Anti-tumor component in ferula sinkiangensis endophyte as well as extraction and separation method and application of anti-tumor component
By activating the strain N93 of Xinjiang asawei endophytic fungi and using multi-step extraction and isolation methods, the anti-tumor component ferupencine B was successfully isolated, which solved the problem of difficulty in effectively utilizing the anti-tumor component of the strain in the prior art, and achieved effective extraction of this component and significant inhibitory effect on breast cancer cells.
Patent Information
- Application Number
- CN202510274609.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-10
- Publication Date
- 2025-06-06
AI Technical Summary
The prior art is difficult to effectively utilize the anti-tumor components in the strain N93 of the Xinjiang Asawei endophytic fungus, and the traditional extraction methods are unsustainable and difficult to meet market demand.
By activating the strain N93 of Xinjiang Avocado endophytic fungi, cultured secondary metabolites using rice culture medium, and using ethanol extraction, ultrasonic reflux, silica gel column chromatography gradient elution and high performance liquid chromatography gradient elution, the anti-tumor component ferupencine B in Xinjiang Avocado endophytic fungi was successfully isolated and purified.
Effective extraction and purification of anti-tumor components in Xinjiang's endophytes was achieved, demonstrating the significant inhibitory effect of this component on MCF-7 cells, and providing a potential application for the preparation of drugs for preventing breast cancer or anti-breast cancer.
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Figure CN120098006A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of separation and purification of Xinjiang Ferula endophytic fungus strain N93, and is an anti-tumor component in Xinjiang Ferula endophytic fungus, and an extraction and separation method and application thereof. Ferupencine B is the abbreviation of the anti-tumor component in Xinjiang Ferula endophytic fungus, and is an alkaloid compound. Background Art
[0002] Endophytic fungi are fungi that live in plant tissues and do not cause obvious disease symptoms. They include saprophytes that live on the surface at a certain stage of their life history, latent pathogens that do not harm the host temporarily, and mycorrhizal fungi. It is a large and special group of fungi, and endophytic fungi are found in almost all plant tissues.
[0003] Extracting endophytic fungi from plants is of great significance for understanding the interaction between endophytic fungi and plants and discovering new drug ingredients. For example, paclitaxel is a precious anticancer drug extracted from Taxus plants, which has significant therapeutic effects; however, Taxus grows slowly and is an endangered species, and traditional extraction methods are unsustainable. The growth of market demand has made it urgent to find alternative production methods. Therefore, scientists have begun to explore the production of paclitaxel by fermentation of endophytic fungi to solve the problem of insufficient drug sources. Therefore, plant endophytic fungi have now become an important source for finding anti-tumor drugs, which has great research significance and economic value.
[0004] Ferula belongs to the Umbelliferae family, also known as aniseed. Because of its unpleasant smell, it is known as the "devil's feces". There are about 150 species in the world, which are mainly distributed in the Mediterranean, Central Asia and its neighboring areas; there are 26 variants in my country, mainly distributed in Xinjiang. This genus has many medicinal species and has a long history of use. It has the functions of stopping diarrhea, eliminating accumulation, and detoxifying. It is often used by the people to treat digestive system diseases. The "Chinese Pharmacopoeia" contains the resins of Xinjiang Ferula Ferulasinkiangensis KM Shen and Fukang Ferula F.fukangensis KMShen. There are a lot of reports on the research of Ferula at home and abroad. In recent years, some of them have attracted much attention because of the discovery that some of them have phytoestrogen active ingredients and anti-cancer substances and anti-HIV activity and substances that prevent the release of cytoplasm.
[0005] In the article "Study on the Chemical Components of the Uyghur Medicine Ferula multiflora" in Journal of Beijing Institute of Technology (1001-0645 (2021) 00-0001-08), 15 monomer components were separated and identified from the ethyl acetate layer extract of Ferula multiflora using various column chromatography separation techniques and modern spectroscopic techniques. Compound 1 was determined to be a new compound based on Scifinder and Reaxy database retrieval, and compound 9 was isolated from this plant for the first time. The absolute configuration of compound 1 was determined for the first time by calculating the ECD method. The results of the activity screening experiment showed that compounds 3, 4, 5 and 15 exhibited potential cytotoxic activity in MCF-7 cells, indicating that sesquiterpenoid compounds containing phenolic hydroxyl groups have a good inhibitory effect on human breast cancer cells, providing certain help for the further development of anti-tumor drugs.
[0006] The pharmacological efficacy of Xinjiang Ferula endophytic fungus strain N93 mainly comes from alkaloid compounds. Therefore, developing and utilizing the alkaloid monomer compounds of Xinjiang Ferula endophytic fungus strain N93, further exploring its potential medicinal value, and determining and characterizing the structure and physicochemical properties of its monomer compounds are of great significance to the development and utilization of Xinjiang Ferula endophytic fungus strain N93. Summary of the invention
[0007] The present invention provides an anti-tumor component in Xinjiang Ferula endophyte and a method for extracting and separating the component and application thereof, which overcomes the deficiencies of the above-mentioned prior art and discloses for the first time the anti-tumor component in Xinjiang Ferula endophyte and its application in preparing a drug for preventing breast cancer or / and preparing an anti-breast cancer drug.
[0008] One of the technical solutions of the present invention is achieved by the following measures: an anti-tumor component in the endophytic bacteria of Xinjiang Ferula, whose chemical structure is .
[0009] The following is a further optimization and / or improvement of one of the above-mentioned technical solutions: The anti-tumor component in the Xinjiang Ferula endophyte is obtained according to the following steps: The first step is to activate the Xinjiang Ferula endophytic fungus strain N93 with anti-tumor activity and use rice culture medium to culture the secondary metabolites of the strain; The second step is to use ethanol to extract the secondary metabolites, soak them at room temperature for 10 to 14 hours, and then perform ultrasonic reflux extraction for 3 times, combine the reflux extracts for each time, recover them under reduced pressure, and concentrate them to obtain the total extract of Xinjiang Ferula endophytic fungus strain N93; The third step is to disperse the total extract of Xinjiang Ferula endophytic fungus strain N93 with water, and extract it with dichloromethane and ethyl acetate in sequence to obtain a dichloromethane extract and an ethyl acetate extract; The fourth step is to take the extract of the dichloromethane part and separate it by gradient elution with silica gel column chromatography to obtain 8 fractions, wherein the eluent used in the gradient elution with silica gel column chromatography includes dichloromethane and methanol; In the fifth step, the fifth fraction of the eight fractions obtained is purified and separated by high performance liquid chromatography gradient elution, and the eluate is collected to obtain the target product, the anti-tumor component of the endophytic bacteria of Ferula xinjiangensis, at 21.3 minutes, wherein the eluent used in the high performance liquid chromatography gradient elution is a mixture of methanol and water.
[0010] In the first step, 8 mL to 12 mL of 95% ethanol was added to every 1 g of Xinjiang Ferula endophytic fungus strain N93.
[0011] In the second step, the ultrasonic reflux operation time is 10 minutes to 20 minutes.
[0012] In the second step, the temperature of the heating reflux operation is 50° C. to 60° C., and the reflux time each time is 1 hour to 3 hours.
[0013] In the fourth step, the volume ratios of dichloromethane and methanol are 1:0, 100:1, 30:1, 20:1, 10:1, 5:1, 2:1 and 0:1 respectively.
[0014] In the fifth step, the volume ratio of methanol to water is 40:60.
[0015] The second technical solution of the present invention is achieved by the following measures: A method for extracting and separating anti-tumor components from Xinjiang Ferula endophytes is carried out according to the following steps: The first step is to activate the Xinjiang Ferula endophytic fungus strain N93 with anti-tumor activity and use rice culture medium to culture the secondary metabolites of the strain; The second step is to use ethanol to extract the secondary metabolites, soak them at room temperature for 10 to 14 hours, and then perform ultrasonic reflux extraction for 3 times, combine the reflux extracts for each time, recover them under reduced pressure, and concentrate them to obtain the total extract of Xinjiang Ferula endophytic fungus strain N93; The third step is to disperse the total extract of Xinjiang Ferula endophytic fungus strain N93 with water, and extract it with dichloromethane and ethyl acetate in sequence to obtain a dichloromethane extract and an ethyl acetate extract; The fourth step is to take the extract of the dichloromethane part and separate it by gradient elution with silica gel column chromatography to obtain 8 fractions, wherein the eluent used in the gradient elution with silica gel column chromatography includes dichloromethane and methanol; In the fifth step, the fifth fraction of the eight fractions obtained is purified and separated by high performance liquid chromatography gradient elution, and the eluate is collected to obtain the target product, the anti-tumor component of the endophytic bacteria of Ferula xinjiangensis, at 21.3 minutes, wherein the eluent used in the high performance liquid chromatography gradient elution is a mixture of methanol and water.
[0016] The third technical solution of the present invention is achieved through the following measures: an anti-tumor component in the endophytic bacteria of Xinjiang Ferula is used as a drug for preparing a breast cancer prevention drug.
[0017] The fourth technical solution of the present invention is achieved through the following measures: an application of an anti-tumor component in the endophytic bacteria of Ferula foetida in Xinjiang as a preparation of an anti-breast cancer drug.
[0018] The present invention discloses for the first time the anti-tumor component alkaloid compound ferupencine B in the endophytic bacteria of Xinjiang Ferula, and conducts an in vitro anti-cancer pharmacodynamic experiment on the compound on MCF-7 (human breast cancer cells). The experiment clearly shows that the compound has a strong inhibitory effect on MCF-7 cells, so that the compound can be used as a drug for preventing breast cancer or / and for preparing anti-breast cancer drugs. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Attached Figure 1 The figure is a chemical structure diagram of ferupencine B, an anti-tumor component in the endophytic bacteria of Xinjiang Ferula described in the present invention.
[0020] Attached Figure 2 The invention discloses a novel anti-tumor component ferupencine B in the endophytic bacteria of Xinjiang Ferula. 1 H-NMR spectrum.
[0021] Attached Figure 3 The invention discloses a novel anti-tumor component ferupencine B in the endophytic bacteria of Xinjiang Ferula. 13 C-APT spectrum. DETAILED DESCRIPTION
[0022] The present invention is not limited by the following embodiments, and the specific implementation method can be determined according to the technical scheme of the present invention and the actual situation. 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; the normal temperature and room temperature in the present invention generally refer to the temperature of 15°C to 25°C, and are generally defined as 25°C.
[0023] The present invention will be further described below in conjunction with embodiments: Example 1: The anti-tumor component in the Xinjiang Ferula endophyte has the chemical structural formula .
[0024] The anti-tumor components in the Xinjiang Ferula endophyte of the present invention were subjected to nuclear magnetic resonance spectroscopy ( 1 H-NMR) and carbon nuclear magnetic resonance spectroscopy ( 13 C-APT) analysis, anti-tumor components 1 H-NMR spectrum Figure 2 As shown, the anti-tumor components 13 C-APT spectrum Figure 3 As shown, Figure 2 and Figure 3 Perform spectrum analysis and compare spectrum 2 and Figure 3 Each peak is assigned Figure 2 and Figure 3 The peak assignments are shown in Table 1. From the data in Table 1, it can be seen that the chemical structural formula of the anti-tumor component of the present invention is as follows Figure 1 As shown, it is easily soluble in chloroform and methanol.
[0025] Embodiment 2: As an optimization of the above embodiment, the following steps are performed: The first step is to activate the Xinjiang Ferula endophytic fungus strain N93 with anti-tumor activity and use rice culture medium to culture the secondary metabolites of the strain; The second step is to use ethanol to extract the secondary metabolites, soak them at room temperature for 10 to 14 hours, and then perform ultrasonic reflux extraction for 3 times, combine the reflux extracts for each time, recover them under reduced pressure, and concentrate them to obtain the total extract of Xinjiang Ferula endophytic fungus strain N93; The third step is to disperse the total extract of Xinjiang Ferula endophytic fungus strain N93 with water, and extract it with dichloromethane and ethyl acetate in sequence to obtain a dichloromethane extract and an ethyl acetate extract; The fourth step is to take the extract of the dichloromethane part and separate it by gradient elution with silica gel column chromatography to obtain 8 fractions, wherein the eluent used in the gradient elution with silica gel column chromatography includes dichloromethane and methanol; In the fifth step, the fifth fraction of the eight fractions obtained is purified and separated by high performance liquid chromatography gradient elution, and the eluate is collected to obtain the target product, the anti-tumor component of the endophytic bacteria of Ferula xinjiangensis, at 21.3 minutes, wherein the eluent used in the high performance liquid chromatography gradient elution is a mixture of methanol and water.
[0026] In the present invention, the Xinjiang Ferula endophytic fungus strain N93 is preserved at the General Microbiological Center of China Microbiological Culture Collection Administration, with the culture collection number of 41708. The biological material sample is classified and named Penicillium sp. The unit that preserves the biological material sample is the General Microbiology Center of China Microbiological Culture Collection Administration. The address of the preservation unit is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The preservation date is December 11, 2024. The preservation number is CGMCC No.41708
[0027] Example 3: As an optimization of the above example, in the second step, 8 mL to 12 mL of 95% by mass ethanol was added to every 1 g of Xinjiang Ferula endophytic fungus strain N93.
[0028] Embodiment 4: As an optimization of the above embodiment, in the second step, the ultrasonic reflux operation time is 10 minutes to 20 minutes.
[0029] Embodiment 5: As an optimization of the above embodiment, in the second step, the temperature of the heating reflux operation is 50°C to 60°C, and the reflux time each time is 1 hour to 3 hours.
[0030] Example 6: As an optimization of the above example, in the fourth step, the volume ratios of dichloromethane and methanol are 1:0, 100:1, 30:1, 20:1, 10:1, 5:1, 2:1, and 0:1, respectively.
[0031] Example 7: As an optimization of the above example, in the fifth step, the volume ratio of methanol to water is 40:60.
[0032] Embodiment 8: The method for extracting and separating the anti-tumor components from the endophytic bacteria of Xinjiang Ferula is characterized by following the following steps: The first step is to activate the Xinjiang Ferula endophytic fungus strain N93 with anti-tumor activity and use rice culture medium to culture the secondary metabolites of the strain; The second step is to use ethanol to extract the secondary metabolites, soak them at room temperature for 10 to 14 hours, and then perform ultrasonic reflux extraction for 3 times, combine the reflux extracts for each time, recover them under reduced pressure, and concentrate them to obtain the total extract of Xinjiang Ferula endophytic fungus strain N93; The third step is to disperse the total extract of Xinjiang Ferula endophytic fungus strain N93 with water, and extract it with dichloromethane and ethyl acetate in sequence to obtain a dichloromethane extract and an ethyl acetate extract; The fourth step is to take the extract of the dichloromethane part and separate it by gradient elution with silica gel column chromatography to obtain 8 fractions, wherein the eluent used in the gradient elution with silica gel column chromatography includes dichloromethane and methanol; In the fifth step, the fifth fraction of the eight fractions obtained is purified and separated by high performance liquid chromatography gradient elution, and the eluate is collected to obtain the target product, the anti-tumor component of the endophytic bacteria of Ferula xinjiangensis, at 21.3 minutes, wherein the eluent used in the high performance liquid chromatography gradient elution is a mixture of methanol and water.
[0033] Example 9: Application of the anti-tumor component in the Xinjiang Ferula endophyte as a drug for the preparation of a breast cancer prevention drug.
[0034] Example 10: Application of the anti-tumor component in the Xinjiang Ferula endophyte as a preparation of anti-breast cancer drugs.
[0035] Example 11: The anti-tumor component in the Xinjiang Ferula endophyte was obtained according to the following method: The first step is to activate the Xinjiang Ferula endophytic fungus strain N93 with anti-tumor activity and use rice culture medium to culture the secondary metabolites of the strain; In the second step, the secondary metabolites were extracted with ethanol, and after being soaked at room temperature for 13 hours, ultrasonic reflux was performed for 15 minutes, and reflux extraction was performed at 55°C for 3 times, each time for 2 hours, and the reflux extracts were combined and recovered under reduced pressure and concentrated to obtain the total extract of Xinjiang Ferula endophytic fungus strain N93; The third step is to disperse the total extract of Xinjiang Ferula endophytic fungus strain N93 with water, and extract it with dichloromethane and ethyl acetate in sequence to obtain a dichloromethane extract and an ethyl acetate extract; The fourth step is to obtain 8 fractions by gradient elution of the extract of the dichloromethane part by silica gel column chromatography, wherein the eluents used in the gradient elution of the silica gel column chromatography include dichloromethane and methanol, and the volume ratios of dichloromethane and methanol are 1:0, 100:1, 30:1, 20:1, 10:1, 5:1, 2:1, and 0:1, respectively; In the fifth step, the fifth fraction of the eight fractions was purified and separated by high performance liquid chromatography gradient elution, and the eluate was collected to obtain the target product, the alkaloid compound ferupencine B, at 21.3 minutes. The eluent used in the high performance liquid chromatography gradient elution was a mixture of methanol and water, and the volume ratio of methanol to water was 40:60.
[0036] The anti-tumor component alkaloid compound ferupencine B in the Xinjiang Ferula endophyte prepared in Example 11 of the present invention was subjected to an in vitro anti-tumor pharmacodynamics experiment, and the in vitro anti-tumor pharmacodynamics experiment used the MTT colorimetric method.
[0037] Ferupencine B was used as the experimental group, and Cisplatin was used as the control group. MCF-7 (human breast cancer cells) were selected as the experimental objects in the experimental and control groups. After the culture medium was diluted, 4×10 5 The density of the cells was inoculated in a 96-well plate, 100 μL per well. After normal culture in an incubator for 24 hours, the corresponding drugs were added to each group, so that the final concentrations of the drugs in each group were 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. There were 5 concentrations in total, and 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 discarded, 150 μL of DMSO was added to each well, and the cells were shaken at a low speed on a shaker for 10 minutes to fully dissolve the crystals. The optical density value was detected at a wavelength of 570 nm using an enzyme-linked immunosorbent assay, and the 50% inhibitory concentration (IC) was calculated based on the optical density value. 50 , μg / mL), optical density value was used to calculate IC 50 The calculation method of IC of MCF-7 cells in the experimental group and the control group is known in the art. 50 As shown in Table 2. From the data in Table 2, it can be seen that the alkaloid compound ferupencine B of the present invention has a certain inhibitory effect on MCF-7 cells.
[0038] In summary, the present invention discloses for the first time the anti-tumor component alkaloid compound ferupencine B in the endophytic bacteria of Xinjiang Ferula. The ferupencine B described in the present invention has a certain inhibitory effect on MCF-7 cells, so that the anti-tumor component in the endophytic bacteria of Xinjiang Ferula can be used as a drug for preventing breast cancer or / and preparing anti-breast cancer drugs.
[0039] The above technical features respectively constitute various embodiments of the present invention, which have strong adaptability and implementation effect. Non-essential technical features can be added or reduced according to actual needs to meet the requirements of different situations.
[0040] Table 1 .
[0041] Table 2 .
Claims
1. An anti-tumor component from an endophytic bacterium of Ferula foetida, characterized in that Its chemical structure is 。 2. The anti-tumor component of the endophytic bacteria of Xinjiang Ferula according to claim 1, characterized in that Follow the steps below to get: The first step is to activate the Xinjiang Ferula endophytic fungus strain N93 with anti-tumor activity, and use rice culture medium to culture the secondary metabolites of the Xinjiang Ferula endophytic fungus strain N93; The second step is to use ethanol to extract the secondary metabolites, soak them at room temperature for 10 to 14 hours, and then perform ultrasonic reflux extraction for 3 times, combine the reflux extracts for each time, recover them under reduced pressure, and concentrate them to obtain the total extract of Xinjiang Ferula endophytic fungus strain N93; The third step is to disperse the total extract of Xinjiang Ferula endophytic fungus strain N93 with water, and extract it with dichloromethane and ethyl acetate in sequence to obtain a dichloromethane extract and an ethyl acetate extract; The fourth step is to take the extract of the dichloromethane part and separate it by gradient elution with silica gel column chromatography to obtain 8 fractions, wherein the eluent used in the gradient elution with silica gel column chromatography includes dichloromethane and methanol; In the fifth step, the fifth fraction of the eight fractions obtained is purified and separated by high performance liquid chromatography gradient elution, and the eluate is collected to obtain the target product, the anti-tumor component of the endophytic bacteria of Ferula xinjiangensis, at 21.3 minutes, wherein the eluent used in the high performance liquid chromatography gradient elution is a mixture of methanol and water.
3. The anti-tumor component in the endophytic bacteria of Xinjiang Ferula according to claim 2, characterized in that In the second step, 8 mL to 12 mL of 95% ethanol was added to every 1 g of Xinjiang Ferula endophytic fungus strain N93.
4. The anti-tumor component in the endophytic bacteria of Xinjiang Ferula according to claim 2 or 3, characterized in that In the second step, the ultrasonic reflux operation time is 10 min to 20 min.
5. The anti-tumor component in the endophytic bacteria of Xinjiang Ferula according to claim 2, 3 or 4, characterized in that In the second step, the temperature of the heating reflux operation is 50° C. to 60° C., and the reflux time each time is 1 hour to 3 hours.
6. The anti-tumor component in the endophytic bacteria of Xinjiang Ferula according to claim 2, 3 or 4, characterized in that In the fourth step, the volume ratios of dichloromethane and methanol are 1:0, 100:1, 30:1, 20:1, 10:1, 5:1, 2:1, and 0:1, respectively.
7. The anti-tumor component in the endophytic bacteria of Xinjiang Ferula according to claim 2, 3, 4, 5 or 6, characterized in that In the fifth step, the volume ratio of methanol to water is 40:
60.
8. A method for extracting and separating anti-tumor components from the endophytic bacteria of Xinjiang Ferula according to any one of claims 1 to 7, characterized in that Follow the steps below: The first step is to activate the Xinjiang Ferula endophytic fungus strain N93 with anti-tumor activity and use rice culture medium to culture the secondary metabolites of the strain; The second step is to use ethanol to extract the secondary metabolites, soak them at room temperature for 10 to 14 hours, and then perform ultrasonic reflux extraction for 3 times, combine the reflux extracts for each time, recover them under reduced pressure, and concentrate them to obtain the total extract of Xinjiang Ferula endophytic fungus strain N93; The third step is to disperse the total extract of Xinjiang Ferula endophytic fungus strain N93 with water, and extract it with dichloromethane and ethyl acetate in sequence to obtain a dichloromethane extract and an ethyl acetate extract; The fourth step is to take the extract of the dichloromethane part and separate it by gradient elution with silica gel column chromatography to obtain 8 fractions, wherein the eluent used in the gradient elution with silica gel column chromatography includes dichloromethane and methanol; In the fifth step, the fifth fraction of the eight fractions obtained is purified and separated by high performance liquid chromatography gradient elution, and the eluate is collected to obtain the target product, the anti-tumor component of the endophytic bacteria of Ferula xinjiangensis, at 21.3 minutes, wherein the eluent used in the high performance liquid chromatography gradient elution is a mixture of methanol and water.
9. Use of the anti-tumor component in the endophytic bacteria of Ferula ferulae according to any one of claims 1 to 7 as a drug for preventing breast cancer.
10. Use of the anti-tumor component in the endophytic bacteria of Ferula ferulae according to any one of claims 1 to 7 as a drug for preparing anti-breast cancer drugs.