C32 cycloartane type triterpenoids containing 3-ketone carbonyl as well as preparation method and application of C32 cycloartane type triterpenoids

Abstract

The invention belongs to the technical field of medicines, and discloses a C32 cycloartane triterpenoid compound containing 3-ketone carbonyl as well as a preparation method and application of the C32 cycloartane triterpenoid compound. The method comprises the following steps: enriching triterpenoids by utilizing various separation technologies and means, taking pond fir fallen leaves as a separation target and utilizing a molecular sieve principle of gel, then sequentially carrying out silica gel column chromatography and ODS column chromatography separation to obtain a target component, and further purifying to obtain the C32 cycloartane triterpenoids with 3-ketocarbonyl groups. An in-vitro anti-neuritis experiment proves that the C32 cycloartane type triterpenoid with the 3-site keto-carbonyl group has remarkable anti-neuritis activity, can be used as an active ingredient of an anti-neuritis medicine, and has wide application.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and relates to a C 32 Cycloartane triterpenoids and preparation method and application thereof. BACKGROUND

[0002] Taxodium distichum is a plant of Taxodiaceae, and is originally from the southeast of the United States. Taxodium distichum was introduced in China in the 1970s, and is now widely distributed in the Yangtze River Basin and wetland ecosystems in southern China, and has significant ecological restoration value and landscape function. The chemical composition research of Taxodium distichum shows that the leaves and mature cones of Taxodium distichum are rich in various terpenoids, mainly including monoterpenes, diterpenes and cycloartane triterpenoids, and exhibit diverse biological activities such as antibacterial, antiviral and antitumor activities. SUMMARY

[0003] Therefore, the present application aims to provide a C 32 Cycloartane triterpenoids and preparation method and application thereof.

[0004] To solve the above problems, the technical scheme provided by the present application is as follows:

[0005] In the first aspect of the present application, the present application provides a C 32 Cycloartane triterpenoids, which are selected from one of the compounds shown in the structural formulae of formula I to formula V:

[0006]

[0007] In the second aspect of the present application, the present application provides a preparation method of the above-mentioned C 32 Cycloartane triterpenoids containing a 3-keto carbonyl group. 32 Cycloartane triterpenoids.

[0008] As a preferred embodiment of the preparation method of the C 32 Cycloartane triterpenoids containing a 3-keto carbonyl group, the method comprises the following steps:

[0009] S1. The dried fallen leaves of Taxodium distichum are extracted with ethanol, and the obtained extract is vacuum concentrated to be free of ethanol to obtain total extract;

[0010] S2. The total extract is suspended in water, and extracted with ethyl acetate to obtain an extract;

[0011] S3. The extract is subjected to gel column chromatography, and methanol is used as an elution solvent, and each 350 mL is a flow fraction to obtain flow fractions Fr.1-8;

[0012] S4. The flow Fr.3 is subjected to ODS column chromatography separation, and the elution solvents are methanol / water with a volume ratio of 80:20, methanol / water with a volume ratio of 85:15, methanol / water with a volume ratio of 90:5, and methanol / water with a volume ratio of 100:0, respectively, to obtain four flow portions, which correspond to flow portions Fr.3A-3D, respectively;

[0013] S5. The flow portions Fr.3A-3C are subjected to silica gel column chromatography separation, respectively, to obtain flow portions Fr.3A-1-Fr.3A-5, Fr.3B-1-Fr.3B-5 and Fr.3C-1-Fr.3C-5, respectively;

[0014] S6. The flow portions Fr.3C-1 and Fr.3C-2 are subjected to ODS column chromatography purification to obtain a compound with a structural formula as shown in formula I; the flow portion Fr.3B-1 is subjected to recrystallization to obtain a compound with a structural formula as shown in formula II; the flow portion Fr.3A-3 is subjected to high performance liquid chromatography preparation to obtain a compound with a structural formula as shown in formula III; the flow portion Fr.3C-3 is subjected to high performance liquid chromatography preparation to obtain a compound with a structural formula as shown in formula IV; and the flow portion Fr.3C-4 is subjected to high performance liquid chromatography preparation to obtain a compound with a structural formula as shown in formula V.

[0015] As the C 32 In the preferred embodiment of the preparation method of the cycloartane type triterpenoid compound, in step S5, the silica gel column chromatography separation is performed by using petroleum ether / ethyl acetate with a volume ratio of 50:1, petroleum ether / ethyl acetate with a volume ratio of 25:1, petroleum ether / ethyl acetate with a volume ratio of 10:1, petroleum ether / ethyl acetate with a volume ratio of 2:1, and petroleum ether / ethyl acetate with a volume ratio of 1:1, in sequence.

[0016] As the C 32 In the preferred embodiment of the preparation method of the cycloartane type triterpenoid compound, in step S6, the ODS column chromatography is performed by using methanol-water mixed solvents with a volume ratio of 94:6 as the elution solvent.

[0017] As the C 32 In the preferred embodiment of the preparation method of the cycloartane type triterpenoid compound, in step S6, the recrystallization is performed by using methanol for multiple times.

[0018] As the C 32 In the preferred embodiment of the preparation method of the cycloartane type triterpenoid compound, the high performance liquid chromatography is performed by using an XB-C18 chromatographic column with a specification of 5 μm and 21.2*250 mm, and the mobile phase of the high performance liquid chromatography is a methanol-water solution, in which the volume ratio of methanol to water is 90:10-100:0.

[0019] In the third aspect of the present application, the present application further provides a C 32 The application of the cycloartane triterpenoid as an active ingredient in the preparation of an anti-neuritis drug.

[0020] In the fourth aspect of the present application, the present application further provides a drug comprising the C 32 cycloartane triterpenoid or a pharmaceutically acceptable salt thereof.

[0021] In some embodiments, the drug further comprises pharmaceutically acceptable excipients and carriers.

[0022] In some embodiments, the drug preparation of the drug is granules, tablets, pills, solutions, capsules, films, tinctures, creams, ointments, aerosols, suppositories, liniments, gels or injections.

[0023] Compared with the prior art, the present application selects the dried fallen leaves of Taxodium ascendens Brongn, reflux extracts with ethanol, extracts multiple times with ethyl acetate, and then obtains the C 32 cycloartane triterpenoid Formula I-V with a 3-keto carbonyl group 32 The cycloartane triterpenoid and its derivatives are the first discovered C 32 and its decarburized triterpenoid derivatives, enrich the skeleton and types of cycloartane triterpenoids, and also provide ideas and sources for the discovery of natural source drugs, and have significant anti-neuritis activity, can be used as an active ingredient of an anti-neuritis drug, and provide alternative compounds for the development of new anti-neuritis drugs. DETAILED DESCRIPTION

[0024] The scheme of the present application will be explained below in conjunction with examples. Those skilled in the art will understand that the following examples are only used to illustrate the present application, and should not be regarded as limiting the scope of the present application. If the specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the art or according to the product instructions are used. If the specific conditions are not specified in the following examples, the conventional conditions or the conditions recommended by the manufacturer are used, and the methods used are the conventional methods known in the art, and the consumables and reagents used are commercially available unless otherwise specified. Unless otherwise specified, the professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any method or material similar or equivalent to that described can also be applied in the present application.

[0025] In terms of chemical structure, the C 32Cycloartane triterpenoids are a rare class of triterpenoids with two additional carbon atoms in the side chain at position 17 of the common triterpenoids. Due to the presence of a three-membered ring at position 19, the chemical shift of hydrogen at position 19 is basically unchanged [δ H 0.78, d (J = 4.2 Hz); 0.57, d (J = 4.2 Hz)], which are very characteristic signals of the three-membered ring. Guided by these hydrogen signals, five C 32 Cycloartane triterpenoids and derivatives thereof.

[0026] Example 1: Preparation of compounds with structural formulae of Formula I-Formula V

[0027] The invention uses the leaves of Taxodium distichum var. imbricatum as raw materials, and 95% ethanol is used for reflux extraction. The compounds are prepared through extraction, enrichment, separation and other steps. The leaves of Taxodium distichum var. imbricatum are collected from Nantong, Jiangsu.

[0028] Taking the leaves of Taxodium distichum var. imbricatum as an example, the preparation process of compounds with structural formulae of Formula I-Formula V is as follows:

[0029] 1) Extraction, extraction, enrichment and initial separation

[0030] The dried leaves of Taxodium distichum var. imbricatum are crushed (25 kg), and 95% ethanol is used for reflux extraction three times, and then vacuum concentrated to remove alcohol, mixed with hot water (50°C), and then extracted with an equal volume of ethyl acetate for 4-5 times. The extraction layer (1.4 kg) is subjected to gel column chromatography, and methanol is used as the elution solvent, and each 350 mL is a flow fraction, to obtain flow fractions Fr. 1-8. Guided by the hydrogen spectrum nuclear magnetic data, the Fr. 3 component is subjected to ODS column chromatography separation, and the elution solvents are methanol / water with a volume ratio of 80:20, methanol / water with a volume ratio of 85:15, methanol / water with a volume ratio of 90:5, and methanol / water with a volume ratio of 100:0, respectively, to obtain four flow fractions, which correspond to Fr. 3A-3D, respectively, and each sub-flow fraction is subjected to hydrogen spectrum detection to lock the target sub-flow fraction Fr. 3A-3C. The three flow fractions Fr3A-3C are subjected to silica gel column chromatography separation, and the same elution solvents and elution ratios are used for elution, and the elution solvents are petroleum ether / ethyl acetate with a volume ratio of 50:1, petroleum ether / ethyl acetate with a volume ratio of 25:1, petroleum ether / ethyl acetate with a volume ratio of 10:1, petroleum ether / ethyl acetate with a volume ratio of 2:1, and petroleum ether / ethyl acetate with a volume ratio of 1:1, respectively, to obtain Fr. 3A-1-Fr. 3A-5, Fr. 3B-1-Fr. 3B-5 and Fr. 3C-1-Fr. 3C-5.

[0031] 2) Purification

[0032] Components Fr.3C-1 and Fr.3C-2 were purified by ODS column chromatography. The preparation conditions were as follows: elution with methanol / water (94:6) yielded Formula I. Component Fr.3B-1 was recrystallized multiple times from methanol to obtain Formula II. Component Fr.3A-3 was prepared by HPLC to obtain Formula III, with the following preparation conditions: XB-C18 (5 μm, 21.2 × 250 mm), methanol-water (94:6, V / V), flow rate 10.0 mL / min, detection wavelength 230 nm. Component Fr.3C-3 was prepared by HPLC to obtain Formula IV, with the following preparation conditions: XB-C18 (5 μm, 21.2 × 250 mm), methanol-water (98:2, V / V), flow rate 10.0 mL / min, detection wavelength 230 nm. Component Fr.3C-4 was prepared into formula V by HPLC under the following conditions: XB-C18 (5 μm, 21.2 × 250 mm), methanol-water (100:0, V / V), flow rate 10.0 mL / min, and detection wavelength 230 nm. The mass spectrometry and wavelength spectrometry data of formulas I to V are shown below.

[0033] Compound I: taxcycloterpene A; optical rotation (c 0.08, MeOH); UV absorption (MeOH)λ max (logε)203(4.14)nm; the main absorption peak in the infrared spectrum is (KBr)ν. max 2961, 2865, 1466, 1378 and 1126cm -1 The high-resolution mass spectrometry m / z was 477.3710 [M+Na]. + (calcd for C 31 H 50 O2Na, 477.3709), the 1H and 1C NMR spectra are shown in Table 1.

[0034] Compound II: taxcycloterpene B; optical rotation (c 0.04, MeOH); UV absorption (MeOH)λ max (logε)203(4.54)nm; the main absorption peak in the infrared spectrum is (KBr)ν. max 2943, 2866, 1709, 1466, 1379, 1341, and 1113cm -1 The high-resolution mass spectrometry m / z was 463.3554 [M+Na]. + (calcd for C 30 H 48 O2Na (463.3552), the 1H and 1C NMR spectra are shown in Table 1.

[0035] Compound III: taxcycloterpene C; optical rotation (c 0.08, MeOH); UV absorption (MeOH) λ max (log ε) 203(4.12) nm; infrared spectrum main absorption peaks (KBr) v max 2960, 2866, 1706, 1466, 1378, and 1126 cm -1 ; high resolution mass spectrum m / z 435.3242 [M + Na] + (calcd for C 28 H 44 O2Na, 435.3239), nuclear magnetic resonance hydrogen spectrum and carbon spectrum data are shown in Table 1.

[0036] Compound IV: taxcycloterpene D; optical rotation (c 0.03, MeOH); UV absorption (MeOH) λ max (log ε) 203(4.30) nm; infrared spectrum main absorption peaks (KBr) v max 2943, 2866, 1709, 1466, 1378, and 1112 cm -1 ; high resolution mass spectrum m / z 493.3662 [M + Na] + (calcd for C 31 H 50 O3Na, 493.3658), nuclear magnetic resonance hydrogen spectrum and carbon spectrum data are shown in Table 2.

[0037] Compound V: taxcycloterpene E; optical rotation (c 0.10, MeOH); UV absorption (MeOH) λ max (log ε) 204(3.99) nm; infrared spectrum main absorption peaks (KBr) v max 2925, 2867, 1466, 1379, and 1098 cm -1 ; high resolution mass spectrum m / z 493.4023 [M + Na] + (calcd for C 32 H 54 O2Na, 493.4022), nuclear magnetic resonance hydrogen spectrum and carbon spectrum data are shown in Table 2.

[0038] Table 1. Nuclear magnetic hydrogen spectrum (600 MHz, CDC13) and carbon spectrum (150 MHz) data of compounds of formula I - formula III

[0039]

[0040]

[0041] The above results show that the obtained compounds of formula I- formula III are correct in structure.

[0042] Table 2. Nuclear magnetic hydrogen spectrum (600 MHz, CDCl3) and carbon spectrum (150 MHz) data of compounds of formula IV- formula V

[0043]

[0044] The above results show that the obtained compounds of formula IV- formula V are correct in structure.

[0045] Example 2: Anti-neurogenic activity of compounds of formula I- formula V

[0046] 1) Experimental materials

[0047] Instruments and reagents: CO2 incubator (Thermo Fisher Scientific, USA); CKX41 microscope (Olympus Optical Industrial Co., Ltd., Japan); Easy pure II laboratory grade ultrapure water instrument (Thermo Fisher Scientific, USA); DMSO (Biosharp, USA); DMEM / RPMI 1640 medium (Invitrogen, USA); DMSO, MTT, etc. (Sigma, USA); fetal bovine serum (Hangzhou Sijiqing Biological Material Research Institute); other commonly used inorganic salts and other reagents are analytical pure (Nanjing Chemical Reagent Co., Ltd.).

[0048] Test cell strain: BV2 cells (mouse microglial cell line), purchased from the China Medical Science Academy Cell Bank.

[0049] Test sample: C from 3-keto carbonyl obtained from Tsuga heterophylla (Raf.) Sarg. 32 Cycloartane triterpenoids of formula I-V with a purity of more than 95%; L-NMMA was selected as a positive control drug, and each compound was dissolved in DMSO and then diluted.

[0050] 2) Experimental method

[0051] a. Cell viability detection: This experiment uses an in vitro MTT assay kit to detect the cytotoxicity of formula I-V on BV-2. BV-2 cells are cultured in 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin DMEM basic medium at 37°C, 5% CO2 cell incubator. When the cell growth density is appropriate, the cells are resuspended to a density of 1.5×10 5A cell suspension of 1 x 104 / mL was prepared. The control group, drug group and blank control group were set up. The cells were inoculated in 96-well plates and cultured until the cell density reached 70% to 80%. Then, a culture solution containing different concentrations of formula I-V (1, 10, 25, 50, 100 μM) was prepared, and the drug group was pretreated with the gradient culture solution of formula I-V for 24 hours. According to the manufacturer's instructions, the MTT stock solution was prepared. After the treatment with formula I-V was completed, the culture solution in the 96-well plate was replaced with 150 mL of DMEM containing 10% MTT, and the incubation was continued in the cell incubator for 4 hours in the dark. After the incubation was completed, 150 μL of DMSO was added to dissolve the deep purple formazan crystals, and the incubation was continued in the constant temperature shaker for 10 min in the dark. Finally, the OD value was detected at 490 nm wavelength using a microplate reader, and the cell survival rate was calculated according to the following formula.

[0052]

[0053] b. Griess method for detecting NO content: In this experiment, a nitric oxide detection kit was used to detect the release of NO from BV-2 cells induced by LPS to reflect the anti-neuroinflammatory effect of formula I-V. The control group, PLS group and PLS+formula I-V (1, 10, 25, 50, 100 μM) group were set up. The cells were inoculated in 96-well plates and cultured until the cell density reached 70% to 80%. In the LPS group, the basic culture medium containing 500 ng / mL LPS was used, and at the same time, the drug group was incubated with different concentrations of formula I-V and 500 ng / mL LPS for 24 hours. According to the manufacturer's instructions, 100 μL of cell culture supernatant was aspirated from each well, Griess reagent was added, and the incubation was continued at room temperature for 10 min. The absorbance value was measured at 540 nm wavelength using a microplate reader. According to the standard curve established in advance, the concentration of NO in the sample was calculated. The experimental data was processed by scientific statistical method, and the concentration of compound required to inhibit 50% NO production (IC 50 value) was used as the index to evaluate the anti-inflammatory activity of the compound.

[0054] 3) Experimental results

[0055] According to the test results of MTT method, the IC 50 value of the test sample on the above-mentioned cells was calculated, and the results are shown in Table 3.

[0056] Table 3. Screening results of in vitro NO inhibitory activity of test samples

[0057]

[0058]

[0059] The results show that the compounds of formula I, formula IV and formula V have good potential anti-neuritis activity and can be used as active ingredients for anti-neuritis.

[0060] Those skilled in the art should understand that the present application is not limited to the above-mentioned embodiments, and the above-mentioned embodiments and descriptions in the specification are only to illustrate the principles of the present application, and various changes and improvements can be made without departing from the spirit and scope of the present application, and these changes and improvements all fall within the scope of the claimed present application. The scope of protection of the present application is defined by the appended claims and their equivalents.

Claims

1. A C-type carbonyl group containing a ketone carbonyl group at the 3-position. 32 Cycloalartin triterpenoids, characterized in that, Select one of the compounds with the structures shown in Formulas I to V:

2. A C-type compound containing a ketone carbonyl group at the 3-position as described in claim 1. 32 A method for preparing cyclic Altyn-type triterpenes, characterized in that, The preparation method includes the following steps: S1. Extract the dried fallen leaves of pond cypress with ethanol, and concentrate the obtained extract under vacuum until no ethanol remains to obtain the total extract; S2. The total extract is suspended in water and extracted with ethyl acetate to obtain the extract; S3. The extract was subjected to gel column chromatography with methanol as the elution solvent, and each fraction was 350 mL to obtain fractions Fr.1-8. S4. The fraction Fr.3 was separated by ODS column chromatography, and the elution solvent was successively methanol / water with a volume ratio of 80:20, methanol / water with a volume ratio of 85:15, methanol / water with a volume ratio of 90:5, and methanol / water with a volume ratio of 100:0, to obtain four fractions, corresponding to fractions Fr.3A-3D respectively. S5. Separate the fractions Fr3A-3C by silica gel column chromatography to obtain fractions Fr.3A-1-Fr.3A-5, Fr.3B-1-Fr.3B-5 and Fr.3C-1-Fr.3C-5; S6. Fractions Fr.3C-1 and Fr.3C-2 were purified by ODS column chromatography to obtain the compound with the structure shown in Formula I; fraction Fr.3B-1 was recrystallized to obtain the compound with the structure shown in Formula II; fraction Fr.3A-3 was prepared by high performance liquid chromatography to obtain the compound with the structure shown in Formula III; fraction Fr.3C-3 was prepared by high performance liquid chromatography to obtain the compound with the structure shown in Formula IV; fraction Fr.3C-4 was prepared by high performance liquid chromatography to obtain the compound with the structure shown in Formula V.

3. The preparation method according to claim 2, characterized in that, In step S5, the silica gel column chromatography separation is performed by eluting sequentially with petroleum ether / ethyl acetate at a volume ratio of 50:1, 25:1, 10:1, 2:1, and 1:

1.

4. The preparation method according to claim 2, characterized in that, In step S6, the elution solvent for the ODS column chromatography is a methanol-water mixture with a volume ratio of methanol to water of 94:

6.

5. The preparation method according to claim 2, characterized in that, In step S6, the recrystallization is performed by recrystallizing multiple times with methanol.

6. The preparation method according to claim 2, characterized in that, The high-performance liquid chromatography (HPLC) uses an XB-C18 column with a specification of 5 μm and 21.2 × 250 mm. The mobile phase of the HPLC is a methanol-water solution, in which the volume ratio of methanol to water is 90:10 to 100:

0.

7. A C-type compound containing a ketone carbonyl group at the 3-position as described in claim 1. 32 Application of cycloaltin triterpenoids as active ingredients in the preparation of antineuritis drugs.

8. A drug, characterized in that, The drug comprises the C3-ketone carbonyl group as described in claim 1. 32 Cycloaltin triterpenoids or their pharmaceutically acceptable salts.

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