A method for converting ferric holly acid into its derivatives using microorganisms and its application in preparing lipid-lowering drugs

Abstract

The present invention discloses a method for converting ferric holly acid into its derivatives using microorganisms and its application in the preparation of lipid-lowering drugs. The microorganism used in the method for converting ferric holly acid into its derivatives using microorganisms of the present invention is the endophytic fungus Lasiodiplodia sp. JBY-33 strain, which can convert ferric holly acid into its derivative M, and the obtained derivative M has a large safety factor, and shows better lipid-lowering activity than the original drug ferric holly acid in the lipid-lowering activity screening, therefore, it can be used to prepare lipid-lowering drugs. In addition, the reaction conditions of the method for converting ferric holly acid into its derivatives using microorganisms of the present invention are mild, the conversion efficiency is high, the price of the culture medium selected in the conversion process is low, the source of raw materials is sufficient, the conversion reaction has high specificity, the separation and purification steps are simple, the product purity is high, and it is easy to quantify production.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and more specifically, relates to a method for converting ferric holly acid into its derivatives by using microorganisms and the application of the method in preparing lipid-lowering drugs. Background Art

[0002] Rotundic acid is an ursane-type triterpenoid saponin isolated from Ilex rotunda Thunb., which has good anti-tumor, hypolipidemic, anti-thrombotic and anti-inflammatory activities, but its poor solubility and bioavailability limit its clinical application to a certain extent. Therefore, it is necessary to modify its structure appropriately in order to improve its solubility and bioavailability and expand its clinical application.

[0003] At present, there are two methods for structural modification of ferric holmic acid, namely chemical method and microbial transformation modification. Although the solubility, bioavailability and antitumor activity of ferric holmic acid derivatives prepared by chemical method are significantly improved compared with ferric holmic acid (He Yufang. Preparation of ferric holmic acid and design, synthesis and antitumor activity of its derivatives [D]. Jilin University, 2013), the chemical method for preparing ferric holmic acid derivatives has a complex reaction process, high requirements for conditions, high toxicity of reagents, more by-products and low yield, which has great limitations in practical applications.

[0004] Microbial transformation refers to the process of selectively modifying the structure of exogenous substrates using a series of enzymes produced during microbial metabolism. It has good stereoselectivity and regioselectivity, can modify the structure of inactive carbon atoms, or can greatly improve the solubility, bioavailability and activity of substrates. At the same time, the reaction conditions of microbial transformation are mild, the cost is low, and the separation and purification of the products are relatively simple, all of which are advantages that chemical methods do not have. At present, there have been studies on the activity of metabolites obtained from fungal transformation culture, which can not only be used to reveal the material basis for drugs to enter the body and exert their effects, but also help to discover derivatives with better pharmacological activity and bioavailability. Therefore, exploring and developing microorganisms that can structurally modify ferric holmic acid to improve its activity is of great significance to improving the pharmacological activity of ferric holmic acid and expanding the scope of clinical application of ferric holmic acid. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the above-mentioned existing technologies and provide a method for converting ferric holly acid into its derivatives using microorganisms and its application in the preparation of lipid-lowering drugs.

[0006] The first object of the present invention is to provide an endophytic fungus Lasiodiplodia sp. JBY-33.

[0007] The second object of the present invention is to provide a ferric holly acid derivative M.

[0008] The third object of the present invention is to provide a method for preparing the ferric holly acid derivative M.

[0009] The fourth object of the present invention is to provide the use of the endophytic fungus Lasiodiplodia sp. JBY-33 in the preparation of ferric holly acid derivative M.

[0010] The fifth object of the present invention is to provide the use of the ferric holly acid derivative M in the preparation of lipid-lowering drugs.

[0011] The sixth object of the present invention is to provide a lipid-lowering drug.

[0012] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0013] The present invention uses a tissue block separation method to separate an endophytic fungus from the fresh bark of the traditional Chinese medicine Jiubiying. After amplifying its ITS region and sequencing and comparison, it is found that the obtained endophytic fungus is Lasiodiplodia sp. fungus, and it is named Lasiodiplodia sp. JBY-33 strain. The present invention finds that the Lasiodiplodia sp. JBY-33 strain can convert ferric holly acid into a ferric holly acid derivative M (19a, 22a, 24-trihydroxy-urs-3-oxo-12-ene-28-oic acid) by adding a ferric holly acid solution to the culture medium. Its structural formula is shown in formula (I), which is a new compound with a large amount and high purity, and in the lipid-lowering activity screening, it is shown that it has better lipid-lowering activity than the original drug ferric holly acid. Therefore, the present invention applies to protect the preparation method and application of the Lasiodiplodia sp. JBY-33 strain and its ferric holly acid derivative.

[0014] The present invention requests protection for the endophytic fungus Lasiodiplodia sp. JBY-33, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on September 2, 2022, with the deposit number GDMCC NO: 62757.

[0015] The present invention also claims protection for a ferric holly acid derivative M (also referred to as "derivative M"), the structural formula of the derivative M is shown in formula (I):

[0016]

[0017] The present invention also provides a method for converting ferric holly acid into its derivatives using microorganisms, namely, a method for preparing the ferric holly acid derivative M described in formula (I), wherein the derivative M is obtained by culturing and isolating the endophytic fungus Lasiodiplodia sp. JBY-33 in a culture medium containing ferric holly acid.

[0018] Specifically, the method for preparing the ferric holly acid derivative M comprises the following steps:

[0019] S1. Activation of fungus Lasiodiplodia sp. JBY-33;

[0020] S2. Preparation of Lasiodiplodia sp. JBY-33 seed culture solution;

[0021] S3. The seed culture solution obtained in step S2 is inoculated into a liquid culture medium, shaken and cultured at 25 to 35°C for 12 to 48 hours, and then the ferric holly acid solution is added, and the transformation culture is carried out under the same conditions for 24 to 168 hours and then filtered;

[0022] S4. Extract the filter residue obtained by filtration in step S3 with 1 to 3 times the mass of methanol, combine the filtrate and the methanol extract, and recover under reduced pressure to obtain a conversion extract;

[0023] S5. The conversion extract obtained in step S4 is extracted with 1 to 3 times the volume of ethyl acetate, and the extract is separated by reverse phase column chromatography, and gradient eluted with 4 to 7 times the column volume of 40%, 50%, 60%, and 70% methanol / water, respectively, and the 70% methanol / water elution portion is collected and recovered, and the ferric holly acid derivative M is obtained by recrystallization with methanol.

[0024] Specifically, after recrystallization, HR-ESI-TOF-MS and NMR methods were used to identify the structure of the conversion product.

[0025] Specifically, the activation in step S1 is to inoculate the cryopreserved Lasiodiplodia sp. JBY-33 on a slant culture medium and culture it statically at 25 to 35° C. for 24 to 96 hours.

[0026] More specifically, cryopreserved Lasiodiplodia sp. JBY-33 was inoculated on a slant culture medium and statically cultured at 28° C. for 48 h.

[0027] Specifically, the seed culture solution in step S2 is obtained by inoculating activated spores of Lasiodiplodia sp. JBY-33 into a liquid culture medium and culturing the culture medium under constant temperature shaking at 25 to 35° C. and 100 to 200 rpm / min for 24 to 48 hours.

[0028] More specifically, the seed culture solution is obtained by inoculating activated spores of Lasiodiplodia sp. JBY-33 into a liquid culture medium and culturing with constant temperature shaking at 28° C. and 160 rpm / min for 24 hours.

[0029] Specifically, in step S3, the inoculation amount of the seed solution is 1-15%, and the final concentration of the added ferric holly acid solution is 0.05-0.50 g / L.

[0030] More specifically, the inoculation amount of the seed solution in step S3 is 5%, and after constant temperature shaking culture at 28°C and 160rpm / min for 24h, a ferric holly acid solution with a final concentration of 0.2g / L is added, and the transformation culture is carried out under the same conditions for 144h and then filtered.

[0031] Specifically, the solvent used to prepare the ferric holly acid solution is any one or more of anhydrous ethanol, dimethyl sulfoxide, and acetone, and the final content of the organic solvent in the culture solution is less than 3%.

[0032] Specifically, the extraction in step S4 is ultrasonic extraction, and the filter residue is ultrasonically extracted with methanol for 3 times, each time for 1 hour, and the amount of methanol used is twice that of the filter residue by mass.

[0033] Specifically, the methanol / water in step S5 refers to a mixture of methanol / water in a volume ratio.

[0034] Specifically, the liquid culture medium used for culturing Lasiodiplodia sp. JBY-33 is PDB culture medium.

[0035] The formula (per liter) of the PDB liquid culture medium is: by mass ratio, 300.0 g of potato (from which the extract powder is extracted) and 20.0 g of glucose.

[0036] In view of the fact that the endophytic fungus Lasiodiplodia sp. JBY-33 described in the present invention can convert ferric holly acid into ferric holly acid derivative M, the present invention also requests protection for the use of the endophytic fungus Lasiodiplodia sp. JBY-33 in the preparation of the ferric holly acid derivative M shown in formula (I).

[0037] The present invention finds that the ferric oleic acid derivative M can effectively reduce the TG content in the FFA-induced HepG2 cell hyperlipidemia model compared with the original drug ferric oleic acid, has good lipid-lowering activity, and can be used to prepare lipid-lowering drugs. Therefore, the present invention also requests protection for the use of the ferric oleic acid derivative M in the preparation of lipid-lowering drugs.

[0038] Specifically, the application is the application of the ferric holly acid derivative M in the preparation of a drug for reducing the TG content in the FFA-induced HepG2 cell hyperlipidemia model.

[0039] The present invention also provides a lipid-lowering drug, which contains the ferric holly acid derivative M shown in formula (I).

[0040] The present invention has the following beneficial effects:

[0041] The present invention provides an endophytic fungus Lasiodiplodia sp. JBY-33, which can convert ferric holly acid into a ferric holly acid derivative M, and its structural formula is shown in formula (I). The ferric holly acid derivative M of the present invention has a large safety factor, and is shown to have better lipid-lowering activity than the original drug ferric holly acid in lipid-lowering activity screening, and can be used to prepare lipid-lowering drugs. The present invention provides a new effective ingredient for the preparation of lipid-lowering drugs, namely, the ferric holly acid derivative M, which has good application prospects in the preparation of lipid-lowering drugs for FFA-induced HepG2 cell hyperlipidemia models.

[0042] Based on the endophytic fungus Lasiodiplodia sp. JBY-33 described in the present invention, the present invention also provides a method for converting ferric holly acid into its derivative M using microorganisms. The reaction conditions of the method are mild, the conversion efficiency of ferric holly acid is high, the culture medium selected in the conversion process is low-priced, the source of raw materials is sufficient, the conversion reaction has high specificity, and the conversion product is separated and purified by reverse phase chromatography and recrystallization. It is found that the purity of the ferric holly acid derivative M in the conversion product is as high as 98.0%, the separation and purification steps are simple, the product purity is high, and it is easy to quantitatively produce. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Schematic diagram of the structure of the conversion of ferric holly acid into ferric holly acid derivative M.

[0044] Figure 2 This is the liquid phase purity chart and retention time of ferroholic acid derivative M.

[0045] Figure 3 This is the high-resolution mass spectrum of the ferric holly acid derivative M.

[0046] Figure 4 This is the effect of ferroic acid derivative M on cell viability.

[0047] Figure 5These are the results of a study on the lipid-lowering activity of ferric oleate and ferric oleate derivative M; NC in the figure is the control group; HC is the model group; ST is the simvastatin-positive drug group; L is the low-dose administration group; M is the medium-dose administration group; H is the high-dose administration group; RA is ferric oleate; M is the ferric oleate derivative M; compared with the normal group, ** in the model group represents P<0.01, which is a significant difference; compared with the model group, # in the administration group represents P<0.05, and ## represents P<0.01, which is a significant difference. DETAILED DESCRIPTION

[0048] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0049] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0050] Example 1 Isolation of endophytic fungus Lasiodiplodia sp. JBY-33

[0051] The invention adopts a tissue block separation method to separate an endophytic fungus from the fresh bark of the traditional Chinese medicine Jiubiying (Ilex oleifera), and the growth temperature range of the endophytic fungus is 25-35 DEG C.

[0052] Bacterial species identification:

[0053] A DNA extraction kit (TSINGKE, catalog number TSP101) was used to extract and separate the genomic DNA of the endophytic fungus according to the steps in the instruction manual. Using DNA as a template, the ITS region was amplified with primers ITS1 and ITS4 (ITS1: 5′-TCCGTAGGTGAACCTGCGG-3′; ITS4: 5′-TCCTCCGCTTATTGATATGC-3′). The ITS-rRNA gene sequence of the fungus was obtained by sequencing, and the fungus was identified by sequence alignment.

[0054] The ITS-rRNA gene sequence of the endophytic fungus obtained by sequencing in the present invention is as follows:

[0055]

[0056]

[0057] The similarity search was performed through the BLAST database. After comparison, the sequence had a homology of 100% with strains such as Lasiodiplodia pseudotheobromae isolate DSS51 and Lasiodiplodia sp. Strain Bpf-20. The isolated endophytic fungus was preliminarily determined to be a Lasiodiplodia sp. fungus, and was named the endophytic fungus Lasiodiplodia sp. JBY-33 strain. The Lasiodiplodia sp. JBY-33 strain was deposited in the Guangdong Provincial Microbiological Culture Collection on September 2, 2022, with a deposit number of GDMCC NO: 62757, and the deposit address is 5th Floor, Building 59, No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province.

[0058] Example 2 Isolation and Identification of Lasiodiplodia sp. JBY-33 Transformation Products

[0059] In this example, the endophytic fungus Lasiodiplodia sp. JBY-33 isolated in Example 1 was used to transform and culture ferric holly acid, and the transformation product obtained by the transformation and culture was isolated and identified.

[0060] 1. Transformation and cultivation

[0061] S1. Activated bacteria: The endophytic fungus JBY-33 strain isolated in Example 1 was inoculated on a slant culture medium and cultured at 28° C. for 24 h;

[0062] S2. Preparation of seed culture medium: The spores on the slant medium in step S1 were inoculated into 30 mL of PDB liquid medium, and cultured at 28 ° C and 160 rpm / min for 24 h to obtain a seed culture medium;

[0063] S3. Transformation culture: The seed culture solution obtained in step S2 was inoculated into 600 mL of PDB liquid culture medium at an inoculum size of 5%, and a total of 25 bottles were inoculated. After pre-culture at 28°C and 160 rpm / min under constant temperature shaking for 24 h, a ferric holly acid solution was added to a final concentration of 0.20 g / L, and a total of 3.0 g of the original drug ferric holly acid was added. Then, the transformation culture was continued under the same conditions for 144 h and then filtered;

[0064] S4. Residue extraction: The residue obtained by filtration in step S3 was ultrasonically extracted with 2 times the amount (by mass) of methanol for 3 times, each time for 0.5 h, and the filtrate and the methanol extract were combined and recovered under reduced pressure to obtain a conversion extract;

[0065] S5. Isolation, purification and structural identification of the conversion product: The conversion extract in step S4 was extracted with 2 volumes of ethyl acetate, and the extract was separated by reverse phase column chromatography, and gradient eluted with 5 column volumes of 40%, 50%, 60%, and 70% methanol / water in sequence, and the 70% methanol / water elution was collected and recovered. The main conversion product was obtained by recrystallization with methanol, which weighed 1.4 g after drying, and the calculated yield was 46.7%.

[0066] Specifically, the formula (per liter) of the slant culture medium in step S1 is: 300.0 g of potato (from which the extract powder is extracted), 20.0 g of glucose, 15.0 g of agar, and 0.1 g of chloramphenicol.

[0067] Specifically, the formula (per liter) of the PDB liquid culture medium in step S2 is: 300.0 g of potato (from which the extract powder is extracted) and 20.0 g of glucose.

[0068] Specifically, the solvent for dissolving ferric holly acid in step S3 is anhydrous ethanol.

[0069] 2. Structural identification of transformation products

[0070] The transformation product obtained by the endophytic fungus Lasiodiplodia sp. JBY-33 strain on ferric holmic acid is colorless needle-shaped crystals (methanol recrystallization). The structure of the obtained transformation product is identified by HR-ESI-TOF-MS and NMR methods, and the main transformation product (with a purity of up to 98%) is named M, i.e. the ferric holmic acid derivative M of the present invention; thin layer detection of 10% sulfuric acid ethanol shows grass green; HR-ESI-TOF-MS m / z 501.3226[MH] - (calcd for C 30 H 45 O 6 , 501.3211).

[0071] The structural formula of the ferric holly acid derivative M obtained in the present invention is shown in formula (I):

[0072]

[0073] The structural diagram of the conversion of ferroic acid (RA) into ferroic acid derivative M is shown in Figure 1 As shown by Figure 1 It can be seen that ferric oxalic acid underwent carbonylation at position 3 and hydroxylation at position 22 under the action of the endophytic fungus Lasiodiplodia sp. JBY-33, and was transformed into a new compound, namely, ferric oxalic acid derivative M.

[0074] The purity chart and retention time of the M liquid phase test of ferroic acid derivatives are as follows Figure 2As shown by Figure 2 It can be seen that except for the fluctuation caused by the solvent effect at about 2.5 minutes, there are no other impurities. The separated derivative M has high purity and can be used for nuclear magnetic resonance spectroscopy testing and pharmacological activity research.

[0075] The high-resolution mass spectrometry data of the ferric holmic acid derivative M are shown in Table 1 and Figure 3 As shown in Table 1 and Figure 3 It can be seen that the quasi-molecular ion peak of derivative M is m / z 501.3226 [MH] - , the calculated values ​​are shown in Table 1, which is C 30 H 45 O 6 501.3211, thus the molecular formula of derivative M is determined to be C 30 H 46 O 6 .

[0076] The high-resolution mass spectrometry data of the ferric holmic acid derivative M are shown in Table 1:

[0077] Table 1

[0078] Formula Calculated m / z(Da) Error(mmu) Error(ppm) RDB <![CDATA[C 30 H 45 O 6 ]]> 501.3211 1.53 3.1 9.0

[0079] The one-dimensional NMR data are shown in Table 2.

[0080] Table 2 NMR data of derivative M (C 5 D 5 N, 400MHz / 100MHz, ppm)

[0081]

[0082] Example 3 Isolation and Identification of Lasiodiplodia sp. JBY-33 Transformation Products

[0083] In this example, the endophytic fungus Lasiodiplodia sp. JBY-33 isolated in Example 1 was used to transform and culture ferric holly acid, and the transformation product obtained by the transformation and culture was isolated and identified.

[0084] 1. Transformation and cultivation

[0085] S1. Activated bacteria: The endophytic fungus JBY-33 strain isolated in Example 1 was inoculated on a slant culture medium and cultured at 28° C. for 24 h;

[0086] S2. Preparation of seed culture medium: The spores on the slant medium in step S1 were inoculated into 30 mL of PDB liquid medium, and cultured at 28 ° C and 160 rpm / min for 24 h to obtain a seed culture medium;

[0087] S3. Transformation culture: The seed culture obtained in step S2 was inoculated into 600 mL of PDB liquid culture medium at an inoculum size of 5%, and a total of 25 bottles were inoculated. After pre-culture at 28°C and 160 rpm / min for 24 h, ferric holly acid solution was added to a final concentration of 0.50 g / L, and then the transformation culture was continued under the same conditions for 24 h and then filtered;

[0088] S4. Residue extraction: The residue obtained by filtration in step S3 was ultrasonically extracted with 2 times the amount (by mass) of methanol for 3 times, each time for 0.5 h, and the filtrate and the methanol extract were combined and recovered under reduced pressure to obtain a conversion extract;

[0089] S5. Isolation, purification and structural identification of the conversion product: The conversion extract in step S4 was extracted with 2 volumes of ethyl acetate, and the extract was separated by reverse phase column chromatography, and gradient eluted with 5 column volumes of 40%, 50%, 60%, and 70% methanol / water in sequence, and the 70% methanol / water elution portion was collected and recovered, and the main conversion product was obtained by recrystallization with methanol.

[0090] The separation and identification results of the main transformation products are the same as those in Example 2.

[0091] Example 4 Lipid-lowering activity of derivative M and its effect on cell viability

[0092] The ferric holly acid derivative M used in this example is obtained by transformation, culture, separation and identification in Example 2.

[0093] 1. Effect of derivative M on cell viability

[0094] 1) Experimental methods

[0095] (1) Principle

[0096] The cell viability is determined by the MTT method. The chemical name of MTT is 3 (4,5-dimethylthiazole 2) 2,5-diphenyltetrazolium bromide, and its trade name is thiazolyl blue. The detection principle is as follows: the difference between living cells and dead cells is that the mitochondria of living cells contain succinate dehydrogenase, which can reduce exogenous MTT to water-insoluble blue crystalline formazan; dimethyl sulfoxide (DMSO) can dissolve the formazan deposited in the cells. Depending on the intervention group and the positive control group with the added compound, the absorbance is measured at a wavelength of 490 or 540 nm, which can indirectly reflect the number of surviving cells.

[0097] (2) Experiment on the effect on cell viability

[0098] HepG2 cell suspension was inoculated into 96-well plates for 10 4 / well, add 200 μL culture medium to each well, 100 μL PBS to each well at the outermost edge of the 96-well plate, the first column without cells but with culture medium as the blank control group, the rest were divided into control group and drug-treated group, with 6 replicates in each group; incubate at 37°C, 5% CO 2 Culture in a saturated humidity incubator.

[0099] When the cell density grew to about 80%, the supernatant was discarded, and the drug-treated group was intervened with 5 different concentrations of the transformation product M (derivative M isolated and identified in Example 2, with concentrations of 25, 50, 100, 200, and 400 μM, respectively) for 24 h, and the non-drug group was not treated; after 24 h of culture, 10 μL of MTT solution (mass concentration of 5 mg / mL) was added to each well except the blank control group, and culture was continued for 4 h.

[0100] Aspirate the culture medium in the wells, add 150 μL of DMSO solution to each well, and shake the 96-well plate on a shaker for 10 minutes at room temperature to completely dissolve the crystals. Use an ELISA reader to measure the OD value of each well at a wavelength of 490 nm. Repeat three times and take the average value to calculate the cell survival rate.

[0101]

[0102] Determine 6 concentrations of derivative M, draw a dose inhibition rate curve, and obtain its IC 50 value.

[0103] 2) Experimental results

[0104] The experimental results of the effect of derivative M on cell viability are as follows Figure 4 As shown by Figure 4 It can be seen that the derivative M has little effect on the viability of HepG2 cells in the concentration range of 25-400 μmol / L, and the cell survival rate is greater than 80%, indicating that the derivative M has a large safety factor.

[0105] 2. Lipid-lowering activity

[0106] 1) Principle

[0107] The lipid-lowering activity experiment adopted the intracellular TG (triglyceride) determination method commonly used in clinical and laboratory settings. TG (triglyceride) can be converted into glycerol-3-phosphate under the action of lipase and glycerol kinase, and then form hydrogen peroxide through 3-phosphoglycerol dehydrogenase. Hydrogen peroxide is oxidized by peroxidase to form a red quinone compound, and its color is proportional to the triglyceride content. The absorbance value is measured and the triglyceride content is calculated using the following formula. The standard concentration is 2.26mmol / L.

[0108] 2) Experimental methods

[0109] HepG2 cell suspension was inoculated in 6-well plates, 5×10 5 / well, at 37°C, 5% CO 2 Culture in a saturated humidity incubator.

[0110] The cells were divided into a normal group (NC), a model group (HC), a simvastatin positive drug group (ST, 10 μmol / L), ferric arachidic acid (RA) and a derivative M group, and low (L, 5 μmol / L), medium (M, 15 μmol / L) and high (H, 50 μmol / L) dose groups were set up. When the cell density grew to about 80%, the cells in the NC group were cultured with a DMEM solution containing 1% BSA, and the cells in the other groups were incubated with FFA. After 24 hours, the culture medium in each well was discarded, and the cells were washed twice with PBS, and then scraped with a cell scraper and transferred to a centrifuge tube to prepare a cell suspension. The cells were centrifuged at 1000 rpm for 10 min, and the supernatant was discarded. The cells were then dispersed with PBS, and the cells were centrifuged at 1000 rpm for 10 min. The supernatant was discarded and the cell pellet was retained.

[0111] The cell pellet was lysed with RIPA lysis buffer for 30 min, and the cell suspension was vortexed every 5 min. The lysed liquid was operated strictly according to the instructions of the TG kit, and TG working solution was added to a 96-well plate in the dark. Each group had 6 replicate wells and repeated three times.

[0112] Incubate the 96-well plate at 37°C for 10 minutes, and measure the absorbance of each well at a wavelength of 510 nm. Take 2 μL of the cell suspension and dilute it 20 times, and measure the protein concentration of each group of cells in the 96-well plate. Strictly follow the instructions of the BCA protein concentration quantification kit, and measure the absorbance of each well at 562 nm after the operation is completed.

[0113]

[0114] 3) Experimental results

[0115] The results of the study on lipid-lowering activity of ferric holmic acid and ferric holmic acid derivative M are as follows Figure 5 As shown in the figure, NC is the control group; HC is the model group; ST is the simvastatin positive drug group; L is the low-dose administration group; M is the medium-dose administration group; H is the high-dose administration group; RA is ferric holmic acid; M is ferric holmic acid derivative M; compared with the normal group, the model group ** represents P<0.01, and there is a significant difference; compared with the model group, the drug administration group # represents P<0.05, and ## represents P<0.01, and there is a significant difference.

[0116] Depend on Figure 5It can be seen that when ferric oleate (RA) and ferric oleate derivative M were administered at low, medium and high doses, the intracellular TG content was significantly reduced compared with the model group, and as the dosage increased, the triglyceride-lowering activity became more significant, with a certain dose dependence. The TG clearance rates of ferric oleate (RA) and ferric oleate derivative M at a concentration of 50 μmol / L were 35.8% and 41.8%, respectively. It can be seen that ferric oleate derivative M showed a better lipid-lowering effect than the original drug ferric oleate (RA) at a concentration of 50 μmol / L.

[0117] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A ferric holly acid derivative M, characterized in that: The structural formula of the derivative M is shown in formula (I):

2. A method for preparing the ferric holmic acid derivative M according to claim 1, characterized in that: The derivative M is obtained by culturing the endophytic fungus Lasiodiplodia sp. JBY-33 in a culture medium containing ferric holly acid and then isolating it; the fungus Lasiodiplodia sp. JBY-33 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 2, 2022, with the deposit number GDMCC NO: 62757.

3. The method according to claim 2, characterized in that: The following steps are involved: S1. Activation of fungus Lasiodiplodia sp. JBY-33; S2. Preparation of Lasiodiplodia sp. JBY-33 seed culture solution; S3. The seed culture solution obtained in step S2 is inoculated into a liquid culture medium, and after shaking culture at 25 to 35° C. for 12 to 48 hours, the ferric holly acid solution is added, and the transformation culture is carried out under the same conditions for 24 to 168 hours and then filtered; S4. Extract the filter residue obtained by filtration in step S3 with 1 to 3 times the mass of methanol, combine the filtrate and the methanol extract, and recover under reduced pressure to obtain a conversion extract; S5. The conversion extract obtained in step S4 is extracted with 1 to 3 times the volume of ethyl acetate, and the extract is separated by reverse phase column chromatography, and gradient eluted with 4 to 7 times the column volume of 40%, 50%, 60%, and 70% methanol / water, respectively, and the 70% methanol / water elution portion is collected and recovered, and the ferric holly acid derivative M is obtained by recrystallization with methanol.

4. The method according to claim 3, characterized in that: The activation in step S1 is to inoculate the frozen Lasiodiplodia sp. JBY-33 on a slant culture medium and culture it statically at 25 to 35° C. for 24 to 96 hours.

5. The method according to claim 3, characterized in that: The seed culture solution in step S2 is obtained by inoculating activated spores of Lasiodiplodia sp. JBY-33 into a liquid culture medium and culturing the culture medium under constant temperature shaking at 25 to 35° C. and 100 to 200 rpm / min for 24 to 48 hours.

6. The method according to claim 3, characterized in that: In step S3, the inoculation amount of the seed solution is 1-15%, and the final concentration of the added ferric holly acid solution is 0.05-0.50 g / L.

7. Use of the endophytic fungus Lasiodiplodia sp. JBY-33 in the preparation of the ferric holly acid derivative M according to claim 1, characterized in that: The fungus Lasiodiplodia sp. JBY-33 was deposited in Guangdong Provincial Microbiological Culture Collection Center on September 2, 2022, with the deposit number GDMCC NO: 62757.

8. Use of the ferric oleate derivative M according to claim 1 in the preparation of lipid-lowering drugs.

9. A lipid-lowering drug, characterized in that: Contains the ferric holly acid derivative M according to claim 1.

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