CN108314618B

CN108314618B - Sesquiterpenoids, extraction method and medical application of sesquiterpenoids in resisting Alzheimer's disease

Info

Publication number: CN108314618B
Application number: CN201810420521.0A
Authority: CN
Inventors: 戚建华; 程丽红; 孙玉娟
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2018-05-04
Filing date: 2018-05-04
Publication date: 2020-05-22
Anticipated expiration: 2038-05-04
Also published as: CN108314618A

Abstract

The invention provides sesquiterpene compounds, and two sesquiterpene compounds are obtained through a series of processes such as solvent distribution, chromatographic column separation, high performance liquid chromatography purification and the like. As a small molecule compound, the naturally separated active compound shows obvious NGF-like activity in an in vitro screening model PC12 cell of senile dementia. The compound is used as a guide to optimize the structure, and has important practical significance in carrying out basic research on the research and development of new drugs for preventing and treating neurodegenerative diseases such as senile dementia and the like. The sesquiterpenoids provided by the invention have obvious activity of the nerve growth factor, and can be applied to medicines and health products for preventing neurodegenerative diseases such as senile dementia and the like. The preparation method is simple and rapid, and the obtained compound has high purity. The structural formula of compound 2 is as follows:

Description

Sesquiterpenoids, extraction method and medical application of sesquiterpenoids in resisting Alzheimer's disease

Technical Field

The invention belongs to the technical field of medicines, and relates to a novel sesquiterpene compound, an extraction method thereof and application thereof in preparing medicines for preventing and treating neurodegenerative diseases such as senile dementia and the like.

Background

Senile dementia (AD) is a degenerative disease of the central nervous system that occurs in the early and old age, mainly in progressive dementia and psychobehavioral abnormalities, and is second to heart disease, tumor and stroke among the causes of death of the elderly, and is the fourth place. AD is a progressively developing lethal neurodegenerative disease with clinical manifestations of progressive deterioration of cognitive and memory functions, progressive decline of daily living capacity, and various neuropsychiatric symptoms and behavioral disorders. In the international alzheimer's association annual report it is stated that in 2015, about 4680 million people worldwide suffer from dementia, which is expected to reach 7470 million people in 2030 and more than 1 hundred and 3150 million people in 2050, of which about half are AD patients. 58% of dementia patients live in today's low-income countries, and this figure will increase to 63% in 2030 and 68% in 2050. The global dementia care costs amounted to $ 8180 billion in 2015. As short as 3 years, this amount will increase to $ 1 million, and more likely to $ 2 million in 2030. The number of Alzheimer's disease patients in China is estimated to be more than 500 ten thousand, which accounts for about 1/4 of the total number of cases in the world; moreover, with the accelerating aging process of the population in China, the number is more huge, and great influence is brought to the social stability and development.

The main pathological features of Senile dementia are Senile Plaque (SP) presented on the surface of nerve cells in brain due to abnormal amyloid plaque deposition, neurofibrillary tangle (NFT), and selective neuronal and synaptic loss, severe decrease of various neurotransmitters, especially acetylcholine, amyloid plaque is mainly formed by extracellular accumulation of β amyloid (A β) which is generated by intracellular abnormality, and the widely accepted "A β hypothesis" at present considers that β amyloid deposited abnormally by extracellular acts on neurons and glial cells directly or indirectly through a series of cellular cascade reactions (including free radical reaction, mitochondrial oxidative damage, inflammatory reaction, etc.), finally resulting in neuronal dysfunction or death, causing cognitive impairment.

Many drugs are used today for the treatment of AD, and are adapted to different mechanisms of action, wherein the pharmacological treatment of AD is mainly to increase acetylcholine levels in patients by inhibiting Acetylcholinesterase (AChE), i.e. AChE inhibitors. It is a medicine for treating AD which is the most mature at the earliest time in clinic, is suitable for treating patients with light and moderate AD, and has 4 kinds of medicines approved by the FDA in the United states, namely Tacrine (Tacrine), Donepezil (Donepezil), Rivastigmine (Rivastigmine) and Galantamine (Galantamine). Although the medicines play a role in relieving the senile dementia to a certain extent, the medicines have certain toxic and side effects, so that the search for new effective treatment medicines and methods aiming at the causes of AD becomes a hotspot and difficulty of current research. Among the numerous candidate drugs, we looked at Nerve Growth Factor (NGF). Animal experiments prove that NGF can promote proliferation and differentiation, regulate the survival and growth of AD neurons, repair and protect damaged neurons, and is an important purpose of treatment. However, as an effective method for treating AD, NGF is expensive, relatively large in molecular mass, unable to permeate the Blood Brain Barrier (BBB), and only available for intracerebroventricular injection, and has many technical problems for long-term treatment. Therefore, the search for small molecule compounds that mimic the activity of nerve growth factor and that can successfully pass through the BBB has been a hot point of research. To date, scientific researchers have discovered nearly a hundred active compounds that mimic nerve growth factor and are effective in promoting nerve cell growth, some of which have been structurally modified or fully synthesized.

China is rich in natural resources, and researches on new components and new pharmacological activities in natural products are increasingly widespread. Wherein the Chinese medicinal composition has remarkable therapeutic effect on senile dementia, and most notably huperzine A (huperzine A), which is an alkaloid compound extracted from Huperzine A (Huperzine A) of huperzia serrata of huperziaceae. It is a high-efficiency high-selectivity reversible acetylcholinesterase (AChE) inhibiting drug which is developed and researched for the first time in China and selectively acts on the brain.

The development and utilization of the components in the body of a microorganism or the metabolites thereof, which are one of the main sources of natural products, are also one of the important research targets. Fusarium graminearum (F.graminearum)Fusarium graminearum) The caused wheat head blight is a devastating disease, and Gibberellin (GA) is found in the research of rice bakanae disease and refers to a class of compounds which have a gibberellin skeleton and can stimulate cell division and elongation. In 1935 Japanese scientists isolated a growth-promoting amorphous solid from a bakanae fungus which was also known as gibberellin. In 1938, the scientist isolated two biologically active crystals, designated "gibberellin A" and "gibberellin B", from the filtrate of the culture medium of gibberellic bacteria. With the progress of research, many types of gibberellins are found and widely distributed in the plant kingdom, and the existence of gibberellins is found in angiosperms, gymnosperms, ferns, brown algae, green algae, fungi and bacteria. Gibberellins are, as it were, the most diverse of the plant hormones. Gibberellin is one of the indispensable plant hormones for regulating plant growth and development, and can regulate seed germination, hypocotyl elongation, leaf extension, flower, fruit and seed development and the likeNumerous physiological processes. The compounds found in gibberella are present in many plants and act similarly to plant hormones, so that the study of this fungus appears to be of great interest.

Disclosure of Invention

The invention aims to provide an active sesquiterpene compound, which has the following structure:

the second purpose of the invention is to provide the extraction method of the sesquiterpenoids, which is a method for extracting new sesquiterpenoids from a gibberellic disease culture solution, and the method is realized by the following technical scheme:

(1) the gibberellin culture solution is distributed by water and ethyl acetate solvent to respectively obtain a water layer and an ethyl acetate layer;

(2) separating the ethyl acetate layer sample by a silica gel open-ended column separation (200 meshes, n-hexane: ethyl acetate =10:0, 9:1, 8:2, 7:3,0: 10), and combining the obtained components to obtain five samples I-1-I-5;

(3) collecting elution parts with combined n-hexane and ethyl acetate of 7:3 to obtain I-4, performing reversed-phase ODS open column separation (methanol: water =6:4,7:3, 8:2, 10: 0) for the second time, and combining the obtained components to obtain 4 samples II-1-4;

(4) collecting and combining methanol and water at the elution part with the ratio of 7:3 to obtain a sample II-2, carrying out HPLC purification (the conditions are: ODS-UG-5, the flow rate is 3 ml/min, the detection wavelength is 210 nm, the mobile phase is a 65-80% methanol water solution gradient, and 45 min), collecting one component every five minutes, and if an obvious peak appears within five minutes, separately collecting the peak to obtain ten samples III-1-10;

(5) subsequently, a second HPLC purification (conditions: PAKC18, flow rate 3 ml/min, detection wavelength 210 nm, mobile phase 30% -80% acetonitrile in water gradient, 60 min) is carried out on the active sample (retention time 20-25 min), and an active fraction is obtained by separation;

(6) subsequently, performing HPLC purification for the third time (conditions are that Ph-UG-5, flow rate is 1 ml/min, detection wavelength is 210 nm, mobile phase is 65% methanol water solution) on the active sample (retention time is 28-32 min), and separating to obtain active compound 1 (retention time is 14 min respectively) and new sesquiterpene compound 2 (retention time is 28 min);

。

the third purpose of the invention is to provide the application of the sesquiterpene compound in the preparation of the medicine for preventing the neurodegenerative diseases such as senile dementia and the like. The sesquiterpenoids are compound 1 and compound 2. The pharmaceutical sesquiterpenoids and a pharmaceutically acceptable carrier.

The fourth purpose of the invention is to provide the application of the sesquiterpene compound extracted by the method in the preparation of the health care product for resisting Alzheimer's disease, wherein the sesquiterpene compound is compound 1 and compound 2. The health care product consists of the sesquiterpene compound and a carrier acceptable for food or health care products.

The pharmaceutically acceptable carrier as used herein refers to a pharmaceutical carrier which is 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.

The sesquiterpene compounds of the present invention may be administered in unit dosage forms, both enterally and parenterally.

The sesquiterpene compound can be administered by intravenous administration. The injection includes intravenous injection, intraperitoneal injection, intramuscular injection, acupoint injection and subcutaneous injection.

The various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional production methods in the pharmaceutical field, for example, mixing cucurbitacin compounds with one or more carriers, and then making the mixture into the desired dosage form.

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

The invention has the advantages that: (1) two sesquiterpene compounds are obtained through a series of processes such as solvent distribution, chromatographic column separation, High Performance Liquid Chromatography (HPLC) purification and the like, wherein the compound 1 is a new compound, and the preparation method has the advantages of simplicity, rapidness, high purity of the obtained compound and the like. (2) As small molecule compound 1 and compound 2, naturally isolated active compounds showed significant NGF-like activity in vitro screening model PC12 cells for senile dementia. The compound is used as a guide to optimize the structure, and has important practical significance in carrying out basic research on the research and development of new drugs for preventing and treating neurodegenerative diseases such as senile dementia and the like. (3) The sesquiterpenoids have obvious activity of nerve growth factor, and can be applied to the preparation of medicines and health products for preventing neurodegenerative diseases such as senile dementia and the like.

Drawings

FIG. 1 COSY and HMBC picture of compound 2.

Figure 2 compounds 1 and 2 significantly increased neurite differentiation rates of PC12 cells.

FIG. 3 is a micrograph of cells exposed to PC12 cells for 48 h for Compounds 1 and 2.

Detailed Description

The present invention is described in further detail with reference to the drawings and examples, but it should not be understood that the scope of the subject matter of the present invention is limited to the following examples, and all technologies implemented based on the above contents of the present invention are within the scope of the present invention.

Example 1 preparation of sesquiterpene Compounds

The preparation method of the compounds 1 and 2 with obvious NGF-like activity obtained from the gibberellin culture solution comprises the following specific steps:

(1) obtaining a culture solution: 100L of CMC gibberellic disease (the gibberellic disease is purchased from China general microbiological culture Collection center) culture solution (the culture condition is that 100 pieces of PH-1 bacterium blocks with the diameter of 0.8 cm are added into 1L of CMC liquid culture medium, shake culture is carried out for 72 h, the rotating speed is 155 rpm, then the culture solution is filtered by a warp cloth to obtain CMC gibberellic disease culture solution, and 100L of CMC gibberellic disease culture solution is co-cultured), and after water and an ethyl acetate solvent are distributed, a water layer and an ethyl acetate layer (1.9 g) are respectively obtained;

(2) separation and purification: separating the ethyl acetate layer crude sample by a silica gel open-ended column (200 meshes and 300 meshes, the following are the volume ratio of the solvent, namely n-hexane: ethyl acetate =10:0, 9:1, 8:2, 7:3 and 0: 10), respectively collecting and combining elution parts (124 mg) of which n-hexane: ethyl acetate is 7:3, and concentrating; the sample was separated again by reverse phase ODS open column (methanol: water =6:4,7:3, 8:2, 10: 0), and elution fractions (66 mg) combined with methanol: water at 7:3 were collected and concentrated, respectively; the sample was purified by first HPLC under the following conditions: ODS-UG-5, flow rate of 3 ml/min, detection wavelength of 210 nm, mobile phase of 65% -80% methanol water solution gradient, 45 min to obtain active fraction (25 mg), and retention time of 20-25 min; the sample was purified by a second HPLC under the following conditions: PAKC18, the flow rate is 3 ml/min, the detection wavelength is 210 nm, the mobile phase is 30% -80% acetonitrile water solution gradient, 60min, active fraction is obtained, and the retention time is 28-32 min; the sample was then purified by a third HPLC under the following conditions: Ph-UG-5 at flow rate of 1 ml/min, detection wavelength of 210 nm, mobile phase of 65% methanol water solution to obtain two active fractions, retention time of 14 min to obtain compound 1 (16 mg), retention time of 28 min to obtain compound 2 (400 min)

) (ii) a Finally obtain 800

Compound 2.

Example 2 structural characterization of compounds 1 and 2 obtained in example 1:

the structure of the compound 1 is shown by LC-MS,¹H NMR and comparison with the literature. Compound 1 is a white solid; ESI-MS [ High-resolution ESI-MS found 275.1626, calculated for C₁₅H₂₄O₃Na (M + Na)275.1618].¹H NMR (500 MHz, CDCl₃)：δ0.84 (d, 3H,J= 6.8), 0.89 (d, 3H,J=6.8), 1.49 (m, 1H), 1.62 (m, 1H), 1.77 (m, 2H), 2.11 (s, 3H), 2.35 (m, 2H),2.54 (m, 4H), 4.92 (br s, 1H), 4.96 (br s, 1H), 5.41 (dd, 1H,J= 16.0, 9.5),5.99 (d, 1H,J= 16.0)。

The structure of the compound 2 is shown by LC-MS,¹H NMR、¹³C NMR，¹H-¹H COSY, HMBC, HSQC after testing. Compound 2 is a white solid; [

]_D ²⁵+7.0 (c0.42, benzene); ESI-MS [High-resolution ESI-MSfound 261.1827, calculated for C₁₅H₂₆O₂Na (M + Na) 261.1825].¹H NMR and¹³the C NMR data are shown in Table 1 below. Further two-dimensional COSY, HSQC, HMBC data demonstrate this hypothesis (COSY and major HMBC correlations are shown in fig. 1). The configuration of the C-6 and C-7 double bond is defined by the relationship between H-6 and H-7J _6-7= 15.8 Hz.

^a500 MHz for¹H NMR and 125 MHz for¹³C NMR, coupling constants (JinHz) are in parentheses.

^bNumbering of 1.

Example 3 biological Activity of sesquiterpene Compounds

The research finds that in an animal model of neurodegeneration, NGF can prevent or reduce the degeneration of neurons, can prevent AD progression to a certain extent, and has the effects of promoting neurotrophic factors and neuroprotection. PC12 cells are a successful in vitro model that is widely used to model neurons. It is derived from pheochromocytoma of rat adrenal medulla, and like sympathetic neurons and sensory neurons, originates in the neural crest, and after NGF treatment, the cells stop proliferating, and neurite outgrowth develops, showing a mature sympathetic neuron-like cell phenotype. Therefore, the PC12 cell bioactivity identification system is adopted to screen the active ingredients with the activity of the nerve growth factor from natural products, and the active ingredients become effective medicaments for preventing and treating the senile dementia.

The experimental method comprises the following steps:

1) culturing of PC12 cells: 20X 10 cells were inoculated into 100 mm dishes containing 10 mL of DMEM medium (10% horse serum, 5% fetal bovine serum)⁴Two days later, the culture medium was replaced for each PC12 cell, and three days later, subculture was performed. Cells were washed twice with 5mL PBS, 10 mL were added to the dish, 5% CO at 37 deg.C₂The cells were incubated in the carbon dioxide incubator of (1) for 10 minutes, lightly purged, and transferred to a 15 mL disposable centrifuge tube, centrifuged at 800 rpm for 5 minutes, and then counted on a hemocytometer. 24-well cell culture plates 1 mL serum-containing DMEM medium was added to each well, and 2X 10 cells were plated to each well⁴Individual cell, CO₂The incubator was ready for activity testing after 24 hours of incubation.

2) And (3) activity test: and (3) preparing cucurbitacin compounds into DMSO solutions with different concentrations by taking 0.5% DMSO as a negative control and NGF 40 ng/mL as a positive control. After the medium in each well of the 24-well cell plate was replaced with 1 mL of DMEM solution (without serum) containing 1% DMSO and the sample, the plate was placed at 37 ℃ in 5% CO₂Cultured in an incubator. The morphological changes of the cells were observed every 24 hours for 3 consecutive days under an inverted microscope, and the neurite differentiation rate (the ratio of the number of cells with neurites longer than one time the cell body diameter to the total number of cells in the visual field) of the cells was recorded, and about 100 cells in each visual field were randomly selected from 3 positions and analyzed by statistical mapping.

3) Experimental results in fig. 2-3, fig. 2 is a graph showing neurite differentiation rates of PC12 cells when different concentrations of compounds 1 and 2 were added. Wherein, 0.5% DMSO is used as a negative Control, and NGF (40 ng/mL) is used as a positive Control.^*** P<0.001. FIG. 3 is a micrograph of PC12 cells after 248 h addition of different concentrations of Compound 1 and Compound. (a) 0.5% DMSO, Control, (b) 40 ng/mL NGF, (c) 1, 1

; (d) 1, 3

; (e) 1, 10

; (f) 2,1

; (g) 2, 3

; (h) 2, 10

; (i) 2, 30

。

The invention provides an extraction method of sesquiterpene compounds, which comprises the steps of extracting a fungus gibberellic disease culture solution by using ethyl acetate and water to obtain a crude extract of an ester layer, and separating and purifying to obtain a target compound. The sesquiterpenoids provided by the invention are novel compounds, and the extraction method is simple and feasible. The sesquiterpene compound provided by the invention can obviously improve the neurite differentiation rate of PC12 cells in an anti-Alzheimer in-vitro screening model, and can be applied to preparation of anti-Alzheimer drugs and health care products. Provides basis for the research and development of new drugs for resisting the Alzheimer disease and basic research, and has important practical significance.

Claims

1. A sesquiterpene compound having the structure:

。

2. the extraction method of the sesquiterpene compound is characterized by comprising the following steps:

(2) separating the ethyl acetate layer sample by a silica gel open-ended column with 200 meshes and 300 meshes, wherein the ratio of n-hexane to ethyl acetate is =10:0, 9:1, 8:2, 7:3 and 0:10, and combining the obtained components to obtain five samples I-1-I-5;

(3) collecting elution parts with combined n-hexane and ethyl acetate of 7:3 to obtain I-4, performing reversed-phase ODS open column separation, performing second separation on methanol and water =6:4,7:3, 8:2, 10:0, and combining the obtained components to obtain 4 samples II-1-4;

(4) collecting combined methanol, namely collecting an elution part with water of 7:3 to obtain a sample II-2, carrying out HPLC purification, collecting one component every five minutes, and if an obvious peak appears within five minutes, separately collecting the peak to obtain ten samples III-1-10;

(5) performing HPLC purification for the second time on the active sample with the retention time of 20-25 min, and separating to obtain active fraction;

(6) performing HPLC purification for the third time on the active sample with the retention time of 28-32 min, and separating to obtain a compound 1 with the retention time of 14 min and a sesquiterpene compound 2 with the retention time of 28 min;

。

3. the extraction method of sesquiterpene compounds according to claim 2, wherein the HPLC purification conditions in step (4) are: ODS-UG-5, flow rate of 3 ml/min, detection wavelength of 210 nm, mobile phase of 65% -80% methanol water solution gradient, 45 min.

4. The extraction method of sesquiterpene compounds according to claim 2, wherein the HPLC purification conditions of step (5) are: PAKC18, flow rate of 3 ml/min, detection wavelength of 210 nm, mobile phase of 30% -80% acetonitrile in water gradient, 60 min.

5. The extraction method of sesquiterpene compounds according to claim 2, wherein the HPLC purification conditions in step (6) are: Ph-UG-5, flow rate of 1 ml/min, detection wavelength of 210 nm, and mobile phase of 65% methanol water solution.

6. The application of the sesquiterpene compound obtained by the method of claim 2 in preparing the medicine for preventing the senile dementia neurodegenerative diseases, wherein the sesquiterpene compound is a compound 1 or a compound 2, and the medicine sesquiterpene compound consists of a pharmaceutically acceptable carrier.

7. The use of sesquiterpenes obtained by the method of claim 2 in the preparation of anti-alzheimer healthcare products, the sesquiterpenes are compound 1 and compound 2, and the healthcare products consist of the sesquiterpenes and a carrier acceptable for healthcare products.

8. The use according to claim 6, wherein the medicament is in a solid or liquid form and is administered enterally or parenterally.

9. The use of claim 7, wherein the nutraceutical dosage form is a solid or a liquid.