CN113817007B

CN113817007B - Gentiopicroside derivative and preparation and application thereof

Info

Publication number: CN113817007B
Application number: CN202111208330.6A
Authority: CN
Inventors: 戚建华; 王颖; 陈丹妮; 向兰; 刘倩
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2021-10-18
Filing date: 2021-10-18
Publication date: 2023-04-07
Anticipated expiration: 2041-10-18
Also published as: CN113817007A

Abstract

The invention discloses a preparation method of gentiopicroside from gentiana rigescens, a gentiopicroside derivative synthesized on the basis of the preparation method and a synthesis method of the gentiopicroside derivative. According to the invention, evaluation of a PC12 cell bioactivity system shows that the compounds have good nerve growth factor simulating activity and can be applied to preparation of anti-Alzheimer disease drugs and health care products. The invention provides a basis for research and development of new anti-Alzheimer's disease drugs and basic research, and has important practical significance.

Description

Gentiopicroside derivative and preparation and application thereof

Technical Field

The invention relates to the technical field of medicines, in particular to gentiopicroside derivatives and preparation and application thereof.

Background

With the improvement of the medical health level of the modern society, the average life of human beings is prolonged, and the aging phenomenon of the society is increasingly serious. Relevant statistical reports predict that by the year 2050, the population over the age of 60 will reach 20 billion, accounting for approximately 22% of the world's general population. With the aging process, the incidence of senile related diseases is also rising year by year, and Alzheimer's Disease (AD), commonly known as senile dementia, is one of the most common diseases. Related reports show that 11.2% of sick old people aged 60 years old and older are caused by senile dementia, and are higher than people suffering from stroke, cardiovascular diseases and cancers. Therefore, intensive research on the pathogenesis of AD and research and development of related drugs have attracted high attention of the scientific community and are of great significance for promoting social scientific progress and human life health.

In general, AD is a chronic neurodegenerative disease, mainly because β -amyloid (a β) and phosphorylated Tau protein accumulate in the nervous system, resulting in synaptic dysfunction, causing impairment of cognitive function. The precise molecular mechanism of AD is not completely understood, and various theories and assumptions based on pathogenic factors exist in the science at present, such as amyloid beta peptide theory, tau protein abnormal phosphorylation theory, cholinergic system injury theory, free radical oxidative stress theory, influence of other diseases such as diabetes or hypertension, immune system dysfunction and gene mutation, etc. Combining all these hypotheses can lead to the conclusion that: AD is a multifactorial disease.

Currently, scientists have developed several broad classes of corresponding therapeutic agents based on different pathological mechanisms. First-line therapeutic drugs against AD in the market today, including acetylcholinesterase inhibitors (AChEI), such as tacrine, rivastigmine, galantamine, donepezil and huperzine a, which is developed and marketed independently in our country, have already passed FDA approval in the united states and are now widely marketed. However, these drugs can only relieve the early symptoms of patients to a certain extent, but cannot cure the diseases radically. Another representative drug, memantine (memantine), delays the release rate of glutamate, an excitatory neurotransmitter that has a toxic effect on nerve cells, mainly by blocking the glutamate NMDA receptor (N-methyl-D-aspartic acid receptor). Currently, memantine is the only drug used for moderate and severe stages of AD. The mechanism of action of memantine differs from that of AChEI, and both may be used in combination to increase efficacy.

While other drugs effective for AD, such as the brain metabolism regulator pyrrolidone derivative piracetam, are representative drugs for promoting cerebral metabolism, or phosphodiesterase inhibitors such as propentofyuine, which is a drug capable of vasodilating, promoting cerebral circulation, and improving cerebral energy metabolism. Similar drugs include pyritinol and cerebrol. In addition, the calcium antagonist nimodipine has certain protective effect on brain circulation damage, expands cerebral blood vessels, improves cerebral blood circulation, and thus improves cognitive dysfunction of AD patients.

In addition, the antioxidant and the free radical scavenger can protect nerve cells from being damaged to a certain extent, and have a certain treatment effect on AD. Experimental study A Ginkgo leaf (Ginkgo biloba L.) extract is used for treating AD patients, and is found to be capable of obviously improving cognitive dysfunction of the patients caused by oxidative stress damage of nerve cells. The centella asiatica (L.) Urban extract resveratrol can relieve cognitive impairment of dementia male rat model, eliminate redundant free radicals in rat brain, and protect nervous system from the influence of the resveratrol. Vitamin C and vitamin E have good antioxidant effect, improve stability of cell membrane, protect hippocampal cells of central nervous system, inhibit and eliminate Abeta deposited in brain, and improve AD symptom.

Nerve Growth Factor (NGF) is produced by the prefrontal brain tissue of the central nervous system, is transported to the cell body after being ingested by cholinergic neurons, is the most effective component for maintaining the growth and differentiation of neurons in the body, and has remarkable effects on repairing and regenerating damaged neurons. Experimental research discusses the influence of NGF on the learning and memory abilities of senile dementia rats, and Morris water maze experiments and Y maze tests find that NGF has a nutritional effect on central cholinergic neurons, can improve the release of synaptic clefts ACh in the central nervous system, activate the activity of AChE, can effectively prevent the degeneration and death of neurons, improve the cognitive dysfunction of AD rats, reverse the dementia symptoms and improve the learning and memory abilities. But because NGF has high polarity, it is difficult to permeate the blood brain barrier, limiting its application in the treatment of AD. Therefore, a small molecular compound which has the quasi-NGF activity and can promote the regeneration of neurons and easily pass through a blood brain barrier is searched, and the research value and the application prospect are high.

As a result of long-term screening and evolutionary selection of nature, the structure and function research of natural products has important scientific significance for new drug discovery, understanding of life science and exploring the mechanism of action of drugs. Small molecules which can be used as a drug lead compound and have biological activity are developed from natural products, and a new idea is provided for drug research and development. In the field of anti-AD drug development, active ingredients in natural products such as terpenes, esters, alkaloids, glycoside steroids and the like have obvious pharmacological activity in the aspect of neuroprotective function scientifically discovered at present.

Gentiana rigescens is a gentianaceae plant, is a perennial herb and is distributed in Guangxi, sichuan, guizhou, yunnan and other places. Studies have shown that gentiana rigescens has antioxidant, antiinflammatory, analgesic, antidiabetic and cognitive ability improving effects. A large number of compounds are found in gentiana rigescens through research at present, and mainly comprise chemical structures such as iridoid, triterpenes, flavonoids, lignin and benzoate. The inventor discovers a benzoate compound (n-GS) with a new chemical structure from a low-polarity part of a Gentiana rigescens Franch extract in 2010, can remarkably promote neurite differentiation of PC12 cells, has the activity of a nerve mimic factor, and proves that the n-GS can relieve symptoms such as memory loss of a dementia model mouse induced by scopolia. On the basis, the structure-activity relationship research finds that the compound represented by ABG-001 has the most remarkable activity and has great potential for being developed into anti-AD medicaments. In addition, the iridoid glycoside compound is one of the main medicinal components in gentian and is also a characteristic component of gentian. One of the main active ingredients, gentiopicroside, is the main content of the study.

PC12 cells (phytochromatoma cells) were cloned from rat adrenal pheochromocytoma cell line as a cell model in this study, and undifferentiated PC12 cells were bright in soma and irregular in morphology. Under the action of Nerve Growth Factor (NGF), the cell has obvious neurite elongation, and is a typical neuron differentiation and NGF action molecular mechanism research model. Thus, PC12 cells are often used as a cell model for screening compounds with NGF-mimetic activity, i.e. for screening lead compounds against AD. The PC12 cell has similar structure and function with nerve cell, and compared with neuron, the PC12 cell has simple culture condition and can be subcultured. As a neuron cell model, the model is widely applied to the research in the fields of neurophysiological biochemistry and the like.

Disclosure of Invention

One of the purposes of the invention is to provide a gentiopicroside derivative which has a structure shown in the following formula (I) or (II):

in the formula (I), R ₁ is-CH ₂ CH ₃ ，R ₂ Is H or-COOCH ₂ CH ₃ Sequentially marked as compounds 2 and 3;

in the formula (II), R ₃ is-COOCH ₂ CH ₃ or-COCH ₃ And are sequentially marked as compounds 4 and 5.

Another object of the present invention is to provide a method for producing gentiopicroside (herein denoted as compound 1) and the gentiopicroside derivatives (i.e., compounds 2 to 5) described above.

The preparation method of gentiopicroside comprises the following steps: soaking and extracting a traditional Chinese medicine gentian in methanol at room temperature, filtering the obtained filtrate, concentrating, dissolving with water, and then sequentially distributing with n-hexane, ethyl acetate and n-butanol to obtain an active n-butanol layer, separating an n-butanol fraction by using an ODS open chromatographic column, and purifying the extract by using a ratio of (20, 30, 70, 40 ₂ Eluting with O to obtain seven fractions, wherein the third fraction is purified by HPLC to obtain gentiopicroside;

the conditions for HPLC purification include: chromatographic column Cosmosil 5C30-UG-5 phi 10X 250mm, mobile phase MeOH/H ₂ O volume ratio of 30.

The preparation method of the compound 2 comprises the following steps:

adding triphenylphosphine rhodium chloride to an anhydrous methanol solution of gentiopicroside at room temperature, and reacting the mixture in the presence of hydrogen ₂ Reacting under atmosphere, filtering, vacuum concentrating, and separating by ODS column chromatography with MeOH/H as eluent ₂ O volume ratio 20;

the conditions for HPLC purification include: chromatographic column SP-ODS-A phi 20X 250mm, mobile phase MeOH/H ₂ And (3) gradient elution is carried out for 60min at an O volume ratio of 15.

The preparation method of the compound 3 comprises the following steps:

adding a compound 2 into an anhydrous toluene/ethanol mixed solution of diethyl pyrocarbonate and scandium trifluoromethanesulfonate at room temperature; the obtained mixture is heated and stirred at 50-60 ℃ for reaction, then cooled to room temperature, and saturated NaHCO is used ₃ The solution was quenched, filtered, concentrated in vacuo, the concentrate extracted with EtOAc and the organic phase over Na ₂ SO ₄ Drying, filtration and concentration, the crude material obtained is separated by silica gel column chromatography eluting with DCM (dichloromethane)/MeOH in a volume ratio of 90, then purified by HPLC to give compound 3;

the conditions for HPLC purification include: chromatographic column 5C18-MS-II phi 10X 250mm, mobile phase MeOH/H ₂ Gradient elution is carried out for 45min at the volume ratio of O to 30.

The preparation method of the compound 4 comprises the following steps:

adding acetic anhydride into anhydrous pyridine solution of compound 3 at room temperature, stirring for reaction, and reacting with saturated NaHCO ₃ The solution was quenched, concentrated in vacuo, the concentrate extracted with EtOAc and the organic phase over Na ₂ SO ₄ Drying, filtering and concentrating, the crude material obtained is separated by silica gel column chromatography, eluting with DCM/MeOH at a volume ratio of 70, then purified by HPLC to obtain compound 4;

the conditions for HPLC purification include: chromatographic column 5C18-MS-II phi 10X 250mm, mobile phase MeOH/H ₂ Gradient elution is carried out for 45min at a flow rate of 3mL/min, the detection wavelength is 210nm, and the retention time is 38.3min according to the volume ratio of O.

The preparation method of the compound 5 comprises the following steps:

adding acetic anhydride into anhydrous pyridine solution of compound 2 at room temperature, stirring for reaction, and reacting with saturated NaHCO ₃ The solution was quenched, concentrated in vacuo, the concentrate extracted with EtOAc and the organic phase over Na ₂ SO ₄ Drying, filtering and concentrating, the crude obtained is separated by silica gel column chromatography eluting with DCM/MeOH in volume ratio 75;

the conditions for HPLC purification include: chromatographic column 5C18-MS-II phi 10X 250mm, mobile phase MeOH/H ₂ Gradient elution is carried out for 50min at an O volume ratio of 30.

The invention also aims to provide application of the gentiopicroside and the gentiopicroside derivatives (namely the compounds 2-5) in preparing medicaments for promoting elongation of cell neurites and preventing and treating neurodegenerative diseases including Alzheimer's disease.

The invention also aims to provide a medicament, which contains at least one of gentiopicroside and gentiopicroside derivatives (namely compounds 2-5) with effective dose, and can be used for promoting the elongation of cell neurites and preventing and treating neurodegenerative diseases including Alzheimer's disease.

The medicine can be solid preparation or liquid preparation, including tablet, capsule, oral liquid, small needle, infusion, ointment, lyophilized powder for injection, liniment or suppository etc.

The medicament can be a medicament in a gastrointestinal administration dosage form or a medicament in a parenteral administration dosage form. The route of administration may be intravenous. The injection includes intravenous injection, intraperitoneal injection, intramuscular injection, acupoint injection, subcutaneous injection, etc.

The medicament may further comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier refers to a pharmaceutical carrier conventional in the pharmaceutical field, such as diluents and the like, fillers such as sucrose, starch and the like; binders such as hydroxypropylcellulose, starch slurry, etc.; wetting agents such as magnesium stearate, aerosil and the like; absorption enhancer such as polysorbate and lecithin, surfactant such as sorbitan fatty acid, poloxamer, etc., and other adjuvants such as sweetener and flavoring agent can also be added into the composition.

Compared with the prior art, the invention has the main advantages that:

1) The invention adopts PC12 cells as an effective activity identification system, and discovers that the active compound 1 obtained by separating the methanol extract of gentiana rigescens can induce the neurite elongation of the PC12 cells and has obvious activity of the pseudonerve growth factor.

2) On the basis of the compounds, structural modification is carried out to synthesize 4 new iridoid glycoside compounds which also have obvious activity of the nerve growth factor. However, the introduction of two groups can increase the metabolism process of the drug in vivo, and is a candidate compound for improving bioavailability.

3) The active compounds 1-5 can be used for preparing medicines for preventing neurodegenerative diseases such as Alzheimer's disease.

Drawings

FIG. 1 shows the structures (A) of compounds 1 to 5 and the effect (B) of neurite elongation after 48h of action on PC12 cells at different concentrations;

FIG. 2 is a photomicrograph of PC12 cells, all on the same scale, after addition of compounds 1 to 5h, wherein (a) 0.5% DMSO (dimethyl sulfoxide), control (negative Control, also denoted by the abbreviation C in the present invention); (b) 40ng/mL NGF (positive control); (c) Compound 1, 10. Mu.M; (d) Compound 2, 10. Mu.M; (e) Compound 3,3. Mu.M; (f) compound 4, 10 μ M; (g) Compound 5,1. Mu.M;

figure 3 is a digital representation of the effect of compounds 1-5 on neurite outgrowth of PC12 cells, with P <0.01 and P <0.001.

Detailed Description

The invention is further described with reference to the following drawings and specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The following examples are conducted under conditions not specified, usually according to conventional conditions, or according to conditions recommended by the manufacturer.

Dried and powdered traditional Chinese medicine gentian (100 g) was extracted in methanol (gentian/methanol =1/10, w/v, g/mL) at room temperature for 24 hours. The methanol extract was filtered, and the filtrate was concentrated, dissolved in water, and partitioned with n-hexane, ethyl acetate and n-butanol in this order to give an active n-butanol layer (7.5 g). Separating the n-butanol fraction by ODS open chromatography and MeOH/H ₂ O (volume ratio 20. A third fraction (30 mg) was purified by HPLC (column Cosmosil 5C30-UG-5 Φ 10X 250mm, mobile phase MeOH/H ₂ O volume ratio 30Time t _R ＝44min)。

By analysis of HR ESI-MS and ¹ h NMR data determined the structure of compound 1, compound 1: white solid, HR ESI-TOF-MS m/z 379.0977, calcd.for C ₁₆ H ₂₀ NaO ₉ (M+Na) ⁺ 379.1000. ¹ H NMR(500MHz,methanol-d ₄ ):δ＝7.44(1H,s),5.75(1H,m),5.66(1H,d,J＝2.9Hz),5.61(1H,m),5.23(1H,m),5.20(1H,m),5.05～4.95(2H,m),4.64(1H,d,J＝7.9Hz),3.89～3.64(2H,m),3.21～3.36(4H,m),3.10～3.14(1H,m)。

Triphenylphosphine rhodium chloride (0.2eq.416mg) in H was added to a solution of gentiopicroside (1eq.800mg) in anhydrous methanol (15 mL) at room temperature ₂ The atmosphere was allowed to react for 48h, after the reaction had stopped it was filtered and the mixture was concentrated in vacuo. The crude sample was chromatographed on an ODS column (eluent MeOH: H) ₂ O volume ratio =20:80 Followed by HPLC (column SP-ODS-A phi 20X 250mm, mobile phase MeOH/H) ₂ Gradient elution with an O volume ratio of 15 _R ＝24.0min)。

To Diethylpyrocarbonate (DEPC) (1.2eq.21.7mg) and scandium trifluoromethanesulfonate (Sc (OTf) at room temperature ₃ ) (0.05eq.2.8 mg) of anhydrous toluene: to the mixed solution of ethanol volume ratio = 4. After the mixture was heated at 55 ℃ and stirred for 5h, the reaction was stopped, cooled to room temperature and quenched with saturated NaHCO ₃ The solution was quenched, filtered, and the mixture was concentrated under vacuum. The concentrate was extracted with EtOAc. The organic phase is passed through Na ₂ SO ₄ Dried, filtered and concentrated. The crude sample was chromatographed on silica gel (eluent DCM: meOH vol = 90) and then by HPLC (column 5C18-MS-II Φ 10 × 250mm, mobile phase MeOH/H ₂ Gradient elution with an O volume ratio of 30 _R ＝36.0min)。

To a solution of compound 3 (20 mg) in anhydrous pyridine (2 mL) was added acetic anhydride (50. Mu.L) at room temperature. Stirring the mixture at room temperature for 15min, stopping the reaction, and adding saturated NaHCO ₃ Solution(s)The reaction was quenched and the mixture was then concentrated under vacuum. The concentrate was extracted with EtOAc. The organic phase is passed through Na ₂ SO ₄ Dried, filtered and concentrated. The crude sample was chromatographed on silica gel (eluent DCM: meOH vol = 70) and then by HPLC (column 5C18-MS-II Φ 10 × 250mm, mobile phase MeOH/H ₂ Gradient elution with an O volume ratio of 20 _R ＝38.3min)。

To a solution of compound 2 (20 mg) in anhydrous pyridine (2 mL) was added acetic anhydride (50. Mu.L) at room temperature. Stirring the mixture at room temperature for 15min, stopping the reaction, and adding saturated NaHCO ₃ The solution quenched the reaction, and then the mixture was concentrated under vacuum. The concentrate was extracted with EtOAc. The organic phase is passed through Na ₂ SO ₄ Drying, filtering and concentrating. The crude sample was chromatographed on silica gel (eluent DCM: meOH vol = 75) and then by HPLC (column 5C18-MS-II Φ 10 × 250mm, mobile phase MeOH/H ₂ Gradient elution with an O volume ratio of 30 _R ＝42.5min)。

By analysis of HR ESI-MS and ¹ h NMR data determined the structure of compound 2, compound 2: white solid, HR ESI-TOF-MS m/z 381.1126, calcd.for C ₁₆ H ₂₂ NaO ₉ (M+Na) ⁺ 381.1156. ¹ H NMR(500MHz,methanol-d ₄ ):δ＝7.40(1H,s),5.66(1H,d,J＝2.9Hz),5.56(1H,m)，5.06～4.98(2H,m),4.62(2H,d,J＝7.9Hz),3.90～3.65(2H,m),3.34(1H,m),3.25(1H,m),3.13(1H,m),2.48(1H,m),1.60(1H,m),1.32(1H,m),0.97(3H,t,J＝7.4Hz)。

By analysis of HR ESI-MS and ¹ h NMR data determined the structure of compound 3, compound 3: white solid, HR ESI-TOF-MS m/z 431.1521, calcd.for C ₁₉ H ₂₇ O ₁₁ (M+H) ⁺ 431.1548. ¹ H NMR(500MHz,methanol-d ₄ ):δ＝7.40(1H,s),5.57(1H,m),5.52(1H,d,J＝2.9Hz)，5.06(1H,m),4.97(1H,m),4.64(1H,d,J＝7.9Hz),4.46～4.26(2H,m),4.20(1H,m),3.52(1H,m),3.36(2H,m),3.28(1H,m),3.14(1H,m),2.49(1H,m),1.61(1H,m),1.34(1H,m),1.29(3H,t,J＝7.1Hz),0.97(3H,t,J＝7.4Hz)。

By analysis of HR ESI-MS and ¹ h NMR data determined the structure of compound 4, compound 4: white solid, HR ESI-TOF-MS m/z 579.1684, calcd.for C ₂₅ H ₃₂ NaO ₁₄ (M+Na) ⁺ 579.1690. ¹ H NMR(500MHz,methanol-d ₄ ):δ＝7.38(1H,s),5.60(1H,m),5.52(1H,d,J＝1.6Hz)，5.28(1H,t,J＝9.5Hz),5.04(2H,m),4.97(2H,m),4.82(1H,m),4.27(2H,m),4.18(2H,m),3.96(1H,m),2.49(1H,m),2.01(3H,s),1.96(3H,s),1.90(3H,s),1.57(1H,m),1.33(1H,m),1.29(3H,t,J＝7.1Hz),0.97(3H,t,J＝7.4Hz)。

By analysis of HR ESI-MS and ¹ h NMR data confirmed the structure of compound 5, compound 5: white solid, HR ESI-TOF-MS m/z 549.1552, calcd.for C ₂₄ H ₃₀ NaO ₁₃ (M+Na) ⁺ 549.1584. ¹ H NMR(500MHz,methanol-d ₄ ):δ＝7.39(1H,s),5.61(1H,m),5.52(1H,d,J＝1.5Hz)，5.28(1H,t,J＝9.6Hz),5.05(2H,m),4.99(2H,m),4.82(1H,m),4.30(1H,m),4.16(1H,m),3.95(1H,m),2.49(1H,m),2.07(3H,s),2.01(3H,s),1.96(3H,s),1.90(3H,s),1.57(1H,m),1.32(1H,m),0.97(3H,t,J＝7.4Hz)。

Biological activity assay of compounds 1 to 5:

(1) PC12 cell recovery process: CM medium (DMEM high-sugar medium containing 10% (v/v) Horse Serum (HS), 5% Fetal Bovine Serum (FBS) and 1% diabody (penicillin-streptomycin)) required for the experiment was preheated for 10min in a 37 ℃ water bath. Taking out the cell freezing tube from the liquid nitrogen tank, rapidly placing the tube into a water bath kettle at 37 ℃ for continuous stirring for a certain time, adding 4mL of CM when a small amount of liquid exists in the freezing tube, transferring the tube into a centrifuge tube, placing the tube into a centrifuge for centrifugation at 800r/min for 3min, simultaneously adding 10mL of CM culture medium into a culture dish, labeling information such as cell generation, culture time and operator, placing the tube at 37 ℃ and 5% CO for certain time ₂ The incubator for use. After cell centrifugation is finished, pouring out supernatant, adding 1-2mL of CM, blowing uniformly, and counting by using a blood counting chamber. 5X 10 portions of CM Medium were added to each dish containing 10mL of CM Medium ⁴ A cell, thenCulturing in an incubator, replacing the CM culture medium on the first day after recovery, replacing the culture medium again on the third day, and subculturing and freezing the cells after six to seven days.

(2) The PC12 cell subculture process comprises the following steps: PBS, CM medium required for the experiment was pre-warmed in a 37 ℃ water bath for 10min. Removing CM medium from PC12 cell culture dish with 5mL pipet twice, adding 5mL PBS, shaking the dish gently, removing PBS, adding 5mL PBS, shaking the dish gently, removing the dish, adding 10mL PBS, placing at 37 deg.C, 5% CO ₂ Standing in the incubator for 10min. Simultaneously, 1mL of preheated CM was added to each well of a 24-well cell culture plate and placed in an incubator for use. The dish was removed, one side of the dish was lifted, the bottom was fully purged, the cell suspension was transferred to a centrifuge tube and centrifuged at 800r/min for 3min. 10mLCM is added into the original culture dish and put into an incubator for standby. Centrifuging, removing supernatant, adding 1-2mL preheated CM, blowing, and mixing. Counting with a hemocytometer plate, 2.5X 10 cells were added to each well of a 24-well cell culture plate ⁴ Individual cells, mixed, labeled with information on passage time, operator, etc., at 37 ℃ and 5% CO ₂ And culturing and preserving in an incubator for sample adding after 24 h. Inoculating 2X 10 to the original culture dish ⁵ And (4) culturing the cells for the next generation. After 48h, CM medium was changed, subcultured on the fifth day, and the cycle was repeated.

(3) The biological activity test method comprises the following steps: test samples were configured with DMSO at the concentrations tested, using 0.5% DMSO as a negative control and NGF (40 ng/mL) as a positive control. Adding DMSO, NGF and sample to be tested into EM culture medium (DMEM high-sugar medium containing 1% double antibody (penicillin-streptomycin)) to obtain 1mL total volume of liquid, replacing CM culture medium in each well of 24-well cell plate, placing 24-well cell plate at 37 deg.C, and 5% CO ₂ The incubator of (2). After 24h, the morphological changes of the cells were observed and the differentiation rate of the neurites of PC12 cells was recorded (cellular neurite differentiation rate = the number of cells with a neurite longer than one time the longest diameter of the soma/total number of cells in the selected field x 100%). And observed again after 48 h. About 100 cells per field, 3 sites were randomly selected and averaged. The data in the experiment is applied to t-test in SPSSl6.0 statistical softwareData processing was performed and statistical results are expressed as mean ± standard deviation (X + SD).

(4) The experimental results are as follows:

in FIG. 1, (A) is the structure of compounds 1 to 5; (B) Is the influence of the compounds 1-5 on the neurite differentiation rate of PC12 cells after the compounds are acted for 48 hours at different concentrations. Among them, DMSO was 0.5% as a negative Control, and NGF (40 ng/mL) as a positive Control. * P <0.001. The results show that the compounds 1 to 5 can obviously prolong the neural process differentiation rate of the PC12 cells.

FIG. 2 is a microphotograph of PC12 cells treated with compounds 1-5 for 48 hours. The results show that the compounds 1 to 5 can obviously prolong the neural process differentiation rate of the PC12 cells under different concentrations.

Figure 3 is a digital representation of the effect of compounds 1-5 on neurite outgrowth of PC12 cells, with P <0.01 and P <0.001. The results show that compounds 1, 2, 4 were most effective at a concentration of 10 μ M, compound 3 was most effective at a concentration of 3 μ M, and compound 5 was most effective at a concentration of 1 μ M.

The invention provides a preparation method of gentianella gentiopicroside (compound 1) from gentiana rigescens and a synthesis method of novel compounds 2-5 synthesized on the basis of the preparation method. The evaluation of a PC12 cell bioactivity system finds that the activity of the nerve growth factor mimic is better, and the nerve growth factor mimic can be applied to the preparation of anti-Alzheimer disease drugs. The research provides basis for the research and development of new drugs for resisting the Alzheimer disease and basic research, and has important practical significance.

Furthermore, it should be understood that various changes and modifications can be made by one skilled in the art after reading the above description of the present invention, and equivalents also fall within the scope of the invention as defined by the appended claims.

Claims

1. A gentiopicroside derivative is characterized by having a structure shown in the following formula (I) or (II):

in the formula (II), R ₃ is-COOCH ₂ CH ₃ And is denoted as compound 4.

2. A method of preparing a gentiopicroside derivative as claimed in claim 1, wherein the gentiopicroside derivative is compound 2, which comprises:

3. A method for preparing the gentiopicroside derivative according to claim 1, wherein the gentiopicroside derivative is compound 3, comprising:

adding a compound 2 into an anhydrous toluene/ethanol mixed solution of diethyl pyrocarbonate and scandium trifluoromethanesulfonate at room temperature; the obtained mixture is heated and stirred at 50-60 ℃ for reaction, then cooled to room temperature, and saturated NaHCO is used ₃ The solution was quenched, filtered, concentrated in vacuo, the concentrate extracted with EtOAc and the organic phase over Na ₂ SO ₄ Drying, filtering and concentrating, the crude material obtained is separated by silica gel column chromatography, eluent DCM/MeOH volume ratio 90;

the above-mentionedConditions for HPLC purification included: chromatographic column 5C18-MS-II phi 10X 250mm, mobile phase MeOH/H ₂ Gradient elution is carried out for 45min at an O volume ratio of 30.

4. A method of preparing a gentiopicroside derivative as claimed in claim 1, wherein the gentiopicroside derivative is compound 4, which comprises:

5. A preparation method of gentiopicroside derivative is characterized in that the gentiopicroside derivative is a compound 5, and the preparation method comprises the following steps:

the conditions for HPLC purification include: chromatographic column 5C18-MS-II phi 10X 250mm, mobile phase MeOH/H ₂ Gradient elution is carried out for 50min at a flow rate of 3mL/min, the detection wavelength is 210nm, and the retention time is 42.5min, wherein the O volume ratio is 30;

compound 2 has the structure shown in formula (I) below:

in the formula (I), R ₁ is-CH ₂ CH ₃ ，R ₂ Is H;

compound 5 has the structure shown in formula (II) below:

in the formula (II), R ₃ is-COCH ₃ 。

6. The use of gentiopicroside, a gentiopicroside derivative according to claim 1, and a compound 5 having a structure represented by the following formula (II) in the preparation of a medicament for promoting elongation of a cell neurite, and preventing and treating neurodegenerative diseases including Alzheimer's disease;

in the formula (II), R ₃ is-COCH ₃ 。

7. The use according to claim 6, wherein the medicament is a solid formulation or a liquid formulation.

8. The use according to claim 6, wherein the medicament is a medicament in a gastrointestinal or parenteral dosage form.

9. A medicament comprising an effective amount of at least one of gentiopicroside and a gentiopicroside derivative as defined in claim 1, which is useful for promoting elongation of cellular neurites, and preventing and treating neurodegenerative diseases including Alzheimer's disease.