Antibacterial activity of selected medicinal plants used by traditional healers in Genta Meyche (Southern Ethiopia) for the treatment of gastrointestinal disorders

Journal Pre-proof Antibacterial activity of selected medicinal plants used by traditional healers in Genta Meyche (Southern Ethiopia) for the treatment of gastrointestinal disorders Awoke Guadie (Conceptualization)methodology)formal analysis)investigation)data curation)writing - original draft)writing - review and editing), Demisse Dakone (Conceptualization)methodology)writing - original draft)validation)software), Dikaso Unbushe (Methodology)writing review and editing)visualization), Aijie WangWriting-review and editing)formal analysis)visualization), Siqing Xia (Conceptualization)formal analysis)supervision)

PII:

S2210-8033(20)30010-5

DOI:

https://doi.org/10.1016/j.hermed.2020.100338

Reference:

HERMED 100338

To appear in:

Journal of Herbal Medicine

Received Date:

19 December 2016

Revised Date:

20 January 2020

Accepted Date:

27 January 2020

Please cite this article as: Guadie A, Dakone D, Unbushe D, Wang A, Xia S, Antibacterial activity of selected medicinal plants used by traditional healers in Genta Meyche (Southern

Ethiopia) for the treatment of gastrointestinal disorders, Journal of Herbal Medicine (2020), doi: https://doi.org/10.1016/j.hermed.2020.100338

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Antibacterial activity of selected medicinal plants used by traditional healers in Genta Meyche (Southern Ethiopia) for the treatment of gastrointestinal disorders

Awoke Guadiea,c,d, Demisse Dakonea, Dikaso Unbusheb, Aijie Wangc, Siqing Xiad,* a

Department of Biology, College of Natural Sciences, Arba Minch University, P.O.Box. 21, Arba

Minch, Ethiopia Department of Biology, College of Natural and Computational Sciences, Jinka University, P.O.Box.

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b

165, Jinka, Ethiopia

Key Laboratory of Environmental Biotechnology, Research Center for Eco-environmental Sciences,

Chinese Academy of Sciences, Beijing (100085), China

State Key Laboratory of Pollution Control and Resource Reuse, College of Environmental Science and

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c

Corresponding author. Tel.: +86 21 65980440

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Engineering, Tongji University, Shanghai (200092), China

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Fax: +86 21 65986313

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E-mail address: [email protected]

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Graphical abstrcat

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Abstract This study was designed to investigate the antibacterial activity of selected medicinal plants used by the Genta Meyche community in Southern Ethiopia. Ethnomedicinal data was collected from 25 herbalists through semi-structured interviews and observations. Based on the highest use value (UV), plants were

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selected to be tested on pathogenic bacteria. A total of 26 ethnomedicinal plant species belonging to 17 families were collected and identified. The extraction of active compounds from the selected plants was carried out using five solvents. Roots (30.8%) and leaves (26.9%) were the plant parts most frequently used for the treatment of gastrointestinal disorders (GIDs). Herbalists mostly prepared medicinal plants as juice (57.7%) and administered the juice through oral routes (92.3%). Solanum incanum L.

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(UV=0.44), Harrisonia abyssinica Oliv. (UV=0.36), Hydnora abyssinia A.Br. (UV=0.36) and Leucas aspera (Willd.) Link. (UV=0.32) were found to be the most frequently used medicinal plants in the study area. These plants showed a promising broad spectrum of activity against Gram-positive and Gramnegative test bacteria with growth inhibition zone and minimum inhibition concentration (MIC) values ranging from 7.40±0.60−16.81±2.03 mm and 156−2500 μg/mL, respectively. Among solvents, ethanol and ethyl acetate extracts showed significantly greater antibacterial activity than aqueous, acetone and chloroform extracts. Steroids, tannins, saponin, flavonoids, alkaloids and terpenoids were identified as 2

bioactive compounds in most of the tested plants. Unused parts of Solanum incanum L. and Harrisonia abyssinica Oliv. were investigated and results exhibited potential candidacy with inhibition zones of 7.1−13.7 and 10.2−15.3 mm, respectively. Overall, the results of this study provide evidence for the treatment of GIDs by using traditional medicinal plants used by the Genta Meyche community to act against pathogenic bacteria.

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Keywords: Antibacterial activity; Genta Meyche; Gastrointestinal disorder; Medicinal plant

Introduction

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Gastrointestinal disorders (GIDs) are a common human health problem in both developing and developed countries (Johns et al., 1995). Among GIDs, functional digestive disorders are the most

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prominent with about 50% of patients who refer to gastroenterology care centers suffering from them (Bahmani et al., 2014). These disorders often lead to diarrhea, nausea, vomiting, fever, abdominal pain,

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intestinal inflammation, bloating and flatulence, or systemic diseases (Ahmed et al., 2013). Diarrhea is a major GID caused by a host of bacterial, viral and parasitic organisms, most of which are spread by contaminated water and food (Bahmani et al., 2014; Kadir et al., 2013; Singh et al., 2016).

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Rotavirus and Escherichia coli are the two most etiological agents of diarrhea in developing countries (WHO, 2013). Some of the other pathogenic bacteria which cause GIDs (diarrhea) include Escherichia

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coli, Salmonella typhi, Bacillus cereus, Staphylococcus aureus, Enterococcus feacalis, Campylobacter jejuni, Aeromonas hydrophila, Shigella spp., Yersinia spp. and Vibrio cholera (Fabry et al., 1998; Pendota et al., 2013; Singh et al., 2016). Giardia intestinalis and Cryptosporidium parvum are also major parasitic organisms that cause diarrheal disease (Johns et al., 1995). The World Health Organization (WHO) estimated approximately 1.7 billion cases of diarrhea worldwide per year in 2013, with these

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causing 6.9% of deaths overall (WHO, 2013). In 2004, diarrheal disease was the third leading cause of death in low-income countries and with this disease still continuing as a major problem today (Kadir et al., 2013). In 2013, the WHO also mentioned that diarrheal disease is the second leading cause of (760, 000) death in children under five years old which is both preventable and treatable (WHO, 2013). The use of medicinal plants for the treatment of human GIDs has been an old tradition in many societies (Chuen et al., 2016; Kadir et al., 2013; Malla et al., 2015; Paksoy et al., 2016; Teklehaymanot, 2009; Vuddanda et al., 2016; Wondimu et al., 2007; Yineger et al., 2007). It has been reported that threequarters of the world’s population is dependent on indigenous medicine (Dey and De, 2012). The WHO 3

also recognizes that the majority of the populations in developing countries are still relying on herbal medicines to meet their health needs (Kadir et al., 2013). Particularly in rural areas, people choose medicinal plants as their primary form of health care due to a number of reasons including inaccessibility of synthetic drugs, unaffordable cost of modern drugs, the fewer side effects of medicinal plants and indigenous knowledge on the efficacy of a particular plant against a particular disease (Bhat et al., 2014). Medicinal plants have a wide variety of bioactive compounds such as alkaloids, tannins, flavonoid, terpenoids, phenolic compounds steroids, resins, and other secondary metabolites which act significantly on parasitic and pathogenic organisms (Ahmed et al., 2013; Do et al., 2014; Kazemi, 2015; Paula-Freire

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et al., 2013; Pendota et al., 2013; Singh et al., 2016). The screening of medicinal plants for antimicrobial activity and phytochemicals is important for finding potential new compounds for therapeutic use (Singh

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et al., 2016). In fact, 25% of all modern drugs prescribed today were developed with a plant related compound as their principal active ingredient (Malla et al., 2015).

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In Ethiopia, there are many plant species (800) used for the traditional treatment of infectious diseases (Teklehaymanot, 2009). Human (80%) and livestock populations (>90%) in the country are

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significantly dependent on this traditional medicine to meet their health care needs (Birhanu et al., 2015). In the study area, medicinal plants are extensively employed in daily life for the treatment and prevention

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of GIDs. However, there has been no previous research work carried out based on an ethnobotanical survey, that included phytochemical screening and the evaluation of antimicrobial activity of these traditional medicinal plants.

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The aim of this study was to evaluate the antimicrobial activity of selected medicinal plant extracts (Harrisonia abyssinica Oliv., Leucas aspera (Willd.) Link., Solanum incanum L., and Hydnora abyssinica A.Br.) used by traditional healers in the Genta Meyche community for the treatment of GIDs. Stem extracts of Harrisonia abyssinica Oliv. and root extracts of Solanum incanum L. (which is commonly used by herbalists in Genta Mecha) have shown relatively greater antimicrobial activity than

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other medicinal plants tested. Based on these results, these plants were selected for further comparative study with other parts of the plant (leaf, fruit and stem of Solanum incanum L., and leaf and root of Harrisonia abyssinica Oliv.). 2. Materials and methods 2.1. Study area Genta Meyche is located in Arba Minch Zuria Woreda (AMZW), the Gamo Gofa Zone of Southern Ethiopia. It lies between latitudes of 05°39′36′′−06°12′20′′N and longitudes of 37°24′36′′−37°33′20′′E. The altitudinal range of the area also lies between 1100-1950 meters above sea 4

level. The mean annual rainfall in the area is 930 mm. The rainfall distribution has a bimodal nature with the first and second rainfall happening during April to May and September to October, respectively. The average maximum and minimum temperature is about 33.3 and 17.4°C, respectively. The area is semi-forest, topographically lying at the Woyna Dega agroecological zone. Although other ethnic groups exist in the area, the dominant one is the Gamo people who follow a traditional farming system. 2.2. Informants and ethnomedicinal data collection Fresh plant parts were collected at different times between 2013-2014 from the tribal villages under the supervision of the 25 traditional medicine healers (Fig. 1) who were involved in this study.

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Before data collection, a brief group discussion (in the local language, Gamogna) was held with the key informants to explain to them the objectives, benefits, risks and general procedures of the survey. This

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was carried out to acknowledge the informants’ cooperation in preserving traditional knowledge of the study area and build their confidence in providing reliable information. Once the objective was briefly

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explained, the respondents were asked to sign a prior informed consent form. Ethical approval was also obtained from the AMZW administration and peasant association head of the village.

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Semi-structured interviews were then conducted following the most frequently used method (Martin, 1995). These interviews were conducted with the help of translators who were conversant in

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Gamogna. The interviews were designed to obtain the informants personal information (age, level of education and occupation), scope of health problems treated with plants, plants used (local name, growth form, source abundance as wild/cultivated, degree of scarcity, part used, methods of preparation, route

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of administration, threats, conservation efforts) and indigenous knowledge transfer. These interviews were done mostly in the field to avoid possible confusion with regard to the identity of the medicinal

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plants.

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70

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Sex, education and age (%)

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0 Male

Female Illitrate Primary Secondary Bachelor <40

Education

41-50

51-60

>60

Age (year)

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Sex

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Demography

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Fig. 1. Respondents demographic characteristics. Morphological characteristics of medicinal plants were observed and recorded during and after the interviews. Photograph and GPS (Magellan GPS-315, Germany) readings of latitudes and longitudes were also taken at the sites where each medicinal plant was collected. As shown in Table 1, identified

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specimens of plants were given a specific code (AMUCC01-26) in the field which was kept at the Arba Minch University Botanical Garden Herbarium (AMU-BGH). Some of the plants were identified in the

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field by a botany specialist while most of the other plants were identified at AMU-BGH through comparison with already identified herbarium specimens and the use of taxonomic keys in the Flora of Ethiopia and Eritrea (Edwards et al., 1995; Edwards et al., 2000; Hedberg and Edwards, 1989; Hedberg and Edwards, 1995). Plant names have also been checked with The Plant List (2013) of the Royal Botanic Gardens, Kew accessed on 19 November 2016. Finally, the use value (UV) that demonstrates

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the relative local importance of each species was calculated according to Eq. (1) (Bhat et al., 2014). UV=U/N (1) where U= number of citations per species; N= number of informants

2.3. Preparation of plant extract The plant parts such as root extract of Solanum incanum L. and Hydnora abyssinia A.Br.; leaf extract of Leucas aspera (Willd.) Link. and stem extract of Harrisonia abyssinica Oliv. were used for in vitro antibacterial screening test. About 100 g of the powdered extract of each plant sample was succesfuly extracted using maceration techniques in five solvent systems (aqueous, chloroform, ethanol, 6

acetone and ethyl acetate) within 72 h. The macerated products of each plant were then filtered using Whatman # 1 filter paper to separate solid and liquid fractions. Each of the filtrates were then concentrated under reduced pressure using a rotary evaporator (Buchi Rota vapor R-205, Switzerland) and crude extracts were stored at -20ºC until analysis. For antibacterial bioassays, 100 mg each of plant crude extract was dissolved in 1 mL of dimethyl sulfoxide (DMSO) to achieve a final concentration of 100 mg/mL solution of the test sample. 2.4. Antibacterial activity test This study evaluated four selected plant species extracted with five solvents on four bacterial

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strains. The bioassay was carried out using Gram-positive [Staphylococcus aureus (ATCC25923) and Enterococcus feacalis (ATCC29212)] and Gram-negative [Escherichia coli (ATCC25922) and

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Salmonella typhimurium (ATCC13311)] bacteria. All strains were obtained from the Arba Minch Hospital Regional Laboratory. For each test organism, the viability test was condcuted by growing each

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strain on nutrient broth. Once the viability of each organism was tested under sterile conditions, the strains were subcultured on nutrient agar medium and incubated at 37ºC for 24 h. The bacteria were also

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maintained on sterile nutrient agar slants in the refrigerator (at -20°C) and used as a stock

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Table 1: List of plants used to treat gastrointestinal disorders in Genta Meyche

Euphorbiaceae

Haydnoraceae

Linaceae Moringaceae Myrtaceae Phytolaccaceae Polygonaceae Ranunculacae Rosaceae

Solanaceae

Moringa stenopetala (Baker f.) Cufod Syzygium guineense (Willd.) DC. Phytolacca dodecandra L,H,er. Rumex abyssinicus Jack. Nigella sativa L. Hagenia abyssinia (Bruce ex Steud.) J.F.Gmel. Citrus aurantifolia (Christm.) Swingle Citrus sinensis (L.) Osbeck Harrisonia abyssinica Oliv. Ruta chalepensis L. Capsicum annuum L. Solanum incanum L. Withania somnifera (L.) Dunal Zingiber officinale Roscoe

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Rutaceae

Zingiberaceae

AMUCC-02 AMUCC-14 AMUCC-13

Growth Habit Herb Herb Herb Shrub Shrub Tree Tree

Parts used Root Root /leaf Leaf/fruit Seed/fruit Leaf Leaf Root

Treatment/ prevention Both Both Prevention Both Both Both Prevention

Frequency of citation 1.0 7.0 3.0 7.0 6.0 3.0 7.0

Use value 0.04 0.28 0.12 0.28 0.24 0.12 0.28

Administration s Oral Oral/Topical Oral Oral/Topical Oral Oral Oral

Shrub Shrub

Leaf/fruit Root

Both Both

3.0 9.0

0.12 0.36*

Oral Oral

Herb Shrub

Leaf Leaf

Both Both

8.0 6.0

0.32* 0.24

Oral Oral

Shrub

Fruit

Both

5.0

0.20

Oral

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Gullo DachiMarache Dortsa Athi-afedale Talba

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Leucas aspera (Willd.) Link. Ocimum lamiifolium Hochst. ex Benth. Linum usitatissimum L.

AMUCC-25 AMUCC-04

AMUCC-17

Halako

Tree

Root

Treatment

3.0

0.12

Oral

AMUCC-23 AMUCC-08 AMUCC-07 AMUCC-19 AMUCC-12

Ochi Hazinche Chole Karte-dale Kosso

Tree Shrub Shrub Herb Tree

Leaf Root Root Fruit Fruit/leaf

Both Both Both Both Both

3.0 5.0 6.0 7.0 7.0

0.12 0.20 0.24 0.28 0.28

Oral Oral Oral Oral Oral

AMUCC-16

Lome

Tree

Fruit

Treatment

6.0

0.24

Oral

AMUCC-15 AMUCC-03 AMUCC-10 AMUCC-20 AMUCC-01 AMUCC1-26 AMUCC1-21

Burtukane Gadase Talata Mitimita Bullo Gsawa Zanjo

Tree Tree Shrub Shrub Shrub Shrub Herb

Fruit Stem Leaf Fruit Root Leaf Root

Treatment Both Both Both Both Both Both

3.0 9.0 5.0 3.0 11.0 1.0 6.0

0.12 0.36* 0.20 0.12 0.44* 0.04 0.24

Oral Oral Oral Oral Oral Oral Oral

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Lamiaceae

Local name Shunkurte Tumo Katikala Feto Natira Gara Chope

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Apiaceae Brassicaceae Compositae

Allium cepa L. Allium sativum L. Foeniculum vulgare Mill. Lepidium sativum L. Artemisia afra Jacq. ex Willd. Vernonia amygdalina Delile Croton macrostachyus Hochst. .ex.Delile Ricinus communis L. Hydnora abyssinia .A.Br.

Collection number AMUCC-24 AMUCC-18 AMUCC-11 AMUCC-06 AMUCC-09 AMUCC-22 AMUCC-05

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Amaryllidaceae

Species name

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Family name

*=Plant species which have relatively high use value selected for further antimicrobial test

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culture when they were required. Disc diffusion antibacterial assays were carried out following the protocol of the Clinical and Laboratory Standards Institute (CLSI) (CLSI, 2009). Fresh test microbial samples were prepared from the stock culture using nutrient broth medium and allowed to grow at 37°C for 24 h. Final bacterial numbers were adjusted to 0.5 McFarland turbidity standard (5×10 5 cfu/mL) with a sterile saline solution. The bacterial suspension was spread over Petri dishes containing Muller Hinton agar (∅ = 90 𝑚𝑚) using a sterile cotton swab. About 50 μL of each crude extract solution was applied onto the surface of sterile discs (∅ = 6 𝑚𝑚) (Whatman # 3 filter paper) and allowed to diffuse for

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five minutes. The discs were then placed on the surface of the Petri dishes containing the test organisms and kept in an incubator at 37°C for 24 h. All of the antibacterial activity experiments were performed in triplicate and results were expressed as diameters (mm) of inhibition surrounding 6 𝑚𝑚) of gentamicin were also used as a positive control.

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the disc. Similarly, 50 μL of 0.2% DMSO were used as negative controls. Antibiotic discs (∅ =

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Based on the extracts with the best inhibition zone results (Table 2), minimum inhibition concentrations (MIC) of the extracts were further evaluated with potential solvents (CLSI, 2006).

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For this purpose, two, four and five extracting solvents (Table 3) were used to extract the leaf of Leucas aspera (Willd.) Link., the root of Hydnora abyssinia A.Br., and the root/stem of Solanum

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incanum L./Harrisonia abyssinica Oliv., respectively. The MIC broth dilution method was carried out by using crude extracts prepared at concentrations of 5000, 2500, 1250, 625, 312, 156, 78 and 39 𝜇g/mL. Nutrient broth samples (10 mL) were inoculated with different concentrations of the

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crude extracts and 100 µL of active inoculums of bacterial suspension in test tubes. The preparation was then incubated for 24 h at 37°C. The MIC was determined as the lowest concentration of the extract which inhibited the growth of the organism. The absence of growth was confirmed by the absence of turbidity and inoculation on agar containing Petri dishes.

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2.5. Screening for the presence of some phytochemicals The plant extracts were screened for the presence of major secondary metabolite classes such as tannins, saponins, flavonoids, alkaloids, steroids, and terpenoids. The phytochemical screenings were performed following a guide to modern techniques of plant analysis (Harborne, 1973). In this study, all the chemicals and solvents used for analysis were of analytical grade and used

without further purification. 2.6. Statistical Analysis All the data was presented as the mean of three determinations ± standard error. The standard error and significance levels were calculated using SPSS version 20.0 software. One-way analysis 9

of variance (ANOVA) followed by post-hoc Tukey's honestly significant difference (HSD) test was performed to calculate the statistical significance between mean values. Differences were considered significant if p<0.05.

3. Results and discussion 3.1. Demographic detail and medicinal plants The information obtained through interviews of the 25 traditional herbalists involved in the study is shown in (Fig. 1). The herbalists that were interviewed were mostly men (72%) with the

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majority of the respondents being between the ages of 51-60 years old. The number of female herbalists (28%) was low which was expected as it was observed in others parts of Ethiopia

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(Teklehaymanot, 2009; Yineger et al., 2007) and the world (Bhat et al., 2014; Malla et al., 2015) that there were a higher number of male traditional healers than females. On the basis of their educational qualifications, most of the herbalists were non-educated (68%) while others had

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education levels ranging from primary school to bachelor (Fig. 1).

A total of 26 plant species belonging to 17 different families were identified as medicinal

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plants used in the Genta Meyche communities to treat primary health problems. The plant families Rutaceae (four species) and Solanaceae (three species) were found to rank first and second in terms

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of frequency of use (15.4 and 11.5%, respectively), followed by the families Euphorbiaceae, Amaryllidaceae, Lamiaceae and Compositae each with two species (7.7% each) that had high frequency of use (Table 1). The plant species that were cited most and had the highest calculated

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UV values (0.44) were Solanum incanum L. (UV=0.44) followed by Harrisonia abyssinica Oliv. (UV=0.36), Hydnora abyssinia A.Br. (UV=0.36) and Leucas aspera (Willd.) Link. (UV=0.36) (Table 1). Most of the plant species identified in this study were also known in the literature as medicinal plants used for the treatment of human and animal diseases elsewhere (Bhat et al., 2014; Birhanu et al., 2015; Kareru et al., 2007; Malla et al., 2015; Paksoy et al., 2016; Teklehaymanot and

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Giday, 2007; Teklehaymanot, 2009; Wondimu et al., 2007; Yineger et al., 2007). Interestingly, Withania somnifera (L.) Dunal. (Solanaceae) was observed to be the least preferred plant species (UV=0.04) in the study area to treat human disease (Table 1) despite the fact that it had been previously reported as the most preferred medicinal plant species in Dheeraa communities in the Arisi Zone (Ethiopia) when compared to other identified plants (Wondimu et al., 2007). Information was gathered from herbalists regarding medicinal plant growth forms, parts used, preparation methods, routes of administration and the purpose of using the plants (Fig. 2). 10

a) 50 F+S L+R L+F Stem (S) Root (R)

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Fruit (F) b) 30 Leaf (L)

40

25

Plant species (%)

Plant species (%)

35 30 25 20 15

20

15

10

10

5

0

0 Shrubs

Trees

Herbs

Root (R) Leaf (L)

Growth form

Stem

L+R

L+F

F + Seed

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100

55

90

50

80 70

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Plant species (%)

40

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d)

45

35 30

50 40

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25

60

20 15

30 20

10

0 Juice

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10

5

Powder

Decoction

0 Oral

Decoction/powder

Oral/Topical

Route of adminstration

Wild

Cultivated

Conservation status

Adminstration and status

Preparation method

Fig.2. Medicinal plant species (a) growth forms, (b) plant parts used, (c) preparation methods, and (d) route of drug administration and conservation status. Shrubs (46.2%) followed by trees and herbs were the growth forms of the medicinal plants

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Plant species (%)

Fruit (F)

Plant parts used

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c)

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used by the respondents (Fig. 2a) which is in line with other studies in Ethiopia (Giday et al., 2016). Shrub leaves, roots, fruits and combinations of leaf/fruit and leaf/stem parts were used. Herbalists mentioned that roots (30.8%) and leaves (26.9%) were the most frequently used plant parts to treat different diseases (Fig. 2b). Although the use of leaves has been found to be a sustainable way of medicinal plant preparation in the study area, using a relatively higher proportion of root was considered to be unsustainable, especially for some of the rarer and slower reproducing medicinal 11

plants which could be threatened in the long run. Previous studies also reported that root is the main medicinal plant part used for remedy preparation (Yineger et al., 2007). However, results reported by Bhat et al. (2014) from India, Malla et al. (2015) from Nepal, and Birhanu et al. (2015) in Western Ethiopia were not in line with this study in which leaves were reported as the most frequently used part. This could most likely be due to the vegetation distribution and abundance, as well as indigenous knowledge of the different study areas. Most of the plants were used for preventive measures (prophylaxis) and treatment of different GIDs (Table 1). According to herbalists, customers choose medicinal plants due to several reasons

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including a lack of modern drugs in rural areas, unaffordable costs of modern drug and fewer side effects when using plants. These reasons were in line with problems mentioned in another study

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(Bhat et al., 2014). Although they prepare the plant materials in various forms (juice, powder, and decoction) and administer the plants through different routes (oral and oral/topical), preparation in the form of juice (57.7%) (Fig. 2c) and administering the plants through the oral route (92.3%) were

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the most frequently used methods (Fig. 2d) (Giday et al., 2016). It was shown that preparing the plant as a juice (through crushing the required plant parts) was the most efficient method in a short

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period of time and that administering the drugs through an oral route have been proven to most efficiently treat customer’s gastrointestinal complications. This research agrees with Malla et al.

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(2015), which studied medicinal plants used by ethnic people in Western Nepal and reported that juice (50.7%) preparation was the most commonly used method followed by paste (36.3%). This previous study also showed that the oral route of herbal drug administration is the dominant way

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followed by most traditional healers.

In terms of conservation status, the proportion of cultivated and wild types of medicinal plants were found to be 42.3 and 57.7%, respectively (Fig. 2d). Although a reasonable amount of medicinal plants were conserved in the study area, the most frequently used medicinal plants [Solanum incanum L., Leucas aspera (Willd.) Link., Harrisonia abyssinica Oliv., Hydnora abyssinia A.Br.,

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Croton macrostachyus Hochst. ex.Delile, Rumex abyssinicus Jack., Phytolacca dodecandra L,H,er., Vernonia amygdalina Delile, Syzygium guineense (Willd.) DC., Ricinus communis L., and Withania somnifera (L.) Dunal] are still wild types, suggesting that the continuous collection of these wild types might threaten the species in the future. Moreover, the use of roots which was the highest part recorded to treat different diseases, could further threaten the local supply of wild medicinal plants (in the long run), particularly those that are rare and slow-reproducing medicinal plants such as Hydnora abyssinia A.Br. and Solanum incanum L. (Malla et al., 2015; Paula-Freire et al., 2013; 12

Yineger et al., 2007). As a result, the government and other organizations working on plant conservation in the study area need to emphasize limits. It is also important for the government to note that the majority of the herbalists in the study area have been found to be of old age and are non-educated and tend to transfer indigenous knowledge orally. For example, most respondents said that they obtained their knowledge from their father, and planned to transfer their knowledge to their first-born sons, which agrees with other studies (Teklehaymanot and Giday, 2007). As a result, comprehensive documentation of transferred knowledge will also be needed. According to the male informants’ response, it was previously stated that collecting wild

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medicinal plants was a matter of going out from home and easily gathering them. However, informant’s have now expressed that these plants have become more difficult to find and they fear

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the need for traveling long distances to collect the same plant that may even be lost in the future. Female respondents said that they are interested in growing some medicinal plants in their home gardens which can help them to treat their children’s health problems. This suggests that female

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respondents may be more equipped in regard to conservation but have limited knowledge regarding medicinal plants (Teklehaymanot, 2009). Both male and female respondents mentioned that

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agricultural expansion, deforestation for firewood, overgrazing and drought are the major reasons for a decrease in conservation of medicinal plants. The same anthropogenic medicinal plant threats

Yineger et al., 2007).

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were also reported in other studies (Birhanu et al., 2015; Paksoy et al., 2016; Wondimu et al., 2007;

3.2. Antibacterial activities and bioactive compounds

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Depending on the type of extracting solvent, the four plant species tested showed a promising broad spectrum of antimicrobial activity against Gram-positive and Gram-negative test bacteria with growth inhibition ranging from 7.40±0.60−16.81±2.03 mm (Table 2). The extracts also displayed a broad spectrum of activity against test bacteria with MIC values ranging from 156−2500 𝜇g/mL (Table 3). Different reports have mentioned different ranges of MIC values of significance against

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disease-causing pathogenic organisms (Fabry et al., 1998; Pendota et al., 2013; Singh et al., 2016). For example, MIC values <8000 𝜇g/mL of the crude extract are considered to be good activity (Fabry et al., 1998), whilst Pendota et al. (2005) suggest that isolated phytochemicals should have MIC values ≤780 𝜇g/mL. A recent review study by Kuete (2010) also classified crude extract activity as significant if MIC values are below 100 μg/mL, moderate if 100625 μg/mL. In this study, MIC results marked in bold (MIC=156−625 𝜇g/mL) were considered good activity against the bacterial strain tested (Table 3). 13

The root of Hydnora abyssinia A.Br. was extracted with acetone/ethanol, aqueous and ethyl acetate and showed a different degree of inhibition zone against three (7.70±0.60−16.70±0.40 mm), two (13.0±1.04−13.40±0.60 mm) and one (15.0±0.40) strains of bacteria, respectively (Table 2). However, Hydnora abyssinia A.Br. extracted with chloroform did not show any antibacterial effect. Additionally, the same plant extract with most solvents showed lower MIC values for Gram-negative bacteria than Gram-positive bacteria. This can be supported by the results shown in Table 3 which show that the thicker peptidoglycan composed cell walls for Gram-positive bacteria (1250 𝜇g/mL) offer a protective advantage for other parts of the cell when compared to Gram-negative bacteria

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which has been affected by lower values of MIC (156−650 𝜇g/mL). Phytochemical analysis of Hydnora abyssinia A.Br. showed the presence of tannins, saponins, flavonoids, alkaloids, and

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terpenoids (Table 4) which are all well studied in many other medicinal plants (Ahmed et al., 2013; Do et al., 2014; Paula-Freire et al., 2013; Pendota et al., 2013; Singh et al., 2016). Recent reports in Kenya (Onyancha et al., 2015) and Sudan (Yagi et al., 2012) also identified the presence of

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alkaloids, tannins, phenols, and flavonoids from the root extract of Hydnora abyssinia A.Br. Phytochemical composition from the studies from these two countried differed from this study in

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that steroids, which were identified in Kenya and Sudan, weren’t detected in the current investigation (Table 4). On the contrary, saponins which had been detected in the current study were not reported

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in studies by Onyancha et al. (2015) and Yagi et al. (2012) in Kenya and Sudan, respectively. This could have been a result of environmental and other factors. Indeed, soil fertility, humidity, solar radiation, the wind, temperature, plant age and collection time were all reported factors that could

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alter the chemical composition of plants (Paula-Freire et al., 2013). The purpose of using the plant in Kenya, Sudan and Ethiopia (the current study area) is almost the same in that the root extract is used for the treatment of stomach ache, fever, diarrhea, typhoid, and throat problems. The ethanolic root extract of Solanum incanum L. showed the best result by inhibiting all test organisms followed by an ethyl acetate extract which was active against three test organisms

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(Escherichia coli, Enterococcus feacalis and Salmonella typhimurium (Table 2). Acetone and chloroform extracts of this plant only showed inhibition zones against the strain of Enterococcus feacalis and Staphylococcus aureus, respectively. The maximum and minimum inhibition zones of Solanum incanum L. were found to be 16.81±2.03 with ethyl acetate extract against Salmonella typhimurium and 7.40±1.30 with chloroform extract against Staphylococcus aureus. Salmonella typhimurium, a pathogenic Gram-negative bacteria which is predominately found in the intestinal lumen and can cause human GIDs was also inhibited by a relatively low dose of Solanum incanum 14

L. extracted with different solvents (MIC=156−625 𝜇g/mL). Similarly, Enterococcus feacalis, a Gram-positive bacteria which inhabits the gastrointestinal tract of humans also displayed a range of sensitivity (MIC=312−1250 𝜇g/mL) for most extracts investigated. The highest MIC (2500 𝜇g/mL) against Staphylococcus aureus (Table 3) was recorded for a chloroform extract of Solanum incanum L. Compared with other solvents investigated in this study, most of the other plant chloroform extracts also showed insignificant antimicrobial activity (Tables 2 and 3). This could be due to the relative weak eluent strength of chloroform. The eluent strength of organic solvents is as follows: ethanol (0.88) > Ethyl acetate (0.58) > acetone (0.56) > chloroform (0.40) (Reichardt, 2003). As

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shown in Table 4, all phytochemical compounds investigated were detected in Solanum incanum L. root extract. There are many reports in different countries about the presence of tannins, saponins,

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flavonoids, alkaloids and steroids in various parts of Solanum incanum L. (Eltayeb et al., 1997; Indhumathi and Mohandass, 2014; Sinani et al., 2016). For example, Indhumathi and Mohandass (2014) from Oman extensively studied the different parts of Solanum incanum L. by taking samples

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at different developmental stages and observing the variation in bioactive compounds. Eltayeb et al. (1997) also reported that the concentration of alkaloid in roots was higher than in the stem while

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both plant organ components showed more alkaloid accumulation with time. In the current study area, Solanum incanum L. root juice is orally taken for the treatment or prevention of various GIDs.

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As such, this plant is often mentioned as being common in Africa and used as treatment of various human diseases (Sinani et al., 2016).

Evaluation of Leucas aspera (Willd.) Link. leaf extract using different solvents showed good

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antibacterial candidacy (Table 2). Particularly, the ethanol (11.0±1.03−16.40±1.10 mm) and ethyl acetate (10.70±1.40−13.01±1.06 mm) extracts of this plant revealed a range of antibacterial activity against all the tested pathogenic organisms. However, acetone, aqueous and chloroform extracts of Leucas aspera (Willd.) Link. did not show any antimicrobial activity against all bacteria tested (Table 2). Except for Salmonella typhimurium, the ethanol leaf extracts of Leucas aspera (Willd.)

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Link. displayed promising antimicrobial activity at lower extract doses (MIC=156−625 𝜇g/mL) for Escherichia coli, Enterococcus feacalis and Staphylococcus aureus (Table 3). Ethyl acetate extracts of this plant also showed notable activity against all tested microorganisms (Table 3). The reason for the medicinal effect of these plants has been found to be the presence of certain bioactive compounds (tannins, saponins, flavonoids, and terpenoids) detected from phytochemical analyses (Table 4). The presence of various phytochemical compounds in Leucas aspera (Willd.) Link. has also been extensively studied by different scholars (Karthikeyan et al., 2013; Rahman et al., 2007). 15

For example, Rahman et al. (2007) studied the ethanol root extract of this plant and detected alkaloids, tannins, reducing sugars, steroids, and gums. Chew et al. (2012) in Malaysia also studied the whole part of Leucas aspera L. and identified alkaloids, steroids and flavonoids that were effective against Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa and Shigella flexneri) pathogenic bacteria which were similar to the results from this study. Harrisonia abyssinica Oliv. stem extracted with acetone, aqueous and chloroform showed antimicrobial activity against the Enterococcus feacalis (7.40±0.60 mm), Staphylococcus aureus

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(9.70±0.60 mm) and Salmonella typhimurium (10.70±2.60 mm) strains, respectively (Table 2). The ethyl acetate and ethanol extract of this plant also showed a range of antimicrobial activities against

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three (Escherichia coli, Enterococcus feacalis and Staphylococcus aureus) and four (Escherichia coli, Salmonella typhimurium, Enterococcus feacalis and Staphylococcus aureus) bacterial strains, respectively (Table 2). Using the same dose (MIC=625 μg/mL), the aqueous and chloroform stem

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extracts of Harrisonia abyssinica Oliv. also showed antibacterial activity against the Enterococcus feacalis and Salmonella typhimurium strains, respectively (Table 3). Ethyl acetate extracts with

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relatively low MIC (<625 𝜇g/mL) and ethanol extracts with MIC values of 625−1250 𝜇g/mL also exhibited potential antimicrobial activities against all the tested microorganisms (Table 3). This

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result is supported by a phytochemical analysis that contains bioactive compounds including tannins, saponins, flavonoids, alkaloids and terpenoids (Table 4). This study is in line with previous research work on the same plant (Fabry et al., 1998; Kareru et al., 2007). Harrisonia abyssinica Oliv. has

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been documented as being used widely in various African folk remedies to treat gonorrhea, dysentry, skin disease, tuberculosis, bilharzia infections and has also been used as an ascaricide throughout Africa (Kareru et al., 2007). In the current study area, the plant stem is used for the treatment of diarrhea and fever. The in vitro results in this study showed that pathogenic Gram-negative and Gram-positive bacteria have been inhibited by the stem extract which agrees with Cyrus et al. (2008)

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who carried out studies on the antibacterial activity of this plant stem and found that the stem inhibits Gram-negative (Pseudomonas aeruginosa and Escherichia coli) and Gram-positive (Bacillus cereus and Staphylococcus aureus) bacteria. Comparing the overall antimicrobial activity results showed that Solanum incanum L.> Harrisonia abyssinica Oliv.>Hydnora abyssinia A.Br.> Leucas aspera (Willd.) Link. through the inhibition of the number of bacterial strains investigated. In terms of the effect of solvents on the extraction of plant parts, the potency of the solvents can be ranked as follows: ethanol/ethyl acetate 16

> aqueous > acetone > chloroform. This was statistically confirmed by the fact that the four plants extracted using ethanol showed significantly greater antimicrobial activity than aqueous (p=0.020), acetone (p=0.001) and chloroform (p<0.05) extracts. Ethyl acetate also showed a significant difference in antimicrobial activity when compared to acetone and chloroform, but insignificant when compared to ethanol extract (p=0.995). Overall, the difference in antibacterial activity of thr different plant extracts was most likely due to (i) the presence of different bioactive compounds in the four plant species (ii) the extraction potential of the five different solvents (iii) the sensitivity of the bacterial strain on the extract that

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could be influenced by the concentration of a given compound and/the type of bioactive compounds present in the crude extract and (iv) the combined effect of the three conditions. Indeed, previous

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studies on various plants and solvents confirmed these differences (Abirami et al., 2012; Ahmed et al., 2013; Chuen et al., 2016; Do et al., 2014; Fabry et al., 1998; Paula-Freire et al., 2013; Pendota et al., 2013; Singh et al., 2016). Do et al. (2014) for example, investigated the effect of three

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extraction solvents on a single plant (Limnophila aromatic) and they observed that ethanol extracts showed higher antimicrobial activity than ethyl acetate and hexane. Similarly, Singh et al. (2016)

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investigated the antimicrobial activity and bioactive compounds of Bergenia ciliata Sternb. using three organic solvents and reported that the methanol extract showed higher antibacterial activity

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than ethyl acetate and hexane which clearly indicated the extraction potentials of the different solvents. In contrast to the current study in which chloroform extracted plant showed the least activity, Abirami et al. (2012) carried out a preliminary study on the antimicrobial activity of

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Enicostemma littorale using different solvents (acetone, hexane and chloroform) and the results were found to be as follows in regard to antimicrobial activity: chloroform>acetone>methanol. This difference suggests that the scientific importance of testing one plant with different solvents and recommending one as the best extracting solvent is crucial. However, comparison should always be made with the extraction methods used traditionally to treat a certain ailment.

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3.3. Antibacterial activity of plant parts unused by herbalists Based on the results of the UV (Table 1), inhibition zone (Tables 2), MIC (Table 3) and

phytochemical (Table 4) tests, two plants (Solanum incanum L. and Harrisonia abyssinica Oliv.) showed better antimicrobial performance and were selected for further analysis of their plant parts not used by the local herbalists. Leaf and root ethyl acetate extracts of Harrisonia abyssinica Oliv., and leaf, fruit and stem ethanol extracts of Solanum incanum L. were assayed for the presence of antimicrobial activity against the test bacteria (Table 5). The use of ethanol and ethyl acetate as 17

extraction solvents were also selected based on their relatively better efficacy on the root (Solanum incanum L.) and stem (Harrisonia abyssinica Oliv.) extracts of the selected plants, respectively. The leaf, fruit and stem ethanol extracts of the Solanum incanum L. showed a range of inhibitory effects (7.1−13.7 mm) on the test organism but these results were relatively lower than the inhibitory effects of the root part (9.8−16.0 mm) used by herbalists (Table 5). The fruit and stem extracts were observed to be more active than the leaf extract by inhibiting two test organisms. However, the leaves and roots of Harrisonia abyssinica Oliv. extracted with ethanol displayed better antibacterial activity (10.2−15.3 mm) than the stem (8.0−13.1), a primary choice of traditional healers (Table 5).

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The leaf extracts of this plant showed antibacterial activity against Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus. This demonstrates comparatively the efficient activity of

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leaf extract in each test organism versus that of the root extract.

In this study, comparing the plant parts commonly used and not used by herbalists in the Genta Meyche community has confirmed two concepts. First, by taking into consideration the

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indigenous knowledge of herbalists about medicinal plant parts used during bioactive compound screening studies helped to ensure the avoidance of wasting time, energy and money that would be

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required for testing all plant parts. For instance, the stem (8.0±0.0−9.7±0.60 mm), fruit (7.7±0.6−10.7±3.0 mm) and leaf (9.4±0.6 mm) extracts of Solanum incanum L. were tested and did

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not show better antimicrobial activity than the commonly used root extract (10.7±0.9−15.4±0.6 mm) of this plant (Table 5). This suggests that herbalists choose the root part of Solanum incanum L. rather than other parts as a result of developed knowledge that was accumulated through a generation

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of trial and error efforts, were scientifically proved in this study. Second, comparing plant parts in this study also gave clue that medicinal plants used by herbalists need scientific conformation. For example, the leaf (11.8±0.1−14.7±0.6 mm) extract of Harrisonia abyssinica Oliv. has shown better inhibitory activity against Gram-positive and Gram-negative pathogenic bacteria than the commonly used stem (10.0±1.0−12.7±0.8 mm) of this plant (Table 5). Using the leaf part of a medicinal plant

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is a more sustainable approach than other parts of a plant. Yineger et al. (2007) mentioned that using roots for the purpose of medicinal value has a negative influence on the survival and continuity of useful medicinal plants and hence affects the

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Table 2: Zone of inhibition (mm) of the different plant part extracts using different solvents

abyssinia A.Br.

Root

Solanum incanum L.a

DMSOc

GENd

15.00±0.40

-

21.00±1.00

-

-

-

25.00±1.00

-

-

-

-

27.00±0.00

-

-

-

-

-

22.00±1.00

14.00±1.05

8.40±0.80

-

10.40±1.60

-

21.00±1.00

13.70±0.40

11.33±1.60

-

16.81±2.03

-

25.00±1.00

15.40±0.60

-

7.40±1.30

-

-

27.00±0.00

7.50±0.60

10.70±0.60

8.70±0.60

-

15.03±0.20

-

22.00±1.00

-

11.00±1.03

-

-

11.04±1.05

-

21.00±1.00

Salmonella typhimurium

-

11.40±1.50

-

-

13.01±1.06

-

25.00±1.00

Staphylococcs aureus

-

16.40±1.10

-

-

12.40±1.30

-

27.00±0.00

Enterococcus feacalis

-

14.00±1.10

-

-

10.70±1.40

-

22.00±1.00

Escherichia coli

-

8.70±0.60

-

-

11.70±1.40

-

21.00±1.00

Salmonella typhimurium

-

-

-

10.70±2.60

10.04±1.02

-

25.00±1.00

Staphylococcs aureus

-

10.00±0.30

9.70±0.60

-

10.40±2.40

-

27.00±0.00

Enterococcus feacalis

7.40±1.60

10.00±1.02

-

-

12.70 ±0.60

-

22.00±1.00

Ethanolb

Escherichia coli

16.70±0.40

13.70±0.40

13.00±1.04

-

Salmonella typhimurium

15.40±0.80

8.00±0.11

13.40±0.60

Staphylococcs aureus

-

7.70±0.60

Enterococcus feacalis

10.70±0.60

Escherichia coli

-

Salmonella typhimurium

-

Staphylococcs aureus

-

Enterococcus feacalis Leaf

(Willd.) Link.

abyssinica Oliv.a

Stem

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Harrisonia

Escherichia coli

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Leucas aspera

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Root

Inhibition effect of extract (mm) Aqueous Chloroform Ethyl acetateb

Acetone

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Hydnora

Test organisms

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Parts use

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Plants

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−= No inhibition zone; a/b=plants/solvents selected to investigate unused parts; c=Dimethyl sulfoxide (DMSO as negative control); d= Gentamicin (GEN as positive control)

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Table 3: Minimal inhibition concentration (𝜇g/mL) of different plant extract using different solvents Plant

Extract

Hydnora abyssinia A.Br.

Acetone

≥1250

−

Ethyl acetate

≥312

−

−

−

Aqueous

≥625

≥625

−

−

Acetone

−

−

≥1250

−

Ethanol

≥312

≥312

≥1250

≥156

Ethyl acetate

≥312

≥156

≥312

≥625

Chloroform

−

−

−

≥2500

Aqueous

≥1250

≥625

Ethanol

≥625

−

Ethyl acetate

≥625

≥312

Acetone

≥1250

−

Ethanol

≥1250

≥625

Ethyl acetate

≥625

Chloroform

−

Aqueous

−

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≥650

≥1250

≥312

≥312

≥156

≥312

≥625

−

−

≥625

≥1250

≥625

≥312

≥625

≥625

−

−

−

≥625

−

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Harrisonia abyssinica Oliv.

≥625

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Leucas aspera (Willd.) Link.

Gram-positive bacteria Enterococcus Staphylococcus feacalis aureus ≥1250 ≥1250

Ethanol

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Solanum incanum L.

Gram-negative bacteria Escherichia Salmonella coli typhimurium ≥156 ≥156

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−= Activity absent; ‘bold’ indicated values are considered good activity against Gram-positive and Gram-negative bacteria

Table 4: Different solvent plant extracts phytochemical compounds Hydnora abyssinia A.Br.a +

Saponins Flavonoids Alkaloids Steroids

a

= Ethanol

Harrisonia abyssinica Oliv.b +

+

+

+

+

+

+

+

+

+

+

−

+

−

+

−

−

+

+

+

+

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Terpenoids

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Phytochem icals Tannins

Plant species Solanum incanum Leucas aspera (Willd.) L.a Link.b + +

extract; b= Ethyl acetate; + = Present; - = Absent

sustainable utilization of the plants. Although a more detailed investigation is required, results observed in Harrisonia abyssinica Oliv. did not support the idea that the plant stem used by traditional healers is as effective as unused parts. The leaf of Harrisonia abyssinica Oliv. which showed higher antimicrobial activity might be a more sustainable alternative to substitute for stems used by traditional healers. In fact, instead of the stem, the Harrisonia abyssinica Oliv. root has been 20

reported as a primary choice of Kenyan people to treat malaria and GIDs (Fabry et al., 1998; Kareru et al., 2007). Although phytochemical tests of the unused parts were not done, Paula-Freire et al. (2013) did compare the chemical composition and biological effects of the roots, branches and leaves of another plant (Heteropterys tomentosa A. Juss) and detected relatively similar bioactive compounds (saponins, hydrolysable tannins, flavonoids, polyphenols, and triterpenes) in the three parts. The current preliminary phytochemical results might be considered sufficient as a basis for further studies for the isolation and identification of the bioactive compounds with different solvents.

Leaf

9.4±0.6

L.

Fruita

10.7±3.0

−

7.7±0.6

−

−

Stema

−

8.0 ±0.0

9.7 ±0.6

−

−

Roota*

14.0±1.0

13.7±0.4

10.7±0.9

15.4±0.6

−

Harrisonia

Leafb

14.7±0.6

12.7± 0.2

−

11.8±0.1

−

abyssinica Oliv.

Rootb

−

10.5 ±0.3

−

10.7±0.6

−

Stemb*

11.7±1.4

10.0±1.0

12.7 ±0.8

10.4±2.4

−

aureus −

-p

−

= Ethanol extract; b= Ethyl acetate extract;*= Parts used by herbalists - = No inhibition zone

4. Conclusions

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a

feacalis −

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Solanum incanum

typhimurium −

a

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coli

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Table 5: Different parts of Solanum incanum L. and Harrisonia abyssinica Oliv. inhibition zone on test bacteria Plants Parts Zone of inhibition (mm) Escherichia Salmonella Enterococcus Staphylococcus Control tested

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In this study, rich indigenous knowledge and a high diversity of medicinal plants to treat GIDs was recorded. However, most of the medicinal plants mentioned were of the wild type that have been under continuous anthropogenic threat. The selected medicinal plants extracted with five solvents showed significant antimicrobial activity. In particular, ethanol and ethyl acetate were found to be the best solvents. The extracts showed a promising broad spectrum of activity against

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Gram-positive and Gram-negative test bacteria with growth MIC values ranging from 7.40±0.60−16.81±2.03 mm and 156−2500 μg/mL, respectively. These efficient antimicrobial activities were supported by the presence of different bioactive compounds detected in the extract including flavonoids, alkaloids, saponins, tannins, steroids and terpenoids. The investigation of plant parts that are unused by herbalists provide valuable clues about the indigenous knowledge during bioactive compound extraction from different plant parts helps to avoid wasting time, energy and money. On the other hand, this study also provides comparative evidence with regard to looking for

21

alternatives that can substitute for traditionally used plant parts that may be considered as destructive. Overall, the plants that had been selected on the basis of their traditional use value in the Genta Meyche community for the treatment of GIDs were confirmed in this study and shown to contain bioactive compounds which significantly inhibit the growth of pathogenic bacteria including Salmonella typhimurium, Enterococcus feacalis, Staphylococcus aureus and Escherichia coli. The traditional practitioners in the study area who use aqueous solvents to prepare medicinal plants (Solanum incanum L., Harrisonia abyssinica Oliv., Hydnora abyssinia A.Br. and Leucas aspera

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(Willd.) Link.) might be most efficient if they use ethanol (e.g. local alcohol) for extract preparation (although this may be difficult in rural communities). Further structural elucidation and clinical trials

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on bioactive compounds is recommended for the study area medicinal plants.

Credit Author Statement

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We declare that we have no conflict of interest.

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Conflict of interest statement

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Awoke Guadie: Conceptualization, methodology, formal analysis, investigation, data curation, writing-original draft, writing-review and editing.

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Demisse Dakone: Conceptualization, methodology, writing-original draft, validation, software. Dikaso Unbushe: Methodology, writing review and editing, visualization. Aijie Wang: Writing-review & editing, formal analysis, visualization.

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Siqing Xia: Conceptualization, formal analysis, supervision

Acknowledgments

This work was supported by the Foundation of State Key Laboratory of Pollution Control and

Resource Reuse (Tongji University) (PCRRT16003), Arba Minch University (AMUBIO132015) and the CAS-President's International Fellowship Initiative (2019PE0033). We are grateful for the traditional healers of the study area for their dedicated contribution to sharing their accumulated indigenous knowledge. We would also like to thank the AMZW administration and peasant association head of the village for allowing us to work in the area and for assisting us with the 22

identification of recognized traditional healers. The constancy of the Biological and Cultural Diversity Research Center of Arba Minch University are also truly thanked. We also thank the staff members of the Applied Microbiology Laboratory for their kind cooperation and help while doing the antimicrobial activity tests. Finally, we are grateful to Mr. Sintayehu Semu and Mr. Gidele Gito for their brilliant help in ethnobotanical data collection and language editing.

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