Purification, characterization and immunostimulatory effects of polysaccharides from Anemarrhena asphodeloides rhizomes.

The crude polysaccharide was extracted from A. asphodeloides rhizomes and further purified to produce two fractions F1 (50.0%) and F2 (19.6%). The chemical constitutions of the polysaccharides were neutral sugars (51.4%-89.7%), uronic acids (1.0%-30.2%) and sulfate esters (3.4%-8.1%), with various ratios of monosaccharides including rhamnose (1.4%-6.1%), arabinose (7.1%-21.2%), xylose (0.2%-4.8%), mannose (39.9%-79.0%), glucose (6.0%-11.1%) and galactose (2.6%-22.0%). The molecular properties of the polysaccharides were investigated by the HPSEC-UV-MALLS-RI system, revealing the Mw 130.0 × 103-576.5 × 103 g/moL, Rg 87.6-382.6 nm and SVg 0.3-54.3 cm3/g. The polysaccharides stimulated RAW264.7 cells to produce considerable amounts of NO and up-regulate the expression of TNF-α, IL-1 and COX-2 genes. Polysaccharides exhibited the growth inhibitory effects on cancer cells lines of AGS, MKN-28 and MKN-45, in which F2 fraction exhibited prominent bioactivities. The AGS cells treated with F2 experienced condensed cytoplasm, shrinkage of nucleus and chromatin marginalization with the highest number of cells at early-stage apoptosis reaching 54.6%. The inhibitory effect of F2 polysaccharide on AGS cells was through MAPKs and STAT3 signaling pathways. The backbone of the F2 was mainly linked by (1 â†’ 4)-linked mannopyranosyl and (1 â†’ 3)-linked galactopyranosyl. Taken together, the polysaccharide from A. asphodeloides rhizomes could be utilized as medicinal, pharmacological and functional food ingredients.

Studies on chemical composition, rheological and antioxidant properties of pectin isolated from Riang (Parkia timoriana (DC.) Merr.) pod.

Although synthetic antioxidant food additives are widely used in a variety of food products, some of them are suspected of having a noxious effect on human health. As a consequence, much research attention has been focused on developing natural antioxidant compounds from plants. Riang (Parkia timoriana (DC.) Merr.) is known as a traditional medicinal plant in which its various parts have been reported to exhibit antioxidant and numerous biological activities. In this study, pectins from Riang pod husk and pod powder were extracted, and their physico-chemical, rheological, and antioxidant properties were characterized. The extracted pectins showed high uronic acid content (> 65%) and high molecular weight (200-250 kDa) and the yields were approximately 15 and 36%w/w (dry basis), for Riang husk pectin (RHP) and Riang pod powder pectin (RPP), respectively. Furthermore, both pectins were classified as a high methoxyl with their DE of ~66%. Rheological measurements revealed a pseudoplastic behavior above 2% w/v. RHP contained higher content of total phenolics, flavonoids and tannin, compared with RPP. Antioxidant activities of RHP were consequently higher than RPP in all studied assays. The highest antioxidant activities of RHP and RPP, obtained from ABTS assay, were 0.95 and 0.24 mmol Trolox equivalents/g, respectively.

Enzymatic preparation of a low-molecular-weight polysaccharide rich in uronic acid from the seaweed Laminaria japonica and evaluation of its hypolipidemic effect in mice.

Here, we describe a method combining thermo-acid pretreatment and alginate lyase hydrolysis to prepare a low-molecular-weight polysaccharide from the seaweed Laminaria japonica (SP). The in vitro results showed that SP displayed obvious absorption of oil (2.95 g g-1) and cholesterol (21.87 g g-1 at pH 2.0). In addition, the in vivo assessment of SP-related anti-obesity effects in C57BL/6J mice fed a high-fat diet and treated with SP for 8 weeks revealed that SP significantly reduced weight gain and lipid accumulation in white adipose and liver tissues, improved serum lipid profiles, and ameliorated intestinal damage. Moreover, SP activated the AMP-activated protein kinase pathway in liver tissues, downregulated sterol regulatory element-binding protein and fatty acid synthase, and suppressed lipid synthesis. These findings indicated that SP extracted from L. japonica might represent a potent functional food exhibiting anti-obesity effects.

Microwave-Assisted Extraction, Purification, Partial Characterization, and Bioactivity of Polysaccharides from Panax ginseng.

Polysaccharides are a main active substance in Panax ginseng; however, microwave-assisted extraction used to prepare P. ginseng polysaccharides (MPPG) has rarely been reported, and knowledge of the bactericidal activity of P. ginseng polysaccharides remains low. Thus, this study was designed to investigate the extraction of P. ginseng polysaccharides by using two methods-hot water extraction and microwave-assisted extraction-and compare their chemical composition and structure. In addition, their antibacterial and antioxidant activities were also determined. The data implied that P. ginseng polysaccharides extracted by microwave-assisted extraction possessed a higher extraction yield than hot water extraction (WPPG) under optimized conditions, and the actual yields were 41.6% ± 0.09% and 28.5% ± 1.62%, respectively. Moreover, the preliminary characterization of polysaccharides was identified after purification. The WPPG with the molecular weight (Mw) of 2.07 × 105 Da was composed of Man, Rib, Rha, GalA, Glu, Gal, and Arab, and the typical characteristics of polysaccharides were determined by IR spectra. Compared with WPPG, MPPG had a higher Mw, uronic acid content, and Glu content. More importantly, the antioxidant activity of MPPG was higher than WPPG, which was probably ascribed to its highly Mw and abundant uronic acid content. Besides, both of them exhibited high bactericidal activity. These results demonstrate that microwave-assisted extraction is an effective method for obtaining P. ginseng polysaccharides, and MPPG could be applied as an antioxidant and antibacterial agent.

Physicochemical, functional, and biological properties of water-soluble polysaccharides from Rosa roxburghii Tratt fruit.

Water-soluble polysaccharides (RTFP) were extracted from Rosa roxburghii Tratt fruit by hot water method. The physicochemical, functional, and hypoglycemic properties of RTFP were investigated. The results revealed that RTFP mainly contained carbohydrates (63.79 ±â€¯0.73%, g/g), uronic acids (14.8 ±â€¯0.06%, g/g), and proteins (4.10 ±â€¯0.58%, g/g). RTFP was composed of arabinose, galactose, glucose, mannose, xylose, and fucose with molar percentages of 33.8, 37.3, 20.7, 1.74, 3.43, and 2.95%, respectively. Functional analyses indicated that RTFP had good oil-holding capacity, foaming properties, and emulsifying capacity. The rheological results showed that RTFP exhibited typical shear-thinning behavior and viscoelastic properties influenced by sample concentration, temperature and inorganic salt. Additionally, RTFP exhibited favorable inhibitory activities against α-glucosidase in a mixed inhibition type, and against α-amylase in an uncompetitive inhibition type. These results suggest that RTFP can be exploited as a multi-functional additive or hypoglycemic agent in foods, pharmaceuticals and cosmetics.

Effects of polysaccharides from abalone viscera (Haliotis discus hannai Ino) on MGC 803 cells proliferation.

The polysaccharides (AVP) was obtained from abalone (Haliotis discus hannai Ino) viscera, using the alkaline protease to enzymolysis, sevage method and repeated freezing and thawing method to remove protein and hydrogen peroxide method to depigment. The total sugar content was 46.27±1.5% and uronic acid, sulfate radical, hexosamine and protein contents were 17.44±0.22%, 16.98±0.15%, 0.65±0.02% and 1.64±0.13% in AVP respectively. The main monosaccharide compositions of AVP were d-galactose, d-xylose, d-mannose, d-glucose and d-glucuronic acid. MTT assay showed AVP had a significant anti-tumor activity to gastric carcinoma cells, especially to MGC 803, while it had no influence upon proliferation of normal stomach cells GES 1. The results of Morphological changes, cell migration ability and AO/EB staining indicated that MGC803 cells underwent apoptosis in a dose-dependent manner induced by AVP. Moreover, the western blotting results showed that the expressions of survivin, Bcl-2 and VEGF were decreased, while the expression of Bax and p53 were increased in a dose-dependent manner of AVP. The results suggested that AVP might be a potential anti-tumor agent securely and naturally.

Unexpected features of exponentially growing Tobacco Bright Yellow-2 cell suspension culture in relation to excreted extracellular polysaccharides and cell wall composition.

This article presents a new insight about TBY-2 cells; from extracellular polysaccharides secretion to cell wall composition during cell suspension culture. In the medium of cells taken 2 days after dilution (end of lag phase), a two unit pH decrease from 5.38 to 3.45 was observed and linked to a high uronic acid (UA) amount secretion (47.8%) while, in 4 and 7 day-old spent media, pH increased and UA amounts decreased 35.6 and 42.3% UA, respectively. To attain deeper knowledge of the putative link between extracellular polysaccharide excretion and cell wall composition, we determined cell wall UA and neutral sugar composition of cells from D2 to D12 cultures. While cell walls from D2 and D3 cells contained a large amount of uronic acid (twice as much as the other analysed cell walls), similar amounts of neutral sugar were detected in cells from lag to end of exponential phase cells suggesting an enriched pectin network in young cultures. Indeed, monosaccharide composition analysis leads to an estimated percentage of pectins of 56% for D3 cell wall against 45% D7 cell walls indicating that the cells at the mid-exponential growth phase re-organized their cell wall linked to a decrease in secreted UA that finally led to a stabilization of the spent medium pH to 5.4. In conclusion, TBY-2 cell suspension from lag to stationary phase showed cell wall remodeling that could be of interest in drug interaction and internalization study.

Minor polysaccharidic constituents from the red seaweed Hypnea musciformis. Appearance of a novel branched uronic acid.

Two polysaccharide fractions isolated from Hypnea musciformis after room temperature- and hot water extraction, soluble after KCl precipitation of the more abundant carrageenans, were subfractionated by ion-exchange chromatography eluting with increasing concentrations of NaCl. The lowest NaCl concentration (0.2M) eluted agarans. The dl-hybrids (or mixtures) eluted at intermediate concentrations of NaCl. The d/l-galactose ratio and the sulfate proportion increased with the NaCl concentration. Different types of substitution were present, mainly at C-3 with sulfate, Xyl and methylated Gal stubs, as well as low amounts of 3,6-AnGal. A novel constituent, identified as 3-C-carboxy-d-erythrose1 in its ß-furanosic form, was found linked to C-6 of ß-Gal units. A search carried out in other species like Iridaea undulosa and Kappaphycus alvarezii also revealed the same constituent. Finally, the late-eluting fractions were mostly carrageenans, with a structure consistent with that of a κ/ι/ν-carrageenan hybrid.

Cell wall glycopolymers of Streptomyces albus, Streptomyces albidoflavus and Streptomyces pathocidini.

The cell wall glycopolymers of three strains of Streptomyces albus and the type strain of Streptomyces pathocidini were investigated. The structures of the glycopolymers were established using a combination of chemical and NMR spectroscopic methods. The cell wall of S. albus subsp. albus VKM Ac-35(T) was found to be comprised of three glycopolymers, viz. unsubstituted 1,5-poly(ribitol phosphate), 1,3-poly(glycerol phosphate) substituted with ß-D-glucopyranose, and the major polymer, a 3-deoxy-D-glycero-D-galacto-non-2-ulosonic acid (Kdn)-teichulosonic acid: ß-D-Glcp-(1 â†’ 8)-α-Kdnp-(2[(→6)-ß-D-Glcp-(1 â†’ 8)-α-Kdnp-(2 â†’] n 6)-ß-D-Glcp-(1 â†’ 8)-ß-Kdnp-(2-OH, where n ≥ 3. The cell walls of 'S. albus' J1074 and 'S. albus' R1-100 were found to contain three glycopolymers of identical structures, viz. unsubstituted 1,3- and 2,3-poly(glycerol phosphates), and the major polymer, a Kdn-teichulosonic acid with an unusual structure that has not been previously described: ß-D-Galp-(1 â†’ 9)-α-Kdnp-(2[(→3)-ß-D-Galp-(1 â†’ 9)-α-Kdnp-(2 â†’] n 3)-ß-D-Galp-(1 â†’ 9)-ß-Kdnp-(2-OH, where n ~ 7-8. The cell wall of S. pathocidini (formerly S. albus subsp. pathocidicus) VKM Ac-598(T) was found to contain two glycopolymers, viz. 1,3-poly(glycerol phosphate) partially O-glycosylated with 2-acetamido-2-deoxy-α-D-glucopyranose and/or O-acylated with L-lysine, and a poly(diglycosyl 1-phosphate) of hitherto unknown structure: -6)-α-D-Glcp-(1 â†’ 6)-α-D-GlcpNAc-(1-P-.

Teichoic, teichulosonic and teichuronic acids in the cell wall of Brevibacterium aurantiacum VKM Ac-2111(Т).

Two different teichoic acids, along with a teichulosonic and a teichuronic acids, were identified in the cell wall of Brevibacterium aurantiacum VKM Ac-2111(Т). One teichoic acid is 1,3-poly(glycerol phosphate) with 2-acetamido-2-deoxy-α-D-galactopyranose and L-glutamic acid as non-stoichiometric substituents at O-2 of the glycerol residue. The second one is a poly(glycosylglycerol phosphate) with -4)-α-D-Galp-(1 → 2)-sn-Gro-(3-P- and/or -6)-α-D-Galp-(1 → 2)-sn-Gro-(3-P- units in the main chain. The structure of the first has not been reported so far, while the latter one is new for actinobacteria. The teichulosonic acid with α-3-deoxy-ß-D-glycero-D-galacto-non-2-ulopyranosonic acid (Kdn) and ß-D-glucopyranose residues in the backbone represents a novel polymer: → 8)-α-Kdn-(2 → 6)-ß-D-Glcp-(1 →. The teichuronic acid has also hitherto unknown structure: → 3)-ß-D-Galf(2OAc)0.3-(1 → 3)-ß-D-GlcpА-(1 → and is found in members of the genus Brevibacterium for the first time. The polymer structures were elucidated using 1D- and 2D-NMR spectroscopy: (1)H,(1)H COSY, TOCSY, ROESY, (1)H,(13)C HSQC, HSQC-TOCSY, and (1)H,(13)C and (1)H,(31)P HMBC.

Purification, structural characterization and anticancer activity of the novel polysaccharides from Rhynchosia minima root.

Three novel acidic polysaccharides termed PRM1, PRM3 and PRM5 were purified from Rhynchosia minima root using DEAE-52 cellulose and sephadex G-150 column chromatography. Their structures were characterized by ultraviolet (UV) and Fourier transform infrared (FTIR) spectrometry, gel permeation chromatography (GPC), gas chromatography-mass spectrometry (GC-MS), and differential scanning colorimeter (DSC) analysis. The uronic acid contents of PRM1, PRM3 and PRM5 were 30.7%, 12.7% and 47.7%, respectively. PRM1 (143.2 kDa), PRM3 (105.3 kDa) and PRM5 (162.1 kDa) were heteropolysaccharides because they were composed of arabinose, mannose, glucose and galactose. Their enthalpy values were 201.0, 111.0 and 206.8 J/g, respectively. PRM3 and PRM1 exhibited strong in vitro anticancer activity against lung cancer A549 and liver cancer HepG2 cells in a dose-dependent manner. These findings suggested that PRM1 and PRM3 could be potentially developed as natural anticancer agents.

Ammonium oxalate-extractable uronic acids positively affect biomass enzymatic digestibility by reducing lignocellulose crystallinity in Miscanthus.

Based on systems biology analyses of total 179 representative Miscanthus accessions, ammonium oxalate (AO)-extractable uronic acids could either positively affect biomass digestibility or negatively alter lignocellulose crystallinity at p<0.01 or 0.05. Comparative analysis of four typical pairs of Miscanthus samples indicated that the AO-extractable uronic acids, other than hexoses and pentoses, play a predominant role in biomass enzymatic saccharification upon various chemical pretreatments, consistent with observations of strong cell tissue destruction in situ and rough biomass residue surface in vitro in the unique Msa24 sample rich in uronic acids. Notably, AO-extraction of uronic acids could significantly increase lignocellulose CrI at p<0.05, indicating that uronic acids-rich polymers may have the interactions with ß-1,4-glucan chains that reduce cellulose crystallinity. It has also suggested that increasing of uronic acids should be a useful approach for enhancing biomass enzymatic digestibility in Miscanthus and beyond.

Characterization and immunomodulatory activities of polysaccharides from Spirogyra neglecta (Hassall) Kützing.

Sulfated polysaccharides (SP) isolated from freshwater green algae, Spirogyra neglecta (Hassall) Kützing, and fractionated SPs were examined to investigate their molecular characteristics and immunomodulatory activity. The crude and fractionated SPs (F1, F2, and F3) consisted mostly of carbohydrates (68.5-85.3%), uronic acids (3.2-4.9%), and sulfates (2.2-12.2%) with various amounts of proteins (2.6-17.1%). D-galactose (23.5-27.3%), D-glucose (11.5-24.8%), L-fucose (19.0-26.7%), and L-rhamnose (16.4-18.3%) were the major monosaccharide units of these SPs with different levels of L-arabinose (3.0-9.4%), D-xylose (4.6-9.8%), and D-mannose (0.4-2.3%). The SPs contained two sub-fractions with molecular weights (Mw) ranging from 164 × 10(3) to 1460 × 10(3) g/mol. The crude and fractionated SPs strongly stimulated murine macrophages, producing considerable amounts of nitric oxide and various cytokines via up-regulation of their mRNA expression by activation of nuclear factor-kappa B and mitogen-activated protein kinases pathways. The main backbone of the most immunoenhancing SP was (1→3)-L-Fucopyranoside, (1→4,6)-D-Glucopyranoside, and (1→4)-D-Galactopyranoside.

Carbohydrate composition of peach palm (Bactris gasipaes Kunth) by-products flours.

The flours obtained from peach palm by-products are rich in dietary fiber (62-71%) and they can be used as food ingredients. The aim of this work was to investigate the carbohydrate composition of the flours processed from the residual parts (stem and median sheath) of a hearts-of-palm industry. The flours were fractionated, based on their solubility, whose monomeric compounds were determined. The fraction containing mostly cellulose (S5) was the most abundant (26-28%), followed by the sum of fractions (S2, 53, S4) extracted with alkaline solutions (21-22%). The S1 fraction contained the highest percentage of uronic acids, which characterizes the presence of pectin. Xylose and arabinose were found in high proportion in S2 and S3 fractions. The S4 and S5 fractions, rich in glucose, were the main portion of the cell wall material and correspond to the insoluble fraction of the dietary fiber.

Structural features of a novel polysaccharide isolated from a New Zealand Maori mushroom Iliodiction cibarium.

A purified water-soluble fraction (ICP5) of a polysaccharide, isolated from a local Maori mushroom Iliodiction cibarium in New Zealand, was investigated for its structural properties. Size exclusion chromatography and dynamic light scattering showed that ICP5 had a large MW of 1.6 × 10(5) Da with a hydrodynamic diameter of 83 ± 8 nm. Particle size measurements also displayed the tendency of ICP5 to aggregate when suspended in water. The results of GC-MS, FTIR and NMR analyses allowed some characteristics of the chemical structure of ICP5 to be determined. GC-MS results showed that ICP5 contained only glucose (81.61%), galactose (12.90%) and mannose (5.49%) monomers. The characterized fragment structures of ICP5 were found to be dominantly consisting of uronic acids, which formed a backbone containing 1,4-ß-D-GlcpA. A small amount of unsaturated uronic acid also appeared to be present.

Optimization of ultrasound assisted extraction of bioactive components from brown seaweed Ascophyllum nodosum using response surface methodology.

The objective of this study was to investigate the effect of key extraction parameters of extraction time (5-25 min), acid concentration (0-0.06 M HCl) and ultrasound amplitude (22.8-114 µm) on yields of bioactive compounds (total phenolics, fucose and uronic acid) from Ascophyllumnodosum. Response surface methodology was employed to optimize the extraction variables for bioactive compounds' yield. A second order polynomial model was fitted well to the extraction experimental data with (R(2)>0.79). Extraction yields of 143.12 mgGAE/gdb, 87.06 mg/gdb and 128.54 mg/gdb were obtained for total phenolics, fucose and uronic acid respectively at optimized extraction conditions of extraction time (25 min), acid concentration (0.03 M HCl) and ultrasonic amplitude (114 µm). Mass spectroscopy analysis of extracts show that ultrasound enhances the extraction of high molecular weight phenolic compounds from A. nodosum. This study demonstrates that ultrasound assisted extraction (UAE) can be employed to enhance extraction of bioactive compounds from seaweed.

Structural characterization and stimulating effect on osteoblast differentiation of a purified heteropolysaccharide isolated from Hedysarum polybotrys.

Radix Hedysari polysaccharides (HPS) is the principal active fraction of Radix Hedysari (RH). The information about HPS3d, the main fraction of HPS3, and its effect on bone is still unknown. In the present study, the purified HPS3d was obtained by anion-exchange column. It consisted of 94.38% polysaccharide, 3.40% protein and 13.30% uronic acid. The molecular weight was measured to be 84.6kDa. The backbone consisted of galactopyranose and galacturonopyranose, and the side chains were composed of glucopyranose, rhamnopyranose and arabinofuranose. The FT-IR and elemental analysis showed that HPS3d was the sulfated polysaccharide. HPS3d upregulated alkaline phosphatase (ALP) activity and the expression of other osteogenic marker genes in osteoblast. In addition, HPS3d increased the expression and transcriptional activity of Runt-related transcription factor 2 (Runx-2) and Osterix, the two master genes of osteoblast differentiation. These findings suggest that HPS3d stimulates osteoblast differentiation by activation of Runx-2 and Osterix.

Comparison of physicochemical properties and immunomodulatory activity of polysaccharides from fresh and dried litchi pulp.

Drying is commonly used for preservation and processing of litchi. However, its polysaccharide structure may be altered by the drying process, resulting in biological activity changes. Polysaccharides from fresh and dried litchi pulp (denoted as LPF and LPD, respectively) were isolated, investigated by GC-MS, GPC and UV/IR spectrum analysis and their antitumor and immunomodulatory activities were evaluated in vitro. LPD, the molecular weight of which was lower than that of LPF, contained more protein, uronic acid, arabinose, galactose and xylose. Compared with LPF, LPD exhibited a higher inhibitory effect on the proliferation of HepG2, Hela and A549 cells from 50-750 µg/mL. LPD was also a better stimulator of spleen lymphocyte proliferation, NK cells cytotoxicity and macrophage phagocytosis from 50-400 µg/mL. In summary, drying could change the physicochemical properties and enhance the bioactivity of polysaccharides from litchi pulp. This finding is supported by the fact that dried litchi pulps are used in Traditional Chinese Medicine.

Studies on Tinospora cordifolia monosugars and correlation analysis of uronic acids by spectrophotometric methods and GLC.

Cold water-soluble (CWSP) and hot water soluble polysaccharides (HWSP) from Tinospora cordifolia stems were isolated and purified in 2.99% and 1.99% yield respectively. Complete hydrolysis followed by paper chromatography and GLC analysis indicated the presence of L-rhamnose, L-arabinose, D-xylose, D-mannose, D-galactose and D-glucose in molar ratio of 0.857, 1.106, 0.727, 0.526, 0.708 and 95.763 in CWSP and 0.697, 0.777, 2.048, 0.777, 0.292 and 95.408 in HWSP. The uronic acid content in the polysaccharide has been studied extensively using assorted approaches. It was quantitatively estimated by GLC analysis and spectrophotometric methods using carbazole, m-hydroxydiphenyl and 3,5-dimethylphenol as colorimetric reagents. GLC analyses indicated galacturonic acid content of 3.06% and 5.16% in CWSP and HWSP respectively. Estimation of uronic acid using 3,5-dimethylphenol corroborated the above analysis. The study resulted in composition of constituent monosugars of CWSP and HWSP and co-relation analysis of uronic acid content, leading to an unambiguous structural analysis.

Fractionation and characterization of hemicelluloses from young bamboo (Phyllostachys pubescens Mazel) leaves.

Bamboo leaves are considered as an important source of bioactive molecules. In this work, leaves from young bamboo (Phyllostachys pubescens Mazel) aged 3 months were subjected to aqueous extraction and 2% NaOH solution extraction followed by precipitation in ethanol-water medium with different ethanol concentrations. The dissolved hemicellulosic polysaccharides presented a total recovery of 67.83% based on the total hemicellulose content in bamboo leaves. Chemical analysis of the fractions was performed by sugar composition analysis, Fourier-transform infrared spectrometry, and 1D nuclear magnetic resonance imaging. The results revealed that all polysaccharide fractions contained xylose, arabinose, glucose, galactose, ribose, and uronic acid. The polysaccharides from young bamboo leaves mainly consisted of arabinoxylans, arabinogalactans, and non-cellulosic ß-D-glucans having (1→3)- and (1→4)-glucosidic linkages. The content of these polysaccharides was found to vary among the fractions depending on the separation method. Finally, the thermal behavior was also discussed.