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1 대한한의학회지제 35 권제 3 호 (2014 년 9 월 ) J Korean Med. 2014;35(3):60-69 pissn eissn Original Article 곽향정기산전탕액의보관기간에따른항염증및항산화효능비교연구 진성은, 김온순, 신현규, 정수진 한국한의학연구원한약방제연구그룹 Comparative Study on Biological Activities of Gwakhyangjeonggi-san Decoction According to the Preservation Periods Seong Eun Jin, Ohn Soon Kim, Hyeun-Kyoo Shin, Soo-Jin Jeong Herbal Medicine Formulation Research Group, Korea Institute of Oriental Medicine, Background: Herbal formulas are generally served as a type of decoction. However, there is no scientific evidence for determining preservation and circulation period of herbal medicine decoctions. Thus, we investigated anti-inflammatory and anti-oxidative effects of the Gwakhyangjeonggi-san decoction according to its preservation period. Methods: Gwakhyangjeonggi-san decoction was stored for 0, 1, 2 or 3 months at room temperature. To evaluate anti-inflammatory effects, enzyme-linked immunosorbent assays (ELISAs) for tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), and nitric oxide (NO) assay were conducted using the culture supernatant from RAW cells stimulated with lipopolysaccharide (LPS). The antioxidant activities were studied by measuring free radical scavenging activities on ABTS and DPPH. Results: Gwakhyangjeonggi-san decoction maintained the inhibitory effects on TNF-α, IL-6 and NO productions after up to 2 months of storage in LPS-treated RAW cells. No inflammatory response was observed in 3 months of storage. In addition, the scavenging activities on ABTS and DPPH of Gwakhyangjeonggi-san decoction were reduced time-dependently and showed less than 50% inhibition after 3 months of storage. Conclusions: Our results suggest that preservation period of Gwakhyangjeonggi-san decoction is recommended within 2 months after storage. Key Words : Gwakhyangjeonggi-san decoction; preservation period; anti-inflammation; anti-oxidation 서론한약의이용이전세계적으로확대되면서한약의유효성및안전성에대한보고가증가하고있지만, 안정성에대한연구는부족한실정이다. 약물의안정성이란약물이일반적인물리적, 화학적혹은생 물학적환경에서도약효를잃지않고유지될수있는기간을말하는것으로약물의개발과생산및투약의모든과정에서매우중요한요소이다 1). 한약은여러가지제형중에서특히탕액이기본으로사용되어왔다. 현재대부분의탕액은레토르트파우치에첨가한전탕팩의형태로공급되고있다. 한약전탕 Received:14 July 2014 Revised:11 September 2014 Accepted:16 September 2014 Correspondence to: 정수진 (Soo-Jin Jeong) 대전광역시유성구유성대로 1672 한국한의학연구원한약연구본부한약방제연구그룹 Tel: , Fax: , sjijeong@kiom.re.kr 60

2 진성은외 3 명 : 곽향정기산전탕액의보관기간에따른항염증및항산화효능비교연구 (311) 팩의보관및유통기간설정과관련한연구로는, 식품의약품안전처에서제시하고있는장기보존시험 2-4) 과한약구성생약의주요성분에대한함량변화를근거로유통기한을예측한연구 5-8) 가보고된바있다. 하등은 6개월및 12개월보관기간에대한평위산의항염증효과를평가함으로써 6개월의보관유효기한을제시하였다 9). 길등은연교패독산전탕팩의냉장보관기간에따른항염및항균효능을평가한결과약리학적효능이확정되는 9일을넘지않아야함을제시하였다 10). 또한김등은인진호탕전탕팩의냉장보관기간에따른실험동물의혈중활성도를관찰하여, 10일이하의보관이안정할것임을보고하였다 11). 그러나, 탕액이얼마나안정적으로유지되는지에대한연구는여전히부족한실정으로, 이에대한연구를통한탕액의보관및유통기간설정에대한과학적근거제시가요구되고있다. 곽향정기산 ( 藿香正氣散 ) 은송대태평혜민화제국방 ( 太平惠民和劑局方 ) 에처음기재된처방으로곽향 ( 藿香 ), 자소엽 ( 紫蘇葉 ), 백지 ( 白芷 ), 대복피 ( 大腹皮 ), 백복령 ( 白茯苓 ), 후박 ( 厚朴 ), 백출 ( 白朮 ), 진피 ( 陳皮 ), 반하 ( 半夏 ), 길경 ( 桔梗 ), 감초 ( 甘草 ), 생강 ( 生薑 ) 및대조 ( 大棗 ) 등 13종의생약으로구성되어있으며해표화습 ( 解表化濕 ) 과이기화중 ( 理氣和中 ) 을통하여병인을다스리는데사용된다 12). 또한외감풍한 ( 外感風寒 ) 과내상습체 ( 內傷濕滯 ) 로인한곽란토사 ( 霍亂吐瀉 ), 발열오한 ( 發熱惡寒 ), 두통 ( 頭痛 ), 흉격비민 ( 胸膈痞悶 ) 및완복창통 ( 脘腹脹痛 ) 등의증상과산람장기 ( 山嵐瘴氣 ) 에사용된다 12). 최근보고에따르면, 곽향정기산은면역향상 13),14), 항고혈압 15) 및위장관보호 16),17) 등의생물학적활성을가지는것으로보고되었다. 이에본연구에서는곽향정기산전탕팩의보관기간차이에따른약리활성을비교평가함으로써전탕팩유통기한설정을위한과학적기초자료를제공하고자한다. Table 1. Composition of Herbal Medicine in Extract of Gwakhyangjeonggi-san Herbal medicine Latin name Original region Company Amount (g) Agastache rugosa (Fisch. et Meyer) O. Kuntze Perilla frutescens var. crispa (Thunb.) H. Deane Angelica dahurica Benth. et Hook. f. Agastachis Herba Perillae Folium Angelicae Dahuricae Radix Areca catechu L. Aracae Pericarpium China Poria cocos F. A. Wolf Magnolia officinalis Rehd. et E. H. Wils. Atractylodes macrocephala Koidz. Citrus reticulata Blanco Poria Sclerotium Magnoliae Cortex Atractylodes Rhizoma Alba Citrus Unshius Pericarpium Andong, Gyeongbuk, Korea Yeongcheon, Gyeongbuk, Korea Uljin, Gyeongbuk, Korea Pyeongchang, Gangwon, Korea China China Jeju, Korea Pinellia ternata Breit. Pinelliae Tuber China Platycodon grandiflorum A. DC. Platycodonis Radix Andong, Gyeongbuk, Korea Glycyrrhiza uralensis Fisch. Ziziphus jujuba var. inermis (Bunge) Rehder Zingiber officinale Rosc. Glycyrrhizae Radix et Rhizoma Zizyphi Fructus Zingiberis Rhizoma Recens China Yeongcheon, Gyeongbuk, Korea Ulsan, Korea

3 (312) 대한한의학회지제 35 권제 3 호 (2014 년 9 월 ) 재료및방법 1. 곽향정기산전탕액의조제및보관곽향정기산의구성약재는광명당제약 (Ulsan, Korea) 을통해구입하여각각전문가감별후사용하였으며 (Table 1), 구성약재의표본 (2013-KE32-1~13) 은한국한의학연구원한약방제연구그룹에보관하였다. 배합한약재무게 (676 g) 의약 10배에해당하는전탕수 (7 L) 를가한뒤, 초고속진공저온농축추출기 (Cosmos 660, Kyungseo machine, Korea) 를이용하여 100 에서가압하여 2시간동안전탕하였다. 전탕액은 3개의 lot로나누어전탕하였으며, 전탕액을자유조절롤포장기 (MH 205 Tower, Kyungseo machine, Incheon, Korea) 를통해레트로트파우치로포장하였다. 파우치는 8~11월에실내 ( 온도 25, 습도 40~60%) 에서보관하면서 0, 1, 2 및 3개월차에개봉하여, 감압건조하여분말화하였다. 최종수득율은 20 ± 3% 로나타났으며, 효능분석에는감압건조된검체분말 100 mg을 PBS 1 ml에용해시킨뒤 0.2 μm filter로여과하여보존용액을 100 mg/ml 농도로조제하고이를희석하여사용하였다. 3. 항염증효능분석 1) 세포배양 Mouse macrophage cell line인 RAW 세포는 American Type Culture Collection(ATCC, Rockville, MD, USA) 으로부터분양받아사용하였으며, 10% FBS가포함된 DMEM 배지에 penicillin (100 U/mL) 과 streptomycin(100 µg/ml) 을첨가하여 37C 및 5% CO 2 조건에서배양하였다. 2) 세포독성평가곽향정기산전탕팩의상온보관기간별세포독성을알아보기위해 RAW 세포를 96 well plate 에 cells/well씩분주하여 18 시간배양한후, 추출물을농도별로처리하여 24시간동안배양하였다. 이후 CCK-8 용액을 10 μl 씩첨가하여 4시간동안배양하고 microplate reader(benchmark Plus, Bio-Rad, Hercules, CA, USA) 를사용하여 450 nm 에서흡광도를측정하였다. 측정값은대조군과비교하여상대적인세포생존율 (% of control) 을계산하였으며, 이후의실험은세포독성이나타나지않는최고농도를기준으로진행하였다. 2. 시약 Dulbecco's modified Eagle's medium(dmem), fetal bovine serum(fbs), penicillin-streptomycin 및 phosphate-buffered saline(pbs) 은 Gibco BRL(Carlsbad, CA, USA) 에서구입하였으며, lipopolysaccharide (LPS), N G -methyl- L -arginine(l-nmma), 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid)(abts) 및 2-2-diphenyl-1-picrylhydrazyl(DPPH) 은 Sigma Chemical Co.(St. Louis, MO, USA) 에서구입하여사용하였다. Cell Counting Kit-8(CCK-8) 은 Dojindo(Kumamoto, Japan) 제품, Griess reagent는 Promega Corporation (Madison, WI, USA) 제품, TNF-α 및 interleukin(il)-6 ELISA kit은 Invitrogen Co.(Camarillo, CA, USA) 제품을구입하여사용하였다. 3) RAW 세포에서의 NO, TNF-α 및 IL-6 분비량측정 RAW 세포를 48 well plate에 cells/well씩분주하여 18 시간배양한후상온보관기간별곽향정기산전탕팩을농도별로처리하고 4 시간후 LPS(1 µg/ml) 를처리하여 20시간동안배양하였다. Griess reagent를이용하여제조사의방법에따라배양상등액내에존재하는 nitrite의분비량을측정하였으며, 양성대조군으로 nitric oxide synthase(nos) inhibitor인 l-nmma(100 M) 를사용하였다. 상등액내의 TNF-α 및 IL-6 분비량을측정하기위해 ELISA kit를사용하여각제조사의방법에따라측정하였다. 62

4 진성은외 3 명 : 곽향정기산전탕액의보관기간에따른항염증및항산화효능비교연구 (313) Fig. 1. Effect of Gwakhyangjeonggi-san stored with different period on LPS-induced NO production in RAW cells. (A) Gwakhyangjeonggi-san of 0 month storage; (B) Gwakhyangjeonggi-san of 1 month storage; (C) Gwakhyangjeonggi-san of 2 month storage; (D) Gwakhyangjeonggi-san of 3 month storage. Cells were pre-treated with Gwakhyangjeonggi-san or l-nmma (100 M) for 4 h and then stimulated with LPS (1 g/ml) for 20 h. NO production was measured in the culture medium with the Griess reaction. The data are mean values of three experiments ± SEM (n=3). # p<0.01 versus vehicle-treated control group; * p<0.05 and ** p<0.01 versus LPS-treated cells. 4. 항산화효능분석 1) ABTS 라디칼소거능측정 ABTS 라디칼소거능은 Re의방법을변형하여측정하였다 18). ABTS(7 mm) 와 potassium perulfate (2.45 mm) 를 PBS에녹여혼합한후차광하여실온에서 24시간동안반응시켜 ABTS + 를형성시킨후 743 nm에서흡광도값이 0.7이나오도록희석하였다. 상온보관기간별곽향정기산전탕팩과희석한 ABTS + 용액을 96 well plate에가한후실온에서 30 분동안반응시키고 microplate reader(benchmark Plus, Bio-Rad, Hercules, CA, USA) 를사용하여 743 nm에서흡광도를측정하였다. 결과값은시료를첨가하지않은대조군에대한 ABTS 라디칼의소거활성으로나타내었으며양성대조군으로 ascorbic acid 를사용하였다. 63

5 (314) 대한한의학회지제 35 권제 3 호 (2014 년 9 월 ) Fig. 2. Effect of Gwakhyangjeonggi-san stored with different period on LPS-induced TNF-α production in RAW cells. (A) Gwakhyangjeonggi-san of 0 month storage; (B) Gwakhyangjeonggi-san of 1 month storage; (C) Gwakhyangjeonggi-san of 2 month storage; (D) Gwakhyangjeonggi-san of 3 month storage. Cells were pre-treated with Gwakhyangjeonggi-san for 4 h and then stimulated with LPS (1 g/ml) for 20 h. The levels of TNF-α released into the culture medium were assessed using commercially available ELISA kit. The data are mean values of three experiments ± SEM (n=3). # p<0.01 versus vehicle-treated control group; ** p<0.01 versus LPS-treated cells. ABTS radical scavenging activity=(1-a sample /A control )100 2) DPPH 라디칼소거능측정 DPPH 라디칼소거능은 Moreno의방법을변형하여실시하였다 19). 즉, 96 well plate에상온보관기간별곽향정기산전탕팩과 DPPH 용액 (0.15 mm) 을가한후실온에서 30분간반응시키고 microplate reader(benchmark Plus, Bio-Rad. Hercules, CA, USA) 를사용하여 517 nm에서흡광도를측정하였 다. 결과값은시료를첨가하지않은대조군에대한 DPPH 라디칼의소거활성으로나타내었으며양성대조군으로 ascorbic acid를사용하였다. DPPH radical scavenging activity=(1-a sample /A control ) 통계처리실험값은 mean ± SEM으로표시하였다. 실험결과에대한통계학적유의성은 ANOVA 검정을적용 64

6 진성은외 3 명 : 곽향정기산전탕액의보관기간에따른항염증및항산화효능비교연구 (315) 하였으며, Dunnet's multiple comparison test를이용하여 p-value가 0.05 미만일경우유의한것으로판정하였다. 결과 1. 항염증효능검색곽향정기산전탕팩의상온보관기간별항염증효능을비교평가하기위하여 LPS로자극시킨 RAW 세포에서각보관기간별전탕팩추출물의 NO, TNF-α 및 IL-6 생성억제효과를검색하였다 (Figs. 1-3). 대조군과비교하여 LPS 처리군은 NO 생성이유의적으로증가한반면 (p<0.01), 양성대조군으로사용한 l-nmma 처리군은 LPS에의한 NO 생성을농도의존적으로억제하는것으로나타났다 (p<0.01). 곽향정기산전탕팩을상온에보관한경우 2개월까지는 62.5 µg/ml 이상의농도에서 NO 생성을억제하는것으로나타났으나, 3개월간상온에보 Fig. 3. Effect of Gwakhyangjeonggi-san stored with different period on LPS-induced IL-6 production in RAW cells. (A) Gwakhyangjeonggi-san of 0 month storage; (B) Gwakhyangjeonggi-sanof 1 month storage; (C) Gwakhyangjeonggi-san of 2 month storage; (D) Gwakhyangjeonggi-san of 3 month storage. Cells were pre-treated with Gwakhyangjeonggi-san for 4 h and then stimulated with LPS (1 g/ml) for 20 h. The levels of IL-6 released into the culture medium were assessed using commercially available ELISA kit. The data are mean values of three experiments ± SEM (n=3). # p<0.01 versus vehicle-treated control group; ** p<0.01 versus LPS-treated cells. 65

7 (316) 대한한의학회지제 35 권제 3 호 (2014 년 9 월 ) 관한경우에는 1000 µg/ml 이하의농도에서유의적인 NO 억제활성이나타나지않았다 (Fig. 1). TNF-α 및 IL-6 생성또한 LPS 처리에의해유의적으로증가하였으며 (p<0.01), 곽향정기산전탕팩을상온에보관한경우 2개월까지 62.5 µg/ml 이상의농도에서 TNF-α 및 IL-6 생성을유의적으로억제하는것으로나타났다. 반면 3개월간상온에보관한곽향정기산전탕팩은모든실험농도에서 TNF-α 및 IL-6 생성에유의적인차이를보이지않았다 (Figs. 1-2) 2. 항산화효능검색곽향정기산전탕팩제조시점의동결건조시료와상온에서 1, 2, 3개월동안보관한전탕팩의동결건조시료를검체로하여 ABTS 및 DPPH 라디칼소거활성을측정함으로써항산화활성을비교하였다. ABTS 라디칼의소거활성을비교한결과, 모든검체에서농도의존적인소거활성이나타났으나 0개월시료에비해보관기간이증가함에따라소거활 성이감소되었다. 대조군의흡광도를 1/2로환원시키는데필요한시료의농도인 RC 50 값은각각보관 0개월 ; 66.96, 1개월 ; 99.65, 2개월 ; 95.90, 3개월 ; µg/ml로, 3개월시료의 ABTS 라디칼소거활성이제조시점에비해 2배이상감소하는것으로나타났다 (Fig. 4). 양성대조군으로사용한 ascorbic acid는 5 µg/ml에서약 72% 의일정한 ABTS 라디칼소거활성을나타내었다. DPPH 라디칼의소거활성또한 ABTS 라디칼의소거활성과유사한결과를나타내었다. 보관기간에따라 0, 1, 2개월곽향정기산의 DPPH 라디칼에대한 RC 50 값은각각 , , µg/ml로측정되었으며, 3개월보관시료의경우실험최고농도인 400 µg/ml에서 40.33% 의라디칼소거활성을나타내었다 (Fig. 5). 양성대조군 ascorbic acid는 20 µg/ml에서약 86% 의일정한 DPPH 라디칼소거활성을나타내었다. Fig. 4. Effect of Gwakhyangjeonggi-san stored with different period on ABTS radical scavenging activity. ABTS radical cation was produced by reacting 7 mm ABTS solution with 2.45 mm potassium persulfate store in the dark at room temperature for 16 h. Then, ABTS radical solution was added to a 96-well plate containing of samples or ascorbic acid (AA, 5 g/ml). After 30 min of incubation, the absorbance was measured at 743 nm using a microplate reader. The data are mean values of three experiments ± SEM (n=3). * p<0.1, ** p<0.01 versus 0 month. 66

8 진성은외 3 명 : 곽향정기산전탕액의보관기간에따른항염증및항산화효능비교연구 (317) Fig. 5. Effect of Gwakhyangjeonggi-san stored with different period on DPPH radical scavenging activity. DPPH radical solution (0.15 mm in ethanol) was added to a 96-well plate containing of samples or ascorbic acid (AA, 20 g/ml). After 30 min of incubation, the absorbance was measured at 517 nm using a microplate reader. The data are mean values of three experiments ± SEM (n=3). ** p<0.01 versus 0 month. 고찰현재한의원이나한방의료기관등에서치료나예방을위해처방되는한약은끓여서파우치에넣어제공하는탕제가대부분이며, 탕제는서늘한곳에보관하도록복약지도하고있지만탕액파우치의보관기간에대한과학적인근거는설정되어있지않다. 본연구에서는곽향정기산전탕팩의보관및유통기한설정을위한과학적근거를제시하고자, 곽향정기산전탕팩을상온에서 0, 1, 2 및 3개월간보관한각각에대한약리효능변화를측정하였다. 항염증효능을비교평가한결과, 상온에서보관한경우 2개월까지는 NO 생성및염증성사이토카인 TNF-α와 IL-6 분비에대한항염증효능에유의적인변화가없었으나, 3개월동안보관한경우항염증효능이나타나지않았다 (Figs. 1-3). 본연구팀은전탕팩유통기간설정을위한연구로서평위산의항염증효능을평가한바있다 9). 실 온에서 0, 6, 12개월동안보관한평위산전탕팩의항염증효능을비교하였을때, 6개월보관시료에서이미효능이상실된것을확인하였다. 이러한연구결과에근거하여본연구에서는시료를보다단기간으로보관하여실험하였으며, 그결과 2개월과 3개월동안보관한전탕팩의항염증효능차이를확인할수있었다. 또한이전의연구는동물모델을사용함으로써 in vitro 실험에서제한된요소를보완하였다는장점이있지만, 평가항목이한가지였던단점이있다. 이를보완하기위하여본연구에서는항염증효능평가항목으로세가지의염증관련인자들을확인하여결과의신뢰도를높이고자하였으며, 이결과를토대로향후동물모델을이용한검증이필요할것으로사료된다. 길등의연구에서는연교패독산전탕팩의항염및항균활성평가를통하여, 냉장에서의보관기간을 9일로설정하였다 10). 이와같은결과는향후한약전탕팩보관기간의연구시, 상온에서의보관뿐만아니라냉장또는냉동보 67

9 (318) 대한한의학회지제 35 권제 3 호 (2014 년 9 월 ) 관등에대한부분이추가되어야할것이며, 처방의종류에따라평가기간도개별적으로설정되어야할것임을제시한다. 최근한약을비롯한여러가지천연물들의항산화효능에대한연구결과들이많이보고되었다 20-22). 다양한질환의원인인산화적스트레스억제와관련하여약물의우수한항산화효능은신약개발에서중요한사항으로고려되고있다. 따라서본연구에서는전탕팩의보관기간의경과에따른항산화효능을측정하였다. ABTS와 DPPH 라디칼소거활성의변화를통해항산화효능을비교한결과, 제조시점의시료에비해보관기간이증가함에따라라디칼의소거활성이감소하여 3개월시료의경우항산화효능이절반이하로감소하는것으로나타났다 (Figs. 4-5). 이러한결과는항염증효능연구결과와함께, 상온에서 2개월동안은전탕팩이안정하게유지되지만, 상온에서의보관기간이 3개월이상경과하면약효의손실이생길수있음을의미한다. 따라서한약탕액을장기간복용하는경우에는전탕후 2개월이내의보관이안정할것으로생각된다. 본연구를비롯한이전연구에서제시하는보관기간에대한자료는한약처방의종류및연구방법에따라상이함을알수있다. 따라서보다정확한유통기한설정을위해서는좀더다양한종류의한약처방에대한연구및평가방법의확립이필요할것이다. 또한보관방법의경우의수를다양화하여효능의변화를관찰하고, 나아가이에따른주요성분의변화및한의서에근거한한약처방의약리학적효능비교연구가병행되어야할것이다. 결론곽향정기산전탕팩에대하여 3개월동안상온보관시항염증및항산화활성을평가함으로써유통기한을예측하였다. 그결과곽향정기산전탕팩을상온에보관한경우 2개월이내에복용했을때약효의손실이없을것으로판단되었다. 이러한결과는곽향정기산전탕팩의유통기한설정을위한기초자 료로이용될수있을것으로사료된다. 감사의글본연구는한국한의학연구원에서지원하는 한약처방의과학적근거기반구축사업 (K14030) 에의해수행되었으며, 이에감사드린다. 참고문헌 1. Adessi C, Soto C. Converting a peptide into adrug: strategies to improve stability and bioavailability. Curr Med Chem. 2002;9(9): 한의과대학방제학교수공편저. 방제학-개정증보판-. Seoul:Yeongnimsa. 2003: Yu C, Zhu L. Explore of immune mechanism of HUOXIANG ZHENGQI ORAL LIQUOR restrain degranulation of mast cell. Chin Traditional Patent Med. 2002;2: Yu C, Zhu L. Experimental researches on inhibitory effect of Huoxiang Zhengqi Liquid ( 藿香正气水 ) on histamine release. Chin J Integrative Med. 2003;9(4): Sun JK, Kim HH, Nam CG, Koo CM. Effects of GwakHyangJungGiSan on the arterial contraction in rabbit. J Korean Oriental Intern. 2003;24(2): Xue X, Huang X, Gao N, Liu E, Ren L. Effects of Huoxiang Zhengqi liquid on the anti-oxidation and expression of EGFR in gastric mucosa of rats with dampness retention syndrome. Chin J Exp Traditional Med Formulae. 2012;18(21): Yuan Y. Study on the effect of Huoxiang Zhengqi liquid on the gastrointestinal tract in rats. J Pract Med Tech. 2007;14(16): Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-evans C. Antioxidant activity applying an improved ABTS radical cation 68

10 진성은외 3 명 : 곽향정기산전탕액의보관기간에따른항염증및항산화효능비교연구 (319) decolorization assay. Free Radical Biol Med. 1999;26: Moreno MI, Isla MI, Sampietro AR, Vattuone MA. Comparison of the free radical-scavenging activity of propolis from several regions of Argentina. J Ethnophamacol. 2000;71: Ministry of Food and Drug Safety. Ministry of Food and Drug Safety Notification Ministry of Food and Drug Safety. Ministry of Food and Drug Safety Notification Ministry of Food and Drug Safety. Ministry of Food and Drug Safety Notification Seo CS, Kim JH, Kim SS, Lim SH, Shin HK. Evaluation of shelf-life of Bojungikgi-tang by long-term storage test. Korean J acogn. 2013;44(2): Seo CS, Kim JH, Lim SH, Shin HK. Establishment of shelf-life of Ssanghwa-tang by long-term storage test. Korean J acogn. 2012;43(3): Seo CS, Kim JH, Lim SH, Shin HK. Estimation of shelf-life by long-term storage test of Pyungwi-san. Korean J Oriental Med Prescription. 2011;19(1): Son JY, Shin JW, Son CG. Stability study for herbal drug according to storage conditions and periods. J Korean Oriental Med. 2009;30(2): Ha H, Shin IS, Lim HS, Jeon WY, Kim JH, Seo CS, et al. Changes in anti-inflammatory effect of Pyungwi-san decoction according to the preservation temperature and period. Formular Sci. 2012;20(2): Kil GJ, Lim DB, Lee YJ. A study on the degraded effect of decocted Yeonkyopaedogsan over a period. Korean J Herbology. 1998;13(1): Kim WG, Choi YB, Lee YJ. A study on the degraded effect of decocted Injinhotang over a period. Koean J Herbology. 1998;13(2): Zhou X, Chan SW, Tseng HL, Deng Y, Hoi PM, Choi PS, et al. Danshensu is the major marker for the antioxidant and vasorelaxation effects of Danshen (Salvia miltiorrhiza) water-extracts produced by different heat water-extractions. Phytomed. 2012;19(14): Shen B, Truong J, Helliwell R, Govindaraghavan S, Sucher NJ. An in vitro study of neuroprotective properties of traditional Chinese herbal medicines thought to promote healthy ageing and longevity. BMC Complement Altern Med. 2013;13: Hilmi Y, Abushama MF, Abdalgadir H, Khalid A, Khalid H. A study of antioxidant activity, enzymatic inhibition and in vitro toxicity of selected traditional Sudanese plants with anti-diabetic potential. BMC Complement Altern Med. 2014;14:

Journal of Life Science 2011, Vol. 21. No μ μ

Journal of Life Science 2011 Vol. 21. No. 8. 1120~1126 ISSN : 1225-9918 DOI : http://dx.doi.org/10.5352/jls.2011.21.8.1120 μ μ μ α β Journal of Life Science 2011, Vol. 21. No. 8 1121 μ μ 1122 생명과학회지 2011,