Abstract
Asparagus africanus Lam., Asparagaceae, is used traditionally as medicinal plant for treatment of various gastrointestinal disorders and for birth related applications. This study aimed to evaluate anti-implantation potential, screening for bioactive phytochemicals and to determine its toxicity. Thirty healthy rats were distributed into five groups (n = 6). Pregnant rats were orally administered vehicle and aqueous extract A. africanus at three different doses thrice daily for seven days. Misoprostol 300 µg/kg bw was used as positive control. All rats were laparotomized 24 h after the last dose and number of live fetuses, implantations and resorption sites were enumerated, and ovaries were harvested for histopathology. The phytochemical analysis was carried out using LC/MS. Acute toxicity was investigated, the animals were randomly grouped into five groups (n = 3); control, four different doses of aqueous extract A. africanus at a single dose treatment and rats were observed for 14 days. Prenatal study demonstrated that 300 mg/kg bw of extract and misoprostol were significantly increased the percentage of anti-implantation as compared to untreated rats. Histopathology of ovaries showed a dose dependent toxicity. LC/MS revealed the presence of steroidal saponins; asparasaponin II, sarsasapogenin, spirostans, and stigmasterol. The mean weight gain of rats treated with 2000 mg/kg bw of aqueous extract was significantly reduced (p = 0.032) compared to control group. In conclusion, the aqueous extract A. africanus has anti-implantation effect in female rats and is safe up to 2000 mg/kg bw. In addition, it contains some potential steroidal saponins, which could be used to explain its anti-implantation activity, however this finding needs further pharmacological studies to confirm the antifertility activities.
Keywords
Anti-implantation; Acute toxicity; Phytochemicals; Interceptive agent; Steroidal saponins
Introduction
It is extremely obvious that plants play cogent role in the well-being of humans and animals. Hundreds, if not thousands of plant materials have been reported to possess medicinal properties owing to their phytochemical constituents. In addition, many plants have been traditionally explored as remedy for various pathologic conditions such as: burning sensation, fever, nephropathy, hepatopathy, bladder irritation, throat infections, tuberculosis, cough, bronchitis, leucorrhoea, hemorrhoids, hypertension, abortion, and cardiac problem (Warrier et al., 1993Warrier et al., 1993 Warrier, R.K., Nambiar, V.P.K., Ramankutty, C., 1993. Economical Importance of Indian Pandanus Species. In: Indian Medicinal Plant: a Compendium of 500 Species, vol. 1. University of Press (Indian). Pvt. Ltd, Chapter 7.). One of such medicinal plants is Asparagus africanus Lam., Asparagaceae, a climbing shrub, which has been used traditionally to treat diverse ailments such as: peptic ulcer, diarrhea (Hutchinson et al., 1963Hutchinson et al., 1963 Hutchinson, J., Dalziel, J., Hepper, F., 1963. Flora of West Tropical Africa, vol. 2. JSTOR.), headache, backache, stomach pain and for easing childbirth (Maroyi, 2011Maroyi, 2011 Maroyi, A., 2011. An ethnobotanical survey of medicinal plants used by the people in Nhema communal area, Zimbabwe. J. Ethnopharmacol. 136, 347-354.). A. africanus has also been used for other non-therapeutic usage such as food and ornamental purposes (Ribeiro et al., 2010Ribeiro et al., 2010 Ribeiro, A., Romeiras, M.M., Tavares, J., Faria, M.T., 2010. Ethnobotanical survey in Canhane village, district of Massingir, Mozambique: medicinal plants and traditional knowledge. J. Ethnobiol. Ethnomed., http://dx.doi.org/10.1186/1746-4269-6-33.
http://dx.doi.org/10.1186/1746-4269-6-33...
). Despite all these aforementioned uses of this plant, there are still insufficient data regarding its abortifacient and toxicological effects.
Geremew et al. (2006)Geremew et al., 2006 Geremew, T., Yalemtsehay, M., Eyasu, M., 2006. Antifertility effect of aqueous and ethanol extracts of the leaves and roots of Asparagus africanus in rats. Afr. Health Sci. 6, 81-85. showed that the root of A. africanus possess hormonal properties which can modulate the reproductive function of the experimental rats and suggested that the contraceptive activity of A. africanus needs further exploration to elucidate the bioactive constituents. In line with this, a previous study has been reported two compounds from the roots of A. africanus namely, spirostanosides and furostanol glycoside using combining MS with two-dimensional NMR technique (Asfaw et al., 1999Asfaw et al., 1999 Asfaw, D., Erst, H., Olaf, K., Gunter, M., Dawit, A., 1999. Steroidal saponins from Asparagus africanus. Phytochemistry 51, 1069-1075.). Furthermore, the structure of isolated compound, sapogenin, from A. africanus has been elucidated by combining X-ray crystallography with MS and 2D NMR (Oketch-Rabah et al., 1997Oketch-Rabah et al., 1997 Oketch-Rabah, H.A., Dossaji, S.F., Christensen, S.B., Frydenvang, K., Lemmich, E., Cornett, C., Theander, T., 1997. Antiprotozoal compounds from Asparagus africanus. J. Nat. Prod. 60, 1017-1022.).
Antifertility of A. africanus extracts has been studied in rats and a single dose of 300 mg/kg bw of leaves and roots extracts showed the possible effect of the plant in hormonal signaling pathway which may be responsible for certain reproductive function (Geremew et al., 2006Geremew et al., 2006 Geremew, T., Yalemtsehay, M., Eyasu, M., 2006. Antifertility effect of aqueous and ethanol extracts of the leaves and roots of Asparagus africanus in rats. Afr. Health Sci. 6, 81-85.). Asparagus africanus has been reported to ease childbirth and expulsion of placenta after birth (Hamill et al., 2003Hamill et al., 2003 Hamill, F., Apio, S., Mubiru, N., Bukenya-Ziraba, R., Mosango, M., Maganyi, O., Soejarto, D., 2003. Traditional herbal drugs of Southern Uganda, II: literature analysis and antimicrobial assays. J. Ethnopharmacol. 84, 57-78.), whereas its aqueous extract had inhibited conception in a dose-dependent manner in Sprague–Dawley (SD) rats (Okidi et al., 2019Okidi et al., 2019 Okidi, O.P.O., David, N., Joseph, O., 2019. Anti-fertility activity of aqueous root bark extracts of Asparagus africanus Lam and Annona senegalensis Pers combination on female Spraugue Dawley rats. Afr. J. Pharm. Pharmacol. 13, 12-16.).
Owing to the fact that chromatography couple to mass spectrometry method is a direct and fast analytical approach for identification of phytocomponents (Goyal, 2014Goyal, 2014 Goyal, A.K., 2014. Phytochemistry and in vitro studies on anti-fertility effect of Ficus religiosa fruits extract on uterine morphology of goat (Capra hircus). Int. J. Drug Dev. Res. 6, 141-158.), this study aimed to screen and identify the major phytochemicals in the aqueous extract of A. africanus root using LC/MS. Subsequently, to determine the in vivo anti-implantation activity and acute toxicity of the aqueous extract of A. africanus in SD rats.
Materials and methods
Chemicals and reagents
Misoprostol was purchased from Cayman (Michigan, USA). Ethanol and methanol were of HPLC grade and obtained from Fisher Scientific (Illkirch, France). Haematoxylin, bluing reagent and eosin were obtained from Leica Microsystems (Milan, Italy). All other chemicals were of analytical grade and obtained mainly from Merck and Friedemann Schmidt, Germany.
Plant material
Asparagus africanus Lam., Asparagaceae, was collected between September 2015 and January 2016 from Sindiri village in Damaturu; local government area of Yobe State, Nigeria. The location is having the bearing of long 110, 44″, 52 s, N. latitude 110, 57″, 35 s, E. The various plant parts were obtained from a healthy plant. The plant was authenticated by taxonomist at Institute of Biological Sciences, Faculty of Science, University of Malaya with voucher specimen No. KLU:48696. The voucher specimen was deposited in Rimba Ilmu Herbarium. The root parts were washed with distilled water and air dried at room temperature. The dried roots were ground with an electric blender into a fine powder to pass through a 0.2 mm sieve (BS 410 Endecott's Ltd., London).
Plant extraction
Approximately 50 g of the fine powder of the roots were extracted using 500 ml of ultra-pure water for 24 h in temperature control wrist shakers at 20 °C (Dunn et al., 2004Dunn et al., 2004 Dunn, J.L., Turnbull, J.D., Sharon, A.R., (Publication no. http://ro.uow.edu.au/scipapers/41)), 2004.
http://ro.uow.edu.au/scipapers/41)...
). The extract was filtered using Whatman filter paper no. 4. The extract was kept in a freezer at −20 °C for a day before being evaporated for 48 h in a vacuum using a freeze dryer (Eyela FDU-1200, Japan). The obtained yield extract was kept in a fridge at 4 °C until used for further analysis.
Anti-implantation experiment
The study was conducted using a healthy female Sprague–Dawley rats weighing 160–200 g obtained from Animal Experimental Unit, Faculty of Medicine, University of Malaya. All experimental procedures were carried out according to the approval of the ethics committee for animal experimentation, Faculty of Medicine, University of Malaya (Ethics Reference no.: 2015-180505/PHAR/AEI).
The anti-implantation activity of aqueous extract of A. africanus was evaluated according to the method described by Salhab et al. (1999)Salhab et al., 1999 Salhab, A.S., Shomaf, M.S., Gharaibeh, M.N., Amer, N.A., 1999. Effects of castor bean extract and ricin a-chain on ovulation and implantation in rabbits. Contraception 59, 395-399., with slight modification. Briefly, female rats were paired overnight with the male that has passed sexual behavioural testing (ratio 1:1) in plastic cage that provide free access to food and water. Pregnancy was confirmed by the presence of vaginal plug and spermatozoa in the vaginal smear that can be observed under the light microscope (Marcondes et al., 2002Marcondes et al., 2002 Marcondes, F.K., Bianchi, F.J., Tanno, A.P., 2002. Determination of the estrous cycle phases of rats: some helpful considirations. Braz. J. Biol. 62, 609-614.). The first day of pregnancy was considered as day 0 of gestation (G0). The pregnant rats were randomized into five groups, consisting of six animals each (n = 6). On G1 to G7, pregnant rats were orally administered vehicle, 300 µg/kg bw misoprostol as positive control and A. africanus extracts at three different doses (50, 150, and 300 mg/kg bw) thrice daily. The rats were weighed weekly, and all rats were laparotomized 24 h after the last dose (day 8), under anesthesia (50 mg/kg ketamine/5 mg/kg xylazine), and the following parameters were recorded: number of implantations and number of resorption sites (Salhab et al., 1999Salhab et al., 1999 Salhab, A.S., Shomaf, M.S., Gharaibeh, M.N., Amer, N.A., 1999. Effects of castor bean extract and ricin a-chain on ovulation and implantation in rabbits. Contraception 59, 395-399.). Gravid ovaries from the females exposed to the different treatments were harvested and proceeded for histopathological examination (Guillaumon and Couto, 2013Guillaumon and Couto, 2013 Guillaumon, A.T., Couto, M.A., 2013. Hystopathological study of the intestinal epithelium submitted to different times of programmed ischemia and twenty four hours reperfusion. Acta Cir. Bras. 28, 288-299.). Anti-implantation activity was expressed as percentage using the following formulae according to Geremew et al. (2006)Geremew et al., 2006 Geremew, T., Yalemtsehay, M., Eyasu, M., 2006. Antifertility effect of aqueous and ethanol extracts of the leaves and roots of Asparagus africanus in rats. Afr. Health Sci. 6, 81-85.:
Histopathology of ovaries
At the end of the study, the ovaries were examined for gross pathological changes and the ovary tissues were collected and fixed in 10% formalin for 12 h. The samples were harvested by cutting 4–5 µm thickness and stained with haematoxylin and eosin stain (Kadagi et al., 2014Kadagi et al., 2014 Kadagi, M., Shridhar, N.B., Jagadeesh, S.S., Narayana, S.H.D., Phani, A.R., Ramachandra, S.G., Isloor, S., 2014. Acute oral toxicity study and LD50 determination of 5-fluorouracil in wister albino rats. Int. J. Pharmacol. Ther. 4.). Changes in follicles and corpora lutea organization and luminal epithelial cell height were investigated and compared with the control untreated rats (Kadagi et al., 2014Kadagi et al., 2014 Kadagi, M., Shridhar, N.B., Jagadeesh, S.S., Narayana, S.H.D., Phani, A.R., Ramachandra, S.G., Isloor, S., 2014. Acute oral toxicity study and LD50 determination of 5-fluorouracil in wister albino rats. Int. J. Pharmacol. Ther. 4.; Shaikh et al., 2015Shaikh et al., 2015 Shaikh, M.V., Kala, M., Ravat, N., Nivsarkar, M., 2015. Abortifacient potential of methanolic extract of Anthocephalus cadamba stem bark in mice. J. Ethnopharmacol. 173, 313-317.).
Liquid Chromatography/Mass Spectrometry (LC/MS Q-TOF)
Agilent technologies with LC/MS:6550 model iFunnel LC/MS-Q-TOF; po.05.01(B5125.3), and MS range (m/z): 100–1700 were used for this analysis. Aqueous extract (5 mg) from the root was redissolved in 5 ml of HPLC grade methanol and filtered using polytetrafluoroethylene (PTFE) syringe filter with 0.2 µm pore size. A column zorbax eclipse plus C18 from Agilent technologies with a dimension of (4.6 m × 100 mm, 3.5 µm) was used for the peaks separation, the initial temperature of the column oven was 32.8 °C and it was ramped up to final temperature of 34.4 °C. The MS and MS/MS scan rate were both at 1spectra/sec with initial pressure of 115.1 bar to final pressure of 114.2 bar at the end of the run.
The instrument was operating at a flow rate of 0.5 ml/min with injection volume of 10.0 µl, and needle wash time of 3 s. The LC grade mobile phase comprises of two solvents: A (0.1% Formic acid in Milli-Q water) and B (100% methanol HPLC grade), with graded elution system. The total run time was 20 min, with a post run time of 10 min (Alali et al., 2008Alali et al., 2008 Alali, F.Q., Tahboub, Y.R., Al-Daraysih, I.S., El-Elimat, T., 2008. LC-MS and LC-PDA vs. Phytochemical analysis of Colchicum brachyphyllum. Pharmazie 63, 860-865.; Agilent, 2015Agilent, 2015 Agilent, T., 2015. Agilent 1220 Infinity LC User Manual. Hewlett-Packard-Strasse 8, Printed in Germany.; Han et al., 2015Han et al., 2015 Han, L., Liu, E., Kojo, A., Zhao, J., Li, W., Zhang, Y., Wang, T., Gao, X., 2015. Qualitative and quantitative analysis of Eclipta prostrata L. by LC/MS. Sci. World J. , http://dx.doi.org/10.1155/2015/980890.
http://dx.doi.org/10.1155/2015/980890...
).
Identification of phytoconstituents
Chromatograms and spectrums obtained were identified using database of National Institute of Standards and Technology Library (NIST). Mass Spectrum Search Interpreter version 11 database was used for possible chemical identification by comparing the spectrum, and m/z of unknown compounds with the range of known phytoconstituents stored in the NIST library, also by molecular feature algorithm in the software package to elucidate the compounds. The probable names of compounds were identified.
Acute toxicity study
Healthy female Sprague-Dawley rats were randomly distributed into five groups (n = 3) and fed with a standard rat pellet and water ad libitum. The animals were housed in plastic cages at 25 ± 2 °C temperature and 50 ± 10% humidity, in a standard light/dark cycle (12 h light/12 h dark cycle) and left for one week to acclimatize to these conditions. Furthermore, administration of extract, blood and organs sampling as well as all surgical procedures were conducted with humane care according to good practice guide to the administration of substances and removal of blood, including the routes and volumes (Diehl et al., 2001Diehl et al., 2001 Diehl, K.H., Hull, R., Morton, D., Pfister, R., Rabemampianina, Y., Smith, D., Vidal, J.M., van de Vorstenbosch, C., 2001. A good practice guide to the administration of substances and removal of blood, including routes and volumes. J. Appl. Toxicol. 21, 15-23.).
The rats were fasted for 3–4 h before oral administration of aqueous extract of A. africanus to the groups, but water was allowed ad libitum. Group I served as normal control, gavaged with distilled water; groups II, III, IV, and V were orally administered single dose of crude extract dissolved in distilled water at 5, 50, 300 and 2000 mg/kg bw, respectively (Kadagi et al., 2014Kadagi et al., 2014 Kadagi, M., Shridhar, N.B., Jagadeesh, S.S., Narayana, S.H.D., Phani, A.R., Ramachandra, S.G., Isloor, S., 2014. Acute oral toxicity study and LD50 determination of 5-fluorouracil in wister albino rats. Int. J. Pharmacol. Ther. 4.). Wellness parameters of animals were observed continuously during the first half hour after dosing and periodically for the next 24 h and then daily for 14 days. All observations were systematically recorded.
At day 15, the animals were fasted overnight prior to being sacrificed under ketamine (150 mg/kg) and xylazine (15 mg/kg) anesthesia. Blood from each animal were collected for biochemical analyses including, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALT), blood urea nitrogen (BUN), uric acid (UA), creatinine (Cr) and total protein (TP). The remaining whole blood was collected in EDTA tubes for haematological parameters (Halpern et al., 2015Halpern et al., 2015 Halpern, V., Randy, M.S., Derek, H.O., Laneta, J.D., Anja, L., Kate, H.R., 2015. Towards the development of a longer-acting injectable contraceptive: past research and current trends. Contraception 92, 3-9.). Liver and kidney were harvested from all rats and examined for gross pathological changes and representative samples were collected in freshly prepared 10% buffered formalin for histopathology examination. Sections of 4–5 µm thickness from liver and kidney were stained with haematoxylin and eosin (H&E) and examined under the microscope (Kadagi et al., 2014Kadagi et al., 2014 Kadagi, M., Shridhar, N.B., Jagadeesh, S.S., Narayana, S.H.D., Phani, A.R., Ramachandra, S.G., Isloor, S., 2014. Acute oral toxicity study and LD50 determination of 5-fluorouracil in wister albino rats. Int. J. Pharmacol. Ther. 4.).
Statistical analysis
Results were expressed as mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) was used for data with normal distribution (body weight, ALP, ALT, AST, TP, Cr, urea, uric acid, HGB, RBC, MCH, RDW, WBC and platelets), while Kruskal–Wallis was used for data with non-normal distribution (HCT, MCHC and MCV). IBM SPSS software version 22 (Chicago, USA) was used to analyze all data. Statistical significant was considered at (p < 0.05).
Results
Evaluation of anti-implantation activity of Asparagus africanus aqueous extract (AEAA) in female SD rats
The results obtained are summarized in Figs. 1–3. The average weight gain for normal control group was lower than treated groups. Significant increase in weight gain was observed in rats treated with 300 µg/kg bw misoprostol (p < 0.009), and 150 mg/kg bw A. africanus (p < 0.025). For 50 mg/kg bw A. africanus and 300 mg/kg bw A. africanus, the difference was not significant when compared with normal control. 150 mg/kg bw A. africanus may be equivalent to effective dose that favour the growth requirement of the animals. Overall the highest mean weight gain throughout the study period was observed in misoprostol treated group (Fig. 1).
Effect of Asparagus africanus on weight gain for pregnant rats. Values are expressed as mean ± SEM, (n = 6), p*< 0.05, **p < 0.005 vs. control. kgbw, kg per body weight; DW, distilled water.
Effect of Asparagus africanus on anti-implantation activity in SD rats. Values are expressed as mean ± SEM, (n = 6), p* < 0.05, **p < 0.005 vs. control. kgbw, kg per body weight; DW, distilled water.
Photomicrographs of ovaries from treated and untreated female rats, (magnification ×100).(A) Normal control 5 mg/kg bw (dH2O), showing small [⇩] and large [⇧] follicles, ovary with multiple follicles at different stages of development, ovulated follicle seen. (B) 300 µg/kg bw misoprostol treated group showing regressive corpora lutea [🡇]. (C) 50 mg/kg bw Asparagus africanus. (D) 150 mg/kg bw A. africanus, showing the histology of ovary, vacuolization and fatty acid deposition [🡅]; (E) 300 mg/kg bw A. africanus, showing regressive corpora lutea, section of ovary also showing decrease in oogenesis [⭧].
For evaluation of anti-implantation (interceptive) activity, the results showed that the A. africanus crude extracts at doses 150 and 300 mg/kg bw significantly (p < 0.05) reduced the number of implantations and exhibited a higher anti-implantation activity as compared to untreated rats (Fig. 2). Findings also showed that there was significant (p = 0.019) increase in the percentage of anti-implantation in Misoprostol treated rats comparing to negative control, whereas there was no significant difference between Misoprostol and the highest dose 300 mg/kg bw of A. africanus treated groups. The extract did not cause premature delivery, post-implantation loss, however the highest dose of A. africanus exhibited 71.85% anti-implantation effect.
The photomicrographs of ovaries tissue obtained from treated female rats with different doses of A. africanus and control are shown in Fig. 3. Ovaries of rats in control group showed normal multiple follicles at different stages of development, while ovaries in misoprostol treated rats showed regressive corpora luteum and few empty follicles. Ovaries of rats treated with 50 mg/kg and 150 mg/kg of A. africanus demonstrated vacuolization and fatty acid deposition as compared to control, while ovaries of rats treated with 300 mg/kg of A. africanus showed regressive corpora luteum and decreased in oogenesis.
LC/MS analysis and phytoconstituents identification
LC/MS chromatogram of aqueous extract of A. africanus is presented in Fig. 4. The mass spectral analysis has identified eleven compounds with some of them having steroidal nucleus such as asparasaponin II, sarsasapogenin, stigmasterol, and spirostan. Other non-steroidal compounds identified are acetylcaranine; 1,3,6,8-naphthalenetetrol; prosopinine; sugeonyl acetate; glutinosone; pandaroside C; and cinncassiol C3 (Table 1).
Phytochemical compounds identified in Asparagus africanus aqueous extract using LC/MS Q-I-TOF.
Acute oral toxicity of Asparagus africanus extract
The results of the effects of aqueous extract of A. africanus on body weight and organs weight, serum biochemistry and haematology are summarized in Tables 2–4. Figs. 5 and 6 show photomicrographs of the liver and kidneys of treated and untreated rats with aqueous extract of A. africanus.
Effect of Asparagus africanus aqueous extract on the body and organs weights of treated and untreated rats.
Effect of Asparagus africanus aqueous extract on biochemical indices of treated and untreated rats.
Effect of Asparagus africanus aqueous extract on haematological parameters of treated and untreated rats.
Photomicrographs of liver sections in rats treated with Asparagus africanus aqueous extract. (H&E stain, magnification ×100). (A) Control: no bile stasis, central veins normal, portal tract unremarkable. (B) 5 mg/kg bw A. africanus and (C) 50 mg/kg bw of A. africanus: Liver in (B) and (C) shows occasional hepatocyte with binucleation [◁], but no bile stasis or steatosis. Perivenous inflammation [⇨] and lytic necrosis was noted in other areas. Portal tracts are unremarkable. (D) 300 mg/kg bw and (E) 2000 mg/kg bw of A. africanus: Liver in (D) and (E) shows moderately swollen (abundant clear cytoplasm) and binucleated hepatocytes [◁]. Perivenous inflammation, lobular hepatitis [🡅] and lytic necrosis noted.
Photomicrographs of kidney sections in rats treated with Asparagus africanus aqueous extract showed kidneys with normal glomeruli, tubules and blood vessels (H&E stain). (A) Control (magnification ×100). (B) 5 mg/kg bw A. africanus (magnification ×40). (C) 50 mg/kg bw of A. africanus (magnification ×100). (D) 300 mg/kg bw of A. africanus (magnification ×200). (E) 2000 mg/kg bw of A. africanus treated rat (magnification ×40).
Body weight gain and organs weight
The mean body weight gain of all treated rats, except 5 mg/kg bw group, were less than the mean weight gain of rats in control group. The average weight gain in 2000 mg/kg bw group was approximately 3.6 times lower than that gain in control group (Table 2). Likewise, for 50 mg/kg bw group was also 1.5 times lower than the control group. However, the difference in the percentage of weight gain in the control and the treated groups were not statistically significant compared to control group, except for the highest dose of 2000 mg/kg bw (p < 0.032). Liver and kidneys mean weights showed no significant difference between treated and untreated rats (Table 2).
Effect of Asparagus africanus aqueous extract on biochemical and haematological parameters
Liver function and kidney function biochemical parameters (ALT, ALP, AST, Cr, urea, uric acid and total protein) were not significantly different between treated and control groups (Table 3). In addition, Table 4 shows the effects of oral administration of the aqueous extract of A. africanus on the haematology parameters compared to control group. The values for HGB, HCT, RBC, MCV, MCH, MCHC, RDW, WBC and platelet between control and treated groups were all not statistically significant.
Histopathology
Liver sections obtained from the rats of control group showed no bile stasis, normal central veins and portal tracts. On the other hand, 5, 50, 300 and 2000 mg/kg bw treated rats showed congested, moderately swollen hepatocytes with binucleation (Fig. 5A–E). Although no bile stasis was seen in all treated groups, perivenous inflammation, and lytic necrosis were observed (Fig. 5C and D). The portal tracts were also not remarkable. For kidney samples, both control and the highest dose group (2000 mg/kg bw) have normal glomeruli, tubules, interstitium and blood vessels (Fig. 5A and B).
Discussion
Asparagus africanus root extract was evaluated for anti-implantation activity. Interestingly, the high dose of A. africanus (300 mg/kg bw) showed significant increase in anti-implantation activity (71.85%) as compared to untreated rats and this indicates a potential interceptive activity of the root extract of A. africanus. For implantation of zygote, it is important that oestrogen and progesterone are in equilibrium and any disturbance in the levels of these hormones can cause failure in implantation activity (Geun-Shik et al., 2005Geun-Shik et al., 2005 Geun-Shik, L., Hoe-Jin, K., Yong-Woo, J., Kyung-Chul, C., Eui-Bae, J., 2005. Estrogen receptor a pathway is involved in the regulation of -calbindin-D9k in the uterus of immature rats. Toxicol. Sci. 84, 270-277.). Our unpublished data demonstrated that the extract produced a dose-dependent increase in the oestrogen levels as compared to untreated rats. The oestrogenic properties of A. africanus could explain the anti-implantation properties of this plant. On the other hand, antagonizing the action of progesterone will result in the down-regulation of progesterone-dependent genes, with decidual necrosis and detachment of the products of conception.
More closely, to our result is the report by Geremew et al. (2006)Geremew et al., 2006 Geremew, T., Yalemtsehay, M., Eyasu, M., 2006. Antifertility effect of aqueous and ethanol extracts of the leaves and roots of Asparagus africanus in rats. Afr. Health Sci. 6, 81-85. on the antifertility of A. africanus using ethanol, and aqueous extracts. Their results further explained the possible hormonal changes as a result of the treatment with this plant, which may be responsible for certain reproductive abnormalities in experimental rats.
Histopathology evaluations were conducted on ovary tissues according to Theise (2007)Theise, 2007 Theise, N.D., 2007. Liver biopsy assessment in chronic viral hepatitis: a personal, practical approach. Mod. Pathol. 20, S3-S14.. Results revealed that the ovaries of rats in negative control showed normal, small and large follicles. In addition, ovary with multiple follicles at different stages of development, and old ovulated follicles were also reported. For positive control misoprostol treatment group, ovary with regressive corpora luteum and empty follicles were clearly seen. Histology of ovaries of rats treated with 50 and 150 mg/kg of A. africanus showed vacuolization and fatty acid deposition. Meanwhile, in 300 mg/kg bw treated rats there were regressive corpora luteum and decreased in oogenesis. However, one of the limitation of this study is that the quantification of follicles and corpora lutea were not reported and instead qualitative histological examinations were carried out.
Several studies have reported that A. africanus plant has potent anti-fertility properties which initiated their pre-natal and post-natal applications among women (Geremew et al., 2006Geremew et al., 2006 Geremew, T., Yalemtsehay, M., Eyasu, M., 2006. Antifertility effect of aqueous and ethanol extracts of the leaves and roots of Asparagus africanus in rats. Afr. Health Sci. 6, 81-85.). This study screened the bioactive phytochemicals responsible for those pharmacological properties of A. africanus plant. The presence of many phytochemicals can be linked to different pharmacological activities exhibited by plant extract. Aqueous extract was used in this study because it is often and the most available solvent used in traditional medicine preparations. In our study, the percentage of yield extract obtained from aqueous extract of the roots of A. africanus was 7%, and this is comparable to that reported by Madikizela (2014)Madikizela, 2014 Madikizela, B., 2014. Pharmacological Evaluation of South African Medicinal Plants Used for Treating Tuberculosis and Related Symptoms. University of KwaZulu-Natal, Pietermaritzburg, South Africa, 209523515. which resulted in 6.8%. On the other hand, this amount of yield extract is more appreciable when compared to that reported by Moshi et al. (2003)Moshi et al., 2003 Moshi, M., Kamuhabwa, A., Mbwambo, Z., De Witte, P., 2003. Cytotoxic screening of some Tanzania medicinal plants. East and Central Afr. J. Pharm. Sci. 6, 52-56., which yielded 1–2% only of starting material of A. africanus. In another study, a modified traditional crude saponin extraction was used and the yield was 3.59% extract from the root of A. africanusAsfaw et al. (1999)Asfaw et al., 1999 Asfaw, D., Erst, H., Olaf, K., Gunter, M., Dawit, A., 1999. Steroidal saponins from Asparagus africanus. Phytochemistry 51, 1069-1075.. Methods of extraction, nature of soil, topography and geographical location are some of the factors that might be responsible for these differences.
The present finding from LC/MS analysis indicated the presence of eleven phytochemical compounds; of which only four of them have a cyclic steroidal nucleus; namely asparasaponin II, sarsasapogenin, stigmasterol, and spirostan. The current result further confirmed and in line with the implicated steroidal saponins reported by Asfaw et al. (1999)Asfaw et al., 1999 Asfaw, D., Erst, H., Olaf, K., Gunter, M., Dawit, A., 1999. Steroidal saponins from Asparagus africanus. Phytochemistry 51, 1069-1075.. These steroidal saponins might have different pharmacological and physiological effects on pregnant rats or might act synergistically to produce the oestrogenic effects of A. africanus. Other non-steroidal compounds identified are: 1,3,6,8-naphthalenetetrol, prosopinine, sugeonyl acetate, glutinosone, pandaroside C, and cinncassiol C3.
The presence of flavonoids is similar to that reported by Charles and Robert (2004)Charles and Robert, 2004 Charles, R.C., Robert, E.S., 2004. Mordern Pharmacology With Clinical Applications, 5th ed. Lippincott Williams and Wilikins, London. in Trifolium pratense known as red clover which shown to contain isoflavones and their effects are similar to that of diethylstilbestrol (Charles and Robert, 2004Charles and Robert, 2004 Charles, R.C., Robert, E.S., 2004. Mordern Pharmacology With Clinical Applications, 5th ed. Lippincott Williams and Wilikins, London.). Unfortunately, the undesirable effect of red clover on sheep fertility endangered the New Zealand economy in the past (Charles and Robert, 2004Charles and Robert, 2004 Charles, R.C., Robert, E.S., 2004. Mordern Pharmacology With Clinical Applications, 5th ed. Lippincott Williams and Wilikins, London.). Sapogenin and furostanol also have been isolated from A. africanus which shown an antiprotozoal activity (Oketch-Rabah et al., 1997Oketch-Rabah et al., 1997 Oketch-Rabah, H.A., Dossaji, S.F., Christensen, S.B., Frydenvang, K., Lemmich, E., Cornett, C., Theander, T., 1997. Antiprotozoal compounds from Asparagus africanus. J. Nat. Prod. 60, 1017-1022.). The reported sapogenin is in line with our study where asparasaponin II and sarsasapogenin were also reported in A. africanus using LC/MS method of analysis.
Findings of this study confirm that A. africanus contains some potential steroidal like compounds, which could explain the traditional uses of the root part of this plant as contraceptive. The antifertility activities of other species of Asparagus has been documented with the suggestion that the root part of A. pubescens has anti-implantation or direct effect on the uterus (Nwafor et al., 1998Nwafor et al., 1998 Nwafor, P.A., Okwuasaba, F.K., Onoruvwe, O.O., 1998. Contraceptive and non-estrogenic effects of methanolic extract of Asparagus pubescens root in experimental animals.. J. Ethnopharmacol. 62, 117-122.). Ethnobotanical studies of 152 medicinal plants of Madagascar used by local people have also revealed eight native species that are widely used by women for treating placental apposition and complications during childbirth. Moreover, it has also been used for the treatment of tropical illnesses such as malaria, filariasis, and sexually transmitted diseases like gonorrhea and syphilis. Among eight widely used plants by women in the region of Madagascar, Asparagaceae was particularly used for birth related properties (Razafindraibe et al., 2013Razafindraibe et al., 2013 Razafindraibe, M., Kuhlman, A.R., Rabarison, H., Rakotoarimanana, V., Rajeriarison, C., Rakotoarivelo, N., Randrianarivony, T., Rakotoarivony, F., Ludovic, R., Randrianasolo, A., Bussmann, R.W., 2013. Medicinal plants used by women from Agnalazaha littoral forest (Southeastern Madagascar). J. Ethnobiol. Ethnomed. 4, 1-13, http://dx.doi.org/10.1186/1746-4269-9-73.
http://dx.doi.org/10.1186/1746-4269-9-73...
).
Investigation of the acute toxicity is often the initial stage in almost all screening of unknown potential medicinal herbal substances. Most acute toxicity data are used to predict the safe dose limit in clinical application because some herbs exert toxic effects even at a very low dose. Hence, multiple doses study is immensely valuable in evaluating the safety profile of medicinal plants (Woode and Abotsi, 2011Woode and Abotsi, 2011 Woode, E., Abotsi, W.K., 2011. Antinociceptive effect of an ethanolic extract of the aerial parts of Hilleria latifolia (Lam.) H. Walt. (Phytolaccaceae). J. Pharm. Bioallied Sci. 3, 384-396.; Abiodun et al., 2015Abiodun et al., 2015 Abiodun, H.A., Oyinlade, C.O., Oluwatobi, S.A., 2015. Sub-acute evaluation of extract of Syzygium malaccense in albino rats. Res. J. Med. Plant. 9, 60-71.).
In current study, acute toxicity of root extract of A. africanus was administered at doses of 5, 50, 300 and 2000 mg/kg bw. At all dosed groups, there were no abnormal changes in the general appearance and behavioural characteristics of animals and no mortality was also recorded for all administered doses.
A study by Kebede et al. (2016)Kebede et al., 2016 Kebede, S., Afework, M., Debella, A., Ergete, W., Makonnen, E., 2016. Toxicological study of the butanol fractionated root extract of Asparagus africanus Lam., on some blood parameter and histopathology of liver and kidney in mice. BMC Res. Notes 9, 1-9, http://dx.doi.org/10.1186/s13104-016-1861-5.
http://dx.doi.org/10.1186/s13104-016-186...
has been reported that the butanol extract of A. africanus showed no toxicity at a tested concentration in mice. However, focal lobular inflammation of liver was noticed in few mice, hence this was attributed to the presence of flavonoid glycoside in the butanol fraction of the extract. They reported that A. africanus at 300 and 600 mg/kg bw has no hepatonephrotoxic and heamatotoxic effects (Kebede et al., 2016Kebede et al., 2016 Kebede, S., Afework, M., Debella, A., Ergete, W., Makonnen, E., 2016. Toxicological study of the butanol fractionated root extract of Asparagus africanus Lam., on some blood parameter and histopathology of liver and kidney in mice. BMC Res. Notes 9, 1-9, http://dx.doi.org/10.1186/s13104-016-1861-5.
http://dx.doi.org/10.1186/s13104-016-186...
).
The mean percentage of weight gain for aqueous extract of the control group was 42.59%, which is higher than the mean weight gain of rats in 50, 300 and 2000 mg/kg bw treated groups. However, these reductions in weight gain in treated groups were not significantly different except for 2000 mg/kg bw (p < 0.032) when compared with that of control group. The biochemistry markers of the liver and kidney function tests showed no significant difference between treated and untreated rats.
Meanwhile, HGB, RBC, RDW, HCT, MCV and MCH were not statistically significant between treated and control groups. In addition, histopathology was conducted on formalin-fixed paraffin-embedded experimental animal tissues according to Theise (2007)Theise, 2007 Theise, N.D., 2007. Liver biopsy assessment in chronic viral hepatitis: a personal, practical approach. Mod. Pathol. 20, S3-S14.. Results showed that liver and kidney appeared normal and showed no remarkable difference between control and treated rats. However, the liver in high doses (300 and 2000 mg/kg bw) treated rats showed congestions with mildly swollen hepatocytes and, occasional binucleation, but no bile stasis or steatosis. Ultimately, A. africanus root extract causes no death or abnormal behavioural effects in rats, however it has an adverse outcome on the implantation and number of implant in female rats.
Conclusion
Asparagus africanus aqueous root extract has anti-implantation effect in female SD rats. A. africanus root extract probably has interceptive effect mainly by inhibiting implantation and the possible mechanisms could be through the oestrogenic and antiprogestin effects. LC/MS analysis revealed the presence of four steroidal saponins spirostan, stigmasterol, asparasaponin II, and sarsasapogenin in the aqueous root extract of A. africanus. This result can be used to justify its various uses in traditional medicine especially in birth related applications. The acute toxicity study showed that A. africanus aqueous root extract is safe up to 2000 mg/kg bw. Further pharmacological studies should be carried out to investigate the estrogenic effect and the antifertility activities of this plant as well as its bioactive steroidal saponins.
-
Ethical disclosuresProtection of human and animal subjects. The authors declare that the procedures used in this research were in accordance with the regulations and approval of the ethics committee for animal experimentation, Faculty of Medicine, University of Malaya (Ethics Reference no.: 2015-180505/PHAR/AEI).
Acknowledgements
The authors would like to express their profound gratitude to University of Malaya, Malaysia, for supporting this study with grant no. PG112-2014B and Federal Polytechnic, Damaturu, Nigeria (Tetfund) for approval of first author study abroad.
References
-
Abiodun et al., 2015Abiodun, H.A., Oyinlade, C.O., Oluwatobi, S.A., 2015. Sub-acute evaluation of extract of Syzygium malaccense in albino rats. Res. J. Med. Plant. 9, 60-71.
-
Agilent, 2015Agilent, T., 2015. Agilent 1220 Infinity LC User Manual. Hewlett-Packard-Strasse 8, Printed in Germany.
-
Alali et al., 2008Alali, F.Q., Tahboub, Y.R., Al-Daraysih, I.S., El-Elimat, T., 2008. LC-MS and LC-PDA vs. Phytochemical analysis of Colchicum brachyphyllum Pharmazie 63, 860-865.
-
Asfaw et al., 1999Asfaw, D., Erst, H., Olaf, K., Gunter, M., Dawit, A., 1999. Steroidal saponins from Asparagus africanus Phytochemistry 51, 1069-1075.
-
Charles and Robert, 2004Charles, R.C., Robert, E.S., 2004. Mordern Pharmacology With Clinical Applications, 5th ed. Lippincott Williams and Wilikins, London.
-
Diehl et al., 2001Diehl, K.H., Hull, R., Morton, D., Pfister, R., Rabemampianina, Y., Smith, D., Vidal, J.M., van de Vorstenbosch, C., 2001. A good practice guide to the administration of substances and removal of blood, including routes and volumes. J. Appl. Toxicol. 21, 15-23.
-
Dunn et al., 2004Dunn, J.L., Turnbull, J.D., Sharon, A.R., (Publication no. http://ro.uow.edu.au/scipapers/41)), 2004.
» http://ro.uow.edu.au/scipapers/41) -
Geremew et al., 2006Geremew, T., Yalemtsehay, M., Eyasu, M., 2006. Antifertility effect of aqueous and ethanol extracts of the leaves and roots of Asparagus africanus in rats. Afr. Health Sci. 6, 81-85.
-
Geun-Shik et al., 2005Geun-Shik, L., Hoe-Jin, K., Yong-Woo, J., Kyung-Chul, C., Eui-Bae, J., 2005. Estrogen receptor a pathway is involved in the regulation of -calbindin-D9k in the uterus of immature rats. Toxicol. Sci. 84, 270-277.
-
Goyal, 2014Goyal, A.K., 2014. Phytochemistry and in vitro studies on anti-fertility effect of Ficus religiosa fruits extract on uterine morphology of goat (Capra hircus). Int. J. Drug Dev. Res. 6, 141-158.
-
Guillaumon and Couto, 2013Guillaumon, A.T., Couto, M.A., 2013. Hystopathological study of the intestinal epithelium submitted to different times of programmed ischemia and twenty four hours reperfusion. Acta Cir. Bras. 28, 288-299.
-
Halpern et al., 2015Halpern, V., Randy, M.S., Derek, H.O., Laneta, J.D., Anja, L., Kate, H.R., 2015. Towards the development of a longer-acting injectable contraceptive: past research and current trends. Contraception 92, 3-9.
-
Hamill et al., 2003Hamill, F., Apio, S., Mubiru, N., Bukenya-Ziraba, R., Mosango, M., Maganyi, O., Soejarto, D., 2003. Traditional herbal drugs of Southern Uganda, II: literature analysis and antimicrobial assays. J. Ethnopharmacol. 84, 57-78.
-
Han et al., 2015Han, L., Liu, E., Kojo, A., Zhao, J., Li, W., Zhang, Y., Wang, T., Gao, X., 2015. Qualitative and quantitative analysis of Eclipta prostrata L. by LC/MS. Sci. World J. , http://dx.doi.org/10.1155/2015/980890
» http://dx.doi.org/10.1155/2015/980890 -
Hutchinson et al., 1963Hutchinson, J., Dalziel, J., Hepper, F., 1963. Flora of West Tropical Africa, vol. 2. JSTOR.
-
Kadagi et al., 2014Kadagi, M., Shridhar, N.B., Jagadeesh, S.S., Narayana, S.H.D., Phani, A.R., Ramachandra, S.G., Isloor, S., 2014. Acute oral toxicity study and LD50 determination of 5-fluorouracil in wister albino rats. Int. J. Pharmacol. Ther. 4.
-
Kebede et al., 2016Kebede, S., Afework, M., Debella, A., Ergete, W., Makonnen, E., 2016. Toxicological study of the butanol fractionated root extract of Asparagus africanus Lam., on some blood parameter and histopathology of liver and kidney in mice. BMC Res. Notes 9, 1-9, http://dx.doi.org/10.1186/s13104-016-1861-5
» http://dx.doi.org/10.1186/s13104-016-1861-5 -
Madikizela, 2014Madikizela, B., 2014. Pharmacological Evaluation of South African Medicinal Plants Used for Treating Tuberculosis and Related Symptoms. University of KwaZulu-Natal, Pietermaritzburg, South Africa, 209523515.
-
Marcondes et al., 2002Marcondes, F.K., Bianchi, F.J., Tanno, A.P., 2002. Determination of the estrous cycle phases of rats: some helpful considirations. Braz. J. Biol. 62, 609-614.
-
Maroyi, 2011Maroyi, A., 2011. An ethnobotanical survey of medicinal plants used by the people in Nhema communal area, Zimbabwe. J. Ethnopharmacol. 136, 347-354.
-
Moshi et al., 2003Moshi, M., Kamuhabwa, A., Mbwambo, Z., De Witte, P., 2003. Cytotoxic screening of some Tanzania medicinal plants. East and Central Afr. J. Pharm. Sci. 6, 52-56.
-
Nwafor et al., 1998Nwafor, P.A., Okwuasaba, F.K., Onoruvwe, O.O., 1998. Contraceptive and non-estrogenic effects of methanolic extract of Asparagus pubescens root in experimental animals.. J. Ethnopharmacol. 62, 117-122.
-
Oketch-Rabah et al., 1997Oketch-Rabah, H.A., Dossaji, S.F., Christensen, S.B., Frydenvang, K., Lemmich, E., Cornett, C., Theander, T., 1997. Antiprotozoal compounds from Asparagus africanus J. Nat. Prod. 60, 1017-1022.
-
Okidi et al., 2019Okidi, O.P.O., David, N., Joseph, O., 2019. Anti-fertility activity of aqueous root bark extracts of Asparagus africanus Lam and Annona senegalensis Pers combination on female Spraugue Dawley rats. Afr. J. Pharm. Pharmacol. 13, 12-16.
-
Razafindraibe et al., 2013Razafindraibe, M., Kuhlman, A.R., Rabarison, H., Rakotoarimanana, V., Rajeriarison, C., Rakotoarivelo, N., Randrianarivony, T., Rakotoarivony, F., Ludovic, R., Randrianasolo, A., Bussmann, R.W., 2013. Medicinal plants used by women from Agnalazaha littoral forest (Southeastern Madagascar). J. Ethnobiol. Ethnomed. 4, 1-13, http://dx.doi.org/10.1186/1746-4269-9-73
» http://dx.doi.org/10.1186/1746-4269-9-73 -
Ribeiro et al., 2010Ribeiro, A., Romeiras, M.M., Tavares, J., Faria, M.T., 2010. Ethnobotanical survey in Canhane village, district of Massingir, Mozambique: medicinal plants and traditional knowledge. J. Ethnobiol. Ethnomed., http://dx.doi.org/10.1186/1746-4269-6-33
» http://dx.doi.org/10.1186/1746-4269-6-33 -
Salhab et al., 1999Salhab, A.S., Shomaf, M.S., Gharaibeh, M.N., Amer, N.A., 1999. Effects of castor bean extract and ricin a-chain on ovulation and implantation in rabbits. Contraception 59, 395-399.
-
Shaikh et al., 2015Shaikh, M.V., Kala, M., Ravat, N., Nivsarkar, M., 2015. Abortifacient potential of methanolic extract of Anthocephalus cadamba stem bark in mice. J. Ethnopharmacol. 173, 313-317.
-
Theise, 2007Theise, N.D., 2007. Liver biopsy assessment in chronic viral hepatitis: a personal, practical approach. Mod. Pathol. 20, S3-S14.
-
Warrier et al., 1993Warrier, R.K., Nambiar, V.P.K., Ramankutty, C., 1993. Economical Importance of Indian Pandanus Species. In: Indian Medicinal Plant: a Compendium of 500 Species, vol. 1. University of Press (Indian). Pvt. Ltd, Chapter 7.
-
Woode and Abotsi, 2011Woode, E., Abotsi, W.K., 2011. Antinociceptive effect of an ethanolic extract of the aerial parts of Hilleria latifolia (Lam.) H. Walt. (Phytolaccaceae). J. Pharm. Bioallied Sci. 3, 384-396.
Publication Dates
-
Publication in this collection
09 Dec 2019 -
Date of issue
Sep-Oct 2019
History
-
Received
27 Oct 2018 -
Accepted
2 June 2019