Antioxidant activity of Camellia sinensis leaves and tea from a lowland plantation in Malaysia

Methanol extracts of fresh tea leaves from a lowland plantation in Malaysia were screened for total phenolic content (TPC) and antioxidant activity (AOA). AOA evaluation included 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical-scavenging ability, ferric-reducing antioxidant power (FRAP), and ferrous-ion chelating (FIC) ability. Ranking, based on TPC and AOA, was as follows: shoots > young leaves > mature leaves. TPC and AOA of lowland leaves were comparable to those of highland plants. A green tea produced by drying young leaves in a household microwave oven for 4 min showed significantly higher TPC and AOA than did four commercial brands of green and black tea.     

Extraction of active ingredients from green tea (Camellia sinensis): Extraction efficiency of major catechins and caffeine

The effect of different extraction set-ups that influence the extraction efficiency of catechins and caffeine from green tea leaves (variety Fanning Belas, China) were studied using different aqueous and pure solvents (acetone, ethanol, methanol, acetonitrile, water), different temperatures (60, 80, 95 and 100 °C) and times (5–240 min). Raw extracts were analysed for contents of major catechins (EC, EGC, ECG, EGCG), caffeine, proanthocyanidins and flavonols (myricetin, caempherol, quercetin). Starting material was found to contain 191 g major catechins/kg material, 36 g caffeine/kg material and 5.2 g flavonols/kg material on a dry mass basis. The content of major catechins in green tea extracts varied from approximately 280–580 g/kg dry extract, with extraction efficiencies of major catechins varying from 61% to almost 100%. Content of caffeine in extract was in the range of 75 g/kg, where its extraction efficiency varied from 62% to 76%. Average extraction yield was 30% with exceptions when using pure acetone and acetonitrile, where extraction yield was about 3%. Contents of flavonols and proanthocyanidins were in the ranges 6–20 and 12–19 g/kg, respectively. Different extraction procedures with water were also investigated and optimal conditions determined: maximum achieved extraction efficiency of catechins with water was obtained at 80 °C after 20 min (97%) and at 95 °C after 10 min of extraction (90%). Degradation of catechins was observed at higher extraction temperatures and with prolonged extraction times. Using a lower ratio of solvent to material, extraction efficiencies were increased by applying a multi-step extraction procedure. Optimal extraction procedure was then performed using decaffeinated green tea leaves, which were obtained by high-pressure extraction with CO₂, when 98% of caffeine was selectively isolated without significant impact on valuable catechins.     

Simultaneous analysis of purine alkaloids and catechins in Camellia sinensis, Camellia ptilophylla and Camellia assamica var. kucha by HPLC

Simultaneous analysis of purine alkaloids and catechins in tea from dry leaves of Camellia sinensis, Camellia ptilophylla and Camellia assamica var. kucha by a reversed-phase high-performance liquid chromatography (RP-HPLC) method. This HPLC method had been proved to be appropriate for the identification and quantification of purine alkaloids and catechins, and exhibited good correlation coefficients, detection levels and recovery rates. Caffeine, theobromine, theacrine, (+)-catechin, (−)-epicatechin, (−)-epigallocatechin, (−)-epigallocatechin gallate, (−)-epicatechin gallate, (−)-gallocatechin gallate and (−)-gallocatechin were identified and quantified in the three species of genus Camellia Sect. Thea. There was 2.72% caffeine and 0.26% theobromine in C. sinensis, 4.85% theobromine in C. ptilophylla, and 1.58% theacrine, 0.94% caffeine and 0.45% theobromine in C. assamica var. kucha. Theacrine in C. sinensis and C. ptilophylla, and caffeine in C. ptilophylla were not detected. These data highlight differences in the relative proportions of purine alkaloids in the three species of Camellia Sect. Thea. In addition, different catechins were identified and quantified. The highest content of catechin in dry leaves was (−)-epigallocatechin gallate (EGCG) 3.51%, (−)-gallocatechin gallate (GCG) 9.88% and (−)-epigallocatechin gallate (EGCG) 6.78% in C. sinensis, C. ptilophylla and C. assamica var. kucha, respectively.     

Isolation and identification of compounds from the ethanolic extract of flowers of the tea (Camellia sinensis) plant and their contribution to the antioxidant capacity

While beneficial health properties of tea leaves have been extensively studied, less attention has been given to that of flowers of the tea (Camellia sinensis) plant. In this work, the ethanolic extract and its ethyl acetate-soluble fraction (EEA) from the tea flowers were found to possess the potent antioxidant activity using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical-scavenging assay. The compounds present in EEA had comparatively strong DPPH scavenging activity and strongly contributed to the antioxidant activity of the tea flowers. From EEA, besides eight catechins, five flavonol glycosides were isolated and their structures were elucidated on the basis of mass spectrometry and nuclear magnetic resonance spectroscopy as myricetin 3-O-β-d-galactopyranoside, quercetin 3-O-β-d-galactopyranoside, kaempferol 3-O-β-d-galactopyranoside, kaempferol 3-O-β-d-glucopyranoside, and kaempferol 3-O-[α-l-rhamnopyranosyl-(1-6)-β-d-glucopyranoside]. In addition, epigallocatechin gallate and epicatechin gallate were found as the major active components responsible for the antioxidant activity of tea flowers through the use of a combination of preparative liquid chromatography separation and DPPH assay.     

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.     

Catechin contents in tea (Camellia sinensis) as affected by cultivar and environment and their relation to chlorophyll contents

The variations of the total and individual catechin contents of tea were evaluated among three cultivars grown in 10 locations; the dynamic changes of chlorophyll and catechin contents in two cultivars during young leaf development were also analysed. The results showed the variations of catechin contents among locations were larger than those among cultivars. Correlation analysis indicated that (+)-catechin was the major catechin fraction significantly correlated with climate factors and chlorophyll contents. Moreover, chlorophyll contents were significantly influenced by climate factors, with the highest correlation coefficient for daily lowest temperature. Furthermore, the rise of chlorophyll a content during young leaf development was observed alongside the increase of (−)-epicatechin, (−)-epigallocatechin and the decline of (+)-catechin, suggesting chlorophyll plays a vital role in the regulation of individual catechins.     

Changes in antioxidant phytochemicals and volatile composition of Camellia sinensis by oxidation during tea fermentation

Monomeric flavonoids (flavan 3-ols or tea catechins) present in Camellia sinensis leaf are transformed to polymeric theaflavin and thearubigin by oxidation occurring during tea fermentation. The distinctive colour, decreased bitterness and astringency, and characteristic flavour are derived from the fermentation process giving fermented teas a marked distinction from non-fermented green tea. Even though teas are available in many different fermentation levels from green to black, the difference in phytochemicals and volatile compounds in teas with different degrees of fermentation has not been fully investigated yet within the same tea leaf. The objective of this study was to observe non-volatile phytochemicals including polyphenolic and volatile compounds changes by oxidation under strict processing control and to evaluate the degree of fermentation for the maximum antioxidant capacity with the same tea material. Harvested tea leaf was immediately processed to different degrees of oxidative fermentation (0%, 20%, 40%, 60%, and 80%). Tea infusions brewed with each processed tea leaf were analysed for polyphenolic profile, total soluble phenolics, antioxidant capacity, and volatile profile using LC–MS, HPLC, Folin–Ciocalteu assay, Oxygen Radical Absorbance Capacity (ORAC), and GC–MS analyses. The flavonoids in non-fermented green tea were significantly lessened during the oxidative fermentation process and the decreased monomeric flavonoids were transformed to polymeric theaflavin and thearubigin as the leaves were more processed. Total soluble phenolics and antioxidant capacity were significantly higher as tea leaves were less processed with a high correlation with individual polyphenolic changes. Volatile compounds present in tea leaf were analysed by GC–MS to observe changes due to processing and were utilised to create a model to differentiate fermentation based on volatile composition. Twenty-four compounds were used to build an initial model which was optimised to 16 compounds with complete separation of the groups using discriminant function analysis. The data suggested that fermentation diminished antioxidant capacity of tea and could result in lowering potential health benefits from flavonoids. This result should be considered for tea manufacturing and the development of functional foods desiring maximum potential health benefits from antioxidant flavonoids in 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).     

Characterization of the constituents and antioxidative activity of cocoa tea (Camellia ptilophylla)

Three main types of cocoa tea derived from Camellia ptilophylla, white cocoa tea (WCT), green cocoa tea (GCT) and black cocoa tea (BCT) were prepared. A comparison between cocoa tea and commonly consumed tea Biluochun cha (BLC), Longjing cha (Lj), Qimenhong cha (Qmh) and Yinghong 9^# (Yh9) was studied. Levels of catechins and purine alkaloids were determined by HPLC. Total polyphenol contents were determined by ferrous tartrate assay. Antioxidative activity was evaluated using ABTS decolorization assay, DPPH radical assay and ferric thiocyanate (FTC) method. The results showed that WCT contained the highest polyphenol content and showed the highest antioxidative activity. The radical scavenging ability of the tea extracts followed this order: WCT > GCT > Blc > Lj > BCT > Yh9 > Qmh. Tea extracts measured by the linoleic acid system also showed the same patterns of activity as the ABTS and DPPH method. A high correlation between polyphenol content and antioxidative activity of tea extracts was observed.     

The relationship between some chemical parameters and sensory evaluations for plain black tea (Camellia sinensis) produced in Kenya and comparison with similar teas from Malawi and South Africa

Reliable and accurately measurable chemical parameters that can be used to estimate black tea quality are desirable in trade, research and breeding programmes. Using plain Kenyan black tea from 11 cultivars, which gave some significant differences in their plain black tea quality parameters, the individual theaflavins composition, total theaflavins, thearubigins, theaflavin digallate equivalent, total colour and brightness were determined. The parameters were regressed against sensory evaluation scores of two tasters A and B. The theaflavin digallate equivalent (TDE) showed the strongest relationship (r = 0.71 (P ? 0.01) and r = 0.80 (P ? 0.001)) for A and B′, respectively. The simple (non gallated) theaflavin and thearubigins did not show significant relationships with sensory evaluation. Of the liquor characteristics, there were significant relationships between liquor brightness and sensory evaluation by A and B (r = 0.58 (P ? 0.06) and r = 0.59 (P ? 0.05)), respectively. In consequence, TDE and brightness can be used in tea breeding programmes as quality indicators or to estimate plain black tea quality potential in the tea trade. Optimising their levels can also help to produce good quality Kenyan black teas during processing. Comparison of these results with work published earlier indicates that, of the individual theaflavins, theaflavin-3,3′-digallate correlates best with tea taster scores for the 11 Kenyan cultivars, whereas the simple theaflavin correlates best with tea tasters’ scores for 40 Malawian cultivars. However, the derived parameter, TDE correlates very well with tea tasters’ scores for all of the above cultivars.     

Levels of essential and non-essential metals in leaves of the tea plant (Camellia sinensis L.) and soil of Wushwush farms,Ethiopia

Five tea clones of the Camellia assamica variety grown in Wushwush tea plantation farms, Ethiopia, were analyzed for their contents of essential, non-essential and toxic metals (K, Ca, Mg, Fe, Mn, Cu, Zn, Na, Cd and Pb) by atomic absorption flame emission spectroscopy. Both the tea leaves and the soils of the study farms showed similar accumulation patterns in their contents of the studied macronutrients. Among the macronutrient metals, K was the most abundant element in the tea leaves (17.7–24.8 mg/g) and the soils (7.14–9.73 mg/g). Mn was the predominant micronutrient heavy metal in the tea leaf tissues ranging between 501 and 1281 mg/kg. Level of Fe (29.6–100 mg/kg) in the leaf tissue was found to be the second most abundant micronutrient next to Mn whereas concentrations of Cu and Co were relatively lower both in the soil and tea samples. The toxic heavy metals Pb and Cd in the leaf tissues were present at levels too low to be detected by the analytical technique used in this study. The soils were found to be acidic (pH 5.04–5.49) with high organic matter (5.48–6.02%). Fe was the most abundant metal followed by Mn, Na and Zn in the soils. Unlike the tea leaves, the soils were found to contain traces of the toxic metal, Cd (0.02–1.10 mg/kg). The levels of most of the metals determined in this study compared well with those reported for tea leaves from some other parts of the world.     

Phenolic antioxidants from Chinese toon (fresh young leaves and shoots of Toona sinensis)

Chinese toon (the fresh young leaves and shoots of Toona sinensis), known as a tree vegetable, belongs to the family of Meliaceae. It is one of the most popular vegetables in China. The 80% acetone extract of Chinese toon exhibited considerable antioxidant activity in the 1,1-diphenyl-2-picryldydrazyl (DPPH) radical-scavenging assay. Bioassay-guided purification of the extract led to the isolation of 12 phenolic compounds (1–12), whose structures were determined by detailed spectroscopic analyses, including UV, IR, MS, 1D and 2D NMR techniques. Of them, compounds 3–8 and 11 were isolated from T. sinensis for the first time. All of the isolated compounds were examined for their antioxidant activities by the DPPH method, and the results showed that gallic acid and its derivatives, gallotannins and flavonoids were the main constituents contributing to the antioxidant activity of Chinese toon. From the health point of view, Chinese toon is an ideal dietary vegetable with natural antioxidants.     

Simultaneous determination of free ergosterol and ergosteryl esters in Cordyceps sinensis by HPLC

The caterpillar-shaped Chinese medicinal mushroom Cordyceps sinensis consists of the fruiting body (CsA) and the host caterpillar (CsB). Ergosterol is a principal sterol in fungi and can indicate the level of mycelia of C. sinensis. Ergosterol is present in two forms, as free ergosterol and esterified ergosterol, which have different physiological functions. The relative abundances of free to esterified ergosterol are different among the various species. In the present study, a gradient reversed-phase high-performance liquid chromatography (HPLC) method was developed for the simultaneous determination of free and esterified ergosterol in CsA and CsB of C. sinensis. The results showed that CsA and CsB had similar ergosterol compositions, but the level of ergosteryl esters in CsB was much higher than that in CsA, indicating that CsA and CsB might be in different growth phase or have different physiological functions for the growth and multiplication of C. sinensis.     

In vitro evaluation of antioxidant activities of aqueous extracts from natural and cultured mycelia of Cordyceps sinensis

Cordyceps sinensis, one of the best known traditional Chinese medicines and health foods, has been highly valued for the treatment of a wide range of diseases and reported to have antioxidant properties. In the present study, the antioxidant activities of hot-water extracts from natural and cultured mycelia of C. sinensis were investigated and evaluated using six in vitro assays, including inhibition of linoleic acid peroxidation; scavenging abilities on DPPH, hydroxyl and superoxide anion radicals; the reducing power and the chelating ability on ferrous ions. Among these assays, the extracts showed the best effect on the inhibition of linoleic peroxidation with the lowest IC50 values and with an inhibition rate over 90% at concentration of 0.8-1.6 mg/ml, more stable than that of α-tocopherol, a recognised natural antioxidant. The scavenging activities on superoxide anion and hydroxyl radicals of the two extracts were slightly lower than that of butylated hydroxytoluene. DPPH scavenging activities of both extracts reached over 80% inhibition at 4-8 mg/ml. Both extracts showed moderate reducing power and ferrous ion chelating activity. The IC50 value of the extract from cultured mycelia in all the tests, except for linoleic acid peroxidation, was significantly lower than that of natural mycelia. There was no evident correlation between the antioxidant activity and the content of protein, polysaccharides and mannitol of extracts from C. sinensis; the antioxidant activity may be due to a combined effect of these or some other compounds. These results suggested that both the extracts from cultured and natural mycelia have direct and potent antioxidant activities and that the cultured mycelia of the fungus could be used for the antioxidant activity to reduce the human demands on the natural resources of the fungus, an endangered species.     

Melatonin contents in mulberry (Morus spp.) leaves: Effects of sample preparation,cultivar, leaf age and tea processing

The effects of sample preparation, cultivar, leaf age and tea processing on melatonin content of mulberry (Morus spp.) leaves were investigated. Sample preparation using ultrasonic technique in combination with solid phase extraction resulted in high recovery rate (76%), when compared to homogenisation in combination with liquid–liquid extraction procedure (12% recovery rate). The melatonin contents in mulberry leaves harvested from three major cultivars (Buriram 60, Sakonnakhon and Khunphai) grown in Thailand were identified and quantified using high-performance liquid chromatography combined with fluorescence detector. The average melatonin content of the mulberry leaves cv. Buriram 60 (279.6 ng/g dry weight (DW)) was higher than those of cv. Sakonnakhon (100.5 ng/g DW) and cv. Khunphai (40.7 ng/g DW). The melatonin contents of all cultivars tested were highest in the tip of the leaves, followed by that in the young leaves, whereas the lowest was found in the old leaves. The melatonin contents of the two types of tea produced from mulberry leaves cv. Buriram 60 were also determined. Heat treatment during tea processing decreased the melatonin content in mulberry leaves cv. Buriram 60 by approximately 87%, when compared to that of the fresh leaves. However, there were no significant differences between the melatonin contents of the mulberry leaf tea produced with blanching (mulberry green tea) and those produced without blanching (mulberry black tea).     

Characterisation of volatile and non-volatile metabolites in etiolated leaves of tea (Camellia sinensis) plants in the dark

Aroma is an essential factor affecting the quality of tea (Camellia sinensis) products. While changes of volatile compounds during tea manufacturing have been intensively studied, the effect of environmental factors on volatile contents of fresh tea leaves has received less attention. We found that C. sinensis var. Yabukita kept in darkness by shading treatment for 3 weeks developed etiolated leaves with significantly increased levels of volatiles, especially volatile phenylpropanoids/benzenoids (VPBs). Upstream metabolites of VPBs, in particular shikimic acid, prephenic acid, and phenylpyruvic acid, showed lower levels in dark treated than in control leaves, whereas the contents of most amino acids including l-phenylalanine, a key precursor of VPBs, were significantly enhanced. In addition, analysis by ultra performance liquid chromatography-time of flight mass spectrometry, capillary electrophoresis–time of flight mass spectrometry, high performance liquid chromatography, and gas chromatography–mass spectrometry indicated that volatile and non-volatile metabolite profiles differed significantly between dark treated and untreated leaves.     

Flow injection spectrophotometry using natural reagent from Morinda citrifolia root for determination of aluminium in tea

A flow injection (FI) spectrophotometric method with using natural reagent extracted from Morinda citrifolia root has been developed for determination of aluminium. The extract contained anthraquinone compounds which could react with Al³⁺ to form reddish complexes which had maximum absorption wavelength at 499.0 nm. The extract could be used as a reagent in FI system without further purification to obtain pure compound. A sensitive method for determination of aluminium in concentration range of 0.1-1.0 mg L⁻¹, with detection limit of 0.05 mg L⁻¹ was achieved. Relative standard deviations of 1.2% and 1.7% were obtained for the determination of 0.1 and 0.6 mg L⁻¹ Al³⁺ (n = 11). Sample throughput of 35 h⁻¹ was achieved with the consumption of 3 mL each of carrier and reagent solutions per injection. The developed method was successfully applied to tea samples, validated by the FAAS standard method. The method is simple, fast, economical and could be classified as a greener analytical method.     

Volatile constituents of Cachucha peppers (Capsicum chinense Jacq.) grown in Cuba

The steam volatile components of three cultivars of Cachucha mature peppers (Capsicum chinense Jacq.) were isolated by steam-distillation-continuous-extraction and analysed using GC and GC-MS. The composition of volatile compounds of the peppers differs clearly for the different cultivars. The content of volatile compounds, responsible for the flavour of Cachucha peppers, varied between 110.71 and 302.53 mg kg⁻¹. One hundred and thirty-six compounds were identified, from which hexyl isopentanoate, hexyl pentanoate, hexyl 2-methylbutanoate, 3,3-dimethylcyclohexanol, γ-himachalene and germacrene D were the major ones.