CN115184483B - Two-dimensional screening method for active ingredients of traditional Chinese medicine - Google Patents
Two-dimensional screening method for active ingredients of traditional Chinese medicine Download PDFInfo
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- CN115184483B CN115184483B CN202210646838.2A CN202210646838A CN115184483B CN 115184483 B CN115184483 B CN 115184483B CN 202210646838 A CN202210646838 A CN 202210646838A CN 115184483 B CN115184483 B CN 115184483B
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Abstract
The invention discloses a two-dimensional screening method of active ingredients of traditional Chinese medicines, which is to use a full two-dimensional chromatographic contour map combining two-dimensional biological activity maps with chemical ingredients for two-dimensional accurate screening of active ingredients of the traditional Chinese medicines of polygonum cuspidatum, thereby providing a novel, rapid and accurate screening method for the active ingredients in natural products; the invention expands by two dimensions of liquid chromatography and reversed phase liquid chromatography, the two chromatographic dimensions have good complementarity, and the related activity screening method comprises the following 5 steps of tyrosinase monophenolase, tyrosinase diphenolase, alpha-glucosidase, DPPH free radical scavenging test and ABTS free radical scavenging test; compared with the traditional activity screening method, the novel two-dimensional activity screening method has the advantages of accurate positioning, rapid screening, high resolution and the like, and is beneficial to rapidly and accurately screening effective active ingredients from traditional Chinese medicines with complex compositions.
Description
Technical Field
The invention relates to the field of screening active ingredients in natural products, in particular to a two-dimensional screening method of active ingredients of traditional Chinese medicines, and the method is applied to traditional Chinese medicine polygonum cuspidatum (Polygonum cuspidatum Sieb. Et Zucc).
Background
Natural products are an important resource for drug development, and almost half of small molecule drugs on the market are approved worldwide and are derived from natural products or modified derivatives thereof, so that the screening of lead compounds from natural products is an effective method. The complex composition of natural products, their high degree of chemical structural complexity and diversity of biological activity, remain a great challenge for rapid screening of active ingredients. Therefore, how to rapidly and effectively screen effective bioactive components from complex natural products is particularly important.
Currently, activity screening strategies for molecular level target proteins are largely divided into two broad categories, namely screening strategies based on affinity and activity assays, which can effectively screen biologically active components from complex natural products. The screening method based on the affinity mainly comprises a magnetic microsphere fishing method, ultrafiltration, front-edge affinity chromatography and the like; the screening method based on the activity test mainly comprises a biological activity tracking method and a post-column biological assay, and a post-column activity screening mode developed by taking liquid chromatography as a platform comprises an online biological assay, micro-fraction activity evaluation and the like.
The core of the micro-fraction activity evaluation method is that the micro-fraction collection system is used for collecting the micro-fraction into a porous plate, after the solvent volatilizes, a proper buffer solution is selected to add target protein and corresponding ligand into the porous plate, the signal of biochemical reaction is monitored, and the off-line biological activity detection is completed. Finally, comparing the liquid phase spectrum with the biological activity detection result to find out the components affecting the enzyme activity; or the corresponding chemical structure can be obtained by separating and purifying the corresponding active components and carrying out structure confirmation. The micro-fraction activity evaluation method is to collect eluent at certain time intervals after chromatographic separation, perform micro-fraction activity evaluation by using 96-hole, 384-hole and even 1536-hole plates to obtain a biological activity chromatogram (biochromatogram), then perform comparison with a chemical component chromatogram (chromatogram) to screen out active components, and finally perform structure confirmation and complete pharmacodynamics study. Compared with an online bioassay method, the offline micro-fraction activity evaluation method has relatively low automation degree, but the stability and accuracy of the method can be relatively improved, so that the influence of the existence of an organic phase on the biological activity of target protein in an online mode is avoided, and the method can be combined with more activity detection technologies more flexibly.
Liquid-liquid chromatography (LLC) is a high-efficiency partition chromatography technology that has been raised in recent years, and is mainly characterized in that both the mobile phase and the stationary phase are liquid, so that the stationary phase does not need a solid carrier, the running and maintenance costs are relatively low, and the preparative separation of samples is easy to realize. Compared with the traditional liquid chromatography and other technologies, the liquid chromatography has the following advantages: ① No irreversible adsorption; ② The requirement on the sample injection sample is low, and complex pretreatment is not needed; ③ The sample injection amount is large, and the conventional sample injection amount can reach hundreds of milligrams to gram level; ④ The separator has good complementarity with the traditional chromatographic technique. Liquid chromatography has been widely used in recent years for extraction, separation and purification of complex natural products by virtue of its unique advantages of large sample injection amount, high recovery rate, no irreversible adsorption, and the like. In recent years, in order to improve the separation efficiency of liquid chromatography, preliminary research work has been carried out on improvement of a two-dimensional liquid chromatography (2D LLC) system device, optimization of a solvent system, and application of various coupling strategies, and the purpose of the research work is to improve the resolution of the two-dimensional liquid chromatography separation so that a larger peak capacity can be generated in a reasonable time.
The research reported in most of the literature for screening active ingredients based on chromatographic techniques is based on one-dimensional liquid chromatographic separation. The invention provides a novel rapid and accurate screening method for screening active ingredients in natural products such as traditional Chinese medicines by combining a two-dimensional biological activity map (2D biochromatogram) with a full two-dimensional chromatographic contour map (2D chromatogram) of chemical ingredients. Firstly, combining liquid chromatography and two key technologies with different separation mechanisms of liquid chromatography to obtain a full two-dimensional chromatographic contour map with chemical dimensions of target medicinal materials. Because of the differential complementarity of the separation mechanism and the mode of the two separation technologies, the sample separation is realized by combining the advantages of the two separation mechanisms and the mode, the method is particularly suitable for separating components with similar polarities, similar properties and similar structures of substances, and the two-dimensional chromatography is taken as the most common multi-dimensional chromatography, so that the peak capacity, the selectivity and the separation degree can be obviously improved, and the obtained two-dimensional chromatography with chemical dimension has strong guidance on subsequent activity evaluation.
Few literature studies report the use of two-dimensional chromatography techniques for the separation and purification of target compounds after active ingredient screening, however this application is mainly applied to preparative separation of target components by centre-cut two-dimensional chromatography. The invention combines the two-dimensional micro-fraction bioactive map with the two-dimensional chromatographic contour map to accurately screen the target active compounds. Based on the established full-two-dimensional chromatographic contour diagram, the key point of the two-dimensional activity screening method provided by the invention is that the related activity of the target medicinal material compound is comprehensively evaluated from the dimension of biological activity evaluation, including activity evaluation indexes such as tyrosinase monophenolase, tyrosinase diphenolase, alpha-glucosidase, DPPH free radical scavenging capacity, ABTS free radical scavenging capacity and the like by adopting a micro-fraction activity evaluation method. Finally, the contour map of chemical dimension and the activity of biological dimension are integrated in a two-dimensional activity spectrogram, and the compound with target activity is precisely positioned by cross positioning of activity peaks, which is the key point unique to the invention. Therefore, the construction of the full-two-dimensional chromatographic separation system based on liquid chromatography with no sample loss, no sample inactivation, large sample injection amount and high separation efficiency is an effective supplement and expansion of the traditional full-two-dimensional liquid chromatography technology, and has important significance in the construction of a novel method for separating and analyzing complex samples and in particular in the screening and discovery of natural active ingredients.
Disclosure of Invention
The invention aims to overcome the defect that the traditional one-dimensional activity screening method is difficult to screen rapidly and accurately, and aims to provide a two-dimensional screening method for active ingredients of traditional Chinese medicines, so that the problems of accurately screening effective bioactive ingredients from traditional Chinese medicines with complex compositions and the like are solved, and the efficiency of activity screening, especially enzyme inhibitor screening and free radical scavenger screening is greatly improved.
The invention provides a novel rapid and accurate screening method for screening active ingredients in natural products such as traditional Chinese medicines by combining a two-dimensional biological activity map (2D biochromatogram) with a full two-dimensional chromatographic contour map (2D chromatogram) of chemical ingredients. Compared with the traditional activity screening method, the novel two-dimensional activity screening method has the advantages of accurate positioning, rapid screening, high resolution and the like.
The technical scheme of the invention is as follows:
A two-dimensional screening method of active ingredients of traditional Chinese medicines comprises the following steps:
(1) Extracting: weighing coarse powder of rhizoma Polygoni Cuspidati, and reflux-extracting with ethanol to obtain ethanol extract;
specifically, reflux-extracting the giant knotweed coarse powder with 70% ethanol for 2 hours, filtering, repeatedly extracting filter residues for 1-2 times, combining the filtrates, and spin-drying to obtain an ethanol extract;
(2) Extraction: dissolving the ethanol extract in water, sequentially extracting with diethyl ether, ethyl acetate and n-butanol, collecting extractive solutions, and spin-drying to obtain diethyl ether extract, ethyl acetate extract and n-butanol extract, and spin-drying the rest water phase to obtain water extract;
(3) IC 50 determination: determining IC 50 values of tyrosinase monophenolase, tyrosinase diphenolase, alpha-glucosidase, ABTS and DPPH of the alcohol extract, diethyl ether extract, ethyl acetate extract, n-butanol extract and water extract, and screening diethyl ether extract with highest biological activity as a target for subsequent two-dimensional activity screening;
(4) Optimizing the liquid chromatography conditions: analyzing the polygonum cuspidatum diethyl ether extract by adopting liquid chromatography, and achieving the aim of separating each component by optimizing detection conditions;
The optimized liquid chromatography conditions were: the chromatographic column is an H & E-C18 column, the mobile phase comprises formic acid water (A) and acetonitrile (B) with the volume fraction of 0.1%, gradient elution conditions are adopted, the flow rate is 1.0mL/min, the detection wavelength is 280nm, the column temperature is 30 ℃, the sample injection concentration is 5.00mg/mL, and the sample injection volume is 20 mu L;
Particularly preferred liquid chromatography conditions are: the chromatographic column is an H & E-C18 column (250 multiplied by 4.6mm,5 mu m), the mobile phase composition is 0.1% formic acid water (A) and acetonitrile (B), the gradient elution condition is that ：0-8min：13％-23％(B),8-15min：23％-23％(B),15-25min：23％-30％(B),25-36min：30％-45％(B),36-40min：45％-50％(B),40-51min：50％-90％(B),51-56min：90％-13％(B); flow rate is 1.0mL/min, the detection wavelength is 280nm, the column temperature is 30 ℃, the sample injection concentration is 5.00mg/mL, and the sample injection volume is 20 mu L;
(5) Optimizing liquid chromatography conditions: separating the ether extract of rhizoma Polygoni Cuspidati by liquid chromatography, and improving the separation degree of each component by screening countercurrent solvent system and elution mode;
The optimized liquid chromatography conditions are as follows: petroleum ether-ethyl acetate-methanol-water is selected as a solvent system, and a gradient elution mode is selected to improve the separation degree of each active compound;
particularly preferred liquid chromatography conditions are: the solvent system is petroleum ether-ethyl acetate-methanol-water, a gradient elution mode is adopted, and countercurrent conditions are as follows: 0-60min: petroleum ether-ethyl acetate-methanol-water (3:5:2:8, v/v); 60-170min: petroleum ether-ethyl acetate-methanol-water (3:5:4:6, v/v); 170-255min, adopting a pushing elution mode, wherein the flow rates are all 2mL/min;
(6) Drawing by utilizing Matlab 2018a script based on the optimized chromatographic conditions in the steps (4) and (5), and establishing and obtaining an off-line full-two-dimensional liquid chromatography-liquid chromatogram;
(7) Establishing a high-resolution liquid chromatography micro-fraction screening and evaluating method: eluting the diethyl ether extract under the optimized liquid chromatography condition in the step (4), collecting the eluent by using a porous plate, and establishing a liquid chromatography dimension biological activity spectrogram through micro-fraction activity screening;
(8) Establishing a high-resolution liquid chromatography micro-fraction screening and evaluating method: eluting the diethyl ether extract under the optimized liquid-liquid chromatography condition in the step (5), collecting the eluent by a fraction collector, concentrating, adding a porous plate, and establishing a liquid-liquid chromatography dimension biological activity spectrogram through micro-fraction activity screening;
(9) Building a two-dimensional activity map: based on the offline full-two-dimensional liquid chromatography-liquid chromatography of the step (6), the liquid chromatography dimension biological activity spectrum of the step (7) and the liquid chromatography dimension biological activity spectrum of the step (8), drawing by an origin Pro 2021, establishing and obtaining a corresponding offline full-two-dimensional biological activity spectrum, and precisely positioning a target compound by cross positioning an activity peak to realize two-dimensional screening of active ingredients.
The invention has the beneficial effects that:
According to the invention, as the two-dimensional spectrum construction requires the combination of liquid chromatography and liquid chromatography, compared with the existing micro-fraction screening technology, the two-dimensional active spectrum screening method adopted by the invention combines the advantages of the two chromatography technologies and the micro-fraction active screening method, so that the effective active ingredients in the complex traditional Chinese medicine polygonum cuspidatum can be rapidly and accurately screened, the spectrum resolution is high, and tyrosinase inhibitors, alpha-glucosidase inhibitors, DPPH and ABTS free radical scavengers can be rapidly and effectively screened.
Drawings
FIG. 1 is a two-dimensional contour diagram of the ether layer liquid chromatography-liquid chromatography of giant knotweed rhizome of example 6.
FIG. 2 is a graph of spatial coverage of the two-dimensional contour map of example 6.
FIG. 3 is a two-dimensional activity pattern of tyrosinase monophenolase of the ether layer of Polygonum cuspidatum of example 7.
FIG. 4 is a two-dimensional activity pattern of tyrosinase diphenolase of the ether layer of Polygonum cuspidatum of example 8.
FIG. 5 is a two-dimensional activity pattern of the alpha-glucosidase of the ether layer of Polygonum cuspidatum of example 9.
FIG. 6 is a two-dimensional activity graph showing the DPPH radical scavenging ability of the ether layer of Polygonum cuspidatum of example 10.
FIG. 7 is a two-dimensional activity graph of the ability of the ether layer of Polygonum cuspidatum to scavenge ABTS free radicals of example 11.
Detailed Description
The present invention is further described below by way of specific examples, but the scope of the present invention is not limited thereto.
The traditional Chinese medicine polygonum cuspidatum used in the examples is purchased from Zhejiang, china, east China.
The extraction method of the giant knotweed comprises the following steps:
Weighing 100.00g of giant knotweed coarse powder, extracting with 2000mL of 70% ethanol under reflux for 2h, repeatedly extracting for 2 times, combining filtrates, and spin-drying to obtain 22.64g of dry giant knotweed extract with a yield of 22.64%;
Weighing 3.01g of the extract, dissolving in 300mL of water, sequentially extracting with diethyl ether, ethyl acetate and n-butanol with equal volume for 3 times, respectively, mixing the upper layer extract, spin drying to obtain corresponding layer extract, and spin drying the rest lower layer extract to obtain water layer.
Example 1: establishment of tyrosinase monophenolase test method and selection of optimal active layer
(1) Preparing a solution: ① PBS buffer: taking phosphate powder package, and preparing PBS buffer solution with pH of 6.8 by a pH meter; ② Tyrosinase monophenolase solution: preparing 71.4U/mL tyrosinase solution by using PBS buffer solution with pH of 6.8; ③ L-tyrosine solution: 1mM L-tyrosine solution was prepared with PBS buffer at pH 6.8; ④ Sample solution: 1.00mg of total ethanol extract, diethyl ether extract, ethyl acetate extract, n-butanol extract and water layer extract of giant knotweed rhizome are respectively precisely weighed, a sample mother solution of 1.00mg/mL is prepared by using 40% of DMSO solution, and sample solutions with proper concentrations are prepared by gradient in sequence.
(2) The experimental conditions for tyrosinase monophenolase inhibitory activity are as follows. For sample groups, 10. Mu.L of sample solution, 90. Mu.L of PBS solution and 50. Mu.L of tyrosinase solution with activity value of 124U/mL are sequentially added into a 96-well plate, incubated in an enzyme-labeled instrument at 35 ℃ for 5min, 50. Mu.L of L-tyrosine solution with concentration of 1mM is added after incubation is completed, monitoring is carried out for 30min at 35 ℃ and once every 1min, the detection wavelength is 492nm, absorbance A is measured, and each sample group level test is repeated three times. Setting a sample group, a sample blank group without enzyme, a control group without sample and a blank control group without sample and enzyme, and recording Slope change values of corresponding absorbance as Slope sample、Slopesample blank、Slopecontrol、Slopecontrol blank in sequence, and calculating tyrosinase inhibition rate according to a formula (1), wherein the specific steps are as follows.
The inhibition rates of the sample solutions with different concentrations were calculated using formula (1), and the obtained data were analyzed with Grafit a 7 to obtain IC 50 values of the corresponding extracts. The inhibition activities of the total extract, the diethyl ether extract, the ethyl acetate extract, the n-butanol extract and the water layer extract of the polygonum cuspidatum are measured through tyrosinase monophenolase test. The enzyme inhibition activity of the five different extracts is enhanced along with the increase of the concentration, and other extracts except the water extract have stronger inhibition activity. IC 50 values of total extract, diethyl ether extract, ethyl acetate extract and n-butanol extract of giant knotweed are 13.06+ -0.50 μg/mL, 6.25+ -0.43 μg/mL, 11.64+ -1.47 μg/mL, 21.73+ -2.92 μg/mL in this order, and the lower activity of the water extract can not accurately calculate the IC 50 value. Therefore, the tyrosinase monophenolase inhibitory activity of the ether layer of giant knotweed is strongest, and the IC 50 value is 6.25+/-0.43 mug/mL.
Example 2: establishment of tyrosinase diphenolase test method and selection of optimal active layer
(1) Preparing a solution: steps ① and ④ are the same as in example 1; ② Tyrosinase diphenolase solution: preparing a 124U/mL tyrosinase solution by using PBS buffer solution with pH of 6.8; ③ L-DOPA solution: a5 mM solution of L-DOPA was prepared in PBS buffer at pH 6.8.
(2) The experimental conditions for tyrosinase diphenolase activity measurement are as follows. The sample group was subjected to the following procedures, 10. Mu.L of the sample solution, 90. Mu.L of the PBS solution and 50. Mu.L of the L-DOPA solution with a concentration of 5mM were sequentially added to a 96-well plate, incubated at 30℃for 10 minutes in an microplate reader, 50. Mu.L of the tyrosinase solution with a concentration of 124U/mL was added after the incubation was completed, monitoring was performed at 30℃for 6 minutes at 1 minute intervals, the detection wavelength was 492nm, and the absorbance A was measured, and the parallel test was repeated three times, and the calculation formula was the same as in example 1.
Through tyrosinase diphenol enzyme test, the inhibition curves of different extracts are analyzed, the enzyme inhibition activities of five different extracts are enhanced along with the increase of concentration, and other extracts except the water extract have stronger inhibition activities. IC 50 values of total extract, diethyl ether extract, ethyl acetate extract and n-butanol extract of giant knotweed are 42.03 + -2.27 μg/mL, 13.69+ -1.19 μg/mL, 26.30+ -1.66 μg/mL, 35.69 + -3.07 μg/mL in sequence, and the IC 50 value of the water extract cannot be accurately calculated due to lower activity. Therefore, the tyrosinase diphenolase inhibitory activity of the ether layer of the giant knotweed is strongest, and the IC 50 value is 13.69+/-1.19 mug/mL.
Example 3: establishment of alpha-glucosidase test method and selection of optimal active layer
(1) Preparing a solution: ① PBS buffer: taking phosphate powder package, and preparing PBS buffer solution with pH of 7.0 by a pH meter; ② Alpha-glucosidase solution: preparing 10U/mL of alpha-glucosidase mother liquor by using PBS buffer solution with pH of 7.0, and finally diluting the alpha-glucosidase mother liquor into 0.1U/mL of alpha-glucosidase solution; ③ p-NPG solution: diluting with PBS buffer solution with pH of 7.0 to obtain 10mM p-NPG solution; ④ Sample solution: respectively precisely weighing 1.00mg of total ethanol extract, diethyl ether extract, ethyl acetate extract, n-butanol extract and water layer extract of rhizoma Polygoni Cuspidati, preparing into 1.00mg/mL of sample mother liquor with DMSO solution, and sequentially preparing into sample solution with proper concentration in gradient.
(2) The experimental conditions for the α -glucosidase inhibitory activity are as follows. For the sample group, 8 μl of sample solution, 92 μl of PBS solution and 50 μl of α -glucosidase solution with activity value of 0.1U/mL were sequentially added into a 96-well plate, incubated at 28deg.C for 10min in a microplate reader, 50 μl of p-NPG solution with concentration of 10mM was added after incubation, monitoring was carried out at 28deg.C for 30min, monitoring was carried out every 5min, detection wavelength was carried out at 405nm, absorbance A was measured, and the parallel test was repeated three times, and the calculation formula was the same as in example 1.
The enzyme inhibition activities of the total extract of polygonum cuspidatum and different extract layers were determined by an alpha-glucosidase assay. The enzyme inhibition activity of the five different extracts is enhanced along with the increase of the concentration, and the water extract also has certain hypoglycemic activity. IC 50 values of total extract, diethyl ether extract, ethyl acetate extract, n-butanol extract, and water layer extract of rhizoma Polygoni Cuspidati were 1.28+ -0.14 μg/mL, 1.39+ -0.08 μg/mL, 1.08+ -0.07 μg/mL, 0.51+ -0.03 μg/mL, and 11.14+ -0.69 μg/mL. According to analysis of the obtained activity results, the alpha-glucosidase inhibitory activity of each layer of the giant knotweed is strong, the alpha-glucosidase inhibitory activity is consistent with the blood glucose reducing activity reported in the literature, the other layers outside the water removing layer have strong alpha-glucosidase inhibitory activity, and the similar difference of the values is small.
Example 4: establishment of DPPH free radical scavenging test method and selection of optimal active layer
(1) Preparing a solution: ① DPPH mother liquor: weighing a proper amount of DPPH powder, dissolving the DPPH powder with methanol, and preparing a DPPH mother solution with the concentration of 32 mu M; ② DPPH working solution: diluting appropriate amount of mother liquor to obtain 6.4 μm DPPH solution (for preparation and preservation in dark place); ③ Sample solution: the procedure is as in step ④ of example 1, except that 50% methanol solution is used for dissolution.
(2) The DPPH radical scavenging test was carried out under the following conditions. 50. Mu.L of sample solution was mixed with 200. Mu.L of DPPH working solution and incubated at room temperature for 6min in the absence of light to perform the reaction, absorbance A was measured at 517nm after completion of the reaction, and the level test was repeated three times for each sample group, with vitamin C as a positive control. Setting a sample group, a sample blank group, a control group and a blank control group, and recording corresponding absorbance differences as As, asb, ac, acb in sequence, wherein a calculation formula (2) of the DPPH free radical scavenging capacity is shown as follows.
Through DPPH free radical scavenging test, IC 50 values of total extract of giant knotweed, diethyl ether extract, ethyl acetate extract, n-butyl alcohol extract and aqueous layer extract are 7.09+/-0.60 mug/mL, 10.68+/-1.03 mug/mL, 3.36+/-0.23 mug/mL, 3.61+/-0.43 mug/mL and 51.55 +/-5.06 mug/mL in sequence, the activity of the aqueous extract is far lower than that of other active layers, and the activity of the ethyl acetate layer and the n-butyl alcohol layer are similar. Therefore, the DPPH free radical scavenging ability of the ethyl acetate layer and the n-butanol layer of the giant knotweed is stronger.
Example 5: establishment of ABTS free radical scavenging test method and selection of optimal active layer
(1) Preparing a solution: ① ABTS mother liquor: dissolving 1.53mM ABTS powder and 0.13mM K 2S2O8 in pure water to obtain ABTS mother liquor, and placing in a dark refrigerator for 12-16 hr. ② ABTS working fluid: 5mL of ABTS mother solution is taken and dissolved in 20mL of PBS buffer solution with pH of 7.3 to prepare an ABTS working solution. ③ Sample solution: step ③ in example 4 is followed.
(2) The conditions for the ABTS radical scavenging assay were as follows. 20. Mu.L of sample solution was mixed with 200. Mu.L of ABTS working solution and incubated at room temperature for 10min in the absence of light to perform the reaction, absorbance A was measured at 734nm after the reaction was completed, and the level test was repeated three times for each sample group, with vitamin C as a positive control. The rest of the experimental design and inhibition rate calculation formula are the same as in example 4.
Through the ABTS free radical scavenging test, the IC 50 values of the total extract of giant knotweed, the ether extract, the ethyl acetate extract, the n-butanol extract and the water layer extract are 1.74+/-0.10 mug/mL, 1.30+/-0.16 mug/mL, 1.12+/-0.06 mug/mL, 1.40+/-0.13 mug/mL and 6.96+/-0.37 mug/mL in sequence. The activity of the water extract is lower than that of other active layers, and the ABTS free radical scavenging ability of the other extraction layers of the giant knotweed is similar, so that the giant knotweed ether layer, the ethyl acetate layer and the n-butyl alcohol layer have better ABTS free radical scavenging activity.
The comprehensive tyrosinase monophenolase and tyrosinase diphenolase activity results can be obtained, the inhibition activity of the ether layer of the polygonum cuspidatum is strongest, and the inhibition activities of the ether layer, the ethyl acetate layer and the n-butanol layer are similar to the inhibition activities of alpha-glucosidase activity, DPPH and ABTS free radical scavenging capacity, so that the ether layer is comprehensively considered to be selected for subsequent two-dimensional chromatographic condition optimization and two-dimensional activity screening.
Example 6: screening and optimization of two-dimensional chromatographic conditions
(1) Establishment of chromatographic analysis conditions of giant knotweed rhizome liquid
The liquid chromatography adopts a head-tail elution mode that an upper organic phase is a stationary phase and a lower aqueous phase is a mobile phase, when the upper organic phase fills the chromatographic column, the control voltage is adjusted to be a proper rotating speed at 40V, and the mobile phase is pumped into the chromatographic column at a flow rate of 2mL/min until the two phases are balanced. The solvent system is petroleum ether-ethyl acetate-methanol-water, a gradient elution mode is adopted, and countercurrent conditions are as follows: 0-60min: petroleum ether-ethyl acetate-methanol-water (3:5:2:8, v/v); 60-170min: petroleum ether-ethyl acetate-methanol-water (3:5:4:6, v/v); 170-255min, adopting a pushing elution mode, wherein the flow rates are all 2mL/min. According to the solvent ratio, each solvent system was formulated in a separatory funnel and vigorously shaken at room temperature, after equilibration the upper and lower phases were separated as stationary and mobile phases, respectively. A sample solution of 0.86mg/mL was prepared by dissolving 5.18mg of the ether extract of Polygonum cuspidatum in 6mL of an equilibrated biphasic solvent system (upper 3mL, lower 3 mL). The stationary phase retention was 52.38%.
(2) Establishment of liquid chromatography analysis conditions of giant knotweed
The 235 components obtained by collection were analyzed sequentially by reversed phase liquid chromatography, the column was H & E-C18 column (250X 4.6mm,5 μm), the mobile phase composition was 0.1% formic acid water (A) and acetonitrile (B), the gradient elution conditions were as follows ：0-8min：13％-23％(B),8-15min：23％-23％(B),15-25min：23％-30％(B),25-36min：30％-45％(B),36-40min：45％-50％(B),40-51min：50％-90％(B),51-56min：90％-13％(B); flow rate 1.0mL/min, the detection wavelength was 280nm, the column temperature was 30 ℃, the sample concentration was 5.00mg/mL, and the sample volume was 20. Mu.L.
(3) Establishment of off-line full-two-dimensional liquid chromatography-liquid chromatogram
The liquid chromatography is used for the first dimension of two-dimensional separation due to the large sample injection amount and the high sample recovery rate, and the liquid chromatography is used for the second dimension of two-dimensional separation due to the high resolution and the short analysis time. And transferring each component collected by the liquid chromatography into the liquid chromatography for analysis, volatilizing the organic reagent and the water of each component, concentrating and redissolving the organic reagent and the water into 1mL by adopting an acetonitrile-water mixed solvent, further analyzing the acetonitrile-water mixed solvent into a second dimension to obtain a liquid chromatogram of the corresponding fraction, drawing a two-dimensional contour map by a Matlab 2018a script, and establishing an off-line full-dimensional liquid chromatography-liquid chromatogram, as shown in figure 1. And a convex hull method is adopted to calculate the two-dimensional space coverage rate to be 88.86 percent, as shown in figure 2.
Example 7: establishment of two-dimensional active fingerprint
(1) Establishment of giant knotweed rhizome liquid chromatographic activity spectrogram
Firstly, 6mL of sample solution with the concentration of 0.86mg/mL is taken, elution is carried out according to the optimized liquid chromatography analysis condition, gradient elution condition and component treatment are as in example 6, and 235 components collected in the time period of 20-255min are all connected into a fraction collector. And (3) using a part of the components obtained by liquid-liquid chromatography for liquid phase analysis, and a part of the components are used for liquid-liquid chromatography activity determination, wherein the resolution of the biological activity separation of the 96-well plate is 5 min/component, 47 components are combined, 24 sample groups and 12 control groups can be respectively arranged in each 96-well plate, and each component is repeated three times.
And secondly, adding a proper volume of concentrated sample solution into a 96-well plate, volatilizing each component by using a solvent volatilizing instrument, using a gun to correspond to reagents required by an activity test in the 96-well plate, and recording absorbance values required in the test process by the specific operation as in examples 1-5.
Thirdly, the formula of the inhibition ratio is the same as that of examples 1 to 5.
(2) Establishment of giant knotweed rhizome liquid phase dimension activity spectrogram
Firstly, 20 mu L of sample solution with the concentration of 5.00mg/mL is taken, elution is carried out according to optimized liquid phase analysis conditions, all components collected by liquid phase separation in the gradient elution condition example 5 and 0-56min are connected into a 96-well plate, 480 components are collected in total, the resolution of biological activity separation of the 96-well plate is 7 s/point, 84 sample groups and 12 control groups are arranged in each 96-well plate.
The second step is the same as the second step of establishing the liquid chromatography activity spectrum in example 7.
In the third step, since the number of samples obtained by the micro fraction is large, the sample blank group of each group is not set singly, and only the blank group of each group is set, so that the calculation formula of the enzyme inhibition rate is adjusted to formula (3).
The formula of the radical removal rate is adjusted to formula (4).
(3) Establishment of high-resolution two-dimensional activity map
An off-line full two-dimensional activity fingerprint for tyrosinase monophenolase was established by mapping, as shown in figure 3. From fig. 3 it can be concluded that compounds 5, 7, 10, 13, 20 have a certain tyrosinase monophenolase inhibitory activity by cross-analysis of the countercurrent and liquid dimensions. Wherein the retention time of the liquid phase is 25.31min, the inhibition rate of the compound 13 in the liquid phase dimension reaches 92.71%, and the compound can be eluted in the countercurrent dimension in petroleum ether-ethyl acetate-methanol-water (3:5:4:6, v/v) to obtain purer substances, namely a countercurrent section III, which shows stronger inhibition activity. Therefore, the compound 13 has strong enzyme inhibition activity, is a monomer component with main inhibition effect in an ether layer, the component is resveratrol of stilbene type through structural identification, after separation and purification, IC 50 of the monomer is measured to be 4.74+/-0.53 mu M, IC 50 of positive control kojic acid and arbutin is respectively 16.65+/-0.89 mu M and 149.45 +/-5.82 mu M, and the in-vitro tyrosinase inhibition activity of the monomer component is higher than that of kojic acid and is far higher than that of arbutin.
Meanwhile, as can be seen from a two-dimensional contour plot, when the liquid phase retention time is 13.30min, the compound 7 and the compound 8 are eluted simultaneously, however, due to the difference of partition coefficients, the compound 7 and the compound 8 are separated into two peaks in liquid-liquid chromatography, and experimental results show that the compound 7 has better inhibitory activity, and through structural identification, the compound 7 is (-) -epicatechin gallate, and the IC 50 of the separated monomers is 35.15 +/-2.35 mu M. Through subsequent separation, purification and structural identification, the compound 5 is polydatin, and the IC 50 of the monomer is 19.07+/-1.68 mu M; compound 10 is polydatin-2' -O-gallate, which is a derivative of compound 5, and the IC 50 of the monomer is 7.80+/-0.51 mu M; compound 20 was vanicoside B and IC 50 of the monomer was 45.32± 24.51 μm. The result is consistent with the cross analysis result of the two-dimensional activity map, and the two-dimensional activity screening method is strongly demonstrated to accurately screen effective active ingredients in the complex traditional Chinese medicine.
Example 8: establishment of two-dimensional active fingerprint
(1) Same as in example 7.
(2) Same as in example 7.
(3) Establishment of high-resolution two-dimensional activity map
An off-line full two-dimensional activity fingerprint for tyrosinase diphenolase was established by plotting as shown in fig. 4. From fig. 4 it can be concluded that compounds 5, 10, 13 have a certain tyrosinase diphenolase inhibitory activity by cross-over analysis. Similar to example 7, wherein compound 13 also had the strongest inhibitory activity, resveratrol of stilbene type, the inhibition rate of this component in the liquid phase dimension reached 56.62% at 25.32min, and eluted in the countercurrent dimension in petroleum ether-ethyl acetate-methanol-water (3:5:4:6, v/v) to give purer species, countercurrent section iii, which exhibited stronger inhibitory activity, however, in IC 50, the IC 50 value of this monomer could not be accurately determined due to the influence of monomer solubility and physicochemical properties. Through structural identification and activity measurement, the compound 10 is polydatin-2' -O-gallate, the IC 50 of the monomer is 17.45+/-1.16 mu M, the IC 50 of positive control kojic acid tyrosinase diphenol enzyme is 42.72+/-1.36 mu M, and the activity of the separated monomer is higher than that of the positive control.
Example 9: establishment of two-dimensional active fingerprint
(1) Same as in example 7.
(2) Same as in example 7.
(3) Establishment of high-resolution two-dimensional activity map
By plotting, an off-line full two-dimensional active fingerprint for alpha-glucosidase was established as shown in fig. 5. From fig. 5 it can be concluded that compounds 2, 3, 5, 7, 10, 13, 16, 20, 22 have a certain α -glucosidase inhibitory activity by cross analysis. As can be seen from FIG. 5, the compound 20 shows strong alpha-glucosidase inhibitory activity, the inhibition rate of the component in the liquid phase dimension reaches 98.64% when the liquid phase retention time is 38.85min, and the component can be eluted in the countercurrent dimension in petroleum ether-ethyl acetate-methanol-water (3:5:4:6, v/v) to obtain a purer substance, namely countercurrent section II, which shows strong inhibitory activity. The component is vanicoside B by structural identification, after separation and purification, the IC 50 of the monomer is 1.42+/-0.05 mu M, the IC 50 of the positive control acarbose is 3.79+/-0.26 nM, and the monomer component has the strongest inhibitory activity in the separated monomer although the in-vitro alpha-glucosidase inhibitory activity is lower than that of acarbose.
Meanwhile, according to two-dimensional activity diagram analysis, the compound 5 polydatin, the compound 7 (-) -epicatechin gallate, the compound 10 polydatin-2' -O-gallate and the compound 13 resveratrol also have certain alpha-glucosidase inhibition activity, and when the liquid phase retention time is respectively 12.48min, 13.18min, 16.57min and 25.08min, the inhibition rate of the component in the liquid phase dimension reaches 58.52%, 99.38%, 62.01% and 74.64%, and the IC 50 corresponding to the compounds 5, 7, 10 and 13 is 149.79 +/-26.61, 37.13+/-7.27, 6.91+/-0.59 and 64.74 +/-26.57 mu M respectively. Wherein the compounds 2 and 3 are respectively (+) -catechin and (-) -epicatechin which are enantiomers of each other through structural identification, and the IC 50 activity is respectively 148.21 +/-3.14 and 118.85 +/-6.02 mu M, and the alpha-glucosidase inhibitory activity (-) -epicatechin is slightly stronger than (+) -catechin.
Example 10: establishment of two-dimensional active fingerprint
(1) 100. Mu.L of the reconstituted sample was added to a 96-well plate and the rest of the procedure was as in example 7.
(2) Same as in example 7.
(3) Data processing and mapping an off-line full two-dimensional activity profile was created for DPPH radical scavenging capacity as in example 7, as shown in fig. 6. From fig. 6 it can be concluded that compounds 5, 7, 10, 13, 14 have a certain DPPH radical scavenging capacity by cross analysis. Wherein, the compound 7 has the strongest inhibitory activity, when the retention time of liquid phase is 13.30min, the compound 7 and the compound 8 are eluted at the same time, the compound 7 and the compound 8 are well separated in the countercurrent dimension, the inhibition rate of the component in the liquid phase dimension reaches 83.19 percent, and the component can be eluted together with the active components 5 and 10 in the countercurrent dimension (3:5:2:8, v/v), the compound 7 has stronger inhibitory activity, and the IC 50 of the compound 5 polygonin, the compound 7 (-) -epicatechin gallate and the compound 10 polygonin-2' -O-gallate are 77.57+/-4.26, 8.66+/-0.74 and 12.25+/-0.89 mu M respectively, and the obtained compound 7 has the strongest inhibitory activity and is consistent with the analysis result of the two-dimensional activity map. In addition, the resveratrol compound 13 has IC 50 of 66.15 +/-6.35 mu M, the positive control vitamin C has IC 50 of 21.61+/-1.33 mu M, the activity of the separated monomers 7 and 10 is higher than that of the positive control, and the activity of the monomers 5 and 13 is slightly weaker than that of the positive control.
Example 11: establishment of two-dimensional active fingerprint
(1) 100. Mu.L of the reconstituted sample was added to a 96-well plate and the rest of the procedure was as in example 7.
(2) Same as in example 7.
(3) Data processing and mapping an off-line full two-dimensional activity profile was created for ABTS radical scavenging capacity as in example 7, as shown in fig. 7. From fig. 7, it can be concluded that, by cross analysis, compounds 2, 3,5, 7, 10, 13, 14, 16, 20, 27 have a certain ABTS radical scavenging ability, which is consistent with the stronger activity results measured by this evaluation method compared to other activity evaluation methods when IC 50 values are measured. The compounds 5, 7, 10 and 13 have the strongest inhibition activities, and when the liquid phase retention time is respectively 12.72min, 13.30min, 16.68min and 25.55min, the inhibition rate of the components in the liquid phase dimension reaches 84.19%, 84.60%, 82.27% and 83.70%, and the IC 50 corresponding to the compounds 5, 7 (-) -epicatechin gallate, 10 polydatin-2' -O-gallate and 13 resveratrol are respectively 4.26+/-0.22, 2.62+/-0.18, 3.63+/-0.32 and 3.15+/-0.21 mu M according to the activity measurement. In addition, compound 27 has no obvious inhibition activity in the evaluation method, but has a certain ABTS free radical scavenging capability, and through structural identification, compound 27 is emodin, and IC 50 corresponding to a monomer is 4.22+/-0.67 mu M. In addition, the enantiomer compound 2 (+) -catechin and compound 3 (-) -epicatechin have the IC 50 of 5.57+/-0.30 mu M and 6.55+/-0.29 mu M respectively, the ABTS free radical scavenging ability of the enantiomer compound 2 (+) -catechin and the compound 3 (-) -epicatechin are similar, the IC 50 of the positive control vitamin C is 11.71+/-0.72 mu M, and the activities of the separated monomers 2, 3,5, 7, 10, 13 and 27 are higher than those of the positive control.
The method comprises the following steps: abbreviations
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Claims (2)
1. A two-dimensional screening method of active ingredients of traditional Chinese medicine is characterized by comprising the following steps:
(1) Extracting: weighing coarse powder of rhizoma Polygoni Cuspidati, and reflux-extracting with ethanol to obtain ethanol extract;
(2) Extraction: dissolving the ethanol extract in water, sequentially extracting with diethyl ether, ethyl acetate and n-butanol, collecting extractive solutions, and spin-drying to obtain diethyl ether extract, ethyl acetate extract and n-butanol extract, and spin-drying the rest water phase to obtain water extract;
(3) IC 50 determination: determining IC 50 values of tyrosinase monophenolase, tyrosinase diphenolase, alpha-glucosidase, ABTS and DPPH of the alcohol extract, diethyl ether extract, ethyl acetate extract, n-butanol extract and water extract, and screening diethyl ether extract with highest biological activity as a target for subsequent two-dimensional activity screening;
(4) Optimizing the liquid chromatography conditions: analyzing the polygonum cuspidatum diethyl ether extract by adopting liquid chromatography, and achieving the aim of separating each component by optimizing detection conditions;
The optimized liquid chromatography conditions were: the chromatographic column is H & E-C18 column 250 multiplied by 4.6mm,5 mu m, the mobile phase composition is formic acid water (A) and acetonitrile (B) with volume fraction of 0.1%, gradient elution conditions are ：0-8min：13％-23％(B),8-15min：23％-23％(B),15-25min：23％-30％(B),25-36min：30％-45％(B),36-40min：45％-50％(B),40-51min：50％-90％(B),51-56min：90％-13％(B);, the flow rate is 1.0mL/min, the detection wavelength is 280nm, the column temperature is 30 ℃, the sample concentration is 5.00mg/mL, and the sample volume is 20 mu L;
(5) Optimizing liquid chromatography conditions: separating the ether extract of rhizoma Polygoni Cuspidati by liquid chromatography, and improving the separation degree of each component by screening countercurrent solvent system and elution mode;
the optimized liquid chromatography conditions are as follows: the solvent system is petroleum ether-ethyl acetate-methanol-water, a gradient elution mode is adopted, and countercurrent conditions are as follows: 0-60min: petroleum ether-ethyl acetate-methanol-water 3:5:2:8, v/v;60-170min: petroleum ether-ethyl acetate-methanol-water 3:5:4:6, v/v;170-255min, adopting a pushing elution mode, wherein the flow rates are all 2mL/min;
(6) Drawing by utilizing Matlab 2018a script based on the optimized chromatographic conditions in the steps (4) and (5), and establishing and obtaining an off-line full-two-dimensional liquid chromatography-liquid chromatogram;
(7) Establishing a high-resolution liquid chromatography micro-fraction screening and evaluating method: eluting the diethyl ether extract under the optimized liquid chromatography condition in the step (4), collecting the eluent by using a porous plate, and establishing a liquid chromatography dimension biological activity spectrogram through micro-fraction activity screening;
(8) Establishing a high-resolution liquid chromatography micro-fraction screening and evaluating method: eluting the diethyl ether extract under the optimized liquid-liquid chromatography condition in the step (5), collecting the eluent by a fraction collector, concentrating, adding a porous plate, and establishing a liquid-liquid chromatography dimension biological activity spectrogram through micro-fraction activity screening;
(9) Building a two-dimensional activity map: based on the offline full-two-dimensional liquid chromatography-liquid chromatography of the step (6), the liquid chromatography dimension biological activity spectrum of the step (7) and the liquid chromatography dimension biological activity spectrum of the step (8), drawing by an origin Pro 2021, establishing and obtaining a corresponding offline full-two-dimensional biological activity spectrum, and precisely positioning a target compound by cross positioning an activity peak to realize two-dimensional screening of active ingredients.
2. The two-dimensional screening method of traditional Chinese medicine active ingredients according to claim 1, wherein in the step (1), the polygonum cuspidatum coarse powder is extracted by reflux with 70% ethanol for 2 hours, filtered, the residue is repeatedly extracted for 1-2 times, and the filtrate is combined and dried by spin to obtain the alcohol extract.
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