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

To find novel functional beverages from folk teas, 33 species of frequently used non-Camellia tea (plants other than Camellia) were collected and compared with Camellia tea (green tea, pu-erh tea and black tea) for the first time. Data are reported here on the quantities of 20 free amino acids (FAAs) and three purine alkaloids (measured by UHPLC), total polyphenols (measured by Folin-Ciocalteu assay), and antioxidant activity (DPPH). The total amounts of FAAs in non-Camellia tea (0.62–18.99 mg/g) are generally less than that of Camellia tea (16.55–24.99 mg/g). However, for certain FAAs, the quantities were much higher in some non-Camellia teas, such as γ-aminobutyric acid in teas from Ampelopsis grossedentata, Isodon serra and Hibiscus sabdariffa. Interestingly, theanine was detected in tea from Potentilla fruticosa (1.16±0.81 mg/g). Furthermore, the content of polyphenols in teas from A. grossedentata, Acer tataricum subsp. ginnala are significantly higher than those from Camellia tea; teas from I. serra, Pistacia chinensis and A. tataricum subsp. ginnala have remarkable antioxidant activities similar to the activities from green tea (44.23 μg/mL). Purine alkaloids (caffeine, theobromine and theophylline) were not detected in non-Camellia teas. The investigation suggest some non-Camellia teas may be great functional natural products with potential for prevention of chronic diseases and aging, by providing with abundant polyphenols, antioxidants and specific FAAs.


Introduction
Our expected lifespan continues increasing, but many of us will lead a higher percentage of our lives in poor health conditions due to aging and increasing threats by many chronic diseases such as hypertension, hyperlipidemia, diabetes, chronic inflammation, and other stressors. As healthcare costs increase, preventive approaches (e.g., supplemental diets, functional foods or drinks) are gaining in popularity. These alternatives represent inexpensive and readily applicable approaches to reduce the incidence of chronic diseases. Such preventive health products have gained a great deal of attention from both the scientific community and the general public. Additionally, exploration of the natural and sustainable resources for healthcare and supplementary nutrition has become crucial for the future of developing countries and for those with large populations such as China.
It is well-known that green teas prepared from leaves of Camellia plants have many important physiological properties and health benefits. Drinking green teas may reduce the risk of many diseases, such as cancers and cardiovascular diseases, as teas have a variety of biological activities including anti-tumor, antioxidation, and anti-obesity [1][2][3] . Previous studies have demonstrated that amino acids, polyphenols and purine alkaloids are important nutritional and active components in green teas. Examples include the nutritional roles for essential amino acids and the pharmacological effects of theanine and γ-aminobutyric acid (GABA). Theanine is a major amino acid uniquely found in green tea which can decrease norepinephrine and serotonin levels in the brain, lower blood pressure and produce neuroprotective and cognitive-enhancing actions 4,5 . GABA is an important inhibitory neurotransmitter in the mammalian central nervous system and is known to exhibit antihypertensive effects 6 . Teas rich in GABA can decrease blood pressure in rats 7 . Amino acids also participate in the biosynthesis of polyphenols and alkaloids 8,9 . The antioxidant and free radical-scavenging abilities of polyphenols in green tea may play an important role in the prevention of cardiovascular disease, chronic gastritis and some cancers 10 . However, purine alkaloids (such as caffeine, theobromine and theophylline), as additional key components of tea and coffee, may have negative impacts on our wellness when the intake is high 11 .
A variety of folk teas originated from plants other than Camellia have been used by various indigenous and minority groups in China for centuries. Their potential nutritional and medical value in preventing chronic diseases has not been widely recognized or compared with common green teas until recent years. Folk teas are also a part of the traditional Chinese tea culture and are widely consumed as beverages and medicines in folk for disease prevention and treatment 12,13 . Recently, our team performed a systematic collection and some chemical and pharmacological investigations of these traditional teas and named them as non-Camellia teas (or in Chinese: Bie-yang-cha) [14][15][16] . Our previous studies demonstrated that many non-Camellia teas contain abundant polyphenols, show striking antioxidant effects in vitro, and have many other health benefits [17][18][19][20] .
Due to the biological significance of the amino acids, polyphenols and alkaloids contained in green tea, for the first time we systematically investigated these important types of constituents and the antioxidant activity of extracts of plants for non-Camellia teas, in order to provide information on the health properties and promote the development of non-Camellia teas as functional beverages. In this study, 33 non-Camellia tea samples were collected based on these criteria: those teas (i) are not originated from Camellia plants; (ii) are popularly used for at least three hundred years in some minorities' areas or indigenous groups in China; and (iii) are presently consumed.
Several well-established biochemical methods were used in our study. The ultra-high performance liquid chromatography (UHPLC) method was used to determine the amino acid content. It is a rapid, modern, and effective method which has been used to detect amino acids in cheese and green tea 21,22 . Precolumn derivatization with 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate (AccQ) is a widely used method for the derivatization of amino acids 21,23,24 , but the use of UHPLC with precolumn derivatization with AccQ to detect 20 amino acids has not been previously reported. UHPLC was also applied to determine the purine alkaloid content. Lastly, the phenolic content and antioxidant activity of these tea products were determined using the Folin-Ciocalteu (F-C) assay and 1,1-diphenyl-2-picryl-hydrazyl (DPPH) method, respectively. The F-C assay and DPPH methods provide convenient, rapid and simple estimation of the total phenols content and antioxidant activity, respectively.

Collection 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

Chemicals and standards
AccQ Á 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 1C. Diluted solutions were freshly prepared weekly.

Preparation 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 1C 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).

Analysis of total polyphenols
The total phenolic content of the tea samples was determined according to a procedure described by Ainsworth and Gillespie 25 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).

Antioxidant assay
The antioxidant activities of the tea samples were evaluated by DPPH method as previously reported, with some modifications [26][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: where A 1 is the absorbance of the test sample, and A 0 is the absorbance of the control. Results are expressed as EC 50 and values are referred to the concentration of the tea infusions required for the 50% of the antioxidant activity (g/mL).

UHPLC 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 Auto-Sampler, 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 Â 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 1C. 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 procedure 29 . Briefly, UHPLC was performed with DIONEX Acclaim PA2 column (150 mm Â 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 1C. 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.

UHPLC 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-tonoise 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.

Statistical 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).

Validation 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 (R 2 Z 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 o3%, whereas the repeatability (RSD) was o5% for all analytes. The stability of amino acid standards (RSD) varied o4%. 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.

Quantitative 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.
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.

Principal 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).

Cluster 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 (o4.0 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.    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),  Fig. 4, V: 3 Camellia teas (green tea, black tea and pu-erh tea). * EC 50 was too high to be detected in this study conditions. 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).

Caffeine, 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.

Conclusions
A variety of teas from non-Camellia plant are popularly consumed in many regions of China for centuries. In this study, we report for the first time, that some non-Camellia teas contain higher amount of some specific amino acids (such as Arg, Pro, Cys, Thea and GABA), polyphenols (particularly, teas from A. grossedentata, A. tataricum subsp. ginnala, M. oblongifolius and P. fruticosa, I. serra and P. chinensis) than found in Camellia tea, Some non-Camellia teas also have remarkable antioxidant activities (particularly, teas from I. serra, P. chinensis, A. tataricum subsp. ginnala, and M. oblongifolius). They do not contain addictive substances such as caffeine, theobromine and theophylline. Furthermore, a UHPLC method with precolumn derivatization with AccQ to detect 20 amino acids in teas has been established. This method compares favorably against the HPLC or UHPLC methods in which less than 20 amino acids are quantified.
In a summary, the discovery that some non-Camellia teas contain abundant amino acids and polyphenols, which have significant antioxidant activities, but lack caffeine, demonstrating that some of these teas have potential to prevent many chronic diseases. Additionally, non-Camellia teas have been frequently used for centuries throughout many regions of China, indicating that they are quite safe to use. Furthermore, since the non-Camellia teas are derived from various families of plants, the unique types of polyphenols and corresponding bioactivities are highly diverse and can be their characteristic features different from common Camellia tea. Some bioactivity profiles of several non-Camellia teas have been recently characterized by several preliminary chemical and pharmacological studies 31,32 . Therefore, our study suggests that non-Camellia teas might be more valuable as a functional beverage than Camellia tea, and could be a beneficial supplement or alternative beverage to Camellia tea. Exploration of the non-Camellia teas should provide a variety of beverage choices for health benefits.