CN1566942A - Method for drafting traditional Chinese medicinal spectrum by employing high-speed reverse-flow chromatogram - Google Patents
Method for drafting traditional Chinese medicinal spectrum by employing high-speed reverse-flow chromatogram Download PDFInfo
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Abstract
This invention relates to a traditional medicine fingerprint pattern preparation method by use of high speed counter-current chromatogram, which comprises the following: to prepare gross extracted materials of each batch of the medicines; to determine the main compound types through TLC; to choose solvent system for HSCCC; to purify the gross extracted materials of the traditional medicines by high speed counter-current chromatogram device; to detect eluent by ultraviolet detector and draw a relationship chart of ultraviolet absorption and elution time; to determine corresponding property of each group of gross extracted materials through HPLC and UV-Vis; to judge common fringe peak; to synthesize traditional medicine fringe atlas methodology reference data ,that is to form fringe atlas. The accuracy and repeatability of this method are apparently superior to TLC and solve the problem that HPLC can barely separate and analyze sample with high viscosity and easy filled and absorbed and expensive devices and materials.
Description
Technical field
The present invention relates to a kind of quality analysis of traditional Chinese medicine method, relate to a kind of method of using high speed adverse current chromatogram to work out traditional Chinese medicine fingerprint specifically.
Technical background
The Chinese crude drug raw material is from natural, mostly being artificial dispersion gathers, processes, be subjected to weather, regional difference and artifical influence factor very big, the standardization of raw material and semi-manufacture and end product quality, standardized management gap are bigger, for the homogeneous that guarantees product quality and stable, need to set up strict quality monitoring standard and good method for supervising.General analytical technology can only characterize the part chemical information, for example comprises not principal component of known active component, known non-active ingredient, a part.Know that now the drug effect of Chinese medicine is not from any single active component, but by the various active composition, even relevant with synergy or " the giving birth to the gram effect " of " non-active ingredient ", so the chemical information of Chinese medicine has certain ambiguity.Traditional Chinese medicine fingerprint is exactly by adopting one or more detection techniques, and the chemical information of the Chinese medicine mode with figure is characterized and described, and promptly draws traditional Chinese medicine fingerprint.Traditional Chinese medicine fingerprint not only possesses individual absolute uniqueness, also possess the uniqueness of species feature and the similarity between the individuality of the same race, the quality control index that makes Chinese medicine rises to detection to whole Chinese medicine interior quality from original mensuration to certain several component content, the interior quality that can show Chinese medicine to greatest extent, although for a certain concrete composition of Chinese medicine be what we may be still unclear, but can be with this important content as control and measurement traditional Chinese medicine quality standard, and make traditional Chinese medicine research more meet the organic conception of the traditional Chinese medical science, improved the good and bad standard of differentiating of the Chinese medicine true and false.
Analytical technology commonly used at present comprises the coupling of spectrum, wave spectrum, chromatogram, nuclear magnetic resonance, X-ray diffraction and various technology etc.Wherein chromatogram is the most frequently used method of working out finger-print.Chang Yong chromatographic technique comprises the most: (1) thin-layer chromatography (TLC): this method is easy and simple to handle, no matter in drug compound have or not uv absorption or volatility, all can carry out qualitatively with TLC, but this method precision is relatively poor; (2) high performance liquid chromatography (HPLC): this method goes for middle drug compound of different nature according to included detecting device difference, for example uses UV-detector to go for the middle drug compound of uv absorption; Use evaporation laser light scattering or differential detecting device go for a little less than the uv absorption or do not have the middle drug compound of uv absorption; This method is applicable to compounds such as analysis/separating bio alkali, glucoside, anthraquinone, organic acid, phenols, lactone, and the precision height, favorable reproducibility; But this method can't the higher sample of analysis of viscosity, analyzes that flavonoids is this easily to be acquired a certain degree of difficulty by the component of filling adsorption, and instrument and consumptive material costliness, is unfavorable for promoting; (3) gas chromatography (GC): the scope of application of this method also is limited, and its range of application and HPLC complementation are applicable to alkaloid, fat, lactone, phenol, sugar, animal kind medicine thing behind volatile ingredient and the derivatization.
High speed adverse current chromatogram (HSCCC) is a kind of chromatographic technique that develops recently.Its principle such as document: Sun Xuefei. the progress of adverse current chromatogram. foreign medical science pharmacy fascicle .1990,18 (1), 40-43 is described, and high speed adverse current chromatogram is made separating column with polytetrafluoroethylhelix helix tube, without any solid-state supporter or filler; According to the physicochemical characteristics of separated potpourri, select a kind of organic/water two phase solvent system or aqueous two-phase dicyandiamide solution, this system can be a binary or polynary; With the going up mutually or, at first it being filled with in the pipe of this system down as the stationary phase of chromatographic process, make tubing string do planetary rotatablely moving then, comprise 2 kinds of motions: a kind of be along self spool rotation---rotation, it makes the abundant mixing of two-phase of distribution; Another kind is the rotation along the centrifugal axis of centres---revolution, and it keeps stationary phase; If at this moment with in the dicyandiamide solution another as moving phase, push the separation tubing string with biased sample by the pressure of pump, sample will pass the whole tubing string space of two liquid phase convection current, each component is distributed on the surface of two-phase particulate, through thousands of time ground, efficiently, extraction continuously, each component is separated by its partition factor (being the ratio of the solubleness of a certain component in moving phase with its solubleness in stationary phase) in two-phase.This technology has been successfully applied to fields such as biological chemistry, pharmacy, agricultural, environment, material, chemical industry, sea life, and has prepared the hundreds of monomer components of class, Polyphenols, catechin, polysaccharide, glycoprotein that comprised flavonoids, anthraquinone class, saponin class, macrolide.
Summary of the invention
The objective of the invention is to overcome prior art when working out traditional Chinese medicine fingerprint, TLC precision and reappearance are relatively poor, HPLC is difficult to analyze/separating high-viscosity and and easily by the defective of the sample of filling adsorption and instrument consumptive material costliness, thereby provide a kind of equipment and consumptive material price economy, be easy to promote, use high speed adverse current chromatogram to work out the method for traditional Chinese medicine fingerprint.
The objective of the invention is to realize by following technical scheme:
The invention provides a kind of method of using high speed adverse current chromatogram to work out traditional Chinese medicine fingerprint, comprise the steps:
1) prepares the crude extract of each batch Chinese crude drug: adopt routine techniques to extract Chinese crude drug, obtain crude extract;
2) determine the main compound type: detect the crude extract that step 1) obtains by thin-layered chromatography (TLC), determine the main compound type that the Chinese crude drug crude extract is comprised;
3) choose dicyandiamide solution: according to step 2) definite main compound type, choose organic phase/organic phase or the organic phase/aqueous phase solvent system that is applicable to HSCCC from following 4 kind solvent systems:
Described 4 kind solvent systems are: the first kind is the hydrophilic solvent system, is made up of the nonaqueous phase and the water of low polarity (polarity is little), and two-phase polarity differs greatly;
Second class is the lipophilic solvent system, is made up of the nonaqueous phase and the water of high polarity, and two-phase polarity is more or less the same;
The 3rd class is the organic phase dicyandiamide solution, constitute, comprise system 1---n-alkane/halogenated hydrocarbons/fatty alcohol or aliphatic ketone, system 2---n-alkane/ethers/fatty alcohol or aliphatic ketone and system 3---n-alkane/fatty ester class/acetonitrile/fatty alcohol or aliphatic ketone fully by organic solvent;
The 4th class is the aqueous two-phase dicyandiamide solution, comprises organic phase/water system: normal hexane/ethanol/water, n-hexane/ethyl acetate/methanol, chloroform/methanol/water, chloroform/methanol/phosphate etc.; Adopt different solvent ratios, go for separating different types of compound, as chloroform: methyl alcohol: water system, when ratio routinely is 13: 7: 8 separable flavonoidss; Be applicable to separating anthraquinone at 6: 3: 2; 5: 4: 3 separable alkaloids; Be applicable to the separation Coumarins at 13: 23: 16;
4) crude extract of use high-speed counter-current chromatograph purifying Chinese crude drug: the solvent that step 3) is selected is fully vibration in separating funnel, and standing demix is told that one deck solvent as stationary phase, and it is pumped into the tubing string of high-speed counter-current chromatograph; Remaining one as moving phase; At 10～30 ℃, open high-speed counter-current chromatograph, adopt the main frame forward or reverse, engine speed is 750～900rpm, and moving phase is pumped in the separating column with the flow velocity of 1.5～2mL/min, whole system is set up mobile equilibrium, from sampling valve implantation step 1) a certain batch of Chinese crude drug crude extract making, each component is separated by its partition factor in two-phase;
5) use UV-detector to detect eluent, draw the uv absorption of each component and the graph of a relation of elution time;
6) in elution process, collect each component simultaneously respectively, determine the correspondence of the each component of each batch Chinese crude drug crude extract again in the retention time on the HPLC and the peak value on the UV-Vis spectrogram and shape by it;
7) judge total fingerprint peaks, measure the retention time of each total fingerprint peaks among the HSCCC, calculate relative standard's variance RSD of corresponding fingerprint peaks, should satisfy RSD≤3%, the non-total peak total area must not be greater than 10% of total peak area;
8), need the sample more than 10 batches is detected according to the requirement of national standard about the methodological study data of finger-print; Same test sample continuous sample introduction is more than 5 times, to investigate the precision of instrument; The test sample of same lot number is more than 5 parts, to investigate the reappearance of experimental technique; Through the HSCCC separation and purification, the RSD of the total fingerprint peaks of gained collection of illustrative plates answers≤3% to same test sample at different time; Last these data promptly constitute traditional Chinese medicine fingerprint and methodological study data thereof.
Described Chinese crude drug comprises anthraquinone analog compound, alkaloid compound, flavone compound, terpenoid and oside compound.
The invention has the advantages that:
Use high speed adverse current chromatogram provided by the invention is worked out the method for traditional Chinese medicine fingerprint, first HSCCC is applied to the formulation of traditional Chinese medicine fingerprint, it is important supplement to existing method, compared with the prior art advantage is: high speed adverse current chromatogram does not have solid support, has avoided the harmful effect such as absorption, sex change, inactivation to sample component; Its precision and reappearance obviously are better than TLC; Solved HPLC and be difficult to analyze/separating high-viscosity and and easily by the sample of filling adsorption, easily remarkable advantages is arranged at separating and purifying flavone etc. by the material of filling adsorption; Can the full-bodied material of separation and purification; The efficient of analytic type HSCCC, sensitivity and instrument repeatability are approaching with HPLC; Preparation type HSCCC can obtain the highly purified component of capacity when working out finger-print, identify and active the detection in order to further structure; Because of its unique separation principle, HSCCC only need change solvent system when separating the variety classes material, but not chromatographic column has been avoided degradation problem under the column efficiency, makes instrument and consumptive material price economy, is easy to promote.
Description of drawings
Fig. 1 is the HSCCC collection of illustrative plates of 3 place of production reds sage root among the embodiment 1;
Fig. 1-the 1st wherein originates from the red sage root in Hebei;
Fig. 1-2 is the red sage root that originates from Shandong;
Fig. 1-the 3rd originates from the red sage root in Jiangsu.
Embodiment
1) prepares the crude extract of each batch Chinese crude drug: 10 batches of reds sage root are all adopted following method roughing out: red sage root or rhizome are ground into powder; The 20g danshen powder be dissolved in the 50mL solvent (normal hexane: ethanol=1: 1, V/V), fully stir 45min, get supernatant after the sedimentation; The precipitation be dissolved in again the 25mL solvent (normal hexane: ethanol=1: 1, V/V), fully stir 45min, get supernatant after the sedimentation; Merge twice supernatant, centrifugal 10000g * 10min abandons precipitation, stays supernatant, constant volume; Add the water of 2 times of volumes, in separating funnel, shake up standing over night;
Tell phase (organic phase), constant volume is with 2 times of phases (water) under the washing of the 30wt% of organic phase volume ethanol, until phase (water) is intimate colourless down, concentrate and go up phase, fully dry in vacuum dryer, obtain the crude extract dry powder of the red sage root, slightly carrying yield is 2wt%;
2) determine the main compound type: detect the crude extract that step 1) obtains by thin-layered chromatography (TLC), determine that the main compound type that the Chinese crude drug crude extract is comprised is an anthraquinone analog compound;
3) choose dicyandiamide solution: according to step 2) definite main compound type, preparation high speed adverse current chromatogram mobile phase solvent system A is a normal hexane: ethanol: water=10: 5.5: 4.5 (V/V), dicyandiamide solution B are normal hexane: ethanol: water=10: 7: 3 (V/V);
4) crude extract of the use high-speed counter-current chromatograph purifying red sage root: with dicyandiamide solution A fully vibration in separating funnel of step 3) preparation, standing demix is told that one deck solvent as stationary phase, and it is added the separating column of high-speed counter-current chromatograph; Following to moving phase; At 10～30 ℃, open high-speed counter-current chromatograph, adopt main frame just to change, engine speed is 900rpm, and with the flow velocity of 2mL/min moving phase is pumped in the tubing string, and whole system is set up mobile equilibrium, from sampling valve implantation step 1) a certain batch of Chinese crude drug crude extract making, 0-470min is a moving phase with the following of system A mutually, and being replaced by the following of system B behind the 470min is moving phase mutually, and each component is separated by its partition factor in two-phase;
5) use UV-detector to detect eluent, draw the uv absorption of each component and the graph of a relation of elution time; Each batch red sage root sample all obtains 12 of eluting peaks through the elution curve of high speed adverse current chromatogram separation and purification;
6) in elution process, collect each component simultaneously respectively, determine the correspondence of the each component of each batch Chinese crude drug crude extract again in the retention time on the HPLC and the peak value on the UV-Vis spectrogram and shape by it;
Figure 1 shows that the HSCCC collection of illustrative plates of the red sage root in 3 places of production.As seen from the figure, through the HSCCC purifying, the red sage root in 3 places of production respectively obtains 12 eluting peaks, the number unanimity at peak.Each eluting peak is analyzed: measure the retention time of each eluting peak on reversed-phase high-performance liquid chromatography (HPLC), list in table 1; In ultraviolet-visible spectrophotometer 900-200nm scope each eluting peak is carried out full wavelength scanner, the spectrum that is absorbed is listed in table 2.
The retention time of table 1 HSCCC elution fraction in the HPLC reversed phase chromatography detects
Red sage root Jiangsu, red sage root Shandong, the eluting peak Hebei red sage root
Retention time retention time retention time
(min) (min) (min)
1 24.887 24.939 24.920
2 24.750 24.893 24.987
3 28.917 28.824 28.911
4 28.292 28.250 28.000
5 28.992 28.998 29.993
6 29.129 29.460 29.129
7 30.702 31.246 30.800
8 30.817 30.817 30.944
9 28.402 28.444 28.399
10 32.294 32.287 32.354
11 34.886 34.781 34.936
12 25.170 25.191 25.202
The ultraviolet absorption peak of table 2 HSCCC elution fraction
Red sage root Jiangsu, red sage root Shandong, the eluting peak ultraviolet absorption peak Hebei red sage root
Number wavelength (nm) wavelength (nm) wavelength (nm)
1 1 276.5 277.4 278
2 1 275.9 276.2 276.7
3 1 233 233.2 233.6
2 269.5 269.5 269.1
4 1 240.5 240.2 240.6
2 291 292 292.6
3 332.5 335.4 336
4 417.5 417 416.8
5 1 223.5 224.6 223.6
2 279.5 276.6 278.6
6 1 221.5 220.4 220.8
2 253.5 252.8 253.8
3 270 271 269.8
7 1 219.5 219 219
2 264.5 264.4 264.2
3 363 363.2 361.6
4 452 457 454
8 1 246.5 246 246.2
2 425 425 424.4
9 1 247 247.2 246.8
2 326 325.4 325.8
3 326 325.4 325.8
4 365.5 365.2 367.4
10 1 227 226.8 226.6
2 293.5 292.4 293.4
3 512 512 512.4
11 1 224 223.8 223.8
2 253 252.6 252.6
3 270.5 269.6 269.6
4 356 356.8 357.2
5 468 468 468.6
12 1 275 276.8 274.2
By table 1 and 2 as seen, the peak value of the eluting peak 1 in 3 HSCCC elution curves and 2 retention time and absorption spectrum is consistent respectively, thinks that tentatively eluting peak 1 and 2 is a same substance; From HPLC retention time and ultra-violet absorption spectrum decidable also, the individual eluting peak of n (n is 3-12) in 3 place of production red sage root elution curves is same component.That is to say, the HSCCC wash-out collection of illustrative plates of 3 place of production reds sage root, not only eluting peak number unanimity, and corresponding eluting peak is same component.
7) judge total fingerprint peaks: 12 11 components that eluting peak comprised in 10 wash-out collection of illustrative plates are corresponding respectively, there is not non-total fingerprint peaks, satisfying the non-total peak total area must not be greater than the requirement of total peak area 10%, measure the retention time of each total fingerprint peaks among the HSCCC, relative standard's variance RSD≤3% of corresponding fingerprint peaks as calculated;
8) according to the requirement of national standard, the sample more than 10 batches is detected about the methodological study data of finger-print; Same test sample continuous sample introduction is more than 5 times, to investigate the precision of instrument; The test sample of same lot number is more than 5 parts, to investigate the reappearance of experimental technique; Through the HSCCC separation and purification, the RSD of the total fingerprint peaks of gained collection of illustrative plates answers≤3% to same test sample at different time.
Last these data promptly constitute the finger-print and the methodological study data thereof of the red sage root;
1) prepare the crude extract of each batch Chinese crude drug: the body surface of 10 batches of red earthworms is cleaned up, put that 2h makes it empty gastrovascular cavity in the distilled water, middlely change distilled water 1 time, control is done, and adds equivalent sucrose, makes the earthworm secreting mucus;
2) determine the main compound type: detect the crude extract that step 1) obtains by thin-layered chromatography (TLC), determine that the main compound type that the Chinese crude drug crude extract is comprised is an alkaloid compound;
3) choose dicyandiamide solution: according to step 2) definite main compound type, preparation high speed adverse current chromatogram mobile phase solvent system chloroform: methyl alcohol: water=5: 5: 3 (V/V);
4) crude extract of use high-speed counter-current chromatograph purifying earthworm mucus: with dicyandiamide solution A fully vibration in separating funnel of step 3) preparation, standing demix is told that one deck solvent as stationary phase, and it is added the separating column of high-speed counter-current chromatograph; Following to moving phase; At 10～30 ℃, open high-speed counter-current chromatograph, adopt main frame just to change, engine speed is 800rpm, and moving phase is pumped in the tubing string with the flow velocity of 1.7mL/min, whole system is set up mobile equilibrium, from sampling valve implantation step 1) a certain batch of Chinese crude drug crude extract making, each component is separated by its partition factor in two-phase;
5) use UV-detector to detect eluent, draw the uv absorption of each component and the graph of a relation of elution time; Each batch earthworm mucus sample all obtains 5 of eluting peaks through the elution curve of high speed adverse current chromatogram separation and purification;
6) in elution process, collect each component simultaneously respectively, determine the correspondence of the each component of each batch Chinese crude drug crude extract again in the retention time on the HPLC and the peak value on the UV-Vis spectrogram and shape by it;
7) judge total fingerprint peaks: 5 eluting peaks in 10 wash-out collection of illustrative plates are corresponding respectively, there is not non-total fingerprint peaks, satisfying the non-total peak total area must not be greater than the requirement of total peak area 10%, measure the retention time of each total fingerprint peaks among the HSCCC, relative standard's variance RSD≤3% of corresponding fingerprint peaks as calculated;
8) according to the requirement of national standard, the sample more than 10 batches is detected about the methodological study data of finger-print; Same test sample continuous sample introduction is more than 5 times, to investigate the precision of instrument; The test sample of same lot number is more than 5 parts, to investigate the reappearance of experimental technique; Through the HSCCC separation and purification, the RSD of the total fingerprint peaks of gained collection of illustrative plates answers≤3% to same test sample at different time.
Last these data promptly constitute the finger-print and the methodological study data thereof of the red sage root;
1) prepare the crude extract of each batch Chinese crude drug: 10 batches of saussurea involucrata discard the sherwood oil leaching liquid, and saussurea involucrata are volatilized with sherwood oil cold soaking 6 days, with 60% ethanol cold soaking 7 days, filter the ethanol leaching liquid, Rotary Evaporators suitably concentrates, remove chlorophyll, be condensed into pale brown look medicinal extract;
2) determine the main compound type: detect the crude extract that step 1) obtains by thin-layered chromatography (TLC), determine that the main compound type that the Chinese crude drug crude extract is comprised is a flavone compound;
3) choose dicyandiamide solution: according to step 2) definite main compound type, preparation high speed adverse current chromatogram mobile phase solvent system chloroform: methyl alcohol: water=4: 3: 2 (V/V);
4) crude extract of use high-speed counter-current chromatograph purifying saussurea involucrata flavone compound: with dicyandiamide solution A fully vibration in separating funnel of step 3) preparation, standing demix, tell that one deck solvent, it is added the separating column of high-speed counter-current chromatograph as stationary phase; Following to moving phase; At 10～30 ℃, open high-speed counter-current chromatograph, adopt main frame just to change, engine speed is 850rpm, and moving phase is pumped in the tubing string with the flow velocity of 2mL/min, whole system is set up mobile equilibrium, from sampling valve implantation step 1) a certain batch of Chinese crude drug crude extract making, each component is separated by its partition factor in two-phase;
5) use UV-detector to detect eluent, draw the uv absorption of each component and the graph of a relation of elution time; Each batch saussurea involucrata flavone compound sample all obtains 6 of eluting peaks through the elution curve of high speed adverse current chromatogram separation and purification;
6) in elution process, collect each component simultaneously respectively, determine the correspondence of the each component of each batch Chinese crude drug crude extract again in the retention time on the HPLC and the peak value on the UV-Vis spectrogram and shape by it;
7) judge total fingerprint peaks: 6 eluting peaks in 10 wash-out collection of illustrative plates are corresponding respectively, there is not non-total fingerprint peaks, satisfying the non-total peak total area must not be greater than the requirement of total peak area 10%, measure the retention time of each total fingerprint peaks among the HSCCC, relative standard's variance RSD≤3% of corresponding fingerprint peaks as calculated;
8) according to the requirement of national standard, the sample more than 10 batches is detected about the methodological study data of finger-print; Same test sample continuous sample introduction is more than 5 times, to investigate the precision of instrument; The test sample of same lot number is more than 5 parts, to investigate the reappearance of experimental technique; Through the HSCCC separation and purification, the RSD of the total fingerprint peaks of gained collection of illustrative plates answers≤3% to same test sample at different time.
Last these data promptly constitute the finger-print and the methodological study data thereof of the red sage root;
1) prepare the crude extract of each batch Chinese crude drug: 10 batches of saussurea involucrata discard the sherwood oil leaching liquid, and saussurea involucrata are volatilized with sherwood oil cold soaking 6 days, with 95% ethanol cold soaking 7 days, filter the ethanol leaching liquid, Rotary Evaporators suitably concentrates, remove chlorophyll, be condensed into pale brown look medicinal extract;
2) determine the main compound type: detect the crude extract that step 1) obtains by thin-layered chromatography (TLC), determine that the main compound type that the Chinese crude drug crude extract is comprised is a terpenoid;
3) choose dicyandiamide solution: according to step 2) definite main compound type, preparation high speed adverse current chromatogram mobile phase solvent system chloroform: methyl alcohol: water=9: 12: 8 (V/V);
4) crude extract of use high-speed counter-current chromatograph purifying saussurea involucrata terpenoid: with dicyandiamide solution A fully vibration in separating funnel of step 3) preparation, standing demix, tell that one deck solvent, it is added the separating column of high-speed counter-current chromatograph as stationary phase; Following to moving phase; At 10～30 ℃, open high-speed counter-current chromatograph, adopt main frame just to change, engine speed is 850rpm, and moving phase is pumped in the tubing string with the flow velocity of 1.5mL/min, whole system is set up mobile equilibrium, from sampling valve implantation step 1) a certain batch of Chinese crude drug crude extract making, each component is separated by its partition factor in two-phase;
5) use UV-detector to detect eluent, draw the uv absorption of each component and the graph of a relation of elution time; Each batch saussurea involucrata terpenoid sample all obtains 14 of eluting peaks through the elution curve of high speed adverse current chromatogram separation and purification;
6) in elution process, collect each component simultaneously respectively, determine the correspondence of the each component of each batch Chinese crude drug crude extract again in the retention time on the HPLC and the peak value on the UV-Vis spectrogram and shape by it;
7) judge total fingerprint peaks: 14 eluting peaks in 10 wash-out collection of illustrative plates are corresponding respectively, there is not non-total fingerprint peaks, satisfying the non-total peak total area must not be greater than the requirement of total peak area 10%, measure the retention time of each total fingerprint peaks among the HSCCC, relative standard's variance RSD≤3% of corresponding fingerprint peaks as calculated;
8) according to the requirement of national standard, the sample more than 10 batches is detected about the methodological study data of finger-print; Same test sample continuous sample introduction is more than 5 times, to investigate the precision of instrument; The test sample of same lot number is more than 5 parts, to investigate the reappearance of experimental technique; Through the HSCCC separation and purification, the RSD of the total fingerprint peaks of gained collection of illustrative plates answers≤3% to same test sample at different time.
Last these data promptly constitute the finger-print and the methodological study data thereof of the red sage root;
1) prepare the crude extract of each batch Chinese crude drug: the pH3.5 ethanol extract of 10 batches of bright balsam pears concentrates;
2) determine the main compound type: detect the crude extract that step 1) obtains by thin-layered chromatography (TLC), determine that the main compound type that the Chinese crude drug crude extract is comprised is an oside compound;
3) choose dicyandiamide solution: according to step 2) definite main compound type, preparation high speed adverse current chromatogram mobile phase solvent system chloroform: methyl alcohol: ethanol: water=5: 3: 1: 3 (V/V);
4) crude extract of use high-speed counter-current chromatograph purifying momordica saponins compounds: with dicyandiamide solution A fully vibration in separating funnel of step 3) preparation, standing demix, tell that one deck solvent, it is added the separating column of high-speed counter-current chromatograph as stationary phase; Following to moving phase; At 10～30 ℃, open high-speed counter-current chromatograph, adopt main frame just to change, engine speed is 900rpm, and moving phase is pumped in the tubing string with the flow velocity of 1.8mL/min, whole system is set up mobile equilibrium, from sampling valve implantation step 1) a certain batch of Chinese crude drug crude extract making, each component is separated by its partition factor in two-phase;
5) use UV-detector to detect eluent, draw the uv absorption of each component and the graph of a relation of elution time; Each batch momordica saponins compounds sample all obtains 5 of eluting peaks through the elution curve of high speed adverse current chromatogram separation and purification;
6) in elution process, collect each component simultaneously respectively, determine the correspondence of the each component of each batch Chinese crude drug crude extract again in the retention time on the HPLC and the peak value on the UV-Vis spectrogram and shape by it;
7) judge total fingerprint peaks: 5 eluting peaks in 10 wash-out collection of illustrative plates are corresponding respectively, there is not non-total fingerprint peaks, satisfying the non-total peak total area must not be greater than the requirement of total peak area 10%, measure the retention time of each total fingerprint peaks among the HSCCC, relative standard's variance RSD≤3% of corresponding fingerprint peaks as calculated;
8) according to the requirement of national standard, the sample more than 10 batches is detected about the methodological study data of finger-print; Same test sample continuous sample introduction is more than 5 times, to investigate the precision of instrument; The test sample of same lot number is more than 5 parts, to investigate the reappearance of experimental technique; Through the HSCCC separation and purification, the RSD of the total fingerprint peaks of gained collection of illustrative plates answers≤3% to same test sample at different time.
Last these data promptly constitute the finger-print and the methodological study data thereof of the red sage root;
Claims (7)
1, a kind of method of using high speed adverse current chromatogram to work out traditional Chinese medicine fingerprint comprises the steps:
1) prepares the crude extract of each batch Chinese crude drug: adopt routine techniques to extract Chinese crude drug, obtain crude extract;
2) determine the main compound type: detect the crude extract that step 1) obtains by thin-layered chromatography, determine the main compound type that the Chinese crude drug crude extract is comprised;
3) choose dicyandiamide solution: according to step 2) definite main compound type, choose organic phase/organic phase or the organic phase/aqueous phase solvent system that is applicable to HSCCC from following 4 kind solvent systems:
Described 4 kind solvent systems are:
The first kind is the hydrophilic solvent system, is made up of nonaqueous phase and water that polarity is little, and two-phase polarity differs greatly;
Second class is close ester dicyandiamide solution, is made up of the nonaqueous phase and the water of high polarity, and two-phase polarity is more or less the same;
The 3rd class is the organic phase dicyandiamide solution, constitute, comprise system 1---n-alkane/halogenated hydrocarbons/fatty alcohol or aliphatic ketone, system 2---n-alkane/ethers/fatty alcohol or aliphatic ketone and system 3---n-alkane/fatty ester class/acetonitrile/fatty alcohol or aliphatic ketone fully by organic solvent;
The 4th class is the aqueous two-phase dicyandiamide solution, comprising: normal hexane/ethanol/water, n-hexane/ethyl acetate/methanol, chloroform/methanol/water, chloroform/methanol/phosphate;
4) crude extract of use high-speed counter-current chromatograph purifying Chinese crude drug: the solvent that step 3) is selected is fully vibration in separating funnel, and standing demix is told that one deck solvent as stationary phase, and it is added the separating column of high-speed counter-current chromatograph; Remaining one as moving phase; At 10～30 ℃, open high-speed counter-current chromatograph, adopt the main frame forward or reverse, engine speed is 750～900rpm, and moving phase is pumped in the separating column with the flow velocity of 1.5～2mL/min, whole system is set up mobile equilibrium, from sampling valve implantation step 1) a certain batch of Chinese crude drug crude extract making, each component is separated by its partition factor in two-phase;
5) use UV-detector to detect eluent, draw the uv absorption of each component and the graph of a relation of elution time;
6) in elution process, collect each component simultaneously respectively, determine the correspondence of the each component of each batch Chinese crude drug crude extract again in the retention time on the HPLC and the peak value on the UV-Vis spectrogram and shape by it;
7) judge total fingerprint peaks, measure the retention time of each total fingerprint peaks among the HSCCC, calculate relative standard's variance RSD of corresponding fingerprint peaks, should satisfy RSD≤3%, the non-total peak total area must not be greater than 10% of total peak area;
8), need the sample more than 10 batches is detected according to the requirement of national standard about the methodological study data of finger-print; Same test sample continuous sample introduction is more than 5 times, to investigate the precision of instrument; The test sample of same lot number is more than 5 parts, to investigate the reappearance of experimental technique; Through the HSCCC separation and purification, the RSD of the total fingerprint peaks of gained collection of illustrative plates answers≤3% to same test sample at different time; Last these data promptly constitute the finger-print and the methodological study data thereof of Chinese medicine.
2, use high speed adverse current chromatogram as claimed in claim 1 is worked out the method for traditional Chinese medicine fingerprint, and it is characterized in that: described Chinese crude drug comprises anthraquinone analog compound, alkaloid compound, flavone compound, terpenoid and oside compound.
3, use high speed adverse current chromatogram as claimed in claim 2 is worked out the method for traditional Chinese medicine fingerprint, and it is characterized in that: described anthraquinone analog compound is the red sage root.
4, use high speed adverse current chromatogram as claimed in claim 2 is worked out the method for traditional Chinese medicine fingerprint, it is characterized in that: described alkaloid compound is the ground abortin.
5, use high speed adverse current chromatogram as claimed in claim 2 is worked out the method for traditional Chinese medicine fingerprint, and it is characterized in that: described flavone compound is the saussurea involucrata flavones.
6, use high speed adverse current chromatogram as claimed in claim 2 is worked out the method for traditional Chinese medicine fingerprint, and it is characterized in that: described terpenoid is the flavones terpene.
7, use high speed adverse current chromatogram as claimed in claim 2 is worked out the method for traditional Chinese medicine fingerprint, and it is characterized in that: described oside compound is a momordica saponins.
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| CN102336825A (en) * | 2011-08-16 | 2012-02-01 | 大连医科大学 | Balsam pear protein as well as preparation method and application of balsam pear protein in preparing antitumor medicament |
| WO2012129847A1 (en) * | 2011-03-25 | 2012-10-04 | 湖南汉森制药股份有限公司 | Methods for simultaneously determinating multi-component of simotang and fingerprint building method thereof |
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| CN112263600B (en) * | 2020-10-27 | 2022-07-05 | 湖南省中医药研究院 | Carpesium abrotanoides extract, preparation method and application thereof in anti-liver cancer active drugs passing through JAK2/STAT3 channel |
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