CYP2C8 (Ki = 4.82 μM); CYP2J2 (Ki = 5.75 μM)

Salvianolic acid C has a Ki of 4.82 and 5.75 μM for CYP2C8 and CYP2J2, respectively, making it a non-competitive corrector of CYP2C8 and a moderate mixed corrector of CYP2J2 [1]. The production of NO produced by LPS was greatly reduced by 5 μM Salvianolic acid C. Salvianolic acid C dramatically lowered iNOS expression. Salvianolic acid C prevents the overproduction of TNF-α, IL-1β, IL-6, and IL-10 that is brought on by LPS. NF-κB activation produced by LPS is inhibited by salvianolic acid C. In BV2 asteroid cells, C can also upregulate the expression of HO-1 and Nrf2 [2].

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Effects of Salvianolic Acid C/SalC on cell viability [2]
The effect of SalC on BV2 microglia cells viability was detected by MTT assay. The results showed that 1 μg/mL LPS had no obvious influence on the viability of BV2 microglia cells. SalC didn't affect cell viability in the concentration range of 0–20 μM (Fig. 1B), and the concentrations of 50 and 100 μM of SalC slightly decreased cell viability.

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Effects of SalC/Salvianolic Acid C on NO, iNOS, PGE2 and COX-2 levels in LPS-treated BV2 cells [2]
The amount of NO release was measured using Griess reagent. As shown in Fig. 2A, the content of NO in supernatant of BV2 cells was significantly upregulated by LPS stimulation compared to the control group. 1 and 5 μM SalC could significantly inhibit the NO production induced by LPS (P < 0.01, P < 0.01). To clarify the reason for the inhibition of NO production, the expression of iNOS was further investigated by immunofluorescence and Western methods. As shown in Fig. 2B–D, the expression of iNOS was very low in normal control group, but LPS stimulation significantly upregulated the expression of iNOS. SalC decreased the expression of iNOS significantly. The above results showed that the inhibitory effect of SalC on NO production is achieved by inhibiting iNOS expression. The effects of SalC on the PGE2 and COX-2 were further examined. The secretion of PGE2 was detected by ELISA method. As shown in Fig. 2E, LPS stimulation resulted in a significant increase of PGE2 content in cells (P < 0.01). 1 and 5 μM SalC significantly decreased LPS-induced PGE2 upregulation (P < 0.05, P < 0.01). Western blot method was used to detect the expression of COX-2. SalC significantly reduced the COX-2 expression induced by LPS stimulation (Fig. 2F). The above results showed that SalC could significantly inhibit the expression of iNOS and COX-2, thereby blocking NO and PGE2 synthesis.

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SalC/Salvianolic Acid C inhibited LPS-induced TNF‑α, IL‑1β, IL‑6 and IL‑10 overproduction [2]
The production of TNF‑α, IL‑1β, IL‑6 and IL‑10 in BV2 microglia cells was then measured. The results showed that LPS stimulation could significantly increase the production of TNF‑α, IL‑1β, IL‑6 and IL‑10 (P < 0.01, Fig. 3A–D), while SalC significantly decreased the production of TNF‑α, IL‑1β, IL‑6 and IL‑10 in BV2 microglia cells. As shown in Fig. 3E–H, LPS significantly stimulated the transcription of TNF‑α, IL‑1β, IL‑6 and IL‑10 mRNA compared to the control group. SalC decreased the transcription of TNF‑α, IL‑1β, IL‑6 and IL‑10 mRNA significantly.

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SalC/Salvianolic Acid C inhibited LPS-induced NF‑κB activation [2]
According to the immunofluorescence results, the nuclear translocation of p‑NF‑κB p65 was significantly increased after LPS stimulation, while SalC could inhibit the nuclear translocation of p‑NF‑κB p65 (Fig. 4A). Western blot assay was used to detect the expression of inflammation related proteins. The activated form p‑NF‑κB p65 was significantly increased by LPS stimulation (P < 0.01), and SalC (0.1, 1 and 5 μM) inhibited the LPS-induced NF‑κB activation in a concentration-dependent manner (P < 0.01, Fig. 4B, C). Fig. 4B, D showed that the p‑IκBα level was significantly increased in LPS group (P < 0.01), while SalC (0.1, 1 and 5 μM) downregulated p‑IκBα level significantly (P < 0.05, P < 0.01 and P < 0.01).

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SalC/Salvianolic Acid C increased the expression of Nrf2 and HO‑1 in BV2 microglial cells [2]
Nrf2 signaling pathway plays an important role in the regulation of inflammatory response and oxidative stress. Fig. 5 showed that the nucleus Nrf2 level in LPS stimulated group showed no significant difference compared with the normal control group (P > 0.05), and the expression of Nrf2 downstream protein HO‑1 also showed no significant difference between the two groups (P > 0.05). But SalC (0.1, 1 and 5 μM) significantly increased the expression of Nrf2 (P < 0.01), as well as its downstream protein HO‑1 (P < 0.05, P < 0.01 and P < 0.01). SalC (1 and 5 μM) also significantly increased the anti-oxidation enzyme NQO1 expression compared with the LPS group (P < 0.01).

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SalC/Salvianolic Acid C inhibited NF‑κB activation via Nrf2 pathway [2]
To clarify whether SalC inhibits NF‑κB activation via Nrf2 pathway. Nrf2 was knocked down using siRNA technology. The results showed that Nrf2 knockdown reversed the inhibitory effect of SalC on TNF‑α, IL‑1β, IL‑6 and IL‑10 production (Fig. 6B–E). In addition, immunofluorescence results also showed that Nrf2 knockdown reversed the inhibitory effects of SalC on NF‑κB nuclear translocation (Fig. 6A).

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AMPK/Nrf2 signaling contributed to the inhibition effect of SalC/Salvianolic Acid C on NF‑κB [2]
For signaling pathway investigation, the specific Nrf2 siRNA was used to clarify the interaction between Nrf2 and NF‑κB p65. The AMPK inhibitor Compound C was used to study the upstream protein of Nrf2. Results showed that the pre-incubated with Compound C reversed the activation effect of SalC on Nrf2 in both BV2 microglia cells and primary microglia cells. What's more, the pre-incubated with Compound C also reversed the inhibition effect of SalC on NF‑κB. And treatment with Nrf2 siRNA reversed the inhibition effect of SalC on NF‑κB (Fig. 10). The above results showed that the AMPK/Nrf2 signaling contributed to the inhibition effect of SalC on NF‑κB and neuroinflammation.

Treatment with salvianolic acid C (20 mg/kg) dramatically lowered the escape latency. Furthermore, as comparison to the LPS model group, the number of plateau crossings was considerably higher in the salvianolic acid C (10 and 20 mg/kg) treatment group. Systemic rat salvianolic acid C reduced brain tissue TNF-α, IL-1β, and IL-6 levels in comparison to the model group. The cerebral cortex and hippocampus were used in the modeling of iNOS and COX-2 levels. Treatment with salvaianolic acid C (5, 10 and 20 mg/kg) significantly reduced the cortical and hippocampal levels, while the levels of p-AMPK, Nrf2, HO-1, and NQO1 were dose-dependently increased in [2].

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SalC/Salvianolic Acid C inhibited rat neuroinflammation and improved memory behavior [2]
Systemic administration of LPS induced neuroinflammation in SD rats. As shown in Fig. 7A, in the Morris water maze test, the escape latency of rat was significantly higher in LPS model group compared with the control group (P < 0.01), while SalC (20 mg/kg) treatment could significantly decreased the escape latency (P < 0.05). In addition, rats in LPS model group crossed the location fewer times compared with rats in the control group. And SalC (10 and 20 mg/kg) treatment significantly increased the platform crossing number compared with the LPS model group (P < 0.05 and P < 0.01, Fig. 7B). These results indicated that rats in LPS model group had impairments in spatial learning and memory, while SalC treatment could attenuate the impairment. Systemic administration of LPS significantly upregulated the brain TNF‑α, IL‑1β and IL‑6 levels, and SalC downregulated the brain TNF‑α, IL‑1β and IL‑6 levels compared with the model group (Fig. 7C). The iNOS and COX‑2 levels in rat brain cortex and hippocampus were higher than that in the control group, while SalC treatment significantly downregulated the iNOS and COX‑2 levels both in the cortex and hippocampus regions (Fig. 7D–F). Further immunofluorescence study showed that LPS increased the GFAP and Iba1 positive cells fluorescence intensity both in the cortex and the hippocampus, SalC treatment inhibited the neuroinflammation induced by LPS, and decreased the GFAP and Iba1 fluorescence intensity (Fig. 7G–I).

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SalC/Salvianolic Acid C activated AMPK/Nrf2 and inhibited NF‑κB signaling pathways in rat brain [2]
As shown in Fig. 8, SalC (5, 10 and 20 mg/kg) treatment dose-dependently increased the p‑AMPK, Nrf2, HO‑1 and NQO1 levels in rat brain cortex and hippocampus. In addition, systemic administration of LPS induced the activation of NF‑κB signaling pathway, while SalC (5, 10 and 20 mg/kg) treatment dose-dependently inhibited the phosphorylation NF‑κB p65 and the phosphorylation of IκBα (Fig. 9).

Enzyme kinetic analysis [1]
Taxol 6-hydroxylation and astemizole O-desmethyastemizole were determined as probe activities for CYP2C8 and CYP2J2, respectively. Incubation mixtures were prepared with a total volume of 200 μL, and the final concentration of organic solvent in the mixtures was less than 1% (v/v). Incubation mixtures were as follows: recombinant P450 enzymes, NADPH-generating system (10 mM glucose 6-phosphate, 0.5 mM NADP, 10 mM MgCl2, 1 unit of glucose 6-phosphate dehydrogenase), 100 mM potassium phosphate buffer (pH 7.4), and a range of concentrations of the probe substrates. Taxol and astemizole were dissolved in methanol. There was a 5-min preincubation period at 37 °C before the reaction was initiated by adding the NADPH-generating system. All incubations were conducted under linear conditions with regard to time and protein concentrations, in accordance with the preliminary experiments (Supplementary Table 1). Different concentrations of taxol (0.2–50 μmol·L−1) and astemizole (0.05–50 μmol· L−1) were added to the incubation mixtures. The Km (the substrate concentration at which the action rate is half its maximum speed) and Vmax (the maximum initial velocity of the enzyme catalysed reaction under the given conditions) values were determined using different concentrations of marker substrates by nonlinear regression analysis of the enzyme activity-substrate concentration data using the Michaelis-Menten model (GraphPad Prism 5, CA, USA). The final concentrations of the metabolites in the incubation mixtures were determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

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Screening the inhibitory effects of Danshen compounds on CYP2C8 and CYP2J2 [1]
The incubation mixture contained CYP2C8 or CYP2J2, an NADPH-generating system, the substrate (final concentration ∼ Km), and different concentrations (0–50 μmol·L−1) of a single test compound, in 100 mM phosphate buffer (pH 7.4). Hydrophilic components (salvianolic acids, SAs, e.g. Salvianolic Acid C) were dissolved in water, and lipophilic components (TSTs) were dissolved in dimethyl sulfoxide, whose final concentration was less than 1% (v/v) of the mixture. Quercetin and danazol were used as the positive controls for CYP2C8 and CYP2J2, respectively. All incubations were performed in triplicate, and mean values were used for analysis. The activities of CYP2C8 and CYP2J2 in the presence of the inhibitors were expressed as percentages of the corresponding controls. From plots of percent inhibition versus logistic inhibitor concentrations, the corresponding IC50 values (the inhibitor concentration required for 50% inhibition of the enzyme) were calculated by fitting the percent remaining enzyme activity (Y) versus the logarithm of the initial inhibitor concentration (X) data to eq. (1) using nonlinear regression analysis. (1)Y = 100/(1 + 10X−LogIC50)

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Inhibition-type study [1]
The inhibition-types and Ki values (inhibitory constant for the inhibitor, equal to the dissociation constant of the enzyme/inhibitor complex) of Danshen components on specific CYPs were determined when their IC50 values were lower than 15 μmol·L−1. Taxol (2.5, 5 and 10 μmol·L−1), astemizole (0.5, 1 and 2 μmol·L−1), and different concentrations of Danshen components were used for the construction of Lineweaver-Burk and Dixon plots and estimation of inhibition-type and Ki values. Other procedures were similar to those of the reversible inhibition studies.

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Time-dependent inhibition study [1]
The time-dependent inhibition of CYP2C8 and CYP2J2 by Danshen components was measured by a traditional IC50 shift method when their IC50 values were lower than 5 μmol·L−1. Several different concentrations of test compounds were preincubated with recombinant CYPs (2 to 10-fold concentration), either with or without an NADPH-generating system, at 37 °C for 15 min. After inactivation incubation, aliquots (20–100 μL) were transferred to fresh incubation tubes (final volume 200 μL) containing an NADPH-generating system and CYPs substrate. The incubating, quenching, sample extracting and analytical procedures were the same as those in the inhibition-type study. IC50 shift was calculated with the IC50 values determined by the presence and absence of a NADPH-generating system (eq. (5)), and an obvious left shift (IC50 shift>2) would indicate a mechanism-based inactivator. (5)IC50shift = IC50(-NADPH) / IC50(+NADPH)

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Assays of time- and concentration-dependent inhibition of DI on CYP2C8 [1]
A preincubation mixture contained 100 mmol·L−1 potassium phosphate buffer (pH 7.4), recombinant CYP2C8 (10 nmol·L−1) and various concentrations (ranged from 0.5 to 8 μmol·L−1) of DI in a total volume of 100 μL. Control samples containing no test compound were prepared by adding solvent alone. While the preincubation mixture was maintained at 37 °C, 20 μL of the NADPH-generating system was added, and the mixture was further incubated for 30 min. At 0, 5, 10, 20 and 30 min of incubation, a 50 μL aliquot of the mixture was collected and added to an assay mixture (50 μL) consisting of 100 mM potassium phosphate buffer (pH 7.4), 10 μmol·L−1 taxol, and the NADPH-generating system for taxol hydroxylase activity determination. After the assay mixture for CYP2C8 activity was incubated for 20 min at 37 °C, 100 μL of midazolam prepared in acetonitrile was added to the system to terminate the reaction. And the concentrations of 6-hydroxytaxol were determined using LC-MS/MS.

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Determination of marker metabolites [1]
The 6-hydroxylation metabolite of taxol was quantitated by a partially validated LC-MS/MS method. After incubation with the recombinant CYP2C8 system at 37 °C, the reaction was stopped by adding 100 μL of 20.16 ng·mL−1 midazolam prepared in acetonitrile (internal standard). The tubes were vortex mixed for 1 min and then centrifuged at 12,000 rpm for 10 min. 5 μL of the resulted supernatant was injected into the LC-MS/MS system consisting of an Agilent 1100 Series HPLC system coupled to an API 4000 mass spectrometer, which equipped with electrospray ionization (ESI) source for ion production. Chromatographic separation was performed on a C18 column (1.8 μm, 100 × 4.6 mm; Agilent, USA) and the analytes were eluted with acetonitrile (A) and 0.1% formic acid aqueous solution (B) at a flow rate of 0.4 mL·min−1 by using the following gradient program: 0–0.3 min, 60% A; 0.3–2 min, 60%–90% A; 2–4 min, 90% A; 4–5.1min, 90%–60% A. The column temperature was maintained at 40 °C. The ion spray voltage was set at 5.0 kV for positive ionization and the heater gas temperature was 550 °C. Nitrogen was used as nebulizing gas (50 p.s.i), auxiliary gas (55 p.s.i) and curtain gas (15 p.s.i). The multiple reaction monitoring (MRM) experiments were conducted by monitoring the precursor ion to product ion transitions for 6-hydroxytaxol m/z 870.4–286.3 with declustering potential (DP) of 60 V and collision energy (CE) of −23 eV, and for midazolam m/z 326.3–291.1 with DP of 60 V and CE of 40 eV, which showed no internal interference. The method was linear in the concentration range of 1.17–117 nmolmL−1 for 6-hydroxytaxol. Intra-day and inter-day precision (CV%) was less than 2.7–8.2% and 4.8–11.4% respectively, and the accuracy ranged from 96.2 to 109.5% and 101.6 to 106.5%, respectively. Average recoveries and matrix effects ranged from 97.6 to 100.3% and 87.3 to 92.4%, respectively, at the concentrations of 2.00, 10.0 and 80.0 nmol·mL−1. 6-hydroxytaxol in treated samples was stable (92.1–103.3%) for at least 24 h in autosampler conditions.

BV2 microglia cells and primary rat microglial cells culture and grouping [2]
Cell injury model was accessed by LPS stimulation with a final concentration of 1 μg/mL. The experiment was divided into the following groups: normal control group (culture medium only), LPS stimulation model group (1 μg/mL LPS), Salvianolic Acid C/SalC low dose group (1 μg/mL LPS + 0.1 μM SalC), SalC middle dose group (1 μg/mL LPS + 1 μM SalC), and SalC high dose group (1 μg/mL LPS + 5 μM SalC). BV2 and primary microglia cells were both pre-incubated with 20 μM AMPK inhibitor Compound C for 1 h and then treated with SalC.

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MTT assay [2]
The effects of Salvianolic Acid C/SalC on cell viability was determined by MTT assay. First, BV2 microglia cells were seeded at a density of 1 × 104 cells/well on a 96-well plate. Then, cells were co-incubated with different concentrations of SalC and 1 μg/mL LPS for 24 h. Subsequently, the supernatant was removed, and MTT was added to each well. After 4 h incubation, 100 μL DMSO was added to each well, and the absorption was measured at 490 nm wavelength on a SpectraMax M5 microplate reader.

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NO detection [2]
Griess method was used to detect the NO levels in the supernatant. At the end of the experiment, 50 μL supernatant was transferred into a new well. Then 50 μL Griess Reagent I was added and mixed. 50 mL of Griess Reagent II was added and mixed. Finally, the absorbance was measured at 540 nm, and the NO concentration was calculated.

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Enzyme linked immunosorbent assay (ELISA) [2]
After LPS stimulation and Salvianolic Acid C/SalC incubation, the supernatants of culture medium were collected. And the concentrations of inflammatory mediators including TNF‑α, IL‑1β, IL‑6, IL‑10 and PGE2 were determined by the corresponding ELISA kits according to the manufacturer's instructions. The rats brain tissue concentrations of inflammatory mediators including TNF‑α, IL‑1β and IL‑6 in control group, LPS model group and LPS + SalC treated groups were also collected and determined according to the above manufacturer's instructions.

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Immunofluorescence assay [2]
The expression or intracellular localization of iNOS and p‑NF‑κB p65 was observed by immunofluorescence assay. First, BV2 microglia cells were seeded at a density of 1 × 104 cells per well on 96-well plates and incubated for 24 h. After co-incubation with different concentrations of Salvianolic Acid C/SalC and LPS for 24 h, the supernatant was removed out. BV2 microglia cells were fixed with 4% paraformaldehyde for 15 min. Cells were incubated with 0.3% Triton X-100 for 10 min, subsequently blocked with 5% BSA for 1 h, and incubated with primary antibodies against iNOS and p‑NF‑κB p65 at 4 °C overnight. Then, the fluorescent secondary antibody was added and incubated for 1.5 h. Hoechst 33258 was added and incubated for 30 min. Finally, the fluorescence images were acquired and measured under the Cellomics ArrayScan® VTI Imaging Platform

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Nrf2 siRNA transfection [2]
Cells were seeded in 6-well plates. After 24 h, 0.8 mL siRNA transfection medium was added to each well. After incubated with Nrf2 siRNA or control siRNA for 6 h, the normal culture medium was added again. Cells were incubated with different concentrations of Salvianolic Acid C/SalC and 1 μg/mL LPS for another 24 h. Finally, the ELISA assay, immunofluorescence assay and Western blot method were carried out.

Systemic neuroinflammation animal model and drug treatment [2]
Sprague–Dawley (SD) rats (210–240 g) were housed under a temperature of 21–24 °C, with a 12 h light/12 h dark cycle and a relative humidity of 50%. Rats were randomly divided into five groups (n = 12 for each group): control group, LPS model group and LPS + Salvianolic Acid C/SalC treated groups (SalC at 5, 10 and 20 mg/kg/day doses). Systemic administration of LPS was used to induce neuroinflammation in rats, which was approved by many previous reported studies. Rat systemic neuroinflammation was induced according to the previous reported procedure. Briefly, to induce systemic inflammation, LPS model group rats were i.p. administered with LPS in normal saline at 500 μg/kg dose for seven days. And during the seven days, the control group rats were i.p. administrated with only normal saline. The LPS + SalC treated groups were i.p. administered with SalC at doses of 5, 10, or 20 mg/kg each day 1 h after LPS injection for also seven days. Morris water maze test was performed 3 h after SalC injection.

[1]. Inhibitory Effects of Danshen components on CYP2C8 and CYP2J2. Chem Biol Interact. 2018 Jun 1;289:15-22.

[2]. Activation of Nrf2 signaling by salvianolic acid C attenuates NF‑κB mediated inflammatory response both in vivo and in vitro. Int Immunopharmacol. 2018 Oct;63:299-310.

Salvianolic acid C is a member of benzofurans.
Salvianolic acid C has been reported in Salvia miltiorrhiza, Origanum vulgare, and other organisms with data available.

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The use of Chinese herbal medicines and natural products has become increasingly popular in both China and Western societies as an alternative medicine for the treatment of diseases or as a health supplement. Danshen, the dried root of Salvia miltiorrhiza (Fam.Labiatae), which is rich in phenolic acids and tanshinones, is a widely used herbal medicine for the treatment of cardio-cerebrovascular diseases. The goal of this study was to examine the inhibitory effects of fifteen components derived from Danshen on CYP2C8 and CYP2J2, which are expressed both in human liver and cardiovascular systems. Recombinant CYP2C8 and CYP2J2 were used, and the mechanism, kinetics, and type of inhibition were determined. Taxol 6-hydroxylation and astemizole O-desmethyastemizole were determined as probe activities for CYP2C8 and CYP2J2, respectively. Metabolites formations were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results demonstrated that salvianolic acid A was a competitive inhibitor of CYP2C8 (Ki = 2.5 μM) and mixed-type inhibitor of CYP2J2 (Ki = 7.44 μM). Salvianolic Acid C had moderate noncompetitive and mixed-type inhibitions on CYP2C8 (Ki = 4.82 μM) and CYP2J2 (Ki = 5.75 μM), respectively. Tanshinone IIA was a moderate competitive inhibitor of CYP2C8 (Ki = 1.18 μM). Dihydrotanshinone I had moderate noncompetitive inhibition on CYP2J2 (Ki = 6.59 μM), but mechanism-based inhibition on CYP2C8 (KI = 0.43 μM, kinact = 0.097 min-1). Tanshinone I was a moderate competitive inhibitor of CYP2C8 (Ki = 4.20 μM). These findings suggested that Danshen preparations appear not likely to pose a significant risk of drug interactions mediated by CYP2C8 after oral administration; but their inhibitory effects on intestinal CYP2J2 mediated drug metabolism should not be neglected when they are given orally in combination with other drugs. Additionally, this study provided novel insights into the underling pharmacological mechanisms of Danshen components from the perspective of CYP2C8 and CYP2J2 inhibition. [1]

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In summary, our data indicate that TI, TIIA, Cry, DI, SAA, SAB and Salvianolic Acid C/SAC have inhibitory effects, to a varying degree, on CYP2C8 or CYP2J2 with IC50 values lower than 50 μmol·L−1. However, the IC50/Ki values determined in the current study are more than fifty times higher than the reported plasma concentration of these compounds in healthy humans following oral dosing of Danshen preparations. Therefore, neither Danshen preparations nor these components appear likely to pose a significant risk of drug interactions mediated by CYP2C8 after oral administration; however, their inhibitory effects on intestinal CYP2J2 mediated drug metabolism should not be neglected when they are given in combination with other drugs. Additionally, this study provided new insights into the underling pharmacological mechanisms of Danshen components from the perspective of CYP2C8 and CYP2J2 inhibition, which are very useful for deep understanding the interactions between Danshen and drug metabolizing enzymes.[1]

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Neurodegenerative diseases are closely related to neuroinflammation. Drugs targeting inflammation have been proved to be effective in many animal models. Salvianolic Acid C (SalC) is a compound isolated from Salvia miltiorrhiza Bunge, a plant with reported effects of inhibiting inflammation. However, the anti-inflammation effects and biological mechanisms of SalC on LPS-stimulated neuroinflammation remain unknown. The aim of this paper was to study its protective effects and its anti-inflammation mechanisms. LPS was used both in vivo and in vitro to induce neuroinflammation in SD rats and microglia cells. MTT assay was carried out to detect cell viability. The levels of TNF‑α, IL‑1β, IL‑6, IL‑10 and PGE2 were detected by ELISA method. The expressions of p‑AMPK, p‑NF‑κB p65, p‑IκBα, Nrf2, HO‑1 and NQO1 proteins were examined by Western blot analysis. The nuclear translocation of NF‑κB p65 was studied by immunofluorescence assay. The specific Nrf2 siRNA was used to clarify the interaction between Nrf2 and NF‑κB p65. The AMPK inhibitor Compound C was used study the upstream protein of Nrf2. Results showed that LPS induced the overexpression of inflammatory cytokines and mediated the phosphorylation and nuclear translocation of NF‑κB p65 in rat brains and microglia cells. SalC reversed the inflammatory response induced by LPS and inhibited the NF‑κB activation. SalC also upregulated the expression of p‑AMPK, Nrf2, HO‑1 and NQO1. But the anti-inflammation and NF‑κB inhibition effects of SalC were attenuated by transfection with specific Nrf2 siRNA or interference with the potent AMPK inhibitor Compound C. In conclusion, SalC inhibited LPS-induced inflammatory response and NF‑κB activation through the activation of AMPK/Nrf2 signaling both in vivo and in vitro. [2]

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In summary, the above results demonstrated that Salvianolic Acid C/SalC inhibited LPS-induced production of inflammatory cytokines through the inactivation of NF‑κB signaling. And the inactivation of NF‑κB was triggered by AMPK/Nrf2 signaling.[2]

C26H20O10

492.4310

492.105

115841-09-3

13991590

Light yellow to yellow solid powder

1.6±0.1 g/cm3

844.2±65.0 °C at 760 mmHg

464.4±34.3 °C

0.0±3.3 mmHg at 25°C

1.752

3.12

6

10

8

36

802

1

C1=CC(=C(C=C1C[C@H](C(=O)O)OC(=O)/C=C/C2=C3C=C(OC3=C(C=C2)O)C4=CC(=C(C=C4)O)O)O)O

GCJWPRRNLSHTRY-VURDRKPISA-N

InChI=1S/C26H20O10/c27-17-5-1-13(9-20(17)30)10-23(26(33)34)35-24(32)8-4-14-2-7-19(29)25-16(14)12-22(36-25)15-3-6-18(28)21(31)11-15/h1-9,11-12,23,27-31H,10H2,(H,33,34)/b8-4+/t23-/m1/s1

(2R)-3-(3,4-dihydroxyphenyl)-2-[(E)-3-[2-(3,4-dihydroxyphenyl)-7-hydroxy-1-benzofuran-4-yl]prop-2-enoyl]oxypropanoic acid

Salvianolic acid C; 115841-09-3; UNII-I16H9Z53ZL; I16H9Z53ZL; (2R)-3-(3,4-dihydroxyphenyl)-2-[(E)-3-[2-(3,4-dihydroxyphenyl)-7-hydroxy-1-benzofuran-4-yl]prop-2-enoyl]oxypropanoic acid; Benzenepropanoic acid, alpha-(((2E)-3-(2-(3,4-dihydroxyphenyl)-7-hydroxy-4-benzofuranyl)-1-oxo-2-propen-1-yl)oxy)-3,4-dihydroxy-, (alphaR)-; Benzenepropanoic acid, alpha-[[(2E)-3-[2-(3,4-dihydroxyphenyl)-7-hydroxy-4-benzofuranyl]-1-oxo-2-propen-1-yl]oxy]-3,4-dihydroxy-, (alphaR)-; BENZENEPROPANOIC ACID, .ALPHA.-(((2E)-3-(2-(3,4-DIHYDROXYPHENYL)-7-HYDROXY-4-BENZOFURANYL)-1-OXO-2-PROPEN-1-YL)OXY)-3,4-DIHYDROXY-, (.ALPHA.R)-;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~101.54 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.08 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.08 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)