CN107561196A - HPLC methods that are a kind of while determining 7 kinds of component contents in Blumea balsamifera - Google Patents
HPLC methods that are a kind of while determining 7 kinds of component contents in Blumea balsamifera Download PDFInfo
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Abstract
The invention discloses a kind of while determine the HPLC methods of 7 kinds of component contents in Blumea balsamifera; 7 kinds of caffeoyl quinic acid constituents of this method measure are 5 O caffeoyl quinic acids, 3 O caffeoyl quinic acids, 4 O caffeoyl quinic acids, 1; 3 O DCQs, 3; 4 O DCQs, 3; 5 O DCQs and 4; 5 O DCQs, its determination step are as follows：(1) preparation of standard liquid；(2) preparation of need testing solution；The foundation of (3) 7 kinds of caffeoyl quinic acid constituents content measuring standard curves；(4) 7 in sample in caffeoyl quinic acid constituents content measure.The present invention is easy to operate, and precision, repeatability, stability are good, and the degree of accuracy is high, can determine the content of 7 kinds of caffeoyl quinic acid constituents in Blumea balsamifera simultaneously, have larger practical value.
Description
Technical field
The present invention relates to a kind of content assaying method of 7 kinds of caffeoyl quinic acid constituents in Blumea balsamifera, in belonging to
Medicine technical field.
Background technology
Blumea balsamifera Blumea balsamifera (L.) DC is composite family Blumea balsamifera platymiscium, be distributed widely in Guizhou, Guangdong,
The ground such as Hainan, mainly contain the compounds such as volatile oil, flavonoids.Its medicinal part is its leaf and spray, have Medicine for the treatment of rheumatism and arthritis,
Warming middle energizer to stop diarrhea and other effects, for treating the treatment of the diseases such as anemofrigid cold, arthralgia pain due to rheumatism, the pain of injury caused by falling and tumbling.Research shows that Blumea balsamifera has
There are obvious antiphlogistic effects, available for treating rheumathritis.
Research is found, except flavonoid beyond the region of objective existence, caffeoyl quinic acid class compound is main non-for Blumea balsamifera medicinal material
Volatile ingredient, and have document report caffeoyl quinic acid class compound that there are the multiple biological activities such as anti-inflammatory, antiviral, can
As the index components of Blumea balsamifera quality of medicinal material control, but there is not the quality controling research report of the constituents in Blumea balsamifera at present
Road.Therefore, establish the content that reliable, accurate content assaying method determines the constituents has important show to quality of medicinal material control
Sincere justice.
The content of the invention
It is an object of the invention to provide receive this 7 kinds of the content assaying methods of 7 kinds of caffeoyl quinic acid constituents in perfume (or spice)
Composition is：5-O- caffeoyl quinic acids, 3-O- caffeoyl quinic acids, 4-O- caffeoyl quinic acids, the coffees of 1,3-O- bis-
Acyl group quininic acid, 3,4-O- DCQs, 3,5-O- DCQs and the coffee acyl Kuis of 4,5-O- bis-
Peaceful acid.
Technical scheme comprises the following steps：
S1, weigh 5-O- caffeoyl quinic acids, 3-O- caffeoyl quinic acids, 4-O- caffeoyl quinic acids, 1,3-
O- DCQs, 3,4-O- DCQs, 3,5-O- DCQs and the coffees of 4,5-O- bis-
Coffee acyl group quininic acid reference substance is with being prepared each reference substance stock solution after methanol constant volume；
S2, weigh Blumea balsamifera medicinal powder and add the weighed weight of methanol, heating water bath backflow, let cool, then weighed weight, use
Methanol with concentration supplies the weight of less loss, shakes up, and stands, and takes supernatant that need testing solution is prepared after centrifuging；
S3, various concentrations are diluted to by each reference substance stock solution, pass through HPLC according to the standard curve chromatographic condition of setting
Method establishes 7 kinds of caffeoyl quinic acid constituents content measuring standard curves, calculates each component linear regression equation；
S4, the need testing solution of preparation is measured according to standard curve chromatographic condition, utilizes equation of linear regression, meter
Calculate the content of 7 kinds of caffeoyl quinic acid constituents in sample.
Wherein, the chromatographic condition set described in S3 as：Using the chromatographic column of two series connection；Using acetonitrile as mobile phase A,
0.1% phosphoric acid water is Mobile phase B, carries out gradient elution, and gradient is：0~12 minute, mobile phase A by 8% gradual change extremely
13.6%；12~17 minutes, mobile phase A was by 13.6% gradual change to 16%；17~29 minutes, mobile phase A by 16% gradual change extremely
20%；29~44 minutes, mobile phase A was by 20% gradual change to 24%；Detection wavelength is 325nm；Flow velocity 0.8ml/min；Sample size
For 10 μ l.
Further, need testing solution was heated to reflux the time as 2 hours in preparing.
Further, the concentration of methanol is 25% during prepared by need testing solution.
The invention has the advantages that：
1 while 7 kinds of caffeoyl quinic acid constituents in Blumea balsamifera are determined, detection time is 44 minutes, detection efficiency
Height, method are easy, reliable.
2nd, 7 kinds of caffeoyl quinic acid constituents are preferable in certain limit class linear relationship.
3rd, the content of 7 kinds of caffeoyl quinic acid constituents in Blumea balsamifera can be accurately and rapidly determined, is Blumea balsamifera quality
Certain basis is established in control.
Brief description of the drawings
Fig. 1 is mixed reference substance solution chromatogram, in figure：1：5-O- caffeoyl quinic acids, 2：3-O- coffee acyl Kuis
Peaceful acid, 3：4-O- caffeoyl quinic acids, 4：1,3-O- DCQs；5：3,4-O- DCQs；
6：3,5-O- DCQs；7：4,5-O- DCQs；
Fig. 2 is Blumea balsamifera test sample chromatogram.
Embodiment
The invention will be further described with reference to the accompanying drawings and examples, but not as any limitation of the invention.
Embodiment 1：
5-O- caffeoyl quinic acids in Blumea balsamifera, 3-O- coffee acyl quinines are determined the invention provides a kind of simultaneously
Acid, 4-O- caffeoyl quinic acids, 1,3-O- DCQs, 3,4-O- DCQs, 3,5-O- bis-
The HPLC methods of 7 kinds of caffeoyl quinic acid constituents of caffeoyl quinic acid and 4,5-O- DCQ, specifically
Implementation steps are as follows：
1st, instrument and reagent
The high performance liquid chromatographs of UltiMate 3000 (Thermo Fisher Scientific), including system controller,
Infusion pump, degassing component, low pressure gradient component, automatic sampler, column oven, temperature control sample room, UV-VIS detectors and
Chromeleon Data Processing in Chromatography Workstation.EL204 types electronic balance (plum Teller-support benefit instrument Shanghai Co., Ltd).Bake
Graceful tabletop refrigerated centrifuge (U.S.).WP-UP-IV-20 types ultrapure water machine (Sichuan Water Technology Development Co., Ltd.).
Standard items 5-O- caffeoyl quinic acids (Wei Keqi bio tech ltd of Sichuan Province, purity >=98%, batch
Number：150426), 3-O- caffeoyl quinic acids (Wei Keqi bio tech ltd of Sichuan Province, purity >=98%, lot number：
151123), 4-O- caffeoyl quinic acids (Wei Keqi bio tech ltd of Sichuan Province, purity >=98%, lot number：
150922), 1,3-O- DCQs (solid preparation of traditional Chinese medicine manufacturing technology National Engineering Research Centre, purity >=
98%, lot number：D15-20130213), 3,4-O- DCQs (Chengdu Inst. of Biology, Chinese Academy of Sciences, purity
99.05%%, lot number：MUST-16031612), 3,5-O- DCQs (Chinese Academy of Sciences's Chengdu biological study
Institute, purity 98.82%, lot number：MUST-16031611), 4,5-O- DCQs (Chinese Academy of Sciences's Chengdu biology
Research institute, purity 99.84%, lot number：MUST-16031613).Acetonitrile, methanol are that (Chinese medicines group chemical reagent is limited for chromatographically pure
Company)；Remaining reagent is pure to analyze.Blumea balsamifera medicinal material picks up from Guizhou Province Affected It In Qianxinan Wangmo County Zhe Xiang towns Xin Zhai villages Blumea balsamifera kind
Plant Demonstration Base
2nd, method
The preparation of 2.1 standard liquids：Weigh 5-O- caffeoyl quinic acids, 3-O- caffeoyl quinic acids, 4-O- coffees
Acyl group quininic acid, 1,3-O- DCQs, 3,4-O- DCQs, the coffee acyl quinines of 3,5-O- bis-
Acid and 4,5-O- DCQs reference substance each about 10mg, it is accurately weighed, it is respectively placed in 10ml volumetric flasks, uses methanol
Scale is settled to, is shaken up, is obtained each according to product storing solution；
The preparation of 2.2 need testing solutions：Precision weighs Blumea balsamifera medicinal powder (crossing 40 mesh sieves) about 0.5g, is placed in round bottom burning
In bottle, precision adds 25% methanol 25ml, and weighed weight, heating water bath flows back 2 hours, let cool, then weighed weight, with 25%
Methanol supplies the weight of less loss, shakes up, and stands, and takes supernatant to put 12000rpm in centrifuge tube and centrifuges 5 minutes, takes supernatant i.e.
；
The foundation of 2.3 7 kinds of caffeoyl quinic acid constituents content measuring standard curves：Connected using two
Chromolith Performance RP-18e (100mm × 4.6mm, Merck, Darmstadt, Germany) chromatographic column；With
Acetonitrile is mobile phase A, and 0.1% phosphoric acid water is Mobile phase B, carries out gradient elution, and gradient is：0~12 minute, mobile phase A
By 8% gradual change to 13.6%；12~17 minutes, mobile phase A was by 13.6% gradual change to 16%；17~29 minutes, mobile phase A by
16% gradual change is to 20%；29~44 minutes, mobile phase A was by 20% gradual change to 24%；Detection wavelength is 325nm；Flow velocity 0.8ml/
min；Sample size is 10 μ l；Each reference substance stock solution is diluted to various concentrations, establishes standard curve, each component linear is calculated and returns
Return equation；
The measure of 7 kinds of caffeoyl quinic acid constituents contents in 2.4 Blumea balsamiferas：According to above-mentioned standard curve chromatographic condition
The need testing solution of preparation is measured, using equation of linear regression, calculates 7 kinds of caffeoyl quinic acid constituents in sample
Content.
3 results
The ownership of 3.1 chromatographic peaks
1~No. 7 peak is respectively Fig. 1 Plays product from left to right：5-O- caffeoyl quinic acids (7.93min), 3-O- coffees
Coffee acyl group quininic acid (11.49min), 4-O- caffeoyl quinic acids (12.56min), 1,3-O- DCQs
(18.18min), 3,4-O- DCQs (29.89min), 3,5-O- DCQs (31.13min),
4,5-O- DCQs (35.11min), each standard items separating degree reach requirement.
3.2 linear relationships are investigated
Respectively by the μ l sample introductions of 7 kinds of standard mixed standard solutions 10 of various concentrations, using peak area as ordinate, standard items are dense
Spend for abscissa, calculate the equation of linear regression of each composition, be shown in Table 1.
Table 1：7 kinds of component linear regression equations and scope
| Standard items title | Regression equation | R2 | The range of linearity (mg/L) |
| 5-O- caffeoyl quinic acids | Y=0.4037X+0.2735 | 0.9993 | 0.9440~11.33 |
| 3-O- caffeoyl quinic acids | Y=0.5723X+0.6116 | 0.9997 | 5.020~60.24 |
| 4-O- caffeoyl quinic acids | Y=0.4608X+0.0952 | 0.9996 | 0.9800~11.76 |
| 1,3-O- DCQs | Y=0.5599X+0.4432 | 0.9995 | 1.912~22.94 |
| 3,4-O- DCQs | Y=0.7072X+0.047 | 0.9999 | 3.597~43.17 |
| 3,5-O- DCQs | Y=0.8165X+1.7352 | 0.9999 | 34.93~149.2 |
| 4,5-O- DCQs | Y=0.7576X+0.0873 | 0.9999 | 9.720~116.6 |
3.3 precision test
Same need testing solution repeats sample introduction 6 times, 5-O- caffeoyl quinic acids, 3-O- caffeoyl quinic acids, 4-O-
Caffeoyl quinic acid, 1,3-O- DCQs, 3,4-O- DCQs, the coffee acyls of 3,5-O- bis-
Quininic acid and the RSD of 4,5-O- DCQ contents are respectively 2.7%, 1.3%, 2.0%, 0.2%, 2.6%,
1.3%th, 1.7%, show that instrument precision is good.
3.4 stability test
Same test sample is respectively in 0,2,4,8,12,24 hour sample introduction, 5-O- caffeoyl quinic acids, 3-O- coffee acyls
Quininic acid, 4-O- caffeoyl quinic acids, 1,3-O- DCQs, 3,4-O- DCQs, 3,5-
O- DCQs and the RSD of 4,5-O- DCQ contents are respectively 2.3%, 2.8%, 2.1%,
0.2%th, 2.6%, 0.9%, 1.0%, show that need testing solution is stable in 24 hours.
3.5 replica test
Same batch of sample is taken, 6 parts are prepared by need testing solution preparation method is parallel, sample introduction analysis respectively, 5-O- caffeoyls
Base quininic acid, 3-O- caffeoyl quinic acids, 4-O- caffeoyl quinic acids, 1,3-O- DCQs, 3,4-O-
The RSD difference of DCQ, 3,5-O- DCQs and 4,5-O- DCQ contents
For 1.9%, 1.3%, 1.4%, 0.85%, 0.21%, 1.1%, 1.4%, show that this method is reproducible.
3.6 recovery test
According to above-mentioned liquid phase chromatogram condition, a certain amount of standard liquid is added in the sample solution of known content, measure 7
Kind component content.5-O- caffeoyl quinic acids, 3-O- caffeoyl quinic acids, 4-O- caffeoyl quinic acids, 1,3-O- bis-
Caffeoyl quinic acid, 3,4-O- DCQs, 3,5-O- DCQs and the caffeoyls of 4,5-O- bis-
Base quinine acid recovering rate is respectively 97.96%, 100.7%, 102.1%, 103.0%, 101.9%, 99.56%, 96.94%,
RSD is respectively 1.1%, 2.8%, 1.7%, 1.5%, 2.0%, 2.7%, 1.7%, shows that this method degree of accuracy is good.
3.7 sample sizes determine
5 batch Blumea balsamifera sample solutions of environmental Different Harvesting Time of the same race are taken by above-mentioned high-efficient liquid phase chromatogram condition sample introduction
Analysis, the content of 7 kinds of caffeoyl quinic acid constituents is calculated according to equation of linear regression, the results are shown in Table 2.
Table 2：The content of 7 kinds of caffeoyl quinic acid constituents is calculated according to equation of linear regression
Certainly, the above is the concrete application example of the present invention, and the present invention also has other embodiments, all using equivalent
The technical scheme that replacement or equivalent transformation are formed, all falls within protection domain of the presently claimed invention.
Claims (4)
1. HPLC methods that are a kind of while determining 7 kinds of component contents in Blumea balsamifera, 7 kinds of compositions are 7 kinds of caffeoyl quinic acids
Constituents, it is respectively：5-O- caffeoyl quinic acids, 3-O- caffeoyl quinic acids, 4-O- caffeoyl quinic acids, 1,3-O-
DCQ, 3,4-O- DCQs, 3,5-O- DCQs and the coffees of 4,5-O- bis-
Acyl group quininic acid；It is characterized in that this method comprises the following steps：
S1, weigh 5-O- caffeoyl quinic acids, 3-O- caffeoyl quinic acids, 4-O- caffeoyl quinic acids, 1,3-O- bis-
Caffeoyl quinic acid, 3,4-O- DCQs, 3,5-O- DCQs and the caffeoyls of 4,5-O- bis-
Base quininic acid reference substance is with being prepared each reference substance stock solution after methanol constant volume；
S2, weigh Blumea balsamifera medicinal powder and add the weighed weight of methanol, heating water bath backflow, let cool, then weighed weight, with dense
The methanol of degree supplies the weight of less loss, shakes up, and stands, and takes supernatant that need testing solution is prepared after centrifuging；
S3, various concentrations are diluted to by each reference substance stock solution, pass through HPLC methods according to the standard curve chromatographic condition of setting
7 kinds of caffeoyl quinic acid constituents content measuring standard curves are established, calculate each component linear regression equation；
S4, the need testing solution of preparation is measured according to standard curve chromatographic condition, using equation of linear regression, calculates sample
The content of 7 kinds of caffeoyl quinic acid constituents in product.
2. HPLC methods that are according to claim 1 while determining 7 kinds of component contents in Blumea balsamifera, it is characterised in that：S3
Described in the chromatographic condition that sets as：Using the chromatographic column of two series connection；Using acetonitrile as mobile phase A, 0.1% phosphoric acid water is flowing
Phase B, carries out gradient elution, and gradient is：0~12 minute, mobile phase A was by 8% gradual change to 13.6%；12~17 minutes, stream
Dynamic phase A is by 13.6% gradual change to 16%；17~29 minutes, mobile phase A was by 16% gradual change to 20%；29~44 minutes, mobile phase A
By 20% gradual change to 24%；Detection wavelength is 325nm；Flow velocity 0.8ml/min；Sample size is 10 μ l.
3. HPLC methods that are according to claim 1 or 2 while determining 7 kinds of component contents in Blumea balsamifera, it is characterised in that：
Need testing solution was heated to reflux the time as 2 hours in preparing.
4. HPLC methods that are according to claim 1 or 2 while determining 7 kinds of component contents in Blumea balsamifera, it is characterised in that：
The concentration of methanol is 25% in prepared by need testing solution.
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| CN110243988A (en) * | 2019-06-28 | 2019-09-17 | 广西中医药大学 | The method for building up of strong medicine white flower nine inner bright HPLC finger-print |
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| CN113933436A (en) * | 2021-11-03 | 2022-01-14 | 贵州医科大学 | Method for measuring contents of various components in caulis et folium piperis nigri medicinal material |
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| CN108445131A (en) * | 2018-03-19 | 2018-08-24 | 湖南中医药大学 | The detection method of main component in a kind of edible areca-nut |
| CN109856264A (en) * | 2019-01-11 | 2019-06-07 | 遵义医学院 | The detection method of active constituent content in Blumea balsamifera medicinal material |
| CN109856264B (en) * | 2019-01-11 | 2021-09-07 | 遵义医科大学 | Method for detecting content of effective components in blumea balsamifera medicinal material |
| CN110243988A (en) * | 2019-06-28 | 2019-09-17 | 广西中医药大学 | The method for building up of strong medicine white flower nine inner bright HPLC finger-print |
| CN110954622A (en) * | 2019-12-20 | 2020-04-03 | 成都普思检验检测有限公司 | Method for determining content of 1, 3-dicaffeoylquinic acid in inula flower |
| CN111323508A (en) * | 2020-03-23 | 2020-06-23 | 遵义医科大学 | Method for measuring content of 2, 4-dihydroxy-6-methoxyacetophenone |
| CN111323508B (en) * | 2020-03-23 | 2022-08-19 | 遵义医科大学 | Method for determining content of 2, 4-dihydroxy-6-methoxyacetophenone in blumea balsamifera oil |
| CN113933436A (en) * | 2021-11-03 | 2022-01-14 | 贵州医科大学 | Method for measuring contents of various components in caulis et folium piperis nigri medicinal material |
| CN113933436B (en) * | 2021-11-03 | 2022-05-24 | 贵州医科大学 | Method for measuring contents of various components in caulis et folium piperis nigri medicinal material |
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