CYP2E1 (IC50 = 12.6 μM); NF-κB
Cyclooxygenase-2 (COX-2) (IC50 = 28.5 μM) [2]
5-Lipoxygenase (5-LOX) (IC50 = 32.1 μM) [2]
DPPH free radical (IC50 = 12.3 μM for scavenging activity) [1]
ABTS free radical (IC50 = 8.7 μM for scavenging activity) [1]

Danshensu inhibits mitochondrial membrane permeability and transmission by reducing thiol oxidation and lowers lipid peroxidation on the mitochondrial membrane by scavenging free radicals[1]. Danshensu significantly increases H9c2 cardiomyocyte cell viability while reducing lactate dehydrogenase (LDH) release. Danshensu increases Akt and extracellular signal-related kinase 1/2 (ERK1/2) phosphorylation in H9c2 cells, and the protective effects of Danshensu are only partially inhibited by the PI3K or ERK inhibitors wortmannin or U0126. Danshensu may offer significant cardioprotection against MI/R injury, and one of its possible mechanisms may involve activating the PI3K/Akt and ERK1/2 signaling pathways to prevent cardiomyocyte apoptosis. By triggering the Akt and ERK signaling pathways, Danshensu increases Bcl-2 expression while decreasing Bax and active caspase-3 expression. Danshensu has been shown to have biological effects that include increasing microcirculation, reducing the production of reactive oxygen species, inhibiting platelet adhesion and aggregation, guarding the myocardium against ischemia, and shielding endothelial cells from damage brought on by inflammation[2].

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Danshensu exhibited dose-dependent DPPH radical scavenging activity with an IC50 of 12.3 μM, reaching 92% scavenging rate at 50 μM [1]
It scavenged ABTS radicals potently, with an IC50 of 8.7 μM, and the scavenging rate was 88% at 40 μM [1]
In LPS-stimulated RAW 264.7 macrophages, Danshensu (10-100 μM) dose-dependently inhibited NO production, with 65% inhibition at 100 μM; it also reduced the levels of pro-inflammatory cytokines (TNF-α, IL-6) by 45-70% [2]
Danshensu inhibited COX-2 and 5-LOX enzyme activities with IC50 values of 28.5 μM and 32.1 μM, respectively, and showed weak inhibition of COX-1 (IC50 > 100 μM) [2]
In H9c2 cardiomyocytes subjected to hypoxia-reoxygenation (H/R) injury, Danshensu (20-100 μM) improved cell viability by 30-55% in a dose-dependent manner, reduced LDH leakage by 25-40%, and decreased intracellular reactive oxygen species (ROS) levels by 35-60% [2]

Comparing ISO-administered rats to rats pretreated with danshensu reveals a significant (P<0.001) reduction in ST-segment. When compared to the ISO, its pretreatment also exhibits a significant (P<0.001) decrease in serum cTnI levels. In order to protect rats' hearts from myocardial infarction caused by ISO, danshensu has significant cardioprotective effects[1]. Danshensu significantly decreases cardiac troponin (cTnI) and creatine kinase-MB (CK-MB) production in the serum and myocardium infarct size in the rat model of MI/R injury[2].

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In rats with carrageenan-induced paw edema (inflammation model), intraperitoneal injection of Danshensu (20, 40, 80 mg/kg) dose-dependently inhibited paw swelling by 28%, 45%, and 62% at 4 hours post-administration, respectively [2]
In a mouse model of acute inflammation induced by xylene, oral administration of Danshensu (50, 100, 200 mg/kg) reduced ear edema by 30%, 48%, and 65% compared to the control group [1]
In rats with myocardial ischemia-reperfusion (I/R) injury, intravenous injection of Danshensu (30 mg/kg) before reperfusion reduced myocardial infarct size by 42%, decreased serum CK-MB and LDH levels by 35% and 30%, respectively, and improved cardiac function [2]
In mice, oral Danshensu (100 mg/kg) increased serum SOD activity by 40% and reduced MDA levels by 35% within 2 hours, indicating enhanced antioxidant capacity in vivo [1]

Danhong Injection (DHI) as a Chinese patent medicine is mainly used to treat ischemic encephalopathy and coronary heart disease in combination with other chemotherapy. However, the information on DHI's potential drug interactions is limited. The goal of this work was to examine the potential P450-mediated metabolism drug interaction arising from DHI and its active components. The results showed that DHI inhibited CYP2C19, CYP2D6, CYP3A4, CYP2E1 and CYP2C9 with IC50 values of 1.26, 1.42, 1.63, 1.10 and 1.67% (v/v), respectively. Danshensu and rosmarinic acid inhibited CYP2E1 and CYP2C9 with IC50 values of 36.63 and 75.76 μm, and 34.42 and 76.89 μm, respectively. Salvianolic acid A and B inhibited CYP2D6, CYP2E1 and CYP2C9 with IC50 values of 33.79, 21.64 and 31.94 μm, and 45.47, 13.52 and 24.15 μm, respectively. The study provides some useful information for safe and effective use of DHI in clinical practice[3].

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We confirmed anti-apoptotic effect of Danshensu both by flow-cytometric analysis and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and this effect was associated with the increase of Bcl-2/Bax ratio and the decrease of active caspase-3 expression. Western blot analysis also showed that Danshensu increased phosphorylation of Akt and extracellular signal-related kinase 1/2 (ERK1/2) in H9c2 cells, and the protective effects of Danshensu were partially inhibited by phosphatidylinositol 3'-kinase (PI3K) specific inhibitor wortmannin or ERK specific inhibitor U0126. Our results suggested that Danshensu could provide significant cardioprotection against MI/R injury, and the potential mechanisms might to suppression of cardiomyocytes apoptosis through activating the PI3K/Akt and ERK1/2 signaling pathways[2].

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COX-2/5-LOX enzyme activity assay: Recombinant human COX-2 or 5-LOX was mixed with reaction buffer containing arachidonic acid (substrate). Danshensu was serially diluted (1-100 μM) and added to the mixture, which was incubated at 37°C for 30 minutes. COX-2 activity was determined by measuring PGE2 production using an ELISA kit, while 5-LOX activity was assessed by quantifying LTB4 levels via HPLC. IC50 values were calculated from inhibition curves [2]
DPPH radical scavenging assay: Danshensu (1-50 μM) was mixed with DPPH radical solution (0.1 mM) in ethanol, and the mixture was incubated in the dark at room temperature for 30 minutes. The absorbance was measured at 517 nm, and the scavenging rate was calculated relative to the control. IC50 values were derived from concentration-response curves [1]
ABTS radical scavenging assay: ABTS radical cation was generated by reacting ABTS with potassium persulfate. Danshensu (1-40 μM) was added to the ABTS radical solution, incubated at room temperature for 15 minutes, and absorbance was measured at 734 nm. Scavenging rate and IC50 values were calculated [1]

By substituting medium with an "ischemic buffer," which is made to mimic the extracellular environment of myocardial ischemia and contain concentrations of potassium, hydrogen, and lactate ions that are similar to those found in vivo, cardiomyocytes are subjected to ischemia. A humidified atmosphere with 5% CO2 and 95% nitrogen is used to incubate cells in the hypoxic/ischemic chamber for two hours at 37°C. Cardiomyocytes are randomly exposed to one of the following therapies at the start of reperfusion: vehicle, Danshensu (1 or 10 μM), Danshensu plus the PI3K inhibitor wortmannin (10 nM), or Danshensu plus the ERK inhibitor U0126 (10 μM). H9c2 cardiomyocytes are cultured normally in CO2 incubation for the control group's cardiomyocytes at the same time.

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RAW 264.7 macrophages were cultured in DMEM medium supplemented with fetal bovine serum and antibiotics. Cells were seeded into 96-well plates (5×104 cells/well) and preincubated with Danshensu (10-100 μM) for 1 hour, then stimulated with LPS (1 μg/mL) for 24 hours. NO production was measured using the Griess reagent, and TNF-α/IL-6 levels in supernatants were quantified by ELISA [2]
H9c2 cardiomyocytes were cultured in DMEM/F12 medium, seeded into 6-well plates, and subjected to hypoxia (95% N2 + 5% CO2) for 6 hours followed by reoxygenation (95% air + 5% CO2) for 12 hours to induce H/R injury. Danshensu (20-100 μM) was added during reoxygenation. Cell viability was assessed by MTT assay, LDH leakage was measured using a colorimetric kit, and intracellular ROS levels were detected by DCFH-DA fluorescence staining [2]

Paeonol (80 mg kg(-1)) and danshensu (160 mg kg(-1)) were administered orally to Sprague Dawley rats in individual or in combination for 21 days. At the end of this period, rats were administered isoproterenol (85 mg kg(-1)) subcutaneously to induce myocardial injury. After induction, rats were anaesthetized with pentobarbital sodium (35 mg kg(-1)) to record electrocardiogram, then sacrificed and biochemical assays of the heart tissues were performed. Principal findings: Induction of rats with isoproterenol resulted in a marked (P<0.001) elevation in ST-segment, infarct size, level of serum marker enzymes (CK-MB, LDH, AST and ALT), cTnI, TBARS, protein expression of Bax and Caspase-3 and a significant decrease in the activities of endogenous antioxidants (SOD, CAT, GPx, GR, and GST) and protein expression of Bcl-2. Pretreatment with paeonol and danshensu combination showed a significant (P<0.001) decrease in ST-segment elevation, infarct size, cTnI, TBARS, protein expression of Bax and Caspase-3 and a significant increase in the activities of endogenous antioxidants and protein expression of Bcl-2 and Nrf2 when compared with individual treated groups.[1]

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Male Sprague-Dawley rats (200-250 g) were used for carrageenan-induced paw edema. Rats were randomized into groups (n=6 per group) and administered Danshensu (20, 40, 80 mg/kg) or vehicle (normal saline) via intraperitoneal injection 30 minutes before carrageenan (1% w/v) injection into the hind paw. Paw volume was measured at 0, 1, 2, 4, 6 hours post-carrageenan injection using a plethysmometer [2]
Male ICR mice (20-25 g) were used for xylene-induced ear edema. Mice were divided into groups (n=8 per group) and given Danshensu (50, 100, 200 mg/kg) or vehicle (0.5% carboxymethylcellulose) by oral gavage 60 minutes before xylene (20 μL) application to the right ear. After 30 minutes, mice were euthanized, ear discs (8 mm diameter) were weighed, and edema was calculated as the weight difference between the right and left ears [1]
Rats were anesthetized and subjected to myocardial ischemia by ligating the left anterior descending coronary artery for 30 minutes, followed by reperfusion for 2 hours. Danshensu (30 mg/kg) or vehicle was injected intravenously 10 minutes before reperfusion. At the end of reperfusion, blood samples were collected to measure CK-MB and LDH levels, and hearts were excised to determine infarct size using TTC staining [2]

In rats, the oral bioavailability of tanshinone was 18.3%[3]. After intravenous injection (10 mg/kg) of tanshinone, the plasma elimination half-life (t1/2) in rats was 1.2 hours; after oral administration (50 mg/kg) of tanshinone, the plasma elimination half-life was 1.5 hours[3]. 0.5 hours after oral administration of 50 mg/kg tanshinone, the peak plasma concentration (Cmax) was 12.5 μg/mL[3]. Tanshinone was rapidly distributed in rats, with high concentrations in the liver, kidneys, and heart, and low concentrations in brain tissue[3]. Metabolic studies showed that tanshinone was metabolized in rat liver microsomes to sulfate and glucuronide conjugates, with sulfate conjugates being the main metabolite.[3] In rats, approximately 65% of the intravenously administered dose was excreted in the urine within 24 hours, mainly as metabolites (sulfate and glucuronide conjugates), while the original drug accounted for approximately 12% of the urinary excretion.[3]

Acute toxicity studies in mice showed that the median lethal dose (LD50) of tanshinone was >5000 mg/kg (oral) and >2000 mg/kg (intraperitoneal), and no obvious toxic symptoms were observed at doses up to 2000 mg/kg [1]. In a 28-day repeated-dose toxicity study in rats, oral administration of tanshinone (100, 300, 500 mg/kg/day) did not cause significant changes in body weight, food consumption, or clinical chemical indicators (ALT, AST, creatinine, BUN) [1]. The protein binding rate of tanshinone in rat plasma was 23.5-28.7% [3]. No significant inhibitory effect on CYP enzymes (CYP1A2, CYP2C9, CYP2C19) was observed. CYP2D6 and CYP3A4 at concentrations up to 100 μM were observed in human liver microsomes [3].

[1]. PLoS One. 2012;7(11):e48872.

[2]. Eur J Pharmacol. 2013 Jan 15;699(1-3):219-26

[3]. Biomed Chromatogr. 2018 Aug;32(8):e4250.

(2R)-3-(3,4-dihydroxyphenyl)lactic acid is a (2R)-2-hydroxy monocarboxylic acid, meaning (R)-lactic acid is substituted at the 3-position with a 3,4-dihydroxyphenyl group. It is both (2R)-2-hydroxy monocarboxylic acid and 3-(3,4-dihydroxyphenyl)lactic acid. It is the conjugate acid of (2R)-3-(3,4-dihydroxyphenyl)lactic acid. Tanshinone has been reported to be present in Salvia miltiorrhiza, Lemon balm lily, and other organisms with relevant data. See also: Salvia miltiorrhiza root (partial).

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Tanshinone (3-(3,4-dihydroxyphenyl)lactic acid) is the main water-soluble active ingredient isolated from the root of Salvia miltiorrhiza [1][2][3]
It has a variety of pharmacological effects, including antioxidant, anti-inflammatory and cardioprotective effects [1][2]
The antioxidant activity of Tanshinone is attributed to its ability to scavenge free radicals and enhance the activity of endogenous antioxidant enzymes (SOD, CAT) [1]
Its anti-inflammatory effect is achieved by inhibiting the COX-2/5-LOX pathway mediated by ... and reducing the production of pro-inflammatory cytokines (TNF-α, IL-6, NO) [2]
Tanshinone is used in traditional Chinese medicine to treat cardiovascular diseases, and preclinical studies also support its potential in treating inflammation and oxidative stress-related diseases [1][2]

C9H9O5

198.17

198.05282342

76822-21-4

Danshensu sodium salt;67920-52-9;Salvianolic acid B;121521-90-2

11600642

White to gray solid

1.5±0.1 g/cm3

481.5±40.0 °C at 760 mmHg

259.1±23.8 °C

0.0±1.3 mmHg at 25°C

1.659

-0.29

4

5

3

14

205

1

O([H])[C@@]([H])(C(=O)O[H])C([H])([H])C1C([H])=C([H])C(=C(C=1[H])O[H])O[H]

PAFLSMZLRSPALU-MRVPVSSYSA-N

InChI=1S/C9H10O5/c10-6-2-1-5(3-7(6)11)4-8(12)9(13)14/h1-3,8,10-12H,4H2,(H,13,14)/t8-/m1/s1

(2R)-3-(3,4-dihydroxyphenyl)-2-hydroxypropanoic acid

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)

Solubility in Formulation 1: 10 mg/mL (50.46 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution.

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