CN110108808A - Method that is a kind of while evaluating oxidation-resistant active ingredient and content in downy rosemyrtle root - Google Patents
Method that is a kind of while evaluating oxidation-resistant active ingredient and content in downy rosemyrtle root Download PDFInfo
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- CN110108808A CN110108808A CN201910295995.1A CN201910295995A CN110108808A CN 110108808 A CN110108808 A CN 110108808A CN 201910295995 A CN201910295995 A CN 201910295995A CN 110108808 A CN110108808 A CN 110108808A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
Abstract
The present invention relates to a kind of methods evaluated simultaneously using inoxidizability and its content of the DPPH-UPLC to effective component gallic acid and ellagic acid in downy rosemyrtle root, comprising the following steps: the sample to be tested before reacting with DPPH and after reaction carries out Ultra Performance Liquid Chromatography instrument detection respectively；With the gallic acid and ellagic acid peak area of test solution after DPPH reaction before the DPPH reaction detected according to UPLC, gallic acid and ellagic acid content and clearance rate are calculated.The present invention can detect the inoxidizability and its content of active principle in downy rosemyrtle root simultaneously；And easy to operate, quick, reagent consumes less, high sensitivity, can be used for evaluating oxidation-resistant active ingredient and its content in downy rosemyrtle root medicinal material, provides theoretical foundation for the screening and further exploitation of hill gooseberry's oxidation-resistant active ingredient.
Description
Technical field
It is specially a kind of golden to peach using DPPH-UPLC the present invention relates to Chinese medicine oxidation-resistant active ingredient analysis field
The method that the inoxidizability and its content of effective component gallic acid and ellagic acid are evaluated simultaneously in ma's root.
Background technique
Principle active component in downy rosemyrtle root is gallic acid and ellagic acid, and gallic acid and ellagic acid have been found
With anti-oxidation efficacy, therefore, oxidation-resistant active ingredient and its content in downy rosemyrtle root medicinal material is evaluated, can be hill gooseberry's antioxygen
Change the screening of active constituent and further develop and theoretical foundation is provided.
Currently, evaluation Chinese medicine in the antioxidative method of effective component be mainly UV method, HPLC-UV-DPPH method,
HPLC-DPPH-UV-MS method etc..UV method is cumbersome, and reagent consumption is big, and time-consuming, poor reproducibility；Time-consuming for HPLC-UV-DPPH method；
HPLC-DPPH-UV-MS method instrument price is expensive, requires height to operator, popularity rate is not high.
Summary of the invention
The technical problem to be solved by the present invention is to, using ultra performance liquid chromatography (UPLC) have ultraspeed, sensitivity,
The principle that decrease is absorbed after the advantages that separating degree and DPPH and radical reaction, based on UPLC establish it is a kind of quickly, it is easy
The method for evaluating oxidation-resistant active ingredient and content in downy rosemyrtle root medicinal material simultaneously.
The present invention solves above-mentioned technical problem and is adopted the technical scheme that: a kind of golden to peach simultaneously using DPPH-UPLC
The method that the inoxidizability and its content of effective component gallic acid and ellagic acid are evaluated in ma's root, comprising the following steps:
(1) downy rosemyrtle root is identified；
(2) preparation of standard solution: precision weighs gallic acid reference substance respectively and ellagic acid reference substance is placed in 50mL palm fibre
In color tolerance measuring bottle, add methanol to dissolve and be diluted to scale, is made containing gallic acid 50~500 μ g/mL and 50~500 μ of ellagic acid
The mixed reference substance solution of g/mL；Face the used time, successively dilute above-mentioned standard solution with methanol respectively, is configured to the mark of various concentration
Quasi- working solution；
(3) preparation of test solution: dry downy rosemyrtle root is beaten into powder, is sieved, obtains downy rosemyrtle root medicinal material to be detected
Sample powder is set in drier and is stored；It accurately weighs 0.1~1.0g downy rosemyrtle root powder to be placed in 100mL stuffed conical flask, essence
Close addition 10~150mL methanol, weighed weight, the ultrasonic extraction 10 under the conditions of 50~1500W, 25~100kHz, 25~60 DEG C
~90min, lets cool, and filtering volatilizes, methanol dissolved residue, and be settled in 25mL volumetric flask.Accurate absorption 5mL is above-mentioned respectively
Solution is set in 10mL brown volumetric flask, and the DPPH solution for being separately added into methanol and 5.04mmol/L is settled to scale, is shaken up, room temperature
Under be protected from light 10~60min, obtain before DPPH reaction and test solution after DPPH reaction, all test solutions cross 0.2 μ
It is measured again after m filter membrane.
(4) ultra performance liquid chromatography detects, and chromatographic condition is as follows:
Chromatographic column: 2.1mm × 100mm, 1.6 μm of WatersT3 chromatographic column；Mobile phase: second
- 0.2% phosphoric acid of nitrile；Condition of gradient elution: 6% acetonitrile 0~2min, 6%~16% 2~3min of acetonitrile, 16%~20% acetonitrile 3
~8min, 6% 8~10min of acetonitrile；Detection wavelength: 240~350nm；Flow velocity: 0.1~0.5mL/min；Column temperature: 25~50 DEG C；
0.5~10 μ L of sampling volume.
(5) component content and its antioxidation evaluation: the DPPH that record UPLC is measured reacts preceding and DPPH reaction
The gallic acid of test solution and ellagic acid peak area afterwards calculate clearance rate: clearance rate=1-A/A according to following formula0, wherein A0
Compound peaks area before reacting for DPPH, A are compound peaks area after DPPH reaction, evaluate gallic acid and tan in downy rosemyrtle root
Spend the content and oxidation resistance of acid.
The present invention using DPPH-UPLC to the inoxidizability of effective component gallic acid in downy rosemyrtle root and ellagic acid and its
The method that content is evaluated simultaneously, has the advantages that compared with prior art:
(1) present invention can detect the inoxidizability and its content of active principle in downy rosemyrtle root simultaneously；
(2) operation of the present invention is easy, quick, reagent consumes less, high sensitivity.
(3) present invention can be used for evaluating oxidation-resistant active ingredient and its content in downy rosemyrtle root medicinal material, can be hill gooseberry
The screening of oxidation-resistant active ingredient and further exploitation provide theoretical foundation.
Detailed description of the invention
After Fig. 1 is test sample before gallic acid in the present invention and ellagic acid mixing reference substance, DPPH reaction and DPPH reaction
The UPLC chromatogram of test solution, wherein A represents gallic acid and ellagic acid mixing reference substance, and B is that DPPH reaction is preceding for examination
Product, C are test sample after DPPH reaction, and peak 1 is gallic acid, and peak 2 is ellagic acid.
Specific embodiment
Below with reference to specific implementation method and attached drawing, technical scheme is described further.
The downy rosemyrtle root medicinal material place of production used by two embodiments as described below is respectively Guangxi Yulin and Guangxi Gold show, warp
Guangxi Plant Inst. Wen Fang assistant researcher is accredited as downy rosemyrtle root.
Embodiment 1
(1) configuration of standard solution: precision weighs appropriate gallic acid reference substance respectively and ellagic acid reference substance is placed in
In 50mL brown volumetric flask, add methanol to dissolve and be diluted to scale, is made containing 98.72 μ g/mL of gallic acid and ellagic acid 74.01
The mixed reference substance solution of μ g/mL；Face the used time, successively dilutes above-mentioned standard solution with methanol respectively, be configured to various concentration
Standard working solution；
(2) sample pre-treatments: the drying downy rosemyrtle root that the place of production is Guangxi Gold show is beaten into powder, is sieved, obtains peach to be detected
Golden ma's root herb sample powder, sets in drier and stores；0.5g fine powder is accurately weighed to be placed in 100mL stuffed conical flask, it is accurate
50mL methanol, weighed weight is added, ultrasonic (350W, 53kHz, 35 DEG C) extracts 40min, let cool, filter, volatilize, methanol dissolution
Residue, and be settled in 25mL volumetric flask.The accurate above-mentioned solution of 5mL of drawing is set in 10mL brown volumetric flask respectively, is separately added into
The DPPH solution of methanol and 5.04mmol/L are settled to scale, shake up, are protected from light 30min at room temperature, before obtaining DPPH reaction
With test solution after DPPH reaction, it is measured again after crossing 0.2 μm of filter membrane.
(3) chromatographic condition: chromatographic column: WatersT3 chromatographic column (2.1mm × 100mm, 1.6 μ
m)；Mobile phase: -0.2% phosphoric acid of acetonitrile；Condition of gradient elution: 0~2min, 6% acetonitrile；2~3min, 6%~16% acetonitrile；3
~8min, 16%~20% acetonitrile；8~10min, 6% acetonitrile；Detection wavelength: 271nm；Flow velocity: 0.25mL/min；Column temperature: 35
℃；5 μ L of sampling volume.
(4) component content and its antioxidation evaluation: clearance rate: clearance rate=1-A/A is calculated according to following formula0(A0For
Compound peaks area before DPPH reacts, A are compound peaks area after DPPH reaction).Gallic acid in Guangxi Gold show downy rosemyrtle root
1 is shown in Table with the content and its clearance rate of ellagic acid.The gallic acid in downy rosemyrtle root and ellagic acid all have relatively strong as the result is shown
Oxidation resistance, wherein the oxidation resistance of ellagic acid be higher than gallic acid.
The content and clearance rate (n=3) of 1 gallic acid of table and ellagic acid
Embodiment 2
(1) configuration of standard solution is the same as embodiment 1.
(2) sample pre-treatments: the drying downy rosemyrtle root that the place of production is Guangxi Yulin is beaten into powder, is sieved, obtains peach to be detected
Golden ma's root herb sample powder, sets in drier and stores；0.5g fine powder is accurately weighed to be placed in 100mL stuffed conical flask, it is accurate
50mL methanol, weighed weight is added, ultrasonic (350W, 53kHz, 40 DEG C) extracts 30min, let cool, filter, volatilize, methanol dissolution
Residue, and be settled in 25mL volumetric flask.The accurate above-mentioned solution of 5mL of drawing is set in 10mL brown volumetric flask respectively, is separately added into
The DPPH solution of methanol and 5.04mmol/L are settled to scale, shake up, are protected from light 30min at room temperature, before obtaining DPPH reaction
With test solution after DPPH reaction, it is measured again after crossing 0.2 μm of filter membrane.
(3) chromatographic condition is the same as embodiment 1.
(4) component content and its antioxidation evaluation: clearance rate: clearance rate=1-A/A is calculated according to following formula0(A0For
Compound peaks area before DPPH reacts, A are compound peaks area after DPPH reaction).Gallic acid in Guangxi Yulin downy rosemyrtle root
2 are shown in Table with the content and its clearance rate of ellagic acid.The gallic acid in downy rosemyrtle root and ellagic acid all have relatively strong as the result is shown
Oxidation resistance, wherein the oxidation resistance of ellagic acid be higher than gallic acid.
The content and clearance rate (n=3) of 2 gallic acid of table and ellagic acid
Claims (4)
1. it is a kind of using DPPH-UPLC simultaneously to the inoxidizability of effective component gallic acid in downy rosemyrtle root and ellagic acid and its
The method that content is evaluated, which comprises the following steps:
(1) downy rosemyrtle root is identified；
(2) preparation of standard solution: precision weighs gallic acid reference substance respectively and ellagic acid reference substance is placed in brown volumetric flask
In, add methanol to dissolve and be diluted to scale, is made mixed containing 50~500 μ g/mL of 50~500 μ g/mL of gallic acid and ellagic acid
Close reference substance solution；Face the used time, successively dilute above-mentioned standard solution with methanol respectively, is configured to the standard work of various concentration
Liquid；
(3) preparation of test solution: dry downy rosemyrtle root is beaten into powder, is sieved, obtains downy rosemyrtle root medicinal material sample to be detected
Powder is set in drier and is stored；It weighs downy rosemyrtle root powder to be placed in stuffed conical flask, weighed weight after methanol dissolution is added,
10~90min of ultrasonic extraction under the conditions of 50~1500W, 25~100kHz, 25~60 DEG C, lets cool, and filtering volatilizes, and first is added
Alcohol dissolved residue and constant volume；
It draws acquired solution respectively again to set in two brown volumetric flasks, is separately added into methanol and DPPH solution constant volume, shakes up, room temperature
Under be protected from light 10~60min, obtain DPPH reaction before and DPPH reaction after test solution；
(4) ultra performance liquid chromatography detects, and chromatographic condition is as follows:
Chromatographic column: 2.1mm × 100mm, 1.6 μm of WatersT3 chromatographic column；Mobile phase: acetonitrile-
0.2% phosphoric acid；Condition of gradient elution: 6% acetonitrile 0~2min, 6%~16% acetonitrile 2~3min, 16%~20% acetonitrile 3~
8min, 6% 8~10min of acetonitrile；Detection wavelength: 240~350nm；Flow velocity: 0.1~0.5mL/min；Column temperature: 25~50 DEG C；Into
0.5~10 μ L of sample volume；
(5) it component content and its antioxidation evaluation: is supplied before the DPPH reaction that record UPLC is measured and after DPPH reaction
The gallic acid and ellagic acid peak area of test sample solution calculate clearance rate: clearance rate=1-A/A according to following formula0；Wherein, A0For
Compound peaks area before DPPH reacts, A are compound peaks area after DPPH reaction；Evaluate gallic acid and tan flower in downy rosemyrtle root
The content and oxidation resistance of acid.
2. the method according to claim 1, wherein in the step (3), concrete operations are as follows: accurately weigh 0.1
~1.0g downy rosemyrtle root powder is placed in 100mL stuffed conical flask, and precision is added 10~150mL methanol, weighed weight, 50~
1500W, 25~100kHz, 10~90min of ultrasonic extraction under the conditions of 25~60 DEG C, let cool, and filter, volatilize, and methanol dissolution is added
Residue is simultaneously settled in 25mL volumetric flask；
Accurate 5mL acquired solution of drawing is set in two 10mL brown volumetric flasks respectively again, is separately added into the DPPH solution of methanol sum
It is settled to scale, is shaken up, is protected from light 10~60min at room temperature, obtains test solution after the preceding reaction with DPPH of DPPH reaction.
3. according to the method described in claim 2, it is characterized in that, the DPPH solution concentration used is 5.04mmol/L.
4. according to the method described in claim 3, it is characterized in that, the test sample before gained DPPH reaction and after DPPH reaction is molten
Liquid is measured again after first passing through 0.2 μm of filter membrane.
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Cited By (1)
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Application publication date: 20190809 |