CN108303485A - A kind of method of winter pear tea quality conformance assessment - Google Patents
A kind of method of winter pear tea quality conformance assessment Download PDFInfo
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Abstract
The invention discloses a kind of method of winter pear tea quality conformance assessment, the appraisal procedure includes：Liquid phase test sample is prepared, is divided into two parts, it is spare；First part of liquid phase test sample is subjected to liquid phase analysis, obtains original chromatogram A, and the peak area of each main peak is calculated；The DPPH clearance rates Q1 of first part of liquid phase test sample is measured using DPPH methods；DPPH alcoholic solutions are added in second part of liquid phase test sample, after reaction, the chromatogram B after liquid phase analysis is reacted, and the peak area of each main peak is calculated；The DPPH clearance rates Q2 of second part of liquid phase test sample after DPPH alcoholic solutions are added is measured using DPPH methods；By establishing the correlation analysis between Δ X and Δ Q.It can more accurately reflect the relationship between " active material " and its " antioxidant activity " using new analysis method, improve traditional Chinese medicine quality evaluation system.
Description
Technical field
The present invention relates to Chinese medicine quality controling research fields, and in particular, to a kind of winter pear tea quality conformance assessment
Method.
Background technology
Chinese pear-leaved crabapple is a kind of fossil plants, is distributed widely in China Guangxi, the southern band in Hunan.The Chinese pear-leaved crabapple system rose family, Malus.At present
Country's distribution has two kind of sharp mouth Chinese pear-leaved crabapple and Fructus Mali formosanae.Fructus Mali formosanae (Malusdoumeri (Bois) chev.) is arbor
Or megaphanerophyte, it is high that up to 15m, sprig is cylindrical, and puce, close quilt and thin villosity, fall off when old when young, taupe,
It is hairless；Blade oblong is to ovum shape lanceolar, and long 9-15cm, wide 4-6cm, apex is tapering, base portion wide wedge shape or subcircular, side
Edge has serrated irregular, and two sides have hair when young gradually falls off.Chinese pear-leaved crabapple is also known as winter pear, wild pears, sugared chestnut, birchleaf pear.
Chinese pear-leaved crabapple effect mainly has the relieving summer-heat that quenches the thirst, changes and stagnant go addiction, control rush down dysentery, lower gas, control that cholera collywobbles, dissolving phlegm, to control all cold
Product lump in the abdomen, the deficiency of qi in middle-jiao change all wind phlegm stagnation of the circulation of vital energys like the non-malaria of malaria.Pertinent literature is reported：Containing much to human body in Fructus Mali formosanae
Vitamin E and vitamin C etc. in beneficial nutritional health ingredient, such as threonine in amino acid, methionine and vitamin,
And a variety of chemical compositions such as flavonoids, Polyphenols, terpene, sterols, saponin(e and organic acid with and iron, copper, zinc multi mineral
Matter ingredient.Also there is Chinese pear-leaved crabapple anti-cancer, anti-aging to be depressured, anti-oxidant, liver protection, be good for the multiple efficacies such as tooth, meanwhile, Chinese pear-leaved crabapple can also carry
High immunity can play certain bacteriums, mould and yeast certain inhibiting effect.
Currently, be widely recognized based on the Chinese medicine quality controling research of " spectrum-effect " relationship, chemically substance
Spectrum and the double angle of bioactivity detection carry out quality analysis research.And how more accurately to embody " active material " and
Relationship between " activity " is still worth further investigation, to develop more accurate appraisement system.
Invention content
The object of the present invention is to provide a kind of methods of winter pear tea quality conformance assessment, solve traditional Chinese medicine fingerprint
The ingredient of the winter pear tea method for evaluating quality detection of atlas calculation is more single, it is difficult to comprehensively be carried out to the ingredient in winter pear tea
The problem of discriminating.
To achieve the goals above, the present invention provides a kind of method of winter pear tea quality conformance assessment, the assessments
Method includes：
(1) winter pear tea and the first alcoholic solution are mixed, obtains filtrate after filtering, filtrate and the second alcoholic solution is mixed to get
Liquid phase test sample, is divided into two parts, spare；
(2) first part of liquid phase test sample is subjected to liquid phase analysis, obtains original chromatogram A, and each main peak is calculated
Peak area；The DPPH clearance rates Q1 of first part of liquid phase test sample is measured using DPPH methods；
(3) DPPH alcoholic solutions, after reaction, the chromatography after liquid phase analysis is reacted are added in second part of liquid phase test sample
Scheme B, and the peak area of each main peak is calculated；Second part of liquid phase test sample after DPPH alcoholic solutions are added is measured using DPPH methods
DPPH clearance rates Q2；
(4) by canonical correlation analysis, the correlation between Δ X and Δ Q is established；Wherein, Δ X indicates each in step (2)
The difference of the peak area of each main peak in the peak area and step (3) of main peak, Δ Q indicate the difference of Q1 and Q2.
Through the above technical solutions, the present invention provides a kind of method of winter pear tea quality conformance assessment, the assessment
Method includes：Winter pear tea and the first alcoholic solution are mixed, filtrate is obtained after filtering, filtrate and the second alcoholic solution is mixed to get liquid
Phase test sample, is divided into two parts, spare；First part of liquid phase test sample is subjected to liquid phase analysis, obtains original chromatogram A, and calculate
Obtain the peak area of each main peak；The DPPH clearance rates Q1 of first part of liquid phase test sample is measured using DPPH methods；In second part of liquid phase
DPPH alcoholic solutions are added in test sample, after reaction, the chromatogram B after liquid phase analysis is reacted, and each main peak is calculated
Peak area；The DPPH clearance rates Q2 of second part of liquid phase test sample after DPPH alcoholic solutions are added is measured using DPPH methods；By more
The correlation between Δ X and Δ Q is established in first linear analysis；This method reacts front and back chromatography by liquid phase test sample with DPPH
Peak area changes difference and carries out modeling analysis with the difference to DPPH clearance rates, can not only find specific activity in winter pear tea
Substance, using correlation analysis can more accurately embody active material with it is anti-oxidant between be associated with, can be more accurate
Relationship between ground quantitative description active material and antioxidant activity provides new for the quality evaluation of winter pear tea and Conformance Assessment
Method and foundation.
Other features and advantages of the present invention will be described in detail in subsequent specific embodiment part.
Description of the drawings
Attached drawing is to be used to provide further understanding of the present invention, an and part for constitution instruction, with following tool
Body embodiment is used to explain the present invention together, but is not construed as limiting the invention.In the accompanying drawings：
Fig. 1 is chromatogram B after being reacted in embodiment 1；
Fig. 2 is chromatogram B after being reacted in embodiment 2；
Fig. 3 is chromatogram B after being reacted in embodiment 3；
Fig. 4 is chromatogram B after being reacted in embodiment 4；
Fig. 5 is content of material and activity relationships analysis chart in test case；
Fig. 6 be in test case active material to active Contribution Analysis.
Specific implementation mode
The specific implementation mode of the present invention is described in detail below.It should be understood that described herein specific
Embodiment is merely to illustrate and explain the present invention, and is not intended to restrict the invention.
The endpoint of disclosed range and any value are not limited to the accurate range or value herein, these ranges or
Value should be understood as comprising the value close to these ranges or value.For numberical range, between the endpoint value of each range, respectively
It can be combined with each other between the endpoint value of a range and individual point value, and individually between point value and obtain one or more
New numberical range, these numberical ranges should be considered as specific open herein.
The present invention provides a kind of method of winter pear tea quality conformance assessment, the appraisal procedure includes：
(1) winter pear tea and the first alcoholic solution are mixed, obtains filtrate after filtering, filtrate and the second alcoholic solution is mixed to get
Liquid phase test sample, is divided into two parts, spare；
(2) first part of liquid phase test sample is subjected to liquid phase analysis, obtains original chromatogram A, and each main peak is calculated
Peak area；The DPPH clearance rates Q1 of first part of liquid phase test sample is measured using DPPH methods；
(3) DPPH alcoholic solutions, after reaction, the chromatography after liquid phase analysis is reacted are added in second part of liquid phase test sample
Scheme B, and the peak area of each main peak is calculated；Second part of liquid phase test sample after DPPH alcoholic solutions are added is measured using DPPH methods
DPPH clearance rates Q2；
(4) by canonical correlation analysis, the correlation between Δ X and Δ Q is established；Wherein, Δ X indicates each in step (2)
The difference of the peak area of each main peak in the peak area and step (3) of main peak, Δ Q indicate the difference of Q1 and Q2.
In a kind of preferred embodiment of the present invention, for the kind of Active substance in accurate discrimination winter pear tea
Class, the substance that Δ X is changed significantly are Active substance, and then identify related activity using efficient liquid phase time-of-flight mass spectrometry (TOFMS)
The type of substance.Here Active substance refers to the free radical scavenger ingredient in winter pear tea, when DPPH is clear with free radical
When being coexisted except agent, the lone electron of DPPH free radicals is paired, and color can decline, the removing of fading extent and free radical scavenger
There are quantitative relationships for ability and quantity, therefore, can be by evaluating the removing feelings of free radical in the measurement of this wavelength absorbance
Condition.
In a kind of preferred embodiment of the present invention, in order to accurately measure the related activity object in winter pear tea sample
Matter, relative to 1g winter pear tea, the dosage of the first alcoholic solution is 9-11mL；Relative to the filtrate of 5mL, the dosage of the second alcoholic solution is
19-21mL。
In a kind of preferred embodiment of the present invention, in order to effectively extract the active ingredient in winter pear tea, the first alcohol
The volumetric concentration of solution is 55-65%.
In a kind of preferred embodiment of the present invention, in order to effectively extract the active ingredient in winter pear tea, the second alcohol
The volumetric concentration of solution is 55-65%.
In a kind of preferred embodiment of the present invention, in order to accurately measure the related activity object in winter pear tea sample
Matter, relative to second part of liquid phase test sample of 0.1mL, the dosage of DPPH alcoholic solutions is 0.18-0.22mL.
In a kind of preferred embodiment of the present invention, in order to accurately measure the related activity object in winter pear tea sample
Matter, DPPH alcoholic solutions are made of the DPPH solution and 0.12-0.13mL alcoholic solutions of a concentration of 3.75mg/mL of 0.07-0.08mL.
In a kind of preferred embodiment of the present invention, in order to accurately measure the related activity object in winter pear tea sample
Matter, alcoholic solution are one or more in ethanol solution, ethylene glycol solution and methanol solution.
In a kind of preferred embodiment of the present invention, in order to enable occurring more peak in liquid phase analysis so that
Baseline is stablized relatively, in liquid phase analysis, with 0.1% formic acid (A)-acetonitrile (B) for mobile phase.
In a kind of preferred embodiment of the present invention, in order to enable occurring more peak in DPPH methods, in DPPH
In method, the ranging from 253-255nm of wavelength selection.
The present invention will be described in detail by way of examples below.
Embodiment 1
The preparation of liquid phase test sample：Winter pear tea sample 1g is weighed, is added in 60% ethyl alcohol to 10mL volumetric flasks, ultrasonic 30min,
Centrifugation filters to get filtered fluid, takes filtered fluid 5mL, add in 60% ethyl alcohol to 20mL volumetric flasks to get liquid phase test sample；
Liquid phase test sample is equally divided into 2 parts, first part of liquid phase test sample 0.1mL is subjected to liquid phase analysis, is obtained original
Chromatogram A, and the peak area of each main peak is calculated；The condition of liquid phase analysis is：Agilent EXPLE chromatographic columns (250mm ×
4.6mm, 5 μm), 0.1% formic acid of mobile phase (A) and acetonitrile (B), gradient elution program：0~24min, 10~18%B；24~
30min, 18~20%B；30~40min, 20~23%B；40~60min, 23~32%B；Column temperature：25 DEG C, volume flow：
1.0mL/mL, sample size：5 μ L, Detection wavelength：254nm is deaerated using preceding ultrasound, and each component in mobile phase is with 0.22 μ
M filtering with microporous membrane, preparation mobile phase are pure water；
The DPPH clearance rates Q1 of first part of liquid phase test sample is measured using DPPH methods；
Take second part of liquid phase test sample 0.1mL, the middle DPPH solution (a concentration of 3.75mg/mL) that 0.075mL is added and
0.125mL methanol, after reaction, chromatogram B (see Fig. 1) after liquid phase analysis is reacted, and the peak face of each main peak is calculated
Product；The condition of liquid phase analysis is same as above.
The DPPH clearance rates Q2 of second part of liquid phase test sample after DPPH alcoholic solutions are added is measured using DPPH methods.
Embodiment 2
It is carried out according to the method for embodiment 1, unlike, second part of liquid phase test sample 0.1mL is taken, middle addition 0.05mL's
DPPH solution (a concentration of 3.75mg/mL) and 0.15mL methanol, after reaction, chromatogram B is (see figure after liquid phase analysis is reacted
2), and the peak area of each main peak is calculated.
Embodiment 3
It is carried out according to the method for embodiment 1, unlike, second part of liquid phase test sample 0.1mL is taken, middle addition 0.1mL's
DPPH solution (a concentration of 3.75mg/mL) and 0.1mL methanol, after reaction, chromatogram B is (see figure after liquid phase analysis is reacted
3), and the peak area of each main peak is calculated.
Embodiment 4
It is carried out according to the method for embodiment 1, unlike, take second part of liquid phase test sample 0.1mL, middle addition 0.125mL
DPPH solution (a concentration of 3.75mg/mL) and 0.075mL methanol, after reaction, the chromatogram B after liquid phase analysis is reacted
(see Fig. 4), and the peak area of each main peak is calculated.
Test case
16 parts of winter pear tea sample is taken, is carried out according to the method for embodiment 1, before respectively obtaining the active material reaction filtered out
Peak area difference afterwards and the difference to DPPH clearance rates, are as a result listed in table 1.Further use polytomy variable homing method pair
Correlation between Δ X and Δ Q is analyzed, and using regression coefficient compare the importance of this six active materials
Compared with as a result such as Fig. 1 and Fig. 2.
The analysis of 1 winter pear tea actual sample of table measures.
The increase with DPPH concentration is can be seen that by the chromatogram in embodiment 1-4, peak disappears more, comprehensive
Consider, the additive amount in embodiment 1 is most preferred scheme.
It can be obtained in conjunction with table 1, Fig. 5 and Fig. 6, by combining forecasts algorithm, we construct between Δ X and Δ Q
Correlation；By multiple linear regression analysis, we can find a linear syntagmatic to reflect " active material-
Relationship between DPPH clearance rates ", to the correlation between clear active material and antioxidant activity.As can be seen from Fig. 5, I
Utilize modeling analysis, obtain the linear model between active material and antioxidant activity, and using 95% confidence level as standard,
Sample in normal line shows preferable antioxidant activity consistency, that is, quality is more consistent.In addition, utilizing structure
Model regression coefficient further can carry out importance evaluation to the active material filtered out, and coefficient is bigger, illustrates the substance pair
Active contribution is bigger.In figure 6 as it can be seen that substance 4 has maximum active contribution degree, thus select this substance as winter pear tea
Quality evaluation index can more accurately reflect the quality of winter pear tea.
This research and utilization sample reacts front and back chromatographic peak area variation difference and the difference to DPPH clearance rates with DPPH
Modeling analysis is carried out, can not only find specific active material in winter pear tea, it can be more accurately using correlation analysis
Embody active material with it is anti-oxidant between be associated with, correlation that can more accurately between qualitative assessment active material and activity
Property, provide new method and foundation for the quality evaluation of winter pear tea and Conformance Assessment.
The preferred embodiment of the present invention has been described above in detail, still, during present invention is not limited to the embodiments described above
Detail can carry out a variety of simple variants to technical scheme of the present invention within the scope of the technical concept of the present invention, this
A little simple variants all belong to the scope of protection of the present invention.
It is further to note that specific technical features described in the above specific embodiments, in not lance
In the case of shield, can be combined by any suitable means, in order to avoid unnecessary repetition, the present invention to it is various can
The combination of energy no longer separately illustrates.
In addition, various embodiments of the present invention can be combined randomly, as long as it is without prejudice to originally
The thought of invention, it should also be regarded as the disclosure of the present invention.
Claims (9)
1. a kind of method of winter pear tea quality conformance assessment, which is characterized in that the appraisal procedure includes：
(1) winter pear tea and the first alcoholic solution are mixed, obtains filtrate after filtering, filtrate and the second alcoholic solution is mixed to get liquid phase
Test sample is divided into two parts, spare；
(2) first part of liquid phase test sample is subjected to liquid phase analysis, obtains original chromatogram A, and the peak face of each main peak is calculated
Product；The DPPH clearance rates Q1 of first part of liquid phase test sample is measured using DPPH methods；
(3) DPPH alcoholic solutions are added in second part of liquid phase test sample, after reaction, the chromatogram B after liquid phase analysis is reacted,
And the peak area of each main peak is calculated；Second part of liquid phase test sample after DPPH alcoholic solutions are added is measured using DPPH methods
DPPH clearance rates Q2；
(4) by canonical correlation analysis, the correlation between Δ X and Δ Q is established；Wherein, Δ X indicates each main peak in step (2)
Peak area and step (3) in each main peak peak area difference, Δ Q indicates the difference of Q1 and Q2.
2. according to the method described in claim 1, wherein, the substance that Δ X is changed significantly is Active substance, utilizes efficient liquid
Phase time-of-flight mass spectrometry (TOFMS) analyzes the type of Active substance.
3. method according to claim 1 or 2, wherein in step (1), relative to 1g winter pear tea, the first alcoholic solution
Dosage is 9-11mL；Relative to the filtrate of 5mL, the dosage of the second alcoholic solution is 19-21mL.
4. according to the method described in claim 3, wherein, the volumetric concentration of the first alcoholic solution is 55-65%；And/or
The volumetric concentration of second alcoholic solution is 55-65%.
5. according to the method described in claim 3, wherein, relative to second part of liquid phase test sample of 0.1mL, DPPH alcoholic solutions
Dosage is 0.18-0.22mL.
6. according to the method described in claim 5, wherein, DPPH alcoholic solutions are by a concentration of 3.75mg/mL's of 0.07-0.08mL
DPPH solution and 0.12-0.13mL alcoholic solutions composition.
7. according to the method described in claim 6, wherein, alcoholic solution is in ethanol solution, ethylene glycol solution and methanol solution
It is one or more.
8. according to the method described in claim 3, being flowing with 0.1% formic acid (A)-acetonitrile (B) in liquid phase analysis wherein
Phase.
9. according to the method described in claim 3, wherein, in DPPH methods, ranging from 253-255nm that wavelength is selected.
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Application publication date: 20180720 |