CN106124684A - A kind of qualitative and quantitative detection method of Pericarpium Granati ZHIXIE SAN - Google Patents
A kind of qualitative and quantitative detection method of Pericarpium Granati ZHIXIE SAN Download PDFInfo
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Abstract
The present invention relates to the qualitative and quantitative detection method of a kind of Pericarpium Granati ZHIXIE SAN.Its feature: use thin layer chromatography, with Pericarpium Granati control medicinal material for comparison, with short-cut method, authenticated characteristic point corresponding in sample；Use high performance liquid chromatography, with acetonitrile methanol 0.1% phosphoric acid that volume ratio is 3: 5.4: 91.6 for flowing phase, at 215 ± 1nm, determine total gallic acid content in sample, with always with gallic acid for compareing, after developing the color with P-Mo-Wo acid, use spectrophotography, the method measuring content of tannin is compared, easy, quick, specificity is strong, highly sensitive, accurate；With acetonitrile methanol 0.1% phosphoric acid that volume ratio is 18: 5: 77 for flowing phase, at 254nm ± 1nm, determine ellagic acid content in sample, revise mutually through flowing, improve crest separating effect, the revision of Extraction solvent, making ellagic acid content increase, the accuracy of method promotes.
Description
Technical field
The present invention relates to the qualitative and quantitative detection method of a kind of Pericarpium Granati ZHIXIE SAN.
Background technology
Punica granatum L. leatherware relieving diarrhea with astringents, hemostasis, effect of anthelmintic.It it is a kind of parts of generic medicinal plants.Pericarpium Granati ZHIXIE SAN is by stone
Pomegranate skin pulverizing medicinal materials become in powder, direct packaging becomes the packed of different size, for animal health medicine, for diarrhea of pigs, HUANGBAI(sic)
Dysentery.Within one day, consumption is 3～15g.
The prescription of Pericarpium Granati ZHIXIE SAN is as follows with preparation technology:
[prescription] Pericarpium Granati 1000g
[preparation method] takes Pericarpium Granati, pulverizes, sieves, and mixing is made powder 1000g, to be obtained final product.
Pericarpium Granati master contains tannin, and how based on hydrolyzable tannin, condensed tannin class is less.Hydrolyzable tannin is divided into again Galla Turcica (Galla Helepensis) tan
Matter and ellagitannin, gallotannin is the esters derivative that gallic acid is formed with saccharide；Ellagitannin is ellagic acid and sugar
The esters derivative that class is formed, meanwhile, gallotannin and ellagitannin all can be esterified or further by oxidation cross-linked shape
Become more complicated hydrolyzable tannin.So Chinese Pharmacopoeia one is from the beginning of version in 2005, have been used up spectrophotography, measure tannin
Content, within 2015, version just increases the HPLC assay of upper ellagic acid.
The concrete assay method of its tannin is: with gallic acid as index, by reacting generation navy blue with P-Mo-Wo acid,
Measuring its total phenol content at 760nm, deducting the polyphenol amount of the non-tannis do not adsorbed by casein with total phenol content, be tannin
Content.Operating procedure is: first have to prepare gallic acid reference substance solution, then prepares standard curve, the 3rd step confession to be prepared
Test sample solution, this solution must stand overnight, and tannin could be extracted completely, the total phenols in the 4th step colorimetric determination need testing solution
Content, the non-tannis polyphenol sample that the 5th step insulation preparation is not adsorbed by casein, and measure its content.Total phenol content deducts not
The polyphenol content adsorbed by casein, is content of tannin.Actually measure: with reference substance gallic acid reactant color
For reference, the content calculated by the trap of color sample, tannin contained in sample on earth and gallic acid are any ratios
Example relation, it is impossible to textual criticism.
The content assaying method of Chinese Pharmacopoeia one ellagic acid of version in 2015 is as follows:
Chromatographic condition and system suitability are with octadecylsilane chemically bonded silica as filler；With acetonitrile-0.2%
Phosphoric acid solution (21: 79) is flowing phase；Detection wavelength is 254nm.Number of theoretical plate is calculated by ellagic acid peak should be not less than 5000.
It is appropriate that the preparation of reference substance solution takes ellagic acid reference substance, adds methanol and makes every 1ml solution containing 20 μ g, to obtain final product.
The preparation of need testing solution takes this product powder (crossing No. 3 sieves) 0.2g, accurately weighed, puts in tool plug conical flask, accurate
Add methanol 50ml, close plug, weighed weight, supersound process (power 150W, frequency 40kHz) 40 minutes, let cool, more weighed heavy
Amount, supplies the weight of less loss, shakes up with methanol, filters, takes subsequent filtrate, to obtain final product.
Algoscopy precision respectively draws reference substance solution 5 μ l, 10 μ l, need testing solution each 5～10 μ l, injects liquid phase color
Spectrometer, measures, and calculates content with external standard two-point method, to obtain final product.
Once measuring the content of Pericarpium Granati ZHIXIE SAN by the method for pharmacopeia ellagic acid, and occurred that ellagic acid crest trailed, peak type is not
Symmetry, and sample concentration is high, PeakArea is big, and flowing phase pH value, below 2, belongs to rapid wear chromatographic column, undesired scope, it is impossible to
Directly use, method need to be improved.
Consulting literatures is learnt, has the report using Syrups by HPLC gallic acid in Punica granatum L. extraction content, but majority is all
It is free gallic acid, the humble about 1mg/g of content in the Pericarpium Granati measured【1】；Detection wavelength is 270nm, 273nm or 280nm,
Its crest height is only 1/3 (see Fig. 2) of 215nm, and flowing uses buffer salt mutually【2】Even if not being buffer salt, organic
The ratio of phase is 3%, flow velocity is 0.8ml/min, does not reaches 5% and 1ml/min scope of pharmacopoeia limit.Thus cause sample
Sampling amount is big, and interference component is many, and buffer salt or relatively low organic facies all can lowering apparatus and the life-spans of chromatographic column.Also there is mensuration total
The report of gallic acid【3】, but sample pre-treatments not science, i.e. take Pomegranate Rind 20g, add hydrochloric acid (1 → 9) 250ml, ether
250ml, back hydrolysis 2 hours, filter, extract 2 times, merging filtrate is evaporated, and methanol constant volume, to 100ml, takes subsequent filtrate, as confession
Test sample solution.The medical material 0.2g Han Pericarpium Granati in quite every 1ml need testing solution.From the word of method, non-specialized-technical personnel are very
The difficult filtrate judging to merge is hydrochloric acid water part, or ether portion.Total Galla Turcica (Galla Helepensis) in 13 place of production difference Pericarpium Granatis measured
Acid content between 1.30～4.64mg/g, average out to 2.92mg/g.
We are exactly under above-mentioned background condition, for controlling Pericarpium Granati ZHIXIE SAN with easy, quick, accurate, high standard
Quality, the discussion research that it has been carried out quality standard, invent the qualitative and quantitative detection method of Pericarpium Granati ZHIXIE SAN.
Summary of the invention
(1) present invention uses thin layer chromatography, with Pericarpium Granati control medicinal material for comparison, authenticated in its powder corresponding
Characteristic point, method is easy, only needs the ultrasonic control medicinal material of 2ml60% methanol and test sample, supernatant point sample, launches, ultra-violet lamp
Inspect under 254nm, get final product (Fig. 1)；
(2) high performance liquid chromatography is used, with acetonitrile-methanol-0.1% phosphoric acid that volume ratio is 3: 5.4: 91.6 for flowing
Phase, at 215 ± 1nm (Fig. 2), determines total gallic acid in six batches of Pericarpium Granati ZHIXIE SAN, content at 3.76mg/g～
Between 13.90mg/g, meansigma methods is 9.08mg/g, and crest separates good, and in three kinds of different chromatographic columns, peak retention time is all
At about 10 minutes, sample introduction one pin, run 15 minutes, gallic acid reference substance sample size 0.05 μ g, its PeakArea is up to 50
Ten thousand, easy, quick, accurate, sensitivity is more than 3 times of report method.
(3) high performance liquid chromatography is used, with acetonitrile-methanol-0.1% phosphoric acid that volume ratio is 18: 5: 77 for the phase that flows,
At 254nm ± 1nm (Figure 10), determine ellagic acid content in sample, flow by acetonitrile-0.2% phosphorus of volume ratio 21: 79
After acid solution is changed to acetonitrile-methanol-0.1% phosphoric acid of volume ratio 18: 5: 77, improves crest symmetry, make flowing phase
PH value rises to more than 2, decreases the damage to chromatographic column；Extraction solvent is changed to 80% methanol, ellagic acid content by methanol
Improving 9.9%, measure numerical value and more they tend to the real content (being shown in Table 8) of sample, the accuracy of method promotes；The sample size of ellagic acid
Being adjusted to 0.04 μ g, PeakArea, in 50 power, had both ensured Numerical accuracy, had extended again the chromatographic column life-span.
By methodological study, the sample size of gallic acid is at 0.012298～0.196773 μ g, with peak area in well
Linear relationship, regression equation is: Y=10149563.8X-3334, r=0.99997 (be shown in Table 1, Fig. 3)；Sample-adding is used to reclaim
Experiment, result shows: the average recovery rate of gallic acid is 99.97% (n=6), and RSD is 0.51% (being shown in Table 2)；Gallic acid
Precision (being shown in Table 3)；Stability (is shown in Table 4)；Repeatability (being shown in Table 5)；Specificity (see Fig. 4,5,6) (is shown in Table with post ruggedness
6, Fig. 7,8,9)；Gallic acid content result (table 7) in Pericarpium Granati ZHIXIE SAN；Extraction solvent affects (table 8) to ellagic acid content；
The sample size of ellagic acid, at 0.01302～0.2083 μ g, is good linear relationship with peak area, and regression equation is: Y=
(13048189.2X-21214r=0.9998 be shown in Table 9, Figure 11)；The average recovery rate of ellagic acid is 99.86% (n=6), RSD
It is 0.55% (being shown in Table 10)；The precision (being shown in Table 11) of ellagic acid；Stability (is shown in Table 12)；Repeatability (being shown in Table 13)；Specificity
(see Figure 12,13,14) and post ruggedness (be shown in Table 14, Figure 15,16,17)；Ellagic acid acid content result (table in Pericarpium Granati ZHIXIE SAN
7)；Methodology requirement is all met etc. experimental data.Gallic acid and the quantitative determination of ellagic acid be applicable to Pericarpium Granati ZHIXIE SAN.
The technical solution adopted for the present invention to solve the technical problems is:
(1) qualitative identification method
Take Pericarpium Granati ZHIXIE SAN 0.5g, finely ground, add 60% methanol 2ml, supersound process 15 minutes, supernatant is as test sample
Solution；Take Pericarpium Granati control medicinal material 0.5g again, prepare control medicinal material solution with method；Draw above-mentioned two kinds of solution each 8～10 μ l, point
Other point is in same silica gel G F254On lamellae, with cyclohexane-ethyl acetate-methyl alcohol-formic acid that volume ratio is 2.5: 5: 0.3: 0.2
For developing solvent, launch, take out, dry, put and inspect under ultra-violet lamp 254nm, in test sample chromatograph, with control medicinal material chromatograph phase
On the position answered, aobvious same color principal spot；
(2). total gallic acid content assay method
A. chromatographic condition and system suitability are with octadecylsilane chemically bonded silica as filler；With volume ratio for 3
: acetonitrile-methanol-0.1% phosphoric acid of 5.4: 91.6 is flowing phase；Detection wavelength: 215nm；Column temperature is 30 DEG C；Number of theoretical plate is by going
Gallic acid peak calculates and is not less than 2500；
B. to take gallic acid reference substance appropriate in reference substance solution preparation, accurately weighed, adds 50% methanol and makes every 1ml and contain
The solution of 2.5 μ g, to obtain final product；
C. need testing solution preparation takes the Pericarpium Granati ZHIXIE SAN powder 0.1g by No. 3 sieves, accurately weighed, puts tool plug cone
In shape bottle, add water 15ml and concentrated hydrochloric acid 6ml, is heated to reflux 3 hours, lets cool, and adds 20% sodium hydroxide 4ml, shakes up, quantitative with water
Being transferred in 50ml measuring bottle to scale, shake up, filter, precision measures subsequent filtrate 2ml, puts in 10ml measuring bottle, adds 50% methanol extremely
Scale, shakes up, and filters, takes subsequent filtrate as need testing solution；
D. algoscopy precision respectively draws reference substance solution and each 20 μ l of need testing solution, injects chromatograph of liquid, surveys
Fixed, to obtain final product；
(3). ellagic acid content assaying method
A. chromatographic condition and system suitability are with octadecylsilane chemically bonded silica as filler；With volume ratio it is
Acetonitrile-methanol-0.1% phosphoric acid of 18: 5: 77 is flowing phase；Detection wavelength: 254nm；Column temperature is 30 DEG C；Number of theoretical plate is by going tan
Flower acid peak calculates and is not less than 4000；
B. the preparation of reference substance solution takes ellagic acid reference substance in right amount, adds methanol and makes every 1ml solution containing 2 μ g, i.e.
?；
C. the preparation of need testing solution takes the Pericarpium Granati ZHIXIE SAN powder 0.1g by No. 3 sieves, accurately weighed, puts tool plug
In conical flask, accurate addition 80% methanol 25ml, close plug, weighed weight, with power 250w, frequency 33kHz, supersound process 40 points
Clock, lets cool, more weighed weight, supplies the weight of less loss with methanol, shakes up, and filters, and precision measures subsequent filtrate 1ml, puts 10ml amount
In Ping, add 80% methanol to scale, shake up, filter with the microporous filter membrane of 0.45 μm, take subsequent filtrate as need testing solution；
Algoscopy precision respectively draws reference substance solution and each 20 μ l of need testing solution, injects chromatograph of liquid, measures,
Obtain.
The principle of the present invention is as follows:
1., according to chemical constitution and the character of each effective ingredient of Chinese medicine, it then follows the extraction principle of similar compatibility, it is suitable to use
Extraction solvent, easy, prepare test sample and control medicinal material solution quickly.Again with the developing solvent of opposed polarity, launch,
Various chemical compositions will be different according to Adsorption and desorption ability attached, that Adsorption and desorption is attached again along with different developing solvents, and
Being able to good separation on respective lamellae, test sample chromatogram presents identical on position corresponding with control medicinal material chromatograph
Thin layer speckle, and differentiated.
2. there is absorption maximum according to gallic acid at 215nm, and ellagic acid has absorption maximum at 254nm, by flowing phase
Component and ratio inquire into, make gallic acid or ellagic acid crest be able to good separation, and crest be symmetrical, retention time is suitable,
In 3 kinds of different chromatographic columns, gallic acid retention time about 10 minutes, ellagic acid retention time 13～15 minutes.Depend on again
According to these 2 kinds of compositions sample size within the specific limits, present good linear relationship with its peak area, and be used for quantitative determining.
The innovative point of the present invention and having the beneficial effect that:
(1) thin layer of the present invention differentiates that a test sample and control medicinal material only step 2ml60% methanol is ultrasonic, supernatant point sample, exhibition
Open, inspect under ultra-violet lamp 254nm, without filtering, concentrate, be evaporated, development step, 15 minutes pre-treatment time, solvent
4ml；Compared with official method, detect a batch sample and can save time for sample pretreatment 2.5 hours, organic solvent 105ml, colour developing
Agent 10ml.
(2) content assaying method of total gallic acid of the present invention, compared with report method, in the preparation of test sample
On, only need sample 0.1g, acid water is solvent, after hydrolyzing 3 hours, in appropriate bases and too high acidity, with the direct constant volume of water in
50ml, take 2ml subsequent filtrate, with 50% methanol dilution to 10ml, filter,；And report method needs sample 20g, sour water
250ml, ether 250ml, repeat to hydrolyze 2 times, each 2 hours, and merging filtrate is evaporated, and methanol constant volume, in 100ml, filters, enters
Sample,.Two methods are compared, this method than report method save organic solvent 596ml, time 2.5 little time；From detection sensitivity,
The concentration of need testing solution of the present invention is 0.0004g/ml medical material, and report method is then 0.2g/ml medical material, the two difference 500
Times, namely report method sample introduction is once, and equal to this method sample introduction 500 times, this method significantly extends instrument and chromatograph
Column life.From method accuracy, the powder that 6 batches of different sources Pericarpium Granatis that this method measures produce, total gallic acid is average
Content is 9.08mg/g, and the average content of 13 batches of Pericarpium Granatis that report method measures is 2.92mg/g, and this method numerical value is report
3 times of road method, reason is that this law acid concentration is high, and hydrolysis is in sour water one phase solvent, uniformly, completely.To sum up the present invention easy,
Fast, accurately, the pollution that low cost, highly sensitive, environmental protection, solvent-free evaporation bring to environment.
(3) compared with the assay of the Ellagic Acid in Granati Cortex that pharmacopeia is recorded:
1) flowing is different mutually, and pharmacopeia is acetonitrile-0.2% phosphoric acid (21: 79), and the concentration of acid first is big, pH value 2.0 with
Under, easily damage instrument and chromatographic column, second the crest hangover of ellagic acid, the symmetry at peak is bad, and being revised as volume ratio is 18:
After acetonitrile-methanol-0.1% phosphoric acid of 5: 77, solving the crest hangover of ellagic acid, peak type is symmetrical, and the pH value of flowing phase also carries
High to more than 2.0；
2) ellagic acid reference substance sample size is different, and pharmacopeia sample size ellagic acid is 0.1～0.2 μ g, and the present invention is 0.04 μ g,
Peak area, still 500,000, can meet the requirement of Accurate Determining content completely, and pharmacopeia sample size is 2.5～5 times of the present invention, ripple
Peak area to reach more than million to two hundred ten thousand, and high concentration of justing think is entered a pin and studied into 3 pins equal to this, says for the chromatographic column life-span,
This research can be extended；
3) Extraction solvent is different, and from ellagic acid flowing mutually, the ratio of organic facies is 21%, illustrates that it is an inclined water solublity
Compound, the dissolubility in methanol should be lower than in aqueous methanol, so studying by contrast, uses 80% methanol extraction, can
Making ellagic acid content improve 9.9% (being shown in Table 8), make measurement result closer to the real content of sample, and then the method that improves is accurate
Really property.
Accompanying drawing explanation
The thin layer of Fig. 1 Pericarpium Granati ZHIXIE SAN differentiates figure
Fig. 2 gallic acid spectral scan figure
Fig. 3 gallic acid linear relationship chart
Fig. 4 gallic acid reference substance HPLC chromatogram
Gallic acid HPLC chromatogram in Fig. 5 Pericarpium Granati ZHIXIE SAN
Fig. 6 gallic acid blank sample HPLC chromatogram
Fig. 7 gallic acid post serviceability test-Shimadzu post HPLC chromatogram
Fig. 8 gallic acid post serviceability test-luna post HPLC chromatogram
Fig. 9 gallic acid post serviceability test-Agilent post HPLC chromatogram
Figure 10 ellagic acid spectral scan figure
Figure 11 ellagic acid linear relationship chart
Figure 12 ellagic acid reference substance HPLC chromatogram
Ellagic acid HPLC chromatogram in Figure 13 Pericarpium Granati ZHIXIE SAN
Figure 14 ellagic acid blank sample HPLC chromatogram
Figure 15 ellagic acid post serviceability test-Shimadzu-VP-ODS post HPLC chromatogram
Figure 16 ellagic acid post serviceability test-luna post HPLC chromatogram
Figure 17 ellagic acid post serviceability test-moon rising sun post HPLC chromatogram
In Fig. 1,1.2.3 is sample, is 4. Pericarpium Granati control medicinal material.
Fig. 2, Tu10Zhong, abscissa be wavelength (nm), vertical coordinate be trap (mAU).
Fig. 3, Tu11Zhong, abscissa be sample size (μ g), vertical coordinate be peak area.
In Fig. 4, Fig. 5, Fig. 6, Fig. 7, Fig. 8, Fig. 9, abscissa is retention time (minute), and vertical coordinate is response value (uV), 1
For gallic acid peak.
Figure 12, Figure 13, Figure 14, Figure 15, Figure 16, Tu17Zhong, abscissa is retention time (minute), and vertical coordinate is response value
(uV), 1 is ellagic acid peak.
The specific embodiment of the invention
Embodiment
(1) qualitative identification method
Take Pericarpium Granati ZHIXIE SAN 0.5g, finely ground, add 60% methanol 2ml, supersound process 20 minutes, supernatant is as test sample
Solution.Take Pericarpium Granati control medicinal material 0.5g again, prepare control medicinal material solution with method.Draw above-mentioned two kinds of solution each 8～10 μ l, point
Other point is in same silica gel G F254On lamellae, with cyclohexane-ethyl acetate-methyl alcohol-formic acid that volume ratio is 2.5: 5: 0.3: 0.2
For developing solvent, launch, take out, dry, put and inspect under ultra-violet lamp 254nm；In test sample chromatograph, corresponding to reference substance chromatograph
Position on, aobvious same color principal spot；
(2). total gallic acid content assay method
A. chromatographic condition and system suitability are with octadecylsilane chemically bonded silica as filler；With volume ratio for 3
: acetonitrile-methanol-0.1% phosphoric acid of 5.4: 91.6 is flowing phase；Detection wavelength: 215nm；Column temperature is 30 DEG C；Number of theoretical plate is by going
Gallic acid peak calculates and is not less than 2500；
B. to take gallic acid reference substance appropriate in reference substance solution preparation, accurately weighed, adds 50% methanol and makes every 1ml and contain
The solution of 2.5 μ g, to obtain final product；
C. need testing solution preparation takes the Pericarpium Granati ZHIXIE SAN powder 0.1g by No. 3 sieves, accurately weighed, puts tool plug taper
In Ping, add water 15ml and concentrated hydrochloric acid 6ml, is heated to reflux 3 hours, lets cool, and adds 20% sodium hydroxide 4ml, shakes up, quantitatively turn with water
Moving in 50ml measuring bottle to scale, shake up, filter, precision measures subsequent filtrate 2ml, puts in 10ml measuring bottle, adds 50% methanol to carving
Degree, shakes up, and filters, takes subsequent filtrate as need testing solution；
D. algoscopy precision respectively draws reference substance solution and each 20 μ l of need testing solution, injects chromatograph of liquid, surveys
Fixed, to obtain final product, to determine 6 batch sample content (being shown in Table 7)；
(3). ellagic acid content assaying method
A. chromatographic condition and system suitability are with octadecylsilane chemically bonded silica as filler；With volume ratio it is
Acetonitrile-methanol-0.1% phosphoric acid of 18: 5: 77 is flowing phase；Detection wavelength: 254nm；Column temperature is 30 DEG C；Number of theoretical plate is by going tan
Flower acid peak calculates and is not less than 4000；
B. the preparation of reference substance solution takes ellagic acid reference substance in right amount, adds methanol and makes every 1ml solution containing 2 μ g, i.e.
?；
C. the preparation of need testing solution takes the Pericarpium Granati ZHIXIE SAN powder 0.1g by No. 3 sieves, accurately weighed, puts tool plug
In conical flask, accurate addition 80% methanol 25ml, close plug, weighed weight, with power 250w, frequency 33kHz, supersound process 40 points
Clock, lets cool, more weighed weight, supplies the weight of less loss with methanol, shakes up, and filters, and precision measures subsequent filtrate 1ml, puts 10ml amount
In Ping, add 80% methanol to scale, shake up, filter with the microporous filter membrane of 0.45 μm, take subsequent filtrate as need testing solution；
Algoscopy precision respectively draws reference substance solution and each 10 μ l of need testing solution, injects chromatograph of liquid, measures,
Obtain, determine 6 batch sample content (being shown in Table 7)；
Table 1 gallic acid sample size and peak area
The recovery test result of gallic acid in table 2 sample
Table 3 gallic acid Precision Experiment result (peak area)
Table 4 gallic acid stability experiment result
Table 5 gallic acid replica test result
Table 6 gallic acid post serviceability test result (mg/g)
Gallic acid content measurement result in table 7 Pericarpium Granati ZHIXIE SAN
Table 8 variable concentrations methanol extraction affects (mg/g) to ellagic acid content
Table 9 ellagic acid sample size and peak area
The recovery test result of ellagic acid in table 10 sample
Table 11 ellagic acid Precision Experiment result (peak area)
Table 12 ellagic acid stability experiment result
Table 13 ellagic acid replica test result
Table 14 ellagic acid post serviceability test result (mg/g)
List of references
[1] Zhang Shuosheng, HPLC method measures gallic acid in Punica granatum L. extraction content before and after concocting, pharmaceutical analysis magazine, [J] .2010.30
(6) 1104～1106
[2] Zhou Benhong, Liu Chun, Wu Zhenhua etc., the content of Gallic Acid in Megranates pericarp by HPLC, [J]. China
Hospital pharmacy's magazine, 2005.25 (12) 1148～1150
[3] Chang Zhanying, horse Ramulus Cinnamomi, Gao Xiaoli, HPLC method measures the content of Xinjiang different sources gallic acid in Punica granatum L. extraction, [J].
Xinjiang Medicine University journal .2009.32 (11) 1552～1553.
Claims (2)
1. the qualitative and quantitative detection method of a Pericarpium Granati ZHIXIE SAN, it is characterised in that:
(1) qualitative identification method
Take Pericarpium Granati ZHIXIE SAN 0.5g, finely ground, add 60% methanol 2ml, supersound process 20 minutes, supernatant is molten as test sample
Liquid.Take Pericarpium Granati control medicinal material 0.5g again, prepare control medicinal material solution with method.Draw above-mentioned two kinds of solution each 8～10 μ l, respectively
Point is in same silica gel G F254On lamellae, with cyclohexane-ethyl acetate-methyl alcohol-formic acid that volume ratio is 2.5: 5: 0.3: 0.2 it is
Developing solvent, launches, and takes out, dries, put and inspect under ultra-violet lamp 254nm；In test sample chromatograph, corresponding with reference substance chromatograph
On position, aobvious same color principal spot；
(2). total gallic acid content assay method
A. chromatographic condition and system suitability are with octadecylsilane chemically bonded silica as filler；With volume ratio for 3: 5.4
: acetonitrile-methanol-0.1% phosphoric acid of 91.6 is flowing phase；Detection wavelength: 215nm；Column temperature is 30 DEG C；Number of theoretical plate presses Galla Turcica (Galla Helepensis)
Acid peak calculates and is not less than 2500；
B. reference substance solution preparation takes gallic acid reference substance in right amount, accurately weighed, adds 50% methanol and makes every 1ml containing 2.5 μ g
Solution, to obtain final product；
C. need testing solution preparation takes the Pericarpium Granati ZHIXIE SAN powder 0.1g by No. 3 sieves, accurately weighed, puts tool plug conical flask
In, add water 15ml and concentrated hydrochloric acid 6ml, is heated to reflux 3 hours, lets cool, and adds 20% sodium hydroxide 4ml, shakes up, quantitatively shift with water
To scale to 50ml measuring bottle, shaking up, filter, precision measures subsequent filtrate 2ml, puts in 10ml measuring bottle, adds 50% methanol to scale,
Shake up, filter, take subsequent filtrate as need testing solution；
D. algoscopy precision respectively draws reference substance solution and each 20 μ l of need testing solution, injects chromatograph of liquid, measures, i.e.
?；
(3). ellagic acid content assaying method
A. chromatographic condition and system suitability are with octadecylsilane chemically bonded silica as filler；With volume ratio for 18: 5:
Acetonitrile-methanol-0.1% phosphoric acid of 77 is flowing phase；Detection wavelength: 254nm；Column temperature is 30 DEG C；Number of theoretical plate is by removing ellagic acid
Peak calculates and is not less than 4000；
B. the preparation of reference substance solution takes ellagic acid reference substance in right amount, adds methanol and makes every 1ml solution containing 2 μ g, to obtain final product；
C. the preparation of need testing solution takes the Pericarpium Granati ZHIXIE SAN powder 0.1g by No. 3 sieves, accurately weighed, puts tool plug taper
In Ping, the accurate 80% methanol 25ml that adds, close plug, weighed weight, with power 250w, frequency 33kHz, supersound process 40 minutes,
Let cool, more weighed weight, to supply the weight of less loss with methanol, shake up, filter, precision measures subsequent filtrate 1ml, puts 10ml measuring bottle
In, add 80% methanol to scale, shake up, filter with the microporous filter membrane of 0.45 μm, take subsequent filtrate as need testing solution；
Algoscopy precision respectively draws reference substance solution and each 20 μ l of need testing solution, injects chromatograph of liquid, measures, to obtain final product.
The qualitative and quantitative detection method of a kind of Pericarpium Granati ZHIXIE SAN the most according to claim 1, it is characterised in that described
Pericarpium Granati ZHIXIE SAN be made up of Pericarpium Granati medical material, every 1g powder Pericarpium Granati Han 1g.
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