CN104280499A - Method for controlling quality of medicinal material, portulaca oleracea L. - Google Patents
Method for controlling quality of medicinal material, portulaca oleracea L. Download PDFInfo
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Abstract
The invention provides a method for controlling the quality of a medicinal material, portulaca oleracea L.. The method is achieved by a high performance liquid chromatography. The medicinal material, portulaca oleracea L., contains complex and various chemical components; ferulic acid is one of active ingredients of portulaca oleracea L.. The ferulic acid is used as a quality control index component in the method. The ferulic acid of the medicinal material, portulaca oleracea L., can be stably and feasibly detected by screening pretreatment methods and chromatographic conditions of the medicinal material.
Description
Technical field
The present invention relates to the method for quality control of purslane medicinal material.
Background technology
Purslane (Classification system: Portulaca oleracea L.) is Portulacaceae annual herb plant.Plump succulence, without hair, high 10 ~ 30cm, is born in the area without shade such as roadside, field and ruins, flower garden.All there is distribution domestic various places.This kind is medicine food dual purpose plant.Herb hyoscine, has clearing heat and promoting diuresis, removing toxicity for detumescence, anti-inflammatory, quenches the thirst, diuresis; Seed improving eyesight.
Containing fatty acid, terpene, alkaloid, cumarin, flavones, phenolic acid and volatilization wet goods number of chemical composition in purslane.Modern study shows, purslane has antibacterial, anti-inflammatory analgetic, relaxed muscle, controls wound and neuroprotection, and anti-oxidant and delaying senility function significantly.
Current in the quality control of purslane medicinal material, be its index components mainly with flavones ingredient.But inventor finds in the research of prior art, be separated from purslane at present and obtained oleracein A ~ E, N-trans asafoetide acyl group tyrasamine etc., be also separated simultaneously and obtain the phenolic acid compounds such as forulic acid.Containing one or two phenolic hydroxyl group in purslane amide structure, the hydrogen atom of phenolic hydroxyl group can by free radical capture, there is certain antioxidation activity, simultaneously, oleracein A ~ D is the derivant of p-Coumaric Acid and forulic acid, report is had to show, p-Coumaric Acid and forulic acid etc. can remove superoxide radical, reduce MDA content, there is very strong antioxidation activity (Yang Zijuan, the chemical composition of purslane and antioxidation activity research, Jilin Agriculture University, 2008. the 2nd, 3,5,13 page).The alkaloid component of purslane---oleracein B, D and N-trans asafoetide acyl group tyrasamine is ferulic acid derivative, has forulic acid to generate after hydrolysis.
Show the research of forulic acid, this compound has spectrum bacteriostasis, anti-oxidant, neuroprotective; platelet aggregation-against, suppresses platelet 5-HT release, suppresses the generation of platelet thrombus element A2 (TXA2); strengthen prostaglandin activity, analgesia, the effects such as alleviating vascular spasm.As can be seen here, the drug activity of forulic acid and the activity of purslane medicinal material have higher consistance.
Therefore, in view of said structure and drug activity feature, inventor thinks, forulic acid and there is the alcaloid-derivatives of forulic acid structure, significant contribution is had to effect of purslane medicinal material, if can detect the ferulic acid and its derivatives in purslane medicinal material, the quality of purslane medicinal material more effectively can be controlled.
Summary of the invention
From prior art, purslane alkaloid has certain biologically active, and as antioxidation, oleracein B, D and N-trans asafoetide acyl group tyrasamine is also ferulic acid derivative, has forulic acid to generate after hydrolysis; And the free forulic acid contained in purslane medicinal material, be the important activity composition in purslane medicinal material too.Therefore, measure the content of forulic acid in purslane medicinal material, just effectively can detect in purslane the total amount of the ferulic acid and its derivatives with physiologically active, thus realize the control to purslane medicinal material quality.
Particularly, the invention provides the method for quality control of purslane medicinal material, it comprises following operation steps:
(1) preparation of need testing solution:
Get purslane, pulverize, accurately weighed, extracting in water, merges extract, upper large pore resin absorption column, use water, 70%v/v ethanol elution successively, collect ethanol eluate, after reclaiming ethanol, after adding strong acid hydrolysis, be extracted with ethyl acetate, collect extraction into ethyl acetate layer, except desolventizing, drying, filters after dissolving, obtains need testing solution;
(2) preparation of reference substance solution: be reference substance with forulic acid, namely obtains reference substance solution after dissolving;
(3) respectively by need testing solution and reference substance solution injection liquid chromatography, external standard method is adopted to calculate ferulaic acid content in purslane; Chromatographic condition is as follows:
Chromatographic column: C18 chromatographic column; Determined wavelength: 316nm; Mobile phase: methyl alcohol-1.0%v/v glacial acetic acid solution=20:80v/v.
The present invention's research shows, only under above-mentioned mobile phase condition, can be separated with assorted peak by forulic acid main peak well, degree of separation is greater than 1.5, ensure that degree of accuracy and the reappearance of assay.
Further, described macroporous absorbent resin is D101, HPD450 or H103.
Further, described macroporous absorbent resin is D101.
Further, adding the strong acid hydrolysis time is 15min ~ 120min.
Further, adding the strong acid hydrolysis time is 30min.
Wherein, strong acid of the present invention is selected from hydrochloric acid, sulfuric acid or phosphoric acid.Wherein, the final concentration of hydrochloric acid is 1% ~ 5%w/v (g/ml).Certainly, the final concentration of sulfuric acid or phosphoric acid also can be converted into according to conventionally calculation mode.
Described final concentration, after namely strong acid joins concentrate, the strong acid concentration calculated according to whole liquid volume.
Wherein, flow rate of mobile phase: 1.0ml/min.
Wherein, chromatographic column temperature: 30 DEG C.
Wherein, in step (1), the solvent of need testing solution is mobile phase.
Wherein, in step (2), the solvent of reference substance solution is 70% methanol solution.
Purslane medicinal material chemical composition complexity is various, and forulic acid is one of its effective constituent, and the present invention chooses the index components of forulic acid as quality control.The present invention, by the examination to medicinal material pre-treating method, chromatographic condition etc., finally achieves and stablizes feasible detection to purslane medicinal material forulic acid.
The embodiment of form by the following examples, is described in further detail foregoing of the present invention again.But this should be interpreted as that the scope of the above-mentioned theme of the present invention is only limitted to following example.All technology realized based on foregoing of the present invention all belong to scope of the present invention.
Accompanying drawing explanation
Fig. 1 reference substance chromatogram
Fig. 2 test sample chromatogram
Fig. 3 is without the test sample chromatogram of macroporous resin treatment
Fig. 4. mobile phase (acetonitrile-0.085% phosphoric acid solution 17:83)
Fig. 5. mobile phase (acetonitrile-0.085% potassium dihydrogen phosphate 17:83)
Fig. 6. mobile phase (methyl alcohol-1.0% glacial acetic acid 30:70)
Embodiment
Embodiment 1 detection method
The extraction of sample forulic acid
(1) extract: precision takes purslane powder (the new lotus prepared slices of Chinese crude drugs in Sichuan 1208021 batches) 5.0g, adds 50ml pure water, adds hot reflux 30min, filter, repeat 3 times, merging filtrate.
(2) purifying: the macroreticular resin (D101) crossing 20ml volume, loading speed≤3BV/hr; Impurity elution, 80ml pure water, elution speed 2BV/hr; Effective constituent wash-out, 80ml 70% ethanol, elution speed 2BV/hr, collects 70% ethanol eluate, and 60 DEG C revolve steaming extremely without ethanol taste, and remaining liq is about 12ml.
(3) acidolysis: add 10ml 2%HCl and be about 1%w/v (g/ml) to hydrochloric acid final concentration, add hot reflux 30min, cooling.
(4) extract: be extracted with ethyl acetate 3 times, each 20ml, combined ethyl acetate liquid, revolve and steam to dry.Dissolve with HPLC mobile phase and be settled to 10ml, crossing 0.45 μm of filter membrane, obtain need testing solution, detect with HPLC.
The preparation of reference substance solution:
Get forulic acid reference substance 9.54mg, put in brown volumetric flask, add 70% methyl alcohol and dissolve and constant volume, be prepared into the reference substance solution of variable concentrations.
HPLC detects the chromatographic condition of ferulaic acid content:
C18 chromatographic column;
Detecting device: UV-detector;
Mobile phase: methyl alcohol-1.0% glacial acetic acid solution (20:80), what wherein (20:80) represented is the volume ratio of mobile phase composition;
Flow rate of mobile phase: 1.0ml/min;
Chromatographic column temperature: 30 DEG C;
Determined wavelength: 316nm;
Sample size: 10 μ l.
Reference substance solution is injected liquid chromatography (chromatogram is shown in Fig. 1), after detecting by above-mentioned condition, obtained typical curve; Need testing solution is injected liquid chromatography (chromatogram is shown in Fig. 2), after detecting by above-mentioned condition, calculate the content of forulic acid according to external standard method.
Embodiment 2 methodological study
1, linear relationship
Precision takes forulic acid reference substance 9.54mg, puts in the brown volumetric flask of 25mL, adds 70% methyl alcohol and dissolves and constant volume, in contrast product storing solution.A certain amount of reference substance storing solution of accurate absorption, puts brown volumetric flask and mixes and add 70% methanol constant volume respectively, and form the reference substance solution of 6 concentration, concentration is as follows:
Table 1 forulic acid sample size and peak area response
| Concentration (μ g mL -1) | Sampling volume (μ L) | Sample size (μ g) | Peak area response |
| 19.080 | 10 | 0.19080 | 790606 |
| 30.528 | 10 | 0.30528 | 1352804 |
| 38.160 | 10 | 0.38160 | 1683455 |
| 45.792 | 10 | 0.45792 | 2091109 |
| 53.424 | 10 | 0.53424 | 2462784 |
| 61.056 | 10 | 0.61056 | 2826070 |
With peak area (y) to concentration (x, μ g mL
-1) carry out linear regression, obtain regression equation: y=48603x-141448, R
2=0.9996.
2 precision tests
(concentration is 29.016 μ g mL to get reference substance solution 10 μ l
-1), continuous sample introduction 5 times, with retention time and calculated by peak area mean value and relative standard deviation.
| Repeat | RT | Area |
| 1 | 54.047 | 1271022 |
| 2 | 54.036 | 1268125 |
| 3 | 54.040 | 1264268 |
| 4 | 54.052 | 1270896 |
| 5 | 54.061 | 1269785 |
| Mean | 54.047 | 1268819 |
| RSD(％) | 0.02％ | 0.22％ |
Result shows, and the retention time of forulic acid main peak and the RSD of peak area are respectively 0.02% and 0.22%, show that the precision of instrument is good.
3 reappearance tests
Get same batch sample totally 6 parts, every part of 5.0g, accurately weighed, prepare sample solution by sample extraction step, sample introduction measures.
| Repeat | Area | C(μg?mL -1) |
| 1 | 1710201 | 38.097 |
| 2 | 1690957 | 37.701 |
| 3 | 1717662 | 38.251 |
| 4 | 1674243 | 37.358 |
| 5 | 1728090 | 38.465 |
| 6 | 1663311 | 37.133 |
| Mean | 1697410 | 37.834 |
| RSD(％) | 1.50％ | 1.27％ |
Result shows, and the RSD of forulic acid concentration is 1.27%, shows this condition favorable reproducibility.
4 sample stabilities
Get same part sample solution, respectively at 2,6,12,18,24h sample introduction 10 μ l measures,
| Time (h) | Area | C(μg?mL -1) |
| 2 | 1727311 | 38.449 |
| 6 | 1728870 | 38.482 |
| 12 | 1721330 | 38.326 |
| 18 | 1716877 | 38.235 |
| 24 | 1714778 | 38.192 |
| RSD(％) | 0.36％ | 0.33％ |
The RSD of result display forulic acid concentration is 0.33%, shows in sample solution stable in 24h.
Brief summary (following result and analysis are all make on the basis of embodiment 1 with batch medicinal material):
(1) during trial test in early stage, also adopt 70% methyl alcohol (10 times of volumes), add hot reflux 1.5h, filter, repeat the method for 3 times.The method is extracted ferulaic acid content in purslane and is not significantly improved, and length consuming time, do not have water extraction to get economical and practical yet.Therefore, the present invention selects extraction process by water.
(2) during preliminary experiment in early stage, without the sample of macroporous resin purification, the concentration of the forulic acid obtained is only 16% (concentration about 6 μ g mL before purifying of sample after purifying
-1, after purifying, concentration is 38 μ g mL
-1), and HPLC chromatogram is mixed, peak is a lot, the severe jamming detection (see Fig. 3) of forulic acid main peak.
(3), during preliminary experiment in early stage, without the sample of acidolysis, ferulaic acid content is very low, and concentration is only 5 μ g mL
-1.The selection of HCl final concentration, has used the variable concentrations of 1%, 2%, 3% and 5%w/v, found that and improves the content that HCl concentration does not improve the forulic acid detected.
In different plant, the existence form of forulic acid is different, and in purslane, forulic acid exists mainly with the form in conjunction with state or derivant, and therefore the present invention needs to carry out acidolysis to extract, thus detects the content of forulic acid to greatest extent.
(4), during preliminary experiment in early stage, the selection of mobile phase has been carried out.Have employed different mobile phases to be separated: acetonitrile-0.085%v/v phosphoric acid solution (17:83), acetonitrile-0.02mol/L potassium dihydrogen phosphate (17:83) (PH2.5), methyl alcohol-1.0%v/v glacial acetic acid (25:75), methyl alcohol-5.0%v/v glacial acetic acid (30:70), under these four kinds of mobile phase conditions, main peak cannot reach baseline separation, degree of separation is all less than 1.5, and its degree of separation and symmetry all cannot reach the corresponding requirement of Chinese Pharmacopoeia.Finally have selected methyl alcohol-glacial acetic acid system, the ratio of 20:80, be separated with assorted peak by forulic acid main peak well, degree of separation is greater than 1.5, meets the requirement (see Fig. 4 ~ 6) of Chinese Pharmacopoeia to degree of separation.
Claims (10)
1. the detection method of purslane medicinal material, is characterized in that: it comprises following operation steps:
(1) preparation of need testing solution:
Get purslane, pulverize, accurately weighed, extracting in water, merges extract, upper large pore resin absorption column, use water, 70%v/v ethanol elution successively, collect ethanol eluate, after reclaiming ethanol, after adding strong acid hydrolysis, be extracted with ethyl acetate, collect extraction into ethyl acetate layer, except desolventizing, drying, filters after dissolving, obtains need testing solution;
(2) preparation of reference substance solution: be reference substance with forulic acid, namely obtains reference substance solution after dissolving;
(3) respectively by need testing solution and reference substance solution injection liquid chromatography, external standard method is adopted to calculate ferulaic acid content in purslane; Chromatographic condition is as follows:
Chromatographic column: C18 chromatographic column; Determined wavelength: 316nm; Mobile phase: methyl alcohol-1.0%v/v glacial acetic acid solution=20:80v/v.
2. detection method according to claim 1, is characterized in that: described macroporous absorbent resin is D101, HPD450 or H103.
3. detection method according to claim 2, is characterized in that: described macroporous absorbent resin is D101.
4. detection method according to claim 1, is characterized in that: the acid hydrolysis time is 15min ~ 120min.
5. detection method according to claim 4, is characterized in that: adding the strong acid hydrolysis time is 30min.
6. detection method according to claim 1 or 5, is characterized in that: described strong acid is selected from hydrochloric acid, sulfuric acid or phosphoric acid.
7. detection method according to claim 6, is characterized in that: the final concentration of hydrochloric acid is 1% ~ 5%w/v.
8. detection method according to claim 1, is characterized in that: flow rate of mobile phase: 1.0ml/min, chromatographic column temperature: 30 DEG C.
9. detection method according to claim 1, is characterized in that: in step (1), the solvent of need testing solution is mobile phase.
10. detection method according to claim 1, is characterized in that: in step (2), the solvent of reference substance solution is 70%v/v methanol solution.
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| CN102526138A (en) * | 2012-01-19 | 2012-07-04 | 贾晓斌 | Composition of active components from fresh purslane for decreasing blood sugar, and preparation method thereof |
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| CN102526138A (en) * | 2012-01-19 | 2012-07-04 | 贾晓斌 | Composition of active components from fresh purslane for decreasing blood sugar, and preparation method thereof |
Non-Patent Citations (4)
| Title |
|---|
| NACIYE ERKAN: "Antioxidant activity and phenolic compounds of fractions from Portulaca oleracea L.", 《FOOD CHEMISTRY》, vol. 133, 4 February 2012 (2012-02-04) * |
| NACIYE ERKAN等: "Antioxidant activities of Sideritis congesta Davis et Huber-Morath and Sideritis arguta Boiss et Heldr: Identification of free flavonoids and cinnamic acid derivatives", 《FOOD RESEARCH INTERNATIONAL》, vol. 44, 31 December 2011 (2011-12-31) * |
| 郑智音: "鲜马齿苋多糖、生物碱和多酚类组分的制备及其降血糖活性研究", 《中草药》, vol. 45, no. 18, 30 September 2014 (2014-09-30) * |
| 陶霞娟等: "烟草木质素合成途径几个中间代谢物HPLC分析", 《北京林业大学学报》, vol. 27, no. 5, 30 September 2005 (2005-09-30), pages 111 - 114 * |
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