Loss of production and productivity in the livestock sector, and public health problems are among the major challenges worldwide. Infectious diseases are the major contributing factors to the overall economic losses in livestock industry. Fungi are parasites that can cause significant harm to agriculture and forestry as well as major illnesses in both humans and animals. Allergies are brought on by their spores. Aspergillosis, candidiasis, and cryptococcosis are invasive fungal illnesses caused by opportunistic organisms from the genera Aspergillus, Candida, and Cryptococcus respectively [1]

The majority of Candida spp. that has been described is saprophytes. Due to their inability to thrive at 37°C, half of the identified species are ineffective as human pathogens. 90–92% of cases of candidiasis are caused by Candida albicans and non-albicans species such Candida glabrata, Candida tropicalis, Candida krusei, and Candida parapsilosis. Common yeast C. tropicalis has characteristics with C. albicans. Compared to other Candida species of medicinal significance, they share a higher genetic similarity with C. albicans [2]. Candida zeylanoides is relatively rare in humans and animals and has been reported on the skin, nails, and blood as an opportunistic yeast pathogen [3].

Aspergillus species can cause serious clinical illness, particularly in immunocompromised people, and they become a significant opportunistic fungal infection [4]. Also, many patients have been diagnosed with chronic pulmonary aspergillosis worldwide following the treatment of pulmonary tuberculosis [5]. Glucose, bran, maltose, xylan, xylose, sorbitol, and lactose are all excellent carbon sources for the A. niger strain, which also thrives well in other mediums [6].

Different conventional medications are used to treat ailments in both animals and people However, traditional medications are less favorable because of drug residues, increasing drug resistance in target species, and their accessibility and price issues for underprivileged farmers. Therefore, another alternative approach has been getting greater attention. Increased use of traditional medicinal plants is due to the acceptability, accessibility, inadequate coverage of the modern medical system, high cost of modern drugs, and other biomedical benefits [7].

The utilization of medicinal plants has been the mainstay of the world's approach to treating illnesses and fighting infections. Plants have long been abundant sources of efficient and secure medications [8]. Eighty percent of the world’s population exclusively relies on traditional medicine; especially in developing countries, where they have the resources to sustain primary healthcare systems. There is considerable global interest in tapping the accumulated knowledge of traditional medicine; research is being carried out in many countries to increase the use of medication for the welfare of the human population [9].

Plants have medical significance because of their chemical components, or phytochemical elements, which have a specific physiological effect on both humans and animals. In herbal remedies of different part of plants, phytochemicals are naturally occurring compounds with defense mechanisms that help plants themselves fighting against numerous ailments. Phytochemicals may include primary or secondary compounds. Chlorophyll, proteins, and common sugars are the common primary constituents with secondary compounds (the most important bioactive constituents of the plants) including alkaloids, tannins, flavonoids, terpenoids, saponins, cardiac glycosides, steroids, anthraquinones and phenolic [10].

They have great roles in health protection, when their dietary intake is significant [10,11]. High concentrations of phytochemicals, which may protect against free radical damage, accumulate in fruits and vegetables [12]. Plants containing beneficial phytochemicals may supplement the needs of the human body by acting as natural antioxidants. Numerous studies have shown that many plants are rich sources of antioxidants. For instance, vitamins A, C, E, and phenolic compounds such as flavonoids, tannins, and lignins, found in plants, all act as antioxidants [12]. 

According to a survey of the literature, Calpurnia aurea species parts have been used to treat various human and animal ailments. They have several ethnoveterinary uses such as treating external parasites, [13], helminthiasis, [14], swelling of the nose in mules, sores and depression, rabies, parasitic leeches, snakebites, [14] anthrax, [15] and blackleg  [16]  Roots are used to treat bloody diarrhea, [13], skin diseases, [17] and stomachache [18].

Calpurnia aurea leaf extracts evaluated for phytochemicals showed the presence of cardiac glycosides, phytosteroids, flavonoids, alkaloids, phenols, and terpenoids [19,20]. A range of quinolizidine alkaloids and pyrrole carboxylic acid ester has been detected in Calpurnia aurea leaves after chemical analysis. Calpurnia possesses a significant amount of flavonoid content, which has been associated with strong antioxidant capabilities [21].

The genus Verbascum (family Scrophulariaceae) contains about 360 species. The plant has historically been used to treat wounds, stomachaches, viral infections, and cancer sunstroke fever, abdominal colic, diarrhea, hemorrhage, and anthrax [22]; hepatitis [23], rheumatoid pain, elephantiasis, Measles [24,25], reported the in vitro broad-spectrum antimicrobial activity: against Gram (+) bacteria such as Staphylococcus aureus and Gram (-) bacteria including Pseudomonas aeruginosa of the methanol extract of V. sinaiticum leaves. Several bioactive principles including iridoid glycosides, flavonoids, phenolic acids, sterols, triterpenes, saponins, polysaccharides, and alkaloids, were found in Verbascum species. Many iridoids have been isolated from Verbascum [26].

In West Shewa Zone, like any other place in Ethiopia, utilization of herbal medicine is limited with traditional practice, serving only the rural community and stock raisers. The indigenous use of different medicinal plants for the management of fungal like infections in Gindeberet, Elifata, Dendi, Ambo, Dire Inchini, Cheliya, and Ilugelan districts of West Shewa Zone, Oromia Regional State were documented [27]. In this study, two plants namely Calpurnia aurea (local name Cheka) and Verbascum sinaiticum (local name Gura Hare) having traditional claims for the treatment of various skin disorders were identified for evaluation against Aspergillus niger, Candida tropicalis, and Candida zeylanoid [28].

 Previously these plants were studied for their different activities (antibacterial, antioxidant, antidiarrheal, anti-trypanosomiasis, etc.) [29,30]. However, the plants were not evaluated for their antifungal activity and there are no articles on their utilization against fungal strain. Therefore, determining the antifungal activities of these medicinal plants and the assessment of their phytochemical components utilizing pharmacological action is essential. So the objective of this study is to determine antifungal activities and phytochemical screening of Calpurnia aurea and Verbascum sinaiticum against selected fungal strain of animal pathogen.

Study Area

The study plants were collected from Toke Kutaye district of West Shewa zone, Ethiopia. Toke Kutaye district lays 12 km west of Ambo Town and around 128 km west of Addis Ababa, the capital of Ethiopia. Geographically, the district lies between 80 47’ to 90 21’ latitudes and 370 32’ to 370 03’E longitude. The district has an average annual temperature of 19.50C, 1100 mm of rainfall, a main rainy season that extends from May to September, and an elevation range of 1500 to 2000 m above mean sea level.  The district's soils ranged from dark reddish-brown loamy sand to sandy clay loams, with thick layers of reddish-brown loamy sand on top. The soils were very deep and well-drained [31].

Study Design

An experimental study was conducted from November 2021 to June 2022 to evaluate the invitro antifungal effects of methanolic extracts of Verbascum sinaiticum leaves and Calpurnia aurea seed and leaves against Aspergillus niger, Candida zeylanoides, and Candida tropicalis. Standard antifungal ketoconazole (15µg Laboratoires Humeau) was taken as positive control and dissolvent 5% dimethyl sulfoxide (DMSO) was used as a negative control.

Plant Collection and Preparation.

Fresh leaves and seeds of Calpurnia aurea and leaves of Verbascum sinaiticum were collected from Toke Kutaye districts of the West Shewa Zone. The collected plants were brought to the Microbiology Laboratory at Ambo University, Guder Mamo Mezemir Campus, after being identified and verified by a botanist Biruk Bedore of Ambo University. To get clear of any remaining dust and grime, the plants were then carefully cleaned with distilled water. The gathered plants were spread out on the paper sheet and allowed to dry for two weeks at room temperature in the shade after they was chopped into little pieces. Lastly, a mortar and pestle were used to coarsely ground the plant parts, which were then kept until they were required for extraction.

Plant Extraction

The powdered plant specimen was then subjected to extraction using 99.5% methanol (Central Drug House LTD). Maceration techniques were used to prepare the extracts according to procedures given by [32]. An automated orbital shaker was used to shake the ground plant materials for 72 hours after they had been separately soaked in methanol (99.5%) at a ratio of 1:4. These were carried out three times in order to give the solvents sufficient time to extract significant amounts of the chemical components from the ground plant materials. The mixture was first filtered using gauze and then the filtrate was passed through sterile Whatman No. 1 filter papers. Then the filtered extract was dried in a warm air oven at a temperature of 40 0C and the resulting crude extracts were transferred into well-labeled beakers, and it was kept in a refrigerator until required for use.

Determination of Extraction Yield

About 120gm leaf of Verbascum sinaiticum, 300gm seed, and 160gm leaf of Calpurnia aurea powdered plant materials were obtained and the powdered plants were extracted using 99.5% methanol. From these the percentage yield of each extract was obtained using the formula;

Where W2 is the weight of the dried extract and the container; W1 is the weight of the container alone; and W0 is the weight of the dried plant material [33].

Test Microorganisms

Microorganisms selected for the experiment were standard fungal strains including Aspergillus niger (EMCC-F3F8), Candida tropicalis (EMCC-F307), and Candida zeylanoides (EMCC-F123) obtained from the Ethiopian Biodiversity Institute and transported to the laboratory of the Veterinary Laboratory Technology, Ambo University, using cold chain system.

Preparation of Discs

Sterile Whatman No. 1 filter paper was used to create the disc. Papers were punched to make 6-mm diameter paper disks using a 6-mm cork-borer. To ensure further usage, the disks were autoclaved and kept sterile. 5% dimethyl sulfoxide (DMSO) was used to dilute and make the concentration of Calpurnia aurea and Verbascum sinaticum extraction. The test involved three sets of concentration dilutions: 200 mg/ml, 100 mg/ml, and 50 mg/ml. Using micropipettes, five microliters (5µl) of extract from Verbascum sinaiticum and Calpurnia aurea at the aforementioned concentrations were aseptically applied to these discs.

Preparation of inoculum

Tested species of fungi (A. niger, C. trolpicalis, and C. zeylanoides) colonies that were stored on Sabouraud Dextrose Agar (SDA) slant were sub cultured on potato dextrose agar (PDA) plate and incubated at 35°C for three days. An A. niger inoculum was facilitated and produced by mixing A. niger colonies that were collected from fresh culture of three days old with one milliliter of sterile saline solution and a drop of Tween 20. After total dissolution, the inoculum supernatant was adjusted using physiological solution and compared to the 0.5 McFarland standard. Antifungal tests were conducted using the supernatant. C. tropicalis and C. zeylanoide inoculum suspensions were prepared by taking a few colonies from fresh cultures grown on the PDA plate. The colonies were suspended in 1 mL of sterile saline. The inoculum suspensions were shaken until the inoculum completely dissolved. The turbidity of the inoculum was then measured using a sterile physiological saline solution and compared to the 0.5 McFarland standards [34].

Agar disk diffusion assay

Invitro antifungal activity of Verbascum sanaiticum and Calpurnia aurea was determined by using the agar disk diffusion assay technique ( Valgas et al., 2007). The test fungi were grown on potato dextrose agar (PDA) medium for three days at 27 °C and the plates were done in triplicates. The positive and negative controls were also carried out to monitor the antifungal activity of the plant’s extraction. Standard antifungal ketoconazole (15µg Laboratoires Humeau) was taken as positive control and dissolvent 5% dimethyl sulfoxide (DMSO) [35], was used as a negative control. Using ethanol dipped and flamed forceps the prepared discs were aseptically placed over PDA plates seeded with the respective test microorganism. The plates were then left at room temperature for 30 minutes to let the extracts and controls diffuse in the agar medium. Finally, the plates were incubated at 37 °C for 48 hr. The antifungal potential of extracts was assessed in terms of the zone of inhibition and the diameters of the inhibition zone in mm were measured [36].

Determination of Minimum Inhibitory Concentration (MIC)

The MIC assay was performed to determine the minimum concentration that inhibits the growth of microorganisms. Plant extracts were subjected to determine their MIC or the lowest concentration that will inhibit the visible growth of fungal strain. In the agar dilution method, the serial dilution approach was employed to find the lowest dosage that could inhibit visible fungal growth. The extract was serially diluted from its 200 mg/ml concentration to 100 mg/ml, 50 mg/ml, 25mg/ml, 12.5mg/ml, 6.25mg/ml, 3.125 mg/ml, and 1.5625 mg/ml. These was done by incorporating 50 µl of each dilution concentrations of plant extracts into a molten agar medium and then tested fungal species were inoculated onto the agar plate surface. The MIC endpoint is recorded as the lowest concentration of antimicrobial agent that inhibits growth under suitable incubation conditions. MIC panel reading was interpreted as [37].

Phytochemical screening of the plant’s extracts was conducted to determine the presence of saponins, steroids, phenolic compounds, flavonoids, alkaloids, and glycosides by using various standard methods. The color change was recorded as present (+) or absent (-) depending on the outcome of the test. Methanol extracts of Calpurnia aurea (Chekaa) and Verbascum sinaiticum (Gura hare) was tested for the presence of mentioned active principles.

Test for flavonoids

Bate-Smith and Metcalf test: Extracts were treated with 0.5ml of concentrated hydrochloric acid (HCL) and warmed for 15 minutes in a water bath. The formation of strong red/ violet color was observed in the presence of flavonoids [38]

Test for Glycosides

Liebermann's test: 2ml of extracts, 2ml of chloroform, and 2ml of acetic anhydride were added together and mixed. The formation of a reddish-brown /blue-green or violet color was observed for the presence of glycosides [38].

Test for phenolic compounds

Ferric Chloride test: 2ml of the extract was treated with 4 drops of concentrated ferric chloride solution. A bluish-black color product was observed for the presence of phenolic compounds (39].

Test for saponins

Foam test: About 0.5mg of extract was mixed with 40 ml of distilled water in a test tube and then agitated forcefully. The formation of persistent foam that remained for 10 minutes was observed for the presence of saponins (39].

Test for steroids

Salkowiski test: 5ml of plant extract was mixed with 3ml of chloroform and then a few drops of concentrated sulfuric acid (H2SO4) were added. Appearance of reddish-brown color was observed for the presence of steroids [40]

Test for alkaloids

Hager's test: 2 mL of extracts were treated with 2 mL of Hager's reagent (saturated picric acid solution). The presence of a yellow color precipitate was observed for the presence of alkaloids [37].

        Percentage Yields

The percentage yields of extracts obtained from the total dried plant material were calculated and shown in (Table 1). The highest percentage of yield was obtained from Calpurnia aurea leaf (31.87%/160gm), Verbascum sinaiticum leaf was followed with (19.16%/120gm) and the least percentage yield extraction had obtained from Calpurnia aurea seed (11.2%300gm).

Table 1: percentage yield of Verbascum sinaiticum and Calpurnia aurea methanol extracts.

W₂gm= weight of beaker and dried extract in grams, W₁gm= weight of beaker alone in grams, W₀gm= weight of dried plant material in grams, %yield= percentage yield.

Invitro Antifungal Activities

The plant extract at lower and higher concentrations showed substantial antifungal activity. Comparatively, all tested fungi were highly susceptible to standard antibiotics with a different zone of inhibition against Aspergillus niger (20±0.577), Candida tropicalis (20±0.67), and Candida zeylanoides (17±0.577). The results of the antifungal assay of the plant extracts are shown below (Table 2) and the evaluated plants were found to be shown a significant antifungal activity against tested strains.

The extract was found to be effective in a concentration-dependent manner against all tested strains. The highest inhibition zone diameter was determined at a concentration of 200 mg/ml and the lowest zone of inhibition diameter was determined at a concentration of 50 mg/ml against all pathogens. The extract of the leaves of Verbascum sinaiticum moderately inhibited the growth of all the tested fungal strains. Calpurnia aurea seed and leaf had shown high inhibition activity against all tested strains.

Table 2: The antifungal activity of C. aurea and V. sinaiticum extracts against tested fungi.

Results in Mean ± Standard error (triplicate test n=3), values indicated with different superscript show the significant mean difference between concentrations, the value was significant difference that, a =when compared to 200mg/ml, b=compared to 100mg/ml, c= compared to 50mg/ml, d= compared to positive control and e= compared to negative control, -ve control= DMSO and +ve control= ketokonazole.

Abbreviations; a. n= Aspergillus niger, c.t= Candida tropicalis, c. z= Candida zeylanoides, v. s= Verbascum sinaiticum, c. a= Calpurnia aurea

The extracts obtained from the seed of Calpurnia aurea were shown strong antifungal activities against all pathogens, with average inhibition zones ranging from 12.33±0.333 to 20±0.577. It had shown a significant antifungal activity within the highest inhibition zone 20±0.577 against Candida zeylanoides and the lowest was shown against Candida tropicalis. Calpurnia aurea leaf was shown the highest inhibition zone against Candida tropicalis and Candida zeylanoides (17.67±0.33 and 20.33± 0.88) respectively and it showed a moderate inhibition zone against Aspergillus niger (14.33±0.33). Verbascum sinaiticum was found to be shown moderate antifungal activity against all tested pathogens.

A comparable result was recorded from the extract of Calpurnia aurea seed to standard antifungal against all tested strains. Candida zeylanoides was more susceptible to the seed and leaf extract of Calpurnia aurea as compared to the standard antifungal drug ketoconazole with the inhibition zone 20±0.57 and 20.33± 0.88, respectively. In contrast, Candida tropicalis and Candida zeylanoides were shown less susceptibility to Verbascum sinaiticum leaf with inhibition zone 14±0.577 and 13±0.000 respectively as compared to standard drug ketoconazole. Similarly, Aspergillus niger showed less susceptibility (14.33±0.33) to Calpurnia aurea leaf extracts as compared to what it had shown against Calpurnia aurea seed.

Minimum Inhibitory Concentration

Plant extracts were subjected to determine their MIC or the lowest concentration that will inhibit the visible growth of fungal strain. It was decided to conduct more tests on the extracts of Calpurnia aurea leaf and seed and Verbascum sinaiticum leaf since they demonstrated a promising inhibitory zone when compared to the standard reference antifungal. In the agar dilution procedures, the serial dilution approach was employed to find the lowest dosage that could inhibit visible fungal growth. The extract was serially diluted from its 200 mg/ml concentration to 100 mg/ml, 50 mg/ml, 25, 12.5 mg/ml, 6.25 mg/ml, 3.125 mg/ml, and 1.5625 mg/ml. Each extract has a different MIC depending on the species. Calpurnia aurea seed had shown the highest antifungal activity against Candida zeylanoides and Candida tropicalis at the lowest concentration (1.5625mg/ml and 3.125mg/ml respectively) (Table 3).

Table 3: The minimum inhibitory concentration of evaluated plants against tested fungi.

1. n= Aspergillus niger, c.t= Candida tropicalis, c. z= Candida zeylanoide, v. s= Verbascum sinaiticum, c. a= Calpurnia aurea.

Phytochemical Screening

The phytochemical screening of the extract of Calpurnia aurea leaf and seed revealed the presence of alkaloids, glycosides, steroids, flavonoids, and saponins. Verbascum sinaiticum leaf had shown the presence of alkaloids, glycosides, steroids, and flavonoids and failed to show the presence of saponins. The phytochemical analysis summary was described in the table 4.

Table 4: Summary of Phytochemical screening.

-= absence, += presence, ++= strong red v. s=Verbascum sinaiticum, c. a=Calpurnia aurea.

The results obtained from this study showed that the plants have significant antifungal activity against tested fungal pathogens. Candida zeylanoides are more susceptible to the extract of Calpurnia aurea seed and leaves as compared to standard antifungal ketoconazole with an average inhibition zone of 20.333± 0.88 and 20±0.577, respectively. This agreed with the investigation of [41], in which Candida albicans showed high susceptibility to plant extract than the positive control. The highest concentration of Calpurnia aurea leaf and seed showed comparable inhibition with positive control. It was observed that the extract inhibited the growth of all tested pathogenic fungi. Increased inhibition was found against Candida zeylanoides and Candida tropicalis [41].

The antifungal activity evaluated in this work could be attributed to the presence of different phytochemical compounds in variable amounts in plant extracts. Phytochemical analysis results suggest that the presence of biologically active compounds (?avonoids, saponins, steroids, tannins, phenol, and alkaloids) in the plant extract could be correlated to the antifungal effects of substances known to possess antimicrobial properties of the plants as shown by [42].

[43] Investigated the antifungal activity of the different plant extracts against pathogenic fungi (Microsporum, Candida albicans, Aspergillus flavus, Fusarium solani, Aspergillus fumigatus and Candida glaberata), and showed their activity may be due to the combined effect of plant bioactive material such as: glycoside, saponins, alkaloid, tannin and flavonoids. These were gone in line with my investigation in which tested plants Calpurnia aurea, and Verbascum sinaiticum revealed the presence of glycoside, saponins, alkaloid, tannin, phenolic compound, and flavonoids, found to show significant antifungal activity against Candida zeylanoid, Candida tropicalis, and Aspergillus niger.

The results of the phytochemical analysis will support the utilization of the respective plants. Saponins, tannins, ?avonoids, alkaloids, and phenols had been reported to have antifungal activity [44,45] Phenolic compounds and flavonoids have demonstrated potential therapeutic activities as antifungal, antibacterial, and antioxidant agents [46]. Flavonoids are effective antifungal agents against a wide range of pathogenic organisms, including aspergillus niger and candida tropicalis [47,48]. They often inhibit fungal growth with various underlying mechanisms, including plasma membrane disruption, the induction of mitochondrial dysfunction, and inhibiting the following: cell wall formation, cell division, RNA and protein synthesis, and the efflux-mediated pumping system [49].

Although the mechanisms underlying the antimicrobial activity of the phenolic compounds are rather variable, many of them act by promoting damage to the function of the cell membrane or cell wall [50]. The antifungal effect of alkaloids may be due to their activity in lyses of the fungal cell wall and cytoplasmic membrane due to the liberation of antimicrobial products and it was also plants used in reported that plant lytic enzymes act on the fungal cell traditional medicine, wall causing breakage of B-1, 3 glycan, B-1, 6, glycan and chitin polymer [51].

Phytochemical analysis of Calpurnia aurea leaves and seed extract showed the presence of all screened bioactive constituents such as steroids, glycoside, phenolic compound, flavonoids, saponins, and alkaloids. These results were supported by several previous findings [52,53] made on the phytochemical screening of Calpurnia aurea were the plants shown presence of different bioactive materials. Verbascum sinaiticum leaves also were subjected to phytochemical screening and the extract had shown the presence of steroids, glycoside, phenolic compound, flavonoids, and alkaloids. However, it failed to show the presence of saponins. These agreed with the result of [33]  in which the leaf methanol extract of Verbascum sinaiticum showed no presence of saponins.

However, the present study was having some limitations such as using only maceration technique and methanol as extraction solvent to extract the plants. In this study also, major phytochemical of the plants was only addressed and plants fungicidal properties have not determined.

The present study has shown that extracts of seed and leaves of Calpurnia aurea and leaves of Verbascum sinaiticum revealed significant antifungal activity. Calpurnia aurea seed proved to be the most effective against all tested fungal strains (Aspergillus niger, Candida tropicalis, and Candida zeylanoides). Calpurnia aurea leaf extract showed a high inhibition against the tested Candida strains and moderately inhibited Aspergillus niger. Verbascum sinaiticum showed moderate antifungal activity against all tested fungal strains. It is suggested that the extracts of Calpurnia aurea and Verbascum sinaiticum have wonderful antifungal activity against a group of fungi and this effect depends on both the plant part and the solvent used for extraction. Based on the results, it is possible to conclude that Calpurnia aurea seed extract has stronger antifungal activity as compared to Verbascum sinaiticum and Calpurnia aurea leaf.

Data Availability

The data used to support the findings of this study are available from the corresponding author up on reasonable request.

Authors’ Contributions

All authors have made substantial, direct, and intellectual contributions to the work.

Acknowledgments

We would like to thank Ambo University for sponsoring our study

1. Mulata N, Seifu D, Umeta M, Wendwesson E and Natesan G. Protective Effects of Calpurnia aurea Seed Extract on HAART Hepatotoxicity. European journal of medicinal plants. 2015; 9: 1-12.
2. Butler G, Rasmussen M, Lin M, Santos M, Sakthikumar S, Munro C. Evolution of Pathogenicity and sexual   reproduction in eight Candida genomes. Nature. 2009; 459: 657-662.
3. Lee Cj, Shin Jh, Kim Jp, Kook H, Suh Sp, Ryang Dw. A Case Of  Mixed Fungemia With Cryptococcus Laurentii And Candida Zeylanoides. Korean Journal Of Clinical Pathology. 2001: 282-286.
4. Chowdhary A, Sharma C, Hagen F, Meis JF. Exploring azole antifungal drug resistance in Aspergillus fumigatus with special reference to resistance mechanisms.  Future Microbiology.  2014; 9: 697-711.
5. Denning D, Pleuvry A and Donald CC. Global burden of chronic pulmonary aspergillosis as a sequel to pulmonary tuberculosis. Bull World Health Organization. 2011; 89: 864-872.
6. Toghueo R, Sahal D, Zabalgogeazcoa Í, Baker B and Boyom F. Conditioned media and Organic Elicitors Underpin the Production of Potent Antiplasmodial Metabolites by Endophytic Fungi from Cameroonian Medicinal Plants. Parasitological Research. 2018; 117: 2473-2485.
7. Zerabruk S and Yirga G. Traditional knowledge of medicinal plants in Gindeberet district, Western Ethiopia. South African Journal of Botany. 2011; 10: 10-13.
8. Russell S, Karunaratne N and Mahindapala R. Rapid inventory of wild medicinal plant populations in Sri Lanka.  Journal of Biological Conservation.  2006; 132:  22-32.
9. Hamilton A. Medicinal Plants and Conservation. issues and approaches, International plant conservation unit. 2003; 29–33.
10. Krishnaiah D, Sarbatly R and Bono A. Phytochemical antioxidants for health and medicine. A move towards nature.  Biotechnology and Molecular Biology Reviews. 2007; 1: 97-104.
11. Koche D, Shirsat R, Syed I and Bhadange D. Phytochemical screening of eight ethnomedicinal plants from Akola District (MS). International Journal of Pharmacology and Bioscience. 2010; 1: 256- 259.
12. Suffredini I, Sader H, Gonçalves A, Reis A, Gales A, Varella A and Younes R. Screening on antibacterial extracts from plants native to the brazilian amazon rainforest and atlantic forest. Brazilian Journal of Medical Biology Research. 2004; 37: 379-384.
13. Chekole G, Asfaw Z and Kelbessa E. Ethnobotanical study of medicinal plants in the environs of Tara-gedam and Amba remnant forests of Libo Kemkem District, northwest Ethiopia. Journal of Ethnobiology Ethnomedicine. 2015; 11: 1–38.
14. Lulekal E, Asfaw Z, Kelbessa E and Van D. Ethnoveterinary plants of Ankober District, North Shewa Zone, and Amhara Region, Ethiopia. Journal of Ethnobiology Ethnomedicine. 2014; 10: 21.
15. Avigdor E, Wohlmuth H, Asfaw Z and Awas T. The current status knowledge of herbal medicine and medicinal plants in Fiche, Ethiopia current status knowledge of herbal medicine and medicinal plants in Fiche, Ethiopia. Journal of Ethnobiology Ethno medicine. 2014; 10.
16. Yigezu Y, Haile D and Ayen W. Ethnoveterinary medicines in four districts of Jimma zone. Ethiopia. cross-sectional survey for plant species and mode of use. Journal of Ethnobiology Ethnomedicine, 2014; 10: 1–12.
17. Abera B. Medicinal plants used in traditional medicine by Oromo people, Ghimbi District, Southwest Ethiopia. Journal of Ethnobiology Ethnomedicine. 2014; 10: 40.
18. Kidane L, Gebremedhin G and Beyene T. Ethnobotanical study of medicinal plants in Ganta Afeshum District, Eastern Zone of Ethiopia. Journal of Ethnobiology Ethnomedicine. 2018; 14: 1–19.
19. Zorloni A, Penzhorn B and Eloff J. Extracts of Calpurnia aurea leaves from southern Ethiopia attract and immobilize or kill ticks. Veterinary Parasitol 2010; 168: 160-164.
20. Umer S, Tekewe A and Kebede N. Antidiarrhoeal and antimicrobial activity of Calpurnia aurea leaf extract. BMC Complementary and Alternative Medicine. 2013; 13: 13.
21. Do QD, Angkawijaya A and Tran-Nguyen P. Effect of extraction solvent on total phenol content, total flavonoid content, and antioxidant activity of Limnophila aromatica. Journal of Food and Drug Analysis. 2014; 22: 296–302
22. Weldegerima B, Teferra A and Muthuswamy R. Ethno-Veterinary use of medicinal plants by traditional healers in Dabat District, Northwestern Ethiopia. Pharmacognosy Magazine 2008; 4: 93.
23. Yineger H, Kelbessa E Bekele T and Lulekal E. Ethnoveterinary medicinal plants at bale mountains national park, Ethiopia. Journal of Ethnopharmacology. 2007; 112: 55-70.
24. Fullas F. Ethiopian Traditional Medicine: Common Medicinal Plants in Perspective. Sioux City. 2001.
25. Tadeg H, Mohammed E, Asres K and Gebre-Mariam T. Antimicrobial activities of some selected traditional Ethiopian medicinal plants used in the treatment of skin disorders. Journal of Ethnopharmacology. 2005; 100: 168–175.
26. Tamiru F, Terfa W, Kebede E, Dabessa G, Rajeeb K and Sorsa M. Ethno knowledge of plants used in veterinary practices in Dabo Hana District, West Ethiopia. Journal of Medicinal Plant Research. 2013; 7: 2960-2971.
27. Birhanu T, Abera D. Survey of ethino-veterinary medicinal plants at selected Horo Guduru Districts, Western Ethiopia. African Journal of plant science. 2015; 9: 185-192.
28. Abebayehu A, Mammo F and Kibret B. Isolation and characterization of terpene from leaves of Croton macrostachyus (Bissana). Journal of Medicinal Plants Research. 2016; 1: 256-269.
29. Mergia E, Shibeshi W, Terefe G and Teklehaymanot T. Phytochemical Screening and In Vitro Antitrypanosomal Activity of Aqueous and Methanol Leaf Extract of Verbascum sinaiticum (Scrophulariaceae) against Trypanosoma congolense Field Isolate. Journal of Clinical and Experimental Pathology. 2014; 4: 4.
30. Natesan G, Haile N, Umeta M and Seifu D. Phytochemical Screening and Assessment of In Vitro Antioxidant Activities of Calpurnia Aurea Seeds and Leaves. Huma. 2015; 2: 1-12.
31. Tolera A. Pedological characterization, fertility status and classification of the soils under maize production of Bako Tibe and Toke Kutaye districts of Western Showa, Ethiopia. Ethiopian Journal of Applied Science and Technology. 2016; 7: 1-17.
32. Azwanida N. A Review on the Extraction Methods Use in Medicinal Plants, Principle, Strength and Limitation. Medicinal and Aromatic Plants. 2015; 4: 196.
33. Anokwuru C, Anyasor G, Ajibaye O, Fakoya O and Okebugwu P. Effect of extraction solvents on phenolic, flavonoid and antioxidant activities of three nigerian medicinal plants. Natural Science. 2011; 9: 53–61.
34. EUCAST (European Committee for Antimicrobial Susceptibility testing). Method for the
35. determination of. Minimum inhibitory concentration (MIC) by broth dilution of fermentative yeasts. Subcommittee on Antifungal susceptibility testing (AFST) of the ESCMID, E.Dis.7.1, Clinical Microbiology Information. 2003; 9: 8.
36. Kwon O, Ju W, Kim H and Kim Y. Antifungal activities of extracts from different parts of mulberry plant against Alternaria alternata and Fusarium species. International Journal of Industrial Entomology. 2019; 38: 6-13.
37. M100-S24 Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Fourth Informational Supplement. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Fourth Informational Supplement. 2014; 34: 1-150.
38. Bandiola B. Extraction and Qualitative Phytochemical Screening of Medicinal Plants. A Brief Summary. International Journal of Pharmacology. 2018; 8:  137-143.
39. Karthikeyan G and Vidya K. phytochemical analysis, antioxidant and antibacterial activity of pomegranate peel. Research journal of life science, bioinformatics, pharmaceutical and chemical science. 2019; 5: 218.
40. Pandey A and Tripathi S. Concept of Standardization, Extraction and Pre-Phytochemical Screening Strategies for Herbal Drug. Journal of Pharmacognosy and Phytochemistry. 2014; 2: 115-119.
41. Malik K, Kolte P, Baruah L, Saravanan M, Bakshi B and Bhatta R. Enteric methane mitigation in sheep through leaves of selected tanniniferous tropical tree species. Livestock  2017; 200: 29-34.
42. Alper S, Basara D. antimicrobial activity of the leaves of Verbascum sinuatum. on microorganisms isolated from urinary tract infection. African Journal of Micro Research. 2009; 3: 778-781.
43. Felhi S, Daoud A, Hajlaoui H, Mnafgui K, Gharsallah N and Kadri A. Solvent extraction effects on phytochemical constituents’ proles, antioxidant and antimicrobial activities and functional group analysis of allium elaterium seeds and peels fruit. Journal of Food Science and Technology (Campinas). 2017; 37 (3): 483-492.
44. Baloch N, Nabi S, Al-Kahraman Y. In vitro antimicrobial, insecticidal, antitumor activities and Their phytochemical estimation of methanolic extract and its fractions of Medicago lupulina leave. World Applied Science Journal. 2013; 23(4): 500-506.
45. Bhattacharya A, Sood P and Citovsky V. The roles of plant phenolics in defense and communication during Agrobacterium and Rhizobium infection. Molecular Plant Pathology. 2010; 11(5): 705-719.
46. Patra A. An overview of antimicrobial properties of di?erent classes of phytochemicals, in Dietary Phytochemicals and Microbes. Springer nature. 2012; 1-32.
47. Orhan D, ¨Ozc¸elik B, ¨Ozgen S and Ergun F. Antibacterial, antifungal, and antiviral activities of some flavonoids. Microbiological Research. 2010; 165: 496-504.
48. Salazar-Aranda R, Granados-Guzmán G, Pérez-Meseguer J, González G, de Torres N. Activity of Polyphenolic Compounds against Candida glabrata. Molecules. 2015; 20: 17903-17912.
49. Baptista R, Madureira A, Jorge R, Adão R, Duarte A, Duarte N, Lopes M and Teixeira G. Antioxidant and Antimycotic Activities of Two Native Lavandula Species from Portugal. Evidence Based Complementary and Alternative Medicine. 2015; 1-10.
50. Cushnie T and Lamb A. Antimicrobial activity of ?avonoids. International Journal of Antimicrobial 2005; 26: 343-356.
51. Al Aboody M and Mickymaray S. Anti-fungal e?cacy and mechanisms of ?avonoids. 2020; 9: 45.
52. Sohn H, Son K, Kwon C, Kwon G and Kang S. Antimicrobial and cytotoxic activity of 18 prenylated flavonoids isolated from medicinal plants.  2004; 11: 666-672.
53. Mebrahtu E, Workineh S and Mirutse G. In vivo antimalarial activity of hydromethanolic leaf extract of Calpurnia aurea (Fabaceae) in Mice infected with chloroquine-sensitive Plasmodium berghei. International Journal of Pharmacology. 2013; 2: 123-142.
54. Birhanu Z, Wuhab A, Abula T. Antimalarial Activity of Calpurnia Aurea Hydro alcoholic Leaf Extract in Mice Infected with Plasmodium berghei. Archives. 2015; 2: 73-79.
55. policy perspective on medicines – Traditional medicine – Growing needs and potentia. World Health Organization, Geneva. 2002; 6.