**5.** *Ex vivo* **model**

*Ex vivo* model is based on primary culture of cells from unaffected human aortic intima that are incubated with blood serum from patients treated with the substance of interest. Therefore, potential anti‐atherogenic properties of substances are evaluated based on their pharmacody‐ namic properties, or the influence on blood serum atherogenicity after digestion and possible metabolic modifications in patient's body. Blood samples are drawn before and after admin‐ istration of single doses of tested substances, and serum obtained from the samples is added to cultured primary cells. *Ex vivo* model can be used for testing drugs with known safety profiles, as well as various natural products.

Several studies have demonstrated successful application of this model for evaluation of anti‐ atherogenic properties of botanicals. Screening studies were performed on volunteers (groups of 4–8 men and women 45–60 years old) with high blood serum atherogenicity. One of the tested natural products with anti‐atherosclerotic properties was encapsulated onion (*Allium cepa*) bulb powder (300 mg) (**Figure 3**). Administration of a single dose of the product resulted in a moderate decrease of blood serum atherogenicity by 12, 28, and 24% from the baseline after 2, 4, and 6 h, respectively. Another tested natural product with anti‐atherosclerotic properties was preparation of wheat seedlings (*Triticum aestivum*). Administration of a single dose of 300 mg of the preparation resulted in a pronounced reduction of blood serum athero‐ genicity after 4 h (**Figure 4**). Moderate but prolonged anti‐atherosclerotic effect was registered for dry beet (*Beta vulgaris*) juice (encapsulated preparation of 300 mg) (**Figure 5**). Garlic (*Allium sativum*) powder possessed a strong and prolonged effect (**Figure 6**). Blood serum atheroge‐ nicity was completely suppressed 4 h after administration of a single dose of 300 mg of the preparation. Several other natural products were screened for potential anti‐atherosclerotic activity using the *ex vivo* model (**Table 3**). The highest activity after a single dose administration was detected for garlic powder and wheat seedlings, with garlic powder providing the strongest effect. Importantly, anti‐atherosclerotic effects of garlic have been reported by several independent groups during the recent years [65–67].

tion was induced by incubation of cells with atherogenic serum obtained from patients with confirmed atherosclerosis. The increase of cellular cholesterol content reached as high as two

Potential anti‐atherogenic substances were evaluated by concomitant incubation of cells with atherogenic serum and aqueous solutions of tested substances. Anti‐atherosclerotic effect was measured as a decrease in the levels of intracellular cholesterol in the cells with test substances compared to the control cells (treated with atherogenic serum only). The described model allowed evaluating a number of different drugs and substances and detecting several novel active molecules with anti‐atherosclerotic potential. Some substances were demonstrated to possess a pro‐atherogenic effect, enhancing intracellular cholesterol accumulation induced by

*Ex vivo* model is based on primary culture of cells from unaffected human aortic intima that are incubated with blood serum from patients treated with the substance of interest. Therefore, potential anti‐atherogenic properties of substances are evaluated based on their pharmacody‐ namic properties, or the influence on blood serum atherogenicity after digestion and possible metabolic modifications in patient's body. Blood samples are drawn before and after admin‐ istration of single doses of tested substances, and serum obtained from the samples is added to cultured primary cells. *Ex vivo* model can be used for testing drugs with known safety

Several studies have demonstrated successful application of this model for evaluation of anti‐ atherogenic properties of botanicals. Screening studies were performed on volunteers (groups of 4–8 men and women 45–60 years old) with high blood serum atherogenicity. One of the tested natural products with anti‐atherosclerotic properties was encapsulated onion (*Allium cepa*) bulb powder (300 mg) (**Figure 3**). Administration of a single dose of the product resulted in a moderate decrease of blood serum atherogenicity by 12, 28, and 24% from the baseline after 2, 4, and 6 h, respectively. Another tested natural product with anti‐atherosclerotic properties was preparation of wheat seedlings (*Triticum aestivum*). Administration of a single dose of 300 mg of the preparation resulted in a pronounced reduction of blood serum athero‐ genicity after 4 h (**Figure 4**). Moderate but prolonged anti‐atherosclerotic effect was registered for dry beet (*Beta vulgaris*) juice (encapsulated preparation of 300 mg) (**Figure 5**). Garlic (*Allium sativum*) powder possessed a strong and prolonged effect (**Figure 6**). Blood serum atheroge‐ nicity was completely suppressed 4 h after administration of a single dose of 300 mg of the preparation. Several other natural products were screened for potential anti‐atherosclerotic activity using the *ex vivo* model (**Table 3**). The highest activity after a single dose administration was detected for garlic powder and wheat seedlings, with garlic powder providing the strongest effect. Importantly, anti‐atherosclerotic effects of garlic have been reported by several

folds after a 24‐h incubation with atherogenic serum.

atherogenic serum (**Table 2**).

24 Cholesterol Lowering Therapies and Drugs

profiles, as well as various natural products.

independent groups during the recent years [65–67].

**5.** *Ex vivo* **model**

**Figure 3.** Anti‐atherosclerotic effect of onion in *ex vivo* model.The study involved four volunteers (three males, one fe‐ male, mean age 57 ± 5 years) whose blood serum induced 1.3–1.5‐fold increase in cholesterol content of cells cultured from unaffected human aortic intima (the average level of serum atherogenicity was 141 ± 4%). Intracellular cholesterol in control cultures was 38.4 ± 1.1 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. Reproduced with permis‐ sion from [30].

**Figure 4.** Anti‐atherosclerotic effect of wheat seedlings in *ex vivo* model.The study involved eight volunteers (five males, three females, mean age 51 ± 2 years) whose blood serum induced 1.7–2.3‐fold increase in cholesterol content of cells cultured from unaffected human aortic intima (the average level of serum atherogenicity was 199 ± 6%). Intracel‐ lular cholesterol in control cultures was 28.0 ± 1.2 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].

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

**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].


\* The integrated effect was calculated as a mean reduction in serum atherogenicity for 6 h after a single oral dose.

**Table 3.** Integral estimation of anti‐atherogenic actions of natural products\*.

The described *ex vivo* model could be used for establishing the effective dose and posology of the potential anti‐atherosclerotic natural products. For this purpose, blood samples were drawn before and after (2 and 4 h) administration of a single dose to patients with high blood serum atherogenicity. Dose dependency was tested by comparison of the effect of two different doses. Each dose was evaluated on at least six different study participants. It was demonstrated that the anti‐atherosclerotic effect of garlic powder was present in the dose range from 50 to 300 mg with half‐maximal effect observed at a dose of 100 mg, and maximal effect—at 150 mg. Therefore, natural products of botanical origin can be regarded as an important source of agents with anti‐atherosclerotic activity that can be used for the development of direct anti‐ atherosclerotic therapy. Based on the obtained results, several dietary supplements were registered and further evaluated in clinical studies presented below.

As any model, cellular models for studying atherosclerosis development have their limita‐ tions [68–71]. Limitations of the experimental models used for atherosclerosis research have been discussed in a number of comprehensive reviews [72–77]. However, the described test system allows performing the initial screening for anti‐atherosclerotic activity that can be further studied and confirmed in pre‐clinical and clinical studies.
