RESUMEN

Ultrasonic-assisted activated carbon separation (UACS) was first employed to improve product quality by regulating adsorption rate and removing bacterial endotoxin from salvia miltiorrhizae injection. The adsorption rate was related to three variables: activated carbon dosage, ultrasonic power, and pH. With the increase of activated carbon dosage from 0.05 % to 1.0 %, the adsorption rates of salvianolic acids and bacterial endotoxin increased simultaneously. The adsorption rates at which bacteria endotoxins increased from 52.52 % to 97.16 % were much higher than salvianolic acids. As the ultrasonic power increased from 0 to 700 W, the adsorption rates of salvianolic acids on activated carbon declined to less than 10 %, but bacterial endotoxin increased to more than 87 %. As the pH increased from 2.00 to 8.00, the adsorption rate of salvianolic acid dropped whereas bacterial endotoxin remained relatively stable. On the basis of response surface methodology (RSM), the optimal separation conditions were established to be activated carbon dose of 0.70 %, ultrasonic power of 600 W, and pH of 7.90. The experimental adsorption rates of bacterial endotoxin were 94.15 %, which satisfied the salvia miltiorrhizae injection quality criterion. Meanwhile, salvianolic acids' adsorption rates were 1.92 % for tanshinol, 4.05 % for protocatechualdehyde, 2.21 % for rosmarinic acid, and 3.77 % for salvianolic acid B, all of which were much lower than conventional activated carbon adsorption (CACA). Salvianolic acids' adsorption mechanism on activated carbon is dependent on the component's molecular state. Under ideal separation conditions, the molecular states of the four salvianolic acids fall between 1.13 % and 6.60 %. The quality of salvia miltiorrhizae injection can be improved while maintaining injection safety by reducing the adsorption rates of salvianolic acids to less than 5 % by the use of ultrasound to accelerate the desorption mass transfer rate on the activated carbon surface. When activated carbon adsorption was used in the process of producing salvia miltiorrhizae injection, the pH of the solution was around 5.00, and the proportion of each component's molecular state was tanshinol 7.05 %, protocatechualdehyde 48.93 %, rosmarinic acid 13.79 %, and salvianolic acid B 10.28 %, respectively. The loss of useful components was evident, and the corresponding activated carbon adsorption rate ranged from 20.74 % to 41.05 %. The average variation rate in plasma His and IgE was significant (P < 0.05) following injection of 0.01 % activated carbon, however the average variation rate of salvia miltiorrhizae injection was dramatically decreased with the use of UACS and CACA (P > 0.05). The ultrasonic at a power intensity of 60 W/L and the power density of 1.20 W/cm2 may resolve the separation contradiction between salvianolic acids and bacterial endotoxin, according to experiments conducted with UACS at different power intensities. According to this study, UACS has a lot of potential applications in the pharmaceutical manufacturing industry and may represent a breakthrough in the field of ultrasonic separation.

Asunto(s)

RESUMEN

Salvia miltiorrhiza Bunge is a herb plant used as a traditional Chinese medicine to cure cardiovascular disease. In December 2018, a root rot disease was observed on S. miltiorrhiza in four surveyed counties (Song, Yuzhou, Fangcheng, and Mianchi) in Henan province in China. The disease incidence ranged from 15 to 50% in 12 surveyed fields. At the early stage, the diseased plants were wilting with purple leaves. Leaves and branches became withered and fibrous roots became brown and rotted. The main roots of severely diseased plants also became rotted. The color of the stem surface turned from red to black, and the color of the stem xylem and phloem turned from dark red to brown. Eventually, the roots of diseased plants became completely rotted and the whole plants became dead, but no stink, which is different from Fusarium solani (Mart.) Sacc. (Yuan et al. 2015). Diseased root tissues (5×5×5 mm in size) were cut from diseased plants, surface-sterilized with 1% sodium hypochlorite for 1 min followed by dipping in 75% alcohol for 30 sec, rinsed in sterile distilled water for 3 times, air-dried on a sterilized filter paper in a laminar flow hood, placed on potato dextrose agar (PDA) containing 250 mg/l of streptomycin sulfate, and incubated at 28℃. Five isolates of Fusarium were obtained and purified using the single-spore isolation method. On PDA plates, the colonies were purple in color with formation of white aerial mycelia and reached 50 to 60 mm in diameter after incubation for 5 days. The colonies produced abundant microconidia on the colonies. The microconidia were 4.3 to 12.3 (10.0) × 2.1 to 3.5 (3.1) µm in size (n = 40), hyaline, ovoid or ellipse in shape. The conidiogenous cells were polyphialides. On mung bean media, the isolates formed macroconidia with 3 to 6 septae, fusiform in shape, slightly curved, 21.8 to 32.7 (31.4) × 2.6 to 4.3 (3.4) µm in size (n = 50). The morphological features of the five isolates were consistent with the description for Fusarium proliferatum (Matsush.) Nirenberg ex Gerlach & Nirenberg (Leslie and Summerell 2006). To further define the identity of the five isolates, molecular phylogenetic analysis was performed. The genomic DNA was extracted from all five isolates using the cetyl trimethylammonium bromide (CTAB) method. Five genes [nuclear ribosomal internal transcribed spacer (ITS) region, translation elongation factor 1-α (EF1α), ß-tubulin gene, partial sequence for calmodulin (PRO), and RNA-dependent DNA polymerase II subunit (RPB2)] in F. proliferatum were amplified using primers pairs ITS1/ITS4, EF1T/2T, ß-tubulin 2a/b, PRO1/2, and RPB2F/R, respectively (Glass and Donaldson 1995; Liu et al. 1999; Mulè 2004; O'Donnell et al. 1998; O'Donnell et al., 2010). The sequences (GenBank accession numbers: MT371373, MT371384, MT925651, MT925652, and MT934441, respectively) showed 99.6 to 100% identities to the corresponding DNA sequences in F. proliferatum (GenBank Acc. Nos. MK243486, MN245720, KJ12896, MN245721, and MK144327, respectively). All five isolates were tested for pathogenicity to fulfill the Koch's postulates. The 45-day-old healthy plants of S. miltiorrhiza grown in sterilized soil in pots (20 cm in diameter), one plant in one pot, were inoculated with conidial suspensions (1.0 × 107 cfu/ml) by pouring 10 ml conidial suspensions around the stem base in one pot. For each isolate, four plants were inoculated. Four plants were treated with sterilized water in the same volume as a control. The tested plants were placed in a growth room at 25°C (RH > 60%) with a 12 h photoperiod of fluorescent light. The pathogenicity assay was repeated for three times. The similar wilt symptoms were observed on the roots in the inoculated plants 30 days after inoculation but were not observed in the control plants. F. proliferatum was re-isolated from the infected roots, and its identity was confirmed by PCR with the primers described above. To our knowledge, this is the first report of F. proliferatum casing root rot disease on S. miltiorrhiza in China.

Asunto(s)

RESUMEN

Development of fast and accurate methods to discover lead compounds for drug candidates is highly important. In this study, a reliable and effective post-column on-flow biochemical assay (POBA) was established to screen potent peroxidase inhibitors from complex chemical mixtures (e.g., natural product extracts). Multiple factors such as flow rate, organic phase, detection wavelength, and reaction coil were carefully investigated. To better understand the features of POBA, another emerging technology of ultrafiltration LC-MS was used for comparison. The result showed that POBA had advantages in saving time, avoiding false positives, and improving the accuracy. To illustrate the practicality of the method, Radix Salvia Miltiorrhizae, a traditional herb for cardiovascular disease treatment, was applied as the research objective. Finally, six compounds including tanshinol, protocatechuic aldehyde, salvianolic acid D, rosmarinic acid, lithospermic acid, and salvianolic acid B were determined as novel peroxidase inhibitors. Their bioactivities were validated by microplate-based assay, molecular docking, and pharmacophore modeling. This study demonstrates a great potential of POBA in the efficient and accurate discovery of drug candidates. Graphical abstract Compared with a classical method of ultrafiltration LC-MS, the newly developed method of on-flow bioassay shows advantages in saving time, avoiding false positives and improving the accuracy.

Asunto(s)

RESUMEN

A simple and rapid gas chromatography-flame photometric detection (GC-FPD) method was developed for the determination of 12 organophosphorus pesticides (OPPs) in Salvia miltiorrhizae by using ultrasonication assisted one-step extraction (USAE) without any clean-up steps. Some crucial parameters such as type of extraction solvent were optimized to improve the method performance for trace analysis. Any clean-up steps were negligent as no interferences were detected in the GC-FPD chromatograms for sensitive detection. Under the optimized conditions, limits of detection (LODs) and quantitation (LOQs) for all pesticides were in the range of 0.001-0.002mg/kg and 0.002-0.01mg/kg and 0.002-0.01mg/kg, respectively, which were all below the regulatory maximum residue limits suggested. RSDs for method precision (intra- and inter-day variations) were lower than 6.8% in approval with international regulations. Average recovery rates for all pesticides at three fortification levels (0.5, 1.0 and 5.0mg/kg) were in the range of 71.2-101.0% with relative standard deviations (RSDs) <13%. The developed method was evaluated for its feasibility in the simultaneous pre-concentration and determination of 12 OPPs in 32 batches of real S. miltiorrhizae samples. Only one pesticide (dimethoate) out of the 12 targets was simultaneously detected in four samples at concentrations of 0.016-0.02mg/kg. Dichlorvos and omethoate were found in the same sample from Sichuan province at 0.004 and 0.027mg/kg, respectively. Malathion and monocrotophos were determined in the other two samples at 0.014 and 0.028mg/kg, respectively. All the positive samples were confirmed by LC-MS/MS. The simple, reliable and rapid USAE-GC-FPD method with many advantages over traditional techniques would be preferred for trace analysis of multiple pesticides in more complex matrices.

Asunto(s)

RESUMEN

Compound Danshen Dripping Pills (CDDP), a herbal patent medicine, is widely used in China for the prevention and treatment of cardiovascular diseases. A simple, sensitive and reliable method for simultaneous determination of danshensu (DSS), protocatechuic aldehyde (PCA), and their related metabolites, 4-hydroxy-3-methyloxyphenyl lactic acid (HMLA) and protocatechuic acid (PAA) in human plasma was developed and validated based on liquid chromatography tandem mass spectrometry (LC-MS/MS). The analytes and internal standard (IS), vanillic acid (VAA), were extracted from plasma with ethyl acetate and separated on a C18 column by using the mobile phase consisted of methanol-0.1% formic acid via gradient elution. The electrospray ionization (ESI) source was applied and operated under the multiple reaction monitoring (MRM) mode. The linear calibration curves were obtained at the concentration ranges of 0.46-1000ng/mL for DSS and PAA, and 1.38-1000ng/mL for PCA and HMLA, respectively. The inter- and intra-day precisions (RSD%) were less than 13.5%, and the accuracy (±RE%) was within 13.4%. The described method was successfully applied for the clinical pharmacokinetics of CDDP in Chinese healthy volunteers.

Asunto(s)

RESUMEN

BACKGROUND: Severe acute pancreatitis (SAP) and obstructive jaundice (OJ) are frequent recurring diseases that bring about huge threat to human health. Some reports have demonstrated that Salviae miltiorrhizae can protect multiple organs of SAP and OJ model animals or patients, but their related mechanisms were not clear. In this study, we observed the effects of Salvia miltiorrhizae injection on apoptosis and NF-κB expression in kidney and explored the protective effect and mechanism of Salvia miltiorrhizae on the kidney of SAP or OJ rats. The results obtained will provide a theoretical basis for clinical application of Salvia miltiorrhizae. MATERIAL AND METHODS: A total of 288 rats were used for SAP -and OJ-associated experiments. The mortality rates of rats, the contents of serum BUN and CREA, the expression levels of Bax, NF-κB proteins and the apoptosis index were observed, respectively. RESULTS: The pathological changes in the kidney of SAP or OJ rats in treated group were mitigated to varying degrees. At 6 and 12 hours after operation in SAP rats or on 21 and 28 days after operation in OJ rats, the contents of serum CREA in treated group were significantly lower than those in model control group; At 3 and 6 hours after operation, the staining intensity of Bax protein of kidney in treated group was significantly lower than that in model control group; on 14 days after operation, the apoptosis index in the kidney of OJ rats in treated group was significantly lower than that in model control group. CONCLUSION: Salvia miltiorrhizae can exert protective effects on the kidney of SAP and OJ rats.

Asunto(s)

RESUMEN

For seeking novel antibacterial agents with high efficacy and low toxicity to deal with drug resistance, the effects of Salvia miltiorrhizae from various sources on Escherichia coli were evaluated by microcalorimetry coupled with chemometrics. Firstly, the heat-flow power-time curves of E. coli growth affected by different S. miltiorrhizae samples were recorded. Then, some crucial quantitative thermo-kinetic parameters including growth rate constant, heat-flow power and heat output, etc. were obtained from theses curves and were further investigated by some powerful chemometric techniques including similarity analysis, multivariate analysis of variance, hierarchical clustering analysis and principle component analysis. By analyzing the principle parameters, growth rate constant of the second exponential phase (k 2) and the heat-flow output powers of the second highest peak (P 2), together with the derived parameter inhibitory ratio (I,  %), it could be quickly concluded that the tested S. miltiorrhizae samples from different sources in China exhibited strong antibacterial effects on E. coli and the samples from Beijing city exhibited the strongest anti-E. coli effects, which might be used as novel and underlying antibacterial candidates for the resistance of E. coli to the existing drugs in practice. This study provides a useful tool and helpful idea to accurately and rapidly evaluate the antibacterial effects of some complex matrices, offering some references for exploring new antibacterial agents.

RESUMEN

Objective To evaluate the quality of six kinds of radix salvia miltiorrhizae tables and verify the methods of the quality evaluation . Methods Identification, detection and quantitative test method in Chinese Pharmacopoeia Part One 2005 edition and the HPLC-FPC analysis method were used to evaluate the quality of six kinds of radix salvia miltiorrhiza tables.Results The content of tanshinone IIA of six samples is not in accordance with that in the Chinese Pharmacopoeia and the content of lithospermate B of three samples is not in accordance with that in the Chinese Pharmacopoeia.Both the content of lip-soluble component and the water-soluble component of all the samples differ from those of standard radix salvia miltiorrhiza tables.Conclusion The HPLC-FPC analysis method is useful and effective to identify and detect the quality of the HPLC-FPC analysis method.More attention shall be paid to the quality of the radix salvia miltiorrhiza tables and the supervision of the radix salvia miltiorrhizae tables market should be strengthened.

RESUMEN

A sensitive and rapid ultra-fast liquid chromatography with tandem mass spectrometry (UFLC-MS/MS) method was developed for simultaneous qualitative and quantitative of four characteristic tanshione components including tanshinone IIA, cryptotanshinone, tanshinone I and dihydrotanshinone I in Salvia miltiorrhizae after ultrasound-assisted extraction. By using a C18 column, the four analytes were separated by gradient elution with acetonitrile and water both containing 0.1% formic acid at the flow rate of 0.3mL/min. Multiple-reaction monitoring (MRM) was used for quantification, and an information-dependent acquisition (IDA) method was used to trigger enhanced product ion scans (EPI) for supplementary characteristic identification for qualitative research. Calibration curves showed good linearities with correlation coefficients (r) higher than 0.9990. The method showed high sensitivity with limits of detection (LODs) and quantification (LOQs) less than 0.0002ng/mL and 0.0008ng/mL, respectively, as well as good precision and reproducibility. Mean recoveries for four analytes ranged from 92.5% to 106.2% with relative standard deviations (RSDs) lower than 14.59%. Real application of the developed method in 32 batches of S. miltiorrhizae samples demonstrated that the total contents of four analytes in all samples were in the range of 2.258-52.342mg/g. Ultrasound-assisted extraction technique took a small amount of sample and low time but giving high extraction efficiency. Combining with UFLC-MS/MS method in MRM-IDA-EPI mode, more components in other complicated matrices can be simultaneously analyzed for qualitation and quantitation in one run.

Asunto(s)

RESUMEN

Objective To explore the effects of salvia miltiorrhizae (SM) injection on the apoptosis of cultured rat neuronal stem cells induced by hypoxia and the activity of Caspase-3, in order to provide the further evidence for the molecular mechanism of neuroprotection of SM injection. Methods The neuronal stem cells from neonatal rat hippocampus were cultured and divided randomly into normal control group, hypoxia group and SM treatment group. After Hoechst staining, the apoptotic morphological change and apoptosis percentage were observed under fluorescence microscope. The activities of Caspase-3 in the 3 groups were evaluated by the colorimetric assay. Results Compared with normal control group [(2.75±0.28)%, 1.16±0.07], the percentage of apoptosis and the activity of Caspase-3 were increased significantly in neuronal stem cells cultured in hypoxia [(30.12%±2.09)%,3.85±0.41, P<0.05). Application of SM injection reduced markedly the percentage of apoptosis and the activity of Caspase-3 of the neuronal stem cells cultured in hypoxia [(9.16±1.34)%, 1.50±0.09, P<0.05].Conclusion SM injection can depress the apoptosis of the rat neuronal stem cells induced by hypoxia,so as to exert the neuroprotection.

RESUMEN

Objective To investigate the effects of radix salvia miltiorrhizae on the apoptosis of hepatoeytes during cold preservation and reperfusion injury in rat donor liver. Methods Forty male SD rats were divided into experimental group, control group and sham operation group. The rat model of liver transplantation was established according to the Kamada method. The grafts were preserved in lactated Ringer's solution with radix salvia miltiorrhizae in experimental group and in lactated Ringer's solution in control group for 5 hours, then they were transplanted orthotopically. Six hours after transplantation, the recipients were sacrificed, and the serum ALT and AST were detected by automatic biochemistry analyzer, the hepatocyte apoptosis by TUNEL, the expression of Bcl-2 and FasL protein by flow cytometry. The histopathological changes of the liver grafts were observed under light microscope. Results The levels of ALT and AST in the experimental group were significantly lower than those in the control group after reperfusion. Compared with that in the control group, the apoptosis index of the hepatoeyte was signifieandy decreased in the experimental group ( F = 133.802, P <0.05 ), while the level of Bel-2 protein expression was increased ( F = 91.063, P < 0.01 ). No statistical difference upon FasL protein expression was detected between the 2 groups( F = 1.329, P >0.05). The histopathological injury of the liver grafts in the experimental group was significantly slighter than that in the control group. Conclusions Radix salvia mihiorrhizae inhibits the apoptosis of hepatocytes by increasing the Bcl-2 protein expression during cold preservation and reperfusion injury, so it has a protective effect on the liver graft against ischemia reperfusion injury.

RESUMEN

Objective To investigate the ex tr action technique for seperating the active components in the root of Salvia mi ltiorrhizae bunge by supercritical fluid, and to analyze the extracted product s by HPLC-MS n . Methods The extraction condition s were established as follows: 950ml?L -1ethanol as the first entrainer, t he pressure of 20.0 MPa, temperature at 45 ℃, and extracting time 1 h; then 100 mL?L -1 ethanol was selected as the second entrainer, pressur e was 30.0 MPa, temperature was 65 ℃, and extracting time was 3 h. Results Compared with traditional refluxing extraction and ultrasonic extraction, supercritical fluid extraction was better and more effect ive. Conclusion Supercritical extraction is simple, highly selec tive and efficient in extracting the active components in Salvia miltiorrhizae bunge.

RESUMEN

AIM: To study the technological parameters of the purification process of total salvianolic acid(TSA) of Radix Salvia Miltiorrhizae(RSM) with D101 macroreticular resin. METHODS: Based on orthogonal experiment,the reservation rate of TSA in macroreticular resin columniation was took as index to investigate the chief influential factors in the purification of TSA. RESULTS: The volume of distilled water and the content of TSA in RSU solution had significant effect on the reservation rate of TSA in macroreticular resin columniation. With the help of macroreticular resin,the reservation rate of TSA could reach 87.52%.Its purity was 77.74%,comparing with crude extract,increased by 3.6 times. CONCLUSION: The method of purifing TSA of RSM with D101 macroreticular resin is feasible,it could be applied to industrialized production of TSA of RSM.

RESUMEN

AIM:A voltammetric method was developed for the determination of salvianolic acid B using a carbon nanotube paste electrode.And the separation process of salvianolic acid B was detected. METHODS: In Britton-Robinson buffer solution of pH(1.81),the square wave stripping voltammetric method was used to determine salvianolic acid B. RESULTS: The proposed method was verified by an established HPLC method,and it was applied to determining salvianolic acid B in eluent of eluting extracts of Radix salvia Miltiorrhiza from SP-207 colunm with ethanol solution,and the results were satisfied. CONCLUSION: A new method for determining salvianolic acid B was developed,and the proposed method would be used as an on-line quality control method for detecting of salvianolic acid B in eluting extracts of Radix salvia Miltiorrhiza in the future.

RESUMEN

AIM:To establish the HPLC-fingerprint of the water-soluble constituents of Salvia miltiorrhizae Bge.on 18 batches from 9 areas in China. METHODS: Samples of Salvia miltiorrhizae Bge.from different areas were determined by Agilent 1100 DAD-HPLC on Sino Chrom ODS-BP column(250 mm?4.6 mm,5 ?m),gradient elution with methanol-water(0.7%H_3PO_4) as mobile phase,column temperature was at 30℃,flow rate was 1 mL/min,wavelength was at 280 nm,and the inject volume was 20 ?L. RESULTS: The HPLC-fingerprints of the water-soluble constituents from Salvia miltiorrhizae Bge.,including 12 mutual peaks,were developed after determination of 10 batches of drugs according to SPSS analysis.,and in which,3 characteristic components were recognized. CONCLUSION: The separation effect of peaks in fingerprints is better and the characteristic is also obvious.The RSD values of precision and reproducibility are less than 5%.This method can be used for the quality control of Salvia miltiorrhizae Bge.

RESUMEN

Objective: To study the effect of different sorts of fertilizer on the content of tanshinone Ⅱ A, the effective element of cultivated Radix Salvia miltiorrhizae. Methods: Fertilize Radix Salvia miltiorrhizae with the several kinds of combination: the oddment cake(round flat cake made of residue of seed after extracting oil form it), pig manure, chicken manure, duck manure, human excrement, plant ashes, dregs of a decoction (residue of Traditional Chinese medicine material after being extracted), phosphoric fertilizer and compound fertilizer; and gather Radix Salvia miltiorrhizae one year later. Determine the content of Tanshinone Ⅱ A in cultivated Radix Salvia miltiorrhizae by HPLC. Results: The contents of Tanshinone Ⅱ A in Radix Salvia miltiorrhizae fertilized by different sorts of fertilizer are of significant difference. Conclusion: Plant ashes, oddment cake and compound fertilizer are good for growing of Radix Salvia miltiorrhizae and raises of the content of Tanshinone Ⅱ A.

RESUMEN

AIM: To apply the solid phase extraction (SPE) on salvianolic acids in Radix Salvia Miltiorrhizae by means of high performance liquid chromatography with mass spectroscopic detector (HPLC MSn). METHODS: A C 18 solid phase column and negative ion electrospray ionization (ESI) mode were used by orthogonal design to optimize four factors which might affect recoveries of samples, which included vacuum tightness, sample size, amount of eluting agent and eluting power. RESULTS: The optimized results were as follows: vacuum tightness was 0.002?106 Pa, sample size was 0.75 mL, amount of eluting agent was 15mL and eluting power was 1.0mL?s -1 . CONCLUSION: The established method is simple, reliable and suitable for the usual quality control method of salviamolic acids in Radix Salvia Miltiorrhizae.

RESUMEN

AIM: To observe the changes of Ca-L current (ICa-L) and to investigate the mechanism of salvia miltiorrhizae (SM) for eliminating Ca~2+ overloaded in cells during acute hypoxia/reoxygenation. METHODS: The whole cell patch clamp technique was applied to study the changes of ICa-L. Different concentrations (32, 320, ~3 200 mg/L) of SM were added to the ventricular myocytes isolated from guinea pigs by enzyme digestion. RESULTS: SM (32, 320, ~3 200 mg/L) decreased the amplitude of ICa-L in a concentration-dependent manner regardless of these cells were under normoxia, hypoxia or reoxygenation. Furthermore, SM at low concentration (32 mg/L) was more effective to hypoxia or reoxygenation-treated cells than that to the cells under normoxia condition. CONCLUSION: These results indicate that SM effectively decreases the abnormal raised amplitude of ICa-L in ventricular myocytes under hypoxia or reoxygenation conditions, preventing Ca~2+ overloaded in the cells. [