New Hydrolyzable Tannin with Potent Antioxidant and α-Glucosidase Inhibitory Activity from Black Tea Produced from Camellia taliensis

Camellia taliensis (W. W. Smith) Melchior, belonging to the genus Camellia sect. Thea., is mainly distributed from northern Myanmar to western and southwestern Yunnan province of China, and its leaves have been used to make various teas by the locals of its growing regions. The chemical constituents of C. taliensis are significantly related to those of cultivated tea plants, C. sinensis and C. sinensis var. assamica. The HPLC-ESI-MS analysis of black tea prepared from the leaves of C. taliensis showed a rich existence of polyphenols. Further comprehensive chemical study led to the separation and recognition of 32 compounds (1–32), including one new hydrolyzable tannin, 1-O-galloyl-4,6-tetrahydroxydibenzofurandicarboxyl-β-D-glucopyranose (1), and one new natural product (24). The known compounds referred to seven hydrolyzable tannins (2–8), 10 flavonols and glycosides (9–18), and 14 simple phenolics (19–32). Their structures were elucidated by comprehensive spectroscopic analyses. Among them, 20 compounds (2, 3, 6, 7, 8, 15, 17, 18, 20–22, 24–32) were isolated from black tea for the first time. Most isolates displayed obvious antioxidant activities on DPPH and ABTS+ assays, and the hydrolyzable tannins 1, 3–5, 7, and 8 exhibited stronger inhibitory activities on α-glycosidase than quercetin and acarbose (IC50 = 5.75 and 223.30 μM, respectively), with IC50 values ranging from 0.67 to 2.01 μM.

C. taliensis (W. W. Smith) Melchior is the most extensively distributed wild tea tree in the genus Camellia sect. Thea. [13,14], and primarily distributed from the north of Myanmar to the west and southwest of Yunnan province, China, with a scattered distribution along the Ailao Mountain and Lancang (Mekong) and Nujiang (Salween) river basins of

HPLC and LC-MS Analysis
The fine powder (4.0 g) of black tea from C. taliensis was ultrasonically extracted twice (20 min each time) during 12 h with 70% MeOH (150 mL) at room temperature. The extract was first dried down to produce a crude residue, which was then dissolved with water and redistributed using CHCl 3 to weaken the interference from caffeine. For further HPLC and LC-MS analyses, the aqueous fraction was filtered over a 0.22 µm nylon membrane.
Then, 20 µL sample solution was applied for the HPLC analysis conducted with an Agilent Zorbax SB C-18 column (4.6 × 250 mm inner diameter, 5 µm), with a gradient elution of 4-40% (45 min) MeOH−H 2 O (containing 1.5‰ HCOOH) solution (1 mL/min) as a mobile phase. The chromatogram was collected at 240, 254, 280, 300, 330, and 350 nm. The temperature of the column was kept at 30 • C. The electrospray ionization (ESI) with two modes (negative and positive ionization), working with a full-scan mode (100−1500 m/z), was applied to perform MS analysis. The following operation parameters were applied: the ion spray voltage, 4 kV; temperature, 400 • C.

Antioxidant Activity
Assays for DPPH and ABTS + were carried out using the previous method with some modifications [19]. The DPPH assay was conducted as follows: the tested compound (final concentration: 0.2-1000 µM in EtOH, 100 µL) and DPPH solution (200 µM in EtOH, 100 µL) were sequentially added to 96-well plates at room temperature, then hid from the light for 15 min. The blank control and positive control (ascorbic acid) were set simultaneously. OD values at 517 nm were detected with a microplate reader. ABTS + assay was carried out as follows: firstly, the ABTS + solution was prepared using the procedure in our previous paper [19]. Then, the tested compound (final concentration: 0.2-1000 µM in EtOH, 10 µL) and ABTS + solution (200 µL) were added to 96-well plates at room temperature in an orderly manner, and then hided from light for 6-8 min. The blank control and positive control (trolox) were set synchronously. OD values at 405 nm were then detected with a microplate reader. All the reactions were set up three replicates. The formula for calculating the scavenging percentage is as follows: inhibition percentages (%) = (OD blank − OD test )/OD blank × 100%, and IC 50 values were computed based on the Reed-Muench method [20].

α-Glucosidase Inhibitory Activity
As recorded in our prior paper [4], 4-nitrophenol-α-D-glucopyranoside (PNPG) was used as the zymolyte for the screening of enzyme inhibitors. Initially, 96-well plates with the following ingredients were added in a proper order: testing compound (50 µM), αglucosidase solution (0.025 U/mL), buffer, and the zymolyte (1 mM), and kept at 37 • C for 50 min. Quercetin and acarbose were used as the positive control. All the reactions were set up with three replicates. OD values at 405 nm were recorded with a microplate reader. The formula for calculating the scavenging percentage is as follows: inhibition percentages (%) = (OD blank − OD test )/OD blank × 100%, and IC 50 values were computed based on the Reed-Muench method [20].

HPLC and LC-MS Analysis
HPLC-ESI-MS analysis of the extract of black tea from C. taliensis using 70% aqueous MeOH revealed a total of 33 chemical constituents, including mainly polyphenols, e.g., 5 catechins, 3 theaflavins, 4 hydrolyzable tannins, 6 simple phenolics, 13 flavonols and their glycosides and coumarin, as well as theanine (Table 1), on the basis of their quasi-molecular ions, fragment ions, UV absorption, and retention times (t R ), combined with standards obtained in our prior studies. Half of them were also isolated in further chemical study. Note: cou, rha, glu, and gal refer to coumaroyl, rhamnosyl, glucosyl, and galactosyl, respectively.

Identification of Compounds 1-32
The extract of black tea from C. taliensis using 60% aqueous acetone was dissolved with water and successively redistributed with CHCl 3 , EtOAc, and n-BuOH. The EtOAc extract was further isolated by various CC on Diaion HP20SS, RP-18, Toyopearl HW-40F, MCI-gel CHP20P, and Sephadex LH-20, to yield 32 compounds, including one undescribed hydrolyzable tannin (1) and one new natural product (24). The compounds, which had were in advance, were recognized as seven hydrolyzable tannins ( Table 2). The HMBC correlation from the anomeric proton (δ H 5.73) to galloyl carboxyl carbon (δ C 166.8) clarified that the galloyl group was connected at glucosyl C-1. The obvious lower field shift of glucosyl C-6 (δ C 67.6), H-4 (δ H 5.00), and H-6 (δ H a, 4.78; b, 4.01) compared with those of 1-O-galloyl-β-D-glucopyranose [52], suggested the HHDP-related acyl group should be attached to the glucosyl C-4 and C-6 positions, analogous to those of 4. However, 1 H and 13 C NMR data assignable the HHDP-related acyl group in 1 were very different to those of the HHDP group in 4, and the molecular formula of 1 was differed from 4 by one H 2 O, indicating that 1 should be a dehydrated derivative of 4. The HHDP-related acyl group at glucosyl C-6 and C-4 in 1 was determined to be 1,1 -(3,3 ,4,4 -tetrahydroxy) dibenzofurandicarboxyl group, compared with the NMR data with those in mallotusinin, the first compound with a 1,1 -(3,3 ,4,4 -tetrahydroxy) dibenzofurandicarboxyl group isolated from Mallotus japonicus [53]. Other 2D NMR correlations (Figure 3 and Figure S3-S6) confirmed the structure of 1. Hence, compound 1 was confirmed to be 1-O-galloyl-4,6-tetrahydroxydibenzofurandicarboxyl-β-D-glucopyranose, as shown in Figure 2.  Table 2). The HMBC correlation from the anomeric proton (δH 5.73) to galloyl carboxyl carbon (δC 166.8) clarified that the galloyl group was connected at glucosyl C-1. The obvious lower field shift of glucosyl C-6 (δC 67.6), H-4 (δH 5.00), and H-6 (δH a, 4.78; b, 4.01) compared with those of 1-O-galloylβ-D-glucopyranose [52], suggested the HHDP-related acyl group should be attached to the glucosyl C-4 and C-6 positions, analogous to those of 4. However, 1 H and 13 C NMR data assignable the HHDP-related acyl group in 1 were very different to those of the HHDP group in 4, and the molecular formula of 1 was differed from 4 by one H2O, indicating that 1 should be a dehydrated derivative of 4. The HHDP-related acyl group at glucosyl C-6 and C-4 in 1 was determined to be 1,1′-(3,3′,4,4′-tetrahydroxy) dibenzofurandicarboxyl group, compared with the NMR data with those in mallotusinin, the first compound with a 1,1′-(3,3′,4,4′-tetrahydroxy) dibenzofurandicarboxyl group isolated    Hydrolyzable tannins are segmented into two major classes based on their structures: gallotannins (GTs) and ETs. GTs are esters with only a galloyl group bounded to glucose, while ETs are esters with hexahydroxydiphenoyl (HHDP) or its similar acyl groups commonly bounded to glucose [50]. Isolates 6-8 were classified as GTs, and 1, 3, 4, and 5 were classified as ETs. ETs are often present as an equilibrium mixture of two isomers with a different configuration at anomeric carbon [50]. For example, compound 3 was isolated as a mixture, due to the chirality of anomeric carbon. It was reported that the original product of the oxidative coupling of two galloyl groups in ETs was the dehydrohexahydroxydiphenoyl (DHHDP) group, which was then reduced to the HHDP group [54], as shown in Figure S10. The DHHDP group in dehydroellagitannins usually comes simultaneously in Hydrolyzable tannins are segmented into two major classes based on their structures: gallotannins (GTs) and ETs. GTs are esters with only a galloyl group bounded to glucose, while ETs are esters with hexahydroxydiphenoyl (HHDP) or its similar acyl groups commonly bounded to glucose [50]. Isolates 6-8 were classified as GTs, and 1, 3, 4, and 5 were classified as ETs. ETs are often present as an equilibrium mixture of two isomers with a different configuration at anomeric carbon [50]. For example, compound 3 was isolated as a mixture, due to the chirality of anomeric carbon. It was reported that the original product of the oxidative coupling of two galloyl groups in ETs was the dehydrohexahydroxydiphenoyl (DHHDP) group, which was then reduced to the HHDP group [54], as shown in Figure S10. The DHHDP group in dehydroellagitannins usually comes simultaneously in six-membered and five-membered hemiacetal rings. However, due to the influence of Gibbs free energy, the stability of the six-membered ring structure of DHHDP is better than that of the five-membered ring structure. Meanwhile, the pressure load carried by ester carbonyl carbons and the flexibility of the macrocylic lactone ring in ETs make a difference to the stability of DHHDP [54]. Consequently, the structure, molecule size, and location of acyl group are all related to the structural diversity of ETs, which will affect the stability of DHHDP. A study found that the DHHDP group, created with pyridine in acetonitrile, was disproportionated by redox reaction to yield the 1,1 -(3,3 ,4,4 -tetrahydroxy) dibenzofurandicarboxyl group as a reduction product [55]. Thus, compound 1 could be a reduction product, reduced through the two galloyl groups of 4 which were converted into DHHDP by the oxidative coupling reaction, followed by redox disproportionation.
Urolithins, as natural metabolites of ETs with better gastrointestinal absorption, were reported to have inhibitory effects on the proliferation of prostate and colon cancer cells as well as anti-inflammation activity [56]. It is reported that ETs were hydrolyzed by intestinal bacteria to produce ellagic acid, which were subsequently converted into urolithins. The presence of compound 31 (resembled a urolithin) demonstrated a possible occurrence of the ester hydrolysis of ET acyl groups and further decarboxylation during the fermentation of black tea made from the leaves of C. taliensis.
Part of the flavonol and its glycosides (11,13,16,17) showed equivalent effects to the positive control (ascorbic acid and trolox). The sequence of activity for inhibiting the DPPH radical was 13 > 16 > 11 > ascorbic acid > 17, and the sequence of activity for inhibiting the ABTS + radical was 16 > 13 > 11 > trolox > 17. The p-coumaryl group has a little positive influence on the antioxidant effects of flavonol and its glycosides, which could be found from 10 (6.28%) and 9 (0.97%), respectively.

α-Glucosidase Inhibitory Activity
The inhibitory activities of hydrolyzable tannins 1-8 and gallic acid (19) on α-glucosidase were investigated. At a concentration of 50 µM, 1, 3-5, 7 and 8 with three or more galloyl groups showed a higher inhibition ratio (>50%) on α-glucosidase. Their IC 50 values were further evaluated, and as shown in Table 4, all showed a stronger inhibitory activity than quercetin and acarbose (IC 50 = 5.75 and 223.30 µM, respectively), with IC 50 values ranging from 0.67 to 2.01 µM. Their activity order was 5 > 3 > 1 > 4 > 7 > 8 > quercetin > acarbose. Compounds 2 and 6 with one or two galloyl groups showed equivalent inhibitory effects to acarbose on α-glucosidase at a concentration of 50 µM, while gallic acid (19) showed almost no inhibitory effect. The results revealed that the number of phenolic hydroxyl group plays a positive role on the α-glucosidase inhibitory activities of hydrolyzable tannins, probably due to their hydrophobic association with α-glucosidase [58].
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/foods12132512/s1, Figure S1: 1 H NMR spectrum of compound 1 in CD 3 OD; Figure S2: 13 C NMR spectrum of compound 1 in CD 3 OD; Figure S3: HSQC spectrum of compound 1 in CD 3 OD; Figure S4: HMBC spectrum of compound 1 in CD 3 OD; Figure S5: COSY spectrum of compound 1 in CD 3 OD; Figure S6: ROESY spectrum of compound 1 in CD 3 OD; Figure S7: HRESI-MS spectrum of compound 1; Figure S8: CD and UV spectra of compound 1 in MeOH; Figure  S9: OR of compound 1 in MeOH; Figure S10: Previous studies on the oxidative coupling of galloyl groups; Figure S11: 1 H NMR spectrum of compound 2 in CD 3 OD; Figure S12: 13