Two candidate peaks F1 and F2 (RT = 14.951, 15.633; protonated/deprotonated ions m/z 1051.2838/1049.2775, 1034.2859/1033.2822) were initially identified from tea product Y303 extracts by UPLC-MS (Fig. 1). To identify their exact structures, these two compounds were isolated by resin column separation and preparative HPLC chromatography. Y303 tea extracts were initially obtained by ultrasound-assisted extraction which can greatly enhance material disruption, solvent penetration, and mass transfer, followed by concentrating and freeze-drying to obtain crude extract I. Considering the highest adsorption and desorption capacities of the two target compounds (Supplemental Table S3), AB-8 resin was selected to separate AFGs-rich fractions from crude extract I as described in Section 4.2. Sequential elution with water and gradient ethanol solutions, selectively removed the impurities such as phenolic acids, minerals, and sugars and the F1 and F2 rich fraction were obtained by eluting with 60% and 80% ethanol solutions. The collected eluates were analyzed by HPLC method and the fractions rich in F1 and F2 were combined, evaporated, and freeze-dried, to obtain crude extract II. This extract was further purified with a preparative HPLC program which showed a good separation of F1 (peak 8) and F2 (peak 11) from other non-target compounds (Supplemental Fig. S1). The separated F1 and F2 fractions were collected with an automatic fraction collector and quantified by the UPLC-MS system. The purified fractions (> 95%) of each monomer were concentrated, evaporated, and freeze-dried. The purity of F1 and F2 were verified as 98.77% and 97.08%, respectively (Fig. 2). The MS/MS data of F1 and F2 in negative mode are shown in Supplemental Table S4. To further explore their exact structures, the isolated compounds were subjected to 1H NMR and 13C NMR analysis (Supplemental Table S5).
UPLC chromatogram (UV detection at 360 nm) of ethanol extracts of tea product Y303 (a) and mass spectrometry scan results of peak F1 (b) and peak F2 (c).
UPLC (UV detection at 360 nm) and MS/MS chromatograms in negative ion mode of purified F1 (a) and F2 (b).
F1: Yellow amorphous powder, HRMS m/z [M-H]- 1049.2904, molecular formula C47H54O27.
13C NMR (125 MHz, MeOD) δ 177.62, 167.36, 164.28, 161.62, 159.86, 157.52, 157.02, 148.25, 145.92, 144.49, 133.56, 129.95, 125.95, 122.09, 121.85, 116.16, 115.38, 114.80, 113.78, 104.48, 104.16, 103.89, 100.90, 99.57, 98.43, 93.44, 83.07, 81.68, 76.12, 75.38, 74.10, 73.15, 72.49, 71.17, 70.83, 69.88, 69.52, 68.77, 68.10, 67.07, 65.81, 60.71, 16.58.
1H NMR (500 MHz, MeOD) δ1.13(d, J = 6.1 Hz), 3.26(m), 3.28(dd, J = 10.6 Hz), 3.3(s), 3.43(d, J = 4.6 Hz), 3.45(s), 3.48(s), 3.50(s), 3.52(m), 3.54(s), 3.55(d, J = 2.0 Hz), 3.55(dd, J = 6.8, 3.9 Hz), 3.55(m), 3.63(s), 3.70(d, J = 4.2 Hz), 3.73(d, J = 4.2 Hz), 3.75(m), 3.86(s), 3.87(s), 3.87(td, J = 4.27, 10.35, 11.99 Hz), 3.99(dd, J = 3.3, 1.7 Hz), 4.34(d, J = 7.0 Hz), 4.45(d, J = 7.7 Hz), 4.61(d, J = 1.6 Hz), 5.23(dd, J = 9.46, 7.94 Hz), 5.56(d, J = 7.90 Hz), 6.14 (d, J = 2.06 Hz), 6.34(d, J = 2.12 Hz), 6.39(d, J = 15.88 Hz), 6.8(d, J = 8.39 Hz), 6.88(d, J = 8.43 Hz), 7.46(d, J = 8.62 Hz), 7.55(dd, J = 8.47, 2.21 Hz), 7.60(d, J = 2.22 Hz), 7.68(d, J = 15.94 Hz).
The 1H and 13C NMR spectral patterns of F1 were identical to a published quercetin tetraglycoside derivative. It was confirmed as quercetin 3-O-[(E) -p-coumaroyl- (1→2)] [α-L-arabinopyranosyl- (1→3)] [β-D-glucopyranosyl- (1→3) -α-L-rhamnopyranosyl (1→6)]-β-D-glucopyranoside, and the structural formula is shown in Fig. 2.
F2: Yellow amorphous powder, HRMS m/z [M-H]- 1033.2948, molecular formula C47H54O26.
13C NMR (125 MHz, MeOD) δ 177.65, 167.21, 164.35, 161.67, 159.95, 159.88, 157.62, 157.07, 145.84, 133.38, 130.94, 129.91, 125.93, 121.47, 115.38, 114.86, 113.78, 104.47, 104.23, 103.91, 100.84, 99.45, 98.49, 93.50, 82.88, 81.71, 76.14, 75.41, 74.08, 73.11, 72.50, 71.14, 70.80, 69.86, 69.46, 68.83, 68.13, 68.09, 67.02, 65.80, 60.67, 16.58.
1H NMR (500 MHz, MeOD) δ1.12(d, J = 6.1 Hz), 3.25(d, J = 6.2 Hz), 3.27(s), 3.39(s), 3.43(d, J = 4.2 Hz), 3.45(s), 3.48(d, J = 3.1 Hz), 3.50(s), 3.52(d, J = 6.0 Hz), 3.54(d, J = 2.0 Hz), 3.55(s), 3.55(s), 3.59(m), 3.60(d, J = 3.2 Hz), 3.70(d, J = 4.1 Hz), 3.73(dd, 7.3, 3.2 Hz), 3.76(d, J = 6.4 Hz), 3.87(s), 3.87(s), 3.88(d, J = 5.9 Hz), 4.00(dd, J = 3.3, 1.7 Hz), 4.37(d, J = 7.0 Hz), 4.45(d, J = 7.7 Hz), 4.62(d, J = 1.7 Hz), 5.21(dd, J = 9.5, 8.0 Hz), 5.61(d, J = 7.90 Hz), 6.19(d, J = 2.1 Hz), 6.35(d, J = 2.11 Hz), 6.37(d, J = 15.9 Hz), 6.39(d, J = 8.8 Hz), 6.39(d, J = 8.8 Hz), 6.84(d, J = 8.6 Hz), 7.49(d = 8.7 Hz), 7.72(d, J = 15.9 Hz), 8.01(d, J = 8.8 Hz), 8.01(d, J = 8.8 Hz).
Therefore, it can be confirmed that F2 is kaempferol 3-O-[(E) -p-coumaroyl- (1→2)] [α-L-arabinopyranosyl- (1→3)] [β-D-glucopyranosyl- (1→3) -α-L-rhamnopyranosyl (1→6)]-β-D-glucopyranoside, which has four glycosyls attached a p-coumaryl moiety. The structural formula is shown in Fig. 2.
Bai et al. extracted Lu'an GuaPian tea (9 kg) with 80% aqueous acetone three times and concentrated to a water-soluble extract. This water-soluble extract was applied to MCI-Gel CHP20P gel column chromatography (CC), Silica gel CC, Sephadex LH-20 CC and Toyopearl CC, then 18.7 mg compound 1 (F1) and 5.6 mg compound 9 (F2) were obtained. In the above study, the workload of initial extracting materials is relatively large. Complex column chromatography is used for separating AFGs with higher purity. In the present work, F1 and F2 with high purity can be obtained through a relatively simple process. High purities of F1 and F2 are helpful to a better quantitative detection with the HPLC or UPLC method. It is also convenient to the quantification of these two AFGs in different tea cultivars and different season samples. Besides, simple experimental steps and instruments make two AFGs in bulk in industrial production possible, large scale production will further promote their application. Using tea as a raw material to extract AFGs with high safety and limited side effects, leads to good practical significance and market prospects.
Seasonal changes in the contents of F1 and F2
The cultivar differences in these two AFGs stimulated our interest to explore their distribution in other tea cultivars. Hence, fresh leaves of fifty tea cultivars widely cultivating in Zhejiang, Fujian, Anhui, Hunan, Guangdong, Guizhou, and Jiangxi provinces and growing in the same tea garden were collected on the same autumn day and dried for further analysis. The contents of F1 and F2 in each cultivar are listed in Supplemental Table S6 and the results are displayed as a heatmap (Fig. 4). This chart highlights the notable variation in F1 and F2 among different tea cultivars. In general, the F1 content ranged from 0−1.35 mg/g and F2 content ranged from 0−1.17 mg/g. 'Zhenghe Dabaicha' had the highest F1 content of 1.35 mg/g. 'Longjing 1', 'Tiantai Nanshanzhong', 'Zhenong 21', 'Ruian Qingmingzao', 'Bibo', 'Maoxie', 'Meizhan', 'Benshan', 'Fujian Shuixian', 'Qimen Fuxikouzhong', 'Hunan Yuntaishan', and 'Lushan Yunwu' had relatively high F1 content at around 1.0 mg/g. In contrast, F1 levels were undetectable in 'Yingshuang', 'Jingfeng', 'Fuding Dahaocha', 'Qilan', 'Lvya Foshou', and 'Ziya Foshou' samples. 'Wannong 95' had the highest F2 level of 1.17 mg/g, followed by 'Zaohuangcha' 0.64 mg/g and 'Zhenghe Dabaicha' 0.55 mg/g, respectively. F2 was undetectable in 'Jingfeng', 'Zisun', 'Fuding Dahaocha', 'Qilan', 'Lvya Foshou', and 'Ziya Foshou'. Generally, the level of F1 was higher than that of F2 in most tested tea cultivars, although 'Wannong 95' had an especially high F2 level which was about 2-fold above its F1 level. The content of tea flavonol glycosides have been analyzed by many researchers. Jiang et al. analyzed the content of flavonol glycosides from green tea, oolong tea and black tea commercial samples by UHPLC. The abundance of kaempferol rhamnodiglucoside varied from 0.392 mg/g to 1.183 mg/g in green tea, quercetin glucorhamnoglucoside changed from 0.759 mg/g to 0.844 mg/g in oolong tea, quercetin glucoside ranged from 0.805 mg/g to 1.410 mg/g in black tea samples. Wu et al. quantified the content of six flavonol glycosides from four tea cultivars, the results showed that myricetin 3-O-galactoside was the major component reaching the highest level at 2.018 mg/g in second leaves of the 'Shuchazao' cultivar. Zheng et al. reported 13 flavonol glycosides in green tea, oolong tea, and black tea made from four cultivars, the highest level of kaempferol-3-O-glucosyl-rhamnosyl-glucoside was observed at 1.251 mg/g. Hence, the content of two AFGs obtained from this work are comparable to those flavonol glycosides.
Heat-map of the content of F1 and F2 in different tea cultivars.
Furthermore, fifty cultivars originated from different provinces and have complex genetic backgrounds, and no other detailed information is available, such as a specialized physiological role of flavonol glycosides in particular cultivars that could explain the observed results. Jungblut et al. found that the acylated flavonol 3-O-glucoside in the epidermal layer of Scots pine (Pinus sylvestris) needles could dramatically increase the absorption of UV-B. Intriguingly, Tohge et al. also found a class of acylated-flavonols in Arabidopsis accessions selected from those grown in natural sunlight and they suggested acylated modification in flavonoids was an adaption strategy to reduce UV stress in plants. The cultivars of 'Ruian Qingmingzao', 'Bibo', 'Maoxie', 'Meizhan', 'Benshan', 'Fujian Shuixian', and 'Hunan Yuntaishan' which have relative high contents of F1 originated from places with high sunlight irradiation (from Chinese meteorological data) and the question of whether the accumulation of AFGs is related to sunlight irradiation requires further investigation.
Antibacterial activities against P. gingivalis and F. nucleatum and biofilm development of F1, F2, and their aglycones
The antibacterial activity of F1 and F2 against two principle periodontopathic bacteria, P. gingivalis and F. nucleatum, was investigated by determining the minimum inhibitory concentration (MIC) for each compound. The MIC values of two purified compounds and their aglycones, quercetin and kaempferol, are shown in Supplemental Table S7. For P. gingivalis, quercetin had the lowest MIC value of 0.03 mM, while kaempferol's MIC value was higher than 0.48 mM. F1 and F2 had the same MICs which were lower than that of kaempferol, but higher than that of quercetin. Thus, F1, F2, and quercetin could efficiently inhibit the growth of P. gingivalis. The MICs of F1, F2, quercetin, and kaempferol against F. nucleatum were 0.12 mM, 0.24 mM, 0.12 mM, and 0.48 mM, respectively. Compared to kaempferol, F1, F2, and quercetin again had higher antibacterial activities against F. nucleatum and these two AFGs had an equivalent inhibitory activity to quercetin.
Based on the MIC values of tested compounds, the biofilm inhibitory rates of four tested compounds on P. gingivalis and F. nucleatum were evaluated by crystal violet staining. The results in Fig. 5a showed that all compounds had a dose dependent effect on P. gingivalis and increasing the concentration of test compounds produced a stronger inhibitory rate. Apart from kaempferol, 0.12 mM of F1, F2, and quercetin strongly inhibited the biofilm formation of P. gingivalis by more than 75% and F1 and quercetin at a concentration of 0.24 mM could inhibit biofilm formation of P. gingivalis by over 80%. As depicted in Fig. 5b, all tested compounds could inhibit over 70% biofilm formation of F. nucleatum at a high concentration of 0.24 mM. F2 at 0.12 mM had the highest inhibitory value (60%) of all tested samples. Compared to the inhibitory values of F. nucleatum, the tested samples seemed to have a greater inhibitory effect on P. gingivalis biofilm.
Effects of F1 and F2 and their aglycones on inhibition of biofilm formation by P. gingivalis (a) and F. nucleatum (b). Control is the treatment without drugs, and the vertical axis is the inhibition rate of biofilm production (%), different letters (a, b, c) for the same compound indicate significant difference (p < 0.05).
Numerous published works have focused on the evaluation of the anticariogenic potential of medicinal plants. The primary factor in periodontal disease is biofilm formation and its dynamic complexity. Theoretically, inhibition of the early steps in this process can result in the prevention of periodontal diseases, as has been the focus in developing vaccines against this pioneer microorganism species. Inhibition of the formation of biofilm is the key step required to reduce the pathogenic effect of bacteria. P. gingivalis is one of the key members for early colonization in biofilm formation. F. nucleatum plays a key role as a 'bridge' between early and late colonizers in the oral biofilm. By bridging the gap between these two separate groups of oral bacteria, the adhesin genes of F. nucleatum act outside the commonly observed narrow range of individual inter-species interactions, and join many bacteria together to form a single community.
To prevent oral disease, the effective removal of plaque biofilm in the oral cavity plays a key role in dental caries prevention. Lots of plant flavonoids were reported to adhere to hard and soft oral tissues, microbes' proteins, and prevent biofilm formation on tooth surfaces[36,37].
In previous studies, flavonol aglycones and their glycosides showed good antibacterial capacities on key oral pathogens. Patra et al. evaluated the antibacterial activities of quercetin and quercetin 3-glucoside, the results showed that they both had strong growth inhibition activity against the oral pathogen S. mutans. Muhammad et al. evaluated the antibacterial potential of quercetin, quercetin-3-O-glucoside, quercetin 3-O-glucuronide, kaempferol 3-O-rutinoside, kaempferol 3-O-glucuronide, kaempferol 7-O-neohesperidoside according to planktonic minimum inhibitory concentration (PMIC) and planktonic minimum bactericidal concentration (PMBC). These compounds showed different antibacterial effects on different stains, and quercetin was proved to be an excellent antibacterial agent which could inhibit periodontal pathogen growth and biofilm formation by affecting the metabolic activity and architecture of mature, multispecies, pathogenic biofilm.
Here, two isolated AFGs were found to have the equivalent inhibitory abilities on the growth of P. gingivalis and F. nucleatum, with a similar effectiveness to quercetin. These two compounds also have good inhibitory activities on the biofilm formation of two oral pathogens in a dose-dependent manner. Different from flavonol glycosides, these two AFGs had a special p-coumaryl moiety. Tiliroside, which has a 6"-O-p-coumaroyl moiety, was reported to have higher antioxidant and cytoprotective activities, and enhanced Fe-chelating ability than astragalin which doesn't have p-coumaroyl moiety. The specific mechanism of p-coumaryl moiety on antibacterial activity is worthy of further research.
Ferric carrier activity of F1 and F2