WO2019071688A1 - 一种具有特定结构的壳寡糖及其制备方法和应用 - Google Patents

一种具有特定结构的壳寡糖及其制备方法和应用 Download PDF

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WO2019071688A1
WO2019071688A1 PCT/CN2017/109990 CN2017109990W WO2019071688A1 WO 2019071688 A1 WO2019071688 A1 WO 2019071688A1 CN 2017109990 W CN2017109990 W CN 2017109990W WO 2019071688 A1 WO2019071688 A1 WO 2019071688A1
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chitosan
deacetylation
chitooligosaccharide
specific structure
degree
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French (fr)
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杜昱光
程功
贾培媛
孙明
焦思明
任立世
冯翠
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中科荣信（苏州）生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
    • C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
    • C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A61K31/722—Chitin, chitosan
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates

Definitions

  • the invention belongs to the technical field of chitosan oligosaccharide application, and particularly relates to a chitosan oligosaccharide having a specific structure and a preparation method and application thereof.
  • Chitooligosaccharides are oligosaccharides with a degree of polymerization of less than 20 formed by glucosamine and N-acetylglucosamine linked by ⁇ -1,4 glycosidic bonds, and have biological activities such as anti-inflammatory, anti-tumor and immunomodulation. Studies have shown that the structure of chitooligosaccharides, including the degree of polymerization, degree of deacetylation and the distribution of acetyl sites in oligosaccharides, play a decisive role in the biological activity.
  • the traditional process for the preparation of chitosan involves high-temperature and strong alkali deacetylation of chitin, and the product chitosan has good acid solubility only when the degree of deacetylation is >80%. Therefore, the degree of deacetylation of the existing industrial chitosan is generally >80%, and the content of acetyl groups in chitosan is small.
  • the degree of deacetylation of chitosan oligosaccharides prepared by using the above chitosan as substrate is also >80%, so the acetylated monosaccharide component contained in the chitosan oligosaccharide chain is correspondingly reduced, and the corresponding number of different chitosan oligosaccharide components is limited. .
  • Homogeneous deacetylation of chitin can be achieved by low temperature alkali method (Kurita K, Sannan T, Iwakura Y. Studies on chitin, .4. Evidence for formation of block and random copolymers of N-acetyl-D-glucosamine and D-glucosamine By heterogeneous and homogeneous hydrolyses [J]. Makromolekulare Chemie-Macromolecular Chemistry and Physics, 1977, 178(12): 3197-3202. Liu Dasheng, Wei Yuanan, Jiang Linbin, et al. Preparation of water-soluble chitosan by deacetylation of ultrafine chitin [J].
  • glycoside hydrolase family 18 GH18
  • GH19 chitinase can only recognize and hydrolyze AA and DA type glycosidic bonds
  • chitosanase based on the difference in glycosidic bond recognition, roughly It is divided into three subtypes (I, II and III): type I recognizes hydrolyzed DD and AD, type II recognizes only DD, and type III recognizes DD and DA glycosidic bonds.
  • the inventors of the present application have also discovered the use of the novel chitosan oligosaccharide in medicine, especially in the field of anti-liver cancer.
  • the chitosan oligosaccharide of the present invention has a degree of deacetylation of 50% to 80%.
  • the chitosan oligosaccharide has a degree of deacetylation of 56% to 78%, and even more preferably, the chitosan oligosaccharide has a degree of deacetylation of 60% to 70%, and a still more preferred degree of deacetylation is 60-65%, the most preferred degree of deacetylation is 62%.
  • the degree of deacetylation refers to a fraction of the total chain segment of the chitosan oligosaccharide structure represented by the formula ( ⁇ ).
  • the corresponding degree of deacetylation is 78%.
  • the low degree of deacetylation chitosan described herein refers to chitosan having a degree of deacetylation of less than 80%.
  • the invention also provides a preparation method of the chitosan oligosaccharide having the specific structure, which comprises: obtaining the chitosan oligosaccharide by enzymatic hydrolysis of a chitosan substrate, the enzyme used can specifically recognize and hydrolyze glucosamine
  • the glycosidic bond formed by the sugar and glucosamine causes all the components of the chitosan oligosaccharide obtained by hydrolysis to be glucosamine at both the reducing end and the non-reducing end.
  • the specific steps of the preparation method of the chitosan oligosaccharide are as follows: (1) deacetylating chitin to obtain a chitosan substrate, and obtaining chitosan with different degrees of deacetylation by controlling the deacetylation reaction time; (2) utilizing The chitosan substrate is hydrolyzed by an enzyme that specifically recognizes and hydrolyzes the DD type glycosidic bond to obtain the chitooligosaccharide product.
  • the chitosan has a degree of deacetylation of 50% to 80%, further preferably 56% to 78%, and even more preferably, the chitosan oligosaccharide has a degree of deacetylation of 60% to 70%.
  • a still more preferred degree of deacetylation is 60-65%, and a most preferred degree of deacetylation is 62%.
  • the degree of deacetylation of chitosan can be controlled by controlling the deacetylation reaction time of chitin.
  • the deacetylation reaction of chitin is carried out under alkaline low temperature conditions.
  • Bases which can be used for deacetylation include sodium hydroxide and potassium hydroxide, and the preferred base is sodium hydroxide.
  • the temperature for deacetylation ranges from 40 ° C to 90 ° C, and the preferred temperature range is from 50 ° C to 60 ° C.
  • the concentration of the alkaline solution ranges from 40% to 50%, preferably 45%.
  • the time for the deacetylation reaction ranges from 0.5 h to 24 h, preferably from 2 h to 6 h.
  • chitin is deacetylated in a 45 wt% sodium hydroxide solution at 60 ° C, and the deacetylation reaction time is controlled in the range of 0.5 to 24 hours, and 50 is obtained. %-80% deacetylated chitosan.
  • the present invention hydrolyzes the chitosan substrate using an enzyme capable of specifically recognizing and hydrolyzing a D-D type glycosidic bond.
  • the enzyme is a neutral protease.
  • the neutral protease used in the examples of the present invention is a neutral protease crude protease derived from Bacillus subtilis.
  • a neutral proteolytic enzyme is preferably used, and the specific conditions for the neutral protease to hydrolyze chitosan are: the amount of neutral protease is from 2% by weight to 25% by weight of the substrate, preferably from 5% by weight to 20% by weight. More preferably, it is 5 wt% to 15 wt%; the hydrolysis temperature is 25-55 ° C, the preferred temperature is 30-45 ° C, more preferably 35-45 ° C; the hydrolysis time is 30-60 h, preferably 40-50 h. .
  • the invention also provides the use of the chitosan oligosaccharide having the specific structure as a medicament.
  • the invention further provides the use of the chitosan oligosaccharide having the specific structure for preparing an anti-liver cancer drug.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the chitooligosaccharide having a specific structure or the pharmaceutically acceptable salt of the chitooligosaccharide as an active ingredient.
  • the pharmaceutical composition of the present invention further comprises, in addition to the chitosan oligosaccharide of the present invention, a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier herein is meant a pharmaceutically acceptable material, ingredient or medium, such as a liquid or solid filler, diluent, adjuvant, solvent or encapsulating material, including from one organ or part of the body to another organ. Or carry or transport the main pharmaceutical agent in a part of the body.
  • Each carrier must be “acceptable” and compatible with other forms of medication without harm to the patient.
  • Some examples of pharmaceutically acceptable carriers include: sugars such as lactose, glucose and sucrose; starches such as wheat starch and potato starch starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl Cellulose, cellulose acetate, powdered tragacanth, malt, gelatin, talcum powder; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and Soybean oil; glycols such as butanediol; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and hydrogen Alumina; alginic acid; pyrogen-free water; physiological saline; Ringer's solution; ethanol; phosphate buffer, and other
  • chitosan hydrolase 1, by using different hydrolysis sites to identify the type of chitosan hydrolase for less than 80% deacetylation
  • the degree of chitosan was hydrolyzed, and on this basis, the structures of the chitosan oligosaccharides were identified and their differences in anticancer activity were evaluated.
  • the inventors have surprisingly found that one of the specific structural types of chitosan oligosaccharides is superior to other chitooligosaccharides in anti-hepatocarcinoma activity, and has great application potential in the field of anti-liver cancer.
  • the structural regularity of the chitosan oligosaccharide of this specific structure is that the reducing end and the non-reducing end of all components of chitosan oligosaccharide are glucosamine.
  • the chitosan oligosaccharide of the specific structure obtained by the present invention has better pharmacological activity and can be used in the field of medicine.
  • the chitooligosaccharide of the specific structure disclosed in the present invention has an activity of inhibiting the growth of liver cancer cells, and can be applied to preparation of an anti-liver cancer drug or a pharmaceutical composition.
  • Fig. 1 is a 1 H-NMR chart of a degree of deacetylation of 62% chitosan in Example 1 of the present invention.
  • Figure 2 is a MALDI-TOF mass spectrum of different types of chitooligosaccharides in Example 2 of the present invention.
  • 3A-3D are 1H-NMR and 13C-NMR spectra of chitooligosaccharides COS-62-PA and COS-62-NP in Example 2 of the present invention.
  • 3A and 3B correspond to 1 H-NMR and 13 C-NMR spectra of COS-62-PA; and
  • FIGS. 3C and 3D correspond to 1H-NMR and 13C-NMR spectra of COS-62-NP.
  • Figure 4 is a bar graph showing the effect of chitosan oligosaccharides of different structure types on the growth of HepG2 cells in Example 3 of the present invention.
  • FIG. 5A-5B are bar graphs showing the effects of different concentrations of chitooligosaccharide COS-62-NP and positive drug 5Fu on the growth of HepG2 cells in Example 4 of the present invention; wherein FIG. 5A corresponds to COS-62-NP inhibiting cell survival rate.
  • FIG. 5B corresponds to a schematic diagram of the positive drug 5Fu inhibiting cell survival.
  • 6A-6E are 1 H-NMR spectra corresponding to different degrees of deacetylation chitosan in Example 5 of the present invention; wherein the degree of deacetylation corresponding to FIG. 6A is 56%, and the degree of deacetylation corresponding to FIG. 6B is 66%.
  • the degree of deacetylation corresponding to Fig. 6C is 70%, the degree of deacetylation corresponding to Fig. 6D is 74%, and the degree of deacetylation corresponding to Fig. 6E is 78%.
  • Figure 7 is a bar graph showing the effect of different degrees of deacetylation chitosan on the growth of HepG2 cells in Example 5 of the present invention.
  • Example 1 Preparation of low deacetylation chitosan oligosaccharides of different structure types
  • the degree of deacetylation was measured by 1 H-NMR (see Fig. 1), and the degree of deacetylation was determined to be 62% based on the 1 H-NMR chart of Fig. 1 .
  • Two portions of the prepared chitosan were weighed and each 10 g was added to a thermostatic reaction vessel containing 200 mL of a 1.5% aqueous acetic acid solution, and thoroughly stirred to completely dissolve.
  • NP neutral protease dry powder
  • PA 1g papain powder
  • COS-62-NP referred to as neutral protease hydrolysis
  • COS-62-PA refers to the chitosan oligosaccharide product after hydrolysis of papain
  • Example 2 Composition and structure identification of different types of chitooligosaccharides
  • the chitooligosaccharide fractions of the above three chitosan oligosaccharides were identified using MALDI-TOF mass spectrometry.
  • the specific method is as follows: three kinds of prepared or purchased chitosan oligosaccharide samples COS-62-NP, COS-62-PA and COS-MP-162 are weighed, and an ultra-pure water is used to prepare an aqueous solution having a concentration of 2 mg/mL, each of which is aspirated.
  • the commercial chitooligosaccharide COS-MP-162 consists mainly of the fully chitosan oligosaccharide component, while the low deacetylation chitosan oligosaccharide (COS-62-NP and COS-62-PA) ) consists of an oligosaccharide component containing more N-acetylglucosamine. Further, according to the mass spectrum of Fig. 2, it is understood that the degree of polymerization of the low deacetylated chitosan oligosaccharides (COS-62-NP and COS-62-PA) is between 2 and 20.
  • oligosaccharides is also significantly different, the degree of deacetylation of chitosan oligosaccharides obtained by neutral protease hydrolysis of low deacetylation chitosan in the detection range Higher, only 1-3 acetyl groups exist in the acetyl group-containing oligosaccharide component, while papain hydrolyzes low deacetylation chitosan to obtain a low degree of deacetylation of chitosan oligosaccharide in the detection range, containing 3- 6 acetyl groups.
  • the chitosan hydrolysis in the neutral protease is a relatively simple enzyme. According to the reducing and non-reducing end properties of the product, it should be a type II shell that can only recognize and hydrolyze DD glycosidic bonds. Carbohydrase. This is in good agreement with the MALDI-TOF mass spectrometry results of COS-62-NP (shown in Figure 2) in which the oligosaccharide component contains at least two D monosaccharides.
  • Example 3 Comparison of anti-hepatocarcinoma activity of chitosan oligosaccharides with different structural types
  • the human hepatoma cell line HepG2 cultured to log phase was digested and added to MEM medium (10% FBS, S/P, 1% NAEE) and diluted to 1 ⁇ 10 4 cells/ml. 100 ⁇ l/well was inoculated with 3 96-well cells. The plates were incubated overnight in a 37 ° C 5% CO 2 incubator until the cells were fully adherent. Three kinds of chitosan oligosaccharides (COS-62-NP, COS-62-PA and COS-MP-162) aqueous solution (10 mg/ml) were prepared and sterilized by filtration through a 0.22 ⁇ m filter in an intercellular clean room.
  • MEM medium % FBS, S/P, 1% NAEE
  • the three aqueous solutions of chitosan oligosaccharides prepared above were diluted to 200 ⁇ g/ml with MEM medium (60 ⁇ l of 10 mg/ml oligosaccharide solution was added to MEM medium 2940 ⁇ l to 3 ml), and 100 ⁇ l/well was inoculated with 3 96-well cell culture plates ( The final concentration of chitooligosaccharide was 100 ⁇ g/ml), and the culture was continued in a 37 ° C 5% CO 2 incubator. At the same time, the same concentration of 5-fluorouracil (5Fu) was used as a positive control, and blank medium MEM was used as a negative control.
  • 5Fu 5-fluorouracil
  • MTT 20 ⁇ l/well was added to the 96-well cell culture plate 72 h after administration, and the culture was continued at 37 ° C for 4 h in a 5% CO 2 incubator.
  • the liquid in each well of the culture plate was aspirated by a lance and discarded, and DMSO 100 ⁇ l/well was added.
  • the OD490 values in each well were determined and plotted with OriginPro 8.5 software.
  • the cell survival rate of three different chitosan oligosaccharides and positive control drug groups was calculated by using MEM medium group as 100% cell survival rate. The specific results are shown in Fig. 4. The results showed that the positive drug 5Fu had the most obvious inhibitory effect on the growth of HepG2 cells.
  • the cell survival rate was only 11.64% of the control group. COS-62-NP also inhibited the growth of HepG2 cells. The cell survival rate was only 69.87. %, while chitosan oligosaccharides COS-62-PA and COS-MP-162 did not significantly inhibit HepG2 cells.
  • Example 4 Effect of concentration on anti-hepatocarcinoma activity of chitosan oligosaccharide COS-62-NP
  • the HepG2 cells cultured to log phase were digested, diluted with MEM medium (10% FBS, S/P, 1% NAEE) to 1 ⁇ 10 4 cells/ml, and 100 ⁇ l/well inoculated with 3 96-well cell culture plates. Incubate overnight in a 37 ° C 5% CO 2 incubator until the cells are fully adherent.
  • the chitosan COS-62-NP aqueous solution (10 mg/ml) was prepared and sterilized by filtration through a 0.22 ⁇ m filter in an intercellular clean room.
  • MTT 20 ⁇ l/well was added to the 96-well cell culture plate 72 h after administration, and the culture was continued for 4 h at 37 ° C in a 5% CO 2 incubator.
  • the liquid in each well of the culture plate was aspirated by a lance and discarded, and DMSO 100 ⁇ l/well was added.
  • the OD490 value in each well was determined and plotted with OriginPro 8.5 software.
  • the cell survival rate of chitosan COS-62-NP and positive control drug group with different concentrations was calculated by using MEM medium group as 100% cell survival rate. The specific results are shown in Fig. 5A and Fig. 5B, wherein Fig. 5A corresponds to COS.
  • Example 5 Effect of degree of deacetylation on the activity of chitosan oligosaccharide against liver cancer
  • the degree of deacetylation corresponding to Fig. 6E is 78%.
  • the degree of deacetylation was determined to be 56% (deacetylation reaction time was 1 h), 66% (deacetylation reaction time was 3 h), and 70% (deacetylation reaction time was 6h), 74% (deacetylation reaction time is 9h) and 78% (deacetylation reaction time is 12h).
  • the above chitosan substrate was hydrolyzed by the method of Reference Example 1 using a neutral protease, and the obtained products were sequentially referred to as DA56, DA66, DA70, DA74 and DA78, and the original COS-62-NP was also recorded as DA62. Evaluation of anti-hepatocarcinoma activity.
  • the specific method for evaluating the anti-hepatocarcinoma activity of the above different degrees of deacetylation chitosan oligosaccharide refers to Example 4, and the final concentration of chitooligosaccharide used is 100 ⁇ g/mL.
  • the same concentration of 5-fluorouracil (5Fu) was used as a positive control, and blank medium MEM was used as a negative control.
  • the results are shown in FIG. It can be seen from the data of Fig.
  • chitosan oligosaccharides with different degrees of deacetylation have certain anti-hepatocarcinogenic activity, and the anti-hepatocarcinoma activity of chitosan oligosaccharides is best when the degree of deacetylation is 62%.

Abstract

提供了一种具有特定结构的壳寡糖及其制备方法和应用。所述壳寡糖的所有组分的还原端及非还原端均为氨基葡萄糖，脱乙酰度为50％-80％。所述壳寡糖可通过酶水解壳聚糖底物获得，所采用的酶能够特异性识别并水解氨基葡萄糖与氨基葡萄糖形成的糖苷键，从而使得水解获得的壳寡糖的所有组分其还原端及非还原端均为氨基葡萄糖。相比普通结构的壳寡糖，特定结构的壳寡糖对肝癌细胞具有更高的抑制活性，可应用于肝癌的预防及辅助治疗等领域。

Description

一种具有特定结构的壳寡糖及其制备方法和应用 技术领域
本发明属于壳寡糖应用技术领域，具体涉及一种具有特定结构的壳寡糖及其制备方法和应用。
背景技术
壳寡糖(Chitooligosaccharides，COS)是氨基葡萄糖及N-乙酰氨基葡萄糖经β-1,4糖苷键连接形成的聚合度小于20的寡聚物，具有抗炎、抗肿瘤及免疫调节等生物活性。研究显示，壳寡糖的结构，包括聚合度、脱乙酰度及寡糖中的乙酰基位点分布等结构特征在其生物活性发挥中起到决定性作用。传统工艺制备壳聚糖过程中涉及甲壳素的高温强碱脱乙酰，产物壳聚糖只有在脱乙酰度>80％时才具有较好的酸溶解性。因此，现有工业壳聚糖的脱乙酰度通常>80％，壳聚糖中的乙酰基含量较少。以上述壳聚糖为底物制备的壳寡糖的脱乙酰度也>80％，故壳寡糖链上含有的乙酰化单糖组分也相应减少，对应的不同壳寡糖组分数量有限。
利用低温碱法可以实现甲壳素的匀相脱乙酰(Kurita K,Sannan T,Iwakura Y.Studies on chitin,.4.Evidence for formation of block and random copolymers of N-acetyl-D-glucosamine and D-glucosamine by heterogeneous and homogeneous hydrolyses[J].Makromolekulare Chemie-Macromolecular Chemistry and Physics,1977,178(12):3197-3202。刘大胜,魏远安,蒋林斌,等.超细甲壳素脱乙酰制备水溶性壳聚糖的研究[J].食品科技,2007,32(9):108-110.)，利用上述方法均可以获得脱乙酰度>50％的低脱乙酰度壳聚糖。由于该类型壳聚糖的糖链上N-乙酰氨基葡萄糖(以下简称A)及氨基葡萄糖(以下简称D)含量均较高，因此可识别A和D的水解酶类，如几丁质酶及壳聚糖酶均可对该类型壳聚糖进行水解。研究发现，不同壳聚糖水解酶类对水解部位的糖苷键识别存在较大差异，如糖苷水解酶家族18(GH18)几 丁质酶只能识别并水解A-A及A-D型糖苷键，而GH19几丁质酶则只能识别并水解A-A及D-A型糖苷键；对于壳聚糖酶，根据对糖苷键识别的差异，大致将其分为三个亚型(I、II及III)：I型可识别水解D-D及A-D，II型仅识别D-D，而III型可识别D-D及D-A糖苷键。即使水解底物是相同的低脱乙酰度壳聚糖，但是由于这些不同的酶类对底物的识别存在差异，因此使用不同的壳聚糖水解酶可以获得不同结构类型的壳寡糖，如聚合度及乙酰基位点的分布不同，这些结构差异的壳寡糖在特定生物活性上可能会存在较大差异。
发明内容
本发明的目的是，提供一种新型的，具有特定结构的壳寡糖及其制备方法。除此之外，本申请的发明人还发现了该新型壳寡糖在医药上的用途，尤其是在抗肝癌领域的应用。
为实现上述目的，本发明所采用的技术方案如下：
一种具有特定结构的壳寡糖，所述壳寡糖的结构如式(Ι)所示，
Figure PCTCN2017109990-appb-000001
其中，n为0-18；R为H或者COCH3。
本发明的壳寡糖的脱乙酰度为50％-80％。优选地，所述壳寡糖的脱乙酰度为56％-78％，更进一步优选地，所述所述壳寡糖的脱乙酰度为60％-70％，更进一步优选的脱乙酰度为60-65％，最优选的脱乙酰度为62％。所述脱乙酰度是指式(Ι)所示的壳寡糖结构中，脱乙酰基链节占总链节的分数。假设壳寡糖的重复结构单元为100个，其中乙酰氨基葡糖糖结构单元为22个，氨基葡萄糖结构单元为78个，则对应的脱乙酰度为78％。通过控制式(Ι)中R为COCH3的数量比例，即可控制对应的脱乙酰度。
本文中所述的低脱乙酰度壳聚糖指的是脱乙酰度小于80％的壳聚糖。
本发明还提供了所述具有特定结构的壳寡糖的制备方法，该方法为：通过酶水解壳聚糖底物获得所述壳寡糖，所采用的酶能够特异性识别并水解氨基葡糖糖与氨基葡萄糖形成的糖苷键，使水解获得的壳寡糖的所有组分其还原端及非还原端均为氨基葡糖糖。所述壳寡糖制备方法的具体步骤为：(1)对甲壳素进行脱乙酰化获得壳聚糖底物，通过控制脱乙酰化反应时间获得不同脱乙酰度的壳聚糖；(2)利用特异性识别并水解D-D型糖苷键的酶对所述壳聚糖底物进行水解，获得所述壳寡糖产品。
优选地，所述壳聚糖的脱乙酰度为50％-80％，进一步优选为56％-78％，更进一步优选地，所述所述壳寡糖的脱乙酰度为60％-70％，更进一步优选的脱乙酰度为60-65％，最优选的脱乙酰度为62％。壳聚糖的脱乙酰度可以通过控制甲壳素脱乙酰化反应时间来控制。
本发明中，甲壳素的脱乙酰化反应在碱性低温的条件下进行。可以用于脱乙酰化的碱包括氢氧化钠和氢氧化钾，优选的碱为氢氧化钠。脱乙酰的温度范围为40℃-90℃,优选的温度范围为50℃-60℃。碱溶液的浓度范围40％-50％，优选的为45％。脱乙酰化反应的时间范围为0.5h-24h，优选的为2h-6h。
在本发明的一个具体的实施例中，甲壳素在45wt％的氢氧化钠溶液中于60℃下进行脱乙酰化反应，控制脱乙酰化反应时间在0.5至24小时的范围内，可获得50％-80％脱乙酰度的壳聚糖。
本发明使用能够特异性识别并水解D-D型糖苷键的酶对所述壳聚糖底物进行水解。优选地，所述酶为中性蛋白酶。本发明的实施例中使用的中性蛋白酶为来源于枯草芽孢杆菌的中性蛋白酶粗酶(Neutral protease)。
不同的酶对壳聚糖底物的水解需要不同的水解条件。根据使用的酶的特性，本领域的技术人员可以通过实验来确定合适的水解条件。在本发明中，优选地使用中性蛋白水解酶，所述中性蛋白酶水解壳聚糖的具体条件为：中性蛋白酶用量是底物的2wt％-25wt％，优选的为5wt％-20wt％，更优选的为5wt％-15wt％；水解温度为25-55℃，优选的温度为30-45℃，更优选的为35-45℃；水解时间为30-60h，优选的为40-50h。
本发明还提供了所述具有特定结构的壳寡糖作为药物的用途。
本发明进一步提供了所述具有特定结构的壳寡糖在制备抗肝癌药物中的应用。
本发明还提供一种药物组合物，该药物组合物含有所述具有特定结构的壳寡糖或所述壳寡糖药学上可接受的盐作为活性组分。
本发明的药物组合物，除了含有本发明的壳寡糖外，进一步含有药物学上可接受的载体。这里“药物学上可接受的载体”是指药学接受的材料、成份或介质，如液体或固体填料、稀释剂、辅料、溶剂或封装材料，包括从一个器官或身体的某部分到另一个器官或身体的某部分携带或运输主要药学试剂。每个载体必须是“可以接受”，能兼容其他形式的药物成而对病人不造成伤害。一些可作为药学上可以接受的载体的例子包括：糖，如乳糖、葡萄糖和蔗糖糖；淀粉，如小麦淀粉和马铃薯淀粉淀粉；纤维素及其衍生物，如钠羧甲基纤维素、乙基纤维素、醋酸纤维素，粉状西黄蓍胶、麦芽、明胶、滑石粉；辅料，如可可黄油和栓剂蜡；油，如花生油、棉籽油、红花油、芝麻油、橄榄油、玉米油和豆油；甘醇，如丁二醇；多元醇，如甘油、山梨醇、甘露醇和聚乙二醇；酯，如油酸乙酯和月桂酸乙酯；琼脂；缓冲剂，如氢氧化镁和氢氧化铝；海藻酸；无热原水；生理盐水；林格氏液；乙醇；磷酸盐缓冲液，以及其他无毒的应用在药物制剂的可兼容物质。
与现有技术相比，本发明的有益效果如下：
1，通过利用不同水解位点识别类型的壳聚糖水解酶对低于80％脱乙酰 度的壳聚糖进行水解，在此基础上对产物壳寡糖的结构进行鉴定并评价它们在抗癌活性方面的差异。发明人惊奇地发现，其中一种特定结构类型的壳寡糖在抗肝癌活性方面明显优于其他壳寡糖，在抗肝癌领域具有较大的应用潜力。该特定结构的壳寡糖其结构规律即是：壳寡糖所有组分的还原端及非还原端均为氨基葡糖糖。
2，本发明研究获得的特定结构的壳寡糖具有更好的药理学活性，能够用于医药领域。具体的来说，本发明披露的特定结构的壳寡糖具有抑制肝癌细胞生长的活性，可应用于制备抗肝癌药物或者药物组合物。
附图说明
图1是本发明实施例1中脱乙酰度62％壳聚糖的1H-NMR图谱。
图2是本发明实施例2中不同类型壳寡糖的MALDI-TOF质谱图。
图3A-图3D是本发明实施例2中壳寡糖COS-62-PA及COS-62-NP的1H-NMR和13C-NMR图谱。其中，图3A和图3B对应COS-62-PA的1H-NMR和13C-NMR谱图；图3C和图3D对应COS-62-NP的1H-NMR和13C-NMR谱图。
图4是本发明实施例3中不同结构类型壳寡糖对HepG2细胞生长影响的条形数据图。
图5A-图5B是本发明实施例4中不同浓度壳寡糖COS-62-NP和阳性药5Fu对HepG2细胞生长影响的条形数据图；其中图5A对应COS-62-NP抑制细胞生存率的示意图，图5B对应阳性药5Fu抑制细胞生存率的示意图。
图6A-图6E是本发明实施例5中不同脱乙酰度壳聚糖对应的1H-NMR图谱；其中图6A对应的脱乙酰度为56％，图6B对应的脱乙酰度为66％，图6C对应的脱乙酰度为70％，图6D对应的脱乙酰度为74％，图6E对应的脱乙酰度为78％。
图7是本发明实施例5中不同脱乙酰度壳聚糖对HepG2细胞生长影响的条形数据图。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1：不同结构类型低脱乙酰度壳寡糖制备
参考刘大胜等(刘大胜,魏远安,蒋林斌,等.超细甲壳素脱乙酰制备水溶性壳聚糖的研究[J].食品科技,2007,32(9):108-110.)的方法制备低脱乙酰度壳聚糖，具体方法如下：称取超微粉碎后的甲壳素粉末30g，加入到10倍的45％氢氧化钠溶液中，搅拌均匀，然后升温至60℃进行脱乙酰反应2h，反应完毕后离心，沉淀物用60％的乙醇-水混合溶液洗涤至无碱性，干燥后得产品。使用1H-NMR测定其脱乙酰度(参见图1)，根据图1的1H-NMR图谱确定其脱乙酰度为62％。称取制备的壳聚糖两份，每份10g，分别加至装有200mL 1.5％乙酸水溶液的恒温反应釜中，充分搅拌使其完全溶解。调节反应温度至40℃，分别加入1克中性蛋白酶干粉(缩写为NP，酶活：5万U/g，购自山东泰安信得利生物工程有限公司)及1g木瓜蛋白酶粉末(缩写为PA，酶活：5万U/g，购自南宁庞博生物工程有限公司)，恒温反应48小时。反应结束后，离心去除不容物，上清经旋转蒸发仪在40℃下浓缩至50-100mL，再经冷冻干燥得成品壳寡糖，依次命名为COS-62-NP(指中性蛋白酶水解后的壳寡糖产品)及COS-62-PA(指木瓜蛋白酶水解后的壳寡糖产品)。
实施例2：不同类型壳寡糖的组成及结构鉴定
使用MALDI-TOF质谱方法对上述三种壳寡糖中的壳寡糖组分进行鉴定。具体方法为：称取三种制备或购买的壳寡糖样品COS-62-NP、COS-62-PA及COS-MP-162，用超纯水配置成浓度为2mg/mL的水溶液，各吸取1μL点样至样品板上，待其自然干燥后，各加入1μL基质2,5- 二羟基苯甲酸(DHB)溶液，待其干燥后使用autoflex Ⅲ smartbeam型MALDI-TOF质谱仪(Bruker公司)进行检测(正离子反射模式)。质谱检测结果如图2所示：分别对应COS-62-NP、COS-62-PA和COS-MP-162的质谱图；为便于区分，在图2中以A代表N-乙酰氨基葡萄糖，D代表氨基葡萄糖，随后的数字代表含有该单糖的个数，二者加和为寡糖的聚合度。从图2的质谱结果来看，商品壳寡糖COS-MP-162主要由全脱乙酰壳寡糖组分组成，而低脱乙酰度壳寡糖(COS-62-NP及COS-62-PA)则由含有较多N-乙酰氨基葡萄糖的寡糖组分组成。另外，根据图2的质谱图可知：低脱乙酰度壳寡糖(COS-62-NP及COS-62-PA)的聚合度在2-20之间。不仅如此，即使是相同的底物，使用不同的酶水解，寡糖的结构也存在明显的差异，中性蛋白酶水解低脱乙酰度壳聚糖获得的壳寡糖在检测范围内的脱乙酰度较高，含有乙酰基的寡糖组分中仅存在1-3个乙酰基，而木瓜蛋白酶水解低脱乙酰度壳聚糖获得在检测范围内的壳寡糖脱乙酰度较低，含有3-6个乙酰基。
由于MALDI-TOF质谱方法只能对分子量2000以下的组分进行较好检测，为进一步对制备的不同类型壳寡糖的结构特征进行进一步确认，使用1H-NMR及13C-NMR对COS-62-PA及COS-62-NP的还原端及非还原端结构特征进行分析，如图3A-图3D所示，其中图3A和图3B对应COS-62-PA的1H-NMR和13C-NMR谱图；图3C和图3D对应COS-62-NP的1H-NMR和13C-NMR谱图。图3A-图3D的谱图结果显示，COS-62-PA的还原端同时包含单糖单元A及D(如图3A所示)，而非还原端也同时包含A及D(如图3B所示)。该结果说明木瓜蛋白酶中可能同时含有多种水解类型的壳聚糖水解酶类，从而产生还原端及非还原端结构较复杂的壳寡糖产物。相对而言，COS-62-NP的结构更有规律可循：还原端及非还原端均由D糖单元构成(如图3C和3D所示)，具有易于分辨的结构特征。这可能是由于中性蛋白酶中起壳聚糖水解作用的是较为单一的酶类，根据产物的还原端及非还原端特性，应该是一种仅能识别并水解D-D糖苷键的II型壳聚糖酶。这与COS-62-NP的MALDI-TOF质谱结果 (如图2所示)中寡糖组分至少含有两个D单糖也十分吻合。
实施例3：不同结构类型壳寡糖抗肝癌活性比较
将培养至对数期的人肝癌细胞系HepG2消化后加入MEM培养基(10％FBS，S/P，1％NAEE)稀释至1×104个/ml，100μl/well接种3块96孔细胞培养板，37℃5％CO2培养箱中过夜培养至细胞完全贴壁。配制三种壳寡糖(COS-62-NP、COS-62-PA及COS-MP-162)水溶液(10mg/ml)，于细胞间超净台内0.22μm滤膜过滤除菌。用MEM培养基将上述配制的三种壳寡糖水溶液稀释至200μg/ml(取60μl 10mg/ml的寡糖溶液加入MEM培养基2940μl至3ml)，100μl/well接种3块96孔细胞培养板(壳寡糖终浓度为100μg/ml)，37℃5％CO2培养箱中继续培养。同时以相同浓度的5-氟尿嘧啶(5Fu)作为阳性对照，以空白培养基MEM作阴性对照。给药后72h的96孔细胞培养板中加入MTT 20μl/well，5％CO2培养箱中37℃下继续培养4h，用排枪吸出培养板中各孔中的液体弃去，加入DMSO 100μl/well，测定各孔中的OD490值，并且用OriginPro8.5软件作图。以MEM培养基组为100％细胞生存率，计算出三种不同壳寡糖及阳性对照药组的细胞生存率，具体结果如图4所示。结果显示，阳性药5Fu对HepG2细胞生长的抑制作用最明显，细胞生存率仅为对照组的11.64％，COS-62-NP对HepG2细胞的生长也有明显抑制作用，细胞生存率仅为对照的69.87％，而壳寡糖COS-62-PA及COS-MP-162对HepG2细胞的抑制不明显。
实施例4：浓度对壳寡糖COS-62-NP抗肝癌活性的影响
将培养至对数期的HepG2细胞消化后加入MEM培养基(10％FBS，S/P，1％NAEE)稀释至1×104个/ml，100μl/well接种3块96孔细胞培养板，37℃5％CO2培养箱中过夜培养至细胞完全贴壁。配制壳寡糖COS-62-NP水溶液(10mg/ml)，于细胞间超净台内0.22μm滤膜过滤除菌。用MEM培养基依次稀释至200μg/ml、100μg/ml、50μg/ml、20μg/ml 及2μg/ml，100μl/well接种3块96孔细胞培养板(壳寡糖终浓度对应减半)，37℃5％CO2培养箱中继续培养。同时以相同浓度的5-氟尿嘧啶(5Fu)作为阳性对照，以空白培养基MEM作阴性对照。给药后72h的96孔细胞培养板中加入MTT 20μl/well，5％CO2培养箱中37℃下继续培养4h，用排枪吸出培养板中各孔中的液体弃去，加入DMSO 100μl/well，测定各孔中的OD490值，并且用OriginPro8.5软件作图。以MEM培养基组为100％细胞生存率，计算出不同浓度壳寡糖COS-62-NP及阳性对照药组的细胞生存率，具体结果如图5A和图5B所示，其中图5A对应COS-62-NP抑制细胞生存率的示意图，图5B对应阳性药5Fu抑制细胞生存率的示意图。结果显示，壳寡糖COS-62-NP随着浓度的加大，对HepG2细胞的抑制活性也相应增加，阳性药5Fu也得到相似的结果，只是抑制效果更加明显。
实施例5：脱乙酰度对壳寡糖抗肝癌活性的影响
为确定脱乙酰度对中性蛋白酶水解低脱乙酰度壳聚糖产物抗肝癌活性的影响，进一步在脱乙酰度62％壳聚糖的基础上，参考刘大胜等(刘大胜,魏远安,蒋林斌,等.超细甲壳素脱乙酰制备水溶性壳聚糖的研究[J].食品科技,2007,32(9):108-110.)的方法，在60℃下通过调整脱乙酰反应时间(反应时间分别有1h、3h、6h、9h、12h)，重新制备不同脱乙酰度的壳聚糖，通过1H-NMR确定脱乙酰度，如图6A-图6E所示，其中图6A对应的脱乙酰度为56％，图6B对应的脱乙酰度为66％，图6C对应的脱乙酰度为70％，图6D对应的脱乙酰度为74％，图6E对应的脱乙酰度为78％。根据图6A-图6E的1H-NMR图谱数据，确定脱乙酰度分别为56％(脱乙酰反应时间为1h)、66％(脱乙酰反应时间为3h)、70％(脱乙酰反应时间为6h)、74％(脱乙酰反应时间为9h)及78％(脱乙酰反应时间为12h)。参考实施例1中的方法使用中性蛋白酶对上述壳聚糖底物进行水解，得到的产物依次记为DA56、DA66、DA70、DA74及DA78，原先的COS-62-NP也记为DA62一起用于抗肝癌活性评价。
上述不同脱乙酰度壳寡糖抗肝癌活性评价的具体方法参考实施例4，使用的壳寡糖终浓度均为100μg/mL。同时以相同浓度的5-氟尿嘧啶(5Fu)作为阳性对照，以空白培养基MEM作阴性对照，结果如图7所示。通过图7的数据结果可知，不同脱乙酰度壳寡糖均具有一定的抗肝癌活性，其中脱乙酰度为62％时壳寡糖的抗肝癌活性最佳。
上述仅为本发明的部分优选实施例，本发明并不仅限于实施例的内容。对于本领域中的技术人员来说，在本发明技术方案的构思范围内可以有各种变化和更改，所作的任何变化和更改，均在本发明保护范围之内。

Claims (11)

  1. 一种具有特定结构的壳寡糖，其特征在于：所述壳寡糖的结构如式(Ι)所示，
    Figure PCTCN2017109990-appb-100001
    其中，n为0-18；R为H或者COCH3。
  2. 根据权利要求1所述的一种具有特定结构的壳寡糖，其特征在于：所述壳寡糖的脱乙酰度为50％-80％。
  3. 根据权利要求1所述的一种具有特定结构的壳寡糖，其特征在于：所述壳寡糖的脱乙酰度为56％-78％。
  4. 权利要求1-3任一项所述的具有特定结构的壳寡糖的制备方法，该方法为：通过酶水解壳聚糖底物获得所述壳寡糖，所采用的酶能够特异性识别并水解氨基葡糖糖与氨基葡萄糖形成的糖苷键，使水解获得的壳寡糖的所有组分其还原端及非还原端均为氨基葡糖糖。
  5. 根据权利要求4所述的具有特定结构的壳寡糖的制备方法，其特征在于：所述壳聚糖的脱乙酰度为50％-80％；通过控制甲壳素脱乙酰化反应时间获得所述不同脱乙酰度的壳聚糖。
  6. 根据权利要求4所述的具有特定结构的壳寡糖的制备方法，其特征在于：所述酶为中性蛋白酶。
  7. 根据权利要求6所述的具有特定结构的壳寡糖的制备方法，其特征在于，所述中性蛋白酶水解壳聚糖的具体条件为：中性蛋白酶用量是底物的 5wt％-15wt％，水解温度为35-45℃，水解时间为30-60h。
  8. 权利要求1-3任一项所述的具有特定结构的壳寡糖在制备药物中的应用。
  9. 根据权利要求8所述的具有特定结构的壳寡糖在制备药物中的应用，其特征在于：所述药物为抗肝癌药物。
  10. 一种药物组合物，该药物组合物含有如权利要求1-3任一项所述的壳寡糖或所述壳寡糖药学上可接受的盐作为活性组分。
  11. 根据权利要求10所述的药物组合物，所述药物组合物进一步含有药物学上可接受的载体。
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