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Ich Tri Vuong capsules (ITV) (components Ginkgo biloba, Radix Salviae miltiorrhi zae, Radix Astragali membranacei and Radix Angelicae sinensis) is used to treat diseases caused by atherosclerosis. This study was designed to assess the effects of ITV on plasma levels of total cholesterol, triglyceride, Low-density lipoprotein (LDL).
JOURNAL OF MEDICAL RESEARCH EFFECT OF ICH TRI VUONG CAPSULE ON PLASMA LIPID LEVEL AND DEGREE OF ATHEROSCLEROSIS IN CHOLESTEROL-FED RABBITS Le Thi Minh Phuong1, Nguyen Tran Thi Giang Huong1, Do Thi Phuong1, Nguyen Thi Tuyet Nga2 Faculty of Traditional Medicine, Hanoi Medical University, Military Institute of Traditional Medicine Ich Tri Vuong capsules (ITV) (components Ginkgo biloba, Radix Salviae miltiorrhi zae, Radix Astragali membranacei and Radix Angelicae sinensis) is used to treat diseases caused by atherosclerosis This study was designed to assess the effects of ITV on plasma levels of total cholesterol, triglyceride, Low-density lipoprotein (LDL), High-density lipoprotein (HDL) and degrees of aorta atherosclerosis in rabbits with induced atherosclerosis by high cholesterol feeds 50 New Zealand White rabbits were divided into groups, 10 per group Experiment conditions were carried out for weeks Normal group: fed with water ml/kg/day Control group: fed cholesterol 0.5 g/kg/day + water ml/kg Atorvastatin group: fed cholesterol 0.5 g/kg/day + Atorvastatin mg/kg/day ITV dose group (60 mg/kg/day): fed cholesterol 0.5 g/kg/day+ ITV 60 mg/kg/day ITV dose group (180 mg/kg/day): fed cholesterol 0.5 g/kg/ngày + ITV 180 mg/kg/day The results suggest that Ich Tri Vuong 180 mg/kg/day may decrease plasma levels of Total cholesterol and LDL and reduce atherosclerotic lesions of rabbits with induced atherosclerosis by high cholesterol feeds when compared to control group These effects were equivalent to Atorvastatin mg/kg/day and better than Ich Tri Vuong 60mg/kg/day There were no significant differences in HDL and Triglyceride of ITV groups when compared to control group Key works: Ich Tri Vuong, Atherosclerosis, plasma lipid I INTRODUCTION Atherosclerosis is a disease of large and medium arteries Lesions begin as fatty streaks, caused by massive accumulation of LDL oxidative product [1; 2] with high blood cholesterol LDL is increased in the plasma as well, and LDL is subject to oxidative modifications in the subendothelial space Monocytes attache to endothelial Corresponding author: Le Thi Minh Phuong, Faculty of Traditional Medicine, Hanoi Medical University Email: lethiminhphuong@hmu.edu.vn Received: 05 June 2017 Accepted: 16 November 2017 JMR 111 E2 (2) - 2018 cells Adherent monocytes migrate into the subendothelial space and differentiate into macrophages Uptake of oxLDL by macrophages via scavenger receptors leads to foam cell formation Interactions between macrophage foam cells establish a chronic inflammatory process Smooth muscle cells migrate to the side of inflammation Necrosis of macrophage and smooth muscle cell-derived foam cells leads to the formation of a necrotic core and accumulation of extracellular cholesterol, creating a plaque Macrophage secretion of matrix metalloproteinases and 27 JOURNAL OF MEDICAL RESEARCH neovascularization contribute to weakening of the fibrous plaque The ruptured plaque is exposed to plasma components, causing the recruitment of platelets, and the formation of a thrombus on the medial portion of the arterial wall The thrombus proliferates and secretes extracellular matrix proteins that form a fibrous plaque [1] Atherosclerosis progresses slowly over years, and can cause stroke, myocardial infarction, and peripheral artery disease These are the mojor causes of disability and mortality Therefore, treatment of lipid metabolism disorders, especilly high special LDL, plays an important role in preventing atherosclerosis requires [2] Recently, research show that many herbal medicines that are used for treating diseases caused by atherosclerosis have an effect on lipid metabolism and preventing atherosclerosis [3; 4] Ich Tri Vuong capsule component include: Folium Ginkgo biloba, Radix Salviae miltiorrhi zae, Radix Astragali membranacei, and Radix Angelicae sinensis This capscule is used in the treatment of chronic vertigo, headache, and insomnia caused by cerebral atherosclerosis Ich tri vuong components were used in the “Angelica decoction for enriching plasma” (Radix Astragali membranacei, Radix Angelicae sinensis), a traditional medicine formula which effects plasma lipid and the expression of genes related to foam cell formation in the early stages of atherosclerosis [5] Other components of Ich Tri Vuong, Folium Ginkgo biloba and Radix Salviae miltiorrhi zae, also effect plasma lipids in in-vitro and in-vivo [6 - 8] Research has shown that Ich 28 Tri Vuong capsule has no acute and sub acute toxicities [9] To hlear more about Ich Tri Vuong capsule is mechanism of action, this study was designed to assess the effect of Ich Tri Vuong capsule on plasma level of total cholesterol, triglyceride, LDL, HDL and the degree of aorta atherosclerosis in an induced atherosclerosis by high cholesterol feeds rabit model II SUBJECTS AND METHODS Animal New Zealand and White rabbits (both male and female gender, body weight 1.8 – 2.5 kg) were supplied by Son Tay Goat and Rabbit Research Center The rabbits were fed with standard rabbit food in the laboratory days prior to the experiment Material Ich Tri Vuong capsule Ich Tri Vuong capsule (ITV) components include 40 mg Folium Ginkgo biloba extract, 150 mg extract of Radix Salviae miltiorrhi zae, and Radix Astragali membranacei, Radix Angelicae sinensis ITV capsules were supplied by the National Institute of Drug Quality Control and stored at room temperature Dose in rabbit was 60 mg/ kg/day, dose in rabbit was 180 mg/kg/day Atorvastatin Atorvastatin 20 mg tablet, product of Standa Vietnam, stored at room temperature Dose in rabbits was 5mg/kg/ day Cholesterol oil Pure cholesterol (product of MerkGermany) was dissolved in peanut oil (product of Tuong An company) JMR 111 E2 (2) - 2018 JOURNAL OF MEDICAL RESEARCH Methods Rabbits were devided to groups of 10 animals each Experiment conditions were carried out for weeks Normal group: fed water ml/kg/day Control group: fed cholesterol oil 0.5 g/ kg/day, 1ml/kg, hours later fed water ml/ kg/day Atorvastatin group: fed cholesterol oil 0.5 g/kg/day, 1ml/kg, hours later fed Atorvastatin 5mg/kg/day ITV dose (60 mg/kg/day): fed cholesterol oil 0.5 g/kg/day, 1ml/kg, hours later fed Ich Tri Vuong 60 mg/kg/day (equivalent to clinical dose) ITV dose (180 mg/kg/day): fed cholesterol oil 0.5 g/kg/day, 1ml/kg, hours later fed Ich Tri Vuong 180 mg/kg/day (three time higher than clinical dose) Determination of plasma lipid levels The plasma triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) were determined by commercial assay kits (Hospitex Diagnostics Italy and DIALAB GmbH Austria.) after weeks and weeks Quantification of gross atherosclerotic lesions At the end of the week - experiment, 30% of the rabbit aorta was collected for analysis of the aortic lesions The progression of atherosclerosis was graded according to American Heart Association guidelines dividing atherosclerotic lesions into I-VI morphological types [10] Statistical analysis way ANOVA test and Mann–Whitney U test was used to analyze data, and p values less than 0.05 were considered statistically significant III RESULTS weeks of the high cholesterol diet induced high blood lipids and atherosclerosis in rabbits of group 2, 3, 4, and with significantly the increases TC, TG, LDL - C, and HDL - C plasma (p < 0.05), and lesions of aorta atherosclerosis seen on histological examination Meanwhile, in group 1, there was no significant difference in lipids plasma levels with comparable to the begin (p > 0.05), and no lesion of atherosclerosis was seen in the aorta on histological examination Plasma Lipids level To assess the effect of Ich Tri Vuong on plasma lipid level, we measured and compared plasma level of TC, TG, LDL - C, HDL - C at the beginning of the experiment (W0), after weeks (W4) and after weeks (W8) JMR 111 E2 (2) - 2018 29 JOURNAL OF MEDICAL RESEARCH Figure Plasma level of Total Cholesterol (TC) In Figure 1, after weeks and weeks, plasma TC level of the Atorvastatin group (10.74 ± 8.18 mmol/l and 10.71 ± 6.17 mmol/l) was significantly lower than the control group (21.13 ± 9.98 mmol/l and 22.35 ± 10,31 mmol/l) and ITV dose group (23.97 ± 14.28 mmol/l and 17.31 ± 9.56 mmol/l) (p < 0.05) ITV dose group (13.91 ± 9.36 mmol/l and 12.76 ± 7,13 mmol/l) was significantly lower than Control group and had no significant difference to Atorvastatin (p > 0.05) The amount TC increased over weeks in the Atorvastatin group (7.74 ± 5.84 mmol/l) and the ITV dose (10.72 ± 6.77 mmol/l) group was significantly lower than the TC increase in the control group (19.57 ± 9.34 mmol/l) (p < 0.05) There was no significantly difference in the amount TC increased over weeks in the ITV dose group when compared to the Atorvastatin group (p > 0.05) Figure Plasma level of LDL In Figure 2, after weeks and weeks, plasma LDL - C level of the Atorvastatin group 30 JMR 111 E2 (2) - 2018 JOURNAL OF MEDICAL RESEARCH (9.35 ± 7.31 mmol/l and 8.98 ± 5.35 mmol/l) were significantly lower than the control group (18.55 ± 9.18 mmol/l and 19.51 ± 9.66 mmol/l) and ITV dose group (21.30 ± 3.12 mmol/l and 14.96 ± 8.58 mmol/l) (p < 0.05) ITV dose group (11.96 ± 8.18 mmol/l and10.59 ± 6.23 mmol/l) was significantly lower than control group (p < 0.05) and had no significant difference to the Atorvastatin group (p > 0.05) After weeks, increasing levels of plasma LDL level of Atorvastatin group (6.64 ± 5.13 mmol/l) and ITV dose (9.11 ± 6.01 mmol/l) group were significantly lower than the control group (17.31 ± 8.82 mmol/l) (p < 0.05) The ITV dose group had no significant difference in amount of LDL - C increase over weeks when compared to the control group (p > 0.05) There was no significant difference in ithe amount LDL - C increased over weeks in the ITV dose group when compared to the Atorvastatin group (p > 0.05) In Figure 3, after weeks and weeks, plasma HDL - C level of the Atorvastatin group (1.39 ± 0.89) and 1.73 ± 0.83 mmol/l) were significantly lower than the control group (2.59 ± 0.91 mmol/l and 2.84 ± 0.69 mmol/l) (p < 0.05) ITV dose group (2.68 ± 1.26 mmol/l and 2.35 ± 1.01 mmol/l) and ITV dose group (1.94 ± 1.20 mmol/l and 2.17 ± 0.94 mmol/l) had no significant difference to the Control group (p > 0.05) After weeks, the amount that HDL - C increase over weeks in the Atorvastatin group (1.10 ± 0.74 mmol/l) was significantly lower when the control group (2.26 ± 0.67 mmol/l) (p > 0.05) however the ratio of HDL/ LDL in the Atorvastain group (0.19) was higher than the Control group (0.15) There was no significant difference the amount HDL - C increase over weeks in the ITV dose group when compared to the Atorvastatin group (p > 0.05) Figure Plasma level of HDL In figure 4, after weeks and weeks, plasma triglyceride level of group had no significantly difference (p > 0.05) JMR 111 E2 (2) - 2018 31 JOURNAL OF MEDICAL RESEARCH Figure Plasma level of Triglyceride In figure 4, after weeks and weeks, plasma triglyceride level of all groups had no significant difference when compared to the control (p > 0.05) Lesions of aortic atherosclerosis In Figure 5, after weeks, all samples of Normal group and 2/3 sample of Atorvastin group and ITV dose group had no aortic atherosclerotic gross lesions, 3/3 samples of Control group and 2/3 sample of ITV dose had a gross lesion of aortic atherosclerosis A 32 Normal Control Atorvastatin ITV dose ITV dose JMR 111 E2 (2) - 2018 JOURNAL OF MEDICAL RESEARCH 100% 80% B 60% Type III 40% Type II 20% Normal 0% Normal group Control group Atorvastatin group ITV dose group ITV dose group Figure Lesion of aortic atherosclerosis Figure 5: Lesion of aortic atherosclerosis (A) Aortic arch and histological examination of groups (B) Degree lesions in aortic arch sections histological examination In Figure 5, after weeks, all samples of Normal group and 2/3 sample of Atorvastin group and ITV dose group had no aortic atherosclerotic gross lesions, all samples of Control group and 2/3 sample of ITV dose had a gross lesion of aortic atherosclerosis Histological examination of aortic arch sections were examined to quantify the degree of atherosclerotic lesions All samples of the Control group and 2/3 samples of ITV dose group had type III lesions There were fewer intimal lession in the Atorvastatin group (with 1/3 sample had type II of lesion, 2/3 sample was normal) and ITV dose group (with 1/3 sample had type II of lesion, 2/3 sample was normal), when compared to the control group IV DISCUSSION Ich Tri Vuong capsule components include Folium Ginkgo biloba, Radix Salviae miltiorrhi zae, Radix Astragali membranacei, and Radix Angelicae sinensis Capsules have been used in the treatment chronic vertigo, headache and insomnia caused by cerebral atherosclerosis The results show that ITV dose 180 mg/kg/day and Atorvastatin mg/kg/day significantly lower plasma level of total cholesterol and LDL when compared with the control group ITV dose 180 mg/kg/ day and 60 mg/kg/day had no significant JMR 111 E2 (2) - 2018 difference in HDL when compared with the control group The increase over week of plasma total cholesterol and LDL of the Atorvastatin group (7.74 ± 5.84 mmol/l and 6.64 ± 5.13 mmol/l) and the ITV dose (10.72 ± 6.77 mmol/l and 9.11 ± 6.01 mmol/l) group were significantly lower than the control group (19.57 ± 9.34 mmol/l and 17.31 ± 8.82 mmol/l) (p < 0.05) ITV dose 60mg/kg/day had plasma levels of Total cholesterol significantly lower than the Control group, but had no significant difference in LDL compared with the Control 33 JOURNAL OF MEDICAL RESEARCH group There was no significant difference of the increase over weeks of plasma lipids of the ITV dose group when compared with the Atorvastatin group (p > 0.05) These effects of Ich Tri Vuong may be due to effects of its components Previous studies have shown that Ich Tri Vuong components had an effect on plasma lipids, expression of genes related to foam cell formation in the early stage of atherosclerosis in a diabetic rat model [5] Folium Ginkgo biloba and Radix Salviae miltiorrhi of Ich Tri Vuong also had effects on plasma lipid in in-vitro and in-vivo model [6 - 8] Hypercholesterolemia is a major risk factor for atherosclerosis [1; 2] In this research ITV dose group and ITV dose group decreased atherosclerosis lesion in histological examination of the aortic arch, when compared with the Control group The decrease in atherosclerosis lesions effected by Ich Tri Vuong 180 mg/ kg/day was equivalent to that of Atorvastatin mg/kg/day Atherosclerotic lesions begin as massive accumulations of LDL oxidative productions So the lowering Total cholesterol and LDL effects of Ich Tri Vuong could mediate the reduction in atherosclerosis Although Atorvastatin mg/kg/day decreased LDL more than Ich Tri Vuong 180 mg/kg/day, the eddect on atherosclerosis lesions of Atorvastatin mg/kg/day was equivalent to the effect of Ich Tri Vuong 180 mg/kg/day Therefore Ich Tri Vuong effect on atherosclerosis pathology may be mediated by more than one pathway 34 V CONCLUSION These results suggest that in an induced - atherosienrosis rabbit model Ich Tri Vuong 180 mg/kg/day may decrease plasma levels of Total cholesterol and LDL, and reduce atherosclerosis lesion when compared to control group The effect of ITV 180 mg/kg/ day was equivalent to Atorvastatin mg/kg/ day and better than Ich Tri Vuong 60 mg/kg/ day There was no significant difference in HDL and Triglyceride of ITV groups when compared to the control group Acknowledgments We would like to thank the staff that participated This work was supported by a research grant of Hanoi Medical University REFERENCE James S Forrester (2008), The Pathogenesis of Atherosclerosis and Plaque Instability, Atherosclerosis and Oxidant Stress: A New Perspective, Springer, – Judith A Berliner, Mohamad Navab, Alan M Fogelman et al (1995), Atherosclerosis: basic mechanisms Oxidation, inflammation, and genetics, Circulation, 91(9), 2488 - 2496 Hao Xu, Dazhuo Shi, Keji Chen (2012), Atherosclerosis: An Integrative East-WestMedicine Perspective, EvidenceBased Complementary and Alternative Medicine, Vol 2012, Article ID 148413, pages, doi:10.1155/2012/148413 Qing Liu, Jianping Li, Adam Hartstone-Rose et al (2015), Chinese Herbal Compounds for the Prevention and Treatment of Atherosclerosis: Experimental JMR 111 E2 (2) - 2018 JOURNAL OF MEDICAL RESEARCH Evidence and Mechanisms, Evidence-Based Complementary and Alternative Medicine, Vol 2015, Article ID 752610, 15 pages, http://dx.doi.org/10.1155/2015/752610 Zhang H, Chen S, Deng X et al (2006), The effects of Danggui-Buxue-Tang on plasma lipid and expression of genes related to foam cell formation in the early stage of atherosclerosis in diabetic GK rats, Diabetes Res Clin Pract, 2006, DOI: http:// dx.doi.org/10.1016/j.diabres.2006.11.005 Kobuchi H, Droy-Lefaix MT, Christen Y et al (1997), Ginkgo bilobu extract (EGb 761): Inhibitory effect on nitric oxide production in the macrophage cell line RAW 264.7, Biochem Pharmacol, 53, 897 903 Barth SA, lnselmann G, Heidemann HT (1991), Influences of Ginkgo bilobu on cyclosporin A induced lipid peroxidation in human liver microsomes in comparison to vitamin E, glutathion and N-acetylcysteine, Biochem Pharmacol, 41, JMR 111 E2 (2) - 2018 1521 - 1526 S Xu, Z Liu, Y Huang et al (2012), Tanshinone II-A inhibits oxidized LDL-induced LOX-1 expression in macrophages by reducing intracellular superoxide radical generation andNF-kB activation, Translational Research, 160 (2), 114 – 124 Tran Viet Hung, Pham Thi Nguyet Hang (2012), Neuroprotective Effect of Combined Crude Extracts (Ginkgo biloba Lin., Radix Astragali, Radix Salviae miltiorhizae, Radix Angelicae sinensis) in Comparison with Ginkgo biloba Extract Against H2O2-Induced NG108-15 Cells Damage, Journal of Medicinal Material, 22 (2), 104 – 109 10 Stary HC, Chandler AB, Dinsmore RE et al (1995), A definition of advanced typees of atherosclerotic lesions and a histological classification of American Heart Association, Circulation, 92, 1355–74 35 ... about Ich Tri Vuong capsule is mechanism of action, this study was designed to assess the effect of Ich Tri Vuong capsule on plasma level of total cholesterol, triglyceride, LDL, HDL and the degree. .. histological examination Plasma Lipids level To assess the effect of Ich Tri Vuong on plasma lipid level, we measured and compared plasma level of TC, TG, LDL - C, HDL - C at the beginning of the experiment... than Ich Tri Vuong 180 mg/kg/day, the eddect on atherosclerosis lesions of Atorvastatin mg/kg/day was equivalent to the effect of Ich Tri Vuong 180 mg/kg/day Therefore Ich Tri Vuong effect on atherosclerosis