Determination of catechins and catechin gallates in biological fluids by HPLC with coulometric array detection and solid phase extraction

Tea polyphenols are well known for their beneficial health effects that involve their anti-carcinogenic, anti-mutagenic, anti-pathogenic and anti-oxidative properties. The main polyphenols of green tea are favan-3-ols (catechins) and their corresponding gallate compounds, which constitute about one-third of the dry weight of tea leaves. Their main ingredients are (+)-catechin (C), (−)-epicatechin (EC), (−)-gallocatechin (GC), (−)-epigallocatechin (EGC), (−)-catechin gallate (CG), (−)-epicatechin gallate (ECG), (−)-gallocatechin gallate (GCG) and (−)-epigallocatechin gallate (EGCG). Each has slightly different biological properties. We have developed a method to simultaneously analyze all these compounds in plasma and urine. The samples were first incubated with β-d-glucuronidase and sulfatase to release the catechin residues from their corresponding conjugates for subsequent extraction by selective solid phase column, Waters Oasis HLB extraction cartridges. The extracted molecules were resolved by reversed phase HPLC and monitored by coulometric chemical detection on a CoulArray detector. All eight catechin compounds were analyzed in a single chromatogram within 25 min. For plasma and urine analyses, good linearity (>0.9950) was validated in the range 10-2000 and 10-5000 ng/ml, respectively. The coefficients of variance (CV) were less than 5%. Absolute recovery was greater than 85% and detection limit was 5 ng/ml. The chromatogram exhibited minimal interference as a result of the highly selective solid phase extraction and CoulArray detection.     

Efficient procedure for isolating methylated catechins from green tea and effective simultaneous analysis of ten catechins,three purine alkaloids,and gallic acid in tea by high-performance liquid chromatography with diode array detection

Monomers of (−)-epigallocatechin (EGC), (−)-epigallocatechin gallate (EGCG), (−)-epicatechin (EC), (−)-epicatechin gallate (ECG), (−)-epigallocatechin 3-O-(3-O-methyl) gallate (EGCG3″Me) and (−)-3-O-methyl epicatechin gallate (ECG3′Me) (purity, >97%) were successfully prepared from extract of green tea by two-time separation with Toyopearl HW-40S column chromatography eluted by 80% ethanol. In addition, monomers of (−)-catechin (C), (−)-gallocatechin (GC), (−)-gallocatechin gallate (GCG), and (−)-catechin gallate (CG) (purity, >98%) were prepared from EC, EGC, EGCG, and ECG by heat-epimerization and semi-preparative HPLC chromatography. With the prepared catechin standards, an effective and simultaneous HPLC method for the analysis of gallic acid, tea catechins, and purine alkaloids in tea was developed in the present study. Using an ODS-100Z C₁₈ reversed-phase column, fourteen compounds were rapidly separated within 15 min by a linear gradient elution of formic acid solution (pH 2.5) and methanol. A 2.5–7-fold reduction in HPLC analysis time was obtained from existing analytical methods (40–105 min) for gallic acid, tea catechins including O-methylated catechins and epimers of epicatechins, as well as purine alkaloids. Detection limits were generally on the order of 0.1–1.0 ng for most components at the applied wavelength of 280 nm. Method replication generally resulted in intraday and interday peak area variation of <6% for most tested components in green, Oolong, black, and pu-erh teas. Recovery rates were generally within the range of 92–106% with RSDs less than 4.39%. Therefore, advancement has been readily achievable with commonly used chromatography equipments in the present study, which will facilitate the analytical, clinical, and other studies of tea catechins.     

Versatile synthesis of epicatechin series procyanidin oligomers, and their antioxidant and DNA polymerase inhibitory activity

Proanthocyanidins, known as condensed tannins or oligomeric flavonoids, exist in many edible plants and show various interesting biological activities. We have developed a simple and versatile method of synthesizing procyanidin oligomers consisting of (−)-epicatechin and (+)-catechin. This method is applicable to the synthesis of various 3-O-substituted oligomers. We report here the stereoselective and length controlled synthesis of [4-8]-condensed (−)-epicatechin series procyanidin oligomers. We described the details of the synthesis of an two tetramers, (−)-epicatechin-(−)-epicatechin-(−)-epicatechin-(−)-epicatechin and (−)-epicatechin-(−)-epicatechin-(−)-epicatechin-(+)-catechin (arecatannin A1), (−)-epicatechin pentamer and two 3,3″,3?-tri-O-galloyl trimers, (−)-epicatechin-(−)-epicatechin-(−)-epicatechin-3,3″,3?-tri-O-gallate and (−)-epicatechin-(−)-epicatechin-(+)-catechin-3,3″,3?-tri-O-gallate with the condensation method using TMSOTf as a catalyst. The ability of DPPH radical scavenging activity and DNA polymerase inhibitory activity of these oligomeric compounds were investigated.     

Degradation effects in the extraction of antioxidants from birch bark using water at elevated temperature and pressure

Experiments with birch bark samples have been carried to enable a distinction between extraction and degradation effects during pressurised hot water extraction. Two samples, E80 and E180, contained birch bark extracts obtained after extraction at 80 and 180 °C for up to 45 min, respectively. Two other samples, P80 and P180, were only extracted for 5 min at the two temperatures and were thereafter filtered and hydrothermally treated at 80 and 180 °C, respectively. During the latter treatment, samples were collected at different times to assess the stability of the extracted compounds. An offline DPPH (2,2-diphenyl-1-picrylhydrazyl) assay, as well as a high performance liquid chromatographic separation coupled to an electrochemical detector, were used to determine the antioxidant capacity of the processed samples. The results obtained with the different techniques were compared to assess the yield of the extraction and degradation processes. In addition, an online hyphenated system comprising high performance liquid chromatography coupled to diode-array; electrochemical; and tandem mass spectrometric detection (HPLC-DAD-ECD-MS/MS) was used to study the compositions of the extracts in more detail. The results for the samples processed at 80 °C showed that the extraction reached a steady-state already after 5 min, and that the extracted compounds were stable throughout the entire extraction process. Processing at 180 °C, on the other hand, gave rise to partly degraded extracts with a multitude of peaks in both the diode array and electrochemical detectors, and a higher antioxidant capacity compared to for the extracts obtained at 80 °C. It is concluded that HPLC-DAD-ECD is a more appropriate technique for the determination of antioxidants than the DPPH assay. The mass spectrometric results indicate that one of the extracted antioxidants, catechin, was isomerised to its diastereoisomers; (+)-catechin, (−)-catechin, (+)-epicatechin, and (−)-epicatechin.     

Separation of proanthocyanidins isolated from tea leaves using high-speed counter-current chromatography

The proanthocyanidin extract from tea (Camellia sinensis) leaves was purified for the further study of the biological role of proanthocyanidins in blister blight leaf disease of tea, which is caused by the fungus Exobasidium vexans. An aqueous acetone extract of proanthocyanidins prepared from healthy tea leaves was partially purified using Sephadex LH-20 chromatography. The crude proanthocyanidin extract obtained was fractionated with high-speed counter-current chromatography (HSCCC) using the solvent system n-hexane–EtOAc–MeOH–water (1:5:1:5). The purity of the each isolated fraction after a single HSCCC run was evaluated by high-performance liquid chromatography (HPLC). Seven fractions of high purity were isolated. The identity of the compound present in each fraction isolated was established using electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR) spectroscopy. Five proanthocyanidins and two flavanol digallates, (−)-epigallocatechin digallate (EGCDG) and (−)-epicatechin digallate (ECDG) were isolated. Comparison of spectral data of the proanthocyanidins isolated with those previously reported indicated that all five were known B-type proanthocyanidins with 2,3-cis stereochemistry in both the upper (u-unit) and the terminal (t-unit) units, and 4R configuration of the C-ring in the u-unit. The proanthocyanidins were established to be dimers composed of (−)-epigallocatechin gallate (EGCG), (−)-epicatechin gallate (ECG) and (−)-epiafzelechin gallate (EAG) units with the following structures: EGCG-(4β → 6)-EGCG, ECG-(4β → 6)-EGCG, EGCG-(4β → 6)-ECG, EAG-(4β → 6)-EGCG, ECG-(4β → 6)-ECG by analysis of spectral data. Therefore HSCCC offers a powerful method for the separation of a group of closely related naturally occurring compounds.     

Scaling up of high-speed countercurrent chromatographic apparatus with three columns connected in series for rapid preparation of (−)-epicatechin

In the present study, compact high-speed countercurrent chromatographic apparatus was constructed with three columns connected in series. Two sets of columns were prepared from 10 mm and 12 mm I.D. tubing to form 12 L and 15 L capacities, respectively. Performance of these columns was compared for the separation of (−)-epicatechin gallate (ECG) from a tea extract by flash countercurrent chromatography (FCCC). In each separation, 200 g of the tea extract in 1600 mL of mobile phase was loaded onto the column. The 12 L column gave 7.5 L (35 g of ECG) and the 15 L column gave 9 L (40 g of ECG) of ECG solution without impurities. The ECG solution was directly hydrolyzed by tannase into (−)-epicatechin. The hydrolysate was purified by flash chromatography on AB-8 macroporous resin to give 52 g of EC (purity 99.1%). This scaled up apparatus could be used for the industrial separation of natural products.     

Borate complexation-assisted field-enhanced sample injection for on-line preconcentration of cis-diol-containing compounds in capillary electrophoresis

A new on-line preconcentration technique called borate complexation-assisted field-enhanced sample injection (BCA-FESI) was proposed for preconcentrating cis-diol-containing compounds (CDCCs) in capillary electrophoresis (CE). The principle relies on amplification of the difference in the electrophoretic mobilities of CDCC in sample matrix and background electrolyte (BGE) through complexation of CDCC with borate in a sample matrix of basic pH and dissociation of the complex in a BGE of acidic pH. Meanwhile, CDCC and borate ions electro-injected into the capillary are finally in neutral state, which maintains the pre-filled low conductivity zone and thus allows for longer injection time. With catechol as a test compound, the principle and effectiveness of BCA-FESI was verified. As compared to conventional sample injection, BCA-FESI allowed for sensitivity enhancement of 1850-fold. The established method was further evaluated with three catechins, including (−)-epicatechin gallate (ECG), (−)-gallocatechin gallate (GCG), and (−)-epigallocatechin (EGC), in a standard mixture of trace content. The limit of detection (LOD) was found to be 1.4, 3.8, 17.5 nM (S/N = 3) for ECG, GCG, EGC, respectively. Finally, the BCA-FESI method was applied to a real sample of diluted tea beverage, in which the three catechins were detected.     

Simultaneous analysis of tea catechins, caffeine, gallic acid, theanine and ascorbic acid by micellar electrokinetic capillary chromatography

A micellar electrokinetic capillary chromatography (MEKC) method for the simultaneous analysis of five tea catechins, theanine, caffeine, gallic acid and ascorbic acid has been developed. The catechins are (-)-epicatechin, (+)-catechin, (-)-epigallocatechin, (-)-epicatechin gallate and (-)-epigallocatechin gallate. p-Nitrophenol serves as both reference and internal standard. All the components are separated within 13 min with a 57 cm uncoated fused-silica column. On-column detection was carried out at 200 nm. This method has been used to measure these compounds in fresh tea leaves and tea liquor. The limit of detection for all analytes ranged from 1 to 20 microg/ml.     

Determination of proanthocyanidins in fresh grapes and grape products using liquid chromatography with mass spectrometric detection

Fresh grapes and grape products, such as grape wine and grape juice, were analyzed for proanthocyanidins (PACs) using liquid chromatography with electrospray ionization mass spectrometric (MS) detection. PACs were successfully separated and analyzed on the basis of their protonated molecules, allowing the identification of PACs in different degrees of polymerization from monomers to oligomers (up to 7 units), and in various isomeric forms. Using reversed-phase high-performance liquid chromatography (HPLC) combined with MS detection, the PAC monomers, (+)-catechin (C), (-)-epicatechin (EC), (-)-catechin gallate (CG), and (-)-epicatechin gallate (ECG), were successfully quantified using selected ion monitoring (SIM) mode. Standard curves were fitted for each PAC ranging from 43.8 to 5600 ng/mL for C, from 42.2 to 5400 ng/mL for EC, from 36.7 to 4700 ng/mL for CG, and from 39.8 to 5100 ng/mL for ECG. Good linearity (r2>0.999) was achieved for each analyte. The accuracy and precision (RSD) were within 10% (n=8) at the limit of detection. This method allows direct quantification of monomeric PACs in fresh grapes and grape-derived products. Additionally, flow injection analysis (FIA) was applied to estimate the concentration levels of PAC oligomers by comparing their FIA-MS peak areas, which were well correlated (r2=0.936) to the total concentrations of PAC monomers.     

Separation of eight selected flavan-3-ols on cellulose thin-layer chromatographic plates

The potential of microcristaline cellulose as sorbent in the separation of eight compounds: (+)-catechin (C), (-)-epicatechin (EC), (-)-gallocatechin (GC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg), (-)-epigallocatechin gallate (EGCg), procyanidin B1 and procyanidin B2 was studied. Cellulose HPTLC plates prewashed in water (not necessary, when water was used as developing solvent) and dried with a hair dryer, bandwise application and development in horizontal developing chamber (sandwich configuration) gave the best results. Detection was performed using vanillin-H3PO4 reagent. Four new developing solvent systems were proposed: water, 1-propanol-water (20:80, v/v), 1-propanol-water-acetic acid (4:2:1, v/v) and 1-propanol-water-acetic acid (20:80:1, v/v), and at least two of them were needed for the differentiation between all eight compounds. Surprisingly, water enabled the separation of epimers C from EC and GC from EGC, as well as the dimers procianidin B1 and B2. Additionally, C, EGC, B1 and B2 were separated from all the other compounds. The best choice for developing solvent is given for each of the studied compounds. The best separation of the five main catechins (EC, GC, EGC, ECg, EGCg) present in green tea extract was achieved using 1-propanol-water-acetic acid (20:80:1, v/v). The chromatograms of oak bark extract developed in solvents with higher water content (1-propanol-water (1:4, v/v) and 1-propanol-water-acetic acid (20:80:1, v/v)) showed less bands than chromatograms developed in solvents with higher organic modifier content (e.g. 1-propanol-water-acetic acid (4:2:1, v/v)). It was proved that such behavior was due to the presence of procyanidins beside the main component catechin.     

Study of the catechins and proanthocyanidins ofQuercus robur

More than 20 compounds have been isolated from the bark ofQuercus robur. Monomers: (–)-epicatechin, (–)-epicatechin gallate, (+)-catechin, (+)-catechin gallate, (+)-gallocatechin, (–)-epigallocatechin, and (–)-epigallocatechin gallate; dimeric proanthocyanidins: (+)-catechin-(4-8)-(+)-catechin, 3-O-galloyl-(+)-catechin-(4-8)-3-O-galloyl-(+)-catechin, 3-O-galloyl-(+)-gallocatechin-(4-8)-(+)-gallocatechin, (–)-epicatechin-(4-8)-3-O-galloyl-(–)-epigallocatechin gallate, 3-O-galloyl-(–)-epicatechin-(4-8)-3-O-galloyl-(–)-epigallocatechin, 3-O-galloyl-(–)-epigallocatechin-(4-8)-(+)-catechin; and oligomeric proanthocyanidins: D14-D19.Materials presented at the IInd International Symposium on the Chemistry of Natural Compounds (SCNC, Eskiehir, Turkey, October, 22–24, 1996).Institute of the Chemistry of Plant Substances, Academy of Sciences of the Republic of Uzbekistan, Tashkent, fax (3712) 40 64 75. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 819–833, November–December, 1997.     

Proanthocyanidins ofZiziphus jujuba

The catechin and proanthocyanidin compositions of the leaves and bark ofZiziphus jujuba have been studied over the vegetation periods. This has led to the isolation of 16 compounds, including 8 monomeric catechins — (–)-epiafzelechin, (–)-epicatechin, (–)-epigallocatechin, (–)-epicatechin gallate, (–)-epigal-locatechin gallate, (+)-catechin, (+)-catechin gallate, and (+)-gallocatechin; 4 dimeric proanthocyanidins — (–)-epiafzelechin-(4-8)-(–)-epicatechin, proanthocyanidin B-2, (–)-epicatechin-(4-8)-(–)-epigallocatechin, and (–)-epiafzelechin-(4-8)-(–)-epigallocatechin; and 4 oligomeric proanthocyanidins consisting of epiafzelechin, epigallocatechin, catechin, and epicatechin with different degrees of polymerization. Their structures have been established by a study of PMR and¹³C NMR spectra and the products of chemical transformation.The materials of this paper were presented at the Second International Symposium on the Chemistry of Natural Compounds (SCNC, Eskiehir, Turkey, October 22–24, 1996).Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 221–231, March–April, 1997.     

Development of HPLC Method for Catechins and Related Compounds Determination and Standardization in Miang (Traditional Lanna Fermented Tea Leaf in Northern Thailand)

High performance liquid chromatography (HPLC) for catechins and related compounds in Miang (traditional Lanna fermented tea leaf) was developed to overcome the matrices during the fermentation process. We investigated a variety of columns and elution conditions to determine seven catechins, namely (+)-catechin, (−)-gallocatechin, (−)-epigallocatechin, (−)-epicatechin, (−)-epigallocatechin gallate, (−)-gallocatechin gallate, (−)-epicatechin gallate, as well as gallic acid and caffeine, resulting in the development of reproducible systems for analyses that overcome sample matrices. Among the three reversed-phase columns, column C (deactivated, with extra dense bonding, double endcapped monomeric C18, high-purity silica at 3.0 mm × 250 mm and a 5 µm particle size) significantly improved the separation between Miang catechins in the presence of acid in the mobile phase within a shorter analysis time. The validation method showed effective linearity, precision, accuracy, and limits of detection and quantitation. The validated system was adequate for the qualitative and quantitative measurement of seven active catechins, including gallic acid and caffeine in Miang, during the fermentation process and standardization of Miang extracts. The latter contain catechins and related compounds that are further developed into natural active pharmaceutical ingredients (natural APIs) for cosmeceutical and nutraceutical products.     

Tannins from Betel Nuts

From the acetone extract of fresh-betel nuts, two procyanidin pentamers, two procyanidin tetramers and three procyanidin trimers, along with (+)-catechin, (-)-epicatechin, procyanidin A-l, procyanidin B-l, procyanidin B-2, and procyanidin B-7 were isolated. Based on ₁H NMR and ₁₃C NMR spectral data as well as the results of thiolysis degradation, the chemical structures of two series of oligomeric arecatannins A and B have been established. The arecatannins from betel nuts were found to be condensed-type tannins which possess (-)-epicatechin unit and (+)-catechin units. All arecatannins had C₄-C₈ linked (-)-epicatechin in the upper units whereas the (+)-catechin in the terminal unit had C₄-C₈ or C₄-C₆ linkage.     

The Qualitative and Quantitative Compositions of Phenolic Compounds in Fruits of Lithuanian Heirloom Apple Cultivars

As the interest in heirloom cultivars of apple trees, their fruit, and processed products is growing worldwide, studies of the qualitative and quantitative composition of biological compounds are important for the evaluation of the quality and nutritional properties of the apples. Studies on the variations in the chemical composition of phenolic compounds characterized by a versatile biological effect are important when researching the genetic heritage of the heirloom cultivars in order to increase the cultivation of such cultivars in orchards. A variation in the qualitative and quantitative composition of phenolic compounds was found in apple samples of cultivars included in the Lithuanian collection of genetic resources. By the high-performance liquid chromatography (HPLC) method flavan-3-ols (procyanidin B1, procyanidin B2, procyanidin C2, (+)-catechin and (−)-epicatechin), flavonols (rutin, hyperoside, quercitrin, isoquercitrin, reynoutrin and avicularin), chlorogenic acids and phloridzin were identified and quantified in fruit samples of heirloom apple cultivars grown in Lithuania. The highest sum of the identified phenolic compounds (3.82 ± 0.53 mg/g) was found in apple fruit samples of the ‘Koštelė’ cultivar     

Structural characterization of a procyanidin tetramer and pentamer from the apple by low-temperature NMR analysis

The structures of a procyanidin tetramer and pentamer from unripe apple (Malus pumila) were elucidated by low-temperature NMR analysis at −34 °C. These structures were [epicatechin-(4β→6)-epicatechin-(4β→8)-epicatechin-(4β→8)-epicatechin (1)] and [epicatechin-(4β→8)-epicatechin-(4β→8)-epicatechin-(4β→8)-epicatechin-(4β→8)-epicatechin (2)].     

Catechin gallates ofRheum maximoviczii A. Los

Summary The roots ofRheum maximoviczii A. Los. contain two catechin gallates: (–)-epicatechin gallate, previously isolated from the leaves of green tea and (±)-catechin gallate found in this plant for the first time. The amount of catechin gallates in the roots is lower in the autumn than in the spring.Khimiya Prirodnykh Soedinenii, Vol. 2, No. 1, pp. 26–29, 1966     

A green analytical procedure for flow-injection determination of nitrate in natural waters

Nitrate determination in waters is generally carried out with cadmium filings and carcinogenic reagents or by reaction with phenolic compounds in highly concentrated sulfuric acid medium. In this work, it was developed a green analytical procedure for nitrate determination in natural waters based on direct spectrophotometric measurements in ultraviolet, using a flow-injection system with an anion-exchange column for separation of nitrate from interfering species. The proposed method employs only one reagent (HClO₄) in a minimum amount (equivalent to 18 μL concentrated acid per determination), and allowed nitrate determination within 0.50-25.0 mg L⁻¹, without interference of up to 200.0 mg L⁻¹ humic acid; 1.0 mg L⁻¹ NO₂⁻; 200.0 mg L⁻¹ PO₄³⁻; 75.0 mg L⁻¹ Cl⁻; 50.0 mg L⁻¹ SO₄²⁻ and 15.0 mg L⁻¹ Fe³⁺. The detection limit (99.7% confidence level) and the coefficient of variation (n = 20) were estimated as 0.1 mg L⁻¹ and 0.7%, respectively. The results obtained for natural water samples were in agreement with those achieved by the reference method based on nitrate reduction with copperized cadmium at the 95% confidence level.     

Determination of psilocybin in hallucinogenic mushrooms by reversed-phase liquid chromatography with fluorescence detection

The determination of psilocybin was carried out by reversed-phase liquid chromatography (HPLC) with fluorescence (FL) detection. Psilocybin was labeled with 5-dimethylaminonaphthalene-1-[N-(2-aminoethyl)]sulfonamide (DNS-ED) at 60 °C for 4 h in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) as the activation reagent. The resulting derivative was separated on a Mightysil RP-18 GP column (150 mm × 4.6 mm, i.d. 3 μm) with the mixture of 50 mM ammonium acetate (AcONH₄) and CH₃CN, and detected at 539 nm (excitation at 321 nm). The structure of the derivative was identified by HPLC-ESI-MS. A good linear relation of the calibration curve of psilocybin was observed under the proposed conditions for labeling, separation and detection. The quantification limit was 4.4 ng in 1 mg dried mushroom. The proposed procedure was successfully used for the determination of psilocybin in real samples. The contents of psilocybin in six magic mushrooms by the proposed HPLC-FL method were less than 20.0 ng in 1 mg dried samples.     

Ultrasound extracted flavonoids from four varieties of Portuguese red grape skins determined by reverse-phase high-performance liquid chromatography with electrochemical detection

Several flavonoids present in red grape skins from four varieties of Portuguese grapes were determined by reverse-phase high-performance liquid chromatography (RP-HPLC) with electrochemical detection (ECD). Extraction of flavonoids from red grape skins was performed by ultrasonication, and hydrochloric acid in methanol was used as extraction solvent. The developed RP-HPLC method used combined isocratic and gradient elution with amperometric detection with a glassy carbon-working electrode. Good peak resolution was obtained following direct injection of a sample of red grape extract in a pH 2.20 mobile phase. Eleven different flavonoids: cyanidin-3-O-glucoside (kuromanin), delphinidin-3-O-glucoside (myrtillin), petunidin-3-O-glucoside, peonidin-3-O-glucoside, malvidin-3-O-glucoside (oenin), (+)-catechin, rutin, fisetin, myricetin, morin and quercetin, can be separated in a single run by direct injection of sample solution. The limit of detection obtained for these compounds by ECD was 20-90 pg/L, 1000 times lower when compared with photodiode array (PDA) limit of detection of 12-55 ng/L. RP-HPLC-ECD was characterized by an excellent sensitivity and selectivity, and appropriate for the simultaneous determination of these electroactive phenolic compounds present in red grape skins.