Amelioration of testosterone induced benign prostatic hyperplasia by Prunus species

Author’s Accepted Manuscript Ameolioration of testosterone induced benign prostatic hyperplasia by Prunus species Ashish Kumar Jena, Karan Vasisht, Neetika Sharma, Ramdeep Kaur, Mamta Sachdeva Dhingra, Maninder Karan www.elsevier.com/locate/jep

PII: DOI: Reference:

S0378-8741(16)30335-X http://dx.doi.org/10.1016/j.jep.2016.05.052 JEP10190

To appear in: Journal of Ethnopharmacology Received date: 10 March 2016 Revised date: 29 April 2016 Accepted date: 21 May 2016 Cite this article as: Ashish Kumar Jena, Karan Vasisht, Neetika Sharma, Ramdeep Kaur, Mamta Sachdeva Dhingra and Maninder Karan, Ameolioration of testosterone induced benign prostatic hyperplasia by Prunus species, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2016.05.052 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Ameolioration of testosterone induced benign prostatic hyperplasia by Prunus species Ashish Kumar Jena, Karan Vasisht, Neetika Sharma, Ramdeep Kaur, Mamta Sachdeva Dhingra and Maninder Karan*

University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, India - 160014

Corresponding author details: *

Professor Maninder Karan University Institute of Pharmaceutical Sciences–UGC Centre of Advanced Study Faculty of Pharmaceutical Sciences Panjab University, Chandigarh-160014, India Tel.: +91–9876057171 fax: +91–172–2543101 [email protected]

Abstract

Ethnopharmacological relevance: Benign prostatic hyperplasia (BPH) is a common urological disorder of men. The ethnomedicinal use of an African plant Prunus africana (Hook.f.) Kalkman (Pygeum) in treating men’s problems made it a popular remedy all over the globe for the treatment of BPH and related disorders. However, rampant collections made from the wild in Africa have pushed the plant to Appendix II of the CITES demanding conservation of the species. Aim of the study: In the present study, the aim was to unearth the protective effect of bark of different species of Prunus against BPH. The five selected Indian plants of family Rosaceae viz. P. amygdalus Stokes, P. armeniaca L., P. cerasoides Buch.-Ham. ex D.Don, P. domestica L. and P. persica (L.) Batsch were evaluated against P. africana (Hook.f.) Kalkman for a suitable comparison of efficacy as antiBPH agents. Materials and methods: The antiBPH activity was evaluated in testosterone (2 mg/kg/day, s.c, 21 days) induced BPH in Wistar rats. The parameters studied were body weights; histopathological examination, immunohistochemistry (PCNA) and biochemical estimations of the prostate; supported by prostatic index, testicular index, creatinine, testosterone levels; antioxidant and anti-inflammatory evaluation. The study also included chemical profiling using three markers (β-sitosterol, docosyl ferulate and ursolic acid) and estimation of βsitosterol content through GC. Results: The Prunus species showed the presence of all the three markers in their TLC fingerprint profile and maximum amount of β-sitosterol by GC was observed in P. domestica. Interestingly, all the species exhibited significant amelioration in testosterone induced 1

parameters with P. domestica showing the most encouraging effect as indicated from histopathological examination, immunohistochemistry and biochemical studies. The Prunus species further showed remarkable anti-inflammatory and antioxidant activity signifying their role in interfering with various possible factors involved in BPH. Conclusions: These findings are suggestive of a meaningful inhibitory effect of testosterone induced BPH by the bark of different species of Prunus in the order of P. domestica, P. persica, P. amygdalus, P. cerasoides and P. armeniaca with an efficacy of P. domestica comparable to P. africana and can be used as the potential backup of Pygeum for the management of BPH. Key words: Benign prostatic hyperplasia, Prunus species, Chemical profiling, 5α-Reductase, Anti-inflammatory, Antioxidant List of chemical compounds studied in this article: β-sitosterol (PubChem CID: 222284), Docosyl ferulate (PubChem CID: 14238616 ), Ursolic acid (PubChem CID: 64945), Finasteride (PubChem CID: 57363), Testosterone (PubChem CID: 6013), Carrageenan (PubChem CID: 101231953)

1. Introduction Benign prostatic hyperplasia (BPH) is a disease of long duration especially in elderly men, characterized by hyperplasia of prostatic stromal and epithelial cells (Berry et al., 1984) . Its etiology is multi-factorial largely affected by the hormonal (Marker et al., 2003), genetic (Sanda et al., 1994) and nutritional factors (Bravi et al., 2006). Although the causes of BPH are not fully understood, but one of the major factor involved is the enzyme 5α-reductase (5αR) that leads to increased level of dihydrotestosterone (DHT) by the conversion of testosterone (Galbraith & Duchesne, 1997). The primary aspects involved are increase in the smooth muscle tone and non-malignant enlargement of the gland (Thiyagarajan et al., 2002), while complications include frequent recurrent urinary retention, urinary tract infections and rare post-renal failure (Homma et al., 2011). A number of surgical and non-surgical approaches including 5α-reductase inhibitors, α-adrenergic receptor blockers and natural substances are available to counteract various complications of BPH. (Power & Fitzpatrick, 2004; Ponholzer et al., 2004; Pagano et al., 2014). According to the US Medicare data, a decrease of more than 65% in prostatectomy cases since 1980s is attributed to the welltolerated and effective medical therapies available in the current time (Kevin & Mc Vary, 2007). Phytotherapy in particular, is very popular as the primary treatment in the USA and European Union (Pagano et al., 2014). 2

The most widely used herbal preparations are based on Saw palmetto, Pygeum and Nettle extract. The well reported plants used in different parts of the world are Prunus africana (Hook.f.) Kalkman (Carani et al., 1991), Hypoxis rooperi T.Moore (Berges et al., 1995), Serenoa

repens W. Bartram (Tacklind et al., 2012), Urtica dioica L. (Safarinejad, 2005), Cucurbita pepo L. (Vahlensieck et al., 2015), Secale cereal L. (Mac Donald et al., 2000). Of all these, P. africana (African cherry, Pygeum) of family-Rosaceae, is a globally famous plant official in United States Pharmacopoeia (USP) (Anonymous, 2011), European Pharmacopoeia (EP) (Anonymous, 2010) and British Pharmacopoeia (BP) (Anonymous, 2008). It was used traditionally in southern, east and central Africa that got attention of Europe in the 17th century about its usefulness in controlling bladder discomfort and the old men’s diseases (Watt & Beyer-Brandwijk, 1962; Kalkman, 1965; ESCOP monographs, 2009). A number of clinical studies have shown promising efficacy and safety of Pygeum against BPH (Bombardelli & Morazzoni, 1997; Awang, 1997; Bassi et al., 1987). The heavy exploitation of P. africana for the commercial production of extract led to its listing in Appendix II of the CITES (Convention of International Trade in Endangered Species of Wild Fauna and Flora) (Stewart, 2003; Nkeng, et al., 2010). Various organizations including UNESCO have recommended a strong need for the alternative sources of Pygeum (Cunningham & Mbenkum, 1993). Two of the recent patents referring to the (i) improved process for Pygeum extraction (Tewari & Sharma, 2013) and (ii) process for Pygeum extraction using stem cuttings/twigs of P. domestica (Tewari & Sharma, 2013a) is in fact a step towards conservation of P. africana using other renewable sources. About thirty six species of the genus Prunus are reported to be growing in India (Ghora & Panigrahi, 1984; Anonymous, 2003) and to the best of our knowledge none of these species have been evaluated as yet for their probable role in BPH. Therefore, in the present study, the bark of five different Indian species of Prunus largely known for edible stones and fruits like, P. amygdalus Stokes (Almond, Sweet almond and Badam), P. armeniaca L. (Apricot, Khurmani and Zardalu), P. cerasoides Buch.-Ham. ex D.Don (Sour cherry, Padam and Paija), P. domestica L. (Wild plum, Alubukhara and Alucha) and P. persica (L.) Batsch (Peach, Nectarine, Shaftalu and Aru) have been investigated and compared with P. africana for their protective effect towards BPH. Some of the important phytoconstituents reported from the bark include β-sitosterol (Stewart, 2003), daucosterol (Catalano & Ferretti, 1984), ursolic acid, oleanolic acid, (Hall et al., 2000), n-docosanol, docosyl ferulate (Cristoni et al., 2000) from P. africana; kaempferol, quercetin 3,4’-diglycoside (Rawat & Pant, 1995) from P. amygdalus; β-sitosterol, chrysophanol (Garg et al., 1985) from P. cerasoides; and 3

persicogenin (Backheet & Farag, 2003) from P. persica. No phytoconstituent is reported from the bark of P. domestica and P. armeniaca, although many compounds have been isolated from other parts of these two plants. It is expected that more species of Prunus will emerge as suitable candidate(s) for the treatment of prostate hyperplasia and related disorders. The study is further aimed to bring therapeutic value to more species of Prunus in the international market which till date are largely known for their nutritional value and a possible solution for conservation of the endangered species of P. africana.

2. Materials and methods 2.1. Chemicals Testosterone, finasteride, β-sitosterol, ursolic acid and carrageenan were obtained from Sigma-Aldrich Chemicals Pvt. Ltd. All other chemicals and solvents (Merck Specialities Pvt. Ltd., S.D. Fine Chemicals and Himedia Laboratories Pvt Ltd, India) were of analytical grade and procured locally. A reference sample of docosyl ferulate was provided by the Medicinal Chemistry Unit of the Division of Pharmaceutical Chemistry, University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, India. 2.2. Plant material and preparation of extract The bark of P. domestica was procured from Kangra and its surrounding areas (Himachal Pradesh, India) in the month of March, 2012. A gift sample of P. africana bark was provided by Chemical Resources, Panchkula, India. The bark of other species of Prunus i.e. (P. amygdalus, P. armeniaca, P. cerasoides and P. persica) collected from the Dr. Y.S. Parmar University of Horticulture & Forestry, Nauni, Solan (Himachal Pradesh, India) in the year 2010-2011 was taken up for the present studies. The authentication of the procured material was done by National Institute of Science Communication and Information Resources (NISCAIR), New Delhi vide ref-no NISCAIR/RHMD/Consult/-2012-13/2031/39 dated 27.06.2012. A voucher specimen of all the authentic samples has been deposited in the Museum-cum-Herbarium of the University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, India under the voucher nos. 236-241 for P. domestica, P. amygdalus, P. armeniaca, P. cerasoides, P. persica and P. africana, respectively. The coarsely powdered bark (10 g) of the plant material was extracted by macerating with 200 mL methanol for 48 h. The extract was filtered, concentrated under reduced pressure to get 1.8 g (P. africana), 2.2 g (P. amygdalus), 1.9 g (P. armeniaca), 2.4 g (P. cerasoides), 2.3 g

4

(P. domestica) and 2.1 g (P. persica) respectively, which was used for phytochemical screening, chemical profiling, toxicity and pharmacological evaluation. 2.3. Phytochemical analysis The methanolic extract of the bark of Prunus species under study was subjected to preliminary phytochemical screening following the standard methods reported in the literature (Farnsworth, 1966). 2.4. TLC fingerprint profile The comparative TLC fingerprint profiles were developed using pre-coated silica gel F254 plates [E. Merck (India) Ltd., alumina base, 0.2 mm thickness]. A number of mobile phases were tried for developing well resolved chromatograms and finally the solvent systems of cyclohexane:chloroform:ethyl acetate:methanol (10:2.5:2:1) for β-sitosterol, hexane:dichloromethane:acetic acid (11:1:2.5) for docosyl ferulate and toluene:ethyl acetate:acetic acid (8:1.8:0.2) for ursolic acid were used in the study. The reference markers and extracts were applied as bands of 1 cm width using Camag Linomat 5. The running distance was kept at 8 cm and anisaldehyde-sulphuric acid reagent was used as derivatizing agent followed by heating at 110 °C for 5 min or till the bands developed colour. The TLC fingerprint profiles were recorded as images under UV before spray for docosyl ferulate and under white light after derivatization for β-sitosterol and ursolic acid on Camag Reprostar fitted with D×A 252 16 mm camera. 2.5. Estimation of β-sitosterol by gas chromatography The estimation of β-sitosterol in different Prunus species was carried out as per the standard procedure given in the USP (Anonymous, 2011). The analysis was performed on an Agilent Technologies 7890A gas chromatograph equipped with HP-5, 30 m × 0.322 mm × 0.25 μm GC column and FID detector. The temperature of column was equilibrated at 250 ºC for 5 min, and increased to 320 ºC at a rate of 5 ºC/min. The injection port and detector temperature was maintained at 285 ºC. Nitrogen was used as the carrier gas with flow rate of 2 mL/min, hydrogen was used as detector gas maintained at 30 mL/min and air flow was programmed at a constant flow of 300 mL/min. A volume of 2 µL of standard and sample solution was injected. The split ratio of 1:50 was used at the injection port and data for each run was acquired for 20 min and analyzed. 5α-Cholestane was used as internal standard and a mixture of bis (trimethylsilyl) acetamide and trimethylchlorosilane was used as the derivatizing agent. 2.6. Experimental animals 5

The animal studies were approved by the Institutional Animal Ethics Committee (IAEC) vide ref no. 536/IEAC dated 20.01.2015 and were used according to the CPCSEA (Committee for the Purpose of Control and Supervision of Experimentation on Animals) guidelines. Male Wistar rats weighing ≈150-250 g were procured from the Central Animal House, Panjab University, Chandigarh, India. The animals were kept in animal cages, 6 rats per cage, on straw bedding in an animal house maintained to natural light and dark cycle under controlled environment of temperature (25 ± 2 °C) and humidity (50 ± 10%). The animals were fed on standard rodent food pellets diet and water ad libitum. After one week of acclimatization, animals were randomly distributed into different experimental groups. The test and standard doses were administered orally with the help of an oral cannula fitted on a tuberculine syringe and testosterone was injected subcutaneously. 2.7. Acute toxicity study The acute toxicity study of methanolic extract of P. domestica was carried out as per the OECD guidelines (OECD, 2008). An oral dose of 2000 mg/kg body weight was administered to the animals for the assessment of toxicity. The animals were observed periodically during the first 24 h with special attention given during the first 4 h and then daily for a total of 14 days for any signs of toxicity and mortality. 2.8. Dose selection and administration Most clinical studies on P. africana (Pygeum) against BPH have been carried out at the dose levels of 100 and 200 mg/day in patients (Bombardelli & Morazzoni, 1997). Hence, using the dose translation conversion formula from human to rats (Reagan-Shaw et al., 2007), initially an oral dose of 10 and 20 mg/kg/day of the tested drugs was selected which was to be administered for 21 days for evaluating antiBPH activity. As 20 mg/kg/day dose showed better activity, so this dose was used subsequently to evaluate the efficacy. Similarly, for antiinflammatory activity, initially a single dose of 20, 100, 200 and 400 mg/kg of the tested drugs was selected. The best activity was observed at 200 mg/kg, and this dose was used subsequently. 2.9. Experimental design and induction of BPH The rats were divided into ten groups of six animals each and received the following treatment for 21 consecutive days: Group 1 (negative control group)-Tween-80 (2% v/v in normal saline, p.o.) and olive oil (1 mL/kg, s.c.); Group 2 (BPH control group)-normal saline (5 mL/kg, p.o.) and testosterone (2 mg/kg, s.c.) prepared in olive oil; Group 3 (positive control group)-finasteride (1 mg/kg, p.o.) and testosterone (2 mg/kg, s.c.) prepared in olive 6

oil; Group 4-extract of P. domestica (10 mg/kg) orally and testosterone (2 mg/kg, s.c.) in olive oil; Group 5 to 10-extract of P. domestica, P. amygdalus, P. armeniaca, P. cerasoides P. persica and P. africana, respectively at a dose of 20 mg/kg orally and testosterone (2 mg/kg, s.c.) in olive oil. All animals were examined every alternate day for any external changes and the body weight was measured weekly. On day 22, the animals were sacrificed humanly by cervical dislocation. Prostate from each animal was excised, weighed and rinsed immediately with ice-cold normal saline. Half of the ventral lobe of the prostate was stored for the histological studies in neutral buffered formalin and the remaining of the prostate was used for biochemical assays. Serum was separated for the measurement of creatinine and testosterone levels. 2.10. Characterization of BPH activity 2.10.1. Prostatic and testicular index determination The prostatic index and testicular index were calculated by the following equations: Prostatic index = prostatic wet weight/animal weight Testicular index = testicle wet weight/animal weight 2.10.2. Percentage inhibition of prostate and testis weight This was calculated using the following formula: Percentage inhibition of prostate weight/testis weight= 100 – [(T–NC)/PC–NC) × 100] Where T = treatment group, NC = negative control group and PC = positive control group 2.10.3.

Measurement of testosterone in the serum and prostate

The testosterone level in the serum and prostate was estimated using a testosterone enzymelinked immunosorbent assay (ELISA) kit (YH Biosearch Laboratory, Sanghai, China) according to the manufacturer’s instructions. The measurements were done using the testosterone ELISA kit and the standard curve of testosterone was prepared by making the dilutions as per the procedure specified by the manufacturer. After completion of the assay procedure, the intensity of the colour was measured with ELISA microreader at 450 nm. 2.10.4. Measurement of serum creatinine The serum creatinine determination was done as it is recommended in the initial evaluation of the disease with LUTS (Gerber et al., 1997) and was measured by Jaffe-S method (Husdan & Rapoporf, 1968). Briefly, the assay mixture consisted 1 mL of 0.75 N NaOH, 1 mL of 1% picric acid and 1 mL of protein free filtrate of serum. The blank solution consisted of 1 mL of

7

distilled water in place of serum. The yellowish orange colour developed within 15 min of keeping the mixture at room temperature and the absorbance was measured at 520 nm. A known concentration of standard creatinine was dissolved in 0.1N HCL as it is insoluble in water. The creatinine concentration was calculated using the following equation: Ctest = Cstd × Atest/Astd Where Ctest & Cstd = concentration of test substance and standard creatinine, Atest & Astd = absorbance of test substance and standard creatinine. 2.11. Histopathological examination The separated ventral lobe of the prostate was fixed with 10% formalin, dehydrated in increasing concentrations of ethanol and embedded in paraffin. The tissue was sectioned at 5 µm and mounted on Mayer’s albumin coated glass slides. The mounted sections were then deparaffinized with xylene, rehydrated with alcohol and water. The rehydrated sections were stained using hematoxylin-eosin (H&E), mounted with DPX, examined under a microscope (Nikon-90i microscope, Japan) and the images were captured. 2.12. Immunohistochemical (IHC) analysis The paraffin embedded tissue sections (5 µm) were dried, deparaffinised and rehydrated. The sections were treated in citrate buffer (pH 6.0) for antigen retrieval and then immersed in 3% H2O2 for 20 min to block the endogenous peroxidase activity. After washing with PBS, the slides were incubated with anti-PCNA antibody (Santa Cruz Biotechnology, USA) and kept at 4 °C overnight. The sections were then incubated with a secondary antibody for 1 h at room temperature, and were developed using commercially available kit Novolink™ polymer detection system (Leica, Milton, Keynes, UK). Finally, the sections counterstained with hematoxylin were observed under microscope (Nikon-90i microscope, Japan) for proliferating cell nuclear antigen analysis and the images were recorded. 2.13. Biochemical assays for oxidative parameters 2.13.1. Sample preparation About 10% (w/v) tissue homogenate was prepared in an ice cold 0.1 M phosphate buffer (pH 7.4) using a Potter Elvenhjem homogenizer (Remi, Mumbai, India). The homogenate was centrifuged at 12,000 g for 15 min at 4 °C to obtain the supernatant, which was separated and used for further enzymatic analysis. A double beam UV–VIS spectrophotometer (Shimadzu UV-1800, Japan) was used for the estimation of biochemical parameters (Dhingra et al., 2014). 2.13.2. Estimation of lipid peroxidation 8

The quantitative measurement of lipid peroxidation in the prostate was performed according to the reported method (Wills, 1966). The malondialdehyde (MDA) content, a measure of lipid peroxidation, was assayed in the form of thiobarbituric acid reacting substances (TBARS). Briefly, 0.5 mL of the sample and 0.5 mL of Tris HCL were incubated at 37 °C for 2 h. After incubation, 1 mL of 10% trichloroacetic acid was added and centrifuged at 1000 × g for 10 min. To 1 mL of the supernatant, 1 mL of 0.67% thiobarbituric acid was added and the tubes were kept in boiling water for 10 min. After cooling, 1 ml of the distilled water was added and absorbance was measured at 532 nm using the double beam UV–VIS spectrophotometer. The values were calculated using the molar extinction coefficient of chromophore (1.56 × 105 M−1 cm−1) and expressed as nanomoles of malondialdehyde per mg of protein. LPO (nanomoles of MDA/mg protein) = 4.8 × OD/mg protein

2.13.3. Estimation of catalase The catalase activity was assayed by a method in which the breakdown of hydrogen peroxide (H2O2) is measured (Luck, 1971). Briefly, the assay mixture consisted of 1.95 mL phosphate buffer (0.05 M, pH 7), 1 mL hydrogen peroxide (0.019 M) and 0.05 mL homogenate supernatant (10%) in a final volume of 3 mL and the change in absorbance was recorded at 240 nm using the double beam UV–VIS spectrophotometer. The catalase activity was expressed as µmoles H2O2 decomposed per mg of protein/min. Catalase (µ moles of H2O2 decomposed/min/mg protein) = 42.95 × ΔOD/mg protein 2.13.4. Estimation of reduced glutathione The reduced glutathione (GSH) in the prostate was measured by a well-known method (Ellman, 1959). In brief, 1 mL of the supernatant was precipitated with 1 mL of 4% sulfosalicylic acid and samples were kept at 4 °C for 1 h. The sample was centrifuged at 1200 rpm for 15 min at 4 °C. To 1 mL of this supernatant, 2.7 mL of 0.1 M phosphate buffer (pH 7.4) and 0.2 mL of 5,5-dithiobis 2-nitrobenzoic acid (DTNB) (40 mg/10 mL of 0.1 M phosphate buffer, pH 7.4) were added. The developed yellow colour was measured immediately at 412 nm using the double beam UV–VIS spectrophotometer. The results were calculated using the molar extinction coefficient of chromophore (1.36 × l03 M−1 cm−1) and were expressed as micromole GSH per mg protein. GSH (µ moles of GSH/mg protein) = 0.05 × O7D/mg protein

9

2.13.5. Estimation of superoxide dismutase Superoxide dismutase activity was assayed according to the method wherein the reduction of nitrobluetetrazolium (NBT) is inhibited by the superoxide dismutase and measured at 560 nm using the double beam UV–VIS spectrophotometer (Kono, 1978). The assay system consisted of 0.1 mM EDTA, 50 mM sodium carbonate and 96 nM of NBT. To 2 mL of above mixture in a cuvette, 0.5 mL of sample and 0.5 mL of hydroxylamine hydrochloride (adjusted to pH 6.0 with NaOH) was added. The auto-oxidation of hydroxylamine was observed by measuring the change in optical density at 560 nm for 2 min at 30 sec intervals. The results were expressed as unit/mg protein. 2 (Δ Non enzymatic reaction - Δ Enzymatic reaction) SOD (units/mg protein) = × mg protein Δ Enzymatic reaction

2.13.6. Estimation of nitrite The nitrite accumulation in the supernatants, an indicator of the production of nitric oxide (NO), was based on colorimetric assay using Greiss reagent (0.1% N-(1-napthyl) ethylenediamine dihydrochloride, 1% sulphanilamide and 2.5% phosphoric acid) as reported in literature (Green et al., 1982). Equal volumes of the supernatants and Greiss reagent were mixed, the mixture was incubated for 10 min at room temperature and the absorbance was measured at 540 nm using the double beam UV–VIS spectrophotometer. The concentration of the nitrite in the supernatant was determined from a sodium nitrite standard curve and was expressed as micromole/mg protein. 2.13.7. Estimation of protein content The protein content was measured by the Biuret method (Ernest, 1996) using bovine serum albumin (BSA) as a standard. In this method, 2.9 mL of normal saline and 3 mL of Biuret reagent was added to 0.1 mL of the sample. The mixture was kept for 10 min at room temperature and absorbance was measured at 540 nm. The concentration of protein in the supernatant was determined from a BSA standard curve and the calculated protein content was used in the estimation of different oxidative enzymatic parameters. 2.14. Anti-inflammatory activity Anti-inflammatory activity was determined using carrageenan-induced paw oedema in rats (Karan et al., 2012; Guan-Jhong et al., 2012). The animals were divided into control, standard 10

(ibuprofen) and test groups of six animals each. The animals were starved overnight. Acute oedema was induced by injecting 0.1 mL of freshly prepared working solution of carrageenan (1%) under plantar region of left hind paw. The control group received only the vehicle while standard and test groups received ibuprofen and test substances, respectively. The paw was marked with ink at the level of the lateral malleolus and immersed in solution up to this mark for noting the paw volume. The increase in paw volume was measured using Plethysmometer (water displacement, UGO-Basile, Varese, Italy) at 0, 1, 3 and 6 h after carrageenan challenge. The per cent protection of paw oedema was calculated from the following formula: % Protection of paw oedema = (Vt - V0)control - (Vt - V0)treated / (Vt - V0)control X 100 (V0) = volume of the paw before treatment with carrageenan (Vt) = volume of paw after carrageenan administration at 0, 1, 3 and 6 h

2.15. Statistical analysis Results were expressed as mean ± SEM. The significance of difference in the response of treated and control group was assessed by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison and two way ANOVA followed by Bonferroni tests. The results were considered significant at p < 0.05. The statistical analysis was done using the Graph Pad Prism statistical software version 5. 3. Results 3.1. Phytochemical screening The methanolic extract of the investigated species of Prunus showed the presence of phytosterols, pentacyclic triterpenoids, glycosides, alkaloids, flavonoids carbohydrates, proteins and tannins. 3.2. TLC fingerprint profile TLC fingerprinting serves as a valuable source of information for the authentication, identification and evaluation of similarities and dissimilarities between the plants based on their chemical profile. The TLC chromatograms were developed in the respective solvent systems for the identification of two official markers given in USP (docosyl ferulate and β-sitosterol) in different species of Prunus. Further, the presence of ursolic acid was also established. It is reportedly accredited with antiandrogenic activity and is considered useful in BPH (In-Sik et al., 2012). For β-sitosterol and ursolic acid, the developed plates were

11

derivatized with anisaldehyde-sulphuric acid reagent followed by heating at 110 ºC till the bands appeared and were visualized in white light. Docosyl ferulate was observed under UV366 as it is sensitive to UV light. All the species showed pink to purple colour band of β-sitosterol at Rf 0.72 and ursolic acid at Rf 0.44 under white light (Fig 1 i & iii) as well as a prominent blue fluorescent band of docosyl ferulate at Rf 0.5 under UV light (Fig 1 ii). 3.3. Estimation of β-sitosterol by gas chromatography The reported literature revealed potent efficacy of this ubiquitous phytosterol in the treatment of BPH (Wilt et al., 1999). USP further specifies estimation of β-sitosterol by GC in P. africana and its extracts for the purpose of quality control. Hence, quantitative estimation of β-sitosterol was done using gas chromatographic analytical method of USP. All the species showed the presence of β-sitosterol at a retention time of 14.61±0.01 min in the GC chromatogram (Fig 2). The amount of β-sitosterol was calculated and was found to be maximum in P. domestica (0.10%) and minimum in P. cerasoides (0.027%) with P. africana showing 0.041% content. The obtained β-sitosterol content of different species of Prunus is shown in Fig 3. 3.4. Evaluation of BPH activity 3.4.1. Body weight The administration of testosterone increased the body weight of all animals but the results were not significant (p < 0.05) when compared to the negative control (Table 1). 3.4.2. Effect on prostate weight (PW) and testicular weight (TW) A significant increase in the prostate and testicular weights was induced by the testosterone treatment (2 mg/kg, s.c., 21 days) when compared to negative control group (p < 0.05). A reduction in the elevation of both prostate and testis weights was observed in all the groups treated with Prunus species as well as finasteride. The maximum percentage inhibition (p < 0.05) of the prostate weight was shown by finasteride (95.92%) followed by P. africana (92.86%) and P. domestica (90.82%) as compared to the BPH control (Table 1). Similarly, a significant (p < 0.05) decrease in the testis weight was observed in the treated groups as compared to the BPH group. The maximum inhibition was found to be 536, 318 and 282% by finasteride, P. africana and P. domestica, respectively when compared to the BPH control animals (Table 1). 3.4.3. Effect on prostatic index (PI) and testicular index (TI) The induction of BPH appreciably (p < 0.05) increased the prostatic and testicular index in testosterone induced BPH animals, as compared to the negative control group. The treatment 12

with finasteride and Prunus species decreased prostatic and testicular index significantly when compared to the BPH control group (p < 0.05). The maximum percent inhibition of PI was found out to be 92.13, 93.16 and 90.1% for finasteride, P. domestica and P. africana respectively, (Table 1), while in case of TI, the maximum inhibition was observed with P. africana (262%) closely followed by P. domestica (245%) and P. cerasoides (241%). The percent inhibition of TI in P. amygdalus, P. armeniaca and P. persica was observed to be 209, 203 and 188%, respectively; and the standard drug finasteride exhibited maximum inhibition of 435% (Table 1). 3.4.4. Effect on testosterone levels in serum and prostate The mode of action of tested drugs was supported by measuring serum and prostate tissue testosterone levels for 5α-reductase inhibitory activity. The administration of finasteride significantly enhanced the serum and prostate testosterone levels as compared to the BPH group, suggesting the inhibition of conversion of testosterone to DHT, which is the key hormone responsible for the progression of disease (Galbraith & Duchesne, 1997). The Prunus species restored the serum and prostate testosterone level in a similar manner as by finasteride signifying that the tested drugs may be acting through inhibition of 5α-reductase enzyme. Among the different species, the serum concentration of testosterone in P. domestica and P. africana treated groups (18.5 and 18.0 nmol/L) was nearly the same (Fig 4A). Similarly, in the prostate tissue, the testosterone concentration of 11.59 and 13.26 nmol/L suggested that the efficacy of 5α-reductase enzyme inhibition of P. domestica was close to P. africana (Fig 4B). 3.4.5. Effect on serum creatinine The serum creatinine level was significantly increased by 66% (p < 0.05) in testosterone induced BPH control group as compared to the negative control group. Finasteride and Prunus species showed significant lowering of the serum creatinine level. The highest reduction was observed in the standard drug finasteride (68%) and P. domestica group (67%) followed by P. africana (61%) and P. armeniaca (54%) as compared to the BPH control group (Fig 4C). 3.4.6. Histopathological studies The effect of different species of Prunus on the morphology of the prostate gland in testosterone induced BPH animals is shown in Fig 5A-J. The prostate tissue in the negative control group consisted of tightly packed, flattened, cuboidal, regular size epithelium with not much change in the histoarchitecture. The animals of the testosterone induced BPH group 13

showed large stromal spaces of prostate and glandular hyperplasia with epithelial proliferation and nuclear stratification. The glandular epithelium became thicker and formed numerous papillae projecting into the glandular lumen, thereby decreasing the glandular luminal area (Fig. 5B). The treatment with finasteride (Fig. 5C) and different species of Prunus i.e, P. domestica 10 mg/kg (Fig. 5D), P. domestica (Fig. 5E), P. amygdalus (Fig. 5F), P. armeniaca (Fig. 5G), P. cerasoides (Fig. 5H), P. persica (Fig. 5I) and P. africana (Fig. 5J) all at a dose of 20 mg/kg, exhibited some features of prostate glandular hyperplasia, but with very much reduced epithelial proliferation and stromal spaces of the prostate as compared to the BPH induced group (Fig. 5B). The administration of finasteride and Prunus species protected the overall morphology and restored the histological features of the prostate to nearly normal.

3.4.7. Immunohistochemical (IHC) analysis The key feature of prostatic hyperplasia is the increased cell proliferation in prostate. The proliferating cell nuclear antigen (PCNA) is considered as a histological marker of the S phase of cell cycle. There is noteworthy immunoreactivity to PCNA (brown colour spot) in the BPH control animals as compared to the negative group (Fig 6A&B). Administration of finasteride and all the species of Prunus attenuated the cell proliferation as compared to the BPH group. All the tested plant species showed a significant decrease in the PCNA positive cells similar to that of finasteride (Fig 6C-J). The maximum inhibition, shown by brown colour spot in the epithelium tissue was observed in P. africana (Fig 6J) followed by P. domestica (Fig 6E) as compared to the standard drug finasteride (Fig 6C). 3.4.8. Evaluation of antioxidant parameters 3.4.8.1. Effect on prostate SOD, catalase and GSH Reactive oxygen species (ROS) are produced during the progression of the disease leading to severe damage to the prostate. The activities of SOD, catalase and GSH play an important role in the oxidative defence of the prostate tissue. Testosterone treatment significantly (p < 0.05) decreased the SOD, catalase and GSH levels by 71, 75 and 87% when compared with negative control animals as shown in Fig 7A-C. The administration of finasteride and different species of Prunus significantly restored all the three enzymes. Finasteride, P. domestica, P. africana, P. persica and P. amygdalus restored SOD by 67, 62, 59, 57 and 53% as compared to BPH control (Fig 7A). The restoration of catalase by finasteride,

14

P. africana, P. amygdalus and P. domestica, was 65, 63, 58 and 54% when compared to BPH control (Fig 7B). Similarly, the GSH level was restored by 87, 86, 85, 82 and 82%, with finasteride, P. domestica, P. africana, P. persica and P. amygdalus as compared to the BPH control group (Fig 7C). 3.4.8.2. Effect on prostate LPO and total nitrite levels The animals with testosterone induced BPH showed a significant (p < 0.05) increase in the prostate LPO-MDA and the nitrite levels going up to 71 and 56% as compared to the negative control. Administration of finasteride and different species of Prunus significantly (p < 0.05) lowered the elevated MDA and nitrite levels. The maximum amelioration of MDA was observed with P. domestica (61%), followed by P. africana (57%) and P. persica (51%) with finasteride showing 64% reversal when compared to the BPH control group (Fig 7D). The maximum improvement in the nitrite level was observed in P. africana (61%) followed by finasteride (57%) and P. domestica (52%), respectively as compared to the BPH control group (Fig 7E). 3.5. Evaluation of anti-inflammatory activity The anti-inflammatory activity of the methanolic extract of different species of Prunus was carried out at a single per oral dose of 20, 100, 200 and 400 mg/kg in carrageenan induced paw oedema model. The different species of Prunus showed statistically significant activity (p ≤ 0.05) as compared to the standard drug ibuprofen. All the species at different doses showed significant decrease in paw volume at variable time intervals as shown in Table 2. The 200 mg/kg dose of both P. domestica and P. africana exhibited significant inhibition of inflammation, 64.43 and 61.32%, respectively at 3 h in comparison to 68.71% inhibition shown by ibuprofen (Table-2). The maximum inhibition of 65.39% however, was observed for P. cerasoides at 6 h. 4. Discussion Benign prostatic hyperplasia is a common health problem in the older men which affects the quality of life. The aetiology of prostatic hyperplasia is multi-factorial and hormonal changes (Marker et al., 2003), biochemical alterations and enhanced tissue proliferation (Li et al., 2007) are believed to play a key role in the development of BPH. Pygeum is one of the top selling herbs in European countries for use as first line treatment for the prevention and cure of BPH. However, owing to over exploitation, the plant has become endangered and is included in Appendix II of the CITES. Based on numerous trials, the effectiveness of Pygeum products is reported to be due to a synergistic interaction of the many constituents of the bark 15

extract and notably whole bark appears to be necessary for the effective treatment (Bassi et al., 1987), thereby putting more pressure on P. africana. Hence, with an exigent need to look for a solution to P. africana problem, an investigation into other well distributed species of Prunus holds promise, which has not been addressed so far. Therefore, in the present study, we selected the bark of five Indian species of Prunus viz. P. amygdalus (Almond, Sweet almond and Badam), P. armeniaca (Apricot, Khurmani and Zardalu), P. cerasoides (cherries), P. domestica (Wild plum, Alubukhara and Alucha) and P. persica (Peach, Nectarine, Shaftalu and Aru) known for wider occurrence across the globe and did a comparative evaluation with the bark of P. africana (African cherry, Pygeum). The selected plants are famous for their edible fruits and stones with high nutritional value. It is believed that the bark of these plants, if proven effective against BPH, will add to their significance in the international market as medicinal agents of importance. We evaluated the ameliorative potential of Prunus species against benign prostatic hyperplasia by studying the effect on prostate size, prostatic epithelial cell proliferation, testosterone and creatinine levels, proliferating cell nuclear antigen (PCNA) and biochemical changes arising out of the oxidative stress in a testosterone induced BPH rat model. Further, the results were supported by determining the presence of different biomarkers through TLC fingerprint profile, estimation of β-sitosterol by GC and evaluation of anti-inflammatory activity. The characterization of the bark extracts was first established through phytochemical screening which showed the presence of similar type of phytoconstituents in the selected plants including P. africana. Phytosterols, pentacyclic triterpenoids, flavonoids, glycosides, alkaloids, carbohydrates, proteins and tannins were present in all the species. The presence of three markers i.e. docosyl ferulate, β-sitosterol (both official USP markers) and ursolic acid believed to be responsible for the activity was confirmed in all the selected Prunus species from the generated TLC fingerprint profile. Further, USP describes GC method for the estimation of β-sitosterol in P. africana and its extracts for the purpose of quality control; hence, its content was estimated in the selected species following the official method. The percent content of β-sitosterol was observed to be 2.5 times higher in P. domestica (0.10) than official drug species of P. africana (0.04). The prostate and testicular enlargement is one of the important indicators of prostatic hyperplasia. The prostate weight measurement is an essential criterion, as BPH involves epithelial and stromal hyperplasia of the prostate with an increase in the prostate weight (Li et al., 2007). The BPH control group showed significantly increased relative prostate and

16

testicular weight as compared with the negative control group; however, finasteride and the Prunus treated animals showed a remarkable reduction in the prostate and testicular weight as compared with the BPH control animals indicating a protective effect in ameliorating the testosterone induced BPH. These results are in confirmity with the antiBPH activity mentioned in an invention on improved process for Pygeum extraction of P. domestica. However, this patent is not on the bark but stem cuttings/twigs of P. domestica and using a dose that is 10-20 times higher than that reportedly used in clinical trials and our study. The present findings were supported by the histopathological and PCNA analysis of the prostate tissue. In the histopathological study, increased glandular hyperplasia with epithelial proliferation was observed in the BPH control group as compared to the negative control group. On the other hand, the animals treated with finasteride and different species of Prunus showed mild prostatic epithelial hyperplasia coupled with a reduction in epithelial thickness as compared to the BPH control group. These results are in agreement with the prostate weight results. PCNA measurement is the most ideal method to evaluate cellular proliferation in benign proliferating tissue and the level is directly correlated to the degree of proliferation. The results showed a significant decrease in PCNA expressions in prostate of Prunus and finasteride treated groups as compared with BPH group. The evidence linking androgens and androgen receptor pathways to BPH has been well established (Marker et al., 2003). The main pharmacological agents for BPH are 5α-reductase inhibitors, which like α-adrenergic blockers regulate the levels of testosterone and DHT (Steers, 2001); the inhibition of 5α-R activity can hence reduce the risk of BPH or even treat the existing disease. The intracellular 5α-R enzyme converts circulating testosterone to dihydrotestosterone (DHT), which plays a major role in the stromal-epithelial interactions. Therefore, 5α-R inhibitory activity was assessed by measuring the testosterone level in serum and prostate. All the species of Prunus, significantly enhanced serum and prostate testosterone concentration as compared to the standard drug finasteride clearly indicating that the test drugs have potent 5α-R inhibitory activity. P. domestica was observed to be as effective as P. africana in ameliorating the testosterone levels. A patient with BPH having impaired renal dysfunctions generally shows elevated serum creatinine level and is mostly recommended for the initial screening of BPH (Gerber et al., 1997). The serum creatinine was determined in all the groups and it was observed that its level increased significantly in BPH control animals with respect to the negative control, while the finasteride and Prunus treated groups showed reduced creatinine level suggesting the reversal of renal dysfunctions

17

arising out of BPH. P. domestica and finasteride normalized the creatinine to same level as the negative control group. There is a direct relationship between the oxidative stress and development of BPH. Androgens enhance the cellular metabolism in prostatic cells resulting in free radicals formation. Tissue damage and oxidative stress may lead to the compensatory cellular proliferation with resulting hyperplastic growth (Fleshner & Klotz, 1998). Antioxidant enzymes such as SOD, GSH, catalase, LPO and nitrite play a significant role in scavenging these free radicals (Aryal et al., 2007). The results of biochemical estimations showed that, the testosterone induced BPH group had significantly decreased SOD, catalase and GSH activities and an increase in the LPO and nitrite levels as compared to the negative control group. Treatment with finasteride and Prunus species significantly restored the SOD, catalase, GSH and ameliorated the nitrite and LPO levels confirming the antioxidant potential of the test drugs. It is expected that flavonoids and ferulic esters identified in the Prunus species might be responsible in protecting the prostate from oxidative injury and possibly suppress the progression of BPH. The prostatic inflammation is considered a crucial factor in persuading the prostatic growth and the progression of symptoms. There is not a single pathomechanism, but inflammation has significant role in the initiation, proliferation, development and evolution of BPH (Kramer & Marberger, 2006). It has been hypothesized that BPH is an immune-mediated inflammatory disease and inflammation may be directly contributing to the prostate growth (Nickel, J.C., 1994). In the carrageenan induced paw oedema model, ibuprofen as well as the different Prunus species showed substantial anti-inflammatory activity supporting the suppression of inflammatory symptoms of BPH. Taking into account different parameters studied viz antiandrogen, oxidative stress and inflammation, the effectiveness of the Prunus species as antiBPH agents is in the order of P. domestica, P. persica, P. amygdalus, P. cerasoides and P. armeniaca. Further, the acute toxicity study showed that P. domestica is safe at 2000 mg/kg. The European studies have shown Pygeum bark to be useful in maintaining a healthy prostate, and numerous clinical trials have demonstrated its usefulness in prostatic hyperplasia as well (Barth, 1981). The plant is official in many Pharmacopoeias. The current trade and uses of the species is highly regulated as it is listed in CITES APPENDIX II. The Pygeum extract contains several pharmacologically active compounds that are known to work synergistically to counteract the structural and biochemical changes associated with

18

BPH. Phytosterols (β-sitosterol) suppresses the inflammatory symptoms associated with BPH and chronic prostatitis through the inhibition of the production of prostaglandins in the prostate. The pentacyclic triterpenes (ursolic acids) inhibit the activity of glucosyltransferase, an enzyme involved in the inflammation process while ferulic esters (ndocosanol) reportedly lower the blood levels of cholesterol, from which testosterone is produced (Awang, 1997; Bassi et al., 1987; Bombardelli and Morazzoni, 1997; Simons et al., 1998). In summary, the investigated species of Prunus have shown the presence of similar type of phytoconstituents as P. africana including the markers β-sitosterol, docosyl ferulate and ursolic acid. They have been shown to exert antiBPH effects via same mechanisms as established for P. africana. First, they restored the testosterone concentration by inhibiting the 5α-reductase enzyme activity. Second, they suppressed the inflammatory symptoms and lastly they exhibited promising antioxidant activity as proven by amelioration of oxidative stress parameters; and well supported by histopathological and immunohistochemistry findings. Although the bark of all the investigated species exhibited significant activity, but P. domestica was most promising and was observed to be as effective as P. africana. It is expected that the current study will help meet the future market demand of Pygeum, as it is aimed at effective utilization of resources available across the globe of both wild-collected and cultivated species other than P. africana. 5. Conclusions The results of the present study provide first report on the comparative efficacy of five species of Prunus with widely used drug P. africana for the treatment of BPH. The different species of Prunus substantially reduced the prostate and testis weights, prostatic hyperplasia, PCNA expression, serum creatinine; improved testosterone levels; restored antioxidant parameters and decreased the inflammation. Their efficacy and quality was supported by the presence of all the three markers in TLC fingerprint profile and determination of β-sitosterol content by GC analysis. All the species showed significant antiBPH activity in the order of P. domestica, P. persica, P. amygdalus, P. cerasoides and P. armeniaca with an efficacy of P. domestica comparable to P. africana. Combined with the safety of the plant, these findings suggest the possible use of other Prunus species and especially P. domestica in place of P. africana for the treatment of BPH and related disorders. The study will be of immense value not only to African countries in protecting their valuable resources of P. africana, but also to many other countries across the globe in utilizing their own resources of other edible

19

species of Prunus. Further, the study will bring value to more species of Prunus in the international market as important therapeutic agents. Conflict of interest The authors declare that no conflict of interest exists in this study. Authors’ contributions Professor Maninder Karan and Professor Karan Vasisht conceptualized, planned and designed the study; Ashish Kumar Jena prepared the extracts, did phytochemical analysis and carried out the biological experiments; Neetika Sharma collected the samples and performed the GC analysis; Ramdeep Kaur developed the fingerprint profiles and Mamta Sachdeva Dhingra provided docosyl ferulate. The authors listed at no.1, 2, 3 and 6 drafted and finalized the manuscript. All the authors have read and approved the final manuscript before submission.

Acknowledgement We acknowledge the contribution of Dr. G.B. Jena, Associate Professor, Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, Mohali, India for assisting in histopathological and immunohistochemical analysis. We are thankful to University Grants Commission, New Delhi, India, for financial support to Ashish Kumar Jena and Neetika Sharma and Chemical Resources, Panchkula, India, for providing genuine sample of Prunus africana.

References Anonymous, 2003. The Wealth of India: A Dictionary of Indian Raw Materials and Industrial Products: First Supplement Series (Raw Materials), Vol. IV. National Institute of Science Communication and Information Resources, New Delhi, pp. 250-282. Anonymous, 2008. British Pharmacopoeia, Vol. II, Market Towers 1, Nine Elms Lane, London, pp. 1852-1853. Anonymous, 2010. European Pharmacopoeia: Directorate for the Quality of Medicines and Healthcare of the Council of Europe, Vol. I, seventh ed. Strasbourg Cedex, France, p. 1221. Anonymous, 2011. The United States Pharmacopoeia: The National Formulary Vol. I, ed. 29, Twinbrook, Parkway, Rockville, pp. 1222-1225. 20

Aryal, M., Pandeya, A., Gautam, N., Baral, N., Lamsal, M., Majhi, S., Chandra, L., Pandit, R., Das, B.K., 2007. Oxidative stress in benign prostate hyperplasia. Nepal Med. Coll. J. 9, 222-224. Awang, D.V.C., 1997. Saw palmetto, African prune and stinging nettle for benign prostatic hyperplasia (BPH). Can. J. Pharm. 130, 37-44. Backheet, E. Y., Farag, S.F., 2003. Flavonoids and cyanogenic glycosides from the leaves and stem bark of Prunus persica (L.) Batsch (Meet ghamr) peach local cultivar in Assuit region. Bull. Pharm Sci. 26, 55-56. Barth, H., 1981. Non-hormonal Treatment of Benign Prostatic Hypertrophy; Clinical Evaluation of the Active Extract of Pygeum africanum. in: Proceedings of the Symposium on Benign Prostatic Hypertrophy, Paris, 45-51. Bassi, P., Artibani, W., De Luca, V., Zattoni, F., Lembo, A., 1987. Standardized extract of Pygeum africanum in the treatment of benign prostatic hypertrophy. Controlled clinical study versus placebo. Minerva Urol. Nefrol. 39, 45-50. Berges RR, Windeler J, Trampisch HJ. (1995) Randomised, placebo-controlled, double-blind clinical trial of beta-sitosterol in patients with benign prostatic hyperplasia. Betasitosterol study group. Lancet 345, 1529-1532. Berry, S.J., Coffey, D.S., Walsh, P.C., Ewing, L.L., 1984. The development of human benign prostatic hyperplasia with age. J. Urol. 132, 474-499. Bombardelli, E., Morazzoni, P., 1997. Prunus africana (Hook. f.) Kalkm. Fitoterapia 68, 205218. Bravi, F., Bosetti, C., Dal Maso, L., Talamini, R., Montella, M., Negri, E., Ramazzotti, V., Franceschi S, La., 2006. Food groups and risk of benign prostatic hyperplasia. Urology 67, 73-79. Carani C, Salvioli V, Scuteri A, et al. (1991). Urological and sexual evaluation of treatment of benign prostatic disease using Pygeum africanum at high doses. Arch. Ital. Urol. Nefrol. Androl. 63, 341-345. Catalano, S., Ferretti, M., 1984. New constituents of Prunus africana bark extract. J. Nat. Prod. 47, 910. Cristoni, A., Di Pierro, F., Bombardelli, E., 2000. Botanical derivatives for the prostate. Fitoterapia 71, S21-S28.

21

Cunningham, A.B., Mbenkum, F.T., 1993. Sustainability of harvesting Prunus africana bark in Cameroon: a medicinal plant in international trade. People and Plants Working Paper 2. UNESCO, Paris. Dhingra, M.S., Dhingra, S., Kumria, R., Chadha, R., Singh, T., Kumar, A., Karan, M., 2014. Effect of trimethylgallic acid esters against chronic stress-induced anxiety-like behavior and oxidative stress in mice. Pharmacol. Rep. 66, 606-612. Ellman, G., 1959. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 70-77. Ernest, R.M.K., 1996. Biochemistry-an Introduction to Dynamic Biology, first ed. The Macmillan Company, New York. ESCOP Monographs., 2009. 2nd ed. Pygeum Africana cortex Monograph. European Scientific Cooperative on Phytochemistry, Georg Thieme Verlag, Stuttgart Supplement. Farnsworth, N.R., 1966. Biological and phytochemical screening of plants. J. Pharm. Sci. 55, 225-276. Fleshner, N.E., Klotz, L.H., 1998. Diet, androgens, oxidative stress and prostate cancer susceptibility. Cancer Metastasis Rev. 17, 325-330. Galbraith, S.M., Duchesne, G.M., 1997. Androgens and prostate cancer: biology, pathology and hormonal therapy. Eur. J. Cancer 33, 545-554. Garg, M., Garg, S.K., Gupta, S.R., 1985. Chemical examination of Carum copticum seeds and Prunus cerasoides stem bark. Proceedings of the National Academy of Sciences, India, Section A: Physical Sciences 55, 95-98. Gerber, G.S., Goldfischer, E.R., Karrrison, Bales, G.T., 1997. Serum creatinine measurements in men with lower urinary tract symptoms secondary to benign prostatic hyperplasia. Urology 49, 697-702. Ghora, C., Panigrahi, G., 1984. Fascicles of flora of India. Rosaceae: Genus Prunus. No. 18, Botanical Survey of India, Calcutta, India. Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnok, J.S., Tannenbaum, S.R., 1982. Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids. Anal. Biochem. 126, 131-138. Guan-Jhong, H., Chun-Hsu, P., Fon-Chang, L., Tian-Shung, Wu., Chieh-His, Wu., 2012. Anti-inflammatory effects of ethanolic extract of Antrodia salmonea in the lipopolysaccharide-stimulated RAW246.7 macrophages and the λ-carrageenan-induced paw edema model. Food Chem. Toxicol. 50, 1485-1493.

22

Hall, J.B., O'Brien, E.M., Sinclair, F.L., 2000. Prunus africana: a Monograph. School of Agricultural and Forest Sciences Publication Number 18, University of Wales, Bangor. 104. Homma, Y., Gotoh, M., Yokoyyama, O., Masumori, N., Kawauchi, A., Yamanishi, T., Ishizuka, O., Seki, N., Kamoto, T., Nagai, A., Ozono, S., 2011. Outline of JUA clinical guidelines for benign prostatic hyperplasia. Int. J. Urol. 18, 741-756. Husdan, H., Rapoporf, A., 1968. Estimation of creatinine by the Jaffe reaction a comparison of three methods. Clin. Chem. 14, 222-238. In-Sik, S., Mee-Young, L., Da-Young, J., Chang-Seob, S., Hye-Kyung, H., Hyeun-Kyoo, S., 2012. Ursolic acid reduces prostate size and dihydrotestosterone level in a rat model of benign prostatic hyperplasia. Food Chem. Toxicol. 50, 884-888. Kalkman, C., 1965. The old world species of Prunus subgenus Laurocerasus including those formerly referred to Pygeum. Blumea 13, 1-115. Karan, M., Sarup, P., Suneja. V., Vasisht, K., 2012. Effect of traditional purification processes of guggulu on carrageenan induced paw oedema in rats. J. Pharm. Biomed. Sci. 21, 1-5. Kevin, T., Mc Vary, M.D., 2007. A review of combination therapy in patients with benign prostatic hyperplasia. Clin. Ther. 29, 387-398. Kono, Y., 1978. Generation of superoxide radical during auto-oxidation of hydroxylamine and an assay for superoxide dismutase. Arch. Biochem. Biophys. 186, 189-195. Li, W., Wu, C., Febbo, P.G., Olumi, A.F., 2007. Stromally expressed c-Jun regulates proliferation of prostate epithelial cells. Am. J. Pathol. 171, 1189-1198. Luck, H., 1971. Catalase. in: Bergmeyer HU, Methods of Enzymatic Analysis. New York, Academic Press, pp. 885-893. Mac Donald R, Ishani A, Rutks I, et al. (2000). A systematic review of Cernilton for the treatment of benign prostatic hyperplasia. BJU Int. 85, 836-841. Marker, P.C., Donjacour, A.A., Dahiya, R., Cunha, G.R., 2003. Hormonal, cellular and molecular control of prostatic development. Dev. Biol. 253, 165-174. Nickel, J.C., 1994. Prostatic inflammation in benign prostatic hyperplasia-the third component? Can. J. Urol. 1, 1-4. Nkeng, P.F., V, Ingram., A, Awono., 2010. Assessment of Prunus africana bark exploitation methods and sustainable exploitation in the South west, North west and Adamaoua regions of Cameroon. Project GCP/ RAF/408/ EC. Mobilisation et Renforcement des

23

Capacités des Petites et Moyennes Entreprises impliquées dans les Filières des Produits Forestiers Non Ligneux en Afrique Centrale. CIFOR. Yaoundé: FAO-CIFOR-SNVWorld Agroforestry CenterCOMIFAC: 57. OECD, 2008. Test no. 425: acute oral toxicity: up-and-down procedure. OECD Publishing, Paris. Pagano, E., Laudato, M., Griffo, M., Capasso, R., 2014. Phytotherapy of benign prostatic hyperplasia: a minireview. Phytother. Res. 28, 949-955. Ponholzer, A., Marszalek, M., Madersbacher, S., 2004. Minimally invasive treatment of BPH: an update. EAU. Update. Ser. 2, 24-33. Power, R.E., Fitzpatrick, J.M., 2004. Medical treatment of BPH: an update on results. EAU. Update Ser. 2, 6-14. Rawat, M.S.M., Pant, G., 1995. A persicogenin-3-glucoside from the stem bark of Prunus amygdalus. Phytochemistry 38, 1519-1520. Reagan-Shaw, S., Nihal, M., Ahmad, N., 2007. Dose translation from animal to human studies revisited. FASEB. J. 22, 1-3. Safarinejad MR. (2005). Urtica dioica for treatment of benign prostatic hyperplasia: a prospective, randomized, double-blind, placebo controlled, crossover study. J. Herb. Pharmacother. 5, 1-11. Sanda, M.G., Beaty, T.H.., Stutzman, R.E., Childs, B., Walsh, P.C., 1994. Genetic susceptibility of benign prostatic hyperplasia. J. Urol. 152, 115-119. Simons, A.J., Dawson, I.K., Duguma, B., Tchoundjeu, Z., 1998. Passing problems: prostate and Prunus. Herbalgram. 43, 49-53. Steers, W.D., 2001. 5α-Reductase activity in the prostate. Urology. 58, 17-24. Stewart, K.M., 2003. The African cherry (Prunus africana): can lessons be learned from an over-exploited medicinal tree? J. Ethnopharmacol. 89, 3-13. Tacklind J, Macdonald R, Rutks I, et al. (2012). Serenoa repens for benign prostatic hyperplasia. Cochrane Database Syst. Rev. 12, CD001423 Tewari, K., Sharma, A., 2013. World Intellectual Property Organisation Patent No. 2013014681 A1. India. Tewari, K., Sharma, A., 2013a. U.S. Patent No. 20130236574 A1. India.

24

Thiyagarajan, M., Kaul, C.L., Ramarao, P., 2002. Enhancement of alpha-adrenoceptormediated responses in prostate of testosterone-treated rat. Eur. J. Pharmacol. 453, 335-344. Vahlensieck W, Theurer C, Pfitzer E, et al. (2015). Effects of pumpkin seed in men with lower urinary tract symptoms due to benign prostatic hyperplasia in the one-year, randomized, placebo-controlled GRANU Study. Urol. Int. 94, 286-395. Watt, J.M., Beyer-Brandwijk, M.M., 1962. Medicinal and Poisonous Plants of Southern and Eastern Africa. London, E & S Livingstone Publishers. Wills, E.D., 1966. Mechanisms of lipid peroxide formation in animal tissues. Biochem. J. 99, 667-676. Wilt, T.J., Macdonald, R., Ishani, A., 1999. β-Sitosterol for the treatment of benign prostatic hyperplasia: a systematic review. BJU Int. 83, 976-983.

Abbreviations: BPH, Benign Prostatic Hyperplasia; GC, Gas Chromatography; 5α-R, 5α-Reductase; DHT, Dihydrotestosterone; LUTS, Lower Urinary Tract Symptoms; CITES, Convention of International Trade in Endangered Species of Wild Fauna and Flora; TLC, Thin Layer Chromatography; USP, United States Pharmacopoeia; LPO, Lipid Peroxidation; MDA, Malonaldehyde; GSH, Reduced Glutathione; SOD, Superoxide Dismutase; ANOVA, Analysis of Variance; UV, Ultra Violet; Rf, Retention factor; PW, prostate weight, TW, Testis weight; PI, Prostatic index; TI, Testicular Index; PCNA, Proliferating Cell Nuclear Antigen; ROS, Reacting Oxygen Species.

Figure Captions Fig 1: TLC fingerprint profile of bark of different species of Prunus. (i): Stdβ-sitosterol, (ii): Std-docosyl ferulate, (iii): Std-ursolic acid. A: P. africana, B: P. amygdalus, C: P. armeniaca, D: P. cerasoides, E: P. domestica and F: P. persica. Fig 2: GC chromatograms of standard β-sitosterol and Prunus species. Fig 3: Per cent content of β-sitosterol in Prunus species. Fig 4: Effect of finasteride and different species of Prunus on the testosterone and creatinine levels in testosterone induced BPH rats. (A) Serum testosterone level (B) Prostate 25

testosterone level and (C) Serum creatinine level. Values are expressed as mean ± SEM, ANOVA followed by Tukey’s test. ‘a’ p < 0.05 as compared to negative control, ‘b’ p < 0.05 as compared to BPH control and ‘c’ p < 0.05 as compared to finasteride. Fig 5: Representative photomicrographs of prostate histology (H&E) and the effects of finasteride and Prunus species (X200 magnification). A: Negative control. B: BPH control. C: Finasteride (2 mg/kg) D: P. domestica 10 mg/kg E: P. domestica F: P. amygdalus G: P. armeniaca H: P. cerasoides I: P. persica J: P. africana (all at a dose of 20 mg/kg). Fig 6: Representative photomicrographs showing positive signal for PCNA expression (brown colour cells) in the ventral prostate and the effect of finasteride and different species of Prunus (X200 magnification). A: Negative control. B: BPH control. C: Finasteride (2 mg/kg) D: P. domestica 10 mg/kg. E: P. domestica F: P. amygdalus G: P. armeniaca H: P. cerasoides I: P. persica J: P. africana (all at a dose of 20 mg/kg). The arrow mark indicating PCNA expression. Fig 7: Effect of finasteride and different species of Prunus on the oxidative stress parameters in testosterone induced BPH rats. (A) SOD, (B) Catalase, (C) GSH, (D) LPO and (E) Nitrite. Values are expressed as mean ± SEM, ANOVA followed by Tukey’s test. ‘a’ p < 0.05 as compared to negative control, ‘b’ p < 0.05 as compared to BPH control and ‘c’ p < 0.05 as compared to finasteride.

Table 1: Effect of Prunus species on the body weight, prostate and testis enlargement. Groups

Body weights (g) ± SEM

PW (g) ± SEM

% Inhibitio n

TW (g) ± SEM

% Inhibitio n

PI × 10-3 ± SEM

% Inhibitio n

TI × 10-3 ± SEM

% Inhibitio n

Initia l

Fina l

Negative control

180 ± 4.47

217 0.31 ± 4± 3.74 0.03

-

2.64 ± 0.08

-

1.45 ± 0.135

-

12.14 ± 0.27

-

BPH control

176 ± 6.78

211 0.51 ± 0± 5.09 0.06

-

3.03 ± 0.09

-

2.42 ± 0.293

-

14.42 ± 0.676

-

a

a

26

Standard Finasteri de

170 ± 5.48

210 0.32 ± 2± 4.47 0.05

95.92

0.94 ± 0.07a,b

536

b

1.53 ± 0.232

92.13

4.49 ± 0.335a,b

435

b

P. domestic a 10 mg/kg

170 ± 3.54

205 0.40 ± 5± 2.24 0.06

53.57

P. domestic a 20 mg/kg

181 ± 7.97

219 ± 4.0

90.82

P. amygdalu s

200 ± 8.37

223 0.40 ± 1± 6.24 0.03

55.61

2.14 ± 0.22b,c

228

1.74 ± 0.132

69.98

9.66 ± 0.702b,c

209

182 ±9.1 7

212 ± 3.74

0.38 2± 0.02

65.31

2.07 ± 0.14b,c

246

1.81 ± 0.073

63.35

9.8 ± 0.738b,c

203

P. cerasoide s 20 mg/kg

180 ± 5.48

222 ± 8.6

0.41 9± 0.04

46.43

1.97 ± 0.19a,b

272

1.90 ± 0.116

54.04

8.90 ± 0.859a,b

241

P. persica

190

214

0.39

61.23

1.82

61.90

±8.3

±

0±

7

8.12

0.02

170 ± 3.16

212 ± 3.4

0.32 8± 0.03

0.33 2± 0.02

2.02 ± 0.10

259

1.97 ± 0.295

46.37

9.84 ± 0.434b,c

201

282

1.52 ± 0.123

93.16

8.83 ± 0.235

245

a,b,c

1.93 ± 0.05a,b ,c

b

a,b,c

b

20 mg/kg P. armeniac a 20 mg/kg

20 mg/kg

P. africana 20 mg/kg

,c

2.17 ±

220

0.10b,c

,c

10.13 ±

188

0.32b,c

± 0.062

92.86

1.79 ± 0.09a,b ,c

b

318

1.55 ± 0.116

90.10

8.44 ± 0.427a,b

262

,c

b

PW: Prostate weight, TW: Testicular weight, PI: Prostate index and TI: Testicular index. Values are expressed as mean ± SEM, one-way ANOVA followed by Tukey’s test. ‘a’ p < 0.05 as compared to negative control, ‘b’ p < 0.05 as compared to BPH control and ‘c’ p < 0.05 as compared to finasteride.

Table 2: Anti-inflammatory activity of different species of Prunus in carrageenan induced paw

oedema model

27

Group

Control

Ibuprofen

P. domestica

P. amygdalus P. armeniaca P. cerasoides P. persica P. africana

Mean paw volume ± SEM (% protection)

Dose (mg/kg) 0h

1h

3h

6h

0.1 mL of control vehicle

0.108 ± 0.006

0.387 ± 0.034

0.973 ± 0.024

0.605 ± 0.032

50

0.065 ± 0.003

0.197 ± 0.013a (47.50)

0.336 ± 0.012a (68.71)

0.281± 0.011a (56.54)

20

0.067 ± 0.004

0.332 ± 0.031b (4.64)

0.763 ± 0.055a,b (19.63)

0.458 ± 0.024a,b (21.33)

100

0.054 ± 0.005

0.282 ± 0.018a,b (18.57)

0.683 ± 0.020ab (33.14)

0.359 ± 0.010a (38.63)

200

0.064 ± 0.004

0.234 ± 0.019a (38.57)

0.372 ± 0.011a (64.43)

0.262 ± 0.017a (60.16)

400

0.072 ± 0.006

0.232 ± 0.019a (42.14)

0.430 ± 0.012a,b (58.64)

0.308 ± 0.011a (52.31)

200

0.072 ± 0.005

0.265 ± 0.023a (31.07)

0.482 ± 0.012a,b (53.02)

0.280 ± 0.012a (58.15)

200

0.073 ± 0.004

0.275 ± 0.017a (22.5)

0.522 ± 0.024a,b (46.74)

0.310 ± 0.034a (49.3)

200

0.065 ± 0.004

0.245 ± 0.020a (35.71)

0.430 ± 0.030a,b (57.85)

0.237 ± 0.023a (65.39)

200

0.072 ± 0.005

0.273 ± 0.019a (29.28)

0.427 ± 0.046ab (59.35)

0.300 ± 0.013a (54.73)

200

0.062 ± 0.003

0.240 ± 0.020 a (36.43)

0.397 ± 0.053a (61.32)

0.272 ± 0.022a (57.75)

Values were expressed as mean ± SEM. Statistical analysis is done by two-way ANOVA followed by Bonferroni- test. ‘a’ p ≤ 0.05 as compared to control, ‘b’ p ≤ 0.05 as compared to ibuprofen

28

Graphical abstract

29

30

31