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.