金钗石斛不同储存期多糖组分、总多糖含量及分子量分布的变化*

姚晓东，朱锡敏，张 渊，吴 芹，石京山，周旭美

(1.遵义医学院药学院，贵州遵义 563099;2.遵义医学院院士工作站糖化学与糖生物学实验室，贵州遵义 563099; 3.遵义医学院基础药理省部共建教育部重点实验室，贵州遵义 563099)

基础医学研究

金钗石斛不同储存期多糖组分、总多糖含量及分子量分布的变化*

姚晓东1，2，朱锡敏1，张 渊1，吴 芹2，3，石京山2，3，周旭美1，2

(1.遵义医学院药学院，贵州遵义 563099;2.遵义医学院院士工作站糖化学与糖生物学实验室，贵州遵义 563099; 3.遵义医学院基础药理省部共建教育部重点实验室，贵州遵义 563099)

目的从金钗石解多糖组分中总多糖含量和分子量分布的角度评价不同储存期金钗石解的品质。方法金钗石解多糖采用水提醇沉法获得，适量双蒸水复溶，再次醇沉，然后采用Savag法除去蛋白得到精制多糖。采用比色法测定总多糖的含量。金钗石解精制多糖的分子量分布采用高效凝胶渗透色谱(HPGPC)测得。结果储存期为1年时，金钗石解总多糖的含量是35.97%，在水中煮沸0.5、1、2、4和8 h后其含量分别为39.07%、45.40%、44.23%、43.07%和45.40%。储存期为3年时，金钗石解总多糖的含量是13.57%，在水中煮沸0.5、1、2、4和8 h后其含量分别为13.40%、13.40%、13.73%、15.73%和16.73%。1年储存期多糖的含量显著高于3年储存期(P＜0.01)。1年储存期多糖主要由2部分组成，其保留时间为9.287～9.326min和14.137～14.452min，分子量为9.0×104～9.2×104Da和6.0×102～9.6×102Da。3年储存期多糖保留时间为14.148～14.158 min，分子量为9.3×102～9.5×102Da。结论无论是1年或3年储存期，水中煮沸至8 h时，金钗石解多糖组分总多糖的含量和分子量没有发生明显改变。随着储存时间的延长，金钗石解总多糖的含量在下降，其原因可能与多糖降解有关。

总多糖含量;分子量分布;HPGPC;多糖;金钗石斛;储存期

Dendrobium nobile Lindl.was one of three dendroium resources in Chinese Pharmacopeia(2010 edition)［1］，which located in Chishui，Guizhou Province，was awarded‘National Productof Geographical Indication’in 2006.Dendrobine，an active alkaloid content extracted from Dendrobium nobile Lindl.，is reported anti Alzheimer’Disease，anti-rat brain apoptosis［2-3］.Polysaccharide of Dendrobium nobile Lindl.is another research hot-spot in recent years，it is a potential antioxidant，antitumor agent，anti-aging，hyperlipidemiamodulator，anti-hyperglycemic，and so on［4-8］.Recently，we have found that the total sugar content of Dendrobium nobile Lindl.in different storage period was varied.The reason was still unknown until now.Hence，we try to explain the phenomena in the perspective of variation of DNP molecular weight distribution in different storage period，in order to establish a theoretical foundation for DNP research and the development of Dendrobium nobile Lindl.

1 Sam ple，equipment and reagents

1.1 Sample Dendrobium nobile Lindl.(Batch No.:CSWL 20140325G-3)was collected in Chishui，Guizhou Province;(Batch No.:CSWL 20120710)was provided by Key Laboratory of Basic Pharmacology of Ministry of Education，Zunyi Medical University，and both of sampleswere identified by Professor Yang Jianwen.The voucher specimen was deposited in the Laboratory of Glycochemistry and Glycobiology，Academic Workstation of Zunyi Medical University(Batch No.:LGGDNL20150414D-1 and LGGDNL20150330D-1).

1.2 Equipment Microplate reader(318 C+)，a product of Shanghai Peiou Analytical Instrument Co.，Ltd.An Agilent 1260 HPLC system was consisted of automatic injector，column oven，quaternary pump and vacuum degasser，and Refractive Index Detector (RID).The data were collected and analyzed by Agilent ChemStation.Rotary evaporator(RE2000A，Shanghai Yarong，Shanghai，China).Freeze dryer (Modulyod-230，Thermoelctron Corporation，USA). 1.3 Reagents Ethanol，Chloroform，n-Butanol，Phenol，Sulfuric acid，Sodium Nitrate，Glucose，Sinopharm Chemical Regent Co.，Ltd products，Analytical Reagents(AR).Dextran serial standards (molecular weight 1 000，5 000，12 000，25 000，50 000 Da)were Fluka products.

2 M ethods

2.1 Crude DNP preparation and purification［9］

Dendrobium nobile Lindl.was dried in an oven set at 60℃for 24 h under(0.08 MPa，then grinded appropriately.A 10 fold(w/v)ethanol was added and extracted 5 d(room temperature).The residue was dried by airing，then extracted by hot water for 5 times，3 h each time.Water solution was filtered with a 4-layer gauze and mixed，then condensed using a rotary evaporator.And 4-fold(v/v)pre-cold 95% ethanolwas added and well-mixed，then stored at4℃for 12 h.The ethanol solution was centrifuged at4 390 g for 10 min，and the residue was dissolved into hot water again，equivalent volume Savag solution (Chloroform:n-Butanol=4:1，v/v)was added，at the same time，mixed evenly.The upper solution was treated twice the way described above，condensed using the rotary evaporator into an appropriatevolume，then，dialyzed using a semi-permeable membrane whose cut-offmolecularweightwas3 500 Da for 24 h.The solution in the semi-permeable membrane was condensed，then dried with a freeze dryer.At last，the purified DNP was obtained and readily for the following procedure.

2.2 Polysaccharide stability in boiling water Polysaccharide extracted from Dendrobium nobile Lindl. both in 1 year store and 3 year store was made 1 mg/ml solution，then put into boilingwater for 0.5，1，2，4 and 8 h.These sampleswere centrifuged at10 140 g for10min for polysaccharide stability analyzing.

2.3 Total sugar content determination Appropriate glucose(pre-dried at105℃for6 h)was added into distilled water tomake the solution concentration as 260μg/ml，then diluted into 208，156，104，52 μg/ml，respectively.200μl standard solutions were added into 400μl 5%Phenol(w/v)，and 2 ml sulfuric acid，after that，mixed evenly.The same volume distilled water instead of sample was as blank control.2 h later，the OD490was determined in the microplate reader.The glucose standard curvewas fitting using glucose concentration(X axis)vs OD490(Y axis).The polysaccharide sample wasmade 1 mg/ml solution，then centrifuged at4 390 g for 15 min，the supernatantwas diluted into 40μg/ml using distilled water.The total sugar contentwas determined as previously described.

2.4 HPLC sample preparation of purified DNP A-bout 2mg purified DNPwas put into an EP tube，and 1 ml deionized water was added，sonicated to dissolve，then centrifuged at 10 140 g for 10 min，supernatantwas HPLC loading sample.

2.5 HPLC analysis procedure The molecular weight distribution of purified DNPwas carried out on the Agilent1260 HPLC coupled with RID.An Ultrahydrogel 250 column(30 cm，i.d.0.78 cm)was used to separate the DNP.Themobile phase was 0.1 mol/L NaNO3，flow ratewas0.6 ml/min，and injection volume was 40μl using the automatic injector. Column temperature was set at30℃.

2.6 Statistical analysis The data were presented as means(n=3).The differences of total sugar content in different year store were analyzed by t-test using SPSS 13.0 software.P＜0.01 was considered statistically significant.

3 Results

3.1 The standard curve of glucose and molecular weight curve of standard dextran fitting The glucose fitting equation was y=0.003 x+0.003 6(R2= 0.999 1)，and the absorbance was in good linear relationship while glucose concentration between 0 and 208μg/ml.The standard curve of glucose was showed in Fig 1，and molecular weight curve of standard dextran was showed in Fig 2.

Fig 2 M olecular weight curve of standard dextran

3.2 Polysaccharide from 1 year store and 3 year store stability in boiling water The stability of polysaccharide was evaluated by total sugar content and polysaccharide molecular weight distribution.In 1 year store，the total sugar content was 39.57%，39.07%，45.40%，44.23%，43.07%and 45. 40%，while in 3 year store，the total sugar content was 13.57%，13.40%，13.40%，13.73%，15. 73%and 16.73%.The resultwas showed in Fig 3. Whether in 1 year store or3 year store，the total sugar contentwas stable in boiling water for 8 h.

Fig 3 Stability of total sugar content in 1 year store and 3 year store

The result of polysaccharide molecular weight from 1 year store and 3 year store was showed in Fig 4.In 1 year store，retention time of the polysaccharide portion was 9.287～9.326 min and 14.137～14.452 min，and the molecular weight was 9.0× 104～9.2×104Da，and 6.0×102～9.6×102Da.While in 3 year store，the polysaccharide compound retention time was 14.148～14.158 min. Themolecular weight was 9.3×102～9.5×102Da.In 1 year store or 3 year store，the polysaccharide molecular weight distribution was unchanged in boiling water for 8 h.

3.3 The total sugar content in 1 year store and 3 year store The total sugar content in 1 year storewas 35.97%，while in 3 year store was 13.57%，total sugar content in 1 year was higher than that in 3 year (P＜0.01)，and the result was showed in Fig 5. Combined with Fig 4，the portion(retention time: about9.3 min)was disappeared as store year going. Because of the polysaccharide was stable in boiling water for 8 h(as showed in Fig.3)，itwas reasonable to speculate that the total sugar content decreased was related to polysaccharide degradation.

Fig 4 The HPLC chromatogram of DNP in 1 store year(A)and 3 store years(B)

Fig 5 The total sugar content in 1 store and 3 year store years

4 Discussion

Diverse biological activities of polysaccharides are related to their primary structure，secondary and tertiary structure［10］.The molecular weight of polysaccharide also plays an important role in the relationship between structure and function.The higher polysaccharide molecular weight，the higher polysaccharide solution viscosity.On the contrary，the lower polysaccharide water-solubility.As bio-macromolecular polysaccharide degrading，their activities havebeen reported.Low molecular weight polysaccharide from Schisandra chinensis(Turcz.)Baill has shown antitumor and immunomodulatory activity［11］.Depolymerization of agar was performed using agarose has shown antitumor activity［12］andα-glucosidase inhibitor activity［13］.

Themain degradation stage of the polysaccharide fractions occurred between 210 and 320℃［14］.Differential scanning calorimetry(DSC)，differential thermal analysis(DTA)and thermos-gravimetric analysis(TGA)were employed to characterize thermal property of natural gums，the result revealed that the natural gumswere thermally stable［15］，all the results above verified our result，i.e.，polysaccharides from Dendrobium nobile Lindl.were stable in boiling water for 8 h.

However，we found that total sugar content in different storage year was varied.We speculated that the total sugar content decreased was related to polysaccharide degradation，the factor induced polysaccharide degradation was deserved to investigate furthermore.

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［收稿2015-06-30;修回2015-07-15］

(编辑:王 静)

Variation of total sugar content and polysaccharidemolecular weight distribution from Dendrobium nobile Lindl.in different storage period

Yao Xiaodong1，2，Zhu Ximin1，Zhang Yuan1，Wu Qin2，3，Shi Jingshan2，3，Zhou Xumei1，2
(1.Pharmacy School，ZunyiMedical University，ZunyiGuizhou 563099，China;2.Laboratory of Glycochemistry and Glycobiology，Academic Workstation of Zunyi Medical University，Zunyi Guizhou 563099，China;3. The Key Laboratory of Basic Pharmacology of Ministry of Education，Zunyi Medical University，Zunyi Guizhou 563099，China)

Objective To evaluate the quality of Dendrobium Nobile Lindl.in different storage period from the perspective of total sugar content and polysaccharide molecular weight distribution.M ethods Polysaccharide of Dendrobium nobile Lindl.(DNP)was obtained by hot-water extracting and high concentration ethanol precipitating，then itwas refined using water dissolving and ethanol precipitating.And the Savagmethod was used for deproteinization.Total sugar contentwas calculated by colorimetric.The polysaccharidemolecular weight distribution was determined by High Performance Gel Permeation Chromatography(HPGPC).Results The total sugar content of DNP via 1 year store was 35.97%，the polysaccharide content was 39.07%，45.40%，44.23%，43.07%and 45.40%in boiling water for 0.5，1，2，4 and 8 h respectively.While the total sugar content via 3 year storewas13.57%，the polysaccharide contentwas13.40%，13.40%，13.73%，15.73%and 16.73% in boiling water for 0.5，1，2，4 and 8 h.Total sugar content via 1 year store was higher than that via 3 yearstore(P＜0.01).The polysaccharide compound in 1 year store was composed of two portion，whose retention time was9.287～9.326min and 14.137～14.452min.And themolecularweightwas9.0×104～9.2×104Da，and 6.0×102～9.6×102Da respectively.While in 3 year store，the polysaccharide compound was mainly composed of one portion，whose retention time was14.148～14.158 min.Themolecular weightwas9.3 ×102～9.5×102Da.Conclusion The total sugar contentandmolecularweightwere stable in boilingwater up to 8 h whether in 1 year store or 3 year store.Total sugar content was reducing as the storage period goes on，which may be related to polysaccharide degradation.

total sugar content;molecular weight distribution;HPGPC;polysaccharide;Dendrobium nobile Lindl.;storage period

Q539

A

1000-2715(2015)04-0341-05

贵州省遵义医学院院士工作站建设项目(NO:C-768);教育部春晖计划项目(NO:B-035);遵义医学院招标课题(NO:F-681);遵义医学院硕士启动基金(NO:F-714)。

周旭美，女，教授，硕士生导师，研究方向:药物质量标准建立及新药研发，E-mail:zmczxm@163.com。