Two Kinds Of Chinese Herbal Medicine Active Ingredient For Anti-prostate Disease

With the global trends in aging, the incidence of prostate diseases is stably increasing. How to prevent and treat these diseases has been an important topic in the medical field for old men. To find safer and more effective agents from natural products attracts more and more attention.In this thesis, we tested parts of traditional Chinese agents on sale used for the treatment of benign prostate hyperplasia (BPH) by evaluating their inhibition effect on secreted prostate specific antigen (PSA) in LNCaP cells with ELISA method, and found ShuLieAn showed strong activity. The raw material plant of Shuliean is Campylotropis hirtella (Franch.) Schindl. We found that the ethyl acetate extract of Campylotropis hirtella is the active fraction of the whole plant, and further carried out active constitutes study on the fraction by bioassay-guided isolation. 43 compounds from the ethyl acetate extract of Campylotropis hirtella were isolated. Their structures were elucidated on the basis ofphysico-chemical property and spectroscopic analysis. The 43 compounds were erythro-Guaiacylglycerol-β-O-4'-(5')-methoxylariciresinol (1*), 4-[(-6-hydroxy-2, 3-dihydro-1-benzofuran-3-yl) methyl]-5-methoxybenzene-1, 3-diol (2*), hedyotisol A (3), hedyotisol C (4), hedyotisol B (5), buddlenol B (6), sesquipinsapols B (7), sesquimarocanol B (8), 5, 5'-dimethoxylariciresinol (9), erythro-guaiacylglycerol-β-O-4'-coniferyl ether (10), threo-Guaiacylglycerol-β-O-4'-coniferyl ether (11), secoisolariciresinol (12), dehydrodiconiferyl alcohol) (13), (-)-secoisolariciresinol-9'-β-D-glucopyranoside (14), 7, 2', 4'-trihydroxy-5-methoxy-3-arylcoumarin) (15*), angelol-M (16*), angelol A (17), angelol B (18), angelol-G (19), xathotoxin (20), isopimpinellin (21), bergapten (22), alloimperatorin (23), lomatin acetate (24), Columbianetin (25), 8-O-Methylmellein (26), 3, 9-dihydroxy coumestan (27), (-) epicatechin (28), (-) catechin (29), isoferreirin (30), genistein (31), Naringenin (32), dihydrokaempferol (33), quercetin (34), kaempferol (35), isovitexin (36), isoorientin (37), quercetin 3-β-D-glucuropyranoside (38), rutin (39), benzyl-β-D-glucopyranoside (40), 9, 10, 13-trihydrosy-(E)-11-octadecenoic acid (41), 4-(2-hydroxyethyl)phenol(42), daucosterol(43), respectively. Compounds 1, 2, 15, 16 were new compounds, compounds 3-14, 17-27, 29-43 were isolated for the first time from the Campylotropisgenus.Among the 43 compounds, 25 compounds showed inhibition effect on PSA secretion in LNCaP cells at different levels. These 25 active compounds belong to three structural types, which were lignans, flavanoids and coumarins. Lignans and flavanoids were more effective than coumarins in general. The values of IC₅₀ for the most of lignans and flavanoids were smaller than 150μM, and for the most of coumarins, the values were more than 150μM.The mechanism of action on secreted PSA inhibition may be resulted from their interrupting androgen/androgen receptor (AR) functional axle. So we further studied the effects of six active lignans on AR and PSA proteins expression in LNCaP cells by western blotting assay. The results showed that compound 13 and 2 significantly down-regulated AR and PSA expression at dose-dependent manner. Compound 10 and 11 suppressed AR expression at the highest test dose, while down-regulated PSA expression at dose-dependent manner. Compound 12 had no obvious effect on AR expression, but dose-dependently down-regulated PSA expression. Compound 9 showed no influence both on AR and PSA expression.Given that AR plays an essential role in the pathogenesis of prostate cancer, and compound 13 exhibited most significant down-regulation effect on AR and PSA expression, we farther performed the study of growth inhibition and apoptosis induced on LNCaP cells by compound 13. The results showed that compound 13 inhibited proliferation of LNCaP cells by MTT assay at dose and time-dependent manner under the concentration range of 0～100μg/ml. Moreover, compound 13 activated Caspase 3 and its downstream substrate PARP, suppressed Bcl expression, and did not influence Bax and Bcl-xl expression. These results indicated that compound 13 inhibited LNcaP cell proliferate through, at least partly, inducing the cells apoptosis, and suggested that compound 13 also could have the potential to prevent or treat prostate cancer.Based on the above results, we further collected variety kinds of natural constitutes that were similar or different from the active compounds isolated from Campylotropis hirtella, and evaluated their activity on growth inhibition of prostate cancer cell lines to fred more potent compounds used for anti-prostate caner research. The results showed that Physalin A and B, from the Physalis alkekengi L. var. franchetii (Mast.) Makino, exhibited significant activity. Accordingly, we further investigated the mechanism of these two compounds involved in this action. The results showed that Physalin A and B significantly decreased the viability of androgen-independent prostate cell lines C42B and CWR22Rvl at time and dose-dependent manner, physalin B exhibited much stronger activity than phyaslin A. These two compounds reduced colony-forming ability of CWR22Rvl, and physalin B also was more effective than physalin A. Western blotting analysis showed that Physalin A and B activated Caspase 3 and its downstream substrate PARP, this result indicated these two compounds induced cells apoptosis. Further study indicated that physalin A and B activated c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) pathways in CWR22Rvl. The results were demonstrated by the experiments that these two compounds increased p-JNK and p-ERK levels, had no obvious effect on JNK and ERK expression, and activated the JNK substrate c-Jun. While in C42B, physalin A and B only activated JNK, and no obvious p-ERK was detected. In these two cell lines, P38 was not activated. These results suggested that physalin A and B induced C42B and CWR22Rvl apoptosis possibly by activating JNK pathway. In CWR22Rvl, p-ERK was also significantly activated, which indicated ERK and JNK both mediated this cell line undergoing apoptosis. P38 was not involved these two cell lines apoptosis process. In addition, physalin A and B down-regulated AR and PSA expression in C42B, and AR expression in CWR22Rvl at dose dependent-manner. To down-regulate AR expression levers is also a promising target for treatment of androgen-independent prostate caner. So these results collectively suggested physalin A and B could have the potential to treat androgen-independent prostate caner.In conclusion, based on the bioassay evaluation, this thesis studied active constitutes of the raw material plant of Shuliean, Campylotropis hirtella, and clarified the main active components of Campylotropis hirtella for treatment of BPH that were lignans, flavanoids and coumarins. The mechanism of action for active lignans on BPH was studied, and further suggested they could have the potential to prevent or treat BPH, even prostate cancer. Physalin A and B, from the Physalis alkekengi, showed strong effects on growth inhibition and induced apoptosis of cell lines C42B and CWR22Rvl. The results supported that physalin A and B could be used for treatment of androgen-independent prostate caner.