Biology, Medicine, & Natural Product Chemistry ISSN 2089-6514 (paper) Volume 12, Number 1, April 2023 | Pages: 281-288 | DOI: 10.14421/biomedich.2023.121.281-288 ISSN 2540-9328 (online) Phytochemical Analysis and Antibacterial Activity of Methanol and Ethyl Acetate Extracts of Detarium microcarpum Guill. & Perr. Muhammad Mubarak Dahiru*, AbdulAzeez Mumsiri Abaka, Susan Pwakangdi Artimas Department of Science Laboratory Technology, School of Science and Technology, Adamawa State Polytechnic, Yola, Jimeta, 640101, Tel. 555-555-1212, Adamawa State, Nigeria. Corresponding author* mubaraq93@gmail.com Manuscript received: 30 January, 2023. Revision accepted: 11 March, 2023. Published: 27 March, 2023. Abstract This research aimed to investigate the phytoconstituents and antibacterial effects of methanol and ethyl acetate stem bark extracts of Detarium microcarpum (DM). The phytochemicals were detected and quantified while the antibacterial activity against Escherichia coli, Staphylococcus aureus, and Salmonella typhi was established determining the zone of inhibition (ZI), minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC). Phytochemical screening showed alkaloids (16.33% ±0.88) were present in the methanol extract only while saponins and flavonoids were detected in concentrations of 31.00% ±2.31 and 21.01% ±2.33 respectively for the methanol extract and 21.67% ±1.76 and 38.01% ±1.16, for the ethyl acetate. The methanol extract exhibited the highest ZI on S. aureus (21.3 mm ±1.11) with its least inhibition observed on E. coli (6.5 mm ±0.77) while the ethyl acetate extract demonstrated the highest ZI on S. typhi (19.1 mm ±2.01). S. typhi exhibited more sensitivity to DM extracts at the least concentrations of 12.5 mg/ml (methanol) and 25 mg/ml (ethyl acetate) while the MBC results showed that the 12.5 mg/ml and 25 mg/ml were the effective respective concentration for methanol and ethyl acetate extracts against S. typhi. Conclusively, DM exhibited an antibacterial effect against the test organisms with notable inhibitory and bactericidal effects. Keywords: Antibacterial activity; Antimicrobial activity; Antibacterial resistance; Detarium microcarpum; Phytochemical analysis. Abbreviations: DM (Detarium microcarpum), ESBE (Ethyl acetate stem bark extract), MSBE (Methanol stem bark extract), MIC (Minimum inhibitory concentration), MBC (Minimum bactericidal concentration), and ZI (Zone of inhibition). INTRODUCTION Medicinal plants have long been reported to be sources of therapeutics and have been utilized in the management of several ailments in traditional and folkloric practices. Different research reported the pharmacological effects of several plants employed to manage ailments including diabetes, cancer, inflammation, parasite, and microbial infections (Prasathkumar et al., 2021). In traditional practice, medicinal plant parts such as roots, leaves, and stem bark are prepared in different forms such as decoction and infusions administered by oral or inhalation as an alternative to modern medicines to achieve therapeutic purposes (Ullah et al., 2020). There are several reasons for the inclination toward the use of medicinal plants as it offers certain advantages such as being cheap, and available with minimized side effects compared to modern drugs (Ahad et al., 2021; Rahayu et al., 2020). In other cases, poverty, poor health facilities, and access may contribute to the recognition of drugs from plant sources as alternatives. The therapeutic use of plants as sources of drugs in disease and infection management has been attributed to their phytochemical components extracted via the preparation method which are associated with several biological effects (George et al., 2021; Hossen et al., 2022; Zheng et al., 2022). Bacterial infections are attributed as the cause of millions of deaths and morbidities worldwide and have so far evolved into a major health concern all over the world (Ji et al., 2016). Although different antibiotics of varying efficacy are employed in the treatment of infections, these antibiotics are losing their efficacy because of antimicrobial resistance which is a major challenge in achieving treatment goals (Maillard et al., 2020). Antimicrobial resistance forms a major problem in treatment of infectious diseases especially in rural communities and developing countries even though this resistance continues to occur every day during treatment with an estimated projected death of up to 4 million by 2050 (Akinde & Taiwo, 2017; Elton et al., 2020; Tong et al., 2019). There is an increase in antimicrobial resistance, especially in Africa, however, phytochemicals from plants serve as long-time sources of therapeutics with proven results in treatments of infectious diseases https://doi.org/10.14421/biomedich.2023.121.281-288 282 Biology, Medicine, & Natural Product Chemistry 12 (1), 2023: 281-288 (Bouyahya et al., 2017). Phytochemicals from plants offer an alternative as utilized in traditional practices as infectious diseases are a worldwide menace and create a burden on an individual and governments. Phytochemicals extracted from different plant preparations exert different pharmacological effects individually or synergistically observed in the folkloric use of plant drugs. Alkaloids are associated with anti- diabetic (Adhikari, 2021), anticancer (Song et al., 2022), and antimicrobial effects (Fan et al., 2022; Jafaar et al., 2021). Flavonoids exert biological and pharmacological activities against oxidative stress, cancer, inflammation, and tumor through different modes of action (Jucá et al., 2020). Flavonoids also exert an antimicrobial effect on antibiotic-resistant microbes (Biharee et al., 2020; Gupta et al., 2022). Glycosides exhibit pharmacological effects including anti-diabetic (Yang et al., 2022) and antimicrobial activities (Tm et al., 2022). Terpenoids exert pharmacological effects against a tumor, cancer, inflammation, malaria, diabetes, and microbial infections (Wu et al., 2020; Yang et al., 2020). Saponins exhibit pharmacological effects against cancer, oxidative stress, inflammation, and microbial infections (Fang et al., 2020). Several phytochemicals exert different pharmacological activities working either individually or in synergy with each other. Thus, this research aimed to establish the phytochemical composition and antibacterial effects of methanol and ethylacetate extracts of DM. MATERIALS AND METHODS Study area Detarium microcarpum (DM) sample collection was carried out in the Girei Local Government Area of Adamawa state, Nigeria. The authentication of the plant sample was done by a Forest Technologist from the Department of Forestry, Adamawa State Polytechnic Yola, where a voucher specimen (ASP/FT/0212) was deposited. The stem bark was dried and ground into powder using a blender. Chemicals and reagents All the chemicals and reagents used for the study were of AnarlaR. Procedures Extract preparation Stem bark powder (300 g) of DM was macerated in 1 L of 70% v/v methanol and ethyl acetate for 48 hours, followed by filtration and concentrated to dryness over reduced pressure (Evans, 2009). Qualitative phytochemical Analysis The detection of phytochemicals in methanol (MSBE) and ethyl acetate (ESBE) stem bark extracts of DM was carried out following the methods previously described (Evans, 2009). Quantitative phytochemical analysis  Alkaloids Alkaloids were quantified as previously described (Harborne, 1998).  Saponins Saponins were estimated by the method previously described (Obadoni & Ochuko, 2002).  Flavonoid Quantitation of flavonoids was carried out by the method previously (Harborne, 1998). Antibacterial activity  Test organisms The bacteria isolates (E. coli, S. aureus, and S Typhi) were collected from Modibbo Adama University Teaching Hospital microbiology laboratory, Yola, Nigeria. The characterization of the test organisms was done through observation of their cultural growth characteristics according to a previously described method (Idris Abubakar & Abubakar Usman, 2016). Confirmation of clinical isolates’ identity was carried out via biochemical tests following the standard method previously described (Cheesbrough, 2002; Talaiekhozani, 2013). The pure cultures were streaked onto a nutrient agar slant followed by 24 h incubation at 37 °C and subsequent storage at 4 °C until needed for use.  McFarland Standard Preparation Exactly 9.95 mL of 1% H2SO4 and 0.05 mL of 1.17% BaCl were mixed to form a precipitated suspension acting as 0.5 McFarland standard set as the turbidity for the test organisms (Cheesbrough, 2002).  Inoculum Standardization The confirmed isolates were subjected to further culturing on nutrient agar containing petri-dishes followed by overnight incubation at 37 °C to form colonies which were transferred into test-tubes / containing 5 mL of 0.9% normal saline adjusted to the turbidity of previously prepared McFarland's standard (Andrews, 2005). Determination of antibacterial Activity A slightly modified agar well diffusion technique was adopted to establish the antibacterial effects of DM (Biradar et al., 2008). The bacterial inoculation was carried out with a sterile swab onto a prepared solidified Mueller-Hinton agar, and allowed to stand for 15 min, followed by the addition of 0.2 mL of the extracts of varied concentrations (100 mg/mL, 50 mg/mL, 25 mg/mL, 12.5 mg/mL and 6.25 mg/mL) to five wells bored with cork borer with the addition of Ciprofloxacin to the other well as a positive control and incubated overnight at 37°C. The diameter of the zones of Dahiru et al. – Phytochemical Analysis and Antibacterial Activity of … 283 inhibition was used to determine the antibacterial effects of the extracts. Determination of Minimum Inhibitory Concentration (MIC) The previously described method was adopted to ascertain the MIC of DM following the procedures described by National Committee for Clinical Laboratory Standards (NCCLS) (Lar et al., 2011). Briefly, one mL of concentrations (100 mg/mL, 50 mg/mL, 25 mg/mL, 12.5 mg/mL, and 6.25 mg/mL) of the extract was introduced into seven tubes with 5 mL of Muller-Hinton broth and thoroughly mixed, then 0.1 mL of broth cultures of the test organism and overnight incubation at 37 °C for bacterial growth to be observed. The minimum extract concentration to inhibit bacterial growth was defined as the MIC of the extracts. Determination of Minimum Bactericidal Concentration (MBC) The evaluation of the MBC was carried out by further culturing the test-tubes with no visible growth in the MIC test on Mueller-Hinton agar by spreading 0.1 mL of the inoculum with a sterile loop, followed by overnight incubation at 37 °C. The minimum concentration with no visible growth was defined as the MCB (De & Ifeoma, 2002). Data analysis Data obtained were expressed as mean ± standard error of triplicate determinations' mean (± SEM) and evaluated with Statistical Package for the Social Sciences (SPSS) version 22 Software. RESULTS AND DISCUSSION The phytochemicals detected in the MSBE and ESBE of DM are presented in Table 1. Alkaloids were detected in only the MSBE while saponins and flavonoids were detected in both the MSBE and ESBE. However, steroids, glycosides, and terpenoids were absent in both the MSBE and ESBE. Table 1. Qualitative phytochemical test of MSBE and ESBE of DM. Phytochemicals Inference MSBE ESBE Alkaloids + - Saponins + + Steroids - - Glycosides - - Terpenoids - - Flavonoids + + Note: + = present, - = absent. The phytochemicals quantified in the MSBE and ESBE are shown in Table 2. Alkaloids were quantified up to 16.33 % ±0.88 in the MSBE while saponins quantified up to 31.00 % ±2.31 and 21.67 % ±1.76 for MSBE and ESBE respectively. The concentration of flavonoids in the MSBE was 21.01 % ±2.33 while that of the ESBE was 38.01 % ±1.16. Table 2. Quantitative phytochemical composition of MSBE and ESBE of DM. Phytochemicals Concentration (%) MSBE ESBE Alkaloids 16.33 ±0.88 - Saponins 31.00 ±2.31 21.67 ±1.76 Steroids - - Glycosides - - Terpenoids - - Flavonoids 21.01 ±2.33 38.01 ±1.16 Note: Values are in triplicate determinations ± SEM The antibacterial activities of MSBE and ESBE showed varying degrees of inhibition (figure 1-3). The MSBE exhibited a maximum mean zone of inhibition against S. aureus (21.3 mm ±1.11 at 100 mg/mL) with its least inhibitory effect observed against E. coli (6.5 mm ±0.77) at 25 mg/mL. The extracts were ineffective against all the isolates at 6.25 mg/mL (figure 1-3). The ESBE demonstrated a maximum mean zone of inhibition against S. typhi (19.1 mm ±2.01 at 100 mg/mL concentrations) with no effect at 12.5 mg/mL and 6.25 mg/mL against E. coli and 6.25 mg/mL against both the S. aureus and S. typhi. 284 Biology, Medicine, & Natural Product Chemistry 12 (1), 2023: 281-288 Figure 1. ZI of S. aureus. Figure 2. ZI of S. typhi. Figure 3. ZI of E. coli. The inhibitory effects of MSBE and ESBE of DM are displayed in Table 3. MIC values for the MSBE and ESBE are between 100 mg/mL to 3.125mg/ml for S. aureus, S. typhi, and E. coli. S. typhi was the most sensitive to the MSBE and ESBE at 12.5 mg/mL and 25 mg/mL respectively. 21,3 17 12,5 9,5 18,8 16,4 12,6 8,5 40 0 5 10 15 20 25 30 35 40 45 100 50 25 12,5 6,25 Z o n e o f In h ib it io n ( m m ) Concentrations (mg/mL) M.E = Methanol Extract E.E = Ethylacetate extract C.F = Ciprofloxacin 19,8 14,5 9,5 7 19,1 15 11,4 7,4 35 0 5 10 15 20 25 30 35 40 100 50 25 12,5 6,25 Z o n e o f In h ib it io n ( m m ) Concentrations (mg/mL) M.E = Methanol Extract E.E = Ethylacetate extract C.F = Ciprofloxacin 12,1 9,5 6,5 13,2 10,4 7,2 25 0 5 10 15 20 25 30 100 50 25 12,5 6,25 Z o n e o f In h ib it io n ( m m ) Concentrations mg/mL M.E = Methanol Extract E.E = Ethylacetate extract C.F = Ciprofloxacin Dahiru et al. – Phytochemical Analysis and Antibacterial Activity of … 285 Table 3. MIC of MSBE and ESBE. Concentrations (mg/mL) S. aureus S. typhi E. coli M.E E.E M.E E.E M.E E.E 100 - - - - - - 50 - -* - - - -* 25 - + - -* -* + 12.5 -* + -* + + + 6.25 + + + + + + 3.125 + + + + + + Note: M.E= Methanol extract, E.E= Ethylacetate extract, * = MIC value, + = Turbidity , - = No turbidity Table 4 displays the results of the MBC which reveal the minimum extract concentration needed to fully kill the bacterial isolates. A concentration of 12.5 mg/mL was required to kill S. typhi for the MSBE While 25 mg/mL was effective for the ESBE. Table 4. MIC of MSBE and ESBE. Test organism MBC (mg/mL) M.E E.E S. aureus 12.0 ±1.34 25.0 ±2.33 S. typhi 12.5 ±1.12 25.0 ±1.89 E. coli 25.0 ±2.21 50.0 ±2.62 Note: Values are in triplicate determinations ± SEM, M.E= Methanol extract, E.E= Ethylacetate extract Discussion The result indicated a variation in the presence of concentrations of phytochemicals in the MSBE and ESBE of DM which might be the difference in the polarity of the two solvents as the extraction process depends on the ability of the solvents to penetrate the extracts and solubilize the phytochemicals. Thus, different solvents extract different phytochemicals with variable concentrations (Aboshora et al., 2014). This might also account for the detection of alkaloids in the MSBE as they are more polar. Flavonoids were previously reported in the ethyl acetate fraction of stem bark of DM with the absence of alkaloids and saponins which were all absent in the methanol extract including flavonoids (Ibrahim et al., 2021). Our report agrees with a previous report (Abdullahi et al., 2021; Mu'Azu et al., 2022) for alkaloids, saponins, and flavonoids detection in MSBE of DM. The phytochemicals detected in our study were previously reported to exert antibacterial effects (Akinpelu et al., 2014; Donadio et al., 2021; Liu et al., 2020; Yan et al., 2021). Alkaloids exert their antibacterial activities through the inhibition of bacterial cell walls, protein, nucleic acid, metabolic pathways, and by changing cellular membrane permeability (Yan et al., 2021). Another mechanism of action for flavonoids (indole) was reported to be by inhibiting efflux pumps, biofilm formation, filamentous temperature-sensitive protein Z, and pyruvate kinase (Liu et al., 2020). The positions of the nitrogen and methylenedioxy of isoquinoline were previously implicated in the antibacterial activity of the alkaloids (Qing et al., 2017). Saponin fractions of Erythrophleum suaveolens exhibited antibacterial effects against some gram-positive cocci and gram-negative organisms indicating a good source of antibiotics (Akinpelu et al., 2014). Flavonoids demonstrated antibacterial activity by inhibiting biofilm formation and attachment, nucleic acid synthesis, energy metabolism, membrane properties, and function, reducing pathogenicity (Xie et al., 2015). Additionally, flavonoids further exhibit antibacterial effects by binding to microbial proteins required for basic cellular functions (Donadio et al., 2021). The antibacterial effects of the MSBE and ESBE were measured at 100, 50, 25, 12.5, and 6.25 mg/mL (figure 1-3). Both of the extracts demonstrated an antibacterial effect against the test organism with the highest mean ZI of 21.3 mm ±1.11 observed against S. aureus at 100 mg/mL by the MSBE. A similar result showed the superior effectiveness of the MSBE against S. aureus against the ESBE (Tiwari et al., 2011). The ZI difference might be attributed to the solvent polarity which influences the types and concentrations of the phytochemical extracted, thus influencing the antibacterial effects of the extracts (Aboshora et al., 2014; Gomashe et al., 2014). The antibacterial effects demonstrated by DM might be attributed to growth- inhibiting phytochemicals such as flavonoids which were reported to possess antibacterial potentials (Sanusi et al., 2022). Although in the traditional and folkloric practice the primary solvent used for extraction is water for preparation, extraction with organic solvents exhibits superior antimicrobial effects compared to aqueous extracts (Gomashe et al., 2014). The best practice for the extraction of broad-spectrum antimicrobial compounds employs alcohol as an extraction solvent as seen in the present study (Gomashe et al., 2014). The effective concentration of the ESBE was against the S. typhi as presented in Table 1. The nature of the Gram-negative bacteria cell wall might contribute to the resistance of the organism to the extract by limiting the penetration of the extract to affect its action due to the thin lipopolysaccharide exterior membrane (Biswas et al., 2013). However, the mesh-like peptidoglycan layer of Gram-positive bacteria allows for permeability and access to the extract (Malanovic & Lohner, 2016). The MIC describes the minimum concentration of antimicrobial agent needed to inhibit microbial growth though not applicable in clinical practice for the determination of antibiotic dosage for a patient. However, it is applicable in the determination of the effective target antidiabetic with a lower chance of developing resistance (Wiegand et al., 2008). The inhibitory effect of methanolic and ethyl acetate extracts DM was observed to be between 3.125mg/ml to 100 mg/ml for S. aureus, S. typhi, and E. coli with S. typhi 286 Biology, Medicine, & Natural Product Chemistry 12 (1), 2023: 281-288 being the most sensitive. This variation in MBC values might be attributed to the phytoconstituents of the extract which vary due to solvent difference, thus, exhibiting inferior different inhibitory and bactericidal effects to the standard (Aboshora et al., 2014; I. Abubakar & A. Usman, 2016; Gomashe et al., 2014; Sanusi et al., 2022). Similar results to the present study were reported on the phytoconstituents and antibacterial effect of stem bark extracts of DM (Sanusi et al., 2022). CONCLUSION The study investigated the phytoconstituents and antibacterial effects of D. microcarpum which exhibited antibacterial effects against E. coli, S. typhi, and S. aureus with notable inhibitory and bactericidal effects. The antibacterial capabilities of D. microcarpum might be due to the phytochemical components of the plants and might be a potential source of a novel antibiotic against antibiotic-resistant bacteria. Acknowledgments: The authors extend their appreciation to the Department of science laboratory technology, Adamawa State Polytechnic Yola, for institutional support. Authors’ Contributions: All authors contributed to the design and writing of the research. S.A.P. collected the plant sample and carried out the extraction while D.M.M. carried out the phytochemical analysis. A.A.M. carried out the antibacterial study. Competing Interests: The authors declare that there are no competing interests. Funding: The authors declare that there was no funding for the research REFERENCES Abdullahi, A. R., Malami, S., & Bichi, L. A. (2021). In vivo antiplasmodial activity of detarium microcarpum (fabaceae) stem bark extract. Thrita, 10(1), 1-9. 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