051~062 음곡(KI10) 금앵자

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1 (KI10) 51 DOI : /KPI KI10 Received : Accepted : Effects of Herbal-Acupuncture with Rosae Laevigatae Fructus Extract at KI10(Umgok) on Osteoporosis in Ovariectomized Mice Kim Dong-soo, Kim Jeoung-ho, Kim Young-il Key Words: Osteoporosis, Herbalacupuncture, Rosae Laevigatae Fructus, KI10(Umgok) * Dept. of Acupuncture & Moxibustion, College of Oriental Medicine, Daejeon University ABSTRACT Objective & Methods : The purpose of this study was to observe the effects of herbal-acupuncture with Rosae Laevigatae Fructus extact(rlf-ha) at KI10(Umgok) on osteoporosis in ovariectomized(ovx) ddy mice. We carried out several experimental items to analyze the changes in body weight, uterine weight, uterus index, tibial length, the ash bone weight, tibial BMD, serum ALP, serum osteocalcin, serum Ca, and the levels of Ca, P, Ca/P ratio in tibia, and we performed histological and histomorphological analysis as well. Results : 1. Herbal-acupuncture with Rosae Laevigatae Fructus at KI10(Umgok) and saline injection at KI10(Umgok) significantly inhibited the reduction of tibial calcium level in ovariectomized mice. 2. Herbal-acupuncture with Rosae Laevigatae Fructus at KI10(Umgok) significantly inhibited the increase of tibial osteoclast cells in ovariectomized mice. 3. Herbal-acupuncture with Rosae Laevigatae Fructus at KI10(Umgok) significantly inhibited the reduction of tibial trabecular bone thickness(tbt) in ovariectomized mice. 4. Herbal-acupuncture with Rosae Laevigatae Fructus at KI10(Umgok) significantly inhibited the overgrowth of tibial growth plate length(gpl) in ovariectomized mice. Conclusion : These results suggested that herbal-acupuncture with Rosae Laevigatae Fructus at KI10(Umgok) has a therapeutic effect on osteoporosis, and to be put to practical use in the future osteoporosis clinic. I Corresponding author : Kim Young-il, Dae-jeon Uni. Department of Acupuncture & Moxibustion, College of Oriental Medicine, Daejeon University Dunsandong Seogu Daejeon, Korea Tel omdkim01@dju.ac.kr

2 Rosae laevigata fructus KI10 ALP, creatinine, phosphorus, calcium, osteocalcin, calcium, phosphorus, Ca/P ratio, osteoclast like cell II ddy mice 22 5g Rosae laevigata fructus 3 1 Table 1 2 Table flask shaking water bath 3 rotary evaporator 95 ethyl alcohol 30 rotary evaporator 85 ethyl alcohol ethyl alcohol 30 ethyl alcohol rotary evaporator N NaOH ph syringe filter PBS 5 2 ddy mice cell/well MTT assay (1) Osteoblast cell ddy mice PBS PBS syringe 1200rpm 5 MSC osteogenic singlequots differentiation Basal medium Cat No. PT-3924, Lonza, USA 3 7 osteoblast cell

3 (KI10) 53 (2) MTT assay osteoblast cell viability trypan blue 96well plate cells/well MSC osteogenic singlequots differaentiation Basal medium Cat No. PT-3924, Lonza, USA MTT solution 5 Cat No , Sigma, USA well incubation 5 well medium 100 solubilizing solution 100 pipetting well dark blue crystals ELISA reader 570 optical densities 3 ddy mouse 4 ovariectomy 3 ddy dorso-lateral abdominal wall 1 abdominal muscle scheme 1 4 KI10 5 ddy 9 normal sham-ovx control HG hand grasping NP needle prick saline saline injection RLF-HA 7 normal sham-ovx control HG NP KI10 26gauge 0.5 saline 0.2 KI10 RLF-HA KI10 5 RLF-HAS 0.2 N.P. saline 8 3 KI Uterus index = uterus g 100 g furnace Lab companion, Korea HCl Ca P Ca/P ratio Ca/P ratio Calcium contents of tibia 100 Phosphorus contents of tibia 4

4 formalin 24 PIXImus Lunar meison,wi DEXA dual-energy x-ray absorptionmetry 5 ethyl ether ALP, calcium osteocalcin Mouse Osteocalcin ELISA Kit Cat No. BT-470, Biomedical Tech. Inc., USA ELISA reader ng 6 osteoclast like cell 15 tube PBS trypan blue cell/ vial FACs tube osteoclast like cell MHC II CD115 flow cytometry 7 sham- OVX HG normal HG SPSS 14.0 KO t-test ANOVA test Scheffe s test 95 p<0.05 III 1. control Fig. 1 7 H&E 10 formalin microtome 5 H&E Histomorphometry H&E ECLIPSE E600 digital camera DXM 1200F ACT-1 program TBV trabecular bone volume TBT trabecular bone thickness GPL growth plate length I-solution trabecular bone volume of sample section TBV 100 total bone volume of sample section 4

5 (KI10) ALP, creatinine, phosphorus, calcium, osteocalcin 1 Alkaline phosphatase ALP ALP Normal control ALP RLF-HA control ALP 2 Creatinine creatinine Normal control creatine RLF-HA control saline creatinine Fig. 2 3 Phosphorus phosphorus Normal control phosphorus RLF-HA control phosphorus Fig. 3 4 Calcium calcium calcium 5 Osteocalcin osteocalcin ELISA osteocalcin Ca P Ca/P ratio 1 2 Calcium calcium Saline RLF-HA control NP calcium Fig. 4 3 Phosphorus phosphorus RLF-HA control phosphorus Fig. 5 4 Ca/P ratio calcium phosphorus 5. osteoclast like cell osteoclast like cell flow cytometry assay Control MHCII + /CD normal RLF-HA control NP saline osteoclast like cell flow cytometry assay control osteoclast like cell normal RLF-HA control Fig H&E Hematoxylin and eosin H&E

6 RLF-HA control NP saline pore normal Fig. 7 2 Histomorphometry (1) TBV(Trabecular Bone Volume, ) H&E TBV (2) TBT trabecular bone thickness, H&E TBT Control normal TBT RLF-HA control TBT Fig. 8 (3) GPL growth plate length, H&E GPL RLF-HA control Fig. 9 IV WHO FDA bisphosphonate estrogen raloxifene, calcitonin teriparatide WHI estrogen KI10 Rosae laevigata fructus

7 (KI10) 57 ALP, creatinine, phosphorus, calcium, osteocalcin, calcium, phosphorus, Ca/P ratio, osteoclast like cell RLF-HA control Fig. 1 5 ALP Creatinine creatinine Phosphorus phosphorus Osteocalcin ALP, phosphorus, calcium, osteocalcin creatinine collagen calcium phosphorus calcium phosphorus Osteoclast like cell calcium phosphorus phosphorus, Ca/P ratio RLF-HA saline calcium RLF-HA osteoclast like cell H&E RLF-HA control NP saline pore Fig. 7 TBV TBT TBT control RLF- HA control TBT Fig. 8 Estrogen GPL

8 RLF-HA control GPL Fig. 9 TBV TBT TBV GPL estrogen calcium osteoclast like cell TBT GPL V 1. saline injection calcium 2. osteoclast like cell VI 1. The Korean Society of Bone Metabolism. Osteoporosis. 3rd Edition. Seoul:Hanmi book. 2006: Kang S.K, Park Y.B, Ahn H.S. The Bibliographical studies on the acupuncture treatment of the osteoporosis. The Journal of Korean Acupuncture&Moxibusion Society. 1996;15(2): Department of Herbology, College of Oriental Medicine. Herbology. Seoul:Younglim book. 1999: Department of Acupuncture&Moxibustion, College of Oriental Medicine. Acupuncture &Moxibustion, Seoul:Jipmoondang, 2001:531-63, Choi C.M, Lee S.J, Park K.H, Kim S.B, Cho H.B. The effect of Woogyuyeum on the Osteoporosis of Rats Caused by Ovariectomy. The Journal of Oriental Gynecology. 2006;19(4) : Lee J.J. The Effects of Dangguijihwangyeum on the Osteoporosis of Rats Caused by Ovariectomy. The Journal of Oriental Gynecology. 2004;17(1): Choi J.K. The Effects of Gami-Joaguiyeum on the Osteoporosis of Rats Caused by Ovariectomy. The Journal of Oriental Gynecology. 2006;19(2): Lee J.A. The Effects of Rehmanniae Radix on the Osteoporosis of Rats Caused by Ovariectomy. The Journal of Oriental Gynecology. 2004;17(4): Jung K.Y. The Effects of Ginseng Radix on the Osteoporosis of Rats Caused by Ovariectomy. The Journal of Herbology. 2004;19(1): Lee J.A. The Effects of Eucommiae Cortex and Chaenomelis Fructus on the aged Ovariectomized Rat of Postmenopausal Osteoporosis. The Journal of Herbology. 2001;16(1): Yim Y.K, Jung Y.T. The Effects of Acanthopanacis Cortex Herbal Acupuncture at KI10 on the Osteopororsis of Rats Caused by Ovariectomy. The Journal of Meridian&Acupoint. 2008;25(1): Seok J.W. A Study on Effects of the

9 (KI10) 59 Pakukchun Aqua- acupuncture on the Osteoporosis Induced by Ovariectomy in Rats. The Journal of Acupuncture&Moxibustion. 2000;17(3): Yook T.H. A Study on the Effects of the Carthami Semen, Cervi Pomtotrichum Cornu, Hominis Placenta Aquacupuncture on the Osteoporosis in the Rats. The Journal of Acupuncture& Moxibustion. 2000;17(3): Park Y.H, Lee E, Lim S.C. Effects of Guemengja(Rosae Laevigatae Michx.) Extracts on Serum Lipid Composition and Antioxidative Capacity in Rats Fed High Oxidized Fat. The Jornal of Plant Resources Society of Korea ;17(3): Lee E, Lee J.M. The Effects of Rosae Lavigatae Fructus on Blood Characteristic in Rat. The Journal of the Korean Academy of Oriental Medicine Prescription. 2004;12(1): Kim K.D, Jeong J.C. The Effects of Rosae Lavigatae Fructus Extract on the Nitric Oxide Synthase Activity and Antioxidan Action in Rats Corpus Cavernosum Penis. The Journal of the Oriental Internal Medicine. 1998;19(1): Lee H.Y. A Study of Correlation among the Knowledge of the Disease, Health Promoting Behaviors and the Quality of Life in the Female Patients with Osteoporosis. The Journal of Rheumatology Health. 2001;8(1): Jeong H.Y. Osteoporosis Diagnosis and Treatment The Journal of Korean Society of Bone Metabolism. 2008;23(2): Kang B.M. Comparison of Anti-osteoporotic Medications and Alternatives. Korean Journal of Obstetrics and Gynecology. 2006;49(12): Lim S.K. Osteoporosis: Recent Trend. The Journal of Korean Society of Biochemistry and Molecular Biology. 2002;22(3): Wang K.W. Hwangje Naikyung Su-wen. Seoul:Seongbobook. 1983:14, 27, 54, 60, 87, 127, 179, 202, 206, 212, 247, 275, Korean Institute of Herbal Acupuncture. Manual of Herbal Acupuncture Seoul: The Journal of Korean Institute of Herbal Acupuncture. 1999; Lee S.I. The Herbology. Seoul:EuiYSa. 1975: Shin G.G. Herbology. Seoul:Soomunsa. 1982: Herbology Society of Chinese Medicine. Chinese Herbology. Shanghai:Shanghai Science& Technology publisher. 1999: An J.H. Oriental Chinese Medicine. Beijing: Publisher of Renmin. 1991: Kasper B, Fauci H, Longo J. Harrison s Principles of Internal Medicine. Seoul:MIP. 1997: , , H. A. Morris, S. J. Porter, T. C. Durbright, R. J. Moore, A. G. Need, B. E. C. Nordin. Effects of cophorectomy on biochemical and bone variables in the rat. Bone and Mineral. 1992;18: Morris Notelovitz. Osteoporosis: screening, prevention, and management. Fertility and Sterility. 1993;59(4): Garnero P, Delmas PD. Assessment of the serum level of bone Alkaline Posphatase with a new immunoradimetric assay in patients with metabolic bone disease. J Clin Endocrinol Metab ;77(4): Doo H.K. The Oriental Nephrology. Seoul:Oriental Medicine Institute. 1991:654-60, J. M. Aitken, E. Armstrong, and J. B. Anderson. Osteoporosis after oophorectomy in the mature female rat and the effect of ostrogen and/or progestogen replacement therapy in its prevention ;55: Anthony M. Frumar, David R. Meldrum, F. Geola, Issa M. Shamonki, Ivanna V. Tataryn, Leonard J. Deftos, et al. Realtionship of fasting urinary calcium to circulating estrogen and

10 body weight in postmenopausal women. JCE& M. 1980;50(1): Lee K.N, Kweon O.H. Clinicopathology. Seoul :Euihak Moonhwasa. 2003:102-5, , 300-8, , 425-7, Yamazaki I, Yamazaki H. Charateristics of an ovaroectomized osteopenic rat model. J Bone Mirer Res. 1989;4(1): Lee W.J. Dictionary of Medine in English. Seoul:Academy Book. 1990: Baek S.H, Kim S.Y. Pathogenesis of Osteoporosis. The Journal of Korean Hip Society. 2006;18(4): Falahati-Nini A, Riggs BL, Atkinson EJ, O`Fallon WM, Eastell R and Khosla S. Relative contributions of testosterone and estrogen in regulating bone resorption and formation in normal elderly men. J Clin invest. 2000;106: Table 1. The List of Reagents that Used in This Study Reagent Manufacturer Nation Ethyl ether Ca kit Phosphorus kit Phosphate buffer saline (PBS) RPMI1640 medium Differentiation Basal medium MSC Osteogenic SingleQuots Mouse Osteocalcin ELISA Kit Samchun Asan CAMBREX LONZA Biomedical Tech. Inc. Korea USA 3-(4,5-dimethylthiazol-2yl)-2,5 diphenyl tetrazolium bromide(mtt) Trypan blue Formalin Sigma Table 2. The List of Devices that Used in This Study Device Manufacturer Nation Rotary evaporator Camera(DXM 1200F) ECLIPSE E600 microscopy Eyela Nikon Japan Syringe Korea vaccine Co. Centrifuge Ice maker Hanil Korea Furnace Daewoong ELISA reader TECAN Canada Bone densitometer (PIXImus) GE Lunar Co. USA

11 (KI10) 61 Ca/ash weight z(mg/g) Normal Control Np Saline RLF-HA Fig. 1 Cell viability rate with various concentrations of RLF-HAS. *** : p 0.001, * 0.05 compared to control by ANOVA test. : p compared to 0.5% RLF-HAS by ANOVA test. : p compared to 1% RLF-HAS by ANOVA test. Fig. 4 Effect of RLF-HA on tibial Ca level in ovariectomized mice. * : p 0.05 compared to normal group by t-test. : p compared to control group by ANOVA test. : p 0.01 compared to N.P. group by ANOVA test. Scheme 1. Experimental Scheme Fig. 5 Effect of RLF-HA on tibial phosphorus level in ovariectomized mice. : p 0.01 compared to control group by ANOVA test. : p 0.05 compared to N.P. group by ANOVA test. Fig. 2 Effect of RLF-HA on serum creatinine level in ovariectomized mice. ** : p 0.01 compared to normal group by t-test. : p 0.001, : p 0.01 compared to control group by ANOVA test. : p 0.01 compared to N.P. group by ANOVA test. ## : p 0.01 compared to saline group by ANOVA test. Fig. 6 Effect of RLF-HA on osteoclast like cell in tibia of ovariectomized mice. * : p compared to normal group by t-test. : p 0.01 compared to control group by ANOVA test Fig. 3 Effect of RLF-HA on serum P level in ovariectomized mice. : p 0.01, : p 0.05 compared to control group by ANOVA test. Fig. 7 Histological analysis (H&E stain) of tibia in ovariectomized mice

12 ash weight z(mg/g) Normal Control Np Saline RLF-HA Fig. 8 Effect of RLF-HA on the trabecular bone thickness of tibia in ovariectomized mice. * : p 0.05 compared to normal group by t-test. : p compared to control group by ANO- VA test. : p compared to N.P. group by ANOVA test ## : p compared to saline group by ANOVA test. Fig. 9 Effect of RLF-HA on growth plate length of tibia in ovariectomized mice. ** : p 0.01 compared to normal group by t-test : p compared to control group by ANOVA test. : p 0.01 compared to N.P. group by ANOVA test. ## : p 0.01 compared to saline group by ANOVA test.

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경락경혈학회지 Vol.27, No.1, pp.87 106, 2010 Journal of Meridian & Acupoint Dept. of 1 Meridian & Acupoint, 3 Acupuncture & Moxibustion, College of Oriental Medicine, Daejeon University 2 Division of Clinical