CN109633002A - A kind of method of Kaempferol-O- rutinoside content in measurement safflower - Google Patents
A kind of method of Kaempferol-O- rutinoside content in measurement safflower Download PDFInfo
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- G01N30/02—Column chromatography
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses a kind of methods of Kaempferol-O- rutinoside content in measurement safflower, belong to effective component of chinese medicine content detection technical field.Effective component Kaempferol-O- rutinoside in safflower is extracted by organic solvent, using as test solution, it is measured again through high performance liquid chromatograph with reference substance solution, the qualitative and quantitative detection to effective component Kaempferol-O- rutinoside in safflower can be achieved, and this detection is limited up to 0.16ug/mL；This method has the characteristics such as easy to operate, stability is high, reproducible, specificity is strong, is the reliable detection method of offers such as the research of the qualitative, quantitative of Kaempferol-O- rutinoside and control of product quality, evaluation index foundation in subsequent safflower.
Description
Technical field
The present invention relates to it is a kind of measurement safflower in Kaempferol-O- rutinoside content method, especially be related to one kind
Using the method for Kaempferol-O- rutinoside content in high effective liquid chromatography for measuring safflower, belongs to effective component of chinese medicine and contain
Measure detection technique field.
Background technique
Safflower, composite family have blood circulation, removing blood stasis and acesodyne and other effects, are generally used for menostasis, dysmenorrhea, lochia, Disorder lump in the abdomen
Lump in the abdomen, chest impediment and cardialgia, stasis of blood, the side of body twinge, injury from falling down and sore swell and ache curative etc..Paper document " Fei Yali, Huang little Min, it is red
Mention in the research of flower Injection in Rats endotoxin-induced acute liver injury protection mechanism ": Sofflower injection can pass through reduction
INOS activity, then reduces Serum concentration of NO in hepatic injury, to play the protective effect to acute liver damage.Paper document
" Ma Junfei, Zhang Shilei, nymphaea tetragona Kaempferol-O- rutinoside cause the protective effect of acute liver to galactosamine "
In mention: Kaempferol-O- rutinoside to D-Gal cause acute liver have protective effect, protective effect and promote
Into antioxidase, inhibit the expression of proinflammatory factor related.Patent document " CN102415569A, it is a kind of with the soft of liver-protecting function
It is disclosed in capsule ": the compound preparation flexible glue being made of safflower seed oil, kudzu root extract, Salvia root P.E, concentrated soybean phospholipid etc.
Capsule, with liver-protecting function.In addition, a variety of safflower preparations such as safflower liver clearing pill, DANHONG ZHUSHEYE, safflower fuling pill etc. show
Safflower has liver-protecting function.It follows that the active constituent in safflower with liver protection effect is very likely Kaempferol-O- rue
Fragrant glucosides.
Meanwhile modern pharmacological studies have shown that in safflower Kaempferol-O- rutinoside have anti-oxidant, antibacterial, it is antiviral,
The multiple biological activities such as anti-inflammatory and antalgic and neuroprotection.
But currently, not yet occurring the side such as being used for the content detection of Kaempferol-O- rutinoside in safflower, isolate and purify
Method, although identification and the assay about safflower and west safflower are recorded in 2015 editions " Chinese Pharmacopoeias ", wherein also not
It is related to the measurement of Kaempferol-O- rutinoside content.
Summary of the invention
The present invention is directed to overcome the deficiencies in the prior art, and propose Kaempferol-O- rutinose in a kind of measurement safflower
The method of glycosides content.The technical program extracts effective component in safflower by organic solvent, using as test solution, then with it is right
It is measured through high performance liquid chromatograph according to product solution, it can be achieved that effective component Kaempferol-O- rutinoside in safflower
Qualitative (identification) and quantitative (content) detection, and the reachable 0.16ug/mL of this detection limit；This method has easy to operate, stable
Property the characteristics such as high, reproducible, specificity is strong, be the research of the qualitative, quantitative of Kaempferol-O- rutinoside in subsequent safflower,
And the reliable detection method of offers such as control of product quality, evaluation index foundation.
In order to achieve the above technical purposes, the following technical solution is proposed:
The method of Kaempferol-O- rutinoside content, includes the following steps: in a kind of measurement safflower
A. Kaempferol-O- rutinoside reference substance is weighed, uses 70% methanol for solvent, prepares at least five parts with concentration gradient
Reference substance solution；
B. at least five parts of reference substance solution difference sample introductions prepared through step A after measured, are obtained respectively in high performance liquid chromatograph
The corresponding chromatogram of reference substance solution；
C. using the concentration of each reference substance solution as abscissa, the peak of Kaempferol-O- rutinoside chromatographic peak in corresponding chromatogram
Area is ordinate, draws standard curve, obtains equation of linear regression；
D. weigh safflower test sample to be determined uses 70% methanol for solvent after dry smashing, carries out ultrasonic extraction, obtains for examination
Product solution；
E. by the resulting test solution sample introduction of step D in high performance liquid chromatograph, then to be carried out with the consistent condition of step B
Measurement, obtains the chromatogram of test solution；
F. the peak area of test solution chromatographic peak is brought into equation of linear regression obtained in step C, is obtained to be determined red
Kaempferol-O- rutinoside content in flower test sample.
Preferably, in step, it is 0.06mg/mL, 0.15mg/mL, 0.30mg/ that the reference substance solution, which includes concentration,
Kaempferol-O- rutinoside the solution of mL, 0.45 mg/mL and 0.60mg/mL.Using the concentration gradient, survey with higher
Determine accuracy.
Preferably, in step D, the preparation method of the test solution specifically includes: weighing safflower test sample, does
It is dry, it smashes as the saffron powder of 50~80 mesh, then 70% methanol 10mL is added with every gram of saffron powder, in 30~60 kHz of frequency
And under the conditions of 15~35 DEG C of temperature, 60~90min of ultrasonic extraction obtains extracting solution；It is again 14000r/min in revolving speed by extracting solution
Under the conditions of, it is centrifuged, taking supernatant is test solution.
Preferably, in step B and E, the high performance liquid chromatograph be UV-DAD detector, use Detection wavelength for
The ultraviolet light of 344nm.
Preferably, in step B and E, chromatographic column filler C18, column length 100mm, internal diameter 4.6mm, filter sizes
For 3.5um.
Preferably, in step B and E, chromatogram column temperature is 20~40 DEG C.
Preferably, in step B and E, the mobile phase is the mixed liquor of acetonitrile and phosphoric acid solution, wherein acetonitrile volume
Score 10~20%, phosphoric acid solution volume fraction 80~90%, and phosphoric acid solution are the phosphate aqueous solution that concentration is 0.2%.
Preferably, in step B and E, the flow velocity of the mobile phase is 0.8~1.2 mL/min.
Preferably, in step B and E, the reference substance solution is identical with the sample volume of test solution, sample volume 8
~12 μ L.
Safflower test sample used is flos carthami, and market can directly buy acquisition.
Using the technical program, bring advantageous effects are as follows:
1) present invention uses high performance liquid chromatography, is positioned with Kaempferol-O- rutinoside reference substance, in safflower
Kaempferol-O- rutinoside carries out qualitative and quantitative detection, and measurement method is easy to operate, specificity, quantitative limit, detection limit, line
Property, precision, accuracy, stability of solution, durability etc. can obtain effective guarantee, and RSD is respectively less than 2%, actually detected
It works well；
2) present invention is practical, and during actual measurement, detection is limited up to 0.16ug/mL, both may determine that safflower medicine
The material true and false, and may determine that the superiority and inferiority of flos carthami quality, detection process is simple, quick；
3) present invention is pair with Kaempferol-O- rutinoside methanol solution using the extracting solution of flos carthami as test solution
It according to product solution, is injected separately into high performance liquid chromatograph and is measured, in the assay method, there is test solution preparation letter
The advantages that single, equipment requirement is lower, and experimental implementation safety, stability is high, reproducible, and specificity is strong and easy to operate.
Detailed description of the invention
Fig. 1 is the canonical plotting of embodiment 1 in the present invention
Fig. 2 is the chromatogram of embodiment 7 in the present invention.
Specific embodiment
Below by technical scheme in the embodiment of the invention is clearly and completely described, it is clear that described reality
Applying example is only a part of the embodiments of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is general
Logical technical staff all other embodiment obtained without making creative work belongs to what the present invention protected
Range.
Test sample source: using the flos carthami of Chengdu three batches of lotus pond Market of Chinese Materia Medica as test sample, lot number is respectively
S201810003, S201810004 and S201810005；
Reference substance source: with Kaempferol-O- rutinoside (also known as " Nicotifiorin ", it is limited by the general think of biotechnology share in Chengdu
Company's production, batch number PS010120, purity 99.54%) it is used as reference substance；
Involved reagent: methanol, acetonitrile, phosphoric acid, ultrapure water (ultrapure water be mainly used for prepare 0.2% phosphate aqueous solution of mobile phase,
And prepare 70% methanol solution)；
Involved instrument and software: ten a ten thousandth electronic balance of Mei Tele SQP, in good HC-2062 centrifuge,
Agilent1100 HPLC and OpenLAB software.
Embodiment 1
A kind of method of Kaempferol-O- rutinoside content in measurement safflower, uses high performance liquid chromatograph to measure safflower medicine
The content of Kaempferol-O- rutinoside in material, the specific steps are as follows:
A. Kaempferol-O- rutinoside reference substance is weighed, using 70% methanol as solvent, being configured to concentration is respectively 0.0634mg/
Five parts of reference substance solutions of mL, 0.1585mg/mL, 0.317mg/mL, 0.4755 mg/mL and 0.634mg/mL；
B. by through the resulting five parts of reference substance solutions of step A respectively with the sample volume sample introduction of 10 μ L in high performance liquid chromatograph,
Obtain the corresponding chromatogram of each reference substance solution, the condition in high performance liquid chromatograph are as follows:
Chromatographic column: C18 chromatographic column, column length 100mm, internal diameter 4.6mm, filter sizes 3.5um；
Chromatographic column column temperature: 35 DEG C；
Mobile phase: -0.2% phosphate aqueous solution of acetonitrile, the two volume ratio are 15:85；
Flow velocity: 1.0ml/min；
Detection wavelength: 344nm, ultraviolet light；
C. using the concentration of each reference substance solution as abscissa, to correspond to Kaempferol-O- rutinoside chromatographic peak in chromatogram
Peak area is ordinate, draws standard curve, obtains equation of linear regression y=14767.46277x-3.61391, R2=0.99998)
(as shown in Figure 1), simultaneously, it is known that: Kaempferol-O- rutinoside content has good within the scope of 0.0634~0.634mg/mL
Good linear relationship；
D. the safflower test sample 2.0g for being S201810005 to lot number is weighed, it is dry, it smashes as the saffron powder of 60 mesh, adds
70% methanol of 20mL, ultrasonic extraction 60min under the conditions of 60 kHz of frequency and 35 DEG C of temperature, obtains extracting solution；Again by extracting solution into
Row 14000r/min centrifugation, taking supernatant is test solution.By resulting test solution sample introduction in high performance liquid chromatograph
In, then to be measured with the consistent condition of step B, the chromatogram of test solution is obtained, it will wherein Kaempferol-O- rutinose
The peak area of glycosides chromatographic peak is brought into equation of linear regression obtained by step C, and the Kaempferol-O- rue in test sample to be determined is obtained
Glycosides Contents are 0.178%.
Meanwhile obtaining: with signal-to-noise ratio be 3 when, corresponding Kaempferol-O- rutinoside concentration be detection limit, i.e., it is dense
Degree is 0.16ug/mL；When with signal-to-noise ratio being 10, corresponding Kaempferol-O- rutinoside concentration is quantitative limit, i.e. concentration is
0.63ug/mL。
Embodiment 2
The present embodiment surveys Kaempferol-O- rutinoside content in the flos carthami that lot number is S201810005 batch
It is fixed, and parallel sample introduction six times, remaining is with processing method same as Example 1, and the results are shown in Table 1:
Learnt by table 1: the relative standard deviation RSD of this measurement content method is 0.161%, and test method precision meets the requirements.
Embodiment 3
The present embodiment surveys Kaempferol-O- rutinoside content in the flos carthami that lot number is S201810005 batch
It is fixed, and six parts of test solutions of parallel processing, sample introduction is primary respectively, as a result remaining is seen with processing method same as Example 1
Shown in table 2:
Learnt by table 2: the relative standard deviation RSD of this measurement content method is 0.870%, and test method repeatability meets the requirements.
Embodiment 4
The present embodiment surveys Kaempferol-O- rutinoside content in the flos carthami that lot number is S201810005 batch
It is fixed, wherein test sample is subjected to ultrasonic extraction, obtains test solution, and sample introduction measures after 0h, 2h, 4h, 6h and 8h respectively,
Remaining the results are shown in Table shown in 3 with processing method same as Example 1:
As shown in Table 3: the relative standard deviation RSD of this measurement content method is 0.251%, stability of solution symbol in test method
It closes and requires.
Embodiment 5
The present embodiment surveys Kaempferol-O- rutinoside content in the flos carthami that lot number is S201810005 batch
It is fixed, wherein A. uses two different high performance liquid chromatographs, and model is respectively Agilent1100 and Waters2487-
2996, remaining the results are shown in Table shown in 4 with processing method same as Example 1:
B. three different chromatographic columns are used, model is respectively Féraud door Phenomenex Luma C18 (4.6*100mm*
3um), Agilent ZORBAX Eclipse Plus(4.6*100mm*3.5um) and Agilent ZORBAX Eclipse Plus
(4.6*100mm*3.5um), remaining the results are shown in Table shown in 5 with processing method same as Example 1:
From table 4-5: the relative standard deviation RSD of this measurement content method is below 2%, which meets
It is required that.
Embodiment 6
During being measured to the Kaempferol-O- rutinoside content in flos carthami, the present embodiment is to Kaempferol-
O- rutinoside carries out mark-on reclaims, and the result to ensure this method measurement is consistent with true value or reference value.Step includes:
Weighing Kaempferol-O- rutinoside 17.92mg is reference substance, is placed in 10mL volumetric flask, and 70% methanol is added, molten
Scale is solved and is diluted to, shaking up to get concentration is 1.792mg/mL reference substance solution.
Weigh known content flos carthami powder 1.0g be test sample, parallel six parts, and be separately added into gained reference substance
Solution 1mL adds 70% methanol of 10mL, ultrasonic extraction 60min；It then, is 14000r/min progress with revolving speed by extracting solution
Centrifugation is to inject in high performance liquid chromatograph, remaining is with processing side same as Example 1 for test solution with supernatant
Method, measurement calculate the rate of recovery, the results are shown in Table shown in 6:
As shown in Table 6: the Kaempferol-O- rutinoside rate of recovery shows 98.52~100.40% in this measurement content method
There is good accuracy of measurement.
Experimental example 7
Safflower test sample is weighed respectively from three batches, prepares test solution by step D in embodiment 1, it is molten to obtain test sample
Liquid；Then, take concentration efficient for Kaempferol-O- rutinoside reference substance solution and the test solution injection of 0.317mg/mL
Liquid chromatograph obtains chromatogram (as shown in Figure 2), calculates Kaempferol-O- rue with the peak area one point external standard method of chromatographic peak
Glycosides Contents the results are shown in Table shown in 7:
Claims (9)
1. a kind of method of Kaempferol-O- rutinoside content in measurement safflower, which comprises the steps of:
A. Kaempferol-O- rutinoside reference substance is weighed, uses 70% methanol for solvent, prepares at least five parts with concentration gradient
Reference substance solution；
B. at least five parts of reference substance solution difference sample introductions prepared through step A after measured, are obtained respectively in high performance liquid chromatograph
The corresponding chromatogram of reference substance solution；
C. using the concentration of each reference substance solution as abscissa, the peak of Kaempferol-O- rutinoside chromatographic peak in corresponding chromatogram
Area is ordinate, draws standard curve, obtains equation of linear regression；
D. weigh safflower test sample to be determined uses 70% methanol for solvent after dry smashing, carries out ultrasonic extraction, obtains for examination
Product solution；
E. by the resulting test solution sample introduction of step D in high performance liquid chromatograph, then to be carried out with the consistent condition of step B
Measurement, obtains the chromatogram of test solution；
F. the peak area of test solution chromatographic peak is brought into equation of linear regression obtained in step C, is obtained to be determined red
Kaempferol-O- rutinoside content in flower test sample.
2. the method for Kaempferol-O- rutinoside content in measurement safflower according to claim 1, which is characterized in that
In step, the reference substance solution include concentration be 0.06mg/mL, 0.15mg/mL, 0.30mg/mL, 0.45 mg/mL and
Kaempferol-O- rutinoside the solution of 0.60mg/mL.
3. the method for Kaempferol-O- rutinoside content in measurement safflower according to claim 1, which is characterized in that
In step D, the preparation method of the test solution specifically includes: safflower test sample is weighed, it is dry, and it smashes as 50~80 mesh
Saffron powder, then 70% methanol 10mL is added with every gram of saffron powder, in 15~35 DEG C of conditions of 30~60 kHz of frequency and temperature
Under, 60~90min of ultrasonic extraction obtains extracting solution；Extracting solution is centrifuged, is taken under the conditions of revolving speed is 14000r/min again
Supernatant is test solution.
4. the method for Kaempferol-O- rutinoside content in measurement safflower according to claim 1, which is characterized in that
In step B and E, chromatographic column filler C18, column length 100mm, internal diameter 4.6mm, filter sizes 3.5um.
5. the method for Kaempferol-O- rutinoside content, feature exist in measurement safflower according to claim 1 or 4
In using Detection wavelength for the ultraviolet light of 344nm in step B and E.
6. the method for Kaempferol-O- rutinoside content in measurement safflower according to claim 5, which is characterized in that
In step B and E, chromatogram column temperature is 20~40 DEG C.
7. the method for Kaempferol-O- rutinoside content in measurement safflower according to claim 1, which is characterized in that
In step B and E, the mobile phase is the mixed liquor of acetonitrile and phosphoric acid solution, wherein acetonitrile volume fraction 10~20%, phosphoric acid
Liquor capacity score 80~90%, and phosphoric acid solution is the phosphate aqueous solution that concentration is 0.2%.
8. the method for Kaempferol-O- rutinoside content, feature exist in measurement safflower according to claim 1 or claim 7
In in step B and E, the flow velocity of the mobile phase is 0.8~1.2 mL/min.
9. the method for Kaempferol-O- rutinoside content in measurement safflower according to claim 1, which is characterized in that
In step B and E, the reference substance solution is identical with the sample volume of test solution, and sample volume is 8~12 μ L.
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Application publication date: 20190416 |