Phytochemical profiling, heavy metals composition, in silico aphrodisiac potential, and ADMET study of Gardenia erubescens

1. Introduction

Impotence otherwise termed erectile dysfunction (ED) is a recurrent and persistent inability to achieve and/or keep sufficient erection for satisfactory intercourse following sexual stimulation (1). Erection or tumescence is a state of engorgement characterized by a flow of blood induced by neurotransmitters released from the cavernous nerves during sexual stimulation, though it occurs spontaneously (1). Causes of ED are classified based on conditions associated with hypoactive and normoactive sexual activity with the former covering attraction toward partners, ailments (including hypogonadism and hyperprolactinemia), and psychogenic conditions (2) while the latter covers metabolic, vascular, neurological, and inflammatory ailments (1). For centuries, the use of pharmaceuticals and aphrodisiacs was employed for the management of ailments, however, the current approach includes improvement in lifestyle and the use of drugs, notably the phosphodiesterase inhibitor sildenafil (1). Other approaches include nutraceuticals and physical and surgical treatments. Sildenafil has been previously associated with visual impairment and hepatotoxicity, stomach upsets, headaches, and nosebleeds (3-5). Medicinal plants with aphrodisiac activities have emerged as alternatives to sildenafil attributed to their minimized side effects (6-9).

Medicinal plants are vital for both traditional and modern medicine, and pharmaceutical industries. In traditional medicine, medicinal plants are utilized in herb forms prepared in different forms taken orally, topically, or through inhalation for the treatment of ailments, especially in rural areas where there is poor healthcare delivery (10, 11). The synergy and low side effects of medicinal plants make them desirable especially considering their affordability compared to synthetic medicines. In modern medicine, different medicinal plants were reported to possess pharmacological properties thus, finding their way for utilization against different conditions such as cancer, diabetes, and bacterial, fungi, and viral infections (12, 13). In the pharmaceutical industries, medicinal plants serve as a vital source of bioactive compounds used in the synthesis of novel therapeutics. Different plants were reported to be associated with aphrodisiac pharmacological properties including Gardenia erubescens (GE) (12, 14).

The therapeutic roles of medicinal plants are credited to their phytochemical components made up of different bioactive compounds working individually or synergistically to produce pharmacological effects (15). Phytochemicals are substances produced by plants to perform important functions other than nourishment such as protection against pathogens (16). GE is a popular plant which is called Gaude in Northern Nigeria. In traditional practice, the root of the plant is utilized as an aphrodisiac while the aerial parts are applied in the management of gonorrhea and insomnia by herbalists (17, 18). The plant was also reported to exert moderate antioxidant, anti-obesity, and anti-plasmodial activity (14, 19). The application of in silico studies including molecular docking, molecular dynamics, and ADMET significantly improves the drug discovery and development process paving the way for wet lab and reducing cost and time in identifying lead compounds from a library of compounds. Additionally, this aspect allows for the improvement of the pharmacological properties of the lead compounds. Thus, in our study, we conducted the phytochemical profiling and determined the heavy metals composition and in silico aphrodisiac potential of ethanol extract of GE seeing it reported aphrodisiac application in traditional ethnomedicine, thus leading to heavy metal poisoning.

2. Experimental Section

2.1 Plant material

A stem bark sample of the GE was collected from Girei Local Government, Adamawa state, Nigeria. A voucher specimen (ASP/FT/111) was deposited after identification by a Forest Technologist from the Forestry Technology Department of Adamawa State Polytechnic, Yola, followed by shade-drying and grinding using a blender.

2.2 Extract preparation

The sample was extracted by maceration of 400 g of bark powder of GE in 1.5 L of 90% (v/v) ethanol for 48 h, followed by filtration and concentration to dryness in a rotary evaporator (Buchi Rotavapor R-200) at 40oC to yield the ethanol stem bark extract (ESBE) of GE (20).

2.3 Qualitative phytochemical analysis

Phytochemicals present in the ESBE of GE were identified using the method reported previously to detect alkaloids, saponins, steroids, glycosides, terpenoids, and flavonoids (20). The chemicals and reagents used in the present were of AnarlaR obtained from Xilong Scientific Co., Ltd. Guangdong, China.

2.4 Quantitative phytochemical analysis

The quantification of phytochemicals in ESBE of GE was carried out by methods reported previously as follows:

Total Alkaloids content

Alkaloids were quantified by the gravimetric method (21). Briefly, 0.5 g extract was introduced into a conical flask and 10 ml of 20% aqueous ethanol was added. The sample was heated over a water bath for 1 h with continuous stirring at about 550°C. The concentrate was transferred into a 250 ml separator funnel and 5 mL of diethyl ether was added and shaken vigorously. The aqueous layer was recovered and the ether layer was discarded. About 10 ml of n-butanol was then added followed by the addition of 2 ml of 5% aqueous NaCl. The remaining solution was heated over a water bath. After evaporation, the sample was dried in the oven to a constant weight and calculated using Equation 1.

% Total metabolites=(Weight of residue)/( Weight of sample)×100% (Equation 1)

Saponins content

Quantification of saponins was done by the method previously described (22). Exactly 0.5 g extract was dispensed into a conical flask and 10 mL of 20% aqueous ethanol was added. The sample was heated over a water bath for 1 h with continuous stirring at about 550C. The concentrate was transferred into a 250 mL separating funnel and 5 mL of diethyl ether was added and shaken vigorously. The aqueous layer was recovered and the ether layer was discarded. Exactly 10 mL of n-butanol was then added followed by the addition of 2 mL of 5% aqueous NaCl. The remaining solution was heated over a water bath. After evaporation, the sample was dried in the oven to a constant weight and calculated using Equation 1.

Total glycosides content

Glycosides were quantified as described previously (23). Exactly 0.5 g of the extract was dispensed into a 100 mL volumetric flask containing 10 mL of 70% of ethanol. It was boiled for 2 minutes in a water bath, filtered and the filtrate was diluted with 20 mL of distilled water. Afterwards, 2 mL of 10% lead acetate was added to this volumetric flask to precipitate the chlorophyll, tannins, and alkaloids, followed by filtration. The filtrate was transferred to a separating funnel containing 10 mL of chloroform. The funnel was shaken by inverting repeatedly. Two layers were formed, and the lower organic layer was collected (chloroform); dried, and weighed. The percentage of total glycosides contents was determined using Equation 1.

Flavonoid content

Quantification of flavonoids was carried out according to a method described previously (21). Exactly 0.5 g of the extract was mixed with 10 ml of 80% aqueous methanol. The whole solution was filtered through Whatman filter paper. The filtrate was transferred to a pre-weighed crucible and evaporated into dryness over a water bath weighed, and calculated using Equation 1.

2.5 Gas chromatography-mass spectrometry (GC-MS) analysis

GC-MS analysis was carried out with a combination of a Gas chromatography-mass spectrophotometer (Agilent 19091-433HP, USA), fitted fused with a silica column while the settings and compound identification were as we previously described (24).

2.6 Determination of heavy metal composition

A gram of the samples was burned to ash at 500ºC for 1 h, dissolved in 25 mL of 10% HCl, and made up to 100 mL (25). Chromium (Cr), cadmium (Cd), and lead (Pb) contents were quantified by the method previously described (25) using an Atomic Absorption Spectrophotometer (AAS) (Buck Scientific AAS210).

2.7 Molecular docking and molecular dynamics simulation

The compounds identified in ESBE of GM were initially screened applying the Lipinski’s rule and Veber filters using the DruLiTo software (https://niper.gov.in/pi_dev_tools/DruLiToWeb) predicting 7 with drug-likeness properties out of the 25. The structures of the 7 compounds and sildenafil citrate (standard drug) were downloaded from the PubChem website (https://pubchem.ncbi.nlm.nih.gov) in SDF format and energy minimized with PyRx virtual screening Tool software (version 0.8). Table 1 shows the list of compounds and sildenafil citrate inclusive of their PubChem ID. The docking targets including Human Arginase II (HMA2) and Phosphodiesterase 5 (PDE5) with PDB IDs of 1PQ3 and 5ZZ2 respectively were downloaded from the RSCB database (https://www.rcsb.org) and prepared by removing identical chains, water molecules, and heteroatoms using AutoDockTools version 1.5.7 (26). The docking pockets (coordinates) for HMA2 (X= 69.73, Y= 54.15, and Z= -4.94) and PDE5 (X= 32.49, Y= -31.77, and Z= -37.40) were identified by the Prankweb online server (https://prankweb.cz) (27). The docking was carried out using the Vina wizard of the PyRx software. The inhibition constant (Ki) was evaluated from the binding affinity (BA) by the equation; Ki = exp ∆G/RT where T=298.15 K (temperature) and R=1.985 x 10-3 kcal-1 mol-1 k-1 (the universal gas constant) and ∆G = binding affinity (28). The 2D and 3D dock poses of the complexes were viewed with the Biovia Discovery Studio visualizer software (version 16.1.0). The docking targets (HMA2 and PDE5) were further subjected to MDS using the Webnm online server (http://apps.cbu.uib.no/webnma3) (29) to identify cluster and residue displacements with their structures.

Table 1. List of Ligands and their PubChem IDs.

2.8 ADMET predictions

The absorption, distribution, metabolism, excretion, and toxicity (ADMET) of the top docked compounds were predicted using the pkCSM online server (https://biosig.lab.uq.edu.au/pkcsm) (30) to further ascertain their pharmacological properties.

2.9 Statistical analysis

Data obtained in the present study were expressed as mean ± standard error of triplicate determinations' mean (± SEM) evaluated with Statistical Package for the Social Sciences (SPSS) version 22 Software.

3. Result

The phytochemicals identified and quantified in ESBE of GE are presented in Table 2. Flavonoids were present in the highest concentration (32.67% ±1.45), followed by alkaloids and saponins with concentrations of 22.33% ±1.45, and 20.17% ±1.88 respectively. Glycosides were detected in the least concentration (0.55% ±0.03), with the absence of steroids and terpenoids.

Table 2. Phytochemical composition of ethyl acetate stembark extract of Gardenia erubescens.

Note: concentration values are in triplicate determinations (± SEM).

Table 3 presents the various compounds identified ESBE of Gardenia erubescens showing their retention times, peak areas, molecular weights, and formulas. The fatty acid linoleic acid had the highest (28.01%) peak, followed by palmitic acid (14.08%), and 9, 17-Octadecadienal (11%). Ethyl palmitate, pentadecanoic acid, and decanoic acid were identified with peak areas of 8.03%, 4.98%, and 4.66% respectively. Other compounds identified were 5-Hydroxymethylfurfural, ethyl stearate, palmitic acid glyceryl ester, squalene, and ethyl icosanoate.

Table 3. Bioactive compounds identified in ethyl acetate stembark extract of Gardenia erubescens

The structures of the identified compounds displaying their functional groups are also shown in Figure 1, while the chromatogram of the GC-MS analysis is present in Figure 2, revealing the retention time and peak areas of the compounds. GC-MS analysis identified 25 compounds in ESBE of G. erubescens. Most of the compounds identified were long-chain fatty acids and a few aromatic compounds, which isn't surprising considering the oily nature of the extract.

Figure 1. Structures of compounds identified in ethyl acetate stembark extract of Gardenia erubescens.

Figure 2. GC-MS chromatogram of ethyl acetate stembark extract of Gardenia erubescens.

The heavy metals present in the ESBE of GE are presented in Table 4. Chromium (Cr) was present in the highest concentration (0.145 ppm ±0.03), followed by lead (Pb) (0.065 ppm ±0.03). Cadmium had the lowest concentration (0.001 ppm ±0.00).

Table 4. Heavy metals composition of ethyl acetate stembark extract of Gardenia erubescens.

Note: concentration values are in triplicate determinations (± SEM).

Table 5 reveals the docking interaction of the top compounds and sildenafil citrate with HMA2 depicting the BA and Ki. Although sildenafil citrate showed the least BA (-8) and Ki (1.35 µM) than the compounds, ethyl D-glucose had the least BA (-6.3) and Ki (23.82 µM) amongst the compounds next to Tyrosinol. Furthermore, Figure 3 shows the docking interaction of sildenafil with HMA2 depicting the binding interactions. Four conventional and carbon-hydrogen bonds (HBs) were observed with additional 3 π-interactions. The binding interactions of HMA2 with ethyl D-glucopyranoside are shown in Figure 3. Exactly 3 conventional and 1 HBs were observed in the interaction with π-interaction with Thr265 acting as an unfavorable donor-donor. Figure 3 depicts the binding interactions of HMA2 with IV showing the HBs and π-interactions. Asp143, 253, and 251 participated in conventional HBs while His145 in π-cation interaction with Asp147 as an acceptor-acceptor.