**3. Results**

### *3.1. Liensinine Scavenges DPPH Free Radicals and Inhibits Serum Lipid Peroxidation*

Liensinine showed concentration-dependent DPPH free radical scavenging activity as displayed in Figure 2a. The inhibitory concentration 50 (IC50) of liensinine was found

to be 1.8 μg/mL. Figure 2b shows the measurement of serum lipid peroxidation. Liensinine at concentrations of 30 and 40 μg/mL showed remarkable reduction of serum lipid peroxidation in terms of TBARS value.

**Figure 2.** The antioxidant potency of liensinine. (**a**) 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity (IC50 = 1.8 μg/mL and r2 = 0.94) (**b**) Serum lipid peroxidation inhibitory activity. N represents the normal group (without copper sulphate). a *p* < 0.05 and b *p* < 0.01 vs. C (the control group; presence of copper sulfate only). n=3replicates.

### *3.2. Liensinine Inhibits VSMC Proliferation*

PDGF-BB is a potent growth factor over-expressed in human coronary arteries during atherosclerosis and restenosis [27]. Figure 3 shows the potent anti-proliferative activity of liensinine against PDGF-BB stimulated VSMC proliferation. Liensinine, at a concentrations of 20 and 30 μg/mL notably decreased the % cell proliferation to 67.16% and 47.02%, respectively, vs. 100% of control (PDGF-BB only).

**Figure 3.** The effect of liensinine on platelet-derived growth factor-BB (PDGF-BB) induced proliferation of human vascular smooth muscle cells (VSMC). VSMC were pretreated with different concentration of liensinine for 1 h followed by stimulation with 20 ng/mL of PDGF-BB for 24 h. The effect on proliferation of VSMC was evaluated by MTT assay. a *p* < 0.05 and b *p* < 0.01 vs. Control (only PDGF).n=3replicates.

### *3.3. Liensinine Inhibits MMP-9 Enzymatic Action*

The degradation of the extracellular matrix by enzymatic action of MMPs (stimulated by various mitogens) is responsible for VSMC migration. TNF-α is one such mitogen/activator that stimulates MMP-9 enzyme activity in VSMCs [28]. The effect of liensinine on the MMP-9 proteolytic degradation of gelatin in VSMCs is displayed in Figure 4. The MMP-9 band was drastically increased by TNF-α (compared to without TNF-α) and decreased concentration dependently by liensinine (compared to TNF-α).

**Figure 4.** The effect of liensinine on matrix metalloproteinase (MMPs) secretion in TNF-α stimulated VSMC. VSMC were pretreated with different concentrations of liensinine and stimulated with TNF-<sup>α</sup>. The effect of liensinine on MMPs proteolytic/enzymatic activity was observed by gelatin zymography. The photographs of the gel were taken after Coomassie Brilliant Blue staining. n=3replicates.

### *3.4. Liensinine Inhibits IL-6 in VSMC*

As shown in Figure 5, TNF-α significantly increased the IL-6 production in VSMC by 3-fold compared to the control (without TNF-α) while liensinine at a concentration of 10, 20, 30 μg/mL significantly inhibited the IL-6 release.

**Figure 5.** The effect of liensinine in IL-6 release in TNF-α stimulated VSMC. VSMC were pretreated with different concentrations of liensinine and stimulated with TNF-<sup>α</sup>. The effect of liensinine on IL-6 release was measured by IL-6 ELISA Kit. Liensinine concentration-dependently inhibited the IL-6 release in VSMC. a *p* < 0.05 and b *p* < 0.01 vs. Control (only TNF-α). n = 3 replicates.

### *3.5. Liensinine Suppresses NO Production and Inhibit Protein Expression of iNOS and COX-2 in RAW264.7*

First, we checked the cytotoxicity of various concentrations of liensinine in RAW264.7 cells. Liensinine up to 20 μM did not exert any significant decrease in RAW264.7 cell viability (Figure 6a). The 24 h treatment of cells with LPS increased the production of NO (95.2 ± 9.8 μM) by approximately 3-fold compared to those without LPS treatment. Liensinine showed inhibition of NO release in a concentration-dependent manner (Figure 6b).

Consistent with NO production, there was an overexpression of iNOS (Figure 6c,d) and COX-2 (Figure 6c,e) proteins after LPS treatment, while liensinine (5–20 μM) treatment resulted in a notable reduction in protein expression.

**Figure 6.** The effects of liensinine on cell viability, nitric oxide (NO) release, and protein expression of iNOS and COX-2. (**a**) Cytotoxicity/cell viability was done by MTT colorimetric assay. (**b**) The effect of liensinine on NO release was determined by Griess reagen<sup>t</sup> assay. (**c**) The expression of the inflammatory proteins iNOS and COX-2 was done by immunoblot. *β*-actin was used as housekeeping/reference control to calculate relative fold change. (**d**) The fold change of iNOS. (**e**) The fold change of COX-2. a *p* < 0.05 and b *p* < 0.01 vs. Control (only LPS).n=3replicates.
