Apoptosis-mediated anticancer activity in prostate cancer cells of a chestnut honey (Castanea sativa L.) quinoline-pyrrolidine gamma-lactam alkaloid.

Prostate cancer (PCa) is the most common malignancy in men and represents the second leading cause of cancer deaths in Western countries. PCa is initially androgen-dependent, however, this tumor inevitably progresses as castration-resistant prostate cancer (CRPC), which represents the most aggressive phase of the pathology. In this work, in two CRPC cell lines (DU145 and PC3), we studied the in vitro inhibitory properties of the tryptophan-derived endogenous metabolite kynurenic acid (KYNA) and of the lactam form of 3-2'-pyrrilonidinyl-kynurenic acid (3-PKA-L), alkaloids usually present in combination in chestnut honey. Cytotoxicity was evaluated by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cell colony formation assay, and Western blot analysis of the major mediator proteins involved in apoptotic processes. In all experiments, KYNA was scarcely or not active while 3-PKA-L showed anticancer activity in the high concentration range (0.01 mM - 1 mM) from 24 to 72 h. The results obtained showed that cell death was induced by extrinsic apoptotic pathway, by cell morphological changes and reduction of cell colonies number. These novel results represent the first promising step to the accurate description of 3-PKA-L cytotoxic effect, not observed with KYNA, paving the way to the search of new anticancer agents, as well as to the better understanding of the physiopathological role of this interesting natural product.

Supplementation of diets with tannins from Chestnut wood or an extract from Stevia rebaudiana Bertoni and effects on in vitro rumen fermentation, protozoa count and methane production.

The aim of the trial was to evaluate the effect of dietary additions of Stevia rebaudiana Bertoni extract (SB) and Chestnut wood tannin (CWT) on the in vitro rumen fermentability, protozoal population and methane yield. Both plant products were tested at 3 different levels of inclusion (0.75, 1.50 and 3.00% of incubated dry matter, DM) in a total mixed ration (TMR) for ruminants by using rumen batch culture systems and a rumen inoculum collected from sheep. Total volatile fatty acid concentration, their proportions and gas production were not modified by the plant extracts inclusion, except a significant linear increment of gas production at 24 hr for SB (p = .049). Ammonia concentration decreased (p < .05) of about 17% when 1.50 or 3.00% of CWT were included into TMR. Rumen protozoa population was depressed by the SB inclusion (p = .002) with a maximum reduction of 40% at the highest SB dosage, whereas CWT negatively affected total protozoa counts (-19%) only at the dose of 3.00%. In vitro DM and NDF degradability were not affected by the supplementation of SB and CWT, as well as the methane yield. Thus, the addition of SB and CWT decreases the in vitro protozoa population of the rumen with different intensity and without effects on fermentation parameters, apart from a reduction of nitrogen degradability caused by CWT. Despite the effect on protozoa, no decreasing effect on methane production was detected.

Development of a natural preservative obtained from male chestnut flowers: optimization of a heat-assisted extraction technique.

The aim of this work was to optimize the conditions for the extraction of phenolic compounds (PC) from male chestnut flowers using heat-assisted extraction in developing extracts rich in PC for potential industrial application as a natural ingredient. The study conditions of time (t), temperature (T), solvent (S, water-ethanol mixtures) and solid-to-liquid ratio (S/L) were optimized. The responses used were obtained from the quantification of the fourteen major individual PC identified by HPLC-DAD-ESI/MS (seven hydrolysable tannins and seven flavonoids). The recovery of hydrolysable tannins was higher than that of flavonoids, with trigalloyl-HHDP-glucoside being the major one. The conditions that maximized the PC content were t = 20.0 ± 37.7 min, T = 25.0 ± 5.7 °C, S = 0.0 ± 8.7% ethanol and S/L = 82.8 g L-1, producing an extract with 86.5 mg PC g-1 of extract. The results highlight the potential of valorising chestnut flower agro-residues as a productive source of PC for the development of bio-based ingredients for food/pharmaceutical/cosmeceutical industrial applications able to compete with synthetic compounds.

Comparison of Physicochemical Properties of Starches from Nine Chinese Chestnut Varieties.

Chestnut is a popular food in many countries and is also an important starch source. In previous studies, physicochemical properties of starches have been compared among different Chinese chestnut varieties growing under different conditions. In this study, nine Chinese chestnut varieties from the same farm were investigated for starch physicochemical properties to exclude the effects of growing conditions. The dry kernels had starch contents from 42.7 to 49.3%. Starches from different varieties had similar morphologies and exhibited round, oval, ellipsoidal, and polygonal shapes with a central hilum and smooth surface. Starch had bimodal size distribution and the volume-weighted mean diameter ranged from 7.2 to 8.2 µm among nine varieties. The starches had apparent amylose contents from 23.8 to 27.3% but exhibited the same C-type crystalline structure and similar relative crystallinity, ordered degree, and lamellar structure. The gelatinization onset, peak, and conclusion temperatures ranged from 60.4 to 63.9 °C, from 64.8 to 68.3 °C, and from 70.5 to 74.5 °C, respectively, among nine starches; and the peak, hot, breakdown, final, and setback viscosities ranged from 5524 to 6505 mPa s, from 3042 to 3616 mPa s, from 2205 to 2954 mPa s, from 4378 to 4942 mPa s, and from 1326 to 1788 mPa s, respectively. The rapidly digestible starch, slowly digestible starch, and resistant starch ranged from 2.6 to 3.7%, from 5.7 to 12.7%, and from 84.4 to 90.7%, respectively, for native starch, and from 79.6 to 89.5%, from 1.3 to 3.8%, and from 7.1 to 17.4%, respectively, for gelatinized starch.

Isolation of quercetin and mandelic acid from Aesculus indica fruit and their biological activities.

BACKGROUND: In this study Aesculus indica fruit was subjected to isolation of phytochemicals. Two antioxidants quercetin and Mandelic acid were isolated in pure state. The free radical scavenging and acetyl choline esterase inhibitory potential of the crude extract and sub fractions were also determined. RESULTS: The antioxidant capacity of crude extract, fractions and isolated compounds were determined by DPPH and ABTS methods. Folin-Ciocalteu reagent method was used to estimate the total phenolic contents and were found to be 78.34 ± 0.96, 44.16 ± 1.05, 65.45 ± 1.29, 37.85 ± 1.44 and 50.23 ± 2.431 (mg/g of gallic acid) in crude extract, ethyl acetate, chloroform, n-hexane and aqueous fractions respectively. The flavonoid concentration in crude extract, ethyl acetate, chloroform, n-hexane and aqueous fraction were; 85.30 ± 1.20, 53.80 ± 1.07, 77.50 ± 1.12, 26.30 ± 1.35 and 37.78 ± 1.25 (mg/g of quercetin) respectively. The chloroform fraction was more potent against enzymes, acetyl choline esterase and butyryl choline esterase (IC50 = 85 and 160 µg/ml respectively). The phenolic compounds in the crude extract and fractions were determined using HPLC standard method. Chlorogenic acid, quercetin, phloroglucinol, rutin, mandelic acid and hydroxy benzoic acid were detected at retention times 6.005, 10.062, 22.623, 30.597, 35.490 and 36.211 in crude extract and different fractions. The ethyl acetate fraction was rich in the targeted compounds and was therefore subjected to column isolation. The HPLC chromatogram of isolated compounds showed single peak at specified retention times which confirms their isolation in pure state. The isolated compounds were then characterized by FTIR and NMR spectrophotometric techniques. CONCLUSION: The Aesculus indica fruit extracts showed antioxidant and anticholine esterase inhibitory potentials. Two bioactive compounds were isolated in the pure form ethyl acetate fraction. From results it was concluded that the fruit of this plant could be used to minimize oxidative stress caused by reactive oxygen species.

Triterpene saponins from Billia rosea.

Five previously undescribed triterpene saponins, billiosides A-E, and a known analogue, were isolated from the seeds of Billia rosea (Planch. & Linden) C. Ulloa & P. Jørg. Their structures were elucidated on the basis of extensive 1D and 2D NMR experiments (1H, 13C, DEPT, COSY, TOCSY, NOESY, ROESY, HSQC, and HMBC) and mass spectrometry as (3ß,21ß,22α)-3-[(2-O-ß-D-glucopyranosyl-O-[α-L-arabinopyranosyl-(1 â†’ 4)]-ß-D-glucopyranosyl)oxy]-21-[((2E,6S)-2,6-dimethyl-6-hydroxyocta-2,7-dienoyl)oxy]-22-(acetyloxy)-24-hydroxyolean-12-en-28-oic acid, (3ß,21ß,22α)-3-[(2-O-ß-D-galactopyranosyl-ß-D-glucopyranosyl)oxy]-21,22-dihydroxyolean-12-en-28-yl O-α-L-arabinopyranosyl-(1 â†’ 4)-ß-D-glucopyranoside, (3ß,21ß,22α)-3-[(2-O-ß-D-galactopyranosyl-O-[α-L-arabinopyranosyl-(1 â†’ 4)]-ß-D-xylopyranosyl)oxy]-21,22-dihydroxyolean-12-en-28-yl O-ß-D-glucopyranoside, (3ß,21ß,22α)-3-[(2-O-ß-D-galactopyranosyl-O-[α-L-arabinopyranosyl-(1 â†’ 4)]-ß-D-glucopyranosyl)oxy]-21,22-dihydroxyolean-12-en-28-yl O-ß-D-glucopyranoside, (3ß,21ß,22α)-3-[(2-O-ß-D-galactopyranosyl-O-[α-L-arabinopyranosyl-(1 â†’ 4)]-ß-D-glucopyranosyl)oxy]-21,22-dihydroxyolean-12-en-28-yl O-ß-D-glucopyranosyl-(1 â†’ 6)-ß-D-glucopyranoside, and dipteroside A. Billiosides B and C exhibited moderate effects when tested as hepatic glucose-6-phosphatase inhibitors and as glucose intestinal absorption inhibitors, using in situ rat intestinal segments.

Procyanidins, from Castanea mollissima Bl. shell, induces autophagy following apoptosis associated with PI3K/AKT/mTOR inhibition in HepG2 cells.

Procyanidins from Castanea mollissima Bl. shell (CSPCs) induced autophagy and apoptosis in HepG2 cells and its mechanism remains to be examined. In this paper, autophagy was measured by the lipid modification of light chain-3 (LC3) and the formation of autophagosomes. Hoechst 33258 staining and flow cytometer analysis were used to measure apoptosis. The western blot analysis was used to examine the effects of CSPCs on the expression of LC3, PI3K, phosphorylation of AKT, mTOR, Bcl-2, Bad, Bax, BID and cleaved caspase 3 in HepG2 cells. The results showed that 3-methyladenine (3-MA) and apoptosis inhibitor (Z-VAD) could inhibited the death of HepG2 induced by CSPCs for 48h (150µg/mL). CSPCs induced the accumulation of autophagosomes and microtubule-associated proteins light chain 3-II (LC3-II, a marker of autophagy). P-AKT, PI3K and mTOR were significantly decreased on CSPCs exposure. However, these phenomena were not observed in the group pretreated with the autophagy inhibitor 3-MA and Z-VAD. CSPCs also induced the expression of Bad, Bax and Beclin-1 proteins and decreased the expression of Bcl-2, which was inhibited by 3-MA and Z-VAD. Moreover the apoptotic cell death could be inhibited by 3-MA. In addition, inhibition of LC3-II by siRNA-dependent knockdown attenuated the cleavage of caspase 3. These results suggested CSPCs could trigger autophagy via inhibition of the PI3K/AKT/mTOR signaling pathway, enhanced apoptosis in HepG2 cells which may be associated with the mitochondria-dependent signaling way.

Kinetics of co-pyrolysis of sawdust, coal and tar.

Two coals, sawdust and coal tar were selected to prepare briquettes. Thermogravimetric analyses at three heating rates (i.e. 10, 20 and 30°C/min) and up to 1000°C were carried out with the briquette components. Four blends were prepared and the experimental decomposition profiles were compared with the calculated data taking into account the amount of each component in the blend. No interaction was found when comparing the experimental and calculated decomposition profiles of the blends. Isoconversional models OFW (Ozawa-Flynn-Wall) and KAS (Kissinger-Akahira-Sunose) were used to obtain the activation energies of the blend components. The activation energies obtained were introduced in the Coats-Redfern (CR) model to derive the pre-exponential factors. The thermal decomposition profiles calculated using the kinetic parameters were in good agreement with the experimental results in the case of the briquette components, but worse results were obtained in the case of the blends due to their greater complexity.

Comparative analysis of the antioxidant and anticancer activities of chestnut inner shell extracts prepared with various solvents.

BACKGROUND: The chestnut inner shell (CIS) has long been used as a medicinal herb for strengthening the antioxidant in the cosmetic industry. However, little is known about the antioxidant and anticancer effects of the CIS. RESULTS: The antioxidant and anticancer effects of CIS extracts (CISEs) were investigated by the use of various methods and cancer cell lines, respectively. The total polyphenol content of CISEs using ethanol, methanol, butanol, ethyl acetate and water were 53.30, 43.98, 32.16, 26.79 and 11.53 mg gallic acid equivalents g(-1), respectively. The CISEs using ethanol and methanol exhibited high antioxidant activities in the 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity, ß-carotene bleaching and ferric reducing ability of plasma assays; the effects were equivalent to those of butylated hydroxytoluene. All CISEs at 2.5 mg mL(-1) were shown to have a cytotoxic effect over 50%, and the CISE using ethyl acetate at 0.6 mg mL(-1) was proved to have 90% cytotoxic effect against the tested cancer cells. CONCLUSION: The ethanol and methanol CISEs had potent antioxidant effects, and the ethyl acetate CISE had the highest cytotoxicity. These results suggest that CISEs could be used as functional ingredients for antioxidant and anticancer effects in foods as extraction solvents.

In vitro antiviral activity of chestnut and quebracho woods extracts against avian reovirus and metapneumovirus.

Field evidences have suggested that a natural extract, containing tannins, could be effective against poultry enteric viral infections. Moreover previous studies have shown that vegetable tannins can have antiviral activity against human viruses. Based on this knowledge three different Chestnut (Castanea spp.) wood extracts and one Quebracho (Schinopsis spp.) wood extract, all containing tannins and currently used in the animal feed industry, were tested for in vitro antiviral activity against avian reovirus (ARV) and avian metapneumovirus (AMPV). The MTT assay was used to evaluate the 50% cytotoxic compounds concentration (CC(50)) on Vero cells. The antiviral properties were tested before and after the adsorption of the viruses to Vero cells. Antiviral activities were expressed as IC(50) (concentration required to inhibit 50% of viral cytopathic effect). CC(50)s of tested compounds were > 200 microg/ml. All compounds had an extracellular antiviral effect against both ARV and AMPV with IC(50) values ranging from 25 to 66 microg/ml. Quebracho extract had also evident intracellular anti-ARV activity (IC(50) 24 microg/ml). These preliminary results suggest that the examined vegetable extracts might be good candidates in the control of some avian virus infections. Nevertheless further in vivo experiments are required to confirm these findings.