Monoterpenoid compounds in Ailanthus altissima and preparation method and application thereof

Abstract

The present invention relates to monoterpenoid compounds in Ailanthus altissima and a preparation method and application thereof, belonging to the field of medical technology, and specifically relates to 5 monoterpenoid compounds extracted and separated from Ailanthus altissima [Ailanthus Desf.] of the family Simaroubaceae, wherein compound 3 has good anti-tyrosinase activity. The preparation method of the present invention is simple and easy to implement, has good reproducibility and high purity.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to five monoterpenoid compounds prepared from Ailanthus altissima and applications of the compounds in preparing tyrosinase, acetylcholinesterase and / or butyrylcholinesterase inhibitors. Background Art

[0002] Ailanthus altissima (Mill.) Swingle is a plant of the genus Ailanthus (Ailanthus Desf.) of the family Simaroubaceae. It is widely distributed in Shaanxi, Gansu, Sichuan, Yunnan and other provinces in my country. Ailanthus bark is the dried root bark or dry bark of Ailanthus altissima, which can also be called white bark of Ailanthus altissima, bitter bark of Ailanthus altissima, and white bark of Ailanthus altissima. It is a commonly used Chinese herbal medicine in my country. Since Ailanthus altissima leaves are usually not used as medicinal parts, there are few studies on the chemical components of Ailanthus altissima leaves. Bitter substances, triterpenes, coumarins, alkaloids and lignan compounds are the main chemical components of Ailanthus altissima; the chemical components in Ailanthus altissima have pharmacological activities such as anti-tumor, anti-malarial, anti-viral and anti-inflammatory.

[0003] Based on the previous research, the present invention systematically studies the chemical components and pharmacological activities of Ailanthus altissima leaves and root barks. Summary of the invention

[0004] The purpose of the present invention is to solve the deficiencies of the prior art and to provide five monoterpenoid compounds with novel structures in Ailanthus altissima and their preparation methods and their applications in the preparation of tyrosinase, acetylcholinesterase and / or butyrylcholinesterase inhibitors.

[0005] In order to achieve the purpose of the present invention, the present invention adopts the following technical scheme:

[0006] The present invention also provides application of the monoterpenoid compound in anti-tumor activity.

[0007] In the first aspect, the present invention provides 5 monoterpenoid compounds isolated from Ailanthus Desf. of the family Simaroubaceae, and the structures are as follows:

[0008]

[0009] In a second aspect, the present invention provides a method for preparing the monoterpenoid compounds in the above Ailanthus altissima, the preparation method comprising the following steps:

[0010] Take dried Ailanthus altissima leaves, extract them with 70% industrial ethanol under reflux, combine the extracts and concentrate to obtain an extract, extract the extract with dichloromethane-n-butanol and subject the obtained components to silica gel column chromatography, perform gradient elution with dichloromethane-methanol system 100:0-1:1, and obtain 4 components Fr.AD;

[0011] The component Fr.D was eluted with an ethanol-water system of 20:80-100:0 using HP20, ODS column chromatography to obtain two components Fr.D1-Fr.D2;

[0012] Silica gel column chromatography was used to elute with dichloromethane-methanol system 10:0-10:1 to obtain Fr.D1.3.1-Fr.D1.3.8. HP20 column chromatography was used to gradiently elute the component Fr.D with ethanol-water system 20:80-90:10 to obtain 4 components Fr.D1-Fr.D2;

[0013] Using ODS column chromatography, component Fr.D1 was gradient eluted with ethanol-water system 10:90-90:10 to obtain 4 components Fr.D1.1-Fr.D1.4; after silica gel column chromatography, elution with dichloromethane-methanol system 10:0-10:1 obtained Fr.D1.3.1-Fr.D1.3.8, Fr.D1.3.1 was separated on preparative reverse-phase HPLC using methanol-water mobile phase to obtain 6 components Fr.D1.3.1.1-Fr.D1.3.1.6, Fr.D1.3.1.1 was separated on semi-preparative reverse-phase HPLC using acetonitrile-water mobile phase to obtain compound 1-2.

[0014] Fr.D1.4 was eluted by silica gel column chromatography with dichloromethane-methanol system 10:0-10:1 to obtain Fr.D1.4.1-Fr.D1.4.6. Fr.D1.4.5 was separated by preparative reverse phase HPLC using methanol-water mobile phase to obtain 6 components Fr.D1.4.5.1-Fr.D1.4.5.6, and Fr.D1.4.5.5 and Fr.D1.4.5.6 were separated by semi-preparative reverse phase HPLC using acetonitrile-water mobile phase to obtain compounds 3-5.

[0015] As an optional mode, in the above preparation method, the Ailanthus altissima is Ailanthus altissima [Ailanthus Desf.] of the genus Simarthaceae.

[0016] As an optional manner, in the above preparation method, the dried Ailanthus altissima leaves are extracted with 70% industrial ethanol under reflux for 3 times, each time for 2-3 hours.

[0017] As an optional method, in the above preparation method, Fr.D1.3.1.1 is separated with an acetonitrile-water mobile phase of 30:70-20:80.

[0018] The obtained compounds were systematically identified and the results are as follows:

[0019] The structures of compounds 1-5 were identified by high-resolution mass spectrometry, one-dimensional NMR and two-dimensional NMR. The corresponding spectra are shown in Figure 1-20 As shown in Table 1-2.

[0020] Chouchunionone A(1): colorless oil. HRESIMS(m / z):[M+Na] + (m / z):291.1567(calcd for C 15 H 24 NaO4:291.1567), by analyzing the 1 HNMR, 13 C NMR, HSQC and HMBC spectra confirmed the structure of Chouchunionone A, which is a new compound.

[0021] Chouchunionone B(2): colorless oil. UV (methanol) λmax (logε) 240nm (2.30); HRESIMS: [M+Na] + (m / z):263.1608(calcd for C 14 H 24 NaO3:263.1618), by analyzing Chouchunionone B 1 H NMR, 13 C NMR, HSQC and HMBC spectra confirmed the structure of Chouchunionone B, which is a new compound.

[0022] Chouchunionone C(3): Pale yellow amorphous powder. UV(methanol)λmax(logε)230nm(2.34),205nm(2.34);HRESIMS:[M+H] + (m / z):371.2063,(calcd forC 19 H 31 O7:371.2064,), by analyzing the 1 HNMR, 13 C NMR, HSQC and HMBC spectra confirmed the structure of Chouchunionone C as a new compound.

[0023] Chouchunionone D(4): colorless oil. UV(methanol)λmax(logε)264(2.29); HRESIMS:[M+Na] + (m / z):393.1884(calcd for C 19 H 30 O7Na,393.1883), by analyzing the 1 HNMR, 13 C NMR, HSQC and HMBC spectra confirmed the structure of Chouchunionone D, which is a new compound.

[0024] Chouchunionone E(5): yellowish white amorphous powder. UV(methanol)λmax(logε)219(1.63); HRESIMS:[M+H] + (m / z):447.2227(calcd for C 21 H 35 O 10 ,447.2225).,Through the analysis of Chouchunionone E 1 H NMR, 13 C NMR, HSQC and HMBC spectra confirmed the structure of Chouchunionone E, which is a new compound.

[0025] In a third aspect, the present invention provides a pharmaceutical composition comprising the monoterpenoid compound prepared from Ailanthus altissima or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient.

[0026] In a fourth aspect, the present invention provides use of the monoterpenoid compound prepared from Ailanthus altissima or a pharmaceutical composition comprising the compound in the preparation of tyrosinase, acetylcholinesterase and / or butyrylcholinesterase inhibitors.

[0027] The anti-tyrosinase, acetylcholinesterase and / or butyrylcholinesterase activities of the five new compounds described in the present invention were investigated, and compound 3 exhibited good anti-tyrosinase activity. Therefore, the monoterpene compounds described in the present invention have the prospect of further developing anti-tyrosinase drugs.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The advantages of the present invention are that the compounds are all new compounds, monoterpene compounds with novel structures, and compound 3 has good anti-tyrosinase activity and is worthy of further development.

[0030] Table 1 Proton spectrum data of compounds 1-5 (600 MHz) (compounds 2, 3, 5 dissolved in deuterated dimethyl sulfoxide solution, compounds 1, 4 dissolved in deuterated chloroform solution)

[0031]

[0032]

[0033] Table 2 Carbon spectrum data of compounds 1-5 (150 MHz) (compounds 2, 3, 5 dissolved in deuterated dimethyl sulfoxide solution, compounds 1, 4 dissolved in deuterated chloroform solution)

[0034]

[0035] BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 HRESIMS spectrum of compound 1;

[0037] Figure 2 Compound 1 1 H-NMR spectrum;

[0038] Figure 3 Compound 1 13 C-NMR spectrum;

[0039] Figure 4 HSQC spectrum of compound 1;

[0040] Figure 5 HMBC spectrum of compound 1;

[0041] Figure 6 HRESIMS spectrum of compound 2;

[0042] Figure 7 Compound 2 1 H-NMR spectrum;

[0043] Figure 8 Compound 2 13 C-NMR spectrum;

[0044] Fig. 9 HSQC spectrum of compound 2;

[0045] Fig.10 HMBC spectrum of compound 2;

[0046] Fig.11 HRESIMS spectrum of compound 3;

[0047] Fig.12 HSQC spectrum of compound 3;

[0048] Fig.13 HMBC spectrum of compound 3;

[0049] Fig.14 HRESIMS spectrum of compound 4;

[0050] Fig.15 Compound 4 1 H-NMR spectrum;

[0051] Fig.16 HSQC spectrum of compound 4;

[0052] Fig.17 HMBC spectrum of compound 4;

[0053] Fig.18 HRESIMS spectrum of compound 5;

[0054] Fig.19 HSQC spectrum of compound 5;

[0055] Fig. 20 HMBC spectrum of compound 5. DETAILED DESCRIPTION

[0056] The following examples are intended to help those skilled in the art to better understand the present invention, but are not intended to limit the present invention in any way.

[0057] Example 1: Preparation of Compound 1-5

[0058] Take dried Ailanthus altissima leaves and extract them with 70% industrial ethanol for 3 times, each time for 2 hours. Combine the extracts and concentrate to obtain an extract, extract the extract with dichloromethane-n-butanol and pass the obtained components through silica gel column chromatography, with dichloromethane-methanol system 100:0-1:1 for gradient elution, to obtain 4 components Fr.AD;

[0059] The component Fr.D was eluted by gradient elution with an ethanol-water system of 20:80-90:10 using HP20 column chromatography to obtain four components Fr.D1-Fr.D2;

[0060] Component Fr.D1 was gradient eluted using an ethanol-water system of 10:90-90:10 using ODS column chromatography to obtain four components Fr.D1.1-Fr.D1.4;

[0061] Fr.D1.3 was eluted by silica gel column chromatography with dichloromethane-methanol system 10:0-10:1 to obtain Fr.D1.3.1-Fr.D1.3.8. Fr.D1.3.1 was separated by preparative reverse-phase HPLC using methanol-water mobile phase to obtain 6 components Fr.D1.3.1.1-Fr.D1.3.1.6. Fr.D1.3.1.1 was separated by semi-preparative reverse-phase HPLC using acetonitrile-water mobile phase to obtain compounds 1-2.

[0062] Fr.D1.4 was eluted by silica gel column chromatography with dichloromethane-methanol system 10:0-10:1 to obtain Fr.D1.4.1-Fr.D1.4.6. Fr.D1.4.5 was separated by preparative reverse-phase HPLC using methanol-water mobile phase to obtain 6 components Fr.D1.4.5.1-Fr.D1.4.5.6. Fr.D1.4.5.5 and Fr.D1.4.5.6 were separated by semi-preparative reverse-phase HPLC using acetonitrile-water mobile phase to obtain compounds 3-5.

[0063] Example 2: Investigation of the anti-tyrosinase activity of compounds 1-5

[0064] Compounds 1-5 were dissolved in DMSO and diluted to 5 series of concentrations with potassium phosphate buffer (KH2PO4 / K2HPO4, 0.1M, pH 7.4). The reaction system contained 40 μL of tyrosine, 40 μL of the test compound or positive drug arbutin, and 80 μL of PBS buffer solution. After adding 40 μL of tyrosinase, the reaction began. At a wavelength of 492 nm, the absorbance was measured 3 times within 10 minutes after adding the enzyme. The response concentration (logarithmic) curve was analyzed by nonlinear regression using the Graph-Pad Prism program to calculate the IC 50 The experimental results are shown in Table 3, where compound 3 has a significant anti-tyrosinase effect.

[0065] Table 3: Inhibitory activity of compounds from Ailanthus altissima against tyrosinase

[0066]

[0067] a The results are expressed as mean ± SD (n = 3).

[0068] Example 3: Investigation of the anti-acetylcholinesterase and butyrylcholinesterase activities of compounds 1-5

[0069] Compounds 1-5 were dissolved in DMSO and diluted to 5 series of concentrations with potassium phosphate buffer (KH2PO4 / K2HPO4, 0.1M, pH 7.4). The reaction system contained 25 μL of the compound to be tested, 12.5 μL of the enzyme, 125 μL of DTNB (dithiobis-p-nitrobenzoic acid), and 50 μL of ATCI (iodinated acetylthiocholine). The reaction started after the enzyme was added. Incubate overnight in the refrigerator. After incubation, 50 μL of ATCI was added to each well to terminate the reaction. The absorbance was measured 3 times within 10 minutes at a wavelength of 412 nm using an enzyme reader. The Graph-PadPrism program was used to perform nonlinear regression analysis on the response concentration (logarithmic) curve to calculate the IC 50 The experimental results are shown in Table 4.

[0070] Table 4: Inhibitory activity of compounds from Ailanthus altissima against acetylcholinesterase

[0071]

[0072]

[0073] a The results are expressed as mean ± SD (n = 3).

[0074] Compounds 1-5 were dissolved in DMSO and diluted to 5 series of concentrations with potassium phosphate buffer (KH2PO4 / K2HPO4, 0.1M, pH 7.4). The reaction system contained 25 μL of the compound to be tested, 12.5 μL of the enzyme, 125 μL of DTNB, and 50 μL of BTCI (S-butyrylthiocholine iodide). The reaction started after the enzyme was added. Incubate overnight in the refrigerator. After incubation, 50 μL of BTCI was added to each well to terminate the reaction. The absorbance was measured 3 times within 10 minutes at a wavelength of 412 nm. The Graph-Pad Prism program was used to perform nonlinear regression analysis on the response concentration (logarithmic) curve to calculate the IC 50 The experimental results are shown in Table 5.

[0075] Table 5: Inhibitory activity of compounds from Ailanthus altissima against butyrylcholinesterase

[0076]

[0077] a The results are expressed as mean ± SD (n = 3).

[0078] 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. A monoterpene compound in Ailanthus altissima, characterized in that: The monoterpene compound contains two isopentenyl groups, and the monoterpene compound is any one of the following structures: 。 2. The monoterpenoid compound in Ailanthus altissima according to claim 1, characterized in that: The Ailanthus altissima is a plant of the genus Ailanthus of the family Simaroubaceae. Ailanthus Desf . ].

3. A method for preparing monoterpenoid compounds in Ailanthus altissima according to claim 1 or claim 2, characterized in that: The preparation method comprises the following steps: Take dried Ailanthus altissima leaves, extract them with 70% industrial ethanol under reflux, combine the extracts and concentrate to obtain an extract, extract the extract with dichloromethane-n-butanol and subject the obtained components to silica gel column chromatography, gradient elution with dichloromethane-methanol system 100:0-1:1, and obtain 4 components Fr. AD; The component Fr. D was eluted by gradient elution with an ethanol-water system of 20:80-90:10 using HP20 column chromatography to obtain four components Fr. D1-Fr. D2; Component Fr. D1 was eluted by ODS column chromatography with an ethanol-water system of 10:90-90:10 gradient to obtain four components Fr. D1.1-Fr. D1.4; Silica gel column chromatography was performed with dichloromethane-methanol system 10:0-10:1 for elution to obtain Fr. D1.3.1-Fr. D1.3.

8. Fr. D1.3.1 was separated on preparative reverse phase HPLC using methanol-water mobile phase to obtain 6 components Fr. D1.3.1.1-Fr. D1.3.1.

6. Fr. D1.3.1.1 was separated on semi-preparative reverse phase HPLC using acetonitrile-water mobile phase to obtain compound 1-2. Fr. D1.4 was eluted by silica gel column chromatography with dichloromethane-methanol system 10:0-10:1 to obtain Fr. D1.4.1-Fr. D1.4.

6. Fr. D1.4.5 was separated by preparative reverse-phase HPLC using methanol-water mobile phase to obtain 6 components Fr. D1.4.5.1-Fr. D1.4.5.

6. Fr. D1.4.5.5 and Fr. D1.4.5.6 were separated by semi-preparative reverse-phase HPLC using acetonitrile-water mobile phase to obtain compounds 3 and 5.

4. The method for preparing monoterpenoid compounds from Ailanthus altissima according to claim 3, characterized in that: The Ailanthus altissima is a plant of the genus Ailanthus altissima of the family Simaroubaceae. Ailanthus Desf . ].

5. The method for preparing monoterpenoid compounds in Ailanthus altissima according to claim 3, characterized in that: Take the dried Ailanthus altissima leaves and extract them with 70% industrial ethanol for 3 times, each time for 2-3 hours.

6. The method for preparing monoterpenoid compounds from Ailanthus altissima according to claim 3, characterized in that: Fr. D1.3.1.1, Fr. D1.4.5.5 and Fr. D1.4.5.6 were separated using 30:70-20:80 acetonitrile-water mobile phase.

7. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the monoterpenoid compound in Ailanthus altissima according to claim 1 or claim 2 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient.

8. Use of the monoterpenoid compound in Ailanthus altissima or a pharmaceutically acceptable salt thereof according to claim 1 or claim 2 in the preparation of a tyrosinase, acetylcholinesterase and / or butyrylcholinesterase inhibitor.

9. Use of the pharmaceutical composition according to claim 7 in the preparation of tyrosinase, acetylcholinesterase and / or butyrylcholinesterase inhibitors.

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