Extraction, selenylation modification and antitumor activity of the glucan from Castanea mollissima Blume.

The present study aimed to characterize the glucan from C. mollissima Blume fruits and its selenium derivative, then investigate their antitumor activity in vitro. A glucan, designated as CPA, was firstly isolated from the fruits of C. mollissima Blume. Structure analysis indicated that CPA was a linear 1,6-α-D-glucan with the average molecular weight about 2.0 × 103 kDa. The selenylation modification derivative of CPA (sCPA), exhibited a stronger antiproliferative effect on tumor cells than CPA in vitro. CPA and sCPA could induce HeLa cells apoptosis and decrease mitochondrial membrane potential. sCPA could also arrest HeLa cells in S phase, promote reactive oxygen species generation and activate caspase-3 activity in HeLa cells. These results manifest that CPA and sCPA inhibit the proliferation of HeLa cells via different mechanisms, which is meaningful for their potential use as antitumor drugs.

Antioxidative activity of the polyphenols from the involucres of Castanea mollissima Blume and their mitigating effects on heat stress.

Polyphenols extracted from plants have multiple functions in animal production. To explore new sources of tannin-rich extracts, which have potential benefits for animal health, this study focused on the effects of polyphenolic extracts from involucres of Castanea mollissima Blume (PICB) on heat-stressed broilers. In vitro experiments were first performed using intestinal cryptlike epithelial cell line-6 (IEC-6) cells to evaluate the effects of PICB on cell proliferation and antioxidative parameters under normal and heat-stress conditions. Then in vivo experiments were carried out with 2 trials: in trial 1, 400 one-d-old male Arbor Acres (AA) broilers were randomly assigned to 5 groups (4 replicates/group, 20 chicks/replicate): group 1 was a normal control group fed the basic ration; groups 2 to 5 were fed the basic ration supplemented with 0.2% vitamin C and 0.2%, 0.3%, and 0.4% PICB, respectively. Trial 1 lasted 42 d, and growth performance was monitored every week. At the end of the trial, the chicks were sacrificed and sampled. In trial 2, 400 twenty-eight-d-old chicks were randomly assigned to 5 groups as described in trial 1. After 1 week of adaptation, heat stress was applied for 7 consecutive days. On days 3 and 7 of heat stress, the chicks were sacrificed and sampled. The results showed that PICB could stimulate IEC-6 cell proliferation and had strong in vitro antioxidant activity. PICB had no effect on the growth performance and carcass parameters of AA broilers in trial 1, whereas in trial 2, group 4 saw improved growth performance and antioxidant activity compared to the first three groups (P < 0.05). In conclusion, PICB had no effects on the growth performance of IEC-6 cells and AA broilers under normal conditions, whereas it could mitigate heat-stress effects on the growth performance and antioxidant activity of IEC-6 cells and AA broilers, implying that PICB could be used as a suitable additive to improve animal production under heat-stress conditions.

Effect of boron toxicity on pollen tube cell wall architecture and the relationship of cell wall components of Castanea mollissima Blume.

Boron (B) is essential to plant development. However, excessive B is toxic to plants. This research was performed to evaluate the effects of B toxicity on cell wall architecture of Chinese chestnut (Castanea mollissima Blume) pollen tubes with emphasis on the relationship among pectins, cellulose, and callose. Results showed that 0.8 mM H3BO3 inhibited pollen germination and led to abnormal morphology of the pollen tubes. B toxicity also affected the distribution of cell wall components of the pollen tube. In control pollen tube, esterified and acid pectins were distributed unevenly, with the former mainly at the tip and the latter on the distal region. Cellulose was distributed uniformly on the surface with less at the tip; callose reduced gradually from base to sub-tip of the pollen tubes and no callose at the tip of the tube was detected. B toxicity led to the deposition of esterified and acid pectins, cellulose, and callose at the tip of the pollen tube. Results from scanning electron microscopy and transmission electron microscopy showed that B toxicity also altered pollen tube wall ultrastructure. The results from enzymatic treatment illustrated that there existed a close relationship among pectins, cellulose, and callose. B toxicity also altered the relationship. In a word, B toxicity altered deposition and relationship of pectins, cellulose, and callose of pollen tube wall.

Morphological observation and protein expression of fertile and abortive ovules in Castanea mollissima.

Chinese chestnuts (Castanea mollissima Blume.) contain 12-18 ovules in one ovary, but only one ovule develops into a seed, indicating a high ovule abortion rate. In this study, the Chinese chestnut 'Huaihuang' was used to explore the possible mechanisms of ovule abortion with respect to morphology and proteomics. The morphology and microstructure of abortive ovules were found to be considerably different from those of fertile ovules at 20 days after anthesis (20 DAA). The fertile ovules had completely formed tissues, such as the embryo sac, embryo and endosperm. By contrast, in the abortive ovules, there were no embryo sacs, and wide spaces between the integuments were observed, with few nucelli. Fluorescence labelling of the nuclei and transmission electron microscopy (TEM) observations showed that cells of abortive ovules were abnormally shaped and had thickened cell walls, folded cell membranes, condensed cytoplasm, ruptured nuclear membranes, degraded nucleoli and reduced mitochondria. The iTRAQ (isobaric tag for relative and absolute quantitation) results showed that in the abortive ovules, low levels of soluble protein with small molecular weights were found, and most of differently expressed proteins (DEPs) were related to protein synthesis, accumulation of active oxygen free radical, energy synthesis and so on. These DEPs might be associated with abnormal ovules formation.

Pilot Study of the Effects of Polyphenols from Chestnut Involucre on Methane Production, Volatile Fatty Acids, and Ammonia Concentration during In Vitro Rumen Fermentation.

Nutritional strategies can be employed to mitigate greenhouse emissions from ruminants. This article investigates the effects of polyphenols extracted from the involucres of Castanea mollissima Blume (PICB) on in vitro rumen fermentation. Three healthy Angus bulls (350 ± 50 kg), with permanent rumen fistula, were used as the donors of rumen fluids. A basic diet was supplemented with five doses of PICB (0%-0.5% dry matter (DM)), replicated thrice for each dose. Volatile fatty acids (VFAs), ammonia nitrogen concentration (NH3-N), and methane (CH4) yield were measured after 24 h of in vitro fermentation, and gas production was monitored for 96 h. The trial was carried out over three runs. The results showed that the addition of PICB significantly reduced NH3-N (p < 0.05) compared to control. The 0.1%-0.4% PICB significantly decreased acetic acid content (p < 0.05). Addition of 0.2% and 0.3% PICB significantly increased the propionic acid content (p < 0.05) and reduced the acetic acid/propionic acid ratio, CH4 content, and yield (p < 0.05). A highly significant quadratic response was shown, with increasing PICB levels for all the parameters abovementioned (p < 0.01). The increases in PICB concentration resulted in a highly significant linear and quadratic response by 96-h dynamic fermentation parameters (p < 0.01). Our results indicate that 0.2% PICB had the best effect on in-vitro rumen fermentation efficiency and reduced greenhouse gas production.

A novel polysaccharide from Castanea mollissima Blume: Preparation, characteristics and antitumor activities in vitro and in vivo.

A new water-soluble polysaccharide, CMP90, with a molecular weight of 23.9 kDa was isolated from Castanea mollissima Blume and the preliminary structural characteristics and antitumor effects of CMP90 in vitro and in vivo were investigated in the research. CMP90 consists of arabinose, galactose, glucose, xylose and mannose (molar ratio: 0.08:0.11:5.14:0.12:0.08) with α- and ß-anomeric units. The results of in vitro experiments indicated that CMP90 exhibited a significant inhibitory effect on the proliferation of HL-60 cells with typical apoptotic characteristics by inducing cell cycle arrested at G1/M phase. Additionally, the results in vivo suggested CMP90 was able to inhibit the growth of S180 solid tumors via protecting immune organs, improving the levels of serum cytokines (TNF-α, IL-2 and IFN-γ), enhancing the activities of immune cells (macrophages, lymphocytes and NK cells) and inducing cell apoptosis or death. Taken together, these combined data clearly indicated that CMP90 may be used as a potential candidate agent for cancer therapy.

Construction of Pseudomolecules for the Chinese Chestnut (Castanea mollissima) Genome.

The Chinese chestnut (Castanea mollissima Bl.) is a woody nut crop with a high ecological value. Although many cultivars have been selected from natural seedlings, elite lines with comprehensive agronomic traits and characters remain rare. To explore genetic resources with aid of whole genome sequence will play important roles in modern breeding programs for chestnut. In this study, we generated a high-quality C. mollissima genome assembly by combining 90× Pacific Biosciences long read and 170× high-throughput chromosome conformation capture data. The assembly was 688.93 Mb in total, with a contig N50 of 2.83 Mb. Most of the assembled sequences (99.75%) were anchored onto 12 chromosomes, and 97.07% of the assemblies were accurately anchored and oriented. A total of 33,638 protein-coding genes were predicted in the C. mollissima genome. Comparative genomic and transcriptomic analyses provided insights into the genes expressed in specific tissues, as well as those associated with burr development in the Chinese chestnut. This highly contiguous assembly of the C. mollissima genome provides a valuable resource for studies aiming at identifying and characterizing agronomical-important traits, and will aid the design of breeding strategies to develop more focused, faster, and predictable improvement programs.

Synthesis and cytotoxicity of selenium nanoparticles stabilized by α-D-glucan from Castanea mollissima Blume.

A 1,6-α-D-glucan (CPA) with a high molecular weight of 2.0 × 103 kDa was isolated from the fruits of Castanea mollissima Blume. CPA showed good water solubility, low side effects on normal cells, and moderate cytotoxicity on cancer cells. Selenium nanoparticles (SeNPs) possess significant anticancer effects, but poor water solubility and dispersity. Therefore, an effective nanocomposite was designed in order to enhance the stability and anticancer effects of SeNPs decorated with CPA. Water soluble and dispersed selenium nanoparticles (CPA-SeNPs) were fabricated using CPA and Na2SeO3 by redox reaction with ascorbic acid. The average diameter of CPA-SeNPs was 53.7 ±â€¯4.0 with a Se content of approximately 171.4 mg/g. CPA-SeNPs showed a significant anti-proliferative effect on HeLa cells in vitro. Since these NPs could induce apoptosis and S phase arrest. The apoptotic pathway triggered by CPA-SeNPs on HeLa cells was also characterized as induced by reactive oxygen species (ROS) overproduction, mitochondrial dysfunction and activation of caspase-3. This study suggested that CPA-SeNPs could be developed as a potential candidate in cancer treatment, especially against human cervical cancer.

Gene expressions and metabolomic research on the effects of polyphenols from the involucres of Castanea mollissima Blume on heat-stressed broilers chicks.

To study the effects of polyphenolic extract from involucres of Castanea mollissima Blume ( PICB: ), a novel approach using gene expression by real time polymerase chain reaction ( REAL-TIME PCR: ) coupled with metabolomic profiling technique was established to explain the mechanism of PICB on heat-stressed broiler chicks. Four thousand 28-day-old male Arbor Acres (AA) broilers were randomly assigned to 5 groups (4 replicates / group, 20 chicks / replicate), in which group 1 was normal control group fed with basic ration; groups 2, 3, 4, and 5 were fed with the basic ration with a supplementation of 0.2% Vitamin C ( VC: ), or 0.2%, 0.3%, or 0.4% of PICB respectively. After 1 wk of adaptation, heat stress was applied for 7 consecutive days. On d 3 and d 7 of heat stress, the chicks were sacrificed and sampled. The mRNA expression of heat stress protein 70 (HSP70), glutathione peroxidase ( GSH-PX: ), ornithine decarboxylase ( ODC: ), epidermal growth factor ( EGF: ) and epidermal growth factor receptor ( EGFR: ) were detected by real-time PCR using samples from jejunum mucosa. The serum and jejunum mucosa metabolomic profiles of PICB group showing best antioxidative effects and control group at d 3 were studied using the method of the gas chromatography - time of flight mass spectrometry ( GT-TOF-MS: ), followed by principal component analysis and partial least squares-discriminate analysis. Potential biomarkers were found using Student's t-test. The results showed mRNA expressions of HSP70, GSH-Px, ODC, EGF, and EGFR were altered by the supplementation of PICB. PICB exhibited antioxidative and growth promoting effects, and 0.3% PICB supplementation level exhibited the best. Three metabolites in the serum and 5 in the jejunum mucosa were identified as potential biomarkers. They were considered to be in accordance with antioxidative and growth promoting effects of PICB, which involved in the energy metabolism (sorbitol, palmitic acid), carbohydrate metabolism, amino acids metabolism (serine, L-ornithine), glutathione metabolism (glutamate, L-ornithine), GnRH signaling pathway (inositol), etc. These findings provided novel insights into our understanding of molecular mechanism of PICB effects on heat-stressed chicks.