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1 Journal of Korean Medicine Rehabilitation Vol. 28 No. 2, April 2018 pissn eissn Original Article ChondroT 구성약재의항응고효과에관한연구 김선길 * 정지원 * 임용하 * 김지훈 * 나창수 김선종 * 동신대학교한의과대학한방재활의학과교실 *, 동신대학교한의과대학경락경혈학회 A Study on the Anti-Condensing Effect of ChondroT Components Sun-Gil Kim, K.M.D.*, Ji-Won Jeong, K.M.D.*, Young-Ha Lim, K.M.D.*, Ji-Hoon Kim, K.M.D.*, Chang-Su Na, K.M.D., Seon-Jong Kim, K.M.D.* Department of Rehabilitation Medicine of Korean medicine, College of Korean Medicine, Dongshin University*, Department of Meridian and Acupoint, College of Korean Medicine, Dongshin University 본연구는보건복지부의재원으로한국보건산업진흥원의보건의료기술연구개발사업지원에의하여이루어진것임 ( 과제고유번호 : HI17C0911) RECEIVED Mar 18, 2018 REVISED Apr 4, 2018 ACCEPTED Apr 7, 2018 CORRESPONDING TO Seon-Jong Kim, Professor Department of Rehabilitation Medicine of Korean medicine, College of Korean Medicine, Dongshin University Mokpo Oriental Hospital of Dongshin University, 313 Baengnyeon-daero, Mokpo 58665, Korea Objectives The objective of this study was to investigate the effect of Anti-condensing on the composition of ChondroT Methods Specimens are divided in 7 groups (Control, ChondroT, Lonicerae Folium (Gumenhwa, GEH), Angelicae Gigantis Radix(Danggui, DG), Phellodendri Cortex(HwangBaek, HB), Osterici Radix(Kanghwal, KH), Clematidis Radix(Weeryungsun, WRS)) Each specimen is subjected to a concentration of 20 %, 10 %, and 5 %, and is administered to collagen and thrombin-stimulated platelets. Results In the anticoagulance effect test, Lonicerae Folium and ChondroT very well. The effect was high in order of Lonicerae Folium Angelicae Gigantis Phellodendri Cortex Osterici Radix and Clematidis Radix. Conclusions ChondroT has anti-condensing effects on blood platelet. (J Korean Med Rehabil 2018;28(2):47-60) Key words ChondroT, Lonicerae Folium, Angelicae Gigantis Radix, Phellodendri Cortex, Osterici Radix, Clematidis Radix. TEL (061) FAX (061) mofoster@hanmail.net Copyright 2018 The Society of Korean Medicine Rehabilitation 서론»»» 血行은혈액이혈관을통하여신체의각부분으로이동하는것을의미한다. 혈액은신체의각조직으로산소와영양분을공급하고세포에서만들어낸노폐물을제거해준다 1). 또한우리몸에필요한호르몬을운반하고, 외부의유해한물질로부터세포를방어하며 2), 일정한체온을유지시켜주고, 지혈작용을하는등신체내항상성을 유지하는역할을한다 3). 따라서혈액의원활한흐름은신체기능을유지하는데매우중요하다 4). 이러한혈액의순환에장애가생길경우뇌졸중, 동맥경화등뇌심혈관질환이발생하는데, 특히뇌혈관질환과심장질환은 2004년이후국내사망원인의 2위와 3위를차지할정도로높은발생빈도를보이고있다 5). 따라서혈액순환의중요도는계속증가되고 6), 血行의이상을초래하는인자들에대한관리또한중요해졌다. 47

2 김선길 정지원 임용하 김지훈 나창수 김선종 한의학에서血은形을成하게하는것으로 7), 營이되어몸속을다스리며五臟을조화롭게하고육부로흩어지며經脈으로들어간다 8). 또한근골, 장부를濡養하는작용을하며전신을순행하여안으로는장부밖으로는피육, 근골까지전신의장부, 형체, 九竅등의조직기관에끊임없이순환하여영양, 자윤작용을하여인체의정상적인생리활동을유지하게한다 9). ChondroT는관절염치료를위해개발중인한약제제로대강활탕과청열사습탕의구성한약제인강활, 당귀, 위령선, 금은화, 황백의조합으로만들어졌다. 강활은袪風除濕止痛散寒의효능으로관절의痺證을해소하고지통하는작용이있으며 10), 당귀는甘溫하며袪風濕藥과배합하여風濕痺痛을치료하고, 항염증및혈액순환개선의작용이있다 11,12). 또한위령선은祛風除濕, 通經活絡의효능을가지며 10) 강활과배합될경우항염증에상승작용을나타내고 13), 금은화는해독작용과소염, 항응고작용을하며 14,15), 황백은淸熱燥濕, 瀉火解毒으로항염증의효과가있다 11,16). 이에본연구는 ChondroT와그구성약재의개별투여에대한항응고효과를확인하기위해실시되었으며, Thrombin, Collagen으로활성화된혈소판에대한응집능반응실험에서 ChondroT의항응고효과에대한유의미한결과를얻었기에보고하는바이다. 2. 약재사용한약재는강활 (Osterici Radix), 금은화 (Lonicerae Folium), 당귀 (Angelicae Gigantis Radix), 위령선 (Clematidis Radix), 황백 (Phellodendri Cortex) 으로 ( 주 ) 옴니허브 ( 대구, 한국 ) 에서구입하여사용하였다 (Table I). 3. 시료추출강활, 금은화, 당귀, 위령선, 황백각단일시료와강활, 금은화, 당귀, 위령선, 황백을배합한복합시료 ChondroT ( 강활 : 당귀 : 금은화 : 위령선 : 황백 = 6 : 4 : 4 : 4 : 3) 를준비하였다. 복합시료 ChondroT 총량을각각 105 g 으로정하였고, 각단일시료는 ChondorT 시료와동량인 105 g으로정하였으며, 각각정제수 1000 ml에넣어 3시간동안열수추출법으로추출하였다. ChondroT 및각재료별시료는농축하여 50 ml이되도록하여실험에적용하였다. 50 ml로농축된것을 1 로하였고, 이를기준으로 0.2, 0.1, 0.05 로희석하여혈소판응집능실험에적용하였다. 혈소판응집능은트롬빈 (thrombin) 과콜라겐 (collagen) 에자극된상태를구분하여측정하였다. 4. 혈소판응집능측정 재료및방법»»» 1. 동물체중이약 1,800 g의뉴질랜드화이트토끼의耳動脈에서채혈을하였으며, 이때항응고제 (ACD (anticoagulant citrate dextrose), citric acid) 를 1/10의비율로사용하였다. 뉴질랜드화이트토끼를쳄버에넣고고정후耳動脈에서혈액을채취하였는데, 이때항응고제로서 ACD(anticoagulant citrate dextrose), citric acid를 1/10의비율로 conical tube에미리채워둔후 40 ml의혈액을얻었다. 채혈한전혈을 230 g로 10분동안원심분리하여혈소판이풍부한상층액 PRP(platelet rich plasma) 를얻은후다시 800 g로 10분동안원심분리하여혈소판이거의존재하지않는상층액 PPP(platelet poor plasma) 을 Table I. Composition of ChondroT and the Used Parts of 5 Individual Herbs Latin name Scientific name Family Used part Rate Source Osterici Radix Ostericum koreanum Maximowicz Umbelliferae Root 6 Korea Lonicerae Folium Lonicera japonica Thunberg Caprifoliaceae Root 4 China Angelicae Gigantis Radix Angelica gigas Nakai Umbelliferae Root 4 Korea Clematidis Radix Clematis manshurica Ruprecht Ranunculaceae Leaf 4 China Phellodendri Cortex Phellodendrom amurense Ruprecht Rutaceae Tree bark 3 China 48 J Korean Med Rehabil 2018;28(2):47-60

3 ChondroT 구성약재의항응고효과에관한연구 얻었다. 이때얻은혈소판은 1차완충액 (137 mm NaCl, 2.7 mm KCl, 1 mm MgCl 2, 5.6 mm glucose, 3.8 mm HEPES, 0.35% BSA, 0.4 mm EGTA, ph 6.5) 으로현탁시킨후다시 3,000 rpm 로 10분간원심분리하여잔사를얻고, 2 차완충액 (137 mm NaCl, 2.7 mm KCl, 1 mm MgCl 2, 5.6 mm glucose, 3.8 mm HEPES, 0.35% BSA, ph 6.5) 으로다시현탁한후 3,000 rpm 로 10분간원심분리하여잔사를얻고, 마지막 3차완충액 (137 mm NaCl, 2.7 mm KCl, 1 mm MgCl 2, 5.6 mm glucose, 3.8 mm HEPES, 0.35% BSA, ph 7.2) 으로재현탁하여얻은후 cells/ml의농도로혈소판수를맞추어 washed platelet (WP) 를제조하였다. 혈소판분리후 Tyrode buffer를이용하여세척후 Aggregometer( 응집능측정기 ) 를이용해광학법 (optical method) 으로혈소판응집능을측정하였다. 광학법은시간에따라혈소판응집으로인해광투과도 (light transmission) 가변화하는정도를측정하여백분율로표시하는방법을사용하였고, 응집유발물질로는혈전생성과정중시작단계인내피세포로부터노출되는콜라겐 (collagen), 활성화된혈소판에서생성되는트롬빈 (thrombin) 을사용하여혈소판활성화를유발하였다. 산출된혈소판응집능은 transmission maximum reduction percent 식에의거하여값을얻었다. 5. 분석방법및통계응집반응시험을시행하여응집반응활성이가장잘발현된경우에대하여각시료별응집능을시간대별응집능발생이완료되는지점의값으로비교하였다. 시작지점을 0분대로설정하였고, 이후시간대별로 3 개지점을설정하였는데 0~90초지점 (1.5 min. phase), 90~180초지점 (3 min. phase), 180 ~ 270초지점 (4.5 min. phase) 으로각각나누었고, 시간대별로 15초간얻어진평균값을구하였다. Thrombin 혹은 Collagen으로자극된상태에대하여시료를처치하지않은상태를대조군으로설정하였고, 강활추출물 (Osterici Radix, Kanghwal, KH), 금은화추출물 (Lonicerae Folium, Gumenhwa, GEH), 당귀추출물 (Angelicae Gigantis Radix, Danggui, DG), 위령선추출 물 (Clematidis Radix, Weeryungsun, WRS), 황백추출물 (Phellodendri Cortex, HwangBaek, HB), 그리고이들을배합한 ChondroT 추출물 (ChondroT) 을각각자극한상태로나누어비교하였다. 각농도별 (0.05, 0.1, 0.2 ) 로얻어진각시간대별지점의모든측정값은 Excel statistic program(microsoft, USA) 을이용하여평균치와표준오차 (mean±standard error) 로표시하였고, 각실험군간의통계학적분석은 SPSS 21.0 ver. for windows를사용하여비모수적방법으로 Mann-Whitney U test를시행하였다. 각실험군은대조군에비하여 α=0.05 수준 (P<0.05) 과 α=0.01 수준 (P<0.01) 에서유의성을검정하였다. 결과»»» 1. 트롬빈 (thrombin) 으로자극된혈소판에대한항응집반응 1) 0.05 농도의 ChondroT 및각시료별반응 Thrombin 으로자극된상태에서 0.05x 농도에서 ChondroT 및각단일시료별항응집활성을측정한결과, 3.5 min phase에서 Control이 65.7±5.6% 를보인것에비하여단일시료와 ChondroT는모두유의한감소를나타내었고 ChondroT가가장낮은수준을보였으며, 4.5 min. phase 에서 Control이 73.6±0.9% 를보인것에비하여단일시료모두와배합시료 ChondroT는유의한감소를나타내었고, ChondroT는금은화 (Lonicerae Folium, GEH) 시료다음으로낮은수준을보였다 (Table II, Fig. 1-1, 1-2). 2) 0.1 농도의 ChondroT 및각시료별반응 Thrombin으로자극된상태에서 0.1x농도에서 ChondroT 및각단일시료별항응집활성을측정한결과, 3.5 min phase에서 Control이 69.2±6.4% 를보인것에비하여단일시료와 ChondroT는모두유의한감소를나타내었고 ChondroT가가장낮은수준을보였으며, 4.5 min. phase 에서 Control이 79.4±1.3% 를보인것에비하여단일시료모두와배합시료 ChondroT는유의한감소를나타내었고, ChondroT는금은화 (Lonicerae Folium, GEH) 시료다음으로낮은수준을보였다 (Table III, Fig. 2-1, 2-2). 49

4 김선길 정지원 임용하 김지훈 나창수 김선종 Table II. Changes on the Aggregation-test of Thrombin after ChondroT and Constituent Materials (Dosage 0.05X) in Rabbit Blood Group Aggregation Rate (%) 1.5 min. phase 3.5 min. phase 4.5 min. phase Control 11.7± ± ±0.9 GEH -0.3± ± ±0.7 DG 0.8± ± ±1.2 KH -0.8± ±11.6* 65.6±0.5 WRS 0.1± ±14.4* 66.4±1.1 HB 0.4± ± ±1.2 CondroT 0.1± ± ±1.5 Values are expressed Mean±SD. Control, Thrombin or Collagen-stimulated blood platelet and saline treated GEH, Thrombin or Collagen-stimulated blood platelet and 0.05x Lonicerae Folium treated DG, Thrombin or Collagen-stimulated blood platelet and 0.05x Angelicae Gigantis Radix treated KH, Thrombin or Collagen-stimulated blood platelet and 0.05x Osterici Radix treated WRS, Thrombin or Collagen-stimulated blood platelet and 0.05x Clematidis Radix treated HB, Thrombin or Collagen-stimulated blood platelet and 0.05x Phellodendri Cortex treated ChondroT, Thrombin or Collagen-stimulated blood platelet and 0.05x ChondroT Table III. Changes on the Aggregation-test of Thrombin after ChondroT and Constituent Materials (Dosage 0.1X) in Rabbit Blood Group Aggregation Rate (%) 1.5 min. phase 3.5 min. phase 4.5 min. phase Control 14.1± ± ±1.3 GEH 0.8± ± ±1.6 DG 0.4± ± ±4.1 KH -0.5± ± ±0.7 WRS -0.2± ± ±0.9 HB 0.5± ± ±1.0 CondroT 0.4± ± ±7.4 Table IV. Changes on the Aggregation-test of Thrombin after ChondroT and Constituent Materials (Dosage 0.2X) in Rabbit Blood Group Aggregation Rate (%) 1.5 min. phase 3.5 min. phase 4.5 min. phase Control 2.6± ± ±1.1 GEH 1.0± ± ±1.2 DG 0.8± ± ±1.9 KH -0.1± ± ±0.4 WRS 1.0± ± ±0.9 HB -0.4± ± ±0.6 CondroT 1.0± ± ±2.8 The groups refer to Table II. Values are expressed Mean±SD. 100 Control GEH-0.05X DG-0.05X KH-0.05X WRS-0.05X HB-0.05X ChondroT-0.05X Aggreation Rate of Thrombin (%) * * (minute) -20 Fig Effects of ChondroT and constituent materials (Dosage 0.05X) on aggregation-test of thrombin in rabbit blood(phase-by-phase summation analysis data). 50 J Korean Med Rehabil 2018;28(2):47-60

5 ChondroT 구성약재의항응고효과에관한연구 The groups refer to Table II. Values are expressed Mean±SD. Fig Effects of ChondroT and constituent materials (Dosage 0.05X) on aggregation-test of thrombin in rabbit blood(original data). [Left figure] Trace1 : Control, saline treated. Trace2 : 0.05x Lonicerae Folium treated. Trace3 : 0.05x Angelicae Gigantis Radix treated. Trace4 : 0.05x ChondroT [Right figure] Trace1 : Control, saline treated. Trace2 : 0.05x Osterici Radix treated. Trace3 : 0.05x Clematidis Radix treated. Trace4 : 0.05x Phellodendri Cortex treated The groups refer to Table III. Values are expressed Mean±SD. 100 Control GEH-0.1X DG-0.1X KH-0.1X WRS-0.1X HB-0.1X ChondroT-0.1X 80 Aggreation Rate of Thrombin (%) (minute) -20 Fig Effects of chondrot and constituent materials (Dosage 0.1X) on aggregation-test of thrombin in rabbit blood(phase-by-phase summation analysis data). 3) 0.2 농도의 ChondroT 및각시료별반응 Thrombin으로자극된상태에서 0.2x농도에서 ChondroT 및각단일시료별항응집활성을측정한결과, 3.5 min phase에서 Control이 69.2±6.4% 를보인것에비하여단일시료와 ChondroT는모두유의한감소를나타내었고 ChondroT가가장낮은수준을보였으며, 4.5 min. phase 에서 Control이 79.4±1.3% 를보인것에비하여단일시료 51

6 김선길 정지원 임용하 김지훈 나창수 김선종 The groups refer to Table III. Values are expressed Mean±SD. Fig Effects of ChondroT and constituent materials (Dosage 0.1X) on aggregation-test of thrombin in rabbit blood(original data). [Left figure] Trace1 : Control, saline treated. Trace2 : 0.1x Lonicerae Folium treated. Trace3 : 0.1x Angelicae Gigantis Radix treated. Trace4 : 0.1x ChondroT [Right figure] Trace1 : Control, saline treated. Trace2 : 0.1x Osterici Radix treated. Trace3 : 0.1x Clematidis Radix treated. Trace4 : 0.1x Phellodendri Cortex treated The groups refer to Table IV. Values are expressed Mean±SD. Aggreation Rate of Thrombin (%) Control GEH-0.2X DG-0.2X KH-0.2X WRS-0.2X HB-0.2X ChondroT-0.2X (minute) -20 Fig Effects of ChondroT and constituent materials (Dosage 0.2X) on aggregation-test of thrombin in rabbit blood(phase-by-phase summation analysis data). 52 J Korean Med Rehabil 2018;28(2):47-60

7 ChondroT 구성약재의항응고효과에관한연구 The groups refer to Table IV. Values are expressed Mean±SD. Fig Effects of ChondroT and constituent materials (Dosage 0.2X) on aggregation-test of thrombin in rabbit blood(original data). [Left figure] Trace1 : Control, saline treated. Trace2 : 0.2x Lonicerae Folium treated. Trace3 : 0.2x Angelicae Gigantis Radix treated. Trace4 : 0.2x ChondroT [Right figure] Trace1 : Control, saline treated. Trace2 : 0.2x Osterici Radix treated. Trace3 : 0.2x Clematidis Radix treated. Trace4 : 0.2x Phellodendri Cortex treated 모두와배합시료 ChondroT는유의한감소를나타내었고, ChondroT는금은화 (Lonicerae Folium, GEH) 시료다음으로낮은수준을보였다 (Table IV, Fig. 3-1, 3-2). 2. 콜라겐 (Collagen) 으로자극된혈소판에대한항응집반응 1) 0.05 농도의 ChondroT 및각시료별반응 Collagen 으로자극된상태에서 0.05x농도에서 ChondroT 및각단일시료별항응집활성을측정한결과, 3.5 min phase에서 Control이 52.8±16.8% 를보인것에비하여당귀 (Angelicae Gigantis Radix, DG) 시료를제외한단일시료군과 ChondroT는모두유의한감소를나타내었고황백 (Phellodendri Cortex,HB) 시료, 강활 (Osterici Radix, KH) 시료다음으로 ChondroT가낮은수준을보였으며, 4.5 min. phase에서 Control이 70.6±1.2% 를보인것에비하여단일시료모두와배합시료 ChondroT는유의한감소를나타내었고, ChondroT는단일시료와비슷한수준을보였다 (Table V, Fig. 1-1, 1-2). Table V. Changes on the Aggregation-test of Collagen after ChondroT and Constituent Materials (Dosage 0.05X) in Rabbit Blood Group Aggregation Rate (%) 1.5 min. phase 3.5 min. phase 4.5 min. phase Control -0.0± ± ±1.2 GEH -1.3± ±15.8* 51.5±0.4 DG -0.4± ± ±0.7 KH 0.3± ±23.2* 57.6±1.8 WRS 0.4± ±23.3* 59.4±1.4 HB 0.3± ± ±3.0 CondroT 0.2± ±23.1* 57.7±1.6 2) 0.1 농도의 ChondroT 및각시료별반응 Collagen 으로자극된상태에서 0.1x농도에서 ChondroT 및각단일시료별항응집활성을측정한결과, 3.5 min phase에서 Control이 65.9±8.8% 를보인것에비하여단일시료와 ChondroT는모두유의한감소를나타내었고 ChondroT가가장낮은수준을보였으며, 4.5 min. phase 에서 Control이 75.5±0.3% 를보인것에비하여단일시료모두와배합시료 ChondroT는유의한감소를나타내었 53

8 김선길 정지원 임용하 김지훈 나창수 김선종 The groups refer to Table V. Values are expressed Mean±SD. 100 Control GEH-0.05X DG-0.05X KH-0.05X WRS-0.05X HB-0.05X ChondroT-0.05X 80 Aggreation Rate of Collagen (%) * * * (minute) -20 Fig Effects of ChondroT and constituent materials (Dosage 0.05X) on aggregation-test of collagen in rabbit blood(phase-by-phase summation analysis data). The groups refer to Table V. Values are expressed Mean±SD. Fig Effects of ChondroT and constituent materials (Dosage 0.05X) on aggregation-test of collagen in rabbit blood(original data). [Left figure] Trace1 : Control, saline treated. Trace2 : 0.05x Lonicerae Folium treated. Trace3 : 0.05x Angelicae Gigantis Radix treated. Trace4 : 0.05x ChondroT [Right figure] Trace1 : Control, saline treated. Trace2 : 0.05x Osterici Radix treated. Trace3 : 0.05x Clematidis Radix treated. Trace4 : 0.05x Phellodendri Cortex treated 54 J Korean Med Rehabil 2018;28(2):47-60

9 ChondroT 구성약재의항응고효과에관한연구 The groups refer to Table VI. Values are expressed Mean±SD. 100 Control GEH-0.1X DG-0.1X KH-0.1X WRS-0.1X HB-0.1X ChondroT-0.1X 80 Aggreation Rate of Collagen (%) * (minute) -20 Fig Effects of ChondroT and constituent materials (Dosage 0.1X) on aggregation-test of collgen in rabbit blood (Phase-by-phase summation analysis data). The groups refer to Table VI. Values are expressed Mean±SD. Fig Effects of ChondroT and constituent materials (Dosage 0.1X) on aggregation-test of collagen in rabbit blood(original data). [Left figure] Trace1 : Control, saline treated. Trace2 : 0.1x Lonicerae Folium treated. Trace3 : 0.1x Angelicae Gigantis Radix treated. Trace4 : 0.1x ChondroT [Right figure] Trace1 : Control, saline treated. Trace2 : 0.1x Osterici Radix treated. Trace3 : 0.1x Clematidis Radix treated. Trace4 : 0.1x Phellodendri Cortex treated 55

10 김선길 정지원 임용하 김지훈 나창수 김선종 Table VI. Changes on the Aggregation-test of Collagen after ChondroT and Constituent Materials (Dosage 0.1X) in Rabbit Blood Group Aggregation Rate (%) 1.5 min. phase 3.5 min. phase 4.5 min. phase Control 5.5± ± ±0.3 GEH -1.0± ± ±0.7 DG 0.7± ± ±0.4 KH 0.1± ± ±0.6 WRS 0.2± ±16.9* 63.3±0.3 HB 0.5± ± ±1.4 CondroT 1.5± ± ±1.7 Table VII. Changes on the Aggregation-test of Collagen after ChondroT and Constituent Materials (Dosage 0.2X) in Rabbit Blood Group Aggregation Rate (%) 1.5 min. phase 3.5 min. phase 4.5 min. phase Control 6.4± ± ±0.6 GEH 0.2± ± ±1.8 DG 0.7± ± ±0.7 KH -0.3± ± ±1.3 WRS -0.0± ± ±0.3 HB 0.4± ± ±2.1 CondroT 1.0± ± ±2.5 고, ChondroT는금은화 (Lonicerae Folium, GEH) 시료다음으로낮은수준을보였다 (Table VI, Fig. 5-1, 5-2). 3) 0.2 농도의 ChondroT 및각시료별반응 Collagen 으로자극된상태에서 0.2x농도에서 ChondroT 및각단일시료별항응집활성을측정한결과, 3.5 min phase에서 Control이 61.0±7.2% 를보인것에비하여단일시료와 ChondroT는모두유의한감소를나타내었고 ChondroT는황백 (Phellodendri Cortex,HB) 시료다음으로가장낮은수준을보였으며, 4.5 min. phase 에서 Control 이 69.6±0.6% 를보인것에비하여단일시료모두와배합시료 ChondroT는유의한감소를나타내었고, ChondroT The groups refer to Table VII. Values are expressed Mean±SD. Group Aggregation Rate (%) 1.5 min. phase 3.5 min. phase 4.5 min. phase Control 2.6± ± ±1.1 GEH 1.0± ± ±1.2 DG 0.8± ± ±1.9 KH -0.1± ± ±0.4 WRS 1.0± ± ±0.9 HB -0.4± ± ±0.6 CondroT 1.0± ± ±2.8 Aggreation Rate of Collagen (%) Control GEH-0.2X DG-0.2X KH-0.2X WRS-0.2X HB-0.2X ChondroT-0.2X (minute) -20 Fig Effects of ChondroT and constituent materials (Dosage 0.2X) on aggregation-test of collagen in rabbit blood(phase-by-phase summation analysis data). 56 J Korean Med Rehabil 2018;28(2):47-60

11 ChondroT 구성약재의항응고효과에관한연구 The groups refer to Table VII. Values are expressed Mean±SD. Fig Effects of ChondroT and constituent materials (Dosage 0.2X) on aggregation-test of collagen in rabbit blood(original data). [Left figure] Trace1 : Control, saline treated. Trace2 : 0.2x Lonicerae Folium treated. Trace3 : 0.2x Angelicae Gigantis Radix treated. Trace4 : 0.2x ChondroT [Right figure] Trace1 : Control, saline treated. Trace2 : 0.2x Osterici Radix treated. Trace3 : 0.2x Clematidis Radix treated. Trace4 : 0.2x Phellodendri Cortex treated 는위령선 (Clematidis Radix, WRS) 시료를제외한나머지시료들과비슷한수준을보였다 (Table VII, Fig. 6-1, 6-2). 고찰»»» 뇌졸중, 동맥경화, 심근경색등의혈액순환장애로발생하는심혈관계질환은유럽, 미국, 아시아등전세계국가의주요사망원인이되고있다 17,18). 2014년한국보건사회연구원의보고에따르면우리나라국민의심혈관질환, 뇌혈관질환등혈액순환과관련질환은점차증가하고있으며, 교통사고를제외한우리나라사망원인의 1, 2위를차지하고있다 19). 인체는혈액순환을통해혈장및혈구세포로구성된혈액이신체각조직에산소와영양분을공급하고세포내대사를통해생성된노폐물을제거하여항상성을유지한다 20,21). 이역할을수행하기위해서는구성세포및조직의기능이정상적으로유지되어야하며혈액의순환이원활히이뤄져야한다 22). 혈액중혈장은혈액응고와관련된 coagulation factors 로구성되어있고, 적혈구, 혈소판, 면역세포등의혈액세포들과상호작용을주고받으며평 형상태를유지한다 23,24). 혈관이손상되면혈관내벽의 von Willebrand factor, collagen, fibronectin 등이노출되면서혈소판의부착을촉진한다 25). 혈소판이활성화되면 Ca2+, thromboxane A2, serotonin 등을유리하여주위의혈소판응집을증가시키고, 혈장에존재하는 coagulation factor와반응하여혈괴를형성하여지혈이더신속하게일어나도록한다 26,27). 지혈작용은손상된부위의혈액손실을최소화하고혈액의정상적인순환을유지하는방어기전이다 28). 그러나과도한지혈작용및혈괴 (clot) 의생성은혈행이상을초래하며혈전 (thrombus) 과같은병변을유발한다 29). 혈전이생성되면정맥에서혈액순환장애로부종이나염증이발생하고동맥에서는허혈이나경색으로심근경색증 30), 뇌졸중 31), 폐동맥경색증 32) 등의질환이초래된다. 이와더불어혈소판활성화시혈관조절인자들 (serotonin, TXA2 등 ) 이유리되어혈관을수축시킨다 33). 이과정에서혈소판의응집반응을지연시키는것이본실험에서확인한 ChondroT의효과이다. 따라서 ChondroT 의항응고효과는현재상용중인항혈소판약물 (anti-platelet drug) 과비교하여살펴볼필요가있다. 대표적인항혈소판약물로는 aspirin, sulfinpyrazone, dipyridamole 및 propranolol 이있으며이약물은장기간복용에대한 57

12 김선길 정지원 임용하 김지훈 나창수 김선종 부작용이있다. aspirin은위장관출혈, 레이증후군, 구토, 의식장애의부작용이있으며 34,35), sulfinpyrazone 은출혈증가, AST, ALT, ALP 상승등의간기능악화, 피부발진, 크레아틴상승으로인한수분나트륨저류의부작용이있고 36,37), dipyridamole은열감, 심계항진, 혈압강하, 구토, 식욕부진, 어지러움의부작용이있으며, propranolol은저혈당, 무력감, 피로감, 무과립구증, 말초동맥순환장애등의부작용이있다 38,39). 따라서이약물은관절염환자와같이장기적인치료가필요한환자에게사용하기부적합하다. ChondroT는대강활탕과청열사습탕 40) 의구성약물인강활, 당귀, 위령선, 금은화, 황백의조합으로만들어진한약제제이다. 대강활탕은위생실록에수록된처방으로 41) 風濕相搏하여肢節腫痛하며不可屈伸한증을치료하며 7,42,43) 去風濕, 通經絡, 지통의효능을가지며 11,12,16) 최근 CFA 유발관절염이있는동물모델에서 inos, nnos의발현을유의하게감소시키는것이확인된처방이다 18). 또한청열사습탕은痺證, 통풍, 역절풍, 관절동통등을치료하는처방으로 7) 최근 carrageenan 으로유발된관절염에부종증가를억제시키며, WBC, ESR, CRP, urine NO 및 synoviocytesno를감소시키는효과가확인된처방이다 44). 한의학에서혈은인체를구성하고인체의생명활동을유지하는기본물질의하나로서 45) 廣義의혈은혈액이가지고있는營養과滋潤작용그리고그물질적기초를의미하며, 이는순환의뜻을내포하고있다 9). 혈액은경맥에서끊임없이흐르면서전신을영양하는작용을담당하기에 ChondroT의通經絡작용은혈액순환의의미와상통하며, 혈액순환의효과를검증하기위해서는혈소판에대한항응고작용이선행되어확인되야한다. ChondroT는관절염치료를목적으로보건산업진흥원한의약선도기술개발한약제제사업에서개발중인처방이다. 하지만본연구는관절염이아닌항응고작용이라는부가적인효능을확인하기위해진행되었다. 기원처방인대강활탕과청열사습탕의通經絡작용을현대과학에서항응고작용과연계하여실험하였고, 각구성약재와혼합물의항응고효과를비교하여수치화하였다. 항응고작용이라는단일효과의관점에서 ChondroT는금은화, 황백에못미치는결과를도출하지만, 관절염치료라는주적응증에항응고작용이라는부가적인효과를가진다는점에서 ChondroT는단일제재보다다양한치료효과를 가진다. 본실험은혈소판의응집에대한실험이기에 5분이라는짧은시간동안반복관찰된결과로항응고에대한효과만확인되었다. 하지만한의학에서通經絡은항응고보다넓은의미인혈액순환개선을포함하는개념으로, 혈액순환효과에대한확인은장기간의추가실험을필요로한다. 본연구는 ChondroT 구성약물의항응고효과를확인하였으며, Collagen과 Thrombin으로활성화된혈소판에대해금은화와 ChondroT가높은항응고효과가있음을확인하였다. 또한 ChondroT와그구성약재의항응고효과를비교하여관찰하였다. 결론»»» ChondroT 구성약재가 Thrombin과 Collagen으로자극된혈소판에미치는항응고효과에대하여다음과같은결론을얻었다. 1. Thrombin으로활성화된혈소판에대한응집능반응에서 ChondroT의농도별 20%, 10%, 5% 응집능은각각 10.3%, 37.0%, 50.2% 를나타내었다. 단일시료와비교하였을때 ChondroT는 3.5 min phase 에서가장낮은응집률을보였으며 4.5 min phase 에서금은화다음으로낮은응집률을보였다. 2. Collagen으로활성화된혈소판에대한응집능반응에서 ChondroT의농도별 20%, 10%, 5% 응집능은각각 35.9%, 48.7%, 57.7% 를나타내었다. 단일시료와비교하였을때 5% 농도에서 ChondroT는단일시료와비슷한수준의응집률을보였으며, 10% 농도에서 ChondroT는 3.5 min phase 에서가장낮은응집률을보였고, 4.5 min phase에서금은화다음으로낮은응집률을보였다. 20% 의농도에서 ChondroT 는 3.5 min phase에서황백다음으로낮은응집률을보였고, 4.5 min phase에서금은화다음으로낮은응집률을보였다. 3. Thrombin과 Collagen으로활성화된혈소판에대한 ChondroT와단일시료들의응집능을측정한결과, 금은화와 ChondroT가높은항응집효과를나타났으며, 금은화-당귀 -황백-강활-위령선의순서로높은항응고효과를나타냈다. 58 J Korean Med Rehabil 2018;28(2):47-60

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