CN114028367A - Application of cajanin derivative in preparation of medicine for preventing and/or treating novel coronavirus infection diseases - Google Patents
Application of cajanin derivative in preparation of medicine for preventing and/or treating novel coronavirus infection diseases Download PDFInfo
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Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/075—Ethers or acetals
- A61K31/085—Ethers or acetals having an ether linkage to aromatic ring nuclear carbon
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C41/00—Preparation of ethers; Preparation of compounds having groups, groups or groups
- C07C41/01—Preparation of ethers
- C07C41/34—Separation; Purification; Stabilisation; Use of additives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C41/00—Preparation of ethers; Preparation of compounds having groups, groups or groups
- C07C41/01—Preparation of ethers
- C07C41/34—Separation; Purification; Stabilisation; Use of additives
- C07C41/36—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C41/00—Preparation of ethers; Preparation of compounds having groups, groups or groups
- C07C41/01—Preparation of ethers
- C07C41/34—Separation; Purification; Stabilisation; Use of additives
- C07C41/40—Separation; Purification; Stabilisation; Use of additives by change of physical state, e.g. by crystallisation
Abstract
The invention discloses an application of a cajanin derivative in preparing a medicine for preventing and/or treating a novel coronavirus infection disease, belonging to the technical field of medicinal chemistry. The name of the cajanin derivative is 3-methoxy-5-hydroxy-2- (3-methyl-2-butenyl) bibenzyl, and the cajanin derivative is used in the inventionIs first discovered in pair Mpro/3CLproThe protease has inhibitory effect, and can be used for treating or preventing the diseases caused by MproProtease or 3CLproThe protease dominates diseases caused by the infection of the propagating viruses, for example, diseases caused by the infection of novel coronavirus, and the application value is high.
Description
Technical Field
The invention belongs to the technical field of medicinal chemistry, and particularly relates to application of a cajanin derivative in preparation of a medicine for preventing and/or treating a novel coronavirus infection disease.
Background
The new type coronavirus (2019-nCoV) belongs to single positive strand RNA virus, has high homology with SARS-CoV and MERS-CoV, after the virus is infected into host cell, under the help of host cell, ORF1a/b of its genetic material RNA first translationally expresses two polyprotein precursors (pp1a and pp1ab), which are in main protease (main protease, abbreviated as M)pro) And papain (papain like proteins) to produce multiple nonstructural proteins. MproAlso known as 3C-like protease (3 CL)pro) Since the cleavage site specificity is similar to that of 3C protease (3C protease) of picornavirus (picornavirus), both are called Mpro/3CLpro. The non-structural protein generated by the polymer protein precursor participates in the generation of virus subgenomic RNA and four structural proteins (envelope/E protein, membrane/M protein, spike/S protein and nucleocapsid/N protein), thereby completing the propagation and release of progeny virus. Due to MproProtease plays a crucial role in the life cycle of viruses, and there is no homologous protein in the human body, so MproThe main protease is an ideal target for developing antiviral drugs.
Disclosure of Invention
A kind of cajanin derivative, named 3-methoxy-5-hydroxy-2- (3-methyl-2-butenyl) bibenzyl, its structure is shown in the following formula I:
the invention has found for the first time that the compound pair M of the formula Ipro/3CLproThe protease has inhibitory effect. On the basis of this, the compounds of the formula I mentioned above can be used for the prophylaxis and/or treatment of the diseases caused by MproProtease and/or 3CLproProteases dominate diseases caused by infections with a propagating virus. Master and slaveThe protease is a key protein in the life cycle of coronavirus, and is expressed in the currently known coronavirus, so that the disease may preferably be a disease caused by coronavirus infection. For example, human coronavirus OC43(HCoV-OC43), human coronavirus HKU1(HCoV-HKU1), middle east respiratory syndrome coronavirus (MERS-CoV), SARS virus (SARS-CoV), novel coronavirus (2019-nCoV) and the like.
The above diseases caused by coronavirus infection include, but are not limited to, respiratory diseases such as pneumonia. The pneumonia can be acute or chronic pneumonia, including mild pneumonia, moderate pneumonia, severe pneumonia or critical pneumonia.
The present invention provides the use of a compound of formula I as described above: for preparing a medicament for the prophylaxis and/or treatment of a disease caused by MproProtease and/or 3CLproProteases dominate the drugs for diseases caused by the infection of a propagating virus. Preferably, the compounds of formula I above are used for the preparation of a medicament for the prophylaxis and/or treatment of diseases caused by coronavirus infections. Further preferably, the above compounds of formula i are used for the preparation of a medicament for the prevention and/or treatment of diseases caused by infection with a novel coronavirus (2019-nCoV).
The medicine prepared by the compound of the formula I can be a single active ingredient medicine consisting of the compound of the formula I only, and can also be a medicine composition consisting of the compound of the formula I and other chemical components.
The other chemical components are selected from single chemical components or compositions.
The other chemical components do not have antagonistic effect with the cajanin derivatives described in formula I. The other chemical component pair consists of MproProtease and/or 3CLproThe protease dominated propagating virus has or does not have inhibitory activity. The other chemical components can assist the treatment or prevention of the disease caused by M of the cajanin derivatives of formula IproProtease and/or 3CLproProteases dominate the infection of the propagating virus.
The medicine prepared from the compound shown in the formula I also comprises one or more of pharmaceutically acceptable carriers, auxiliary materials and excipients.
The medicament prepared from the compound shown in the formula I is prepared into a dosage form selected from capsules, granules, tablets, pills, sprays, suppositories, aerosols, powder aerosols, patches, oral liquid or injection and the like.
The above-mentioned novel coronaviruses (2019-nCoV) also include, but are not limited to, the following variants: alpha, Beta, Gamma, Zeta, Delta, Lambda, and Omicron, among others.
A process for the preparation of a compound of formula I as described above, comprising the steps of:
pulverizing semen Cajani branches and leaves, soaking and extracting with ethanol, and concentrating under reduced pressure to obtain thick total extract. Extracting the total extract with water and ethyl acetate, stirring, standing for layering, removing water layer, and concentrating ethyl acetate layer under reduced pressure to obtain ethyl acetate extract. Performing MCI column chromatography on the ethyl acetate part extract, and performing extraction with ethanol: water (v/v) as mobile phase, gradient eluting with 50% ethanol, 70% ethanol and 95% ethanol respectively, and collecting 70% ethanol flow section. The 70% ethanol stream was subjected to silica gel column chromatography using a petroleum ether: and ethyl acetate (v/v) is used as a mobile phase for gradient elution, and the gradient is set to be 50:1, 25:1, 10:1, 5:1, 2:1 and 1:1 in sequence. And (3) carrying out Sephadex LH-20 gel column chromatography on the 2:1 flow section to remove pigments. The 2:1 fraction was then purified by preparative HPLC to give the compound of formula I.
The invention has the beneficial effects that:
the cajanin derivative has good novel coronavirus inhibitory activity, and has important application value in the aspect of preparing medicaments for treating or preventing diseases caused by novel coronavirus infection.
Drawings
FIG. 1 is an HPLC evaporation light scattering spectrum of an ethyl acetate fraction of cajan branches and leaves;
FIG. 2 shows retention time tRESI of Compounds at 22.38min (a) and 22.66min (b)+And (4) mass spectrum.
Detailed Description
Terms used in the present invention have generally meanings as commonly understood by one of ordinary skill in the art, unless otherwise specified. The present invention will be described in further detail with reference to the following data in conjunction with specific examples. The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.
A cajanin derivative has a structure shown in formula I:
the cajanin derivatives are derived from cajan leaves, which are collected from Honghe county of Yunnan province of China in 2017 and 9 months, and are identified as the leaves of Cajanus cajan (Cajanus cajan) of Leguminosae (Leguminosae) by Wujiwei vice investigator of Guangzhou university of traditional Chinese medicine.
Preparation of Compounds of formula I
(1) Extraction and extraction
Pulverizing 20kg dried Cajanus cajan branches and leaves into small branch segments, soaking in 95% ethanol (preferably submerging the medicinal materials) at room temperature, and extracting for three times (each time for 7 days); mixing the three extractive solutions, and concentrating under reduced pressure until no ethanol smell is detected to obtain 3.7kg thick total extract. Suspending the total extract with water, placing in a 20L dragon mouth bottle, adding ethyl acetate for extraction, stirring continuously, after liquid layering, respectively concentrating ethyl acetate layer and water layer under reduced pressure to obtain 2.1kg ethyl acetate part extract and 1.6kg water part extract.
(2) Screening of stilbene Compounds
1mg of ethyl acetate fraction was dissolved in 1mL of chromatographic methanol, filtered, and 10. mu.L of the filtrate was analyzed by LC-MS. The analysis results are shown in fig. 1 and 2.
FIG. 1 is an HPLC evaporation light scattering spectrum of ethyl acetate extract, each chromatographic peak shown in FIG. 1 represents a corresponding compound, and the structural type of the compound can be identified by analyzing the chromatographic peak in FIG. 1 by mass spectrometry. For retention time t in FIG. 1RESI was performed at 22.38min and 22.66min chromatographic peaks+Mass spectrometry analysis, as shown in figure 2. As can be seen from fig. 2, the molecular ion peak of the two chromatographic peaks is 295, i.e., the molecular weight thereof corresponds to 294, which is the molecular weight of cajanin a and cajanin C. Therefore, the LC-ELSD analysis result shows that the retention time of the stilbene compounds is 22.00-24.00 min.
(3) Enrichment of stilbenes
Performing MCI column chromatography on the ethyl acetate part extract, and performing extraction with ethanol: water (v/v) is used as a mobile phase for gradient elution to enrich stilbene compounds, the gradient is respectively 50% ethanol, 70% ethanol and 95% ethanol, and MCI column chromatography enrichment obtains 3 components in total: and performing LC-MS analysis on the component A (50% ethanol flow section), the component B (70% ethanol flow section) and the component C (95% ethanol flow section), wherein the results show that the compounds with the retention time of 22.00-24.00 min are enriched in the component B, so that the enrichment of stilbene compounds is completed by MCI column chromatography technology.
(4) Purification and separation of stilbene compounds
Subjecting fraction B to silica gel column (300-400 mesh) chromatography with a mixture of petroleum ether: and ethyl acetate (v/v) is used as a mobile phase for gradient elution, and the gradient is set to be 50:1, 25:1, 10:1, 5:1, 2:1 and 1:1 in sequence. And (3) carrying out Sephadex LH-20 gel column chromatography (the mobile phase is 100% methanol) on the 2:1 flow section to remove pigments. The 2:1 stream fragment was then subjected to preparative HPLC (CH)3CN/H2O, 50% to 80%) to obtain compound 1(33mg), compound 2(101mg), compound 3(1g), compound 4(212mg) and compound 5(42 mg).
In this example, the operating conditions for preparative HPLC were:
WATERS high performance liquid (equipped with Alltech 3300ELSD detector), operating system using Varian PrepStar, separation column using WatersRP C185 μm, 30 mm. times.150 mm, the fluidity system being CH3CN/H2O, the flow rate is 25.0mL/min, and the preparation time is 120 min. The retention time in the preparation process of the compounds 1-5 is respectively as follows: compound 1(81.28min), compound 2(83.55min), compound 3(92.11min), compound 4(85.20min), and compound 5(97.48 min).
Compound 1 is the compound of formula i according to the present invention. The structures of the compounds 2-5 are shown as follows:
the identification data for compounds 1-5 are shown below:
compound 1: a light yellow powder;1H NMR(400MHz,CDCl3):δH 7.32(2H,m,H-3″,5″),7.21(3H,m,H-2″,4″,6″),6.31(1H,d,J=2.5Hz,H-4),6.26(1H,d,J=2.5Hz,H-6),5.06(1H,m,H-2'),3.79(3H,s,-OCH3),3.29(2H,d,J=6.7Hz,H-1'),2.85(4H,s,H-7/H-8),1.74(3H,s,H-5'),1.67(3H,s,H-4');13C NMR(101MHz,CDCl3):δC 158.81(C-3),154.38(C-5),142.20(C-1),142.13(C-1″),130.83(C-3'),128.52(C-3″,C-5″),128.50(C-2″,C-6″),126.08(C-4″),123.97(C-2'),120.86(C-2),108.04(C-6),97.12(C-4),55.76(3-OCH3),37.67(C-2),35.33(C-1),25.89(C-4'),24.53(C-1'),18.07(C-5')。
compound 2: a white powder;1H NMR(400MHz,CDCl3):δH 7.48(2H,d,J=7.6Hz,H-2',H-6'),7.34(2H,m,H-3',H-5'),7.24(1H,m,H-4'),6.93(1H,J=16.1Hz,H-7),7.29(1H,J=16.1Hz,H-8),6.69(1H,s,H-2),6.37(1H,s,H-6),5.11(1H,t,J=6.8Hz,H-2″),3.78(3H,s,-OCH3),3.41(2H,d,J=6.8Hz,H-1″),1.80(3H,s,H-4″),1.67(3H,s,H-5″);13C NMR(101MHz,CDCl3):δC 158.59(C-3),154.48(C-5),137.96(C-1'),137.60(C-1),130.96(C-3″),130.54(C-7),128.71(C-3',-5'),127.65(C-4″),126.60(C-2',-6'),126.52(C-8),123.51(C-2″),121.03(C-4),104.15(C-6),98.62(C-2),55.75(3-OCH3) 25.80(C-4 '), 24.47(C-1 '), 17.99(C-5 '), identified as cajanin A.
Compound 3: a white powder;1H NMR(400MHz,CDCl3):δH 11.53(br,s,-COOH),7.83(1H,d,J=16.1Hz,H-7),7.52(2H,d,J=6.8Hz,H-10/14),7.38(2H,d,J=6.8Hz,H-11/13),7.29(1H,m,H-12),6.82(1H,d,J=16.1Hz,H-8),6.66(1H,s,H-6),5.21(1H,m,H-17),3.95(3H,s,-OCH3),3.37(2H,d,J=6.8Hz,H-16),1.79(3H,s,H-19),1.68(3H,s,H-20);13C NMR(101MHz,CDCl3):δC 175.72(COOH),162.44(C-5),162.28(C-3),141.92(C-1),137.28(C-9),131.98(C-17),130.82(C-8),130.38(C-7),128.75(C-13,11),127.89(C-12),126.81(C-10,14),121.90(C-18),116.76(C-4),103.33(C-2),103.05(C-6),55.74(5-OCH3) 25.84(C-19),22.10(C-16),17.81 (C-20). Identified as cajanin.
Compound 4: a white powder;1H NMR(400MHz,CDCl3):δH 7.51(2H,J=6.8Hz,H-2'/6'),7.35(2H,J=6.8Hz,H-3'/5'),7.34(1H,m,H-4'),7.03(2H,d,J=3.7Hz,H-4/6),7.26(1H,d,J=14.6Hz,H-7),6.64(1H,d,J=14.6Hz,H-8),5.24(1H,t,J=7.2Hz,H-2″),3.87(3H,s,-OCH3),3.40(2H,d,J=7.2Hz,H-1″),1.82(3H,s,H-4″),1.74(3H,s,H-5″);13C NMR(101MHz,CDCl3):δC 158.07(C-3),155.48(C-5),137.27(C-1'),136.64(C-1),134.52(C-3″),128.67(C-7),128.64(C-3',5'),128.36(C-8),127.5(C-4')7,126.48(C-2',6'),121.82(C-2″),115.01(C-2),107.13(C-6),101.56(C-4),55.82(3-CH3) 25.83(C-1 '), 22.37(C-4 '), 17.87(C-5 '). Identified as cajanin C.
Compound 5: a yellow powder;1H NMR(400MHz,CDCl3):δH 11.62(br,s),7.29(2H,m,H-11/13),7.21(2H,m,H-10/14),7.20(1H,m,H-12),6.20(1H,s,H-6),5.20(1H,t,J=7.2Hz,H-17),3.96(3H,s,-OCH3),3.34(2H,d,J=7.2Hz,H-16),3.26(2H,m,H-7),2.92(2H,m,H-8),1.79(3H,s,H-19),1.68(3H,s,H-20);13C NMR(101MHz,CDCl3):δC 175.29(C-15),163.03(C-5),162.25(C-3),145.79(C-9),142.04(C-1),131.90(C-18),128.66(C-11,13),128.50(C-12),126.09(C-10,14),122.35(C-17),115.52(C-4),106.57(C-6),103.77(C-2),55.70(5-OCH3),39.36(C-7),38.32(C-8),25.97(C-CH3) 22.13(C-16),17.94 (C-19). Identified as 2-hydroxy-4-methoxy-3-phenyl-6-phenylenebenzoic acid.
Novel coronavirus main protease inhibitory activity test
(1) Preparation of samples and reagents
Sample solution: the compounds 1-5 were dissolved in DMSO to prepare a sample stock solution with a concentration of 32 mM. Diluting the mother liquor, and preparing to-be-detected sample solutions with the concentrations of 100, 33, 11, 3.7 and 1.23 mu g/mL in sequence for later use.
Positive control: ebselen (Ebselen) was dissolved in DMSO to prepare a drug stock solution with a concentration of 10mM, and the drug stock solution was diluted to prepare drug solutions with concentrations of 5, 2.5, 1.25, 0.625, and 0.3125 μ M in this order for use.
Enzyme solution: according to the volume ratio of 1:92 (v: v), the 2019-nCoV Mpro/3CLproThe protease is uniformly mixed in the Assay Buffer, and is stored at low temperature and is ready for use.
2019-nCoV Mpro/3CLproThe protease is a new type of natural coronavirus main protease (main protease, abbreviated as M)proAlso known as 3C-like protease or 3CLpro) A novel coronavirus M having the same amino acid sequence as that of (A)pro/3CLproProtease without any additional tags and amino acids.
Substrate (Substrate): MCA fluorogenic substrates, i.e. MCA-AVLQSGFR-Lys (Dnp) -Lys-NH2(coronavirus main protease fluorogenic substrate).
(2) Principle of testing
MCA is a fluorescence Donor (Donor), Dnp is a fluorescence Acceptor (Acceptor) or a quenching group (Quencher), absorption spectra of the two fluorophores have certain overlap, and when the distance between the two fluorophores is within a certain range (generally 7-10 nm), fluorescence energy is transferred from the Donor to the Acceptor, so that the fluorescence intensity of the Donor fluorescent molecule is attenuated.
MCA and Dnp are connected to 2019-nCoV Mpro/3CLproFormation of MCA-AVLQSGFR-Lys (Dnp) -Lys-NH on the natural Substrate (Substrate) of the protease2A substrate. When 2019-nCoV Mpro/3CLproWhen the substrate is not cleaved by the protease, the two groups are close enough for fluorescence resonance energy transfer to occur, i.e., Dnp can quench MCA fluorescence resulting in fluorescence not being detected; when the substrate is 2019-nCoV Mpro/3CLproAfter protease cleavage, the head and tail ends of the polypeptide are separated, the two groups are separated, MCA fluorescence is not quenched by Dnp any more, and then MCA fluorescence can be detected, so 2019-nCoV M can be detected very sensitively through fluorescence detectionpro/3CLproThe enzymatic activity of the protease. If 2019-nCoV M is added into the reaction systempro/3CLproThe generation of fluorescence is suppressed, and the fluorescence intensity is inversely proportional to the suppression effect of the Inhibitor, so that 2019-nCoV M can be detectedpro/3CLproInhibitory effect of protease inhibitors.
The maximum excitation wavelength of MCA was 325nm and the maximum emission wavelength was 393 nm.
(3) Determination of sample inhibitory Activity
Using a 96-hole blackboard, setting each group according to the table 1, sequentially adding the corresponding components, and uniformly mixing. Each group was provided with 3 parallel holes. Incubate at 37 ℃ for 10min to allow the enzyme to act sufficiently with the compound. Then, 2. mu.L of Substrate (Substrate) was quickly added to each well, mixed well, incubated at 37 ℃ in the dark for 10 min. And detecting the fluorescence value of each hole at the excitation wavelength of 325nm and the emission wavelength of 393nm of the multifunctional microplate reader.
TABLE 1
The mean fluorescence values for each sample well and control well were calculated and scored as RFU blank, RFU 100% enzyme activity control, RFU positive control, and RFU sample, respectively. RFU, Relative Fluorescence Unit. The inhibition rate of the sample was then calculated according to the following formula: inhibition (%) - (RFU 100% enzyme activity control-RFU sample)/(RFU 100% enzyme activity control-RFU blank) × 100%. Finally, IC is obtained through IBM SPSS statics 26 software50The value is obtained. The test results are finally shown in table 2.
TABLE 2
Compound (I) | IC50±SD(μM) |
1 | 2.443±0.867 |
2 | >100 |
3 | >100 |
4 | >100 |
5 | >100 |
Ebselen | 0.468±0.008 |
The test data of the compound 1, such as fluorescence value and inhibition rate, are shown in the following table 3:
TABLE 3
As can be seen from Table 2, compounds 2 to 5 did not inhibit 2019-nCoV Mpro/3CLproProtease activity, whereas Compound 1 shows better inhibitory Activity, IC50The value was 2.443. + -. 0.867. mu.M. Therefore, the compound 1 has wide application prospect in inhibiting coronavirus, including but not limited to preparing the compound into a medicament for treating diseases caused by coronavirus.
The foregoing is directed to preferred embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.
Claims (8)
2. use according to claim 1, characterised in that the M is a member of the groupproProtease and/or 3CLproThe protease dominated virus is a coronavirus.
3. Use according to claim 1, characterized in that the coronavirus is selected from the group consisting of the novel coronavirus (2019-nCoV), the human coronavirus OC43(HCoV-OC43), the human coronavirus HKU1(HCoV-HKU1), the middle east respiratory syndrome coronavirus (MERS-CoV) and the SARS virus (SARS-CoV).
4. The use according to any one of claims 1 to 3, wherein the disease is acute or chronic pneumonia.
5. For the prevention and/or treatment of a disease caused by MproProtease and/or 3CLproA medicament for treating a disease caused by infection with a propagating virus by a protease, comprising a compound of formula i.
6. The medicament of claim 5, further comprising one or more of pharmaceutically acceptable carriers, excipients and excipients.
7. The medicament of claim 5 or 6, wherein the medicament is in a dosage form selected from the group consisting of capsules, granules, tablets, pills, sprays, suppositories, aerosols, dusts, patches, oral liquids, and injections.
8. A process for the preparation of a compound of formula i for use in the preparation of a medicament as claimed in claim 5, which comprises the steps of:
pulverizing semen Cajani branches and leaves, soaking and extracting with ethanol, and concentrating under reduced pressure to obtain thick total extract; extracting the total extract with water and ethyl acetate, stirring, standing for layering, removing water layer, and concentrating ethyl acetate layer under reduced pressure to obtain ethyl acetate extract; performing MCI column chromatography on the ethyl acetate part extract, and performing extraction with ethanol: performing gradient elution with water (v/v) as mobile phase, wherein the gradient is 50% ethanol, 70% ethanol and 95% ethanol respectively, and collecting 70% ethanol flow section; the 70% ethanol stream was subjected to silica gel column chromatography using a petroleum ether: performing gradient elution by taking ethyl acetate (v/v) as a mobile phase, wherein the gradient is set to be 50:1, 25:1, 10:1, 5:1, 2:1 and 1:1 in sequence; carrying out Sephadex LH-20 gel column chromatography on the 2:1 flow section to remove pigments; the 2:1 fraction was then purified by preparative HPLC to give the compound of formula I.
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