박치영논문-1

HK-1000 55 DOI:10.3831/KPI.2008.11.3.055 HK-1000 A Study on the Effect of Herbal-acupuncture with HK-1000 Solution on Hyperlipidemia and Arteriosclerosis Induced Rats Park Chi-Young, Hong Kwon-Eui Dept. of Acupuncture & Moxibustion, College of Oriental Medicine, Daejeon University ABSTRACT Objective&Methods The purpose of this study is to observe the effects of HK-1000 Herbal-acupuncture solution at GB34(Yangleungchean) on hyperlipidemia and arteriosclerosis in rats. The author performed several experimental items to analyze the levels of various components and enzymes in serum, urine and liver, as well as the histological change of liver and aorta. Results 1. HK-1000 herbal-acupuncture infusion increased the cell viability rate, DPPH radical scavenging activity and HMG-CoA reductase inhibition rate in rat liver cells. 2. The levels of total cholesterol, free cholesterol, LDL-cholesterol, phospholipid in serum and AI (atherogenic index) were decreased, and the ratio of HDL to TCL(HDL/TCL) and the level of TG in serum were increased as compared with those of the control group. 3. In the HK-1000 group, serum AST and ALT were significantly lower than those of the HG group and saline group. 4. Hepatic GSH and catalase activities were significantly increased as compared with those of the saline group. Conclusions From the above results, it is suggested that HK-1000 herbal-acupuncture at GB34 has a therapeutic effect on hyperlipidemia and arteriosclerosis. Key words Hyperlipidemia, Arteriosclerosis, Herbal-acupuncture, HK-1000, GB34(Yangleungchean) I 22-5 Tel. 042-229-6816 Fax. 042-254-3403 E-mail : hkeacu@dju.ac.kr 2007 Received 2008.7.14, Accepted 2008.8.25

56 11 3 2008 9 HK-1000 2 1 HK-1001 flask 5000 ml 3 shaking water bath 3 rotary evaporator 95 ethyl alcohol 30 ml 85 ethyl alcohol 30 ml 75 ethyl alcohol 30 ml 2 ethyl alcohol rotary evaporator 20 ml 1N NaOH ph 6.8 4 12 syringe filtering PBS 1 II 1 1 6 200 20 g AIN-76A diet USA 22 2 2 HK-1000 HK-1000 Table 1 Table 1. Composition of HK-1000 g Saururus chinensis Baill 60 Mori Folium 60 Atractylodis 30 macrocephalae Rhizoma Akebiae Caulis 20 170 2 1 10 5 cell well 5 1 0.5 HK-1001 MTT assay 3 DPPH 1,1-Diphenyl-2-Picryl-Hydrazyl DPPH Sigma USA 80 mg 50 ml 50 ml 96 well 180 L HK-1001 30 517 nm O D activity 4 cholesterol 7 AIN-76A diet Dyet INC., PA, USA HMG-CoA reductase 5 1 2.5 0.1 HK-1001 control DMSO 20 L 340 nm 5 5 alloxan 150 mg/kg

HK-1000 57 180 220 mg/dl AIN-76A diet 4 4 Total cholesterol-hdl cholestrol HDL cholestrol 4 urine glucose ph uriscan 4 hemogph 6 8 normal control HG hand grasping NP needle prick saline saline injection HK-1000 HK-1000 6 GB34 HG Saline GB34 saline 0.2 ml NP GB34 26 gauge 0.5 cm HK-1000 GB34 1 HK-1000 0.2 ml H.G., N.P., saline HK- 1000 alloxan 4 3 7 1 1 liver index 2 ethyl ether Reference laboratory DaeJeon, Korea total cholesterol free cholesterol LDL HDL cholesterol AST ALT LDH ALP 3 Atherosclerosis index Total cholesterol HDL cholesterol 5 HMG-CoA reductase HMG-CoA reductase 1 ml cuvette 0.5mM phosphate buffer ph 7.0 20mM DTT100 L 3 mm NADPH100 L 100 L 37 10 preincubation 3 mm HMG-CoA 100 L 340 nm 5 Glutathione, superoxide dismutase(sod) and catalase activity Bansal 1g 4 150mM KCl homogeniger 600 g 10 mitochondrial fraction, cytosolic fraction, microsomal fraction microsomal fraction kit Dojindo Japan 405 nm glutathion cytosolic fraction SOD assay kit Dojindo Japan 50 nm SOD activity Mitochondrial fraction Catalase assay kit Oxford Biomedical Research USA 520 nm Catalase 6 10 formalin gum-sucrose 24 4 10 m 100% propylene glycol 60 oven Sudan black B 85 propylene glycol Nuclear fast red ECLIPSE E600 Nikon Japan

58 11 3 2008 9 ACT-1 program 7 10 formalin gumsucrose 24 4 10 m 100 propylene glycol 60 oven oil red O 85 propylene glycol 1 Harris' hematoxylin ECLIPSE E600 Nikon Japan ACT-1 program 2 DPPH DPPH HK- 1000 DPPH Fig. 2 9 SPSS 12.0 KO ANOVA 95 p 0.05 Figure 2. Effect of various concentrations of HK-1000- HAS on DPPH radical scavenging activity. Values represent the means SEM of 3 independent experiments. **: p 0.01, ***: p 0.001 compared to 5% HK-1000 HAS. : p 0.01, : p 0.001 compared to 2.5% HK-1000 HAS. : p 0.001 compared to 1% HK-1000 HAS. III 1 Fig. 1 3 HMG-CoA reductase HMG-CoA reductase HK-1000 HMG-CoA reductase Fig. 3 Figure 1. Cell viability rate with various concentrations of HK-1000 HAS. Values represent the means SEM of 3 independent experiments. ***: p 0.001 compared to control. : p 0.001 compared to 5% HK-1000 HAS. Figure 3. Hepatic HMG-CoA reductase inhibition rate of various concentrations of HK-1000-HAS ***: p 0.001 compared to 5% HK-1000 HAS. : p 0.001 compared to 2.5% HK-1000 HAS. : p 0.001 compared to 1% HK-1000 HAS. 4 1

HK-1000 59 HK-1000 Fig. 4 3 HK-1000 Fig. 6 Figure 4. Effect of HK-1000 HA at GB34 on body weight in rats with hyperlipidemia. Values represent the mean SD(n=8). 2 HK-1000 Fig. 5 Figure 6. Effect of HK-1000 HA at GB34 on the liver index in rats with hyperlipidemia. Values represent the mean SD(n=8). ***: p 0.001 compared to normal group. 5 Figure 5. Effect of HK-1000 HA at GB34 on liver weight in rats with hyperlipidemia. Values represent the mean SD(n=8). *: p 0.05, **: p 0.01, ***: p 0.001 compared to normal group. 1 total cholesterol, free cholesterol, HDL, LDL, triglyceride phospholipid Table 2 Table 2. Effect of HK-1000 HA at GB34 on various components in serum of rats with hyperlipidemia. Parameter Normal Control H.G. N.P. Saline HK-1000 Total cholesterol 59.2 7.694 214.4 23.093 125 13.398 105.6 9.788 130.6 38.102 109.8 18.377 Free cholesterol 3.8 2.775 19 3.162 18.6 7.57 11 2 16 4.637 12.4 3.362 HDLcholesterol 40.6 5.497 84.2 5.11 73.2 7.121 67.92 6.736 66.724 8.75 68.44 11.501 LDLcholesterol 8 1.871 34.4 3.362 32.75 3.961 24.8 4.324 32 2.345 27.25 1.785 Triglyceride 25.2 6.943 14.6 4.561 27.8 8.04 26.2 7.497 26 5.148 21.8 0.837 Phospholipid 95.6 6.465 137.2 15.32 133 14.265 118.8 7.95 121.8 9.783 122.8 19.79 Data were expressed as mean SD(n=5)

60 11 3 2008 9 1 Total cholesterol total cholesterol HG NP saline HK- 1000 2 Free cholesterol free cholesterol NP saline HK-1000 3 HDL cholesterol HDL-cholesterol 4 HDL/Total cholesterol HDL / total cholest erol HG NP saline HK-1000 5 LDL cholesterol LDL cholesterol NP HK-1000 LDL-cholesterol 6 Triglyceride triglyceride HG NP saline HK-1000 triglyceride 7 Phospholipid phospholipid NP saline HK-1000 phospholipid 8 atherogenic index AI NP HK-1000 2 ALP, AST, ALT, LDH Table 3 1 Alkaline phosphatase ALP ALP level 2 AST HG saline AST HK- 1000 AST HG saline 3 ALT HG ALT HK-1000 ALT HG saline Table 3. Effect of HK-1000 HA at GB34 on various enzymes in serum of rats with hyperlipidemia. Parameter Normal Control H.G. N.P. Saline HK-1000 ALP 253.6 76.402 322.6 16.682 267.2 19.33 306.6 34.428 340.4 39.539 330.4 44.73 AST 344.2 81.3 367.6 110.19 392.8 38.713 347.6 65.854 426.8 51.183 229 58.116 ALT 68 12.43 90.6 19.45 150.6 43.24 121.2 35.39 148.8 49.93 69.8 18.42 LDH 1908 167.22 2206.8 199.68 2263 210.66 2372 125.11 2120.4 276.26 2352.2 193.759 4 LDH NP LDH 6 glucose HG NP 1 mg/ml Saline HK-1000

HK-1000 61 glucose urine ph 7 9 HK-1000 urine ph 8.5 Table 4 7 HMG- CoA reductase SOD activity, glutathione catalsase activity Table 5 1 HMG-CoA reductase activity HMG-CoA reductase activity HK-1000 HMG-CoA reductase activity saline 2 GSH GSH activity HK-1000 saline Table 4. Effect of HK-1000 HA at GB34 on urine glucose and ph in rats with hyperlipidemia. Parameter Normal Control H.G. N.P. Saline HK-1000 glucose - - - ph 9 7 8.5 9 9 8.5 Data were expressed as mean SD(n=3). Glucose; :1, +:2.5, ++:5, +++:10, ++++:20(mg/mL) Table 5. Effect of HK-1000 HA at GB34 on various hepatic enzymes in rats with hyperlipidemia Parameter Normal Control H.G. N.P. Saline HK-1000 HMG-CoA. reductase activity (%) 54.321 5.685 91.124 5.685 85.185 7.407 79.012 8.553 94.443 1.852 86.42 5.658 SOD activity (%) 92.522 6.148 93.439 6.432 97.137 5.687 91.02 5.928 101.835 4.96 78.176 12.655 Glutathione Conc.(umol/L) 155.19 4 15.416 118.931 10.418 134.778 11.338 132.125 14.108 112.389 8.361 162.736 1.459 Catalase activity (U/mL) 176.26 0.087 126.02 3.007 141.267 0.785 138.207 4.627 128.877 0.894 154.367 4.437 Data were expressed as mean SD (n=5). 3 SOD HG NP saline SOD activity HK-1000 SOD activity saline 4 Catalase catalase activity HK-1000 catalase activity HG NP saline 8 1 HK-1000 Fig. 7 Figure 7. Histological analysis of liver in rats with hyperlipidemia(sbb stain)

62 11 3 2008 9 2 HK-1000 Fig. 8 Figure 8. Histological analysis of aorta in rats with hyperlipidemia(oil red O stain) IV cholesterol, triglyceride, phospholipid, free fatty acid cholesterol triglyceride 35 LDL cholesterol 220 mg/dl 3-6 LDL cholesterol 190 mg/dl 2 LDL cholesterol 160 mg/dl GB34 HK-1000 HK-1000

HK-1000 63 HK-1000 HK-1000 Fig. 1 HK-1000 DPPH DPPH DPPH Fig. 2 DPPH HK-1000 HMG-CoA reductase HK-1000 HMG- CoA reductase HMG-CoA reductase Fig. 3 HMG-CoA reductase cholesterol HMG-CoA reductase cholesterol HK-1000 cholesterol 4 HK-1000 Fig. 4 HK-1000 Fig. 5 HK-1000 Fig. 6 Triglyceride, Total cholesterol, LDL cholesterol HK- 1000 total cholesterol level total cholesterol HG NP saline HK-1000 free cholesterol level NP saline HK-1000 HK-1000 HDL-cholesterol level HDL-cholesterol total cholesterol HDLcholesterol HG NP saline HK-1000 HDL/total cholesterol HDL cholesterol LDL-cholesterol level LDL-cholesterol NP HK-1000 LDL-cholesterol LDLcholesterol LDL Triglyceride LDL HDL triglyceride level

64 11 3 2008 9 triglyceride HG NP saline HK-1000 triglyceride cholesterol TG phospholipid level NP saline HK-1000 phospholipid phospholipid cholesterol Total phospholipid/total cholesterol total cholesterol HDL cholesterol NP HK-1000 HK-1000 total cholesterol, free cholesterol, LDL-cholesterol, phospholipid, AI TG ALP ALP level AST ALT level AST HG saline HK-1000 HG saline ALT HG HK-1000 HG saline AST ALT LDH level NP LDH LDH lactate pyruvate HK-1000 HMG-CoA reductase activity HMG- CoA reductase activity HK-1000 HMG-CoA reductase activity saline HMG- CoA reductase cholesterol GSH level GSH activity HK-1000 GSH activity saline glutathione peroxidase glutathione hydrogen peroxidase lipid peroxidase SOD activity HG NP saline SOD activity HK-1000 SOD activity saline SOD scavenger superoxide anion radical O2 2 O 2 O 2 H 2 O 2 Catalase activity HK-1000 catalase activity HG NP saline Catalase H 2 O 2 H 2 O O 2 2 HK-1000 GSH catalase activity HMG-CoA reductase activity HK-1000

HK-1000 65 Fig. 7 HK-1000 Fig. 8 HK-1000 HK-1000 HK- 1000 V HK-1000 1 HK-1000 DPPH HMG -CoA reductase 2 HK-1000 total cholesterol, free cholesterol, LDL-cholesterol, phospholipid, AI HDL-cholesterol, HDL/total cholesterol, TG 3 HK-1000 AST ALT HG saline 4 HK-1000 GSH activity catalase activity VI 1. Medical Postgraduates, 31:160-165, 2003. 2. http://www.mohw.go.kr/user.tdf, 2006. 3. pp.209-214, 2001 4. pp.214-215. 1995 5. pp.1-5, 1997 6. pp.648-649, 1457-1467, 2000 7. 13(2):97-108, 2004 8. 13(2):109-123, 2004 9. 27(1):16-26, 2006 10. 10(2):99-106, 2006 11. 34(1):42-56,2005 12. Xie W, Wang W, Su H, Xing D, Cai G, Du L : Hypolipidemic mechanisms of Ananas comosus L. leaves in mice: different from fibrates butsimilar to statins, J pharmacol Sci 103:267-274, 2007. 13. Bansal VS, Kanfer JN: The phospholipid-nmethyltransferase of rat brain microsomes, Biochim Biophys Acta, 836(1):73-79, 1985. 14. 1992 :27, 38-53 15. p.261, 2005 16. p11, 83, 135, 2003 17. pp.995-1005 1991. 18. 1 Workshop, pp.41-49, 1994 19.

66 11 3 2008 9 p.545, 1998 20. Gastrin 21(3):179-191, 2004 21. pp.1379-1348, 1707-1717, 2124-2125, 2175-2179, 1997 22. pp.145-146, 315-316, 536-537, 2000 23. NO DPPH IL-4 20(2):184-194 2003 24. LEE, M. K., Moon, S. S., Lee, S. E., Bok, S. H., Jeong, T. S., Park, Y. B., Choi, M. S. : Naringenin 7-Ocetyl ether as inhibitor of HMG-CoA reductase and modulator of plasma and hepatic lipids in high cholesterol-fed rats. Bioorganic and medicinal chemistry, 11:393-398, 2003 25. Bocan, T. M., Mueller, S. B., Brown, E. Q., Lee, P., Bocan, M. J., Rea, T., Pape, M. E. : HMG-CoA reductase and ACAT inhibitors act synergistically to lower plasma cholesterol and limit atherosclerotic lesion development in the cholesterol-fed rabbit. Atherosclerosis, 139:21-30, 1998 26. Martin MJ. : Serum cholesterol, blood pressure, and mortality: implications from a cohort of 361662 men. Lancet, 2:933-939, 1986 27. pp.273-276, 281-283, 303-315, 2001 28. Total cholesterol HDL-cholesterol, triglyceride LDL-cholesterol 1987 29. Berard AM., Foger B., Remaley A., Shamburek R., Vaisman BL., Talley G., Paigen B, Hoyt RF Jr., Marcovina S., Brewer HB jr., Santamarina-Fojo S. High plasma HDL concentrations associated with enhanced atherosclerosis intransgenic mice overexpressing lecithin-cholesteryl acyltransferase. Nat Med. 3:744-749, 1997. 30. Jurgens G., Taddei-Peters WC., Koltringer P., Petek W., Chen Q., Greilberger J., Macomber PF., Butman BT., Stead AG., Ransom JH. Lipoprotein(a) serum concentration and apolipoprotein(a) phenotype correlate with severity and presence of ischemic cerebrovascular disease. Stroke. 26:1841-1848, 1995.. 31. Cristopher, KG and Joseph, IW : Atherosclerosis : The road ahead. Cell 104:503-516, 2001. 32. Anber V, Griffin BA, McConnel M, Packard CJ, Shepherd J. Role of plasma triglyceride in the regulation of plasma lowdensity lipoprotein (LDL) subfractions: relative contribution of small, dense LDL to coronary heart disease risk, Atherosclerosis, 106:241-253, 1994. 33. 24(4):297-300, 1994 34. 5(2):61-69, 1995 35. 2 31(2):190-195, 2000 36. Ozturk O, Gumuslu S. Changes in glucose-6- phosphate dehydrogenase. copper. zinc-superoxide dismutase and catalase activities. glutathione and its metabolizing enzymes. and lipid peroxidation in rat erythrocytes with age. Experimental gerontology. 39(2):211-216, 2004 37. 25(4):147-157, 2004. 38. Kim, S. M., Cho, Y. S. and Sung, S. K. : The antioxidant and nitrite scavening ability of waste resource(crop shell, sesame meal, korean tangrin peal) extracts. J. Korean Soc. Food Sci. Nutr. 30:589-593, 2001