A kind of enantiotesenane-type norditerpene lactone compound and its preparation method and application
By extracting and purifying enantiosenoxanyl-type hypoditerpene lactone compounds from Euphorbia, the problem of difficult to extract acetylcholinesterase activity from herbs in the prior art was solved, and a new drug development was realized for the treatment of Alzheimer's disease.
Patent Information
- Application Number
- CN202310901532.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-07-21
- Publication Date
- 2025-04-22
- Estimated Expiration
- 2043-07-21
AI Technical Summary
The prior art is difficult to extract natural products with inhibitory acetylcholinesterase activity from herbs for the treatment of Alzheimer's disease.
Enantiosenoxanyl-type diterpene lactone compounds were extracted from dried roots of Euphorbia, and the compounds with anti-acetylcholinesterase activity were purified by diafiltration extraction, extraction, column chromatography gradient elution and other steps.
The extraction of enantiosenoxane-type hypoditerpene lactone compounds with inhibitory acetylcholinesterase activity from Euphorbia was achieved, providing a way to develop novel anti-Alzheimer's disease drugs.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of natural medicines, and in particular to an enantiotesenane-type norditerpene lactone compound and a preparation method and application thereof. Background Art
[0002] In recent years, with the increase in life expectancy, the aging of the global population has gradually intensified. Various geriatric diseases have brought a lot of pressure to the social security system and personal family life. One of the most common is Alzheimer's disease. Alzheimer's disease (AD) is a common and complex multifactorial degenerative disease in the central nervous system. It not only leads to the degeneration of intelligence, memory, personality, etc., but also brings a huge economic burden. Although the pathogenesis of AD has not been fully elucidated, the cholinergic hypothesis is currently the most widely accepted pathological theory. Many studies have found that cholinergic disorders are closely related to memory and cognitive disorders. Under the guidance of this theory, acetylcholinesterase inhibitors are currently the most studied and most active anti-Alzheimer's drugs. This class of drugs increases the level of acetylcholine in the patient's brain by inhibiting acetylcholinesterase, thereby improving cognitive function. Therefore, finding effective acetylcholinesterase inhibitors to treat AD is a major public health issue in the medical community in my country and even the world today. At the same time, traditional Chinese medicine provides an important source and new opportunity for modern drug development. The active ingredients of natural products have become an important source for new drug research and development due to their multi-target, high efficiency and low toxicity.
[0003] Euphorbia chamaejasme, a perennial herbaceous plant of the Euphorbiaceae family, has a thick fleshy root with white latex and is cylindrical in shape. It is one of the source plants of the traditional Chinese medicine wolfsbane. It tastes spicy, flat, and toxic. It enters the liver and spleen meridians, has the effect of dispersing knots and killing insects, and is widely used in Chinese patent medicines and clinical prescriptions. In traditional medicine, wolfsbane is used for phlegm, food, and insect accumulation, edema and abdominal distension, heart and abdominal pain, accumulation of symptoms, lymph tuberculosis, scabies, etc. Modern research results show that the traditional Chinese medicine wolfsbane has a variety of pharmacological activities, such as anti-tuberculosis, anti-HIV virus, anti-tumor, antibacterial, anti-inflammatory and insecticidal effects. Modern research shows that the chemical components of wolfsbane euphorbia mainly include two major categories: phenolic acids and terpenes. Among them, terpenes are mainly rich and diverse diterpenes, which are also considered to be the main material basis for the many pharmacological activities of wolfsbane euphorbia. Enantiopyranoid-type norditerpenes are important chemical components in wolfsbane euphorbia.
[0004] Therefore, whether it is possible to extract natural products that inhibit acetylcholinesterase from herbal plants has become a technical problem that needs to be solved urgently by those skilled in the art. Summary of the invention
[0005] The invention provides an enantiotesenane-type norditerpene lactone compound and a preparation method thereof. The compound has good anti-acetylcholinesterase activity and can be used for preparing anti-acetylcholinesterase drugs.
[0006] An enantiotesenane-type norditerpene lactone compound, wherein the enantiotesenane-type norditerpene lactone compound has a structure shown in the following formula I:
[0007]
[0008] A method for preparing the enantiotesenane-type norditerpene lactone compound represented by the above formula I, characterized in that it comprises the following steps: using the dried root of Euphorbia chamaejasme as raw material, extracting and concentrating the dried root to obtain an extract, suspending the extract in water to obtain a suspension, extracting and concentrating the extract;
[0009] The extract is subjected to gradient elution by neutral alumina column chromatography, silica gel column chromatography, and MCI resin column chromatography, and then eluted by ODS medium pressure column chromatography to obtain Fr.1 to Fr.10. Fr.6 is purified by ODS liquid chromatography to obtain the enantiotherm-type norditerpene lactone compound shown in formula I.
[0010] Further, the neutral alumina column chromatography is subjected to gradient elution to obtain a first eluate, the eluent is petroleum ether-ethyl acetate-methanol, and the volume ratio of the petroleum ether, ethyl acetate and methanol is 100:0:0 to 0:0:100. Specifically, the eluent includes an eluent having a volume ratio of petroleum ether-ethyl acetate-methanol of 100:0:0, 90:10:0, 80:20:0, 50:50:0, 30:70:0, 27:75:0, 0:100:0, 0:50:50, and 0:0:100, and 9 eluates are collected segmentally by gradient elution with the eluent.
[0011] Further, the first eluate is subjected to gradient elution through the silica gel column chromatography to obtain a second eluate, the eluent is petroleum ether-acetone-methanol, and the volume ratio of the petroleum ether, acetone and methanol is 100:0:0-0:100:0-0:0:100. Specifically, the eluent includes an eluent having a volume ratio of petroleum ether-acetone-methanol of 100:0:0, 95:4.5:0.5, 90:9.5:0.5, 85:14:1, 80:19:1, 48:48:2, 20:75:5, and 0:0:100, and 8 eluates are collected segmentally through gradient elution with the eluent.
[0012] Further preferably, the first eluate is obtained by eluting with an eluent having a volume ratio of petroleum ether, ethyl acetate and methanol of 25:75:0.
[0013] Further, the second eluate is subjected to gradient elution through the MCI resin column chromatography to obtain a third eluate, the eluent is methanol-water, and the volume ratio of the methanol to water is 0:100 to 100:0. Specifically, the eluent includes eluents with a volume ratio of methanol-water of 0:100, 20:80, 40:60, 60:40, 80:20, and 100:0, and 6 eluates are collected segmentally through gradient elution with the eluent.
[0014] Further preferably, the second eluate is obtained by eluting with an eluent having a volume ratio of petroleum ether, acetone and methanol of 85:14:1.
[0015] Furthermore, the third eluate is eluted through the ODS medium pressure column chromatography to obtain Fr.1 to Fr.10, the mobile phase is methanol-water, and the volume ratio of methanol to water is 50:50-60:40.
[0016] Further preferably, the third eluate is obtained by combining eluates obtained by eluting with eluents having a methanol-water volume ratio of 60:40 and 80:20.
[0017] Furthermore, Fr.6 was purified by ODS liquid chromatography to obtain the compound of formula I, the mobile phase was acetonitrile-water, and the volume ratio of acetonitrile to water was 45:55.
[0018] Furthermore, the diafiltration extraction step is as follows: taking ethanol to diafiltration extract the dry root of the wolfberry Euphorbia at room temperature, obtaining an extract-like ethanol extract after concentration, and suspending the ethanol extract in water to obtain a suspension; the ethanol concentration is 95%, the diafiltration extraction time is 120h, and the ethanol diafiltration extraction flow rate is 0.5L / h.
[0019] Furthermore, the extraction step is as follows: adding dichloromethane in an equal volume to the suspension, and concentrating after extraction to obtain the extract.
[0020] A use of the enantiotesenane-type norditerpene lactone compound represented by the above formula I in the preparation of a drug for preventing or treating Alzheimer's disease.
[0021] Furthermore, the enantiotesenane-type norditerpene lactone compound is used as a raw material to prepare a pharmaceutical composition for preventing or treating Alzheimer's disease or any pharmaceutically acceptable salt thereof.
[0022] Furthermore, the pharmaceutical composition or any pharmaceutically acceptable salt is in the form of tablets, capsules, granules, oral liquids, granules, pills or pellets.
[0023] One or more technical solutions provided in the embodiments of the present invention have at least the following technical effects or advantages:
[0024] The present invention realizes for the first time the extraction of enantiotesenane-type norditerpene lactone compounds with the ability to inhibit acetylcholinesterase from Euphorbia chamaejasma. Such compounds have great development value as new drugs for the prevention or treatment of Alzheimer's disease. The design ideas of such compounds also provide new ideas and approaches for the development of new drugs for the prevention or treatment of Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 The present invention is the enantiopyrane-type norditerpene lactone compound 1 H-NMR spectrum;
[0026] Figure 2 The present invention is the enantiopyrane-type norditerpene lactone compound 13 C-NMR spectrum;
[0027] Figure 3 It is a dot plot of the inhibitory effect of the enantiotesenane-type norditerpene lactone of the present invention on acetylcholinesterase in a concentration-dependent manner;
[0028] Figure 4 The figure is a kinetic curve of inhibition of acetylcholinesterase by the enantiotesenane-type norditerpene lactone of the present invention;
[0029] Figure 5 is a Lineweaver–Burk plot of the inhibitory effect of the enantiotesenane-type norditerpene lactone of the present invention on acetylcholinesterase;
[0030] Figure 6 The present invention uses the Lineweaver-Burk curve to determine the inhibition kinetic parameter (Ki) curve of the slope of the enantiotesenane-type norditerpene lactone concentration. DETAILED DESCRIPTION
[0031] In order to better understand the above technical solution, the above technical solution will be described in detail below in conjunction with the accompanying drawings and specific implementation methods.
[0032] An enantiotesenane-type norditerpene lactone compound has a structure shown in the following formula I:
[0033]
[0034] The method for preparing the enantiotesenane-type norditerpene lactone compound includes the following embodiments:
[0035] Example 1
[0036] 1 kg of dried medicinal materials of wild Euphorbia chamaejasma root from high altitude was used as raw material, and the material was extracted by filtration with 95% ethanol for 120 hours at a flow rate of 0.5 L / h. The material was concentrated by rotary evaporation to obtain an ethanol extract in the form of an extract. The ethanol extract was suspended in water to obtain a suspension, and dichloromethane with a volume equal to that of the suspension was added for extraction. The extract solution was concentrated to obtain an extract. The dichloromethane extract was subjected to neutral alumina column chromatography and gradient eluted with a petroleum ether-ethyl acetate-methanol solution with a volume ratio of 100:0:0 to 0:0:100. Specifically, the eluent included petroleum ether-ethyl acetate-methanol with a volume ratio of 100:0:0, 90:10:0, 80:20:0, 50:50:0, 30:70:0, 27:75:0, 0:100:0, 0:50:50, and 0:0:100. After gradient elution with the eluent, 9 eluates were collected in segments. The eluate obtained by elution with an eluent having a volume ratio of petroleum ether-ethyl acetate-methanol of 25:75:0 is subjected to silica gel column chromatography and gradient elution is performed with a petroleum ether-acetone-methanol solution having a volume ratio of 100:0:0 to 0:100:0 to 0:0:100. Specifically, the eluent includes eluents having a volume ratio of petroleum ether-acetone-methanol of 100:0:0, 95:4.5:0.5, 90:9.5:0.5, 85:14:1, 80:19:1, 48:48:2, 20:75:5, and 0:0:100. After gradient elution with the eluent, 8 eluates are collected in sections. The eluate eluted with an eluent having a volume ratio of petroleum ether-acetone-methanol of 85:14:1 was subjected to MCI resin column chromatography and gradient eluted with a methanol-water solvent having a volume ratio of 0:100 to 100:0. Specifically, the eluent includes eluents having a volume ratio of methanol-water of 0:100, 20:80, 40:60, 60:40, 80:20, and 100:0. Six eluates were collected in segments after gradient elution with the eluent. The eluates eluted with methanol-water volume ratios of 60:40 and 80:20 were combined and subjected to ODS medium pressure column chromatography with a gradient elution of methanol-water solvent in a volume ratio of 50:50-60:40 at a flow rate of 20 mL / min and an elution time of 6 hours to obtain 10 components Fr.1 to Fr.10. The Fr6 portion was prepared by preparative ODS liquid chromatography using acetonitrile-water solution in a volume ratio of 45:55 as the mobile phase to obtain a compound of formula I.
[0037] The embodiment of the present invention also provides a use of the enantiotesenane-type norditerpene lactone compound represented by Formula I in the preparation of a drug for preventing or treating Alzheimer's disease.
[0038] Specifically, the enantiotesenane-type norditerpene lactone compound is used as a raw material to prepare a pharmaceutical composition or any pharmaceutically acceptable salt for preventing or treating Alzheimer's disease. The pharmaceutical composition is added with an excipient, which is not particularly limited in the present invention and can be an applicable excipient well known to those skilled in the art.
[0039] The pharmaceutical composition or any pharmaceutically acceptable salt is in the form of tablets, capsules, granules, oral liquids, granules, pills or pellets.
[0040] Embodiment 2:
[0041] The compound of formula I prepared in Example 1 was subjected to structural identification.
[0042] The identification results are as follows:
[0043] The compound of formula I is a colorless columnar crystal (methanol), and 10% sulfuric acid ethanol solution is purple-red. High-resolution mass spectrometry gives the quasi-molecular ion peak of the compound [M+Na] + m / z 325.1774 (calcd. for C 19 H 26 O3Na, 325.1780), and combined with NMR data analysis, it is inferred that its molecular formula is C 19 H 26 O3, its unsaturation is calculated to be 7. Figure 1 As shown in Table 1, 1 In the H-NMR (400MHz, CDCl3) spectrum, three methyl proton signals are given in the high field region, namely, δ H 1.42 (3H, s, Me-18), 1.31 (3H, s, Me-19), 0.85 (3H, brs, Me-20); a pair of terminal double bond olefin proton signals δ H 4.91-4.93 (1H, m, H-17a), 4.69-4.70 (1H, m, H-17b), as shown in Table 1. Figure 2 As shown in Table 1, 13 In the C-NMR (100 MHz, CDCl3) spectrum, a total of 19 carbon signals are given, among which δ C 146.4 (C-16) and 107.8 (C-17) are a pair of olefin carbon signals, δ C 169.8 (C-2) and 215.9 (C-14) are respectively a carboxyl group and a keto carbonyl carbon signal, δ C86.1 (C-4) is an oxygen-substituted carbon signal. The carbon-hydrogen signals of the compound were fully assigned according to the HSQC spectrum. Based on the above spectral information, it was found that the compound is similar to the known seco-atisane diterpenoid compound sapinsigin A, the difference is that it lacks a methoxy group and an oxygen-containing methylene group. It is speculated that it is a demethylated norditerpenoid derivative of sapinsigin A. In the HMBC spectrum, H-1a (δ H 2.51) and C-2(δ C 169.8), C-5(δ C 51.9), C-9(δ C 49.8), C-10(δ C 37.4) has telecorrelation, Me-18(δ H 1.42) and C-4(δ C 86.1), C-5(δ C 51.9), C-19(δ C 25.1) has a long-range correlation, combined with the degree of unsaturation, confirming the speculation that the compound is a 3-position cyclopentadiene lactone compound. The relative configuration was determined by NOESY, H-5 (δ H 1.51) and H-9(δ H 1.68) have NOE correlation, indicating that H-5 and H-9 are located on the same side; H-12 (δ H 2.74-2.77) and Me-20 (δ H 0.85) has NOE correlation, indicating that H-12 and Me-20 are located on the same side. The absolute configuration of the compound is determined by calculating ECD and. The ECD spectra of the two diastereomers are calculated using TD-DFT theory. Finally, according to the agreement between the ECD experimental value and the calculated value, the absolute configuration of the compound is determined to be 5S, 8S, 9S, 10S, 12R. To ensure the accuracy of the absolute configuration of compound 1, X-ray single crystal diffraction analysis was used to further determine the result, which was consistent with the calculated ECD.
[0044] Through the above analysis, it is finally determined that the compound of formula I is the enantiotesenane-type norditerpenoid having a carbon-reducing and a lactone fragment in its structure of the present invention.
[0045] Table 1 Compounds of Formula I 1 H-NMR (600MHz, CDCl3) and 13 C-NMR (150MHz, CDCl3) data
[0046]
[0047]
[0048] Example 3: Pharmacological activity of compounds of formula I
[0049] Test methods and results
[0050] 1. Improved Ellman colorimetric assay to test the effect of the compound of formula I on acetylcholinesterase activity in vitro
[0051] Add 155 μL PBS, 5 μL compound of formula I, 10 μL (0.2U / mL) acetylcholinesterase to a 96-well plate, incubate at 37°C for 20 min, add 10 μL (4 mM) iodinated acetylthiocholine and 10 μL (5 mM) 5,5′-dithiobisnitrobenzoic acid, and incubate at 37°C for 60 min. Add 10 μL sodium dodecyl sulfate (0.4%) to terminate the reaction, and measure the absorbance (OD value) at 405 nm. The acetylcholinesterase inhibition rate of the test compound is as follows: Inhibition rate (%) = [(A0-A1 / A0] × 100 (where A0 is the absorbance of the blank, and A1 is the absorbance of the compound)
[0052] 2. Results of the inhibition of acetylcholinesterase activity by the compound of formula I
[0053] The test data results are as follows Figure 3-Figure 6 The results shown in the figure show that, through preliminary screening of the effect of the compound of formula I on the activity of acetylcholinesterase, it was found that the compound of formula I had significant inhibitory activity. When the concentration of the compound of formula I was 50 μM, it had a significant inhibitory effect on acetylcholinesterase, and its inhibition rate was 98%. Figure 3 The data results of the concentration-dependent inhibitory effect of the enantiopyrane-type norditerpene lactone on acetylcholinesterase show that the IC of the compound of formula I for inhibiting acetylcholinesterase is 50 The value is 2.13±0.06μM. Figure 4 Further in vitro inhibition kinetic studies showed that Figure 5 The Lineweaver-Burk plot shows that the compound of formula I can competitively inhibit the activity of acetylcholinesterase. Figure 5 a Lineweaver–Burk plot of the inhibitory effect of the enantiotesenane-type norditerpene lactones of the present invention on acetylcholinesterase, and Figure 6 The Lineweaver-Burk curve is used to determine the inhibition kinetic parameter (Ki) of the concentration of the enantiopyrane-type norditerpene lactone of the present invention. The data shown show that K i The value is 0.058μM.
[0054] In summary, the enantiotesenane-type norditerpene lactone compounds prepared by the present invention have good anti-acetylcholinesterase activity and can be used to prepare anti-acetylcholinesterase drugs. Such compounds have great development value as new drugs for the prevention or treatment of Alzheimer's disease, and the design ideas of such compounds also provide new ideas and approaches for the development of new drugs for the prevention or treatment of Alzheimer's disease.
[0055] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.
[0056] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.
Claims
1. An enantiotesenane-type norditerpene lactone compound, characterized in that: The enantiotesenane-type norditerpene lactone compound has a structure shown in the following formula (I):
2. A method for preparing the enantiotesenane-type norditerpene lactone compound according to claim 1, characterized in that: The following steps are involved: The dried root of Euphorbia chamaejasme is used as a raw material, and the extract is obtained after ethanol percolation extraction and concentration, and the extract is suspended in water to obtain a suspension, and then the extract is extracted and concentrated to obtain an extract; The extract is subjected to gradient elution through neutral alumina column chromatography to obtain a first eluate, the eluent is petroleum ether-ethyl acetate-methanol, and the volume ratio of the petroleum ether, ethyl acetate and methanol is 100:0:0 to 0:0:100; The first eluate is subjected to gradient elution by silica gel column chromatography to obtain a second eluate, wherein the eluent is petroleum ether-acetone-methanol, and the volume ratio of the petroleum ether, acetone and methanol is 100:0:0 to 0:100:0 to 0:0:100; The second eluate is subjected to gradient elution by MCI resin column chromatography to obtain a third eluate, wherein the eluent is methanol-water, and the volume ratio of the eluent methanol to water is 0:100 to 100:0; The third eluate is taken and subjected to ODS medium pressure column chromatography to obtain Fr.1-Fr.10, the mobile phase is methanol-water, and the volume ratio of the mobile phase methanol to water is 50:50-60:40; Fr.6 was purified by ODS liquid chromatography with acetonitrile-water as the mobile phase to obtain the enantiotesenane-type norditerpene lactone compound shown in formula I.
3. The method for preparing the enantiotesenane-type norditerpene lactone compound according to claim 2, characterized in that: The volume ratio of acetonitrile to water is 45:
55.
4. The method for preparing the enantiotesenane-type norditerpene lactone compound according to claim 2 or 3, characterized in that: The diafiltration extraction steps are as follows: taking ethanol to diafiltration extract the dry root of the wolfberry Euphorbia at room temperature, obtaining an extract-like ethanol extract after concentration, and suspending the ethanol extract in water to obtain a suspension; the ethanol concentration is 95%, the diafiltration extraction time is 120h, and the ethanol diafiltration extraction flow rate is 0.5L / h.
5. The method for preparing the enantiotesenane-type norditerpene lactone compound according to claim 2 or 3, characterized in that: The extraction step is as follows: adding dichloromethane in an equal volume to the suspension, extracting and concentrating to obtain the extract.
6. Use of the ent-atisenane-type norditerpene lactone compound according to claim 1 in the preparation of a drug for inhibiting acetylcholinesterase activity.
7. The use according to claim 6, characterized in that: The enantiotesenane-type norditerpene lactone compound is used as a raw material to prepare a pharmaceutical composition for inhibiting acetylcholinesterase activity or any pharmaceutically acceptable salt thereof.