CN110261525A - The method for building up of precious jade medicinal material benefit female ginger methylene chloride position HPLC finger-print - Google Patents
The method for building up of precious jade medicinal material benefit female ginger methylene chloride position HPLC finger-print Download PDFInfo
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- CN110261525A CN110261525A CN201910510442.3A CN201910510442A CN110261525A CN 110261525 A CN110261525 A CN 110261525A CN 201910510442 A CN201910510442 A CN 201910510442A CN 110261525 A CN110261525 A CN 110261525A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8686—Fingerprinting, e.g. without prior knowledge of the sample components
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
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Abstract
The invention discloses the method for building up of precious jade medicinal material benefit female ginger methylene chloride position HPLC finger-print, the following steps are included: (1) prepares test solution, (2) using diosgenin as standard items, reference substance solution is prepared, (3) HPLC detection: the HPLC map in record 95min.(4) multiple test samples are detected, utilizes " similarity evaluation " software, all chromatographic peaks is matched by the method for Supplements, reference fingerprint R will be automatically generated as reference fingerprint.The finger-print that the present invention passes through the female ginger of foundation benefit, it can more fully reflect the relativeness of beneficial female ginger ingredient, effectively the medicinal material of different sources can also be accurately distinguished, preferably its quality is evaluated and controlled, may advantageously facilitate the development and utilization of the female ginger of Guangxi precious jade medicine benefit.
Description
Technical field
The present invention relates to Fingerprint of Chinese medicine materia technical field, specifically precious jade medicinal material benefit mother ginger methylene chloride position HPLC refers to
The method for building up of line map.
Background technique
Beneficial mother ginger is zingiberaceous plant Costus speciosusCostus speciosus(Koen.) SmithRhizome, perennial root grass
This, is to commonly use one of precious jade medicine.The Yao nationality is civil often as the nursing medication of puerpera, good to the rehabilitation efficacy of puerpera, therefore precious jade
The people are referred to as " benefit mother ginger ", " kwan-yin ginger ".It is born in the mountain valley below 1700 m of height above sea level, hayashishita or border roadside, small stream side shrub grass
At the humidity such as clump.It is distributed mainly on the provinces and regions such as Guangxi, Guangdong, Yunnan, Fujian, Taiwan, Southeast Asia and South Asia subtropical zone
It is distributed.The Guangxi female ginger of benefit be distributed mainly on guest, Baise, Guilin, Pingxiang, Guiping, Guigang, Qiezhou, Nanning, Yulin, Liuzhou,
The ground such as Wuzhou are born at the lower hayashishita of height above sea level or barren hill humidity more.Beneficial mother ginger can treat a variety of diseases, and it is few to be not only China
The national conventional Chinese medicine of number, also there is very long medicinal history, therefore the quality evaluation of the medicine in states such as India, Bhutan, Cambodia
Once became the hot spot studied both at home and abroad.
The nature and flavor of beneficial mother ginger, toxicity, Habitat producing aspect, each ancient books and records record is different, goes through version " Chinese Pharmacopoeia " and precious jade medicine mark
Uncharged this product, lacks unified standard in standard, this is easy for the confusion for causing this product nature and flavor, habitat processing method, medicinal material
Quality be difficult to be effectively controlled, to influence its clinical efficacy.Domestic Qiao Chunfeng and Su new people etc. have carried out respectively to be closed
The identification research and dai medicine material Costus speciosus quality research of sheath ginger class medicinal material, Pratap Singh etc. is also to the Costus speciosus of India locality
Pharmacognostical study is carried out.However the present inventor by comparison, it was found that, though the female ginger of precious jade medicinal material benefit and Costus speciosus are same base source medicine
Material, but because of reasons such as the weather of each department, soil property differences, there are larger differences for the content of benefit mother ginger, so that existing identification
Method cannot control effectively to the quality of beneficial female ginger medicinal material, and it is accurate to carry out to the quality of the female ginger medicinal material of Guangxi province benefit
Evaluation.The female ginger of precious jade medicine benefit is widely used, but due to a lack of the feasible quality evaluating method of specification, so that its Different therapeutical effect is larger, shadow
It is rung further to develop and utilize.
The finger-print of Chinese medicine is a kind of characteristic spectrum with height specificity, can than more fully characterize medicinal material at
The relativeness divided has become the effective means of generally acknowledged quality control at present.The growing environment of same base medicinal material, harvesting
Season and holding conditions are different, and finger-print also can difference.
It there is no the relevant report of beneficial female ginger finger-print in the prior art both at home and abroad at present.
Summary of the invention
The object of the present invention is to provide a kind of quality evaluating methods of female ginger of precious jade medicinal material benefit, by the fingerprint for establishing the female ginger of benefit
Map can more fully reflect the relativeness of beneficial female ginger ingredient, can also effectively to the medicinal material of different sources into
Row accurately distinguishes, and preferably its quality is evaluated and controlled, and promotes the development and utilization of the female ginger of Guangxi precious jade medicine benefit.
In order to effectively control the quality of the female ginger of precious jade medicinal material benefit, original of the present invention to the 11 batches of female ginger medicinal materials of different sources benefit in Guangxi
Phytomorph, Medicinal Materials Characters, microscopic features, thin layer identification, item inspection, assay etc. carry out quality evaluation, establish
Its quality standard, the further development and utilization for the female ginger of Guangxi precious jade medicinal material benefit provide the foundation of science.
The invention is realized by the following technical scheme:
The method for building up of precious jade medicinal material benefit female ginger methylene chloride position HPLC finger-print, comprising the following steps:
(1) it prepares test solution: accurately weighing beneficial female 2.0000 g of ginger medicinal material, the ethyl alcohol that volume fraction is 70% is added in precision
Solution 50ml, weighed weight, 60 min of ultrasound weigh after letting cool, supply the weight of less loss, and filtration, filtrate precision is added 3
20 ml of hydrochloric acid solution of mol/ml, water-bath flow back 2 h, after letting cool, are adjusted to the sodium hydroxide solution that mass fraction is 40%
Property, water bath method, residue is with 30 ml hot water dissolvings and is transferred in separatory funnel with chloroform extraction, extraction 2 times, every time
30 ml merge chloroform part, volatilize, residue methanol constant volume is into 10 ml brown volumetric flasks, with 0.22 μm before sample introduction
The filtration of organic filter membrane to get；
(2) prepare reference substance solution: accurately weighed diosgenin standard items are appropriate, dissolved with methanol, and 1 ml is made containing Chinese yam
The reference substance solution of 0.428 mg of sapogenin, stores in the volumetric flask of brown, is filtered before sample introduction with 0.22 μm of organic filter membrane,
To obtain the final product；
(3) HPLC is detected: above-mentioned reference substance solution and test solution are drawn respectively, are injected separately into high performance liquid chromatograph,
High performance liquid chromatography detection is carried out according to chromatographic condition, records the HPLC map in 95min；
Wherein, the chromatographic condition are as follows: permaphase ODS-SP C18(250 × 4.6 nm, 5M)；Detection wavelength 203
nm；0.8 ml/min of flow velocity；30 DEG C of column temperature；Sample volume 10l；Mobile phase A: acetonitrile；Mobile phase B: 0.1% phosphoric acid；Gradient
Elution requirement is as follows: 0~10 min, 5%A+95%B；10~40 min, 20%A+80%B；40~65 min, 70%A+30%B；65
~95 min, 95%A+5%B；Percentage indicates percent by volume shared by mobile phase A and B.
(4) multiple test samples are detected, utilizes " similarity evaluation " software, passes through
The method of Supplements matches all chromatographic peaks, will automatically generate reference fingerprint R as reference fingerprint.
One, the chromatographic condition of finger-print of the present invention is investigated as follows:
1, the selection of mobile phase
The chemical component of beneficial mother's ginger medicinal material is complex, and present invention employs the methods of gradient elution respectively to methanol-water, first
- 0.1% phosphoric acid of alcohol, acetonitrile-water, acetonitrile four kinds of flow phase systems of -0.1% phosphoric acid are investigated, and are with the separating effect of chromatographic peak
Inspection target finds suitable mobile phase.As a result as shown in Figure 1.
Experimental result is shown, when using methanol as mobile phase, chromatogram baseline seriously drifts about, and the number of chromatographic peak compared with
It is few；More with the chromatogram Interference Peaks that acetonitrile-water flow phase system affords, separating degree is poor.Comprehensively consider.Finally select
Gradient elution is carried out with -0.1% phosphoric acid flow phase system of acetonitrile.
2, the selection of Detection wavelength
In order to obtain the chemical information of the female ginger medicinal material of more benefits, the selection of Detection wavelength is particularly important.The present invention is with chromatogram peak out
Quantity, abundance, baseline drift situation etc. be inspection target, respectively to 196 nm, 203 nm, 206 nm, 208 nm, 225 nm
Five kinds of different wave lengths are investigated, and suitable Detection wavelength is selected.As a result as shown in Figure 2.
Experimental result shows that Detection wavelength is smaller, and the peak height of chromatographic peak is higher, but baseline can also drift about increase.Work as detection
When wavelength is 196 ~ 208 nm, the peak number amount of each chromatogram is consistent, and when using 225 nm as Detection wavelength, chromatographic peak number is obvious
It reduces.In conjunction with the chromatogram under each Detection wavelength, final choice is using 203 nm as Detection wavelength.
3, sample volume, column temperature, flow velocity, the selection of analysis time
The present invention is also to the different column temperatures of chromatographic condition (25,30,25 DEG C)；Different sample injection volumes (5,10,15,20
L)；(0.5,0.8,1.0,1.2 ml/min) different in flow rate is investigated respectively, and the chromatogram under the conditions of comparison is each, synthesis is examined
Consider the factors such as peak type, retention time, separating degree, peak height, determines with 20L is sample volume, 30 DEG C of column temperature, flow velocity 0.8
Ml/min carries out the measurement of the female ginger finger-print of benefit.As a result as shown in Fig. 3,4,5.
It is found according to the chromatography that identical chromatographic condition carries out 120 min to sample solution, chromatogram after 95 min
Occur again without lag peak, therefore selects the analysis time using 95 min as the finger-print.
To sum up, the female ginger finger-print optimum chromatogram condition of the benefit finally determined are as follows: C18(250 × 4.6 permaphase ODS-SP
Nm, 5M)；Detection wavelength: 203 nm；Flow velocity: 0.8 ml/min；Column temperature: 30 DEG C；Sample volume: 10l。
Two, to the investigation of the method for the present invention
1, precision is investigated
Same test solution is taken, repeats sample introduction 6 times, is measured by chromatographic condition of the invention.Calculate each main chromatographic peak
Relative retention time and relative peak area and its RSD value, the results are shown in Table 1, table 2.
1 precision test of table-relative retention time (n=6)
2 precision tests of table-relative peak area (n=6)
By table 1, table 2 it is found that the relative retention time of each main chromatographic peak of 6 sample introductions and the RSD value of relative peak area are respectively less than
3.00%, show that instrument precision is good.
2, repetitive test
Female 6 parts of medicinal material of the ginger of same batch (YMJ-6,20180920) benefit is taken, is prepared by the preparation method of test solution of the present invention
Test liquid, successively sample introduction, is detected with chromatographic condition of the invention.Calculate the opposite of each main chromatographic peak of 6 parts of finger-prints
Retention time and its RSD value, the results are shown in Table 3, table 4.
3 repetitive tests of table-relative retention time
4 repetitive tests of table-relative peak area
The results show that the RSD value of the relative retention time of each chromatographic peak is respectively less than 3.00%, show that the sample preparation methods repeat
Property is good.
Stability test
According to a test liquid of the fresh preparation of the preparation method of test solution of the present invention, with chromatographic condition of the invention respectively at
0,2,4,8,12,24 h carry out HPLC detection, calculate the relative retention time of each main chromatographic peak of 6 time point finger-prints,
And its RSD value is calculated, it the results are shown in Table 5,6.
5 stability tests of table-relative retention time
6 stability tests of table-relative peak area
Experiment shows that the RSD value of each main chromatographic peak relative retention time and relative peak area is respectively less than 1.00%, shows for examination
Product solution is basicly stable in 24 h.
Beneficial effects of the present invention:
1, the present invention studies the HPLC finger-print of beneficial female ginger, provides reference for the formulation of its quality standard.By building
The finger-print of the vertical female ginger of benefit, can more fully reflect the relativeness of beneficial female ginger ingredient, can also be effectively to not
Medicinal material with the place of production is accurately distinguished, and preferably its quality is evaluated and controlled.
2, the method for building up of precious jade medicinal material benefit of the present invention female ginger methylene chloride position HPLC finger-print, repeatability, precision
Well, high stability illustrates that measuring method operation is feasible.
3, test sample of the present invention is simple for production, and chromatographic condition is easy to accomplish, can be used for the female ginger true and false of precious jade medicinal material benefit, the place of production
With the identification and control of quality, the development and utilization of the female ginger of Guangxi precious jade medicine benefit may advantageously facilitate.
Detailed description of the invention
Fig. 1 is the chromatogram of different mobile phases；Wherein, A, acetonitrile-water；B, -0.1% phosphoric acid of acetonitrile；C, methanol-water；D, first
- 0.1% phosphoric acid of alcohol.
Fig. 2 is the chromatogram under different Detection wavelengths；Wherein, A, 196 nm, B, 203 nm, C, 206 nm, D, 208 nm,
E、225 nm。
Fig. 3 is the chromatogram under different column temperatures.
Fig. 4 is the chromatogram under different sample volumes.
Fig. 5 be it is different in flow rate under chromatogram.
Fig. 6 is the finger-print of the ten female ginger medicinal materials of a batch benefit；Wherein, S1, Bobai, S2, respectful city, S3, Guiping, S4, Heng County,
S5, Jin Xiu, S6, the Lingshan, S7, Pingguo, S8, Pingnan County, S9, Pingxiang, S10, three rivers, S11, XiLin；R is to generate after Supplements
Reference fingerprint.
Fig. 7 is the characteristic spectrum at beneficial female ginger methylene chloride position；Wherein, the female ginger test solution of A, benefit, B, reference substance are molten
Liquid C, methanol solution, S, diosgenin control.
Specific embodiment
In order to keep technical solution of the present invention and advantage clearer, below with reference to the embodiment of the present invention, to the present invention
Technical solution be clearly and completely described.
Embodiment 1
The method for building up of precious jade medicinal material benefit female ginger methylene chloride position HPLC finger-print, comprising the following steps:
(1) it prepares test solution: accurately weighing beneficial female 2.0000 g of ginger medicinal material, the ethyl alcohol that volume fraction is 70% is added in precision
Solution 50ml, weighed weight, 60 min of ultrasound weigh after letting cool, supply the weight of less loss, and filtration, filtrate precision is added 3
20 ml of hydrochloric acid solution of mol/ml, water-bath flow back 2 h, after letting cool, are adjusted to the sodium hydroxide solution that mass fraction is 40%
Property, water bath method, residue is with 30 ml hot water dissolvings and is transferred in separatory funnel with chloroform extraction, extraction 2 times, every time
30 ml merge chloroform part, volatilize, residue methanol constant volume is into 10 ml brown volumetric flasks, with 0.22 μm before sample introduction
The filtration of organic filter membrane to get；
(2) prepare reference substance solution: accurately weighed diosgenin standard items are appropriate, dissolved with methanol, and 1 ml is made containing Chinese yam
The reference substance solution of 0.428 mg of sapogenin, stores in the volumetric flask of brown, is filtered before sample introduction with 0.22 μm of organic filter membrane,
To obtain the final product；
(3) HPLC is detected: above-mentioned reference substance solution and test solution are drawn respectively, are injected separately into high performance liquid chromatograph,
High performance liquid chromatography detection is carried out according to chromatographic condition, records the HPLC map in 95min；
Wherein, the chromatographic condition are as follows: permaphase ODS-SP C18(250 × 4.6 nm, 5M)；Detection wavelength 203
nm；0.8 ml/min of flow velocity；30 DEG C of column temperature；Sample volume 10l；Mobile phase A: acetonitrile；Mobile phase B: 0.1% phosphoric acid；Gradient
Elution requirement is as follows: 0~10 min, 5%A+95%B；10~40 min, 20%A+80%B；40~65 min, 70%A+30%B；65
~95 min, 95%A+5%B；Percentage indicates percent by volume shared by mobile phase A and B.
(4) multiple test samples are detected, utilizes " similarity evaluation " software, passes through
The method of Supplements matches all chromatographic peaks, will automatically generate reference fingerprint R as reference fingerprint.
Determining fingerprint pattern is carried out to the female ginger medicinal material of the benefit in 11 places of production according to the fingerprint spectrum method of above-mentioned foundation, as a result sees figure
6。
The characteristic spectrum at beneficial mother's ginger methylene chloride position is as shown in Figure 7.As shown in Figure 7, benefit female ginger methylene chloride position is special
10 characteristic peaks can be observed in sign map, and by being compared with reference substance, having pointed out No. 5 peaks is diosgenin chromatographic peak,
Therefore using diosgenin peak as the reference peak S of beneficial female ginger methylene chloride genius loci map.
The quality evaluation of beneficial mother's ginger methylene chloride position finger-print is as follows:
" similarity evaluation 2012 editions " software is utilized, by the method for Supplements, to 11
The chromatographic peak for criticizing the female ginger medicinal material of benefit, which is matched and automatically generates reference fingerprint R(, sees Fig. 6).With the control fingerprint image of generation
Composing R is control, carries out similarity calculation to the finger-print at 11 batches of benefit female ginger methylene chloride positions, as a result as shown in table 7 below.
The female ginger fingerprint similarity calculated result of 7 benefit of table
By similarity result it is found that in addition to the similarity of the female ginger of the Pingguo place of production (YMJ-10) benefit is 0.89, the female ginger of remaining place of production benefit
The similarity of finger-print 0.90 or more, show except the female ginger content of Pingguo place of production benefit extremely in addition to, different sources benefit in Guangxi is female
The chemical composition consistency of ginger is preferable.The harvest date of 11 batches of medicinal materials is not much different, it may be possible to the place of production (soil, weather) difference etc.
This exception caused by reason.
Using the retention time at reference peak and peak area as 1, the relative retention time at shared peak is calculated separately and with respect to peak
Area is shown in Table 8, table 9.
The relative retention time at 80 one places of production of table each shared peak of the female ginger finger-print of benefit
The relative peak area at 90 one places of production of table each shared peak of the female ginger finger-print of benefit
By table 8, table 9 it is found that the RSD value of the relative retention time at 11 batches of each shared peaks of the female ginger map of benefit is respectively less than 3.0%, relatively
The RSD value of peak area is 0.53~141.29%, shows that the appearance time of beneficial mother's Jiang Gongyou ingredient is close, the female ginger of different sources benefit
Chemical component consistency it is good.
Claims (2)
1. the method for building up of precious jade medicinal material benefit female ginger methylene chloride position HPLC finger-print, it is characterised in that: the following steps are included:
(1) it prepares test solution: accurately weighing beneficial female 2.0000 g of ginger medicinal material, the ethyl alcohol that volume fraction is 70% is added in precision
Solution 50ml, weighed weight, 60 min of ultrasound weigh after letting cool, supply the weight of less loss, and filtration, filtrate precision is added 3
20 ml of hydrochloric acid solution of mol/ml, water-bath flow back 2 h, after letting cool, are adjusted to the sodium hydroxide solution that mass fraction is 40%
Property, water bath method, residue is with 30 ml hot water dissolvings and is transferred in separatory funnel with chloroform extraction, extraction 2 times, every time
30 ml merge chloroform part, volatilize, residue methanol constant volume is into 10 ml brown volumetric flasks, with 0.22 μm before sample introduction
The filtration of organic filter membrane to get；
(2) prepare reference substance solution: accurately weighed diosgenin standard items are appropriate, dissolved with methanol, and 1 ml is made containing Chinese yam
The reference substance solution of 0.428 mg of sapogenin, stores in the volumetric flask of brown, is filtered before sample introduction with 0.22 μm of organic filter membrane,
To obtain the final product；
(3) HPLC is detected: above-mentioned reference substance solution and test solution are drawn respectively, are injected separately into high performance liquid chromatograph,
High performance liquid chromatography detection is carried out according to chromatographic condition, records the HPLC map in 95min；
(4) multiple test samples are detected, utilizes " similarity evaluation " software, passes through multiple spot
The method of correction matches all chromatographic peaks, will automatically generate reference fingerprint R as reference fingerprint.
2. the method for building up of precious jade medicinal material benefit according to claim 1 female ginger methylene chloride position HPLC finger-print, special
Sign is: the chromatographic condition are as follows: permaphase ODS-SP C18(250 × 4.6 nm, 5)；203 nm of Detection wavelength；Stream
0.8 ml/min of speed；30 DEG C of column temperature；Sample volume 10；Mobile phase A: acetonitrile；Mobile phase B: 0.1% phosphoric acid；Gradient elution item
Part is as follows: 0~10 min, 5%A+95%B；10~40 min, 20%A+80%B；40~65 min, 70%A+30%B；65~95
Min, 95%A+5%B；Percentage indicates percent by volume shared by mobile phase A and B.
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| CN110596298A (en) * | 2019-10-15 | 2019-12-20 | 韦必爱 | Quality evaluation method of Yao medicine material Zingiber zerumbet |
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