The health benefits of green tea consumption have been reported to include: cancer inhibition [1
], allergy relief effects [3
], cognitive dysfunction [4
], and preventive effects on metabolic syndrome [5
]. In a large epidemiological study, which was based on a follow-up investigation of 82,369 Japanese people for 13 years, green tea consumption also showed positive effects such as lowering the risk of cardiovascular diseases and stroke [7
]. Moreover, another follow-up study of 90,914 Japanese people for 18.7 years suggested that the consumption of green tea reduces the risk of total mortality and three leading causes of death: heart disease, cerebrovascular disease, and respiratory disease [8
]. This evidence of health benefits should attract the attention of health-conscious people.
There are two prominent methods for serving green tea. The general serving method consists of steeping the leaves in hot water and filtering them through a tea strainer. In contrast, in traditional Japanese tea ceremony, fine powdered green tealeaves (matcha) are foamed with a tea whisk in hot water. Ingestion of matcha likely allows for the introduction of a large amount of tealeaf components into our body.
Matcha is typically produced from shade-grown green tealeaves that are steamed, dried, and then ground with a set of millstones. According to Sawamura et al.
], it is unknown when millstones came into use. However, since ancient documents have reported the use of millstones for tea leaves in the 11th century in China [9
] and in the 14th century in Japan [9
], it is likely that the ingestion of powdered green tealeaves may have been a habit for a long time.
Major components of matcha [12
] are catechins, which are known for their anti-oxidant activity [13
], amino acids, and saponins, which contribute to the foaming property of matcha [14
]. Using high-performance liquid chromatography (HPLC) analysis, Saijo et al.
] showed that catechins make up 4.92% of the dry weight of matcha. In addition, liquid chromatography–mass spectrometry (LC-MS) quantification of catechins in commercially available green tea by Goto et al.
] showed that epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG), and epigallocatechin gallate (EGCG) were the four main types of catechin present in matcha with an average content of 11.19%, while the caffeine content was 3.77%. Moreover, EGCG has predominant functions as a highly anti-oxidant compound [17
] among green tea catechins.
Studies on the physical properties of matcha have revealed differences in particle sizes, shapes, and foaming properties as the result of diverse powdering methods. Onishi et al.
] reported matcha particles of several micrometers by using electron microscopy, while Sawamura et al.
reported median diameters of 15–20 µm (millstone and ball mill), or ≤5 µm (jet mill) using laser diffraction analysis [20
Although there are many reports on the chemical and physical properties of powdered green tea, changes in its properties before and after powdering process using the same tealeaves have not been revealed. Since changes in the powdering process may alter the chemical components and functionality of the green tea, thus contributing to expand its health-promoting benefits, we investigated the differences in physical property, catechin concentration, and reactive oxygen species (ROS) inhibitory effect of green tea prepared from whole leaves or powdered leaves.
4. Materials and Methods
4.1. Tea Preparation and Powdering Process
Green tea leaves (second picked tea leaves) were purchased from a tea specialty shop in Tokyo, Japan. For powdering process of green tea leaves, we used a set of ceramic mills (Figure 5
) in a tea maker, Healsio Ocha Presso (TE-GS10A, Sharp, Osaka, Japan), a ball mill with an alumina ball, and a mixer with stainless cutter for cooking use for 3–4 min (mill-contact time of tea leaf was approximately 30 s), 420 min, and 5 min, respectively.
Because Shishikura et al.
] mentioned that most individuals in their laboratory (n
= 34) spend less than 1 min preparing a cup of tea in their paper, we adopted a 1-min brewing time for green tea preparation. Then hot tap water after boiling (60 °C, 80 mL) was added to the tea powder or leaf (0.36 g or 0.96 g) in a conical flask and stirred for 1 min with a magnetic stirrer (SW-RS077, Nisshin Rika, Tokyo, Japan). As for regular tea, hot tap water after boiling (60 °C, 80 mL) was added to tealeaf (2.0 g) in a conical flask and placed for 1 min (no stirring). The aim of regular-tea investigation was the comparison with the general green tea, which we drink daily. After being placed for 1-min on ice, all the tea solutions were transferred into a light-shielding glass bottle using a 10-mL pipet and refrigerated at −20 °C until examination. For investigating the particle distribution (Section 4.4
.), the regular tea was filtered with a tea strainer after brewing instead of 10-mL pipet transfer.
(−)-Epigallocatechin (98%), (−)-Epicattechin (98%), Caffeine anhydrous (98.5%), Phosphoric Acid, Ortho (85%), Acetonitrile (99.8%, used for HPLC analysis in this paper) were purchased from Nacalai tesque, Kyoto, Japan. (+)-Catechin Hydrate (>95.0%), (−)-Epicatechin Gallate (>98.0%), (−)-Epigallocatechin Gallate Hydrate (>98%), tert-butyl hydroperoxide (70% in Water) were purchased from Tokyo Chemical Industry, Tokyo, Japan. Acetonitrile for High Performance Liquid Chromatography (99.8+%, used for LC-MS/MS analysis in this paper) and Sodium Carbonate (99.8+%) were purchased from Wako Pure Chemical Industries, Tokyo, Japan. Folin–Denis’ reagent and 2’,7’-dichlorofluorescein diacetate (≥97%, DCFDA) was purchased from Merck KGaA (Sigma-Aldrich), Darmstadt, Germany. Anmonium formate solution, and Formic acid-Acetonitrile were purchased from Kanto Kagaku, Tokyo, Japan. Sodium hexametaphosphate was purchased from Mitejima Chemical, Osaka, Japan. SOD Assay Kit-WST was purchased from Dojindo Molecular Technologies, Kumamoto, Japan.
4.3. Observation of Tea Powders and Leaves with a SEM and a Multifocal Optical Microscope
For the observation with an electron microscope, tea powders and leaves were scattered on a carbon tape, followed by blowing the excess powders or leaves. Then powders and leaves were observed with SEM (JSM-5800LV, JEOL, Tokyo, Japan) using accelerating voltage of 15 kV at magnification of 250 and 1000 times. For the multifocal optical observation, 10 µL of tea was injected into a plastic slide and then images were recorded with an automated cell counter (TC20, Biorad, CA, USA). The optical images were analyzed with an image analysis software (WinRoof 2013, Mitani Corporation, Tokyo, Japan) for counting the particles in the picture (around 4 mm2).
4.4. Size Distribution in Tea Liquid
The size distribution of tea powders was measured with laser diffraction particle size analyzer (SALD 1100, Shimadzu Corporation, Kyoto, Japan) after dispersion in 1% sodium hexametaphosphate in water. For the measurement of particles size in the regular tea, tea liquid after filtration with a tea strainer (Section 4.1
.) was used for exclusion of large tealeaves.
4.5. Catechin and Caffeine Analysis with HPLC System
For quantification of major catechins and caffeine, EC, ECG, EGC, EGCG and caffeine were analyzed in the method refer to the paper of Terada et al.
] and partially modified. The peak of catechins and caffeine was detected at 280 nm absorbance with a HPLC system (LC-10A, Shimadzu corporation, Kyoto, Japan) equipped with CAPCELL Pack C18 (UG120S-5, 4.6 × 150 mm, Shiseido). Column oven temperature was at 43 °C. In reference to the previous paper, the tea samples were mixed with triple volume of acetonitrile then filtered with PVDF membrane (Acrodisc LC PVDF, 0.45 µm, 13 mm) quickly. Gradient separation was carried with eluent A (0.1% acetonitrile and 5% N,N
-dimethylformamide in 0.1% phosphoric acid water solution) and eluent B (acetonitrile). The volume of the injected sample was 10 µL and flow rate was 1.0 mL/min. The gradient program was as follows: Rate of elute B: 0–30 min: 1%→15%; 30–40 min: 15%→90%; and 40–40.1 min: 90%→1%. As the standard, (−)-epigallocatechin, epicatechin, (−)-epicatechin gallate (−)-Epigallocatechin Gallate Hydrate, and Caffeine anhydrous were used in the range of 0–500 µg/mL.
4.6. Total Polyphenol Concentration
The total phenol of teas was quantified with the Folin–Denis’ reagent assay [34
] (refer to our previous paper [35
]). Each sample was diluted with purified water (1/5 dilution) and added to the Folin–Denis’ reagent and 10% sodium carbonate solution (Diluted sample: Folin–Denis’ reagent: 10% sodium carbonate solution = 1:1:1). After the solution was placed for 20 min at room temperature, the solution was centrifuged at 10,000 g and room temperature for 1 min. Then, 700 nm absorbance was measured with a multimode plate reader (EnSpire, PerkinElmer, Waltham, MA, USA). (+)-Catechin Hydrate was used as standard in the range 0–1000 μg/mL. The phenol contents of the teas were calculated as catechin equivalents (mg CE/mL).
4.7. Chemical Components Analysis with LC-MS/MS System
The chemical components of strong powder tea and strong leaf tea were analyzed with LC-MS/MS system (Maxis 3G, Bruker, MA, USA) equipped with ACQUITY UPLC BEH C8 Column (1.7 µm, 2.1 mm × 50 mm, Waters, MA, USA). Column oven temperature was 40 °C. Mass spectra were acquired in ESI negative modes. The tea samples were filtrated with the Acrodisc PVDF membrane filter. The binary gradient 10 mM ammonium formate in water (eluent A) and acetonitrile containing 10 mM ammonium formate (eluent B) in the negative mode was applied with injection volume of 5 μL at a constant flowrate of 0.2 mL/min. The gradient program was as follows: Rate of elute A: 0–10 min: 0%→95%; 10–13 min: 95%; and 13–13.1 min: 95%→0%. The detected molecular masses were found in the Human Metabolome Database [36
] using MS Search.
4.8. Measurement of SOD-Like Activity
In reference to our previous paper [35
] and manufacturers’ protocol, the tea samples (no pretreatment) were diluted in the range of 1–32,000 dilution and analyzed with SOD Assay Kit-WST (Inhibition assay of xanthine oxidase). Inhibition rate (%) was calculated by the ratio of the control (water).
4.9. Inhibition of Intracellular ROS in a T Cell Line
Jurkat human leukemic T cell line was used for the measurement of intracellular ROS. Referring to the method by Bass et al.
] and partially modified, 20 µM 2’,7’-dichlorofluorescein diacetate was added to 1.25 × 105
cells/mL of the cells, then incubated at 37 °C for 30 min (5% CO2
). Next, the tea samples were added to the cells at the 500 times dilution followed by 30-min incubation. After the incubation, 200 µM tert-butyl hydroperoxide was added and incubated for 1 h. Then the fluorescent of intracellular ROS was measured with the Enspire plate reader (Ex 485 nm/Em 535nm). Inhibition rate (%) was calculated by the ratio of control (water).
4.10. Data Analysis
All statistical analyses were carried out in triplicate. The mean ± standard deviation (SD) (three measurements) was presented. Statistical significance was calculated in Bonferroni–Dunn multiple comparison test or Welch’s t test with the Microsoft Office Excel 2013 (Microsoft, Redmond, WA, USA) and the add-in software Statcel 3 (OMS publishing Inc., Saitama, Japan). Significance level was set at 5%.