**6. Clinical studies**

**Figure 5.** Anti‐atherosclerotic effect of beet juice in *ex vivo* model.The study involved eight volunteers (six males, two females, mean age 53 ± 5 years) whose blood serum induced 1.3–2.2‐fold increase in cholesterol content of cells cul‐ tured from unaffected human aortic intima (the average level of serum atherogenicity was 161 ± 8%). Intracellular cho‐ lesterol in control cultures was 37.0 ± 3.6 mg/mg cell protein. Baseline serum atherogenicity was taken as 100%. The average values of changes of serum atherogenicity with indication of standard errors are presented. \*, Significant de‐

**Figure 6.** Anti‐atherosclerotic effect of garlic powder in the *ex vivo* model.The study involved eight volunteers (six males, two females, mean age 53 ± 5 years) whose blood serum induced 1.3–2.7‐fold increase in cholesterol content of cells cultured from unaffected human aortic intima (the average level of serum atherogenicity was 164 ± 9%). Intracel‐ lular cholesterol in control cultures was 39.0 ± 4.2 mg/mg cell protein. Baseline serum atherogenicity was taken as 100%. The average values of changes of serum atherogenicity with indication of standard errors are presented. \*, Sig‐

nificant decrease of serum atherogenicity, p < 0.05. Reproduced with permission from [30].

crease of serum atherogenicity, p < 0.05. Reproduced with permission from [30].

26 Cholesterol Lowering Therapies and Drugs

Tests on cellular models demonstrated that garlic powder preparations possessed a pro‐ nounced anti‐atherosclerotic activity. Based on the obtained results, a garlic‐based dietary supplement (Allicor, INAT‐Farma, Russia) was developed. The effect of the supplement on carotid intima‐media thickness (cIMT) was evaluated in an open‐label prospective pilot study conducted on 28 men (46–58 years old, mean age 52.0, SD = 9.0). The study participants had no signs of coronary heart disease, no chronic diseases requiring treatment with vasoactive drugs, diuretics, lipid‐lowering or antidiabetic drugs and were normolipidemic or mildly hyperlipi‐ demic. Study subjects were analysed for presence of diffuse intimal thickening by ultrasound imaging of common carotid arteries [65]. The cut‐off cIMT value of 0.7 mm in the distal segment of at least one common carotid artery was set up to diagnose diffuse intimal thickening. The mean cIMT value at the baseline was 0.832 ± 0.024 mm. Study participants were divided into two groups. Subjects from Allicor group (n = 16) received 600 mg of Allicor daily, and subjects from the control group (n = 12) received no treatment. The total duration of the study was 12  months, with interviews and ultrasound assessment of cIMT every 3 months. No adverse effects were observed during the follow‐up period, and the product was demonstrated to have good tolerability. The results of cIMT assessments are presented on **Figure 7**.

**Figure 7.** The effects of garlic‐based drug Allicor on atherosclerosis determined by cIMT. Open circles, Allicor recipi‐ ents; solid circles, control subjects. Presented are mean values ±S.E.M. Reproduced from [30].

No statistically significant changes of cIMT were observed after 12 months, and the value was not significantly different between the two groups. However, regression analysis revealed a significant difference between the trends of cIMT dynamics (p < 0.05). In the control group, a tendency to cIMT increase was detected, which was significantly different from that of null hypothesis of no change (F‐test, 31.72; p = 0.011). In the Allicor‐treated group, the tendency to cIMT decrease was revealed, which was also significantly different from that of null hypothesis (F‐test, 28.81; p = 0.013). These results indicate that treatment with Allicor may potentially halter the development and induce the regression of subclinical atherosclerosis. The statistical power of this pilot study was insufficient to avoid type 2 error. Therefore, the pilot study was followed by a larger prospective clinical study, in which a number of clinical and biochemical parameters associated with atherogenesis were taken into account. The dynamic of serum atherogenicity was also assessed. This double‐blind placebo‐controlled clinical study evalu‐ ated the effect of garlic powder tablets Allicor on the progression of cIMT in 211 men (40–74  years old) with no symptoms of atherosclerosis (ClinicalTrials.gov identifier, NCT01734707). The primary outcome was the progression of subclinical atherosclerosis evaluated by B‐mode ultrasonography as the increase of cIMT. By the end of the first 12‐month follow‐up period, a decrease of cIMT by 0.028 ± 0.008 mm was observed in the Allicor group. At the same time, moderate increase of 0.014 ± 0.009 mm was observed in the placebo group (p = 0.002). Serum atherogenicity was decreased in the Allicor group by 45% from the baseline and remained unaltered in the placebo group. Therefore, long‐term treatment with Allicor had a direct anti‐ atherosclerotic effect in patients with subclinical atherosclerosis associated with decreased serum atherogenicity [78]. By the end of the 24‐month follow‐up period, the mean rate of cIMT was decreased in the Allicor group by 0.022 ± 0.007 mm per year, which was significantly different (p = 0.002) from the placebo group, in which there was a moderate but statistically significant progression of 0.015 ± 0.008 mm at the overall mean baseline cIMT of 0.931 ± 0.009  mm [37, 39]. A significant reduction of cIMT was observed in 47.3% of study subjects from the Allicor group vs 30.1% in the placebo group (p < 0.05). Further significant increase of cIMT was registered in 32.2% study participants in Allicor‐treated group vs 47.3% in placebo group (p  < 0.05). Study of blood serum atherogenicity demonstrated a 1.56‐fold increase of intracellular cholesterol accumulation in the cellular test at the baseline. Study participants from Allicor group had an average 30% decrease of blood serum atherogenicity, while in the placebo group, this parameter remained unaltered during the study. A significant correlation was observed between changes of blood serum atherogenicity and intima‐media thickness of common carotid arteries (r = 0.144, p = 0.045) (**Figures 8** and **9**). Therefore, it was demonstrated that garlic‐based food supplement Allicor possessed a direct anti‐atherosclerotic effect at the subclinical stage of the disease, which could be attributed to the decrease of blood serum atherogenicity [37, 39].

diuretics, lipid‐lowering or antidiabetic drugs and were normolipidemic or mildly hyperlipi‐ demic. Study subjects were analysed for presence of diffuse intimal thickening by ultrasound imaging of common carotid arteries [65]. The cut‐off cIMT value of 0.7 mm in the distal segment of at least one common carotid artery was set up to diagnose diffuse intimal thickening. The mean cIMT value at the baseline was 0.832 ± 0.024 mm. Study participants were divided into two groups. Subjects from Allicor group (n = 16) received 600 mg of Allicor daily, and subjects from the control group (n = 12) received no treatment. The total duration of the study was 12  months, with interviews and ultrasound assessment of cIMT every 3 months. No adverse effects were observed during the follow‐up period, and the product was demonstrated to have

**Figure 7.** The effects of garlic‐based drug Allicor on atherosclerosis determined by cIMT. Open circles, Allicor recipi‐

No statistically significant changes of cIMT were observed after 12 months, and the value was not significantly different between the two groups. However, regression analysis revealed a significant difference between the trends of cIMT dynamics (p < 0.05). In the control group, a tendency to cIMT increase was detected, which was significantly different from that of null hypothesis of no change (F‐test, 31.72; p = 0.011). In the Allicor‐treated group, the tendency to cIMT decrease was revealed, which was also significantly different from that of null hypothesis (F‐test, 28.81; p = 0.013). These results indicate that treatment with Allicor may potentially halter the development and induce the regression of subclinical atherosclerosis. The statistical power of this pilot study was insufficient to avoid type 2 error. Therefore, the pilot study was followed by a larger prospective clinical study, in which a number of clinical and biochemical parameters associated with atherogenesis were taken into account. The dynamic of serum atherogenicity was also assessed. This double‐blind placebo‐controlled clinical study evalu‐ ated the effect of garlic powder tablets Allicor on the progression of cIMT in 211 men (40–74  years old) with no symptoms of atherosclerosis (ClinicalTrials.gov identifier, NCT01734707). The primary outcome was the progression of subclinical atherosclerosis evaluated by B‐mode

ents; solid circles, control subjects. Presented are mean values ±S.E.M. Reproduced from [30].

good tolerability. The results of cIMT assessments are presented on **Figure 7**.

28 Cholesterol Lowering Therapies and Drugs

**Figure 8.** The dynamics of cIMT in double‐blind placebo‐controlled study on anti‐atherosclerotic effects of garlic‐based drug Allicor. Hatched bars, Allicor recipients; open bars, placebo recipients. Presented are mean values ±S.E.M. \*, sig‐ nificant difference between groups, p < 0.05. Reproduced with permission from [30].

Another clinical study was focused on the evaluation of potential anti‐atherosclerotic activity of herbal products with anti–inflammatory effects. Atherosclerosis is tightly associated with the inflammatory process at all stages of the disease development [79, 80]. Substances with systemic anti‐inflammatory properties can therefore be regarded as potential therapeutic agents for treatment and prevention of atherosclerosis.

**Figure 9.** The dynamics of serum atherogenicity in double‐blind placebo‐controlled study on anti‐atherosclerotic ef‐ fects of garlic‐based drug Allicor. Hatched bars, Allicor recipients; open bars, placebo recipients. Presented are mean values ±S.E.M. \*, significant difference between groups, p < 0.05. Reproduced with permission from [30].

**Figure 10.** The changes of cIMT in double‐masked placebo‐controlled study on anti‐atherosclerotic effects of Inflami‐ nat. Presented are mean values ±S.E.M. \*, significant difference between groups, p < 0.05. Reproduced with permission from [30].

Several natural compounds, such as calendula (*Calendula officinalis*), elder (*Sambucus nigra*) and violet (*Viola* sp.), were demonstrated to possess not only anti‐inflammatory, but also anti‐ atherosclerotic effects [81–83]. The combination of these herbs was used for the development of a novel dietary supplement (Inflaminat, INAT‐Farma, Russia) [84]. The effect of Inflaminat on cIMT dynamics was evaluated in a pilot placebo‐controlled double‐blinded study per‐ formed on 67 asymptomatic men (ClinicalTrials.gov Identifier, NCT01743404) [39, 85]. The protocol of the 12‐month study was similar to that described for Allicor food supplement. Administration of Inflaminat induced cIMT regression in subclinical atherosclerosis, with statistically significant difference between the baseline as the placebo group (**Figure 10**). Therefore, Inflaminat was demonstrated to possess anti‐inflammatory and anti‐atherosclerotic effects at the cellular level and to induce regression of subclinical atherosclerotic lesions in asymptomatic men.

**Figure 11.** The changes of cIMT in double‐masked placebo‐controlled study on anti‐atherosclerotic effects of Karinat. The data are presented in the terms of means and S.D. \*, significant difference between groups, p < 0.05. Reproduced with permission from [30].

Finally, several phytoestrogen‐rich natural substances were evaluated for potential anti‐ atherosclerotic activity using the described *in vitro* and an *ex vivo* models [86–88]. The most promising of these compounds were garlic powder, extract of grape seeds, green tea leaf and hop cones. All these substances possessed a significant anti‐atherogenic effect. A combination of these compounds was used for development of a novel isoflavonoid‐rich dietary supplement (Karinat, INAT‐Farma, Russia). The resulting supplement is a source of biologically active polyphenols, including resveratrol, genisteine and daidzeine that are claimed to produce beneficial effects on atherosclerosis development. The efficiency of Karinat was evaluated in a randomized double‐blind placebo‐controlled 12‐month clinical study conducted on 157 asymptomatic postmenopausal women (ClinicalTrials.gov Identifier, NCT01742000) [89, 90]. The primary endpoint was the annual rate of cIMT change. The protocol of the study was similar to that reported above. An annual increase of mean cIMT of more than 100 μm (13% per year) was observed in the placebo group, indicative of a high rate of cIMT progression in postmenopausal women. Growth of atherosclerotic plaques was estimated to be 40% per year. In the Karinat group, mean cIMT value remained unaltered, with a statistically insignificant increase of 6 μm per year, *that is* <1% (**Figure 11**). Therefore, phytoestrogen‐rich substances were proven to possess beneficial effects on the dynamics of subclinical atherosclerosis progression in postmenopausal women [39, 91].

#### **7. Conclusions**

systemic anti‐inflammatory properties can therefore be regarded as potential therapeutic

**Figure 9.** The dynamics of serum atherogenicity in double‐blind placebo‐controlled study on anti‐atherosclerotic ef‐ fects of garlic‐based drug Allicor. Hatched bars, Allicor recipients; open bars, placebo recipients. Presented are mean

**Figure 10.** The changes of cIMT in double‐masked placebo‐controlled study on anti‐atherosclerotic effects of Inflami‐ nat. Presented are mean values ±S.E.M. \*, significant difference between groups, p < 0.05. Reproduced with permission

Several natural compounds, such as calendula (*Calendula officinalis*), elder (*Sambucus nigra*) and violet (*Viola* sp.), were demonstrated to possess not only anti‐inflammatory, but also anti‐ atherosclerotic effects [81–83]. The combination of these herbs was used for the development of a novel dietary supplement (Inflaminat, INAT‐Farma, Russia) [84]. The effect of Inflaminat on cIMT dynamics was evaluated in a pilot placebo‐controlled double‐blinded study per‐ formed on 67 asymptomatic men (ClinicalTrials.gov Identifier, NCT01743404) [39, 85]. The protocol of the 12‐month study was similar to that described for Allicor food supplement. Administration of Inflaminat induced cIMT regression in subclinical atherosclerosis, with statistically significant difference between the baseline as the placebo group (**Figure 10**). Therefore, Inflaminat was demonstrated to possess anti‐inflammatory and anti‐atherosclerotic

values ±S.E.M. \*, significant difference between groups, p < 0.05. Reproduced with permission from [30].

agents for treatment and prevention of atherosclerosis.

30 Cholesterol Lowering Therapies and Drugs

from [30].

Introduction of the concept of blood serum atherogenicity allowed creating cell model suitable for screening of substances with potential anti‐atherosclerotic activity. Such models helped revealing several novel compounds of botanical origin that could be used for the development of dietary supplements for treatment of subclinical (asymptomatic) atherosclerosis. The effect of "direct" anti‐atherosclerotic therapy can be observed at the level of the arterial wall cells by a decrease of intracellular lipid accumulation. Therapy of patients with established athero‐ sclerosis should induce regression of the existing plaques or hinder the progression of novel lesions. Introduction of food supplements from botanicals with anti‐atherosclerotic properties and suitable for long‐term consumption is an important step toward the improvement of the preventive treatment of atherosclerosis. Further studies will help revealing natural products with anti‐atherogenic and anti‐atherosclerotic effects that can be used for the development of novel cardiovascular drugs possessing mechanistic mode of action. Despite the unavoidable limitations of the described models, the obtained results have demonstrated that cultured arterial wall cells offer a suitable instrument for initial analysis of drug effects. The discovery of anti‐atherosclerotic activity of natural products opens great opportunities for prevention and treatment of atherosclerotic disease, reducing cardiovascular morbidity and mortality.
