Extraction and deproteinization of pumpkin polysaccharide
GANGLIANG HUANG,YA CHEN,&XUEQIN WANG
College of Chemistry,Chongqing Normal University,Chongqing 400047,China
Abstract
Extraction and deproteinization methods of polysaccharide from pumpkin were studied.We extracted crude polysaccharide by the method of hot water extraction.The percentages of deproteinization and polysaccharide loss were compared as indexes using the trichloroacetic acid (TCA)method,the NaCl method,the and CaCl 2method,respectively.The results showed that the TCA method exhibited the lowest percentage of deproteinization,and a little higher percentage of polysaccharide loss than the other two methods.The CaCl 2method excelled over the NaCl method in deproteinization.
Keywords:Pumpkin,polysaccharide,extraction,deproteinization,comparison
Introduction
Polysaccharides,present in almost all organisms,have many biological functions,such as energy storage,
structure support,antigenic determinant,and so on.Because the separation,purification,composition determination,and structural analysis of polysacchar-ides have made remarkable progress,as well as their biological functions now being further understood,research of polysaccharides is catching researchers’attention again (Huang 2008).Polysaccharides and their derivatives have special functions;for example,anti-inflammation,anti-radicalization,lowering blood lipid,acting as an anticoagulant,reactive oxygen species scavengers (Nishizawa et al.2008),and biological response modifier.They have therefore become the one of the research hotspots of natural medicine in recent years.
The pumpkin is a gourd-like squash of the genus Cucurbita and the family Cucurbitaceae (which also includes gourds).It generally contains large amounts of polysaccharide,amino acid,and vitamins such as B 1,B 2,and C,as well as several active ingredients,including protein and carotenoid (Nara et al.2009).Pumpkin polysaccharide is a kind of non-specific immunopotentiator.It can improve the immune function and promote the production of cytokine.By activating the pathway of complement and the other
pathways,pumpkin polysaccharide plays a variety of adjusting functions to the immune system.Pumpkin polysaccharide is composed of glucose,glucuronic acid,galactose,arabinose,xylose a
nd rhamnose,and it has antioxidative activity (Yang et al.2007).Based on the important biological functions of pumpkin poly-saccharide,its extraction and deproteinization have been studied.The aim of the present work was to provide an appropriate approach to obtain more pure pumpkin polysaccharide for further tests in in vivo and in vitro experiments.Three methods of deproteiniza-tion were investigated,including the trichloroacetic acid (TCA)method,the NaCl method,and the CaCl 2method.
Materials and methods General
Pumpkin was purchased from the local vegetable market.Infrared (IR)spectra were recorded with Fourier transform IR apparatus,and wavenumbers are reported per centimeter.The concentration of proteins (C )was determined by ultraviolet absorption using the relationship:
C ¼1:45A 28020:74A 260;
ISSN 0963-7486print/ISSN 1465-3478online q 2011Informa UK,Ltd.DOI:
10.3109/09637486.2011.560566
Correspondence:Gangliang Huang,College of Chemistry,Chongqing Normal University,Chongqing 400047,China.E-mail:huangdoctor226@163
International Journal of Food Sciences and Nutrition ,September 2011;62(6):568–571
where A280and A260are the absorbances at280and 260nm,respectively.This method will correct for any interfering absorbance due to nucleic acid present in the solution.The concentration of pumpkin poly-saccharide was determined by the phenol–sulfuric acid method using glucose as standard(Huang et al. 2010).
Extraction of crude pumpkin polysaccharide
The water-soluble pumpkin polysaccharide was obtained from200g pumpkin by extraction with water (2l)at1008C for0.5h,then608C for2h,and cooled to room temperature.After centrifugation and super-natant liquor concentration,the pumpkin polysacchar-ide was precipitated by adding200ml(4volumes)of absolute ethanol.The precipitate was washed with absolute ethanol and diethyl ether,respectively. Deproteinization by the TCA method
The concentrated solution of crude pumpkin poly-saccharide was adjusted to pH3with10%TCA solutio
n,and kept overnight.The sample was centrifuged for10min at5,000rpm,and the precipitate was discarded to obtain the deproteinized solution.This procedure was repeated three or four times.The pumpkin polysaccharide was precipitated with3–4volumes of ethanol from the aqueous phase. Deproteinization by the NaCl method
The concentrated solution of crude pumpkin poly-saccharide was adjusted to pH9–10with2%NaOH solution under the boiling condition.The NaCl solid was added up to the concentration of5%(w/v),then mixed round and boiled for30min.After cooling to room temperature andfiltering,thefiltrate was adjusted to pH7with dilute HCl.The pumpkin polysaccharide was precipitated by adding4volumes of absolute ethanol.This procedure was repeated two or three times.Afterfiltration,the precipitate was washed with absolute ethanol and diethyl ether, respectively.Drying at low temperature and abrading into dust,the product was obtained. Deproteinization by the CaCl2method
The concentrated solution of crude pumpkin poly-saccharide was adjusted to pH8–9with2%NaOH solution,and was heated to858C.The CaCl2solid was added up to the concentration of5%(w/v),then mixed round,cooled andfiltrated.The latter process was like the NaCl method.
Separation of deproteinized polysaccharide
The deproteinized polysaccharide was separated by column chromatography on a Sephadex G200 (Pharmacia,Uppsala,Sweden)and washed with distilled water.The polysaccharide solution was collected,which was identified to contain a single composition by the phenol–sulfuric acid method.The more pure polysaccharide was obtained according to the above-mentioned process.
Results and discussion
IR spectra analysis of pumpkin polysaccharide deproteinized by three methods
The crude pumpkin polysaccharide was extracted by the hot water extraction method.Starting with a boiling step in water for its initial extraction,this would possibly denature all proteins and remove them by centrifugation.The crude polysaccharide was deproteinized by the TCA method,NaCl method,and CaCl2method,respectively.The deproteinized polysaccharide was separated by col-umn chromatography on a Sephadex G200to give water-soluble pumpkin polysaccharide with better purity.Figure1shows the IR spectra of pumpkin polysaccharide.The spectra contain absorption bands arising from the n(CC)and the n(COC)stretching vibrations at1,126cm21,a band at890cm21 assigned to the corresponding b-glycosidic bond (C12H)deformation mode,a band at835cm21 assigned to the corresponding a-glycosidic bond deformation mode,and the highest intensity of the n(OH)band at lowe
r frequency(3,450cm21).The presence of a carbonyl group band at1,650cm21 proved the residual protein in the pumpkin poly-saccharide samples.
Comparison of the three methods for deproteinization TCA,NaCl,and CaCl2as the reagents were investigated for deproteinization of crude pumpkin polysaccharide.The principle of the TCA method is that the protein cation can bind the TCA to form an insoluble salt for precipitation at pH,pI(isoelectric point).The NaCl method and CaCl2method are used for deproteinization because the protein can be salted out to precipitate with them.The results are shown in T able I.They indicate that the TCA method exhibited the lowest percentage of deproteinization,and a slightly higher percentage of polysaccharide loss than the other two methods,which may be due to more evident damage of pumpkin polysaccharide caused by TCA.At the same time,they prove that the CaCl2 method excelled over the NaCl method in deprotei-nization.The Sevage method(Staub1965)was not used to deproteinize herein,because it contains poisonous chloroform that is environmentally disadvantageous.
Conclusions
The pumpkin polysaccharide was extracted by the hot water extraction method,and indicated that
Extraction and deproteinization of pumpkin polysaccharide569
70(a)
(b)
reactive carbonyl species
(c)
T /(%)
T /(%)
T /(%)
6050403020100
907560453015
908070605040
30
375035003250300027502500Wave number/(1/cm)225020001750150012501000750
500
375035003250300027502500Wave number/(1/cm)
225020001750150012501000750
500
375035003250300027502500Wave number/(1/cm)
225020001750150012501000750
500
Figure 1.IR spectrum (in KBr)of pumpkin polysaccharide deproteinized by (a)the TCA method,(b)the NaCl method,and (c)the CaCl 2method.
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the CaCl2method had good ability for deproteini-zation.
Declarations of interest:The present work was supported by Foundation Project of Chongqing Normal University(No.10XLZ004),Natural Science Foundation Project of CQ CSTC(numbers CSTC, 2009BB5238and2009JJ1075),and Chongqing Education Commission Foundation(number KJ080810),China.
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Nishizawa A,Y ukinori Y,Shigeoka S.2008.Galactinol and raffinose as a novel function to protect plants from oxidative damage.
Plant Physiol147:1251–1263.
Staub AM.1965.Removal of proteins:Sevage method.Methods Carbohydr Chem5:5–6.
Y ang XB,Zhao Y,Lv Y.2007.Chemical composition and antioxidant activity of an acidic polysaccharide extracted from Cucurbita moschata duchesne ex poiret.J Agric Food Chem55: 4684–4690.
T able I.Comparison of the three techniques for deproteinization.
Method
Percentage
of deproteinization(%)
Percentage of pumpkin
polysaccharide loss(%)
TCA92.8^0.510.9^0.6
NaCl96.6^0.4  5.0^0.9
CaCl297.3^0.7  4.8^1.2
Data presented as the mean^standard deviation of three separate
determinations.
Extraction and deproteinization of pumpkin polysaccharide571
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