Zambia. Its common English name is "damson" [99,100]. In Senegal pulped leaves are applied to sores and ulcers [101]. Fruit fractions and a purified compound (2,3,6-trihydroxymethyl benzoate) from S. juglandifolia is reported to have an antimycobacterial activity against M. ulcerans strain 1615 with MIC value of 62.5 g/mL and minimal bactericidal concentration (MBC) values of 250 and 125 g/mL, respectively [11].

Annickia chlorantha (Oliv.) Setten & Maas.
Annickia chlorantha is a tree up to 30 m tall commonly known as "Yello Wood" found in dense forests in Cameroon, Nigeria, and Gabon. In the southern forest zone of Cameroon, it is used for the traditional treatment of stomach problems, jaundice, urinary tract infections, malaria, tuberculosis, hepatitis, and some forms of ulcer [3,15]. The stem bark and stem preparations of A. chlorantha have been shown to have high inhibition against the growth of M. ulcerans strain 1615 with respective MIC values of 1.95 and 7.81 g/mL ( Table 1). The chemistry of A. chlorantha formally Enantia chlorantha has been extensively studied. Berberine and protoberberine alkaloids [15] with antibacterial [102] properties have been isolated from the stem bark of A. chloranta. A mixture of protoberberine alkaloids from A. chlorantha containing palmatine, jatrorrhizine, and columbamine was shown to prevent liver injury from chemically induced traumatization and also promoted the healing process after injury in experimental mice [16].

Greenwayodendron suaveolens (Engl. & Diels) Verdc.
Greenwayodendron suaveolens is a deciduous, medium-sized to fairly large tree up to 35 (to 45) m tall that is widespread from Southern Nigeria, East to Western Uganda, Northern Tanzania, and Southern Democratic Republic of Congo and Cabinda (Angola) and commonly known in English as "Molinda." Various plant parts are used in traditional medicine to treat stomach ache and other pains, gonorrhoea, psychosis, rheumatism, epilepsy and toothache, malaria, liver complaints and headache, helminths, oedema and swollen glands, and hepatitis; it is used to manage infertility, as diuretic, purgative, an aphrodisiac, and to facilitate childbirth [17]. Extracts and fractions from G. Suaveolens (cited as Polyalthia suaveolens) have been shown to inhibit M. ulcerans with moderate MIC and MBC values of 3,125 g/mL (Table 1) [11]. Many compounds such as polysin, greenwayodendrin-3-one, 3-O-acetyl greenwayodendrin, N-acetylpolyveoline, polyveoline [18], and several alkaloids including indolosesquiterpenes and aporphines have been isolated from the plant [17].

Phyllanthus amarus Schumach. & Thonn. Phyllanthus
amarus is a small herb well known for its medicinal properties and widely used worldwide [44]. It is useful in gastropathy, diarrhoea, dysentery, intermittent fevers, ophthalmopathy, scabies, ulcers, and wounds [44].  [34] and Buruli ulcer. A decoction of the stem bark of the plant is administered orally or used topically to disinfect the ulcer or the ulcer is covered with fine powder of the stem bark and bandaged [33]. The aqueous extract of S. gabonensis showed promising activity against M. ulcerans in vitro (MIC = 780 g/mL) [33] (Table 1). Bergenin, an isocoumarin, was identified as the main active compound of the stem bark extract of S. gabonensis [34]. Further analyses of stem bark extract have shown tannins, sterols, polyterpenes, polyphenols, flavonoids, and alkaloids in appreciable amounts with a trace of saponins. The stem bark also contains 2 cis/trans isomers of lignans (calopiptin and galgravin) [34]. Sofowora [35] has also reported that these compounds are responsible for the biological activities of the plant. Kamanzi [105] also argued that plants show antibacterial activity when they contain flavonoids, tannins, saponins, and alkaloids. (i) Spigelia anthelmia L. Spigelia anthelmia is a common annual weed that grows in open regrowths, on unused land in towns and on road sides [106]. The plant is reputed to be useful in wound healing. A decoction of leaves and twigs is used to wash the wound, which is then dressed with a powder of the bark of the plant [107]. Phytochemical investigation has described the isolation of the alkaloid spiganthine, volatile alkaloids, isoquinoline and actinide isomer, three quaternary alkaloids, choline, benzoylcholine and 2,3-dimethylacrolyl choline, phenylcarboxylic acids, and flavonoids [54,55] from the plant.
(ii) Zea mays L. Zea mays is a robust annual grass up to 4(-6) m tall [108] that possesses wound healing activity [109]. Z. mays corn silk is rich in phenolic compounds, particularly flavonoids. It also consists of proteins, vitamins, carbohydrates, calcium, potassium, magnesium and sodium salts, volatiles oils, and steroids such as sitosterol and stigmasterol, alkaloids, and saponins [56] that might promote the wound healing and antibacterial activities of the mixture. (i) Citrus aurantifolia (Christm.) Swingle. Citrus aurantifolia is widespread in tropical and subtropical regions around the world and it is known for its nutritional values and flavour [110]. The plant and fruit of C. aurantifolia have been commonly used in traditional medicine to treat various diseases [110] including Buruli ulcer. It is used either alone as decoction or mixed with G. barbadense leaves or S. campanulata as a bandage for ulcer [4,5,20,68,88]. The essential oil of C. aurantifolia fruits contain mainly limonene, betapinene, gamma-terpinene, and citral [111]. The methanolic extract of C. aurantifolia contains alkaloids, flavonoids, tannins, saponins, steroids, cardiac glycosides, and reducing sugar [57]. The hexane extract of the fruit peels fractionated by column chromatography yielded the following major compounds: 5-geranyloxypsoralen, 5-geranyloxy-7-methoxycoumarin, 5,7-dimethoxycoumarin, 5-methoxypsoralen, and 5 M. ulcerans could be attributed to these compounds and might have significantly contributed to the observed potency of the mixture.
(ii) Gossypium barbadense L. G. barbadense is an annual herb that has been reported to have many therapeutic effects including treatment of cutaneous and subcutaneous parasitic infections are mostly attributed to its active constituent gossypol [58].
(i) Jatropha curcas L. See the above.
(ii) Physalis angulata L. Physalis angulata is a much branched annual shrub, perennial in subtropical zones, and can grow until it reaches 1.0 m. It is used in several countries of tropical and subtropical regions of the world as medicinal and fruit tree. Recent ethnopharmacological studies show that P. angulata leaf paste is used as an external application for wounds [59]. The major identifiable phytochemical constituents of medicinal importance are physalins and withanolides [59,60]. The anti-inflammatory, antimycobacterial, antinociceptive, and antitumor activities together with the inhibitory effect on M. Ulcerans support its traditional uses for Buruli ulcer treatment.
(iii) Gossypium hirsutum L. Gossypium hirsutum is a perennial shrubs also known as upland cotton or Mexican cotton. It is native to Mexico. Extract from cotton plant, which would be primarily gossypol, has been used as traditional medicine. Cotton leaves have been used as a treatment for nausea during pregnancy, for "proud flesh" (swollen tissue around a wound) or for fungal infections. Cotton tissue, particularly the seeds, can be toxic if ingested in excessive quantities because of the presence of antinutritional and toxic factors including gossypol and cyclopropenoid fatty acids (including dihydrosterculic, sterculic, and malvalic acids) [61,62].

Conclusion and Future Perspectives
In this review, we have discussed medicinally significant plant species from Sub-Saharan Africa and showed that many have activity against M. ulcerans. Currently, there are only two studies that have reported the purification of active compounds against M. ulcerans. This highlights the poor emphasis given to research into new chemotherapeutic agents against one of the world most neglected diseases: Mycobacterium ulcerans disease. The present review can be used to validate ethnomedicinal knowledge and bioactivities. Unfortunately, most of the species that are claimed to contain antimycobacterial activities have not been studied in vivo. Screening with in vitro assays has little meaning if there is no clear evidence of effectiveness of the extracts in vivo. Therefore, further in vivo studies of preparations from the identified plant species are required. This should be followed by systematic phytochemical studies of plants that contain bioactive antimycobacterial activities, isolation, and characterization of the anti-M. ulcerans chemical entities. These can provide the needed validation before such chemical entities can be used as sustainable cheaper/alternative medicines for management of Buruli ulcer disease.