CN105092744A - Characteristic spectrum of total flavonoid extracts in snowbellleaf tickclover herbs as well as establishment method and application of characteristic spectrum - Google Patents
Characteristic spectrum of total flavonoid extracts in snowbellleaf tickclover herbs as well as establishment method and application of characteristic spectrum Download PDFInfo
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Abstract
The invention discloses a method for extracting total flavonoid extracts from snowbellleaf tickclover herbs, a method for establishing a characteristic spectrum of the total flavonoid extracts in the snowbellleaf tickclover herbs, a method for determining flavonoid material composition in the snowbellleaf tickclover herbs and a method for determining the source of the snowbellleaf tickclover herbs. According to the method for extracting total flavonoids from the snowbellleaf tickclover herbs, the total flavonoids can be extracted efficiently from the snowbellleaf tickclover herbs, and the resolution of a liquid chromatographic peak of the total flavonoids is good. The method for establishing the characteristic spectrum of the total flavonoid extracts in the snowbellleaf tickclover herbs, the method for determining the flavonoid material composition in the snowbellleaf tickclover herbs and the method for determining the source of the snowbellleaf tickclover herbs have the characteristics of stability and repeatability.
Description
Technical field
The present invention relates to biomedicine field, concrete, the present invention relates to a kind of from Desmodium styracifolium, extract extractive of general flavone method and a kind of Desmodium styracifolium extractive of general flavone characteristic spectrum and method for building up thereof and application.
Background technology
Desmodium styracifolium is a kind of medicinal plant, has effect of dampness removing removing jaundice, inducing diuresis for treating strangurtia.Containing abundant Flavonoid substances in Desmodium styracifolium.Desmodium styracifolium extractive of general flavone is the Flavonoid substances that a class has identical mother nucleus structure 2-phenyl chromone.Effect of flavones is many-sided, it can oxygen radical effectively in purged body, improve blood circulation, reduce cholesterol, reduce blood sugar, promote wound healing and pain relieving.But, the extraction process of Desmodium styracifolium flavones and determine that the method for wherein Flavonoid substances composition still haves much room for improvement.
Meanwhile, Desmodium styracifolium is widely distributed, is distributed in the provinces and regions such as Fujian, Hunan, Guangxi and Guangdong, therefore, how effectively to determine Desmodium styracifolium source and set up Desmodium styracifolium extractive of general flavone characteristic spectrum be one wait solve problem.
Summary of the invention
The present invention is intended at least to solve one of technical matters existed in prior art.For this reason, one object of the present invention be to propose a kind of from Desmodium styracifolium, extract extractive of general flavone method, a kind of determine Flavonoid substances composition in Desmodium styracifolium method and a kind ofly determine the method that Desmodium styracifolium is originated.
In a first aspect of the present invention, the present invention proposes a kind of method extracting extractive of general flavone from Desmodium styracifolium.According to embodiments of the invention, the method comprises: mixed with ethanol by Desmodium styracifolium sample, and extracted by backflow or ultrasonic method, to obtain extractive of general flavone, wherein, the concentration of described ethanol is 25 ~ 95%, and the weight of described Desmodium styracifolium and the volume ratio of described ethanol are 25mg:25 ~ 100ml, preferred 25mg:50ml.By said extracted method, both can guarantee to extract the general flavone in Desmodium styracifolium completely, extraction efficiency is high, and the degree of separation at peak in later stage liquid-phase chromatographic analysis can be made again good.
According to embodiments of the invention, the above-mentioned method extracting extractive of general flavone from Desmodium styracifolium may further include following additional technical feature one of at least:
According to embodiments of the invention, said method adopts ultrasonic method to carry out extraction 10 ~ 40 minutes, preferably 20 minutes.By above-mentioned ultrasonic extracting method, both saved extraction time, and in turn ensure that the general flavone extracted completely in Desmodium styracifolium, make extraction efficiency high, be beneficial to the medicinal of flavones and liquid-phase chromatographic analysis.
According to embodiments of the invention, the concentration of above-mentioned ethanol is 75%.The discovery that inventor is surprised, Desmodium styracifolium sample extraction efficiency inside 75% ethanol is the highest, and in liquid-phase chromatographic analysis, the degree of separation at peak is good.
In a second aspect of the present invention, the present invention proposes a kind of method setting up Desmodium styracifolium extractive of general flavone characteristic spectrum, according to embodiments of the invention, the method comprises: according to foregoing method, from Desmodium styracifolium, extract extractive of general flavone; Liquid-phase chromatographic analysis is carried out to described extractive of general flavone, and sets up described Desmodium styracifolium extractive of general flavone characteristic spectrum based on obtained liquid-phase chromatographic analysis result.Inventor finds, by said extracted method, the general flavone in Desmodium styracifolium can by high efficiency extraction.Liquid-phase chromatographic analysis is carried out to above-mentioned extractive of general flavone and obtains stratographic analysis figure, thus the characteristic spectrum of Desmodium styracifolium extractive of general flavone can be set up.According to embodiments of the invention, above-mentioned method accuracy of setting up Desmodium styracifolium extractive of general flavone characteristic spectrum is high, stability is high.
According to embodiments of the invention, the above-mentioned method setting up Desmodium styracifolium extractive of general flavone characteristic spectrum may further include following additional technical feature one of at least:
According to embodiments of the invention, described liquid-phase chromatographic analysis adopts following condition: adopt methyl alcohol-0.1% formic acid system to carry out gradient elution, chromatographic column is WatersBEHC
18(2.1mm × 100mm, 1.8 microns), column temperature is 25 degrees Celsius, and flow velocity is 0.2ml/min; Sample size: 1 microlitre, determined wavelength is 272nm, elution requirement:
| Time (min) | 0.1% formic acid | Methyl alcohol |
| 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, thus sets up the characteristic spectrum of Desmodium styracifolium extractive of general flavone.According to embodiments of the invention, the method setting up Desmodium styracifolium extractive of general flavone characteristic spectrum under above-mentioned liquid-phase chromatographic analysis condition has the feature of high stability and accuracy.In a third aspect of the present invention, the present invention proposes a kind of method determining Flavonoid substances composition in Desmodium styracifolium, according to embodiments of the invention, the method comprises: according to the method described above, from Desmodium styracifolium, extract extractive of general flavone; Liquid-phase chromatographic analysis is carried out to extractive of general flavone, and determines the composition of Flavonoid substances in described Desmodium styracifolium based on obtained liquid-phase chromatographic analysis result.Inventor finds, by said extracted method, the general flavone in Desmodium styracifolium can by high efficiency extraction.Liquid-phase chromatographic analysis is carried out to above-mentioned extractive of general flavone and obtains stratographic analysis figure, thus accurately can determine the composition of Flavonoid substances in Desmodium styracifolium.According to embodiments of the invention, the above-mentioned method accuracy determining that in Desmodium styracifolium, Flavonoid substances forms is high, stability is high.
According to embodiments of the invention, above-mentionedly determine that the method for the composition of Flavonoid substances in Desmodium styracifolium may further include following additional technical feature one of at least:
According to embodiments of the invention, the liquid-phase chromatographic analysis in the method adopts following condition: adopt methyl alcohol-0.1% formic acid system to carry out gradient elution, chromatographic column is WatersBEHC
18(2.1mm × 100mm, 1.8 microns), column temperature is 25 degrees Celsius, and flow velocity is 0.2ml/min, sample size: 1 microlitre, and determined wavelength is 272nm, and elution requirement is:
| Time (min) | 0.1% formic acid | Methyl alcohol |
| 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, thus effectively determines Flavonoid substances composition in Desmodium styracifolium.According to embodiments of the invention, in the determination Desmodium styracifolium under above-mentioned liquid-phase chromatographic analysis condition, the method for Flavonoid substances composition has the feature of high stability and accuracy.
In a fourth aspect of the present invention, the present invention proposes a kind of method that Desmodium styracifolium is originated of determining, according to embodiments of the invention, the method comprises: according to the method for extractive of general flavone in said extracted Desmodium styracifolium, from Desmodium styracifolium, extract extractive of general flavone; And liquid-phase chromatographic analysis is carried out to described extractive of general flavone, and determine the source of described Desmodium styracifolium based on obtained liquid-phase chromatographic analysis result.Inventor finds, by said extracted method, the general flavone in Desmodium styracifolium can by high efficiency extraction.Liquid-phase chromatographic analysis is carried out to above-mentioned extractive of general flavone and obtains stratographic analysis figure, thus accurately can determine the source of Desmodium styracifolium.According to embodiments of the invention, the above-mentioned method accuracy determining that Desmodium styracifolium is originated is high, stability is high.
According to embodiments of the invention, the above-mentioned method determining that Desmodium styracifolium is originated may further include following additional technical feature one of at least:
According to embodiments of the invention, the determined wavelength that the liquid-phase chromatographic analysis in the method adopts is 272nm.Under this determined wavelength condition, chromatographic peak can reach good separating effect, thus effectively determines that Desmodium styracifolium is originated.
According to embodiments of the invention, the liquid-phase chromatographic analysis in the method adopts following condition: adopt methyl alcohol-0.1% formic acid system to carry out gradient elution, chromatographic column is WatersBEHC
18(2.1mm × 100mm, 1.8 microns), column temperature is 25 degrees Celsius, and flow velocity is 0.2ml/min, sample size: 1 microlitre, and elution requirement is:
| Time (min) | 0.1% formic acid | Methyl alcohol |
| 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, thus effectively determines that Desmodium styracifolium is originated.According to embodiments of the invention, the method in the determination Desmodium styracifolium source under above-mentioned liquid-phase chromatographic analysis condition has the feature of high stability and accuracy.
According to embodiments of the invention, above-mentionedly determine that the source of described Desmodium styracifolium comprises based on obtained liquid-phase chromatographic analysis result: the chromatogram of described liquid-phase chromatographic analysis result is compared with the predetermined collection of illustrative plates that contrasts by (a); And (b) judges described chromatogram and the described similarity contrasting collection of illustrative plates, and determine the source of described Desmodium styracifolium based on described similarity.By the similarity of comparative analysis result chromatogram and contrast color spectrogram, the source of Desmodium styracifolium effectively can be determined.According to embodiments of the invention, the above-mentioned method determining that Desmodium styracifolium is originated has the feature of high stability and accuracy.
According to embodiments of the invention, in step (b), whether described similarity exists peak based on one of at least position following in collection of illustrative plates and determines: 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 representative of described position is as follows: 15.01min represents vicenin-2, 18.04min represents cyanidenon-6-C-α-L-arabopyranose-8-C-β-D-glucopyranoside, 19.77min represent vicenin-1, 23.18min represents cyanidenon-6-C-β-D-glucopyranose-8-C-β-xylopyranose glucosides, 23.73min represent Schaftoside, 27.20min represents genistein-7-O-β-D-furans celery glycosyl-(1 → 6)-O-β-D-glucopyranoside, 31.08min represent new Schaftoside, 31.72min represent Saponaretin, 32.98min represent Vicenin-3.According to embodiments of the invention, peak shape and the relative retention time of above-mentioned position are stablized, and by contrasting chromatogram peak shape and the contrast color spectrogram peak shape of Desmodium styracifolium to be determined, effectively can determine the source of Desmodium styracifolium.
According to embodiments of the invention, in step (b), whether described similarity exists peak based on column position lower in collection of illustrative plates and determines: 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 described position is as follows: 15.01min represents vicenin-2, 18.04min represents cyanidenon-6-C-α-L-arabopyranose-8-C-β-D-glucopyranoside, 19.77min represent vicenin-1, 23.18min represents cyanidenon-6-C-β-D-glucopyranose-8-C-β-xylopyranose glucosides, 23.73min represent Schaftoside, 27.20min represents genistein-7-O-β-D-furans celery glycosyl-(1 → 6)-O-β-D-glucopyranoside, 31.08min represent new Schaftoside, 31.72min represent Saponaretin, 32.98min represent Vicenin-3.According to embodiments of the invention, peak shape and the relative retention time of above-mentioned position have stability, by contrasting chromatogram peak shape and the contrast color spectrogram peak shape of Desmodium styracifolium to be determined, effectively can determine the source of Desmodium styracifolium.
It will be appreciated by those skilled in the art that method and the effect of the determination at peak, position in above-mentioned collection of illustrative plates are equally applicable to set up the method for Desmodium styracifolium extractive of general flavone characteristic spectrum and determine the method for Flavonoid substances composition in Desmodium styracifolium.
Additional aspect of the present invention and advantage will part provide in the following description, and part will become obvious from the following description, or be recognized by practice of the present invention.
Accompanying drawing explanation
Fig. 1 is the reference substance UV scanning figure according to the embodiment of the present invention 1;
Fig. 2 be according to the embodiment of the present invention 1 liquid-phase chromatographic analysis is carried out to Desmodium styracifolium extractive of general flavone time, different formic acid concn is as the chromatogram of chromatogram flow phase;
Fig. 3 be according to the embodiment of the present invention 1 liquid-phase chromatographic analysis is carried out to Desmodium styracifolium extractive of general flavone time, the chromatogram under different column temperature;
Fig. 4 be according to the embodiment of the present invention 1 liquid-phase chromatographic analysis is carried out to Desmodium styracifolium extractive of general flavone time, the chromatogram of different brands chromatographic column;
Fig. 5 is chromatogram Desmodium styracifolium extractive of general flavone being carried out to liquid-phase chromatographic analysis according to the embodiment of the present invention 1;
When Fig. 6 is the investigation Desmodium styracifolium total flavone extracting method according to the embodiment of the present invention 2, the comparatively large and good characteristic peak of degree of separation of five peak areas as evaluation index of selection;
Fig. 7 be according to the embodiment of the present invention 3 superelevation liquid chromatography (UPLC) peak attribution analysis experiment in, reference substance and sample chromatogram figure;
Fig. 8 is the total ion current figure of the detection sample according to the embodiment of the present invention 3;
Fig. 9 is the total ion current figure of the reference substance Vicenin-2 according to the embodiment of the present invention 3;
Figure 10 is the mass spectrogram of sample according to the embodiment of the present invention 3 and reference substance Vicenin-2;
Figure 11 is the total ions chromatogram of the reference substance cyanidenon-6-C-α-L-arabopyranose-8-C-β-D-glucopyranoside according to the embodiment of the present invention 3;
Figure 12 is the mass spectrogram of sample according to the embodiment of the present invention 3 and reference substance cyanidenon-6-C-α-L-arabopyranose-8-C-β-D-glucopyranoside;
Figure 13 is the total ion current figure of the reference substance Vicenin-1 according to the embodiment of the present invention 3;
Figure 14 is the mass spectrogram of sample according to the embodiment of the present invention 3 and reference substance Vicenin-1;
Figure 15 is the total ion current figure of the reference substance cyanidenon-6-C-β-D-glucopyranose-8-C-β-xylopyranose glucosides according to the embodiment of the present invention 3;
Figure 16 is the mass spectrogram of sample according to the embodiment of the present invention 3 and reference substance cyanidenon-6-C-β-D-glucopyranose-8-C-β-xylopyranose glucosides;
Figure 17 is according to the total ion figure of the reference substance Schaftoside of the embodiment of the present invention 3;
Figure 18 is the mass spectrogram of sample according to the embodiment of the present invention 3 and reference substance Schaftoside;
Figure 19 is the total ion current figure of reference substance genistein-7-O-β-D-furans celery glycosyl-(1 → the 6)-O-β-D-glucopyranoside according to the embodiment of the present invention 3;
Figure 20 is the mass spectrogram of sample according to the embodiment of the present invention 3 and reference substance genistein-7-O-β-D-furans celery glycosyl-(1 → 6)-O-β-D-glucopyranoside;
Figure 21 is the total ions chromatogram according to the new Schaftoside of the reference substance of the embodiment of the present invention 3;
Figure 22 is the mass spectrogram of sample according to the embodiment of the present invention 3 and the new Schaftoside of reference substance;
Figure 23 is the reference substance Saponaretin total ion current figure according to the embodiment of the present invention 3;
Figure 24 is the mass spectrogram of sample according to the embodiment of the present invention 3 and reference substance Saponaretin;
Figure 25 is the total ion current figure of the reference substance Vicenin-3 according to the embodiment of the present invention 3;
Figure 26 is the mass spectrogram of sample according to the embodiment of the present invention 3 and reference substance Vicenin-3;
Figure 27 is the mass spectrogram at five unknown peaks of 15.71min, 16.60min, 21.38min, 22.78min and 29.51min in retention time according to the embodiment of the present invention 3;
Figure 28 be according to the embodiment of the present invention 4 investigate detection system stability test in, as the characteristic spectrum with 12 characteristic peaks of inspection target;
Figure 29 be according to the different batches Desmodium styracifolium medicinal material of the embodiment of the present invention 5 and extractive of general flavone thereof before extraction after stratographic analysis figure;
Figure 30 is the liquid chromatogram of the Desmodium styracifolium extractive of general flavone sample of ten batches according to the embodiment of the present invention 6; And
Figure 31 take Schaftoside as the characteristic spectrum of Desmodium styracifolium extractive of general flavone of contrast according to the embodiment of the present invention 7.
Embodiment
The object of the invention is to propose a kind of from Desmodium styracifolium, extract extractive of general flavone method, a kind of determine Flavonoid substances composition in Desmodium styracifolium method and a kind ofly determine the method that Desmodium styracifolium is originated.
The method of extractive of general flavone is extracted from Desmodium styracifolium
In a first aspect of the present invention, the present invention proposes a kind of method extracting extractive of general flavone from Desmodium styracifolium.According to embodiments of the invention, the method comprises: mixed with ethanol by Desmodium styracifolium sample, and extracted by backflow or ultrasonic method, to obtain extractive of general flavone, wherein, the concentration of described ethanol is 25 ~ 95%, and the weight of described Desmodium styracifolium and the volume ratio of described ethanol are 25mg:25 ~ 100ml, preferred 25mg:50ml.Inventor finds, ethanol contend is lower than 25ml, extraction efficiency then can be caused not high, extract not exclusively, ethanol contend, higher than 100ml, can cause again extraction solution excessive, easily cause the determination that flux residual interference extractive of general flavone forms, but consider cost-saving but extract completely, the volume of preferred alcohol is 50ml.In above-mentioned concentration of alcohol and volume range, both can guarantee to extract the general flavone in Desmodium styracifolium completely, extraction efficiency is high, and the degree of separation at peak in later stage liquid-phase chromatographic analysis can be made again good.
According to embodiments of the invention, said method adopts ultrasonic method to carry out extraction 10 ~ 40 minutes, preferably 20 minutes.Inventor finds, extraction time, easily cause and extract not exclusively, extraction time was greater than 40 minutes, can lose time again, and therefore extraction time controls at 10 ~ 40 minutes lower than 10 minutes, considers to save time and guarantee to extract completely, preferably ultrasonic 20 minutes simultaneously.By above-mentioned ultrasonic extracting method, both saved extraction time, and in turn ensure that the general flavone extracted completely in Desmodium styracifolium, make extraction efficiency high, be beneficial to the medicinal of flavones and liquid-phase chromatographic analysis.
According to embodiments of the invention, in said method, the concentration of ethanol is 75%.The discovery that inventor is surprised, Desmodium styracifolium sample peak shape in 95% ethanol is poor, and degree of separation is lower, and therefore select Desmodium styracifolium sample to extract inside 75% ethanol, extraction efficiency is the highest, and in liquid-phase chromatographic analysis, the degree of separation at peak is good.
Set up the method for Desmodium styracifolium extractive of general flavone characteristic spectrum
In a second aspect of the present invention, the present invention proposes a kind of method setting up Desmodium styracifolium extractive of general flavone characteristic spectrum, according to embodiments of the invention, the method comprises: according to foregoing method, from Desmodium styracifolium, extract extractive of general flavone; Liquid-phase chromatographic analysis is carried out to described extractive of general flavone, and sets up Desmodium styracifolium extractive of general flavone characteristic spectrum based on obtained liquid-phase chromatographic analysis result.Inventor finds, by said extracted method, the general flavone in Desmodium styracifolium can by high efficiency extraction.Liquid-phase chromatographic analysis is carried out to above-mentioned extractive of general flavone and obtains stratographic analysis figure, thus the characteristic spectrum of Desmodium styracifolium extractive of general flavone can be set up.According to embodiments of the invention, above-mentioned method accuracy of setting up Desmodium styracifolium extractive of general flavone characteristic spectrum is high, stability is high.
According to embodiments of the invention, described liquid-phase chromatographic analysis adopts following condition: adopt methyl alcohol-0.1% formic acid system to carry out gradient elution, chromatographic column is WatersBEHC
18(2.1mm × 100mm, 1.8 microns), column temperature is 25 degrees Celsius, and flow velocity is 0.2ml/min; Sample size: 1 microlitre, determined wavelength is 272nm, elution requirement:
| Time (min) | 0.1% formic acid | Methyl alcohol |
| 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, thus sets up the characteristic spectrum of Desmodium styracifolium extractive of general flavone.According to embodiments of the invention, the method setting up Desmodium styracifolium extractive of general flavone characteristic spectrum under above-mentioned liquid-phase chromatographic analysis condition has the feature of high stability and accuracy.
Determine the method for Flavonoid substances composition in Desmodium styracifolium
In a third aspect of the present invention, the present invention proposes a kind of method determining Flavonoid substances composition in Desmodium styracifolium, according to embodiments of the invention, the method comprises: according to a first aspect of the present invention, from Desmodium styracifolium, extract extractive of general flavone; And liquid-phase chromatographic analysis is carried out to extractive of general flavone, and determine the composition of Flavonoid substances in described Desmodium styracifolium based on obtained liquid-phase chromatographic analysis result.Inventor finds, by said extracted method, the general flavone in Desmodium styracifolium can by high efficiency extraction.Liquid-phase chromatographic analysis is carried out to above-mentioned extractive of general flavone and obtains stratographic analysis figure, thus accurately can determine the composition of Flavonoid substances in Desmodium styracifolium.According to embodiments of the invention, the above-mentioned method accuracy determining that in Desmodium styracifolium, Flavonoid substances forms is high, stability is high.
According to embodiments of the invention, the liquid-phase chromatographic analysis in the method adopts following condition: adopt methyl alcohol-0.1% formic acid system to carry out gradient elution, chromatographic column is WatersBEHC
18(2.1mm × 100mm, 1.8 microns), column temperature is 25 degrees Celsius, and flow velocity is 0.2ml/min, sample size: 1 microlitre, and determined wavelength is 272nm, and elution requirement is:
| Time (min) | 0.1% formic acid | Methyl alcohol |
| 0～28 | 82→74 | 18→26 |
| 28～40 | 74 | 26 |
| 40～41 | 74→82 | 26→18 |
| 41～50 | 82 | 18 |
Inventor is surprised to find, and at investigation three kinds of different formic acid concns as in the experiment of mobile phase, formic acid concn is respectively 0.05%, 0.1%, 0.2%, considers the impact of low PH on chromatographic column, have chosen 0.1% formic acid-methyl alcohol and detects as mobile phase; Inventor finds simultaneously, and choosing of chromatographic column is also most important, and the chromatographic behavior impact of chromatographic column on sample of different size is very large, except choosing WatersBEHC
18outside chromatographic column, all the other chromatographic columns all can not better be separated; Meanwhile, inventor also finds, the chromatographic behavior impact of column temperature on sample of chromatographic column is very large, and temperature is strict controlled in 25 degrees Celsius, the chromatogram peak energy of sample obtains very satisfied separating effect.According to embodiments of the invention, sample concentration is 0.5g/L, sample size 1 microlitre.For the determination of determined wavelength, inventor carries out Sample Scan by 210 ~ 400nm ultraviolet, finds that sample has maximum absorption band at 272nm and 330nm place, with reference to the existing quality standard of Desmodium styracifolium extractive of general flavone, determined wavelength is decided to be 272nm.Under above-mentioned chromatographic condition, chromatographic peak can reach good separating effect, thus effectively determines Flavonoid substances composition in Desmodium styracifolium.According to embodiments of the invention, in the determination Desmodium styracifolium under above-mentioned liquid-phase chromatographic analysis condition, the method for Flavonoid substances composition has the feature of high stability and accuracy.
Determine the method that Desmodium styracifolium is originated
In a fourth aspect of the present invention, the present invention proposes a kind of method that Desmodium styracifolium is originated of determining, according to embodiments of the invention, the method comprises: according to the method for extractive of general flavone in said extracted Desmodium styracifolium, from Desmodium styracifolium, extract extractive of general flavone; And liquid-phase chromatographic analysis is carried out to described extractive of general flavone, and determine the source of described Desmodium styracifolium based on obtained liquid-phase chromatographic analysis result.Inventor finds, by said extracted method, the general flavone in Desmodium styracifolium can by high efficiency extraction.Desmodium styracifolium wide material sources, obtaining stratographic analysis figure by carrying out liquid-phase chromatographic analysis to above-mentioned extractive of general flavone, accurately can determine the source of Desmodium styracifolium.According to embodiments of the invention, the above-mentioned method accuracy determining that Desmodium styracifolium is originated is high, stability is high.
According to embodiments of the invention, the determined wavelength that the liquid-phase chromatographic analysis in the method adopts is 272nm.As mentioned above, inventor carries out Sample Scan by 210 ~ 400nm ultraviolet, finds that sample has maximum absorption band at 272nm and 330nm place, with reference to the existing quality standard of Desmodium styracifolium extractive of general flavone, determined wavelength is decided to be 272nm.Under this determined wavelength condition, chromatographic peak can reach good separating effect, thus effectively determines that Desmodium styracifolium is originated.
According to embodiments of the invention, the liquid-phase chromatographic analysis in the method adopts following condition: adopt methyl alcohol-0.1% formic acid system to carry out gradient elution, chromatographic column is WatersBEHC
18(2.1mm × 100mm, 1.8 microns), column temperature is 25 degrees Celsius, and flow velocity is 0.2ml/min, sample size: 1 microlitre, and elution requirement is:
| Time (min) | 0.1% formic acid | Methyl alcohol |
| 0～28 | 82→74 | 18→26 |
| 28～40 | 74 | 26 |
| 40～41 | 74→82 | 26→18 |
| 41～50 | 82 | 18 |
As mentioned above, consider the impact of low PH on chromatographic column, inventor have chosen 0.1% formic acid-methyl alcohol and detects as mobile phase; Simultaneously chromatographic column choose also most important, the chromatographic behavior impact of chromatographic column on sample of different size is very large, except choosing WatersBEHC
18outside chromatographic column, all the other chromatographic columns all can not better be separated; Inventor also finds, the chromatographic behavior impact of column temperature on sample of chromatographic column is very large, and temperature is strict controlled in 25 degrees Celsius, the chromatogram peak energy of sample obtains very satisfied separating effect.According to embodiments of the invention, sample concentration is 0.5g/L, sample size 1 microlitre.Under above-mentioned chromatographic condition, chromatographic peak can reach good separating effect, thus effectively determines that Desmodium styracifolium is originated.According to embodiments of the invention, the method in the determination Desmodium styracifolium source under above-mentioned liquid-phase chromatographic analysis condition has the feature of high stability and accuracy.
According to embodiments of the invention, above-mentionedly determine that the source of Desmodium styracifolium comprises based on obtained liquid-phase chromatographic analysis result: the chromatogram of liquid-phase chromatographic analysis result is compared with the predetermined collection of illustrative plates that contrasts by (a); And (b) judges chromatogram and the similarity contrasting collection of illustrative plates, and determine the source of Desmodium styracifolium based on this similarity.The chromatogram of Desmodium styracifolium extractive of general flavone is the characteristic spectrum of Desmodium styracifolium, therefore passes through the similarity of comparative analysis result chromatogram and contrast color spectrogram, effectively can determine the source of Desmodium styracifolium.According to embodiments of the invention, the above-mentioned method determining that Desmodium styracifolium is originated has the feature of high stability and accuracy.
According to embodiments of the invention, in step (b), whether this similarity exists peak based on one of at least position following in collection of illustrative plates and determines: 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 representative of described position is as follows: 15.01min represents vicenin-2, 18.04min represents cyanidenon-6-C-α-L-arabopyranose-8-C-β-D-glucopyranoside, 19.77min represent vicenin-1, 23.18min represents cyanidenon-6-C-β-D-glucopyranose-8-C-β-xylopyranose glucosides, 23.73min represent Schaftoside, 27.20min represents genistein-7-O-β-D-furans celery glycosyl-(1 → 6)-O-β-D-glucopyranoside, 31.08min represent new Schaftoside, 31.72min represent Saponaretin, 32.98min represent Vicenin-3.The peak of above-mentioned position is determined by LC-MS analytical approach, and the flavones wherein having the peak of nine positions to represent is known, and the flavones of the peak representative of five positions is unknown, but its position and specific charge are for surveying feature.According to embodiments of the invention, the peak shape of above-mentioned position and relative retention time are stablized, the chromatogram of Desmodium styracifolium extractive of general flavone is the characteristic spectrum of Desmodium styracifolium, by the parameter such as position, retention time, specific charge of the chromatogram peak shape and contrast color spectrogram peak shape and peak that contrast Desmodium styracifolium to be determined, the source of Desmodium styracifolium effectively can be determined.
According to embodiments of the invention, in step (b), whether described similarity exists peak based on column position lower in collection of illustrative plates and determines: 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 described position is as follows: 15.01min represents vicenin-2, 18.04min represents cyanidenon-6-C-α-L-arabopyranose-8-C-β-D-glucopyranoside, 19.77min represent vicenin-1, 23.18min represents cyanidenon-6-C-β-D-glucopyranose-8-C-β-xylopyranose glucosides, 23.73min represent Schaftoside, 27.20min represents genistein-7-O-β-D-furans celery glycosyl-(1 → 6)-O-β-D-glucopyranoside, 31.08min represent new Schaftoside, 31.72min represent Saponaretin, 32.98min represent Vicenin-3.The peak of above-mentioned position is determined by liquid phase chromatography analytical method, and the flavones wherein having the peak of nine positions to represent is known, and the flavones of the peak representative of three positions is unknown, but its position is for surveying feature.According to embodiments of the invention, peak shape and the relative retention time of above-mentioned position have stability, the chromatogram of Desmodium styracifolium extractive of general flavone is the characteristic spectrum of Desmodium styracifolium, by the parameter such as position, retention time of the chromatogram peak shape and contrast color spectrogram peak shape and peak that contrast Desmodium styracifolium to be determined, the source of Desmodium styracifolium effectively can be determined.
Be described below in detail embodiments of the invention, these embodiments are exemplary, only for explaining the present invention, and can not be interpreted as limitation of the present invention.
It should be noted that, unless explicitly stated otherwise, the materials and methods do not described in detail in embodiment to be below in this area conventional to use.
Conventional method:
Unless expressly stated, following instrument is adopted in the examples below that:
Instrument: WatersUPLCACQUITYH-class Ultra Performance Liquid Chromatography instrument; WatersACQUITYBEHC
18chromatographic column (2.1mm × 100mm, 1.8 microns); CQ250 ultrasonoscope (Shanghai Ultrasonic Instrument Factory); Plum Teller-Toronto XP205 type analysis balance; Plum Teller-Toronto XS204 type analysis balance.
In the examples below that, the reference substance of employing 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 institute | 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-glucopyranose-8-C-β-xylopyranose glucosides | Self-control | 85.6 |
| DS-11 | Genistein-7-O-β-D-furans celery glycosyl-(1 → 6)-O-β-D-glucopyranoside | Self-control | 95.7 |
| DS-14 | Cyanidenon-6-C-α-L-arabopyranose-8-C-β-D-glucopyranoside | Self-control | 91.3 |
| DS-15 | New Schaftoside | Self-control | 83.8 |
Above-mentioned all self-control reference substances are Waters and prepare liquid phase acquisition, use front phosphorus pentoxide drying under reduced pressure 12h; In stratographic analysis process, methyl alcohol used is chromatographically pure rank, and water is Millipore ultrapure water, and it is pure that reagent is analysis.
In the examples below that, sample is detected as shown in table 2:
Table 2
| Lot number | The place of production | Corresponding medicinal material lot number |
| 141201 | Guangxi Yulin Zham Zhen | 141106 |
| 141202 | Resource vanilla village, Guangxi | 141101 |
| 141203 | Five-way town, Lingui, Guangxi | 141102 |
| 141204 | Golden mean of the Confucian school town, Lingui, Guangxi | 141103 |
| 141205 | Si Ding town, Rong'an, Guangxi | 141104 |
| 150101 | The honest and kindhearted town of Guangxi Yulin | 141105 |
| 150102 | Shaoguan, Guangdong | 20120903 |
| 150103 | Cheng Yue town, Suixi, Guangdong | 141107 |
| 150104 | Guangxi Luzhai | 20131127 |
| 140504 | Guilin | 141108 |
In the examples below that, the compound method detecting sample solution is as follows:
Precision takes the sample of 25mg, puts in 50ml measuring bottle, and add 75% ethanol to nearly scale mark, ultrasonic 20min, lets cool, constant volume.Each detection sample introduction 1 microlitre.
Embodiment 1 investigates liquid phase chromatogram condition
The selection of 1.1 determined wavelength: get Schaftoside and all the other eight kinds self-control reference substances appropriate, respectively at ultrasonic dissolution in 75% ethanol, UV scanning is carried out at 210 ~ 400nm, absorption maximum is had at 272nm and 330nm place, with reference to the existing quality standard of Desmodium styracifolium extractive of general flavone, determined wavelength is decided to be 272nm, UV scanning figure as shown in Figure 1, wherein Fig. 1-1 is the full wavelength scanner figure of DS-1, Fig. 1-2 is the full wavelength scanner figure of DS-3, Fig. 1-3 is full wavelength scanner figure of DS-4, Fig. 1-4 is full wavelength scanner figure of DS-5, Fig. 1-5 is full wavelength scanner figure of DS-8, Fig. 1-6 is full wavelength scanner figure of DS-11, Fig. 1-7 is full wavelength scanner figure of DS-14, Fig. 1-8 is full wavelength scanner figure of DS-15, Fig. 1-9 is full wavelength scanner figure of DS-2.Fig. 1 result shows nine kinds of control samples under 210 ~ 400nm UV scanning, has absorption maximum at 272nm and 330nm place.
1.2 the selection of elution requirement
The selection of mobile phase: adopt WatersBEHC
18(2.1mm × 100mm, 1.7 microns) chromatographic column, find by comparing the systems such as methyl alcohol-0.1% formic acid, acetonitrile-0.1% formic acid, methanol-acetonitrile-0.1% formic acid, methyl alcohol-0.1% phosphoric acid, Desmodium styracifolium extractive of general flavone is in the system of methyl alcohol-0.1% formic acid, the chromatographic peak obtained is more, degree of separation is better, therefore selects methyl alcohol-0.1% formic acid system to carry out gradient elution.
Simultaneously, configure the mobile phase of three kinds of different formic acid concns respectively, 0.05% formic acid water-methanol, 0.1% formic acid water-methanol, 0.2% formic acid water-methanol, found that, the chromatographic behavior impact of above-mentioned three kinds of formic acid concns on Desmodium styracifolium extractive of general flavone is little, consider the impact of low pH on chromatographic column, choosing 0.1% formic acid water-methanol is that mobile phase detects, different formic acid concn as chromatogram flow phase chromatogram as shown in Figure 2, wherein Fig. 2-1 is 0.05% formic acid water-methanol is the chromatogram of mobile phase, Fig. 2-2 is 0.1% formic acid water-methanols is the chromatogram of mobile phase, Fig. 2-3 is 0.2% formic acid water-methanols is the chromatogram of mobile phase.
Condition of gradient elution is as shown in table 3,
Table 3
| Time (min) | 0.1% formic acid | Methyl alcohol |
| 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 respectively, 25 DEG C and 30 DEG C, these three kinds different column temperatures are on the impact of chromatographic behavior, found that, peak 5 and peak 6 to need under comparatively low temperature 20 DEG C, reasonable separating effect could be obtained, and peak 10 and peak 11 are higher temperature 30 DEG C, reasonable separating effect could be obtained, 25 DEG C time, peak 5 and peak 6, peak 10 and peak 11 all can obtain satisfied separating effect, can find that the chromatographic behavior of sample is very responsive to temperature, therefore needing strictly to control column temperature is 25 DEG C, under different column temperature condition, chromatogram as shown in Figure 3, wherein Fig. 3-1 is the chromatogram at column temperature 20 DEG C, Fig. 3-2 is the chromatograms at column temperature 25 DEG C, Fig. 3-3 is the chromatograms at column temperature 30 DEG C.
The selection of 1.4 chromatographic columns
The present embodiment adopts WatersBEHC respectively
18(2.1mm × 100mm, 1.7 microns), Shiseido CapellcoreC
18(2.1mm × 100mm, 2.7 microns) and Shimadzu Shim-packXR-ODSII (2.0mm × 75mm, 2.2 microns), found that, the chromatographic behavior impact of chromatographic column on sample of different brands specification is very large, except WatersBEHC
18(2.1mm × 100mm, 1.8 microns) chromatographic column can reach outside desirable baseline separation, all the other chromatographic columns all can not better be separated, therefore, answer brand and the model of stationary chromatographic post, different chromatographic column detects the chromatogram of Desmodium styracifolium chromocor extract as shown in Figure 4, and wherein Fig. 4-1 is WatersBEHC
18the chromatogram of (2.1mm × 100mm, 1.7 microns), Fig. 4-2 is Shiseido CapellcoreC
18(2.1mm × 100mm, 2.7 microns) chromatogram, Fig. 4-3 is Shimadzu Shim-packXR-ODSII (2.0mm × 75mm, 2.2 microns) chromatograms.
To sum up under liquid-phase chromatographic analysis condition, adopt methyl alcohol-0.1% formic acid system to carry out gradient elution, chromatographic column is WatersBEHC18 (2.1mm × 100mm, 1.8 microns), and column temperature is 25 degrees Celsius, and flow velocity is 0.2ml/min; Sample size: 1 microlitre, determined wavelength is 272nm, elution requirement:
| Time (min) | 0.1% formic acid | Methyl alcohol |
| 0～28 | 82→74 | 18→26 |
| 28～40 | 74 | 26 |
| 40～41 | 74→82 | 26→18 |
| 41～50 | 82 | 18 |
As shown in Figure 5, Fig. 5 result shows the chromatogram of the Desmodium styracifolium general flavone sample obtained, and under above-mentioned chromatographic condition, the chromatographic peak degree of separation of Desmodium styracifolium general flavone sample is good.
Embodiment 2 investigates the method extracting Desmodium styracifolium general flavone
Owing to being mainly the Flavonoid substances that a class has identical mother nucleus structure in Desmodium styracifolium extractive of general flavone, therefore inventor mainly to choose peak area in sample larger, and good five chromatographic peaks of degree of separation are evaluated as index, investigate the extracting method of sample, as evaluation index five peaks as shown in Figure 6.
The selection of 2.1 extracting modes
Precision takes the Desmodium styracifolium extractive of general flavone sample of 25mg, precision adds 50% ethanol 50ml, adopt ultrasonic (250KW respectively, 33khz) and backflow two kinds of extracting method extract, the chromatographic peak map analysis result of the chromocor extract that two kinds of extracting modes obtain is as shown in table 4, and table 4 result shows, both extraction efficiency there was no significant differences, but consider the simplicity of operation, choose ultrasonic as extracting method.
Table 4
The selection of 2.2 extraction times
Precision takes 25mg sample, precision adds 50% ethanol 50ml, ultrasonic 10min, 20min, 30min, 40min respectively, compare, the chromatogram result of the general flavone obtained under different extraction time is as shown in table 5, and table 5 result shows, and extraction time is to extraction efficiency there was no significant difference, but consider to save time but guarantee again to extract completely, choosing ultrasonic 20min.
Table 5
The selection of 2.3 solvent loads
Precision takes 25mg sample, precision adds the ultrasonic extraction of 50% ethanol 25ml, 50ml, 75ml, 100ml respectively, with 50ml solvent extraction for benchmark, carry out conversion respectively by volume to compare, the chromatogram result of the general flavone that different solvents measures is as shown in table 6, and table 6 result shows, and solvent load is to extraction efficiency there was no significant difference, but consider cost-saving but guarantee again to extract completely, choosing 50ml and extract.
Table 6
The selection of 2.4 solvent strengths
Precision takes 25mg sample, precision adds 25% ethanol, 50% ethanol, 75% ethanol, each 50ml of 95% ethanol respectively, ultrasonic extraction, the chromatogram result of the chromocor extract obtained under variable concentrations solvent is as shown in table 7, table 7 result shows, sample extraction efficiency inside 75% ethanol is the highest, and degree of separation is good; In 95% ethanol, peak shape is poor, and degree of separation is lower, therefore selects 75% alcohol extract.
Table 7
To sum up, finally determine that the preparation method of the detection sample solution of Desmodium styracifolium extractive of general flavone is: precision takes the Desmodium styracifolium extractive of general flavone sample of 25mg, puts in 50ml measuring bottle, add 75% ethanol to nearly scale mark, ultrasonic 20min, lets cool, constant volume.
The analysis of embodiment 3 synergy
3.1 superelevation liquid chromatography (UPLC) peak attribution analysises
Get appropriate Schaftoside, Vicenin-3, Vicenin-1, Saponaretin, Vicenin-2, cyanidenon-6-C-β-D-glucopyranose-8-C-β-xylopyranose glucosides, genistein-7-O-β-D-furans celery glycosyl-(1 → 6)-O-β-D-glucopyranoside, cyanidenon-6-C-α-L-arabopyranose-8-C-β-D-glucopyranoside respectively, newly Schaftoside reference substance is appropriate, add 75% ethanol, be made into nine kinds of reference substance solution, get each reference substance solution more appropriate, be made into mixing reference substance solution, the detection sample solution of Desmodium styracifolium extractive of general flavone is obtained with legal system, injection liquid chromatography, relatively reference substance and sample chromatogram figure, nine chromatographic peaks in sample are belonged to, result as shown in Figure 7, wherein Fig. 7-1 is DS-5 and sample liquid chromatogram, Fig. 7-2 is DS-2 and sample liquid chromatogram, Fig. 7-3 is DS-3 and sample liquid chromatogram, Fig. 7-4 is DS-8 and sample liquid chromatogram, Fig. 7-5 is DS-2 and sample liquid chromatogram, Fig. 7-6 is DS-11 and sample liquid chromatogram, Fig. 7-7 is DS-15 and sample liquid chromatogram, Fig. 7-8 is DS-4 and sample liquid chromatogram, Fig. 7-9 is DS-1 and sample liquid chromatogram, Fig. 7-10 is mixing reference substance solution liquid chromatograms, Fig. 7-11 is samples and mix reference substance solution liquid chromatogram.Can be drawn by Fig. 7, peak 1 represents Vicenin-2, peak 2 represents cyanidenon-6-C-α-L-arabopyranose-8-C-β-D-glucopyranoside, peak 3 represents Vicenin-1, peak 4 represents cyanidenon-6-C-β-D-glucopyranose-8-C-β-xylopyranose glucosides, peak 5 represents Schaftoside, peak 6 represents genistein-7-O-β-D-furans celery glycosyl-(1 → 6)-O-β-D-glucopyranoside, peak 7 represents new Schaftoside, peak 8 represents Saponaretin, and peak 9 represents Vicenin-3.
3.2 LC-MS peak attribution analysises
In order to carry out attribution analysis and checking to the liquid chromatography peak detecting sample further, inventors performed LC-MS experiment.
3.2.1 instrument and chromatographic condition
Instrument: LC-MS instrument WatersxevoG2Q-TofLC/MS;
Chromatographic column AcquityUPLCBEHC
18(1.7 μm, 2.1*100mm);
Chromatographic condition: mobile phase: A:0.1% formic acid solution; B: methyl alcohol;
Condition of gradient elution is as shown in table 3;
Flow velocity: 0.2mL/min;
Column temperature: 25 DEG C;
Sample size 1 μ L.
Mass Spectrometry Conditions: ion gun: ESI (-); Scan pattern: MSE; Capillary voltage: 2.6kV; One-level taper hole voltage: 12V; Secondary taper hole voltage: 4.0V; Cracked voltage: 15-60V; Source temperature: 100 DEG C; Desolventizing temperature: 350 DEG C; Desolventizing gas: 600L/h; Taper hole blowback air: 50L/h.
3.2.2 obtain solution
It is appropriate that preparation reference substance solution takes above-mentioned nine kinds of reference substances respectively, adds 75% ethanol and make the reference substance solution that concentration is 30 mcg/ml;
Preparation detects the Desmodium styracifolium extractive of general flavone sample that sample solution precision takes 25mg, puts in 50ml measuring bottle, and add 75% ethanol to nearly scale mark, ultrasonic 20min, lets cool, constant volume.
3.2.3 peak attribution analysis
By above-mentioned two kinds of solution respectively injection liquid matter combined instrument detect, gained total ion current figure and mass spectrogram are as shown in Fig. 8 ~ Figure 27, Fig. 8 is the total ion current figure detecting sample, Fig. 9 is the total ion current figure of reference substance Vicenin-2, Figure 10 is the mass spectrogram of sample and reference substance Vicenin-2, Figure 11 is the total ions chromatogram of reference substance cyanidenon-6-C-α-L-arabopyranose-8-C-β-D-glucopyranoside, Figure 12 is the mass spectrogram of sample and reference substance cyanidenon-6-C-α-L-arabopyranose-8-C-β-D-glucopyranoside, Figure 13 is the total ion current figure of reference substance Vicenin-1, Figure 14 is the mass spectrogram of sample and reference substance Vicenin-1, Figure 15 is the total ion current figure of reference substance cyanidenon-6-C-β-D-glucopyranose-8-C-β-xylopyranose glucosides, Figure 16 is the mass spectrogram of sample and reference substance cyanidenon-6-C-β-D-glucopyranose-8-C-β-xylopyranose glucosides, Figure 17 is the total ion figure of reference substance Schaftoside, Figure 18 is the mass spectrogram of sample and reference substance Schaftoside, Figure 19 is the total ion current figure of reference substance genistein-7-O-β-D-furans celery glycosyl-(1 → 6)-O-β-D-glucopyranoside, Figure 20 is the mass spectrogram of sample and reference substance genistein-7-O-β-D-furans celery glycosyl-(1 → 6)-O-β-D-glucopyranoside, Figure 21 is the total ions chromatogram of the new Schaftoside of reference substance, Figure 22 is the mass spectrogram of the new Schaftoside of sample and reference substance, Figure 23 is reference substance Saponaretin total ion current figure, Figure 24 is the mass spectrogram of sample and reference substance Saponaretin, Figure 25 is the total ion current figure of reference substance Vicenin-3, Figure 26 is the mass spectrogram of sample and reference substance Vicenin-3, Figure 27 is 15.71min in retention time, 16.60min, 21.38min, the mass spectrogram at five unknown peaks of 22.78min and 29.51min.The retention time of 14 chromatographic peaks and Information in Mass Spectra belong to as shown in table 8.
Table 8
Note: * represents molecular ion peak
Embodiment 4 investigates the stability of detection system
The ownership of comprehensive above-mentioned separating effect and characteristic peak, finally determines 12 characteristic peaks and evaluates.Choose retention time moderate, the Schaftoside that peak area is larger is S peak, carries out characteristic spectrum methodology study on the stability.Characteristic spectrum as shown in figure 28.
4.1 precision tests get the detection sample solution (lot number 140504) of Desmodium styracifolium extractive of general flavone, by above-mentioned chromatographic condition, and continuous sample introduction 6 times, record chromatogram.The relative retention time RSD at each peak is less than 1.5%, and relative peak area RSD is less than 3.1%, and analysis result is as shown in table 9, wherein shows the relative retention time that 9-1 is peak, and table 9-2 is the relative peak area at peak.
Table 9-1
Note: chronomere is min;
Table 9-2
Note: non-resolved peaks 5 is split to it peak area obtained with chromatographic work station software with peak 11 with peak 6, peak 10 and calculated
The detection sample solution (lot number 140504) of Desmodium styracifolium extractive of general flavone is got in 4.2 stability tests, by above-mentioned chromatographic condition, measures respectively at 0,5,10,15,20h, record chromatogram.The relative retention time RSD% at each peak is all less than 1%; Relative peak area RSD is less than 3.2%, and analysis result is as shown in table 10, wherein shows the relative retention time that 10-1 is peak, and table 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 measures;
Table 10-2
Note: non-resolved peaks 5 is split to it peak area obtained with chromatographic work station software with peak 11 with peak 6, peak 10 and calculated
4.3 replica tests get the detection sample solution (lot number 140504) 6 parts of Desmodium styracifolium extractive of general flavone, detect sample solution, measure by above-mentioned chromatographic condition, record chromatogram according to above-mentioned compound method preparation.The relative retention time at each peak, RSD% is all less than 0.2%; Relative peak area RSD is all less than 2%, and analysis result is as shown in table 11, wherein shows the relative retention time that 11-1 is peak, and 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 is split to it peak area obtained with chromatographic work station software with peak 11 with peak 6, peak 10 and calculated
To sum up precision test, stability test and replica test result, known detection system stability of the present invention is high, and the characteristic spectrum methodology of Desmodium styracifolium extractive of general flavone is stablized.
Embodiment 5 investigates the composition transfer before and after the extraction of Desmodium styracifolium medicinal material and its extractive of general flavone
In order to study Desmodium styracifolium medicinal material and its extractive of general flavone before extraction after composition transfer, inventor respectively by Desmodium styracifolium extractive of general flavone and Desmodium styracifolium medicinal material under above-mentioned same chromatographic condition, inject Ultra Performance Liquid Chromatography instrument, compare analysis, it is as follows that its preparation detects sample solution:
Desmodium styracifolium medicinal material: the Desmodium styracifolium medicinal material taking about 0.2g, add 75% ethanol in 25ml measuring bottle, ultrasonic 20min, lets cool, and constant volume to obtain final product;
Desmodium styracifolium extractive of general flavone: precision takes the sample of 25mg, puts in 100ml measuring bottle, and add 75% ethanol to nearly scale mark, ultrasonic 20min, lets cool, constant volume.
The chromatogram obtained as shown in figure 29, wherein Figure 29-1 is 141201 batches of extractive of general flavone collection of illustrative plates corresponding to its crude drug, Figure 29-2 is 141202 batches of extractive of general flavone collection of illustrative plates corresponding to its crude drug, Figure 29-3 is 141203 batches of extractive of general flavone collection of illustrative plates corresponding to its crude drug, Figure 29-4 is 141204 batches of extractive of general flavone collection of illustrative plates corresponding to its crude drug, Figure 29-5 is 141205 batches of extractive of general flavone collection of illustrative plates corresponding to its crude drug, Figure 29-6 is 150101 batches of extractive of general flavone collection of illustrative plates corresponding to its crude drug, Figure 29-7 is 150102 batches of extractive of general flavone collection of illustrative plates corresponding to its crude drug, Figure 29-8 is 150103 batches of extractive of general flavone collection of illustrative plates corresponding to its crude drug, Figure 29-9 is 150104 batches of extractive of general flavone collection of illustrative plates corresponding to its crude drug, Figure 29-10 is 140504 batches of extractive of general flavone collection of illustrative plates corresponding to its crude drug.
Figure 29 chromatogram result shows, and extractive of general flavone is after medicinal material extract, and material base does not change, thus ensure that extractive of general flavone clinical effectiveness.
Embodiment 6 measures the liquid chromatogram of Desmodium styracifolium extractive of general flavone sample
Under above-mentioned chromatographic condition, the sample taking the Desmodium styracifolium extractive of general flavone of ten batches respectively measures, injection liquid chromatography, obtain the chromatogram of ten batches, chromatogram as shown in figure 30, the relative retention time at peak is as shown in table 12, and the Desmodium styracifolium extractive of general flavone through comparing ten batches all has this 12 characteristic peaks, and relative retention time is stablized.
Table 12
Embodiment 7 sets up the characteristic spectrum of Desmodium styracifolium chromocor extract
Chromatographic condition: with octadecyl silane WatersACQUITYBEHC
18(2.1mm × 100mm, 1.7 microns) are filling agent, and take methyl alcohol as mobile phase A, the formic acid solution with 0.1% is Mobile phase B, and condition of gradient elution is as shown in table 3; Column temperature is 25 DEG C; Determined wavelength is 272nm.Theoretical cam curve calculates should be not less than 5000 by Schaftoside.
It is appropriate, accurately weighed that preparation object of reference solution gets Schaftoside reference substance, adds 75% ethanol and make the solution of every 1ml containing Schaftoside 20ug, to obtain final product.
Preparation detection sample solution takes Desmodium styracifolium extractive of general flavone and is about 25mg, and add 75% ethanol and put 50ml measuring bottle to nearly scale mark, ultrasonic 20min, lets cool, constant volume.
Measure characteristic spectrum difference accurate absorption object of reference solution and detect each 1ul of sample solution, injection Ultra Performance Liquid Chromatography instrument, to obtain final product.
Detect in sample collection of illustrative plates and should have 12 characteristic peaks, the peak corresponding to object of reference is S peak, calculates the relative retention time at each characteristic peak and S peak, its relative retention time should setting ± 5% within.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 (peaks 9), 1.308 (peaks 10), 1.333 (peaks 11), 1.385 (peaks 12).Desmodium styracifolium extractive of general flavone contrast characteristic spectrum as shown in figure 31, wherein peak 1 is Vicenin-2, peak 3 is cyanidenon-6-C-α-L-arabopyranose-8-C-β-D-glucopyranosides, peak 4 is Vicenin-1, peak 6 is cyanidenon-6-C-β-D-glucopyranose-8-C-β-xylopyranose glucosides, peak 7 is Schaftosides, peak 8 is genistein-7-O-β-D-furans celery glycosyl-(1 → 6)-O-β-D-glucopyranosides, peak 10 is new Schaftosides, peak 11 is Saponaretins, and peak 12 is Vicenin-3.
In the description of this instructions, specific features, structure, material or feature that the description of reference term " embodiment ", " some embodiments ", " example ", " concrete example " or " some examples " etc. means to describe in conjunction with this embodiment or example are contained at least one embodiment of the present invention or example.In this manual, identical embodiment or example are not necessarily referred to the schematic representation of above-mentioned term.And the specific features of description, structure, material or feature can combine in an appropriate manner in any one or more embodiment or example.
Although illustrate and describe embodiments of the invention, those having ordinary skill in the art will appreciate that: can carry out multiple change, amendment, replacement and modification to these embodiments when not departing from principle of the present invention and aim, scope of the present invention is by claim and equivalents thereof.
Claims (10)
1. from Desmodium styracifolium, extract a method for extractive of general flavone, it is characterized in that, comprising:
Desmodium styracifolium sample is mixed with ethanol, and is extracted by backflow or ultrasonic method, to obtain extractive of general flavone,
Wherein, the concentration of described ethanol is 25 ~ 95%, and the weight of described Desmodium styracifolium and the volume ratio of described ethanol are 25mg:25 ~ 100ml, preferred 25mg:50ml.
2. the method extracting extractive of general flavone from Desmodium styracifolium according to claim 1, is characterized in that, adopts ultrasonic method to carry out extraction 10 ~ 40 minutes, preferably 20 minutes.
3. the method extracting extractive of general flavone from Desmodium styracifolium according to claim 1, is characterized in that, the concentration of described ethanol is 75%.
4. set up a method for Desmodium styracifolium extractive of general flavone characteristic spectrum, it is characterized in that, comprising:
According to the method described in any one of claims 1 to 3, from Desmodium styracifolium, extract extractive of general flavone;
Liquid-phase chromatographic analysis is carried out to described extractive of general flavone, and sets up described Desmodium styracifolium extractive of general flavone characteristic spectrum based on obtained liquid-phase chromatographic analysis result,
Optionally, described liquid-phase chromatographic analysis adopts following condition:
Methyl alcohol-0.1% formic acid system is adopted to carry out gradient elution,
Chromatographic column is WatersBEHC
18(2.1mm × 100mm, 1.8 microns),
Column temperature is 25 degrees Celsius,
Flow velocity is 0.2ml/min;
Sample size: 1 microlitre,
Determined wavelength is 272nm,
Elution requirement:
。
5. determine a method for Flavonoid substances composition in Desmodium styracifolium, it is characterized in that, comprising:
According to the method described in any one of claims 1 to 3, from Desmodium styracifolium, extract extractive of general flavone;
Liquid-phase chromatographic analysis is carried out to described extractive of general flavone, and determines the composition of Flavonoid substances in described Desmodium styracifolium based on obtained liquid-phase chromatographic analysis result,
Optionally, described liquid-phase chromatographic analysis adopts following condition:
Methyl alcohol-0.1% formic acid system is adopted to carry out gradient elution,
Chromatographic column is WatersBEHC
18(2.1mm × 100mm, 1.8 microns),
Column temperature is 25 degrees Celsius,
Flow velocity is 0.2ml/min;
Sample size: 1 microlitre,
Determined wavelength is 272nm,
Elution requirement:
。
6. determine to it is characterized in that the method that Desmodium styracifolium is originated, comprising:
According to the method described in any one of claims 1 to 3, from Desmodium styracifolium, extract extractive of general flavone; And
Liquid-phase chromatographic analysis is carried out to described extractive of general flavone, and determines the source of described Desmodium styracifolium based on obtained liquid-phase chromatographic analysis result.
7. according to claim 6ly determine the method that Desmodium styracifolium is originated, it is characterized in that, the determined wavelength that described liquid-phase chromatographic analysis adopts is 272nm,
Optionally, described liquid-phase chromatographic analysis adopts following condition:
Methyl alcohol-0.1% formic acid system is adopted to carry out gradient elution,
Chromatographic column is WatersBEHC
18(2.1mm × 100mm, 1.8 microns),
Column temperature is 25 degrees Celsius,
Flow velocity is 0.2ml/min,
Sample size: 1 microlitre,
Elution requirement:
。
8. according to claim 6ly determine the method that Desmodium styracifolium is originated, it is characterized in that, determine that the source of described Desmodium styracifolium comprises based on obtained liquid-phase chromatographic analysis result:
A the chromatogram of described liquid-phase chromatographic analysis result is compared with the predetermined collection of illustrative plates that contrasts by (); And
B () judges described chromatogram and the described similarity contrasting collection of illustrative plates, and determine the source of described Desmodium styracifolium based on described similarity.
9. according to claim 8ly determine the method that Desmodium styracifolium is originated, it is characterized in that, in step (b), whether described similarity exists peak based on one of at least position following in collection of illustrative plates and determines: 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 representative of described position is as follows: 15.01min represents vicenin-2, 18.04min represents cyanidenon-6-C-α-L-arabopyranose-8-C-β-D-glucopyranoside, 19.77min represent vicenin-1, 23.18min represents cyanidenon-6-C-β-D-glucopyranose-8-C-β-xylopyranose glucosides, 23.73min represent Schaftoside, 27.20min represents genistein-7-O-β-D-furans celery glycosyl-(1 → 6)-O-β-D-glucopyranoside, 31.08min represent new Schaftoside, 31.72min represent Saponaretin, 32.98min represent Vicenin-3.
10. according to claim 8ly determine the method that Desmodium styracifolium is originated, it is characterized in that, in step (b), whether described similarity exists peak based on column position lower in collection of illustrative plates and determines: 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 described position is as follows: 15.01min represents vicenin-2, 18.04min represents cyanidenon-6-C-α-L-arabopyranose-8-C-β-D-glucopyranoside, 19.77min represent vicenin-1, 23.18min represents cyanidenon-6-C-β-D-glucopyranose-8-C-β-xylopyranose glucosides, 23.73min represent Schaftoside, 27.20min represents genistein-7-O-β-D-furans celery glycosyl-(1 → 6)-O-β-D-glucopyranoside, 31.08min represent new Schaftoside, 31.72min represent Saponaretin, 32.98min represent Vicenin-3.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105758980A (en) * | 2016-04-27 | 2016-07-13 | 广西壮族自治区梧州食品药品检验所 | Method for measuring total flavones in wild chrysanthemum flowers |
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| CN110470773A (en) * | 2019-09-20 | 2019-11-19 | 广东一方制药有限公司 | A kind of the UPLC characteristic spectrum construction method and discrimination method of Desmodium styracifolium medicinal material and Desmodium styracifolium standard decoction |
| CN110470773B (en) * | 2019-09-20 | 2022-03-29 | 广东一方制药有限公司 | UPLC characteristic spectrum construction method and identification method of desmodium styracifolium medicinal material and desmodium styracifolium standard decoction |
| CN114235980A (en) * | 2021-11-16 | 2022-03-25 | 武汉人福创新药物研发中心有限公司 | Method for detecting content of flavonoid compounds in desmodium styracifolium extract |
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