E-ISSN: 2814 – 1822; P-ISSN: 2616 – 0668
ORIGINAL RESEARCH ARTICLE
1M. Mukhtar
,2
HM Adamu, 2AM Shibdawa, and 2DA
Ajiya,
1Department of Science Laboratory Technology, Binyaminu Usman Polytechnic Hadejia Jigawa State, Nigeria
2Department of Chemistry, Abubakar Tafawa Balewa University. Bauchi, Nigeria
Correspondence: kabirummukhtar@gmail.com
The increasing resistance to existing antimalarial drugs has elevated malaria to a critical global health concern, underscoring the urgent need for novel, safe, and effective therapeutic solutions. This study evaluated the phytochemical composition, antiplasmodial activity, and antibacterial properties of Bauhinia rufescens root extract. The roots were extracted using 85% methanol and screened for phytochemicals using standard methods. Antimicrobial activity was assessed using the Agar Well Diffusion Method, while the antiplasmodial efficacy was tested in Plasmodium berghei-infected male Swiss albino mice at doses of 150, 300, and 600 mg/kg. Phytochemical screening identified alkaloids, flavonoids, tannins, saponins, terpenoids, steroids, phlobatannins, and anthraquinones. The extract exhibited dose-dependent antiplasmodial activity, significantly reducing parasitaemia levels and extending the survival of infected mice. Additionally, antimicrobial assays demonstrated moderate inhibitory effects against Staphylococcus aureus and Escherichia coli. These findings support the traditional use of Bauhinia rufescens in managing malaria and associated infections, highlighting its potential as a promising source for developing multi-target therapies.
Keywords: Antiplasmodial activity, Antimicrobial properties, and Phytochemical analysis
Malaria remains a significant public health challenge in developing nations, contributing substantially to illness and mortality despite global efforts to implement effective control measures, including enhanced vector management systems (Aliyu, 2022). In 2020, there were an estimated 241 million malaria cases and 627,000 deaths across 85 endemic countries, with sub-Saharan Africa (SSA) bearing the greatest burden (WHO, 2021). Nigeria, in particular, accounts for 39% of global malaria deaths among children under five years of age, with an estimated 55 million cases and nearly 90,000 deaths annually (Shekarau et al., 2024). This has highlighted the critical need for improved malaria management strategies.
The 2021 World Malaria Report revealed that the global impact of malaria is more severe than previously estimated and that malaria-related deaths have been underestimated (Mahamat & Kenyatta, 2021). This persistent burden underscores the urgent need for innovative malaria control and treatment solutions, particularly as resistance to widely used antimalarial drugs becomes more prevalent.
The emergence of chloroquine (CQ)-resistant strains of Plasmodium falciparum is a major contributor to the global malaria crisis, driven by the extensive use of CQ for prevention and treatment. The Resistance against chloroquine and other antimalarial drugs significantly complicates disease management in endemic areas (Peters, 1982). The World Health Organization (WHO) recommends artemisinin-based combination therapies (ACTs) such as artemether-lumefantrine (AL) and artesunate-amodiaquine (AS-AQ) as the first-line treatments for uncomplicated Plasmodium falciparum malaria (Ringwald et al., 2005). However, resistant strains of the parasite continue to emerge and spread rapidly (Ross & Fidock, 2019; Takala-Harrison & Laufer, 2015; Wellems & Plowe, 2001). Given the growing prevalence of resistance, medicinal plants are gaining attention as potential sources of novel antimalarial drugs. The effectiveness of quinine and its derivatives, including artemisinin, has spurred interest in plant-based compounds for treating resistant malaria (Olasehinde et al., 2014). As drug resistance to affordable and accessible antimalarials spreads globally, innovative malaria control and treatment approaches are urgently needed.
Bauhinia species (Fabaceae) are frequently referred to as "cow's hoofs." Due to the shape of its leaves. Most tropical nations, particularly those in Africa, Asia, and South America, are home to the species. Typically, bauhinia trees grow 6–12 m tall, with branches that extend 3–6 m from the trunk. The lobed leaves typically measure 10-15 cm in width (Aminu, 2013). In folk medicine, the plant is used for the treatment of gout, gingivitis, diarrhea, dysentery, diabetes, leprosy, and malaria (Compaoré et al., 2011). The extract of Bauhinia was reported to show significant activity against Salmonella spp (Alamin et al., 2021).
This study aims to evaluate the phytochemical composition, antiplasmodial, and antimicrobial effects of Bauhinia rufescens root extract against Plasmodium berghei in a model. The primary goal is to assess the extract's ability to reduce parasitemia and improve survival rates in infected mice. The findings from this study could provide scientific validation for the traditional use of B. rufescens and contribute to the search for new, plant-based antimalarial agents.
Fresh roots of Bauhinia rufescens were collected from the Hadejia-Nguru Wetland in Jigawa State, Nigeria. The plant was authenticated at the Herbarium of Ahmadu Bello University, Zaria, Nigeria, where reference specimens (ABU0900230) were stored for future use. The collected plant materials were washed with deionized water to remove impurities and air-dried for 21 days. Subsequently, the dried materials were pulverized using a mortar and pestle. The pulverized plant bark was then soaked in 85% methanol and filtered using Whatman No. 1 filter paper. The resulting filtrate was dried under reduced pressure at 40°C.
phytochemical tests were conducted on the extract to identify the bioactive constituents of the plant material, following the procedures outlined by Sofowora ( 1993) and Trease & Evans (1989).
The antimicrobial properties of the extracts were evaluated using the agar well diffusion method, following the protocols described by Ara et al., (2012) and Artizzu et al., (1995). A sterile petri dish containing 0.1 ml of various organisms cultured in nutrient broth was filled with melted nutrient agar for bacteria and Sabouraud Dextrose Agar (SDA) for fungi and allowed to set. Using a sterile maize borer, wells were created in the solidified agar. Each well was filled with 0.1 ml of the extract solution at varying concentrations. The plates were left to pre-diffuse for 30 minutes before incubation. Dimethyl sulfoxide (DMSO) served as the negative control, while ketoconazole and Ciprofloxacin were the positive controls for antifungal and antibacterial activities. The bacterial plates were incubated at 37°C for 24 hours, and the fungal plates were incubated at 25°C for 27 hours. The inhibition zones, measured in millimetres, were recorded to assess the antimicrobial activity of each extract.
Thirty-six male swiss albino mice (6-8 weeks) were obtained from the Department of Pharmacology Bayero University Kano. They were randomly divided into six groups, each consisting of six mice. The mice were housed and fed according to the recommended standard (NIH 2007).
The study utilized plasmodium berghei NK65 obtained from Aminu Kano teaching hospital, which is sensitive to chloroquine. Mice were infected with plasmodium berghei for the survey. All the mice used in the study received an intraperitoneal inoculation of 1+107 p. berghei parasitized erythrocytes on day 1. The parasitized erythrocytes were obtained by suitable dilution with 0.9% saline from a donor-infected mouse through cardiac puncture.
With some slight modifications, the approach described by (Akuodor et al., 2011; Peters et al., 2002) was used to evaluate the efficacy of Bauhinia rufescens root extract in treating a condition. In this investigation, a blood suspension (0.2 ml) containing 1x107 Plasmodium berghei was intraperitoneally administered to thirty mice on the first day of the experiment. The experimental animals were split into six groups, with 6 mice in each cage, 72 hours after the inoculation. Bauhinia rufescens root extract was administered in varying quantities to groups 1-3 (180, 300, and 600 mg/kg), while groups 4 and 5 received attermeter (5 ml/kg) and chloroquine (10 ml/kg) as positive controls. Group 6 received 10 millilitres per kilogramme of distilled water as a negative control. There were four, five, and six days of oral therapy. Each mouse's 16mm blood film was produced and dried on the seventh day. The dried films were fixed in methanol and dyed with Giemsa. Later, the parasite density was investigated under a microscope by counting the parasitised red blood cells in ten distinct fields. The average time of the mice in each group over a 30-day period was calculated to estimate each group's mean survival time (in days).
\(parasitemia\ (\%) = (\frac{Number\ of\ parasitized\ red\ blood\ cell}{Total\ Number\ of\ red\ blood\ cell})\) x 100
\[Mean\ survival\ time\ = \frac{Total\ survival\ time\ in\ all\ mice\ in\ the\ group}{Total\ number\ of\ mice\ in\ the\ group}\]
\((\%)inhibition = (\frac{Mean\ parasitemia\ of\ control\ group - Mean\ parasitemia\ of\ treatment\ group}{Total\ Number\ of\ mice\ in\ the\ group})\) x 100
The data obtained were analyzed using the Statistical Package for the Social Sciences (SPSS) Version 20. Differences between the means were evaluated using one-way analysis of variance (ANOVA), followed by Tukey’s post-hoc test. Values of p < 0.05 were considered statistically significant, and the results were expressed as mean ± standard error of the mean (SEM).
Phytochemical screening of the root bark of methanol extract of Bauhinia rufucens showed the presence of alkaloids, flavonoids, tannins, saponins, terpenoids, steroids, phlobatanins, and anthraquinines
TABLE 1: Zone of inhibition (mm) of crude Bauhinia rufucens root extract at different concentrations (µg/ml) against clinical isolate
| Organism/con. µg/ml | 4000 | 3000 | 1000 | 500 | Cipro | Keto |
|---|---|---|---|---|---|---|
| Styhyloccus aureus | 14.00 | 11.00 | 9.00 | 8.00 | 27.00 | - |
| Streptococcus pyogenes | 11.00 | 10.00 | 8.00 | 0.00 | 24.00 | - |
| Escherichia Coli | 15.00 | 13.00 | 11.00 | 9.00 | 30.00 | - |
| klebsiella pneumoniae | 14.00 | 12.00 | 10.00 | 8.00 | 22.00 | - |
| Candida albicans | 13.00 | 12.00 | 10.00 | 8.00 | - | 20.00 |
| aspergillus fumigatus | 12.00 | 11.00 | 9.00 | 0.00 | - | 18.00 |
Table 2: Effect of Methanol Root Extract of Bauhinia rufucenson Parasitaemia Level of Plasmodium berghei Infected Mice in Rane’s Test
| Treatment | Dose (mg/kg) | Parasitaemia level | % Inhibition |
|---|---|---|---|
|
10 mL/kg |
|
|
|
150 |
|
|
|
300 |
|
|
|
600 |
|
|
|
10 |
|
|
|
5 |
|
|
Values are presented as Mean ± S.E.M., * = p<0.001 as compared to distilled water group – One way ANOVA followed by Tukey’s post hoc test, n = 6, MEAR = Methanol root extract of Bauhinia rufucens
Table 3: Effect of Methanol Root Extraction Survival Time of Plasmodium berghei
Infected Mice in Rane’s Test
| Treatment | Dose (mg/kg) | Survival time (days) |
|---|---|---|
|
10 mL/kg |
|
|
150 |
|
|
300 |
|
|
600 |
|
|
10 |
|
|
5 |
|
Values are presented as Mean ± S.E.M., No significant difference when compared to the distilled water group – One way ANOVA followed by Tukey’s post hoc test, n = 6, MEAR = Methanol root extract of Bauhinia rufucens
The phytochemical screening of Bauhinia rufescens revealed a rich diversity of bioactive compounds, including alkaloids, flavonoids, tannins, saponins, terpenoids, steroids, phlobatannins, and anthraquinones. These results align with those of Garbi et al., (2015), who identified flavonoids, tannins, triterpenes, saponins, and alkaloids in the leaves of Bauhinia rufescens. Such secondary metabolites are well-documented for their medicinal properties, including antimicrobial, antimalarial, and other therapeutic activities (Hassan et al., 2009; Hostettmann & Marston, 1995).
The antimicrobial activity of the root extract highlighted its therapeutic potential. The extract exhibited significant inhibition against Staphylococcus aureus (14 mm zone of inhibition at 4000 µg/ml), indicating its efficacy against Gram-positive bacteria commonly associated with skin and soft tissue infections. In contrast, Streptococcus pyogenes showed lower sensitivity, with no inhibition observed below 500 µg/ml. This variability may be attributed to differences in bacterial cell wall structures or efflux mechanisms. The extract also demonstrated activity against Gram-negative pathogens such as Escherichia coli (15–9 mm) and Klebsiella pneumoniae (14–8 mm), suggesting potential efficacy against multidrug-resistant infections. These findings corroborate the antimicrobial potential of Bauhinia rufescens, as reported by (Hassan et al., 2009).
The antifungal activity of the extract, evident from its inhibition of Candida albicans (13–8 mm) and Aspergillus fumigatus (12 mm at 4000 µg/ml), further supports its broad-spectrum antimicrobial capabilities. This aligns with prior studies on the antimicrobial activity of B. rufescens (Issa et al., 2021) and related species such as B. Purpurea (Negi et al., 2012), reinforcing its therapeutic promise.
Additionally, the methanol root extract (MEAR) exhibited significant antiplasmodial activity. In Plasmodium berghei-infected mice, MEAR showed dose-dependent parasitaemia reduction, achieving 63.30% inhibition at 600 mg/kg, although this was lower than chloroquine (93.07%) and artemether (90.45%). The antimalarial activities observed in Bauhinia rufucens extract may be attributed to various phytochemicals, including alkaloids, terpenes, flavonoids, xanthones, anthraquinones, and phenolic compounds, which are well-documented for their bioactivities (Batista et al., 2009; Mazid et al., 2011). However, these findings differ from previous reports, such as the study by Nadège Bonkian et al. (2017), which indicated that Bauhinia rufescens Lam. lacks significant antiplasmodial activity. In this study, MEAR-treated mice exhibited extended survival times, with a maximum mean survival time (MST) of 18.83 days at 600 mg/kg compared to 13.83 days in the control group. Although the increase was not statistically significant, the extract's potential to reduce parasitic load and mitigate malaria pathology is noteworthy. This prolonged MST aligns with findings by Chutoam & Klongthalay (2015), who reported significantly extended MST in Bauhinia strychnifolia treated mice compared to untreated controls. These findings highlight the extract's potential as a complementary therapeutic agent in malaria management.
In conclusion, Bauhinia rufescens root extract demonstrates significant potential as a natural therapeutic agent, offering both antiplasmodial and antimicrobial benefits. The dose-dependent reduction of parasitemia in Plasmodium berghei-infected mice and its ability to inhibit bacterial strains linked to secondary infections underscore the plant's dual bioactivity. The phytochemicals identified in the extract, including alkaloids, flavonoids, tannins, and saponins, likely contribute to its effectiveness. These findings support the traditional use of Bauhinia rufescens in malaria treatment and suggest that the plant could be a valuable source for developing novel, multi-target therapies against malaria, particularly in the face of growing resistance to conventional antimalarial drugs. Further studies on the isolation of bioactive compounds and their mechanisms of action are recommended to fully realize their therapeutic potential.
Akuodor, G., Essien, A., & Ibrahim, J. (2011). Phytochemi cal and antimicrobial properties of the methanol extracts of Bombax buonopozense leaf and root. Asian J Med Sci 2011, 2(2), 190–194. [Crossref]
Alamin, N. A., Koya, M. S., & Mus, M. (2021). Evaluation of Antimicrobial activity of Leaves extracts of Bauhinia rufescens Lam . ( Fabaceae ) on Salmonella Spp . isolated from Chicken intestine. 8(9), 624–633.
Aliyu, I. A. (2022). Geospatial Analysis of Malaria Prevalence among Children Under Five Years in Jigawa State , North. 1–18. [Crossref]
Aminu, M., & Sirat, H. (2013). Antimicrobial , antityrosinase and brine shrimp lethality test of Bauhinia rufescens L am ( F abaceae ). 1(2), 135–140. [Crossref]
Ara, I., Bukhari, N. A., Solaiman, D., & Bakir, M. A. (2012). Antimicrobial effect of local medicinal plant extracts in the Kingdom of Saudi Arabia and search for their metabolites by gas chromatography-mass spectrometric (GC-MS) analysis. Journal of Medicinal Plants Research, 6(45), 5688–5694. [Crossref]
Artizzu, V., Bonsignore, L., Cottiglia, F., & Loy, G. (1995). Studies of the diuretic and antimicrobial activity of Cynodon dactylon essencial oil. Fitoterapia, 174–175.
Batista, R., De Jesus Silva, J. A., & De Oliveira, A. B. (2009). Plant-derived antimalarial agents: New leads and efficient phytomedicines. Part II. Non-alkaloidal natural products. Molecules, 14, 3037–3072. [Crossref]
Chutoam, P., & Klongthalay, S. (2015). Effect of Crude Leaf Extract of Bauhinia strychnifolia in BALB/c Mice Infected with Plasmodium berghei. Malaria Control & Elimination, s1, 1–5. [Crossref]
Compaoré, M., Lamien, C. E., Lamien-Meda, A., Vlase, L., Kiendrebeogo, M., I., & Onescu, C. (2011). Antioxidant, xanthine oxidase and lipoxygenase inhibitory activities and phenolics of Bauhinia rufescens Lam. (Caesalpiniaceae). , Nat. Prod. Res., 26, 1069-74. [Crossref]
Garbi, I. M., Kabbashi, S. A., Osman, E. E., Dahab, M. M., Koko, W. ., & Ahmed, I. F. (2015). Antioxidant activity and phytochemical screening of methanolic leaves extract of Bauhinia rufescens (Lam). International Invention Journal of Biochemistry and Bioinformatics., 3(23), 27.
Hassan, H. S., Sule, M. I., Usman, M., & Ibrahim, A. (2009). Preliminary Phytochemical and Antimicrobial Screening of the Stem Bark Extracts of Bauhinia Rufescens Lam Using Some. Bayero Journal of Pure and Applied Sciences, 2(2), 53–55. [Crossref]
Hostettmann, K., & Marston, A. (1995). Saponins. Cambridge University Press. [Crossref]
Issa, E., Fouda Abderrazzack, A., Anani, K., & Yaovi, A. (2021). Antimicrobial Properties of the Hydroethanolic Extract of &lt;i&gt;Bauhinia rufescens&lt;/i&gt; L. and &lt;i&gt;Euphorbia hirta&lt;/i&gt; L., Two Plants of the Traditional Chadian Pharmacopoeia. Journal of Diseases and Medicinal Plants, 7(2), 30. [Crossref]
Mahamat, H. E. M. F., & Kenyatta, H. E. P. U. (2021). Malaria progress report. 1–2.
Mazid, M., Khan, T., & Mohammad, F. (2011). Role of secondary metabolites in defense mechanisms of plants. Biol. Med., 3, 232–249.
Nadège Bonkian, L., Yerbanga, R. S., Traoré Coulibaly, M., Lefèvre, T., Sangaré, I., Ouédraogo, T., Traore, O., Bosco Ouédraogo, J., Robert Guiguemde, T., Roch Dabiré, K., Serge Yerbanga, R., Traoré, M., Lefevre, T., Traoré, O., Robert Guiguemdé, T., & Rakiswendé Serge Yerbanga, C. (2017). Plants against Malaria and Mosquitoes in Sahel region of Burkina Faso: An Ethno-botanical survey. International Journal of Herbal Medicine, 5(3). https://hal.science/hal-02411013
National Institutes of Health. (2007). Guidelines for the care and use of laboratory animals (8th ed.). National Academies Press.
Negi, B. S., Dave, B. P., & Agarwal, Y. K. (2012). Evaluation of Antimicrobial Activity of Bauhinia purpurea Leaves Under In Vitro Conditions. Indian Journal of Microbiology, 52(3), 360–365. [Crossref]
Olasehinde, G. I., Ojurongbe, O., Adeyeba, A. O., Fagade, O. E., Valecha, N., Ayanda, I. O., Ajayi, A. A., & Egwari, L. O. (2014). In vitro studies on the sensitivity pattern of Plasmodium falciparum to anti-malarial drugs and local herbal extracts. Malaria Journal, 13(1), 1–7. [Crossref]
Peters, W. (1982). Antimalarial drug resistance: an increasing problem. Br Med Bull, 32, 187–192. [Crossref]
Peters, W., Fleck, S., Robinson, B., Stewart, L., & Jefford, C. (2002). The chemotherapy of rodent malaria. LX. The importance of formulation in evaluating the blood schizontocidal activity of some endoperoxide antimalarials. . . Ann Trop Med Parasitol, 96(6), 559–573. [Crossref]
Ringwald, P., Shallcross, L., Miller, J. M., & Seiber, E. (2005). Susceptibility of Plasmodium falciparum to Antimalarial Drugs: Report On Global Monitoring 1996–2004( WHO 2005).
Ross, L. S., & Fidock, D. A. (2019). Elucidating mechanisms of drug-resistant Plasmodium falciparum. Cell Host Microbe, 26, 35–47. [Crossref]
Shekarau, E., Uzoanya, M., Ogbulafor, N., Malaria, S., & Group, W. (2024). Severe malaria intervention status in Nigeria : workshop meeting report. 1–18. [Crossref]
Sofowora A. (1993). Medicinal Plant and Traditional Med. In Africa (2nd ed.). Spectrum Books Ltd.
Takala-Harrison, S., & Laufer, M. K. (2015). Antimalarial drug resistance in Africa: key lessons for the future. Ann. N. Y. Acad. Sci., 1342, 62–67. [Crossref]
Trease, G. E., & Evans, M. C. (1989). Textbook of Pharmacognosy (13th ed.). Bailliere Tindall Ltd.
Wellems, T. E. & Plowe, C. V. (2001). Chloroquine‐resistant malaria. J. Infect. Dis., 184, 770–776. [Crossref]
WHO. (2021). World malaria report 2021.