Isolation,Purification, and Structural Features of a Polysaccharide from Phellinus linteus and Its Hypoglycemic Effect in Alloxan‐Induced Diabetic Mice

Phellinus linteus is a medicinal mushroom that has been used in Oriental countries for centuries for its antitumor, antioxidant, immunomodulatory, and biological activity on hyperglycemia. A water‐soluble crude polysaccharide was extracted using hot water from P. linteus mycelia grown under submerged culture. An orthogonal experiment was used to optimize the extraction conditions of P. linteus mycelia polysaccharides (PLP). The crude polysaccharide was purified using DEAE Sephadex A‐50 and Sephadex G‐200 chromatography. Fourier transform infrared (FT‐IR) spectroscopy and nuclear magnetic resonance (¹H NMR) spectroscopy were used to investigate the structure of the purified P. linteus polysaccharide (PLP‐I), revealing that it was mainly a branched‐type glycan with both α‐ and β‐linkages and a pyranoid sugar ring conformation. PLP orally administered at 100 mg/kg body weight/d could significantly reduce the blood glucose level by 35.60% in alloxan‐induced diabetic mice. The results of an oral glucose tolerance test (OGTT) revealed that PLP had an effect on glucose disposal after 28 d of treatment. The result revealed that PLP from a submerged culture of P. linteus mycelia possessed potent hypoglycemic properties. The polysaccharide may be useful as a functional food additive and a hypoglycemic agent.     

PURIFICATION AND CHARACTERIZATION OF A SERINE PROTEINASE FROM THE TUNA PYLORIC CAECA

A 15.0 kDa serine proteinase with collagenase activity from pyloric caeca of tuna, Thunnus thynnus, was purified in four steps; acetone precipitation, gel filtration chromatography on a Sephadex G‐100, ion‐exchange chromatography on a DEAE‐Sephadex α‐50 and gel filtration chromatography on a Sephadex G‐75 column. The purification and yield were 30.5‐fold and 0.023%, respectively, as compared with those in the starting crude extract. The optimum pH and temperature for the purified collagenolytic enzyme were around pH 7.5 and 55C, respectively. The purified proteinase was strongly inhibited by metal ions (Hg²⁺ and Zn²⁺) and serine proteinase inhibitors (PMSF, TLCK and soybean trypsin inhibitor) suggesting it is a serine protease. The K_m and V_max of the purified enzyme for collagen type I were approximately 3.82 mM and 851.5 U, respectively.     

Chemical characteristics and antioxidant activities of polysaccharide purified from the seeds of Plantago asiatica L.

BACKGROUND: A water‐soluble polysaccharide from the seeds of Plantago asiatica L. (P. asiatica L. polysaccharide, PLP) was extracted with hot water and purified by gel filtration chromatography. The chemical characteristics of PLP were determined by high‐performance gel permeation chromatography (HPGPC) and Fourier transform infrared (FTIR) spectroscopy. In addition, the antioxidant activities of PLP in vitro were evaluated using various test systems, including scavenging of 1,1‐diphenyl‐2‐picryl‐hydrazyl (DPPH) radicals, scavenging of superoxide radicals generated by 1,2,3‐phentriol autoxidation, scavenging of hydroxyl radicals and inhibition of lipid peroxidation. RESULTS: The molecular weight of PLP was determined by HPGPC to be about 1894 kDa. PLP contained 29.2 g kg^?1 protein and 145.8 g kg^?1 uronic acid. The FTIR spectrum of PLP also revealed typical characteristics of a polysaccharide containing protein and uronic acid. Moreover, the results showed that PLP possessed antioxidant activities, but lower than those of ascorbic acid. CONCLUSION: PLP is an acid protein‐bound polysaccharide of high molecular weight, but its structure needs further study. The present results suggest that PLP could potentially be used as a natural antioxidant. Copyright © 2009 Society of Chemical Industry     

基于酶活力模型和细胞模型分析红毛藻多糖降血脂活性

目的：对红毛藻（Bangia fusco-purpurea）中多糖组分进行分离纯化，分析多糖组分的体外降血脂活性，阐明红毛藻的降血脂活性功效机理，为红毛藻的精深加工和高值化应用提供科学依据。方法：采用热水提取-乙醇沉淀法从红毛藻中提取粗多糖；除去蛋白质后，通过Sephadex G75葡聚糖凝胶柱层析纯化后得到红毛藻多糖（B. fusco-purpurea polysaccharide，BFP）；采用DEAE-cellulose 52柱层析对BFP进一步分级纯化得到3 个多糖组分F1、F2和F3；通过酶动力学分析各组分对胰脂肪酶活性的影响；采用噻唑蓝法测定BFP组分F1、F2和F3对HepG2细胞和Caco-2细胞活力的影响；通过高血脂/高胆固醇HepG2细胞模型、油酸诱导Caco-2细胞模型分析BFP组分F1、F2和F3的体外降血脂活性。结果：酶动力学分析结果表明，BFP组分F3显著抑制了胰脂肪酶活性，且为可逆竞争性抑制；细胞模型结果表明，F1、F2和F3在质量浓度0～500 mg/mL范围内对所试细胞均无显著毒性作用；F1显著抑制Caco-2细胞对游离脂肪酸的吸收；F3对高血脂HepG2细胞模型中甘油三酯的合成和高胆固醇HepG2细胞模型中脂类的合成均有显著抑制作用。结论：BFP组分可有效抑制胰脂肪酶活性和细胞对游离脂肪酸的吸收、降低胆固醇和甘油三酯的合成，具有潜在的降血脂活性。     

Structural characterization and antioxidant activities of 2 water-soluble polysaccharide fractions purified from tea (Camellia sinensis) flower

Abstract: The water‐soluble crude polysaccharide tea flower polysaccharide (TFP), obtained from tea (Camellia sinensis) flower by boiling‐water extraction and ethanol precipitation, was fractionated by Sephadex G‐100 column chromatography, giving 2 polysaccharide fractions termed TFP‐1 and TFP‐2. The structural features of TFP‐1 and TFP‐2 were investigated by high‐performance liquid chromatography (HPLC), gel‐permeation chromatography (GPC), rheometer, infrared (IR) spectra, nuclear magnetic resonance (NMR) spectroscopy, atomic force microscope (AFM), and scanning electron microscope (SEM). Results indicated that TFP‐1 was composed of glucose: xylose: rhamnose: galactose = 1.0:1.2:0.81:0.98 with a molecular weight of 167.5 KDa, while TFP‐2 comprised glucose: xylose: rhamnose: arabinose = 1.0:0.76:2.3:2.3 with a molecular weight of 10.1 KDa. The ¹H NMR revealed that TFP‐1 contained α‐L‐Rhap, α‐D‐Galp, α‐D‐GalpNAc, α‐D‐Xylp, α‐D‐Glcp, and β‐D‐Glcp residues, while TFP‐2 was illustrated to have α‐L‐Rhap, α‐L‐Arap, α‐D‐Xylp, α‐D‐Glcp, and α‐D‐GlcpNAc residues. Antioxidant activities of these fractions were investigated using various in vitro assay systems compared with ascorbic acid. In conclusion, TFP‐2 exhibited the higher antioxidant activities and could be explored as a novel potential antioxidant. Practical Application: At present, commonly low‐grade tea is preferred to extract the tea polysaccharide, to take full advantage of tea flower resource to extract polysaccharides can greatly reduce the cost of tea products. Thus, the search for plant‐derived biomaterials from this study could generate natural value‐added products from underutilized tea plant waste and used as a medicinal agent against chronic health problems, such as cancers, aging, and atherosclerosis caused by reactive free radicals that produced from oxidation.     

Structural characterisation and immunomodulatory effects of polysaccharides isolated from Dendrobium aphyllum

A crude polysaccharide extract of Dendrobium aphyllum (cDAP, yield 38.15 ± 0.20%) was generated. The D. aphyllum polysaccharide (DAP, M_w 471.586 kDa), purified by DEAE‐Sepharose and Sephadex‐G200 Fast Flow, was composed of mannose (71.3%) and glucose (28.7%), according to GC–MS analysis. Its backbone was composed of β‐d ‐mannopyranose and β‐d ‐glucopyranose residues, as revealed by infrared spectroscopic analysis. Its glycosidic bond was mainly 1, 4‐linked, and the O‐acetyl groups were mainly linked to mannose residues, according to periodate oxidation and Smith degradation analysis. The DAP units polymerised into a filiform‐shaped spatial pattern, as characterised by atomic force microscopy and scanning electron microscopy. DAP treatment enhanced cytokine secretion (nitric oxide, interleukin‐6 and tumour necrosis factor‐α) and pinocytic and phagocytic capacities of RAW 264.7 mouse macrophages. The complement receptor 3 and mannose receptor were identified to be the receptors of DAP on RAW 264.7 cells, indicating that the Akt/mTOR/MAPK and IKK/nuclear factor‐?B pathways could be involved in DAP‐activated immunomodulation.     

Evaluation of antioxidative and hypolipidemic properties of a novel functional diet formulation of Auricularia auricula and Hawthorn

Auricularia auricula and hawthorn are well known for both traditional food and folk medicine. To develop a novel healthy functional diet (FD), the formulation of A. auricula polysaccharide (AAP) and hawthorn flavones were postulated in present study. In vitro, FD significantly increased the radical-scavenging effects and inhibited LDL oxidation, compared with AAP. In vivo, the hyperlipidemic mice, induced by cholesterol-enriched diet, were provided either 150, 300, 450 mg/kg day of FD or 300 mg/kg day of AAP for 12 weeks to evaluate their expected hypolipidemic activity. Compared with CED, both FD and AAP groups could lower serum TC and atherogenic index (AI), improve serum and hepatic antioxidant status. Especially, formulating with hawthorn could enhance hepatic antioxidant status in dose-dependent manner, compared with AAP. Thus, it can be concluded that FD may act as a potent hypolipidemic and powerful antioxidant formulation via improving serum lipid profile and inhibiting lipid peroxidation in mice.Industrial relevanceWith the development of modern living standard, dyslipidemia has become one of major causes leading to antioxidant stress and atherosclerosis. It is of great significance to maintain the normal body functions by reducing the elevated serum cholesterol to an adequate level. In view of side effects of recent therapies, it is urgent to produce certain health-promoting functional diet against dyslipidemia. Therefore, we studied on a novel functional formulation diet (Auricularia auricula and Hawthorn) against hyperlipidemia, using the traditional both edible and medicinal herbs. The antioxidant and hypolipidemic properties are roundly evaluated in vitro and in vivo with the appropriate animal model. Our research also suggests the optimum dosage with potent nutritional property. It could provide the future practical application to produce this functional diet as an adjuvant dietetic food.     

Production of Agaricus brasiliensis mycelium from food industry residues as a source of antioxidants and essential fatty acids

Agaricus brasiliensis is a medicinal mushroom traditionally employed in prevention and treatment of cancer, used as immunostimulant and as a source of antioxidants. We investigated the chemical composition of the mycelium produced by submerged (SF) and solid‐state fermentation (SSF), using residues from food industry as substrates. After fermentation, antiradical activity and levels of antioxidants were enhanced, indicating that the micro‐organism produces these metabolites. For myceliated substrate (mushroom mycelia grown around and into the substrate particles) obtained by SSF, phenolics ranged from 18.57 to 70.46 mg g^?1 and flavonoids from 0.83 to 4.51 mg g^?1. For myceliated substrate obtained by SF, the variation was 27.19 to 66.99 mg g^?1 and 0.75 to 5.34 mg g^?1 for phenolics and flavonoids, respectively. The fatty acid profile determined by FT‐ICR MS and UPLC‐MS showed predominance of palmitic, linoleic, linolenic and oleic acids. Our findings indicated that mycelium has nutraceutical potential and can be incorporated in food supplements.     

裂褶菌胞内多糖的提取纯化及生物活性分析

为提高裂褶菌胞内多糖得率,并研究裂褶菌多糖纯化组分的生物活性,本研究采取超声波破碎和热水浸提相结合的方法提取裂褶菌胞内粗多糖,利用响应面法对提取条件进行优化,通过DEAE-52离子交换柱层析和Sephadex G-100凝胶柱层析对提取的粗多糖进行分离纯化,并进行结构表征、抗氧化性和抑菌性试验。结果表明,裂褶菌胞内粗多糖的最优提取条件为:提取温度90 ℃,水料比30:1,超声时间30 min,超声功率230 W,裂褶菌胞内粗多糖得率达到了18.14%±0.33%。纯化多糖组分NSPG-1为β型吡喃糖,分子量为1.05×106 Da,NSPG-1多糖具有较好的抗氧化性和抑菌性,其对DPPH自由基、羟基自由基和超氧阴离子自由基的IC₅₀值分别为6.97、1.07和11.41 mg/mL,对大肠杆菌、枯草芽孢杆菌和金黄色葡萄球菌的IC₅₀值分别为7.56、12.54和10.42 mg/mL。本研究结果为裂褶菌多糖的工业化生产和开发利用提供了基础。     

Purification and some properties of a cysteine proteinase from sorghum malt variety SK5912

A cysteine proteinase from sorghum malt variety SK5912 was purified by a combination of 4 M sucrose fractionation, ion‐exchange chromatography on Q‐ and S‐Sepharose (fast flow), gel filtration chromatography on Sephadex G‐100 and hydrophobic interaction chromatography on Phenyl Sepharose CL‐4B. The enzyme was purified 8.4‐fold to give a 13.4% yield relative to the total activity in the crude extract and a final specific activity of 2057.1 U mg^?1 protein. SDS—PAGE revealed two migrating protein bands corresponding to apparent relative molecular masses of 55 and 62 kDa, respectively. The enzyme was optimally active at pH 6.0 and 50 °C, not influenced across a relatively broad pH range of 5.0–8.0 and retained over 60% activity at 70 °C after 30‐min incubation. It was highly significantly (P < 0.001) inhibited by Hg²⁺, appreciably (P < 0.01) inhibited by Ag⁺, Ba²⁺ and Pb²⁺ but highly significantly (P < 0.001) activated by Co²⁺, Mn²⁺ and Sr²⁺. The proteinase was equally highly significantly (P < 0.001) inhibited by both iodoacetate and p‐chloromercuribenzoate and hydrolysed casein to give the following kinetic constants: K_m = 0.33 mg ml^?1; V_max = 0.08 µmol ml^?1 min^?1. Copyright © 2004 Society of Chemical Industry     

Antigenotoxicity of extracts from Pleurotus citrinopileatus

While Pleurotus citrinopileatus is a widely used edible mushroom, little is known about its physiological effects. Extracts, including aqueous extract, water‐soluble polysaccharide (WSP), crude protein solution (CPS) and residue from chloroform–ethyl acetate–methanol elution (CEM), were obtained first from fruiting bodies, through a solid‐state culture, and then from the mycelium, through a submerged culture. This study explored the antigenotoxicity effects of these extracts from Pleurotus citrinopileatus via the Ames test and a spore rec‐Assay. The results showed that, regardless of where the extract came from, the fruiting body or the mycelium, the antigenotoxicity effect was highest for CEM, followed by CPS, aqueous extract and WSP. The results of the Ames test indicated that, among several mutagens, CEM had the highest inhibition rate against AFB_l in TA98 and TA100 and the lowest inhibition rate against NQNO. The concentrations of the various extracts were as follows: water extracts were 1 mg ml^?1 and 5 mg ml^?1 WSP, while CPS and CEM were 0.4 mg ml^?1 and 2 mg ml^?1, respectively; the higher the concentration of the extract, the higher the antimutagenicity effect. The results of the rec‐Assay indicated that CEM had the highest anti‐DNA‐damaging activity with or without the S9 mixture; the higher the concentration, the more significant the effect (p < 0.05). The anti‐DNA‐damaging activities were lower in the water extract concentrations, at 30 µg disc^?1 dry weight^?1, while the WSP, CPS and CEM at 12, 150 and 60 µg disc^?1, respectively, were high. Copyright © 2004 Society of Chemical Industry     

Recent developments in Hericium erinaceus polysaccharides: extraction,purification, structural characteristics and biological activities

Abstract

Hericium erinaceus (H. erinaceus), an edible mushroom with medicinal value, has a long history of usage in China and other oriental countries. Polysaccharide is supposed to be one of the major bioactive compounds in H. erinaceus, which possesses immunomodulating, anti-cancer, antioxidant, gastroprotection and intestinal health promotion, neuroprotective, hepatoprotective, antihpyerglycemic and hypolipidemic activities. In this review, the current advancements on extraction, purification, structural characteristics and biological activities of polysaccharide from different sources (fruiting body, mycelium and culture broth) of H. erinaceus were summarized. Among these aspects, summaries of the structural characteristics focused on the purified polysaccharides. Meanwhile, comparisons on the structural characteristics among the purified polysaccharides obtained from above three sources were made. Moreover, their biological activities were introduced on the basis of in vivo and in vitro experiments, and some possible action mechanisms were listed. Furthermore, the structure-activity relationship of the polysaccharide was discussed. New perspectives for the future work of Hericium erinaceus polysaccharide were also proposed.

• HIGHLIGHTS

• Extraction, purification, structural characteristics and biological activities of Hericium erinaceus polysaccharide (HEP) were summarized.

• Structural characteristics of the purified polysaccharides from different sources (fruiting body, mycelium and culture broth) of Hericium erinaceus were summarized and compared.

• Structure-activity relationship of HEP was discussed, and new perspectives for the future work of this polysaccharide were proposed.

     

Chemical characterisation and hypolipidaemic effects of two purified Pleurotus eryngii polysaccharides

This study is focused on the isolation and characterisation of two purified polysaccharide fractions (namely PPEP‐1 and PPEP‐2) from Pleurotus eryngii (P. eryngii) and evaluation of their hypolipidaemic effects. The Congo red analysis indicated that PPEP‐2 but not PPEP‐1 possessed a triple‐helix conformation. The atomic force microscope analysis revealed that PPEP‐1 and PPEP‐2 showed different polysaccharide chain conformations. Importantly, the mice treated with PPEP‐1 showed significantly lowered serum levels of total cholesterol (TC), triglyceride (TG) and low‐density lipoprotein cholesterol (LDL‐c) and increased high‐density lipoprotein cholesterol when compared with the hyperlipidaemia mice induced by the high‐fat diet. However, PPEP‐2 revealed less hypolipidaemic effect than PPEP‐1. Additionally, both PPEP‐1 and PPEP‐2 demonstrated remarkable hypolipidaemic effects by decreasing levels of serum TC, TG and LDL‐c in the hyperlipidaemic model induced by P‐407. Taken together, our findings suggest that the P. eryngii polysaccharides, especially PPEP‐1, could be developed as a natural functional food supplement for preventing hyperlipidaemia.     

Purification and Some Properties of a Protease from Sorghum Malt Variety KSV8–11

A protease from sorghum malt variety KSV8–11 was purified by a combination of dialysis against 4 M sucrose, ion‐exchange chromatography on Q‐Sepharose (Fast flow), gel filtration chromatography on Sephadex G‐100 and hydrophobic interaction chromatography on Phenyl Sepharose CL‐4B. The enzyme was purified 5‐fold to give a 14.1% yield relative to the total activity in the crude extract and a final specific activity of 1348.9 U mg^?1 protein. SDS‐PAGE revealed a single migrating protein band corresponding to a relative molecular mass of 16 KDa. Using casein as substrate, the purified protease had optimal activity at 50°C and maximal temperature stability between 30°C and 40°C but retained over 64% of its original activity after incubation at 60°C for 30 min. The pH optimum was 5.0 with maximum stability at pH 6.0 but 60% of the activity remained after 24 h between pH 5.0 and 8.0. The protease was inhibited by Ag⁺, Ca²⁺, Co²⁺, Fe²⁺, Mg²⁺, iodoacetic acid (IAA) and p‐chloromercuribenzoate (p‐CMB), stimulated by Cu²⁺, Sr²⁺, phenylmethylsulfonyl‐fluoride (PMSF) and 2‐mercaptoethanol (2‐ME) while Mn²⁺ and ethylenediaminetetraacetic acid (EDTA) had no effect. The purified enzyme had a K_m of 18 mg·mL^?1 and a V_max of 11.1 μmol · mL^?1 · min^?1 with casein as substrate.     

Production,partial purification and properties of β‐mannanases obtained by solid substrate fermentation of spent soluble coffee wastes and copra paste using Aspergillus oryzae and Aspergillus niger

In order to achieve a higher added value of two galactomannan‐containing wastes, copra paste and spent coffee from the soluble coffee industry (SCW), solid substrate fermentation (SSF) was used. Filamentous fungi Aspergillus oryzae and A niger were used to evaluate the feasibility of producing β‐mannanase by SSF. A 2³ factorial design was used to select the best interaction among the two fungi, the two substrates and two fermentation times. The treatment ‘A niger–copra–2.5 days’ produced a significantly higher (p < 0.05) β‐mannanase activity, having five different isoforms of the enzyme, one of which was partially purified to a specific activity of 764 U mg⁻¹ (U = nmol of mannose released per second from a galactomannan substrate). Copra paste had a higher mannose/galactose ratio (14:1) than SCW (6:1), and low oil content, which led to higher β‐mannanase production from SSF. A β‐mannanase from SSF of copra produced by A oryzae was highly purified using acetone precipitation and cation exchange and size exclusion chromatographies. This enzyme had an MW of 110 kDa, a pI between 3.5 and 4.5 and a specific activity of 1760 U mg⁻¹; purification achieved was 90.7 times. The temperature and pH for optimal activity were 40 °C and 6.0 respectively. The optimal temperature was lower and the optimal pH higher than others previously reported (produced by submerged fermentation), which could be important for viscosity reduction of concentrated coffee extract in instant coffee manufacture. Copra is an interesting alternative for β‐mannanase production, since it is readily available in Mexico; moreover, the residue after SSF has a reduced galactomannan content and may be used for monogastric animal feed. © 2000 Society of Chemical Industry     

Extracellular polysaccharides from suspension-cultured cells of Rubus fruticosus. Structure of a galactoglucomannan

Carbohydrate analysis of the cell walls from suspension-cultured cells of Rubus fruticosus between day 10 and day 40 after inoculation revealed an overall increase of the net content of neutral sugars. However the composition of the extracellular polysaccharides showed only discrete variation. A galactoglucomannan containing galactose, glucose and mannose in the ratio 1.0:1.4:1.2 respectively, could be purified by barium hydroxide precipitation both in the extracellular medium and from the alkaline extract of the cell walls. Methylation analysis and ¹³C-NMR spectroscopy demonstrated that the extracellular form is the counterpart of the wall polysaccharide.     

榛仁降脂活性肽分离纯化及结构鉴定

采用超滤、Sephadex G-25、Sephadex G-15、反相高效液相色谱及质谱对榛仁分离蛋白降脂活性肽进行分离纯化及结构鉴定,并通过测定3T3-L1前脂肪细胞诱导分化过程中脂质积累、总胆固醇及甘油三酯水平,筛选出具有较高降脂活性的肽段。结果表明,经Sephadex G-15分离得到的C3组分的胰脂肪酶抑制、胆固醇胶束吸附及细胞降脂活性均显著高于其他组分。进一步经质谱解析筛选出的肽段Phe-Leu-Leu-Pro-His（FLLPH）与模型组相比,可抑制26.31%的总脂形成,降低32.67%胆固醇和23.87%甘油三酯水平。FLLPH具有较好的降脂活性,本研究可为榛仁降脂活性肽的开发提供理论参考。     

PURIFICATION AND CHARACTERIZATION OF A CATALASE FROM THE LIVER OF BULLFROG, RANA CATESBEIANA SHAW

A catalase was purified from the liver of bullfrog, Rana catesbeiana Shaw, after extraction, ammonium sulfate precipitations, DEAE‐Sephadex A‐50 chromatography, gel filtration chromatography on a Sephadex G‐150 column, ion exchange chromatography on a DEAE‐Sephacel column and Sephacryl S‐300 column. The yield and purification from the starting crude extract were 0.25% and 73.57‐fold, respectively. The purified catalase with an apparent molecular mass of 186 kDa was shown to be composed of four identical subunits of apparent molecular mass of 47.7 kDa. The purified catalase is active over a broad pH range of 6.0–10.0, and it has an isoelectric point of 6.3. The enzyme showed a K_mfor H₂O₂of 20 mM and an apparent V_maxof 51.91 U/mg, and its maximum absorption was at 408 nm in the visible portion of the spectrum. In addition, the purified enzyme was markedly inhibited by azide, cyanide and hydroxylamine.     

Isolation,purification and properties of the peroxidase from the hull of Glycine max var HH2

Soybean hull peroxidase (EC 1.11.1.7), an acidic peroxidase isolated from soybean (Glycine max var HH2) hulls was purified to electrophoretic homogeneity by a combination of ammonium sulphate fractionation, DEAE‐Sephadex A‐50 chromatography, concanavalin A‐Sepharose 4B affinity chromatography and Bio‐Gel P‐60 gel filtration. The specific activity of purified peroxidase was about 57‐fold higher than that of crude extract. The yield was about 16.4%. The molecular weight of the enzyme was estimated to be 38 000 by SDS‐polyacrylamide gel electrophoresis. The peroxidase was a glycoprotein containing about 18.7% carbohydrate, approximately one‐quarter of which was shown to be glucosamine residues. It was found to have an isoelectric point of 3.9. The enzyme was most active at pH 4.6 and 45°C, and was stable in the pH range 2.5–11.5. The enzyme could tolerate heating for 10 min at 75°C without being inactivated, and at 85°C, it took 40 min to inactivate the enzyme 50%, confirming that the peroxidase was a novel thermostable enzyme. Fe ²⁺, Fe³⁺, Sn²⁺, CN ⁻ and N₃⁻ inhibited enzyme activity, while Hg²⁺, Ag⁺, Pb ²⁺, Cr³⁺, EDTA and SDS were not significantly inhibitory. © 1999 Society of Chemical Industry     

Isolation and chemical characterisation of a polysaccharide from green tea (Camellia sinensis L.)

BACKGROUND: To contribute towards understanding the relationship of structure and bioactivity, a protein‐bound acidic polysaccharide named TPC3‐1 was isolated and purified from low‐grade green tea (Camellia sinensis L.). The homogeneity and weight average molecular weight of TPC3‐1 was determined by agarose gel electrophoresis and high‐performance gel permeation chromatography. The monosaccharide and amino acid composition of TPC3‐1 were analysed by gas chromatography and an amino acid analyser. The molecular structure of TPC3‐1 was characterised by Fourier transform infrared spectroscopy, ¹³C nuclear magnetic resonance spectroscopy and atomic force microscopy. RESULTS: Based on the data obtained, the average peak molecular weight of TPC3‐1 was about 120 kDa. TPC3‐1 was composed of L ‐arabinose, D ‐ribose, D ‐xylose, D ‐glucose and D ‐galactose with a molar ratio of 4.9:2.2:3.1:1.8:1.0. Fifteen amino acids were identified as components of the polymer. The TPC3‐1 molecule was found to have an anomeric carbon sign of both α and β configurations and high‐branched chains. The network structure of TPC3‐1 was observed. CONCLUSION: The tea polysaccharide TPC3‐1 was an acid protein‐bound polysaccharide with an image of network structure. The results presented here will facilitate further study of the relationship between the chemical structure and biological role of tea polysaccharide. Copyright © 2008 Society of Chemical Industry