CN106706532A - Detection method of total flavonoid content in Momordica charantia L. leaves - Google Patents
Detection method of total flavonoid content in Momordica charantia L. leaves Download PDFInfo
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Abstract
The invention discloses a detection method of total flavonoid content in Momordica charantia L. leaves. The detection method comprises the following steps: (1) establishing a standard curve: a, preparing a reference liquid: b, determining the reference liquid: (2) determining the total flavonoid content in a sample to be tested: c, preparing a test sample liquid: and d, determining the test sample solution. According to the detection method of the total flavonoid content in the Momordica charantia L. leaves disclosed by the invention, the content of total flavonoids in the Momordica charantia L. leaves can be accurately, reliably and conveniently detected through the screening of multiple processes to effectively monitor the quality of Chinese medicinal herbs, namely the Momordica charantia L. leaves so as to provide effective guarantee for clinical effects of the Momordica charantia L. leaves.
Description
Technical field
The present invention relates to the detection method of quality of medicinal material, and in particular to the detection method of general flavone content in balsampear leaf.
Background technology
Balsampear leaf is the annual herbaceous plant balsam pear of Curcurbitaceae Momordica (Momordica charantia L.)
Dried leaf.Cool in nature, bitter.Have clearing heat and detoxicating, sterilized, stop dysentery, effect of analgesic, stomachache, dysentery, furuncle swelling toxin can be treated.It is bitter
Melon leaf, using extensive, is usually used in red and swollen heat pain, child miliaria, the heat toxin of skin after treatment bite by mosquitos as conventional Folk medicine
Sore swells, syphilis, dog sting and the fungal infection of the hand.
Balsampear leaf aboundresources, determined curative effect, among the people using extensive, degree of recognition is high and is conveniently easy to get, with preferably opening
Make an offer value.But at present, domestic and international multipair balsam pear fruit carries out multi-angle research and the research of its leaf is very few, still lack to balsampear leaf
Quality evaluating method and standard.
Therefore, in order to ensure the quality and safety of balsampear leaf are, it is necessary to a kind of detection method, the matter for assessing balsampear leaf
Amount.
The content of the invention
It is the invention provides a kind of detection method of general flavone content in balsampear leaf including following to solve the above problems
Step：
(1) foundation of standard curve
The preparation of a, reference substance solution：
Control substance of Rutin is taken, plus ethanol is configured to reference substance solution；
The measure of b, reference substance solution：
Reference substance solution is taken, 5% sodium nitrite solution, 10% aluminum nitrate solution, sodium hydroxide solution, ethanol is sequentially added
Constant volume, in mensuration absorbance under 506nm in UV detector, standard curve is obtained according to testing result；
(2) in testing sample general flavone content measure：
The preparation of c, need testing solution：
Balsampear leaf sample to be measured is taken, 60 DEG C~90 DEG C of petroleum ether refluxing extraction, filtering discards filtrate, is repeated once, takes
Residue, 60%~90% EtOH Sonicate or refluxing extraction, filtering supplies institute's weight loss with the ethanol of respective concentration, obtains for examination
Product solution；
The measure of d, need testing solution：
Need testing solution is taken, is measured in the method for step b, the standard curve according to step (1) obtains testing sample
The content of middle general flavone.
Further, in step a, the ethanol is 50% ethanol.
Further, in step b, 5% sodium nitrite solution is 1 with the volume ratio of reference substance solution：1.
Further, in step b, the 10% aluminum nitrate solution solution is 1 with the volume ratio of reference substance solution：1.
Further, in step b, the sodium hydroxide solution is 10 with the volume ratio of reference substance solution：1.
Further, in step c, the ethanol is 80% ethanol.
Further, in step c, the ethanol is extracted as refluxing extraction.
Further, the time that the alcohol reflux is extracted is 0.5~3 hour, preferably 1.5 hours.
Further, the temperature that the alcohol reflux is extracted is 60 DEG C~80 DEG C.
Further, the temperature that the alcohol reflux is extracted is 70 DEG C.
Further, the ethanol and the volume mass ratio of balsampear leaf sample to be measured are 100mL：1g~160mL：1g, it is excellent
Select 140mL：1g.
The detection method of general flavone content in balsampear leaf of the present invention, by the screening of multiple techniques, can accurately, it is reliable,
The content of general flavone in balsampear leaf is easily detected, so that the quality of effective monitoring balsampear leaf medicinal material, is the clinical treatment of balsampear leaf
Effect provides effective guarantee.
Obviously, the above of the invention, according to the ordinary technical knowledge and customary means of this area, is not departing from
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification of other diversified forms can also be made, is replaced or is changed.
The specific embodiment of form, remakes further specifically to the above of the invention by the following examples
It is bright.But this scope for being interpreted as above-mentioned theme of the invention should not be only limitted to following example.It is all based on the above of the present invention
The technology realized belongs to the scope of the present invention.
Brief description of the drawings
Fig. 1 balsam pear leaf flavonoids ultraviolet determination standard curves.
Fig. 2 different sources balsampear leaf determination of total flavonoids.
Specific embodiment
Experiment material used by following embodiments is as follows：
Difference harvesting places samples picked up from respectively before and after in August, 2011 in Sichuan Province Chengdu, Pengzhou City, Jianyang City,
The ground such as Neijiang City, Zigong City, Yibin City and Baoding, Zhejiang Jiaxing, Foshan.Collection fresh leaf, removes impurity, dries in the shade
Obtain final product.Above laboratory sample is accredited as cucurbitaceous plant balsam pear through pharmaceutical college of Chengdu University of Traditional Chinese Medicine professor Jiang Guihua
The dried leaf of Momordica charantia L..Sample collection place, time and growing environment are shown in Table 1.
Adopted in Sichuan Province Pengzhou City Ao Ping towns in the counterpart on the 20th of August in 2011, through pharmaceutical college of Chengdu University of Traditional Chinese Medicine Jiang Gui
Professor Hua is accredited as the dried leaf of Curcurbitaceae balsam pear platymiscium balsam pear Momordica charantia L., used as control medicinal material；
It is pure that agents useful for same is analysis.
The different sources balsampear leaf sample of table 1
Instrument is as follows：
Ultraviolet specrophotometer (Japanese Shimadzu UV-1600 types), vacuum drying chamber (the gloomy letter limited public affairs of laboratory apparatus in Shanghai
Department, DZG-6090 types), ten a ten thousandth electronic balances (German SartoriusBP211D), (city of Kunshan surpasses ultrasonic cleaner
Sound Instrument Ltd., KQ5200E types), electronic balance (Shanghai Precision Scientific Apparatus Co., Ltd, YP601N) etc..
Reagent is as follows with reagent：
Rutin (Nat'l Pharmaceutical ＆ Biological Products Control Institute 100080-201009)；Petroleum ether, ethanol, natrium nitrosum, nitric acid
Aluminium, NaOH etc. are AR.
Embodiment 1
(1) investigation of Determination Method of Flavone Content
1 reference substance solution
Reference substance solution：Take the dry control substance of Rutin to constant weight appropriate, it is accurately weighed, 100ml volumetric flasks are placed in, add
50% ethanol, constant volume is made the rutin solution of 0.2mg/ml.
2 Detection wavelengths are investigated
Precision measures rutin solution 1.0ml, is placed in 25ml volumetric flasks, adds 5% sodium nitrite solution 1.0ml, 6min
After add 10% aluminum nitrate solution 1.0ml, after 6min add 1mol/L sodium hydroxide test solution 10ml, 50% ethanol is used after 15min
Constant volume.
The rutin solution that will be made, is put into ultraviolet specrophotometer, in being scanned in 200~600nm wave-length coverages, as a result shows
Show, rutin solution has absorption maximum at 506nm.Therefore, wavelength will be determined and is set to 506nm.
3 linear relationships are investigated
According to certain gradient, precision measures rutin solution in right amount respectively, and in 25ml volumetric flasks, colour developing, constant volume determines extinction
Degree A.With absorbance A as ordinate, rutin solution concentration C is abscissa, draws standard curve.Obtain linear equation A=32.173x
+ 0.0066, R2=0.9977.In 0.005~0.025mg/ml concentration ranges, absorbance A and rutin solution concentration C is in good
Good linear relationship.The results are shown in Table 2 and Fig. 1.
The absorbance of table 2 and reference substance concentration relationship
4 precision are investigated
Precision measures rutin solution in right amount, 7 parts, is placed in 25ml volumetric flasks, develops the color, constant volume, mensuration absorbance A.As a result
It is shown in Table 3.
The precision of table 3 is investigated
Experimental result shows that the absorbance A stabilizations of rutin solution calculate its RSD value, is 0.12%, illustrates the precision
Degree is good, can carry out the measure of the absorbance A of rutin solution.
5 need testing solution preparation methods are investigated
5.1 extracting methods are investigated
Same batch (No. 7 samples in table 1) balsampear leaf sample powder 0.5g is taken, it is 6 parts, accurately weighed, it is placed in 250ml conical flasks
In, plus petroleum ether (60~90 DEG C) 50ml, water-bath backflow 1h, filter, discard filtrate.It is repeated once.Residue adds different extractions
Solvent 50ml, weighs, and is extracted 1 hour using under Different Extraction Method, is mended to original weight with corresponding Extraction solvent, and filtering takes filtrate
4.0ml, is placed in the volumetric flask of 25ml, colour developing, adds Extraction solvent constant volume, and mensuration absorbance A, experimental result is shown in Table 4.
The need testing solution extracting method of table 4 is investigated
Experimental result shows：The absorbance A that refluxing extraction is determined is apparently higher than ultrasonic extraction；With the liter of concentration of alcohol
Height, the absorbance A for being determined also is increased, and peak is reached when concentration of alcohol is 80%, is then declined slightly.80% ethanol
Absorbance A measured during refluxing extraction is maximum, is 0.870.Therefore, the extracting method of balsam pear leaf flavonoids is set to 80% second
Alcohol reflux is extracted.
5.2 reflux temperatures are investigated
Same batch (No. 7 samples in table 1) balsampear leaf sample powder 0.5g is taken, it is 6 parts, accurately weighed, it is placed in 250ml conical flasks
In, plus petroleum ether (60~90 DEG C) 50ml, water-bath backflow 1h, filter, discard filtrate.It is repeated once.Residue adds 80% ethanol
50ml, weighs, and is extracted 1 hour under different Extracting temperatures, is mended to original weight with 80% ethanol, and filtering takes filtrate 4.0ml in 25ml
Volumetric flask in, colour developing is settled to 25ml with 80% ethanol, mensuration absorbance, as a result such as table 5.
The Extracting temperature of table 5 is investigated
Result shows：The absorbance A for obtaining is determined during 70 DEG C of refluxing extractions maximum, therefore selection refluxing extraction temperature is 70
℃。
5.3 extraction times were investigated
Same batch (No. 7 samples in table 1) balsampear leaf sample powder 0.5g is taken, it is 6 parts, accurately weighed, it is placed in 250ml conical flasks
In, plus petroleum ether (60~90 DEG C) 50ml, water-bath backflow 1h, filter, discard filtrate.It is repeated once.Residue adds 80% ethanol
50ml, weighs, 70 DEG C of refluxing extraction different times, is mended to original weight with 80% ethanol, and filtering takes filtrate 4.0ml in the appearance of 25ml
In measuring bottle, colour developing is settled to 25ml, mensuration absorbance, as a result such as table 6 with 80% ethanol.
The extraction time of table 6 is investigated
Experimental result shows：With the extension of reflux extracting time, the absorbance A surveyed is in rising trend, when backflow is carried
When taking the time for 1.5h, absorbance A is maximum, then tends to be steady.That is backflow 1.5h just can be by Flavonoid substances base in balsampear leaf
This extraction is completely.Therefore, the test sample extraction time of balsampear leaf determination of total flavonoids is set to 1.5h.
5.4 solid-liquid ratios are investigated
Same batch (No. 7 samples in table 1) balsampear leaf sample powder 0.5g is taken, it is 6 parts, accurately weighed, it is placed in 250ml conical flasks
In, plus petroleum ether (60~90 DEG C) 50ml, water-bath backflow 1h, filter, discard filtrate.It is repeated once.Residue adds 80% ethanol not
Same volume, weighs, 70 DEG C of refluxing extraction 1.5h, is mended to original weight with 80% ethanol, and filtering takes filtrate 4.0ml in the capacity of 25ml
In bottle, colour developing is settled to 25ml, mensuration absorbance, as a result such as table 7 with 80% ethanol.
The solid-liquid ratio of table 7 is investigated
Result shows：Extraction solvent can fully extract the general flavone in balsampear leaf when being 50ml, when Extraction solvent is
Extraction efficiency highest during 70ml.
The determination of the optimal need testing solution preparation method of 5.5 present invention
According to foregoing investigation result, it is determined that the optimal need testing solution preparation method of the present invention is：Take balsampear leaf powder
0.5g, it is accurately weighed, it is placed in 250ml conical flasks, plus petroleum ether (60~90 DEG C) 50ml, water-bath backflow 1h, filter, discard filter
Liquid.It is repeated once.Residue adds 80% ethanol 50ml, weighs, 70 DEG C of refluxing extraction 1.5h, is mended to original weight with 80% ethanol, filters,
Obtain final product need testing solution.
6 stability experiments are investigated
(No. 7 samples in table 1) balsampear leaf medicinal powder 0.5g is taken, it is accurately weighed, according to balsampear leaf general flavone content under " 5.5 " item
Determine need testing solution preparation method and prepare need testing solution.Parallel precision measures totally 6 parts of need testing solution 4.0ml, is placed in
In 25ml volumetric flasks, develop the color respectively, 80% ethanol constant volume, timing determines an absorbance A at interval of 10min.Result such as table
8。
The balsampear leaf Determination of Total Flavonoids stability test of table 8
Experimental result shows：60min internal absorbances A without significant change, between 0.546~0.550.Calculate RSD values
It is 0.26%, illustrates under this experiment condition, need testing solution colour developing 60min internal absorbance values A stabilizations, this stable experiment is good
It is good.
7 replica tests are investigated
Take same batch (No. 7 samples in table 1) balsampear leaf sample powder 0.5g, it is 6 parts, accurately weighed, by under " 5.5 " item for examination
Product solution manufacturing method prepares need testing solution.Need testing solution 4.0ml is taken in the volumetric flask of 25ml, 80% second is used in colour developing
Alcohol is settled to 25ml, mensuration absorbance.Calculate general flavone content in balsampear leaf medicinal material.Result such as table 9.
The balsam pear leaf flavonoids replica test of table 9
Result shows：The absorbance of 1~6 parallel need testing solution is 0.617~0.636, by calibration curve equation meter
Calculation show that 1~6 parallel test sample general flavone content is 1.19%~1.21%, and average content is 1.20%, calculates its RSD
It is 0.68% to be worth, and illustrates that this experimental repeatability is preferable.
8 rate of recovery are tested
Same batch (in table 1 No. 7) balsampear leaf sample powder 0.5g, 6 parts, accurately weighed, addition rutin 1mg are taken, is pressed
Balsampear leaf determination of total flavonoids need testing solution preparation method prepares need testing solution under " 5.5 " item.Take need testing solution
4.0ml develops the color in 25ml volumetric flasks by color condition, 80% ethanol constant volume, mensuration absorbance, by regression equation calculation balsam pear
Leaf flavonoids content, the results are shown in Table 10.
The balsam pear leaf flavonoids rate of recovery of table 10 is investigated
Experimental result shows：1~No. 6 average recovery of test sample is 99.94%~100.44%, and average is
100.13%, it is 0.22% to calculate its RSD, illustrates that this experimental technique average recovery is good.
6.4 balsampear leaf determination of total flavonoids
10 crowdes of balsampear leaf medicinal powder 0.5g are respectively taken in table 1, it is accurately weighed, according to balsampear leaf general flavone content under " 5.5 " item
Determine need testing solution preparation method and prepare need testing solution.Need testing solution 4.0ml is taken in 25ml volumetric flasks, by reference substance
Solution color condition develops the color, and 80% ethanol constant volume, mensuration absorbance, by regression equation calculation general flavone content, the results are shown in Table 11
And Fig. 2.
The balsampear leaf determination of total flavonoids (n=2) of table 11
In sum, in balsampear leaf of the present invention general flavone content detection method, by the screening of multiple techniques, can be with standard
Really, content that is reliable, easily detecting general flavone in balsampear leaf, so that the quality of effective monitoring balsampear leaf medicinal material, is balsampear leaf
Clinical efficacy provide effective guarantee.
Claims (11)
1. in balsampear leaf general flavone content detection method, it is characterised in that：Comprise the following steps：
(1) foundation of standard curve
The preparation of a, reference substance solution：
Control substance of Rutin is taken, plus ethanol is configured to reference substance solution；
The measure of b, reference substance solution：
Reference substance solution is taken, 5% sodium nitrite solution, 10% aluminum nitrate solution, sodium hydroxide solution is sequentially added, ethanol is fixed
Hold, in mensuration absorbance under 506nm in UV detector, standard curve is obtained according to testing result；
(2) in testing sample general flavone content measure：
The preparation of c, need testing solution：
Balsampear leaf sample to be measured is taken, 60 DEG C~90 DEG C of petroleum ether refluxing extraction, filtering discards filtrate, is repeated once, takes residual
Slag, 60%~90% EtOH Sonicate or refluxing extraction, filtering supplies institute's weight loss with the ethanol of respective concentration, obtains test sample
Solution；
The measure of d, need testing solution：
Need testing solution is taken, is measured in the method for step b, the standard curve according to step (1) obtains total in testing sample
The content of flavones.
2. detection method according to claim 1, it is characterised in that：In step a, the ethanol is 50% ethanol.
3. detection method according to claim 1 and 2, it is characterised in that：In step b, 5% sodium nitrite solution with
The volume ratio of reference substance solution is 1：1.
4. detection method according to claim 1 and 2, it is characterised in that：In step b, 10% aluminum nitrate solution is molten
Liquid is 1 with the volume ratio of reference substance solution：1.
5. detection method according to claim 1 and 2, it is characterised in that：In step b, the sodium hydroxide solution with it is right
Volume ratio according to product solution is 10：1.
6. the detection method according to claim any one of 1-5, it is characterised in that：In step c, the ethanol is 80%
Ethanol.
7. the detection method according to claim any one of 1-6, it is characterised in that：In step c, the ethanol is extracted as back
Stream is extracted.
8. detection method according to claim 7, it is characterised in that：The time that the alcohol reflux is extracted is 0.5~3
Hour, preferably 1.5 hours.
9. the detection method according to claim any one of 1-8, it is characterised in that：The temperature that the alcohol reflux is extracted is
60 DEG C~80 DEG C.
10. detection method according to claim 9, it is characterised in that：The temperature that the alcohol reflux is extracted is 70 DEG C.
11. detection methods according to claim 10, it is characterised in that：The volume of the ethanol and balsampear leaf sample to be measured
Mass ratio is 100mL：1g~160mL：1g, preferably 140mL：1g.
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| CN109187387A (en) * | 2018-08-31 | 2019-01-11 | 成都大学 | The method for evaluating quality of American lotus a kind of sedge |
| CN111812046A (en) * | 2020-07-01 | 2020-10-23 | 云南省烟草质量监督检测站 | Method for measuring content of total flavonoids in tobacco |
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