CN112538100B - Isoquinoline alkaloid glycoside compound extracted from cortex Phellodendri and having anti-inflammatory activity, and its preparation method and application - Google Patents
Isoquinoline alkaloid glycoside compound extracted from cortex Phellodendri and having anti-inflammatory activity, and its preparation method and application Download PDFInfo
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Abstract
The invention relates to an isoquinoline alkaloid glycoside compound with anti-inflammatory activity extracted from phellodendron amurense, a preparation method and application thereof, which can effectively solve the problem of extracting isoquinoline alkaloid glycoside compound with anti-inflammatory activity from phellodendron amurense and realize the application in anti-inflammatory drugs, the compound is (1S) -1,2,3,4-tetrahydro-7-hydroxy-1- [ (4-hydroxyphenyl) methyl ] -2-methyl-8-O-isoquinonyl- [3-hydroxy-3-methylglutaryl ] -beta-D-glucopyranoside, which is named as phellodendrin A (PDA), the phellodendron amurense is extracted by a solvent, decompressed and condensed into a crude extract, the crude extract is extracted by an organic solvent in a water suspension or is eluted by macroporous resin, then the eluent of a resin column chromatography is carried out, the eluent is eluted and condensed into an extract, sequentially performing gel column chromatography and reverse column chromatography to obtain gel column chromatography isolate, i.e. isoquinoline alkaloid glycoside compounds with antiinflammatory activity; the invention has rich raw materials and simple preparation method, exploits the commercial value and the medicinal value of the phellodendron, and has obvious economic and social benefits.
Description
Technical Field
The invention relates to a medicine, in particular to an isoquinoline alkaloid glycoside compound with anti-inflammatory activity extracted from phellodendron amurense, a preparation method and application thereof.
Background
Phellodendron bark, cortex phellodendri is the dry bark of Phellodendron chinense (Phellodendron chinense) which is one of the commonly used Chinese medicines for clearing heat and removing toxicity in clinic, and has the efficacies of clearing heat and drying dampness, purging fire and removing steam, removing toxicity and curing sore. Modern pharmacological research proves that the traditional Chinese medicine composition has the effects of resisting bacteria, inflammation and fever, resisting cancers, protecting heart and cerebral vessels and the like.
Macrophages are activated by a variety of inflammatory factors, such as cytokines, bacterial Lipopolysaccharides (LPS), extracellular matrix proteins, and other chemical mediators. LPS is an important inflammation-causing factor, and can stimulate macrophages in vivo to synthesize and release various endogenous active factors, thereby inducing inflammation. The model of inflammation of RAW264.7 cells by LPS is a common model of inflammation in vitro.
At present, the non-steroidal anti-inflammatory drugs and the steroidal anti-inflammatory drugs which are commonly used clinically have good clinical application effects, but a series of side effects or adverse reactions, such as liver injury, kidney injury, gastric mucosa injury and the like, can be generated in the long-term large-scale use process, and can generate tolerance along with the increase of the use time. Therefore, the search for new anti-inflammatory drugs with high efficacy, low toxicity and even no toxicity is still one of the hot spots for anti-inflammatory drug research.
The traditional Chinese medicine has more than five thousand years of clinical application experience and is a treasure for preventing and treating diseases. The search for lead compounds from Chinese medicine has become an important source for new drug research in recent years. The invention separates an isoquinoline alkaloid glycoside compound with the effect of resisting RAW264.7 cell inflammation caused by LPS from Chinese medicinal phellodendron bark, thereby developing a new anti-inflammatory drug candidate with high efficiency and low toxicity, but no public report is found so far.
Disclosure of Invention
In view of the above situation, in order to overcome the defects of the prior art, the present invention aims to provide an isoquinoline alkaloid glycoside compound with anti-inflammatory activity extracted from phellodendron amurense, and a preparation method and an application thereof, which can effectively solve the problems of extracting the isoquinoline alkaloid glycoside compound with anti-inflammatory activity from phellodendron amurense and realizing the application thereof in anti-inflammatory drugs.
The technical scheme of the invention is that the isoquinoline alkaloid glycoside compound with anti-inflammatory activity is (1S) -1,2,3,4-tetrahydro-7-hydroxy-1- [ (4-hydroxybenzyl) methyl ] -2-methyl-8-O-isoquinonyl- [3-hydroxy-3-methylglutaryl ] -beta-D-glucopyranoside, named phellodendrin glycoside A, PDA, and has a molecular structural formula as follows:
the preparation method comprises the following steps:
(1) extracting cortex Phellodendri with solvent at a mass ratio of 1: 8-16 for 2 times, filtering, mixing filtrates, and concentrating under reduced pressure to obtain crude extract with relative density of 1.2-1.4 at 50 deg.C;
the solvent is any one of ethanol, methanol or acetone;
the extraction is cold leaching method, percolation method, microwave extraction method, ultrasonic extraction method, reflux extraction method or continuous reflux extraction method;
(2) suspending the crude extract obtained in the step (1) with water, extracting with an organic solvent, filtering the residual water solution with diatomite, and concentrating to obtain a raffinate extract with the relative density of 1.2-1.4 at 50 ℃; or carrying out macroporous resin elution on the crude extract obtained in the step (1), and concentrating the eluent to obtain a macroporous resin elution extract with the relative density of 1.2-1.4 at 50 ℃;
the organic solvent is petroleum ether, ethyl acetate and n-butanol;
the macroporous resin is one of D101, XDA-8, XDA-1 or LSA-8, and is eluted by water, 10% -95% ethanol or methanol in a gradient way, and the elution is carried out for 2-3 column volumes respectively according to the volume fraction;
(3) subjecting the extract residue or macroporous resin eluate to MCI resin column chromatography, concentrating the eluate to obtain extract with relative density of 1.2-1.4 at 50 deg.C, sequentially subjecting to gel column chromatography and reverse column chromatography, and separating with gel column chromatography to obtain isoquinoline alkaloid glycoside compounds with antiinflammatory activity;
the MCI resin column is one of GEL CHP 55A, GEL CHP 70Y, GEL CHP 2MG, GEL CHP 20 or GEL CHP 30;
the MCI column chromatography adopts water, 10-95% ethanol or methanol for gradient elution, and finally adopts a plurality of or one fixed concentration of 30-100% acetone solution for elution, and the elution is carried out for 2-3 column volumes respectively according to volume fraction;
the gel column is one of HW-40F, HW-40C or HW-75;
gradient elution is carried out on the eluent of the gel column chromatography, wherein the eluent is pure water, 10% -60% ethanol or methanol solution is subjected to gradient elution, and 1 column volume is eluted respectively according to volume fraction;
the reverse column chromatography is eluted by 20-60% methanol or ethanol solution, and the elution is carried out by 1-2 column volumes respectively according to volume fraction.
The isoquinoline alkaloid glycoside compound extracted from phellodendron amurense has the functions of inhibiting the release of NO, IL-1 beta, IL-6 and TNF-alpha in RAW264.7 cells induced by LPS, reducing the over-expression of iNOS and COX-2 proteins in RAW246.7 cells induced by LPS, showing good anti-inflammatory activity, can be used for developing anti-inflammatory related medicaments, has rich RAW materials and simple preparation method, can be effectively used for preparing anti-inflammatory medicaments, develops the commercial value and medicinal value of the phellodendron amurense, and has obvious economic and social benefits.
Drawings
FIG. 1 is a molecular structural diagram of the compound of the present invention.
FIG. 2 is a 1H-NMR spectrum of the compound of the present invention.
FIG. 3 is a drawing of a compound of the present invention13C-NMR spectrum
FIG. 4 is a graph showing the effect of compounds of the present invention on NO production.
FIG. 5 is a graph showing the effect of compounds of the present invention on IL-1 β production.
FIG. 6 is a graph showing the effect of compounds of the present invention on IL-6 production.
FIG. 7 is a graph showing the effect of compounds of the present invention on TNF- α production.
FIG. 8 is a graph showing the effect of the compounds of the present invention on the expression of iNOS proteins.
FIG. 9 is a graph showing the effect of compounds of the present invention on COX-2 protein expression.
Detailed Description
The following examples and specific examples are given to illustrate specific embodiments of the present invention.
In particular, the invention may be embodied as set forth in the following examples.
Example 1
The invention relates to a preparation method of an isoquinoline alkaloid glycoside compound with anti-inflammatory activity, which is characterized by comprising the following steps:
(1) extracting 20kg of phellodendron amurense with 10 times of ethanol solution with mass concentration of 50% at room temperature for 2 times and 7 days at each time, filtering, and concentrating the filtrate under reduced pressure to obtain a crude extract with relative density of 1.3 at 50 ℃;
(2) suspending the crude extract obtained in the step (1) with water, loading the suspension on a D101 type macroporous resin column, filling the suspension and resin according to a volume ratio of 1: 3, and sequentially using water and the following components in mass concentration: eluting with 10% ethanol, 20% ethanol, 30% ethanol, 50% ethanol, 70% ethanol, and 95% ethanol, eluting 3 column volumes per gradient in terms of mass fraction, mixing 10%, 20% and 30% ethanol elution parts, and concentrating to obtain macroporous resin elution sample with relative density of 1.3 at 50 deg.C;
(3) and (3) subjecting the macroporous resin eluted sample in the step (2) to MCI column chromatography, and sequentially treating with water and the following components in mass concentration: eluting with 20% ethanol, 30% ethanol, 50% ethanol, 70% ethanol, 30% acetone, and 60% acetone by mass fraction, wherein each gradient elutes 3 column volumes to obtain 7 fractions A, B, C, D, E, F, G; subjecting fraction G to HW-40C column chromatography, eluting with 30% ethanol and 50% ethanol by gradient, and eluting 1 column volume per gradient in terms of mass fraction; obtaining sub-flow components GA, GB and GC; eluting the sub-stream GB by reversed phase column chromatography C18 with eluent of 30% ethanol, 40% ethanol, 50% ethanol and 60% ethanol, and performing gradient elution for 2 column volumes each to obtain sub-stream GB-A, GB-B, GB-C, GB-D, GB-E; subjecting the sub-stream GB-D to HW-40C gel column chromatography, eluting with pure water and 50% methanol by mass concentration, eluting 1 column volume per gradient by mass fraction, and collecting the 50% methanol eluate fraction to obtain isoquinoline alkaloid glycoside compounds (7.8mg) with antiinflammatory activity and purity of 98.2%.
Example 2
The invention relates to a preparation method of an isoquinoline alkaloid glycoside compound with anti-inflammatory activity, which is characterized by comprising the following steps:
(1) extracting 20kg of phellodendron amurense with 14 times of acetone solution with the mass concentration of 60% at room temperature for 2 times and 6 days each time, filtering, and concentrating the filtrate under reduced pressure to obtain a crude extract with the relative density of 1.2 at 50 ℃;
(2) suspending the crude extract obtained in the step (1) by using water, sequentially extracting the crude extract by using petroleum ether, ethyl acetate and n-butanol which have the same volume at room temperature, extracting the crude extract twice by using each solvent, then combining the extracted solutions, and concentrating to obtain an extract residue with the relative density of 1.2 at 50 ℃;
(3) subjecting the raffinate obtained in the step (2) to MCI column chromatography, eluting with water, 20% ethanol by mass concentration, 30% ethanol, 50% ethanol, 70% ethanol, 60% acetone and 100% acetone in sequence, and eluting by mass fraction of 2 column volumes each to obtain 7 fractions A, B, C, D, E, F, G; subjecting the fraction G to HW-40F column chromatography under the elution conditions of 20% methanol and 50% methanol in mass concentration, and eluting for 1 column volume in mass fraction to obtain sub-fractions GA, GB, GC and GD; eluting the sub-stream GC by using reverse phase column chromatography C18, wherein the eluent comprises 30% methanol, 40% methanol, 50% methanol and 60% methanol by mass concentration, and the elution is carried out for 2 column volumes in terms of mass fraction, so as to obtain sub-stream GC-A, GC-B, GC-C, GC-D, GC-E, GC-E; subjecting sub-fraction GC-D to HW-40F gel column chromatography, eluting with pure water and 60% methanol at mass concentration, collecting 60% methanol eluate of 1 column volume by mass fraction, and collecting the eluate to obtain isoquinoline alkaloid glycoside compounds (8.9mg) with antiinflammatory activity and purity of 98.6%.
Example 3
The invention relates to a preparation method of an isoquinoline alkaloid glycoside compound with anti-inflammatory activity, which is characterized by comprising the following steps:
(1) heating and reflux-extracting 20kg of cortex Phellodendri with 10 times of methanol with mass concentration of 60% at 75 deg.C for 2 times, 40min each time, filtering, and concentrating the filtrate under reduced pressure to obtain crude extract with relative density of 1.4 at 50 deg.C;
(2) suspending the crude extract obtained in the step (1) with water, sequentially extracting for 3 times at room temperature with equal volume of ethyl acetate and n-butanol respectively, and then concentrating the extracted solution to obtain an extract residue with a relative density of 1.4 at 50 ℃;
(3) subjecting the raffinate obtained in the step (2) to MCI column chromatography, and eluting with water, 30% methanol by mass concentration, 50% methanol, 70% methanol, 30% acetone and 50% acetone in sequence to obtain A, B, C, D, E, F, wherein each elution is 2 column volumes by mass fraction; subjecting the fraction E to HW-75 column chromatography, wherein eluents comprise 20% methanol and 40% methanol in mass concentration, and each eluent is eluted by 1 column volume in mass fraction to obtain sub-fractions EA, EB and EC; subjecting the sub-stream EC to C18 reverse phase column chromatography, and eluting with methanol with mass concentration of 40%, 50% and 60% in a gradient manner, wherein each elution is 1 column volume in terms of mass fraction, to obtain sub-stream EC-A, EC-B, EC-C; subjecting the sub-flow EC-C to HW-75 gel column chromatography, eluting with pure water and 50% methanol by mass concentration, eluting 1 column volume each, and collecting 50% methanol eluate to obtain isoquinoline alkaloid glycoside compounds (8.7mg) with purity of 98.6% and antiinflammatory activity.
Use of the isoquinoline alkaloid glycosides compounds with anti-inflammatory activity described in examples 1-3 above in the preparation of anti-inflammatory drugs.
The isoquinoline alkaloid glycoside compounds with anti-inflammatory activity disclosed in the above embodiments 1 to 3 are applied to the preparation of anti-inflammatory drugs, wherein the anti-inflammatory drugs are tablets, capsules, injections, powder injections, granules, microcapsules, dropping pills, ointments or transdermal controlled release patches.
Use of the isoquinoline alkaloid glycosides compounds with anti-inflammatory activity described in examples 1-3 above in the preparation of herbal extracts or mixtures containing the compounds.
The preparation method is simple, the RAW materials are rich, the isoquinoline alkaloid glycoside compound with anti-inflammatory activity can be effectively extracted from the phellodendron, the levels of NO, IL-1 beta, IL-6 and TNF-alpha in mouse macrophage RAW246.7 caused by Lipopolysaccharide (LPS) can be obviously inhibited, the over-expression of iNOS and COX-2 protein in RAW246.7 cells induced by LPS is reduced, and a dose dependence relationship is formed, so that the compound has obvious anti-inflammatory activity and can be used for preparing anti-inflammatory drugs. And the experiment results show that the method has very good technical effect, and the related data are as follows (taking example 2 as an example):
first, structural identification
The compound is prepared by mass spectrum, nuclear magnetic resonance (MS, NMR, etc.),1H-NMR、13C-NMR, 2D-NMR) and its structure, and its molecular weight is precisely determined by high resolution mass spectrometry (HR-ESI-MS type mass spectrometer). The analytical process and spectral data are as follows:
pale yellow solid, easily soluble in water and methanol. TLC 10% sulfuric acid-ethanol is brown after heating. ESI-MS showed the peak of the excimer ion to be 592.2[ M + H ]]+Is combined with1H-NMR (FIG. 2) and13C-NMR (FIG. 3) spectrum data, and the molecular formula of the compound is determined to be C29H37NO12. It is composed of1The aromatic proton [ delta ] is shown in the H-NMR dataH7.05(d, J ═ 8.5Hz, H-5) and δH 7.00 (d,J＝8.5Hz,H-6)；δH7.18(2H, d, J-8.4 Hz, H-13, H-17) and δH6.90(2H, d, J. 8.4Hz, H-14, H-16) demonstrate the presence of a 1,2,3, 4-tetrasubstituted phenyl ring and a 1, 4-disubstituted phenyl ring. DeltaH2.78(3H, s, H-18) is the N-methyl proton signal, δH1.18(3H, s, H-6') is a monomodal methyl signal and deltaH5.10(1H, dd, J ═ 9.4,4.4Hz, H-1) is a methine signal. In addition to this, δH4.97(d, J ═ 7.0Hz,1H, H-1') is the proton signal on the anomeric carbon, δH 3.57(m,4H,H-2',3',4',5'),δH4.46(d, J ═ 11.7,1H, H-6'a) and 4.24(dd, J ═ 12.4,4.5,1H, H-6' b) are proton signals for 6 vicinal oxygens, and these data indicate that this compound is a glycoside compound.13The C-NMR spectrum showed 29 carbon signals including a glycosyl moiety (. delta.)C103.19,73.77,73.83,69.25,75.41,63.05), two phenyl rings, two carbonyl carbons (δ)C172.48,179.42). The compound was then acid hydrolyzed and subjected to TLC control with monosaccharide standards to determine that glucose was attached to the compound.
In HMBC spectrogram, C-10 is related to H-3, H-4, H-6, and C-8 is related to H-1, H-6; the correlation of C-1 and H-3, H-11 and the correlation of C-11 and H-13, H-17 prove that the compound is a benzylisoquinoline alkaloid. H-3 and H-1 with C-Me (. delta.)C40.05) showed that only one N-methyl group was present. Also, the correlation of H-1 and H-13 with C-11 indicates that the 1, 4-disubstituted benzene ring is attached at the C-1 position. From H-1' (delta)H4.97) and H-1 (. delta.))H5.10) remote correlation peak with C-8, determination of the glucose attachment position at C-8, coupling constant (J-7.0 Hz) and C-1' (δ) according to the sugar end group proton (J-7.0 Hz)C103.19) to determine the configuration of glucose as the beta configuration.
In addition to this, a monomodal methyl group (. delta.)H1.18, H-6') and 2 methylene groups (. delta.)H2.37 and 2.34; 2.57 and 2.48) with the vicinal oxygen quaternary carbon C-3' (delta)C70.2) indicates the presence of a 3-hydroxy-3-methylglutaryl group (HMG) in the molecule. At the same time, H-6' (delta)H4.46,4.24) and C-1' (delta)H172.48) indicating that the HMG group is attached at the C-6' position of glucose.
In the NOESY spectrum, H-6 (. delta.) (H7.00) correlation with H-4 indicates that the positions of C5-C6 and C7-C8 cannot be interchanged. By combining the above analysis, the structure of the compound is deduced to be (1S) -1,2,3, 4-tetrahydroxy-7-hydroxy-1- [ (4-hydroxybenzyl) methyl]-2-methyl-8-O-isoquinolinyl-[3-hy droxy-3-methylglutaryl]-beta-D-glucopyranoside, named phellodendrine glycoside (PDA).
Spectral data are shown in table 1:
TABLE 1 Popp data for compounds
a1HNMR(400MHz)and b13CNMR(100MHz)measured in D2O
The products of examples 1 and 3 were also subjected to the same structural identification, and the identification results showed that the structures were the same, (1S) -1,2,3,4-tetrahydro-7-hydroxy-1- [ (4-hydroxybenzyl) methyl ] -2-methyl-8-O-isoquinonyl- [3-hydroxy-3-methylglutaryl ] - β -D-glucopyranoside, which was named phellodendrin glycoside (PDA), and the molecular structural formula was:
second, pharmacodynamic experiment
An experimental model of LPS induced mouse mononuclear macrophage RAW264.7 inflammation is adopted to investigate the effect of the compound on inflammation.
1 method of experiment
1.1 cell culture of mouse mononuclear macrophage RAW264.7 DMEM culture solution containing 10% fetal calf serum and 100mg/L streptomycin at 37 deg.C and 5% CO2Subculturing in a constant temperature incubator.
1.2 MTT method for detecting influence of compound on viability of RAW264.7 cells in logarithmic growth phase are taken, and cell density is adjusted to 5 x 104200. mu.L/mL of each well was inoculated into a 96-well plate. The total number is 5: normal control groups, groups dosed with different concentrations of compound (5, 10, 25, 50 μmol/L), each group having 6 replicate wells. After the cells were attached, the medium was changed to DMEM with or without 5, 10, 25, 50. mu. mol/L of the compound, and MTT experiments were performed after 24h and 48h of culture. 4h before the experiment, adding 10 mu L of MTT into each well for continuingCulturing for 4h, discarding the supernatant, adding 150 μ L of dimethyl sulfoxide (DMSO) into each well, placing on a shaker, shaking for 10min, dissolving the crystal completely, and measuring absorbance A at 490 nm. The experiment was repeated 3 times. The cell viability was calculated as (a detection well/a blank well) x 100%.
1.3 Effect of compounds on LPS-induced release of NO, IL-1 β, IL-6 and TNF- α from RAW264.7 cells. Taking RAW264.7 cell suspension in logarithmic growth phase, adjusting cell density to 2 × 105each/mL, 200 μ L per well, was inoculated into 96-well plates, divided into 5 groups: normal control group, LPS group (1. mu.g/mL), administration groups of different concentrations of LPS + compound (5, 10, 25. mu. mol/L), 6 replicate wells per group. After 12h of adherence, the cells were pretreated with different concentrations (5, 10, 25. mu. mol/L) of the compounds for 1h, then treated with LPS for 24h and cultured, the supernatant of each well was collected, the content of NO in the supernatant was determined by Griess method, and the secretion of IL-1 beta, IL-6 and TNF-alpha in the supernatant was determined by ELISA kit.
Effect of Compounds on LPS-induced expression of iNOS and COX-2 proteins in RAW264.7 cells at 1X 106each/mL density was inoculated in 6-well plates with 3mL of medium per well, divided into 5 groups: normal control group, LPS group (1. mu.g/mL), administration groups of different concentrations of LPS + compound (5, 10, 25. mu. mol/L), 6 replicate wells per group. After 12h overnight incubation, after 1h pretreatment with different concentrations (5, 10, 25. mu. mol/L) of the compound, followed by 1. mu.g/mL LPS for 24h, cells were lysed on ice for 15 min with RIPA lysis buffer containing phenylmethylsulfonyl fluoride and phosphatase inhibitor. After incubation, the lysate was centrifuged at 12000rpm for 10 minutes and the supernatant was collected. Protein concentration was quantified using BCA protein assay kit. An equal amount of protein sample (40. mu.g) was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane. Membranes were dissolved in triple buffered saline with 5% dry defatted powder with 0.1% tween-20 TBST for 4 hours and blocked at room temperature. The membranes were then incubated with the corresponding antibodies overnight in a shaking incubator at 4 ℃, washed 3 times with TBST, and incubated with horseradish peroxidase (HRP) -conjugated secondary antibody for 1 hour at room temperature. After washing, detection of the test protein by chemiluminescenceThe abundance of (a).
1.4 statistical analysis SPSS 18.0 software was used, experimental results in
S, between groups, using one-way anova, the difference was statistically significant when P < 0.05, P < 0.01, P < 0.001; in comparison with the LPS group,#P＜0.05、##P＜0.01、###the difference is statistically significant when P is less than 0.001.
2 results of the experiment
2.1 Effect of Compounds on the viability of RAW264.7 cells after incubation of the compounds at different concentrations (5, 10, 25, 50. mu. mol/L) with cells for 24h and 48h, the cell viability was not reduced, and still could reach more than 95%, compared with the blank group, the difference was not statistically significant. The cell morphology was good under the microscope, no significant difference was observed, and no cytotoxic effect was observed. The results show that the concentrations of the compounds administered in the experiments were within safe concentration ranges.
TABLE 2 Effect of Compounds on 24h and 48h survival of RAW264.7 cells
2.2 Effect of the compounds on LPS-induced NO, IL-1. beta., IL-6, TNF-. alpha.release from RAW264.7 cells the effect of the compounds on the inhibition of NO release from LPS-induced RAW264.7 cells is shown in FIG. 5, where LPS treatment caused a significant increase in NO release compared to the control group, and the compound treatment inhibited LPS-stimulated NO production from RAW264.7 cells dose-dependently. The inhibition rates of the compounds on NO production at three concentrations of 5, 10 and 25. mu.M were 21.74%, 33.24% and 70.51%, respectively. As shown in FIGS. 6-8, the amount of IL-1. beta., IL-6, TNF-. alpha.released in the supernatant was significantly increased after LPS stimulation (P < 0.01) compared to the blank group. Compared with LPS group, each concentration group of the compound can inhibit the release amount of NO, IL-1 beta, IL-6 and TNF-alpha in cell supernatant and show dose dependence (P < 0.05 and P < 0.01).
TABLE 3 Effect of Compounds on NO, TNF-alpha, IL-6 and IL-1 beta in LPS-induced RAW264.7 cells
2.3 Effect of Compounds on LPS-induced release of iNOS and COX-2 protein from RAW264.7 cells As shown in FIG. 9, over-expression of iNOS and COX-2 protein was significantly reduced in a dose-dependent manner after treatment with the compounds compared to the control group.
TABLE 4 Effect of Compounds on iNOS and COX-2 proteins in LPS-induced RAW264.7 cells
As is clear from the above, the invention relates to the application of a novel isoquinoline alkaloid glycoside compound in the preparation of anti-inflammatory drugs, and also relates to a preparation method for extracting, separating and purifying the compound from traditional Chinese medicine phellodendron. Pharmacological research shows that the novel compound can obviously inhibit the levels of NO, IL-1 beta, IL-6 and TNF-alpha in mouse macrophage RAW246.7 caused by Lipopolysaccharide (LPS), reduce the over-expression of iNOS and COX-2 proteins in RAW246.7 cells induced by LPS, and is in a dose-dependent relationship. The preparation method is simple and feasible, the obtained compound has high purity which is more than 98 percent, is an innovation in preparing anti-inflammatory drugs, exploits the medicinal value and the commercial value of the phellodendron, has wide application prospect, and has remarkable economic and social benefits.
Claims (7)
1. An isoquinoline alkaloid glycoside compound with anti-inflammatory activity is characterized in that the compound is named as phellodendrine glycoside, and the molecular structural formula is as follows:
2. the method of preparing isoquinoline alkaloid glycosides having anti-inflammatory activity of claim 1, comprising the steps of:
(1) extracting cortex Phellodendri with solvent at a mass ratio of 1: 8-16 for 2 times, filtering, mixing filtrates, and concentrating under reduced pressure to obtain crude extract with relative density of 1.2-1.4 at 50 deg.C;
the solvent is any one of ethanol, methanol or acetone;
the extraction is cold leaching method, percolation method, microwave extraction method, ultrasonic extraction method and reflux extraction method;
(2) suspending the crude extract obtained in the step (1) with water, extracting with an organic solvent, filtering the residual water solution with diatomite, and concentrating to obtain a raffinate extract with the relative density of 1.2-1.4 at 50 ℃; or carrying out macroporous resin elution on the crude extract obtained in the step (1), and concentrating the eluent to obtain a macroporous resin elution extract with the relative density of 1.2-1.4 at 50 ℃;
the organic solvent is petroleum ether, ethyl acetate and n-butanol;
the macroporous resin is one of D101, XDA-8, XDA-1 or LSA-8, and is eluted by water, 10% -95% ethanol or methanol in a gradient way, and the elution is carried out for 2-3 column volumes respectively according to the volume fraction;
(3) subjecting the extract residue or macroporous resin eluate to MCI resin column chromatography, concentrating the eluate to obtain extract with relative density of 1.2-1.4 at 50 deg.C, sequentially subjecting to gel column chromatography and reverse column chromatography, and separating with gel column chromatography to obtain isoquinoline alkaloid glycoside compounds with antiinflammatory activity;
the MCI resin column is one of GEL CHP 55A, GEL CHP 70Y, GEL CHP 2MG, GEL CHP 20 or GEL CHP 30;
the MCI column chromatography adopts water, 10-95% ethanol or methanol for gradient elution, and finally adopts a plurality of or one fixed concentration of 30-100% acetone solution for elution, and the elution is carried out for 2-3 column volumes respectively according to volume fraction;
the gel column is one of HW-40F, HW-40C or HW-75;
gradient elution is carried out on the eluent of the gel column chromatography, wherein the eluent is pure water, 10-60% ethanol or methanol solution, and 1 column volume is eluted respectively according to volume fraction;
the reverse column chromatography is eluted by 20-60% methanol or ethanol solution, and the elution is carried out by 1-2 column volumes respectively according to volume fraction.
3. The method of claim 2, comprising the steps of:
(1) extracting 20kg of phellodendron amurense with 10 times of ethanol solution with mass concentration of 50% at room temperature for 2 times and 7 days at each time, filtering, and concentrating the filtrate under reduced pressure to obtain a crude extract with relative density of 1.3 at 50 ℃;
(2) suspending the crude extract obtained in the step (1) by using water, feeding the suspended extract to a D101 type macroporous resin column, filling the suspended extract and resin according to the volume ratio of 1: 3, and sequentially using water and the following components in mass concentration: eluting with 10% ethanol, 20% ethanol, 30% ethanol, 50% ethanol, 70% ethanol, and 95% ethanol, eluting 3 column volumes per gradient in terms of mass fraction, mixing 10%, 20% and 30% ethanol elution parts, and concentrating to obtain macroporous resin elution sample with relative density of 1.3 at 50 deg.C;
(3) and (3) subjecting the macroporous resin eluted sample in the step (2) to MCI column chromatography, and sequentially treating with water and the following components in mass concentration: eluting with 20% ethanol, 30% ethanol, 50% ethanol, 70% ethanol, 30% acetone, and 60% acetone by mass fraction, wherein each gradient elutes 3 column volumes to obtain 7 fractions A, B, C, D, E, F, G; subjecting fraction G to HW-40C column chromatography, eluting with 30% ethanol and 50% ethanol by gradient, and eluting 1 column volume per gradient in terms of mass fraction; obtaining sub-flow components GA, GB and GC; eluting the sub-stream GB by reversed phase column chromatography C18 with eluent of 30% ethanol, 40% ethanol, 50% ethanol and 60% ethanol, and performing gradient elution for 2 column volumes each to obtain sub-stream GB-A, GB-B, GB-C, GB-D, GB-E; subjecting the sub-flow GB-D to HW-40C gel column chromatography, eluting with pure water and 50% methanol by mass concentration, eluting 1 column volume per gradient by mass fraction, and collecting 50% methanol elution part to obtain isoquinoline alkaloid glycoside compounds with anti-inflammatory activity.
4. The method of claim 2, comprising the steps of:
(1) extracting 20kg of phellodendron amurense with 14 times of acetone solution with the mass concentration of 60% at room temperature for 2 times and 6 days each time, filtering, and concentrating the filtrate under reduced pressure to obtain a crude extract with the relative density of 1.2 at 50 ℃;
(2) suspending the crude extract obtained in the step (1) by using water, sequentially extracting by using petroleum ether, ethyl acetate and n-butanol with the same volume at room temperature for 2 times by using each solvent, combining the extracted solutions, and concentrating to obtain raffinate with the relative density of 1.2 at 50 ℃;
(3) subjecting the raffinate obtained in the step (2) to MCI column chromatography, and eluting with water, 20% ethanol by mass concentration, 30% ethanol, 50% ethanol, 70% ethanol, 60% acetone and 100% acetone sequentially, wherein each elution is 2 column volumes in terms of mass fraction, and 7 fractions A, B, C, D, E, F, G are obtained by elution; subjecting the fraction G to HW-40F column chromatography under the elution conditions of 20% methanol and 50% methanol in mass concentration, and eluting for 1 column volume in mass fraction to obtain sub-fractions GA, GB, GC and GD; eluting the sub-stream GC by a reversed phase column chromatography C18, wherein the eluent comprises 30% methanol, 40% methanol, 50% methanol and 60% methanol by mass concentration, and the elution is carried out for 2 column volumes in terms of mass fraction, so as to obtain sub-stream GC-A, GC-B, GC-C, GC-D, GC-E, GC-E; subjecting the sub-stream GC-D to HW-40F gel column chromatography, eluting with pure water and 60% methanol at mass concentration, eluting 1 column volume per column by mass fraction, and collecting the 60% methanol eluate to obtain isoquinoline alkaloid glycoside compounds with anti-inflammatory activity.
5. The method of claim 2, comprising the steps of:
(1) heating and refluxing 20kg of phellodendron amurense with 10 times of methanol with the mass concentration of 60% at 75 ℃ for 2 times, each time for 40min, filtering, and concentrating the filtrate under reduced pressure to obtain a crude extract with the relative density of 1.4 at 50 ℃;
(2) suspending the crude extract obtained in the step (1) with water, sequentially extracting with equal volume of ethyl acetate and n-butanol at room temperature for 3 times, respectively, and concentrating the extracted solution to obtain raffinate with relative density of 1.4 at 50 ℃;
(3) subjecting the raffinate obtained in the step (2) to MCI column chromatography, and eluting with water, 30% methanol by mass concentration, 50% methanol, 70% methanol, 30% acetone and 50% acetone in sequence to obtain A, B, C, D, E, F, wherein each elution is 2 column volumes by mass fraction; subjecting the fraction E to HW-75 column chromatography, wherein eluents comprise 20% methanol and 40% methanol in mass concentration, and each eluent is eluted by 1 column volume in mass fraction to obtain sub-fractions EA, EB and EC; subjecting the sub-stream EC to C18 reverse phase column chromatography, and eluting with methanol with mass concentration of 40%, 50% and 60% in a gradient manner, wherein each elution is 1 column volume in terms of mass fraction, to obtain sub-stream EC-A, EC-B, EC-C; performing HW-75 gel column chromatography with sub-flow part EC-C, eluting with pure water and 50% methanol at mass concentration, eluting 1 column volume each, and collecting 50% methanol elution part to obtain isoquinoline alkaloid glycoside compounds with anti-inflammatory activity.
6. Use of an isoquinoline alkaloid glycoside compound with anti-inflammatory activity as claimed in claim 1 in the preparation of anti-inflammatory drugs.
7. The application of isoquinoline alkaloid glycoside compounds with anti-inflammatory activity in the preparation of anti-inflammatory drugs, which is characterized in that the anti-inflammatory drugs are tablets, capsules, injections, powder injections, granules, microcapsules, dropping pills, ointments or transdermal controlled release patches.
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|---|---|---|---|---|
| WO2001068609A1 (en) * | 2000-03-14 | 2001-09-20 | Actelion Pharmaceuticals Ltd. | 1,2,3,4-tetrahydroisoquinoline derivatives |
| CN112010849A (en) * | 2019-05-29 | 2020-12-01 | 北京中医药大学 | Flavonoid glycoside and isoquinoline alkaloid complex for inhibiting multiple drug-resistant staphylococcus aureus and preparation of carrier-free nano-drug thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001068609A1 (en) * | 2000-03-14 | 2001-09-20 | Actelion Pharmaceuticals Ltd. | 1,2,3,4-tetrahydroisoquinoline derivatives |
| CN112010849A (en) * | 2019-05-29 | 2020-12-01 | 北京中医药大学 | Flavonoid glycoside and isoquinoline alkaloid complex for inhibiting multiple drug-resistant staphylococcus aureus and preparation of carrier-free nano-drug thereof |
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| "A new alkaloid glycoside from the rhizomes of Aristolochia fordiana";Zhongbo Zhou et al.;《Natural Product Research》;20140409;第28卷(第14期);第1065-1069页 * |
| "Isolation and characterization of phellodendronoside A, a new isoquinoline alkaloid glycoside with anti-inflammatory activity from Phellodendron chinense Schneid";Yanpo Si et al.;《Fitoterapia》;20210814;第154卷;第105021号 * |
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