Preparation Of Zelkova Serrata Alcohol Extract Cream And Its Pharmacological Effect On Benign Prostatic Hyperplasia

Benign prostatic hyperplasia(BPH)is a progressive disease of middle-aged and elderly men.The incidence of BPH increases with age,and the incidence of60-year-old men is as high as 50%.BPH may lead to a series of lower urinary tract symptoms that eventually develops into varying degrees of bladder outlet obstruction.The pathogenesis of BPH is related to chronic inflammation,imbalance of estrogen and androgen,epithelial-mesenchymal transition,cell proliferation and apoptosis.Both5-alpha-reductase inhibitor and alpha blockers are commonly used clinically,however,these coumpounds cause postural hypotension,hypersensitivity,and sexual dysfunction.It is a folk custom to use the sap of the roots and twigs of Zelkova serrata(ZS)to treat the pain and swelling of the prostate.Previous research by our group showed that ZS is rich in flavonoids such as quercetin,luteolin and apigenin,these compounds could significantly inhibit BPH in rats model by alleviating inflammation,reducing levels of dihydrotestosterone(DHT),and androgen receptor(AR)competition.However,the effects of these constituents vary widely.In order to explore the overall effect and drug safety of folk external use of ZS sap,this study intends to prepare ZS alcohol extract cream and evaluate the feasibility from two aspects:in-vitro transdermal study and BPH model study.Our study provides a scientific basis for the clinical application of ZS sap in the treatment of BPH and lays a data foundation for the development of new drugs.The research methods included four aspects:1)Study on formulation and technology:the selection of the cream base was based on the single factor selection with the same dosage range of other factors,and then the prescription and preparation technology of the alcohol extract of ZS were optimized by the orthogonal design of3-factors and 3-levels;2)Quality standard study:HPLC method was established for the determination of quercetin,luteolin and apigenin in ZS alcohol extract cream,and the quality control was carried out by appearance,pH,particle size and the content of three components.3)In vitro transdermal test:Franz diffusion cell was used to investigate the transdermal permeation of the cream,and the cumulative permeation amount and permeation rate of total flavonoids were obtained.4)With finasteride as positive control,the effects of ZS alcohol extract cream on BPH rats were investigated:The BPH rat model was induced by subcutaneous injection of testosterone propionate(5 mg/kg/d)for four weeks.The finastide group was given finastide 1 mg/kg/d,the blank group was treated with blank cream,and ZS group was treated with 4%,6%and10%,respectively for 4 weeks.Then serum DHT and prostate specific antigen(PSA)levels were detected by ELISA.HE,immunohistochemistry and Western Blot were used to investigate the pathological changes,apoptosis and expression of proteins related to epithelial-mesenchymal transition of prostate tissue to evaluate the inhibitory effect of the cream on BPH rats.The results are as follows:1.Study on the prescription technology of ZS alcohol extract creamTaking appearance,spreading ability,centrifugal stability,heat stability and cold stability as indexes,the optimal prescription was obtained as follows:ZS alcohol extract 5 g,white vaseline 2.5 g,liquid paraffin 2.5 g,stearic acid 4.0 g,SDS 0.75 g,monostearin 1.75 g,glycerol 6.5 g,laurocapram 1.5 g,ethyl nipagin 0.005 g,add water up to 50 g.The preparation method is to add extract powder to the water phase,and the upper load is 10%.2.Study on the quality of ZS alcohol extract creamThe ZS alcohol extract cream prepared according to the optimized prescription was brown,with moderate texture and fine spreading.The pH value of the cream is about 5.4,the particle size is about 120 nm,and the physical stability is good.A HPLC method for the determination of ZS alcohol extract cream was established and the method was investigated.The contents of quercetin,luteolin and apigenin in the cream were 0.58±0.04 mg/g,0.49±0.03 mg/g and 0.34±0.04 mg/g respectively.3.In vitro transdermal test of ZS alcohol extract creamThe results of in vitro transdermal test showed that the content of total flavonoids increased with time,the initial release was slow,the increase was obvious after 6-9 h,and the increase was slow after 9-12 h.The cumulative transdermal permeability per unit area was 55.34±1.62μg/cm~2at 12 h,and the transdermal permeability of each component was quercetin>apigenin>luteolin,which indicated that the self-made cream could penetrate through skin.4.Pharmacodynamic study of ZS alcohol extract cream on BPH ratsZS alcohol extract cream down-regulated the prostate volume and relative weight in BPH rats,and decreased the serum levels of PSA and DHT.The papillary protrusion of prostate tissue and the thickness of glandular epithelial tissue were reduced,and the pathological features of prostate tissue were improved.The expression of key proteins TGFβ1 and Bcl-2/Bax were down-regulated,showing anti-proliferation effect.Up-regulation of E-cadherin and down-regulation of N-cadherin and Vimentin in prostate tissue inhibited the process of epithelial-mesenchymal transition(EMT)to a certain extent,thus inhibiting the further development of BPH.In this study,a simple preparation process and stable quality of ZS alcohol extract cream were developed.The cream could inhibit the levels of PSA and DHT,the relative expression of Bcl-2/Bax and the process of epithelial-mesenchymal transition in BPH rats through transdermal processes.This may be the reason for the folk external use of Zelkova serrata sap in the treatment of BPH.