Radix asparagi oligosaccharide with anti-depression effect and preparation method thereof

Abstract

The invention belongs to the technical field of medicine, and discloses radix asparagi oligosaccharide with an anti-depression effect and a preparation method thereof.The radix asparagi oligosaccharide is formed by polymerizing glucose and fructose, a sugar chain of the radix asparagi oligosaccharide is formed by connecting T-D-glucose and 2, 1-D-fructose, the molar ratio of the glucose to the fructose is 1: 9.4, the molar ratio of the T-D-glucose to the 2, 1-D-fructose is 1: 9.4, and the molar ratio of the T-D-glucose to the 2, 1-D-fructose is 1: 9.4. The molar ratio of the 1-D-fructose is 1: 10. The radix asparagi polysaccharide is obtained through water extraction, alcohol precipitation, membrane separation and other methods, the radix asparagi polysaccharide is subjected to acid hydrolysis to obtain the radix asparagi oligosaccharide polymerized by glucose and fructose, the obtained radix asparagi oligosaccharide has a good anti-depression effect, the preparation method is simple, nontoxic and pollution-free, industrial production can be achieved, and the radix asparagi oligosaccharide has wide market prospects.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and more specifically, to an asparagine oligosaccharide with antidepressant effects and a preparation method thereof. Background Art

[0002] Depression (major depressive disorder, MDD) is a common mental disorder characterized by significant and persistent low mood. Currently, except for cardiovascular diseases, depression has become one of the main killers of human health. The WHO has listed antidepressants as one of the drugs that need to be urgently developed in the 21st century. Currently, the drugs used to treat depression mainly include: selective 5-hydroxytryptamine reuptake inhibitors (SSRIs), tricyclic antidepressants, monoamine oxidase inhibitors, and norepinephrine reuptake inhibitors. However, the anti-effects of these drugs usually cannot be sustained, and are accompanied by side effects such as apathy, sedation, cognitive impairment, sleep disorders, and sexual dysfunction, and they are expensive and have strong drug dependence. In recent years, some researchers have begun to focus on the study of the antidepressant activity of carbohydrates. With the in-depth understanding of the pathogenesis of depression, the research and development of natural antidepressant drugs have also become a new hot spot and development trend. Therefore, it is particularly important to develop new, highly effective, and low-toxic antidepressant drugs.

[0003] Asparagus is the tuberous root of the plant Asparagus cochinchinensis (Lour.) Merr. of the Liliaceae family. Asparagus was first recorded in "Shennong Ben Cao Jing" and listed as a top-grade medicine, also known as Dian Le. It tastes sweet and bitter, and is cold in nature. It belongs to the lung and kidney meridians. It is used to treat symptoms such as yin deficiency fever, cough with hemoptysis, lung abscess, sore throat, thirst, constipation, etc. Asparagus mainly contains components such as steroidal saponins, oligosaccharides, polysaccharides, and amino acids. Among them, asparagine oligosaccharide is an important pharmacodynamic component and has important research value in the research fields of carbohydrate chemistry, glycobiology, carbohydrate engineering, and carbohydrate drugs. More and more research focuses on the pharmacological activities of asparagine oligosaccharides, especially in the aspects of antidepressant and antianxiety effects. Summary of the Invention

[0004] The purpose of the present invention is to solve the disadvantages existing in the prior art, and to propose an asparagine oligosaccharide with antidepressant effects and a preparation method thereof.

[0005] To solve the above problems, the present invention adopts the following technical solutions: An asparagine oligosaccharide with antidepressant effects, wherein the asparagine oligosaccharide is polymerized from glucose and fructose, and the sugar chain of the asparagine oligosaccharide is connected by T-D-glucose and 2,1-D-fructose.

[0006] As a further aspect of the present invention: the molar ratio of glucose to fructose is 1:9.4, and the molar ratio of T-D-glucose to 2,1-D-fructose is 1:10.

[0007] As a further solution of the present invention: the D-glucose exists in the structure in the anomeric form, and the D-fructose exists in the structure in the 2,1-D-fructose linkage mode.

[0008] As a further solution of the present invention: the aspartic oligosaccharide sugar chain is a straight chain, the non-reducing end is D-glucose, and the D-fructose is connected in the 2→1 glycosidic bond linkage mode. The more 2,1-D-fructose, the higher the degree of polymerization of the oligosaccharide. The structural formula is as follows: , In the formula, n is one or more integers selected from 1-10, for example, n is one or more integers selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0009] As a further solution of the present invention: in the formula, n is selected from 2-8.

[0010] As a further solution of the present invention: in the formula, n is selected from 3-5.

[0011] As a further solution of the present invention: the aspartic oligosaccharide has a good antidepressant effect.

[0012] The present invention also provides a preparation method of an aspartic oligosaccharide with antidepressant effect, comprising the following steps: (1) Extracting asparagus with an aqueous solution once or multiple times to obtain an asparagus extract; (2) Concentrating the extract in (1), and then adding an organic solvent to make the concentration of the organic solvent reach 60-90% (±5), and centrifuging to obtain a precipitate; (3) Redissolving the precipitate in (2) with water, adding acid for hydrolysis, ending the reaction, and evaporating the hydrolysis solution to dryness; (4) Passing the hydrolysis solution in (3) through a 3K ultrafiltration membrane, collecting the permeate, concentrating and drying to obtain aspartic oligosaccharide.

[0013] As a further solution of the present invention: in the step (1), the water extraction temperature is 60-100°C, the extraction times are 1-2 times, the extraction time is 0.5-3 h, and the solid-liquid ratio is 5:1 to 30:1.

[0014] As a further solution of the present invention: in the step (2), the organic solvent is selected from methanol, ethanol, propanol, acetone or a mixture thereof, and ethanol is preferred.

[0015] As a further solution of the present invention: in the step (5), the acid is selected from trifluoroacetic acid, hydrochloric acid, sulfuric acid or glacial acetic acid, and trifluoroacetic acid is preferred.

[0016] Compared with the prior art, the advantages of the present invention are: The present invention uses methods such as water extraction and alcohol precipitation, and membrane separation to obtain asparagus polysaccharide. The asparagus polysaccharide is hydrolyzed by acid to obtain asparagus oligosaccharide polymerized from glucose and fructose. The obtained asparagus oligosaccharide has a good antidepressant effect. Its preparation method is simple, non-toxic and pollution-free, can be industrially produced, and has broad market prospects. Description of the Drawings

[0017] Figure 1 It is the HPLC chromatogram of asparagus oligosaccharide 4; Figure 2 It is the monosaccharide composition of asparagus oligosaccharide 4; Figure 3 It is the graph of the effect of continuous oral gavage administration of asparagus oligosaccharide 4 on the immobile time of mice in forced swimming; Figure 4 It is the graph of the effect of continuous oral gavage administration of asparagus oligosaccharide 4 on the sucrose preference index of mice; Figure 5 It is the graph of the effect of continuous oral gavage administration of asparagus oligosaccharide 4 on the hair score of mice. Detailed Embodiments

[0018] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work belong to the scope of protection of the present invention.

[0019] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the present invention. The preferred implementation methods and materials described in the text are only for demonstration purposes, but cannot limit the content of this application.

[0020] The experimental methods in the following embodiments are all conventional methods unless otherwise specified; the test materials and test strains used in the following embodiments are all obtained from commercial channels unless otherwise specified.

[0021] Example 1 An asparagus oligosaccharide with antidepressant effects, the asparagus oligosaccharide is polymerized from glucose and fructose, with a molar ratio of 1:9.4. The sugar chain of the asparagus oligosaccharide is connected by T-D-glucose and 2,1-D-fructose, with a molar ratio of 1:10. The sugar chain of the asparagus oligosaccharide is a straight chain, the non-reducing end is D-glucose, and the D-fructose is connected in a 2→1 glycosidic bond connection mode. The more 2,1-D-fructose, the higher the degree of polymerization of the oligosaccharide. The structural formula is as follows: , Wherein, n is one or more integers selected from 3 to 5.

[0022] Example 2 Preparation method of an aspartic oligosaccharide with antidepressant effect Take 400 g of asparagus, add 4 L of H2O, extract twice, 30 min each time, extraction temperature 60 °C. After combining the extracts, filter through a 50-mesh sieve. Add ethanol to the extract to make the alcohol content reach 90%, centrifuge to obtain a precipitate. Redissolve the precipitate in water, add 0.1 M hydrochloric acid for hydrolysis to a final concentration of 5 mg / mL, hydrolyze for 2 h at a temperature of 85 °C. After the reaction ends, pass the hydrolyzate through a 3K ultrafiltration membrane, collect the permeate, concentrate and dry to obtain aspartic oligosaccharide 1.

[0023] Example 3 An aspartic oligosaccharide with antidepressant effect and its preparation method Take 400 g of asparagus, add 4 L of H2O, extract twice, 1 h each time, extraction temperature 100 °C. After combining the extracts, filter through a 50-mesh sieve. Add ethanol to the extract to make the alcohol content reach 70%, centrifuge to obtain a precipitate. Redissolve the precipitate in water, add 0.1 M sulfuric acid for hydrolysis to a final concentration of 5 mg / mL, hydrolyze for 2 h at a temperature of 85 °C. After the reaction ends, pass the hydrolyzate through a 3K ultrafiltration membrane, collect the permeate, concentrate and dry to obtain aspartic oligosaccharide 2.

[0024] Example 4 An aspartic oligosaccharide with antidepressant effect and its preparation method Take 400 g of asparagus, add 4 L of H2O, extract twice, 2 h each time, extraction temperature 100 °C. After combining the extracts, filter through a 50-mesh sieve. Add ethanol to the extract to make the alcohol content reach 70%, centrifuge to obtain a precipitate. Redissolve the precipitate in water, add 0.1 M trifluoroacetic acid for hydrolysis to a final concentration of 5 mg / mL, hydrolyze for 2 h at a temperature of 85 °C. After the reaction ends, pass the hydrolyzate through a 3K ultrafiltration membrane, collect the permeate, concentrate and dry to obtain aspartic oligosaccharide 3.

[0025] Example 5 An aspartic oligosaccharide with antidepressant effect and its preparation method Take 400 g of asparagus, add 4 L of H2O, extract twice, 2 h each time, extraction temperature 100 °C. After combining the extracts, filter through a 50-mesh sieve. Add ethanol to the extract to make the alcohol content reach 65%, centrifuge to obtain a precipitate. Redissolve the precipitate in water, add 0.1 M trifluoroacetic acid for hydrolysis to a final concentration of 5 mg / mL, hydrolyze for 2 h at a temperature of 85 °C. After the reaction ends, pass the hydrolyzate through a 3K ultrafiltration membrane, collect the permeate, concentrate and dry to obtain aspartic oligosaccharide 4.

[0026] The present invention selects aspartic oligosaccharide 4 prepared in Example 5 for structure characterization, including the following steps: (1) The monosaccharide composition determination method is as follows: Ion chromatography was used to determine the monosaccharide composition of asparagine oligosaccharide. The sample was prepared into a 10 mg / mL solution, 0.5 mL was taken and placed in an ampoule, 0.5 ml of 6MTFA solution was added, the bottle was sealed with an alcohol lamp, and hydrolyzed in an oven at 105°C for 4 hours. Then 0.5 ml of 6MTFA solution was added, the bottle was sealed with an alcohol lamp, and hydrolyzed in an oven at 105°C for 4 hours. Methanol was added, and the sample was dried with a nitrogen blower. Repeat 3-5 times. The mixed standard sample was prepared into a 1.0 mg / mL solution.

[0027] Chromatographic conditions: Chromatographic column: Dionex CarboPacTM PA10 BioLCTM 4×250mm; Mobile phase: A: H2O, B: 10 mM NaOH (0:100); Column temperature: 30°C; Flow rate: 0.8 mL / min; Injection volume: 20 μL; Detector: Ampereometric detector; Data collection time: 60 minutes.

[0028] Asparagine oligosaccharide 4 HPLC chromatogram Figure 1 The results of the 4 monosaccharide composition of asparagine oligosaccharide are shown in Figure 2. It is mainly composed of glucose and fructose with a molar ratio of 1:9.4.

[0029] (2) The connection method is as follows: Methylation reaction: Accurately weigh 1 mg of the sample to be tested and add 500 μl DMSO to dissolve. Add 1 mg NaOH and incubate for 30 min. Add 50 μl iodomethane solution and react for 1 h. Add 1 ml water and 2 ml dichloromethane, vortex to mix, centrifuge, and discard the aqueous phase. Repeat the water wash 3 times. Aspirate the lower dichloromethane phase and evaporate to dryness.

[0030] Hydrolysis reaction: Add 100 μl 2M TFA, react at 121°C for 90 min, and evaporate to dryness at 30°C.

[0031] Reduction reaction: Add 50 μl 2 M ammonia water and 50 μl 1 M NaBD4, mix well, and react at room temperature for 2.5 h. Add 20 μl acetic acid to terminate the reaction, blow dry with nitrogen, wash twice with 250 μl methanol, and blow dry with nitrogen.

[0032] Acetylation reaction: Add 250 μl of acetic anhydride, vortex to mix well, and react at 100 °C for 2.5 h. Add 1 ml of water and let stand for 10 min. Add 500 μl of dichloromethane, vortex to mix well, centrifuge, and discard the aqueous phase. Repeat the water washing 3 times. Take the lower dichloromethane phase and detect it by machine.

[0033] Chromatographic conditions: Chromatographic column: HP-5MS fused silica capillary column 30m * 250μm * 0.25μm. The carrier gas is helium. Inject with a split ratio of 100:1, the inlet temperature is 250 °C, the flow rate is 1 mL / min, and the injection volume is 1 μL.

[0034] GC method: Maintain at 50 °C for 1.0 min, increase the temperature to 130 °C at a rate of 50 °C / min, then increase the temperature to 220 °C at a rate of 3 °C / min and maintain for 2 min.

[0035] MS acquisition method: Quadrupole temperature: 150 °C, ion source temperature: 230 °C, scan type: full scan (scan), scan rate: N = 1.

[0036] The 4-linkage mode of asparto-oligosaccharide consists of T-D-Glc and 2,1-D-Fru, and the molar ratio is 1:10.

[0037] Experimental Example 1 Antidepressant effect experiment: Experimental drug: Asparto-oligosaccharide 4, administered at doses of 25 mg / kg (low dose) and 75 mg / kg (high dose) respectively.

[0038] Experimental animals: SPF-grade C57BL / 6J mice (body weight 18 - 22 g, provided by Zhejiang Vital River Laboratory Animal Technology Co., Ltd.).

[0039] Experimental instruments: Electronic balance, produced by Adam Equipment (Wuhan) Co., Ltd.; Animal behavior video analysis software, produced by Noldus Information Technology, Wageningen, Netherlands.

[0040] Experimental method: Keep the mouse breeding environment at a temperature of 19 - 26 °C, humidity of 40 - 70%, with a 12-hour light-dark cycle, and let the mice freely ingest filtered water and autoclaved mouse food. The experiment is carried out in 2 batches: The first batch of experiments was divided into 3 groups, with 10 mice in each group, namely the control group, the asparto-oligosaccharide 4 25 mg / kg group, and the asparto-oligosaccharide 475 mg / kg group. Each experimental group was orally administered (p.o.) the corresponding medicinal liquid at a dose of 0.1 ml / 10 g body weight once a day for 35 consecutive days, while the control group was given ddH2O. The body weight was measured once a week. 30 minutes after administration on the 26th day, a pre-swimming test was conducted. The mice were placed in a plexiglass round cylinder with a diameter of ø9 cm and a height of h20 cm and a water depth of 11 cm (water temperature 24°C), and their swimming time was 15 minutes. The test was conducted 24 hours later, with other conditions the same as the pre-swimming test, and the swimming duration was 6 minutes. The camera system automatically collected data, and the time when the mice stopped swimming and remained motionless within the last 4 minutes was statistically analyzed. The swimming immobility time of the depressive mice increased significantly. After the experiment, the mice were deeply anesthetized with CO2 and sacrificed.

[0041] Experimental method: The experiment was divided into 3 groups, with 10 mice in each group, namely the control group, the model group, and the asparto-oligosaccharide 4 75 mg / kg group. Except for the control group mice which were intraperitoneally injected with phosphate buffer solution, the other groups of mice were intraperitoneally injected with 5 mg / kg lipopolysaccharide once. 48 hours after the lipopolysaccharide injection, each experimental group was orally administered the corresponding medicinal liquid at a dose of 0.1 mL / 10 g body weight once a day until the end of the experiment. One week after the lipopolysaccharide injection, except for the control group mice which were handled 2 times a day, the other groups of mice were given 4 h of restraint stress every day (a 50 mL centrifuge tube with 3 air holes opened at the bottom) for 30 consecutive days. The sucrose preference test was conducted on the day after the end of the restraint.

[0042] The sucrose preference test method was divided into an adaptation period and a test period. During the adaptation period, the mice were housed individually in cages, and each cage was provided with a bottle of 1% (w / v) sucrose solution and a bottle of pure water. After 24 hours, their positions were exchanged and this was continued for 48 hours. After the adaptation ended, the mice were fasted and water-deprived for 24 hours. During the test period, each mouse was placed in two pre-weighed bottles, one containing 1% sucrose solution and the other containing pure water. After 24 hours, the two bottles were removed and weighed, and the total liquid consumption, sucrose solution consumption, and pure water consumption of the mice were recorded, and the sucrose preference index was calculated (sucrose preference index = sucrose solution consumption / total liquid consumption * 100%). Since depressive mice have anhedonia, their preference for sucrose solution decreases. At the same time, the hair condition and mental state of the mice were scored, and the higher the score, the worse the state. After the experiment, the mice were deeply anesthetized with CO2 and sacrificed.

[0043] Experimental results: (1) Effect of asparto-oligosaccharide 4 on the immobility time of mice in forced swimming As shown in Table 1 and Figure 3: Compared with the control group, the immobility time of mice in the 4 25mg / kg asparto-oligosaccharide group decreased, with statistical significance (P<0.05 v.s control group); the immobility time of mice in the 4 75mg / kg asparto-oligosaccharide group decreased significantly, with extremely significant statistical significance (P<0.001 v.s control group), indicating that asparto-oligosaccharide 4 reduces the despair immobility time of mice in the forced swimming test and has a certain antidepressant effect.

[0044] Table 1 Effects of continuous oral gavage administration of asparto-oligosaccharide 4 on the immobility time of mice in the forced swimming test (Mean±SEM, n=10) Note: Compared with the control group, # P<0.05, ### P<0.001 (unpaired t-test) (2) Effects of asparto-oligosaccharide 4 on the sucrose preference index and hair condition score of mice As shown in Table 2 and Figure 4: Compared with the control group, the sucrose preference index of mice in the model group decreased, with statistical significance (P<0.01 vs. control group), suggesting that the depression model was successfully established. Compared with the model group, asparto-oligosaccharide 4 increased the sucrose preference index of mice, with statistical significance (P<0.01 vs. model group), indicating that 75mg / kg asparto-oligosaccharide 4 has a certain improvement effect on the mouse depression model.

[0045] Table 2 Effects of asparto-oligosaccharide 4 on the sucrose preference index of mice (Mean±SEM) Note: Compared with the control group, **P<0.01; compared with the model group, ##P<0.01, analyzed by unpaired t-test. As shown in Table 3 and Figure 5: Compared with the control group, the hair condition of mice in the model group deteriorated significantly, the score increased with statistical significance (P<0.001 vs. control group), suggesting that the depression model was successfully established. Compared with the model group, asparto-oligosaccharide 4 improved the hair condition of mice, reduced the hair score, with statistical significance (P<0.05 vs. model group), indicating that 75mg / kg asparto-oligosaccharide 4 has a certain improvement effect on the mouse depression model.

[0046] Table 3 Effects of continuous oral gavage administration of asparto-oligosaccharide 4 on the hair score of mice (Mean±SEM) Experimental conclusion: At the tested dosage, asparto-oligosaccharide 4 at 75 mg / kg showed a certain improvement effect on the mouse depression model in the sucrose preference test.

[0047] The above is only a preferred specific implementation manner of the present invention; however, the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, making equivalent substitutions or changes according to the technical solution and its improvement concept of the present invention, shall be covered by the protection scope of the present invention.

Claims

1. An aspartic oligosaccharide with antidepressant effects, characterized in that: The aspartic oligosaccharide is polymerized from glucose and fructose, and the sugar chain of the aspartic oligosaccharide is composed of T-D-glucose and 2,1-D-fructose linked together.

2. An aspartic oligosaccharide having an antidepressant effect according to claim 1, characterized in that: The D-glucose exists in the structure in the anomeric form, and the D-fructose exists in the structure in the 2,1-D-fructose linkage mode.

3. An asparagine oligosaccharide with an antidepressant effect according to claim 1, characterized in that: The molar ratio of glucose to fructose is 1:9.4, and the molar ratio of T-D-glucose to 2,1-D-fructose is 1:

10.

4. An aspartic oligosaccharide having an antidepressant effect according to claim 1, characterized in that: The sugar chain of the aspartic oligosaccharide is a straight chain, the non-reducing end is D-glucose, and the D-fructose is linked in a 2→1 glycosidic bond linkage mode. The structural formula is as follows: ， In the formula, n is one or more integers selected from 1 to 10.

5. An aspartic oligosaccharide having an antidepressant effect according to claim 4, characterized in that: In the structural formula, n is selected from 2 to 8.

6. The aspartic oligosaccharide with antidepressant effect according to claim 5, characterized in that: In the structural formula, n is selected from 3 to 5.

7. The preparation method of an aspartic oligosaccharide having an antidepressant effect according to any one of claims 1-6, characterized in that: It includes the following steps: (1) Extract asparagus once or multiple times with an aqueous solution to obtain an asparagus extract; (2) Concentrate the extract in (1), then add an organic solvent to make the concentration of the organic solvent reach 60-90% (±5), and centrifuge to obtain a precipitate; (3) Redissolve the precipitate in (2) with water, then add acid for hydrolysis. After the reaction ends, evaporate the hydrolysis solution to dryness; (4) Pass the hydrolysis solution in (3) through a 3K ultrafiltration membrane, collect the permeate, concentrate and dry it to obtain aspartic oligosaccharide.

8. The preparation method of an aspartic oligosaccharide with antidepressant effect according to claim 7, characterized in that: In step (1), the water extraction temperature is 60-100°C, the extraction times are 1-2 times, the extraction time is 0.5-3 h, and the solid-liquid ratio is 5:1 to 30:

1.

9. The preparation method of an aspartic oligosaccharide with antidepressant effect according to claim 7, characterized in that: In step (2), the organic solvent is selected from methanol, ethanol, propanol, acetone or a mixture thereof.

10. The preparation method of an aspartic oligosaccharide having an antidepressant effect according to claim 7, characterized in that: In step (5), the acid is selected from trifluoroacetic acid, hydrochloric acid, sulfuric acid or glacial acetic acid.

Images