CN109856265A - Longan study on chemical compositions of leaves evaluation method based on anti-oxidant and hypoglycemic spectrum effect relationship - Google Patents
Longan study on chemical compositions of leaves evaluation method based on anti-oxidant and hypoglycemic spectrum effect relationship Download PDFInfo
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Abstract
The invention discloses a kind of longan study on chemical compositions of leaves evaluation methods based on anti-oxidant and hypoglycemic spectrum effect relationship, this method is by establishing the HPLC finger-print at longan leaf opposed polarity position, common characteristic peaks are demarcated, the anti-oxidant and hypoglycemic effect of chemical component in spectrum effect mathematical analysis model evaluation longan leaf is passed through.The method of the present invention has convenient succinct, objectivity is strong, reproducibility is strong, the advantages that characteristic peak is clear, the chemical component in longan leaf with anti-oxidant and hypoglycemic effect can quickly and accurately be evaluated, scientific and effective method is provided for the effective substance research and quality control of longan leaf, provides reference for anti-oxidant and hypoglycemic effect drug or health food for further exploitation longan leaf.
Description
Technical field
The present invention relates to a kind of methods for evaluating chemical composition of Chinese materia medica drug activity, and in particular to one kind is based on anti-oxidant
With the longan study on chemical compositions of leaves evaluation method of hypoglycemic spectrum effect relationship.
Background technique
Longan leaf is Chinese medicine simply with Guangxi national characters, is recorded in " this sketch of the southern regions of the Yunnan Province is said " earliest.It is famous
Subtropical fruit -- longan (Dimocarpus longan Lour.) tree leaf or tender shoots.Its nature and flavor is sweet, light, flat, has
Deliver heat-clearing, eliminating damp, detoxifying and other effects.It is mainly used for treating cold, fever, malaria, furunculosis, eczema etc..Longan originates in China
The south subtropics region in south and North Vietnam, has more than 2000 years cultivation histories in China.China's Longan Cultivation area and production
Amount occupies first place in the world.South China longan leaf resource reserves are abundant, and after annual longan is plucked, orchard worker cuts trees
Branch, a large amount of leaf is abandoned or burns, and results in waste of resources and environmental pollution.
Longan leaf mainly contains the ingredients such as tannin, phenolic acid class, flavonoids and volatile oil.It is domestic at present to longan leaf chemistry at
The research report divided is few, in terms of being concentrated mainly on flavones ingredient.Inventor once to longan leaf carried out system chemistry at
Divide research, it is found that its main active is flavone component.Although it is apparent hypoglycemic that existing research shows that longan leaf has, resist
The pharmacological actions such as oxidation, but relevant research both at home and abroad report it is less, inventor once to its hypoglycemic effect, antioxidation into
Gone research, find its type II diabetes resisting, inhibit alpha-glucosidase activity, the active component of antioxidation is mainly second
Acetoacetic ester position, and its main active be lupeol, progallin A, Kaempferol, luteolin, Quercetin,
Astragalin.The longan leaf hypoglycemic effect that inventor once carried out is studied, the results showed that separation is extracted from longan leaf
The ingredients such as resulting lupeol, progallin A, Kaempferol, luteolin, Quercetin, astragalin are to alpha-glucosaccharase
Enzyme all has a strong inhibitory effect.Inventor has also carried out content assaying method research to these active constituents, it is determined that simultaneously
The content assaying method of this 5 kinds of ingredients is measured, while also to its chief active position --- the finger-print of ethyl acetate extract
It is studied, establishes the method for building up and its finger-print of longan leaf ethyl acetate extract finger-print.
Traditional Chinese medicine fingerprint can control traditional Chinese medicine quality on the whole, and finger-print is mutually tied with herbal medicine efficacy index
It closes, passes through some Mathematical Statistic Analysis Methods such as Partial Least Squares, Bivariate analysis method and grey Relational Analysis Method
Deng doing correlation analysis to finger-print and pharmacodynamics index, potential effective substance is found.The present inventor is in early-stage study
Middle discovery longan leaf has the function of inhibiting animal body lipid peroxidating and hypoglycemic, but the result of study of related effective substance
It is still indefinite.Therefore, the present inventor is on the basis of early period is to the pharmacology of longan leaf, medicine and quality controling research, with dragon
Based on the HPLC finger-print at eye leaf opposed polarity position, using grey relational grade analysis and Pearson relevant function method, grind
Study carefully that longan leaf is anti-oxidant, hypoglycemic spectrum effect relationship.Relevant report is had no at present.
The disclosure of background above technology contents is only used for auxiliary and understands inventive concept and technical solution of the invention, not
The prior art for necessarily belonging to present patent application shows above content in the applying date of present patent application in no tangible proof
In the case where having disclosed, above-mentioned background technique should not be taken to the novelty and creativeness of evaluation the application.
Summary of the invention
In order to solve the above technical problems, clear longan leaf effective substance, the present invention provides one kind based on anti-oxidant
With the longan study on chemical compositions of leaves evaluation method of hypoglycemic spectrum effect relationship, can comprehensively and systematically evaluate has antioxygen in longan leaf
The chemical component and active ingredient screening of change and hypoglycemic effect mention for the effective substance research and quality control of longan leaf
For scientific and effective method.
The technical solution adopted in the present invention is as follows:
A kind of longan study on chemical compositions of leaves evaluation method based on anti-oxidant and hypoglycemic spectrum effect relationship, this method pass through foundation
The HPLC finger-print at longan leaf opposed polarity position demarcates common characteristic peaks, imitates mathematical analysis model evaluation longan by spectrum
The anti-oxidant and hypoglycemic effect of chemical component in leaf.
Further, the above-described longan study on chemical compositions of leaves evaluation side based on anti-oxidant and hypoglycemic spectrum effect relationship
Method includes the following steps:
S1. longan leaf opposed polarity position is prepared；
S2. the HPLC finger-print at longan leaf opposed polarity position is established；
S3. common characteristic peaks are demarcated；
S4. the DPPH free radical scavenging activity at longan leaf opposed polarity position is measured；
S5. the alpha-glucosaccharase enzyme inhibition rate at longan leaf opposed polarity position is measured；
S6., the hypoglycemic activity anti-oxidant with it by the HPLC Fingerprints peak data at longan leaf opposed polarity position
Data, it is for statistical analysis through chemometrics method, evaluate the drug activity of chemical component in longan leaf.
Further, the above-described longan study on chemical compositions of leaves evaluation side based on anti-oxidant and hypoglycemic spectrum effect relationship
Method prepares the method at longan leaf opposed polarity position in the step S1 are as follows:
R1. longan leaf coarse powder is taken, with successively heating and refluxing extraction 2 times of 95% ethyl alcohol, 50% ethyl alcohol, each 2h is filtered, and is closed
And filtrate, ethyl alcohol is recycled to no alcohol taste, obtains total extract part；
R2. total extract part is taken, adds water to be suspended in separatory funnel, successively uses opposed polarity solvent: petroleum ether, acetic acid respectively
Ethyl ester, n-butanol are extracted, until the extract liquor clear of every kind of solvent, the extraction for merging variant polar solvent respectively are molten
Liquid, it is spare through the solvent-extracted aqueous solution of opposed polarity；
R3. by the extraction solution recycling design of the variant polar solvent after merging, volatilize, respectively obtain ethyl acetate portion
Position, n-butanol portion；The solvent-extracted aqueous solution of opposed polarity of learning from else's experience, is evaporated, the position get Shui；
R4. longan leaf coarse powder is separately taken, water is added, heating and refluxing extraction 2h is filtered, and filtrate concentration volatilizes, and is obtained decocting liquid and is mentioned
Extracting portion position.
Further, the above-described longan study on chemical compositions of leaves evaluation side based on anti-oxidant and hypoglycemic spectrum effect relationship
Method, the HPLC finger-print for establishing longan leaf opposed polarity position, includes the following steps:
T1. the preparation of the test solution at opposed polarity position
Precision weighs total extract part, ethyl acetate extract, n-butanol portion respectively, respectively with methanol ultrasonic dissolution, from
The heart takes supernatant, compound concentration 50mgmL-1The total extract part of longan leaf, ethyl acetate extract, n-butanol portion for examination
Product solution；
Precision weighs water position, decocting liquid extract position respectively, respectively with 50% methanol ultrasonic dissolution, be centrifuged, take on
Clear liquid, compound concentration 50mgmL-1The test solution at longan leaf water position, decocting liquid extract position；
T2. the preparation of mixed reference substance solution
Precision weighs progallin A, astragalin, Quercetin, luteolin reference substance, and methanol is added to dissolve, constant volume,
Progallin A 0.10mgmL must be contained-1, 0.15mgmL containing astragalin-1, 0.12mgmL containing Quercetin-1, contain sweet-scented osmanthus
Careless element 0.85mgmL-1Mixed reference substance solution；
T3. chromatographic condition
Chromatographic column: using octadecylsilane chemically bonded silica as filler, 4.60mm × 250mm, 5 μm of granularity；Flow velocity:
1.0mL·min-1；Detection wavelength: 280nm；Column temperature: 30 DEG C；Sample volume: 10 μ L；Mobile phase A is methanol, Mobile phase B 0.2%
Phosphoric acid solution, gradient elution program are as follows: 0~30min, 10% → 20%A, 30~90min, 20% → 55%A；
T4. finger-print is established
The test solution for taking the opposed polarity position obtained under step T1, by step T3 chromatographic condition, sample introduction is analyzed, obtains
To corresponding chromatogram, preliminary treatment is carried out using liquid phase work station, it is soft to reuse chromatographic fingerprints of Chinese materia medica similarity evaluation
Part carries out Auto-matching to the relevant parameter of variant polar fraction HPLC finger-print: total extract part HPLC map, which is arranged, is
Referring to map, control HPLC finger-print is generated with median method, time window width is set as 0.5, full peak is selected after Supplements
Match pattern obtains control map and matched data.
Further, the above-described longan study on chemical compositions of leaves evaluation side based on anti-oxidant and hypoglycemic spectrum effect relationship
Method, it is described calibration common characteristic peaks retention time be respectively as follows: 3.107min, 5.829min, 8.325min, 12.271min,
13.323min、16.274min、19.802min、23.253min、24.61min、26.808min、29.003min、
32.066min、34.366min、35.662min、36.455min、46.922min、62.873min、63.998min、
66.628min、70.843min、79.594min、81.897min。
Further, the above-described longan study on chemical compositions of leaves evaluation side based on anti-oxidant and hypoglycemic spectrum effect relationship
Method, No. 14 peaks are progallin A in common characteristic peaks, and No. 20 peaks are astragalin, and No. 22 peaks are Quercetin.
Further, the above-described longan study on chemical compositions of leaves evaluation side based on anti-oxidant and hypoglycemic spectrum effect relationship
Method brings the finger-print chemical information at longan leaf opposed polarity position and drug activity information into mathematical model and carries out spectrum effect phase
The analysis of closing property, including grey relational grade and Pearson Bivariate analysis method, the drug effect for evaluating chemical component in longan leaf are living
Property, study its effective substance.
Further, the above-described longan study on chemical compositions of leaves evaluation side based on anti-oxidant and hypoglycemic spectrum effect relationship
Method, the grey Relational Analysis Method are as follows: the IC of DPPH free radical will be removed in pharmacodynamic test part50, inhibit alpha-glucosaccharase
The IC of enzymatic activity50Respectively as system action characteristic sequence X01、X02, by the quantization at 5 longan leaf opposed polarity genius loci peaks
Peak area is set as system factor Xi, and the observation data on observation object serial number k are Xi (k), Xi=(xi (1), xi
(2) ..., xi (n)) be Xi behavior transverse direction sequence, calculate X01、X02With the degree of association of Xi (k), and to degree of association size carry out
Sequence is to differentiate each peak area to the influence power of pharmacodynamics index；The Pearson bivariate correlation analysis method are as follows: with quantization
Peak area is independent variable, and pharmacodynamics index is dependent variable, is carried out using the Bivariate analysis method of SPSS24.0 statistical software
Data processing obtains the Pearson correlation coefficient of each chromatographic peak and drug effect in finger-print.
The application of the above-described longan study on chemical compositions of leaves evaluation method based on anti-oxidant and hypoglycemic spectrum effect relationship, can
For evaluating the anti-oxidant and hypoglycemic effect of longan leaf or the leaf preparation containing longan.
The beneficial effects of the present invention are:
1, the present invention has carried out the correlation research of longan leaf Fingerprints and drug activity, is known using chemical model
Other method establishes the variation for the chemical component that longan leaf extract is embodied on finger-print to the variation of drug effect related
Property, the quality control for longan leaf provides more fully and accurate spectrum imitates basis.
2, the method for the present invention can quickly and accurately evaluate in longan leaf have it is anti-oxidant and hypoglycemic effect chemistry at
Point, scientific and effective method is provided for the effective substance research and quality control of longan leaf, for further exploitation longan leaf
Reference is provided for anti-oxidant and hypoglycemic drug or health food.
3, the present invention has the advantages that convenient succinct, objectivity is strong, and reproducibility is strong, and characteristic peak is clear, makes the matter of longan leaf
Amount control is more scientific perfect.
4, method of the invention can mention for researchs such as the evaluation of the chemical component of other Chinese medicines or medicinal material, screening and quality controls
For reference.
Detailed description of the invention
Fig. 1 is longan leaf opposed polarity position finger-print stacking chart
Fig. 2 is DPPH canonical plotting
Fig. 3 is alpha-glucosidase and PBS fluorescence peak figure
Fig. 4 is the fluorescence pattern of alpha-glucosidase+D1
Fig. 5 is the fluorescence pattern of alpha-glucosidase+D2
Fig. 6 is the fluorescence pattern of alpha-glucosidase+D3
Fig. 7 is the fluorescence pattern of alpha-glucosidase+D4
Fig. 8 is the fluorescence pattern of alpha-glucosidase+D5
Fig. 9 is D1 inhibition dynamics analysis chart
Figure 10 is D2 inhibition dynamics analysis chart
Figure 11 is D3 inhibition dynamics analysis chart
Figure 12 is D4 inhibition dynamics analysis chart
Figure 13 is D5 inhibition dynamics analysis chart
Specific embodiment
The invention will be further described combined with specific embodiments below, but does not limit the scope of the invention and apply
Range.
One, instrument and material
1. instrument
Agilent1100 high performance liquid chromatograph (Agilent company, the U.S.), UV-1780 type UV, visible light spectrophotometric
It counts (Shimadzu instrument (Suzhou) Co., Ltd), INFINITE 200PRO type microplate reader (Austrian TECAN company), Sorvall
ST16R type high speed freezing centrifuge (Thermo Fisher scientific & technical corporation, the U.S.), KQ-500DA Ultrasound Instrument (city of Kunshan's Ultrasound Instrument
Device Co., Ltd), RF-6000 sepectrophotofluorometer (Shimadzu Corp).
2. material
Longan leaf sample: being collected in Guangxi Wuzhou, is accredited as through Guangxi University of Chinese Medicine professor Teng Jianbei
The leaf of Dimocarpus longan Lour..
Reference substance: astragalin (upper Hiroad standing grain Pharmaceutical Technology Co., Ltd, lot number: 170326)；Quercetin (Chinese food
Drug assay research institute, lot number: 100081-201610)；Luteolin (upper Hiroad standing grain Pharmaceutical Technology Co., Ltd, lot number:
170626)；Kaempferol (upper Hiroad standing grain Pharmaceutical Technology Co., Ltd, lot number: 170517).
3. reagent
Methanol, acetonitrile are chromatographically pure, and water is ultrapure water, other reagents are that analysis is pure.2,2- xenyl -1- picrylhydrazyl
(DPPH, Shanghai Mike's woods biochemical technology Co., Ltd, lot number M72518024)；Glucuroide (G5003-100UN Sigma
Company, lot number SLBV6748)；P-nitrophenyl-α-D- glucopyranoside (PNPG, Sigma company, lot number BCBT2911)；
Acarbose (Acarbose, Beyer Co., Ltd, lot number BJ33686).
Two, longan leaf opposed polarity position is prepared
1. breaking into coarse powder after longan leaf medicinal material is cleaned drying, 640g is weighed, is successively added with 6 times of 95%, 50% ethyl alcohol of amount
Circumfluence distillation 2 times, each 2h, filtration, merging filtrate is waved to no alcohol taste after recycling ethyl alcohol to get total extract part (D1)
99.82g；
2. taking D1, suitable quantity of water is added, is suspended in separatory funnel, successively uses opposed polarity solvent: petroleum ether, acetic acid second respectively
Ester, n-butanol are extracted, until the extract liquor clear of every kind of solvent, the extraction for merging variant polar solvent respectively are molten
Liquid, it is spare through the solvent-extracted aqueous solution of opposed polarity；
3. by the extraction solution recycling design of the variant polar solvent after merging, volatilize, respectively obtain petroleum ether part,
Ethyl acetate extract, n-butanol portion, wherein petroleum ether part obtains blackish green medicinal extract 5.28g, and ethyl acetate extract (D2) obtains
To pale yellow powder 22.52g, n-butanol portion (D3) obtains orange-yellow powder 27.72g；Opposed polarity of learning from else's experience is solvent-extracted
Aqueous solution is evaporated, and the position get Shui (D4) is crocus colloid 30.78g；
4. separately taking longan leaf coarse powder, suitable quantity of water is added, heating and refluxing extraction 2h is filtered, and filtrate concentration volatilizes, and obtains decocting
Liquid extract position (D5).
By above-mentioned longan leaf opposed polarity position D1, D2, D3, D4, D5 being prepared, further tested.
Three, the HPLC finger-print at longan leaf opposed polarity position is established
1. the preparation of the test solution at opposed polarity position
It is appropriate to weigh each extractive part D1-D3 of opposed polarity for precision respectively, uses methanol ultrasonic dissolution, 13000r respectively
min-1Centrifugation 10min, supernatant, compound concentration 50mgmL are taken-1The total extract part of longan leaf, ethyl acetate extract, positive fourth
The test solution at alcohol position.
Precision weighs each extractive part D4~appropriate D5 of opposed polarity respectively again, molten with appropriate 50% methanol ultrasound respectively
Solution, 13000rmin-1Centrifugation 10min, supernatant, compound concentration 50mgmL are taken-1Longan leaf water position, decocting liquid are extracted
The test solution at object position.
2. the preparation of mixed reference substance solution: precision weighs progallin A, astragalin, Quercetin, luteolin
Appropriate reference substance adds methanol to dissolve, constant volume, obtains 0.10mgmL containing progallin A-1, 0.15mgmL containing astragalin-1, 0.12mgmL containing Quercetin-1, 0.85mgmL containing luteolin-1Mixed reference substance solution.
3. chromatographic condition
Chromatographic column Phenomenex Gemini 5u C18 column (4.60mm × 250mm, 5 μm), flow velocity 1.0mLmin-1,
Detection wavelength 280nm, column temperature: 30 DEG C, 10 μ L of sample volume, mobile phase methanol (A) -0.2% phosphoric acid (B) gradient elution (0~
30min, 10% → 20%A, 30~90min, 20% → 55%A).
4. methodological study
4.1 precision test
D2 test solution is taken, by 3 lower chromatographic conditions, continuous sample introduction 6 times, records map, the results showed that, share peak
The value of relative retention time RSD is between 0%~0.26906%；Peak relative peak area RSD value is shared 0%~7.80693%
Between, show that the precision of instrument is good.
4.2 repetitive test
D1 test solution is taken, chromatographic condition is measured under 3, is recorded map, is investigated the repeatability of experimental method.
The result shows that 6 parts of samples share the relative retention time RSD value at peak between 0~1.10506%, peak relative peak area is shared
RSD value shows that experimental method repeatability is good between 0~4.83483%.
4.3 stability test
D5 test solution is taken, by 3 lower fingerprint chromatogram conditions, was detected, is examined at 0,2,4,8,16,24 hour respectively
Examine the stability of test solution.The result shows that the relative retention time RSD value at shared peak is between 0~0.67783%, it is total
There is the relative peak area RSD value at peak between 0~4.9334%, shows that test sample for 24 hours stablize by interior quality.
4.4 establish finger-print
Taking each test solution of D1~D5 by 3 lower chromatographic conditions, sample introduction is analyzed, obtains corresponding chromatogram, uses
Agilent ChemStation liquid phase work station carries out preliminary treatment, and it is soft to reuse chromatographic fingerprints of Chinese materia medica similarity evaluation
Part (2012 editions) carries out Auto-matching to the relevant parameter of each polar fraction finger-print: setting D1 is referring to map, with middle position
Number method generates reference fingerprint, and time window width is set as 0.5, full peak match mode is selected after Supplements, obtains comparative diagram
Spectrum and matched data, are shown in Table 1, Fig. 1.
Each polar fraction HPLC of 1 longan leaf of table quantifies peak area tables of data
Four, the common characteristic peaks of opposed polarity position finger-print are demarcated
According to Data Matching as a result, in conjunction with chromatographic peak separation situation, in D1 manually calibration select peak shape preferably, peak
22 moderate Characteristic chromatographic peaks of area, retention time (tR) be respectively as follows: 3.107min, 5.829min, 8.325min,
12.271min、13.323min、16.274min、19.802min、23.253min、24.61min、26.808min、
29.003min、32.066min、34.366min、35.662min、36.455min、46.922min、62.873min、
63.998min,66.628min,70.843min,79.594min,81.897min.According to the retention time of this 22 characteristic peaks,
22,16,9,18 characteristic peaks are marked in D2, D3, D4, D5 same position respectively, wherein No. 14 peaks are galla turcica ethyl ester, No. 20 peaks
For astragalin, No. 22 peaks are Quercetin.The ratio for calculating each genius loci peak area and sample introduction concentration seeks longan leaf not homopolarity
Property position HPLC characteristic peak quantify peak area, be shown in Table 1.
Five, the DPPH free radical scavenging activity at longan leaf opposed polarity position is measured
1. the preparation of solution
The preparation of test solution: precision weighs the opposed polarity position test sample obtained under " two " item and vitamin C is each
0.1g with 95% ethyl alcohol dissolution (water position and decocting liquid extract position with 50% ethyl alcohol dissolved) and is settled to 100mL,
13000r·min-1Be centrifuged 10min, take supernatant, it is accurate respectively to measure 1,2,4,6,8 and be diluted to 10mL, both 0.1,0.2,
0.4、0.6、0.8mg·mL-1Test solution.
The preparation of DPPH solution: precision weighs 60mg DPPH, and 200mL is dissolved and be settled to 95% ethyl alcohol, is obtained
0.3mg·mL-1DPPH solution, take and appropriate 0.03,0.06,0.09,0.12,0.15mgmL be configured to by dilution-1Gradient
Solution is for drawing standard curve.
2. measuring method
0.5mL sample, 0.7mLDPPH solution are added in clean tube, is denoted as A1Pipe；The anhydrous second of 0.5mL sample, 0.7mL
Alcohol is denoted as A2Pipe；0.7mLDPPH solution, 0.5mL dehydrated alcohol, are denoted as A0Pipe.Each pipe shaking is uniformly mixed solution, is protected from light anti-
30min is answered, A is measured517, calculate the DPPH clearance rate and IC of each sample50。
3. result
3.1DPPH standard curve
DPPH standard curve shows DPPH concentration in about 0.03~0.15mgmL-1It is linear good in range, see Fig. 2.
3.2 longan leaf opposed polarity position DPPH free radical scavenging activities
As shown in Table 2, longan leaf opposed polarity position all has significant Scavenging activity to DPPH free radical, when sample is dense
Degree > 0.2mgmL-1, D2 reduces (P > 0.01 or P > 0.05) but each group with D3 clearance rate difference and extremely shows with positive group compared to having
It writes sex differernce (P < 0.01), according to IC50Value judges the Scavenging activity of each polar fraction, IC50The smaller Scavenging activity that represents is stronger,
The result shows that the IC of ethyl acetate extract and n-butanol portion50It is minimum, therefore the Scavenging activity of each polar fraction DPPH free radical
Sequence are as follows: D2 > D3 > D5 > D1 > D4, the positive group excessively high IC of clearance rate in test scope50Value can not be found out, and data are shown in Table 2.
Each polar fraction DPPH clearance rate of 2 longan leaf of table
Note: ▲ x: with concentration and position x two-by-two compared with P>0.05, x:0.05>P>0.01 * do not make marks and represents P<0.01,
Following table is same.
Six, the alpha-glucosaccharase enzyme inhibition rate at longan leaf opposed polarity position is measured
1. spectrofluorimetry of the longan leaf opposed polarity position to alpha-glucosidase
Longan leaf opposed polarity extractive part is weighed respectively, is dissolved with appropriate 95% ethyl alcohol, adds 0.1mmol/LPBS slow
It rushes solution (pH=6.8) and is diluted to 0.2mgmL-1, obtain each sample solution, 0.2UmL-1Alpha-glucosidase, by enzyme solutions
The piping and druming of the μ L each sample solution of 1mL and 50,100,150,200,250 mixes, and reacts 10min at 37 DEG C, then carries out fluorescent emission
Spectral scan.The a length of 200nm of excitation light wave, wavelength of transmitted light range are 300~400nm, scanning speed 6000nmmin-1, swash
It shines and the bandwidth width of transmitting light is set as 10.0nm.
2. longan leaf opposed polarity position is to the inhibiting rate of alpha-glucosidase
A certain amount of longan leaf opposed polarity position sample and positive control acarbose are weighed respectively, with a small amount of 95% ethyl alcohol
Dissolution, adds 0.1mmol/LPBS buffer solution (pH=6.8) to be diluted to 0.2,0.4,0.6,0.8,1.0mgmL-1.It is accurate to draw
PBS buffer solution, longan leaf opposed polarity position sample various concentration sample solution, 0.2UmL-1Alpha-glucosidase, by table
3 are added PNPG50 μ L37 DEG C and react 15min in 37 DEG C of reaction 10min on 96 orifice plates, then every hole, and concentration is added after reaction is
The Na of 0.1mol/L2CO3100 μ L of solution colour developing measures A using microplate reader405, calculate each longan leaf opposed polarity position sample pair
Alpha-glucosaccharase enzyme inhibition rate and IC50。
3 alpha-glucosidases of table-example reaction system
3. longan leaf opposed polarity position inhibits type analysis to alpha-glucosidase
Using Lineweaver-Burk double reciprocal curve method analysis longan leaf opposed polarity position to alpha-glucosidase
Inhibit type.By the reaction solution volume, reaction step method of B in table, the dense of the variant polar fraction of longan leaf is measured respectively
Degree is respectively 0.2,0.4,0.6mgmL-1When, PNPG concentration is 5.0,6.7,10.0,20.0mmolL-1Under enzyme reaction speed
Rate.It is mapped with inverse (1/S) of the inverse (1/V) of each position reaction rate to PNPG concentration, the intercept on X, Y-axis respectively represents
The inverse of Km and Vm.
Lineweaver-Burk equation:
4. result
4.1 alpha-glucosidase fluorescent quenching spectrum analyses
From the figure 3, it may be seen that the Fluorescent peal of PBS is in 310nm, the fluorescence of alpha-glucosidase when excitation wavelength is 200nm
Peak is located near 335nm, and the fluorescence spectra of longan leaf opposed polarity position sample is shown in Fig. 4-8.It follows that with sample is added
The increase alpha-glucosidase fluorescent emission intensity of product amount reduces, and solvent PBS fluorescence peak is increase accordingly, illustrate each sample with α-
Interaction is produced between glucuroide, is quenched so as to cause the fluorescence of alpha-glucosidase.
Rejection ability of the 4.2 longan leaf opposed polarity positions to alpha-glucosidase activity
As shown in Table 4, each polar fraction of longan leaf can inhibit the activity of alpha-glucosidase, and when compared with positive group
All have extremely significant sex differernce (P < 0.01)；Compare inhibiting rate when with concentration, there is extremely significant sex differernce (P < between group two-by-two
0.01), judge variant polar fraction to the rejection ability of alpha-glucosidase activity according to IC50 value, the results showed that n-butanol
The IC50 at position is minimum, each polar fraction rejection ability sequence are as follows: D3 > D1 > D2 > D5 > D4.
Inhibiting rate of the 4 longan leaf opposed polarity position of table to alpha-glucosidase
4.3 longan leaf opposed polarity positions are analyzed by alpha-glucosaccharase enzyme inhibition dynamics
The characteristics of according to inhibitor in conjunction with enzyme can will inhibit type be divided into can not retroactive inhibition and two kinds of reversible inhibition, wherein
Most of to inhibit all to be reversible inhibition, reversible inhibition is divided into Reverse transcriptase, Noncompetition inhibition and uncompetitive inhibitor again.
Reverse transcriptase enzyme cannot be simultaneously in conjunction with substrate and inhibitor, and Km increases at this time, and Vm is constant；Noncompetition inhibition enzyme can be simultaneously
In conjunction with substrate and inhibitor, Km is constant, Vm decline；Uncompetitive inhibitor refers to that after enzyme and Binding Capacity, inhibitor could be with
Enzyme combines, and forms the compound that can not be decomposed, Km, Vm change at this time.According to enzyme inhibition dynamics characteristic analysis
Lineweaver-Burk hyperbolic chart (Fig. 9-Figure 13), can determine whether out the bond type of D1, D2, D3, D5 and alpha-glucosidase
Inhibit type for the mixing of Reverse transcriptase and Noncompetition inhibition, D4 shows as typical Reverse transcriptase.
Seven, spectrum effect correlation analysis
Present invention employs grey correlation analysis, Pearson correlation coefficient analysis methods to carry out comprehensive analysis, discloses multiple
Relationship between independent variable and a dependent variable, the effective substance for clear Chinese medicine.
1 method
1.1 grey relational grade analysis
The IC of DPPH free radical will be removed in pharmacodynamic test part50, inhibit alpha-glucosidase activity IC50Make respectively
For system action characteristic sequence X01、X02, by the quantization peak area at 5 longan leaf opposed polarity genius loci peaks be set as system because
Plain Xi, the observation data on observation object serial number k are Xi (k), and Xi=(xi (1), xi (2) ..., xi (n)) is the row of Xi
For lateral sequence, X is calculated01、X02With the degree of association of Xi (k), and degree of association size is ranked up to differentiate each peak area to medicine
Imitate the influence power of index.
1.2Pearson correlation analysis
To quantify peak area as independent variable, pharmacodynamics index is dependent variable, using the bivariate phase of SPSS24.0 statistical software
It closes analysis method and carries out data processing, obtain the Pearson correlation coefficient of each chromatographic peak and drug effect in finger-print.
2 results
2.1 grey relational grade analysis
2.1.1 calculate correlation coefficient
Due to the IC of free radical scavenging activity and enzyme inhibition rate50With pharmaceutical efficacy negative correlation, i.e. IC50Smaller drug effect
It acts on stronger.Therefore first by pharmacodynamics index IC50Value is converted into inverseization picture, and data are shown in Table 5.And data matrix is used into first value
Method carries out nondimensionalization processing, seeks X0With the initial value of Xi as the absolute difference sequence of respective components:
Δ i (k)=│ X '0(k)-X ' i (k) │, i=1,2...5；K=1,2...21
Ask the maximum difference of second level and lowest difference:
M=max max Δ i (k), m=min min Δ i (k)
Calculate correlation coefficient:
ξ takes 0.5
2.1.2 calculating correlation
The degree of association is X0With the average value of each corresponding incidence coefficient of Xi.The degree of association is bigger, and chromatographic peak and corresponding drug effect are made
Correlation is bigger.As degree of association > 0.6, then determine that there is correlation at the peak and drug effect；As degree of association > 0.8, then determine
The peak and drug effect have high correlation, the results show that 18 in longan leaf, 1, No. 22 peak (Quercetin) and anti-oxidant association sequence
Near preceding, show that Quercetin has compared with strong anti-oxidation, thus speculates No. 18, chemical component equally has representated by No. 1 peak
Stronger antioxidant activity；It is more with the higher chromatographic peak of the inhibition of enzyme activity degree of association, illustrate each position enzymatic activity suppression of longan leaf
It is multicomponent collaboration as a result, wherein 5,6,8,16, No. 7 peaks association sequences are near preceding that production, which is used, it may be possible to the master to play a role
Want ingredient.Data are shown in Table 6.
The IC50 of table 5 longan leaf opposed polarity extractive part free radical scavenging activity and enzyme inhibition rate-1
Table 6 imitates correlation analysis based on the spectrum of grey relational grade
2.2Pearson correlation analysis
As can be seen from Table 7, making with having 18 peak areas and drug effect in anti-oxidant correlation analysis in 22 characteristic peaks
With being positively correlated, 4 negatively correlated, and it is 14 (progallin As), 18, No. 13 peaks, No. 14 chromatographies that wherein correlation is higher
The related coefficient at peak is 0.927, and removing with DPPH has significant correlation (P < 0.05), is positively correlated, shows gallic acid second
Ester, which has, removes DPPH, antioxidation.Inhibit in correlation analysis with enzyme, 16,8,18,7, No. 5 peaks all have significant correlation
Property (P < 0.05), and be positively correlated, illustrate that these chromatographic peaks and inhibition of enzyme activity have substantial connection.Data are shown in Table 7.
Table 7 imitates correlation analysis based on the spectrum of Pearson correlation coefficient
Note: for * at 0.05 rank (double tails), correlation is significant.
In summary the analysis of two kinds of analytic approach is as a result, the spectrum based on grey correlation analysis model is imitated analysis shows to anti-oxidant
Biggish contribution is 18,1,22 (Quercetin) number peaks, and biggish to inhibition of enzyme activity contribution is 5,6,8,16, No. 7 peaks；And
Pearson correlation analysis shows that stronger with anti-oxidant correlation is 14 (progallin As), No. 18 peaks, with inhibition of enzyme activity
Stronger correlation is 16,8,18,7, No. 5 peaks.These relevant peaks are mainly enriched in ethyl acetate and n-butanol portion, this and phase
It closes pharmacodynamic test result to be consistent, shows that the effective substance of longan leaf is mainly moderately polar ingredient.
In conclusion the present invention passes through the fingerprint characteristic of effective component in longan leaf and anti-oxidant between hypoglycemic activity
Correlation conduct a research, finger-print and pharmacy effect research on the basis of, obtain 22 spies of longan leaf HPLC finger-print
Peak data and anti-oxidant and hypoglycemic activity quantized data are levied, grey relational grade and Pearson Bivariate analysis are passed through
Two kinds of relevant function methods, chemical component representated by integrated survey longan leaf HPLC Fingerprints is to anti-oxidant and drop blood
The size degree of sugared activity pharmacodynamics index contribution, specifies chemical component more close with medicine efficacy relation in longan leaf, more comprehensively
The correlation reflected in longan leaf between spectrum effect, controlled for the research of its effective substance and quality provide it is scientific and effective
Method provides reference for anti-oxidant and hypoglycemic drug or health care product for further exploitation longan leaf, can quickly, accurately
Ground, which is evaluated, has anti-oxidant and hypoglycemic effect chemical component and active ingredient screening in longan leaf, be the drug effect object of longan leaf
Matter basic research and quality control provide scientific and effective method, have in terms of the health care of development and utilization longan leaf and medical value
It is significant.
Claims (9)
1. a kind of longan study on chemical compositions of leaves evaluation method based on anti-oxidant and hypoglycemic spectrum effect relationship, which is characterized in that pass through
The HPLC finger-print at longan leaf opposed polarity position is established, common characteristic peaks are demarcated, mathematical analysis model evaluation is imitated by spectrum
The anti-oxidant and hypoglycemic effect of chemical component in longan leaf.
2. the longan study on chemical compositions of leaves evaluation method according to claim 1 based on anti-oxidant and hypoglycemic spectrum effect relationship,
It is characterized by comprising the following steps:
S1. longan leaf opposed polarity position is prepared；
S2. the HPLC finger-print at longan leaf opposed polarity position is established；
S3. the common characteristic peaks of opposed polarity position finger-print are demarcated；
S4. the DPPH free radical scavenging activity at longan leaf opposed polarity position is measured；
S5. the alpha-glucosaccharase enzyme inhibition rate at longan leaf opposed polarity position is measured；
S6. spectrum effect correlation analysis.
3. the longan study on chemical compositions of leaves evaluation method according to claim 2 based on anti-oxidant and hypoglycemic spectrum effect relationship,
It is characterized in that, the method for preparing longan leaf opposed polarity position in the step S1 are as follows:
R1. longan leaf coarse powder is taken, with successively heating and refluxing extraction 2 times of 95% ethyl alcohol, 50% ethyl alcohol, each 2h, filtration merges filter
Liquid recycles ethyl alcohol to no alcohol taste, obtains total extract part；
R2. total extract part is taken, adds water to be suspended in separatory funnel, successively uses opposed polarity solvent: petroleum ether, acetic acid second respectively
Ester, n-butanol are extracted, until the extract liquor clear of every kind of solvent, the extraction for merging variant polar solvent respectively are molten
Liquid, it is spare through the solvent-extracted aqueous solution of opposed polarity；
R3. by the extraction solution recycling design of the variant polar solvent after merging, volatilize, respectively obtain ethyl acetate extract,
N-butanol portion；The solvent-extracted aqueous solution of opposed polarity of learning from else's experience, is evaporated, the position get Shui；
R4. longan leaf coarse powder is separately taken, water is added, heating and refluxing extraction 2h is filtered, and filtrate concentration volatilizes, and obtains decocting liquid extract
Position.
4. the longan study on chemical compositions of leaves evaluation side according to claim 1 or 2 based on anti-oxidant and hypoglycemic spectrum effect relationship
Method, which is characterized in that the HPLC finger-print for establishing longan leaf opposed polarity position includes the following steps:
T1. the preparation of the test solution at opposed polarity position
Precision weighs total extract part, ethyl acetate extract, n-butanol portion respectively, respectively with methanol ultrasonic dissolution, be centrifuged, take
Supernatant, compound concentration 50mgmL-1The total extract part of longan leaf, ethyl acetate extract, the test sample of n-butanol portion are molten
Liquid；
Precision weighs water position, decocting liquid extract position respectively, respectively with 50% methanol ultrasonic dissolution, be centrifuged, take supernatant,
Compound concentration is 50mgmL-1The test solution at longan leaf water position, decocting liquid extract position；
T2. the preparation of mixed reference substance solution
Precision weighs progallin A, astragalin, Quercetin, luteolin reference substance, and methanol is added to dissolve, and constant volume must contain
Progallin A 0.10mgmL-1, 0.15mgmL containing astragalin-1, 0.12mgmL containing Quercetin-1, contain luteolin
0.85mg·mL-1Mixed reference substance solution；
T3. chromatographic condition
Chromatographic column: using octadecylsilane chemically bonded silica as filler, 4.60mm × 250mm, 5 μm of granularity；Flow velocity: 1.0mL
min-1；Detection wavelength: 280nm；Column temperature: 30 DEG C；Sample volume: 10 μ L；Mobile phase A is methanol, and Mobile phase B is that 0.2% phosphoric acid is molten
Liquid, gradient elution program are as follows: 0~30min, 10% → 20%A, 30~90min, 20% → 55%A；
T4. finger-print is established
The test solution for taking the opposed polarity position obtained under step T1, by step T3 chromatographic condition, sample introduction is analyzed, obtains phase
The chromatogram answered carries out preliminary treatment using liquid phase work station, reuses chromatographic fingerprints of Chinese materia medica similarity evaluation software pair
The relevant parameter of variant polar fraction HPLC finger-print carries out Auto-matching: it is reference that total extract part HPLC map, which is arranged,
Map generates control HPLC finger-print with median method, and time window width is set as 0.5, full peak match is selected after Supplements
Mode obtains control map and matched data.
5. the longan study on chemical compositions of leaves evaluation side according to claim 1 or 2 based on anti-oxidant and hypoglycemic spectrum effect relationship
Method, which is characterized in that it is described calibration common characteristic peaks retention time be respectively as follows: 3.107min, 5.829min, 8.325min,
12.271min、13.323min、16.274min、19.802min、23.253min、24.61min、26.808min、
29.003min、32.066min、34.366min、35.662min、36.455min、46.922min、62.873min、
63.998min、66.628min、70.843min、79.594min、81.897min。
6. the longan study on chemical compositions of leaves evaluation side according to claim 1 or 2 based on anti-oxidant and hypoglycemic spectrum effect relationship
Method, which is characterized in that No. 14 peaks are progallin A in common characteristic peaks, and No. 20 peaks are astragalin, and No. 22 peaks are quercitrin
Element.
7. the longan study on chemical compositions of leaves evaluation method according to claim 2 based on anti-oxidant and hypoglycemic spectrum effect relationship,
It is characterized in that, by the finger-print chemical information at longan leaf opposed polarity position and drug activity information bring into mathematical model into
Row spectrum effect correlation analysis, including grey relational grade and Pearson Bivariate analysis method, evaluate chemical component in longan leaf
Drug activity, study its effective substance.
8. the longan study on chemical compositions of leaves evaluation method according to claim 2 based on anti-oxidant and hypoglycemic spectrum effect relationship,
It is characterized in that, the grey Relational Analysis Method are as follows: the IC of DPPH free radical will be removed in pharmacodynamic test part50, inhibit α-
The IC of glucosidase activity50Respectively as system action characteristic sequence X01、X02, by 5 longan leaf opposed polarity genius locis
The quantization peak area at peak is set as system factor Xi, the observation data on observation object serial number k be Xi (k), Xi=(xi (1),
Xi (2) ..., xi (n)) be Xi behavior transverse direction sequence, calculate X01、X02With the degree of association of Xi (k), and to degree of association size into
Row sequence is to differentiate each peak area to the influence power of pharmacodynamics index；The Pearson bivariate correlation analysis method are as follows: with amount
Change peak area be independent variable, pharmacodynamics index is dependent variable, using SPSS24.0 statistical software Bivariate analysis method into
Row data processing obtains the Pearson correlation coefficient of each chromatographic peak and drug effect in finger-print.
9. the longan study on chemical compositions of leaves evaluation side according to claim 1 or 2 based on anti-oxidant and hypoglycemic spectrum effect relationship
The application of method, which is characterized in that for evaluating the anti-oxidant and hypoglycemic effect of longan leaf or the leaf preparation containing longan.
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