Kor. J. Microbiol. Biotechnol. Vol. 30, No. 3, 270 281(2002) ROS 제거기능을갖는천연산물로부터장내유용세균 Lacobacillus 속의성장을촉진시키는조합의구성 김종덕 * ㆍ김민용 1 ㆍ서효진ㆍ김봉조 2 ㆍ김대현 3 ㆍ김은옥ㆍ정해영 3 ㆍ공재열 2 여수대학교생명ㆍ화학공학부, 1 냉동공학과, 2 부경대학교식품생명공학부, 3 부산대학교약학과 Combination of Natural Products Removing ROS for Growth Promoting Effects of the Useful Enterobacteria Lacobacillus sp. Kim, Jong-Deog *, Min-Yong Kim 1, Hyo-Jin Seo, Bong-Jo Kim 2, Dae-Hyun Kim 3, Eun-Ok Kim, Hae-Young Chung 3, and Jai-Yul Kong 2. Department of Biotechnology, 1 Department of Referigeration Engineering, Yosu National University, Yosu, Korea, 2 Department of Faculty of Food Science and Biotechnology, Pukyong National University, Pusan, Korea, 3 College of Pharmacy, Pusan National University, Pusan, Korea The growth of Enterobacteria, Lactobacillus acidophilus and L. plantarum was promoted by natural products themselves bearing antioxidative capacity and combined two, three and four kinds of them. L. acidophilus was promoted by Paeonia japonica, Acori graminei rhizoma, Aurantii nobilis pericarpium, alone, and two mixed combinations were composed of Acori graminei rhizoma and Cinnamomi cortex, Aurantii nobilis pericarpium and Puerariae radix, and three mixed combinations were formed with Cimicifugae rhizoma, Cinnamomi cortex and Glycyrrhizae radix, and four mixed combinations were made up Glycyrrhizae radix, Theae folium, Scutellaria baicalensis george and Cinnamomi cortex. There four combination promoted growth of L. acidophilus with 1.3 times than that of control, and their antioxidative capacity also higher than that of 4 times, the ratio of elimination of hydroxyl radical was more than 70% in dilution rate of 100 times. In the case of L. plantarum was promoted by Corni fructus, Nelumbo nuclfera gaertner, Sophorae flos, alone, and two mixed combinations were organized with Schizandrae fructus and Theae folium, Paeonia japonica and Epimedii herba, and three mixed combinations were combined with Corni fructus and Theae folium and Paeonia japonica, Corni fructus and Coptidis rhizoma and Schizandrae fructus, and four mixed combinations were composed of Corni fructus, Theae folium, Coptidis rhizoma, Scutellaria baicalensis george, and Corni fructus, Schizandrae fructus, Paeonia japonica, Theae folium. These four combinations assisted growth of L. plantarum with 1.5 times than that of control, and their antioxidative capacity also higher than that of 4-5 times, the ratio of elimination of hydroxyl radical was more than 75% in dilution rate of 100 times. As these combinations of natural products could activate some parts of body, they might be applied pharmaceuitcal sources, functional foods, and expected to fermentative beverages. Key words : Antioxidative capacity, natural product, combination, growth promotion, Lactobacillus sp 우리의생체에서는호흡을통한에너지의생산, 면역활동을통한병의예방, 독성물질이생체내에투여되었을때간에서의무독화과정, 생체의균형을유지하는활동, 그리고운동등과같이일상생활의한부분인정상적인생활의활동임에도불구하고이런활동으로부터 ROS(Reactive Oxygen Species) 가생산되어정상적인세포를공격하여산화시킴으로써사멸되며이러한결과로각세포는제기능을다하지못하여생체는주름지고늙어가고병들어간다 [9,12,21]. ROS 는산소의부대전자에의하여생산되며 superoxyradical *Corresponding author Tel. 061-659-3305 Fax. 061-659-3305 E-mail: pasteur@yosu.ac.kr 이외에도 hydroxyradical, singlet oxygen 등의독성이아주강력한 ROS 가생성되고있다. 이들 ROS 의강산화력에의하여생성될수있는질병을살펴보면세포산화에의한세포의사멸, DNA 공격하여유전자의이상을일으켜암의발생, 불포화지방산이많은세포막을공격하여물질수송시스템의고장으로인한세포의사멸, LDL 산화에의한동맥경화, 간염, 아토피성피부염, 란게르한스섬의공격에의한당뇨병의발생, 수정체단백질손상으로인한백내장, 뇌세포공격에의한치매, 반점및주름등을들수있다 [8,14,15,17]. 이러한질병들은어떻게 ROS 를제거할수있는가에의존되며 SOS 항산화제 (Superoxide Scavenging Antioxidants) 를사용함으로써제거할수있다 [8]. 천연산물은그종류와성질및역할이매우다양하여옛부터아주중요한치료제로써사용되어왔다 [2,4,6,10,20,22]. 항산화력을
GROWTH PROMOTING EFFECTS BY NATURAL PRODUCTS 271 검증할수있는방법을사용하여 ROS 의제거기능이강한천연산물들을탐색하고이들을이용하여장내세균중에서 Lactobacillus 속 [1,16,19] 과같은세균의기능을향상시켜생체내에 probiotic 효과를증가시키도록한다면 ROS 를제거효과와더불어장내세균의균형의유지가가능하며또한천연산물자체의약리효과를이용할수있어여러가지의장점을가질것으로사료된다. Lactobacillus 속의세균은 Staphylococcus aureus, Salmonella typhimurium, Yersinia enterocolitica 및 Clostridium perfringens 등과같은병원성미생물의성장시길항효과를발휘하며, 프로바이오틱미생물은항미생물기작을경유하여장내서식하고있는병원성미생물에대한저항성을증진시키는역할도하며, 경쟁적인집락화과정을통하여위장내병원성세균이장점막에점착하는것을저해하는작용을하는것으로알려져있다 [3,11]. Lactobacillus 속의세균에의하여생성한젖산과초산같은유기산은장내 ph 를낮추어병원성미생물의성장에있어간접적으로영향을끼치며, 이들유기산에의해장의연동운동이자극을받아장관으로부터병원성미생물을제거하는역할도한다. 그리고 Lactobacillus 에의해생성되는과산화수소 (H 2 O 2 ) 는 lactoperoxidasethiocyanate system 을통하여식중독성병원성미생물의증식을억제하는 hydrocyanic acid 를방출하기도하며, 이산화탄소와 diacetyl 또한병원성미생물의성장을저해한다 [13]. 따라서본연구에서는 ROS 제거기능이강력한항산화력을가지고있는천연산물들의선별과이를이용한배합에의하여항산화력이강하면서생체내의 Lactobacillus 속의장내세균을활성화시킬수있는천연산물의배합을구성하여장의활성화를도모하고장내질환을예방할수있는천연산물의조합을구성하고자한다. 재료및방법 사용균주장내유용균주 Lactobacillus acidophilus(kctc 3145) 및 Lactobacillus plantarum(kctc 3108) 들은 KTCC 로부터분양받아사용하였다. 재료및시약균주의성장에필요한배지로서 L. acidophilus는 Difco사로부터구입한 Lactobacilli MRS broth(casein peptone 10.00 g, meat extract 10.00 g, yeast extract 5.00 g, glucose 20.00 g, tween 80 1.00 g, K 2 HPO 4 2.00 g, Naacetate 5.00 g, (NH 4 ) 2 citrate 2.00 g, MgSO 4 7H 2 O 0.20 g, MnSO 4 H 2 O 0.05 g, distilled water 1000 ml, ph 6.5) 를사용하였고, L. plantarum은 MRS broth에 0.5% lactose medium을혼합한배지를사용하였으며, 250 ml의 flask로 37 o C에서 150 rpm으로교반하면서배양하였다. 유용천연산물의선별천연산물의선별은동의보감 [4], 증맥 방약합편 [6], 한방처방의구성과적용 [10], 도해상용한방처방 [2], 현대한방강좌 [20], 현대생약학 [22] 등의여러고서에수록된고처방으로부터소화기계에많이사용되는처방중에서생체를보하는기능, 해독작용, 수렴, 조혈작용, 항균작용을가지는천연산물의역할을바탕으로 30 종류를선택하여사용하였다. 천연산물의추출및조합천연산물의시료는각각의천연산물 300 g 에 3 차증류수 1500 ml 를넣어 1 시간동안열수추출한여액을 500 ml 로농축하여사용하였다. 한가지의천연산물을사용할때는각천연산물추출액을사용하였고, 두가지의천연산물의조합은각각의천연산물을 1:1 로조합한혼합액을, 세가지의천연산물의조합은각각의천연산물을 1:1:1 로조합한혼합한액을, 네가지의천연산물의조합은각각의천연산물을 1:1:1:1 로조합한혼합액을실험의시료로하고필요한량에따라사용하였다. 장내유용세균 L. acidophilus 및 L. plantarum 의활성을증가시키는천연산물군의탐색 L. acidophilus 및 L. plantarum 의배양은 250 ml 삼각플라스크에한가지, 두가지, 세가지및네가지종류의천연산물의조합시료 3ml 와세균의특성에맞는배지를 97 ml 첨가하여전체량을 100 ml 로하여멸균한후, 질소가스로충진된 glove box(jisco, J-926) 내에서, 제균필터가부착된 needle 로배양액내로질소를불어넣어탈기시키고, 전배양한균주를 0.1% 접종하여 37 o C 에서혐기배양하면서 6 시간간격으로질소가스가충진된 glove box 내에서시료를채취한후 20 배희석하여 spectrophotometer(shimadzu, UV- 2101PC) 를이용하여 660 nm 에서흡광도를측정하였고, 세균의성장정도가천연산물을넣지않은대조군보다높은천연산물을선택하였다. 용존산소분석법 (D.O. analysis method) 에의한 ROS 제거활성의측정용존산소분석법 [14] 에의한 ROS 의제거활성의측정은 100 ml 의 flask 에 linoleic acid 1 g 과 tween 40 용액을 2ml 넣고, 천연산물의조합시료 3ml 를첨가하고증류수로써최종량을 30 ml 로조정한후, 37 o C 의항온조에서 Fe 2+ ion 을첨가하고 D.O. meter(tps, Model WP-82) 를사용하여 D.O. 의감소를측정하였으며, 표준물질로서는합성항산화제인 BHA(tertbutylhydroxyanisol) 및 BHT(tert butylhydroxytoluene), 그리고 β-carotene 을사용하여천연산물의열수추출액의항산화력을 AUC(Area Under Curve) 로써비교하였다. Hydroxyl radical 소거활성측정 Hydroxyl radical 은생체의대사과정에서생산되는지질의
272 Kim et al. 과산화물이나 H 2 O 2 가 Fe 2+ 나 Cu 2+ ion 의존재하에서생산되며가장독성이강한 free radical 이므로이라디칼을소거하는정도를측정하였다 [5,7,18]. Hydroxyl radical 소거활성은 2-deoxyribose oxidation method 을사용하였으며즉, 시험관에 0.1 mm FeSO 4 /EDTA 용액 0.2 ml, 10 mm 2-deoxyribose 0.2 ml, 및천연산물의조합시료 0.2 ml 와 0.1 M phosphate buffer (ph 7.4) 1.2 ml, 10 mm H 2 O 2 0.2 ml 를가하고 37 o C 의항온조에서 4 시간반응시킨후, 2.8% 의 TCA(trichloroacetic acid) 용액 1ml 를가하여반응을중지시키고, 생성되는 malondialdehyde 를 1.0% 의 TBA (thiobarbituric acid) 용액 1ml 를가하여 100 o C 에서 10 분간가열시킨후급속냉각하고 532 nm 에서흡광도를측정하였다. 총 phenol 함량및방향족의측정항산화력은 phenol 성화합물에서강하며, 또한방향족화합물의량에의하여결정되므로천연산물의조합시료의일정량을분광광도계를이용하여 280nm 에서흡광도측정하여방향족화합물의함량을측정하였고, 총 phenol 함량의분석은 Folin-Denis 법을개량하여측정한후 tannic acid 의함량으로나타내었다. 결과및고찰 용존산소분석법 (D.O. analysis method) 에의한천연산물의 ROS 제거활성용존산소분석법에의하여 30 종류의천연산물을분석한후 AUC 를계산하여기존의합성항산화제인 0.02% 의 BHA 및 0.02% 의 BHT 등과비교하였을때승마 (Cimicifugae rhizoma), 음양곽 (Epimedii herba) 은항산화력이비슷함을보 Fig. 1. Comparision of antioxidantive capacity with AUC(Area Under Curve) of synthetic antioxidants(bha, BHT, β-carotene) and natural products. :0.02% BHA(1656.8), :0.02% BHT(1516.6), :0.02% β-carotene (485.3), :Cimigugae rhizoma(1045.2), :Epimedii herba (783.2). Numerics are designated as AUC values. 였으며 (Fig. 1), Table 1 에 30 종류의천연산물의 AUC 를나타내었다. 현초 (Geranii herba), 백작약 (Paeonia japonica), 천문동 (Asparagi tuber), 상백피 (Mori cortex radicis), 연자육 (Nelumbo nuclfera gaertner), 백굴채 (Chelidonium majus L.), 황련 (Coptidis rhizoma), 백출 (Atractylodis rhizoma alba), 진피 (Aurantii nobilis pericarpium), 오배자 (Galla rhois), 두충 (Eucommiae cortex), 황백 (Phellodendri cortex), 계피 (Cinnamomi cortex), 구기자 (Lycii fructus), 녹차 (Theae folium), 오가피 (Acanthodanacis cortex), 사간 (Beiamcanda chinensis), 고삼 (Sophorae radix) 및당귀 (Angelicae gigantis radix) 등은 0.02% 의 β-carotene 보다나은항산화력을나타 Table 1. Comparison of the AUC values of natural products analyzed by dissolved oxygen method No Natural products AUC No Natural products AUC 1 B H A 1656.80 18 Cinnamomi cortex 592.23 2 B H T 1516.58 19 Lycii fructus 568.86 3 Cimicifugae rhizoma 1045.24 20 Theae folium 557.93 4 Corni fructus 984.50 21 Acanthodanacis cortex 555.90 5 Geranii herba 941.58 22 Beiamcanda chinensis 539.44 6 Paeonia japonica 788.73 23 Sophorae radix 537.52 7 Asparagi tuber 787.92 24 Angelicae gigantis radix 532.32 8 Epimedii herba 783.20 25 Sophorae flos 509.67 9 Mori cortex radicis 723.28 26 Acori graminei rhizoma 497.97 10 Nelumbo nuclfera gaertner 695.86 27 Puerariae radix 495.21 11 Chelidonium majus L. 695.86 28 Astragali radix 492.97 12 Coptidis rhizoma 682.05 29 β-carotene 485.28 13 Atractylodis rhizoma alba 665.05 30 Schizandrae fructus 484.36 14 Aurantii nobilis pericarpium 658.23 31 Glycyrrhizae radix 450.27 15 Galla rhois 652.37 32 Scutellaria baicalensis george 429.41 16 Eucommiae cortex 640.47 33 Crataegi fructus 363.42 17 Phellodendri cortex 601.10
GROWTH PROMOTING EFFECTS BY NATURAL PRODUCTS 273 Table 2. Comparison of the eliminative activity of hydroxyl radical with natural products analyzed by TBA method. No Natural products Elimination (%) No Natural products Elimination (%) 1 0.02% BHA 91.86 18 Theae folium 68.21 2 0.02% BHT 91.23 19 Corni fructus 64.95 3 0.02% β-carotene 90.86 20 Puerariae radix 63.57 4 Schizandrae fructus 90.73 21 Asparagi tuber 62.45 5 Geranii herba 82.47 22 Cimicifugae rhizoma 61.95 6 Galla rhois 82.35 23 Lycii fructus 58.57 7 Phellodendri cortex 81.97 24 Astragali radix 56.94 8 Angelicae gigantis radix 81.72 25 Aurantii nobilis pericarpium 51.94 9 Scutellaria baicalensis george 79.47 26 Crataegi fructus 51.81 10 Paeonia japonica 77.47 27 Coptidis rhizoma 46.55 11 Epimedii herba 77.47 28 Cinnamomi cortex 45.18 12 Glycyrrhizae radix 75.97 29 Mori cortex radicis 40.67 13 Sophorae radix 75.97 30 Beiamcanda chinensis 39.54 14 Chelidonium majus l. 73.84 31 Acori graminei rhizoma 38.92 15 Nelumbo nuclfera gaertner 72.96 32 Eucommiae cortex 38.17 16 Acanthodanacis cortex 72.59 33 Atractylodis rhizoma alba 3.88 17 Sophorae radix 70.83 Table 3. Amounts of aromatic compounds of natural products measured at 280 nm with spectrophotometer. Natural products Absorbance at 280 nm Natural products Absorbance at 280 nm Natural products Absorbance at 280 nm Asparagi tuber 2.98 Sophorae radix 2.50 Angelicae gigantis radix 2.48 Chelidonium majus 2.80 Theae folium 2.50 Puerariae radix 2.48 Beiamcanda chinensis 2.78 Acanthodanacis cortex 2.50 Crataegi fructus 2.48 Glycyrrhizae radix 2.76 Cinnamomi cortex 2.49 Mori cortex radicis 2.48 Geranii herba 2.76 Epimedii herba 2.49 Nelumbo nuclfera gaertner 2.48 Astragali radix 2.76 Eucommiae cortex 2.49 Acori graminei rhizoma 2.48 Schizandrae fructus 2.76 Corni fructus 2.49 Paeonia japonica 2.47 Coptidis rhizoma 2.75 Phellodendri cortex 2.49 Aurantii nobilis pericarpium 2.46 Scutellaria baicalensis george 2.59 Cimicifugae rhizoma 2.48 Lycii fructus 2.41 Galla rhois 2.51 Sophorae flos 2.48 Atractylodis rhizoma alba 1.62 내었다. 그리고, 괴화 (Sophorae flos), 석창포 (Acori graminei rhizoma), 황기 (Astragali radix) 등은 β-carotene 의항산화력과비슷한효과를나타내었으며, 오미자 (Schizandrae fructus), 감초 (Glycyrrhizae radix), 갈근 (Puerariae radix), 황금 (Scutellaria baicalensis george) 및산사자 (Crataegi fructus) 등은 0.02% 의 β-carotene 보다약한항산화력을나타내었다. 천연산물중에서도항산화력이 0.02% 의 β- carotene 보다강한제제들이많아합성항산화제의대용뿐만아니라, ROS 제거를위한생체투여가가능한물질도많음을알수있었으며, 식품보존제, 항노화제제등의다방면으로사용이가능할것으로사료된다. Hydroxyl radical 소거능력의비교각각의천연산물의 hydroxyl radical 소거정도는각각의천연산물을 100 배희석하여 TBA 법으로써측정하였고그소거율을 Table 2 에나타내었다. 항산화능력을나타내고자하는 AUC 의서열과차이가있었으며, 이는 TBA 의방법으로는 free radical 의모두를측정할수없기때문으로사료된다. 총 phenol 함량및방향족의측정항산화력이높은것으로나타난 30 종류의천연산물의방향족화합물의함량을 Table 3 에나타내었으며, 총 phenol 함량은 Table 4 에나타내었다. 천연산물에따라방향족의함
274 Kim et al. Table 4. Amounts of total phenols of various natural products Natural products Amounts of total phenols(µg/100 g) Natural products Amounts of total phenols(µg/100 g) Natural products Amounts of total phenols(µg/100 g) Nelumbo nuclfera gaertner 20.33 Geranii herba 19.40 Galla rhois 19.22 Phellodendri cortex 20.22 Sophorae radix 19.36 Crataegi fructus 19.21 Corni fructus 19.97 Scutellaria baicalensis george 19.36 Lycii fructus 18.96 Puerariae radix 19.94 Theae folium 19.34 Eucommiae cortex 18.92 Angelicae gigantis radix 19.70 Chelidonium majus 19.33 Astragali radix 18.55 Paeonia japonica 19.68 Sophorae flos 19.32 Cinnamomi cortex 18.48 Asparagi tuber 19.61 Glycyrrhizae radix 19.30 Atractylodis rhizoma alba 18.38 Epimedii herba 19.58 Aurantii nobilis pericarpium 19.28 Schizandrae fructus 17.81 Cimicifugae rhizoma 19.49 Coptidis rhizoma 19.28 Mori cortex radicis 17.67 Beiamcanda chinensis 19.41 Acanthodanacis cortex 19.22 Acori graminei rhizoma 17.66 Fig. 2. The growth of L. acidophilus by addition of one kind of natural product alone, designated panel (a) to (d). Panel (a), :Paeonia japonica, :Atractylodis rhizoma alba, :Epimedii herba, :Acori graminei rhizoma, :control. Panel (b), :Aurantii nobilis pericarpium, :Angelicae gigantis radix, :control. Panel (c), :Acanthodanacis cortex, :Lycii fructus, :Cinnamomi cortex, :Sophorae flos, :control. Panel (d), :Phellodendri cortex, :Nelumbo nuclfera gaertner, :Asparagi tuber, :Mori cortex radicis, :control. 량과총 phenol 함량은일치하지않은부분이있었으며, 또한항산화력과도반드시일치하지않는것으로나타났다. 이것은방향족의화합물을가지고있다하더라도그구조상항산화력을나타낼수있는 phenolic acid 나 flavonoid 등의구조가아닌것으로사료되며이에따라항산화력도차이가나 는것으로사료된다. L. acidophilus 의성장촉진효과를갖는천연산물의조성이균주를 MRS Medium 에 30 종류의천연산물을첨가하여배양하였을때백작약, 백출, 음양곽, 석창포, 진피, 당귀,
GROWTH PROMOTING EFFECTS BY NATURAL PRODUCTS 275 오가피, 구기자, 계피, 괴화, 황백, 연자육, 천문동및상백피등을첨가한세균의성장정도가대조군보다성장이좋은것으로나타나, 이들천연산물은 L. acidophilus 균주의성장을촉진하는것으로판단되었고그결과를 Fig. 2 에나타내었다. L. acidophilus 균주의성장을촉진하는각각의천연산물들을장에대한기능은백작약은해독작용과통증완화작용을하며, 백출은소변이잘나오지않는것을치료하며, 음양곽은정자의생산, 건망증의해소, 소변이잘나오지않음을치료하고, 석창포는간, 심장, 비장, 폐, 신장의오장을도우고, 전간, 건망증을치료하며, 진피는음식을먹은후의체한것과, 또한위암을치료하고, 당귀는장에활력주며, 생리불순을치료하고, 오가피는사지불수및滋補肝腎작용을, 구기자는간과신장그리고益精을, 계피는혈액을잘돌게하며, 괴화는오장의기생충을죽이고, 痔血을치료하며, 황백은해독, 살충, 황달및여성의대하를치료하고, 연자육은비장의활성을도 우며, 장의활성을, 천문동은폐의기능을도우고, 소변이잘나오게하며, 그리고상백피는토하고설사하며위의경련에각각효과가있는것으로알려져있으며 [2,4,6,10,20,22], 이들의공통작용은신체의간, 심장, 비장, 폐신장의오장에작용하여그활력을도우는것이며상승시키는작용을한다. 따라서이들천연산물들이적어도장내유용세균의성장에도움을주며이에따라장의활성화에도기여하는것으로사료된다. 또한이러한천연산물은장내의 L. acidophilus 균주의성장을활성화하고, 이에따라오장의기능을상승시키는것으로보아도무방할것으로판단된다. 한가지의천연산물로써성장의촉진효과가있었던천연산물들을두종류씩조합한석창포와계피, 석창포와괴화, 석창포와황기, 석창포와승마, 진피와갈근, 진피와계피, 진피와녹차및갈근과계피등의두가지의천연산물의조합 Fig.3.The growth of L. acidophilus by addition of two combinations of natural products, designated panel (a) and (b). Panel (a), :combination of Acori graminei rhizoma and Cinnamomi cortex, :Acori graminei rhizoma and Sophorae flos, :Acori graminei rhizoma and Astragali radix, :Acori graminei rhizoma and Cimicifugae rhizoma, :control. Panel (b), :combination of Aurantii nobilis pericarpium and Puerariae radix, :Aurantii nobilis pericarpium and Cinnamomi cortex, :Aurantii nobilis pericarpium and Theae folium, :Puerariae radix and Cinnamomi cortex, :control. Fig. 4. The growth of L. acidophilus by addition of three combinations of natural products showed panel (a) and (b). Panel (a), :combination of Cimicifugae rhizoma, Cinnamomi cortex and Theae folium, :Cimicifugae rhizoma, Cinnamomi cortex and Glycyrrhizae radix, :Cimicifugae rhizoma, Astragali radix and Aurantii nobilis pericarpium, :control. Panel (b), :combination of Cimicifugae rhizoma, Aurantii nobilis and Theae folium, :Cimicifugae rhizoma, Cinnamomi cortex and Scutellaria baicalensis george, :Cimicifugae rhizoma, Acori graminei rhizoma and Aurantii nobilis pericarpium, :control.
276 Kim et al. Fig.5.The growth of L. acidophilus by addition of four combinations of natural products. :combination of Glycyrrhizae radix, Theae folium, Scutellaria baicalensis george and Cinnamomi cortex, : Cimicifugae rhizoma, Glycyrrhizae radix, Scutellaria baicalensis george and Cinnamomi cortex, : Cimicifugae rhizoma, Glycyrrhizae radix, Acori graminei rhizoma and Theae folium, : Glycyrrhizae radix, Scutellaria baicalensis george, Aurantii nobilis pericarpium and Cinnamomi cortex, :control. Fig. 6. Comparison of antioxidative capacity with AUC of four combinations of natural antioxidants used for culturing L. acidophilus. :combination of Glycyrrhizae radix, Theae folium, Scutellaria baicalensis george and Cinnamomi cortex (495.48) :Cimicifugae rhizoma, Glycyrrhizae radix, Acori graminei rhizoma and Theae folium(855.97), :Cimicifugae rhizoma, Glycyrrhizae radix, Scutellaria baicalensis george and Cinnamomi cortex(518.76), :Glycyrrhizae radix, Scutellaria baicalensis george, Aurantii nobilis pericarpium and Cinnamomi cortex(502.36), :control (116.61). Numerics in brackets are designated as AUC values of each combination. 에의한성장촉진효과는뚜렷하지않았으나 L. acidophilus 의성장이촉진되었으며, 그결과를 Fig. 3 나타내었다. 각천연산물은서로의상승효과및제어효과로작용하는것으로사료된다. 2 종류의천연산물의조합으로부터성장촉진효과가있는조합을근간으로 3 종류씩조합한승마와계피그리고녹차, 승마와계피그리고감초, 승마와황기그리고진피, 승마와진피그리고녹차, 승마와계피그리고황금, 승마와석창포그리고진피등의세가지의천연산물의배합에서 L. acidophilus 의성장이촉진되었으며결과를 Fig. 4 에나타내었다. 2 가지의조합보다는성장촉진효과가나은것은천연산물각각의고유의약리작용에따라상승및제어효과에기인되는것으로사료된다. 3 종류의천연산물의조합으로부터성장촉진효과가있는조합을근간으로 4 종류씩조합한감초, 녹차, 황금및계피의조합, 그리고승마, 감초, 황금및계피의조합, 승마, 감초, 석창포및녹차의조합, 그리고감초, 황금, 진피및계피의조합등의 4 가지의배합에서 L. acidophilus 의성장이현저히촉진되었으며, 이중감초, 녹차, 황금및계피의조합조성물이대조군보다 1.3 배의성장촉진효과를보였으며, 그결과를 Fig. 5 에나타내었다. 생체의기능을향상시켜야할부위에따라천연산물의조합을선택할수있을것으로사료되며많은다른분야로의응용을기대할수있을것으로사료된다. L. acidophilus 의성장촉진효과를갖는 4 가지조합의항산화력용존산소분석법에의한 4 차배합조성물의항산화력은승마, 감초, 석창포및녹차의조합이 855.97 로써가장강한것으로나타났고, 다음으로승마, 감초, 황금및계피의조 Fig.7.The ratio of OH radical elimnation by four combinations of natural antioxidants used for culturing L. acidophilus. A: Cimicifugae rhizoma, Glycyrrhizae radix, Acori graminei rhizoma and Theae folium, B: Cimicifugae rhizoma, Glycyrrhizae radix, Scutellaria baicalensis george and Cinnamomi cortex, C: Glycyrrhizae radix, Scutellaria baicalensis george, Aurantii nobilis pericarpium and Cinnamomi cortex, D: Glycyrrhizae radix, Theae folium, Scutellaria baicalensis george and Cinnamomi cortex.
GROWTH PROMOTING EFFECTS BY NATURAL PRODUCTS 277 Table 5. Amounts of aromatic compounds and total phenols of four combinations of natural products used for culturing L. acidophilus. Combinations Absorbance at 280 nm Amounts of total phenols (µg/100 g) Cimicifugae rhizoma, Glycyrrhizae radix, Acori graminei rhizoma and Theae folium 1.92 16.77 Cimicifugae rhizoma, Glycyrrhizae radix, Scutellaria baicalensis george and Cinnamomi cortex 1.93 17.08 Glycyrrhizae radix, Scutellaria baicalensis george, Aaurantii nobilis pericarpium and Cinnamomi cortex 1.95 17.49 Glycyrrhizae radix, Theae folium, Scutellaria baicalensis george and Cinnamomi cortex 1.95 16.05 Fig.8.The growth of L. plantarum by addition of one kind of natural product alone, designated panel (a) to (d). Panel (a), :Phellodendri cortex, :Nelumbo nuclfera gaertner, :Asparagi tuber, :Mori cortex radicis, :control. Panel (b), :Acanthodanacis cortex, :Lycii fructus, :Cinnamomi cortex, :Sophorae flos, :control. Panel (c), :Cimicifugae rhizoma, :Corni fructus, :Theae folium, :control. Panel (d), :Eucommiae cortex, :Galla rhois, :Sophorae radix, :Geranii herba, :control. 합 (518.76), 감초, 황금, 진피및계피의조합 (502.36), 감초, 녹차, 황금및계피의조합 (495.48) 의순서였으며 (Fig. 6), L. acidophilus 의성장촉진효과가가장나았던조합은감초, 녹차, 황금및계피의조합으로대조군의 AUC 116.61 보다 4.2 배정도항산화력이강한것으로나타났다. 항산화력이큰조합이라고해서세균의성장성장촉진효과와일치하는것은아닌것으로나타났으나조합의약리적효과에따라다양하게사용될수있을것으로사료된다. 또한, Hydroxyl radical 의강한독성의제거능력을 TBA 를사용하여측정하였다. 네가지조합의혼합액 1ml 를취하여 50, 100, 200 및 300 배로희석하여 hydroxyl radical 의소거활성을검토하였으며, 조합의특징에따라 50 배, 100 배, 200 배의희석액에서특징있는소거율을보였다 (Fig. 7). 승마, 감초, 석창포및녹차 (A) 의조합은다른조합보다전반적으로낮은소거율을보였고, 50 배의희석액에서 64.87% 의소거율을보였다. 다른 3 종류의 B, C 및 D 조합은 50, 100 및 200 배등의높은희석액에서도 70% 이상의높은소거율을보여기능성항산화차로서의사용가능성을나타내었다. 승마, 감초, 황금및계피의조합 (B), 그리고감초, 황금, 진피및계피의조합 (C) 은 50 배의희석액에서각각 85.8% 및 83.5% 의높은소거율을나타내었고, 감초, 녹차, 황금및계피의조합 (D) 은 200 배의희석액에서 84.5% 의소거율을보였다. 이것은천연산물의추출액이 hydroxyl radical 을소거하기위해서는일정량의희석이필요하며이것은적당한농도의물이존재하여야소거
278 Kim et al. Fig.9.The growth of L. plantarum by addition of two combinations of natural products described panel (a) to (d). Panel (a), :combination of Paeonia japonica and Corni fructus, :Paeonia japonica and Epimedii herba, :Paeonia japonica and Coptidis rhizoma, :Paeonia japonica and Schizandrae fructus. Panel (b), :combination of Schizandrae fructus and Glycyrrhizae radix, :Schizandrae fructus and Theae folium, :Glycyrrhizae radix and Theae folium, :control. Panel (c), :combination of Coptidis rhizoma and Schizandrae fructus, :Coptidis rhizoma and Glycyrrhizae radix, :Coptidis rhizoma and Theae folium, :control. Panel (d), :combination of Geranii herba and Schizandrae fructus, :Geranii herba and Glycyrrhizae radix, :Geranii herba and Theae folium, :control. 활성이증가하는것으로사료된다. 이처럼희석배수가높아음료등의제품화에적당할것으로판단된다. 그리고, 각조합에따른방향족의함량및총 phenol 함량을 Table 5 에나타내으며, 4 가지의조합에의한총 phenol 함량및방향족의량은한가지단독일때보다감소하는경향을나타내었으며, 이는 4 종류의천연산물이조합을이룸에따라각성분의희석률에기인되는것으로판단된다. L. plantarum 의성장촉진효과를갖는천연산물의조성이균주를 MRS Medium 에서천연산물을첨가하여 L. plantarum 을배양하였을때, 황백, 연자육, 천문동, 상백피, 오가피, 구기자, 계피, 괴화, 산수유및현초등은대조군보다성장이좋은것으로나타났으며, 이들천연산물은 L. plantarum 균주의성장을촉진하는것으로판단되었고그결과를 Fig. 8 에나타내었다. L. plantarum 균주의성장을촉진하는각각의천연산물들을장에대한기능을살펴보면황백, 연자육, 천문동, 상백피, 오가피, 구기자, 계피및괴화는 L. acidophilus 의효능에서살펴보았으며, 산수유는정액을고이게하고, 장과위의나쁜기운을제거하는역할을 하며, 현초는설사와이질을치료하는역할을하는것으로알려져있다 [2,4,6,10,20,22]. L. acidophilus 의활성을증가시키는천연산물들과는많은부분이중복되는것을볼수있으며, Lactobacillus sp. 는비슷한종류의천연산물에의하여성장이촉진되는것는흥미로운일이며, 장내세균의조절에유용하게응용될수있을것으로사료된다. 그리고, 한가지의천연산물로써성장의촉진효과가있었던천연산물들을두종류씩조합한백작약과산수유, 백작약과음양곽, 백작약과황련, 백작약과오미자, 오미자와감초, 오미자와녹차, 감초와녹차, 황련과오미자, 황련과감초, 황련과녹차, 현초와오미자, 현초와감초및현초와녹차등의조합은대조군보다성장이좋은것으로나타났으며, 이균주의성장정도를아래의 Fig. 9 에나타내었다. 또한, 두가지의천연산물조합을근간으로세가지종류의천연산물의조합을구성하였으며, 산수유와녹차및황련, 산수유와녹차및오미자, 산수유와녹차및백출, 산수유와녹차및백작약, 산수유와녹차및현초, 산수유와녹차및황금, 산수유와황련및오미자, 산수유와황련및백출, 산수유와황련및백작약, 산수유와황련및현초,
GROWTH PROMOTING EFFECTS BY NATURAL PRODUCTS 279 Fig. 10. The growth of L. plantarum by addition of three combinations of natural products showed panel (a) and (b). Panel (a), :combination of Corni fructus, Theae folium and Coptidis rhizoma, :Corni fructus, Theae folium and Schizandrae fructus, :Corni fructus, Theae folium and Atractylodis rhizoma alba, :Corni fructus, Theae folium and Paeonia japonica, :control. Panel (b), :combination of Corni fructus, Theae folium and Geranii herba, :Corni fructus, Theae folium and Baicalensis george, :Corni fructus, Coptidis rhizoma and Schizandrae fructus, :Corni fructus, Coptidis rhizoma and Atractylodis rhizoma alba, :control. 산수유와황련및황금, 산수유와오미자및백출, 산수유와오미자및백작약, 산수유와오미자및현초, 그리고산수유와백출및백작약등의조합은대조군보다성장이좋은것으로나타났으며, 이균주의성장정도를아래의 Fig. 10 에나타내었다. 그리고, 세가지의천연산물조합을근간으로네가지종류의천연산물의조합을구성하였으며, 산수유, 녹차, 황련및백작약의조합, 산수유, 녹차, 황련및오미자의조합, 산수유, 녹차, 황련및백출의조합, 산수유, 녹차, 백작약및오미자의조합, 산수유, 황련, 백작약및황금의조합, 산수유, 백작약, 현초및녹차의조합, 산수유, 백작약, 현초및황련의조합, 그리고산수유, 백작약, 현초및오미자등의조합은대조군보다성장이좋은것으로나타났으며, 이균주의성장정도를아래의 Fig. 11 에나타내었다. 성장의촉진효과가좋은산수유, 백작약, 현초및녹차의조합은대조군보 Fig. 11. The growth of L. plantarum by addition of four combinations of natural products described panel (a) and (b). Panel (a), :combination of Corni fructus, Theae folium, Coptidis rhizoma and Paeonia japonica, :Corni fructus, Theae folium, Coptidis rhizoma and Schizandrae fructus, :Corni fructus, Theae folium, Coptidis rhizoma and Atractylodis rhizoma, :Corni fructus, Coptidis rhizoma, Paeonia japonica and Schizandrae fructus, :control. Panel (b), :combination of Corni fructus, Coptidis rhizoma, Paeonia japonica and Scutellaria baicalensis, :Corni fructus, Paeonia japonica, Geranii herba and Theae folium, :Corni fructus, Paeonia japonica, Geranii herba and Coptidis rhizoma, :Corni fructus, Paeonia japonica, Geranii herba and Schizandrae fructus, :control. 다 1.6 배의성장촉진효과를보였다. 정자를고이게하고장과위의나쁜기운을치료하는산수유, 불필요한생체의열을식히며, 정신을맑게하는녹차, 그리고해독및통증을가라앉히고혈액의생성을돕는역할을하는백작약 [2,4,6,10,20,22] 등의천연산물조합에서성장촉진효과가현저함을보여그응용가능성을보인것으로사료된다. L. plantarum 의성장촉진효과를갖는 4 가지조합의항산화력용존산소분석법에의한 4 차배합조성물의항산화력은산수유, 백작약, 현초및황련의조합이 650.2 로써가장높
280 Kim et al. Fig. 13. The ratio of OH radical elimnation by four combinations of natural antioxidants used for coulturing L. plantarum. A:combination of Corni fructus, Paeonia japonica, Geranii herba and Coptidis rhizoma, B:combination of Corni fructus, Paeonia japonica, Geranii herba and Theae folium, C:combination of Corni fructus, Paeonia japonica, Geranii herba and Schizandrae fructus, D:combination of Corni fructus, Theae folium, Coptidis rhizoma and Paeonia japonica. Fig. 12. Comparison of antioxidative capacity with AUC of four combinations of natural antioxidants used for coulturing L. plantarum described panel (a) and (b). Panel (a), :combination of Corni fructus, Theae folium, Coptidis rhizoma and Schizandrae fructus(477.2), :Corni fructus, Theae folium, Coptidis rhizoma and Atractylodis rhizoma alba(522.3), :Corni fructus, Theae folium, Paeonia japonica and Schizandrae fructus(503.8), :Corni fructus, Theae folium, Coptidis rhizoma and Paeonia japonica(553.3), :control (116.61). Panel (b), :combination of Corni fructus, Paeonia japonica, Geranii herba and Schizandrae fructus(600.70), :Corni fructus, Coptidis rhizoma, Paeonia japonica and Scutellaria baicalensis george(521.15), :Corni fructus, Paeonia japonica, Geranii herba and Theae folium(620.19), :Corni fructus, Paeonia japonica, Geranii herba and Coptidis rhizoma(650.2), :control (116.61). Numerics in brackets are designated as AUC values of the each combination. 게나타났고, 다음으로는산수유, 백작약, 현초및녹차의조합 (620.19), 산수유, 백작약, 현초및오미자의조합 (600.70), 산수유, 녹차, 황련및백작약의조합 (553.3), 산수유, 녹차, 황련및백출의조합 (522.3), 산수유, 황련, 백작약및황금의조합 (521.15), 산수유, 녹차, 백작약및오미자의조합 (503.3), 그리고산수유, 녹차, 황련및오미자의조합 (477.2) 의순서로나타났으며 (Fig. 12), 산수유, 백작약, 현초및녹차의조합 (620.19), 산수유, 녹차, 황련및오미자의조합 (477.2), 그리고산수유, 녹차, 황련및백출의조합 (522.3) 이 L. plantarum 의성장을현저하게촉진시켰으며, 이들의항산화력은대조군의 AUC 116.61 보다 4.1~5.3 배정도강한것으로나타났고, 항산화력과성장촉진효과는일치하지는않았으며, 세균에대한작용의다양함을보였다. 각조합을 50, 100, 200 및 300 배로희석하여 hydroxyl radical 의소거활성을검토하였으며, 4 가지의조합은모두 50 배, 100 배및 200 배의희석액에서 75% 이상의높은소거율을보였으며 (Fig. 13), 산수유, 백작약, 현초및오미자 (C) 의조합은 200 배의희석액에서 91.62% 의소거율을나타내어아주가능성있는조합으로보여진다. 산수유, 백작약, 현초및황련 (A) 의조합과산수유, 백작약, 현초및녹차 (B) 의조합은 50 배의희석액에서각각 86.25% 및 81.87% 의높은소거율을보였고, 산수유, 녹차황련및백작약 (D) 의조합은 100, 200 및 300 배의희석액에서다른조합에서보다는비교적낮은소거율을보였으나 77% 이상의제거효율을보여 L. plantarum 을성장시키는시킴과동시에 free radical Table 6. Amounts of aromatic compounds and total phenols of four combination of natural products used for culturing L. plantarum. Combination Absorbance at 280 nm Amounts of total phenols (µg/100 g) Corni fructus, Peonia japonica, Geranii herba and Coptidis rhizoma 1.86 16.26 Corni fructus, Paeonia japonica, Geranii herba and Theae folium 1.85 15.53 Corni fructus, Paeonia japonica, Geranii herba and Schizandrae fructus 1.89 16.15 Corni fructus, Theae folium, Coptidis rhizoma and Paeonia japonica 1.82 15.64
GROWTH PROMOTING EFFECTS BY NATURAL PRODUCTS 281 을제거할수있는좋은조합으로사료된다. 각조합에따른방향족의함량및총 phenol 함량을 Table 6 에나타내으며, 4 가지의조합에의한총 phenol 함량및방향족은 L. acidophilus 와같은경향을나타내었다. 요 장내유용세균인 L. acidophilus 및 L. plantarum 은선택되어진천연산물의한종류단독으로서도촉진되었으며, 또한두종류, 세종류및네종류의배합에의하여특징적으로성장이촉진되었으며, L. acidophilus 는이들천연산물의배합중에서한종류의천연산물로서는백작약, 석창포, 진피등이좋은효과를보였고, 두종류천연산물의배합에서는석창포와계피, 진피와갈근등의조합이, 세종류의천연산물배합에서는승마와계피및감초등의조합이, 네종류의천연산물배합에서는감초, 녹차, 황금및계피등의조합에서잘성장함을보였으며, 네종류의조합에의한세균의성장촉진효과는대조군보다는 1.3 배이상의효과를보였으며이때의항산화력은대조군보다도 4 배이상의강함을보였고, hydroxyl radical 의소거활성은천연산물을 100 배희석하였을때 70% 이상의소거능력을보였다. 또한, L. plantarum 은한종류의천연산물로서는산수유, 연자육, 괴화등이좋은효과를보였고, 두종류의천연산물의배합에서는오미자와녹차, 백작약과음양곽, 현초와녹차등의조합이, 세종류의천연산물배합에서는산수유와녹차및백작약, 산수유와황련및오미자, 산수유와오미자및백작약등의조합이, 4 종류의천연산물배합에서는산수유, 녹차, 황련및황금의조합, 산수유, 오미자, 백작약및녹차의조합, 산수유, 녹차, 황련및현초등의조합에서 L. plantarum 가잘성장함을보였고, 이네종류의조합에의한세균의성장촉진효과는대조군보다 1.5 배이상의효과를보였고, 항산화력은 4~5 배이상으로나타났으며, hydroxyl radical 의소거활성은천연산물을 100 배희석하였을때 75% 이상의소거능력을보였다. 이러한천연산물의배합으로생체의특정부위를활성화시킬수있고, 항산화력으로유해산소를제거할수있으며, 천연산물고유의약성을활용할수있고, 여러가지의유용세균의성장촉진효과를기대할수있어생체에긍정적인효과를제공할수있을것으로사료되며, 기능성항산화차등의건강식품의원료로사용할수있을것으로판단된다. 약 감사의말 본연구는농림기술연구센터에서지원한농림기술개발과제 ( 관리번호 200005-2) 결과의일부이며, 이에감사합니다. REFERENCES 1. Ahrné, S., S. Nobaek, B. Jeppsson, and I. Adlerberth. 1998. The normal Lactobacillus flora of healthy human rectal and oral mucosa. Journal of Applied Microbiology. 85: 88-94. 2. Gan, K. K. 1982. Zugai ZouyouKanbouSyohou, pp. 15-190. Yakukyosihosya, Japan.18 3. Gusils, C., A. P. Chaia, S. Gonzalez, and G. Oliver. 1999. Lactobacilli isolated from chicken intestines: tial use as probiotics. J Food Prot. 62: 252-256.18 4. Huh, J. 1999. Dongeuibogam, pp. 21-164. Bobinmunwhasa, Korea. 5. Kawagan, S. 1996. Protocol for control of body functional material in food, pp. 8-15, Kakuen press 18 center, Japan. 6. Kawagdo, Y. 1989. Proof of Pulse-Bangyakhappyeon, pp.15-70. Namsandang, Korea. 7. Kogukuchi, N. 1999. Protocol for free radical experiments, pp. 40-45. suiyoonsa. Japan. 8. Li, H.C., S. Yashiki, J. Sonoda, H. Lou, S. K. Ghosh, J. J. Byrnes, C. Lema, T. Fujiyoshi, M. Karasuyama, and S. Sonoda. 2000. Green tea polyphenols induce apoptosis in vitro in peripheral blood T lymphocytes of adult T-cell leukemia patients. Jpn. J. Cancer Res. 91:34-40. 9. Lunec, J. 1995. Oxygen Radical Activity-Detection and Measurment in vivo, pp. 3679-3688, In Townshend, A. (ed.), Encyclopedia of Analytical Science, vol. 6, Academic Press, U.S. 10. Mori, Y. Z. 1986. KanbouSyohounoKouseitoTekiyou, pp. 20-140. Hakubustusyokan, Japan. 11. Naidu, A. S., W. R. Bidlack, and R. A. Clemens. 1999. Probiotic spectra of lactic acid bacteria (LAB). Rev Food Sci Nutr. 39:13-126. 12. Nakada, C. 1997. Talk for Free Radical, pp. 56-102. Koudansya. Japan. 13. Nemcova, R. 1997. Criteria for selection of lactobacilli for probiotic use. Vet Med (Praha). 42:19-27. 14. Outa, S. 1987. Food and Antioxidant, pp 1-38. Syokuhinzairyokenkyukai, Japan. 15. Peter T.P. 1998. The skin's Antioxidant Systems. Dermatology nursing. 10: 401-406. 16. Saavedra, J. M. 1999. Probiotics plus antibiotics. The Journal of Pediatrics.135: 535-537. 17. Serafini, M., A. Ghiselli, and A. Ferro-Luzzi. 1994. Red wine, tea, and antioxidants. The Lancet. 344:626-629. 18. Torizaki, K. 1995. Protocol for lipid peroxide and free radical, pp. 145-147. Kakuen press center, Japan. 19. Vanderhoof, J. A. and R. J. Young. 1998. Use of probiotics in childhood gastrointestinal disorders. J Pediatr.Gastroenterol Nutr. 27:323-332. 20. Yeom, T.H. and S. S. Park. 1975. Lecture of modern Hanbang, pp.101-300, Haenglimseowon. 21. Yosikawa, M., W. Kawano, and I. Yano. 2000. All of reactive Oxygen and Free Radical, pp.8-75. Marusen L.T.D., Japan.18 22. Yuk, C. S. and H. S. Yang. 1999. Hyundai Saengyakhak, pp.133-581, Hakchangsa. 23. Zoubi, H. 1998. Methods for not to lose against free radicals, pp. 13-52. Noubunkyo, Japan. (Received May 30, 2002/Accepted Aug. 19, 2002)