Journal of Medicinal Plant Research Volume 11 Number 16, 25 April, 2017 ISSN 1996-0875
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Associate Editors Dr. Ravichandran Veerasamy AIMST University Dr. Ugur Cakilcioglu Faculty of Pharmacy, AIMST University, Semeling - Elazıg Directorate of National Education 08100, Turkey. Kedah, Malaysia.
Dr. Jianxin Chen Dr. Sayeed Ahmad Information Center, Herbal Medicine Laboratory, Department of Beijing University of Chinese Medicine, Pharmacognosy and Phytochemistry, Beijing, China Faculty of Pharmacy, Jamia Hamdard (Hamdard 100029, University), Hamdard Nagar, New Delhi, 110062, China. India.
Dr. Hassan Sher Dr. Cheng Tan Department of Botany and Microbiology, Department of Dermatology, first Affiliated Hospital College of Science, of Nanjing Univeristy of King Saud University, Riyadh Traditional Chinese Medicine. Kingdom of Saudi Arabia. 155 Hanzhong Road, Nanjing, Jiangsu Province, China. 210029 Dr. Jin Tao Professor and Dong-Wu Scholar, Dr. Naseem Ahmad Department of Neurobiology, Young Scientist (DST, FAST TRACK Scheme) Medical College of Soochow University, Plant Biotechnology Laboratory 199 Ren-Ai Road, Dushu Lake Campus, Department of Botany Suzhou Industrial Park, Aligarh Muslim University Suzhou 215123, Aligarh- 202 002,(UP) P.R.China. India.
Dr. Pongsak Rattanachaikunsopon Dr. Isiaka A. Ogunwande Department of Biological Science, Dept. Of Chemistry, Faculty of Science, Lagos State University, Ojo, Lagos, Ubon Ratchathani University, Nigeria. Ubon Ratchathani 34190, Thailand.
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Prof Hatil Hashim EL-Kamali Dr. Arash Kheradmand Omdurman Islamic University, Botany Department, Lorestan University, Sudan. Iran.
Prof. Dr. Muradiye Nacak Prof Dr Cemşit Karakurt Department of Pharmacology, Faculty of Medicine, Pediatrics and Pediatric Cardiology Gaziantep University, Inonu University Faculty of Medicine, Turkey. Turkey.
Dr. Sadiq Azam Samuel Adelani Babarinde Department of Biotechnology, Department of Crop and Environmental Protection, Abdul Wali Khan University Mardan, Ladoke Akintola University of Technology, Pakistan. Ogbomoso Nigeria. Kongyun Wu Department of Biology and Environment Engineering, Dr.Wafaa Ibrahim Rasheed Guiyang College, Professor of Medical Biochemistry National Research Center China. Cairo Egypt. Prof Swati Sen Mandi Division of plant Biology, Bose Institute India.
Dr. Ujjwal Kumar De Indian Vetreinary Research Institute, Izatnagar, Bareilly, UP-243122 Veterinary Medicine, India.
Journal of Medicinal Plants Research
Table of Contents: Volume 11 Number 16 25 April, 2017
ARTICLES
Secondary metabolites from endophytic fungus from Lippia sidoides Cham. 296 Talita Pereira de Souza Ferreira, Gil Rodrigues dos Santos, Ilsamar Mendes Soares, Sergio Donizeti Ascêncio, Tarso da Costa Alvim, Chrystian de Assis Siqueira and Raimundo Wagner de Souza Aguiar
Ethnobotanical study of ethnoveterinary plants in Kelem Wollega Zone, Oromia Region, Ethiopia 307 Tolera Fufa Feyissa, Moa Melaku shigut, Tilahun Bekele Hailemariam, Tena Regassa and Nebiyu Kassa Kergano
Vol. 11(16), pp. 296-306, 25 April, 2017 DOI: 10.5897/JMPR2017.6371 Article Number: 8E7286764097 ISSN 1996-0875 Journal of Medicinal Plants Research Copyright © 2017 Author(s) retain the copyright of this article http://www.academicjournals.org/JMPR
Full Length Research Paper
Secondary metabolites from endophytic fungus from Lippia sidoides Cham.
Talita Pereira de Souza Ferreira1*, Gil Rodrigues dos Santos2, Ilsamar Mendes Soares1, Sergio Donizeti Ascêncio3, Tarso da Costa Alvim4, Chrystian de Assis Siqueira1 and Raimundo Wagner de Souza Aguiar1
1Bioprocess Engineering and Biotechnology, Federal University of Tocantins, University Campus of Gurupi, 77410-530 Gurupi, TO, Brazil. 2Agronomic Engineering, Federal University of Tocantins, University Campus of Gurupi, 77410-530 Gurupi, TO, Brazil. 3Medicine, Federal University of Tocantins, University Campus of Palmas, 77001-090 Palmas, TO, Brazil. 4Food Engineering, Federal University of Tocantins, University Campus of Palmas, 77001-090 Palmas, TO, Brazil.
Received 14 March, 2017; Accepted 18 April, 2017
Lippia sidoides Cham. (Verbenaceae) is a species native to the Brazilian northeast, widely used in popular medicine. Its leaves were used for the isolation of endophytic fungi and extraction of metabolites. Among them, three were selected according to fungitoxicity tests against the maize phytopathogenic fungus, Curvularia lunata (Wakker). However, the objective of this study was to identify the role of L. sidoides extracts associated with their endophytic fungi, necessary to reduce excess of fungicides applied on the maize crop. Metabolites were evaluated for antioxidant activity by 2.2-diphenyl-1-picrylhydrazyl (DPPH), phenols, total flavonoids and one of it endophytic fungus were evaluated for synergism (Verticilium sp. and plant extracts). The endophytic fungi and plant extracts evaluated for phenolic content ranged from 0.29 ± 0.05 to 96.94 ± 11.86 mgEAG/g, the content of flavonoids from 14.31 ± 1.56 to 192.33 ± 4.58 mgER/g, and antioxidant activity could only be observed for the plant extract with EC50 81 ± 0.3%. The secondary metabolites identified by HPLC in the plant extract were catechin, quercetin, gallic acid and naringin. Naringenin, catechin, epigallocatechin gallate and quercetin were identified in the extract of the fungi viz. Verticillium sp. and Fusarium sp. Synergistic analysis between a 1:1 proportion of plant and fungal extracts has shown more efficient (79.0%) inhibition of C. lunata. Thus, alternative control of phytopathogenic fungi can be accomplished using plant extracts associated with their endophytic fungi, reducing the excess of fungicides applied on the maize crop.
Key words: Curvularia lunata, endophytic fungal, HPLC, extract, Verticillium sp., Fusarium sp., Colletrotrichum sp.
INTRODUCTION
Endophytic fungi isolated from plants belonging to important new sources of bioactive compounds for tropical regions produce more active secondary applications in agriculture, medicine, and the food metabolites than those from temperate regions, because industry (Yadav et al., 2014b; Sadananda et al., 2011). they are exposed to an environment of greater biodiversity Over time, some endophytic fungi have developed the (Bhardwaj et al., 2015). These fungi are recognized as ability to produce the same or similar bioactive Ferreira et al. 297
substances to those produced by host plants (Sharma et 1990s, it has caused economic loss in maize crop al., 2016). This lends a great advantage to the study of productivity in China (Gao et al., 2015) and has been the relationships between endophytes and their host progressing in recent years in Brazil (Assunção et al., plants (Fouda et al., 2015). Nevertheless, most of the 2006). The pathogen mainly infects maize leaves, leaf time, these valuable bioactive compounds are scarce in sheaths, and husks and causes soaked or yellow necrotic nature; therefore, the development of a substitute spots in the early stages, which then expand to round, approach for their efficient production is warranted oval, spindle-shaped, or strip lesions (Hou et al., 2013). (Kusari et al., 2013). The green leaf area of maize is considered the main More recent estimates based on high-throughput source of photoassimilates for the plant (Akram et al., sequencing methods suggest that as many as 5.1 million 2014) and, according to Liu et al. (2016), a loss in this fungal species exist (Taylor et al., 2014). The estimate of source may reflect the development of the plant and the known species has almost tripled in the period between production of grains. 1943 (38000 described species) and the present, In this paper, the objective was the isolation and amounting to an increase of more than 60000 described identification of the endophytic fungus from L. sidoides. species (Blackwell, 2011). Accordingly, endophytes are To this end, antioxidant activity, phenols and total good sources of genetic diversity and new species flavonoids, and the evaluated metabolites were identified belonging to a large group of fungi that colonize tissues by HPLC. The extracts of endophytic fungi and plants of healthy plants without causing apparent symptoms were evaluated by the selective test of fungitoxicity and (Fatima et al., 2016). They protect plants against synergism against C. lunata. herbivores, insect attack, or invading pathogens by entering this mutually beneficial relationship, according to host habitat conditions (Bhardwaj et al., 2015). MATERIALS AND METHODS Bioactive products that can be produced by endophytes Collection of the vegetal material and preparation of the can be classified as saponins, phenols, flavonoids, vegetal extract steroids, tannins, alkaloids, anthraquinones, terpenoids, and others (Li et al., 2015). These compounds defend the L. sidoides from Ceará was collected in Gurupi (11°44'48"S latitude, plant against phytopathogenic fungi. For example, 49°02'55" Longitude O), Tocantins, Brazil. It was then identified and flavonoids are well-known substances, and their deposited at the Herbarium of the Federal University of São João Del Rei under the reference number 8303. The plant material was production involves a variety of processes such as cell sent to the Laboratory Integrated Pest Management of the Gurupi signaling, plant growth, and reproduction (Garrido- Campus– UFT, where the extracts were dried and obtained Arandia et al., 2016). according to methodology of Al-Marby et al. (2016). The leaves Lippia sidoides Cham. (Verbenaceae), also known as were dried in the shade at room temperature, subsequently cut and “rosemary pepper” is an aromatic plant of popular subjected to extraction with cold solvent. Thirty grams of plant were medicinal use found in the Caatinga region of the used for 1.5 L of methanol in each extraction for a period of 7 days. After this period, the mixture was filtered and evaporated under Brazilian Northeast (Santos et al., 2016). It presents reduced pressure to obtain the extracts. insecticidal activity against Tenebrio molitor (Lima et al., 2011) and larvae of Aedes aegypti (Lima et al., 2013) and acaricide against Tetranychus urticae Koch (Soares et Cultures of endophytic fungi al., 2016). When tested against different pathogenic For isolation, fresh leaves of L. sidoides were surface washed in tap bacteria, such as Staphylococcus aureus, Pseudomonas water and soap to remove impurities. As described by Banhos et al. aeruginosa, and Escherichia coli (Ratnaweera et al., (2014), leaves were successively immersed in 70% alcohol (1 min), 2015) and different fungi and yeasts, including Candida 2.5% sodium hypochlorite (4 min), and 70% alcohol (30 s), followed albicans (Premjanu et al., 2016), it demonstrated a strong by washing with autoclaved distilled water (6 min) and drying on antimicrobial action. Thus, compounds produced by sterile filter paper. After washing, 50 μL of the distilled water used in the asepsis of the plant material was removed, then this volume endophytic fungi of plants with recognized biological was added (inoculated) in a Petri dish with PDA culture medium activity for biotechnological purposes can be applied in (potato, dextrose, and agar) in order to verify the efficiency of the the alternative control of urban and agricultural pests asepsis process. The leaves were fragmented and inoculated into (Dutta et al., 2014), in place of the toxic chemicals sold 9-cm diameter Petri dishes containing PDA culture medium. To the on the commercial market. culture medium were added 50 μg/mL tetracycline or 100 μg/mL chloramphenicol (antibiotics). For the isolation of pure cultures, On the other hand, the Curvularia spot, a disease sequential peaks were made in Petri dishes containing the same caused by the fungus Curvularia lunata (Wakker) Boed., culture medium, using the streaking technique. Subsequently, they has a high incidence in maize (Zea mays L.). Since the were stored in tubes for centrifugation with glycerol at -80ºC for the
*Corresponding author. E-mail: [email protected]. Tel: +55 63 33113571, +55 63 984621838.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License 298 J. Med. Plants Res.
preservation of pure cultures. For tentative identification, maintained for 1 h in the dark, and the absorbances were measured microscopic slides of each endophyte were prepared (Kohlmeyer at 420 nm in a spectrophotometer (BioSpectro model SP-220). The and Kohlmeyer, 1972) and examined under a light microscope. total flavonoid contents were determined by means of an analytical curve containing standards at concentrations of 1 to 10 μg mL−1 of rutin, and the results were expressed in microgram equivalents of Curvularia lunata strain rutin per milligram of dry extract (μgER/mg).
C. lunata fungus was obtained from Federal University of Tocantins, in our laboratory culture collection, previously identified based on Antioxidant activity morphological and molecular characters. The initial isolation of the fungus was obtained by growth on PDA medium (39 g L−1) The antioxidant capacity was measured by the DPPH method, as supplemented with ampicillin (500 mg L−1) for 7 days. Petri dishes described by Soares et al. (2014) with modifications. The extracts were monitored daily and fungal colonies that did not present obtained were prepared using methanol as the solvent at contaminants were picked and transferred onto new plates with the concentrations of 20 to 180 μg mL−1, w/v. In triplicate, 0.5 mL of the same culture medium. extract was added to a methanolic solution of DPPH (3 mL at 40 μg mL−1, w/v). White (reference solution) was made by replacing the DPPH with methanol in each reaction. All test tubes containing the Extraction of secondary metabolites of endophytic fungi reactions were stirred and held in the dark for 30 min. An analytical curve was performed for this activity using standard (positive Two 7-mm diameter mycelial agar discs of the endophytes were controls) ascorbic acid and rutin (20-180 μg mL−1, w/v) also adding inoculated into 200 mL of Czapek fermentation medium to which it to a methanolic solution of DPPH. Absorbance readings were was added 100 μg mL−1 chloramphenicol (Plotnikov et al., 2016). taken at 517 nm in a spectrophotometer (BioSpectro model SP- After 21 days of incubation under constant stirring (120 rpm) at 28 ± 220). The absorbance of the DPPH solution at 40 μg mL−1 was also 1°C, the culture fluids were separated from the mycelial masses by measured and used as a negative control. The antioxidant activity vacuum filtration, and the extract was obtained following the of free radical removal was expressed as the percentage of methodology of Dhankhar et al. (2012). For extractions, organic inhibition determined by the equation solvents such as hexane and ethyl acetate were used in order to obtain the largest number of active substances. ( ) [ ]
Determination of total phenolic compounds Where %AA is the percentage of antioxidant activity; ABSnc is the absorbance of the negative control; ABS is the absorbance of The content of phenolic compounds of both the plant extract and sample the sample; and ABSwhite is the absorbance of white. three extracts of the endophytic fungi with the best results for Using calibration curves obtained by plotting the different fungitoxic activity was determined by the Folin-Ciocalteu method, concentrations in relation to the %AAs, the efficient concentration, according to Sánchez-Rangel et al. (2013) with modifications, the amount of sample required to decrease the initial concentration where gallic acid was used as the standard. Methanol solutions of −1 −1 of DPPH by 50% (EC 50), which was expressed in μg mL . the extracts were prepared at a concentration of 1 mg mL . Then, in 15 mL vials, 0.5 mL of the extract, 5 mL of Mili-Q water, 0.5 mL of
Folin-Ciocalteu reagent (1 M) and 0.5 mL of sodium carbonate were Analysis by high-performance liquid chromatography (HPLC) added (20%, m/v). The blank was prepared by placing 0.5 mL of distilled water in place of the Folin-Ciocalteu reagent. An analytical High-performance liquid chromatography (HPLC) was developed in curve was also prepared for the standard gallic acid reagent at −1 the Laboratory of Scientific Instrumentation, Federal University of concentrations from 10 to 100 μg mL . For the blank (reference Tocantins on a Shimadzu® LC-10 Series Avp Chromatograph, solution) of the curve, 50 μL of distilled water was added instead of equipped with a pump (LC-10AD), degasser (DGU-14A), UV-VIS gallic acid. All tubes were homogenized with the aid of the test tube detector (SPD-10A) (CTO-10A), Rheodyne hand injector (20 μL agitator. All reactions were performed in triplicate and kept under loop), and CLASS integrator (LC-10A). Separation was performed dark incubation for 1 h. The readings were performed in a by the gradient elution method using a Phenomenex Luna C18 5μ spectrophotometer (BioSpectro model SP-220) at 765 nm (Zhou et (2) (250 × 4.6 mm) reverse-phase column and pre-column al., 2009). The results obtained by spectrophotometric analyses of Phenomenex C18 (4 × 3.0 mm) filled with material similar to that in total phenols were expressed as milligram equivalents of gallic acid the main column. Mobile phase A was 0.1% phosphoric acid in Milli- per gram of extract (mgEAG/g). Q water and mobile phase B was 0.1% phosphoric acid in Milli-Q water/acetonitrile/methanol (54:35:11). Program gradient: 0 to 0.01 min, 0% B; 0.01-5 min, 0% B, 5-10 min, 30% B, 10-20 min 40% B, Determination of total flavonoids 20-29 min, 40% B, 29-30 min 50% B, 30-50 min 100% B, 50-80 min, 100% B. Flow rate: 1 ml/min; temperature: 22°C. UV detection The determination of the total flavonoid content was performed was done at 280 nm. The compounds were identified by comparing according to the methodology developed by Da Silva et al. (2015). the retention times of samples with the authentic standards, such The solutions of the plant extract and the three extracts of as gallic acid, catechin, epigallocatechin gallate, naringin, quercetin, endophytic fungi with the best results in the fungitoxic activity were −1 and naringenin (Sigma®). The quantities of the compounds were prepared using 1 mg mL of methanol as the solvent. Then, in a expressed in micrograms per milligram of extract (μg/mg) by 15-mL vial filled with aluminum foil, 0.5 mL of the extract, 0.5 mL of correlating the area of the analyte with the calibration curve of aqueous acetic acid (60%, v/v), 2 mL of pyridine methanolic standards built in concentrations of 4.5 to 18 μg mL-1. solution (20%, v/v), 1 mL of aluminum chloride (5%, w/v) and 6 mL of Milli-Q water were combined. The blank (reference solution) was made with all the above-mentioned reagents replacing the aluminum Selection according to fungitoxicity against C. lunata chloride with methanol. All reactions were carried out in triplicate and homogenized in a tube shaker. The reactions were In order to select the endophytic fungi with the best antibiotic
Ferreira et al. 299
Table 1. Mycelial inhibition screening of Lippia sidoides Cham. endophytic fungi against the plant pathogen Curvularia lunata Wakker.
Fungus Extract from ethyl acetate Extract from hexane LS–1 (u.f.) + +++ Colletotrichum sp. + = Verticillium sp. +++ = Alternaria sp. + = LS-5 (u.f.) + + LS-6 (u.f.) +++ = LS-7 (u.f.) ++ = LS-8 (u.f.) = ++ Colletotrichum sp. +++ = Phomopsis sp. = + Fusarium sp. = ++ Fusarium sp. 1 +++ = Fusarium sp. 2 +++ = LS-14 (u.f.) +++ = LS-15 (u.f.) = ++
LS, Initials of the L. sidoides plant to identify the endophytic fungus in the isolation. u.f., Morphologically unidentified fungus. They were visually classified as mycelial diameter equal to the control (=), smaller than the control (+), much smaller than the control (++), and much, much smaller than the control (+++).
activity, the in vitro mycelial growth inhibition capacity of the Statistical analysis phytopathogenic C. lunata fungus was previously isolated and identified. According to Carotenuto et al. (2015) with modifications, The data obtained in the experiments were subjected to statistical the evaluation of inhibition of mycelial growth of the phytopathogen analysis, such as analysis of variance (ANOVA) and Tukey’s test, was carried out by adding to the surface of the 9 cm diameter Petri with a significance level of α = 0.05, and linear regression. dish containing approximately 20 ml of BDA culture medium already Calculations were performed using Sigmaplot® 12.0 and Assistat® solidified, 100 μL of the extracts of the endophytic fungi. The software. extracts were diluted in Tween 80 and water. Discs 7 mm in diameter containing the mycelia of phytopathogenic fungus from pure colonies with approximately twelve days of growth in PDA RESULTS were placed in the center of the plates. Plates were incubated in biochemical oxygen demand (BOD) at 25 ± 1ºC and a photoperiod of 12 h of light and 12 h of darkness. Fifteen morphologically distinct endophytic fungi were After growth, the presence or absence of zones of inhibition was obtained from the fresh leaf fragments of L. sidoides. observed. Biological tests were carried out using extracts made The bioassays were performed in triplicate, and a plate from both hexane and ethyl acetate against the containing only one disk of micelium agar of the phytopathogen phytopathogenic fungus of maize plants, C. lunata. The without extract served as a positive control. results of the selection according to fungitoxicity are
presented in Table 1. Three extracts of different
Synergism evaluation endophytes extracted with ethyl acetate were selected for the analysis of secondary metabolite synthesis. This After selection of endophytic fungi by the in vitro diffusible analysis included the methanolic extract of L. sidoides metabolite test according to the methodology to Mutawila et al. leaves. These fungi were analyzed morphologically and (2015), only one sample was chosen for synergism evaluation. The identified at the genus level as Verticilium sp., extract of endophytic fungus was used together with extract of L. Colletotrichum sp., and Fusarium sp. 1 (Table 1). sidoides. In accordance with the methodology of Tadtong et al. The three fungi with fungistatic capacity against C. (2014), five different proportions of the two substances were used: 0:1; 1:3; 1:1; 3:1, and 1:0. Thus, the concentration of each lunata in this work were evaluated in order to identify the substance was fixed at 7500 μg mL−1. In Petri dishes with already antioxidative profile of the respective fungal extracts. The solidified BDA culture medium were placed 100 μL of each results obtained by spectrophotometric analyzes of total proportion, scattered with a handle Drigalsky. Then, a mycelium- phenols were expressed as milligrams of gallic acid agar disk of C. lunata fungus was placed in the center of the equivalents per gram of extract (mgEAG/g). In this way, plaque. An evaluation was performed after 10 days of incubation, and the the total phenolic content for the plant extract, and mycelial diameter was measured for comparison with the control extracts of the endophytic fungi Verticilium sp., (water and Tween 80). Colletotrichum sp., and Fusarium sp. 1 were 96.94 ± 300 J. Med. Plants Res.
Table 2. Mean values and standard deviations of total phenol content and flavonoids found in Lippia sidoides Cham., and endophytic fungi Verticilium sp., Colletotrichum sp. and Fusarium sp. 1.
Extracts Total phenols (mgEAG/g)* Total flavonoids (mgER/g) ** L. sidoides 96.94 ± 11.86a 192.33 ± 4.58a Verticilium sp. 6.38 ± 1.27b 21.56 ± 1.14b Colletotrichum sp. 0.29 ± 0.05c 14.31 ± 1.56c Fusarium sp. 1 10.54 ± 1.69b 64.59 ± 1.15b
*mgEAG/g, Milligram equivalents of gallic acid per gram of extract. **mgER/g, milligram equivalents of rutine per gram of extract. Means followed by the same letter in the column do not differ statistically from each other by the Tukey test (p > 0.05).
100
80
(%)
50 L. sidoides Rutine 60 Ascórbico ac. Verticillium Colletotrichum Fusarium 40
20
Antioxidant activity ECactivity Antioxidant
0 0 20 40 60 80 100 120 140 160 180 200 220
-1 Extract concentrations ( g mL )
Figure 1. Percentage of antioxidant activity (%AA) of of L. sidoides endophytes fungi Verticillium sp., Colletotrichum sp., and Fusarium sp. 1, and positive controls (rutine and ascorbic acid) by the DPPH method.
11.86, 6.38 ± 1.27, 0.299 ± 0.05 and 7.60 ± 1.69 is, the amount of sample required to decrease the initial mgEAG/g respectively (Table 2). concentration of DPPH by 50% was also expressed in μg -1 −1 It was verified that the total flavonoid content of extracts mL . The EC50 for the plant extract was 81.03 μg mL of the fungi Verticilium sp., Colletotrichum sp., and (Figure 1). The %AA for the highest concentration (200 Fusarium sp. 1 were 21.56 ± 1.14, 14.31 ± 1.56 and μg mL-1) for the fungi Verticillium sp., Colletotrichum sp., 64.59 ± 1.15 mgER/g, respectively (Table 2). These and Fusarium sp. was 19, 17 and 21%, respectively. values are lower than that of the plant extract, which was Therefore, the EC50 calculation could not be performed. 192.33 ± 4.58 mgER/g (Table 2). From the HPLC analyses performed for detection and The percentage of antioxidant activity (%AA) was also quantification, fingerprints were obtained (Figure 2), determined by the DPPH method. The curves are shown which revealed matrices of phenolic compounds and in Figure 1, indicating an increase only for the plant flavonoids. In Figure 2B, the phenolic compound gallic extract, with the increase of the amount of extract in the acid 0.37 μg mL-1 (Rt 16.4 min) was detected, along with reaction medium. The results were higher than 65% from the flavonoids catechin 1.67 μg mL-1 (Rt 23.7 min), the concentration of 120 μg mL-1, reaching a maximum of naringin 44.77 μg mL-1 (Rt 43.2), and quercetin 3.08 μg 95% for the concentration of 200 μg mL-1 for L. sidoides mL-1 (Rt 52.2 min). A high concentration of the compound extract (Figure 1). The effective concentration (EC50), that naringin in the methanolic extract of the plant is
Ferreira et al. 301
z
mAU
mAU mAU
Minutes
mAU mAU
Minutes
Figure 2. High-performance liquid chromatography (HPLC) fingerprints of authentic standards of phenolic compounds mixture (A), methanolic extract of the leaves of L. sidoides (B) and isolated endophytic fungi: Verticilium sp. (C), Colletotrichum sp. (D), Fusarium sp. (E) detected at 280 nm, as described in material and methods. Peak 1: Gallic acid; peak 2: Catechin; peak 3: Epigallocatechin gallate; peak 4: Naringin; peak 5: Quercetin; peak 6: Naringenin.
noteworthy. to compare any peak with laboratory standards. As It was also verified by HPLC in this study which shown in Figure 2D, the visualization of many uniform flavonoids could be synthesized by the endophytic fungi peaks indicated that many compounds were synthesized. and which could be identified and quantified according to The fungus Fusarium sp. synthesized (Figure 2E) three the standards in the laboratory where the analysis was substances identified as catechin 0.31 μg mL-1 (Rt 23.3), performed. According to Figure 2C, it can be seen that epigallocatechin gallate, and quercetin 19.73 μg mL−1 (Rt the extract of the endophytic fungus Verticilium sp. 29.1 min). synthesized a compound identified as naringenin 0.61 μg A synergism test was also performed between L. mL−1 (Rt 55.0 min) in addition to other compounds that sidoides extract and the endophytic extract of Verticillium could not be identified. sp. (Table 3). The curve was fitted to a 2nd degree The fungus Colletotrichum sp. also produced secondary equation with R2 = 0.9999. The highest inhibitory effect metabolites of the flavonoid class, yet it was not possible was in the proportion of 50% essential oil and 50% fungal 302 J. Med. Plants Res.
Table 3. Effect of synergism between the Lippia sidoides (Cham.) plant extract and the endophytic extract of Verticillium sp. against mycelial growth of Curvularia lunata (Wakker) in vitro.
Treatment (proportions of extracts) Inhibition (%) Control 0 ± 0.0 L. sidoides + Verticillium sp. (0:1) 54.8 ± 1.2 L. sidoides + Verticillium sp. (1:3) 43.5 ± 1.2 L. sidoides + Verticillium sp. (1:1) 79.0 ± 1.6 L. sidoides + Verticillium sp. (3:1) 71.3 ± 2.7 L. sidoides + Verticillium sp. (1:0) 22.4 ± 4.6
extract, both of which were at a concentration of 7500 μg A549 (lung) cells from a secondary metabolite called mL−1. Proportions of 0:1, 1:3, 1:1, 3:1 and 1:0 (v/v) beauvericin isolated from the endophytic Fusarium inhibited 54.8, 43.5, 79.0, 71.3 and 22.4% of the mycelial oxysporum of the Cinnamomum kanehirae plant. This growth of C. lunata in vitro (Table 3). same endophyte, F. oxysporum, nonpathogenic strains, isolated by Kundu et al. (2016), produced , bikaverin (1), 3-O-methyl-8-O-methyl fusarubin (2), 8-O-methyl DISCUSSION fusarubin (3), anhydrofusarubin (4) and fusarubin (5). In addition, the methanolic extract of Fusarium Some previous works found the same endophytic fungi. proliferatum prepared by Mohana et al. (2012), You et al. (2009) isolated the endophytic fungus endophytic of Dysoxylum binectariferum demonstrated Verticillium sp. from the roots of Rehmannia glutinosa by cytotoxic activity in HCT-116 (colon) and MCF-7 (breast) identifying two compounds produced by this fungus: 2,6- human cancer cells. dihydroxy-2-methyl-7-(prop-1E-eny)-1-benzofuran-3(2H)- The results confirm that both the plant and endophytic one, reported for the first time, and the ergosterol fungi produce compounds of a phenolic nature. Some peroxide, which inhibited the growth of pathogenic fungi. studies corroborate our results, even though some Suradkar et al. (2014) were able to isolate the endophyte previously reported fungal extracts contained more total Verticillium albo-atrum from Withania somnifera (L.) and phenols than ours. Yadav et al. (2014a) verified the Ocimum sanctum L. phenolic compound content of endophytic fungi extracts The fungi Colletotrichum sp. and Fusarium sp. were obtained from ethyl acetate. The highest concentration of also found in other studies isolating endophytic fungi. phenols was observed in the extract of Chaetomium sp. Alternaria alternata, Colletotrichum gloeosporioides, (60.13 ± 0.41 mgEAG/g), followed by that of Aspergillus Drechslera dematioidea, Guignardia bidwelli, Fusarium niger. The total phenol concentration values of the 21 lateritium, and Phomopsis archeri were endophytic fungi endophytic fungi studied ranged from 4.20 to 60.13 isolated from L. sidoides and identified by Siqueira mgEAG/g. Srinivasan et al. (2010) obtained values of (2011). In this work, the author isolated 15 endophytic 18.33 ± 0.68 mgEAG/g of total phenols from the extract fungi from L. sidoides, the same number of isolates in the of the endophytic fungus Phyllosticta sp. present work. Similar results were verified, where the In order to confirm the synthesis of total flavonoids by fungi Fusarium, Colletotrichum, Phomosis, and Alternaria the endophytic fungi present in the L. sidoides and plant were also present in the work of Siqueira (2011). extract (Table 2), extracts were obtained with the solvent Among the metabolites produced by Colletorichum sp.: ethyl acetate (AcOEt) to extract compounds with fusarentine 6,7-dimethyl ether, monocerin, and recognized antimicrobial activity, such as phenols and colletotrialide (Tianpanich et al., 2011), stigmasterol, flavonoids (Baba and Malik, 2015). sitostenone, squalene, ergosterol and ergosterol peroxide However, even if they are smaller, it can be verified that (Carvalho et al., 2016) were identified. Colletotrichum the endophytic fungi contribute to the plant in the species have been identified to produce a variety of production and synthesis of secondary metabolites for its secondary metabolites genes, including flavones, protection. Hence, an appreciable amount of flavonoids peptides and terpenes (Crouch et al., 2014) and many produced by the endophytic fungi may be present in the other metabolites as Jayawardena et al. (2016) shows. quantity verified in the plant; moreover, they contributed The genus Fusarium is also widely known as a to its synthesis. producer of a variety of chemical compounds derived Huang et al. (2007) isolated 42 endophytic fungi from from its secondary metabolism (Nongalleima et al., Nerium oleander and found flavonoids among the main 2013). Wang et al. (2011) reported anticancer activity bioactive compounds of some cultures. They also against human PC-3 (prostate), PANC-1 (pancreas) and clarified that some phenolic compounds are produced so
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that certain endophytic fungi survive along with their host phenolic constituents described in the literature as plants. Qiu et al. (2010) found flavonoid contents of important in several biological functions. Phenolic acid 0.01162 ± 0.0014 and 0.01256 ± 0.00378 mgER/mL in has a strong anti-inflammatory, antimutagenic, and extracts of Aspergilus nidulans, Aspergilus oryzae, and antitumor action, inhibiting genes related to the cell cycle, Ginkgo biloba endophytes, respectively. Their values metastasis, angiogenesis, and apoptosis (Verma et al., were lower than those of the present work; however, 2013). The detection of three types of flavonoids, different fungi were used. catechin, naringin, and quercetin, in the L. sidoides plant The data obtained in the determination of this (Figure 2B) also allows presenting and confirm the bioactivity is significant when compared with others possibility of harnessing its leaves for biological obtained in the work of Almeida et al. (2010). At a applications. Several studies have already demonstrated concentration of 100 μg mL−1 (or 1 mg mL−1 as reported), the potential and ability of these metabolites to act to there was an antioxidative activity of 99.5% and an EC50 reduce the risks associated with pathologies such as of 16.3 μg mL−1 in the ethanolic extract of L. sidoides. intestinal inflammation, bacterial infections, diabetes, The results obtained in the present study were lower; cardiovascular diseases, and cancer, among others however, the solvent used for the extraction may have (Clemensen et al., 2017; Macheleidt et al., 2016). In the been the source of this difference. They also assayed the present study, however, antifungal activities of the DPPH activity of plant-isolated substances such as extracts are related to the antioxidative activity of tecomaquinone and naringenin. At a concentration of 1 flavonoids present in considerable amounts (Funari et al., −1 -1 mg mL , %AA was 64.7% and the EC50 was 720 μg mL 2012). for the flavonoid naringenin. The compound Many studies report the capacity of flavonoid tecomaquinone did not display any activity. production by plants, such as those found in the leaves of As for the antioxidant activity of the extracts of Euphorbia neriifolia (pencil-tree), where Sharma et al. endophytic fungi, they showed a low capacity to (2014) identified a flavonoid as quercetin by HPLC. This sequester free radicals, as there was no negative effect substance and other flavonoids are credited with on the measured absorbance. Initial values remained antioxidative and anticarcinogenic activity. Naringenin is essentially unchanged at all concentrations tested. It can a flavonoid of the flavanone subclass, found mainly in be said that there was activity; however, compared with citrus fruits (Dou et al., 2013), with a beneficial the results obtained from the plant extract, it was much antioxidative and neuroprotective effect (Raza et al., lower than expected. It is believed that perhaps the 2013). cultivation conditions used did not promote the Among these, the flavonoids catechin and quercetin were biosynthesis of antioxidant molecules that are detectable also found in plant extracts. Thus, it can be confirmed by this method. In addition, there was no change in the that these two metabolites were probably synthesized by amount of free radicals as the concentration of fungal the endophytic fungi in conformation with the plant- extract varied. fungus mutualistic interactions. In addition, as endophytic Observing the work of Devi and Singh (2015), they also fungi produce the same compounds present in the plant, did not obtain positive results of a significant amount of it can be inferred that fungi produce these compounds for free radical sequestration by DPPH in extracts of the plant as a way of assisting them in their defense. Verticillium. However, they reported an antioxidative These substances contribute greatly to the antioxidative activity 95% in Alternaria sp. 2 made from ethyl acetate. activities credited to L. sidoides plants (Funari et al., Yadav et al. (2014a) carried out tests with extracts of 2012). endophytic fungi, including the genus Fusarium sp. They Kumar et al. (2013) characterized the vinblastine identified antioxidant activity by DPPH of Aspergillus alkaloid from the extract of the endophytic fungus F. peyronelii and Aspergillus niger of 71 and 72%, oxysporium, isolated from Catharanthus roseus. By respectively, but identified low activity in extracts of HPLC analyzes, they obtained 76 μg L-1 of the Fusarium sp. Nath et al. (2014) also reported a strong compound. Also Zaiyou et al. (2015) found in the extract activity in the sequestration of free radicals in ethanolic of the endophyte Fusarium sp. The compound paclitaxel extracts of Colletotrichum gloeosporioides of 0.67 ± 0.05, at the concentration of 0.0153 mg L-1. 47.89 ± 0.06, 73.84 ± 0.08, 60.09 ± 0.08, and 52.77% ± Besides, Chapla et al. (2014) also identified eight 0.06% at concentrations of 10, 25, 50, 75 and 100 μg mL- compounds synthesized by the endophyte Colletotrichum 1, respectively. gloeosporioides, a new compound 2-phenylethyl 1H- Endophytic fungi and plants can produce flavonoids indol-3-yl-acetate (1) and seven other known compounds: together, varying their biological activities, such as the Uracil (2), cyclo-(S*(S)-Pro-S*-Val) (4), 2-(2-aminophenyl) genus Bauhinia variegata (cow's foot), which had acetic acid (5), 2-(4-hydroxyphenyl) acetic acid (6), 4- antidiabetic activity confirmed by the metabolic profiles of hydroxy-benzamide (7), and 2-(2-hydroxyphenyl) acetic both the plant and the associated endophytic fungi acid (8). Table 4 shows the compounds identified by (Costa, 2005). HPLC from extracts of both the plant L. sidoides and the These analyzes by HPLC allowed us to identify endophytic fungi isolated from it. 304 J. Med. Plants Res.
Table 4. Identified compounds from samples of plant extracts and endophytic fungi identified and analyzed by HPLC with their respective peaks, concentrations, and retention times.
Sample (extract) Compound identified Peak Concentration (mg ml−1) Retention time (min) Gallic acid 2 0.37 16.4 Catechin 8 1.67 23.7 L. sidoides Naringin 21 44.77 43.2 Quercetin 27 3.08 52.0
Verticillium sp. Naringenin 11 0.61 55.0 Colletotrichum sp. N.I. * - - -
Catechin 8 0.31 23.7 Fusarium sp. 1 Epigallocatechin gallate 10 0.94 28.9 Quercetin 17 19.73 52.2
N.I.* = compound not identified
The production of compounds that possess biological joint action of the extract of L. sidoides and extract of the activity by endophytic fungi can be stimulated in the plant, endophytic fungus Verticillium sp. However, it was only a by the host plant extract, and in this case, by the extracts preliminary test, and other tests should be performed in of fermented fungi without contact with any part or extract order to obtain results on the best concentration of the of the plant (Dos Santos et al., 2015). When grown in metabolites, test the efficiency of the other fungi along vitro, fungi continued to produce metabolites. Further with the plant extract, and add another type of extract. studies must be carried out to find out if the fungi would Recent discoveries, as exemplified in this work, have cease production for some time or if, in the presence of associated endophytic fungi with medicinal plants, the plant extract or any part of it, the synthesis would important sources of secondary metabolites of continue, that is, what factors could encourage this pharmaceutical, agricultural, and industrial interest. Brazil production.No reports were found in the scientific has a wide potential source of plants, due to the varied literature on the inhibition of mycelial growth of biomes, and a study of plant-endophytic interactions is phytopathogenic fungi using extracts of endophytic fungi necessary. The discovery of metabolites with recognized and of plants with synergistic effects. However, in the bioactive action produced by endophytes can help to stop work of Oliveira et al. (2014), synergistic activity was the excessive exploitation of plants and consequently, the found between a butanolic fraction of Lippia alba extract whole set of endophytes associated with it (Pusztahelyi et with commercial antifungal agents against Candida al., 2015).Thus, the study of fungal endophytes is glomerata. The minimum inhibitory concentration was becoming a great necessity, because, as in the present 0.062 μg mL-1. This may be a potential alternative in the work, they assist their host plants in the synthesis of treatment for candidemia caused by yeast species. metabolites with recognized bioactivity. This property of The synergistic effect of the endophytic fungal extracts endophytes will help to diminish the exploitation of plant of Aspergillus awamori, Penicillium sp., and C. biodiversity for drug extraction. Consequently, gloeosporioides with standard antibiotics was studied conservation strategies will be more efficient. against the four test bacterial strains viz. Streptococcus pyogenes, Escherichia coli, Enterococcus faecalis, and Salmonella enterica ser paratyphi. A significant increase Conclusions in the diameter of inhibition zones was observed when the fungal extracts in combination with the antibiotic The analyses of the study showed the presence of Norfloxacin was used against the test pathogens. Crude phenols and flavonoids in all extracts and high extract of A. awamori in combination with Norfloxacin antioxidant activity was only observed in the plant extract. showed antimicrobial activity (25 and 31-mm inhibition Secondary metabolites were identified and quantified by zones) against E. coli and E. faecalis, respectively (Nath HPLC. The best result for mycelial inhibition by the et al., 2013). synergism test was determined by the joint action of the The secondary metabolites act on the fungus through extract of L. sidoides and that of the endophytic fungus cytoplasmic granulation, disorganization of cellular Verticillium sp. against C. lunata. contents, and inactivation of enzymes, which inhibits germination, germinative tube elongation, and mycelial CONFLICT OF INTERESTS growth (Lo et al., 1996). Thus, it can be inferred that the best result of mycelial inhibition was determined by the The authors have not declared any conflict of interests.
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Vol. 11(16), pp. 307-317, 25 April, 2017 DOI: 10.5897/JMPR2016.6200 Article Number: B58A46C64104 ISSN 1996-0875 Journal of Medicinal Plants Research Copyright © 2017 Author(s) retain the copyright of this article http://www.academicjournals.org/JMPR
Full Length Research Paper
Ethnobotanical study of ethnoveterinary plants in Kelem Wollega Zone, Oromia Region, Ethiopia
Tolera Fufa Feyissa1, Moa Melaku shigut1*, Tilahun Bekele Hailemariam1, Tena Regassa2 and Nebiyu Kassa Kergano3
1Jimma University College of Agriculture and Veterinary Medicine 2Wollega University Departments of Biology and Botanical science 3Chena woreda livestock and fishery resource development office
Received 14 July, 2016; Accepted 15 September, 2016
Questionnaire based cross sectional study design was conducted from November 2015 to April 2016 in Dale Sadi district area, Kellem Wollega Zone of Oromia regional state of Ethiopia, to identify potential medicinal plants used for treatment of the livestock ailments. In this study 50 species of medicinal plant species were identified which were categorized under 32 different families. Among the medicinal plants 45(90%) were used for curative purpose, 2(4%) for only prophylactic purpose and 3(6%) for both curative and prophylactic activities. Shrubs 29(58%), herbs 10(20%) and tree 8(16%) were the main habitat of the herbal plants. The main routes of administration were oral and topical, 30(60%) and 9(18%) respectively. Leaves 18(36%) and roots 7(14%) were the main parts of the plant used as medicinal values. The results of this study play a significant role in encouraging further investigations by extracting and identifying bioactive constituents of those herbal medicines for the antimicrobial effect. It is recommended that further detailed examination should be conducted to investigate the medical principles and pharmaceutical activity found in these plants.
Key words: Livestock, traditional healers, medicinal plants.
INTRODUCTION
Medicinal plants have served through ages as a constant pharmacopoeias (Kim, 2005). Medicinal plants play a key source of medicament for treatment of a variety of role in the development and advancement of modern diseases (Okoli et al., 2007). The history of herbal studies by serving as a starting point for the development medicine is almost as old as human civilization of novelties in drug (Wright, 2005). Tropical plants have (Choudhary et al., 2015). In ancient cultures people been used for medicinal purposes since the evolution of developed their own herbal pharmacopoeias based on man. This knowledge is still alive and several hundred information gained through experience and in our today’s species are used in herbal remedies in indigenous scientific pharmacopoeia much of the information on system of medicines, where the whole plant or plant part scientific medicine is derived from those herbal or its extraction is used (Alawa et al., 2002; Okoli et al.,
*Corresponding author. E-mail: [email protected].
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License 308 J. Med. Plants Res.
2002; Ogbuewu et al., 2015). Medicinal plants are the traditional healers and veterinarian. “backbone” of traditional medicine, which means more than 3.3 billion people in the less developed countries Study design and sampling methods utilize medicinal plants on a regular basis (Davidson- Hunt, 2000). Primitive people learned by trial and error to Cross sectional study design was conducted to identify traditional distinguish useful plants with beneficial effects from those plants used in animals disease healing. Primary data were collected that were toxic or non-active and also which by questionnaire survey from purposively selected elders, combinations or processing methods had to be used to especially traditional healer’s livelihoods which depend gain consistent and optimal results (Jagessar et al., predominantly on traditional plants for curing their livestock and their own health. At the same time voluntary animal owners, animal 2008). health practitioners were interviewed using focus group discussions In comparison with modern medicine, herbal medicines and field observation. Non-probability sampling method was carried cost less, are more often used to treat chronic diseases out to collect information related to medicinal plants for livestock in and the occurrence of undesirable side effects seems to the study area. All volunteer traditional healers selected based on be less frequent (Jagessar et al., 2007). Natural products recommendation from elders of the study area. have long been regarded as excellent sources for drug discovery, given their structural diversity and a wide Study methodology variety of biological activities (Fu et al., 2008). Due to easy availability and low cost of ethno veterinary The structured questionnaire was used to collect information related medicinal plants, the livestock owners of the remote to medicinal plants used to treat livestock and Information regarding local name of the plant, its habit, part(s) of plants used, route of areas use them as a first aid for their animals (Jabbar et administration, methods of preparation, local name of the animal al., 2006). diseases treated, dosage used and species of animals treated was Ethno veterinary knowledge is acquired by communities recorded at spot. Based on ethno botanical information acquired over many years and passed between generations from informants, the plants were collected from the surrounding through oral tradition. Today, with rapid cultural changes, forests and other parts of the study areas with the people who know this knowledge is being lost, necessitating its scientific the local name of the plants. Pictures (Figure 1) of voucher specimens were captured and their leaves were collected, pressed documentation (Mathias, 2001). Thus, saving the species, and dried to identify the scientific names of the collected medicinal documenting and preserving indigenous knowledge is plants at the National Herbarium of Biology Department of Natural essential (Alam and Ali, 2010). There is a lack of ethno Science Faculty, Wollega University, Ethiopia. botanical survey carried out in most parts of the country. In view of these, documentation of the traditional use of medicinal plants is important to preserve the knowledge Data analysis and management regarding the traditional plants. Once these local ethno The information that was gathered through questionnaire survey medical preparations are scientifically evaluated and was coded and entered in to Microsoft Excel spreadsheet. disseminated properly, people will be better informed Descriptive statics and chi-square were employed by SPSS version regarding efficacious drug treatment and improved health 20 software for analyzing of ethno botanical data. status. Therefore, this study was attempted with objectives: RESULTS 1. To identify and document potential medicinal plants for the livestock ailment treatments at the study area. The total numbers of species of plants collected during 2. To investigate the scientific name of those medicinal the study period were fifty (50). These were categorized plants used by the local traditional healers. under 32 different types of families. From the total 32 families of the plant-samples, 6(12%) plant species belong to family Asteraceae and 4(8%) plant species MATERIALS AND METHODS belong to family Solanaceae. Those family including Zygophyllaceae Fabaceae, Rosaceae, Musaceae, Study area and study population Euphorbiaceae, Moraceae, Rutaceae, Malvaceae, Brassicaceae and Cucurbitaceae each contains 2(4%) The study was conducted from November 2015 to April 2016 on species of plants. Each of the remaining twenty (20) some traditional medicinal plants found in Dale Sadi district. Dale Sadi is one of the districts in Kellem Wollega zone of Oromia families was represented by single species of plants Regional state. It is 510 km far from Addis Ababa capital city of (Table 1). Ethiopia. The area lies at average altitude of 1150 m above sea The identified traditional medicinal plants were used level. The area has temperature range of 33 of 35°C with more traditionally to cure livestock ailments by local agricultural crops. The climatic condition alternates with long communities. It was found that the medicinal plants were summer May to August and short rainy seasons from March to used as anti-bacterial, anti-parasitic, anti-viral. At the April. The winter dry seasons (November to February) with mean annual rain fall of 1200 mm (DDBOA, 2013). The target populations same time those medicinal plants could be used for for this study were voluntary livestock’s owners such as farmers, haemostatic, retention of fetal membrane, suturing the Feyissa et al. 309
A B
C D
Figure 1. Some of the identified medicinal plants in the study area. (A). L. actucain ermis; (B) Bersama abyssinica; (C). Datura arborea; (D). Justiciasc himperiana
physically damaged part, snake bites, fracture, snake seed, bark, root, sap and latex were widely used for allergy, poison consumed, free gas bloat, tympanic bloat, treating animal disease. Leaf 18(36%) were the most and others (Tables 2 to 6). frequently used plant parts followed by root 7(14%) and seed 4(8%) and the minimum used part was pod (2%) (Table 8). Routes and dosage of the administered medicinal The investigated plants were fall under 5 groups. plants Shrubs are highest 29(58%), followed by herbs 10(20%), tree 8(16%) and epiphyte is the least 1(2%) (Table 9). Numerous techniques of administrations routes were employed during administering the remedies. The highest numbers of medicinal plants were given orally 30(60%), DISCUSSION followed by topical application 9(18%) (Table 7). According to study area much of recorded medicinal Medicinal plants play a key role in the development and plants were found not to be toxic to animals even given at advancement of modern studies by serving as a starting higher doses, except some plants like Euphorbia point for the development of novelties in drug. Herbal Ampliphylla, Zehneriascabra, Capparistomentosa, medicine has been widely formulated and used as an Capsicum annuum and Eulophia that were reported to integral part of primary health care in Nigeria, China, be toxic at higher concentration (Table 7). Ethiopia and Argentina (Ogbuewu et al., 2015). Among the plant families, Asteraceae was the dominant medicinal plant (12%) followed by family Parts of plant used as medicinal values and their Solanaceae (8%). This finding was in line with Getaneh growth form et al. (2014), in which Asteraceae was found to be the most dominant (11.2%) and Solanaceae was the second The plants part used to treat animal disease varied from dominant (7.4%). This result is different with the finding of species to species and from diseases to diseases. Leaf, Zewdu et al. (2015), in which Euphorbiaceae was 310 J. Med. Plants Res.
Table 1. Lists of plant’s family frequently used by traditional healers in the study area.
Family name No. of species of plants under the family Percentage (%) Asteraceae 6 12.0 Zygophyllaceae 2 4.0 Apocynaceae 1 2.0 Scrophulariaceae 1 2.0 Fabaceae 2 4.0 Urticaceae 1 2.0 Combretaceae 1 2.0 Solanaceae 4 8.0 Cactaceae 1 2.0 Simaroubiaceae 1 2.0 Scrophulariaceae 1 2.0 Unknown 1 2.0 Rosaceae 2 4.0 Musaceae 2 4.0 Capparaceae 1 2.0 Loranthaceae 1 2.0 Agavaceae 1 2.0 Liliaceae 1 2.0 Euphorbiaceae 2 4.0 Moraceae 2 4.0 Rutaceae 2 4.0 Phytolaccaceae 1 2.0 Malvaceae 2 4.0 unknown 1 2.0 mucronata 1 2.0 Brassicaceae 2 4.0 Curbitaceae 2 4.0 unknown 1 2.0 Amaryllidaceae 1 2.0 Francoaceae 1 2.0 Acanthaceae 1 2.0 Zingiberaceae 1 2.0
reported to have the highest number of species employed Khare., 2007; Akhtar., 2013). Similar to the reports of in the treatment of diseases fallowed by Cucurbitaceae, Firaol et al. (2013), Euphorbia ampliphylla plant was Lamiaceae, Solanaceae and Verbenaceae underscoring found to have the ability to cure the wart by topical the significance of these species in the ethnomedicine of application. Gondar town. At the study area, Phytolacca dodecandra have Citrus aurantifolia is a traditional medicinal plants also medicinal values for internal parasites, abdominal aches identified in this study which have insecticidal property and washing of the wound. However, the study results of against lice infestation. Similar result was reported by Abebe et al. (2003) indicated that P. dodecandra used as Parle and Chaturvedi (2012), who indicated that orange taenicidal and molluscicidal activity and other study peel oil produces lethal effect on fleas, fire ants, and conducted in Hadya Zone, Ethiopia also revealed that houseflies due to its 90 to 95% limonene. Other than this plant’s root was useful for treating Anthrax and using as insecticides, it was reported that the juice of C. itchiness (Habtamu et al., 2014). aurantifolia is considered as tonic for libido and as In this study finding the Gossypium herbaceum plant antidote for poison and the diluted form of the C. have medicinal values for treating of ocular problem. In aurantifolia fruit juice is used for mouth wash to treat sore contrast to this Dhamija et al. (2011) reported that mouth, sore throat and useful to treat irritation, diarrhea aqueous extract of G. herbaceum showed significant anti- and swelling due to mosquito bites (Aibinu et al., 2007; depressant like effect due to activation of adenyl cyclase Feyissa et al. 311
Table 2. Summary of general characteristics of the identified medicinal plants under family Asteraceae’s family.
Plant local Plant Part Scientific name Adm. Route Method of preparation Indication Animal’s species name type used Its leaves are chopped and given with feed. It is also Crassocephalum Oral, auricular and For dulled and emaciated Lomi simbira H Leaf squeezed and the drop applied in both eyes and Bovine sarcobasis nasally animals noses
The roots finely chopped and given with feed. Its root Oral and nasal For abdominal aches, circling Kabaricho Echinops S Root is also chopped and added to burning fire and the All mammals disease and dry coughing vapor of the smoke is inhaled
Some of the leaves added to their feed with salt and For washing and healing the Ebicha Vernonia amygdalina S Leaf Oral and topical All mammals wash the wound locally wound
Its root are inserted into the needle for suturing of All domestic Ada Guizotiascabra S Root Topical Suturing the wounded body the wounded part animals
Ageratum leaves are squeezed and dropped to the bleeding All domestic Kasi S Leaf Topical For haemostasis conyzoides area Animals
After rooted off, whole parts are chopped together For killing maggots found in All domestic Koricha ramo Lactucainermis S Whole Oral and given with their feed open wound. animals
S, Shrub; H, herb.
Table 3. Summary of general characteristics of the identified medicinal plants under family Solanaceae.
Plant local plant Scientific lame Part used Adm. Route Method of preparation Indication Animal’s species name types Oral 2-3 pods are grinded with the bulb of Allium sativum then For abdominal aches and removal of Mimita Capsicum annuum H Pod All mammals &Topical drenched. It also applied topically for leech treatment Leech from gum Leaves are given orally with the bulb of Zingiber officinal for For Black leg, dullness, ocular and nasal Asangira gibe Datura arborea H Leaf Oral ruminants. But for equines it should be chopped and drench by All mammals discharges bottle Solanum Hidi warabesa S Root Oral Some of its roots are chopped and added to their feed Black leg Ruminants marginatum For coughing & killing of Internal Tambo Nicotiana tobacum S Leaf Oral The leaves are squeezed into throats of sheep and cattle Ovine and bovine parasites
cAMP pathway in signal transduction system and diuretic activity. In addition, the Allium cepa is common ailments like cold, allergies, toothaches, hence protecting the neurons from the lesion and another type of traditional medicinal plants useful laryngitis and cough. Narasimha et al. (2008) also emphasized that G. for treating Foot and mouth viral diseases. This In this study, the whole parts of Bersama herbaceum seeds to have antioxidant activity, result is different from the finding of Yusha’u et al. abyssinica were identified having the ability to anti-diarrheic, wound healing, anti-migraine, and (2008), in which A. cepa is used in treatment of treat internal parasites. This is in agreement with 312 J. Med. Plants Res.
Table 4. Summary of general characteristics of the identified medicinal plants under families of Zygophyllaceae, Fabaceae, Rosaceae, Musaceae, Euphorbiaceae
Plant local Plant Adm. Animal’s Scientific name Family name Part used Method of preparation Indication name types route species A cup of seed is grinded then the powder mixed with chopped For taeniasis, treat and Hagenia form of A. sativum and given for taeniasis infected animals. It prevention and from any kind of All mammals Koso abyssinica Rosaceae S Seed Oral also grinded with ant’s clustered on a tree then drench for infections in donkey. donkey
For emaciation, coughing, black Facah NI Zygophyllaceae S Root Oral Root are chopped and given with feed Ruminants leg, trypanosomiasis
Barks and Leaves and bark are chopped together and applied topically to To kill maggot larvae found in All domestic Ambalta Entada abyssinica Fabaceae T Topical leaves wound containing maggot’s larvae the wound animals
The immature parts of leaves are given with its fruits for Croton Oral and Bakanisa Euphobiaceae T Latex bloated animal. Its latex is also directly painted on the affected For bloats, dandruff macrostachyus topical All mammals area
Its sap was collected to clean container and a few drops are Ensete Kacho Musaceae H Stem Oral drenched orally For diarrhea and cough Poultry ventricosum
The sap is collected and given with their feed and it is also Euphorbia Oral and For any internal parasites, All domestic Adami Euphorbiaceae S Latex applied to the local warts. ampliphylla topical Lameness, wart. animals
Its sap is collected to clean container and washing an animal All domestic Warke Musa sapientum Musaceae H Stem Topical Mange mites, ticks and lice body with a medicinal infusion animals
For prolonged emaciated Leaf and All stem and leaves are chopped together and given with their Tumjo NI Rosaceae C Oral animal, lameness, Anthrax, All ruminants stem feed for 2 times per year coughing, Black leg
Some of the leaves juice are squeezed to their body and For all ecto-parasites, and All domestic Ceka Calpurnia aurea Fabaceae S Leaves Topical washed thoroughly alopcia animals
Koricha Porlieria All domestic Zygophaceae S Leaves Oral The leaves is chopped added to their feed For snake bite bofa hygrometra animal
S, Shrub; H, herb; T, tree; C, climber;NI, not identified.
report of Mathewos et al. (2015) and Zewdu et al. conducted on Ethnobotanical study of traditional officinale have medicinal values for treating any (2015), that the extracts of B. abyssinica which medicinal plants in and around Fiche District, internal parasites. This is similar with the finding of could be administered orally for treating Ethiopia. Anthelmintics use of H. abyssinica in Iqbal et al. (2006), who investigated the dysentery, stomach disorders such as abdominal ruminants has been in agreement with the finding anthelmintic activity of crude powder and crude pain, colic, diarrhoea, intestinal worms, of Abebe et al. (2000). However it was reported aqueous extract of dried Zinger (1 to 3 g/kg) in amoebiasis. Justicia schimperiana have medicinal that H. abyssinica also used against tapeworms in sheep naturally infected with mixed species of values for prevention rabies. The result was in line humans in Ethiopia (Giday et al., 2003, Yayesh et gastrointestinal nematodes. Contrary to this with other finding of Abiyu et al. (2014) that was al., 2015). According to local herbalists Zingiber finding specific aspects of Zinger’s actions has Feyissa et al. 313
Table 5. Summary of general characteristics of the identified medicinal plants under families of Moraceae, Rutaceae, Malvaceae, Brassicaceae, Cuccurbitaceae.
Plant local Plant Adm. Animal’s Scientific name Family name Part used Method of preparation Indication name types route species Whole The whole plant is chopped and the powdered form is For dry cough, emaciated, Hida refa Zehneriascabra Cucurbitacea C Oral Equine parts dissolved in water and drenched by bottle. lameness, alopecia, colic
Kiltu Ficusvasta Moraceae T Sap Oral Decocted leaf is collected and drunk by cup For GIT parasites All mammals Lomi Citrus aurantifolia Rutaceae S Fruit Oral A few drops of fresh juice are squeezed orally to throat Coughing and lice infestation Poultry
Ruta The leaf are chopped are squeezed to the bottle and drunk All domestic Ciladami Rutaceae H Leaves Oral For poisoned animals chalepensis the poisoned animals animal
The leaves are pinched and squeezed and dropped to All domestic Karabi Sidarhombifolia Malvaceae S Leaves Topical Wound wound topically. animal
A cup of its Seed are grinded and mixed with water and Sanaficha Brassica nigra Brassicaceae H Seed Oral For frothy and free gas bloat All mammal drunk by bottle
Some of its upper part are chopped and mixed with water H Leaves Oral and Prevention of disease from Ruminants Cucurbita then drench the neonates half of a bottle. Its fruit is roasted Buke Cucurbitaceae and fruit topical neonates like Orf Pepe and applied topically to infection found on the nose
Gossypiumherbace Some of its seed are grinded and the dust particles are Jirbi Malvaceae S Seeds Ocular For ocular diseases All animal um applied to the infected eye only once
Lepidium A cup of its seed juices is mixed with water and drunk only For both productive & dry Shinfa Brassicaceae S Seed Oral All mammals sativum once coughing
A few drops of its latex are pasted with ash of the burned All domestic Odaa Ficussycomorus Moraceae T Sap Topical Aspiliamossambicensis (Oliv.) and applied to the wound until For wound healing animal it dry
S, Shrub; H, herb; T, tree; C, climber; NI, not identified.
been practiced for centuries in Chinese, Ayurvedic Echinops is a well-known medicinal plant which is medicinal values in health care of livestock for and Tibb-Unani fields of study as herbal used for treating abdominal ache, dry cough and treatment of multiple ailments. This was also medicines for the treatment of catarrh, circling diseases. Similarly Hymete et al. (2007) reported in different countries (Deeba et al., 2009; rheumatism, nervous diseases, gingivitis, tooth reported that the root of Echinops is chewed and Shinwari, 2010). ache, asthma, stroke, constipation and diabetes used to alleviate stomach ache. The anti-microbial (Wang and Wang, 2005; Tapsell et al., 2006). On activities of medicinal plants are varying; this is in the other hand (Grzanna et al., 2005) indicated agreement with (Yusha’u et al., 2008) who Conclusion the anti-inflammatory action of ginger, while reported that antibacterial activity may vary from Shukla and Singh (2007) dealt with the cancer one plant to another. Generally herbal medicines The results of the present study show presence of prevention properties of the Zinger crude drug. collected during the study were found to have wide range of herbal medicine used for treating 314 J. Med. Plants Res.
Table 6. Summary of general characteristics of the remaining identified medicinal plants.
Plant local Plant Part Adm. Animal’s Scientific name Family name Method of preparation Indication name types used route species Kulubi Allium cepa Amaryllidaceae S Bulb Oral Roots are chopped and given with salt for 2 days Foot and mouth disease Ruminants warabesa
Two bulbs are chopped with salt and given orally for 2 All domestic Jijimbila Zingiber officinale Zingiberaceae S Roots Oral Ascariosis, stomach disorder days animal
Justicia S The juice of 2-5 leaves are squeezed to their feed and Dumuga Acanthaceae Leaves Oral Rabies All mammals schimperiana given for 3 days
Black legs, chronically diseased, All of its parts are grinded together and mixed with water Lolchisa Bersama abyssinica Francoaceae T Whole Oral paralyzed animal that unable to Ruminants and drench a bottle of the mixture stand
Leaves The odd’s number of its seed (either 3, 5 or 7) are given Asangira Snake bite, for retained fetal All domestic Eulophia spp. Orchidaceae S and Oral orally and some of its leaves are chopped then given with guracha membrane, abdominal aches animal seed their feed
Some of its leaves are chopped and grinded together with Aleltu mucronata mucronata T Leaves Oral Justicia schimperiana’s leaves and drench a bottle of the Rabies prevention All mammals filtrate
Acokanthera Its bark grinded with leaf of Vernonia Tabanae Apocynaceae T Bark Oral For healing of swelling All mammals schimperi amygdalina and added to its feed
Root is covered by clean cloth and applied locally to Arangama Capparistomentosa Capparaceae S Root Topical For treat local swelling All mammals swelled area
Phragmantheramacr Clinical mastitis, prolonged Loranthaceae E Leaves Oral The leaves are chopped and given with their feed Ruminants Dertu osolen emaciated animals
Kicu Agave americana Agavaceae H Bulb Oral The bulbs are chopped and given with salt For Black leg Ruminants warabesa
Basango Canna indica Cannaceae S Root Oral The roots are given with the leaves of Datura arborea. Black leg Ruminants
All domestic Kulubi adi Allium sativum Liliaceae H Bulb Oral The bulb is chopped with salt and drenched orally Abdominal aches and bloat animal Feyissa et al. 315
Table 6 cont’d
Brucea Ocular and Some of its leaves are chopped and squeezed. Then its For blinded and eye discharged All domestic Komogno antidysenterica Simaroubaceae S Leaves auricular drops applied at the side of infected eye animals animal
Verbascumsinaiticum Its leaves are chopped with the bulb of Allium sativum and Gura harre Scrophulariaceae S Oral For coughing All mammals Benth. Leaves drenched orally
For retention of fetal membrane, Harkis Opuntia ficus-indica Cactaceae S Steam Oral Some of steam parts are chopped and given with salt All mammals and against bloat
Table 7. Lists of administration route(s) for medicinal plant(s) in study area.
Routes of administration of medicinal plant (s) Number of responders Percentage Oral 30 60.0 Topical 9 18.0 Ocular 1 2.0 Oral and nasal 1 2.0 Oral and topical 6 12.0 Oral, auricular and nasal 1 2.0 Ocular and auricular 1 2.0 Nasal and ocular 1 2.0
Table 8. Summary of plant part(s) used as medicinal values.
Parts of medicinal value(s) Number of species Percentage (%) Leaf 18 36.0 Root 7 14.0 Bark 2 4.0 Stem 3 6.0 Leaf and stem 1 2.0 Bulb 3 6.0 Latex 2 4.0 Sap 2 4.0 Fruit 1 2.0 Bark and leaf 1 2.0 316 J. Med. Plants Res.
Table 8. Cont’d
Seed 4 8.0 Pod 1 2.0 Leaf and seed 1 2.0 Leaf and fruit 1 2.0 Whole parts 3 6.0
Table 9. Distribution of the medicinal plants by their growth forms.
Habit of the plants Frequency Percentage (%) Herb 10 20.0 Shrubs 29 58.0 Tree 8 16.0 Climber 2 4.0 Epiphyte 1 2.0 Total 50 100.0
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