Rapid discovery of antioxidants in Salvia miltiorrhiza by UPLC-DPPH- PDA-ESI-TOF/MS online technique / 中草药

Objective: To establish a method for rapid screening and identifying antioxidants in natural products by ultra high performance liquid chromatography-(1,1-dipheny l-2-trinitrophenylhydrazine)-photo diode array-electrospray-time-of-flight mass spectrometry (UPLC-DPPH-PDA-ESI-TOF/MS) and apply it to the rapid discovery of antioxidants from Salviamiltiorrhiza extracts. Methods: The Waters ACQUITY UPLC system and a Waters ACQUITY UPLC HSS T3 column (100 mm×3.0mm,1.8 μm; Waters Technologies, USA) were used to elute the sample at a low flow rate of 0.35 mL/min, which could achieve the rapid separation of S. miltiorrhiza extracts. The elution was joined with DPPH solution (0.3 mL/min) and reacted in the PEEK coil, and then flew into the detector. The anti-oxidation components in S. miltiorrhiza extracts were quickly screened by comparing the chromatograms at 517 nm and 254 nm. Results: The UPLC technology was adopted to realize the efficient separation of samples at low volume flow rate, which avoided the baseline noise caused by high volume flow rate, significantly improved the resolution and sensitivity of the online screening method, and reduced the solvent consumption.The results showed that 31 antioxidant active compounds were screened from S. miltiorrhiza extracts, and 11 of them were identified by PAD-ESI-TOF/MS. Activity verification experiments showed that caffeic acid, salvianolic acid B, salvianolic acid A and danshensu had better antioxidant activity. The IC50 value ofcaffeic acid was 29.00 μmol/L, which was significantly better than the positive control (vitamin C). Conclusion: The method is rapid and efficient, which provides methods and technical support for the rapid discovery of antioxidant components from complex natural products.

Effect of influencing factors on stability of Guanxinning Injection / 中草药

Objective: To identify the influencing factors (light, temperature, etc) of Guanxinning Injection (GXNI) in the storage and transportation process, and lay the foundation for the storage and transportation of GXNI. Methods: Taking the content of sodium danshensu, protocatechuic aldehyde, ferulic acid, rosmarinic acid, and salvianolic acid B as quantitative indicators, combined with the common peak similarity analysis, principal component analysis, and relative content comparison in the fingerprint to evaluate the effect of different influencing factors on the main chemical composition of GXNI. Results: After low temperature treatment, the content of four compounds, such as senkyunolide I and senkyunolide H, were increased, the content of five compounds, such as vanillic acid and vanillin aldehyde, were reduced, the content of 13 compounds, such as protocatechuic aldehyde and ferulic acid, showed no significant changes. Under the freezing-thawing conditions, the content of four compounds, such as 5-hydroxymethylfurfural, were increased, the content of seven compounds, such as vanillin acid and chlorogenic acid, was reduced, the content of 11 compounds, such as sodium danshensu and ferulic acid, showed no significant changes. Under high temperature, the content of seven compounds, such as sodium danshensu and protocatechuic aldehyde, were increased, the content of 11 compounds, such as ferulic acid and rosmarinic acid, were reduced, the content of four compounds, such as chlorogenic acid and caffeic acid, showed no significant changes. Under strong light conditions, the content of two compounds, such as senkyunolide H, were increased, the content of 16 compounds, such as ferulic acid and salvianolic acid B, were reduced, the content of four compounds, such as sodium danshensu and protocatechuic aldehyde, showed no significant changes. Conclusion: High temperature and strong light will affect the components of GXNI, and some compounds will also be affected under freezing-thawing conditions. Thus, the GXNI should be stored under low temperature, but higher than 0 ℃.

Construction of aseptic cultural system of Salvia miltiorrhiza and determination of active components by HPLC analysis / 中草药

Objective: The regeneration system of Salvia miltiorrhiza was constructed and contents of active components of the sterile materials were determined, so as to provide the basis for the rapid propagation and secondary metabolism regulation of S. miltiorrhiza. Methods: The optimum hormone ratio for inducing callus was screened by orthogonal test. The buds and roots induction were conducted to estabilish the tissue culture system. The contents of effective components were evaluated by HPLC analysis. Results: The suitable medium for callus induction was MS+6-BA (2.0 mg/L)+NAA (1.0 mg/L)+2,4-D (0.5 mg/L). The preferred enrichment medium of adventitious bud induction was MS+6-BA (2.0 mg/L)+NAA (1.0 mg/L). And rooting medium was 1/2 MS+NAA (0.5 mg/L). Seven active components in aseptic seedlings, callus, and regenerated seedlings could be detected with significant differences in different aseptic materials (P < 0.05). The contents of rosmarinic acid and salvianolic acid B were higher than the others. Conclusion The culture system of S. miltiorrhiza was successfully established, and vigor regenerated seedlings were also obtained. The accumulation of active components in the three sterile materials showed difference, laying a foundation for further study in S. miltiorrhiza.

Investigation of Systematic Stability of Danshen Capsules / 中国实验方剂学杂志

Objective:To prepare Danshen extract and Danshen capsules, and study on its stability under high humidity, high temperature and light conditions.To determine the hygroscopicity of Danshen capsules and its intermediates. Method:The contents of danshensu, rosmarinic acid, lithospermic acid and salvianolic acid B in Danshen extract and Danshen capsules were determined by UPLC, the detection wavelength was 280 nm and the mobile phase was 0.05% phosphoric acid aqueous solution(A)-acetonitrile(B)for gradient elution(0-2 min, 93% -79.2% A;2-6 min, 79.2% -75% A;6-9 min, 75% -65% A;9-10.5 min, 65% -10% A;10.5-11 min, 10% -93% A).The critical relative humidity(CRH) was calculated and hygroscopic isothermal curve was drawn by determining the moisture absorption and weight gain of Danshen capsules and its intermediates. Result:The fluctuation of contents of these four phenolic acids in Danshen extract and Danshen capsules was within±10% and no significant change trend after placing at temperature of 40℃, relative humidity of 75% and 92.5%, light intensity of (4 500±500) Lx for 10 days.The change rates of danshensu in Danshen extract and Danshen capsules under 60℃ were 47.45% and 32.24%, and change rates of salvianolic acid B were -6.39% and -9.64%, respectively.The hygroscopic investigation showed that CRH of starch-based pellets was 58.5%, CRH of Danshen extract was 72.34%, CRH of coated pills was 72.85%, and CRH of Danshen capsules was 73.55%. Conclusion:High temperature has effect on stability of phenolic acids in Danshen extract and Danshen capsules, in order to ensure the quality of them, high temperature environment should be avoided.In order to prevent excessive moisture absorption of Danshen capsules and its intermediates, the relative humidity in the production and storage environment should be controlled below the corresponding CRH.

Quality Evaluation of Standard Decoction of Wine-processing Salviae Miltiorrhizae Radix et Rhizoma / 中国实验方剂学杂志

Objective:To establish the quality control method of the wine-processing Salviae Miltiorrhizae Radix et Rhizoma standard decoction,in order to provide reference for the quality evaluation of the wine-processing Salviae Miltiorrhizae Radix et Rhizoma formula granules and other related products. Method:Totally 15 batches of representative wine-processing Salviae Miltiorrhizae Radix et Rhizoma pieces were collected to prepare the standard decoction, establish the HPLC fingerprint and determine the content of five components(sodium danshensu,caffeic acid,rosmarinic acid,lithospermic acid,salvianolic acid B). The main common peaks in fingerprint were identified to define the main chemical constituents in the standard decoction, the parameters,such as dry extract rate,transfer rate of index components and pH of the standard decoction were calculated, and the comprehensive evaluation index was established to evaluate the stability of the preparation process. Result:The main component standard decoction was phenolic acids. The concentrations of five components(sodium danshensu,caffeic acid,rosmarinic acid,lithospermic acid,salvianolic acid B)in the standard decoction were 0.21%-0.37%,0.03%-0.10%,0.08%-0.18%,0.07%-0.13%,2.68%-4.34%, the dry extract rates of standard decoction were 71.8%-85.4%,50.0%-71.4%,68.2%-81.0%,66.7%-84.6%,67.5%-79.6%,the transfer rates were between 45.1%-55.3%,and pH value was between 5.91-6.05. The fingerprint similarities of the 15 batches of standard decoction with reference fingerprints were>0.98,the fingerprint showed 12 common peaks,7 of which were considered to be sodium danshensu,protocatechuic aldehyde,caffeic acid,ferulic acid,rosmarinic acid,lithosperic acid and salvianolic acid B. Conclusion:The established systematic evaluation for the quality of wine-processing Salviae Miltiorrhizae Radix et Rhizoma standard decoction is stable and feasible,and provides a reference for the quality control of relevant preparations of wine-processing Salviae Miltiorrhizae Radix et Rhizoma decoction.

Protective effects of compatibility of effective constituents in Salvia miltiorrhiza and Ligusticum chuanxiong on hippocampal neurons injured by oxygen glucose deprivation / 中草药

Objective: To observe the protective effect of Salvia miltiorrhizae and Ligusticum chuanxiong effective constituents: danshensu, protocatechuic aldehyde, ligustrazine, and ferulic acid combination on primary cultured hippocampal neurons injured by oxygen glucose deprivation, and find out an optimized combination. Methods Primary cultured rats hippocampal neurons waschosen as research objects by adopting immunohistochemistry of the neuron-specific enolase IgG to authenticate, then the OGD model of the hippocampal neurons injured by oxygen glucose deprivation was established. The non-cytotoxic dose range of danshensu, protocatechuic aldehyde, ligustrazine, ferulic acid, and nimodipine was studied by MTT method. The compatibility of components was arranged by L9 (34) orthogonal design. Primary cultured rats hippocampal neurons was divided into 12 groups: control group, model group, Nimodipine positive control group, and orthogonal design 1-9 group. The activity of LDH was measured by colorimetry, the activity of SOD was tested by WST-1 and the levels of MDA were examined by TBA. The levels of TNF-α, IL-1β, and IL-6 in cell culture supernate were examined by ELISA, the apoptosis of hippocampal neurons was detected by Fluorochrome Hoechst33258 staining and the cell early apoptosis rate was detected with flow cytometry. The results of orthogonal test was analyzed by using range analysis method. Results The orthogonal compatibility of reagents played significant roles against the hypoxia damage of hippocampal neurons, improved the cellular morphology obviously, reduced the activity of LDH, increased the activity of SOD and decreased the content of MDA significantly, inhibited the release of TNF-α, and reduced the content of IL-1β and IL-6, reduced the apoptosis of cells apparently. The effect of active ingredients of S. miltiorrhiza and L. Chuanxiong on LDH activity was danshensu > ligustrazine > protocatechuic aldehyde > ferulic acid, and the best combination was danshensu (120 μg/mL), protocatechuic aldehyde (120 μg/mL), ligustrazine (80 μg/mL), and ferulic acid (20 μg/mL). The effect on SOD activity was ferulic acid > ligustrazine > danshensu > protocatechuic aldehyde. The best combination was danshensu (120 μg/mL), protocatechuic aldehyde (120 μg/mL), ligustrazine (80 μg/mL), and ferulic acid (40 μg/mL). The order of influence on MDA content was danshensu > protocatechuic aldehyde > ferulic acid > ligustrazine, and the best combination was danshensu (60 μg/mL), protocatechuic aldehyde (60 μg/mL), ligustrazine (80 μg/mL), and ferulic acid (20 μg/mL). The effect on TNF-α content were: ligustrazine > protocatechuic aldehyde > danshensu > ferulic acid, and the best combination was danshensu (60 μg/mL), protocatechuic aldehyde (60 μg/mL), ligustrazine (40 μg/mL), and ferulic acid (10 μg/mL). The order of influence on the content of IL-1β was ligustrazine > ferulic acid > danshensu > protocatechuic aldehyde, and the best combination was danshensu (30 μg/mL), protocatechuic aldehyde (30 μg/mL), ligustrazine (80 μg/mL), and ferulic acid (20 μg/mL). The effect on the content of IL-6 was protocatechuic aldehyde > ligustrazine > ferulic acid > danshensu, and the best combination was danshensu (120 μg/mL), protocatechuic aldehyde (120 μg/mL), ligustrazine (80 μg/mL), and ferulic acid (10 μg/mL). The effect on the early apoptosis rate was ferulic acid > protocatechuic aldehyde > ligustrazine > danshensu, and the best combination was danshensu (60 μg/mL), protocatechuic aldehyde (30 μg/mL), ligustrazine (20 μg/mL), and ferulic acid (40 μg/mL). Conclusion The protective mechanism of the effective constituents of S. miltiorrhizae and L. chuanxiong were relied on reducing the oxidative damage, reducing inflammation damage, and antagonizing cell apoptosis. According to the experimental results, we need to change the prescription ratio and guide clinical medication for different clinical courses.

Based on fingerprint and multi-index components to evaluate difference of Salvia miltiorrhiza between microwave extraction method and traditional extraction method / 中草药

Objective: To establish the UPLC fingerprint of Salvia miltiorrhiza microwave extraction method and traditional extraction method and identify the main chromatographic peaks, the chemometrics method was used to compare the chemical differences of different extraction methods. Methods: The separation was performed on a chromatographic Waters ACQUITY UPLC BEH sheid C18 column (100 mm × 2.1 mm, 1.7 μm), with acetonitrile (A)-0.1% aqueous formic acid (B) as the mobile phase gradient elution. The flow rate was 0.3 mL/min, the detection wavelength was 270 nm, and the fingerprints of traditional extraction method and microwave extraction method of S. miltiorrhiza; The content of eight index components was determined, and SPSS 22.0 software was used for principal component analysis and t-test analysis to further evaluate the difference between microwave extraction method and traditional extraction method. Results: The traditional extraction method and the microwave extraction method respectively calibrate 16 and 17 common peaks, and the content of eight index components was different. In the similarity evaluation, the fingerprints of different extraction methods of S. miltiorrhiza were compared, and the similarity was > 0.945. The content of the index components and the comprehensive mass fraction of the index components in the microwave extraction method were higher than the traditional extraction method, and the mean value of the eight index components showed that the microwave extraction method was higher than the traditional extraction method. Compared with the traditional extraction method, there were significant differences in the content of salvianic acid A sodium, protocatechuic aldehyde, rosmarinic acid, lithospermic acid, salvianolic acid B, cryptotanshinone, and tanshinone IIA (P < 0.05, 0.01). Conclusion: Through the comparison of fingerprints and chemometric methods, the overall chemical composition of S. miltiorrhiza under different extraction methods is similar. The content of chemical components is different by t-test, and the content of S. miltiorrhiza microwave extraction method is higher than traditional extraction method. It is concluded that the microwave extraction method has certain advantages compared with the traditional extraction method. The establishment of this method can lay a foundation for the wide application of microwave extraction method in the field of traditional Chinese medicine.

Chemical comparison of Salvia miltiorrhiza phloem and xylem based on fingerprint analysis and quantitative analysis of multi-components / 中草药

Objective: To comprehensively compare and evaluate the composition of Salvia miltiorrhiza phloem and xylem samples based on fingerprint analysis and quantitative analysis of multi-components. Methods: In the present study, an accurate and reliable fingerprint approach was developed using high performance liquid chromatography for chemical comparison ofphloem and xylem samples of S. miltiorrhiza. Furthermore, eight bioactive compounds including four salvianolic acids and four tanshinones in S. miltiorrhiza phloem and xylem samples were simultaneously quantified. Moreover, chemometrics methods were performed to compare and discriminate the phloem and xylem based on the quantitative data. Results: The specific fingerprints of phloem and xylem of S. miltiorrhiza were obtained, and a total of 10 common peaks were marked. The quantitative and chemometrics analysis results indicated the content of chemical components in phloem and xylem samples of S. miltiorrhiza were notably different. Obviously, the content of tanshinones were notably different between phloem and xylem samples. The content of tanshinones were significant higher in phloem compared with xylem in S. miltiorrhiza. Conclusion: The fingerprint analysis and quantitative analysis of multi-components could be a well-acceptable strategy for chemical comparison of S. miltiorrhiza phloem and xylem.

The clinical effect of citicoline sodium combined with Shenxiong glucose in the treatment of senile hypertensive cerebral infarction in the elders / 中国医师杂志

Objective To investigate the clinical therapeutic effect of citicoline sodium combined with Shenxiong glucose in the treatment of senile hypertensive cerebral infarction in the elders.Methods 80 elderly patients with senile hypertensive cerebral infarctiontreated in our hospital were selected and randomly divided into the single treatment group and the combination treatment group,40 cases in each group.Both groups received the routine treatment.The single treatment group additionally received Shenxiong glucose injection (100 ml/d),while patients in the combination treatment group additionally received Shenqiong glucose injection combined with citicoline sodium intravenous infusion therapy (0.5 g/d),both groups were treated for 2 weeks.The levels of serum inflammatory factors,the neurological deficit score,the cognitive function score were compared and analyzed before and after treatment between two groups.Results After systemic treatment,the blood pressure and blood lipid levels of two groups were significantly improved,but there was no significant difference between the two groups (P ＞ 0.05);The serum levels of interleukin (IL)-6,IL-8 and tumor necrosis factor-α (TNF-α) of the combination treatment group improved more significantly (P ≤0.05).After treatment,the oxidative stress indexes were significantly improved in the two groups (P ≤ 0.05).The content of malondialdehyde (MDA) was decreased,while the superoxide dismutase (SOD) activity and nitric oxide (NO) content were increased significantly (P ≤ 0.05);and the improvement degree in the combination treatment group was better than in the single treatment group (P ≤0.05).The degree of improvement in the Modified Edinburgh-Scandinavia Stroke Scale (MESSS) and Hasegawa Dementia Scale (HDS) scores of the combination treatment group was more significant than those in the single treatment group (P ≤ 0.05).The total effective rate of the combination treatment group was 92.5％,which was significantly higher than that of the single group (75.0％),with statistically significant difference (P ≤ 0.05).No obvious adverse reactions happened in two groups during treatment.Conclusions Combination of citicoline sodium and shenxiang glucose on the basis of routine treatment can significantly reduce oxidative stress and inflammation levels,promote the recovery of neurological and cognitive functions,and improve the clinical efficacy and safety.It is worth popularizing and applying in the clinical treatment of senile hypertensive cerebral infarction.

Identification of metabolites of Danshensu in rats / 中国中药杂志

To identify the metabolites of Danshensu in plasma and urine in rats by using UHPLC-LTQ-Orbitrap method. After oral gavage of Danshensu CMC-Na suspension in SD rats, urine and plasma samples were collected and processed by solid phase extraction. ACQUITY UPLC BEH C₁₈ column (2.1 mm×100 mm, 1.7 μm) was utilized, with 0.1% formic acid (A)-acetonitrile (B) solution as the mobile phase for gradient elution. Negative electrospray ion mode based data-acquisition method was established to collect the mass spectrometry data of biological samples. As a result, Danshensu and 21 Danshensu Ⅰ phase and Ⅱ phase metabolites were finally identified according to the accurate mass measurements, mass fragmentation behaviors and comparing with the reference standards. The main metabolic pathways included dehydration, methylation, glucuronide conjugation, sulfate conjugation and their composite reactions. Consequently, our study expounded metabolites of Danshensu in rats based on UHPLC-LTQ-Orbitrap method and provided a reference for further researches on therapeutic material basis and mechanism of Danshensu.

Effect of 60Co-γ Ray Irradiation on the Four Components in Tongmai Granules / 中国药师

Objective: To study the effect of 60Co-γ ray irradiation on the contents of danshensu,protocatechuic aldehyde,puerarin and salviamolic acid B in Tongmai granules by HPLC. Methods: An Agilent ZORBAX Eclipse XDB C18(2) column (250 mm × 4. 6 mm,5 μm) was adopted and the wavelength of UV detection was 280 nm at the flow rate of 1. 0 ml·min-1. The mobile phase consisted of 0. 1% phosphoric acid (B) and acetonitrile (A) with gradient elution, and the column temperature was 35℃. Tongmai granules were irradiated by 60Co-γ ray respectively at 0, 2, 5,10 and kGy,the contents of the active ingredients were compared before and after the irradiation. Results: The linear range of danshensu,protocatechuic aldehyde,puerarin and salviamolic acid B was 0. 098-4. 925 μg, 0. 028-1. 411 μg, 0. 378-18. 882 μg and 0. 218-10. 888 μg, respectively. The average recovery was 99. 8% , 97. 7% , 99. 9% and 99. 9% , respectively. When the radiation dose was not more than 2 kGy, the contents of the five components did not change significantly (P>0. 05). After 5 kGy radiation, the contents of protocatechuic aldehyde and salviamolic acid B were signifi-cantly different (P <0. 05). Conclusion: The dose of 60Co ray should be controlled not more than 2 kGy, and the sterilization method is safe and effective for Tongmai granules.

Optimization of extraction and purification technology for phenolic acids and flavonoids in stems and leaves of Salvia miltiorrhiza / 中草药

Objective To study the optimum condition for the extraction and purification of phenolic acids and flavonoids in the stems and leaves of Salvia miltiorrhiza. Methods The contents of phenolic acids and flavonoids were determined by ultra performance liquid chromatography (UPLC). The extraction process was evaluated by single factor test and orthogonal design with yield of dry extract and the content of phenolic acids (including danshensu, protocatechuic acid, protocatechualdehyde, caffeic acid, salvianolic acid B, rosmarinic acid, and lithospermic acid) and flavonoids (including rutin, isoquercitrin, kaempferol-3-O-rutinoside, and astragalin) as index. The effects of extraction method, extraction solvent, ratio of material to liquid, extracting time, and extracting times on the extraction of stems and leaves of S. miltiorrhiza were investigated. Macroporous adsorption resin was used to purify the sample, and the purification process parameters were investigated to determine the optimum purification process. Results The optimum condition for the extraction of the stems and leaves of S. miltiorrhiza is that eight times of 50% ethanol for three times reflux extraction and 1 h for each time and AB-8 macroreticular resin was selected for the purification. Optimum process was as following:The concentration of sample solution was 1.0 g/mL; The loading quantity of the sample was 0.15 g dried extract of per gram; The resin column chromatography was eluted with 3 BV of 40% ethanol. Under these conditions, the total purity of phenolic acids and flavonoids could reach 40.83%. Conclusion The optimum technology is stable and feasible for the extraction and purification of phenolic acids and flavonoids in the stems and leaves of S. miltiorrhiza, and can provide reference for further development and utilization.

Quality marker research of monarch herb Salvia miltiorrhiza in Compound Danshen Dripping Pills / 中草药

To explore the chemical ingredients of Salvia miltiorrhiza in Compound Danshen Dripping Pills (CDDP) based on the concept of quality marker (Q-marker). Methods The main salvianolic acids of S. miltiorrhiza and CDDP were determined by UPLC method. According to the extraction process of CDDP chemical transformation of salvianolic acids, including lithospermic acid, salvianolic acids B, E, U, and T were studied. Results Salvianolic acid B and rosmarinic acid, more than 90.0% and 5.0% of total salvianolic acid, were the main salvianolic acids in S. miltiorrhiza. But in CDDP, eight salvianolic acids (danshensu, protocatechuic aldehyde, salvianolic acids A, B, D, T, and U) were the main salvianolic acids. And the contents of danshensu and protocatechuic aldehyde were higher than the other six salvianolic acids. Through the study on chemical transformation of salvianolic acids, it was proved that lithospermic acid, salvianolic acids B, E, U, and T could transform into other salvianolic acids with smaller molecular weight, and danshensu and protocatechuic aldehyde were the main end products. Conclusion It is scientific to choose salvianolic acid B as the Q-marker of salvianolic acids of S. miltiorrhiza, and danshensu as the primary Q-marker. During the preparation of CDDP, salvianolic acids from S. miltiorrhiza have chemical chages, eight main salvianolic acids of which have been produced. The contens of danshensu and protocatechuic aldehyde are the highest in the eight salvianolic acids. From the chemical composition level, it is scientific and reasonable to choose danshensu as the Q-marker of monarch herb S. miltiorrhiza in CDDP.

Pharmacological mechanism of main ingredients of Guanxinning Injection treating cardiovascular diseases by computer simulation study / 中草药

Objective: To study network pharmacology mechanism of main ingredients of Guanxinning Injection treating cardiovascular diseases by computer simulation. Methods: Danshensu, salvianolic acid B, protocatechuic aldehyde, rosmarinic acid, tanshinone IIA, lithospermic acid, ferulic acid, senkyuno lide I, ligustrazine, butylphthalide, and ligustilide from Guanxinning Injection were used to predict and screen the targets by reverse molecular docking technology, and were used to study pharmacological mechanism of main ingredients of Guanxinning Injection treating cardiovascular diseases relying on protein-protein interaction network, GO biological processes enrichment, and KEGG signaling pathways enrichment. Results: There were total 11 active ingredients acting INS, Akt1, TNF, MAPK1, ESR1, F2, SERPINE1, and 142 cardiovascular diseases related targets in Guanxinning Injection. These targets were mainly involved in steroid hormone mediated signaling pathway, glutathione metabolic process, positive regulation of smooth muscle cell proliferation and other biological processes, and regulation of coagulation, inflammation and immune, endocrine, and 20 relevant pathways. Conclusion: Guanxinning Injection participated in the treatment of the cardiovascular diseases by anti-inflammatory, anti-oxidant, anticoagulation, promoting fibrinolysis, regulating hormones, and maintaining cardiovascular homeostasis.

Effect of Danshensu borneol esteron P-glycoprotein expression level in rat brain / 中国药理学通报

Aim To investigate the relationship between the brain targeting effect and P-glycoprotein(P-gp)expression level of Danshensu borneol ester(DBE)and the combination use of sodium Danshensu and borneol(SDSS-B).Methods The liquid chromatography mass spectrometry(LC-MS)method was applied to investigate the accumulation of Danshensu(DSS)in rat brain tissues after intravenous injection of DBE,SDSS-B and SDSS.Also their effect on regulating the expression level of P-gp in rat hippocampus was investigated using Western blot.Results The brain targeting effect of DBE,SDSS-B was qualitatively analyzed through the brain distribution of DSS,and the result was DBE(SDSS-B)>SDSS(P0.05 at 5,15,45,60 min,vs control group).Conclusion An attenuated expression level of P-gp can be realized by DBE and SDSS-B,which is advantageous to their brain targeting.

Optimization of the extraction technology of ganglouyuchuang lotion by orthogonal test / 医学研究生学报

Objective Ganglouyuchuang lotion was used to dress the wound for Anal fistula in Crohn's disease, control inflammation and promote wound healing.This paper studied the extraction techonology of ganglouyuchuang lotion.Methods Four factors of ganglouyuchuang lotion, including volume of water, extraction time, extraction times and liquid ratio, were studied by the orthogonal test, and three levels were selected for each factor.The content of sodium danshensu, the active component contained in Chinese herbal medicine, was regarded as evaluating indicator, and the content of Danshensu Sodium was determined by HPLC.The water extraction and alcohol precipitation technology of ganglouyuchuang lotion was optimized according to the results of measurement.Results The optimum extraction technology of ganglouyuchuang lotion was as follows: four herbs, including Salvia miltiorrhiza, Radix Astragali, Radix sanguisorbae and Senecio were added with8 times amount of water overnight and decocted 3 times with 2 h, 1.5 h, and 1.5 h respectively, and then the extraction was concentrated to the ratio of herbs and concentrate of 1∶1.5.The results showed that the contents of Danshensu Sodium from the three examples were 0.520, 0.498, and 0.521 mg/mL, and the RSD were 0.34%, 0.41%, and 0.29%.Conclusion The optimum extraction technology is feasible and applicable for the preparation of ganglouyuchuang lotion.

Quantitative research on multiple active components in Dandeng Tongnao Soft Capsules by UPLC-MS/MS-pattern recognition / 中草药

Objective: To establish a rapid MS method for the determination of nine principal active components (danshensu, caffeic acid, rosmarinic acid, salvianolic acid B, salvianolic acid A, tanshinone I, cryptotanshinone, tanshinone IIA, and ursolic acid) in Dandeng Tongnao Soft Capsules (DTSC), in order to make a comprehensive evaluation of the quality of drugs by pattern recognition technology. Methods: The UPLC-MS/MS method was used and the chromatographic conditions were as follows: The column was Acquity UPLC® BEH C18 (50 mm × 2.1 mm, 1.8 μm); The mobile phase was consisted of acetonitrile-water (containing 0.1% formic acid) at a flow rate of 0.2 mL/min with gradient elution; Mass spectrometer conditions: a triple quadrupole mass spectrometer equipped with electrospray ionization source (ESI) was used in positive and negative ion mode, and multiple reaction monitoring (MRM) was performed except ursolic acid in selected ion recording (SIR) for quantitative analysis of these compounds; The results of determination were calculated by the pattern recognition function of multivariate data processing software SIMCA 14.0 to evaluate the quality of DTSC. Results: Under the optimized conditions, danshensu, caffeic acid, rosmarinic acid, salvianolic acid B, salvianolic acid A, tanshinone I, cryptotanshinone, tanshinone IIA, and ursolic acid all showed good liners in the ranges of 100.0-1 000.0, 1.0-10.0, 8.0-80.0, 120.0-1 200.0, 15.0-150.0, 40.0-400.0, 10.0-100.0, 10.0-100.0, and 1.2-12.0 μg/mL, respectively (r ≥ 0.999 6). The recoveries were ranged from 98% to 101%, and RSDs were below 3%. The average contents in 10 batches of DTSC were (4.854 ± 0.314), (0.063 ± 0.005), (0.764 ± 0.070), (12.937 ± 0.648), (1.954 ± 0.178), (3.623 ± 0.221), (0.720 ± 0.062), (1.437 ± 0.116), and (0.073 ± 0.007) mg/g, respectively. The data were analyzed by SIMCA 14.0 software, and the results showed that the quality deviation of 10 batches of DTSC was below 2 SD (standard deviation, SD) range. Conclusion: The result shows that the UPLC-MS/MS method is simple, sensitive, and accurate for the rapid determination of main active components in the DTSC; It also shows that the quality of DTSC is stable in the different batches and displays an overall comprehensive evaluation of the drug quality using the multivariate data pattern recognition method, and provides a scientific basis and data processing method for the quality control of this drug.

Identification of water-soluble phenolic acids from Clerodendranthus spicatus / 中草药

Objective: To investigate the water-soluble phenolic acids from Clerodendranthus spicatus of Dai medicine. Methods: The chemical constituents were separated and purified by D101, Sephadex LH-20, MCI, and preparative HPLC column chromatographies. Their structures were determined by physicochemical properties and spectral data analyses. Results: Sixteen constituents were isolated and elucidated as protocatechualdehyde (1), protocatechuic acid (2), 3, 5-dihydroxybenzaldehyde (3), 4-hydroxybenzoic acid (4), 4-hydroxybenzaldehyde (5), vanillic acid (6), danshensu methyl ester (7), 3, 5-O-dimethyl gallic acid (8), helisterculins A (9), lithospermic acid (10), methyl lithospermate (11), dimethy lithospermate (12), helisterculins B (13), ethyl dihydrocaffeate (14), ferulic acid (15), and dihydroferulic acid (16). Conclusion: Compounds 3, 6-9, and 13-16 are obtained from Clerodendranthus Kudo for the first time.

Spectra-effect relationship of anti-inflammation of Salvia miltiorrhiza extract based on gray correlation analysis / 中草药

Objective: To study the relationship between UPLC and anti-inflammatory effects of different extracts of Salvia miltiorrhiza, and to provide a basis for clarifying the material basis of anti-inflammatory effects of S. miltiorrhiza. Methods: UPLC was used to analyze the different extraction sites of S. miltiorrhiza. The anti-inflammatory effect of anti-bronchial epithelial cell inflammation model was studied by different extraction sites. The spectral relationship was established by gravitational correlation analysis (GRA). Results: 50% grams of ethanol extract (S3), 80% ethanol eluent (S4), filtered precipitate (S5), and extracted aqueous layer extract (S9) had strong anti-inflammatory ability to inflammatory cells. The 50% ethanol eluate (S3) of the ethanol extract of Danshen was the strongest. The effect of chemical composition on the anti-inflammatory efficacy of each characteristic peak was as the following order: A9>A1>A2>A6>A15>A11>A12>A7>A10>A5>A14>A3>A18>A4>A13>A16>A8, and the top four peaks with strong contribution to anti-inflammatory effects were peaks 9, 1, 2, and 6, respectively. The results were as follows: tanshinone IIA, danshensu, salvianolic acid B, and 3’-methyl salvianolic acid B. Conclusion: The anti-inflammatory efficacy of S. miltiorrhiza is the result of combination of various components. It is clear that the four components are tanshinone IIA, danshensu, salvianolic acid B, and 3’-methyl salvianolic acid B. The efficacy of the largest contribution to the future to explore the efficacy of anti-inflammatory effects of S. miltiorrhiza provides new ideas.

Study on chemical constituents and dose-effect correlation of anti-oxidative activities among extracts of stems and leaves of Salvia miltiorrhiza / 中草药

Objective To evaluate the anti-oxidative effect and chemical constituents of stems and leaves of Salvia miltiorrhiza (SMSL) collected in July and December, water extracts and alcohol extracts of SMSL collected in July and December were taken as the subject, therefore provide scientific basis for the comprehensive development and utilization of SMSL. Methods The chemical constituents in the extracts were identified and determined by Ultra-high performance liquid chromatography combined with triple quadrupole electrospray tandem mass spectrometry (UPLC-TQ/MS), then confirmed the main salvianolic acids (danshensu, caffeic acid, rosmarinic acid, and salvianolic acid B) as the material basis of anti-oxidant activity of SMSL. Moreover, based on the anti-oxidant activity evaluation index: 1,1-diphenyl-2-picryl-hydrazyl (DPPH), 2,2'-azinobis-(3-ethylbenzthiazoline- 6-sulphonate) (ABTS) free radical scavenging and iron reduction/anti-oxidant capacity (FRAP), anti-oxidant activity of SMSL was evaluated. Meanwhile Salvia Miltiorrhiza Radix et Rhizoma (SM) from market was used to be control. Results It showed that the water extracts of SMSL in July possessed strong anti-oxidant activities, and the total salvianolic acids with the content of 75.663 mg/g was the highest; Followed by the alcohol extracts of SMSL in July, anti-oxidant activity and total phenolic acid contents of SM extracts were all lower than that of SMSL in July. Danshensu, caffeic acid, rosmarinic acid, and salvianolic acid B showed obvious anti-oxidative activities and significant dose-dependent effect in scavenging free radicals. Conclusion It revealed that SMSL possessed strong in vitro anti-oxidant activity. Additonally, it is shown that SMSL was rich in salvianolic acids, in which danshensu, caffeic acid, rosmarinic acid, and salvianolic acid B also had obvious anti-oxidant activity.