Gaphical Abstract

INTRODUCTION:

Ongoing advancements in both pharmacological and non-pharmacological strategies are dedicated to enhancing prognosis, quality of life, morbidity, and mortality associated with benign prostate hyperplasia (BPH), obesity, and other related conditions. At present, mild cases of BPH are commonly managed through observation, while moderate-to-severe cases are typically addressed through medical and surgical interventions. However, several studies have shown that the use of these interventions for obesity and BPH is often associated with relatively high costs.¹ Medications, such as finasteride, which require prolonged usage can also adversely affect patients’ quality of life due to the necessary minimum duration of 6 months.^2,3 According to previous studies, BPH is often exacerbated by its coincidence with obesity. This shows that the management of both conditions necessitates the adoption of a holistic lifestyle, including making nutritious dietary selections, participating in consistent physical exercise, and actively managing weight.^4,5 Although its adoption seems challenging for many individuals, this lifestyle is essential for the treatment process. Consequently, several studies have been carried out to develop alternative therapies that offer safer, more cost-effective, and efficient solutions. In recent years, pharmaceuticals derived from plants in diverse geographical areas have proven to be effective alternatives.

Picriafel-terrae Merr. (PF) also known as Puguntano, is an indigenous plant of the Sumatra family that has historically been used by communities for the management of obesity. PF leaves have also been extensively used for their putative medicinal properties across various cultures. In the Diari Regency of North Sumatra Province, TigaLingga Village has traditionally employed leaves through infusion methods. Consequently, several pharmacological investigations have been carried out to validate these age-old applications, showing the antiproliferative, antioxidant, diabetic, and anti-inflammatory properties of PF leaf extract.^6–8 Previous studies have also reported an increase in the cultivation of the plant by local communities, primarily due to its inherent medicinal properties.^9,10 These medicinal properties can be attributed to the presence of several phytochemicals, including terpenoids, tannins, saponins, and flavonoids. Flavonoids and tannins are known to have the ability to mitigate inflammation and oxidative stress. However, the potential therapeutic implications of PFleaf extract for BPH, specifically in addressing inflammation induced by adiposity, remain largely unexplored. Flavonoids, a class of polyphenolic compounds frequently found in fruits, vegetables, and plants, have garnered significant scientific attention owing to their potential therapeutic applications, particularly in the regulation of VEGF expression and mitigation of inflammatory responses.¹¹

Flavonoids are characterized by a fundamental flavan nuclear structure and typically show an extensive array of biological activities. In addition, their therapeutic effectiveness and mechanisms of action are determined by structural variations, specifically the substitution pattern in the flavan nucleus.¹² In addition, these compounds possess significant anti-inflammatory and analgesic characteristics by modulating several signalling pathways and molecular targets associated with pain and inflammation.¹³ Flavonoids are also known as potential therapeutic agents for conditions marked by persistent inflammation and aberrant angiogenesis. This is primarily due to their ability to specifically modulate the expression of VEGF, an essential regulator of angiogenesis.¹⁴ Another group of essential compounds in PF leaves are tannins belonging to the polyphenol subclass, which are known to possess anti-inflammatory, neuroprotective, and cardioprotective properties.¹⁵ Apart from providing a financially viable substitute, these compounds also have a commendable safety record, as substantiated by comprehensive preclinical and clinical assessments.¹⁶ Despite the therapeutic potential of flavonoids, their clinical efficacy largely depends on various factors, such as bioavailability and precise mode of action, which require additional investigation to optimize clinical applicability.¹⁷ Therefore, this study aims to systematically investigate the effect of PF leaf extract on various biomarkers and histopathological features of BPH in obese rats. Vascular Endothelial Growth Factor (VEGF), Interleukin-6 (IL-6), prostate index, and prostate histopathology were used as key indicators of disease progression and response to treatment.

MATERIALS AND METHODS:

Materials:

The material used in this study included PF leaves, 2 types of diets (a standard diet with 42% fat, 60% carbohydrate, and 14% protein, and a high-fat diet with 43% fat, 35% carbohydrate, and 18% protein), citrate buffer (Sigma Aldrich), xylocaine spray (ASPEN), ketamine (KTM-100), ELISA kits for Interleukin-6 (Bioenzy), VEGF (Bioenzy), and prostate tissue staining kit (Merck). Meanwhile, the tools used were rat cages and feeding equipment, tweezers (GOOI), scissors (GOOI), well plates (Bioenzy), pipettes (Eppendorf), and ELISA Reader (Thermo).

The Preparation of Ethanol Extract from PFLeaves:

Extractionof PFleaves was carried out in a subsequent approach using a percolation procedure with 70% ethanol as the solvent. A covered container was initially filled with 300g of dried powdered plant material and a sufficient quantity of 70% ethanol was added to the mixture. The concoctions obtained were then macerated for a total of 3hours. Subsequently, the bulk was systematically transferred into a percolator, and intermittent pours of the solvent ensured that the plant material was completely submerged in the liquid. The mélange was then left to rest for 24hours, followed by the addition of the solvent into the system at a rate of 1 ml per minute through a trickle. Further solvent was also added to preserve the liquid layer above the plant material. The percolation process was terminated at the point of colourlessness in the final percolate, which was later subjected to concentration using a rotavapor. After the acquisition of the concentrated extract, it was subjected to additional freeze-drying.^18–20

Phytochemical analysis using GC-MS:

Gas chromatography-mass spectrometry (GC-MS) analysis was performed using a mass spectrophotometer and a 7890A gas chromatograph instrument (Agilent 19091-433HP, USA). In addition, the system comprised a 5675.0m 250μm fused silica column (5% phenyl methyl siloxane, 30.0m × 250μm, film thickness 0.25 μm) and a 5675.00C Inert MSD with Triple-Axis detector. Helium gas was modified to a column velocity flow rate of 1.0ml/min. The GC-MS parameters comprised a 250°C ion source temperature, a 300°C interface temperature, a 16.2 psi pressure, a 1.8mm discharge time, and an injector operating in split mode with a 1:50 split ratio. The temperature of injection was maintained at 300°C with a rate of 4°C/min, and the column temperature increased from 36°C for 5 minutes to 150°C within this time range. The temperature was maintained at 250°C for 5 minutes after being increased at a rate of 20°C/min. The elution duration was 47.5 minutes in total, and the relative percentage of each component was determined by comparing the average peak area of each component to the total area. MS Solution software, which was supplied by the vendor, was used for system control and data acquisition.^21–24

Animal Preparation and Dietary:

Male Wistar rats (Rattus norvegicus) were used in this study, and the samples were selected randomly, adhering to strict inclusion and exclusion criteria. The inclusion criteria were male rats, aged 10 weeks, and had a body weight ranging from 180 to 270g while maintaining good health. Meanwhile, samples with pre-existing diseases or injuries were excluded from the procedures. Rats that did not survive until the completion of the study were categorized as dropout samples and were excluded from the subsequent analysis. The experimental protocol was approved by the Research Ethics Committees University of Sumatera Utara under approval number: 44/KEP/USU/2021.

Treatment Regime:

After the acclimation period, particular dietary and hormonal interventions were implemented in rodents to induce BPH and obesity. Group 1 (G1) comprised 6 male Wistar rats, which served as the normal control, while Group 2 (G2) consisted of 6 male Wistar rats, serving as the obesity-induced BPH group without treatment (negative control). To induce obesity in G2, G3, G4, and G5 groups, a daily high-calorie diet of 1235 kJ was provided. The diet administered contained 13.78% proteins, 42.69% fat, and 34.55% carbohydrates. Meanwhile, the control normal group (G1) had access to purified potable water and a standard diet consisting of 733 kJ of daily energy. The group induced with obesity was administered purified drinking water containing an extra 30% sucrose. Consistency in diet was maintained throughout the 8-week duration of the study through daily monitoring and restocking of food and water supply.

After 4 weeks of exposure to a high-calorie diet, a subset of samples from the groups that had developed obesity was subjected to a 1-night fast. Rats were then administered subcutaneous testosterone injections at a rate of 10mg/kg body weight (BW) weekly for a total of 4 weeks. For an additional 4 weeks, the remaining samples were provided a high-calorie diet, bringing the total duration of dietary exposure in these groups to 12 weeks. After 12 weeks, G2, G3, G4, and G5 were subjected to another 1-night fasting. In G3, male Wistar rats were orally administered finasteride at a dose of 1 mg/kg body weight (BW) for 10 days, while G4 was given PF extract at a dose of 100mg/kg bw/day for 10 days. Furthermore, G5 was treated with PF extract at a dose of 200mg/kg bw/day for the same duration. All rat groups were sacrificed under appropriate anesthesia (ketamine + HCl) for VEGF, IL6, and histopathological analyses. VGEF and IL-6 levels were assessed in the serum of experimental animals using the ELISA method, while the prostate index was the weight of the prostate in rats compared to the body weight.^25,26

Prostate Histopathology:

Prostate tissues were subjected to histological processing by placing the fixed specimens in an Automatic Tissue Processor (Leica TP 1020). The procedure comprised immersing the specimen in a 10% formalin buffer for 1 h, followed by dehydration using 70%, 80%, and 96% alcohol for 1 h and 30 min each. The specimen was then treated with pure alcohol for 1 h to eliminate any remaining water. Subsequently, the tissues were treated with xylene for 1 h to remove any residual alcohol. The process of embedding was carried out by using liquid paraffin (Merck, Darmstadt, Germany) at a temperature of 56°C for 2 h. After the chilling process, sections were sliced to a precise thickness of 4μm using a Leica microtome. The sections obtained were immersed in a water bath and affixed to glass slides coated with glycerine. The samples were then deparaffinized in xylene (three changes, with each change lasting 15min) and rehydrated in 96%, 80%, and 50% alcohol for 15 min each, followed by rinsing with running water. The staining process consisted of immersing the samples in Haematoxylin Mayer's solution for 5 min, followed by rinsing in running water for 10 min and incubation in a 1% eosin solution for 1 min. The dehydration process was carried out through immersion in 80%, 96%, and 100% alcohol for 1 minute each, with 1-minute immersion in xylene. Subsequently, cover glasses were placed, and histological analysis was conducted at 100x magnification using an Olympus light microscope.^27,28

Statistical Analysis:

The acquired data were subjected to statistical analyses using one-way ANOVA under the assumption that the distribution followed a normal shape. Subsequently, for post hoc analysis, the Least Significant Difference (LSD) test was applied. When the distribution of the data deviated from normality, post hoc analysis was performed using the Mann-Whitney test, followed by non-parametric analysis using the Kruskal-Wallis test. A significance level of p<0.05 was established for the results.

RESULT:

Phytochemical Screening :

The results of the phytochemical screening of PF leaves are presented in Tables 1 and 2. This screening was essential for determining the presence of secondary metabolites in leaves. In addition, the presence of these metabolites often suggests their potential medicinal, therapeutic, or other beneficial properties.

Figure 1: GC-MS chromatogram

In table 1, table 2, and figure 2, the phytochemical screening of PF leaves showed the presence of several secondary metabolites, including alkaloids, flavonoids, glycosides, saponins, tannins, and triterpenes/steroids. Furthermore, the GCMS analysis identified various compounds, such as hydroxy-acetic acid, n-hexadecenoic acid, and 2-nitro-cyclohexanone, showing the diverse chemical composition of leaves. This study showed the potential therapeutic and industrial applications of the compounds found in the test sample.

VEGF examination

Based on examination using the ELISA method assay, the mean measurement results of VEGF levels in G2 (343.83±4.69 pg/L) were higher compared to G1 (239.5 ±42.75pg/L). In addition, G2 showed the highest VEGF levels, showing that the occurrence of obesity and BPH led to high VEGF levels. The mean VEGF levels in G3 (300.5±44.51 pg/L), G4 (313.33±46.25pg/L), and G5 (289.6±48.2 pg/L) were lower compared to G2 (343.83 ±4.69ng/L). The results also showed that the mean value obtained in G5 (289.6±48.2pg/L) was lower than G3 (300.5±44.51pg/L), while the mean in G4 (313.33± 46.25ng/L) was higher than G5 (289.6±48.2pg/L). These resultsshowed that the administration of PF extract reduced VEGF levels. Statistical analysis of mean VEGF levels using the one-way ANOVA parametric test showed no significant difference between the treatment groups (p<0.05), as shown in Figure 2.

Figure 2: VEGF concentration, G1, Normal Group; G2 negative control; G3,treatment Group finasteride 1 mg/kgBW; G4, PF extract100 mg/kgBW; G5, PF extract 200 mg/kgBW, **p≤0.01; NS, not significant (p>0.05)

Based on the results of this study, the administration of PF extract could increase the average levels of VEGF in obese rats with BPH. The dose of PF extract (100 mg/KgBW) could reduce VEGF levels in obese model rats with BPH. Meanwhile, the 200mg/KgBWPF extract dose group was closer to the average value of VEGF levels in the normal group and better compared to the samples given finasteride 1mg/KgBW and 100 mg/KgBW PF extract.

Interleukin – 6 Level:

The examination of interleukin-6 levels was measured in blood samples using ELISA. The average measurement of interleukin-6 levels in G2 (2606.67±445.8pg/L) was higher compared to G1 (243.17±277.24pg/L), and it had the highest value. The mean interleukin-6 levels in G3 (1962.67±728.57pg/L), G4 (2141.83±862.49pg/L) and G5 (1155.4±724.1pg/L) were lower compared to G2 (2606.67±445.8pg/L). The mean value obtained in G5 (1155.4±724.1pg/L) was lower than G3 (1962.67± 728.57pg/L), while G4 (2141.83±862.49pg/L) was higher than G5 (1155.4±724.1pg/L). These results showed that the administration of PF extract could reduce interleukin-6 levels. The results of statistical analysis of the mean interleukin-6 levels using a one-way ANOVA parametric test showed that there were significant differences in the values obtained between the treatment groups (p<0.05), as shown in Figure 3.

Figure 3: Interleukin-6 concentration, G1, Normal control group; G2 Negative contol group; G3,treatment Group finasteride 1 mg/kgBW; G4, PF extract 100 mg/kgBW; G5, PF extract 200 mg/kgBW.*p ≤ 0.05, **p ≤ 0.01, *** p ≤ 0.001, ****p ≤ 0.0001; NS, not significant (p> 0.05)

In this study, the different doses led to varying effects in each treatment group. The results of the post hoc test with the LSD on the mean interleukin-6 levels showed a significant difference between G2 and G5, G3 and G5, and G4 and G5 (p<0.05). However, there was no significant difference in the between G2, G3, G2, G4, G3, and G4 (p>0.05). Based on these results, the administration of PF extract could increase the mean levels of interleukin-6 in obese rats with BPH. The dose of PF extract (100mg/KgBW) could reduce interleukin-6 levels in obese model rats with BPH. The results showed that the 200mg/KgBW PF extract dose group was closer to the average value of interleukin-6 levels in the normal group and better than samples given finasteride 1 mg/KgBW and 100mg/KgBW PF extract.

Prostate index:

The prostate index was the weight of the prostate in rats compared to the total body weight. The mean prostate index in G2 (6.46±0.08mg/gr) was higher compared to G1 (2.45±0.03mg/gr), with G2 having the highest value. This showed that obesity associated with BPH led to a high prostate index. In addition, the mean prostate index in G3 (5.07±0.11mg/gr), G4 (5.33±0.11mg/gr), and G5 (4.93±0.11mg/gr) was lower compared to G2 (6.46± 0.08mg/gr). The value obtained in G5 (4.93±0.11mg/gr) was lower compared to G3 (5.07±0.11mg/gr), while G4 (5.33±0.11mg/gr) was higher than G5 (4.93±0.11 mg/gr). This showed that the administration of PF extract could reduce the prostate index. Statistical analysis of the mean prostate index using a one-way ANOVA parametric test showed that there was a significant difference between the treatment groups (p<0.05). The difference in dose showed varying effects in each treatment group. The results of the post hoc test with LSD on the mean prostate index showed a significant difference between G2 and G3, G2 and G4, G2 and G5, G3 and G4, G3 and G5, and G4 and G5 (p<0.05), as shown in Figure 4.

Figure 4: Prostate index, G1, Normal Group; G2 negative control; G3,treatment Group finasteride 1 mg/kgBW; G4, PF extract100 mg/kgBW; G5, PF extract 200 mg/kgBW. *p ≤ 0.05, **p ≤ 0.01, *** p ≤ 0.001, ****p ≤ 0.0001; NS, not significant (p> 0.05)

The results of this study showed that the injection of PF extract could effectively increase the average prostate index value in obese rats with BPH. In addition, the administration of 100mg/KgBWof PF extract was sufficient to decrease the prostate index value. The group receiving a dose of 200mg/KgBW was more similar to the average prostate index value of the normal group and superior to samples receiving a dose of 1 mg/KgBW of finasteride and 100mg/kg of PF extract.

Prostate histopathology analysis:

Prostate tissue changes in each group of rats were examined under a light microscope (Olympus). Prostate tissue staining was carried out using haematoxylin and eosin staining. Furthermore, histopathology of the normal prostate in the G1/normal group (magnification 100 times) contained tubule-shaped glands coated with cellular columnar epithelium, with the stroma consisting of fibromuscular connective tissue. The ratios of epithelium, stroma, and lumen were 12.17%, 11%: 76,83%. The results showed that there were no chronic inflammatory cells or signs of malignancy (Figure 5A).

In this study, G2 (negative control group) obtained a histopathological picture of the prostate containing glandular proliferation that was close to each other, tubular in shape, and lined with proliferative columnar epithelium, forming a papillary structure into the lumen. The stroma consisted of proliferating fibromuscular connective tissues and numerous inflammatory lymphocytes. The ratios of epithelium, stroma, and lumen were 42.5%: 24.67%: and 32.83%, and there were no signs of malignancy (Figure 5B).

Figure 5: Histopathologic staining of prostate tissue with haematoxylin-eosin, magnification 100x. (A) no inflammatory cells were seen; (B) many inflammatory cells of lymphocytes were found (arrow); (C) inflammatory cells were found (arrow); (D) inflammatory cells of lymphocytes were found (arrow); (E) minimal inflammatory cells of lymphocytes were found (arrow).

In the G3 (finasteride-treated group), the histopathological picture of the prostate contained proliferative glands coated with a layer of columnar epithelium, partially coated with columnar epithelium that grew papillary into the lumen. Furthermore, the ratios of epithelium, stroma, and lumen were 23.67%: 22,5%: 53,83%. The stroma consisted of fibromuscular connective tissue and lymphocyte inflammatory cells, with no signs of malignancy (Figure 5C).

For G4 (PF extract 100mg/kgBW), a histopathological picture of the prostate containing glands coated with hyperplastic cellular columnar epithelium, papillary growth into the lumen, and partial coating with cellular columnar epithelium was obtained. In addition, the ratios of epithelium, stroma, and lumen were 33.67%: 22.5%: and 43.83%. The stroma consisted of fibromuscular connective tissue and lymphocyte inflammatory cells, with no signs of malignancy (Figure 5D).

In the histopathological picture of G5 (PF extract 200 mg/kgBW), the prostate contained glands lined with multilayered columnar epithelium and partially lined with columnar epithelium that grew papillary into the lumen. The ratios of epithelium, stroma, and lumen were 21.6%: 13.68%: and 64.6%. The stroma consisted of fibromuscular connective tissue, minimal lymphocyte inflammatory cells, and no signs of malignancy (Figure 5E).

DISCUSSION:

The results of this study showed that there were no differences in VEGF levels between the treatment groups (p=0.274) based on a one-way ANOVA parametric test. An improvement in the average PF extract dose at 100mg/KgBW and 200mg/KgBW was also observed. The treatment group 200mg/KgBW had a mean value of VEGF levels, which was closer to the normal group and better compared to the samples given finasteride 1mg/KgBW (G3) and PF extract 100 mg/KgBW (G4). In another study, VEGF levels decreased after treatment with Tribulus terrestris extract, which was rich in saponins. Several studies have shown that saponins worked by inhibiting glucose absorption, increasing PI3K levels, and suppressing adenosine monophosphate-activated protein kinase (AMPK), leading to a decrease in eNOS and NOS. This condition suppressed the occurrence of oxidative stress and low levels of VEGF. In addition, oxidative stress triggered the release of arachidonic acid, which was converted by cyclooxygenase (COX) into prostaglandins known to play an important role in regulating cell proliferation.²⁹ Another study showed a decrease in VEGF levels following the administration of Rhizoma Dioscoreae Nipponicae saponins in rat models of rheumatoid arthritis (RA). Hypoxia triggered the release of VEGF and growth factors, such as FGF-7, TGF, FGF-2, and interleukin-8, which stimulated neo-angiogenesis and fibroblast differentiation.³⁰ Androgen stimulation caused rapid growth of the prostate tissue, leading to hypoxic conditions that induced angiogenesis. Hypoxic conditions typically stimulated processes that engaged the hypoxia-inducible factor 1 (HIF-1). HIF-1 is a heterodimeric transcription factor composed of two polypeptides, namely HIF-1a and HIF-1b. HIF-1afunctions as a regulator of oxygen homeostasis and accumulates in the cytosol. Through translocation into the nucleus, HIF-1b activated genes sensitive to hypoxic conditions, leading to the formation of pro-angiogenesis cytokines, such as VEGF. Furthermore, one of the most important molecules in the process of angiogenesis is the vascular permeability factor (VPF), VEGF. VEGF plays an essential role in endothelial cell differentiation, migration, proliferation, tubularisation, and blood vessel formation. Several studies also showed that there was a positive correlation between VEGF and blood vessel density in BPH.³¹

In this study, IL-6 levels analysed using one-way ANOVA showed that there were significant differences between the groups (p<0.001). An improvement was observed in the values obtained for samples given PF extract dose at 100mg/KgBW(G4) and 200 mg/KgBW(G5). In the PF extract dose group, 200 mg/KgBW(G5) was closer to the average value of interleukin-6 levels obtained in the normal group (G1) and better compared to samples administered finasteride 1mg/KgBW(G3) and PF extract 100mg/KgBW(G4). Based on a study conducted by Kwak et al. (2021), adiponectin was closely related to IL-6, where low levels of adiponectin were found in high-adipocyte cells. Therefore, the formation of fatty acid synthesis inhibited the production of lipolytic enzymes, decreased fat oxidation, and reduced fat cell apoptosis. This condition increased the level of IL-6, which was a marker of inflammation associated with increased adipocyte cells in obesity.³²

According to Garmsiri et al. (2020), obesity correlated with high oxidative stress and inflammatory markers, such as IL-6, which affected glucose tolerance. In experimental animal models, there was an increase in triglyceride levels, gluconeogenesis, and glycogenolysis, but glycogenesis was inhibited, leading to hepatic damage. Several IL-6 studies have also established correlations with the occurrence of BPH.³³ In rats injected with testosterone, the expression of inflammatory markers, including IL-6, TNF-α, COX-2, and iNOS increased. The largest increase was shown by IL-6 in prostate tissue by 275% compared to control values by immunohistochemistry.³⁴ Interleukin-6 was expressed in BPH stromal and epithelial cells, while interleukin-6 receptor (IL-6R) was detected in both stromal and epithelial cells. The main role of these components was to control or regulate paracrine or autocrine epithelial cell growth. Previous studies also showed that interleukin-6 increased the density and responsiveness of androgen receptors and activated the androgen response in tumour cells. These conditions made IL-6 a candidate for further investigation as a potentially important biomarker, found in high concentrations in seminal plasma and systemic circulation.³⁵

In this study, the prostate index value was significantly different between the groups (p<0.001). The results of the one-way ANOVA test showed an improvement in the mean value obtained for samples given PF extract at doses of 100mg/KgBW(G4) and 200mg/KgBW(G5). In PF extract dose group, 200mg/KgBW(G5) was closer to the average PSA level in the normal group (G1) and better compared to samples administered finasteride 1 mg/KgBW(G3) and a dose of PF extract of 100 mg/KgBW(G4). Previous studies have also shown a positive relationship between obesity and prostate index. The prostate index was greater in obese individuals compared to non-obese individuals with BPH. Furthermore, aetiology was focused on the role of sex steroid hormones, namely androgens and oestrogens that stimulate prostate growth through aromatization. This caused changes in the balance between testosterone and oestrogen levels in prostate tissue, contributing to the occurrence of BPH.³⁶ In 2007, Xieab et al. showed that obese Chinese men had a larger prostate index compared to non-obese Chinese men.³⁷

The results of this study also showed that prostate cell hyperproliferation with HE staining prostate histopathology examination in the BPH group with obesity contained glandular proliferation that was close to each other, tubule shaped, lined with proliferative growing layered columnar epithelium, forming papillary structures into the lumen. The results of histopathological examination of the prostate showed that the ratio of stroma and epithelium of the BPH model group with obesity was the highest compared to others. Histopathology examination of G4 showed a decrease in the ratio of epithelium, stroma, and lumen by 33.67%: 22.5%: and 43.83%. A decrease in the ratio of epithelium, stroma, and lumen was observed in the positive control group (CTF) G3 by 23.67%: 22.5%: and 53.83%. G5 also showed a ratio of epithelium, stroma, and lumen approaching the normal group with a ratio of 21.6%: 13.68%: 64.6%. Stromal hyperproliferation in the form of smooth muscle cells in BPH affected the size of the prostate, which was the static component, as well as the tension or contractility of the smooth muscle, serving as the dynamic component. These static and dynamic components played a role in the clinical symptoms of BPH due to bladder outlet obstruction (BOO).³⁸ Inflammatory processes were found in 90% of histopathologic transurethral resection of prostate preparations. Angiogenesis was important for inflammatory processes in response to tissue damage. Prostaglandins were inflammatory mediators derived from arachidonic acid mediated by COX enzymes, specifically COX-2. An increase in COX-2 mRNA expression was reported in BPH, specifically in the epithelial cells. The role of COX-2 in BPH was to increase proliferation and inhibit apoptosis in prostate cells. Several types of stimuli could induce chronic prostate inflammation, leading to tissue damage followed by a continuous healing process.³⁹ Descazeaud found that inflammation was present in approximately 78% of BPH cases. Furthermore, almost all patients had inflammatory cells in BPH tissue. The results showed that 81%, 52%, and 82% had T lymphocytes, B-lymphocyte markers, and macrophage markers, respectively.⁴⁰ The present study showed that extract from PF leaves had promising efficacy in the treatment of BPH.

CONCLUSION:

In conclusion, this study found a significant increase in interleukin-6 levels, prostate index values, and changes in prostate histopathological examination of epithelial and stromal cells with the use of PF extract. However, the extract did not affect the VEGF levels of the samples.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors are grateful to Universitas Sumatera Utara for providing the technical facilities.

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Received on 14.12.2023 Modified on 15.03.2024

Research J. Pharm. and Tech 2024; 17(10):5046-5054.

DOI: 10.52711/0974-360X.2024.00776

No.	Secondary metabolite	Reactor	Results	Desc.	PF Ethanol Extract
1	Alkaloid	Dragendroff + Bouchardat Meyer	+	Positive	Formation of orange precipitate in Dragendorff's. Formation of yellow precipitate in Mayer's.
2	Flavonoid	Mg Powder + n-Amyl Alcohol + HCl	+	Positive	Formation of yellow color and white precipitate.
3	Glycoside	Molish + H₂SO₄	+	Positive	Formation of green or blue color.
4	Saponin	Hot water/shaked	+	Positive	Formation of stable foam.
5	Tannin	FeCl₃	+	Positive	Formation of dark green color.
6	Triterpene/Steroid	Lieberman-Bourchat	+	Positive	Formation of green color.

S. No.	Chemical name	Molecular weight (g/mol)	Molecular formula	Retention time (Min)
1.	Acetic acid, hydroxy-	76.05	HOCH₂COOH	1.444
2.	n-Hexadecanoic acid	256.42	C₁₆H₃₂O₂	14.932
3.	Cyclohexanone, 2-nitro-	143.14	C₆H₉NO₃	16.888
4.	Undecanoic acid, hydroxy-, l	202.29	C₁₁H₂₂O₃	17.638
5.	11-Bromoundecanoic acid	265.19	C₁₁H₂₁BrO₂	17.915
6.	Hexadecanoic acid, 2,3-dihyd	288.423	C₁₆H₃₂O₄	23.368
7.	Pyridine, 2-ethenyl-	105.14	H₂C=CHC₅H₄N	28.327
8.	4-Pyridineethanesulfonic acid	187.22	C₇H₉NO₃S	28.579