Isolation of Hydrolyzable Tannins from Castanea sativa Using Centrifugal Partition Chromatography.

Castanea sativa wood is a rich source of hydrolyzable tannins, known for their diverse bioactivities. To investigate these bioactive properties further, it is crucial to isolate and characterize hydrophilic compounds effectively. To address this issue, we developed a centrifugal partition chromatography (CPC) method and applied it to an aqueous C. sativa wood extract. We determined the partition coefficients (KD) of the six major compounds using four butanol-/water-based biphasic solvent systems. Initially, we utilized the n-butanol/propanol/water (3:1:4, v/v/v) systems for the first fractionation step. Subsequently, we employed the water/methyl tert-butyl ether/butanol/acetone (8:5:3:4, v/v/v/v) system to fractionate moderately and highly hydrophilic fractions. We calculated the KD values for major compounds of the most hydrophilic fractions using the butanol/ethanol/water (4:1:5, v/v/v) and butanol/isopropanol/water (2:1:3, v/v/v) systems. In total, we isolated 23 compounds through a combination of CPC, size exclusion chromatography, and preparative HPLC. Among these compounds, six have never been previously described. We characterized them by 1D and 2D NMR experiments and high-resolution mass spectroscopy acquisitions.

Investigation into the Anti-Acne Effects of Castanea sativa Mill Leaf and Its Pure Ellagitannin Castalagin in HaCaT Cells Infected with Cutibacterium acnes.

Acne vulgaris is a prevalent skin disorder affecting many young individuals, marked by keratinization, inflammation, seborrhea, and colonization by Cutibacterium acnes (C. acnes). Ellagitannins, known for their antibacterial and anti-inflammatory properties, have not been widely studied for their anti-acne effects. Chestnut (Castanea sativa Mill., C. sativa), a rich ellagitannin source, including castalagin whose acne-related bioactivity was previously unexplored, was investigated in this study. The research assessed the effect of C. sativa leaf extract and castalagin on human keratinocytes (HaCaT) infected with C. acnes, finding that both inhibited IL-8 and IL-6 release at concentrations below 25 µg/mL. The action mechanism was linked to NF-κB inhibition, without AP-1 involvement. Furthermore, the extract displayed anti-biofilm properties and reduced CK-10 expression, indicating a potential role in mitigating inflammation, bacterial colonization, and keratosis. Castalagin's bioactivity mirrored the extract's effects, notably in IL-8 inhibition, NF-κB inhibition, and biofilm formation at low µM levels. Other polyphenols, such as flavonol glycosides identified via LC-MS, might also contribute to the extract's biological activities. This study is the first to explore ellagitannins' potential in treating acne, offering insights for developing chestnut-based anti-acne treatments pending future in vivo studies.

Utilization of Response Surface Methodology in Optimization of Proline Extraction from Castanea sativa Honey.

Proline constitutes approximately 85 % of the amino acid composition of honey. Therefore, the quantitative determination of this amino acid in honey samples is used by many national/international authorities to evaluate the quality of honey types. In this study, it was aimed to achieve maximum proline amino acid extraction from honey samples whose botanical origins were confirmed by melissopalynological analysis. For this reason, based on three different spectrophotometric methods used in the literature for proline analysis, proline extraction was optimized with the Response Surface Method (RSM) and Box-Behnken experimental design. Three independent variables were determined as treatment time (2, 6, and 10 min), treatment temperature (22, 46, and 70 °C), and cooling time (5, 25, and 45 min). As a result of the optimization, it was seen that only significantly effective independent variable on the proline content of honey was the processing temperature. The optimum conditions obtained as a result of the RSM were found to be 2 min for the treatment time, 70 °C for the treatment temperature and 45 min for the cooling time. The composite desirability of the optimum conditions (R2 ) was found to be 1.00. It was determined that the method proposed by International Honey Commission (IHC) is efficient for proline analysis, but it provides more proline extraction by reducing of time from 10 min to 2 min in hold time in boiling water bath only during the extraction step. As a result, the conditions to be used in order to achieve maximum proline extraction with different spectrophotometric methods were determined and optimum values were determined. In addition, since the botanical origin of honey samples significantly affects the proline content of honey, it can be suggested that this study be optimized for different monofloral honey samples as well.

Identification and Quantification of the Major Phenolic Constituents in Castanea sativa and Commercial Interspecific Hybrids (C. sativa x C. crenata) Chestnuts Using HPLC-MS/MS.

Due to the lack of studies on chestnut metabolites, this study was conducted to identify and quantify the major phenolic constituents in chestnuts. Data were compared with the three most commonly grown interspecific hybrids of C. sativa and C. crenata ('Bouche de Betizac', 'Marsol', and 'Maraval') and three "native" accessions of C. sativa. High-performance liquid chromatography coupled with mass spectrometry was used to identify and quantify these compounds. Four dicarboxylic acid derivatives, five hydroxybenzoic acids, nine hydroxycinnamic acids, and three flavanols were identified and quantified, most of them for the first time. Hydroxybenzoic acids were the major phenolic compounds in all chestnut cultivars/accessions, followed by flavanols, dicarboxylic acid derivatives, and hydroxycinnamic acids. Of all the compounds studied, the (epi)catechin dimer was the most abundant in chestnut. The assumption that cultivars from commercial hybrids have a better and different metabolic profile than "native" accessions was refuted.

Proanthocyanins and anthocyanins in chestnut (Castanea mollissima) shell extracts: biotransformation in the simulated gastrointestinal model and interaction with gut microbiota in vitro.

BACKGROUND: Chestnut (Castanea mollissima) shell is rich in flavonoids and our previous studies showed that proanthocyanins and anthocyanins were the two markedly varied flavonoids in chestnut shell extracts (CSE) during digestion. Here, the biotransformation of proanthocyanins and anthocyanins in a simulated gastrointestinal model, and the interactions between non-absorption CSE (NACSE) and gut microbiota in vitro, were investigated by ultra-high-performance liquid chromatography combined with triple-quadrupole mass spectrometry and 16S rRNA sequencing. RESULTS: Chestnut shell was richer in proanthocyanins and anthocyanins, while the loss of proanthocyanins was greater after digestion. Additionally, the content of anthocyanin decreased after gastric digestion but increased after intestinal digestion and remained stable after fermentation. After fermentation, delphinidin-3-O-sambubioside and pelargonidin-3-O-galactoside were newly formed. Furthermore, microbiome profiling indicated that NACSE promoted the proliferation of beneficial bacteria, while inhibiting pathogenic bacteria. CONCLUSION: All these data suggest that CSE may be a promising candidate to protect gut health. © 2023 Society of Chemical Industry.

Castanea sativa Mill. Leaf: UHPLC-HR MS/MS Analysis and Effects on In Vitro Rumen Fermentation and Methanogenesis.

Castanea sativa Mill. (Fagaceae) is a deciduous tree grown for its wood and edible fruits. Chestnut processing produces residues (burs, shells, and leaves) exploitable for their diversity in bioactive compounds in animal nutrition. In fact, plant-specialized metabolites likely act as rumen modifiers. Thus, the recovery of residual plant parts as feed ingredients is an evaluable strategy. In this context, European chestnut leaves from northern Germany have been investigated, proving to be a good source of flavonoids as well as gallo- and ellagitannins. To this purpose, an alcoholic extract was obtained and an untargeted profiling carried out, mainly by means of ultra-high-performance liquid chromatography/high-resolution tandem mass spectrometry (UHPLC-HR MS/MS) techniques. To better unravel the polyphenol constituents, fractionation strategies were employed to obtain a lipophilic fraction and a polar one. This latter was highly responsive to total phenolic and flavonoid content analyses, as well as to antiradical (DPPH● and ABTS+●) and reducing activity (PFRAP) assays. The effect of the alcoholic extract and its fractions on rumen liquor was also evaluated in vitro in terms of fermentative parameter changes and impact on methanogenesis. The data acquired confirm that chestnut leaf extract and the fractions therefrom promote an increase in total volatile fatty acids, while decreasing acetate/propionate ratio and CH4 production.

A Joint Approach of Morphological and UHPLC-HRMS Analyses to Throw Light on the Autochthonous 'Verdole' Chestnut for Nutraceutical Innovation of Its Waste.

Nowadays, chestnut by-products are gaining a lot of interest as a low-cost raw material, exploitable for developing added-value products. This is in line with suitable chestnut by-products' management, aimed at reducing the environmental impact, thus improving the chestnut industry's competitiveness and economic sustainability. In this context, with the aim of valorizing local cultivars of European chestnuts (Castanea sativa Mill.), our attention focused on the Verdole cultivar, which has been characterized by using the UPOV guidelines for its distinctness, homogeneity, and stability. After harvesting, Verdole chestnuts were properly dissected to collect the outer and inner shells, and episperm. Each chestnut part, previously crushed, shredded, and passed through diverse sieves, underwent ultrasound-assisted extraction. The extracts obtained were evaluated for their total phenolic, flavonoid, and tannin content. The antiradical capacity by DPPH and ABTS assays, and the Fe(III) reducing power, were also evaluated. Although all the samples showed dose-dependent antioxidant efficacy, plant matrix size strongly impacted on extraction efficiency. LC-HRMS-based metabolic profiling highlighted the occurrence of different polyphenol subclasses, whose quantitative ratio varied among the chestnut parts investigated. The outer shell was more chemically rich than inner shell and episperm, according to its pronounced antioxidant activity. The polyphenol diversity of Verdole by-products is a resource not intended for disposal, appliable in the nutraceutical sector, thus realizing a new scenario in processing chestnut waste.

Optimization of activated charcoal detoxification and concentration of chestnut shell hydrolysate for xylitol production.

OBJECTIVES: To increase xylose concentration of the chestnut shell hemicellulosic hydrolysate with an acceptable phenolic compound level in order to enhance xylitol production by Candida tropicalis M43. RESULTS: The xylose concentration and total phenolic compound concentration of the hydrolysate were obtained as 33.68 g/L and 77.38 mg gallic acid equivalent/L, respectively by optimization of detoxification parameters and concentration level (60 °C, 115 min contact time, 5.942% (w/v) dosage of activated charcoal, 120 strokes/min shaking rate and 0.2 volume ratio). Xylitol production was achieved in the hydrolysate by using Candida tropicalis M43. The maximum xylitol concentration was 6.30 g/L and productivity, yield and percentage of substrate conversion were calculated as 0.11 g/L h, 19.13% and 97.79%, respectively. In addition, the chestnut shell hydrolysate fortified with xylose and the maximum xylitol concentration increased to 18.08 g/L in the hydrolysate-based medium containing 80 g/L xylose. CONCLUSIONS: Optimizing detoxification conditions with concentration level was found to be useful for enhancing xylitol production. In addition, fortification of the hydrolysate caused a three fold increase in maximum xylitol concentration.

Effect of Hydrolyzable Tannins on Glucose-Transporter Expression and Their Bioavailability in Pig Small-Intestinal 3D Cell Model.

Intestinal transepithelial transport of glucose is mediated by glucose transporters, and affects postprandial blood-glucose levels. This study investigates the effect of wood extracts rich in hydrolyzable tannins (HTs) that originated from sweet chestnut (Castanea sativa Mill.) and oak (Quercus petraea) on the expression of glucose transporter genes and the uptake of glucose and HT constituents in a 3D porcine-small-intestine epithelial-cell model. The viability of epithelial cells CLAB and PSI exposed to different HTs was determined using alamarBlue®. qPCR was used to analyze the gene expression of SGLT1, GLUT2, GLUT4, and POLR2A. Glucose uptake was confirmed by assay, and LC-MS/ MS was used for the analysis of HT bioavailability. HTs at 37 µg/mL were found to adversely affect cell viability and downregulate POLR2A expression. HT from wood extract Tanex at concentrations of 4 µg/mL upregulated the expression of GLUT2, as well as glucose uptake at 1 µg/mL. The time-dependent passage of gallic acid through enterocytes was influenced by all wood extracts compared to gallic acid itself as a control. These results suggest that HTs could modulate glucose uptake and gallic acid passage in the 3D cell model.

Dietary inclusion of chestnut (Castanea sativa) polyphenols to Nile tilapia reared in biofloc technology: Impacts on growth, immunity, and disease resistance against Streptococcus agalactiae.

A feeding trial was carried out to examine the effects of adding chestnut (Castanea sativa) polyphenols (CSP) on the growth, skin mucus and serum immune parameters of Nile tilapia (Oreochromis niloticus). Five experimental diets with inclusion levels of 0, 1, 2, 4, and 8 g kg-1 of CSP were fed to Nile tilapia fingerlings (12.77 ± 0.17 g fish-1) during an eight-week trial. Fish were analyzed on the fourth and eighth week to determine the influences of CSP on growth, skin mucus, and serum immune parameters. Challenging test versus Streptococcus agalactiae was evaluated at the end of the trial. Fish fed with CSP enriched diets displayed a significant increase (P ≤ 0.05) in growth and a decline in feed conversion ratio (P ≤ 0.05). Similarly, skin mucus and serum immune parameters were significantly increased (P ≤ 0.05) in fish fed CSP with respect to the control. The effects were already evident four weeks after the CSP administration. The disease protection test displayed that the fish's survival rate was significantly higher (P < 0.05) in CSP diets over the control. The relative percentage of survival (RSP) was 62.5, 75.0, 58.3, and 37.5 in fish fed diets contained 1, 2, 4, and 8 g kg-1 CSP, respectively. The best effect on growth, immune response, and disease resistance were shown in Nile tilapia fed with a diet supplementation of 2 g kg-1 CSP.

Glucose Uptake-Stimulating Galloyl Ester Triterpenoids from Castanopsis sieboldii.

Using molecular networking-guided isolation, three new galloyl ester triterpenoids (1-3), two new hexahydroxydiphenic acid-conjugated triterpenoids (6 and 7), and four known compounds (4, 5, 8, and 9) were isolated from the fruits and leaves of Castanopsis sieboldii. The chemical structures of 1-3, 6, and 7 were elucidated on the basis of interpreting their NMR, HRESIMS, and ECD spectra. All compounds (1-9) were evaluated for their glucose uptake-stimulating activities in differentiated adipocytes using 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-d-glucose as a fluorescent-tagged glucose probe. Compounds 2 and 9 resulted in a 1.5-fold increase in glucose uptake. Among them, compound 2 from the fruits showed an upregulation of p-AMPK/AMPK ratio in differentiated C2C12 myoblasts to support the mechanism proposed of glucose uptake stimulation.

Effects of feeding a quebracho-chestnut tannin extract on lactating cow performance and nitrogen utilization efficiency.

The effects of feeding a quebracho-chestnut tannin extract mixture on performance and nitrogen (N) utilization were assessed with 36 multiparous lactating Holstein cows (mean ± standard deviation; 706 ± 59 kg of body weight; 126 ± 20 d in milk) randomly assigned to 3 dietary treatments in a randomized complete block design. Following a 2-wk covariate adjustment period, cows were fed their assigned treatment diets for 13 wk. Rice hulls were removed from a total mixed ration with a 54:46 forage:concentrate ratio (% of dry matter; DM), and a tannin extract mixture from quebracho and chestnut trees (2:1 ratio) was included at 0, 0.45, and 1.80% of dietary DM. There was no interaction between dietary treatments and experimental week for the reported measurements except milk lactose percentage. Overall, treatments did not affect milk yield (48.6 ± 7.8 kg/d), fat- and protein-corrected milk (46.1 ± 7.6 kg/d), milk fat content (3.88 ± 0.65%) and yield (1.85 ± 0.38 kg/d), and true protein yield (1.45 ± 0.21 kg/d). However, incremental levels of tannin extracts in the diet produced a linear increase in DM intake (29.2 to 30.9 kg/d) and a linear decrease in kilograms of milk per kilogram of DM intake (1.67 to 1.57 kg/kg) and MUN (12.2 to 10.8 mg/dL). Furthermore, there was a quadratic effect of tannin extracts on milk true protein content (2.96, 3.13, and 3.00% for 0, 0.45, and 1.80% tannin extract, respectively) and a tendency for linear and quadratic response for body weight gain (0.31, 0.16, and 0.44 kg/d for 0, 0.45, and 1.80% tannin, respectively). Intake of N increased linearly (782, 795, and 820 g/d) and N utilization efficiency (milk N/intake N) decreased linearly (0.300, 0.301, and 0.275 for 0, 0.45, and 1.80% tannin, respectively). Relative to the 0% diet, 1.80% tannin extract reduced estimated urinary N excretion by 11%. In this study, adding 0.45% tannin extract to the diet reduced feed efficiency but had a positive effect on milk protein content. Feeding a tannin extract mixture from quebracho and chestnut may reduce environmental labile urinary N excretion without affecting milk yield but at the expense of a lower feed utilization efficiency.

Allelopathic Effects of Castanea henryi Aqueous Extracts on the Growth and Physiology of Brassica pekinensis and Zea mays.

The present study investigated the allelopathic effects of aqueous extracts of Castanea henryi litter on the growth and physiological responses of Brassica pekinensis and Zea mays. Treatment with high concentrations of leaf extract (0.05 g/ml for B. pekinensis and 0.10 g/ml for Z. mays) significantly increased malonaldehyde content and reduced seed germination, seedling growth, chlorophyll content, and the activity levels of antioxidant enzymes. These effects generally increased with increasing extract concentration. However, in Z. mays, low extract concentrations actually promoted seed germination, shoot growth, chlorophyll content, and antioxidant enzyme activity. The allelopathic effects of the various C. henryi extracts decreased as follows: leaf extract > twig extract > shell extract. Eleven potential allelochemicals including rutin, quercetin, luteolin, procyanidin A2, kaempferol, allantoin, propionic acid, salicylic acid, jasmonic acid, methylmalonic acid, and gentisic acid were identified in the leaves of C. henryi which were linked to the strongest allelopathic effects. These findings suggest that the allelopathic effects of C. henryi differ depending on receptor plant species, and that leaves are the most allelopathic litter in C. henryi.

Comparison of Biochemical Constituents and Contents in Floral Nectar of Castanea spp.

Pollination is essential for efficient reproduction in pollinator-dependent crops that rely on the attraction of pollinators to flowers. Especially, floral nectar is considered to be an important factor attracting pollinator like honey bees, but differences among major chestnut species (Castanea crenata, C. mollissima, C. dentata, and C. sativa) are still little explored. This study aims to evaluate the value of honey source by analyzing floral nectar characteristics and comparing the composition of volatile organic compounds (VOCs) that mediate plant-pollinator interaction. In this study, we analyzed nectar samples obtained from male flowers using HPLC and HS-SPME/GC-MS. The five chestnuts showed significant differences between the volume of secreted nectar, free sugar composition, amino acid content and VOCs composition. Furthermore, C. crenata (Japanese cultivar 'Ungi') was revealed to emit the highest total amounts of VOCs and high levels of benzenoid compounds that are generally associated with flower-visiting insects. The sugar content per catkin, which is used to determine the honey yield, was the highest in C. crenata, suggesting that C. crenata 'Ungi' can be highly valued as a honey tree. Therefore, a better understanding of the relationship between pollinator and nectar characteristics of C. crenara could contribute to a prospective honey plant.

Phytochemical Profiles and Cellular Antioxidant Activities in Chestnut (Castanea mollissima BL.) Kernels of Five Different Cultivars.

In this study, the phytochemical profiles, total and cellular antioxidant activities of five different Chinese chestnut (Castanea mollissima BL.) cultivars were analyzed. Phenolics, flavonoids as well as phytochemical compounds in five cultivars of chestnut kernels were determined. Results showed that the free forms played a dominant role in total phenolics, flavonoids and antioxidant activities of all five cultivars of chestnut kernels. The cultivar 'Fyou' showed the highest total and free phenolic contents, 'Heguoyihao' showed the highest total and free flavonoids contents, and 'Chushuhong' showed the highest total and cellular antioxidant activities. Eight phenolic compounds were detected, and chlorogenic acid, gallic acid, and quercetin were shown as three predominant components in all five cultivars. These results provide valuable information which may be a guidance for selection of good chestnut variety to be used as functional food.

Untargeted Characterization of Chestnut (Castanea sativa Mill.) Shell Polyphenol Extract: A Valued Bioresource for Prostate Cancer Cell Growth Inhibition.

Chestnut seeds are used for fresh consumption and for the industrial preparation of derivatives, such as chestnut flour. During industrial processing, large amounts of by-products are generally produced, such as leaves, flowers, shells and burs. In the present study, chestnut shells were extracted by boiling water in order to obtain polyphenol-rich extracts. Moreover, for the removal or non-phenolic compounds, a separation by preparative reverse phase chromatography in ten fractions was carried out. The richest fractions in terms of phenolic content were characterized by means of untargeted high-resolution mass spectrometric analysis together with a dedicated and customized data processing workflow. A total of 243 flavonoids, phenolic acids, proanthocyanidins and ellagitannins were tentatively identified in the five richest fractions. Due its high phenolic content (450.03 µg GAE per mg of fraction), one tumor cell line (DU 145) and one normal prostate epithelial cell line (PNT2) were exposed to increasing concentration of fraction 3 dry extract for 24, 48 and 72 h. Moreover, for DU 145 cell lines, increase of apoptotic cells and perturbation of cell cycle was demonstrated for the same extract. Those outcomes suggest that chestnut industrial by-products could be potentially employed as a source of bioresources.

Assessment of emulsion gels formulated with chestnut (Castanea sativa M.) flour and chia (Salvia hispanica L) oil as partial fat replacers in pork burger formulation.

BACKGROUND: The aim of this work was to evaluate the effect on chemical composition, physico-chemical properties, cooking characteristics, fatty acid profile, lipid oxidation, and sensory acceptability of an oil-in-water emulsion gel that was prepared with chestnut flour, chia oil, gellan gum, and water (CEG), used as a fat replacer in pork burgers. The original mixture was used as a control sample (CS). The other samples were formulated partially replacing pork backfat with 5% of CEG (CEG5%) and 10% of CEG (CEG10%). RESULTS: Proximate analysis of samples showed several differences between samples. The CEG addition was found to be effective for improving the cooking yield while diameter reduction and thickness increase were positively affected. As regards lipid oxidation, in cooked burger, the 2-thiobarbituric acid (TBA) values for CS, CEG5% and CEG10% were 0.46, 0.57, and 0.59 mg malonaldehyde/kg sample, respectively. The linolenic and linolenic acid content of pork burger increased as CEG addition increased. Sensory properties for CS and CEG5% were similar whereas CEG10% showed the highest sensory scores. CONCLUSIONS: A combination of chestnut flour and chia oil could be used as a novel ingredient to develop pork burgers with a better nutritional profile without diminishing their sensory and physico-chemical properties. © 2019 Society of Chemical Industry.

Molecular analysis of semen-like odor emitted by chestnut flowers using neutral desorption extractive atmospheric pressure chemical ionization mass spectrometry.

Knowledge about the chemical composition of floral volatile organic compounds (VOCs) is valuable in biological studies as well as for the flavor, cosmetic, and fragrance industries. The flowers of Chinese chestnut (Castanea mollissima) emit a distinctive semen-like odor; however, the chemical composition and biological role for the semen-like odor of chestnut flowers remain scarcely studied. Herein, we report the floral VOCs and the pollinators of chestnut flowers. A fast method based on a neutral desorption (ND) device coupled to extractive atmospheric pressure chemical ionization mass spectrometry (EAPCI-MS) was developed for the rapid identification of VOCs from freshly collected chestnut flowers without any chemical pretreatment. Chemical identification was performed using high-resolution MS analysis in combination with tandem MS analysis and whenever possible was confirmed by the analysis of standard reference compounds. Twenty volatiles were identified, most of which are nitrogen-containing. Out of the identified volatiles, 1-pyrroline is known to have a semen-like odor and is probably also responsible for the semen-like odor of the chestnut flowers. Four nitrogenous VOCs of chestnut flowers, including 1-pyrroline, 1-piperideine, 2-pyrrolidone, and phenethylamine, were also common in other semen-like odor flowers such as Photinia serrulata, Castanopsis sclerophylla, and Stemona japonica, suggesting similar chemical origin. The main visitors of chestnut flowers were dipteran species, such as Eristalis tenax, Eristalis arvorum, Episyrphus balteatus, Lucilia sericata, Chrysomya megacephala, Chrysochus asclepiadeus, and Adalia bipunctata. Our results suggest that the chestnut flowers and other semen-like odor flowers may present a new type of sapromyophily. This study also indicates that ND-EAPCI-MS provides more sensitive and simpler detection of many VOCs (particularly nitrogen-containing VOCs) than GC-MS and therefore can be used to complement traditional approaches for the higher chemical coverage of VOCs analysis. Graphical abstract ᅟ.

One New Phenolic Compound from Castanea mollissima Shells and its Suppression of HepatomaCell Proliferation and Inflammation by Inhibiting NF-κB Pathway.

Shells of Castanea mollissima (CMS), an agricultural remain and often considered waste from chestnut processing industry, have been proven a resource for traditional Chinese medicine. One new phenol, named castanolB(1), andsix known phenolic compounds (2⁻7) were isolated froma water-soluble extract of CMS. Their chemical structures were determined using preparative HPLC and various spectral analyses, and then were compared to literatures, which indicated the first identification of the seven compounds from C. mollissima. The physicochemical property of compound (2) was also reported for the first time. After antiproliferative screening of compounds (1⁻7) on LPS-induced SMMC-7721 and HepG2 hepatoma cells, castanolB (1) showed the best suppression. CastanolB(1) also significantly induced cell apoptosis. Furthermore, castanolB (1) decreasedsecretion of TNF-α and IL-6. Mechanistically, TLR4⁻NF-κB pathway was inhibited bycastanolB (1) with downregulation of TLR4, IKKß, and NF-κB p65. This study presents a new phenol and shows its profiles of anticancer and anti-inflammation via inhibiting the TLR4⁻NF-κB pathway.

Reductive Metabolism of Ellagitannins in the Young Leaves of Castanopsis sieboldii.

The leaves of Castanopsis sieboldii (Fagaceae) contain characteristic hexahydroxydiphenoyl (HHDP) esters of 28-O-glucosyl 2α,3ß,23,24-tetrahydroxyolean- and urs-12-en-28-oic acids. In this study, uncharacterized substances were detected in the young leaves, which are not observed in the mature leaves. Preliminary HPLC analyses indicated that the substances had dehydro-HHDP (DHHDP) ester groups; however, the esters were unstable and decomposed during extraction. Therefore, the compounds were isolated as their stable phenazine derivatives by extracting the young leaves with acidic aqueous EtOH containing o-phenylenediamine. The structures of the phenazine derivatives indicated that the unstable metabolites of the young leaves were 3,24-DHHDP esters of the abovementioned triterpenes. Extraction of the young leaves with 80% acetonitrile containing reducing agents, ascorbic acid or dithiothreitol afforded the corresponding HHDP esters. Furthermore, heating of the young leaves in 80% acetonitrile also yielded the same HHDP esters as the reduction products. The results suggested that the HHDP esters are reductively produced from DHHDP esters in the young leaves. In addition, the structures of five previously reported triterpene HHDP esters were revised.