CN105866321B - A kind of thin-layer identification method of CORTEX KALOPANACIS granule - Google Patents
A kind of thin-layer identification method of CORTEX KALOPANACIS granule Download PDFInfo
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The present invention relates to Pharmaceutical Analysis technical field, specially a kind of thin-layer identification method of CORTEX KALOPANACIS granule.This method is：Take 0.5g particles finely ground, plus ultrasonically treated, filtration after methanol, filtrate adds to be heated to reflux 1 hour after hydrochloric acid, is evaporated, residue adds methanol 1ml to make dissolving, as need testing solution, separately take oleanolic acid, hederagenin reference substance appropriate, plus reference substance solution is made in methanol.Each 3 μ l of above two solution are drawn, are put respectively on same silica gel g thin-layer plate, using cyclohexane ethyl acetate glacial acetic acid as solvent, deploys, takes out, dry, spray with ethanol solution of sulfuric acid, spot development is heated at 105 DEG C clearly, puts and is inspected under ultraviolet lamp.In test sample chromatogram, on position corresponding with reference substance chromatogram, show the fluorescence spot of same color.As a result thin-layer chromatography clear spot, reappearance is preferable.
Description
Technical field
The present invention relates to Pharmaceutical Analysis technical field, specially a kind of thin-layer identification method of CORTEX KALOPANACIS granule.
Background technology
CORTEX KALOPANACIS be Araliaceae kalopanax septemlobus, or burr Chinese catalpa dry bark.The bark can be harvested whole year, strip bark,
Dry, can wind-damp dispelling, dredging collateral, analgesic.For rheumatoid arthritis.Lumbocrural pain；It is used for traumatic injury outside.It is frequently grown on mountain
In ground sparse woods, Anhui, Hubei, Hunan or Guangxi are originated in.
For the domestic rare report of correlative study of CORTEX KALOPANACIS, current pharmacopeia and each province's Chinese medicine standard are without CORTEX KALOPANACIS
Thin layer differentiates project appearance, and CORTEX KALOPANACIS granule similarly lacks thin layer and differentiates item, and only indivedual research and development units have it certainly
Oneself discrimination method.Contain the compositions such as saponin(e, polyyne, phenylpropyl alcohol alkanes, lignanoid and simple phenolic glycoside in CORTEX KALOPANACIS granule.Soap
Methods of glycosides is its main component, and wherein kalopanax septemlobus saponin A has significant anti-inflammatory activity, and it can obtain ivy soap after hydrolyzing
Aglycon and oleanolic acid.In existing discrimination method：The preparation process of need testing solution contains ultrasound, water-saturated n-butanol extraction
Take 3 times, backflow, chloroform extraction 2 times, operating process is cumbersome, it is a large amount of to use the organic toxic agent chloroform of the first kind.Together
When this method only pointed out a principal component-hederagenin.The carry out thin layer discriminating that can not be completely determined.
The content of the invention
The present invention exactly for above technical problem there is provided it is a kind of can quickly, multi information enters to CORTEX KALOPANACIS granule
Row thin layer mirror method for distinguishing.
The concrete technical scheme of the present invention is as follows：
A kind of thin-layer identification method of CORTEX KALOPANACIS granule, this method comprises the following steps：
（1）Take CORTEX KALOPANACIS medicinal material granule appropriate, proper amount of methanol is added after grinding, uses power for 600W, frequency is
Filtered after 40kHz ultrasonically treated certain times, preferably 30 minutes time, appropriate hydrochloric acid, the matter of hydrochloric acid are added in obtained filtrate
Amount concentration is 36%-38%, is evaporated after being heated to reflux, and methanol dissolving is added in the residue after being evaporated, need testing solution is made；
（2）It is another to take oleanolic acid, hederagenin reference substance appropriate, plus every 1ml is made after methanol respectively containing the molten of 0.3mg
Liquid, is used as reference substance solution；
（3）On silica gel g thin-layer plate, using cyclohexane-ethyl acetate-glacial acetic acid as solvent, deploy, take out, dry, spray
With 10v/v% ethanol solution of sulfuric acid；
（3）Spot development is heated at 100-108 DEG C clear, preferably 105 DEG C, puts and inspected under 365nm ultraviolet lamps.
Preferably, step（1）Specially：Take CORTEX KALOPANACIS medicinal material granule appropriate, it is finely ground, take 0.5g, plus methanol
20ml, uses power for 600W, and frequency is 40kHz ultrasonically treated 30 minutes, filtration, and filtrate adds hydrochloric acid（The quality percentage of hydrochloric acid
Concentration is 36%-38%）2ml, is heated to reflux 1 hour, is evaporated, residue adds methanol 1ml to make dissolving, as need testing solution,
Described solvent is cyclohexane-ethyl acetate-glacial acetic acid, with volume basis, hexamethylene：Ethyl acetate：Ice vinegar
Acid=4-7：2-5：0.2-0.35, preferably with volume basis, hexamethylene：Ethyl acetate：Glacial acetic acid=6：4：0.25.
The ultrasonically treated power is 600W, and frequency is 40kHz.
The positive effect of the present invention is embodied in：
（One）, can quickly, multi information to CORTEX KALOPANACIS granule carry out thin layer discriminating.
（Two）, test sample solvent preparation only comprising two processes of ultrasound and backflow, operating process is simple and convenient.
（Three）, need testing solution preparation and solvent in not use the big organic solvent of toxicity, practical guarantor
The healthy of operating personnel has been protected, the pollution of environment is reduced.
（Four）, the present invention pointed out two known chemical compositions, i.e. oleanolic acid and hederagenin, greatly increase
The specificity of method is added.
（Five）, temperature on CORTEX KALOPANACIS granule sample solution thin layer differentiate without influence, illustrate that the thin-layer identification method is resistance to
It is good with property；Humidity differentiates without influence on CORTEX KALOPANACIS granule sample thin layer, illustrates the thin-layer identification method good tolerance；
Thin-layer identification method has preferable durability, and favorable reproducibility.
Brief description of the drawings
Fig. 1 is that the thin layer of test sample and reference substance in embodiment differentiates chromatogram；
Wherein 1 it is oleanolic acid, 2 is that hederagenin, 3-5 are CORTEX KALOPANACIS granule 1409065.
Fig. 2 is that the thin layer that CORTEX KALOPANACIS granule point sample amount different with reference substance is investigated differentiates chromatogram；
Wherein 1 it is the μ l of reference substance solution 1,2 be the μ l of reference substance solution 2,3 be the μ l of reference substance solution 3,4 is reference substance solution 4
μ l, 5 be the μ l of CORTEX KALOPANACIS granule 1,6 be the μ l of CORTEX KALOPANACIS granule 2,7 be the μ l of CORTEX KALOPANACIS granule 3,8 be that CORTEX KALOPANACIS is matched somebody with somebody
The square μ l of particle 4.
Fig. 3 is CORTEX KALOPANACIS granule thin-layer chromatogram, and its T is 21 DEG C, and RH is 45%；
Wherein, 1 it is reference substance solution, 2 is that negative sample, 3-5 are CORTEX KALOPANACIS granule 1409065.
Fig. 4 is the thin layer discriminating chromatogram of CORTEX KALOPANACIS granule under normal temperature condition；Its T is 21 DEG C, and RH is 45%；
Wherein 1 is that reference substance, 2-4 are CORTEX KALOPANACIS particle 1409065.
Fig. 5 is CORTEX KALOPANACIS granule thin layer discriminating chromatogram under cryogenic conditions；Its T is 6 DEG C, and RH is 31%；
Wherein 1 is that reference substance, 2-4 are CORTEX KALOPANACIS particle 1409065.
Fig. 6 is CORTEX KALOPANACIS granule thin layer discriminating chromatogram under super-humid conditions；Its T is 21 DEG C, and RH is 75%；
Wherein 1 is that reference substance, 2-4 are CORTEX KALOPANACIS particle 1409065.
Fig. 7 is CORTEX KALOPANACIS granule thin layer discriminating chromatogram under low humidity conditions；Its T is 21 DEG C, and RH is 20%；
Wherein 1 is that reference substance, 2-4 are CORTEX KALOPANACIS particle 1409065.
Fig. 8 difference silica G plates（Qingdao Haiyang）Thin layer differentiates chromatogram, and its T is 21 DEG C, and RH is 45%；
Wherein 1 is that reference substance, 2-4 are CORTEX KALOPANACIS particle 1409065.
Fig. 9 is different silica G plates（Qingdao spectrum section）Thin layer differentiates chromatogram, and its T is 21 DEG C, and RH is 45%；
Wherein 1 is that reference substance, 2-4 are CORTEX KALOPANACIS particle 1409065.
Figure 10 is each validation batches TLC figures of CORTEX KALOPANACIS granule,
Wherein 1 be reference substance solution, 2 be 1109015,3 be 1103042,4 be 1401019,5 be 1409065.
Figure 11 is the thin layer discriminating chromatogram that embodiment part changes need testing solution after solvent.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to embodiment pair
The present invention is described in further detail, but this should not be interpreted as to the scope of above-mentioned theme of the invention is only limitted to following implementations
Example.
1st, instrument and reagent
Instrument：Thin layer automated imaging instrument（CAMAG TLC VISUALIZER）, the semi-automatic point samples of linamat5- CAMAG
Instrument, a ten thousandth balance（Sai Duolisi BS110S）, KQ-250B type ultrasonic cleaning machines（Kunshan ultrasonic electronic Co., Ltd）、
Testing chamber for medicine stability（Shanghai Yiheng Scientific Instruments Co., Ltd）, silica gel g thin-layer plate（The limited public affairs of Tianjin Si Lida science and technology
Department, can clipboard, lot number：130508）, silica gel g thin-layer plate（Qingdao makes people rich source silica gel chemical reagent work, glass plate, lot number：
141201）, silica gel g thin-layer plate（Subsidiary factory of Haiyang Chemical Plant, Qingdao, glass plate, lot number：20130701）.
Reagent：CORTEX KALOPANACIS granule（Lot number：1409065,1401019,1109015,1409065, Sichuan new green medicine
Industry sci-tech development Co., Ltd.）；
Oleanolic acid（110709-201206, Chinese pharmaceutical biological product identifies institute）；Hederagenin（111733-
201205, Chinese pharmaceutical biological product identifies institute）；
Hexamethylene, ethyl acetate, glacial acetic acid etc. are that analysis is pure.
2nd, the preparation of solution
The preparation of 2.1 need testing solutions
Take this product appropriate, it is finely ground, 0.5g, plus methanol 20ml are taken, power is used for 600W, frequency is at 40kHz ultrasound
Reason 30 minutes, filtration, filtrate adds hydrochloric acid（Mass percentage concentration is 36%-38%）2ml, is heated to reflux 1 hour, is evaporated, residue adds
Methanol 1ml makes dissolving, is used as need testing solution.
The preparation of 2.2 reference substance solutions
Take oleanolic acid, hederagenin reference substance appropriate, plus every 1ml respectively solution containing 0.3mg is made in methanol, as
Reference substance solution.
It is prepared by 2.3 negative control solutions
Take maltodextrin 0.5g, plus methanol 20ml, it is ultrasonically treated（Power 600W, frequency 40kHz）30 minutes, filter, filter
Liquid adds hydrochloric acid（36%-38%）2ml, is heated to reflux 1 hour, is evaporated, residue adds methanol 1ml to make dissolving, be used as negative sample solution.
3rd, thin-layer chromatography condition
Lamellae：Silica gel g thin-layer plate
Solvent：Cyclohexane-ethyl acetate-glacial acetic acid (6：4：0.25)
Inspect：Spray is heated to spot development at 105 DEG C clearly, is put ultraviolet lamp (365nm) with 10% ethanol solution of sulfuric acid
Under inspect.
As a result：In test sample chromatogram, with reference substance chromatogram on a corresponding position, show the fluorescence spot of same color（See
Fig. 1）.
4th, different point sample amounts are investigated
Draw CORTEX KALOPANACIS granule need testing solution（Lot number：1409065）The different μ l of point sample amount 1,2,3,4, reference substance
The μ l of solution difference point sample amount 1,2,3,4, point sample is on same silica gel g thin-layer plate respectively, with cyclohexane-ethyl acetate-glacial acetic acid
Volume ratio be 6：4：0.25 is solvent, is deployed, and takes out, dries, and spray is with 10wt% ethanol solution of sulfuric acid, in 105 DEG C of heating
It is clear to spot development, inspected under the ultraviolet lamp for putting 365nm, experimental result is shown in Fig. 2, from Figure 2 it can be seen that CORTEX KALOPANACIS granule
In need testing solution chromatogram, the spot of same color is shown on position corresponding with reference substance chromatogram.CORTEX KALOPANACIS granule is supplied
When the point sample amount of test sample solution is that 3 μ l, the point sample amount of CORTEX KALOPANACIS reference substance solution are 3 μ l, clear spot and do not trail.Therefore,
The point sample amount of CORTEX KALOPANACIS granule need testing solution is 3 μ l, and the point sample amount of reference substance solution is 3 μ l.
5th, CORTEX KALOPANACIS granule specificity is investigated
Draw respectively CORTEX KALOPANACIS granule need testing solution, reference substance solution and negative control solution difference point sample in
On same silica gel g thin-layer plate, deploy by above-mentioned thin-layer chromatography condition, take out, dry, spray with 10wt% ethanol solution of sulfuric acid,
105 DEG C are heated to spot development clearly, are inspected under the ultraviolet lamp for putting 365nm, experimental result is shown in Fig. 3.As seen from Figure 3, river paulownia
In skin granule need testing solution chromatogram, the spot of same color is being shown with the relevant position of reference substance reference substance chromatogram, and
Negative sample is noiseless.
Further, since the medicinal material containing hederagenin includes：Field pennycress, the root of Chinese clematis, honeysuckle, Akebia Fruit etc., if
Only the hederagenin in CORTEX KALOPANACIS granule is considered, although even if the figure that last thin layer is identified is represented
Go out, the relevant position of need testing solution and reference substance reference substance chromatogram shows the spot of same color, still not can determine that and identifies
The medicinal material come is CORTEX KALOPANACIS granule.
6. different temperatures is investigated
CORTEX KALOPANACIS granule need testing solution and reference substance solution point are drawn respectively on same silica G plate, by upper
Thin-layer chromatography condition is stated, is deployed respectively under normal temperature and cryogenic conditions, takes out, dries, is sprayed with 10wt% ethanol solution of sulfuric acid,
105 DEG C are heated to spot development clearly, are inspected under the ultraviolet lamp for putting 365nm, thin-layer chromatogram is shown in Fig. 4-Fig. 5.By Fig. 4 and figure
5 is visible, under normal temperature and cryogenic conditions, in CORTEX KALOPANACIS granule test sample chromatogram, shows phase in relevant position with reference substance chromatogram
With the fluorescence spot of color.And CORTEX KALOPANACIS granule test sample chromatogram spot separating effect is preferable.Test result indicates that, temperature
CORTEX KALOPANACIS granule sample solution thin layer is differentiated without influence, illustrates the thin-layer identification method good tolerance.
7. different humidity is investigated
CORTEX KALOPANACIS granule need testing solution and CORTEX KALOPANACIS reference substance solution point are drawn respectively in same silica G plate
On, by above-mentioned thin-layer chromatography condition, by above-mentioned thin-layer chromatography condition, deploy respectively under the conditions of 75% and 20% different humidity,
Take out, dry, spray with 10wt% ethanol solution of sulfuric acid, spot development is heated at 105 DEG C clearly, 365nm ultraviolet lamp is put
Under inspect, test result and see Fig. 6~Fig. 7.Under Fig. 6 and Fig. 7, high humidity and low-moisture conditions, CORTEX KALOPANACIS granule is for examination
In product chromatogram, show the fluorescence spot of same color in relevant position with reference substance chromatogram.CORTEX KALOPANACIS granule under different humidity
Sample chromatogram spot separating effect is all preferable.Test result indicates that, humidity differentiates without shadow to CORTEX KALOPANACIS granule sample thin layer
Ring, illustrate the thin-layer identification method good tolerance.
8th, the comparison of different lamellaes
CORTEX KALOPANACIS granule solution, CORTEX KALOPANACIS reference substance solution point are drawn respectively in different manufacturers（Qingdao Haiyang chemical industry
Subsidiary factory of factory, Tianjin ENBREL reach Science and Technology Ltd.）On silica gel g thin-layer plate, deploy by above-mentioned thin-layer chromatography condition, take out, dry,
Spray is heated to spot development at 105 DEG C clearly, inspected under the ultraviolet lamp for putting 365nm with 10% ethanol solution of sulfuric acid；Thin layer color
Spectrogram is shown in Fig. 8~Fig. 9.From the silica gel g thin-layer plate of Fig. 8-9, two different manufacturers production to the equal energy of CORTEX KALOPANACIS granule
Play good separating effect.And in CORTEX KALOPANACIS granule test sample chromatogram, phase is shown on position corresponding with reference substance chromatogram
With the fluorescence spot of color.Experimental result illustrates that the thin-layer identification method has preferable durability.
9. different batches sample thin layer differentiates comparative study
The CORTEX KALOPANACIS granule of different batches is differentiated using thin-layer identification method described in text, experimental result table
Bright, in test sample chromatogram, each batch CORTEX KALOPANACIS granule shows the glimmering of same color on position corresponding with reference substance chromatogram
Hot spot point, reappearance is preferable.
10. the comparative study that different solvents differentiate to sample thin layer
Will with afore-mentioned test with batch CORTEX KALOPANACIS granule using preceding method carry out need testing solution, method with
In the case that step is consistent, only changes the composition of solvent, i.e., the n-hexane in solvent is changed to the hexamethylene of equivalent, then
Thin layer discriminating is carried out to need testing solution, experimental result is shown in Figure 11：Test result indicates that, the thin-layer chromatogram shows ivy soap
Aglycon principal spot with it is following close to spot be not totally separated from, and its Rf value be 0.18, less than 2015 versions《Middle traditional Chinese medicines
Allusion quotation》Regulation of the Rf value between 0.2-0.8 in 4th general rule 0502.Therefore development system n-hexane-acetic acid second in the document
Ester-glacial acetic acid is not equal with cyclohexane-ethyl acetate-glacial acetic acid, and n-hexane can not directly replace with hexamethylene.
The foregoing description of the disclosed embodiments, enables professional and technical personnel in the field to realize or using the present invention.
A variety of modifications to these embodiments will be apparent for those skilled in the art, as defined herein
General Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore, it is of the invention
The embodiments shown herein is not intended to be limited to, and is to fit to and principles disclosed herein and features of novelty phase one
The most wide scope caused.
Claims (2)
1. a kind of thin-layer identification method of CORTEX KALOPANACIS granule, it is characterised in that this method comprises the following steps：
（1）Take CORTEX KALOPANACIS medicinal material granule appropriate, proper amount of methanol is added after grinding, filtered after ultrasonically treated certain time,
To filtrate in plus appropriate hydrochloric acid, be evaporated after being heated to reflux, in the residue after being evaporated plus methanol dissolving, be made need testing solution；
（2）It is another to take oleanolic acid, hederagenin reference substance appropriate, plus it is made every 1ml respectively solution containing 0.3mg after methanol,
It is used as reference substance solution；
（3）On silica gel g thin-layer plate, using cyclohexane-ethyl acetate-glacial acetic acid as solvent, deploy, take out, dry, spray with
10wt% ethanol solution of sulfuric acid；
（4）Spot development is heated at 100 DEG C -108 DEG C clear, puts and inspected under 365nm ultraviolet lamps；
Step（1）In take CORTEX KALOPANACIS medicinal material granule appropriate, it is finely ground, take 0.5g, plus methanol 20ml, ultrasonically treated 30 minutes,
Filtration, filtrate adds hydrochloric acid 2ml, is heated to reflux 1 hour, is evaporated, residue adds methanol 1ml to make dissolving, is used as need testing solution；
Described solvent is cyclohexane-ethyl acetate-glacial acetic acid, with volume basis, hexamethylene：Ethyl acetate：Glacial acetic acid=
4-7：2-5：0.2-0.35；The ultrasonically treated power is 600W, and frequency is 40kHz.
2. the thin-layer identification method of CORTEX KALOPANACIS granule according to claim 1, it is characterised in that：Described solvent is
Cyclohexane-ethyl acetate-glacial acetic acid, with volume basis, hexamethylene：Ethyl acetate：Glacial acetic acid=6：4：0.25.
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