Novel Salt Cocrystal of Chrysin with Berberine: Preparation, Characterization, and Oral Bioavailability

A novel salt cocrystal of chrysin (ChrH) with berberine (BerbOH), [Berb⁺–Chr^–]-2ChrH (<b>1</b>), was prepared. Crystal structure analysis shows one chrysin lost its proton in 7-hydroxyl group and turned into a chrysin anion. Two neutral chrysin molecules interact simultaneously with chrysin anion through charge-assisted strong hydrogen-bonding interactions between phenolic anion and hydroxyl groups of chrysin to give rise to a 1:3 salt cocrystal based on berberine and chrysin. Density functional theoretical calculations indicate that the 1:3 stoichiometry of berberine and chrysin is more stable due to its largest interaction energies. The bioavailability of chrysin in the form of cocrystal <b>1</b> in rats is about 1.7 times than that of pure chrysin

Combined Salvianolic Acid B and Ginsenoside Rg1 Exerts Cardioprotection against Ischemia/Reperfusion Injury in Rats

<div><p>Lack of pharmacological strategies in clinics restricts the patient prognosis with myocardial ischemia/reperfusion (I/R) injury. The aim of this study was to evaluate the cardioprotection of combined salvianolic acid B (SalB) and ginsenoside Rg1 (Rg1) against myocardial I/R injury and further investigate the underlying mechanism. I/R injury was induced by coronary artery ligation for Wistar male rats and hypoxia/reoxygenation injury was induced on H9c2 cells. Firstly, the best ratio between SalB and Rg1was set as 2:5 based on their effects on heart function detected by hemodynamic measurement. Then SalB-Rg1 (2:5) was found to maintain mitochondrial membrane potential and resist apoptosis and necrosis in H9c2 cell with hypoxia/reoxygenation injury. Companying with same dose of SalB or Rg1 only, SalB-Rg1 showed more significant effects on down-regulation of myocardial infarct size, maintenance of myocardium structure, improvement on cardiac function, decrease of cytokine secretion including TNF-α, IL-1β, RANTES and sVCAM-1. Finally, the SalB-Rg1 improved the viability of cardiac myocytes other than cardiac fibroblasts in rats with I/R injury using flow cytometry. Our results revealed that SalB-Rg1 was a promising strategy to prevent myocardial I/R injury.</p></div

SalB-Rg1 protected H9c2 cells against apoptosis and necrosis.

<p>(A) The protection of SalB-Rg1 against H9c2 apoptosis was detected by mitochondrial depolarization using JC-1 stain. (B) Quantification of the ratio for JC-1 multimer to JC-1 monomer fluorescence intensity (red:green). (C) The apoptosis or necrosis of H9c2 cell was evaluated using Annexin V-FITC/PI Apoptosis Detection Kit. Apoptotic cells were stained green (Annexin V-FITC) and necrosis cells were stained red (PI). At least three time experiments were repeated and the representative figures were shown.</p

Salvianolic Acid A, a Novel Matrix Metalloproteinase-9 Inhibitor, Prevents Cardiac Remodeling in Spontaneously Hypertensive Rats

<div><p>Cardiac fibrosis is a deleterious consequence of hypertension which may further advance to heart failure and increased matrix metalloproteinase-9 (MMP-9) contributes to the underlying mechanism. Therefore, new therapeutic strategies to attenuate the effects of MMP-9 are urgently needed. In the present study, we characterize salvianolic acid A (SalA) as a novel MMP-9 inhibitor at molecular, cellular and animal level. We expressed a truncated form of MMP-9 which contains only the catalytic domain (MMP-9 CD), and used this active protein for enzymatic kinetic analysis and Biacore detection. Data generated from these assays indicated that SalA functioned as the strongest competitive inhibitor of MMP-9 among 7 phenolic acids from <i>Salvia miltiorrhiza</i>. In neonatal cardiac fibroblast, SalA inhibited fibroblast migration, blocked myofibroblast transformation, inhibited secretion of intercellular adhesion molecule (ICAM), interleukin-6 (IL-6) and soluble vascular cell adhesion molecule-1 (sVCAM-1) as well as collagen induced by MMP-9 CD. Functional effects of SalA inhibition on MMP-9 was further confirmed in cultured cardiac H9c2 cell overexpressing MMP-9 <i>in vitro</i> and in heart of spontaneously hypertensive rats (SHR) <i>in vivo</i>. Moreover, SalA treatment in SHR resulted in decreased heart fibrosis and attenuated heart hypertrophy. These results indicated that SalA is a novel inhibitor of MMP-9, thus playing an inhibitory role in hypertensive fibrosis. Further studies to develop SalA and its analogues for their potential clinical application of cardioprotection are warranted.</p> </div

SalB-Rg1 (2:5) improved cardiac function.

<p>10mg/kg SalB-Rg1 significantly improved ventricular function by evaluation of hemodynamic parameters including +dP/dt_max,-dP/dt_min, EDP and LVSP. No improvement on cardiac function was found with the treatment of SalB or Rg1 at 10 mg/kg only. All the values are expressed as mean ± S.E. *p<0.05, **p<0.01versus Sham; #p<0.05, ##p<0.01 versus I/R; n≥6 for every group.</p

The ratio for SalB to Rg1 was set as 2:5 based on ventricular function.

<p>I/R injury was induced by 40 minutes coronary artery ligation followed by 1 hour reperfusion. Ventricular function was evaluated by hemodynamic parameters including maximum rate of pressure development for contraction (+dP/dt_max), maximum rate of pressure development for relaxation (-dP/dt_min), left ventricular systolic pressure (LVSP) and end-diastolic pressure (EDP). All the values were expressed as mean ± S.E. *p<0.05, **p<0.01, ***p<0.001 versus Sham; #p<0.05 versus I/R; n≥6 for every group.</p

SalA inhibited myofibroblast transformation induced by MMP-9 CD.

<p>Fluorescence immunohistochemistry, using a specific α-SMA first antibody following by a second antibody conjugated with FITC, was performed to demonstrate myofibroblast transformation induced by MMP-9. Nuclei were stained with DAPI and images are shown at 400x. MMP-9/SalA(1) or MMP-9/SalA(10) denoted 1 µmol/L SalA or 10 µmol/L SalA was used to detect the inhibitory effect of SalA on MMP-9.</p

SalA inhibited the secretion of cytokines and collagen induced by MMP-9 CD.

<p>(A) MMP-9 CD stimulated the secretion of IL-6, ICAM, sVCAM-1, TNF-α and SalA reversed the effects of MMP-9 CD partially. (B) SalA inhibited collagen secretion induced by MMP-9 CD. MMP-9/SalA(1) or MMP-9/SalA(10) denoted 1 µmol/L SalA or 10 µmol/L SalA was used to detect the inhibitory effects of SalA on MMP-9. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 versus Con; #<i>p</i><0.05, ### <i>p</i><0.001 versus MMP-9.</p

SalA inhibited cardiac fibroblast migration induced by MMP-9 CD.

<p>(A) The representative pictures for migration of cardiac fibroblast detected by transwell migration assay, the magnification was 200 X. (B) The quantitative data for migration of cardiac fibroblast. (C) Proliferation of cardiac fibroblast detected by CCK-8. Triplicate determinations were performed for each experimental condition and data are expressed as mean±SE. *<i>p</i><0.05, ***<i>p</i><0.001 versus Con; #<i>p</i><0.05 versus MMP-9.</p

Effects of SalA on basic cardiovascular characteristics of SHR.

<p>All the values are expressed as mean ± S.E. Heart weight divided by body weight (HW/BW), lung weight divided by body weight (LW/BW) and kidney weight divided by body weight (KW/BW) were also calculated. #<i>p</i><0.05 versus SalA (0 mg/kg). n = 20 per each group. Results are expressed as mean±S.E.</p