Improving flavor of summer Keemun black tea by solid-state fermentation using Cordyceps militaris revealed by LC/MS-based metabolomics and GC/MS analysis.

Cordyceps militaris (C. militaris) has been approved and widely used in healthy food. The present study aimed to improve the flavor of summer Keemun black tea (KBT) using C. militaris solid-state fermentation. Combined with sensory evaluation, the volatile and non-volatile components of solid-state fermentation of KBT (SSF-KBT) and KBT were analyzed. The results showed that after the solid-state fermentation, the contents of total polyphenol, total flavonoid, and total free amino acids were significantly reduced. Further non-targeted metabolomics analysis revealed that the contents of non-galloylated catechins and d-mannitol increased, while the galloylated catechins and flavonoid glycosides decreased as did the bitterness and astringency of KBT. Dihydro-ß-ionone and ß-ionone (OAV = 59321.97 and 8154.17) were the aroma-active compounds imparting woody and floral odors in SSF-KBT, respectively. Current study provides a new avenue to develop summer-autumn KBT.

Cytosolic Nudix Hydrolase 1 Is Involved in Geranyl ß-Primeveroside Production in Tea.

Geraniol is a potent tea odorant and exists mainly as geranyl glycoside in Camellia sinensis. Understanding the mechanisms of geraniol biosynthesis at molecular levels in tea plants is of great importance for practical improvement of tea aroma. In this study, geraniol and its glycosides from tea plants were examined using liquid chromatography coupled with mass spectrometry. Two candidate geraniol synthase (GES) genes (CsTPS) and two Nudix hydrolase genes (CsNUDX1-cyto and CsNUDX1-chlo) from the tea genome were functionally investigated through gene transcription manipulation and gene chemical product analyses. Our data showed that in tea leaves, levels of geranyl ß-primeveroside were dramatically higher than those of geranyl ß-glucoside, while free geraniol was undetectable in this study. A tempo-spatial variation of geranyl ß-primeveroside abundance in tea plants existed, with high levels in young and green tissues and low levels in mature or non-green tissues. Cytosolic CsNUDX1-cyto showed higher hydrolysis activity of geranyl-pyrophosphate to geranyl-monophosphate (GP) in vitro than did chloroplastidial CsNUDX1-chlo. A transgenic study revealed that expression of CsNUDX1-cyto resulted in significantly more geranyl ß-primeveroside in transgenic Nicotiana benthamiana compared with non-transgenic wild-type, whereas expression of CsNUDX1-chlo had no effect. An antisense oligo-deoxynucleotide study confirmed that suppression of CsNUDX1-cyto transcription in tea shoots led to a significant decrease in geranyl ß-primeveroside abundance. Additionally, CsNUDX1-cyto transcript levels and geranyl ß-primeveroside abundances shared the same tempo-spatial patterns in different organs in the tea cultivar "Shucha Zao," indicating that CsNUDX1-cyto is important for geranyl ß-primeveroside formation in tea plants. Results also suggested that neither of the two candidate GES genes in tea plants did not function as GES in transgenic N. benthamiana. All our data indicated that CsNUDX1-cyto is involved in geranyl ß-primeveroside production in tea plants. Our speculation about possible conversion from the chemical product of CsNUDX1-cyto to geranyl ß-primeveroside in plants was also discussed.

EGCG-derived polymeric oxidation products enhance insulin sensitivity in db/db mice.

The present study investigated the influence of epigallocatechin-3-gallate (EGCG) and its autoxidation products on insulin sensitivity in db/db mice. Compared to EGCG, autoxidation products of EGCG alleviated diabetic symptoms by suppressing the deleterious renal axis of the renin-angiotensin system (RAS), activating the beneficial hepatic axis of RAS, and downregulating hepatic and renal SELENOP and TXNIP. A molecular weight fraction study demonstrated that polymeric oxidation products were of essential importance. The mechanism of action involved coating polymeric oxidation products on the cell surface to protect against cholesterol loading, which induces abnormal RAS. Moreover, polymeric oxidation products could regulate RAS and SELENOP at doses that were far below cytotoxicity. The proof-of-principal demonstrations of EGCG-derived polymeric oxidation products open a new avenue for discovering highly active polymeric oxidation products based on the oxidation of naturally occurring polyphenols to manage diabetes and other diseases involving abnormal RAS.

A new anti-proliferative acylated flavonol glycoside from Fuzhuan brick-tea.

Fuzhuan brick-tea (FBT) is unique for a fungal fermentation stage in its manufacture process and is classified in dark tea. A new acylated flavonol glycoside, kaempferol 3-O-[E-p-coumaroyl-(→2)][α-l-arabinopyranosyl-(1→3)][α-l-rhamnopyranosyl(1→6)]-ß-d-glucopyranoside, which was trivially named as camellikaempferoside A (1), was isolated from FBT along with camelliquercetiside C (2). Their structures were unambiguously elucidated by combination of spectroscopic and chemical methods. Compound 1 showed anti-proliferative activity against MCF-7 and MDA-MB-231 cells with IC50 values of 7.83 and 19.16 µM, respectively.

Changes of major tea polyphenols and production of four new B-ring fission metabolites of catechins from post-fermented Jing-Wei Fu brick tea.

HPLC analysis of samples from four major fermentation procedures of Jing-Wei Fu brick tea showed that the level of major tea catechins epigallocatechin gallate (EGCG) and epicatechin gallate (ECG) dropped increasingly to about 1/3 in the final product. Phytochemical study of the final product led to the discovery of four new B-ring fission metabolites of catechins (BRFCs) Fuzhuanin C-F (1-4) together with three known BRFCs (5-7), six known catechins (8-13), five simple phenols (14-18), seven flavones and flavone glycosides (19-25), two alkaloids (26, 27), three triterpenoids (28-30) and one steroid (31). The structures were elucidated by spectroscopic methods including 1D and 2D NMR, LC-HR-ESI-MS, IR, and CD spectra. Five compounds (16-18, 28, 29) were reported for the first time in tea. Possible pathways for the degradation of major tea catechins and the generation of BRFCs were also provided.

Fuzhuanins A and B: the B-ring fission lactones of flavan-3-ols from Fuzhuan brick-tea.

Fuzhuan brick-tea is a special dark tea prepared from the leaves of Camellia sinensis var. sinensis. Its production involves a fungal fermentation stage, which forms the unique flavors and functions by a series of biochemical reactions. Our phytochemical research of the material led to the isolation of two new B-ring fission lactones of flavan-3-ols, fuzhuanins A (1) and B (2). In addition, three other flavan-3-ol derivatives (3-5), three flavone C-glycosides (6-8), eight flavonoid O-glycosides (10-17), five simple phenolics (19-23), two norisoprenoid glycosides (24, 25), two sesquiterpenoids (26, 27), and theobromine (28), as well as two flavonoid anions (9 and 18), were also identified. The structures of these compounds were determined by spectroscopic methods. Compounds 4, 19, 20, 22-24, 26, and 27 were reported for the first time in Camellia spp. and tea. Furthermore, HPLC analysis method was performed to compare the chemical constituents of the before/after fungal fermentation Fuzhuan brick-teas. Compound 1 was indicated as one of the major characteristic constituents generated in the fungal fermentation process. The IC50 value of the antiproliferative activity of 2 on HeLa cells was assayed as 4.48 µM. None of the isolated compounds showed any inhibition activity against the enteric pathogenic microbes at 800 µg/mL by the hole plate diffusion method.

A new saponin from tea seed pomace (Camellia oleifera Abel) and its protective effect on PC12 cells.

A new triterpenoid saponin, oleiferasaponin A1, was isolated from tea seed pomace (Camellia oleifera Abel). The structure of oleiferasaponin A1 was elucidated on the basis of chemical and physicochemical evidence and was found to be 22-O-cis-2-hexenoyl-A1-barrigenol 3-O-[ß-D-galactopyranosyl(1→2)] [ß-D-glucopyranosyl(1→2)-α-L-arabinopyranosyl(1→3)]-ß-D-glucopyranosiduronic acid. PC12 cells injured with H2O2 were used as the model to test the protective effects of oleiferasaponin A1. The results indicated that oleiferasaponin A1 can potentially prevent the H2O2-induced cell death of PC12 cells.

α-Tocopherol quinone inhibits ß-amyloid aggregation and cytotoxicity, disaggregates preformed fibrils and decreases the production of reactive oxygen species, NO and inflammatory cytokines.

Alzheimer's disease (AD) is a complex, multifactorial neurodegenerative disease. The aggregation of beta-amyloid (Aß) into extracellular fibrillar deposition is a pathological hallmark of AD. The Aß aggregate-induced neurotoxicity, inflammatory reactions and oxidative stress are linked strongly to the etiology of AD. The currently available hitting-one-target drugs are insufficient for the treatment of AD. Therefore, finding multipotent agents able to modulate multiple targets simultaneously is attracting more attention. Previous studies indicated that vitamin E or its constituent such as α-tocopherol (α-T) was able to attenuate the effects of several pathogenetic factors in AD. However, ineffective or detrimental results were obtained from a number of clinical trials of vitamin E. Here, we showed that naturally synthesized RRR-α-tocopherol quinone (α-TQ), a main derivative of α-T, could inhibit Aß42 fibril formation dose-dependently. Further investigations indicated that α-TQ could attenuate Aß42-induced neurotoxicity toward SH-SY5Y neuroblastoma cells, disaggregate preformed fibrils and interfere with natural intracellular Aß oligomer formation. Moreover, α-TQ could decrease the formation of reactive oxygen species (ROS) and NO, and modulate the production of cytokines by decreasing TNF-α and IL-1ß and increasing IL-4 formation in microglia. Taken together, α-TQ targeting multiple pathogenetic factors deserves further investigation for prevention and treatment of AD.

Diverse ecdysterones show different effects on amyloid-ß42 aggregation but all uniformly inhibit amyloid-ß42-induced cytotoxicity.

Amyloid-ß (Aß) plays a pivotal role in Alzheimer's disease (AD) pathogenesis and in toxic mechanisms such as oxidative stress, mitochondrial dysfunction, calcium turbulence, and apoptosis induction. Therefore, interfering with Aß aggregation has long been one of the most promising strategies for AD treatment. Ecdysterones (ECRs) are steroidal hormones in insects and terrestrial plants that have high structural diversity and multiple beneficial pharmacological activities. Here, we studied the effects of six ECRs on Aß aggregation and cytotoxicity. Two ECRs with an acetoxyl group at the 2 or 3 position and saturated chains as side groups showed apparent promotion of Aß42 fibrilization, resulting in less Aß42 oligomers in the samples. Another three with unsaturated side chains clearly inhibited Aß aggregation and disaggregated preformed fibrils, but increased the Aß42 oligomer levels. Nevertheless, our MTT results showed that all ECRs tested inhibited Aß42-induced cytotoxicity. This protective activity may be partly attributable to ECR-mediated amelioration of A&beta42-induced release of reactive oxygen species. Taken together, our findings suggest that ECRs, a series of natural compounds in many plants and insects, have therapeutic potential in AD and that the deduced structure-activity relationships may be beneficial in drug design for the treatment of AD and other amyloidoses.