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We predicted the anti-hepatitis B virus (HBV) active components and mechanism of Salvia miltiorrhiza based on network pharmacology. The active components of S. miltiorrhiza were obtained through TCMSP, PubChem database and literature research. The potential targets of the active components and HBV infection were predicted by SwissTargetPrediction and GeneCards databases, respectively. The protein-protein interaction (PPI) network was constructed by String database. Cytoscape software was adopted to construct a visual network of active component-disease target and perform topological analysis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using DAVID platform. The molecular docking of key components and core targets was carried out by AutoDock Vina software. We screened out a total of 38 active components and 178 disease-component overlapping targets. Enrichment analyses obtained 405 related GO items and 68 signaling pathways, such as T/B cell receptor signaling pathways, PI3K/AKT signaling pathway, and mTOR signaling pathway. According to the results of molecular docking, most characteristic components of S. miltiorrhiza (miltionone Ⅱ, miltirone, protocatechuic acid, lithospermic acid, protocatechualdehyde) showed good affinity with the key targets (PIK3CA, APP, STAT3,AKT1 and mTOR). Furthermore, the anti-HBV activity of lithospermic acid, the representative active component of S. miltiorrhiza, and its regulation on PI3K/AKT and mTOR signaling pathways were investigated in an HBV replicating mouse model. Animal welfare and experimental procedures follow the regulations of the Animal Ethics and Welfare Committee of Hubei University. The results showed that lithospermic acid significantly inhibited HBV DNA replication, reduced serum HBsAg and HBeAg levels, and decreased the phosphorylation protein expression levels of AKT and mTOR in liver, indicating that lithospermic acid might exert the anti-HBV activity by regulating PI3K/AKT and mTOR signaling pathways.
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Objective::To establish an LC-MS/MS method for the determination of alcohol-soluble components and water-soluble components in Arnebiae Radix(L-shikonin, β, β′-dimethylacrylshikonin, β-acetoxy-isovalerylshikonin, lithospermic acid, caffeic acid, rosmarinic acid), for the purpose of quality control of the herb. Method::Chromatographic separation was carried out at 35 ℃ on Agilent ZORBAX SB C18 (4.6 mm×50 mm, 1.8 μm), with 0.1% formic acid acetonitrile (A)-0.1% formic acid solution (B) as the mobile phase for gradient elution (0-3 min, 5%-95%A; 3-7 min, 95%A; 7-7.01 min, 95%-5%A; 7.01-8.5 min, 5%A). The flow rate was 0.2 mL·min-1, and the injection volume was 5 μL. In a negative ionization mode, MRM scanning mode was adopted for quantification. Result::The calibration curves of L-shikonin, β, β′-dimethylacrylshikonin, β-acetoxy-isovalerylshikonin, lithospermic acid, caffeic acid, rosmarinic acid showed a good linearity, and the linear ranges of the above six compounds were 10.12-1 012 μg·L-1(r=0.998 1), 10.88-1 088 μg·L-1(r=0.991 2), 10.08-806.4 μg·L-1(r=0.997 6), 20.32-1 016 μg·L-1(r=0.996 6), 10.37-1 037 μg·L-1(r=0.999 6), 10.26-1 026 μg·L-1(r=0.997 8), respectively. The average recoveries of the analysts were 95.8%(RSD 3.2%), 103.5%(RSD 2.3%), 105.3%(RSD 2.1%), 96.1%(RSD 3.3%), 98.9%(RSD 2.7%), 100.8%(RSD 3.4%). The contents of six components in 13 batches of samples showed significant differences. Conclusion::The established method is feasible and simple, and provides a basis for comprehensive quality evaluation of Arnebiae Radix.
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Objective: To optimize the water-precipitation in the pretreatment manufacture process of Salviae Miltiorrhizae (Danshen) and Ligustrazine Hydrochloride Injection (SMLHI) based on the quality by design (QbD) concept and the sequential design. Methods: A design space was obtained by the global-type sequential design methodology combined with the fishbone-diagram risk analysis and central composite experiment based on global type sequential design method. The preliminary optimized zone of critical process parameters was extended in axial direction on the basis of the statistical result gained from the first central composite design. Then an updated multivariate linear regression model was fitted and a design space was delineated and verified. Results: According to the design space, the recommended operation space of the water-precipitation was as follows: when the pH value of the material was between 3.1 and 3.4, 3.25-5.00 times of water adding into the solution was recommened, the standing time was 7-17 h and the standing temperature was 7 ℃. Conclusion: This study introduces the sequential design into the process optimization of traditional Chinese medicine pharmaceuticals. Compared with the experimental design methods such as uniform space grid design and orthogonal design with a large number of samples, while samples cover the same feasible zone in a continuous operation form, sequential design methodology can effectively reduce the workload of the optimization experiment, avoid the waste of manpower and resources as well as guarantee the prediction ability of the fitting model.
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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.
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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.
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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.
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Based on the concept of quality marker (Q-marker) and the application of Q-marker in traditional Chinese medicine, the quality control methods of Chinese materia medica injections were put forward, taking salvianolic acids for injection as an example. Through the analysis of components, efficacy and pharmacokinetics, it has been identified that the Q-marker of salvianolic acids for injection were salvianolic acid B, rosmarinic acid, lithospermic acid, salvianolic acid D, and salvianolic acid Y. The quality control system of multi-index content determination, fingerprint, near infrared on-line control, and biological quality control was established with Q-marker as the core. It provides a reference for the new ideas of quality control of Chinese materia medica injection.
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Objective: By investigating the material composition, anti-oxidant and hypoglycemic activity of acetonitrile-water extracts of Origanum vulgare leaf (OL) in different harvest time, to acquire the scientific data for the utilization of OL. Methods: OL was extracted with acetonitrile and water (1: 1). The contents of total phenols and total flavonoid were measured by Foline-Phenol reagent method and AlCl3 colorimetry. The main compositional analysis was performed using LC-QTOF-MS technology. Meanwhile, the anti-oxidation of OL extracts in different harvest time was evaluated by total anti-oxidant capacity assay kit with ABTS method and ferric reducing anti-oxidant potential assay (FRAP). The methods of 2-NBDG glucose uptake and α-glycosides inhibition were applied to evaluate hypoglycemic activity of OL extracts. Results: The six main components in OL extract were identified to be origanoside I, luteolin 7-O-glucuronide, apigenin 7-O-glucuronide, rosmarinic acid, lithospermic acid and origanoside I derivative. The contents of total phenols and the total flavonoids were the highest in OL extracts harvested in July and September, respectively. There was good in vitro anti-oxidant and hypoglycemic activity for OL extracts harvested in different times. Among them, the best anti-oxidant activity was observed in OL extracts harvested in July, while the best hypoglycemic activity was observed in OL extracts harvested in October. Conclusion: OL has potential anti-oxidant and hypoglycemic activity, which is affected by the harvest time.
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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.
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Objective To investigate the chemical constituents from salvia polyphenolic acids. Methods Various column chromatographic techniques were used to separate and purify the chemical constituents and their structures were identified by spectral analysis. Results Seven compounds were isolated and obtained from salvia polyphenolic acids, which were identified as (2R)-2-[(3-(7-hydroxy-2-oxo-2,3-dihydrobenzofuran-4-yl)-(2E)-acryloyl) oxy]-3-(3,4-dihydroxyphenyl)-propanoic acid (1), salvianolic acid D (2), protocatechuic aldehyde (3), rosmarinic acid (4), lithospermic acid (5), salvianolic acid B (6), and salvianolic acid Y (7). Conclusion Compound 1 is a new compound, which is named as salvianolic acid D lactone.
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OBJECTIVE:To investigate the compatibility stability of Salvianolic acid for injection (SAFI) combined with 8 kinds of common solvents. METHODS:Referring to package inserts,SAFI was collected and combined with 0.9% Sodium chlo-ride injection,5% Glucose injection,10% Glucose injection,Compound sodium chloride injection,Mannitol injection,Glycerol fructose and sodium chloride injection,Sodium lactate Ringer's injection,Glucose and sodium chloride injection,250 mL each re-spectively,and then sealed in the dark at the room temperature. The appearance of mixtures were observed,pH value,the number of insoluble particles,maximal absorption wavelength and maximal absorbance were detected,and the contents of salvianolic acid B,rosmarinic acid,lithospermic acid and salvianolic acid Y in mixtures were determined by HPLC at 0,1,2,4,8 h after mix-ing. RESULTS:Under above condition,no obvious change was found in appearance or pH values of the mixtures within 8 h. Maxi-mal absorption wavelength ranged 284.5-286.0 nm. After mixed with Mannitol injection,the number of particles≥10μm(1-8 h af-ter mixing)and particles ≥25 μm(4-8 h after mixing)exceeded the scope of Chinese Pharmacopoeia(2015 edition);the maxi-mal absorbance changed significantly(RSD=9.17%,n=5);the relative content of salvianolic acid B,rosmarinic acid,lithosper-mic acid and salvianolic acid Y decreased by more than 10%(RSD=14.65%,6.45%,8.97%,12.49%,n=5);after mixed with Sodium lactate Ringer's injection,the relative content of rosmarinic acid and lithospermic acid changed greatly (RSD=14.57%, 7.28%,n=5);after mixed with 5% Glucose injection(4-8 h after mixing)and Glycerol fructose and sodium chloride injection(8 h after mixing),the relative content of rosmarinic acid were less than 90%(RSD=6.30%,4.86%,n=5);and the number of particles ≥25μm exceeded the scope of phamcopoeia after mixing with Glycerol fructose and sodium injection(0 h). The number of insoluble particles in other mixtures were in line with the standard of pharmacopoeia;maximal absorbance had no significant change(RSD<5%,n=5),and the relative content change of analytes were all less than 10%. CONCLUSIONS:Clinical appli-cation of SAFI combined with Mannitol injection,Sodium lactate Ringe's injection and Glycerol fructose and sodium injecrion should be avoided. After mixed with 5% Glucose injection,SAFI should be used within 4 h. SAFI can be compatible with 0.9%Sodium chloride injection,10% Glucose injection,Compound sodium chloride injection and Glucose and sodium chloride injec-tion.
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OBJECTIVE:To establish a method for the simultaneous determination of lithospermic acid and rosmarinic acid in Guanxin danshen capsule. METHODS:UPLC was performed on the column of ACQUITY UPLC-BEH C18 with mobile phase of 0.1% formic acid-methanol solution (gradient elution) at a flow rate of 0.3 ml/min,detection wavelength was 280 nm,column temperature was 30 ℃,and the injection volume was 5 μl. RESULTS:The linear range was 0.019-0.76 mg/ml for lithospermic ac-id(r=0.999 9)and 0.005-0.2 mg/ml for rosmarinic acid(r=0.999 7);RSDs of precision,stability and reproducibility tests were lower than 2%;recoveries were 97.34%-101.24%(RSD=1.57%,n=6) and 98.30%-100.39%(RSD=0.86%,n=6). CONCLU-SIONS:The method is simple,accurate and reproducible,and can be used for the contents determination of lithospermic acid and rosmarinic acid in Guanxin danshen capsule.
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Objective: To establish a blood-brain barrier (BBB) model for screening the effective ingredients of total salvianolic acids components probably effecting central nervous system (CNS), so as to provide basis for the rapid high throughput screening of CNS drugs. Methods: The permeability parameters and transendothelial electrical resistance (TEER) values showed that the co-cultured human brain microvascular endothelial cells (HBMEC)/HA (human astrocytes) model was of integrity and vitality. The active components in total salvianolic acids were screened with this in vitro BBB model, and then analyzed by HPLC-MS. Results: HA induced HBMEC to produce higher TEER in the first 10 d up to 300 Ω/cm2, sodium fluorescein was used to detect the formation of tight junctions of endothelial cells, and the permeability coefficient of its cross-vitro BBB model was (2.659 ± 0.730) × 10-3 cm/min. We found that six compounds at least from total salvianolic acids could permeate across BBB model, in which danshensu, protocatechuic acid, caffeic acid, isoferulic acid, lithospermic acid, and rosmarinicacid were identified. Conclusion: The co-cultured HBMEC/HA model combined with HPLC-MS analysis is a potential tool for screening of BBB permeable drug candidates from plant-derived materials.
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BACKGROUND: Lithospermic acid B (LAB), an active component isolated from Salvia miltiorrhizae, has been reported to have renoprotective effects in type 1 and type 2 diabetic animal models. We examined the effects of LAB on the prevention of diabetic nephropathy compared with amlodipine, a calcium channel blocker, and losartan, an angiotensin receptor blocker, in Otsuka Long-Evans-Tokushima Fatty (OLETF) rats, an animal model of type 2 diabetes. METHODS: LAB (20 mg/kg), amlodipine (10 mg/kg), or losartan (10 mg/kg) was given orally once daily to 10-week-old male OLETF rats for 28 weeks. RESULTS: None of LAB, losartan, and amlodipine exhibited effects on blood glucose levels. Treatment with amlodipine or losartan resulted in similar reductions in blood pressure; however, LAB was less effective in lowering blood pressure. Albuminuria was markedly suppressed by losartan and LAB, but not by amlodipine. LAB treatment decreased levels of renal lipid peroxidation, monocyte chemoattractant protein-1 (MCP-1), and transforming growth factor-beta1 (TGF-beta1). CONCLUSION: These results suggest that LAB has beneficial effects on the diabetic nephropathy in OLETF rats by decreasing oxidative stress and inflammation as potent as losartan.