Pulsatilla chinensis:phytochemistry, pharmacology and new drug development / 中国药理学与毒理学杂志

Pulsatilla chinensis is a widely used traditional Chinese herb, which contains 56 types of chemical constit?uents, mainly including triterpenoid saponins, organic acids, coumarins and lignans. The largest portion of the ingredi?ents in Pulsatilla chinensis is the family of triterpenoid saponins, in which anemoside B4 is the major effective compound and indexing component. The main components of Pulsatilla chinensis can metabolize into a vast array of active prod?ucts in vivo, which play vital roles in its biological activity. Mounting evidence reveals that Pulsatilla chinensis exerts a wide range of therapeutic activities, such as anti-cancer, immunoregulation, anti-inflammation and anti-schistosome, with fewer adverse reactions, via various signaling pathways and multiple targets. It was documented that the active ingre?dient of Pulsatilla chinensis can lessen the drug resistance and synergize the effects of other natural products includ?ing paclitaxel, as well as ameliorate the clinical efficacy of chemical drugs, such as adriamycin. However, Pulsatilla chi?nensis was also reported to be possibly the main cause of hemolysis and chronic liver injury. The efforts should be made to deeply investigate the pharmacological actions and underlying mechanisms of Pulsatilla chinensis, with a focus on the anti-cancer efficacy, and develop new drugs based on the components of Pulsatilla chinensis for future utilization in the clinical setting.

Network pharmacological analysis and experimental study of Pulsatilla chinensis against inflammatory injury caused by pneumonia in mice infected with influenza virus FM_1 / 中国中药杂志

Network pharmacology and the mouse model of viral pneumonia caused by influenza virus FM_1 were employed to explore the main active components and the mechanism of Pulsatilla chinensis against the inflammatory injury of influenza virus-induced pneumonia. The components and targets of P. chinensis were searched from TCMSP, and the targets associated with influenza virus-induced pneumonia were searched from GeneCards. The common targets between P. chinensis and influenza virus-induced pneumonia were identified with Venn diagram established in Venny 2.1. The herb-component-disease-target(H-C-D-T) network was constructed by Cytoscape 3.7.2. The above data were imported into STRING for PPI network analysis. Gene Ontology(GO) enrichment and KEGG pathway enrichment were performed with DAVID. BALB/cAnN mice were infected with the influenza virus FM_1 by nasal drip to gene-rate the mouse model of pneumonia. Immunohistochemistry was adopted to the expression profiling of inflammatory cytokines in the lung tissues of mice in the blank group, model group, and P. chinensis group 1, 3, 5, and 7 days after infection. The pathological changes of lung and trachea of mice in blank group, model group, and P. chinensis group were observed with light microscope and scanning electron microscope at all the time points. The network pharmacological analysis indicated that 9 compounds of P. chinensis were screened out, with a total of 57 targets, 22 of which were overlapped with those of influenza virus-induced pneumonia. A total of 112 GO terms(P<0.05) were enriched, including 81 terms of biological processes, 11 terms of cell components, and 20 terms of molecular functions. A total of 53 KEGG signaling pathways(P<0.05) were enriched, including TNF signaling pathway, influenza A signaling pathway, NF-κB signaling pathway, MAPK signaling pathway and other signaling pathways related to influenza/inflammation. In the P. chinensis group, the expression of TNF-α and IL-1 in the lung tissue was down-regulated on the 3 rd day after infection, and that of IL-6 in the lung tissue was down-regulated on the 5 th day after infection. Light microscopy and scanning electron microscopy showed that P. chinensis significantly alleviated the pathological damage of lung and trachea compared with the model group. This study reflects the multi-components, multi-targets, and multi-pathways of P. chinensis against influenza virus-induced pneumonia. P. chinensis may reduce the production of proinflammatory cytokines and mediators and block the pro-inflammatory signaling pathways to alleviate viral pneumonia, which provides reference for future research.

Alpha-hederin induces the apoptosis of oral cancer SCC-25 cells by regulating PI3K/Akt/mTOR signaling pathway

BACKGROUND: Oral cancer is one of the common malignant tumors of the head and neck. However, current treatments have numerous side effects, and drugs from natural sources may have better therapeutic potential. This research investigated the induction of apoptosis by α-hederin (α-HN), a constituent of Pulsatilla chinensis (Bunge) Regel, in the oral cancer cell line SCC-25 and its underlying mechanism. RESULTS: SCC-25 cells were treated with 50, 100, and 200 µmol/L α-HN. Cell proliferation; extent of apoptosis; activities of caspases-3, 8, and 9; and the expression of Bcl-2, Bax, phosphorylated (p)-phosphoinositide 3-kinase (PI3K), p-Akt, and p-mammalian target of rapamycin (mTOR) proteins were determined using the 3-(4,5)-2-thiazole-(2,5)-diphenyl tetrazolium bromide, flow cytometry, caspase activity detection kits, and western blot assays, respectively. The results showed that the proliferation of SCC-25 cells in the α-HN-treated groups decreased significantly, and the inhibitory effect was time and concentration dependent. Compared with cells in the control group, the extent of apoptosis increased significantly, caspase-3 and -9 activities were significantly enhanced, and the Bcl-2 level was lowered and the Bax level was elevated significantly in SCC-25 cells treated with α-HN for 48 h (P b 0.05). The expression of p-PI3K, p-Akt, and p-mTOR was also significantly lower in SCC-25 cells treated with α-HN than that in the control group (P b 0.05). CONCLUSION: These results indicate that α-HN can inhibit proliferation and induce apoptosis of SCC-25 cells and may exert these effects by inhibiting the PI3K/Akt/mTOR signaling pathway.

Alkali hydrolysate of total saponins from Pulsatilla chinensis inhibits hepatic carcinoma cell growth by inducing apoptosis through mitochondrial pathway / 中草药

Objective: To investigate the anti-hepatocarcinoma effect and mechanisms of the alkali hydrolysate of total saponins from Pulsatilla chinensis (PAHS). Methods: MTT assay was used to evaluate the effect of PAHS on proliferation of human liver cancer cell line SMMC-7721 in vitro; Cell morphology was observed by Giemsa staining. The effects of PAHS on apoptosis, cell cycle, and mitochondrial membrane potential of SMMC-7721 were detected by Hoechst 33258 staining and flow cytometry assay; Western blotting was employed to detect the protein expression of Cytochrome C, Caspase-3, cleaved Caspase-3, and Bcl-2 in SMMC-7721 cells. An in vivo liver cancer model was established using ICR mice subcutaneously received H22 hepatoma carcinoma cells to detect tumor growth inhibitory rate. Morphological changes of the tumor samples were observed by HE staining and transmission electron microscope. Results: MTT assay showed that PAHS could inhibit proliferation and increase apoptosis of SMMC-7721 cells in dose-and time-dependent manners in vitro, block the cell cycle in S phase, and decrease mitochondria membrane potential; PAHS could significantly increase the expression of Cytochrome C and cleaved Caspase-3 and decrease the expression of Bcl-2 and Caspase-3. PAHS dramatically decreased the weight of tumor tissue in a dose-dependent manner. Histopathological examination showed that large necrosis area and apoptotic cells were found in tumor tissues of mice in PAHS administrated group. Conclusion: PAHS exerts antitumor activity in vitro and in vivo by inducing apoptosis, and its mechanism is related to regulation of the mitochondrial pathway.

Study on DNA molecular identification of mix samples of five species of Baitouweng medicinal materials based on high-throughput sequencing technology / 药学学报

Traditional Chinese medicine Baitouweng have a long history of application. The pharmacopoeia included dry roots of Pulsatilla chinensis (Bge.) Regel of Ranunculaceae. There are easily confused species in the market circulation, such as P. cernua (Thunb.) Bercht. et Opiz., P. dahurica (Fisch.) Spreng., P. turczaninovii Kryl. et Serg., and P. chinensis (Bge.) Regel var. kissii (Mandl) S. H. Li et Y. H. Huang, etc. In this study, using the method of metagenomics, based on high-throughput sequencing technology, the ITS2 sequence of mixed samples of five species of Baitouweng medicinal materials was sequenced. First, the total DNA extraction of medicinal materials mixing powder, and the ITS2 fragment of total DNA was amplified by PCR. Second, the Illumina MiSeq platform was used to carry out Paired-end sequencing for DNA fragments. Last, using FLASH, QⅡME and GraPhlAn software to arrange and analyze, and clustering analysis with the sequences of uploaded to GenBank by our group in the early stage. The results showed that a total of 53 024 sequences of ITS2 were obtained from the mixed samples, there are 52 295 effective sequences, there are a total of 49 079 of five species of medicinal materials of P. Miller. After the representative sequences and the sequence of uploaded to GenBank by our group in the early stage were clustering analysis, 5 species of Baitouweng medicinal materials were clustered into one branch separately, presenting monophyletic. The results showed that using the high-throughput sequencing technology, using ITS2 sequence as DNA barcode, the mix powder of 5 species of Baitouweng medicinal materials could be effectively identified. It provides a new method and thought for the origin identification of mixed Chinese medicinal materials.

Study on antidiabetic effect of aqueous extract from Pulsatilla chinensis / 中草药

Objective: To study the blood glucose lowering activity of aqueous extract from Pulsatilla chinensis (AEPC) on Streptozocin (STZ)-induced type-2 diabetic rats. Methods: AEPC was obtained through traditional aqueous extraction procedure; Oral glucose tolerance test was employed for the preliminary study of AEPC's blood glucose lowering activity; For further study, STZ-induced neonatal diabetic rat model was used, and the serum SGPT, SGOT, ALP, serum urea, triglycerides, total cholesterol, HDLC, total protein, and serum insulin were tested; The Kunming rats were used for acute toxicity experiments, with the highest dose of 1 000 mg/kg body weight; Insulin release from RIN 5F cells was performed to study the mechanism of AEPC on antidiabetes effect. Results: Oral glucose tolerance test showed AEPC could lower the blood sugar, and the optimal activity was observed at a dose of 20 mg/kg body weight; In neonatal STZ-induced type-2 diabetic rats, treatement with 20 mg/kg AEPC for 20 d resulted in significant blood glucose lowering activity, liver glycogen and serum biological parameters back to normal; The acute toxicity results showed that no obvious toxicity was observed even at 1 000 mg/kg dose; At last, AEPC could promote the secretion of insulin from RIN 5F cells. Conclusion: AEPC shows the anti-diabetes activity on STZ-induced type-2 diabetic rats, which may be the result of its effect on promoting insulin release from pancreatic α-cells.

Regulation of saponins from Pulsatilla chinensis on energy metabolism of Bel-7402 xenograft in nude mice / 中草药

Objective: To investigate the antirumour activity of saponins from Pulsatilla chinensis against the Bel-7402 human hepatocellular carcinoma xenograft in nude mice, and to explore the mechanism of energy metabolism. Methods: The antitumor activity in vivo was estimated using a Bel-7402 xenograft model. The tumor volume was measured, the tumor inhibitory rate was calculated, the levels of lactic acid and ATP were assessed in Bel-7402 xenograft in nude mice, energy metabolic enzymes in Bel-7402 xenograft in nude mice were detected by ELISA assay, and hypoxia inducible factor-1 (HIF-1α) was detected by Western blotting assay. Results: The sapoinins from P. chinensis could inhibit the growth of Bel-7402 xenograft in a dose-depedent manner. The tumor inhibitory rates at the dose of 400, 200, and 100 mg/kg were 42.8%, 31.5%, and 14.9%. They had no side effects on white blood cell (WBC) in blood; The saponins from P. chinensis could decrease the contents of lactic acid and ATP. They also could decrease the levels of glycolytic enzymes phosphofructokinase (PFK), hexokinase-II (HK-II), and pyruvate kinase (PK), while increase the level of succinatedehydrogenase (SDH) that was the key enzyme during tricarboxylic acid cycle. Western blotting indicated that the expression of HIF-1α protein was significantly decreased in Bel-7402 xenograft in nude mice. Conclusion: The saponins from P. chinensis could inhibit the growth of Bel-7402 xenograft in nude mice in vivo, the mechanism is related with regulating energy metabolism through the HIF-1α pathway.

Simultaneous determination of eight saponins in alkali hydrolysate of total saponins from Pulsatilla chinensis by HPLC-ELSD / 中草药

Objective: To develop an HPLC method for the simultaneous determination of eight saponins in alkali hydrolysate of total saponins from Pulsatilla chinensis. Methods: HPLC was performed on a Kromasil C18 analytical column (250 mm × 4.6 mm, 5.0 μm) at 35°C with MeOH-0.2% HCOOH solution as the mobile phase by gradient elution and the step gradients were as follows: 0-30 min, 70%-100% MeOH; The flow rate was 1.0 mL/min; ELSD gasification chamber temperature was 40°C; Gas pressure of carrier gas N2 was 350 kPa. Results: The linear response (the log values of peak areas with corresponding log values of sample introducing amounts) ranges were 0.799-4.568 μg for pulsatilla saponin D, 0.563-6.756 μg for hederagenin 3-O-β-D-glucopyranosyl-(1→4)-α-L-arabinopyranoside, 0.431-2.683 μg for pulsatilla saponin A, 0.894-7.826 μg for hederacolchiside A1, 0.643-7.504 μg for pulsatilla saponin F, 1.351-7.822 μg for oleanolic acid 3-O-β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl - (1→3)-α-L-rhamnopyranosyl-(1→2) - α-L-arabinopyranoside, 0.629-2.515 μg for oleanolic acid 3-O-β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl - (1→2)-α-L-arabinopyranoside, and 0.698-2.794 μg for oleanolic acid 3-O-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside, respectively (n = 5). The average recoveries of the eight saponins were between 99.0% and 101.0%, and RSD values were less than 1.5%. Conclusion: The results demonstrate that the established method has the adequate accuracy and selectivity for the quality control of alkali hydrolysate of total saponins from P. chinensis.

Comparison of Content of Pulchinenoside B4 from Different Sources of Pulsatilla Chinensis / 中国中医药信息杂志

Objective To investigate the quality of Pulsatilla chinensis from different sources on reference of pulchinenoside B4 as the index. Methods Five kinds of Pulsatilla chinensis herbs and cut crude drug were purchased from Chinese herbal medicine market in Bozhou,and Wild Pulsatilla chinensis were collected from Chuzhou. The content of pulchinenoside B4 were determined by HPLC. Results Linear relationship was good within 0.312 5～10 ?g,the regression equation was Y=276 350X -12 180,R2= 0.999 6. Conclusion The differences of pulchinenoside B4 from different sources of Pulsatilla chinensis varied greatly.