Spectroscopic Analysis, Aphrodisiac Potential of Carapa Procera Stem Bark Extract in Male Wistar Rats and In Silico Studies of Hexadecanoic and Oleic Acids on Phosphodiesterase-5 and Adenylcyclase Enzymes Volume 54- Issue 5

Azibanasamesa D C Owaba^1,2*, Frank Arueniobebh³, Samuel J Bunu¹, Raji O Rafiu¹, Ekarika C Johnson² and Emmanuel I Etim²

• ¹Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmacy, Niger Delta University, Nigeria
• ²Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmacy, University of Uyo, Nigeria
• ³Department of Pharmacognosy and Herbal Medicine, Niger Delta University, Nigeria

Received: January 10, 2024;   Published: February 02, 2024

*Corresponding author: Azibanasamesa D C Owaba, Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmacy, Niger Delta University, Wilberforce Island Bayelsa State, Nigeria, Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmacy, University of Uyo, Uyo, Akwa-Ibom State, Nigeria

DOI: 10.26717/BJSTR.2024.54.008609

Abstract PDF

Background: Carapa procera D.C Meliaecae is a medicinal plant used by the Ijaw in the management of erectile dysfunction in men. The aim was to assess the aphrodisiac effect of Carapa procera in male Wistar rats.
Materials and Method: The crude stem bark was assessed for elemental content and aphrodisiac effect using the physical behavioral mating method. The extracts were administered mg/kg/day for 7 days. On the 8th day male Wistar rats were sacrificed, liver, kidney, testis, seminal vesicle, epididymis, and vas deferens were harvested, weighed, and testes were subjected to histological appraisal. Purification of the dichloromethane fraction of the stem bark using chromatographic techniques yielded Sample A₁.
Results: The aphrodisiac assay in male Wistar rats showed that the extracts reduced mount latency at p<0.05-0.001. It also affects intromission latency at p<0.001. The DCMb, HDcb, and STD significantly reduced PEI at p<0.05-0.01, this proved the aphrodisiac potential of the extracts. It displayed 16 carbon atoms as revealed by 13C NMR spectroscopy. Fourteen methylene, methyl (Sp3), and quaternary carbon (Sp2) signals. The 1H-NMR further confirmed the assignment of these signals; 2.73 (J = 8.0) showed triplet assigned to C-2, multiplet at 1.65 ppm assigned to C-3 position, and due to 2H and intense peak appearing as multiplet at 1.27 ppm integrated for 20 protons assigned (C-4 to C-13) position and triplet at 0.90 ppm assigned to C-16. The spectrum showed a carbonyl group of carboxylic acid appearing at δ 179.9 ppm affording the most deshielded carbon. The IR spectra also revealed a diagnostic peak at 1781 cm-1 due to the carbonyl group of carboxylic acid and a signal at 2914.8 and 2847.7 cm^-1 due to the C-H stretch. The GC-MS analysis showed a molecular ion peak of 256 due to C₁₆H₃₂O₂.
Conclusion: The extract of Carapa procera enhanced sexual indices in male albino Wistar rats. This corroborates the use of Carapa procera stem bark in ethnomedicine as an aphrodisiac agent Sample A1 (-4.4) and had a binding affinity lower than the standard drug sildenafil (-6.5) against the Phosphodiesterase enzyme while in adenyl-cyclase enzyme model; Alprostadil, Oleic acid and hexadecanoic acid had a binding affinity (-6.35, -2.61 and -1.48) respectively.

Keywords: Carapa Procera; Aphrodisiac; Phosphodiesterase; Adenyl Cyclase; Hexadecanoic Acid; Spectroscopic Analysis

Abbreviations: BM: Basal Membrane; cAMP: Cyclic Adenosine Monophosphate; CDCl3 Deutrated Chloroform; DCMb: Dichloromethane Fraction; DEPT: Distortionless Enhanced Polarisation Transfer; EF: Erection Frequency; EL: Ejaculatory Latency; HDcb: High Dose Crude Extract; HMBC: Heteronuclear Multiple Bond Correlation; HMQC: Heteronuclear Multiple Quantum Coherence; IL: Intromission Latency; IF: Intromission Frequency; ISS: Interstitial Spaces; MDcb: Median Dose Crude Extract; MF: Mount Frequency; ML: Mount Latency; NIST: National Institute of Standard and Technology; PE: Penile Erection; PEI: Post Ejaculation Interval; VLC: Vacuum Liquid Chromatography

Carapa procera D.C Meliaecae is a species of forest tree of about 17 m high in swampy forest. These species are widely distributed from Senegal to Angola and East Africa, as well as in tropical America in the Amazon [1]. The stem bark is used in folkloric medicine to treat paralysis, epilepsy, skeletal spasms, and eye problems and as a genital stimulant [1,2]. All parts of the plants are bitter which is due to terpene called meliacins [3]. Male impotence also called sexual dysfunction is a common medical condition that affects the sexual life of millions of men worldwide and is a serious medical and social problem that occurs in 10-52% of men. This could be a result of social or biological issues such as; loss of libido, problems with ejaculation, and failure of the testicles to produce the normal quantity of male sex hormones. This could be a very distressing condition for men socially, and biologically and may lead to a loss of self-esteem [4]. The use of Carapa procera for the management of impotence in folkloric medicine could lead to the isolation of novel or existing compounds with a new mechanism of action [5].

Materials

Chemicals/Reagents: All chemicals and reagents used are of analytical grade: sigma and JHD products and reputable Pharmaceutical companies. Methanol (Sigma-Aldrich U.K), Dichloromethane (JHD) n-Hexane (Sigma-Aldrich U.K) Ethylacetate (Sigma-Aldrich U.K), Dimethylsulphoxide (Sigma-Aldrich U.K), Sephadex LH-20 (Sigma-Aldrich U.K), Silica gel 200-400 (Sigma-Aldrich U.K), Tween 80 (JHD), Testosterone (Testost™, Embassy Pharmaceutical, Nigeria), Oestradiol benzoate 10 mg/ml (Naman Pharma Drugs, India), Progesterone 25 mg (Pauco Pharmaceutical, Nigeria), Corn oil (Atara edible oil Ltd).

Methods

Collection and Identification of Plant Materials: The fresh stem bark was collected from the wild at Otabi Community in Ogbia Local Government Area of Bayelsa State. It was identified and authenticated at the Forestry Research Institute of Nigeria, Ibadan, and Herbarium number FHI 112975 was assigned.

Extraction: About 3271 g of the powder stem bark was extracted successively using n-hexane, dichloromethane, and 70% methanol (4x2.5L) respectively, for The extracts were concentrated at 50oC in vacuo and were subjected to phytochemical screening using standard procedures (Figure 1).

Figure 1

Experimental Animals: Forty-eight matured male albino rats of about twelve weeks old weighing between 130-281 g were used for the experiment. They were kept in a well-ventilated conventional cage 28-31oC, photoperiod of darkness for 12 hours and 12 hours of natural light. The animals were allowed to acclimatize for two weeks were fed on a standard diet and had free access to water. The experiment was carried out according to the standard laboratory conditions as approved by the animal’s ethical committee of the University [5-7]. The animals were divided into eight groups of six animals per group.

Aphrodisiac Assay: Based on the LD50, the crude extract was administered at a dose of 44.72, 89.44, and 134.16 mg/ kg, and the fractions given a median dose (89.44 mg/kg) of the fractions were administered. Group I and II were given 10 mL and 1 mg/kg of distilled water and testosterone administered subcutaneously respectively. Groups III to V were administered crude extract at 44.72, 89.44, and 134.16 mg/kg respectively. Groups VI to VIII were given 89.44 mg/kg of n-hexane, dichloromethane, and 70% methanol fractions daily for 7 days respectively. The female rats were brought to oestrus by the sequential administration of 17β-oestradiol (8 ug/kg, and progesterone 500 μg/kg) were given through subcutaneous injections, 48hrs and 4 hrs respectively before pairing [7,8]. Sexual behavior assessment was conducted using male rats on receptive females and the male exhibiting low sexual activity was excluded from the experiment. The following parameters were determined, Mount Latency (ML), Intromission Latency (IL), Ejaculatory Latency (EL), Mount Frequency (MF), Intromission Frequency (IF), Penile Erection (PE), Post Ejaculation Interval (PEI), Erection Frequency (EF), by adopting standard procedures [7,9]. The organs and tissues were collected, weighed, and tested preserved in 10% (v/v) formalin were subjected to histological analysis [10].

Isolation of Dichloromethane Fraction: About 20 g of dichloromethane fraction was subjected to Vacuum Liquid Chromatography and the following solvent system was gradiently eluted from n-hexane (100%) to methanol 100%. Fractions 4-5 gave a similar spot on TLC in a solvent system n-hexane: ethylacetate (7:3) and were pooled together and weighed 3.327g. This was subjected to gel filtration using a column (72.5 cm x 1.3 cm) and Sephadex LH-20 (25 g) as the stationary phase. Elution commences isocratically, using dichloromethane, 20 mL of the eluent was collected and 21 fractions were obtained based on the TLC in a solvent system n-hexane; ethylacetate (5:1), fraction 8-14 were pooled together weighed 0.563 g. This was further purified using silica gel (120 g, 200-400 mesh) in column chromatography and elution commences gradiently using n-Hexane; Ethylacetate (95:5) to Ethyl acetate (100%), 20 mL of the eluent was collected. Fractions 8-11 were pooled together and weighed about 0.191 g. It was further subjected to purification using a silica gel (20 g, 200-400 mesh) in a column (72.5 x 1.3 cm) and elution commenced gradiently from n-hexane (100%) to ethylacetate (100%). Fraction 19 gave a single spot in a solvent system n-Hexane: Ethylacetate (9:4), and weighed 26.1 mg. It was subjected to spectroscopic studies to elucidate the structure.

GC-MS and IR Analyses of the Samples: n- Hexane fraction, Fractions A2, A3, and A4 dichloromethane extract of the Stem bark obtained from VLC, were subjected to Gas Chromatography Mass Spectrometry for determination of chemical constituents as shown in Table 1-3.

Table 1: GC-MS analysis of fraction A2 obtained from dichloromethane (DCMb) extract.

Table 2: GC-MS analysis of fraction A₃ of dichloromethane extract of stem bark of C. procera.

Table 3: GC-MS Analysis of A₄ (16-19) obtained from dichloromethane.

Molecular Docking of the Isolated Compounds: The isolated compounds were subjected to molecular docking against the phosphodiesterase enzyme. Hexadecanoic acid, Oleic acid, and Sildenafil Citrate were designed with ChemDraw Pro 12.0 (Cambridge Soft Corporation, USA) and saved in SDF format. The Phosphodiesterase V enzyme was downloaded in PDB format from the Protein data bank (http://www.rcsb.org/pdb/home/home.do). Ligands and targets were converted to pdbqt format using PyRx (https://pyrx.sourceforge. io/). Molecular docking of the ligands with each of the target proteins was done using Autodock Vina (http://vina.scripps.edu/), to obtain their respective binding affinity. Discovery Studio (Dassault Systèmes), and Ligplot (https://www.ebi.ac.uk/thorntonsrv/ software/LIGPLOT/) were used to analyze ligand-protein binding interactions. Calculated molecular properties were obtained from the molinspiration website (https://www.molinspiration.com/cgibin/ properties), while pharmacokinetic inetic properties from pKCM website (http://biosig.unimelb.edu.au/pkcsm/prediction); [11]. The ligand alprostadil, oleic, and hexadecanoic acids against Enzyme/protein ID: 8COT and docking score recorded respectively as shown in Table 4 [12].

Table 4: GC-MS analysis of n-hexane extracts of stem bark.

Statistical Analysis

The results obtained were expressed as multiple comparisons of Mean ± S.E.M. Significance was determined using one-way ANOVA followed by Tukey Kramer multiple comparison post-test with a p < 0.05 was considered significant [5,7,13].

Extraction and Phytochemical Analysis

The yield obtained from stem bark extracts were 0.5%, 0.83%, and 1.0% for n-hexane, dichloromethane, and 70% methanol fractions respectively, while the stem bark crude extract yielded 4%. Phytochemical screening of the stem bark revealed the presence of coumarin, cardiac glycoside; terpenes, tannins, and saponins however, alkaloids, carbohydrates, and flavonoids were absent in stem bark extracts [14,15].

Aphrodisiac Assessment

Aphrodisiac evaluation of the stem bark extracts revealed an increase in sexual indices of vigor, libido, and potency. The standard drug (testosterone); LDcb, HDcb, and MTb fraction significantly reduced the mount latency at p<0.01 and 0.001 when compared to control as shown in Table 5. The STD and MDcb significantly reduced the mount frequency at p< 0.05. The intromission latency; STD, LDcb, MDcb, and HDcb significantly decreased the IL at p< 0.001 compared to the control. This showed that Carapa procera stem bark extracts significantly enhanced sexual function [8]. STD, LDcb, MDcb and dichloromethane fraction significantly reduced the ejaculation latency at p<0.05-0.001 when compared to control respectively. These are in line with results obtained from the literature [7,9,16,17]. The post-ejaculatory interval is the time taken for the male animal to recover from the depressive effect of ejaculation and subsequent intromission with the female rats. The STD, LDcb, MDcb, HDcb, and DCMb significantly reduced the post-ejaculation interval at p < 0.05 and 0.001 when compared to control as shown in Table 5 [7,16,18]. This ratify that the extracts could reduce the depressive effect of ejaculation and would make the animals have erections for intromission and increase pleasure and satisfaction by both partners [19]. The increase in sexual activity could be due to an increase in the concentration of several anterior pituitary hormones, and dilation of the blood vessel to supply the penile organ which could be due to the inhibition of phosphodiesterase enzyme and potentiates serum testosterone which could stimulate dopamine receptor and sexual behavior [20-21]. Penile erection, the time required for the experimental rats to have an erection after ejaculation characterized by licking of the penile organ is significantly decreased at p<0.05-0.001 for LDcb, MDcb, HDcb, n-Hexane, dichloromethane, and 70% Methanol fractions when compared to control as shown in Table 5.

Table 5: Aphrodisiac assay of stem bark extracts of Carapa procera.

Note: Values represent Mean ± SEM, Significance relative to control; ap<0.05, bp<0.01, cp<0.001, (n = 6)
Keys; VEH= Distilled water (10ml /kg), STD = Standard drug (Testosterone 1 mg/kg), LDcb = Low Dose Crude Extract (44.72 mg/kg), MDcb = Median Dose Crude Extract (89.44 mg/kg), HDcb = High Dose Crude Extract (134.16 mg/kg), n-Hex = n-Hexane fraction (89.44 mg/kg), DCMb = Dichloromethane
fraction (89.44 mg/kg) and MTb = 70% Methanol fraction (89.44mg/kg).

The High dose of the crude extract significantly increased the erection frequency at p<0.01 these are following standard literature [7,22,23]. The presence of 3-acetoxy-20-hydroxypregnane, linolenic acid, Furan, 2,5-dione, oleic acid hexylester, andrographolide and 3-Hydroxycholestan-8,24-diene, 4-decenylfuran-2,5-dione, 7,9-diisobutyloxaspiro( 4,5)-deca-6,9-diendione and 20-hydroxylpregnane- 3-acetate could enhance the sexual effect of DCMb as revealed by the GC-MS analysis of n-hexane extract (Table 4) and A2 (Table 3), A3 (Table 1), A4 (Table 2) fractions obtained from chromatographic fractionation of DCMb extracts. The median dose of the crude extracts significantly increased the weight of the animals at p<0.05 compared to the control, which could be due to the anabolic effect of the extracts (Table 6). The standard drug (Testosterone), Low-dose crude extract and dichloromethane fraction significantly increased the weight of the liver at p<0.05, 0.001, and 0.01 respectively which could be a sign of toxicity. The stem bark extract did not have any significant effect on the weight of the testes, epididymis, kidneys, and vas deferens. The extracts showed an insignificant effect on the weight of the seminal vesicle, however, dichloromethane increased significantly the weight of the seminal vesicle at p<0.05. This could be due to enhanced sperm production due to the effect of steroids and triterpenes presence [7,19].

Table 6: Effect of stem bark extract of Carapa procera on organs and tissues.

Note: Values represent Mean ± SEM, Significance relative to control; ap<0.05, bp<0.01, cp<0.001, (n = 6).

Histological Assessment of Stem Bark

The histological assessment of the testis showed the presence of spermatogonia, spermatozoa in seminiferous tubules, and Leydig cells, but interstitial space is normal compared to control (distilled water), and the standard drug (testosterone) administered daily for 7 days (Figure 2).

Figure 2

Figure 3

Spectroscopic Analysis

The proton NMR spectrum of the compound (Sample A1) exhibited four prominent peaks. A signal at 2.37(J = 8.0 H3) integrating for 2H showed as a triplet and assigned to CH2- attached to the C-2 position. This downfield chemical shift value is due to the deshielding effect by the neighboring carboxylic acid group. A multiplet at 1.65 was assigned to the CH2 of carbon (C-3) position and integrated for 2H. An intense peak appearing as a multiplet at δ 1.27 integrated for 20 protons, representing long chain (CH2)n and assigned to position (C-4 to C-13) protons. A triplet at δ 0.90 integrating for 3H representing Sp3 hybridized proton was assigned to position C-16. This confirmed the presence of the alkyl chain in fatty acid molecules. The 13C NMR spectrum displayed 16 carbon atoms; Ten methylene (CH2), Methyl, and a quarternary carbon signals. The spectrum showed a carbonyl group appearing at 179.9 ppm the most downfield carbon (C-1), this also showed an important correlation in H-C HMBC spectra, that the carbonyl carbon correlates with proton signal at 2.37 ppm assigned and attached to carbon at position C-2 Signal at δ 34.0 was assigned to carbon (C-2), signal δ 31.9, was assigned to C-3, signal δ 29.1-29.7 assigned to C-4 to C-13, a signal at δ 24.68 and 22.69 assigned to carbon (C14-15) respectively. The signal at δ 14.1 was assigned to C-16, terminating the alkyl fatty acid. This is also supported by the result of IR analysis showed prominent peaks at 1781 cm^-1 due to the carbonyl group of carboxylic acid, 3004.2 cm^-1 due to =CH₂ stretch, 2914.8 and 2847.7 cm^-1 due to CH₂ and CH₃ stretch.

Sample A1 was subjected to GC-MS analysis revealed the presence of two peaks with a retention time (16.204 and 17.631) and percentage area (98.20 and 1.80) percent. This further revealed that sample A1 contained hexadecanoic acid as the major constituent (98.20) and oleic acid (1.80) as a contaminant. This was ratified by the 1H and 13C-NMR spectra using CDCl3 as a solvent which also served as an internal reference (1H NMR shift value of residual proton at δ 7.29 ppm) as shown in Table 7 [24]. DEPT-45 displayed methine (CH), methylene (CH₂), Methyl (CH₃), and the absence of carbonyl or quarternary carbon. The 1H NMR further revealed the presence of olefinic protons resonates at a narrow chemical shift δ 5.369 and δ 5.367 ppm [25]. This is also reflected on the 13C-NMR spectra at δ 130.03 and δ 129.73 ppm due to the unsaturated ethylene group (CH=CH) due to the unsaturated oleic acid group. This was also reflected in the H-C HMQC corresponding to δ 5.369 and δ 5.367 ppm respectively. H-C HMQC further, revealed the absence of H-C correlation at δ 7.29 ppm as stated by Knothe and Kenar, 2004 [24]. The HMQC also revealed that carbon 34.02 and 31.93 ppm is attached to the H2 proton at 2.37 ppm, and multiplet at δ 1.6 ppm to carbon δ 31.93 ppm. The intense peak at 1.27 ppm of 1H-NMR is due to long-chain fatty acid corresponding to δ 29.6-29.06 ppm of the 13C NMR. The carbon peak at δ 14.1 ppm is attached by (H3) to 0.90 ppm of the proton which is triplet [26].

Table 7: ¹H and ¹³C-NMR of Sample A1 [26, 28, 30, 31, 32, 33, 34].

These are in line with H-H COSY. The oleic acid is a minor contaminant because it is colorless liquid oil at room temperature with a melting point (13-14oC) while hexadecanoic acid (60-62oC) melting point determined using gallenkamp melting point apparatus while the reference standard (62.9oC). The GC-MS analysis of sample A1 (Table 4), gives a molecular ion peak of 256 due to C16H32O2 and the elimination of C2H5 gives m/z = 29, and gives 227, and the elimination of methylene ion (CH2 + = 14); yield; 213; 199; 185; 171; 157; 143; 129; 115 and elimination of (OH+ = 17) to give m/z = 98 [27-29]. This was compared with the NIST library. Based on the foregoing, sample (A1) was proposed as hexadecanoic acid, and it was compared to previous literature (Figure 3) [26,28,30-34].

Molecular Docking

Molecular docking is used to predict the affinity of ligands against target proteins [35]. The binding affinity (kcal/mol) and interaction of sildenafil, hexadecanoic, and oleic acids are; -6.5, -4.4, and -4.5 respectively. The calculated molecular properties and interaction of the ligand with amino acid residues of phosphodiesterase enzyme as shown in Table 8, and Figure 4. This implies that sildenafil had a higher binding affinity when compared to hexadecanoic and oleic acid in inhibiting phosphodiesterase 5 enzyme [11,36,37] However, the Adenyl cyclase model, alprostadil may directly stimulate this enzyme by binding as an agonist on the EP2 receptor, which in turn activates adenylate cyclase leading to accumulation of 3’5’-cAMP which is responsible for therapeutic effect which include, smooth muscle relaxation and increasing peripheral blood flow. Alprostadil had a binding affinity of -6.35 when compared to oleic and hexadecanoic acid with a docking score of -2.61 and -1.46 respectively. This implies that alprostadil had a higher binding affinity compared to oleic and hexadecanoic acid in stimulating adenyl cyclase enzyme which is implicated in male erection as shown in Figure 5 [38]. The pharmacokinetics and calculated molecular properties (Table 9) [39], of the samples obeyed the Lipinski rule of five and implied the potential drug-like molecule of the samples [38].

Figure 4

Figure 5

Table 8: Calculated molecular properties and binding affinity.

Table 9: Pharmacokinetic Properties.

The extract of Carapa procera enhanced sexual indices in male albino Wistar rats. This corroborates the use of Carapa procera stem bark in ethnomedicine as an aphrodisiac agent. It also ratifies that the best solvent for extraction to obtain an optimum aphrodisiac effect is 70% methanol.

ADCO, ECJ, and EIM designed the research, the experiments were carried out by ADCO and AF while ECJ, and EIM supervised the studies. Molecular docking was performed by SJB. AF and ROR reviewed the manuscript. ADCO wrote the manuscript and performed data analysis. Vetted by ECJ and EIM, and proof read by all the authors.

ADCO, ECJ, and EIM designed the research, the experiments were carried out by ADCO and AF while ECJ, and EIM supervised the studies. Molecular docking was performed by SJB and PAC, Data analysis and the manuscript were written by ADCO, Vetted by ECJ and EIM, and proofread by all the authors.

This research was funded by Tetfund, Nigeria.

Further data that support the findings of this study are available with the corresponding author, upon reasonable request.

Ethics approval and consent to participate: Ethical approval was obtained from the animal ethical committee of the Department of Pharmacology and Toxicology, Faculty of Pharmacy, Niger Delta University on the Use of Laboratory, Animals and in line with international standard guidelines.

Consent for Publication

All Authors agreed to publish this article.

Conflict of Interest

No conflict of interest.

Guideline and Legislation on the Use of Medicinal Plants

Not applicable.

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