[Analysis and evaluation of dynamic accumulation of multiple bioactive constituents in Spatholobi Caulis].

A method was established for simultaneous determination of 21 active constituents including flavanols, isoflavones, flavonols, dihydroflavones, dihydroflavonols, chalcones, pterocarpan, anthocyanidins and phenolic acids in Spatholobi Caulis by ultra fast liquid chromatography with triple quadrupole linear ion trap mass spectrometry(UFLC-QTRAP-MS/MS). Then, it was employed to analyze and evaluate the dynamic accumulation of multiple bioactive constituents in Spatholobi Caulis. The chromatographic separation was performed on a XBridge®C_(18)(4.6 mm×100 mm, 3.5 µm) at 30 ℃ with a gradient elution of 0.3% formic acid aqueous solution-methanol, and the flow rate was 0.8 mL·min~(-1), using multiple-reaction monitoring(MRM) mode. A comprehensive evaluation of the multiple bioactive constituents was carried out by gray correlation analysis(GRA). The 21 target components showed good linearity(r>0.999 0) in the range of the tested concentrations. The average recovery rates of the 21 components were from 97.46% to 103.6% with relative standard deviations less than 5.0%. There were differences in the contents of 21 components in Spatholobi Caulis at diffe-rent harvest periods. Spatholobi Caulis had high quality from early November to early December, which is consistent with the local tradi-tional harvest period. This study reveals the rule of the dynamic accumulation of 21 components in Spatholobi Caulis and provides basic information for the suitable harvest time. At the same time, it provides a new method reference for the comprehensive evaluation of the internal quality of Spatholobi Caulis.

[Research progress in molecular biology of Lonicerae Japonicae Flos].

Molecular biology is a new subject that clarifies the phenomena and nature of life at the molecular level. Its development provides new biotechnology and methods for the study of traditional pharmacognosy. The formation of molecular biology has brought the development of pharmacognosy into a new era of gene research. Lonicerae Japonicae Flos is a classical Chinese medicine. Many scholars of home and abroad have carried out relevant studies on its molecular biology on the basis of the in-depth study with traditional methods, and have achieved certain results. In order to provide references on the method, technical for promoting the modernization of Lonicerae Japonicae Flos, and the development, protection, and utilization of other traditional Chinese medicine resources. This article summarized the application status of molecular biology methods and techniques on the identification, biosynthesis of active constituents, and molecular mechanism of secondary metabolite under stress conditions of Lonicerae Japonicae Flos in recent years. In hybridization technology of tag(RFLP), molecular markers based on PCR(RAPD, AFLP, SSR and ISSR), based on DNA sequence analysis of SNP and DNA barcode for the variety identification, diagnosis, identification of Lonicerae Japonicae Flos, and so forth in detail. At the same time, it is proposed that multi-omics technology can be used to build systems biology technology and platforms, and establish related models of secondary metabolite biosynthesis, so as to deepen acknowledge the molecular mechanism of the active component biosynthesis of Lonicerae Japonicae Flos and the accumulation of metabolites, life activities of other medicinal plants under adverse environment, then to regulate them.

[Stimultaneous determination of multiple bioactive constituents in Panacis Japonici Rhizoma processed by different methods and grey relational analysis].

A method, for determination of saponins, amino acids and nucleosides in Panacis Japonici Rhizoma of ultra fast liquid chromatography with triple quadrupole linear ion trap mass spectrometry (UFLC-QTRAP-MS/MS), was established to investigate the effect of different processing methods on the target components of Panacis Japonici Rhizoma. The chromatographic separation was performed on a XBridgeC18(4.6 mm×100 mm, 3.5 µm) at 30 °C with a gradient elution of 0.1% formic acid solution-0.1% formic acid acetonitrile, and the flow rate was 0.8 mL·min⁻¹, using multiple-reaction monitoring (MRM) mode. The grey relational analysis was adopted for the analysis of different processing samples. The results showed that the thirty-three constituents were in a good linear range and the correlation coefficient was greater than 0.999 0; the precision, repeatability and stability were good; the average recovery rates were between 95.33% and 101.8%, and the relative standard deviations were less than 5%. The result of grey relational analysis showed that the complete rhizomes without peeling, which were adopted for the microwave dried method, had the best quality. The established method was accurate and reliable, which could be used to appraise the quality of Panacis Japonici Rhizoma. Our study may lay the way for the processing method of Panacis Japonici Rhizoma in optimization,normalization and standardization.

[Comparative analysis of multiple index constituents in Ophiopogonis Radix and Liriopes Radix].

An analytical method based on UFLC-QTRAP-MS/MS was established for simultaneous determination of thirty-three components including steroidal saponins, homoisoflavonoids, amino acids and nucleosides in Ophiopogonis Radix. Thirty-three target components of commercial medicinal materials of Maidong were comparative analysis. Synergi™ Hydro-RP 100 column (2.0 mm × 100 mm, 2.5 µm) was used with 0.1% formic acid solution-0.1% formic acid acetonitrile for gradient elution at a flow rate of 0.4 mL·min⁻¹. In addition, multiple reaction monitoring (MRM) mode was employed. The data were comprehensively processed and analyzed with hierarchical clustering analysis(HCA), principal component analysis(PCA) and partial least squares discriminant analysis(PLS-DA) methods. All components showed good linearity(r>0.999 0) within the tested ranges. The average recoveries were between 96.23%-102.0%, and the relative standard deviation(RSD) were less than 5%. The results showed that there were significant differences in components between Ophiopogonis Radix and Liriopes Radix, with seven components obviously different. This method was useful for providing basis for the comprehensive evaluation and intrinsic quality control of Ophiopogonis Radix and Liriopes Radix , and may provide a new method reference for the identification of Ophiopogonis Radix and Liriopes Radix.

[Thoughts of omics research on quality formation in Dao-di herbs].

Dao-di herbs have been recognized as "quality models" with a firmly stable status. The formation of Dao-di herbs quality is involved from the genetic inheritance on the molecular level to the metabolic phenotype of final products, and the full material-based biosynthetic pathway remains unknown. In recent years, an increasing variety of omics technologies has provided new methods and ideas for the analysis of complex life systems and are suitable for explanation of quality formation in Dao-di herbs as well. In order to alleviate the scarcity of natural resources and offer scientific guidance of transplanting varieties, achievements of omics in the aspects of Dao-di herbs from genetics to phenotyping, the biosynthetic pathway of secondary metabolites, the interaction with human body and the new methods of quality evaluation have been summarized. It will be a fundamental work for protection and utilization of Chinese medicine resources.

[Effects of tetramethylpyrazine on trigeminal neuralgia induced by chronic constriction injury of infraorbital nerve in rats].

Trigeminal neuralgia (TN) is a kind of recurrent transient and severe pain that is limited to the trigeminal nerve in one or more branches. The clinical incidence of TN is high, which seriously affects the quality of life of the patients and is difficult to cure. The present study investigated the effects of tetramethylpyrazine (TMP) on TN induced by chronic constriction injury of the infraorbital nerve (ION-CCI) in rats. Adult male Sprague-Dawley rats were randomly assigned to four groups: sham, sham treated with TMP (Sham+TMP), TN model (TN), and TN treated with TMP (TN+TMP). The rat TN model was established by ION-CCI and TMP (50 mg/kg) was injected intraperitoneally once a day for 2 weeks after operation. The mechanical response threshold was tested by the electronic von Frey filaments. The expression of CGRP in the trigeminal ganglia (TGs) of rats on the operative side was detected by RT-PCR, immunohistochemical staining and Western blot. In 15 days after operation, TN group showed a robust decrease in mechanical response threshold as compared with sham group. From day 9 to day 15 after operation, TMP treatment significantly suppressed the TN-induced mechanical hyperalgesia (P < 0.05). On day 15 after operation, RT-PCR, immunohistochemical staining and Western blot analysis showed an obvious increase in expression level of CGRP in TGs of TN group compared with sham group, which was downregulated by TMP treatment (P < 0.05). These results suggested that TMP might have a therapeutic potential for the treatment of TN through regulating CGRP expression in the TGs.

Emodin inhibits the expression of receptor and calcitonin-gene-related peptide release in trigeminal ganglia of trigeminal neuralgia rats.

Trigeminal neuralgia (TN) is one of the most intense forms of facial pain. It has been reported that the P2X3 receptor plays a crucial role in facilitating pain transmission, and the calcitonin-gene-related peptide (CGRP) from trigeminal ganglia (TGs) might perform differing function in nociceptive afferent input transmission. The present study investigated whether emodin can affect TN pain transmission by suppressing the expression of P2X3 receptors and CGRP in TGs. Chronic constriction injury of the infraorbital branch of the trigeminal nerve (CCI-ION) was used as TN model. The TN rats were randomly divided into the following 4 groups: (1) a sham group (Sham), (2) a sham rats treated with emodin group (TN + E), (3) a TN rats treated with 0.5% sodium carboxymethyl cellulose (CMC) as vehicle group (TN) and (4) a TN rats treated with emodin group (TN + E). The mechanical hyperalgesia threshold of TN rats was tested by Electric Von Frey filaments. The change of the expression of P2X3 receptors and CGRP in rat's TG was detected with RT-PCR, immunohistochemical staining, and Western blotting. The phosphorylation of p38 and ERK1/2 pathway of TG was detected by Western blotting. After CCI-ION injury, the threshold of mechanical hyperalgesia for the territory of ligated infraorbital nerve in TN group decreased significantly compared with that in sham group. On day 14 after operation of CCI-ION, there was also an evident increase in the expression of P2X3 receptors and CGRP in the TG of TN group. However after treatment with emodin, the response of mechanical hyperalgesia of TN rats was clearly increased while the enhanced expression of P2X3 receptor and CGRP in TN rats was significantly decreased. The phosphorylation of p38 and ERK1/2 in TN group was stronger than that in Sham group. But these phosphorylation changes in the TN rats were much weaker after treatment with emodin. In conclusion, P2X3 receptor may cooperate with CGRP in the pain transmission of TN, and emodin can inhibit the expression and activation of P2X3 receptor and CGRP in TG to relieve TN.