Cyanidin-3-O-glucoside and its derivative vitisin A alleviate androgenetic alopecia by exerting anti-androgen effect and inhibiting dermal papilla cell apoptosis.

Androgenetic alopecia (AGA), one of the most common forms of hair loss, lacks satisfactory treatment methods in modern society. This study employed an experimental design combining in vitro and in vivo approaches to explore the effects of Cyanidin-3-O-glucoside (C3G) and Carboxypyranocyanidin-3-O-glucoside (Vitisin A) on AGA. In human dermal papilla cells (HDPCs), both anthocyanins demonstrated inhibitory effects on androgen receptors, significantly reduced dihydrotestosterone (DHT) induced apoptosis of HDPCs, and regulated the secretion of Fibroblast growth factor 7 and Transforming growth factor beta 1. In vitro transdermal experiment revealed that both C3G and Vitisin A could penetrate mice skin, aided by the application of cream. Furthermore, in vivo experiments with mice indicated that application of C3G or Vitisin A cream effectively improved hair follicles miniaturization, regression, and apoptosis caused by DHT. The repression of Wnt10b and ß-catenin expression induced by DHT was prevented by C3G and Vitisin A in both cell and mouse model. Consequently, these findings suggest that C3G and Vitisin A could be considered as alternative methods for alleviating AGA.

The promising stability of carboxylpyranocyanidin-3-O-glucoside during food processing and simulated digestion and its bioavailability research.

BACKGROUND: Pyranoanthocyanins are stable anthocyanin derivatives. Carboxylpyranoanthocyanin is one of the simplest pyranoanthocyanin, among which the production of carboxylpyranocyanidin-3-O-glucoside (crboxyl-pycy-3-gluc) is most feasible as a result of the abundance of its reactant, cyanidin-3-O-glucoside (Cy-3-gluc). RESULTS: In the present study, carboxyl-pycy-3-gluc was synthesized and its stability during processing and after ingestion as well as its bioavailability in vivo were comprehensively evaluated. Our results indicated that the color of carboxyl-pycy-3-gluc remained more stable compared to Cy-3-gluc when facing the large-span pH variation. The high retention of anthocyanin symbolized the superb stability under thermal processing, sulfur dioxide bleaching and ultrasonic treatment of carboxyl-pycy-3-gluc. Because of the stability under the alkaline condition, carboxyl-pycy-3-gluc is more stable after oral-gastrointestinal digestion. After in vitro gut microbiota fermentation, the retention of carboxyl-pycy-3-gluc was significantly higher than that of Cy-3-gluc. The larger molecular size made absorption of carboxyl-pycy-3-gluc into blood more difficult than its precursor. CONCLUSION: The present study demonstrated the promising stability of carboxyl-pycy-3-gluc during food processing and after digestion, confirming the potential of carboxyl-pycy-3-gluc as a colorant. © 2023 Society of Chemical Industry.

Vitisin A, as a Type of Pyranoanthocyanin, Suppresses Inflammation by Restricting Hematopoietic Stem Cell Differentiation toward Monocytes in Bone Marrow.

Hematopoietic stem and progenitor cells (HSPCs) could be differentiated into mature myeloid and lymphoid cells, maintaining the requirements of immune cells. Atherosclerosis and ulcerative colitis (UC) drive HSPC homeostasis destruction, which triggers expansive HSPC proliferation and Ly6Chi monocyte production, contributing to aggravated inflammation. Vitisin A belongs to the anthocyanin derivatives with excellent stability and bioactivity in vitro. However, there is no report about the anti-inflammation of Vitisin A via reprogramming HSPC differentiation toward monocytes. In this study, we found that Vitisin A presents anti-inflammatory ability during the development of atherosclerosis and UC by depressing Ly6Chi monocyte production from bone marrow. This performance depended on restricted HSPC differentiation, which suggested that Vitisin A participated in monocyte generation and carried out the immunomodulation. Together, Vitisin A ameliorates inflammation during atherosclerosis and UC via the suppressed differentiation of HSPCs toward monocytes, which could be considered an ideal functional component with immunomodulatory effects.

Vitisin B, a resveratrol tetramer from Vitis thunbergii var. taiwaniana, ameliorates impaired glucose regulations in nicotinamide/streptozotocin-induced type 2 diabetic mice.

Background and aim: In Taiwan, Vitis thunbergii var. taiwaniana (VTT) is used in traditional medicine and as a local tea. VTT rich in resveratrol and resveratrol oligomers have been reported to exhibit anti-obesity and anti-hypertensive activities in animal models; however, no studies have investigated type 2 diabetes mellitus (T2DM) treatments. This study aimed to investigate the anti-T2DM effects of resveratrol tetramers isolated from the VTT in nicotinamide/streptozotocin (STZ)-induced Institute of Cancer Research (ICR) mice. Experimental procedure: The oral glucose tolerance test (OGTT) was used to imitate postprandial blood glucose (BG) regulations in mice by pre-treatment with VTT extracts, resveratrol tetramers of vitisin A, vitisin B, and hopeaphenol 30 min before glucose loads. Vitisin B (50 mg/kg) was administered to treat T2DM-ICR mice once daily for 28 days to investigate its hypoglycemic activity. Results and conclusion: Mice pre-treated with VTT-S-95EE, or vitisin B (100 mg/kg) 30-min before glucose loading showed significant reductions (P < 0.001) in the area under the curve at 120-min (BG-AUC0-120) than those without pre-treatment with VTT-S-95 E E or vitisin B. Vitisin B-treated T2DM mice showed hypoglycemic activities via a reduction in plasma dipeptidyl peptidase (DPP)-IV activities to maintain insulin actions and differed significantly than those of untreated T2DM mice (P < 0.05), and also reduced BG-AUC0-120 and insulin-AUC0-120 in the OGTT.These in vivo results showed that VTT containing vitisin B would be beneficial for developing nutraceuticals and/or functional foods for glycemic control in patients with T2DM, which should be investigated further.

The central administration of vitisin a, extracted from Vitis vinifera, improves cognitive function and related signaling pathways in a scopolamine-induced dementia model.

Neurodegenerative disorders, such as Alzheimer's disease (AD), are characterized by cognitive function loss and progressive memory impairment. Vitis vinifera, which is consumed in the form of fruits and wines in various countries, contains several dietary stilbenoids that have beneficial effects on neuronal disorders related to cognitive impairment. However, few studies have investigated the hypothalamic effects of vitisin A, a resveratrol tetramer derived from V. vinifera stembark, on cognitive functions and related signaling pathways. In this study, we conducted in vitro, ex vivo, and in vivo experiments with multiple biochemical and molecular analyses to investigate its pharmaceutical effects on cognitive functions. Treatment with vitisin A increased cell viability and cell survival under H2O2-exposed conditions in a neuronal SH-SY5 cell line. Ex vivo experiments showed that vitisin A treatment restored the scopolamine-induced disruption of long-term potentiation (LTP) in the hippocampal CA3-CA1 synapse, indicating the restoration of synaptic mechanisms of learning and memory. Consistently, central administration of vitisin A ameliorated scopolamine-induced disruptions of cognitive and memory functions in C57BL/6 mice, as evidenced by Y-maze and passive avoidance tests. Further studies showed that vitisin A upregulates BDNF-CREB signaling in the hippocampus. Together, our findings suggest that vitisin A exhibits neuroprotective effects, at least partially, by upregulating BDNF-CREB signaling and LTP.

Cholesterol-Lowering Activity of Vitisin A Is Mediated by Inhibiting Cholesterol Biosynthesis and Enhancing LDL Uptake in HepG2 Cells.

Pyranoanthocyanins have been reported to possess better chemical stability and bioactivities than monomeric anthocyanins in some aspects. The hypocholesterolemic activity of pyranoanthocyanins is unclear. In view of this, this study was conducted to compare the cholesterol-lowering activities of Vitisin A with the anthocyanin counterpart Cyanidin-3-O-glucoside(C3G) in HepG2 cells and to investigate the interaction of Vitisin A with the expression of genes and proteins associated with cholesterol metabolism. HepG2 cells were incubated with 40 µM cholesterol and 4 µM 25-hydroxycholeterol with various concentrations of Vitisin A or C3G for 24 h. It was found that Vitisin A decreased the cholesterol levels at the concentrations of 100 µM and 200 µM with a dose-response relationship, while C3G exhibited no significant effect on cellular cholesterol. Furthermore, Vitisin A could down-regulate 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR) to inhibit cholesterol biosynthesis through a sterol regulatory element-binding protein 2 (SREBP2)-dependent mechanism, and up-regulate low-density lipoprotein receptor (LDLR) and blunt the secretion of proprotein convertase subtilisin/kexin type 9 (PCSK9) protein to promote intracellular LDL uptake without LDLR degradation. In conclusion, Vitisin A demonstrated hypocholesterolemic activity, by inhibiting cholesterol biosynthesis and enhancing LDL uptake in HepG2 cells.

Role of Oak Ellagitannins in the Synthesis of Vitisin A and in the Degradation of Malvidin 3-O-Glucoside: An Approach in Wine-Like Model Systems.

Recent studies highlight the influence that oak ellagitannins can have on wine astringency and color. Direct reactions between flavanols or anthocyanins with vescalagin have been reported to occur, but participation of these compounds in the formation of other types of derivatives has only been suggested but not demonstrated. This study aims at evaluating, in wine-like model systems, the possible different roles of the main oak ellagitannins, castalagin and vescalagin, alone or combined, in the synthesis of vitisin A and in the degradation of malvidin 3-O-glucoside. In the presence of pyruvic acid, the anthocyanin disappeared mainly as a result of the synthesis of vitisin A, whereas in its absence, degradation reactions prevailed. In general, ellagitannins increased the synthesis of vitisin A, decreased the total content of degradation products, and changed the degradation profile, with differences observed between castalagin and vescalagin. The results of the study revealed that the fate of malvidin 3-O-glucoside is conditioned by the presence of ellagitannins.

Vitisin A, a Resveratrol Tetramer, Improves Scopolamine-Induced Impaired Learning and Memory Functions in Amnesiac ICR Mice.

Resveratrol has been reported to exhibit neuroprotective activities in vitro and in vivo. However, little is known about resveratrol tetramers of hopeaphenol, vitisin A, and vitisin B with the same molecular mass in the improvement of degenerative disorders. In this study, two 95% ethanol extracts (95EE) from stem parts of Vitis thunbergii Sieb. & Zucc. (VT-95EE) and from the root (R) parts of Vitis thunbergii var. taiwaniana (VTT-R-95EE) showed comparable acetylcholinesterase (AChE) inhibitory activities. It was found that VT-95EE and VTT-R-95EE showed different distribution patterns of identified resveratrol and resveratrol tetramers of hopeaphenol, vitisin A, and vitisin B based on the analyses of HPLC chromatographic profiles. The hopeaphenol, vitisin A, and vitisin B, showed AChE and monoamine oxidase-B inhibitions in a dose-dependent manner, among which vitisin B and vitisin A exhibited much better activities than those of resveratrol, and had neuroprotective activities against methylglyoxal-induced SH-SY5Y cell deaths. The scopolamine-induced amnesiac ICR mice treated with VT-95EE and its ethyl acetate-partitioned fraction (VT-95EE-EA) at doses of 200 and 400 mg/kg, or vitisin A at a dose of 40 mg/kg, but not vitisin B (40 mg/kg), were shown significantly to improve the impaired learning behaviors by passive avoidance tests compared to those in the control without drug treatments (p < 0.05). Compared to mice in the control group, the brain extracts in the vitisin A-treated mice or donepezil-treated mice showed significant reductions in AChE activities and malondialdehyde levels (p < 0.05), and elevated the reduced protein expressions of brain-derived neurotrophic factor (BDNF) and BDNF receptor, tropomyosin receptor kinase B (TrkB). These results revealed that vitisin A was the active constituent in the VT-95EE and VTT-95EE, and the VT medicinal plant and that the endemic variety of VTT has potential in developing functional foods for an unmet medical need for neurodegenerative disorders.

Pulsed Electric Fields to Improve the Use of Non-Saccharomyces Starters in Red Wines.

New nonthermal technologies, including pulsed electric fields (PEF), open a new way to generate more natural foods while respecting their organoleptic qualities. PEF can reduce wild yeasts to improve the implantation of other yeasts and generate more desired metabolites. Two PEF treatments were applied; one with an intensity of 5 kV/cm was applied continuously to the must for further colour extraction, and a second treatment only to the must (without skins) after a 24-hour maceration of 17.5 kV/cm intensity, reducing its wild yeast load by up to 2 log CFU/mL, thus comparing the implantation and fermentation of inoculated non-Saccharomyces yeasts. In general, those treated with PEF preserved more total esters and formed more anthocyanins, including vitisin A, due to better implantation of the inoculated yeasts. It should be noted that the yeast Lachancea thermotolerans that had received PEF treatment produced four-fold more lactic acid (3.62 ± 0.84 g/L) than the control of the same yeast, and Hanseniaspora vineae with PEF produced almost three-fold more 2-phenylethyl acetate than the rest. On the other hand, 3-ethoxy-1-propanol was not observed at the end of the fermentation with a Torulaspora delbrueckii (Td) control but in the Td PEF, it was observed (3.17 ± 0.58 mg/L).

Pulsed Electric Field Processing of Red Grapes (cv. Rondinella): Modifications of Phenolic Fraction and Effects on Wine Evolution.

Pulsed electric field (PEF) is a non-thermal technology able to promote color and polyphenols extraction from grape skins. Most of the publications about PEF in winemaking report data concerning international varieties, poorly considering minor cultivars and the medium/long-term effects of the treatment on wine composition during storage. PEF was applied at different specific energies (2, 10, and 20 kJ kg-1) on grapes of the low-color red cv. Rondinella, after crushing-destemming. Pressing yield, the evolution of color, and total phenolic index (TPI) were measured during skin maceration. Moreover, the wines were characterized for basic compositional parameters, color, anthocyanin profile, phenolic composition (glories indices), metal content (Fe, Cr, and Ni), and sensory characters, two and twelve months after the processing, in comparison with untreated samples and pectolytic enzymes (PE). PEF did not affect fermentation evolution, nor did it modify wine basic composition or metal content. Treatments at 10 and 20 kJ kg-1 led to higher color and TPI in wines, in comparison to PE, because of increased content of anthocyanins and tannins. The sensory evaluation confirmed these findings. Modifications remained stable in wines after twelve months. Glories indices and vitisin A content highlighted greater potential stability of wine color in PEF-treated wines.

Pyranoanthocyanins in bilberry (Vaccinium myrtillus L.) wines fermented with Schizosaccharomyces pombe and their evolution during aging.

Fifteen vitisin A-type pyranoanthocyanins (vAPs) were determined in bilberry wines fermented with Saccharomyces cerevisiae and Schizosaccharomyces pombe by HPLC-DAD and UPLC-DAD-ESI-MS/MS. The fermentation involving S. pombe enhanced the production of vAPs compared to the fermentation with pure S. cerevisiae. The formation of vAPs correlated significantly with the decrease in the content of monomeric anthocyanins and pyruvic acid during 12 months of aging. vAPs were more stable than their corresponding monomeric anthocyanins. Methylation in the B-ring and glycosylation with galactose and arabinose further improved the stability of vAPs. Aging for 12 months led to depletion of pyruvic acid and reduction of over 50% of monomeric anthocyanins. The content of vAPs increased by 26-54% during the first six months of aging, followed by a 2.2-10.2% reduction over the following six months. More residual pyruvic acid in S. pombe wines after fermentation consequently enhanced the generation of vAPs during aging.

Effect of the pre-treatment and the drying process on the phenolic composition of raisins produced with a seedless Brazilian grape cultivar.

The grape is an important fruit regarding economic and health benefit parameters, because of its large consumption around the world and their bioactive phenolic compounds. The drying process of BRS Morena grapes, whether pre-treated or not with olive oil for producing raisins, resulted in qualitative and quantitative changes in their phenolic composition (anthocyanins, flavonols, stilbenes, hydroxycinammic acid derivatives, flavan-3-ols and proanthocyanidins). The raisins with the pre-treatment preserved more anthocyanins and proanthocyanidins than the raisins not pre-treated. Moreover, the total dehydration time accelerated by approximately 40% in the raisins pre-treated. Therefore, the production of raisins of BRS Morena grapes pre-treated with olive oil as a natural surfactant constitutes an interesting process from both the industrial and health points of view, because of the remarkable reduction in the processing time and the preservation of high concentrations of flavonoids, which have important claims to health benefits from biological activities.

Application of ultrasound to improve lees ageing processes in red wines.

Ageing on lees (AOL) is a technique that increases volatile compounds, promotes colour stability, improves mouthfeel and reduces astringency in red wines. The main drawback is that it is a slow process. Several months are necessary to obtain perceptible effects in wines. Different authors have studied the application of new techniques to accelerate the AOL process. Ultrasound (US) has been used to improve different food industry processes; it could be interesting to accelerate the yeast autolysis during AOL. This work evaluates the use of the US technique together with AOL and oak chips for this purpose studying the effects of different oenological parameters of red wines. The results obtained indicate an increase of polysaccharides content when US is applied in wine AOL. In addition, total polyphenol index (TPI) and volatile acidity were not affected. However, this treatment increases the dissolved oxygen affecting the volatile compounds and total anthocyanins.

Formation of polymeric pigments in red wines through sequential fermentation of flavanol-enriched musts with non-Saccharomyces yeasts.

Non-Saccharomyces yeasts may contribute to enrich wine aroma while promoting the formation of stable pigments. Yeast metabolites such as acetaldehyde and pyruvate participate in the formation of stable pigments during fermentation and wine aging. This work evaluated the formation of polymeric pigments in red musts added with (+)-Catechin, ProcyanidinB2 and ProcyanidinC1. The non-Saccharomyces yeasts used were Lachancea thermotolerans, Metschnikowia pulcherrima and Torulaspora delbrueckii in sequential fermentation with Saccharomyces cerevisiae and Schizosaccharomyces pombe. Use of Lachancea thermotolerans led to larger amounts of polymeric pigments in sequential fermentation. (+)-Catechin is the flavanol prone to forming such pigments. The species Metschnikowia pulcherrima produced higher concentration of esters and total volatile compounds. The sensory analysis pointed out differences in fruitiness and aroma quality. The results obtained strengthen the fact that metabolites from non-Saccharomyces yeasts may contribute to form stable polymeric pigments while also influencing wine complexity.

Yeast influence on the formation of stable pigments in red winemaking.

The anthocyanin profile of a wine greatly varies over time depending on many factors. In addition to color modifications due to changes in the chemical composition of wine, there may be some influence of the yeast strain used in fermentation. The main aim of this study is to identify and quantify the different ways in which yeast may influence on wine color and its stability, during red winemaking. Hydroxycinnamate decarboxylase activity was measured by the ability to transform the p-coumaric acid (HPLC-DAD). Acetaldehyde (GC-FID) and pyruvic acid (Y15 enzymatic autoanalyser) contents were monitored along fermentation. Stable pigments formation, including vitisins, vinylphenolic pyranoanthocyanins and flavanols-anthocyanins adducts, were analyzed by HPLC-DAD/ESI-MS. Moreover, the ability of adsorbing color molecules by yeasts' cell walls was assessed. It could be concluded that the strain used has substantial influence on the formation of stable pigments, and therefore, proper yeast selection is important to ensure the stability of the wine coloring matter.

Studying the effect of storage conditions on the metabolite content of red wine using HILIC LC-MS based metabolomics.

The main aim of this work was to develop an untargeted normal phase LC-MS method, starting from a targeted method already validated for the analysis of 135 polar metabolites. Since the LC instrument and column were the same, most of the chromatographic conditions remained identical, while the adaptations focused on maintaining the ionic strength of the eluents constant. The sample preparation was simplified and the effectiveness of LC-MS for long batches was evaluated, in order to record the maximum number of metabolites with good chromatographic resolution and the best MS stability and accuracy. The method was applied to study the influence of storage conditions on wine composition. Slightly sub-optimum storage conditions had a major impact on the polar metabolite fingerprint of the red wines analysed and the markers revealed included phenolics, vitamins and metabolites indentified in wine for the first time (4-amino-heptanedioic acid and its ethyl ester).

Vitis thunbergii var. taiwaniana Extracts and Purified Compounds Ameliorate Obesity in High-Fat Diet-Induced Obese Mice.

The increasing prevalence of obesity continues to gain more attention worldwide. In this study, diet-induced obese mice were used to evaluate the antiobesity effects of extracts, fractions, and purified compounds from Vitis thunbergii var. taiwaniana (VTT). The C57BL/6J mice were fed a 5-week high-fat diet (HF) concurrently with ethanol extracts (Et-ext, 80 mg/kg) from roots (R), stems (S), and leaves (L) by oral gavage daily. Only R-Et-ext interventions showed significant weight reduction in mice compared with those in the HF group; however, mouse plasma contents of total cholesterols (TC), total triglycerides (TG) and low-density lipoproteins (LDL) of all three Et-ext intervened groups showed significant reductions compared with those in the HF group. Furthermore, intervention with the ethyl acetate-partitioned fraction (EA-fra, 60 mg/kg) from R-Et-ext but not the n-butanol-partitioned fraction or water fraction from R-Et-ext showed significant weight reduction in mice compared with those in the HF group. The same molecular weights of three resveratrol tetramers, (+)-hopeaphenol, (+)-vitisin A, and (-)-vitisin B, were isolated from the EA-fra of VTT-R. The (+)-vitisin A and fenofibrate (25 mg/kg) but not the (+)-hopeaphenol and (-)-vitisin B interventions showed significant weight reduction in mice compared with those in the HF group. The total feed intake among the HF groups with or without interventions showed no significant differences. The mouse plasma contents of TC, TG, LDL, free fatty acid, and plasma lipase activity of the three resveratrol tetramer-intervened groups showed reductions in the mice compared with those in the HF group. It was proposed that the lipase inhibitory activities of VTT extracts and purified resveratrol tetramers might contribute in part to the antiobesity effect, and these results suggested that VTT may be developed as functional food for achieving antiobesity objectives and requires further investigation.

Anti-α-glucosidase and Anti-dipeptidyl Peptidase-IV Activities of Extracts and Purified Compounds from Vitis thunbergii var. taiwaniana.

Ethanol extracts (Et) from the stem (S) and leaf (L) of Vitis thunbergii var. taiwaniana (VTT) were used to investigate yeast α-glucosidase and porcine kidney dipeptidyl peptidase-IV (DPP-IV) inhibitory activities. Both VTT-Et showed complete α-glucosidase inhibition at 0.1 mg/mL; VTT-S-Et and VTT-L-Et showed 26 and 11% DPP-IV inhibition, respectively, at 0.5 mg/mL. The VTT-Et interventions (20 and 50 mg/kg) resulted in improvements in impaired glucose tolerance of diet-induced obese rats. (+)-Hopeaphenol, (+)-vitisin A, and (-)-vitisin B were isolated from the ethyl acetate fractions of S-Et and showed yeast α-glucosidase inhibition (IC50 = 18.30, 1.22, and 1.02 µM) and porcine kidney DPP-IV inhibition (IC50 = 401, 90.75, and 15.3 µM) compared to acarbose (6.39 mM) and sitagliptin (47.35 nM), respectively. Both (+)-vitisin A and (-)-vitisin B showed mixed noncompetitive inhibition against yeast α-glucosidase and porcine kidney DPP-IV, respectively. These results proposed that VTT extracts might through inhibitions against α-glucosidase and DPP-IV improve the impaired glucose tolerance in diet-induced obese rats.

Pre-drying and submerged cap winemaking: Effects on polyphenolic compounds and sensory descriptors. Part II: BRS Carmem and Bordô (Vitis labrusca L.).

Brazilian wine production is dominated by the use of American grape cultivars (Vitis labrusca L.) and their hybrids. In this context, this study analyzed the phenolic composition and sensory profile of Bordô and BRS Carmem red wines elaborated from traditional and two alternative winemaking technologies: grape pre-drying and submerged cap of chaptalized musts. Anthocyanins and pyranoanthocyanins apparently seemed to be affected by the thermal process (60°C), causing their degradation. In addition, the decrease of the concentration of these compounds could be suggested as a result of possible oxidation and hydrolysis reactions of anthocyanin 3-glucosides. Stilbenes were also affected by thermal degradation; however, flavan-3-ols and HCAD seemed to be less affected by the drying process. Submerged cap winemaking resulted in an increase of the anthocyanin and pyranoanthocyanin compounds due to the constant contact between the must and pomace during the alcoholic fermentation. The antioxidant capacity seemed not to be affected by thermal degradation, since the products of Maillard reaction also present antioxidant properties. Pre-dried wines were described as structured due to their higher flavan-3-ols content, and with high color intensity probably due to the formation of Maillard reaction products. The submerged cap wines presented an intense violet hue due to their high anthocyanin derivative concentrations and showed strong correlation with all other classes of the phenolic compounds.