대한한의학회지제 37 권제 1 호 (2016 년 3 월 ) J Korean Med. 2016;37(1):21-33 pissn 1010-0695 eissn 2288-3339 Original Article 반하사심탕전탕액의보관온도및기간에따른안정성및유효성연구 진성은 1, 김온순 2, 서창섭 1, 신현규 1, 정수진 2,3 1 한국한의학연구원 K-herb 연구단, 2 한의약융합연구부, 3 한국과학기술연합대학원대학교, 한의생명과학전공 Comparative study on stability and efficacy of Banhasasim-tang decoction depending on the preservation temperature and periods Seong Eun Jin 1, Ohn Soon Kim 2, Chang-Seob Seo 1, Hyeun-Kyoo Shin 1, Soo-Jin Jeong 2,3 1 K-herb Research Center, 2 KM Convergence Research Division, Korea Institute of Oriental Medicine, Daejeon 34054, Republic of Korea 3 Korean Medicine Life Science, University of Science & Technology, Daejeon 34113, Republic of Korea Objectives: Banhasasim-tang (BHSST) has been used for the treatment of the digestive and gastric diseases in Korea. This study aimed to investigate the stability and biological activities of BHSST decoction depending on the preservation temperature and periods. Methods: BHSST decoction was preserved at room temperatures (R/T, 23±1 ) or refrigeration (4 ) for 0, 30, 60 and 90 days. To evaluate the stability of BHSST decoction, ph and sugar content were estimated. In addition, high-performance liquid chromatography (HPLC) analysis was performed to determine marker compounds of BHSST decoction. To evaluate anti-inflammatory effect, nitric oxide (NO) and prostaglandin E 2 (PGE 2 ) productions were measured in LPS-stimulated RAW 264.7 macrophages. Antioxidant activity was examined using the assays for 3-ethyl-benzothiazoline-6-sulfonic acid (ABTS) and 1-1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activities. Results: There was no change in ph and sugar content depending on the preservation temperature and periods of BHSST decoction. Among the major components of BHSST, contents of liquiritin, baicalein and wogonin was reduced time-dependently both at R/T and 4. Inhibitory effects of BHSST decoction on NO and PGE2 productions were slightly decreased in a time-dependent manner by 90 days of preservation. In addition, BHSST decoction maintained ABTS and DPPH radical scavenging activities by 60 days while significantly reducing the activities in 90 days of preservation at R/T. By contrast, BHSST decoction had no significant change of ABTS and DPPH radical scavenging activities by 90 days at 4. Conclusions: Our results suggest that the stability and efficacy of BHSST decoction are maintained for 60 days at 4 rather than R/T. Key Words : Banhasasim-tang decoction; preservation temperature; preservation period: anti-inflammation; anti-oxidation 서론 한약의수요가지속적으로증가함에따라한약의효능 (efficacy) 과안전성 (safety) 및표준화와관련 된안정성 (stability) 에대한과학적연구가요구되고있다 1-2). 한약은환제나분말, 건조엑스및탕약등의형태로사용되어왔으며, 그중다양한물질들이액상형태로추출된탕약이가장많이사용된다. Received:27 January 2016 Revised:14 March 2016 Accepted:17 March 2016 Correspondence to: 정수진 (Soo-Jin Jeong) 대전광역시유성구유성대로 1672 한국한의학연구원한의약융합연구부 Tel:+82-42-868-9651, Fax:+82-42-864-2120, E-mail:sjijeong@kiom.re.kr 21
(22) 대한한의학회지제 37 권제 1 호 (2016 년 3 월 ) 일반적으로임상에서는탕약을 2~4주이내에실온이나저온에서보관하도록권장하고있지만 3), 실제로탕약의안정성이어떤조건에서얼마동안유지되는지에대한근거및관련연구는부족하다. 따라서탕약의보관방법및기간에따른성분함량및약효변화에대한연구가필요하다. 반하사심탕 ( 半夏瀉心湯, Banhasasim-tang, Hange -shashin-to, Ban xia xie xin tang) 은상한론 ( 傷寒論 ) 4) 과금궤요략 ( 金匱要略 ) 5) 에수록된처방으로소화기질환의증상인심하비 ( 心下痞 ) 를치료하는데사용되며, 화위강역 ( 和胃降逆 ), 개결제비 ( 開結除痞 ), 진토 ( 鎭吐 ), 해열 ( 解熱 ), 소염 ( 消炎 ) 및진통 ( 鎭痛 ) 등의작용이있는것으로알려져있다 6). 실험연구로는진정, 진통, 항부종및위액분비억제 7), 총산도억제 8), 항위궤양 9-11) 및위운동능촉진 12) 등의효과가있다고보고되었으며, 임상연구로는급성위장염, 소화불량증, 위산과다및위십이지장궤양에효과가있는것으로보고되었다 13). 본연구에서는반하사심탕전탕팩의안정성및유효성을확인함으로써유효기한설정을위한과학적근거를제공하기위하여반하사심탕전탕팩의보관온도및기간에따른성분함량및약리활성변화를평가하였다. 재료및방법 1. 실험재료반하사심탕을구성하는 8종의생약은광명당제약 (Ulsan, Korea) 에서규격품을구입하여본초학전문가로부터감정후실험에사용하였다. 각구성생약의표본 (2012-KE38-1 KE38-8) 은한국한의학연구원 K-herb연구단에보관하였다. 2. 시약및기기표준품인 liquiritin, baicalin, glycyrrhizin 및 wogonin 은 Wako Pure Chemical Industries Ltd. (Osaka, Japan) 에서구입하였으며, liquiritigenin, wogonoside 및 baicalein은 Biopurify Phytochemicals Ltd. (Chengdu, China), Tauto Biotech (Shanghai, China) 및 Sigma-Aldrich (St. Louis, MO, USA) 에서각각구입하였다 (Fig. 1). 이들표준품의순도는모두 98.0% 이상이었다. Water, acetonitrile 및 methanol 은 HPLC급으로 J.T. Baker (Phillipsburg, NJ, USA) 로부터구입하였다. Trifluoroacetic acid는 Sigma -Aldrich (St Louis, MO, USA) 에서특급시약을구입하여사용하였다. 함량분석을위한고성능액체크로마토그래피 (high-performance liquid chromatography, HPLC) 는 solvent delivery unit (LC-20AT), online degasser (DGU-20A 3 ), column oven (CTO-20A), auto sample injector (SIL-20AC) 및 photodiode array (PDA) detector (SPD-M20A) 로구성된 Shimadzu Prominence LC-20A series HPLC system (Kyoto, Japan) 을사용하였으며, 분석데이터수집과처리는 LC solution software (version1.24, Kyoto, Japan) 를이용하였다. 당도와 ph는 Atago사의 Pal-α 디지털굴절계 (Tokyo, Japan) 와 Metrohm사의 692 ph/ion meter (Herisau, Switzerland) 를각각사용하여측정하였다. 전탕과포장은경서기계산업의초고속진공저온농축추출기 (Cosmos 660, Incheon, Korea) 와자유조절롤포장기 (MH 205 Tower, Kyungseo machine, Korea) 를이용하였다. 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), indomethacin, 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) 에서구입하였다. Cell Counting Kit-8 (CCK-8), Griess reagent 및 prostagladin E 2 (PGE 2 ) enzyme-linked immunosorbent assay (ELISA) kit은각각 Dojindo (Kumamoto, Japan), Promega Corporation (Madison, WI, USA) 및 Cayman Chemical Co. (Ann Arbor, MI) 제품을사용하였다. 22
진성은외 4 명 : 반하사심탕전탕액의보관온도및기간에따른안정성및유효성연구 (23) Fig. 1. Chemical structure of Banhasasim-tang. 3. 추출물조제및보관반하사심탕을구성하는 8종의생약을 Table 1의비율로배합 ( 약 750g; 37.5g 20) 하여약 10배수의물을넣어초고속진공저온농축추출기를이용하여추출하였다. 위와같은방법으로 4개의 lot로나누어추출물을조제한후상온 (23±1 C) 과냉장 (4 C) 에보관하였다. 4. ph 및당도측정조제된반하사심탕전탕액을보관방법및보관기간에따라 ph와당도를측정하였다. 5. HPLCSisudomyeong 분석 Shimadzu Prominence LC-20A series HPLC system을이용하여반하사심탕의주요성분인 liquiritin, baicalin, liquiritigenin, wogonoside, baicalein, glycyrrhizin 및 wogonin 등 7종에대하여함량분석을실시하였다. 이들성분들은 Phenomenex사의 Gemini C 18 칼럼 (250 4.6mm, 5μm, Torrance, CA, USA) 을이용하여 40 에서분리하였다. 이동상은 0.1% (v/v) trifluoroacetic acid가함유된물 (A) 과 acetonitrile (B) 을이용하여다음과같은기울기용매조건으로흘려주었다 ; 10-60% B (0-30분), 23
(24) 대한한의학회지제 37 권제 1 호 (2016 년 3 월 ) 60-100% B (30-40분), 100% B (40-45분), 100-10% B (45-50분) 및 10% B (50-60분). 유속은 1.0 ml/min으로흘려주었으며주입량은 10 μl였다. 함량분석을위해조제된전탕액 1 ml을정확히취하여물을이용하여 10 ml로한후 HPLC 주입전에 0.2 μm syringe filter (PALL Life Sciences, Ann Arbor, MI, USA) 로여과하여검액으로하였다. 6. 항염증효능평가 1) 세포배양 RAW 264.7 대식세포 (mouse macrophage cell line) 는 American Type Culture Collection (ATCC, Rockville, MD, USA) 으로부터분양받았으며, DMEM 배지에 5.5% FBS, penicillin (100 U/mL) 및 streptomycin (100 μg/ml) 을첨가하여 37 C 및 5% CO 2 조건에서배양하였다. 2) 세포독성평가반하사심탕전탕팩의상온및냉장보관기간에따른세포독성을확인하기위하여 RAW 264.7 대식세포를 96 well plate에 3 10 3 cells/well씩분주하여 18시간동안배양하였다. 이후추출물을농도별 (62.5-1000 μg/ml) 로처리하여 24시간동안배양하고, Cell Counting Kit-8 (CCK-8, Dojinho, Kumamoto, Japan) 용액을 10 μl씩첨가하여 4시간동안배양하였다. Cell proliferation을확인하기위하 여 microplate reader (Benchmark Plus, Bio-Rad, Hercules, CA, USA) 를사용하여 450 nm에서흡광도를측정하였다. 대조군과비교하여상대적인세포생존율 (% of control) 을계산하였으며, 이후의실험은세포독성이나타나지않는최고농도를기준으로진행하였다. 3) RAW 264.7 대식세포에서의 NO 및 PGE2 분비량측정 RAW 264.7 대식세포를 48 well plate에 2.5 10 5 cells/well씩분주하여 18시간배양한후추출물을농도별 (125-500 μg/ml) 로처리하여 4시간동안배양하였다. 이후 LPS (1 μg/ml) 를처리하여 20시간동안배양하고상등액내에존재하는 nitrite 및 PGE 2 의분비량을각각 Griess reagent (Promega Corporation, Madison, WI, USA) 및 ELISA kit (Cayman Chemical Co., Ann Arbor, MI) 을사용하여제조사의방법에따라측정하였다. 양성대조군으로각각 l-nmma 및 indomethacin을사용하였다. 7. 항산화활성평가 1) ABTS 라디칼소거활성측정 3-ethyl-benzothiazoline-6-sulfonic acid (ABTS, Sigma Chemical Co. St. Louis, MO) 라디칼을이용한항산화활성측정은 ABTS + cation decolorization assay방법을 96 well plate에맞게수정하여실시하 Table 1. Composition of Banhasasim-tang Herbal medicine Scientific name Amount (g) Origin Pinelliae Tuber Pinellia ternata 7.500 China Scutellariae Radix Scutellaria baicalensis 5.625 Gurye, Korea Ginseng Radix Panax ginseng 5.625 Yeongju, Korea Glycyrrhizae Radix et Rhizoma Glycyrrhiza uralensis 5.625 China Zingiberis Rhizoma Zingiber officinale 3.750 Taean, Korea Coptidis Rhizoma Coptis japonica 1.875 China Zingiberis Rhizoma Crudus Zingiber officinale 3.750 Ulsan, Korea Zizyphi Fructus Zizyphus jujuba 3.750 Yeongcheon, Korea Total amount 37.50 24
진성은외 4 명 : 반하사심탕전탕액의보관온도및기간에따른안정성및유효성연구 (25) 였다. 7 mm ABTS와 2.45 mm potassium persulfate 를최종농도로혼합하여실온인암소에서 24시간동안방치하여 ABTS + 를형성시킨후 743 nm에서 0.7의흡광도값을갖도록 PBS로희석하였다. 96 well plate에 ABTS + 용액과처방추출물을농도별 (50, 100, 200, 400 μg/ml) 로혼합하여실온에서 30 분간반응시킨후, microplate reader (Benchmark Plus, Bio-Rad. USA) 를사용하여 743 nm에서흡광도를측정하였다. 시료를첨가하지않은대조구의흡광도를 1/2로환원시키는데필요한시료의농도인 RC 50 값을계산하여처방추출물의라디칼소거활성을비교하였다. 2) DPPH 라디칼소거활성측정 1-1-diphenyl-2-picrylhydrazyl (DPPH, Sigma Chemical Co.) 라디칼을이용한항산화활성측정은 96 well plate을이용하여실시하였다. 96 well plate 에 0.15 mm의 DPPH 용액과처방추출물을혼합하여실온에서 30분간반응시킨후, 517 nm에서흡광도를측정하였다. ABTS와마찬가지로 RC 50 값을계산하여처방추출물의항산화활성을비교하였다. 8. 통계처리실험값은 mean ± SEM으로표시하였다. 대조군과 LPS 처리군간의비교를위해ANOVA 검정을실시한후 Dunnet s multiple comparison test를이용 하여모든그룹간의차이를비교하였으며, p-value 가 0.05 미만일경우유의한것으로판정하였다. 결과 1. ph 및당도측정반하사심탕전탕액의최초 ph는 4.70으로나타났으며, 보관방법및기간에따른 ph를측정한결과상온에서는 4.60-4.70, 냉장에서는 4.69-4.71로나타났다 (Table 2). 당도는최초 3.13 브릭스로측정되었으며, 보관방법및기간에따른당도측정결과상온과냉장모두 2.77-3.13으로나타났다 (Table 2). 2. 반하사심탕중주요성분의함량분석설정된 HPLC 분석법은반하사심탕의 7가지주요성분의함량분석에적용되었다. Liquiritin, baicalin, liquiritigenin, wogonoside, baicalein, glycyrrhizin 및 wogonin 등 7가지주요성분에대한검량선작성결과상관계수가 0.9998 이상으로양호한직선성을나타냈으며, 이들성분의머무름시간은 15.20, 19.36, 21.38, 22.25, 25.83, 27.18 및 30.49 분이었다 (Fig. 2). 설정된분석법을이용하여상온에서보관기간에따른반하사심탕에서 liquiritin, baicalin, liquiritigenin, wogonoside, baicalein, glycyrrhizin 및 wogonin의함량분석결과 3.18-3.74, Table 2. ph and sugar content of Banhasasim-tang by preservation temperature and periods Storage method Room temperature Refrigeration *day Lot ph Sugar content (Brix) 0* 30 60 90 0 * 30 60 90 1 4.70 4.65 4.63 4.61 3.13 3.10 2.90 2.87 2 4.70 4.65 4.62 4.61 3.13 3.10 2.93 2.77 3 4.70 4.65 4.62 4.60 3.13 3.10 2.90 2.83 4 4.70 4.65 4.62 4.60 3.13 3.07 2.90 2.87 1 4.70 4.71 4.70 4.70 3.13 2.87 2.90 2.87 2 4.70 4.71 4.71 4.70 3.13 2.83 2.87 2.90 3 4.70 4.70 4.70 4.70 3.13 2.77 2.87 2.87 4 4.70 4.70 4.70 4.69 3.13 2.93 2.87 2.87 25
(26) 대한한의학회지제 37 권제 1 호 (2016 년 3 월 ) Fig. 2. Typical HPLC chromatogram of Banhasasim-tang decoction at wavelength 254 (A) and 275 (B) nm. Liquiritin (1), baicalin (2), liquiritigenin (3), wogonoside (4), baicalein (5), glycyrrhizin (6), and wogonin (7). Fig. 3. Cytotocicity of Banhasasim-tang stored with different preservation conditions and periods in RAW 264.7 macrophages. BHSST: Banhasasim-tang. 47.51-52.62, 3.69-3.97, 14.38-15.77, 0.04-0.80, 5.26-5.99 및 0.03-0.77 mg/ml로나타났으며 (Table 3), 냉장에서는 3.28-3.77, 45.80-55.63, 3.68-3.95, 10.78-16.60, 0.12-0.80, 5.37-5.99 및 0.14-0.77 mg/ml으로나타났다 (Table 4). 3. 세포독성 RAW 264.7 대식세포에서반하사심탕의세포독성을확인한결과, 상온및냉장보관한경우모두 90일까지 500 μg/ml 이하의농도에서세포독성이 26
진성은외 4 명 : 반하사심탕전탕액의보관온도및기간에따른안정성및유효성연구 (27) Table 3. Content of the seven marker compounds in Banhasasim-tang by preservation periods in room temperature Lot 1 2 3 4 *day Compound Content (mg/g) 0 * 30 60 90 R R R R Liquiritin 3.74 0.01 0.19 3.50 0.03 0.79 3.32 0.12 3.51 3.18 0.06 1.75 Baicalin 51.82 0.22 0.43 51.52 0.05 0.10 50.39 0.12 0.24 51.34 1.24 2.41 Liquiritigenin 3.94 0.01 0.29 3.94 0.04 0.91 3.90 0.01 0.16 3.70 0.02 0.61 Wogonoside 14.42 0.05 0.35 15.15 0.06 0.39 14.70 0.03 0.22 15.77 0.03 0.22 Baicalein 0.80 0.00 0.63 0.12 0.00 3.94 0.06 0.00 3.08 0.05 0.00 8.34 Glycyrrhizin 5.61 0.06 1.06 5.97 0.08 1.35 5.38 0.02 0.42 5.32 0.08 1.50 Wogonin 0.77 0.00 0.32 0.62 0.01 2.36 0.11 0.00 0.90 0.03 0.00 7.04 Liquiritin 3.74 0.01 0.19 3.46 0.01 0.36 3.47 0.08 2.42 3.18 0.13 4.03 Baicalin 51.82 0.22 0.43 52.13 0.04 0.08 49.66 0.04 0.07 47.51 0.35 0.74 Liquiritigenin 3.94 0.01 0.29 3.94 0.00 0.04 3.89 0.05 1.28 3.69 0.01 0.36 Wogonoside 14.42 0.05 0.35 15.30 0.05 0.30 14.53 0.01 0.08 14.60 0.03 0.21 Baicalein 0.80 0.00 0.63 0.14 0.00 1.82 0.05 0.00 7.22 0.05 0.01 16.27 Glycyrrhizin 5.61 0.06 1.06 5.93 0.07 1.18 5.57 0.03 0.63 5.28 0.05 0.88 Wogonin 0.77 0.00 0.32 0.58 0.02 2.70 0.22 0.00 0.01 0.06 0.00 1.71 Liquiritin 3.74 0.01 0.19 3.51 0.01 0.23 3.43 0.04 1.05 3.24 0.02 0.54 Baicalin 51.82 0.22 0.43 52.62 0.14 0.26 49.55 0.08 0.17 51.12 0.87 1.70 Liquiritigenin 3.94 0.01 0.29 3.95 0.00 0.12 3.97 0.06 1.46 3.70 0.04 1.11 Wogonoside 14.42 0.05 0.35 15.57 0.07 0.44 14.44 0.09 0.61 15.45 0.02 0.11 Baicalein 0.80 0.00 0.63 0.14 0.00 1.10 0.06 0.00 6.43 0.04 0.00 8.17 Glycyrrhizin 5.61 0.06 1.06 5.94 0.06 0.98 5.52 0.05 0.97 5.36 0.02 0.38 Wogonin 0.77 0.00 0.32 0.59 0.01 2.22 0.19 0.00 0.79 0.06 0.00 0.19 Liquiritin 3.74 0.01 0.19 3.48 0.02 0.66 3.42 0.04 1.26 3.26 0.12 3.71 Baicalin 51.82 0.22 0.43 49.84 0.06 0.12 49.50 0.23 0.46 51.36 0.97 1.89 Liquiritigenin 3.94 0.01 0.29 3.93 0.01 0.26 3.90 0.01 0.34 3.79 0.03 0.82 Wogonoside 14.42 0.05 0.35 14.95 0.01 0.09 14.38 0.04 0.29 15.60 0.04 0.23 Baicalein 0.80 0.00 0.63 0.15 0.00 0.40 0.05 0.00 1.43 0.05 0.00 3.87 Glycyrrhizin 5.61 0.06 1.06 5.99 0.06 1.04 5.46 0.02 0.46 5.26 0.08 1.43 Wogonin 0.77 0.00 0.32 0.60 0.02 2.75 0.18 0.00 1.41 0.10 0.00 0.08 나타나지않았다 (Fig. 3). 4. 항염증효능반하사심탕전탕팩의상온및냉장보관기간에따른항염증효능을비교평가하기위하여 RAW 264.7 대식세포에 LPS를처리하여자극을유도하고, 보관온도및기간별전탕팩추출물의 NO 및 PGE 2 생성에대한억제효과를검색하였다. LPS 처리군은대조군과비교하여 NO 및 PGE 2 생성을증가시킨반면 (p<0.01), 양성대조군으로사용한 l-nmma (100 μm) 및 indomethacin (2.5 ng/ml) 에의해 NO 및 PGE 2 생성이유의적으로억제되었다 (p<0.01). 반하사심탕을상온및냉장보관한경우모두 90일까지 125 μg/ml 이상의농도에서 NO 및 PGE 2 생성을유의적으로억제하는것으로나타났다 27
(28) 대한한의학회지제 37 권제 1 호 (2016 년 3 월 ) Table 4. Content of the seven marker compounds in Banhasasim-tang by preservation periods in refrigeration Lot 1 2 3 4 *day Compound Content (mg/g) 0 * 30 60 90 R Liquiritin 3.74 0.01 0.19 3.69 0.02 0.58 3.71 0.01 0.34 3.48 0.01 0.41 Baicalin 51.82 0.22 0.43 49.00 0.08 0.17 46.95 0.05 0.11 55.63 0.73 1.32 Liquiritigenin 3.94 0.01 0.29 3.92 0.02 0.39 3.94 0.01 0.32 3.68 0.04 1.11 Wogonoside 14.42 0.05 0.35 12.96 0.05 0.36 11.35 0.00 0.03 16.60 0.10 0.63 Baicalein 0.80 0.00 0.63 0.68 0.02 2.78 0.14 0.01 3.91 0.39 0.00 0.76 Glycyrrhizin 5.61 0.06 1.06 5.99 0.03 0.44 5.72 0.04 0.76 5.49 0.04 0.79 Wogonin 0.77 0.00 0.32 0.61 0.00 0.21 0.66 0.01 1.21 0.37 0.00 0.06 Liquiritin 3.74 0.01 0.19 3.67 0.01 0.31 3.69 0.01 0.40 3.28 0.01 0.38 Baicalin 51.82 0.22 0.43 48.56 0.10 0.21 47.06 0.08 0.17 54.82 0.80 1.45 Liquiritigenin 3.94 0.01 0.29 3.94 0.02 0.48 3.80 0.01 0.38 3.74 0.02 0.51 Wogonoside 14.42 0.05 0.35 12.43 0.04 0.32 12.29 0.00 0.02 15.50 0.01 0.09 Baicalein 0.80 0.00 0.63 0.66 0.01 2.18 0.12 0.00 3.41 0.25 0.01 3.68 Glycyrrhizin 5.61 0.06 1.06 5.97 0.02 0.28 5.66 0.04 0.71 5.39 0.03 0.47 Wogonin 0.77 0.00 0.32 0.69 0.00 0.27 0.56 0.00 0.35 0.16 0.00 1.06 Liquiritin 3.74 0.01 0.19 3.76 0.01 0.34 3.71 0.04 1.06 3.30 0.05 1.66 Baicalin 51.82 0.22 0.43 49.81 0.06 0.11 45.80 0.36 0.78 54.94 0.28 0.52 Liquiritigenin 3.94 0.01 0.29 3.95 0.02 0.44 3.94 0.03 0.87 3.68 0.06 1.70 Wogonoside 14.42 0.05 0.35 13.23 0.04 0.29 10.78 0.02 0.21 15.74 0.02 0.13 Baicalein 0.80 0.00 0.63 0.73 0.02 2.20 0.12 0.00 3.37 0.24 0.02 7.91 Glycyrrhizin 5.61 0.06 1.06 5.99 0.06 0.94 5.65 0.05 0.81 5.42 0.02 0.36 Wogonin 0.77 0.00 0.32 0.73 0.00 0.39 0.57 0.00 0.53 0.14 0.00 0.63 Liquiritin 3.74 0.01 0.19 3.68 0.01 0.28 3.77 0.10 2.64 3.32 0.07 1.99 Baicalin 51.82 0.22 0.43 46.90 0.06 0.13 46.62 0.05 0.11 55.42 0.33 0.60 Liquiritigenin 3.94 0.01 0.29 3.94 0.01 0.31 3.84 0.12 3.22 3.74 0.03 0.93 Wogonoside 14.42 0.05 0.35 11.96 0.04 0.31 11.52 0.01 0.06 15.93 0.02 0.11 Baicalein 0.80 0.00 0.63 0.66 0.02 2.46 0.14 0.00 1.29 0.26 0.00 1.40 Glycyrrhizin 5.61 0.06 1.06 5.99 0.02 0.25 5.66 0.06 1.07 5.37 0.07 1.21 Wogonin 0.77 0.00 0.32 0.73 0.00 0.35 0.60 0.00 0.24 0.17 0.00 0.32 R R R (Fig. 4, p<0.05). 한편, 반하사심탕 125, 250 및 500 μg/ml 처리군간에는 NO 및 PGE 2 생성억제에유의적인차이가없는것으로나타났다. 5. 항산화활성반하사심탕전탕팩의상온및냉장보관기간에따른항산화활성을비교평가하기위하여 ABTS와 DPPH 라디칼소거활성을비교분석하였다. 그결과, 상온보관의경우는 0개월시료에비해보관기간이증가함에따라라디칼소거활성이감소되었다. 0, 30, 60 및 90일동안상온보관된반하사심탕의 ABTS 라디칼 RC 50 값은 122.97, 125.80, 136.74 및 157.92 μg/ml, DPPH 라디칼 RC 50 값은 165.44, 190.97, 205.13 및 265.66 μg/ml로측정되었다. 특히 90일상온보관시료의경우제조시점 (0개월) 28
진성은외 4 명 : 반하사심탕전탕액의보관온도및기간에따른안정성및유효성연구 (29) Fig. 4. Effect of Banhasasim-tang stored with different preservation conditions and periods on LPS-induced NO (A) and PGE 2 (B) production in RAW 264.7 macrophages. Cells were pre-treated with Banhasasim-tang for 4 h and then co-stimulated with LPS (1 μg/ml) for 20 h. The levels of NO and PGE 2 released into the culture supernatant were measured using Griess reagent and ELISA kit, respectively. The data are presented as the means ± SEM (n = 3). ## p<0.01 versus vehicle-treated control group; *p<0.05 and **p<0.01 versus LPS-treated group. BHSST: Banhasasim-tang, ID: indomethacin. 에비해 ABTS와 DPPH 라디칼소거활성이유의적으로감소함을알수있었다. 냉장보관전탕팩의경우 90일까지두라디칼모두 RC 50 값의유의적인변화가없었다 (Fig. 5). 고찰한약의안정성및유효성에대한과학적근거마 련은한약에대한복약지도마련과신뢰도향상을위해중요하며, 한약의표준화에대한기초적인자료가될수있다. 그러나현재까지한약의보관및유통기한설정에대한명확한기준은없으며, 이에대한연구또한부족한실정이다. 특히한방병원또는한의원에서처방되는한약의대부분은끓여서제공하는탕제의형태이므로한약처방전탕팩의보관기간설정에대한연구가무엇보다시급하다. 29
(30) 대한한의학회지제 37 권제 1 호 (2016 년 3 월 ) Fig. 5. Effect of Banhasasim-tang stored with different preservation conditions and periods on ABTS (A) and DPPH (B) radical scavenging activity. ABTS or DPPH radical solution was added to a 96-well plate containing of several concentrations (50, 100, 200, 400 μg/ml) of Banhasasim-tang. After 30 min of incubation, the absorbance (ABTS; 734 nm, DPPH; 517nm) was measured using a microplate reader. RC50 is concentration of the sample which is required to scavenge 50% of radicals. The data are presented as the means ± SEM (n = 3). **p<0.01 versus 0 days. 이와관련하여식품의약품안전처에서는장기보존시험및한약구성생약의주요성분에대한함량변화를근거로유통기한에대해연구한바있다 14-16). 최근본연구팀의곽향정기산을이용한연구를비롯하여 17), 보중익기탕, 쌍화탕및평위산등의보관기간에따른주요성분및약리학적효능변화에대한연구가보고된바있다 18-21). 본연구팀의곽향정기산전탕팩보관에관한연구에서는전탕액파우치를실온에서각각 0, 1, 2 및 3개월간보관후개봉하여항염증및항산화효능에대한평가를실시하였다 17). 그러나실제전탕팩의보관은실온뿐만아니라냉장에서보관하는빈도가더높은것으로판단되어, 본연구에서는반하사심탕의보관온도를실온과냉장의두가지조건으로설정하였으며, 안정성에대한실험을추가하였다. 반하사심탕전탕팩의보관방법및기간에따른성분변화를분석한결과상온및냉장에서 90일까지보관했을때, 저장온도및기간에따른탕액의 ph와당도는변화가없는것으로나타났다. 그러나지표성분 7종의함량을비교한결과, 상온에보관 하는기간이길어짐에따라 liquiritin, baicalein 및 wogonin의함량은감소하는경향이나타났다. 또한 30일이상냉장보관한결과 baicalein과 wogonin의함량이감소하였으며, 냉장에서 90일까지보관한경우 liquiritin의함량역시감소하는것으로나타났다. 반하사심탕은소화기질환에다빈도로처방되는한약처방이다. 특히역류성식도염 (reflux esophagitis) 으로인한식도의점막자극에의한염증이나아스피린장기복용, 심한스트레스, 담즙산역류및 prostaglandin 합성억제등에의한급 만성위염등에적용된다. 이와같은사실에근거하여본연구에서는반하사심탕전탕액의항염증효능측정을통하여, 전탕팩의보관조건에따른변화를비교분석하였다. 적절한농도의 NO는조직손상및감염성병원체의침입등으로부터인체를보호하기위한면역조절및신경전달등에서중요한역할을하지만, NO가만성적으로과잉생성되면류마티스관절염, 동맥경화증, 위염및천식등염증성질환을유발할수있다 22). 또다른중요한염증인자중하나인 30
진성은외 4 명 : 반하사심탕전탕액의보관온도및기간에따른안정성및유효성연구 (31) PGE 2 역시혈관의투과성을높이기때문에염증과정에서발열및통증등을유발할수있다 23). 따라서활성화된대식세포에서 NO 및 PGE 2 의과잉생성을막는것은염증반응을조절하는데중요하다. 본연구에서 LPS로염증반응을유도한 RAW 264.7 대식세포에반하사심탕추출물을처리하였을때, 90일보관시까지 NO 및 PGE 2 생성억제효능이유의적으로지속되었다. 그러나 30일보관과비교하여 60일과 90일보관시에는 NO 및 PGE 2 억제효능이낮은것으로관찰되었다. 상온과냉장보관에따른항염증효능은차이가없었다. 산화적스트레스는생체내단백질, 지질, DNA 및당질등거대분자를불활성화시키고세포구조를붕괴시켜세포를사멸시키는염증반응에관여한다 24-25). 이러한염증반응시대식세포와같은염증관련세포에서면역반응으로인해 reactive oxygen species (ROS) 및 reactive nitrogen species (RNS) 가생성되고, 반복되는조직의손상이나재생에의해염증반응이지속되면 ROS와 RNS가과다형성되어만성염증성질환이유발된다 26-27). 즉세포가적정수준의항산화물질로부터지속적으로보호받지못하면 free radical에의한산화적스트레스에의해질병을초래하게된다 28). 따라서본연구에서는반하사심탕전탕액의 ABTS 및 DPPH 라디칼소거활성측정을통한항산화활성을평가하여전탕팩의보관조건에따른변화를비교분석하였다. 반하사심탕전탕팩의상온보관은 90일보관시항산화활성의유의적인감소를나타내었다. 반면에냉장보관의경우는 90일보관시까지활성이유지되었다. 이상의결과를종합하였을때, 전탕팩을 90 일까지상온에보관한경우항산화활성이전탕한날에비해유의적으로감소하는것으로보아전탕팩을상온에서보관할때는 60일이내에복용하는것이안정할것으로사료된다. 이전연구에서반하사심탕의지표성분 liquiritin, baicalin, liquiritigenin, wogonoside, baicalein, glrcyrrhyzin 및 wogonin은항염증 29-33) 및항산화효능 34-37) 이있다고보고된바있다. 따라서비록 liquiritin, baicalein 및 wogonin의함량이보관기간이길어짐에따라감소하지만, 지표성분중함량이가장높은 baicalin을비롯한 liquiritigenin, wogonoside 및 glycyrrhyzin에의해반하사심탕전탕팩의항염증활성은유지되는것으로판단된다. 본연구와관련하여, 한약을장기간복용하는경우탕액보다도안정성이확보된제형을고려할필요가있는것으로생각되며, 보다정확한평가를위해서는한약의제형에따른다양한조건에서의연구가요구된다. 결론본연구에서상온및냉장보관기간에따른반하사심탕전탕팩의성분함량변화, 항염증및항산화효능을비교함으로써유통기한을예측한결과, 상온및냉장에서 60일까지보관해도약효의손실이없는것으로판단되었다. 그러나상온에서 90일동안보관한경우항산화효능이감소하는것으로보아전탕팩의장기보관시상온보다는냉장보관하는것이더안전할것으로사료된다. 참고문헌 1. Jang IS, Yang CS, Lee, Han CH. A review of herbal medicine products associated with toxic events in Korea. J Korean Oriental Med. 2007;28(1):1-10. 2. Man SC, Durairajan SS, Kum WF, Lu JH, Huang JD, Cheng CF, et al. Systematic review on the efficacy and safety of herbal medicines for Alzheimer s disease. J Alzheimer s Dis. 2008;14(2):209-23. 3. Son JY, Shin JW, Son CG. Stability study for herbal drug according to storage conditions and periods. J Korean Oriental Med. 2009;30(2): 127-32. 4. Jang JK. Shang HL. Seoul:DS print. 1984:188, 31
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