Effects of cosolvents on the decaffeination of green tea by supercritical carbon dioxide

Due to the adverse effects of the caffeine in a variety of plant products, many methods have been explored for decaffeination, in efforts to remove or reduce the caffeine contained in plant materials. In this study, in order to remove caffeine from green tea (Camellia sinensis) leaves, we have employed supercritical carbon dioxide (SC–CO₂), which is known to be an ideal solvent, coupled with a cosolvent, such as ethanol or water. By varying the extraction conditions, changes not only in the amount of caffeine, but also in the quantities of the principal bioactive components of green tea, including catechins, such as epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG) and epicatechin (EC), were determined. The extraction conditions, including temperature, pressure and the cosolvent used, were determined to affect the efficacy of caffeine and catechin extraction. In particular, the type and concentration of a cosolvent used constituted critical factors for the caffeine removal, combined with minimal loss of catechins, especially EGCG. When the dry green tea leaves were extracted with SC–CO₂ modified with 95% (v/v) ethanol at 7.0 g per 100 g of CO₂ at 300 bar and 70 °C for 120 min, the caffeine content in the decaffeinated green tea leaves was reduced to 2.6% of the initial content. However, after the SC–CO₂ extraction, a substantial loss of EGCG, as much as 37.8% of original content, proved unavoidable.     

Decaffeination of fresh green tea leaf (Camellia sinensis) by hot water treatment

Hot water treatment was used to decaffeinate fresh tea leaf in the present study. Water temperature, extraction time and ratio of leaf to water had a statistically significant effect on the decaffeination. When fresh tea leaf was decaffeinated with a ratio of tea leaf to water of 1:20 (w/v) at 100 °C for 3 min, caffeine concentration was decreased from 23.7 to 4.0 mg g⁻¹, while total tea catechins decreased from 134.5 to 127.6 mg g⁻¹; 83% of caffeine was removed and 95% of total catechins was retained in the decaffeinated leaf. It is considered that the hot water treatment is a safe and inexpensive method for decaffeinating green tea. However, a large percentage of tea catechins was lost if rolled leaf and dry tea were decaffeinated by the hot water treatment and so the process is not suitable for processing black tea.     

Determination of catechins in green tea infusions by reduced flow micellar electrokinetic chromatography

A sulfated-β-cyclodextrin (s-β-CD) modified reduced flow micellar electrokinetic chromatography (RF-MEKC) method was developed and validated for the determination of catechins in green tea. The optimal electrolyte consisted of 0.2% triethylamine, 50 mmol/L SDS and 0.8% s-β-CD (pH = 2.9), allowing baseline separation of five catechins in 4 min. The samples and standards were injected at 0.6 psi for 5 s under constant voltage of −30 kV. Sample preparation simply involved extraction of 2 g of tea with 200 mL water at 95 °C under constant stirring for 5 min. The method demonstrated excellent performance, with limits of detection (LOD) and quantification (LOQ) of 0.02–0.1 and 0.1–0.5 μg/mL, respectively, and recovery percentages of 94–101%. The method was applied to six samples of Brazilian green tea infusions. Epigallocatechin gallate (23.4–112.4 μg/mL) was the major component, followed by epigallocatechin (18.4–78.9 μg/mL), epicatechin gallate (5.6–29.6 μg/mL), epicatechin (4.6–14.5 μg/mL) and catechin (3.2–8.2 μg/mL).     

Impact of heating on chemical compositions of green tea liquor

Heating pasteurization changed flavour of green tea liquor and it was a technical barrier in ready-to-drink tea production. The effect of heating on chemical compositions of green tea liquor was investigated by high performance liquid chromatography and gas chromatography–mass spectrometry. With increase of heating temperature from 85 °C to 120 °C, the green tea liquor became darker and less green, but deeper yellow in colour. During the heating, epigallocatechin gallate, epigallocatechin, epicatechin and epicatechin gallate partially epimerized, and concentration of total catechins decreased. Twenty volatiles were identified and concentrations of pentanol, cis-3-hexenol, linalool oxide I, linalool oxide II and β-ionone were decreased while phenylacetaldehyde, linalool, linalool oxide III, α-terpineol and indole increased. The decline of volatiles with pleasant odours and increase of some volatiles with unpleasant odours, such as indole (animal-like) and α-terpineol (faint ammoniacal), was considered to be responsible for the change in flavour of green tea liquor. Treatment at 85 °C caused fewer changes in liquor colour and concentrations of catechins and volatiles and it is recommended that extraction and pasteurization of canned ready-to-drink green tea should be carried out at 85 °C or less.     

Green tea: A promising anticancer agent for renal cell carcinoma

Renal cell carcinoma (RCC) is one of the most lethal amongst the urologic malignancies, comprising three percent of all human neoplasms, and its incidence appears to be rising. RCC is refractory to both chemotherapy and radiotherapy. Therefore, the discovery of new strategies for therapeutic intervention remains a priority. Green tea (Camellia sinensis) and tea polyphenols have been proposed to exert protective effects against several types of cancer, based on preclinical and clinical trial data; however, the anticarcinogenic activity of green tea towards RCC is unknown. In this study, a targeted metabolite analysis on a green tea leaves methanolic extract was performed by HPLC/DAD and the antiproliferative activity of the extract was assayed using human renal cancer cell lines A-498 and 769-P. The total phenolic content was very high (31.8% of methanolic extract), and the main compounds were flavan-3-ols (94.3% of the total phenolic content), and especially (−)-epigallocatechin-3-gallate (35.9% of the total phenolic content). In addition, two methylxanthines – theophylline and caffeine – were also present in the extract, caffeine being the most abundant. Green tea extract strongly inhibited the growth of both RCC cell lines in a concentration-dependent manner, with IC₅₀ values of 54 ± 10 and 129 ± 28 μg/ml for A-498 and 769-P cells, respectively. This is the first report showing that green tea is likely to be an effective anticancer agent for renal cell carcinoma.     

Albumin causes a synergistic increase in the antioxidant activity of green tea catechins in oil-in-water emulsions

Model oil-in-water emulsions containing epicatechin (EC) and epigallocatechin gallate (EGCG) showed a synergistic increase in stability in emulsions containing added albumin. EGCG showed a stronger synergy (35%) with ovalbumin than did EC. Oxidation of the oil was monitored by determining peroxide values and hexanal contents. The effect of bovine serum albumin (BSA) on model oil-in-water emulsions containing each of the green tea catechins [epicatechin gallate (ECG), EGCG, EC and epigallocatechin (EGC)] was studied during storage at 30 °C. The green tea catechins showed moderate antioxidant activity in the emulsions with the order of activity being ECG ≈ EGCG > EC > EGC. Although BSA had very little antioxidant activity in the absence of phenolic antioxidants, the combination of BSA with each of the catechins showed strong antioxidant activity. BSA, in combination with EC, EGCG or EGC, showing the strongest antioxidant activity with good stability after 45 days storage. Model experiments with the catechins stored with BSA in aqueous solutions confirmed that protein–catechin adducts with antioxidant activity were formed between the catechins and protein. The antioxidant activity of the separated protein–catechin adducts increased strongly with storage time and was stronger for EGCG and ECG than for EC or EGC.     

Optimal extraction conditions for simultaneous determination of catechins and caffeine in green tea leaves

The amount of catechins and caffeine in green tea are considered a standard of quality evaluation for green tea. The objectives of this study were to investigate the optimal conditions for simultaneous extraction of catechins and caffeine and to compare their quantity and composition within the exotic tea varieties. Moreover, 8 tea varieties of exotic origin were tested with the optimal extraction conditions found in this study. Regarding the caffeine and catechins contents, a 2 h room temperature extraction using a 2% phosphoric acid-40% EtOH solution was the most suitable simultaneous extraction method. The total catechins and caffeine contents of the tea leaves ranged 44.25–64.80 and 9.26–13.11 mg/g, respectively. The optimal extraction method of catechins and caffeine in tea leaves may be used for further studies and breeding of high quality tea plants.     

Analysis of some selected catechins and caffeine in green tea by high performance liquid chromatography

Green tea seems to have a positive impact on health due to the catechins-found as flavanols. Thus, the present study was aimed to develop a low cost reversed phase high performance liquid chromatographic (HPLC) method for simultaneous determination of flavanol contents, namely catechin (C), epicatechin (EC), epigallocatechin (EGC), epicatechin 3-gallate (ECG) and epigallocatechin 3-gallate (EGCG) and caffeine in 29 commercial green tea samples available in a Saudi Arabian local market. A C-18 reversed-phase column, acetonitrile–trifluoroacetic acid as a mobile phase, coupled with UV detector at 205 nm, was successfully used for precise analysis of the tested analytes in boiled water of digested tea leaves. The average values of N (No. of theoretical plates), HETP (height equivalent of theoretical plates) and R_s (separation factor) (at 10 μg ml⁻¹ of the catechins EC, EGC, EGCG and ECG) were 2.6 × 10³ ± 1.2 × 10³, 1.7 × 10⁻³ ± 4.7 × 10⁻⁴ cm and 1.7 ± 5.53 × 10⁻², respectively. The developed HPLC method demonstrated excellent performance, with low limits of detection (LOD) and quantification (LOQ) of the tested catechins of 0.004–0.05 μg ml⁻¹ and 0.01–0.17 μg ml⁻¹, respectively, and recovery percentages of 96–101%. The influence of infusion time (5–30 min) and temperature on the content of the flavanols was investigated by HPLC. After a 5 min infusion of the tea leaves, the average concentrations of caffeine, catechin, EC, EGC, ECG and EGCG were found to be in the ranges 0.086–2.23, 0.113–2.94, 0.58–10.22, 0.19–24.9, 0.22–13.9 and 1.01–43.3 mg g⁻¹, respectively. The contents of caffeine and catechins followed the sequence: EGCG > EGC > ECG > EC > C > caffeine. The method was applied satisfactorily for the analysis of (+)-catechin, even at trace and ultra trace concentrations of catechins. The method was rapid, accurate, reproducible and ideal for routine analysis.     

Comparison of extraction and isolation efficiency of catechins and caffeine from green tea leaves using different solvent systems

Catechins from green tea (Camellia sinensis L.) have received considerable attention due to their beneficial effects on human health, such as antioxidant and anticancer activities. Optimisation of extraction conditions of the catechins from green tea leaves was performed using different solvents (ethanol or distilled water), different extraction methods (ultrasound‐assisted, room temperature or reflux extractions) and various extraction times (0.5–24 h). The optimal extraction conditions were determined using 40% ethanol with ultrasound‐assisted extraction method for 2 h at 40 °C. In addition, two isolation methods for the recovery of catechins from green tea extracts were compared using different solvent combinations (chloroform/ethyl acetate versus ethyl acetate/dichloromethane). The results showed that the ethyl acetate/dichloromethane system could achieve much higher content of catechins than the other isolation approaches, indicating the method that extract catechins first with organic solvent such as ethyl acetate before removing caffeine is much effective than removing caffeine first when organic solvents are used for the recovery of catechins without caffeine from green tea extracts.     

Characterization of anthocyanins in Kenyan teas: Extraction and identification

Characterization and quantification of anthocyanins in selected tea cultivars processed into black (aerated) and green (unaerated) tea products was carried out in this study. The anthocyanins were extracted from tea products processed from a number of newly bred purple leaf coloured Kenyan tea cultivars (Camellia sinensis) using acidified methanol/HCl (99:1 v/v). Extracted anthocyanins were purified by C₁₈ solid phase extraction (SPE) catridges and characterised by HPLC-UV-Visible. They were identified according to their HPLC retention times, elution order and comparison with authentic standards that were available. Total monomeric anthocyanins were determined by the pH-differential method. Although the tea cultivars gave different yields of anthocyanins, the unaerated (green) teas had significantly (p ? 0.05) higher anthocyanin content than the aerated (black) teas. This was attributed to the degradation of anthocyanins by polyphenol oxidase products (catechin O-quinones) formed during the auto-oxidation (fermentation) process of black tea manufacture. Of the six most common natural anthocyanidins, five were identified in the purified extracts from purple leaf coloured tea, in both aerated (black) and unaerated (green) teas namely; delphinidin, cyanidin, pelargonidin, peonidin and malvidin. The most predominant anthocyanidin was malvidin in both tea products. In addition, two anthocyanins namely, cyanidin-3-O-galactoside and cyanidin-3-O-glucoside were also identified. Tea catechins were also identified in the tea products derived from the purple coloured tea cultivars namely, epigallocatechin (EGC), catechin (+C), epicatechin (EC), epigallocatechin gallate (EGCG), and epicatechin gallate (ECG). Correlation between the total catechins versus the total anthocyanins and anthocyanin concentration in unaerated teas revealed significant negative correlations (r = −0.723^∗ and r = −0.743^∗∗, p ? 0.05 and p ? 0.01, respectively). However, in aerated (black) tea the correlations were insignificant (r = −0.182 and r = −0.241, p > 0.05).     

Analysis of catechins from milk–tea beverages by enzyme assisted extraction followed by high performance liquid chromatography

Extraction and analysis of physiologically significant tea catechins from complex food matrices is complicated by strong association of tea catechins with macronutrients such as proteins. Dependable extraction methods are required to accurately assess and validate levels of bioactive tea catechins in new products. The objective of this work was to investigate recovery of tea catechins from dairy matrices and evaluate pepsin treatment as an enzymatic step to enhance catechin recovery from milk and other protein rich formulations. Brewed green tea was combined with skim milk to produce test solutions ranging from 10% to 50% milk. Samples were treated by either acid (0.1 N HCl), methanol, or by pepsin (40.0 mg/mL). Following treatments, samples were centrifuged and supernatants analyzed for tea catechins by reversed phase C18 HPLC with photodiode array detection. Recovery of total catechins was highest for pepsin treated samples (89–102%), followed by methanol deproteination (78–87%) and acid precipitation (20–74%) with values decreasing with increased milk content. Individual recovery of gallated catechins, namely epigallocatechin-gallate (EGCG) and epicatechin-gallate (ECG), was most affected by the presence and level of milk. The usefulness of pepsin treatment for enhancing recovery of tea catechins was further demonstrated in analysis of commercial soy and milk–tea beverages.     

Characterisation,extraction efficiency,stability and antioxidant activity of phytonutrients in Angelica keiskei

Phytonutrients in Angelica keiskei, a dark green leafy vegetable, were characterised and their extraction efficiency by different compositions of water/ethanol as well as stability at different temperatures was determined. A range in the content of lutein (205–265 mg/kg dry wt), trans-β-carotene (103–130 mg/kg dry wt), and total phenols (8.6–9.7 g/kg) was observed amongst Angelica keiskei grown in three different conditions. LC-ESI-MS/MS analysis identified chlorogenic acid, chalcones and the glucosides of luteolin and quercetin as the major phenolic compounds in Angelica keiskei. Only 46% of lutein was extracted by 70% of ethanol and no carotenoid was detected in 40% ethanol and 100% water extracts of Angelica keiskei. Major phytonutrients in Angelica keiskei were stable at −80 °C up to 12 months whilst β-carotene was significantly degraded at room temperature and 4 °C within 2 months and lutein at room temperature within 12 months.     

Effect of green and black teas (Camellia sinensis L.) on the characteristic microflora of yogurt during fermentation and refrigerated storage

The effect of tea on the fermentation and survival of yogurt microorganisms was studied. Green and black teas were added to milk at the beginning of fermentation. Acidity of yogurt products and survival of their microflora were studied during 42 days at 4 °C. Results showed that the presence of tea did not significantly (P < 0.05) influence the yogurt characteristic microorganisms. HPLC studies demonstrated that yogurt bacteria did not affect tea catechins when they were incubated together for 48 h. Indeed, all five products reached about 10⁹ CFU/ml after 6 h of fermentation. Viability during 6 weeks storage at 4 °C varied very little (8.35 < log CFU/ml < 8.65). Similarly, green and black teas had no effect on lactic acid levels of the final products (after 6 weeks of storage, acidity remained above 80 °D). According to these findings, addition of teas or tea catechins to yogurt can be recommended to take advantage of their beneficial properties on human health attributed to their antioxidant and antimicrobial activities.     

Microwave-assisted extraction of chlorogenic acids from green coffee beans

Microwave-assisted extraction (MAE) has been considered as a potential alternative to conventional solvent extraction for the isolation of phenolic compounds from plants. Aqueous and alcoholic extracts of green coffee bean obtained by MAE were quantitatively analysed for total yield of extracts, chlorogenic acids, caffeine and total polyphenol content. The extracts were also evaluated for radical-scavenging activity, using 1,1-diphenyl-β-picrylhydrazyl radical. Under optimum conditions of time (5 min), temperature (50 °C) and wattage (800 W), the maximum chlorogenic acids and caffeine could be extracted with water as solvent. The extracts contained chlorogenic acids and caffeine in the ranges of 31–62% and 22–40%, respectively. The yields of MAE under optimum conditions were higher than those from the conventional solvent extraction at 5 min and 50 °C and the extracts showed radical-scavenging activity of >75%, even at the concentration of 25 ppm. The MAE process can thus be predicted and controlled for industrial application.     

Effect of tannase treatment on protein-tannin aggregation and sensory attributes of green tea infusion

Effect of tannase enzymatic treatment on protein-tannin aggregation and sensory attributes of green tea infusion was investigated. Green tea leaves were extracted with hot water at 85 °C for 20 min, the tea infusion was then treated with tannase. Results showed that both EGCG and ECG of the tea catechins were hydrolyzed by tannase into EGC and EC, respectively, accompanied by production of gallic acid. The tannase-treated tea infusion had a relatively lower binding ability with protein. Changes in the content of tea catechins, formation of tea cream, and turbidity of tea infusion with or without tannase treatment were measured after 4 weeks. Content of catechins in the tannase-modified tea remained almost unchanged, while those without tannase treated (control) decreased significantly (p < 0.05). Meanwhile, better color appearance and less tea cream formation were observed for the tannase-treated green tea, and tea cream formed for the control after storage. Results of the sensory evaluation showed that mouth feeling, taste and the overall acceptance of the tannase-treated green tea infusion were all better than those of the control.     

原料对茶绿色素提取率的影响

分别以鲜茶、杀青叶、干茶为试材,采用四因素四水平正交试验设计对茶绿色素浸提工艺参数进行了系统的研究。试验结果表明,杀青叶是提取茶绿色素最好的原料;采用100%的乙醇,4.5mL∶1g的醇茶比,75℃水浴浸提25min,浸提效果最佳,提取率为31.50%。茶绿色素含有丰富的茶多酚、咖啡碱、氨基酸等有效成分,是一种优良的天然食用色素。     

Extraction behaviors of caffeine and chlorophylls in supercritical decaffeination of green tea leaves

The decaffeination of green tea using supercritical carbon dioxide (SC-CO₂) was optimized by response surface methodology (RSM) for the maximal removal of caffeine, and the coextration of chlorophylls was also monitored during decaffeination. The experimental conditions for the SC-CO₂ extraction of caffeine were set up according to the Box-Behnken design of RSM. The relationships between the extraction yield of caffeine and various parameters used for the SC-CO₂ extraction such as pressure, temperature and concentration of ethanol were studied at a fixed CO₂ flow rate. The extraction yields of caffeine and total chlorophyll were significantly influenced by extraction pressure, temperature and concentration of cosolvent, and their extraction yields behaved almost in parallel at different extraction conditions that were obtained by varying pressure, temperature and ethanol cosolvent concentration. At the optimal decaffeination conditions such as 3.0 g of 95% (v/v) ethanol cosolvent per 100 g of CO₂, 23 MPa, 63 °C and an extraction duration of 120 min for 10 g of green tea leaves, the extraction yields for caffeine and catechins were 96.60% (w/w) and 40.61% (w/w), respectively, and the substantial coextraction of total chlorophyll (43.09% of the total amount) was also observed during the decaffeination process.     

Quantitative analysis of acrylamide in tea by liquid chromatography coupled with electrospray ionization tandem mass spectrometry

An effective sample preparation procedure was optimized and a liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed for the quantitative analysis of acrylamide in tea. [¹³C₃]-acrylamide was used as internal standard. Acrylamide was extracted at 25 °C for 20 min by 10 ml water followed by 10 ml acetonitrile, and then 4 g of magnesium sulfate and 0.5 g of sodium chloride were added to the above mixture under stirring thoroughly. In order to increase the response of acrylamide, 9 ml acetonitrile layer was taken and concentrated to 0.5 ml. Solid-phase extraction with an Oasis MCX cartridge was carried out for clean-up. The limit of detection (LOD) and limit of quantification (LOQ) were 1 and 5 ng/ml, respectively. The recovery efficiency of the extraction procedure ranged between 74% and 79%. The levels of acrylamide in 30 tea samples were less than 100 ng/g. Black, oolong, white and yellow tea samples had quite low acrylamide contents (<20 ng/g). Higher acrylamide levels occurred in baked, roasted, and one sun-dried green tea samples (46–94 ng/g).     

Cream formation in a semifermented tea

The formation of cream in Paochung tea, a popular semifermented tea, which undergoes a lesser degree of enzymic oxidation during manufacture, was investigated at various extraction temperatures, extraction times, pHs and leaf/water ratios. The primary components of Paochung tea cream were catechins (30%), caffeine (20%) and protein (16%). (−)-Epigallocatechin gallate and (−)-epicatechin gallate were the major catechins precipitated during creaming, constituting 19% and 5% of the tea cream respectively. The amount of tea cream produced and its composition were influenced by extraction temperature and pH. The tea leaf/water ratio determined the amount of tea cream formed but did not affect the cream composition. Catechins were considered to be the key component in tea cream. They interacted with caffeine and protein to induce tea cream formation. © 1999 Society of Chemical Industry     

HPLC Analysis of Catechins, Theaflavins, and Alkaloids in Commercial Teas and Green Tea Dietary Supplements: Comparison of Water and 80% Ethanol/Water Extracts

ABSTRACT:  To help meet the needs of consumers, producers of dietary tea supplements, and researchers for information on health-promoting tea compounds, we compared the following conditions for the extraction of tea leaves and green tea-containing dietary supplements: 80% ethanol/water at 60 °C for 15 min and boiled water for 5 min. The following 7 catechins, 4 theaflavins, and 3 alkaloids were separated in a 70-min single HPLC analysis: (−)-epigallocatechin, (−)-catechin, (+)-epicatechin, (−)-epigallocatechin-3-gallate, (−)–gallocatechin-3-gallate, (−)-epicatechin-3-gallate, (−)-catechin-3-gallate, theaflavin, theaflavin-3-gallate, theaflavin-3'-gallate, theaflavin-3,3'-digallate, caffeine, theobromine, and theophylline. The following ranges of concentrations of flavonoids (catechins plus theaflavins) in the tea leaves extracted with 80% ethanol were observed (in mg/g): in 32 black teas, 19.8 to 115.1; in 24 green teas, 12.3 to 136.3; in 14 specialty teas, 4.9 to 118.5; in 7 herbal teas, 0 to 46.0. Total alkaloids in all teas ranged from 0 to 32.6 mg/g. Significantly greater amounts of flavonoids were extracted from the tea leaves with aqueous ethanol than with boiled water. Levels of tea catechins in 10 capsules sold as dietary supplements were about 50 to 75% lower than the amounts listed on the labels. Catechin content of 4 commercial green tea extracts ranged from 96 to 696 mg/g. The results make it possible to maximize the extraction of tea compounds to better relate the flavonoid and alkaloid content of teas and dietary tea supplements to their health-promoting effects.