An alkaloid compound with 14,21 oxygen bridge structure and a preparation method and application thereof

Abstract

The scheme discloses an alkaloid compound with 14, 21 oxygen bridge structures and a preparation method and application thereof in the field of traditional Chinese medicine extraction and separation. The ethyl acetate extraction part of ethanol extracts of toad skin is repeatedly separated and purified by combining various chromatography technologies; 1D and 2D nuclear magnetic and high resolution mass spectrometry are used to identify the structures of two new compounds, and the molecular formula and structure formula of the two new compounds are deduced. In vitro pharmacodynamic studies show that the two alkaloid compounds with 14, 21 oxygen bridge structures have good inhibition on colon cancer cells (HCT-116), and IC 50 The two alkaloid compounds with 14, 21 oxygen bridge structures have good inhibition on colon cancer cells (HCT-116), and IC 50 values are 0.92 μM and 10.91 μM respectively. The present application is helpful for the development and utilization of natural compounds from toad.

Description

Technical Field

[0001] This invention belongs to the field of extraction and separation of traditional Chinese medicine. Specifically, it relates to an alkaloid compound with a 14,21 oxygen bridge structure, its preparation method, and its application. Background Technology

[0002] Toad skin is a traditional Chinese medicine, and its medicinal value was first recorded in the "Mingyi Bielu" written by Tao Hongjing during the Liang Dynasty. According to the "Chinese Materia Medica Dictionary", toad skin is the dried skin of the Chinese giant toad (Bufo bufo gargarizans Cantor) or the black-rimmed toad (Bufomelanosticus Schneider) after removing its internal organs. It has the effects of clearing heat and detoxifying, and promoting diuresis and reducing swelling.

[0003] Modern pharmacological studies have shown that toad skin possesses anti-tumor and anti-hepatitis B virus pharmacological effects, and is mainly used clinically to treat hepatitis B, chronic bronchitis, sore throat, carbuncles, and boils. Chemical composition studies indicate that the main chemical components of toad skin include bufotalene, indolealkylamine alkaloids, nucleosides, and amino acids.

[0004] Currently, the variety of chemical components found in toad skin is limited, and its medicinal value has not been fully realized. Therefore, the development of new compounds from toad skin needs further investigation. In particular, the two alkaloid compounds with 14,21 oxygen bridge structures described in this application, along with their preparation methods and applications, have not yet been reported. Summary of the Invention

[0005] The purpose of this invention is to provide an alkaloid compound with a 14,21 oxygen bridge structure, its preparation method, and its application.

[0006] This scheme includes an alkaloid compound with a 14,21 oxygen bridge structure, with the molecular formula: C 24 H 33 NO5, chemical structural formula as shown in Bufogarlides D below, or molecular formula C 24 H 37 NO3, the chemical structural formula is shown in Bufogarlides E below:

[0007]

[0008] This application also provides a method for preparing the alkaloid compound having a 14,21 oxygen bridge structure, comprising the following steps:

[0009] S1. Take the skin of the Chinese toad, crush it, add 90% to 95% ethanol, heat and reflux to extract, combine the extracts after multiple extractions, concentrate to obtain an extract, suspend the extract in water and extract it with petroleum ether and ethyl acetate in sequence, concentrate to obtain the corresponding extract fraction.

[0010] S2. The ethyl acetate fraction obtained from S1 was initially separated by silica gel column chromatography. The petroleum ether-ethyl acetate gradient elution was used with a volume ratio of 1:0 to 0:1. After thin-layer chromatography identification, similar components were combined to obtain Fr.1 to Fr.16.

[0011] S3. Take the Fr.12 obtained from S2 and separate it by ODS column chromatography. Elute it with methanol-water gradient with a volume ratio of 1:1 to 1:0 to obtain Fr.12.1 to Fr.12.6.

[0012] Fr.12.3 was further subdivided using a preparative HPLC XB-C8 column to obtain Fr.12.3.1 to Fr.12.3.6;

[0013] Among them, Fr.12.3.2 was prepared by semi-precipitation HPLC Waters sunfire C 18 Column purification yielded the Bufogarlides D;

[0014] Fr.10 obtained from S2 was separated by Sephadex LH-20 column chromatography and eluted with methanol to obtain Fr.10.1 to Fr.10.10; Fr.10.4 was separated by silica gel column chromatography and eluted with a dichloromethane-methanol gradient at a volume ratio of 100:1 to 1:1 to obtain Fr.10.4.1 to Fr.10.4.6.

[0015] Among them, Fr.10.4.4 was separated by ODS column chromatography and eluted with a methanol-water gradient of volume ratio of 4:6 to 1:0 to obtain Bufogarlides E.

[0016] The preferred specifications of the HPLC XB-C8 column are 30×150mm, 5μm, 64% methanol / water, 8ml / min.

[0017] The preferred HPLC Waters sunfire C 18 The column specifications were 10×250mm, 5μm, 23% acetonitrile / water + 0.5% acetic acid, 2.5mL / min.

[0018] Furthermore, the concentration of ethanol in S1 is preferably 95%. The extraction is performed by reflux three times, each time for 2 hours, and the mass-to-volume ratio of toad skin to ethanol is preferably 1:1.

[0019] The beneficial effects of this invention are as follows: The ethyl acetate extract of toad skin ethanol extract is repeatedly separated and purified using a combination of various chromatographic techniques. The structures of the two new compounds are identified using 1D and 2D NMR and high-resolution mass spectrometry (HRESIMS), leading to the derivation of their molecular and structural formulas. In vitro pharmacodynamic studies have shown that the two alkaloid compounds with 14,21 oxygen bridge structures exhibit good inhibitory effects on colon cancer cells (HCT-116), with an IC50 concentration of [missing information]. 50 The values ​​were 0.92 μM and 10.91 μM, respectively.

[0020] Therefore, extracts containing alkaloids with a 14,21 oxygen bridge structure as described in this invention, or pharmaceutical compositions containing alkaloids with a 14,21 oxygen bridge structure as described in claim 1, or pharmaceutically acceptable salts thereof, all possess potential antitumor effects. Based on this invention, a new pharmaceutical compound with pharmacological activity can be obtained; the preparation method is simple, rapid, and easy to control. Attached Figure Description

[0021] Figure 1 This is a flowchart of the extraction and separation process.

[0022] Figure 2 This is a structural diagram of the compound.

[0023] Figure 3 (A) is the HMBC (→) and COSY correlation (──) of compound Bufogarlides D; (B) is the NOESY correlation of compound Bufogarlides D. Related.

[0024] Figure 4 (A) is the HMBC (→) and COSY correlation (──) of compound Bufogarlides E; (B) is the NOESY correlation of compound Bufogarlides E. Related. Detailed Implementation

[0025] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, equipment and methods used in the present invention are conventional reagents, equipment and methods in this technical field.

[0026] Example 1: Preparation and structural identification of the novel alkaloid compound with a 14,21 oxygen bridge structure according to the present invention.

[0027] 1. Extraction and Separation

[0028] Extraction and separation process combination Figure 1As shown, 20 kg of Chinese toad skin was pulverized and extracted by reflux with 95% ethanol (3 × 2 h × 20 L). The extracts were combined and concentrated under reduced pressure to obtain 1 kg of extract. The extract was then suspended in water and extracted sequentially with petroleum ether (3 × 10 L) and ethyl acetate (3 × 10 L). After concentration under reduced pressure, the corresponding extract fractions were obtained. 200 g of the ethyl acetate fraction was taken and preliminarily separated by silica gel column chromatography using a petroleum ether-ethyl acetate (V:V, 1:0 to 0:1) gradient elution. After identification by thin-layer chromatography, similar components were combined to obtain Fr.1 to Fr.16.

[0029] Fr.12 (26g) was partially separated by ODS column chromatography, followed by gradient elution with methanol-water (V:V, 1:1 to 1:0) to obtain Fr.12.1 to Fr.12.6; Fr.12.3 was further subdivided by preparative HPLC using an XB-C8 column (30×150mm, 5μm, 64% methanol / water, 8ml / min) to obtain Fr.12.3.1 to Fr.12.3.6; among which, Fr.12.3.2 was further subdivided by semi-preparative HPLC using Waters Sunfire C 18 Purification was performed using a column (10×250 mm, 5 μm, 23% acetonitrile / water + 0.5% acetic acid, 2.5 mL / min) to obtain Bufogarlides D (10 mg, t R =86min).

[0030] Fr.10 (14.8 g) was separated by Sephadex LH-20 column chromatography and eluted with methanol to obtain Fr.10.1 to Fr.10.10; Fr.10.4 was separated by silica gel column chromatography and eluted with a dichloromethane-methanol (V:V, 100:1) gradient to obtain Fr.10.4.1 to Fr.10.4.6; among which, Fr.10.4.4 was separated by ODS column chromatography and eluted with a methanol-water (V:V, 4:6 to 1:0) gradient to obtain Bufogarlides E (1.5 mg).

[0031] 2. Structural identification

[0032] This invention isolated and identified two new alkaloid compounds with 14,21 oxygen bridge structures, with the molecular formulas C14,21 and C21, respectively. 24 H 33 NO5 (named Bufogarlides D) and C 24 H 37 NO3 (named Bufogarlides E), see Figure 2 .

[0033] 2.1 Structural identification of the new compound Bufogarlides D

[0034]

[0035] Bufogarlides D: White amorphous powder. ESIMS gives a quasi-molecular ion peak at m / z 438 [M+Na]. + And 853[2M+Na] + HRESIMS m / z 438.2254 [M+Na] + (calcd.for C 24 H 33 NO5Na 438.2251), indicating that its molecular composition is C 24 H 33 NO5. 1 H NMR and 13 C10 NMR spectroscopy revealed the presence of a six-membered lactam fragment in the structure [δ]. H 1.63(m,H-20),4.64(d,9.0,H-21),1.64(m,H-22a),1.58(m,H-22b),2.44(m,H2-23); δ C 38.8(C-20), 82.0(C-21), 22.2(C-22), 32.2(C-23), 174.8(C-24)]; two angular methyl groups [δ H 1.18(s,H-18),δ C 14.1 and δ H 1.29(s,H-19),δ C 27.5]; a hydroxymethyl group [δ] H 3.97(m,H-3),δ C 67.3]; a quaternary carbon atom [δ] C 86.7 (C-14)]; a carbonyl quaternary carbon [δ C 200.0(C-12)]; two unsaturated double-bonded quaternary carbons [δ C[133.2 (C-9) and 142.1 (C-11)]. Based on the above information, combined with the previous identification of compounds isolated from toad skin, it is preliminarily inferred that this compound is a bufos sterene analogue. In the HMBC spectrum, correlation signals can be observed between H-21 and C-17, C-22, and C-24, indicating that the hexa-lactam fragment is located at C-17; at the same time, the HMBC spectrum also shows correlation signals between H-21 and C-14, H-18 and C-12, and C-14 and C-17, indicating that C-14 and C-21 are connected by an oxygen bridge; the HMBC spectrum also shows correlations between H-8 and C-11, and H-19 and C-10, indicating that an unsaturated double bond is formed between C-11 and C-12; in addition, H-2 and H-5 are connected to C-3, indicating that one hydroxyl group is substituted at the C-3 position; to meet the requirements of molecular weight and two quaternary carbon double bonds, the other hydroxyl group can only be substituted at the C-11 position. In summary, the planar structure of this compound is as follows: Figure 3 As shown in the NOESY spectrum, correlation signals can be observed between H-4a and H-3 and H-6a, and between H-6a and H-17, indicating that the above protons are all in the α configuration; at the same time, correlation signals can be observed between H-5 and H-19 and H-18, and between H-18 and H-21, indicating that the above protons are in the β configuration. Based on the above spectral data, the compound is named Bufogarlides D.

[0036] 2.2 Structural identification of the new compound Bufogarlides E

[0037]

[0038] Bufogarlides E: White amorphous powder. ESIMS gives a quasi-molecular ion peak at m / z 388 [M+H]. + HRESIMS m / z 410.2663[M+Na] + (calcd.for C 24 H 37 NO3Na 410.2666), indicating that its molecular composition is C 24 H 37 NO3. 1 HNMR and 13 C10 NMR spectroscopy revealed the presence of a six-membered lactam fragment, two angular methyl groups, one hydroxymethyl group, and one hydroxyquaternary carbon group in the structure, suggesting it is an analogue of Bufogarlides D. HMBC spectroscopy confirmed the planar structure (see [link to HMBC spectrum]). Figure 4In the NOESY spectrum, correlation signals were observed between H-2a and H-3 and H-9, and between H-17 and H-9 and H-21, indicating that the above protons are all in the α configuration; at the same time, correlation signals were observed between H-19 and H-18, indicating that the above protons are in the β configuration. Based on the above spectral data, the compound was named Bufogarlides E.

[0039] Table 1. NMR data for Bufogarlides D and Bufogarlides E

[0040]

[0041]

[0042] Example 2: In vitro anti-colon cancer activity study of compounds Bufogarlides D and Bufogarlides E

[0043] 1. Cell viability assay

[0044] Using 5-fluorouracil (5-Fu) as a positive control, HCT-116 colon cancer cells in logarithmic growth phase were harvested at a concentration of 8 × 10⁻⁶ cells / year. 3 The cells were seeded into 96-well plates and cultured at 37°C with 5% CO2. After 24 hours of incubation, the plates were removed and 200 μL of culture medium containing Bufogarlides D and Bufogarlides E were added to each well, with three replicates per group. After incubation, pre-chilled 10% TCA was added, and the plates were fixed at 4°C for 2 hours. The TCA fixative was discarded, and the plates were washed five times with deionized water to remove excess liquid and air-dried at room temperature. After drying, 100 μL of 0.4% SRB dye was added to each well and incubated at room temperature for 20 minutes. Unbound SRB dye was then washed five times with 0.1% acetic acid solution, and the plates were air-dried at room temperature. 100 μL of 10 mM Tris buffer was added to each well to dissolve the dye completely. The IC50 was measured using an ELISA reader, and the IC50 was calculated. 50 The results are shown in Table 2.

[0045] Table 2. Cytotoxic activity of Bufogarlides D and Bufogarlides E against HCT-116 colon cancer cells.

[0046]

[0047] Note: Compared with the positive control, # P<0.05

[0048] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. An alkaloid compound having a 14,21 oxygen bridge structure, characterized in that, The molecular formula is: C 24 H 33 NO5, chemical structural formula as shown in Bufogarlides D below, or molecular formula C 24 H 37 NO3, the chemical structural formula is shown in BufogarlidesE below: 。 2. The method for preparing an alkaloid compound having a 14,21 oxygen bridge structure as described in claim 1, characterized in that, Includes the following steps: S1. Take the skin of the Chinese toad, crush it, add 90%~95% ethanol, heat and reflux to extract, combine the extracts after multiple extractions, concentrate to obtain an extract, suspend the extract in water and extract it with petroleum ether and ethyl acetate in sequence, concentrate to obtain the corresponding extract fraction. S2. The ethyl acetate fraction obtained from S1 was initially separated by silica gel column chromatography. A gradient elution of petroleum ether-ethyl acetate with a volume ratio of 1:0 to 0:1 was used. After thin-layer chromatography identification, similar components were combined to obtain Fr.1 to Fr.

16. S3. Take the Fr.12 obtained from S2 and separate it by ODS column chromatography. Elute it with methanol-water gradient with a volume ratio of 1:1 to 1:0 to obtain Fr.12.1 to Fr.12.

6. Fr.12.3 was further subdivided using a preparative HPLC XB-C8 column to obtain Fr.12.3.1~Fr.12.3.6; Among them, Fr.12.3.2 was prepared by semi-precipitation HPLC Waters sunfire C 18 Column purification yielded the Bufogarlides D; Fr.10 obtained from S2 was separated by Sephadex LH-20 column chromatography and eluted with methanol to obtain Fr.10.1~Fr.10.10; Fr.10.4 was separated by silica gel column chromatography and eluted with a dichloromethane-methanol gradient at a volume ratio of 100:1~1:1 to obtain Fr.10.4.1~Fr.10.4.

6. Among them, Fr.10.4.4 was separated by ODS column chromatography and eluted with a methanol-water gradient of volume ratio of 4:6 to 1:0 to obtain Bufogarlides E.

3. The preparation method according to claim 2, characterized in that: The HPLC XB-C8 column has specifications of 30×150mm, 5 μm, and a methanol aqueous solution with a concentration of 64% at a flow rate of 8 ml / min.

4. The preparation method according to claim 2, characterized in that: The HPLC Waters sunfire C 18 The column specifications were 10×250 mm, 5 μm, 23% acetonitrile / water + 0.5% acetic acid, 2.5 mL / min.

5. The preparation method according to claim 2, characterized in that: The concentration of ethanol in S1 is preferably 95%.

6. A pharmaceutical composition, characterized in that: The pharmaceutical composition contains an alkaloid compound having a 14,21 oxygen bridge structure as described in claim 1, or a pharmaceutically acceptable salt thereof.

7. The use of the alkaloid compound having a 14,21 oxygen bridge structure as described in claim 1, or a pharmaceutically acceptable salt thereof, in the preparation of an anti-colon cancer drug.

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