CN108548884A - The method that HPLC methods detect Rosmarinic acid, carnosic acid and ursolic acid in rosemary alcohol extract - Google Patents
The method that HPLC methods detect Rosmarinic acid, carnosic acid and ursolic acid in rosemary alcohol extract Download PDFInfo
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- CN108548884A CN108548884A CN201810517748.7A CN201810517748A CN108548884A CN 108548884 A CN108548884 A CN 108548884A CN 201810517748 A CN201810517748 A CN 201810517748A CN 108548884 A CN108548884 A CN 108548884A
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- acid
- carnosic
- rosmarinic
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Abstract
The present invention provides a kind of methods that HPLC detects Rosmarinic acid, carnosic acid and ursolic acid in rosemary alcohol extract.Detection method of the present invention is to carry out sample detection based on Waters e2695 high performance liquid chromatographs (Waters, US).It is an advantage of the invention that：(1) ultrasonic wave added solvent extraction rosmarinic acid in Rosmarinus officinalis L, carnosic acid and ursolic acid are used, effectively improves extraction efficiency, and environmentally protective convenient for recycling.(2) 4.6 × 250mm is used, Rosmarinic acid, carnosic acid and ursolic acid in two isocratic chromatographic condition analysis rosemary alcohol extracts is arranged in 5 μm of Symmetry C18 chromatographic columns, the time for analyzing Rosmarinic acid is only 8min, and the time for analyzing carnosic acid and ursolic acid is only 12min.(3) mobile phase is arranged in pairs or groups in varing proportions using 0.5% formic acid water and methanol and acetonitrile, and mobile phase process for preparation is easier.
Description
Technical field
The present invention relates to detection technique field, more particularly to a kind of HPLC methods detection rosmarinic acid in Rosmarinus officinalis L, Salvia japonica
The method of acid and ursolic acid.
Background technology
Rosemary is as a kind of natural perfume plant, containing plurality of active ingredients, extracted in production at present use it is main
For Rosemary Oil and rosemary natural anti-oxidant.The safety of rosemary antioxidant and its in animal and plant fat antioxygen
Protrusion effect in terms of change has obtained international food circle and has generally acknowledged that Rosmarinus officinalis extract has been put into European Union's organic food additive
List.
Rosemary is rich in polyphenolic substance：Phenolic acid compound, such as Rosmarinic acid and caffeic acid；Phenolic Diterpenoids,
Such as carnosic acid and carnosol；Triterpene compound such as ursolic acid.Rosmarinic acid (Rosmarinic acid), molecular formula
C18H16O8, molecular weight 360.318g/mol, No. CAS：20283-92-5.Carnosic acid (Carnosic acid), molecular formula
C20H28O4, molecular weight 332.44g/mol, No. CAS：3650-09-7.Ursolic acid (Ursolic acid), molecular formula C30H48O3,
Molecular weight 456.711g/mol, No. CAS：77-52-1.Rosmarinic acid and carnosic acid have been eaten as natural
Product industrial input uses, and ursolic acid has been demonstrated there is good antitumor, antiviral effect, relatively low water-soluble in having
Five ring structures, therefore be short of in terms of bioavilability, the UV absorption of ursolic acid is weaker, and baseline easily occurs when detection
Drift, causes absorbance relatively low, is unfavorable for its analysis detection.In recent years use HPLC measure three kinds of active principles method compared with
It is few, and phase composition complexity is flowed, appearance time is long, and peak shape and separating degree are not satisfactory.
Invention content
The object of the present invention is to provide a kind of HPLC to detect Rosmarinic acid, carnosic acid and black bearberry in rosemary alcohol extract
The method of acid.
The purpose of the present invention is achieved through the following technical solutions：Rosemary in a kind of HPLC detection rosemary alcohol extract
The method of acid, carnosic acid and ursolic acid, using Waters e2695 high performance liquid chromatographs (Waters, US),
It is of the present invention that detection method includes the following steps：
(1) standard solution is prepared：Take the standard items of Rosmarinic acid, carnosic acid and ursolic acid that mark is made with methanol respectively
Quasi- product solution；
(2) rosemary alcohol extract is prepared：It takes rosemary cured leaf to carry out ultrasonic wave added solvent extraction, obtains alcohol extract；
(3) standard curve is drawn：Take the standard solution progress of the Rosmarinic acid, carnosic acid and ursolic acid of various concentration
HPLC is detected, and obtains the peak area of the lower three kinds of substances of various concentration, draws Rosmarinic acid, carnosic acid and ursolic acid respectively
Standard curve；
(4) alcohol extract is detected：It takes alcohol extract to carry out HPLC detections, obtains Rosmarinic acid, carnosic acid and bear in alcohol extract
Then the peak area of tartaric acid obtains rosemary in alcohol extract according to the standard curve of Rosmarinic acid, carnosic acid and ursolic acid
The content of acid, carnosic acid and ursolic acid.
In the step (1), Rosmarinic acid, carnosic acid and ursolic acid standard items 5.0mg are accurately weighed respectively, uses first
Alcohol is configured to the mother liquor of 1mg/mL, and gradient dilution is that 25,50,100,200,400,800 μ g/mL, HPLC measure peak area,
And using mass concentration as abscissa, peak area is that ordinate draws standard curve respectively.
In the step (2), rosemary cured leaf is crushed, 40 mesh sieve is crossed, weighs 0.2g powder samples and be dissolved in 4mL volumes
The ethanol solution of score 90% impregnates 15min, ultrasonic extraction 30min at 40 DEG C, ultrasonic power 150W, 5000 r/min centrifugation
Supernatant, residue is taken to repeat to extract once, merge supernatant, 25mL is settled to, with 0.45 μm of organic system filter mistake after 10min
HPLC detections are carried out after filter.
Chromatographic condition in the step (3) and (4)：4.6 × 250mm, 5 μm of Symmetry C18 chromatographic columns, sample introduction
Amount：10 μ L, volume flow：1mL/min, 35 DEG C of column temperature.Rosmarinic acid Detection wavelength is 328nm, mobile phase：Containing 0.5% formic acid
Water-acetonitrile (68:32, V/V)；Carnosic acid and ursolic acid Detection wavelength are 210nm, mobile phase：Water-containing 0.5% formic acid
Methanol (10:90, V/V).The wavelength for analyzing Rosmarinic acid is 328nm, and retention time is 4.83 min；It is ultraviolet due to ursolic acid
Absorption is very weak, in order to reduce baseline drift, and keeps absorbance as high as possible, reduction analytical error, wavelength selective 2 10nm,
Retention time is 10.08min；The absorbance of carnosic acid at 210 nm is more than 328nm, and can be preferable with other substances
Separation, is detected with ursolic acid under same chromatographic condition, retention time 5.84min.
Compared with prior art, the invention has the advantages that：
1. the present invention uses ultrasonic wave added solvent extraction rosmarinic acid in Rosmarinus officinalis L, carnosic acid and ursolic acid, extraction effect
Rate is high, and environmentally protective convenient for recycling.
2. the present invention uses 4.6 × 250mm, 5 μm of Symmetry C18 chromatographic columns, the isocratic chromatographic condition inspection of setting two
Three kinds of active principles in rosemary alcohol extract are surveyed, the time for analyzing Rosmarinic acid is only 8min, analyzes carnosic acid and black bearberry
The time of acid is only 12min, can effectively improve analysis efficiency.
3. mobile phase is arranged in pairs or groups in varing proportions using 0.5%- formic acid waters and methanol and acetonitrile, mobile phase process for preparation is simpler
Just.
Description of the drawings
Fig. 1 is Rosmarinic acid, carnosic acid and ursolic acid the standard items (a under two kinds of wavelength conditions：328nm, b：210nm)
Chromatogram.
Fig. 2 is (a under two kinds of wavelength conditions：328nm, b：210nm) the chromatogram of rosemary alcohol extract.
Specific implementation mode
Embodiment 1
(1) standard solution is prepared：Rosmarinic acid, carnosic acid and ursolic acid standard items 5.0mg are accurately weighed respectively, are used
Methanol is configured to the mother liquor of 1mg/mL, and gradient dilution is 25,50,100,200,400,800 μ g/mL.
(2) rosemary alcohol extract is prepared：Rosemary cured leaf is crushed, 40 mesh sieve is crossed, weighs 0.2g powder samples and be dissolved in 4mL
The ethanol solution of volume fraction 90% impregnates 15min, ultrasonic extraction 30min, ultrasonic power 150W, 5000r/min at 40 DEG C
Supernatant, residue is taken to repeat to extract once, merge supernatant, be settled to 25mL, filtered with 0.45 μm of organic system after centrifugation 10min
HPLC detections are carried out after head filtering.
(3) standard curve is drawn：HPLC measures Rosmarinic acid under various concentration, carnosic acid and ursolic acid standard solution
Peak area, and using mass concentration as abscissa (X), peak area is that ordinate (Y) draws standard curve respectively.Rosemary love song
Line equation is：Y=28874X-131123, R2=0.9979；Carnosic acid curvilinear equation is：Y=28387X+1 × 106, R2=
0.9967；Ursolic acid curvilinear equation is：Y=3992.6X+92240, R2=0.9986；Rosmarinic acid, carnosic acid and black bearberry
Acid is in good linear relationship in 100~800 μ g/mL.
Chromatographic condition：4.6 × 250mm, 5 μm of Symmetry C18 chromatographic columns, sample size：10 μ L, volume flow：1mL/
Min, 35 DEG C of column temperature.Rosmarinic acid Detection wavelength is 328nm, mobile phase：Water-acetonitrile (68 containing 0.5% formic acid:32, V/V)；
Carnosic acid and ursolic acid Detection wavelength are 210nm, mobile phase：Water-methanol (10 containing 0.5% formic acid:90, V/V).
(4) alcohol extract is detected：Chromatographic condition is identical as step (4), obtains Rosmarinic acid, carnosic acid and bear in alcohol extract
Then the peak area of tartaric acid is calculated according to the standard curve of Rosmarinic acid, carnosic acid and ursolic acid in rosemary alcohol extract
The content of Rosmarinic acid, carnosic acid and ursolic acid.
(5) sample measures：Three parts of powder from rosemary are weighed, sample introduction extraction detection, as a result as follows as stated above：
Each substance yield of 1 repeated experiment of table
The present invention can be summarized with others without prejudice to the concrete form of the spirit or essential characteristics of the present invention.The present invention's
The embodiment above can only all be considered the description of the invention rather than limit, therefore every substantive skill according to the present invention
Art belongs to the range of technical solution of the present invention to any subtle modifications, equivalent variations and modifications made by above example
It is interior.
Claims (4)
1. a kind of method of Rosmarinic acid, carnosic acid and ursolic acid in HPLC detections rosemary alcohol extract, characterized in that including
Following steps：
(1) standard solution is prepared：Take the standard items of Rosmarinic acid, carnosic acid and ursolic acid that standard items are made with methanol respectively
Solution；
(2) rosemary alcohol extract is prepared：It takes rosemary cured leaf to carry out ultrasonic wave added solvent extraction, obtains alcohol extract；
(3) standard curve is drawn：Take the Rosmarinic acid of various concentration, the standard solution progress HPLC of carnosic acid and ursolic acid
Detection obtains the peak area of the lower three kinds of substances of various concentration, and the standard for drawing Rosmarinic acid, carnosic acid and ursolic acid respectively is bent
Line；
(4) alcohol extract is detected：It takes alcohol extract to carry out HPLC detections, obtains the peak area of Rosmarinic acid, carnosic acid and ursolic acid,
Then Rosmarinic acid, carnosic acid and bear in alcohol extract are obtained according to the standard curve of Rosmarinic acid, carnosic acid and ursolic acid
The content of tartaric acid.
2. the side of Rosmarinic acid, carnosic acid and ursolic acid in HPLC detections rosemary alcohol extract according to claim 1
Method, characterized in that the chromatographic condition in the step (3) and (4)：4.6 × 250mm, 5 μm of Symmetry C18 chromatographic columns, into
Sample amount：10 μ L, volume flow：1mL/min, 35 DEG C of column temperature.Rosmarinic acid Detection wavelength is 328nm, mobile phase：Containing 0.5% first
Water-acetonitrile (68 of acid:32, V/V)；Carnosic acid and ursolic acid Detection wavelength are 210nm, mobile phase：Containing 0.5% formic acid
Water-methanol (10:90, V/V).
3. the side of Rosmarinic acid, carnosic acid and ursolic acid in HPLC detections rosemary alcohol extract according to claim 1
Method, characterized in that in the step (1), accurately weigh Rosmarinic acid, carnosic acid and ursolic acid standard items 5.0mg respectively, use
Methanol is configured to the mother liquor of 1mg/mL, and 25,50,100,200,400,800 μ g/mLHPLC of gradient dilution measure peak area, and
Using mass concentration as abscissa, peak area is that ordinate draws standard curve respectively.
4. the side of Rosmarinic acid, carnosic acid and ursolic acid in HPLC detections rosemary alcohol extract according to claim 1
Method, characterized in that in the step (2), rosemary cured leaf is crushed, 40 mesh sieve is crossed, weighs 0.2g powder samples and be dissolved in 4mL bodies
The ethanol solution of fraction 90% impregnates 15min, ultrasonic extraction 30min at 40 DEG C, ultrasonic power 150W, 5000r/min centrifugation
Supernatant, residue is taken to repeat to extract once, merge supernatant, be settled to 25mL, filtered with 0.45 μm of organic system filter after 10min
HPLC detections are carried out afterwards.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110632190A (en) * | 2019-08-28 | 2019-12-31 | 湖南先伟实业有限公司 | Method for simultaneously measuring rosmarinic acid, carnosic acid and carnosol in rosemary |
| CN115724901A (en) * | 2022-12-09 | 2023-03-03 | 湖南一科生物科技有限公司 | Method for extracting rosmarinic acid, carnosic acid and ursolic acid from rosemary |
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| CN101744877A (en) * | 2010-01-21 | 2010-06-23 | 广州星群（药业）股份有限公司 | Selfheal quality control method |
| CN102286059A (en) * | 2011-09-08 | 2011-12-21 | 南京泽朗医药科技有限公司 | Method for extracting ursolic acid, carnosic acid and rosmarinic acid from rosmarinus officinalis |
| US20130315983A1 (en) * | 2012-05-24 | 2013-11-28 | The Research Foundation Of The City University Of New York | Methods and compositions for the treatment of cancer |
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Patent Citations (3)
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| CN101744877A (en) * | 2010-01-21 | 2010-06-23 | 广州星群（药业）股份有限公司 | Selfheal quality control method |
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| US20130315983A1 (en) * | 2012-05-24 | 2013-11-28 | The Research Foundation Of The City University Of New York | Methods and compositions for the treatment of cancer |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110632190A (en) * | 2019-08-28 | 2019-12-31 | 湖南先伟实业有限公司 | Method for simultaneously measuring rosmarinic acid, carnosic acid and carnosol in rosemary |
| CN115724901A (en) * | 2022-12-09 | 2023-03-03 | 湖南一科生物科技有限公司 | Method for extracting rosmarinic acid, carnosic acid and ursolic acid from rosemary |
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Application publication date: 20180918 |