Fresh leaves of the F. benghalensis (5 kg) were collected from Chakwal, Pakistan. The plant was authenticated by a taxonomist from the Department of Botany, University of Sargodha (UOS), Pakistan. The leaves were washed and dried in the air, followed by the production of coarse powder using a mechanical grinder. Powdered leaves (20 g) were soaked in 200 ml of either solvent—petroleum ether (PE), ethyl acetate (EtOAc), and 70% v/v aqueous ethanol (aq. EtOH) labeled as FBLE1-3. The suspensions were placed on an orbital shaker for 5 days. Later, the mixtures were filtered, subjected to evaporation, concentrated using porous aluminum foil, and finally kept at 4°C until further use (Handa et al., 2008).

The inhibition of the catalytic activity of SRD5AII was studied by initially mincing 4 g of the prostate gland of a male goat and crushing it under liquid nitrogen. The tissue was further homogenized using a handled homogenizer in 20 ml of sodium phosphate buffer (SPB) (pH 6.5, 50 mM) containing 1 mM EDTA (200 µl), 100 mM sucrose (64 ml), 1 mM sodium azide (200 µl), and a protease inhibitor tablet (Roche Pharma, Mannheim, Germany). Homogenate was centrifuged at 15,000 rpm for 20 min at 4°C, separating the supernatant for further use as a crude enzyme source. The soluble protein was tested by Bradford assay (Rehman et al., 2009).

For the reverse-phase high-performance liquid chromatography (RP-HPLC; Shimadzu, Japan), Testovirone ampoule (testosterone enanthate, 250 mg/ml; Bayer Pharma, Leverkusen, Germany) was taken as standard. Six reaction mixtures (RMs) were prepared as positive control (PC), reaction control (standard), complete reaction (internal standard, IS), and others with FBLEs (1–3) (Table 1). The RMs were incubated at 37°C for 30 min, and the reaction was terminated by adding 2 ml of EtOAc followed by the addition of 150 µl prednisolone 250 μg/ml ethanol as an IS. The RMs were mixed for 1 min and centrifuged at 5,000 rpm (10 min, 4°C). Subsequently, the water phase was frozen at −80°C, and the organic layer was decanted and evaporated. The concentrated residue (1.5 ml) was later diluted in 3 ml methanol. Finally, RMs were syringe filtered (0.2 µm) before HPLC run to avoid contamination (Sher et al., 2015; Kumar et al., 2011).

The RP-HPLC was performed using Chromatographic Column C18-ODS type (250 mm × 4.6 mm), maintaining system conditions at 40°C and flow rate 10 μl/min for 30 min, while absorbance was recorded at 254 nm. Isocratic elution was performed, making a mobile phase with methanol and water (80:20) and filtering it to remove any contamination. The percentage inhibition of SRD5AII was observed from the peak height and the peak area ratio (r) applying the following formula (Kumar et al., 2011): ( % ) = r   s a m p l e   −   r   r e a c t i o n r   s t a n d a r d   −   r   r e a c t i o n × 100   r = Area of sample Area with least value ; Peak area (mm²) = height × width_1/2.

To determine the ligand–receptor binding interactions, we docked SD5ARII with minoxidil and reported bioactive constituents: (-)-Gallocatechin, Rhein, and Mucusoside of FBLEs using AutoDock Vina and AutoDock Tools 1.5.6. The X-ray crystal structure of the target mammalian’s SD5ARII with a resolution of 2.8 Å was retrieved from Protein Data Bank (PDB ID: 7BW1). After docking, the binding energies (BEs) were noted, and the lowest values were chosen to view the interactions in PyMol (Bhaskara Rao et al., 2014; Hassan et al., 2020; Bilal et al., 2021; Rehman et al., 2021).

The female albino rabbits (6–11 months old; 1–2 kg) were purchased from the University of Veterinary and Animal Sciences, Lahore, Pakistan. Prior to proceeding further, rabbits were authorized by the ethical committee of the College of Pharmacy, UOS, providing approval no. 70B18 IAEC/UOS. Animals were acclimatized for 3 days providing standard food and water in an animal housing facility at the College of Pharmacy, UOS. Rabbits were divided into eight groups, shaved at the dorsal side area (5 × 4 cm), using a razor, and left for 24 h to observe any edema or erythema. HG formulations (500 µl) were sprayed once a day on the nude area, and eventually HG was observed. On the 28th day, rabbits were sacrificed, and skin biopsies were performed while skin tissues were kept in 10% formalin for histological study (Roy et al., 2008; Imtiaz et al., 2017). Ten hair strands were randomly plucked every week, and an average length of each hair was noted in mm using a digital Vernier caliper (Adhirajan et al., 2003). Histology was performed using subcutaneous hematoxylin and eosin staining (Lin et al., 2015) to analyze HFs, observed at ×10 magnification using a digital microscope (Bresser, Rhede, Germany).

Phytochemical analysis was performed to analyze various phytoconstituents in crude extracts like phenolic compounds and tannins, alkaloids, saponins and glycosides, terpenoids and steroids, anthocyanins, flavonoids, coumarins, and quinones (Harborne, 1998). The results of qualitative analysis were noted with color change, foamy appearance, or precipitates formation.

The FBLEs were analyzed for calcium, iron, zinc, and potassium using FAAS. The powdered leaves (50 mg) were digested using 15 ml of HCl and HNO3 (3:1) to extract metal ions. The acid mixture was poured into the beaker having powdered leaves and placed on a hot plate until the sample got mixed thoroughly. The acidic mixture was allowed to cool, then filtered, and finally diluted up to 100.0 ml using distilled water. Stock solutions of 3, 6, 9, 12, and 15 ppm of metal salts were prepared for mineral detection, and concentrations were noted in mg/g (Ahmad et al., 2014). Chemical characterization of FBLEs was done through Fourier transform infrared (FTIR) spectroscopy. The greasy extracts were dried using KBr pellets and analyzed in the mid-range, 4,000–600 cm-1 (Gopukumar et al., 2016).

Total polyphenol content (TPC) was estimated by modifying a method (Abbas et al., 2021), taking gallic acid as a standard, and FBLEs (50 mg) diluted in 5 ml methanol were used to determine TPC. The absorbance was noted at 765 nm in triplicate. Total flavonoid content (TFC) was estimated by aluminum chloride colorimetric assay, taking catechin as a standard, using FBLE dilutions prepared for TPC. Each reading was measured in triplicate at 510 nm.

Radical scavenging activity (RSA) was assessed using DPPH (1,1-diphenyl-2-picrylhydrazyl) assay, taking 100 µl FBLE dilutions, added to 3 ml of DPPH (0.1 mM), followed by incubation at room temperature in the dark for 30 min. The absorbance was recorded in triplicate at 517 nm while taking 3 ml of DPPH as control (Abbas et al., 2021). Data were recorded as % RSA, and IC₅₀ (minimum inhibitory concentration) was calculated using MS Excel 2016. The percentage of RSA was calculated using the following formula; RSA ( % ) = A b s   c o n t r o l   –   A b s   s a m p l e A b s   c o n t r o l × 100

The statistical analysis was scrutinized by one-way ANOVA via Tukey test using SigmaPlot ver. 14.0. The Pearson correlation coefficient (p) was applied to correlate HG results among all test groups at significance level p < 0.05 or highly significant p < 0.01 using SPSS version 21.

The percentage yield of FBLEs (PE, EtOAc, and 70% aq. EtOH) was calculated as 3.9%, 4.8%, and 20.6%, respectively.

The quantified protein from the male goat prostate gland was 7.99 mg/ml of BSA. In the RP-HPLC charts, a prominent peak (b) at retention time (RT) around 6 min was entitled as the testosterone’s peak (Figure 5D). Results are justified by the peak height intensity (mAU), which determines how much testosterone has been left behind in the RM. The results of (r) and peak height revealed more unconverted testosterone in hydroalcoholic FBLE, favoring sufficient percentage inhibition for SRD5AII, than Minoxin 5% (Table 2). No nicotinamide adenine dinucleotide hydrogen phosphate (NADPH) was added in the “reaction control” to compare the peak height of the PE 1-3 and PC. However, the minimum peak height a) observed in the complete reaction with IS indicated more testosterone conversion to DHT. A delayed peak c) refers to SRD5AII due to the hydrophobic nature of both steroid enzyme and stationary phase.

Docking studies showed a strong binding interaction of Rhein (BE; −7.4 kcal/mol) with the enzyme’s active sites, revealing a prompt inhibitory potential against SD5ARII. In comparison, the binding energies (Bes) of phytoconstituents of crude FBLEs showed significant interactions with the receptor protein than Minoxidil. Figure 1 and Table 3 show protein–ligand interactions and values of their BEs in Kcal/mol and type of binding interaction.

No skin irritation was observed in the rabbits, and rapid hair regrowth was noticed in the first week that varied subsequently Figure 2. The hair texture appeared to be rough and weak in PC and NCs but smooth in others. The average hair length confirms that weekly HG was approximately 1–2 mm Figure 3. The Pearson correlation (p < 0.05) revealed an exceptional HG rate in group 5. From the photomicrographs of the longitudinal section, HF phases and DPCs were observed in treated tissues (Figure 4). The pH outcome was 7.6, 4.3, and 6.4 for FBLE1-3, respectively.

The screening of the preliminary phytochemicals has shown the presence of carbohydrates, proteins, phenolic compounds, flavonoids, alkaloids, saponins, glycosides, steroids, and tannins. The results of all phytochemicals are represented in Table 4.

FAAS showed the mineral concentrations in mg/g (copper = 13; zinc = 9; iron = 23.4; potassium = 21.8) found in powdered leaves. In FTIR fingerprints, peaks are attributed to stretching and bending vibrations, characterizing functional groups in essential metabolites (Figures 5A,C).

FBLEs showed concentrations for TPC, TFC, and RSA, presented in Figure 5B.

Different concentrations of FBLEs against DPPH were tested to find the inhibitory potential of antioxidants present in the crude extracts. It has been observed that %RSA increased as the concentrations of FBLEs increased. Data were recorded as mean ± SD. Table 5 shows the maximum %RSA and IC₅₀ values for FBLEs.