Alzheimer’s disease (AD) pathology is characterized by Aβ protein aggregation and senile plaque accumulation in the brain [1
]. Aβ peptide is neurotoxic and plays a role in neuronal cell death in AD [2
]. Tea is one of most popular beverages globally and has been ascribed a number of health benefits [3
]. The relationship between exposure to tea extracts and neuronal protective effects has been widely investigated in both in vivo and in vitro models [4
]. Many compounds in tea have been found to have neuroprotective effects in vitro associated with detoxifying Aβ fibrils and aggregates, such as catechins, flavonol glycosides and gallic acid, amongst others [6
]. Clinical studies have also revealed that people who consume tea may have a slower decline in cognitive function [10
According to the different manufacturing methods, tea can be divided into six types: white tea (WT), green tea (GT), oolong tea (OT), black tea (BT), yellow tea and dark tea. WT is a type mainly produced in Fujian, China which has the least processing steps comprising only withering and drying. The feature process of WT is prolonged withering, which reduces the moisture content from 75% in the tea leaves to 20% and allows slight fermentation by endogenous enzymes [13
]. Studies have already uncovered some relatively potent bioactivities of WT, such as antioxidant [15
] and anti-inflammatory actions [16
]. However, whether white tea can protect neuronal cells from Aβ-evoked toxicity has not been investigated.
In this study, one batch of tea leaves from Camellia sinensis (Jin Guanyin) was used to produce GT, OT, BT, and WT, the four main tea types produced in Fujian Province, China. Neuronal cell protective effects of the tea extracts were then investigated under oxidative stress conditions (t-bhp and H2O2) or following neurotoxic Aβ exposure. In addition, the anti-amyloid effect of each tea was compared using the ThT assay of Aβ fibrillization kinetics and TEM was used to investigate the physical structure of Aβ aggregates. Finally, analyses by UPLC-QTOF-MS and UPLC-QqQ-MS, in combined with chemometrics, were used to compare leaf constituents among the four tea types and dried leaves (DL) to provide insight regarding potential neuroprotective compounds.
WT and other tea extracts failed to rescue PC-12 cells from toxicity mediated by exposure to the oxidative stressors t
-bhp and H2
. These results are consistent with the effects of major constituent neuroprotective bioactive compounds in tea, such as EGCG, as shown from previous studies [4
]. Although many studies have demonstrated the antioxidant activity of tea and its catechins in vitro
], the time exposed to oxidative stress is usually short (a few hours). Longer times of exposure to ROS, as in the current study, may be more effective at mimicking the constant ROS toxicity occurring in AD-affected brain neurons. Furthermore, EGCG has also been reported to induce cell death via oxidative stress through H2
-dependent T-type Ca2+
channel opening, or by cell autophagy [20
However, WT and other tea extracts significantly prevented the loss of cell viability following Aβ treatment. ThT and TEM results also further supported the anti-aggregative ability of each tea type. TEM further supported that the most effective inhibition of the aggregation effect of WT and GT is by modifying Aβ into seldom aggregate morphology. However, only under WT treatment does the Aβ morphology became amorphous and punctate, indicating a different anti-aggregative effect of WT under TEM observation.
Distinct chemical profiles in tea which have been reported to possess Aβ anti-aggregative or neuroprotective effects mostly comprise catechins, theaflavins, amino acids and flavonol or flavone glycosides [5
]. Catechins are representative of the main chemical profiles of tea, which usually account for 30–42% of the dry weight, while EGCG and ECG are the two main polyphenols that account for 50–80% of total tea catechins. Studies have shown that gallated catechins like EGCG and ECG were able to protect neuronal cells by inhibiting Aβ aggregation, but this does not extend to non-gallated catechins like EC or EGC [4
]. Further reports demonstrated that EGCG converts toxic Aβ into SDS-stable, off-pathway and non-toxic oligomers by rotating the galloyl moiety in the B-ring and D-ring [24
]. Interestingly, EGCG and ECG levels in WT were not significantly different with OT, but non-gallated catechins were. This is similar to a previous study that found whole WT processing decreased EGCG and ECG levels by 10% when compared to fresh leaves, while EC, EGC, C and GC decreased by 50%, 40%, 60% and 60%, respectively [27
]. Although EGCG content in WT was higher reserved than non-gallated catechin, it was still lower than that in GT and OT. Although EGCG and ECG retained in WT may contribute to its anti-aggregative ability, they may not the key compounds in WT that differentiate the neuroprotective activity of WT from other tea types. It should be noted that although BT was comparatively deficient in the gallated catechins notable for their neuroprotective effects and inhibition of amyloid β aggregation, it still afforded the same degree of neuroprotection as other tea types. This may be due to the higher levels of theaflavin gallates and free gallic acid, which also share similar neuroprotective and anti-amyloid properties [5
]. This may serve to highlight the general health benefits of broader tea consumption generally.
EGCG or ECG in tea can also form derivatives or oxides into catechin derivatives or theaflavins [29
]. ECG‘’3Me and 8-C
-ascorbyl-EGCG are two catechin derivatives that have similar structures with their catechin precursors, and were shown to have the highest levels in WT (Figure 7
and Figure S2
). Catechin derivatives with new structures and activities have been occasionally reported from diverse tea cultivars or tea types [31
]. Interestingly, in a recent study, two N
-ethyl-2-pyrrolidinone-substituted catechin derivatives were found in aged white teas for the first time [33
]. They possess similar structures with their catechin precursors. Whether they are also relevant to inhibiting Aβ aggregation by WT requires further experimentation. We speculate that the high content of catechin derivatives in WT may play a key role in its special anti-aggregate ability formation.
Twelve flavonol or flavone glycosides have been identified in different tea types. Reports showed that many flavonol or flavone glycosides possess anti-aggregative and neuroprotective effects similar to their aglycones [34
]. A majority of flavonol or flavone glycosides in this study were found at their highest levels in WT. Studies reported that fixed, rotated, fermented and dried steps during tea processing can cause the loss of flavonol or flavone glycosides [36
]. Thus, the accumulation of flavonol or flavone glycosides in WT may also render this tea type with a favorable flavonoid composition conferring anti-amyloid and neuroprotective effects.
Many amines and amino acids were found at their highest levels in WT, including GABA and Gln. GABA is a major inhibitory neurotransmitter in the central nervous system, while GABA derived from natural products has neuroprotective actions [23
]. GABA normally occurs in plants at low levels, but increases following exposure to a range of stressors [39
]. GABA has been shown to protect PC-12 cells from kainic acid excitotoxicity by depressing caspase-3 expression [41
]. Additionally, Aβ has been found to elicit GABA-A receptor endocytosis [42
] and GABA-A receptor subunit loss is found in AD-affected brains [43
]. GABA can also downregulate Aβ-evoked endocytosis to protect neuronal cells [44
]. Gln has been reported to activate expression of Hsp70 to protect PC-12 cells from α-synuclein toxicity [45
]. Hsp70 activation can also partly mitigate Aβ-evoked cell toxicity [46
], while Neuro 2A cells low in Gln are more sensitive to the neurotoxic effects of Aβ [47
] and Gln conjugated nanoparticles inhibit amyloidogenesis [48
]. Therefore, accumulation of amine-containing compounds like GABA and glutamine in WT may provide a basis for the varying anti-aggregative effect not seen in other tea types.
4. Materials and Methods
Human Aβ1–42 protein was obtained from rPeptide (Bogart, GA, USA) and Merck Millipore (Bayswater, VIC, Australia). ThT, MTT, Trypan Blue, DMSO, RPMI-1640 medium and FCS were obtained from Sigma-Aldrich (St. Louis, MO, USA). NEAA, penicillin/streptomycin, 10 trypsin EDTA and PBS at pH 7.4 were obtained from Thermo Fisher Scientific (Scoresby, VIC, Australia).
EGCG, EGC, C, ECG, EC, GC, GCG, que-3-glu, gallic acid and amino acids (all with purity ≥ 95%) were obtained from Sigma-Aldrich (St. Louis, MO, USA). EGCG3’’Me (≥95%) and kae-3-rut (≥98%) were purchased from ChemFaces (Wuhan, China). Acetonitrile (MS grade) and methanol (HPLC grade) were obtained from Sigma-Aldrich. Deionized water was produced by a Milli-Q water purification system (Millipore, Billerica, MA, USA).
4.2. Tea Samples Preparation for Cell Culture and Chemical Analysis
Twenty-five kilograms of fresh tea leaves of the Camellia sinensis
(Jin Guanyin) variety (one bud with two or three leaves) were collected from the tea garden at Qilin Mountain Tea Factory, Fujian, China. These tea leaves were then divided into five portions for the manufacturing of DL, GT, OT, BT and WT at the tea factory of Fujian Agriculture and Forestry University by the manufacturing procedures illustrated in Figure S1
For cell culture experimentation, 25 g of dried tea powder were extracted with water for 30 min at 80 °C and then freeze-dried into powder after filtering. The tea extracts were stored at −80 °C and diluted by PBS until use.
Chemical profiling analysis was performed according to previous methods [49
]. Briefly, freeze-dried tea leaves were individually ground to fine powders using precooled mortar and pestle. Following lyophilization, 30 mg (±0.5 mg) of ground samples were weighed and 1.2 mL of 70% (v
) methanol was added for metabolite extraction. Samples were vortexed, sonicated at 25 °C for 20 min and centrifuged (10 min, 12,000 g). Supernatants were diluted 50-fold with 70% (v
) methanol, filtered through a 0.22 µm PVDF filter (Millipore) and stored at −20 °C until analyzed. Three biological sample replicates were prepared for each tea types.
4.3. Cell Culture
PC-12 cells (Ordway) displaying a semi-differentiated phenotype with neuronal projections were kindly donated by Professor Jacqueline Phillips (Macquarie University, NSW, Australia) [51
]. Cells were maintained in RPMI-1640 media with 10% FCS, 1% Gln, 1% NEAA and 1% penicillin/streptomycin. Cells were seeded at 2 × 104
cells per well in RPMI-1640 with 10% FCS. PC-12 cells were equilibrated for 24 h before treatment with test compounds and/or Aβ.
4.4. Cell Treatment and Cell Viability Measurements
Ninety-six well plates containing 2 × 104
PC-12 cells per well were treated with each of the tea extracts (50, 100 or 150 µg/mL) or vehicle (PBS) in appropriate wells. The plates were then incubated for 15 min at 37 °C with 5% CO2
, with or without tea extracts. Plates were then incubated with either t
-bhp (50-400 μM) or H2
(400 µM) for 24 h respectively (n
= 4 each), or with Aβ (0.5 µM) for 48 h (n
= 6). The concentration setting of t
and Aβ were according to previous studies used in PC-12 cells model [4
PC-12 cell viability was determined using the MTT assay. After incubation, removed culture media was replaced with serum-free media containing 0.25 mg/mL of MTT. The plate was further incubated for 2 h, MTT solution removed and cells lysed with DMSO. Absorbance was measured at 570 nm using a Synergy MX microplate reader (Bio-Tek, Bedfordshire, UK).
4.5. Aβ Preparation
Native, monomeric pre-fibrillar Aβ was prepared by dissolving in 1% DMSO to yield a protein concentration of 3.8 mM. Sterile PBS was added to prepare a final concentration of 100 μM. Aβ protein was then dispensed into aliquots and immediately frozen at −80 °C until required.
4.6. ThT Assay and TEM of Aβ Fibril and Aggregate Formation
ThT (10 µM in PBS) was added to wells on a black microplate with or without Aβ and tea extracts (100 μg/mL, n = 4). Fluorescence was then measured at 37 °C every 10 min for 48 h using a microplate reader (Bio-Tek, Bedfordshire, UK) with excitation and emission wavelengths at 446 nm and 490 nm respectively. ThT output from all treatment groups was normalised to appropriate blanks (ThT with or without tea extracts).
TEM samples were prepared by incubating native Aβ with or without tea extracts (100 μg/mL) for 48 h at 37 °C. A 400 mesh formvar carbon-coated nickel electron microscopy grid (Proscitech, Kirwan, QLD, Australia) was used. A 5 µL sample was placed onto this grid and after 1 min this sample was blotted off using filter paper. Ten microliters of contrast dye containing 2% uranyl acetate was then placed onto the grid, left for one minute and blotted off with filter paper. Grids were then loaded onto a specimen holder and then into a FEI Tecnai G2 Spirit Transmission Electron Microscope (FEI, Milton, QLD, Australia). Sample grids were then viewed using a magnification of 34,000–92,000×. Grids were extensively scanned manually in search of fibrils and representative images were taken.
4.7. UPLC-QqQ-MS Based Targeted Quantification of Catechins, Que-3-Rut, Caffeine and Amino Acids in Different Tea Types
Two microliters of tea extracts or range of the calibration curve were injected on a Waters Acquity UPLC system with Waters photodiode array detector and a XEVO TQ-S MS triple quadrupole mass spectrometer (Waters, Milford, MA, USA).
To detect catechins, que-3-rut and caffeine, chromatographic separation was achieved on a Waters Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.7 µm) at 40 °C with water containing 0.1% formic acid (phase A) and acetonitrile containing 0.1% formic acid (phase B) for chromatographic elution: as previously described [54
]. The flow rate was set at 0.3 mL/min. Mass spectrometry was performed in the ESI-
for catechins and que-3-rut, ESI+
for caffeine under the same settings as previously described [54
]. Collision energy and cone voltage were optimized for above compounds with multiple reaction monitoring for quantification. Calibration curves generated by injecting increasing concentrations of chemical standards were used to determine the absolute concentrations of catechins, que-3-rut and caffeine.
Amino acids were detected in the same manner except that the chromatographic separation was achieved on a Merck SeQuant ZIC-HILIC column (2.1 × 100 mm, 5 µm) at 40 °C with water containing 5 mM ammonium acetate (phase A) and acetonitrile containing 0.1% formic acid (phase B) following our previously published protocol [54
]. The flow rate was set at 0.4 mL/min. Mass spectrometry was performed in the ESI+
mode using the same setting for caffeine. The MassLynx software (version 4.1, Waters, Milford, MA, USA) was used for instrument control and data acquisition.
4.8. UPLC-QTOF MS-Based Non-Targeted Metabolite Analysis of Different Tea Types
The metabolomics measurements and analysis were carried out according to our previous method [54
]. Briefly, one microliter of the metabolite extract was injected into an Acquity UPLC system coupled in tandem to a photodiode array detector and a SYNAPT G2-Si HDMS QTOF mass spectrometer (Waters, Milford, MA, USA). Separation was achieved on a Waters Acquity UPLC HSS T3 column (2.1 × 100 mm, 1.8 µm) thermostat controlled at 40 °C using a gradient from solvent A (water with 0.1% formic acid) to solvent B (acetonitrile with 0.1% formic acid). The flow rate was set at 0.3 mL/min. Data was collected in the ESI mode, scanning from 50–1200 m
. QC samples were prepared by mixing an equal amount of each sample to become a combined sample, and were injected every five samples throughout the runs to monitor the instrument performance. The MassLynx software (version 4.1, Waters, Milford, MA, USA) was used to control of the instruments. Each tea sample was analyzed in triplicates.
4.9. Data Processing, Metabolite Identification, and Statistical Analysis
Data obtained from the MTT assay was analyzed via a two-way analysis of variance (ANOVA) to assess neuronal cell viability arising from incubation in H2O2, t-bhp or Aβ, alone or in the presence of tea extracts, with a Bonferroni’s post hoc test used to determine the significance level for each tea extract treatment. Area under the curve analysis for ThT fluorescence data was analyzed using one-way ANOVA with a Dunnett’s multiple comparisons test used for determining the significance of each extract vs. Aβ. Data of chemical profiles of tea was analyzed via Tukey’s HSD test. Resulting chromatograms from UPLC-QTOF MS were processed in Progenesis QI. After filtering, a total of 381 single molecular features were used as inputs for PCA and Loading Plot to observe intrinsic metabolite variances between tea types using Progenesis QI extension EZinfo after Pareto scaling. Differential metabolites among tea samples with VIP >1 and p value < 0.05 were identified. Data analysis and production of graphs was performed in GraphPad Prism 6 for Windows (GraphPad Software, San Diego, USA) or SPSS (version 19.0, Chicago, IL, USA).
In summary, WT and other tea types failed to protect neuronal cells from oxidative stress. However, each tea type significantly increased neuronal cell viability following Aβ exposure. ThT fluorescence kinetics and TEM observation showed that Aβ aggregate formation was dramatically inhibited by each tea type. TEM also supported that the most effective inhibition of the aggregation effect of WT and GT was by modifying Aβ into seldom aggregate morphology. However, amorphous and punctate Aβ morphology was observed only following WT treatment, indicating a different anti-aggregative effect of WT under TEM observation. Chemical analysis revealed that EGCG and ECG in WT were significantly lower than in GT, but accumulation of other bioactive compounds such as ECG3′’Me, 8-C-ascorbyl-EGCG, que-3-glu-rut, myr-3′-glu, myr-3-rob or 3-neo, eri 5,3′-di-glu and other flavonol or flavone glycosides may underlie the anti-aggregative and neuroprotective effect of WT. Particularly, GABA and glutamine levels were much higher in WT than other tea types, and this may also facilitate the neuroprotective effect of this tea type. Further studies are needed to characterize any discrete neuroprotective effects of some of these compounds found in WT and in vitro findings need to be substantiated via appropriate in vivo paradigms.