Specific Combination of Salvianolic Acids As Core Active Ingredients of Danhong Injection for Treatment of Arterial Thrombosis and Its Derived Dry Gangrene

Abstract

Although single-targeting anti-platelet agents are used extensively in clinics, their limitations in resistance and bleeding have started a trend of combination therapy. Danhong injection (DHI) is a widely prescribed injection medicine for cardiovascular and cerebrovascular diseases in China. However, its precise clinical efficacy and functional components remain unexplored. In this study, we investigated the anti-thrombotic role and its chemical basis of DHI. In a photochemically-induced thrombosis model, DHI effectively dissolved thrombus and ameliorated its derived dry gangrene. DHI inhibited multiple GPCR agonists-induced platelet adhesion, aggregation and downstream Ca²⁺ and cAMP signaling pathways. A functional screen of DHI library identified its major active components as a cluster of seven salvianolic acids. A combination of salvianolic acid A and C synergistically inhibited platelet aggregation in vitro while salvianolic acid B antagonized this effect. Our study revealed the anti-thrombotic activity of DHI. The multi-targeting mechanism of DHI proves the effectiveness of a natural anti-thrombotic combination therapy. The identification of salvianolic acids as a core anti-thrombotic activity of DHI and the discovery that their different combinations could either synergistically or antagonistically provide a better guidance for safer clinical application and paves the way for further development of DHI.

Keywords: G-protein-coupled receptors; anti-platelet therapy; danhong injection; gangrene; salvianolic acid.

Figure 1

DHI dissolved laser-induced arterial thrombus in a rat model. (A) Photomicrographs of arteria thrombosis. Laser activation of rose Bengal dye caused a clearly visible blood clot in the rat iliac arteries. Three doses of DHI (1.75, 5.25, and 15.75 mL/kg) dissolved the clots. Heparin (2,500 U/kg) was used as a positive control. DHI or heparin was injected for 7 days as follow-up treatment. (B) Bar graph quantifying the arterial clot area. (Data are expressed as mean ± S.D. of 8 rats per group, ^**P < 0.01).

Figure 2

DHI ameliorates symptoms of dry gangrene caused by artery occlusion. There was obvious necrosis of the tail in rats that without treatment. The three examples were individual animals by the same group setting. Symptom of dry gangrene was reduced by treatment with different dose of DHI. Low dose DHI (1.75 mL/kg) relieved the tissue ulcer. Medium dose of DHI (5.25 mL/kg) reduced the lesions to dry and shrunken disposition. The symptom of dry gangrene was resolved by high dose of DHI (15.75 mL/kg). As a positive control, heparin (2,500 U/kg) also reduced the lesions to dry and shrunken disposition similar to medium dose of DHI (8 rats per group).

Figure 3

DHI inhibited platelet adhesion. (A) Platelets (1 × 10⁸ platelets/mL) were pre-incubated for 10 min with a series of DHI concentration (1:20–1:160) and allowed to adhere to fibrinogen-coated wells containing ADP (10 μM) for 15 min. (B) Platelets (1 × 10⁸ platelets/mL) were pre-incubated for 10 min with a series of DHI concentration (1:10–1:640) and allowed to adhere to fibrinogen-coated wells containing thrombin (0.1 U/mL) for 15 min. Platelet adhesion was shown as a percentage of the maximal signal induced by ADP or thrombin in control conditions. EGTA was used as a positive control (n = 3, ^*P < 0.05, ^**P < 0.01, compared with ADP or thrombin group. ^##P < 0.01, compared with the rest of the groups. Data are expressed as mean ± S.D.).

Figure 4

DHI inhibited platelet aggregation. Platelets (1 × 10⁸ platelets/mL) were pre-incubated for 10 min with different concentrations of DHI or vehicle. (A) Platelets aggregation was initiated with 25 μM ADP inhibited by series dilutions of DHI (1:120–1:20). (B) Platelets aggregation induced by 0.5 U/mL thrombin was inhibited by different dilutions of DHI (1:80 and 1:40). (C) Platelets aggregation induced by 11.4 μM U46619 was inhibited by a series dilutions of DHI (1:1,280–1:20, ^**P < 0.01, All data are expressed as mean ± S.D. n = 3).

Figure 5

DHI reversed [Ca²⁺]_i and inhibition of cAMP production in ADP-activated platelets. (A) Time course of different concentrations of DHI on 15 μM ADP induced [Ca²⁺]_i. (B) The maximum intracellular Ca²⁺ produced by 15 μM ADP and inhibited by different concentrations of DHI. (C) Reversal of ADP-induced inhibition of cAMP production by DHI. Platelets were incubated with DHI and the control group was incubated with solvent, for 10 min at 37°C, then exposed to ADP (20 μM) for 8 min to cause activation as the control group. Forskolin (10 μM) was used as positive control for cAMP elevation. The cAMP levels were measured by ELISA assay. Previous result has shown that DHI at highest concentration (1:20) had no obvious effect on cAMP in the resting platelet. (All data are expressed as mean ± S.D., ^**P < 0.01 compared with control, ^##P < 0.01 compared with resting group, n = 3).

Figure 6

Screening for anti-platelet aggregation activities of DHI fractions. A component library containing 25 fractions of DHI was screened by platelet aggregation assay. (A) ADP (25 μM)-induced platelet aggregation of each fraction were measured and plotted. ADP and ticagrelor (Tic) were used as controls indicating maximal activation (66.79%, redline) and inhibition (10.76%), respectively. (B) Thrombin (0.5 U/mL) -induced platelet aggregation of each fraction were measured and plotted. Thrombin was used as controls indicating maximal activation (86.86%, redline).

Figure 7

Solid content analysis via LC-MS for identification of the active compounds present in DHI. (A) A representative LC-MS chromatogram of fraction no. 21. Polyphenolic compounds, including RA (1), LA (2), SAB (3), SA A (4), 9”- methy lithospermate B/isomer (5,6), and SAC (7) were simultaneously identified. (B) A representative UPLC chromatogram of fraction no. 21. SAA (4) and SAC (7) are the main compounds by quantity. (C) UPLC chromatogram of SAA, SAB, and SAC reference compounds. (D) Chemical structures of the major constituents identified in fraction no. 21.

Figure 8

Interactions between active constituents of DHI in platelet aggregation assay. (A) Dose-dependent (10–320 μM) inhibition of platelets aggregation induced by 25 μM ADP by SAA, 1:1-ratio mixture of SAA + SAC (AC) or 1:1:1-ratio mixture of SAA + SAB + SAC (ABC). (B) SAA (10–320 μM) dose dependently inhibited platelets aggregation induced by 0.5 U/mL thrombin. Also, 1:1-ratio mixture of SAA + SAC (AC, 10–40 μM) or 1:1:1-ratio mixture of SAA + SAB + SAC (ABC, 10–320 μM) dependently inhibited platelets aggregation. (C) A combination index (CI)/Fa plot of ADP-induced platelet aggregation. CompuSyn™ software was used to determine the synergy/ additivity/antagonism between SAA, SAB, and SAC. The CI of SAA + SAC or SAA + SAB at Fa₉₀ is 1.178 or 0.91, respectively, indicating the interaction between SAA and SAC is antagonism whereas between SAA and SAB is additivity. (D) A CI /Fa plot of thrombin-induced platelet aggregation. The CI of SAA + SAC or SAA + SAB at Fa₉₀ is 0.325 or 1.60 indicating the interaction between SAA and SAC is synergy and between SAA and SAB is antagonism.