Antibacterial and Antifungal Activities of Ethiopian Medicinal Plants: a Systematic Review

Antibacterial and antifungal activities of Ethiopian medicinal plants: a systematic review

Article (Accepted Version)

Nigussie, Dereje, Davey, Gail, Beyenee, Takele Beyene, Brewster, Malcolm, Legesse, Belete Adefris, Fekadu, Abebaw and Makonnen, Eyasu (2021) Antibacterial and antifungal activities of Ethiopian medicinal plants: a systematic review. Frontiers in Pharmacology, 12. a633921 1-17. ISSN 1663-9812

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http://sro.sussex.ac.uk 1 Title: Antibacterial and antifungal activities of Ethiopian medicinal plants: a systematic 2 review

3 Dereje Nigussiea,b, Gail Daveyb,d, Takele Beyenee, Malcolm Brewsterf, Belete Adefris

4 Legessea, Abebaw Fekadua,b, Eyasu Makonnena,c

6 a Centre for Innovative Drug Development and Therapeutic Trials for Africa (CDT-Africa),

7 College of Health Sciences, Addis Ababa University. P.O. Box 9086, Addis Ababa, Ethiopia;

8 b Centre for Global Health Research, Brighton and Sussex Medical School, University of

9 Sussex, Brighton, BN1 9PX, United Kingdom;

10 c Department of Pharmacology and Clinical Pharmacy, College of Health Sciences, Addis

11 Ababa University, Addis Ababa, Ethiopia;

12 d School of Public Health, Addis Ababa University, Addis Ababa, Ethiopia

13 e Department of Biomedical Sciences, College of Veterinary Medicine and Agriculture, 14 Addis Ababa University

15 f Rye Medical Centre, Rye, East Sussex, TN31 7SQ, UK

18 19 20 21 22 23 24 25 26 27 28 29

Page | 1 30 Abstract 31 32 Background: Podoconiosis and lymphatic filariasis are the most common causes of lower 33 limb lymphoedema in the tropics. Many sufferers experience frequent painful episodes of 34 acute bacterial infection. Plant based traditional medicines are used to treat infections in 35 many countries and are culturally established in Ethiopia. Ethiopian medicinal plants found to 36 have antibacterial and antifungal activities were reviewed with the aim of increasing 37 information about the treatment of wound infections in patients with lymphoedema. 38 Methods: This study collates data from published articles on medicinal plants with 39 antibacterial and antifungal activities in Ethiopia. A systematic search of Scopus, EMBASE, 40 PUBMED/MEDLINE and Google Scholar was undertaken. The Preferred Reporting Items 41 for Systematic Reviews and Meta-analysis (PRISMA) guidelines were followed. The 42 protocol was registered on PROSPERO with registration number CRD42019127471. All 43 controlled studies of in vitro antibacterial and antifungal activities were considered. All 44 articles containing the descriptors published until June 28, 2019 were included. The outcome 45 was measured as percent inhibition of microbial growth. For quality assessment of individual 46 in vitro studies, OECD guidelines and the WHO-Good Laboratory Practice (GLP) handbook 47 were used. 48 Results: Seventy-nine studies met the inclusion criteria. A total of 150 plant species and 3 49 compounds had been tested against 42 species of bacteria, while 43 plant species had been 50 tested against 22 species of fungus. 51 Conclusions: Materials derived from several Ethiopian medicinal plants have been shown to 52 have promising activity against a variety of bacteria and fungi. Those derived from 53 Azadiractha indica and Lawsonia inerms are the most extensively studied against a wide 54 range of gram-negative and positive bacteria, and fungal species. 55 56 Key words: Ethiopia, antibacterial, antifungal, systematic literature review, medicinal 57 plants 58 59 60 61 62

Page | 2 63 64

65 1. Background 66

67 Lymphoedema is a chronic illness that has a major physical and psychological effect on 68 patients and lowers the quality of patient life substantially. Neglected tropical diseases 69 (NTDs) such as podoconiosis, lymphatic filariasis, and leprosy are the most common causes 70 of lower limb lymphoedema in the tropics. Suffering can be aggravated by frequent painful 71 episodes of acute bacterial limb infection known as ‘acute attacks’(1). Cellulitis and 72 erysipelas are typical wound complications, with the majority of infections caused by group 73 A, C, or G streptococci and staphylococcus aureus species (2). However, Aeromonas 74 hydrophila/caviae, Acinetobacter lwoffii, Escherichia coli, Klebsiella pneumoniae, 75 Pseudomonas aeruginosa, Shewanella algae, Staphylococcus aureus, Streptococcus 76 pyogenes, Streptococcus. dysgalactiae, Staphylococcus haemolyticus, Streptococcus 77 agalactiae and Staphylococcus. simulans have recently been found to be involved in 78 colonising wounds of lymphoedematous limbs in patients from Ethiopia (3).

79 Lymphoedema caused by the aforementioned NTDs is progressive if not treated. In the early 80 stages oedema can be reversed overnight, but with disease progression there can be serious 81 impairment and loss of independence. Patients are excluded from society because of their 82 incapacitating and stigmatized impairments which cause significant economic effects, 83 intergenerational poverty, and alienation from society. In Ethiopia, an estimate of 5.6 million 84 and 34.9 million peoples are at risk of lymphatic filariasis and podoconiosis respectively (4). 85 There are 1.5 million cases of podoconiosis across 345 districts of Ethiopia, and the country 86 has the highest prevalence of podoconiosis in the world (5).

87 The use of medicinal plants has a long history in the treatment of a range of diseases, 88 including infectious diseases, and these days hundreds of thousands of plant species have 89 been tested for their medicinal properties (6). However, the phytochemical and 90 pharmacological activities of many more plants remain to be studied. Plant-derived 91 substances are tolerated and accepted by patients and seem a reliable source of antimicrobial 92 compounds (6).

93 Medicinal plants are commonly used worldwide as alternative treatments for mental and 94 physical illnesses. Herbal formulated medicines and traditional health practices are

Page | 3 95 considered more affordable and accessible to most rural societies than modern drugs (7). The 96 World Health Organization (WHO) estimated that about 65% of the world population use 97 medicinal plants for their primary health care. In addition, approximately 39% of the drugs 98 developed since 1980 have been derived from plants and their derivatives (8).

99 Traditional medicine has long been established in the culture of Ethiopian communities (9). 100 In rural areas this includes the use of plant-based treatments of inflammation, wounds and 101 infection (10). Records from as far back as the fifteenth century detail traditional medical 102 practices and remedies obtained from oral tales, early medico-religious manuscripts, and 103 traditional pharmacopeia (7). The antimicrobial activities of these traditional medicinal 104 plants are based on their secondary metabolites such as alkaloids, terpenoids, flavonoids, 105 tannins and glycosides (11).

106 Many in vitro antibacterial and antifungal studies have been conducted on the safety and 107 efficacy of Ethiopian medicinal plants used to treat bacterial and fungal infections. However, 108 data on the efficacy and safety of these medicinal plants in the management of wound 109 infection have not yet been summarised. This systematic review draws together up-to-date 110 information on Ethiopian medicinal plants used as antibacterial and antifungal agents that 111 might potentially be used for the management of wound infections in lymphoedema.

112 The aim of this systematic literature review was to evaluate Ethiopian medicinal plants found 113 to have antibacterial and antifungal properties in in vitro studies.

114 In the context of this review, terms are defined as follows:

115 ‘Ethiopian medicinal plants’ refer to plants that are found in Ethiopia and have been utilized 116 traditionally for medicinal purposes by societies in Ethiopia and elsewhere.

117 ‘In vitro anti-infective activity tests’ involve direct culture of the microorganisms in media 118 and application of plant extracts to the media to evaluate their activity.

119 ‘Anti-infective agents’ refer to agents (medicinal plant extracts, fractions, and/or compounds) 120 that act against infective agents (bacteria, fungi and others) either by inhibiting the agent’s 121 growth or by killing it.

122 2. Methods 123

Page | 4 124 To ensure inclusion of relevant information, the study was undertaken based on the guideline 125 of Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) (9). The 126 protocol for this review was registered on PROSPERO, with registration number 127 CRD42019127471 (12).

128 2.1. Study design 129

130 This systematic review considered all controlled in vitro antibacterial and antifungal studies 131 the activities of Ethiopian medicinal plants. The components population, exposure 132 (intervention), comparator and outcome (PICO) of this review are given underneath:

133 Study subjects: Microorganisms (bacteria and fungi) that were used for anti-infective 134 efficacy tests of medicinal plants.

135 Intervention: Medicinal plants as whole plants or their adjuncts: seed, root, flower, bud and 136 leaf extracts used in the experimental groups. The intervention products used were 137 manufactured from a single or complex medicinal plant, plant extracts, or plant preparations, 138 regardless of the types of the preparations (extracts, decoctions, tablets, capsules, pills, 139 powders, injections or other types of preparations), but not synthesized compounds. There 140 were no restrictions on dosage form, concentration, frequency of administration, dose, 141 intensity or duration of medicinal plants used.

142 Comparator: Placebo (no intervention) or conventional (reference) drugs used for treatment 143 of controls.

144 Outcomes: Outcomes were the rate of response to treatment, such as efficacy of medicinal 145 plants in inhibiting or killing microbial growth (in culture media) in comparison with 146 conventional drugs.

147 2.2. Eligibility criteria 148

149 Inclusion criteria: Published works including theses, articles and proceedings in English 150 until June 2019 that deal with efficacy evaluation of antibacterial and antifungal activities in 151 in vitro studies.

152 Exclusion criteria: Newspapers and reviews,

Page | 5 153 2.3. Information sources 154 Electronic databases were looked at using a combination of free text keywords and Medical 155 Subject Heading (MeSH) terms related to Ethiopian restorative plants investigated to have 156 anti-inflammatory, anti-infective and wound healing activities. Scopus, Embase, 157 PubMed/Medline and Google Scholar were utilized as sources of data for the search. Grey 158 literature such as theses, technical reports, working papers, evaluation reports, conference 159 proceedings, patents, and preprints was too included in the review.

160 2.4. Search strategy 161 The search approach covered all articles containing descriptors available till June 28, 2019. 162 Only articles composed in English have been used on this study. Structured search methods 163 have been developed using the lexical terms of each database and targeting the “title” and 164 “abstract” fields. We additionally searched manually using the references of already 165 distributed works. The following search terms were used: Ethiopia, medicinal plants, 166 Ethiopian medicinal plants, herbal products, care, management, therapeutic, lymphoedema, 167 lymphedema, swelling, podoconiosis, elephantiasis, bacteria, anti-bacterial, fungi, anti- 168 infective, antimicrobial, anti-fungal and different associated words or phrases.

169 2.5. Selection of studies

170 After electronic searching, the records were uploaded to Mendeley. Some studies were 171 screened before being totally selected. All medicinal drug and antifungal studies were 172 screened severally by two investigators (DN and TB by scanning the titles and abstracts of 173 the articles that supported the inclusion criteria. For documents that fitted the inclusion 174 criteria, the investigators scan the whole article to substantiate whether or not it met the 175 criteria. Disagreements were resolved by discussion between the two investigators.

176 2.6. Data extraction and management 177 Two reviewers (DN and TB) independently extracted data using a data extraction form. We 178 performed a standardization exercise before beginning the review to make ensure consistency 179 between the reviewers. The subsequent information were extracted: title, author, year of 180 publication, statistical methods used, study duration, type of microorganism used for the 181 study (clinical isolates or reference strains), concentrations of the plant extracts used for 182 activity evaluation, reference drugs used, minimum inhibitory concentration (MIC) of 183 extracts/fractions (µg/mL, mg/mL), zone of inhibition (mm), concentrations that inhibit 184 microbial growth of the extracts/fractions, type of solvent extracts and fractions used for

Page | 6 185 activity and safety, parts of plant used, extraction type, sources of the plants, place of 186 collection, traditional use, scientific names of the plants, local names of the plants, voucher 187 numbers, types and number of compounds isolated (if any). When individual studies had 188 multiple treatments, groups were combined to avoid the possibility of introducing bias caused 189 by multiple statistical comparisons with one control group (9).

190 2.7. Outcomes measured

191 For the in vitro studies of antibacterial and antifungal activities, the most outcomes measured 192 were the percent of inhibition of growth of microorganisms, minimum inhibitory 193 concentration, and concentration needed to inhibit multiplication of micro-organisms by 50%

194 (IC50).

195 2.8. Assessment of risk of bias 196

197 Two review authors (DN and TB) independently assessed the risk of bias for each included 198 study. The internal and external validity of each study was evaluated using this tool. For the 199 in vitro antibacterial and antifungal studies, a good practice for pharmaceutical microbiology 200 laboratories guidelines (WHO) was used for quality assessment (13,14). Criteria used to 201 assess the quality of in vitro individual studies were reported in Nigussie et al. (12).

202 Then, the risk of bias criteria was judged as ‘low’, ‘high’ or ‘unclear’. Studies with a low and 203 medium risk of bias were considered for analysis, whereas high risk of bias studies were 204 omitted from the analysis.

205 2.9. Data synthesis and analysis 206 All studies included for data synthesis were classified into two different experimental models, 207 i.e., antibacterial and antifungal activity studies, in keeping with the kind and purpose of the 208 studies. Heterogeneity was evaluated descriptively from the narrative synthesised data, and 209 potential reasons for heterogeneity were identified by examining individual study and 210 subgroup characteristics. Interventional, methodological and statistical heterogeneity was 211 apparent among the studies. Consequently, statistical pooling of studies for meta-analysis was 212 not possible.

213 Instead, a narrative (qualitative) overview of the studies was conducted using textual 214 descriptions of studies, grouping, and tabulation. Then, a detail of the characteristics of the 215 studies compared the effect of each plant extract relative to controls, the main parameters

Page | 7 216 measured/analysed, the quality of included studies and the risk of bias of all studies was 217 described.

218 3. Results 219 220 3.1. Literature search results and description of study characteristics 221

222 In the search of Ethiopian medicinal plants used for their anti-inflammatory, wound healing 223 or anti-infective activities, a total of 2330 relevant articles were independently identified by 224 two reviewers for preliminary review from electronic and manual searches. After removal of 225 duplicates by reviewing relevant titles and abstracts, a total of 330 articles on antibacterial 226 and antifungal activities were retrieved for full text review. After a detailed review of each 227 article, 234 articles were excluded and a total of 96 articles were retained: anti-bacterial 228 activity (n=79) and anti-fungal activity (n=17) (Figure 1 and supplementary table 1, 2 and 3).

229 3.2. Excluded studies

230

231 In this review, we have identified that there were many studies conducted in these areas. 232 However, most of the published articles did not meet the inclusion criteria due to i) 233 Incomplete information: not reported the concentration of plant extracts used, the number of 234 experimental duplicates, the method of outcome measurement, the negative and positive 235 controls used, the sources and quality control of micro-organism, the time at which the 236 outcomes were measured, the sources of the cell lines, and statistical methods used for data 237 analysis; ii) Not relevant studies: clinical trials; studies conducted on medicinal plants which 238 are not growing in Ethiopia, activity was not conducted for human pathogens (animal and 239 plant pathogens).

240

241 3.3. Studies included for the antibacterial activity of medicinal plants

242 i. Characteristics of the studies 243

244 For the anti-bacterial studies, 79 studies were eligible for data extraction. The year of 245 publication ranged from 2003 to 2019. A total of 76 peer-reviewed full articles and 3 MSc 246 theses were included. The seventy-nine studies were conducted in Ethiopia (48), India

Page | 8 247 (eight), Kenya (seven), Iran (three), Sudan (three), Cameroon and South Africa (two each), 248 China (one), Oman (one), Malaysia (one), Nigeria (one), Pakistan (one), The Netherlands 249 (one) and Tunisia (one) (Supplementary table 1 and 3).

250 All the studies designs met the inclusion criteria and tests were performed according to the 251 procedures described in the national, regional and international guidelines. The titles of the 252 studies met the objectives stated in the studies. Of the 79 studies, 36 used agar well diffusion 253 techniques with micro-dilution assay (MIC and MBC), and 28 used paper disc diffusion 254 method with micro-dilution assay (MIC and MBC). Two of the studies used agar-well 255 diffusion alone, while 9 used microdilution methods for minimum inhibitory concentration 256 and minimum bactericidal assays together with other methods, colorimetric assay and crystal 257 violet assay methods.

258 A total of 150 plant species and 4 compounds were tested and all except two plant species 259 were identified and authenticated by a botanist. Leaves were the most used plant parts for 260 anti-bacterial tests (n=82) (Table 1). All essential oils were extracted by steam distillation 261 with a Clevenger-type apparatus, while maceration and Soxhlet techniques were the most 262 frequently used techniques to extract plant materials.

263 A total of 25 gram-negative and 17 gram-positive bacteria were tested in the studies. Most of 264 the microorganisms tested were American Type Culture Collection (ATCC) reference 265 microorganisms and some were clinical isolates from samples. Among the gram-negative 266 bacteria, Escherichia coli was tested against more than 70 types of medicinal plants, followed 267 by Pseudomonas aeruginosa, Klebsiella pneumoniae and Salmonella typhi tested, which 268 were tested against thirty-nine, twenty-eight and twenty-two medicinal plants, respectively. 269 Among the gram-positives, Staphylococcus aureus was the most tested bacteria and was 270 tested against sixty-six medicinal plants. Others included Bacillus subtilis (twelve), 271 Streptococcus pyogenes (ten), and Enterococcus faecalis (eight) (Supplementary table 1).

272 Twenty-six studies used SPSS statistical software for data analysis. One-way ANOVA was 273 used to test the existence of statistically significant differences between mean zones of 274 inhibition of controls and test substances. However, 45 studies did not report the statistical 275 method used. The rest used the unpaired Student t-test to test the differences between 276 treatment and control arms (Supplementary table 1).

Page | 9 277 278 ii. Main parameters analysed 279

280 For agar well and paper disc diffusion assay methods, the outcome measured at each test 281 level was the diameter (mm) of the zone of inhibition of the control and experimental tests 282 using a calibrated distance measuring instrument. The time of measurement for all included 283 studies was after 24h exposure to reference and test substances. For the microdilution 284 methods (MIC and MBC), colour change for colorimetric assays or bacterial growth for non- 285 colorimetric methods (visually identified as clear or turbid solution in test tubes after 24h 286 treatment) were used.

287 A wide range of concentrations and different types of units of measurements were used 288 across the studies. Among the medicinal plants tested against the microorganisms, at least 289 one plant constituent was active against bacteria. The units used to describe MIC were 290 µg/mL, µL/mL, mg/mL, µg/disc, %(w/v) and ppm. Similarly, for the measurement of zone of 291 inhibition of bacterial growth, millimetre (mm) and millimetre squared (mm2) were used.

292 The lowest concentration (8µg/mL) of plant material that inhibited the growth of 293 microorganisms was reported by Chaieb et al. (15). Eleven human pathogenic strains were 294 tested against thymoquinone, a constituent of the black seed of Nigella sativa L. It was shown 295 to inhibit the growth of Bacillus cereus ATCC 14579 and S. epidermidis CIP 106510 at 8 296 µg/mL and S. aureus ATCC 25923 16µg/mL (15). Ameya et al. (16) reported the minimum 297 inhibitory concentration of the alcoholic extract of Echinops kebericho Mesfin against S. 298 aureus, E. coli and E. faecalis, which ranged from 3.12 µg/mL to 12.5 µg/mL (17). Similarly, 299 Oumer et al. reported the MIC values of latex of Aloe trichosantha Berger , which ranged 300 from 10µg/mL to 100µg/mL for bacterial species such as bacillus species, E. coli species, 301 Salmonella species, Shigella species, S. aureus and V. cholerae (16) (Supplementary table 1).

302 Minale et al. reported the MIC values of Aloe sinana Reynolds and its compounds, 303 Microdontn, Aloin and Aloinoside ranged from 10 to 50 µg/mL for the leaf latex, 10-25 304 µg/mL for Aloinoside, 5-200 µg/mL for Microdontn, 10-200 µg/mL for Aloin against gram- 305 negative and gram-positive bacterial species. This was shown to have a strong anti-bacterial 306 activity in comparison to the positive control, ciprofloxacin (18). Gadissa et al. tested the 307 essential oils of Blephari scuspidata, Boswellia ogadensis Vollesen and Thymus schimperi 308 Ronniger (19). The MICs of Blepharis cuspidate against S. aureus (ACCT & MRSA), E. coli

Page | 10 309 (MDR) and K. pneumonia (MDR) were 1.56 µL/mL, 12.5µL/mL and 3.12µL/mL, 310 respectively, which is comparable to ciprofloxacin activity (19).

311 Similarly, Thymus schimperi Ronniger was reported to have MICs of 3.12µL/mL, 6.5l/mL, 312 3.12µL/mL against S. aureus (ATCC & MRSA), E. coli (ACCT & MDR) and K. pneumoniae 313 (ATCC & MDR), respectively, and the minimum bactericidal concentration (MBC) ranged 314 from 3.12-12.5µL/mL. In addition, Boswellia ogadensis Vollesen was shown to have MIC 315 values of 3.12 µL/mL, 6.25 µL/mL, 3.12 µL/mL against S. aureus (ATCC & MRSA), E. coli 316 (ACCT & MDR) and K. pneumoniae (ATCC & MDR), respectively, and MBC ranged from 317 6.25 µL/mL to 12.5 µL/mL (19).

318 The MIC values of Boswellia ogadensis Vollesen and T.schimper essential oil combination 319 against S. aureus (ATCC & MRSA), E. coli (ACCT & MDR), K. pneumoniae (ATCC & 320 MDR) were 3.12, 6.25 and 1.56 µL/mL, respectively. The MBC ranged from 1.56 µL/mL to 321 25 µL/mL. Similarly, the combination of essential oil of T. Schimper and B. cuspidate 322 showed significant activity against S. aureus (ATCC & MRSA), E. coli (ACCT & MDR) and 323 K. pneumoniae (ATCC & MDR) with MIC of 0.39, 1.56, and 0.39 µL/mL, respectively, and 324 with MBC values ranges from 0.39µL/mL to 3.12µL/mL. The combined activities of 325 essential oils of B. cuspidate and B. ogadensis showed similar activity against S. aureus 326 (ATCC & MRSA), E. coli (ACCT & MDR) and K. pneumoniae (ATCC & MDR) with MICs 327 of 1.56, 6.25, and 0.78µL/mL, respectively. The MBC ranged from 1.56µL/mL to 25µL/mL. 328 These essential oils were shown to have comparable activity to ciprofloxacin (19).

329 Habbal et al. reported that 50% ethanol extracts of Lawsonia inermis L. demonstrated 330 antibacterial activity against a wide range of gram-negative and positive bacterial strains with 331 the highest antibacterial activity against P. aeruginosa (20). Nagarajan et al. reported that 332 ethanol, chloroform, hexane and methane extracts of L. inermis showed nearly equal zones of 333 inhibition against B. subtitles, S. aurous, and E. coli at 400mg/kg comparable to that of 334 tetracycline (21). Ethanol, methanol, and ethyl acetate extracts of Azadiractha indica were 335 reported by Maleki et al. to have a wider zone of inhibition against P. aeruginosa, S. aureus 336 and E. faecalis at 300mg/mL. The extracts had bactericidal activity against both reference 337 and clinical isolates of S. aureus and P. aeruginosa, and bacteriostatic activity against E. 338 faecalis (22).

339 The degree of bacterial growth inhibition, as determined by values of diameter of inhibition 340 zone (IZ) of the respective plant extracts, varied among the extracts and microorganisms. The

Page | 11 341 widest inhibition was reported by Bacha et al. who showed the inhibitory zones of petroleum 342 ether extract (500 mg/mL) of seeds of Nigella sativa L. to be 44 ± 0.31 mm against Bacillus 343 cereus and 40 ± 2.33 mm against B. cereus ATCC 10987 compared to that of gentamycin (29 344 mm) (23). Wide zones of inhibition were recorded for the petroleum ether extract of stem of 345 Kosteletzkya begonifolia and stem of Leucas marthineensis against E. coli, S. typhimurium, S. 346 aureus and P. aeruginosa at all concentrations, comparable to ciprofloxacin (24). In another 347 study, acetone extract of Capsicum frutescens L. against ATCC S. aureus at a concentration 348 of 0.1 mg/mL was reported to produce an inhibitory zone of 28 mm (25).

349

350 iii. Quality of included studies (bias analysis) 351

352 Critical appraisal of the studies included was done using the checklist for Good In Vitro 353 Method Practices (OECD) and the WHO Good Practice for Microbiology Laboratory. Seven 354 main criteria were used to evaluate the validity of methodological and reporting qualities 355 (details are in the Methods section) (Supplementary 1).

356 Under the main checklist there were thirty criteria to evaluate the internal validity of the 357 studies. Studies with unacceptable levels of bias were excluded. However, the studies 358 included still had some weaknesses in reporting the status of microbiology facilities, regular 359 equipment, apparatus maintenance and calibration. In addition, there was lack of clarity as to 360 whether the test methods were validated or not; and there was also lack of evidence as to 361 whether the microbiological tests were performed and supervised by an experienced person 362 qualified in microbiology or equivalent, and whether the opinions and interpretations of test 363 results in reports were done by authorized personnel with suitable experience and relevant 364 knowledge.

365 There was also some methodological weakness. For instance, the number of replicates for 366 each testing condition, including concentration level(s) used for the reference and control 367 item(s), and test items were not specified in some studies. None of the studies reported the 368 applicability domain of the in vitro methods or any limitations or exceptions to the methods. 369 Four studies did not report complete information about the degree of inhibition of bacterial 370 growth and the concentrations by the respective medicinal plants, and one study did not 371 mention the unit of measurement of the zone of inhibition by plant extracts.

Page | 12 372 We categorized the judgment of bias as ‘yes’, ‘no’ or ‘unclear’. A “yes” judgement indicated 373 a low risk of bias; and a “no” judgment indicated high risk of bias; the judgment was 374 “unclear” if insufficient details were reported to assess the risk of bias properly.

375 3.4. Studies included on anti-fungal activity of medicinal plants 376 i. Characteristics of the studies 377

378 Seventeen studies that evaluated anti-fungal activities of Ethiopian medicinal plants were 379 included. The year of publication of the studies included ranged from 2000 to 2018, and 380 studies were conducted in six different countries, Ethiopia (n=7), Kenya (n=1), India (n=5), 381 Colombia (n=1), Lithuanian (n=1), Republic of Korea (n=1), and Romania (n=1). Sixteen 382 studies were peer reviewed full articles and one was an MSc thesis. Five studies used 383 microdilution assay, two used agar well diffusion method, and twelve used both methods 384 (Supplementary table 2).

385 Medicinal plants claimed to have anti-fungal activities were tested against different fungal 386 species and one species of yeast. These were Candida albicans, Aspergillus species, 387 Trichophyton species, Microscopium species, Penicillium species, Fusarium species, 388 Epidermophyton species and Rhodotorula rubra. Aspergillus species (n=18) were the most- 389 studied fungi, followed by Trichophyton species (n=13) and Candida albicans (n=10).

390 A total of forty-three different species of medicinal plants were tested against different fungi, 391 and all of them identified and authenticated by botanists and with voucher numbers (Table2).

392 Leaves (n=21), seeds (n=6), roots (n=4), stem bark (n=2), aerial parts (n=2), cloves (n=2), 393 bulbs (n=1), fruits (n=4), were the plant parts used (Table 2). The maceration technique was 394 the most frequently used method of extraction for plant extracts, followed by Soxhlet. Steam 395 distillation with Clevenger-type apparatus was used for extraction of essential oils. Hydro- 396 alcohol solvents were the most frequently used solvents for the extraction of plant materials 397 followed by aqueous solvents.

398 Six studies used the agar well diffusion (AWD) method, six the micro dilution (MID) method 399 and five both methods. For both experimental methods, the duration of exposure of the 400 microorganisms to the extracts ranged from 2-7 days incubation time; and outcomes were 401 measured after this. Zone of inhibition of fungal growth, turbidity (visually) and anti-fungal 402 activity index (%) were the outcomes measured in the included studies. All the measurements 403 were replicated three times and the results were presented as mean ± SD. One-way ANOVA

Page | 13 404 followed by Tukey’s test was used to compare extraction solvents and the difference in the 405 sensitivity of the test microorganisms.

406 i. Main parameters

407 The antifungal activity of plant extracts was measured in a similar way as that of the anti- 408 bacterial activity. These were zone of inhibition of fungal growth for the agar well and paper 409 disc diffusion methods and fungal growth which distinguished clear and turbid solutions for 410 the micro-dilution methods, measured after incubation periods.

411 A wide range of concentrations and units of measurement were used across the studies. For 412 the MIC and minimum fungicidal concentration (MFC) mg/mL, µg/mL and activity index in 413 percent (%), and mm was used for the measurements of ZI in AWD assays.

414 The activity index of the extracts was determined using the following formula:

415 Ameya et al. (16) reported that the methanol extract of Echinops kebericho Mesfin against 416 Aspergillus flavus and Candida albicans had MICs of 6.25 µg/mL and 3.12 µg/mL, 417 respectively; and the MFC of methanol extract to be 12.5 µg/mL and 6.25 µg/mL against A. 418 flavus and C. Albicans, respectively. The ethanol extract had MICs of 12.5 µg/mL and 6.25 419 µg/mL against A. flavus and C. albicans, respectively with fungicidal activity of 22.92 µg/mL 420 and 12.50 µg/mL, respectively. The zone of inhibition of the methanol extract against C. 421 albicans and A. flavus were 18.6 ± ... mm and 20.33 ± 0.57mm, respectively. In this study, 422 ethanol and methanol extracts of E. kebericho were shown to have comparable activity with 423 ketoconazole (Supplementary table 2).

424 Kasparaviciene et al. evaluated the activity of oleo-gels formulated with different 425 concentrations of thyme essential oil. The MIC value of 0.25% essential oil of thyme in oleo- 426 gels against C. albicans was 0.05% (26). In another study, the antifungal activity of T. 427 vulgaris essential oil against dermatophytic fungi was reported by Neetu et al. to have a very 428 strong antifungal activity at low concentrations. The MIC values ranged from 0.05µL/mL to 429 0.1µL/mL; and the MFC ranged from 0.05 µg/mL to 2 µg/mL against the dermatophtic fungi 430 (27) (Supplementary table 2 and 3).

431 The seed extracts of Trachyspermum ammi (L.) Sprague (0.2 mg/mL) and the leaf extract of 432 Cestrum nocturnum L. (0.2 mg/mL) exhibited the widest zones of inhibition, at 38.3 mm and 433 31.3 mm, respectively against C. albicans. Similarly, the methanol extract of E. kebericho 434 exhibited ZI of 20.33 ± 0.57 mm against C. albicans and 18.6 mm against A. flavus

Page | 14 435 (Supplementary table 2). Salazar et al showed that leaf and seed oil extracts of neem tree 436 inhibited the growth of Trichophyton menta, Trichophyton rubrum, Epidermophyton floccos 437 and Microsporumcanis. Whereas Simhadri et al reported that the aqueous extract of 438 Azadirachta indica A.Juss. leaves had superior activity against T. rubrum, M. gypseum, E. 439 floccosum, and Candida species (28).

440 ii. Quality of included studies (Bias analysis)

441 Checklists employed for antibacterial studies were also used in the antifungal studies. There 442 were gaps in methodology as well as in reporting and interpreting the outcomes. Validation 443 of the test methods before conducting the experiments was not reported for all included 444 studies, and eight studies did not report the statistical methods used.

445 There was no evidence that the anti-fungal activity tests were performed or supervised by an 446 experienced person qualified in microbiology or equivalent. Similarly, there was no report on 447 whether the microbiology facilities were fit for purpose or detailed description of the 448 workflow for the microbiology methods and related processes. Furthermore, nine studies did 449 not report the statistical methods used, not expressed an estimate of the uncertainty of the test 450 result on the test report, and limitations of the test were not reported clearly.

451 4. Discussion 452

453 The purpose of this review was to demonstrate the activities of Ethiopian medicinal plants as 454 antimicrobial agents that might potentially be used for limb care (particularly, of tropical 455 lymphoedema and associated wounds). This section discusses the efficacy of plant extracts 456 and their secondary metabolites investigated as antibacterial and antifungal, and the most 457 frequently used models.

458 This systematic review identified a total of 96 articles covering two different experimental 459 models, i.e., 79 antibacterial activity and 17 antifungal activity models. Overall, medicinal 460 plant extracts tested for these two conditions in in vitro were shown to have good activity. 461 Despite the heterogeneity of the studies, all plant extracts investigated succeeded in inhibiting 462 bacterial and fungal growth.

463 In this review of antibacterial activity, a total of 171 medicinal plants and three compounds 464 were investigated against 25 gram-negative and 17 gram-positive bacteria using agar well 465 diffusion, paper disc diffusion, broth micro/macro-dilution and agar dilution method. A

Page | 15 466 summary of plant species whose extracts and their isolated compounds were shown to have 467 significant in vitro activity against bacteria is the focus for our discussion.

468 Chaieb et al. reported the MIC of thymoquinone, constituent of N. sativa, which was shown 469 to have MIC of 32 µg/mL against V. parahaemolyticus ATCC 17802 and E. faecalis ATCC 470 29212, 16 µg/mL against L. monocytogene ATCC 19115, 8 µg/mL against B. cereus ATCC 471 14579, S epidermidis CIP 106510, M. luteus NCIMB 8166, S. aureus ATCC 25923 and S. 472 epidermidis CIP 106510 in a broth microdilution assay method. Its activity was shown to be 473 similar to the standard drugs gentamycin and erythromycin (15). This finding agrees with the 474 report of Kokoska et al., who tested the essential oil of N. sativa seed against gram-positive 475 bacteria. Thymoquinone, the main constituent of the essential oil, was shown to have a potent 476 bacteriostatic effect with MIC ranging from 8 to 64 µg/mL in broth microdilution method 477 (29). However, E. coli ATCC 35218, S. enterica serovar Typhimurium ATCC 14028, and P. 478 aeruginosa ATCC27853 were found to be resistant to this compound (MIC > 512 µg/mL) 479 (29).

480 Ameya et al. tested the alcoholic extract of E. kebericho against S. aureus, E. coli and E. 481 faecalis and demonstrated significant activity with MIC ranged from 3.12 µg/mL to 12.5µ 482 g/mL using AWD, while E. coli was found to be resistant (16). Anwar et al. assessed the 483 antimicrobial activity of latex of Aloe trichosantha A. Berger and its compounds (aloin A/B 484 and aloin-6’-O- acetate A/B), which were effective against E. coli, Salmonella and V. 485 cholerae strains with an average MIC value of 25 µg/ml. The activities of the test substances 486 could be due to changes to cell wall integrity, enzymatic activity and protein inactivation in 487 the microorganisms (30).

488 Minale et al. performed anti-bacterial activity tests on Aloe sinana and its compounds 489 (Microdontin, Aloin and Aloinoside) against 21 strains of bacteria using the disk diffusion 490 method. The leaf latex showed high inhibitory activities against B. pumillus 82, B. subtilis 491 ATCC 6633 and S. aureus ML 267, E. coli K99, E. coli K88, E. coli CD/99/1, E. coli LT37, E. 492 coli 306, E. coli 872, E. coli ROW 7/12, E. coli 3:37C, S. enterica TD 01, S. typhi Ty2, S. 493 boydii D13629, S. dysentery 8, S. flexneri Type 6, S. soneii 1, V. cholerae 85, V. cholerae 494 293, V. cholerae 1313 and V. cholerae 1315 at a concentration of 200 µg/mL, which showed 495 comparable activity to the standard drug ciprofloxacin. Similarly, compounds isolated from 496 Aloe sinana were shown to have high activity against E. coli, S. typhi Typ 2, Shigella, S. 497 aureus and V. cholerae, comparable to the reference drug, ciprofloxacin (18). The leaf latex’s

Page | 16 498 action was due to the secondary metabolite anthraquinones, which possess a range of 499 functional groups and have the ability to disrupt bacterial cell wall permeability and inhibit 500 nucleic acid synthesis and then cause death of the microorganism (31,32).

501 According to Gadisa et al., combined essential oils of oregano-basil, basil-bergamot, 502 oregano-bergamot and oregano-perilla have significant activity against S. aureus, E. coli, B. 503 subtilis and S. cerevisiae, respectively. The synergistic effect of these essential oils may be 504 due to synergistic or additive interactions between different classes of compounds such as 505 phenols, aldehydes, ketones, alcohols, esters, ethers or hydrocarbons, which might act on the 506 same target or different targets (19). This finding is consistent with Nasir et al. (33), who 507 postulated that the ability of plant extracts to act synergistically with antibiotics and other 508 plant extracts could be considered a new approach to combat antimicrobial resistance. There 509 is low risk of bacterial resistance in plant extracts and antibiotics combinations, due to the 510 varied modes of action of the compounds present in the extracts, to which the organism had 511 never been exposed before (33) .

512 Antibacterial activity of Lawsonia inermis L. was also reported against wide range of gram- 513 positives and gram-negatives (34,35). This could be due to the presence of a compound, 2- 514 hydroxy-1,4- naphthoquinone. Quinones are the main constituent in the leaves of L. Inermis 515 L. and are known in making complexing irreversibly with nucleophilic amino acids, leading 516 to inactivation of the protein and loss of function in microorganisms. Cell wall adhesions, 517 polypeptides and membrane bound enzymes are the targets in microbial cells (35).

518 In another anti-microbial study, the leaf and stem bark extracts of Azadirachta indica A.Juss. 519 exhibited significant antibacterial activity against a wide range of bacteria due to the tricyclic 520 diterpenoids isolated from stem bark, and azadirachtins, quercetin and β- sitosterol isolated 521 from the leaves (36).

522 Bacha et al tested 18 plant extracts against E. coli K12 DMS 498, S. aureus DMS 346, B. 523 cereus ATCC 10987, B. cereus, Lab strain and P. aeruginosa 1117 using AWD and MID 524 methods. The highest ZI was recorded with petroleum ether extract of N. Sativa against B. 525 cereus and B. cereus ATCC 10987; and mature unripe fruit oil of Aframomum corrorima (A. 526 Braun) P. C. M. Jansen against S. aureus. The activities of petroleum ether extract of seed of 527 N. sativa against both laboratory isolated and reference strain of B. cereus were greater than 528 the activity of gentamycin sulphate. The oil extract of unripe fruit of A. corrorima was shown 529 to have an activity comparable to the reference drug gentamycin sulphate. P. aeruginosa was

Page | 17 530 the most resistant to all the plant extracts tested in this study (23). The antimicrobial 531 activities of extracts of A. corrorima, Nigella sativa L., A. angustifolium and V. amygdalina 532 were due to the presence of phenol, tannin, saponin and flavonoids, flavonoids and terpenoids 533 compounds and their combinations (23). The antibacterial activity of flavonoids is well 534 documented and found in almost all parts of the plants, which inhibit the energy metabolism 535 and synthesis of nucleic acids of different microorganisms (37). Furthermore, tannins were 536 reported to have antibacterial activity against S. aureus, acting by inducing complexation 537 with enzyme or substrates and causes toxicity; and altering the membrane of the microbes 538 (38).

539 Many studies have been carried out to screen medicinal plants for their antifungal activity, 540 and various groups of researchers have initiated antifungal programs for traditionally used 541 plants. Classes of compounds from plant metabolites, such as terpenoids (isoprenoids), 542 saponins, phenolic compounds, flavonoids, coumarins, alkaloids, proteins and peptides 543 showed anti-fungal activity against different fungal species (39,40). Under this review, 15 544 studies were included comprising 46 species of plant extracts against 50 species of fungus 545 using agar well diffusion, disc diffusion, macro/microdilution and agar dilution methods.

546 Alcoholic extracts (methanol and ethanol) of E. kebericho were tested by Ameya et al. 547 against A. flavus and C. albicans using disc diffusion and agar dilution methods, and shown 548 to cause significant inhibition at low concentration, comparable to the reference drug 549 ketoconazole. The alcoholic solvents have the ability to extract phenolic compounds such as 550 flavonoids, anthocyanins and phenolic acids which may contribute to the antifungal activity 551 of the extracts (17).

552 Kasparaviciene et al. tested the activity of oleo-gels, formulated with different concentrations 553 of thyme essential oil against C. albicans by broth dilution method, which showed significant 554 activity with MIC value of 0.25%. Thymol was reported the major constituent of the thyme 555 essential oil in this study. The biological activity of thyme essential oil depends on its yield 556 and chemical composition, and the essential oils have several chemical names depending on 557 the main constituents they have, such as thymol, carvacrol, terpineol, and linalool (26).

558 Similarly, Jain et al. reported the antifungal activity of T. vulgaris essential oil against T. 559 mentagrophytes MTCC 7687, M. gypseum MTCC 452, M. fulvumMTCC2837, T. rubrum 560 MTCC 296, T. soudanense (isolate) and T. interdigitale (isolate) using macro-dilution 561 method. T. vulgaris essential oil was shown to have significant activity against the

Page | 18 562 dermatophytes with MIC ranges from 0.02 to 0.1μl/mL (27). These activities could be due to 563 high content of phenolic compounds and potent vapour activity against dermatophytes (41). 564 This finding agrees with the report of Marina et al., which showed the activity of T.s vulgaris 565 essential oil against Alternaria alternata, Fusarium tricinctum, all Aspergillus species and 566 dermatomycetes at concentration of 0.25 µL/mL and Phomopsis helianthi and Cladosporium 567 cladosporioides at 0.125 µL/mL by macro-dilution method (41).

568 In another study, T. ammi seed extract exhibited potent antifungal efficacy, with a maximum 569 MZ of 38.3 mm diameter against C. albicans using the AWD method (42). This is in 570 agreement with the finding of Sharifzadeh et al., which evaluated T ammi essential oil against 571 C. albicans, which were fluconazole-resistant, with MIC values ranging from 300 to 400 572 µg/mL (43). The extracts of A. indica was also shown to have antifungal activity, attributable 573 to the terpenoids. The fractions of A. indica have complex mixtures of compounds reported to 574 have synergistic and additive effect of against fungus (44).

575 5. Conclusion 576

577 The present study showed that plant extracts and compounds traditionally used in Ethiopia 578 are promising anti-infective agents. In this review Calpurnia aurea (Aiton) Benth., Croton 579 macrostachyus Hochst. ex Delile, Withaniasomnifera, Achyranthes aspera L., Datura 580 stramonium L., Solanum incanum L., Verbascum erianthum Benth., Nigella sativa L., 581 Gymnanthemum amygdalinum (Delile) Sch.Bip., Oliniarochetiana, Sidarhombifolia, 582 Bersama abyssinica and Azadirachta indica A.Juss are the most studied plants species against 583 bacteria, and Azadirachta indica A.Juss and Lawsonia inerms against fungal species. 584 Thymoquinone, a constituent of the black seed of Nigella sativa L., alcoholic extract of 585 Echinop skebericho, Aloe sinana and its compounds (Microdontin, Aloin and Aloinoside), 586 alcoholic extract of Azadiractha indica and Lawsonia inerms are the most effective plant 587 materials against gram negative and gram-positive species. In addition, Azadiractha indica 588 and Lawsonia inerms have activity against a wide range of gram-negative and positive 589 bacterial strains. Similarly, methanol extract of Echinops kebericho Mesfin and oleo-gels 590 formulated with different concentrations of thyme essential oil are the most effective against 591 different fungal species.

592 5.1. Implications for future research and recommendations 593

Page | 19 594 It is vital to systematically summarize, and document medicinal plants tested against different 595 disease agents and used traditionally for treatment, and to test further their effectiveness 596 against a range of disease-related pathology such as wounds in patients with lymphoedema. 597 Information about many medicinal plants is fragmented, meaning that systematic compilation 598 and synthesis is important. The findings of this systematic review helped us in identifying 599 and prioritizing medicinal plants species for further investigation to determine their efficacy 600 as alternative therapy to microbial infections associated with lymphoedema.

601 Acknowledgements: We would like to thank CDT-Africa, Addis Ababa University and the 602 Ethiopian Public Health Institute for organising and sponsoring training on systematic review 603 and meta-analysis; and our special gratitude to Clare Callow, Grit Gansch and Manuela 604 McDermid for administrative support.

605 Author contributions: DN, GD, EM, TB, MB, BAL and AF were involved in 606 conceptualization and design of the study, and in collection, analysis and interpretation of the 607 data. DN wrote the first draft of the manuscript and DN, GD, EM, TB, MB, BAL and AF 608 critically reviewed the manuscript for intellectual content. All authors have read and 609 approved the final manuscript. 610 Funding: This research was funded by the National Institute for Health Research (NIHR) 611 Global Health Research Unit on NTDs at BSMS using UK aid from the UK Government to 612 support global health research. The views expressed in this publication are those of the author 613 (s) and not necessarily those of the NIHR or the UK Department for Health and Social Care.

614 Competing interests: None to be declared.

615 Ethics approval: Not applicable

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1023

1024 Table 1. Summary of common medicinal plants identified from literature search as antibacterial

S/N Plant species Family Life form Parts used Numb Ref. er of citatio ns 1 Aloe trichosantha Aloaceae Herb L latex 1 (30) A.Berger 2 Huernia hystrix (Hook.f.) Apocynaceae Herb S, R, W 1 (45) N.E. Br 3 Entada abyssinica Steud. Fabaceae Shrub Sb 1 (46) ex A.Rich. 4 Entada africana Guill. & Fabaceae Tree Sb 2 (46,47) Perr. 5 Carica papaya L. C(48–50)aricaceae Tree S 1 (51) 6 Persea americana Mill. Lauraceae Tree F 2 (46,47) 7 Calpurnia aurea (Aiton) Fabaceae Tree L, Ait, S, 3 (52–54) Benth. R, 8 Croton macrostachyus Euphorbiaceae Tree L, Sb 5 (55–57) Hochst. ex Delile 9 Withania somnifera (L.) Solanaceae Herb L 2 (52,58,59) Dunal 10 Achyranthes aspera L. Amaranthaceae Weed L 3 (50,60,61) 11 Brucea antidysenterica Simaroubaceae Tree R 1 (50) J.F.Mill. 12 Datura stramonium L. Solanaceae Weed L 3 (48–50) 13 Acokanthera schimperi Apocynaceae Tree L 2 (50,62) (A.DC.) Schweinf. 14 Phytolacca dodecandra Phytolaccaceae Tree R, F 2 (50,62) L’Hér. 15 Millettia ferruginea Fabaceae Tree L 1 (50) (Hochst.) Hochst. ex Baker 16 Solanum incanum L. Solanaceae Herb L 3 (50,63,64) 17 Cuminum cyminum L.. Umbelliferae Herb S 1 (65)

18 Trachyspermum ammi (L.) Apiaceae Weed S 2 (66,67) Sprague (L.) Sprague

Page | 34 19 Verbascum erianthum Scrophulariaceae Weed R, L 3 (53,68) Benth. 20 Kosteletzkya begonifolia Malvaceae Weed L 1 (24) (Ulbr.) Ulbr 21 Leucas martinicensis Lamiaceae Herb L 1 (24) (Jacq.) R.Br. 22 Ranunculus multifidus Ranunculaceae Herb L 1 (24) Forssk. 23 Eugenia caryophyllata Myrtaceae Tree L 1 (69) Thunberg 24 Origanum vulgare L. Lamiaceae Herb L 1 (69)

25 Thymus vulgaris L. Lamiaceae Herb L 1 (69) 26 Nigella sativa L. Ranunculaceae Herb S 3 (15,29,70)

27 Anthocleista schweinfurthii Gentianaceae Tree B, F & L 1 (71) Gilg 28 Caucalis melanantha Apiaceae Weed B, F & L 1 (71) (Hochst.) Hiern 29 Zehneria scabra (L.f.) Cucurbitaceae Herb W 1 (72) Sond. (L.f.) Sond. (L.f.) Sond. 30 Combretum molle R.Br. ex Combretaceae Shrub B 2 (73,74) G.Don 31 Rotheca myricoides Lamiaceae shrub L 1 (75) (Hochst.) Steane & Mabb. 32 Ficus palmata Forssk. Moraceae Shrub L 1 (75) 33 Grewia ferruginea A. Tiliaceae Shrub L 1 (75) Rich. 34 Periploca linearifolia Asclepediaceae Shrub L 1 (75) Quart. -Dill. & A.Rich. Quart. -Dill. & A.Rich. 35 Vernonia amygdalina (Syn: Asteraceae Shrub F 5 (9,23,70,76,77) Gymnanthemum amygdalinum) (Delile) Sch.Bip. 36 Clematis hirsuta Guill. & Ranunculaceae Climbers L 2 (78,79) Perr. 37 Cuminum cyminum L. Umbellifers Herb L 1 (80) 38 Calpurnia aurea (Aiton) Fabaceae Shrub L 3 (54,81,82) Benth. 39 Delonix elata (L.) Gamble Fabaceae Tree L 1 (83) 40 Spathodea campanulata Bignoniaceae Shrub L 1 (83) P.Beauv. 41 Kalanchoe petitiana A. Crassulaceae Shrub L 1 (53) Rich. 42 Lippia adoensis Hochst Verbenaceae Herb L 2 (53,64)

43 Olinia rochetiana A.Juss. Oliniaceae Shrub L, SB 3 (53,64,84) 44 Malva parviflora L. Malvaceae Herb R 1 (53) 45 Prunus africana (Hook.f.) Rosaceae Tree Sb 1 (85) Kalkman 46 Warburgia ugandensis Canellaceae Shrub Sb 1 (85) Sprague

Page | 35 47 Plectranthus barbatus Lamiaceae Shrub Sb 1 (85) Andrews 48 Embelia schimperi Vatke Myrsinaceae Shrub Sb, S 2 (9,86)

49 Clausena anisata (Willd.) Rutaceae Shrub St 1 (48) Hook.f. ex Benth. 50 Clematis simensis Fresen. Theaceae Shrub St, L 2 (76,87)

51 Rotheca myricoides Verbanaceae Shrub St 1 (76) (Hochst.) Steane & Mabb. 52 Juniperus procera Hochst. Cuprassaceae Shrub St 1 (76) ex Endl. 53 Justicia schimperiana Acanthaceae Shrub St 1 (76) (Hochst. ex Nees) 54 Olea europaea L. Oleaceae Shrub St 1 (76)

55 Phoenix reclinata Jacq. Arecaceae Tree St 1 (76)

56 Rubus apetalus Poir. Rosaceae shrub St 1 (76) 57 Sesbania sesban (L.) Merr. Leguminosae Tree St 1 (76)

58 Sida rhombifolia L. Malvaceae Shrub St, W, R 3 (64,76,88) 59 Spilanthes mauritiana Asteraceae Shrub F 1 (48) (Rich. ex Pers.) DC. 60 Stereospermum Bignoniaceae Shrub St 1 (48) kunthianum Cham. 61 Cymbopogon citratus Poaceae Herb L 3 (89–91) (DC.) Stapf 62 Echinops kebericho Mesfin Asteraceae Herb R, Tu 2 (16,92)

63 Costus speciosus (Koenig) Costaceae Herb Rh 1 (93) J.E. Smith 64 Taverniera abyssinica Fabaceae Herb R 1 (17) A.Rich. 65 Bersama abyssinica Melianthaceae Shrub L, S, Sb 3 (64,78,94) Fresen. 66 Carissa spinarum L. Apocynaceae Shrub R 1 (9) 67 Clutia abyssinica Jaub. & Euphorbiaceae Shrub R 1 (8) Spach 68 Cyathula cylindrica Moq. Amaranthaceae Herb R 1 (8) 69 Dodonaea viscosa Jacq. Sapindaceae Shrub L 1 (8) subsp. angustifolia (L.f.) J.G.West 60 Jasminum abyssinicum Oleaceae Shrub L 2 (9,95) Hochst. ex DC. 70 Maesa lanceolata Forssk. Primulaceae Tree L 1 (9)

71 Ocimum lamiifolium Lamiaceae Herb L 2 (9,96) Hochst. ex Benth. 72 Aframomum corrorima Zingiberaceae Herb F 1 (23) (A.Braun) P.C.M.Jansen 73 Albizia schimperiana Oliv. Mimosaceae Tree R 1 (23) 74 Curcuma longa L. Zingiberaceae Herb Rh 1 (23) 75 Erythrina brucei Schweinf. Fabaceae Herb Sb 1 (23) emend. Gillett 76 Justicia schimperiana Acanthaceae Herb S 1 (23)

Page | 36 (Nees) T. Anders. 77 Ocimum gratissimum Lamiaceae Herb L 2 (70) subsp. gratissimum 78 Ruta graveolens L. Rutaceae Herb L 1 (70)

79 Premna resinosa (Hochst.) Lamiaceae Shrub R 1 (97) Schauer 80 Hagenia abyssinica Rosaceae Herbal L, Sb 1 (98) (Bruce) J.F.Gmel. 81 Fuerstia africana Lamiaceae Herb Ae 1 (98) T.C.E.Fr. 82 Ekebergia capensis Sparrm Meliaceae Herb R 1 (98) 83 Asparagus racemosus Asparagaceae Herb Sb 1 (98) Willd. 84 Brassica nigra (L.) Brassicaceae Herb S 2 (99,100) W.D.J.Koch 85 Thymus schimperi Lamiaceae Herb L 1 (101) Ronnigeri Ronniger 86 Ocimum basilicum L. Lamiaceae Herb L 2 (102,103) 87 Syzygium aromaticum (L.) Lamiaceae Herb F 1 (69) Merr. & L.M.Perry (L.) Merr. & L.M.Perry 88 Elettaria cardamomum Zingiberaceae Herb F 1 (104) (L.) Maton 89 Cinnamomum verum Lauraceae Herb Sb 1 (105) J.Presl 90 Kniphofia isoetifolia Asphodelacea Weed R 1 (106) Hochst 91 Blepharis cuspidata Acanthaceae Herb L 1 (19) Lindau 92 Boswellia ogadensis Burseraceae Herb L 1 (19) Vollesen 93 Thymus schimperi Lamiaceae Herb L 3 (101,107,108) Ronniger 94 Artemisia abyssinica Asteraceae Herb L 1 (92) Sch.Bip. ex A.Rich. 95 Satureja punctata (Benth.) Lamiaceae Herb Ber 1 (91) R.Br. ex Briq.

96 Schrebera alata (Hochst.) Oleaceae Herb B 1 (109) Welw. 97 Ormocarpum kirkii Fabaceae Herb Ae 1 (109) S.Moore 98 Cussonia holstii Harms ex Araliaceae Tree B 1 (109) Engl. 99 Helichrysum forskahlii Asteraceae Herb W 1 (109) (J.F.Gmel.) Hilliard & B.L.Burtt 100 Aloe macrocarpa Tod. Aloaceae Herb L 1 (110)

101 Moringa stenopetala Moringaceae Tree L 1 (111) (Baker f.) Cufod. 102 Nicotiana tabacum L. Euphorbiaceae Herb L 2 (60,112) 103 Zehneria scabra (L.f.) Curbitaceae Herb L 1 (113) Sond. (L.f.) Sond. 104 Ricinus communis L. Euphorbiaceae Herb L 1 (113)

Page | 37 105 Cissus quadrangularis L. Vitaceae Herb Ae 1 (63)

106 Commelina benghalensis Commelinaceae Herb L 1 (62) L. 107 Euphorbia heterophylla L. Euphorbiaceae Herb R 1 (62) 108 Euphorbia prostrata Aiton Euphorbiaceae Herb W 1 (62)

109 Grewia villosa Willd. Malvaceae Shrub L 1 (62) 110 Momordica foetida Cucurbitaceae Shrub F 1 (62) Schumach. 111 Trianthema Aizoaceae Weed Ae 1 (62) portulacastrum L. 112 Schinus molle L. Anacardiaceae Tree L 1 (62) 113 Aloe sinana Reynolds Asphodelaceae Shrubs L 1 (18)

114 Maerua oblongifolia Capparaceae Shrub L, St 1 (114) (Forssk.) A.Rich. 115 Clematis longicauda Ranunculaceae Shrub L 1 (115) Steud. ex A.Rich. 116 Clematis hirsuta Guill. & Ranunculaceae Shrub L 1 (114) Perr.

117 Capsicum frutescens L. Solanaceae Herb F 1 (25) 118 Apodytes dimidiata E.Mey. Icacinaceae Tree Sb 1 (64) ex Arn. 119 Asparagus africanus Lam. Asparagaceae Herb L 1 (63) 120 Cucumis ficifolius A.Rich. Cucurbitaceae Herb R 1 (63) 121 Gladiolus abyssinicus Iridaceae Herb/shru Bu 1 (63) (Brongn. ex Lem.) b 122 Guizotia schimperi Asteraceae Herb L 1 (63) Sch.Bip. 123 Pavonia urens Cav. Malvaceae Herb/shru L 1 (63) b 124 Premna schimperi Engl. Verbenaceae Tree L 1 (63) 125 Pittosporum viridiflorum Pittosporaceae Shrub L 1 (63) Sims 126 Polygala sadebeckiana Polygalaceae. Herb R 1 (63) Gürke 127 Aloe harlana Reynolds Asphodelaceae Shrub L 1 (116) 128 Ficus carica L. Moraceae Tree L 1 (60) 129 Malva parviflora L. Malvaceae, Herb L 1 (60)

130 Solanum hastifolium Solanaceae Shrub L 1 (60) Hochst. ex Dunal 131 Calpurnia aurea (Aiton) Fabaceae Shrub L 1 (60) Benth. 132 Ziziphus spina-christi (L.) Rhamnaceae Shrub L 1 (60) Willd. 133 Hibiscus micranthus L.f. Malvaceae Shrub L 1 (117)

134 Linum usitatissimum L. Linaceae Herb S 1 (118)

135 Vernonia auriculifera Compositae Shrub L 1 (119) Hiern

Page | 38 136 Aloe pulcherrima Asphodelaceae Shrub L 1 (120) M.G.Gilbert & Sebsebe 137 Lepidium sativum L. Cruciferae Herb S 1 (121) 138 Foeniculum vulgare Mill. Umbelliferae Herb L 1 (122)

139 Solanecio gigas (Vatke) Asteraceae Shrub L 1 (95) C.Jeffrey 140 Lagenaria siceraria Cucurbitaceae Herb L, S, F 1 (94) (Molina) Standl.

141 Aloe trigonantha Aloaceae Shrub L 1 (123) L.C.Leach 142 Pycnostachys eminii Gürke Laminaceae Herb L, S, R 1 (124)

143 Moringa oleifera Lam. Moringaceae Tree S 1 (125)

144 Azadirachta indica Meliaceae Tree L 3 (36,126,127) A.JussA.Juss. 145 Pimenta racemosa (Mill.) Myrtaceae Tree L 1 (69) J.W.Moore 146 Nauclea latifolia Sm. Rubiacea Shrub B, F, L 1 (71)

147 Boehmeria virgata var. Urticaceae Herb W 1 (71) macrostachya (Wight) Friis & Wilmot-Dear

148 Erigeron floribundus Asteraceae Weed W 1 (71) (Kunth) Sch.Bip. 159 Zehneria scabra (L.f.) Cucurbitaceae Herb W 1 (71) Sond. (L.f.) Sond. 1025 Leaves = L, root = R, Stem Bark =SB, Fruits =F, Bark =B, Aerial = Ae, Flower = Fl, Stem =St, Rhizome = Rh, 1026 Bulbs = Bu, Seed = S, Berries = Be, Whole =W

1027

1028 Table 2: Summary of common medicinal plants identified from literature search for anti-fungal activity

S/N Plant species Family Life Parts Number Ref. form used of citations 1 Combretum molle R.Br. ex G. Combretaceae Tree Sb 1 (73) Don 2 Clerodendrum myricoides R. Laminaceae Tree L 1 (75) Br. 3 Ficus palmata Forssk. Moraceae Tree L 1 (75) 4 Grewia ferruginea A. Rich. Tiliaceae Tree L 1 (75) 5 Periploca linearifolia Quart. - Asclepeiaceace Shrub Ae 1 (75) Dill. & A.Rich. 6 Allium sativum L. Liliaceae Herb C 1 (128) 7 Allium schoenoprasum L. Liliaceae Herb C 1 (127) 8 Allium cepa L. Liliaceae Herb Bu 1 (127) 9 Acalypha indica L. Meliaceae Herb L 1 (127) 10 Azadirachta indica A.Juss Euphorbiaceae, Tree L 3 (28,44,129) 11 Camellia sinensis (L.) Kuntze Theaceae, Shrub L 1 (128) 12 Senna alata (L.) Roxb. Caesalpiniaceae, Tree L 1 (127) 13 Cassia fistula L. Caesalpiniaceae Shrub L 1 (127) 14 Senna occidentalis (L.) Link Caesalpiniaceae weed L 1 (127) 15 Coffea arabica L. Rubiaceae Shrub S 1 (127)

Page | 39 16 Curcuma longa L. Zingiberaceae Shrub R 1 (127) 17 Lawsonia inermis L. Lythraceae Shrub L 2 (35,128,130) 18 Murraya koenigii (L.) Rutaceae Herb L 1 (127) 19 Ocimum tenuiflorum L. Labiatae Herbal L 1 (127) 20 Piper betle L. Piperaceae Herb L 1 (127) 21 Cullen corylifolium (L.) Medik. Papilionaceae Herb S 1 (127) 22 Cinnamomum porrectum Lauraceae Herb S 1 (131) (Roxb.) Kosterm. 23 Phyla nodiflora (L.) Greene Verbenaceae Shrub L 1 (131) 24 Cestrum nocturnum L. Solanaceae Shrub F 1 (131) 25 Trachyspermum ammi (L.) Apiaceae Herb S, F 2 (42,43) Sprague 26 Leptospermum petersonii Myrtacea Shrub S 1 (132) F.M.Bailey 27 Syzygium aromaticum (L.) Myrtacea Herb S 2 (132,133) Merr. & L.M.Perry 28 Echinops kebericho Mesfin Asteraceae Shrub R 1 (16) 29 Taverniera abyssinica A.Rich. Fabaceae Herb R 1 (16) 30 Cymbopogon martini (Roxb.) Poaceae Herb Ae 1 (42) W.Watson 31 Foeniculum vulgare Mill. Apiaceae Herb Ae 1 (42) 33 Dodonaea viscosa Jacq. Sapindaceae Shrub L 1 (39) 34 Rumex nervosus Vahl Polygonaceae Herb R 1 (39) 35 Rumex abyssinicus Jacq. Lamiaceae Herb R 1 (39) 36 Thymus vulgaris L (Oleogel) Lamiaceae Herb L 1 (134) 37 Juniperus communis L. Cupressaceae Tree F 1 (135) 38 Bersama abyssinica Fresen. Francoaceae Tree Sb 1 (64) 39 Thymus vulgaris L. Lamiaceae Herb L 1 (27) 40 Inula confertiflora A.Rich. Compositae, Tree L, F 1 (136) 41 Clematis simensis Fresen. Ranunculaceae Tree L 1 (135) 42 Zehneria scabra (L.f.) Sond. Cucurbitacea Herb L 1 (135) 43 Pycnostachys abyssinica Labiatae Herb L 1 (135) Fresen. 1029 Leaves = L, root = R, Stem Bark =SB, Fruits =F, Bark =B, Aerial = Ae, Flower = Fl, Stem =St, Rhizome = Rh, 1030 Bulbs = Bu, Seed = S, Berries = Be, Whole =W, Clove =C 1031

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