Natural product

(Rac)-Salvianic acid A ((Rac)-Danshensu) is more effective than vitamin C at scavenging free radicals, superoxide anion radicals (O2), 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radicals, and 2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radicals[1].

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Danshensu (3-(3,4-dihydroxyphenyl) lactic acid) and salvianolic acid B, two natural phenolic acids of caffeic acid derivatives isolated from Salvia miltiorrhiza root of the most widely used traditional Chinese medicine for the treatment of various cardiovascular diseases, have been reported to have potential protective effects from oxidative injury. To better understand their biological functions, the in vitro radical scavenging and antioxidant activities of danshensu and salvianolic acid B were evaluated along with vitamin C. Both danshensu and salvianolic acid B exhibited higher scavenging activities against free hydroxyl radicals (HO()), superoxide anion radicals (O(2)(-)), 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radicals and 2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radicals than vitamin C. In contrary, danshensu and salvianolic acid B showed weaker iron chelating and hydrogen peroxide (H(2)O(2)) scavenging activities than vitamin C. As expressed as vitamin C equivalent capacity (VCEAC), the relative VCEAC values (mg/100ml) were in the order of salvianolic acid B (18.59) > danshensu (12.89) > vitamin C (10.00) by ABTS radical assay. The protective efficiencies against hydrogen peroxide induced human vein vascular endothelial cell damage were correlated with their antioxidant activities. Analysis of structure-activity relationship of these two compounds showed that the condensation and conjugation of danshensu and caffeic acid appears important for antioxidant activity. These results indicated that danshensu and salvianolic acid B are efficient radical scavengers and antioxidants, and salvianolic acid B is superior to danshensu. Their radical scavenging and antioxidant properties might have potential applications in food and healthcare industry. [1]

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Effects of Danshensu on SARS-CoV-2 and S protein-pseudo-typed virus in vitro [3]
As shown in Fig. 1a, e, Danshensu showed the potent antiviral activity with EC50 of 0.97 μM and a concentration-dependent manner in inhibiting SARS-CoV-2. Danshensu was also identified to potently inhibit the entry of SARS-CoV-2 S protein-pseudo-typed virus (SARS-CoV-2 S) into ACE2-overexpressed HEK-293T cells (IC50 = 0.31 μM) and Vero-E6 cell (IC50 = 4.97 μM) (Fig. 1b, c), but it had no significant inhibitory effect on VSV-G pseudo-virus (Fig. 1d). The S protein could only be detected from supernatants containing SARS-CoV-2 S to confirm the pseudo-typed SARS-CoV-2 as observed in Fig. 1f. In total, Danshensu has shown a potential antiviral ability against SARS-CoV-2.

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Background: Oral squamous cell carcinoma (OSCC) that comprises about 90% of all oral cancer cases is associated with poor prognosis due to its highly metastatic nature. The majority of OSCC treatment options are related detrimental side-effects. Hypothesis/Purpose: The present study aimed at deciphering the effects of a bioactive phytochemical, sodium Danshensu, on human oral cancer cell metastasis. Methods and Results: The treatment of FaDu and Ca9-22 cells with different doses of sodium danshensu (25, 50, and 100 μM) caused a significant reduction in cellular motility, migration, and invasion, as compared to the untreated cells. This effect was associated with a reduced expression of MMP-2, vimentin and N-cadherin, together with an enhanced expression of E-cadherin and ZO-1. Further investigation on the molecular mechanism revealed that treatment with sodium danshensu caused significant reduction in p38 phosphorylation; however, phosphorylation of ERK1/2 significantly decreased only in FaDu cells, whereas p-JNK1/2 did not show any alteration. A combination of p38 and JNK1/2 inhibitors with sodium danshensu also reduced the migration in the FaDu and Ca9-22 cell lines. Conclusion: Collectively, the present study findings reveal that sodium danshensu execute anti-metastatic effect by suppressing p38 phosphorylation in human oral cancer. The study identifies sodium danshensu as a potential natural anticancer agent that can be used therapeutically to manage highly metastatic OSCC.[4]

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The effect of Danshensu on cisplatin-induced oxidative stress [5]
The antioxidant effect of Danshensu was verified by mouse kidney injury induced by cisplatin and HK-2 cells. Lipid peroxides such as MDA is considered as an easily accessible biomarker of ROS damage. Moreover, cells have developed various endogenous antioxidant enzymes (e.g. SOD, CAT, and GSH-Px) to detoxify ROS and prevent oxidative stress. The changes of total reactive oxygen species (tROS), MDA and antioxidant enzymes were assayed to evaluate the effect of Danshensu on cisplatin-induced oxidative stress in vivo and in vitro. In vivo, cisplatin significantly increased the levels of tROS and MDA compared to the control group in kidney tissues. Danshensu groups can attenuate the levels of tROS and MDA at three doses (Fig. 3A and B). Meanwhile, reduced activities of the renal antioxidant enzymes were observed in cisplatin group. Danshensu treated groups showed an increase in the activities of antioxidant enzymes compared to cisplatin group in kidney tissues (Fig. 3C–E).

In vitro, the cell model, made of HK-2 cells stimulated by cisplatin, was used to evaluate the effect of Danshensu on cell activity and the production of reactive oxygen species. First, cytotoxicity test was carried out. It was confirmed that the concentration of Danshensu between 2.5 and 20 μM was non-cytotoxic and the IC50 of cisplatin was 20 μM (Fig. 4A and B). The results showed that Danshensu could attenuate the cytotoxicity of cisplatin at the concentration of 5 μM and 10 μM (Fig. 4C). High level of ROS was also found in HK-2 cells incubated by cisplatin, however, Danshensu could reduce the level of ROS at dose of 10 μM (Fig. 4D). It can be concluded through the results that Danshensu has antioxidant effect on oxidative stress both in mice and in HK-2 cells caused by cisplatin.

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The effects of Danshensu on oxidative stress through Nrf2/ HO-1 pathway [5]
The Nrf-2 was found to be an important regulator of body resistance to oxidative stress. The expression of Nrf2, HO-1 and NQO1 was assayed to discuss the mechanism of Danshensu on anti-oxidative stress induced by cisplatin through the activation of Nrf2/ HO-1pathway. As shown in Fig. 5, Compared to the control group, cisplatin group exhibited increases in the expression of Nrf2, HO-1 and NQO1 in both kidney tissues and HK-2 cells. Danshensu groups dose-dependently up-regulated the expression of Nrf2 and HO-1 to activate Nrf2/ HO-1 pathway.

In rabbits, (Rac)-Salvianic acid A ((Rac)-Danshensu; iv; 30 mg/kg) exhibits a T1/2 of 16.6 min. Mice received SARS-CoV-2 S via trachea to induce ALI, while the VSV-G treated mice served as controls. The mice were administered Danshensu (25, 50, 100 mg/kg, i.v., once) or Danshensu (25, 50, 100 mg·kg-1·d-1, oral administration, for 7 days) before SARS-CoV-2 S infection. We showed that SARS-CoV-2 S infection induced severe inflammatory cell infiltration, severely damaged lung tissue structure, highly expressed levels of inflammatory cytokines, and activated TLR4 and hyperphosphorylation of the NF-κB p65; the high expression of angiotensinogen (AGT) and low expression of ACE2 at the mRNA level in the lung tissue were also observed. Both oral and intravenous pretreatment with Danshensu dose-dependently alleviated the pathological alterations in mice infected with SARS-CoV-2 S. This study not only establishes a mouse model of pseudo-typed SARS-CoV-2 (SARS-CoV-2 S) induced ALI, but also demonstrates that Danshensu is a potential treatment for COVID-19 patients to inhibit the lung inflammatory response.[3]

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Danshensu could prevent SARS-CoV-2 S protein-induced acute lung inflammation [3]
H&E staining was conducted to observe the protective effect of Danshensu on physiological impairment. As revealed in Fig. 3, pathological changes in lung tissue of mice in each group were observed with a microscopic analysis after H&E staining. The alveolar structure of mice in the VSV-G group was normal, without the hemorrhages, shrink of alveolar space, noticeable thickening of the alveolar septum or inflammatory cell infiltration. After establishing the model, mice in the SARS-CoV-2 S group exhibited a thickened alveolar septum, collapsed and reduced alveoli, and marked inflammatory cell infiltration. In contrast, these changes were obviously alleviated by Danshensu in a dose-dependent manner. In the Danshensu 50 and 100 mg/kg groups, the alveolar septum was slightly thickened, a few inflammatory cells could be seen and some of the alveoli were shrunken in appearance (Fig. 3c, d). Lung histological scores in the SARS-CoV-2 S group were significantly higher than the blank group and VSV-G group, while lung histological scores in the Danshensu 50 and 100 mg/kg groups and Danshensu i.v. 100 mg/kg groups were significantly lower than in the SARS-CoV-2 S group (Fig. 3a, d) (all P < 0.05). Analytical results demonstrated that Danshensu obviously ameliorated the histopathological condition in SARS-CoV-2 S-induced acute lung inflammation.

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Danshensu ameliorated inflammatory cytokines in serum and lung tissue [3]
The effects of Danshensu on serum cytokines including IL-6, IL-1β, and TNF-α were examined with ELISA kits. As revealed in Fig. 4a–c, the levels of TNF-α, IL-1β, and IL-6 in serum samples presented apparent increases with SARS-CoV-2 S stimulation, compared to the blank and VSV-G groups. However, Danshensu significantly reduced the levels of TNF-α, IL-1β and IL-6 in the serum, compared with those in the SARS-CoV-2 S group. Expression of the mRNA for TNF-α, IL-1β, and IL-6 was detected in the lung of all group mice, as shown in Fig. 4d–f. TNF-α, IL-1β, and IL-6 were significantly reduced by the treatment with Danshensu 50 and 100 mg/kg, whereas SARS-CoV-2 S elevated the mRNA levels of these pro-inflammatory biomarkers.

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The antioxidant system could be protected by treatment with Danshensu [3]
Fig. 4 illustrated the effects of Danshensu on oxidative stress. SARS-CoV-2 S administration decreased CAT (Fig. 4h) and GPx (Fig. 4i) activities. In contrast, pretreatment with Danshensu enhanced the activities of CAT and GPx.

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The regulation of p-NF-κB p65 and TLR4 expression in SARS-CoV-2 S-induced lung tissues by Danshensu [3]
TLR4 is the traditional regulating receptor in the NF-κB p65 pathway and, indirectly, have operated an important role in the upregulation of TNF-α, IL-1β, and IL-6. As depicted in Fig. 5a, SARS-CoV-2 S-treated mice showed obvious upregulation of pneumonic expressions of TLR4 and p-NF-κB p65. However, Danshensu effectively suppressed the activations of TLR4 and NF-κB p65 in the lung tissues of SARS-CoV-2 S-treated mice.

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Danshensu could reverse the mRNA expression of ACE2, AGT, and inflammatory cytokines in SARS-CoV-2 S-induced lung tissues [3]
The lung of mice was analyzed by qPCR, to assess the induction of mRNA for ACE2 and AGT. Expression of the mRNA for ACE2 and AGT was detected in the lungs of all mice, as shown in Fig. 5b, c. AGT was significantly reduced by the treatment of Danshensu (Fig. 5c), whereas SARS-CoV-2 S elevated the mRNA levels of AGT. The mRNA level of the ACE2 was significantly promoted by the treatment with Danshensu (Fig. 5b). Danshensu i.v. at the dose of 100 mg/kg and pretreatment with Danshensu effectively reduced the mRNA expression of pro-inflammatory genes AGT to the normal levels and elevated the mRNA expression of ACE2. Danshensu i.v. at 50 mg/kg did also diminish the mRNA expression of AGT in some cases.

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The clinical application of cisplatin was mainly limited by severe nephrotoxicity. Danshensu was the main pharmacological active diterpenoids which extracted from the roots of Salvia milthiorriza Bunge. This study is aimed to investigate the protective effects and potential mechanisms of Danshensu against cisplatin-induced nephrotoxicity. After fasting for 12 h, all mice groups except the control group were administered a single intraperitoneal injection of 25 mg/kg cisplatin. 1 h later, cisplatin (25 mg/kg) + Danshensu (15 mg/kg, 30 mg/kg, 60 mg/kg) groups were treated with corresponding doses of Danshensu once a day for 7 consecutive days. Blood urea nitrogen (BUN), creatinine, reactive oxygen species (ROS), superoxide dismutase (SOD), Glutathione peroxidase (GPx), Catalase (CAT) and malondialdehyde (MDA) were assayed in this study. The expression of inflammatory cytokines TNF-α, IL-6 and IL-1β were examined by ELISA. The results showed that Danshensu could improve kidney damage, attenuate serum BUN, creatinine, cytokines and oxidative stress markers. Further studies showed that Danshensu can induce Nrf2/HO-1 activation and inhibition of NF-κB pathway. In conclusion, Danshensu exerts the protective effects on cisplatin-induced nephrotoxicity, which may be related to the activation of Nrf2/HO-1 and inhibition of NF-ĸB pathway. [5]

Metal chelating activity assay [1]
The chelating activity for ferrous ions was measured according to the method of Dinis et al. (1994). The reaction mixture contained 0.5 ml of various concentrations of test compounds, 1.6 ml of deionized water and 0.05 ml of 2 mM of FeCl2 solution. After 30 s, 0.1 ml of 5 mM ferrozine solution was added. Fe2+-Ferrozine magenta complex was very soluble and stable in water. After 10 min at room temperature, the absorbance at 562 nm was measured. The relative activities of test compounds to chelate ferrous iron were expressed as percentage (%) of absorbance disappearance [(Acontrol−Asample)/Acontrol × 100].

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Hydrogen peroxide scavenging activity assay [1]
Hydrogen peroxide scavenging activity was measured by replacement titration (Zhao et al., 2006). A solution of 1.0 ml of 0.1 mM H2O2 and 1.0 ml of various concentrations of test compounds were mixed, followed by 2 drops of 3% ammonium molybdate, 10 ml of 2 M H2SO4 and 7.0 ml of 1.8 M KI. The mixed solution was titrated with 5.09 mM NaS2O3 until yellow color disappeared. The relative activities of test compounds to scavenge hydrogen peroxide were expressed as percentage (%) of the titer volume change [(Vcontrol−Vsample)/Vcontrol × 100].

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Hydroxyl radicals scavenging activity assay [1]
The assay of scavenging activity for hydroxyl radicals was based on Fenton reaction (Yu et al., 2004). Reaction mixture contained 0.02 mM FeCl2, 0.03 mM 1,10-phenanthroline, 0.16 M phosphate buffer (pH 7.8), 8.5 mM H2O2, and various concentrations of test compounds. The total volume of the mixture was 3 ml. The reaction was started by adding H2O2. After incubation at room temperature for 5 min, the absorbance at 560 nm was measured. The relative hydroxyl radicals scavenging activities were calculated similarly to that of metal chelating activity assay.

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Superoxide anion scavenging activity assay [1]
Superoxide anions were generated in a non-enzymatic PMS–NADH system and measured by the reduction of NBT according to the method of Liu et al. (1997). A 0.75 ml of 120 μM PMS solution was added to 3 ml of 100 mM Tris–HCl buffer (pH 7.4) containing 0.75 ml of 300 μM NBT solution, 0.75 ml of 936 μM NADH solution and 0.3 ml of different concentrations of test compounds. After 5 min of incubation at room temperature, the absorbance at 560 nm was measured. The results were expressed as percentage (%) of superoxide anion scavenging calculated similarly to that of metal chelating activity assay.

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Vitamin C equivalent antioxidant capacity (VCEAC) assay [1]
Total antioxidant capacity of test compounds was expressed as VCEAC using DPPH radicals and ABTS radicals. VCEAC was determined and calculated as the method of Kim et al., 2002, Kim and Lee, 2004.
DPPH free radicals scavenging activity assay. [1]
An aliquot of 2.9 ml of 0.1 μM DPPH solution in 80% methanol and 0.1 ml of test compounds at various concentrations were mixed. The mixture was shaken vigorously and allowed to reach a steady state at room temperature in the dark for 30 min. Decolorization of DPPH was determined by measuring the absorbance at 517 nm.

ABTS free radical scavenging activity assay. [1]
A mixture of 1.0 mM AAPH and 2.5 mM ABTS in 100 mM phosphate buffer saline (PBS) solution (pH 7.4, containing 150 mM NaCl) was prepared and incubated in a water bath at 68 °C for 13 min to generate the colored ABTS radicals. The concentration of the resulting ABTS radical solution was adjusted with PBS to an absorbance of 0.650 ± 0.020 at 734 nm. Aliquots of 60 μl testing samples at various concentrations was added to 2.94 ml ABTS radical solution and allowed to react in a water bath at 37 °C for 10 min. The decrease of absorbance at 734 nm was measured. Standard curves of absorbance reduction of ABTS radicals and DPPH radicals versus vitamin C concentration were generated. The absorbance decreases at 734 nm for ABTS radicals and 517 nm for DPPH radicals by salvianolic acid and Danshensu were measured at various concentrations. They were converted into VCEAC using their respective vitamin C standard curves. The scavenging activities of tested compounds against ABTS and DPPH radicals were calculated as VCEAC in mg/100 ml.

Cell culture and cell viability assay using MTT [1]
Human umbilical vein endothelial cells were isolated from freshly obtained human umbilical cords by collagenase type II treatment as previously described (Ding et al., 2005a). Endothelial cells were counted and seeded at a density of 5 × 104 cells/well onto gelatin-coated 96-well plates containing endothelial growth medium supplemented with 15% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin, and cultured at 37 °C in a humidified environment containing 5% CO2. After treatment with various concentrations of salvianolic acid or danshen for 4 h followed with 0.4 mM H2O2 for 18 h, each well was washed twice with PBS to remove the medium. One hundred microliter of MTT (0.5 mg/ml) was added to each well and incubated at 37 °C for an additional 4 h. Finally, 150 μl dimethyl sulphoxide (DMSO) was added to each well and the absorbance at 570 nm was read. The absorbance was used as a measurement of cell viability. It was normalized by the absorbance of cells incubated in control medium, which were considered 100% viable.

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Antiviral activity experiments in vitro [3]
Vero-E6 cells were infected with SARS-CoV-2 (MOI = 0.05) at 37 °C for 1 h before the pre-incubation of Danshensu and Vero-E6 cells for 1 h. After that, fresh medium with Danshensu was added, 150 μL supernatant was collected for testing after 24 h, the copy number of the virus was determined by qRT-PCR, and the ability of Danshensu resisting SARS-CoV-2 was evaluated.

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SARS-CoV-2 S was mixed with Danshensu in different concentrations in advance at 37 °C for 30 min, and the 100 μL mixture was added to ACE2 HEK-293T and Vero-E6 cells with ACE2 overexpression. After co-incubation for 12 h, the supernatant was discarded, and the same volume of fresh medium was replenished. After 48 h, the cells were washed with PBS and lysed. The Luciferase level in cell lysates was detected as previously described to evaluate the anti-SARS-CoV-2 S ability of Danshensu.

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MTT Assay [4]
The viability of FaDu and Ca9-22 cells was determined using MTT assay. For the experiment, the cells were seeded onto 24-well plates and treated with different concentrations of sodium Danshensu (0–100 μM) for 24 h. After the treatments, the cell viability was assessed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, as defined earlier.

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Wound Healing Assay [4]
For self-insertion, appropriate amounts of FaDu and Ca9-22 cells were seeded onto culture-insert wells and incubated overnight. Next, the cells were treated with 0, 25, 50, and 100 μM of sodium Danshensu for 0, 3, 6, 8, and 24 h after creating the wound. The cells were then photographed and the mean crawling distance was measured.

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Cell line and procedures [5]
Renal proximal tubular epithelial cells (HK-2 cells) of human were brought from ScienCell Research Laboratories, America. HK-2 cells were cultured in DMEM medium with 1.0% penicillin-streptomycin solution and 10% heat-inactivated FBS in a 5.0% CO2 humidified incubator at 37 °C. Cells from passages 3–5 were utilized throughout research after recovery. During the experiment, HK-2 cells were seeded at a density of 104 cells/well in 96-well plates. After 24 h of culture, the cells were treated with Danshensu (2.5–10 µM) and cisplatin for 18 h. Then, Cell suspensions from each group were collected for analysis viability, antioxidant, anti-inflammatory and so on.

A total of 48 mice were used in this study and randomly separated into 6 groups (n = 8), including blank group (24 h and 72 h), VSV-G group (24 h and 72 h), SARS-CoV-2 S group (24 h and 72 h). ALI model was induced by SARS-CoV-2 S via trachea in SARS-CoV-2 S groups, while blank groups received DMEM via trachea and VSV-G groups received VSV-G via trachea.
Another total of 72 mice were used in this study and randomly separated into 9 groups (n = 8), including blank group, VSV-G group, SARS-CoV-2 S group, Danshensu 25 mg/kg group, Danshensu 50 mg/kg group, Danshensu 100 mg/kg group, Danshensu 25 mg/kg i.v. group, Danshensu 50 mg/kg i.v. group, Danshensu 100 mg/kg i.v. group. Blank group received distilled water orally, VSV-G group received distilled water orally, SARS-CoV-2 S group received distilled water orally. Danshensu i.v. groups received distilled water orally, and only received Danshensu (i.v. 25, 50, or 100 mg/kg, dissolved in normal saline for tail vein injection) once before the intratracheally received SARS-CoV-2 S at the 7th day; Danshensu groups received Danshensu (Oral administration. 25, 50, or 100 mg/kg/d, dissolved in distilled water) orally. All groups except Danshensu (i.v. 25, 50, or 100 mg/kg) were administrated once daily for 7 continuous days, Danshensu (i.v. 25, 50, or 100 mg/kg) groups just received distilled water orally for 7 continuous days before the tail vein injection. ALI model was induced by SARS-CoV-2 S via trachea after the last treatment, blank groups received DMEM via trachea and VSV-G groups received VSV-G via trachea. [3]

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After a 7-day period of acclimation to the experimental area, the mice were randomly assigned to 5 groups (n = 10/group) as follows: control group, cisplatin group (25 mg/kg), cisplatin (25 mg/kg) + Danshensu (15 mg/kg), cisplatin (25 mg/kg) + Danshensu (30 mg/kg) and cisplatin (25 mg/kg) + Danshensu (60 mg/kg). Before the in vivo experiments, all the mice were starved for 12 h. After fasting, all other groups except the control group were administered a single intraperitoneal injection of 25 mg/kg cisplatin. 1 h later，cisplatin (25 mg/kg) + Danshensu (15 mg/kg, 30 mg/kg, 60 mg/kg) groups were treated with corresponding doses of Danshensu once a day for 7 consecutive days. After the last of administration, blood samples were collected to prepare serum, the serum was stored at −80 °C in polystyrene tubes until analysis. Both kidneys were quickly dissected and weighed immediately following sacrifice. The kidneys and the serum were processed and stored for later various items analysis.[5]

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Assessment antioxidant activity of Danshensu [5]
The active oxygen scavenging activity of Danshensu was determined as the protocol described below. The kidney tissues in each group were homogenized in ice-cold phosphate buffer saline (PBS) (0.05 M, PH 7) and centrifuged at 10,000g for 10 min at 4 ℃. The supernatants were collected for the estimation of malondialdehyde (MDA), reactive oxygen species (ROS) levels and superoxide dismutase (SOD), Glutathione peroxidase (GPx), Catalase (CAT) activities determined using commercial kits according to the manufacturer’s instructions. HK-2 cells were seeded at a density of 104 cells /well in 96-well plates. After 24 h of culture, the cells were treated with Danshensu and cisplatin for 18 h. Then, the cells were incubated with 2, 7-dichlorofluorescein diacetate (DCF-DA) and Hoechst33342 for 20 min the result was expressed as percentage change fluorescence, where the control group was taken as 100%.

[1]. Characterization of the radical scavenging and antioxidant activities of danshensu and salvianolic acid B. Food Chem Toxicol. 2008 Jan;46(1):73-81.

[2]. Danshen: an overview of its chemistry, pharmacology, pharmacokinetics, and clinical use. J Clin Pharmacol. 2005 Dec;45(12):1345-59.

[3].Danshensu alleviates pseudo-typed SARS-CoV-2 induced mouse acute lung inflammation. Acta Pharmacol Sin. 2022 Apr;43(4):771-780.

[4].Sodium Danshensu Inhibits Oral Cancer Cell Migration and Invasion by Modulating p38 Signaling Pathway. Front Endocrinol (Lausanne). 2020 Sep 30:11:568436.

[5].Danshensu attenuates cisplatin-induced nephrotoxicity through activation of Nrf2 pathway and inhibition of NF-κB. Biomed Pharmacother. 2021 Oct:142:111995.

3-(3,4-dihydroxyphenyl)lactic acid is a 2-hydroxy monocarboxylic acid and a member of catechols. It is functionally related to a rac-lactic acid. It is a conjugate acid of a 3-(3,4-dihydroxyphenyl)lactate.
3-(3,4-Dihydroxyphenyl)-2-hydroxypropanoic acid has been reported in Salvia miltiorrhiza, Salvia officinalis, and other organisms with data available.
See also: 3-(3,4-Dihydroxyphenyl)lactate (annotation moved to).

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In conclusion, salvianolic acid B and Danshensu of caffeic acid derivatives isolated from S. miltiorrhiza were shown to be efficient antioxidants. Both salvianolic acid B and danshensu showed higher free radical scavenging activity than vitamin C, but lower iron chelating and hydrogen peroxide scavenging activities. Their antioxidant capacities are in agreement with their protective effects against cell injury from oxidative stresses. Condensation and conjugation of caffeic acid and its derivatives appears important for antioxidant activity. Furthermore, salvianolic B was a better antioxidant than danshensu. These results suggested that phenolic antioxidants isolated from natural resources could also have potential applications in food industry.[1]

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Danshen, the dried root of Salvia miltiorrhiza, has been widely used in China and, to a lesser extent, in Japan, the United States, and other European countries for the treatment of cardiovascular and cerebrovascular diseases. In China, the specific clinical use is angina pectoris, hyperlipidemia, and acute ischemic stroke. The current review covers its traditional uses, chemical constituents, pharmacological activities, pharmacokinetics, clinical applications, and potential herb-drug interactions based on information obtained in both the English and Chinese literature. Although numerous clinical trials have demonstrated that certain Danshen products in China are effective and safe for the treatment of cardiovascular diseases, most of these lack sufficient quality. Therefore, large randomized clinical trials and further scientific research to determine its mechanism of actions will be necessary to ensure the safety, effectiveness, and better understanding of its action.[2]

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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can induce acute inflammatory response like acute lung inflammation (ALI) or acute respiratory distress syndrome, leading to severe progression and mortality. Therapeutics for treatment of SARS-CoV-2-triggered respiratory inflammation are urgent to be discovered. Our previous study shows that Salvianolic acid C potently inhibits SARS-CoV-2 infection. In this study, we investigated the antiviral effects of a Salvia miltiorrhiza compound, Danshensu, in vitro and in vivo, including the mechanism of S protein-mediated virus attachment and entry into target cells. In authentic and pseudo-typed virus assays in vitro, Danshensu displayed a potent antiviral activity against SARS-CoV-2 with EC50 of 0.97 μM, and potently inhibited the entry of SARS-CoV-2 S protein-pseudo-typed virus (SARS-CoV-2 S) into ACE2-overexpressed HEK-293T cells (IC50 = 0.31 μM) and Vero-E6 cell (IC50 = 4.97 μM).
Therefore, we confirmed that Danshensu could induce the expression of antioxidant enzyme and suppress the production of inflammatory mediator as a beneficial anti-inflammatory agent in vivo. ALI in mice could be induced by SARS-CoV-2 S and reversed in biochemical and pathological indexes by Danshensu. Meanwhile, our results revealed that the model induced by SARS-CoV-2 S via trachea could be used for the experiment in vivo of anti-inflammatory drug for the inflammatory response of COVID-19.[3]

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In conclusion, the present study demonstrates the effect of sodium Danshensu, which is a bioactive, water soluble, phenolic compound found in Salvia miltiorrhiza (Danshen), on cancer cell migration and invasion in human OSCC. The study findings reveal that sodium danshensu significantly reduces the motility and metastatic ability of oral cancer cells by reducing the phosphorylation of p38 MAPK. However, sodium danshensu-mediated alteration in ERK1/2 phosphorylation was observed only in FaDu cells. Collectively, the present study identifies sodium danshensu as a potential natural anticancer agent that can be used therapeutically to manage highly metastatic OSCC. [4]

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Both inflammatory cytokines and NLRP3 inflammasome can be regulated by the NF-κB signaling pathway. Moreover, accumulating evidence suggests that there is crosstalk between the Nrf2 and the NF-ĸB signaling pathway. Some studies have observed that the activation of Nrf2 retarded the NF-ĸB-mediated proinflammatory reaction. Nrf2 knockout mouse showed increased activation of NF-ĸB. Nrf2 is an oxidative stress axis factor, which plays a key role in attenuating the oxidant-induced injury. While oxidative stress occurs, Nrf2 dissociated from Keap1, transferred from the cytoplasm to the nucleus and combined with antioxidant response elements to activate the antioxidant enzyme genes (such as HO-1, NQO1) transcription and expression. It is revealed in this study that Danshensu upregulated the expression of Nrf2 and HO-1 proteins and suppressed the phosphorylation and subsequent translocation of nuclear transcription factor-kappa B (NF-κB) to the nucleus, through the inhibition of the IκB kinase complex (IKK) activation and IκBα protein phosphorylation. Therefore, it can be inferred that Danshensu may alleviate oxidative stress and inflammation to exert a protective effect on cisplatin-induced nephrotoxicity by activating Nrf2 pathway and inhibiting NF-ĸB signaling pathway.[5]

C9H10O5

198.172

198.053

C, 54.55; H, 5.09; O, 40.37

23028-17-3

Danshensu sodium salt;67920-52-9

439435

Light yellow to yellow solid powder

1.54g/cm3

480.3ºC at 760mmHg

258.4ºC

0.085

4

5

3

14

205

0

OC(C(=O)O)CC1C=CC(=C(C=1)O)O

PAFLSMZLRSPALU-UHFFFAOYSA-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)

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

23028-17-3; 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoic acid; 3,4-Dihydroxyphenyllactic acid; (Rac)-Salvianic acid A; 3-(3,4-dihydroxyphenyl)lactic acid; Benzenepropanoic acid, alpha,3,4-trihydroxy-; Sodium Danshensu; NA8H56YM3K;

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.)