野木瓜水溶性多糖的分离纯化及抗氧化活性研究

王文平 ，田 亮，吴国卿，唐维媛，王明力，郭祀远

1贵州大学贵州省发酵工程与生物制药重点实验室，贵阳 550003;2 华南理工大学轻工与食品学院，广州 510640

Introduction

Chaenomeles cathayensis is known as Chinese quince in China.As one of the most important economic plants，the fruit of C.cathayensis is a specialized natural resource of Zhengan county (Guizhou，China).It can be used to produce various types of foods，beverages，drinks，preserved fruit，wine and fruit vinegar，etc.Owing to being abundant in carbohydrates，organic acids，amino acids，proteins，minerals and vitamins as well as some bioactive components such as trierpenoid saponins，flavonoids，polysaccharides，pectin and superoxide dismutase (SOD)［1］，C.cathayensis is a valuable plant used for both food and medicine.

The chemical constituents of C.cathayensis have been also investigated within the last few years，but only some constituents of low molecular weight had been isolated and identified［2］.Polysaccharides are one group of important macromolecular compounds in the fruit of C.cathayensis，however，few studies have been reported on them.In recent years，plant polysaccharides have emerged as an important class of bioactive natural products.A wide range of polysaccharides were reported to exhibit a variety of antioxidant ability［3，4］.In addition，further investigations were also shown that polysaccharides were associated with anticancer，antivirus，reducing blood-glucose and blood-fat，anti-inflammatory，antibacterial and immunological activities，etc［5，6］.

The isolation，structure and molecular weight，as well as the anticomplement activity of the water-soluble polysaccharides from C.cathayensis were discussed in our previous research［7，8］.The aims of the present study were to isolate and purify polysaccharide from C.cathayensis and to evaluate the antioxidant activities of the isolated component as well as its concentration-activity relationship.It is expected that C.cathayensis can be used as a natural antioxidant agent or functional food.

Materials and Methods

Plant materials and chemicals

Fresh fruits of C.cathayensis were purchased from Guizhou Tianlou Food Co.，Ltd.(Zunyi，China).Sepharose CL-6B was purchased from Fluka (Neu-Ulm，Switzerland).DEAE-Cellulose was obtained from Shanghai Reagents Company (Shanghai，China).Other reagents were of analytical grade and purchased from local markets.All authentic standards were obtained from Sigma (St.Louis，USA).

Extraction and preparation of crude polysaccharides

The dried sample was grinded and subsequently treated with hot distilled water to extract water-soluble polysaccharides.The extracts were concentrated by a rotary evaporator at 50 ℃.The water-soluble polysaccharides were precipitated out from the concentrated extracts with 95% ethanol.The isolated crude water-soluble polysaccharides were dried in vacuum after being sequentially washed by ethanol，acetone and ethyl ether.Crude C.cathayensis polysaccharides (CCCPs)were prepared and kept at 5 ℃for further purification.

Purification of CCCPs

Sevage reagent［9］was added to the crude polysaccharides solution to denature and precipitate protein and D101 macroporous resin was experimentally selected to remove pigment，and then dialyzed against distilled water in a dialysis bag (molecular weight cut off=10000 μ)under magnetic stirring for 72 h.The dialysate was vacuum-concentrated，precipitated by 95%ethanol，and then vacuum-dried.The fine CCPs (FCCPs)was obtained finally.FCCPs was fractionated by anion-exchange chromatography on a pretreated DEAEcellulose column.The sample was dissolved in distilled water and applied onto the column.The column was first eluted with distilled water at a flow rate of 1.0 mL/min followed by NaCl solution (0(1 mol/L).Fractions of 10 mL was collected and monitored for the presence of polysaccharides using the phenol-sulfuric acid method［10］.The elution profile was drawn according with the number of testing-tube (X-axis)as well as the absorbance (Y-axis).FCCPs were thus separated into a main fraction CCP1.CCP1was further purified on a Sepharose CL-6B column，eluted with 0.1 mol/L of NaCl.Eluent of 5 mL was collected and monitored by phenol-sulfuric acid method.The absorbance (Y-axis)was plotted against the number of testing-tube (X-axis)to build the elution profile.

Determination for monosaccharide composition

The monosaccharide components analysis was determined by high performance liquid chromatography(HPLC)analysis.Different monosaccharide standards were dissolved in distilled water at a concentration of 1% (w/v).20 mg of CCP1sample was subjected to hydrolyze with 2 mol/L of H2SO4in a dry oven for 8 h at 110 ℃［11］.BaCO3was used for neutralization of the hydrolysate followed by centrifugation.The supernatant was filtered through a 0.45 μm membrane filter before sample injection，and the monosaccharide composition and molar ratio of CCP1were determined according to retention time and peak area.

Antioxidant activity of polysaccharides

Assay of hydroxyl radical scavenging activity［12］

Hydroxyl free radical (·OH)were produced by Fenton reaction under weak basic environment.In the system，3.0 mL of 100 mmol/L phosphate buffer solution(pH 7.4)，1.0 mL of 5.5 mmol/L FeSO4，1.0 mL of 6.5 mmol/L EDTA-2Na，1.0 mL of 0.2% H2O2，1.0 mL of 10 mmol/L sodium salicylate and 0.5 mL of sample solution with different concentrations were mixed and topped up to 8.0 mL using distilled water.No H2O2but only solvent was added in the control group.The mixture was incubated in water bath at 37℃for 1 h，and then the absorbance AXof tube-sample and ACof tube-control were determined at 510 nm.The clearance rate of ·OH was calculated according to the following formula:

Hydroxyl free radicals clearance rate=(AC-AX)/AC×100%

Assay of superoxide anion free radical scavenging activity［13］

The superoxide anion free radical ()were generated by pyrogallol autoxidation method.The amount ofwas accumulated along with time，so the absorbance at 325 nm increased continuously.After added the sample，a part ofwas scavenged.The variation rate of the absorbance with time △BCof tube-control and △BXof tube-sample were determined.The clearance rate ofwas calculated according to the following formula:

Superoxide anion free radical clerarance rate=(ΔBCΔBX)/ΔBC×100%

4.5 mL of Tris-HCl buffer solution (pH 8.2)，1.0 mL of 1.0 mmol/L pyrogallol (prepared by 10 mmol/L HCl)and 0.5 mL of sample solution with different concentrations were mixed and topped up to 9.0 mL using distilled water.The mixture was incubated in water bath at 25 ℃for 20 min.The control group was prepared using solvent instead of sample.The absorbance variation rate with time △BCof tube-control and △BXof tube-sample were determined at 325 nm every 30 s within 5 min.

Data analysis

All experimental results were expressed in terms of mean ± standard deviation.The UV and IR spectra were obtained using the computer interface with the origin software.

Results and Discussion

Isolation of polysaccharide fractions

CCCPs yielded approximately 17.42% (w/w)according to the procedure as described previously.FCCPs were separated into several fractions by ion exchange column chromatography on a DEAE-cellulose column.The main fraction CCP1was eluted with 0.2 mol/L NaCl solution.The eluting profile for CCP was shown in Fig.1(a).

CCP1was further purified on a Sepharose Cl-6B column.The purifying identification profile is a single symmetrical peak as shown in Fig.1(b).

Fig.1 Fractionation profile of CCP on DEAE-Cellulose column (a)and CCP1on Sepharose CL-6B column (b)

Monosaccharide composition of CCP1

According to HPLC conditions described previously，5 monosaccharide standards were subjected to HPLC analysis.The retention time of rhamnose，arabinose，fructose，mannitose，glucose was 8.112，11.971，13.106，14.780 and 17.908 min，respectively.The HPLC chromatograms of standards and CCP1were shown in Fig.2(a)and Fig.2(b)，respectively.The experimental results indicated that CCP1consisted of rhamnose，arabinose，fructose，mannitose，glucose in a molar ratio of 0.034∶0.228∶0.045∶0.055∶0.638.

Fig.2 HPLC chromatograms of mixed standards of 5 monosaccharides and CCP1

Antioxidant activity of polysaccharides

Scavenging activity of hydroxyl radical

The results showed that C.cathayensis polysaccharides with different purity had certain effect on scavenging·OH.The ·OH clearance rate was in positive correlation with concentration of different purity C.cathayensis polysaccharides.In this experiment，the ·OH scavenging activity of CCCPs was the highest，followed by the FCCPs samples and lowest in the CCP1samples.On the basis of IC50value，the ·OH scavenging activity of C.cathayensis polysaccharides with different purity was lower than that of ascorbic acid as shown in Table 1.Scavenging activity of superoxide anion free radical

Table 1 The scavenging effect of water-soluble polysaccharides from C.cathayensis on ·OH

The A325nmvalues significantly decreased after treatments with different purities of C.cathayensis polysaccharides.The O-·2 clearance rate was in positive correlation with concentration of different purity C.cathayensis polysaccharides.Superoxide anion free radical scavenging activity of C.cathayensis extracts followed the order:CCCPs ＞FCCPs ＞CCP1.On the basis of IC50value，thescavenging activity of C.cathayensis polysaccharides with different purity was lower than that of ascorbic acid as shown in Table 2.

Table 2 The scavenging effect of water-soluble polysaccharides from C.cathayensis on

Table 2 The scavenging effect of water-soluble polysaccharides from C.cathayensis on

aValues were presented as the means of three independent experiments±standard deviation.bIC50，the half maximal inhibitory concentration，represented the concentration of an inhibitor that was required for 50% scavenging effect on

Conclusion

The results showed that crude polysaccharides had the highest scavenging efficiency on superoxide anion free radical and hydroxyl radical than FCCPs and CCP1.This implied that there were other components such as vitamin C，polypeptides and oligosaccharides which may be responsible for the antioxidant activity，and the purification process resulted in a decrease of the antioxidant activities to some extent.Moreover，crude polysaccharides had higher scavenging efficiency on superoxide anion free radical than on hydroxyl radical.The fruit of C.cathayensis may be a new source of natural antioxidants.

1 Sancheti S，Sancheti S，Bafna M，et al.Antihyperglycemic，antihyperlipidemic，and antioxidant effects of Chaenomeles sinensis fruit extract in streptozotocin-induced diabetic rats.Eur Food Res Tech，2010，231:415-421.

2 Sun LN，Hong YF.Chemical constituents of Chaenomeles sinensis (Thouin.)Koehne.J Chin Pharm Sci，2000，9:6-9.

3 Hokputsa S，Gerddit W，Pongsamart S，et al.Water-soluble polysaccharides with pharmaceutical importance from Durian rinds (Durio zibethinus Murr.):isolation，fractionation，characterization and bioactivity.Carbohydr Polym，2004，56:471-481.

4 Lee JM，Kwon H，Jeong H，et al.Inhibition of lipid peroxidation and oxidative DNA damage by Ganoderma lucidum.Phytother Res，2001，15:245-249.

5 Wang WP，Guo SY，Li L，et al.Isolation，purification and anticomplement activity of water-soluble polysaccharides from Chaenomeles cathayensis.Food Sci，2008，29:120-124.

6 Wang WP，Guo SY，Li L，et al.Extraction，separation and structural analysis of water-soluble polysaccharides from Chaenomeles cathayensis.J South China Univ Tech，2008，36:128-133.

7 Dourado F，Madureira P，Carvalho V，et al.Purification，structure and immunobiological activity of an arabinan-rich pectic polysaccharide from the cell walls of Prunus dulcis seeds.Carbohydr Res，2004，399:2555-2566.

8 Xing JM，Li FF.Purification of aloe polysaccharides by usingaqueous two-phase extraction with desalination.Nat Prod Res，2009，23:1424-1430.

9 Huang G，Chen Y，Wang X.Extraction and deproteinization of pumpkin polysaccharide.Int J Food Sci Nutr，2011，62:568-571.

10 Dubois M，Gilles KA，Hamilton J，et al.Colorimetric method for determination of sugars and related substances.J Anal Chem，1956，8:350-356.

11 Hokputsa S，Harding SE，Inngjerdingen K，et al.Bioactive polysaccharides from the stems of the Thai medicinal plant Acanthus ebracteatus:their chemical and physical features.Carbohydr Polym，2004，339:753-762.

12 Nergard CS，Diallo D，Michaelsen TE，et al.Isolation，partial characterisation and immunomodulating activities of polysaccharides from Vernonia kotschyana Sch.Bip.exWalp.J Ethnopharmacol，2004，91:141-152.

13 Santhiya D，Subramanian S，Natarajan KA，et al.Surface chemical studies on sphalerite and galena using extracellular polysaccharides isolated from Bacillus polymyxa.J Colloid Interf Sci，2002，256:237-248.