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Assortment of Stauntonia Hexaphylla and Cornus Officinalis Protect Against Testosterone

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Assortment of Stauntonia Hexaphylla and Cornus Officinalis Protect Against Testosterone bioRxiv preprint doi: https://doi.org/10.1101/819474; this version posted October 25, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Research article 2 Assortment of Stauntonia hexaphylla and Cornus officinalis protect against testosterone- 3 induced benign prostatic hyperplasia through anti-inflammatory and anti-proliferative 4 activity 5 6 7 Shanika Karunasagara1, Geum-Lan Hong1, Da-Young Jung1 , Kyung-Hyun Kim1, Eun-Jeong 8 Koh2, Kyoung-won Cho2, Sung-Sun Park2 , Ju-Young Jung1* 9 10 11 1Department of Veterinary Medicine & Institute of Veterinary Science, Chungnam National 12 University, Daejeon, Republic of Korea 13 2 Chong Kun Dang Healthcare Corporation, Seoul, South Korea 14 15 #a Department of Veterinary Medicine & Institute of Veterinary Science, Chungnam National 16 University, Yusung-gu, Dae-Jeon, South Korea 17 #b Chong Kun Dang Healthcare Corporation, Yeongdeungpo-gu, Seoul, South Korea 18 19 * Corresponding author 20 E-mail: [email protected] (JY) 21 1 bioRxiv preprint doi: https://doi.org/10.1101/819474; this version posted October 25, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 22 Abstract 23 Benign prostatic hyperplasia (BPH) is a progressive pathological condition associated with 24 proliferation of prostatic tissues, prostate enlargement, and lower-urinary tract symptoms. 25 However, the mechanism underlying the pathogenesis of BPH is not clear. The aim of this 26 study was to investigate the protective effects of Stauntonia hexaphylla and Cornus officinalis 27 (SC extract) on a testosterone propionate (TP)-induced BPH model. For in vitro experiments, 28 a human prostate adenocarcinoma cell line was used to perform western blotting for androgen 29 receptor (AR), prostate specific antigen (PSA), and 5α-reductase type 2. Male Sprague-Dawley 30 rats were randomly divided into 8 groups as follows for the in vivo experiments: control, BPH, 31 Fina, Saw, SC25, SC50, SC100, and SC200. To induce BPH, all rats, except those in the control 32 group, were daily administered with subcutaneous injections of TP (5 mg/kg), and orally 33 treated with appropriate PBS/drugs for 4 consecutive weeks. Our findings indicated that the 34 SC treatment significantly reduced the prostate size and downregulated the serum testosterone 35 and DHT levels in BPH rats. The histological examination revealed that SC treatment markedly 36 recovered the TP-induced abnormalities and reduced the prostatic hyperplasia. In addition, in 37 vitro and in vivo western blotting indicated that SC treatment significantly downregulated the 38 AR, PSA, and 5α-reductase type 2 expression, while an immunohistochemistry examination 39 revealed that the SC extract significantly reduced the expression of type 2 5α-reductase and 40 proliferating cell nuclear antigen positive cell count. Collectively, our findings demonstrated 41 that SC extract attenuates BPH through anti-proliferative and anti-inflammation activities and 42 might be useful in the clinical treatment of BPH. 43 Key words: BPH, SC extract, testosterone, DHT, 5α-reductase type 2 44 45 2 bioRxiv preprint doi: https://doi.org/10.1101/819474; this version posted October 25, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 46 Introduction 47 Many plants have been identified as good sources of natural antioxidants, which protect against 48 BPH and prostate cancer [1, 2]. In this study, we investigated the protective effect of a 9:1 49 mixture of Stauntonia hexaphylla and Cornus officinalis, called the SC extract, on BPH. 50 Stauntonia hexaphylla belongs to the family Lardizabalaceae, which is native to Southern 51 Japan and Korea and has been used in medicine owing to its analgesic, sedative, diuretic, and 52 anti-cancer properties [3]. Cornus officinalis, native to Korea, Japan, and China comprises, 53 compounds including terpenoids, flavonoids, sterols, carboxylic acids, polysaccharides, and 54 phenylpropanoids that have been isolated and identified. Owing to its chemical constituents, 55 C. officinalis displays diverse pharmacological activities such as hypoglycemic activity and 56 protective activity toward diabetic target organs, and antioxidant, anti-inflammatory, and 57 anticancer activity [4, 5]. 58 Benign prostatic hyperplasia (BPH) is the most frequent, non-cutaneous form of cancer among 59 elderly men and is characterized by progressive glandular and stromal tissue hyperplasia, which 60 leads to an enlarged prostate [6]. The rapid growth of stromal and epithelial elements result in 61 BPH in the prostate, along with lower urinary tract symptoms (LUTS), including obstructive 62 symptoms such as hesitancy, poor intermittent stream, feeling of incomplete bladder emptying, 63 and irritative symptoms such as frequency, urgency, and nocturia [7, 8]. Various molecular 64 etiologies of BPH have suggested that hormones, oxidative stress, chronic inflammation, and 65 aging may play a crucial role in BPH development, while researchers consider androgens, 66 especially testosterone-related hormones to be the major contributory factors in the 67 development and progression of BPH [9, 10]. The growth and development of prostate gland 68 depends on androgen stimulation, especially through DHT [11, 12]; accumulation of DHT with 69 aging results in rapid growth and hyperplasia of prostatic cells [13]. DHT is an active metabolic 3 bioRxiv preprint doi: https://doi.org/10.1101/819474; this version posted October 25, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 70 product of the conversion of testosterone by steroid 5α-reductase [14]. Finasteride, a drug that 71 is used as a steroid 5α-reductase Type 2 inhibitor, can be used for the treatment for BPH [15]. 72 It inhibits the conversion of testosterone to DHT, thereby preventing prostatic hyperplasia. 73 Furthermore, saw palmetto has been widely used as a therapeutic remedy for urinary 74 dysfunction due to BPH and works by ceasing the breakdown of testosterone into its byproduct 75 DHT [16]. Race, family history of prostate cancer, and environmental factors act as possible 76 risk factors of BPH [17- 19]. 77 The failure and unpleasant adverse effects of conventional anti-BPH drugs have led to the 78 search for phytotherapeutic solutions as a safer and less toxic alternative. Recently 79 phytotherapeutics have become popular in the treatment of BPH worldwide [20, 21]. This study 80 was aimed to investigate the protective role of SC extract in the development of BPH in 81 testosterone-induced BPH. 82 83 Materials and method 84 Plant material 85 SC extract (CKDHC-P29) was provided from Chong Kun Dang Healthcare (Seoul, Korea). 86 Reference compounds, hederacoside D (purity ≥98.0%, ChemFace, Wuhan, China) and 87 morroniside (purity ≥98.0%, ChemFace, Wuhan, China) were analyzed in the CKDHC-P29 88 sample. Reagents including acetonitrile, methanol, and ethanol were purchased from Burdick 89 & Jackson (Muskegon, MI, USA) and formic acid (HPLC grade) was purchased from Sigma- 90 Aldrich (St. Louis, Mo, USA). Water was purified using a Milli-Q system (Sinhan, Seoul, 91 Korea). 92 4 bioRxiv preprint doi: https://doi.org/10.1101/819474; this version posted October 25, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 93 Preparation of SC extract 94 The SC extract consist of Stauntonia hexaphylla leaves (Lot number: 20181116) and Cornus 95 officinalis Siebold & Zucc furits (Lot number: 20181112) mixed into 9:1 ratio. The leaves of 96 Stauntonia hexaphylla were harvested in the area of Goheung-gun, Jeollanam-do, Korea and 97 dried at 60 ℃ for 12h. Next, Dried leaves were extracted with 70% ethanol at 75℃ for 12h 98 and proceeded spray drying with 30% dextrin. The dried fruit of Cornus officinalis Siebold & 99 Zucc fruit was harvested in the area of Gurye-gun, Jeollanam-do, Korea. Dried fruit was 100 extracted with 70% ethanol at 75℃ for 12h and proceeded spray drying with 50% dextrin. 101 High performance liquid chromatography (HPLC) of SC extract 102 HPLC was used to identify the compounds, hederacoside D (C53H86O22) and morroniside 103 (C17H25O11) in the SC extract. The constituent of SC extract was performed by HPLC equipped 104 with photodiode array detector (PDA, Berlin, Germany) using Agilent Zorbax eclipse plus C18 105 column (4.6 x 250 mm, 5 μm). The elution was performed using a linear gradient from 12 to 106 0.1% formic acid in acetonitrile for detection of hederacoside D and morroniside and the 107 injection volume was 10 μL. PDA detector was set at 205 nm and 240 nm for appropriate 108 hederacoside D (205 nm) and morroniside (240 nm). 109 Cell culture and cell viability assay 110 Human prostate adenocarcinoma cells (LNCaP) were purchased from the American Type 111 Culture Collection (ATCC, Manassas, VA, USA), seeded on to 6-well plates (5×105 cells/well) 112 and fed with a medium containing 1 µM testosterone (Tokyo Chemical Ins. Co., Tokyo, Japan), 113 and Finasteride (10 µM, Sigma, USA), Saw palmetto extract (100 µg/mL) or SC extract (25, 114 50 µg/mL). After 72 hours post treatment (72 hpt), cells were harvested for the preparation of 115 total protein and subjected to western blot analysis.
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