Alkaloid compound in ferula sinkiangensis endophyte as well as preparation method and application of alkaloid compound
By isolating and purifying the alkaloid compound ferupencine A in Xinjiang endophytes, the problem of the endangered extinction of the asafoetida resource in Xinjiang has been solved, and the effective preparation of this alkaloid compound and the potential application of anti-tumor drugs have been achieved.
Patent Information
- Application Number
- CN202510276001.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-10
- Publication Date
- 2025-06-13
AI Technical Summary
Xinjiang's asafoetida resources are on the verge of extinction, and the existing technology is difficult to effectively utilize the alkaloid compounds in its endophytic fungi, resulting in the failure to fully utilize its medicinal value.
The alkaloid compound ferupencine A in Xinjiang endophytes was isolated and purified, and the alkaloid compound was successfully prepared by using rice culture medium and ethanol extraction method, combined with silica gel column chromatography and high performance liquid chromatography technology.
Effective isolation and purification of alkaloid compounds in Xinjiang asaveophageal bacteria, has an inhibitory effect on MCF-7 human breast cancer cells, and can be used to prepare tumor prevention and anti-tumor drugs.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of isolation and purification of endophytic fungal strains of Ferula, and is an alkaloid compound in endophytes of Ferula sinkiangensis in Xinjiang, its preparation method and application. The alkaloid compound in endophytes of Ferula sinkiangensis is abbreviated as ferupencine A. Background Art
[0002] Ferula is a kind of gum secreted from the flower stems of plants of the genus Ferula (Ferula L.) in the Umbelliferae family, with a special smell of onion and garlic. Medicinal Ferula is a kind of plant resource with important economic value. There are about 150 species of Ferula plants in the world, mainly distributed in the Mediterranean, Central Asia and its adjacent regions. There are 26 species and 1 variety in China, mainly distributed in Xinjiang. The plant origin recorded in the Pharmacopoeia of the People's Republic of China is Ferula sinkiangensis K. M. Shen and Ferula fukanggensis K. M. shen, which have the effects of treating malaria, eliminating indigestion, detoxifying, killing insects, reducing phlegm, dispelling wind, promoting blood circulation and dredging channels, and are commonly used in folk medicine to treat digestive system diseases. Modern research shows that Ferula has a wide range of pharmacological activities such as anti-fertility, immunosuppression, antipyretic, analgesic, anti-inflammatory, and anti-tumor, and has attracted much attention in recent years due to the discovery of anti-cancer substances in it.
[0003] As one of the medicinal material sources of genuine Ferula, the perennial herb Ferula sinkiangensis has a narrow resource distribution. Also, due to its special biological characteristics, that is, the production of gum only occurs during the flowering and fruiting period of Ferula, and the plant dies after flowering and fruiting once, resulting in a shortage in the Ferula resin market and the resource being on the verge of extinction. It is included in both the China Species Red List and the White Paper. Therefore, it is extremely urgent to find alternative resources for Ferula sinkiangensis.
[0004] Endophytic fungi are a type of fungi that live inside plants for part or all of their life cycle without causing obvious disease symptoms in the host plants. During the long process of symbiosis with the host plants, they have established a mutually beneficial relationship. In 1993, Stierle isolated an active strain Taxomyces andreanae from the phloem of Taxus brevifolia, and then isolated an important anti-cancer drug, taxol, from it. Since then, the research on searching for and discovering new bioactive substances from endophytic fungi has received increasing attention from scientific researchers. The secondary metabolites of endophytic fungi have rich structural types, including alkaloids, polyketides, terpenoids, sterols, anthraquinones, flavonoids, xanthines, phenols, perylene derivatives, furandiones, and cyclic peptides, etc. At the same time, the biofunctional activities of these metabolites are also diverse, including antibacterial, antiviral, anti-cancer, antioxidant, insecticidal, anti-diabetic, and immunosuppressive activities. A large number of studies have shown that endophytic bacteria can not only participate in the secondary metabolism and transformation synthesis of plants, promote plant growth, but also produce active ingredients that are the same or similar to those of the host plants, and are an important source of natural drugs. In addition, the characteristics of fast reproduction, easy cultivation, easy control, high yield, low cost, and not affected by seasonal changes of endophytic fungi provide the possibility for them to become reasonable substitutes for medicinal plants. Therefore, the active metabolites of endophytic fungi can be used as potential leads for antibiotics, anti-cancer drugs, or pesticides, and have great research significance and economic value.
[0005] The medicinal efficacy of the endophytic fungus strain N93 of Ferula sinkiangensis mainly comes from alkaloid compounds. Therefore, developing and utilizing the monomeric alkaloid compounds of the endophytic fungus strain N93 of Ferula sinkiangensis, further exploring its potential medicinal value, and determining and characterizing the structure and physicochemical properties of its monomeric compounds are of great significance for the development and utilization of the endophytic fungus strain N93 of Ferula sinkiangensis. Summary of the Invention
[0006] The present invention provides an alkaloid compound in endophytic bacteria of Ferula sinkiangensis, its preparation method, and its application as a preparation for preventing and treating tumors, overcoming the above-mentioned deficiencies of the prior art. The present invention discloses for the first time an alkaloid compound (denoted as ferupencine A) in endophytic bacteria of Ferula sinkiangensis, enabling it to be applied in the preparation of drugs for preventing tumors and treating tumors.
[0007] One of the technical solutions of the present invention is achieved by the following measures: An alkaloid compound in endophytic bacteria of Ferula sinkiangensis, with the chemical structural formula: .
[0008] The following is a further optimization and / or improvement of one of the above-mentioned inventive technical solutions: The alkaloid compound (ferupencine A) in the endophyte of Ferula sinkiangensis is prepared according to the following steps: First step: Activate the endophytic fungus strain N93 of Ferula sinkiangensis, culture the secondary metabolites of the strain using a rice medium, extract the secondary metabolites with ethanol, and concentrate the extract to obtain an extract of the endophytic fungus strain N93 of Ferula sinkiangensis. Second step: Disperse the extract of the endophytic fungus strain N93 of Ferula sinkiangensis in water to form a suspension, and then extract it successively with dichloromethane and ethyl acetate. After concentrating the dichloromethane extract and the ethyl acetate extract respectively, an extract of the dichloromethane fraction and an extract of the ethyl acetate fraction are obtained. Third step: Perform gradient elution on the extract of the dichloromethane fraction using silica gel column chromatography, and separate it to obtain 8 fractions. Fourth step: Separate and purify the 5th fraction by high performance liquid chromatography to obtain the alkaloid compound (ferupencine A) in the endophyte of Ferula sinkiangensis.
[0009] In the above first step, the composition of the rice medium includes: adding 100 mL of water to every 80 g of rice, and the culture conditions are: culturing in the dark at 26 °C.
[0010] In the above first step, the specific operation of extracting the secondary metabolites with ethanol is as follows: Step 1: Immerse the cultured endophytic fungus strain N93 of Ferula sinkiangensis in 95% ethanol (volume fraction) for 10 to 14 hours, and perform ultrasonic extraction for 15 min. Step 2: Repeat Step 1 two to four times, and combine the extracts.
[0011] In the above Step 1, add 8 mL to 12 mL of 95% ethanol to every 1 g of the endophytic fungus strain N93 of Ferula sinkiangensis.
[0012] In the above third step, the eluent for gradient elution is a mixed solution of dichloromethane and methanol, and the volume ratios of dichloromethane and methanol for gradient elution are successively 1:0, 100:1, 30:1, 20:1, 10:1, 5:1, 2:1, 0:1.
[0013] In the above fourth step, the chromatographic conditions for high performance liquid chromatography separation and purification are: the chromatographic column is a Sephadex LH-20 column, the column temperature is 25 °C, the eluent is a mixed solution of methanol and water, the volume ratio of methanol and water is 25:75, and the flow rate is 2 mL / min.
[0014] The second technical solution of the present invention is achieved by the following measures: A method for preparing alkaloid compound (ferupencine A) from endophytic fungi of Ferula sinkiangensis, which is carried out according to the following steps: First step, activate the endophytic fungus strain N93 of Ferula sinkiangensis, cultivate the secondary metabolites of the strain using rice medium, extract the secondary metabolites with ethanol, and obtain the extract of the endophytic fungus strain N93 of Ferula sinkiangensis after concentrating the extract. Second step, disperse the extract of the endophytic fungus strain N93 of Ferula sinkiangensis into a suspension with water, and then extract it successively with dichloromethane and ethyl acetate. After concentrating the dichloromethane and ethyl acetate extracts respectively, obtain the dichloromethane fraction extract and the ethyl acetate fraction extract. Third step, perform gradient elution on the dichloromethane fraction extract using silica gel column chromatography, and obtain 8 fractions after separation. Fourth step, separate and purify the 5th fraction by high performance liquid chromatography to obtain the alkaloid compound (ferupencine A) from the endophytic fungi of Ferula sinkiangensis.
[0015] The third technical solution of the present invention is achieved by the following measures: An application of the alkaloid compound (ferupencine A) from the endophytic fungi of Ferula sinkiangensis in the preparation of anti-tumor prevention drugs.
[0016] The fourth technical solution of the present invention is achieved by the following measures: An application of the alkaloid compound (ferupencine A) from the endophytic fungi of Ferula sinkiangensis in the preparation of anti-tumor drugs.
[0017] The alkaloid compound (ferupencine A) from the endophytic fungi of Ferula sinkiangensis in the present invention has a certain inhibitory effect on MCF-7 human breast cancer cells, so that the alkaloid compound from the endophytic fungi of Ferula sinkiangensis in the present invention can be applied in the preparation of anti-tumor prevention drugs and anti-tumor drugs. Description of the Drawings
[0018] Attached Figure 1 is the 1 1H-NMR spectrum of the alkaloid compound (ferupencine A) from the endophytic fungi of Ferula sinkiangensis in the present invention.
[0019] Attached Figure 2 is the 13 13C-APT spectrum of the alkaloid compound (ferupencine A) from the endophytic fungi of Ferula sinkiangensis in the present invention. Detailed Embodiments
[0020] The present invention is not limited by the following embodiments, and specific implementation manners can be determined according to the technical solution of the present invention and actual situations. Various chemical reagents and chemical supplies mentioned in the present invention are well-known and commonly used chemical reagents and chemical supplies in the prior art unless otherwise specified; the percentages in the present invention are mass percentages unless otherwise specified; normal temperature and room temperature in the present invention generally refer to the temperature from 15°C to 25°C, and are generally defined as 25°C.
[0021] The present invention will be further described below in conjunction with embodiments: Example 1: The alkaloid compound (denoted as: ferupencine A) in the endophyte of Ferula sinkiangensis has the following chemical structural formula: .
[0022] The systematic name of the alkaloid compound (denoted as: ferupencine A) in the endophyte of Ferula sinkiangensis of the present invention is: (3 R , 5 S , 6 S ) 6-methyl-2H-pyrrolo-oxazin-10-propanoic acid; the Chinese name is: (3 R , 5 S ,6 S ) 6-methyl-2H-pyrrolo-oxazine-10-propanoic acid.
[0023] In the present invention, the alkaloid compound (ferupencine A) in the endophyte of Ferula sinkiangensis is isolated and purified from the endophytic fungus strain N93 of Ferula sinkiangensis. The preservation location of the endophytic fungus strain N93 of Ferula sinkiangensis is the General Microbiology Center of the China Committee for Culture Collection of Microorganisms; the strain preservation number is 41708.
[0024] Example 2: As an optimization of the above example, the alkaloid compound (ferupencine A) in the endophyte of Ferula sinkiangensis is prepared according to the following steps: First step, activate the endophytic fungus strain N93 of Ferula sinkiangensis, and use rice medium to culture the secondary metabolites of the strain. Extract the secondary metabolites with ethanol, and concentrate the extract to obtain the extract of the endophytic fungus strain N93 of Ferula sinkiangensis; Second step, disperse the extract of the endophytic fungus strain N93 of Ferula sinkiangensis in water to form a suspension, and then extract it successively with dichloromethane and ethyl acetate. After concentrating the dichloromethane and ethyl acetate extracts respectively, obtain the dichloromethane fraction extract and the ethyl acetate fraction extract; Third step, perform gradient elution on the dichloromethane fraction extract with silica gel column chromatography, and separate to obtain 8 fractions; Step 4: Subject the 5th fraction to high-performance liquid chromatography for separation and purification to obtain the alkaloid compound (ferupencine A) from the endophytic fungi of Ferula sinkiangensis.
[0025] Example 3: As an optimization of the above example, in the first step, the composition of the rice medium includes: adding 100 mL of water to every 80 g of rice, and the culture conditions are: culturing in the dark at 26 °C.
[0026] Example 4: As an optimization of the above example, in the first step, the specific operation of extracting the secondary metabolites using ethanol is as follows: Step 1: Immerse the cultured endophytic fungus strain N93 of Ferula sinkiangensis in 95% ethanol (volume fraction) for 10 to 14 hours, and perform ultrasonic extraction for 15 min; Step 2: Repeat Step 1 two to four times, and combine the extracts.
[0027] Example 5: As an optimization of the above example, in Step 1, add 8 mL to 12 mL of 95% ethanol to every 1 g of the endophytic fungus strain N93 of Ferula sinkiangensis.
[0028] Example 6: As an optimization of the above example, in the third step, the eluent for gradient elution is a mixed solution of dichloromethane and methanol, and the volume ratios of dichloromethane to methanol for gradient elution are 1:0, 100:1, 30:1, 20:1, 10:1, 5:1, 2:1, 0:1 in sequence.
[0029] Example 7: As an optimization of the above example, in the fourth step, the chromatographic conditions for high-performance liquid chromatography separation and purification are: the chromatographic column is a Sephadex LH-20 column, the column temperature is 25 °C, the eluent is a mixed solution of methanol and water, the volume ratio of methanol to water is 25:75, and the flow rate is 2 mL / min.
[0030] Example 8: Application of the alkaloid compound (ferupencine A) from the endophytic fungi of Ferula sinkiangensis in the preparation of drugs for preventing tumors.
[0031] Example 9: Application of the alkaloid compound (ferupencine A) from the endophytic fungi of Ferula sinkiangensis in the preparation of anti-tumor drugs.
[0032] Example 10: The alkaloid compound (ferupencine A) from the endophytic fungi of Ferula sinkiangensis is prepared according to the following steps: First step: Activate the endophytic fungus strain N93 of Ferula sinkiangensis, and use a rice culture medium (80 g of rice : 100 mL of water) to culture the secondary metabolites of the strain (cultivate in the dark at 26 °C), and extract the secondary metabolites with 95% ethanol. Add 10 mL of 95% ethanol to every 1 g of the endophytic fungus strain N93 of Ferula sinkiangensis. The extraction steps are as follows: soak at room temperature for 12 hours, then ultrasonicate for 15 min, repeat the soaking extraction three times, combine the ultrasonic extracts each time and recover and concentrate under reduced pressure to obtain the extract of the endophytic fungus strain N93 of Ferula sinkiangensis.
[0033] Second step: Disperse the extract of the endophytic fungus strain N93 of Ferula sinkiangensis into a suspension with water, and then extract it successively with dichloromethane and ethyl acetate. After concentrating the dichloromethane and ethyl acetate extracts respectively, obtain the dichloromethane fraction extract and the ethyl acetate fraction extract.
[0034] Third step: Perform gradient elution on the dichloromethane fraction extract using silica gel column chromatography, and 8 fractions are obtained after separation; the eluent is 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, 0:1 in sequence.
[0035] Fourth step: Separate and purify the 5th fraction by high-performance liquid chromatography. The chromatographic conditions are as follows: the chromatographic column is an ephadexLH-20 column, the column temperature is 25 °C, the eluent is a mixed solution of methanol and water, the volume ratio of methanol and water is 25:75, and the flow rate is 2 mL / min. After separation by high-performance liquid chromatography, the alkaloid compound (ferupencine A) in the endophytic fungus of Ferula sinkiangensis is obtained at 29.2 minutes.
[0036] Perform nuclear magnetic resonance hydrogen spectrum ( 1 H-NMR) and nuclear magnetic resonance carbon spectrum ( 13 C-APT) analysis on the alkaloid compound (ferupencine A) in the endophytic fungus of Ferula sinkiangensis prepared in this example. The 1 H-NMR spectrum of (ferupencine A) described in this example is as shown in Figure 1 . The 13 C-APT spectrum of the alkaloid compound (ferupencine A) in the endophytic fungus of Ferula sinkiangensis is as shown in Figure 2 . Perform spectral analysis on Figure 1 and Figure 2 , assign each peak, and the peak assignments of Figure 1 and Figure 2 are shown in Table 1. From the data in Table 1, it can be seen that the chemical structural formula of the alkaloid compound (ferupencine A) in the endophytic fungus of Ferula sinkiangensis is as follows, and it is easily soluble in chloroform and methanol.
[0037] The alkaloid compound (ferupencine A) in the endophyte of Ferula sinkiangensis of the present invention was subjected to in vitro anti-tumor pharmacodynamic experiments, and the MTT colorimetric method was used for the in vitro anti-tumor pharmacodynamic experiments.
[0038] Taking the alkaloid compound (ferupencine A) in the endophyte of Ferula sinkiangensis as the experimental group, Cisplatin as the control group, and a blank group was established at the same time. The MCF-7 (human breast cancer cells) cells were selected as the experimental objects for the experimental group, the control group and the blank group. After dilution with the culture medium, they were inoculated into a 96-well plate at a density of 4×10 5 , 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), 200 μg / mL (Group 5), a total of 5 concentrations were set, and there were 3 replicates for each concentration; after culturing for 48 hours, 10 μL of MTT was added to each well for staining; after continuing to culture for four hours, the original culture medium was aspirated, 150 μL of DMSO was added to each well, and it was placed on a shaker and shaken at a low speed for 10 min to fully dissolve the crystals, and the optical density value was detected at a wavelength of 570 nm by an enzyme-linked immunosorbent detector. The 50% inhibitory concentration (IC 50 , μg / mL) was calculated according to the optical density value. The calculation method of the optical density value for calculating IC 50 is a publicly known technology. The IC 50 of the experimental group and the control group on MCF-7 cells is shown in Table 2. It can be seen from the data in Table 2 that the alkaloid compound (ferupencine A) in the endophyte of Ferula sinkiangensis of the present invention has a certain inhibitory effect on MCF-7 cells.
[0039] In summary, the present invention discloses for the first time the alkaloid compound (ferupencine A) in the endophyte of Ferula sinkiangensis. The alkaloid compound (ferupencine A) in the endophyte of Ferula sinkiangensis has a certain inhibitory effect on MCF-7 cells, so that the alkaloid compound (ferupencine A) in the endophyte of Ferula sinkiangensis of the present invention can be used as an application for preparing anti-tumor drugs and anti-tumor drugs.
[0040] The above technical features respectively constitute the embodiments of the present invention, which have strong adaptability and implementation effects. Non-essential technical features can be added or reduced according to actual needs to meet the requirements of different situations.
[0041]
Claims
1. An alkaloid compound in an endophytic bacterium of Xinjiang Ferula, characterized in that The chemical structure is: 。 2. The alkaloid compound in the endophytic bacteria of Xinjiang Ferula according to claim 1, characterized in that Prepared according to the following steps: The first step is to activate the Xinjiang Ferula endophytic fungus strain N93, and use rice culture medium to culture the secondary metabolites of the strain, use ethanol to extract the secondary metabolites, and concentrate the extract to obtain the Ferula endophytic fungus strain N93 extract; The second step is to disperse the extract of the endophytic fungus strain N93 of Ferula into a suspension with water, extract it with dichloromethane and ethyl acetate in sequence, and concentrate the dichloromethane and ethyl acetate extracts to obtain dichloromethane extract and ethyl acetate extract respectively; The third step is to perform gradient elution on the extract of the dichloromethane part by silica gel column chromatography to obtain 8 fractions after separation; The fourth step is to separate and purify the fifth fraction by high performance liquid chromatography to obtain the alkaloid compounds in the endophytic bacteria of Ferula xinjiangensis.
3. The alkaloid compound in the endophytic bacteria of Xinjiang Ferula according to claim 2, characterized in that In the first step, the rice culture medium composition includes: adding 100 mL of water to every 80 g of rice, and the culture conditions are: culture in the dark at 26°C.
4. The alkaloid compound in the endophytic bacteria of Xinjiang Ferula according to claim 2 or 3, characterized in that In the first step, the specific operation of extracting secondary metabolites using ethanol is as follows: Step 1, soaking the cultured Xinjiang Ferula endophytic fungus strain N93 in 95% ethanol for 10 to 14 hours, and ultrasonically extracting for 15 minutes; Step 2: Repeat step 1 2 to 4 times and combine the extracts.
5. The alkaloid compound in the endophytic bacteria of Xinjiang Ferula according to claim 4, characterized in that In step 1, 8 mL to 12 mL of 95% ethanol was added to every 1 g of the endophytic fungus strain N93 of Ferula.
6. The alkaloid compound in the endophytic bacteria of Xinjiang Ferula according to any one of claims 2 to 5, characterized in that In the third step, the eluent for gradient elution is a mixture of dichloromethane and methanol, and the volume ratios of dichloromethane and methanol for gradient elution are 1:0, 100:1, 30:1, 20:1, 10:1, 5:1, 2:1, and 0:1, respectively.
7. The alkaloid compound in the endophytic bacteria of Xinjiang Ferula according to any one of claims 2 to 6, characterized in that In the fourth step, the chromatographic conditions for separation and purification by HPLC are as follows: the chromatographic column is a Sephadex LH-20 column, the column temperature is 25° C., the eluent is a mixture of methanol and water, the volume ratio of methanol to water is 25:75, and the flow rate is 2 mL / min.
8. A method for preparing alkaloid compounds from the endophytic bacteria of Xinjiang Ferula according to any one of claims 1 to 7, characterized in that Follow these steps: The first step is to activate the Xinjiang Ferula endophytic fungus strain N93, and use rice culture medium to culture the secondary metabolites of the strain, use ethanol to extract the secondary metabolites, and concentrate the extract to obtain the Ferula endophytic fungus strain N93 extract; The second step is to disperse the extract of the endophytic fungus strain N93 of Ferula into a suspension with water, extract it with dichloromethane and ethyl acetate in sequence, and concentrate the dichloromethane and ethyl acetate extracts to obtain dichloromethane extract and ethyl acetate extract respectively; The third step is to perform gradient elution on the extract of the dichloromethane part by silica gel column chromatography to obtain 8 fractions after separation; The fourth step is to separate and purify the fifth fraction by high performance liquid chromatography to obtain the alkaloid compounds in the endophytic bacteria of Ferula xinjiangensis.
9. Use of the alkaloid compound in the endophytic bacteria of Ferula foetida according to any one of claims 1 to 7 in the preparation of a tumor prevention drug.
10. Use of the alkaloid compound in the endophytic bacteria of Ferula foetida according to any one of claims 1 to 7 in the preparation of anti-tumor drugs.