DNP and ATP modulate the pulp softening and breakdown in fresh longan by acting on the antioxidant system and the metabolisms of membrane lipids and cell wall polysaccharides.

Compared to the control longan, DNP treatment elevated pulp breakdown index, reduced the values of pulp firmness, CSP, ISP, cellulose, and hemicellulose by enhancing the activities of PE, PG, Cx, XET, and ß-Gal. Additionally, DNP treatment increased the levels of PLD, lipase, LOX, PA, and SFA, and decreased the values of PC, PI, USFA, U/S, and IUFA, displaying higher cell membrane permeability and more severe cell membrane damage in longan pulp. Furthermore, DNP treatment weakened the levels of SOD, CAT, APX, AsA, GSH, TP, and TF, thereby exacerbating ROS outbreak and MDA production. These results indicate that DNP treatment destroyed the antioxidant system to cause ROS eruption. This disruption further disturbed the metabolisms of membrane lipids and cell wall polysaccharides, leading to the breakdown of cell membrane and cell wall, and eventually aggravated longan pulp softening and breakdown. However, ATP treatment exhibited the opposite effects of DNP treatment.

Longan pulp polysaccharides regulate gut microbiota and metabolites to protect intestinal epithelial barrier.

Longan pulp polysaccharide is a bioactive component with prebiotic activity and intestinal barrier protection. This study aimed to evaluate the influence of digestion and fermentation on the bioavailability and intestinal barrier protection of polysaccharide LPIIa from longan pulp. The molecular weight of LPIIa didn't change significantly after gastrointestinal digestion in vitro. After fecal fermentation, 56.02% of LPIIa was consumed by gut microbiota. The short-chain fatty acid level in LPIIa group was 51.63% higher than that in blank group. LPIIa intake also increased short-chain fatty acid production and G-protein-coupled receptor 41 expression in the colon of mice. Moreover, LPIIa improved the relative richness of Lactobacillus, Pediococcus, and Bifidobacterium in colon content. Compared to LPIIa, fecal fermented LPIIa better protected intestinal epithelial barrier by increasing Zonula occludens-1 expression. These results provided an important basis for the design of functional food based on longan polysaccharides to prevent intestinal barrier damage related diseases.

DNP and ATP regulate the breakdown occurrence in the pulp of Phomopsis longanae Chi-infected longan fruit through modulating the metabolism of membrane lipid.

This study aimed to illustrate how DNP and ATP affected the pulp breakdown occurrence in P. longanae-infected longan and their relationship with the membrane lipid metabolism. Compared with P. longanae-inoculated samples, the pulp of DNP-treated P. longanae-infected longan exhibited higher cellular membrane permeability, breakdown index, activities of PI-PLC, PLD, PC-PLC, LOX, and lipase, and values of SFAs, PA, and DAG, while lower levels of PI, PC, USFAs, IUFA and U/S. However, the opposite findings were observed in ATP-treated P. longanae-infected longan. The data manifested that DNP-increased the pulp breakdown occurrence in P. longanae-inoculated samples was due to the elevated MLDEs activities that reduced the contents of phospholipids (PI, PC) and USFAs, disrupting the cell membrane structures. Nevertheless, ATP decreased the pulp breakdown occurrence in P. longanae-inoculated samples, which was ascribed to the reduced MLDEs activities that raised phospholipids (PI, PC) and USFAs contents, thus maintaining the cell membrane structures.

DNP and ATP modulate the developments of pulp softening and breakdown in Phomopsis longanae Chi-infected fresh longan through regulating the cell wall polysaccharides metabolism.

Compared with P. longanae-infected longan, 2, 4-dinitrophenol (DNP) treatment for P. longanae-infected longan displayed the lower levels of pulp firmness, cell wall materials, ionic-soluble pectin, covalent-soluble pectin, hemicellulose, or cellulose, but the higher amount of water-soluble pectin, the higher activities of cell wall-degrading enzymes (CWDEs) (PG, ß-Gal, PME, Cx, and XET), and the higher transcript levels of CWDEs-related genes (DlPG1, DlPG2, Dlß-Gal1, DlPME1, DlPME2, DlPME3, DlCx1, and DlXET30). On the contrary, ATP treatment for P. longanae-infected longan exhibited opposite effects. The above results imply that DNP accelerated P. longanae-induced pulp softening and breakdown of fresh longan, which was because DNP up-regulated the transcript levels of CWDEs-related genes, enhanced the CWDEs activities, and accelerated the degradation of cell wall polysaccharides (CWP). However, ATP suppressed longan pulp softening and breakdown caused by P. longanae, because ATP down-regulated the transcript levels of CWDEs-related genes, lowered the CWDEs activities, and reduced the CWP degradation.

DNP and ATP regulate the pulp breakdown development in Phomopsis longanae Chi-infected longan fruit through modulating the ROS metabolism.

Compared with the P. longanae-infected longan, the DNP-treated P. longanae-infected fruit represented a higher pulp breakdown index, a higher O2 -. production rate, and a higher MDA content, but the lower activities of APX, SOD and CAT, the lower transcript levels of DlAPX6, DlSOD1, DlSOD2, DlSOD3 and DlCAT1, the lower values of AsA, GSH, flavonoid and total phenolics, a lower scavenging ability of DPPH radical, and a lower value of reducing power. Whereas, the ATP-treated P. longanae-infected samples showed the contrary results. The above findings indicated that the DNP-promoted the pulp breakdown in P. longanae-infected longan was because DNP weakened the capacity of scavenging ROS, raised the O2 -. level, and accelerated the membrane lipids peroxidation. However, the ATP-suppressed the pulp breakdown in P. longanae-infected longan was because ATP improved the capacity of scavenging ROS, reduced the O2 -. level, and reduced the membrane lipids peroxidation.

Structural elucidation, anti-inflammatory activity and intestinal barrier protection of longan pulp polysaccharide LPIIa.

In this study, LPIIa, a purified polysaccharide from longan pulp, was isolated. Its anti-inflammatory activity and intestinal barrier protection were investigated with LPS-treated co-culture model of Caco-2 cells and RAW 264.7 macrophages. The average molecular weight LPIIa was 159.3 kDa. Its detailed structure was shown below. The backbone of LPIIa was composed of (1→3,4)-linked-α-Rhap, (1→4)-linked-ß-Galp, (1→6)-linked-ß-Galp, and (1→3,6)-linked-ß-Galp, with branches at the O-4 of Rha and O-3 of Gal, consisting of side chains of α-Araf, ß-Galp, and α-Glcp. In LPS-induced RAW 264.7 macrophages, LPIIa suppressed the production of inflammatory mediators, including TNF-α, IL-6, NO, and PGE2, and inhibited iNOS and COX-2 gene expression. In addition, LPIIa attenuated intestinal tight-junctional channel protein Claudin-2 expression and increased tight-junctional barrier protein ZO-1 expression in Caco-2 cells. Knowing the structural features and activities of longan polysaccharide gives insights into longan polysaccharide application as an anti-inflammatory agent or adjuvant in curing the intestinal inflammation.

Longan pulp polysaccharides relieve intestinal injury in vivo and in vitro by promoting tight junction expression.

The integrity of the intestinal mucosal barrier is important for the health of the host. In this study, longan pulp polysaccharides (LP) prevented the intestinal mucosal injury by increasing the expression of mucin 2, tight junction proteins zonulae occludens-1 (ZO-1), claudin-1, claudin-4, and adherens junction E-cadherin in cyclophosphamide-treated mice. To further identify the principle bioactive component of LP, four acidic polysaccharides (LPIa, LPIIa, LPIIIa, and LPIVa) were purified, and their intestinal protection activity in vitro was compared. LPIa, LPIIa, and LPIIIa displayed an ability to increase mRNA expression of ZO-1, claudin-1, occludin, and E-cadherin in differentiated Caco-2 cells, especially LPIa. LPIa has specific structure characteristics: porous surface structure, a high molecular weight (1.47 × 105 Da), and two specific glycosidic linkages of α-Araf-(1→ and →5)-α-Araf-(1→. These structure characteristics might primarily contribute to greater intestinal barrier protective effect of LPIa.

Effects of a Lysine-Involved Maillard Reaction on the Structure and In Vitro Activities of Polysaccharides from Longan Pulp.

The effects of amino acid-involved Maillard reactions (MRs) on the structure and activities of longan pulp polysaccharides (LPs), which were heteropolysaccharides mainly composed of glucose, galactose, mannose, rhamnose, glucuronic acid, ribose, and galacturonic acid, were investigated. The changes of browning degree and molecular weight (Mw) distribution in the MR systems containing LPs and amino acids (lysine, proline, or glycine) indicated that lysine was more active in conjugating with LPs. The MR-modified LPs (MLPs) obtained via a 4 h MR between LPs and lysine showed obvious structural differences from LPs. Specifically, particle-like LPs contained 94% fractions with a Mw less than 7.07 kDa, by contrast, network-like MLPs contained 45% fractions with a Mw larger than 264.1 kDa. Moreover, MLPs showed stronger radical scavenging abilities and macrophage immunostimulating effects, but weaker cancer cell growth-inhibitory abilities. The results indicate that the amino acid-involved MR is a promising method to modify native polysaccharides for better biological properties.

Investigation of the Maillard Reaction between Polysaccharides and Proteins from Longan Pulp and the Improvement in Activities.

The purpose of this study was to investigate the Maillard reaction between polysaccharides and proteins from longan pulp and the effects of reaction on their in vitro activities. The polysaccharide-protein mixtures of fresh longan pulp (LPPMs) were co-prepared by an alkali extraction-acid precipitation method. They were then dry-heated under controlled conditions for monitoring the characterization of the Maillard reaction by the measurement of the free amino group content, ultraviolet-visible spectrum, Fourier transform infrared spectrum and molecular weight distribution. All the physicochemical analyses indicated the development of the Maillard reaction between polysaccharides and proteins. The in vitro activity evaluation indicated that the Maillard reaction could effectively enhance the antioxidant, antitumor and immunostimulating activities of LPPMs. The enhancement of 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity and ferric reducing antioxidant power displayed both a positive correlation with the reaction time (p < 0.05). LPPMs dry-heated for three days obtained relatively strong inhibitory activity against HepG2 cells and SGC7901 cells, as well as strong immunostimulating effects on the nitric oxide production and tumor necrosis factor α secretion of macrophages. Maillard-type intermacromolecular interaction is suggested to be an effective and controllable method for improving the functional activities of polysaccharides and proteins from longan pulp.

Ultrahigh pressure-assisted enzymatic extraction maximizes the yield of longan pulp polysaccharides and their acetylcholinesterase inhibitory activity in vitro.

An extraction method employing ultrahigh pressure-assisted enzymatic treatment was developed and optimized by response surface methodology to increase the yield of longan pulp polysaccharides (LP-UE). A maximum polysaccharides yield of 8.55% was obtained under the optimal conditions of 407MPa ultrahigh pressure maintained for 6min with an enzyme to pretreated material ratio of 1:100, an enzymolysis time of 1.7h and a water to pretreated material ratio of 42ml/g. Subsequently, the physicochemical properties and acetylcholinesterase (AChE) inhibitory activity of LP-UE were compared to those of longan pulp polysaccharides (LP) extracted by hot water (LP-H), ultrahigh pressure (LP-U) or enzymatic treatment (LP-E). Results demonstrated that the extraction yield, hexuronic acid content and AChE inhibitory activity of LP-UE was the highest among the four LP samples. LP-UE was primarily made up of arabinose, glucose, and galactose and was linked mainly by ß-type glycosidic linkage. The FTIR spectrum of LP-UE was very similar to those of LP-H, LP-U, and LP-E. In summary, ultrahigh pressure-assisted enzymatic treatment is a more efficient technique for extracting LP with considerable improvement of both yield and memory enhancement function.

Extraction, molecular weight distribution, and antioxidant activity of oligosaccharides from longan (Dimocarpus Longan Lour.) pulp.

Ultrasonic-microwave synergistic extraction (UMSE) was optimized for the extraction of oligosaccharides from longan pulp (OLP). Box-Behnken design was used to evaluate the effects of temperature (35-55°C), ultrasonic time (5-25 min), and water to material ratio (10-30 mL/g) on the extraction efficiency of crude OLP. A regression model was developed and its validity was statistically demonstrated. Significant interaction between temperature and water to material ratio was observed. The following optimal conditions for the extraction yield of crude OLP were determined: extraction temperature 55°C, ultrasonic time 18.52 min, and water to material ratio 10 mL/g. The extracted OLP were purified for the determination of molecular weight distribution and antioxidant activity. Results of matrix-assisted laser desorption ionization time-of-flight mass spectrometry revealed that the molecular weight distribution of the purified OLP ranged from m/z 495.138 to 795.511. The purified OLP exhibited a dose-dependent behavior in 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity.

Microanalysis, Pharmacokinetics and Tissue Distribution of Polysaccharide-Protein Complexes from Longan Pulp in Mice.

A high performance size exclusion-fluorescence detection (HPSEC-FD) method combined with fluorescein isothiocyanate (FITC) prelabeling was established for the microanalysis of polysaccharide-protein complexes from longan pulp (LPP). FITC-labeled LPP (LPPF) was fractionated by gel filtration chromatography. The weight-average molecular weight and FITC substitution degree of LPPF were 39.01 kDa and 0.20%, respectively. The HPSEC-FD calibration curves linear over the range of 1-200 µg/mL in mouse plasma, spleen and lung samples with correlation coefficients greater than 0.995. The inter-day and intra-day precisions of the method were not more than 6.9%, and the relative recovery ranged from 93.7% to 106.4%. The concentration-time curve of LPPF in plasma following intravenous (i.v.) administration at 40 mg/kg body weight well fitted to a two-compartment model. LPPF rapidly eliminated from plasma according to the short half-lives (t1/2α=2.23 min, t1/2ß=39.11 min) and mean retention times (MRT0-t=1.15 h, MRT0-∞=1.39 h). After administration over 5 to 360 min, the concentration of LPPF in spleen homogenate decreased from 7.41 to 3.68 µg/mL; the concentration in lung homogenate decreased from 9.08 to 3.40 µg/mL. On the other hand, the increasing concentration of LPPF fraction with low molecular weight in heart homogenate was observed.

Prebiotic activity score and bioactive compounds in longan (Dimocarpus longan Lour.): influence of pectinase in enzyme-assisted extraction.

The optimal extraction of bioactive compounds from longan fruit pulp using Pectinex® Ultra SP-L pectinase hydrolysis of the fruit homogenate was evaluated. The highest degree of hydrolysis (DH), as determined by the amount of reducing sugars released from the longan pulp, was obtained at a pectinase concentration of 2.5 % (v/w) (257 polygalacturonase units/g fruit) for 4 h. The level of bioactive compounds obtained from the pectinase-treated longan pulp increased with increasing DH to a maximum at the highest DH (21 %) obtained, with an antioxidant activity of 0.083 EC50 µg fresh mass (FM)/µg diphenyl-(2,4,6-trinitrophenyl)iminoazanium and 92.7 µM Trolox equivalent/g FM, respectively. The total phenolic and flavonoid contents in the 21 % DH extract were 196.0 mg gallic acid equivalents/g FM and 19.6 mg catechin equivalents/g FM, respectively. The 21 % DH longan extract showed an enhanced (3.6- to 4.0-fold) inhibition of lipid peroxidation of oil compared to the untreated (0 % DH) extract. In addition, the 21 % DH longan extract had the highest soluble dietary fiber content, which was related to the decreased particle size of 345 µM, and displayed enhanced prebiotic activity scores of 1.69 and 1.44 for Lactobacillus acidophilus La5 and Bifidabacterium lactis Bb12, respectively. Most of the 33 detected volatile compounds differed in their relative proportions after enzymic extraction (15 increased, 15 decreased with three showing no significant change) with the 0 % and 21 % DH hydrolysates exhibiting 25 and 22 different volatile compounds, respectively, with 11 and eight unique compounds between them, respectively.

Structural features and immunomodulatory activities of polysaccharides of longan pulp.

An aqueous extract of polysaccharides from longan pulp was chromatographed on a DEAE anion-exchange column to yield four fractions (LPI-IV). Immunomodulatory activities of these polysaccharides were also evaluated in vitro. The purified products, neutral polysaccharide LPI, polysaccharide-protein complex LPII and acidic polysaccharides LPIII and LPIV, exhibited conspicuous differences in their monosaccharide composition, molecular mass and glycosidic linkages. Except for LPI, the other three significantly stimulated lymphocyte proliferation in the dose range of 100-400µg/mL compared with the normal control (P<0.05), and might electively stimulate B cells, but not T cells. Furthermore, their stimulations on normal/lipopolysaccharide-induced proliferation and depressions on concanavalin A-induced proliferation could be ordered as LPIII>LPIV>LPII>LPI. All the fractions had the optimal dose of 100µg/mL on enhancing macrophage phagocytosis. Among them, LPII had the considerable yield and activity for exploiting as a potential immunoadjuvant.