Investigation of free amino acid, total phenolics, antioxidant activity and purine alkaloids to assess the health properties of non-Camellia tea

2.1Collection of tea samples

The 33 non-Camellia teas (119 accessions) were collected in China from 2008 through 2013, the 9 Camellia teas (3 green teas, 3 pu-erh teas and 3 black teas, respectively) were purchased from local retailor shops (Table 1). Except for the teas from Hibiscus sabdariffa, Chrysanthemum indicum, Dendranthema morifolium and Coreopsis tinctoria are derived from flowers, Fagopyrum tataricum and Cassia obtusifolia are from seeds, the plant part of all other teas are from leaves. All samples were kept sealed and stored in dry and cool places before testing.

The plant species of the tea samples were authenticated by Dr. Peigen Xiao and all species were validated taxonomically as described on the web site www.theplantlist.org. The voucher specimens were deposited in Xiao׳s laboratory at the Institute of Medicinal Plant Development of the Chinese Academy of Medical Sciences in Beijing, China.

2.2Chemicals and standards

AccQ·Tag™ Ultra UPLC™ amino acid analysis derivatization kits were purchased from Waters (Milford, MA, USA). The 20 FAAs, caffeine, theobromine, theophylline and gallic acid standards were purchased from Winherb Medical Technology Co., Ltd. (Shanghai, China). Mixed amino acid standard stock solutions of 2.5 mmol/L were prepared in 0.1 mol/L HCl and stored at 4 °C. Diluted solutions were freshly prepared weekly.

HPLC-grade acetonitrile was purchased from Honeywell Burdick & Jackson (Morristown, NJ, USA). HPLC-grade methanol was from Fisher Scientific (Atlanta, GA, USA) and hydrochloric acid, formic acid, and ortho-phosphoric acid were from CNW Technologies GmbH (Dusseldorf, Germany). Sodium acetate and triethylamine were purchased from Guangfu Technology Development Co. (Tianjin, China). Sodium azide, α-aminobutyric acid (AABA, internal standard), 1,1-diphenyl-2-picryl-hydrazyl (DPPH) and Folin-Ciocalteu phenol reagent were from Sigma-Aldrich (St. Louis, MO, USA). Sodium carbonate was purchased from Beijing Chemical Works (Beijing, China). Trolox solution (10 mmol/L) was purchased from Beyotime Biotechnology (Haimen, Jiangsu, China). All reagents used were of analytical grade. All solutions were made with Milli-Q water (Millipore, Bedford, MA). Eluent A concentrate for gradient elution contained sodium acetate (1.4 mol/L), sodium azide (1.5 mmol/L), disodium EDTA (2.6 mmol/L) and tri-ethyl amine (170 mmol/L) in water, and was titrated to pH 4.95 with phosphoric acid.

2.3Preparation of tea samples

Tea samples were prepared as follows: 250 mg (for amino acid and antioxidant analysis) and 150 mg (for total phenolics and purine alkaloids analysis) of teas were extracted with 10 mL of distilled water at 80 °C for 30 min respectively. After the tea water extract was cooled to room temperature, the volume was brought back to 10 mL with water. The sample solutions were filtered through a 0.22 μm nylon membranes purchased from Jinteng experiment equipment Co., Ltd. (Tianjin, China).

2.4Analysis of total polyphenols

The total phenolic content of the tea samples was determined according to a procedure described by Ainsworth and Gillespie25 with slight modifications. One hundred microliters of each filtered sample solution was mixed with 200 μL of 10% (v/v) F-C phenol reagent and 800 μL of 700 mmol/L sodium carbonate solution in a 2-mL microtube for 2 h before spectrometric analysis. Finally, 200 μL of the sample, standard or blank was transferred to clear 96-well microplates, and the absorbances read at 765 nm using the Quant MQX200 microplate reader from Biotek Instruments, Inc. (Winooski, VT, USA). Gallic acid was used as standards for quantification, and the results were expressed as percent gallic acid equivalents (GAE).

2.5Antioxidant assay

The antioxidant activities of the tea samples were evaluated by DPPH method as previously reported, with some modifications26, 27, 28. Briefly, 7 μL of the diluted sample or Trolox (six different concentrations) or ethanol was added to 193 μL of 0.2 mmol/L DPPH (freshly prepared prior to assay), then left at ambient temperature in the dark for 30 min. The absorbance was measured at 517 nm. Trolox and ethanol were used as the reference and control, respectively. The DPPH radical scavenging activity was calculated using the following formula:(1)DPPHRadicalScavengingActivity(%)=[(A0-A1)/A0]×100where A₁ is the absorbance of the test sample, and A₀ is the absorbance of the control. Results are expressed as EC₅₀ and values are referred to the concentration of the tea infusions required for the 50% of the antioxidant activity (µg/mL).

2.6Derivatization reaction of free amino acids

Precolumn 6-aminoquinolyl-N-hydrosysuccinimidyl carbamate (AQC) derivatization of FAAs was accomplished using a Waters AccQ·Tag™ reagent kit. Amino acid standards or tea samples (10 μL) were derivatized directly by mixing with 70 μL AccQ·Tag™ borate buffer. After adding 20 μL derivatizing reagent (10 mmol/L AccQ·Tag™ reagent), the mixtures were immediately vortexed, left to rest for 1 min at room temperature and finally heated for 10 min at 55 °C to complete the derivatization. Derivatized sample solutions were then subjected to chromatographic analysis. The concentration level of each analyte was approximately as follows: 0, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, 50, 100, 200 and 400 pmol/μL.

2.7UHPLC instrument and chromatographic conditions

The analysis of FAAs and alkaloids was carried out by using the DIONEX Ultimate 3000 UHPLC system equipped with a HPG3400 RS Pump, SRD-3400 degasser, WPS-3000T RS AutoSampler, TCC-3000RS Column Compartment, DAD-3000RS Diode Array Detector and Chromeleon chromatography software package (6.8 version).

UHPLC separation of AQC-derivatized amino acids was performed with a Waters Acquity UPLC™ column (BEH C18, 100 mm×2.1 mm, 1.7 μm). The mobile phase A was Eluent A concentrate diluted 1:10 with ultrapure water while B was 60% (v/v) acetonitrile. The temperature of the column oven was set at 37 °C. The elution program is described in Table 2. After the program, the initial conditions were regenerated within 0.5 min and maintained for another 4.5 min, resulting in a net separation time of 11.5 min and a 16 min total cycle time. The injection volume was 2 μL, and UV detection of AQC amino acid derivatives was performed at 254 nm. Data were archived using Chromeleon chromatography software package. All of the operations and the data acquisition were controlled by a DIONEX ChemStation.

The caffeine, theobromine and theophylline content of the tea samples were determined according to our previously reported procedure29. Briefly, UHPLC was performed with DIONEX Acclaim PA2 column (150 mm×2.1 mm, 2.2 μm). The mobile phase A was acetonitrile, while B was ultrapure water. The flow rate was set to 0.5 mL/min, and the temperature of column oven was set at 30 °C. Elution was accomplished with a linear gradient of phase A from 5% to 15% in 10 min. The injection volume was 10 μL, and UV detection of these alkaloids was performed at 272 nm.

2.8UHPLC method validation

The precision, repeatability, stability, detection and quantification limits, linearity ranges and recovery of the proposed method were validated following the guideline of the International Conference on Harmonisation (ICH)30. To evaluate the precision of the method, a standard solution was injected six times successively. To get the repeatability of the method, six replicate analyses of a standard solution were performed to determine both the retention time and the peak area of the amino acid standards. The stability was assessed by injecting a standard solution six times within 0, 2, 4, 6, 8, 16, and 24 h, respectively. Detection and quantification limits (LOD and LOQ) were calculated at the respective signal-to-noise ratios of 3 and 10. Linear calibration curves were calculated over a concentration range of 0.001–1600 pmol per 2 μL injection volume. The recovery was assessed by the experiments in which the known amounts of AMQ (6-aminoquinoline)–amino acid standards (0.01, 0.50 and 1 mmol/L) were added to the tea samples. Each standard was analyzed in triplicate, and the peak area was plotted against the corresponding concentrations.

2.9Statistical analysis

Mean values of each sample were obtained from at least three replications. The hierarchical cluster analysis was conducted by R 3.1.0 with package Pheatmap 0.7.7 (Free Software Foundation). The Python 2.7.8 software (Python Software Foundation) was used for principal component analysis (PCA). Pearson correlation was performed by SPSS 17.0 (SPSS Inc., Chicago, IL, USA).

3.1Validation of FAA analysis method

The gradient elution program of the UHPLC method was optimized, and the final analytical method achieved a high resolution allowing the identification of target compounds in a short time (Fig. 1A). All analyzed AQC derivatives exhibited appropriate linearity (R²≥0.9950) within the applied calibration range (10–400 pmol per 2 μL injection volume) at 254 nm.

For most of the amino acids, the instrumentation precision of relative standard deviation (RSD) of amino acid standards varied <3%, whereas the repeatability (RSD) was <5% for all analytes. The stability of amino acid standards (RSD) varied <4%. Limits of detection and quantification (LOD and LOQ) were calculated at the respective signal-to-noise ratios of 3 and 10, with on-column amounts of AQC derivatives ranging between 0.05–0.10 pmol and 0.10–0.50 pmol (each per 2 μL injection volume), respectively. The recovery rate was calculated by comparing the obtained amounts with those added, and values ranged between 80% and 105%. Therefore, the overall UHPLC analytical procedure was fast, accurate and suitable for the quantitative analysis of a large number of samples.

3.2Quantitative analysis of selected FAAs in various non-Camellia teas

The new analytical method was subsequently applied to simultaneously determine 20 FAAs levels of 119 samples from 33 non-Camellia teas representing a variety of types. Green tea (34), black tea (35) and pu-erh tea (36) from Camellia leaves were also analyzed at the same time for comparison. The individual contents of the 20 FAAs in these samples are listed in Table S1.

The results showed that most non-Camellia teas had relatively moderate levels of the 20 FAAs (Table S2). The total amino acid content in green tea (34) (24.99 mg/g) was the highest followed by pu-erh tea (36), black tea (35), teas from Lycium barbarum (25), H. sabdariffa (5), Gynostemma pentaphyllum (23) and Scutellaria baicalensis (32), with contents ranging from 10.41 to 18.69 mg/g. Teas from C. tinctoria (30), Pistacia chinensis (29), Forsythia suspensa (18), Litsea coreana var. lanuginose (14), Adinandra nitida (12), Acer tataricum subsp. ginnala (28), D. morifolium (20), Rubus chingii var. suavissimus (6), C. indicum (19), and Scoparia dulcis (26) were at intermediate levels ranging from 5.24 to 9.70 mg/g. The other teas had lower concentrations ranging from 0.62 to 4.67 mg/g (Fig. 2A). Moreover, essential amino acid (EAAs) concentrations were high in black tea (35, 18.29 mg/g). Pu-erh tea (36), teas from H. sabdariffa (5), L. barbarum (25), P. chinensis (29), A. nitida (12) and green tea (34) with intermediate levels (4.62–8.17 mg/g). Other teas had lower levels (less than 4.50 mg/g) (Fig. 2B). HEAs are considered conditionally essential in the human diet, meaning their synthesis can be limited under special pathophysiological conditions. The content of HEAs was highest in tea from L. barbarum (25, 6.24 mg/g), and followed by green tea (34, 5.74 mg/g), teas from C. tinctoria (30, 4.86 mg/g) and S. baicalensis (32, 4.70 mg/g). Teas from G. pentaphyllum (23, 3.03 mg/g), P. chinensis (29, 2.91 mg/g), Orthosiphon aristatus (31, 2.88 mg/g), C. indicum (19, 2.54 mg/g), R. chingii var. suavissimus (6, 2.31 mg/g). Pu-erh tea (36, 2.13 mg/g) contained relatively large amounts of HEA ranging from 2.13 to 3.03 mg/g, while other teas had less than 1.60 mg/g (Fig. 2C). The three NEAs, which are dispensable in humans and can be synthesized in the body, existed at relatively high amounts in teas from L. barbarum (25, 3.08 mg/g), G. pentaphyllum (23, 3.12 mg/g), H. sabdariffa (5, 3.14 mg/g) and green tea (34, 2.85 mg/g), and less than 1.5 mg/g in the other teas (Fig. 2D).

Although the total content of amino acids in each non-Camellia tea is lower than the content in the three common Camellia teas, the contents of some specific amino acids are significantly higher in non-Camellia teas than in green tea. In terms of the essential amino acids, the respective Thr contents of each of the non-Camellia were: P. chinensis (29, 3.85 mg/g), L. barbarum (25, 2.67 mg/g), S. dulcis (26, 3.66 mg/g), S. baicalensis (32, 2.68 mg/g), L. coreana var. lanuginose (14, 2.57 mg/g) and D. morifolium (20, 2.75 mg/g). The respective Val contents were L. barbarum (25, 0.60 mg/g), S. baicalensis (32, 0.76 mg/g), R. chingii var. suavissimus (6, 0.35 mg/g), and A. nitida (12, 0.52 mg/g). Met contents were F. suspensa (18, 2.09 mg/g). The Lys content in F. suspensa was (18, 0.51 mg/g). The Phe contents respectively were L. barbarum (25, 1.33 mg/g), A. nitida (12, 3.03 mg/g) and G. pentaphyllum (23, 1.65 mg/g), significantly higher than in green tea (34, 1.95 mg/g, 0.17 mg/g, 0.56 mg/g, 0.29 mg/g, 0.53 mg/g, respectively). In terms of NEA, the content of Asp in teas from H. sabdariffa (5, 2.61 mg/g) and G. pentaphyllum (23, 2.75 mg/g) were significantly higher than in green tea (34, 1.24 mg/g).

In terms of HEA, Gly showed low contents in all the samples but tea from H. sabdariffa showed the highest content (0.21 mg/g). The content of Arg in tea from P. chinensis (29, 1.93 mg/g) was relatively higher than that in three Camellia teas. The content of Pro in teas from R. chingii var. suavissimus (6, 2.03 mg/g), C. indicum (19, 1.40 mg/g), L. barbarum (25, 3.91 mg/g), C. tinctoria (30, 4.41 mg/g), Orthosiphon aristatus (31, 2.48 mg/g) and S. baicalensis (32, 3.17 mg/g), were significantly higher than in three Camellia teas which ranged from 0.05 to 0.31 mg/g. Except for nine teas (including 4–8, 15, 16, 22, 33), Cys content in most non-Camellia teas also showed higher concentration than in the three Camellia teas.

Of note, GABA was detected in all teas except tea from Sarcandra glabra (15, Fig. 2E). Thea was detected in approximately half of the teas (Fig. 2F). The GABA content in teas from Ampelopsis grossedentata (10), Isodon serra (17) and H. sabdariffa (5) was the highest (all above 1.02 mg/g), much higher than that in green tea (34, 0.28 mg/g). For Thea, green tea (34) was found to have significantly higher content (10.61 mg/g) than the others. Pu-erh tea (36) was the second highest at approximately 4.59 mg/g. The only other tea with a Thea content above 1.0 mg/g was tea from Potentilla fruticosa (21, 1.16 mg/g). All others had less than 0.5 mg/g. We are the first group reporting these results, and further studies on more tea samples remain to be performed.

3.3Principal component analysis (PCA) of teas

The potential utility of employing PCA using a combination of the key parameters and the contents of the 20 FAAs as a means of classifying the teas was explored. The PCA provided three eigenvalues, which were analyzed to obtain three factors (Z1–Z3, Table S3). The proportion of each eigenvalue was computed, and the cumulative proportion of the three factors was found to total 90.61%. The three factors were retained for further analyses because their cumulative proportions were higher than 90%, which is considered adequate for the estimation of FAAs patterns. The eigenvectors of the patterns of the 20 FAAs used to obtain the three factors are summarized in Table S4. The plot of the PCA scores, as shown in Fig. 3A, was readily divided into two relative clusters, which indicates that the content and distribution of the 20 FAAs are highly varied in the three Camellia teas and 33 non-Camellia teas. The plots of the PCA loadings were utilized to identify the differential FAAs for the discrimination of groups. Gly and Arg were largely influenced by the first factor (PC1), whereas His was dominant by the second factor (PC2). Try, Cys and Arg were mainly influenced by the third factor (PC3, Fig. 3B).

A similar PCA was performed to analyze 33 non-Camellia teas based on content of the 20 FAAs. The cumulative proportion of the three factors was found to total 82.51% (Table S5), and the eigenvectors of the patterns of the 20 FAAs used to obtain the three factors are summarized in Table S6. The plot of the PCA scores, as shown in Fig. 3C, was readily divided into four relative clusters, indicating that the content and distribution of the 20 FAAs is highly varied in the different non-Camellia teas. The plots of the PCA loadings were utilized to identify the differential FAAs for the discrimination of groups. Gly, Arg and Try are largely influenced by the first factor (PC1), whereas His and Thr are dominant by the second factor (PC2). Try and Arg are mainly influenced by the third factor (PC3, Fig. 3D).

3.4Cluster analysis of non-Camellia tea based on FAA level

Hierarchical cluster analysis (HCA) was used to confirm the results of the PCA analysis. The content of the 20 FAAs obtained for the 33 non-Camellia teas was standardized into the Euclid equation to obtain the Euclidean distances between the samples. The cluster analysis provided a dendrogram, and Ward's minimum-variance cluster analysis (WMVCA) produced two large sub-clusters, which were denoted A and B (Fig. 4).

Cluster B was further divided into two sub-clusters, which were denoted II and III. Sub-cluster II, which is comprised of 3, 5, 14, 20, 26 and 29, was defined by relatively high contents of Gly and Thr. Sub-cluster III also consisted of six non-Camellia teas, including 6, 19, 25, 30, 31 and 32; this sub-clusters contained a high amount of Pro, Thr and Glu. Cluster I included 19 non-Camellia teas have relatively low content of the total amino acid (<4.0 mg/g) except 10, 18, and 28 (4.26, 6.77 and 5.91 mg/g, respectively). Among these teas, 10 and 17 with high content of GABA could be considered together for a separate class. Cluster IV included two non-Camellia teas (12 and 23) which exhibited relatively high contents of Phe, Asp and Glu.

HCA was also conducted to accurately describe the content characters among the 20 FAAs (Fig. 4). Thr was the FAA with the highest average content of (1.42 mg/g), followed by Pro (0.69 mg/g). GABA, Phe, Glu and Asp were at intermediate levels in all 20 FAAs ranging from 0.27 to 0.40 mg/g, while the other FAAs had a lower content ranging from 0.09 to 0.25 mg/g.

3.5Total phenolic content of non-Camellia tea

Fig. 5A shows the total phenolic content of the non-Camellia tea samples (data presented in Table S7). Most of the samples had high polyphenol content. Tea from A. grossedentata (10) had the highest total phenolic content (177.25 mg GAE/g) among all of the non-Camellia teas. It had over 2 times higher phenolic content than green tea (34, 80.07 mg GAE/g), black tea (35, 39.77 mg GAE/g) and pu-erh tea (36, 67.82 mg GAE/g). Teas from A. tataricum subsp. ginnala (28), Mallotus oblongifolius (3) and P. fruticosa (21) all had a high phenolic content over 100.00 mg GAE/g. The total phenolic content of teas from I. serra (17, 97.84 mg GAE/g) and P. chinensis (29, 96.86 mg GAE/g) was relatively lower but still higher than that of green tea. Furthermore, teas from C. tinctoria (30, 81.92 mg GAE/g) and Engelhardtia roxburghiana (1, 82.62 mg GAE/g) had a similar level of polyphenolics as green tea. The polyphenol content of some samples of teas from Ligustrum robustum (11), A. nitida (12), Ilex latifolia (13) and L. coreana var. lanuginose (14) were much higher than comparable values from green tea, potentially due to different collection times or/and areas. The polyphenol content of teas from R. chingii var. suavissimus (6), F. suspensa (18), Malus hupehensis (22), Cratoxylum cochinchinense (24), S. baicalensis (32) and Lithocarpus litseifolius (33) was slightly lower than that of green tea. The other teas had much lower polyphenol content than found in green tea. Tea from C. obtusifolia (16) had the lowest polyphenol content (6.27 mg GAE/g).

3.6Antioxidant potential of non-Camellia tea

As shown in Fig. 5B (and Table S7), there were remarkable differences in the DPPH-scavenging capacity among non-Camellia teas. The EC₅₀ values varied from 38.41 μg/mL to 724.13 μg/mL. According to their antioxidant power, 33 non-Camellia teas can be divided in three groups: (a) strong (EC₅₀<100 μg/mL); (b) intermediate (EC₅₀, 100–500 μg/mL); (c) weak (EC₅₀>500 μg/mL). The EC₅₀ values of teas from I. serra (17, 38.41 μg/mL), P. chinensis (29, 41.83 μg/mL), and A. tataricum subsp. ginnala (28, 48.27 μg/mL) were significantly lower than those of the remaining non-Camellia teas and two Camellia teas, black tea (35, 176.23 μg/mL), pu-erh tea (36, 108.10 μg/mL), and had no significant difference with the green tea (34, 44.23 μg/mL) and the Trolox (17.67 μg/mL). In addition, correlation analysis showed that the DPPH-scavenging capacity of the non-Camellia teas was significantly correlated with their phenolic content (r=−0.5552; P<0.01) (Fig. 5C).

3.7Caffeine, theobromine, and theophylline in non-Camellia tea

The presence of caffeine, theobromine, and theophylline in the non-Camellia tea samples was determined by comparison to alkaloid standard chromatograms. The results show that these alkaloids were not present in any of the non-Camellia tea samples. In contrast, in the three common Camellia teas, caffeine and theobromine were detected, but theophylline was not detected (Fig. 6). Therefore, non-Camellia tea may be a more suitable beverage in some cases than Camellia tea due to the very low amounts of addictive and potentially harmful purine alkaloids.