Preliminary phytochemical screening and antibacterial effects of root bark of Ferula communis (Apiaceae)

Abstract Introduction Plants are widely used in traditional medicine because they contain a high concentration of antimicrobial agents, serving as the foundation for medicines. The aim of this study was preliminary identification of phytochemicals and assesses the antimicrobial activity of extracts of Ferula communis root bark. Methods Plant was collected, and standard qualitative procedures were conducted. The plant samples were extracted with 99.9% methanol and 80% ethanol. To identify phytochemicals found in plants, a preliminary phytochemical analysis was performed. Agar diffusion tests, minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were performed to evaluate antibacterial activity. Result The preliminary phytochemical analysis of the ethanol and methanol extract revealed positive results for flavonoids, coumarins and tannins. Terpenoids and anthraquinones were detected only in the methanol extract. The extract of Ferula communis showed an antibacterial effect on both gram‐negative and gram‐positive bacteria in a concentration‐dependent manner. The average zone of inhibition for gram‐positive bacteria was 11 mm, whereas for gram‐negative bacteria, it was 9 mm. The MIC and MBC values also varied with the type of bacteria. In all bacterial species tested, the mean MBC value was similar to the MIC. Conclusion Different phytochemicals were detected in extracts of the root bark of F. communis and extracts showed antibacterial effects in a concentration‐dependent manner. Therefore, further purification and evaluation of the extracts and antioxidant activity of the plant should be investigated.

identified as having pharmacological activities, and the active ingredients are primarily extracted from different parts of the plant, which are then processed and administered via appropriate routes (Yigezu et al., 2014).
Traditional animal healthcare practices called ethno veterinary medicine provide low cost alternatives in the situation where drug and veterinary services are not available or are too expensive. These practices were developed and practice through trial and error method. However, they have not been evaluated, standardized and documented well. For this reason, appropriate place has not been given in the mainstreaming of veterinary medicine (Dayal et al., 2012). Plants are quite commonly used in traditional practices since medicinal plants represent a rich source of antimicrobial agents. Plants are the source of many potent and powerful drugs. A wide variety of parts have been used for extract as raw drugs, and they all have different medicinal properties. The various parts (root, stem, bark, leaf, seed, flower, fruit and twig exudates) are used. Some of these raw drugs are collected in small quantities and used to treat certain diseases within communities, whereas others are collected in large quantities and traded on the market as raw materials for many herbal industries (Abdallah et al., 2017).
Plants are the basis for medicines by containing natural source of antimicrobial drugs that provide novel or lead compounds for the fight against diseases (Mengiste et al., 2014). These bioactive compounds of plants include alkaloids, flavonoids, tannins and phenolic compounds.
Phytochemicals isolated from the medicinal plants show different antimicrobial activities. Because phytochemicals differ in structure from antibiotics, they have different modes of action (Bhatnager et al., 2015).
Ferula is a food plant that is also used in traditional medicine to treat animal diseases. Ferula communis L., also known as giant fennel, has long been used to treat a variety of ailments in traditional medicine.
Fresh plant materials, crude extracts and isolated components of F. communis are used as drugs (Gamal & Atraiki, 2015). Several authors studied F. communis' botanical properties, photochemistry, pharmacology and toxicology to determine its therapeutic and toxic potential.
The phytochemical component and antimicrobial effect of the plant may vary depending on the soil and climate in which it grows (Mamoci et al., 2011). There was no attempt to determine phytochemicals and antibacterial activity of the plant in Northwest part of Ethiopia.

Plant collection, identification and characterization
The plant was collected in the Gondar Zuria district of north-western Ethiopia. An experienced botanist identified and characterized the plant. This study made by using of the plant's root bark.

Plant preparation for extraction and maceration
After the plant was collected, the root bark was used for work. To remove the dirt and soil, it was washed with water. The root was pulverized and stored in a shed. It was then cut into small pieces with a knife and a scalpel blade. In vitro antibacterial activities of 80% methanol crude extracts were prepared from the root bark of F. communis (Nn, 2015). In this study, we socked plant material with 80% methanol and 80% ethanol for three days. The mixture was then filtered through double layer gauze into another container. To obtain a clear extract, the filtration process was repeated three times. Finally, it was kept at +4 • C until it was used.

Antibacterial assay
The initial screening of antimicrobial activity and determination of minimum inhibitory concentration (MIC) for different extracts were performed by Mueller Hinton agar plate diffusion method and macrobroth dilution method, respectively (Shubha & Hiremath, 2010). The organisms spread by pour plate method using sterile cotton swab. In each plate wells of 6 mm diameter were made using a sterile borer.
Bacterial concentration of 1 × 10 8 CFU/mL was used for antibacterial activity. The extracts were freshly reconstituted with dimethyl sulphide. The wells were filled with 50 µL of diluted extracts at 500 mg/mL surrounding each well was recorded (Patil et al., 2015).

Determination of minimum inhibitory concentration
The MICs of plant extracts were determined using the Mueller Hinton Broth micro-dilution method in 96-well microtitre plates. Micro culture tetrazolium assay reagent has been used effectively to differentiate between live and dead bacteria because only live bacteria convert the dye into an insoluble purple formazan measured at 560 nm. One ml of broth was mixed with 100 µL of bacterial suspension, of which 50 µL was used during transferred to 96 well microtitre plat for one plate. First 50 µL broth added to the plat up to 12 well then 50 µL plant extract was added with it up to 11 well then it was made a dilution supplemented by serial doubling dilutions of the extract. Finally, 50 µL of bacterial suspension (108 CFU/mL) was mixed up to 10 well. The plates were wrapped loosely with cling film to ensure that the bacteria did not get dehydrated and then they were placed in an incubator at 37 • C for 24 h. After 24 h of incubation, the plats were removed from incubator and adding of tetrazolium chloride by preparing as liquid and again incubated and was kept for 30 min then observe the colour change visually or subculture with plate count agar and observe growth.

Data analysis
The

Preliminary phytochemical screening
The result of preliminary phytochemical screening test is shown in   (Tables 2 and 3).

Minimum inhibitory concentration and minimum bactericidal concentration
The MIC and MBC of methanol extract were lower in concentration than ethanol extract except S. aureus and Citrobacter. The mean MIC and MBC (in mg/mL) has almost similar amount (Figures 1 and 2). Table 4 F I G U R E 1 Photo taken from minimum bactericidal concentrations of ethanol extract within 96 well plates.

DISCUSSION
Plants have been found to contain over 2000 phytochemicals. The medicinal value of plants is determined by the chemicals found in them (Nn, 2015). F. communis is a medicinal plant that is used to treat a variety of diseases. In this study, the methanol and ethanol extracts of F. communis root bark were positive for flavonoid, tannin and coumarin in this study, whereas the methanol solvent fraction was positive for tannins, flavonoids, coumarin, terpenoid and anthraquinone but negative for saponins, steroid and cardiac glycosides. According to other reports, the phytochemical analysis of F. communis Ethyl acetate extract and n-butanol extract revealed the presence of flavonoids, alkaloids, terpenoid, diterpenes, glycosides, terpenoid, phlobatannins and tannins (Gamal & Atraiki, 2015;Maggi et al., 2009). The presence of different chemicals may be due to differences in the type of solvent, extraction method, soil and age of the plant. According to (Hu et al., 2017), the presence of coumarin, terpenoid, essential oils and highly lipophilic TA B L E 2 Zone of inhibition of the different concentrations of methanol extracts.     compounds in plants causes, antibacterial activity to be more effective (Kang et al., 2012;Waksmundzka-hajnos et al., 2012).

Concentration
The present study was undertaken to determine on which extract do the constituents of the root bark of F. communis responsible for its antibacterial activity. The antibacterial activities of F. communis plant extracts had inhibitory effect against S. aureus and gram-negative bacteria. According to (Akaberi et al., 2015;Gamal & Atraiki, 2015 aeruginosa was more susceptible in methanol extract than ethanol extract and had a lower effective than other gram-negative bacteria in the current study. This could be due to the extract's mechanism of action on bacteria. In this study, F. communis was more potent against gram positive bacteria than that of gram-negative bacteria. MIC was 16.67 mg/mL in ethanol extract and 18.66 mg/mL in methanol extract of minimum inhibition in S. aureus but in gramnegative bacteria average MIC above 30 mg/mL. In other study better antibacterial effect of 12 mg/mL and antimycobacterial effect of 8 mg/mL with ethyl acetate and n-butanol extract (Gamal & Atraiki, 2015). The difference may be due to extraction solvent variation and bacterial strain difference. The ethyl acetate and n-butanol extracts of Ferula asafoetida had substantial antibacterial activity that is MIC and MBC against S. aureus was 1.25 mg/mL (Shubha & Hiremath, 2010) which shows more antimicrobial activities than F. communis. The difference may be due to specious of plant, solvent extraction or type of soil.

CONCLUSIONS AND RECOMMENDATIONS
Medicinal plant species are used to treat diseases of infectious origin.

AUTHORS CONTRIBUTION
In this research paper, Dr. Betelihem Yirdaw participated in conceptualization, data collection, laboratory work and manuscript write-up, whereas Dr. Temesgen Kassa contribute to data analysis, edition and validation.

FUNDING INFORMATION
This research did not receive a specific fund

DATA AVAILABILITY STATEMENT
Data will be available when necessary