CN109503613B - Gryllus chinensis bipolaris staurosporine I and preparation method and application thereof, and Gryllus chinensis bipolaris staurosporine J and preparation method and application thereof - Google Patents
Gryllus chinensis bipolaris staurosporine I and preparation method and application thereof, and Gryllus chinensis bipolaris staurosporine J and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to yard grass bipolaris sacchari I and a preparation method and application thereof, and yard grass bipolaris sacchari J and a preparation method and application thereof, belonging to the field of chemical engineering. Wherein the chemical structural formula of the yard grass bipolaris I is shown in the specification
The chemical structural formula of yard grass bipolaris sacchari J is shown as
The preparation method of the two comprises fermenting Achillea sinensis, extracting the fermentation product with ethyl acetate, and separating and purifying the ethyl acetate extract. The method is simple and easy to operate, and the obtained compound has high purity. The yard grass bipolaris sacchari I and yard grass bipolaris sacchari J both have certain effect of inhibiting alternaria solani, and the yard grass bipolaris sacchari I can also inhibit fusarium oxysporum.
Description
Technical Field
The invention relates to the field of chemical engineering, and in particular relates to yard grass bipolaris sacchari I and a preparation method and application thereof, and yard grass bipolaris sacchari J and a preparation method and application thereof.
Background
Potatoes (the name of the science: Solanum tuberosum L.) belong to annual herbaceous plants of the solanaceae family, tubers are edible, are the fourth most important grain crops in the world, and are second only to wheat, rice and corn. The potato is also called as ground egg, potato, yam, etc., the tuber of solanaceae plant, wheat, rice, corn and sorghum are combined into five crops in the world.
The fresh potatoes generally contain the following components: 9-20% of starch, 1.5-2.3% of protein, 0.1-1.1% of fat and 0.6-0.8% of crude fiber. 100g of the potato contains the following nutrients: 318 kilojoule of energy, 5-8mg of calcium, 15-40mg of phosphorus, 0.4-0.8 mg of iron, 340mg of potassium 200-sodium bicarbonate, 0.8-1.2 mg of iodine, 12-30mg of carotene, 0.03-0.08mg of thiamine, 0.01-0.04mg of riboflavin and 0.4-1.1mg of nicotinic acid, and is rich in nutrition.
The traditional Chinese medicine considers that the potatoes are neutral in nature, sweet in taste and non-toxic, and can invigorate the spleen and stomach, replenish qi and regulate middle warmer, relieve spasm and pain and relieve constipation. Has obvious effect on patients with weakness of the spleen and the stomach, dyspepsia, incoordination between the intestines and the stomach, epigastric pain and abdominal pain and unsmooth defecation. Modern researches prove that the potato has special effect on the regulation of dyspepsia, and is a good medicine and a high-quality health-care product for patients with stomach diseases and heart diseases. In addition, the potato is rich in nutrition and is one of anti-aging foods.
At present, scholars at home and abroad make certain progress on the chemical components and application research of potatoes, but further intensive research is needed.
Disclosure of Invention
One of the purposes of the invention is to provide a yard grass bipolaris cephalosporin I, the chemical structural formula of which is shown in the specification
The second purpose of the invention is to provide a yard grass bipolaris sacchari J, the chemical structural formula of which is as follows
The invention also aims to provide a preparation method of yard grass bipolaris staurosporine I and yard grass bipolaris staurosporine J, which is simple and easy to operate and can effectively prepare yard grass bipolaris staurosporine I or yard grass bipolaris staurosporine J with higher purity.
The fourth purpose of the invention is to provide yard grass bipolaris cephalosporin I which can be used for inhibiting alternaria alternata and/or fusarium oxysporum of potato early blight.
The fifth purpose of the invention is to provide application of yard grass bipolaris cephalosporin J, which can be used for inhibiting alternaria solani.
The technical problem to be solved by the invention is realized by adopting the following technical scheme:
the invention provides a yard grass bipolaris staurosporine I, the chemical structural formula of the yard grass bipolaris staurosporine I is as follows:
the invention provides a yard grass bipolaris rhzomorph J, the chemical structural formula of the yard grass bipolaris rhzomorph J is as follows:
the invention also provides a preparation method of the yard grass bipolaris sacchari I and yard grass bipolaris sacchari J, which comprises the following steps:
fermenting yard grass bipolaris, extracting a fermentation product by using ethyl acetate, carrying out first normal phase silica gel column separation on an ethyl acetate extract, carrying out second normal phase silica gel column separation on a third elution component after the first normal phase silica gel column separation, carrying out gel column separation on a second sub-component after the second normal phase silica gel column separation, carrying out preparative liquid chromatography purification on a substance obtained after the gel column separation, collecting two chromatographic peak components with the highest content, and obtaining yard grass bipolaris J with the front retention time and yard grass bipolaris I with the rear retention time.
Wherein, the conditions of the first normal phase silica gel column separation comprise: chloroform and methanol are used as eluent, and the concentration gradient of the chloroform and the methanol in the eluent is 100:0, 50: 1: gradient elution is carried out at the ratio of 20:1, 10:1, 5:1, 1:1 and 0:1, the volume of eluent used in each gradient is 4.5-5.5L, and seven elution components are respectively obtained.
The conditions of the second normal phase silica gel column separation include: petroleum ether and acetone are used as eluent, the volume ratio of the petroleum ether to the acetone is 10-14:1, isocratic elution is carried out, and every 180-220mL is used as an elution stage, so that six sub-elution components are respectively obtained.
The conditions for gel column separation include: and eluting with 600mL of methanol as eluent and Sephadex LH-20 as a filler.
The conditions for preparative liquid chromatography purification include: eluting with acetonitrile and water as eluent at initial concentration of 30:70 and final concentration of 60:40 for 20-30 min.
The invention also provides application of the yard grass bipolaris cephalosporin I, and the yard grass bipolaris cephalosporin I can be used for inhibiting alternaria solani and/or fusarium oxysporum.
The invention also provides application of the yard grass bipolaris rhzomorph J, and the yard grass bipolaris rhzomorph J can be used for inhibiting alternaria solani.
The yard grass bipolaris sacchari I and the preparation method and the application thereof, and the yard grass bipolaris sacchari J and the preparation method and the application thereof have the beneficial effects that:
the raw materials of the yard grass bipolaris sacchari I and the yard grass bipolaris sacchari J provided by the scheme of the invention are natural products, are green and safe, and have small toxic and side effects. And the yard grass bipolaris sacchari I and the yard grass bipolaris sacchari J are new substances which are separated from the fermentation product of yard grass bipolaris sacchari for the first time.
The yard grass bipolaris sacchari I and yard grass bipolaris sacchari J both have certain effect of inhibiting alternaria solani. Also, yard grass bipolaris I can be used to inhibit fusarium oxysporum.
In addition, the preparation method of the yard grass bipolaris sacchari I and the yard grass bipolaris sacchari J provided by the preferred embodiment of the invention is simple and easy to operate, and the yard grass bipolaris sacchari I or the yard grass bipolaris sacchari J with higher purity and higher yield can be effectively prepared from the fermentation product of the yard grass bipolaris sacchari.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 is a microscopic morphological view of hyphae of an endophytic fungus in test example 1 of the present application;
FIG. 2 is a microscopic morphological view of spores of an endophytic fungus in test example 1 of the present application;
FIG. 3 is a molecular phylogenetic tree of the 18S rDNA sequence analysis of the endophytic fungi of test example 1 of the present application.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The yard grass bipolaris sacchari I and the preparation method and application thereof, and yard grass bipolaris sacchari J and the preparation method and application thereof will be specifically described.
The chemical structural formula of the yard grass bipolaris I provided by the application is
The chemical structural formula of yard grass bipolaris sacchari J is shown as
The above-mentioned process for producing yard grass bipolaris I and yard grass bipolaris J may comprise, for example, the steps of: fermenting yard grass bipolaris, extracting a fermentation product by using ethyl acetate, carrying out first normal phase silica gel column separation on an ethyl acetate extract, carrying out second normal phase silica gel column separation on a third elution component after the first normal phase silica gel column separation, carrying out gel column separation on a second sub-component after the second normal phase silica gel column separation, carrying out preparative liquid chromatography purification on a substance obtained after the gel column separation, collecting two chromatographic peak components with the highest content, and obtaining yard grass bipolaris J with the front retention time and yard grass bipolaris I with the rear retention time.
Wherein, the extraction frequency of the ethyl acetate can be only 1 time, or can be multiple times, such as 2 times, 3 times and the like, when the extraction frequency is multiple times, the ethyl acetate extract used for the first normal phase silica gel column separation is the combined solution of the ethyl acetate extracts obtained after each extraction. In some embodiments, the volume of ethyl acetate used is the same as the volume of ethyl acetate fermentation product at each extraction.
In some embodiments, the conditions of the first normal phase silica gel column separation comprise: chloroform and methanol are used as eluent, and the concentration gradient of the chloroform and the methanol in the eluent is 100:0, 50: 1: gradient elution is carried out at ratios of 20:1, 10:1, 5:1, 1:1 and 0:1, and the volume of eluent used in each gradient can be 4.5-5.5L (preferably 5L), so that seven elution components are obtained respectively.
It should be noted that the concentration gradient of chloroform to methanol is not limited to 100:0, 50: 1: 20:1, 10:1, 5:1, 1:1 and 0:1, and an error of 100mL is allowed to be added or subtracted in the actual configuration process. Taking the volume of the eluent used in each gradient as 5L and the concentration gradient of chloroform and methanol as 100:0 as an example, setting the volume of chloroform to 4900mL-5100mL in the configuration process is within the protection scope of the scheme of the application. In addition, the volume of the eluent used for each gradient is also allowed to be plus or minus 100mL, that is, the total volume of the eluent used for each gradient is 4900mL-5100mL in the configuration process, which is within the protection scope of the scheme of the application. The following references to the volume of eluent are all within a certain range of tolerances.
In some embodiments, the elution flow rate during the first normal phase silica gel column separation process can be 140-160mL/min, such as 140mL/min, 145mL/min, 150mL/min, 155mL/min, 160mL/min, etc., and furthermore, the elution flow rate can be any flow rate value within the range of 140-160 mL/min.
In some embodiments, the conditions of the second normal phase silica gel column separation comprise: the elution is carried out with petroleum ether and acetone as eluent and with the volume ratio of the petroleum ether to the acetone being 10-14:1, for example, the volume ratio of the petroleum ether to the acetone is 10:1, 11:1, 12:1, 13:1 or 14:1, etc., or 10.5:1, 11.5:1, 12.5:1 or 13.5:1, etc., or any volume ratio within the range of 10-14: 1. In some preferred embodiments, the volume ratio of petroleum ether to acetone is maintained at 12: 1.
In the second normal phase silica gel column separation process, for example, 220mL (preferably 200mL) can be used as an elution stage, and six sub-elution components are respectively obtained.
Optionally, the silica gel used for the first normal phase silica gel column separation is 200-300 meshes, and/or the silica gel used for the second normal phase silica gel column separation is 200-300 meshes.
In the present application, the first normal phase silica gel column separation and the second normal phase silica gel column separation can be performed in the same separation column, that is, only the eluent and the elution conditions are different in the two separation processes. Furthermore, it is also possible to carry out the separation in two different separation columns.
In some embodiments, the conditions for gel column separation comprise: and eluting with 600mL of methanol as eluent and Sephadex LH-20 as a filler.
In some embodiments, the conditions of preparative liquid chromatography purification include: taking acetonitrile and water as eluent, and carrying out gradient elution for 20-30min according to the initial concentration of the acetonitrile and the water of 30:70 and the final concentration of the acetonitrile and the water of 60:40, wherein the process is directly carried out by setting in a preparative liquid chromatography program.
Alternatively, the elution flow rate during preparative liquid chromatography purification can be 8-12mL/min, such as 8mL/min, 8.5mL/min, 9mL/min, 9.5mL/min, 10mL/min, 10.5mL/min, 11mL/min, 11.5mL/min, or 12mL/min, etc., preferably 10 mL/min.
Alternatively, in the purification by preparative liquid chromatography, the sample amount may be, for example, 80 to 120. mu.L (preferably 100. mu.L) and the column temperature set at 25 to 27 ℃ (preferably 26 ℃).
During the purification process of the preparative liquid chromatography, two most obvious chromatographic peaks appear in the chromatogram, namely two chromatographic peaks with the highest content, wherein the chromatographic peak with the retention time at the first (the first peak) is yard grass bipolaris J, and the chromatographic peak with the retention time at the last (the last peak) is yard grass bipolaris I. For reference, the retention time of yard grass bipolaris sacchari J was about 16.7min and the retention time of yard grass bipolaris sacchari I was about 18.3min in the above manner.
In the present application, bipolaris eleusines may be bipolaris eleusines related to the prior art, or may be isolated and purified as follows.
Preferably, the yard grass bipolaris in the present application is isolated from potato, and the obtaining method thereof comprises the following steps: culturing potato in culture medium to grow colony, separating and purifying colony.
Wherein, the potatoes are provided by the research institute of potatoes of Yunnan agricultural university, are respectively collected from three places of Dehong, Dali and Lincang in Yunnan province, are fresh and healthy, and have no obvious plant diseases and insect pests.
The potato raw material is cultured in different culture media, and the target strain can grow in which culture medium. Specifically, the roots, stems, leaves and tubers of healthy picked potato plants are cleaned and wiped under pure tap water, the roots, stems and tubers are cut into small segments of 2-4cm, the leaves are cut into small pieces of (2-4) cm x (2-4) cm, and then surface sterilization is carried out according to the following steps: rinsing each separated part with 75 wt% ethanol for 1min on an ultra-clean bench (under aseptic condition), and washing with aseptic water for 3 times; sterilizing with 2 wt% sodium hypochlorite (sterilization time: root 3min, stem 2.5min, leaf 2min, tuber 1.5min) or 0.1 wt% mercuric chloride (sterilization time: root 40s, stem 30s, leaf 20s, tuber 10s), and rinsing with sterile water for 3 times; then placed on sterile filter paper and the water blotted dry. Sterilizing the surface, cutting off surface cuts of root, stem and tuber, cutting the rest part from the middle, and cutting into 0.3-0.5cm3The small blocks are inoculated and cultured, the leaves are cut off at the periphery, and the rest parts are cut into small blocks of (0.2-0.4) cm multiplied by (0.2-0.4) cm for inoculation and culture.
The different media used for the inoculation culture include water agar (non-nutrient) medium, normal medium, modified PDA medium, Peter medium and BPA (meat peptone) medium.
The preparation method of the water agar (without nutrition) culture medium comprises the following steps: heating and boiling 20g of agar powder and 1000mL of deionized water, subpackaging after agar is melted, and sterilizing for 15-20min by high-pressure steam at 121 ℃.
The preparation method of the common culture medium comprises the following steps: taking 1.0g of glucose, 0.03g of pork peptone, 0.01g of monopotassium phosphate, 0.1g of yeast powder, 0.01g of anhydrous magnesium sulfate, 20g of agar powder and 1000mL of deionized water, heating and boiling, subpackaging after the agar is melted, and sterilizing for 15-20min by high-pressure steam at 121 ℃.
The preparation method of the improved PDA culture medium comprises the following steps: peeling potato 200g (slice, slightly thick), deionized water 1000mL, heating and boiling for 20min, filtering with double-layer gauze to remove residue, adding deionized water to 1000mL, adding glucose 20g, anhydrous magnesium sulfate 1.5g, potassium dihydrogen phosphate 3.0g, pork peptone 1.0g, agar powder 20g, vitamin B1Heating to dissolve 10mg, adjusting pH to 6.0-6.5 with citric acid, packaging, and sterilizing with high pressure steam at 121 deg.C for 15-20 min.
The preparation method of the Peter culture medium comprises the following steps: 20g of glucose, 3.0g of yeast extract powder, 3.0g of malt extract powder, 5.0g of peptone, 20g of agar powder and 1000mL of deionized water, heating for dissolving, adjusting the pH value to 7.2-7.4 by using citric acid, subpackaging, and sterilizing for 15-20min by high-pressure steam at 121 ℃.
The preparation method of the BPA (meat peptone) culture medium comprises the following steps: taking 3.0g of beef extract, 5.0g of peptone, 1.0g of yeast extract, 10g of glucose, 20g of agar and 1000mL of deionized water, heating for dissolving, adjusting the pH value to 7.2 by using citric acid, subpackaging, and sterilizing for 15-20min by using high-pressure steam at 121 ℃.
By culturing in a 22-25 ℃ incubator (by isothermal culture herein is meant that the temperature of the incubation can be set to any temperature value within the range of 22-25 ℃), only the modified PDA medium is able to grow species that may be the target species.
Further, when the macroscopic colonies are observed to grow to the periphery of the improved PDA culture medium in different tissues of the potato, single colonies with obvious differences are picked up to be subjected to purification culture in the new improved PDA culture medium according to the colony morphology, the color and the growing time. A top end hypha purification method is adopted, the growing condition of endophytic fungi of potato plant segments inoculated on a new improved PDA culture medium needs to be regularly observed, the top end part of the newly grown hypha at a cut is picked up after the endophytic fungi grow out, the top end part of the newly grown hypha is transferred to a new PDA culture medium, and purification culture is continuously carried out until pure endophytic fungi strains are obtained.
Adopting a bevel low-temperature preservation method. Inoculating 3 activated purified strains of each strain of the pure strains to a slant test tube culture medium, and culturing in a constant temperature incubator at 22-25 ℃. After the endophytic fungi grow on the whole inclined plane, the mixture is placed in a refrigerator at 4 ℃ for storage, and the storage time is 3-6 months.
Further, the fermentation of yard grass bipolaris comprises: the yard grass bipolaris is cultured for the first time in a potato glucose agar (PDA) solid culture medium, and then transferred to a liquid fermentation culture medium containing potato glucose (PDA) for the second time.
Wherein the first culture can be carried out at 23-25 deg.C for 8-12 days. The second culture can be carried out at 27-29 deg.C for 1-3 days, and then at 24-26 deg.C for 19-21 days. In some embodiments, the second culturing process is performed at a rotation speed of 150-.
It is worth mentioning that the yard grass bipolaris fermentation broth can be concentrated before extraction in order to reduce the amount of the extraction agent and shorten the extraction time.
In addition, the application also provides application of the yard grass bipolaris sacchari I and yard grass bipolaris sacchari J, wherein the yard grass bipolaris sacchari I can be used for inhibiting alternaria solani and/or fusarium oxysporum, and the yard grass bipolaris sacchari J can be used for inhibiting alternaria solani.
Example 1
Cleaning roots, stems, leaves and tubers of healthy potato plants provided by the potato research institute of Yunnan agricultural university in pure tap water, wiping the roots, stems and tubers to be dry, cutting the roots, stems and tubers into small segments of 3cm, cutting the leaves into small pieces of 3cm multiplied by 3cm, and then carrying out surface sterilization according to the following steps: rinsing each separated part with 75 wt% ethanol for 1min on an ultra-clean bench (under aseptic condition), and washing with aseptic water for 3 times; sterilizing with 2 wt% sodium hypochlorite (sterilization time: root for 3min, stem for 2.5 min)2min for leaves and 1.5min for tubers), rinsing with sterile water for 3 times; then placed on sterile filter paper and the water blotted dry. Sterilizing the surface, cutting off surface cuts of root, stem and tuber, cutting the rest part from the middle, and cutting into 0.4cm3The small pieces are inoculated and cultured, the leaves are cut off at the periphery, and the rest parts are cut into small pieces of 0.3cm multiplied by 0.3cm for inoculation and culture. The different media used for the inoculation culture include water agar (non-nutrient) medium, normal medium, modified PDA medium, Peter medium and BPA (meat peptone) medium. The inoculation culture is carried out in a constant temperature incubator at a temperature of 24 ℃.
When macroscopic colonies grow to the periphery of the improved PDA culture medium in different tissues of the potato, selecting single colonies with obvious differences to perform purification culture in a new improved PDA culture medium, and then continuously performing purification culture by adopting a top hypha purification method until an endophytic fungi pure strain (yard grass bipolaris) is obtained.
The yard grass bipolaris is cultured for the first time in a potato glucose agar solid culture medium at 24 ℃ for 10 days, then transferred into a 500nL specification fermentation shake flask filled with 100mL of PDA nutrient solution, suspended and shaken for 2 days at 28 ℃ and 160r/min, and then transferred into 120 bottles in a mode of transferring 25mL of fermentation broth after 25mL of shake flask fermentation into a 250mL specification fermentation shake flask, and the 120 bottles of inoculation shake flasks are all subjected to shake culture at 25 ℃ and 160r/min for 20 days.
Taking 20L of yard grass bipolaris fermentation liquor after shaking culture, concentrating to 3L, extracting for 3 times with equal volume of ethyl acetate, mixing extractive solutions, and concentrating to obtain 18g extract.
Mixing the extract with 54g normal phase silica gel, performing first normal phase silica gel column separation, performing second normal phase silica gel column separation on the third elution component (1.2g) after the first normal phase silica gel column separation, performing gel column separation on the second sub-component (90mg) after the second normal phase silica gel column separation, performing preparative liquid chromatography purification on the substance after the gel column separation, and collecting two chromatographic peak components with the highest content to obtain yard grass bipolaris J (3.5mg) with the retention time of 16.7min and yard grass bipolaris I (4.2mg) with the retention time of 18.3 mg.
Wherein, the conditions of the first normal phase silica gel column separation comprise: silica gel of 200 meshes is used as a filler, chloroform and methanol are used as eluent, and the ratio of the concentration gradient of the chloroform to the concentration gradient of the methanol in the eluent is 100:0, 50: 1: gradient elution is carried out at the ratio of 20:1, 10:1, 5:1, 1:1 and 0:1, the volume of eluent used in each gradient can be 5L, the elution flow rate is 150mL/min, and seven elution components are respectively obtained.
The conditions of the second normal phase silica gel column separation include: taking 200-mesh silica gel as a filler, taking petroleum ether and acetone as eluent, carrying out isocratic elution with the volume ratio of the petroleum ether to the acetone being 12:1, and using 1.2L of eluent in total to respectively obtain six sub-elution components.
The conditions for gel column separation include: and eluting with 600mL of methanol as eluent and Sephadex LH-20 as a filler.
The conditions for preparative liquid chromatography purification include: taking acetonitrile and water as eluent, carrying out gradient elution for 30min according to the initial concentration of the acetonitrile and the water of 30:70 and the final concentration of 60:40, wherein the elution flow rate is 10mL/min, the sample injection amount is 100 mu L, and the column temperature is 26 ℃.
Example 2
Cleaning roots, stems, leaves and tubers of healthy potato plants provided by the potato research institute of Yunnan agricultural university in pure tap water, wiping the roots, stems and tubers to be dry, cutting the roots, stems and tubers into small segments of 2cm, cutting the leaves into small pieces of 2cm multiplied by 2cm, and then carrying out surface sterilization according to the following steps: rinsing each separated part with 75 wt% ethanol for 1min on an ultra-clean bench (under aseptic condition), and washing with aseptic water for 3 times; sterilizing with 0.1 wt% mercuric chloride (sterilizing time: root 40s, stem 30s, leaf 20s, and tuber 10s), and rinsing with sterile water for 3 times; then placed on sterile filter paper and the water blotted dry. Sterilizing the surface, cutting off surface cuts of root, stem and tuber, cutting the rest part from the middle, and cutting into 0.3cm3The small pieces are inoculated and cultured, the leaves are cut off at the periphery, and the rest parts are cut into small pieces of 0.2cm multiplied by 0.2cm for inoculation and culture. The different culture media used for the inoculation culture include water agar (non-nutrient) culture medium, and common cultureMedium, modified PDA medium, Peter medium and BPA (meat peptone) medium. The inoculation culture is carried out in a constant temperature incubator at 22 ℃.
When macroscopic colonies grow to the periphery of the improved PDA culture medium in different tissues of the potato, selecting single colonies with obvious differences to perform purification culture in a new improved PDA culture medium, and then continuously performing purification culture by adopting a top hypha purification method until an endophytic fungi pure strain (yard grass bipolaris) is obtained.
Culturing yard grass bipolaris in potato glucose agar solid culture medium at 23 deg.C for 12 days, transferring into 500nL fermentation shake flask containing 100mL PDA nutrient solution, suspending and shaking 3 natural at 27 deg.C and 150r/min, transferring 25mL fermentation broth into 250mL fermentation shake flask, transferring into 120 bottles, and shake-culturing 120 bottles at 24 deg.C and 150r/min for 21 days.
Taking 20L of yard grass bipolaris fermentation liquor after shaking culture, concentrating to 3L, extracting for 2 times with equal volume of ethyl acetate, mixing extractive solutions, and concentrating to obtain extract.
Mixing the extract with normal phase silica gel, performing first normal phase silica gel column separation, performing second normal phase silica gel column separation on the third elution component after the first normal phase silica gel column separation, performing gel column separation on the second sub-component after the second normal phase silica gel column separation, performing preparative liquid chromatography purification on the substance after the gel column separation, and collecting two chromatographic peak components with the highest content to obtain yard grass bipolaris J with retention time of 16.7min and yard grass bipolaris I with retention time of 18.3 mg.
Wherein, the conditions of the first normal phase silica gel column separation comprise: using 250-mesh silica gel as a filler, using chloroform and methanol as eluent, and performing concentration gradient of the chloroform and the methanol in the eluent at a ratio of 100:0 and 50: 1: gradient elution was performed at 20:1, 10:1, 5:1, 1:1 and 0:1 ratios, the volume of eluent used for each gradient was 4.5L, and the elution flow rate was 140mL/min, to obtain seven fractions, respectively.
The conditions of the second normal phase silica gel column separation include: taking 250-mesh silica gel as a filler, taking petroleum ether and acetone as eluent, carrying out isocratic elution with the volume ratio of the petroleum ether to the acetone being 10:1, and using 1.08L of eluent in total to respectively obtain six sub-elution components.
The conditions for gel column separation include: and eluting with 600mL of methanol as eluent and Sephadex LH-20 as a filler.
The conditions for preparative liquid chromatography purification include: taking acetonitrile and water as eluent, performing gradient elution for 20min according to the initial concentration of the acetonitrile and the water of 30:70 and the final concentration of the acetonitrile and the water of 60:40, wherein the elution flow rate is 8mL/min, the sample injection amount is 80 mu L, and the column temperature is 25 ℃.
Example 3
Cleaning roots, stems, leaves and tubers of healthy potato plants provided by the potato research institute of Yunnan agricultural university in pure tap water, wiping the roots, stems and tubers to be dry, cutting the roots, stems and tubers into 4cm small segments, cutting the leaves into 4cm multiplied by 4cm small pieces, and then carrying out surface sterilization according to the following steps: rinsing each separated part with 75 wt% ethanol for 1min on an ultra-clean bench (under aseptic condition), and washing with aseptic water for 3 times; sterilizing with 2 wt% sodium hypochlorite (sterilization time: root 3min, stem 2.5min, leaf 2min, tuber 1.5min), and rinsing with sterile water for 3 times; then placed on sterile filter paper and the water blotted dry. Sterilizing the surface, cutting off surface cuts of root, stem and tuber, cutting the rest part from the middle, and cutting into 0.5cm3The small pieces are inoculated and cultured, the leaves are cut off at the periphery, and the rest parts are cut into small pieces of 0.4cm multiplied by 0.4cm for inoculation and culture. The different media used for the inoculation culture include water agar (non-nutrient) medium, normal medium, modified PDA medium, Peter medium and BPA (meat peptone) medium. The inoculation culture is carried out in a constant temperature incubator at a temperature of 25 ℃.
When macroscopic colonies grow to the periphery of the improved PDA culture medium in different tissues of the potato, selecting single colonies with obvious differences to perform purification culture in a new improved PDA culture medium, and then continuously performing purification culture by adopting a top hypha purification method until an endophytic fungi pure strain (yard grass bipolaris) is obtained.
The yard grass bipolaris is cultured for the first time in a potato glucose agar solid culture medium at 25 ℃ for 8 days, then transferred into a 500nL specification fermentation shake flask filled with 100mL of PDA nutrient solution, suspended and shaken for 1 natural at 29 ℃ and 170r/min, and then transferred into 120 bottles in a mode of transferring 25mL of fermentation broth after 25mL of shake flask fermentation into a 250mL specification fermentation shake flask, and the 120 bottles of inoculation shake flasks are all subjected to shake culture at 26 ℃ and 170r/min for 19 days.
Taking 20L of yard grass bipolaris fermentation liquor after shaking culture, concentrating to 3L, extracting for 1 time with equal volume of ethyl acetate, concentrating the extract to obtain extract.
Mixing the extract with normal phase silica gel, performing first normal phase silica gel column separation, performing second normal phase silica gel column separation on the third elution component after the first normal phase silica gel column separation, performing gel column separation on the second sub-component after the second normal phase silica gel column separation, performing preparative liquid chromatography purification on the substance after the gel column separation, and collecting two chromatographic peak components with the highest content to obtain yard grass bipolaris J with retention time of 16.7min and yard grass bipolaris I with retention time of 18.3 mg.
Wherein, the conditions of the first normal phase silica gel column separation comprise: using 300-mesh silica gel as a filler, using chloroform and methanol as eluent, and performing concentration gradient of the chloroform and the methanol in the eluent at a ratio of 100:0 and 50: 1: gradient elution is carried out at the ratio of 20:1, 10:1, 5:1, 1:1 and 0:1, the volume of eluent used in each gradient can be 5.5L, the elution flow rate is 160mL/min, and seven elution components are respectively obtained.
The conditions of the second normal phase silica gel column separation include: taking 300-mesh silica gel as a filler, taking petroleum ether and acetone as eluent, carrying out isocratic elution with the volume ratio of the petroleum ether to the acetone being 14:1, and using 1.32L of eluent in total to respectively obtain six sub-elution components.
The conditions for gel column separation include: and eluting with 600mL of methanol as eluent and Sephadex LH-20 as a filler.
The conditions for preparative liquid chromatography purification include: taking acetonitrile and water as eluent, carrying out gradient elution for 25min according to the initial concentration of the acetonitrile and the water of 30:70 and the final concentration of the acetonitrile and the water of 60:40, wherein the elution flow rate is 12mL/min, the sample injection amount is 120 mu L, and the column temperature is 27 ℃.
Test example 1
The examples 1 to 3 were repeated, and the shapes of the hyphae and spores of the endophytes obtained after purification in examples 1 to 3 were observed by the mycological patch culture method. Inoculating endophytic fungi into a PDA culture dish, obliquely inserting a sterilized cover glass at an angle of 45 degrees at a position close to 1/3 parts of the edge of the culture dish, placing the cover glass in a constant-temperature incubator at 25 ℃ for inverted culture for 4 days, and taking out and placing the cover glass on a glass slide to a microscope to observe the hypha form when the hypha grows to the cover glass. By observation, the endophytic fungi grow faster on the PDA culture medium, and after 4 days of culture, the endophytic fungi appear as dark brown colonies with the diameter of 4.2cm and the back dark brown to black, the aerial hyphae of the endophytic fungi are developed, and a picture of the hyphae under a microscope is shown in figure 1.
The endophytic fungi were inoculated on a spore production medium, spores were induced, and the spore morphology was observed on a microscope, and the results are shown in fig. 2. Wherein the spore production culture medium is OA (oat agar) spore-promoting culture medium, and the preparation method comprises: heating 30g of oatmeal and 1000mL of deionized water in a water bath at 60 ℃ for 1h, filtering with double-layer gauze to remove residues, adding deionized water to 1000mL of filtrate and 20g of agar powder, heating and boiling, subpackaging after agar is melted, and sterilizing with high-pressure steam at 121 ℃ for 20 min.
The purified endophytic fungi are preliminarily classified and identified by a classification system such as Ainsworth and the like according to methods such as 'fungi identification handbook', fungi taxonomy and the like, wherein a molecular phylogenetic tree of 18S rDNA sequence analysis of the endophytic fungi is shown in figure 3, so that the endophytic fungi can be proved to have high homology with yard grass Bipolaris and can be determined to be yard grass Bipolaris (Bipolaris eleusines) by combining hyphal morphological characteristics and spore characteristics of the endophytic fungi.
Test example 2
The examples 1 to 3 were repeated to obtain a sufficient amount of yard grass bipolaris I and yard grass bipolaris J.
The resultant yard grass bipolaris sacchari I and yard grass bipolaris sacchari J were subjected to structural assays including nuclear magnetic resonance assay, high resolution mass spectrometry (HRESIMS) assay, ultraviolet assay, and infrared assay.
The nmr test results are shown in table 1:
TABLE 1 preparation of yard grass bipolaris sacchari I and yard grass bipolaris sacchari J1H and13c NMR data (delta ppm; JinHz)
aData is sourced from 500MHz1H NMR spectrum and 150MHz13C NMR spectrum (solvent CDCl)3)。
The high resolution mass spectrometry result is: the molecular weight of yard grass bipolaris sacchari I is M/z575.2250[ M + Na [)]+(calculated value: C)31H36O9Na, 585.2252); the molecular weight of yard grass bipolaris sacchari J is M/z 567.2240[ M-H]-(calculated value: C)31H35O10，567.2236)。
The ultraviolet measurement result is as follows: the main uv data for yard grass bipolaris sacchari I include (MeOH): lambda [ alpha ]max(log ε)203.0(3.65),250.5(3.66),329(2.88) nm; the principal uv data for yard grass bipolaris sacchari J include (MeOH): lambda [ alpha ]max(logε)203.0(3.53),246(3.52),329.5(2.58),382.5(2.37)nm。
The infrared measurement result is as follows: the main infrared data of yard grass bipolaris sacchari I comprises (KBr) vmax：3440，2924，1745，1661，1635，1619，1462，1383，1208cm-1(ii) a The main infrared data of yard grass bipolaris sacchari J comprises (KBr) vmax：3441，2923，1740，1632，1462，1384，1206，1061cm-1。
In addition, the method can be used for producing a composite materialThe yard grass bipolaris I is light yellow oily substance, is easily soluble in organic solvent such as chloroform, acetone and methanol, and has specific optical rotation [ α]21.7 DGryllus chinensis bipolaris Merrill J is light yellow jelly, is easily soluble in organic solvents such as chloroform, acetone and methanol, and has specific optical rotation of [ α ]]21.5 D＝+22.0(c 0.2，MeOH)。
Through the determination, the chemical structural formulas of the yard grass bipolaris sacchari I and the yard grass bipolaris sacchari J obtained in the scheme are respectively shown as
Test example 3
The antibacterial tests of yard grass bipolaris I and yard grass bipolaris J obtained in the examples were carried out by the following test methods.
The test method comprises the following steps: the inhibitory effect on the following phytopathogens, i.e. the Minimum Inhibitory Concentration (MIC), was determined by the broth dilution method. The strains comprise: phytophthora infestans (Phytophthora infestane), Alternaria solani (Alternaria solani), Rhizoctonia solani (Rhizoctonia solani), Fusarium oxysporum (Fusarium oxysporum).
The minimum inhibitory concentrations of yard grass bipolaris sacchari I and yard grass bipolaris sacchari J against the four phytopathogens were determined in 96-well plates. The designed concentration was 256. mu.g/L, the procedure was as follows:
taking a 96-well plate, respectively adding 5uL of nutrient broth (PDA) into seven wells, adding 5uL of 256 mu g/mL compound to be detected, namely the yard grass bipolaris sacchari I or the yard grass bipolaris sacchari J into the first well, uniformly mixing, sucking 5 mu L of nutrient broth, adding the nutrient broth into the second well, uniformly mixing, sucking 5 mu L of nutrient broth into the second well, adding the nutrient broth into the third well, repeating the steps until the nutrient broth reaches the seventh well, sucking 5 mu L of nutrient broth into the seventh well, and discarding. And respectively adding 5 mu L of bacterial liquid into seven holes, uniformly mixing, placing into an incubator for 20h, taking out and observing until a certain hole is clarified, wherein the drug concentration of the hole is the minimum inhibitory concentration MIC, the test result is shown in Table 2, and Hygromycin B in the Table 2 represents Hygromycin B and serves as a positive control.
TABLE 2 inhibition of plant pathogenic bacteria (MIC, μ g/mL) by yard grass bipolaris I and yard grass bipolaris J
As can be seen from Table 2, the MICs of yard grass bipolaris sacchari I and yard grass bipolaris sacchari J on Alternaria solani (Alternaria solani) are 8 μ g/mL and 16 μ g/mL respectively, and the MIC of yard grass bipolaris sacchari I on Fusarium oxysporum (Fusarium oxysporm) is 64 μ g/mL, which indicates that yard grass bipolaris sacchari I can be used for inhibiting Alternaria solani and/or Fusarium oxysporum, and yard grass bipolaris sacchari J can be used for inhibiting Alternaria solani.
In summary, the raw materials of the yard grass bipolaris sacchari I and the yard grass bipolaris sacchari J provided by the application are natural products, the green and safe effects are achieved, the yard grass bipolaris sacchari I can be used for inhibiting alternaria solani and/or fusarium oxysporum, and the yard grass bipolaris sacchari can be used for inhibiting alternaria solani. In addition, the preparation method of the yard grass bipolaris sacchari I and the yard grass bipolaris sacchari J provided by the application is simple and easy to operate, and the yard grass bipolaris sacchari I and the yard grass bipolaris sacchari J with higher purity and higher yield can be effectively prepared from the yard grass bipolaris sacchari fermentation liquor.
The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Claims (9)
1. The yard grass bipolaris staurosporine I is characterized in that the chemical structural formula of the yard grass bipolaris staurosporine I is shown in the specification
2. The yard grass bipolaris sacchari J is characterized in that the chemical structural formula of the yard grass bipolaris sacchari J is shown as
3. A process for the production of cricket grass bipolaris sacchari I according to claim 1 and cricket grass bipolaris sacchari J according to claim 2, comprising the steps of:
fermenting yard grass bipolaris, extracting a fermentation product by using ethyl acetate, carrying out first normal phase silica gel column separation on an ethyl acetate extract, carrying out second normal phase silica gel column separation on a third elution component after the first normal phase silica gel column separation, carrying out gel column separation on a second sub-component after the second normal phase silica gel column separation, carrying out preparative liquid chromatography purification on a substance obtained after the gel column separation, collecting two chromatographic peak components with the highest content, and obtaining yard grass bipolaris J with the front retention time and yard grass bipolaris I with the rear retention time;
wherein, the conditions of the first normal phase silica gel column separation comprise: taking chloroform and methanol as eluent, and carrying out concentration gradient of the chloroform and the methanol in the eluent at a ratio of 100:0 and 50: 1: gradient elution is carried out at the ratio of 20:1, 10:1, 5:1, 1:1 and 0:1, the volume of eluent used in each gradient is 4.5-5.5L, and seven elution components are respectively obtained;
the conditions of the second normal phase silica gel column separation include: petroleum ether and acetone are used as eluent, the volume ratio of the petroleum ether to the acetone is 10-14:1, isocratic elution is carried out, and every 180-220mL is used as an elution stage to respectively obtain six sub-elution components;
the conditions for gel column separation include: eluting with 600mL of methanol as eluent and Sephadex LH-20 as filler;
the conditions for preparative liquid chromatography purification include: taking acetonitrile and water as eluent, and carrying out gradient elution for 20-30min according to the initial concentration of the acetonitrile and the water of 30:70 and the final concentration of 60: 40.
4. The method as claimed in claim 3, wherein the silica gel used for the first normal phase silica gel column separation is 200-300 mesh and/or the silica gel used for the second normal phase silica gel column separation is 200-300 mesh.
5. The method according to claim 3, wherein the elution flow rate in the preparative liquid chromatography purification is 8 to 12 mL/min.
6. The method of claim 3, wherein the fermentation of yard grass bipolaris comprises: the yard grass bipolaris is cultured for the first time in a potato glucose agar solid culture medium, and then transferred to a liquid fermentation culture medium containing potato glucose for the second time.
7. The method according to claim 6, wherein the first culturing is carried out at 23 to 25 ℃ for 8 to 12 days;
and/or the second culturing is carried out at 27-29 deg.C for 1-3 days, and then at 24-26 deg.C for 19-21 days.
8. The method as claimed in claim 7, wherein the second culturing is performed at a rotation speed of 150-170 r/min.
9. The method according to claim 3, wherein the yard grass bipolaris is isolated from potato.
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