INTRODUCTION:

Libido, erection, orgasm, ejaculation and detumescence are five interrelated events that must occur in a defined sequence for a normal male sexual response cycle. Any disturbance in the effective performance/completion of this sexual response cycle or disruption of the sequence order is defined as male sexual dysfunction.¹ The majority of medicines now on the market focus on the intromission phase of sexual dysfunction, which is the most prevalent stage. The most often used medications are sildenafil citrate, vardenafil, and tadalafil citrate, which decrease phosphodiesterase type 5 (PDE-5), increasing penile blood flow and tumescence in the process. Unfortunately, these drugs can have severe adverse effects like infertility, myalgia, distorted vision, and rapid hypotension².

A fresh approach is necessary because the present treatment medications do not target all stages of the sexual response cycle and have negative side effects. Aphrodisiacs have long been employed in traditional folklore to increase sexual desire and performance. They can exert their effects on a variety of targets, including the neuroendocrine system, which is important.³

Large perennial tree Ficus religiosa, which is glabrous when young and can be found all throughout the Indian subcontinent up to 170 meters above sea level, can be found.⁴ Traditional uses of the leaves and bark include the treatment of skin conditions, gonorrhoea, ulcers, and antibacterial, antiprotozoal, antiviral, astringent, and antidiarrheal properties.⁵The menstrual cycle is controlled by the leaves, which also claimed antivenom action. Infectious diseases, cancer, and inflammation are just a few of the illnesses for which it has been utilized as a treatment.⁶ This plant is available in entire India and traditional system has been reported to possess various activities like fertility agent, feet cracks, eye diseases, heart strengthen, anti-inflammatory, antileprosy, spleen and hepatoprotective activity, hiccups, antiasthamatic agent, antiemetic and improve the semen secretion etc⁷

The presence of saponin, flavonoids, steroids, phenols, glycosides responsible for the aphrodisiac activity. According to Unani system of medicine, root, bark is aphrodisiac. The fruit powder is also given to enhance fertility and used as aphrodisiac. The leaf juice is used for treatment of sexual disorders^.4,8The present study was therefore aimed to evaluate Aphrodisiac activity the and its underlying mechanism of action.

MATERIALS AND METHODS:

Preparation of drug solution:

Sildenafil citrate was procured as a gift sample from Research-lab Fine Chem. For oral administration to animals, distilled water was used to make a solution of sildenafil citrate.

Collection and authentication of plant material:

The Fresh bark of the Ficus religiosa plant were collected during the month of February and March 2021. Authentication of plant was done at Kasturba Walchand College, Sangli, by Dr. Wadmare Sir, HOD of Botany department.

Preparation of extract:

The resultant dried powder of FR bark extract (30gm) was defatted using petroleum ether maceration. In a Soxhlet apparatus, the powder was extracted using ethanol as the solvent. The obtained ethanol extract was dried and concentrated at room temperature. Prior to being used for animal experiments, the test sample of ethanol extract was prepared using distilled water in the proper concentrations.

Preliminary qualitative phytochemical analysis:

Different classes of photochemical constituents such as alkaloids, steroids, tannins, flavonoids, saponins, cardiac glycosides and triterpenoids were detected by subjecting extract to preliminary qualitative screening.⁹

Procurement of animals and ethics approval:

Swiss albino male wistar rats weighing (200-250gm) was used in present study. The experiment was carried out after approval and clearance from IAEC constituted in accordance of CPCSEA, India (Protocol no – IAEC/ABCP/11/2022-23) of Appasaheb Birnale College of Pharmacy, Sangli, Maharashtra. (843/PO/Re/S/04/ CPCSEA) India.

In silico molecular docking:

Computational docking was carried out using VlifeMDS to assess the affinity of several ligands towards the phosphodiesterase enzyme Receptor (PDE5).^10,11 The structures of all the ligands were drawn by using Chemdraw application. The structure of the phosphodiesterase enzyme Receptor (PDE5) was retrieved from the PDB database (PDB ID-1UDT). Protein structures were prepped for docking by removing water molecules and adding hydrogen.

Behavioural tests:

Experimental design:

To investigate the Aphrodisiac like effects of plant extract, the animals were divided into four groups (n = 6 per group): control (10mg/kg distilled water), sildenafil citrate (5mg/kg) treated, FRBE (200, 400mg/kg) treated. The extract and sildenafil citrate were orally administered using a gavage needle. The treatment was given for 21 days. The Aphrodisiac activity was evaluated on 1^st, 7^th, 14^th and 21^stday. After 21-day treatment the rats were sacrificed, accessory organs were isolated and total cholesterol, sperm count, testosterone levels were estimated.

For evaluation of aphrodisiac activity following models were used.

Physical methods:

A] Test for libido:

Sexually experienced male albino rats should be kept singly in separate cages during the experiment. The female rats should be made receptive by hormonal treatment and all the animals should be accustomed to the testing condition as previously presented in mating behavior test.¹²The animals should be observed for the Mounting Frequency MF, IF, ML, IL on the specific day according to the design of the experiment (likely 7th, 14^th, 21^st day.) Selected test groups received the prescribed oral medication of Ficus religiosa bark extract for 21 days. The number of mountings, intromission should be noted. The animals should also be observed for intromission and mount latency.¹³Evaluations were made on the mounting and intromission frequencies. It was remarked how many mountings and introductions were there.^14,15

B] Orientation behavior test:

Throughout the trial, the male animals should be housed alone in separate cages. At least 30 minutes to an hour prior to the start of the experiment, the extracts must be delivered. The effect of Ficus religiosa bark extract on male albino rats was evaluated using a variety of criteria in this orientation behaviour test. Effects of Ficus religiosa bark extract on male rats' behaviour were assessed using three different parameters: behaviour towards females, which included licking and sniffing at the female anogenital organ;¹⁵ self-exploratory behaviour, which included non-genital and genital grooming; and general behaviour, which included exploring, rearing, and climbing.¹⁵We counted the total number of incidents for each behaviour attribute. The episodes of the rats were assessed, and a specific number of points were assigned to each episode^.15

C] Determination of hesitation time of male rat and attraction of male towards female:

In order to conduct the experiment successfully, the male animals must be housed separately in single cages. At least 30 minutes to an hour before the experiment is to start, the extracts must be administered. A female rat was put in a cage with a 15 cm-high wooden partition between the male and female sections. The amount of time the male rat needed to hesitate before attempting to pass the barrier was measured in seconds.^16,17

Similar to this, during a 15-minute observation session, a score between 0 and 5 was used to rate the subject's attraction to women. The male rat was given a score of -5 for successfully crossing the partition each time, a score of -2 for trying to climb, and a score of 0 for showing no interest in climbing. On days one, seven, fourteen, and twenty-one of treatment, the readings were recorded. By measuring a male rat's readiness to move past an unpleasant or impeding situation, this test can reveal whether or not he is attracted to another person sexually. Male rats from all groups were utilized in the experiment, and scores for attraction and hesitation time were recorded for each animal.^15,16,17

Effect of test extracts on sexual organ weight and histological studies:

After the mating behaviour analysis and serum cholesterol level evaluation, the next morning on (Day 22), all selected control, standard and experimental groups of male albino rats were evaluated for their body weight and histological studies. Body weight of all selected experimental animals was recorded. The animals were completely anaesthetized with anaesthetic ether for blood withdrawal then sacrificed by using a CO2 chamber. Testis, seminal vesicles, epididymis, vas-deference, penis and prostate glands along with vital organ like liver, kidney, adrenal gland, and spleen were carefully removed and weighed using 4- digital electronic balance. weight of each organ was determined.^15,18

In -vivo sperm count:

All of the animals were put down using a CO2 chamber on day 22. The left and right epididymis were inspected after being removed from the surrounding fat. The cauda part of the epididymis was thoroughly squeezed until milky suspension was obtained using a needle and forceps. Sperm suspension was created by mixing sperm with diluted fluid (formalin, sodium bicarbonate, and distilled water). This diluted liquid was vigorously stirred before being put onto Neubauer's haemocytometer as a droplet and counted the typical number of sperm cells in each chamber.^15,19 Calculation for sperm count Total sperm count in the four squares (each of 0.1mm³) =N Sperm count/cauda epididymis =N ×1.25×10⁵

Biochemical studies:

Determination of Total Serum Cholesterol:

Cholesterol is the precursor in the synthesis of many physiologically important steroids such as bile acids, steroid hormones and vitamin D and its requirement for normal testicular activity. blood was withdrawn by retro orbital blood withdrawal technique. Ether was used for anaesthesia. Total serum cholesterol concentrations were determined by the Chod-PAP method. After mixing 0.01 ml of serum with 1 ml of cholesterol reagent, and the mixture was incubated for 5 minutes at 37°C. Absorbance was measured at 505nm.^15,18,20

Preparation of serum for testosterone analysis:

Testosterone is one of the important male gonadal hormones and is essential for the development of male sex organs and secondary sexual characteristics in men.¹⁷The blood was collected via cardiac puncture using Ether anaesthesia. Using sterilized syringe, 3ml of blood was collected in appropriately labelled blank tubes containing no anti-coagulant. The tubes were given 5 to 10 min for coagulation before they were subjected to centrifugation. The needles were changed for every single animal. After coagulation these tubes were centrifuged at 3000 rpm for 10 min. The supernatant formed after centrifugation was sucked and collected in a clean, empty tube for testosterone assay¹⁵

Testosterone analysis:

Quantitative determination of total testosterone was done by the competitive fluorescence immunoassay method mentioned in the manufacturer’s test procedure (Finecare^TM) testosterone ELISA kit). The testosterone level was measured in ng/ml^15,20

Statistical Analysis:

The date was stated as mean±SEM. From the study, the obtained biochemical parameters were statistically analysed using Two-way ANOVA followed by Dunnett’s test. The software GraphPad Prism was used for analysis of result.

RESULTS:

Phytochemical analysis of plant extract:

Qualitative phytochemical screening revealed the presence of alkaloids, flavonoids, triterpenoids, coumarin glycosides, tannins and saponins.²¹

In silico molecular docking

To predict the potential aphrodisiac mechanism of action, therefore, the identified compounds of Ficus religiosa were obtained by virtual screening and docked with enzymes or targets related to erectile dysfunction i.e., PDE5. In order to evaluate the affinity of different ligands towards phosphodiesterase 5 enzyme Receptor (PDE5), computational docking was performed using VlifeMDS. The structures of all the ligands were drawn by using Chemdraw application. From the PDB database (PDB ID 1UDT), the crystal structure of the Phosphodiesterase 5 receptor was downloaded. Protein structures were prepared for docking by removing water molecules and adding hydrogen. The 8 identified bioactive molecules were docked against the phosphodiesterase type 5 (PDE5) enzyme and compared with the standard PDE5 inhibitor (sildenafil^).8,21,22

Behavioral activity of ethanolic extract of Ficus religiosa bark:

Test for libido:

Mount frequency:

Table no. 1: Effect of Ficus religiosa bark ethanolic extract on mount frequency in experimental rats.

Groups	Day 1	Day 7	Day 14	Day 21
Control	4.33±0.42	4.33±0.42	5.16±1.10	7.33±1.47
Sildenafil citrate 5mg/kg	12.83±0.82*	15.0±1.98**	18.3±2.02***	20.3±1.87***
FR ethanolic extract 200mg/kg	10.83±0.87*	11.33±1.47*	14.66±2.23*	16.6±2.41**
FR ethanolic extract 400mg/kg	10.16±0.83*	13.16±2.53*	16.93±1.03**	20.5±2.42***

Intromission frequency:

Table no. 2: Effect of Ficus religiosa bark ethanolic extract on intromission frequency in experimental rats-

Groups	Day 1	Day 7	Day 14	Day 21
Control	3.17±0.47	3.33±0.3	4.33± 0.84	6.167±0.74
Sildenafil citrate 5mg/kg	10.67±0.8*	12±1.032*	13.83±0.9*	17.67±1.14***
FR ethanolic extract 200mg/kg	6.17±0.65*	9.5±1.05*	11.7±0.76*	15.17±1.30 **
FR ethanolic extract 400mg/kg	8.67±0.71*	10.16±0.90 *	11.5±0.67*	14.5±1.11**

Mount latency:

Table no 3: Effect of Ficus religiosa bark ethanolic extract on mount latency in experimental rats.

Groups	Day 1	Day 7	Day 14	Day 21
Control	241.6 ±14.4	203.37 ±37.2	219.3 ±30.3	200.6 ±27.7
Sildenafil citrate 5mg/kg	196.3 ±24.6*	154.3 ±20.3***	144.8 ±11.9***	136.1 ±13.03***
FR ethanolic extract 200mg/kg	222.3 ±27.4*	215.0 ±40.4**	198.3 ±29.0**	189.83 ±15.6**
FR ethanolic extract 400mg/kg	210.8 ±16.4*	196.3 ±37.4**	178.1 ±25.32**	161.16 ±12.5**

Intromission latency:

Table no. 4: Effect of Ficus religiosa bark ethanolic extract on intromission latency in experimental rats.

Groups	Day 1	Day 7	Day 14	Day 21
Control	289±18.8	277.5±25	257.83±13.5	247.83±30.82
Sildenafil citrate 5mg/kg	265.8±7.12 **	275±6.09 **	181.16±6.441**	173.16±10.23 ***
FR ethanolic extract 200mg/kg	272.17±5.4 *	265±6.557*	204.83±5.08**	198±5.24 **
FR ethanolic extract 400mg/kg	181.6±7.0*	229.833±5.86**	189.833±5.86**	178±11.45***

Orientation Behaviour Test:

Sniffing:

Table no. 5: Effect of Ficus religiosa bark extract on Sniffing in experimental rats

Groups	Day 1	Day 7	Day 14	Day 21
Control	5.67±0.55	5.83±0.94	8.67±1.14	8.17±0.71
Sildenafil citrate 5mg/kg	11.167±0.47*	12.83±0.74*	19.0±1.39**	22.1±0.94***
FR ethanolic extract 200mg/kg	8.67±0.6*	11.00±0.55*	14.83±0.47**	17.83±1.13**
FR ethanolic extract 400mg/kg	9.5±0.56*	12.33±0.66*	17.5±1.17**	20.16±1.01**

Licking:

Table no. 6: Effect on licking behaviour in experimental rats after the oral administration of sildenafil citrate and both extracts.

Groups	Day 1	Day 7	Day 14	Day 21
Control	5.8±0.90	8.5±1.28	11.1±0.30	7.50±0.67
Sildenafil citrate 5mg/kg	13.3±0.80*	14.16±1.57938*	18.6±0.6146**	22.1±0.60**
FR ethanolic extract 200mg/kg	8.6±0.49*	12.5±0.9*	15.16±0.65 *	18.3±0.8**
FR ethanolic extract 400mg/kg	9.83±0.54*	12.6±1.11*	16.00±1.36 *	17.3±1.02**

Rearing:

Table no 7: Effect of Ficus religiosa bark extract on rearing in experimental animals.

Groups	Day 1	Day 7	Day 14	Day 21
Control	9.67±0.8	10.5±0.42	11.50±0.6	12.00±0.9
Sildenafil citrate 5mg/kg	13.83±0.94**	17.0±0.73***	19.1667±0.30***	22.6±0.66***
FR ethanolic extract 200mg/kg	12.16±0.47**	12.8±0.70**	14.16±1.19**	17.8±0.83***
FR ethanolic extract 400mg/kg	11.7±0.66**	14.00±0.36**	16.00±0.57**	19.8±0.65***

Climbing:

Table no 8: Effect of Ficus religiosa bark extract on climbing in experimental animals.

Groups	Day 1	Day 7	Day 14	Day 21
Control	7.167±0.6	9.16±0.47	11.3±0.76	11.5±0.76
Sildenafil citrate 5mg/kg	13.16±0.87 **	14.1±0.30**	19.3±1.11***	23.00±0.57****
FR ethanolic extract 200mg/kg	9.83±0.60 *	11.83±0.4**	14.167±0.87**	18.16±0.54***
FR ethanolic extract 400mg/kg	11.5±0.67 **	13.0±0.36**	15.0±1.09**	21.5±0.42***

Determination of Hesitation time of male and attraction of male towards female: -

Hesitation time of male:

Table no 9 Effect of Ficus religiosa bark extract of hesitation time in experimental animals.

Groups	Day 1	Day 7	Day 14	Day 21
Control	30.3±1.42	28.00±2.04	26.16±1.27	25.83±0.10
Sildenafil citrate 5mg/kg	13.3±0.66**	12.00±2.4**	11.6±1.13***	9.83±0.60****
FR ethanolic extract 200mg/kg	17.67±1.14*	14.16±1.5**	13.16±0.90**	11.1±1.1***
FR ethanolic extract 400mg/kg	16.00±0.3 *	15.33±2.45*	14.5±1.05**	12.16±2.4***

Attraction of male towards female:

Table no 10 Effect of Ficus religiosa bark extract on attraction of male towards female in experimental animals.

Groups	Day 1	Day 7	Day 14	Day 21
Control	19.66±10.6	21.3 ±12.84	22.66±13.57	25.66±14.38
Sildenafil citrate 5mg/kg	24.16±7.09 *	50.0±14.28**	68.8±20.88***	89.0±20.97 ***
FR ethanolic extract 200mg/kg	22.16±5.64*	46.0±44.25**	55.0±29.25**	58.0±30.55**
FR ethanolic extract 400mg/kg	23.66±12.39 *	48.42±49.1 **	67.25±12.6 ***	75.00±45.41 ***

In vivo Sperm count Analysis:

Table No 12 Effect of Ficus religiosa bark extract on sperm counts in experimental animals.

Groups	Sperm count (millions /ml)
Control	40.833±0.8333
Sildenafil citrate 5mg/kg	69.3333±1.47573
FR ethanolic extract 200mg/kg	52.6667±2.135676
FR ethanolic extract 400mg/kg	57.6667±1.584649

Estimation of serum total cholesterol and Testosterone level:

Table No 13,14 Effect of Ficus religiosa bark extract on total cholesterol level andTestosterone level in experimental animals.

Groups	Testosterone ng/ml
Control	0.828±0.464
Sildenafil citrate 5mg/kg	5.083±0.58****
FR ethanolic extract 200mg/kg	2.993±0.3501**
FR ethanolic extract 400mg/kg	3.25±0.89***

Groups	Cholesterol mg/dl
Control	119.833±4.1985
Sildenafil citrate 5mg/kg	158.1667±6.5950***
FR ethanolic extract 200mg/kg	134.1667±3.7454**
FR ethanolic extract 400mg/kg	138.6667±5.4569**

DISCUSSION:

Significant public health issue and a serious medical one is sexual dysfunction^1,23Aside from that, several drugs can diminish libido by sedating, depressing, or slowing ejaculation in their users. Digoxin, cimetidine, antipsychotic, antihypertensive, and a variety of hormonal and antineoplastic drugs are all associated with sexual dysfunction. The intricate process of sexual function includes the brain, hormones, emotions, neurons, muscles, and blood vessels. Due to its high sensitivity to stress, sexual dysfunction is commonly brought on by stressful circumstances or ways of living.^8,23,24Preliminary phytochemical analysis of plant ethanolic extracts of Ficus religiosa bark in the present research reveals the presence of alkaloids, saponins, steroids, tannins, phenolics, amino acids, flavonoids, methionine, terpenoids, and glycoside. Steroids and saponin components have been proven to have fertility-promoting effects and to be helpful in the treatment of impotence.²⁵

The ethanolic extracts of Ficus religiosa at the doses of 400mg/kg and 200mg/kg significantly (p<0.001) increased the libido parameters in the test for libido, according to the results. In a similar way, sildenafil, the conventional medication, also exhibits a statistically significant increase (p<0.001) in the MF and IF at the dose of 5mg/kg on the 7^th, 14^th and 21^st day as compared to the control animals. The current study's observation of an increase in libido may be attributable to the steroid, flavonoids as quercetin and saponins contained in the bark of the Ficus religiosa plant. There was highly significant decreased (p<0.001) in ML and IL observed in animals treated with standard Sildenafil citrate. The study revealed significant increase in no. of genital sniffing, penile licking, rearing and climbing after the administration of extracts, which demonstrate the direct impact of extracts on sexual pleasure.^22,25Administration of ethanolic extract of Ficus religiosa bark showed statistically significant decrease in hesitation time and consequently increased in the barrier crossing frequency (p<0.001) while standard group (p<0.0001) when compared with the control group on 7th and 14th day and 21^st day. In the present study, ethanolic extract of Ficus religiosa bark at the dose level of 200mg/kg and 400mg/kg body weight of extract resulted in weight gain in treated animals. The weight of the reproductive organs likes testes, seminal vesicle, penis, epididymis, vas-deference and prostrate also increased significantly along with that of vital organs like liver, kidney, spleen and adrenal glands. Genesis of steroids is one of the causes of increased body and sexual organ weight and an increase in these parameters could be regarded as a biological indicator for effectiveness of the plant extract in improving the genesis of steroidal hormones.²⁶Presence of saponins in test extracts may play a role in testosterone secretion, allowing improved availability of hormone to gonads. Amini and Kamkar also earlier reported that androgenic action is attributable to testosterone level in blood.²⁶Significant increase in Sperm count was observed in animals treated with ethanolic extract of Ficus religiosa bark at a higher concentration of 400mg/kg body weight. Ethanolic extract of Ficus religiosa bark at lower concentrations of 200mg/kg body weight were found to be less active compared to higher dose. Similarly, the standard drug sildenafil shows statistically significant increase (p< 0.001) in the sperm count as compared to control. Increased testis weight could indicate the high spermatic production due to increase in length of seminiferous tubules and proliferation of germ cell.²⁷The present results clearly indicates that administration of FR extracts has effect on spermatogenesis in rats. significant increase in serum cholesterol levels was observed Accordingly, such an increase may indicate stimulation of steroid genesis, which could result in an increase in testosterone concentration and a rise in testosterone levels may reflect an increase in libido performance.^25,27.

The effect of Ficus religiosa bark extract on total testosterone levels are presented in table no. Generally elevated testosterone levels enhance sexual behaviour in humans.³²The Ficus religiosa extract shows significant effect on serum testosterone level as compared to vehicle control animals. There was dose dependent increased in the level of serum testosterone drug treated animals. Ficus religiosa at 400mg/kg produce more increased in serum testosterone which was nearly equal to the value of testosterone of standard Sildenafil citrate. According to the study findings, the eight ß sitosterol, ß lanosterol, ß stigmasterol, bornanol, bergaptol, bergapten top binding molecules may be used as potent and safe PDE5 inhibitors and could potentially be used in the treatment of ED.

CONCLUSION:

The current study findings show that ethanolic extracts of Ficus religiosa bark significantly improved sexual function, and the higher dose (400mg/kg) extract is more effective than the lower dose (200mg/kg), It also significantly enhances testosterone level, cholesterol level and sperm count, suggesting that it may be useful for improving sexual performance. The available data imply possible usefulness in treating sexual dysfunction. It might have an impact on the intensity and effectiveness of the sex. The presence of alkaloids, saponins, and flavonoids via a central and peripheral channel may be the cause of the aphrodisiac action of the FR Bark extract. According to the study findings, the eight ß sitosterol, ß lanosterol, ß stigmasterol, bornanol, bergaptol, bergapten top binding molecules may be used as potent and safe PDE5 inhibitors and could potentially be used in the treatment of ED. All these effects could be attributed a number of phytoconstituents including flavonoids, triterpenoids and phytosterols which were found to be present in the extract. Overall, the findings show that the ethanolic extract of Ficus religiosa bark has claiming effects. However, additional research is required to isolate and characterize the phytoconstituents present in the plant extract as well as the aphrodisiac properties.

ABBREVATIONS:

MI-Mount frequency, IM -Intromission frequency

ACKNOWLEDGMENTS:

We thankful to Dr. Smt T. S. Shikalgar, and Dr. Smt. N. S. Naikwade for their valuable suggestions and guidance. We also thankful to Research Journal of Pharmacy and Technology for supporting this work.

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Received on 26.09.2023 Modified on 17.01.2024

Research J. Pharm. and Tech 2024; 17(9):4363-4369.

DOI: 10.52711/0974-360X.2024.00674