CN108414630A - The multicomponent content assaying method of Ramulus Taxilli - Google Patents
The multicomponent content assaying method of Ramulus Taxilli Download PDFInfo
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- CN108414630A CN108414630A CN201810097439.9A CN201810097439A CN108414630A CN 108414630 A CN108414630 A CN 108414630A CN 201810097439 A CN201810097439 A CN 201810097439A CN 108414630 A CN108414630 A CN 108414630A
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention belongs to technical field of chemistry, and in particular to the multicomponent content assaying method of Ramulus Taxilli.The multicomponent content assaying method is to carry out liquid chromatogram assay under same chromatographic condition, while to 3 kinds of chemical compositions of Quercetin, quercitin, avicularins contained by Ramulus Taxilli.The assay method of the present invention utilizes high performance liquid chromatography (HPLC), the content of Quercetin in Ramulus Taxilli, quercitin He 3 kinds of active ingredient of avicularin is measured simultaneously, the detection method is simple, accurate, favorable reproducibility, can be thoroughly evaluating and controls Ramulus Taxilli quality of medicinal material and provide foundation.
Description
【Technical field】
The present invention relates to technical field of chemistry, and in particular to the multicomponent content assaying method of Ramulus Taxilli.
【Background technology】
Ramulus Taxilli is the drying stem and branch with leaf of Loranthaceae plant Ramulus Taxilli Taxillus chinensis (DC.) Danse.
Winter to secondary spring is tapped, and removes thick stem, segment is dry, or drying after steaming.Ramulus Taxilli be used as medicine first recorded in《Sheng Nong's herbal classic》, name
" parasitic on mulberry ", is included in top grade.《Mingyi Bielu》Cloud：" creeping plant, raw to expand on the paddy mulberry tree of agriculture river, March 3 adopted cauline leaf, cloudy
It is dry.”《Tang materia medica》It carries：" on the trees such as this more raw Mongolian oaks, beech, willow, bigcatkin willow, maple, son is yellow, as large as small Chinese date.Only Guo Zhou has on mulberry
Person, sub- juice is very viscous, and core is big like red bean；Leaf is thick without negative and positive, such as thin willow leaf；Late stem thickness is short.People from Jiangnan Xiang Cheng is with for teasel root, very not
It is related.And the parasitism real beginning in September is ripe and yellow.”《Another name for Sichuan Province book on Chinese herbal medicine》Cloud：" there are parasitism, cauline leaf simultaneously similar by Zhu Shuduo." and cloud：" leaf is such as
Tangerine and it is thick soft, stem such as Chinese scholartree and fertilizer is crisp, the present has everywhere.The Fan family only must person on mulberry, it is so non-to be difficult to not, to break stem and regard it from adopting,
It is to test with color depth Huang person.《Figure is through book on Chinese herbal medicine》Leaf is thick wealthy like rough gentian, and stem is short like chicken feet, makees tree-like.March, flower in April, yellow redness of the skin or complexion,
June, knot yellow green in July, such as red bean, with juice, sticky person is good.", Ramulus Taxilli has as China's tradition parts of generic medicinal plants
Filling liver kidney, wind-damp dispelling, strengthening the bones and muscles, tocolysis and other effects.
In China, Loranthaceae plant is widely used as medicinal material, in addition to《Pharmacopeia》The Ramulus Taxilli recorded and mistletoe
Outside, according to statistics, Loranthaceae plant at least further includes having Scurrula Parasitica L Scurrula parasitica during actual circulation
L., blunt fruit parasitism Taxillus nigrans (Hance) Danser of hair leaf, the blunt fruit parasitism Taxillus delavayi of willow leaf
(Van Tiegh.) Danser etc., it is even more up to 39 kinds to be used as medicinal material in civil Loranthaceae plant, more complicated.I.e.
Make to belong to《Pharmacopeia》The drug effect of the Ramulus Taxilli recorded, different host sources Ramulus Taxilli is all different.
It is only single to investigate containing for Quercetin and/or quercitin currently, fairly simple for the quality control of Ramulus Taxilli medicinal material
Amount, cannot clearly evaluate the quality of Ramulus Taxilli medicinal material, such as document comprehensively《The research of Ramulus Taxilli quality standard》(quotient is beautiful etc.,
《China Health industry》, 2015,12 (27)：The content that HPLC methods measure Quercetin in Ramulus Taxilli 126-128) is disclosed, only
Ramulus Taxilli and mistletoe can be distinguished.Document《RP-HPLC methods measure carrying for quercitin and quercetin content in Ramulus Taxilli
Method is taken to compare》(Su Benwei etc.,《Guangxi traditional Chinese medicine》,2012,35(4):It 53-55) discloses while measuring quercitrin in Ramulus Taxilli
The method of glycosides and quercetin content, is merely capable of detection two ingredients of quercitin and Quercetin, and different host sources Ramulus Taxilli
Due to the difference of Qi avicularin content, curative effect difference in clinical application.
Therefore, if plurality of active ingredients in Ramulus Taxilli can be measured simultaneously, such as Quercetin, 3 kinds of quercitin, avicularins are effectively
The content of ingredient more can more comprehensively and accurately evaluate the quality of Ramulus Taxilli, ensure that its clinical efficacy.
【Invention content】
In order to solve the above technical problem, the present invention provides the multicomponent content assaying method of Ramulus Taxilli, the measurement
Method utilizes high performance liquid chromatography (HPLC), while measuring Quercetin in Ramulus Taxilli, quercitin He 3 kinds of active ingredients of avicularin
Content, the detection method is simple, accurate, favorable reproducibility, can be thoroughly evaluating and controls Ramulus Taxilli quality of medicinal material and provide foundation.
To achieve the goals above, the technical solution adopted by the present invention is as follows：
The multicomponent content assaying method of Ramulus Taxilli, the multicomponent content assaying method be under same chromatographic condition,
Liquid chromatogram assay is carried out to 3 kinds of chemical compositions of Quercetin, quercitin, avicularins contained by Ramulus Taxilli simultaneously.
Further, the multicomponent content assaying method carries out in the steps below：
(1) chromatographic condition：Chromatographic column is C18Column；Mobile phase is acetonitrile：Water=22:78；Flow velocity is 1.0ml/min；Column temperature
It is 30~40 DEG C；Detection wavelength is 254nm；
(2) preparation of reference substance solution：Quercetin, quercitin, avicularin reference substance are weighed respectively, and step (1) institute is added
The flowing phased soln stated, obtains reference substance solution；
(3) preparation of test solution：Ramulus Taxilli medicinal powder is taken, adds methanol and 5% hydrochloric acid solution, ultrasonic wave extraction,
Supernatant is taken, test solution is obtained；
(4) measuring method：According to step (1) chromatographic condition measure to get.
Further, step (1) chromatographic condition is：
Instrument is Agilent1260 high performance liquid chromatographs；Chromatographic column is AgilentC18Liquid-phase chromatographic column, chromatographic column rule
Lattice be 150 × 4.6mm, 5 μm；Mobile phase is acetonitrile：Water=22：78；Flow velocity is 1.0ml/min；Column temperature is 35 DEG C；Detection wavelength
For 254nm.
Further, the step (2) reference substance solution is prepared as：Take Quercetin reference substance appropriate, it is accurately weighed,
It is placed in 25ml measuring bottles, adds Quercetin reference substance solution of the mobile phase dissolved dilution to scale to get a concentration of 0.44mg/ml；
Take quercitin reference substance appropriate, it is accurately weighed, it is placed in 25ml measuring bottles, adds mobile phase dissolved dilution to scale to get a concentration of
The quercitin reference substance solution of 0.48mg/ml；Take avicularin reference substance appropriate, it is accurately weighed, it is placed in 25ml measuring bottles, adds flowing
Phased soln is diluted to scale to get the avicularin reference substance solution of a concentration of 0.53mg/ml.
Further, the step (3) test solution is prepared as：Ramulus Taxilli medicinal powder 2.0g, precision is taken to claim
It is fixed, add methanol 20ml, adds 5% hydrochloric acid 10ml, weighed weight, ultrasonic wave extraction lets cool, supplies weight with methanol, shake up,
It stands, takes 1ml to centrifuge, take supernatant to get test solution.
In conclusion by adopting the above-described technical solution, the beneficial effects of the invention are as follows：
The assay method of the present invention realizes single injected sampling, while measuring Quercetin in Ramulus Taxilli, quercitin He avicularin 3
The content of kind of active ingredient, chromatographic peak peak shape is good, and separating degree is higher, can accurately measure in Ramulus Taxilli medicinal material 3 kinds effectively at
The content divided, shortens detection time, improves work efficiency.This method is easy to operate, and precision, reproducibility and stability are good
It is good, foundation can be provided for thoroughly evaluating and control Ramulus Taxilli quality of medicinal material.
【Description of the drawings】
Fig. 1：Blank solvent high-efficient liquid phase chromatogram.
Fig. 2：Avicularin reference substance high-efficient liquid phase chromatogram.
Fig. 3：Quercitin reference substance high-efficient liquid phase chromatogram.
Fig. 4：Quercetin reference substance high-efficient liquid phase chromatogram.
Fig. 5：Test solution high-efficient liquid phase chromatogram (254nm).Chromatographic peak corresponds to respectively：A Wei avicularins, B are quercitrin
Glycosides, C are Quercetin.
【Specific implementation mode】
Embodiment 1：The high performance liquid chromatography of Quercetin, quercitin contained in Ramulus Taxilli He 3 kinds of active ingredient of avicularin
Assay.
1. instrument and reagent
1260 high performance liquid chromatographs of Agilent；Agilent C18 liquid-phase chromatographic columns (150 × 4.6mm, 5 μm)；Germany
Sai Duolisi companies SQP electronic analytical balances；Millipore simplicity-185 Superpure water machines；City of Kunshan's ultrasonic instrument has
Limit company KQ5200E ultrasonic cleaners；L600 low speed centrifuges；Hunan Xiang Yi Laboratory Instruments development corporation, Ltd. TGL-
20M table-type high-speed refrigerated centrifuges；DHG-9146A electric heating constant-temperature blowing drying boxes；HWS-26 electric-heated thermostatic water baths；Acetonitrile
(chromatographically pure, Sinopharm Chemical Reagent Co., Ltd.)；Methanol (analyzes pure, Sinopharm Chemical Reagent Co., Ltd.)；It is ultrapure
Water；Other reagents are that analysis is pure.10 batches, the Ramulus Taxilli sample in different host plant sources, each sample specifying information such as 10 institute of table
Show, is that the blunt fruit parasitism of Loranthaceae belongs to wide parasitic through pharmaceutical college of Guangxi University of Chinese Medicine Li Yonghua researcher identification
The stem and branch with leaf of Taxilluschinensis (DC.) Danser plant dryings.Quercetin reference substance (Sichuan Province Wei Keqi biologies
Science and Technology Ltd., lot number：Wkq16063005, purity >=98%)；(Sichuan Province's Wei Keqi biotechnologies have quercitin reference substance
Limit company, lot number：Wkq16080402, purity >=98%)；Avicularin reference substance (the limited public affairs of Sichuan Province's Wei Keqi biotechnologies
Department, lot number：Wkq16050902, purity >=98%).
2. chromatographic condition and system suitability test
Chromatographic column：Agilent C18 liquid-phase chromatographic columns (column specification 150 × 4.6mm, 5 μm)；
Mobile phase：Acetonitrile：Water=22:78；
Flow velocity：1.0ml/min；
Column temperature：35℃；
Detection wavelength：254nm.
3. the preparation of test solution
The preparation of 3.1 reference substance solutions
Take Quercetin reference substance appropriate, it is accurately weighed, it is placed in 25ml measuring bottles, adds mobile phase dissolved dilution to scale, i.e.,
Obtain the Quercetin reference substance solution of a concentration of 0.44mg/ml；Take quercitin reference substance appropriate, it is accurately weighed, it is placed in 25ml measuring bottles
In, add quercitin reference substance solution of the mobile phase dissolved dilution to scale to get a concentration of 0.48mg/ml；Quercitin is taken to compare
Appropriate product, it is accurately weighed, it is placed in 25ml measuring bottles, adds mobile phase dissolved dilution to scale to get the flat of a concentration of 0.53mg/ml
Store glycosides reference substance solution.
The preparation of 3.2 test solutions
Ramulus Taxilli medicinal powder (crossing No. three sieves) 2.0g is taken, it is in duplicate, accurately weighed, 100ml tool plug tapers are set respectively
In bottle, adding methanol 20ml, add 5% hydrochloric acid 10ml, weighed weight, ultrasonic 2.5h lets cool, supplies weight with methanol, shake up,
It stands, takes 1ml to centrifuge 15min (rotating speed 13000rpm), take supernatant to get test solution.
The preparation of 3.3 blank solutions
With mobile phase acetonitrile：Water=22:78 be blank solution.
4. system suitability test
It is accurate respectively to draw blank solution, reference substance solution, each 10 μ l of test solution, high performance liquid chromatograph is injected,
Chromatogram is recorded, as shown in Figures 1 to 5.The result shows that：Blank solvent is noiseless.
5. linear relationship is investigated
It is accurate respectively to draw Quercetin reference substance solution (0.44mg/mL) 0.5 μ l, 1 μ l, 2 μ l, 3 μ l, 5 μ l, 8 μ l, 10 μ
L injects liquid chromatograph, measures peak area by above-mentioned chromatographic condition, is returned to sample size C (μ g) with peak area A, as a result
As shown in table 1, calibration curve equation is obtained：A=3461.9C+16.714, R=1.Show Quercetin in 0.22-4.4 μ g ranges
It is in good linear relationship with peak area.
1 Quercetin linear relationship of table investigates data result
It is accurate respectively to draw quercitin reference substance solution (0.48mg/mL) 0.2 μ l, 0.5 μ l, 1 μ l, 2 μ l, 3 μ l, 5 μ l, 8 μ
L injects liquid chromatograph, measures peak area by above-mentioned chromatographic condition, is returned to sample size C (μ g) with peak area A, as a result
As shown in table 2, calibration curve equation is obtained：A=3011.5C+104.23；R=0.9996.Show quercitin in 0.096-3.84 μ g
With peak area in good linear relationship in range.
2 quercitin linear relationship of table investigates data result
It is accurate respectively to draw avicularin reference substance solution (0.53mg/mL) 0.2 μ l, 0.5 μ l, 1 μ l, 2 μ l, 3 μ l, 5 μ l, 8 μ
L, 10 μ l inject liquid chromatograph, measure peak area by above-mentioned chromatographic condition, are returned to sample size C (μ g) with peak area A
Return, the results are shown in Table 3, obtains calibration curve equation：A=2122.1C+10.716；R=0.9999.Biao Ming avicularins exist
With peak area in good linear relationship in 0.106-5.3 μ g ranges.
3 avicularin linear relationship of table investigates data result
6. precision test
People face tree host source Ramulus Taxilli medicinal powder (crossing No. three sieves) 2.0g is taken, it is accurately weighed, by 3.2 lower test samples
Preparation method carries out sample preparation, and precision draws 10 μ l, injects liquid chromatograph, and peak area is measured by above-mentioned chromatographic condition, will
Peak area substitutes into linear equation and carries out assay, and continuous sample introduction 6 times, the results are shown in Table 4：Quercetin reference substance peak area
RSD values are 0.61%；Quercitin reference substance peak area RSD values are 0.53%；Avicularin peak area RSD values are 1.35%.
4 precision test data result (n=2) of table
7. stability test
People face tree host source Ramulus Taxilli medicinal powder (crossing No. three sieves) 2.0g is taken, it is accurately weighed, by 3.2 lower test samples
Preparation method carries out sample preparation, and respectively in 0,1,2,5,10,12 hour 10 μ l of accurate sample introduction, peak is measured by above-mentioned chromatographic condition
Area, the results are shown in Table 5：Quercetin reference substance peak area RSD values are 2.24% (n=6)；Quercitin reference substance peak area
RSD values are 1.87% (n=6)；Avicularin peak area RSD values are 2.66% (n=6).Show test solution in 12 hours
Stablize.
5 stability test data result (n=2) of table
8. repetitive test
Take people face tree host source Ramulus Taxilli medicinal powder (crossing No. three sieves) 2.0g, accurately weighed, 6 parts of a formula, by 3.2
Lower test sample preparation method carries out sample preparation, and precision draws 10 μ l, injects liquid chromatograph, and peak is measured by above-mentioned chromatographic condition
Peak area is substituted into linear equation and carries out assay by area, specific as shown in table 6：Quercetin reference substance content RSD values are
2.72%；Quercitin reference substance content RSD values are 2.76%；Avicularin content RSD values are 2.89%.Show this method reproducibility
Well.
6 repetitive test data result (n=2) of table
9. being loaded recovery test
Take Ramulus Taxilli medicinal material sample powder (crossing No. three sieves) 1.0g of known content, accurately weighed, 6 parts of a formula, by 3.2
Lower test sample preparation method carries out sample preparation, and precision draws 10 μ l, injects liquid chromatograph, and peak is measured by above-mentioned chromatographic condition
Peak area is substituted into linear equation and carries out assay by area, and specific data are as shown in table 7 to table 9：Quercetin reference substance
Average recovery rate is that 99.32%, RSD values are 2.36%；The average recovery rate of quercitin reference substance is that 100.53%, RSD values are
2.77%；The average recovery rate of avicularin reference substance is that 98.96%, RSD values are 1.93%.
7 Quercetin of table is loaded recovery test data result (n=6)
8 quercitin of table is loaded recovery test data result (n=6)
9 avicularin of table is loaded recovery test data result (n=6)
10. different hosts source Ramulus Taxilli assay
Ramulus Taxilli medicinal powder (the crossing No. three sieves) 2.0g in variant host source in table 10 is taken respectively, it is accurately weighed, one
Two parts of formula carries out sample preparation by 3.2 lower test sample preparation methods, and precision draws 10 μ l, liquid chromatograph is injected, by above-mentioned
Chromatographic condition measures peak area, and peak area is substituted into linear equation and carries out assay, specific data are as shown in table 11.
The Ramulus Taxilli sample collection information table of the different host plants of table 10
The different host plants of table 11 source Ramulus Taxilli sample size determination data table
As it can be seen that Quercetin, quercitin and avicularin content are different in the Ramulus Taxilli of different host plants source, Er Qiehan
It is all very big to measure difference.Host plant has larger impact for the quality of Ramulus Taxilli quality, should reinforce Ramulus Taxilli quality control, focuses on
The plantation and utilization of genuine Ramulus Taxilli.Therefore, the selection and breeding of kind to be carried out in conjunction with different host plant sources.
Above description is the detailed description for the present invention preferably possible embodiments, but embodiment is not limited to this hair
Bright patent claim, it is all the present invention suggested by technical spirit under completed same changes or modifications change, should all belong to
In the covered the scope of the claims of the present invention.
Claims (5)
1. the multicomponent content assaying method of Ramulus Taxilli, which is characterized in that the multicomponent content assaying method is with of the same colour
Under spectral condition, while liquid chromatogram is carried out containing measurement to 3 kinds of chemical compositions of Quercetin, quercitin, avicularin contained by Ramulus Taxilli
It is fixed.
2. the multicomponent content assaying method of Ramulus Taxilli according to claim 1, which is characterized in that the multicomponent content
Assay method carries out in the steps below：
(1) chromatographic condition：Chromatographic column is C18 columns；Mobile phase is acetonitrile：Water=22:78；Flow velocity is 1.0ml/min；Column temperature is 30
~40 DEG C；Detection wavelength is 254nm；
(2) preparation of reference substance solution：Quercetin, quercitin, avicularin reference substance are weighed respectively, are added described in step (1)
Phased soln is flowed, reference substance solution is obtained；
(3) preparation of test solution：Ramulus Taxilli medicinal powder is taken, methanol and 5% hydrochloric acid solution, ultrasonic wave extraction is added to take
Clear liquid obtains test solution；
(4) measuring method：According to step (1) chromatographic condition measure to get.
3. the multicomponent content assaying method of Ramulus Taxilli according to claim 2, which is characterized in that step (1) color
Spectral condition is：
Instrument is Agilent1260 high performance liquid chromatographs；Chromatographic column is AgilentC18 liquid-phase chromatographic columns, and chromatographic column specification is
150 × 4.6mm, 5 μm；Mobile phase is acetonitrile：Water=22：78；Flow velocity is 1.0ml/min；Column temperature is 35 DEG C；Detection wavelength is
254nm。
4. the multicomponent content assaying method of Ramulus Taxilli according to claim 2, which is characterized in that the step (2)
Reference substance solution is prepared as：Take Quercetin reference substance appropriate, it is accurately weighed, it is placed in 25ml measuring bottles, adds flowing phased soln dilute
Quercetin reference substance solution to get a concentration of 0.44mg/ml is released to scale；Take quercitin reference substance appropriate, it is accurately weighed, it sets
In 25ml measuring bottles, add quercitin reference substance solution of the mobile phase dissolved dilution to scale to get a concentration of 0.48mg/ml；It takes
Avicularin reference substance is appropriate, accurately weighed, is placed in 25ml measuring bottles, adds mobile phase dissolved dilution to scale to get a concentration of
The avicularin reference substance solution of 0.53mg/ml.
5. the multicomponent content assaying method of Ramulus Taxilli according to claim 2, which is characterized in that the step (3)
Test solution is prepared as：Ramulus Taxilli medicinal powder 2.0g is taken, it is accurately weighed, add methanol 20ml, adds 5% hydrochloric acid
10ml, weighed weight, ultrasonic wave extraction are let cool, and are supplied weight with methanol, are shaken up, and are stood, are taken 1ml to centrifuge, take supernatant, i.e.,
Obtain test solution.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112903868A (en) * | 2021-02-04 | 2021-06-04 | 中山市中医院 | Method for measuring contents of various chemical components in compound guava preparation |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112903868A (en) * | 2021-02-04 | 2021-06-04 | 中山市中医院 | Method for measuring contents of various chemical components in compound guava preparation |
| CN112903868B (en) * | 2021-02-04 | 2022-06-28 | 中山市中医院 | Method for measuring contents of various chemical components in compound guava preparation |
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