Fusarium moniliforme and application thereof in extraction of quassin

Abstract

The invention provides a Fusarium moniliforme and application thereof in extraction of quassin. The Fusarium moniliforme is used for extracting quassin with insecticidal activity in quassia. In the mixed fermentation process of the picrasma quassioides powder leach liquor and the osteomyelia decumbens culture solution, the osteomyelia decumbens can generate cellulase and pectinase which can destroy cell walls, so that the puzzle structure of the cell walls of the picrasma quassioides is destroyed, and separation of quassin compounds in the picrasma quassioides is facilitated. Meanwhile, protein and polysaccharide generated in the growth process of the decumbent fungus sclerotin for many years can influence the activity of different quassin compounds, the aim of preliminarily separating a quassin crude extract is achieved, quassin components with insecticidal activity can be released, and the fermented liquid is beneficial to subsequent extraction and elution separation. When being used for extracting quassin with insecticidal activity from quassia, the decubitus sclerotin has the characteristics that the purity and the yield of the quassin are improved, and impurities are easy to separate.

Description

Technical Field

[0001] The invention belongs to the technical field of effective ingredients of natural products, and relates to a polypore and application thereof in extracting quassin. Background Art

[0002] Quassia is a deciduous tree of the genus Quassia of the family Simosamineaceae. It has the effects of clearing away heat and detoxifying, drying dampness and reducing swelling. It is mainly used clinically to treat colds, sore throats and other symptoms. It is mainly used to treat various inflammations such as respiratory tract infections and pneumonia. It is a traditional folk medicine in my country.

[0003] Pharmacological studies have shown that the main active ingredients of Quassin are quasin, quasin alkaloids, triterpenes, etc., which have antibacterial and anti-inflammatory, antihypertensive, anti-tumor, anti-snake venom, dehumidification and insecticidal effects. The research reports on the active ingredients of Quassin are mainly quasin alkaloids and triterpenes, but there are few reports on quasin. As a common natural compound, quasin plays an important role in inhibiting cell apoptosis and downregulating caspase-3 activation. It has antipyretic, anthelmintic, antihypertensive and anti-tumor functions, and has a protective effect on nerve cell damage.

[0004] The existing extraction methods of quassin are mainly based on traditional extraction and separation methods, such as using various chromatographic techniques such as silica gel column chromatography, small pore resin, C18 reverse phase column, Sephadex LH-20 and preparative HPLC to extract quassin compounds and improve the separation degree of quassin compounds. However, such methods are complicated to operate, have a small extraction amount, and require further identification to determine the components of various quassin compounds. Summary of the invention

[0005] The purpose of the present invention is to provide a polypore and an application thereof in extracting quassin, so as to solve the problem of low quassin content extracted by the existing extraction method.

[0006] To achieve the above object, the present invention adopts the following technical solutions: The present application provides a porphyra, which is a bone pseudo-perennial porphyra. Perenniporiopsis sp. ) was deposited in the General Microbiology Center of China Microorganism Culture Collection on November 8, 2023, with the deposit number CGMCC No. 40976. The address of the deposit unit is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101.

[0007] The bone pseudo-perennial porphyra provided in the present application is used to extract quasin with insecticidal activity from quasin. Specifically, the bone pseudo-perennial porphyra can produce cellulase and pectinase that destroy the cell wall during the fermentation process, thereby destroying the mysterious structure of the cell wall of quasin, which is conducive to the precipitation of quasin compounds in quasin. At the same time, the proteins and polysaccharides produced during the growth of the bone pseudo-perennial porphyra can affect the activity of different quasin compounds, achieve the purpose of preliminary separation of the quasin crude extract, and be conducive to releasing the quasin component with insecticidal activity, so that the fermented liquid is conducive to subsequent extraction and elution separation.

[0008] Specifically, the method for extracting quassin with insecticidal activity from quassin by using Pseudomonas aeruginosa comprises: S01: Under sterile conditions, inoculate Pseudomonas aeruginosa into a culture medium and culture on a shaking table to obtain Pseudomonas aeruginosa culture fluid.

[0009] Under sterile conditions, take an appropriate amount of culture medium and inoculate the bone-based pseudopora in the culture medium, wherein the volume of the bone-based pseudopora is 1-10% of the volume of the culture medium. The bone-based pseudopora is cultured in a shaking table in the culture medium for 7 days to obtain a bone-based pseudopora culture solution containing protein and polysaccharide, wherein the shaking table temperature is 28°C and the rotation speed is 180r / min.

[0010] In the present application, the composition of the culture medium includes, by mass percentage, 43% glucose, 17.7% corn flour, 10.8% maltose, 10.8% peptone, 4.3% potassium dihydrogen phosphate, 2.2% magnesium sulfate, 10.8% yeast powder, 11.1% vitamin B1, and the rest is distilled water.

[0011] S02: The extract is obtained by ethanol extraction and vacuum distillation of the quassia wood powder.

[0012] Fresh quassin slices were dried at 46-52°C, crushed with a high-speed grinder, and passed through a 40-mesh sieve to obtain quassin powder. The quassin powder was added to 80% ethanol at a weight ratio of 1:8, mixed, and heated to reflux at 105°C for 2-4 times, each time for 3 hours. The heating and reflux treatment can help to separate quassin with insecticidal effect and increase the yield of quassin. After each reflux, filter, mix all the filtrates, and recover ethanol through vacuum distillation to obtain an extract without alcohol taste.

[0013] S03: The extract is diluted with distilled water and added into the culture solution of the Pseudomonas aeruginosa, and aerobically fermented to obtain a fermentation solution.

[0014] The extract was diluted with distilled water to form a crude extract. The culture solution of Pseudomonas aeruginosa was added to the crude extract, and after sufficient stirring, the fermentation temperature was 28°C, the fermentation pH was 5-6, and the ventilation volume was 0.6m 3 / min, and then aerobic fermentation was performed for 96 hours to obtain a fermentation liquid with a pH of 5.2-5.3. The mass ratio of the bitterwood powder to the culture liquid of the bone-like pseudo-perennial porphyra was 1: (5-10).

[0015] S04: The fermentation broth is vacuum filtered and centrifuged to obtain a supernatant which is extracted with petroleum ether and ethyl acetate to obtain an ethyl acetate extract.

[0016] In order to better extract the effective ingredients such as quassin in the fermentation broth, petroleum ether with the lowest polarity is first used for extraction to extract quassin to the greatest extent; then ethyl acetate is used for extraction to obtain an ethyl acetate extract. Specifically, the fermentation broth is filtered with a vacuum filtration pump and centrifuged for 15 minutes at a speed of 3500r / min to obtain a supernatant. The supernatant is mixed with petroleum ether in a volume ratio of 1:1, and a first extraction is performed to obtain a petroleum ether extract. The petroleum ether extract is mixed with ethyl acetate in a volume ratio of 1:1, and a second extraction is performed to obtain an ethyl acetate extract.

[0017] S05: The ethyl acetate extract is loaded onto a macroporous resin column and eluted with methanol. The eluted component is concentrated under reduced pressure and crystallized to obtain quassin crystals.

[0018] The present invention has the following beneficial effects: (1) After the quassin powder is heated and refluxed with ethanol, it is beneficial to separate the quassin with insecticidal effect and increase the yield of quassin.

[0019] (2) During the mixed fermentation process of the extract of quassia wood powder and the culture medium of Pseudomonas aeruginosa, Pseudomonas aeruginosa can produce cellulase and pectinase that destroy the cell wall, thereby destroying the mysterious structure of the quassia wood cell wall, which is conducive to the precipitation of quassin compounds in quassia wood.

[0020] (3) The proteins and polysaccharides produced during the growth of Pseudomonas aeruginosa can affect the activity of different quassin compounds, thereby achieving the purpose of preliminary separation of the crude quassin extract, which is beneficial to the release of quassin components with insecticidal activity, making the fermented liquid more conducive to subsequent extraction and elution separation.

[0021] (4) The fermentation liquid formed by mixing the extract of quassin powder and the culture medium of Pseudomonas aeruginosa with petroleum ether and ethyl acetate can extract quassin to the greatest extent.

[0022] (5) When Pseudomonas aeruginosa is used to extract quassin with insecticidal activity from quassin, it has the characteristics of improving the purity and yield of quassin and facilitating the separation of impurities. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a graph showing the effect of different volumes of Pseudomonas aeruginosa on the extraction rate of quassin. Figure 2 This is a graph showing the effect of different volume ratios of Pseudomonas aeruginosa culture solution and Quassin powder on the extraction rate of Quassin. Figure 3 This is a graph showing the effect of different ventilation rates on the extraction rate of quassin. DETAILED DESCRIPTION

[0024] The technical solution of the present invention is further explained and illustrated by means of specific embodiments below.

[0025] Example 1 The present application provides a method for using a bone-based pseudo-perennial porphyra to extract quassin with insecticidal activity from quassin, comprising: S101: Under sterile conditions, take an appropriate amount of culture medium, the composition of which includes, by mass percentage, 43% glucose, 17.7% corn flour, 10.8% maltose, 10.8% peptone, 4.3% potassium dihydrogen phosphate, 2.2% magnesium sulfate, 10.8% yeast powder, 11.1% vitamin B1, and the rest is distilled water. Inoculate the bone pseudo-perennial porphyra in the culture medium, wherein the volume of the bone pseudo-perennial porphyra is 8% of the volume of the culture medium. Under the conditions of a shaking table temperature of 28°C and a rotation speed of 180r / min, the bone pseudo-perennial porphyra is shaken and cultured in the culture medium for 7 days to obtain a bone pseudo-perennial porphyra culture solution containing protein and polysaccharide.

[0026] S102: Cut fresh quassin into slices, dry them at 48°C, crush them with a high-speed grinder, and pass them through a 40-mesh sieve to obtain quassin powder. Add the quassin powder to 80% ethanol at a weight ratio of 1:8, mix them well, and heat and reflux them at 105°C for 3 times, each time for 3 hours. After each reflux, filter them, mix all the filtrates, and distill them under reduced pressure to recover the ethanol, and obtain an extract without alcohol taste.

[0027] S103: Dilute the extract with distilled water to form a crude extract. Add the culture solution of Pseudomonas aeruginosa to the crude extract, stir it thoroughly, and ferment it at a temperature of 28°C, a pH of 5.6, and an aeration rate of 0.6 m 3 / min, and then aerobic fermentation was performed for 96 hours to obtain a fermentation liquid with a pH of 5.2. The mass ratio of the quassia powder to the culture liquid of the bone-like Pseudomonas aeruginosa was 1:8.

[0028] S104: After the fermentation liquid is filtered by a vacuum filtration pump, it is centrifuged at a speed of 3500r / min for 15min to obtain a supernatant. The supernatant is mixed with petroleum ether at a volume ratio of 1:1, and a first extraction is performed to obtain a petroleum ether extract. The petroleum ether extract is mixed with ethyl acetate at a volume ratio of 1:1, and a second extraction is performed to obtain an ethyl acetate extract.

[0029] S105: The ethyl acetate extract is loaded onto a macroporous resin column and eluted with methanol. The eluted component is concentrated under reduced pressure and crystallized to obtain quassin crystals.

[0030] Example 2 The present application provides a method for using a bone-based pseudo-perennial porphyra to extract quassin with insecticidal activity from quassin, comprising: S201: Under sterile conditions, take an appropriate amount of culture medium, the composition of which includes, by mass percentage, 43% glucose, 17.7% corn flour, 10.8% maltose, 10.8% peptone, 4.3% potassium dihydrogen phosphate, 2.2% magnesium sulfate, 10.8% yeast powder, 11.1% vitamin B1, and the rest is distilled water. Inoculate the bone pseudo-perennial porphyra in the culture medium, wherein the volume of the bone pseudo-perennial porphyra is 7% of the volume of the culture medium. Under the conditions of a shaking table temperature of 28°C and a rotation speed of 180r / min, the bone pseudo-perennial porphyra is shaken and cultured in the culture medium for 7 days to obtain a bone pseudo-perennial porphyra culture solution containing protein and polysaccharide.

[0031] S202: Cut fresh quassin into slices, dry them at 52°C, crush them with a high-speed grinder, and pass them through a 40-mesh sieve to obtain quassin powder. Add the quassin powder to 80% ethanol at a weight ratio of 1:8, mix them well, and heat and reflux them at 105°C for 3 times, each time for 3 hours. After each reflux, filter them, mix all the filtrates, and distill them under reduced pressure to recover the ethanol, and obtain an extract without alcohol taste.

[0032] S203: Dilute the extract with distilled water to form a crude extract. Add the culture solution of Pseudomonas aeruginosa to the crude extract, stir it thoroughly, and ferment it at a temperature of 28°C, a pH of 5.5, and an aeration rate of 0.6 m 3 / min, and then aerobic fermentation was performed for 96 hours to obtain a fermentation liquid with a pH of 5.3. The mass ratio of the quassia powder to the culture liquid of the bone-like Pseudomonas aeruginosa was 1:5.

[0033] S204: After the fermentation liquid is filtered by a vacuum filtration pump, it is centrifuged at a speed of 3500 r / min for 15 minutes to obtain a supernatant. The supernatant is mixed with petroleum ether at a volume ratio of 1:1, and a first extraction is performed to obtain a petroleum ether extract. The petroleum ether extract is mixed with ethyl acetate at a volume ratio of 1:1, and a second extraction is performed to obtain an ethyl acetate extract.

[0034] S205: The ethyl acetate extract is loaded onto a macroporous resin column and eluted with methanol. The eluted component is concentrated under reduced pressure and crystallized to obtain quassin crystals.

[0035] Example 3 The present application provides a method for using a bone-based pseudo-perennial porphyra to extract quassin with insecticidal activity from quassin, comprising: S301: Under sterile conditions, take an appropriate amount of culture medium, the composition of which includes, by mass percentage, 43% glucose, 17.7% corn flour, 10.8% maltose, 10.8% peptone, 4.3% potassium dihydrogen phosphate, 2.2% magnesium sulfate, 10.8% yeast powder, 1.1% vitamin B1, and the rest is distilled water. Inoculate the bone pseudo-perennial porphyra in the culture medium, wherein the volume of the bone pseudo-perennial porphyra is 5% of the volume of the culture medium. Under the conditions of a shaking table temperature of 28°C and a rotation speed of 180r / min, the bone pseudo-perennial porphyra is shaken and cultured in the culture medium for 7 days to obtain a bone pseudo-perennial porphyra culture solution containing protein and polysaccharide.

[0036] S302: Cut fresh quassin into slices, dry them at 50°C, crush them with a high-speed grinder, and pass them through a 40-mesh sieve to obtain quassin powder. Add the quassin powder to 80% ethanol at a weight ratio of 1:8, mix them well, and heat and reflux them at 105°C for 2 times, each time for 3 hours. After each reflux, filter them, mix all the filtrates, and distill them under reduced pressure to recover the ethanol, and obtain an extract without alcohol taste.

[0037] S303: Dilute the extract with distilled water to form a crude extract. Add the culture solution of Pseudomonas aeruginosa to the crude extract, stir it thoroughly, and ferment it at a temperature of 28°C, a pH of 6, and an aeration rate of 0.6 m 3 The fermentation was carried out aerobically for 96 hours under the condition of 1:10, and a fermentation liquid with a pH of 5.3 was obtained.

[0038] S304: After the fermentation liquid is filtered by a vacuum filtration pump, it is centrifuged at a speed of 3500r / min for 15min to obtain a supernatant. The supernatant is mixed with petroleum ether at a volume ratio of 1:1, and a first extraction is performed to obtain a petroleum ether extract. The petroleum ether extract is mixed with ethyl acetate at a volume ratio of 1:1, and a second extraction is performed to obtain an ethyl acetate extract.

[0039] S305: The ethyl acetate extract is loaded onto a macroporous resin column and eluted with methanol. The eluted component is concentrated under reduced pressure and crystallized to obtain quassin crystals.

[0040] Example 4 The present application provides a method for using a bone-based pseudo-perennial porphyra to extract quassin with insecticidal activity from quassin, comprising: S401: Under sterile conditions, take an appropriate amount of culture medium, the composition of which includes, by mass percentage, 43% glucose, 17.7% corn flour, 10.8% maltose, 10.8% peptone, 4.3% potassium dihydrogen phosphate, 2.2% magnesium sulfate, 10.8% yeast powder, 11.1% vitamin B1, and the rest is distilled water. Inoculate the bone pseudo-perennial porphyra in the culture medium, wherein the volume of the bone pseudo-perennial porphyra is 10% of the volume of the culture medium. Under the conditions of a shaking table temperature of 28°C and a rotation speed of 180r / min, the bone pseudo-perennial porphyra is shaken and cultured in the culture medium for 7 days to obtain a bone pseudo-perennial porphyra culture solution containing protein and polysaccharide.

[0041] S402: Cut fresh quassin into slices, dry them at 46°C, crush them with a high-speed grinder, and pass them through a 40-mesh sieve to obtain quassin powder. Add the quassin powder to 80% ethanol at a weight ratio of 1:8, mix them well, and heat and reflux them at 105°C for 4 times, each time for 3 hours. After each reflux, filter them, mix all the filtrates, and then distill them under reduced pressure to recover the ethanol, and obtain an extract without alcohol taste.

[0042] S403: Dilute the extract with distilled water to form a crude extract. Add the culture solution of Pseudomonas aeruginosa to the crude extract, stir it thoroughly, and ferment it at a temperature of 28°C, a pH of 5.4, and an aeration rate of 0.6 m 3 The fermentation was carried out aerobically for 96 hours at a temperature of 100 °C / min to obtain a fermentation liquid with a pH of 5.2. The mass ratio of the quassia powder to the culture liquid of the bone-like Pseudomonas aeruginosa was 1:6.

[0043] S404: After the fermentation liquid is filtered by a vacuum filtration pump, it is centrifuged at a speed of 3500r / min for 15 minutes to obtain a supernatant. The supernatant is mixed with petroleum ether at a volume ratio of 1:1, and a first extraction is performed to obtain a petroleum ether extract. The petroleum ether extract is mixed with ethyl acetate at a volume ratio of 1:1, and a second extraction is performed to obtain an ethyl acetate extract.

[0044] S405: The ethyl acetate extract is loaded onto a macroporous resin column and eluted with methanol. The eluted component is concentrated under reduced pressure and crystallized to obtain quassin crystals.

[0045] Comparative Example 1 The comparative example of the present application provides a method for using yeast to extract quassin with insecticidal activity from quassin. The specific process of the method is the same as that of Example 1, except that the bone-like Pseudomonas aeruginosa is replaced by yeast.

[0046] Comparative Example 2 The comparative example of the present application provides a method for using Aspergillus fumigatus to extract quassin with insecticidal activity from quassin. The specific process of the method is the same as that of Example 1, except that Pseudomonas aeruginosa is replaced by Aspergillus fumigatus.

[0047] The yields of quassin in Example 1, Comparative Example 1 and Comparative Example 2 were calculated to show that the yield of quassin extracted in Example 1 was 2.6%, the yield of quassin extracted in Comparative Example 1 was 1.56%, and the yield of quassin extracted in Comparative Example 2 was 1.69%. Thus, relative to Example 1, the yields of quassin extracted in Comparative Examples 1 and 2 were reduced by 40% and 35%, respectively, which indicates that Pseudomonas aeruginosa can increase the yield of quassin extracted from quassin.

[0048] In addition, in order to explore the effects of the proportion of Pseudomonas aeruginosa in the culture medium, the ratio of Pseudomonas aeruginosa to quassin powder, and the ventilation volume on the yield of quassin, experiments were conducted separately in the embodiments of the present application, and the specific contents are as follows.

[0049] 1. Effect of the proportion of Pseudomonas aeruginosa in the culture medium on the yield of quassin The method for extracting quassin with insecticidal activity from quassin by using Pseudomonas australis in Example 1 was used to extract quassin, and the volume proportion of Pseudomonas australis in the culture medium was set to 4%, 6%, 8% and 10%, respectively. The yields of quassin were calculated and plotted to obtain the attached Figure 1 .

[0050] By the attached Figure 1It can be seen that when the volume proportion of Pseudomonas australis in the culture medium is 8%, the yield of quassin is the highest, reaching 2.6%. Therefore, it is determined that when using Pseudomonas australis to extract quassin from quassin, the optimal volume proportion of Pseudomonas australis in the culture medium is 8%.

[0051] 2. Effect of the ratio of Pseudomonas aeruginosa to Quassin powder on the yield of quassin The method for extracting quassin with insecticidal activity from quassin in Example 1 was used to extract quassin, and the weight ratio of quassin to quassin powder was set to 4 times, 6 times, 8 times and 10 times, respectively. The yield of quassin was calculated and plotted to obtain the attached Figure 2 .

[0052] By the attached Figure 2 It can be seen that when the weight ratio of Pseudopora sibiricum to Quassia powder is 10 times, the yield of quassin is the highest, reaching 3.8%. Therefore, it is determined that when using Pseudopora sibiricum to extract quassin from Quassia, the optimal weight ratio of Pseudopora sibiricum to Quassia powder is 10 times.

[0053] 3. Effect of ventilation volume on quassin yield The method for extracting quassin with insecticidal activity from quassin by using Pseudomonas aeruginosa in Example 1 was used to extract quassin, and the ventilation volume during aerobic fermentation was set to 0.2 m 3 / min、0.4m 3 / min, 0.6m 3 / min and 0.8m 3 / min, calculate the yield of quassin and draw a graph to obtain the attached Figure 3 .

[0054] By the attached Figure 3 It can be seen that when the ventilation volume during aerobic fermentation is 0.6m 3 / min, the yield of quassin was the highest, reaching 2.1%. It was determined that the optimal ventilation volume for aerobic fermentation when using Pseudomonas aeruginosa to extract quassin from quassin was 0.6m 3 / min.

[0055] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A polypore fungus, characterized in that: The inoculum is a bone pseudo-perennial inoculum, and the bone pseudo-perennial inoculum ( Perenniporiopsis sp. ) was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on November 6, 2023, with the deposit number CGMCC No. 40976.

2. The Pseudomonas aeruginosa described in claim 1 is used to extract quassin with insecticidal activity from quassin.

3. The use according to claim 2, characterized in that: The extraction method comprises: Under sterile conditions, inoculate the bone-bearing Pseudomonas australis into the culture medium, and culture on a shaking table to obtain the bone-bearing Pseudomonas australis culture fluid; The extract was obtained by ethanol extraction and vacuum distillation of the quassia wood powder; The extract is diluted with distilled water and added into the culture solution of the Pseudomonas aeruginosa, and aerobically fermented to obtain a fermentation solution; The fermentation broth is vacuum filtered and centrifuged to obtain a supernatant which is extracted with petroleum ether and ethyl acetate to obtain an ethyl acetate extract; The ethyl acetate extract is loaded onto a macroporous resin column and eluted with methanol. The eluted component is concentrated under reduced pressure and crystallized to obtain quassin crystals.

4. The use according to claim 3, characterized in that: The mass ratio of the bitterwood powder to the culture solution of the bone-bearing pseudopora is 1:(5-10).

5. The use according to claim 3, characterized in that: The fermentation temperature is 28℃, the fermentation pH is 5-6, and the ventilation volume is 0.6m 3 / min, and the pH of the fermentation liquid after fermentation was 5.2-5.

3.

6. The use according to claim 3, characterized in that: The volume of the bone-like Pseudomonas aeruginosa is 1-10% of the volume of the culture medium.

7. The use according to claim 3, characterized in that: The shaking temperature was 28°C, the rotation speed was 180 r / min, and the shaking culture time was 7 days.

8. The use according to claim 3, characterized in that: The volume ratio of the supernatant to the petroleum ether is 1:1, and the volume ratio of the petroleum ether extract extracted from the petroleum ether to the ethyl acetate is 1:

1.

9. The use according to claim 3, characterized in that: The ethanol extraction of the quassia powder comprises: adding the quassia powder to 80% ethanol in a weight ratio of 1:8, mixing, heating and refluxing at 105° C. for 2-4 times, each time for 3 hours, filtering and mixing the filtrate.

10. The use according to claim 3, characterized in that: The preparation method of the quassia powder comprises: cutting fresh quassia into slices, drying at 46-52° C., crushing, and passing through a 40-mesh sieve to obtain the quassia powder.

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