Natural product

SA-B/salvianolic acid-B 0.1, 1, 10, and 100 micromol/L suppressed HSC proliferation concentration-dependently as determined by [3H]TdR uptake by 94.1 %, 82.4 %, 62.7 %, and 4 % of the control respectively (P<0.05 or P<0.01). SA-B 1, 10, and 100 micromol/L inhibited soluble type I collagen secretion by 75.3 %, 69.8 %, and 63.5 % of the control and decreased the matrix collagen deposition to 86.2 %, 75.4 %, and 73.4 % (P<0.05 or P<0.01). SA-B 1 and 10 micromol/L decreased the cell active TGF-beta1 secretion by 63.3 % and 15.6 % of the control, down-regulated pro-collgen alpha1(I) mRNA expression to 77.0 % and 51.8 % respectively (P<0.05). SA-B 1 and 10 micromol/L also inhibited MAPK activity by 1 to 2 fold respectively.
Conclusion: salvianolic acid-B/SA-B inhibited HSC proliferation and collagen production as well as decreased the cells' TGF-beta1 autocrine and MAPK activity, which might contribute to the mechanism of SA-B action against hepatic fibrosis.[1]

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Effect of salvianolic acid-B/SA-B on HSC proliferation [1]
SA-B 0.1–100 mmol/L inhibited the HSC intracellular uptake of [ 3H]TdR in a concentration-dependent manner and the inhibitory rates were 94.1 %, 82.4 %, 62.7 %, and 4 % of the control, respectively (Tab 1).

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Effect of salvianolic acid-B/SA-B on HSC collagen production [1]
Overproduction of extracellular matrix, particularly collagen, is an important feature of liver fibrosis. SA-B 1, 10, and 100 mmol/L suppressed soluble type I collagen to 75.3 %, 69.8 %, and 63.5 % of the control, and inhibited HSC total collagen deposition to 86.2 %, 75.4 %, and 73.4 % of the control, respectively (Tab 2). Also RT-PCR analysis showed that SA-B 1 and 10 mmol/L down-regulated the expression of type I procollagen mRNA to 77.0 % and 51.8 % of the control (Tab 3, Fig 1).

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Effect of salvianolic acid-B/SA-B on HSC TGF-b 1 secretion [1]
TGFb1 is one of the most potent cytokines for extracellular matrix synthesis and HSC is main effector and producer of TGF-b1 in the liver. On acidification, the active TGF-b1 was released from the complex of TGFb1 and latency-associated peptide (LAP), while SA-B 1 and 10 mmol/L significantly decreased the active TGFb1 content in the culture medium (Tab 3).

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Effect of salvianolic acid-B/SA-B on HSC MAPK activity [1]
Ras/ MAPK is an important pathway of TGF-b signaling in HSC. To elucidate the possible effects of SA-B onTGFb1 signaling in HSC, we examined MAPK activity. As shown in Fig 2, the TGF-b1 5 mg/L increased MAPK phosphorylation level remarkably, while SA-B 1 mmol/ L and 10 mmol/L reduced TGF-b1 stimulated-MAPK phosphorylation level by 1- and 2-fold respectively.

The aim of the present study was to examine the effect and possible mechanism of salvianolic acid B (SalB) on pulmonary microcirculation disturbance induced by lipopolysaccharide (LPS) in rat. Male Sprague-Dawley rats were subjected to thoracotomy under continuous anesthesia and mechanical ventilation. Albumin leakage from pulmonary capillary and the numbers of leukocytes adherent to the pulmonary capillary wall were determined for 60 min by an upright microscope upon LPS (2 mg · kg(-1) · h(-1)) infusion with or without administration of SalB (5 mg · kg(-1) · h(-1)). Pulmonary tissue wet-to-dry weight ratio, tumor necrosis factor α, and interleukin 8 in plasma and bronchoalveolar lavage fluid were measured. In addition, the expressions of E-selectin, intercellular adhesion molecule 1, and myeloperoxidase in pulmonary tissue were assessed by immunohistochemistry. The expressions of aquaporin 1 (AQP-1), AQP-5, metalloproteinase 2 (MMP-2), and MMP-9 were assessed by Western blot assay. Pretreatment with SalB significantly attenuated LPS-induced pulmonary microcirculatory disturbance, including the increase in leukocyte adhesion and albumin leakage. In addition, LPS increased pulmonary tissue wet-to-dry weight ratio and tumor necrosis factor α and interleukin 8 levels in plasma and bronchoalveolar lavage fluid enhanced the expression of E-selectin, intercellular adhesion molecule 1, myeloperoxidase, MMP-2, and MMP-9, whereas it decreased the expression of AQP-1 and AQP-5 in pulmonary tissue, all of which were attenuated by SalB pretreatment. Salvianolic acid B pretreatment improves pulmonary microcirculation disturbance and lung injury on LPS exposure. More studies are required to evaluate the potential of SalB as an option for protecting lung from endotoxemia. [2]

MAPK activity assay [1]
HSC was treated with TGF-b1 5 mg/L for 1 h and then incubated with salvianolic acid B/SA-B. The cytoplasmic proteins were lysed and immunoprecipitated with p44/p42 MAPK antibody. After reacting with EIK-1 and ATP substrate, the immunoprecipitated antigen was fractionated by SDS/polyacrylamide gel electrophoresis, and were transferred onto PVDF membranes. After blocking with 5 % skimmilk, the blot was incubated with p44/p42 MAPK primary antibody (1:1000) overnight at 4 . Then the blot was washed and incubated with HRP-coupled second antibody and signals were detected by ECL film.

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Pro-collagen type I mRNA level analysis [1]
Total RNA was extracted from the cells by the acid-guandium -phenol-chloroformmethod as described . mRNA levels of TGF-b1 and pro-collagen type I were analyzed by reverse-transcription polymerase chain reaction (RTPCR), while house keeping gene-"b-actin" was used as the internal standard. The left and right primersfor bactin were respectively as follows: 5’-ACA TCT GCT GGA AGG TGG AC-3’ and 5’-GGT ACC ACC ATG TAC CCA GG-3’ (the expected product size 163 bp) . The left and right primers for type I pro-collagen a1 (I): 5’-CACCCT CAA GAG CCT GAGTC-3’ and 5’-GTT CGG GCT GAT GTA CCA GT-3’ (the expected product size 253 bp). The RT-PCR reaction was carried on with Access RT-PCR kit. The products were fractionated in 2 % agarose electrophoresis containing ethidium bromide (EB) 0.5 mg/L and photographed under UV transilluminator. Their density was semiquantitatively analyzed with MPIAS-500 image system

Cell proliferation assay [1]
HSC proliferation was measured by [ 3H]TdR uptake. Confluent HSC in 24- well plates wasincubated with salvianolic acid B/SA-B for 48 h (the following as the same), and impulsed with 74 kBq/well [ 3H]TdR among the last 24 h. Then the cells were harvested by trypsinization and collected onto 0.45 mm membrane. The radioactivity of intracellular [ 3H]TdR incorporation was counted by Beckman Wallac 1410 liquid-scintillator.

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Collagen deposition measurement [1]
After treated with salvianolic acid B/SA-B, cells on the 24 well-plate were washed with phosphate buffer saline (PBS), fixed with 10 % formalin, dehydrated with 70 % ethanol, and stained with Victorial blue for 15 min. After washing with distilled water twice, cells were stained with Ponceau S for 2 min and dehydrated with ethanol twice. The collagen deposition was analyzed according to its optical density with computer MPIAS-500 image analytic system.

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Type I collagen and active TGF-b 1 content assay [1]
After drug treatment, cells were incubated with serum free M199 for 24 h and the supernatants were collected. Type I collagen contents were assayed with ELISA according to Rennard’s method. For active TGF-b1 content assay, the supernatants were acidified with HCl 1 mol/L and then measured by ELISA with the TGF-b1 immunoassay system kit according to the manufacturer's instructions.

A total of 90 rats were divided into three groups, six animals in each group in evaluation of each parameter (see Table 1 for further detail). For the animals in the control group, saline (1 mL kg−1 · h−1) was continuously infused via the left jugular vein catheter until the end of the observation. The animals in the LPS group were continuously infused with LPS in saline (2 mg · kg−1 · h−1) via the left jugular vein catheter until the end of the observation. In the animals in the salvianolic acid B/SalB pretreatment group (SalB + LPS group), the SalB (5 mg · kg−1 · h−1) was continuously infused via the left jugular vein catheter starting from 20 min before LPS administration until the end of the observation. The doses of SalB and LPS used in the present study were selected based on the report of Guo et al. (9), wherein LPS at a dose of 2 mg · kg−1 · h−1 was shown to induce obvious microcirculatory disturbance without affecting the systematic circulation. For each variable, an equilibration period of 10 min was allowed before recording, and thereafter, observation was carried out every 15 min for a total period of 1 h. [1]

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Evaluation of pulmonary edema [1]
In another set of experiments, we used the same rat model to determine the lung wet-to-dry weight (W/D) ratio to evaluate the effect of salvianolic acid B/SalB on LPS-induced pulmonary edema. Briefly, 1 h after reagent administration, rats were killed, and lungs were removed. The right main bronchus was ligated, and 2 mL of saline was injected via a tracheal cannula to the left lung, and 1.2 mL of fluid was collected as bronchoalveolar lavage fluid (BALF) (18). The left lung then was excised to determine the levels of interleukin 8 (IL-8) and TNF-α, whereas the right lung was excised to measure the pulmonary W/D ratio. The lung tissue samples were weighted and dried at 80°C for 48 h. Dry weights were measured, and the W/D ratio of lung tissue was calculated. [1]

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IL-8 in plasma and BALF [1]
The IL-8 concentration in rat blood plasma and BALF was measured quantitatively using an enzyme-linked immunoabsorbent assay. Briefly, the wells of a 96-well immunoplate were coated with capture antibody (goat anti–rat antibody diluted at 1:396 in coating buffer) and incubated overnight at 4°C. Nonspecific binding sites were blocked with blocking buffer containing 5% nonfat dry milk in sterile PBS. The wells were washed with PBS. Bronchoalveolar lavage fluid and blood plasma from the LPS and salvianolic acid B/SalB + LPS groups were diluted to 1:5 in blocking buffer. Bronchoalveolar lavage fluid and blood plasma from the control group were not diluted. The IL-8 standard peptide was diluted from 0 to 25 ng · mL−1 in blocking buffer for generating a standard curve. The samples were added to the wells and incubated overnight at room temperature. After washing, the specific antibody (rabbit anti-rat) diluted to 1:20,000 in blocking buffer was added and incubated at room temperature for 2 h. Thereafter, the horseradish peroxidase–conjugated goat anti–rabbit antibody was applied and incubated for 30 min at room temperature. An immunopure tetramethyl benzidine substrate kit was used for detection. The reaction was stopped with 1 mol H2SO4 after 15 min and read at 492 nm.

[1]. Effect of salvianolic acid B on collagen production and mitogen-activated protein kinase activity in rat hepatic stellate cells. Acta Pharmacol Sin. 2002 Aug;23(8):733-8.
[2]. Salvianolic acid B protects from pulmonary microcirculation disturbance induced by lipopolysaccharide in rat. Shock. 2013 Mar;39(3):317-25.

Salvianolic acid B is a member of the class of 1-benzofurans that is an antioxidant and free radical scavenging compound extracted from S. miltiorrhiza It has a role as a plant metabolite, an anti-inflammatory agent, an antioxidant, a hypoglycemic agent, an osteogenesis regulator, an apoptosis inducer, a hepatoprotective agent, a neuroprotective agent, a cardioprotective agent, an autophagy inhibitor, an antidepressant and an antineoplastic agent. It is a polyphenol, a member of 1-benzofurans, an enoate ester, a dicarboxylic acid and a member of catechols.
Lithospermic acid B has been reported in Salvia miltiorrhiza, Celastrus hindsii, and other organisms with data available.
See also: Salvia Miltiorrhiza Root (part of).

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Aim: To investigate the mechanism of salvianolic acid B (SA-B) action against liver fibrosis relating to mediating hepatic stellate cell (HSC) activation and transforming growth factor-beta1(TGF-beta1) intracellular signal transduction. Methods: HSC was isolated from normal rat through in situ perfusion of liver with pronase E and density-gradient centrifugation with 11 % nycondenz, then cells were subcultured. Cell proliferation was observed by [3H]TdR uptake. Cellular collagen deposition was measured with Ponceau S stain and semi-quantified with image analytic system. Type I collagen secretion in the supernatant was detected with ELISA. The gene expression of type I pro-collagen was analyzed by RT-PCR. The supernatant was acidified and active TGF-beta1 contents were assayed with ELISA. Mitogen-activated protein kinase (MAPK) activity was analyzed with immunoprecipitation and Western blot. Results: SA-B 0.1, 1, 10, and 100 micromol/L suppressed HSC proliferation concentration-dependently as determined by [3H]TdR uptake by 94.1 %, 82.4 %, 62.7 %, and 4 % of the control respectively (P<0.05 or P<0.01). SA-B 1, 10, and 100 micromol/L inhibited soluble type I collagen secretion by 75.3 %, 69.8 %, and 63.5 % of the control and decreased the matrix collagen deposition to 86.2 %, 75.4 %, and 73.4 % (P<0.05 or P<0.01). SA-B 1 and 10 micromol/L decreased the cell active TGF-beta1 secretion by 63.3 % and 15.6 % of the control, down-regulated pro-collgen alpha1(I) mRNA expression to 77.0 % and 51.8 % respectively (P<0.05). SA-B 1 and 10 micromol/L also inhibited MAPK activity by 1 to 2 fold respectively. Conclusion: SA-B inhibited HSC proliferation and collagen production as well as decreased the cells' TGF-beta1 autocrine and MAPK activity, which might contribute to the mechanism of SA-B action against hepatic fibrosis.[1]

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In summary, salvianolic acid B/SA-B could effectively inhibit HSC proliferation and collagen production and interfere with TGF-b1/MAPK signal transduction. These actions underlie the mechanism of SA-B effect against hepatic fibrosis. [1]

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The present study has some limitations. For example, the observation time in the experiments was 60 min, at a very early phase of the development of ALI and ARDS; thus, the outcome of ALI and ARDS after salvianolic acid B/SalB treatment needs further exploration. Yet, the present study examined the effect of SalB pretreatment on LPS-induced pulmonary injury; it remains unclear whether this agent will have any salutary effects on ALI if administered following endotoxemia, which is more clinically relevant, particularly in the intensive care unit, and remains to be studied. In summary, the present study demonstrated that pretreatment with SalB significantly improved the LPS-induced pulmonary microcirculatory dysfunction and lung injury. The potential of SalB as a prophylaxis for the patients with the risk of ALI and ARDS is worthy to be explored. [2]

C36H30O16

718.6138

718.153

115939-25-8

6451084

Off-white to yellow solid powder

1.6±0.1 g/cm3

1020.3±65.0 °C at 760 mmHg

322.1±27.8 °C

0.0±0.3 mmHg at 25°C

1.739

2.14

9

16

14

52

1290

4

O1C2=C(C([H])=C([H])C(C([H])=C([H])C(=O)OC([H])(C(=O)O[H])C([H])([H])C3C([H])=C([H])C(=C(C=3[H])O[H])O[H])=C2C([H])(C(=O)OC([H])(C(=O)O[H])C([H])([H])C2C([H])=C([H])C(=C(C=2[H])O[H])O[H])C1([H])C1C([H])=C([H])C(=C(C=1[H])O[H])O[H])O[H]

SNKFFCBZYFGCQN-VWUOOIFGSA-N

InChI=1S/C36H30O16/c37-20-6-1-16(11-24(20)41)13-27(34(45)46)50-29(44)10-5-18-3-9-23(40)33-30(18)31(32(52-33)19-4-8-22(39)26(43)15-19)36(49)51-28(35(47)48)14-17-2-7-21(38)25(42)12-17/h1-12,15,27-28,31-32,37-43H,13-14H2,(H,45,46)(H,47,48)/b10-5+/t27-,28-,31+,32-/m1/s1

(2R)-2-[(E)-3-[(2S,3S)-3-[(1R)-1-carboxy-2-(3,4-dihydroxyphenyl)ethoxy]carbonyl-2-(3,4-dihydroxyphenyl)-7-hydroxy-2,3-dihydro-1-benzofuran-4-yl]prop-2-enoyl]oxy-3-(3,4-dihydroxyphenyl)propanoic acid

115939-25-8; Salvianolic-acid-B; Salvianic acid B; (R)-2-(((2R,3R)-4-((E)-3-((R)-1-Carboxy-2-(3,4-dihydroxyphenyl)ethoxy)-3-oxoprop-1-en-1-yl)-2-(3,4-dihydroxyphenyl)-7-hydroxy-2,3-dihydrobenzofuran-3-carbonyl)oxy)-3-(3,4-dihydroxyphenyl)propanoic acid; (2R)-2-[(E)-3-[(2R,3R)-3-[(1R)-1-carboxy-2-(3,4-dihydroxyphenyl)ethoxy]carbonyl-2-(3,4-dihydroxyphenyl)-7-hydroxy-2,3-dihydro-1-benzofuran-4-yl]prop-2-enoyl]oxy-3-(3,4-dihydroxyphenyl)propanoic acid; 1607436-77-0; MFCD23115783; CHEMBL3747259;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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