Objective To establish the quality control method of Yinqiao Chaihuang Granules.Methods Lonicerae Japonicae Flos, Bupleuri Radix, Scutellariae Radix and Glycyrrhizae Radix et Rhizoma in Yinqiao Chaihuang Granules were identified by TLC, and the content of forsythoside A was detected by HPLC. Results Under the selected chromatographic conditions, TLC spots were clear, the separating was good, and non-interference was found in negative control. Forsythoside A in calibration curve showed good linearity in the range of 0.029 6–0.474 μg, with average recovery of 99.73%, RSD=1.18%. Conclusion The method is accurate and simple, which can be used for the quality control of Yinqiao Chaihuang Granules.

Administration, Topical ; Alopecia/drug therapy* ; China ; Double-Blind Method ; Fibroblast Growth Factors/therapeutic use* ; Hair ; Humans ; Male ; Minoxidil/therapeutic use* ; Treatment Outcome

In this study, citrate synthase gene(CIT2), and malate synthase gene(MLS1) were successfully knocked out in β-amyrin-producing yeast cells by using CRISPR/CAS9. The promoter of phosphoglucose isomerase gene(PGI1) was replaced by that of cytochrome c oxidase subunit Ⅶa(Cox9)to weaken its expression, aiming to channel more carbon flux into the NADPH-producing pathway. The fermentation results showed that CIT2 deletion had no effect on the β-amyrin production. Compared with the control strain, the production of β-amyrin was increased by 1.85 times after deleting MLS1, reaching into 3.3 mg·L~(-1). By replacing the promoter of PGI1, the β-amyrin yield was 3.75 times higher than that of the control strain, reaching up to 6.7 mg·L~(-1). This study successfully knocked out the CITT2 and MLS1 genes and weakened the PGI1 gene by using CRISPR/CAS9, which directly influenced the production of β-amyrin and provided some reference for the the metabolic engineering of triterpernoid producing strain.
Ethanol ; Fermentation ; Metabolic Engineering ; Saccharomyces cerevisiae ; genetics

In this study, the synthetic pathway of β-amyrin was constructed in the pre-constructed Saccharomyces cerevisiae chassis strain Y0 by introducing β-amyrin synthase from Glycyrrhiza uralensis, resulting strain Y1-C20-6, which successfully produced β-amyrin up to 5.97 mg·L~(-1). Then, the mevalonate pyrophosphate decarboxylase gene(ERG19), mevalonate kinase gene(ERG12), 3-hydroxy-3-methylglutaryl-CoA synthase gene(ERG13), phosphomevalonate kinase gene(ERG8) and IPP isomerase gene(IDI1)were overexpressed to promoted the metabolic fluxto the direction of β-amyrin synthesis for further improving β-amyrin production, resulting the strain Y2-C2-4 which produced β-amyrin of 10.3 mg·L~(-1)under the shake flask fermentation condition. This is 100% higher than that of strain Y1-C20-6, illustrating the positive effect of the metabolic engineering strategy applied in this study. The titer of β-amyrin was further improved up to 157.4 mg·L~(-1) in the fed-batch fermentation, which was almost 26 fold of that produced by strain Y1-C20-6. This study not only laid the foundation for the biosynthesis of β-amyrin but also provided a favorable chassis strain for elucidation of cytochrome oxidases and glycosyltransferases of β-amyrin-based triterpenoids.
Fermentation ; Glycyrrhiza uralensis ; enzymology ; genetics ; Industrial Microbiology ; Intramolecular Transferases ; genetics ; Metabolic Engineering ; Oleanolic Acid ; analogs & derivatives ; biosynthesis ; Saccharomyces cerevisiae ; metabolism

To study the quality control of three traditional Chinese medicines derived from Gleditsia sinensis [Gleditsiae Sinensis Fructus(GSF), Gleditsiae Fructus Abnormalis(GFA), and Gleditsiae Spina(GS)], this paper established a multiple reaction monitoring(MRM) approach based on ultra-high performance liquid chromatography-triple quadrupole-linear ion-trap mass spectrometry(UHPLC-Q-Trap-MS). Using an ACQUITY UPLC BEH C_(18) column(2.1 mm × 100 mm, 1.7 μm), gradient elution was performed at 40 ℃ with water containing 0.1% formic acid-acetonitrile as the mobile phase running at 0.3 mL·min~(-1), and the separation and content determination of ten chemical constituents(e.g., saikachinoside A, locustoside A, orientin, taxifolin, vitexin, isoquercitrin, luteolin, quercitrin, quercetin, and apigenin) in GSF, GFA, and GS were enabled within 31 min. The established method could quickly and efficiently determine the content of ten chemical constituents in GSF, GFA, and GS. All constituents showed good linearity(r>0.995), and the average recovery rate was 94.09%-110.9%. The results showed that, the content of two alkaloids in GSF(2.03-834.75 μg·g~(-1)) was higher than that in GFA(0.03-10.41 μg·g~(-1)) and GS(0.04-13.66 μg·g~(-1)), while the content of eight flavonoids in GS(0.54-2.38 mg·g~(-1)) was higher than that in GSF(0.08-0.29 mg·g~(-1)) and GFA(0.15-0.32 mg·g~(-1)). These results provide references for the quality control of G. sinensis-derived TCMs.
Flavonoids/analysis* ; Alkaloids ; Chromatography, High Pressure Liquid/methods* ; Mass Spectrometry ; Drugs, Chinese Herbal

Taking lipophagy as the breakthrough point, we explored the mechanism of Zexie Decoction(ZXD) in improving lipid metabolism in the hepatocyte model induced by palmitic acid(PA) and in the animal model induced by high-fat diet(HFD) on the basis of protein kinase B(Akt)/transcription factor EB(TFEB) signaling pathway. Co-localization was carried out for the microtubule-associated protein light chain 3(LC3) plasmid labeled with green fluorescent protein(GFP) and lipid droplets(LDs), and immunofluorescence co-localization for liver LC3 of HFD mice and perilipin 2(PLIN2). The results showed that ZXD up-regulated the expression of LC3, reduced lipid accumulation in hepatocytes, and increased the co-localization of LC3 and LDs, thereby activating lipo-phagy. Western blot results confirmed that ZXD increased autophagy-related protein LC3Ⅱ/LC3Ⅰ transformation ratio and lysosome-associated membrane protein 2(LAMP2) in vivo and in vitro and promoted the degradation of sequestosome-1(SQSTM1/p62)(P<0.05). The results above jointly explained that ZXD regulated lipophagy. Furthermore, ZXD activated TFEB expression(P<0.05) and reversed the PA-and HFD-induced decrease of TFEB nuclear localization in hepatocytes(P<0.05). Meanwhile, ZXD activated liver TFEB to up-regulate the expression of the targets Lamp2, Lc3 B, Bcl2, and Atg5(P<0.05). Additionally, ZXD down-regulated the protein level of p-Akt upstream of TFEB in vivo and in vitro. In conclusion, ZXD may promote lipophagy by regulating the Akt/TFEB pathway.
Animals ; Mice ; Autophagy/drug effects* ; Hepatocytes/metabolism* ; Microtubule-Associated Proteins/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Signal Transduction ; Drugs, Chinese Herbal/pharmacology*

Elaphurus davidianus( Milu),a rare animal unique to China,has been used as medicine for more than a thousand years,but the extinction of Milu in modern times resulted in the unavailability of related medical products. Today,the reintroduction of Milu population makes it possible to restore its medicinal usage. The resource reserves of Cervi Cornu,the natural shedding product from Milu,are increasing with the expansion of the population,allowing it to be fully utilized in the medical field. Mijiao Pills,first recorded in Important Prescriptions Worth a Thousand Gold for Emergency( Bei Ji Qian Jin Yao Fang) by Sun Simiao in the Tang Dynasty,is the first Chinese medicinal prescription with Cervi Cornu as the sovereign medicinal,which is effective in tonifying. Its composition,preparation,efficacy and indications,and administration are described in detail in the Important Prescriptions Worth a Thousand Gold for Emergency,which however,have changed significantly over the thousands of years,seriously affecting the clinical application of this classical prescription and related product development. Therefore,the key information of this prescription should be systematically collated and summarized. According to the principles of textual research on key information in ancient classical prescriptions promulgated by relevant authorities,this paper reviewed ancient Chinese medical books of the past dynasties,modern literature,as well as the Pharmacopoeia of the People's Republic of China( 2020 Version) to figure out such key information as the source,historical evolution,original plants and animals and their processing,dosage,preparation,and usage of Mijiao Pills. This paper aimed to provide a basis for the clinical application of Mijiao Pills and subsequent product development,thus facilitating the development and utilization of this precious medicinal animal resource.
Books ; China ; Cornus ; Humans ; Medicine, Chinese Traditional ; Prescriptions