ANTIBACTERIAL ACTIVITIES OF ETHANOL EXTRACTS FROM Croton macrostachyus (Hochst. Ex Delile) AND Justicia schimperiana

Our study was designed to evaluate antibacterial activities of ethanol extracts from Croton macrostachyus and Justicia schimperiana bark and leaf against certain bacterial pathogens such as E. coli O157 H:7 ATCC 25922, Salmonella Typhi ATCC 13311, Staphylococcus aureus ATCC 25923 and Streptococcus pyogenes ATCC 196151. Qualitative and quantitative methods were followed to screen the phytochemicals in the plants extracts and to estimate total phenolic content (TPC) and terpenoids. Antibacterial activity was evaluated by disc diffusion method and by determination of Minimum Inhibitory Concentration (MIC). The results of the investigation revealed that both the plants were found to contain alkaloids, saponin, steroids, tannins and terpenoids. Highest TPC (3.82±0.82 mg/g Tannic Acid Equivalent) and highest Terpenoid content (118.33±4.4 mg/g) were observed in the leaf extract of C . macrostachyus . Bark extract of C . macrostachyus displayed the strongest activity against the gram-positive bacteria S. aureus with an inhibition zone of 16.17±0.67 mm. E. coli O157 H:7 and S . Typhi were found more resistant to bark extract of J. schimperiana at the concentration of 100 mg/ml. The potent plant part extract should be subjected for further phytochemical analysis and bioactive investigations.


INTRODUCTION
The infections caused by bacterial pathogens that are resistant to drugs are a foremost cause of mortality across the world. The antimicrobial resistance of bacteria led to death of 1.27 million people around the world in 2019 (Antimicrobial Resistance Collaborators, 2022). Among the bacterial pathogens, Salmonella Typhi (Ali Shah et al., 2020), Staphylococcus aureus (Foster, 2017;Guo et al., 2020), Streptococcus pyogenes (Pieretti et al., 2017) have emerged highly resistant to the antibacterial drugs (Ali Shah et al., 2020). Further, several side effects of the antibacterial drugs have been identified (Tsuruga, 2007). Hence, researchers have been hunting for alternative medicine from plants as antibacterial agents (Sasikumar et Mechesso et al., 2016). Cytotoxic diterpenes were isolated from the seeds of this plant (Yong et al., 2017). Some phenolic compounds were also obtained (Tala et al., 2013). A few studies were reported on antibacterial activity of the plant (Geyid et al., 2005;Mesfin et al., 2010;Tene et al., 2009;Wagate et al., 2010). The leaves and stem bark of J. schimperiana are used to treat wound, gonorrhoea, malaria, rabies, a burning and arthritis, jaundice, and headache (Habtamu et al., 2014, Tamrat et al., 2015. Antidiarrheal activity of the leaf extract of this plant was investigated (Mekonnen et al., 2018). A study from Ethiopia reported the hepatoprotective activity of J. schimperiana (Umer et al., 2010). Earlier, essential oil from leaves of J. schimperiana were tested for antibacterial activity (Abebe et al., 2018). Despite some antibacterial activities of C. macrostachyus and J. schimperiana from different areas in the country and overseas, environmental factors in the locality of the plant collection have an impact on the chemical composition and bioactivity (De Zoysa et al., 2019) and the plants could exhibit better activity than the other reported areas. Thus, our work aimed to evaluate antibacterial activity of ethanol extracts from C. macrostachyus and J. schimperiana bark and leaf against certain bacterial pathogens such as E. coli O157 H:7, S. Typhi, S. aureus and S. pyogenes.

Ethanol extract preparation
The leaves and bark of C. macrostachyus and J. schimperiana were collected from villages near Haramaya University, Ethiopia. Plant Taxonomists assisted in identification of the plants and the specimens were deposited in Haramaya University herbarium. The leaves and bark of the plants were dried and pulverized with the help of homogeniser. The leaf and bark powder (80 g each) were extracted with 400 ml of (1:5 w/v) 95% Ethanol. The extracts were evaporated to dryness at 40 ± 10 o C by rotary flash evaporator (Buchi type rotavapor) under reduced pressure. The crude ethanol extracts of leaf and bark were stored at 4 0 C until further use.

Total phenolic content (TPC)
The total phenolic content (TPC) was measured by spectrophotometric method (Amorim et al., 2012). In brief, the ethanol extracts (500 μl) were added with Folin-Ciocalteu reagent (500 μl) and sodium carbonate solution (1 ml). Then, the final volume was adjusted to 10 ml by adding 8 ml of distilled water. The samples were kept at room temperature for 30 minutes and their absorptions were measured at 760 nm using distilled water as blank. The TPC was calculated as tannic acid equivalent (TAE) by the following equation: Where, T is the TPC in mg/g of the extracts as Tannic Acid Equivalence (TAE), C is the concentration of tannic acid obtained from the calibration curve in mg/ml, V is the volume of the extract in ml and M is the weight of the extract used in g.

Terpenoid content
The terpenoid content was quantified following the method of (Furguson, 1956). The plant powder (2 g) was soaked with 97% ethanol (50 ml) for 4 hours. The extracts were filtered by Whatman No 1 filter paper and the filtrate was added into a separating funnel (250 ml) and then 50 ml of petroleum ether was added. The resultant mixture was shaken and allowed to stay for 5 minutes for layer formation. Then, the bottom layer was discarded while the top petroleum ether layer was collected and concentrated to dryness using rotary evaporator at 40 ºC for 18 hrs. The mass of dry extract, which was considered to represent crude terpenoids, was measured and its content was calculated as mg per the sample powder used.

Bacterial pathogens
For antibacterial activity study, two Gram negative pathogenic bacteria Escherichia coli O157 H:7 (ATCC 25922), Salmonella Typhi (ATCC 13311) and Gram-positive pathogenic bacteria Staphylococcus aureus (ATCC 25923) and Streptococcus pyogenes (ATCC 196151) were obtained from Ethiopian Public Health Institute (EPHI) and Ethiopian Biodiversity Institute (EBDI), Addis Ababa, Ethiopia. The guidelines of Clinical and Laboratory Standard Institute were employed to prepare the bacterial turbidity of the bacteria (CLSI, 2012).

Disc diffusion assay
The antibacterial effect of ethanol extracts of C. macrostachyus and J. schimperiana bark and leaf was determined by disc diffusion method against the bacterial pathogens E. coli, S. Typhi, S. aureus and S. pyogenes (CLSI, 2012). The bacterial strains were cultured in 25 ml nutrient agar plates prepared by transferring 25 ml of nutrient agar into a pre-sterilized Petri dish and allowed to solidify at room temperature. Muller Hinton agar (MHA) was used to smear 0.1ml suspension cultures of bacterial pathogens on plates. Sterile Whatman No. 1 paper discs (6 mm) were used to impregnate different concentrations (100 mg/ml, 125 mg/ml, 150 mg/ml) of ethanol extracts (0.1 ml/disc). The extract loaded discs were placed onto the agar surface at equidistance and the Petri dishes were incubated at 37°C for 24 hrs. Amoxicillin (25 μg/disc) was used as positive control. The experiment was performed in triplicates under sterile conditions. The antibacterial activity of the extracts was tested by measuring diameter of clear inhibition zones.

Determination of Minimum Inhibitory Concentration (MIC)
The ethanol extracts of C. macrostachyus and J. schimperiana bark and leaf were subjected for the minimum inhibitory concentrations (MIC) using broth dilution technique based on CLSI (2012). The concentrations of the extracts ranged from 100 mg/ml -6.25 mg/ml and where 0.1 ml of each dilution were added to the different test tubes.

Statistical analysis
The experiments were performed in triplicate and results were presented as mean values ± Standard deviation (SD.) Differences between means were measured by one-way analysis of variance (ANOVA) using SPSS. P-values ≤ 0.05 were considered as showing significance difference.

Preliminary screening and Quantification
The results of preliminary phytochemical analysis are shown in Table 1. In both C. macrostachyus and J. schimperiana, ethanol extracts of both leaves and bark were found to show positive results for alkaloids, saponin, steroids, tannins and terpenoids. Flavonoids and phlobatannins were found absent in the screened plants.
The quantification of phytochemicals in both the plants showed that ethanol extracts of C. macrostachyus leaves (3.82±0.82 mg/g Tannic acid equivalents) and bark (mg/g 3.01±0.07 Tannic acid equivalents) were found to contain significantly (P <0.05) higher TPC than that of ethanol extracts of J. schimperiana leaves (2.92±0.63 mg/g Tannic acid equivalents) and bark (mg/g 32. 14±0.13 mg/g Tannic acid equivalents). The ethanol leaf extract of C. macrostachyus significantly (P < 0.05) had the highest content of terpenoids (118.33±4.40 mg/g).
indicates the presence of the phytochemicals while "−" indicates the absence of the phytochemicals.

Disc diffusion test
The effects of ethanol extracts of C. macrostachyus and J. schimperiana leaves and bark against the pathogenic bacteria were investigated and the results are exhibited (Table 3.). Our findings displayed that the plants' extracts possessed highest to moderate antibacterial activities against the tested bacteria. A significantly (P< 0.05) highest activity was shown by ethanol extract of C. macrostachyus leaf against the Gram-positive bacteria S. aureus with an inhibition of 16.17±0.67 mm at the concentration of 150 mg/ml. Among the four bacterial strains tested, S. aureus was found more susceptible to the extracts at the concentration of 150 mg/ml of ethanol extracts of leaf and bark of both plants. Interestingly, at 100 mg/ml concentration, J. schimperiana showed no activity against the Gram-negative bacteria E. coli O157 H:7 and S. Typhi. The leaf and bark extracts of C. macrostachyus had highest antibacterial activity against E. coli and S. Typhi with the inhibition zones of 14.70±0.15 and 13.33±0.60 mm respectively at 150 mg/ml concentration. The positive control Amoxicillin displayed significantly higher activity against the tested bacterial pathogens than the plant extracts.

MIC values of the extracts
The values of MIC of the ethanol extracts of C. macrostachyus and J. schimperiana leaves and bark against the pathogenic bacteria are recorded in Table 4. The ranges of MIC values were 25±0.00 to 100±0.00 mg/ml. The leaf extract of C. macrostachyus exhibited the lowest MIC value (25±0.00) against S. aureus followed by the MIC value (41.67±8.33) of leaf extract of C. macrostachyus against S. pyogenes. Both Gram negative E. coli O157 H:7 and S. Typhi were observed to be more resistant to the ethanol extracts of the plant parts.

DISCUSSION
The present investigation reported qualitative phytochemical analysis, quantification of total phenolics and terpenoids and antibacterial activity ethanol extracts from C. macrostachyus and J. schimperiana leaves and bark against the pathogenic bacteria include Gram negative E. coli O157 H:7 and S. Typhi and Gram-positive S. aureus and S. pyogenes. With regard to qualitative phytochemical analysis, extracts of both plants contain alkaloids, saponin, steroids, tannins and terpenoids. Our results are in covenant with the previously published reports in the case of C. macrostachyus (Kibret et al., 2018) and for J. schimperiana (Mekonnen et al., 2018). The quantification of phytochemicals revealed that C. macrostachyus contained high TPC and terpenoid contents which could be attributed to its antibacterial potential. The phenolic terpenoid compound has been proved to disturb cell membrane integrity and ion leakage of gram positive and gram-negative bacteria (Erguden, 2021). The evaluation of antibacterial activity of the studied plants revealed a strong activity against the tested pathogens. The antibacterial activities of the ethanol extracts from leaf and bark of the investigated plants were found inferior than the standard control Amoxicillin which is due to the crude nature of the plant extracts (Mengiste et al., 2015). Concerning to C. macrostachyus, the ethanol extract of the leaves and bark displayed strong antibacterial activity against the gram-positive bacteria. Regarding J. schimperiana, the antibacterial activity of the ethanol leaf extract was found stronger against the gram-positive bacteria. Surprisingly, ethanol extract from J. schimperiana bark failed to exhibit any antibacterial activity against E. coli O157 H:7 and S. Typhi. The stronger antibacterial activity of ethanol extracts both the plants against gram positive bacteria S. aureus and S. pyogenes than the gramnegative bacteria such as E. coli O157 H:7 and S. Typhi was due to cell wall structure of the gram-positive bacteria with thick layer of peptidoglycan sheets in the outer wall (Kitonde et al., 2013). However, gram positive bacteria have been posing threat to humans as they are developing resistance to antibacterial drugs. For instance, Streptococcus pyogenes has been known to cause pharyngitis among the paediatric group and infection of skin and soft tissue which is resistant to erythromycin and clindamycin (Kebede et al., 2021). Similarly, S. aureus cause soft tissue infections. bacteraemia, and fatal pneumonia and this bacterium is resistant to methicillin (Guo et al., 2020). The gram-negative bacteria S. Typhi, causative agent of typhoid disease, has become resistant to chloramphenicol and ampicillin (Dyson et al., 2019). The enterohemorrhagic E. coli O157 H:7 causes food borne and water borne diseases like bloody or non-bloody diarrhea in humans (Dean-Nystrom et al., 1997) and this strain was reported to show resistant against antibiotics (Ferdous et al., 2016). Furthermore, the increasing side effects caused by the synthetic antibacterial agents jeopardizes the human health (Mohsen et al.,  2020). To overcome above mentioned issues, development of alternative drugs is essential. Thus, the current study was conducted. In our study, the ethanol extracts of C. macrostachyus and J. schimperiana leaves and bark displayed a potent activity against these bacteria with good inhibition zone and least MIC value. Hence, further studies are warranted to isolate phenolics and terpenoids using bioactivity-directed fractionation from the currently studied plants to combat drug resistant pathogenic bacteria.

CONCLUSION
The findings of the study revealed that ethanol extract of C. macrostachyus and J. schimperiana leaves and bark showed significant antibacterial effects against Gram negative E. coli O157 H:7 and S. Typhi and Gram-positive S. aureus and S. pyogenes. Gram negative bacteria were more resistant than the gram-positive bacteria. E. coli O157 H:7 and S. Typhi were found to show strong resistance to plant extract at low concentrations. Among the pathogens, S. aureus was found to me most sensitive to the plant extracts. The antibacterial properties of the two plants against the tested pathogenic bacteria could be attributed to the total phenolic and terpenoids contents. Further in vivo antibacterial activity and isolation of active components by bioactivity-guided fractionation is vital.