CN110361469A - A kind of Chinese medicine mildew detection method - Google Patents
A kind of Chinese medicine mildew detection method Download PDFInfo
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- CN110361469A CN110361469A CN201910644142.4A CN201910644142A CN110361469A CN 110361469 A CN110361469 A CN 110361469A CN 201910644142 A CN201910644142 A CN 201910644142A CN 110361469 A CN110361469 A CN 110361469A
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Abstract
A kind of mildew detection method of Chinese medicine, it is characterised in that the following steps are included: 1, mildew marker preparation: taking mildew Chinese medicine or mildewed bacterium, extract, filter, volatilize solvent, solubilizer dissolution, in liquid chromatograph sample introduction, it is enriched with target peak fraction, is concentrated to give mildew marker；2, prepared by test solution: the Chinese materia medica preparation sample for taking Chinese medicine or powder to be used as medicine, and solubilizer is extracted, filtered, filtrate is evaporated, and the dissolution of residue solubilizer obtains test solution；3, it analysis sample to be tested mildew situation: in sample chromatogram, goes mouldy with aobvious same color fluorescence spot on mildew marker corresponding position or identical chromatographic peak, i.e. sample.The present invention can effectively characterize the quality of Chinese medicine (in addition to mushroom drug) and the Chinese materia medica preparation being used as medicine with powder, avoid mildew Chinese medicine from being used as medicine, improve the safety of clinical use.The method of the present invention has the advantages that easy, sensitive, at low cost.
Description
Technical field
The present invention relates to a kind of substance mildew detection methods, more particularly, to a kind of mildew detection method of Chinese medicine.
Background technique
Application in TCM is extensive, by multicomponent, multiple target point play curative effect, with its in-depth study obtained it is more and more
The approval of people.Traditional Chinese medicine ingredients are complicated, store, transport, dealing with improperly and easily cause mildew, mildew is influence Chinese native medicine security and has
The principal element of effect property.The common pathogen of Chinese medicine (such as aflatoxin) has potential hazard to human body, directly affects use
The safety of medicine and clinical efficacy.Even if removing mildew medicinal material surface mould, ingredient can also change a lot.
Currently, it is more deep for the Chinese medicine mildew researchs such as influence factor and control measure, but national standard, place mark
Quasi- even international standard there is no perfect detection method for the mildew inspection of Chinese medicine and related preparations, only can by naked eyes, show
Micro- or aflatoxin inspection.Since certain mildew medicinal material (such as dried orange peel) aflatoxin inspections may be feminine gender, tradition is with meat
Eye, micro- sem observation properties and characteristics mildew inspection knowledge method accurate judgement is difficult to for internal subtle mildew, make Chinese medicine and correlation
The safety of preparation clinical use is seriously threatened.
The detection method of Chinese medicine mildew is established, lays the foundation for Chinese medicine and the perfect of related preparations quality evaluation system, is
Chinese medicine safe handling provides safeguard.
Summary of the invention
For prior art defect, the present invention provides the Chinese medicines a kind of Chinese medicine (in addition to mushroom drug) and be used as medicine with powder
The mildew detection method of preparation has the advantages that highly sensitive, efficient, low cost, can control Chinese medicine by means of the present invention
(in addition to mushroom drug) and the Chinese materia medica preparation quality being used as medicine with powder, guarantees the safety and validity of clinical application.
For achieving the above object, the present invention provides a kind of mildew detection method of Chinese medicine, it is characterised in that including with
Lower step:
1, mildew marker preparation:
Mildew Chinese medicine or mildewed bacterium is taken, extracts, filter, volatilizing solvent, solubilizer dissolves, in liquid chromatograph sample introduction,
It is enriched with target peak fraction, is concentrated to give mildew marker；
2, prepared by test solution:
The Chinese materia medica preparation sample for taking Chinese medicine or powder to be used as medicine, solubilizer is extracted, is filtered, filtrate is evaporated, and residue solubilizer is molten
Solution, obtains test solution；
3, analysis sample to be tested mildew situation:
In sample chromatogram, and same color fluorescence spot or identical chromatographic peak are shown on mildew marker corresponding position, i.e.,
Sample goes mouldy.
In the step 1, mildew herb powder or mildewed bacterium are taken, adds 5-50 times to measure solvent extraction, solvent is methanol, second
Alcohol, acetone, any one in ethyl acetate or two or more mixtures, extracting mode be ultrasonic extraction, continuous circumfluence extraction,
Refluxing extraction, Soakage extraction, extraction time are 10-180 minutes.
In the step 1, after volatilizing solvent, using petroleum ether as solvent-loaded column, loading, elution, tracking mildew is inspected
Marker is collected and merges the eluent with mildew marker, recycling design.
The tracking mildew marker of inspecting, by eluent point in silica gel g thin-layer plate, is set ultraviolet by thin-layer chromatography
It is inspected under light, required solvent is that the ratio of toluene, ethyl acetate or chloroform, formic acid or acetic acid or phosphoric acid composition is (5-
10): (3-0.7): the mixed solvent of (0.5-0.1) or by petroleum ether and ethyl acetate with (20-1): the expansion that 1 ratio forms
Agent.
The elution be successively with petroleum ether, 20:1 or 40:1 petroleum ether: ethyl acetate mixture, 10:1 or
The petroleum ether of 20:1: ethyl acetate mixture elution.
In the step 1, the solvent of dissolution is methanol or ethyl alcohol, and volume is to take the weight of mildew Chinese medicine or mildewed bacterium
1/30-1/2。
In the step 2, the Chinese materia medica preparation sample for taking Chinese medicine or powder to be used as medicine adds 5-50 times to measure solvent, solvent be methanol,
Ethyl alcohol, acetone, any one in ethyl acetate or two or more mixtures, extracting mode are that ultrasonic extraction, continuous backflow mention
It takes, refluxing extraction, Soakage extraction, extraction time is 10-180 minutes, and residue, which adds, extracts solvent for use 0.5-10ml dissolution.
It is to draw mildew marker, confession respectively using thin layer chromatography analysis sample to be tested mildew situation in the step 3
Test sample solution 1-20 μ l, for point in same silica gel g thin-layer plate, solvent is toluene, ethyl acetate or chloroform, formic acid or acetic acid or phosphorus
Acid composition ratio be (5-10): (3-0.7): (0.5-0.1) mixed solvent or by petroleum ether and ethyl acetate with (20-1): 1
The solvent of ratio composition, sets 365nm under ultraviolet light and inspects.
In the step 3, using high efficiency liquid phase chromatographic analysis method analysis sample to be tested mildew situation, mildew mark is drawn respectively
Will object, test solution inject high performance liquid chromatograph, record chromatogram.
The high efficiency liquid phase chromatographic analysis method is accurate absorption mildew marker, each 5-20 μ l of test solution, injection
High performance liquid chromatograph, using acetonitrile or methanol and water composition ratio 95:5-5:95 as mobile phase, flow velocity 0.7-1.2ml/min, column
Warm 20-35 DEG C, 254nm, 365nm dual-wavelength simultaneous detection.
The preparation method of mildew marker established by the present invention, can obtain the mildew mark with specific recognition effect
Object, by with sample to be tested Comparative result, judgement sample go mouldy situation.Chinese medicine (in addition to mushroom drug) established by the present invention and
With the mildew detection method for the Chinese materia medica preparation that powder is used as medicine, it can effectively characterize Chinese medicine (in addition to mushroom drug) and be entered with powder
The quality of the Chinese materia medica preparation of medicine avoids mildew Chinese medicine from being used as medicine, improves the safety of clinical use.The method of the present invention have it is easy,
Advantage sensitive, at low cost.
Detailed description of the invention
Fig. 1 is that different sources mildew sample thin layer inspects result comparative diagram；
Fig. 2 is that mildew and the sunflower disk thin layer that do not go mouldy inspect result comparative diagram；
Fig. 3 is that different proportion mildew inspects result comparative diagram；
Fig. 4 is mildew marker thin-layer chromatogram；
Fig. 5 is mildew marker minimum detectable activity thin layer figure；
Fig. 6-Fig. 7 is the thin layer figure of different medicinal material mildews front and back；
The three-dimensional map of green fluorescence ingredient is shown in Fig. 8-Fig. 9 mildew dried orange peel；
Figure 10 is Chinese materia medica preparation Er Chen Pill thin-layer chromatogram；
Figure 11 A- Figure 11 B is mildew marker and mildew sample chromatograms.
Specific embodiment
Embodiment 1
Step 1: the preparation of mildew marker
Mildew the mildewed bacterium 1g of Chinese medicine is taken, adds 50 times of amount ethyl acetate ultrasonic extraction 10 minutes, filters, is spin-dried for, is added a small amount of
Ethyl acetate makes to dissolve, and adds silica gel (100-200 mesh) in right amount, mixes thoroughly, and water-bath volatilizes solvent, fills by solvent wet process of petroleum ether
Column, dry method loading are successively mixed with petroleum ether, petroleum ether and ethyl acetate 20:1 mixed solution, petroleum ether and ethyl acetate 10:1
Solution elution is closed, eluent point is set ultraviolet in silica gel g thin-layer plate using toluene-ethyl acetate-formic acid 24:7:1 as solvent
It is inspected under light lamp (365nm), tracking mildew marker is collected and merges the eluent containing target component, recycling design, must go mouldy mark
The crude extract of will object.
Take crude extract that methanol 0.5ml is added to leach, 0.22 μm of membrane filtration (is matched in 1260 high performance liquid chromatograph of Agilent
Have DAD detector) sample introduction, using acetonitrile and water 35:65 as mobile phase, flow velocity 1.0ml/min, 25 DEG C of column temperature, 254nm, 365nm are bis-
Wavelength detects simultaneously.It is enriched with target peak fraction, is concentrated to give mildew marker.
Step 2: prepared by test solution
Sample to be tested powder 10g is taken, 5 times of amount ethyl acetate is added to be ultrasonically treated 20 minutes, filtration, filtrate is evaporated, and residue adds
Ethyl acetate 10ml dissolution, obtains test solution.
The situation step 3: thin-layered chromatography analysis sample goes mouldy
Step 2 draws mildew to the 10 mildew sample preparation test solutions not gone mouldy and different location acquires respectively
Each 10 μ l point of marker, test solution is expansion with toluene-ethyl acetate-formic acid 24:7:1 on same silica gel g thin-layer plate
Agent is unfolded, and takes out, dries, set and inspect under ultraviolet lamp (365nm), and mildew sample thin-layer chromatography is shown and mildew marker phase
Same green fluorescence spot, the result is shown in Figure 1.In figure 1~10 be different batches mildew sunflower disk sample, 11 is do not go mouldy sunflower
Disk sample, 12 be mildew marker.
Embodiment 2
Step 1: the preparation of mildew marker
Mildew sample powder 50g is taken, adds 15 times of amount ethyl alcohol continuous circumfluence extraction 60 minutes, is filtered, is spin-dried for, adds ethyl alcohol 5ml
Make to dissolve, 0.22 μm of membrane filtration, in 1260 high performance liquid chromatograph of Agilent (be furnished with DAD detector) sample introduction, with acetonitrile and
Water 35:65 be mobile phase, flow velocity 0.9ml/min, 30 DEG C of column temperature, 254nm, 365nm dual-wavelength simultaneous detection.Enrichment target peak evaporates
Point, it is concentrated to give mildew marker.
Step 2: prepared by test solution
Sample to be tested powder 20g is taken, adds handle within 10 times of amount ethyl alcohol continuous circumfluence extraction 60 minutes, filtration, filtrate is evaporated, residual
Slag adds ethyl alcohol 10ml to dissolve, and obtains test solution.
The situation step 3: thin-layered chromatography analysis sample goes mouldy
By second step to the sample preparation test solution that do not go mouldy and go mouldy, it is molten that mildew marker, test sample are drawn respectively
Each 1 μ l point of liquid is on same silica gel g thin-layer plate, and using toluene-ethyl acetate-acetic acid 10:0.7:0.1 as solvent, expansion is taken
Out, it dries, sets and inspected under ultraviolet lamp (365nm), the aobvious green identical with mildew marker of mildew sample thin-layer chromatography is glimmering
Hot spot point.
Embodiment 3
Step 1: the preparation of mildew marker
Mildew sample powder 100g is taken, 5 times of amount EtOH Sonicates is added to extract 60 minutes, filtration is spin-dried for, adds a small amount of ethyl alcohol to make molten
Solution, adds silica gel (100-200 mesh) in right amount, mixes thoroughly, and water-bath volatilizes solvent, using petroleum ether as solvent wet method dress post, dry method loading, according to
Secondary petroleum ether, petroleum ether and ethyl acetate 20:1 mixed solution, petroleum ether and the elution of ethyl acetate 10:1 mixed solution, will wash
Liquid point is taken off in silica gel g thin-layer plate, using toluene-chloroform-phosphoric acid 5:3:0.5 as solvent, is set and is inspected under ultraviolet lamp (365nm),
Tracking mildew marker, collects and merges the eluent containing target component, recycling design, the crude extract for the marker that must go mouldy.
Take crude extract that ethyl alcohol 10ml is added to leach, 0.22 μm of membrane filtration (is furnished in 1260 high performance liquid chromatograph of Agilent
DAD detector) sample introduction, using acetonitrile and water 30:70 as mobile phase, flow velocity 0.9ml/min, 30 DEG C of column temperature, 254nm, 365nm double wave
It grows while detecting.It is enriched with target peak fraction, is concentrated to give mildew marker.
Step 2: prepared by test solution
Sample to be tested powder 5g is taken, 10 times of amount alcohol dippings is added to extract 180 minutes, filtration, filtrate is evaporated, and residue adds ethyl alcohol
5ml dissolution, obtains test solution.
The situation step 3: thin-layered chromatography analysis sample goes mouldy
By second step to the sample preparation test solution that do not go mouldy and go mouldy, it is molten that mildew marker, test sample are drawn respectively
Each 5 μ l point of liquid using toluene-chloroform-phosphoric acid 5:3:0.5 as solvent, is unfolded on same silica gel g thin-layer plate, takes out, dry,
It sets and is inspected under ultraviolet lamp (365nm), the aobvious green fluorescence spot identical with mildew marker of mildew sample thin-layer chromatography.
Embodiment 4
Step 1: the preparation of mildew marker
Mildew the mildewed bacterium 300g of Chinese medicine is taken, 30 times of amount methanol eddies is added to extract 90 minutes, filtration is spin-dried for, adds a small amount of first
Alcohol makes to dissolve, and adds silica gel (100-200 mesh) in right amount, mixes thoroughly, and water-bath volatilizes solvent, using petroleum ether as solvent wet method dress post, dry method
Loading is successively washed with petroleum ether, petroleum ether and ethyl acetate 20:1 mixed solution, petroleum ether and ethyl acetate 10:1 mixed solution
It is de-, by eluent point in silica gel g thin-layer plate, using petroleum ether and ethyl acetate 20:1 as solvent, set under ultraviolet lamp (365nm)
It inspects, tracking mildew marker is collected and merges the eluent containing target component, recycling design, the crude extract for the marker that must go mouldy.
Take crude extract that methanol 10ml is added to leach, 0.22 μm of membrane filtration (is matched in 1260 high performance liquid chromatograph of Agilent
Have DAD detector) sample introduction, using acetonitrile and water 40:60 as mobile phase, flow velocity 1.2ml/min, 35 DEG C of column temperature, 254nm, 365nm are bis-
Wavelength detects simultaneously.It is enriched with target peak fraction, is concentrated to give mildew marker.
Step 2: prepared by test solution
Sample to be tested powder 2g is taken, adds 20 times of amount methanol ultrasonic extraction 10 minutes, is filtered, filtrate is evaporated, and residue adds methanol
3ml dissolution, obtains test solution.
The situation step 3: thin-layered chromatography analysis sample goes mouldy
By second step to the sample preparation test solution that do not go mouldy and go mouldy, it is molten that mildew marker, test sample are drawn respectively
Each 10 μ l point of liquid using petroleum ether and ethyl acetate 20:1 as solvent, is unfolded on same silica gel g thin-layer plate, takes out, dry,
It sets and is inspected under ultraviolet lamp (365nm), the aobvious green fluorescence spot identical with mildew marker of mildew sample thin-layer chromatography.
Embodiment 5
Step 1: the preparation of mildew marker
Mildew sample powder 500g is taken, 50 times of amount acetone soaks is added to extract 180 minutes, filtration is spin-dried for, a small amount of acetone is added to make
Dissolution, adds silica gel 100-200 mesh appropriate, mixes thoroughly, and water-bath volatilizes solvent, using petroleum ether as solvent wet method dress post, dry method loading, according to
Secondary petroleum ether, petroleum ether and ethyl acetate 40:1 mixed solution, petroleum ether and the elution of ethyl acetate 20:1 mixed solution, will wash
Liquid point is taken off in silica gel g thin-layer plate, using petroleum ether and ethyl acetate 1:1 as solvent, is set and is inspected under ultraviolet lamp (365nm), chase after
Track mildew marker, collects and merges the eluent containing target component, recycling design, the crude extract for the marker that must go mouldy.
Take suitable crude extract that methanol 20ml is added to leach, 0.22 μm of membrane filtration, in 1260 high performance liquid chromatography of Agilent
Instrument, (being furnished with DAD detector) sample introduction, using acetonitrile and water 30:70 as mobile phase, flow velocity 1.0ml/min, 25 DEG C of column temperature, 254nm,
365nm dual-wavelength simultaneous detection.It is enriched with target peak fraction, is concentrated to give mildew marker.
Step 2: prepared by test solution
Sample to be tested powder 1g is taken, adds 10 times of amount acetone ultrasonic extraction 60 minutes, is filtered, filtrate is evaporated, and residue adds acetone
1ml dissolution, obtains test solution.
The situation step 3: thin-layered chromatography analysis sample goes mouldy
By second step to the sample preparation test solution that do not go mouldy and go mouldy, it is molten that mildew marker, test sample are drawn respectively
Each 20 μ l point of liquid using petroleum ether and ethyl acetate 1:1 as solvent, is unfolded on same silica gel g thin-layer plate, takes out, dry, set
It is inspected under ultraviolet lamp (365nm), the aobvious green fluorescence spot identical with mildew marker of mildew sample thin-layer chromatography.
Embodiment 6
Step 1: the preparation of mildew marker
Mildew sample powder 750g is taken, adds 20 times of amount ethyl acetate ultrasonic extraction 60 minutes, is filtered, is spin-dried for, adds a small amount of second
Acetoacetic ester makes to dissolve, and adds silica gel 100-200 mesh appropriate, mixes thoroughly, and water-bath volatilizes solvent, using petroleum ether as solvent wet method dress post, does
Method loading, successively with petroleum ether, petroleum ether and ethyl acetate 40:1 mixed solution, petroleum ether and ethyl acetate 20:1 mixed solution
Elution, by eluent point in silica gel g thin-layer plate, is solvent as solvent using toluene-ethyl acetate-formic acid 24:5:0.5, sets
It is inspected under ultraviolet lamp (365nm), tracking mildew marker is collected and merges the eluent containing target component, recycling design obtains mould
Become the crude extract of marker.
Take crude extract that methanol 30ml is added to leach, 0.22 μm of membrane filtration (is matched in 1260 high performance liquid chromatograph of Agilent
Have DAD detector) sample introduction, using first alcohol and water 95:5 as mobile phase, flow velocity 0.7ml/min, 20 DEG C of column temperature, 254nm, 365nm are bis-
Wavelength detects simultaneously.It is enriched with target peak fraction, is concentrated to give mildew marker.
Step 2: prepared by test solution
Sample to be tested powder 0.5g is taken, adds 10 times of amount ethyl acetate ultrasonic extraction 30 minutes, is filtered, filtrate is evaporated, residue
Add ethyl acetate 0.5ml to dissolve, obtains test solution.
The situation step 3: liquid chromatography analysis sample goes mouldy
0.22 μm of membrane filtration of test solution, using octadecylsilane chemically bonded silica as stationary phase, precision is drawn mould
Become marker, each 20 μ l of test solution, injection 1260 high performance liquid chromatograph of Agilent (is furnished with DAD detector), with methanol
With water 95:5 be mobile phase, flow velocity 0.7ml/min, 20 DEG C of column temperature, 254nm, 365nm dual-wavelength simultaneous detection.Test sample map
In show and the chromatographic peak for the identical retention time of marker of going mouldy.
Embodiment 7
Step 1: the preparation of mildew marker
Mildew sample powder 1000g is taken, 40 times of amount methanol eddies is added to extract 60 minutes, filtration is spin-dried for, a small amount of methanol is added to make
Dissolution, adds silica gel 100-200 mesh appropriate, mixes thoroughly, and water-bath volatilizes solvent, using petroleum ether as solvent wet method dress post, dry method loading, according to
Secondary petroleum ether, petroleum ether and ethyl acetate 20:1 mixed solution, petroleum ether and the elution of ethyl acetate 10:1 mixed solution, will wash
Liquid point is taken off in silica gel g thin-layer plate, is solvent as solvent using toluene-ethyl acetate-acetic acid 10:0.7:0.1, is set ultraviolet lamp
It is inspected under (365nm), tracking mildew marker is collected and merges the eluent containing target component, recycling design obtains mildew marker
Crude extract.
Take crude extract that methanol 35ml is added to leach, 0.22 μm of membrane filtration (is matched in 1260 high performance liquid chromatograph of Agilent
Have DAD detector) sample introduction, using acetonitrile and water 5:95 as mobile phase, flow velocity 1.2ml/min, 35 DEG C of column temperature, 254nm, 365nm are bis-
Wavelength detects simultaneously.It is enriched with target peak fraction, is concentrated to give mildew marker.
Step 2: prepared by test solution
Sample to be tested powder 4g is taken, adds 50 times of amount methanol continuous circumfluence extraction 90 minutes, is filtered, filtrate is evaporated, and residue adds
Methanol 2ml dissolution, obtains test solution.
The situation step 3: liquid chromatography analysis sample goes mouldy
0.22 μm of membrane filtration of test solution, using octadecylsilane chemically bonded silica as stationary phase, precision is drawn mould
Become marker, each 5 μ l of test solution, injection 1260 high performance liquid chromatograph of Agilent (be furnished with DAD detector), with acetonitrile and
Water 5:95 be mobile phase, flow velocity 1.2ml/min, 35 DEG C of column temperature, 254nm, 365nm dual-wavelength simultaneous detection.In test sample map
The chromatographic peak of aobvious retention time identical as mildew marker.
Test example 1: mildew front and back thin layer is inspected
It takes the sample that do not go mouldy, mildew sample appropriate, 50 times of methanol is added to be ultrasonically treated 30 minutes, filtration, filtrate is evaporated, residue
Add methanol 1ml to dissolve, obtains test solution.It is appropriate that test sample, mildew marker are drawn respectively, are put in same silica gel g thin-layer plate
On, using toluene-ethyl acetate-formic acid 24:7:1 as solvent, it is unfolded, takes out, dry, set and inspected under ultraviolet lamp (365nm),
As a result see Fig. 2.Wherein 1 be sunflower disk control medicinal material, 2,3 be the sunflower disk that do not go mouldy for being collected in Taonan urban district, and 4,5 is acquire
Mildew sunflower disk in Taonan urban district, 6,7 be the sunflower disk that do not go mouldy for being collected in the township Er Long, and 8,9 be to be collected in the mould of the township Er Long
Become sunflower disk.It goes mouldy inconsistent with thin-layer chromatography spot behavior of not going mouldy, with the sample ratio that do not go mouldy, sample principal spot color of going mouldy
It shoals and occurs with green fluorescence spot, it is believed that green fluorescence spot has certain correlation with sample mildew.
Test example 2: the relevance verification of green fluorescence spot and Chinese medicine mildew
Experimental formula different proportion mildew sample, carries out thin layer according to 1 method of experimental example and inspects.As a result Fig. 3 is seen, wherein 1 is
Do not go mouldy sunflower disk, and 2~7 be respectively different mildew ratios mildew sunflower disk samples.Increase with mildew ratio, sample Central Plains is deposited
The fluorescence intensity of bluish violet spot declines, green fluorescence spot intensity enhancing on the contrary, it was demonstrated that green fluorescence spot and Chinese medicine go mouldy
It is closely related.
Test example 3: the selection for the marker that goes mouldy
Removing mildew surface mould, according to 1 method of experimental example preparation mould extracting solution, do not go mouldy extracting solution and mildew extraction
Liquid is drawn 10 μ l points respectively and is inspected in progress thin layer on silica gel g thin-layer plate.As a result see Fig. 4, wherein 1, No. 2 is mildew marker,
3, No. 4 are mildew sunflower disk, and No. 5 are the sunflower disk that do not go mouldy.On position corresponding with mildew medicinal material chromatography, mould shows single
Green fluorescence spot, mildew sample still show other spots in addition to spot corresponding with mould.Speculate green fluorescence caused by going mouldy
One of them is to have specificity caused by mould to spot, determines the ingredient for mildew marker；Another may be certain in medicinal material
Caused by kind chemical component conversion.The sample thin-layer chromatography that goes mouldy is shown and mildew marker same color fluorescence spot, and do not go mouldy sample
Then without this spot, it was demonstrated that the method is capable of detecting when that Chinese medicine goes mouldy.
Test example 4: the measurement of mildew sample minimum detectable activity
Mildew sample 0.5g is taken, prepares test solution according to 1 method of experimental example, gradually dilutes test solution concentration difference
Point using toluene-ethyl acetate-formic acid 24:7:1 as solvent, is unfolded on same silica gel g thin-layer plate, takes out, dry, set purple
It is inspected under outer smooth lamp (365nm), situation is detected by observation mildew marker and investigates mildew sample minimum detectable activity.As a result it shows
Show, mildew minimum detectable activity is 4.11 μ g, and this method is sensitive.As a result see Fig. 5, each spot mildew content is shown in Table 1.1~11 in Fig. 5
Respectively various concentration mildew sunflower disk test sample.
Test example 5: thin-layered chromatography analyzes sample mildew situation
(1) 12 kinds of mildew medicinal materials such as radix bupleuri, rhizoma alismatis, pueraria lobata, cimicifugae foetidae are collected, it is not mould to variety classes by 1 method of experimental example
Become and mildew medicinal material is tested.As a result pueraria lobata, Radix Astragali, rhizoma polygonati, the sophora bud, dried orange peel, ginseng, rhizoma anemarrhenae, campanulaceae mildew sample thin layer
Chromatography is shown and mildew marker same color fluorescence spot, and the medicinal material that do not go mouldy is without this spot；Cimicifugae foetidae, radix bupleuri, rhizoma alismatis and Poria cocos
Do not go mouldy and go mouldy sample micro- hypofluorescence compared with the marker that goes mouldy, thus it is speculated that may be for sample storage time is too long and breeds
Naked eyes are not easy the mould observed；Poria cocos is the dry sclerotia of polyporaceae fungus, and the possible original of mildew marker is stored in Poria cocos
Equal fungus medicinal materials.This method is sensitive effectively, can be used for judging the mildew situation of non-mushroom Chinese medicine.As a result see Fig. 6,7.Wherein Fig. 6
In 1 not go mouldy the sophora bud, 2 be the mildew sophora bud, and 3 be the rhizoma polygonati that do not go mouldy, and 4 be mildew rhizoma polygonati, and 5 be the dried orange peel that do not go mouldy, and 6 is old to go mouldy
Skin, 7 be the ginseng that do not go mouldy, and 8 be mildew ginseng, and 9 be the rhizoma alismatis that do not go mouldy, and 10 be mildew rhizoma alismatis, and 11 be the pueraria lobata that do not go mouldy, and 12 be mould
Become pueraria lobata, 13 be mildew marker；1 is the cimicifugae foetidae that do not go mouldy in Fig. 7, and 2 be mildew cimicifugae foetidae, and 3 be the radix bupleuri that do not go mouldy, and 4 be mildew bavin
Recklessly, 5 be the campanulaceae that do not go mouldy, and 6 be mildew campanulaceae, and 7 be the rhizoma anemarrhenae that do not go mouldy, and 8 be mildew rhizoma anemarrhenae, and 9 be the Radix Astragali that do not go mouldy, and 10 be mildew
Radix Astragali, 11 be the Poria cocos that do not go mouldy, and 12 be mildew Poria cocos, and 13 be mildew marker.
(2) three-dimensional map verifying: taking mildew dried orange peel appropriate, add 30 times of amount ethyl acetate ultrasonic extraction 30 minutes, filter, filter
Liquid is evaporated, with silica gel 100-200 mesh wet method dress post, dry method loading, respectively with petroleum ether, petroleum ether and ethyl acetate 20:1, stone
Oily ether and the elution of ethyl acetate 20:2 mixed solvent, thin-layered chromatography tracking show identical fluorescence with mildew marker corresponding position
Eluent merges eluent, and concentration, with 0.22 μm of membrane filtration, using octadecylsilane chemically bonded silica as stationary phase, precision is inhaled
Mildew marker, each 20 μ l of test solution are taken, injection 1260 high performance liquid chromatograph of Agilent (is furnished with DAD detector), with
Acetonitrile and water 40:60 are mobile phase, flow velocity 1.0ml/min, 25 DEG C of column temperature.Observation mildew dried orange peel and mildew marker three-dimensional figure
Spectrum, absorption curve trend is identical, is determined as same constituents substantially, and this method is accurate, and three-dimensional spectrum is shown in Fig. 8,9.
Test example 6: thin-layered chromatography analyzes Er Chen Pill mildew situation
According to one Er Chen Pill preparation method of " Chinese Pharmacopoeia " version in 2015, respectively using dried orange peel, mildew dried orange peel as raw material, system two is old
The Er Chen Pill sample of ball, the dried orange peel containing mildew.It crushes, the sample thin-layer chromatography by 1 method test of experimental example, as a result containing the dried orange peel that goes mouldy
Aobvious fluorescence identical as mildew marker, for the sample thin layer that do not go mouldy then without this spot, this method can be used for judging the mould of Chinese materia medica preparation
Become situation.The result is shown in Figure 10, wherein 1,2 be Er Chen Pill thin layer figure made from mildew dried orange peel, 3,4 be not go mouldy two made from dried orange peel
Old ball thin layer figure.Whether detection Chinese medicine and related preparations go mouldy, the spot compared with the marker that goes mouldy.
Test example 7: liquid chromatography analysis sample mildew situation
0.22 μm of membrane filtration of test solution, using octadecylsilane chemically bonded silica as stationary phase, precision is drawn mould
Become marker, each 20 μ l of test solution, injection 1260 high performance liquid chromatograph of Agilent (is furnished with DAD detector), with acetonitrile
With water 40:60 be mobile phase, flow velocity 1.0ml/min, 25 DEG C of column temperature, 254nm, 365nm dual-wavelength simultaneous detection.As a result see figure
11A is mildew marker chromatograms, and Figure 11 B is mildew sunflower disk chromatograms, when retaining in Figure 11 B with mildew marker
Between corresponding position show identical chromatographic peak, it was demonstrated that the method is effective for judging Chinese medicine mildew.
Claims (10)
1. a kind of mildew detection method of Chinese medicine, it is characterised in that the following steps are included:
(1) mildew marker preparation
Mildew Chinese medicine or mildewed bacterium are taken, extracts, filter, volatilizing solvent, solubilizer dissolution, in liquid chromatograph sample introduction, enrichment
Target peak fraction is concentrated to give mildew marker；
(2) prepared by test solution
The Chinese materia medica preparation sample for taking Chinese medicine or powder to be used as medicine, solubilizer is extracted, is filtered, filtrate is evaporated, and the dissolution of residue solubilizer obtains
Test solution；
(3) analysis sample to be tested mildew situation
In sample chromatogram, with aobvious same color fluorescence spot or identical chromatographic peak, i.e. sample on mildew marker corresponding position
It goes mouldy.
2. the mildew detection method of a kind of Chinese medicine according to claim 1, it is characterised in that: in step 1, take mildew traditional Chinese medicine powder
End or mildewed bacterium add 5-50 times to measure solvent extraction, and solvent is methanol, ethyl alcohol, acetone, any one in ethyl acetate or two kinds
Above mixture, extracting mode are ultrasonic extraction, continuous circumfluence extraction, refluxing extraction, Soakage extraction, extraction time 10-
180 minutes.
3. the mildew detection method of a kind of Chinese medicine according to claim 1, it is characterised in that: in the step 1, volatilize molten
After agent, using petroleum ether as solvent-loaded column, loading, elution, tracking mildew marker is inspected, collects and merges with mildew marker
Eluent, recycling design.
4. the mildew detection method of a kind of Chinese medicine according to claim 3, it is characterised in that: described inspects tracking mildew mark
Will object, by eluent point in silica gel g thin-layer plate, is set and is inspected under ultraviolet light by thin-layer chromatography, required solvent be toluene,
The ratio of ethyl acetate or chloroform, formic acid or acetic acid or phosphoric acid composition is (5-10): (3-0.7): the mixed solvent of (0.5-0.1)
Or by petroleum ether and ethyl acetate with (20-1): the solvent that 1 ratio forms.
5. the mildew detection method of a kind of Chinese medicine according to claim 3, it is characterised in that: the elution is successively to use stone
The petroleum ether of oily ether, 20:1 or 40:1: the petroleum ether of ethyl acetate mixture, 10:1 or 20:1: ethyl acetate mixture
Elution.
6. the mildew detection method of a kind of Chinese medicine according to claim 1, it is characterised in that: in the step 1, dissolution
Solvent is methanol or ethyl alcohol, and volume is the weight 1/30-1/2 for taking mildew Chinese medicine or mildewed bacterium.
7. the mildew detection method of a kind of Chinese medicine according to claim 1, it is characterised in that: in the step 2, take Chinese medicine or
The Chinese materia medica preparation sample that powder is used as medicine adds 5-50 times to measure solvent, and solvent is methanol, ethyl alcohol, acetone, any one in ethyl acetate
Or two or more mixtures, extracting mode are ultrasonic extraction, continuous circumfluence extraction, refluxing extraction, Soakage extraction, extraction time
It is 10-180 minutes, residue, which adds, extracts solvent for use 0.5-10ml dissolution.
8. the mildew detection method of a kind of Chinese medicine according to claim 1, it is characterised in that: in the step 3, same color
Fluorescence spot analyzes sample to be tested mildew situation using thin layer chromatography, i.e., draws mildew marker, test sample respectively
Solution 1-20 μ l, for point in same silica gel g thin-layer plate, solvent is toluene, ethyl acetate or chloroform, formic acid or acetic acid or phosphoric acid group
At ratio be (5-10): (3-0.7): (0.5-0.1) mixed solvent or by petroleum ether and ethyl acetate with (20-1): 1 ratio
The solvent of composition is set 365nm under ultraviolet light and is inspected.
9. the mildew detection method of a kind of Chinese medicine according to claim 1, it is characterised in that: in the step 3, identical chromatography
Peak is that it is molten to draw mildew marker, test sample respectively using high efficiency liquid phase chromatographic analysis method analysis sample to be tested mildew situation
Liquid injects high performance liquid chromatograph, records chromatogram.
10. the mildew detection method of a kind of Chinese medicine according to claim 9, it is characterised in that: the high performance liquid chromatography
Analytic approach is accurate absorption mildew marker, each 5-20 μ l of test solution, high performance liquid chromatograph is injected, with acetonitrile or methanol
With water composition ratio 95:5-5:95 be mobile phase, flow velocity 0.7-1.2ml/min, 20-35 DEG C of column temperature, 254nm, 365nm dual wavelength
It detects simultaneously.
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