CN104897797A - Method for determining oleanolic acid content and ursolic acid content in perilla frutescens oil through high performance liquid chromatography method - Google Patents
Method for determining oleanolic acid content and ursolic acid content in perilla frutescens oil through high performance liquid chromatography method Download PDFInfo
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- CN104897797A CN104897797A CN201510189020.2A CN201510189020A CN104897797A CN 104897797 A CN104897797 A CN 104897797A CN 201510189020 A CN201510189020 A CN 201510189020A CN 104897797 A CN104897797 A CN 104897797A
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Abstract
The present invention discloses a method for determining the oleanolic acid content and the ursolic acid content in perilla frutescens oil. According to the method, mainly the reverse-phase high performance liquid chromatography method is used; the chromatographic conditions comprise that a, the chromatographic column is a C18 column (250*4.6 mm, 5 [mu]m), b, the mobile phase comprises 265% by volume of methanol, 35% by volume of water, 0.1% by volume of glacial acetic acid and 0.05% by volume of triethylamine and is formed from 265 mL of methanol, 35 mL of water, 0.05 mL of glacial acetic acid and 0.05 mL of triethylamine, c, the detection wavelength of the used ultraviolet detector is 210 nm, and d, the flow rate is 0.6 mL/min, the sample injection volume is 20 [mu]L, and the column temperature is 20 DEG C; and the sample solution treatment comprises: taking about 1.0-2.5 g of a perilla frutescens oil sample, precisely weighing, placing into a conical flask having a cork, precisely adding 25 ml of methanol, tightly covering the cork, weighing, carrying out microwave extraction for 10-30 min, weighing again, complementing the weight loss with methanol, uniformly shaking, filtering, and taking the subsequent filtrate so as to obtain the finished product. In order to effectively control the quality, the method of the present invention is sensitive, efficient, accurate and stable, and can effectively control the oleanolic acid content and the ursolic acid content in the perilla frutescens oil.
Description
Technical field
The present invention relates to a kind of method of oleanolic acid, ursolic acid content in high effective liquid chromatography for measuring perilla herb oil, art: two, biological and new medical technology (seven) modern agricultural technology 4, agricultural product intensive processing and modern accumulating: agricultural product quality and safety evaluation, detect fast, the technology such as whole-course quality control.
Background technology
HPLC is a high pressure, efficient, highly sensitive isolation technics, can be used for that compartment analysis higher boiling, relative molecular weight are large, the organic compound of poor heat stability, is widely used in chemistry, chemical industry, macromolecule, biology, food, medicine and other fields.
Oleanolic acid, ursolic acid are mainly present in perilla herb oil and oil foot thereof, are pentacyclic triterpenoid; Character is close, is difficult to be separated; It is a kind of active component; Protect the liver, anti-inflammatory, antitumor, reducing blood lipid, the effect such as enhancing immunity and Immunosuppression (immune two-way conciliation effect).
At present these 2 kinds of materials can only extract from plant, cannot chemosynthesis; Detection method has spectrophotometric method, thin layer chromatography scanning and high performance liquid chromatography, and the former measures after usually adopting chromogenic reagent, and because their character is close, spectrophotometric method records its triterpenes total amount usually, and method lacks specificity; And common thin-layer chromatography is difficult to make OA with UA be separated; Comparatively speaking, HPLC is easier to the separation of both enforcement, and UV detect has good reappearance relative to Evaporative light scattering detector.Look in scope existing, oleanolic acid, ursolic acid in HPLC Simultaneously test perilla herb oil is utilized to have no report, adopt reversed-phased high performace liquid chromatographic diode array detector to determine oleanolic acid in perilla herb oil and ursolic acid content, the quality be intended to for evaluating perilla herb oil provides scientific basis.
Summary of the invention
Technical matters to be solved by this invention is a kind of method providing oleanolic acid, ursolic acid content in high effective liquid chromatography for measuring perilla herb oil, thus its content of Accurate Determining, method is simple to operate, highly sensitive, favorable reproducibility.
The present invention adopts following technical scheme to achieve these goals:
A method for oleanolic acid, ursolic acid content in high effective liquid chromatography for measuring perilla herb oil, is characterized in that comprising the following steps:
(1) the preparation precision of reference substance solution takes oleanolic acid reference substance 10.20mg, ursolic acid reference substance 8.54mg, puts respectively in 10ml measuring bottle, adds methyl alcohol and dissolves and be diluted to scale, shake up, and obtains oleanolic acid, ursolic acid reference substance stock solution.
(2) process of sample solution: get perilla herb oil sample and be about 1.0-2.5g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 25ml, close plug, weighed weight, Microwave Extraction 10-30min, more weighed weight, supply the weight of less loss with methyl alcohol, shake up, filter, get subsequent filtrate, to obtain final product;
(3), after filtrate being crossed 0.40-0.50 μm of miillpore filter, under experiment chromatographic condition, carry out high effective liquid chromatography for measuring, obtain mensuration numerical value; Described experiment chromatographic condition is: chromatographic column: C
18post, 250 × 4.6mm, 5 μm; Mobile phase: 265% methyl alcohol-35% water-0.1% glacial acetic acid-0.05% triethylamine, wherein, mobile phase is configured by methyl alcohol 265mL, water 35mL, glacial acetic acid 0.05mL and triethylamine 0.05mL; The determined wavelength of UV-detector: 210nm; Flow rate of mobile phase is 0.6mL/min, column temperature 20 DEG C, and sampling volume is 20 μ L;
(4) content of oleanolic acid, ursolic acid in perilla herb oil is calculated according to external standard method.
The method of oleanolic acid, ursolic acid content in described high effective liquid chromatography for measuring perilla herb oil, is characterized in that: described experiment chromatographic condition is: chromatographic column: C
18post, 250 × 4.6mm, 5 μm; Mobile phase: 265% methyl alcohol-35% water-0.1% glacial acetic acid-0.05% triethylamine, wherein, mobile phase is configured by methyl alcohol 265mL, water 35mL, glacial acetic acid 0.05mL and triethylamine 0.05mL; The determined wavelength of UV-detector: 210nm; Flow rate of mobile phase is 0.6mL/min, column temperature 20 DEG C, and sampling volume is 20 μ L.
Beneficial effect of the present invention: the present invention can the content of oleanolic acid, ursolic acid in Accurate Determining perilla herb oil, and method is simple to operate, highly sensitive, favorable reproducibility.
Accompanying drawing explanation
The chromatogram of oleanolic acid, ursolic acid in Fig. 1 embodiment of the present invention 2 reference substance, a oleanolic acid, b ursolic acid.
The chromatogram of oleanolic acid, ursolic acid in Fig. 2 embodiment of the present invention 2 perilla herb oil, a oleanolic acid, b ursolic acid.
Embodiment
Embodiment 1: the method for oleanolic acid, ursolic acid content in high effective liquid chromatography for measuring perilla herb oil, comprises the following steps:
(1) the preparation precision of reference substance solution takes oleanolic acid reference substance 10.20mg, ursolic acid reference substance 8.54mg, puts respectively in 10ml measuring bottle, adds methyl alcohol and dissolves and be diluted to scale, shake up, and obtains oleanolic acid, ursolic acid reference substance stock solution.
(2) process of sample solution: get perilla herb oil sample and be about 0.5-1.5g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 25ml, close plug, weighed weight, Microwave Extraction 10-30min, more weighed weight, supply the weight of less loss with methyl alcohol, shake up, filter, get subsequent filtrate, to obtain final product;
(3), after filtrate being crossed 0.40-0.50 μm of miillpore filter, under experiment chromatographic condition, carry out high effective liquid chromatography for measuring, obtain mensuration numerical value; Described experiment chromatographic condition is: chromatographic column: C
18post, 250 × 4.6mm, 5 μm; Mobile phase: 265% methyl alcohol-35% water-0.1% glacial acetic acid-0.05% triethylamine, wherein, mobile phase is configured by methyl alcohol 265mL, water 35mL, glacial acetic acid 0.05mL and triethylamine 0.05mL; The determined wavelength of UV-detector: 210nm; Flow rate of mobile phase is 0.6mL/min, column temperature 20 DEG C, and sampling volume is 20 μ L;
(4) content of oleanolic acid, ursolic acid in perilla herb oil is calculated according to external standard method.
Described experiment chromatographic condition is: chromatographic column: C
18post, 250 × 4.6mm, 5 μm; Mobile phase: 265% methyl alcohol-35% water-0.1% glacial acetic acid-0.05% triethylamine, wherein, mobile phase is configured by methyl alcohol 265mL, water 35mL, glacial acetic acid 0.05mL and triethylamine 0.05mL; The determined wavelength of UV-detector: 210nm; Flow rate of mobile phase is 0.6mL/min, column temperature 20 DEG C, and sampling volume is 20 μ L.
Embodiment 2: specific experiment data of the present invention:
1 instrument and reagent
Instrument: 1100 high performance liquid chromatographs (comprise G1311A pump, G1322 degasser, G1313AZ automatic sampler, G1313A column oven, G1315 DAD; Agilent 1100 ChemStation); BP221D electronic balance (Sartorius company); KQ-250DB numerical control ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); Boil high purity water extraction apparatus (Jiangsu Jin Cheng Educational Instrument Factory) in quartz Asia.
Reagent: oleanolic acid, ursolic acid reference substance (110709-200505,110742-200516, for assay) are provided by Nat'l Pharmaceutical & Biological Products Control Institute; Methyl alcohol is chromatographically pure, and it is pure that perilla herb oil (Yuexi Glorifies Sage Tea Oil Ltd. chrysanthemum contains board perilla herb oil) other reagent are analysis.
Mobile phase is prepared: respectively according to 265mL methyl alcohol, 35mL water, 0.05mL glacial acetic acid and 0.05mL triethylamine proportional arrangement mobile phase.
Oleanolic acid, ursolic acid standard solution: precision takes oleanolic acid reference substance 10.20mg, ursolic acid reference substance 8.54mg, puts respectively in 10ml measuring bottle, adds methyl alcohol and dissolves and be diluted to scale, shake up, obtain oleanolic acid, ursolic acid reference substance stock solution.
The process of sample solution: get perilla herb oil sample and be about 0.5-1.5g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 25ml, close plug, weighed weight, Microwave Extraction 10-30min, more weighed weight, supply the weight of less loss with methyl alcohol, shake up, filter, get subsequent filtrate, to obtain final product, after crossing 0.45 μm of miillpore filter, under experiment imposes a condition, carry out HPLC mensuration.
The foundation of 2 content assaying methods
2.1 wavelength chooses
Oleanolic acid and ursolic acid have absorption maximum at 206nm place, this experiment adopts DAD detecting device, carry out multi-wavelength to same perilla herb oil test liquid in 205,210,215 and 220nm place to detect simultaneously, result display is except 205nm has solvent to disturb and end absorbs, other wavelength place is separated all ideal, but all comparatively 210nm wavelength place response intensity is low for 215nm and 220nm, and detection sensitivity is low, therefore this experiment adopts 210nm to be determined wavelength.
The selection of 2.2 mobile phases
Multiple flow phase system such as methanol-water, methanol-water-phosphoric acid, methanol-water-glacial acetic acid, methanol-acetonitrile-water, methanol-water-glacial acetic acid-triethylamine are investigated, adjusted by proportioning, find that methanol-water-glacial acetic acid-triethylamine (265: 35: 0.1: 0.05) easily obtains satisfied and stable separating resulting in the chromatographic system determined; Flows decrease is beneficial to separation, and this experimental flow rate is 0.6ml/min.
2.3 qualitative test
Utilize relative retention time method and add standard substance legal.Chromatogram under identical chromatographic condition of reference standard solution, mark-on sample solution and sample solution and relative retention time.Experimental result shows, in three kinds of solution, the appearance time of oleanolic acid, ursolic acid is consistent.
2.4 typical curves and related coefficient
Precision measures oleanolic acid reference substance stock solution 0.04ml, 0.22ml, 0.42ml, 0.62ml, 0.82ml, ursolic acid reference substance stock solution 0.05ml, 0.28ml, 0.52ml, 0.80ml, 1.00ml, put in 5 10ml measuring bottles respectively, add methanol dilution to scale, shake up, obtain oleanolic acid and ursolic acid mixing reference substance solution series.The each mixing reference substance solution 20 μ l of accurate absorption respectively, injection liquid chromatography, measures according to above-mentioned chromatographic condition.With chromatographic peak area (y) for ordinate, reference substance concentration (x, mg/ml) is horizontal ordinate, drawing standard curve, and obtaining regression equation is oleanolic acid: y=16782.3x+2.17 (r=0.9998); Ursolic acid: y=15682.0x+11.58 (r=0.9994).From table, oleanolic acid sample size is at 0.162 ~ 3.31 μ g, and ursolic acid sample size presents good linear relationship between 0.195 ~ 3.91 μ g.
2.5 reference substances, sample tests and replica test
Get above-mentioned treated reference substance solution and sample solution, according to above-mentioned chromatographic condition, squeezed into by sample in high performance liquid chromatograph, its chromatogram is shown in accompanying drawing 1,2.
After sample introduction 6 times, according to chromatogram, record experimental data, its sample introduction result: in perilla herb oil, the average content of oleanolic acid, ursolic acid is respectively: 0.125%, 0.353%; RSD is respectively 1.27%, and 1.03%.
2.6 stability test
Get same perilla herb oil sample according to above-mentioned disposal route, produce sample solution, measure at 0,4,8,12,24h sample introduction respectively, the RSD of peak area is 1.05%, and result shows that oleanolic acid in sample solution, ursolic acid are stablized at 24H intensive amount.
Experimental result shows: to mensuration oleanolic acid, ursolic acid accurately and reliably, favorable reproducibility, can be used for the mensuration of oleanolic acid in perilla herb oil, ursolic acid content in the present invention.
Claims (1)
1. the method for oleanolic acid, ursolic acid content in high effective liquid chromatography for measuring perilla herb oil, is characterized in that comprising the following steps:
(1) the preparation precision of reference substance solution takes oleanolic acid reference substance 10.20mg, ursolic acid reference substance 8.54mg, puts respectively in 10ml measuring bottle, adds methyl alcohol and dissolves and be diluted to scale, shake up, and obtains oleanolic acid, ursolic acid reference substance stock solution.
(2) process of sample solution: get perilla herb oil sample and be about 1.0-2.5g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 25ml, close plug, weighed weight, Microwave Extraction 10-30min, more weighed weight, supply the weight of less loss with methyl alcohol, shake up, filter, get subsequent filtrate, to obtain final product;
(3), after filtrate being crossed 0.40-0.50 μm of miillpore filter, under experiment chromatographic condition, carry out high effective liquid chromatography for measuring, obtain mensuration numerical value; Described experiment chromatographic condition is: chromatographic column: C
18post, 250 × 4.6mm, 5 μm; Mobile phase: 265% methyl alcohol-35% water-0.1% glacial acetic acid-0.05% triethylamine, wherein, mobile phase is configured by methyl alcohol 265mL, water 35mL, glacial acetic acid 0.05mL and triethylamine 0.05mL; The determined wavelength of UV-detector: 210nm; Flow rate of mobile phase is 0.6mL/min, column temperature 20 DEG C, and sampling volume is 20 μ L;
(4) content of oleanolic acid, ursolic acid in perilla herb oil is calculated according to external standard method.
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Cited By (2)
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| CN105732762A (en) * | 2016-03-31 | 2016-07-06 | 王婧 | Method for extracting perilla oil and ursolic acid from perilla seeds |
| CN111707738A (en) * | 2020-05-07 | 2020-09-25 | 安徽国风塑业股份有限公司 | Method for determining purity of pyromellitic dianhydride by liquid chromatography |
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Cited By (2)
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| CN111707738A (en) * | 2020-05-07 | 2020-09-25 | 安徽国风塑业股份有限公司 | Method for determining purity of pyromellitic dianhydride by liquid chromatography |
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Application publication date: 20150909 |