In vitro antibacterial and antifungal activities of extracts and fractions of leaves of Ricinus communis Linn against selected pathogens

Abstract Introduction Infectious disease impacts are reduced due to the development of antimicrobial agents. However, the effectiveness of antimicrobial agents is reduced over time because of the emergence of antimicrobial resistance. To overcome these problems, scholars have been searching for alternative medicines. Ricinus communis is used as a traditional treatment for bovine mastitis, wound infection, and other medicinal purposes. Objective The objective of the present study was to further evaluate the antimicrobial activities of R. communis leaf extracts and fractions. Methods R. communis leaves were macerated in methanol and acetone. The methanol extract showed better antimicrobial activity and subjected to further fractionation via increasing polarity of solvents (n‐hexane, chloroform, ethyl acetate, and aqueous). Test microorganisms included in the study were six laboratory reference bacteria (Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, Kleibsella pneumoniae, Pseudomonas aeruginosa and Streptococcus pyogenes), two clinical isolate bacteria (E. coli and S. aureus), and Candida albicans. The agar well diffusion method was employed to determine antimicrobial activity. The minimum inhibitory concentrations (MIC) and minimum bactericidal/fungicidal concentrations (MBC/MFC) were determined through broth microdilution. Results The results indicated that the best antimicrobial activity for ethyl acetate fraction ranged from 14.67 mm (clinical E. coli) to 20.33 mm (S. aureus) at 400 mg/ml, however, n‐hexane exhibited the lowest antimicrobial activity. Among the tested fractions, ethyl acetate fraction showed the lowest MIC values ranged from 1.5625 mg/ml (S. aureus) to 16.67 mg/ml (Candida albicans). The ethyl acetate fraction showed bactericidal activity against all tested microorganisms. Conclusion Hence, ethyl acetate fraction of crude methanol extract exhibited the best antimicrobial activity.


INTRODUCTION
Infectious diseases are exacerbated due to the existence of zoonotic diseases and antimicrobial resistance (Rwego et al., 2008;Uchil et al., 2014). Hence, several surveillances have been conducted on antimicrobial resistance in different countries that indicated the development of drug resistance by different pathogens to the same or different drugs are increasing from time to time with variations from region to region (Berhe et al., 2021;Brzychczy-Wock et al., 2013;Mshana et al., 2013;Ventola, 2015). The augmentations of antimicrobial resistances have harmed both human and animal health, exposing to longer periods of hospitalisation and affecting treatment costs (Bedasa et al., 2018;Getahun et al., 2008;Kerro & Tareke, 2003). Alternative medicines have been screened from a variety of plants for their pharmacological potential as secondary metabolites are less in drug adverse effects, resistance and residues (Felhi et al., 2017;Helander et al., 1998;Puupponen-Pimia et al., 2001;Zgurskaya et al., 2015). The examples of modern drugs that come from medicinal plant studies are vinblastine, artemisinin, topotecan, teniposide, anisodamine, 3-n-butylphthalide, indirubin, huperzine, acetyldigoxin, theobromide, physostigmine, digitoxin and ephedrine (Helmenstine, 2021;Kong et al., 2003;Tu, 2016;Zhang, 2002).

Ricinus communis Linn taxonomically belongs to the family of
Euphorbiaceae and it is a sole species in the monotypic genus Ricinus.
The vernacular names are 'Qobboo' (in Afan Oromo), 'Castor oil plant or castor bean' (in English), and 'Gulo' (in Amharic). It grows in altitude ranging from 400 to 4500 m above sea level in tropics and temperate regions of the world. The plant grows perennially as high as 5-10 m with a 15 cm thick and hollow trunk and leaves. It has a green or reddish colour, alternate, stipulate, long petiolate and a membranous lobe of a leaf and fruit with a thorny capsule covering a seed. It has been reproduced with mixed pollination of self-pollination (geitonogamy) and outcrossers by wind pollination (anemophily) or insect pollination (or entomophily) (Edwards et al., 1995;Neelam & Singh, 2015).
R. communis in Ethiopia is used in the treatment of blackleg and actinomycosis (Bayecha et al., 2018), diarrhoea, wound and skin rashes/dermatitis (Gijan & Dalle, 2019;Mengesha & Dessie, 2018) and bovine mastitis (Romha et al., 2015). The studies on validation of antimicrobial activity of R. communis leaf extracts were conducted using different solvents in Pakistan and Ghana, and methanol extract was reported to have a promising antimicrobial potential (Naz & Bano, 2012;Suurbaar et al., 2017). According to a study report on antibacterial activity of R. communis leaf in Ethiopia, organic solvent extracts exhibited better activity than the aqueous one (Abew et al., 2014).
However, methanol was not used for extraction in Abew et al. (2014) and none has been done on the antimicrobial activities of solvent fractions of R. communis leaf.  (Liu et al., 2016). The test pathogens were selected based on their ability of causing a variety of diseases in humans and animals and the traditional claims on usage of R. communis leaf as ethnomedicine in the country. Therefore, the current study was intended to compare antimicrobial activities of methanol and acetone extracts, evaluate antimicrobial activities of solvent fractions of the best performed crude extract among the two extracts and characterise phytochemical constituents of the solvent fractions.

Plant authentication and collection
The experimental plant was verified based on the works of Edwards et al. (1995) on the description of flora of Ethiopia and Eritrea before collection at the field and then authenticated by a Plant Taxonomist, Mr.
Melaku Wondafrash, at the national herbarium of the College of Natural and Computational Sciences, Addis Ababa University. The plant was collected from the Sululta district, Finfinne city surrounding special zone, Oromia regional state, Ethiopia which is located at about 25 km from the capital city in October, 2019.

Extraction of the plant
The extraction was performed according to Ogbiko et al. (2018

Crude extract solvent fractionation
The crude methanol extract was subjected to further solvent fractionation by increasing polarity including n-hexane, chloroform, ethyl acetate and aqueous. Voukeng et al.'s (2017)

Test organisms
Microorganisms selected for the experiment were standard strains

Standard drugs
Gentamicin 10 μg disc was used as positive control against bacteria and brought from Animal Products, Veterinary Drug, and Ani-

Antibacterial activity
The brain heart infusion (BHI) broth was prepared for streptococcal species and nutrient broth for other test bacteria. Overnight cultured 3-5 distinct colonies of bacteria based on their colony size were inoculated into 4 ml broth media and incubated at 37 • C overnight. The nutrient or BHI broth was added to the overnight incubated bacterial suspension and vortexed on a vortex mixer (Fisher Scientific Ltd., England) for 1 min to attain uniform distribution. The vortexed bacterial suspension was adjusted to 0.5 McFarland standards (Remel, Lenexa Kansas 66215, USA) (equivalent to 1-2 × 10 8 CFU/ml) through contrasting against white paper black line striped and was used for experiment within 15 min (CLSI, 2015).
The 100 μl of adjusted bacterial suspension was pipetted using a micropipette and applied on the surface of Mueller Hinton agar and was swabbed at 60 o rotation to uniformly distribute bacteria throughout media surface using a cotton swab. The swabbed Mueller Hinton agar stood for 15 min to provide time for the attachment of bacteria on the media. After that, the sterilised cork borer of 6 mm diameter was perforated with the swabbed media to create 6 mm diameter wells. At the time of punching media for different test bacteria, the cork borer was sterilised by immersing in alcohol and burning with Bunsen burner flames (Gonelimali et al., 2018;Umer et al., 2013). The concentration of extracts for the experiment was determined based on a previous study on the plant (Abew et al., 2014). The created wells were filled with 50 μl extracts or fractions at a concentration of 400, 200 and 100 mg/ml, and negative control, but the positive control disc (gentamicin) was placed on the media surface. After all the wells on the Petri dishes were filled, and the positive control was placed on Petri dishes, then the Petri dishes were placed in the refrigerator at 4 • C for 2 h to facilitate diffusion of extracts or fractions in the media. Subsequently, Petri dishes were incubated at 37 • C for 24 h in the incubator (BioTechnics India).
The inhibition zone diameter after 24 h incubation was measured by a ruler in millimetre and recorded (Abew et al., 2014;Ohikhena et al., 2017;Suurbaar et al., 2017). The experiment was done in triplicate.

Antifungal activity
The Candida albicans was cultured on sabouraud dextrose agar and incubated overnight. The overnight incubated yeast culture was inoculated into normal saline (0.85%). The inoculated normal saline was vortexed on a vortex mixer and adjusted to 0.5 McFarland standards (equivalent to 1-5 × 10 6 cells/ml) by contrasting against white paper black line striped (EUCAST, 2003). The 100 μl adjusted Candida albicans suspension was pipetted using a micropipette and applied on the surface of sabouraud dextrose agar and swabbed at 60 o rotation to uniformly distribute yeast throughout the media surface using a cotton swab. The swabbed sabouraud dextrose agar stood for 15 min to provide time for the attachment of yeast on the media. After that, the sterilised 6 mm diameter cork borer was used to perforate the swabbed media to create a 6 mm diameter of wells (Ohikhena et al., 2017). The concentration of extracts for the experiment was determined based on a previous study on the plant (Suurbaar et al., 2017). The created wells were filled with the 50 μl extracts or fractions at 400, 200 and 100 mg/ml, negative, and positive control. The inoculated Petri dishes were placed in the refrigerator at 4 • C for 2 h to facilitate diffusion of extracts or fractions in the media. Next to that, Petri dishes were incubated at 37 • C for 24 h in the incubator. The inhibition zone diameter after 24 h incubation was measured by a ruler in millimetre and recorded (Abew et al., 2014;Ohikhena et al., 2017;Suurbaar et al., 2017). The experiment was done in triplicate.

Determination of minimum inhibitory concentration (MIC)
Minimum inhibitory concentration is the minimum concentration of extracts or fractions which have inhibited the growth of microorganisms. The minimum inhibitory concentrations were determined using the broth microdilution technique for extracts or solvent fractions as their inhibition zones equal to or greater than 7 mm in agar well diffusion techniques (Taye et al., 2011).

Determination of minimum inhibitory concentration for pathogenic bacteria
The overnight cultured 3-5 distinct bacterial colonies were inoculated into 4 ml Mueller Hinton broth and incubated at 37 • C overnight.
Overnight incubated bacterial suspension that had been adjusted (0.5 McFarland standards) was diluted at a ratio of 1:20 with Mueller Hinton broth (0.5 ml bacterial suspension was added to 9.5 ml broth) and vortexed to have uniformly distributed bacterial suspension (5 × 10 6 CFU/ml). The UV radiated sterile microtitre plate (Greiner Bio-One, Germany) wells were filled with 100 μl Mueller Hinton broth which commenced from well 1 to 12. The serial double dilution technique was employed for extracts and fractions in broth filled wells. The serial double dilution was performed as 100 μl extracts or fractions were added to the first well and thoroughly mixed for five times by rinsing using micropipette and 100 μl of the mixture was transferred to the second well using a new micropipette tip and thoroughly mixed as above. A 100 μl of the second well mixture was pipetted using a new micropipette tip and transferred to the third well, and then thoroughly mixed as above.
The process was continued until the tenth well and 100 μl mixture of the tenth well was pipetted and discarded to have an equal volume of fluid in wells (CLSI, 2015).The twofold serially diluted concentrations of extracts for the experiment were determined from a previous study on the plant. The serially diluted concentrations used in the experiment were 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125 and 0.3906 mg/ml (Abew et al., 2014). The 100 μl broth-filled 11th and 20th wells were used as growth and sterility control, respectively. The 10 μl diluted bacterial suspension (10% of 100 μl well volume) was pipetted to wells from eleventh to first wells to reduce contamination to sterility control and attained a final concentration of 5 × 10 5 CFU/ml bacteria in each well, but 10 μl broth was pipetted to the 12th well. Finally, microtitre plates were sealed using parafilm and incubated at 37 • C for 24 h (CLSI, 2015). The incubated microtitre plate wells were filled with 0.01% resazurin sodium salt indicator from 12th to 1st well and incubated for 2 h at 37 • C. The resazurin sodium salt reaction with actively growing microorganisms produces colour changes which are important to determine the MIC of extracts or fractions based on colour changes. The blue or purple colour appears if the growth of microorganisms is inhibited, while pink or colourless change is observed for those actively growing cells which reduced resazurin sodium salt to resorufin. Resazurin sodium salt solution was prepared by dissolving 0.01 g in 100 ml sterile distilled water and filtered through a 0.2 μm pore size filter paper and stored in a dark container at 4 • C refrigerator until use (Blazic et al., 2019;Ohikhena et al., 2017). The experiment was performed in triplicate.

Determination of minimum inhibitory concentration for pathogenic fungi
Overnight cultured colonies of yeast were inoculated into sabouraud dextrose broth and incubated at 37 • C overnight. Overnight incubated yeast suspension which had been adjusted (0.5McFarland standard) was diluted at a ratio of 1:20 with sabouraud dextrose broth (0.5 ml yeast suspension was added to 9.5 ml broth) and vortexed to have uniformly distributed yeast suspension (0.5-2.5 × 10 5 CFU/ml). The sterile microtitre plate wells were filled with 100 μl broth of sabouraud dextrose from well one to twelve. The serial double dilution technique was employed for extracts and fractions in broth filled wells commenced from the first to tenth wells. The serial double dilution was performed as 100 μl extracts or fractions were added to the first well and thoroughly mixed five times by rinsing using a micropipette and 100 μl of the mixture was transferred to the second well using a new micropipette tip and thoroughly mixed as above. A 100 μl of the second well mixture was pipetted using a new micropipette tip and transferred to the third well and thoroughly mixed as above. The process was continued until the tenth well and 100 μl mixture of the tenth well was pipetted and discarded to have an equal volume of fluid in the wells (EUCAST, 2003). The twofold serially diluted concentrations of extracts for the experiment were determined from a previous study on the plant. The serial double dilution concentrations used in the experiment were 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125 and 0.3906 mg/ml (Suurbaar et al., 2017). The 100 μl broth-filled 11th and 20th wells were used as growth and sterility control, respectively. The 10 μl diluted yeast suspension (10% of 100 μl broth volume) was pipetted to wells from the eleventh to first wells to reduce contamination on sterility control and the attained final concentration of yeast suspension (2.5 × 10 4 CFU/ml) in each well, but 10 μl broth was pipetted to the 12th well. The filled microtitre plate wells were sealed by parafilm and incubated at 37 • C for 24 h (CLSI, 2015;EUCAST, 2003).
The incubated microtitre plate wells were filled with 0.01% resazurin sodium salt indicator from the 12th to the 1st well and incubated for 2 h at 37 • C. The MIC of extracts and fractions were determined as blue or purple resazurin colour changed to pink or colourless (Blazic et al., 2019;Ohikhena et al., 2017). The experiment was done in triplicate.

Determination of minimum bactericidal concentration (MBC)
The minimum bactericidal concentration was determined through subculturing of 10 μl content of microtitre plate well which is greater or equal to the lowest minimum inhibitory concentration on the Mueller Hinton agar and incubated for 24 h. After 24 h incubation, the Petri dish was assessed for the presence of growth, and the minimum concentration of extracts or fractions with no visible growth was taken as a minimum bactericidal concentration (Akinduti et al., 2019). The experiment was done in triplicate.

Determination of minimum fungicidal concentration (MFC)
The minimum fungicidal concentration was determined through subculturing of 10 μl content of microtitre plate well which is greater or equal to the lowest minimum inhibitory concentration on the sabouraud dextrose agar and incubated for 24 h. After 24 h incubation, the Petri dish was assessed for the presence of growth, and the minimum concentration of extracts or fractions with no visible growth was taken as minimum fungicidal concentration (Akinduti et al., 2019). The experiment was done in triplicate.

Data analysis
The data were entered into an excel spreadsheet for statistical analysis using Statistical Package for Social Science (SPSS) version 20. The descriptive statistics, one-way ANOVA, Tukey's post hoc test and linear regression R 2 (Coefficient of determination) were utilised for statistical analysis and inference. The descriptive statistics were employed for calculation of group mean of inhibition zone diameter as mean ± SEM.
The one-way ANOVA was performed to determine the significant difference among group means. Whereas, Tukey's post hoc test followed one-way ANOVA to determine the significant difference between each group mean. The linear regression R 2 was calculated to determine the concentration dependence of extracts and fractions on antimicrobial activities against test microorganisms. Statistically significant differences were declared at a p value of less than 0.05.

Antibacterial activity
3.1.1 Agar well diffusion assay  (Table 5). Values expressed as mean ± SEM for n = 3. The mean comparisons for different extracts and Gentamicin 10 μg (control) were performed by one-way ANOVA followed by Tukey's HSD post hoc multiple comparison test. Where, compared to a positive control, b 100 mg/ml, c 200 mg/ml and d 400 mg/ml. 1 p < 0.05, 2 p < 0.01, 3 p < 0.001. R 2 = coefficient of determination. Values expressed as mean ± SEM for n = 3. The mean comparisons for different extracts, crude methanol extract's fractions and amphotericin-B 20 μg/ml (control) were performed by one-way ANOVA followed by Tukey's HSD post hoc multiple comparison test. Where, compared to a positive control, b 100 mg/ml, c 200 mg/ml and d 400 mg/ml. 1 p < 0.05, 2 p < 0.01, 3 p < 0.001. No activity = -, R 2 = coefficient of determination.

Agar well diffusion assay
The assay determined inhibition zone diameter for extracts, fractions and positive control, but not for negative control. The aqueous fraction exhibited the highest inhibition zone diameter of 21 mm, but no inhibition zone diameter was observed for n-hexane and chloroform fractions against C. albicans (Table 6).

Determination of minimum inhibitory concentration of extracts and fractions of methanol extract against pathogenic bacteria
The minimum inhibitory concentration of the methanol extract ranged from 6.25 mg/ml (S. aureus) to 25 mg/ml (E. coli, K. pneumoniae, P. aeruginosa) and the acetone extract ranged from 8.33 mg/ml (S. pyogenes) to 100 mg/ml (K. pneumoniae). Also, the minimum inhibitory concentration of the ethyl acetate fraction ranged from 1.5625 mg/ml (S. aureus) to 12.5 mg/ml (P. aeruginosa) and for the aqueous fraction ranging from 6.25 mg/ml (S. aureus and S. pyogenes) to 66.67 mg/ml (K. pneumoniae) (Tables 7 and 8). The minimum inhibitory concentration for the clinical isolate bacteria ranged from 3.125 mg/ml of ethyl acetate fraction (S. aureus) to 100 mg/ml of n-hexane and chloroform fractions (S. aureus and E. coli) (Table 9).

3.2.3
Determination of minimum bactericidal concentration of extracts and fractions of methanol extract  (Table 10).

3.2.5
Physical characteristics and preliminary screening of phytochemical constituents of Ricinus communis leaf The physical characteristics of Ricinus communis leaf extracts and fractions of the methanol extract were dark green and reddishbrown, sticky solid inconsistency and per cent of the yield ranged from 7.5% to 41.67%. The phytochemical screening detected alkaloids, flavonoids, terpenoids, tannins, cardiac glycosides, steroids, anthraquinones, saponins and phenols in the crude methanol extract and ethyl acetate fraction of Ricinus communis leaf (Tables 11   and 12).

DISCUSSION
The current study aimed to investigate antimicrobial activities of extracts and fractions of methanol extract of R. communis leaf against pathogenic bacteria and Candida albicans. However, antibacterial activity had been done in a previous study from Gonder, Ethiopia, but this study did not include antifungal activity and methanol in extraction (Abew et al., 2014). Furthermore, previous findings have reported that methanol solvent extract exhibited the best antimicrobial activities from Ghana and Pakistan (Naz & Bano, 2012;Suurbaar et al., 2017). Both methanol and acetone extracts were also assessed for their antimicrobial activities to select the one which exhibited bet-ter antimicrobial activity for further solvent fractionation. There was a difference in the antimicrobial activities of the two extracts for the presence and concentration of secondary metabolites which could be affected by the type of solvent used for extraction (Liu et al., 2016).
The current study indicated that ethyl acetate fraction exhibited the highest antimicrobial activities in all tested microorganisms.
Crude extracts were tested for their effects against Gram-positive and Gram-negative bacteria for their antimicrobial activities. Methanol extract revealed higher antimicrobial activity than acetone extract at the same concentrations. This finding agrees with that of the previous studies of Chandrasekaran and Venkatesalu (2004), Naz and Bano (2012) and Suurbaar et al. (2017). It is probably due to the    Molla et al. (2016). The resistance mechanisms such as efflux pumps, β-lactamase production and biofilm formation could have hindered the effectiveness of antibacterial in clinical isolates than laboratory strains (Kapoor et al., 2017).
The broth microdilution technique revealed the lowest minimum inhibitory concentration for ethyl acetate fraction against pathogenic bacteria whereas the aqueous fraction was against yeast. The experiment indicated that the minimum inhibitory concentration of the broth microdilution technique was inversely proportional to the inhibition zone of the agar well diffusion technique. This is an indication of the reproducibility of an experiment (Scorzoni et al., 2007).
The ethyl acetate fraction also exhibited minimum bactericidal and fungicidal concentration against all tested microorganisms. Apart from this, n-hexane and chloroform fractions were devoid of bactericidal and fungicidal activity. This could be due to the concen-  (Cowan, 1999;Felhi et al., 2017;Palmer-Young et al., 2017;Shafique et al., 2011).

LIMITATION OF STUDY
The current study limitation was the small number of tested microorganisms.

CONCLUSION AND RECOMMENDATIONS
The R. communis leaf was subjected to different solvents for extraction whereas the methanol solvent yielded more crude extract. The methanol extract contained all the screened secondary metabolites and exhibited the best antimicrobial properties against all tested microorganisms in a concentration-dependent manner. The methanol extract exposed to different solvents for solvent fractionation indicated that the ethyl acetate fraction contained all the screened secondary metabolites and revealed the most pronounced antimicrobial activity than the extracts and other fractions, but the aqueous fraction exhibited better anticandidal activity. The current study supports the claims of use of R. communis leaf as traditional medicine for the treatment of infectious diseases caused by bacterial and fungal pathogens.
Based on the current study findings the following points were forwarded.
• Studies should be conducted on ethyl acetate fraction to further isolate, purify, and identify bioactive principle(s) responsible for antibacterial and antifungal activities of the plant. • Further study should be conducted on antimicrobial activities of the plant on other bacterial and fungal pathogens apart from the currently tested microorganisms.
• Mechanistic studies on isolated, purified and identified bioactive principle(s) of ethyl acetate fraction against bacterial and fungal pathogens should be conducted.
• In vivo antimicrobial study should be conducted to confirm the in vitro antimicrobial activities of the extracts and fractions of plant against the selected bacterial and fungal pathogens.