Maclura tricuspidata (Carr.) Bureau ex Lavallee (formerly Cudrania tricuspidata) is mainly distributed in the East Asia region including Korea, China and Japan. The whole part of this plant, including the root, bark, stem, twig and leaves have been used as a traditional herbal medicine due to bioactivities such as antioxidant, anti-inflammatory, antiumor, hepatoprotective, neuroprotective, anti-obesity, anti-diabetic, anti-atherosclerotic, immunomodulatory and antimicrobial effects. The diversity of bioactivities in this plant can be due to the presence of phytochemicals such as xanthones and flavonoids, phenylpropanoids, phenolic acid and polysaccharides. The ripe Maclura tricuspidata (MT) fruits are also being used to make fresh juice, jam, concentrate, fermented wine and vinegar in Korea. In particular, because the ripe MT fruit has a high free sugar content and sweet taste with a desirable flavor and distinct red color, it has a high potential as a raw material for fermented vinegar preparation. Vinegars are rich in nutrients and bioactive compounds. The compositions and amounts of these compounds in vinegars are closely related to the raw materials, microorganisms used, fermentation process and aging conditions. These compounds not only contribute to overall flavors of vinegars, but also play important roles in the expression of health benefits. However, not sufficient study has been performed on production, chemical constituents and their bioactivities of MT fruit vinegar. Therefore, the objectives of this study were (1) to explore new bioactive compounds in MT fruit, (2) to investigate the potential of MT fruit as a raw material to produce fermented vinegar, (3) to investigate physicochemical composition of prepared MT fruit vinegar (4) to explore anti-obesity activity of MT fruit vinegar, and finally (5) to investigate the effect of addition of MT twig extracts and chip as supplement raw material for MT fruit vinegar. In the first chapter of this study, eighteen compounds were identified from MT fruit extract by HPLC and HPLC/QTOF-MS analyses and of them, five parishin derivatives (gastrodin, parishin A, B, C, E) were positively identified for the first time in this plant. Parishin A was the most abundant constituent, followed by chlorogenic acid, gastrodin, eriodictyol glucoside, parishin C, parishin E and parishin B. The contents of all the compounds were higher at the immature and premature than at fully mature and overmature stages. On the other hand, the ripe MT fruit has a distinct red color due to carotenoids, their chemical nature and composition has not been elucidated in detail so far. In this study, chemical structures and composition of carotenoids in the ripe MT fruit were investigated. After oleoresin isolated from the fruit with solvent extraction was saponified, and then two free carotenoids were isolated by preparative TLC and repeated column chromatography on silica gel. The major carotenoids isolated were determined as capsanthin and cryptocapsin, respectively, by spectroscopic analysis. These ketocarotenoids were presented mono- or di-esterified forms with saturated fatty acids rather than free forms and the results showed that esterification of these compounds occurred at early stage (yellow-brownish stage) of maturation. Considering the high cryptocapsin content, these results also suggest that the ripe MT fruit can be used as a potentially valuable source of cryptocapsin for food and cosmetic application as well as a source of provitamin A. In the second chapter, it was investigated for a potential of MT fruit as a raw material to make fermented vinegar. When MT fruit juice were fermented with 5 kinds of yeast including commercial Fermivin, the alcohol content after fermentation for 10 days at 26℃ was between 15 - 16%. A large amount of chlorogenic acid and gastrodin {(4-(β-D-glucopyranosyloxy)benzyl alcohol}in the MT fruit juice during the alcoholic fermentation were biotransformed into caffeic acid and 4-hydroxybenzyl alcohol (4-HBA), respectively. Total phenol content was about 1 mg/g (dw) in the MT fruit juice (control), but increased to 4.5 mg/g (dw)l after 10 day-fermentation with Fermivin, and total flavonoid content was also increased in a similar manner with total phenol during alcoholic fermentation. A new acetic acid bacteria having excellent acetic fermentation ability was isolated from the brown rice vinegar produced in some farmhouse. The nucleotide sequence of this strain was almost identical (99%) with that of A. pasteurianus subsp. pasteurianus LMG 1262 (NBRC 106471) and therefore isolated acetic acid bacteria was named as A. pasteurianus subsp. YJ17. Acetic acid fermentation of MT fruit wine was performed by mixing seed culture of YJ17 and MT fruit wine (alcohol content 13%) with a ratio of 1:1 (v/v). During stationary culture at 30℃, fresh MT fruit wine was periodically supplemented to increase the total acidity content. After fermentation for 28 days, the total acidity increased to 7.4%, but no further increase was observed until 35 days. In the third chapter, physicochemical properties such as free sugars, organic acids, free amino acids, polyphenols and parishin derivatives, composition of volatile aroma compounds and antioxidant properties of MT fruit vinegar were investigated. Fructose and glucose as free sugars, oxalic, citric, malic, succinic and lactic acid as non-volatile organic acids, and arginine, GABA, lysine and proline as free amino acids, were detected as major constituents in the MT fruit vinegar. A total of 57 compounds including 15 alcohols, 15 esters, 12 aldehydes and ketones, 7 acids and 8 miscellaneous compounds were identified in the MT fruit wine and/or vinegar by gas chromatography and gas chromatography-mass spectrometry analysis. Among them, ethyl hydrogen succinate, phenylacetic acid, 4-HBA, p-hydroxybenzaldehyde, p-hydroxyphenylacetic acid, tyrosol (p-hydroxyphenethyl alcohol), dihydroactinidiolide, 3-hydroxy-β-ionone and tryptophol have not been previously reported so far as volatile flavor constituents of vinegar, but only in the MT fruit vinegar. DPPH, ABTS and FRAP activities as antioxidant capacities of MT fruit vinegar (TA 7.4% and TA 11.1%) were 2.08±0.01?2.47 ± 0.00 mM TE/mL, 8.37 ± 0.09 ? 10.6 ± 0.46 mM TE/mL and 4.30 ± 0.17 ? 6.53±0.12 mM TE/mL, respectively, while total phenol and total flavonoid content of two MT fruit vinegar were 333.4 ±19.3 ? 426.3 ± 18.1 mg GAE/100 mL and 8.5 ± 0.5 ? 11.6 ± 1.0 mg QUE/100 mL, respectively. In the fourth chapter, the effect of MT fruit vinegar on anti-obesity activity was investigated. MT fruit vinegar attenuated the increases of lipid accumulation, triglyceride and cholesterol content and leptin release with the suppression of adipogenesis related protein expression in a dose-dependent manner. Therefore, the result suggest that MT fruit vinegar has anti-obesity efficacy. Finally, in the fifth chapter, the content of polyphenolic compounds and parishin derivatives are higher in the MT twig than in the MT fruit. Therefore, it was investigated the effect of water extract, 70% ethanol extract, and chip of MT twig as supplement raw material for production of enriched functional MT fruit vinegar. When water extract, alcohol extract or chips of MT twig were added to the at the stage of alcoholic fermentation with a certain concentration (2, 5, 10, 15%), the contents of 4-HBA and polyphenolic compounds in the vinegar products were increased in a dose-dependent manner. Therefore, the result also suggest that MT twig can be used as a supplement for the production of fermented vinegar with enriched bioactivities.
List of figures viiiList of tables xiiiABSTRACT xvChapter I. 꾸지뽕 열매의 기능성 성분Ⅰ. 서론 1Ⅱ. 재료 및 방법 51. 시약 52. 시료 63. 폴리페놀류 화합물 분석 73.1. 시료 추출 73.2. HPLC 분석 73.3. HPLC/QTOF-MS 분석 74. Carotenoid 분석 84.1. 시료 추출 및 검화 84.2. 불검화한 올레오레진에서 esterified carotenoids 분획의 분리 94.3. 검화한 올레오레진에서 capsanthin 및 cryptocapsin의 분리 및 정제 94.4. TLC, HPLC 및 QTOF-MS 분석 104.5. HPLC 분석용 시료 추출 114.6. GC에 의한 fatty acid 조성 분석 114.7. 분광학적 분석 124.8. 검량선 작성 용액의 준비 125. 통계분석 12Ⅲ. 결과 및 고찰 131. 폴리페놀 화합물 131.1. HPLC/QTOF-MS에 의한 성분 동정 131.2. HPLC에 의한 분석 및 분석법의 validation 201.3. 꾸지열매의 숙도에 따른 폴리페놀 화합물의 차이 비교 222. 꾸지열매 색소성분 동정 242.1. 꾸지열매에서 carotenoid류의 형태 242.2. 검화한 꾸지열매의 올레오레진에서 ketocarotenoids의 동정 282.3. 꾸지열매에 함유된 esterfied ketocarotenoids의 동정 372.4. 꾸지열매와 고추속 식물에 함유된 capsanthin과 cryptocapsin 함량 비교 42Ⅳ. 요약 51Ⅴ. 참고 문헌 53Chapter II. 꾸지열매를 이용한 발효식초 제조Ⅰ. 서 론 65Ⅱ. 재료 및 방법 691. 시약 692. 재료 693. 사용 균주 694. 발효 714.1. 알코올발효 714.2. 초산발효 715. 분석 715.1. 이화학성 및 성분분석 715.2. 유리당 함량 725.3. 유리 아미노산 함량 725.4. Polyphenol 및 parishin 유도체 725.5. UPLC/QTOF-MS 분석 73Ⅲ. 결과 및 고찰 741. 효모를 이용한 꾸지열매 주스의 알코올 발효 741.1. 가용성 고형분, pH 및 알코올 함량 변화 741.2. 유리당, phenolic acids, flavonoids 및 parishin 유도체의 변화 772. 초산발효 862.1. 우수 초산균의 선발 및 염기서열 분석 862.2. 선발된 초산균 (YJ17)을 이용한 초산발효 88Ⅳ. 요 약 90Ⅴ. 참고 문헌 91Chapter III. 꾸지열매 발효식초의 품질 특성Ⅰ. 서 론 95Ⅱ. 재료 및 방법 971. 재료 972. 시약 973. 사용기기 984. 분석 994.1. 이화학적 특성 994.2. 시료 준비 994.3. 유리당 994.4. 유기산 1004.5. 유리 아미노산 1004.6. 휘발성 성분 1004.6.1. 분리 1004.6.2. GC 및 GC-MS 분석 1024.7. Total phenol 및 total flavonoid 함량 1034.7.1. Total phenol 1034.7.2. Total flavonoid 1034.8. Phenolic acid 및 flavonoid 분석 1034.8.1. 추출 1034.8.2. 분석 1044.8.2. UPLC/QTOF-MS 분석 1044.9. Carotenoid 분석 1044.9.1. 추출 1044.9.2. 분석 1055. 통계분석 105Ⅲ. 결과 및 고찰 1061. 이화학적 특성 1062. 유리당 함량 1073. 유기산 함량 1084. 유리 아미노산 함량 1095. 휘발성 향기성분 1116. Total phenol 및 total flavonoid 함량 1187. Phenolic acid, flavonoid 및 parishin 유도체 분석 1208. UPLC/QTOF-MS 분석 1239. 발효 단계별 ketocarotenoid의 변화 12810. 항산화 활성 132Ⅳ. 요약 135Ⅴ. 참고문헌 137Chapter IV. 꾸지열매 발효식초의 항비만 활성Ⅰ. 서론 147Ⅱ. 재료 및 방법 1501. 재료 및 시약 1502. 꾸지열매 와인 및 발효식초 준비 1503. 항비만 활성 측정 1513.1. 세포 배양 1513.2. 3T3-L1 세포의 분화유도 1513.3. 세포 생존율 측정 1523.4. Oil Red O 염색 1523.5. Triglyceride 및 cholesterol 함량 분석 1523.6. Leptin 농도 1533.7. 관련된 유전자 발현 1534. 통계분석 153Ⅲ. 결과 및 고찰 1541. 세포 생존율 변화 1542. 지방세포 내 지방구 함량 1553. Triglyceride 및 cholesterol 함량 1564. Leptin 함량 1595. 관련 유전자의 발현 변화 160Ⅳ. 요약 163Ⅴ. 참고문헌 164Chapter V. 꾸지뽕 나무 잔가지 (twig) 추출물 첨가 후 발효식초 제조Ⅰ. 서론 169Ⅱ. 재료 및 방법 1701. 재료 1702. 시약 1703. 방법 1713.1. 꾸지뽕 나무 잔가지 추출물 제조 1713.2. 알코올 및 초산발효 1723.2.1. 알코올 발효 1723.2.2. 초산발효 1723.3. 시료 추출 1723.3.1. 부위별 시료 추출 1723.3.2. 와인 및 발효식초 추출 1733.4 분석 1733.4.1. HPLC 1733.4.2. HPLC/QTOF-MS 및 UPLC/QTOF-MS 분석 1733.5. 총 폴리페놀 및 총 플라보노이드 함량 1743.5.1. Total phenol 함량 1743.5.2. Total flavonoid 함량 1744. 통계분석 174Ⅲ. 결과 및 고찰 1751. 꾸지뽕 잔가지 중의 성분분석 1752. 꾸지뽕 잔가지 추출물 첨가효과 1782.1. 알코올 발효 1782.2. 초산발효 186Ⅳ. 요약 192Ⅴ. 참고문헌 193