Phytochemical Screening, Toxic Effects, and Antimicrobial Activity Studies of Digitaria abyssinica (Hochst. ex A.Rich.) Stapf (Poaceae) Rhizome Extracts against Selected Uropathogenic Microorganisms

In Kenya, the D. abyssinica rhizome's decoction is traditionally used to treat urinary tract infections (UTIs), mainly gonorrhea and candidiasis. UTIs are the most severe public health problems that affect over one hundred and fifty million people worldwide annually. They are caused by a wide range of microorganisms where Escherichia coli is known to be the main causative pathogen. Medicinal plants are used in traditional Kenya set up for treatment and most recently as an alternative source of treatment for UTIs due to the increased cost of treatment and many challenges experienced with antibiotic therapy. The current study is designed to investigate the phytochemical composition, acute oral toxicity, and antimicrobial activity of Digitaria abyssinica rhizome extracts against Staphylococcus aureus, Escherichia coli, Neisseria gonorrhea, and Candida albicans. The rhizomes of D. abyssinica were obtained, dried, ground, and extracted using water and organic solvents. The phytochemical assay was carried out using standard phytochemical screening methods. Single-dose toxicity studies were done to determine LD50 while disk diffusion and microbroth dilution techniques were used to determine antimicrobial activity. Results revealed that saponins, phenolics, alkaloids, cardiac glycosides, tannins, flavonoids, steroids, and terpenes were present in the powder, aqueous, methanol, and dichloromethane : methanol extracts. All the extracts had an LD50 of above 2,000 mg/kg of body weight and there was no observation of behavioral changes. Also, the aqueous and methanol extracts revealed antifungal activity against Candida albicans with the lowest average minimum zone of inhibition at MIC of 31.25 mg/ml. The study did not reveal antibacterial activity for any extract against the studied uropathogenic bacteria, Staphylococcus aureus, Escherichia coli, and Neisseria gonorrhoeae. The results from the current study suggested that D. abyssinica rhizome aqueous and methanol extracts have potential antifungal activity against C. albicans, thus validating the folklore of its use to treat candidiasis.


Introduction
Urinary tract infections (UTIs) are termed severe public health problems and afect over one hundred and ffty million people worldwide per year [1]. UTIs are known to afect more than half of women at least once in their lives and reinfection is reported to be more recurrent in young women [2]. Urinogenital infections are caused by bacteria and fungi, the most causative agent is Escherichia coli usually called uropathogenic Escherichia coli (UPEC) [3]. Other pathogenic microorganisms are Klebsiella pneumoniae, Proteus mirabilis, Enterococcus faecalis, group B Streptococcus (GBS),

Staphylococcus saprophyticus, Staphylococcus aureus, and
Candida species [4]. Te most recommended antibiotics for the treatment of UTIs are trimethoprim, sulfamethoxazole, ciprofoxacin, and ampicillin. Nevertheless, increasing rates of antibiotic resistance and high recurrent rates and the spread of multidrug resistance (MDR) threaten to greatly enhance the burden that these common infections place on society [5]. Tere is a continuous efort toward the development of alternative therapies that can be used to manage drug resistance [3,6]. A large population of over 80% in lowincome countries uses medicinal plants as an alternative source of antibiotics and other conditions for primary health care [7][8][9]. Te World Health Organization advocates the use of medicinal and aromatic plants as an alternative source of drugs that can be potentially efective in the treatment of uropathogens [10,11]. Some plants have demonstrated antimicrobial activities [8,[10][11][12][13][14].
Scientifc literature has been published about the plant extracts of the Poaceae family that have chemicals with antimicrobial activity [15][16][17][18]. Some of the Poaceae plants that have demonstrated in vitro antibacterial activities are Cynodon dactylon, Cymbopogon citratus, Triticum aestivum, Bambusa vulgaris, Dichanthium annulatum, Dactyloctenium aegyptium, Imperata cylindrica, Eleusine indica, Saccharum spontaneum, and Vetiveria zizanioides [19][20][21]. In addition, Panicum maximum and Cymbopogon citratus have that they comprise secondary metabolites that are active against fungal strains [22,23]. Te activity of Zea mays silk extracts is also known for the treatment of uropathogenic microorganisms [24]. Te current study focuses on D. abyssinica (Poaceae) referred to as East African couch grass [25]. It is a common weed that is used as food for livestock characterized by low nutritional value [26,27]. Diferent parts of the plant are used traditionally to treat fu and diarrhea [28], liver diseases [29], hernia [30], malaria, yellow fever, and wound healing [28,31]. In Kenya, the D. abyssinica rhizome's decoction has been traditionally used to treat urinary tract infections, mainly gonorrhea and candidiasis [32]. Tere is limited scientifc evidence on the biological activities of D. abyssinica. Based on the ethnomedicinal claims of D. abyssinica, the current study seeks to evaluate the safety, phytochemistry, and antimicrobial activity of D. abyssinica rhizome extracts against the selected uropathogenic microorganisms, Staphylococcus aureus, Escherichia coli, Neisseria gonorrhea, and Candida albicans. Te study aims to validate the ethnomedicinal claims of using the extracts of D. abyssinica rhizome by traditional medicine practitioners and herbalists.

Extraction of the Plant Material.
Te fresh rhizome of D. abyssinica was cleaned with clean water, then air-dried in a well-ventilated, insect-and rodent-free at room temperature. Te rhizomes were pulverized into powder by use of an electric mill. Te resulting powder was kept in a welllabeled manilla sack and kept in a cool and nonhumid place awaiting extraction.
Aqueous extraction was prepared by cold maceration by soaking 300 g of ground powder in 2.5 liters of distilled water. Te extraction mixture was stirred continuously and allowed to macerate for 48 hours and then fltered through a Whatman No. 1 flter paper. Te resultant fltrate was then lyophilized using a freeze dryer. Te obtained freeze-dried product was weighed and stored in airtight plastic vials at 4°C in a refrigerator awaiting further analysis.
Te methanolic extract was prepared by measuring 200 g of D. abyssinica rhizome extract into an extraction jar, adding 1 liter of analytical methanol gradually and then shaking vigorously for 48 hours to macerate. Te process was repeated for another batch of 200 g of rhizome powder. Te resultant mixture was then decanted and fltered through cotton gauze to remove course residues. Te resultant fltrate was then fltered through a Whatman No. 1 flter paper. Te resultant fltrate was then combined and reduced in a vacuum at 50°C using a rotary evaporator. To further remove the solvent and concentrate the extract, the extract was placed in a clean, dry, and light-resistant bottle and placed in a sand bath set at 35°C. Finally, it was weighed using an analytical balance, and the percentage yield of the extract was calculated and stored at 4°C in a refrigerator awaiting further analysis.
Te dichloromethane : methanol (1 : 1) extract was prepared by measuring 200 g of rhizome powder into an extraction jar. An equal amount of dichloromethane and methanol was mixed to make a solution of 1 liter. Te mixture was then gradually added to the powder. Te mixture was shaken vigorously for 48 hours and allowed to macerate. Te abovementioned process was then repeated for another batch of 200 g of the rhizome powder. Te Evidence-Based Complementary and Alternative Medicine resultant mixture was then decanted and then fltered through gauze. Te resultant mixture was then fltered through a Whatman No. 1 flter paper. Te resultant mixture from the frst and the second batch was combined and reduced in a vacuum (in vacuo) at 50°C using a rotary evaporator. To further remove the solvent and concentrate the extract, the extract was placed in a clean, dry, and lightresistant bottle and placed in a sand bath set at 35°C. Finally, it was weighed using an analytical balance and stored at 4°C in a refrigerator until use.

Experimental Animals. Fifteen 8-10-week, female
Wistar albino rats weighing 125 ± 45 grams were used to assess the acute oral toxicity of Digitaria abyssinica rhizome extracts. Tese animals were purchased from the Kabete Vet Lab animal house. Tey were transported to the Public Health, Pharmacology, and Toxicology (PHPT) Department animal house where they were housed for 5 days to acclimatize following Biosafety, Animal Use and Ethical Committee (BAUEC) guidelines. All the experimental animals were nulliparous and nonpregnant. Tey were housed at a temperature of 25 ± 3°C and 56-60% relative humidity. A 12-hour day and night cycle were maintained, and the animals were fed on standard rat pellets from a commercial feed supplier (Unga Feeds). Water was provided ad libitum.

Phytochemical
Screening. Te analysis of the phytochemical groups of compounds, namely, plant favonoids, tannins, saponins, phenols, coumarins, steroids, terpenoids, glycosides and alkaloids of water, methanol, and dichloromethane : methanol (1 : 1) extracts of D. abyssinica rhizomes were done using standard phytochemical screening procedures described by [33,34] as modifed by [35]. Te tests were performed in triplicates to ensure the results' accuracy and were examined by visual observations.

Test for Saponins (Froth Test)
. About 0.1 g of the water, methanol, and dichloromethane : methanol (1 : 1) extracts of D. abyssinica rhizome were added to 10 ml of distilled water in separate test tubes, respectively. Te mixtures were boiled for 10 minutes, and they were fltered using Whatman flter paper No.1. A mixture of 3 ml distilled water and 5 ml of the fltrate was agitated vigorously for 15 seconds and left to stand for 10 minutes. Frothing which persisted for about 3 minutes was an indication of saponins [36].

Test for Alkaloids. Two tests, namely, Mayer's and
Dragendorf's tests, were done to detect alkaloids in the extracts.

Test for Flavonoids (Sodium Hydroxide Reagent Test).
Approximately, 0.1 g of the water, methanol, and dichloromethane : methanol (1 : 1) extracts of D. abyssinica rhizome were warmed in 10 ml of 70% ethanol and thereafter hydrolyzed with 10% hydrochloric acid. Sodium hydroxide (10%; 1 ml) was added to the mixture and the appearance of yellow color was a positive test for the presence of favonoids [39,40].

Test for Phenolics.
Approximately, 0.1 g of the water, methanol, and dichloromethane : methanol (1 : 1) extracts of D. abyssinica rhizome were measured and put into separate test tubes and 10 ml of 70% ethanol were added. Te mixtures were boiled using water for fve minutes. Te extracts were then cooled, and they were fltered through Whatman flter paper No.1. Five drops of 5% of ferric chloride were added into 2 ml of each respective extract. Te formation of a green precipitate indicates the presence of phenols [36].

Test for Glycosides.
(1) Keller-Killiani Test. Glacial acetic acid (4.0 ml) solution with 1 drop of 2.0% FeCl 3 mixture was added to the 10 ml water, methanol, and dichloromethane : methanol (1 : 1) extracts of D. abyssinica rhizome in separate test tubes. One milliliter of concentrated sulphuric acid was added to the mixture and a reddishbrown ring formed between the layers which progressively turned blue indicating the presence of steroidal glycosides with deoxy sugars [36].
(2) Kedde Test. One milliliter of 2% solution of 3,5dinitrobenzoic acid in 95% alcohol was added to the 2 ml of the water, methanol, and dichloromethane : methanol (1 : 1) extracts of D. abyssinica rhizome. Te solution was made alkaline with 5% sodium hydroxide. Te appearance of a purple-blue color indicates the presence of an unsaturated lactone ring in cardenolides [36].

Test for Steroids.
(1) Salkowski's Test. Approximately, 2 mg of the water, methanol, and dichloromethane : methanol (1 : 1) extracts of D. abyssinica rhizome were dissolved in 1 ml of chloroform and then shaken gently. Five drops of concentrated sulphuric acid were added along the side of the test tube. A reddish-brown color that was formed at the interface indicated steroids [39,41].
(2) Liebermann-Burchard Test. About 2 mL of acetic acid was added to 1 mL of the water, methanol, and dichloromethane : methanol (1 : 1) extracts. After cooling the solution in an ice bath, concentrated sulphuric was added Evidence-Based Complementary and Alternative Medicine carefully. Te development of violet to blue or bluish-green color confrms the test for steroids [42,43].
2.5.8. Test for Coumarins. Approximately, 0.5 g of the extracts and powder of D. abyssinica were added into separate test tubes. Te test tubes were covered with flter paper which was moistened with 1 N NaOH. Te tubes were warmed in a hot water bath and then allowed to cool. Yellow fuorescent color was an indication of coumarins [44].
2.5.9. Test for Tannins. About 0.5 g of the sample was boiled in 20 ml of water and fltered. 0.1% of ferric chloride was added to the fltrate. Te formation of a brownish-green or blue-black color was an indication of the presence of tannins [44].

Single-Dose Toxicity Study.
Te up-and-down procedure for acute oral toxicity described by the Organization for Economic Cooperation and Development (OECD), [45] Document No. 425 was used to determine the safety of the aqueous, methanol, and dichloromethane : methanol (1 : 1) extracts of D. abyssinica rhizome. Five female Wistar rats were used to perform the limit tests for each of the aqueous extracts. A dose of 2000 mg/kg body weight of each extract was given orally to one female rat, and fatalities were not observed; thereafter, four additional animals were dosed sequentially. In addition, the rats were observed for wellness parameters that included the skin and fur appearance, fecal matter consistency, urination and urine appearance, itching, salivation, convulsions, tremors, breathing, coma somatomotor activity, aggression, grooming, eyes, and unconsciousness or death.

Preparation of Inoculums.
To prepare the stock cultures, CLSI guidelines and procedures were used. Te method as described by [46] was used with modifcations. In brief, bacterial stock cultures were subcultured on respective media and incubated at 37°C for 24 hours. To obtain young growing culture, Neisseria gonorrhea isolates were cutured on Tayer Martin agar while isolates of Escherichia coli and Staphylococcus aureus were cultured on MacConkey and sheep blood agar, respectively. Candida albicans were subcultured in Sabouraud Dextrose Agar (SDA) at 37°C for 48 hours. Te test strains were suspended in Mueller-Hinton broth (MHB) to give a fnal density of 1.5 × 10 6 bacteria colony-forming units and 1.5 × 10 5 fungal colonyforming units.

Preparation of the Stock Solutions.
Six diferent concentrations of 15.625 mg/ml, 31.25 mg/ml, 62.50 mg/ ml, 125 mg/ml, 250 mg/ml, and 500 mg/ml of each of the three extracts (aqueous, methanol, and dichloromethane : methanol (1 : 1) were prepared for susceptibility testing using 1% DMSO as a diluent. A vortex mixer was used to facilitate the dissolving of extracts into the 1% DMSO. A constant volume of 20 μl of each of the individual stock solutions was pipetted using a micro titer-pipette onto sterile flter paper disks measuring 6 mm to prepare the respective concentrations of the plant extracts. 20 μl of 1% DMSO was impregnated onto sterile flter paper disks which were used as the negative control for the experiment.
Te Mueller-Hinton agar plates inoculated with S. aureus and those inoculated with E. coli were incubated at 37°C for 24 hours. Tayer Martin Media inoculated with Neisseria gonorrhoeae was incubated at 5% CO 2 for 48 hours while Sabouraud Dextrose Agar inoculated with Candida albicans was incubated at 37°C for 24 hours. Generally, the antimicrobial agent difuses into the agar and inhibits the germination and growth of the test microorganism.
After the incubation period, the diameters of the inhibition zones were measured in millimeters using a transparent ruler. All the tests were done in triplicates and the means were calculated as the results. Evidence-Based Complementary and Alternative Medicine 2.7.5. Broth Macrodilution Technique. Te broth macrodilution procedure as described in 2021 by Mailu et al. [46] with modifcation was used to determine the minimum inhibitory concentration for the active crude extracts against the test microorganisms. Six culture tubes with 2 ml sterile Mueller-Hilton broth were prepared. From the stock solution, two-fold serial dilutions were prepared. 0.1 ml of each microorganism was inoculated into each tube of diluted plant extract using a micropipette. Te bacterial organisms and the fungal organism were then incubated for 24 hours at 37°C. Te extract's minimum inhibitory concentration (MIC) value was determined by observing the lowest concentration of plant extracts that prevented the visible growth of microorganisms resulting in no visible growth (turbidity).
To determine MBC, all broth in tubes with no visible bacterial growth was aseptically cultivated in sterile agar using the streak-plate method and incubated at appropriate temperatures and conditions. Te MIC value is the lowest concentration of the plant extract that demonstrates no visible bacterial growth. All tubes with no visible fungal growth were aseptically cultured in sterile molten agar and incubated using the streak-plate method to determine the minimum bactericidal concentration (MBC). Te minimal fungicidal concentration (MFC) value was defned as the lowest plant extract concentration that shows no visible fungal growth. Tubes that were just inoculated with microorganisms and tubes that were only inoculated with media served as controls. All the experiments were carried out in triplicate, and the results were recorded in a table.

Statistical Analysis.
All experiments were performed in triplicates. Data were analyzed by GraphPad Prism version 9.0.0 and the results are provided as mean ± SEM. One-way analysis of variance (ANOVA) and post hoc ANOVA using Tukey's HSD test with a 95% confdence level was used to compare the diferences in the mean zone of inhibitions among and between the groups, respectively. Diferences among groups were statistically signifcant at p < 0.05.

Phytochemical Composition.
Aqueous extracts had the highest yield value (7%), followed by methanol with 2.3% and the mixture of dichloromethane : methanol (1 : 1) recorded the least (1.89% yield value). Te three extracts and the powder of D. abyssinica rhizome possessed secondary metabolites (Table 1). Except for the aqueous extract, saponins were detected in the powder and all other extracts. Alkaloids, glycosides, phenolics, coumarins, tannins, favonoids, steroids, and terpenoids were present in the powder and all other extracts (aqueous dichloromethane : methanol (1 : 1) and methanol) from D. abyssinica rhizome. Mayer's test for alkaloids was negative while Dragendorf's test was positive for all the samples.

Efects of Single-Dose Toxicity.
Te study of the toxic efects of D. abyssinica aqueous, methanol, and dichloromethane : methanol extracts after oral single-dose administration revealed no signs of unwellness (Table 2). In addition, there wasn't any rat mortality that was observed at a single dose of 2,000 mg/kg body weight (bw). Terefore, the extracts were classifed as nontoxic according to the OECD 425 guidelines. Te LD 50 of the extracts was found to be more than 2000 mg/kg. To the best of our knowledge, this was the frst time the toxicity profle of D. abyssinica is being reported.

Antimicrobial Activity.
Te crude extracts of D. abyssinica rhizome were tested for antimicrobial efcacy against four pathogenic microorganisms utilizing disk diffusion and microdilution techniques. Te study revealed that the extracts had antifungal activity and did not demonstrate antibacterial activity against the studied microorganisms. Tables 3 and 4 summarize that the aqueous, methanol, and

Discussion
Medicinal plants are endowed with secondary metabolites. Tese plants continue to provide solutions to human and livestock ailments in traditional medicine systems for primary health care. In the current study, chemical compounds known to have antimicrobial activities were detected in the rhizome extracts of D. abyssinica. Phenols, tannins, favonoids, terpenoids, saponins, alkaloids, and coumarins which were positively indicated in the aqueous, methanol, and dichloromethane : methanol (1 : 1) extracts are known to have antimicrobial activities and may be explored to develop botanicals that can be used to combat antimicrobial resistance [48]. To the best of our knowledge, this is the frst published report on the qualitative phytochemical composition of the powder and extracts from D abyssinica rhizome. Also, toxic efects and antimicrobial activity studies have not been reported in the literature. However, reports exist for several plants from the Digitaria genus that possesses active phytochemicals with antimicrobial activities and other varied biological activities including analgesic, antiviral, anti-infammatory, antitumor, anthelmintic action, and effects on the central nervous system [49,50]. All female Wistar rats in this study, survived after administration of a single dose of 2000 mg/kg of the aqueous, methanol, and dichloromethane : methanol (1 : 1) extracts of D. abyssinica to the Wistar rats orally. Terefore, the dose of the D. abyssinica rhizome extracts that can kill half of the tested animals was more than 2000 mg/kg. According to the Organization for Economic Cooperation and Development (OECD) test no 425, the fndings in this study indicate that the extracts are not toxic to the female Wistar rats as in many other acute toxicity studies of herbal medicines [45,51,52]. Te current research indicates that D. abyssinica rhizome extracts have antifungal activity against C. albicans and that the strength of the activity was directly proportional to the concentration of the extract loaded to the disk. Te size of the zones of inhibition of the aqueous extract was more than 15 mm at concentrations 125 mg/ml, 250 mg/ml, and 500 mg/ml (Table 3) which is interpreted as potential antifungal activity [53,54]. Tere was no statistical diference in activity at concentrations more than 125 mg/ml at p < 0.05 (Table 3). Tough there was noted low activity at 15.625 mg/ml, 31.25 mg/ml, and 62.50 mg/ml of zones of inhibition between 6.5 mm and 9.67 mm, there were statistical diferences in activity at p < 0.05.
Te methanol extract also had high antifungal activity (15.17 ± 0.75 mm) at a concentration of 500 mg/ml and is statistically diferent from the activity of 11.67 ± 0.52 mm exerted by the extract at a concentration of 250 mg/ml at p < 0.05. Subsequently, zones of inhibition of 11.50 ± 0.55, 8.83 ± 0.75, 8.33 ± 0.52, and 6.83 ± 0.41 mm at concentrations of 125, 62.50, 31.25, and 15.625 mg/ml, respectively. Tere were notable statistical diferences between the antifungal activities of the methanol extract at concentrations between 15.625 and 31.25 mg/ml (p < 0.05) and between 62.50 and 125 mg/ml. However, there were no statistical diferences in activities between concentrations 31.25 and 62.50 mg/ml and between concentrations 125 and 250 mg/ ml at p < 0.05. Te zones of inhibition that revealed the activity of the mixture of dichloromethane : methanol D. abyssinica rhizome extract similarly refected proportionality to extract concentrations (Table 3). Te aqueous, methanol, and dichloromethane : methanol (1 : 1) extracts of D. abyssinica did not show any zone of inhibition against the bacterial strains, E. coli, N. gonorrhoeae, and S. aureus, in this study.

Conclusion and Recommendation
Based on the current study, it was concluded that the aqueous, methanol, and mixture of dichloromethane : methanol (1 : 1) extracts of D. abyssinica rhizome possess phytochemicals compounds with anticandidal activity. Te extracts can be classifed as nontoxic and therefore safe for female Wistar rats when administered orally at a single dose. It was further deduced that the extracts had low antifungal efects. Nevertheless, herbalists and traditional medicine practitioners have used the plant material in wide cultural setups. Given that herbalist frequently uses aqueous extracts in their practice, these study fndings provide scientifc evidence to validate the use of the extracts in the management of candidiasis. Bioassay-guided fractionation was recommended to isolate anticandidal compounds from D. abyssinica rhizome extracts. In addition, quantitative phytochemical studies and subchronic and chronic toxicity assays were recommended.