E-ISSN: 2814 – 1822; P-ISSN: 2616 – 0668
ORIGINAL RESEARCH ARTICLE
*1,2Lawal, S. M., 2Inabo, H. I., 2Ella, E. E. and 3Okubanjo, O. O.
1Department of Microbiology, Faculty of Science, Kaduna State University, Kaduna, Nigeria
2Department of Microbiology, Faculty of Life Sciences, Ahmadu Bello University, Zaria, Nigeria
3Department of Parasitology, Faculty of Veterinary Medicine, Ahmadu Bello University, Zaria, Nigeria
The development of resistance by trypanosomes to existing trypanocidal drugs necessitates the need to search for safer and more effective alternative drugs with a broad spectrum of activity. Hence, this study was carried out to evaluate the in vitro anti-trypanosomal activity of ethanolic and n-hexane extracts of Hymenocardia acida stem bark against Trypanosoma congolense and Trypanosoma brucei brucei. The stem bark of H. acida was collected, identified, dried and then ground into fine powder. The powdered H. acida stem bark was extracted successively using n-hexane and ethanol to obtain n-hexane and ethanolic extracts, respectively. Phytochemical screening of the extracts was carried out using standard procedure. In vitro, anti-trypanosomal activity of different concentrations of the stem bark extracts (2.5 mg/mL to 40.0 mg/mL) were determined using motility assay with diminazeneaceturate (100 µg/mL) and phosphate buffered saline used as positive and negative controls, respectively. Results of the phytochemical screening revealed that flavonoids, alkaloids, saponins, and tannins were present in the ethanolic and n-hexane extracts. In vitro, the anti-trypanosomal activity of ethanolic and n-hexane extracts was observed against T. brucei brucei and T. congolense. Cessation of trypanosome motility was observed after 30 and 50 minutes of exposure to 2.5 mg/mL of ethanolic extract and n-hexane extract, respectively, for both T. brucei brucei and T. congolense. In conclusion, ethanolic and n-hexane extracts of H. acida exhibited anti-trypanosomal activity against Trypanosoma brucei brucei and Trypanosoma congolense. Hence, the plant could serve as a source of new trypanocidal drugs.
Keywords: Anti-trypanosomal activity, Extracts, H. acida, T. brucei brucei, T. congolense
Trypanosomiasis is a protozoan parasitic disease caused by species of trypanosomes, which are blood and tissue-dwelling unicellular protozoa. Trypanosomes have a complex life cycle that alternates between the insect vector (tsetse fly) and the host (mammals) (Ogbadoyi et al., 2011; Sani et al., 2018). Trypanosoma brucei brucei and Trypanosoma congolense are protozoa that cause trypanosomiasis in cattle and other domestic animals. It is transmitted by tsetse flies belonging to the family Glossinidae and Genus Glossina. In cyclical transmission, the trypanosomes multiply actively in the tsetse flies (Sani et al., 2018).
Trypanosomiasis mostly affects poor populations that live in the remote areas of Africa. Trypanosomiasis is usually fatal especially when not treated. Travelers are at risk of being infected when they travel in tsetse fly prevalent regions. The disease is generally not commonly found in urban areas. However, there have been reports of some cases in suburban areas, especially in trypanosomiasis-endemic countries (Simarro et al., 2011). Occurrence of sleeping sickness is restricted to only 36 countries in sub-Saharan Africa where tsetse flies that can transmit trypanosomiasis are found. Estimates by the WHO Expert Committee in 1995 project that 60 million individuals were at risk, and there are approximately 300,000 new cases annually in Africa, with less than 30,000 diagnosed and treated cases (WHO, 2012; Madaki et al., 2016).
Plants have served as a remedy for illnesses since pre-historical times, and the use of medicinal plants as phytomedicines is increasing globally. Plants are vital in pharmacological research as well as in drug development. They serve as a reservoir of bioactive compounds; hence, they can directly serve as therapeutic agents; so also, plants serve as starting materials in the synthesis of drugs and as models for pharmacologically bioactive compounds (Deepika et al., 2016).
Plants are considered valuable sources of natural therapeutic agents with promising potential for the treatment of infectious diseases and with fewer side effects compared to synthetic drugs. In the past three decades, several studies have been conducted focusing on the antimicrobial activity of plants. Research on medicinal plants is a re-emerging health aid and is fueled by the increasing cost, adverse drug reactions, serious side effects, and toxicity of synthetic drugs, as well as the prospect of novel drugs derived from plants (Ehiagbonare and Onyibe, 2008).
The fight against trypanosomiasis relies majorly on vector control and chemotherapies. The effectiveness of the chemotherapies and their safety are a source of concern because of the side effects of the drugs and the resistance developed by trypanosomes to the drugs (Yusuf et al., 2012; Madaki et al., 2016). These dilemmas call for continued efforts to develop novel bioactive drugs for the treatment of trypanosomiasis.
Hymenocardia acida has been used in traditional medicine to treat various illnesses in Nigeria and some African countries. It is distributed widely within the savanna region of Nigeria. It is known as "Heartfruit" in English, "janyaro" among Hausas, "yawasatoje" among Fulanis, "ikalaga" among Igbos, "Orunpa” among Yorubas and “Enache” among Idomas (Haruna et al., 2017; Sabo et al., 2017), “ii-kwarto” in Tiv, "emela" in Etulo, "Uchuo" in Igede (Agishi et al., 2004), "enanche" in Idoma (Abu and Uchendu, 2011). Decoction of its stem bark or powders of its roots is used in the treatment of fever, diarrhoea, jaundice, dysentery, muscle pains, and sexual incapacity (Sabo et al., 2017).
In Nigeria, trypanosomiasis is considered a re-emerging disease, causing a major, clinically important disease in small ruminants and spreading to zones designated previously as tsetse-free (Majekodunmi et al., 2013; Nwodo et al., 2015).
The chemotherapy of trypanosomiasis is confronted with problems of unavailability of drugs, resistance to available anti-trypanosomal agents, unacceptable toxicity, and long treatment protocols. The rural poor in Africa are the worst hit by the disease, and without adequate treatment, it is fatal (Ogbadoyi et al., 2011).
Despite major advances in drug development in recent decades, essential medicines used in the treatment of trypanosomiasis and other diseases that affect the world’s poor are either too expensive, no longer produced, highly toxic, or ineffective, hence the need to develop new treatments for these diseases (Johnson and Omoniwa, 2014). Hymenocardia acida has been reported to contain a variety of phytochemicals and possess antimicrobial activity. Hence, this study aimed to assess the anti-trypanosomal activity of H. acida extracts.
Stem bark samples of Hymenocardia acida were collected within the environs of Zaria, Kaduna State, Nigeria, and identified at the herbarium, Department of Botany, Ahmadu Bello University, Zaria. The leaves and stems were used for identification, while the stem bark was used in the extraction and bioassay for in vitro anti-trypanosomal activity.
The stem bark of Hymenocardia acida was cleaned, washed, air dried at ambient room temperature (28 oC – 30oC) for seven days, pulverized, ground to fine powder, and stored at room temperature in air-tight containers until required. The powdered stem bark of Hymenocardia acida was macerated with n-hexane and ethanol to obtain n-hexane and ethanolic extracts, respectively. One hundred grams (100 g) of the powdered material was then macerated with 1000 mL of n-hexane for 72 hours, filtered, and the filtrate was evaporated using a rotary evaporator to dryness in a water bath set 50 oC to obtain n-hexane extract. The residue was macerated with 1000 mL of ethanol for 72 hours, filtered, and the filtrate was evaporated using a rotary to dryness in a water bath set at 50 oC to obtain ethanolic extract (Simorangkir et al., 2019).
The phytochemical constituents of the extracts were determined as described by Yadav and Agarwala (2011), Wadood et al. (2013), and Chechet et al. (2018). The extracts were screened for the presence of flavonoids, alkaloids, terpenoids, tannins, steroids, saponins, and anthraquinones.
Five different concentrations (2.5, 5.0, 10.0, 20.0and 40.0 mg/mL) of the ethanolic and n-hexane stem bark extracts were prepared using phosphate-buffered saline (PBS) as diluent. Ten microliters (10µL) of each concentration was mixed with 60 μL of trypanosome-infected blood in wells of microtitre plates, and then the mixture was incubated for 10 minutes at 37oC. Ten microliters (10 μL) of PBS mixed with 60 μL of trypanosome-infected blood served as negative, while for positive controls, 10 μLof diminazene aceturate (100 µg/mL) was used. The trypanosomes were then observed microscopically (× 400magnification) for drop-in motility or cessation of motility at 5-minute intervals for 60 minutes (Abu et al., 2009).
Results of the phytochemical screening reveal the presence of flavonoids, alkaloids, terpenoids, saponins, tannins, steroids, anthraquinones, carbohydrates, and cardiac glycoside in the ethanolic extract of H. acida stem bark. However, terpenoids, steroids, anthraquinones, and cardiac glycoside were not detected in the n-Hexane extract of H. acida stem bark (Table 1).
Table 2 shows the in vitro anti-trypanosomal activity of Hymenocardia acida ethanolic and n-hexane extracts against Trypanosoma brucei brucei. The ethanolic extract exhibited higher activity against T. brucei brucei, with all the parasites dead after 30 minutes of exposure. Parasite exposure to n-hexane extract was still active after 30 minutes of exposure. Diminazeneaceturate at 100 µg/mL resulted in cessation of motility within 10 minutes of exposure. Trypanosomes exposed to phosphate-buffered saline were active even after 60 minutes of exposure.
In vitro anti-trypanosomal activity of Hymenocardia acida extracts against Trypanosoma congolense is shown in Table 3. At 2.5 mg/mL, ethanolic extract and n-hexane killed all the trypanosomes after 30 and 50 minutes of exposure, respectively. Exposure of the trypanosomes to Diminazeneaceturate at 100 µg/mL resulted in cessation of motility within 10 minutes of exposure. Trypanosomes exposure to phosphate-buffered saline did not result in motility cessation even after 60 minutes of exposure.
| Phytochemical Constituents | Ethanolic Extract | n-Hexane Extract |
|---|---|---|
| Flavonoids | + | + |
| Alkaloids | + | + |
| Terpenoids | + | - |
| Tannins | + | + |
| Steroids | + | - |
| Saponins | + | + |
Anthraquinones Carbohydrate Cardiac glycoside |
+ + + |
- + - |
Key: + = present
- = absent
| Extracts | Concentrations (mg/mL) | Time of Exposure (Minutes) / Motility 5 10 15 20 25 30 35 40 45 50 55 60 |
|||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| -ve control (PBS) | 5+ | 5+ | 5+ | 5+ | 5+ | 5+ | 4+ | 4+ | 4+ | 4+ | 4+ | 3+ | |
| +ve control DA(100µg/mL) | 2+ | * | |||||||||||
| Ethanolic | 2.5 | 5+ | 4+ | 4+ | 3+ | 2+ | * | - | - | - | - | - | - |
| 5.0 | 5+ | 4+ | 4+ | 3+ | 2+ | * | - | - | - | - | - | - | |
| 10.0 | 4+ | 3+ | 2+ | 3+ | * | - | - | - | - | - | - | - | |
| 20.0 | 4+ | 3+ | 2+ | 1+ | * | - | - | - | - | - | - | - | |
| 40.0 | 4+ | 2+ | 1+ | 1+ | - | - | - | - | - | - | - | - | |
| n-Hexane | 2.5 | 5+ | 5+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 3+ | 2+ | 2+ |
| 5.0 | 5+ | 5+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 3+ | 3+ | 2+ | 2+ | |
| 10.0 | 5+ | 5+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 3+ | 3+ | 2+ | 2+ | |
| 20.0 | 5+ | 5+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 3+ | 3+ | 2+ | 2+ | |
| 40.0 | 5+ | 5+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 3+ | 3+ | 2+ | 2+ | |
Key: 5+ = Extremely active; 4+ = very active; 3+ = slowly active; 2+ = sluggish; + = slow ;* = motility ceases;-ve control = negative control; +ve control = positive control; PBS = phosphate buffered saline; DA = Diminazene aceturate
| Extracts | Concentrations (mg/mL) | Time of Exposure (Minutes) / Motility 5 10 15 20 25 30 35 40 45 50 55 60 |
|||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| -ve control (PBS) | 5+ | 5+ | 5+ | 5+ | 5+ | 5+ | 4+ | 4+ | 4+ | 4+ | 4+ | 3+ | |
| +ve control DA(100µg/mL) | 2+ | * | |||||||||||
| Ethanolic | 2.5 | 3+ | 2+ | 2+ | 2+ | 1+ | * | - | - | - | - | - | - |
| 5.0 | 3+ | 2+ | 2+ | 2+ | 1+ | * | - | - | - | - | - | - | |
| 10.0 | 3+ | 2+ | 2+ | 1+ | * | - | - | - | - | - | - | - | |
| 20.0 | 2+ | 2+ | 2+ | 1+ | * | - | - | - | - | - | - | - | |
| 40.0 | 2+ | 2+ | 1+ | * | - | - | - | - | - | - | - | - | |
| n-Hexane | 2.5 | 5+ | 5+ | 4+ | 4+ | 3+ | 3+ | 2+ | 1+ | 1+ | * | - | - |
| 5.0 | 5+ | 5+ | 4+ | 3+ | 3+ | 3+ | 2+ | 1+ | 1+ | * | - | - | |
| 10.0 | 4+ | 4+ | 3+ | 3+ | 3+ | 3+ | 1+ | 1+ | * | - | - | - | |
| 20.0 | 3+ | 3+ | 3+ | 2+ | 2+ | 2+ | 1+ | 1+ | * | - | - | - | |
| 40.0 | 2+ | 2+ | 2+ | 2+ | 1+ | 1+ | * | - | - | - | - | - | |
Key: 5+ = Extremely active; 4+ = very active; 3+ = slowly active; 2+ = sluggish; + = slow ; * = motility ceases; -ve control = negative control; +ve control = positive control; PBS = phosphate buffered saline; DA = Diminazeneaceturate
Phytochemical compounds detected in the ethanolic extract of H. acida stem bark were saponins, tannins, flavonoids, terpenoids, steroids, anthraquinones, carbohydrates, cardiac glycoside, and alkaloids. Terpenoids, steroids, anthraquinones, and cardiac glycoside were absent in the n-hexane extract. The variation observed in the phytochemical constituents of the extracts might be linked to differences in the polarity of the solvents used in the extraction. These phytochemicals have been reported to exhibit trypanocidal or trypanosomatic activity either as individual or in synergy (Mergia et al., 2014), and they act on a single site or multiple sites that are associated with the physiological process trypanosomes (Maikai, 2011). The anti-trypanosomal activity of flavonoids might be linked to their ability to serve as a scavenger of free radicals and metal chelators (Mergia et al.,2014). Terpenoids exert their anti-trypanosomal activity by reacting with sulfur-containing cellular components, leading to the production of aldehyde-thiol adducts and subsequently decreasing the buffering agents, leading to the creation of oxidative stress in the trypanosome (Nibret and Wink, 2010). Saponins exert their anti-trypanosomal activity through stimulation of cell death (Johnson and Omoniwa, 2014).
Similar to this observation, the presence or absence of anthraquinones and cardiac glycosides in stem bark extract of Hymenocardia acida have been observed to vary based on the solvent of extraction. Usman et al.(2021) reported that anthraquinones and cardiac glycosides were absent in the methanol extract of H. acida stem bark. However, the presence of glycosides and the absence of anthraquinones in the hydroethanolic stem bark of Hymenocardia acida was reported by Abu et al. (2011). Anthraquinones and cardiac glycosides were found in the stem bark of Hymenocardia acida by Iyadi et al. (2003).
In vitro anti-trypanosomal activity of the extracts observed against T. brucei brucei and T. congolense in this study implies that the extracts contain compounds with trypanocidal activity. This was demonstrated by complete cessation or reduction in motility of trypanosome when exposed to different concentrations of the extracts. The motility of trypanosomes has been reported to constitute a reliable indicator of trypanosome viability, where complete cessation or reduction in trypanosome motility compared to the control serves as an index of trypanocidal activity (Abdeta et al., 2020). Inhibition or killing of the trypanosomes when exposed to extracts is indicated by loss of motility. This suggests that the extract interferes with essential trypanosome functions or processes.
In vitro anti-trypanosomal activity of ethanolic extract of H. acida stem bark was observed against T. brucei brucei and T. congolense. Similar to this finding, in vitroantitrypanosomal activity of ethanolic extract of H. acida stem bark against T. brucei brucei at 40.0 mg/mL – 2.5 mg/mL concentrations was also reported by Abu et al. (2009).
Higher in vitro anti-trypanosomal activity was exhibited by ethanolic extract against T. brucei brucei and T. congolense compared to n-hexane extract. The difference observed in the anti-trypanosomal activity of the extracts might be due to differences in the phytochemical constituents of the extracts as well as the quantity of the phytochemicals.
Ethanolic and n-hexane extracts of H. acida stem bark exhibited invitro anti-trypanosomal activity against Trypanosoma brucei brucei and Trypanosoma congolense. These extracts can be exploited for novel anti-trypanosomal drugs.
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