CN112973189B - Method for preparing Chinese medicinal extract absorption component group - Google Patents
Method for preparing Chinese medicinal extract absorption component group Download PDFInfo
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- CN112973189B CN112973189B CN201911282477.2A CN201911282477A CN112973189B CN 112973189 B CN112973189 B CN 112973189B CN 201911282477 A CN201911282477 A CN 201911282477A CN 112973189 B CN112973189 B CN 112973189B
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
- B01D15/18—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns
- B01D15/1807—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns using counter-currents, e.g. fluidised beds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/23—Apiaceae or Umbelliferae (Carrot family), e.g. dill, chervil, coriander or cumin
- A61K36/236—Ligusticum (licorice-root)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/55—Liquid-liquid separation; Phase separation
Abstract
The invention discloses a method for preparing a traditional Chinese medicine extractA method for absorbing component groups belongs to the technical field of analytical chemistry, and can solve the problem that the prior art is difficult to integrally prepare multi-component compounds which take effect in traditional Chinese medicine extracts. The method comprises preparing a liquid-liquid two-phase system I; obtaining the distribution coefficient logK of the absorption component group to be prepared in the liquid-liquid two-phase system I1A range value; according to the distribution coefficient logK1The range value and the stationary phase retention value of the liquid-liquid two-phase system I in the high-speed counter-current chromatograph are used for obtaining the collection time of the absorption component group to be prepared; preparing the absorption component group in the traditional Chinese medicine extract by adopting a high-speed counter-current chromatography according to the collection time of the absorption component group to be prepared. The method of the invention obtains the distribution coefficient logK of the absorption component group to be prepared in the liquid-liquid two-phase system I1The range value is that the compounds in the Chinese medicinal extract are divided into absorption component group and non-absorption component group, and the absorption component group of the Chinese medicinal extract is prepared by high performance counter current chromatography.
Description
Technical Field
The invention relates to the technical field of analytical chemistry, in particular to a method for preparing an absorption component group of a traditional Chinese medicine extract.
Background
The traditional Chinese medicine, especially the compound, has numerous and complicated compositions, and the knowledge mastered at present shows that the components cannot all take effect, but the knowledge that the components take effect is not completely known.
At present, the technology widely applied to the separation and preparation of traditional Chinese medicines mostly comes from the column chromatography technology of western medicines, such as C18Columns, gel columns, ion exchange columns, macroporous resins, polyamide columns, and the like. The column chromatography technology is a technology for separating compounds according to different adsorption capacities of the compounds on fillers, the compounds in the traditional Chinese medicine extract have a complex content proportion relationship, and the nature of multi-component effect of the traditional Chinese medicine cannot be reduced by a monomer preparation and combination administration mode, so that the column chromatography technology is difficult to integrally prepare the multi-component effect-taking compounds in the traditional Chinese medicine extract.
Disclosure of Invention
In view of the above, the invention provides a method for preparing an absorption component group of a traditional Chinese medicine extract, which can solve the problem that the prior art is difficult to integrally prepare a multi-component compound which jointly acts in the traditional Chinese medicine extract.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for preparing Chinese medicinal extract absorption component group comprises the following steps:
and 4, preparing the absorption component group in the traditional Chinese medicine extract by adopting a high-speed counter-current chromatography according to the collection time of the absorption component group to be prepared.
As a still further scheme of the invention: the liquid-liquid two-phase system I comprises the following components in proportion:
the volume ratio of the component A to the component B to the component C is (0-25): (25-7): 25;
wherein the component A is acetonitrile and/or isopropanol; the component B is n-octanol; the component C is 0.1-0.3 mol/L phosphate buffer solution.
Preferably, the component C is independently selected from 0.1mol/L phosphate buffer solution, 0.12mol/L phosphate buffer solution, 0.15mol/L phosphate buffer solution, 0.18mol/L phosphate buffer solution, 0.2mol/L phosphate buffer solution, 0.23mol/L phosphate buffer solution, 0.25mol/L phosphate buffer solution, 0.27mol/L phosphate buffer solution and 0.3mol/L phosphate buffer solution.
Specifically, the upper phase of the liquid-liquid two-phase system I is a mixed solution of acetonitrile and/or isopropanol, n-octanol and water; the lower phase of the liquid-liquid two-phase system I is a mixed solution of water and phosphate, and the distribution coefficient logK1The value is the ratio of the concentration of the upper phase to the concentration of the lower phase of the liquid-liquid two-phase system I.
Preferably, the component A: and B component: the volume ratio of the component C is independently selected from 0:25:25, 5:20:25, 10:15:25, 15:10:25, 25:15:25, 25:10:25 and 25:7: 25.
Preferably, the liquid-liquid two-phase system I is isopropanol-n-octanol-0.1 mol/L phosphate buffer solution, and the ratio of isopropanol: n-octanol: the volume ratio of 0.1mol/L phosphate buffer solution is (0-25): (25-7): 25.
as a still further scheme of the invention: the step 2 specifically comprises the following steps:
step 2.1, measuring the distribution coefficient logK of the compound in the liquid-liquid two-phase system I2；
In particular, the distribution coefficient logK of the compound in a liquid-liquid two-phase system I2The measuring method comprises the following steps: respectively analyzing the concentrations of the compounds in the upper phase and the lower phase of a liquid-liquid two-phase system I, logK by high performance liquid chromatography2The value is the ratio of the concentration of the upper phase to the concentration of the lower phase.
Step 2.2, obtaining the distribution coefficient logP of the compound in the liquid-liquid two-phase system II2；
In particular, the distribution coefficient logP of the compound in a liquid-liquid two-phase system II2The method for obtaining the (B) has two methods: one is to analyze the concentration of the compound in the upper phase and the lower phase of a liquid-liquid two-phase system II, logP, respectively by high performance liquid chromatography2The value is the ratio of the concentration of the upper phase to the concentration of the lower phase; one is to calculate and obtain logP by utilizing the structure of a compound2The value is obtained.
Step 2.3, fitting logK2And logP2A linear relationship of values;
step 2.4, according to the theoretical distribution coefficient logP of the absorption component group in the liquid-liquid two-phase system II1Value, log K of the absorption component group to be prepared in the liquid-liquid two-phase system I is obtained from the linear relationship obtained in step 2.31A range value.
Specifically, the theoretical distribution coefficient logP of the absorption component group in the liquid-liquid two-phase system II1Distribution coefficient logP for optimal absorption component group1Values, e.g. logP of the best intestinal absorption component group1Is 1.35; logP of optimal oral absorption component group1Is 1.8; log P of optimal intestinal absorption component group1Is 1.3; logP of optimal neural absorptive component population1Was 2.7.
As a still further scheme of the invention: the liquid-liquid two-phase system II is an n-octanol/water two-phase system.
Specifically, the upper phase of the liquid-liquid two-phase system II is n-octanol, and the lower phase of the liquid-liquid two-phase system II is water.
Preferably, the theoretical partition coefficient logP in the liquid-liquid two-phase system II is determined according to the absorption component group1Setting the proportion of A, B, C three components in the liquid-liquid two-phase system I;
theoretical partition coefficient logP of absorption component group in liquid-liquid two-phase system II1When the volume ratio of the component A to the component B to the component C in the liquid-liquid two-phase system I is more than 2, the volume ratio of the component A to the component B to the component C is 25:7: 25;
theoretical partition coefficient logP of absorption component group in liquid-liquid two-phase system II1When the volume ratio of the component A to the component B to the component C in the liquid-liquid two-phase system I is less than 1, the volume ratio is 7:25: 25.
Preferably, the theoretical partition coefficient logP of the absorption component group in the liquid-liquid two-phase system II1When the volume ratio of the component A to the component B to the component C in the liquid-liquid two-phase system I is more than 1 and less than 3, the volume ratio is selected from (0-25): (25-7): 25, in any ratio.
As a still further scheme of the invention: the Chinese medicinal extract is mixture of Chinese medicinal standard substances, or one or more of secondary metabolites extracted from Chinese medicinal materials, Chinese medicinal compound or non-medicinal plants.
Preferably, the Chinese medicinal extract is obtained by extracting with methanol solution or ethanol solution.
As a still further scheme of the invention: before the preparation of the liquid-liquid two-phase system I, at least the following steps are included:
step S1, determining the distribution coefficient logK of at least 20 compounds in the preset liquid-liquid two-phase system I2A value;
step S2, obtaining the distribution coefficient logP of the at least 20 compounds in the liquid-liquid two-phase system II2A value;
step S3, fitting logK2Value sum logP2Linear relation of values, when the coefficient of correlation R of said linear relation2When the current value is greater than the first preset value, continuing the next step; when the correlation coefficient R of the linear relation2When the value is less than or equal to the first preset value, repeating the step S1;
step S4, measuring the stationary phase retention rate of the preset liquid-liquid two-phase system I in the high-speed counter-current chromatography, and when the stationary phase retention rate of the preset liquid-liquid two-phase system I in the high-speed counter-current chromatography is larger than a second preset value, determining that the preset liquid-liquid two-phase system I is the liquid-liquid two-phase system I in the method for preparing the absorption component group in the traditional Chinese medicine extract; and when the stationary phase retention rate of the preset liquid-liquid two-phase system I in the high-speed counter-current chromatography is less than or equal to a second preset value, repeating the step S1.
As a still further scheme of the invention: the first preset value is 0.6.
As a still further scheme of the invention: the second preset value is 20%.
Specifically, an upper phase and a lower phase in a liquid-liquid two-phase system I are respectively a mobile phase and a stationary phase. The elution method of the high-speed counter-current chromatography is two types: one is that the upper phase is a stationary phase, the lower phase is a mobile phase, and the elution is carried out from the tail end to the head end; the other one is that the lower phase is a stationary phase, the upper phase is a mobile phase, and the elution is carried out from the tail end to the head end; determining an elution mode according to the composition and the proportion of the liquid-liquid two-phase system I so as to obtain more than 20% of stationary phase retention; preferably, the second elution mode is adopted, and the stationary phase retention rate is higher.
As a still further scheme of the invention: the high-speed countercurrent chromatograph is a J-type high-speed countercurrent chromatograph, and the ratio of the rotation radius and the revolution radius of the spiral pipe is 0.5-0.8.
Specifically, the high-speed countercurrent chromatograph is a J-type semi-preparative high-speed countercurrent chromatograph, and is provided with three spiral tubular columns connected in series, wherein the volume of each column is 280 mL.
When in use, the high-speed counter-current chromatograph is filled with a liquid-liquid two-phase system I, then a main machine of the high-speed counter-current chromatograph rotates, and a mobile phase is pumped into the spiral pipe column; injecting sample by a sample injection valve; and determining the fraction receiving start-stop time according to the relation between the K value and the retention time.
Wherein, the solvent of the sample injection is isopropanol solution; preferably a 50% isopropanol solution.
Specifically, the relationship between the K value and the retention time is as follows:
Rt＝Vr/v＝(VsK+Vm)/v
Vs＝280×Sf；
Vm＝280-Vs；
wherein Rt is the compound retention time; vr: compound retention volume; v: a mobile phase flow rate; vs: the stationary phase retention volume; k: the partition coefficient of the compound in the system is defined by logK1Conversion is carried out to obtain; vm: volume of mobile phase in chromatography column; sf: a stationary phase retention value;
the starting and ending time of collection can be determined by substituting the K value, stationary phase retention value (Sf) and mobile phase flow rate (v) into the formula.
In the present application, all conditions relating to a numerical range may be independently selected from any intermediate range within said numerical range.
In this application, all conditions relating to numerical ranges are inclusive of the endpoints unless specifically stated otherwise.
The beneficial effects of the invention include but are not limited to:
(1) the method for preparing the absorption component group of the traditional Chinese medicine extract comprises the steps of firstly preparing a liquid-liquid two-phase system I, and then obtaining the distribution coefficient logK of the absorption component group to be prepared in the liquid-liquid two-phase system I1Range value according to distribution coefficient logK1And (3) obtaining the collection time of the absorption component group to be prepared by the range value and the stationary phase retention value of the liquid-liquid two-phase system I in the high-speed counter-current chromatograph, and finally preparing the absorption component group in the traditional Chinese medicine extract by adopting the high-speed counter-current chromatography according to the collection time of the absorption component group to be prepared. The method of the invention obtains the distribution coefficient logK of the absorption component group to be prepared in the liquid-liquid two-phase system I1Range value, dividing the compounds in the Chinese medicinal extract into absorption component group and non-absorption component group, and preparing the absorption component by high performance countercurrent chromatographyAnd (5) collecting the components.
(2) In the method, the retention time of the high-speed countercurrent chromatography is only related to the distribution coefficient, and the compounds are sequentially eluted according to the distribution coefficient and are collected in sections to realize the integral preparation of the absorption component group. The method has the characteristic of integral preparation, has no solid support in high-speed countercurrent chromatography, ensures no loss of compound amount and unchanged proportion, has simple and quick operation process, and is suitable for research on multi-component pharmacodynamic substances in the traditional Chinese medicine extract and improvement research on pharmaceutical technology.
Drawings
FIG. 1 shows logK of the extract of the Chinese herbs in example 1 of the present invention in isopropanol-n-octanol-0.1 mol/L phosphate buffer 25:7:25(v/v/v)2Value sum logP2Linear relationship of values (theoretical calculations);
FIG. 2 shows logK of the extract of the Chinese herbs in example 2 of the present invention in isopropanol-n-octanol-0.3 mol/L phosphate buffer 25:7:25(v/v/v)2Value and logP in n-octanol/water 1:1 system2A linear relationship of values;
FIG. 3 shows logK of the extract of Chinese herbs in isopropyl alcohol-n-octanol-0.1 mol/L phosphate buffer 0:25:25(v/v/v) according to example 3 of the present invention2Value and logP in n-octanol/water 1:1 system2Linear relationship of values.
Detailed Description
The present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.
The raw materials in the examples of the present application were all purchased commercially, unless otherwise specified.
Example 1:
this example is the bulk preparation of an orally absorbed population of ingredients in a herbal extract.
(1) Preparation of Chinese medicinal extract
Weighing the standard substance of the traditional Chinese medicine compound (table 1), dissolving in 75% methanol solution to prepare about 1mg/mL stock solution, and mixing 200 μ L of the stock solution to prepare the mixed solution of the traditional Chinese medicine compound.
TABLE 1 list of Chinese medicinal compounds
(2) Selection and preparation of a liquid/liquid two-phase System I
According to empirical values in the prior art, the logP of orally absorbed ingredients1The liquid/liquid two-phase system i was determined to be isopropanol-n-octanol-0.1 mol/L phosphate buffer ratio 25:7:25 (v/v/v).
Respectively taking isopropanol, n-octanol and 0.1mol/L phosphate buffer solution, adding the isopropanol, the n-octanol and the phosphate buffer solution into a separating funnel according to a proportion, shaking up, standing and layering.
(3) Distribution coefficient logK of Chinese medicinal compound2And logP2
Taking 50 mu L of the traditional Chinese medicine compound mixture solution, drying by nitrogen drying, adding 2mL of each of the upper phase and the lower phase in the liquid/liquid two-phase system I, ultrasonically dissolving, centrifuging at 6000rpm for 10 minutes, and respectively transferring 1.4mL of each of the upper layer solution and the lower layer solution in a centrifuge tube to a new centrifuge tube. Nitrogen was blown dry, 1mL of methanol was added, dissolved by sonication, vortexed for 30 seconds, centrifuged at 6000rpm for 10 minutes, and 0.7mL of the supernatant in the centrifuge tube was transferred to a fresh tube. Drying with nitrogen, adding 100 μ L methanol, ultrasonic dissolving, centrifuging at 6000rpm for 10 min, transferring 50 μ L supernatant in centrifuge tube to liquid phase analysis bottle, and analyzing with 5 μ L sample to obtain logK of each compound of Chinese medicinal materials2Values, as shown in table 1.
logP is obtained by calculating the structure of the compound2Values, in particular, logP of each compound of the Chinese medicine was obtained from the database "national library of medicine of America2Theoretical predicted values are shown in table 1.
Fitting logK2And logP2As shown in fig. 1:
Y＝2.7862X-0.0395 (1)
R2＝0.9354wherein Y is logP and X is logK
logP according to optimal oral absorption1Substituting the value 1.8 into the above equation (1) to determine logK1When K is 0.66, K is 4.6, and K is 4.6 + -0.9 when K value is 20% of the range.
(4) High-speed countercurrent chromatography parameter optimization
Injecting the liquid/liquid two-phase system I in the step (2) into a high-speed counter-current chromatograph, wherein the column volume is 280mL, and the optimization conditions are as follows: the upper phase is a mobile phase, the lower phase is a stationary phase, the rotating speed of a host is 800rpm, the flow rate (v) of the mobile phase is 2mL/min, after the dynamic balance of the whole system is established, 5mL of a traditional Chinese medicine standard mixture is injected through a sample injection valve, and the solvent is 50% isopropanol. Stationary phase retention (Sf) is: 20 percent.
(5) Intestinal absorption component group collection of Chinese medicinal compounds
According to the formula: rt Vr/v (VsK + Vm)/v (2)
Vs＝280×Sf；
Vm＝280-Vs；
Wherein Rt is the compound retention time; vr: compound retention volume; v: a mobile phase flow rate; vs: the stationary phase retention volume; k: the partition coefficient of the compound in the system is defined by logK1Conversion is carried out to obtain; vm: volume of mobile phase in chromatography column; sf: a stationary phase retention value;
and substituting the K value, the stationary phase retention value (Sf) and the mobile phase flow rate (v) into the formula (2) to determine that the starting and ending time of collection is 216-266 minutes.
TABLE 2 Overall preparation of oral absorption component groups of Chinese medicinal compounds
| Collection time (min) | K | Vm(mL) | Vs(mL) | Vr(mL) | V(mL/min) | Rt(min) |
| Starting time | 3.7 | 224 | 56 | 431 | 2 | 216 |
| End time | 5.5 | 224 | 56 | 532 | 2 | 266 |
Example 2:
this example is an integrated preparation of small intestine absorptive component groups of Chinese herbal compounds.
(1) Preparation of Chinese medicinal extract
Weighing the standard substance of the traditional Chinese medicine compound (table 3), dissolving in 75% methanol solution to prepare about 1mg/mL stock solution, and mixing 200 μ L of the stock solution to prepare the mixed solution of the traditional Chinese medicine compound.
TABLE 3 list of Chinese medicinal compounds
| Name of Chinese | English | Molecular formula | Mass to charge ratio | logP2 | logK2 |
| 3, 5-Dihydroxybenzoic acid | 3,5-Dihydroxybenzoic acid | C7H6O4 | 155.0339 | 0.33 | 1.07 |
| 3, 4-Dihydroxybenzoic acid | 3,4-Dihydroxybenzoic acid | C7H6O4 | 155.0339 | 0.48 | 1.16 |
| 2, 3-dihydroxy benzoic acid | 2,3-Dihydroxybenzoic acid | C7H6O4 | 155.0339 | -0.09 | 1.12 |
| 2, 6-dihydroxy benzoic acid | 2,5-Dihydroxybenzoic acid | C7H6O4 | 155.0339 | -0.74 | 0.26 |
| P-coumaric acid | p-Coumaric acid | C9H8O3 | 163.0401 | 0.25 | 0.95 |
| Caffeic acid methyl ester | Methyl caffeate | C10H10O4 | 193.0555 | 1.28 | 1.54 |
| Apigenin | Apigenin | C15H10O5 | 269.0518 | 1.32 | 2.00 |
| Emodin | Emodin | C15H10O5 | 269.0518 | 0.97 | 1.63 |
| Kaempferol | Kaemferol | C15H10O6 | 285.0463 | 1.33 | 2.17 |
| Eriodictyol | Eriodictyol | C15H12O6 | 287.0616 | 1.20 | 2.02 |
| Hesperidin | Hesperetin | C16H14O6 | 301.041 | 1.22 | 1.94 |
| Gastrodin | Gastrodine | C13H18O7 | 331.1102 | -2.11 | -0.44 |
| Chlorogenic acid | 3-caffeoylquinic acid | C16H18O9 | 353.0945 | -1.18 | 0.73 |
| Ginkgolide A | Ginkgolide A | C20H24O9 | 407.1348 | 0.19 | 1.32 |
| Syringin | Syringin | C17H24O9 | 417.1478 | -1.44 | 0.18 |
| Ginkgolide B | Ginkgolide B | C20H24O10 | 423.1375 | 0.34 | 1.48 |
| Ginkgolide C | Ginkgolide C | C20H24O11 | 439.1322 | -0.19 | 0.30 |
| Robinin | Robinin | C33H40O19 | 739.2091 | -0.94 | 0.19 |
| Cytosine nucleotide | Cytidine | C9H13N3O5 | 244.0937 | -2.07 | -1.07 |
| Osthole | Osthole | C15H16O3 | 245.1215 | 1.45 | 2.23 |
| Adenine nucleotide | Adenosine | C10H13N5O4 | 268.1062 | -0.93 | 0.15 |
| Guanine nucleotide | Guanosine | C10H13N5O5 | 284.0999 | -1.47 | -0.73 |
| Tanshinone IIA | Tanshinone IIA | C19H18O3 | 317.1949 | 1.13 | 2.76 |
| Bavachidin | Psoralidin | C20H16O5 | 359.2038 | 0.07 | 2.24 |
| Icariin | Icariin | C33H40O15 | 677.2518 | 0.97 | 1.71 |
| Andrographolide | Andrographolide | C20H30O5 | 701.4309 | 1.58 | 2.30 |
| Epimedin B | Epimedin B | C38H48O19 | 809.2917 | 0.48 | 1.15 |
| Epimedin C | Epimedin C | C39H50O19 | 823.3071 | 0.57 | 1.13 |
(2) Preparation of liquid/liquid two-phase System I
According to empirical values in the prior art, the logP of the absorbed components of the small intestine1The liquid/liquid two-phase system i was determined to be isopropanol-n-octanol-0.3 mol/L phosphate buffer ratio 25:7:25 (v/v/v).
Respectively taking isopropanol, n-octanol and 0.3mol/L phosphate buffer solution, adding the isopropanol, the n-octanol and the phosphate buffer solution into a separating funnel according to a proportion, shaking up, standing and layering.
(3) Preparation of liquid/liquid two-phase System II
In this example, the liquid/liquid two-phase system II is an n-octanol/water two-phase system.
(4) Distribution coefficient logK of Chinese medicinal compound2And logP2
Taking 50 mu L of the traditional Chinese medicine compound mixture solution, drying by nitrogen drying, adding 2mL of each of the upper phase and the lower phase in the liquid/liquid two-phase system I, ultrasonically dissolving, centrifuging at 6000rpm for 10 minutes, and respectively transferring 1.4mL of each of the upper layer solution and the lower layer solution in a centrifuge tube to a new centrifuge tube. Nitrogen was blown dry, 1mL of methanol was added, dissolved by sonication, vortexed for 30 seconds, centrifuged at 6000rpm for 10 minutes, and 0.7mL of the supernatant in the centrifuge tube was transferred to a fresh tube. Nitrogen is present inDrying by blowing, adding 100 μ L methanol, ultrasonic dissolving, centrifuging at 6000rpm for 10 min, transferring 50 μ L supernatant in centrifuge tube to liquid phase analysis bottle, injecting 5 μ L sample, and analyzing to obtain logK of each compound of Chinese medicinal materials2Values, as shown in table 3.
Determining logP of Chinese medicinal compound in liquid/liquid two-phase system II by the above method2The value is obtained.
Fitting logK2And logP2As shown in fig. 2:
Y＝2.3383X-0.4261 (3)
R20.6365, wherein Y is logP and X is logK
logP according to optimal intestinal absorption1Substituting equation (3) above for 1.35, logK is determined1When K is 0.76, K is 5.8, and K is 5.8 + -1.2 when K value is 20% of the range.
(5) High-speed countercurrent chromatography parameters in accordance with example 1
(6) Collecting Chinese medicinal small intestine absorption component group
In accordance with example 1, the starting and ending time of collection was determined to be 241 to 308 minutes by substituting the K value, stationary phase retention value (Sf), and mobile phase flow rate (v) into equation (2).
TABLE 4 Overall preparation of Chinese medicinal Compound intestinal absorption component groups
| Collection time (min) | K | Vm(mL) | Vs(mL) | Vr(mL) | V(mL/min) | Rt(min) |
| Starting time | 4.6 | 224 | 56 | 482 | 2 | 241 |
| End time | 7.0 | 224 | 56 | 610 | 2 | 308 |
Example 3:
this example is the bulk preparation of a population of intestinal absorption components of a Chinese medicinal compound.
(1) Preparation of Chinese medicinal extract
Weighing the standard substance of the traditional Chinese medicine compound (shown in table 5), dissolving in 75% methanol solution to prepare about 1mg/mL stock solution, and mixing 200 μ L of the stock solution to prepare the mixed solution of the traditional Chinese medicine compound.
TABLE 5 list of Chinese medicinal compounds
| Name of Chinese | Name of English | Molecular formula | Mass to charge ratio | logP2 | LogK2 |
| Cytosine nucleotide | Cytidine | C9H13N3O5 | 244.0928 | -2.1 | -1.9 |
| Adenine nucleotide | Adenosine | C10H13N5O4 | 268.1040 | -1.1 | -1.3 |
| Guanine nucleotide | Guanosine | C10H13N5O5 | 284.0989 | -2.7 | -2.0 |
| 3, 5-Dihydroxybenzoic acid | 3,5-Dihydroxybenzoic acid | C7H6O4 | 155.0339 | -1.8 | -1.4 |
| 3, 4-Dihydroxybenzoic acid | 3,4-Dihydroxybenzoic acid | C7H6O4 | 155.0339 | -0.8 | -1.2 |
| 2, 3-dihydroxy benzoic acid | 2,3-Dihydroxybenzoic acid | C7H6O4 | 155.0339 | -0.1 | -1.5 |
| 2, 6-dihydroxy benzoic acid | 2,5-Dihydroxybenzoic acid | C7H6O4 | 155.0339 | 0.9 | 1.0 |
| 2, 5-Dihydroxybenzoic acid | 2,6-Dihydroxybenzoic acid | C7H6O4 | 155.0339 | -0.7 | -1.7 |
| Apigenin | Apigenin | C15H10O5 | 271.1338 | 1.7 | 1.5 |
| Kaempferol | Kaemferol | C15H10O6 | 287.1348 | 1.9 | 1.6 |
| Eriodictyol | Eriodictyol | C15H12O6 | 289.1441 | 2.0 | 2.5 |
| Quercetin | Quercetin | C15H10O7 | 303.1713 | 2.4 | 1.9 |
| Robinin | Robinin | C33H40O19 | 741.4328 | -0.2 | -0.7 |
| Osthole | Osthole | C15H16O3 | 245.1172 | 3.8 | 3.8 |
| Emodin | Emodin | C15H10O5 | 271.0601 | 1.7 | 1.7 |
| Tanshinone IIA | Tanshinone IIA | C19H18O3+Na | 317.1949 | 4.3 | 4.3 |
| P-coumaric acid | p-Coumaric acid | C9H8O3 | 165.0546 | 0.0 | -0.8 |
| Caffeic acid methyl ester | Methyl caffeate | C10H10O4 | 195.0652 | 1.5 | 1.2 |
| Gastrodin | Gastrodine | C13H18O7+Na | 309.0548 | -0.8 | -1.5 |
| Chlorogenic acid | 5-caffeoylquinic acid | C16H18O11 | 355.1024 | -1.3 | -1.6 |
| Syringin | Syringin | C17H24O9+Na | 395.1313 | 1.7 | -1.1 |
| Icariin | Icariin | C33H40O15 | 677.244 | 0.1 | 1.9 |
| Epimedin B | Epimedin B | C38H48O19 | 809.2717 | 0.6 | 1.5 |
| Epimedin C | Epimedin C | C39H50O19 | 823.3013 | 0.7 | 1.0 |
| Ginkgolide A | Ginkgolide A | C20H24O9 | 409.3344 | 0.3 | 0.6 |
| Ginkgolide B | Ginkgolide B | C20H24O10 | 425.1442 | 2.2 | 0.2 |
| Ginkgolide C | Ginkgolide C | C20H24O11 | 441.3362 | 5.3 | -0.1 |
| Andrographolide | Andrographolide | C20H30O5+Na | 373.1985 | 4.7 | - |
| Lang Du Ning B | Chamaejasmenin B | C32H26O10 | 569.1453 | 5.9 | - |
| Bavachidin | Psoralidin | C20H16O5 | 337.1071 | -2.1 | - |
| Tripterine | Celastrol | C29H38O4 | 451.2843 | -1.1 | - |
(2) Selection and preparation of a liquid/liquid two-phase System I
According to empirical values in the prior art, the logP of the intestinal absorption component1The liquid/liquid two-phase system i was determined to be isopropanol-n-octanol-0.1 mol/L phosphate buffer ratio 0:25:25 (v/v/v).
Respectively taking isopropanol, n-octanol and 0.1mol/L phosphate buffer solution, adding the isopropanol, the n-octanol and the phosphate buffer solution into a separating funnel according to a proportion, shaking up, standing and layering.
(3) Distribution coefficient logK of Chinese medicinal compound2And logP2
Taking 50 mu L of the traditional Chinese medicine compound mixture solution, drying by nitrogen drying, adding 2mL of each of the upper phase and the lower phase in the liquid/liquid two-phase system I, ultrasonically dissolving, centrifuging at 6000rpm for 10 minutes, and respectively transferring 1.4mL of each of the upper layer solution and the lower layer solution in a centrifuge tube to a new centrifuge tube. Nitrogen was blown dry, 1mL of methanol was added, dissolved by sonication, vortexed for 30 seconds, centrifuged at 6000rpm for 10 minutes, and 0.7mL of the supernatant in the centrifuge tube was transferred to a fresh tube. Drying with nitrogen, adding 100 μ L methanol, ultrasonic dissolving, centrifuging at 6000rpm for 10 min, transferring 50 μ L supernatant in centrifuge tube to liquid phase analysis bottle, and analyzing with 5 μ L sample to obtain logK of each compound of Chinese medicinal materials2Values, as shown in table 5.
logP is obtained by calculating the structure of the compound2Values, in particular, logP of each compound of the Chinese medicine was obtained from the database "national library of medicine of America2Theoretical predicted values are shown in table 5.
Fitting logK2And logP2As shown in fig. 3:
Y＝0.7003X+0.04 (4)
R20.6299, wherein Y is logP and X is logK
logP according to intestinal absorption1Substituting the above equation (4) for a value of 1.32, logK is determined1When K is 1.83, K is 67.6, and K is 67.6 + -13.5 when K value is 20% of the range.
(4) High-speed countercurrent chromatography parameter optimization is consistent with example 1
Except that the mobile phase flow rate (v) was 3 mL/min.
(5) Intestinal absorption component group collection of Chinese medicinal compounds
In accordance with example 1, the starting and ending time of collection was determined to be 1085 to 1589 minutes by substituting the K value, the stationary phase retention value (Sf), and the mobile phase flow rate (v) into the above equation (2).
TABLE 6 Overall preparation of oral absorption component groups of Chinese medicinal compounds
| Collection time (min) | K | Vm(mL) | Vs(mL) | Vr(mL) | V(mL/min) | Rt(min) |
| Starting time | 54.1 | 224 | 56 | 4144 | 3 | 1085 |
| End time | 81.1 | 224 | 56 | 6104 | 3 | 1589 |
Example 4:
this example is the overall preparation of the absorption component group of the nervous system of the chuanxiong rhizome extract.
(1) Preparation of Ligusticum wallichii extract
Pulverizing rhizoma Ligustici Chuanxiong 2kg, soaking in 8 times of 70% ethanol solution overnight, reflux extracting for 2 hr, filtering, extracting residue once again, filtering, mixing extractive solutions, and concentrating under reduced pressure to obtain rhizoma Ligustici Chuanxiong extract.
(2) Preparation of liquid/liquid two-phase System I
logP of the neuroabsorptive component according to empirical values in the prior art1The liquid/liquid two-phase system i was determined to be isopropanol-n-octanol-0.1 mol/L phosphate buffer ratio 25:7:25 (v/v/v).
Respectively taking isopropanol, n-octanol and 0.1mol/L phosphate buffer solution, adding the isopropanol, the n-octanol and the phosphate buffer solution into a separating funnel according to a proportion, shaking up, standing and layering.
(3) Partition coefficient logK of compound in rhizoma Ligustici Chuanxiong extract2And logP2The linear relationship of (c).
Since the liquid-liquid two-phase system I of this example is identical to the liquid-liquid two-phase system I of example 1, logK in this example2And logP2The linear relationship of (a) is in accordance with example 1.
logP according to optimal nervous system uptake1Substituting equation (1) for 2.7, logK is determined1When K is 0.98, K is 9.5, and K is within 9.5 +/-1.9 to prepare oral absorption component group.
(4) High-speed countercurrent chromatography parameters in accordance with example 1
(5) Collecting nervous system absorption component groups of rhizoma Ligustici Chuanxiong extract
In accordance with example 1, the starting and stopping time of collection was determined to be 325 to 431 minutes by substituting the K value, stationary phase retention value (Sf), and mobile phase flow rate (v) into equation (2).
TABLE 7 Whole preparation of Ligusticum chuanxiong Hort extract nervous system absorption component groups and collection start and stop time
| Collection time (min) | K | Vm(mL) | Vs(mL) | Vr(mL) | V(mL/min) | Rt(min) |
| Starting time | 7.6 | 224 | 56 | 655 | 2 | 325 |
| End time | 11.4 | 224 | 56 | 868 | 2 | 431 |
Finally, 13 ligustilide components are prepared integrally, and the compounds are reported to be absorbed by the nervous system and are main drug-effect substances of the ligusticum wallichii.
TABLE 8 Overall preparation of Ligusticum chuanxiong Hort extract with nervous system absorptive component group
| Serial number | Name (R) | Molecular formula | | logP | 2 |
| 1 | senkynolide A | C12H16O2 | 192.2542 | 2.7 | |
| 2 | senkyunolide E | C12H12O3 | 204.0786 | 2.7 | |
| 3 | senkyunolide F | C12H14O3 | 206.0943 | 2.7 | |
| 4 | senkyunolide G | C12H16O3 | 208.1099 | 2.7 | |
| 5 | senkyunolide N | C12H18O4 | 226.1205 | 2.7 | |
| 6 | senkyunolide L | C12H15ClO3 | 242.0710 | 2.7 | |
| 7 | senkyunolide M | C16H22O4 | 278.1518 | 2.7 | |
| 8 | senkyunolide Q | C16H22O4 | 278.1518 | 2.56 | |
| 9 | senkyunolide R | C16H22O5 | 240.0998 | 2.7 | |
| 10 | butylphthalide | C12H14O2 | 190.0994 | 2.9 | |
| 11 | (3S)-3-butyl-4-hydroxyphthalide | C12H14O3 | 206.0943 | 2.77 | |
| 12 | 4,7-dihydroxy-3-butyl Phthalide | C12H14O4 | 222.0892 | 2.27 | |
| 13 | Cnidilide | C12H18O2 | 194.1307 | 2.45 |
The above description is only for the purpose of illustrating the present invention and is not intended to limit the present invention in any way, and the present invention is not limited to the above description, but rather should be construed as being limited to the scope of the present invention.
Claims (8)
1. A method for preparing a Chinese medicinal extract absorption component group is characterized by comprising the following steps:
step 1, preparing a liquid-liquid two-phase system I;
step 2, obtaining the distribution coefficient logK of the absorption component group to be prepared in the liquid-liquid two-phase system I1A range value;
step 3, according to the distribution coefficient logK1The range value and the stationary phase retention value of the liquid-liquid two-phase system I in the high-speed counter-current chromatograph are used for obtaining the collection time of the absorption component group to be prepared;
step 4, preparing the absorption component group in the traditional Chinese medicine extract by adopting a high-speed counter-current chromatography according to the collection time of the absorption component group to be prepared;
the step 2 specifically comprises the following steps:
step 2.1, measuring the distribution coefficient logK of the compound in the liquid-liquid two-phase system I2；
Step 2.2, obtaining the distribution coefficient logP of the compound in the liquid-liquid two-phase system II2；
Step 2.3, fitting logK2And logP2A linear relationship of values;
step 2.4, according to the theoretical distribution coefficient logP of the absorption component group in the liquid-liquid two-phase system II1Obtaining the distribution coefficient logK of the absorption component group to be prepared in the liquid-liquid two-phase system I through the linear relation obtained in the step 2.31A range value.
2. The method for preparing Chinese medicinal extract absorbable component groups according to claim 1, wherein the liquid-liquid two-phase system I comprises three components, and each component satisfies the following proportional relationship:
the volume ratio of the component A to the component B to the component C is (0-25): (25-7): 25;
wherein the component A is acetonitrile and/or isopropanol; the component B is n-octanol; the component C is 0.1-0.3 mol/L phosphate buffer solution.
3. The method for preparing a herbal extract absorbing ingredient set as claimed in claim 1, wherein the liquid-liquid two-phase system II is n-octanol/water two-phase system.
4. The method of claim 1, wherein the herbal extract is a mixture of herbal standards or one or more of a mixture of secondary metabolites extracted from herbs, herbal compounds or non-medicinal plants.
5. The method for preparing herbal extract absorptive component groups according to claim 1, wherein the preparation of the liquid-liquid two-phase system I is preceded by at least the following steps:
step S1, determining the distribution coefficient logK of at least 20 compounds in the preset liquid-liquid two-phase system I2A value;
step S2, obtaining the distribution coefficient logP of the at least 20 compounds in the liquid-liquid two-phase system II2A value;
step S3, fitting logK2Value sum logP2Linear relation of values, when the coefficient of correlation R of said linear relation2When the current value is greater than the first preset value, continuing the next step; when the correlation coefficient R of the linear relation2When the value is less than or equal to the first preset value, repeating the step S1;
step S4, measuring the stationary phase retention rate of the preset liquid-liquid two-phase system I in the high-speed counter-current chromatography, and when the stationary phase retention rate of the preset liquid-liquid two-phase system I in the high-speed counter-current chromatography is larger than a second preset value, determining that the preset liquid-liquid two-phase system I is the liquid-liquid two-phase system I in the method for preparing the absorption component group in the traditional Chinese medicine extract; and when the stationary phase retention rate of the preset liquid-liquid two-phase system I in the high-speed counter-current chromatography is less than or equal to a second preset value, repeating the step S1.
6. The method of claim 5, wherein the first predetermined value is 0.6.
7. The method of claim 5, wherein the second predetermined value is 20%.
8. The method for preparing a group of absorption components of chinese herbal medicine extract as set forth in claim 5, wherein the high-speed countercurrent chromatography is a J-type high-speed countercurrent chromatography, and a ratio of a rotation radius and a revolution radius of the spiral tube is 0.5 to 0.8.
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| CN101249316A (en) * | 2007-11-20 | 2008-08-27 | 浙江大学 | Back-stepping eluting adverse current chromatograph method |
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| CN101249316A (en) * | 2007-11-20 | 2008-08-27 | 浙江大学 | Back-stepping eluting adverse current chromatograph method |
| CN104707358A (en) * | 2013-12-13 | 2015-06-17 | 中国科学院大连化学物理研究所 | Novel double water phase high-speed countercurrent chromatography |
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