CN115181083B - Preparation method of compound Cyophiobiolins A-B and application of compound Cyophiobiolins A-B in preparation of anti-inflammatory drugs - Google Patents
Preparation method of compound Cyophiobiolins A-B and application of compound Cyophiobiolins A-B in preparation of anti-inflammatory drugs Download PDFInfo
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Abstract
The application discloses a compound Cyophiobionics A-B, a preparation method thereof and application thereof in preparing anti-inflammatory drugs. The compound Cyophiobiolins A-B is prepared by separating from a fermentation culture of the morinda officinalis endophytic fungus Cytospora rhizophorae A761. Experiments prove that the compound cytohiobiolins A-B inhibits the generation of nitric oxide NO by using a lipopolysaccharide LPS induced mouse macrophage RAW264.7 inflammation model 50 The values are 66.3+/-3.3 mu M and 53.3+/-4.6 mu M respectively, and the positive control is 20.9+/-1.2 mu M, so that the positive control shows more obvious anti-inflammatory activity and can be used for preparing anti-inflammatory medicaments.
Description
Technical Field
The application belongs to the technical field of biological medicines, and particularly relates to a compound Cyophiobolins A-B, a preparation method thereof and application thereof in preparing anti-inflammatory medicines.
Background
The morinda officinalis (Gynochthodes officinalis) is a plant of morinda genus of Rubiaceae family, is perennial vine shrub, is a whole body medicine, and is a clinically common blood pressure reducing traditional Chinese medicine. In addition, morinda officinalis can also treat rheumatalgia, promote secretion of body adrenocortical hormone, strengthen tendons and bones, warm and tonify kidney yang, improve reproductive system functions, resist inflammation and the like.
The plant endophytic fungi are various in species and low in natural toxicity, solves the problems of resource shortage, ecological damage and species extinction caused by extracting products from rare plant resources, greatly shortens the cultivation period, and the generated bioactive substances play roles in various aspects. It plays an important role in medicine research and development, environmental waste degradation, energy production, agricultural pest control and the like. The microbial source is rich and varied, the metabolite is novel and rich, and the metabolite covers various structural types. The chemical backbone is unique and complex, such as polyketides, alkaloids, terpenes, quinones, coumarins, esters, phenol derivatives, sterols, polypeptides, and the like. In terms of biological activity, the compounds are remarkable and various, such as antiviral, cytotoxic, antibacterial, antioxidant, enzyme inhibition activity, anti-inflammatory and the like.
Due to the wide variety of pressure sources in the world today, a wide variety of stress sources are applied to our body, putting us in an "inflammatory mode" and at any time compromising our immune system. The anti-inflammatory drugs in the current market mainly comprise cephalosporins, quinolones, macrolides and the like; the cephalosporins can produce side effects such as nausea, vomiting and abdominal distension after being used at high dosage or for a long time; quinolones can produce serious side effects such as dysbacteriosis, mental confusion, muscle damage, cardiac discomfort, etc. over time; macrolides also have serious side effects on the intestinal tract and liver. In summary, current drugs for treating inflammation are still relatively short, and related researchers are required to continue to conduct intensive researches on the inflammatory and pharmacological mechanisms. Currently, the market demand for anti-inflammatory drugs is increasing dramatically every day. However, the efficacy of today's anti-inflammatory agents is far from optimal, plus a significant portion of the drugs have serious side effects. With the development of organic chemical synthesis and the exploration and understanding of inflammatory mechanisms by researchers, many chemical synthesis drugs continue to appear in the pharmaceutical market, but these drugs have many adverse reactions. Therefore, the development of anti-inflammatory drugs with good curative effect, small side effect, safety and reliability is urgent. The widest clinical application prospect of the anti-inflammatory drugs in the future still needs to depend on natural products with low toxicity and derivatives thereof, and the anti-inflammatory drugs of the natural products can be expected in the future.
The application comprises the following steps:
it is a first object of the present application to provide compounds cytohiobiolins A-B having anti-inflammatory activity.
The structure of the compound Cyophiobions A-B is shown as a formula (I):
the second object of the present application is to provide a method for preparing the compound Cyophiobiolins A-B, wherein the compound Cyophiobiolins A-B is isolated from the fermentation culture of the plant endophytic fungus Cytospora rhizophorae A761 of Morinda citrifolia.
The method specifically comprises the following steps:
(1) Preparing a solid fermentation culture of the morinda officinalis endophytic fungi Cytospora rhizophorae A761, extracting the solid fermentation culture with methanol, and concentrating a methanol extract to obtain an extract;
(2) Subjecting the extract to silica gel column chromatography, and performing gradient elution by using n-hexane-ethyl acetate as eluent in a volume ratio of 100:1, 50:1, 30:1, 20:1, 10:1,5:1,2:1,1:1,1:2,0:1 and dichloromethane-methanol 10:1,5:1 and 1:1v/v respectively; the petroleum ether-ethyl acetate volume ratio 0:1 and dichloromethane-methanol 10:1 were collected and eluted and purified by TLC thin layer chromatography on chloroform: methanol=10:1v/v to obtain component fr.g with rf=0.3-0.5; dichloromethane-methanol 5:1 eluted was collected and purified by TLC thin layer chromatography on chloroform: methanol=5:1v/v to give component fr.i with rf=0.3-0.5;
component fr.g was subjected to column chromatography on RP C18 silica gel with methanol: gradient elution with water (50%. Fwdarw.100%v/v) and methanol collection: the water 90:10v/v eluted fraction Fr.G-6, fr.G-6 was purified by Sephadex LH-20 in chloroform: under an elution system of methanol=1:3v/v, the intermediate fraction fr.g-6-2 eluted by Sephadex LH-20 through a Sephadex column was collected by TLC thin layer chromatography with chloroform: developing methanol=10:1v/v to obtain Rf=0.4-0.5, wherein Rf=0.4-0.5 is not shown under ultraviolet of 254nm, and obtaining a compound Cyophiobiolin A through preparation and purification of a point with a green main component of vanillin color development;
component fr.i was subjected to column chromatography on RP C18 silica gel with methanol: gradient elution with water (60%. Fwdarw.100%v/v) and methanol collection: the component Fr.I-2 eluted with water 60:40 to 70:30v/v, fr.I-2 eluted with Sephadex LH-20CC gel column chromatography with chloroform-methanol=1:1 v/v, fr.I-2-3 eluted with Sephadex LH-20 by TLC thin layer chromatography with ethyl acetate to obtain Rf=0.5-0.9, fr.I-2-3 again eluted with Sephadex LH-20CC gel column chromatography with chloroform-methanol=1:1 v/v, the intermediate Fr.I-2-3-2 eluted with Sephadex LH-20 by TLC thin layer chromatography with ethyl acetate to obtain Rf=0.6-0.8, which is not shown under 254nm ultraviolet, with a deep blue spot of the main component, fr.I-2-3-2 again eluted with Sephadex LH-20CC gel column chromatography with chloroform-methanol=1:1 v/v, with ethyl acetate, and with a gradient of ethyl acetate to obtain Cylinb-2, and eluting with ethyl acetate with a deep blue gradient of Rf=0.8:1:1 v/v.
Preferably, the compound Cyophiobiolin A obtained through preparation and purification is prepared by semi-preparative HPLC using YMC-Pack C18 column, wherein the mobile phase is acetonitrile/water with volume ratio of 60:40, the flow rate is 2mL/min, the eluted components with retention time of 8.0-8.5min are collected, TLC thin layer chromatography is performed by chloroform: the compound of Rf=0.4 developed with methanol=10:1 and developed green with vanillin gave the compound cytohiobiolin a.
Preferably, the step (1) of preparing the solid fermentation culture of the morinda officinalis endophytic fungus Cytospora rhizophorae A761 comprises the following specific steps: picking mycelium of endophytic fungi Cytospora rhizophorae A761, inoculating the mycelium into potato dextrose liquid culture medium, culturing for 5 days at 28 ℃ and 120r/min to obtain seed liquid, inoculating the seed liquid into rice culture medium according to the inoculum size of 0.1mL/g, and culturing for 30 days at 28 ℃ to obtain solid fermentation culture of A761, wherein each liter of potato dextrose liquid culture medium is prepared by the following steps: boiling 200g of potato with 300mL of pure water for 20min, and filteringObtaining potato juice, adding 20g glucose and KH 2 PO 4 3g、MgSO 4 1.5g, vitamin B 1 Adding water to 10mg to 1000mL, and sterilizing; the rice culture medium is prepared by the following method: mixing 250g rice with 350mL water solution, and sterilizing.
The third object of the application is to provide application of the compounds Cyophiobiolins A and B or pharmaceutically acceptable salts thereof in preparing anti-inflammatory drugs.
Experiments show that the compound cytohiobiolins A-B inhibits the generation of nitric oxide NO in a lipopolysaccharide LPS induced mouse macrophage RAW264.7 inflammation model 50 The values were 66.3.+ -. 3.3,53.3.+ -. 4.6, respectively. Positive control indomethacin inhibits generation of nitric oxide NO (NO) by using lipopolysaccharide LPS-induced mouse macrophage RAW264.7 inflammation model 50 The value was 20.9.+ -. 1.2. Mu.M. This result shows that: the compound Cyophiobiolins A-B has relatively obvious anti-inflammatory activity.
A fourth object of the present application is to provide an anti-inflammatory agent comprising the compounds cytohiobiolins a, B or pharmaceutically acceptable salts thereof as an active ingredient.
A fifth object of the present application is to provide the use of morinda citrifolia plant endophytic fungus Cytospora rhizophorae A761 for the preparation of compound cytohiobiolins a or B.
Compared with the prior art, the application has the advantages that:
the compound Cyophiobiolins A-B is separated from the morinda officinalis plant endophytic fungi Cytospora rhizophorae A761, has relatively obvious anti-inflammatory activity, can be used for preparing anti-inflammatory drugs, provides candidate compounds for researching and developing new anti-inflammatory drugs, and provides scientific basis for developing and utilizing natural active substances derived from plant endophytic fungi.
The morinda officinalis endophytic fungi Cytospora rhizophorae A761 disclosed in NCBIhttps://www.ncbi.nlm.nih.gov/nuccore/KR056292.1/) The strain is held by the inventors and is guaranteed to be provided to the public within 20 years from the date of application.
Drawings
FIG. 1 is a diagram of the compound Cyophiobulin A 1 H NMR spectroscopy;
FIG. 2 is a diagram of the compound Cyophiobulin A 13 C NMR spectrum;
FIG. 3 is a diagram of the compound Cyophiobulin A 1 H- 1 H COSY profile;
FIG. 4 is an HSQC spectrum of the compound Cyophiobiolin A;
FIG. 5 is an HMBC spectrum of compound Cyophiobiolin A;
FIG. 6 is a NOESY spectrum of the compound Cyophiobiolin A;
FIG. 7 is an HR-ESIMS spectrum of compound Cyophiobiolin A;
FIG. 8 is a CD spectrum of the compound Cyophiobulin A;
FIG. 9 is a UV spectrum of the compound Cyophiobulin A;
FIG. 10 is an IR spectrum of the compound Cyophiobulin A.
FIG. 11 is a diagram of Cyophiobulin B 1 H NMR spectroscopy;
FIG. 12 is a diagram of Cyophiobulin B 13 C NMR spectrum;
FIG. 13 is a diagram of Cyophiobulin B 1 H- 1 H COSY profile;
FIG. 14 is an HSQC spectrum of compound Cyophiobiolin B;
FIG. 15 is an HMBC spectrum of compound Cyophiobulin B;
FIG. 16 is a NOESY spectrum of the compound Cyophiobiolin B;
FIG. 17 is an HR-ESIMS spectrum of compound Cyophiobiolin B;
FIG. 18 is a CD spectrum of compound Cyophiobulin B;
FIG. 19 is a UV spectrum of compound Cyophiobulin B;
FIG. 20 is an IR spectrum of the compound Cyophiobulin B.
Detailed Description
The following examples are further illustrative of the application and are not intended to be limiting thereof.
Example 1
1. Isolation, purification and identification of morinda officinalis plant endophytic fungus Cytospora rhizophorae A761
The morinda officinalis endophytic fungus Cytospora rhizophorae A761 is 1 month in 2015, is separated from medicinal morinda officinalis collected from the high-importance area of Zhaoqing city in Guangdong, and is identified by ITS sequence analysis, and GenBank gene accession number is: KR056292 (https:// www.ncbi.nlm.nih.gov/nuccore/KR056292.1 /), identified by blast comparison, by homology analysis, as Cytospora rhizophorae M225,225, designated A761, designated Cytospora rhizophorae A761.
2. Solid state fermentation of Cytospora rhizophorae A761
Inoculating activated endophytic fungus Cytospora rhizophorae A761 mycelium of Morinda citrifolia plant into potato glucose liquid medium (prepared by decocting 200g potato with 300mL pure water per liter of medium, boiling for 20min, filtering to obtain potato juice, adding glucose 20g and KH 2 PO 4 3g、MgSO 4 1.5g, vitamin B 1 10mg, water was added to make up to 1000mL, and sterilized), and the mixture was cultured at 28℃for 5 days under 120r/min to obtain a seed solution, and then the seed solution was inoculated into a rice medium (the medium was prepared by: 250g of rice was mixed with 350mL of the aqueous solution, autoclaved at 121℃for 20min, cooled to prepare) and cultured at 28℃for 30 days to prepare a solid fermentation culture of A761.
3. Preparation of Cyophiobiolins A-B
(1) Methanol was added to the solid fermentation culture of A761, followed by soaking and extraction for 24 hours, repeated extraction was performed 3 times, and the extracts were combined and concentrated to obtain an extract (268 g).
(2) Subjecting the extract to silica gel column chromatography, and performing gradient elution by using n-hexane-ethyl acetate as eluent in a volume ratio of 100:1, 50:1, 30:1, 20:1, 10:1,5:1,2:1,1:1,1:2,0:1 and dichloromethane-methanol 10:1,5:1 and 1:1v/v respectively; the petroleum ether-ethyl acetate volume ratio 0:1 and dichloromethane-methanol 10:1 were collected and eluted and purified by TLC thin layer chromatography on chloroform: methanol=10:1v/v to obtain component fr.g with rf=0.3-0.5; dichloromethane-methanol 5:1 eluted was collected and purified by TLC thin layer chromatography on chloroform: methanol=5:1v/v to give component fr.i with rf=0.3-0.5;
component fr.g was subjected to column chromatography on RP C18 silica gel with methanol: gradient elution with water (50%. Fwdarw.100%v/v) gives 8 subfractions (Fr. G-1-Fr. G-8), collecting methanol: the water 90:10v/v eluted fraction Fr.G-6, fr.G-6 was purified by Sephadex LH-20 in chloroform: four subfractions (fr.g-6-1-fr.g-6-4) were obtained in an elution system of methanol=1:3 v/v, the middle fraction fr.g-6-2 eluted by Sephadex LH-20 was collected (rf=0.4-0.5 developed by TLC thin layer chromatography with chloroform: methanol=10:1 v/v, which was not shown under uv light at 254nm, with a green spot of vanillin as the main component), and the eluted fraction was collected by TLC thin layer chromatography with chloroform using YMC-Pack C18 column with a volume ratio of 60:40 in a mobile phase of acetonitrile/water at a flow rate of 2mL/min, with a retention time of 8.0-8.5 min: a compound of which rf=0.4 is developed with methanol=10:1 and which is green by vanillin development gives a compound of Cyophiobiolin a;
component fr.i was subjected to column chromatography on RP C18 silica gel with methanol: gradient elution with water (60%. Fwdarw.100%v/v) gives 5 subfractions (Fr.I-1-Fr.I-5), collecting methanol: the fraction Fr.I-2 eluted with water 60:40 to 70:30v/v, fr.I-2 was subjected to Sephadex LH-20CC gel column chromatography with chloroform-methanol=1:1 v/v elution system to obtain three subfractions (Fr.I-2-1-Fr.I-2-3), the last fraction Fr.I-2-3 eluted with Sephadex LH-20 by Sephadex LH-20 was collected (Rf=0.5-0.9 by TLC thin layer chromatography with ethyl acetate), fr.I-2-3 was subjected to Sephadex LH-20CC gel column chromatography with chloroform-methanol=1:1 v/v elution system to obtain three subfractions (Fr.I-2-3-1-Fr.I-2-3-3), the intermediate fraction Fr.I-2 eluted with Sephadex LH-20 was collected (Rf.I-2 by TLC thin layer chromatography with ethyl acetate=0.5-0.9, the fraction of ethyl acetate was not eluted with ethyl acetate by TLC thin layer chromatography with chloroform-1:1 v/v, and no ethyl acetate was eluted with ethyl acetate by TLC-1:1:1 v/v, the fraction was subjected to obtain the fraction was subjected to ethyl acetate elution with ethyl acetate by TLC-0.0:2:2:2.0.0.0.0.0.2.9.
4. Structural identification of Cyophiobiolins A-B
1 H-NMR、 13 C-NMR and HMBC nuclear magnetic resonance spectrograms are measured by a Bruker Advance-500 nuclear magnetic resonance spectrometer, and Tetramethylsilane (TMS) is used as an internal standard; ESI-MS data were measured using a VG Autospec-3000 type mass spectrometer; the UV spectrum was measured with a Shimadzu UV-2600 spectrophotometer and its structure was identified as follows:
as shown in FIGS. 1-20, FIG. 1 is a compound Cyophiobulin A 1 H NMR spectroscopy; FIG. 2 is a diagram of the compound Cyophiobulin A 13 C NMR spectrum; FIG. 3 is a diagram of the compound Cyophiobulin A 1 H- 1 H COSY profile; FIG. 4 is an HSQC spectrum of the compound Cyophiobiolin A; FIG. 5 is an HMBC spectrum of compound Cyophiobiolin A; FIG. 6 is a NOESY spectrum of the compound Cyophiobiolin A; FIG. 7 is an HR-ESIMS spectrum of compound Cyophiobiolin A; FIG. 8 is a CD spectrum of the compound Cyophiobulin A; FIG. 9 is a UV spectrum of the compound Cyophiobulin A; FIG. 10 is an IR spectrum of the compound Cyophiobulin A. FIG. 11 is a diagram of Cyophiobulin B 1 H NMR spectroscopy; FIG. 12 is a diagram of Cyophiobulin B 13 C NMR spectrum; FIG. 13 is a diagram of Cyophiobulin B 1 H- 1 H COSY profile; FIG. 14 is an HSQC spectrum of compound Cyophiobiolin B; FIG. 15 is an HMBC spectrum of compound Cyophiobulin B; FIG. 16 is a NOESY spectrum of the compound Cyophiobiolin B; FIG. 17 is an HR-ESIMS spectrum of compound Cyophiobiolin B; FIG. 18 is a CD spectrum of compound Cyophiobulin B; FIG. 19 is a UV spectrum of compound Cyophiobulin B; FIG. 20 is an IR spectrum of the compound Cyophiobulin B.
Compound cytohiobiolin a is a brown bulk crystal (its nuclear magnetic data are shown in table 1); according to HRESIMS M/z419.2780[ M+H ]] + (calculated value 419.2792), the molecular formula of the compound was determined to be C 25 H 38 O 5 The unsaturation degree is 7; 1 H-NMR spectra show the presence of two olefin proton signals delta H 7.10 (1H, m, H-8) and delta H 5.30 (1H, brs, H-13) and 5 methyl [ delta ] H 0.82(3H,d,J＝7.5Hz,H-20),0.90(3H,s,H-22),1.03(3H,d,J＝6.8Hz,H-23),1.14(3H,s,H-24),1.18(3H,s,H-25)]。 13 The C-NMR spectrum and HSQC spectrum showed 25 carbon signals (Table 1), including 6 quaternary carbons, 8 methine carbons, 6 methylene carbons, and 5 methyl carbons. The planar structure of the compound Cyophiobilin A is determined to be an ophiobolane ester terpene skeleton by further analyzing two-dimensional spectrograms such as COSY, HSQC and HMBC, and the absolute configuration of the compound Cyophiobolin A is determined by X-ray single crystal diffraction analysis to be 2S,3S,5R,6R,10R,11S,15R and 18R.
Compound cytohiobiolin B is a brown bulk crystal (its nuclear magnetic data are shown in table 1); according to HRESIMS M/z435.2739[ M+H ]] + (calculated value 4352741), the molecular formula of the compound was determined to be C 25 H 38 O 6 The unsaturation degree is 7; the significant difference between Cyophiobiolin B and Cyophiobiolin A was logically attributed to the substitution of C-20 methyl groups in Cyophiobiolin A by hydroxymethyl groups in Cyophiobiolin B, as found by analysis of its 1D and 2D spectra, with the spectra of Cyophiobiolin B and Cyophiobiolin A having a great similarity. By passing through 13 C NMR 1 H NMR chemical shift [ delta ] H 3.53(1H,dd,J＝6.4,10.6Hz(H-20A),3.27(1H,H-20B)；δ C 61.0(C-20)]And important HMBC correlation from H 2 This hypothesis was further validated by 20 through C-2 and C-4 (FIG. 15), which initially established that the planar structure of Cyyobilin B is the opioborane ester terpene skeleton. The absolute configuration of the compound Cyophiobiolin B was determined by X-ray single crystal diffraction analysis and was determined to be 2S,3S,5R,6R,10R,11S,15R,18R.
Nuclear magnetic data of the compounds Cyophiobionins A and B of Table 1 (500 MHz/125MHz, δin ppm, J in Hz)
The structural formula of the target compound Cyophiobiolins A-B separated by the method is shown as the formula (I):
example 2
The anti-inflammatory activity of the compounds Cyophiobiolins A-B was tested using the Griess method.
1. Test reagent: the compound Cyophiobiolins A-B prepared by the application is dissolved by dimethyl sulfoxide (DMSO) to obtain a mother solution with the concentration of 10mM, and then the mother solution is diluted to the required concentration by DMEM culture medium. The positive control was indomethacin aqueous solution.
The cell line used in this experiment was mouse macrophage RAW264.7.
2. The experimental method comprises the following steps: the anti-inflammatory activity of the compounds was evaluated by determining the effect of the compounds on Nitric Oxide (NO) release induced by bacterial Lipopolysaccharide (LPS) in the RAW264.7 cell inflammation model using the Griess method. Taking RAW264.7 cells in logarithmic growth phase, digesting with pancreatin, staining and counting with trypan blue, detecting cell activity greater than 95% by trypan blue exclusion experiment, and adjusting cell concentration to 5×10 with fresh DMEM medium 5 Cells were seeded in 96-well plates at a volume of 180. Mu.L of cell suspension per well and 3 blanks Kong Diaoling were set at 37℃in 5% CO 2 Culturing in an incubator. After 24h, the stock culture was discarded and 200. Mu.L of Cyophiobiolins A-B with a certain concentration and 1. Mu.g/mL of LPS medium were added, and 200. Mu.L of 1. Mu.g/mL of LPS medium was added to the negative control, and 200. Mu.L of indomethacin with a certain concentration and 1. Mu.g/mL of LPS medium was added to the positive control. Placing at 37deg.C and 5% CO 2 After 24h incubation in incubator, 50. Mu.L of supernatant from each well was aspirated, transferred to a new 96-well plate, assayed for effect of compound on Nitric Oxide (NO) release using Griess method, 50. Mu.L of Griess A solution and 50. Mu.L of Griess B solution were added to each well, mixed well, and OD value of each well was measured with an ELISA at 540nm wavelength. Each test was repeated 3 times and its IC was calculated 50 Values. The inhibition of NO production by the drug was calculated using the following formula: NO generation inhibition rate (%) = (1-a) Sample group /A Control group )×100％。
3. Experimental results: IC for inhibiting generation of nitric oxide NO by using compound Cyophiobabilins A-B induced by lipopolysaccharide LPS on mouse macrophage RAW264.7 inflammation model 50 The values were 66.3.+ -. 3.3. Mu.M, 53.3.+ -. 4.6. Mu.M, respectively. Positive control indomethacin inhibits generation of nitric oxide NO (NO) by using lipopolysaccharide LPS-induced mouse macrophage RAW264.7 inflammation model 50 The value was 20.9.+ -. 1.2. Mu.M. This result shows that: the compound Cyophiobionins A-B has relatively obvious anti-inflammatory activity, so the application provides a candidate compound for researching and developing new anti-tumor drugs and provides scientific basis for developing and utilizing natural active substances derived from plant endophytic fungi.
The foregoing is merely a preferred embodiment of the present application, and it should be noted that the above-mentioned preferred embodiment should not be construed as limiting the application, and the scope of the application should be defined by the appended claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the application and these modifications and adaptations are intended to be comprehended within the scope of the application.
Claims (5)
1. Any compound as shown in formula (i):
2. a method of preparing the compound of claim 1, wherein the compound cytoiobilin a-B is isolated from a fermentation culture of a fungal endophyte Cytospora rhizophorae A761 of a morinda officinalis plant;
the method specifically comprises the following steps:
(1) Preparing a solid fermentation culture of the morinda officinalis endophytic fungi Cytospora rhizophorae A761, extracting the solid fermentation culture with methanol, and concentrating a methanol extract to obtain an extract;
(2) Subjecting the extract to silica gel column chromatography, and performing gradient elution by using n-hexane-ethyl acetate as eluent in a volume ratio of 100:1, 50:1, 30:1, 20:1, 10:1,5:1,2:1,1:1,1:2,0:1 and dichloromethane-methanol 10:1,5:1 and 1:1v/v respectively; the n-hexane-ethyl acetate volume ratio 0:1 and dichloromethane-methanol 10:1 were collected and eluted and purified by TLC thin layer chromatography on chloroform: methanol=10:1v/v to obtain component fr.g with rf=0.3-0.5; dichloromethane-methanol 5:1 eluted was collected and purified by TLC thin layer chromatography on chloroform: methanol=5:1v/v to give component fr.i with rf=0.3-0.5;
component fr.g was subjected to column chromatography on RP C18 silica gel with methanol: water 50% → 100% v/v gradient elution, methanol was collected: the water 90:10v/v eluted fraction Fr.G-6, fr.G-6 was purified by Sephadex LH-20 in chloroform: under an elution system of methanol=1:3v/v, the intermediate fraction fr.g-6-2 eluted by Sephadex LH-20 through a Sephadex column was collected by TLC thin layer chromatography with chloroform: developing methanol=10:1v/v to obtain Rf=0.4-0.5, wherein Rf=0.4-0.5 is not shown under ultraviolet of 254nm, and obtaining a compound Cyophiobiolin A through preparation and purification of a point with a green main component of vanillin color development;
component fr.i was subjected to column chromatography on RP C18 silica gel with methanol: water 60% -100% v/v gradient elution, methanol was collected: the method comprises the steps of (1) collecting a component Fr.I-2 eluted by water 60:40 to 70:30v/v, wherein Fr.I-2 is subjected to Sephadex LH-20CC gel column chromatography with chloroform-methanol=1:1 v/v, fr.I-2-3 eluted by Sephadex LH-20 is subjected to TLC thin layer chromatography with ethyl acetate to obtain Rf=0.5-0.9, fr.I-2-3 is subjected to Sephadex LH-20CC gel column chromatography again with chloroform-methanol=1:1 v/v, the intermediate Fr.I-2-3-2 eluted by Sephadex LH-20 is collected and subjected to TLC thin layer chromatography with ethyl acetate to obtain Rf=0.6-0.8, the intermediate Fr.I-3-2 is subjected to TLC thin layer chromatography with ethyl acetate to obtain a deep blue dot, the main component of the liquid vanillin-ethyl vanillin is subjected to TLC thin layer chromatography with ethyl vanillin-2, the liquid vanillin-ethyl acetate is subjected to liquid chromatography with Rf-1:1 v/v=1:1 v/v, and the liquid vanillin-ethyl acetate is eluted with a thin layer chromatography with ethyl vanillin-2:1:0.8;
the compound Cyophiobiolin A obtained through preparation and purification is prepared by semi-preparative HPLC (high performance liquid chromatography) through YMC-Pack C18 column, wherein the mobile phase is acetonitrile/water with the volume ratio of 60:40, the flow rate is 2mL/min, the elution component with the retention time of 8.0-8.5min is collected, and the TLC thin layer chromatography is performed by using chloroform: a compound of which rf=0.4 is developed with methanol=10:1 and which is green by vanillin development gives a compound of Cyophiobiolin a;
the solid fermentation culture of the morinda officinalis endophytic fungi Cytospora rhizophorae A761 prepared in the step (1) comprises the following specific steps: picking mycelium of endophytic fungi Cytospora rhizophorae A761, inoculating the mycelium into potato dextrose liquid culture medium, culturing for 5 days at 28 ℃ and 120r/min to obtain seed liquid, inoculating the seed liquid into rice culture medium according to the inoculum size of 0.1mL/g, and culturing for 30 days at 28 ℃ to obtain solid fermentation culture of A761, wherein each liter of potato dextrose liquid culture medium is prepared by the following steps: boiling 200g of potato with 300mL of pure water for 20min, filtering to obtain potato juice, and adding 20g of glucose and KH 2 PO 4 3g、MgSO 4 1.5g, vitamin B 1 Adding water to 10mg to 1000mL, and sterilizing; the rice culture medium is prepared by the following method: mixing 250g rice with 350mL water solution, and sterilizing.
3. Use of cytopeniolin a, cytopeniolin B or a pharmaceutically acceptable salt thereof in a compound according to claim 1 for the manufacture of an anti-inflammatory medicament.
4. An anti-inflammatory agent comprising, as an active ingredient, cytohiobiolin a, cytohiobiolin B, or a pharmaceutically acceptable salt thereof, of the compounds of claim 1.
5. Use of morinda citrifolia plant endophytic fungus Cytospora rhizophorae A761 for the preparation of cyopobrines a-B in a compound as claimed in claim 1.
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| CN109970538A (en) * | 2019-04-17 | 2019-07-05 | 中山大学 | The Dimeric sesquiterpene compound in a kind of marine fungi source and preparation method thereof and application in preparing anti-inflammatory drugs |
| CN112812125A (en) * | 2020-10-14 | 2021-05-18 | 广西师范大学 | Novel skeleton heteroterpene compound and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109970538A (en) * | 2019-04-17 | 2019-07-05 | 中山大学 | The Dimeric sesquiterpene compound in a kind of marine fungi source and preparation method thereof and application in preparing anti-inflammatory drugs |
| CN112812125A (en) * | 2020-10-14 | 2021-05-18 | 广西师范大学 | Novel skeleton heteroterpene compound and preparation method and application thereof |
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| Title |
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| Ophiobolin Sesterterpenoids and Farnesylated Phthalide Derivatives from the Deep Sea Cold-Seep-Derived Fungus Aspergillus insuetus SD-512;Lu-Ping Chi等;Journal of Natural Products;第83卷;3652−3660 * |
| Ophiobolin-Type Sesterterpenoids from the Mangrove Endophytic Fungus Aspergillus sp. ZJ-68;Runlin Cai等;Journal of Natural Products;第82卷;2268-2278 * |
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