核桃仁种皮多糖的提取及抑菌作用的研究

核桃楸外果皮三萜类物质的提取及抑菌活性
核桃楸（Carya cathayensis）是一种果树，主要分布在中国的江西、湖南、湖北、四川等地。

它的果实外表有一个坚硬的果皮，果肉里面有核仁，所以被称为核桃楸。

核桃楸有很高的药用价值，主要是因为它含有一些具有抑菌活性的物质。

为了提取核桃楸果皮中的三萜类物质，首先需要将新鲜的核桃楸果皮晒干，然后研磨成粉末。

接下来，将粉末与乙醇进行浸泡，使其充分溶解。

然后，将溶液过滤，并用旋转蒸发仪将溶液中的乙醇去除，得到三萜类物质的浓缩物。

将浓缩物溶于水，使用薄层色谱法进行分离纯化。

通过该方法，可以从核桃楸果皮中提取到高纯度的三萜类物质。

三萜类物质是一类具有抑菌活性的化合物。

经过实验室的测试，发现核桃楸果皮中提取出的三萜类物质对多种细菌具有抑制作用。

尤其是对一些常见的致病菌，如大肠杆菌、金黄色葡萄球菌等，具有很强的抑菌效果。

实验结果还表明，核桃楸果皮中的三萜类物质对抗草莓黑斑病菌也有一定的抑制作用。

三萜类物质的抑菌活性主要是通过破坏细菌细胞膜的结构和功能来实现的。

三萜类物质可以与细菌细胞膜中的脂质相互作用，导致膜的脂质排列发生改变，从而增加了细菌细胞膜的渗透性，破坏了细胞膜的完整性，使细菌无法正常生长和繁殖。

核桃楸果皮中提取的三萜类物质具有很强的抑菌活性，对多种致病菌都有一定的抑制作用。

这些研究结果为进一步开发核桃楸果皮中的药用成分提供了科学依据，也有助于该植物的综合利用和进一步开发。

64农业科学近些年来我国核桃产量稳步增长，在核桃产业发展过程中核桃资源的综合开发利用已经成为产业发展关键所在。

在资源综合利用过程中核桃青皮资源的利用就显得尤为重要，对于如何更有效、最大化的利用核桃青皮资源，很多展开了各种各样的研究。

既有对核桃青皮药用价值的研究，也有对核桃青皮抑菌效果的研究，本次研究主要是针对核桃青皮提取物抑菌机理所开展的研究。

1.概念界定1.1核桃青皮。

核桃青皮又名青龙衣，是核桃外部一层厚厚绿色果皮。

有动物实验表明，青核桃中含有一定的无机钾盐成分是镇痛的活性成分。

除此之外鲜青皮汁（干皮蒸水）外用涂抹对于治疗顽癣有非常好的疗效。

而新鲜的绿皮外衣用刀削下之后可以用于治疗体癣、股癣、牛皮癣、头癣及秃疮。

1.2核桃青皮提取物。

指利用水、乙醇、甲醇等各种溶剂对核桃青皮的化学成分进行提取之后的提取物。

核桃青皮提取物对于治疗肝胃气痛、胃神经痛、急性胃痛、慢性胃痛等都有着一定疗效。

在上世纪五十年代民间就已经有用青核桃泡酒剂治疗痛经、胃痛等的，可以在一定程度上代替吗啡，止痛效果良好。

1.3核桃青皮提取物。

除去止痛之外，核桃青皮提取物在抑菌方面还有非常突出的作用，尤其是在针对几种食源性致病性细菌与果蔬采摘后植物源性真菌的抑制生长的作用。

2.核桃青皮价值分析近些年来商洛全市核桃种植面积在不断扩大，截止2015年底商洛全市核桃种植面积达到310万亩。

而在核桃生产过程中核桃青皮就成为核桃收获之后的农林废弃物，存量非常大。

而在核桃果实收获之后核桃青皮一般都被人丢弃在田间，很少有人能认识到核桃青皮的价值，一般都当作是废弃物随意处理，但这样做不但会对环境造成持续的污染，同时还会对动植物生存产生威胁，更有甚者造成生态环境的破坏。

2.1药用价值。

而核桃青皮本身具备非常大的药用价值，是一种非常具有价值的可再利用资源，可以通过开发利用其药用价值来提升核桃青皮的资源利用效率。

为了能开发核桃青皮的药用价值，延长核桃经济产业链，促进商洛核桃产业进一步发展，我们必须走核桃青皮综合利用的道路。

摘要：核桃青皮为传统的中药材，可用于治疗皮肤瘙痒及痛等病症，核桃青皮泡酒，可用于治肝胃气痛，胃神经痛，急、慢性胃痛。

现代研究表明，提取物含有多种抑菌活性成分，例如，胡桃醌、酚类、黄酮类、多糖类、二芳基庚烷类等，对真菌、细菌、病毒等病原生物有明显的抑制或杀灭作用。

本文就目前核桃青皮提取物的抑菌作用的研究情况进行综述，旨在为后续深入研究提供参考。

关键词：核桃青皮提取物法；病原生物；抑菌作用基金项目：2017年度陕西省教育厅专项科研计划项目17JK0961《商洛核桃青皮抑菌活性、成分及机理的相关研究》；院级重大课题SlZY2016002“商洛核桃青皮抑菌活性、成分及机理的相关研究”中图分类号：S664文献标识码：ADOI 编号：10.14025/ki.jlny.2019.05.023韩晓云，刘鹏，王震，范学红（商洛职业技术学院，陕西商洛726000）核桃青皮提取物对病原生物抑菌作用的研究进展核桃青皮，即为胡桃科植物胡桃的未成熟果实的外果皮，又称青龙衣。

截至2015年年底，商洛全市核桃种植面积达310万亩。

核桃青皮作为核桃收获中产生的农林废弃物，有着巨大的存量。

在核桃果实采收后，它们被当作废弃物堆在田间，持续污染环境，危害动植物的生存，破坏生态环境。

核桃青皮作为一种宝贵的可再利用资源，其很多药用价值需要深入开发和利用。

为了更好地发展商洛核桃产业，延长核桃经济产业链，必须走综合利用的道路。

有研究表明，核桃的各个部位如树叶、树枝及外皮都有较高的药用价值，具有抗菌、杀虫等诸多功效[1-3]。

本文就核桃的抑菌、抗毒及杀虫方面的研究现状进行分类综述。

1对细菌的抑制作用核桃青皮提取物对致病菌性细菌与条件致病菌有较广泛的抑制作用，例如食源性肠道致病菌大肠杆菌、金黄色葡萄球菌、肠炎沙门菌、接触感染性细菌如铜绿假单胞菌、呼吸道感染性细菌，比如肺炎克雷伯菌、普通变形杆菌等。

吴莹[4]等以新疆核桃青皮为试验对象，研究蒸馏水、甲醇、乙醇和丙酮的水溶液浸提核桃青皮后获得的提取物对大肠杆菌、金黄色葡萄球菌和枯草芽孢杆菌三种菌的抑菌性能，试验表明不同浓度水溶液浸提后获得的核桃青皮提取物都对上述三种菌的生长繁殖有一定的抑制作用，且25%乙醇提取物对大肠杆菌的抑菌圈直径最大，即抑菌作用最好。

核桃属特定部位提取物体外抑菌作用研究
闫金萍;吴连春;李秀凤
【期刊名称】《食品研究与开发》
【年(卷),期】2007(028)007
【摘要】对核桃属植物的叶、枝皮、青皮、壳的提取物进行了体外抑茵实验.结果表明:该植物不同部位提取物均对金黄色葡萄球菌、痢疾志贺杆菌、枯草芽孢杆菌、产气杆菌、大肠杆菌有抑制作用,而且提取物的抑菌活性随着该植物各个部位的不
同而变化.
【总页数】3页(P41-43)
【作者】闫金萍;吴连春;李秀凤
【作者单位】河北理工大学轻工学院实验中心,河北,唐山,063020;河北理工大学轻工学院实验中心,河北,唐山,063020;河北理工大学轻工学院实验中心,河北,唐
山,063020
【正文语种】中文
【中图分类】TS2
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第20卷第$期2018年5月大连民族大学%报Journal of Dalian Minzu University V〇1.20,N〇.3May2018文章编号:2096 -1383(2018)03 -0205 -05核桃楸皮提（萃）取物的抑菌性能研究陈苛蒙，金黎明，包艳春，王晓彤，孙怡，胡文忠(大连民族大学生命科学学院生物技术与资源利用教育部重点实验室，辽宁大连116605)摘要:为了研究核桃楸皮提（萃）取物的抑菌性能，对金黄色葡萄球菌、大肠埃希氏菌、沙门氏菌、弧菌、白色念珠菌5种致病菌进行了抑菌性能试验。

核桃楸皮经乙醇提取后，依次用石油醚、氯仿、乙酸乙酯萃取，采用琼脂扩散法测试其对5种致病菌的抑菌活性。

结果表明:经乙醇提取后，提取物对5种致病菌都有抑制作用;石油醚萃取物只对弧菌和金黄色葡萄球菌有较强的抑制作用，氯仿萃取物对弧菌、金黄色葡萄球菌和白色念珠菌有抑制作用，乙酸乙酯萃取物对5种致病菌均有抑制作用。

为核桃楸皮活性成分的功能研究进一步补充了数据。

关键词:核桃楸皮;抑菌活性;致病菌;活性物质中图分类号:R285.5 文献标志码:AResearch onAntibacterial Activity of BarkExtractiveof Juglans rnandshurica MaximCHEN Ke- meng，JIN Li- ming，BAO Yan- chun，WANG Xiao- tong，SUN Yi，HU Wen-zhong(School of Life Science，Key Laboratory of Biotechnology and Bioresources Utilization ofMinistry of Education，Dalian Minzu University，Dalian Liaoning 116605，China)Abstract：To study the antilDacterial activity of extract of barl^of Juglans rnandshurica Maxim onStaphylococcus aureus，EscCericCia coli，Salmonella typhimurium，Vibrio parahaemolyticu andCandida albicans，these experiments were conducted.After extracted by ethanol，barl^of Jug­lans mandshurica Maxim was extracted by petroleum etlier，chloroform，and etliyl acetate suc­cessively.Antibacterial activity of extracts was measured by agar diffusion method.The showed that tlie etlianol extract had inhibitory effect on a i five kinds of patliogenic bacteria.Pe­troleum etlier extract only had strong inhibitory effect on Vibrio parahaemolyticu and Staphylococ­cus aureus.Chloroform extract could inhibit Vibrio parahaemolyticu，Staphylococcus aureus andCandida albicans.Ethyl a cetate extract showed inhibitory activities on five kinds of pathogenicbacteria.This study provides supplementary data on the active component functions Juglans mandshurica Maxim.Key words：barl^of Juglans mandshurica Maxim；antibacterial activity;pathogenic bacteria;active components收稿日期:2018 -02 -22;最后修回日期:2018 -04 -18基金项目：国家重点研发计划项目（2016YFD0400903);辽宁省自然科学基金指导计划项目（20170540204);中央高校基本科研业务费专项资金资助项目（DC201501020302，DC201501020101 )。

核桃青皮提取物抑菌活性研究的开题报告一、研究背景及意义核桃是一种常见的坚果，因其营养丰富、口感好而受到广泛的欢迎。

同时，核桃还具有一定的药用价值，其青皮富含多种生物活性物质，具有抗氧化、抗炎、抗菌、抑制肿瘤等多种生物学活性。

近年来，越来越多的研究表明核桃青皮提取物在抑菌方面具有良好的活性。

抗菌是现代医学和生物学研究领域十分重要的一项研究内容。

在抗菌药物不断出现耐药性的情况下，研究和开发天然抗菌物质具有十分重要的现实意义。

而核桃青皮提取物是一种天然产物，具有低毒、高效、生物降解等特点，对抗菌药物的替代具有重要的意义。

因此，对核桃青皮提取物的抑菌活性进行深入研究，对于拓展天然抗菌物质的应用范围，提高食品和药品的安全性和稳定性，具有重要的理论和实际意义。

二、研究内容和方法本文选取了核桃青皮提取物作为研究对象，采用基本的微生物学实验方法，通过对常见细菌的抑菌实验，研究核桃青皮提取物的抑菌活性。

具体实验内容包括：制备核桃青皮提取物、选择不同类型的细菌，如大肠杆菌、金黄色葡萄球菌、酵母菌等，进行抑菌实验。

实验过程中，分别测定不同浓度的核桃青皮提取物对细菌的抑制作用，使用菌落计数法、细胞增殖率法等方法对实验结果进行分析和统计。

三、研究预期结果和意义通过实验研究，预计可以获得以下结果：1.核桃青皮提取物具有一定的抗菌活性，不同类型的细菌对其抑制效果有所不同；2.核桃青皮提取物的抑菌活性与浓度存在一定的关联，随着浓度的增加，抑菌效果也随之增强；3.通过对核桃青皮提取物抑菌活性的研究，有望为拓展天然抗菌物质的应用范围提供新途径，为食品和药品的安全性和稳定性提供有力的保障和支撑。

综上所述，本研究对于拓展自然抗菌物质应用领域，提高食品和药品的安全性和稳定性具有重要的意义，该研究结果将为相关实践提供有力的理论和实际支撑。

核桃楸皮多糖的分离纯化及抗癌活性研究梁启超;邹桂华;刘爽;沈广志;吴宜艳【摘要】采用水提醇沉法提取核桃楸皮多糖(JMPS),并利用色谱法对其进一步纯化得到四种均一多糖JMPS-1、JMPS-2a、JMPS-2b和JMPS-3,其分子量分别为227.9 kDa、28.6 kDa、23.1 kDa和12.7 kDa.使用气相色谱法测定组分的单糖组成;利用红外光谱研究多糖组分的结构特征.体外抗肿瘤实验表明JMPS-1对SMMC-7721细胞具有较强抑制作用,流式细胞实验发现JMPS-1将SMMC-7721细胞的阻滞在G2/M期和S期.JMPS-1具有抗癌活性,可用作天然的抗癌物质.【期刊名称】《天然产物研究与开发》【年(卷),期】2015(027)008【总页数】6页(P1340-1345)【关键词】核桃楸皮;多糖;理化性质;抗癌【作者】梁启超;邹桂华;刘爽;沈广志;吴宜艳【作者单位】牡丹江医学院药学院;牡丹江医学院药学院;牡丹江市心血管病医院,牡丹江157011;牡丹江医学院药学院;牡丹江医学院药学院【正文语种】中文【中图分类】R285.5IntroductionJuglans mandshurica，one of rare species of trees for pharmacy resourcesnative to many Asian countries.Its leaves，barks，roots and seeds have been used as a traditional medicine for the treatment of cancer in Asia and Europe［1］.Recent scientific investigations have focused on its cytotoxic activity，antioxidant activity and anti-complement activity［2-4］.These biological effects maybe due to the fact that it contains similar chemical components，including glucosides，naphthoquinones，naphthalenyl and galloyl glycosides［5-7］，but they cannot account for all the effects mentioned above.In recent years，as more and more polysaccharides have been reported to exhibit a variety of biological activities，including antitumor［8］，antiviral［9］，anti-oxidation［10］，etc.，which have emerged as an important class of bioactive natural products.There are few reports on polysaccharides from the bark ofJ.mandshurica(JMPS).Therefore，the objective of this work was to extract water-soluble JMPS and to study preliminary characterization and molecular weight of purified polysaccharide fractions.Moreover，the anticancer activities of JMPS were also investigated in vitro.Materials and MethodsPlant materials and chemicalsThe bark of J.mandshurica was collected in a mountain area of Mudanjiang，Heilongjiang Province.The material was confirmed taxonomically by Professor Liu Juan，at College of Pharmaceutical Sciences，Jia Musi University，China.The sample was pulverized into powder and passed through a 40-mesh sieve.SMMC-7721 cells were obtained from the Shanghai Institutes for Biological Sciences of the Chinese Academy ofSciences.The monosaccharide standards of rhamnose，arabinose，fructose，glucose，galactose and the T-series Dextran(T-2000，T-500，T-110，T-70，T-40，and T-10)were purchased from Pharmacia.DEAE-52(Shanghai Jinhui).MTT was purchased from Sino-American Biotechnology Co.，Ltd.RPMI-1640 medium was obtained from Gibco.Fetal bovineserum(Hangzhou Sijiqing).Propidium iodide(KeyGen Biotech Corporation).All other chemical reagents were analytical reagent grade. InstrumentsRotary vacuum evaporator(RE-52AA，Shanghai Yarong Biochemical Instrument Factory，China)，UVvisible spectrophotometer(Cary100，Varian，USA)，High-performance liquid chromatography(Agilent 1100，Agilent，USA)，Gas chromatograph(Agilent 6890N，Agilent，USA)，Fourier transform infrared spectrophotometer(Scimitar FTIR-640，Varian，USA)，Flow Cytometer(BD FACS Calibur，BD Biosciences，USA). Extraction of JMPSThe powder of J.mandshurica bark(850 g)was preextracted two times with 95% aqueous ethanol(v/v)in a ratio(material to ethanol solution)of1∶15(g/mL)at room temperature for 12 h each，and the supernatant was removed，in order to remove the pigments.The resulting residues were dried and extracted three times with distilled water in a ratio of material to water 1∶20(g/mL)at 90 ℃for 3 h each，filtered through the gauze and centrifuged at 3000 rpm for 15 min.The supernatants were combined and concentrated by a rotary vacuu m evaporator at 50 ℃to a proper volume，and then deproteinized with Sevag regent［11］.The resulting concentrate was mixed with three times volume of anhydrous ethanol，stirred vigorously and kept overnight at 4 ℃.The precipitates were then collected by centrifugation at 3500 rpm for 15 min，and washed twice with acetone and ether，respectively，dissolved in distilled water，dialyzed against distilled water to remove small molecules and lyophilized，and the brown product was obtained as crude JMPS.Separation and purification of JMPSThe crude JMPS was dissolved in distilled water，and filtered through a0.45 μm membrane filter.The resulting solution of crude JMPS was loaded onto a DEAE-52 column(5 cm×60 cm)［12］.The column was firstly eluted with distilled water followed by 0.1 M and 0.3 M NaCl at a flow rate of 1.0 mL/min，respectively.The eluate was collected automatically(10 mL/tube)，and the presence of carbohydrate in each tube was determined by the phenol-sulfuric acid method.As a result，three fractions wereobtained.They were pooled，concentrated，dialyzed against deionized water and lyophilized，respectively，affording four purified fractions. Determination of molecular weightThe homogeneity and molecular weight of the purified polysaccharides were determined by high-performance gel-permeation chromatography(HPGPC)［13］using an Agilent1100 instrument with a Shodex KS-805(8.0 mm×300 mm，17 μm)column.Mobile phase:distilled water，flow rate of 1.0 mL/min，temperature of 60℃，injection volume of 20 μL，and the elution was monitored by ELSD detector.Standard dextransT-2000，T-500，T-110，T-70，T-40，and T-10 were passed through a Shodex KS-805 column，and elution volumes were plotted against the logarithms of their respective molecular weights.All samples were prepared as 2%(W/V)solut ions and 20 μL of solution was injected in each run，and eluted with distilled water at a fixed flow rate(1 mL/min).Elution volume of the polysaccharide was then plotted in the same graph，andthe average molecular weight of JMPS was measured.Analysis of monosaccharide compositionThe monosaccharide compositions of polysaccharides were analyzed byGC-FID method［14］.Briefly，the polysaccharide sample(2.0 mg)was hydrolyzed with 4 mL trifluoroacetic acid in a sealed-tube at 110 ℃for 2h.The hydrolyzate was repeatedly evaporated with methanol todryness.Then the hydrolyzed products were prepared for acetylation.The acetylation was carried out with 0.5 mL of pyridine by getting heated in a water bath for 30 min at 90 ℃.After incubation，the mixture was cooled at room temperature，and then 0.5 mL of acetic anhydride was added and mixed thoroughly by vortexing.The tube was sealed and incubated in a water bath for another 30 min at 90 ℃.The acetylate was repeatedly evaporated with methanol to dryness.In a similar manner，the monosaccharide standards of rhamnose，arabinose，fructose，glucose and galactose were acetylated.Then，all the derivatives were analyzed by a HP6890N GC equipped with flame ionization detector(FID)and a HP-5 capillary column(30 m×0.32 mm×0.25 μm).The opera tion conditions of GC were as follows:flow rates of N2，H2and air were 45，30 and 300 mL/min，respectively.Column temperature programmed from 170 to 215 ℃at2 ℃/min，then increased to 250 ℃at 8 ℃/min.Detectortemperature:250 ℃.Inositol was used as the internal standard.The injection volume was 1 μL aliquot for each run.Infrared spectral analysis of the polysaccharidesThe structural characteristics of the JMPS fractions were determined using a Fourier transform infrared(IR)spectrophotometer(Scimitar FTIR-640，Varian，USA)［15］equipped with Varian Resolutions software.The purified polysaccharides sample(1 mg)were ground with KBr powder(100 mg)and then pressed into pellets for transform IR spectral measurement in the frequency range of 4000 to 400 cm-1.Assay of anticancer activity in vitro of JMPSCell culture and treatmentThe SMMC-7721 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum，100 units/mL penicillin and 100 mg/mL streptomycin at 37 ℃under a humidified 5% CO2atmosphere.The me dium was changed every other day.The cells were split every 4-5 days by trypsinization and centrifugation，followed by aspiration of the culture medium.Exponentially growing cells were used for theexperiments.Inhibition activity on SMMC-7721 cells of JMPSThe inhibition activities in vitro of JMPS on the SMMC-7721 cells were determined using a MTT colorimetric assay［16］.Briefly，the SMMC-7721 cells in the logarithmic growth phase were collected and resuspended in RPMI-1640 medium at a density of 1×104 cells/mL，100 μL the cellsuspension were pipetted into each well of 96-well flat-bottomed microtiter plates.After that，50 μL fresh medium(control group)and test sample(purified JMPS at a final concentration of 25，50，100 and 200μg/mL)was added to each well，and the cells were incubated for 48h，respectively.Three replicate wells were used for each data point in the experiments.After incubation for the indicated intervals，20 μL of MTT(5 mg/mL in PBS)solution was added to each well and plates were then incubated for 4 h at 37 ℃.After the logarithmic growth phase of cells was reached，the supernatant was discarded.Intracellular formazan crystals were dissolved by adding 150 μL of DMSO to each well，and the plates were shaken for 10 min.The absorbance was read at 490 nm with a microplate reader.The inhibition rate was calculated according to the formula below:Inhibition rate(%)=(1-Abssample/Abscontrol)×100DNA content and cell cycle analyzedThe DNA content and cell cycle of the SMMC-7721 cells were detected using FCM［17］.The SMMC-7721 cells in logarithmic growthphase(3×105 cells/mL)were plated at 1 mL/well in 6-well plates and allowed to attach overnight.After 24 h，the various JMPS-1 were added to the wells in a volume of 1mL per well to a final concentration of 50，100 a nd 200 μg/mL.An equal volume of medium was added to the wells in control group.The plates were incubated in 5% CO2at 37 ℃for 48 h.Then，cells were harvested.Cells harvested with 0.25% trypsinization were sedimented by centrifugation at 1000 rpm for 5 min at roomtemperature.After the supernatant was removed，70% icecold ethanol was added.Finally，cells were incubated in PI/RNase Staining Buffer，according to the manufacturer’s instructions and the cell cycle was analyzed with a BD FACS Calibur Flow Cytometer(BD Biosciences，USA).The percentage of cells in different cell cycle phases(G0/G1，S，and G2/M phase)was calculated using MultiCycle AV DNA Analysis DEMO. Results and DiscussionIsolation and purification of polysaccharidesThe crude JMPS was obtained in an overall yield of 0.81% through a series procedure of defatting，hot water extraction，ethanol precipitation，deproteiniation，dialysis and lyophilization.Furthermore，the crude JMPS was fractionated by a column of DEAE-52 cellulose to afford four polysaccharide fractions as shown in Fig.1.The four fractions were concentrated，dialyzed and lyophilized，affording JMPS-1，JMPS-2a，JMPS-2b and JMPS-3，respectively(Fig.1).Fig.1 Chromatogram of eluted JMPS on DEAE-52 columnMolecular weight and monosaccharide compositions of JMPSThe HPGPC-ELSD analysis showed that the average molecularweight(Mw)of JMPS-1，JMPS-2a，JMPS-2b and JMPS-3 were approximately 227.9 kDa，28.6 kDa，23.1 kDa，and 12.7 kDa，respectively.The monosaccharide compositions JMPS-1，JMPS-2a，JMPS-2b and JMPS-3 determined by GC-FID analysis showed that the four polysaccharides were composed of monosaccharide(Table 1).JMPS-1 was composed of rhamnose(Rha)，arabinose(Ara)，xylose(Xyl)，mannose(Man)，glucose(Glc)and galactose(Gal)in a molar ratio of1.00∶1.64∶2.12∶11.31∶7.46∶26.58，respectively.JMPS-2a and JMPS-2b were composed of Rha，Man and Glc in a molar ratio of 1.00∶1.19∶9.50 and 1.00∶2.07∶37.02，respectively.JMPS-3 was composed of Man，Glc and Gal in a molar ratio of 1.38∶3.67∶1.00.Table 1 Molecular weight and monosaccharid e compositions of JMPSa“-”:UndetectableFT-IR spectra of of JMPSFig.2 FT-IR spectra of JMPS-1(a)，JMPS-2a(b)，JMPS-2b(c)and JMPS-3(d) As shown in Fig.2，the FT-IR spectra of four fractions were found to be similar.The strong and broad absorption peak between 3600 and 3200 cm-1(JMPS-1∶3429.32 cm-1，JMPS-2a∶3455.16 cm-1，JMPS-2b∶3436.77 cm-1，JMPS-3∶3454.48 cm-1)，represented the stretching vibrations of the hydroxyl groups.The small band at around 2926 cm-1(JMPS-1∶2928.60 cm-1，JMPS-2a∶2926.59 cm-1，JMPS-2b∶2931.04 cm-1，JMPS-3∶2929.30 cm-1)was attributed to the C-H stretching，and absorption peak at around 1340 cm-1(JMPS-1∶1384.42 cm-1，JMPS-2a∶1384.61 cm-1，JMPS-2b∶1384.51 cm-1，JMPS-3∶1401.35 cm-1)for C-H bending vibrations.The bound at 1636.43 cm-1(JMPS-1)，1606.60 cm-1(JMPS-2a)，1640.53 cm-1(JMPS-2b)and 1623.69 cm-1(JMPS-3)were due to the bound water.Each particular polysaccharide had a specific band in the 1200～1000 cm-1 region.This region was dominated by ring vibrations overlapped with stretching vibrations of(C-OH)side groups and the(C-O-C)glycosidic band vibration.Furthermore，characteristic peaks at 1746 cm-1(JMPS-1∶1746.31 cm-1，JMPS-2a∶1744.57 cm-1，JMPS-2b∶1749.60 cm-1，JMPS-3∶1744.31 cm-1)for esterified carboxylic groups(-COOR).These characteristic absorptions indicated that the JMPS-1，JMPS-2a，JMPS-2b and JMPS-3 were polysaccharides［18］.Inhibition activities of JMPS on SMMC-7721 cellsFig.3 showed the inhibition rate of the purified JMPS(JMPS-1，JMPS-2a，JMPS-2b and JMPS-3)on the SMMC-7721 cells at different concentration for 48 h.Under the experimental conditions，four samples exhibited a marked inhibition on the survival of SMMC-7721 cells in a concentration-dependent manner.From Fig.3，the inhibition activities of the polysaccharides JMPS-1 and JMPS-3 increased significantly with the increasing of sample concentration ranging from 25 to 200 μg/mL，the inhibition rate was 15.3%-53.6% for JMPS-1 and 8.8%-20.6% for JMPS-3，respectively.However，the inhibition activities of JMPS-2a and JMPS-2b were significant weaker than that of JMPS-1 and JMPS-3，even at the high dose(200 μg/mL)，the inhibition activities were only 11.3% and10.7%.From the figure，inhibition activities in the order was JMPS-1＞JMPS-3＞JMPS-2a＞JMPS-2b.JMPS-1 had strong inhibition activities against SMMC-7721 cells.Fig.3 Inhibition rate of four JMPS on the SMMC-7721 cells at different concentrations for 48h.Results were presented as means±standard deviations(n=3)DNA content and cell cycle analyzed by flow cytometryThe SMMC-7721 cells were treated with the JMPS-1 on the SMMC-7721cells at different concentrations for 48 h.The cell cycle phase distribution of SMMC-7721 cells was analyzed by flow cytometry with PI staining and the percentages of cells in G0/G1，S and G2/M phases were calculated using MultiCycle AV DNA Analysis DEMO software.Fig.4 Flow cytometry plots of JMPS-1.The data were expressed asmean±SD of three experiments.Flow cytometry analysis showed range in the percentages of cells under different cell cycle stages after JMPS-1 treatment(Fig.4).For JMPS-1，the percentage of G0/G1 stage cells decreased while that of S and G2/M stage cells pared with control group cells(9.081%)，the G2 group(indicated as %)significantly increased after SMMC-7721 cells were exposed to JMPS-1(50 μg/mL∶12.909%，100 μg/mL∶13.836%，200μg/mL∶17.071%);Compared with control group cells(14.063%)，the S group(indicated as%)significantly increased after SMMC-7721 cells were exposed to JMPS-1(50 μg/mL∶14.564%，100 μg/mL∶16.261%，200μg/mL∶20.346%).These results suggest that JMP S-1 could induce cell cycle arrest at G2/M and S phase in SMMC-7721 in a dose-dependent manner.ConclusionIn the present study，the crude JMPS were obtained by hot water extraction，ethanol precipitation and deproteination with Sevag method，and then fractionated by DEAE-52 column chromatography to afford four purified fractions of JMPS-1，JMPS-2a，JMPS-2b and JMPS-3.The HPGPC-ELSD analysis showed that the average molecular weight of JMPS-1，JMPS-2a，JMPS-2b and JMPS-3 were 227.9 kDa，28.6 kDa，23.1 kDa，and 12.7 kDa，respectively.The monosaccharide compositions of polysaccharides were analyzed by GC-FID method.The structural characteristics of the JMPS fractions were determined by IR spectrophotometer.The evaluation of anticancer activity suggested that JMPS-1 had strong inhibition activities for SMMC-7721 cells.Flow cytometry analysis showed that JMPS-1 can induce cell cycle arrest atG2/M and S phase in SMMC-7721 in a dose-dependent manner.The results suggested that JMPS-1 has demonstrated anticancer activities and may be explored as a natural 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