Wei Qin, Sunantha Ketnawa, Yukiharu Ogawa*

Graduate School of Horticulture, Chiba University, 648, Matsudo, Matsudo 271-8510, Japan

Keywords:

Green tea

Polyphenols

Antioxidant activity

Digestive enzyme

Bioavailability

A B S T R A C T

To exam ine the effect of digestive attributes such as digestive enzymes and pH on changes in phenolic compound content and antioxidant activity during digestion, the bioavailability of green tea infusion was investigated using a simulated in vitro gastrointestinal digestion model.The total polyphenol content (TPC)decreased to 65%–70% throughout the mimicked normal digestion (MD) compared to the initial value.The total flavonoid content (TFC) decreased to approximately 25% after starting the gastric stage (pH 1.2);however, it regained to approximately 60% in the intestinal stage (pH 6.8).The mimicked digestive condition without digestive enzymes (WOE), which followed only the pH conditions of MD, showed significantly lower TPC and TFC values than MD.The percentage of antioxidant activity based on the initial values indexed by DPPH, ABTS, and FRAP gradually declined from approximately 60% at the gastric stage to approximately 40% at the final digestion stage.Meanwhile, the percentage of residual MIC was around 50% at the gastric stage.However, it gradually increased at the intestinal stage.The significantly lower antioxidant activity showed for WOE than MD throughout the simulated digestion.This study demonstrated that digestive enzymes and pH play a crucial role in the bioavailability of green tea infusion.

1.Introduction

Simulatedin vitrogastrointestinal digestion techniques have recently been applied to evaluate bioaccessibility and bioavailability because of the simple, cost-effective, and reproducible models available to simulate physiological conditions such as temperature,agitation, pH, enzymes, and chemical composition [1].The usual simulatedin vitrodigestion technique consists of two digestion steps (gastric and intestinal) using a single static bioreactor, which is normally used to determine the digestibility and soluble components of homogenized foods [2].The digestion model conditions may result in structural changes that affect the bioactivity, stability,bioaccessibility, and bioavailability of bioactive compounds [1-3].

It is widely known that the consumption of plant-based functional foods rich in phenolic compounds helps ameliorates free radicalinduced diseases [4-7].This therapeutic activity is actualized by:1) accessible phenolic compounds that are released from the food matrix to digestion fluids (bioaccessibility), and 2) available phenolic compounds that can be used for metabolic or endocrine-like activities in the human body (bioavailability) [4].Any compounds available in the human body needs to be accessible from the food matrix during gastrointestinal digestion.Meanwhile, bioactive ingredients in a beverage could be regarded as directly available compounds during digestion, although digestive conditions such as the concentration of digestive enzymes may result in a molecular-level structural transformation that affects the bioactivity, stability, and bioavailability of the compounds [1].Such bioavailability can be computed as the ratio between the concentration of compounds found during or after digestion and that originally present in the initial state before digestion [4].

Tea (Camellia sinensis) is receiving growing interest and high consumption worldwide because of its rich polyphenol content,which is associated with human health.Green tea has been studied for its biological effects over the past decades because of the health benefits of bioactive substances, including flavan-3-ols, especially catechins, epicatechin, and galloylated derivatives, and other polyphenols, which are mainly glycosylated derivatives of quercetin and kaempferol [8,9].Green tea also contains pigments, amino acids, vitamins, carbohydrates, minerals, and purine alkaloids [10].The health-promoting effects of green tea have been documented to protect against oxidative stress and prevent and/or manage several pathological conditions, such as neurodegenerative diseases,cardiovascular diseases, diabetes, high blood pressure, scurvy, and certain types of cancer [5].

Several studies have reported the evolution of the bioavailability of polyphenols and the antioxidant activity of teas during the digestion process using a simulated digestion model.One study documented that both the number of phenolics and the antioxidant activity ofJasonia glutinosaherbal tea infusion were lower and highlighted that the bioavailability of phenolics is drastically reduced after simulated digestion [11].In addition, a loss of approximately 93% of total phenolics and 91% of antioxidant activity in the duodenal stage has been reported [8], as well as a very low intestinal bioavailability(2%-15% of intestinal levels) of polyphenols in several types of tea infusions using a simulated GI digestion protocol [9].In contrast,phenolic compounds in green tea, predominately catechins,appear to be highly stable under gastric conditions [4].However,they are somewhat more sensitive to near-neutral conditions(pH 6-7.5) present in the small intestine because of the epimerization and auto-oxidation of catechins.They may also be sensitive to digestive enzymes.

As mentioned above, the antioxidant properties and changes in phenolic compounds of several kinds of teas or herbal drinks during simulated digestion from the health benefits of tea and comparison purposes have been published [8-12].However, it is hard to consider that accurate basis information on the effect of digestive conditions,such as enzymes and pH on the phenolic compounds and antioxidant activity of green tea during digestion, and specifically Japanese green tea has been provided.Thus, this study aimed to examine the effect of digestive enzymes and pH on the bioavailability of polyphenolic compounds and the residual antioxidant activity of Japanese green tea usingin vitrosimulated gastrointestinal digestion.

2.Materials and methods

2.1 Materials

Dried green tea leaves (Camellia sinensisvar.sinensiscv.Yabukita), which were derived from the first flush, were obtained from the National Agriculture and Food Research Organization(NARO) experimental tea field in Shizuoka, Japan in May 2019.Pepsin from porcine gastric mucosa (EC 3.4.23.1, 800-2 500 U/mg protein), pancreatin from porcine pancreas (EC 232-468-9, 8 × USP specifications), bile extract, 1,1-diphenyl-2-picrylhydrazyl (DPPH),2,4,6-tri(2-pyridyl)-s-triazine (TPTZ?), and 4-benzoylamino-2,5-dimethoxybenzenediazonium chloride hemi (zinc chloride) salt(FBBB) were all purchased from Sigma-Aldrich Chemical Co.(St.Louis, MO, USA).Iron sulfate (FeSO4), 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p’-disulfonic acid monosodium salt hydrate(FerroZine?), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox?) , iron chloride (FeCl2), and all other chemicals of analytical grade were purchased from Wako Pure Chemical Corporation (Tokyo, Japan).

2.2 Preparation of tea infusion

The moisture content of the tea leaves was measured according to the standard AOAC method [13]and calculated on a dry basis(d.b.).Tea infusion was prepared according to a previously described method [14].Five grams of tea leaves (d.b.) were adjusted for each processed sample in a beaker, water at a temperature of 95 °C was added to a total weight of 500 g, and the infusion was left to soak for 5 min.Tea infusion was then filtered using Whatman no.1 filter paper (GE Healthcare UK, Buckinghamshire, UK), and kept at 37 °C in a water bath (NTT-20S; Eyela, Tokyo, Japan) until subjected to simulatedin vitrogastrointestinal digestion within 30 min.

2.3 Simulated static two-stage in vitro gastrointestinal digestion of tea infusion

The simulated static two-stagein vitrogastrointestinal digestion model was applied following a previously described method [15]with slight modifications.Tea infusion (150 mL) was placed in a glass reactor, agitated continuously with a magnetic stirrer, and the temperature was maintained at 37 °C in a water bath (NTT-20S;Eyela) throughout the digestion.The pH of the tea infusion was adjusted to 1.20, by adding an appropriate combination of 1, 3, and 6 mol/L HCl solution, and the gastric stage (G) was initiated by the addition of 19 mL pepsin solution (4.8 mg pepsin/mL).The pH of the mixture was maintained at 1.20 ± 0.01 throughout the G stage.The intestinal digestion stage (GI) was initiated after 1 h of the G stage.To cease the enzyme reaction for the G stage, the pH of each sample infusion was increased to 4.0, by adding 1 mol/L NaHCO3and then to 6.8, by adding an appropriate combination of 1, 3, and 6 mol/L NaOH solution, followed by addition of 25 mL of intestinal enzyme solution (8 mg pancreatin/mL).The sample was maintained in these conditions for 2 h and the pH was kept at 6.80 ± 0.01 throughout GI stage.This digestion protocol was named a mimicked digestion in normal simulated digestive condition (MD).Simulated digestion without digestive enzymes that followed only pH conditions of MD was also conducted and was named a mimicked digestion without digestive enzymes (WOE).All simulations were performed in duplicate, which is consistent with the processes discussed above.The volume of the addition of HCl or NaOH solutions to maintain the pH and sampling volume was recorded and used as the dilution factor for calculation.Tea infusions from each digestion reactor at designated times were sampled and labeled as followed: 1) IT refers to initial tea infusion before digestion; 2) G0 refers to tea infusion at pH 1.2; 3) G1 refers to tea infusion after G stage for 1 h at pH 1.2;4) G1I0 refers to tea infusion after G stage for 1 h at neutral pH (pH 6.8);5) G1I1 refers to tea infusion after G stage for 1 h and I stage for 1 h at pH 6.8; and 6) G1I2 refers to tea infusion after G stage for 1 h and I stage for 2 h at pH 6.8, after digestion.The digestive mixture collected in a test tube was immediately placed in an ice bath for 10 min, followed by centrifugation at 4 000 ×gat 4 °C for 10 min.The supernatant was collected and stored at ?40 °C until further analysis.The determination of phenolic compounds and bioactivity was carried out after thawing the supernatant and maintaining it at 10 °C during measurement.All samples were analyzed in triplicate for the following analyses.

2.4 Determination of bioactive compounds and antioxidant activity

2.4.1 Total phenolic content (TPC) via Fast Blue BB (FBBB) assay

TPC was measured by the FBBB assay according to a previous study [16], with slight modification employing a 96 well-plate.Sample extract (0.1 mL) diluted with distilled water (1:20) was mixed with 0.1% FBBB solution (0.01 mL), mixed for 30 s, followed by 0.5% NaOH (0.01 mL), and allowed to stand for 90 min at room temperature.Absorbance was measured at 420 nm, and water was used as a blank.TPC was expressed as mg of gallic acid equivalent per g of dry matter of the initial tea leaves for infusion.

2.4.2 Total flavonoid content (TFC)

TFC was measured by a colorimetric assay developed in a previous study [17], with slight modification using a 96 well-plate.Diluted sample extract (250 μL) and standard solutions of catechin were added to the plate.0.01 mL of NaNO2(5%,m/V) was added to the sample, and the plate was incubated at room temperature for 5 min.After 5 min, 0.01 mL of AlCl3(10%,m/V) was added, and the plate was incubated at room temperature for 6 min, followed by the addition of NaOH (1 mol/L, 0.06 mL).Absorbance was determined at 520 nm and water was used as a blank.TFC was expressed as mg of catechin equivalent per g of dry matter of the initial tea leaves for infusion.

2.4.3 Radical scavenging activity for DPPH

The DPPH activity of the samples was measured according to a previously described method [15], with slight modifications using a 96 well-plate.0.02 mL sample extract was added to 0.30 mL 60 μmol/L DPPH solution, and mixed for 30 min in the dark at room temperature.The absorbance of the resulting solution was measured at 520 nm using 70% methanol as a blank.DPPH was calculated by a Trolox standard curve (0–1 000 μmol/L) and expressed as μmol of Trolox equivalent per g of dry matter of initial tea leaves for infusion.

2.4.4 Radical scavenging activity for ABTS

ABTS was tested according to the method of previous study [15], with slight modifications.ABTS radicals were produced by the reaction of 7 mmol/L ABTS dissolved in 2.45 mmol/L potassium persulfate, allowing the mixture to react in the dark at room temperature for 15 h before use.0.01 mL of the sample was mixed with 0.32 mL of diluted ABTS solution in a 96 well-plate.Absorbance was measured at 740 nm after 10 min of incubation at 30 °C in the dark using water as a blank.ABTS was expressed as mg of ascorbic acid equivalent per g of dry matter of the initial tea leaves for infusion.

2.4.5 Ferric reducing antioxidant power (FRAP) activity

The capacity of the digested fractions to reduce the ferric–TPTZ complex was evaluated by the FRAP assay as described previously [15],with slight modifications.Freshly prepared FRAP reagent(10 mmol/L TPTZ solution in 40 mmol/L HCl, 20 mmol/L FeCl3·6H2O solution, and 300 mmol/L acetate buffer (pH 3.6),at a ratio of 1:1:10 (V/V/V)) (0.26 mL) was incubated at 37 °C before being mixed with 0.04 mL sample extract.The mixture was incubated at 37 °C for 30 min in the dark, and the absorbance at 590 nm was recorded after 30 min using water as a blank.FRAP was calculated from the FeSO4standard curve (0–100 μmol/L) and expressed as μmol of FeSO4equivalent per g of dry matter of initial tea leaves for infusion.

2.4.6 Metal chelating activity (MIC)

MIC was measured using the method described in a previous study [15], with slight modifications.Sample extract (0.30 mL) was mixed with 2 mmol/L FeCl2·3H2O (5 μL) and 5 mmol/L FerroZine?(0.01 mL) in a 96 well-plate.The reaction mixture was incubated for 10 min at room temperature, and the absorbance was measured at 560 nm and water was used as a blank.MIC was expressed as micromoles of EDTA equivalent per gram of dry matter of initial tea leaves for infusion.

2.5 Assessment of bioavailability of phenolic compounds and residual antioxidant activity

To assess changes in TPC, TFC, and antioxidant activity, the bioavailability and residual antioxidant activity were calculated and reported as a percentage (%) according to Equ (1) and Equ (2),respectively:

wherePCrefers to phenolic compounds, which were quantified in each sample at each simulated digestion stage,AArefers to the antioxidant activity that was quantified in each sample at each simulated digestion stage, andBDis the bioactive compound content or antioxidant activity quantified in the initial tea infusion before digestion.Bioavailability is related to the percentage of phenolic compounds present during the digestion process at each sampling point and defines the proportion of compounds that could become available in systematic circulation.Residual antioxidant activity is defined as the percentage of residual antioxidant activity present in the digestion process at each sampling point compared to the activity of the tea infusion before digestion.

2.6 Statistical analysis

The Statistical Package for the Social Sciences (SPSS?,IBM, Chicago, IL, USA) was applied to analyze the data.Mean comparisons were performed using Tukey’s multiple comparison test and least significant difference (LSD) test.Differences were considered significant atP＜ 0.05.Microsoft Excel?was used to calculate the correlation coefficient (R2) of the standard curve.

3.Results and discussion

3.1 Changes in phenolic compound content during simulated digestion

The phenolic compounds (TPC and TFC) of tea infusions during both conditions of simulated digestion were quantified and are reported in Table 1.TPC was (68.66 ± 0.02) mg gallic acid equivalent/g sample (d.b.) and TFC was (4.02 ± 0.48) mg catechin equivalent/g sample (d.b.) presented in initial tea infusion (IT),respectively.Table 1 shows that the disappearance can be observed between IT (pH 5.98) and G0 (pH 1.2) when the pH was adjusted sharply from the native condition.The disappearance of phenolic compounds was observed from the G0 stage as calculated by the ratio of the value for IT to G0 and reported as a decrement fold: 1.69-fold for TPC, 4.28-fold for TFC.This shows that pH plays a significant role in changing the content of bioactive compounds.The depletion in TPC could be associated with the instability of larger molecules of phenolic compounds in an unstable pH environment [18].Besides,it has been suggested that different pH conditions during simulated digestion result in the distribution of –OH radicals on the rings of the phenolic molecules [18].After adding pepsin as a normal digestive condition and allowing G stage for 1 h (G1), there was an increase in phenolic compound content.The results also showed that the amount of TPC and TFC in each stage of the WOE set was lower than that of the MD set.Enzymatic reactions could cause disruption of chemical bonds among phenolics and proteins, carbohydrates, and lipids, thus promoted the solubilization of phenolics [19].It indicated that digestive enzymes could play a role in the bioavailability of green tea infusions.Meanwhile, previous studies [8,9,20,21]have shown a decrease in various polyphenols of a variety of tea beverages following simulated digestion.Therefore, it was also considered that digestive enzymes could play an important role in the release and/or stability of phenolic compounds during digestion, especially in the intestinal stage.

Table 1TPC and TFC of green tea infusions at each simulated digestion stage with different digestive conditions.

The depletion of phenolic compounds at the late I stage (G1I2)may be due to the generation of other complex phenolic derivatives with poor metabolism, namely quinones and chalcones, and could not be detected by spectrophotometry methods [22].According to a previous study [21], the majority of the phenolics were degraded or structural transformed into new compounds during incubation with pancreatin-bile salts at neutral or slightly basic pH, regarded as under intestinal conditions.Therefore, it could be considered that the loss of phenolic compounds after simulated digestion is due to the change in pH, which may cause structural transformations and interactions among the phenolic compounds and digestive mixtures connected to enzymatic reactions, which requires further investigation.

3.2 Variation in antioxidant activity during simulated digestion

Plant polyphenols such as phenolic acids and flavonoids are usually very effective antioxidants because of their ability to act as 1) reducing agents, 2) free radical scavengers by donating an electron or a hydrogen atom, and 3) chelators of metal ions (e.g., iron and copper), which decrease the catalytic formation of free radicals[23].Thus, assays with different mechanisms were assigned to determine the antioxidant activity.The antioxidant activity of green tea infusions at each digestion stage for both the MD and WOE sets assessed by DPPH, ABTS, FRAP, and MIC are reported in Table 2.Overall, the antioxidant activity decreased by around half to one-third of the activity of the initial tea infusion (IT) to the last stage (G1I2)during simulated digestion.The stability of each index for both the MD and WOE sets was observed in the G stage; thereafter, DPPH,ABTS, and FRAP decreased slightly throughout stage I, while MIC showed the opposite trend.After adjusting the pH to 1.2, before the addition of pepsin (G0), the antioxidant activity decreased by approximately half to two-thirds of the initial activity (IT).Both the MD and WOE sets showed the same trend but with comparatively lower values of antioxidant activity in the WOE set, corresponding to a smaller amount of phenolic compounds (Table 1).

Table 2Changes in indices of antioxidant activity of green tea infusion at each simulated digestion stage with different digestive conditions.

It can be assumed that the action of digestive enzymes, as well as changes in pH, caused degradation, alteration, or transformation of the chemical structure of phenolic compounds [8,11,24].Bioactive molecules form two chiral enantiomers called racemization in the digestion environment, leading to various reactivities in the respective digested fractions [25].Additionally, as mentioned previously, the release of these associated compounds correlated positively with the amount of soluble active phenolic compounds, thus influencing the same trends in antioxidant activity [26].Some studies reported that the antioxidant capacity of most tea beverages decreased rapidly during the first 10 min of the gastric stage with a decrease in TPC,then increased and remained relatively stable throughout the intestinal stage as TPC increased [21].However, other studies have shown that simulated digestion reduces the antioxidant activity of green tea infusions, while the TPC in green tea infusions is relatively stable throughout digestion [14].Thus, studies on the correlation between phenolic profile, structure, antioxidant phenolic compounds, exposed activity, and bioavailability need to be carried out in the future.

3.3 Assessment of bioavailability during simulated digestion

Utilization of anin vitrodigestion model allows effective measurement of the bioavailability of polyphenols during and after digestion.Fig.1 depicts the bioavailability of phenolic compounds in green tea infusion at the end of gastric (G) and gastrointestinal(GI) digestion stages during simulated digestion, expressed as bioavailability (%) compared to that of the initial tea infusion (BD).The bioavailability of TPC and TFC at both the G and GI digestion stages for the MD set was significantly higher than that for the WOE set.It has been mainly explained by the fact that the bioavailability of phenolic compounds in green tea during the intestinal digestion phase is remarkably lower than that of non-digested samples [8].Some previous studies also reported that cinnamon polyphenols in cinnamon beverages at the post-pancreatic stage show 79.9% bioavailability because digestive enzymes cause tannin to precipitate [27].The results of this study showed similar trends in both the MD and WOE sets.

Fig.1 Bioavailability of phenolic compounds in green tea infusion at different digestion stages expressed as recovery (%) compared to the initial tea infusion before digestion (BD).Bars represent the standard deviation of triplicate determinations.Capital letters indicate a significant difference (P ＜ 0.05) between the MD and WOE sets within the same phenolic compound groups.Different lowercase letters indicate significant differences (P ＜ 0.05) between the different digestion stages.

The residual bioactivity of green tea infusion during simulated digestion, expressed as residual activity (%) compared to that of the initial tea infusion (BD), is demonstrated in Fig.2.Even with a loss of antioxidant activity, approximately 60% of DPPH, ABTS,and FRAP activities were retained at the G stage.It decreased slightly at the start of the stage I and then decreased to 30%-50%in the final digestion stage.These results are similar to those of a previous study that mentioned the disappearance of DPPH and FRAP from green tea infusion during simulated digestion [14].Interestingly, residual MIC activity was retained in the G stage,but increased from the beginning of the stage I.Besides, the WOE set exhibited a similar trend for both phenolic compounds and antioxidant activity.It implies that certain compounds with the potential to reduce and trap metal ions were generated in the later digestion stage.As mentioned before, the digestive conditions from the G stage to the I stage, especially the elevated pH, can cause changes in the structure of polyphenol compounds, which could promote several reactions during the I stage, including epimerization and auto-oxidation of catechins [28,29].

Fig.2 Residual bioactivity of green tea infusion during simulated digestion assessed by (A) DPPH, (B) ABTS, (C) FRAP, and (D) MIC expressed as residual activity (%) compared to the initial tea infusion before digestion (BD).Bars represent the standard deviation of triplicate determinations.Lowercase letters indicate significant differences among samples within the digestion stage (G0, G1, G1I0, G1I1, and G1I2) (P ＜ 0.05).Uppercase letters show significant differences between MD and WOE in the same digestion stage(P ＜ 0.05).

This study provides convincing evidence thatin vitrogastrointestinal digestion reduces the bioavailability of phenolic compounds and residual antioxidant activity of green tea infusions with or without digestive enzymes.In addition, the stability of antioxidant activity (DPPH, ABTS, and FRAP) under gastric conditions was found to be better than that of the intestinal stage.In general, the pH of the environment induces changes in the concentration and structural transformation of polyphenolic compounds, which affect antioxidant activity [11].Under pH elevation, structural transfiguration of polyphenolic compounds occurred and caused various forms with wide-ranging chemical properties [30].Apart from an elevation in pH, previous studies documented that the auto-oxidation and epimerization of catechins in the intestinal lumen are also accelerated by reactive oxygen species and residual dissolved oxygen [28].In addition to the chemical structures of phenolic compounds, their bioavailability could be modified using isomeric configurations [31].Structural differences in catechin derivatives in green tea influence the radical scavenging activity.Epigallocatechin gallate and epicatechin gallate with galloyl substituents are related to higher scavenging activity than epigallocatechin and epicatechin [26].Therefore, the occurrence of different individual polyphenol structures may reflect residual antioxidant activity, which needs to be investigated in the future.

4.Conclusions

This study showed that the adjustment of pH to acidic conditions caused a major loss of phenolic compounds, which affected residual antioxidant activity.Meanwhile, it was also identified a relative increase in the phenolic compound content and stability of antioxidant activity during digestion by digestive enzymes.These results suggest that digestive enzymes and pH play significant roles in the digestion of green tea.Our work could also be regarded as valuable knowledge of the functional properties of green tea.This may contribute to the development of new types of functional foods or ingredients.

Conflict of interest

The authors declare no conflict of interest.This study was funded by The Tojuro Iijima Foundation for Food Science and Technology,and the International Kyowa Scholarship Foundation, but it had no role in the study design, data collection, or analysis.The authors alone are responsible for the content and writing of this paper.

Acknowledgments

The authors appreciate the National Agriculture and Food Research Organization (NARO), Kanaya, Shizuoka, Japan for providing tea leaf samples.This research was also supported by funding received from the Tojuro Iijima Foundation for Food Science and Technology and the International Kyowa Scholarship Foundation.

Ethics statement

This study did not include any human subjects or animal experiments.