Daphne-type diterpenoid cleaved daphne diterpenoid and its preparation method and application

Abstract

The present invention relates to the technical field of preparation of daphne-type compounds, and specifically relates to a daphne-type diterpene schizodaphnein, a preparation method thereof, and applications thereof. The present invention discloses for the first time a daphne-type diterpene schizodaphnein compound. The daphne-type diterpene schizodaphnein compound described in the present invention has a certain inhibitory effect on human HGC-27 gastric cancer cells, thereby making it a potential anti-tumor drug.

Description

Technical Field

[0001] The invention relates to the technical field of preparation of daphne-type compounds, in particular to daphne-type diterpenoid cleaved daphne diterpenoid and a preparation method and application thereof. Background Art

[0002] Stelleropsistianschanica is a plant of the genus Stelleropsis in the family Thymelaeaceae. It is 15 to 30 cm tall, with a head inflorescence or a very short spike inflorescence. The rhizome is woody and yellowish-brown or light brown. It grows on hillside grasslands at an altitude of 1,700 to 2,000 meters and is only distributed in Zhaosu County, Xinjiang Uygur Autonomous Region, my country and Kyrgyzstan abroad. Due to the unique geographical environment and growth area, people have done little research on the effective ingredients of Stelleropsistina chamaejasme. The main active ingredients are daphne-type diterpenes and guaiacane-type sesquiterpenes, which have multiple effects such as anti-tumor and anti-inflammatory. Clinically, it is mainly used for bronchitis, cough, asthma, tuberculosis and other diseases.

[0003] Malignant tumors pose a serious threat to human life and health. Due to the high cost of many new anticancer drugs and the high risk of drug resistance and side effects with long-term use, the search for highly effective and low-toxic anticancer drugs is crucial for cancer treatment. Gastric cancer is one of the most common malignant tumors in my country, with the highest incidence rate among all types of cancer. Approximately 170,000 people die from gastric cancer annually, accounting for nearly a quarter of all deaths from malignant tumors. Furthermore, over 20,000 new cases are diagnosed annually, making it a serious threat to public health. Gastric cancer can occur at any age and in any part of the stomach, but is most common in the antrum, particularly the lesser curvature. Gastric cancer is categorized into early-stage and advanced (intermediate and late-stage) stages based on the depth of cancer invasion. Early symptoms of gastric cancer are often subtle, such as vague upper abdominal discomfort, dull pain, belching, heartburn, loss of appetite, and mild anemia, some of which can mimic symptoms of gastric or duodenal ulcers or chronic gastritis. Some patients experience pain relief or relief with painkillers, anti-ulcer drugs, or dietary adjustments, often leading to their condition being overlooked and not further investigated. As the disease progresses, gastric symptoms become more pronounced, including upper abdominal pain, loss of appetite, emaciation, weight loss, and anemia. In later stages, the cancer often metastasizes, leading to abdominal masses, left supraclavicular lymphadenopathy, melena, ascites, and severe malnutrition. Because gastric cancer is extremely common and carries a high risk of disease in my country, research suggests that its causes are related to dietary habits and gastric disorders. Therefore, the effectiveness of gastric cancer treatment depends on early diagnosis and treatment.

[0004] The 5 / 7 / 6-tricyclic diterpenoids in Stellera jasminoides possess potent antitumor activity, are inexpensive, and widely available, attracting the interest of numerous researchers. The efficacy of Stellera jasminoides stems primarily from its terpenoids. Therefore, developing and utilizing the daphne-type diterpenoid monomers from Stellera jasminoides, further exploring their potential medicinal value, and determining and characterizing their structures and physicochemical properties, is crucial for the treatment of gastric cancer with Stellera jasminoides. Summary of the Invention

[0005] The present invention provides a daphne-type diterpene secoepin and a preparation method thereof, which overcomes the shortcomings of the above-mentioned prior art. The present invention discloses for the first time a daphne-type diterpene secoepin, which can be used as a drug for preventing and treating gastric cancer and a drug for inhibiting and / or killing gastric cancer HGC-27 cells.

[0006] One of the technical solutions of the present invention is achieved by the following measures: a daphne-type diterpene cleaved daphne diterpenoid, whose chemical structure is

[0007] .

[0008] The following is a further optimization and / or improvement of one of the above-mentioned technical solutions:

[0009] The above-mentioned daphne-type diterpenoid cleavage daphne diterpenoids are obtained according to the following method: the first step is to crush the root of Stellera chamaejasme and add methanol, soak it at room temperature for 3 to 4 hours, and then heat it under reflux extraction at 50°C to 60°C for 3 times, each time for 1 to 3 hours, combine the reflux extracts of each time, and reduce the pressure to recover and concentrate to obtain Stellera chamaejasme extract; the second step is to disperse the Stellera chamaejasme extract into a suspension with water, extract it with dichloromethane and ethyl acetate in sequence, concentrate the extract, and obtain dichloromethane extract, acetic acid extract, and the like in sequence. The extract of the ethyl ester part; the third step, taking the extract of the dichloromethane part and separating it by gradient elution on a silica gel column chromatography to obtain 8 fractions, wherein the silica gel column chromatography gradient eluent includes dichloromethane and methanol, and the volume ratios of dichloromethane and methanol are 1:0, 100:1, 30:1, 20:1, 10:1, 5:1, and 2:1 respectively; the fourth step, purifying and separating the fourth fraction of the obtained 8 fractions by gradient elution on a high performance liquid chromatography, and collecting the eluate to obtain the daphne-type diterpenoid cleaved daphne diterpenoid at 32.8 minutes.

[0010] In the first step, 8 ml to 12 ml of methanol was added to every 1 g of Stellera chamaejasme.

[0011] In the fourth step, the HPLC eluent is a mixture of methanol and water, wherein the volume ratio of methanol to water is 65:35.

[0012] The second technical solution of the present invention is achieved by the following measures: a preparation method of a daphne-type diterpene cleavage daphne diterpenoid, which is carried out according to the following method: the first step is to crush the root of Stellera chamaejasme and add methanol, soak it at room temperature for 3 to 4 hours, and then heat it under reflux extraction at 50°C to 60°C for 3 times, each time for 1 to 3 hours, combine the reflux extracts of each time and reduce the pressure to recover and concentrate to obtain Stellera chamaejasme extract; the second step is to disperse the Stellera chamaejasme extract into a suspension with water, extract it with dichloromethane and ethyl acetate in sequence, concentrate the extract, and The extract of the dichloromethane part and the extract of the ethyl acetate part are obtained in the third step; the extract of the dichloromethane part is separated by gradient elution on a silica gel column chromatography to obtain 8 fractions, wherein the gradient eluent of the silica gel column chromatography includes dichloromethane and methanol, and the volume ratios of dichloromethane and methanol are 1:0, 100:1, 30:1, 20:1, 10:1, 5:1, and 2:1, respectively; the fourth step is to purify and separate the fourth fraction of the obtained 8 fractions by gradient elution on a high performance liquid chromatography, and the eluate is collected to obtain the daphne-type diterpenoid cleaved daphne diterpenoid at 32.8 minutes.

[0013] The following is a further optimization and / or improvement of the second technical solution of the above invention:

[0014] In the first step, 8 ml to 12 ml of methanol was added to every 1 g of Stellera chamaejasme.

[0015] In the fourth step, the HPLC eluent is a mixture of methanol and water, wherein the volume ratio of methanol to water is 65:35.

[0016] The third technical solution of the present invention is achieved through the following measures: application of a daphne-type diterpene cleaved daphne diterpenoid in the preparation of a drug for preventing gastric cancer.

[0017] The fourth technical solution of the present invention is achieved through the following measures: application of a daphne-type diterpene cleaved daphne diterpenoid in the preparation of a drug for treating gastric cancer.

[0018] The fifth technical solution of the present invention is achieved through the following measures: use of a daphne-type diterpene cleocyclic daphne diterpenoid in the preparation of a drug for inhibiting or / and killing gastric cancer HGC-27 cells.

[0019] The present invention discloses for the first time a compound daphne-type diterpene secoecylindole. The compound daphne-type diterpene secoecylindole has a certain inhibitory effect on human HGC-27 gastric cancer cells, thereby being used as a potential anti-tumor drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Attachment Figure 1 The present invention is the daphne type diterpene cleavage daphne diterpenoid 1H-NMR spectrum.

[0021] Attachment Figure 2 The present invention is the daphne type diterpene cleavage daphne diterpenoid 13 C-APT spectrum. DETAILED DESCRIPTION

[0022] The present invention is not limited to the following embodiments, and specific implementation methods can be determined based on the technical solutions of the present invention and actual conditions. Unless otherwise specified, the various chemical reagents and chemicals mentioned in the present invention are all chemical reagents and chemicals known and used in the prior art.

[0023] The present invention will be further described below in conjunction with the embodiments:

[0024] Example 1: The daphne-type cleaved daphne diterpenoid is characterized by its chemical structural formula being

[0025] .

[0026] Example 2: As an optimization of the above example, the daphne-type diterpene cleavage daphne diterpenoids are obtained according to the following method: the first step is to crush the root of Stellera chamaejasme and add methanol, soak it at room temperature for 3 to 4 hours, and then heat it under reflux extraction at 50°C to 60°C for 3 times, each time for 1 to 3 hours, combine the reflux extracts, reduce pressure, recover and concentrate to obtain Stellera chamaejasme extract; the second step is to disperse the Stellera chamaejasme extract into a suspension with water, extract it with dichloromethane and ethyl acetate in sequence, concentrate the extracts to obtain dichloromethane and ethyl acetate in sequence. Part extract, ethyl acetate part extract; the third step, taking the dichloromethane part extract and separating it by silica gel column chromatography gradient elution to obtain 8 fractions, wherein the silica gel column chromatography gradient eluent includes dichloromethane and methanol, and the volume ratios of dichloromethane and methanol are 1:0, 100:1, 30:1, 20:1, 10:1, 5:1, and 2:1 respectively; the fourth step, purifying and separating the fourth fraction of the obtained 8 fractions by high performance liquid chromatography gradient elution, and collecting the eluate to obtain the daphne-type diterpenoid cleaved daphne diterpenoid at 32.8 minutes.

[0027] Example 3: As an optimization of the above example, in the first step, 8 ml to 12 ml of methanol was added to every 1 g of Stellera chamaejasme.

[0028] Example 4: As an optimization of the above example, in the fourth step, the HPLC eluent is a mixture of methanol and water, wherein the volume ratio of methanol to water is 65:35.

[0029] Example 5: The preparation method of the daphne-type cleaved daphne diterpenoids is carried out as follows: the first step is to crush the root of Stellera chamaejasme and add methanol, soak it at room temperature for 3 to 4 hours, and then heat and reflux it at 50°C to 60°C for 3 times, each time for 1 to 3 hours, combine the reflux extracts, recover them under reduced pressure, and concentrate them to obtain Stellera chamaejasme extract; the second step is to disperse the Stellera chamaejasme extract into a suspension with water, extract it with dichloromethane and ethyl acetate in sequence, concentrate the extracts, and obtain dichloromethane extracts in sequence. ointment, and ethyl acetate extract; the third step, taking the dichloromethane extract and separating it by silica gel column chromatography gradient elution to obtain 8 fractions, wherein the silica gel column chromatography gradient eluent includes dichloromethane and methanol, and the volume ratios of dichloromethane and methanol are 1:0, 100:1, 30:1, 20:1, 10:1, 5:1, and 2:1 respectively; the fourth step, purifying and separating the fourth fraction of the obtained 8 fractions by high performance liquid chromatography gradient elution, and collecting the eluate to obtain the daphne-type diterpenoid cleaved daphne diterpenoid at 32.8 minutes.

[0030] Example 6: Use of the daphne-type diterpenoid cleoside in the preparation of drugs for preventing gastric cancer.

[0031] Example 7: Use of the daphne-type diterpenoid secoecymidone in the preparation of drugs for treating gastric cancer.

[0032] Example 8: Use of the daphne-type diterpenoid secoecymidone in the preparation of drugs for inhibiting or / and killing gastric cancer HGC-27 cells.

[0033] The daphne-type diterpenoid cleaved daphne diterpenoid of the present invention is subjected to an in vitro anti-tumor pharmacodynamics experiment, and the in vitro anti-tumor pharmacodynamics experiment utilizes the MTT colorimetric method.

[0034] The experimental group was composed of daphne-type diterpenoid cleaved daphne diterpenoids, while the control group was composed of cisplatin. A blank group was also established. HGC-27 (human gastric cancer cell) cells were used as experimental subjects for the experimental, control, and blank groups. After dilution of the culture medium, the cells were seeded in a 96-well plate at a density of 4×105, with 100 μL per well. After normal culture in an incubator for 24 hours, the corresponding drugs were added to each group to make the final concentrations of the drugs in each group 12.5 μg / mL (group 1), 25 μg / mL (group 2), 50 μg / mL (group 3), 100 μg / mL (group 4), and 200 μg / mL (group 5), respectively. A total of 5 concentrations were set, with 3 replicates for each concentration. After culture for 48 hours, 10 μL of MTT was added to each well for staining. After further culture for four hours, the original culture medium was aspirated and 150 μL of DMSO was added to each well. The plates were shaken at low speed for 10 min. The crystals were fully dissolved for 1 minute, and the optical density was measured at 570 nm on an enzyme-linked immunosorbent assay (ELISA) instrument. The 50% inhibitory concentration (IC50, μg / mL) was calculated based on the optical density. The IC50 calculation method for the optical density value is known in the art. The IC50 values for the experimental and control groups on HGC-27 cells are shown in Table 3. As can be seen from the data in Table 3, the daphne-type diterpenoid schizodaphne of the present invention has a certain inhibitory effect on HGC-27 cells.

[0035] In summary, the present invention discloses for the first time the compound daphne-type diterpenoid secoebiformis. The compound daphne-type diterpenoid secoebiformis described in the present invention has a certain inhibitory effect on human HGC-27 gastric cancer cells, thereby enabling the application of this type of compound in Stellera chamaejasme as a potential anti-tumor drug.

[0036] The above technical features constitute the embodiments of the present invention, which have strong adaptability and implementation effect. Non-essential technical features can be added or removed according to actual needs to meet the requirements of different situations.

[0037]

Claims

1. A method for preparing daphne-type cleaved daphne diterpenoids, characterized in that The method is as follows: first, crushing the root of Stellera chamaejasme and adding methanol, soaking it at room temperature for 3 to 4 hours, heating and refluxing it at 50 to 60°C for 3 times, each time for 1 to 3 hours, combining the reflux extracts of each time, recovering them under reduced pressure, and concentrating them to obtain Stellera chamaejasme extract; second, dispersing the Stellera chamaejasme extract into a suspension with water, extracting it with dichloromethane and ethyl acetate in turn, concentrating the extracts to obtain dichloromethane extract and ethyl acetate extract in turn; third, taking the dichloromethane extract and purifying it; The extract is separated by gradient elution on a silica gel column chromatography to obtain fractions, wherein the silica gel column chromatography gradient eluent includes dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 1:0, 100:1, 30:1, 20:1, 10:1, 5:1, and 2:1, respectively; in the fourth step, the fourth fraction of the obtained fractions is purified and separated by gradient elution on a high performance liquid chromatography, and the eluate is collected to obtain daphne-type diterpenoid cleavage daphne diterpenoid at 32.8 minutes. The chemical structure of the daphne-type diterpenoid cleavage daphne diterpenoid is 。 2. The method for preparing the diterpenoids of claim 1, wherein In the first step, 8 ml to 12 ml of methanol was added to every 1 g of Stellera chamaejasme.

3. The method for preparing the diterpenoids of claim 1 or 2, wherein In the fourth step, the HPLC eluent is a mixture of methanol and water, wherein the volume ratio of methanol to water is 65:35.

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