Method for extracting 3 beta-acetoxy-15 alpha, 22-dihydroxyhopane from Metarhizium anisopliae

Abstract

The invention discloses a method for extracting 3 beta-acetoxy-15 alpha, 22-dihydroxyhopane from metarhizium anisopliae, which comprises the following steps: firstly, performing activated culture on the metarhizium anisopliae in a PDA (potato dextrose agar) culture medium, then transferring the metarhizium anisopliae into a PDB (potato dextrose agar) culture medium for primary culture, then inoculating the metarhizium anisopliae into a rice culture medium for secondary culture, performing ultrasonic extraction in combination with ethyl acetate extraction and concentration to obtain a crude extract, and performing freeze-drying to obtain the 3 beta-acetoxy-15 alpha, 22-dihydroxyhopane. The crude extract is subjected to separation and purification through a reduced pressure column and a normal phase silica gel column in sequence, and therefore the 3 beta-acetoxy-15 alpha, 22-dihydroxyhopane is obtained. According to the invention, the 3 beta-acetoxy-15 alpha, 22-dihydroxyhopane is separated from the Metarhizium anisopliae for the first time, and it is proved that the 3 beta-acetoxy-15 alpha, 22-dihydroxyhopane can be used for biological control of tea geometrid.

Description

Technical Field

[0001] The present invention belongs to the technical field of extraction and separation of active substances, and particularly relates to a method for extracting 3β-acetoxy-15α,22-dihydroxyhopane from Metarhizium pingshaense. Background Art

[0002] With the continuous iteration and upgrading of research means and the continuous in-depth exploration of the activities and mechanisms of natural products, more and more new compounds and their new activities are being discovered. At present, a variety of preparations have been developed for the control of agricultural and forestry pests. Metarhizium pingshaense is one of the most widely used groups of biocontrol fungi, and scholars have long been committed to studying its mode of action against pests. Metarhizium has strong lethality to various agricultural and forestry pests, and its lethal effects on various insects such as Choristoneura fumiferana, Manduca sexta, and Helicoverpa armigera have been verified through various methods such as feeding, injection, and artificial spraying, indicating its great potential application value in biological pesticides or medicine.

[0003] 3β-acetoxy-15α,22-dihydroxyhopane is a hopane-type triterpenoid compound, which is a white powder, soluble in dichloromethane, and insoluble in solvents such as methanol and water. Its hopane skeleton is equipped with functional groups such as 3β-acetoxy, 15α-hydroxy, and 22-hydroxy, and this structure endows it with unique chemical properties and biological activities. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for extracting 3β-acetoxy-15α,22-dihydroxyhopane from Metarhizium pingshaense and its application in the biological control of Ectropis obliqua Prout.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions: A method for extracting 3β-acetoxy-15α,22-dihydroxyhopane from Metarhizium pingshaense, which comprises the following steps: 1) Activate and culture Metarhizium pingshaense in PDA medium for 7 days, then take the mycelial block and inoculate it into PDB medium, and continuously culture it for 5-10 days under the conditions of 160-180 r / min, 24-28 °C, and relative humidity (RH) of 50-60%. Then transfer it to rice medium according to an inoculation amount of 15wt% - 20wt%, and continue to statically culture it for 28 days under the conditions of 24-28 °C and relative humidity of 50-60% to obtain mycelia; 2) Grind and crush the mycelium obtained in step 1), add an equal volume of ethyl acetate, first perform ultrasonic extraction and then perform leaching, and then filter to collect the extract; repeat the extraction 3 times according to the aforementioned steps, combine the extracts, rotate and evaporate to remove ethyl acetate, and concentrate to obtain a crude extract; 3) Separate the crude extract obtained in step 2) by a reduced-pressure column. After concentrating the collected eluate under reduced pressure, component A is obtained; 4) Rinse and filter the component A obtained in step 3) with methanol to obtain a large amount of white insoluble substance Aa; 5) Perform column chromatography separation on the component Aa obtained in step 4) using a normal-phase silica gel column to obtain a white powder, which is 3β-acetoxy-15α,22-dihydroxyhopane, and its structural formula is as follows: 。

[0006] Further, in step 1), the bacterial blocks with a size of 1×1 cm are cut into 16 pieces and inoculated into the PDB medium.

[0007] Further, the rice medium in step 1) is prepared by stirring and mixing 85 g of rice and 2 g of mealworm powder obtained by grinding liquid nitrogen in 90 mL of ultrapure water, and then autoclaving at 121 °C for 20 min.

[0008] Further, in step 2), the power of the ultrasonic extraction is 500 W, the frequency is 40 KHz, and the time is 60 - 80 minutes.

[0009] Further, the time of the leaching in step 2) is 3 - 6 hours.

[0010] Further, when performing separation by a reduced-pressure column in step 3), petroleum ether, dichloromethane, ethyl acetate, and methanol are used for elution in sequence, and the ethyl acetate elution part is collected.

[0011] Further, when performing column chromatography separation in step 5), a mixed solution of petroleum ether / ethyl acetate is used for gradient elution.

[0012] Furthermore, when performing gradient elution, the volume ratio of petroleum ether to ethyl acetate is 7:1, 6:1, 5:1.

[0013] Further, when performing column separation, the silica gel used is 200 - 300 mesh.

[0014] The prepared 3β-acetoxy-15α,22-dihydroxyhopane can be used for the biological control of Ectropis obliqua Prout.

[0015] The remarkable advantages of the present invention are as follows: (1) 3β-acetoxy-15α,22-dihydroxyhopane was extracted from the metabolites of Metarhizium pingshaense for the first time in this invention, which plays an important role in enhancing the application value of Metarhizium pingshaense and also expands the application fields of 3β-acetoxy-15α,22-dihydroxyhopane.

[0016] (2) The separation and purification process of this invention is simple, low-cost and easy to operate; the obtained compound has high purity and good repeatability.

[0017] (3) The 3β-acetoxy-15α,22-dihydroxyhopane obtained in this invention has a good killing effect on Ectropis obliqua Prout, and thus can be used for the biological control of Ectropis obliqua Prout. Brief Description of the Drawings

[0018] Figure 1 1H NMR (CDCl 1 - d 3 ) spectrum of 3β-acetoxy-15α, 22-dihydroxyhopane prepared in the example. 1 ) spectrum.

[0019] Figure 2 13C NMR (CDCl 13 - d 3 ) spectrum of 3β-acetoxy-15α, 22-dihydroxyhopane prepared in the example. 1 ) spectrum.

[0020] Figure 3 HMBC (CDCl 3 - d 1 ) spectrum of 3β-acetoxy-15α, 22-dihydroxyhopane prepared in the example.

[0021] Figure 4 COSY (CDCl 3 - d 1 ) spectrum of 3β-acetoxy-15α, 22-dihydroxyhopane prepared in the example. Detailed Embodiments

[0022] In order to make the content of this invention easier to understand, the technical solutions of this invention are further described below in conjunction with specific embodiments, but this invention is not limited thereto.

[0023] The Metarhizium pingshaense strain used in the examples was registered and preserved at the China General Microbiological Culture Collection Center in July 2022 (address: Datun Road, Chaoyang District, Beijing), and its preservation number is CGMCC 3.23778.

[0024] Example 1 Isolation of Active 3β-acetoxy-15α,22-dihydroxyhopane 1) Using Metarhizium pingshaense as the material, it was activated by the streaking method in PDA medium (200 g of peeled potatoes, cut into small pieces, added with 1000 ml of water, boiled for 20 - 30 min, filtered through gauze to remove potato residues, then added with 20 g of glucose and 18 g of agar, heated to melt and mix evenly, made up to 1000 ml with water, and sterilized at 121 °C under high pressure for 20 min). After culturing for 7 days, a 1×1 cm mycelial block was picked under sterile conditions, cut into 16 pieces and inoculated into PDB medium (200 g of peeled potatoes, cut into small pieces, added with 1000 ml of water, boiled for 20 - 30 min, filtered through gauze to remove potato residues, then added with 20 g of glucose, mixed evenly, made up to 1000 ml with water, and sterilized at 121 °C under high pressure for 20 min). It was continuously cultured for 5 - 10 days under the conditions of 16 - 180 r / min, 24 - 28 °C, and relative humidity (RH) of 50 - 60%. Then, it was transferred to rice medium (85 g of rice, 2 g of mealworm powder (pre-ground with liquid nitrogen), 90 mL of ultrapure water; sterilized at 121 °C under high pressure for 20 min) at an inoculation amount of 15 wt% - 20 wt%, and continued to be statically cultured for 28 days at 24 - 28 °C and relative humidity of 50 - 60% to obtain mycelia.

[0025] 2) The obtained mycelia were collected, dried at room temperature, ground and pulverized, added with an equal volume of ethyl acetate, ultrasonically extracted for 60 - 80 minutes (power: 500 W, frequency: 40 KHz), then extracted for 3 - 6 hours, and the extract was filtered and collected; the above steps were repeated 3 times, the extracts were combined, and ethyl acetate was removed by rotary evaporation to obtain a crude extract by concentration.

[0026] 3) The obtained crude extract was dissolved in a mixed solution of dichloromethane and methanol (2:1, v / v). Then, flash column chromatography (silica gel fineness: 200 - 300 mesh) was used, and petroleum ether, dichloromethane, ethyl acetate, and methanol were used for elution in turn according to increasing polarity. The ethyl acetate elution fraction was collected to obtain fraction A.

[0027] 4) The obtained fraction A was rinsed and filtered with methanol to obtain a large amount of white insoluble substance Aa.

[0028] 5) The obtained component Aa was subjected to normal-phase silica gel column (silica gel fineness: 200 - 300 mesh), and gradient elution was carried out with a petroleum ether / ethyl acetate mixture (the volume ratio of elution was 7:1 → 6:1 → 5:1). The eluate was collected and concentrated under reduced pressure to obtain a white powder, which was 3β-acetoxy-15α,22-dihydroxyhopane, and its yield was 3.23%.

[0029] Example 2 Application of 3β-acetoxy-15α,22-dihydroxyhopane in the biological control of Ectropis obliqua The insecticidal activity of the obtained 3β-acetoxy-15α,22-dihydroxyhopane against Ectropis obliqua was investigated. Specifically, the product obtained in Example 1 was dissolved in 10% dimethyl sulfoxide to prepare a solution with a concentration of 1 μg / mL for standby. Azadirachtin with the same concentration was used as the positive control, and 10% dimethyl sulfoxide solution was used as the negative control. A hydraulic nano-injection system was used to inject 150 nL into each larva of Ectropis grisescens. 20 larvae were injected for different treatments, and 3 replicates were set. After injection, observe for 10 min. If the larva has no vital signs or body fluid flows out from the wound, it should be discarded and reinjected. Observe the death situation of Ectropis obliqua 168 hours after application, and record the number of dead and surviving individuals. Then calculate the corrected mortality rate according to the following formula: Corrected mortality rate = (treatment mortality rate - control mortality rate) / (1 - control mortality rate) × 100%.

[0030] The results showed that at a concentration of 1 μg / mL, the corrected lethal rate of 3β-acetoxy-15α,22-dihydroxyhopane was 51.11%, which was better than the effect of azadirachtin (the corrected lethal rate was 37.78%), indicating that it had good insecticidal activity.

[0031] The above are only the preferred embodiments of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope covered by the present invention.

Claims

1. A method for extracting 3β-acetoxy-15α,22-dihydroxyhopane from Metarhizium anisopliae, characterized in that: The following steps are involved: 1) The green anisopliae was activated and cultured in PDA medium for 7 days, and then the bacterial block was inoculated into PDB medium, and cultured continuously for 5-10 days at 160-180 r / min, 24-28°C, and relative humidity of 50-60%. After that, the inoculum was transferred to rice medium at a rate of 15wt%-20wt%, and static culture was continued at 24-28°C and relative humidity of 50-60% for 28 days to obtain mycelium; 2) grinding the mycelium obtained in step 1), adding an equal volume of ethyl acetate, first ultrasonically extracting and then leaching, and then filtering and collecting the extract; after repeating the extraction steps for 3 times, combining the extracts, removing the ethyl acetate by rotary evaporation, and concentrating to obtain a crude extract; 3) The crude extract obtained in step 2) is separated by using a vacuum column, and the collected eluate is concentrated under reduced pressure to obtain component A; 4) The component A obtained in step 3) is washed with methanol and filtered to obtain a white insoluble substance Aa; 5) Aa obtained in step 4) is separated by column chromatography using a normal phase silica gel column to obtain a white powder, namely 3β-acetoxy-15α,22-dihydroxyhopane.

2. The method according to claim 1, characterized in that: 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 sterilization at 121° C. for 20 min.

3. The method according to claim 1, characterized in that: The power of the ultrasonic extraction in step 2) is 500 W, the frequency is 40 KHz, and the time is 60 to 80 minutes.

4. The method according to claim 1, characterized in that: The extraction time in step 2) is 3 to 6 hours.

5. The method according to claim 1, characterized in that: Step 3) When separation is performed using a vacuum column, petroleum ether, dichloromethane, ethyl acetate, and methanol are used for elution in sequence, and the ethyl acetate elution portion is collected.

6. The method according to claim 1, characterized in that: Step 5) During column chromatography separation, a mixture of petroleum ether / ethyl acetate is used for gradient elution.

7. The method according to claim 6, characterized in that: During gradient elution, the volume ratios of petroleum ether to ethyl acetate were 7:1, 6:1, and 5:

1.

8. The method according to claim 1, characterized in that: When column separation is performed, the silica gel used is 200-300 mesh.

9. Use of 3β-acetoxy-15α,22-dihydroxyhopane prepared by the method of claim 1 in biological control of tea geometrid.

Images