CN102599519A - Method for preparing peony stamen extract - Google Patents
Method for preparing peony stamen extract Download PDFInfo
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- CN102599519A CN102599519A CN2012100951667A CN201210095166A CN102599519A CN 102599519 A CN102599519 A CN 102599519A CN 2012100951667 A CN2012100951667 A CN 2012100951667A CN 201210095166 A CN201210095166 A CN 201210095166A CN 102599519 A CN102599519 A CN 102599519A
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Abstract
The invention relates to a method for preparing a peony stamen extract, and particularly researches an extraction, separation and purification method for functional substances with antioxidant ability in peony stamens and prepares an extract with antioxidant ability. When the peony stamen extract prepared by the method is used in detection of scavenging of ABTS (2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate)) free radicals, the inhibitory concentration (IC50) of the extract is 0.914mg/mL when 50% of ABTS free radicals are scavenged, and therefore, the peony stamen extract prepared by the method has antioxidant ability and is beneficial for delaying senescence, enhancing immunity, beautifying and caring skin and the like.
Description
(1) technical field
The present invention relates to a kind of method for preparing peony stamen medicinal extract, specifically studied the extraction, the isolation and purification method that have the effect material of oxidation resistance in the peony stamen, and prepared medicinal extract, belong to the new resource for food development field with oxidation resistance.
(2) background technology
Tree peony
Paeonia suffruticosa, Paeoniaceae, Paeonia originate in the western part of China Qinling Mountains and Daba Mountain one band mountain area.Tree peony is the distinctive woody rare flower of China; Large flower and brilliant color, elegant and poised, gorgeous dignity, fragrant strong; And various in style, have " aromatic ", " king's in spending " laudatory title, for a long time by people as rich and honour lucky, flourishing symbol.Most prestigious with Luoyang tree peony, Heze tree peony.Tree peony is removed outside the ornamental value, and its petal was used as food materials one over thousands of year, and the root of tree peony is called " root bark of tree peony " through processing, is a kind of important medicinal material, goes into the heart, liver, kidney three warps, heat-clearing is arranged, invigorate blood circulation, change function such as silt.Yet the stamen of peony but is not fully utilized always.After the annual florescence, a large amount of tree peony stamens are dropped with plant, have caused huge waste.Present technique is to extraction, the separation and purification of effect material in the peony stamen of local flavor delicate fragrance, golden yellow color and prepare medicinal extract research, in the hope of better development and use peony stamen, realizes the great development of tree peony industry and flowers health functional food.
(3) summary of the invention
In order better to develop the peony stamen; Realize the great development of tree peony industry and flowers health functional food; The invention provides a kind of method for preparing peony stamen medicinal extract, purpose is extraction medium, the method for separation and purification and the IC50 value of condition and removing ABTS free radical thereof of effect material in the research peony stamen.
A kind of method for preparing peony stamen medicinal extract may further comprise the steps:
1) the peony stamen is obtained peony stamen effect material in 40-60 minute according to the mass volume ratio 1:16-18 immersion with pure water and slightly carry solution.
2) with step 1 obtain slightly carry solution through Rotary Evaporators after being concentrated into the 1/3-1/4 of original volume under 50 ℃ of temperature, extract 3-5 time under normal temperature condition through benzinum, ethyl acetate, totally until the material removal that is dissolved in benzinum, ethyl acetate.Thaw after 12 hours and filter and ethanol evaporation is got extract carrying out freezing alcohol precipitation below-18 ℃ with absolute ethyl alcohol then.
3) extract that step 2 is obtained is through high performance liquid chromatography, and chromatographic condition is: methyl alcohol: water (containing 0.1% glacial acetic acid) (v/v)=3:7, flow velocity 1.0 mL/min, sample size 10.0 uL detect wavelength 266 nm, temperature is a room temperature, times 80 min; Regather extract.
4) extract that step 3 is obtained carries out column chromatographic isolation and purification.Macroreticular resin AB-8 is adopted wet method dress post, go up appearance towards waiting for to water pH 7.0 backs with deionized water.After adopting deionized water, 30% ethanol, 60% ethanol and absolute ethyl alcohol elution samples after the appearance on the extract respectively, flow out component through efficient liquid phase chromatographic analysis; Liquid phase chromatogram condition is: methyl alcohol: water (containing 0.1% glacial acetic acid) (v/v)=3:7; Flow velocity 1.0 mL/min, sample size 10.0 uL detect wavelength 266 nm; Temperature is a room temperature, times 80 min.Whether the liquid chromatogram collection of illustrative plates decision set according to different component divides contained material identical, and the liquid that will contain same substance merges, with the liquid after merging through Rotary Evaporators under 50 ℃ of temperature, be concentrated into organic solvent-free steam concentrate.
5) concentrate that step 4 is obtained advances half preparative high-performance liquid chromatographic appearance and prepares peony stamen medicinal extract, and the preparative chromatography condition is: Jia Chun ︰ water (containing 0.1% glacial acetic acid)=3 ︰ 7 (v/v), wavelength 266 nm; Flow velocity 10.0 mL/min; Sample size 2mL, temperature is a room temperature, the time is 70min.When half preparative chromatograph figure composes the peak, collect the effect material, the effect material liq after collecting is concentrated into organic solvent-free through Rotary Evaporators steams and promptly obtain peony stamen medicinal extract under 50 ℃ of temperature.
The anti-at low temperatures preservation of the medicinal extract that utilizes the present invention to prepare is drunk after can reconstituting with hot water, and tempting, the golden color of fragrance uniqueness is drunk the back and gladdened the heart and refresh the mind.The peony stamen medicinal extract that utilizes the present invention to obtain is removed the ABTS free radical detect, the concentration (IC50) that draws medicinal extract when removing 50% ABTS free radical is 0.914 mg/mL.The peony stamen medicinal extract that this explanation utilizes the present invention to prepare has oxidation resistance, will help human body to delay senility, increase immunity, beauty care etc.
Beneficial effect of the present invention: this method is a raw material with the peony stamen, the research of starting extraction, separation and purification and the oxidation resistance of peony stamen effect material, and obtain peony stamen medicinal extract with health role.This method is that the comprehensive utilization and the peony health functional food of peony stamen opened up new field.
This method has been confirmed extraction medium, solid-liquid ratio, liquid chromatogram, half preparative high-performance liquid chromatographic condition and the separation and purification of effect material in the peony stamen with column material and oxidation resistance etc., for exploitation peony stamen new type of health functional food has been established theoretical foundation.
The specific embodiment:
The extraction medium of effect material research in the embodiment 1 peony stamen
Get 4 parts on 0.500g peony stamen; Place four small beakers, add 8mL pure water, ethanol, benzinum, ethyl acetate respectively, soak after 1 hour; Observe the color of solution, can find the yellow that pure water solution shows ethanolic solution petroleum ether solution ethyl acetate solution.4 kinds of solution that filtration obtains carry out full wavelength scanner respectively, then dilute the back repetitive operation if the tack peak occurs, in full wavelength scanner figure till the no tack peak.Drawing pure water behind the result of 4 samples of analysis is optimum solvent.Peony stamen and pure water ratio are 1:16 (m/v).
The high performance liquid chromatography of effect material separates and the research of half preparative chromatography preparation condition in the embodiment 2 peony stamens
Confirm that by the full wavelength scanner result detecting wavelength is 266nm.Set 90 minutes single task time; Sample size 10 μ L, flow velocity 1mL/min, temperature room temperature; Attempt methyl alcohol respectively: the water ratio is the flowing phase of 5 ︰, 5,4 ︰ 6,3 ︰, 7,2 ︰ 8 and 9 ︰ 1; Add 0. 1% glacial acetic acid in the water, mixing before flowing phase is used, sonicated 10 minutes is to remove bubble wherein.Find that 3 ︰ 7 are optimal proportion after analyzing each liquid chromatogram, and no longer go out the peak after 60 minutes.Further find the methyl alcohol of 3 ︰ 7 after the ratio of trial 3.5 ︰ 6.5,2.5 ︰ 7.5: the water ratio still is best flowing phase ratio.And then draw the liquid phase chromatogram condition that finally is used for various sample analysis: 60 minutes single task time, sample size 10 μ L, flow velocity 1mL/min, the temperature room temperature, flowing phase is the methyl alcohol of 3 ︰ 7: add the pure water solution of 0.1% glacial acetic acid, the detection wavelength is 266nm.The condition that half preparative chromatography prepares effect material in the peony stamen is: Jia Chun ︰ water (containing 0.1% glacial acetic acid)=3 ︰ 7, wavelength 266nm, and flow velocity 10mL/min, sample size 2mL, temperature is a room temperature, the time is 70min.
The screening of the different column materials of effect separating substances purifying in the embodiment 3 peony stamens
Water behind pure water crude extract process benzinum, the ethyl acetate extraction is waited for appearance on the column chromatography.Present technique adopts polyamide, macroreticular resin AB-8, three kinds of column chromatography materials of macroreticular resin XAD-16 to carry out the test of separation and purification effect comparison.Appearance on the wet method is adopted in experiment; Get appearance on the sample after 20mL concentrates; Use pure water, 30% ethanol, 60% ethanol and absolute ethyl alcohol to carry out wash-out successively; Elution speed is controlled at 40-50 and drips/min, and the below connects liquid according to the wash-out pigment zone with test tube, indicates that eluent concentration and numbering back employing high performance liquid chromatography carry out separating effect relatively.Testing result is a macroreticular resin AB-8 separation and purification best results.
Embodiment 4 peony stamen medicinal extract are removed the IC50 value of ABTS free radical and are confirmed
Extract is carried out sample according to concentration gradient 1,1/2,1/5,1/10,1/20 to be prepared.Ready sample adds 3.6mL ABTS solution, uses the oscillator mixing, reacts 6 minutes, processes negative controls as specimen by aforesaid operations with 0.4mL water simultaneously, and 3 of making are parallel under every kind of concentration.With sample behind the ultraviolet specrophotometer test reaction and the absorbance of negative controls under the 734nm wavelength, the mean value of getting three parallel laboratory test gained results calculates.By formula (A negative control-A) * 100/A negative control is tried to achieve ABTS free radical scavenging activity (%).With ABTS free radical scavenging activity (%) is the longitudinal axis, and the concentration of specimen is the transverse axis mapping, draws regression straight line, and ABTS free radical scavenging activity (%) is that 50 o'clock sample concentration is the 503nhibiting concentration of sample to the ABTS free radical, IC50.The result is 0.914 mg/mL for the IC50 value.
The preparation of embodiment 5 peony stamen medicinal extract
1) peony stamen and pure water are soaked according to mass volume ratio 1:16 obtained peony stamen effect material in 40 minutes and slightly carry solution;
2) with step 1 obtain slightly carry solution through Rotary Evaporators be concentrated under 50 ℃ of temperature original volume 1/3 after extract 3 times under 25 ℃ of conditions through benzinum, ethyl acetate, remove totally until the material that is dissolved in benzinum, ethyl acetate.Then with absolute ethyl alcohol carry out below-18 ℃ freezing alcohol precipitation thaw after 12 hours filter and with ethanol evaporation fall extract;
3) extract that step 2 is obtained is through half preparative high-performance liquid chromatographic appearance, and chromatographic condition is: methyl alcohol: water (containing 0.1% glacial acetic acid) (v/v)=3:7, flow velocity 1.0 mL/min; Sample size 10.0 uL; Detect wavelength 266 nm, 25 ℃ of temperature, times 80 min.Regather extract.
4) extract with 3 acquisitions carries out column chromatographic isolation and purification.Macroreticular resin AB-8 is adopted wet method dress post, go up appearance towards waiting for to water pH 7.0 backs with deionized water.After adopting deionized water, 30% ethanol, 60% ethanol and absolute ethyl alcohol elution samples after the appearance on the extract respectively, flow out component through efficient liquid phase chromatographic analysis; Liquid phase chromatogram condition is: methyl alcohol: water (containing 0.1% glacial acetic acid) (v/v)=3:7; Flow velocity 1.0 mL/min, sample size 10.0 uL detect wavelength 266 nm; 25 ℃ of temperature, times 80 min.Whether the liquid chromatogram collection of illustrative plates decision set according to different component divides contained material identical, and the liquid that will contain same substance merges, with the liquid after merging through Rotary Evaporators under 50 ℃ of temperature, be concentrated into organic solvent-free steam concentrate.
5) concentrate that step 4 is obtained advances half preparative high-performance liquid chromatographic appearance and prepares effect material in the peony stamen; The preparative chromatography condition is: Jia Chun ︰ water (containing 0.1% glacial acetic acid)=3 ︰ 7 (v/v); Wavelength 266 nm, flow velocity 10.0 mL/min, sample size 2mL; 25 ℃ of temperature, the time is 70min.Collect the effect material at every turn when half preparative chromatograph collection of illustrative plates goes out the peak, the effect material liq after collecting is concentrated into organic solvent-free through Rotary Evaporators steams and obtain peony stamen medicinal extract under 50 ℃ of temperature.
Benzinum used in the present invention, ethyl acetate are that analysis is pure.
Claims (1)
1. method for preparing peony stamen medicinal extract is characterized in that may further comprise the steps:
1) the peony stamen is obtained peony stamen effect material in 40-60 minute according to the mass volume ratio 1:16-18 immersion with pure water and slightly carry solution;
2) with step 1 obtain slightly carry solution through Rotary Evaporators after being concentrated into the 1/3-1/4 of original volume under 50 ℃ of temperature, extract 3-5 time under normal temperature condition through benzinum, ethyl acetate, totally until the material removal that is dissolved in benzinum, ethyl acetate; Thaw after 12 hours and filter and ethanol evaporation is got extract carrying out freezing alcohol precipitation below-18 ℃ with absolute ethyl alcohol then;
3) extract that step 2 is obtained is through high performance liquid chromatograph, and chromatographic condition is: methyl alcohol: water (containing 0.1% glacial acetic acid) (v/v)=3:7, flow velocity 1.0 mL/min, sample size 10.0 uL detect wavelength 266 nm, temperature is a room temperature, times 80 min; Regather extract;
4) extract that step 3 is obtained carries out column chromatographic isolation and purification; Macroreticular resin AB-8 is adopted wet method dress post, go up appearance towards waiting for to water pH 7.0 backs with deionized water; After adopting deionized water, 30% ethanol, 60% ethanol and absolute ethyl alcohol elution samples after the appearance on the extract respectively, flow out component through the high performance liquid chromatograph analysis; Liquid phase chromatogram condition is: methyl alcohol: water (containing 0.1% glacial acetic acid) (v/v)=3:7; Flow velocity 1.0 mL/min, sample size 10.0 uL detect wavelength 266 nm; Temperature is a room temperature, times 80 min; Whether the liquid chromatogram collection of illustrative plates decision set according to different component divides contained material identical, and the liquid that will contain same substance merges, with the liquid after merging through Rotary Evaporators under 50 ℃ of temperature, be concentrated into organic solvent-free steam concentrate;
5) concentrate that step 4 is obtained advances half preparative high-performance liquid chromatographic appearance and prepares peony stamen medicinal extract, and the preparative chromatography condition is: Jia Chun ︰ water (containing 0.1% glacial acetic acid)=3 ︰ 7 (v/v), wavelength 266 nm; Flow velocity 10.0 mL/min; Sample size 2mL, temperature is a room temperature, the time is 70min; When half preparative chromatograph figure composes the peak, collect the effect material, the effect material liq after collecting is concentrated into organic solvent-free through Rotary Evaporators steams and promptly obtain peony stamen medicinal extract under 50 ℃ of temperature.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103524578A (en) * | 2013-10-11 | 2014-01-22 | 青岛农业大学 | Method for extraction and separation of paeoniflorin compounds from stamens of peony |
| CN103960729A (en) * | 2014-05-19 | 2014-08-06 | 山东金保罗食品饮料有限公司 | Low-sugar black garlic flower beverage and preparation method thereof |
| CN110507622A (en) * | 2019-09-20 | 2019-11-29 | 洛阳国花坊牡丹生物科技有限公司 | A kind of manufacture craft of peony bud piece |
| CN111616362A (en) * | 2020-06-29 | 2020-09-04 | 山东省经济林管理站 | Walnut male flower antioxidant and preparation method and application thereof |
| CN116143651A (en) * | 2023-03-08 | 2023-05-23 | 西北农林科技大学 | Efficient separation and purification method of peony stamen tri-tonkinensis spermidine |
| CN116850221A (en) * | 2023-07-20 | 2023-10-10 | 山东省科学院菏泽分院 | Peony stamen and semen cuscutae compound and preparation method and application thereof |
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2012
- 2012-03-31 CN CN2012100951667A patent/CN102599519A/en active Pending
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103524578A (en) * | 2013-10-11 | 2014-01-22 | 青岛农业大学 | Method for extraction and separation of paeoniflorin compounds from stamens of peony |
| CN103524578B (en) * | 2013-10-11 | 2016-06-08 | 青岛农业大学 | A kind of method of extraction and isolation paeoniflorin compound from tree peony stamen |
| CN103960729A (en) * | 2014-05-19 | 2014-08-06 | 山东金保罗食品饮料有限公司 | Low-sugar black garlic flower beverage and preparation method thereof |
| CN103960729B (en) * | 2014-05-19 | 2015-09-30 | 山东金保罗食品饮料有限公司 | The black garlic drink made from flower of low sugar and preparation method |
| CN110507622A (en) * | 2019-09-20 | 2019-11-29 | 洛阳国花坊牡丹生物科技有限公司 | A kind of manufacture craft of peony bud piece |
| CN111616362A (en) * | 2020-06-29 | 2020-09-04 | 山东省经济林管理站 | Walnut male flower antioxidant and preparation method and application thereof |
| CN111616362B (en) * | 2020-06-29 | 2022-03-08 | 山东省林业保护和发展服务中心 | Walnut male flower antioxidant and preparation method and application thereof |
| CN116143651A (en) * | 2023-03-08 | 2023-05-23 | 西北农林科技大学 | Efficient separation and purification method of peony stamen tri-tonkinensis spermidine |
| CN116143651B (en) * | 2023-03-08 | 2024-07-05 | 西北农林科技大学 | Efficient separation and purification method of peony stamen tri-tonkinensis spermidine |
| CN116850221A (en) * | 2023-07-20 | 2023-10-10 | 山东省科学院菏泽分院 | Peony stamen and semen cuscutae compound and preparation method and application thereof |
| CN116850221B (en) * | 2023-07-20 | 2024-06-11 | 山东省科学院菏泽分院 | Peony stamen and semen cuscutae compound and preparation method and application thereof |
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Application publication date: 20120725 |