CN102890126A - Method for simultaneously measuring content of multiple active ingredients in eucommia ulmoides - Google Patents
Method for simultaneously measuring content of multiple active ingredients in eucommia ulmoides Download PDFInfo
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Abstract
The invention discloses a method for simultaneously measuring the content of multiple active ingredients in eucommia ulmoides. The method adopts high performance liquid chromatography for analysis; the chromatographic condition is taking octadecylsilane bonded silica as a filler; the mobile-phase gradient elution process is: the mobile phase A is methanol, and the mobile phase B is an aqueous solution of phosphoric acid with mass concentration of 0.1%; the content of the mobile phase B is increased from 30% to 70% by mass according to the first-order linear gradient in 0.01-30.00min, reduced to 30% in 30.00-30.01min and kept at 30% in 30.01-40.00min; the flow rate is 0.8ml/min; the column temperature is 30 DEG C; the sample injection volume is 5mul; the detection wavelength detection process is: the 254nm wavelength is selected in 0.01-7.00min, the 277nm wavelength is selected in 7.00-12.50min, and the 254nm wavelength is selected in 12.50-40.00min; and multiple active ingredients include geniposidic acid, chlorogenic acid, pinoresinol diglucoside and rutin. According to the invention, the method is verified by linear relation survey, stability test, precision test, reproducibility test and sample injection recycling test, and the analysis method is proved to be accurate and reliable.
Description
Technical field
The present invention relates to the content assaying method of various active composition in a kind of bark of eucommia, particularly measure simultaneously the assay method of Geniposidic acid (GPA), chlorogenic acid (CA), Pinoresinol diglucoside (PDG) and rutin (Rutin) content.
Background technology
The bark of eucommia (Eucommia ulmoides OLiv) belongs to perennial deciduous tree for the Eucommiaceae bark of eucommia, it is the traditional economy crop of China, the title that quaternary glacier " living fossil " arranged, middle reaches, distribution the Changjiang river and southern each province, all there is cultivation on the ground such as Henan, Shaanxi, Gansu.The bark of eucommia has filling liver kidney, strengthening the bones and muscles, antiabortive, hypotensive effect.Its main active component has lignanoids, iridoids, Phenylpropanoid Glycosides class, flavonoids etc.Main representative composition is respectively Pinoresinol diglucoside (lignanoids), Geniposidic acid (iridoids), chlorogenic acid (Phenylpropanoid Glycosides class), rutin (flavonoids)
UV-VIS spectrophotometry (UV) is generally adopted in above active component quality control, thin layer chromatography scanning (TLCS) and high performance liquid chromatography (HPLC).
The content assaying method of Pinoresinol diglucoside is high performance liquid chromatography in 2010 editions Chinese Pharmacopoeia regulation barks of eucommia, is fixing phase with octadecyl base silane bonded silica gel, take methanol-water (25: 75) as mobile phase, and detection wavelength 277nm.The content assaying method of regulation Chlorogenic Acid in Eucommia ulmoides Oliv. Leaves is for for high performance liquid chromatography, is fixing phase with octadecyl base silane bonded silica gel, take acetonitrile-0.4% phosphate aqueous solution (13: 87) as mobile phase, and detection wavelength 327nm.
In the prior art, detecting components in Eucommia ulmoides HPLC method is the normal analysis means that uses.But can only control simultaneously 1～3 kind of composition in document and the national standard report at present.We find in practical operation, and Pinoresinol diglucoside has absorption maximum at the 277nm place, and significantly weakens in 254nm place absorption peak strength; Geniposidic acid then has absorption maximum at the 254nm place, and significantly weakens in 277nm place absorption peak strength, so the two is difficult to measure simultaneously; And the polarity of chlorogenic acid and Pinoresinol diglucoside is close, uses the separating effect of conventional isocratic elution method chromatographic peak not good.
Summary of the invention
For the deficiencies in the prior art, the present invention has adopted and has changed the method that wavelength combines with linear gradient elution method, proposed a kind of method of measuring simultaneously Pinoresinol diglucoside, Geniposidic acid, chlorogenic acid and rutin content in bark of eucommia and products thereof, and the chromatographic condition that adopts is fully not identical with method in the present pertinent literature of having reported.The present invention has carried out respectively the linear relationship investigation, stability test, and precision test, reappearance test and application of sample recovery test are verified method, prove that this analysis method is accurate, and be reliable.
Technical scheme of the present invention is: a kind of method of measuring simultaneously various active component content in the bark of eucommia, adopt high-efficient liquid phase chromatogram technique analysis, and chromatographic condition is: be filling agent with octadecylsilane chemically bonded silica; Eluent gradient elution program (seeing Table 1) is: mobile phase A is methyl alcohol, and Mobile phase B is that mass concentration is 0.1% phosphate aqueous solution; In the 0.01min-30.00min volumn concentration of Mobile phase B from 30% according to first-order linear step increase to 70%; When 30.00min-30.01min, be reduced to 30%, and maintenance 30% is constant in 30.01min-40.00min; Flow velocity: 0.8ml.min
-1Column temperature: 30 ℃; The sample introduction volume is 5 μ l;
Detecting wavelength option program (seeing Table 2) is: 0.01min-7.00min selects 254nm, and 7.00min-12.50min selects 277nm, and 12.50min-40.00min selects 254nm;
Wherein said various active composition is Geniposidic acid (retention time 4.7～5.0min detects wavelength 254nm); Chlorogenic acid (retention time 8.0～9.0min detects wavelength 277nm); Pinoresinol diglucoside (retention time 9.5～10.5min detects wavelength 277nm); And rutin (retention time 20.0～21.0min detects wavelength 254nm).
The eluent gradient elution program sees Table 1, detects the wavelength program and sees Table 2:
Table 1 mobile phase program
Table 2 detects the wavelength program
Compared with prior art, advantage of the present invention is:
1, assay method provided by the present invention can be measured various active component content in the bark of eucommia simultaneously, can measure simultaneously representative effective constituent Pinoresinol diglucoside, Geniposidic acid, chlorogenic acid and rutin in bark of eucommia and products thereof, has improved detection efficiency.
2, the present invention has done a large amount of methodology demonstration tests, has carried out respectively the linear relationship investigation, stability test, and precision test, reappearance test and application of sample recovery test are verified method, prove that this assay method is accurate, and be reliable.
Description of drawings:
The HPLC figure of Fig. 1 mixing control sample under chromatographic condition of the present invention;
Fig. 2 adopts the inventive method to separate the HPLC design sketch of effective constituent in the folium cortex eucommiae;
Fig. 3 adopts the inventive method to separate the HPLC design sketch of effective constituent in the Bark of Eucommia Ulmoides;
Fig. 4 adopts the inventive method to separate the HPLC design sketch of effective constituent in the eucommia leaf extract;
Wherein, each peak sign among the figure: No. 1 peak is Geniposidic acid; No. 2 peaks are chlorogenic acid; No. 3 peaks are Pinoresinol diglucoside; No. 4 peaks are rutin.
Embodiment
Below in conjunction with embodiment the present invention is described further.
Embodiment 1 method validation is learned experiment
1 linear relationship is investigated
It is an amount of that precision takes by weighing Pinoresinol diglucoside, Geniposidic acid, chlorogenic acid, control substance of Rutin, and being mixed with concentration with the methyl alcohol dissolving is 0.11mgmL
-1, 0.11mgmL
-1, 0.2mgmL
-1, 0.1mgmL
-1Mix reference substance solution, and the solution that is mixed with different series concentration injects high performance liquid chromatograph, measure peak area (chromatogram is seen Fig. 1) by above-mentioned chromatographic condition, take each reference substance sample size (μ g) as horizontal ordinate, peak area A is ordinate, the drawing standard curve, the results are shown in Table 3, the result shows that each reference substance peak area and concentration are the good linear relation
Table 3 linear relationship is investigated the result
3. Precision Experiment
The above-mentioned same reference substance solution 5 μ L of accurate absorption repeat sample introduction 6 times, record each composition peak area, and RSD all surpasses 3%, show that precision is good.The results are shown in Table 4:
Table 4 Precision Experiment result
| Mixed mark sampling volume (ul) | The GPA peak area | The CA peak area | The PDG peak area | The Rutin peak area |
| 5 | 627264 | 882242 | 837693 | 1254052 |
| 5 | 648236 | 890531 | 859230 | 1287373 |
| 5 | 620999 | 872635 | 829345 | 1245458 |
| 5 | 623683 | 872371 | 824917 | 1255959 |
| 5 | 609339 | 867938 | 854052 | 1213190 |
| 5 | 619346 | 871132 | 866713 | 1269238 |
| Mean value | 624811.1667 | 876141.5 | 845325 | 1254211.67 |
| Standard deviation | 12959.94268 | 8522.6583 | 17070.7375 | 24851.3469 |
| RSD(％) | 2.074217518 | 0.9727491 | 2.01942891 | 1.98143165 |
4. accuracy testing
The method accuracy verifies that by recovery of standard addition RSD% is all less than 5%, and the method for proof accuracy is high.The results are shown in Table 5:
Table 5 recovery of standard addition experimental result
The test of active component in embodiment 2 Bark of Eucommia Ulmoides
Be filling agent with octadecylsilane chemically bonded silica; Mobile phase A is methyl alcohol, and Mobile phase B is mass concentration 0.1% phosphate aqueous solution.Eluent gradient elution program and detect the wavelength program and see Table 1 and table 2: flow velocity: 0.8ml.min
-1Column temperature: 30 ℃; The sample introduction volume is 5 μ l.
The preparation of reference substance solution: it is an amount of that precision takes by weighing Pinoresinol diglucoside, Geniposidic acid, chlorogenic acid, control substance of Rutin, and being mixed with concentration with the methyl alcohol dissolving is 0.11mgmL
-1, 0.11mgmL
-1, 0.2mgmL
-1, 0.1mgmL
-1Mixing reference substance solution sample introduction, degree of separation is not less than 1.5, theoretical tray is not less than 5000.
The preparation of need testing solution: precision took by weighing bark of eucommia rawhide 5g behind 40 mesh sieves, put to add methyl alcohol 50mL in the round-bottomed flask extract 30min in the Microwave Extraction device, and extracting temperature is 60 ℃, and suction filtration is concentrated and be settled to 25mL, and HPLC measures.The result is measured as GPA, CA, PDG and Rutin content and is respectively 0.112%, 0.048%, 0.076% and 0.01%, and the chromatic graph spectrum is seen Fig. 3.
The test of active component in embodiment 3 folium cortex eucommiaes
Be filling agent with octadecylsilane chemically bonded silica; Mobile phase A is methyl alcohol, and Mobile phase B is 0.1% phosphate aqueous solution.The eluent gradient elution program detects the wavelength program and sees Table 1 and table 2: flow velocity: 0.8ml.min
-1Column temperature: 30 ℃; The sample introduction volume is 5 μ l.Column temperature is 30 ℃.
The preparation of reference substance solution: it is an amount of that precision takes by weighing Pinoresinol diglucoside, Geniposidic acid, chlorogenic acid, control substance of Rutin, and being mixed with concentration with the methyl alcohol dissolving is 0.11mgmL
-1, 0.11mgmL
-1, 0.2mgmL
-1, 0.1mgmL
-1Mixing reference substance solution sample introduction, degree of separation is not less than 1.5, theoretical tray is not less than 5000.
The preparation of need testing solution: precision took by weighing folium cortex eucommiae 2g behind 40 mesh sieves, put to add methyl alcohol 30mL in the round-bottomed flask extract 10min in the Microwave Extraction device, and extracting temperature is 60 ℃, and suction filtration is concentrated and be settled to 50mL, and HPLC measures.The result is measured as GPA, CA, PDG and Rutin content and is respectively 0.315,1.415%, 0.023% and 0.223%, and the chromatic graph spectrum is seen Fig. 2.
The test of active component in embodiment 4 eucommia leaf extracts
Be filling agent with octadecylsilane chemically bonded silica; Mobile phase A is methyl alcohol, and Mobile phase B is 0.1% phosphate aqueous solution.The eluent gradient elution program detects the wavelength program and sees Table 1 and table 2: flow velocity: 0.8ml.min
-1Column temperature: 30 ℃; The sample introduction volume is 5 μ l.Column temperature is 30 ℃.
The preparation of reference substance solution: it is an amount of that precision takes by weighing Pinoresinol diglucoside, Geniposidic acid, chlorogenic acid, control substance of Rutin, and being mixed with concentration with the methyl alcohol dissolving is 0.11mgmL
-1, 0.11mgmL
-1, 0.2mgmL
-1, 0.1mgmL
-1Mixing reference substance solution sample introduction, degree of separation is not less than 1.5, theoretical tray is not less than 5000.
The preparation of need testing solution: precision takes by weighing eucommia leaf extract 2g, puts to add methyl alcohol 50mL backflow 60min in the round-bottomed flask, filters and be settled to 50mL, dilutes 50 times again, and HPLC measures.The result is measured as GPA, CA, PDG and Rutin content and is respectively 7.85%, 8.9%, 0.16% and 0.257%, and the chromatic graph spectrum is seen Fig. 4.
Claims (1)
1. method of measuring simultaneously various active component content in the bark of eucommia adopts high-efficient liquid phase chromatogram technique analysis, it is characterized in that chromatographic condition is: be filling agent with octadecylsilane chemically bonded silica; The eluent gradient elution program is: mobile phase A is methyl alcohol, and Mobile phase B is that mass concentration is 0.1% phosphate aqueous solution; In the 0.01min-30.00min volumn concentration of Mobile phase B from 30% according to first-order linear step increase to 70%; When 30.00min-30.01min, be reduced to 30%, and maintenance 30% is constant in 30.01min-40.00min; Flow velocity: 0.8ml.min
-1Column temperature: 30 ℃; The sample introduction volume is 5 μ l;
Detecting the wavelength option program is: 0.01min-7.00min selects 254nm, and 7.00min-12.50min selects 277nm, and 12.50min-40.00min selects 254nm;
Wherein said various active composition is Geniposidic acid, chlorogenic acid, Pinoresinol diglucoside and rutin.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105044225A (en) * | 2015-05-12 | 2015-11-11 | 广西壮族自治区梧州食品药品检验所 | Test method of content of pinoresinol diglucoside in eucommia ulmoides |
| CN106124639A (en) * | 2016-03-31 | 2016-11-16 | 贵州医科大学 | The multicomponent content assaying method of Eucommia ulmoides |
| CN108627599A (en) * | 2018-07-31 | 2018-10-09 | 四川新绿色药业科技发展有限公司 | A kind of Cortex Eucommiae is the content assaying method that raw material is prepared into drug containing Cortex Eucommiae |
| CN111122745A (en) * | 2020-02-13 | 2020-05-08 | 江西普正制药股份有限公司 | Detection method of whole eucommia ulmoides capsules |
| CN112858530A (en) * | 2021-03-16 | 2021-05-28 | 江西普正制药股份有限公司 | Eucommia ulmoides leaf and eucommia ulmoides male flower extract and method for detecting multi-index components in products thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105044225A (en) * | 2015-05-12 | 2015-11-11 | 广西壮族自治区梧州食品药品检验所 | Test method of content of pinoresinol diglucoside in eucommia ulmoides |
| CN106124639A (en) * | 2016-03-31 | 2016-11-16 | 贵州医科大学 | The multicomponent content assaying method of Eucommia ulmoides |
| CN108627599A (en) * | 2018-07-31 | 2018-10-09 | 四川新绿色药业科技发展有限公司 | A kind of Cortex Eucommiae is the content assaying method that raw material is prepared into drug containing Cortex Eucommiae |
| CN111122745A (en) * | 2020-02-13 | 2020-05-08 | 江西普正制药股份有限公司 | Detection method of whole eucommia ulmoides capsules |
| CN111122745B (en) * | 2020-02-13 | 2022-04-19 | 江西普正制药股份有限公司 | Detection method of whole eucommia ulmoides capsules |
| CN112858530A (en) * | 2021-03-16 | 2021-05-28 | 江西普正制药股份有限公司 | Eucommia ulmoides leaf and eucommia ulmoides male flower extract and method for detecting multi-index components in products thereof |
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