CN104374855B - The method of woodruff thuja acid and 10-deacetyl asperulosidic thuja acid content in HPLC Simultaneously test Noni juice - Google Patents
The method of woodruff thuja acid and 10-deacetyl asperulosidic thuja acid content in HPLC Simultaneously test Noni juice Download PDFInfo
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- CN104374855B CN104374855B CN201410413733.8A CN201410413733A CN104374855B CN 104374855 B CN104374855 B CN 104374855B CN 201410413733 A CN201410413733 A CN 201410413733A CN 104374855 B CN104374855 B CN 104374855B
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- thuja acid
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Abstract
The present invention relates to a kind of method of woodruff thuja acid and 10-deacetyl asperulosidic thuja acid content in HPLC Simultaneously test Noni juice.Concrete technical scheme is: preparation standard product solution and sample solution respectively, carries out gradient elution, woodruff thuja acid and 10-deacetyl asperulosidic thuja acid content in Simultaneously test Noni juice by HPLC method.The inventive method is easy, reliable, rapid, highly sensitive, favorable reproducibility, well can be separated woodruff thuja acid in Noni juice with interfering material with 10-deacetyl asperulosidic thuja acid, and its content of Simultaneously test, significant to the research of the both effectiveness composition of Noni juice.
Description
Technical field
The present invention relates to the detection method of woodruff thuja acid and 10-deacetyl asperulosidic thuja acid content in a kind of HPLC Simultaneously test Noni juice.
Background technology
Nuo Ni (Noni), formal name used at school morinda citrifolia (
morindacitrifolialinn
.), in May, 2010, the official approval of promise Buddhist nun pulp is new resource food by ministry of Health of China.Noni juice has nutrition widely and medical value, has the functions such as antitumor, anti-oxidant, develop immunitypty, reducing blood lipid and protection be cardiovascular, has preventive and therapeutic action to various diseases.
Woodruff thuja acid and 10-deacetyl asperulosidic thuja acid are iridoids materials, are active components important in Noni juice, have antitumor, antiviral, anti-oxidant, anti-distortion isoreactivity, closely related with health, become study hotspot gradually.But there is no the method utilizing HPLC to measure woodruff thuja acid and 10-deacetyl asperulosidic thuja acid in Noni juice at present, limit the research of the both effectiveness composition of Noni juice.Therefore, the high performance liquid chromatography setting up woodruff thuja acid and 10-deacetyl asperulosidic thuja acid in a kind of Noni juice of detection is simultaneously needed badly.
Summary of the invention
In order to solve the problems of the technologies described above, the invention provides a kind of method using woodruff thuja acid and 10-deacetyl asperulosidic thuja acid content in HPLC Simultaneously test Noni juice, for the efficacy study of Noni juice is laid a good foundation.Realizing concrete technical scheme of the present invention is: with woodruff thuja acid and 10-deacetyl asperulosidic thuja acid content in HPLC Simultaneously test Noni juice, it is characterized in that:
In the present invention, the preparation method of hybrid standard product solution is:
Take woodruff thuja acid reference substance 5mg, 10-deacetyl asperulosidic thuja acid reference substance 25mg, is placed in 10mL volumetric flask, is diluted with water to scale, shake up, must mix reference substance storing solution, through 0.22 μm of filtering with microporous membrane before measuring.
In the present invention, the preparation method of sample solution is:
Get the centrifugal 5min of 20mL Noni juice 10000r/m, Aspirate supernatant 10mL, in 100mL volumetric flask, is diluted with water to scale, shakes up, through 0.22 μm of filtering with microporous membrane before measuring.
Chromatographic condition of the present invention is:
Chromatographic column is Shim-packVP-ODS-C18 (250mm × 4.6mm, 5 μm); VWD-3000 UV-detector; Column temperature is 35 DEG C; Mobile phase A is 0.1% phosphate aqueous solution, and Mobile phase B is acetonitrile; Condition of gradient elution is 0-10min, 4%B; 10-22,15%B; 22-40min, 4%B; Flow velocity is 0.5mL/min; Determined wavelength is 237nm; Under above-mentioned chromatographic condition, get reference substance solution and standard solution sample introduction respectively, record chromatogram.
In the present invention, said Noni juice is pure Noni juice or the other products containing promise Buddhist nun composition.
The present invention adopts HPLC method, gradient elution, records woodruff thuja acid and 10-deacetyl asperulosidic thuja acid content in Noni juice simultaneously.Result: woodruff thuja acid is have good linear relation within the scope of 5-50 μ g/mL in concentration, and average recovery rate is 99.02%, RSD is 1.58%; 10-deacetyl asperulosidic thuja acid is have good linear relation within the scope of 25-250 μ g/mL in concentration, and average recovery rate is 102.15%, RSD is 2.40%.The inventive method is easy, reliable, rapid, highly sensitive, favorable reproducibility, for the efficacy study of Noni juice provides scientific basis.
four, accompanying drawing explanation
Fig. 1 is the chromatogram of woodruff thuja acid and 10-deacetyl asperulosidic thuja acid hybrid standard product in the present invention.
Fig. 2 is the chromatogram of Noni juice sample solution in the present invention.
five, embodiment
Below in conjunction with instantiation, the invention will be further described, but the present invention is not limited to following examples.
embodiment 1:
1, instrument and material
1.1 instrument
ThermoUltiMate3000 type high performance liquid chromatograph (VWD-3000 UV-detector, Chromeleon7 chromatographic work station, ThermoFisher company), Shim-packVP-ODS-C18 (250mm × 4.6mm, 5 μm) chromatographic column; Minispin hydro-extractor (eppendorf company).
1.2 material
Reference substance woodruff thuja acid is purchased from Chengdu Pu Feide Bioisystech Co., Ltd, and 10-deacetyl asperulosidic thuja acid is purchased from Beijing flourishing age Kang Pu Chemical Engineering Technology research institute, and acetonitrile, phosphoric acid are chromatographically pure.Xisha Noni juice is produced by Hainan Noni Bioengineering Development Co., Ltd., and product batches is 20120911,20130618,20131206 and 20140122.
method and result
The selection of 2.1 chromatographic conditions
Detecting device: ThermoVWD-3000; Determined wavelength: 237nm; Chromatographic column: Shim-packVP-ODS (250 × 4.6mm, 5 μm); Column temperature: 35 DEG C; Mobile phase A is 0.1% phosphoric acid solution, and Mobile phase B is acetonitrile, and carry out gradient elution, gradient elution program is as shown in table 1, flow velocity: 0.5mL/min, and determined wavelength is 237nm, and column temperature optimum is 35 DEG C.
Table 1 gradient elution program
| Time (min) | 0.1 % phosphoric acid solution (A) (%) | Acetonitrile (B) (%) |
| 0-10 | 96 | 4 |
| 10-22 | 85 | 15 |
| 22-40 | 96 | 4 |
2.2 standard solutions and sample solution preparation
Take woodruff thuja acid reference substance 5mg, 10-deacetyl asperulosidic thuja acid reference substance 25mg, is placed in 10mL volumetric flask, is diluted with water to scale, shake up, and must mix reference substance storing solution (mass concentration is respectively 0.5mg/mL and 2.5mg/mL).
Get the centrifugal 5min of 20mL Noni juice 10000r/m, Aspirate supernatant 10mL, in 100mL volumetric flask, is diluted with water to scale, shakes up.
The standard solution of above-mentioned preparation and sample solution all use 0.22 μm of filtering with microporous membrane, and sample size is 5 μ l, and writing time is 40min.
2.3 sample sizes measure
Get four batches of Xisha Noni juices, prepare sample solution according to 2.2, injection liquid chromatography, each sample feeding three pin, according to peak area calibration curve method calculation sample content, average, the results are shown in Table 2.
Table 2 Xisha Noni juice woodruff thuja acid and 10-deacetyl asperulosidic thuja acid measurement result
embodiment 2:
methodological study
1, linear relationship is investigated
Absorption mixing reference substance storing solution 0.1,0.2,0.4,0.6,0.8 and 1.0mL, in 10mL volumetric flask, are diluted with water to scale, shake up.Draw respectively in above-mentioned reference substance solution 10 μ L injection liquid chromatography, with peak area (y), linear regression carried out to mass concentration (x):
Woodruff thuja acid: y=4.405x+0.139, R2=0.9997, the range of linearity is 5-50 μ g/mL.
10-deacetyl asperulosidic thuja acid: y=5.030x+0.578, R2=0.9998, the range of linearity is 25-250 μ g/mL.
2, instrument precision test
Get Xisha Noni juice (batch 20131206) a, continuous sample introduction 6 times under above-mentioned chromatographic condition, record woodruff thuja acid and 10-deacetyl asperulosidic thuja acid mass concentration, its RSD value is respectively 0.44% and 0.31%.
Table 3 precision measurement result
3, reappearance test
Get with a collection of Xisha Noni juice (batch 20131206) 6 parts, analyze by above-mentioned chromatographic condition sample introduction, measure woodruff thuja acid and 10-deacetyl asperulosidic thuja acid mass concentration, its RSD value is respectively 1.28% and 0.79%.
Table 4 reappearance measurement result
4, stability test
Get Xisha Noni juice (batch 20131206) room temperature to place, respectively 0,2,4,6 and 8h sample introduction, record woodruff thuja acid and 10-deacetyl asperulosidic thuja acid mass concentration, its RSD value is respectively 1.57% and 0.78%, shows that need testing solution is stablized in 8h.
Table 5 Stability Determination result
5, recovery test
Draw same batch of Xisha Noni juice (batch 20131206) 6 parts, every part of 1mL, add woodruff thuja acid and 10-deacetyl asperulosidic thuja acid mixing reference substance storing solution 0.5mL respectively, sample introduction analysis, calculate woodruff thuja acid and 10-deacetyl asperulosidic thuja acid average recovery with external standard method.Woodruff thuja acid and 10-deacetyl asperulosidic thuja acid average recovery rate and RSD value are respectively 99.02% and 1.58%, 102.15% and 2.40%.
Table 6 determination of recovery rates result
To sum up, the inventive method be under same condition simultaneously to Noni juice in woodruff thuja acid and 10-deacetyl asperulosidic thuja acid carry out assay.Consider the factors such as the degree of separation of these two kinds of materials, peak symmetry and post effect, final employing Shim-packVP-ODS (250mm × 4.6mm, 5 μm) chromatographic column, acetonitrile-phosphate aqueous solution is mobile phase, gradient separations is carried out to target substance, flow velocity is 0.5mL/min, VWD-3000 UV-detector, and determined wavelength is 237nm.Tested component is separated completely with can reach between interference component, and post effect is high, and component retention time is suitable, and this law is quick, easy, accurate, can be used for Simultaneously test Noni juice woodruff thuja acid and 10-deacetyl asperulosidic thuja acid content.
Claims (3)
1.HPLC the method for woodruff thuja acid and 10-deacetyl asperulosidic thuja acid content in Simultaneously test Noni juice, is characterized in that:
(1) reference substance solution preparation is mixed
Take woodruff thuja acid reference substance 5mg, 10-deacetyl asperulosidic thuja acid reference substance 25mg, is placed in 10mL volumetric flask, is diluted with water to scale, shake up, must mix reference substance storing solution; Draw respectively 0.1,0.2,0.4,0.6,0.8 and 1.0mL mixing reference substance storing solution in 10mL volumetric flask, be diluted with water to scale, shake up, obtain series mixing reference substance solution;
(2) sample preparation
Get the centrifugal 5min of 20mL Noni juice 10000r/m, Aspirate supernatant 10mL, in 100mL volumetric flask, is diluted with water to scale, shakes up;
(3) high performance liquid chromatography is separated testing conditions
Adopt C18 reverse-phase chromatographic column, with mobile phase A phosphate aqueous solution; Mobile phase B acetonitrile, adopts gradient elution: 0 ~ 10min, 4%B; 10 ~ 22min, 4%B ~ 15%B; 22 ~ 40min, 15%B ~ 4%B; Employing UV-detector detects, and column temperature is 30-40 DEG C, and flow velocity is 0.2-1.2mL/min;
(4) sample qualitative and quantitative analysis
Series is mixed reference substance solution by step (3) chromatographic condition sample introduction; With peak area y, linear regression is carried out to mass concentration x, obtain the regression equation of woodruff thuja acid and 10-deacetyl asperulosidic thuja acid reference substance; After each sample carries out pre-treatment by step (2), by the respectively sample introduction analysis of step (3) chromatographic condition, sample peak and the comparison of reference substance retention time qualitative, it is quantitative that sample peak area substitutes into corresponding typical curve regression equation.
2. method according to claim 1, is characterized in that: mobile phase A is phosphate aqueous solution optium concentration is 0.1%, and best detection wavelength is 237nm, and optimum column temperature is 35 DEG C, and optimum flow rate is 0.5mL/min.
3. method according to claim 1, is characterized in that: Noni juice is the juice product containing promise Buddhist nun fermented juice composition.
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Citations (2)
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|---|---|---|---|---|
| CN101874841A (en) * | 2009-04-28 | 2010-11-03 | 仇鑫 | Total glycosides extractive of morinda plants, as well as preparation method and application thereof |
| CN103859546A (en) * | 2014-04-08 | 2014-06-18 | 杜道林 | Natural multifunctional Noni compound drink and preparation method thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101874841A (en) * | 2009-04-28 | 2010-11-03 | 仇鑫 | Total glycosides extractive of morinda plants, as well as preparation method and application thereof |
| CN103859546A (en) * | 2014-04-08 | 2014-06-18 | 杜道林 | Natural multifunctional Noni compound drink and preparation method thereof |
Non-Patent Citations (6)
| Title |
|---|
| Determination and Comparative Analysis of Major Iridoids in Different Parts and Cultivation Sources of Morinda citrifolia;Shixin Deng等;《Phytochemical Analysis》;20110826;第22卷;第26–30页 * |
| Effect of Morinda citrifolia fruit extract and its iridoid glycosides on blood fluidity;Kazuya Murata 等;《Journal of Natural Medicine》;20140307;第69卷;第498–504页 * |
| HPLC法测定白花蛇舌草注射液中去乙酰基车叶草苷酸和鸡屎藤次苷;姚鑫宁 等;《中草药》;20100430;第41卷(第4期);第580－582页 * |
| Noni juice reduces lipid peroxidation–derived DNA adducts in heavy smokers;Mian-Ying Wang 等;《Food Science & Nutrition》;20131231;第1卷(第4期);第141–149页 * |
| Thin Layer Chromatography Methods for Rapid Identity Testing of Morinda citrifolia L. (Noni) Fruit and Leaf;Brett J. West 等;《Advance Journal of Food Science and Technology》;20100925;第2卷(第5期);第298-302页 * |
| 西沙诺尼果汁的营养成分分析;李金霞 等;《食品科技》;20140531;第39卷(第5期);第56－59页 * |
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