CN112142826A - Rubia cordifolia cyclopeptide compound and preparation method thereof - Google Patents
Rubia cordifolia cyclopeptide compound and preparation method thereof Download PDFInfo
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- C07K9/00—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof
- C07K9/006—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof the peptide sequence being part of a ring structure
- C07K9/008—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof the peptide sequence being part of a ring structure directly attached to a hetero atom of the saccharide radical, e.g. actaplanin, avoparcin, ristomycin, vancomycin
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Abstract
The invention discloses a rubia cordifolia cyclopeptide compound and a preparation method thereof. The preparation method of the rubia cordifolia cyclopeptide compound comprises the following steps: taking stem and/or leaf of Rubia plants of Rubiaceae as raw material, obtaining extract, and separating and purifying the obtained extract with macroporous resin column, silica gel column, and high speed countercurrent chromatography. The test result of the applicant shows that the compound has stronger binding capacity with coronavirus targets (S protein, 3CL protein, DC-SIGN receptor protein, ACE2 receptor protein), AIDS virus targets (HIV-1 protein) and hepatitis B virus targets (Bcl-2, Bcl-xL), has potential medicinal value for novel coronavirus, AIDS virus and hepatitis B virus, and is expected to be used for preparing anti-coronavirus medicines and/or anti-AIDS virus and/or anti-hepatitis B virus medicines.
Description
Technical Field
The invention relates to an active ingredient extracted and separated from plants, in particular to an madder cyclopeptide compound extracted from a madder plant of rubiaceae, namely YOUMA, and a preparation method thereof.
Background
Cyclic peptides are a class of cyclic compounds formed primarily by amino acids joined by peptide bonds. The macrocyclic molecule, cyclic peptide, has a well-defined fixed conformation that fits well with receptors, and the absence of free amino and carboxy termini in the molecule greatly reduces the sensitivity to aminopeptidases and carboxypeptidases. In general, the metabolic stability and bioavailability of cyclic peptides are much higher than that of linear peptides. At present, about 500 kinds of cyclic peptide compounds are found in nature, and many cyclic peptides have good biological activity.
Rubiaceae-type cyclopeptides (rubicin cyclopeptides) are a class of bicyclic or monocyclic cyclohexapeptides found in Rubia cordifolia, and currently, about 40 of the Rubiaceae-type cyclopeptide compounds have been found, including monoglycoside, dimer, and the like, such as Yayehuanin A, RA-V, RA-II, RA-VII, RA-I, RA-III, RA-X, RA-XI, RA-XII, RA-XVI, RY-II, and the like. The rubiaceae cyclopeptide has good biological activity on inflammatory diseases, immune diseases, tumor diseases and herpes viruses, and has high medical application value. The representative is as follows:
the invention patent with the publication number CN104829696A discloses the application of rubicordilin A, rubicordilin B and rubicordilin C, which are rubicin-type cyclopeptide compounds, in the preparation of medicines for treating related inflammation and cancers with abnormally activated NF-kappa B signaling pathways.
The invention patent with the publication number of CN109134618A discloses madder cyclopeptide diglycoside Yayehuanganin A extracted and separated from madder Yayehua (Rubia hangii), and finds that the compound can be used for preparing medicines for treating or preventing inflammatory diseases, immune diseases and cancers related to abnormal activation of NF-kB signaling pathway.
The invention patent with publication number CN103877562A discloses the application of rubiaceae cyclic peptide compounds RA-V, RA-I, RA-VII, RA-XII and the like in the preparation of drugs for treating cancers related to abnormal activation of Hedgehog signaling pathway.
The invention patent with publication number CN101921319A discloses the application of rubiaceae cyclic peptide compounds RA-V, RA-I, RA-XX IV, RA-XII and the like in the preparation of medicines for treating herpes simplex virus I related diseases.
At present, no report related to potential drugs for preventing and treating coronavirus diseases, drugs for preventing and treating AIDS and drugs for preventing and treating hepatitis B exists in madder cyclopeptide compounds.
Disclosure of Invention
The invention aims to solve the technical problem of providing a rubia cordifolia cyclopeptide compound with a novel structure and a preparation method thereof.
The madder cyclopeptide compound is a compound with a structure shown in the following formula (I) or a pharmaceutically acceptable salt thereof:
the molecular formula of the compound with the structure shown in the formula (I) is C52H68N6O21Accurate molecular weight 1112.4438, HRESIMS [ M-H ]]-1111.4423，[M+H]+1113.4534 average molecular weight 1113.1. The physical and chemical properties are as follows: white amorphous powder, soluble in methanol, ethanol, pyridine, water, insoluble in cyclohexane and petroleum ether. Named Yayehuanin B in this application, is also referred to as Compound 1 in this application.
The preparation method of the compound with the structure shown in the formula (I) mainly comprises the following steps:
1) taking stems and/or leaves of Rubia plants of Rubiaceae genus YOUYEHUANGYI (Rubia hangii) as raw materials to obtain extract;
2) passing the obtained extract through a macroporous resin column, and separating by volume ratio of 0: 100-100: 0, low carbon alcohol/water gradient elution, and collecting the mixture with the volume ratio of 90: 10-100: eluting the part with 0 of low carbon alcohol/water, and recovering the solvent to obtain a purified product A;
3) the purified product A is applied to a silica gel column and is prepared by mixing the following components in a volume ratio of 100:0 to 0:100 chloroform/methanol gradient elution, collecting volume ratio 80:20, recovering the solvent from the chloroform/methanol elution part to obtain a purified product B;
4) separating the purified product B by high-speed countercurrent chromatography, collecting fractions by stages, detecting and identifying (by UPLC-MS) and combining the fractions, and recovering solvent to obtain compound with structure shown in formula (I); wherein the content of the first and second substances,
the stationary phase and the mobile phase when the high-speed countercurrent chromatography separation is carried out are two-phase systems which are composed of water and one or more than two of the following organic solvents and have a distribution coefficient of the rubia-type cyclopeptide compound of 0.2-10:
ester solvents, alcohol solvents, ketone solvents, ether solvents, halogenated hydrocarbon solvents, alkane solvents, nitrile solvents, and acid solvents.
In step 1) of the above preparation method, branch and/or leaf of the genus rubia of the family rubiaceae is/are usually extracted with water and/or low carbon alcohol as a solvent to obtain an extract of the genus rubia; wherein said branches and/or leaves of said Yttrium acremonium can be fresh or dried. The lower alcohol can be methanol and/or ethanol. The concentration of the methanol and the ethanol can be 10-100 v/v%, preferably 50-100 v/v%. The operation (such as the manner of extraction, the number of times of extraction, and the time for each extraction) and the like in the extraction are the same as those in the prior art, and it is preferable to use heat extraction or reflux extraction. Concentrating the obtained extracting solution to remove the solvent to obtain an extract which is the extract of herba Yttuyniae.
In order to reduce the burden of the subsequent macroporous resin, the extract obtained in the step 1) is preferably subjected to extraction and impurity removal and then is subjected to enrichment by a macroporous resin column. The extraction operation is to extract the extract with petroleum ether or n-hexane to remove impurities such as chlorophyll and sterol.
In the step 2) of the preparation method, the macroporous resin is medicinal grade macroporous adsorption resin capable of adsorbing and separating rubiadin, and the preferable model is Amberlite XAD16, X-5, AB-8 or DA101 and the like. When the macroporous resin column is eluted, the collection volume ratio is preferably 100:0 lower alcohol/water elution fraction. The lower alcohol in this step may be methanol or ethanol. Before eluting the resin column with the mixed solvent of lower alcohol and water, the column is preferably washed with water to remove some water-soluble impurities.
In the step 4) of the preparation method, among the solvents of the stationary phase and the mobile phase when the high-speed countercurrent chromatography is carried out, the ester solvent is preferably one or a combination of more than two selected from ethyl acetate, butyl acetate and amyl acetate; the alcohol solvent is preferably one or the combination of more than two selected from methanol, ethanol, propanol, isopropanol, n-butanol, isobutanol, n-pentanol and 2-pentanol; the ketone solvent is preferably one or the combination of more than two of acetone, butanone, cyclopentanone, methyl isobutyl ketone and cyclohexanone; the ether solvent is preferably one or the combination of more than two of diethyl ether, methyl tert-butyl ether, dioxane and tetrahydrofuran; the halogenated hydrocarbon solvent is preferably one or the combination of more than two of dichloromethane, trichloromethane and carbon tetrachloride; the alkane solvent is preferably one or a combination of more than two of n-hexane, n-heptane, isooctane, petroleum ether (including petroleum ether with a boiling range of 30-60 ℃ and petroleum ether with a boiling range of 60-90 ℃) and gasoline; the nitrile solvent is preferably acetonitrile; the acid solvent is preferably selected from formic acid and/or acetic acid.
In the step 4) of the preparation method, the proportion of the high-speed countercurrent chromatography two-phase solvent system is related to the proportion of the two phases, so the total composition proportion of the two-phase solvent can be changed according to the proportion of the upper phase and the lower phase without influencing the composition of the upper phase or the composition of the lower phase and the distribution coefficient of the two-phase solvent. The test results of the applicant show that the stationary phase and the mobile phase for performing the high-speed countercurrent chromatography are preferably selected from a two-phase system consisting of ethyl acetate-alcohol-water, wherein the alcohol is one or a combination of two or more selected from methanol, ethanol, propanol, isopropanol, n-butanol, isobutanol, n-pentanol and 2-pentanol. More preferably, the alcohol is n-butanol, and in this case, the volume ratio of ethyl acetate, n-butanol and water is preferably 3:1: 4.
in the step 4) of the preparation method, the solvent system for high-speed countercurrent chromatography separation can be a solvent system with an upper phase as a stationary phase and a lower phase as a mobile phase; alternatively, the lower phase may be a stationary phase and the upper phase a mobile phase. When the separation is carried out by a high-speed counter-current chromatograph, the temperature is usually 10-35 ℃, the main machine can rotate forwards or backwards, the rotating speed is preferably 750-900 rpm, and the flow rate is preferably 0.5-15.0 mL/min. The operations of preparation, sample loading, sectional collection and the like of taking a sample to be separated by the high-speed countercurrent chromatography are the same as those of the prior art. The following are preferred:
preparation of a sample to be separated by high-speed countercurrent chromatography: weighing the purified product B obtained in the step 3), taking the upper phase solution or the lower phase solution or both the upper phase solution and the lower phase solution, dissolving the dried product with the volume equivalent to 100 times of the mass number of the dried product, and centrifuging to obtain the supernatant.
Taking 10-17 mL of a sample to be separated, the volume of which is not more than the volume of a sample inlet cup of the high-speed countercurrent chromatograph, injecting the sample into the high-speed countercurrent chromatograph, and collecting 1 fraction every 5-15 mL; and (3) detecting by UPLC-MS, combining the pure fractions of the cyclic peptide compound, and recovering the solvent under reduced pressure to obtain a counter-current chromatography purified product.
Compared with the prior art, the madder cyclopeptide Yayehuanin B is separated from the branches and leaves of the wild aconite bracteata of the madder for the first time by adopting high-speed countercurrent chromatography combination, and the test result of the applicant shows that the compound has stronger combination capacity with coronavirus targets (S protein, 3CL protein, DC-SIGN receptor protein, ACE2 receptor protein), HIV-1 protein and hepatitis B virus targets (Bcl-2 and Bcl-xL), has potential medicinal value for novel coronavirus, HIV and hepatitis B virus, and is expected to be used for preparing anti-coronavirus medicines and/or anti-AIDS virus medicines and/or anti-hepatitis B virus medicines.
Detailed Description
The present invention will be better understood from the following detailed description of specific examples, which should not be construed as limiting the scope of the present invention.
Example 1: preparation of the target Compound
1) Taking dried stems and leaves (15.0kg) of Rubia genus Yamada (Rubia hangii), crushing, extracting with 80 v/v% methanol under reflux for 3 times (60L multiplied by 3 times) for 3 hours each time, and concentrating the extract under reduced pressure to obtain an extract;
2) adding 15L methanol/water (the volume ratio of methanol to water is 50:50) into the obtained extract, suspending, and extracting with petroleum ether for 3 times, wherein the amount of extraction solvent is 15L each time. Extracting, collecting methanol/water phase (lower phase), filtering, removing organic solvent under reduced pressure, adding water 9L and methanol 3L, and purifying with XAD-16 macroporous resin column
After this, the column was washed with water, then with 30 v/v% methanol and finally with 100 v/v% methanol. Collecting 100 v/v% methanol elution part, and concentrating under reduced pressure to obtain Fr.2 (purified product A);
3) and mixing the Fr.2 with silica gel, performing silica gel column chromatography, and performing silica gel column chromatography by using a solvent with a volume ratio of 100: 0-0: 100 chloroform/methanol gradient elution (100:0, 95:5, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 0:100, volume ratio), LC-MS detection method of conjugated cyclopeptide, wherein the 80:20 elution part is determined as the part containing the cyclopeptide diglycoside Yayehuanin A and Yayehuanin B, collecting and merging the corresponding parts, concentrating to dryness, and obtaining Fr.2-2 (namely, purified product B).
3) High-speed countercurrent chromatographic separation of Fr.2-2
3.1) measuring 600mL of ethyl acetate, 200mL of n-butanol and 800mL of pure water, preparing a mixed solution with a volume ratio of 3:1:4, fully shaking, standing for 0.5h, completely layering and clarifying an upper layer and a lower layer, separating the upper layer and the lower layer to obtain an upper phase solution and a lower phase solution, and respectively carrying out ultrasonic degassing for 10min for later use;
3.2) taking Fr.2-2, taking 15mL of the lower phase solution obtained in the step 3.1), dissolving Fr.2-2, centrifuging the solution, and taking the supernatant for later use;
3.3) taking the upper phase solution obtained in the step 3.1) as a stationary phase, pumping into a separation tube of a high-speed counter-current chromatograph, adjusting a main machine to rotate forward after the stationary phase is filled in the whole separation tube to reach the maximum rotation speed (900r/min), simultaneously pumping the lower phase solution obtained in the step 3.1) as a mobile phase, taking 15mL of supernatant obtained in the step 3.2) after the mobile phase flows out from a column port and the stationary phase does not flow out, and injecting a two-phase solvent into a sample injection valve after the two-phase solvent reaches dynamic balance in the separation tube, detecting at the wavelength of 210nm under the condition of the flow rate of 2mL/min, separating a sample, wherein the total separation time is 350min, and 1 fraction is collected every 7 min. And (3) carrying out spin-drying on each fraction by using a vacuum centrifugal concentrator, respectively taking each spin-dried fraction, dissolving the fractions by using chromatographic alcohol methanol, and detecting by using UPLC-MS. Wherein the fraction 18-19 is molecular weight [ M-H ]]-1095 the content of the compound was 95.2% by HPLC as a white solid, 53.8mg by total weight, which was confirmed to be Yayehuanin A by 1HNMR and 13 CNMR. Fraction 21 is divided intoQuantum amount [ M-H]-1111 of the compound (Yayehuanin B) was detected by HPLC to be 93.8% as white powder, and the total weight was 3.4 mg.
For the molecular weight [ M-H ] obtained in this example]-A 1111 white powder was characterized:
yayehuanin B: white amorphous powder; UV (MeOH) λ max 215nm (shoulder), 274 nm;1h (500MHz) and13c (125MHz) NMR data are shown in Table 1; electrospray high-resolution mass spectrum (negative ion mode) HRESIMS M/z 1111.4458[ M-H [ ]]-, formula C52H68N6O20Calculated 1111.4488, unsaturation 22.
Table 1: molecular weight [ M-H]-Of 1111 white powders1H NMR and13c NMR data
Note: the solvent is deuterated methanol, the hydrogen spectrum is 500MHz, and the carbon spectrum is 125MHz
Thus, the molecular weight [ M-H ] obtained in this example was determined]-The 1111 white powder is Yayehuanin B, the structure is shown as the following formula, and the connection sequence of the structures obtains the confirmation of high resolution tandem mass spectrum ESI/MS/MS:
example 2: preparation of the target Compound
Example 1 was repeated, except that,
in the step 1), the extraction solvent is changed into 70 v/v% ethanol; the extract was suspended with 15L ethanol/water (50: 50 volume ratio of ethanol to water) before extraction with petroleum ether.
In the step 2), the model of the macroporous resin is changed to X-5, after the macroporous resin is loaded on the column, the column is washed by water, then eluted by 20 v/v% ethanol, and finally eluted by 100 v/v% ethanol. Collect 100 v/v% ethanol elution site.
In the step 3.1), 600mL of n-hexane, 900mL of ethyl acetate, 600mL of methanol and 900mL of pure water are measured, a mixed solution with the volume ratio of 2:3:2:3 is prepared, the mixed solution is fully shaken and then stands for 0.5h, the upper layer and the lower layer are completely layered and clarified, the upper layer and the lower layer are separated to obtain an upper phase solution and a lower phase solution, and ultrasonic degassing is respectively carried out for 10min for later use.
Finally obtaining the fraction 18-19 with molecular weight [ M-H ]]-1095 the content of the compound was 95.0% by HPLC as a white solid, and the total weight was 48.7mg, which was confirmed to be Yayehuanin A by 1HNMR and 13 CNMR. Fraction 21 is molecular weight [ M-H ]]-1111 of the compound, detected by HPLC, in an amount of 91.7% as a white powder, with a total weight of 3.2 mg.
For the molecular weight [ M-H ] obtained in this example]-1111 of white powder is respectively characterized by ultraviolet and nuclear magnetic resonance hydrogen spectra, carbon spectra and electrospray high-resolution mass spectra, and is determined to be the target compound Yayehuanin B of the application.
Example 3: preparation of the target Compound
Example 1 was repeated, except that,
in step 1), the extraction solvent is changed into water.
In the step 2), the model of the macroporous resin is changed to DA101, after the column is loaded, the column is washed by water, then is eluted by 50 v/v% methanol, and finally is eluted by 90 v/v% methanol. 90 v/v% methanol elution fractions were collected.
Finally obtaining the fraction 18-19 with molecular weight [ M-H ]]-1095 the content of the compound was 94.6% by HPLC as a white solid, and the total weight was 45.3mg, which was confirmed to be Yayehuanin A by 1HNMR and 13 CNMR. Fraction 21 is molecular weight [ M-H ]]-1111 of the compound, detected by HPLC, in an amount of 92.3% in the form of a white powder, weighing 3.3 mg.
For the molecular weight [ M-H ] obtained in this example]-1111 white powder is respectively characterized by ultraviolet and nuclear magnetic resonance hydrogen spectrum, carbon spectrum and electrospray high-resolution mass spectrum, and is determined as the applicationThe target compound Yayehuanin B.
Example 4: molecular docking of compound Yayehuanin B and S protein
The molecular structure of the compound Yayehuanin B was plotted using Kingdraw software and stored in mol format, and then converted to mol2 format using Open babel software and stored. Thereafter, the 3D conformation was optimized with the avigadro software and saved in mol2 format, followed by viewing of the 3D structure with pymol software and saved in pdb format. And finally, distributing related charges through Autodock Tools, automatically setting a twistable key, and exporting the twistable key into a pdbqt format file.
The S protein adopts a pdbqt format file downloaded from a new coronavirus drug target structure information sharing platform system in Qingdao ocean science and technical pilot national key laboratories, and autodock vina is operated to carry out molecular docking, wherein docking parameters are as follows:
center_x＝-32.81
center_y＝25.66
center_z＝4.79
size_x＝23
size_y＝21
size_z＝21
seed＝2020
energy_range＝5
exhaustiveness＝400
num_modes＝20
the docking results are shown in table 2 below.
Table 2: molecular docking result of Yayehuanin B and new coronavirus potential target, hepatitis B virus potential target and AIDS virus potential target
Example 5: molecular docking of compound Yayehuanin B with Ace2 receptor protein (binding region)
The pdbqt format molecular structure of compound Yayehuanin B was the same as that stored in example 4.
The Ace2 receptor protein adopts pdb format files downloaded from a new coronavirus drug target structure information sharing platform system in key laboratories of Qingdao ocean science and technology pilot countries and combined with the Ace2 receptor protein and the S protein, deletes the S protein, and hydrogenates and distributes related charges through Autodock Tools. The added atom type is AD4, and the added atom type is exported to be a pdbqt format file. The RBD binding site of the pro Ace2 receptor protein to S protein was selected as the docking active pocket.
And (3) operating autodock vina to carry out molecular docking, wherein docking parameters are as follows:
center_x＝-32.81
center_y＝25.66
center_z＝4.79
size_x＝23
size_y＝21
size_z＝21
seed＝2020
energy_range＝5
exhaustiveness＝400
num_modes＝20
the docking results are shown in table 2 above.
Example 6: molecular docking of compound Yayehuanin B and new coronavirus 3CL protein
The pdbqt format molecular structure of compound Yayehuanin B was the same as that stored in example 4.
The 3CL protein adopts a pdbqt format file downloaded from a new coronavirus drug target structure information sharing platform system in Qingdao ocean science and technical pilot national key laboratories, autodock vina is operated to carry out molecular docking, and docking parameters are as follows:
center_x＝-9.267
center_y＝11.516
center_z＝68.612
size_x＝19.228
size_y＝28.944
size_z＝20.254
seed＝2020
energy_range＝5
exhaustiveness＝400
num_modes＝20
the docking results are shown in table 2 above.
Example 7: molecular docking of compound Yayehuanin B and new coronavirus receptor DC-SIGN protein
The pdbqt format molecular structure of compound Yayehuanin B was the same as that stored in example 4. DC-SIGN proteins were downloaded from the RCSD protein database (https:// www.rcsb.org /) (PDB ID: 6 GHV). The water molecules of the crystal structure and the original ligand EZ8 were deleted using pymol software and the associated charges were distributed by Autodock Tools hydrogenation. The added atom type is AD4, and the added atom type is exported to be a pdbqt format file. The pro-ligand inhibitor EZ8 was selected as the docking active pocket at the binding site of the DC-SIGN protein F chain.
And (3) operating autodock vina to carry out molecular docking, wherein docking parameters are as follows:
center_x＝4.799
center_y＝9.951
center_z＝79.263
size_x＝22.5
size_y＝22.5
size_z＝22.5
seed＝2020
energy_range＝5
exhaustiveness＝400
num_modes＝20
the docking results are shown in table 2 above.
Example 8: molecular docking of compound Yayehuanin B with HIV-1 protease the pdbqt-formatted molecular structure of compound Yayehuanin B was as described in example 4.
The PDB structure of the HIV-1 protease was downloaded from the RCSD protein database (https:// www.rcsb.org /) (PDB ID: 1 HSG). The water molecules of the crystal structure and the protoligand MK1 were deleted in pymol software and the associated charges were added by Autodock Tools and assigned. The added atom type is AD4, and the added atom type is exported to be a pdbqt format file. The pro-ligand inhibitor MK1 was chosen to act as a docking active pocket at the binding site of the HIV-1 protease.
And (3) operating autodock vina to carry out molecular docking, wherein docking parameters are as follows:
center_x＝16
center_y＝25
center_z＝4
size_x＝30
size_y＝30
size_z＝30
seed＝2020
energy_range＝5
exhaustiveness＝400
num_modes＝20
the docking results are shown in table 2 above.
Example 9: molecular docking of compound Yayehuanin B and hepatitis B virus Bcl-2 protein
The pdbqt format molecular structure of compound Yayehuanin B was the same as that stored in example 4.
PDB structures of Bcl-2 proteins were downloaded from the RCSD protein database (https:// www.rcsb.org /) (PDB ID: 6gl 8). The water molecules of the crystal structure and the protoligand F3Q were deleted using pymol software and the associated charges were distributed by Autodock Tools hydrogenation. The added atom type is AD4, and the added atom type is exported to be a pdbqt format file. Proligand inhibitor F3Q was selected as the docking active pocket at the binding site of the Bcl-2 protein.
And (3) operating autodock vina to carry out molecular docking, wherein docking parameters are as follows:
center_x＝17.806
center_y＝2.92
center_z＝15.208
size_x＝15
size_y＝15
size_z＝18.75
seed＝2020
energy_range＝5
exhaustiveness＝400
num_modes＝20
the docking results are shown in table 2 above.
Example 10: molecular docking of compound Yayehuanin B and hepatitis B virus Bcl-xL protein
The pdbqt format molecular structure of compound Yayehuanin B was the same as that stored in example 4. PDB structures of Bcl-xL proteins were downloaded from the RCSD protein database (https:// www.rcsb.org /) (PDB ID: 3sp 7). The water molecules of the crystal structure and the primary ligand 03B were deleted using pymol software and the associated charges were distributed by Autodock Tools hydrogenation. The added atom type is AD4, and the added atom type is exported to be a pdbqt format file. Proligand inhibitor 03B was selected as the docking active pocket at the binding site of the Bcl-xL protein.
And (3) operating autodock vina to carry out molecular docking, wherein docking parameters are as follows:
center_x＝-3.361
center_y＝5.194
center_z＝-5.139
size_x＝28.5
size_y＝28.5
size_z＝15
seed＝2020
energy_range＝5
exhaustiveness＝400
num_modes＝20
the docking results are shown in table 2 above.
Claims (9)
1. A compound having a structure represented by the following formula (I):
2. a process for the preparation of a compound according to claim 1, essentially comprising the steps of:
1) taking stem and/or leaf of Rubia of Rubiaceae as raw material to obtain extract;
2) passing the obtained extract through a macroporous resin column, and separating by volume ratio of 0: 100-100: 0, low carbon alcohol/water gradient elution, and collecting the mixture with the volume ratio of 90: 10-100: eluting the part with 0 of low carbon alcohol/water, and recovering the solvent to obtain a purified product A;
3) the purified product A is applied to a silica gel column and is prepared by mixing the following components in a volume ratio of 100:0 to 0:100 chloroform/methanol gradient elution, collecting volume ratio 80:20, recovering the solvent from the chloroform/methanol elution part to obtain a purified product B;
4) separating the obtained purified product B by a high-speed counter-current chromatograph, collecting fractions in sections, identifying and combining the fractions, and recovering the solvent to obtain a compound with a structure shown in the formula (I); wherein the content of the first and second substances,
the stationary phase and the mobile phase when the high-speed countercurrent chromatography separation is carried out are two-phase systems which are composed of water and one or more than two of the following organic solvents and have a distribution coefficient of the rubia-type cyclopeptide compound of 0.2-10:
ester solvents, alcohol solvents, ketone solvents, ether solvents, halogenated hydrocarbon solvents, alkane solvents, nitrile solvents, and acid solvents.
3. The preparation method according to claim 2, wherein in the step 1), the stalk and/or leaf of the illicium henryi of rubiaceae is used as a raw material, and water and/or low carbon alcohol is used as a solvent for extraction to obtain the illicium henryi extract.
4. The production method according to claim 3, characterized in that: in the step 1), the lower alcohol is methanol and/or ethanol.
5. The preparation method as claimed in claim 2, wherein in step 2), the macroporous adsorbent resin is Amberlite XAD16, X-5, AB-8 or DA 101.
6. The method according to claim 2, wherein in the step 2), the lower alcohol is methanol or ethanol.
7. The method according to claim 2, wherein in the step 4),
the ester solvent is one or the combination of more than two of ethyl acetate, butyl acetate and amyl acetate;
the alcohol solvent is one or the combination of more than two of methanol, ethanol, propanol, isopropanol, n-butanol, isobutanol, n-pentanol and 2-pentanol;
the ketone solvent is one or the combination of more than two of acetone, butanone, cyclopentanone, methyl isobutyl ketone and cyclohexanone;
the ether solvent is one or the combination of more than two of diethyl ether, methyl tert-butyl ether, dioxane and tetrahydrofuran;
the halogenated hydrocarbon solvent is one or the combination of more than two of dichloromethane, trichloromethane and carbon tetrachloride;
the alkane solvent is one or the combination of more than two of n-hexane, n-heptane, isooctane, petroleum ether and gasoline;
the nitrile solvent is acetonitrile;
the acid solvent is selected from formic acid and/or acetic acid.
8. The method according to any one of claims 2 to 7, wherein in the step 4), the stationary phase and the mobile phase for the high-speed countercurrent chromatography are a two-phase system consisting of ethyl acetate-alcohol-water, wherein the alcohol is one or a combination of two or more selected from methanol, ethanol, propanol, isopropanol, n-butanol, isobutanol, n-pentanol and 2-pentanol.
9. The preparation method according to claim 8, wherein in the step 4), the stationary phase and the mobile phase for the high-speed countercurrent chromatography are a two-phase system consisting of ethyl acetate-n-butanol-water, and the volume ratio of ethyl acetate, n-butanol and water is 3:1: 4.
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| CN109134618A (en) * | 2018-09-04 | 2019-01-04 | 广西壮族自治区中国科学院广西植物研究所 | Rubiaceae type cyclic peptide bioside and its preparation method and application |
| CN112868647A (en) * | 2021-01-21 | 2021-06-01 | 广西壮族自治区中国科学院广西植物研究所 | Application of perfume compound in preparation of sanitary articles for inhibiting infection of novel coronavirus |
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| CN101921319A (en) * | 2010-07-21 | 2010-12-22 | 中国科学院昆明植物研究所 | Rubiaceae-type cyclopeptides, medicinal compositions using same as active ingredients, and preparation method and application thereof |
| CN104829696A (en) * | 2015-05-12 | 2015-08-12 | 中国药科大学 | Open-loop rubiaceae-type cyclopeptide, pharmaceutical composition taking open-loop rubiaceae-type cyclopeptide as active ingredient as well as preparation method and application of open-loop rubiaceae-type cyclopeptide |
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| CN101921319A (en) * | 2010-07-21 | 2010-12-22 | 中国科学院昆明植物研究所 | Rubiaceae-type cyclopeptides, medicinal compositions using same as active ingredients, and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109134618A (en) * | 2018-09-04 | 2019-01-04 | 广西壮族自治区中国科学院广西植物研究所 | Rubiaceae type cyclic peptide bioside and its preparation method and application |
| CN112868647A (en) * | 2021-01-21 | 2021-06-01 | 广西壮族自治区中国科学院广西植物研究所 | Application of perfume compound in preparation of sanitary articles for inhibiting infection of novel coronavirus |
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