Health Bene � cial Effects and Chemical Pro � les of Special Oolong Teas

Narawadee Rujanapun Medicinal Plant Innovation Center of Mae Fah Luang University, Mae Fah Luang University Wuttichai Jaidee Medicinal Plant Innovation Center of Mae Fah Luang University, Mae Fah Luang University Thidarat Duangyod School of Integrative Medicine, Mae Fah Luang University Pravaree Phuneerub School of Integrative Medicine, Mae Fah Luang University Chakree Wattanasiri School of Integrative Medicine, Mae Fah Luang University Napassawan Paojumroom Medicinal Plant Innovation Center of Mae Fah Luang University, Mae Fah Luang University Tharakorn Maneerat School of Science, Mae Fah Luang University Chuchawal Pringpuangkeo Doi Chang Tea Co., Ltd Rawiwan Charoensup (  rawiwan.cha@mfu.ac.th ) School of Integrative Medicine, Mae Fah Luang University

pressure of 45 psig. Agilent Mass Hunter Qualitative Analysis Software, version 8.00, was used for the initial processing of the LC/MS data. Compounds were revealed using the Molecular Feature Extractor Tool (MFE) tool in the software. CSV les were directly imported into Mass Hunter Pro ler Professional (MPP), version 15.1, to generate the statistical analysis required to pro le the samples such as HCA and PCA. One-way analysis of variance (ANOVA) was used to limit the set to those compounds that varied at the P<0.05 level, and the sample set produced a list of thirty compounds that were used for statistical analysis.
Antioxidant activities 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay The assay was measured using the modi ed method reported by Duangyod et al. [6]. Five hundred microlitres of tea extract solution was mixed with 500 µL of 59 µM DPPH solution in methanol. The mixtures were kept in the dark for 30 min, and the optical density was measured at 517 nm. (+)-Catechin hydrate was used as a positive control, and triplicate measurements were carried out. The percentage of scavenging activity was calculated by the formula given below: Scavenging activity (%) = ([absorbance control -absorbance sample ]/absorbance control ) × 100 where the concentrations of the extract required to scavenge 50% of DPPH scavenging activity under the assay conditions are de ned as the IC 50 .
2,2´-Azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) assay The ABTS assay was based on the ability of different substances to scavenge 2,2´-azino-bis-3ethylbenzthiazoline-6-sulfonic acid (ABTS •+ ) radical cations. The radical cation was prepared by mixing 7 mM ABTS stock solution with 2.4 mM potassium persulfate (1/1, v/v) and leaving the mixture for 12 h in the dark. ABTS •+ was diluted with methanol to an absorbance of 0.706 ± 0.001 at 734 nm for measurement. The photometric assay was conducted on a mixture of 500 µL of ABTS solution and 500 µL of tea extract/control solution. After 7 min, the measurement was performed at 734 nm using a spectrophotometer. (+)-Catechin hydrate was used as a positive control, and triplicate measurements were carried out. The percentage of scavenging activity was calculated. The concentration of extract required to scavenge 50% of ABTS scavenging activity under the assay conditions is de ned as the IC 50 .

Total phenolic content
The total phenolic content was measured using a method reported by Duangyod et al. [6]. using the Folin-Ciocalteu reagent. Then, 800 µL of sample extracts and 200 µL of 15% Folin-Ciocalteu reagent were added to the test tube, and the volume was adjusted to 2.0 mL with water. The mixture was left for 5 min. Next, 1.0 mL of Na 2 CO 3 (0.106 g/mL) was added. The mixture was kept in the dark at room temperature for 60 min. The absorbance was measured at 756 nm. The results were expressed as micrograms of (+)-catechin hydrate equivalents per milligram of crude extract and micrograms of gallic acid equivalents (GAE) per milligram of crude extract. α-Glucosidase inhibition assay α-Glucosidase inhibition activity was performed under the following procedures. Fifty microlitres of tea extract/control was mixed with 100 µL of α-glucosidase (0.35 U/mL) and incubated at 37 °C. After 10 min, 100 µL of p-NPG (1.5 mM) was added into the mixture and incubated at 37 °C for 20 min. The reaction was terminated by the addition of 1,000 µL of Na 2 CO 3 . Two hundred microlitres of the mixture was measured at 405 nm on a microplate reader by measuring the quantity of p-nitrophenol released from p-NPG. Acarbose was used as a positive control for the α-glucosidase inhibitor. The concentration of the extract required to inhibit 50% of α-glucosidase activity under the assay conditions was de ned as the IC 50 value [1].

α-Amylase inhibition assay
The activity was measured using the method reported by Kusano et al., with slight modi cations. Acarbose was used as a positive control. The substrate solution was prepared as follows: soluble starch (500 mg) was dissolved in 25 mL of 0.4 M NaOH and heated for 5 min at 100 °C. After cooling in water, the solution was adjusted to pH 7 with 2 M HCl, and DI water was added to adjust the volume to 100 mL. Tea extract solutions were prepared by dissolving each sample in 0.2 M acetate buffer (pH 7). Sixty microlitres of tea extract/control solution was mixed with 120 µL of starch solution and incubated at 37°C for 10 min. Then, 180 µL of α-amylase (1 U/mL) was added to the solution and incubated for 30 min.
The reaction was terminated by adding 240 µL of 0.1 M HCl; then, 300 µL of 0.1 mM iodine solution was added. After that, the solution was mixed with 500 µL of 0.2 M acetate buffer, and the absorbance was measured at 412 nm using a spectrophotometer. Finally, the inhibitory activity (%) was calculated. The concentration of extract required to inhibit 50% of the α-amylase activity under the assay conditions is de ned as the IC 50 value [7].
Glucose consumption 3T3-L1 preadipocyte cell lines were maintained in DMEM culture medium containing 10% FBS, 2 mM glutamine, 100 kU/L penicillin, 100 mg/L streptomycin, and a high glucose concentration (4.5 g/L) at 37°C and 5% CO 2 . Cells were detached from the culture ask with a solution of 0.25% trypsin and 1 mM EDTA. The trypsin digestion was stopped by PBS. The cells were washed twice and resuspended in low glucose (1.0 mg/mL) detection medium. The cell density was adjusted to a concentration of 1 × 10 5 cells/mL, and cells were spread onto 96-well microtiter plates (100 μL per well). The cells were cultured with serial samples at 37 °C and 5% CO 2 for 4−48 h. Insulin and metformin were used as positive controls in this experiment. At the end of incubation, 10 μL of suspension or glucose standard medium (0-1,000 mg/L) was moved to another 96-well plate well by well. The glucose concentration remaining in the suspension was measured by the glucose oxidase-peroxidase (GOD-POD) assay. Brie y, the reaction lasted 30 min at room temperature, and the absorbance at 510 nm was determined.

Glucose uptake
Cell culture and maintain 3T3-L1 preadipocyte and L6 myoblasts cells were obtained from American Type Culture Collection (ATCC, USA). All cells were incubated 37 C in a humidi ed incubator in an atmosphere with 5% CO2 Cell differentiation L6 myoblasts were grown in DMEM containing 10% (v/v) FBS, 1100 kU/L penicillin and 100 mg/L streptomycin in a humidi ed atmosphere of 95% air and 5% CO 2 at 37 °C. Cells were reseeded in six-well plates or 24-well plates (for glucose uptake) at a density of 2×10 4 cells/mL. After 48 h (∼80% con uence), the medium was switched to DMEM with 2% (v/v) FBS and replaced after two, four, and six days of culture. Experiments were initiated on day seven when myotube differentiation was complete.
Glucose uptake assay Anti-adipogenesis assay 3T3-L1 preadipocyte cell lines were differentiated into adipocyte cells. The cells were treated with various concentrations of tea extracts and cultured in DMEM supplemented with 1 μM dexamethasone, 10 μg/mL insulin, and 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) for 2 days. After 2 days, the medium was changed to DMEM containing 10 μg/mL insulin for 2 days. At the end of incubation, the medium was changed to DMEM until day 10. Lipid accumulation was assessed using oil red O staining. The samples were observed and recorded under a microscope. After that, the cells were dissolved in DMSO, and the absorbance was measured at 510 nm. GraphPad Prism 6.0 software. In addition, the statistics were analysed to compare two means between the control and sample exposure groups. The statistics were analysed by one-way ANOVA with the Dunnett method using SPSS software.

Results
Liquid chromatography mass spectrometry analysis The UHPLC-QTOF-MS data of NR, PN, and OL showed in Table S1-S3, their samples were acquired in positive ionization mode. The metabolite features were identi ed based on database searching and compared MS/MS spectra with literatures [5,8,9]. Comparison the UHPLC-LCMS pro les obtained from each teas, it was clear that PR extract have a difference metabolite with content of catechin dimer and avonoids ( Figure S1). These ndings corroborate with their anti-diabetic activities. The ET and Oolong pro les were similar in the number of metabolite which can be attributed to the alkaloid and catechin derivative in both extract ( Figure S2-S3 and Table S2-S3). Principal component analysis (PCA) was used to visualize the abundance variations for the 30 unidenti ed metabolites with signi cant differences across the three subtypes of oolong tea (P < 0.05) (Figure 1). In PCA score plot, each spot represents a sample ( Figure 1A). The PCA analysis of oolong tea revealed that the samples can be divided to two groups: EN, PR and oolong. The PCA analysis data suggests that the catechin and avonoid compounds in the PR may be most abundant components. EN and oolong samples can be assigned to one group, indicating that the catechin derivative compounds were highly similar in terms of types and content.

Effect of tea extracts on Antioxidant activities
The antioxidant activities of tea extracts were determined by the most common radical scavenging assays using ABTS and DPPH radicals. These assays are the most popular for assessing antioxidant-rich fruits, vegetables, and beverages in the US [10]. The results presented in Table 1 demonstrates that the antioxidant capacity detected by the DPPH assay was higher for all samples than that detected by the ABTS assay. The results reveal that all tea sample extracts have strong antioxidant activity.
Effect of tea extracts on Total phenolic content All samples of tea extract were found to have more than 60 µg of gallic acid equivalents (GAE) per milligram of crude extract and more than 20 µg of (+)-catechin hydrate per milligram of crude extract ( Table 1).
Effect of tea extracts on α-glucosidase and α-amylase inhibition assay Aqueous extract of PR exerted the highest inhibitory activity against both enzymes compared to the acarbose, with IC 50 values of 1.22 µg/mL against α-glucosidase and less than 0.03 µg/mL against αamylase ( Table 2). Ethanol extract of PR exhibited less activity on α-amylase (IC 50 of 40.49 µg/mL), while a good activity was found on α-glucosidase (IC 50 value of 10.60 µg/mL) ( Table 2). These results were related to a previous study that showed good α-glucosidase and α-amylase inhibition activities of black tea [1].

Effect of tea extracts on the glucose consumption of 3T3-L1 preadipocyte cells
Glucose consumption was used in insulin screening and was shown to increase after treatment with tea extracts. The results presented with PR aqueous extracts (50,100,250 and 500 µg/ml) showed the greatest enhancement of glucose consumption by 3T3-L1 preadipocyte cells in a dose-dependent manner compared with other tea extracts and metformin (Fig. 2).

Effect of tea extracts on Glucose uptake
All tea extracts stimulated glucose uptake in L6 myotube cells. Cells were incubated with each tea extract, and glucose uptake was measured with 2NBDG uorescence. The results showed that all tea extracts at 100 μg/mL enhanced glucose uptake in L6 cells, especially the PR aqueous extract with 1.72±0.16 fold as compared to the control (Fig. 3).
Effect of tea extracts on intracellular lipid accumulation 3T3-L1 preadipocyte cells were differentiated into adipocytes. This experiment investigated the effect of tea water extracts on intracellular lipid accumulation in adipocyte cells. The cells were treated with 50 μg/mL tea water extracts for 10 days. Lipid droplets were stained with oil red O dye. The results revealed that all tea aqueous extracts reduced intracellular lipid accumulation in adipocyte cells, especially PR aqueous extract (Fig. 4A). Additionally, the PR aqueous extract showed the strongest reduction in intracellular lipid accumulation in adipocyte cells compared with the other tea extracts (Fig. 4B).
Cytotoxicity of tea extracts in RAW 264.7 cells using the MTT assay RAW cells were investigated for viability using the MTT assay. The viability of cells was slightly decreased after treatment with tea extracts. Thus, the tea extracts (6.25-100 µg/ml) did not affect RAW cells. Regarding this evidence, the tea extract is non-toxic to normal cells, such as RAW cells (Fig. 5A and  5B).
Effect of tea extracts on in ammation in RAW 264.7 cells using nitric oxide (NO) inhibition assays The in ammatory was detected nitric oxide secretion in RAW cells. The cells were investigated antiin ammatory with tea extracts (6.25, 12.5, 25, 50 and 100 µg/mL). The results showed that the extracts decreased nitric oxide in dose-dependent manner. In addition, PR aqueous extracts showed the lowest IC 50 with 43.82 µg/mL followed by EN and oolong aqueous extracts with IC 50 of 48.23 and 59.57 µg/mL respectively.

Effect of tea extracts on cancer cell growth inhibition
The IC 50 values of the eternity tea aqueous extract, peaceful rest tea aqueous and ethanolic extract were 490.91, 489.78, and 157.56 µg/mL, respectively. (Fig. 6).

Discussion
The results showed that the rst principal component (PC1) accounted for 84.71% of the variability in the dataset, and the second PC (PC2) accounted for 4.44% of the variance in the data. PR showed negative loadings on PC1 and positive on PC2; however, EN showed positive loadings on PC1 and negative loadings PC2 and Oolong showed positive loadings on PC1 and PC2. The results indicate that their chemical pro les are different. Moreover, the PR extract showed interesting compounds: 1-Caffeoylquinic acid (t R 15.24 min), Kaempferol 3-O-galactoside (t R 19.54 min), and Procyanidin B2 (t R 16.11 min); however, these compounds were not found in other teas (Fig. S1-S3 and Table S1-S3). These compounds stimulated antidiabetic and anticancer activity [11][12][13][14].
The tea samples showed high antioxidant activities compared to (+)-catechin hydrate (Table 1). This might be due to the phenolic content in those samples. The profound antioxidant activity of tea could be attributed to catechins and avonoids. These results agree with previous reports that a greater amount of phenolic compounds leads to a more potent radical scavenging effect [1,6,15]. α-Amylase and α-glucosidase are important enzymes involved in starch breakdown and intestinal glucose absorption, respectively. The inhibition of these enzymes can slow the passage of carbohydrates into the bloodstream, signi cantly decreasing the postprandial increase in blood glucose levels after a mixed carbohydrate diet and therefore can be an important strategy in the management of type 2 diabetes [16]. The PR extract showed high inhibitory activity on α-glucosidase and α-amylase ( Table 2). The chemical pro le of PR revealed the occurrence of alkaloids, catechins, and avonoids (Table S3). The inhibitory activity of the both enzymes may be due, mainly, to the presence of catechins, and avonoids [17][18][19][20][21][22][23][24][25][26][27][28]. Additionally, procyanidin B2 and 5-O-caffeoylquinic were found in PR. Procyanidin B2 effect on a diabetic, that involved in glucose homeostasis [29]. Moreover, 5-O-caffeoylquinic regulated glucose transport which promote glucose plasma reduction thus 5-O-caffeoylquinic increased glucose translocation into cells [30]. Regarding in this evidence, both of these compounds might increase glucose consumption percentage in PR-treated-cells In addition, 5-O-Caffeoylquinic acid also enhances glucose uptake in L6 Cells in dose and time dependent manner via AMPK activation that leads to GLUT4 and PPAR-γ stimulation. [31,32] Therefore PR was the strongest glucose uptake activity. Normally, patients with type 2 diabetes have impaired insulinstimulated glucose uptake into cells; thus, patients with type 2 diabetes have a high level of blood sugar [33] The results suggest that PR might decrease blood glucose levels via glucose uptake stimulation (Fig.  3). The inhibitory activity of the glucose uptake may be due, mainly, to the presence of kaempferol and catechins derivatives [34][35][36]. Not only 5-O-Caffeoylquinic acid promote glucose uptake but also affect lipid metabolism. de Sotillo study reported fasting plasma cholesterol, triacylglycerol and liver triacylglycerols were reduced by 44%, 58% and 24% respectively in 5-O-caffeoylquinic acid-treated-rat [37]. 5-O-Caffeoylquinic acid upregulated PPAR-γ expression which plays a key role in glucolipid regulation [38]. Thus 5-O-Caffeoylquinic acid in PR might enhances lipid decreasing.
In ammatory nitric oxide secretion was detected in RAW cells, and the anti-in ammatory effects of tea extract treatment were investigated. The results showed that both PR and EN water extracts decreased nitric oxide in a dose-dependent manner. In addition, the IC 50 values of the EN and PR water extracts were 48.87 and 42.56 µg/mL, respectively. The results suggest that EN and PR water had a strong antiin ammatory activity. (Fig. 5B).
The results reveal that none of the tea extracts showed strong cancer cell growth inhibition activity against the K562 lymphoblastoid human erythroleukemia cell line (Fig. 6). However, the PR tea extracts showed better cancer cell growth inhibition activity than the Oolong extract. Studies have shown that PN tea contains procyanidin B2, a compound that inhibits cancer cell proliferation [39,40]. According to this previous study, PR tea presented strong cell growth inhibition.

Conclusion
Oolong tea is a popular beverage consumed daily worldwide. In the present study, we investigated the bene cial health effects and chemical pro les of several oolong teas. Peaceful rest and eternity tea were blended with Thai medicinal plants. Our results showed that all teas show anti-diabetic, antioxidant, antiin ammatory, anti-cancer, and anti-adipogenesis effects in a safe manner. All of the tea sample extracts have strong antioxidant activity. The results related to previous studies showed that some catechins present in oolong tea or other polyphenols resulted in decreased free radical scavenging activity [41]. Another study reported that polyphenols not only induced antioxidants but also stimulated apoptosis in cancer cells [42]. These anticancer effects were also found in our results, especially peaceful rest tea. PR contains procyanidin B2, which was not found in other teas. This compound inhibited cancer proliferation. Thus, it showed the highest cancer cell growth inhibition compared to other teas. In addition, Peaceful rest tea has high potential to inhibit α-glucosidase, α-amylase, glucose consumption, and glucose uptake. A previous study reported that quinolones increase insulin release from rat pancreatic islets via blockade of adenosine tri-phosphate (ATP)-sensitive potassium channels [1]. In addition, other previous studies have investigated the antidiabetic activity of gallocatechin in a rat model. A study reported that gallocatechin also stimulated an increase in insulin [5]. Regarding this evidence, Peaceful rest tea might enhance blood sugar reduction. Therefore, in vivo and clinical studies are therefore recommended to provide implications for the potential anti-diabetic properties of special oolong teas.. The data associated with this study is available from corresponding author or the rst authors upon request.
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Competing interests
The authors claimed no con icts of interest.   Effect of tea water extracts on glucose consumption in 3T3-L1 cells.

Figure 3
Effect of tea water extracts on glucose uptake in L6 cells.