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1 생약학회지 Kor. J. Pharmacogn. 46(2) : (2015) 흑미 (Oryza sativa L.) 호분층추출물이난소절제흰쥐의골밀도및골대사지표에미치는영향 남송이 1 김남석 1 이영민 2 최봉겸 1 이선혜 1 장환희 1 김정봉 1 이해정 3 이성준 4 이성현 1 * 1 농촌진흥청국립농업과학원기능성식품과, 2 서울여자대학교식품영양학과, 3 을지대학교식품영양학과, 4 고려대학교식품공학과 Effects of Oryza sativa L. Aleurone Layer Extract on Bone Mineral Density and Bone-related Markers in the Ovariectomized Rat Song-Yee Nam 1, Nam-Seok Kim 1, Young-Min Lee 2, Bong-Kyoum Choi 1, Seon-Hye Lee 1, Hwan-Hee Jang 1, Jung-Bong Kim 1, Hae-Jeung Lee 3, Sung-Joon Lee 4, Sung-Hyen Lee 1 * 1 Functional Food & Nutrition Division, Department of Agro-food Resources, Rural Development Administration, Wanju-gun , Korea 2 Department of Food Science and Nutrition, Seoul Women s University, Seoul , Korea 3 Department of Food and Nutrition, Eulji University, Seongnam-si, Gyeonggi-do , Korea 4 Department of Food Bioscience & Technology, Korea University, Seoul , Korea Abstract Aleurone layer of Black rice (Oryza sativa L.) is enriched with anthocyanin that could increase bone density. This study was conducted to investigate the osteoporosis-preventing effects of the aleurone layer extract (BRE) on bone loss of ovariectomized (OVX) rats. OVX (or sham-operated) rats were assigned to three groups (n=8 per group): sham operated group (Sham); OVX control group (OVX); OVX-BRE group, OVX rats treated with BRE at 90 mg/kg B.W. The deionized water alone or deionized water with BRE was orally administrated to Sham, OVX or OVX-BRE groups, respectively for 12 weeks. High fat diet with 45 kcal% fat and water were fed to all rats ad libitum. Body weight was significantly decreased in the OVX- BRE group compared to the OVX group (p<0.05). The bone mineral density and bone length of tibia were significantly higher in the OVX-BRE group compared to the OVX group and breaking force was significantly higher for the both tibia and femur bones. Serum estradiol concentration and calcium concentration of femur were higher in the OVX-BRE group than those of OVX group. However, serum alkaline phosphatase activity and parathyroid hormone concentration were decreased in the OVX- BRE group compared to the OVX group. The results suggest that aleurone layer of Black rice is a potentially useful ingredient to protect against estrogen deficiency or menopause related osteoporosis. Key words Oryza sativa L., Aleurone layer, Bone mineral density, Bone mineral content, Ovariectomized (OVX) rats 과학과문명의발달로점차인간의평균수명이연장되고중년이후여성의삶중에서약 1/3 을갱년기로보내게되기때문에, 이시기의삶의질을향상시키기위해다양한노력들이이루어지고있다. 1,2) 폐경이진행되는여성은골대사이상에따른골다공증이문제되는데, 골다공증은골구성성분의감소로정상인에비해골밀도와골강도가현저히감소된상태를이르는대사성골질환으로, 연령증가에따 Equal contribution * 교신저자 ( ) : lshin@korea.kr (Tel): 른골흡수와골형성의불균형, 특히폐경으로인한에스트로겐결핍에따라갱년기여성에게서큰문제가되고있다. 3-5) 에스트로겐은조골세포에작용하여파골세포활성을억제하는물질을합성함으로써골격대사에관여하는데, 6) 골소실속도는폐경후에스트로겐결핍으로인해 5~10 년후빨라지며, 노화에의해서서히골밀도가감소된다. 7) 골량의감소는개인차또는다른여러가지원인으로인해그정도의차이가있지만, 병적으로과다하게골량이감소하여일정치이하로저하되면작은충격에도쉽게골절이생기게된다. 8) 골다공증은그증세자체보다는골의약 167

2 168 Kor. J. Pharmacogn. 화에따라용이하게초래되는각종골절, 특히대퇴골골절등으로장기간활동을제한하여건강한생활을영위할수없고, 결과적으로노인층사망의 15% 에대한원인이되는것으로알려져있다. 과도한파골세포의활성에의해유발된골질환의치료나예방의목적으로내인성조절인자들이외에도여러약물들이이용되고있다. 그러나이러한약물들은여러가지부작용과복용상의어려움을갖고있음이알려져있어, 9-12) 새로운작용및약물구조를가지면서독성과부작용이적으며골다공증의예방또는치료에효과적인신물질의개발이요구되고있다. 또한이러한신물질은민간요법으로예로부터사용되어온독성이없는천연물에서발견될가능성이높기때문에천연물로부터신약을창출하려는시도가활발히진행되고있다. 13,14) 민간에서뼈건강을목적으로사용되는식품중에흑미는우리나라에서전국적으로재배및생산량이증가하고있으며, 흑미소재를이용한다양한제품이개발되고있다. 흑미의안토시아닌은수용성의플라보노이드계천연색소로, 다양한형태의식품으로도가공되며진한검붉은색을띄는몸에좋은자연식품이라하여 black food 로많은관심을받고있다. 15) 또한, phenolics hydroxyl 기를갖기때문에거대분자들과결합하는성질을지니고있어 16) 다양한생리작용을지니는것으로알려져있는데, 흑미는항산화 17), 항당뇨, 18) 염증완화, 19) 지방간개선, 20) 동맥경화예방및치료, 21) 면역기능증진 22) 및골다공증예방효과가있는것으로최근보고되었다. 23) 따라서본연구에서는우리가주식으로섭취하는곡류중에서뼈건강에도움을줄수있는흑미에서안토시아닌의함량을많이추출할수있는방법을개발하여특허출원하였고, 이방법에의해추출된흑미추출물을이용하여, 난소절제모델에서뼈의골밀도와골강도및뼈건강관련호르몬에미치는효과를검토하였다. 재료및방법 흑미추출물제조 흑미 (Oryza sativa L.) 는진도소재정미소에서구입하였고, 호분층을분쇄하여 50% 에탄올로 40 o C 에서 48 시간동안추출하였다. 추출물을여과지로거른후에, 농축하고동결건조하여시료로사용하였다. 실험동물및실험식이 실험동물은 Sprague-Dawley 계암컷흰쥐 (5 주령 ) 로, 난소절제군 (ovariectomy, OVX) 은양측난소를절제하였으며, 비난소절제군 (Sham) 은개복수술만시행한것을중앙실험동물 ( 주 ) 에서구입하여사용하였다. 실험동물 (Sham 및 OVX 흰쥐 ) 은 3 주간의회복기간을거친후, Sham 군, 난소절제 (OVX) 군, 난소절제 + 흑미호분층추출물 90 mg/kg BW 투여군 (OVX-BRE) 의총 3 개군으로나누어실험에사용하였고, 증류수및추출물을매일 1 회씩 Table I. Composition of experimental diet Ingredient g/kg diet Casein, 30 Mesh 200 L-Cystine 3 Corn Starch 72.8 Maltodextrin Sucrose Cellulose, BW Soybean Oil 25 Lard Mineral Mix S DiCalcium Phosphate 13 Calcium Carbonate 5.5 Potassium Citrate, 1 H2O 16.5 Vitamin Mix V Choline Bitartrate 2 FD&C Red Dye # 경구투여하였다. 실험식이 (Research Diets, USA) 는지방함량이에너지의 45% 수준에서포함된고지방식이 (Table I) 를 12 주간공급하였는데, 실험식이및식수는실험기간동안자유급여하였으며, 식이섭취량은주 3 회, 체중은주 1 회씩측정하였다. 실험동물은개별사육하였고, 실내온도 20±2 o C, 습도 50±5%, 명암 12 시간을주기로자동조절되는환경에서사육되었으며, 모든실험동물의사육및관리절차는국립농업과학원동물실험윤리위원회로부터승인을받아수행하였다 ( 승인번호 NAAS 1408). 골밀도, 강도및무기질함량측정 실험동물의대퇴골 (femur) 및경골 (tibia) 에서골밀도 (bone mineral density: BMD) 와골무기질함량 (bone mineral content: BMC) 을동물전용골밀도측정기 (PIXImus , LUNAR, USA) 를사용하여측정하였다. 대퇴골과경골의강도는조직특성분석기 (Texture analyzer, System micro systems, UK) 를이용하여분석하였는데, pre speed 0.6 mm/sec, test speed 0.6 mm.sec, force kg 조건으로뼈의중간부분에서일정하게측정하였다. 경골의칼슘과인함량을식품공전시험법에의하여분석하였는데, 24) 경골을 600 o C 회화로에서건식회화시키고, 질산용액에분해시킨후분석용시료로이용하였다. 칼슘의함량은원자방출분광기 (ICP-OES, Perkinelmer, USA) 를이용하여분석하였고, 인의함량은몰리브덴청비색법에따라분광광도계 (UV/VIS spectrophotometer, Mecasys, Korea) 를이용하여측정하였다. 생화학적분석 혈액은실험동물을탄산가스 (CO 2 ) 로마취시킨후심장으로부터채취하였고, 실온에 30 분방치후

Vol. 46, No. 2, 2015 169 3,000 rpm에서 20분간원심분리하여얻은혈청을칼슘대사및관련호르몬분비분석에사용하였다.

3 Vol. 46, No. 2, ,000 rpm에서 20분간원심분리하여얻은혈청을칼슘대사및관련호르몬분비분석에사용하였다. 생화학적항목으로혈중 alkaline phosphatase(alp) 활성도 ( 아산제약, Korea) 와 parathyroid hormone(pth ELISA Kit, USCN life science, China), osteoclacin(osteocalcin ELISA kit, Biomedical technologies Inc., USA) 및 estradiol(estradiol ELISA kit, Biovision., USA) 의농도를측정하였다. 통계분석 실험결과는측정후 Mean±SEM로나타내었고, 처리간의차이는 SPSS 프로그램에서 ANOVA 를실시한후사후검정으로 Duncan's multiple range test에의하여 p<0.05 수준에서검증하였다. 결과및고찰 식이섭취량과체중에미치는영향 식이섭취량은난소절제군 (OVX) 에서약 16.4 g 으로정상대조군 (Sham) 의 14.4 g 에비하여증가하였으나두난소절제군사이에는유의한차이가없었다 ( 자료제시하지않음 ). 체중은 OVX 에서 Sham 에비하여증가하였으나, 흑미호분층추출물 (BRE) 을섭취한 OVX-BRE 에서 OVX 군보다유의한수준에서체중이감소하였다 (Fig. 1). 따라서난소절제는식이섭취량및체중증가에영향을주지만, BRE 를섭취한경우 OVX 군과같은식이섭취량임에도불구하고, 체중증가를억제하는효과가있는것으로나타났다. 골밀도, 골무기질함량, 강도및길이에미치는영향 대퇴골과경골의골밀도 (BMD) 와골무기질함량 (BMC) 을 Table II 에나타내었다. 대체적으로대퇴골과경골의골밀도가골다공증모델에서 5~6% 씩감소하였는데, 흑미호분층추출물을섭취한실험군 (OVX-BRE) 에서대퇴골과경골의 Fig. 1. Effect of Oryza sativa L. aleurone layer extract on body weight of experimental rats. Rats were treated with distilled water for Sham and OVX groups or BRE at 90 mg/kg body weight for OVX-BRE group for 12 weeks. Each bar represents the mean±sem value (n=8). Bars not sharing the same letters are significantly different according to the Duncan s multiple range test at P<0.05. 골밀도가 OVX 군보다각각 2% 와 8% 가증가하였고, 경골의경우, OVX 군과유의한차이를보였다. 골무기질함량 (BMC) 도난소절제한동물모델에서감소하는경향을보였고, BRE 를급여한경우, 대퇴골과경골에서각각 3% 와 18% 가증가하는경향을보였으나실험군사이에유의한차이를보이지는않았다 (Table II). 특히난소절제에의해경골의칼슘함량이유의하게감소되었으나, BRE 를급여한경우 Sham 군과가까운수준으로증가하였다 (Fig. 2). 그러나경골의인함량은모든실험군사이에유의한차이를보이지않았다 ( 결과표시하지않음 ). 따라서 BRE 공급은경골에서칼슘함량을증가시킴으로써골무기 Table II. Effect of Oryza sativa L. aleurone layer extract on bone mineral density (BMD), mineral content (BMC), strength, and length of experimental rats Sham OVX OVX - BRE Femur BMD, g/cm ±0.04 NS 1.88± ±0.01 BMC, g 4.67±0.11 NS 4.57± ±0.12 Strength, kg 21.68±0.98 b 20.37±1.27 b 25.31±0.84 a Length, cm 5.98±0.07 NS 6.09± ±0.07 Tibia BMD, g/cm ±0.03 ab 1.60±0.04 b 1.72±0.02 a BMC, g 3.30±0.37 NS 2.86± ±0.13 Strength, kg 18.74±0.20 ab 17.81±0.68 b 19.77±0.53 a Length, cm 6.25±0.36 ab 5.77±0.16 b 6.65±0.15 a Rats were treated with distilled water for Sham and OVX groups or BRE at 90 mg/kg body weight for OVX-BRE group for 12 weeks. Values are means±sem (n=8). Different letters within each line are significantly different according to the Duncan's multiple range test at P<0.05. NS: Not significantly different

170 Kor. J. Pharmacogn. Fig. 2. Effect of Oryza sativa L. aleurone layer extract on Ca concentration in femur of experimental rats.

Bars not sharing the same letters are significantly different according to the Duncan's multiple range test at P<0.05. Fig. 3. Effect of Oryza sativa L.

05. 질함량에영향을줄수있는것으로보인다. 뼈의강도 (strength) 는 OVX 군에서 Sham 군에비해대퇴골과경골에서각각 6% 와 5% 가감소하였으나 (p>0.05), BRE 를투여한 OVX-BRE 군에서 OVX 군보다각각 24% 와 11% 가유의하게증가하였고 (p<0.05), 정상군인 Sham 군보다높게나타났다 (Table II).

4 170 Kor. J. Pharmacogn. Fig. 2. Effect of Oryza sativa L. aleurone layer extract on Ca concentration in femur of experimental rats. Rats were treated with distilled water for Sham and OVX groups or BRE at 90 mg/kg body weight for OVX-BRE group for 12 weeks. Each bar represents the mean±sem value (n=8). Bars not sharing the same letters are significantly different according to the Duncan's multiple range test at P<0.05. Fig. 3. Effect of Oryza sativa L. aleurone layer extract on serum alkaline phosphatase activity of experimental rats. Rats were treated with distilled water for Sham and OVX groups or BRE at 90 mg/kg body weight for OVX-BRE group for 12 weeks. Each bar represents the mean±sem value (n=8). Bars not sharing the same letters are significantly different according to the Duncan's multiple range test at P<0.05. 질함량에영향을줄수있는것으로보인다. 뼈의강도 (strength) 는 OVX 군에서 Sham 군에비해대퇴골과경골에서각각 6% 와 5% 가감소하였으나 (p>0.05), BRE 를투여한 OVX-BRE 군에서 OVX 군보다각각 24% 와 11% 가유의하게증가하였고 (p<0.05), 정상군인 Sham 군보다높게나타났다 (Table II). 그러므로흑미호분층의추출물은폐경기여성에서자주일어나는골절의예방에도움을줄수있을것으로기대된다. 또한 BRE 를급여한경우경골의길이가 OVX 군보다 15% 증가하였는데 (p<0.05), BRE 의공급은대체적으로대퇴골보다는경골의골밀도와골무기질함량및발달에영향을줄수있는것으로보인다 (Table II). 혈중생화학적지표에미치는영향 Alkaline phosphatase (ALP) 는정상적인혈액에서는적은농도로존재하는데, 조직에서이상이생기거나골육종 (osteosarcoma) 의경우혈청내에서 ALP 가증가하게된다 25-28). Fig. 3 은흑미호분층추출물이혈청 ALP 활성에미치는영향을보여주고있는데, OVX 군이난소를절제하지않은 Sham 군에비해혈청 ALP 활성이높아졌으나, BRE 의투여로인해 OVX-BRE 군은 OVX 군보다 ALP 활성이유의적으로감소하였다 (p<0.05). ALP 는조골대사와관련이깊어대사성골질환등조골세포의활동이증가하여골교체율이빠를때혈액내에서농도가증가하는데, 29) BRE 투여군에서 OVX 군보다 ALP 활성이낮아져골대사에유익한효과가있는것으로보인다. Osteocalcin(OC) 은골아세포에서합성되어뼈의세포내기질에침착되고, 새로합성된일부가혈액내로분비되므로뼈의무기질대사지표로사용된다. 30) 특히혈청 OC 농도는폐경후에증가되며폐경후에나타나는골소실율을 Fig. 4. Effect of Oryza sativa L. aleurone layer extract on serum osteocalcin level of experimental rats. Rats were treated with distilled water for Sham and OVX groups or BRE at 90 mg/kg body weight for OVX-BRE group for 12 weeks. Each bar represents the mean±sem value (n=8). Bars not sharing the same letters are significantly different according to the Duncan's multiple range test at P<0.05. 잘예측할수있는지표로알려져있다. 31,32) 본실험의경우 OVX군의혈중 OC농도는 Sham군보다유의적으로증가하였으나 BRE 투여에따른영향은나타나지않았는데 (Fig. 4), 이는식물추출물이다른생화학지표에는영향을주었으나 OC에는영향을주지못했던이등 (2005) 의보고와일치하는결과이다. 33) 골다공증의진단지표로사용되는혈중 estradiol 함량은 OVX군에서 Sham군에비하여 45% 감소하였으나, BRE를급여한 OVX-BRE은 OVX군에비하여 23% 증가하여유의한효과를보였다 (Fig. 5). 이것은홍삼추출물을이용하여

Vol. 46, No. 2, 2015 171 결 론 Fig. 5. Effect of Oryza sativa L. aleurone layer extract on serum estradiol level of experimental rats.

Bars not sharing the same letters are significantly different according to the Duncan's multiple range test at P<0.05. 흑미호분층추출물은난소절제로인해골소실이일어나는실험동물모델에서, 골밀도, 골무기질함량및뼈의강도에영향을줌으로써, 골소실을억제하는효과가있었다.

5 Vol. 46, No. 2, 결 론 Fig. 5. Effect of Oryza sativa L. aleurone layer extract on serum estradiol level of experimental rats. Rats were treated with distilled water for Sham and OVX groups or BRE at 90 mg/kg body weight for OVX-BRE group for 12 weeks. Each bar represents the mean±sem value (n=8). Bars not sharing the same letters are significantly different according to the Duncan's multiple range test at P<0.05. 흑미호분층추출물은난소절제로인해골소실이일어나는실험동물모델에서, 골밀도, 골무기질함량및뼈의강도에영향을줌으로써, 골소실을억제하는효과가있었다. 이것은흑미호분층추출물이골대사와관련된각종혈중생화학적지표에영향을주고이것이골대사에영향을주기때문인것으로분석된다. 따라서흑미호분층추출물은폐경후여성의골다공증예방및뼈건강개선을위해이용될수있을것으로보이며, 임상실험을통한인체에서의적용및확인실험연구가필요하다. 사 본연구는농촌진흥청의공동연구사업 흑미의 in vivo 뼈건강과비만동시조절개선기전연구 (PJ ) 연구비의지원으로이루어졌으며, 동물실험과정에서도움을준조수연, 조수정선생, 뼈의강도측정에도움을준최수영선생에게감사드립니다. 사 인용문헌 Fig. 6. Effect of Oryza sativa L. aleurone layer extract on serum parathyroid hormone level of experimental rats. Rats were treated with distilled water for Sham and OVX groups or BRE at 90 mg/kg body weight for OVX-BRE group for 12 weeks. Each bar represents the mean±sem value (n=8). Bars not sharing the same letters are significantly different according to the Duncan's multiple range test at P<0.05. 골다공증모델의혈중 estradiol 수준을높일수있었던연구결과와비교할때, 34) 흑미호분층추출물도혈중 estradiol 수준에영향을줌으로써폐경후여성의뼈건강개선에도움을줄수있을것으로기대된다. 부갑상선호르몬 (PTH) 은혈중칼슘농도에따라분비가조절되는데, 칼슘농도가낮아지면부갑상선에서이호르몬의분비를증가시킨다. 본연구에서 Sham 군에비해 OVX 군의혈중 PTH 농도가증가하였으나, 흑미추출물 (BRE) 의공급으로혈중 PTH 수준이유의적으로감소하였고 Sham 군과유의한차이를보이지않았다 (Fig. 6). 1. Kwon, S. C. (1998) Effects of continuously added oral progestin (medroxy progesterone acetate) on the levels of serum lipid and lipoprotein during estrogen replacement therapy in postmenopausal women. Korean Soc. Obstetrics & Gynecology 41: Han, S. H. (2004) A Study on the menopausal symptoms, menopausal managementand quality of life in middle aged women. MA thesis. Ewha womon's university. 3. Lee, E. S. and Kang, B. H. (1997) Biochemiccal bone markers in postmenopausal osteoporotic Women. Korean Soc. obstetrics and Gynecology 40: Clark, J. H. (2006) A critique of Women s Health Initiative Studies ( ). Nucl. Recept. Signal. 4, e Kim, J., Lee, J. H., Kim, K. W., Lee, K. W., Han, S. B. and Kim, H. M. (2002) Effect of serum isolated from osteoporotic human on the growth and differentiation of normal human osteoblast. J. Korean Assoc. Maxillofac. Plast. Reconstr. Surg. 24: Mo, S. M., Lee, Y. S., Ku, J. O., Son, S. M., Seo, J. S., Yoon, E. K., Lee, S. K. and Kim, W. K. (2003) Dietary therapy. 2 nd ed. Kyomun Press; Seoul Yoo, S. W., Jo, H. H., Yeo, K. A., Lew, Y. O., Kwon, D. J., Chung, G. W., Kim, J. H. and Kim J. H. (2000) The effect of vitamin D 3 addition on bone mineral density in postmenopausal hormone replacement. Kor. J. Obst. Gyne. 43: Wasnich, R. (1996) What is an osteoporotic fracture? In

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(01) hwp Journal of Life Science 2013 Vol. 23. No. 2. 157~166 ISSN (Print) 1225-9918 ISSN (Online) 2287-3406 DOI : http://dx.doi.org/10.5352/jls.2013.23.2.157 α μ δ κ 158 생명과학회지 2013, Vol. 23. No. 2 Journal of