CN107300590A - A kind of HPLC detection methods that polygonin and resveratrol are extracted from giant knotweed - Google Patents
A kind of HPLC detection methods that polygonin and resveratrol are extracted from giant knotweed Download PDFInfo
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Abstract
The invention discloses a kind of HPLC detection methods that polygonin and resveratrol are extracted from giant knotweed.The detection method of the present invention includes step：1) polygonin and resveratrol HPLC chromatogram analysis method are set up；2) enzymolysis and extraction process lowest optimization.Cellulase degradation optimum condition is pH7.5,55 DEG C of hydrolysis temperature, enzymolysis time 6 hours.After enzymolysis, the content of resveratrol improves 2.85 times.The present invention is established while determining the HPLC detection methods of polygonin and resveratrol, and for the measure of polygonin after optimal extraction technology and resveratrol, the process stabilizing is reliable, and easy to operate time saving, energy consumption is relatively low, and reference is provided for giant knotweed technical study.
Description
Technical field
The present invention relates to a kind of while the foundation of measure polygonin and Resveratrol content assay method, belongs to chemicals
Analysis technical field, its set up method be applied to giant knotweed optimal extraction technology result constituent analysis, compare draw polygonin and
The HPLC assay methods of Resveratrol content.
Background technology
Giant knotweed is polygonaceae plant giant knotweed Polygonum cuspidatum Sieb.Et.Zucc. dry rhizome and root, property
Be slightly cold, mildly bitter flavor, tool normalizing gallbladder to cure jaundice, clearing heat and detoxicating, blood stasis removing analgesic, preventing phlegm from forming and stopping coughing the effects such as.Giant knotweed contains anthraquinone, talan
The Multiple components such as class, phenols, flavonoids, wherein diphenylethylene are mainly resveratrol and polygonin, and Anthraquinones are mainly with big
Flavine, Physcion are representative, also containing a variety of anthraquinone glycosides.
《Pharmacopoeia of People's Republic of China》Version in 2010 is only used as the index of giant knotweed assay using rheum emodin and polygonin
Thing, and it is not directed to resveratrol.It is that a class has special health care work(but resveratrol content in giant knotweed is of a relatively high
, there can be the polyphenol substance of remarkable effect to health.Resveratrol is a kind of extremely promising pharmaceutical intermediate, to tumour
With elemental abundances, and there is selective killing power to cancer cell；There is good curative effect to treatment cardiovascular and cerebrovascular disease,
And side effect very little, it is easy to long-term taking.
But how by the foundation of polygonin and resveratrol HPLC detection methods, and using set up method to giant knotweed
Optimal extraction technology result is measured, and is the technical barrier that those skilled in the art are badly in need of solving.
The content of the invention
The HPLC detection methods of polygonin and resveratrol are extracted from giant knotweed it is an object of the invention to provide a kind of, and are adopted
Giant knotweed optimal extraction technology result is measured with the method set up, reference is provided for giant knotweed resource complete utilization, and be it
Polygonin and the resveratrol detection of his matrix provide reference.
To achieve these goals, the technical scheme that the present invention takes is as follows：
A kind of HPLC detection methods that polygonin and resveratrol are extracted from giant knotweed, the condition of efficient liquid phase chromatographic analysis
Including：Using reverse phase filler chromatographic column；Detection wavelength is 306nm and full wavelength scanner, and flow velocity is 0.8~1.2ml/min, is entered
Sample amount is 2~20 μ l, and column temperature is 25~35 DEG C, and mobile phase is acetonitrile-water, the formic acid water of methanol -0.1%, the formic acid of acetonitrile -0.1%
Water, methanol-water, the phosphoric acid water of methanol -0.1%, the phosphoric acid water of acetonitrile -0.1%, isocratic elution.
A kind of HPLC detection methods that polygonin and resveratrol are extracted from giant knotweed, the condition of efficient liquid phase chromatographic analysis
For：Using Phenomenex5 μm of EVO C18 (250mm × 4.6mm, 5 μm) chromatographic columns, use wavelength for 306nm,
Flow velocity is 1.0ml/min, and sample size is 10 μ l, and column temperature is 25 DEG C, and analysis time is 40min, and mobile phase is acetonitrile-water (23:
77), isocratic elution.
A kind of HPLC detection methods that polygonin and resveratrol are extracted from giant knotweed, pre-treating method, which investigates object, to be had：
Extraction solvent (absolute ethyl alcohol, methanol, ethyl acetate, Diluted Alcohol)；Extracting method (ultrasonic extraction, be heated to reflux, cold soaking)；Extract
Gu material is than (1:10~1:100)；Extraction solvent volume fraction (30%, 50%, 75%, 95%, 100%)；Extraction time
(30min、60min、90min)。
A kind of HPLC detection methods that resveratrol is extracted from giant knotweed, pre-treating method is：Take Rhizoma Polygoni Cuspidati powder (mistake
No. three sieves) about 0.5g, it is accurately weighed, put in conical flask with cover, precision adds 50% ethanol 20mL；Close plug, is heated to reflux 30 points
Clock, takes out, lets cool to room temperature, the weight lost is refilled with methanol.Filtering, takes subsequent filtrate, produces.
According to actual production conditions and economic benefits, giant knotweed cellulase degradation extraction process is optimized including：Enzyme
Solve liquid pH value, hydrolysis temperature, enzymolysis time.
According to actual production conditions and economic benefits, to giant knotweed cellulase degradation extraction process most preferably：Enzymolysis liquid
PH7.5,55 DEG C of hydrolysis temperature, enzymolysis time 6 hours.
A kind of HPLC assay methods that Resveratrol content is detected from giant knotweed, to giant knotweed cellulase degradation extraction process
Optimum results composition is measured.
Compared with prior art, the invention has the advantages that and beneficial effect：
The present invention determines polygonin and resveratrol while providing a kind of practical, reliable and stable, favorable reproducibility
HPLC detection methods, are that giant knotweed process optimization result composition polygonin and resveratrol are measured, and pass through extraction process
Investigate, while using polygonin, resveratrol as the preferred optimum extraction process of index components.
Brief description of the drawings
Fig. 1 a and 1b are respectively the full wavelength scanner collection of illustrative plates of polygonin and resveratrol；Fig. 1 c and 1d are respectively reference substance
Scanning spectra (sweep by DAD all-wave lengths for (giant knotweed test sample chromatogram (306nm)) and need testing solution (giant knotweed test sample chromatogram)
Retouch)；
Fig. 2 a, 2b and 2c are respectively that reference substance that mobile phase is acetonitrile-water is (polygonin reference substance collection of illustrative plates (acetonitrile-water), white
Veratryl alcohol reference substance collection of illustrative plates (acetonitrile-water)) and test sample (giant knotweed need testing solution collection of illustrative plates (acetonitrile-water)) collection of illustrative plates；
Fig. 3 a and 3b are respectively the canonical plotting of polygonin and resveratrol；
Fig. 4 is different sample size collection of illustrative plates；
Fig. 5 is that different column temperatures investigate collection of illustrative plates；
Fig. 6 is Different Extraction Method collection of illustrative plates；
Fig. 7 is solid material than investigating collection of illustrative plates；
Fig. 8 is different volumes fraction chromatogram；
Fig. 9 is different extraction time collection of illustrative plates.
Embodiment
Experiment material of the invention used is the giant knotweed that is provided by Zhaoqing Huaiji base.
This experiment will be by producing Rhizoma Polygoni Cuspidati (lot number to Zhaoqing Huaiji：20140412) analyzed and studied, set up
Specificity is strong, favorable reproducibility while determine polygonin and resveratrol detection method, including step：1) polygonin and white black false hellebore
Alcohol HPLC chromatogram analysis method is set up；2) enzymolysis and extraction process lowest optimization.Cellulase degradation optimum condition is pH7.5, enzymolysis
55 DEG C of temperature, enzymolysis time 6 hours.After enzymolysis, the content of resveratrol improves 2.85 times.Utilize what is set up simultaneously
Content assaying method, single factor exploration is carried out to cellulase degradation process optimization, investigates hydrolysis temperature, enzymolysis liquid pH value, enzyme
Influence of the solution time to giant knotweed active component (key targets are resveratrols) yield.Pass through the total score to result of the test
Analysis, and then respective more excellent extraction process is drawn, provide reference for the resource complete utilization of giant knotweed.
Instrument of the present invention includes：
High performance liquid chromatograph：The high performance liquid chromatographs of Thermo Ultimate 3000 (the silent winged generation that science and technology of U.S.'s match
Company)：Quaternary pump, PDAD (DAD), automatic sampler, Chromelen chromatographic work stations.
Chromatographic column：Phenomenex5 μm of EVO C18 chromatographic columns (4.6mm × 250mm, 5 μm), balance：Thousand
/ mono- balance (the permanent flat JA2003 in Shanghai), a ten thousandth balance (Sartorius BS224S), ten a ten thousandth balances
(Precisa XR205SM DR)；Ultrapure water system (U.S. Millipore (Mi Libo) company).
Agents useful for same of the present invention includes with reagent：Methanol, ethanol, formic acid, phosphoric acid etc. are that domestic analysis is pure；HPLC first
Alcohol, acetonitrile are chromatographically pure (German Merck companies, Darmstadt, Gemany)；Water is ultra-pure water (resistivity 18.2m Ω
.cm)。
To make the present invention easier to understand, the present invention is expanded on further with reference to specific embodiment.It should be understood that these
Embodiment is only illustrative of the invention and is not intended to limit the scope of the invention, NM specific experiment side in the following example
Method, is generally carried out according to normal experiment method.
Embodiment 1
A kind of HPLC detection methods that polygonin and resveratrol are extracted from giant knotweed, comprise the following steps：
1st, the preparation of need testing solution
1.1 elemental method
Giant Knotweed Rhizome (crossing No. three sieves) 1g is weighed, it is accurately weighed, it is placed in dry conical flask with cover.Precision pipettes methanol
50ml, weighed weight, ultrasonically treated 30min.Take out, let cool to room temperature, the weight lost is refilled with methanol.Filtering, takes continuous
Filtrate, produces test sample.
The investigation of 1.2 pairs of different Detection wavelengths
306nm and full wavelength scanner have been investigated in this experiment.
Chromatographic condition：
Chromatographic column：Phenomenex5 μm of EVO C18 (250mm × 4.6mm, 5 μm)；
Column temperature：25℃；
Mobile phase：Acetonitrile-water (23:77).
Volume flow：1.000ml/min；
Sample size：10μL；
As is shown in figs. la to ld, by the visible polygonin of full wavelength scanner collection of illustrative plates and resveratrol between 280nm~310nm
There is larger absorption.And in 306nm collection of illustrative plates, the peak shape of polygonin and resveratrol preferably, therefore selects 306nm to be detection
Wavelength.
The investigation of 1.3 pairs of different mobile phases
The aqueous formic acid of acetonitrile -0.1%, the aqueous formic acid of methanol -0.1%, acetonitrile-aqueous solution, first have been investigated in this experiment
Alcohol-water solution, the phosphate aqueous solution of methanol -0.1%, the phosphate aqueous solution of acetonitrile -0.1%.
Chromatographic condition：
Chromatographic column：Phenomenex5 μm of EVO C18 (250mm × 4.6mm, 5 μm)；
Column temperature：25℃；
Mobile phase：Above-mentioned solution；
Volume flow：1.000ml/min；
Sample size：10μL；
Detection wavelength：306nm；
Experiment finds that methanol is poor compared with the elution resveratrol of acetonitrile and the ability of polygonin, therefore organic phase selection second
Nitrile.In addition, only acetonitrile-water system is preferable compared with the theoretical cam curve and dissymmetry factor of the mobile phase of other acid addings, and collection of illustrative plates
Peak shape is preferable.Therefore from mobile phase constituent of the acetonitrile-water system as the experiment.As shown in figs. 2 a-2 c, i.e., from second
Nitrile-aqueous solution is used as mobile phase.
The investigation of 1.4 different sample sizes
This experiment has investigated the different sample sizes of 2 μ L, 5 μ L, 8 μ L, 10 μ L, 20 μ L and has separated polygonin and white lamb's-quarters to giant knotweed sample
The effect of reed alcohol.
Chromatographic condition：
Chromatographic column：Phenomenex5 μm of EVO C18 (250mm × 4.6mm, 5 μm)；
Column temperature：25℃；
Mobile phase：Acetonitrile-water (23:77) solution；
Volume flow：1.000ml/min；
Sample size：2、5、8、10、20μL；
Detection wavelength：306nm；
Experiment finds that 2 μ L and 5 μ L are good with respect to 8 μ L, 10 μ L, 20 μ L peak shapes, and without splitting peak.As shown in figure 4,2 μ L and 5 μ L colors
Spectrogram is more or less the same, in order to which assay is more accurate, therefore is used as sample size from 5 μ L.
The investigation of 1.5 different chromatogram column temperatures
This experiment investigated 25 DEG C, 30 DEG C, 35 DEG C of different column temperatures the effect of polygonin and resveratrol is separated to giant knotweed sample
Really.
Chromatographic condition：
Chromatographic column：Phenomenex5 μm of EVO C18 (250mm × 4.6mm, 5 μm)；
Column temperature：25、30、35℃；
Mobile phase：Acetonitrile-water (23:77) solution；
Volume flow：1.000ml/min；
Sample size：5μL；
Detection wavelength：306nm；
Experiment finds that different temperatures influences little to chromatogram, as shown in figure 5, in order to protect efficient liquid phase instrument, therefore select
25 DEG C are column temperature.
The investigation of 1.6 different column flow rates
It is different in flow rate that 0.8ml/min, 0.9ml/min, 1.0ml/min, 1.1ml/min, 1.2ml/min have been investigated in this experiment
To the effect of separation giant knotweed sample polygonin and resveratrol.
Chromatographic column：Phenomenex5 μm of EVO C18 (250mm × 4.6mm, 5 μm)；
Column temperature：25℃；
Mobile phase：Acetonitrile-water (23:77) solution；
Volume flow：0.8、0.9、1.0、1.1、1.2ml/min；
Sample size：5μL；
Detection wavelength：306nm；
Experiment finds that the system suitability result of each flow velocity is good, and difference less, therefore selects conventional 1.0mL/min to be stream
Speed.
1.7 chromatographic conditions determined
Chromatographic condition：
Chromatographic column：Phenomenex5 μm of EVO C18 (250mm × 4.6mm, 5 μm)；
Column temperature：25℃；
Mobile phase：Acetonitrile-water (23:77) solution；
Volume flow：1.000ml/min；
Sample size：5μL；
Detection wavelength：306nm；
2nd, the investigation for the method extracted to polygonin and resveratrol
The investigation of 2.1 extracting methods
This experiment investigate ultrasonic extraction, be heated to reflux, the extraction efficiency of cold soaking, it is as shown in table 1 below.
Precision weighs 6 parts of Giant Knotweed Rhizome (crossing No. three sieves), and every part of 2g is accurately weighed, is placed in dry conical flask with cover, essence
It is close to pipette methanol 25mL, weighed weight, respectively ultrasonically treated 30min, be heated to reflux 30min and cold soaking extracts 12h.Take out, put
It is cooled to after room temperature, the weight lost is refilled with methanol.Filtering, takes subsequent filtrate, produces test sample.
The Different Extraction Method of table 1 investigates result
| Extracting method | Polygonin average content (mg/g) | Resveratrol average content (mg/g) | Total content (mg/g) |
| Ultrasound | 16.20 | 1.51 | 17.71 |
| It is heated to reflux | 16.59 | 1.83 | 18.42 |
| Cold soaking | 14.38 | 1.29 | 15.66 |
Table 1, Fig. 6 result is shown, is heated to reflux with the extraction efficiency more increased.
The investigation of 2.2 Extraction solvents
Absolute ethyl alcohol, methanol, ethyl acetate, the extraction efficiency of several different solvents of Diluted Alcohol are investigated in this experiment.Such as table 4 below
It is shown.
Giant Knotweed Rhizome (crossing No. three sieves) 2g is weighed, it is accurately weighed, it is placed in dry conical flask with cover.Precision pipettes first respectively
Alcohol 25mL, ethanol 25mL, ethyl acetate 25mL and Diluted Alcohol 25mL, weighed weight, are heated to reflux 30min.Take out, let cool to
After room temperature, the weight lost is refilled with methanol.Filtering, takes subsequent filtrate, produces test sample.
The different solvents of table 2 investigate result
The result of table 2 is shown, when ethyl acetate is as Extraction solvent, and polygonin is adjacent miscellaneous peak, therefore excludes.Methanol, second
Alcohol, Diluted Alcohol are more or less the same as chromatogram during Extraction solvent, and Diluted Alcohol is carried as Extraction solvent resveratrol and polygonin
Take total content highest.Therefore selection Diluted Alcohol is used as Extraction solvent.
2.3 extract the investigation for expecting ratio admittedly
Different extraction material ratios, material such as table 5 have been investigated in this experiment.
The different material admittedly that extract of table 3 compare investigation table
Weigh Giant Knotweed Rhizome, respectively in the material ratio ratio of table 3 carry out it is accurately weighed and it is accurate pipette Diluted Alcohol, it is weighed heavy
Amount, is heated to reflux 30min.Take out, let cool to room temperature, the weight lost is refilled with Diluted Alcohol.Filtering, takes subsequent filtrate, produces
Test sample.
The different solid material of table 4 are than investigating result
| Gu material is than (g/mL) | Polygonin average content (mg/g) | Resveratrol average content (mg/g) | Total content (mg/g) |
| 2-20 | 17.26 | 2.09 | 19.35 |
| 2-25 | 19.00 | 2.06 | 21.05 |
| 2-50 | 22.57 | 2.09 | 24.66 |
| 1-20 | 22.08 | 2.06 | 24.14 |
| 1-25 | 22.34 | 2.04 | 24.38 |
| 1-50 | 22.57 | 2.06 | 24.62 |
| 0.5-20 | 23.27 | 2.05 | 25.32 |
| 0.5-25 | 23.05 | 2.05 | 25.10 |
| 0.5-50 | 22.97 | 2.07 | 25.04 |
Experimental result finds that as shown in table 4, Fig. 7, different material is more or less the same than the chromatogram of gained, 1:40(g/mL)
It is used as the extracted amount highest for extracting material ratio resveratrol and polygonin.Therefore selection 1:40 (g/mL) are extracted as material ratio.
The investigation of 2.4 Extraction solvent volume fractions
This study tour Extraction solvent different volumes fraction, 30%, 50%, 75%, 95%, 100%.
Giant Knotweed Rhizome (crossing No. three sieves) 0.5g is weighed, it is accurately weighed, it is placed in dry conical flask with cover.Precision is pipetted respectively
Different volumes fraction ethanol 20mL, weighed weight is heated to reflux 30min.Take out, let cool to room temperature, refilled with coordinative solvent
The weight lost.Filtering, takes subsequent filtrate, produces test sample.
The different solvents volume fraction of table 5 investigates result
| Extraction solvent (ml) | Polygonin average content (mg/g) | Resveratrol average content (mg/g) | Total content (mg/g) |
| 30% ethanol | 21.18 | 0.99 | 22.1713 |
| 50% ethanol | 22.36 | 2.08 | 24.4338 |
| 75% ethanol | 21.95 | 2.04 | 23.9972 |
| 95% ethanol | 15.37 | 1.77 | 17.1368 |
| 100% ethanol | 16.56 | 1.98 | 18.5423 |
Experimental result is shown, as shown in table 5, Fig. 8, and chromatogram peak shape difference is little obtained by each volume fraction, 50% conduct
The volume fraction resveratrol of Extraction solvent and the extracted amount highest of polygonin.Therefore selection 50% is used as the volume of Extraction solvent
Fraction.
The investigation of 2.5 extraction times
This study tour extraction time, 30min, 60min, 90min extracted amount.
Giant Knotweed Rhizome (crossing No. three sieves) 0.5g is weighed, it is accurately weighed, it is placed in dry conical flask with cover.Precision is pipetted respectively
50% ethanol 20mL, weighed weight is heated to reflux 30min, 60min, 90min respectively.Take out, let cool to room temperature, with 50%
Refill the weight lost.Filtering, takes subsequent filtrate, produces test sample.
The different extraction times of table 6 investigate result
| Extraction time (min) | Polygonin average content (mg/g) | Resveratrol average content (mg/g) | Total content (mg/g) |
| 30 | 23.11 | 2.19 | 25.3032 |
| 60 | 20.63 | 1.83 | 22.4563 |
| 90 | 22.24 | 1.94 | 24.1746 |
Experimental result is shown, as shown in table 6, Fig. 9, and less, 30min is as carrying for chromatogram difference obtained by different extraction times
Take the extraction total content highest of time resveratrol and polygonin.Therefore selection 30min is used as extraction time.
It is prepared by 2.6 test samples
Rhizoma Polygoni Cuspidati powder (crossing No. three sieves) about 0.5g is taken, it is accurately weighed, put in conical flask with cover, precision adds 50% second
Alcohol 20mL.Close plug, is heated to reflux 30 minutes, takes out, lets cool to room temperature, the weight lost is refilled with methanol.Filtering, takes continuous filter
Liquid, produces test sample.
According to method determined above, methodological study is carried out to method and assay is carried out to sample.
3rd, methodological study
3.1 system suitability test
Appropriate need testing solution and reference substance solution are taken, by being measured under 1.7 lower chromatographic conditions, chromatogram is recorded,
System suitability is investigated, as a result as shown in figure 1, test sample chromatogram has corresponding absorption with reference substance chromatogram in same time
Peak, shows that system suitability is good.
The investigation of 3.2 linear relationships
The mixed reference substance solution of concentration known, plus methanol is taken to be diluted to the solution of various concentrations respectively：
①409.00μg·mL-1Polygonin；②204.50μg·mL-1Polygonin；③163.60μg·mL-1Tiger
Cane glycosides；④81.80μg·mL-1Polygonin；⑤20.45μg·mL-1Polygonin；⑥16.36μg·mL-1Polygonin；⑦
8.18μg·mL-1Polygonin；⑧4.09μg·mL-1Polygonin.
①411.00μg·mL-1Resveratrol；②205.50μg·mL-1Resveratrol；③164.40μg·mL-1's
Resveratrol；④82.20μg·mL-1Resveratrol；⑤20.55μg·mL-1Resveratrol；⑥16.44μg·mL-1's
Resveratrol；⑦8.22μg·mL-1Resveratrol；⑧4.11μg·mL-1Resveratrol.
Peak area is determined by the chromatographic condition under 1.7, and with peak area (Y) for ordinate, concentration (X) is abscissa,
Draw standard curve and carry out linear regression calculating, the regression line equation for obtaining polygonin is Y=220.89X-0.4549, (r2=
0.9996)；The regression equation of resveratrol is Y=421.22X-0.7082 (r2=0.9997)
Fig. 3 a-3b are linear relationship collection of illustrative plates, show polygonin in the μ gmL of concentration 4.09-1~409.00 μ gmL-1Scope
Interior is in good linear；Resveratrol is in the μ gmL of concentration 4.11-1~411.00 μ gmL-1In the range of be in good linear.
3.3 replica test
6 parts of Giant Knotweed Rhizome is taken, is prepared by the method under 2.6 is parallel, it is as a result as shown in table 7 below.
The replica test result of table 7
It is respectively 0.68% and 1.24% that replica test, which measures polygonin and the RSD of Resveratrol content, shows the party
Method repeatability is good.
3.4 precision test
Precision is drawn with a μ L of need testing solution 5, repeats continuous sample introduction 6 times by above-mentioned chromatographic condition, experimental result is such as
Shown in table 8 below.
The Precision test result of table 8
It is respectively 0.71% and 1.36% to measure polygonin and resveratrol peak area RSD, shows that precision is good.
3.5 stability test
Precision is drawn with a μ L of need testing solution 5, is entered by above-mentioned chromatographic condition respectively at 0,4,8,12,16,20,24h
Sample, experimental result see the table below shown in 9.
The stability test result of table 9
It is respectively 0.34% and 1.43% to measure polygonin and resveratrol peak area RSD, shows need testing solution in 24h
It is interior basicly stable.
3.6 sample-adding recovery tests
6 parts of the giant knotweed of above-mentioned known content is taken, taking about 0.25g, (known Determination of Polydatin 21.77mg/g, resveratrol contain
Measure 1.98mg/g), it is accurately weighed, put in conical flask with cover, accurate add is separately added into polygonin and resveratrol mixing respectively
Reference substance solution (polygonin 5.512mg/ml, resveratrol 0.4745mg/ml) 1ml, prepares test sample, by 1.7 by 2.6
Lower chromatographic condition is determined, and the rate of recovery is calculated, as a result as shown in table 10 below, 11.
The polygonin of table 10 sample-adding, which is reclaimed, determines knot
The resveratrol of table 11 sample-adding reclaims measurement result
Sample-adding recovery test proves that the polygonin rate of recovery is 102.73%, and the resveratrol rate of recovery is 99.45%, is met
Rate of recovery 95%-105% scope, shows that this method is reliable and stable.,
It is a kind of while determining the foundation of polygonin and resveratrol HPLC detection methods, this method is reliable and stable, applicability
By force.Wherein polygonin and resveratrol reference substance are shown in Fig. 1 a-1d with need testing solution collection of illustrative plates, as seen from the figure, polygonin and white lamb's-quarters
Reed alcohol reference substance has absworption peak with need testing solution in corresponding retention time, and peak type is preferable.
Embodiment 2
1st, a kind of polygonin and resveratrol detection method of determining simultaneously is in giant knotweed optimal extraction technology result constituent analysis
Application：
It is prepared by the need testing solution of 1.1 giant knotweed optimal extraction technologies：
According to production application, reagent is used as technical study for the ethanol commonly used in production used in the research of this part
Reagent.Investigation factor such as table 12.
The enzymatic isolation method single factor exploration table of table 12
| Title | Hydrolysis temperature (DEG C) | Enzymolysis time (h) | PH value |
| 1 | 35 | 1 | 4.5 |
| 2 | 40 | 3 | 5.0 |
| 3 | 45 | 6 | 5.5 |
| 4 | 50 | 9 | 6.0 |
| 5 | 55 | 24 | 6.5 |
| 6 | 60 | 48 | 7.0 |
| 7 | 7.5 | ||
| 8 | 8.0 |
Rhizoma Polygoni Cuspidati powder (crossing No. three sieves) about 0.5g is weighed, it is accurately weighed, it is placed in dry conical flask with cover.It is smart respectively
It is close to pipette enzyme solutions 25mL, it is adjusted to different pH, different hydrolysis temperatures, certain enzymolysis time.After enzymolysis, 100 DEG C of water
Bath, enzyme activity of going out.Precision adds absolute ethyl alcohol 50mL, is heated to reflux 30min.Take out, let cool to room temperature, filter, be evaporated, residue
Plus 50% ethanol dissolve and be settled to 25mL volumetric flasks, shake up, with 0.45 μm of filtering with microporous membrane, take subsequent filtrate, produce.
1.2 is a kind of while determining the application of polygonin and resveratrol detection method
The chromatographic condition of resveratrol detection (determines polygonin resveratrol detection side while foundation with reference to the present invention
Method)
1.3 is a kind of while determining the interpretation of result of polygonin and the application of resveratrol detection method
The composition of optimal extraction process is analyzed, Fig. 6, table 13 is as a result seen.As seen from Figure 6, after the process optimization, in vain
The peak of veratryl alcohol absorbs corresponding increase, and peak type is preferable.
The hydrolysis temperature optimum results of table 13
The enzymolysis time optimum results of table 14
| Enzymolysis time (h) | Polygonin average content (mg/g) | Resveratrol average content (mg/g) | Total content (mg/g) |
| 1 | 33.94 | 5.19 | 39.13 |
| 3 | 31.03 | 6.64 | 37.67 |
| 6 | 26.14 | 8.96 | 35.10 |
The different PH of table 15 investigates result
| PH value | Polygonin average content (mg/g) | Resveratrol average content (mg/g) | Total content (mg/g) |
| 4.5 | 35.59 | 3.91 | 39.51 |
| 5.0 | 35.28 | 3.94 | 39.23 |
| 5.5 | 34.11 | 3.89 | 38.00 |
| 6.0 | 34.33 | 3.88 | 38.21 |
| 6.5 | 32.08 | 4.65 | 36.73 |
| 7.0 | 31.42 | 4.83 | 36.26 |
| 7.5 | 30.91 | 5.65 | 36.56 |
| 8.0 | 30.24 | 5.58 | 35.82 |
From result, hydrolysis temperature is at 40 DEG C, and resveratrol extracted amount is relatively low with respect to other hydrolysis temperatures, its reason
When being that to prepare hydrolysis temperature be 40 DEG C of test sample, though being 1.5mg/g principles according to enzyme concentration, but still cellulase is being weighed
Amount has deviation, and hydrolysis temperature resveratrol extracted amount at 55 DEG C is reached after maximum, 55 DEG C, and zymoprotein starts denaturation, enzyme activity
Property decline, resveratrol extracted amount is on a declining curve.It is 55 DEG C that the optimal hydrolysis temperature of this technique, which can be primarily determined that,.
In enzymolysis time 6h, resveratrol extracted amount shows linear upward trend always, within the time point of setting,
6h is optimal enzymolysis time.
Digest pH interval in 4.5-6, there is no considerable influence to the activity of cellulase, the extracted amount difference of resveratrol is not
Greatly.PH is in 6-7.5, as pH value increases, and ascendant trend is presented in the extracted amount of resveratrol.During pH=7.5, resveratrol
Extracted amount reaches maximum 5.6533mg/g (improving 2.85 times than original 1.9783mg/g).After pH ＞ 7.5, resveratrol
Extracted amount is on a declining curve.It is 7.5 that can primarily determine that this technique most preferably digests pH value.Giant knotweed optimum extraction process, can be stated such as
Under：
Rhizoma Polygoni Cuspidati powder (crossing No. three sieves) about 0.5g is weighed, it is accurately weighed, it is placed in dry conical flask with cover, precision is moved
It is 7.5 to take enzyme solutions 25mL, pH, enzymolysis is carried out under the conditions of 55 DEG C 6 hours.After enzymolysis, 100 DEG C of water-baths, enzyme activity of going out.Precision adds
Enter absolute ethyl alcohol 50mL, be heated to reflux 30min.Take out, let cool to room temperature, filter, be evaporated, residue adds the dissolving of 50% ethanol simultaneously
25mL volumetric flasks are settled to, are shaken up, with 0.45 μm of filtering with microporous membrane, subsequent filtrate is taken, produces.
The present invention is established while determine the HPLC detection methods of resveratrol, for polygonin after optimal extraction technology and
The measure of resveratrol, the process stabilizing is reliable, and easy to operate time saving, energy consumption is relatively low, and reference is provided for giant knotweed technical study.
The above described is only a preferred embodiment of the present invention, any formal limitation not is made to the present invention, therefore
It is every without departing from technical solution of the present invention content, what the technical spirit according to the present invention was made to above example any simply repaiies
Change, equivalent variations and modification, in the range of still falling within technical solution of the present invention.
Claims (8)
1. a kind of HPLC detection methods that polygonin and resveratrol are extracted from giant knotweed, it is characterised in that comprise the following steps：
1) preparation of need testing solution
Giant Knotweed Rhizome is weighed, it is accurately weighed, it is placed in dry conical flask with cover；Precision pipettes methanol, and weighed weight is ultrasonically treated
30min；Take out, let cool to room temperature, the weight lost is refilled with methanol；Filtering, takes subsequent filtrate, produces test sample；
2) efficient liquid phase chromatographic analysis
Chromatographic column：Phenomenex5 μm of EVO C18 (250mm × 4.6mm, 5 μm)；
Column temperature：25℃；
Detection wavelength：306nm；
Mobile phase：Acetonitrile-water (23：77), isocratic elution；
Flow velocity：1mL/min；
Sample size：10μL.
2. the HPLC detection methods according to claim 1 that resveratrol is extracted from giant knotweed, it is characterised in that：Step 2)
In, the condition of the efficient liquid phase chromatographic analysis includes：Using reverse phase filler chromatographic column；Detection wavelength is 306nm and all-wave
Long scan, flow velocity be 0.8~1.2ml/min, sample size be 2~20 μ l, column temperature be 20~35 DEG C, mobile phase be acetonitrile-water and
Other, isocratic elution.
3. the HPLC detection methods according to claim 1 that resveratrol is extracted from giant knotweed, it is characterised in that：Step 2)
In, analysis time is 40min.
4. the HPLC detection methods according to claim 1 that resveratrol is extracted from giant knotweed, it is characterised in that：Step 1)
In, described pre-treating method, which is investigated, to be included：Absolute ethyl alcohol, methanol, ethyl acetate, Diluted Alcohol is respectively adopted in Extraction solvent；Carry
Method is taken to include ultrasonic extraction, heating reflux method, cold-maceration；Extraction is expected than (1 admittedly:10~1:100)；Extraction solvent volume integral
Number is respectively 30%, 50%, 75%, 95%, 100%；Extraction time is respectively 30min, 60min, 90min.
5. the HPLC detection methods according to claim 1 that resveratrol is extracted from giant knotweed, it is characterised in that：Step 1)
In, described pre-treating method includes：Rhizoma Polygoni Cuspidati powder (crossing No. three sieves) about 0.5g is taken, it is accurately weighed, put conical flask with cover
In, precision adds 50% ethanol 20mL；Close plug, is heated to reflux 30 minutes, takes out, lets cool to room temperature, refilled and lost with methanol
Weight.Filtering, takes subsequent filtrate, produces.
6. the HPLC detection methods according to claim 5 that polygonin and resveratrol are extracted from giant knotweed, its feature exists
In：The pre-treating method, with reference to practical condition, giant knotweed extraction process is optimized including：Hydrolysis temperature；During enzymolysis
Between；Enzymolysis liquid pH value.
7. the HPLC detection methods according to claim 6 that polygonin and resveratrol are extracted from giant knotweed, its feature exists
In：The giant knotweed cellulase degradation process optimization is most preferably：Enzymolysis liquid pH7.5,55 DEG C of hydrolysis temperature, enzymolysis time 6 hours.
8. the HPLC detection methods according to claim 1 that polygonin and resveratrol are extracted from giant knotweed, its feature exists
In：Giant knotweed optimal extraction technology result composition is measured.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109522672A (en) * | 2018-11-30 | 2019-03-26 | 华南理工大学 | A kind of continuous flow upstream two-phase mass transport process stable state solute concentration distribution model |
| CN109633038A (en) * | 2019-01-31 | 2019-04-16 | 北京中医药大学 | The detection method of polygonin and its metabolite in biological sample |
| CN109709226A (en) * | 2018-12-29 | 2019-05-03 | 中国农业科学院油料作物研究所 | A method of trans-resveratrol content in measurement peanut dry seeds |
| CN110041384A (en) * | 2019-04-29 | 2019-07-23 | 苏州海科医药技术有限公司 | A kind of preparation method of II phase metabolin trans-resveratrol glucuronic acid and its application in the identification of rat cylinder metabolism-ure |
| CN110542732A (en) * | 2019-09-04 | 2019-12-06 | 湖南宇山玉月农业科技有限公司 | Finger print detection method of unicorn |
| CN110967414A (en) * | 2018-09-28 | 2020-04-07 | 四川新绿色药业科技发展有限公司 | High performance liquid chromatography method for detecting fingerprint of giant knotweed medicinal material, decoction pieces, standard decoction and formula granules |
| CN111595983A (en) * | 2020-07-03 | 2020-08-28 | 天津中医药大学 | Method for measuring chemical component content in veratrum nigrum |
| CN113336805A (en) * | 2021-06-07 | 2021-09-03 | 湖南绿蔓生物科技股份有限公司 | Extraction and purification method of polydatin |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103695479A (en) * | 2013-12-25 | 2014-04-02 | 吉首大学 | High-purity resveratrol preparation method |
| CN106018629A (en) * | 2016-08-09 | 2016-10-12 | 云南海沣药业有限公司 | Bushy knotweed leaf fingerprint HPLC method and its use in bushy knotweed leaf capsule quality control |
-
2017
- 2017-06-27 CN CN201710500484.XA patent/CN107300590A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103695479A (en) * | 2013-12-25 | 2014-04-02 | 吉首大学 | High-purity resveratrol preparation method |
| CN106018629A (en) * | 2016-08-09 | 2016-10-12 | 云南海沣药业有限公司 | Bushy knotweed leaf fingerprint HPLC method and its use in bushy knotweed leaf capsule quality control |
Non-Patent Citations (5)
| Title |
|---|
| 向海艳等: "酶法提取虎杖中白藜芦醇新工艺研究", 《林产化学与工业》 * |
| 张明等: "西南5个产区虎杖中虎杖苷和白藜芦醇含量比较", 《中国药房》 * |
| 赵鸿宾等: "酶解法提取虎杖中的白藜芦醇", 《贵州师范大学学报(自然科学版)》 * |
| 邓梦茹等: "酶法提取虎杖中的白藜芦醇", 《中南药学》 * |
| 黄志芳等: "酶解法提取纯化虎杖提取物中白藜芦醇的工艺研究", 《天然产物研究与开发》 * |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110967414A (en) * | 2018-09-28 | 2020-04-07 | 四川新绿色药业科技发展有限公司 | High performance liquid chromatography method for detecting fingerprint of giant knotweed medicinal material, decoction pieces, standard decoction and formula granules |
| CN109522672A (en) * | 2018-11-30 | 2019-03-26 | 华南理工大学 | A kind of continuous flow upstream two-phase mass transport process stable state solute concentration distribution model |
| CN109522672B (en) * | 2018-11-30 | 2020-11-24 | 华南理工大学 | Steady-state solute concentration distribution model in continuous countercurrent two-phase mass transfer process |
| CN109709226A (en) * | 2018-12-29 | 2019-05-03 | 中国农业科学院油料作物研究所 | A method of trans-resveratrol content in measurement peanut dry seeds |
| CN109633038A (en) * | 2019-01-31 | 2019-04-16 | 北京中医药大学 | The detection method of polygonin and its metabolite in biological sample |
| CN109633038B (en) * | 2019-01-31 | 2021-09-14 | 北京中医药大学 | Method for detecting polydatin and metabolite thereof in biological sample |
| CN110041384A (en) * | 2019-04-29 | 2019-07-23 | 苏州海科医药技术有限公司 | A kind of preparation method of II phase metabolin trans-resveratrol glucuronic acid and its application in the identification of rat cylinder metabolism-ure |
| CN110542732A (en) * | 2019-09-04 | 2019-12-06 | 湖南宇山玉月农业科技有限公司 | Finger print detection method of unicorn |
| CN111595983A (en) * | 2020-07-03 | 2020-08-28 | 天津中医药大学 | Method for measuring chemical component content in veratrum nigrum |
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