Characterization of phenolic compounds in rooibos tea

Polyphenols present in rooibos, a popular herbal tea from Aspalathus linearis, were isolated in two steps. First, phenolic ingredients were separated by multilayer countercurrent chromatography (MLCCC). Preparative high-performance liquid chromatography (HPLC) was then applied to obtain pure flavonoids. The purity and identity of isolated compounds was confirmed by different NMR experiments, HPLC-diode array detector (DAD), or gas chromatography-mass spectrometry (GC-MS) analysis. This strategy proved to be valid to isolate material in up to gram quantities and to verify known and previously not published polyphenol structures. In addition the chemistry of dihydrochalcones and related intermediates was studied. The dihydrochalcone aspalathin was oxidized to the corresponding flavanone- C-glycosides (( R)/( S)-eriodictyol-6- C-beta- D-glucopyranoside and ( R)/( S)-eriodictyol-8- C-beta- D-glucopyranoside). Flavanone-6- C-beta- D-glucopyranosides were further degraded to flavones isoorientin and orientin.     

Enhancement of Rooibos (Aspalathus linearis) aqueous extract and antioxidant yield with fungal enzymes

The leaves and stems of the Rooibos plant ( Aspalathus linearis) are used for the production of an herbal tea known for its health promoting properties, which have been linked to its flavonoid content but which is substantially reduced by the traditional processing method employed. Selected food-grade fungi were screened for their potential to improve the yield of soluble matter extracted from rooibos plant material. Fungal cocktails of hydrolyzing enzymes enhanced either the yield of soluble solids ( Lentinula edodes and Rhizopus oryzae cultured in yeast peptone-wheat straw medium) or the yield in antioxidants from fermented rooibos ( R. oryzae cultured in potato dextrose or yeast peptone-wheat straw medium). When applied to green rooibos, L. edodes (cultured in yeast peptone-wheat straw medium) enhanced the release of soluble solids as well as color formation, leading to semifermented rooibos with a relatively high aspalathin content, compared to fermented rooibos.     

Unfermented rooibos tea: quantitative characterization of flavonoids by HPLC-UV and determination of the total antioxidant activity

Unfermented rooibos originates from the leaves and the stems of the indigenous South African plant, Aspalathus linearis, and it has been reported to have a higher content of flavonoids compared to that of fermented rooibos. The HPLC/UV method developed in our laboratory for the analysis of the fermented rooibos was applied to the quantitative characterization of the major flavonoids present in the unfermented rooibos. Main compounds determined were aspalathin (49.92 +/- 0.80 mg/g), isoorientin (3.57 +/- 0.18 mg/g), orientin (2.336 +/- 0. 049 mg/g), and rutin (1.69 +/- 0.14 mg/g), followed in order by isovitexin, vitexin, isoquercitrin and hyperoside, quercetin, luteolin and chrysoeryol. The identity of detected flavonoids was confirmed by comparing their retention times and UV spectra with those of corresponding standards. The total antioxidant activity (TAA) of the tea infusions was measured by the ABTS*+ radical cation decolorization assay. The TAA of unfermented rooibos (0.8 Trolox meq/g) resulted 2-fold higher than that of the fermented rooibos. When compared with different water infusions of Camellia sinensis (green and black tea), this TAA value was about 50% lower.     

Effect of heat on aspalathin, iso-orientin, and orientin contents and color of fermented rooibos (Aspalathus linearis) iced tea

The phenolic quality of commercial South African fermented rooibos iced teas in terms of aspalathin, iso-orientin, and orientin contents in comparison to a "cup of tea" was shown to be inferior. The role of the different manufacturing stages of powdered extract used in iced tea formulation and, more specifically, the impact of pasteurization and sterilization on the color and phenolic content of the beverage, were assessed as potential causes of its inferior phenolic quality. Aspalathin and its corresponding flavones, iso-orientin and orientin, were found to be present at all stages of the powdered extract production process. Spray-drying did not significantly (P ≥ 0.05) alter the aspalathin, iso-orientin, or orientin content of concentrates. Simulated normal-temperature sterilization (NTS at 121 °C/15 min) and high-temperature sterilization (HTS at 135 °C/4 min), but not necessarily pasteurization (93 °C/30 min), significantly (P < 0.05) reduced the aspalathin, iso-orientin, and orientin contents of different iced tea formulations. Heat-induced losses of iso-orientin and orientin were lower than those for aspalathin. Conversion of aspalathin to the flavones is implicated. The addition of ascorbic acid and/or citric acid to the base iced tea formulation containing only rooibos extract and sugar proved to be beneficial, especially for the retention of aspalathin. Browning, that is, absorbance at 420 nm, was significantly (P < 0.05) increased in the base formulation. In the case of the formulations also containing ascorbic acid and/or citric acid, absorbance remained unchanged or decreased when subjected to NTS and HTS treatments. This was attributed to removal of brown polymers from solution as the pH values of these formulations were lower than that of the base formulation.     

Antioxidant and pro-oxidant activities of aqueous extracts and crude polyphenolic fractions of rooibos (Aspalathus linearis)

Unfermented rooibos tea is known to contain higher levels of total polyphenols and flavonoids than its fermented counterpart, making it the obvious choice for the preparation of flavonoid-enriched fractions. Evaluation of aqueous extracts and crude polyphenolic fractions of unfermented and fermented rooibos showed anti- and/or pro-oxidant activities, using a linoleic acid-Tween-buffer emulsion for lipid peroxidation and the deoxyribose degradation assay, based on a Fenton reaction model system containing FeCl3-EDTA and H2O2 for the generation of hydroxyl radicals. Except for the ethyl acetate fraction, with the highest total polyphenol (TP) content and offering the least protection presumably due to pro-oxidant activity, the inhibition of lipid peroxidation by the samples correlated moderately with their TP content in a linear relationship (r = 0.896, P < 0.01). Using the deoxyribose degradation assay, the pro-oxidant activity of the aqueous extracts and their crude polymeric fractions (0.1 mg/mL in the reaction mixture) was linear with respect to their dihydrochalcone (aspalathin and nothofagin) (r = 0.977, P = 0.023) and flavonoid (r = 0.971, P = 0.029) content. Pro-oxidant activity was demonstrated for pure aspalathin. Using the same assay, but with ascorbate added to regenerate Fe3+ to Fe2+, the aqueous extract and crude polymeric fraction of fermented rooibos displayed hydroxyl radical scavenging activity. Fermentation (i.e., oxidation) of rooibos decreased the pro-oxidant activity of aqueous extracts, which was contributed to a decrease in their dihydrochalcone content. The in vitro pro-oxidant activity displayed by flavonoid-enriched fractions of rooibos demonstrates that one must be aware of the potential adverse biological properties of potent antioxidant extracts utilized as dietary supplements.     

Variation in phenolic content and antioxidant activity of fermented rooibos herbal tea infusions: role of production season and quality grade

Data are required to calculate the dietary exposure to rooibos herbal tea flavonoids and phenolic acids. Representative content values for the principal phenolic compounds and total antioxidant capacity of fermented rooibos infusion, taking into account variation caused by production seasons (2009, 2010, and 2011) and quality grades (A, B, C, and D), were determined for samples (n = 114) from different geographical areas and producers. The major phenolic constituents were isoorientin and orientin (>10 mg/L), with quercetin-3-O-robinobioside, phenylpyruvic acid glucoside, and aspalathin present at >5 mg/L. Isovitexin, vitexin, and hyperoside were present at <3 mg/L. Rutin, ferulic acid, and isoquercitrin were present at <2 mg/L. Nothofagin was present at <1 mg/L. Only traces of luteolin-7-O-glucoside and the aglycones quercetin, luteolin, and chrysoeriol were present. Substantial variation was observed in the individual content values of the phenolic compounds and total antioxidant capacity within production seasons and quality grades.     

Modulation of hepatic drug metabolizing enzymes and oxidative status by rooibos (Aspalathus linearis) and Honeybush (Cyclopia intermedia), green and black (Camellia sinensis) teas in rats

Rooibos and honeybush teas significantly (P < 0.05) enhanced the activity of cytosolic glutathione S-transferase alpha. A significant (P < 0.05) to marginal (P < 0.1) increase in the activity of the microsomal UDP-glucuronosyl transferase was obtained with unprocessed rooibos and honeybush teas, respectively. Oxidized glutathione (GSSG) levels were significantly (P < 0.05) reduced in the liver of all tea treated rats while reduced glutathione (GSH) was markedly increased in the liver of the herbal tea treated rats. These changes resulted in a significant (P < 0.05) increase in the GSH/GSSG ratio by the unprocessed, processed rooibos and unprocessed honeybush teas. Green and black teas markedly to significantly decreased the oxygen radical absorbance capacity in liver homogenates, respectively. Modulation of phase II drug metabolizing enzymes and oxidative status in the liver may be important events in the protection against adverse effects related to mutagenesis and oxidative damage.     

Phenolic constituents and antioxidant activity of Wendita calysina leaves (Burrito), a folk Paraguayan tea

Burrito tea originates from the leaves of Wendita calysina, an indigenous Paraguayan plant, which is commonly consumed in South America and in Western countries. Phytochemical investigation of this species has led to the isolation of 14 constituents, among them 2 new flavanonols, dihydroquercetagetin (1) and 3,5,6,7,4'-pentahydroxyflavanonol (2), in addition to several known methoxyflavones, methoxyflavonols, phenylethanoid glycosides, and benzoic acid derivatives (4-14). All structures were elucidated by ESI-MS and NMR spectroscopic methods. Quantitative determination of phenolic constituents from burrito water infusions has been performed by HPLC-UV-DAD. The total antioxidant activity of the tea was measured by the ABTS(*)(+) radical cation decolorization and chemiluminescence (CL) assays and compared with the values of other commonly used herbal teas (green and black teas, mate, and rooibos).     

Identification and comparison of phenolic compounds in the preparation of oolong tea manufactured by semifermentation and drying processes

Oolong tea manufactured via a semifermentation process possesses a taste and color somewhere between green and black teas. Alteration of constituents, particularly phenolic compounds, in the infusion of oolong tea resulting from its manufacture, was analyzed by high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry. The identified constituents contained 2 alkaloids, 11 flavan-3-ols, 8 organic acids and esters, 11 proanthocyanidin dimers, 3 theaflavins, and 22 flavonoid glycosides, including 6 novel acylated flavonol glycosides. The tentative structures of these 6 novel compounds were depicted according to their mass fragmentation patterns in MS(n) (n = 1-4). In comparison with caffeine as an internal standard, relative contents of the constituents in the infusions of fresh tea shoot and different oolong tea preparations were examined. Approximately, 30% catechins and 20% proanthocyanidins were oxidized during the manufacture of oolong tea from fresh tea shoots, and 20% of total flavonoids were decomposed in a follow-up drying process. Gallocatechin-3-O-gallate and theaflavins putatively produced in the semifermentation process of oolong tea were not detected in fresh tea shoots, and the majority of theaflavins were presumably transformed into thearubigins after drying.     

Two types of oxidative dimerization of the black tea polyphenol theaflavin.

Theaflavin and its galloyl esters are polyphenolic pigments of black tea. In the course of studies on the oxidation mechanism of tea polyphenols, two theaflavin oxidation products named bistheaflavins A and B were isolated, and their structures were elucidated on the basis of MS and NMR spectroscopic analyses. Treatment of a mixture of (-)-epicatechin and (-)-epigallocatechin with banana fruit homogenate yielded bistheaflavin A together with theaflavin and theanaphthoquinone. The symmetrical structure of bistheaflavin A suggested that this compound was formed by oxidative C [bond] C coupling of two theaflavin molecules. In contrast, theaflavin in phosphate buffer (pH 7.3) was gradually oxidized to give bistheaflavin B and theanaphthoquinone. Bistheaflavin B possesses a bicyclooctane skeleton probably formed by intermolecular cyclization between dehydrotheaflavin and dihydrotheanaphthoquinone.     

Total phenolics and antioxidant activities of fenugreek, green tea, black tea, grape seed, ginger, rosemary, gotu kola, and ginkgo extracts, vitamin E, and tert-butylhydroquinone

The total phenolics and antioxidant activities of fenugreek, green tea, black tea, grape seed, ginger, rosemary, gotu kola, and ginkgo extracts, vitamin E, and tert-butylhydroquinone, were determined. Grape seed and green tea were analyzed for their phenolic constituents using high-performance liquid chromatography. The total phenolics of the plant extracts, determined by the Folin-Ciocalteu method, ranged from 24.8 to 92.5 mg of chlorogenic acid equivalent/g dry material. The antioxidant activities of methanolic extracts determined by conjugated diene measurement of methyl linoleate were 3.4-86.3%. The antioxidant activity of the extracts using chicken fat by an oxidative stability instrument (4.6-10.2 h of induction time) followed a similar trend in antioxidant activity as determined by the Folin-Ciocalteu method. Seven phenolics in grape seed and green tea extracts were identified that ranged from 15.38 to 1158.49 and 18.3 to 1087.02 mg/100 g of extract, respectively. Plant extracts such as green tea and grape seed extracts can be used to retard lipid oxidation in a variety of food products.     

Polyphenol composition of a functional fermented tea obtained by tea-rolling processing of green tea and loquat leaves

Phenolic constituents of a new functional fermented tea produced by tea-rolling processing of a mixture (9:1) of tea leaves and loquat leaves were examined in detail. The similarity of the phenolic composition to that of black tea was indicated by high-performance liquid chromatography comparison with other tea products. Twenty-five compounds, including three new catechin oxidation products, were isolated, and the structures of the new compounds were determined to be (2R)-2-hydroxy-3-(2,4,6-trihydroxyphenyl)-1-(3,4,5-trihydroxyphenyl)-1-propanone 2-O-gallate, dehydrotheasinensin H, and acetonyl theacitrin A by spectroscopic methods. In addition, theacitrinin A and theasinensin H were obtained for the first time from commercial tea products. Isolation of these new and known compounds confirms that reactions previously demonstrated by in vitro model experiments actually occur when fresh tea leaves are mechanically distorted and bruised during the production process.     

Analysis of tea shoot catechins: spectrophotometric quantitation and selective visualization on two-dimensional paper chromatograms using diazotized sulfanilamide.

A highly specific and sensitive diazotized sulfanilamide reagent is synthesized for determination of tea catechins. The reagent is employed both as spray reagent for selective visualization of tea catechins on two-dimensional paper chromatograms (sensitivity <1 microg of d-(+)-catechin) and for their spectrophotometric quantification in the crude extracts of tea polyphenols isolated from fresh or dried tea shoots. The formation of yellow color (lambda(max) = 425 nm) between catechins and diazotized sulfanilamide was investigated and made the basis of a simple and sensitive spectrophotometric method for estimation of the total and individual catechins in different tea cultivars. At 425 nm, the absorbance was linear (r = 0.999) over the (0.4-8.0 microg/mL) concentration range of d-(+)-catechin.     

Characterization of epoxydecenal isomers as potent odorants in black tea (Dimbula) infusion

In a black tea (Dimbula) infusion, the potent "sweet and/or juicy" odorants were identified as the cis- and trans-4,5-epoxy-(E)-2-decenals by comparison of their gas chromatography retention indices, mass spectra, and odor quality to those of the actual synthetic compounds. Of the two odorants, cis-4,5-epoxy-(E)-2-decenal has been identified for the first time in the black tea. On the basis of the aroma extract dilution analysis on the flavor distillate obtained using the solvent-assisted flavor evaporation technique from the black tea infusion, these isomers showed higher flavor dilution (FD) factors. The FD factors and concentrations of these odorants in the black tea infusion were observed to be much higher than those from Japanese green tea. In addition, the model studies showed that these odorants were generated from linoleic acid and its hydroperoxides by heating, but the generated amounts of these odorants from linoleic acid were much less than those of its hydroperoxides. It can be assumed from these results that the withering and fermentation, which are characteristic processes during the manufacturing of the black tea, which includes the enzymatic reaction such as lipoxygenase, is one of the most important factors for the formation of the epoxydecenal isomers.     

Size-exclusion chromatography of tea tannins and intercepting potentials of peptides for the inhibition of trypsin-caseinolytic activity by tea tannins

Molecular-weight distribution and characterization of tea tannin were investigated by high-performance liquid chromatography and the equivalent preparative exclusion gel chromatography using Sephadex G-25. The characteristics of the fractions were studied regarding the amounts of terminal catechin, sugar, and gallic acid, the color reaction of the Folin-Chiocalteu reagent, the UV absorbance, and the inhibition activity for the trypsin-caseinolytic activity per weight. Furthermore, we investigated the intercepting activities of the inhibition by the amino acids, peptides, their analogues, poly(ethylene glycol)s (PEGs), and histatin 5 using the inhibition of trypsin-caseinolytic activity by tea. Arg, Lys, and their peptides had strong intercepting activities for the inhibition, but only a weak activity was detected in the Pro peptides or gelatin-like peptides of (Pro-Pro-Gly)(n) (n = 5 or 10). The guanidyl group of Arg and the amino methylene group of Lys were important for the intercepting activity, but the activity was weakly dependent upon the peptide bond formation. The intercepting activity of the peptides or PEG exponentially increased with the number of polymerizations. Histatin 5 did not have a remarkably strong intercepting activity considering the peptide length. The activity of the synthetic histatin 5 in which all of the Lys and Arg were substituted by Ala was at the same level as histatin 5.     

Characterization of tyrosinase- and polyphenol esterase-catalyzed end products using selected phenolic substrates

The oxidative end products that result from the biocatalysis of tyrosinase (PPO) and/or a polyphenol esterase (PPE) extract have been investigated simultaneously in model systems containing selected phenolic compounds as substrates. The spectrophotometric scanning of brown color, formed in the presence of both PPO and PPE, showed a decrease in the absorbance compared to that obtained with PPO only. Graphical analyses of the iterative spectra of oxidized phenolic end products by PPO confirmed the presence of, at least, three kinetically related absorbing species. HPLC analyses of the end products, obtained by the biocatalysis of PPE or PPO activity, indicated the presence of two main groups of compounds: colored ones of lambda(max) at 294-324 nm and colorless products of lambda(max) at 264-290 nm. PPE produced both compounds when selected substrates were used as substrates, whereas PPO produced only one type of oxidation product. However, when both enzymes were incubated together, the nature of the end products was similar to that obtained with PPE only.     

The protective role of different green tea extracts after oxidative damage is related to their catechin composition

The antioxidant activities of three different green tea extracts were investigated and compared by two different methods. By the first method, which evaluated the direct protective effect of the green tea extracts on lipid peroxidation, the extracts were added, at different concentrations, to a lipid model system, made by refined peanut oil, freshly submitted to a further bleaching and subjected to forced oxidation at 98 degrees C, by an oxidative stability instrument. By the second method, the effectiveness of the same extracts was checked in cultures of neonatal rat cardiomyocytes exposed to a free radical-generating system by evaluating conjugated diene production and lactate dehydrogenase release. All of the extracts revealed a strong antioxidant activity by both the methods, and a particular effectiveness was demonstrated by the extracts having higher amounts of (-)-epigallocathechin-3-gallate and (-)-epigallocathechin, as analyzed by reverse-phase HPLC analysis.     

Interactions between cyanidin 3-O-glucoside and furfural derivatives and their impact on food color changes

The reaction between (+)-catechin and cyanidin 3-O-glucoside was investigated in the presence of furfural and 5-(hydroxymethyl)furfural using LC/DAD and LC/MS analysis, and the obtained results were compared with those recorded with malvidin 3-O-glucoside. The appearance of colorless and red and yellow compounds was observed showing that the two polyphenols competed in the condensation process with a predominant formation of the reddish adducts. The colored compounds formed in the case of cyanidin 3-O-glucoside seemed to be more stable than those formed when the reaction was conducted with malvidin 3-O-glucoside. The detection of these reddish and yellowish compounds constitutes a new support for the contribution of this kind of reaction in the color evolution of fruit-derived beverages. In addition, other unidentified compounds were also detected, showing the occurrence of other interaction pathways in addition to the polymerization process yielding oligomeric bridged derivatives and opening perspectives of further investigations of these model solutions.     

采用PLS-DA分析毛火方式对工夫红茶品质的影响

为探究不同毛火方式对工夫红茶品质的影响,明确新型电磁内热式滚筒-热风耦合干燥设备的毛火效果,该研究以一芽一二叶初展嫩度的"福鼎大白"品种为原料进行工夫红茶加工,设定电磁滚筒-热风耦合(Rotary pot-Hot air coupling First-Drying with electromagnetic heat,RHFD)、链板热风(Chain plate Hot air First-Drying,CHFD)、箱式热风(Box Hot air First-Drying,BHFD)、滚筒式滚炒(Rotary pot First-Drying,RFD)等4种毛火方式,比较所制茶样的茶多酚、儿茶素、茶色素、可溶性糖、咖啡碱、氨基酸等29个非挥发性指标,114个气相色谱-质谱技术(GasChromatography-Mass Spectrometry,GC-MS)检测的挥发性香气指标,10个外形和汤色色泽客观评价指标,同时进行了毛火方式的热效率、生产效率、生产成本等性能指标的分析比较,通过偏最小二乘判别统计(PartialLeastSquaresDiscriminationAnalysis,PLS-DA)分析毛火方式对优质工夫红茶品质的影响,并获得标志性差异化合物。结果表明:电磁内热式滚筒-热风耦合毛火处理下茶多酚和儿茶素总量显著最低(P<0.05),简单儿茶素含量较高,茶红素和可溶性糖含量、茶黄素综合指标TDE和茶色素综合指标10TFRB最高(P<0.05),毛火方式对茶黄素总量影响不显著(P>0.05);挥发性化合物总量以RHFD方式最高,RFD方式次之,CHFD方式最低;RHFD毛火方式芳香类、萜烯类等化合物含量最高。电磁内热式滚筒-热风耦合毛火升温快、温度分布均匀且稳定性好,热效率和生产效率高(分别为50.0%、220 kg/h),生产成本较低(仅0.32元/kg),预热时间仅14min;所制红茶在汤色透亮度、香气甜久度、滋味甜醇度等方面均得到提升,感官总分最高(P<0.05),达88.1。PLS-DA分析从挥发性和非挥发性角度均可将工夫红茶4种毛火方式显著区分,并分别获得了43种和18种差异化合物,结合差异性分析获得标志性差异化合物,2,4,6-三(1,1-二甲基乙基)-4-甲基环己-2,5-二烯-1-酮、香叶醇、3-辛酮、水杨酸甲酯、茶黄素、茶褐素、可溶性糖、表儿茶素等,可作为区分工夫红茶毛火方式,以及定向加工甜香、甜醇、高亮等优质工夫红茶的指标物质。该研究为红茶加工基础和品质提升提供技术参考和理论指导。     

Oxidative deamination of benzylamine and lysine residue in bovine serum albumin by green tea, black tea, and coffee

Oxidative deamination by various polyphenolic compounds is presumed to be due to the oxidative conversion of polyphenols to the corresponding quinones through autoxidation. Here we examined the oxidative deamination by the polyphenol-rich beverages green tea, black tea, and coffee at a physiological pH and temperature. Green tea, black tea, and coffee extracts oxidatively deaminated benzylamine and the lysine residues of bovine serum albumin to benzaldehyde and alpha-aminoadipic delta-semialdehyde residues, respectively, in sodium phosphate buffer (pH 7.4) at 37 degrees C in both the presence and absence of Cu2+, indicating the occurrence of an amine (lysyl) oxidase-like reaction. We also examined the effects of pH and metal ions on the reaction. The possible biological effects of drinking polyphenol-rich beverages on human are also discussed.