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

Abstract 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)]-β-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.


Introduction
Tea is one of the most popular beverages all around the world. Chinese commercial teas are generally classified as green tea, black tea, white tea, yellow tea, oolong tea and dark tea. Among them, dark tea is the only one that involves microbial fermentation in its manufacturing process and is also called post-fermented tea (Wan 2003). Dark tea is further divided into Pu-erh tea, Fuzhuan brick-tea (FBT), and Qingzhuan brick-tea etc., according to the method of microbial fermentation (Jin et al. 2003;Zhang et al. 2013;Zheng et al. 2015).
FBT is generally made from mature leaves of Camellia sinensis var. sinensis. Some health benefits, i.e. anti-enteropathogen (Keller et al. 2013), anti-diarrhea (Yu et al. 2009), anti-hyperlipidaemic (Wu et al. 2010;Fu et al. 2011) were reported as unique functions of FBT. The manufacture of FBT was described in order of panning, rapid pile-fermentation, rolling, drying, softening with steam, piling, tea brick pressing, fungal fermentation and drying (Jin et al. 2003). The process is characterised by the fungal fermentation stage, in which a mixture of fungi, including Eurotium spp., Debaryomyces spp. and Aspergillus spp. are identified (Xu et al. 2011). Therefore, the key compounds contributing to the special flavour and functions of FBT are considered to be the results of fungal fermentation. The conjecture conducted many investigations on the FBT natural products in recent years (Ling et al. 2010;Luo et al. 2012Luo et al. , 2013Huang et al. 2013;Zhu et al. 2015).
In the present paper, we report the isolation and identification of a new anti-proliferative acylated flavonol glycoside from FBT.

Isolation and identification
The 70% aqueous acetone extract of commercial FBT was separated successively by partitioning with petroleum ether, CHCl 3 and n-BuOH. The n-BuOH fraction was separated by repeated Sephadex LH-20, MCI, ODS and polyamide column chromatography to afford compound 1 and camelliquercetiside C (2) (Manir et al. 2012).

Anti-proliferative activity
A growing body of evidence indicated that kaempferol and its derivatives exert anti-tumour activity efficiently (Chen & Chen 2013). In the present work, MTT assays were performed to determine the viabilities of MCF-7 and MDA-MB-231 cells in the presence or absence of 1 or 5-fuorouracil (5-FU, as positive control). The results showed that 1 has a similar inhibitory effect with 5-FU on the cell growth of MCF-7 and MDA-MB-231 cells (Figure 2(A)). The inhibition activities of 1 with different concentrations (0.0001-10 μM) were then tested on the MCF-7 and MDA-MB-231 cells (Figures 2(B) and (C)) to calculate the IC 50 values, which were 7.83 and 19.16 μM, respectively. In addition, the MCF-7 and MDA-MB-231 cells treated with 1 underwent morphological change, including rounding up and losing their pseudopodia (Figure 2(D)).

Experimental
Please see the online Supplementary Material for the 'experimental' section.

Conclusions
FBT is a unique post-fermented tea product which is fermented with fungi during the manufacturing process. The fungi were considered to have a significant influence on the biochemical profiles of FBT and consequently decide its unique quality (Xu et al. 2015). Many recent phytochemical investigations were focused on the structural special compounds in FBT, such as Fuzhuanins A-F, which were supposed to be generated from flavan-3-ols by fungal fermentation (Luo et al. 2013;Zhu et al. 2015).
On the other hand, it should be noted that tea leaves contain 2-5% of flavonols and their glycosides (FGs) (Wan 2003), which are also potentially important for flavour and biofunction research of dark tea (Scharbert et al. 2004;Graf et al. 2005). In addition, our HPLC analysis also discovered some interesting features with respect to the contents of some FGs in before fungal fermentation tea and after fungal fermentation tea samples (see Supplementary Material). Like flavan-3-ol derivatives, the FGs should also be one of the most important bioactive non-nutrition compositions in tea and deserve an extensive phytochemical investigation (Hilal & engelhardt 2009;Lakenbrink et al. 2000).

Supplementary material
experimental, HPLC analysis of Some FGs in FBT, and Figures S1-S16, as well as Tables S1-S5 are included in the supplementary material and are available online.