CN114957358B

CN114957358B - Lignan glycoside compound, and preparation method and application thereof

Info

Publication number: CN114957358B
Application number: CN202011277987.3A
Authority: CN
Inventors: 崔龙; 李志�; 赵一名; 修孟雪; 张悦
Original assignee: Beihua University
Current assignee: Beihua University
Priority date: 2020-11-16
Filing date: 2020-11-16
Publication date: 2023-11-03
Anticipated expiration: 2040-11-16
Also published as: CN114957358A

Abstract

The invention provides a lignan glycoside compound, a preparation method and application thereof, wherein the lignan glycoside compound is obtained by taking acanthopanax stems as raw materials and extracting and separating the acanthopanax stems by a solvent. The compound can remarkably improve the lactic acid rate, reduce the lactic acid content of a matrix and reduce the BUN and CK levels of body serum after exercise, thereby achieving the effects of relieving and treating fatigue.

Description

Lignan glycoside compound, and preparation method and application thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a lignan glycoside compound and a preparation method and application thereof.

Background

The life rhythm of people in the modern society is accelerated, the working pressure is increased, and fatigue is used as a chronic disease and gradually erodes the health of people in the modern society. Fatigue is a subjective discomfort, mainly manifested as tiredness, lassitude, perceived under prolonged labor, strenuous exercise or prolonged mental stress, which is a normal physiological protective response that suggests to the body that the body should be allowed to recover through rest, thereby avoiding further injury to the body. When the automobile is in a fatigue state for a long time, the physical strength and physical ability of a human body are damaged, and the body is unbalanced, so that a person is difficult to engage in or complete certain work with great physical exertion or fine and exquisite action, thereby reducing the working efficiency; on the other hand, long-term fatigue can also lead the skin of a person to be loose, the complexion is lusterless, the symptoms of premature senility are not recovered after long-term fatigue, the immune system of the person is dysfunctional, even the immunity is low, the barrier of the body against diseases is broken, and the probability of illness of the person is increased.

Fatigue is caused by a plurality of reasons, mainly energy source material exhaustion, metabolite accumulation, neurotransmitter imbalance, aggravation of free radical generation and the like. Mental and physical fatigue is accompanied by various physiological and biochemical indexes. Exercise-induced fatigue recovery requires rapid repair of lesions occurring in the body or promotion of removal of metabolic products accumulated during exercise. Based on this, it is required to find an anti-fatigue substance to delay the occurrence of fatigue caused by long-time labor, intense exercise or excessive mental stress, or to restore the fatigue state to a normal state as soon as possible, in order to restore the sub-health state of people or to exert a therapeutic effect on fatigue syndrome.

Disclosure of Invention

Based on the above, it is necessary to provide a lignan glycoside compound, and a preparation method and application thereof, wherein the lignan glycoside compound can remarkably improve the lactic acid rate, reduce the lactic acid content of a matrix, and reduce the serum BUN and CK levels of a body after exercise, thereby achieving the effects of relieving and treating fatigue.

The invention provides a lignan glycoside compound, which is obtained by taking acanthopanax stems as raw materials and extracting and separating the acanthopanax stems by a solvent.

The invention also provides a lignan glycoside compound, which has a structure shown in any one of formulas (I) - (IV):

the invention also provides a lignan glycoside compound selected from compounds shown in a formula (IV).

The invention also provides a preparation method of the lignan glycoside compound, which comprises the following steps:

s1, taking acanthopanax stems, soaking in an alcohol solution, carrying out ultrasonic extraction, concentrating an extracting solution, adding alcohol, uniformly mixing, standing, separating out a precipitate, separating the precipitate from the solution, retaining the precipitate, and drying the precipitate to obtain an acanthopanax extract;

s2, carrying out gradient elution on the acanthopanax root extract by using flowing, respectively collecting each eluent, concentrating, separating and identifying to obtain the lignan glycoside compound.

In one embodiment, the step S1 specifically includes: taking dried and crushed acanthopanax stems, adding 40-60% ethanol with the volume of 4-6 times to soak for 0.5-1.5 hours, extracting for 2-3 times by utilizing a microwave ultrasonic wave synergistic method, merging extracting solutions, concentrating the extracting solutions to 0.2-0.4 times by using a rotary evaporator, cooling, adding 2-4 times of ethanol, stirring, standing for 20-28 hours at the temperature of 10-15 ℃, standing for precipitation, separating the precipitate, heating and drying to remove a solvent, thus obtaining the acanthopanax extract.

In one embodiment, the step S2 specifically includes: separating the acanthopanax extract by utilizing hydroxypropyl sephadex, performing gradient elution on mobile phases of which the volume ratio of methanol to water is 0:10,1:9,2:8,3:7,4:6,5:5,6:4,7:3,8:2,9:1 and 10:0, respectively collecting eluent, concentrating to obtain components 1 to 11, and separating and identifying the components 1 to 11 to obtain the lignan glycoside compound.

In one embodiment, in the step S2, the specific step of separating the component 3 is: taking the component 3, performing semi-preparative reverse-phase high performance liquid chromatography, using a C18 column, and performing isocratic elution for 55-65 min by using a 14-16% ethanol solution as a mobile phase to obtain the compounds with the structures shown in the formulas (I) and (II).

In one embodiment, in the step S2, the specific step of separating the component 4 is: taking the component 4, performing semi-preparative reverse-phase high performance liquid chromatography, using a C18 column, and performing gradient elution for 65-75 min by using 16-18% to 20-22% ethanol solution as a mobile phase to obtain the compounds with the structures of formula (III) and formula (IV).

The invention also provides a pharmaceutical composition, which comprises any one of the lignan glycoside compounds and pharmaceutically acceptable auxiliary materials, diluents or carriers.

By "pharmaceutically acceptable" is meant those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio, and are effective for their intended use.

The pharmaceutical composition may preferably be: comprises a compound shown in any one of the structural formulas (I) - (IV), and pharmaceutically acceptable auxiliary materials, diluents or carriers. It may also be preferable that: comprises a compound with a structure shown as a formula (I) and a formula (II), and pharmaceutically acceptable auxiliary materials, diluents or carriers. More preferably: comprises compounds with the structures of formula (III) and formula (IV), and pharmaceutically acceptable auxiliary materials, diluents or carriers.

The invention also provides an application of the lignan glycoside compound or the pharmaceutical composition in preparing a medicament for relieving or treating fatigue.

Compared with the prior art, the invention has the following beneficial effects:

the lignan glycoside compound can obviously improve the lactic acid rate, reduce the lactic acid content of a matrix and reduce the serum BUN and CK levels of a body after exercise, thereby achieving the effects of relieving and treating fatigue.

Drawings

FIG. 1 is a diagram of Compound 1 ¹ H-NMR spectrum;

FIG. 2 shows that Compound 1 is in the range of +7.2ppm to +4.2ppm ¹ H-NMR spectrum;

FIG. 3 shows that Compound 1 is in the range of +4.3ppm to +2.1ppm ¹ H-NMR spectrum;

FIG. 4 is a diagram of Compound 1 ¹³ C-NMR spectrum;

FIG. 5 is a HMBC spectral diagram of Compound 1;

FIG. 6 is a NOE spectrum of Compound 1;

FIG. 7 is a diagram of Compound 2 ¹ H-NMR spectrum;

FIG. 8 shows compound 2 in the range of +7.5ppm to +4.4ppm ¹ H-NMR spectrum;

FIG. 9 shows compound 2 in the range of +4.4ppm to +1.4ppm ¹ H-NMR spectrum;

FIG. 10 is a diagram of Compound 2 ¹³ C-NMR spectrum;

FIG. 11 is a HMBC spectral diagram of Compound 2;

FIG. 12 is a NOE spectrum of Compound 2;

FIG. 13 is a diagram of Compound 3 ¹ H-NMR spectrum;

FIG. 14 shows that Compound 3 ranges from +7.2ppm to +4.7ppm ¹ H-NMR spectrum;

FIG. 15 shows compound 3 in the range of +4.4ppm to +1.9ppm ¹ H-NMR spectrum;

FIG. 16 is a diagram of Compound 3 ¹³ C-NMR spectrum;

FIG. 17 is a HMBC spectral diagram of Compound 3;

FIG. 18 is a NOE spectrum of Compound 3;

FIG. 19 is a diagram of Compound 4 ¹ H-NMR spectrum;

FIG. 20 shows that Compound 4 ranges from +7.3ppm to +4.3ppm ¹ H-NMR spectrum;

FIG. 21 shows that Compound 4 is in the range of +5.0ppm to +1.5ppm ¹ H-NMR spectrum;

FIG. 22 is a diagram of Compound 4 ¹³ C-NMR spectrum;

FIG. 23 is a HMBC spectral diagram of Compound 4;

FIG. 24 is a NOE spectrum of Compound 4.

Detailed Description

In order that the invention may be understood more fully, a more particular description of the invention will be rendered by reference to the preferred embodiments that are now set forth. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

For convenience of description, the compounds represented by the formulae (I) to (IV) in the present invention are named compound 1, compound 2, compound 3, and compound 4, respectively.

Example 1

The extraction and separation method of the components 1-11.

Weighing 1.0kg of dried and crushed acanthopanax stems, adding 5.0L of 50% ethanol, soaking for 1 hour, and extracting for 2 times by utilizing a microwave ultrasonic synergistic method for 1 hour. Mixing the extractive solutions, concentrating to 1/5 volume with rotary evaporator, cooling, adding 3 times of ethanol, stirring, standing at low temperature for 24 hr, separating out precipitate, filtering or centrifuging to obtain precipitate, heating the precipitate in a surface dish, removing ethanol and water until no ethanol smell exists, and obtaining radix Acanthopanacis Senticosi extract (357.0 g). Separating radix Acanthopanacis Senticosi extract with hydroxypropyl dextran gel (LH-20), gradient eluting with mobile phases of methanol to water volume ratio of 0:10,1:9,2:8,3:7,4:6,5:5,6:4,7:3,8:2,9:1, 10:0, eluting with 500ml each, collecting different gradient eluents 500ml, and concentrating to obtain component 1 to component 11 respectively.

Example 2

Lactic acid clearance test for Components 1-11.

130 healthy clean Kunming male mice are taken, after the basic feed is fed for 7 days in a balanced mode, 70 mice with the weight of 20-25g are selected and randomly grouped into blank groups, experimental groups are 1-11, and a Hongshan capsule control group of the Tibetan domain card of the department of Central science. From the beginning to the end of the experiment, the experimental components 1-11 groups were lavaged (600 mg/kg); the Hongtian capsule control group of the Tibetan domain card (600 mg/kg); the blank group and the model group are filled with normal saline with the same volume, and the stomach is continuously filled for 20 days. After the last administration for 1h, each mouse was put into an organic glass jar with a height of 50cm, a diameter of 50cm and a water depth of 30cm for swimming for 10min, and the water temperature was 25+ -1deg.C. Before swimming, immediately after swimming and after resting for 20min, each mouse was needled with tail vein to collect blood, and the blood lactate value was measured. The area under the blood lactic acid curve was calculated from the blood lactic acid values at the respective time points. The experimental results are shown in Table 1.

TABLE 1 area under lactic acid curve results for fatigue test in mice

The experimental results show that: compared with the blank group, the area under the blood lactic acid curve of the experimental components 1-7 is reduced. Wherein, the area under the blood lactic acid curve of the experimental component 3-4 is obviously reduced compared with that of the positive control group. The separation, identification and testing was selected at component 3 and component 4.

Example 3

(1) Methods for isolation of Compounds 1-4.

Component 3 (3.5 g) was subjected to semi-preparative reverse phase high performance liquid chromatography using a C18 column (10 mm. Times.250 mm,5 μm) and isocratically eluted with 15% ethanol as the mobile phase for 60min to give compounds 1 (1.3 g) and 2 (0.8 g).

Component 4 (4.3 g) was subjected to semi-preparative reverse phase high performance liquid chromatography using a C18 column (10 mm. Times.250 mm,5 μm) and gradient eluted with 17% to 21% ethanol as the mobile phase for 70min to give compounds 3 (1.1 g) and 4 (0.9 g).

(2) Compounds 1-4 were subjected to spectral data and structural formula.

Compound 1, white powder; optical rotation-11.2 (c 0.01, meOH); MS m/z 522.2106[ M ]] ⁺ (calcd for C ₂₆ H ₃₄ O ₁₁ ,522.2101)；UV(MeOH)λ _max (logε):228(3.96),279(3.28)nm；IR(KBr)ν _max :3419,1599,1514,1267,1053； ¹ H(500MHz,acetone-d ₆ ) And ¹³ C-NMR(125MHz,acetone-d ₆ ) HMBC and NOE maps are shown in figures 1-6; the chemical structural formula is as follows.

Compound 2, white powder; optical rotation-7.2 (c 0.01, meoh); MS m/z 522.2105[ M] ⁺ (calcd for C ₂₆ H ₃₄ O ₁₁ ,522.2103)；UV(MeOH)λ _max (logε):226(3.12),287(3.08)nm；IR(KBr)ν _max :3415,1596,1512,1259,1049； ¹ H(500MHz,acetone-d ₆ ) And ¹³ C-NMR(125MHz,acetone-d ₆ ) HMBC and NOE maps are shown in figures 7-12; the chemical structural formula is as follows.

Compound 3, white powder; optical rotation-15.2 (c 0.01, meoh); MS m/z 522.2104[ M] ⁺ (calcd for C ₂₆ H ₃₄ O ₁₁ ,522.2101)；UV(MeOH)λ _max (logε):233(2.96),273(2.37)nm；IR(KBr)ν _max :3417,1585,1519,1253,1041； ¹ H(500MHz,acetone-d ₆ ) And ¹³ C-NMR(125MHz,acetone-d ₆ ) HMBC and NOE maps are shown in figures 13-18; the chemical structural formula is as follows.

Compound 4, white powder; optical rotation-1.2 (c 0.01, meoh); MS m/z 522.2109[ M ]] ⁺ (calcd for C ₂₆ H ₃₄ O ₁₁ ,522.2102)；UV(MeOH)λ _max (logε):225(3.61),284(3.11)nm；IR(KBr)ν _max :3431,1590,1517,1232,1046； ¹ H(500MHz,acetone-d ₆ ) And ¹³ C-NMR(125MHz,acetone-d ₆ ) HMBC and NOE maps are shown in figures 19-24; the chemical structural formula is as follows.

Example 4

Lactic acid clearance test for Compounds 1-4.

100 healthy clean-grade Kunming male mice are taken, and after 7 days of balanced feeding by basic feed, the mice are randomly grouped into blank groups, 1-4 groups of compounds and a red day capsule control group of the Tibetan domain card of the department of Central science. From the beginning to the end of the experiment, groups 1-4 were gastrected (40 mg/kg,80 mg/kg); the Hongtian capsule control group of the Tibetan domain card (600 mg/kg); the blank group is filled with equal volume of normal saline for 20 days. After the last administration for 1h, each mouse was put into an organic glass jar with a height of 50cm, a diameter of 50cm and a water depth of 30cm for swimming for 10min, and the water temperature was 25+ -1deg.C. Before swimming, immediately after swimming and after resting for 20min, each mouse was needled with tail vein to collect blood, and the blood lactate value was measured. The area under the blood lactic acid curve was calculated from the blood lactic acid values at the respective time points. The experimental results are shown in Table 2.

TABLE 2 area under lactic acid curve results for fatigue test in mice

The experimental results show that: the areas under the blood lactic acid curve were significantly reduced with increasing doses for each of the compound 1-4 groups compared to the blank group. The area under the blood lactic acid curve of the compound 2-4 group is significantly reduced (P < 0.5) compared with the positive control group at the dose of 80 mg/kg.

Example 5

Post-exercise urea nitrogen (BUN) and Creatine Kinase (CK) content assays for compounds 1-4 in serum.

60 healthy clean Kunming male mice are taken, and after 7 days of basic feed balance feeding, the mice are randomly grouped into blank groups, 1-4 groups of compounds and a red day capsule control group of the Tibetan domain card of the department of Central science. From the beginning to the end of the experiment, compound 1-4 groups were intragastrically (80 mg/kg); the Hongtian capsule control group of the Tibetan domain card (600 mg/kg); the blank group is filled with equal volume of normal saline for 20 days. After the last administration for 1h, each mouse was put into a plexiglass jar with a height of 50cm, a diameter of 50cm and a water depth of 30cm for swimming, and the water temperature was 25+ -1deg.C. After each mouse swims for 90min and has a rest for 60min, the eyeballs are taken out to obtain blood, and serum urea nitrogen (BUN) and Creatine Kinase (CK) are measured by a blood biochemical analyzer. The experimental results are shown in Table 3.

TABLE 3 BUN and CK results in blood of mice

The experimental results show that: both serum BUN and CK were significantly lower in the compound 1-4 groups than in the blank group at the 80mg/kg dose. Of these, the effect of groups 3-4 was most pronounced.

Example 6

Weight-bearing swim test for groups 1-4.

60 healthy clean Kunming male mice are taken, and after 7 days of basic feed balance feeding, the mice are randomly grouped into blank groups, 1-4 groups of compounds and a red day capsule control group of the Tibetan domain card of the department of Central science. From the beginning to the end of the experiment, compound 1-4 groups were intragastrically (80 mg/kg); the Hongtian capsule control group of the Tibetan domain card (600 mg/kg); the blank group is filled with equal volume of normal saline for 20 days. After the last administration for 1h, each group of mice was placed in a plexiglass jar with a height of 50cm, a diameter of 50cm and a water depth of 30cm for swimming at a water temperature of 25+ -1deg.C with 5% weight of lead wire loaded at the root of the mice. The mice were submerged in the water bottom 8s and no longer emerged from the water surface as the exhaustion standard. The time from the swimming start to the exhaustion of each mouse is recorded as the swimming time of the mouse. The experimental results are shown in Table 4.

TABLE 4 results of fatigue test run-out time in mice

The experimental results show that: the exhaustion time of each administration group is obviously higher than that of the blank control group at the dose of 80 mg/kg.

The results of the above examples show that: the components 3-4 screened in the acanthopanax can obviously reduce the lactic acid value of the organism and obviously improve the lactic acid clearance rate; the compounds 1-4 can obviously reduce the lactic acid value of the organism at the dosage of 80mg/kg, obviously improve the lactic acid clearance rate, obviously reduce the serum BUN and CK levels of the organism after exercise and obviously increase the stress exhaustion time.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims

1. The preparation method of the lignan glycoside compound is characterized by comprising the following steps of:

step S1, taking dried and crushed acanthopanax stems, adding 5L of 50% ethanol into each kilogram of acanthopanax stems, soaking for 1 hour, extracting for 2 times by utilizing a microwave ultrasonic wave synergistic method, mixing the extracting solutions, concentrating the extracting solutions to 0.2 times of volume by using a rotary evaporator, cooling, adding 3 times of volume of ethanol, stirring, standing for 24 hours at 10-15 ℃, standing for precipitation, separating the precipitate, heating and drying to remove a solvent, thus obtaining the acanthopanax extract;

s2, separating the acanthopanax extract by utilizing hydroxypropyl sephadex, performing gradient elution on mobile phases of which the volume ratio of methanol to water is 0:10,1:9,2:8,3:7,4:6,5:5,6:4,7:3,8:2,9:1 and 10:0, respectively collecting eluent, concentrating to respectively obtain a component 1 to a component 11, and separating and identifying the component 1 to the component 11 to obtain the lignan glycoside compound, wherein the specific steps of separating the component 3 are as follows: taking the component 3, performing semi-preparative reverse-phase high performance liquid chromatography, using a C18 column, and performing isocratic elution for 60min by using a 15% ethanol solution as a mobile phase to obtain a compound with a structure shown in a formula (II); the specific steps of separating component 4 are: taking the component 4, performing semi-preparative reverse-phase high performance liquid chromatography, using a C18 column, and performing gradient elution for 70min by using 17-21% ethanol solution as a mobile phase to obtain compounds with structures shown in formula (III) and formula (IV);

the structures of the compounds of the formulas (II) - (IV) are shown as follows:

（Ⅱ），

（Ⅲ），/>（Ⅳ）。

2. the use of the lignan glycoside compound obtained by the preparation method of claim 1 in the preparation of a medicament for relieving or treating fatigue.