巴戟天水溶性多糖分离纯化的研究

第2l卷 第3期 2006年 6月 云南农业大学学报 Journal of Yunnan Agricultural University Vol_21 No．3 Jun．2006 Isolation of W ater Soluble P0lysaccharides from Morinda officinalis How HE Chuan bo’，CHEN Ling ，Ll Lin ，GUO Si—yuan‘，LI Bing ，WU Shao-xiong (1．School of Light Chemistry and Food Science，South China University of Technology，Guangzhou 5 10640，China； 2．Kunming Medical College，Kunming 65003 1；China) Abstract： I1le water soluble polysaccharides were extracted with hot water and subsequently precipita— ted with ethano1 from Morinda officinalis How(MOP)．And the crude polysaccharides were then isola- ted by anion-exchange chromatography．Effect of different chromatography conditions on adsorption and ehtion of MOP were discussed，and the results showed that，the adsorbent exhibited better adsorption performance for MOP under higher pH and lower ion strength of buffer solutionn．Suitable column chromatography condition obtained was：Tris concentration of buffer 0．02 mol／L，pH of buffer 8．0～ 8．5 and flow rate 1．5 mL／min．Four polysaccharide fractions。named MOHP．I，MOHP-Ⅱ，MOHP— m and MOHP-IV respectively were obtained using step-wise elution manner． Key words：Morinda offwinalis How；polysaccharides；isolation；effect CLCN：Q 539 Document code：A Artide ID：1004—390X(2006)03—0320—04 巴戟天水溶性多糖分离纯化的研究 何传波 ，陈 玲 ，李 琳 ，郭祀远 ，李 冰 ，吴少雄 (1．华南理工大学轻工与食品学院，广东 广州510640；2．昆明医学院，云南 昆明650031) 摘要：采取水提醇沉工艺获得了巴戟天粗多糖MOP，用离子交换柱层析方法对粗多糖进行了分级纯化，考察了 不同层析缓冲液条件下，巴戟天多糖的动态吸附情况。结果表明：较高 pH值和低离子强度的缓冲液有利于巴 戟天多糖的吸附，确定层析用缓冲液pH值为8．0—8．5，Tris浓度为0．02 mol／L，流速1．5 mL／min；在此层析条件 下，采用阶段梯度洗脱方式，在NaCI浓度为0，0．1和0．5 mol／L时分别获得巴戟天多糖组分 MOHP—I，MOHP — II，MOHP一Ⅲ，用NaOH洗脱得到了MOHP一1V组分。 关■词：巴戟天；多糖；分离；影响 Polysaccharide iS another important macromolec． ular compound apart from nucleic acid and protein． Within the last feW years。tremendous advances have been made in the research of p0lvsaccharide8．Later WOrk has shown that polysaccharides are involved in a number of biochemically important functions，such as cel1．cell interaction and communication．attachment for infectious bacteria，viruses，toxins and hormones， to mention just a few．Recent research of polysaccha- tides mainly focus on the isolation，structure，phar- macology and structure·activity relation，among of which，isolation of polysaccharides is a prerequisite f0r other research． Morinda officinalis How(family Rubiaceae)is a 收稿日期：2005—11—04 基金项目：广东省科技攻关项目资助(2003c104025) 作者简介：何传波(1978一)，山东茌平人，博士研究生，主要从事多糖物质及其综合利用研究。 维普资讯 http://www.cqvip.com 第3期 何传波，等：巴戟天水溶性多糖分离纯化的研究 321 plant extensively used as a Yang—tonic agent for about 2 000 years in China．The plant grows mainly in hu- mid areas of southeast China，such as Guangdong，Fu- jian，Guangxi Province．Previous research showed that【 ．the water or ethanolic extracts of Morinda offi— cinalis exhibited anti-aging，antidepressant and immu- nocompetence promotion action．Owing to being abun— dant in carbohydrates，anthraqui none，amino acid， organic acid and vitamin，Morinda officinalis is a valu— able plant suitable for both medicinal and food use． Carbohydrates，accounting for 49．79％ ～58．25％ ，is a significant constituent of Morinda oJ)i~：inalis．CAI et alI 3 ． researched the water extracts of Morinda offici— nalis．Using chemical and spectroscopic methods，they obtained four inulin-type oligosaccharides-nystose，1 F- fructofuranosylnystose(O·B-D-fructofuranosyl一[(2---~ 1)·O·B-D fructofuransy1]3- -D-ghcopyranoside)， inulin-type hexasaccharide (O-B-D-fructofuranosyl- [(2-÷1)-O-B-D-fructofuransy1]4- ·D-glucopyrano- side)and inulin-type heptasaccharide(O-B-D-fructo- furanosyl_[(2-÷1)一O·B-D-fructofuransy1]5⋯ot D glu- copyranoside)respectively．Through animal experi- ments．ZHANG et al E ． drew the c0nclusion that，the water extracts of Morinda offwinalis mainly including these oligosaccharides possessed antidepressant effect． This study is concerned with the isolation of wa- ter soluble polysaccharides present in Morinda offici— nalis，which has not been reported previously．Effects of various chromatography conditions such as pH，ion strength and velocity of mobile phase on polysaccha rides adsorption are investigated in detail．The optimi- zation of protocol for the isolation will help to prepare well—defined polysaccharides fractions in sufficient a- mount to ensure further research in structure and pharmacology． 1 M aterlal and Methods 1．1 M aterials The Morinda offwinalis were purchased from the planter in Deqing County， Guangdong Province，Chi- na．DEAE Sepharose CL-6B was obtained from Phar- macia Biotech(Uppsala，Sweden)．All other rea- gents were of analytical grade． 1．2 Methods 1．2．1 Extraction of polysaccharides from Morinda officinalis The ground dried roots of Morinda officinal& (200 g)were defatted with acetone．The defatted meal was extracted with distilled water(3 L)at 60℃ stirring for 3 h．Th e extractive was centrifugated at 8000 r／min for 20 min in a high—speed centrifuge (model 3K3D，Sigma，Germany)．The pellet was re- suspended，and this procedure was repeated again． All the supernatants were collected，and concentrated to 1／5 volume under vacuum subsequently．The resi· due was extracted with Sevag reagent(CHC13： BuOH =4：1)five times to deproteinize．After removing the Sevag reagent，nine volumes of absolute ethanol was added to the extract and kept at 4℃ overnight in re- frigerator to precipitate polysaccharides．The precipi— tate was redissolved in distilled water，then added to absolute ethano1．This procedure must be repeated three times to obtain polysaccharides as more as possi— ble．Lastly．the precipitate was frozen at一80℃ o- vernight and lyophilized in vacuum freeze dryer (model ALPHA 2—4，Christ，Germany)to obtain 10．6 g brown polysaccharides powder，named MOP． 1．2．2 Fractional isolation of MOP Solutions of MOP were fractionated on a column (50 cm×2．6 cm)of DEAE Sepharose CL-6B ] previously equilibrated with the start buffer． Elution was carried out under different velocity using Tris-HC1 buffer solution with different Tris concentration and pH as eluent．Fractions were collected(5 mL)and analysed for total carbohydrate content and protein by the phenol--sulfuric acid method and spectrophotomet-- ric．Th e breakthrough curve of MOP was drawn ac- cording with the total carbohydrate content of each fraction，as well as the adsorption capacity of MOP on the anion-exchange adsorbent was calculated． 1．2．3 Determ ination of total carbohydrate content Total carbohydrate content was determ ined by the modified phenol-sulfuric acid method as follows．2 mL of appropriate dilute sample(concentrations between 20—100 tLS／mL 1 was mixed with 1 mL of 80％ 维普资讯 http://www.cqvip.com 322 云南农业大学学报 第21卷 (W／W)phenol solution ，then incubated with 5 mL sulfuric acid for 20 min at room temperature．The absorbance of the solution was measured at 490 nm a。 gainst the water blank．A calibration curve using glu- cose as standard was estabhshed． 1．2．4 Determination of protein The fractions were determ ined the protein content using spectrophotometric analysis measured at 280 nm against the water blank． 2．1 Em ct 0f I 8 concentration of ehent on MOP adsorption rIIe water extracts of Morinda officinalis．MOP were dissolved in the Tris—HC1 buffer of pH =8．0 which Tris concentration was 0．02。0．05。0．1 and O．2 mol／L respectively．and continuously pumped on— to the column packed with DEAE Sepharose CL B at a flow rate of 1．5 mL／min．Th e breakthrough curve was drawn according with the total carbohydr~e con． tent change of each fraction，and shown as Fig．1． And the adsorption capacity under each s concen- tration was 0．11，0．053，0．O44 and 0．047 mg MOP／ mL adsorbent． O 5 lO l5 2O 25 eluent number 图1不同Trls浓度下巴戟天多糖的穿透曲线 Fig．1 Breakthrough curves ofMOP with different"Iris concentration of eluent 2．2 Effect of pH of eluent on MOP adsorption Fig．2 presented the changes in breakthrough curve with different pH(7．0，7．5，8．0，8．5 and 9．0)of ehent buffer，and other conditions were： 0．02 mol／L of Tris concentration，1．5 mL／min of flow rate． Corresponding adsorption capacity 0．12，0．11，0．14 and 0．17 mg MOP／ mL adsorbent． eluent num ber 图2不同pH下巴戟天多糖的穿透曲线 Fig．2 Breakthrough curves ofMOPwithdifferentpHofeluent 2．3 Effect of velocity of ehent on MOP adsorption The influence of different velocity(0．5。1．0。 1．5 and 2．0 mL／min)of ehent on MOP adsorption was illuminated by the breakthrough curves in Fig．3， and used ehent was Tris．HCI solution of pH =8．0 with Tris concen~ation 0．02 mol／L．Th e calculated adsorption capacity data was 0．13，0．12，0．12 and 0．096 mg MOP／mL adsorbent respectively． O 5 lO l5 2O 25 ehentnum ber ． 图3不同流速下巴戟天多糖的穿透曲线 Fig．3 Breakthrough curvesofMOP trader differentvelocity of~iucnt 2．4 Elution profile for the isolation of MOP On the basis of the above adsorption expefi． ments，we discussed the ehtion conditions of the MOP．Solutions(5 mL)of MOP(50 mg)in 0．02 mol／L Tris—HCl buffer of pH =8．0 were loaded onto the column．Elution was carried out with the salne buffer containing 0，0．1，O．2，O．5，1．O，2．0 mol／L NaCl successively and finally with 1．0 moVL NaOH at a flow rate of 1．5 mL／min．Th e column fractions (5 mL)were monitored for total carbohyrdrates con— tent and protein using the phenol—sulfuric acid and 6 5 4 3 2 l O O O O O O O 0 日 —P—口晌。对 6 5 4 3 2 l O O O O O O O 03I器 0s案 维普资讯 http://www.cqvip.com 第3期 何传波，等：巴戟天水溶性多糖分离纯化的研究 323 UV-spectrophotometric．The elution profile was shown in Fig．4． 图 4 Fig．4 3 Discussion eluent numble 巴戟天多糖在DEAE Sepharose CL．6B柱上的洗脱曲线 Elution profile 0f MOP on DEAE Sepharose CL-6B column It is known to us all that the primary principle of ion-exchange chromatography is the ion exchange of adsorbent and the isolated sample，so the presence of ion is prerequisite．Polysaccharides are often weakly ionized in alkaline solution[ ， and the ionization will weaken when the quantity of free ion in solution in crease，which is disadvantageous to the adsorption of polysaccharides on adsorbent．So there should be less free ion in buffer as possible as can in order to benefit for the adsorption of polysaccharides．The data of ad— sorption capacity under different Tris concentration al- so verified that，the adsorption capacity had a down— trend with the increase of Tris concentration，and had a highest value when the Tris concentration was 0．02 m0l／／L_But the breakthrough curves in Fig．1 dis- played that，with the decrease of Tris concentration， the time of reaching adsorption equilibrium had a slight increase．Besides，a certain numble of free ion was essential to maintain the buffer capacity of solu- tion．Th erefore．0．02 m0l／／L of Tris concentration was appropriate to adsorption of MOP． pH of buffer solution had great effect on the ad- sorption of MOP．The creased with the incre adsorption capacity of MOP in- ment of pH，while the break- through curves in Fig．2 fight moved obviously，predi- cating the prolong of adsorption equilibrium time． This is due to the alkaline environment is advantage to ionize of polysaccharide，which will reinforce the ion exchange，resulting in more polysaccharides being ad- sorbed onto adsorbent．On the other hand，exorbitant pH will bring too firm attraction between polysaccha- rides and adsorbent，therefore，resulting in difficulty for the sequent elution．We determined the recovery polysaccharides after chromatography only 50％ of the initial polysaccharides．So，there was a balanced pH to guarantee both adsorption capacity of MOP and fa- cility of elution operation．In this experiment，8．0— 8．5 was the exact pH range for adsorption of MOP． Velocity of eluent adopted in chromatography al- so influences adsorption of polysaccharides．High ve- locity will enhance the radial and axial diffusion in chromatography column，hence，broaden flow unit and increase the height equivalent to one theoretical plate，which resuh in the reduce of the adsorption of polysaccharides on adsorbent． Low velocity can im- prove adsorption capacity，but will increase the no- lecular diffusion and prolong elution time．In the H-u curve of Van Deemter equation，there is a optimal ve- locity for chromatography．The curves in Fig．3 illu- minated obvious prolong of time for adsorption equi- librium， and data of adsorption capacity changed slightly．Taken together，we choose 1．5 mL／min as proper velocity． Following the above adsorption experiments，the elution conditions were researched． Using step—wise elution manner，we respectively obtained MOHP-I (21．8 mg)，MOHP—I1(6．9 mg)，MOHP·11I(12．2 mg)with 0，0．1，0．5 mol／L of NaC1 concentration and MOHP-IV(3．7)with NaOH．Elution profile Fig．4 showed that，there were no fractions under high NaCl concentrations such as 1．0 and 2．0 mol／L．Ab， sorbance curve at 280 nm revealed that there con． tained protein in all fractions except for MOHP-I． Experiments data also showed that the DEAE Sepha- rose CL-6B was reused up to five cycles without de— crease in its capacity． (下转第 328页) 维普资讯 http://www.cqvip.com 328 云南农业大学学报 第2l卷 (上接第323页) In conclusion，a simple，easy and cheap proto- c0l for the isolation of polysaccharides from Morinda officinalis using anion-exchange chromatography was achieved．The acquirement of purified polysaccharide could assure the demand for next research in structure and pharmacology of Morinda off~cinatis． [References] [1] ZHANG Z Q，LI Y．The effect of Morinda offi,einalis How，a Chinese traditional medicinal plant，on the DRL 72一S schedule in rats and the forced swimming test in mice[J]．Pharmacology，Biochemistry and Behavior， 2002，72 ：39—43． [2] CAI B，CUI c B．Antidepressant effect of inulin-type ol- igosaccharides from Monnda D伽 协 How in mice [J]．Chin J Pharmacol Toxicol，1996，10：109—112． [3] CHEN Q S．Morinda officinalis How[A]．In：WANG Y． S，DENG W-L，XUE CS，editors． Pharmacology an d [4] [5] [6] [7] applications of Chinese materia medica[c]．Bering： People S Health Press。l988：227． NAVARNI L，GILLI R，GOMBAC V．Polysaecharides from hot water extracts of masted Coff~a arabica beans： isolation and characterization[J]．Carbohydrate Poly· melYi，l999，40：71． SAROJ K M，BIMAIENDU R．Cell-wall pulysaceharides from the fruits of L／mon／ta 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