초록 ▼

○ 연구결과
- 산초나무와 초피나무 부위별 추출물과 분획물을 조제하여 유효생리활성 분획을 확인
- 유효생리활성분획으로부터 생리활성물질로서 17종의 화합물을 분리, 구조 결정
- 산초와 초피나무 부위별 추출물의 항산화, 항당뇨, 항혈전, 항염증 효능이 in vitro로 검색되었고, in vivo 동물실험에서 산초잎 추출물의 효능이 확인되었으며 1일 유효 안전 섭취수준은 1차 알콜 추출물은 1-2 g, 이차 용매분획물은 0.4-0.5 g으로 산정
- 산초잎 추출물과 초피정유를 0.25-1.00 ppm 함유시킨 꽁치

○ 연구결과
- 산초나무와 초피나무 부위별 추출물과 분획물을 조제하여 유효생리활성 분획을 확인
- 유효생리활성분획으로부터 생리활성물질로서 17종의 화합물을 분리, 구조 결정
- 산초와 초피나무 부위별 추출물의 항산화, 항당뇨, 항혈전, 항염증 효능이 in vitro로 검색되었고, in vivo 동물실험에서 산초잎 추출물의 효능이 확인되었으며 1일 유효 안전 섭취수준은 1차 알콜 추출물은 1-2 g, 이차 용매분획물은 0.4-0.5 g으로 산정
- 산초잎 추출물과 초피정유를 0.25-1.00 ppm 함유시킨 꽁치과메기의 품질 보존성 향상
- 유효분획 또는 정제물질을 이용한 항미생물활성 효능 검정 및 각 병원성 미생물에 대한 MIC 결정
- Salmonella typhimurium TA 100, TA 1538 mutant strains을 이용하여 돌연변이 활성을 검정완료
- 세포독성 유효 성분의 분자, 세포생물학적 작용기전 규명 완료
- 신기능성 성분의 실용화를 위하여 초피 정유성분 함유 치약의 제조, 생물 농약제조, 항균화장품 이용가능성 검토
- 국내산의 우수성 확보

Abstract ▼

Ⅳ. RESULTS AND APPLICATIONS
Results
1st Sub-project: Functionality and safety of the extracts and isolated compounds from various parts of Zanthoxylum schinifolium and Zanthoxylum piperitum
1. Physiological activities of the extracts from Z. schinifolium and Z. piperitum
a. Antioxidant a

Ⅳ. RESULTS AND APPLICATIONS
Results
1st Sub-project: Functionality and safety of the extracts and isolated compounds from various parts of Zanthoxylum schinifolium and Zanthoxylum piperitum
1. Physiological activities of the extracts from Z. schinifolium and Z. piperitum
a. Antioxidant activities measured by microsome lipid oxidation and DPPH radical scavenging activity were high in the order of leaf > stem > root of the two plants extracted with methanol, methylene chloride and ethyl acetate and the activities were dose dependent at the concentrations from 0.125 to 5 mg/mL. When the initial ethanol extract of Z. schinifolium leaves and the methylene chloride and n-butanol fractions by second extraction were fed rats on high fat diets at the levels of 0.25 to 2 %. Hepatic levels of lipid peroxide, lipofuscin and carbonyl values were decreased while activities of glutathione peroxidase (GSH-Px) and paraxoanase activities were increased. Activities of the GSH-Px and superoxide dismutase were also increased in red blood cells.
b. Anti-thrombotic activity measured in vitro by activated partial thromboplastin times were highest in n-butanol fractions obtained from leaves of the two plants. This was confirmed in vivo with n-butanol fraction of Z. schinifolium leaves at the level of 0.5 % in diet.
c. Anti-inflammatory activities measured in vitro by measuring soybean lipoxygenase activity were high in ethyl acetate and n-butanol fractions of the two plants. This was confirmed in vivo by 5'-lipoxygenase activities and leukotriene B4 contents of polynuclear white blood cells of rats fed methylene chloride and n-butanol fractions of Z. schinifolium leaves.
d. Anti-diabetic activities measured in vitro by inhibition of α-glucosidase were shown in 22 subfractions of n-butanol extracts of Z. schinifolium leaves which had no antioxidant activities.
e. Improvement of body lipid status were shown in rats fed the ethanol extract of Z. schinifolium leaves at the level of 0.5∼1.0 % via reducing serum total cholesterol and triglyceride and increasing HDL-cholesterol, resulting in lowering atherogenic indice.
f. Serum GOT and GPT levels were not increased in rats fed the ethanol extract of Z. schinifolium leaves up to 2.0 % in diet, while glutathione S-transferase ctivity and GSH/GSSG ratio, the detoxification indice were normalized in the extract fed rats, suggesting that the extract was safe at the level used in the present feeding study.
2. Quality improvement of Coloabis saira kwamaegi by adding the components obtained from Z. schinifolium and Z. piperitum
a. Peroxide, TBA and acid values were decreased as well as diemethyamine and trimethylamine contents of kwamaegi prepared with the ethanol extract of Z. schinifolium leaves or the essential oil obtained from pericarp of Z. piperitum at the level of 0.125∼2 ppm during storage up to 7 days at 4 ℃
b. Sensory evaluation of the kwamaegi exhibitied a slightly higher preference with 0.25∼0.5 ppm treated with the two types of test materials.
2nd Sub-project: Separation and structure identification of bio-active substances from Zanthoxylum schinifolium and Zanthoxylum piperitum
1. We used cut and dried roots and stems of Z. schinifolium and Z. piperitum collected at Palgong Mt. in Gyeongsang-bukdo in 2003. We used freeze-dried leaves and dried fruits of Z. schinifolium and Z. piperitum bought at Dong-A Plant in Gyeongsang-bukdo.
The roots, stems, leaves, pericarp and seeds of Z. schinifolium and Z. piperitum were each extracted with methanol, methanol, 80 % methanol, methanol and methanol, and subsequently fractionated into four parts respectively; methylene chloride, ethyl acetate, n-butyl alcohol and water fractions. These extracts were examined to determine diverse activity.
2. For anti-microbial experiment, we refluxed leaves of Z. schinifolium and Z. piperitum with 100 % and 95 % methanol at room temperature and 60 ℃, respectively. And the pericarp of Z. piperitum and powdered seeds of Z. piperitum were extracted with hexane, methylene chloride, ethyl acetate and methanol at 25 ℃ and 30 ℃ respectively.
3. For comparative study of pericarp of Chinese and Korean Z. piperitum, we bought Chinese and Korean Z. piperitum at Herb market (Yang-nyung-si) in Daegu. The powdered pericarp of Z. piperitum were extracted with methanol, and subsequently fractionated into four parts; methylene chloride, ethyl acetate, n-butyl alcohol and water fractions. These extracts were used to determine diverse activity. The Z. schinifolium was not studied comparatively because of its unavailability.
4. Freeze-dried and powdered leaves of Z. schinifolium collected in June 2004, were extracted with 80 % methanol, and subsequently fractionated into four parts; methylene chloride, ethyl acetate, n-butyl alcohol and water fractions. The methylene chloride and n-butyl alcohol fractions were offered to 1st and 3rd sub-projects to do animal and cytotoxicity tests etc.
5. For anti-microbial test, we extracted pericarp and seeds of Z. piperitum with hexane, methylene chloride, ethyl acetate and methanol at 25 ℃ (pericarp) and 30 ℃ (seeds) and concentrated. The pericarp of Z. schinifolium and pericarp and seeds of Z. piperitum were also steam-distillated.
6. From the flash column chromatography of n-butanol fraction of Z. schinifolium leaves, 23 fractions (SL-MC-A to SL-MC-W) were obtained. SL-Bu-O, -U and -C having strong DPPH radical scavenging activity were subjected to SiO₂, RP C-18 and Sephadex LH-20 column chromatographies to give compounds 1∼5. From the flash column chromatography of methylene chloride fraction with cytotoxicity, 23 fractions, from SL-MC-A to SL-MC-W, were given.
By the activity-guided isolation of SL-MC-O, -R and -S, we isolated compounds 6∼17. Their structures were identified by physical and spectroscopic data such as UV, IR, 1H-NMR and 13C-NMR (1D and 2D) and Mass spectrum.
7. Compounds 1∼14 were identified as 4-[(1E)-3-hydroxy-1-propenyl]-2,6-dimethoxyphenol 1-O-β-D-glucopyranoside (syringin, 1), 2-propyl alcohol β-Dglucopyranoside (2), (6S,7E,9S)-6,9-dihydroxy-4,7-megastigmadien-3-one-9-O-β-dglucopyranoside (vomifoliol 9-O-β-D-glucopyranoside, 3), (3R,6E)-3,7-dimethyl-3, 8-dihydroxy-oct-1,6-dien-8-O-β-D-glucopyranoside (4), (3R,6E)-3,7-dimethyl-3,8-dihydroxyoct-1,6-dien-3-O-β-D-glucopyranoside (cnidioside C, 5), 20(29)-lupen-3-ol (lupeol, 6), 20(29)-lupen-3-one (lupeone, 7), bergapten (8), β-sitosterol (9),7-[(3,7-Dimethyl-2,6-octadienyl)oxy]-coumarin (Auraptene, 10), 6,7-Dimethoxy-2Hnaphthalen-1-one (11), 4-[3-(3-Hydroxy-but-2-enyloxy)-propenyl]-phenol (12), 4-[3-(3-Hydroxy-but-2-enyloxy)-propyl]-phenol (13), 2-Hydroxy-1-(2-methoxyphenyl)-dec-1-en-3-one (14), respectively.
3rd Sub-project: Screening of novel bioactive substances from the Zanthoxylum schinifolium and Zanthoxylum piperitum DC, determination of the stability of the bio-active substances, and industrial applications
1. Effect of Z. schinifolium and Z. piperitum DC on the fermentation of Kimchi
a. We intended to find the advantages of adding the bioactive compounds from Z. schinifolium and Z. piperitum DC for the fermentation of Kimchi that holds an important position in Korean diet. The growth of most bacteria as well as E. coli was inhibited by adding the leaves of Z. schinifolium. The leaves of Z. piperitum DC enhanced the growth of lactic acid bacteria, and this result was consistent with the sensory test, in that the Kimchi containing the leaves of Z. piperitum was more sour than the control. The leaves of Z. schinifolium, however, possessed no effect on the growth of most bacteria, but they enhanced the growth of lactic acid bacteria.
b. Effect of the methanol or ethanol extracts from leaves of Z. schinifolium and Z. piperitum DC on the growth of Leuconostoc mesenteriodes and Lactobacillus plantarum was investigated. In the presence of the methanol or ethanol extracts from leaves of Z. piperitum DC, the growth of L. mesenteriodes and L. plantarum were enhanced than the control. Although the addition of the methanol or ethanol extracts from leaves of Z. schinifolium also enhanced the growth of the lactic acid bacteria, the addition of leaves powder (0.2 %) reduced the growth of Lactobacillus plantarum.
2. Chracterization of antibiotic substances as chemical ingredients of Z. schinifolium and Z. piperitum
a. To isolate anti-microbial ingredients in Z. schinifolium and Z. piperitum DC, the leaves, stems, roots, seeds, or pericarp were extracted with methanol, methylene chloride, ethyl acetate and n-butanol. The anti-microbial activity of individual extracts were tested against several pathogenic microorganisms, including A. hydrophila, B. pumilus, P. vulgaris, S. aureus, S. epidermis, and E. coli O-157. All the extracts from Z. schinifolium did not show antimicrobial activity, whereas the solvent extracts of the seeds of Z. piperitum DC showed anti-microbial activities. In particular, the methylene chloride extract of the seeds of Z. piperitum DC exhibited an inhibitory activity against E. coli O-157.
b. Anti-microbial activities of the essential oil of Z. schinifolium and Z. piperitum DC were investigated against human pathogenic bacteria-Proteus vulgaris, S. aureus, S. epidermis, E. coli O-157 and H. pylori. The essential oil from the seeds of Z. piperitum DC were more bacteriocidal than those of the pericarp, and the minium inhibitory concentrations (MIC) appeared to be from 125 μg/ml to 3 mg/ml. The anti-microbial activities of seeds and pericarp of Z. schinifolium were weaker than those of Z. piperitum DC. This result confirmed that the essential oils from the seeds of Z. piperitum DC had the most potent antibacterial activity against human infectious disease bacteria.
c. Chemical components of the essential oil from seeds and pericarp of Z. piperitum DC, which appeared to possess the strong anti-bacterial activity, were analyzed by GC-MS. The compositions and quantities of main constituents in the two samples were different. The most abundant compounds in the seeds were γ -terpinene (19.1 %), citronellal (12.2 %), lavandulyl acetate (11.1 %), 1-β-pines (7.5 %) and citronellyl acetate (5.0 %), corresponding to almost 67.3 % of all the volatile compounds. On the contrary, γ-terpine (42.2 %), geranyl acetone (14.4 %) and citronellal (10.7 %) were detected as the main constituents in the pericarp.
d. The essential oil from the seeds, which appeared to possess more potent anti-microbial activity than those of pericarp, were fractionated by Thin Layer Chromatography (TLC), and the components in the individual spots were eluted, and assessed for anti-microbial activity. Several spots on TLC exhibited antimicrobial activity, indicating that the anti-microbial activity of the essential oil was not due to a single component, but due to the combined action of several anti-microbial components.
e. The essential oil from the seeds of Z. piperitum DC, which showed the strong anti-bacterial activity against S. mutans, have been examined whether they can be applicable to development of a functional toothpaste possessing antiplaque activity. We have had a toothpaste company make the prototypes of the toothpaste containing the essential oil, and have compared the antibacterial effect of the prototypes with those of three different toothpaste on the market. The anti-microbial activity of the prototype appeared to be stronger than any other toothpaste tested.
f. The essential oil from the seeds of Z. piperitum DC also showed a potent suppressive effect on the growth of Candida albicans.
g. The mutagenic activity from Z. piperitum DC was investigated. The essential oil from the seeds and the pericarp of Z. piperitum DC suppressed the mutagenic activity against S. typhimurium mutant strains, S. typhimurium TA 100 and S. typhimurium TA 1538, at the levels of 88 % to 99 %.
3. Screening of biological active components from Z. schinifolium and Z. piperitum DC and test for their physiological activity
a. To examine antitumor activity of Z. schinifolium or Z. piperitum DC, the leaves, stems, roots, seeds, or pericarp of individual plants were extracted with methanol, methylene chloride, ethyl acetate, and n-butanol. The individual extracts were tested against human acute leukemia Jurkat T cells in order to investigate the antitumor activity. Among these solvent extracts, the methylene chloride extract from the leaves of Z. schinifolium possessed the strongest cytotoxicity toward Jurkat T cells, and the methylene chloride extracts from the stems and seeds of Z. schinifolium appeared to be the next leading samples and showed similar cytotoxicity. The methylene chloride extracts from the roots and pericarps exhibited barely detectable levels of cytotoxicity toward Jurkat T cells.
b. The antitumor activity of the methylene chloride extract of the leaves of Z. schinifolium was also tested against human breast cancer cell line MDA361 (estrogen receptor positive) and MDA438 (estrogen receptor negative). The methylene chloride extract showed more potent cytotoxicity in MDA438 than in MDA361.
c. Antitumor activity of the methylene chloride extract from the leaves of Z. schinifolium appeared to be associated with the apoptogenic activity mediated by mitochondrial cytochrome c release and subsequent activation of caspase cascade including caspase-9 and caspase-3, leading to PARP degradation. The induced apoptosis was negatively regulated by anti-apoptotic protein Bcl-xL.
d. The major components of the methylene chloride extract from the leaves of Z. schinifolium were 9,19-cyclolanost-24-en-3-ol (17.8 %), 2-α-methyl-17, β-hop- 21-ene (17.8 %), 15-methyl-2,3-dihydro-1H benzazepin (14.0 %), phytol (12.2 %), lupeol (11.7 %), 12-methyl-benzofuran (10.0 %), 9,12-octadecadienoic acid (6.7 %), 9,12,15-octadeca-trienoic acid-methylester (4.2 %), 15-methyl-4-(1-methylethylidene)-2-(4-nitrophenyl) (4.0 %), hexadecanoic acid (2.3 %), vitamine E (2.2 %), β-amyrin (1.1 %), auraptene (1.0 %).
e. To purify apoptogenic ingredient of the methylene chloride extract from the leaves of Z. schinifolium, the methylene chloride extract was further fractionated by silica gel column chromatography. An apoptogeneic component isolated from the methylene chloride extract turned out to be auraptene, and its IC50 value against Jurkat T cells was 16.5 μg/mL.
f. Apoptosis induced by auraptene (10∼20 μg/mL) appeared to be provoked by ER-stress-mediated activation of caspase-12 and caspase-8, Bid cleavage, and then excuted by mitoch-ondria cytochrome c-dependent or mitochondria-independent caspase cascade, and PARP degradation, which led to apoptotic DNA fragmentation in a dose-dependent fashion.
g. Auraptene-mediated apoptosis was completely inhibited by overexpression of anti-apoptotic protein Bcl-xL, pan-caspase inhibitor z-VAD-fmk, or caspase-8 inhibitor z-IETD-fmk. On the contrary, the apoptosis was not inhibited by concomitant treatment with the calpain inhibitor (E64d), the JNK inhibitor (SP600125), and the mitochondrial permeability transition pore inhibitor (cyclosporin A, CsA) or by alone. In the presence of caspase-8 inhibitor (z-IETC-fmk), all of the auraptene-induced apoptotic events except caspase-12 activation were abrogated suggesting that caspase-12 activation was upstream of caspase-8 activation.
h. A human peripheral T cells and activated T cells were less sensitive to the cytotoxic activity of auraptene than was human Jurkat T cells. These results demonstrate that auraptene from the leaves of Z. schinifolium can be applicable as antitumor agent.
i. When the effect of the individual extracts from Z. schinifolium and Z. piperitum DC on human Jurkat T cells, the ethyl acetate extract (SL-15) from the leaves of Z. schinifolium and n-butanol extract (SL-16) from the leaves of Z. schinifolium promoted the cell viability measured by MTT assay. Since both SL-15 and SL-16 appeared to possess potent antioxidant activities, the stimulatory effect of SL-15 and SL-16 on the cell viability was attributable to their antioxidant activities.
Applications
1. The ethanol extract of Z. schinifolium leaves can be utilized as a component of multifunctional foods for preventing peroxidation, inflammation and thrombosis in the body at the level of 1∼2 g per day while its secondary methylene chloride or n-butanol fraction for a function-specific food component at the level of 0.4∼0.5 g per day.
2. The ethanol extract of Z. schinifolium leaves and the essential oil obtained from pericarp of Z. piperitum can be utilized as a natural preservative for improving quality of fish and meat products and other parts of the two plant are expected to be.
3. In collaboration with a toothpaste company, we were able to make a prototype of the functional toothpaste using the essential oil from pericarp of Z. piperitum DC, which appeared to have the strongest antibacterial activity against S. mutans. When the antibacterial activity of the prototypes was compared with those of three commercially available toothpaste, the antibacterial activity of our prototype exhibited the strongest activity. One of the commercially available toothpaste was known to contain anti-microbial extracts of green tea. These results indicate that the essential oils from seeds of Z. piperitum DC can be utilized for manufacturing a new functional toothpaste, which is adequate for oral hygiene of immunocompromized patients such as transplant patients or cancer patients.
4. Antibacterial substances of the essential oil from Z. piperitum DC appeared to possess wide-range antibiotic activities from gram-negative bacteria to gram-positive bacteria. These antibacterial substances can be used as pro-environmental biological pesticides which can prevent the growth of phytopathogenic bacteria in the growing fruits and vegetables.
5. The essential oil from seeds and pericarp of Z. piperitum DC can also be utilized in the field of bio-industry to manufacture a plastic wrap possessing anti-microbial activity, or functional soap with anti-microbial activity.
6. The methylene chloride extract of the leaves of Z. schinifolium DC, and the auraptene appeared to have a strong potential to be applicable as antitumor agent.

목차 Contents

• 제출문 ... 1
• 요약문 ... 2
• SUMMARY ... 19
• CONTENTS ... 31
• 목차 ... 37
• 제1장 연구개발과제의 개요 ... 44
• 1. 기술적 측면 ... 46
• 2. 경제·산업적 측면 ... 47
• 3. 사회·문화적 측면 ... 49
• 제2장 국내외 기술개발 현황 ... 50
• 제3장 연구개발 수행 내용 및 결과 ... 52
• 1절. 동물실험을 통한 생리활성 물질의 효능 및 작용기작 규명 (제1세부과제) ... 52
• 1. 산초 및 초피나무 추출물의 만성질환 예방 생리활성 및 안전성 조사 ... 52
• 가. 연구배경 ... 52
• 나. 연구방법 ... 52
• 1) 산초 및 초피나무 추출물의 in vitro 및 in vivo 항산화 작용 측정 ... 52
• 2) In vitro 및 in vivo 항혈전, 항염증 및 항당뇨 활성 측정 ... 53
• 3) 동물실험 사육 및 식이 실험 (in vivo) ... 53
• 가) 동물 사육 및 식이 ... 53
• (1) 1차 동물실험 ... 53
• (2) 2차 동물실험 ... 53
• 나) 분석시료의 준비 ... 55
• 다) 항산화 효소 활성 측정 ... 55
• 라) Oxidative stress 완화 및 free radical scavenging 작용 ... 55
• 마) 지질대사 개선 효과 측정 ... 55
• 바) 해독 및 안전성 지표측정 ... 55
• 4) 통계처리 ... 56
• 다. 연구결과 ... 56
• 1) 산초나무와 초피나무 추출물의 in vitro 생리활성 검색 ... 56
• 가) 항산화 활성 ... 56
• 나) 항혈전 활성 : Activated partial thromboplastin times (APTT) ... 59
• 다) 항염증 활성 ... 61
• 라) 항당뇨 활성 ... 62
• 2) 동물실험에서의 산초 잎 추출물의 효능 및 안전성 규명 (in vivo) ... 65
• 가) 항산화 활성 ... 65
• (1) 간조직과 적혈구의 SOD 및 Catalase 활성 ... 65
• (2) 간조직의 Catalase 활성 ... 66
• (3) 간조직의 PON 활성 및 arylesterase 활성 ... 68
• (4) 간조직과 적혈구 과산화지질 함량 ... 69
• (5) Lipofuscin 함량 ... 70
• (6) 카보닐가 ... 70
• 나) 항혈전 효능 : 혈장 APTT 및 Thrombin times (TT) ... 71
• 다) 항염증 효능 : 다형핵성 백혈구 5‘-lipoxygenase 활성과 leukotriene B4 ... 72
• 라) Oxidative stress 완화 및 free radical scavenging 작용 ... 74
• (1) 간조직 Cytochrome P450 함량 측정 ... 74
• (2) 간조직 Superoxide radical 함량 측정 ... 74
• (3) 간조직 Hydrogen peroxide (H2O2) 함량 측정 ... 75
• (4) 혈장 Paraoxonase 및 arylesterase 활성 측정 ... 76
• 마) 지질대사 개선 효과 측정 : 2차 동물실험 ... 76
• (1) 혈청 지질 수준 ... 76
• (2) 혈청과 간조직의 과산화지질 수준 ... 77
• 바) 산초 잎 추출물의 해독 및 안전성 지표측정 ... 78
• (1) 혈청 GOT와 GPT 활성 ... 78
• (2) 간조직 glutathione S-transferase 활성과 글루타치온 함량 ... 79
• 2. 산초 및 초피 성분의 어류가공품 보존제로서의 활용 ... 80
• 가. 연구배경 ... 80
• 나. 연구방법 ... 81
• 1) 산초 잎 추출물 및 초피 과피 정유 처리 과메기 제조 ... 81
• 2) 산화억제 효과 측정 ... 81
• 3) 아민류 생성 및 관능검사 ... 81
• 4) 통계처리 ... 81
• 다. 연구결과 ... 82
• 1) 과메기 제조 후 외관 보존 ... 82
• 2) 과메기의 산화억제 효과 ... 83
• 3) 과메기의 아민 생성 및 관능검사 ... 85
• 3. 산초추출물의 안전 유효 허용양 산정 ... 87
• 가. 동물실험 결과에서 산정 ... 87
• 나. 과메기 제품에 함유된 유효물질의 안전도 ... 89
• 제2절. 산초나무와 초피나무로부터 생리활성 물질의 분리 및 구조 결정 (제2세부과제) ... 89
• 1. 연구배경 ... 89
• 2. 재료 및 방법 ... 91
• 가. 재료 ... 91
• 나. 기기 ... 91
• 다. 시약 ... 91
• 3. 결과 및 고찰 ... 91
• 가. 산초시료의 부위별 추출 및 분획과정 ... 91
• 1) 산초 뿌리 ... 91
• 2) 산초 줄기 ... 92
• 3) 산초 잎 ... 92
• 4) 산초 종자 ... 93
• 5) 산초 과피 ... 93
• 나. 초피시료의 부위별 추출 및 분획과정 ... 96
• 1) 초피 뿌리 ... 96
• 2) 초피 줄기 ... 97
• 3) 초피 잎 ... 97
• 4) 초피 종자 ... 97
• 5) 초피 과피 ... 98
• 다. 중국산 초피 과피와 한국산 초피 과피의 비교실험 ... 101
• 1) 중국산 초피 과피의 추출 및 분획과정 ... 101
• 2) 한국산 초피 과피의 추출 및 분획과정 ... 101
• 라. 산초 잎과 초피 잎의 용매별 추출실험 ... 103
• 마. 초피 과피와 초피 종자의 용매별 추출실험 ... 104
• 바. 산초, 초피 유래의 치아우식균 (S. mutans), 식중독 및 장내 병원성 미생물(Aeromonas hydrophila, Chryseobacterium joosteii, Bacillus pumilus)에 대한 항균활성 측정을 위한 시료 조제 ... 105
• 1) 산초 과피 및 초피 과피 methylene chloride 층의 preparative TLC 분획실험 ... 105
• 가) 산초 과피 methylene chloride 층 ... 105
• 나) 초피 과피 methylene chloride 층 ... 106
• 2) 산초 과피와 초피 과피 및 종자의 용매별 추출실험과 수증기증류실험 ... 107
• 가) 산초 과피 수증기증류 ... 107
• 나) 초피 과피 수증기증류 ... 107
• 다) 초피 종자 수증기증류 ... 107
• 사. 과메기 가공 시 보존제로의 활용을 위한 산초 잎과 초피 과피의 시료조제 ... 109
• 1) 산초 잎 80 % ethanol extract 조제 ... 109
• 2) 초피 과피 정유 성분 조제 ... 109
• 아. 동물실험 및 활성물질 분리를 위한 시료 조제 ... 110
• 1) 산초 잎의 추출 및 분획 활성 실험 ... 110
• 2) Flash column chromatography 분획실험 ... 110
• 가) n-BuOH 분획의 flash column chromatography 실험 ... 110
• 나) Methylene chloride 분획의 flash column chromatography 실험 ... 112
• 자. 우수 생리활성 분획으로부터 활성 물질의 분리ㆍ정제 및 구조결정 ... 113
• 1) 실험 배경 ... 113
• 2) n-butanol 분획으로부터 활성물질 분리 ... 113
• 3) Methylene chloride 분획으로부터 활성물질 분리 ... 114
• 4) 분리된 화합물의 구조 ... 118
• 5) 분리한 화합물의 물리화학적 및 분광학적 data ... 122
• 6) 분리한 화합물의 구조 결정 ... 127
• 제3절. 산초나무와 초피나무로부터 신 기능성 생리활성 물질의 탐색과 안정성연구 및 제품 개발(제3세부과제) ... 138
• 1. 김치 숙성에 대한 산초와 초피의 영향 ... 138
• 가. 연구배경 ... 138
• 나. 연구방법 ... 139
• 1) 산초 잎과 초피 잎 가루 준비 ... 139
• 2) 산초 잎과 초피 잎 가루 첨가 김치 제조 ... 139
• 3) 일반세균수 측정 ... 140
• 4) 유산균수 측정 ... 140
• 5) 대장균수 측정 ... 140
• 6) 공시균주 ... 140
• 7) 관능검사 ... 140
• 다. 연구결과 ... 140
• 1) 김치발효시간에 따른 일반세균, 유산균, 대장균 수의 변화 ... 140
• 2) 초피 추출물과 산초추출물의 유산균(L. mesenteriodes, L. plantarum) 생육에 대한 영향 조사 결과 ... 143
• 2. 산초와 초피 유래 항균활성 성분에 관한 연구 ... 146
• 가. 연구배경 ... 146
• 나. 연구방법 ... 149
• 1) 각 부위별 추출물 준비 ... 149
• 2) 공시균주 및 배지 ... 149
• 3) 초피 추출물의 Disc 확산법에 의한 항균활성 검정 ... 149
• 4) 활성 성분의 분석 ... 150
• 5) 액체 배양법을 이용하여 S. mutants 외 병원성 미생물에 대한 초피 종자와 과피 정유성분의 최소 저해농도 (Minimum Inhibitory Concentration) 측정 ... 150
• 6) 열안정성 측정 ... 150
• 7) 시제품의 Disc 확산법에 의한 S. mutans에 대한 항균활성 검정 ... 150
• 8) 시제품에 대한 관능검사 ... 151
• 9) Salmonella typhimurium TA 100, 1538 균주에 대한 초피 종자와 과피 정유 성분의 세포 독성 측정 ... 151
• 10) 돌연변이 유발성 측정 ... 151
• 다. 연구결과 ... 151
• 1) 산초와 초피로부터 항균물질의 탐색과 동정 ... 151
• 2) 초피의 항균력을 이용한 신기능성 항균 치약으로의 실용화 방안 ... 169
• 3) 항균 크림 및 화장품 보존료로의 실용화 방안 ... 171
• 4) 초피나무 유래의 항균활성 물질의 돌연변이 유발성 조사를 통한 안정성 측정 ... 173
• 3. 산초나무 유래의 Mitochondoria-defendant Apoptosis 유도 물질의 탐색과 그 작용기전 연구 ... 174
• 가. 연구배경 ... 174
• 나. 연구방법 ... 176
• 1) 산초의 획득 ... 176
• 2) 시약 및 기기 ... 176
• 3) 실험에 이용한 세포주 및 세포배양 ... 176
• 4) 유효물질 추출 및 분획 ... 176
• 5) MTT assay를 통한 viability 조사 ... 176
• 6) Apoptosis 에 의한 DNA fragmentation 추출 ... 177
• 7) Flow cytometry 분석 ... 177
• 8) Cell lysate 조제 ... 177
• 9) Western blot analysis ... 178
• 10) 활성 성분 분석 ... 178
• 다. 실험결과 ... 178
• 1) 산초나무로 부터 세포독성 분획의 탐색 ... 178
• 2) 산초 잎 유래의 암세포독성물질의 탐색 ... 179
• 3) 산초 잎 유래의 암세포독성물질의 작용 기전 ... 180
• 4) 산초 잎의 methylene chloride 분획 활성 성분의 분석 ... 183
• 5) 산초 잎의 methylene chloride 분획 중에서 암세포독성물질 탐색 ... 185
• 4. 산초 (Z. schinifolium)의 세포독성 성분인 auraptene의 인체 백혈병 세포주 Jurkat T 세포에 대한 세포자살 유도 활성 및 그 분자기전 : ER stress-매개 caspase-12 및 caspase-8 활성화 ... 187
• 가. 연구배경 ... 187
• 나. 재료 및 방법 ... 189
• 1) 시약, 항체 및 배지 ... 189
• 2) Z. schinifolium로부터 세포독성성분의 분리 ... 190
• 3) Cytotoxicity assay ... 190
• 4) Apoptotic DNA fragmentation 분석 ... 190
• 5) Flow cytometry 분석 ... 191
• 6) DAPI 염색 방법 ... 191
• 7) Cell lysate 조제 ... 191
• 8) Western blot analysis ... 191
• 9) 마이토콘드리아 cytochrome c의 세포질로의 방출 조사 ... 192
• 다. 실험결과 ... 192
• 1) 산초 (Z. schinifolium) 잎에 함유된 세포자살 유도물질로서의 auraptene의 확인 ... 192
• 2) Auraptene에 의해 유도되는 세포자살에 있어서 미토콘드리아 cytochrome c방출 및 이에 의한 caspase 연쇄반응의 활성화 ... 196
• 3) Auraptene에 의한 세포자살에 있어서 Fas-사망신호를 차단하는 anti-Fas 항체의 영향 ... 200
• 4) Auraptene에 의한 세포자살에 미치는 pan-caspase inhibitor, z-VAD-fmk 또는 caspase-8 inhibitor, z-IETD-fmk의 영향 ... 201
• 5) Cytotoxic effect of auraptene on human peripheral T cells ... 204
• 제4장 목표달성도 및 관련분야에의 기여도 ... 207
• 1절. 세부과제별 평가착안점에 입각한 목표달성도 ... 207
• 2절. 관련분야에의 기여도 ... 208
• 제5장 연구개발결과의 활용계획 ... 209
• 1절. 활용 계획 ... 209
• 2절. 본 연구과제 수행으로 발표된 또는 발표예정인 논문 및 특허건 ... 212
• 3절. 추가연구의 필요성 ... 214
• 제6장 연구개발과정에서 수집한 해외과학기술정보 ... 215
• 1절. 해외과학기술정보 ... 215
• 제7장 참고문헌 ... 218
• 끝페이지 ... 233