Alkaloid compounds in calophyllum inophyllum and preparation method and application thereof

Abstract

This invention discloses alkaloid compounds from Brucea javanica, their preparation methods, and applications, belonging to the field of pharmaceutical technology. Specifically, it relates to two alkaloid compounds extracted and isolated from the fruit of Brucea javanica, a plant of the genus Brucea in the family Simaroubaceae. These compounds have good acetylcholinesterase inhibitory activity and can be used to prepare acetylcholinesterase inhibitors, showing good development prospects and application potential.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to novel alkaloid compounds prepared from the fruit of the plant Brucea javanica and the application of these compounds as acetylcholinesterase inhibitors. Background Technology

[0002] Brucea javanica ( Brucea javanica ) belongs to the genus *Brucea* of the family Simaroubaceae. Brucea This plant, *Brucea javanica*, is widely distributed in Fujian, Taiwan, and Guangdong provinces of my country, and also found in Southeast Asia and northern Oceania. It is bitter, cold in nature, and poisonous. It has the effects of clearing heat and drying dampness, killing parasites, detoxifying, stopping dysentery, and treating malaria, and can be used to treat amoebic dysentery, malaria, and other diseases. Externally, it has a corrosive effect and is used for warts, corns, etc. Modern chemical and pharmacological studies have shown that *Brucea javanica* contains bitter substances, steroids, coumarins, lignin, and other components, possessing various activities such as antitumor, antiviral, anti-inflammatory, and antioxidant properties. A search revealed that compared to the extensive research on quassinolides, there are fewer reports on alkaloid components in *Brucea javanica*, and systematic research remains relatively weak. Summary of the Invention

[0003] The purpose of this invention is to provide an alkaloid compound obtained from Brucea javanica, its preparation method, and its application in inhibiting acetylcholinesterase activity.

[0004] This invention relates to *Brucea javanica*, a plant belonging to the genus *Brucea* of the family Simaroubaceae. Brucea javanica The structural formulas of the two alkaloid compounds isolated from the sample are as follows:

[0005] The present invention provides a technical solution for preparing alkaloid compounds from Brucea javanica, comprising the following steps: The dried fruit of Brucea javanica was extracted with ethanol, and the extracts were combined and concentrated to obtain an extract. The extract was extracted with dichloromethane and n-butanol, and the obtained components were subjected to silica gel column chromatography with a dichloromethane-methanol system of 100:1 to 1:1 for isocratic gradient elution. A total of 6 components Fr. A to Fr. F were collected. The fraction Fr. B was separated by HP20 column chromatography and ODS column chromatography, and gradient elution was performed with an ethanol-water system of 20:80 to 90:10 to obtain 6 fractions Fr. B1 to Fr. B6. Fr. B6 was eluted by silica gel column chromatography with a petroleum ether-ethyl acetate system of 100:1 to 10:1, and then separated by preparative reversed-phase high-performance liquid chromatography with acetonitrile-water as the mobile phase to obtain compounds 1 to 2.

[0006] Preferably, the method for preparing the alkaloid compounds in *Brucea javanica* uses the dried fruit of *Brucea javanica*, a plant belonging to the genus *Brucea* of the family Simaroubaceae. Brucea javanica ].

[0007] Preferably, the method for preparing the alkaloids in Brucea javanica involves extracting dried Brucea javanica fruit by reflux extraction with 70%~80% ethanol (by volume) 2~3 times, each time for 2~3 hours.

[0008] Preferably, the method for preparing the alkaloid compounds in Brucea javanica involves separating Fr. B6 using an acetonitrile-water mobile phase of 70:30 to 30:70.

[0009] A pharmaceutical composition comprising an alkaloid compound or a pharmaceutically acceptable salt thereof prepared from Brucea javanica and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition can be formulated into clinically acceptable dosage forms such as tablets, capsules, granules, powders, oral liquids, injections, ointments, gels, or patches.

[0010] The present invention also provides the use of the alkaloid compounds or their pharmaceutically acceptable salts, or pharmaceutical compositions containing the above compounds, prepared from Brucea javanica in the preparation of medicaments for the prevention and / or treatment of diseases related to acetylcholinesterase activity.

[0011] Compared with the prior art, the present invention has the following advantages: the alkaloid compounds described in the present invention are all new compounds with novel structures, and at the same time, they have good acetylcholinesterase inhibitory activity, which can be used to prepare acetylcholinesterase inhibitors, and have good development prospects and application potential. Attached Figure Description

[0012] Figure 1 HRESIMS spectrum of compound 1; Figure 2 Compound 1 1 H-NMR spectrum; Figure 3 Compound 1 13 C-NMR spectrum; Figure 4 HMBC spectrum of compound 1; Figure 5 HRESIMS spectrum of compound 2; Figure 6 Compound 2 1 H-NMR spectrum; Figure 7 Compound 2 13 C-NMR spectrum; Figure 8 HMBC spectrum of compound 2. Detailed Implementation

[0013] The embodiments listed below are intended to help those skilled in the art better understand the present invention, but do not limit the invention in any way.

[0014] Example 1

[0015] Preparation of compounds 1-2: The dried fruit of *Brucea javanica* was extracted by reflux with 70%–80% ethanol 2–3 times, each time for 2–3 hours. The extracts were combined and concentrated to obtain an extract. The extract was extracted with dichloromethane and n-butanol. The obtained fractions were subjected to silica gel column chromatography with isocratic gradient elution using a dichloromethane-methanol system of 100:1–1:1 (v / v). A total of 6 fractions, Fr. A to Fr. F, were collected. Fr. B was separated by HP20 column chromatography and ODS column chromatography, and gradient elution was performed with an ethanol-water system of 20:80 to 90:10 to obtain 6 fractions Fr. B1 to Fr. B6. Fr. B6 was eluted by silica gel column chromatography with a petroleum ether-ethyl acetate system of 100:1 to 10:1, and then separated by preparative reversed-phase high performance liquid chromatography with acetonitrile-water of 70:30 to 30:70 as the mobile phase to obtain compounds 1 to 2.

[0016] The results of systematic structural identification of compounds 1-2 are as follows: The structures of compounds 1-2 were identified using high-resolution mass spectrometry, one-dimensional NMR, two-dimensional NMR, computational NMR, computational ECD, and X-ray single-crystal diffraction. The corresponding structural characterization data are shown in Table 1, and the spectra are shown in [missing data]. Figures 1-8 As shown.

[0017] Bruceaine I (1): Yellow powder; measured at 260.1643 [M + H] + (calcd for C 16 H 22 NO2, 260.1645) was determined to have the molecular formula C 16 H 21 NO2; By analyzing bruceaine I 1 H NMR, 13 The structure of bruceaine I was determined by C NMR, HSQC, and HMBC spectra, identifying it as a new compound.

[0018] Bruceaine J (2): Yellow powder; via HRESIMS: m / z 326.1353 [M + Na] + (calcdfor C 17 H 21 NO4Na, 326.1363) has been determined to have the molecular formula C 17 H 21 NO4; By analyzing bruceaine J 1 H NMR, 13 CNMR, HSQC, and HMBC spectra confirmed the structure of bruceaine J, identifying it as a novel compound.

[0019] Table 1. Compounds 1-2 1 H NMR and 13 C10 NMR data (CDCl3)

[0020] Example 2

[0021] Investigation of the inhibitory activity of compounds 1-2 on acetylcholinesterase: Compounds 1 and 2 were dissolved in DMSO and diluted with potassium phosphate buffer (KH₂PO₄ / K₂HPO₄, 0.1 M, pH 7.4) to five series concentrations (25, 12.5, 6.25, 3.725, 1.8625 μM). The reaction system contained 25 μL of the analyte, 12.5 μL of acetylcholinesterase (AChE), 125 μL of DTNB, and 50 μL of ATCI. The reaction started immediately after enzyme addition. The absorbance was measured three times within 10 minutes after enzyme addition at 412 nm. The response concentration (logarithmic) curve was analyzed nonlinearly using the Graph-Pad Prism program to calculate the IC₂ concentration. 50 value.

[0022] Table 2. Inhibitory activity of compounds 1-2 against acetylcholinesterase

[0023]

[0024] The acetylcholinesterase inhibitory activity of compounds 1-2 of the present invention was investigated. The results showed that compounds 1 and 2 exhibited good acetylcholinesterase inhibitory activity, indicating that compounds 1 and 2 have the potential to be further developed into acetylcholinesterase inhibitors and can be used to prepare drugs for the prevention and / or treatment of diseases related to abnormal acetylcholinesterase activity.

[0025] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of this invention and its equivalents, this invention also intends to include these modifications and variations.

Claims

1. An alkaloid compound or a pharmaceutically acceptable salt thereof from *Brucea javanica*, characterized in that, The alkaloid compound is any one of the structures shown below: 。 2. The alkaloid compound or its pharmaceutically acceptable salt in *Brucea javanica* according to claim 1, characterized in that, The croton seed mentioned is *Brucea javanica*, a plant belonging to the genus *Brucea* in the family Simaroubaceae. Brucea javanica ].

3. A method for preparing an alkaloid compound or a pharmaceutically acceptable salt thereof from *Brucea javanica* as described in claim 1 or 2, characterized in that, Includes the following steps: The dried fruit of Brucea javanica was extracted with ethanol, and the extracts were combined and concentrated to obtain an extract. The extract was extracted with dichloromethane and n-butanol, and the obtained components were subjected to silica gel column chromatography with dichloromethane-methanol system at 100:1 to 1:1 for isocratic gradient elution. A total of 6 components Fr. A to Fr. F were collected. The fraction Fr. B was separated by HP20 column chromatography and ODS column chromatography, and gradient elution was performed with an ethanol-water system of 20:80 to 90:10 to obtain 6 fractions Fr. B1 to Fr. B6. Fr. B6 was eluted by silica gel column chromatography with a petroleum ether-ethyl acetate system of 100:1 to 10:1, and then separated by preparative reversed-phase high-performance liquid chromatography with acetonitrile-water as the mobile phase to obtain compounds 1 to 2.

4. The method for preparing alkaloids or their pharmaceutically acceptable salts from *Brucea javanica* according to claim 3, characterized in that, The croton seeds used are those from the genus *Brucea* of the Simaroubaceae family. Brucea javanica ].

5. The method for preparing alkaloids or their pharmaceutically acceptable salts from *Brucea javanica* according to claim 3, characterized in that, Take dried Brucea javanica fruit and extract it by reflux with 70%~80% ethanol 2~3 times, each time for 2h~3h.

6. The method for preparing alkaloids or pharmaceutically acceptable salts of *Brucea javanica* according to claim 3, characterized in that, Fr. B6 was separated using an acetonitrile-water mobile phase of 70:30 to 30:

70.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises an alkaloid compound or a pharmaceutically acceptable salt thereof from the croton seed as described in claim 1 or 2, and a pharmaceutically acceptable carrier or excipient; the dosage form of the pharmaceutical composition includes tablets, capsules, granules, powders, oral liquids, injections, ointments, gels, and patches.

8. The use of the alkaloids from *Brucea javanica* as described in claim 1 or 2, or pharmaceutically acceptable salts thereof, in the preparation of medicaments for the prevention and / or treatment of diseases related to acetylcholinesterase activity.

9. Use of the pharmaceutical composition of claim 7 in the preparation of a medicament for the prevention and / or treatment of diseases related to acetylcholinesterase activity.

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