Method for extracting Dustanin from Metarhizium anisopliae
By using PDA and PDB culture media for activation culture in Metarhizium pingshaensis, combined with ethyl acetate ultrasonic extraction and normal phase silica gel column separation and purification, high-purity Dustanin was successfully extracted from Metarhizium pingshaensis, solving the problem of lack of extraction methods in the existing technology and promoting its application in biomedicine and pesticides.
Patent Information
- Application Number
- CN202510758923.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-09
- Publication Date
- 2025-09-16
AI Technical Summary
There is no method for isolating Dustanin from Metarhizium anisopliae in the prior art, which limits its research and application in biomedicine and pesticides.
Metarhizium anisopliae was activated and cultured in PDA and PDB medium, and then statically cultured in rice medium. Dustanin was obtained by gradient elution combined with ultrasonic extraction with ethyl acetate and separation and purification with normal phase silica gel column.
The efficient extraction of Dustanin was achieved with high purity and good reproducibility, providing a guarantee for its further research and application.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of active substance extraction and separation, and particularly relates to a method for extracting Dustanin from Metarhizium anisopliae. Background Art
[0002] Metarhizium anisopliae is one of the earliest entomogenous fungi used in biological control and is widely used to control agricultural and forestry pests. Studies have found that both Metarhizium anisopliae spore suspensions and metabolites have a significant inhibitory effect on corn stalk rot pathogens. With continued in-depth research, a variety of formulations have been developed for the control of agricultural and forestry pests, suggesting the potential application of this fungus in biological pesticides or medicines.
[0003] Dustanin is a pentacyclic triterpenoid compound, a white solid, easily soluble in dichloromethane, and poorly soluble in solvents such as methanol and water. The compound has 8 methyl carbon signals and 6 methine carbon signals, and δ C Dustanin is a fungal metabolite that may play a specific physiological role in fungal growth, development, or survival. However, there are currently no reports of dustanin being isolated from Metarhizium anisopliae. Summary of the Invention
[0004] The purpose of the present invention is to provide a method for extracting Dustanin from Metarhizium anisopliae to promote its research and development in biomedicine and pesticides.
[0005] To achieve the above object, the present invention adopts the following technical solutions: A method for extracting Dustanin from Metarhizium anisopliae comprises the following steps: 1) Metarhizium anisopliae was activated and cultured in PDA medium for 7 days. The bacterial mass was then inoculated into PDB medium and cultured continuously at 160-180 rpm, 24-28°C, and 50-60% relative humidity (RH) for 5-10 days. The inoculum was then transferred to rice medium at a rate of 15-20 wt% and cultured at 24-28°C and 50-60% relative humidity for 28 days to obtain a fermentation product containing mycelium. 2) drying the fermentation product obtained in step 1) and grinding it into powder, adding an equal volume of ethyl acetate, performing ultrasonic extraction followed by leaching, and then filtering and collecting the extract; repeating the extraction process three times, combining the extracts, and concentrating under reduced pressure to remove the ethyl acetate to obtain a crude extract; 3) The crude extract was dissolved in a mixed solvent of dichloromethane and methanol, and then separated and purified using a normal phase silica gel column to obtain component A; 4) Component A was subjected to secondary separation and purification using a normal phase silica gel column to obtain a white solid, dustanin, whose structural formula is as follows: .
[0006] Furthermore, in step 1), the bacterial block with a size of 1×1 cm is cut into 16 pieces and inoculated into PDB culture medium.
[0007] Furthermore, the rice culture medium in step 1) is prepared by stirring and mixing 85 g of rice and 2 g of mealworm powder (previously ground with liquid nitrogen) in 90 mL of ultrapure water, and then sterilizing by high pressure at 121° C. for 20 min.
[0008] Furthermore, the drying temperature in step 2) is 25-40°C.
[0009] Furthermore, the power of the ultrasonic-assisted extraction in step 2) is 500 W, the frequency is 40 KHz, and the extraction time is 60 to 80 minutes.
[0010] Furthermore, the extraction time in step 2) is 3 to 6 hours.
[0011] Furthermore, in step 3), the volume ratio of dichloromethane to methanol in the mixed solvent is 80:20.
[0012] Furthermore, the fineness of the silica gel in the forward silica gel column used in steps 3) and 4) is 200-300 mesh.
[0013] Furthermore, in steps 3) and 4), the separation and purification is performed by gradient elution using petroleum ether and ethyl acetate as eluents.
[0014] Furthermore, in step 3) when gradient elution is performed, the volume ratios of petroleum ether to ethyl acetate used are 2:1, 1:1, 1:2, and 1:3, respectively.
[0015] Furthermore, in step 4), when gradient elution is performed, the volume ratios of petroleum ether to ethyl acetate used are 6:1 and 7:1, respectively.
[0016] The significant advantages of the present invention are: (1) This invention is the first to extract Dustanin from the metabolites of Metarhizium anisopliae, which can provide a guarantee for the further research and application of Dustanin.
[0017] (2) The separation and purification process of the present invention is simple, low-cost and easy to operate.
[0018] (3) The prepared compounds are of high purity and good reproducibility. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 Dustanin prepared in Example 1 H NMR (DMSO- d 6 ) spectrum.
[0020] Figure 2 Dustanin prepared in Example 13 C NMR (DMSO- d 6 ) spectrum. DETAILED DESCRIPTION
[0021] In order to make the contents of the present invention easier to understand, the technical solutions of the present invention are further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.
[0022] Metarhizium anisopliae used in the examples Metarhiziumpingshaense ) strain was registered and deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC) in July 2022 (address: Datun Road, Chaoyang District, Beijing), and its deposit number is CGMCC 3.23778.
[0023] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.
[0024] Example 1 Isolation of Active Dustanin 1) Metarhizium anisopliae was used as the material and activated by streak method in PDA medium (200 g peeled potatoes, cut into small pieces, added to 1000 ml of water, boiled for 20-30 min, filtered with gauze to remove potato residue, then added 20 g glucose and 18 g agar, heated to melt and mix, then added water to make up to 1000 ml, and then sterilized under autoclave at 121°C for 20 min). After 7 days of culture, 1×1 cm-sized blocks were picked under sterile conditions, cut into 16 pieces, and inoculated into PDB medium (200 g peeled potatoes, cut into small pieces, added to 1000 ml of water, boiled for 20-30 min, filtered with gauze to remove potato residue, then added 20 g glucose, mixed, then added water to make up to 1000 ml, and then sterilized under autoclave at 121°C for 20 min). r / min, 24-28°C, and relative humidity (RH) 50-60% for continuous culture for 5-10 days (i.e., primary culture). Then, the culture medium was transferred to a modified rice culture medium (85 g of rice and 2 g of mealworm powder obtained by grinding with liquid nitrogen were mixed in 90 mL of ultrapure water and sterilized by high pressure at 121°C for 20 min) at an inoculum size of 15-20 wt%, and static culture was continued at 24-28°C for 28 days (i.e., secondary culture) to obtain a fermentation product containing mycelium.
[0025] 2) The resulting fermentation product containing mycelium was collected, dried at room temperature, and then ground into powder. An equal volume of ethyl acetate was added and extracted under ultrasonic conditions at 500 W and 40 kHz for 60-80 minutes, followed by leaching for 3-6 hours. The extract was then filtered and collected. After repeating the above extraction steps three times, the extracts were combined and concentrated under reduced pressure to remove the ethyl acetate to obtain a crude extract. 3) The crude extract was diluted with dichloromethane and methanol (80:20, v / v ) was dissolved in a mixed solvent, and silica gel with a fineness of 200-300 mesh was added at an amount of 0.1 g / mL to mix the sample. Then, it was introduced into a normal phase silica gel column (silica gel fineness was 200-300 mesh) by dry loading. Gradient elution was performed with petroleum ether and ethyl acetate in a volume ratio of 2:1, 1:1, 1:2, and 1:3, respectively. The eluates obtained with a volume ratio of petroleum ether to ethyl acetate of 2:1 were collected, combined, and concentrated under reduced pressure to remove the solvent, thereby obtaining a large amount of white insoluble substance component A; 4) Component A was mixed with dichloromethane and methanol (80:20, v / v) in a mixed solvent, 0.1 g / mL of silica gel with a fineness of 200-300 mesh was added for sample mixing. The sample was then reintroduced into a normal phase silica gel column (silica gel with a fineness of 200-300 mesh) by dry loading, and gradient eluted with petroleum ether and ethyl acetate in a volume ratio of 6:1 and 7:1, respectively. The eluates obtained in a volume ratio of petroleum ether to ethyl acetate of 7:1 were collected, combined, and concentrated under reduced pressure to remove the solvent to obtain a white solid, namely dustanin, with a yield of 4% and a purity of >98%.
[0026] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.
Claims
1. A method for extracting Dustanin from Metarhizium anisopliae, characterized in that: The following steps are involved: 1) Metarhizium anisopliae was activated and cultured in PDA medium for 7 days. The bacterial mass was then inoculated into PDB medium and cultured continuously at 160-180 rpm, 24-28°C, and a relative humidity of 50-60% for 5-10 days. The inoculum was then transferred to rice medium at a rate of 15-20% and cultured at 24-28°C and a relative humidity of 50-60% for 28 days to obtain a fermentation product containing mycelium. 2) drying the fermentation product obtained in step 1) and grinding it into powder, adding an equal volume of ethyl acetate, performing ultrasonic-assisted extraction followed by leaching, and then collecting the extract by filtration; repeating the extraction process three times, combining the extracts, and concentrating under reduced pressure to remove the ethyl acetate to obtain a crude extract; 3) The crude extract was dissolved in a mixed solvent of dichloromethane and methanol, and then separated and purified using a normal phase silica gel column to obtain component A; 4) Component A was subjected to secondary separation and purification using a normal phase silica gel column to obtain a white solid, which is Dustanin.
2. The method for extracting Dustanin from Metarhizium anisopliae according to claim 1, wherein: The rice culture medium described in step 1) is prepared by mixing 85 g of rice and 2 g of mealworm powder obtained by grinding with liquid nitrogen in 90 mL of ultrapure water, and then sterilizing by high pressure at 121°C for 20 min.
3. The method for extracting Dustanin from Metarhizium anisopliae according to claim 1, wherein: The power of the ultrasonic-assisted extraction in step 2) is 500 W, the frequency is 40 kHz, and the extraction time is 60 to 80 minutes.
4. The method for extracting Dustanin from Metarhizium anisopliae according to claim 1, wherein: The extraction time in step 2) is 3 to 6 hours.
5. The method for extracting Dustanin from Metarhizium anisopliae according to claim 1, wherein: Step 3) The volume ratio of dichloromethane to methanol in the mixed solvent is 80:
20.
6. The method for extracting Dustanin from Metarhizium anisopliae according to claim 1, wherein: The fineness of the silica gel in the forward silica gel column used in steps 3) and 4) is 200-300 mesh.
7. The method for extracting Dustanin from Metarhizium anisopliae according to claim 1, wherein: Steps 3) and 4) are used for separation and purification by gradient elution using petroleum ether and ethyl acetate as eluents.
8. The method for extracting Dustanin from Metarhizium anisopliae according to claim 7, wherein: In step 3), when performing gradient elution, the volume ratios of petroleum ether to ethyl acetate are 2:1, 1:1, 1:2, and 1:3, respectively.
9. The method for extracting Dustanin from Metarhizium anisopliae according to claim 7, wherein: In step 4), when performing gradient elution, the volume ratios of petroleum ether to ethyl acetate used were 6:1 and 7:1, respectively.