An indole diterpenoid compound penrhizovarin A with antitumor cytotoxic activity, its preparation method and application
The preparation of indole diterpene compound penrhizovarin A through endophytic fungi of Penicillium was solved, and the problem of lack of efficient anti-tumor drugs in the prior art was achieved, and the preparation and application of environmentally friendly and efficient tumor cytotoxic active compounds were achieved.
Patent Information
- Application Number
- CN202310365106.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-04-07
- Publication Date
- 2025-08-01
- Estimated Expiration
- 2043-04-07
AI Technical Summary
There is a lack of efficient and low-toxic anti-tumor drugs in the prior art, especially the development of compounds with tumor cytotoxic activity from microbial resources, and traditional methods may have negative effects on the ecological environment.
Penrhizovariin A was prepared by fermentation of pendylville endophytic fungi. Compounds with tumor cytotoxic activity were obtained by solid fermentation, ultrasonic extraction and chromatography purification, and used in the preparation of anti-tumor drugs.
It provides a compound with significant tumor cytotoxic activity, penrhizovarin A, which can effectively inhibit a variety of human cancer cells, and is environmentally friendly and easy to produce on a large scale, meeting the needs of modern environmental protection and low-carbon economy.
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Figure CN116535425B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to an indole diterpenoid compound penrhizovarin A with tumor cytotoxic activity, and a preparation method and application thereof. Background Art
[0002] Cancer is a major disease endangering human health. In recent decades, researchers in various countries have been committed to developing highly efficient and low-toxic cancer treatment drugs. Fungi are producers of many alkaloids. Among them, indole diterpenes (IDTs) are the most representative fungal secondary metabolites. The core structure of indole diterpenes consists of a cyclic diterpene fused with indole, and it has been proven to be a secondary metabolite with various biological activities. For example, it has significant cytotoxicity. According to literature reports, there are about 9 genera of fungi that can produce indole diterpenes. Among them, Penicillium.sp is one of the most common genera that produce indole diterpenes. Microorganisms, due to their characteristics of not being restricted by the growth season and being able to be cultured artificially on a large scale, do not cause ecological damage compared with other biological resources and are easy to solve the problem of drug sources. Therefore, they have unique advantages in subsequent development and research. Summary of the Invention
[0003] The object of the present invention is to overcome the defects of the prior art and provide an indole diterpenoid compound penrhizovarin A with tumor cytotoxic activity.
[0004] Another object of the present invention is to provide a preparation method of an indole diterpenoid compound penrhizovarin A with tumor cytotoxic activity.
[0005] Another object of the present invention is to provide an application of an indole diterpenoid compound penrhizovarin A with tumor cytotoxic activity in the preparation of anti-tumor drugs.
[0006] One technical solution to achieve the above object is: an indole diterpenoid compound penrhizovarin A with tumor cytotoxic activity, having the structure shown in Formula I:
[0007]
[0008] The present invention also provides a preparation method of an indole diterpenoid compound penrhizovarin A with tumor cytotoxic activity, comprising the following steps:
[0009] S1. Inoculate the endophytic fungus of Penicillium into a fermentation medium for fermentation to obtain a fermentation product of Penicillium fungus. The endophytic fungus of Penicillium was deposited at the China Center for Type Culture Collection on March 27, 2023, with the deposit number CCTCC NO: M2023416;
[0010] S2. Mix the fermentation product of Penicillium fungus obtained in step S1 with alcohol and perform ultrasonic extraction to obtain a crude extract;
[0011] S3. Purify the crude extract by chromatography to obtain the indole diterpenoid compound penrhizovarin A with tumor cell cytotoxic activity.
[0012] In the above preparation method of the indole diterpenoid compound penrhizovarin A with tumor cell cytotoxic activity, in step S1, the raw materials for making the fermentation medium contain potatoes, and the fermentation method is solid fermentation.
[0013] In the above preparation method of the indole diterpenoid compound penrhizovarin A with tumor cell cytotoxic activity, the fermentation temperature in step S1 is 20 - 25 °C, and the fermentation time is 28 - 30 d.
[0014] In the above preparation method of the indole diterpenoid compound penrhizovarin A with tumor cell cytotoxic activity, in step S2, the mass - volume ratio of the fermentation product of Penicillium fungus to alcohol is (60 - 90) g:(100 - 160) mL.
[0015] In the above preparation method of the indole diterpenoid compound penrhizovarin A with tumor cell cytotoxic activity, in step S3, the chromatography purification is as follows:
[0016] Using a dichloromethane - methanol solution with a volume ratio of (100:0) - (3:1) as the elution solvent, elute the crude extract by normal - phase silica gel column chromatography, and then purify the obtained eluate by chromatography to obtain the indole diterpenoid compound penrhizovarin A with tumor cell cytotoxic activity.
[0017] In the above preparation method of the indole diterpenoid compound penrhizovarin A with tumor cell cytotoxic activity, the silica gel column chromatography elution is as follows:
[0018] Dissolve the crude extract with dichloromethane-methanol solution to obtain a crude extract solution; mix the crude extract solution with silica gel in an amount 0.9 - 1.1 times the dry weight of the crude extract, remove the solvent, load the column, and perform gradient elution successively with dichloromethane-methanol solutions with volume ratios of 100:0, 30:1, and 3:1 to obtain the elution fractions of dichloromethane-methanol solutions with volume ratios of 100:0, 30:1, and 3:1 respectively.
[0019] Take the elution fraction of the dichloromethane-methanol solution with a volume ratio of 30:1, use the dichloromethane-methanol solution with a volume ratio of (80:0) - (30:1) as the elution solvent, and perform normal-phase silica gel column chromatography elution to obtain the indole diterpenoid compound penrhizovarin A with tumor cell cytotoxic activity.
[0020] The present invention also provides the use of the above indole diterpenoid compound penrhizovarin A with tumor cell cytotoxic activity or the indole diterpenoid compound penrhizovarin A with tumor cell cytotoxic activity obtained by the above preparation method in the preparation of anti-tumor drugs.
[0021] The indole diterpenoid compound penrhizovarin A with tumor cell cytotoxic activity provided by the present invention has a structure as shown in Formula I. The indole diterpenoid compound penrhizovarin A of the present invention has significant cytotoxic activity against human breast cancer cells MCF-7, lung cancer cells A-549, colon cancer cells SW-480, liver cancer cells SMMC-7721, and leukemia cells HL-60. Among them, the cytotoxic activity of penrhizovarin A against leukemia cells HL-60, lung cancer cells A-549, and colon cancer cells SW-480 is superior to that of the positive control cisplatin. The cytotoxic activity of penrhizovarin A against liver cancer cells SMMC-7721 and breast cancer cells MCF-7 is comparable to that of the positive control cisplatin. This lays a foundation for anti-tumor activity research and the preparation of drugs for preventing or treating lung cancer.
[0022] The indole diterpenoid compound penrhizovarin A with tumor cell cytotoxic activity provided by the present invention is a metabolite of the endophytic Penicillium fungus of Illigera latiflora, and can be prepared by microbial fermentation. The preparation method has the advantages of mild culture conditions, simple and easy preparation of the culture medium, short fermentation time, and easy large-scale production. It not only meets the requirements of modern environmental protection and low-carbon economy, but also can be developed into drugs for preventing or treating human cancers to solve the raw material problem.
[0023] The indole diterpenoid compound penrhizovarin A with tumor cytotoxic activity provided by the present invention can be applied to the preparation of anti-tumor drugs, providing new compounds or lead compounds for the specific prevention or treatment of tumors, and also providing new drug source molecules with application value for the medical industry.
[0024] Biological Deposit Description
[0025] The endophytic fungus Penicillium.sp was deposited in the China Center for Type Culture Collection (CCTCC for short); the address of the depositary institution: within Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province; the deposit time: March 27, 2023, the deposit number: CCTCC NO: M2023416. Description of the Drawings
[0026] Figure 1 It is the HR-ESI-MS spectrum of the indole diterpenoid compound penrhizovarin A with tumor cytotoxic activity of the present invention;
[0027] Figure 2 It is the 1 1H-NMR spectrum of the indole diterpenoid compound penrhizovarin A with tumor cytotoxic activity of the present invention;
[0028] Figure 3 It is the 13 13C-NMR and DEPT spectra of the indole diterpenoid compound penrhizovarin A with tumor cytotoxic activity of the present invention;
[0029] Figure 4 It is the 1 1H- 1 1H COSY spectrum of the indole diterpenoid compound penrhizovarin A with tumor cytotoxic activity of the present invention.
[0030] Figure 5 It is the HMBC spectrum of the indole diterpenoid compound penrhizovarin A with tumor cytotoxic activity of the present invention;
[0031] Figure 6 It is the HSQC spectrum of the indole diterpenoid compound penrhizovarin A with tumor cytotoxic activity of the present invention;
[0032] Figure 7 It is the NOESY spectrum of the indole diterpenoid compound penrhizovarin A with tumor cytotoxic activity of the present invention. DETAILED DESCRIPTION
[0033] In order to enable those skilled in the art to better understand the technical solution of the present invention, the specific implementation methods thereof are described in detail below with reference to the accompanying drawings:
[0034] Endophytic fungi of the genus Penicillium (Penicillium.sp) were screened by the following method:
[0035] (1) Pretreatment
[0036] Freshly collected roots of Sinomenium sinomenium were taken (collected in Shiping County, Yunnan Province, and immediately stored in sealed bags at 4°C after sample collection; endophytic fungi were isolated within 72 h after collection), the surface sediment was removed, and then rinsed with tap water for about 2 h;
[0037] (2) Surface disinfection
[0038] In a clean bench, soak the tissue in a sodium hypochlorite solution with a 50% available chlorine content, then soak it in 75% ethanol for 30 seconds, rinse it with sterile water three times, soak it in 75% ethanol for 30 seconds, rinse it with sterile water four times, and then use sterile filter paper to absorb the moisture before use. (In order to check whether the tissue surface is thoroughly disinfected, a control experiment is required; take 50 μL of the sterile water used for the last wash and add it directly to the separation plate, then spread it on the culture medium with a coating rod and culture it under the same conditions as the control group; if no colonies grow in all the control groups, it proves that the surface disinfection in the above steps is thorough, thus ensuring that the isolated microorganisms are endophytes rather than microorganisms from the air or attached to the tissue surface).
[0039] (3) Isolation of endophytic fungi
[0040] Cut the root of the broadleaf sedge into several 0.5×0.5cm pieces with sterilized scissors. 2 Place the processed samples onto pre-prepared PDA culture plates. Divide each plate into four areas: A, B, C, and D. Place 3 to 4 sample segments into each area. Seal the culture dish with sealing film, number it, and invert it in a 28°C incubator. Check it daily. If new colonies are found, inoculate them onto new plates immediately.
[0041] (4) Purification of endophytic fungi (plate streak method)
[0042] After hyphae grow on the PDA culture medium plate, the grown hyphae are repeatedly streaked and separated and cultured until a pure strain is obtained.
[0043] (5) Preservation of endophytic fungi
[0044] The short-term preservation of the purified fungal strain can be directly inoculated on the slant. After the strain grows out, seal the test tube with sealing film and store it in a 4°C refrigerator.
[0045] (6) Identification of endophytic fungi
[0046] Extract the DNA of the obtained fungi (using conventional methods), perform PCR amplification, and finally perform sequencing (send the samples to Qingke Biotechnology Co., Ltd. for sequencing). Input the sequencing results into GenBank (www.ncbi.nlm.nih.gov), use the BLAST program to search for homologous sequences for comparison, and find the most similar sequences (according to the currently commonly used criteria, that is: similarity between 80% and 90% is regarded as the same phylum level; similarity between 90% and 95% is regarded as the same family level; similarity between 95% and 97% is regarded as the same genus level; similarity above 97% is regarded as the same species level). The sequencing results show that an endophytic fungus of the genus Penicillium (Penicillium.sp) is obtained.
[0047] An indole diterpenoid compound penrhizovarin A with tumor cell cytotoxic activity, the preparation method is as follows:
[0048] S1, inoculate the endophytic fungus of the genus Penicillium in a fermentation medium for fermentation to obtain a fermentation product of the fungus of the genus Penicillium, which specifically includes the following steps:
[0049] S11, strain activation: The endophytic fungus of the genus Penicillium was deposited in the China Center for Type Culture Collection on March 27, 2023, with the deposit number CCTCC NO: M2023416. Inoculate the endophytic fungus of the genus Penicillium on a PDA slant medium, incubate at 28°C for 3 days, and then store it in a 4°C refrigerator for standby.
[0050] S12, preparation of the fermentation medium: Take the washed potatoes, cut them into potato pieces with a diameter of 1 cm 3 and place them in a tissue culture flask (100 g / bottle). Cover the tissue culture flask and sterilize it at 120°C for 30 minutes, then cool to obtain the fermentation medium.
[0051] S13, inoculate the activated endophytic fungus of the genus Penicillium in step 1 into the fermentation medium prepared in step 2 at an inoculation amount of 1%, cover it, and incubate at 20 - 25°C for 28 - 30 days to obtain a fermentation product of the fungus of the genus Penicillium.
[0052] S2, mix 100 g of the fermentation product with 150 ml of methanol, ultrasonicate it at 40 kHz for 30 minutes, filter, and take the filtrate for vacuum concentration until there is no alcohol smell to obtain 15.5 g of the crude extract of penrhizovarin A.
[0053] S3. Dissolve 15.5 g of the crude extract of penrhizovarin A in 20 ml of a dichloromethane - methanol solution with a volume ratio of 3:1, mix it with 15.0 g of silica gel (200 mesh), concentrate under reduced pressure to remove the solvent, and load it onto a column. Gradient elution is carried out successively with dichloromethane - methanol solutions with volume ratios of 100:0, 30:1, and 3:1. Take the eluted part with a dichloromethane - methanol solution with a volume ratio of 30:1, and use a dichloromethane - methanol solution with a volume ratio of 80:0 to 30:1 as the eluent for normal - phase silica gel column chromatography elution to obtain the indole diterpenoid compound penrhizovarin A with tumor cell cytotoxic activity.
[0054] Please refer to Figures 1 to 7 , take the prepared indole diterpenoid compound penrhizovarin A, and conduct structure identification by 1D / 2D NMR (one - dimensional nuclear magnetic resonance spectroscopy and two - dimensional nuclear magnetic resonance spectroscopy) and HR - ESI - MS (high - resolution electrospray ionization mass spectrometry).
[0055] From the HSQC spectrum combined with the carbon spectrum, the chemical shift data of H and the connected C of the compound penrhizovarin A can be obtained, as shown in Table 1.
[0056] Table 1: 1 H (400 MHz) and 13 C (100 MHz) NMR data
[0057]
[0058]
[0059] Please refer to Figures 1 to 7 , the indole diterpenoid compound penrhizovarin A: yellow powder. Its HR - ESI - MS m / z: [M + H] + The quasi - molecular ion peak is 648.2718 (calculated value: C 37 H 43 ClNO7 + , 648.2723), and its molecular formula is inferred to be C 37 H 42 ClNO7, indicating that it has 17 degrees of unsaturation. The 1 H, 13 C NMR and HSQC data (see Table 1) of the compound penrhizovarin A show that it has 37 carbons, including 1 carbonyl group (δ C 213.5), 9 sp 2 hybridized quaternary carbons (δ C(144.5, 141.6, 134.8, 130.9, 129.2, 125.6, 120.0, 119.7, 112.6), 2 sp 2 Hybridized methylene (δ C 111.2, 110.6; δ H 5.04, 5.02, 4.96, 4.87), 1 sp 2 Hybridized methine (δ C 109.5; δ H 7.21), 5 oxygenated sp 3 Hybridized methine (δ C 94.0, 70.5, 73.5, 65.2, 61.2; δ H 6.48, 4.26, 4.06, 4.04, 3.55), 4 oxygenated sp 3 Hybridized quaternary carbon (δ C 76.1, 71.5, 70.8, 65.0), 6 sp 3 Hybridized methylene (δ C 33.0, 31.0, 26.3, 26.0, 24.6, 24.2; δ H 3.66, 3.12, 2.59, 2.59, 2.35, 2.16, 2.11, 1.98, 1.87, 1.71, 1.22), 2 sp 3 Hybridized quaternary carbon (δ C 60.2, 46.3), 2 sp 3 Hybridized methine (δ C 48.4, 42.7,; δ H 3.37, 2.29) and 5 methyls (δ C 31.0, 24.0, 23.8, 22.3, 18.3; δ H 1.93, 1.70, 1.69, 0.98, 0.89). The above data are similar to those of rhizovarin C, indicating that compound penrhizovarin A has a similar carbon skeleton to rhizovarin C and both belong to indole diterpenoids. A careful comparison of their NMR data reveals that compound penrhizovarin A has one more carbonyl group (δ C 213.5) and one less quaternary carbon attached to a double oxygen. Therefore, it is speculated that the F ring of compound rhizovarin C undergoes hydrolysis and ring opening, and then C-19 is oxidized to a carbonyl group to generate compound penrhizovarin A. This speculation is supported by the correlations of H3-40, H-20, and H-21 with C-19 (δ CSupport related to HMBC at 213.5). In addition, further analysis of its HMBC data revealed that H-18 (δ H 6.48) had a strong HMBC correlation with C-5 (δ C 71.5), indicating that there was an ether bond between C-18 and C-15, thus forming a structural unit of dioxabicyclo[3.3.1]nonane. Combining with 1 H- 1 H COSY and the remaining HMBC correlations fully confirmed the planar structure of compound penrhizovarin A.
[0060] The relative configuration of compound penrhizovarin A was determined by NOESY experiment. The NOE correlations of H-18 / H-14, H-18 / H3-35, and H-18 / H3-39 indicated that the spatial orientations of H-18 with H-14, H-35, and H3-39 were the same. The absolute configuration of compound penrhizovarin A was determined by comparing its experimental ECD and calculated ECD spectra.
[0061] The indole diterpenoid compound penrhizovarin A has the structure shown in Formula I:
[0062]
[0063] Antitumor activity detection experiment:
[0064] The prepared indole diterpenoid compound penrhizovarin A was subjected to an antitumor activity detection experiment. The principle of detecting cell activity by the MTS method is as follows: MTS is a brand-new MTT analog and is a yellow dye; succinate dehydrogenase in the mitochondria of living cells can metabolically reduce MTS to generate soluble formazan compounds, and the content of formazan can be measured by an enzyme-linked immunosorbent assay (ELISA) reader at 490 nm. Under normal circumstances, the amount of formazan generated is proportional to the number of living cells, so the number of living cells can be inferred based on the optical density OD value.
[0065] Experimental method:
[0066] 1. Inoculate cells: Prepare a single-cell suspension with a culture medium (DMEM or RMPI1640) containing 10% fetal bovine serum, and inoculate 3000 - 15000 cells per well into a 96-well plate, with a volume of 100 μl per well. The cells are inoculated and cultured 12 - 24 hours in advance.
[0067] 2. Add the test compound solution: The compound is dissolved in DMSO, and the compound is initially screened at a concentration of 40 μM, with a final volume of 200 μl per well. Each treatment is set with 3 replicates.
[0068] 3. Color development: After culturing at 37 °C for 48 hours, discard the culture medium in the wells of adherent cells, add 20 μl of MTS solution and 100 μl of culture medium to each well; for the suspended cells HL-60, discard 100 μl of the culture supernatant, and add 20 μl of MTS solution to each well; set up 3 blank replicate wells (a mixture of 20 μl of MTS solution and 100 μl of culture medium), continue to incubate for 2 - 4 hours, and measure the optical absorption value after the reaction proceeds fully.
[0069] 4. Colorimetry: Select a wavelength of 492 nm, and use a multi-functional microplate reader (MULTISKAN FC) to read the optical absorption values of each well, record the results, and after data processing, plot the inhibition rate graph of the cells with the compound number as the abscissa and the cell inhibition rate as the ordinate.
[0070] 5. Positive control compounds: For each experiment, set two positive compounds, cisplatin (DDP) and paclitaxel (Taxol). Plot the cell growth curve with the concentration as the abscissa and the cell survival rate as the ordinate, and use the two-point method (Reed and Muench method) to calculate the IC 50 value, and the results are shown in Table 2.
[0071] Table 2, Screening of the antitumor cytotoxic activity of penrhizovarin A (IC 50 ):
[0072]
[0073] The experimental results show that in the screening test of the inhibitory activity of the indole diterpenoid compound penrhizovarin A against five kinds of tumor cells in vitro (positive controls: cisplatin, paclitaxel), penrhizovarin A has significant antitumor cytotoxic activity against human breast cancer cells MCF-7, lung cancer cells A-549, colon cancer cells SW-480, liver cancer cells SMMC-7721, and leukemia cells HL-60. Among them, the antitumor cytotoxic activity of the indole diterpenoid compound penrhizovarin A against leukemia cells HL-60, lung cancer cells A-549, and colon cancer cells SW-480 is better than that of the positive control cisplatin. The antitumor cytotoxic activity of the indole diterpenoid compound penrhizovarin A against liver cancer cells SMMC-7721 and breast cancer cells MCF-7 is comparable to that of the positive control cisplatin (see Table 2). Therefore, the indole diterpenoid compound penrhizovarin A has research value for developing antitumor drugs as a lead compound.
[0074] Those of ordinary skill in the art should recognize that the above embodiments are only used to illustrate the present invention and are not intended to limit the present invention. As long as it is within the spirit of the present invention, changes and modifications to the above-described embodiments will fall within the scope of the claims of the present invention.
Claims
1. An indole diterpenoid compound penrhizovarin A with antitumor cytotoxic activity, characterized in that, It has the structure shown in Formula I:
2. A method for preparing indole diterpenoid compound penrhizovarin A with antitumor cytotoxic activity as described in claim 1, characterized in that, It includes the following steps: S1. Inoculate the endophytic fungus of Penicillium in a fermentation medium for fermentation to obtain a Penicillium fungal ferment. The endophytic fungus of Penicillium was deposited at the China Center for Type Culture Collection on March 27, 2023, with the deposit number CCTCC NO: M2023416. In step S1, the raw materials for preparing the fermentation medium contain potatoes, and the fermentation method is solid fermentation; the fermentation temperature is 20 - 25°C, and the fermentation time is 28 - 30 d; S2. Mix the Penicillium fungal ferment obtained in step S1 with alcohol and perform ultrasonic extraction to obtain a crude extract; the mass - volume ratio of the Penicillium fungal ferment to alcohol is (60 - 90) g:(100 - 160) mL; S3. Purify the crude extract by chromatography to obtain the indole diterpenoid compound penrhizovarin A with tumor cell cytotoxic activity; the chromatographic purification is as follows: Using a dichloromethane - methanol solution with a volume ratio of (100:0) - (3:1) as the elution solvent, elute the crude extract by normal - phase silica gel column chromatography, and then purify the obtained eluate by chromatography to obtain the indole diterpenoid compound penrhizovarin A with tumor cell cytotoxic activity.
3. The preparation method of an indole diterpenoid compound penrhizovarin A with tumor cell cytotoxic activity according to claim 2, characterized in that, The silica gel column chromatography elution is as follows: Dissolve the crude extract with a dichloromethane - methanol solution to obtain a crude extract solution; mix the crude extract solution with silica gel with a mass 0.9 - 1.1 times that of the dry weight of the crude extract, remove the solvent, load the column, and perform gradient elution successively with dichloromethane - methanol solutions with volume ratios of 100:0, 30:1, and 3:1 to obtain the elution fractions of dichloromethane - methanol solutions with volume ratios of 100:0, 30:1, and 3:1 respectively; Take the elution fraction of the dichloromethane - methanol solution with a volume ratio of 30:1, and use a dichloromethane - methanol solution with a volume ratio of (80:0) - (30:1) as the elution solvent to elute by normal - phase silica gel column chromatography to obtain the indole diterpenoid compound penrhizovarin A with tumor cell cytotoxic activity.
4. Use of the indole diterpenoid compound penrhizovarin A with tumor cell cytotoxic activity as described in claim 1 or the indole diterpenoid compound penrhizovarin A with tumor cell cytotoxic activity obtained by using the preparation method described in any one of claims 2 - 3 in the preparation of anti - tumor drugs.