Effect of Ich Tri Vuong capsule on plasma lipid level and degree of atherosclerosis in cholesterol fed- rabbits

JOURNAL OF MEDICAL RESEARCH<br /> <br /> EFFECT OF ICH TRI VUONG CAPSULE ON PLASMA<br /> LIPID LEVEL AND DEGREE OF ATHEROSCLEROSIS IN<br /> CHOLESTEROL-FED RABBITS<br /> <br /> Le Thi Minh Phuong1, Nguyen Tran Thi Giang Huong1,<br /> Do Thi Phuong1, Nguyen Thi Tuyet Nga2<br /> 1<br /> Faculty of Traditional Medicine, Hanoi Medical University,<br /> 2<br /> Military Institute of Traditional Medicine<br /> <br /> Ich Tri Vuong capsules (ITV) (components Ginkgo biloba, Radix Salviae miltiorrhi zae, Radix Astragali<br /> membranacei and Radix Angelicae sinensis) is used to treat diseases caused by atherosclerosis. This<br /> study was designed to assess the effects of ITV on plasma levels of total cholesterol, triglyceride,<br /> Low-density lipoprotein (LDL), High-density lipoprotein (HDL) and degrees of aorta atherosclerosis in<br /> rabbits with induced atherosclerosis by high cholesterol feeds. 50 New Zealand White rabbits were<br /> divided into 5 groups, 10 per group. Experiment conditions were carried out for 8 weeks. Normal group:<br /> fed with water 2 ml/kg/day. Control group: fed cholesterol 0.5 g/kg/day + water 2 ml/kg. Atorvastatin<br /> group: fed cholesterol 0.5 g/kg/day + Atorvastatin 5 mg/kg/day. ITV dose 1 group (60 mg/kg/day):<br /> fed cholesterol 0.5 g/kg/day+ ITV 60 mg/kg/day. ITV dose 2 group (180 mg/kg/day): fed cholesterol<br /> 0.5 g/kg/ngày + ITV 180 mg/kg/day. The results suggest that Ich Tri Vuong 180 mg/kg/day may<br /> decrease plasma levels of Total cholesterol and LDL and reduce atherosclerotic lesions of rabbits<br /> with induced atherosclerosis by high cholesterol feeds when compared to control group. These effects<br /> were equivalent to Atorvastatin 5 mg/kg/day and better than Ich Tri Vuong 60mg/kg/day. There were<br /> no significant differences in HDL and Triglyceride of ITV groups when compared to control group.<br /> <br /> Key works: Ich Tri Vuong, Atherosclerosis, plasma lipid<br /> <br /> I. INTRODUCTION<br /> Atherosclerosis is a disease of large and<br /> medium arteries. Lesions begin as fatty<br /> streaks, caused by massive accumulation<br /> of LDL oxidative product [1; 2] with high<br /> blood cholesterol LDL is increased in<br /> the plasma as well, and LDL is subject to<br /> oxidative modifications in the subendothelial<br /> space. Monocytes attache to endothelial<br /> Corresponding author: Le Thi Minh Phuong, Faculty<br /> of Traditional Medicine, Hanoi Medical University<br /> Email: lethiminhphuong@hmu.edu.vn<br /> Received: 05 June 2017<br /> Accepted: 16 November 2017<br /> <br /> JMR 111 E2 (2) - 2018<br /> <br /> cells. Adherent monocytes migrate into<br /> the subendothelial space and differentiate<br /> into macrophages. Uptake of oxLDL by<br /> macrophages via scavenger receptors<br /> leads to foam cell formation. Interactions<br /> between macrophage foam cells establish<br /> a chronic inflammatory process. Smooth<br /> muscle cells migrate to the side of<br /> inflammation. Necrosis of macrophage<br /> and smooth muscle cell-derived foam<br /> cells leads to the formation of a necrotic<br /> core and accumulation of extracellular<br /> cholesterol, creating a plaque. Macrophage<br /> secretion of matrix metalloproteinases and<br /> 27<br /> <br /> JOURNAL OF MEDICAL RESEARCH<br /> neovascularization contribute to weakening<br /> of the fibrous plaque. The ruptured plaque<br /> is exposed to plasma components,<br /> causing the recruitment of platelets, and<br /> the formation of a thrombus on the medial<br /> portion of the arterial wall. The thrombus<br /> proliferates and secretes extracellular<br /> matrix proteins that form a fibrous plaque<br /> [1]. Atherosclerosis progresses slowly over<br /> years, and can cause stroke, myocardial<br /> infarction, and peripheral artery disease.<br /> These are the mojor causes of disability<br /> and mortality. Therefore, treatment of lipid<br /> metabolism disorders, especilly high special<br /> LDL, plays an important role in preventing<br /> atherosclerosis requires [2]. Recently,<br /> research show that many herbal medicines<br /> that are used for treating diseases caused<br /> by atherosclerosis have an effect on lipid<br /> metabolism and preventing atherosclerosis<br /> [3; 4].<br /> Ich Tri Vuong capsule component<br /> include: Folium Ginkgo biloba, Radix Salviae<br /> miltiorrhi zae, Radix Astragali membranacei,<br /> and Radix Angelicae sinensis. This capscule<br /> is used in the treatment of chronic vertigo,<br /> headache, and insomnia caused by cerebral<br /> atherosclerosis. Ich tri vuong components<br /> were used in the “Angelica decoction<br /> for enriching plasma” (Radix Astragali<br /> membranacei, Radix Angelicae sinensis),<br /> a traditional medicine formula which<br /> effects plasma lipid and the expression<br /> of genes related to foam cell formation in<br /> the early stages of atherosclerosis [5].<br /> Other components of Ich Tri Vuong, Folium<br /> Ginkgo biloba and Radix Salviae miltiorrhi<br /> zae, also effect plasma lipids in in-vitro and<br /> in-vivo [6 - 8]. Research has shown that Ich<br /> 28<br /> <br /> Tri Vuong capsule has no acute and sub<br /> acute toxicities [9]. To hlear more about Ich<br /> Tri Vuong capsule is mechanism of action,<br /> this study was designed to assess the effect<br /> of Ich Tri Vuong capsule on plasma level of<br /> total cholesterol, triglyceride, LDL, HDL and<br /> the degree of aorta atherosclerosis in an<br /> induced atherosclerosis by high cholesterol<br /> feeds rabit model.<br /> <br /> II. SUBJECTS AND METHODS<br /> 1. Animal<br /> New Zealand and White rabbits (both<br /> male and female gender, body weight 1.8<br /> – 2.5 kg) were supplied by Son Tay Goat<br /> and Rabbit Research Center. The rabbits<br /> were fed with standard rabbit food in the<br /> laboratory 7 days prior to the experiment.<br /> 2. Material<br /> Ich Tri Vuong capsule<br /> Ich Tri Vuong capsule (ITV) components<br /> include 40 mg Folium Ginkgo biloba extract,<br /> 150 mg extract of Radix Salviae miltiorrhi<br /> zae, and Radix Astragali membranacei,<br /> Radix Angelicae sinensis. ITV capsules<br /> were supplied by the National Institute of<br /> Drug Quality Control and stored at room<br /> temperature. Dose 1 in rabbit was 60 mg/<br /> kg/day, dose 2 in rabbit was 180 mg/kg/day.<br /> Atorvastatin<br /> Atorvastatin 20 mg tablet, product<br /> of Standa Vietnam, stored at room<br /> temperature. Dose in rabbits was 5mg/kg/<br /> day.<br /> Cholesterol oil<br /> Pure cholesterol (product of MerkGermany) was dissolved in peanut oil<br /> (product of Tuong An company).<br /> JMR 111 E2 (2) - 2018<br /> <br /> JOURNAL OF MEDICAL RESEARCH<br /> 3. Methods<br /> Rabbits were devided to 5 groups of 10<br /> animals each. Experiment conditions were<br /> carried out for 8 weeks.<br /> <br /> Normal group: fed water 2 ml/kg/day.<br /> Control group: fed cholesterol oil 0.5 g/<br /> kg/day, 1ml/kg, 2 hours later fed water 2 ml/<br /> kg/day.<br /> Atorvastatin group: fed cholesterol oil<br /> 0.5 g/kg/day, 1ml/kg, 2 hours later fed<br /> Atorvastatin 5mg/kg/day.<br /> ITV dose 1 (60 mg/kg/day): fed<br /> cholesterol oil 0.5 g/kg/day, 1ml/kg, 2<br /> hours later fed Ich Tri Vuong 60 mg/kg/day<br /> (equivalent to clinical dose).<br /> ITV dose 2 (180 mg/kg/day): fed<br /> cholesterol oil 0.5 g/kg/day, 1ml/kg, 2 hours<br /> later fed Ich Tri Vuong 180 mg/kg/day (three<br /> time higher than clinical dose).<br /> Determination of plasma lipid levels<br /> The plasma triglycerides (TG), total<br /> <br /> cholesterol (TC), high-density lipoprotein<br /> cholesterol (HDL-C) and low-density<br /> lipoprotein cholesterol (LDL-C) were<br /> determined by commercial assay kits<br /> (Hospitex Diagnostics Italy and DIALAB<br /> GmbH Austria.) after 4 weeks and 8 weeks.<br /> Quantification of gross atherosclerotic<br /> lesions<br /> At the end of the 8 week - experiment,<br /> 30% of the rabbit aorta was collected<br /> for analysis of the aortic lesions. The<br /> progression of atherosclerosis was graded<br /> according to American Heart Association<br /> guidelines dividing atherosclerotic lesions<br /> into I-VI morphological types [10].<br /> 4. Statistical analysis<br /> 2 way ANOVA test and Mann–Whitney U<br /> test was used to analyze data, and p values<br /> less than 0.05 were considered statistically<br /> significant.<br /> <br /> III. RESULTS<br /> 8 weeks of the high cholesterol diet induced high blood lipids and atherosclerosis in rabbits<br /> of group 2, 3, 4, and 5 with significantly the increases TC, TG, LDL - C, and HDL - C plasma<br /> (p < 0.05), and lesions of aorta atherosclerosis seen on histological examination. Meanwhile,<br /> in group 1, there was no significant difference in lipids plasma levels with comparable to<br /> the begin (p > 0.05), and no lesion of atherosclerosis was seen in the aorta on histological<br /> examination.<br /> Plasma Lipids level<br /> To assess the effect of Ich Tri Vuong on plasma lipid level, we measured and compared<br /> plasma level of TC, TG, LDL - C, HDL - C at the beginning of the experiment (W0), after 4<br /> weeks (W4) and after 8 weeks (W8).<br /> <br /> <br /> JMR 111 E2 (2) - 2018<br /> <br /> 29<br /> <br /> JOURNAL OF MEDICAL RESEARCH<br /> <br /> <br /> <br /> Figure 1. Plasma level of Total Cholesterol (TC)<br /> In Figure 1, after 4 weeks and 8 weeks, plasma TC level of the Atorvastatin group (10.74<br /> ± 8.18 mmol/l and 10.71 ± 6.17 mmol/l) was significantly lower than the control group (21.13<br /> ± 9.98 mmol/l and 22.35 ± 10,31 mmol/l) and ITV dose 1 group (23.97 ± 14.28 mmol/l<br /> and 17.31 ± 9.56 mmol/l) (p < 0.05). ITV dose 2 group (13.91 ± 9.36 mmol/l and 12.76 ±<br /> 7,13 mmol/l) was significantly lower than Control group and had no significant difference to<br /> Atorvastatin (p > 0.05). The amount TC increased over 8 weeks in the Atorvastatin group<br /> (7.74 ± 5.84 mmol/l) and the ITV dose 2 (10.72 ± 6.77 mmol/l) group was significantly lower<br /> than the TC increase in the control group (19.57 ± 9.34 mmol/l) (p < 0.05). There was no<br /> significantly difference in the amount TC increased over 8 weeks in the ITV dose 2 group<br /> when compared to the Atorvastatin group (p > 0.05).<br /> <br /> Figure 2. Plasma level of LDL<br /> In Figure 2, after 4 weeks and 8 weeks, plasma LDL - C level of the Atorvastatin group<br /> 30<br /> <br /> JMR 111 E2 (2) - 2018<br /> <br /> JOURNAL OF MEDICAL RESEARCH<br /> (9.35 ± 7.31 mmol/l and 8.98 ± 5.35 mmol/l) were significantly lower than the control group<br /> (18.55 ± 9.18 mmol/l and 19.51 ± 9.66 mmol/l) and ITV dose 1 group (21.30 ± 3.12 mmol/l<br /> and 14.96 ± 8.58 mmol/l) (p < 0.05). ITV dose 2 group (11.96 ± 8.18 mmol/l and10.59 ± 6.23<br /> mmol/l) was significantly lower than control group (p < 0.05) and had no significant difference<br /> to the Atorvastatin group (p > 0.05). After 8 weeks, increasing levels of plasma LDL level<br /> of Atorvastatin group (6.64 ± 5.13 mmol/l) and ITV dose 2 (9.11 ± 6.01 mmol/l) group were<br /> significantly lower than the control group (17.31 ± 8.82 mmol/l) (p < 0.05). The ITV dose<br /> 1 group had no significant difference in amount of LDL - C increase over 8 weeks when<br /> compared to the control group (p > 0.05). There was no significant difference in ithe amount<br /> LDL - C increased over 8 weeks in the ITV dose 2 group when compared to the Atorvastatin<br /> group (p > 0.05).<br /> In Figure 3, after 4 weeks and 8 weeks, plasma HDL - C level of the Atorvastatin group<br /> (1.39 ± 0.89) and 1.73 ± 0.83 mmol/l) were significantly lower than the control group (2.59 ±<br /> 0.91 mmol/l and 2.84 ± 0.69 mmol/l) (p < 0.05). ITV dose 1 group (2.68 ± 1.26 mmol/l and<br /> 2.35 ± 1.01 mmol/l) and ITV dose 2 group (1.94 ± 1.20 mmol/l and 2.17 ± 0.94 mmol/l) had<br /> no significant difference to the Control group (p > 0.05). After 8 weeks, the amount that HDL<br /> - C increase over 8 weeks in the Atorvastatin group (1.10 ± 0.74 mmol/l) was significantly<br /> lower when the control group (2.26 ± 0.67 mmol/l) (p > 0.05) however the ratio of HDL/<br /> LDL in the Atorvastain group (0.19) was higher than the Control group (0.15). There was no<br /> significant difference the amount HDL - C increase over 8 weeks in the ITV dose 2 group<br /> when compared to the Atorvastatin group (p > 0.05).<br /> <br /> Figure 3. Plasma level of HDL<br /> In figure 4, after 4 weeks and 8 weeks, plasma triglyceride level of 5 group had no<br /> significantly difference (p > 0.05).<br /> <br /> JMR 111 E2 (2) - 2018<br /> <br /> 31<br /> <br />