Alkaloid compound in endophytic fungi of ferula sinkiangensis as well as preparation method and application of alkaloid compound

Abstract

The invention relates to the technical field of ferula asafoetida endophytic fungi separation and purification, in particular to alkaloid compounds in ferula asafoetida endophytic fungi as well as a preparation method and application of the alkaloid compounds in the ferula asafoetida endophytic fungi. And extracting with dichloromethane to obtain dichloromethane part extract, and purifying and separating the dichloromethane part extract by adopting silica gel column chromatography and high performance liquid chromatography to obtain the alkaloid compounds in the endophytic fungi of ferula sinkiangensis. The alkaloid compound in the ferula sinkiangensis endophytic fungi is disclosed for the first time, the compound is subjected to an in-vitro anti-tumor pharmacodynamic experiment, and according to the experiment result, the alkaloid compound has a certain inhibition effect on MCF-7 cells; therefore, the alkaloid compound can be used for preparing the medicine for preventing and treating the breast cancer, and a new medicine is provided for preventing and treating the breast cancer.

Description

Technical Field

[0001] The present invention relates to the technical field of isolation and purification of endophytic fungi of Ferula, and provides an alkaloid compound in endophytic fungi of Ferula sinkiangensis in Xinjiang, a preparation method thereof, and applications thereof. The applications include: the application of the alkaloid compound in endophytic fungi of Ferula sinkiangensis in the preparation of drugs for preventing and / or treating breast cancer, and the application of the alkaloid compound in endophytic fungi of Ferula sinkiangensis in the preparation of drugs for preventing and / or treating human breast cancer. 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, and the 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, resolving phlegm, expelling wind, promoting blood circulation and dredging menstruation, 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, anti-tumor, etc., and has attracted much attention in recent years due to the discovery of anti-cancer substances therein.

[0003] As one of the medicinal material sources of the 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, which is recorded 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 some or all of their life cycles 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

[16] isolated an active strain, Taxomyces andreanae, from the phloem of Taxus brevifolia, and then isolated an important anti-cancer drug, taxol (Stierle A, Strobel G, Stierle D. Taxol and taxane production by Taxomyces andreanae, an endophytic fungus of Pacific yew [J]. Science, 1993, 260: 214-216). Since then, the research on finding and discovering new bioactive substances from endophytic fungi has received increasing attention from scientific researchers. The secondary metabolites of plant endophytic fungi have rich structural types, including alkaloids, polyketones, terpenoids, sterols, anthraquinones, flavonoids, xanthines, phenols, perylene derivatives, furandiones, cyclic peptides and other structural types. At the same time, the biological functional activities of these metabolites are also diverse, including antibacterial, antiviral, anti-cancer, antioxidant, insecticidal, anti-diabetic and immunosuppressive. A large number of studies have shown that plant endophytes can not only participate in the transformation and synthesis of plant secondary metabolites and components, promote plant growth, but also produce active ingredients identical 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 insensitivity to seasonal changes of endophytic fungi provide the possibility for them to become reasonable substitutes for medicinal plants. Therefore, the active metabolites of plant 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 mainly comes from alkaloid compounds. Therefore, developing and utilizing the monomeric alkaloid compounds of the endophytic fungus strain N93 of Ferula, further exploring its potential medicinal value, and determining and characterizing the structures and physicochemical properties of its monomeric compounds are of great significance for the development and utilization of the endophytic fungus strain N93 of Ferula.

[0006] The patent application document with the publication number CN116535318A discloses an extract of Ferula sinkiangensis, its preparation method and application. The disclosed compounds (7R,8R)-sinkiangenone E and (7R,8S)-sinkiangenone E show good anti-inflammatory activity. Compared with L-NAME, they can significantly inhibit the expression of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 produced by LPS-induced RAW264.7 cells. The above two compounds can be used to prepare TNF-α, IL-1β and IL-6 inhibitors, which play an important role in the research and development of anti-inflammatory drugs and can be used to treat chronic inflammatory diseases such as gastritis, enteritis and rheumatoid arthritis, etc.

[0007] The patent application document with the publication number CN116514757A discloses sesquiterpene coumarin compounds in Ferula sinkiangensis, its preparation method and application. It first provides a method for preparing and identifying 10 sesquiterpene coumarin derivatives using the root of Ferula sinkiangensis as the raw material, and systematically evaluates their neuroprotective activity, and clarifies their application in the development and treatment of drugs for neurodegenerative diseases. Summary of the Invention

[0008] The present invention provides an alkaloid compound in endophytic fungi of Ferula sinkiangensis, its preparation method and application, overcoming the deficiencies of the above-mentioned prior art. The present invention first discloses an alkaloid compound (abbreviated as ferupencine C) in endophytic fungi of Ferula sinkiangensis, and first discloses the application of ferupencine C in the preparation of drugs for preventing and treating breast cancer.

[0009] One of the technical solutions of the present invention is achieved by the following measures: An alkaloid compound in endophytic fungi of Ferula sinkiangensis, its chemical structural formula is: .

[0010] The alkaloid compound in endophytic fungi of Ferula sinkiangensis, its chemical name is (3 R , 5 R , 6 S , 8 R , 10 S ,12 S )-8,12-dihydroxy-10-methyloctahydrocyclopenta[b]pyrrolo[2,1-f][1,2,4]oxazin-1-one. The English name is (3 R , 5 R , 6 S , 8 R ,10 S , 12 S)-8,12-dihydroxy-10-methyloctahydrocyclopenta-pyrrolo-oxazin-1-one。

[0011] The following is a further optimization and / or improvement of one of the above-mentioned technical solutions of the invention: The alkaloid compounds in the endophytic fungi of Ferula sinkiangensis are prepared according to the following steps: First step, activate the strain of endophytic fungi N93 of Ferula sinkiangensis, culture the secondary metabolites of the strain, use ethanol to ultrasonically extract the cultured strain of endophytic fungi N93 of Ferula sinkiangensis and its secondary metabolites at least three times, combine the ultrasonic extracts each time and recover and concentrate under reduced pressure to obtain an extract of the strain of endophytic fungi N93 of Ferula sinkiangensis; Second step, disperse the total extract of the strain of endophytic fungi N93 of Ferula sinkiangensis in water to form a suspension, and extract with dichloromethane to obtain an extract of the dichloromethane part; Third step, take the extract of the dichloromethane part and separate it by gradient elution on a silica gel column chromatography to obtain 8 fractions. Among them, the gradient elution solution of the silica gel column chromatography includes dichloromethane and methanol, and the volume ratios of dichloromethane to methanol are 1:0, 100:1, 30:1, 20:1, 10:1, 5:1, 2:1, 0:1 in sequence; Fourth step, purify and separate the 5th fraction by gradient elution on a high performance liquid chromatography, collect the eluate, and obtain the alkaloid compounds in the endophytic fungi of Ferula sinkiangensis at 22.4 minutes.

[0012] In the above first step, 8 ml to 12 ml of 95% ethanol is added to every 1 g of the strain of endophytic fungi N93 of Ferula sinkiangensis.

[0013] In the above first step, the time of each ultrasonic extraction is 10 minutes to 15 minutes, and before each ultrasonic extraction, soak at room temperature for 12 hours to 13 hours.

[0014] In the above first step, use a rice medium to culture the secondary metabolites of the strain, and the culture conditions are: avoiding light, 26 °C.

[0015] The above rice medium is prepared by mixing 80 g of rice with 100 ml of water.

[0016] In the above fourth step, the high performance liquid chromatography eluent is a mixed solution of methanol and water, and among them, the volume ratio of methanol to water is 25:75.

[0017] The second technical solution of the present invention is achieved by the following measures: A preparation method of alkaloid compounds in endophytic fungi of Ferula sinkiangensis 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, use ethanol to ultrasonically extract the cultivated endophytic fungus strain N93 of Ferula sinkiangensis and its secondary metabolites at least 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; Second step: Disperse the total extract of the endophytic fungus strain N93 of Ferula sinkiangensis into a suspension with water, and then extract with dichloromethane to obtain the extract of the dichloromethane part; Third step: Take the extract of the dichloromethane part and separate it by gradient elution on a silica gel column chromatography to obtain 8 fractions. Among them, the gradient elution solution of the silica gel column chromatography includes dichloromethane and methanol, and the volume ratios of dichloromethane to methanol are 1:0, 100:1, 30:1, 20:1, 10:1, 5:1, 2:1, 0:1 in sequence; Fourth step: Purify and separate the 5th fraction by gradient elution on a high performance liquid chromatography, collect the eluate, and obtain the alkaloid compounds in the endophytic fungus of Ferula sinkiangensis at 22.4 minutes.

[0018] The following is the further optimization and / or improvement of the second technical solution of the above invention: In the above first step, 8 ml to 12 ml of 95% ethanol is added to every 1 g of the endophytic fungus strain N93 of Ferula sinkiangensis.

[0019] In the above first step, the time for each ultrasonic extraction is 10 minutes to 15 minutes, and before each ultrasonic extraction, soak at room temperature for 12 hours to 13 hours.

[0020] In the above first step, use a rice medium to cultivate the secondary metabolites of the strain, and the cultivation conditions are: keep away from light, 26 °C.

[0021] The above rice medium is prepared by mixing 80 g of rice with 100 ml of water.

[0022] In the above fourth step, the high performance liquid chromatography eluent is a mixed solution of methanol and water, and among them, the volume ratio of methanol to water is 25:75.

[0023] The third technical solution of the present invention is achieved by the following measures: An application of the alkaloid compounds in the endophytic fungus of Ferula sinkiangensis in the preparation of drugs for preventing and / or treating breast cancer.

[0024] The fourth technical solution of the present invention is achieved by the following measures: An application of the alkaloid compounds in the endophytic fungus of Ferula sinkiangensis in the preparation of drugs for preventing and / or treating human breast cancer.

[0025] The present invention discloses for the first time the alkaloid compound (ferupencine C), and conducts in vitro anti-tumor pharmacodynamic experiments on this compound. According to the experimental results, it is obvious that the alkaloid compound ferupencine C has a certain inhibitory effect on MCF-7 cells, so that the alkaloid compound ferupencine C can be used to prepare drugs for preventing and treating breast cancer, providing new drugs for the prevention and treatment of breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Attached Figure 1 is the chemical structure diagram of the alkaloid compound ferupencine C of the present invention.

[0027] Attached Figure 2 is the 1 1H-NMR spectrum of the alkaloid compound ferupencine C of the present invention.

[0028] Attached Figure 3 is Figure 2 the enlarged schematic diagram of part A in

[0029] Attached Figure 4 is Figure 2 the enlarged schematic diagram of part B in

[0030] Attached Figure 5 is the 13 C-APT spectrum of the alkaloid compound ferupencine C of the present invention.

[0031] Attached Figure 6 is Figure 5 the enlarged schematic diagram of part C in

[0032] Attached Figure 7 is Figure 5 the enlarged schematic diagram of part D in DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention is not limited by the following embodiments, and the specific implementation manners can be determined according to the technical solutions of the present invention and the actual situation.

[0034] All 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; the solutions in the present invention are aqueous solutions with water as the solvent unless otherwise specified. For example, a hydrochloric acid solution is an aqueous solution of hydrochloric acid; normal temperature and room temperature in the present invention generally refer to a temperature range of 15°C to 25°C, and are generally defined as 25°C.

[0035] The present invention will be further described below in conjunction with the embodiments: Example 1: The alkaloid compounds in the endophytic fungi of Ferula sinkiangensis have the following chemical structural formula: .

[0036] The alkaloid compounds in the endophytic fungi of Ferula sinkiangensis have the chemical name of (3 R , 5 R , 6 S , 8 R , 10 S ,12 S )-8,12-dihydroxy-10-methyloctahydrocyclopenta[b]pyrrolo[2,1-f][1,2,4]oxazin-1-one.

[0037] Example 2: The alkaloid compounds in the endophytic fungi of Ferula sinkiangensis are prepared according to the following steps: First step, activate the strain of endophytic fungi of Ferula sinkiangensis N93, and use rice medium (80 g of rice: 100 ml of water) to culture the secondary metabolites of the strain (conditions: 26 °C, in the dark). Use ethanol to ultrasonically extract the cultured strain of endophytic fungi of Ferula sinkiangensis N93 and its secondary metabolites at least three times. Combine the ultrasonic extracts each time and recover and concentrate under reduced pressure to obtain an extract of the strain of endophytic fungi of Ferula sinkiangensis N93; Second step, disperse the total extract of the strain of endophytic fungi of Ferula sinkiangensis N93 in water to form a suspension, and then extract with dichloromethane to obtain an extract of the dichloromethane fraction; Third step, take the extract of the dichloromethane fraction and separate it by gradient elution on a silica gel column chromatography to obtain 8 fractions. Among them, the gradient elution solution of the silica gel column chromatography includes dichloromethane and methanol, and the volume ratios of dichloromethane to methanol are 1:0, 100:1, 30:1, 20:1, 10:1, 5:1, 2:1, 0:1 in sequence; Fourth step, purify and separate the 5th fraction by gradient elution on a high performance liquid chromatography, and collect the eluate to obtain ferupencine C at 22.4 minutes.

[0038] In the present invention, the preservation location of the strain of Penicillium sp. of the endophytic fungi of Ferula sinkiangensis N93: China General Microbiological Culture Collection Center (CGMCC), preservation number: CGMCC No. 41708, preservation date: December 11, 2024.

[0039] Example 3: As an optimization of the above Example 2, in the first step, 8 ml to 12 ml of 95% ethanol is added to every 1 g of the strain of endophytic fungi of Ferula sinkiangensis N93.

[0040] Example 4: As an optimization of Example 2 above, in the first step, the time for each ultrasonic extraction is 10 to 15 minutes, and before each ultrasonic extraction, it is first soaked at room temperature for 12 to 13 hours; the strain is cultured for secondary metabolites using a rice medium, and the culture conditions are: light avoidance, 26 °C; the rice medium is prepared by mixing 100 ml of water with 80 g of rice.

[0041] Example 5: As an optimization of Example 2 above, in the fourth step, the high-performance liquid chromatography eluent is a mixed solution of methanol and water, wherein the volume ratio of methanol to water is 25:75.

[0042] Example 6: Application of the alkaloid compounds in the endophytic fungi of Ferula sinkiangensis described in the above examples in the preparation of drugs for preventing and / or treating breast cancer.

[0043] Example 7: Application of the alkaloid compounds in the endophytic fungi of Ferula sinkiangensis described in the above examples in the preparation of drugs for preventing and / or treating human breast cancer.

[0044] Example 8: Ferupencine C is obtained according to the following method: First step, activate the strain N93 of endophytic fungi of Ferula sinkiangensis screened by the research group in the early stage, and use a rice medium (80 g of rice: 100 ml of water) to culture the secondary metabolites of the strain (conditions: 26 °C, dark place), and use 95% ethanol to ultrasonically extract the cultured strain N93 of endophytic fungi of Ferula sinkiangensis and its secondary metabolites at least three times, combine the ultrasonic extracts each time and recover and concentrate under reduced pressure to obtain an extract of strain N93 of endophytic fungi of Ferula sinkiangensis; Second step, disperse the total extract of strain N93 of endophytic fungi of Ferula sinkiangensis into a suspension with water, and then extract with dichloromethane to obtain an extract of the dichloromethane part; Third step, take the extract of the dichloromethane part and separate it by gradient elution on a silica gel column chromatography to obtain 8 fractions. Among them, the gradient elution solution of the silica gel column chromatography includes dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 1:0, 100:1, 30:1, 20:1, 10:1, 5:1, 2:1, 0:1 in turn; Fourth step, purify and separate the 5th fraction by gradient elution on a high-performance liquid chromatography, collect the eluate, and obtain ferupencine C at 22.4 minutes.

[0045] In this Example 8, in the first step, 8 to 12 ml of 95% ethanol is added to every 1 g of strain N93 of endophytic fungi of Ferula sinkiangensis; before each ultrasonic extraction, it is first soaked at room temperature for 12 to 13 hours; in the fourth step, the high-performance liquid chromatography eluent is a mixed solution of methanol and water, wherein the volume ratio of methanol to water is 25:75.

[0046] Perform nuclear magnetic resonance hydrogen spectrum on the alkaloid compound ferupencine C described in this Example 8 (1 H-NMR) and carbon nuclear magnetic resonance ( 13 C-APT) analysis, the 1 H-NMR spectrum of ferupencine C described in Example 8 is as Figure 2 shown, and the 13 C-APT spectrum of ferupencine C described in Example 8 is as Figure 5 shown. For Figure 2 and Figure 5 for spectral analysis, assign the peaks in Spectrum 2 and Figure 5 respectively, and the peak assignments of Figure 2 and Figure 5 are shown in Table 1. From the data in Table 1, it can be seen that the chemical structural formula of ferupencine C described in this example is as Figure 1 shown, and it is soluble in chloroform and methanol.

[0047] Perform in vitro anti-tumor pharmacodynamic experiments on the alkaloid compound ferupencine C of the present invention. The in vitro anti-tumor pharmacodynamic experiments use the MTT colorimetric method.

[0048] Take ferupencine C as the experimental group, Cisplatin as the control group, and set up a blank group at the same time. The experimental group, the control group and the blank group select MCF-7 (human breast cancer cells) as the experimental objects. After dilution with the culture medium, inoculate them 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, add the corresponding drugs to each group so that the final concentrations of the drugs in each group are 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), with a total of 5 concentrations, and 3 replicates for each concentration; after culturing for 48 hours, add 10 μL of MTT to each well for staining; continue culturing for four hours, then aspirate the original culture medium, add 150 μL of DMSO to each well, place it on a shaker and oscillate at a low speed for 10 min to fully dissolve the crystals, and detect the optical density value at a wavelength of 570 nm with an enzyme-linked immunosorbent assay detector. Calculate the 50% inhibitory concentration (IC 50 , μg / mL). The calculation method of calculating IC 50 from the optical density value is a well-known prior art. The IC 50 of the experimental group and the control group on MCF-7 cells is shown in Table 2. From the data in Table 2, it can be seen that ferupencine C of the present invention has a certain inhibitory effect on MCF-7 cells.

[0049] 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 increased or decreased according to actual needs to meet the requirements of different situations.

Claims

1. An alkaloid compound in an endophytic fungus of Ferula xinjiangensis, characterized in that: Its chemical structure is: 。 2. The alkaloid compound in the endophytic fungus 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 No. N93, culture the secondary metabolites of the strain, use ethanol to ultrasonically extract the cultured Xinjiang Ferula endophytic fungus strain No. N93 and its secondary metabolites for at least three times, combine the ultrasonic extracts of each time, recover and concentrate under reduced pressure to obtain the Ferula endophytic fungus strain No. N93 extract; the second step is to disperse the total extract of the Ferula endophytic fungus strain No. N93 into a suspension with water, and then extract it with dichloromethane to obtain a dichloromethane extract; the third step is to extract the cultured Xinjiang Ferula endophytic fungus strain No. N93 and its secondary metabolites for at least three times, and then use ethanol to ultrasonically ... The first step is to obtain 8 fractions after separation by gradient elution of the dichloromethane extract by silica gel column chromatography, wherein the silica gel column chromatography gradient eluent comprises 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; the fourth step is to purify and separate the fifth fraction by gradient elution of high performance liquid chromatography, and collect the eluate to obtain alkaloid compounds in endophytic fungi of Ferula xinjiangensis at 22.4 minutes.

3. The alkaloid compound in the endophytic fungus of Xinjiang Ferula according to claim 2, characterized in that: In the first step, 8 ml to 12 ml of 95% ethanol was added per 1 g of the endophytic fungus strain N93 of Ferula.

4. The alkaloid compound in the endophytic fungus of Xinjiang Ferula according to claim 2 or 3, characterized in that: In the first step, the time of each ultrasonic extraction is 10 minutes to 15 minutes, and before each ultrasonic extraction, it is soaked at room temperature for 12 hours to 13 hours; or / and, in the first step, the secondary metabolites of the strain are cultured using rice culture medium, and the culture conditions are: protected from light, 26°C.

5. The alkaloid compound in the endophytic fungus of Xinjiang Ferula according to claim 4, characterized in that: The rice culture medium was prepared by adding 100 ml of water to every 80 g of rice and mixing them.

6. The alkaloid compound in the endophytic fungus of Xinjiang Ferula according to claim 2, 3 or 5, characterized in that: In the fourth step, the HPLC eluent is a mixture of methanol and water, wherein the volume ratio of methanol to water is 25:

75.

7. The alkaloid compound in the endophytic fungus of Xinjiang Ferula according to claim 4, characterized in that: In the fourth step, the HPLC eluent is a mixture of methanol and water, wherein the volume ratio of methanol to water is 25:

75.

8. A method for preparing alkaloid compounds from the endophytic fungus of Xinjiang Ferula according to any one of claims 3 to 7, characterized in that: Follow the steps below: The first step is to activate the Xinjiang Ferula endophytic fungus strain No. N93, culture the secondary metabolites of the strain, use ethanol to ultrasonically extract the cultured Xinjiang Ferula endophytic fungus strain No. N93 and its secondary metabolites for at least three times, combine the ultrasonic extracts of each time, recover and concentrate under reduced pressure to obtain the Ferula endophytic fungus strain No. N93 extract; the second step is to disperse the total extract of the Ferula endophytic fungus strain No. N93 into a suspension with water, and then extract it with dichloromethane to obtain a dichloromethane extract; the third step is to extract the cultured Xinjiang Ferula endophytic fungus strain No. N93 and its secondary metabolites for at least three times, and then use ethanol to ultrasonically ... The first step is to obtain 8 fractions after separation by gradient elution of the dichloromethane extract by silica gel column chromatography, wherein the silica gel column chromatography gradient eluent comprises 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; the fourth step is to purify and separate the fifth fraction by gradient elution of high performance liquid chromatography, and collect the eluate to obtain alkaloid compounds in endophytic fungi of Ferula xinjiangensis at 22.4 minutes.

9. Use of the alkaloid compound in the endophytic fungus of Xinjiang Ferula as claimed in any one of claims 1 to 7 in the preparation of a drug for preventing and / or treating breast cancer.

10. Use of the alkaloid compound in the endophytic fungus of Ferula foetida as claimed in any one of claims 1 to 7 in the preparation of a drug for preventing and / or treating human breast cancer.

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