CN105092744B - The characteristic spectrum and its method for building up of Desmodium styracifolium extractive of general flavone and application - Google Patents
The characteristic spectrum and its method for building up of Desmodium styracifolium extractive of general flavone and application Download PDFInfo
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Abstract
The invention discloses a kind of method that extractive of general flavone is extracted from Desmodium styracifolium, Flavonoid substances are constituted in a kind of method for setting up Desmodium styracifolium extractive of general flavone characteristic spectrum, a kind of determination Desmodium styracifolium method and a kind of method in determination Desmodium styracifolium source.By the method that general flavone is extracted from Desmodium styracifolium of the present invention, the general flavone in Desmodium styracifolium can be good by high efficiency extraction, and the separating degree at general flavone liquid chromatogram peak.The method proposed by the present invention for setting up Desmodium styracifolium extractive of general flavone characteristic spectrum, determine method that Flavonoid substances in Desmodium styracifolium constitute and determine the characteristics of method in Desmodium styracifolium source has stability and repeatability.
Description
Technical field
The present invention relates to biomedicine field, carried specifically, extracting general flavone from Desmodium styracifolium the present invention relates to one kind
Take method and a kind of Desmodium styracifolium extractive of general flavone characteristic spectrum and its method for building up and the application of thing.
Background technology
Desmodium styracifolium is a kind of medicinal plant, has effects that removing dampness through diuresis and removing jaundice, inducing diuresis for treating strangurtia.Containing abundant in Desmodium styracifolium
Flavonoid substances.Desmodium styracifolium extractive of general flavone is the flavonoids thing that a class has identical mother nucleus structure 2- phenyl chromones
Matter.Effect of flavones is many, and it can effectively remove internal oxygen radical, improve blood circulation, reduction cholesterol,
Reduce blood glucose, promote wound healing and analgesic.However, the extraction process of Desmodium styracifolium flavones and determination wherein Flavonoid substances group
Into method still have much room for improvement.
Meanwhile, how effectively Desmodium styracifolium is widely distributed, is distributed in the provinces and regions such as Fujian, Hunan, Guangxi and Guangdong, therefore,
Determine the source of Desmodium styracifolium and set up the problem of Desmodium styracifolium extractive of general flavone characteristic spectrum is one to be resolved.
The content of the invention
It is contemplated that at least solving one of technical problem present in prior art.Therefore, one object of the present invention
It is to propose Flavonoid substances group in a kind of method for extracting extractive of general flavone from Desmodium styracifolium, a kind of determination Desmodium styracifolium
Into method and a kind of determination Desmodium styracifolium source method.
In the first aspect of the present invention, the present invention proposes a kind of side that extractive of general flavone is extracted from Desmodium styracifolium
Method.Embodiments in accordance with the present invention, this method includes：Desmodium styracifolium sample is mixed with ethanol, and passes through backflow or ultrasound side
Method is extracted, to obtain extractive of general flavone, wherein, the concentration of the ethanol is 25~95%, the Desmodium styracifolium
Weight and the volume ratio of the ethanol are 25mg：25~100ml, preferably 25mg：50ml., both can be with by said extracted method
Ensure to extract the general flavone in Desmodium styracifolium completely, extraction efficiency is high, point at peak in later stage liquid-phase chromatographic analysis can be caused again
It is good from degree.
Embodiments in accordance with the present invention, the above-mentioned method that extractive of general flavone is extracted from Desmodium styracifolium can be wrapped further
Include at least one following additional technical feature：
Embodiments in accordance with the present invention, the above method carries out extracting 10~40 minutes, preferably 20 minutes using ultrasonic method.
By above-mentioned ultrasonic extracting method, extraction time has both been saved, the general flavone extracted completely in Desmodium styracifolium is in turn ensured that so that
Extraction efficiency is high, beneficial to the medicinal and liquid-phase chromatographic analysis of flavones.
Embodiments in accordance with the present invention, the concentration of above-mentioned ethanol is 75%.Inventor has surprisingly found that, Desmodium styracifolium sample
The extraction efficiency highest inside 75% ethanol, and in liquid-phase chromatographic analysis peak separating degree it is good.
In the second aspect of the present invention, the present invention proposes a kind of Desmodium styracifolium extractive of general flavone characteristic spectrum of setting up
Method, embodiments in accordance with the present invention, this method includes：Method as described above, extracts general flavone from Desmodium styracifolium
Extract；Liquid-phase chromatographic analysis is carried out to the extractive of general flavone, and is set up based on resulting liquid-phase chromatographic analysis result
The Desmodium styracifolium extractive of general flavone characteristic spectrum.Inventor has found, by said extracted method, total Huang in Desmodium styracifolium
Ketone can be by high efficiency extraction.Liquid-phase chromatographic analysis is carried out to above-mentioned extractive of general flavone and obtains chromatography figure, so as to set up
The characteristic spectrum of Desmodium styracifolium extractive of general flavone.Embodiments in accordance with the present invention, it is above-mentioned to set up the extraction of Desmodium styracifolium general flavone
The method degree of accuracy of thing characteristic spectrum is high, stability is high.
Embodiments in accordance with the present invention, the above-mentioned method for setting up Desmodium styracifolium extractive of general flavone characteristic spectrum can enter one
Step includes at least one following additional technical feature：
Embodiments in accordance with the present invention, the liquid-phase chromatographic analysis uses following condition：Using the formic acid body of methanol -0.1%
System carries out gradient elution, and chromatographic column is Waters BEH C18(2.1mm × 100mm, 1.8 microns), column temperature is 25 degrees Celsius, stream
Speed is 0.2ml/min；Sample size：1 microlitre, Detection wavelength is 272nm, elution requirement：
| Time (min) | 0.1% formic acid | Methanol |
| 0~28 | 82→74 | 18→26 |
| 28~40 | 74 | 26 |
| 40~41 | 74→82 | 26→18 |
| 41~50 | 82 | 18 |
Under above-mentioned chromatographic condition, chromatographic peak can reach good separating effect, so as to set up Desmodium styracifolium general flavone
The characteristic spectrum of extract.Under the conditions of embodiments in accordance with the present invention, above-mentioned liquid-phase chromatographic analysis to set up Desmodium styracifolium always yellow
The characteristics of method of ketone extract characteristic spectrum has high stability and accuracy.In the third aspect of the present invention, the present invention is carried
A kind of method for determining that Flavonoid substances are constituted in Desmodium styracifolium is gone out, embodiments in accordance with the present invention, this method includes：According to
The above method, extracts extractive of general flavone from Desmodium styracifolium；Liquid-phase chromatographic analysis is carried out to extractive of general flavone, and based on institute
Obtained liquid-phase chromatographic analysis result determines the composition of Flavonoid substances in the Desmodium styracifolium.Inventor has found, by above-mentioned
General flavone in extracting method, Desmodium styracifolium can be by high efficiency extraction.Liquid-phase chromatographic analysis is carried out to above-mentioned extractive of general flavone
Chromatography figure is obtained, so as to the accurate composition for determining Flavonoid substances in Desmodium styracifolium.Embodiments in accordance with the present invention, on
State and determine that Flavonoid substances are constituted in Desmodium styracifolium method degree of accuracy height, stability are high.
The method that Flavonoid substances are constituted in embodiments in accordance with the present invention, above-mentioned determination Desmodium styracifolium can be wrapped further
Include at least one following additional technical feature：
Liquid-phase chromatographic analysis in embodiments in accordance with the present invention, this method uses following condition：Using methanol -0.1%
Formic acid system carries out gradient elution, and chromatographic column is Waters BEH C18(2.1mm × 100mm, 1.8 microns), column temperature is 25 Celsius
Degree, flow velocity is 0.2ml/min, sample size：1 microlitre, Detection wavelength is 272nm, and elution requirement is：
| Time (min) | 0.1% formic acid | Methanol |
| 0~28 | 82→74 | 18→26 |
| 28~40 | 74 | 26 |
| 40~41 | 74→82 | 26→18 |
| 41~50 | 82 | 18 |
Under above-mentioned chromatographic condition, chromatographic peak can reach good separating effect, so as to effectively determine in Desmodium styracifolium
Flavonoid substances are constituted.Flavones in determination Desmodium styracifolium under the conditions of embodiments in accordance with the present invention, above-mentioned liquid-phase chromatographic analysis
The characteristics of method of class material composition has high stability and accuracy.
In the fourth aspect of the present invention, the present invention proposes a kind of method for determining Desmodium styracifolium source, according to the present invention
Embodiment, this method includes：According to the method for extractive of general flavone in said extracted Desmodium styracifolium, extracted from Desmodium styracifolium
Extractive of general flavone；And liquid-phase chromatographic analysis is carried out to the extractive of general flavone, and based on resulting liquid chromatogram point
Analysis result determines the source of the Desmodium styracifolium.Inventor has found that, by said extracted method, the general flavone in Desmodium styracifolium can
With by high efficiency extraction.Liquid-phase chromatographic analysis is carried out to above-mentioned extractive of general flavone and obtains chromatography figure, so as to accurate determination
The source of Desmodium styracifolium.Embodiments in accordance with the present invention, the method degree of accuracy height in above-mentioned determination Desmodium styracifolium source, stability
It is high.
Embodiments in accordance with the present invention, the method in above-mentioned determination Desmodium styracifolium source may further include following additional skill
At least one art feature：
The Detection wavelength that liquid-phase chromatographic analysis in embodiments in accordance with the present invention, this method is used is 272nm.Examine herein
Survey under wavelength condition, chromatographic peak can reach good separating effect, so as to effectively determine Desmodium styracifolium source.
Liquid-phase chromatographic analysis in embodiments in accordance with the present invention, this method uses following condition：Using methanol -0.1%
Formic acid system carries out gradient elution, and chromatographic column is Waters BEH C18(2.1mm × 100mm, 1.8 microns), column temperature is 25 Celsius
Degree, flow velocity is 0.2ml/min, sample size：1 microlitre, elution requirement is：
| Time (min) | 0.1% formic acid | Methanol |
| 0~28 | 82→74 | 18→26 |
| 28~40 | 74 | 26 |
| 40~41 | 74→82 | 26→18 |
| 41~50 | 82 | 18 |
Under above-mentioned chromatographic condition, chromatographic peak can reach good separating effect, so as to effectively determine that Desmodium styracifolium comes
Source.The method in the determination Desmodium styracifolium source under the conditions of embodiments in accordance with the present invention, above-mentioned liquid-phase chromatographic analysis has high steady
The characteristics of qualitative and accuracy.
Embodiments in accordance with the present invention, it is above-mentioned that the Desmodium styracifolium is determined based on resulting liquid-phase chromatographic analysis result
Source includes：(a) chromatogram of the liquid-phase chromatographic analysis result is compared with the predetermined collection of illustrative plates that compares；And (b) sentences
Break the chromatogram and the similitude for compareing collection of illustrative plates, and determine based on the similitude source of the Desmodium styracifolium.
By the similitude of comparative analysis result chromatogram and control chromatogram, the source of Desmodium styracifolium can be effectively determined.According to this
The embodiment of invention, the characteristics of method in above-mentioned determination Desmodium styracifolium source has high stability and accuracy.
Embodiments in accordance with the present invention, in step (b), the similitude is to be based at least one following position in collection of illustrative plates
Determined with the presence or absence of peak：15.01min、15.71min、16.60min、18.04min、19.77min、21.38min、
22.78min, 23.18min, 23.73min, 27.2min, 29.51min, 31.08min, 31.72min, 32.98min, wherein,
The peak of the position represents as follows：15.01min represents vicenin-2, and 18.04min represents cyanidenon -6-C- α-L- pyrans
Arabinose -8-C- β-D- glucopyranosides, 19.77min represents vicenin-1, and 23.18min represents cyanidenon -6-C-
β-D- glucopyranoses -8-C- β-xylopyranose glucosides, 23.73min represent Schaftoside, and 27.20min represents genistein -7-
O- β-D- furans celerys glycosyl-(1 → 6)-O- β-D- glucopyranosides, 31.08min represents new Schaftoside, 31.72min generations
Table Saponaretin, 32.98min represents Vicenin-3.Embodiments in accordance with the present invention, the peak shape of above-mentioned position and relative reservation
Time is stable, by contrasting the chromatogram peak shape and control chromatogram peak shape of Desmodium styracifolium to be determined, can effectively determine wide gold
The source of money grass.
Embodiments in accordance with the present invention, in step (b), the similitude is to be based in collection of illustrative plates lower column position whether there is
Peak and determine：15.01min、15.71min、18.04min、19.77min、22.78min、23.18min、23.73min、
27.2min, 29.51min, 31.08min, 31.72min, 32.98min, wherein, the peak representative of the position is as follows：
15.01min represents vicenin-2, and 18.04min represents cyanidenon -6-C- α-L- arabopyranose -8-C- β-D- pyrans
Glucoside, 19.77min represents vicenin-1,23.18min represent cyanidenon -6-C- β-D- glucopyranose -8-C- β -
Xylopyranose glucosides, 23.73min represents Schaftoside, 27.20min represent genistein -7-O- β-D- furans celerys glycosyl-(1 →
6)-O- β-D- glucopyranosides, 31.08min represents new Schaftoside, and 31.72min represents Saponaretin, 32.98min generations
Table V icenin-3.Embodiments in accordance with the present invention, the peak shape and relative retention time of above-mentioned position have stability, by right
Than the chromatogram peak shape and control chromatogram peak shape of Desmodium styracifolium to be determined, the source of Desmodium styracifolium can be effectively determined.
It will be appreciated by those skilled in the art that the method and effect of the determination at position peak are equally applicable to build in above-mentioned collection of illustrative plates
The method of vertical Desmodium styracifolium extractive of general flavone characteristic spectrum and the method for determining Flavonoid substances composition in Desmodium styracifolium.
The additional aspect and advantage of the present invention will be set forth in part in the description, and will partly become from the following description
Obtain substantially, or recognized by the practice of the present invention.
Brief description of the drawings
Fig. 1 is according to embodiments of the present invention 1 reference substance UV scanning figure；
Fig. 2 is according to embodiments of the present invention 1 when carrying out liquid-phase chromatographic analysis to Desmodium styracifolium extractive of general flavone, different
Formic acid concn as chromatogram flow phase chromatogram；
Fig. 3 is according to embodiments of the present invention 1 when carrying out liquid-phase chromatographic analysis to Desmodium styracifolium extractive of general flavone, different
Chromatogram under column temperature；
Fig. 4 is according to embodiments of the present invention 1 when carrying out liquid-phase chromatographic analysis to Desmodium styracifolium extractive of general flavone, different
The chromatogram of brand chromatographic column；
Fig. 5 is according to embodiments of the present invention 1 chromatogram that liquid-phase chromatographic analysis is carried out to Desmodium styracifolium extractive of general flavone
Figure；
When Fig. 6 is according to embodiments of the present invention 2 investigation Desmodium styracifolium total flavone extracting method, the conduct evaluation of selection refers to
Five peak areas of target are larger and the preferable characteristic peak of separating degree；
Fig. 7 be according to embodiments of the present invention 3 superelevation liquid chromatogram (UPLC) peak attribution analysis experiment in, reference substance and
Sample chromatogram figure；
Fig. 8 is the total ion current figure of according to embodiments of the present invention 3 detection sample；
Fig. 9 is according to embodiments of the present invention 3 reference substance Vicenin-2 total ion current figure；
Figure 10 is the mass spectrogram of according to embodiments of the present invention 3 sample and reference substance Vicenin-2；
Figure 11 is according to embodiments of the present invention 3 reference substance cyanidenon -6-C- α-L- arabopyranose -8-C- β-D-
The total ion chromatogram of glucopyranoside；
Figure 12 is according to embodiments of the present invention 3 sample and reference substance cyanidenon -6-C- α-L- arabopyranoses -8-
The mass spectrogram of C- β-D- glucopyranosides；
Figure 13 is according to embodiments of the present invention 3 reference substance Vicenin-1 total ion current figure；
Figure 14 is the mass spectrogram of according to embodiments of the present invention 3 sample and reference substance Vicenin-1；
Figure 15 is according to embodiments of the present invention 3 reference substance cyanidenon -6-C- β-D- glucopyranoses -8-C- β-pyrans
The total ion current figure of xyloside；
Figure 16 is according to embodiments of the present invention 3 sample and reference substance cyanidenon -6-C- β-D- glucopyranoses -8-C-
The mass spectrogram of β-xylopyranose glucosides；
Figure 17 is the according to embodiments of the present invention 3 total ion figure of reference substance Schaftoside；
Figure 18 is the mass spectrogram of according to embodiments of the present invention 3 sample and reference substance Schaftoside；
Figure 19 is according to embodiments of the present invention 3 reference substance genistein -7-O- β-D- furans celerys glycosyl-(1 → 6)-O-
The total ion current figure of β-D- glucopyranosides；
Figure 20 be according to embodiments of the present invention 3 sample and reference substance genistein -7-O- β-D- furans celerys glycosyl-(1 →
6) mass spectrogram of-O- β-D- glucopyranosides；
Figure 21 is the total ion chromatogram of the according to embodiments of the present invention 3 new Schaftoside of reference substance；
Figure 22 is the mass spectrogram of according to embodiments of the present invention 3 sample and the new Schaftoside of reference substance；
Figure 23 is according to embodiments of the present invention 3 reference substance Saponaretin total ion current figure；
Figure 24 is the mass spectrogram of according to embodiments of the present invention 3 sample and reference substance Saponaretin；
Figure 25 is according to embodiments of the present invention 3 reference substance Vicenin-3 total ion current figure；
Figure 26 is the mass spectrogram of according to embodiments of the present invention 3 sample and reference substance Vicenin-3；
Figure 27 be according to embodiments of the present invention 3 retention time be 15.71min, 16.60min, 21.38min,
The mass spectrogram at 22.78min and 29.51min five unknown peaks；
Figure 28 be according to embodiments of the present invention 4 investigate detecting system stability test in, be used as inspection target
Characteristic spectrum with 12 characteristic peaks；
Figure 29 be according to embodiments of the present invention 5 different batches Desmodium styracifolium medicinal material and its extractive of general flavone before extraction
Chromatography figure afterwards；
Figure 30 is the liquid chromatogram of the Desmodium styracifolium extractive of general flavone sample of according to embodiments of the present invention 60 batches
Figure；And
Figure 31 is according to embodiments of the present invention 7 feature using Schaftoside as the Desmodium styracifolium extractive of general flavone of control
Collection of illustrative plates.
Embodiment
It is an object of the invention to propose that a kind of method that extractive of general flavone is extracted from Desmodium styracifolium, a kind of determination are wide
Flavonoid substances are constituted in desmodium method and a kind of method in determination Desmodium styracifolium source.
The method that extractive of general flavone is extracted from Desmodium styracifolium
In the first aspect of the present invention, the present invention proposes a kind of side that extractive of general flavone is extracted from Desmodium styracifolium
Method.Embodiments in accordance with the present invention, this method includes：Desmodium styracifolium sample is mixed with ethanol, and passes through backflow or ultrasound side
Method is extracted, to obtain extractive of general flavone, wherein, the concentration of the ethanol is 25~95%, the Desmodium styracifolium
Weight and the volume ratio of the ethanol are 25mg：25~100ml, preferably 25mg：50ml.Inventor has found that ethanol volume is less than
25ml, then can cause extraction efficiency not high, extract incomplete, ethanol volume is higher than 100ml, can cause to extract solution excess again,
The determination of flux residual interference extractive of general flavone composition is easily caused, but in view of cost-effective but extraction completely, preferably
The volume of ethanol is 50ml.In above-mentioned concentration of alcohol and volume range, it both may insure to extract total in Desmodium styracifolium completely
Flavones, extraction efficiency is high, can cause that the separating degree at peak in later stage liquid-phase chromatographic analysis is good again.
Embodiments in accordance with the present invention, the above method carries out extracting 10~40 minutes, preferably 20 minutes using ultrasonic method.
Inventor has found that extraction time is less than 10 minutes, easily causes extraction not exclusively, extraction time is more than 40 minutes, can waste again
Time, therefore extraction time control was at 10~40 minutes, while consider the saving time and ensure to extract complete, preferred 20 points of ultrasound
Clock.By above-mentioned ultrasonic extracting method, extraction time has both been saved, the general flavone extracted completely in Desmodium styracifolium is in turn ensured that,
So that extraction efficiency is high, beneficial to the medicinal and liquid-phase chromatographic analysis of flavones.
The concentration of ethanol is 75% in embodiments in accordance with the present invention, the above method.Inventor has surprisingly found that, wide money
Careless sample peak shape in 95% ethanol is poor, and separating degree is relatively low, therefore selection Desmodium styracifolium sample is extracted inside 75% ethanol,
Extraction efficiency highest, and in liquid-phase chromatographic analysis peak separating degree it is good.
The method for setting up Desmodium styracifolium extractive of general flavone characteristic spectrum
In the second aspect of the present invention, the present invention proposes a kind of Desmodium styracifolium extractive of general flavone characteristic spectrum of setting up
Method, embodiments in accordance with the present invention, this method includes：Method as described above, extracts general flavone from Desmodium styracifolium
Extract；Liquid-phase chromatographic analysis is carried out to the extractive of general flavone, and is set up based on resulting liquid-phase chromatographic analysis result
Desmodium styracifolium extractive of general flavone characteristic spectrum.Inventor has found that, by said extracted method, the general flavone in Desmodium styracifolium can
With by high efficiency extraction.Liquid-phase chromatographic analysis is carried out to above-mentioned extractive of general flavone and obtains chromatography figure, so as to set up wide gold
The characteristic spectrum of money grass extractive of general flavone.Embodiments in accordance with the present invention, it is above-mentioned to set up Desmodium styracifolium extractive of general flavone spy
Method degree of accuracy height, the stability for levying collection of illustrative plates are high.
Embodiments in accordance with the present invention, the liquid-phase chromatographic analysis uses following condition：Using the formic acid body of methanol -0.1%
System carries out gradient elution, and chromatographic column is Waters BEH C18(2.1mm × 100mm, 1.8 microns), column temperature is 25 degrees Celsius, stream
Speed is 0.2ml/min；Sample size：1 microlitre, Detection wavelength is 272nm, elution requirement：
| Time (min) | 0.1% formic acid | Methanol |
| 0~28 | 82→74 | 18→26 |
| 28~40 | 74 | 26 |
| 40~41 | 74→82 | 26→18 |
| 41~50 | 82 | 18 |
Under above-mentioned chromatographic condition, chromatographic peak can reach good separating effect, so as to set up Desmodium styracifolium general flavone
The characteristic spectrum of extract.Under the conditions of embodiments in accordance with the present invention, above-mentioned liquid-phase chromatographic analysis to set up Desmodium styracifolium always yellow
The characteristics of method of ketone extract characteristic spectrum has high stability and accuracy.
Determine the method that Flavonoid substances are constituted in Desmodium styracifolium
In the third aspect of the present invention, the present invention proposes a kind of side for determining that Flavonoid substances are constituted in Desmodium styracifolium
Method, embodiments in accordance with the present invention, this method includes：According to the first aspect of the present invention, general flavone is extracted from Desmodium styracifolium
Extract；And liquid-phase chromatographic analysis is carried out to extractive of general flavone, and determined based on resulting liquid-phase chromatographic analysis result
The composition of Flavonoid substances in the Desmodium styracifolium.Inventor has found, by said extracted method, the general flavone in Desmodium styracifolium
Can be by high efficiency extraction.Liquid-phase chromatographic analysis is carried out to above-mentioned extractive of general flavone and obtains chromatography figure, so as to accurate true
Determine the composition of Flavonoid substances in Desmodium styracifolium.Flavonoid substances in embodiments in accordance with the present invention, above-mentioned determination Desmodium styracifolium
The method degree of accuracy of composition is high, stability is high.
Liquid-phase chromatographic analysis in embodiments in accordance with the present invention, this method uses following condition：Using methanol -0.1%
Formic acid system carries out gradient elution, and chromatographic column is Waters BEH C18(2.1mm × 100mm, 1.8 microns), column temperature is 25 Celsius
Degree, flow velocity is 0.2ml/min, sample size：1 microlitre, Detection wavelength is 272nm, and elution requirement is：
| Time (min) | 0.1% formic acid | Methanol |
| 0~28 | 82→74 | 18→26 |
| 28~40 | 74 | 26 |
| 40~41 | 74→82 | 26→18 |
| 41~50 | 82 | 18 |
It is surprisingly found by the inventors that, in experiment of three kinds of different formic acid concns as mobile phase is investigated, formic acid concn point
Wei 0.05%, 0.1%, 0.2%, it is contemplated that influences of the low PH to chromatographic column, 0.1% formic acid-methanol is have chosen as flowing
Mutually detected；Inventor has found that the selection of chromatographic column is also most important simultaneously, the chromatogram of the chromatographic column of different size to sample
Behavioral implications is very big, except choosing Waters BEH C18Outside chromatographic column, remaining chromatographic column can not be separated preferably；Meanwhile, invention
People also found that the column temperature of chromatographic column influences very big to the chromatographic behavior of sample, and temperature is strict controlled in into 25 degrees Celsius, sample
Chromatogram peak energy obtains very satisfied separating effect.Embodiments in accordance with the present invention, sample concentration is 0.5g/L, and sample size 1 is micro-
Rise.For the determination of Detection wavelength, inventor carries out Sample Scan with 210~400nm is ultraviolet, find sample in 272nm and
There is maximum absorption band at 330nm, with reference to the existing quality standard of Desmodium styracifolium extractive of general flavone, Detection wavelength is set to
272nm.Under above-mentioned chromatographic condition, chromatographic peak can reach good separating effect, so as to effectively determine yellow in Desmodium styracifolium
Letones are constituted.Flavonoids in determination Desmodium styracifolium under the conditions of embodiments in accordance with the present invention, above-mentioned liquid-phase chromatographic analysis
The characteristics of method of material composition has high stability and accuracy.
The method for determining Desmodium styracifolium source
In the fourth aspect of the present invention, the present invention proposes a kind of method for determining Desmodium styracifolium source, according to the present invention
Embodiment, this method includes：According to the method for extractive of general flavone in said extracted Desmodium styracifolium, extracted from Desmodium styracifolium
Extractive of general flavone；And liquid-phase chromatographic analysis is carried out to the extractive of general flavone, and based on resulting liquid chromatogram point
Analysis result determines the source of the Desmodium styracifolium.Inventor has found that, by said extracted method, the general flavone in Desmodium styracifolium can
With by high efficiency extraction.Desmodium styracifolium wide material sources, chromatogram point is obtained by carrying out liquid-phase chromatographic analysis to above-mentioned extractive of general flavone
Analysis figure, can accurately determine the source of Desmodium styracifolium.Embodiments in accordance with the present invention, the method in above-mentioned determination Desmodium styracifolium source
The degree of accuracy is high, stability is high.
The Detection wavelength that liquid-phase chromatographic analysis in embodiments in accordance with the present invention, this method is used is 272nm.As above institute
State, inventor carries out Sample Scan with 210~400nm is ultraviolet, it is found that sample has maximum absorption band at 272nm and 330nm, join
According to the existing quality standard of Desmodium styracifolium extractive of general flavone, Detection wavelength is set to 272nm.Under the conditions of this Detection wavelength,
Chromatographic peak can reach good separating effect, so as to effectively determine Desmodium styracifolium source.
Liquid-phase chromatographic analysis in embodiments in accordance with the present invention, this method uses following condition：Using methanol -0.1%
Formic acid system carries out gradient elution, and chromatographic column is Waters BEH C18(2.1mm × 100mm, 1.8 microns), column temperature is 25 Celsius
Degree, flow velocity is 0.2ml/min, sample size：1 microlitre, elution requirement is：
| Time (min) | 0.1% formic acid | Methanol |
| 0~28 | 82→74 | 18→26 |
| 28~40 | 74 | 26 |
| 40~41 | 74→82 | 26→18 |
| 41~50 | 82 | 18 |
As described above, it is considered that influences of the low PH to chromatographic column, inventor have chosen 0.1% formic acid-methanol as flowing
Mutually detected；The selection of chromatographic column is also most important simultaneously, and the chromatographic column of different size influences very on the chromatographic behavior of sample
Greatly, except choosing Waters BEH C18Outside chromatographic column, remaining chromatographic column can not be separated preferably；Inventor also found, chromatographic column
Column temperature it is very big on the influence of the chromatographic behavior of sample, temperature is strict controlled in 25 degrees Celsius, the chromatogram peak energy of sample obtains non-
The separating effect being often satisfied with.Embodiments in accordance with the present invention, sample concentration is 0.5g/L, 1 microlitre of sample size.In above-mentioned chromatostrip
Under part, chromatographic peak can reach good separating effect, so as to effectively determine Desmodium styracifolium source.According to the implementation of the present invention
The characteristics of method in the determination Desmodium styracifolium source under the conditions of example, above-mentioned liquid-phase chromatographic analysis has high stability and accuracy.
Embodiments in accordance with the present invention, the above-mentioned source that Desmodium styracifolium is determined based on resulting liquid-phase chromatographic analysis result
Including：(a) chromatogram of liquid-phase chromatographic analysis result is compared with the predetermined collection of illustrative plates that compares；And (b) judges chromatogram
Similitude with compareing collection of illustrative plates, and determine based on the similitude source of Desmodium styracifolium.Desmodium styracifolium extractive of general flavone
Chromatogram is the characteristic spectrum of Desmodium styracifolium, therefore by the similitude of comparative analysis result chromatogram and control chromatogram, can
Effectively to determine the source of Desmodium styracifolium.Embodiments in accordance with the present invention, the method in above-mentioned determination Desmodium styracifolium source has height
The characteristics of stability and accuracy.
Embodiments in accordance with the present invention, in step (b), the similitude is to be based at least one following position in collection of illustrative plates to be
It is no to there is peak and determine：15.01min、15.71min、16.60min、18.04min、19.77min、21.38min、
22.78min, 23.18min, 23.73min, 27.2min, 29.51min, 31.08min, 31.72min, 32.98min, wherein,
The peak of the position represents as follows：15.01min represents vicenin-2, and 18.04min represents cyanidenon -6-C- α-L- pyrans
Arabinose -8-C- β-D- glucopyranosides, 19.77min represents vicenin-1, and 23.18min represents cyanidenon -6-C-
β-D- glucopyranoses -8-C- β-xylopyranose glucosides, 23.73min represent Schaftoside, and 27.20min represents genistein -7-
O- β-D- furans celerys glycosyl-(1 → 6)-O- β-D- glucopyranosides, 31.08min represents new Schaftoside, 31.72min generations
Table Saponaretin, 32.98min represents Vicenin-3.The peak of above-mentioned position determined by LC-MS analysis method, its
In have nine positions the flavones that represents of peak, it is known that the flavones that represents of the peak of five positions is unknown, but its position and charge-mass ratio are
Feature can be surveyed.Embodiments in accordance with the present invention, the peak shape and relative retention time of above-mentioned position is stable, and Desmodium styracifolium general flavone is carried
The chromatogram for taking thing is the characteristic spectrum of Desmodium styracifolium, by the chromatogram peak shape and control chromatogram that contrast Desmodium styracifolium to be determined
The parameters such as position, retention time, the charge-mass ratio at figure peak shape and peak, can effectively determine the source of Desmodium styracifolium.
Embodiments in accordance with the present invention, in step (b), the similitude is to be based in collection of illustrative plates lower column position whether there is
Peak and determine：15.01min、15.71min、18.04min、19.77min、22.78min、23.18min、23.73min、
27.2min, 29.51min, 31.08min, 31.72min, 32.98min, wherein, the peak representative of the position is as follows：
15.01min represents vicenin-2, and 18.04min represents cyanidenon -6-C- α-L- arabopyranose -8-C- β-D- pyrans
Glucoside, 19.77min represents vicenin-1,23.18min represent cyanidenon -6-C- β-D- glucopyranose -8-C- β -
Xylopyranose glucosides, 23.73min represents Schaftoside, 27.20min represent genistein -7-O- β-D- furans celerys glycosyl-(1 →
6)-O- β-D- glucopyranosides, 31.08min represents new Schaftoside, and 31.72min represents Saponaretin, 32.98min generations
Table V icenin-3.The peak of above-mentioned position is determined by liquid phase chromatography analytical method, wherein what the peak for having nine positions was represented
Flavones is, it is known that the flavones of the peak representative of three positions is unknown, but its position is that can survey feature.Embodiments in accordance with the present invention, on
The peak shape and relative retention time that rheme is put have stability, and the chromatogram of Desmodium styracifolium extractive of general flavone is Desmodium styracifolium
Characteristic spectrum, during by contrasting the chromatogram peak shape of Desmodium styracifolium to be determined and the position at control chromatogram peak shape and peak, retaining
Between etc. parameter, can effectively determine the source of Desmodium styracifolium.
Embodiments of the invention are described below in detail, these embodiments are exemplary, are only used for explaining the present invention, without
It is understood that as limitation of the present invention.
It should be noted that unless explicitly stated otherwise, the material and method not being described in detail in embodiment below are this
It is conventional use of in field.
Conventional method：
Unless expressly stated, in the examples below that using following instruments：
Instrument：Waters UPLC ACQUITY H-class Ultra Performance Liquid Chromatography instruments；Waters ACQUITY BEH
C18Chromatographic column (2.1mm × 100mm, 1.8 microns)；CQ250 ultrasonoscopes (Shanghai Ultrasonic Instrument Factory)；Plum Teller-Toronto
XP205 type analysis balances；Plum Teller-Toronto XS204 type analysis balances.
In the examples below that, the reference substance of use is as shown in table 1,
Table 1
| Numbering | Title | Source | Content (%) |
| DS-1 | Vicenin-3 | Self-control | 96.1 |
| DS-2 | Schaftoside | Zhong Jian institutes | 92.5 |
| DS-3 | Vicenin-1 | Self-control | 95.4 |
| DS-4 | Saponaretin | Self-control | 99.7 |
| DS-5 | Vicenin-2 | Self-control | 98.0 |
| DS-8 | Cyanidenon -6-C- β-D- glucopyranoses -8-C- β-xylopyranose glucosides | Self-control | 85.6 |
| DS-11 | Genistein -7-O- β-D- furans celerys glycosyl-(1 → 6)-O- β-D- glucopyranosides | Self-control | 95.7 |
| DS-14 | Cyanidenon -6-C- α-L- arabopyranose -8-C- β-D- glucopyranosides | Self-control | 91.3 |
| DS-15 | New Schaftoside | Self-control | 83.8 |
Above-mentioned all self-control reference substances are that Waters prepares liquid phase acquisition, and 12h is dried under reduced pressure using preceding phosphorus pentoxide；
Methanol is chromatographically pure rank used in the process of chromatography, and water is Millipore ultra-pure waters, and reagent is that analysis is pure.
In the examples below that, detection sample is as shown in table 2：
Table 2
| Lot number | The place of production | Correspondence medicinal material lot number |
| 141201 | Guangxi Yulin Zham Zhen | 141106 |
| 141202 | Guangxi resource vanilla village | 141101 |
| 141203 | Guangxi Lingui Wu Tongzhen | 141102 |
| 141204 | Guangxi Lingui golden mean of the Confucian school town | 141103 |
| 141205 | Guangxi Rong'an Si Ding towns | 141104 |
| 150101 | The honest and kindhearted town of Guangxi Yulin | 141105 |
| 150102 | Guangdong Shaoguan | 20120903 |
| 150103 | Guangdong Suixi Cheng Yue towns | 141107 |
| 150104 | Guangxi Luzhai | 20131127 |
| 140504 | Guilin | 141108 |
In the examples below that, the compound method of detection sample solution is as follows：
Precision weighs 25mg sample, puts in 50ml measuring bottles, adds 75% ethanol to nearly graduation mark, ultrasonic 20min is put
It is cold, constant volume.1 microlitre of sample introduction is detected every time.
Embodiment 1 investigates liquid phase chromatogram condition
The selection of 1.1 Detection wavelengths：Take Schaftoside and remaining eight kinds self-control reference substances appropriate, respectively in 75% ethanol
Ultrasonic dissolution, UV scanning is carried out in 210~400nm, there is absorption maximum at 272nm and 330nm, always yellow with reference to Desmodium styracifolium
The existing quality standard of ketone extract, is set to 272nm, UV scanning figure as shown in figure 1, wherein Fig. 1-1 is DS- by Detection wavelength
1 full wavelength scanner figure, Fig. 1-2 is DS-3 full wavelength scanner figure, and Fig. 1-3 is DS-4 full wavelength scanner figure, and Fig. 1-4 is
DS-5 full wavelength scanner figure, Fig. 1-5 is DS-8 full wavelength scanner figure, and Fig. 1-6 is DS-11 full wavelength scanner figure, Fig. 1-7
It is DS-14 full wavelength scanner figure, Fig. 1-8 is DS-15 full wavelength scanner figure, and Fig. 1-9 is DS-2 full wavelength scanner figure.Figure
1 result shows that nine kinds of control samples under 210~400nm UV scannings, have absorption maximum at 272nm and 330nm.
The selection of 1.2 elution requirements
The selection of mobile phase：Using Waters BEH C18(2.1mm × 100mm, 1.7 microns) chromatographic column, by comparing first
The systems such as the formic acid of alcohol -0.1%, the formic acid of acetonitrile -0.1%, the formic acid of methanol-acetonitrile -0.1%, the phosphoric acid of methanol -0.1% are found, wide golden
Money grass extractive of general flavone is in the system of the formic acid of methanol -0.1%, and obtained chromatographic peak is more, and separating degree preferably, therefore is selected
The formic acid system of methanol -0.1% carries out gradient elution.
Meanwhile, it is respectively configured the mobile phase of three kinds of different formic acid concns, 0.05% formic acid water-methanol, 0.1% formic acid water-
Methanol, 0.2% formic acid water-methanol, as a result find, chromatogram row of the above-mentioned three kinds of formic acid concns to Desmodium styracifolium extractive of general flavone
Little for influence, it is contemplated that influences of the low pH to chromatographic column, it is that mobile phase is detected to choose 0.1% formic acid water-methanol, different
Formic acid concn is as the chromatogram of chromatogram flow phase as shown in Fig. 2 it is mobile phase that wherein Fig. 2-1, which is 0.05% formic acid water-methanol,
Chromatogram, Fig. 2-2 is the chromatogram that 0.1% formic acid water-methanol is mobile phase, and Fig. 2-3 is 0.2% formic acid water-methanol for stream
The chromatogram of dynamic phase.
Condition of gradient elution is as shown in table 3,
Table 3
| Time (min) | 0.1% formic acid | Methanol |
| 0~28 | 82→74 | 18→26 |
| 28~40 | 74 | 26 |
| 40~41 | 74→82 | 26→18 |
| 41~50 | 82 | 18 |
The selection of 1.3 column temperatures
The present embodiment has investigated 20 DEG C, 25 DEG C and 30 DEG C respectively, these three influences of different column temperatures to chromatographic behavior, knot
Fruit finds, peak 5 and peak 6 need compared with 20 DEG C under low temperature, to obtain relatively good separating effect, and peak 10 and peak 11 compared with
30 DEG C of high-temperature, could obtain relatively good separating effect, at 25 DEG C, and peak 5 and peak 6, peak 10 can be compared with peak 11
Satisfied separating effect, it can be found that the chromatographic behavior of sample is very sensitive to temperature, therefore need to strictly control column temperature to be 25 DEG C,
Chromatogram under the conditions of different column temperatures is as shown in figure 3, wherein Fig. 3-1 is the chromatogram at 20 DEG C of column temperature, at Fig. 3-2 is 25 DEG C of column temperature
Chromatogram, Fig. 3-3 is the chromatogram at 30 DEG C of column temperature.
The selection of 1.4 chromatographic columns
Waters BEH C are respectively adopted in the present embodiment18(2.1mm × 100mm, 1.7 microns), Shiseido Capell core
C18(2.1mm × 100mm, 2.7 microns) and Shimadzu Shim-pack XR-ODS II (2.0mm × 75mm, 2.2 microns), as a result send out
Existing, the chromatographic column of different brands specification influences very big to the chromatographic behavior of sample, except Waters BEH C18(2.1mm×100mm,
1.8 microns) chromatographic column can reach outside preferable baseline separation that remaining chromatographic column can not be separated preferably, therefore, answer stationary chromatographic
The brand and model of post, the chromatogram of different chromatogram post detection Desmodium styracifolium chromocor extracts is as shown in figure 4, wherein Fig. 4-1 is
Waters BEH C18The chromatogram of (2.1mm × 100mm, 1.7 microns), Fig. 4-2 is Shiseido Capell core C18
(2.1mm × 100mm, 2.7 microns) chromatogram, Fig. 4-3 is that (2.0mm × 75mm, 2.2 is micro- by Shimadzu Shim-pack XR-ODS II
Rice) chromatogram.
To sum up under the conditions of liquid-phase chromatographic analysis, gradient elution is carried out using the formic acid system of methanol -0.1%, chromatographic column is
Waters BEH C18 (2.1mm × 100mm, 1.8 microns), column temperature is 25 degrees Celsius, and flow velocity is 0.2ml/min；Sample size：1
Microlitre, Detection wavelength is 272nm, elution requirement：
| Time (min) | 0.1% formic acid | Methanol |
| 0~28 | 82→74 | 18→26 |
| 28~40 | 74 | 26 |
| 40~41 | 74→82 | 26→18 |
| 41~50 | 82 | 18 |
The chromatogram of obtained Desmodium styracifolium general flavone sample is as shown in figure 5, Fig. 5 results are shown, in above-mentioned chromatographic condition
Under, the chromatographic peak separating degree of Desmodium styracifolium general flavone sample is good.
Embodiment 2 investigates the method for extracting Desmodium styracifolium general flavone
Due to being mainly that a class has the Flavonoid substances of identical mother nucleus structure in Desmodium styracifolium extractive of general flavone, therefore
It is larger that inventor mainly chooses peak area in sample, and preferable five chromatographic peaks of separating degree are evaluated as index, investigates
The extracting method of sample, it is as shown in Figure 6 as five peaks of evaluation index.
The selection of 2.1 extracting modes
Precision weighs 25mg Desmodium styracifolium extractive of general flavone sample, and precision adds 50% ethanol 50ml, is respectively adopted
Ultrasonic (250KW, 33khz) and two kinds of extracting methods of backflow are extracted, the color for the chromocor extract that two kinds of extracting modes are obtained
As shown in table 4, the result of table 4 shows that there was no significant difference for both extraction efficiencies to spectral peak map analysis result, but considers the letter of operation
Just property, chooses ultrasound and is used as extracting method.
Table 4
The selection of 2.2 extraction times
Precision weighs 25mg samples, and precision adds 50% ethanol 50ml, respectively ultrasound 10min, 20min, 30min,
40min, is compared, and as shown in table 5, the result of table 5 shows the chromatogram result of the general flavone obtained under different extraction times
Show, extraction time, there was no significant difference to extraction efficiency, but consider the saving time but ensure to extract complete again, choose ultrasound
20min。
Table 5
The selection of 2.3 solvent loads
Precision weighs 25mg samples, accurate respectively to add 50% ethanol 25ml, 50ml, 75ml, 100ml ultrasonic extraction, with
On the basis of 50ml solvent extractions, conversion comparison, the chromatogram point for the general flavone that different solvents are measured are carried out by volume respectively
Analyse result as shown in table 6, the result of table 6 show, and there was no significant difference to extraction efficiency for solvent load, but consideration it is cost-effective but
Ensure to extract complete, choose 50ml and extract.
Table 6
The selection of 2.4 solvent strengths
Precision weighs 25mg samples, and 25% ethanol of accurate addition, 50% ethanol, 75% ethanol, 95% ethanol are each respectively
The chromatogram result of the chromocor extract obtained under 50ml, ultrasonic extraction, various concentrations solvent is as shown in table 7, the result of table 7
Show, sample extraction efficiency highest inside 75% ethanol, and separating degree is good；Peak shape is poor in 95% ethanol, separating degree
It is relatively low, therefore selection 75% ethanol extraction.
Table 7
To sum up, the preparation method of the final detection sample solution for determining Desmodium styracifolium extractive of general flavone is：Precision is weighed
25mg Desmodium styracifolium extractive of general flavone sample, puts in 50ml measuring bottles, 75% ethanol of addition to nearly graduation mark, ultrasonic 20min,
Let cool, constant volume.
The synergy of embodiment 3 is analyzed
3.1 superelevation liquid chromatogram (UPLC) peak attribution analysises
Appropriate Schaftoside, Vicenin-3, Vicenin-1, Saponaretin, Vicenin-2, cyanidenon -6- are taken respectively
C- β-D- glucopyranoses -8-C- β-xylopyranose glucosides, genistein -7-O- β-D- furans celerys glycosyl-(1 → 6)-O- β-D- pyrroles
Glucopyranoside glycosides, cyanidenon -6-C- α-L- arabopyranose -8-C- β-D- glucopyranosides, the control of new Schaftoside
Appropriate product, add 75% ethanol, are made into nine kinds of reference substance solutions, then take each reference substance solution appropriate, are made into mixing reference substance molten
Liquid；The detection sample solution of Desmodium styracifolium extractive of general flavone is obtained with legal system, liquid chromatograph is injected, compares reference substance and sample
Chromatogram, has belonged to nine chromatographic peaks in sample, as a result as shown in fig. 7, wherein Fig. 7-1 is DS-5 and sample liquid chromatogram
Figure, Fig. 7-2 is DS-2 and sample liquid chromatogram, and Fig. 7-3 is DS-3 and sample liquid chromatogram, and Fig. 7-4 is DS-8 and sample
Liquid chromatogram, Fig. 7-5 is DS-2 and sample liquid chromatogram, and Fig. 7-6 is DS-11 and sample liquid chromatogram, and Fig. 7-7 is
DS-15 and sample liquid chromatogram, Fig. 7-8 are DS-4 and sample liquid chromatogram, and Fig. 7-9 is DS-1 and sample liquid chromatogram
Figure, Fig. 7-10 is mixed reference substance solution liquid chromatogram, and Fig. 7-11 is sample and mixed reference substance solution liquid chromatogram.By
Fig. 7 can show that peak 1 represents Vicenin-2, and peak 2 represents cyanidenon -6-C- α-L- arabopyranose -8-C- β-D- pyrroles
Glucopyranoside glycosides, peak 3 represents Vicenin-1, and peak 4 represents cyanidenon -6-C- β-D- glucopyranoses -8-C- β-xylopyranose
Glycosides, peak 5 represents Schaftoside, and peak 6 represents genistein -7-O- β-D- furans celerys glycosyl-(1 → 6)-O- β-D- glucopyranoses
Glycosides, peak 7 represents new Schaftoside, and peak 8 represents Saponaretin, and peak 9 represents Vicenin-3.
3.2 LC-MS peak attribution analysises
In order to which further to detecting that the liquid chromatogram peak of sample carries out attribution analysis and checking, inventor has carried out liquid matter connection
With experiment.
3.2.1 instrument and chromatographic condition
Instrument：LC-MS instrument Waters xevo G2Q-Tof LC/MS；
Chromatographic column Acquity UPLC BEH C18(1.7μm,2.1*100mm)；
Chromatographic condition：Mobile phase：A：0.1% formic acid solution；B：Methanol；
Condition of gradient elution is as shown in table 3；
Flow velocity：0.2mL/min；
Column temperature：25℃；
The μ L of sample size 1.
Mass Spectrometry Conditions：Ion gun：ESI(-)；Scan pattern：MSE；Capillary voltage：2.6kV；One-level taper hole voltage：
12V；Two grades of taper hole voltages：4.0V；Fragmentation voltage：15-60V；Source temperature：100℃；Remove solvent temperature：350℃；Remove solvent gas：
600L/h；Taper hole blowback air：50L/h.
3.2.2 solution is prepared
Prepare reference substance solution and weigh above-mentioned nine kinds of reference substances respectively in right amount, it is 30 micro- to add 75% ethanol and concentration is made
The reference substance solution of grams per milliliter；
The Desmodium styracifolium extractive of general flavone sample that detection sample solution precision weighs 25mg is prepared, is put in 50ml measuring bottles,
75% ethanol is added to nearly graduation mark, ultrasonic 20min is let cool, constant volume.
3.2.3 peak attribution analysis
Above two solution is injected separately into LC-MS instrument to be detected, gained total ion current figure and mass spectrogram such as Fig. 8
Shown in~Figure 27, Fig. 8 is the total ion current figure for detecting sample, and Fig. 9 is reference substance Vicenin-2 total ion current figure, Tu10Shi
The mass spectrogram of sample and reference substance Vicenin-2, Figure 11 is reference substance cyanidenon -6-C- α-L- arabopyranoses -8-C-
The total ion chromatogram of β-D- glucopyranosides, Figure 12 be sample with reference substance cyanidenon -6-C- α-L- pyrans I
The mass spectrogram of primary sugar -8-C- β-D- glucopyranosides, Figure 13 is reference substance Vicenin-1 total ion current figure, and Figure 14 is sample
The mass spectrogram of product and reference substance Vicenin-1, Figure 15 is reference substance cyanidenon -6-C- β-D- glucopyranoses -8-C- β-pyrrole
Mutter the total ion current figure of xyloside, Figure 16 is sample and reference substance cyanidenon -6-C- β-D- glucopyranoses -8-C- β-pyrans
The mass spectrogram of xyloside, Figure 17 is the total ion figure of reference substance Schaftoside, and Figure 18 is the mass spectrum of sample and reference substance Schaftoside
Figure, Figure 19 is total ion of reference substance genistein -7-O- β-D- furans celerys glycosyl-(1 → 6)-O- β-D- glucopyranosides
Flow graph, Figure 20 is sample and reference substance genistein -7-O- β-D- furans celerys glycosyl-(1 → 6)-O- β-D- glucopyranosides
Mass spectrogram, Figure 21 is the total ion chromatogram of the new Schaftoside of reference substance, and Figure 22 is sample and the new Schaftoside of reference substance
Mass spectrogram, Figure 23 is reference substance Saponaretin total ion current figure, and Figure 24 is the mass spectrogram of sample and reference substance Saponaretin, figure
25 be reference substance Vicenin-3 total ion current figure, and Figure 26 is the mass spectrogram of sample and reference substance Vicenin-3, Figure 27 be
Retention time is the mass spectrogram at 15.71min, 16.60min, 21.38min, 22.78min and 29.51min five unknown peaks.
The retention time and Information in Mass Spectra ownership of 14 chromatographic peaks are as shown in table 8.
Table 8
Note：* molecular ion peak is represented
Embodiment 4 investigates the stability of detecting system
The ownership of summary separating effect and characteristic peak, finally determines 12 characteristic peaks to be evaluated.Choose and protect
Stay the time moderate, the larger Schaftoside of peak area is S peaks, carries out characteristic spectrum methodology study on the stability.Characteristic spectrum is such as
Shown in Figure 28.
4.1 precision tests take the detection sample solution (lot number 140504) of Desmodium styracifolium extractive of general flavone, by above-mentioned
Chromatographic condition, continuous sample introduction 6 times records chromatogram.The relative retention time RSD at each peak is less than 1.5%, relative peak area RSD
Less than 3.1%, as shown in table 9, wherein table 9-1 is the relative retention time at peak to analysis result, and table 9-2 is the relative peak face at peak
Product.
Table 9-1
Note：Chronomere is min；
Table 9-2
Note：Non-resolved peaks 5 carries out splitting obtained peak face with chromatographic work station software with peak 6, peak 10 and peak 11 to it
Product is calculated
4.2 stability tests take the detection sample solution (lot number 140504) of Desmodium styracifolium extractive of general flavone, by above-mentioned
Chromatographic condition, is measured respectively at 0,5,10,15,20h, records chromatogram.The relative retention time RSD% at each peak is respectively less than
1%；Relative peak area RSD is less than 3.2%, and as shown in table 10, wherein table 10-1 is the relative retention time at peak, table to analysis result
10-2 is the relative peak area at peak.
Table 10-1
Note：Chronomere is min；Sequence number 1-5 represents 0,5,10,15,20h and is measured；
Table 10-2
Note：Non-resolved peaks 5 carries out splitting obtained peak face with chromatographic work station software with peak 6, peak 10 and peak 11 to it
Product is calculated
4.3 replica tests take 6 parts of the detection sample solution (lot number 140504) of Desmodium styracifolium extractive of general flavone, according to upper
State compound method and prepare detection sample solution, determined by above-mentioned chromatographic condition, record chromatogram.The relative retention time at each peak,
RSD% is respectively less than 0.2%；Relative peak area RSD is respectively less than 2%, and as shown in table 11, wherein table 11-1 is the phase at peak to analysis result
To retention time, table 11-2 is the relative peak area at peak.
Table 11-1
Note：Chronomere is min；
Table 11-2
Note：Non-resolved peaks 5 carries out splitting obtained peak face with chromatographic work station software with peak 6, peak 10 and peak 11 to it
Product is calculated
To sum up precision test, stability test and replica test result, it is known that detecting system stability of the invention
Height, the characteristic spectrum methodology of Desmodium styracifolium extractive of general flavone is stable.
Embodiment 5 investigates Desmodium styracifolium medicinal material and the composition transfer before and after the extraction of its extractive of general flavone
For the composition transfer after studying Desmodium styracifolium medicinal material and its extractive of general flavone before extraction, inventor respectively will
Desmodium styracifolium extractive of general flavone and Desmodium styracifolium medicinal material inject Ultra Performance Liquid Chromatography instrument under above-mentioned same chromatographic condition,
Analysis is compared, it is as follows that it prepares detection sample solution：
Desmodium styracifolium medicinal material：About 0.2g Desmodium styracifolium medicinal material is weighed, 75% ethanol is added in 25ml measuring bottles, ultrasound
20min, is let cool, and constant volume is produced；
Desmodium styracifolium extractive of general flavone：Precision weighs 25mg sample, puts in 100ml measuring bottles, adds 75% ethanol extremely
Nearly graduation mark, ultrasonic 20min is let cool, constant volume.
As shown in figure 29, wherein Figure 29-1 is 141201 batch extractive of general flavone and its crude drug pair to obtained chromatogram
Collection of illustrative plates is answered, Figure 29-2 is 141202 batch extractive of general flavone collection of illustrative plates corresponding with its crude drug, and Figure 29-3 is that 141203 batches are total
Chromocor extract collection of illustrative plates corresponding with its crude drug, Figure 29-4 is 141204 batch extractive of general flavone and its crude drug corresponding diagram
Spectrum, Figure 29-5 is 141205 batch extractive of general flavone collection of illustrative plates corresponding with its crude drug, and Figure 29-6 is 150101 batch general flavones
Extract collection of illustrative plates corresponding with its crude drug, Figure 29-7 is 150102 batch extractive of general flavone collection of illustrative plates corresponding with its crude drug, figure
29-8 is 150103 batch extractive of general flavone collection of illustrative plates corresponding with its crude drug, and Figure 29-9 is 150104 batch extractive of general flavone
Collection of illustrative plates corresponding with its crude drug, Figure 29-10 is 140504 batch extractive of general flavone collection of illustrative plates corresponding with its crude drug.
Figure 29 chromatogram results show that extractive of general flavone is after medicinal material extract, and material base does not change,
So as to ensure that extractive of general flavone clinical effectiveness.
Embodiment 6 determines the liquid chromatogram of Desmodium styracifolium extractive of general flavone sample
Under above-mentioned chromatographic condition, the sample that the Desmodium styracifolium extractive of general flavone of ten batches is weighed respectively is surveyed
It is fixed, liquid chromatograph is injected, the chromatogram of ten batches is obtained, chromatogram is as shown in figure 30, the relative retention time such as table at peak
Shown in 12, there are this 12 characteristic peaks through comparing the Desmodium styracifolium extractive of general flavone of ten batches, and relative retention time is steady
It is fixed.
Table 12
Embodiment 7 sets up the characteristic spectrum of Desmodium styracifolium chromocor extract
Chromatographic condition：With octadecyl silane Waters ACQUITY BEH C18(2.1mm × 100mm, 1.7 is micro-
Rice) it is filler, using methanol as mobile phase A, using 0.1% formic acid solution as Mobile phase B, condition of gradient elution is as shown in table 3；
Column temperature is 25 DEG C；Detection wavelength is 272nm.Theoretical cam curve is calculated by Schaftoside should be not less than 5000.
Preparing reference solution takes Schaftoside reference substance appropriate, accurately weighed, plus every 1ml Buddhists containing summer are made in 75% ethanol
Tower glycosides 20ug solution, is produced.
Prepare detection sample solution and weigh Desmodium styracifolium extractive of general flavone about 25mg, add 75% ethanol and put 50ml measuring bottles
To nearly graduation mark, ultrasonic 20min is let cool, constant volume.
Characteristic spectrum accurate absorption reference solution and detection each 1ul of sample solution respectively are determined, ultra high efficiency liquid phase is injected
Chromatograph, is produced.
There should be 12 characteristic peaks in detection sample collection of illustrative plates, peak corresponding with object of reference is S peaks, calculates each characteristic peak and S peaks
Relative retention time, its relative retention time should be within ± the 5% of setting.Setting is 0.628 (peak 1), 0.656
(peak 2), 0.757 (peak 3), 0.831 (peak 4), 0.957 (peak 5), 0.972 (peak 6), 1.000 (peak S), 1.138 (peaks 8), 1.240
(peak 9), 1.308 (peaks 10), 1.333 (peaks 11), 1.385 (peaks 12).Desmodium styracifolium extractive of general flavone compare feature collection of illustrative plates is such as
Shown in Figure 31, wherein peak 1 is Vicenin-2, and peak 3 is cyanidenon -6-C- α-L- arabopyranose -8-C- β-D- pyrans Portugal
Polyglycoside, peak 4 is Vicenin-1, and peak 6 is cyanidenon -6-C- β-D- glucopyranoses -8-C- β-xylopyranose glucosides, and peak 7 is
Schaftoside, peak 8 is genistein -7-O- β-D- furans celerys glycosyl-(1 → 6)-O- β-D- glucopyranosides, and peak 10 is new
Schaftoside, peak 11 is Saponaretin, and peak 12 is Vicenin-3.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means to combine specific features, structure, material or the spy that the embodiment or example are described
Point is contained at least one embodiment of the present invention or example.In this manual, to the schematic representation of above-mentioned term not
Necessarily refer to identical embodiment or example.Moreover, specific features, structure, material or the feature of description can be any
One or more embodiments or example in combine in an appropriate manner.
Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that：Not
In the case of departing from the principle and objective of the present invention a variety of change, modification, replacement and modification can be carried out to these embodiments, this
The scope of invention is limited by claim and its equivalent.
Claims (10)
1. a kind of method for setting up Desmodium styracifolium extractive of general flavone characteristic spectrum, it is characterised in that including:
According to the method that extractive of general flavone is extracted from Desmodium styracifolium, extractive of general flavone is extracted from Desmodium styracifolium；
Liquid-phase chromatographic analysis is carried out to the extractive of general flavone, and sets up described based on resulting liquid-phase chromatographic analysis result
Desmodium styracifolium extractive of general flavone characteristic spectrum,
Wherein, the liquid-phase chromatographic analysis uses following condition：
Gradient elution is carried out using the formic acid system of methanol -0.1%,
Chromatographic column is Waters BEH C18, 2.1mm × 100mm, 1.8 microns,
Column temperature is 25 degrees Celsius,
Flow velocity is 0.2ml/min；
Sample size：1 microlitre,
Detection wavelength is 272nm,
Elution requirement：
Wherein, the method that extractive of general flavone is extracted from Desmodium styracifolium is as described below：
Desmodium styracifolium sample is mixed with ethanol, and extracted by backflow or ultrasonic method, is extracted to obtain general flavone
Thing,
The concentration of the ethanol is 25~95%, and the volume ratio of the weight of the Desmodium styracifolium and the ethanol is 25mg：25~
100ml。
2. a kind of method for determining that Flavonoid substances are constituted in Desmodium styracifolium, it is characterised in that including：
According to the method that extractive of general flavone is extracted from Desmodium styracifolium, extractive of general flavone is extracted from Desmodium styracifolium；
Liquid-phase chromatographic analysis is carried out to the extractive of general flavone, and it is described based on resulting liquid-phase chromatographic analysis result determination
The composition of Flavonoid substances in Desmodium styracifolium,
Wherein, the liquid-phase chromatographic analysis uses following condition：
Gradient elution is carried out using the formic acid system of methanol -0.1%,
Chromatographic column is Waters BEH C18, 2.1mm × 100mm, 1.8 microns,
Column temperature is 25 degrees Celsius,
Flow velocity is 0.2ml/min；
Sample size：1 microlitre,
Detection wavelength is 272nm,
Elution requirement：
Wherein, the method that extractive of general flavone is extracted from Desmodium styracifolium is as described below：
Desmodium styracifolium sample is mixed with ethanol, and extracted by backflow or ultrasonic method, is extracted to obtain general flavone
Thing,
The concentration of the ethanol is 25~95%, and the volume ratio of the weight of the Desmodium styracifolium and the ethanol is 25mg：25~
100ml。
3. a kind of method for determining Desmodium styracifolium source, it is characterised in that including：
According to the method that extractive of general flavone is extracted from Desmodium styracifolium, extractive of general flavone is extracted from Desmodium styracifolium；And
Liquid-phase chromatographic analysis is carried out to the extractive of general flavone, and it is described based on resulting liquid-phase chromatographic analysis result determination
The source of Desmodium styracifolium,
Wherein, the Detection wavelength that the liquid-phase chromatographic analysis is used for 272nm,
Gradient elution is carried out using the formic acid system of methanol -0.1%,
Chromatographic column is Waters BEH C18, 2.1mm × 100mm, 1.8 microns,
Column temperature is 25 degrees Celsius,
Flow velocity is 0.2ml/min,
Sample size：1 microlitre,
Elution requirement：
Wherein, the method that extractive of general flavone is extracted from Desmodium styracifolium is as described below：
Desmodium styracifolium sample is mixed with ethanol, and extracted by backflow or ultrasonic method, is extracted to obtain general flavone
Thing,
The concentration of the ethanol is 25~95%, and the volume ratio of the weight of the Desmodium styracifolium and the ethanol is 25mg：25~
100ml。
4. the method according to claim 3 for determining Desmodium styracifolium source, it is characterised in that based on resulting liquid phase color
Analysis of spectrum result determines that the source of the Desmodium styracifolium includes：
(a) chromatogram of the liquid-phase chromatographic analysis result is compared with the predetermined collection of illustrative plates that compares；And
(b) judge the chromatogram and the similitude for compareing collection of illustrative plates, and the wide money is determined based on the similitude
The source of grass.
5. the method according to claim 4 for determining Desmodium styracifolium source, it is characterised in that in step (b), the phase
Determined like property based at least one following position in collection of illustrative plates with the presence or absence of peak：15.01min、15.71min、
16.60min、18.04min、19.77min、21.38min、22.78min、23.18min、23.73min、27.2min、
29.51min, 31.08min, 31.72min, 32.98min,
Wherein, the peak of the position represents as follows：15.01min represents vicenin-2, and 18.04min represents cyanidenon -6-C-
α-L- arabopyranose -8-C- β-D- glucopyranosides, 19.77min represents vicenin-1, and 23.18min represents sweet-scented osmanthus
Careless element -6-C- β-D- glucopyranoses -8-C- β-xylopyranose glucosides, 23.73min represents Schaftoside, and 27.20min represents dye
Expect lignin -7-O- β-D- furans celerys glycosyl-(1 → 6)-O- β-D- glucopyranosides, 31.08min represents new Schaftoside,
31.72min represents Saponaretin, and 32.98min represents Vicenin-3.
6. the method according to claim 4 for determining Desmodium styracifolium source, it is characterised in that in step (b), the phase
Determined like property based on lower column position in collection of illustrative plates with the presence or absence of peak：15.01min、15.71min、18.04min、
19.77min、22.78min、23.18min、23.73min、27.2min、29.51min、31.08min、31.72min、
32.98min,
Wherein, the peak of the position represents as follows：15.01min represents vicenin-2, and 18.04min represents cyanidenon -6-C-
α-L- arabopyranose -8-C- β-D- glucopyranosides, 19.77min represents vicenin-1, and 23.18min represents sweet-scented osmanthus
Careless element -6-C- β-D- glucopyranoses -8-C- β-xylopyranose glucosides, 23.73min represents Schaftoside, and 27.20min represents dye
Expect lignin -7-O- β-D- furans celerys glycosyl-(1 → 6)-O- β-D- glucopyranosides, 31.08min represents new Schaftoside,
31.72min represents Saponaretin, and 32.98min represents Vicenin-3.
7. the method according to any one of claims 1 to 3, it is characterised in that the weight of the Desmodium styracifolium and the second
The volume ratio of alcohol is 25mg：50ml.
8. the method according to any one of claims 1 to 3, it is characterised in that carry out extracting 10~40 using ultrasonic method
Minute.
9. the method according to any one of claims 1 to 3, it is characterised in that carry out extracting 20 minutes using ultrasonic method.
10. the method according to any one of claims 1 to 3, it is characterised in that the concentration of the ethanol is 75%.
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