CN112043713B

CN112043713B - Application of betulinic acid derivative in preparing medicine for treating nerve injury diseases

Info

Publication number: CN112043713B
Application number: CN202010960220.4A
Authority: CN
Inventors: 陈广通; 储呈娇; 张永珍; 宋开南; 宋妍; 孙颖
Original assignee: Nantong University
Current assignee: Nantong University
Priority date: 2020-09-14
Filing date: 2020-09-14
Publication date: 2021-10-12
Anticipated expiration: 2040-09-14
Also published as: CN112043713A

Abstract

The invention belongs to the field of medicines, and discloses an application of betulinic acid derivatives or pharmaceutically acceptable salts thereof in preparing medicines for treating nerve injury diseases. The invention successfully carries out structural modification on the betulinic acid by utilizing a microbial transformation technology to obtain a plurality of novel compounds, and in-vitro nerve cell injury protection tests prove that the compounds have better nerve cell protection activity, can be used as active ingredients of medicaments for treating neurodegenerative diseases, traumatic brain injury and stroke, and have wide application.

Description

Application of betulinic acid derivative in preparing medicine for treating nerve injury diseases

Technical Field

The invention relates to the field of medicines, in particular to application of betulinic acid derivatives in preparing medicines for treating nerve injury diseases.

Background

The incidence of nervous system diseases such as neurodegenerative diseases, traumatic brain injury and stroke rises year by year, and a heavy burden is brought to the society and families. At present, clinical medicines can only relieve part of symptoms, and cannot effectively prevent diseases, improve cognitive states of patients and prevent disease development. The research on the pathogenesis of these common nervous system diseases and the search for active and effective treatment methods have become important tasks in related disciplines such as neuroscience. During the occurrence and development of the above diseases, damage, loss or death of nerve cells in the brain is the most basic pathological change, and the nerve function is often seriously damaged, so that hemiplegia, aphasia, dysnoesia or coma and even death are caused. The importance of preventing cranial nerve injury has attracted the attention of the medical community at home and abroad, and medical scientists are exploring preventive measures from various aspects.

Betulinic acid is a kind of pentacyclic triterpenoid with special structure. The structure is characterized in that the E ring is a five-membered carbon ring, isopropyl at the 19-position of the E ring is substituted by alpha-configuration, and five rings are arranged in a trans-form. The ingredients are mainly distributed in birch bark, spina date seed, radix asparagi, Chinese pulsatilla root and other traditional Chinese medicines and are main effective ingredients of the traditional Chinese medicines. Modern pharmacological research finds that the betulinic acid compound has the effects of resisting bacteria, viruses and tumors, reducing fat, protecting liver and protecting kidney. However, pentacyclic triterpenoid has special structure, lack of active group in the parent nucleus and few reaction sites, and the derivative with hydroxyl, carbonyl and other modifications on the parent nucleus is difficult to obtain by adopting a conventional chemical reaction method. Therefore, the chemical and pharmacological studies of betulinic acid derivatives with parent nucleus structure modification are not complete, and no literature reports about the neuroprotective effect.

Disclosure of Invention

In view of the above, the present invention aims to provide an application of betulinic acid derivatives or pharmaceutically acceptable salts thereof in the preparation of drugs for treating nerve injury diseases, and the betulinic acid derivatives provided by the present invention have good nerve cell repair and protection activity, can be used as active ingredients of drugs for treating neurodegenerative diseases, stroke, brain injury, etc., and have wide applications.

In the invention, the betulinic acid derivative is a compound with a structural formula of formula I-formula VI:

the betulinic acid derivative or its pharmaceutically acceptable salt can be used for preparing medicine for treating diseases related to nerve injury.

The betulinic acid derivative or the pharmaceutically acceptable salt thereof provided by the invention can be used for preparing medicines for treating central nerve injury and the like caused by neurodegenerative diseases, trauma or stroke.

In the present invention, the neurodegenerative disease includes alzheimer's disease, parkinson's disease, huntington's disease or amyotrophic lateral sclerosis.

The invention also provides a preparation method of the betulinic acid derivative, which comprises the following steps:

1) fermenting and culturing microorganisms, adding betulinic acid into a culture medium, then performing transformation culture, and removing mycelium to obtain a fermentation broth, wherein the microorganisms are strains of the genus Torulopsis (Circinella); preferably, the strain is Byssochlamus terricola moscae CGMCC 3.2695.

2) Extracting the fermentation liquor, and evaporating the extract to obtain a converted crude extract;

3) and purifying the transformed crude extract by using reverse phase high performance liquid chromatography to obtain a product. Wherein, the preparation conditions of the reversed-phase high performance liquid chromatography are that A semi-preparative chromatographic column YMC ODS-A is 10.0 I.D. times.250 mm, acetonitrile-water (30:70, V/V), the flow rate is 2.5mL/min, and the detection wavelength is 203 nm.

The concentration of betulinic acid in the culture medium in the step 1) of the method is 2-5000 mug/mL.

The extraction solvent in the step 2) of the method is a conventional organic solvent, preferably ethyl acetate.

In the medicine taking the betulinic acid derivative or the pharmaceutically acceptable salt thereof as the active ingredient, one or more pharmaceutically acceptable carriers can be added when needed. The carrier comprises a diluent, an excipient, a filler, an adhesive, a wetting agent, a disintegrating agent, an absorption enhancer, a surfactant, an adsorption carrier, a lubricant and the like which are conventional in the pharmaceutical field, and can be prepared according to a conventional method in the pharmaceutical field.

The invention successfully carries out structural modification on the betulinic acid by utilizing a microbial transformation technology to obtain a new betulinic acid derivative, and the compounds have better nerve cell repairing and protecting activity through in vivo animal experiments and in vitro nerve cell injury protection experiments, can be used as active ingredients of medicaments for treating central nerve injury and the like caused by neurodegenerative diseases, trauma or stroke, and have wide application.

Drawings

FIG. 1 is an HPLC liquid chromatogram of betulinic acid derivatives according to the present invention.

Detailed Description

Example 1 preparation of Compounds of formula I-VI

The compound is prepared by adopting a microbial conversion method and taking betulinic acid as a raw material through the steps of fermentation, extraction, separation and the like. The strain of Torulopsis (Circinella) can be purchased from China academy of sciences microorganism culture Collection management center (CGMCC), and is preserved in a solid slant culture medium at 4 deg.C in a refrigerator.

Taking Circinella muscsae CGMCC 3.2695 as an example, the process for preparing the compound with the structural formula I-VI is as follows:

1) fermentation, transformation and extraction

The Circinella muscae CGMCC 3.2695 was inoculated into 2 250mL triangular flasks (containing 100mL potato medium) as seed solutions. After shaking culture on a shaking table at 160rpm and 26 ℃ for 1 day, 1mL of seed solution was aspirated by a sterile pipette and added to 20 1000mL shake flasks (containing 400mL potato medium) until the hyphae growth was in vigorous phase. After 1 day of shake culture, 20mg betulinic acid (0.2mL, 100mg/mLDMSO solution) was added to each flask, sharing 400mg substrate. Continuing to transform for 7 days under the same conditions, filtering the fermentation broth, filtering to remove mycelium, extracting the filtrate with equal volume of ethyl acetate for 3 times, and concentrating the extractive solution under reduced pressure to dryness to obtain about 0.56g of crude extract of the transformed matter.

2) Purification by reversed phase high performance liquid chromatography

The combined fractions were purified by reverse phase high performance liquid chromatography. The preparation conditions were a semi-preparative column YMC ODS A-5 μm, 10.0X 250mm, acetonitrile-water (30:70, V/V), flow rate 2.5mL/min, and detection wavelength 203 nm. To obtain the compound with the structural formula I-VI6 transformation products, chromatogram shown in FIG. 1, which¹³The C-NMR data are shown in Table 1.

TABLE 1 carbon spectra data (deuterated pyridines) of Compounds I, II, III, IV, V and VI

The above results indicate that the obtained compound has the correct structure.

EXAMPLE 2 repair and protective Activity of Compounds I-VI on nerve cells damaged by Hydrogen peroxide

(1) Experimental Material

CO₂Incubator (Joean IGO 150); microplate reader (Bio-TEK ELx 800); fluorescence inverted microscope (Olympus IX 51); MTT cell proliferation and cytotoxicity detection kit (Biyuntian Biotech research institute), DMEM high-sugar medium (Gibcol BRL), fetal bovine serum, dimethyl sulfoxide (DMSO), trypsin (Shanghai bioengineering Co., Ltd.), and 30% hydrogen peroxide (H)₂O₂) (Renjite Chemicals, Technology, Tianjin) and SH-SY5Y cells (tumor research institute, national academy of medical sciences).

(2) Experimental methods

Determination of H for each test Compound pair Using the MTT method₂O₂Effect of damaged SH-SY5Y cell activity: after digestion with pancreatin, cell counts were performed, and the cell density of the cell suspension was adjusted to 5X 10⁴one/mL, adding 200. mu.L/well of 96-well culture plate, and placing in 5% CO₂Constant temperature of 37 ℃ CO₂Culturing in an incubator for 12 h. Grouping treatment after cell adherence: control group, model group (H)₂O₂700. mu. mol/L lesion 4h), model + test compound (1, 10, 20. mu.M) groups. The final volume of each well was 200. mu.L, 3 replicates for each concentration. 24h after drug treatment, 10. mu.L (5mg `) of MTT solution is added to each wellmL, i.e., 0.5% MTT), and incubation continued for 4 h. Measuring the light absorption value of each hole at 490nm of the microplate reader, and calculating the cell survival rate: the cell survival rate is the OD value of the drug-added group/the OD value of the control group.

(3) Results of the experiment

According to the MTT method test result, calculating the betulinic acid derivative I-VI to H₂O₂The results of the viability of damaged SH-SY5Y cells are shown in Table 2: (^###P＜0.001，^*P＜0.05，^**P＜0.01，^***P＜0.001)。

TABLE 2

Comparison with the control group, H₂O₂The cell survival rate of the treated group was significantly reduced, indicating successful cell modeling. And H₂O₂Compared with treatment groups, the donepezil hydrochloride (positive drug) and the betulinic acid derivatives I-VI can both remarkably improve the survival rate of cells, and the betulinic acid derivatives I-VI have remarkable effect of repairing nerve cell injury, show good dose dependence in a certain dose range and can be used as active ingredients of drugs for treating neurodegenerative diseases, stroke and traumatic brain injury.

EXAMPLE 3 Effect of Compound VI of the invention on learning and memory Capacity of aging mouse model

(1) Establishment of aging mouse model

40 SPF male ICR mice with the weight of 22-26 g are randomly divided into 4 groups, namely a control group, a model group and a compound VI group. The mice of the other groups were intraperitoneally injected with D-galactose at a dose of 250mg/kg once a day, except for the administration of the normal saline to the control group, and at the same time, the compound was intragastrically administered at a dose of 10mg/kg to the drug group, and the normal saline was intragastrically administered at an equal volume to the control group and the model group once a day for 6 weeks. Mice developed overt aging characteristics and were used in later experiments.

(2) Experimental methods

The water maze laboratory is an experiment for forcing an experimental animal to swim and searching for a platform hidden in water, the experimental animal needs to memorize the space position and direction to position the platform, and the learning and memory ability of the experimental animal is evaluated by testing the time for finding the platform. The invention uses a circular water pool with the diameter of 1 meter, the water temperature is 20 ℃, and a platform with the diameter of 8cm and 0.9cm lower than the water surface is placed in the water pool. Model mice were trained in the water pool for 4 days prior to the experiment. The head of the mouse is placed in water facing to the wall of the pool, and the position of the mouse randomly takes four directions of east, west, south and north. The time for the animal to find the platform within 60 seconds is recorded, if 60 seconds are exceeded the animal is guided onto the platform and stays for 10 seconds. Each animal was trained 5 times per day, with 30 minute intervals between each training. After the last training, the platform is removed, the experimental animal is placed in the water pool, and the times and time of entering and staying in the quadrant where the platform is originally placed within 120 seconds are recorded. 4 replicates were set for each test group.

(3) Results of the experiment

Compared with the control group, the residence time of the model group mice in the platform quadrant is obviously reduced, and the residence time of the compound VI group is obviously improved, and the results are shown in a figure 1. The result shows that the compound VI can obviously improve the learning and memory ability of mice and can be used as an active ingredient of a medicament for treating neurodegenerative diseases, stroke and traumatic brain injury.

It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims

1. The application of betulinic acid derivatives or pharmaceutically acceptable salts thereof in preparing the medicines for treating nerve injury diseases is that the betulinic acid derivatives are selected from one of the formulas I to VI:

2. the use of claim 1, wherein the nerve injury disease is a neurodegenerative disease, trauma-induced central nerve injury, or stroke-induced central nerve injury.

3. The use of claim 2, wherein the neurodegenerative disease comprises alzheimer's disease, parkinson's disease, huntington's disease, or amyotrophic lateral sclerosis.

4. The use of claim 1, wherein the medicament comprises a betulinic acid derivative or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, wherein the betulinic acid derivative is one or more of formula I to formula VI,

5. the use of claim 4, wherein the pharmaceutically acceptable carrier is selected from one or more of diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorptive carriers, and lubricants.

6. The use according to any one of claims 1 to 5, wherein the medicament is a medicament having a repairing and protecting effect on damaged nerve cells.