A New Norisoprenoid and Other Compounds from Fuzhuan Brick Tea

Fuzhuan brick tea, a kind of dark tea consumed mainly in the border regions of Southwestern and Northwestern China since the 1860s, is produced from the leaves of Camellia sinensis var. sinensis by microbial fermentation. From this special fermented tea, a new norisoprenoid, 3R,9R-oxido-5-megastigmene, was isolated, together with α-linolenic acid, strictin, isovitexin, astragalin, (+)-catechin, (−)-epicatechin, (−)-epicatechin gallate, (+)-gallocatechin, (−)-epigallocatechin, (−)-epigallocatechin gallate and gallic acid. The structures of the compounds were identified by spectroscopic means. The new compound didn’t show any inhibition activity against the tested enteric pathogenic microorganisms at a concentration of 800 μg/mL by the hole plate diffusion method.


Introduction
Chinese commercial teas are classified into six categories: i.e., green tea, oolong tea, black tea, white tea, yellow tea and dark tea, according to the different manufacturing process.Among them, dark tea is the only one that involves microbial fermentation in its manufacturing process [1].Fuzhuan brick tea is one of the major brands of dark tea.It is mainly produced in Hunan province of China, and has been consumed by ethnic groups in the border regions of Southwestern and Northwestern China since the 1860s.
Fuzhuan brick tea is produced from the leaves of Camellia sinensis var.sinensis.The entire manufacturing process of the tea has been described in detail by Mo et al. and Xu et al. [2,3].The fresh tea leaves are first pretreated to be raw dark green tea, which was then softened with steam and pressed into brick shapes before being placed in a fungal fermentation workshop for about 15-17 days.The "fungal fermentation" stage is unique to the manufacturing process of Fuzhuan brick tea.In the fermentation stage, many fungi grow within the tea leaves under controlled temperature and moisture conditions.The fungi growing during this stage were characterized as a mixture of several microorganisms with Eurotium spp., Debaryomyces spp.and Aspergillus spp.predominating [3].Fungal growth is the key stage responsible for the unique functions of Fuzhuan brick tea, including anti-dysentery, anti-hyperlipidemia and anti-hyperglycemia [4][5][6].Furthermore, it is interesting that the anti-dysentery activity of the tea is increased with the course of the fermentation [2,7].Therefore, the bioactive compounds in the tea were implied to be formed by fermentation of these microbes [3,6,8].Some rare 6-hydroxy-7-one oleanolic triterpenoids, and a epicatechin derivative with a 3,6-dihydro-6oxo-2H-pyran-2-carboxylic acid moiety, were previously reported from Fuzhuan brick tea [9,10].In our continuing research for the bioactive compounds from Fuzhuan brick tea, a new norisoprenoid was discovered, along with other 11 known compounds.We report herein the isolation and structural elucidation of these compounds.
Compound 1a was reported as an aroma precursor from the fresh tea leaves [19], while compound 1 was the C-3/C-9 ether derivative of 1a after the sugar moiety was removed.It is interesting that 1 may be formed by microbial fermentation of Fuzhuan brick tea with dehydration of 3-OH and 9-OH on 3-hydroxy-7,8-dihydro-β-ionol, which is liberated from its precursor (1a) by endogenous enzymes during the manufacturing process of fresh tea leaves [19].O O

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Since Fuzhuan brick tea is produced by microbe fermentation, the isolated new compound 1 was evaluated for its antibacterial activity against four enteric pathogenic microorganisms, i.e., enteropathogenic Escherichia coli (EPEC), Staphyloccocus aureus, Shigella dysenteriae, and Salmonella typhi, by the hole plate diffusion method [9].However, the compound did not exhibit any activity against the above microorganisms.

Materials
Fuzhuan brick tea (produced in December, 2006) was purchased from Yiyang Fu Cha Industry Development Co. Ltd. (Yiyang, Hunan Province, China).Ampicillin sodium was purchased from Sigma Chemical Co. Ltd. (St. Louis, Missouri, USA).The test microorganisms were obtained from School of Basic Medical Sciences, Anhui Medical University (Hefei, China).