ORIGINAL ARTICLE Year : 2022  |  Volume : 21  |  Issue : 1  |  Page : 40-45

Anticancer and antibacterial potentials of methanolic extracts of the leaf and stem bark of Afzelia africana

Mathew O Oni1, Olorunjuwon O Bello2, Rachael A Ademola1, Uchechi D Mba1, Vincent O Oni3
1 Department of Microbiology, Adeleke University, Ede, Osun State, Nigeria
2 Department of Biological Sciences, University of Medical Sciences, Ondo City, Ondo State, Nigeria
3 Ekiti State COVID Laboratory, Ekiti State University Teaching Hospital, Ado-Ekiti, Ekiti State, Nigeria

Date of Submission30-Jul-2021Date of Decision13-Oct-2021Date of Acceptance26-Oct-2021Date of Web Publication07-Mar-2022

Correspondence Address:
BSc, MSc, PhD Olorunjuwon O Bello
Department of Biological Sciences, University of Medical Sciences, Ondo City, Ondo State
Nigeria

Source of Support: None, Conflict of Interest: None

Check

DOI: 10.4103/epj.epj_47_21

Context Afzelia africana is one of the most important woody fodder plants in many parts of Africa.
Aims This study evaluated the anticancer and antibacterial potentials of A. africana leaf and stem bark in Nigeria.
Patients and methods The methanolic extracts were filtered and concentrated in vacuo according to standard procedures. The extracts were screened for phytochemical properties in accordance with standard methods. The antibacterial potentials of the plant extracts against 10 selected clinical bacterial isolates were evaluated using the agar well diffusion method. Anticancer potential using Human Embryonic Kidney cells and therapeutic index (TI) of the plant extracts were determined by standard procedures.
Statistical analysis used The χ2 test was used to determine the correlation between the antibacterial activities exerted by different concentrations of the stem bark and leaf extracts.
Results Both plant parts contained alkaloids, anthraquinones, flavonoids, glycosides, saponins, tannins, triterpenes, and xanthoproteins. The anticancer test with Human Embryonic Kidney cells showed that the leaf and stem bark extracts elicited cytotoxic activity. The leaf extract had TI ranging from 0.23 to 0.97 whereas that of the stem bark ranged from 0.24 to 0.75, against the selected test organisms. There was no significant difference (P=0.077) in the TI between the leaf and the stem bark extracts. There were no significant differences between the antibacterial activities exerted by the leaf and stem bark extracts and within the antibacterial activities exerted by the control antibiotics (P=0.073).
Conclusions This study revealed that the stem bark and leaf extracts of A. africana possess anticancer and antibacterial properties.

Keywords: Afzelia africana, antibiotics, anticancer, Fabaceae, phytochemicals, therapeutic index

How to cite this article:
Oni MO, Bello OO, Ademola RA, Mba UD, Oni VO. Anticancer and antibacterial potentials of methanolic extracts of the leaf and stem bark of Afzelia africana. Egypt Pharmaceut J 2022;21:40-5
How to cite this URL:
Oni MO, Bello OO, Ademola RA, Mba UD, Oni VO. Anticancer and antibacterial potentials of methanolic extracts of the leaf and stem bark of Afzelia africana. Egypt Pharmaceut J [serial online] 2022 [cited 2022 May 18];21:40-5. Available from: http://www.epj.eg.net/text.asp?2022/21/1/40/339111   Introduction 

Medicinal plants are extensively employed in the treatment of various diseases and disorders all over the world. Historically, the use of herbal medicine dates back to antiquity. In African traditional medicine, several plant species have been claimed to possess medicinal properties and are therefore employed in the treatment of many ailments [1]. Afzelia africana Sm., an African legume tree in Fabaceae family, is used in traditional medicine for the treatments of pain, malaria, gonorrhea, and leprosy; is known for its socio-economic importance; and is well distributed in West African savannahs and forests [2]. The leaves are used in human feeding and forage for livestock. The wood is overexploited and commercialized for various industrial purposes, whereas the barks have several medicinal properties [3],[4]. The stem bark decoction of A. africana is used for treatments of malaria, rheumatism, paralysis, and constipation. The maceration is used for the treatment of leprosy. The bark is burnt, and its ash is mixed with shea butter for soap, which is used to treat lumbago (back pain). Its root is blended with millet-beer for the treatment of hernias, or the root decoction with pimento is taken as treatment for gonorrhea and stomachache. The mixture of A. africana and Syzygium guineense leaves plus Xylopia fruit is used to treat edema [4]. This study, therefore, was designed to investigate the anticancer and antibacterial properties of the leaves and stem bark of A. africana.

  Materials and methods 

Collection and authentication of plant samples

The fresh leaf and stem bark of A. africana were collected at the herbarium of the Department of Botany, Obafemi Awolowo University, Ile-Ife.

Preparation of methanolic extracts of leaf and stem bark of Afzelia africana

The plant parts were air-dried and separately ground into powder. Each sample material (200 g) was soaked into 80% methanol for 72 h and then filtered. The filtrates were concentrated in vacuo to obtain crude methanolic leaf and stem bark extracts [5].

Phytochemical screening of the plant extracts

The plant extracts were screened for phytochemical constituents according to the methods of Harbones [6], Trease and Evans [7], and Sofowora [8].

Flavonoids: a 5-ml portion of the extract was added and mixed with 0.1 g of metallic zinc and 7 ml of concentrated sulfuric acid. The appearance of red color was indicative of flavonols [6].

Tannins: this was determined using the Braymer’s test. Three drops of 5% ferric chloride were added to a mixture containing 2 ml of the extract and distilled water. The appearance of brownish green or dark-blue coloration indicated the presence of tannins [7].

Alkaloids: the Mayer’s test was used to determine the presence of alkaloids in the extract. A 1-ml portion of the Mayer’s reagent was added to 1 ml of the extract. The production of a whitish or cream colored precipitate indicated the presence of alkaloids [6].

Saponins: 2 g of the sample was added to 20 ml of distilled water and boiled in a water bath and filtered. Five milliliters of distilled water was added to 10 ml of the filtrate and shaken vigorously. The appearance of a stable froth signified the presence of saponins [6],[8].

Steroids: the extract (1 ml) was dissolved in 2.0 ml of chloroform in a test tube, and then 1 ml of conc. H2SO4 was added. Formation of reddish-brown color at the interphase confirms the presence of steroids [8].

Cardiac glycoside: this was determined using the Keller-Kilani test. A 5-ml portion of the sample was mixed with 2 ml of glacial acetic acid containing one drop of ferric chloride solution. This mixture was underlaid with 1 ml of concentrated H2SO4. The appearance of a brown-ring interface was indicative of a deoxysugar characteristic of cardenolides [7].

Triterpenes: a 20-mg portion of the extracts was suspended in 10 ml of chloroform. This was warmed and then filtered. A 4-ml portion of concentrated sulfuric acid was mixed to the chloroform filtrate and mixed properly. The formation of red coloration indicated a positive result.

Xanthoproteins: a few drops of nitric acid were mixed with 1 ml of aqueous extract. Some drops of ammonia solution were added, and the mixture was observed for the formation of reddish or light brown precipitate, indicating the presence of xanthoproteins. Three drops of 10% (v/v) of ammonia solution were introduced to the mixture and observed for the formation of pink, violet, or red coloration in the ammonia layer to signify the presence of xanthoproteins in the extract.

Anthraquinones: a 0.5 g portion of the extract was mixed in 2 ml of diluted sulfuric acid, boiled, and filtered while still hot. About 3 ml of benzene was added to the filtrate, mixed thoroughly, and the benzene layer separated.

Phlobatannins: a 0.5 g portion of the extracts was heated with 10% (v/v) HCl in a boiling water bath. The formation of red precipitate was observed signifying the presence of phlobatannins.

Anticancer test: XTT cytotoxicity assay

The anticancer activity of the plant extracts was tested on Human Embryonic Kidney (HEK) 293 cells as reported by Zanariah et al. [9]. Exactly 100 µl of HEK cell suspension (1×105 cells/ml) was added to the inner walls of a 96-well plate and 200 µl of incomplete medium (Dulbecco’s modified eagle’s medium) to the outer wells. The plates were incubated for 24 h to permit adherence of the cells to the base of the wells. Serial dilution of the plant extracts, which had already been dissolved in dimethyl sulfoxide, the negative control (DMSO), and positive control (Actinomycin D), and with the complete medium (10% fetal bovine serum+Dulbecco’s modified eagle’s medium+penicillin/streptomycin) was done in a 24-well plate. This was carried out to give eight different concentrations of each sample and to obtain a final volume of 1 ml per well. Then, 100 µl of each concentration from the 24-well plate was added to the 96-well plate in triplicate, and there was a triplicate medium control and a DMSO control for each extract. To account for the color of the plant extracts, a reference plate was considered. This plate comprised the media and plant extracts in duplicate without cells. The plates were incubated for 72 h, after which 50 µl of XTT reagent (Roche, Charles Avenue, Burgess Hill RH15, UK) was added to all of the wells, which were incubated for about 2 h and 30 min. The plates were then read on an ELISA plate with KC junior software to read the absorbance at 450 and 690 nm as the reference wavelength.

Source of microorganisms

The microorganisms used for this study, most of which have been implicated in infectious diseases, were collected from National Collection of Industrial Bacteria (NCIB) and Locally Isolated Organisms (LIO). These include Klebsiella pneumoniae (NCIB 418), Escherichia coli (NCIB 86), Pseudomonas aeruginosa (NCIB 950), Staphylococcus aureus (NCIB 8588), Bacillus subtilis (NCIB 3610), Bacillus cereus (NCIB 6349), Bacillus stearothermophilus (NCIB 8222), Enterococcus faecalis (NCIB 775), Proteus vulgaris (LIO), and Shigella sp. (LIO).

Preparation of bacterial cultures

The different bacterial cultures were prepared according to the method of Jorgensen et al. [10]. The fresh cultures of the bacterial were prepared from the original stock by subculturing each bacterial culture into freshly prepared nutrient agar and incubated at 37°C for 24 h. The fresh cultures were transferred into freshly prepared nutrient broth and standardized to 0.5 McFarland turbidity standards using a spectrophotometer to obtain the desired cell density of 1.5×108 (cells/ml).

Antibacterial activity of leaf and stem bark extracts using the agar well diffusion method

The sensitivity of the bacterial species to the plant extracts was tested in vitro following the method of Jorgensen et al. [10]. The 24-h-old broth was streaked homogenously on the nutrient agar medium by using a sterile glass spreader, and the plates were incubated at 37°C for 24 h. Each of the crude extracts was prepared in DMSO to obtain a final concentration of 25 mg/ml and screened for antibacterial activity. The sterile Petri dish containing the nutrient agar was allowed to solidify. Then, 0.1 ml of the test organism was smeared on the surface of nutrient agar and allowed to stand for 5 min in aseptic condition. Five wells of 6 mm in diameter each were made on the agar with a cork borer. Six drops of the working solution were transferred into each well and labeled. A prediffusion time of 30 min was allowed before incubation at 37°C for 24 h. After incubation, the plates were examined and the diameters of the zone of complete inhibition were observed and measured using a meter rule. Streptomycin and ampicillin were used as positive controls at 1 mg/ml. All the tests were aseptically performed in triplicates.

Determination of minimum inhibitory concentration

The minimum inhibitory concentration (MIC) of the plant extracts that exhibited antibacterial activities against the test organisms was determined using the microplate dilution method [11]. The plant extracts were separately diluted in DMSO, whereas the inocula of organisms were prepared in Mueller-Hinton broth with the turbidity adjusted to 0.5 McFarland standards in preparation of 1.0×108 bacterial cells/ml. Then, 150 µl of plant extract was added to each well of the 96-well microplate. The bacterial suspension (50 µl) was added to each well except the negative controls. Streptomycin and ampicillin were used as the positive controls. DMSO (10%) and plant extracts without bacterial suspension were used as the negative controls. The microtiter plates were incubated at 37°C for 24 h. The highest dilution which had the lowest concentration of the plant extract that inhibited the growth of the test microorganism was taken as the MIC.

Determination of the minimum bactericidal concentration

The wells showing no growth from the lowest concentrations in the MIC assay were selected for minimum bactericidal concentration (MBC) determination. Bacterial cells (100 µl) from the MIC test plate were subcultured on freshly prepared solid nutrient agar by making streaks on the surface of the agar. The plates were incubated at 37°C for 24 h overnight. The concentration of the plant extract in the plate that showed no microbial growth was considered to be the MBC of the extract [12]. All the tests were performed in triplicate.

Data analyses

Data were collated and statistically analyzed using IBM SPSS software, version 25. A χ2 test was used to test the correlation of the antibacterial activities exerted by different concentrations of the stem bark and leaf extracts. The value of α was set at less than or equal to 0.05. The P value was considered statistically significant if less than the value of α.

  Results 

Phytochemical constituents of the stem bark and leaf extracts of Afzelia africana

Both stem bark and leaf extracts contained alkaloids, anthraquinones, flavonoids, glycosides, saponins, tannins, triterpenes, and xanthoproteins ([Table 1]).

Table 1 Phytochemical constituents of the stem bark and leaf extracts of Afzelia africana

Click here to view

Anticancer potentials, therapeutic index, and effective concentration (EC50) of the stem bark and leaf extracts of Afzelia africana

The anticancer test with HEK cells showed that the leaf and stem bark extracts elicited cytotoxic activity at 0.65 and 0.54 µg/ml. The leaf extract had TIs of 0.23, 0.97, 0.25, 0.27, and 0.41 against S. aureus, E. coli, Klebsiella oxytoca, Salmonella enterica, and Shigella sonnei, respectively. Similarly, the stem bark had TIs of 0.35, 0.75, 0.24, 0.32, and 0.57, respectively, for the same organisms. There was no significant difference (P=0.077) in the therapeutic index (TI) between the leaves and the stem bark extracts ([Table 2]).

Table 2 Cytotoxicity and therapeutic index of the leaf and stem bark extracts of Afzelia africana

Click here to view

Antibacterial sensitivity of test organisms to plant extracts

The antibacterial activity of stem and leaf methanol extracts of A. africana was dictated by the sizes of the zones of inhibition (mm) shown by the test pathogens. The zones of inhibition exerted by the stem bark extract against the test pathogens ranged from 15 to 20 mm, whereas that of leaf extract ranged from 15 to 29 mm. Streptomycin and ampicillin exerted inhibiton zones ranging from 15 to 29 mm and 15 to 21 mm, respectively, against the test pathogens. There were no significant differences (P=0.073) between the antibacterial activities exerted by the leaf and stem bark extracts and within the antibacterial activities exerted by the antibiotics ([Table 3]).

Table 3 Antibacterial activity of stem and leaf methanol extracts of Afzelia africana against selected pathogens

Click here to view

Minimum inhibitory concentration and minimum bactericidal concentration of extracts against test organisms

The MIC of stem bark and leaf methanol extracts of A. africana against the test pathogens ranged from 0.391 to 1.562, whereas that of leaf extract ranged from 0.271 to 1.987. The MBC of the stem bark and leaf extracts of the plant ranged from 0.391 to 6.25 and 0.232 to 6.36, respectively, against the test pathogens, and there were no significant differences (P=0.065) between MICs of the plant parts ([Table 4]).

Table 4 Minimum inhibitory concentration and minimum bactericidal concentration of stem bark and leaf methanol extracts of Afzelia africana

Click here to view

  Discussion 

For a drug substance or herbal preparation to be adopted in clinical applications, it must be selectively toxic to the target microorganisms or must, at least, interfere directly with a reaction pathway with minimal or no toxic effects to the host cells [13]. The antibacterial and anticancer potentials of A. africana detected in this study give a fresh insight into the application of these plant parts with the intent of discovering new leads for fighting infectious diseases including cancer.

Phytochemical constituents such as flavonoids, tannins, alkaloids, saponins, glycosides, triterpenes, xanthoproteins, and anthraquinones were detected in both stem bark and leaf extracts of the plant. Tannins and alkaloids have been earlier reported to possess strong antibacterial activity [14],[15]. Tannins provoke antimicrobial activity by binding and precipitating extracellular and intracellular proteins. Similarly, alkaloids interrupt several molecular targets including cell membranes and nucleic acids, thus inhibiting microbial growth [16]. Selective inhibition of more than one molecular target is essential for combating multidrug and pandrug resistance among bacterial species and cancer cells. The antibiotics and anticancer activities shown by the leaf and stem bark of A. africana are consistent with some other plant materials [15],[17].

Akinpelu et al. [18] also reported the presence of a number of phytochemical compounds in the crude extract of A. africana, and these were tannins, flavonoids, steroids, alkaloids, and saponins. These phytochemical compounds are biologically active and therefore aid the antimicrobial activities of A. africana. Phytochemicals utilize different modes of action to exert antimicrobial activities. For instance, tannins act not only by iron deprivation but by hydrogen bonding or interactions with specific proteins like enzymes in microbial cells. Alkaloid has also been reported in the stem bark extract of A. africana and has been well studied to exhibit anti-asthmatic, anti-inflammatory, and anti-anaphylactic properties [19]. Tannins and alkaloids have remarkable activity in the prevention and treatment of cancer [20]. Flavonoids, in A. africana, elicit analgesic, anti-inflammatory, antimicrobial, anti-allergic, cytostatic, anti-angiogenic, and antioxidant properties [21],[22]. Flavonoids in human diets could prevent cancers and menopausal symptoms [23]. Saponins possess numerous pharmacological properties and had been reported in A. africana stem bark extract [24]. Saponins are major ingredients in traditional Chinese medicine and are associated with biological effects in medicinal plants [25]. Saponins elicit anti-inflammatory activities. The literature cited on phytochemical compounds buttressed our findings on the anticancer and antibacterial potentials of the methanolic extracts of stem bark and leaf of A. africana.The antibacterial activity exerted by leaf and stem bark extracts against K. pneumoniae and B. subtilis was higher than that exerted by streptomycin and ampicillin. As no significant difference occurred between antibacterial activities exerted by the stem bark and leaf extracts, it could be deduced that either of the plant parts could be used for the treatment of bacterial infections.

The anticancer test with HEK cells also showed that both the leaf and stem bark extracts elicited cytotoxic activity. Although the stem bark extract showed stronger cytotoxic activity against HEK cell lines, there was no significant difference in the TI between the two plant parts. TI represents the blood concentration at which the plant extract is relatively toxic, and the larger the TI, the safer the drug. However, in this study, the TI of the plant parts was less than 1.0, suggesting that precautions are required when using the plant remedy in disease treatment. This is consistent with the study of Igwenyi and Akubugwo [26], who also reported that A. africana possesses some toxicity.

Agents with cytotoxic properties were initially introduced in medicine for their ability to interrupt nucleic acid and protein synthesis in malignant cells. Subsequently, they were found to possess immune-suppressing capacities. For this reason, cytotoxic agents are now used in medicine for preventing the rejection of transplanted organs. The further realization that these agents exhibit immunomodulatory capacities led to their use in autoimmune diseases such as systemic lupus erythematosus. Ever since their introduction, the management and prognosis of these diseases have greatly improved. As a result of their adverse effects, cytotoxic therapy is reserved only for patients with moderate to severe disease cases. Therefore, A. africana is found to not only elicit antibiotic activity but also cytotoxic activity, which has implicated its potential use for the treatment of autoimmune diseases and organ rejection.

  Conclusion 

This study revealed that the stem bark and leaf extracts of A. africana possess antibacterial and anticancer properties. The plant material could be exploited for alternative anticancer and antibiotic drugs discovery toward combating the global disease burden.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 

  References 
1.Osuntokun OT. Evaluation of inhibitory zone diameter (IZD), phytochemical screening, elemental composition and proximate analysis of crude cleistopholis patens (Benth) on infectious clinical isolates. J Mol Biomark Diagn 2018; 9:385.  
    2.Gérard J, Louppe D. Afzelia africana Sm. ex Pers. In: Lemmens RHMJ, Louppe D, Oteng-Amoako AA, (editors). PROTA (Plant Resources of Tropical Africa/Ressources végétales de l’Afrique tropicale). Wageningen, Netherlands: Pudoc Scientific Publishers; 2011; pp. 34–91.  
    3.Tamou C, de Boer IJM, Ripoll-Bosch R, Oosting SJ. Traditional ecological knowledge underlying herding decisions of pastoralists. Animal 2018; 12:831–843.  
    4.Balima LB, Nacoulma BMI, Ekué MRM, Kouamé FN, Thiombiano A. Use patterns, use values and management of Afzelia africana Sm. in Burkina Faso: implications for species domestication and sustainable conservation. J Ethnobiol Ethnomed 2018; 14:23.  
    5.Akinpelu BA, Godwin A, Aderogba MA, Makinde AM, Azeez SO, Oziegbe M. Evaluation of anti-inflammatory and genotoxicity potentials of the fractions of Archidium ohioense (Schimp. ex Mull) extract. Ife J Sci 2018; 20:487–495.  
    6.Harbones JB. Textbook of phytochemical method. London: Champman and Hall Ltd; 1973. pp. 49–55.  
    7.Trease G, Evans SM. Pharmacognosy. 5th ed. London: Bailer Tindal; 2002. pp. 23–67.  
    8.Sofowora A. Medicinal plants and traditional medicine in Africa. 2nd ed, Ibadan, Nigeria: Spectrum Books Limited; 2006. pp. 150–153.  
    9.Zanariah BU, Thavamanithevi S, Mazita MD, Harmayumi BW, Badariah BA, Ahmad HB et al. Bioguided fractionation and purification of natural bioactives obtained from alpinia conchigera water extract with melanin inhibition activity. J Biomater Nanobiotechnol 2013; 4:265–272.  
    10.Jorgensen JH, Howell AW, Maher LA. Antimicrobial susceptibility testing of less commonly isolated Haemophilus species using Haemophilus test medium. J Clin Microbiol 1990; 28:985–988.  
    11.Mishra MP, Rath S, Swain SS, Ghosh G, Das D, Padhy RN. In vitro antibacterial activity of crude extracts of 9 selected medicinal plants against UTI causing MDR bacteria. J King Saudi Univ Sci 2015; 29:84–95.  
    12.Chan LW, Cheah EL, Saw CL, Weng W, Heng PW. Antimicrobial and antioxidant activities of cortex Magnoliae officinalis and some other medicinal plants commonly used in South-East Asia. Chin Med 2008; 3:15–21.  
    13.Sigidi MT, Anokwuru CP, Zininga T, Tshisikhawe MP, Shonhai A, Ramaite IDI et al. Comparative in vitro cytotoxic, antiinflammatory and anti-microbiological activities of two indigenous Venda medicinal plants. Transl Med Commun 2016; 1:9.  
    14.Igwenyi IO, Azoro BN. Proximate and phytochemical compositions of four indigenous seeds used as soup thickeners in Ebonyi State Nigeria. IOSR-JESTFT 2014; 8:35–40.  
    15.Umar AT, Muhammad Y, Mohammed B, Salawudeen A, Puma HU. Synergistic antibacterial effect of stem bark extracts of Faidherbia albida and Psidium guajava against methicillin resistant Staphylococcus aureus. BAJOPAS 2017; 10:112–115.  
    16.Osho A, Bello OO, Fayemi SO, Adetunji T. In-vitro screening of some selected Nigerian medicinal plants (Fagara zanthoxyloides, Vernonia amygdalina, Prosopis africana and Azadirachta indica) for antibacterial activity. AdvBiores 2011; 2:190–195.  
    17.Fashola MO, Opere BO, Saibu GM, Bello OO, Yovoyan TS, Usman OF. Antibacterial effects of aqueous extract of onion (Allium cepa) and garlic (Allium sativum) on some clinical bacterial isolates. J Res Rev Sci 2018; 5:1–7.  
    18.Akinpelu DA, Aiyegoro OA, Okoh AI. The in vitro antioxidant property of methanolic extract of Afzelia africana (Smith.). J Med Plant Res 2010; 4:2021–2027.  
    19.Friday C, Akwada U, Igwe OU. Phytochemical screening and antimicrobial studies of Afzelia africana and Detarium microcarpum seeds. Chem Int 2018; 4:170–176.  
    20.Abubakar S. Antibacterial studies of the stem bark of Detarium microcarpum guill and perr (Fabaceae). Int J Nanotechnol Eng Med 2017; 2:32–41.  
    21.David J, Afolabi EO, Olotu PN, Ojerinde SO, Agwom FM, Ajima U. Phytochemical analysis, antidiabetic and toxicity studies of the methanolic leaf extract of Detarium microcarpum guil. and perr. in wistar albino rats. J Chem Pharm Res 2017; 9:55–60.  
    22.Olorunmaiye KS, Apeh LE, Madandola HA, Oguntoye MO. Proximate and phytochemical composition of African Mahogany (Afzelia africana) seed and African mesquite (Prosopis africana) pod. J Appl Sci Environ Manage 2019; 23:249–252.  
    23.Manzo LM, Mousa I, Ikhiri K. Phytochemical screening of selected medicinal plants used in Niger, West Africa. Int J Herb Med 2017; 5:32–38.  
    24.Moghimipour E, Handalu S. Saponin: properties, methods of evaluation and applications. Annu Res Rev Biol 2015; 5:207–220.  
    25.Yassin NZ, Melek FR, Selim MA, Kassem IAA. Pharmacological activities of saponin-containing fraction derived from Gleditsia caspica desf. methanolic fruit extract. Der Pharma Lett 2013; 5:247–253.  
    26.Igwenyi IO, Akubugwo EI. Analysis of four seeds used as soup thickeners in the South Eastern part of Nigeria. Intenational Conference Chemical Chemistry, England; August 1–3, 2010:. Kyoto, Japan.  
    

 
 

  [Table 1], [Table 2], [Table 3], [Table 4]