Study On Production Process Of Penthorum Chinense Pursh Granule And Its Mechanism Of Inducing HepG2 Cell Apoptosis

Liver plays an important role in body,and liver is vulnerable to damage.Various factors such as drug abuse,viral infections,and disease deterioration can easily induce liver cancer.With the development of the pet industry,the rate of liver cancer in animals is increasing.Traditional Chinese veterinarians believe that the cause of liver cancer is mostly damp-heat toxins that invade the body,or damage to the spleen and stomach caused by improper diet,or dysfunction of yin and yang due to weak viscera,stagnation of qi and blood in the flanks,accumulation of Zhongjiao.The disease is located in the liver and spleen,also related to the gallbladder,stomach,and kidneys.The initial course of the disease is relatively insidious,and the clinical manifestations are not obvious.In the early stage,it often manifests as pain and fullness in the right side,weak appetite,weight loss,or jaundice.Therefore,the treatment criteria are soothing the liver and strengthening the spleen,removing dampness and removing blood stasis,and clearing away heat and toxins.The Penthorum Chinense Pursh compound is a self-made compound in our laboratory.The best ratio of this compound was obtained in the preliminary study,which preliminarily proves its antibacterial and anti-inflammatory effects.On the basis of the preliminary study,it is preliminary explored whether the compound has an effect on the growth of HepG2 cells,laid the foundation for the later experiment.Then,according to the requirements for the preparation of new Chinese veterinary medicine in the country,the optimal preparation process and molding process were prepared the compound into granules,and the mechanism of the granules to induce HepG2 cell’s apoptosis was explored.Provided a certain theoretical basis for the clinical application of Penthorum Chinense Pursh Granules.1.This study explored the effects of Penthorum Chinense Pursh compound on the growth of HepG2.The CCK-8 method was used to explore the effect of Penthorum Chinense Pursh compound on the proliferation of HepG2.The results show that Penthorum Chinense Pursh compound can effectively inhibit the growth of HepG2,which is dose-dependent and time-dependent.And the Hoechst 33342 /PI double staining method was used to explore whether the Penthorum Chinense Pursh compound could induce apoptosis and even necrosis of HepG2.It is preliminarily confirmed that Penthorum Chinense Pursh compound can induce apoptosis or necrosis of HepG2,it is dose-dependent and time-dependent.It is preliminarily proved that this compound can induce HepG2 cell apoptosis,and this compound has certain research significance.2.Quercetin is the main component of Penthorum Chinense Pursh compound,we explored the HPLC determination conditions of quercetin by control variate method,and obtained the HPLC conditions as follows: chromatographic column is C18 column(250mm × 4.6 mm,5 μm),detection wavelength:254 nm,volume flow rate: 1 m L/min,Column temperature: 40 ℃,Injection volume: 15 μL,The mobile phase is acetonitrile(A)-0.4 % phosphoric acid water(B)binary linear gradient elution(0～30 min,the organic phase increases to 75 % firstly,then gradually return to the initial state in 30～40min).Under this condition,the peak shape of the target peak is sharp and symmetrical,and the separation effect is good.The tailing factor,the number of theoretical plates and the resolution meet the requirements.It can be used as a suitable HPLC condition for quercetin in the Penthorum Chinense Pursh compound.3.The optimum extraction and concentration technology of Penthorum Chinense Pursh compound was studied.In this study,a combination of steam distillation,water decocting and ethanol ultrasonic extraction was used for extraction.Orthogonal design was used to carry out the extraction process screening test to investigate the influence of main factors on the retention rate of quercetin.Finally,the optimal extraction process was obtained,which proved to be feasible.4.The molding process of Penthorum Chinense Pursh Granule was explored,and the quality control of the granules was studied.Firstly,a single excipient is investigated,the best type of excipient is selected,then the ratio of excipients is screened to determine the adjuvant composition of the granule,finally,the mixture design is optimized to obtain the shaped granule.After obtaining the shaped granules,in order to effectively control the quality of the granules and maintain their effectiveness,the properties,qualitative and quantitative identification of the Penthorum Chinense Pursh Granule were investigated,and the quality control was studied.Research on the quality control of granules found that Penthorum Chinense Pursh Granule is brown-yellow granule.When using thin-layer chromatography to identify the active ingredient quercetin,it has good reproducibility and high specificity.When using HPLC to determine the content of quercetin in Penthorum Chinense Pursh Granule,the precision,repeatability,stability,sample recovery,and specificity of the established method are in line with the pharmacopoeia for HPLC.The final optimized preparation method of Penthorum Chinense Pursh Granule is reliable.5.This study explored the mechanism of Penthorum Chinense Pursh Granule on HepG2 cell apoptosis.The experiment selected the key apoptotic genes Bcl-2,Bax and Caspase-3 genes in the process of apoptosis to explore.ELISA was used to determine the content of Bcl-2,Bax and Caspase-3 in Penthorum Chinense Pursh Granule on HepG2 cells,and the expression of Bcl-2,Bax and Caspase-3 genes in Penthorum Chinense Pursh Granule was detected by fluorescence q RT-PCR.The effect of Penthorum Chinense Pursh Granule on the apoptosis of HepG2 cell.The results of ELISA found that Penthorum Chinense Pursh Granule can reduce Bcl-2 levels,increase Bax and Caspase-3 levels,and effectively induce apoptosis.The results of q RT-PCR method showed that Penthorum Chinense Pursh Granule can effectively reduce the expression of Bcl-2 gene in HepG2,increase the expression of Bax and Caspase-3 genes,and promote apoptosis by regulating these apoptotic genes.It was speculated that the mechanism of apoptosis might be related to the regulation of Bax/Bcl-2/Caspase-3 signaling pathwayIn summary,the HPLC determination conditions for quercetin in the Penthorum Chinense Pursh compound prescription established in this experiment are stable and reliable,and the optimal extraction,concentration and molding process of Penthorum Chinense Pursh granule has been determined.The quality standard of Penthorum Chinense Pursh granule has been initially established.In addition,Penthorum Chinense Pursh granules can inhibit the proliferation of HepG2 and induce apoptosis.It laid the foundation for the development and application of Penthorum Chinense Pursh granules.