Introduction

Currently, herbal drinks are in great demand due to increased public awareness of a healthy lifestyle. Herbal beverages contain phytochemical compounds that are good for health. Phytochemical compounds such as phenolic compounds, phenolic acids, carotenoids and flavonoids are a good source of natural antioxidants.¹ The natural antioxidant compounds in foods can inhibit the oxidation process in the food itself or prevent diseases caused by oxidative stress.² Scientific research shows that daily consumption of herbal teas has potential health benefits, including lowering serum markers of diabetes, weight loss and increasing antioxidant activity.^3,4

Wedang uwuh is an Indonesian traditional beverage that has been consumed for generations. Wedang uwuh comes from a mixture of several herbal, that are sappan wood, ginger, cinnamon leaves, nutmeg leaves, clove stems, and rock sugar. Consumption of wedang uwuh every day is believed to have health effects on the body.⁵ Each ingredient in wedang uwuh contains antioxidants and other compounds that have beneficial effects. The ginger extracts (Zingiber officinale) contain gingerols, shogaols, and zingerone, which have high antioxidant activity.⁶ Brazilin is a major homoisoflavonoid constituent of sappan wood (Caesalpinia sappan L) and has the potential as an antioxidant, anti-inflammation, and antibacterial.^7,8 Eugenol is a phenolic compound found in clove, nutmeg leave, and cinnamon, which has the ability to reduce ferric ions and radical scavenging activity.⁹ Several studies have shown that wedang uwuh has great potential health benefits that are as an immunomodulator,¹⁰ antioxidant,¹¹ and blood glucose levels control.¹²

The wedang uwuh beverage is usually prepared in traditional way i.e. boiling or brewing. The heating process and boiling time affect the amount of phenolic compounds and antioxidants extracted into the drink.¹³ The previous study showed that different ways of preparing wedang uwuh resulted in different antioxidant capacities and total phenolics.¹¹ The highest value of antioxidant activity and phenolic content of basil was achieved in 5 minutes boiling process, while rosemary was obtained after boiling for 15 minutes.¹⁴ In beverage products, the temperature and length of time of heating will affect the phenolic and antioxidant components that can be extracted.¹⁵ In addition, the heating process can cause chemical structural transformations that affect bioaccessibility.¹⁶

The antioxidant capacity calculated in food materials does not always describe the amount of antioxidant compounds available for absorption by the small intestine (bioaccessibility).¹⁷ The environment along the digestive tract, such as pH conditions and digestive enzyme activities, can cause chemical structure modification from antioxidant compounds that affect the level of their ability as antioxidants and bioaccessibility.¹⁸ Determination of the bioaccessibility can be evaluate by in vitro gastrointestinal digestion assay.¹⁹ The researches using the gastrointestinal digestion model on vegetable juices,²⁰ fruit extracts,²¹ rock tea,²² and green tea infusions¹⁵ showed that total phenolic content and antioxidant capacity changed after in vitro gastrointestinal digestion. However, there is no research on the impact of the boiling time on the bioaccessibility of bioactive components in wedang uwuh beverage after gastrointestinal digestion. Therefore, this study evaluates the impact of boiling time on the antioxidant bioaccessibility in wedang uwuh beverage after in vitro gastrointestinal digestion. 

Materials and Methods

Materials

The wedang uwuh used in this study is a commercial product with the highest level of sales and has a good level of satisfaction from consumers. The analytical materials that are porcine pepsin (from gastric mucosa 700 FIP-U g^-1) purchased from Merck, porcine pancreatic (800 U mL^-1 according to trypsin activity), porcine bile extract, and other analytical chemicals were purchased from Sigma.

Praparation of wedang uwuh beverage

This research used the wedang uwuh with composition as described in Table 1. One serving of wedang uwuh is boiled in 250 ml of boiling water for three different boiling times (5, 10 and 15 minutes). The boiling time was selected based on product preparation instructions and household practices. After boiling, the filtrates were separated and stored in the amber bottles at a temperature -20°C.

Table 1: Composition of wedang uwuh per serving size (5 grams)

Total phenolic content measurement

Folin-ciocalteu method was used to determine total phenolic content in the sample.²⁰ First, 0.2 mL of wedang uwuh beverage sample was reacted with 1.5 mL of folin–ciocalteu reagent 10% in vol (prepared in water) and incubated for 5 minutes. Afterwards, 1.31 mL of sodium carbonate solution (60 gL-1) was added to the solution and incubated for 90 minutes at room temperature. Afterward the absorbance of the solution is measured at the wavelength of 725 nm. Blanks are made from solvents. The calibration standard curve (R²=0.996) used a standard gallic acid solution with a concentration of 0 – 500 ppm, and the data were presented in GAE/serving (mg of gallic acid equivalent per serving).

Ferric-ion (Fe³⁺) reducing antioxidant power assay (FRAP)

Measuring the power of the sample to reduce metal ions was carried out using the FRAP method.^20,23 FRAP reagent was prepared by reacting 300 mM acetate buffer (pH3.6), 20 mM ferric chloride, and 10 mM of 2,4,6-tripyridyl-s-triazine (TPTZ) prepared in HCl (40 mM), following proportion: 10:1:1, respectively. Then to calculate the antioxidant capacity, 25 μL sample of wedang uwuh beverage was reacted with 1mL reagent and 1 mL of warm water (37^oC). The solution was incubated at 37°C for 4 minutes. After incubation, the absorbance of the solution was measured at a wavelength of 593 nm. The blank was made from distilled water and reagent. Antioxidant capacity was presented as mg of Trolox equivalent (TE) per serving using the Trolox standard curve (R²=0.976) with a concentration of 0 – 250 ppm.

DPPH^• radical scavenging activity assay (DPPH)

Antioxidant properties were also determined by measurement the scavenging capacity against DPPH^• free radical.^20,24 0.1 mL of the wedang uwuh beverage sample was reacted with 3.9 mL of DPPH reagent (75 µM, in methanol). The mixture was incubated for 30 minutes at 37°C. After that, the absorbance was measured at wavelength 517 nm, and results were reported in mg of Trolox equivalent (TE) per serving. The blank was made from methanol and reagent. A calibration curve (R2=0.984) was used a solution of Trolox with the concentration of 0 – 250 ppm.

ABTS^•+ radical cation scavenging activity Antioxidant assay (ABTS)

The antioxidant capacity of samples was assessed by ABTS assay.^20,25 Briefly, to prepare the ABTS reagent, 44 µL of potassium persulfate (2.45 nM) as an oxidant was reacted with 2.5 mL of the solution of ABTS (pH 4.57, 7 mM prepared in 20 mM sodium acetate buffer). The mixture was incubated for 12-16 hours to form a radical solution (dark blue-green colour). Then radical solution dilution is carried out to form absorbance of 0.7±0.01 (at wavelength 734 nm) to be used as ABTS reagent. The antioxidant capacity was determined by mixing the wedang uwuh beverage sample (20 μL) and reagent (3 mL), followed by incubation in water bath shaker for 30 minutes at 30°C. Absorbance measurement was carried out at a wavelength of 734 nm. The results were converted to mg Trolox equivalent per serving using the Trolox standard curve (R²=0.97) with concentration 0 – 250 ppm.

Antioxidant bioaccessibility assay

The measurement of the bioaccessibility of antioxidants in wedang uwuh beverage was carried out using in vitro gastric and intestinal digestion followed a protocol of harmonized INFOGEST.²⁶ Gastric digestion was simulated using pepsin enzyme and simulated gastric fluid (SGF), while intestine digestion was simulated using pancreatin solution, bile extract and simulated intestinal fluid (SIF). SGF and SIF were made from stock solutions at the same molarity indicated in the protocol²⁶ and shown in Table 2.

Table 2: The Composition of SGF and SIF stock solutions

In the first stage, gastric digestion simulation was carried out by mixing 20 mL sample with 3.2 mL pepsin solution (25000 U mL^-1), 15 mL of SGF solution, 10 μL of CaCl₂ (0.3 M), and 1.39 mL of water then the pH was adjusted to pH 3.0 by adding HCl (1 M). The mixture was incubated at 37^oC using a water bath shaker (200 rpm) for 2 h. After incubation was complete, 20 mL of gastric chyme was taken to enter the intestinal digestion stage, and the remaining solution was kept in a freezer (−20°C).

Before simulating intestinal digestion, the pH of the gastric chyme was adjusted to pH 7.0 by adding NaOH (1 M). Afterwards, gastric chyme solution (20 mL) was reacted with SIF (11 mL), pancreatin solution (5 mL, with activity 800 U mL^-1 based on trypsin activity), fresh bile extract (2.5 mL, 160 mM), CaCl₂ (40 μL, 0.3 M) and 1.31 mL of water. The solution was incubated at 37^oC for 2 h in a water bath shaker (200 rpm). Intestinal digestion simulation was completed after incubation. Furthermore, to determine the dissolved fraction (bioaccessible fraction), 10 mL solution from intestinal digestion was taken to be centrifuged at 4900 rpm at 37^oC and the supernatant was taken. The fractions generated at each digestion stage (gastric, intestine, and supernatant phase) were stored at −20°C for further analysis (total phenolic content, FRAP, DPPH and ABTS). The bioaccessibility index (BI) value was measured with the following equation.²⁷

BI (%) =  , where A is total phenolic content or antioxidant capacity of wedang uwuh beverage before gastrointestinal digestion, and B is total phenolic content or antioxidant capacity from bioaccessible fraction.

Statistical analyzed

The Minitab version 20.3 software was used to evaluate the data using One-Way analyzed of variance (ANOVA). Tukey’s test was carried out to verify the significance difference between the means at p<0.05. All data were presented as average ± SD (n=2).

Results

The composition of the wedang uwuh

This study used commercial wedang uwuh product which has the highest sales level and good consumer acceptance reviews. The ingredients of wedang uwuh consist of dried emprit ginger (Zingiber officinale var amarum), sappan wood (Caesalpinia sappan L), clove leaves and stems (Syzygium aromaticum), cinnamon leaves (Cinnamomum zeylanicum), and nutmeg leaves (Myristica fragrans) as shown in Figure 1 with the formulation as shown in Table 1.

Figure 1: The ingredients of wedang uwuh. Ingredients in one serving size (A); and dried herbs, i.e., gingers (B); sappanwood (C); clove stems (D); cinnamon leaves (E); nutmeg leaves (F); and clove leaves (G). Click here to view Figure

Phenolic content and antioxidants capacity prior to digestion

Table 3 shows the value of antioxidant capacity and phenolic content of wedang uwuh beverage before going through digestion at different boiling times (5, 10 and 15 minutes). In general, the results of analysis showed that antioxidant capacity and phenolic content of wedang uwuh beverage significantly increased according to the boiling time (p<0.05). Boiling time for 15 minutes produced the highest total phenolic content of 65.48 ± 0.6 mg GAE/serving and the highest antioxidant capacity of 62.25 ± 0.1 mg TE/serving (FRAP) and 96.17 ± 0.1 mg TE/serving (DPPH). However, the ABTS method produced the highest antioxidant capacity at 10 minutes of boiling and was not significantly different from 15 minutes of boiling (p>0.05).

Table 3: Phenolic content (TPC) dan antioxidant capacity wedang uwuh beverage prior to digestion.

Total phenolic content and antioxidant capacity of wedang uwuh beverage (R) before digestion at different boiling times (5,10,15 min). Significantly different (p<0.05) measurement results between boiling time variables are represented by differences in superscript letters in the same column. The data values are expressed in mean±SD (n=2).

Phenolic content and antioxidants capacity after digestion

Table 4 and Figure 2 show the value of antioxidant capacity and phenolic content from wedang uwuh beverage after in vitro gastrointestinal digestion. After gastric digestion there was a significant reduction (p<0.05) in antioxidant capacity and number of phenolic contents, as shown in Figure 2. In this phase, the boiling time still showed a significant (p<0.05) influence of antioxidant capacity and phenolic content in the wedang uwuh beverage. The data in Table 5 shows that boiling for 15 minutes produced the highest total phenolic content of 45.85±1.7 mg GAE/serving and the highest antioxidant capacity of 57.25±0.4 mg TE/serving (FRAP) and 76.27±0.9 mg TE/serving (DPPH), except the ABTS method showed no significant difference in antioxidant capacity (p>0.05) at different boiling times.

Table 4: Total phenolic content dan antioxidant capacity wedang uwuh beverage during the digestion process

Total phenolic content and antioxidant capacity of wedang uwuh beverage (R) during gastrointestinal digestion at different boiling times (5,10,15 min). Significantly different (p<0.05) measurement results between boiling time variables are represented by differences in superscript letters in the same column. Data values are expressed in mean±SD (n=2).

After passing through the intestinal digestion there was a greater decrease in antioxidant capacity and number of phenolic contents, while the length of boiling time had no significant effect (p>0.05). The bioaccessible fraction is described by the supernatant resulting from the intestinal digestion which is the soluble fraction generated from the in vitro gastrointestinal digestion. In general, boiling time for 5 minutes showed antioxidant capacity and number of phenolic contents which was not significantly different (p>0.05) from the longer boiling time. The values of phenolic content and capacity of antioxidant in boiling 5 minutes after digestion were 17.58±1.1 mg GAE/serving (total phenolic content), 20.90±1.8 mg TE/serving (FRAP), and 21.93±1.3 mg TE/serving (DPPH). The antioxidant capacity using ABTS method showed higher results than other methods of 96.26 ± 0.9 – 100.60 ± 1.1 mg TE/serving.

Figure 2: Total phenolic content and antioxidant capacity of wedang uwuh beverage (R). Wedang uwuh beverage showed a decrease in number of phenolic content and antioxidant capacity at the gastric digestion stage (GS) Click here to view Figure

Antioxidant bioaccessibility of wedang uwuh beverage

Table 5 shows the bioaccessibility index of antioxidants and phenolics in wedang uwuh beverage after in vitro gastrointestinal digestion. Boiling time of 5 minutes showed phenolic and antioxidant bioaccessibility index of 32.25± 1.5 % (total phenolic content), 37.25 ± 3.2 % (FRAP), 25.88 ± 1.6 % (DPPH), and 83.45 ± 1.0 (ABTS) that were not significantly different with longer boiling times. In addition, the data in Table 5 shows that boiling for more than 5 minutes produces a lower bioaccessible fraction of phenolic compounds and antioxidants.

Table 5: Bioaccessibility index wedang uwuh beverage after gastrointestinal digestion

Bioaccessibility index of wedang uwuh extracts (R) at different boiling times (5, 10, 15 min). Significantly different (p<0.05) measurement results between boiling time variables are represented by differences in superscript letters in the same column. Data values are expressed in mean±SD (n=2).

Discussion

Wedang uwuh is a traditional beverage originating from Yogyakarta, Indonesia. This beverage is reddish color with a pungent and warm taste. The red color comes from the brazilein compound which is extracted from the sappan wood. Meanwhile, the flavor and warm taste come from other ingredients such as ginger, cloves, nutmeg and cinnamon leaves.⁵ This study used wedang uwuh with the composition as in Table 1 without using rock sugar. The addition of sugar can affect the test results for total phenolic content because sugar can reduce the folin-ciocalteu reagent, so that it can result in false positives.²⁸ In addition, the added sugar in beverage products has an effects on the level of phenolic compounds and antioxidants bioaccessibility. The addition of sugar can increase the stability of the phenolic components to digestive conditions.²⁹ In cinnamon drinks, sugar causes an increase in the bioaccessibility of polyphenols because it can reduce the formation of pepsin-tannin precipitates.³⁰ However, research on grape juice stated that the bioavailability of quercetin decreased in samples with added sugar, because the sugar moiety attached to quercetin interfered with the absorption of quercetin.³¹

Wedang uwuh is the best source of phenolic and antioxidant compounds, which are obtained from the ingredients. Dry emprit ginger is the ingredient with the highest percentage. Dried ginger contains gingerol and shogaol compounds as bioactive compounds, which have high antioxidant activity.³² The second most composition is sappan wood. Sappan wood contains the active compound brazilin which forms a red color in the aqueous extract when oxidized to brazilein, due to the release of H⁺ ions from the hydroxyl group of the benzene ring.^7,33 Scientific studies show that brazilein has activity as an antioxidant.⁸ Other ingredients in wedang uwuh are clove stem and leaves. Cloves contain eugenol as bioactive compound which has strong antioxidant activity in reducing ferric ions and scavenging free radicals.⁹ In addition, nutmeg and cinnamon leaves are also good sources of phenolic compounds and antioxidants.⁵ Cinnamon leaves contain cinnamaldehyde and tannins as the major phenolic compounds.²⁶ Eugenol is also abundantly present in the cinnamon and nutmeg leaves.^34,35 Eugenol is found in the four ingredients of wedang uwuh that are clove (stems and leaves), cinnamon leaves, and nutmeg leaves. The antioxidant capacity of wedang uwuh beverage is the result of the interaction of the antioxidant compounds from the constituent ingredients.

Food processing can also affect antioxidant capacity both before and after digestion. Previous studies have shown that wedang uwuh beverage with a longer boiling time produces greater number of phenolic content and activity of antioxidant.¹¹ This is in line with the results of this study which showed that the longer boiling time significantly increased the number of phenolic content (p<0.05) as well as antioxidant capacity in wedang uwuh beverages. Boiling for 15 minutes resulted in the highest antioxidant capacity and phenolic content in the wedang uwuh beverages, as shown in Table 2. The longer boiling time also increased epicatechin and catechin in hawthorn berry extract.³⁶ In addition, the total phenolic content in the infusion of black tea and white tea also increased up to 15 minutes of brewing time.^37,38 This proves that the longer boiling time will increase the antioxidant capacity because the longer the contact time of the material with the solvent will increase the amount of dissolved antioxidant compounds. The increase in antioxidant capacity and total phenolic content occurs because the heating process during boiling can damage the cell walls and cause changes in cellular structure, which results in the dissolving of phenolic and antioxidant components into the solvent.³⁶

A food ingredient must pass through the digestive system and be able to survive with the changing conditions in digestion, such as digestive enzyme activity and pH, before it can be absorbed and utilized by the body.³⁹ Wedang uwuh beverage contains bioactive compounds which can act as antioxidants in the body.⁵ The results showed that the capacity of antioxidant and phenolic content of wedang uwuh beverage decreased significantly after passing through the simulated gastric digestion process (p<0.05). The decrease in total phenolic after gastric digestion was also reported to occur in infusions of moringa leaves and persimmon leaves.^27,40 This occurs because pH conditions and digestive enzyme activity can cause changes in chemical structure and degradation of several phenolic compounds.^36,41 Decreases in total phenolic content and antioxidant capacity may occur because some phenolic compounds are unstable and degraded during gastric digestion. The acidic pH in the gastric environment causes hydrolysis and transformation of the chemical structure of phenolic compounds and fat-soluble vitamins.¹⁸ Although there was a decrease in the capacity of antioxidant and number of phenolic content from wedang uwuh beverage after gastric digestion, boiling time for 15 minutes still had significant impact (p<0.05) on capacity of antioxidant and total phenolic. This may be due to the amount of initial components extracted during sample preparation.

After the gastric digestion phase, food is transferred to the intestinal digestive phase and then absorbed and used by the body. The alkaline environment in intestinal digestion may cause the phenolic chemical structure to be unstable.⁴² Data from the analysis showed that there was a sharper decrease compared to the gastric digestion phase in the value of antioxidant capacity as well as phenolic content in wedang uwuh beverage after passing through the intestinal digestion, as Figure 2. The boiling process for 15 minutes showed the highest reduction in antioxidant capacity and phenolic content of 14 – 82% and 70%, respectively (data not shown). The heat processing may increase in extractability of phenolic compounds, but it also destroys some thermo-labile compounds.³⁶ This causes phenolic compounds and antioxidants to become more susceptible and easily damaged by different pH levels during gastric and intestinal digestion. Similar results were also reported for persimmon leaves infusion,²⁷ green tea infusions,¹⁵ and hawthorn berry decoction³⁶ that there was a decrease in capacity of antioxidant and phenolic content after in vitro digestion. Conversely, an increase in capacity of antioxidant and phenolic content after simulated digestion was reported in jackfruit extract,²¹ fruit juice,²⁸ and vegetable juices.²⁰ This occurs because the pH conditions and digestive enzyme activity causes the food matrix to decompose thereby increasing the release of phenolic compounds.^27,43 A decrease or increase in antioxidant capacity and total phenolic content after digestion indicates a chemical change in phenolic and antioxidant compounds during the gastrointestinal digestion process due to pH conditions and digestive enzyme activity.^36,43

The decrease in number of phenolic content and capacity of antioxidant in wedang uwuh beverage after going through the in vitro gastrointestinal digestion may be due to a modification of the chemical structure from the extracted ingredients. Previous research reported that the concentration of gingerol in ginger extract decreased after passing through the in vitro gastrointestinal digestion.⁴⁴ The cinnamon beverage has lower phenolic content and capacity of antioxidant after gastric-pancreatic digestion because of tannins precipitation, which interacts with pepsin in the gastric phase.³⁰ Scientific research reported that brazilin and phenolic compounds in sappan wood extract showed the highest stability in acid environment, but easily oxidized by adding NAOH (0.1 N) which causes deprotonation of the -OH group and changes in the structure of the brazilein molecule.^7,45 Additionally, clove and nutmeg were reported to be stable during digestion.⁴⁶

The boiling process for 15 minutes resulted in the highest antioxidant capacity and total phenolic before consumed, but there was a significant decrease (p<0.05)          after the simulated gastrointestinal digestion process. After passing through the gastric and intestinal digestion, the boiling time did not significantly affect (p>0.05) the bioaccessible fraction of antioxidant and phenolic compounds in wedang uwuh beverage. The bioaccessible fraction was calculated from the supernatant resulting from the centrifugation of the digestive solution in intestine. The supernatant describes the fraction that can be digested and contains bioactive components that are able to survive digestive conditions so that they can be absorbed by the body.²² The boiling process for 5 minutes resulted in a higher number of bioaccessible fractions and antioxidant bioaccessibility index but not significantly different (p>0.05) from the longer boiling time. This shows that boiling time which is longer than 5 minutes causes greater damage to phenolic and antioxidants compounds so that they are more susceptible to damage during the digestion process. The results of previous studies on ready-to-drink wedang uwuh showed that there was thermal degradation of antioxidant activity at higher pasteurization temperatures.⁴⁷ Similar results were also reported that the lowest bioaccessibility of phenolic compounds occurred in fruit drink products with heat treatment compared to non-thermal methods.⁴⁸ In hawthorn berry fruit extract, there was a decrease in antioxidant activity and phenolic bioaccessibility in boiling for more than 15 minutes.³⁶ The heating process can affect the stability of phenolic compounds before and after digestion, thereby reducing their antioxidant activity.^15,49 Furthermore, the heating process can change the chemical structure of phenolic compounds causing an increase or decrease in bioaccessibility by promoting the release of phenolic compounds from the food matrix, chemical structural degradation, or polymerization.⁴⁸

The ABTS test resulted in the highest bioaccessibility index and antioxidant capacity compared to the FRAP and DPPH methods. This can occur due to differences in the mechanism of the test method.²² The digestion process followed by a structural transformation of phenolic compounds and antioxidants which causes interactions between sample constituents, thus influencing their detection using similar or more sensitive methods.⁵⁰ In addition, the results of other studies have reported that the test method with ABTS does indeed show better sensitivity than DPPH for food samples that are hydrophilic, lipophilic, and contain high pigment components.⁵¹ From the analysis it can be concluded that boiling for 5 minutes is the best way for preparing wedang uwuh beverage, with antioxidant bioaccessibility index of 37.25% (FRAP), 25.88% (DPPH) and 83.45% (ABTS). Previous studies reported variations in the bioaccessibility of phenolic and antioxidant compounds in different beverages, i.e. 18.0 – 25.9% for the phenolic compounds bioaccessibility of fruit juice drinks,⁴⁸ 24.6% for the antioxidants bioaccessibility of persimmon leaves infusion²⁷ and 10% for bioactivity of mono-caffeoylquinic acids from J. glutinosa infusion.²² In addition, differences in the chemical structure of phenolic and antioxidant compounds, as well as the food matrix, may also affect behavior and stability under in vitro gastrointestinal digestion.^18,21

Conclusion

This study concluded that phenolic and antioxidant compounds in wedang uwuh beverages were sensitive to the gastrointestinal environment. Before gastrointestinal digestion, boiling for 15 minutes produced the highest number of phenolic content and capacity of antioxidant in wedang uwuh beverage. However, there was a decrease in the number of phenolic content and capacity of antioxidant in wedang uwuh beverages after the in vitro gastrointestinal digestion due to digestive conditions such as pH and enzyme activity. A boiling time of 5 minutes gave the highest phenolic content, capacity of antioxidant and bioaccessibility index of antioxidant in wedang uwuh beverages after the in vitro gastrointestinal digestion. Therefore, 5 minutes of boiling time is recommended as the best way to prepare wedang uwuh beverage. Further research is envisaged to investigate the composition changes of phenolic and antioxidant compounds of wedang uwuh during the digestion process.

Acknowledgement

This research was supported by Department food science and technology – IPB University, National Research and Innovation Agency (BRIN), and Ministry of Research and Technology of Indonesia.

Conflict of Interest

The authors declare that there is no conflict of interest.

Funding Sources

This research was funded by the Deputy for Strengthening Research and Development, Ministry of Research and Technology of Indonesia – National Research and Innovation Agency (BRIN), in accordance with contract numbers 082/E5/PG.02.00.PT/2022 and 3838/IT3.L1/PT.01/03.

References

1. Chandrasekara A., Shahidi F. Herbal Beverages: Bioactive Compounds and Their Role in Disease Risk Reduction – A review. J Tradit Complement Med. 2018;8(4):451-458.
2. Cömert E.D., Gökmen V. Antioxidants Bound to an Insoluble Food Matrix: Their Analysis, Regeneration Behavior, and Physiological Importance. Compr Rev Food Sci Food Saf. 2017;16(3):382-399.
3. Zemestani M., Rafraf M., Asghari-Jafarabadi M. Chamomile Tea Improves Glycemic Indices and Antioxidants Status in Patients with Type 2 Diabetes Mellitus. Nutrition. 2016;32(1):66-72.
4. Etheridge C.J., Derbyshire E. Herbal Infusions and Health: A Review of Findings from Human Studies, Mechanisms and Future Research Directions. Nutr Food Sci. 2020;50(5):969-985.
5. Setyowati N., Masyhuri, Mulyo J.H., Irham, Yudhistira B. The Hidden Treasure of Wedang Uwuh, An Ethnic Traditional Drink From Java, Indonesia: Its Benefits and Innovations. Int J Gastron Food Sci. 2023;31(February):100688.
6. Semwal R.B., Semwal D.K., Combrinck S., Viljoen A.M. Gingerols and Shogaols: Important Nutraceutical Principles from Ginger. Phytochemistry. 2015;117:554-568.
7. Rajput M.S., Nirmal N.P., Nirmal S.J., Santivarangkna C. Bio-actives from Caesalpinia sappan Linn: Recent Advancements in Phytochemistry and Pharmacology. South African J Bot. 2021;000.
8. Nirmal N.P., Rajput M.S., Prasad R.G.S.V., Ahmad M. Brazilin from Caesalpinia sappan Heartwood and Its Pharmacological Activities: A review. Asian Pac J Trop Med. 2015;8(6):421-430.
9. Gülçin I. Antioxidant Activity of Eugenol: A Structure-Activity Relationship Study. J Med Food. 2011;14(9):975-985.
10. Widyaningsih T.D. Traditional Drink of Black Cincau (Mesona palustris BL) -Based Wedang Uwuh As Immunomodulator on Alloxan-Induced Diabetic Rats. 2020;0034:6659.
11. Widanti Y.A., Nuraini V., Ariyanto SD. Sifat Sensoris dan Aktivitas Antioksidan Wedang Uwuh Kelor Dengan Variasi Cara Penyeduhan. Res Fair Unisri. 2019;3(1).
12. Harijono, Mualimin L., Estiasih T., Wulan S.N., Pramita H.S. Potensi Minuman Fungsional Wedang Uwuh Sebagai Kontrol Berat Badan dan Kontrol Kadar Glukosa Darah. J Pangan dan Agroindustri. 2021;9(3):155-164.
13. Francenia Santos-Sánchez N., Salas-Coronado R., Villanueva-Cañongo C., Hernández-Carlos B. Antioxidant Compounds and Their Antioxidant Mechanism. Antioxidants. Published online 2019:1-28.
14. Salamatullah A.M., Hayat K., Arzoo S., Alzahrani A., Ahmed M.A., Yehia H.M., Alsulami T., Al-Badr N., Al-Zaied B.A.M., Althbiti M.M. Boiling Technique-Based Food Processing Effects on The Bioactive and Antimicrobial Properties of Basil and Rosemary. Molecules. 2021;26(23).
15. Donlao N., Ogawa Y. Impacts of Processing Conditions on Digestive Recovery of Polyphenolic Compounds and Stability of The Antioxidant Activity of Green Tea Infusion During In vitro Gastrointestinal Digestion. LWT – Food Sci Technol. 2018;89(September 2017):648-656.
16. Bilal Hussain M., Hassan S., Waheed M., Javed A., Adil Farooq M., Tahir A. Bioavailability and Metabolic Pathway of Phenolic Compounds. Plant Physiol Asp Phenolic Compd. Published online 2019:1-18.
17. Gutiérrez-Grijalva E.P., Ambriz-Pérez D.L., Leyva-López N., Castillo-López R.I., Heredia J.B. Review: Dietary Phenolic Compounds, Health Benefits and Bioaccessibility. Arch Latinoam Nutr. 2016;66(2):87-100.
18. Mosele J.I., Macià A., Romero M.P., Motilva M.J. Stability and metabolism of Arbutus unedo Bioactive Compounds (Phenolics and Antioxidants) Under In vitro Digestion and Colonic Fermentation. Food Chem. 2016;201:120-130.
19. Szawara-Nowak D., Bączek N., Zieliński H. Antioxidant Capacity and Bioaccessibility of Buckwheat-Enhanced Wheat Bread Phenolics. J Food Sci Technol. 2016;53(1):621-630.
20. Wootton-Beard P.C., Moran A., Ryan L. Stability of The Total Antioxidant Capacity and Total Polyphenol Content of 23 Commercially Available Vegetable Juices Before and After In vitro Digestion Measured by FRAP, DPPH, ABTS and Folin-Ciocalteu Methods. Food Res Int. 2011;44(1):217-224.
21. Pavan V.Ô., Sancho R.A.S., Pastore G.M. The Effect of Invitro Digestion on The Antioxidant Activity of Fruit Extracts (Carica papaya, Artocarpus heterophillus and Annona marcgravii). LWT – Food Sci Technol. 2014;59(2P2):1247-1251.
22. Ortega-Vidal J., Ruiz-Riaguas A., Fernández-de Córdova M.L., Ortega-Barrales P., Llorent-Martínez E.J. Phenolic Profile and Antioxidant Activity of Jasonia glutinosa Herbal Tea. Influence of Simulated Gastrointestinal In vitro Digestion. Food Chem. 2019;287(October 2018):258-264.
23. Benzie I.F., Strain J. The Ferric Reducing Ability of Plasma (FRAP) as a Measure of ‘“Antioxidant Power”’: The FRAP Assay. Anal Biochem. 1996;239:70-76.
24. Brand-Williams W., Cuvelier M.E., Berset C. Use of A Free Radical Method to Evaluate Antioxidant Activity. LWT – Food Sci Technol. 1995;28(1):25-30.
25. Ozgen M., Reese R.N., Tulio A.Z., Scheerens J.C., Miller A.R. Modified 2,2-Azino-bis-3-ethylbenzothiazoline-6-sulfonic Acid (ABTS) Method to Measure Antioxidant Capacity of Selected Small Fruits and Comparison to Ferric Reducing Antioxidant Power (FRAP) and 2,2′-Diphenyl-1-picrylhydrazyl (DPPH) Methods. J Agric Food Chem. 2006;54(4):1151−1157.
26. Minekus M., Alminger M., Alvito P., Ballance S., Bohn T., Bourlieu C., Carri`ere F., Boutrou R., Corredig M., Dupont D., Dufour C., Egger L., Recio I., Santos C.N., Singh R.P., Vegarud G.E., Wickham M.S.J., Macierzanka A., Mackie A., Marze S., McClements D. J., M´enard O, Golding M., Karakaya S., Kirkhus B., Le Feunteun S., Lesmes U., Weitschies W., Brodkorb A. A standardised Static In vitro Digestion Method Suitable for Food – an International Consensus. Food Funct. 2014;5(6):1113-1124.
27. Martínez-Las Heras R., Pinazo A., Heredia A., Andrés A. Evaluation Studies of Persimmon Plant (Diospyros kaki) for Physiological Benefits and Bioaccessibility of Antioxidants by In vitro Simulated Gastrointestinal Digestion. Food Chem. 2017;214:478-485.
28. Quan W., Tao Y., Lu M., Yuan B., Chen J., Zeng M., Qin F., Guo F., He. Stability of The Phenolic Compounds and Antioxidant Capacity of Five Fruit (Apple, Orange, Grape, Pomelo and Kiwi) Juices During In vitro-Simulated Gastrointestinal Digestion. Int J Food Sci Technol. 2018;53(5):1131-1139.
29. Helal A., Tagliazucchi D. Impact of In-vitro Gastro-Pancreatic Digestion on Polyphenols and Cinnamaldehyde Bioaccessibility and Antioxidant Activity in Stirred Cinnamon-Fortified Yogurt. Lwt. 2018;89(October 2017):164-170.
30. Helal A., Tagliazucchi D., Verzelloni E., Conte A. Bioaccessibility of Polyphenols and Cinnamaldehyde in Cinnamon Beverages Subjected to In vitro Gastro-Pancreatic Digestion. J Funct Foods. 2014;7(1):506-516.
31. Meng X., Maliakal P., Lu H., Lee M.J., Yang C.S. Urinary and Plasma Levels of Resveratrol and Quercetin in Humans, Mice, and Rats after Ingestion of Pure Compounds and Grape Juice. J Agric Food Chem. 2004;52(4):935-942.
32. Ko M.J., Nam H.H., Chung M.S. Conversion of 6-Gingerol to 6-Shogaol in Ginger (Zingiber officinale) Pulp and Peel During Subcritical Water Extraction. Food Chem. 2019;270(July 2018):149-155.
33. Ngamwonglumlert L., Devahastin S., Chiewchan N., Raghavan G.S.V. Color and Molecular Structure Alterations of Brazilein Extracted from Caesalpinia sappan L. Under Different pH and Heating Conditions. Sci Rep. 2020;10(1):1-10.
34. Sharma U.K., Sharma A.K., Pandey A.K. Medicinal Attributes of Major Phenylpropanoids Present in Cinnamon. BMC Complement Altern Med. 2016;16(1):1-11.
35. Ashokkumar K., Vellaikumar S., Muthusamy M., Dhanya M.K., Aiswarya S. Compositional Variation in The Leaf, Mace, Kernel, and Seed Essential Oil of Nutmeg (Myristica fragrans Houtt.) from The Western Ghats, India. Nat Prod Res. 2021;36(1):432-435.
36. Lou X., Guo X., Wang K., Wu C., Jin Y., Lin Y., Xu H., Hanna M., Yuan L. Phenolic Profiles and Antioxidant Activity of Crataegus pinnatifida Fruit Infusion and Decoction and Influence of In vitro Gastrointestinal Digestion on Their Digestive Recovery. Lwt. 2021;135:110171.
37. Ramalho S.A., Nigam N., Oliveira G.B., de Oliveira P.A., Silva T.O.M., dos Santos A.G.P., Narain N. Effect of Infusion Time on Phenolic Compounds and Caffeine Content in Black Tea. Food Res Int. 2013;51(1):155-161.
38. Pérez-Burillo S., Giménez R., Rufián-Henares J.A., Pastoriza S. Effect of Brewing Time and Temperature on Antioxidant Capacity and Phenols of White Tea: Relationship With Sensory Properties. Food Chem. 2018;248(December 2017):111-118.
39. Carbonell-Capella J.M., Buniowska M., Barba F.J., Esteve M.J., Frígola A. Analytical Methods for Determining Bioavailability and Bioaccessibility of Bioactive Compounds from Fruits and Vegetables: A review. Compr Rev Food Sci Food Saf. 2014;13(2):155-171.
40. Caicedo-Lopez L.H., Luzardo-Ocampo I., Cuellar-Nuñez M.L., Campos-Vega R., Mendoza S., Loarca-Piña G. Effect of The In vitro Gastrointestinal Digestion on Free-Phenolic Compounds and Mono/Oligosaccharides from Moringa oleifera Leaves: Bioaccessibility, Intestinal Permeability and Antioxidant Capacity. Food Res Int. 2019;120(July 2018):631-642.
41. Spínola V., Llorent-Martínez E.J., Castilho P.C. Antioxidant Polyphenols of Madeira sorrel (Rumex maderensis): How Do They Survive to In vitro Simulated Gastrointestinal Digestion? Food Chem. 2018;259(November 2017):105-112.
42. Carbonell-Capella J., Blesa J., Frígola A., Esteve M. Study of The Interactions of Bioactive Compounds and Antioxidant Capacity of An Exotic Fruits Beverage That Sweetened with Stevia. MOJ Food Process Technol. 2019;7(3):79-86.
43. Moyo S.M., Serem J.C., Bester M.J., Mavumengwana V., Kayitesi E. The Impact of Boiling and In vitro Human Digestion of Solanum nigrum complex (Black nightshade) on Phenolic Compounds Bioactivity and Bioaccessibility. Food Res Int. 2020;137(September):109720.
44. Wang J., Chen Y., Hu X., Feng F., Cai L., Chen F. Assessing The Effects of Ginger Extract on Polyphenol Profiles and The Subsequent Impact on The Fecal Microbiota by Simulating Digestion and Fermentation In vitro. Nutrients. 2020;12(10):1-13.
45. Hemthanon T., Ungcharoenwiwat P. Antibacterial Activity, Stability, and Hemolytic Activity of Heartwood Extract from Caesalpinia sappan for Application on Nonwoven Fabric. Electron J Biotechnol. 2022;55:9-17.
46. Baker I., Chohan M., Opara E.I. Impact of Cooking and Digestion, In Vitro, on the Antioxidant Capacity and Anti-Inflammatory Activity of Cinnamon, Clove and Nutmeg. Plant Foods Hum Nutr. 2013;68(4):364-369.
47. Herdiana D.D., Utami R., Anandito R.B.K. Kinetika Degradasi Termal Aktivitas Antioksidan pada Minuman Tradisional Wedang Uwuh Siap Minum. J Teknosains Pangan. 2012;2 No 2 Apr(1):41-48.
48. Rodríguez-Roque M.J., de Ancos B., Sánchez-Moreno C., Cano M.P., Elez-Martínez P., Martín-Belloso O. Impact of Food Matrix and Processing on The In vitro Bioaccessibility of Vitamin C, Phenolic Compounds, and Hydrophilic Antioxidant Activity from Fruit Juice-Based Beverages. J Funct Foods. 2015;14:33-43.
49. Hans W.B., Serges Cyrille N.H., Ghislain M.N., Audrey Therese N.M., Eric Serge N. Effect of Boiling on the Phenolic Content and Antioxidant Activity of Tomato (Lycopersicon esculentum L.) fruits. J Food Stab. 2018;1(1):28-35.
50. Ma Y., Yang Y., Gao J., Feng J., Shang Y., Wei Z. Phenolics and Antioxidant Activity of Bamboo Leaves Soup as Affected by In Vitro digestion. Food Chem Toxicol. 2020;135(September 2019):110941.
51. Floegel A., Kim D.O., Chung S.J., Koo S.I., Chun O.K. Comparison of ABTS/DPPH Assays to Measure Antioxidant Capacity in Popular Antioxidant-Rich US Foods. J Food Compos Anal. 2011;24(7):1043-1048.