Materials and Methods
Equipment
Stat Fax 3300 chemistry analyzer, Precision pipette, Beakers, Digital balance (model: SF-400), Blender, Incubator, Improved Neuber counting chamber, Glass column, Binocular Microscope (Olumpus),Test tubes (anticoagulant free),General laboratory glass wares, Test tube rack, Syringe and needle, Conical flask, Separation funnel, Glass wool, Spatula, Aluminum foil and Kitchen Knife etc.
Chemicals/Reagents
Enzyme Immunoassay Test kits, Silica gel, Hexane, Distilled water, Chloroform, Ethyl acetate, Methanol etc.
Methods
Fresh leaves material of J. tanjorensis was collected in a home garden, at the University of Agriculture Makurdi and authenticated by the Department of Botany, College of Science, Federal University of Agriculture, Makurdi and Voucher specimen of the plant deposited in the Department’s herbarium. The leaves were thoroughly washed with clean water and allowed to drain, air-dried at room temperature. The crispy leaves were pulverized using an electric blender and preserved in a moisture-free airtight laboratory containers for extraction. The homogenized J. tanjorensis was subjected to extraction as described by Agarwal et al., 2007 1. Thus 100 g of the powdered material was macerated in 1000 ml of 80% (v/v) methanol and allowed to stay for 48 hours with intermittent agitation at a cool temperature of 4ºC. The mixture was first filtered with cheese cloth, then filter paper (Whatman No.1) and the methanol evaporated using a rotary evaporator. The concentrate was allowed for complete dryness using a thermostatically controlled water bath at 42 ºC, this yielded 250 g crude extract.
The methanol extract was chromatographed on silica gel (60-120 mesh size) to separate its component fractions and eluted with different solvent combinations based on increasing polarity beginning from Hexane, chloroform, ethyl acetate and methanol as described by Hostettmann et al., 19997. The following ratios of solvent combinations were sequentially used.
Hexane: Ethyl acetate 9.5:0.5, 7.0:3.0, 6.0:4.0, 5.0:5.0
Chloroform: Methanol 9.5:0.5, 9.0:1.0, 7.0:3.0, 1.0:1.0
Thin layer chromatography was also carried out as described by Tor-Anyiin et al., 2016 26.
The thin layer chromatography (TLC) tank was prepared: A Hexane /Methanol mixture was prepared in the ratio of 9:1 (10ml) because it gave the best resolution on the TLC plates. Spots were put neatly and deftly to ensure uniform and tidy application of the fraction materials on the TLC plates. The developed plates were charred in a hot frying pan for two minutes and the pooling of the fractions was done on the basis of the similarities of the spots, color and the retention factor on the TLC plates. The individual bioactive compounds from the fractions were determined using Nuclear Magnetic Resonance (NMR) technique
Nuclear magnetic resonance was carried out as described by Komiya et al., 1999 10 Always a dilute solution is analyzed. The compound to be studied was generally mixed with solvent (CC14 or etramclhyl silane) and the dilute solution was filled in a tube. The sample under investigation was placed in the magnetic field and subjected to RF field of oscillator the particular combination of the oscillator frequency and strength, the RF energy was absorbed by certain nuclei and an RF signal was picked up by the detector.
Twenty five male wistar rats were procured from the animal house of College of Health Sciences, Benue state University, Makurdi. The animals were acclimatized for one week, housed in cages under room temperature (25±2ºC) , and relative humidity (55±5%) and a 12 hour light/ dark cycle in animal house of laboratory department, College of Veterinary Medicine, University of Agriculture, Makurdi. The animals were allowed free access to chow and tap water ad libitum with all the experimental procedures approve by the university research and ethics committee. Oral acute toxicity was carried out as described by Lorke, 1983 13.
Experimental Design
The design consisted of 25 male wistar rats grouped into five (5) groups of five (5) rats each. The groups were normal control (NC), positive control (PC), standard control (SC) and two treatment groups. The doses used were based on the LD50 determined for the fractions and the predetermined LD50 for the crude extract obtained from preliminary studies. The normal control group received normal feed and water, Positive control group received 25 mg/kg body weight of Sustanon, the standard control group received 10 mg/kg standard drug Amlodipine. The treatment groups’ received 25 mg/kg body weight of phytol and lupeol through oral intubation daily for 14 days.
Collection and Preparation of Sera Samples
The rats were anesthetized with phenobarbital, cardiac puncture performed and blood sample collected. The sera samples were separated, then assayed for Testosterone, follicle stimulating hormone and luteinizing hormone using enzyme-link immune-absorbent assay (ELISA) as described by Marshall, 1975; Knobil, 1980 and Chen et al., 1991 1495 methods.
Cauda epididymis sperm concentration and motility was assessed according to the method employed by Prasad et al., 2008 21
Statistical Analysis
Experimental data were expressed as mean± standard error of mean (SEM) and analyzed using one-way analysis of variance using Statistical Package for Social Sciences (SPSS) version 17. The post hoc test was assessed using Duncan Multiple Test Range (DMRT) at p ≤ 0.05
Discussion
Column chromatography of the leaf extract of Jatropha tanjorensis was done with solvent system of gradually increasing polarity, beginning from Hexane, chloroform, ethyl acetate and methanol, thereby confirming earlier studies 7.
The thin layer chromatography (TLC) of fractions collected from the column chromatography of Jatropha tanjorensisleaf extract showed retention factor of 0.4 for lupeol and 0.90 for phytol, which is within the range of values (0.31 and 0.45) obtained by 24 for lupeol, and 0.80 for phytol 3.The differences in the results obtained by 243 is chiefly due to solvents combination in the mobile phase. Better resolutions are obtained when a combination of polar and non-polar solvents are used.
The structure of J25 was identified based on spectroscopic analysis (1H NMR, 13C NMR, 2D NMR) and by comparison of it spectral data with those reported previously in the literature 10 (Table 4). The proton NMR of J25 revealed the presence of three distinct regions: an olefinic region (5.38-5.42 ppm), an alcohol region (4.14-4.15 ppm) and an aliphatic region (0.78-2.09 ppm). The olefinic signal [5.40 (td, J = 6.3, 3.1 Hz, 2H)] showed characteristics (an ABX coupling pattern), while the alcohol signals [4.14 (d, J = 7.0 Hz, 2H)].
The number of carbons in J25 was revealed to be twenty by 13CNMR data. Prominent HMQC correlations showed that the proton at δ 5.40 ppm was attached to the carbon at δ 123.19 ppm other correlations showed features consistent with those of a diterpene alcohol.
Prominent HMBC correlations showed correlation between protons at C-1 to carbons C-2 (2J)( δ 123.20 ppm) and C-3 (3J)( δ 140.40 ppm) while the proton at C-2 was correlated to C-4 (δ 40.01 ppm) and C-20 (δ 16.31 ppm) proving that C-20 was a substituent at C-3. Literature comparison with the work of 10 confirmed the compound was phytol (Structure 1), a diterpene alcohol and was in full accord with other previously reported values 23.
1H-NMR data of J7 revealed two signals olefinic protons at δH 4.56 (d, J = 2.5 Hz, H-29a) and 4.68 (d, J = 2.6 Hz, H-29b) characteristic of lupine-type triterpenes like lupeol and betulin. A proton signal at δH 4.46 (dd, J = 10.7, 5.5 Hz, 1H) showed that the proton at C-3 was not a carbinol proton while signals at 2.04 (s, 3H) consistent with those of an acetyl methyl group showed that there was an acetyl group attached through an oxygen at position C-3. A literature search showed that the HNMR spectra data of J7 was consistent with that of lupeol acetate in the literature 11.
Acute toxicity study is ˃ 5000 mg/kg because neither death nor any other sign of toxicity (mortality, fast respiratory rate, convulsion and dullness) was observed during the period of the experiment. Earlier studies have proven that LD50 of the aqueous extract of J. tanjorensiswas ˃ 5000 mg/kg 2, confirming its ameliorative effects on phenylhydrazine induced anaemia.
Follicle stimulating hormone (FSH) recorded significant decrease (p < 0.05) in treatment groups when compared to the control rats. The findings of Udoh et al., 2009 27, however, disagree with the submission of Modares and Heidari, 2015 16. FSH regulates the development, growth, pubertal maturation and reproductive process of the human body. In both male and female FSH stimulates the maturation of germ cells. In male induces sertolic cell to secrete androgen-binding protein (ABP), regulated by inhibin’s negative feedback mechanism on anterior pituitary. Specifically, activation of sertolic cell by FSH sustains spermatogenesis and stimulates inhibin-B secretion 4. It is suggested that the low sperm count recorded in this study may have been orchestrated through this mechanism.
Luteinizing hormone (LH) recorded a significant decrease (p < 0.05) in all the treatment groups when compared to the control group. Udoh et al., 27 also recorded reducing effects in the LH levels at 150 mg/kg body weight in non-induced rats, which decreased significantly (p < 0.05) when compared to the control group. Contrary to these findings of Modares and Heidari 2015 16 observed that Allium Sativum at 800 mg/kg body weight increased LH levels on heat stress female mice (3.2 Iμ/ml). These variations in the levels of the hormones may be as the result of varying bioactive compounds in the various plants used by the researchers. Also, sex is another determining factor in organisms’ reaction to drugs or substances as in the case of 16. LH is produced by gonadotropic cells in the anterior pituitary gland. In females, an acute rise of LH ("LH surge") triggers ovulation and development of the corpus luteum 6. In males, LH in synergy with FSH, stimulate Leydig cell production of testosterone 16.Suggestion is therefore made that, the reduction created by the treatment substances on the levels of these hormones may be the reason for the reduction in the testosterone levels recorded in treated rats.
It was observed that the testosterone levels in treated rats decrease significantly (p < 0.05) with lupeol orchestrating the highest reducing effect when compared to the control rats. The also agrees with the findings of Udoh et al., 200927 on Caricpryl-99 seed alkaloid extract on the serum of testosterone, which showed that the extract decreased the hormone levels significantly in non-induced rats.
The findings in some gonadal hormones (Testosterone, Follicle stimulating hormones and Luteinizing hormones) decreasing significantly (p < 0.05) as recorded in all the treatment groups when compared to the control rats may be the likely cause of spermatogenic arrest and failure of spermatogenesis in the histology of rats’ testes. Oluwole et al., 201219 in his work on assessment of hepatic and renal functions administered methanolic leaf extract of Jatropha tanjorensisalsorecorded a disruption in hepatic and renal functions in rats. The findings collaborates the ascension that LH through specific receptors controls the production and secretion of testosterone and testosterone is critical for the completion of meiosis and entry into progress through spermatogenesis in rats 15. This also agrees with the earlier works that, the crude extract of Allium sativum (Garlic) caused a decrease in serum testosterone levels with effects being evoked at a very low dose 18.
The testicular weights of the wistar rats induced and treated with phytol and lupeol in leaf extract of Jatropha tanjorensis recorded no significant decrease (p < 0.05) when the treatment groups was compared to the control. Contrary to these findings of Munir et al., 2017 17 who observed a significant decrease (P ˂ 0.05) in the testicular weight of rats treated with Bisphenol® as compared to the control group. These findings however agree with the findings in this study with reference to amlodipine control group. Amlodipine and bisphenol are both antihypertensive drugs 17. The findings of Rabia et al. (2008) also agree with the findings in this study that amlodipine significantly decreased (P < 0.05) the testicular weights of albino rats. However, this contradicts the findings in this study on the isolated secondary metabolites; the reason may simply be time dependent.
The percentage motility of rats treated with phytol and lupeol recorded a significant decrease (P ˂ 0.05) when compared to the control group. The reduction in the percentage motility of sperm and its total count may chiefly be as a result of the secondary metabolites present in the methanolic leaf extract of J. tanjorensis affecting the leydig cells which are responsible for production of testosterone, and testosterone is responsible for the completion of spermatogenetic process 12.