CN113372320B - Locust endophytic fungus secondary metabolite and extraction method and application thereof - Google Patents
Locust endophytic fungus secondary metabolite and extraction method and application thereof Download PDFInfo
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Abstract
Locust endophytic fungus secondary metabolite and extraction method and application thereof. The invention discloses a locust endophytic fungusParaconiothyriumbrasilienseTerpenoids from sources, extraction method thereof and application of the terpenoids in resisting platelet aggregation. The compounds have platelet aggregation inhibiting activities of different degrees, have obvious inhibition effects on platelet aggregation, the inhibition rates are respectively 11.51% and 15.91%, one of the compounds is superior to the inhibition rate of positive aspirin by 14.73%, and the compounds can be used for developing drugs for treating platelet aggregation. Therefore, the compound of the invention has the potential of clinical application of resisting platelet aggregation.
Description
Technical Field
The invention relates to 2 compounds derived from locust endophytic fungus Paraconiothyrium brasiliense, an extraction method thereof and application of the compounds in resisting platelet aggregation, and belongs to the technical field of biological pharmacy.
Background
Thrombosis is the result of blood flowing in a blood vessel or heart becoming solid. Thrombosis can cause blood flow obstruction, cause thrombotic diseases such as myocardial infarction or apoplexy and the like, and cause serious harm to the health of people [ Olas B. biochemistry of blood platelet activation and the following specific role of cells in cardiovascular diseases, adv Clean Chem, 2020,95: 219. 243. ]; the platelet is an anucleate cell, comes from bone marrow megakaryocyte, and has important functions of adhesion, aggregation and release on the formation of thrombus in blood vessels. Processes affecting platelet adhesion, aggregation, etc. are also the mainstream targets for development of antithrombotic drugs [ mask HMM, Helmy MS, Darwish DA, et al, Apyrase with anti-platelet aggregation activity from the nyph of the cam gel notebook hydro-damerii. exp Appl Acarol, 2020, 80(3):349-361. Wuyan, Lemin, Lihongxiang, etc.. research progress of ischemic stroke antiplatelet therapy, Chinese general medicine, 2015, 18(23):2865-2869. Qinhao, Yangling, Liqing, etc.. research progress of antithrombotic drugs, pharmaceutical progress, 2014, 38(3): 174. 184 ]. Aspirin, clopidogrel and other chemical drugs have the effect of resisting platelet aggregation, are platelet aggregation inhibitors commonly used clinically at present, and are widely applied to the treatment of thrombotic diseases [ Zhu jin Kun, Mao Hua, Yi Yang Guang, etc.. the influence of aspirin and clopidogrel on the activity of in vitro platelet-adhesive endothelial cell matrix and the mechanism research thereof, Chinese general medicine 2015, 18(3):283-287 ]. However, these drugs often have the disadvantages of single action mechanism and large adverse reaction, and are difficult to be accepted by patients who need to take the drugs for a long time. Therefore, the screening of safe and effective new anti-platelet aggregation drugs has very important significance. The terpenoid has certain effect on resisting platelet aggregation [ Pengzhixiang, plum snow, Wangziman, and the like, the research on the effect of natural products on resisting platelet aggregation progresses, Shenyang pharmaceutical university reports, 2019,36(12):1133 and 1144 ], the natural effective terpenoid from the existing plant sources has limited sources, but the diversity and novelty of endophytic fungi metabolites are fully expressed through metabolic regulation, and a rich lead compound library is provided for screening the medicines with good platelet aggregation resistance.
Disclosure of Invention
The invention aims to provide 2 novel compounds in a locust endophytic fungus, and a preparation method and application thereof. 2 compounds have certain anti-platelet aggregation activity. The preparation method disclosed by the invention is simple to operate, scientific and reasonable, can obtain the compounds with stable physicochemical properties and higher purity, and has certain development value.
The structure of the compound is not reported in documents at present, activity determination tests show that 2 compounds show certain anti-platelet aggregation activity, the inhibition rate of one compound is 15.91 +/-0.12% and is better than that of positive aspirin (the inhibition rate is 14.73 +/-0.52%) under the same concentration, and the other compound also shows a certain anti-aggregation effect.
The structure of the terpenoid compounds derived from the locust endophytic fungus Paraconiothyrium brasiliensis is shown as follows:
the application of the terpenoid derived from the locust endophytic fungus Paraconiothyrium brasiliensis in preparing the anti-platelet aggregation medicines.
The locust endophytic fungus Paraconiothyrium brasiliense is classified and named as Paraconiothyrium brasiliense MZ-1, and the Paraconiothyrium brasiliense strain is deposited in China center for type culture Collection at 5/25 2016, with the unit address: wuhan university in Wuhan, China, the preservation name is: the small round spore Brazilian MZ-1, the preservation number: CCTCC NO: m2016279.
The locust endophytic fungus Paraconiothyrium brasiliensis is used for preparing the terpenoid through fermentation.
A method for producing terpenoid through fermentation metabolism comprises the step of inoculating the locust endophytic fungus Paraconiothyrium brasiliensis into a suitable fermentation culture medium for fermentation culture, wherein the fermentation culture medium is a PDA (potato dextrose agar) solid culture medium to obtain seed blocks, the seed blocks are inoculated into a PDB culture medium for culture to obtain seed liquid, the seed liquid is inoculated into the PDB culture medium for culture, L-tryptophan is added in the culture process, and fermentation is continued to obtain a fermentation product.
The L-tryptophan is added in three batches when cultured for 4-5 days, the final concentration is 0.8-1mmol/L, and the culture lasts for 25-30 days.
The method also comprises the following steps of separating and extracting the fermentation product:
filtering the fermentation product to obtain mycelium and fermentation liquor, wherein the fermentation liquor is extracted and concentrated; mycelium is dried, extracted and concentrated, extraction products are combined, and the compound is obtained through chromatographic preparation and separation. The separation and extraction steps are as follows:
(1) drying the mycelium, extracting by using dichloromethane and leaching by using dichloromethane to obtain a coarse substance 1, and extracting the fermentation liquor by using dichloromethane and leaching by using dichloromethane to obtain a coarse substance 2; mixing the crude product 1 and the crude product 2, separating by normal phase silica gel chromatography, eluting with dichloromethane and dichloromethane/methanol mixed solution, and collecting eluate; and in the process of eluting the dichloromethane/methanol mixed solution, gradient concentration is adopted for eluting, and the volume ratio of dichloromethane/methanol in the gradient concentration is 9:1, 8:1, 7:1, 6:1, 4:1 and 3: 1.
(2) Concentrating the eluate, dissolving with methanol solvent, performing Sephadex LH-20 gel chromatography, eluting with dichloromethane/methanol mixed solvent (dichloromethane/methanol volume ratio is 8: 1), and collecting as a fragment every 10 min, wherein the flow rate is 1 ml per minute; dissolving the 20 th fragment with chromatographic methanol, filtering, separating with preparative reverse phase high performance liquid chromatograph (C18 reverse phase column), collecting peak at retention time of 16 min to obtain compound 1; dissolving the 35 th fragment by using chromatographic methanol, filtering, separating by using a preparative reverse phase high performance liquid chromatograph, wherein a chromatographic column adopts a C18 reverse phase column, a mobile phase is a mixed solvent with the acetonitrile/water volume ratio of 65:35, and collecting a peak at the retention time of 12 minutes to obtain the compound 2.
Compared with the prior art, the invention has the following advantages and effects:
the invention provides 2 brand-new compounds from endophytic fungi, wherein part of the compounds in the compounds have certain anti-platelet aggregation activity, and the activity of the compound 2 is superior to that of positive aspirin, so that the compound has application potential in developing drugs for treating cardiovascular diseases.
Drawings
FIG. 1 shows Compound 1 obtained in example 11H-NMR spectrum.
FIG. 2 shows Compound 1 obtained in example 113C-NMR。
FIG. 3 is a DEPT135 plot of Compound 1 obtained in example 1.
FIG. 4 shows Compound 1 obtained in example 11H-1H COSY picture.
FIG. 5 is a HSQC plot of Compound 1 obtained in example 1.
FIG. 6 shows HMBC patterns of Compound 1 obtained in example 1.
FIG. 7 is a NOESY diagram of Compound 1 obtained in example 1.
FIG. 8 is a HR-ESI diagram of Compound 1 obtained in example 1.
FIG. 9 shows Compound 2 obtained in example 11H-NMR spectrum.
FIG. 10 shows Compound 2 obtained in example 113C-NMR。
FIG. 11 is a DEPT135 plot of Compound 2 obtained in example 1.
FIG. 12 shows Compound 2 obtained in example 11H-1H COSY picture.
FIG. 13 is a HSQC plot of Compound 2 obtained in example 1.
FIG. 14 shows the HMBC pattern of Compound 2 obtained in example 1.
FIG. 15 is a NOESY chart of Compound 2 obtained in example 1.
FIG. 16 is a HR-ESI graph of Compound 2 obtained in example 1.
Detailed Description
The present invention is further illustrated by the following examples, which are not intended to limit the invention in any way, except as otherwise indicated, and that the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Unless otherwise indicated, reagents and materials used in the present invention are commercially available.
Example 1
A preparation method of 2 compounds derived from locust endophytic fungi is provided, wherein the 2 compounds are separated from fermentation liquor of a P.brasiliensis strain of small round brazilian spore separated from intestinal tracts of the locust with the angle of Chinese sword in Shennongjia region.
The locust endophytic fungus Paraconiothyrium brasiliensis, classified and named as Paraconiothyrium brasiliensis MZ-1, the strain Paraconiothyrium brasiliensis is deposited in the China center for type culture Collection at 2016, 5, 25 days, and the unit address is: wuhan university in Wuhan, China, the preservation name is: the small round spore Brazilian MZ-1, the preservation number: CCTCC M2016279.
The specific preparation method of the 2 compounds is as follows:
inoculating the frozen endophytic fungus Paraconiothyrium brasiliensis into a PDA solid culture medium for resuscitation, obtaining seed fungus blocks after culturing for 3 days, and respectively inoculating the seed fungus blocks with the size of 0.5cm multiplied by 0.5cm into 3 bottles (500ml conical bottles, the culture medium loading amount in each bottle is 150ml) of PDB fermentation culture medium for culturing for 3 days to obtain seed liquid; 2ml of the seed solution was pipetted into 500ml conical flasks (150 ml of PDB medium per flask) to inoculate 200 flasks altogether. After shaking culture for 4 days at 28 ℃ and 180rpm, adding L-tryptophan liquid (DMSO as solvent) into each bottle for 3 times on average, wherein the final concentration is 1mmol/L, and continuing culture for 30 days to obtain a fermentation product;
filtering a fermentation product to obtain mycelium and fermentation liquor, extracting and concentrating the fermentation liquor by using dichloromethane to obtain 15g of extract, drying the mycelium, performing ultrasonic extraction and concentration by using dichloromethane to obtain 20g of extract, combining the two parts of extract to obtain 35g of crude extract, separating the 35g of crude extract by using normal phase silica gel chromatography (the model of the normal phase silica gel is 200-mesh and 300-mesh in reagent level), wherein the using amount of the normal phase silica gel is 700g, the model of a glass chromatography column is 10cm multiplied by 70cm, eluting by using dichloromethane, dichloromethane/methanol mixed liquor, and the concentration gradient proportion of an eluent is (dichloromethane: methanol/volume ratio): 9:1, 8:1, 7:1, 6:1, 4:1, 3:1, 2:1, 1:1, wherein the elution volume of each concentration gradient is 2 column volumes, and the eluates of all concentration gradients are collected; concentrating 1.2g of elution fragments of dichloromethane and methanol in a ratio of 3:1, dissolving the elution fragments into 2ml of sample solution by using a methanol solvent, selecting Sephadex LH-20 gel chromatography, eluting by using a mixed solvent of dichloromethane and methanol in a ratio of 8:1 (volume ratio), collecting the elution fragments into one fragment every 10 minutes at a flow rate of 1 ml per minute, and collecting 40 fragments in total; dissolving 50mg of the 20 th fragment in chromatographic methanol, filtering, and separating by using a preparative reverse phase high performance liquid chromatograph, wherein a C18 reverse phase column is adopted as a chromatographic column, a mobile phase is a mixed solvent of acetonitrile and water in a volume ratio of 65:35, and a peak is collected at a retention time of 16 minutes to obtain a compound 1(20 mg); after 80mg of the 35 th fragment was dissolved in methanol for chromatography and filtered, the resulting solution was separated by preparative reverse phase high performance liquid chromatography using a C18 reverse phase column as a column chromatography and a mixed solvent of acetonitrile and water at a volume ratio of 45:55, and a peak was collected at a retention time of 12 minutes to obtain compound 2(24 mg).
Example 2
Compound 1: a pale yellow powder. By high resolution mass spectrum HR-ESI-MS M/z 223.0963[ M + H ]]+(C12H15O4Calcd. 223.0970) to determine its molecular formula as C12H14O4The unsaturation degree was 6. The planar structure of Compound 1 is mainly by HMBC and1H-1h COSY relationship determination: in the HMBC spectrum, remote correlation exists between H-9 and C-7, between H-5 and C-7, between C-6 and C-4a, between H-6 and C-8, between H-4 and C-4a, between C-8a, between H-11 and C-3, between C-4 and between H-10 and C-8; in the 1H-1H COSY spectrum, there is a correlation between H-4 and H-3, H-10 (HMBC spectrum of compound 1. sup. and1H-1the H COSY spectrum is shown in figure 3). General analysis of Compound 11H-1H COSY and HMBC related signals, and the structural formula of the compound 1 is determined as follows:
TABLE 1 NMR hydrogen and carbon spectra data for Compound 1 (solvent DMSO-d)6)
Compound 2: a pale yellow oily liquid. By high resolution mass spectrum HR-ESI-MS M/z 217.1592[ M + H ]]+(C15H23O3Calcd.217.1597), the molecular formula of which is determined to be C15H22O3The unsaturation degree is 5. The planar structure of Compound 2 is mainly determined by HMBC and1H-1h COSY relationship determination: in the HMBC spectrum, remote correlation exists between H-14 and C-1, C-10 and C-9, between H-3 and C-2 and C-1, between H-15 and C-3, C-4 and C-5, between H-6 and C-5 and C-8, between H-7 and C-9, between H-13 and C-7, and between H-12 and C-11 and C-13; in the 1H-1H COSY spectrum, there are correlations between H-2 and H-3, between H-9 and H-8, between H-7 and between H-6 (HMBC spectrum of compound 2 and1H-1see fig. 12 for H COSY spectrum) to determine its planar structure.
While the NOESY pattern found that H-14a and H-9b, and H-9a and H-7 are on one side of the ring, confirmed that the C-7 and C-10 configurations of Compound 2 are both S. Analysis of Compound 21H-1H COSY, HSQC and HMBC related signals, the structure of compound 2 was determined as follows:
TABLE 2 NMR hydrogen and carbon spectra data for Compound 2 (solvent DMSO-d)6)
Example 3: determination of anti-platelet aggregation Activity of Compounds 1, 2
Direct antiplatelet aggregation inhibitor color development assay: aspirin is used as a positive drug, endophytic fungi Paraconiothyrium brasiliensis crude body, the positive drug and the compounds 1 and 2 are dissolved in DMSO physiological saline solution with the mass concentration of 5%, the sample concentration of 1mmol/L is selected for determination, an experimental group and a blank group are set in an experiment, fresh plasma and sodium citrate (9:1, V/V) are mixed, the mixture is centrifuged for 10 minutes at 1500g to obtain supernatant, the supernatant is preheated at 37 ℃ in a constant temperature box, 50 microliters of the compounds and 450 microliters of the plasma supernatant are uniformly mixed, 20 microliters of ADP (10umol/L) is added after the mixture reacts for 5 minutes at 37 ℃, and the OD value is determined at 405 wavelengths. Three replicates were run and the average was taken as the final result. The formula for calculating the inhibition rate is as follows:
inhibition was compared for each compound using GraphPad Prism 8.0 software.
The results of the inhibition rates of the compounds 1 and 2 are shown in table 3, and the anti-platelet aggregation inhibition rate of the positive aspirin is 14.73%. The compounds 1 and 2 have different degrees of inhibition, wherein the aggregation inhibition rate of the compound 2 is better than that of positive aspirin.
Table 3.2 anti-platelet aggregation inhibition (%) of compounds (i.e., (ii))
n＝3)
At least 40 compounds were collected in the procedure of example 1, but only two compounds described in this case had a significant effect on anti-platelet aggregation. As in example 1, 60mg of the 27 th fragment was dissolved in methanol and filtered, and then separated by preparative reverse phase high performance liquid chromatography using a C18 reverse phase column as a chromatographic column, a mixed solvent of acetonitrile and water 38:62 (by volume) as a mobile phase, and a peak was collected at a retention time of 13 minutes to obtain compound 3 (18.0mg), compound 3 having a structural formula of compound 3
After the detection is carried out by adopting the method described in the embodiment 3, the platelet aggregation inhibition rate is-8.83 +/-0.36**And have no statistically significant effect, and are not considered to be pharmaceutically acceptable as a viable effect.
Claims (9)
1. Terpenoid derived from locust endophytic fungus Paraconiothyrium brasiliensis, characterized in that the structure of the compound is shown as follows:
2. use of terpenoids derived from the locust endophytic fungus Paraconiothyrium brasiliensis according to claim 1 for the preparation of a medicament for the anti-platelet aggregation.
3. The application of terpenoid derived from locust endophytic fungus Paraconiothyrium brasiliensis in preparing anti-platelet aggregation medicines is characterized in that the terpenoid has the following structural formula:
4. the use according to claim 2 or 3, wherein the endophytic fungus Paraconiothyrium brasiliense, classified as Paraconiothyrium brasiliense MZ-1, is deposited at the China center for type culture Collection at 25/5/2016, with the unit addresses: wuhan university in Wuhan, China, the preservation name is: the small round spore Brazilian MZ-1, the preservation number: CCTCC NO: m2016279.
5. Use of the locust endophytic fungus Paraconiothyrium brasiliensis according to claim 4 for the fermentative preparation of a terpenoid according to claim 1.
6. A method for producing terpenoid through fermentation metabolism is characterized by comprising the steps of inoculating the locust endophytic fungus Paraconiothyrium brasiliensis of claim 4 into a suitable fermentation culture medium for fermentation culture, wherein the fermentation culture medium is a PDA (potato dextrose agar) solid culture medium to obtain seed blocks, the seed blocks are inoculated into a PDB culture medium for culture to obtain seed liquid, the seed liquid is inoculated into the PDB culture medium for culture, L-tryptophan is added in the culture process, and fermentation is continued to obtain a fermentation product;
and (3) separating and extracting fermentation products: filtering the fermentation product to obtain mycelium and fermentation liquor, wherein the fermentation liquor is extracted and concentrated; mycelium is dried, extracted and concentrated, extraction products are combined, and the compound is obtained through chromatographic preparation and separation, wherein the separation and extraction steps are as follows:
(1) drying the mycelium, extracting by using dichloromethane and leaching by using dichloromethane to obtain a coarse substance 1, and extracting the fermentation liquor by using dichloromethane and leaching by using dichloromethane to obtain a coarse substance 2; mixing the crude product 1 and the crude product 2, separating by normal phase silica gel chromatography, eluting with dichloromethane and dichloromethane/methanol mixed solution, and collecting eluate;
(2) concentrating the eluate, dissolving with methanol solvent, performing Sephadex LH-20 gel chromatography, eluting with dichloromethane/methanol mixed solvent at flow rate of 1 ml per minute, and collecting as a fragment every 10 min; dissolving the 20 th fragment with chromatographic methanol, filtering, separating with preparative reverse phase high performance liquid chromatograph (C18 reverse phase column), collecting peak at retention time of 16 min to obtain compound 1; dissolving the 35 th fragment by using chromatographic methanol, filtering, separating by using a preparative reverse phase high performance liquid chromatograph, wherein a chromatographic column adopts a C18 reverse phase column, a mobile phase is a mixed solvent with the acetonitrile/water volume ratio of 65:35, and a peak is collected at the position of 12 minutes of retention time to obtain a compound 2;
the structural formula of the compound 1 is
The structural formula of the compound 2 is
7. The method of claim 6, wherein the L-tryptophan is added in three portions at a final concentration of 0.8 to 1mmol/L for 25 to 30 days after culturing for 4 to 5 days.
8. The method according to claim 6, wherein in the step (1), the dichloromethane/methanol mixed solution is eluted by gradient concentration, and the volume ratio of dichloromethane/methanol in the gradient concentration is 9:1, 8:1, 7:1, 6:1, 4:1 and 3: 1.
9. The method according to claim 6, wherein in the step (2), the volume ratio of dichloromethane/methanol is 8: 1.
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| US4845242A (en) * | 1987-04-28 | 1989-07-04 | Georgia Tech Research Corporation | Isocoumarins with basic substituents as serine proteases inhibitors, anticoagulants and anti-inflammatory agents |
| CN106010980A (en) * | 2016-06-08 | 2016-10-12 | 三峡大学 | Endophytic fungus paraconiothyrium brasiliense strain and application thereof |
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| US4845242A (en) * | 1987-04-28 | 1989-07-04 | Georgia Tech Research Corporation | Isocoumarins with basic substituents as serine proteases inhibitors, anticoagulants and anti-inflammatory agents |
| CN106010980A (en) * | 2016-06-08 | 2016-10-12 | 三峡大学 | Endophytic fungus paraconiothyrium brasiliense strain and application thereof |
Non-Patent Citations (3)
| Title |
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| Cheng-Xiong Liu et al..Paraconfuranones A–H, eight new furanone analogs from the insect-associated fungus Paraconiothyrium brasiliense MZ-1.《Magn. Reson. Chem.》.2015,第53卷第317-322页. * |
| Fu-MEI KIANG et al..NAPHTHALENE AND ISOCOUMARIN DERIVATIVES FROM THE LIVERWORT WETTSTEINIA INVERSA.《Phytochemitry》.1994,第37卷(第5期),第1459-1461页. * |
| Shradha M. Patel et al..Potential of neem (Azadirachta indica L.) for prevention and treatment of oncologic diseases.《Seminars in Cancer Biology》.2016,第40-41卷第100-115页. * |
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