Ruirong Zheng,Mooseob Kim,Zhengtao Wang,Li Yang

a The MOE Key Laboratory for Standardization of Chinese Medicines and the SATCM Key Laboratory for New Resources and Quality Evaluation of Chinese Medicines,Institute of Chinese Materia Medica,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;

b Institute of Interdisciplinary Integrative Medicine Research,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China

Abstract Benign prostatic hyperplasia (BPH) is a common disease in men,and is known to be related to 5α-reductase,which can affect steroid metabolism.Under the promotion of 5α-reductase,testosterone can be converted into dihydrotestosterone (DHT),and excessive DHT will cause related diseases.Since BPH seriously affects the quality of life of patients,it is essential to discover effective 5α-reductase inhibitors.In this study,the simple analytical method of high performance thin layer chromatography(HPTLC) was used to screen active compounds,and seven compounds with strong activity were screened out.This research will provide a reference for studying the material basis of drugs for the treatment of BPH.

Keywords:5α-reductase inhibitor;benign prostatic hyperplasia;HPTLC;steroid hormones;dihydrotestosterone

1 Introduction

Benign prostatic hyperplasia (BPH) is a common urinary disease in men.The prevalence of BPH is positively correlated with age.Its pathological feature is progressive hyperplasia of the gland and interstitial tissues,followed by enlargement of the prostate gland,which causes urethral contraction,further leading to serious urinary problems,including weak urine stream,dysuria,frequent urination,bladder outlet obstruction and difficulty emptying bladder [1].At present,surgical treatment and drug treatment are the two main treatment strategies for BPH [2-5].However,surgery can make a patient feel psychologically stressed.Recent studies have shown that dysuria and hematuria may recur 4-5 y after surgery.What’s worse,if PSA≥ 0.2 ng/mL,it may lead to prostate cancer [6-8].As the elderly population grows,so does BPH.Therefore,we need to prevent and control BPH.

Although the molecular mechanism of BPH has not been clearly revealed,most studies have shown that its pathogenesis is closely related to hormone metabolism [9-11].Among them,testosterone (T)and dihydrotestosterone (DHT) are the key factors affecting the growth and development of prostate.Excessive accumulation of DHT in prostate,a 5

α

-reductase-mediated metabolite of T,induces the occurrence of BPH [12-15].5

α

-reductase is a highly lipophilic enzyme found in the inner membrane of cells.At present,the 5

α

-reductase inhibitors used in clinics mainly include finasteride and dutasteride.It has been reported that these two drugs have obvious side effects in practical application [16-18].Therefore,it is necessary to discover new natural inhibitors with strong efficacy and few side effects for the treatment of BPH.At present,the methods for screening 5

α

-reductase inhibitors

in vitro

mainly include ultraviolet spectrophotometry and isotope labeling [19-21].However,these traditional methods are not suitable for high-throughput screening due to their complex operation,serious pollution,and long time consumption.This study established an

in vitro

model to screen a series of Chinese herbal medicine with active compounds to inhibit 5

α

-reductase in the clinical treatment of BPH.This study provides a theoretical basis for the development of new Chinese medicine for the treatment of BPH.Herein,HPTLC method was applied to analyze the changes of DHT easily and rapidly,which expressed 5

α

-reductase in HaCaT cells under the influence of tested drugs.DHT is a substrate of 5

α

-reductase,so the increase or decrease of DHT indicates the pharmacological activity of the tested drugs.The inhibition rate of the compounds was calculated by TLC scanning.The compounds with higher activity were screened out.To our knowledge,there is no report on the related activities of these compounds.Our report provides a theoretical support for the basic research on the clinical treatment of BPH with traditional Chinese medicine.The results showed that there are still many compounds in nature with the potential to be developed into new drugs for the treatment of BPH.

2 Experimental

2.1 Chemicals and materials

Ultra-pure water was produced by Millipore Milli-Q system (Merck,Germany).All reagents involved were of analytical grade unless otherwise specified.Dulbecco’s modified Eagle medium (DMEM) with high glucose (4.5 g/L)(C11995500BT),penicillin,streptomycin 0.25%Trypsin-EDTA (25200-072),fetal bovine serum(FBS) (10099-141),and Dulbecco’s phosphate buffered saline (DPBS) (14190-144) were obtained from Gibco (Grand Island,NY,USA).HaCaT cells were from Institute of Biochemistry and Cell Biology (CAS,Shanghai,China).NADPH was obtained from Roche Diagnostics (Indianapolis,IN,USA).T-25 cell culture flasks (0030710.126) were obtained from Eppendorf (Hamburg,Germany)and 96-well flat bottoms (#3599) were abtained from Corning Costar (ME,USA).Testosterone-d3 and stanolone (DHT,97.5%) were from Sigma (St.Louis,MO,USA) and testosterone (99.5%) was from Dr.Ehrenstorfer (Augsburg,Germany).Ethyl acetate,acetone,methanol,toluene,glacial acetic acid,formic acid,ethanol,chloroform,triethylamine,phosphoric acid and acetonitrile were obtained from Sinopharm Chemical Reagent Co.Ltd.(Shanghai,China).Curcumin,quercetin,kaempferol,osthole and epicatechin were from Shanghai R&D Center for Standardization of Chinese Medicines (Shanghai,China).Dutasteride and finasteride were from Shanghai Yuanye Biotechnology Co.,Ltd (Shanghai,China).The instruments and equipment used in HPTLC experiments,such as automatic developing chamber,automatic TLC sampler,TLC visualizer,TLC plate heater and twin trough chamber,were all purchased from CAMAG (Muttenz,Switzerland).HPTLC plates precoated with Silica G Fwere from Merck (Darmstadt,Germany).

2.2 Detection of 5α-reductase activity

When the density of HaCaT cells reached 80%,they were digested with pancreatin for 4 min,and then stopped by adding twice the volume of fresh medium.Centrifugation was carried out at 800 r/min for 4 min.The upper medium was discarded and fresh medium was added for suspension.2 ×10cells/well were inoculated into the 24-well plate and cultured in the incubator for 24 h.When the cell adhered to the wall,500

μ

L of the compound to be tested was added (the same volume of fresh medium was added for the control group).After 30 min of interaction with the cells,50

μ

L of the medium containing 25

μ

M T and 250

μ

M NADPH was added.After reaction in the incubator for 24 h,the culture medium was taken out and placed in a 10 mL centrifuge tube.Internal standard t-d3 was added to make the final concentration 10 ng/mL.Then 3 times the volume of ethyl acetate was added,shaken to make it mix well,and centrifugated at 10000 g speed for 5 min.The same volume of supernatant was taken out and dried with nitrogen.Samples were stored at–20 °C.The dried sample was redissolved with 200

μ

L methanol and then put into HPTLC for detection.The concentration of each compound to be tested was determined according to the results of cytotoxic test (see Appendix II).The specific concentration results are shown in Table 1.

Table 1 Results of 5α-reductase inhibitory activity of compounds

2.3 Different extraction methods of DHT

Method 1:25

μ

M T and 25

μ

M DHT were added into 1 mL medium and 2 mL ethyl acetate was added too.The medium was then centrifugated at the speed of 10000 g for 5 min,blew dry with nitrogen,and dissolved with 200

μ

L methanol.Method 2:25

μ

M T and 25

μ

M DHT were added into 1 mL medium and 2 mL methanol was added too.The medium was then centrifugated at the speed of 10000 g for 5 min,blew dry with nitrogen,and dissolved with 200

μ

L methanol.Method 3:25

μ

M T and 25

μ

M DHT were added into 1 mL medium and 2 mL acetonitrile was added too.The medium was then centrifugated at the speed of 10000 g for 5 min,blew dry with nitrogen,and dissolved with 200

μ

L methanol.Method 4:25

μ

M T and 25

μ

M DHT were added into 1 mL medium and 2 mL methanol and 4 mL chloroform were added too.The medium was centrifugated at the speed of 10000 g for 5 min,blew dry with nitrogen,and dissolved with 200

μ

L methanol.Method 5:25

μ

M T and 25

μ

M DHT were added into 1 mL medium and 2 mL methyl tertbutyl ether was added too.The medium was then centrifugated twice at the speed of 10000 g for 5 min,blew dry with nitrogen,and redissolved with 200

μ

L methanol.Method 6:25

μ

M T and 25

μ

M DHT were added into 1 mL medium and 2 mL methanol and 4 mL methyl tertbutyl ether were added too.The medium was then centrifugated at the speed of 10000 g for 5 min,blew dry with nitrogen,and dissolved with 200

μ

L methanol.Method 7:25

μ

M T and 25

μ

M DHT were added into 1 mL medium and 2 mL of methanol and acetonitrile (1:1) was added too.The medium was centrifugated at the speed of 10000 g for 5 min,blowed dry with nitrogen,and dissolved with 200

μ

L methanol.

2.4 High performance thin layer chromatography detection of steroid hormones

First,the automatic TLC sampler was used to spot the samples on the thin-layer plate,and the thin-layer plate was pre-saturated in the automatic developing chamber for 15 min to control the temperature and humidity.The plate was then developed with a mixture of cyclohexane-ethyl acetate-triethylamine (1.5:1:0.2) to a distance of about 7.5 cm and dried in air.The plate was derivatized by spraying with 10% sulfuric acid ethanol reagent and heating at 105 °C for 5 min.After derivatization of the plates,they were documented and evaluated under UV (365 nm).

2.5 Semi quantitative detection of compound activity by HPTLC scanning

HPTLC scanning technology was used to spot the substrate and product,and the peak area of each band was calculated to compare the inhibition rate of the compounds.Inhibition rate (%)=DHT (peak area of administration group)/ DHT (peak area of control group) × 100.

3 Results and discussion

3.1 Selection of the extraction method of DHT

HPTLC is a convenient and fast analytical method with low requirements for sample preparation and can effectively reduce the workload of sample pretreatment.Due to the large number of natural compounds,the selection of appropriate and fast detection methods is conducive to improving the efficiency of screening compounds.HPTLC was selected to screen 5

α

-reductase inhibitors.The different influencing factors of TLC were investigated according to reference [22].In the enzyme reaction,under the optimal condition of substrate and cell number,it is necessary to ensure that T and DHT can be clearly observed,which is a great challenge for TLC with low sensitivity.Therefore,different extraction methods were investigated in the experiment.The same amount of the specifically extracted 7 samples were placed on the same glassed plate (10 cm × 20 cm) coated with silica gel 60 F,developed with the developer cyclohexane-ethyl acetate-triethylamine (1:1:0.1),sprayed with 40%phosphoric acid aqueous solution,heated at 105 °C for 20 min,and examined under 366 nm ultraviolet light.Results showed that ethyl acetate was the best extraction solvent,which could not only screen out the unnecessary components in the culture medium,but also reduce the pretreatment time.Therefore,method 1 was finally selected,as shown in Fig.1.

Fig.1 Comparison of different extraction methods in HPTLC

3.2 Selection of the developing solution of DHT

Samples of T and DHT standards were selected on the TLC plate and the separation of T and DHT was investigated by different developing agents,such as cyclohexane-ethyl acetate (1:1);cyclohexanemethanol (1:1);petroleum ether-ethyl acetate (1:2);cyclohexane-ethyl acetate-triethylamine (1.5:1:0.2);cyclohexane-ethyl acetate (1.5:1);toluene-acetoneglacial acetic acid (8:2:0.2);cyclohexane-ethyl acetate-triethylamine (1:1:0.1),etc.The spray was colored with 40% phosphoric acid aqueous solution,heated at 105 °C for 20 min,and observed under ultraviolet light at 366 nm.As shown in Fig.2,developing agent C,cyclohexane-ethyl acetate-triethylamine (1.5:1:0.2) could well separate T and DHT,with moderate RF value.

Fig.2 Comparison of different development systems in HPTLC

3.3 Selection of the derivatization reagents of DHT

Samples of T and DHT standards were selected on the thin-layer plate and developed with the developer cyclohexane-ethyl acetate-triethylamine(1.5:1:0.2).The color development of T and DHT with different spray reagents was investigated.The color development effects of 40% phosphoric acid aqueous solution and 10% sulfuric acid ethanol solution were compared (Fig.3).Results showed that the fluorescent spots for 10% sulfuric acid ethanol are more obviously observed.In the previous research,the main detection method for T and DHT was 40% phosphoric acid ethanol,which was easy to cause phosphorus pollution.The color development configuration of 10% sulfuric acid ethanol is more convenient and simpler than the others,with obvious spots and good repeatability.Therefore,10% sulfuric acid ethanol solution was finally determined as the color development agent.

Fig.3 Comparison of different derivatization reagent in HPTLC

3.4 Detection limit of T and DHT on TLC

Different concentrations of T and DHT samples were spotted on the pre-coated TLC plate:concentration gradient for T is 20

μ

g,10

μ

g,5

μ

g,1

μ

g,500 ng,250 ng,and 50 ng;concentration gradient for DHT is 20

μ

g,10

μ

g,5

μ

g,1

μ

g,500 ng,250 ng,and 50 ng.The color was developed by directly spraying 10% sulfuric acid ethanol solution and heating at 105 °C for 20 min,and the development of spots was observed under 366 nm ultraviolet light.As shown in Fig.4,T and DHT spots were clearly observed at the concentration of more than 500 ng.During the experiment,in order to more accurately observe the effect of the compound on 5

α

-reductase,the conversion amount of the product should be more than 500 ng.

Fig.4 LOD of testosterone and DHT in HPTLC

The conditions of extraction,developing mobile phase and color development reagent were explored by using reference T and DHT.According to the results of sample detection after the enzyme reaction,the spots of substrate T and product DHT are well observed under this condition,and the active compounds can be screened out.

3.5 Screening the inhibitory activity of the compounds on 5α-reductase

HPTLC was used to screen the inhibitory activity of the compounds on 5

α

-reductase by observing the spots of DHT.Different bands represent different compounds.After the components in the culture medium were extracted,the samples were spotted on the thin-layer plate and developed and the effects of various compounds on 5

α

-reductase in cells were observed.When the DHT spots for a certain compound were significantly smaller or darker than that for the control group,the inhibitory effect of the compound on 5

α

-reductase was stronger.The DHT spots were almost not observed in the positive drug group,indicating that the inhibition effect was better.From the brightness of the spots in the picture,it can be roughly determined that the compounds with strong activity are quercetin (Fig.5),psoralen,Panax notoginseng saponins (PNS) (Fig.6),curcumin,corylin,lutein and zeaxanthin (Fig.7).The scanning results are consistent with the results observed by the naked eye.Curcumin is the most active one.Although it has also been reported in the literature that curcumin and quercetin can inhibit 5

α

-reductase activity [23],no other compounds have been reported.Therefore,this study can be used for further pharmacodynamic study of animal experiments.

Fig.5 Results of HPTLC screening of 5α-reductase inhibitors in vitro

Fig.6 Results of HPTLC screening of 5α-reductase inhibitors in vitro

Fig.7 Results of HPTLC screening of 5α-reductase inhibitors in vitro

4 Conclusion

In this study,a rapid HPTLC method for the screening of anti-5

α

-reductase drugs was developed and validated.Moreover,the inhibitory effect of the compounds on 5

α

-reductase was determined based on the concentration of DHT in each group.Through HPTLC method,seven chemical constituents of Chinese herbal medicines,including curcumin,were screened for their inhibitory effects on 5

α

-reductase.

Acknowledgements

This study was financially supported by the Program of Shanghai Municipal Commission of Health and Family Planning (ZY(2018-2020)-CCCX-5002).

Conflict of interest

The authors declare no competing financial interest.