Study On Bioactive Components Of Rhizoma Coptidis And Molecular Mechanism Of Inhabiting Androgen Synthesis In Sebaceous Gland Cells

Objective:Acne is a chronic skin disease that occurs in hair follicles and sebaceous glands.It has a high incidence rate about 90% during puberty.At present,traditional Chinese medicine have been used for the treatment of acne,but their active ingredients and mechanisms have not been futher studied.As a representative of traditional Chinese medicine for clearing-heat and quenching-fire-toxins,Rhizoma Coptidis(RC)was reported to exert ameliorative effect on acne.The Pharmacopoeia of China recorded that RC has functions of clearing-heat,drying-dampness,quenching-fire-toxins.Therefore,it is necessary to clarify the active substance of RC during the treatment of acne,further research molecular mechanism of its pharmacological activation,finally develope a new topical treatment drug for acne.Methods and results:The extraction of alkaloid constituents from RC was extracted by percolation with acid water.Next,the extraction was extracted with petroleum ether,dichloromethane,ethyl acetate and n-butanol to get various parts.15 compounds were isolated and identified from the nbutanol parts by the comprehensive application of chromatography and spectroscopy technology such as silica gel,macroporus resin,Sephadex LH20,preparative HPLC and TLC,NMR.The names of the isolated compounds are as follow: berberine(1),palmatine(2),columbamine(3),coptisine(4),epiberberine(5),jateorhizine(6),magnoflorine(7),groenlandicine(8),8-oxocoptistine(9),worenine(10),ferulic acid(11),4-O-feruloyl-D-quinic acid(12),5-hydroxymethylfurfural(13),dimethyl phthalate(14),dibutyl phthalate(15).The isolated seven major alkaloid monomers were screened on inhibiting the synthesis of androgen in vitro.The level of 5?-dihydrotestosterone(5?-DHT)and the expression of androgen receptor in human sebaceous gland cells(SZ95 cells)were detected by enzyme linked immunosorbent assay.The results showed that palmatine(PAL)could significantly inhibit synthesis of 5?-DHT in SZ95 cells.And PAL obviously suppressed the expression of androgen receptor at high concentration(P < 0.05).The mechanism on suppressing synthesis of 5?-DHT in SZ95 cells was further investigated.The effects of PAL on cell apoptosis and cycle were detected by flow cytometry.The expression of 5?-reductase ? type(Srd5?1)m RNA in SZ95 cells was detected by real-time PCR.We found that there was not effcte on cell apoptosis or cycle after treatment with different concentration of PAL.The expression of Srd5a1 m RNA was significantly increased by testosterone-stimulating,while PAL at high concentrations could obviously down-regulate the expression of Srd5a1 m RNA.Additionally,the effect of PAL on lipid accumulation in cytoplasm was observed by Nile red staining.Western blot assay was usd to verify whether PAL inhibits cytoplasmic lipid accumulation based on PI3K/AKT and MAPK pathway.The results were as follows: the fluorescence was significantly enhanced by co-stimulation of testosterone and linoleic acid using Nile red staining,the result showed lipogenesis was increased.But the accumulation of cytoplasmic lipids in SZ95 cells was significantly decreased after treatment with PAL.In this experiment,western blot assay was used to detect the expression of related proteins,and it was found that PAL could significantly down-regulate expression of SREBP-1c,PI3 K,p-AKT,p-P38,p-JNK and p-ERK proteins(P < 0.05).Therefore,PAL inhibited lipid synthesis in SZ95 cells through the PI3K/AKT and MAPK pathway.Active fraction of RC rich in PAL was extracted by water extraction and alcohol precipitation,then used to prepare Huanglian anti-acne cream(HAC)according to the effects inhibiting androgen synthesis in SZ95 cells.The apperance and stability of product were tested.The results showed HAC has good ductility and stability.To investigate the effects of PAL and HAC,the testosterone-induced sebaceous gland spots on the back of golden hamsters were used as the model to detecte the changes in the area and tissue morphology in sebaceous gland spots,the expression of androgen receptor(AR),the content of DHT in the serum,and the lipid synthesis in the sebaceous glands.The results of animal experiment indicated that PAL and HAC could significantly reduce the area of sebaceous glands(P < 0.05).H&E staining showed that the sebaceous gland cells in the HAC group became smaller,thinner,and loosely arranged.AR expression and lipid synthesis in PAL and HAC groups were significantly suppressed(P < 0.01)compared with the model group.There were no significant changes in DHT content among the groups.These results suggested that PAL and HAC could reduce the area of sebaceous glands,change the morphology of sebaceous glands,inhibit the lipid synthesis of sebaceous gland cells.Conclusion:In summary,the extraction,separation and purification of the alkaloids of RC were carried out,and the obtained compounds were identified.The effects of alkaloids on inhibiting androgen synthesis were found through vitro experiments.Meanwhile,the preliminary mechanism on inhibiting androgen synthesis was demonstrated.Through vitro experiments,we found that PAL can siginificantly inhibit synthesis of 5?-DHT by down-regulating the expression of Srd5a1 m RNA,rather than apoptosis or cycle.Otherwise,PAL inhibited lipid synthesis in SZ95 cells through down-regulating the related protein of SREBP-1c pathway.Through vivo experients,we found that PAL and HAC have anti-acne effect on testosteroneinducing acne model.These results provided theoretical basis for the treatment acne of RC.