A diterpenoid compound, preparation method and use thereof

Abstract

The invention discloses a novel terpenoid compound and a preparation method and application thereof. The terpenoid compound is a natural compound obtained by fermenting and culturing Micromonospora sp. HM134 and then extracting and separating the terpenoid compound, and the terpenoid compound has good anti-tumor activity.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a new diterpenoid compound, a preparation method thereof and an application of the compound in terms of its anti-tumor activity. Background Art

[0002] Terpenoids are compounds composed of isoprene structural units. In vivo, isoprene can be linked head to head, head to tail, and in different numbers, and with the addition of various group modifications, it constitutes a class of natural products with structural diversity. Terpenoids have a wide range of physiological activities, including cytotoxicity, anti-tumor activity, bactericidal, nematocidal, malarial parasite killing, analgesic, platelet aggregation inhibition, blood pressure reduction, arrhythmia regulation, etc.

[0003] Terrestrial terpenoids are mainly derived from plants and exist in the form of resins, lactones or glycosides. Due to the different synthesis pathways of secondary metabolites in the marine environment, marine terpenes have structures different from those from terrestrial sources. Marine terpenoids are the largest class of compounds in marine natural products, accounting for almost more than 1 / 3 of marine natural products. They can be found in various marine organisms. Currently, they are mainly reported to exist in marine plants, marine invertebrates, sponges and corals. Extracting such substances from plants involves multiple problems such as resource limitations and complex processes, which ultimately hinder the application of such compounds. Therefore, the discovery of such resources in marine microorganisms has important application value. Summary of the invention

[0004] The present invention provides a new diterpenoid compound, the molecular structure of which is shown in formula (I):

[0005]

[0006] The present invention also provides a method for preparing the diterpenoid compound of formula (I), wherein the method uses Micromonas as a starting strain to ferment and produce the diterpenoid compound, and then extracts and separates the diterpenoid compound.

[0007] Furthermore, the fermentation medium used in the fermentation production contains a carbon source and a nitrogen source, and the specific producing bacteria is Micromonospora sp. HM134, which is deposited in CGMCC with a deposit number of CGMCC No.15401.

[0008] Furthermore, the method for preparing the diterpenoid compound of formula (I) comprises the following steps:

[0009] (1) fermenting and culturing Micromonospora sp. HM134 (whose deposit number is CGMCC No. 15401) in a medium containing a carbon source and a nitrogen source, and collecting the fermentation broth;

[0010] (2) collecting the fermentation liquid obtained in step (1), separating the mycelium and the supernatant, extracting the mycelium with an organic solvent A to obtain an extract; adsorbing the supernatant with a resin, and then eluting with an organic solvent B to obtain an eluate; combining the extract and the eluate to obtain a combined solution;

[0011] (3) The combined solution obtained in step (2) is sequentially concentrated, extracted, concentrated, subjected to silica gel column chromatography, and subjected to gel column chromatography to obtain a fraction sample containing diterpenoid compounds;

[0012] (4) Subjecting the fraction sample obtained in step (3) to reverse phase column chromatography to obtain diterpenoid compounds.

[0013] The strain Micromonospora sp.HM134 involved in the present invention is a micromonospora producing diterpenoid compounds isolated from a seawater sample collected from a mangrove forest in Fujian. The bacterium was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration (address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences) on February 27, 2018, with a deposit number of CGMCC No.15401, and was classified and named Micromonospora sp., and registered to prove its survival.

[0014] In the above method, the carbon source in step (1) is selected from glucose, soluble starch, dextrin, corn starch, industrial molasses, glycerol, sucrose, sorbitol, mannitol, lactose, maltose, xylan or a combination thereof, preferably a combination of soluble starch and glucose; the nitrogen source is selected from yeast powder, yeast extract, soybean cake powder, soybean meal, peanut cake powder, cottonseed cake powder, malt extract, peptone, beef extract, yeast extract, corn steep powder, bran, gluten powder, urea, ammonium salt or a combination thereof, preferably soybean cake powder, yeast extract, malt extract or a combination thereof.

[0015] In the above method, the resin in step (2) is a macroporous adsorption resin, preferably HP-20 macroporous adsorption resin, the organic solvent A is selected from lower ketones, C1-C3 alcohols or mixtures thereof, preferably acetone, methanol, ethanol or a combination of two or more thereof; the organic solvent B is selected from lower ketones, C1-C3 alcohols or mixtures thereof, preferably acetone, methanol, ethanol or a combination of two or more thereof.

[0016] In the above method, the silica gel particle size used in the silica gel column chromatography in step (3) is selected from 100-200 mesh, the elution solvent used in the silica gel column chromatography is a mixed solvent system of C5-C8 alkanes and fatty acid esters, and the elution method used in the silica gel column chromatography is preferably gradient elution; further, the C5-C8 alkane is preferably selected from n-heptane, n-hexane or a mixture thereof, and the fatty acid ester is preferably ethyl acetate.

[0017] As a further preferred embodiment, the elution method used in the silica gel column chromatography in step (3) is a gradient elution of n-heptane:ethyl acetate = 100:0-80:20 (V / V); or a gradient elution of n-hexane:ethyl acetate = 100:0-80:20 (V / V).

[0018] In the above method, the gel used in the gel column chromatography in step (3) is gel LH-20, the elution solvent used in the gel column chromatography is a mixed solvent system of chloroalkane and C1-C3 alcohol, and the elution method used in the gel column chromatography is preferably isocratic elution; the chloroalkane is preferably selected from chloroform, dichloromethane or a mixture thereof, and the C1-C3 alcohol is preferably methanol, ethanol or a mixture thereof.

[0019] As a further preferred embodiment, the elution solvent used in the gel column chromatography in step (3) is a chloroform:methanol system (90:10-10:90, V / V) or a dichloromethane:methanol system (90:10-10:90, V / V); preferably, chloroform:methanol = 50:50 (V / V) or dichloromethane:methanol = 50:50 (V / V).

[0020] In the above method, the reverse phase chromatography column used in the reverse phase column chromatography in step (4) is a reverse phase C18 column, and the elution solvent used in the reverse phase column chromatography is selected from acetonitrile, methanol, an aqueous solution of acetonitrile, an aqueous solution of methanol, or an aqueous solution of a mixed solvent of methanol and acetonitrile; preferably, the volume ratio of acetonitrile to water in the aqueous solution of acetonitrile is 20:80-100:0, the volume ratio of methanol to water in the aqueous solution of methanol is 20:80-100:0, and the volume ratio of the mixed solvent of methanol and acetonitrile to water in the aqueous solution of the mixed solvent of methanol and acetonitrile is 20:80-100:0, more preferably, the volume ratio of acetonitrile to water in the aqueous solution of acetonitrile is 25:75-90:10, the volume ratio of methanol to water in the aqueous solution of methanol is 25:75-90:10, and the volume ratio of the mixed solvent of methanol and acetonitrile to water in the aqueous solution of the mixed solvent of methanol and acetonitrile is 25:75-90:10.

[0021] Furthermore, the present invention provides a pharmaceutical composition, which contains an effective dose of a diterpenoid compound of formula (I) and a pharmaceutically acceptable carrier, excipient or a combination thereof.

[0022] Furthermore, the present invention provides the use of a diterpenoid compound of formula (I) or a pharmaceutical composition thereof in the preparation of a drug for treating cancer; wherein the cancer is preferably selected from lung cancer, intestinal cancer, liver cancer, prostate cancer, gastric cancer, breast cancer, ovarian cancer, kidney cancer, lymphoma or leukemia.

[0023] The present invention provides a new diterpenoid compound, a preparation method and use thereof, which provide a material basis for developing and utilizing natural active substances derived from microorganisms. Meanwhile, the diterpenoid has anti-tumor activity and can be used clinically to treat various tumors. DETAILED DESCRIPTION

[0024] The following examples are intended to further illustrate the present invention but are not intended to limit the present invention.

[0025] Example 1: Fermentation culture of strain Micromonospora sp. HM134

[0026] (1) Fermentation strain: The fermentation strain is Micromonospora sp. HM134.

[0027] (2) Slant culture: Solid medium (g / L) (composition: yeast extract 4.0, malt extract 10.0, glucose 4.0, CoCl2·6H2O 0.005, agar 18.0, distilled water, pH 7.0-7.2) was sterilized at 121°C for 20 min and cultured at 28°C for 6-8 days after inoculation.

[0028] (3) Seed culture: The seed culture medium composition (g / L) (glucose 4.0, malt extract 10.0, yeast extract 4.0, CaCO3 2.0, distilled water, pH 7.2-7.4) was sterilized at 121°C for 20 min and dispensed into 1000 ml Erlenmeyer flasks, 250 ml per bottle. The Micromonospora spores on the slope were then washed off with sterile water to prepare a spore suspension with a concentration of 1 × 10 7 ~1×10 8 Add 2 ml of spore suspension to each bottle, place on a shaker, rotate at 250 r / min, and culture at 28°C for 48 hours to obtain seed solution.

[0029] (4) Fermentation: The fermentation medium composition (g / L) (soluble starch 60, glucose 20, soybean cake powder 15, yeast extract 5, malt extract 5, CaCO32, NaCl3, distilled water, pH 7.2-7.4) was sterilized at 121° C. for 20 min. The seed solution was inoculated into a 50 L fermentation tank (30 L capacity) at a 5% (volume percentage) inoculation amount, and cultured at 28° C., with a stirring speed of 200-300 r / min and a ventilation volume of 1000 L / h for 7 days.

[0030] Example 2: Extraction and separation of new diterpenoids

[0031] Fermentation was performed according to the method of Example 1 to obtain 30 L of fermentation liquid, which was centrifuged at 4000 rpm for 10 min to separate mycelium and supernatant. The mycelium was washed with 3 L of deionized water and then soaked in 10 L of industrial methanol overnight, and the methanol extract was filtered. The supernatant was adsorbed by Diaion HP-20 resin and eluted with 5 L of ethanol to obtain an ethanol eluate. The obtained methanol extract and ethanol eluate were combined, concentrated under reduced pressure at 45°C to remove the organic phase to 1 L, and then extracted with an equal volume of ethyl acetate 3 times. The obtained ethyl acetate extract was concentrated under reduced pressure at 45°C to remove the ethyl acetate phase until dry, and 22 g of an oily substance was obtained.

[0032] The obtained oily substance was subjected to column chromatography on a silica gel column (particle size 100-200 mesh), and gradient elution was performed in sequence with n-heptane:ethyl acetate (98:2, 95:5, 90:10, 85:15 and 80:20, V / V) and relevant components were collected and detected by TLC to obtain the target component. The target component was separated by gel LH-20 column chromatography (chloroform:methanol=50:50, V / V), and then further purified by reverse phase column chromatography to obtain the target product.

[0033] The reverse phase column chromatography conditions are as follows:

[0034] Liquid phase system: Shimadzu LC-8A preparative high pressure liquid chromatograph

[0035] Chromatographic column: Shimadzu-C18 (250mm*20mm)

[0036] Eluent: Volume ratio of acetonitrile to water = 25:75-40:60 (20min) 40:60-90:10 (50min)

[0037] Flow rate: 20mL / min

[0038] Detection wavelength: λ = 205nm / 220nm

[0039] The peak with a retention time of 66.7 min was collected to obtain a diterpenoid compound (8.4 mg).

[0040] Example 3: Structural identification of new diterpenoid compounds

[0041] The structure of the new diterpenoid compound was determined by 1D and 2D NMR, MS and other spectral analyses as shown in formula (I):

[0042]

[0043] Appearance: Light yellow amorphous oil

[0044] Solubility: easily soluble in chloroform, acetone, methanol, insoluble in water

[0045] Molecular formula: C 21 H 30 O5

[0046] Specific rotation: (c 0.009, EtOH)

[0047] ESI-MS m / z:361.16[MH] -

[0048] UV absorption spectrumλ max (EtOH) nm (logε): 200 (4.04)

[0049] IR absorption spectrumV max cm -1 : 3659, 2955, 1704, 1625, 1438, 1409, 1306, 1253, 1231, 1211, 1142, 1044, 911, 733

[0050] 1 H and 13 C NMR (CDCl3) data are shown in Table 1.

[0051] Table 1 NMR data of compound of formula (I) in CDCl3 (hydrogen spectrum, 400 MHz; carbon spectrum, 100 MHz)

[0052]

[0053] Example 4: Tumor inhibitory activity of diterpenoid compounds of formula (I)

[0054] The CCK-8 method was used to determine the proliferation inhibition rate of the diterpenoid compound of formula (I) on human colon cancer cell HCT-116 and human liver cancer cell HepG2:

[0055] Human colon cancer cells HCT-116 and human liver cancer cells HepG2 in logarithmic growth phase were digested with 0.25% trypsin and then prepared into single cell suspensions with DMEM culture medium. 4 The cells were inoculated in a 96-well culture plate, with a volume of 90 μl per well. The culture plate was moved into a CO2 incubator, and after static culture for 4 hours at 37°C, 5% CO2 and saturated humidity, 10 μl of doxorubicin and diterpenoid compounds of formula (I) were added respectively, with a gradient of 100 μg / ml, 50 μg / ml, 25 μg / ml, 12.5 μg / ml, 6.25 μg / ml, 3.12 μg / ml, 1.56 μg / ml, 0.78 μg / ml, 0.39 μg / ml, and 0.19 μg / ml, with 2 replicate wells in each group. After continuing to culture in the cell culture incubator for 48 hours, 10 μl of CCK-8 reagent was added, incubated at 37°C for 2 hours, and the absorbance at 490 nm was measured. At the same time, tumor cells, drugs and blank culture medium were set as controls.

[0056] The inhibitory rate of the drug on the corresponding tumor cells is calculated by the measured absorbance value:

[0057] Tumor inhibition rate = (OD control group - OD experimental group) / (OD control group - OD blank group) × 100%

[0058] The results are shown in Table 2.

[0059] Table 2: Inhibitory effect of diterpenoid compounds of formula (I) on tumor cells

[0060]

[0061] Experiments have shown that the diterpenoid compound of formula (I) has good activity in inhibiting the proliferation of human colon cancer cells HCT-116 and human liver cancer cells HepG2.

Claims

1. A diterpenoid compound, characterized in that: Its chemical structure is shown in formula (I):

2. A method for preparing the diterpenoid compound according to claim 1, comprising the following steps: (1) fermenting and culturing Micromonospora sp. HM134, whose deposit number is CGMCC No. 15401, in a medium containing a carbon source and a nitrogen source, and collecting the fermentation broth; (2) collecting the fermentation liquid obtained in step (1), separating the mycelium and the supernatant, extracting the mycelium with an organic solvent A to obtain an extract; adsorbing the supernatant with a resin, and then eluting with an organic solvent B to obtain an eluate; combining the extract and the eluate to obtain a combined solution; (3) The combined solution obtained in step (2) is sequentially concentrated, extracted, concentrated, subjected to silica gel column chromatography, and subjected to gel column chromatography to obtain a fraction sample containing diterpenoid compounds; (4) Subjecting the fraction sample obtained in step (3) to reverse phase column chromatography to obtain diterpenoid compounds.

3. The method of claim 2, wherein the carbon source in step (1) is selected from glucose, soluble starch, dextrin, industrial molasses, glycerol, sucrose, sorbitol, mannitol, lactose, maltose, xylan or a combination thereof; and the nitrogen source is selected from yeast powder, yeast extract, soybean cake powder, soybean powder, peanut cake powder, cottonseed cake powder, malt extract, peptone, beef extract, yeast extract, corn steep liquor, bran, gluten powder, urea, ammonium salt or a combination thereof.

4. The method of claim 2, wherein the carbon source in step (1) is selected from glucose, dextrin, corn starch, industrial molasses, glycerol, sucrose, sorbitol, mannitol, lactose, maltose, xylan or a combination thereof.

5. The method according to claim 2, wherein the carbon source in step (1) is a combination of soluble starch and glucose.

6. The method according to claim 2, wherein the nitrogen source in step (1) is selected from soybean cake powder, yeast extract, malt extract or a combination thereof.

7. The method according to claim 2, wherein the resin in step (2) is a macroporous adsorption resin, the organic solvent A is selected from acetone, C1-C3 alcohol or a mixture thereof; the organic solvent B is selected from acetone, C1-C3 alcohol or a mixture thereof.

8. The method according to claim 2, wherein the resin in step (2) is HP-20 macroporous adsorption resin.

9. The method according to claim 2, wherein the organic solvent A in step (2) is selected from acetone, methanol, ethanol or a combination of two or more thereof.

10. The method according to claim 2, wherein the organic solvent B in step (2) is selected from acetone, methanol, ethanol or a combination of two or more thereof.

11. The method according to claim 2, wherein the silica gel particle size used in the silica gel column chromatography described in step (3) is selected from 100-200 mesh, and the elution solvent used in the silica gel column chromatography is a mixed solvent system of C5-C8 alkane and fatty acid ester.

12. The method according to claim 2, wherein the elution method used in the silica gel column chromatography in step (3) is gradient elution.

13. The method of claim 11, wherein the C5-C8 alkane is selected from n-heptane, n-hexane or a mixture thereof.

14. The method of claim 11, wherein the fatty acid ester is ethyl acetate.

15. The method according to claim 2, wherein the elution method used in the silica gel column chromatography in step (3) is n-heptane:ethyl acetate = 100:0-80:20 V / V gradient elution; or n-hexane:ethyl acetate = 100:0-80:20 V / V gradient elution.

16. The method according to claim 2, wherein the gel used in the gel column chromatography in step (3) is gelatin LH-20, and the elution solvent used in the gel column chromatography is a mixed solvent system of chloroalkane and C1-C3 alcohol.

17. The method according to claim 2, wherein the elution method used in the gel column chromatography in step (3) is isocratic elution.

18. The method of claim 16, wherein the chloroalkane is selected from chloroform, dichloromethane or a mixture thereof.

19. The method of claim 16, wherein the C1-C3 alcohol is selected from methanol, ethanol or a mixture thereof.

20. The method of claim 2, wherein the elution solvent used in the gel column chromatography in step (3) is a chloroform:methanol system of 90:10-10:90 V / V or a dichloromethane:methanol system of 90:10-10:90 V / V.

21. The method according to claim 2, wherein the elution solvent used in the gel column chromatography in step (3) is chloroform:methanol = 50:50 V / V or dichloromethane:methanol = 50:50 V / V.

22. The method of claim 2, wherein the reverse phase chromatography column used in the reverse phase column chromatography in step (4) is a reverse phase C18 column, and the elution solvent used in the reverse phase column chromatography is selected from acetonitrile, methanol, an aqueous solution of acetonitrile, an aqueous solution of methanol, or an aqueous solution of a mixed solvent of methanol and acetonitrile.

23. The method of claim 22, wherein the volume ratio of acetonitrile to water in the aqueous solution of acetonitrile is 20:80-100:0, the volume ratio of methanol to water in the aqueous solution of methanol is 20:80-100:0, and the volume ratio of the mixed solvent of methanol and acetonitrile to water in the aqueous solution of the mixed solvent of methanol and acetonitrile is 20:80-100:

0.

24. The method of claim 22, wherein the volume ratio of acetonitrile to water in the aqueous solution of acetonitrile is 25:75-90:10, the volume ratio of methanol to water in the aqueous solution of methanol is 25:75-90:10, and the volume ratio of the mixed solvent of methanol and acetonitrile to water in the aqueous solution of the mixed solvent of methanol and acetonitrile is 25:75-90:

10.

25. A pharmaceutical composition comprising an effective dose of the diterpenoid compound according to claim 1 and a pharmaceutically acceptable carrier, excipient or a combination thereof.

26. Use of the diterpenoid compound according to claim 1 or the pharmaceutical composition according to claim 25 in the preparation of a medicament for treating cancer.

27. The use according to claim 26, wherein the cancer is selected from lung cancer, intestinal cancer, liver cancer, prostate cancer, stomach cancer, breast cancer, ovarian cancer, kidney cancer, lymphoma or leukemia.

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