Regulation of Dracocephalum Moldavica total flavonoids on foaming and inflammatory response of RAW264. 7 maerophages / 中国药理学通报

Aim To investigate the effeets of Dracocephalum Moldavica total flavonoids (TFDM) on the foaming lipoprotein (ox-LDL), and to further elucidate the mechanism of anti-atherosclerosis (AS) of TFDM. Methods RAW264. 7 maerophages were cultured in vitro and induced to become foam cells by ox-LDL stimulation, and inflammation of mouse monocyte macrophage leukemia cells (RAW264. 7) induced by oxi-dized low density TFDM(25, 50, 100 mg • L"

Total flavonoids of Dracocephalum moldavica L. inhibit oxidative stress against H9c2 cell ischemia-reperfusion injury via VEGF-B/AMPK pathway / 药学学报

Total flavonoids of Dracocephalum moldavica L. (TFDM) is an effective component extracted and isolated from the traditional Uighur medicinal herb Cymbidium fragrans. Cymbidium fragrans has the effects of tonifying the heart and brain, promoting blood circulation and resolving blood stasis, and has been widely used in the treatment of cardiovascular and cerebrovascular diseases for a long time. The purpose of this study was to determine the effect of total flavonoids from Cymbidium fragrans on hypoxia/re-oxygenation (H/R) injury in H9c2 (rat cardiomyocytes) cells and its mechanism. A model (H/R) of hypoxia/re-oxygenation injury in H9c2 cells was established using hypoxia and glucose deprivation for 9 h combined with re-oxygenation and rehydration for 2 h to simulate myocardial ischemia-reperfusion injury. The effects of total flavonoids from Cymbidium fragrans on cell viability, markers of myocardial cell damage, oxidative stress levels, and reactive oxygen radical (ROS) content were investigated, Western blot was used to detect the expression of vascular endothelial growth factor B (VEGF-B) and adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) pathway related proteins. The results showed that the total flavonoids of Cymbidium fragrans significantly increased the viability of myocardial cells after H/R injury, and decreased the content of lactate dehydrogenase (LDH) and creatine kinase isozyme (CK-MB) in the cell supernatant. It significantly reduced malondialdehyde (MDA), increased superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities, and decreased intracellular ROS and nitric oxide (NO) content. Western blot analysis showed that the total flavonoids of Cymbidium fragrans decreased Bax levels in H9c2 cells damaged by H/R and increased Bcl-2 expression. Total flavones of Cymbidium fragrans upregulate VEGF-B/AMPK pathway related proteins VEGF-B, vascular endothelial growth factor receptor 1 (VEGFR-1), neuropilin 1 (NRP-1), peroxisome-proliferator-activated receptor γ coactivator-1α (PGC-1α), phosphorylated adenosine monophosphate activated protein (p-AMPK) and phospho mechanistic target of rapamycin (p-MTOR) levels. The above research results indicate that the total flavonoids of Cymbidium can significantly reduce the H/R injury of myocardial cells, which may be related to the upregulation of VEGF-B/AMPK pathway and inhibition of oxidative stress response.

Identification of components，fingerprint and content determination of the effective parts of Dracocephalum moldavica / 中国药房

OBJECTIVE To provide a reference for comprehensive quality evaluation and control of the effective parts of Dracocephalum moldavica （EPDM）. METHODS A total of 10 batches of EPDM were prepared， and chemical information of EPDM was collected by HPLC-Q-Exactive-MS. EPDM components were identified by literature search， database comparison and manual analysis. HPLC fingerprints of 10 batches of EPDM were established by using the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprint （2004 A edition）； the similarity evaluation and common peak identification were carried out， and the contents of 5 index components were determined by HPLC. RESULTS A total of 11 compounds in EPDM were identified. The fingerprint similarities of EPDM samples from 10 batches were all above 0.96. Among 11 chromatographic peaks， 5 peaks were identified， such as luteolin-7-O-β-D-glucuronide（LG）， apigenin-7-O-glucuronide（APG）， rosmarinic acid（RA）， diosmetin-7-O-β-D-glucuronide（DG）， tilianin（TL） . The results of the quantitative analysis showed that all above 5 components had good linearity （R2≥0.999）， and the average recoveries were in the range of 95.12%-98.37%. The contents of LG， APG， RA， DG， TL were 21.268 3-29.243 9， 6.365 4-7.771 7， 37.327 4-45.671 2， 17.169 9-21.985 9， 66.940 4-91.206 3 mg/g， respectively. CONCLUSIONS The established method of component identification， fingerprint and content determination is stable， feasible and reliable， which is suitable for the comprehensive quality evaluation and control of EPDM.

Effect of effective parts of Dracocephalum moldavica on the necroptosis of HBMECs after OGD/R injury / 药学学报

We investigated the ability of Dracocephalum moldavica (EPDM) flavonoids to protect human brain microvascular endothelial cells (HBMECs) from necroptosis induced by ischemia-reperfusion injury. To mimic the process of cerebral ischemia-reperfusion injury, a necroptosis model was established by treatment with the pan-cysteine aspartic acid protease (caspase) inhibitor Z-VAD-FMK combined with oxygen-glucose deprivation/re-oxygenation (OGD/R) injury using HBMECs. Cell proliferation and cytotoxicity (cell counting kit-8, CCK-8) was used to measure cell viability. A Hoechst33342/PI fluorescent double-staining method was exploited to determine the rate of cell necroptosis. A commercial kit was used to detect lactate dehydrogenase in the cell culture supernate. DCFH-DA probes, calcein AM and JC-1 probes were used to measure changes in ROS production, mitochondrial membrane permeability transformation pore (MPTP) opening and mitochondrial membrane potential (MMP), respectively. Enzyme-linked immunosorbent assay (ELISA) kits were chosen to detect the release of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6). Western blotting was used to detect necroptosis-related proteins. The results show that relative to control group, Z-VAD-FMK combined with OGD/R injury reduced cell viability, increased the necroptosis rate and the levels of LDH and ROS in HBMECs. The MPTP of the model group cells opened and the MMP reduced. TNF-α, IL-1β, and IL-6 levels were significantly elevated. Furthermore, the expression of receptor-interacting protein kinase 3 (RIP3) and mitochondrial phosphoglycerate mutase 5 (PGAM5) was significantly increased, accompanied by an increase of phosphorylated mixed-lineage kinase domain-like protein (p-MLKL)/MLKL. EPDM partially reversed the changes of the above-mentioned factors in HBMECs induced by Z-VAD-FMK plus OGD/R injury. These results indicate that EPDM may protect HBMECs from cerebral ischemia-reperfusion injury by inhibiting the RIP3/MLKL/PGAM5 pathway and MPTP opening to maintain mitochondrial function, thereby providing a scientific basis for the use of EPDM in the treatment of cerebral ischemia-related diseases.

Study on Protective Mechanism of Dracocephalum moldavica Total Flavonoids against Myocardial Ischemia- reperfusion Injury in Rats Based on AMPK/SIRT 1/PGC-1α Signaling Pathway / 中国药房

OBJECTIVE：To s tudy the effects of Dracocephalum moldavica total flavonoids （TFDM）on AMPK/SIRT 1/PGC-1α signaling pathway ，and to explore the mechanism of its protective effect on myocardial ischemia reperfusion injury （MIRI）rats. METHODS：Totally 50 healthy male SD rats were randomly divided into sham operation group ，model group ，TFDM group [ 60 mg/（kg·d），by extract] ，Compound C+TFDM group [ig administration of 60 mg/（kg·d）TFDM+intravenous injection of 250 μg/kg Compound C（AMPK inhibitor ）via tail vein 15 min before reperfusion] ，EX-527+TFDM group [ig administration of 60 mg/ （kg·d）TFDM+ip injection of 5 mg/kg EX- 527（SIRT1 inhibitor）20 min before reperfusion] ，with 10 rats in each group. They were given relevant medicine intragastrically ，once a day ，for consecutive 7 days. After last ig administration ，sham operation group underwent sham operation ，other 4 groups were established MIRI model by ligating left anterior descending coronary artery ， ischemia for 30 min and reperfusion for 2 h. After reperfusion ，the myocardial histopathological changes were observed by HE staining；RP-HPLC method was used to determine the contents of ATP ，ADP，AMP and NAD + in cardiac tissue. mRNA expressions of AMPK ，SIRT1 and PGC- 1α were detected by quantitative real-time PCR assay. Western blotting assay was the expressions of SIRT 1 and PGC- 1α protein in myocardium. RESULTS： Compared with sham operation group ， model group showed myocardial fib ers arranged disorder and horizontal stripes disappearance ，cell swelling burst and necrosis ，and nuclei deformation displacement ；the contents of ATP and NAD+，mRNA expression of AMPK ，SIRT1 and PGC- 1α，protein expression of SIRT 1 and PGC- 1α in cardiac tissue were decreased significantly （P＜0.05 or P＜0.01）；the contents of ADP and AMP ，the phosphorylation level of AMPK protein were increased significantly （P＜0.01）. Compared with model group ，myocardial pathological morphology were improved significantly in TFDM group ；the contents of ATP and NAD + in cardiac tissue ，mRNA expression of AMPK ，SIRT1 and PGC- 1α，the phosphorylation level of AMPK protein ，the protein expression of SIRT 1 and PGC- 1α were increased significantly（P＜0.05 or P＜ 0.01），while the contents of ADP and AMP were decreased significantly （P＜0.01）. Compared with TFDM group ，improvement effects of Compound C + TFDM group and EX- 527 + TFDM group on above indexes were reversed （P＜0.05 or P＜0.01）. CONCLUSIONS：TFDM may play a protective role on myocardium by activating AMPK/SIRT 1/PGC-1α signaling pathway and regulating energy metabolism.

Study on Inhibitory Activity of Dracocephalum moldavica Extracts to Clinical Pathogenic Bacteria and Its Bioin- formatics / 中国药房

OBJECTIVE：To study inhibitory activity of different extracts of Dracocephalum moldavica to clinical pathogenic bacteria，and to excavate its possible antibacterial mechanism. METHODS ：After extraction by 65％ ethanol and extraction by petroleum ether ，dichloromethane，ethyl acetate ，n-butanol，the different polar fractions of D. moldavica were obtained . Taking Klebsiella pneumoniae ，Staphylococcus aureus and other clinical multiple resistant pathogens as objects，the diameter of inhibition zone of different extraction fractions was measured by paper diffusion method ，and the antibacterial active fraction was screened. The minimal inhibitory concentration （MIC）of antibacterial active fraction to common clinical pathogens was determined by agar dilution method ；the growth curve of MRSA was drawn by turbidimetric method. The differentially expressed protein between antibacterial active fraction group and control group was screened by PEAK ® Q 8.5 software，and the gene ontology （GO）analysis and KEGG signaling pathway enrichment analysis were carried out by Blast 2 GO and KOBAS 3.0 online software. RESULTS ： Petroleum ether ，dichloromethane，ethyl acetate and n-butanol fraction of D. moldavica had no significant inhibitory effect on Gram-negative bacteria. The ethyl acetate fraction of D. moldavica had antibacterial activity in varying degrees against several kinds of Gram-positive bacteria as Staphylococcus aureus ，S. epidermidis；the diameter of inhibition zone was 10-16 mm，which was the active fraction. MICs of ethyl acetate fraction to S. aureus ，S. epidermidis and S. hominis were all 0.781 3 mg/mL；MIC to S. saprophyticus was 0.390 7 mg/mL；MICs to S. saprophyticus and standard strain of S. aureus were both 1.562 5 mg/mL. The 1， 2 times MIC of ethyl acetate could inhibit the growth of MRSA ，and the inhibitory activity increased with the increase of dose. A total of 300 differentially expressed proteins were screened （P＜0.01），of which 239 were up-regulated and 61 were down-regulated. The differentially expressed proteins were （No.81260478，No.816- mainly concentrated in cell sites such as cells ，cellular com- 60048） ponent，etc.，and in metabolic process such as cell process ， biological process and molecular functions such as catalytic activity，protein binding ，etc. They were mainly concentratedwang.zhanli@hotmail.com in microbial metabolism in different environments ，fructose and mannose metabolism signaling pathway （P＜0.05）. CONCLUSIONS ：The ethyl acetate fraction of D. Moldavica is the antibacterial active fraction ，and its activity may be related to the microbial metabolism and cell glycometablism.

Research on separation and purification of extract from Dracocephalum moldevica with macroporous resins / 中草药

Objective: To investigate the technology for the separation and purification of extract in Dracocephalum moldevica (EDM) by macroporous resin. Methods: Static and dynamic adsorption-desorption were used to select the best one from seven different type macroporous resins; With the content of total flavonoids, tilianin, luteolin-7-O-β-D-glucuronide, and rosmarinic acid as indexes, the purification technology parameters of EDM were optimized. Results: HPD600 resin showed the best purifying profile, its optimum technology conditions were as follows: The optimum concentration of the sample liquid was 0.08 g/mL equivalent to raw material, the resin column diameter-height ratio was 1:9, the amount of used adsorption was 0.32 g dried medicinal herb/mL resin, sample flow rate was 1.5 BV/h, and adsorption time was 12 h. In the course of elution, the resin column chromatography was eluted with 6 BV of 70% ethanol after removing impurities with 4 BV of water by flow rate of 1.5 BV/h. The contents of total flavonoids, tilianin, luteolin-7-O-β-D-glucuronide, and rosmarinic acid were more than 53%, 5.5%, 4.7%, and 2.5%. Conclusion: Macroporous resin HPD600 is suitable to separate and purify EDM.

Basic physicochemical properties of extract from Dracocephalum moldavica / 中草药

Objective: Taking luteolin-7-O-β-D-glucuronide, rosmarinic acid, and tilianin as indexes, the concentration of extracts from Dracocephalum moldavica (EDM) and their index components in various media, surfactant solution, and apparent oil/water partition coefficient (P) was detected in different pH values, then the stability of EDM in artificial gastrointestinal fluid was investigated. This research would give the reference for the selection and preparation of further novel formulation. Methods: Apparent solubility of EDM in various media and surfactant solution was determined by precipitation method and a shake flask method was used to determine the P of octanol-water/phosphate buffer salt, the stability of EDM in artificial gastrointestinal fluid was investigated. HPLC was adopted to determine the concentration of the index components with Shim-pack ODS column (250 mm×4.6 mm, 5 μm), mobile phase of acetonitrile (A)-0.5% formic acid aqueous solution (B) for gradient elution (0-30 min, 15% A; 30-55 min, 15%→25% A; 55-80 min, 25%→35% A) as the mobile phase at a flow rate of 1.0 mL/min. The detection wavelength was set at 324 nm and the column temperature was maintained at 35℃. Results: At 37℃, the equilibrium solubility of the three index components in acid buffer solution increased obviously in water, but in alkaline the buffer solution of the equilibrium solubility decreased with pH increasing. In 32 g/L sodium dodecyl sulfate solution, the equilibrium solubility of luteolin, rosmarinic acid, and tilianin increased to 1679.61, 1249.2, and 2765.27 μg/mL. The P of luteolin-7-O-β-D-glucuronide, rosmarinic acid, and tilianin were 0.1731 (lgP=-0.7618), 0.068 4 (lgP=-1.1650), and 1.0829 (lgP=0.0346), and increased as pH rising. EDM in artificial gastrointestinal fluid was stable (except luteolin). Conclusion: The methods can be used to determine the apparent solubility, the P value of the extracts and their index components; The EDM in artificial gastrointestinal fluid is stable. The research would give the reference for the selection and preparation of the further novel formulation.

Simultaneous determination the contents of phenylpropanoids and flavonoids in Dracocephalum moldavica L. extract / 中国药学杂志

OBJECTIVE: To establish an HPLC method for simultaneous detemination the contents of rosmarinic acid (phenylpropanoids), luteolin-7-O-β-D-glucuronide and tilianin (flavonoids) in Dracocephalum moldavica L. Extract. METHODS: The samples were separated on Purospher® STARLP RP-18 endcapped column(4.6 mm × 250 mm, 5 μm) by gradient elution with acetonitrile (A)-0.5% formic acid solution(B) (0-28 min, 20% A; 28-55 min, 20%→28% A; 55-80 min, 28%→30% A)as the mobile phase at a flow ratio of 1.0 mL · min-1. The detection wavelength was set at 330 nm and the column temperature was maintained at 35℃. RESULTS: The calibration curves of rosmarinic acid, luteolin-7-O-β-D-glucuronide, tilianin were in good linearity over the ranges of 2.184-21.84 μg · mL-1 (r =0.999 6), 3.14-31.84 μg · mL-1(r =0.999 6), 7.9-39.5 μg · mL-1(r =0.999 5) respectively. The average recoveries were 99.62%, 99.64% and 100.12% with RSD values of 1.27%, 1.05% and 1.09%. CONCLUSION: The method is reliable, simple, and accurate, and can be used for the comprehensive quality control of Dracocephalum moldavica L. extract.

The Identification of Uygur Medicine Dracocephalum Moldavica L. and Its Sibling by ITS2 Sequence / 世界科学技术-中医药现代化

Objective: To establish the DNA barcode identification method of Uygur Medicine Dracocephalum mol-davica L. Methods: D. moldavica L. and its ten sibling species of twenty five samples was amplified by ITS2 se-quence, after sequencing and comparing the intraspecific and interspecific variation, we using K2P and NJ meth-ods to analysis the their relationship, then compare the secondary structure. Results: D. Moldavica (KF041160, KF041163, KF041168, KF041169) from Xinjiang without variation in intraspecies, but there are two 2 variation sites in D. moldavica (AY506659) from GenBank. By NJ method, D. moldavica can be distinguished with their sibling species. Also, D. nutans L., D. bipinnatum Rupr. and D. integrifolium Bge. can be distinguished with oth-er sibling species. Conclusion: ITS2 barcode sequence was able to identify D. moldavica and its sibling species, which provides an effective way for the molecular identification of Uygur Medicine D. moldavica.