대한한의학회지제 39 권제 1 호 (2018 년 3 월 ) J Korean Med. 2018;39(1):22-34 pissn 1010-0695 eissn 2288-3339 Original Article 가미소요산전탕팩의보관온도및기간에따른지표성분함량및항염증효능비교연구 진성은 1, 서창섭 1, 이나리 1, 신현규 1, 하혜경 1* 1 한국한의학연구원한약연구부 Comparative study on the contents of marker compounds and anti-inflammatory effects of Gamisoyo-san decoction according to storage temperature and periods Seong Eun Jin 1, Chang-Seob Seo 1, Nari Lee 1, Hyeun-Kyoo Shin 1, Hyekyung Ha 1* 1 Herbal Medicine Research Division, Korea Institute of Oriental Medicine Objectives: The purpose of this study is to investigate changes of the marker compounds and anti-inflammatory effect of Gamisoyo-san decoction (GMSYS) depending on storage temperature and periods. Methods: GMSYS was stored at room temperature or refrigeration for 12 months. According to storage temperature and periods, ph and sugar content of GMSYS were measured. To determine the marker compounds of GMSYS, high-performance liquid chromatography analysis was performed. To estimate the anti-inflammatory effect of GMSYS, LPS-induced pro-inflammatory mediators and cytokines were measured in RAW 264.7 cells. Results: There was no change in ph and sugar content depending on storage temperature and periods of GMSYS. The contents of gallic acid and mangiferin in both of room temperature and refrigerated decoctions reduced with increasing storage periods. Chlorogenic acid was time-dependently decreased in case of stored at room temperature. GMSYS significantly inhibited the LPS-induced production of nitric oxide, prostaglandin E 2 (PGE 2) and IL-6 in RAW 264.7 cells. These effects equally maintained up to 3 months at both of room temperature and refrigeration. Since 4 months, the inhibitory effect of GMSYS on LPS-induced PGE 2 production was time-dependently reduced, and the decrease in PGE 2 inhibitory effect of decoction stored at refrigeration was lower than that of stored at room temperature. Conclusions: Our results indicate that the anti-inflammatory effect of GMSYS are maintained up to 12 months, but it shows optimal efficacy up to 3 months. It is recommended to store in a refrigeration for short periods since some components decrease as storage periods becomes longer. Key Words : Gamisoyo-san decoction; storage temperature; storage period; high performance liquid chromatography; anti-inflammation 서론 한약의여러제형중여러약재를물에넣고끓인탕약은체내흡수가빨라약효가신속하게나타나며, 임상에서가장많이사용된다 1). 그러나탕약은수용액상태이기때문에건조된제형인산제및환제에비해안정성이떨어진다는단점이있다 2). 또한탕약을장기간복용하기위해파우치에보관할경우역 Received:20 December 2017 Revised:15 March 2018 Accepted:16 March 2018 Correspondence to: 하혜경 (Hyekyung Ha) 대전유성구유성대로 1672 한국한의학연구원한약연구부 Tel:+82-42-868-9513, Fax:+82-42-864-2120, E-mail:hkha@kiom.re.kr 22
가미소요산전탕팩의보관온도및기간에따른지표성분함량및항염증효능비교연구 (23) 가가높아지거나산도가높아지는등의문제가발생할수있다 2). 그러나탕약의보관조건및기간등에대한정확한정보가부족한상황에서잘못된보관으로인해탕약의유효성분이변하여효능이감소할수있다. 따라서탕약의안전하고효과적인복용을위해정확한보관방법및기간에대한가이드라인이요구된다. 본연구진은이전연구에서다빈도한약처방중평위산 2-3), 쌍화탕 4), 보중익기탕 5), 곽향정기산 6), 반하사심탕 7) 및청심연자음 8) 등의보관기간에따른전탕액의 ph, 당도, 지표성분함량및생리활성변화등에대해보고한바있다. 박 9) 등은사물탕의보관온도및기간에따른안정성을평가하기위하여부유물및침전물관찰, 흡광도및 ph 변화, 세균검출유무, 구성성분함량및유효활성변화에대해보고하였으며, 도 10) 등은당귀수산의보관조건설정을위한연구의일환으로보관온도및기간에따른총페놀과플라보노이드및지표성분함량변화에대해보고하였다. 그러나한약처방에함유된지표성분의특성이다르기때문에각처방마다안정성이유지되는기간에차이가있을수있다. 가미소요산 ( 加味逍遙散, Gamisoyo-san, Jiaweixiaoyao san, Kamishoyo-san) 은송대진사문의태평혜민화제국방 ( 太平惠民和劑局方 ) 에최초로수록된처방으로, 본실험에서는시호, 박하를거하고지모, 지골 피, 생지황, 치자, 황백, 길경을가미한동의보감 ( 東醫寶鑑 ) 에기재된처방을기준으로하였다 11-12). 가미소요산은소요산 ( 逍遙散 ) 에청열 ( 淸熱 ) 효능이증가되어있어간화상염 ( 肝火上炎 ) 으로인한제반증상, 우울증, 갱년기장애에사용된다 12). 또한부인혈허로번열이나며조열이있고식은땀이나며가래가끓고기침하는질환에처방한다 13). 가미소요산의실험적연구에서항산화, 항염증및관절염효과에대해보고되었다 13-14). 본연구에서는이전연구에서 14) 항염증효능이있는것으로확인된가미소요산의장기보관시안정성을확인하기위해포장한가미소요산전탕팩을상온및냉장에서보관하여 ph, 당도, 지표성분함량및항염증효능변화를조사하였다. 재료및방법 1. 실험재료 Table 1과같이가미소요산을구성하는 12종의한약재는모두의약품용한약재전문제조회사인 내몸에닿 (Ulsan, Korea) 에서규격품을구입후, 본초학전문가인토마토요양병원이제현박사 (Jeju, Korea) 와대전대학교서영배교수 (Daejeon, Korea) 로부터관능검사후실험에사용하였다. 본처방의 12가지구성생약의표본 (2012 KE45 1 2012 Table 1. Composition of Gamisoyo-san Latin name Scientific name Amount (g) Origin Paeoniae Radix Paeonia lactiflora Pallas 4.500 Uiseong, Korea Atractylodis Rhizoma Alba Atractylodes macrocephala Koidzumi 4.500 China Anemarrhenae Rhizoma Anemarrhena asphodeloides Bunge 3.750 Kangjin, Korea Lycii Radicis Cortex Lycium chinense Miller 3.750 China Angelicae Gigantis Radix Angelica gigas Nakai 3.750 Bonghwa, Korea Poria Sclerotium Poria cocos Wolf 3.000 Pyeongchang, Korea Liriope Tuber Liriope platyphylla Wang et Tang 3.000 Miryang, Korea Rehmanniae Radix Recens Rehmannia glutinosa Liboschitz var. purpurea Makino 3.000 Gunwi, Korea Gardeniae Fructus Gardenia jasminoides Ellis 1.875 Gurye, Korea Phellodendri Cortex Phellodendron amurense Ruprecht 1.875 China Platycodonis Radix Platycodon grandiflorum A. De Candolle 1.125 Muju, Korea Glycyrrhizae Radix et Rhizoma Glycyrrhiza uralensis Fischer 1.125 China Total 35.250 23
(24) Journal of Korean Medicine 2018;39(1) Fig. 1. Chemical structures of the ten marker components in Gamisoyo-san. KE45 12) 은한국한의학연구원 K-herb연구단에보관하였다. 2. 시약및기기가미소요산의주요성분인 gallic acid, chlorogenic acid, mangiferin, geniposide, paeoniflorin, berberine chloride, liquiritin apioside, nodakenin, benzoic acid 및 glycyrrhizin 등 10종의표준품은 Wako (Osaka, Japan), Sigma-Aldrich (St. Louis, MO, USA), Chengdu Biopurify Phytochemicals (Chengdu, China), Acros Origanics (Pittsburgh, PA, USA), Shanghai Sunny Biotech (Shanghai, China) 및 NPC Bio Technology (Yeongi, Korea) 에서각각구입하여사용하였으며, 이들표준품의순도는모두 98.0% 이상이었다 (Fig. 1). 표준품과시료의용해, 분리및정량분석에사용된 distilled water, acetonitrile 및 methanol 은 HPLC용으로 J.T. Baker (Phillipsburg, NJ, USA) 로부터구입하였으며, 개미산은 Merck KGaA (Darmastadt, Germany) 에서특급시약을구입하여사용하였다. 가미소요산전탕액중주요성분의함량분석을위한고성능액체크로마토그래피 (high-performance liquid chromatography, HPLC) 는 Shimadzu Prominence 24
가미소요산전탕팩의보관온도및기간에따른지표성분함량및항염증효능비교연구 (25) LC-20A series HPLC system (Kyoto, Japan) 을사용하였으며, 측정된데이터는 Lab Solution software (version 5.53, SP3) 를이용하여수집, 처리및분석하였다. 당도와 ph 측정은각각 Atago사의 Pal-α 디지털굴절계 (Tokyo, Japan) 와 Metrohm 사의 692 ph/ion meter (Herisau, Switzerland) 를사용하였다. 시료는경서E&P 의초고속진공저온농축추출기 (COSMOS 660, Incheon, Korea) 를사용하여전탕하였으며, 전탕액은자유조절롤포장기 (MH 205 Tower, Kyungseo machine, Korea) 를사용하여포장하였다. 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 및 triprolidine은 Sigma -Aldrich에서구입하였다. Cell Counting Kit-8 (CCK-8) 는 Dojindo (Kumamoto, Japan) 제품을사용하였으며, Griess reagent와 prostaglandin E 2 (PGE 2) enzyme-linked immunosorbent assay (ELISA) kit은각각 Promega Corporation (Madison, WI, USA) 와 Cayman Chemical Co. (Ann Arbor, MI) 제품을사용하였다. Tumor necrosis factor-alpha (TNF-α) 및 interleukin-6 (IL-6) ELISA kit은 BD Biosciences (San Jose, CA, USA) 제품을사용하였다. 3. 추출물조제및보관가미소요산의물추출물은 12종의구성생약을 Table 1과같은무게비율로섞어총시료양을약 3.0 kg (1첩 35.25 g 85.10배 ) 으로맞춘후물을시료의 10배 (30 L) 로첨가하였다. 혼합된시료를초고속진공저온추출기를이용하여 98 kpa 압력으로 100 에서 2시간전탕한후표준체 (No. 270, 53 μ m, Chung Gye Sang Gong Sa, Seoul, Korea) 를이용하여여과하였다. 여과액은포장기를이용하여포장하였다. 이렇게조제된시료전탕팩은상온 (23± 1 ) 과냉장 (4 ) 에각각보관하면서실험에사용하였다. 4. ph 및당도측정가미소요산전탕팩의보관온도및기간에따라주기적으로 ph와당도를측정하였다. 5. HPLC 분석 HPLC PDA를사용하여가미소요산전탕액중주요성분인 gallic acid, chlorogenic acid, mangiferin, geniposide, paeoniflorin, berberine chloride, liquiritin apioside, nodakenin, benzoic acid 및 glycyrrhizin 등 10종에대하여보관온도및기간에따라함량분석을실시하였다. 성분의분리는 40 로유지된 Water 사의 SunFire C 18 칼럼 (250 4.6 mm, 5 μm, Milford, MA, USA) 을이용하였으며, 이동상은 0.1% (v/v) 개미산이함유된물 ( 이동상 A) 과 0.1% (v/v) 개미산이함유된 acetonitrile ( 이동상 B) 을이용하여다음과같은기울기용매조건으로흘려주었다 ; 5 60% B (0-30분), 60 100% B (30 40분), 100% B (40 45분 ) 및 100 5% B (45 50분). 유속은분당 1.0 ml 의속도로흘려주었으며주입량은 10 μl로하였다. Geniposide와 paeoniflorin의함량분석은상온과냉장에각각보관중인전탕액 1.0 ml을정확히취하여총 25 ml이되도록물을첨가하고 HPLC 분석전에 0.2 μm syringe filter (PALL Life Sciences, Ann Arbor, MI, USA) 로여과한후측정하였다. 나머지성분들의함량분석은전탕액 5.0 ml을정확히취하여총 10 ml이되도록물을첨가한후분석하였다. 6. 항염증효능평가 1) 세포배양마우스대식세포주 RAW 264.7은 American Type Culture Collection (ATCC, Rockville, MD, USA) 으로부터분양받았으며, 5.5% FBS 및 1% penicillin -streptomycin 이포함된 DMEM으로 37 C 및 5% CO 2 조건에서배양하였다. 2) 세포독성평가 25
(26) Journal of Korean Medicine 2018;39(1) 가미소요산전탕액의세포독성을확인하기위하여 RAW 264.7 대식세포를 96 well plate에 3 10 3 cells/well씩분주하여 18시간동안배양한후각추출물을 62.5, 125, 250, 500 및 1000 μg/ml의농도로처리하였다. 이후 24시간동안배양하고 CCK-8 용액을 10 μl씩첨가하여 4시간동안배양하였다. Microplate reader (Benchmark Plus, Bio-Rad, Hercules, CA, USA) 를사용하여 450 nm에서흡광도를측정하고대조군과비교하여상대적인세포생존율 (% of control) 을계산하였다. 이후실험은세포독성이나타나지않는농도로진행하였다. 3) NO, PGE 2 및염증성사이토카인분비량측정보관온도및기간별가미소요산전탕액의항염증효능을확인하기위하여 RAW 264.7 대식세포를 48 well plate에 2.5 10 5 cells/well씩분주하여 18시간동안배양한후각추출물을 62.5, 125, 250, 500 및 1000 μg/ml의농도로처리하고 4시간동안배양하였다. 이후 LPS (1 μg/ml) 를처리하여 20시간동안배양하고상등액을수거하여 nitric oxide (NO) 분비량은 Griess reagent로, PGE 2 및염증성사이토카인 TNF-α와 IL-6 분비량은 ELISA kit을사용하여각제조사의방법에따라측정하였다. NO 및 PGE 2 분비억제에대한양성대조군으로각각 L-NMMA 및 indomethacin을사용하였으며, TNF-α 및 IL-6 분비억제에대한양성대조군으로 triprolidine을사용하였다. 7. 통계처리실험값은 mean±standard error of the mean (SEM) 으로표시하였으며, 실험결과에대한통계학적분석은 SYSTET (Version 10.0, Systat Software Inc., San Jose, CA, USA) 을사용하여 ANOVA 실시후 Bonferroni 사후검정을통해 p 값이 0.05 미만일경우유의한것으로판정하였다. 1. ph 및당도측정 결과 가미소요산전탕액의 ph는 Table 2와같이최초 5.17 5.19로나타났으며, 보관온도및기간에따라주기적으로 ph를측정한결과상온에서는 4.50 5.19, 냉장에서는 4.56 5.19로나타났다. 당도는최초 3.70 3.80 Brix로측정되었으며, 보관기간에따라상온에서는 3.70 4.10, 냉장에서는 3.70-4.03 Brix로나타났다 (Table 3). 2. 가미소요산지표성분의함량분석 HPLC를이용하여확립된분석법을이용하여가미소요산전탕액중 gallic acid, chlorogenic acid, mangiferin, geniposide, paeoniflorin, berberine chloride, liquiritin apioside, nodakenin, benzoic acid 및 glycyrrhizin 등 10가지주요성분의함량분석을실시하였으며, 10종성분모두 30분이내로양호한분리가이루어졌다. 이들성분에대한검량선작성결과상관계수가 0.9996 이상으로양호한직선성을나타내었으며 (Table 4), gallic acid, chlorogenic acid, Table 2. ph of Gamisoyo-san by storage temperature and periods Storage period (months) Storage method ph 0 5.18±0.01 1 Room temperature 4.65±0.00 Refrigeration 4.72±0.02 2 Room temperature 4.50±0.00 Refrigeration 4.58±0.02 3 Room temperature 5.03±0.01 Refrigeration 5.13±0.01 4 Room temperature 5.01±0.01 Refrigeration 5.12±0.01 5 Room temperature 5.00±0.01 Refrigeration 5.09±0.02 6 Room temperature 4.96±0.02 Refrigeration 5.11±0.01 12 Room temperature 4.97±0.01 Refrigeration 5.07±0.01 Data are presented as mean ± SEM (n = 3). 26
가미소요산전탕팩의보관온도및기간에따른지표성분함량및항염증효능비교연구 (27) Table 3. Sugar content of Gamisoyo-san by storage temperature and periods Storage period (months) Storage method Brix 0 3.77±0.03 1 Room temperature 3.83±0.02 Refrigeration 4.00±0.02 2 Room temperature 3.91±0.01 Refrigeration 3.92±0.03 3 Room temperature 3.92±0.01 Refrigeration 3.89±0.01 4 Room temperature 3.99±0.01 Refrigeration 3.93±0.02 5 Room temperature 3.98±0.03 Refrigeration 3.98±0.01 6 Room temperature 3.91±0.03 Refrigeration 3.87±0.00 12 Room temperature 4.08±0.01 Refrigeration 3.90±0.00 Data are presented as mean ± SEM (n = 3). mangiferin, geniposide, paeoniflorin, berberine chloride, liquiritin apioside, nodakenin, benzoic acid 및 glycyrrhizin 등 10가지주요성분은 6.26, 12.53, 13.00, 13.56, 15.19, 15.63, 16.22, 17.27, 20.30 및 29.69분에서각각검출되었다 (Fig. 2). 설정된분석법을이용하여보관기간에따른가미소요산전탕액내주요성분의함량분석결과상온에서는 0.01-2.85 mg/ml으로나타났으며 (Table 5), 냉장에서는 0.04-3.01 mg/g으로나타났다 (Table 6). 이중 gallic acid 및 mangiferin의함량이상온과냉장모두에서보관기간이길어질수록감소하는것으로나타났으며, chlorogenic acid는상온에서만보관기간이길어짐에따라함량이감소하였다. 3. 세포독성 RAW 264.7 대식세포에서제조시점의가미소요산전탕액은 1000 μg/ml의농도까지세포독성이없는것으로나타났다 (data not shown). 따라서이후실험은세포독성이없는 1000 μg/ml 이하의농도에서수행하였다. 4. 항염증효능정상대조군과비교하여 LPS 처리에의해 NO, PGE 2, TNF-α 및 IL-6 생성이통계적으로유의성있게증가하였다. 반면, 양성대조군으로사용한 L-NMMA, indomethacin 및 triprolidine은 LPS 처리군에비해각각 NO, PGE 2, TNF-α 및 IL-6의생성을농도의존적으로억제하였다 (Fig. 3). 제조시점의가미소요산전탕액은 LPS 처리군과비교하여 NO 생성을농도의존적으로억제하였으며, 1000 μg/ml에 Table 4. Linear range, regression equation, correlation coefficients, LOD and LOQ for the ten marker compounds Compound Linear range Regression equation a Correlation LOD b LOQ c (ng/ml) coefficient (ng/ml) (ng/ml) Gallic acid 0.63 40.00 y=38259.49x 10572.16 0.9999 0.05 0.15 Chlorogenic acid 0.63 40.00 y=41318.61x 24432.53 0.9996 0.01 0.03 Mangiferin 0.63 40.00 y=50951.35x 13475.92 0.9999 0.01 0.04 Geniposide 0.63 40.00 y=16710.90x 141.25 1.0000 0.04 0.11 Paeoniflorin 0.63 40.00 y=10839.56x+774.96 0.9997 0.08 0.23 Berberine 1.56 100.00 y=60688.22x 23759.65 1.0000 0.00 0.01 Liquiritin apioside 0.63 40.00 y=15209.48x 2448.89 1.0000 0.05 0.16 Nodakenin 1.56 100.00 y=32877.31x 14561.30 1.0000 0.01 0.03 Benzoic acid 0.31 20.00 y=35372.59x 5310.55 0.9999 0.02 0.07 Glycyrrhizin 0.63 40.00 y=8282.39x 1435.76 1.0000 0.09 0.27 a y: peak area (mau) of compounds; x: concentration (mg/ml) of compounds. b LOD = 3.3σ S. c LOQ = 10σ S. σ is the standard deviation of the blank response and S is the slope of the calibration curve. 27
(28) Journal of Korean Medicine 2018;39(1) Fig. 2. Typical HPLC chromatogram of standard solution (A) and Gamisoyo-san decoction (B) at UV wavelength 230 (I), 240 (II), 254 (III), 270 (IV), 275 (V), 325 (VI), 335 (VII) and 345 (VIII). Gallic acid (1), chlorogenic acid (2), mangiferin (3), geniposide (4), paeoniflorin (5), berberine chloride (6), liquiritin apioside (7), nodakenin (8), benzoic acid (9) and glycyrrhizin (10). 서 22.13% 의억제율을나타내었다 (Fig. 3). 이효능은상온및냉장보관시 12개월까지유지되었다. LPS 처리군과비교하여제조시점의가미소요산전탕액은 PGE 2 생성을농도의존적으로유의성있게억제하였으며 (IC 50 = 442.55 μg/ml), 1000 μg/ml에서 77.78% 의억제율을나타내었다 (Fig. 3). 이는상온및냉장보관시 3개월까지동일하게나타났다 (Table 7). 보관기간 4개월부터상온및냉장보관한경우모두 PGE 2 억제효능이감소하는경향이나타났으며, 상온보관시료 (IC 50 = 624.98 μg/ml) 가냉장보관시료 (IC 50 = 576.33 μg/ml) 보다효능이더크게감소하였다 (Table 7). 제조시점의가미소요산전탕액은 LPS 처리군과비교하여 IL-6 생성을고농도에서통계적으로유의성있게억제하였으며, 1000 μg/ml에서 44.57% 의억제율을나타내었다 (Fig. 3). 이러한양상은 12개월보관시까지동일하게나타났다. 한편, TNF-α 생성억제에대해제조시점의가미소요산전탕액은 1000 μg/ml에서 12.77% 의억제율을가지며통계적으로유의성있는억제를나타내지않아이후보관시료에대한 TNF- 28
가미소요산전탕팩의보관온도및기간에따른지표성분함량및항염증효능비교연구 (29) Table 5. The content of the five marker compounds in Gamisoyo-san by storage periods in room temperature Compound Content (mg/g) 0 * 1 2 3 4 5 6 12 Gallic acid 0.32 0.10±0.003 0.03±0.003 0.02±0.000 0.01±0.003 0.01±0.006 0.01±0.003 0.01±0.003 Chlorogenic acid 0.27 0.25±0.000 0.23±0.000 0.21±0.003 0.17±0.003 0.18±0.000 0.18±0.000 0.14±0.000 Mangiferin 0.34 0.25±0.000 0.20±0.003 0.17±0.000 0.09±0.000 0.08±0.000 0.12±0.000 0.07±0.013 Geniposide 2.85 2.82±0.030 2.79±0.058 2.98±0.013 2.77±0.007 2.10±0.021 2.12±0.007 2.17±0.000 Paeoniflorin 2.66 2.58±0.006 2.61±0.021 2.62±0.003 2.66±0.012 2.50±0.029 2.54±0.009 2.58±0.003 Berberine 0.53 0.42±0.003 0.40±0.000 0.40±0.003 0.31±0.003 0.47±0.006 0.31±0.003 0.31±0.006 Liquiritin apioside 0.39 0.34±0.007 0.30±0.003 0.32±0.003 0.31±0.003 0.32±0.000 0.32±0.000 0.32±0.006 Nodakenin 0.32 0.31±0.003 0.32±0.000 0.33±0.000 0.33±0.003 0.33±0.000 0.33±0.003 0.34±0.000 Benzoic acid 0.27 0.29±0.003 0.28±0.003 0.27±0.000 0.27±0.000 0.27±0.000 0.27±0.003 0.29±0.000 Glycyrrhizin 0.42 0.37±0.003 0.37±0.000 0.38±0.000 0.36±0.000 0.37±0.006 0.37±0.003 0.37±0.000 *month Table 6. The content of the five marker compounds in Gamisoyo-san by storage periods in refrigeration Compound Content (mg/g) 0 * 1 2 3 4 5 6 12 Gallic acid 0.32 0.28±0.003 0.24±0.003 0.20±0.006 0.16±0.003 0.14±0.010 0.12±0.006 0.05±0.003 Chlorogenic acid 0.27 0.27±0.000 0.26±0.003 0.26±0.000 0.25±0.000 0.26±0.000 0.25±0.003 0.23±0.000 Mangiferin 0.34 0.28±0.003 0.29±0.003 0.27±0.003 0.20±0.000 0.24±0.006 0.24±0.000 0.19±0.003 Geniposide 2.85 2.79±0.009 2.70±0.067 2.93±0.072 2.75±0.041 2.08±0.010 2.07±0.015 2.03±0.000 Paeoniflorin 2.66 2.57±0.015 2.57±0.012 2.58±0.003 2.64±0.006 2.46±0.015 2.49±0.013 2.63±0.000 Berberine 0.53 0.40±0.003 0.45±0.003 0.40±0.015 0.32±0.007 0.52±0.015 0.30±0.003 0.31±0.006 Liquiritin apioside 0.39 0.34±0.009 0.34±0.007 0.33±0.009 0.31±0.003 0.31±0.006 0.31±0.007 0.29±0.000 Nodakenin 0.32 0.31±0.000 0.30±0.003 0.31±0.000 0.31±0.003 0.31±0.000 0.31±0.000 0.31±0.000 Benzoic acid 0.27 0.28±0.000 0.27±0.000 0.26±0.003 0.26±0.000 0.26±0.000 0.26±0.003 0.27±0.003 Glycyrrhizin 0.42 0.36±0.000 0.38±0.000 0.36±0.009 0.36±0.003 0.37±0.000 0.34±0.000 0.33±0.000 *month α 농도는측정하지않았다 (Fig. 3). 고찰한약은예로부터치료, 보기 ( 補氣 ) 및보양 ( 補陽 ) 을위해복용되고있으며, 안전성및유효성에대한연구가증가하고있는추세이다 15). 그러나처방되는한약은끓여서파우치에제공하는탕약이대부분인데, 이러한한약파우치의장기보관에대한연구는부족한실정이다. 각한약을구성하는약재마다효능이유지되는조건이다르기때문에처방에따른보관온도및기간등에대한과학적인근거설정이필요하다. 본연구팀의이전연구결과에따르면평위산전 탕팩을 8개월동안보관했을때지표성분인 hesperidin의함량이냉장보관시료보다상온보관시료에서더크게감소하였으며 2), 냉장및냉동보관시 6개월까지항염증효능이유지되었으나 6개월동안상온보관한경우에는효능이상실되었다 3). 곽향정기산전탕팩의유통기한설정연구에서는전탕팩보관기간이길어질수록제조시점에비해항산화활성이감소하였으며, 상온에서의보관기간이 3 개월이상경과하면항염증효능이감소하였다 6). 반하사심탕전탕팩보관에관한연구에서는보관기간이길어짐에따라지표성분인 liquiritin, baicalein 및 wogonin의함량이감소하였으며이는냉장보관시료보다상온보관시료에서더크게감소하였다 7). 반하사심탕의항산화활성은전탕팩을상온보다냉 29
(30) Journal of Korean Medicine 2018;39(1) Fig. 3. Effect of Gamisoyo-san on LPS-induced NO (A), PGE 2 (B), TNF-α (C) and IL-6 (D) production in RAW 264.7 cells at the time of extraction. NO level in supernatant was measured using Griess reagent. PGE 2, TNF-α and IL-6 in supernatant were measured by ELISA. L-NMMA and indomethacin were used as positive controls for NO and PGE 2, respectively. Triprolidine was used as a positive control for TNF-α and IL-6. The data are presented as mean ± SEM (n = 3). ## p < 0.01 versus negative control cells; * p < 0.05 and * p < 0.01 versus LPS-treated cells. 장에보관할경우더오래유지되었으며, 장기간보관시효능이점차적으로감소하였다 7). 이처럼같은전탕팩포장을사용하였음에도각처방에따라전탕팩의안정성이유지되는온도및기간에차이가있는것으로확인되어본연구에서는가미소요산전탕팩보관온도를상온과냉장두가지로설정하고, 성분함량및효능이감소되는시점을보다정확하게설정하기위하여 0, 1, 2, 3, 4, 5, 6 및 12개월간보관후개봉하여안정성을평가하고자하였다. 염증은체내에세균이나바이러스등의외부물질이유입되는경우면역세포가이를인지하여염증매개물질을분비함으로써생체방어및항상성유지기능을조절하는기전이다 16). 면역세포가운데대식 세포는동물체내모든조직에분포하며외부물질을탐지및포식하여제거하고, 염증반응이일어나면 NO, PGE 2, TNF-α 및 IL과같은염증매개물질들을분비한다 17). NO는 nitric oxide synthase (NOS) 에의해 L-arginine으로부터생성되는인자로신경전달, 면역조절및생체내항상성조절에중요한역할을한다 18). 그러나 NO의과도한생성은혈관투과성및부종등의염증반응유발로인해조직및신경손상과유전자변이등을초래할수있다 19). 또한대식세포가활성화되어염증반응이과도하게일어나면 cyclooxygenase-2 (COX-2) 에의해 arachidonic acid 로부터 prostaglandin이생성되며 20), 특히 PGE 2 는혈관생성을유도하여종양생성에기여한다 21). TNF-α 30
가미소요산전탕팩의보관온도및기간에따른지표성분함량및항염증효능비교연구 (31) 및 IL-6는염증반응을조절하는전염증성사이토카인으로다양한염증매게물질들을유도하고면역반응의조절에중요한역할을하지만, TNF-α 및 IL-6 역시과도하게생성되면암세포성장및동맥경화, 만성염증질환등이유발된다 22). LPS는그람음성균의세포외막에존재하며염증매개물질들을분비하도록대식세포를자극하는내독소로알려져있다 23-24). 따라서본연구에서는마우스대식세포인 RAW 264.7에서 LPS로염증반응을유도하여가미소요산전탕팩의보관온도및기간별로 NO, PGE 2, TNF-α 및 IL-6 분비량을측정하여효능이안정하게유지되는기한을설정하였다. 다양한성분들은안정성이유지되는 ph 및온도등의조건이다르기때문에탕액의 ph 및온도변화는탕액에함유된성분의변화를초래할가능성이있다. 따라서가미소요산전탕팩의보관온도및기간에따른 ph, 당도및지표성분의함량을확인함으로써장기보관에따른안정성을확인하였다. 가미소요산전탕팩을상온에보관할경우냉장보관하는것보다탕액의 ph 및당도의변화가큰폭으로나타났지만보관온도및기간에따른유의성은없는것으로나타나가미소요산전탕액의 ph 및당도가지표성분함량및항염증효능변화에영향을주지않을것으로사료되었다. 가미소요산의지표성분 gallic acid 25), chlorogenic acid 26), mangiferin 27), geniposide 28), paeoniflorin 29), berberine chloride 30), liquiritin apioside 31), nodakenin 32) 및 glycyrrhizin 33) 의항염증효능은이미알려진바있다. 특히가미소요산내비교적함량이높은 geniposide 및 paeoniflorin은 RAW 264.7 대식세포에서 LPS에의해생성된 NO와 PGE 2 생성및 IL-6 의발현을억제한다고보고되었으며 28-29), 본연구에서가미소요산전탕팩을상온및냉장에 12개월까지보관하더라도제조시점에비해함량의유의적인감소가나타나지않았다. 반면, gallic acid 및 mangiferin 의함량은전탕팩을상온및냉장보관하는기간이길어짐에따라점차적으로감소하였으며, chlorogenic acid의함량은전탕팩을상온에보관하는기간이길 어질수록감소하였다. 이러한사실로미루어보아가미소요산전탕팩을상온및냉장에보관하더라도함량변화가없는 geniposide 및 paeoniflorin에의해항염증효능이유지되기는하나보관기간이길어짐에따라 gallic acid, mangiferin 및 chlorogenic aicd 의함량이감소하면서항염증효능이점차적으로감소하는것으로판단된다. 또한가미소요산전탕팩의상온보관시냉장보관에비해항염증활성성분의함량이큰폭으로감소하는것으로나타나전탕팩을냉장보관할경우항염증효능이더오래유지되는것으로사료된다. 결론가미소요산전탕팩을상온및냉장보관할경우보관기간 4개월부터항염증효능이점차적으로감소하여 3개월이내에복용하는것이약효의손실이없을것으로판단된다. 특히보관기간 4개월이후부터상온에보관할경우냉장보관한것에비해항염증효능이큰폭으로감소하여장기보관시상온보다냉장보관하는것이약효유지에더이로울것으로사료된다. 이러한연구결과는향후가미소요산전탕팩의유통기한설정시기초자료로활용될수있을것으로기대된다. 감사의글본연구는한국한의학연구원에서지원하는 한약처방의과학적근거기반구축사업 (K17251) 에의해수행되었으며, 이에감사드린다. 참고문헌 1. The co-textbook publishing committee of Korean Medicine College. Formula Science. Seoul: Younglimsa. 2009. 2. Seo CS, Shin HK, Kim JH, Shin KS. Changes of Principal Components and Microbial Population 31
(32) Journal of Korean Medicine 2018;39(1) in Pyungwi-san Decoction According to the Preservation Temperature and Period. J Korean Oriental Med. 2011;32(5):41-9. 3. Ha H, Shin IS, Lim HS, Jeon WY, Kim JH, Seo CS, Shin HK. Changes in Anti-inflammatory Effect of Pyungwi-san Decoction According to the Preservation Temperature and Period. Formula Science. 2012;20(2):29-35. 4. Seo CS, Kim JH, Lim SH, Shin HK. Establishment of Shelf-Life of Ssanghwa-tang by Long-term Storage Test. Kor J Pharmacogn. 2012;43(3):257-64. 5. Seo CS, Kim JH, Kim SS, Lim SH, Shin HK. Evaluation of Shelf-life of Bojungikgi-tang by Long-term Storage Test. Kor J Pharmacogn. 2013;44(2):200-8. 6. Jin SE, Kin OS, Shin HK, Jeong SJ. Comparative Study on Biological Activities of Gwakhyangjeonggi-san Decoction According to the Preservation Periods. J Korean Med. 2014; 35(3):60-9. 7. Jin SE, Kim OS, Seo CS, Shin HK, Jeong SJ. Comparative study on stability and efficacy of Banhasasim-tang decoction depending on the preservation temperature and periods. J Korean Med. 2016;37(1):21-33. 8. Yoo SR, Ha H, Lee NR, Shin HK, Seo CS. A Study on Change of Marker Compounds and Biological Activity in Chungsimyeonja-eum Decoction Depending on A Storage Temperature and Periods. Kor J Herbol. 2017;32(4):25-32. 9. Park IH, Kim YH, Choi SH, Yu SN, Kim SH, Ahn SC, Cho SI, Lee I. Effect of Preservation Conditions on the Stability of Samul-tang Decoctions. J Life Sci. 2015;25(10):1124-31. 10. Do HJ, Shim YS, Lee JH, Ahn YJ, Ha IH, Lee YJ, et al. Stability of Danggwisu-san (Dangguixu-san) Water Extract, a Herbal Medicine, Under Various Storage Conditions. Journal of Oriental Rehabilitation Medicine. 2016;26(4):1-8. 11. Jin SM. Taepyunghyeminhwajekukbang. Inminweeseng Publications. 1985;308. 12. Sim TK, Jung IC, Lee SR. The Effect of Gami soyo-san(jiaweixiaoyaosan) on Serotonin Metabolism. Journal of Oriental Neuropsychiatry. 2011;22(1): 37-51. 13. Lee SL, Lee IS, Soh KS. Effects of Gamisoyosan(Jiaweixiaoyaosan 加味逍遙散 ) on Type Ⅱ Collagen-Induced Arthritis. J Oriental Rehab Med. 2001;12(4):167-76. 14. Jin SE, Kim OS, Yoo SR, Seo CS, Kim Y, Shin HK, et al. Anti-inflammatory effect and action mechanisms of traditional herbal formula Gamisoyo-san in RAW 264.7 macrophages. BMC Complement Altern Med. 2016;16:219. doi: 10.1186/s12906-016-1197-7. 15. Choi HM, Kim SJ, Kim IS, Lee JB, Kim JB, Moon SO, et al. Evaluation on Anti-oxidant Activity and Anti-inflammatory Effects for the New Formulation of Gamisoyosan. Kor J Herbol. 2016;31(6):1-9. 16. Willoughby DA. Human arthritis applied to animal models. Towards a better therapy. Ann Rheum Dis. 1975;34:471-8. 17. Lee ES, Ju HK, Moon TC, Lee E, Jahng Y, Lee SH, et al. Inhibition of nitric oxide and tumor necrosis factor α (TNF-α) production by propenone compound through blockade of nuclear factor (NF)-κB activation in cultured murine macrophages. Biol Pharm Bull. 2004; 27:617-20. 18. Wink DA. Chemical biology of nitric oxide: Insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide. Free Radic Biol Med. 1998;25(4-5):434-56. 32
가미소요산전탕팩의보관온도및기간에따른지표성분함량및항염증효능비교연구 (33) 19. Ryu JH, Ahn H, Kim JY, Kim YK. Inhibitory activity of plant extracts on nitric oxide synthesis in LPS-activated macrophages. Phytother Res. 2003;17(5):485-9. 20. Funk CD, Fitzgerald GA. Human platelet, erythroleukemia cell prostaglandin G, H synthase: cdna cloning, expression, and gene chromosomal assignment. FASEB J. 1991;5: 2304-12. 21. Bishop-Bailey D, Calatayud S, Warner TD, Hla T, Michell A. Prostaglandins and the regulation of tumor growth. J Environ Pathol Tox Oncol. 2002;21:93-101. 22. Feldmann M. Role of cytokines in rheumatoid arthritis. Annu Rev Immunol. 1996;14:397-440. 23. Mann JR, Backlund MG, DuBois RN. Mechanism of disease: Inflammatory mediators and cancer prevention. Nat Clin Pract Oncol. 2005;2:202-10. 24. Guzik TJ, Korbut R, Adamek-Guzik T. Nitric oxide and superoxide in inflammation and immune regulation. J Physiol Phamacol. 2003; 43:469-87. 25. Chiu CS, Deng JS, Chang HY, Chen YC, Lee MM, Hou WC, et al. Antioxidant and anti-inflammatory properties of Taiwanese yam (Dioscorea japonica Thunb. var. pseudojaponica (Hayata) Yamam.) and its reference compounds. Food Chem. 2013;141:1087-96. 26. Hwang SJ, Kim YW, Park Y, Lee HJ, Kim KW. Anti-inflammatory effects of chlorogenic acid in lipopolysaccharide-stimulated RAW 264.7 cells. Inflamm Res. 2014;63:81-90. 27. Garrido G, Delgado R, Lemus Y, Rodríguez J, García D, Núñez-Sellés AJ. Protection against septic shock and suppression of tumor necrosis factor alpha and nitric oxide production on macrophages and microglia by a standard aqueous extract of Mangifera indica L. (VIMANG). Role of mangiferin isolated from the extract. Pharmacol Res. 2004;50:165-72. 28. Shi Q, Cao J, Fang L, Zhao H, Liu Z, Ran J, et al. Geniposide suppresses LPS-induced nitric oxide, PGE 2 and inflammatory cytokine by downregulating NF-kB, MAPK and AP-1 signaling pathways in macrophages. Int Immunopharmacol. 2014;20:298-306. 29. Wang QS, Gao T, Cui YL, Gao LN, Jiang HL. Comparative studies of paeoniflorin and albiflorin from Paeonia lactiflora on anti-inflammatory activities. Pharm Biol. 2014;52(9):1189-95. 30. Jeong HW, Hsu KC, Lee JW, Ham M, Huh JY, Shin HJ, et al. Berberine suppresses proinflammatory responses through AMPK activation in macrophages. Am J Physiol Endocrinol Metab. 2009;296:E955-64. 31. Guan Y, Li FF, Hong L, Yan XF, Tan GL, He JS, et al. Protective effects of liquiritin apioside on cigarette smoke-induced lung epithelial cell injury. Fundam Clin Pharmacol. 2012;26(4): 473-83. 32. Rim HK, Cho W, Sung SH, Lee KT. Nodakenin suppresses lipopolysaccharide-induced inflammatory responses in macrophage cells by inhibiting tumor necrosis factor receptor-associated factor 6 and nuclear factor-kb pathways and protects mice from lethal endotoxin shock. J Pharmacol Exp Ther. 2012;342:654-64. 33. Fu Y, Zhou E, Wei Z, Liang D, Wang W, Wang T, et al. Glycyrrhizin inhibits the inflammatory response in mouse mammary epithelial cells and a mouse mastitis model. FEBS J. 2014;281:2543-57. 33
(34) Journal of Korean Medicine 2018;39(1) ORCID Seong Eun Jin https://orcid.org/0000-0002-9224-0735 Chang-Seob Seo https://orcid.org/0000-0002-8156-4464 Nari Lee https://orcid.org/0000-0001-7447-3044 Hyeun-Kyoo Shin https://orcid.org/0000-0003-2319-6678 Hyekyung Ha https://orcid.org/0000-0002-7326-6366 34