Establishment of Specific Chromatogram and Thin-layer Identification Method for Kaixinsan / 中国实验方剂学杂志

ObjectiveTo establish the specific chromatogram and thin layer chromatography（TLC） identification method of Kaixinsan（KXS） samples， in order to clarify the key quality attributes and provide reference for the quality evaluation of KXS. MethodHigh performance liquid chromatography（HPLC） specific chromatogram of KXS was developed with YMC Hydrosphere C18 column（4.6 mm×250 mm， 5 μm）， the mobile phase was acetonitrile（A）-0.2% formic acid aqueous solution（B） for gradient elution（0-15 min， 2%-20%A； 15-25 min， 20%-25%A； 25-30 min， 25%-30%A； 30-45 min， 30%-31%A； 45-50 min， 31%-44%A； 50-65 min， 44%-45%A； 65-73 min， 45%-75%A； 73-95 min， 75%-100%A； 95-105 min， 100%A； 105-105.1 min， 100%-2%A； 105.1-120 min， 2%A）， the detection wavelength was 320 nm. Ultra high performance liquid chromatography-linear ion trap-electrostatic field orbitrap mass spectrometry（UHPLC-LTQ-Orbitrap MS） was used to identify the chemical components of KXS with electrospray ionization（ESI）， negative ion mode and scanning range of m/z 50-2 000. TLC identification methods for Poria and Ginseng Radix et Rhizoma in KXS were established. ResultThere were 11 common peaks in the specific chromatogram of KXS， attributed to Polygalae Radix， Poria and Acori Tatarinowii Rhizoma. Taking peak 9（α-asarone） as the reference peak， the relative standard deviations of the retention times of 15 batches of KXS samples were<0.2%. A total of 34 compounds were identified by UHPLC-LTQ-Orbitrap MS， including terpenoids， phenylpropanoids， oligosaccharides and ketones. The established TLC had good separation and was rapid， reliable， simple， feasible， suitable for the identification of Poria and Ginseng Radix et Rhizoma in KXS. ConclusionThe specific chromatogram and TLC of KXS are stable and reproducible. The material basis of KXS is basically clarified by MS， which can provide a reference for the development and quality control of KXS.

Establishment of Specific Chromatogram and TLC Identification for Qingxin Lianziyin / 中国实验方剂学杂志

ObjectiveTo establish the specific chromatogram and thin layer chromatography（TLC） of Qingxin Lianziyin（QXLZY） benchmark samples， in order to clarify the key quality attributes and provide a reference for the quality evaluation of QXLZY. MethodHigh performance liquid chromatography（HPLC） specific chromatogram of QXLZY benchmark samples was developed by using a YMC Hydrosphere C18 column（4.6 mm×250 mm， 5 μm） with the mobile phase of acetonitrile（A）-0.2% formic acid aqueous solution（B） for gradient elution（0-10 min， 5%-20%A； 10-20 min， 20%A； 20-25 min， 20%-24%A； 25-40 min， 24%-30%A； 40-55 min， 30%-50%A； 55-65 min， 50%-100%A； 65-75 min， 100%A； 75-75.1 min， 100%-5%A； 75.1-90 min， 5%A）， and the detection wavelength was 360 nm. Ultra-high performance liquid chromatography-linear ion trap/orbitrap mass spectrometry（UHPLC-LTQ-Orbitrap MS） with electrospray ionization（ESI） was used to identify the components of QXLZY benchmark samples by accurate relative molecular weight and multilevel MS fragment ion information， the detection conditions were positive and negative ion modes and data dependency scanning mode. TLC identification methods for Ophiopogonis Radix， Lycii Cortex， Nelumbinis Semen， Poria， Astragali Radix and Ginseng Radix et Rhizoma in QXLZY were established. ResultA total of 15 characteristic peaks were identified from Glycyrrhizae Radix et Rhizoma， Plantaginis Semen and Scutellariae Radix， and the relative standard deviations of the retention times of 15 characteristic peaks in 15 batches of QXLZY benchmark samples were≤3% with peak 8（baicalin） as the reference peak. A total of 100 compounds， including flavonoids， organic acids， saponins， amino acids and others， were identified in the benchmark samples by UHPLC-LTQ-Orbitrap MS. The established TLC had good separation and was suitable for the identification of Ophiopogonis Radix， Lycii Cortex， Nelumbinis Semen， Poria， Astragali Radix and Ginseng Radix et Rhizoma in QXLZY. ConclusionThe material basis of QXLZY benchmark samples is basically determined by MS designation and source attribution. The established specific chromatogram and TLC of QXLZY are simple， stable and reproducible， which can provide a reference for the development and quality control of QXLZY.

Establishment of a Method for Determination of Amygdalin in Quality Control System of Qingfei Paidu Granules / 中国实验方剂学杂志

ObjectiveAlmond， which is bitter in taste， contains traces of toxic substances. For the sake of the safety of prescriptions containing this medicinal material， the processing method of "soaking in boiling water" was selected. Moreover， through literature research and network pharmacology， the characteristic index of this medicinal material was determined. On this basis， a method was established for the determination of amygdalin in Qingfei Paidu Granules （QFPD） and the transfer rate of it in the processing of this prescription was monitored， aiming at improving the quality control system of QFPD. MethodThe high performance liquid chromatography conditions are as follows： YMC Triart C18 column （4.6 mm × 150 mm， 5 µm）， mobile phase of methanol-water with flow of 1.0 mL·min-1， column temperature of 35 ℃， and detection wavelength of 210 nm. ResultThe linear curve fitted well and the average recovery of amygdalin was 97.74% with RSD of 4.3%. The transfer rates of amygdalin from the medicinal material to the extract， from extract to concentrate， and from concentrate to granules were investigated with this method. The result showed that the average transfer rate from the medicinal material to the granules was （60±3.91）%. The comparison of transfer rate between the processes suggesting that the extraction of the medicinal material might be the key part influencing the prescription preparation. ConclusionThe method is simple， sensitive， reproducible， stable， and accurate， and the index is reasonable. Thus， the method can be used for the quality control of QFPD and determination of transfer rate of components in the preparation of QFPD. This study further improves the quality control standard of almond in QFPD， which can serve as a reference for the clinical application of QFPD.

Optimization of Ultrasonic-Assisted Extraction and Purification of Rhein from Cassia fistula Pod Pulp.

Rhein is used as an active ingredient in laxatives in medicinal herbal products and is a chemical marker for quality control purposes. Thus, a simple and effective method for the optimized extraction of a high amount of rhein from the fruit pulp of Cassia fistula was investigated using ultrasonic-assisted extraction (UAE). The response surface methodology was applied to find the most suitable parameters for optimizing the extraction process and to study the factors' relationships with each other. The best conditions for ultrasonic extraction were the application of 1:40 g/mL solid-to-liquid ratio and 10% EtOH-H2O as a solvent at 75 °C for 40 min. This method was compared to a conventional decoction in two variations. In these experiments, it was confirmed that the UAE was more favorable than the decoction methods. The resulting crude extract was further purified by liquid-liquid extraction with a basic pH adjustment, followed by recrystallization. High-purity rhein was obtained by using chromatographic techniques and nuclear magnetic resonance spectroscopy. Therefore, this study suggests that UAE is an efficient alternative method for the extraction of rhein from C. fistula pod pulp. The resulting optimized conditions can be applied as a useful tool for the large-scale industrial production of a rhein-rich plant extract.

The effect of acteoside on puromycin nephropathy and podocyte injury model / 中华肾脏病杂志

Objective To explore the effect of acteoside (one of the ingredients of Total Glycosides Extracted from Rehmannia capsules) on treatment of proteinuria and protection of podocytes.Methods In this study,puromycin nephropathy rat model was successfully established.After detecting the degree of proteinuria,the expression of podocyte injury markers and the degree of podocyte foot process fusion were investigated by electron microscope.In addition,puromycin treated podocyte injury model was also successfully established in vitro.Podocyte viability,migration,cytoskeleton and injury marker were detected.Results In vivo study showed that acteoside could effectively reduce proteinuria (P ＜ 0.05),restore the expression of podocyte injury markers such as nephrin and synaptopodin (all P ＜ 0.05),and alleviate the degree of podocyte foot process fusion.In vitro study showed that acteoside could effectively restored podocyte viability (P ＜ 0.05),reduce abnormal migration ability (P ＜ 0.05),protecte cytoskeleton and restore the expression of podocyte injury marker nephrin (P ＜ 0.05).Conclusions This study confirms that acteoside can reduce the degree of proteinuria in puromycin nephropathy rat model in vivo and alleviate the degree of podocyte injury in vitro as well as enrich the molecular mechanism of Total Glycosides Extracted from Rehmannia capsules in treatment of proteinuria.

Identification of bufadienolides profiling in cinobufacino by HPLC-DAD-FT-ICR-MS method / 药学学报

Cinobufacino injection is a significant anti-tumor medicine for the treatment of various tumors in clinic, which was made from water extraction of the skin of Bufo bufo gargarizans. In present paper, HPLC-DAD-FT-ICR-MS method was used to identify the major bufadienolides in cinobufacino for the first time. Solid-phase extraction with dichloromethane and silica was used to enrich the total bufadienolides in cinobufacino. Based on the UV and high resolution MS/MS data, 33 bufadienolides were analyzed and characterized. Among them, eight compounds were identified by comparing with standard references unambiguously. This study elucidated the major bufadienolides in cinobufacino, which provided material foundation of cinobufacino and will be benefit for the further pharmacological research.

LC-FT-ICR-MS analysis of the prototypes and metabolites in rat plasma after administration of huang-lian-jie-du decoction / 药学学报

To obtain chemical constituent information of rat plasma after oral administration of Huang-Lian-Jie-Du Decoction (HLJDD), a LC-FT-ICR-MS method has been established, and both positive and negative ions scan modes were include in the analysis. By comparing their retention time, high resolution mass data of HLJDD extracts, blank plasma and dosed plasma, 38 constituents, including 22 prototype compounds and 16 metabolites, were detected in rat plasma after oral administration of HLJDD. In the 22 prototype compounds, 16 constituents were determined unambiguously by comparing with references. In the analysis of metabolites, phase II reactions like glucuronidation and sulfation were the major biotransformation pathways of HLJDD. M11 was observed as the only phase I metabolite in present experiment. The results will be beneficial for the further pharmacokinetics and pharmacological evaluations of HLJDD.

Comparison of the chemical composition between fresh and dried venenum bufonis by UPLC-Orbitrap MS / 药学学报

To identify the active components in Bufo melanostictus Schneider and clarify the difference between fresh and dried Venenum Bufonis, a UPLC-Orbitrap MS method has been established. The separation was performed with gradient elution of acetonitrile and water (with 0.1% formic acid) as mobile phase. By comparing their retention time and high resolution mass data of Venenum Bufonis extracts, 39 effective components were primarily identified by MS/MS analysis in positive ion mode. Twenty-six of them were bufadienolides. There were significant differences in the main composition between fresh and dried Venenum Bufonis. There are fewer bufadienolides in fresh toad venom.

Chemical constituents of bufadienolides in cinobufacino injection / 药学学报

Cinobufacino injection is purified from water extraction of the skin of Bufo bufo gargarizans, which has been widely used for various cancers in clinic with significant anti-tumor effects. Bufadienolides were regarded as the main active constituents of cinobufacino injection in previous reports. In present study, 6 bufadienolides were isolated and purified from Cinobufacino injection. Their structures were identified as 3-epi-ψ-bufarenogin (1), ψ-bufarenogin (2), 3-epi-arenobufagin (3), arenobufagin (4), 3-epi-gamabufotalin (5), and 3-oxo-arenobufagin (6), separately. Among them, 1 and 3 were new compounds, 5 and 6 were new natural products. Compounds 1, 2 and compounds 3, 4 were two pairs configuration isomers at C-3, separately.

In vivo comparison analysis of scandoside methyl ester metabolites in four kinds of liver microsomes using ultra-performance liquid chromatography combined with electrospray ionization tandem orbitrap mass spectrometry / 药学学报

In order to clarify the metabolism pathways of scandoside methyl ester, the analysis of metabolites profiling in four kinds of liver microsomes was performed by using an ultra-performance liquid chromatography/ electrospray-tandem mass spectrometry (UPLC-ESI-MS). The data obtained from the 0 h-incubation and the 2 h-incubation were compared and analyzed. After incubation, 5 metabolites of scandoside methyl ester were found in rat, Beagles, rhesus monkey and human liver microsome. The results showed that scandoside methyl ester's major metabolic pathway in the liver microsomes is hydrolysis, oxidation and reduction reactions, and there are certain kinds differences between species. The study provides a research base for further research about iridoid compounds in vivo metabolic pathways.

In vitro anti-proliferation effect of peptides from cinobufacini injection / 药学学报

Cinobufacini is an aqueous extract of Bufo bufo gargarizans Cantor dried skin, which has been widely used for cancer therapy in China. So far, its active components are still not very clear. In previous reports, bufadienolides with low-concentration were usually studied because of their anticancer effects. However, the high polarity constituents in cinobufacini are less investigated. The present study found that more than 50% contents of cinobufacini were water-soluble peptides. Then, in vitro anticancer experiments were carried out, including human stomach cancer cell lines BGC823 and MCG803, human colon cancer cell lines DLD-1 and HT-29, and human pancreatic cancer cell line MIAPACA-2. The IC50 for these cell lines model were ranged from 25-123 microgmL(-1). The results indicated that these peptides showed similar activity with cinobufacini injection. As a conclusion, this study provides a new and further understanding of anticancer components in cinobufacini injection.

Pharmacokinetics and bioavailabilities of geniposide in Beagle dogs after oral administration Xingnaojing / 中国中药杂志

<p><b>OBJECTIVE</b>To establish a method for determination of geniposide in Beagle dogs plasma by high performance liquid chromatography (HPLC), and study the pharmacokinetics and bioavailability of geniposide in Beagle dogs after oral administration Xingnaojing.</p><p><b>METHOD</b>To determine the geniposide in Beagle dogs plasma by HPLC after oral administration or intravenous injection Xingnaojing, and the pharmacokinetic parameters were calculated by the software of Kinetica.</p><p><b>RESULT</b>The good linearity range of geniposide was 1.24 - 158.88 mg x L(-1). The main pharmacokinetic parameters after oral administration was as follows: Cmax (11.8 +/- 0.6) mg x L(-1), Tmax (52.0 +/- 4.5) min, AUC(1280.8 +/- 172.0) mg x min x L(-1), MRT(118.7 +/- 25.4) min, and these parameters after intravenous injection was follows: Cmax 107.4 +/- 6.3) mg x L(-1), AUC(7930.1 +/- 670.0) mg x min x L(-1), MRT(92.4 +/- 5.1) min. The bioavailability of geniposide in Beagle dogs after oral administration Xingnaojing was (6.46 +/- 0.87)%.</p><p><b>CONCLUSION</b>The HPLC method had good applicability. The extract recovery, method recovery, intra-day precision and inter-day precision of the method were all met the requirements. The stability in conditions of room temperature and freeze-thaw cycle was good. The results indicated that the oral administration bioavailability of geniposide was in low degree.</p>

Optimal processing techniques of preparing evodiae juice by orthognal experiment / 中国中药杂志

<p><b>OBJECTIVE</b>To study and establish the optimal technology for the preparation of evodiae juice.</p><p><b>METHOD</b>The contents of evodiamine, rutaecarpine and evodin were simultaneously determined with HPLC, and each yield of the three compounds were chosen as the evaluating indicator. The orthogonal test coupled with the weighted coefficient method were adopted to acquire the optimal technology for the preparation of evodiae juice.</p><p><b>RESULT</b>The study showed that the optimal technology for the preparation of evodiae juice was as follows: decocted three times while the first time was with 12-fold of water socked 30 minutes and decocted 45 minutes, the second time was with 8-fold of water decocted 20 minutes and the third time was with 6-fold of water decocted 20 minutes.</p><p><b>CONCLUSION</b>This method is simple and accurate. The optimal technology is suitable for industry manufacture of evodiae juice.</p>