[High performance liquid chromatographic method for determination of active components in lithospermum oil and its application to process optimization of lithospermum oil prepared by supercritical fluid extraction].

Lithospermum erythrorhizon has the functions of cooling blood, activating blood, as well as detoxifying and penetrating rash. Lithospermum oil extracted from Lithospermum erythrorhizon can prevent and treat diaper rash, skin ulceration, eczema, and other skin diseases. Supercritical fluid extraction is the optimal method for the extraction of active components from lithospermum. In this study, an analytical method was established for simultaneously determination of six active components in lithospermum oil with high performance liquid chromatography (HPLC), and the contents of the active components as the evaluation index were used to investigate several important factors in the preparation of lithospermum oil by supercritical fluid extraction. The optimized HPLC conditions were as follows: separation column, Diamonsil C18 (250 mm×4.6 mm, 5 µm); mobile phases, acetonitrile containing 0.1% (v/v) formic acid-0.1% (v/v) formic acid aqueous solution containing 5 mmol/L ammonium formate (75∶25, v/v); flow rate, 1 mL/min; injection volume, 15 µL; room temperature; photodiode array detector (PAD); detection wavelength, 275 nm. The supercritical fluid extraction was optimized for ensuring stability of the amounts of effective components and the reliability of the quality of lithospermum oil. This will serve as the basis for preparation and quality control processes. Three factors and three levels orthogonal tests were adopted to investigate the important factors, viz. the pressure, temperature and CO2 flow rate in the preparation of lithospermum oil. The results showed that the developed HPLC-PAD method can be used for the simultaneous determination of shikonin, acetylshikonin, ß-acetoxyisovaleryl akanin, isobutyryl shikonin, ß,ß-dimethylacryl shikonin, and 2-methylbutyryl shikonin in 30 min. The method has good precision, accuracy and repeatability. The contents of the active components were the highest when the extraction pressure, extraction temperature, and CO2 flow rate were 23 MPa, 40 ℃, and 27 L/h, respectively. The optimized conditions are suitable for the preparation and actual production of lithospermum oil. The HPLC-PAD method is simple, feasible, accurate, and reliable. It can be used for the preparation and quality control of lithospermum oil by supercritical fluid extraction. Thus, with this method, the stability of the contents of active ingredients and the reliability of the quality of lithospermum oil can be ensured; moreover, safe and effective drug use can be realized. The established method has obvious advantages over the traditional process and is a good candidate for widespread use.

PRP1, a heteropolysaccharide from Platycodonis Radix, induced apoptosis of HepG2 cells via regulating miR-21-mediated PI3K/AKT pathway.

Two polysaccharides (PRP1 and PRP2) were isolated from Platycodonis Radix. Preliminary structural analysis indicated that PRP1 was composed of glucose, fructose, and arabinose in a molar ratio of 1:1.91:1.59 with a molecular weight of 440 kDa, whereas PRP2 was composed of arabinose, fructose, and galactose in a molar ratio of 1:1.39:1.18 with a molecular weight of 2.85 kDa. Compared with PRP2, PRP1 exerted stronger anticancer activity in vitro. Treatment with 5-30 µg/ml of PRP1 significantly inhibited the proliferation of HepG2 cells in vitro, and oral administration at the doses of 75-300 mg/kg also reduced the tumor growth in vivo. The miRNA expression patterns of human liver cancer cells HepG2 in vivo under PRP1 treatment were established, and microRNA-21 (miR-21) as the onco-miRNA was appreciably downregulated. PRP1 repressed the expression of miR-21, which directly targeted and suppressed PTEN (a negative regulator of the PI3K/Akt signaling cascade), and subsequently upregulated the expression of PTEN but downregulated the PI3K/AKT pathway, thereby promoting liver cancer cell apoptosis. These findings indicated that PRP1 inhibited the proliferation and induced the apoptosis of HepG2 mainly via inactivating the miR-21/PI3K/AKT pathway. Therefore, PRP1 could be used as a food supplement and candidate for the treatment of liver cancer.

Development and application of a rapid HPLC method for simultaneous determination of hyperoside, isoquercitrin and eleutheroside E in Apocynum venetum L. and Eleutherococcus senticosus.

Apocynum venetum L. and Eleutherococcus senticosus have been used for hundreds of years to treat hypertension in China. In previous research, there was not a suitable quality control of method for the formulas of Apocynum venetum L. and Eleutherococcus senticosus. It is urgent and essential to develop modern analytical methods for Apocynum venetum L. and Eleutherococcus senticosus to ensure the quality of the formulas. A rapid approach for simultaneous determination of hyperoside, isoquercitrin and eleutheroside E in Apocynum venetum L. and Eleutherococcus senticosus by high-performance liquid chromatography with a diode array detector was described and validated. The full method validation, including the linearity, limits of detection and quantification, precision, repeatability, stability and recovery, was examined. All target components, including isomers of hyperoside and isoquercitrin, were baseline separated in 35 min. The developed method was sensitive, reliable and feasible. With this method, the optimal decoction conditions of Apocynum venetum L. and Eleutherococcus senticosus were selected, and their quality analysis was carried out. Furthermore, an herbal compatibility study of Apocynum venetum L. and Eleutherococcus senticosus based on detecting variations in the content of their active ingredients was performed by the developed HPLC method. It could be an alternative for the quantitative analysis of herbs that contain hyperoside, isoquercitrin or (and) eleutheroside E in the future.

A Retrospective Study on the Use of Chinese Patent Medicine in 24 Medical Institutions for COVID-19 in China.

Objective: This research aims to analyze the application regularity of Chinese patent medicine during the COVID-19 epidemic by collecting the names of the top three Chinese patent medicines used by 24 hospitals in 14 provinces of China in four time periods (January 20-22, February 16-18, March 01-03, April 01-03, 2020), and explore its contribution to combating the disease. Methods: 1) We built a database of the top three Chinese patent medicines used by 24 hospitals. 2) The frequency and efficacy distribution of Chinese patent medicine were analyzed with risk areas, regions, and hospitals of different properties as three factors. 3) Finally, we analyzed the differences in the use of heat-clearing and non-heat-clearing medicines among the three factors (χ2 test) and the correlation between the Chinese patent medicine and COVID-19 epidemic (correlation analysis) with SPSS 23.0 statistical software. Results: 1) The heat-clearing medicine was the main use category nationwide during January 20-22, 2020. Meanwhile, there was a significant difference in the utilization rate of heat-clearing and non-heat-clearing medicine in different risk areas (p < 0.01). 2) The variety of Chinese patent medicine was increased nationwide during February 16-18, 2020, mainly including tonics, blood-activating and resolving-stasis, and heat-clearing medicines. Meanwhile, there was a significant difference in the utilization rate of heat-clearing and non-heat-clearing medicine in the southern and northern regions (p < 0.05). 3) Tonics, and blood-activating and resolving-stasis medicines became the primary use categories nationwide during March 01-03, 2020. 4) The tonics class, and blood-activating and resolving-stasis medicine were still the primary categories nationwide during April 01-03, 2020. Meanwhile, there was a significant difference in the utilization rate of heat-clearing and non-heat-clearing medicine in different risk areas (p < 0.01). Conclusion: Chinese patent medicine has a certain degree of participation in fighting against the COVID-19. The efficacy distribution is related to the risk area, region, and hospital of different properties, among which the risk area is the main influencing factor. It is hoped that future research can further collect the application amount of Chinese patent medicine used in hospitals all over the country, so as to perfectly reflect the relationship between Chinese patent medicine and the epidemic situation.

Flavanones from Sedum sarmentosum Bunge Alleviate CCl4-Induced Liver Fibrosis in Rats by Targeting TGF-ß1/TßR/Smad Pathway In Turn Inhibiting Epithelial Mesenchymal Transition.

OBJECTIVE: The aim of the study is to evaluate the therapeutic effects of flavanones from Sedum sarmentosum Bunge (FSSB) on CCl4-induced liver fibrosis in rats and the underlying mechanisms of action. METHODS: An experimental model of liver fibrosis was established by subcutaneous injection of rats with CCl4 (40% v/v, 3 ml/kg) twice per week for six weeks. FSSB (100, 200, and 400 mg/kg) was intragastrically administered once per day consecutively for five weeks. RESULTS: Our results showed that FSSB significantly attenuated CCl4-induced liver fibrosis as evidenced by reducing the elevated levels of serum biochemical indexes and improving the histological changes, including decreasing the elevation in serum alanine transaminase (ALT), aspartate transaminase (AST), hyaluronic acid (HA), and laminin (LN) level, reducing infiltration of inflammatory cells and collagen fibers in liver tissue. In addition, compared to the model group, FSSB markedly downregulated the protein and mRNA expression of TGF-ß1, TGF-ß1 receptors I and II (TßRI and TßRII), Smad2, Smad3, and Vimentin in liver tissue, at the mean time upregulating the expression of Smad7 and E-cadherin. CONCLUSIONS: The results suggest that FSSB alleviated CCl4-induced liver fibrosis probably through inhibition of TGF-ß/TßR/Smad pathway in turn inhibiting epithelial mesenchymal transition.

Analysis of active components in Salvia miltiorrhiza injection based on vascular endothelial cell protection.

Correlation analysis based on chromatograms and pharmacological activities is essential for understanding the effective components in complex herbal medicines. In this report, HPLC and measurement of antioxidant properties were used to describe the active ingredients of Salvia miltiorrhiza injection (SMI). HPLC results showed that tanshinol, protocatechuic aldehyde, rosmarinic acid, salvianolic acid B, protocatechuic acid and their metabolites in rat serum may contribute to the efficacy of SMI. Assessment of antioxidant properties indicated that differences in the composition of serum powder of SMI caused differences in vascular endothelial cell protection. When bivariate correlation was carried out it was found that salvianolic acid B, tanshinol and protocatechuic aldehyde were active components of SMI because they were correlated to antioxidant properties.

[Analysis and identification of glycosides in Paeonia lactiflora by UPLC-MS/MS].

OBJECTIVE: To investigate the monoterpene glycosides in Paeonia lactflora by UPLC-MS/MS. METHOD: An Acquity UPLC BEH C18 column (2.1 mm x 50 mm) with 1.7 microm particle size was used. The mobile phase was composed of acetonitrile and 0.1% formic acid in gradient mode. The flow rate was 0.4 mL x min and the chromatographic run time was 9 min for one run. The mass spectrometer equipped with an eletrospray ion source in negative ion mode. RESULTS: Totally six glycosides were analyzed and identified by the established UPLC-MS/MS method. CONCLUSION: The method was rapid, sensitive, and extremely useful for rapid identification of glycosides in P. lactiflora.

Rapid determination of polysaccharides in BianTi Soft Extract by spectrophotometry coupled with gas chromatography-mass spectrometry.

A simple approach for the rapid determination of polysaccharides in BianTi Soft Extract using spectrophotometry coupled with gas chromatography-mass spectrometry (GC-MS) was developed. The mixed standard solution composed of D-glucose, D-mannose, galactose and D-xylose in different proportions (1.00: 1.01: 0.12: 0.05) was prepared according to the monosaccharide composition analysis of the polysaccharides by GC-MS. The determination of polysaccharides by UV-Vis spectrophotometer was performed after 35-min color reaction, in which 1 ml 5% phenol and 4 ml sulfate was used. The assay of the method validation has shown that the method was stable, reliable and feasible. Furthermore, the proposed method was successfully applied in the preparation procedure of BianTi Soft Extract, selecting out optimal decoction conditions and suitable decoction container. It suggests that the convenient method could be useful for the quality control of BianTi Soft Extract. Meanwhile, it may be an alternative for polysaccharides determination of other formulations.

[Study on intermolecular interaction between 4-aminopyridine and methacrylic acid using two dimensional FTIR spectroscopy].

A two-dimensional infrared (2D IR) correlation spectroscopy technique has been introduced to study the intermolecular interaction between 4-aminopyridine and methacrylic acid. The type and relationship of the bond between 4-aminopyridine and methacrylic acid molecule could not be characterized directly using the one-dimensional FTIR spectroscopy. On the contrary, the interactions between 4-aminopyridine and methacrylic acid molecule were determined using 2D IR correlation spectroscopy. The synchronous cross peaks exist between stretching vibrations of hydroxyl group of methacrylic acid at 1298 and 1202 cm(-1) and C=N group of 4-aminopyridine at 1531 cm(-1), and between carbonyl group of methacrylic acid at 1705 cm(-1) and N-H group of 4-aminopyridine at 3382 and 3212 cm(-1), respectively. According to the two-dimensional correlation rules, the interactions of static electricity and hydrogen bonding exist between 4-aminopyridine and methacrylic acid molecule. Such successful applications demonstrate that two-dimensional infrared correlation spectroscopy may be a convenient and effective means in the study of the intermolecular and intramolecular interaction.

[Study on effect of heating rate on thermal decomposition of HMX energetic materials by in-situ diffuse reflection FTIR spectrum].

In-situ FTIR spectroscopy is a rising and dynamic technique. This technique combines the advantages of investigation in-situ and accurate structure analysis by FTIR spectroscopy, and can detect real time chemical change in materials at different temperatures and obtain the relationship between micromechanism of materials and temperature. In the present paper, thermal decomposition of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) heated with a rate of 5, 10, 20 and 40 degrees C x min(-1), respectively, was investigated by in-situ diffuse reflection FTIR spectroscopy. The results demonstrate that intramolecular cyclization occurs in breakage of HMX with a heating rate of 5 degrees C x min(-1), while intermolecular cyclization appears with a heating rate of 10, 20 and 40 degrees C x min(-1). With increasing temperature, the rupture rate of C-N bond is faster than that of N-N bond, which verifies that the cleavage of C-N bond is the dominant rupture form. With increasing heating rate, the decomposition beginning temperature of C-N bond increases. Eight kinds of gaseous products such as CO2, N2O, CO, NO, HCHO, HONO, NO2 and HCN were determined. The decomposition mechanism of HMX was not affected with changing heating rate.

[Study on thermal decomposition of HMX energetic materials by in-situ FTIR spectroscopy].

In-situ FTIR spectroscopy is a rising and dynamic technique. This technique, which combines the advantages of investigation in-situ and accurate structure analysis by FTIR spectsoscopy, can detect the chemical change of materials in different temperature with real time and obtain the relationship between micromechanism of materials and temperature. In the present paper, thermal decomposition of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) heated with 5 degrees C x min(-1) was investigated by in-situ FTIR spectroscopy. The results demonstrate that C-N bond cleavage and N-N bond cleavage of HMX occur at 205 degrees C. With increasing temperature, the rupture rate of C-N bond is faster than that of N-N bond, which verifies that the cleavage of C-N bond is the dominant rupture form. The augment in tension of HMX cyclic confirms that intramolecular cyclization occurs in breakage of HMX. Eight kinds of gaseous products such as CO2, N2O, CO, NO, HCHO, HONO, NO2 and HCN were determined. According to the structure change in condensed state and gaseous products, the decomposition mechanism was deduced that HCHO, N2O, HONO and HCN were released due to the cleavage of C-N bond, and NO2 was released due to the cleavage of N-N bond.