Xylopia aethiopica Attenuates Oxidative Stress and Hepatorenal Damage in Testosterone Propionate-Induced Benign Prostatic Hyperplasia in Rats

Abstract Objectives   Xylopia aethiopica (XAE), commonly known as African pepper or Ethiopian pepper, is a plant native to West Africa and known for its aromatic and medicinal properties. It was used to investigate the antioxidative, antihepatotoxic, and antinephrotoxic potentials of XAE in benign prostatic hyperplasia (BPH) in Wister albino rats. Methods  The proximate, and vitamin composition, oxidative stress indicators, and indices of kidney and hepatic functions were performed by standard methods. Results  The proximate composition of the XAE leaf showed varied concentrations of Mg, Ca, Na, Zn, Se, and Cl, as well as vitamins A, E, B3, D, C, K, B2, and Bi. The activities of catalase glutathione, superoxide dismutase, malondialdehyde levels, K, Na, Cl − , urea, uric acid, and creatinine in the kidney were increased in testosterone propionate (Tp)-induced BPH compared with the control groups. Total protein levels significantly decreased in Tp-induced BPH compared with XAE-treated groups increased on XAE treatment. The aspartate transaminase, alanine aminotransferase, and alkaline phosphatase activities were not significantly different in Tp-induced BPH, XAE, and normal controls. Conclusion  The study revealed that XAE can be used in the management of oxidative stress and hepatorenal damage in BPH condition.


Introduction
Benign prostatic hyperplasia (BPH) is a common condition that affects older men, involving the noncancerous enlargement of the prostate gland.It is believed to be caused by hormonal changes, such as an increase in dihydrotestosterone levels, as well as genetics and lifestyle.It can cause bothersome urinary symptoms. 1Oxidative stress has been suggested to play a role in the development and progression of BPH, and it may also contribute to hepatorenal implications associated with the condition.The liver and kidneys are important organs for detoxification and elimination of reactive oxygen species and their by-products. 2However, excessive oxidative stress can overwhelm their antioxidant defense mechanisms, leading to hepatic and renal damage. 2 Studies have shown that patients with BPH have increased levels of liver enzymes, such as alanine aminotransferase and aspartate aminotransferase, indicating liver dysfunction.In addition, BPH has been associated with an increased risk of chronic kidney disease and renal dysfunction, possibly due to oxidative stress and inflammation. 2ylopia aethiopica (XAE) is a plant commonly found in West Africa and is known for its medicinal properties. 3Tall, slender, aromatic, and evergreen, XAE is of the family Annonaceae, and can reach heights of 15 to 30 m with a diameter of 60 to 70 cm. 4The plant naturally grows in Savanna region of Africa. 4The fruit of XAE, sometimes known as "Guinea pepper" or "Negro pepper," has a variety of recognized uses in folk medicine. 4It has been traditionally used to treat various ailments such as malaria, fever, and gastrointestinal disorders. 5,6Recent studies have shown that XAE has unique properties that make it useful in the management of metabolic complications. 7The antioxidant properties of XAE are based on the rich presence of antioxidants, such as flavonoids and phenolic compounds. 7These antioxidants help to scavenge free radicals and prevent oxidative stress, which can lead to various diseases, including cancer, diabetes, and cardiovascular diseases.
XAE has earlier been used in the management of BPH. 3 XAE has anti-inflammatory properties, which help to reduce inflammation in the body. 7Chronic inflammation is associated with various diseases including cancer, diabetes, and cardiovascular disease.
Oxidative stress is linked to the development and progression of BPH, and this research has identified antioxidative and hepatorenal protective effects of XAE in animal models.

Methodology Reagents and Chemicals
Chemicals and reagents used were from Sigma-Aldrich, Span Diagnostics, UWI, MONZA, Plasmatec and Elab Science.

Collection of Xylopia aethiopica
The leaves of XAE were harvested from the wild in the early hours of May 9, 2022 at the onset of wet season at 8.30 AM at Enugu State, Nigeria's Obukpa, Nsukka, Igboeze South Local Government Area.Prof. J.C. Okafor verified the harvested XAE leaves, and it was then processed into fine powder before sieving after drying at room temperature varying between 25 and 29°C.The extract was then filtered and concentrated using a rotary evaporator for 3 days at 65°C.The yield of the extract was 15% of the original sample used for extraction.The product was stored in a refrigerator until use.The time and season of plant leaf collection reported in this study are vital for accurate interpretation of data, understanding the effects of environmental factors on biological processes, and ensuring the reproducibility and reliability of scientific studies in plant biology.

Procurement of Drugs and Chemicals
Renhoks Pharmaceuticals supplied testosterone propionate (Tp) and finasteride of analytical grade.

Proximate Analysis of Plant Extract
The following proximate analyses were performed using standard procedures.
The Association of Official Agricultural Chemists (AOAC) method was used to determine crude fat, and crude fat content was obtained using gravimetric measurement of N-hexane or petroleum ether. 8Moisture content was determined by the method of Aguinaldo et al. 9 Ash was determined using the method of Harbone. 10 However, crude proteins were determined by the method of Hussain et al. 11 Crude fiber was determined according to the method of Alberts et al. 12 The gravimetric method was used to determine the percent of indigestible carbohydrate in a sample.However, Clegg's method was used to calculate total carbohydrate, which involved perchloric acid digestion and colorimetric measurement of soluble sugars using the Anthronic method of Clegg. 13The total available glucose was determined as % of glucose.

Determination of Vitamins Content in X. aethiopica
Fat-soluble and water-soluble soluble vitamins were determined using standard procedures following the methods as listed below: Vitamin D was estimated by the modified method of AOAC, 8 vitamin A according to the method of Kumura and Itokawa, 14 tocopherol (vitamin E) following the method of Jargar et al, 15 ascorbic acid (vitamin C) and thiamine (vitamin B1) to the method of AOAC, 16 riboflavin (vitamin B2), and phylloquinone (vitamin K) as described in AOAC, 17 vitamin B3 and B6 according to the method of Zhang et al. 18

Determination of Mineral Content
The mineral content of XAE was determined using the method described by AOAC. 16The atomic absorption spectrophotometer quantitatively measured the concentration of elements present in the liquid sample.XAE extract was placed into a crucible and incinerated in a muffle furnace.After boiling, it was cooled and filtered into a 200 mL volumetric flask.The absorbance values of the minerals were recorded and the percentage of elements in the extract was calculated.

Experimental Animals
A total of 200 male Wistar albino rats were procured from the animal house at the Department of Biochemistry, University of Nigeria, Nsukka.The rats that ranged in weight from 250 to 400 g were 16 weeks old.They were kept in regular laboratory conditions and had unrestricted access to food and water.

Experimental Design and Animal Treatment
Ibiam et al 3 studied how XAE can modulate Tp)-induced BPH in rats.The study was performed in phases as shown in ►Table 1.In this phase, the rats were acclimatized for 2 weeks and they were divided into 12 groups with 12 rats in each group.They were weighed weekly during the experiment.Group A1 served as control and they received only the vehicle, olive oil.Tp, 14 mg kg À1 body weight, was administered intraperitoneally daily to group A2 for 4 weeks to induce BPH.For preventive studies Finasteride (F), a potent and specific 5α-reductase inhibitor, 10 mg kg À1 was administered along with Tp (14 mg kg À1 ) for 8 weeks to group B 1 rats.Groups B 2 , B 3 , B 4, and B 5 were pretreated with ethanolic extract of XAE doses of 100, 200, 300, and 400 mg kg À1 respectively for four weeks.It was followed by Tp(14 mg kg À1 ) along with (XAE), doses of 100, 200, 300, and 400 mg kg À1 treatments for 8 weeks.
In the curative studies, Tp(14 mg kg À1 ) was administered to group C 1 for 4weeks to induce BPH after which F (10 mg kg À1 ) was given for 8 weeks.BPH was induced in group C 2 , C 3 , C 4 , and C 5 with Tp(14 mg kg À1 ) for 4 weeks after which (XAE) doses of 500, 600, 700, and 800 mg kg À1 treatments for 8 weeks.They were all fed with water and feeds. 3

Preparation of Tissue Homogenate
Rats were sacrificed and the prostates were washed, blotted, and weighed.Ten percent of the homogenate was prepared in a 0.05M phosphate buffer and centrifuged.The supernatant was used to measure oxidative stress indicators.

Oxidative Stress Indicators
The superoxide dismutase activity was determined by the method of McCord and Firdorick. 19The catalase activity was determined by the method of Beers and Sizer. 20The reduced glutathione in the tissue was determined by the method of Moron et al. 21The malondialdehyde of the tissue was determined as earlier reported by Udeozor et al. 22

Renal Function Tests
The following renal function tests were performed.
Serum urea was determined using the diacetyl monoxide urea method described by Wybenga. 23Serum creatinine was determined by the alkaline picrate creatinine method described by Husdan and Rapoport. 24The potassium ion was determined using the method of sodium tetraphenyl boron described by Chessbrough. 25The concentration of Na þ was determined by the method of Chessbrough. 25The concentration of chloride ion (Cl À ) was determined by the method of Chessbrough. 25The carbon dioxide content was estimated by a method of Forester et al. 26

Liver Function Indices
The following liver function parameters were performed.Serum protein was determined by means of biuret reaction as described by Sánchez et al. 27 Aspartate transaminase (AST) also known as serum glutamate oxaloacetate transaminase activity was determined by the method of Huang et al. 28 Alanine transaminase (ALT) also called serum glutamate pyruvic transaminase activity was determined by the method of Huang et al. 28 Alkaline phosphatase (ALP) activity was determined by the method of Talib and Khurana 29 Xylopia aethiopica and Benign Prostatic Hyperplasia Ibiam et al.

Determination of Some Metals in Prostate Homogenate
The processes utilized by Alexaris and Lazos 30 to digest the homogenate sample are the most essential elements in this work.The flask was heated, diluted with deionized water, and analyzed using an atomic absorption spectrophotometer.The standard curve was then used to read the metal concentrations.

Results
Values with different superscripts in the same column are significantly different (p 0.05).

Discussion
The results of the proximate composition of XAE showed the following order of occurrence; carbohydrates, ash, fiber, moisture, protein, and fat.The result is consistent with the report researchers in Europe, Japan, India, and Nigeria who found that polysaccharide in XAE reduced tumor and cancers. 31This finding is in line with earlier report that dietary fiber through the consumption of fruits and vegetables has been shown to be preventive and associated with decreased incidence of BPH.XAE ethanol leaf extract contains minerals such as Mg, Ca, Na, Zn, Se, and Cl that can be administered to Tp-induced BPH in albino rats to reduce the levels of BPH.Mg levels were found to be significantly lowered in patients with BPH, while Ca levels in BPH patients have not been associated with the condition. 32XAE ethanol leaf extract contains vitamins A, E, B3, D, C, K, B2, and B that are anti-BPH and protect against growth and viability of BPH cells.National Health and Nutritional Examination survey showed that vitamin D deficiency is associated with lower urinary track symptoms, while intake of vitamin D supplement and vitamin D analog has been shown to decrease BPH prevalence and prostate size. 33It has been widely reported that some antioxidants such as vitamin A, E, and C found also in XAE play key roles in the prevention of BPH by ameliorating oxidative stress, which otherwise results in DNA damage and increases the risk of mutation and malignant transformation. 34PH decreased minerals in prostate homogenates compared with the normal control group, while these were significantly increased in finasteride and XAE treatment groups.XAE treatments can enhance the levels of prostatic fluid minerals to promote prostate health.Zinc is an important constituent of prostatic fluid and is known to play an important role in the development and functioning of prostate. 35It is suggested that the ameliorative effects of  However, it is important to note that these markers are not specific to BPH and can be influenced by various factors, including diet, hydration, and other medical conditions.BPH-induction significantly decreased antioxidant activities and increased lipid peroxidation, consistent with previous reports. 37In this study, the activities were, however, found to be significantly (p < 0.05) increased in finasteride and XAE treatment groups in both studies.Thus, the initial decline in the activities of CT, SOD, GSH and the increased concentration of MDA in Tp-induced BPH rats were substantially restored by XAE treatments well above the control values.This indicates protection of prostate glands against free radicals damage thereby suppressing the development of BPH.This finding can be attributed to the antioxidant properties of XAE based on its phytochemical constituent.
XAE studies showed that Tp-induced BPH had higher levels of potassium, sodium, chloride, bicarbonate, urea, uric acid, and creatinine than control groups.Finasteride and XAE treatment groups had lower levels.Without relief, BPH can lead to asphyxiation, hydroureters, hydronephrosis, obstructive nephropathy, renal insufficiency, and kidney failure. 38XAE and finasteride treatment in both studies exhibited a significant improvement in these major pathological problems observed in Tp-induced BPH rats.Reduction in the levels of serum urea, uric acid, electrolytes, and creatinine by XAE and finasteride treatments in both studies may be due to decreased prostate size, return of urinary flow rate to control values, and recovery of kidney from BPH damage.The findings of this study are in agreement with previous reports that urine output decreased drastically in testosterone treatment group due to enlargement of prostate gland. 39he total protein level in rats with Tp-induced BPH was found to have decreased compared with a control group.However, finasteride and XAE treatment groups showed a significant increase in protein levels.Most plant extracts possess antioxidant, hepatoprotective, and nephroprotective functions.Oxidative stress can lead to tissue damage, including damage to organs involved in protein metabolism.Certain plant extracts are rich in antioxidants that can help reduce oxidative stress, potentially leading to normalized serum protein levels as observed in XAE treatments in this study.The liver plays a crucial role in protein synthesis and metabolism.Some plant extracts are known to have hepatoprotective effects, supporting liver health and function.Improved liver function could positively affect serum protein levels.While kidneys are also involved in maintaining proper protein levels in the blood, some plant compounds possess diuretic and nephroprotective properties, which might impact kidney function and subsequently influence serum protein levels.These could be attributed to the observations with XAE and finasteride in this study.Carbimazole and selenium treatment restored to normal the total protein levels of the prostate epithelial cells in previous studies. 36AE treatments in this study restored the depleted protein content level caused by Tp-induced BPH, and did not affect the activities of AST, ALT, and ALP in the Tp-induced BPH group, finasteride and XAE treatment groups.Thus, XAE aside from inhibiting BPH development was well tolerated by the rats in both studies.

Table 3
Effect of XAE on prostate tissue mineral levels in Tp-induced benign prostatic hyperplasia in rats (preventive studies) groups.Also the levels of the prostate minerals were higher in the XAE groups than in the finasteride treated group.The results showed that the levels of K, Na, Cl, HCO 3 À , urea, uric acid, and creatinine were significantly (p < 0.05) higher in the TP only group compared with the control group.However, the levels were reversed significantly (p < 0.05) lower in the finasteride and XAE treatment groups relative to the Tp only group for both studies.At high doses of XAE 700,800 mg kg À1 , the values were quite lower compared with values obtained in the control group.
Abbreviations: F, finasteride; Tp, testosterone propionate; XAE, Xylopia aethiopica ethanol leaf extracts.The results are presented as mean AE standard deviation of three replicate measurements.A 1 -Olive oil (vehicle only), A 2 -Tp (14 mg kg À1 ), B 1 -Tp (14 mg kgÀ1 ) þ F (10 mg kg À1 ), B 2 -(XAE) 100 mg kg À1 þ Tp (14 mg kg À1 ) þ (XAE) 100 mg kg À1 , B 3 -(XAE) 200 mg kg À1 þ Tp (14 mg kg À1 ) þ (XAE) 200 mg kg À1 , B 4 -(XAE) 300 mg kg À1 þ Tp (14 mg kg À1 ) þ (XAE) 300 mg kg À1 , B 5 -(XAE) 400 mg kg À1 þ Tp (14 mg kg À1 ) þ (XAE) 400 mg kg À1 .Values with different superscripts in the same column are significantly different (p 0.05).treatmentEffect of Xylopia aethiopica on Antioxidant Activities in Tp-Induced Benign Prostatic Hyperplasia in RatsThe results of XAE on antioxidant activities in Tp-induced BPH in rats are shown in ►Tables 5 and 6.The activities of catalase (CT), glutathione (GSH), superoxide dismutase (SOD), and malondialdehyde (MDA) decreased significantly (p < 0.05) in Tp-induced BPH group compared with control group.Thus, the trend was reversed (significantly (p < 0.05) increased) in finasteride and XAE treatment groups.However, the activities increased more significantly (p < 0.05) in XAE treated groups than in finasteride groupEffect of Xylopia aethiopica on Kidney Biochemical Parameters in Tp-Induced Benign Prostatic Hyperplasia in RatsThe results of XAE on kidney biochemical parameters in Tp-induced BPH in rats are shown in ►Tables 7 and 8.Effect of X. aethiopica on Liver Function Parameters in Tp-Induced Benign Prostatic Hyperplasia in RatsThe result of XAE on liver function parameters in Tp-induced BPH in rats are shown in ►Tables 9 and 10.The results showed a significant (p < 0.05) decrease in the total protein level in the Tp-induced BPH rats relative to control group.The trend changed on treatment with finasteride and XAE; it significantly (p < 0.05) increased in finasteride and XAE treatment groups compared with the Tp only group in both studies.The levels of AST, ALT, and ALP were not significantly (p < 0.05) different in the Tp-induced BPH group, finasteride, and XAE treatment groups compared control in both studies (preventive and curative)

Table 8
Effect of XAE on kidney biochemical parameters in Tp-induced benign prostatic hyperplasia in rats (curative studies) AE 0.04 a 146.40 AE 1.20 a 105.95AE 1.65 a 27.60 AE 1.50 a 11.40 AE 1.35 a 3.90 AE 0.01 a 111.35AE 1.25 a AE 0.16 b 130.45 AE 0.95 c 88.20 AE 2.25 b 18.00 AE 2.25 c 2.40 AE 0.40 c 1.20 AE 0.06 c 30.45 AE 2.15 c

Table 10
32fect of XAE on liver function parameters in Tp-induced benign prostatic hyperplasia in rats (curative studies) -Control, A 2 -Tp (14 mg kg À1 ), C 1 -Tp (14 mg kg À1 ) AI þ F (10 mg kg À1 ), C 2 -Tp(14 mg kg À1 ) AIþ (XAE) 500 mg kg À1 , C 3 -Tp (14 mg kg À1 ) AI þ (XAE) 600 mg kg À1 , C 4 -Tp (14 mg kg À1 ) AI þ (XAE) 700 mg kg À1 , C 5 -Tp (14 mg kg À1 ) AI þ (XAE) 800 mg kg À1 .Values with different superscripts in the same column are significantly different (p 0.05).seleniumagainst the histological and histochemical changes of the prostate induced by carbimazole may be due to its antioxidant properties from a previous study 36 this could have been responsible for the observed therapeutic potentials of XAE in this study due to the significant presence of Se.Ca is an important nutrient for prostate health, with a positive correlation between it and Mg and Zn in hyperplasia of the prostate gland.Age-related changes in Ca levels are linked to BPH.32Calcium (Ca) is an essential mineral that plays a crucial role in various bodily functions, including bone health, muscle contraction, blood clotting, and nerve transmission.While calcium is important for overall health, its relevance specifically to prostate health is not as direct as it is for other areas of the body.Prostate health is more closely associated with other nutrients and factorsThe results of XAE on kidney parameters in Tp-induced BPH in rats showed that the levels of K, Na, Cl, HCO 3 À , urea, uric acid, and creatinine were significantly higher in the TP only group compared with the control group.However, the levels were lowered significantly in the finasteride and XAE treatment groups relative to the Tp only group for both studies.Basically, potassium, sodium, chloride, and bicarbonate are electrolytes that play crucial roles in maintaining the balance of fluids and ions in the body.Abnormal levels of these electrolytes can indicate disruptions in kidney function, fluid balance, or other physiological processes.If these levels are consistently elevated in individuals with BPH, it might suggest an underlying kidney or metabolic issue.