AU2017101852A4

AU2017101852A4 - Chitooligosaccharide with a specific structure and preparation method and application thereof

Info

Publication number: AU2017101852A4
Application number: AU2017101852A
Authority: AU
Inventors: Gong CHENG; Yuguang Du; Cui FENG; Peiyuan Jia; Siming JIAO; Lishi REN; Ming Sun
Original assignee: Zhongke Runxin Suzhou Biological Technology Co Ltd
Current assignee: Zhongke Runxin (suzhou) Biological Technology Co Ltd
Priority date: 2017-10-11
Filing date: 2017-11-08
Publication date: 2019-05-30
Anticipated expiration: 2025-11-08
Also published as: CN108003257A; CA3041417A1; CA3041417C; WO2019071688A1; CN108003257B

Abstract

Abstract The present invention discloses a chitooligosaccharide having a specific structure and a preparation method and application thereof. Both the reducing ends and the non-reducing ends of all the components of the chitooligosaccharide are glucosamine, and the degree of deacetylation is 50%-80%. The chitooligosaccharide can be obtained by enzymatic hydrolysis of a chitosan substrate, and the enzyme used can specifically recognize and hydrolyze the glycosidic bond formed by glucosamine and glucosamine, so that all the components of the chitooligosaccharide obtained by hydrolysis have both the reducing end and non-reducing end thereof being glucosamine. Compared with the chitooligosaccharide of a conventional structure, the chitooligosaccharide having a specific structure of the present invention has higher inhibitory activity on liver cancer cells, and can be applied to the fields of prevention and adjuvant treatment of liver cancer. C) co D ICTo Figure-

Description

CHITOOLIGOSACCHARIDEWITH A SPECIFIC STRUCTURE AND

PREPARATION METHOD AND APPLICATION THEREOF

Technical Field

The present invention belongs to the technical field of chitooligosaccharides application, and particularly relates to a chitooligosaccharide having a specific structure and a preparation method and application thereof.

Background Art

Chitooligosaccharides (COS) are oligomers with a degree of polymerization of less than 20 formed by linking glucosamine and N-acetylglucosamine by β-1,4 glycosidic bonds, and have anti-inflammatory, anti-tumor and immunoregulation and other biological activities. Studies show' that the structure of chitooligosaccharide, including the degree of polymerization, degree of deacetylation and the distribution of acetyl sites in oligosaccharides and other structural features, plays a decisive role in the exerting of its biological activity. Traditional processes for the preparation of chitosan involve high-temperature and strong alkali deacetylation ofchitin, and the product chitosan has better acid solubility only in the case that the degree of deacetylation is >80%. Therefore, 20 the degree of deacetylation of chitosan in the existing industry is generally >80%, and chitosan contains a small amount of acetyl groups therein. The degree of deacetylation of chitooligosaccharide prepared by using the above chitosan as a substrate is also >80%, so the acetylated monosaccharide component contained in the chitooligosaccharide chain is also reduced correspondingly, and the 25 corresponding number of different chitooligosaccharide components is limited.

Homogeneous deacetylation ofchitin can be achieved by a 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. Study on the

2017101852 08 Nov 2017 preparation of water-soluble chitosan by deacetylation of ultrafine chitin [J], Food Science and Technology, 2007, 32(9): 108-110.), by which a low degree of deacetylation chitosan with a degree of deacetylation >50% can be obtained. Due to higher contents of both N-acetylglucosamine (hereinafter referred to as A) and 5 glucosamine (hereinafter referred to as D) in the sugar chain of this type of chitosan, hydrolase enzymes such as chitinase and chitosanase capable of recognizing A and D can hydrolyze this type of chitosan. Studies have found that different chitosan hydrolases have large difference in glycosidic bond recognition at the hydrolysis site. For example, glycoside hydrolase family 18 (GH18) 10 chitinase can only recognize and hydrolyze A-A and A-D type glycosidic bonds, while GH19 chitinase can only recognize and hydrolyze A-A and D-A type glycosidic bonds; and as for chitosanase, according to the difference in glycosidic bond recognition, it is generally classified into three subtypes (I, II and III): type I can recognize and hydrolyze D-D and A-D, type II can only recognize D-D, and 15 type III can recognize D-D and D-A glycosidic bonds. Even if the hydrolysis substrate is the same low-degree of deacetylation chitosan, because of the difference in the recognition of the substrate by these different enzymes, chitooligosaccharides having different structure types, such as different degrees of polymerization and distribution of acetyl sites, can be obtained using different 20 chitosan hydrolases, and these structurally different chitooligosaccharides may have large difference in specific biological activities.

Description of the Invention

It is an object of the present invention to provide a novel 25 chitooligosaccharide having a specific structure and a preparation method thereof.

In addition, the present inventors have also found the use of the novel chitooligosaccharide in medicine, especially in the field of anti-hepatoma.

In order to achieve the above object, the technical solution of the present invention is as follows:

a chitooligosaccharide having a specific structure, the structure of the chitooligosaccharide being as shown in formula (I),

2017101852 08 Nov 2017

(i) wherein n is 0-18; and R is H or COCH3.

The chitooligosaccharide of the present invention has a degree of deacetylation of 50% to 80%. Preferably, the chitooligosaccharide has a degree of deacetylation of 56% to 78%, and more preferably, the chitooligosaccharide has a degree of deacetylation of 60% to 70%, further more preferred degree of deacetylation is 60-65%, and the most preferred degree of deacetylation is 62%. The degree of deacetylation refers to a fraction of deacetylated chain segments accounting for the total chain segments in the chitooligosaccharide structure represented by the formula (I). Assuming that the number of the repeating structural units of chitooligosaccharide is 100, in which there are 22 acetylglucosamine structural units and 78 glucosamine structural units, the corresponding degree of deacetylation is 78%. By controlling the quantitative proportion of R being COCH3 in the formula (I), the corresponding degree of deacetylation can be controlled.

The low-degree of deacetylation chitosan described herein refers to chitosan having a degree of deacetylation of less than 80%.

The present invention also provides a preparation method of the chitooligosaccharide having a specific structure, which comprises: obtaining the chitooligosaccharide by enzymatic hydrolysis of a chitosan substrate, the enzyme used can specifically recognize and hydrolyze the glycosidic bond formed by glucosamine and glucosamine, so that all the components of the chitooligosaccharide obtained by hydrolysis have both the reducing ends and non-reducing ends thereof being glucosamine. The specific steps of the preparation method of the chitooligosaccharide are as follows: (1) deacetylating chitin to obtain a chitosan substrate, and obtaining chitosans with different degrees of deacetylation by controlling the deacetylation reaction time; and (2)

2017101852 08 Nov 2017 using enzymes specifically recognizing and hydrolyzing D-D type glycosidic bonds to hydrolyze the chitosan substrate to obtain the chitooligosaccharide product.

Preferably, the chitosan has a degree of deacetylation of 50% to 80%, 5 further preferably 56% to 78%, and even more preferably, the chitooligosaccharide has a degree of deacetylation of 60% to 70%, and a still more preferred degree of deacetylation is 60-65%, and the most preferred degree of deacetylation is 62%. The degree of deacetylation of chitosan can be controlled by controlling the reaction time of deacetylation of chitin.

In the present invention, the deacetylation reaction of chitin is carried out under alkaline and 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 preferably, the temperature ranges 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.

In a specific embodiment of the present invention, chitin is deacetylated in a 45 wt% sodium hydroxide solution at 60°C, and the deacetylation reaction time is 20 controlled to be from 0.5 to 24 hours, and chitosan having a degree of deacetylation of 50%-80% can be obtained.

The present invention uses an enzyme capable of specifically recognizing and hydrolyzing D-D type glycosidic bonds to hydrolyze the chitosan substrate. Preferably, the enzyme is a neutral protease. The neutral protease used in the 25 examples of the present invention is a neutral protease crude protease derived from Bacillus subtilis.

Different enzymes require different hydrolysis conditions for the hydrolysis of chitosan substrate. Depending on the nature of the enzyme used, one skilled in the art can experiment to determine suitable hydrolysis conditions. In the present 30 invention, a neutral protease is preferably used, and the specific conditions for the neutral protease to hydrolyze chitosan are as follows: the amount of the neutral

2017101852 08 Nov 2017 protease is 2 wt% to 25 wt% of the substrate, preferably 5 wt% to 20 wt%, and more preferably 5 wt% to 15 wt%; the hydrolysis temperature is 25-55°C, preferably 30-45°C, and more preferably 35-45°C; and the hydrolysis time is 30-60 h, and preferably 40-50 h.

The present invention also provides the use of the chitooligosaccharide having a specific structure as a medicine.

The present invention further provides the use of the chitooligosaccharide having a specific structure in the preparation of an anti-hepatoma medicine.

The present invention also provides a pharmaceutical composition 10 comprising the chitooligosaccharide having a specific structure or a pharmaceutically acceptable salt of the chitooligosaccharide as an active ingredient.

The pharmaceutical composition of the present invention further comprises, in addition to the chitooligosaccharide of the present invention, a 15 pharmaceutically acceptable carrier. By pharmaceutically acceptable carrier herein is meant a pharmaceutically acceptable material, ingredient or medium, such as liquid or solid fillers, diluents, adjuvants, solvents or encapsulating materials, including carrying or transporting the main pharmaceutical agents from one organ or part of the body to another organ or part of the body. Each carrier 20 must be acceptable and compatible with other forms of medicines without causing 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; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, cellulose acetate, powdered tragacanth, 25 malt, gelatin, talcum powder; adjuvants such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com 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 aluminum hydroxide; alginic 30 acid; pyrogen-free water; physiological saline; Ringer's solution; ethanol;

phosphate buffer, and other non-toxic compatible substances applied in

2017101852 08 Nov 2017 pharmaceutical preparations.

Compared with the prior art, the present invention has the following advantageous effects.

1. The chitosan having a degree of deacetylation of less than 80% was hydrolyzed by chitosan hydrolase of different hydrolysis site recognition types, on the basis of which the structure of the product chitooligosaccharide was identified and evaluated for the difference in the anti-cancer activities thereof. The inventors have surprisingly found that one chitooligosaccharide having a specific structural type is superior to other chitooligosaccharides in terms of 10 anti-hepatoma activity, and has great application potential in the field of anti-hepatoma. The chitooligosaccharide having a specific structure has the structural regularity that, both the reducing ends and the non-reducing ends of all the components of the chitooligosaccharide are glucosamine.

2. The chitooligosaccharide having a specific structure obtained by the present invention has better pharmacological activity and can be used in the field of medicine. Specifically, the chitooligosaccharide having a specific structure disclosed in the present invention has an activity of inhibiting the growth of liver cancer cells, and can be applied to the preparation of anti-hepatoma medicines or pharmaceutical compositions.

Brief Description of the Drawings

Figure 1 is the 'H-NMR spectrum of the chitosan having a degree of deacetylation of 62% in Example 1 of the present invention.

Figure 2 is the MALDI-TOF mass spectra of different types of 25 chitooligosaccharides in Example 2 of the present invention.

Figures 3A-3D are 'H-NMR and ¹³C-NMR spectra of chitooligosaccharides COS-62-PA and COS-62-NP in Example 2 of the present invention. Therein, Figures 3A and 3B correspond to the 'H-NMR and ¹³C-NMR spectra of COS-62-PA; and Figures 3C and 3D correspond to the 'H-NMR and ¹³C-NMR 30 spectra of COS-62-NP.

Figure 4 is a bar data graph showing the effect of chitooligosaccharides

2017101852 08 Nov 2017 having different structural types on the growth of HepG2 cells in Example 3 of the present invention.

Figures 5A-5B are bar data graphs showing the effects of different concentrations of chitooligosaccharide COS-62-NP and positive drug 5Fu on the 5 growth of HepG2 cells in Example 4 of the present invention; wherein Figure 5 A corresponds to a schematic diagram of COS-62-NP inhibiting cell survival rate, and Figure 5B corresponds to a schematic diagram of the positive drug 5Fu inhibiting cell survival rate.

Figures 6A-6E are the 'H-NMR spectra corresponding to chitosans having 10 different degrees of deacetylation in Example 5 of the present invention; wherein the degree of deacetylation corresponding to Figure 6A is 56%, the degree of deacetylation corresponding to Figure 6B is 66%, the degree of deacetylation corresponding to Figure 6C is 70%, the degree of deacetylation corresponding to Figure 6D is 74%, and the degree of deacetylation corresponding to Figure 6E is 15 78%.

Figure 7 is a bar data graph showing the effects of chitosans having different degrees of deacetylation on the growth of HepG2 cells in Example 5 of the present invention.

Detailed Description

The embodiments of the present invention will be described in detail below with reference to the following examples. However, one skilled in the art will understand that, the following examples are intended to only illustrate the invention but not intended to limit the scope of the present invention. Those 25 examples, in which the specific conditions are not specified therein, are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, for which the manufacturers are not specified, are all conventional products that are commercially available.

Example 1: Preparation of low-degree of deacetylation chitooligosaccharides of different structural types

Eow-degree of deacetylation chitosan was prepared by reference to the

2017101852 08 Nov 2017 method by Liu Dasheng et al. (Liu Dasheng, Wei Yuanan, Jiang Linbin, et al. Study on the preparation of water-soluble chitosan by deacetylation of ultrafine chitin [J], Food Science and Technology, 2007, 32(9): 108-110.), and the specific method is as follows: weighing 30 g of ultrafinely pulverized chitin powder, adding it to 10 times of 45% sodium hydroxide solution, stirring evenly, then heating up to 60°C for deacetylation reaction for 2 h, centrifuging after the reaction completion, washing the precipitate with a 60% ethanol-water mixed solution until it was non-alkaline, and drying it to obtain a product. The degree of deacetylation was measured by 'H-NMR (see Figure 1), and the degree of deacetylation was determined to be 62% based on the ¹ H-NMR spectrum in Figure 1. Two portions of the prepared chitosan were weighed, each portion of 10 g, were each added to a thermostatic reaction vessel containing 200 mL of 1.5% aqueous acetic acid solution, and thoroughly stirred to completely dissolve it. The reaction temperature was adjusted to 40°C, 1 g of neutral protease dry powder (abbreviated as NP, enzyme activity: 50,000 U/g, purchased from Shandong Taian Xindeli Bioengineering Co., Ltd.) and 1 g of papain powder (abbreviated as PA, enzyme activity: 50,000 U/g, purchased from Nanning Pangbo Biological Engineering Co., Ltd.) were added therein respectively, and constant temperature reaction was carried out for 48 hours. After the reaction was completed, insoluble substance was removed by centrifugation, and the supernatant was concentrated to 50-100 mL by a rotary evaporator at 40°C, and then freeze-dried to obtain the finished product chitooligosaccharides, which were named as COS-62-NP (referring to the chitooligosaccharide product obtained after hydrolysis by neutral protease) and COS-62-PA (referring to the chitooligosaccharide product obtained after hydrolysis by papain).

Example 2: Composition and structure identification of different types of chitooligosaccharides

The chitooligosaccharide composition of the above three chitooligosaccharides were identified using MALDI-TOF mass spectrometry. The specific method is as follows: weighing three prepared or purchased chitooligosaccharide samples COS-62-NP, COS-62-PA and COS-MP-162,

2017101852 08 Nov 2017 preparing each of them into an aqueous solution having a concentration of 2 mg/mL with ultrapure water, sucking out 1 pL for each sample, spotting onto the sample plate, after natural drying, each adding 1 pL of matrix 2,5-dihydroxybenzoic acid (DHB) solution, and after drying, using an autoflex III 5 smartbeam type MALDI-TOF mass spectrometer (Bruker corporation) for detection (positive ion reflection mode). The results of mass spectrometry were shown in Figure 2: respectively correspond to the mass spectra of COS-62-NP, COS-62-PA and COS-MP-162; and for easy distinction, in Figure 2, A represents N-acetylglucosamine, D represents glucosamine, the subsequent numbers 10 represent the numbers of such monosaccharides contained, and the sum of the two is the degree of polymerization of the oligosaccharide. From the results of mass spectrometry in Figure 2, it can be seen that the commercial product chitooligosaccharide COS-MP-162 consists mainly of fully deacetylated chitooligosaccharide component, while the low-degree of deacetylation 15 chitooligosaccharides (COS-62-NP and COS-62-PA) consist of oligosaccharide components containing more N-acetylglucosamine. Further, it can be known from the mass spectra in Figure 2 that the degrees of polymerization of the low-degree of deacetylation chitooligosaccharides (COS-62-NP and COS-62-PA) were between 2 and 20. Moreover, even with the same substrate, when using 20 different enzymes for hydrolysis, oligosaccharides had their structures significantly different. The degree of deacetylation of the chitooligosaccharide obtained by neutral protease hydrolysis of low-degree of deacetylation chitosan in the detection range is higher, only 1-3 acetyl groups existing in the acetyl group-containing oligosaccharide component, while the degree of deacetylation 25 of the chitooligosaccharide obtained by papain hydrolysis of low-degree of deacetylation chitosan in the detection range is lower, containing 3-6 acetyl groups.

Because MALDI-TOF mass spectrometry can only better detect the components with a molecular weight of below 2000, for further confirmation of 30 the structural characteristics of different types of chitooligosaccharides prepared, the structural characteristics of the reducing ends and non-reducing ends of

2017101852 08 Nov 2017

COS-62-PA and COS-62-NP were analyzed by using ¹H-NMR and ¹³C-NMR, as shown in Figures 3A-3D, wherein Figures 3A and 3B correspond to the 'H-NMR and ¹³C-NMR spectra of COS-62-PA; and Figures 3C and 3D correspond to the 'H-NMR and ¹³C-NMR spectra of COS-62-NP. The results of the spectra of 5 Figures 3A-3D showed that, the reducing end of COS-62-PA contained both monosaccharide units A and D (as shown in Figure 3A), while the non-reducing end also contained both A and D (as shown in Figure 3B). The results indicated that papain may contain multiple hydrolysis types of chitosan hydrolase enzymes at the same time, thereby producing chitooligosaccharide products with complex 10 reducing end and non-reducing end structures. Relatively speaking, the structure of COS-62-NP is more regular: both the reducing end and the non-reducing end were composed of D sugar units (as shown in Figures 3C and 3D), and had easily distinguished structural characteristics. This may be due to the fact that, that plays the role of chitosan hydrolysis in the neutral protease is a relatively single 15 enzyme, and according to the reducing end and non-reducing end properties of the product, it should be a type II chitosanase that can only recognize and hydrolyze D-D glycosidic bonds. This is in good agreement with the MALDI-TOF mass spectrometry results of COS-62-NP (as shown in Figure 2) in which the oligosaccharide component contained at least two D monosaccharides.

Example 3: Comparison of anti-hepatoma activity of chitooligosaccharides of different structural types

Human hepatoma cell line HepG2 cultured to log phase was digested and then added to MEM medium (10% FBS, S/P, 1%NAEE), and diluted to lx 10⁴cells/ml, three 96-well cell culture plates were inoculated at 100 μΙ/well, and 25 culturing was carried out at 37°C in 5% CO2 incubator overnight until the cells were completely attached. The aqueous solutions (10 mg/ml) of three chitooligosaccharides (COS-62-NP, COS-62-PA and COS-MP-162) were prepared and sterilized by filtration through a 0.22 pm filter in an intercellular super clean bench. The three aqueous solutions of chitooligosaccharides prepared 30 above were diluted to 200 pg/ml with MEM medium (60 μΐ of 10 mg/ml oligosaccharide solution was added to MEM medium 2940 μΐ to 3 ml), and three

2017101852 08 Nov 2017

96-well cell culture plates were inoculated at 100 μΐ/well (the final concentration of chitooligosaccharide was 100 pg/ml), and culturing was continued at 37°C in 5% CO2 incubator. At the same time, 5-fluorouracil (5Fu) of the same concentration was used as a positive control, and a blank medium MEM was used 5 as a negative control. MTT was added at 20 μΐ/well into the 96-well cell culture plates 72 h after administration, and the culturing was continued at 37°C in 5% CO2 incubator for 4 h. The liquid in each well of the culture plate was sucked out by a lance and discarded, and DMSO was added at 100 μΐ/well. The OD490 values in each well were determined and plotted with OriginPro 8.5 software. 10 The cell survival rates of the three different chitooligosaccharides and the positive control drug groups were calculated by using MEM medium group as 100% cell survival rate, and the specific results were shown in Figure 4. The results showed that the positive drug 5Fu had the most obvious inhibitory effect on the growth of HepG2 cells, and the cell survival rate was only 11.64% of the 15 control group; COS-62-NP also had obvious inhibitory effect on the growth of

HepG2 cells, and the cell survival rate was only 69.87% of the control group; while the inhibitory effects of the chitooligosaccharides COS-62-PA and COS-MP-162 on HepG2 cells were not obvious.

Example 4: Effect of concentration on anti-hepatoma activity of 20 chitooligosaccharide COS-62-NP

The HepG2 cells cultured to log phase were digested and then added to MEM medium (10% FBS, S/P, 1% NAEE), and diluted to lx 10⁴ cells/ml, three 96-well cell culture plates were inoculated at 100 μΙ/well, and culturing was carried out at 37°C in 5% CO2 incubator overnight until the cells were completely 25 attached. An aqueous solution of the chitooligosaccharide COS-62-NP (10 mg/ml) was prepared and sterilized by filtration through a 0.22 pm filter in an intercellular super clean bench. It was diluted to 200 pg/ml, 100 pg/ml, 50 pg/ml, 20 pg/ml and 2 pg/ml in sequence with MEM medium, three 96-well cell culture plates were inoculated at 100 μΙ/well, and culturing was continued at 37°C in 5% 30 CO2 incubator. At the same time, 5-fluorouracil (5Fu) of the same concentration was used as a positive control, and a blank medium MEM was used as a negative

2017101852 08 Nov 2017 control. MTT was added at 20 μΐ/well into the 96-well cell culture plates 72 h after administration, and the culturing was continued at 37°C in 5% CO2 incubator for 4 h. The liquid in each well of the culture plate was sucked out by a lance and discarded, and DMSO was added at 100 μΐ/well. The OD490 values in each well were determined and plotted with OriginPro 8.5 software. The cell survival rates of different-concentration chitooligosaccharide COS-62-NP and the positive control drug groups were calculated by using MEM medium group as 100% cell survival rate. The specific results were shown in Figure 5A and Figure 5B, wherein Figure 5A corresponded to a schematic diagram of COS-62-NP inhibiting cell survival rate, and Figure 5B corresponded to a schematic diagram of the positive drug 5Fu inhibiting cell survival rate. The results showed that the inhibitory activity of chitooligosaccharide COS-62-NP on HepG2 cells increased with the increase of concentration, and similar results were also obtained for the positive drug 5Fu, but the inhibitory effect was more obvious.

Example 5: Effect of degree of deacetylation on the anti-hepatoma activity of chitooligosaccharide

In order to determine the effect of degree of deacetylation on the anti-hepatoma activity of the product obtained by neutral protease hydrolysis of low-degree of deacetylation chitosan, further on the basis of chitosan having a 20 degree of deacetylation of 62%, by reference to the method by Eiu Dasheng et al (Eiu Dasheng, Wei Yuanan, Jiang Einbin, et al. Study on the preparation of water-soluble chitosan by deacetylation of ultrafine chitin [J], Food Science and Technology, 2007, 32(9): 108-110.), by adjusting the deacetylation reaction time at 60°C (reaction time of lh, 3h, 6h, 9h, 12h respectively), chitosans having 25 different degrees of deacetylation were re-prepared, and the degrees of deacetylation were determined by ¹H-NMR, as shown in Figure 6A to Figure 6E, wherein the degree of deacetylation corresponding to Figure 6A was 56%, the degree of deacetylation corresponding to Figure 6B was 66%, the degree of deacetylation corresponding to Figure 6C was 70%, the degree of deacetylation 30 corresponding to Figure 6D was 74%, and the degree of deacetylation corresponding to Figure 6E was 78%. According to the 'H-NMR spectrum data

2017101852 08 Nov 2017 of Figure 6A to Figure 6E, the degrees of deacetylation were determined to be 56% (deacetylation reaction time was 1 h), 66% (deacetylation reaction time was 3 h), 70% (deacetylation reaction time was 6 h), 74% (deacetylation reaction time was 9 h) and 78% (deacetylation reaction time was 12 h) respectively. The above 5 chitosan substrates were hydrolyzed by reference to the method in Example 1 using a neutral protease, and the obtained products were sequentially recorded as DA56, DA66, DA70, DA74 and DA78, and the original COS-62-NP was also recorded as DA62, for the evaluation of anti-hepatoma activity.

Example 4 was referred to for the specific method for evaluating the 10 anti-hepatoma activity of the above chitooligosaccharides of different degrees of deacetylation, and the final concentration of chitooligosaccharide used was 100 pg/mE. At the same time, 5-fluorouracil (5Fu) of the same concentration was used as a positive control, and a blank medium MEM was used as a negative control, and the results were shown in Figure 7. It can be seen from the data 15 results of Figure 7 that chitooligosaccharides having different degrees of deacetylation all had certain anti-hepatoma activity, and the anti-hepatoma activity of chitooligosaccharide was best when the degree of deacetylation was 62%.

The above is only some of the preferred embodiments of the present invention, and the present invention is not limited to the contents of the embodiments. It will be apparent to those skilled in the art that various changes and modifications may be made within the scope of the inventive concept of the technical solution of the present invention, and any changes and modifications made are within the scope of the present invention.

Claims

Claims

1. A chitooligosaccharide having a specific structure, characterized in that the structure of the chitooligosaccharide is as shown in formula (I), (I) wherein n is 0-18; and R is H or COCH3.
2. A chitooligosaccharide having a specific structure according to claim 1, characterized in that, the chitooligosaccharide has a degree of deacetylation of 50% to 80%.
3. A chitooligosaccharide having a specific structure according to claim 1, characterized in that, the chitooligosaccharide has a degree of deacetylation of 56% to 78%.
4. A preparation method of the chitooligosaccharide having a specific structure according to any one of claims 1 to 3, the preparation method comprises: obtaining the chitooligosaccharide by enzymatic hydrolysis of a chitosan substrate, the enzyme used can specifically recognize and hydrolyze the glycosidic bond formed by glucosamine and glucosamine, so that all the components of the chitooligosaccharide obtained by hydrolysis have both the reducing ends and non-reducing ends thereof being glucosamine.
5. The preparation method of the chitooligosaccharide having a specific structure according to claim 4, characterized in that, the chitosan has a degree of deacetylation of 50% to 80%; and chitosans having different degrees of deacetylation are obtained by controlling the deacetylation reaction time of chitin.
6. The preparation method of the chitooligosaccharide having a specific structure according to claim 4, characterized in that, the enzyme is a neutral protease.

2017101852 08 Nov 2017
7. The preparation method of the chitooligosaccharide having a specific structure according to claim 6, characterized in that, the specific conditions for the neutral protease to hydrolyze chitosan are as follows: the amount of the neutral protease is 5 wt% to 15 wt% of a substrate; the hydrolysis temperature is 35-45°C; and the hydrolysis time is 30-60 h.
8. Use of a chitooligosaccharide having a specific structure according to any one of claims 1 to 3 in the preparation of a medicine.
9. The use of a chitooligosaccharide having a specific structure in the preparation of a medicine according to claim 8, characterized in that, the medicine is an anti-liver cancer medicine.
10. A pharmaceutical composition, comprising the chitooligosaccharide according to any one of claims 1 to 3 or a pharmaceutically acceptable salt of the chitooligosaccharide as an active ingredient.
11. The pharmaceutical composition of claim 10, further comprising a pharmaceutically acceptable carrier.