The authors have declared that no competing interests exist.
The use of medicinal plants in industrialized societies for extraction and development of many drugs and other chemotherapeutics and traditionally for herbal remedies has increased in recent times. Plant–based medicine is essential in health care services with about 80% global population relying on it because of its cheap source and availability.
The secondary metabolites were isolated, characterized, and identified using nuclear magnetic resonance. The experiment was conducted using 25 male wistar rats weighing between 180-200 g randomized into 5 groups, 3 controls and 2 treatment groups of 5 rats each. The treatment groups received 25 mg/kg body weight of phytol and lupeol orally by gastric lavage for 14 days. The animals were anaesthetized and blood samples collected for hormonal assay.
The experimental data was analyzed using one-way analysis of variance (ANOVA) with Statistical Package for Social Sciences (SPSS) version 17.0, while the post hoc test assessed using Duncan Multiple Range Test at p ≥ 0.05. There was a significant decrease (p ˂ 0.05) in the levels of FSH, LH and TST in the treatment groups when compared to the control groups. The motility and sperm count decrease significantly (p ˂ 0.05) when treatment groups were compared to the control animals. The secondary metabolites, phytol and lupeol present in the leaf extract of
An increasing reliance on the use of
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.
Enzyme Immunoassay Test kits, Silica gel, Hexane, Distilled water, Chloroform, Ethyl acetate, Methanol etc.
Fresh leaves material of
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
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
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
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
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.
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
Cauda epididymis sperm concentration and motility was assessed according to the method employed by Prasad
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
The result showed lethal dose determination of methanolic leaf extract of
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Dose (mg/kg) | Number of animal | % mortality |
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Group 1 | 3500 | 3 | 0 |
Group 2 | 5000 | 3 | 0 |
Group 3 | 6500 | 3 | 0 |
Toxicity sign: no-toxicity sign observed within the 48 hours of observation LD50 > 5000 mg/kg
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1 | J1- J5 | 0.9 | J 25 |
4 | J6- J8 | 0.9 | J25 |
4 | J9- J11 | 0.9 | J25 |
2 | J12- J14 | 0.7 | J10 |
3 | J15- J20 | 0.4 | J7 |
1 | J21- J26 | 0.4 | J7 |
1 | J27- J29 | 0.4 | J7 |
2 | J30- J39 | 0.4 | J7 |
Formula for RF=Distance travelled by the spot/Distance travelled by solvent front
KEY: RF=Retention Factor
J1- J5 =
J6- J8 =
J9- J11 =
J12- J14 =
J15- J20 =
J21- J26 =
J27- J29 =
J30- J39 =
13C-NMR spectra were taken with JEOL, JNM-400. Structural determination of J25 was done using 1H-NMR (CDCl3), 13C-NMR (CDC13), 'H-'H-COSY (
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1
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13
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1 |
13 |
1 | 4.14 (d, J = 6.9 Hz, 2H) | 59.53 | 4.09 | 59.4 |
2 | 5.40 (dt, J = 7.9, 3.8 Hz,1H) | 123.20 | 5.34 | 123.0 |
3 | - | 140.40 | - | 140.4 |
4 | 1.98 | 40.01 | - | 39.9 |
5 | 0.84 | 25.27 | - | 25.1 |
6 | 1.37 | 36.80 | - | 36.7 |
7 | 1.52 | 32.83 | - | 32.7 |
8 | 1.21 | 37.56 | - | - |
9 | 1.25 | 24.61 | - | 24.5 |
10 | 1.26 | 37.49 | - | 37.4 |
11 | 1.57 | 32.93 | - | 32.8 |
12 | 1.25 | 37.42 | - | 37.3 |
13 | 1.26 | 24.94 | - | 24.8 |
14 | 1.25 | 39.50 | - | 39.4 |
15 | 1.25 | 28.11 | - | 27.9 |
16 | 0.79 (d, 3H) | 22.77 | - | 22.6 |
17 | 0.84 (d, 3H) | 22.87? | - | 22.7 |
18 | 0.84 (s, 6H) | 19.85 | - | 19.7 |
19 | 0.84 | 19.89 | - | 19.7 |
20 | 1.66 (s, 3H) | 16.31 | 1.60 | 16.2 |
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1
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13
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1 | 4.14 (d, J = 6.9 Hz, 2H) | 59.53 | C-2, 3 | C-1 |
2 | 5.40 (dt, J = 7.9, 3.8 Hz, 1H) | 123.20 | C-4, 20 | C-2 |
3 | - | 140.40 | ||
4 | 1.98 | 40.01 | C-4, 20 | C-4 |
5 | 0.84 | 25.27 | C-4, 7, 8 | C-5 |
6 | 1.37 | 36.80 | ||
7 | 1.52 | 32.83 | ||
8 | 1.21 | 37.56 | ||
9 | 1.25 | 24.61 | C-9 | |
10 | 1.26 | 37.49 | ||
11 | 1.57 | 32.93 | ||
12 | 1.25 | 37.42 | ||
13 | 1.26 | 24.94 | ||
14 | 1.25 | 39.50 | C-14 | |
15 | 1.25 | 28.11 | C-15 | |
16 | 0.79 (d, 3H) | 22.77 | C-20 | |
17 | 0.84 (d, 3H) | 22.87? | C-17 | |
18 | 0.84 (s, 6H) | 19.85 | C-18 | |
19 | 0.84 | 19.89 | C-19 | |
20 | 1.66 (s, 3H) | 16.31 | C-4, 20 | C-20 |
The Carbon-13 NMR of J25 showed the following signals: 13C NMR (101 MHz, Chloroform-
The fraction J25 had the following
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1 | Phytol (2 |
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2 | Lupeol acetate |
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Normal Control | 5.96 ± 0.81a | 7.45 ± 0.66a | 0.45 ± 0.13a |
Positive Control | 7.52 ± 0.02a | 6.48 ± 0.57a | 20.88 ± 0.04b |
Standard Control | 3.40 ± 0.99b | 2.87 ± 0.17b | 13.30 ± 0.63c |
Sus + 25 mg/ phytol | 3.22 ± 0.13b | 2.38 ± 0.29b | 13.67 ± 0.74c |
Sus + 25 mg/kg lupeol | 3.00 ± 0.24b | 3.08 ± 0.13b | 12.72 ± 0.79c |
Data are expressed as mean ± standard error of mean (SEM) with n: 5, values with different superscripts as alphabet down the columns are considered statistically significant (p ˂ 0.05).
Groups | Weight (g) |
Normal Control | 1.20 ± 0.03a |
Positive Control | 1.67 ± 0.05b |
Standard Control | 1.24 ± 0.08b |
Sus + 25 mg/kg phytol | 1.47 ± 0.13b |
Sus + 25 mg/kg lupeol | 1.19 ± 0.11b |
Data are expressed as mean ± standard error of mean (SEM) with n: 5, values with different superscripts as alphabet down the columns are considered statistically significant (p ˂ 0.05).
The Carbon-13 NMR of J25 showed the following signals: 13C NMR (101 MHz, Chloroform-
The proton NMR of J7 revealed the following data:
Groups | % Motility |
Normal Control | 61.64 ± 2.15a |
Positive Control | 66.67 ± 2.36a |
Standard Control | 40.33 ± 2.62b |
Sus + 25 mg/kg phytol | 39.32 ± 2.26b |
Sus + 25 mg/kg lupeol | 41.61 ± 2.24b |
Column chromatography of the leaf extract of
The thin layer chromatography (TLC) of fractions collected from the column chromatography of
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
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 (2
1H-NMR data of J7 revealed two signals olefinic protons at δH 4.56 (d,
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
Follicle stimulating hormone (FSH) recorded significant decrease (p < 0.05) in treatment groups when compared to the control rats. The findings of Udoh
Luteinizing hormone (LH) recorded a significant decrease (p < 0.05) in all the treatment groups when compared to the control group. Udoh
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
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
The testicular weights of the wistar rats induced and treated with phytol and lupeol in leaf extract of
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
In the study, the secondary metabolites; phytol and lupeol were isolated and characterized from the methanolic leaf extract of
We are very grateful to the staff of the Department of Biochemistry, Federal University of Agriculture Makurdi and Professor John Igoli of the Department of Chemistry Federal University of Agriculture Makurdi and Mrs Regina Akighir for their assistance in making sure that research gained the light of the day.