CN110346492A - A kind of glycyrrhiza extract fingerprint atlas detection method - Google Patents
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Abstract
The present invention provides a kind of glycyrrhiza extract fingerprint atlas detection methods, characterized by comprising the following steps: control sample pre-processes, glycyrrhiza extract sample pretreatment to be measured, control sample and glycyrrhiza extract sample detection to be measured, establish reference fingerprint, glycyrrhiza extract sample fingerprint map analyzing to be measured, reference sample preparation, reference sample chromatogram, control sample Principle components analysis, glycyrrhiza extract sample Principle components analysis to be measured, glycyrrhiza extract sample quality to be measured determine.The beneficial effects of the present invention are: a kind of glycyrrhiza extract fingerprint atlas detection method provided by the invention has the advantage that a kind of glycyrrhiza extract fingerprint atlas detection method that invention provides has the fingerprint characteristic of feature, obtain the chromatographic condition of contained compound in glycyrrhiza extract, establish fingerprint file, and the method is easy, stablize, precision is high, favorable reproducibility, the quality of energy Efficient Characterization glycyrrhiza extract, is advantageously used for the quality of monitoring product.
Description
Technical field
The invention belongs to Pharmaceutical Analysis detection and quality control technology, and in particular to a kind of glycyrrhiza extract finger-print inspection
Survey method.
Background technique
Radix Glycyrrhizae also known as state be old, Herba Hedyotis cantonensis, Glycyrrhiza Uralensis, sweet tea root.Pulse family, Glycyrrhiza, perennial herb, root and root shape
Stem thickness is strong, is a kind of help Chinese herbal medicine.Medicinal part is root and rhizome, and Medicinal Materials Characters root is cylindrical, 25-100 centimetres long, directly
0.6-3.5 centimetres of diameter.Crust is elastic different, surface rufous or taupe brown.Rhizome is cylindrical, and there is a bud scar on surface, in section
There is marrow in portion.Major function is clearing heat and detoxicating, expelling phlegm and arresting coughing, stomach duct and abdomen etc., is a kind of important and common Chinese herbal medicine.
At present " Chinese Pharmacopoeia " 2015 version Radix Glycyrrhizae it is lower using high performance liquid chromatography to liquiritin in Radix Glycyrrhizae and sweet
Oxalic acid content is measured, and establishes corresponding quality standard.But it is detected and controlled without the total quality for hay slag
Method, and in view of the ingredient of Chinese herbal medicine is various, it is easy to influence each other between each ingredient, only containing with one or two effective component
Amount is difficult to the real quality of thoroughly evaluating Chinese herbal medicine.Therefore, it is necessary to develop a kind of inspection that can monitor multiple ingredients in glycyrrhiza extract
Survey method, to monitor the quality condition of glycyrrhiza extract more fully hereinafter.
With the development of modern medicines analytical technology, fingerprint pattern technology is current internationally recognized control traditional Chinese medicine quality
Quality Control mode, it crude drug cultivation, introduce a fine variety, the specification and optimization of Chinese patent drug production technology, the formulation of target level of product quality
Etc. comprehensive quality assurance is all provided.
In recent years, the external quality evaluation to herbal product is also advocating finger-print.With various analytical technologies
Innovation and raising and the continuous development of Computer Applied Technology, countries in the world are to the understanding and research of natural drug continuous deep
Entering, traditional Chinese medicine fingerprint technology at home and abroad has become a kind of development trend, and the methods and techniques of research are more mature perfect,
As internationally recognized control Chinese medicine and crude drug quality most efficient method and means.Research of the China to traditional Chinese medicine fingerprint
It is still in the junior stage, there is certain gap compared with developed countries.Chinese medicine and system predominantly are established using various methods at present
The finger-print of agent, and digitized processing is carried out to corresponding information, it can evaluate and control the matter of Chinese medicine and preparation
Amount.
In view of the above problems, we are badly in need of connecting finger-print and research method and the control of the quality of glycyrrhiza extract,
A kind of easy, efficient, practical glycyrrhiza extract fingerprint atlas detection method is established, for the quality evaluation to glycyrrhiza extract.
Summary of the invention
In order to make up for the deficiencies of the prior art, the present invention provides a kind of easy, efficient, practical glycyrrhiza extract finger-prints
Detection method.
A kind of glycyrrhiza extract fingerprint atlas detection method provided by the invention, comprising the following steps:
(1) control sample pre-process: take control glycyrrhiza extract sample sufficiently crushes, cross 80 meshes obtain compare Radix Glycyrrhizae ground-slag,
It takes aforementioned control Radix Glycyrrhizae ground-slag appropriate, adds the 75-80% ethyl alcohol interval ultrasound extraction of 23 times of amounts, mended later with 75-80% ethyl alcohol
Foot loss amount of solution, shakes up, is filtered with 0.2 μm of miillpore filter, obtain control sample solution；Aforementioned interval ultrasound extracts specific
Method is ultrasound 3mim under the conditions of power 50W, frequency 45kHz first, is quickly cooled down, then in power 60W, frequency 55kHz
Under the conditions of ultrasound 5mim, be cooled to room temperature；
(2) glycyrrhiza extract sample pretreatment to be measured: glycyrrhiza extract sample to be measured is taken sufficiently to crush, 80 meshes excessively obtain Radix Glycyrrhizae to be measured
Ground-slag takes aforementioned Radix Glycyrrhizae ground-slag to be measured appropriate, adds the 75-80% ethyl alcohol interval ultrasound extraction of 23 times of amounts, uses 75-80% second later
Alcohol supplies loss amount of solution, shakes up, and is filtered with 0.2 μm of miillpore filter, obtains glycyrrhiza extract sample solution to be measured；Aforementioned interval ultrasound
The specific method of extraction is ultrasound 3mim under the conditions of power 50W, frequency 45kHz first, is quickly cooled down, then in power
Ultrasound 5mim, is cooled to room temperature under the conditions of 60W, frequency 55kHz；
(3) control sample and glycyrrhiza extract sample detection to be measured: respectively it is accurate draw aforementioned control sample solution and it is aforementioned to
Each 10 μ L of glycyrrhiza extract sample solution is surveyed, injection high performance liquid chromatograph is detected, and records the chromatogram in 100min, is obtained
Control sample chromatogram and glycyrrhiza extract sample chromatogram figure to be measured；Aforementioned high performance liquid chromatograph testing conditions are as follows:
Chromatographic column: being filling phase with octadecylsilane chemically bonded silica；
Detection wavelength: 335-340nm；
Mobile phase: using acetonitrile as mobile phase A, using glacial acetic acid solution as Mobile phase B；
Gradient elution program is as follows:
Flow velocity: 1.2-1.5mL/min；
Column temperature: 26-28 DEG C；
(4) establish reference fingerprint: aforementioned control sample chromatogram is reference fingerprint, aforementioned control fingerprint image
Spectrum includes 11 characteristic peaks, and the relative retention time at preceding feature peak is respectively as follows: peak 1:0.117 ± 0.005；Peak 2:0.517
±0.005；Peak 3:0.638 ± 0.005；Peak 4:0.655 ± 0.005；Peak 5:0.707 ± 0.005；Peak 6:0.793 ± 0.005；
Peak 7:0.965 ± 0.005；Peak 8:1.00 ± 0.005；Peak 9:1.052 ± 0.005；Peak 10:1.241 ± 0.005；11:1.259
±0.005；
(5) glycyrrhiza extract sample fingerprint map analyzing to be measured: aforementioned glycyrrhiza extract sample chromatogram figure to be measured is glycyrrhiza extract to be measured
Aforementioned glycyrrhiza extract sample finger-print to be measured is compared with aforementioned reference fingerprint, utilizes fingerprint by sample finger-print
Map software calculates similarity, determines similarity numerical value；
(6) prepared by reference sample: extracting liquorice chalcone A, liquiritin, glycyrrhizic acid and onocerin reference substance are appropriate, add 23
Mixed solution is made as reference sample solution in the 75-80% ethyl alcohol of amount again；
(7) reference sample chromatogram: precision draws 10 μ L of reference sample solution, injects high performance liquid chromatograph according to step
(3) testing conditions of aforementioned high performance liquid chromatograph are detected in, and record the chromatogram in 100min, obtain reference sample
Chromatogram；
(8) control sample Principle components analysis: control sample finger-print is compared with reference sample chromatogram, really
Determine the content of extracting liquorice chalcone A, liquiritin, glycyrrhizic acid and onocerin in control sample；
(9) glycyrrhiza extract sample Principle components analysis to be measured: by glycyrrhiza extract sample finger-print to be measured and reference sample chromatography
Figure is compared, and determines extracting liquorice chalcone A, liquiritin, glycyrrhizic acid and onocerin component content in glycyrrhiza extract sample to be measured；
(10) glycyrrhiza extract sample quality to be measured determines: when glycyrrhiza extract sample finger-print to be measured is similar to reference fingerprint
Degree value is greater than 0.85, and the deviation of glycyrrhiza extract sample to be measured and each main component of control sample all controls within 5%, aforementioned
Glycyrrhiza extract sample to be measured is qualified samples.
As a preferred solution, the column model of high performance liquid chromatograph testing conditions described in step (3) is
GRACE Prevail C185μ4.6×250mm。
It is further preferred that the Detection wavelength of high performance liquid chromatograph testing conditions described in step (3) is 338nm.
It is further preferred that the mobile phase of high performance liquid chromatograph testing conditions described in step (3) are as follows: with acetonitrile be stream
Dynamic phase A, using 0.5% glacial acetic acid solution as Mobile phase B.
It is further preferred that the flow velocity of high performance liquid chromatograph testing conditions described in step (3) is 1.2mL/min, column
Temperature is 28 DEG C.
A kind of glycyrrhiza extract fingerprint atlas detection method above-mentioned is used for the detection of glycyrrhiza extract finger-print.
A kind of glycyrrhiza extract fingerprint atlas detection method above-mentioned determines for glycyrrhiza extract sample quality.
The beneficial effects of the present invention are: a kind of glycyrrhiza extract fingerprint atlas detection method provided by the invention has following excellent
Gesture:
1. a kind of glycyrrhiza extract fingerprint atlas detection method for inventing offer has the fingerprint characteristic of feature, glycyrrhiza extract is obtained
In contained compound chromatographic condition, establish fingerprint file, provide technical support for the quality testing of medicinal material；
2. invent offer a kind of glycyrrhiza extract fingerprint atlas detection method have appearance is more, separating degree is good, it is reproducible,
The high feature of precision, the quality for overall monitor glycyrrhiza extract provide effective guarantee；
3. a kind of glycyrrhiza extract fingerprint atlas detection method that offer is provided can also detect licochalcone A in glycyrrhiza extract,
The content of liquiritin, glycyrrhizic acid and onocerin realizes that multi-angle controls the quality of glycyrrhiza extract；
4. a kind of glycyrrhiza extract fingerprint atlas detection method for inventing offer is easy, stablize, precision is high, favorable reproducibility, energy
The quality of Efficient Characterization glycyrrhiza extract is advantageously used for monitoring product quality.
Detailed description of the invention
Fig. 1 is the reference fingerprint in specific embodiments of the present invention；
Fig. 2 is the reference sample chromatogram in specific embodiments of the present invention.
Specific embodiment
In order to make the object, technical scheme and advantages of the embodiment of the invention clearer, to the technology in the embodiment of the present invention
Scheme is clearly and completely described, it is clear that and described embodiments are some of the embodiments of the present invention, rather than whole
Embodiment.Based on the embodiments of the present invention, those of ordinary skill in the art are obtained without creative efforts
The every other embodiment obtained, shall fall within the protection scope of the present invention.
Embodiment 1
A kind of glycyrrhiza extract fingerprint atlas detection method, comprising the following steps:
(1) control sample pre-processes: control glycyrrhiza extract (Xinjiang Quanan Pharmaceutical Co., Ltd.'s offer) sample is taken sufficiently to crush,
It crosses 80 meshes and obtains control Radix Glycyrrhizae ground-slag, take control Radix Glycyrrhizae ground-slag appropriate, add the 75% ethyl alcohol interval ultrasound extraction of 23 times of amounts, it
Loss amount of solution is supplied with 75% ethyl alcohol afterwards, is shaken up, is filtered with 0.2 μm of miillpore filter, obtains control sample solution；Interval ultrasound
The specific method of extraction is ultrasound 3mim under the conditions of power 50W, frequency 45kHz first, is quickly cooled down, then in power
Ultrasound 5mim, is cooled to room temperature under the conditions of 60W, frequency 55kHz；
(2) glycyrrhiza extract sample pretreatment to be measured: glycyrrhiza extract sample to be measured (specific sample source is shown in Table 1) is taken sufficiently to crush,
It crosses 80 meshes and obtains Radix Glycyrrhizae ground-slag to be measured, take Radix Glycyrrhizae ground-slag to be measured appropriate, add the 75% ethyl alcohol interval ultrasound extraction (side of 23 times of amounts
The same step of method (1)), loss amount of solution is supplied with 75% ethyl alcohol later, is shaken up, is filtered, is obtained to be measured sweet with 0.2 μm of miillpore filter
Careless slag sample solution；
The glycyrrhiza extract sample source table to be measured of table 1
(3) control sample and glycyrrhiza extract sample detection to be measured: accurate respectively to draw control sample solution and glycyrrhiza extract to be measured
Each 10 μ L of sample solution, injection high performance liquid chromatograph is detected, and records the chromatogram in 100min, obtains control sample
Chromatogram and glycyrrhiza extract sample chromatogram figure to be measured；High performance liquid chromatography detection condition is as follows:
Chromatographic column: octadecylsilane chemically bonded silica is filling phase, and concrete model is GRACE Prevail C185 μ 4.6
×250mm；
Detection wavelength: 338nm；
Mobile phase: using acetonitrile as mobile phase A, using 0.5% glacial acetic acid solution as Mobile phase B；
Gradient elution program is as follows:
Flow velocity: 1.2mL/min；
Column temperature: 28 DEG C；
(4) establish reference fingerprint: control sample chromatogram is reference fingerprint, is specifically shown in Fig. 1, compares fingerprint
Map includes 11 apparent characteristic peaks, and the relative retention time of characteristic peak is respectively as follows: peak 1:0.117 ± 0.005, peak 2:
0.517 ± 0.005, peak 3:0.638 ± 0.005, peak 4:0.655 ± 0.005, peak 5:0.707 ± 0.005, peak 6:0.793 ±
0.005, peak 7:0.965 ± 0.005, peak 8:1.00 ± 0.005, peak 9:1.052 ± 0.005, peak 10:1.241 ± 0.005,11:
1.259±0.005；
(5) glycyrrhiza extract sample fingerprint map analyzing to be measured: glycyrrhiza extract sample chromatogram figure to be measured is glycyrrhiza extract sample to be measured
Glycyrrhiza extract sample finger-print to be measured is compared finger-print with reference fingerprint, is calculated using finger-print software
Its similarity determines that similarity numerical value, concrete outcome are shown in Table 2；
The glycyrrhiza extract sample similarity numerical result to be measured of table 2
(6) prepared by reference sample: extracting liquorice chalcone A, liquiritin, glycyrrhizic acid and onocerin reference substance are appropriate, add 23
Mixed solution is made as reference sample solution in 75% ethyl alcohol of amount again；
(7) reference sample chromatogram: precision draws 10 μ L of reference sample solution, injects high performance liquid chromatograph according to step
(3) testing conditions of high performance liquid chromatography are detected in, and record the chromatogram in 100min, obtain reference sample chromatography
Figure, is specifically shown in Fig. 2；
(8) control sample Principle components analysis: control sample finger-print is compared with reference sample chromatogram, really
Determining each Contents of Main Components in control sample is respectively the 5.2% of licochalcone A, liquiritin 0.6%, glycyrrhizic acid 2.0%, awns
Handle florigen 0.9%；
(9) glycyrrhiza extract sample Principle components analysis to be measured: by glycyrrhiza extract sample finger-print to be measured and reference sample chromatography
Figure is compared, and determines extracting liquorice chalcone A, liquiritin, glycyrrhizic acid and onocerin component content in glycyrrhiza extract sample to be measured,
And the deviation of each main component of glycyrrhiza extract sample and control sample to be measured is calculated, concrete outcome is shown in Table 3；
The bias contribution of table 3 glycyrrhiza extract sample to be measured and each main component of control sample
(10) glycyrrhiza extract sample quality to be measured determines: when glycyrrhiza extract sample finger-print to be measured is similar to reference fingerprint
Degree value is greater than 0.85, and the deviation of glycyrrhiza extract sample to be measured and each main component of control sample all controls and is within 5%
Qualified samples are failed test sample according to this standard determination glycyrrhiza extract sample 6 to be measured, remaining is qualified samples.
The methodology reliability of one of embodiment 1 glycyrrhiza extract fingerprint atlas detection method is investigated, item is investigated
Mesh includes precision, reproducibility, stability:
(1) precision is investigated
It takes control sample, glycyrrhiza extract sample 1 to be measured, glycyrrhiza extract sample 10 to be measured, investigates withinday precision, for three days on end
Day to day precision calculates its similarity using finger-print software, and calculated result is shown in Table 4.The result shows that: similarity value is big
In 0.990, the method precision is good.
4 precision of table investigates result
(2) study on the stability
Take control sample, glycyrrhiza extract sample 1 to be measured, 10 each 1 parts of glycyrrhiza extract sample to be measured, respectively close to 0h, 3.5h,
7h, 11h, 15h, for 24 hours sample introduction measure finger-print, calculate its similarity using finger-print software, calculated result is shown in Table 5.Knot
Fruit shows: it is good that similarity value is all larger than sample stability in 0.990,24 hours.
5 study on the stability result of table
(3) reproducibility is investigated
It takes control sample, glycyrrhiza extract sample 1 to be measured, 10 each 6 parts of glycyrrhiza extract sample to be measured, measures finger-print, using referring to
Line map software calculates its similarity, and calculated result is shown in Table 6.The result shows that: similarity value is all larger than 0.990, and the method is reappeared
Property is good.
6 reproducibility of table investigates result
It should be appreciated that described above, the specific embodiments are only for explaining the present invention, is not intended to limit the present invention.By
The obvious changes or variations that spirit of the invention is extended out are still in the protection scope of this invention.
Claims (7)
1. a kind of glycyrrhiza extract fingerprint atlas detection method, which comprises the following steps:
(1) control sample pre-process: take control glycyrrhiza extract sample sufficiently crushes, cross 80 meshes obtain compare Radix Glycyrrhizae ground-slag, take institute
It states that control Radix Glycyrrhizae ground-slag is appropriate, adds the 75-80% ethyl alcohol interval ultrasound extraction of 23 times of amounts, supply damage with 75-80% ethyl alcohol later
Amount of solution is lost, is shaken up, is filtered with 0.2 μm of miillpore filter, obtains control sample solution；The specific method of the interval ultrasound extraction
For ultrasound 3mim under the conditions of power 50W, frequency 45kHz first is quickly cooled down, then in power 60W, frequency 55kHz condition
Lower ultrasound 5mim, is cooled to room temperature；
(2) glycyrrhiza extract sample pretreatment to be measured: glycyrrhiza extract sample to be measured is taken sufficiently to crush, 80 meshes excessively obtain glycyrrhiza extract to be measured
Powder takes the Radix Glycyrrhizae ground-slag to be measured appropriate, adds the 75-80% ethyl alcohol interval ultrasound extraction of 23 times of amounts, uses 75-80% ethyl alcohol later
Loss amount of solution is supplied, is shaken up, is filtered with 0.2 μm of miillpore filter, obtains glycyrrhiza extract sample solution to be measured；The interval ultrasound leaching
The specific method mentioned is ultrasound 3mim under the conditions of power 50W, frequency 45kHz first, be quickly cooled down, then power 60W,
Ultrasound 5mim, is cooled to room temperature under the conditions of frequency 55kHz；
(3) control sample and glycyrrhiza extract sample detection to be measured: respectively it is accurate draw the control sample solution with it is described to be measured sweet
Careless each 10 μ L of slag sample solution, injection high performance liquid chromatograph is detected, and records the chromatogram in 100min, is compareed
Sample chromatogram figure and glycyrrhiza extract sample chromatogram figure to be measured；The high performance liquid chromatograph testing conditions are as follows:
Chromatographic column: being filling phase with octadecylsilane chemically bonded silica；
Detection wavelength: 335-340nm；
Mobile phase: using acetonitrile as mobile phase A, using glacial acetic acid solution as Mobile phase B；
Gradient elution program is as follows:
Flow velocity: 1.2-1.5mL/min；
Column temperature: 26-28 DEG C；
(4) establish reference fingerprint: the control sample chromatogram is reference fingerprint, the reference fingerprint packet
11 characteristic peaks are included, and the relative retention time of the characteristic peak is respectively as follows: peak 1:0.117 ± 0.005；Peak 2:0.517 ±
0.005；Peak 3:0.638 ± 0.005；Peak 4:0.655 ± 0.005；Peak 5:0.707 ± 0.005；Peak 6:0.793 ± 0.005；Peak
7:0.965 ± 0.005；Peak 8:1.00 ± 0.005；Peak 9:1.052 ± 0.005；Peak 10:1.241 ± 0.005；11:1.259 ±
0.005；
(5) glycyrrhiza extract sample fingerprint map analyzing to be measured: the glycyrrhiza extract sample chromatogram figure to be measured is glycyrrhiza extract sample to be measured
The glycyrrhiza extract sample finger-print to be measured is compared with the reference fingerprint, utilizes finger-print by finger-print
Software calculates similarity, determines similarity numerical value；
(6) prepared by reference sample: extracting liquorice chalcone A, liquiritin, glycyrrhizic acid and onocerin reference substance are appropriate, add 23 times of amounts
75-80% ethyl alcohol mixed solution is made as reference sample solution；
(7) reference sample chromatogram: precision draws 10 μ L of reference sample solution, injects high performance liquid chromatograph according to step (3)
Described in the testing conditions of high performance liquid chromatograph detected, and record the chromatogram in 100min, obtain reference sample color
Spectrogram；
(8) control sample Principle components analysis: control sample finger-print is compared with reference sample chromatogram, and determining pair
In the same old way in product extracting liquorice chalcone A, liquiritin, glycyrrhizic acid and onocerin content；
(9) glycyrrhiza extract sample Principle components analysis to be measured: by glycyrrhiza extract sample finger-print to be measured and reference sample chromatogram into
Row compares, and determines extracting liquorice chalcone A, liquiritin, glycyrrhizic acid and onocerin component content in glycyrrhiza extract sample to be measured；
(10) glycyrrhiza extract sample quality to be measured determines: when glycyrrhiza extract sample finger-print to be measured degree similar to reference fingerprint
Value is greater than 0.85, and the deviation of glycyrrhiza extract sample to be measured and each main component of control sample all controls within 5%, described to be measured
Glycyrrhiza extract sample is qualified samples.
2. a kind of glycyrrhiza extract fingerprint atlas detection method according to claim 1, which is characterized in that described in step (3)
The column model of high performance liquid chromatograph testing conditions is 5 4.6 × 250mm of μ of GRACE Prevail C18.
3. a kind of glycyrrhiza extract fingerprint atlas detection method according to claim 1, which is characterized in that described in step (3)
The Detection wavelength of high performance liquid chromatograph testing conditions is 338nm.
4. a kind of glycyrrhiza extract fingerprint atlas detection method according to claim 1, which is characterized in that described in step (3)
The mobile phase of high performance liquid chromatograph testing conditions are as follows: using acetonitrile as mobile phase A, using 0.5% glacial acetic acid solution as Mobile phase B.
5. a kind of glycyrrhiza extract fingerprint atlas detection method according to claim 1, which is characterized in that described in step (3)
The flow velocity of high performance liquid chromatograph testing conditions is 1.2mL/min, and column temperature is 28 DEG C.
6. a kind of glycyrrhiza extract fingerprint atlas detection method according to claim 1, which is characterized in that the detection method is used
In the detection of glycyrrhiza extract finger-print.
7. a kind of glycyrrhiza extract fingerprint atlas detection method according to claim 1, which is characterized in that the detection method is used
Determine in glycyrrhiza extract sample quality.
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