Abstract
The significant production of tea waste globally raises environmental concerns. Tea waste can be valorized by extracting its phytochemicals. In this study, the recovery of phenolic substances, flavonoids, and antioxidants from the black tea wastes using two types of hydrogen-rich water (HRW): hydrogen bubbling and magnesium-water reaction (Mg water), besides ethanol/water (50/50, % vol), ethanol/Mg water (50/50, % vol), and pure water (control) was investigated. The best extraction yield was obtained for HRW (30.13%). The best levels of phenolics (TPC), flavonoids (TFC), and antioxidants (DPPH and ABTS) were found for HRW extracts, followed by ethanol/Mg water (50/50). The levels of TPC, TFC, DPPH, and ABTS increased by 193.05, 210.56, 49.21, and 86.60%, and by 59.70, 33.46, 28.66, and 58.25% when HRW and Mg water was used as a solvent instead of pure water, respectively, in the extraction. The maximum levels of phenolic acids (p-coumaric acid, chlorogenic acid, gallic acid) and flavonoids (rutin and epicatechin) were found in HRW extracts. Hydrogen extraction can be proposed as a sustainable method to extract phenolic substances from agri-food waste.
Keywords: Tea waste, Hydrogen-rich water, Molecular hydrogen, Extraction, Sustainability, Phenolic recovery
Highlights
Huge production of tea generates significant quantities of phenolic-rich tea waste.
The highest extraction yield was for HRW.
The highest phenolics, flavonoids, and antioxidants were for HRW extracts.
The levels of TPC, TFC, and antioxidants increased by about 2 folds by HRW.
HRW is a green method for extracting phenolic substances from agri-food wastes.
Introduction
A timely and sustainable solution seems necessary in the face of the global food security crisis, exacerbated by climate change and its devastating effects on crop production and food availability. Additionally, due to the gradual increase in the world’s population and ever-growing consumption, a significant amount of agri-food waste, such as tea waste, is produced (Gunduzalp 2016).
Tea, a globally popular beverage, is a significant contributor to the food industry’s agri-food waste. The 2021 global tea production reached a staggering 6.33 million tons (Nations 2022), resulting in 5 million tonnes substantial waste production (Kumar et al. 2023). The research findings can potentially revolutionize the tea industry by offering a sustainable solution for utilizing tea waste. Tea waste has been proposed as agricultural fertilizer and raw material to produce caffeine, catechin, and other phenolic substances (Ozarslan et al. 2022).
Tea leaves contain more than 4000 chemical components and are one of the plants containing vast amounts of flavonoids (Gübür 2015). The amount of polyphenols in tea leaves varies depending on the oxidation process applied during tea processing (Okinda Owuor et al. 2006).
Different extraction methods are used to enhance the recovery of phytochemicals, such as phenolic compounds. Some methods need costly equipment, while others necessitate additional preparation steps, increasing energy, solvent, cost, and time consumption. Various extraction methods have been used to recover phytochemicals from tea and tea waste, such as classical (Mariya John et al. 2006), microwave-assisted (Li and Jiang 2010), ultrasonic-assisted (Horzic et al. 2012), high-pressure (Jun 2009), and supercritical fluid (Icen and Guru 2009) methods. However, using sustainable and cost-effective techniques is critical to preserving the planet’s natural resources.
Hydrogen (H2) is a non-toxic, inert, and reducing gas. H2 possesses more than 36 physical, chemical, and biological properties, of which the antioxidant property is the most important in food processing (Alwazeer 2024a). Due to its small size and high diffusibility, H2 can penetrate many membranes, tissues, and plastic materials. These specific properties of H2 opened the door for researchers to evaluate its potential applications in various fields, including health (Alwazeer 2024b; Xie and Song 2024), food and crops (Alwazeer and Engin 2022; Russell et al. 2024), agriculture (Zulfiqar et al. 2021; Alwazeer and Çiğdem 2022), ecology and environment (Köktürk et al. 2022a, b), and, of course, energy. Recently, the ability of H2 to enhance the phenolic, flavonoid, anthocyanin, and antioxidant extraction from different plant materials has been reported (Alwazeer and Elnasanelkasim 2023; Alwazeer et al. 2023a, b, c; d; Ceylan et al. 2023; Alwazeer 2023). Due to the massive generation of tea wastes worldwide, this study aimed to valorize them by evaluating the efficiency of hydrogen-rich water extraction as a sustainable method to recover phenolic substances from black tea wastes. The results can help develop a bio-based circular carbon economic model and a Bio-E3 concept, creating technology promoting the economy, environment, and employment (Hsiao and Hu 2024).
Materials and methods
Chemicals
The chemicals (DPPH, ABTS, Trolox, ascorbic acid, quercetin, 3,4,5 trihydroxybenzoic acid, potassium persulfate, Folin–Ciocalteu reagent were purchased from Merck (Darmstadt, Germany) and Sigma (MO, USA).
Black tea waste preparation
The black tea waste was prepared in the laboratory, as shown below. A dry black tea was obtained from a local market. The black tea infusion was prepared by boiling 25 g of dry black tea leaves in one liter of water for 5 min. The tea leaves (wastes) were then separated and dried at 35 °C. The dried tea wastes were milled into powder and stored at -80 °C.
Phenolic extraction
The extraction procedure was performed according to the method described by Alwazeer and co-workers (Alwazeer et al. 2023d). Briefly, 1 g tea waste powder was mixed with 20 mL of solvent [(pure water prepared by Millipore Milli-Q® Direct 8 Water Purification System, USA), hydrogen-rich water (HRW), magnesium water (Mg water), ethanol/Mg water (50/50, % vol), and ethanol/pure water (50/50, % vol)], followed by a 24-hr incubation at 35 °C. The evaporation of solvents was then performed.
Determination of total phenolic content
The total phenolic content (TPC) in tea waste extracts was determined according to the method described by Alwazeer and co-workers (Alwazeer et al. 2023d).
Determination of total flavonoid content
The total flavonoid content (TFC) in tea waste samples was measured according to the method described by Alwazeer and co-workers (Alwazeer et al. 2023d).
DPPH scavenging activity
The DPPH scavenging activity of tea waste samples was measured according to Alwazeer and co-workers (Alwazeer et al. 2023d).
ABTS scavenging activity
The ABTS scavenging activity of tea waste samples was measured according to the method described by Alwazeer and co-workers (Alwazeer et al. 2023d).
HPLC analysis
The profile of phenolic substances was determined by the HPLC method described by Alwazeer and co-workers (Engin et al. 2025). An HPLC-DAD instrument (1260 Infinity, Agilent Technologies, USA) and a reverse phase C18 column (ACE Generix, particle size 250 × 4.6 mm, 5 μm particle size) were used in phenolic profile analysis. A volume of 20 µL of the filtered tea extract solution (0.45 μm membrane filter) was injected at a flow rate of 0.8 mL/min. A gradient elution system with a mobile phase composed of two solvents, A (1% vol, orthophosphric acid in water) and B (acetonitrile), at a total run time of 40 min, was applied. The gradient program started with 83% A and then changd to 60% and 83% at 30 mn and 35 min, respectively. Phenolic compounds were detected at 350/200 nm wavelength.
Statistical analysis
Data were analyzed by one-way ANOVA using GraphPad Prism 9 Software. Turkey’s post hoc tests were followed by Duncan’s multiple comparison test using the IBM SPSS Statistics 26 package program. The significant differences were statistically considered at a level lower than 0.05. The whole experiment was duplicated, and the measurements were triplicated (n = 3).
Results and discussion
Extract yield
The extraction yield varied according to the type of solvent. The maximum extraction yield was obtained for HRW tea waste extracts at 30.13%, followed by ethanol/Mg water (50/50) at 21.86%, whereas the minimum yield was noticed for pure water at 16.23% (P < 0.05) (Table 1). Similar results were reported for olive leaves, where the maximum extraction yield was shown for HRW by an increase rate of 34.42%, followed by ethanol/Mg water (50/50) with an increase rate of 29.33%, whereas the minimum was shown for pure water by an increase rate of 19.27% (Alwazeer et al. 2023d).
Table 1.
The extraction yield of the black tea waste using different solvents
| Solvent | Extraction yield | |
|---|---|---|
| Extract (mg) | Extract (%) | |
| Pure water | 162.3e | 16.23 |
| Mg water | 207.4c | 20.74 |
| Hydrogen-rich water | 301.3a | 30.13 |
| Ethanol/Mg water (50/50) | 218.6b | 21.86 |
| Ethanol/water (50/50) | 185.8d | 18.58 |
a−d different lowercase letters mean a significant difference between samples at P < 0.05
Hydrogen gas can easily pass through the plant cell’s cytoplasm membrane due to its high diffusion rate, reaching all compartments and organelles of the plant cell and increasing the separation of phenolics from plant materials via different possible mechanisms (Alwazeer 2023). The extraction yield of tea waste obtained with Mg water, in which the amounts of Mg2+ were high, was lower than that of HRW extracts. Tea phenolic substances can be partially retained by combining with ions such as Ca2+ and Mg2+ (Danrong et al. 2009).
Total phenolic content (TPC)
The TPC values of tea waste samples prepared using different solvents are shown in Table 2. HRW tea waste extracts exhibited the highest TPC values, followed by ethanol/Mg water (50/50) samples with 163.70 and 134.06 mg GAE/g, respectively (P < 0.05). Additionally, using aqueous ethanol as a solvent increased the extraction of phenolics compared with pure water.
Table 2.
Effect of solvent type on the recovery of phenolics, flavonoids, and antioxidants of black tea waste extracts
| Parameter | Pure water | Mg water | Hydrogen-rich water | Ethanol/water (50/50) | Ethanol/Mg water (50/50) |
|---|---|---|---|---|---|
| Total phenolic content (mg GAE/g extract) | 55.86 ± 0.64e | 89.21 ± 0.99c | 163.70 ± 1.98a | 75.50 ± 1.65d | 134.06 ± 1.96b |
| Total flavonoid content (mg QE/g extract) | 19.04 ± 0.44e | 25.41 ± 1.03c | 59.13 ± 0.76a | 22.29 ± 0.24d | 40.66 ± 0.52b |
| DPPH scavenging activity (mg AAE/g extract) | 15.18 ± 0.30e | 19.53 ± 0.04c | 22.65 ± 0.06a | 19.01 ± 0.14d | 21.99 ± 0. 04b |
| ABTS scavenging activity (mg TE/g extract) | 14.25 ± 0.34e | 22.55 ± 0.14c | 26.59 ± 0.19a | 21.89 ± 0.02d | 24.58 ± 0. 05b |
a−d different lowercase letters show a significant difference between samples at P < 0.05
Results show that the infusion of H2 into water by bubbling method for preparing HRW provided better TPC levels than the Mg-water reaction method. The TPC values increased by 193.05% for HRW samples compared to the pure water ones. However, using Mg water instead of pure water to prepare aqueous ethanol effectively enhanced the phenolic extraction (Fig. 1; Table 2). This led to a rise in the TPC level by only 77.56% (P < 0.05) (Table 3). The results show that the use of HRW alone or with ethanol effectively improved the recovery of phenolic compounds.
Fig. 1.
Phytochemicals of black tea waste extracts prepared by various solvents [(pure water), hydrogen-rich water (HRW), magnesium water (Mg water), ethanol/water (50/50, % vol), and ethanol/Mg water (50/50, % vol)] (****P < 0.0001)
Table 3.
Effect of solvent type on the percent change of the recovery of phenolics, flavonoids, and antioxidants of black tea waste extracts
| Solvent | TPC change (%) |
|---|---|
| Pure water | - |
| Mg water | 59.70c |
| Hydrogen-rich water | 193.05a |
| Ethanol/water (50/50) | - |
| Ethanol/Mg water (50/50) | 77.56b |
| TFC change (%) | |
| Pure water | - |
| Mg water | 33.46 c |
| Hydrogen-rich water | 210.56a |
| Ethanol/water (50/50) | - |
| Ethanol/Mg water (50/50) | 82.41 b |
| DPPH change (%) | |
| Pure water | - |
| Mg water | 28.66 b |
| Hydrogen-rich water | 49.21a |
| Ethanol/water (50/50) | - |
| Ethanol/Mg water (50/50) | 15.68 c |
| ABTS change (%) | |
| Pure water | - |
| Mg water | 58.25 b |
| Hydrogen-rich water | 86.60a |
| Ethanol/water (50/50) | - |
| Ethanol/Mg water (50/50) | 12.29 c |
a−d different lowercase letters show a significant difference between samples at P < 0.05
Molecular hydrogen dissolved in HRW possesses several specific physical, chemical, and physiological properties, such as antioxidant and redox homeostasis regulator (Alwazeer 2024a). Since the oxidation of phenolic substances can be restricted by reducing agents, such as hydrogen gas, the phenolic substances in HRW extracts were better protected and extracted by HRW than pure water. Similar results have been obtained by Ryu and co-workers (2019), who found that using hydrogen gas was more potent in recovering phenolic substances from fresh green tea leaves compared with other gases, including nitrogen, oxygen, carbon dioxide, and air (Ryu et al. 2019). The authors examined the morphological structure of tea leaves at the end of extraction, reporting that HRW-treated leaves have undergone a surface modification manifested by noticeable flexibility and smoothness characteristics that were not shown in other gases.
Many possible mechanisms were proposed to explain the improved impact of hydrogen-rich solvents on the recovery of phytochemicals, such as phenolic substances. These mechanisms include 1) the protection of phenolics from oxidative reactions, 2) the reduction of quinones and semiquinones thanks to the reducing property of H2, 3) scavenging of the most reactive free radicals, i.e., hydroxyl radical (•OH) and peroxynitrite (ONOO−), 4) scavenging of the polyphenol oxidase reaction products thanks to the selective antioxidant property of H2, 5) degassing dissolved oxygen, 6) redox homeostasis of the plant cell cytoplasm, 7) modification of the physical structure of water, 8) activation of polysaccharide monooxygenases liberating the entrapped phenolic acids, and 9) the modification of the surface structure of plant material (Alwazeer 2023). Hydroxyl radicals, formed via the Fenton reaction in the presence of ferrous, are strong oxidants that can almost immediately and unselectively oxidize all antioxidants and phenolics in the medium (Waterhouse and Laurie 2006). H2 has been proven to selectively scavenge hydroxyl radicals (Ohsawa et al. 2007).”.
Using Mg water that contains dissolved hydrogen, like HRW, led to an increase in the extraction of phenolic substances compared to pure water but less than HRW, with 59.70 and 193.05%, respectively (P < 0.05)(Table 3). This means that the presence of Mg2+ ions in the medium negatively influenced phenolic extraction, possibly due to the phenolic-Mg complex precipitation (Alwazeer et al. 2023d). Other possible mechanisms describing the negative impact of Mg on the recovery of phenolic substances from olive leaves were described in detail in a previous study (Alwazeer et al. 2023d).
Total flavonoid content (TFC)
The TFC values of tea waste extracts prepared using different solvents are shown in Table 2. The solvent type significantly affected the TFC value (P < 0.05) (Table 3). Similar to the total phenolic content values, the best TFC levels were shown for HRW extracts (59.13 mg QE/g extract), followed by Mg water ones (25.41 mg QE/g extract)(P < 0.05). Additionally, using aqueous ethanol instead of pure water as a solvent effectively improved the recovery of flavonoids. Moreover, TFC values of ethanol/Mg water (50/50) (40.66 mg QE/g extract) were better than those of ethanol/water (50/50) (22.29 mg QE/g extract)(Fig. 1; Table 2).
Results showed that using HRW prepared by the infusion of H2 into water was the best method for extracting flavonoids from tea wastes, which increased by 210.56% compared with pure water extracts (P < 0.05). The method of Mg-water reaction for preparing HRW was also effective in extracting flavonoids but was less effective than the first method because it increased TFC by only 33.46%. However, the use of Mg water, which contains dissolved H2, instead of pure water in preparing aqueous ethanolic solvent (50/50, %vol) improved the extraction of flavonoids by 82.41% (P < 0.05) compared to ethanol/water (50/50, %vol) (Table 3). The above results show that the most effective method was using HRW prepared by H2 infusion for extracting flavonoids from tea wastes. The possible mechanisms behind this positive impact of H2 on the extraction of flavonoids can be explained similarly to those discussed above for phenolic substances since flavonoids form a subgroup of phenolic compounds.
DPPH scavenging activity
The DPPH scavenging activity of tea waste extracts is shown in Table 2. Results show that the solvent type significantly affected the DPPH scavenging activity of tea extracts (P < 0.05). The tea waste extracts prepared using HRW showed the highest DPPH scavenging activity (22.65 mg AAE/g extract)(P < 0.05). Using aqueous ethanol instead of pure water as a solvent effectively increased the DPPH activity of tea waste extracts. Moreover, the use of HRW prepared by Mg-water reaction (Mg water) also showed higher DPPH scavenging activity than pure water (19.53 vs. 15.18 mg AAE/g extract, respectively) (Table 2).
Similar to phenolic and flavonoid results, HRW prepared by H2 infusion was more effective than that prepared by the Mg-water reaction method for extracting antioxidants from tea waste. Using HRW instead of pure water increased the DPPH scavenging activity of tea waste by 49.21% (P<0.0001), while the use of Mg water instead of pure water increased it by only 15.68% (P<0.0001) (Table 3). Additionally, using Mg water instead of pure water to prepare an aqueous ethanolic solvent positively improved the DPPH scavenging activity of tea waste extracts.
Again, these results of the DPPH scavenging activity of tea wastes can be explained by the positive impact of hydrogen on the stability of different antioxidants found in the plant cells thanks to the reducing properties of H2. The lower levels of DPPH scavenging activity of tea waste extracts in Mg water samples compared to HRW ones can be explained by the negative impact of Mg ions on the stability of many antioxidants like phenolics (Alwazeer et al. 2023d).
ABTS scavenging activity
The ABTS scavenging activity of tea waste extracts prepared using different solvents is shown in Table 2. Results show that the tea waste extracts prepared by HRW had the highest ABTS scavenging activity (26.56 mg TE/g extract), followed by Mg water (22.55 mg TE/g extract) (P < 0.05). Additionally, using aqueous ethanol instead of pure water as a solvent effectively improved the ABTS scavenging activity of tea waste extracts. Moreover, the use of Mg water instead of pure water in the preparation of aqueous ethanolic solvent highly improved the ABTS scavenging activity of samples (Table 2). While Mg water increased the ABTS scavenging activity of tea waste samples by 58.25%, this value was increased by 86.60% when HRW was used instead of pure water. However, replacing pure water with Mg water for preparing an aqueous ethanolic solvent increased the ABTS scavenging activity by only 12.29% (P < 0.05). Results show that using HRW prepared by H2 infusion in pure water was better than HRW prepared by Mg-water reaction for extracting antioxidants. This may be due to the negative impact of Mg ions on the stability of some antioxidants, such as phenolic substances, as explained above.
HPLC analysis
The phenolic profile of tea waste is shown in Table 4. Results show that some phenolic compounds, such as trans-ferulic acid, p-coumaric acid, and rutin, increased when HRW was used as a solvent in the extraction method (Fig. 2).
Table 4.
Phenolic compounds of black tea waste extracts prepared by different solvents
| Solvent | Phenolic Compounds (µg/g) | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Gallic acid | Chlorogenic acid | P-coumaric acid | Trans- ferulic acid | Rosmaric acid | Caffeic acid | Catechin | epicatechin | Rutin | Quercetin | |
| Pure water | 15.787 | nd | 3.319 | nd | 4.207 | 6.345 | nd | 270.86 | 56.515 | nd |
| Mg water | 72.733 | 550.475 | 2.516 | 4.640 | 3.077 | 5.806 | nd | 247.986 | 54.417 | nd |
| Hydrogen-rich water | 109.407 | 904.325 | 4.687 | 1.626 | 4.836 | 11.211 | 11.232 | 612.418 | 108.87 | nd |
| Ethanol/water (50/50) | 37.554 | 81.673 | 4.262 | nd | 10.099 | 9.31 | nd | 492.579 | 66.43 | 1.749 |
| Ethanol/Mg water (50/50) | 46.086 | 882.59 | 1.562 | nd | 2.33 | 1.813 | nd | 561.327 | 77.785 | 1.980 |
Fig. 2.
HPLC chromatogram of phenolic substances of tea waste samples
On the other hand, other phenolic substances, such as catechin, appeared only in HRW extracts. Moreover, while chlorogenic acid was not found in the pure water extracts of tea waste, it appeared in HRW and Mg water samples with 904.32 and 550.47 µg/g extract, respectively. Additionally, when Mg water was used as a solvent, gallic acid levels increased by 360.71 µg/g, while when pure water was replaced with Mg water for preparing aqueous ethanolic solvent, the gallic acid increased by 22.72%.
Conclusion
Tea waste forms a rich source of phenolic substances. The results showed that using hydrogen-rich water as a solvent can efficiently enhance the recovery of phytochemicals, including phenolic substances, flavonoids, and antioxidants from tea wastes. Using aqueous ethanol instead of pure water effectively improved the recovery of all phytochemicals from tea waste samples. Additionally, although using aqueous ethanol (50/50) could improve the phytochemical extraction, using Mg water instead of pure water for preparing the aqueous ethanol was more effective. The extraction yield was also improved by about two folds when HRW was used. HPLC analysis confirmed the spectrophotometric results, showing a significant increase in the levels of phenolics in HRW extracts, and some phenolics appeared only in this solvent’s extracts. The use of HRW to extract phytochemicals from agri-food wastes such as tea waste can be suggested as an eco-friendly and sustainable method. One of the limitations of the hydrogen extraction method is the potential explosion risk associated with hydrogen gas when it comes into contact with air (oxygen) at certain ratios. This risk can be managed with appropriate safety measures. Another challenge of this novel method is its non-selectivity towards various biochemicals, which necessitates post-treatment of the extract to remove undesirable substances. Further studies are required to address these challenges and to develop specific methods that target both the raw materials and the desired substances.
Acknowledgements
Authors Tunahan Engin and Ayhan Çiğdem are 100/2000 Ph.D. Scholars of the Council of Higher Education (CoHE) in the Bioengineering and Sciences program in Türkiye. This study was financially supported by the Presidential of Turkish Strategy due to Regional development and mission differentiation title for Igdir University.
Author contributions
Conceptualization, D.A.; methodology, D.A.; validation, D.A. and T.E.; formal analysis, M.E.; investigation, M.E., T.E., A.C.; data curation, M.E. and T.A.; writing—original draft preparation, M. E. and D.A.; writing—review and editing, D.E.; visualization, M.E. and D.A.; supervision, D.A.; project administration, D.A.
Funding
Open access funding provided by the Scientific and Technological Research Council of Türkiye (TÜBİTAK).
This research received no external funding.
Data availability
The datasets during and/or analyzed during the current study are available from the corresponding author upon reasonable request.
Code availability
Not applicable.
Declarations
Ethics approval
Not applicable.
Consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare no conflicts of interest.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The datasets during and/or analyzed during the current study are available from the corresponding author upon reasonable request.
Not applicable.