Enantiobehenane diterpenoid compound as well as preparation method and application of enantiobehenane diterpenoid compound

Abstract

The invention discloses an enantiobeyanane diterpenoid compound as well as a preparation method and application thereof, and relates to the technical field of natural medicines. The structure of the enantiobetiane diterpenoid compound is shown as a formula I. The preparation method of the enantiobetiane diterpenoid compound comprises the following steps: by taking a traditional Chinese medicine Aleuritopis argentea (Gmel.) Fe as a raw material, carrying out percolation extraction and concentration to obtain an extract, suspending the extract in water to obtain a suspension, and carrying out extraction and concentration to obtain an extract; and taking the extract, and carrying out normal and reverse phase silica gel column chromatography, Toyopearl HW-40C gel column chromatography, Si-H silica gel column chromatography separation and ODS semi-preparative high performance liquid chromatography purification on the extract to obtain the compound shown in the formula I. According to the invention, the enantiobeyeane diterpenoid compound capable of inhibiting cancer cells is extracted from the traditional Chinese medicine aleuritopteris argentea for the first time, the compound has great development value as a novel anti-cancer drug, and the design thought of the compound also provides a new thought and approach for the development of the novel anti-cancer drug.

Description

Technical Field

[0001] The present invention relates to the technical field of natural medicines, and in particular to an enantiobayerene-type diterpenoid compound, a preparation method thereof, and an application thereof. Background Art

[0002] Cancer is one of the most serious diseases threatening human health. Its mortality rate is second only to cardiovascular and cerebrovascular diseases, and its incidence is increasing year by year. Although chemotherapy, the mainstay of tumor treatment, is highly effective, it often causes severe side effects, making it difficult for patients to adhere to treatment. Furthermore, most cancers lack effective targeted therapies, are insensitive to endocrine therapy and conventional chemotherapy, are highly invasive, and have a poor prognosis, making their clinical treatment a very challenging problem. Therefore, it is necessary to seek treatments and drugs with good efficacy and minimal toxic side effects. In recent years, Traditional Chinese Medicine (TCM) has provided an important source and new opportunities for modern drug development. Active ingredients from natural products, with their multi-target, high efficiency, and low toxicity, have become a key source for new drug research and development.

[0003] The Chinese medicine Tongjingcao is the dried whole herb of the Chinese plant Aleuritopteris argentea (Gmel.) Fee, which belongs to the genus Aleuritopteris of the family Pteridaceae. It has the effects of promoting blood circulation and regulating menstruation, relieving cough, promoting diuresis, detoxifying and reducing swelling. It is mainly used to treat irregular menstruation, amenorrhea and abdominal pain, leucorrhea with red and white discharge, hemoptysis caused by tuberculosis, diarrhea, painful and difficult urination, lung abscess, mastitis, rheumatic joint pain, traumatic injury, intercostal neuralgia, sudden onset of conjunctivitis, and sores. Modern research shows that the chemical components of Tongjingcao mainly include two major categories: terpenes and steroids. Among them, terpenes are mainly rich and diverse diterpenes, which are also considered to be the main material basis for Tongjingcao to exert its many pharmacological activities. As for the new enantiomeric beyane-type diterpenoid compound and its activity involved in the present invention, there has been no patent or literature report so far. Summary of the Invention

[0004] The present invention provides an enantiobayane-type diterpenoid compound and a preparation method thereof. The compound has a good inhibitory effect on four types of cancer cells: A549, HCT116, SW1990 and MHC97H.

[0005] To achieve the above object, the technical solution of the present invention is as follows:

[0006] An enantiobayerene-type diterpenoid compound, wherein the enantiobayerene-type diterpenoid compound has a structure shown in the following formula I:

[0007]

[0008] The present invention also aims to provide a method for preparing the enantiomeric beylane-type diterpenoid compound represented by the above formula I, comprising the following steps:

[0009] S1. Using the Chinese herbal medicine Tongjingcao as raw material, extracting by diafiltration and concentrating to obtain an extract, suspending the extract in water to obtain a suspension, extracting the suspension with ethyl acetate, and concentrating to obtain an ethyl acetate extract;

[0010] S2. Using a first eluent, subjecting the obtained ethyl acetate extract to gradient elution via silica gel column chromatography to obtain a first eluate, wherein the first eluent is petroleum ether-ethyl acetate-methanol;

[0011] S3, using a second eluent to gradiently elute the obtained first eluate through silica gel column chromatography to obtain a second eluate, wherein the second eluent is dichloromethane-methanol;

[0012] S4, using a third eluent, subjecting the obtained second eluate to gradient elution through ODS reverse column chromatography to obtain a third eluate, wherein the third eluent is methanol-water;

[0013] S5. Using a fourth eluent, isocratically eluting the obtained third eluate through a Toyopearl HW-40 gel column chromatography to obtain a fourth eluate, wherein the fourth eluent is dichloromethane-methanol;

[0014] S6. Isocratically eluting the fourth eluate through Si-H silica gel column chromatography to obtain Fr.1 to Fr.8. In this case, the mobile phase is dichloromethane-methanol.

[0015] S7. The eluate Fr.4 obtained in step S6 is subjected to semi-preparative ODS high performance liquid chromatography using a fixed ratio mobile phase to obtain the compound represented by formula I. At this time, the mobile phase is methanol-water.

[0016] Furthermore, in step S1, the diafiltration extraction step is specifically as follows: extracting the Chinese medicinal herb Tongjingcao by diafiltration with ethanol at room temperature, obtaining an extract-like ethanol extract after concentration, and suspending the ethanol extract in water to obtain a suspension; the ethanol concentration is 95%, the diafiltration extraction time is 120 hours, and the ethanol diafiltration flow rate is 0.5 L / h;

[0017] The extraction step comprises: adding ethyl acetate in a volume equal to that of the suspension, extracting and concentrating the suspension to obtain the ethyl acetate extract.

[0018] Furthermore, in the first eluent, the volume ratio of petroleum ether, ethyl acetate and methanol is 8:1:0 to 0:0:1; preferably, the volume ratio of petroleum ether, ethyl acetate and methanol is 8:1:0, 7:1:0, 4:1:0, 3:1:0, 2:1:0, 1:1:0, 1:2:0, 1:3:0, 1:4:0, 0:1:0, 0:3:1, 0:0:1.

[0019] Further, in step S3, the first eluate is the eluate when the volume ratio of petroleum ether, ethyl acetate and methanol in the first eluent is 4:1:0;

[0020] In the second eluent, the volume ratio of dichloromethane to methanol is 100:0 to 0:100; preferably, the volume ratio of dichloromethane to methanol is 100:0, 98:2, 97:3, 96:4, 95:5, or 0:100.

[0021] Further, in step S4, the second eluate is the eluate when the volume ratio of dichloromethane to methanol in the second eluent is 98:2;

[0022] In the third eluent, the volume ratio of methanol to water is 40:60 to 100:0; preferably, the volume ratio of methanol to water is 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, or 100:0.

[0023] Furthermore, in step S5,

[0024] The third eluate is an eluate in which the volume ratio of methanol to water in the third eluent is 90:10;

[0025] In the fourth eluent, the volume ratio of dichloromethane to methanol is 2:1;

[0026] During isocratic elution, the elution rate was 1 mL / min, the elution time was 10 h, and the eluate was collected every 2 h to obtain 5 portions of the fourth eluate.

[0027] Furthermore, in step S6,

[0028] The fourth eluate is the eluate collected in the third time period in step S5;

[0029] The volume ratio of dichloromethane and methanol in the mobile phase was 60:1;

[0030] During isocratic elution, the elution time is 8 hours, and the eluate is collected every 1 hour, and Fr.1 to Fr.8 are collected in sections;

[0031] And / or in step S7, the volume ratio of methanol to water is 82:18.

[0032] The present invention also aims to provide the use of the enantiobayane-type diterpenoid compound represented by formula I in the preparation of anticancer drugs.

[0033] Furthermore, the enantiobayane-type diterpenoid compound is used as a raw material to prepare an anticancer drug composition or any pharmaceutically acceptable salt thereof.

[0034] Furthermore, the pharmaceutical composition or any pharmaceutically acceptable salt is in the form of tablets, capsules, granules, oral liquids, granules, pills or pellets.

[0035] Furthermore, the cancer includes at least one of lung cancer, colon cancer, pancreatic cancer and liver cancer.

[0036] The beneficial effects of the present invention include at least:

[0037] This study, published in Nature Communications, is the first to extract enantio-bayane-type diterpenoids from Herba Dysmenorrhoeae, which exhibit anticancer properties. These compounds demonstrate potent inhibitory effects against four cancer cell lines: A549, HCT116, SW1990, and MHC97H. These compounds hold great promise as novel anticancer drugs, and their design offers new insights and avenues for their development. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The enantiomeric beryllane-type diterpenoid compound of the present invention 1 H-NMR spectrum.

[0039] Figure 2 The enantiomeric beryllane-type diterpenoid compound of the present invention 13 C-NMR spectrum.

[0040] Figure 3 This is a bar graph showing the inhibitory effect of the enantiobayane-type diterpenoid compounds of the present invention on A549 cells.

[0041] Figure 4 This is a bar graph showing the inhibitory effect of the enantiobayane-type diterpenoid compounds of the present invention on HCT116 cells.

[0042] Figure 5 This is a bar graph showing the inhibitory effect of the enantiobayane-type diterpenoid compounds of the present invention on SW1990 cells.

[0043] Figure 6 This is a bar graph showing the inhibitory effect of the enantiobayane-type diterpenoid compounds of the present invention on MHC97H cells. DETAILED DESCRIPTION

[0044] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0045] In addition, the technical solutions between the various embodiments of the present invention can be combined with each other, but it must be based on the fact that ordinary technicians in this field can implement it. When the combination of technical solutions is mutually contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0046] The solution proposed by the present invention is described in detail below through specific embodiments:

[0047] Example 1 Preparation of Enantiobayane-Type Diterpenoids

[0048] The method uses 1 kg of dried Chinese medicinal material of Tongjingcao as raw material, extracts it with 95% ethanol by percolation for 120 hours at a flow rate of 0.5 L / h, and concentrates it by rotary evaporation to obtain an ethanol extract in the form of an extract. The ethanol extract is suspended in water to obtain a suspension, and ethyl acetate is added with an equal volume of the suspension for extraction. The extract solution is concentrated to obtain an ethyl acetate extract. The ethyl acetate extract is subjected to silica gel column chromatography and gradient elution using a petroleum ether-ethyl acetate-methanol solution with a volume ratio of 8:1:0 to 0:0:1. The eluent includes petroleum ether-ethyl acetate-methanol with a volume ratio of 8:1:0, 7:1:0, 4:1:0, 3:1:0, 2:1:0, 1:1:0, 1:2:0, 1:3:0, 1:4:0, 0:1:0, 0:3:1, and 0:0:1. Twelve eluates are collected in stages after gradient elution.

[0049] The eluate eluted with an eluent having a volume ratio of petroleum ether-ethyl acetate-methanol of 4:1:0 was subjected to silica gel column chromatography and gradient elution was performed with a dichloromethane-methanol solution having a volume ratio of 100:0 to 0:100. The eluents included eluents having a volume ratio of dichloromethane-methanol of 100:0, 98:2, 97:3, 96:4, 95:5, and 0:100. Six eluates were collected segmentally after gradient elution with the eluent.

[0050] The eluate eluted with the dichloromethane-methanol volume ratio of 98:2 was subjected to ODS reverse silica gel column chromatography and gradient eluted with a methanol-water solvent with a volume ratio of 40:60 to 100:0. The eluents included eluents with methanol-water volume ratios of 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, and 100:0. Seven eluates were collected segmentally after gradient elution with the eluent.

[0051] The eluate, eluted with a methanol-water ratio of 90:10 by volume, was subjected to isocratic elution using a Toyopearl HW-40C gel column with a dichloromethane-methanol ratio of 2:1 by volume. The eluate was eluted at a rate of 1 mL / min for 10 hours, with fractions collected every 2 hours to yield five fractions.

[0052] The eluate eluted with the eluent in the third time period (collected for 4-6 hours) was subjected to isocratic elution by Si-H silica gel column chromatography with a dichloromethane-methanol solvent with a volume ratio of 60:1. Specifically, the elution time was 8 hours, and the eluate was collected every 1 hour. Fr.1 to Fr.8 were collected in segments after elution with the eluent.

[0053] Fr.4 was subjected to semi-preparative ODS liquid chromatography using a methanol-water solution with a volume ratio of 82:18 as the mobile phase to obtain the compound represented by the following formula I:

[0054]

[0055] Example 2 Structural Identification of Enantiobayane-Type Diterpenoids

[0056] The compound extracted from Example 1 was tested by infrared, nuclear magnetic resonance, mass spectrometry, optical rotation, infrared spectroscopy, ultraviolet spectroscopy, and X-ray single crystal diffraction. The results are as follows:

[0057] Physical and chemical data: colorless needle-shaped crystals; [α]25D: +19.0 (methanol, c 0.1); UV (methanol) λ max (logε): 200(4.26)nm; IR (potassium bromide)ν max :3421,2939,2846,1660,1461,1386,1033cm –1 ;(+)-HRESIMS m / z327.2286[M+Na] + (C 20 H 32 O2Na + Calcd for m / z 327.2300).

[0058] X-ray single crystal diffraction data of compound I: (C 20 H 32 O2)3·H2O,M=931.38,monoclinic,spacegroup P21(no.4), α=90°, β=112.117(2)°, γ=90°, Z=2, T=100.00(10)K, μ(Cu Kα)=0.579mm -1 ,Dcalc=1.181g / cm 3 ,40185reflections measured(4.332°≤2Θ≤148.672°),10204unique(R int =0.0543,R sigma=0.0488)which were used in all calculations.Thefinal R1 was 0.0445(I>2σ(I))and wR2 was 0.1107(all data).The goodness of fiton F 2 was 1.028.Flack parameter=0.0.06(11)(CCDC 2389689).

[0059] 1 H and 13 C NMR data are shown in Figure 1 、 Figure 2 and Table 1.

[0060] Table 1 Compound I 1 H-NMR (600 MHz, chloroform-d) and 13 C-NMR (150 MHz, chloroform-d) data

[0061]

[0062] The compound extracted in Example 1 is colorless needle-shaped crystals (methanol) and is highly soluble in dichloromethane. A 10% sulfuric acid ethanol solution exhibits a purple-red color. The compound extracted in Example 1 was subjected to nuclear magnetic resonance, mass spectrometry, optical rotation, infrared spectroscopy, ultraviolet spectroscopy, and X-ray single crystal diffraction analysis, confirming that the compound extracted in Example 1 is a novel enantiobayerene-type diterpene compound of Formula I.

[0063] Example 3 Pharmacological activity of enantiobayane-type diterpenoid compounds

[0064] Test methods and results

[0065] 1. The MTT assay was used to detect the effect of the compound of formula I on the proliferation of four cancer cells: A549, HCT116, MHCC-97H and SW1990.

[0066] HCT116, SW1990, and MHCC-97H cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS). A549 cells were cultured in RPMI1640 medium supplemented with 10% FBS. All cells were placed in a 37°C, 5% CO2, saturated CO2 incubator.

[0067] Four types of cancer cells in the logarithmic growth phase were selected and digested with trypsin. The cells were then diluted with the corresponding culture medium containing 10% fetal bovine serum to a cell concentration of 5 × 10 3 Cell suspension of 5 × 10 cells / mL was plated (100 μL, 5 × 10 3 / well), fill the edge of the 96-well plate with sterile PBS, and incubate at 37℃ 5% CO2 for 24 hours to allow the cells to adhere to the wall. The next day, observe the state of the cells in the 96-well plate under a microscope. If the cell growth is in good condition, proceed to the next step. The experimental group replaced 100 μL of test compound culture medium with different concentrations in each well, and the content in the DMSO system was controlled within 0.5%. Each concentration was repeated in 3 replicates. The control group replaced the blank culture medium containing the same proportion of DMSO, and repeated 3 wells were used as the background. The cell-free wells were incubated under the above conditions for 24 hours. After incubation for 24 hours, the 96-well plate was placed in an enzyme reader, and the OD value at 450nm was measured. The CCK-8 method was used to detect cell viability (BMU106, Abbkine). The formula for calculating the tumor cell growth inhibition rate of the test compound is: Cell growth inhibition rate % = (A (negative control group) - A (drug group)) / A (negative control group) × 100%

[0068] The experimental data were analyzed using SPSS 17.0 statistical software, and the cell proliferation inhibitory activity of the samples was evaluated using the half-maximal inhibitory concentration.

[0069]

[0070] A G is the average OD value of the drug-treated group, A K is the average OD value of the blank control group, A M is the average OD value of the model group.

[0071] 2. Anticancer activity results of compounds of formula I

[0072] By preliminary screening of the effects of the compound of formula I on the proliferation of four cancer cells, A549, HCT116, MHCC-97H and SW1990, it was found that the compound of formula I had significant inhibitory activity against all four cancer cells. Figure 3-Figure 6 The data show that the IC values ​​of the compound of formula I against the four cancer cells are 50 The values ​​were 5.58±0.21, 14.82±0.74, 14.10±0.97 and 17.63±0.74 μM, respectively.

[0073] In summary, this invention, for the first time, has extracted enantiobayane-type diterpenoid compounds from the traditional Chinese medicine Tongjingcao (Tongjingcao), which have potent anticancer properties. These compounds exhibit potent inhibitory effects against four cancer cell lines: A549, HCT116, MHCC-97H, and SW1990. These compounds hold great promise as novel anticancer drugs, and their design strategy provides new insights and approaches for their development.

[0074] It should be noted that, in this document, the terms "comprises," "includes," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a process, method, article, or apparatus comprising a series of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of other identical elements in the process, method, article, or apparatus comprising the element.

[0075] The serial numbers of the above embodiments of the present invention are for description only and do not represent the advantages or disadvantages of the embodiments.

[0076] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, and all of these should be included in the scope of the claims of the present invention.

Claims

1. An enantiobayane-type diterpenoid compound, characterized in that: The enantiomeric beryllane-type diterpenoid compound has a structure shown in the following formula (I):

2. A method for preparing the enantiobayane-type diterpenoid compound according to claim 1, characterized in that: The following steps are involved: S1. Using the Chinese herbal medicine Tongjingcao as raw material, extracting by diafiltration and concentrating to obtain an extract, suspending the extract in water to obtain a suspension, extracting the suspension with ethyl acetate, and concentrating to obtain an ethyl acetate extract; S2. Using a first eluent, subjecting the obtained ethyl acetate extract to gradient elution via silica gel column chromatography to obtain a first eluate, wherein the first eluent is petroleum ether-ethyl acetate-methanol; S3, using a second eluent to gradiently elute the obtained first eluate through silica gel column chromatography to obtain a second eluate, wherein the second eluent is dichloromethane-methanol; S4, using a third eluent, subjecting the obtained second eluate to gradient elution through ODS reverse column chromatography to obtain a third eluate, wherein the third eluent is methanol-water; S5. Using a fourth eluent, isocratically eluting the obtained third eluate through a Toyopearl HW-40 gel column chromatography to obtain a fourth eluate, wherein the fourth eluent is dichloromethane-methanol; S6. Isocratically eluting the fourth eluate through Si-H silica gel column chromatography to obtain Fr.1 to Fr.

8. In this case, the mobile phase is dichloromethane-methanol. S7. The eluate Fr.4 obtained in step S6 is subjected to semi-preparative ODS high performance liquid chromatography using a fixed ratio mobile phase to obtain the compound represented by formula I. At this time, the mobile phase is methanol-water.

3. The method for preparing enantiobayane-type diterpenoid compounds according to claim 2, characterized in that: In step S1, the diafiltration extraction step is specifically as follows: extracting the Chinese medicinal herb Tongjingcao by diafiltration with ethanol at room temperature, obtaining an extract-like ethanol extract after concentration, and suspending the ethanol extract in water to obtain a suspension; the ethanol concentration is 95%, the diafiltration extraction time is 120 hours, and the ethanol diafiltration flow rate is 0.5 L / h; The extraction step comprises: adding ethyl acetate in a volume equal to that of the suspension, extracting and concentrating the suspension to obtain the ethyl acetate extract.

4. The method for preparing enantiobayane-type diterpenoid compounds according to claim 2, characterized in that: In the first eluent, the volume ratio of petroleum ether, ethyl acetate and methanol is 8:1:0 to 0:0:

1.

5. The method for preparing enantiobayane-type diterpenoid compounds according to claim 4, characterized in that: In step S3, the first eluate is the eluate when the volume ratio of petroleum ether, ethyl acetate and methanol in the first eluent is 4:1:0; In the second eluent, the volume ratio of dichloromethane to methanol is 100:0 to 0:

100.

6. The method for preparing enantiobayane-type diterpenoid compounds according to claim 5, characterized in that: In step S4, the second eluate is the eluate when the volume ratio of dichloromethane to methanol in the second eluent is 98:2; In the third eluent, the volume ratio of methanol to water is 40:60 to 100:

0.

7. The method for preparing enantiobayane-type diterpenoid compounds according to claim 6, characterized in that: In step S5, The third eluate is an eluate in which the volume ratio of methanol to water in the third eluent is 90:10; In the fourth eluent, the volume ratio of dichloromethane to methanol is 2:1; During isocratic elution, the elution rate was 1 mL / min, the elution time was 10 h, and the eluate was collected every 2 h to obtain 5 portions of the fourth eluate.

8. The method for preparing enantiobayane-type diterpenoid compounds according to claim 7, characterized in that: In step S6, The fourth eluate is the eluate collected in the third time period in step S5; The volume ratio of dichloromethane and methanol in the mobile phase was 60:1; During isocratic elution, the elution time is 8 hours, and the eluate is collected every 1 hour, and Fr.1 to Fr.8 are collected in sections; And / or in step S7, the volume ratio of methanol to water is 82:

18.

9. Use of the enantiobayane-type diterpenoid compound according to claim 1 in the preparation of anticancer drugs.

10. The use according to claim 9, characterized in that The anticancer drug uses an enantiobayane-type diterpenoid compound or any pharmaceutically acceptable salt as an active ingredient, and the anticancer drug can be in the form of tablets, capsules, granules, oral solutions, granules, pills or pellets.

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