CN103926212A - Determination method for content of total phenols in callicarpa nudiflora - Google Patents
Determination method for content of total phenols in callicarpa nudiflora Download PDFInfo
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Abstract
The invention relates to a determination method for the content of total phenols in callicarpa nudiflora. The determination method employs a spectrophotometric process to determine the content of total phenols in callicarpa nudiflora, and is beneficial for quantitative control on the callicarpa nudiflora medicinal material, callicarpa nudiflora extracts and callicarpa nudiflora preparations.
Description
Technical field
The present invention relates to the field of Chinese medicines, be specifically related to callicarpa nudiflora middle total phenol content assay method.
Background technology
Callicarpa nudiflora (
callicarpa nudiflorahook.et Arn.) be Verenaceae (Verbenaceae) Callicarpa plant, main product, in China Hainan, Guangdong, Guangxi, is the genunie medicinal materials in Hainan, has the effects such as convergence, hemostasis, analgesia, anti-inflammatory.Existing listing kind is as callicarpa nudiflora, callicarpa nudiflora capsule, the preparations such as LUOHUAZIZHU SHUAN, mainly with general flavone, be quality control standard at present, general flavone in seven kinds of Callicarpa low cost vegetable plant water extracts of < < GUIHAIA > > 11 phase < < in 2012, the mensuration > > of total phenolic acid and antioxidation activity thereof has recorded the content assaying method of total phenol, but total phenol content assay method provided by the invention, the methodological studies such as extraction conditions and content assaying method are further studied, optimized correlation parameter, further improved quality standard.
Summary of the invention
The object of the invention is to set up the content assaying method of total phenol in a kind of accurate, reliable callicarpa nudiflora medicinal material, extract.
Of the present invention callicarpa nudiflora in the content assaying method of total phenol comprise the following steps:
(1). the preparation of need testing solution:
Get callicarpa nudiflora medicinal material appropriate, with alcoholic solvent or the ultrasonic extraction of ketone solvent, extract is centrifugal, take supernatant as need testing solution;
(2). the making of typical curve: precision takes the about 10mg of gallic acid product in contrast, put and in 10 ml volumetric flasks, add 50% methyl alcohol and dissolve and be diluted to scale, shake up, obtaining concentration is the reference substance storing solution of 1 mg/ml; Precision pipettes 1 ml reference substance storing solution and is placed in 10 ml volumetric flasks, with 50% methyl alcohol, is diluted to scale, shakes up, and obtains the reference substance solution that concentration is 0.1 mg/ml; The accurate reference substance solution of drawing is placed in the standard solution that 5ml measuring bottle is mixed with gradient respectively again, add chromogenic reagent, take 50% methyl alcohol as blank, according to UV-VIS spectrophotometry, under 760nm wavelength, measure respectively absorbance, take gallic acid content as horizontal ordinate, take its absorbance as ordinate, obtain gallic acid content bioassay standard curve;
(3). the assay of the total phenol of sample solution: precision measures the described need testing solution of 0.1ml step (1), add chromogenic reagent, take corresponding reagent as blank, according to UV-VIS spectrophotometry, under 760nm wavelength, measure absorbance, according to this absorbance, by typical curve, obtain corresponding concentration, this concentration value is the content of the total phenol of need testing solution;
(4). the calculating of content in medicinal material: according to the content of total phenol in the need testing solution of step (3) gained, converse the content of total phenol in callicarpa nudiflora medicinal material.
Wherein, the preparation of step (1) need testing solution can be preferably: the methyl alcohol that alcoholic solvent is 40 ~ 60% or ethanol, and ketone solvent is 40 ~ 60% acetone, the liquid ratio of ultrasonic extraction is 120:1 ~ 250:1, centrifugation rate is 10000 ~ 15000r/min, extraction time 0.5 ~ 1.5h; More preferably 50% the ultrasonic extraction of acetone, extraction time is 1 hour, liquid ratio is 250:1, extract is centrifugal with 10000r/min, and after centrifugal 1 min, precision measures supernatant 1-5ml and puts in 25ml measuring bottle, add 50% methyl alcohol and be diluted to scale, shake up, make need testing solution.
In a kind of beautyberry extract of the present invention, the content assaying method of total phenol, comprises the steps:
(1). the preparation of need testing solution:
Precision takes 0.01 ~ 0.05g beautyberry extract powder, puts in 25ml measuring bottle, adds 50% methyl alcohol 10 ~ 20ml ultrasonic dissolution, with 50% methyl alcohol, is diluted to scale, shakes up, and filters, and gained filtrate is made need testing solution;
(2). the making of typical curve: precision takes the about 10mg of gallic acid product in contrast, put and in 10 ml volumetric flasks, add 50% methyl alcohol and dissolve and be diluted to scale, shake up, obtaining concentration is the reference substance storing solution of 1 mg/ml; Precision pipettes 1 ml reference substance storing solution and is placed in 10 ml volumetric flasks, with 50% methyl alcohol, is diluted to scale, shakes up, and obtains the reference substance solution that concentration is 0.1 mg/ml; The accurate reference substance solution of drawing is placed in the standard solution that 5ml measuring bottle is mixed with gradient respectively again, add chromogenic reagent, take 50% methyl alcohol as blank, according to UV-VIS spectrophotometry, under 760nm wavelength, measure respectively absorbance, take gallic acid content as horizontal ordinate, take its absorbance as ordinate, obtain gallic acid content bioassay standard curve;
(3). the assay of the total phenol of sample solution: precision measures the described need testing solution of 0.1ml step (1), add chromogenic reagent, take corresponding reagent as blank, according to UV-VIS spectrophotometry, under 760nm wavelength, measure absorbance, according to this absorbance, by typical curve, obtain corresponding concentration, this concentration value is the content of the total phenol of need testing solution;
(4). the calculating of content in extract: according to the content of total phenol in the need testing solution of step (3) gained, converse the content of total phenol in beautyberry extract.
In a kind of callicarpa nudiflora solid pharmaceutical preparation of the present invention, the content assaying method of total phenol, is characterized in that comprising the steps:
(1). the preparation of need testing solution:
Get callicarpa nudiflora solid pharmaceutical preparation, precision takes 0.05 ~ 0.1g internal drug, pulverizes, and puts in 25ml measuring bottle, adds 50% methyl alcohol 10 ~ 20ml ultrasonic dissolution, with 50% methyl alcohol, is diluted to scale, shakes up, and filters, and gained filtrate is made need testing solution;
(2). the making of typical curve: precision takes the about 10mg of gallic acid product in contrast, put and in 10 ml volumetric flasks, add 50% methyl alcohol and dissolve and be diluted to scale, shake up, obtaining concentration is the reference substance storing solution of 1 mg/ml; Precision pipettes 1 ml reference substance storing solution and is placed in 10 ml volumetric flasks, with 50% methyl alcohol, is diluted to scale, shakes up, and obtains the reference substance solution that concentration is 0.1 mg/ml; The accurate reference substance solution of drawing is placed in the standard solution that 5ml measuring bottle is mixed with gradient respectively again, add chromogenic reagent, take 50% methyl alcohol as blank, according to UV-VIS spectrophotometry, under 760nm wavelength, measure respectively absorbance, take gallic acid content as horizontal ordinate, take its absorbance as ordinate, obtain gallic acid content bioassay standard curve;
(3). the assay of the total phenol of sample solution: precision measures the described need testing solution of 0.1ml step (1), add chromogenic reagent, corresponding reagent is blank, according to UV-VIS spectrophotometry, under 760nm wavelength, measure absorbance, according to this absorbance, by typical curve, obtain corresponding concentration, this concentration value is the content of the total phenol of need testing solution;
(4). the calculating of content in preparation: according to the content of total phenol in the need testing solution of step (3) gained, converse the content of total phenol in callicarpa nudiflora preparation.
In a kind of callicarpa nudiflora liquid preparation of the present invention, the content assaying method of total phenol, is characterized in that comprising the steps:
(1). the preparation of need testing solution:
Precision measures the callicarpa nudiflora liquid preparation of 1-5ml, puts in 25ml measuring bottle, with 50% methyl alcohol, is diluted to scale, shakes up, and filters, and gained filtrate is made need testing solution;
(2). the making of typical curve: precision takes the about 10mg of gallic acid product in contrast, put and in 10 ml volumetric flasks, add 50% methyl alcohol and dissolve and be diluted to scale, shake up, obtaining concentration is the reference substance storing solution of 1 mg/ml; Precision pipettes 1 ml reference substance storing solution and is placed in 10 ml volumetric flasks, with 50% methyl alcohol, is diluted to scale, shakes up, and obtains the reference substance solution that concentration is 0.1 mg/ml; The accurate reference substance solution of drawing is placed in the standard solution that 5ml measuring bottle is mixed with gradient respectively again, add chromogenic reagent, take 50% methyl alcohol as blank, according to UV-VIS spectrophotometry, under 760nm wavelength, measure respectively absorbance, take gallic acid content as horizontal ordinate, take its absorbance as ordinate, obtain gallic acid content bioassay standard curve;
(3). the assay of the total phenol of sample solution: precision measures the described need testing solution of 0.1ml step (1), add chromogenic reagent, corresponding reagent is blank, according to UV-VIS spectrophotometry, under 760nm wavelength, measure absorbance, according to this absorbance, by typical curve, obtain corresponding concentration, this concentration value is the content of the total phenol of need testing solution;
(4). the calculating of content in preparation: according to the content of total phenol in the need testing solution of step (3) gained, converse the content of total phenol in callicarpa nudiflora preparation.
Folin-phenol test solution and 1.2 ~ 2.1mlNa that in the present invention, the developer described in above-mentioned content assaying method is 0.45 ~ 0.6ml
2cO
3solution, temperature of reaction is 40 ~ 45 ℃, and the reaction time is 10 ~ 15min, and developing time is 45 ~ 60min, and colour temp is 20 ~ 30 ℃, Na
2cO
3concentration of polymer solution is 5.0% ~ 7.5%; Developer and concentration, the consumption Na that more preferably the Folin-phenol test solution of 0.45ml and the mass concentration of 1.5ml are 7.5%
2cO
3solution, temperature of reaction is preferably 40 ℃, and the reaction time is preferably 10min, and developing time is preferably 45min, and colour temp is preferably 25 ℃.
The extraction process of inventor to callicarpa nudiflora medicinal material, in callicarpa nudiflora medicinal material, extract, the assay method of total phenol content has carried out optimal reaction temperature, best developing time, stability, reappearance, the recovery etc. and has investigated, proved method has good stability and reliability, has further improved the quality control standard of callicarpa nudiflora medicinal material and callicarpa nudiflora preparation.
Below the detailed description of methodological study:
one, content assaying method methodological study:
1, the preparation of callicarpa nudiflora dry extract:
Get the callicarpa nudiflora medicinal material of 10kg, boiling 2 times, add water 80kg at every turn, each 2 hours, merge decocting liquid twice, filter, the upper HP-20 macroporous absorbent resin of gained filtrate, the blade diameter length ratio of resin column is 1:8, water washing cylinder with 6 times of column volumes, discard cleansing solution, then use 20% methyl alcohol, 40% methyl alcohol, 60% methyl alcohol, 80% methanol-eluted fractions, each gradient is all eluted to without till color, collecting each eluent merges, after recovery methyl alcohol, being evaporated to relative density is 1.25, and freeze drying, obtains callicarpa nudiflora dry extract.
, optimum measurement wavelength and best reference substance solution selection
The need testing solution (1mg/ml) that pipettes respectively the callicarpa nudiflora dry extract dissolving of 0.1ml and obtain and gallic acid (0.1mg/ml), acteoside (0.168mg/ml), three groups of reference substance solution of protocatechuic acid (0.184mg/ml), add 0.3mlFolin-phenol test solution, under dark condition, place 2min, then add 2ml7.5%Na
2cO
3solution, adding distil water, to 5ml, shakes up, and after 40 ℃ of water-bath 10min, 30min is secretly put in 25 ℃ of water-baths.Blank tube pipettes 0.1ml coordinative solvent and is placed in 5ml measuring bottle, adds 0.3mlFolin-phenol test solution, under dark condition, places 2min, and then adding 2ml mass concentration is 7.5% Na
2cO
3solution, adds water to scale, shakes up, and after 40 ℃ of water-bath 10min, 30min is secretly put in 25 ℃ of water-baths.With ultraviolet-visible pectrophotometer, in 400～800nm wavelength coverage, every 20nm, survey light absorption value, draw and obtain length scanning figure (accompanying drawing 1), determine optimum detection wavelength and best reference substance solution.
As shown in Figure 1, four all have absorption maximum at 760nm wavelength place, therefore select 760nm, are optimum detection wavelength.In addition, gallic acid is more consistent with dry extract trend, and gallic acid reference substance is easier to obtain, therefore select gallic acid, is that total phenol detects best reference substance solution.
, the determining of sodium carbonate liquor mass concentration
Pipette respectively the best reference substance solution of 21 parts of 0.1ml and be placed in 5ml measuring bottle, be divided into three groups, every group adds 0.3mlFolin-phenol test solution, under dark condition, places 2min, and then adding respectively 2ml mass concentration is 5.0%, 7.5%, 10.0%, 12.5%, 15.0%, 17.5%, 20.0% Na
2cO
3solution, adds water to scale, shakes up, and after 40 ℃ of water-bath 10min, 30min is secretly put in 25 ℃ of water-baths, and at optimum detection wavelength, its absorbance is measured at place, determines Na
2cO
3the best in quality concentration of solution.Blank tube pipettes 0.1ml coordinative solvent and is placed in 5ml measuring bottle, adds 0.3mlFolin-phenol test solution, under dark condition, places 2min, and then adding 2ml mass concentration is 7.5% Na
2cO
3solution, adds water to scale, shakes up, and after 40 ℃ of water-bath 10min, 30min is secretly put in 25 ℃ of water-baths.
Experimental result is in Table 1: therefore experimental selection Na
2cO
3concentration of polymer solution is 7.5%.
the investigation of table 1 sodium carbonate liquor mass concentration
4, the mensuration of sodium carbonate liquor consumption
Pipette respectively the best reference substance solution of 27 parts of 0.1ml and be placed in 5ml measuring bottle, be divided into three groups, every group adds 0.3mlFolin-phenol test solution, under dark condition, places 2min, then adds the Na of best in quality concentration
2cO
3solution, consumption is respectively 0.15,0.30,0.60,0.90,1.20,1.50,1.80,2.10,2.40ml, adds water to scale, shakes up, and after 40 ℃ of water-bath 10min, 30min is secretly put in 25 ℃ of water-baths, and at optimum detection wavelength, its absorbance is measured at place, determines Na
2cO
3the optimum amount of solution.Blank tube pipettes 0.1ml coordinative solvent and is placed in 5ml measuring bottle, adds 0.3mlFolin-phenol test solution, under dark condition, places 2min, and then adding 2ml mass concentration is 7.5% Na
2cO
3solution, adds water to scale, shakes up, and after 40 ℃ of water-bath 10min, 30min is secretly put in 25 ℃ of water-baths.Experimental result is in Table 2: therefore experimental selection Na
2cO
3solution usage is 1.50ml.
the investigation of table 2 sodium carbonate liquor consumption
5, the selection of Folin-phenol test solution consumption
Pipette respectively the best reference substance solution of 24 parts of 0.1ml and be placed in 5ml measuring bottle, be divided into three groups, every group adds Folin-phenol test solution, consumption is respectively 0.15,0.30,0.45,0.60,0.75,0.90,1.20,1.50ml, under dark condition, place 2min, then add the Na of best in quality concentration and optimum amount
2cO
3solution, adds water to scale, shakes up, and at optimum detection wavelength place measures its absorbance after secretly putting 30min after 40 ℃ of water-bath 10min, determines the optimum amount of Folin-phenol test solution.Blank tube pipettes 0.1ml coordinative solvent and is placed in 5ml measuring bottle, adds 0.3mlFolin-phenol test solution, under dark condition, places 2min, and then adding 2ml mass concentration is 7.5% Na
2cO
3solution, adds water to scale, shakes up, and after 40 ℃ of water-bath 10min, 30min is secretly put in 25 ℃ of water-baths.Experimental result is in Table 3: therefore experimental selection Folin-phenol solution consumption is 0.45ml.
the investigation of table 3 Folin-phenol test solution consumption
6, optimal reaction temperature determines
Pipette respectively the best reference substance solution of 24 parts of 0.1ml and be placed in 5ml measuring bottle, be divided into three groups, every group adds best Folin-phenol test solution consumption, under dark condition, places 2min, then adds the Na of best in quality concentration and optimum amount
2cO
3solution, adds water to scale, shakes up, and selects temperature of reaction to be respectively after 25,30,35,40,45,50,55,60 ℃ of water-bath 10min after 30min is secretly put in 25 ℃ of water-baths and measures its absorbance at optimum detection wavelength place, determines optimal reaction temperature.Blank tube pipettes 0.1ml coordinative solvent and is placed in 5ml measuring bottle, adds 0.3mlFolin-phenol test solution, under dark condition, places 2min, and then adding 2ml mass concentration is 7.5% Na
2cO
3solution, adds water to scale, shakes up, and after 40 ℃ of water-bath 10min, 30min is secretly put in 25 ℃ of water-baths.Experimental result is in Table 4: therefore experimental selection optimal reaction temperature is 40 ℃.
the investigation of table 4 optimal reaction temperature
7, optimum reacting time determines
Pipette respectively the best reference substance solution of 15 parts of 0.1ml and be placed in 5ml measuring bottle, be divided into three groups, every group adds best Folin-phenol test solution consumption, under dark condition, places 2min, then adds the Na of best in quality concentration and optimum amount
2cO
3solution, adds water to scale, shakes up, and after 25 ℃ of water-baths are secretly put 30min after water-bath 5,10,15,20,25min respectively under optimal reaction temperature, at optimum detection wavelength place, measures its absorbance, determines optimum reacting time.Blank tube pipettes 0.1ml coordinative solvent and is placed in 5ml measuring bottle, adds 0.3mlFolin-phenol test solution, under dark condition, places 2min, and then adding 2ml mass concentration is 7.5% Na
2cO
3solution, adds water to scale, shakes up, and after 40 ℃ of water-bath 10min, 30min is secretly put in 25 ℃ of water-baths.Experimental result in Table 5:10min after, light absorption value changes little, therefore experimental selection optimum reacting time is 10min.
the investigation of table 5 optimum reacting time
8, best colour temp determines
Pipette respectively the best reference substance solution of 24 parts of 0.1ml and be placed in 5ml measuring bottle, be divided into three groups, every group adds best Folin-phenol test solution consumption, under dark condition, places 2min, then adds the Na of best in quality concentration and optimum amount
2cO
3solution, adds water to scale, shakes up, and selects after optimal reaction temperature water-bath 10min, and in 0,5,10,15,20,25,30,35 ℃ of colour developing 30min, with optimum detection wavelength, its absorbance is measured at place, determines best colour temp.Blank tube pipettes 0.1ml coordinative solvent and is placed in 5ml measuring bottle, adds 0.3mlFolin-phenol test solution, under dark condition, places 2min, and then adding 2ml mass concentration is 7.5% Na
2cO
3solution, adds water to scale, shakes up, and after 40 ℃ of water-bath 10min, 30min is secretly put in 25 ℃ of water-baths.Experimental result is in Table 6: therefore select best colour temp, be 25 ℃.
the investigation of the best colour temp of table 6
9, best developing time determines
Pipette respectively the best reference substance solution of 3 parts of 0.1ml and be placed in 5ml measuring bottle, be divided into three groups, every group adds respectively best Folin-phenol test solution consumption, under dark condition, places 2min, then adds the Na of best in quality concentration and optimum amount
2cO
3solution, adds water to scale, shakes up, and selects after optimal reaction temperature water-bath 10min, and colour developing 15,30,45,60,90,120 is secretly put in 25 ℃ of water-baths, 150min sentences optimum detection wavelength place and measures its absorbance, determines best developing time.Blank tube pipettes 0.1ml coordinative solvent and is placed in 5ml measuring bottle, adds 0.3mlFolin-phenol test solution, under dark condition, places 2min, and then adding 2ml mass concentration is 7.5% Na
2cO
3solution, adds water to scale, shakes up, and after 40 ℃ of water-bath 10min, 30min is secretly put in 25 ℃ of water-baths.Experimental result is in Table 7: in experiment, find, after developing time 45min, light absorption value changes little, considers the factors such as ageing, therefore the best developing time of experimental selection is 45min.
the investigation of the best developing time of table 7
10, Folin-phenol colourimetry is evaluated
stability test:precision pipettes same sample solution 0.1ml and measures absorbance after by the colour developing completely of best color condition, successively 0.5,1,2,4,6,8,12h measures absorbance, results sample is stablized in 12h.The results are shown in Table 8.
the investigation of table 8 stability experiment
reappearance test:precision pipettes 6 parts of 0.1ml solution to be measured, by measuring absorbance after best color condition colour developing completely, and calculates RSD value.Result shows that the method reappearance is good, in Table 9.
the investigation of table 9 reappearance experiment
precision test:precision pipettes 1 part of 0.1ml solution to be measured, by measuring absorbance, METHOD FOR CONTINUOUS DETERMINATION 6 times after best color condition colour developing completely.The results are shown in Table 10.
the investigation of table 10 Precision Experiment
application of sample recovery test: precision takes gallic acid 10.01mg, puts in 10ml volumetric flask, to add 50% methyl alcohol and dissolve and be diluted to scale, shakes up, and obtaining concentration is the reference substance storing solution of 1.001mg/ml.Precision pipettes 1ml reference substance storing solution and is placed in 10ml volumetric flask, with 50% methyl alcohol, is diluted to scale, shakes up, and obtains the reference substance solution that concentration is 0.1001mg/ml.Accurate 0,30,60,90,120,150, the 180 μ l reference substance solution of drawing are in 5ml measuring bottle respectively, add Folin-phenol test solution 0.45ml, mix rear standing 2min, add 7.5% sodium carbonate liquor 1.5ml, add water to scale, after 40 ℃ of water-bath 10min, 45min is secretly put in 25 ℃ of water-baths.Get 0.1ml solution to be measured simultaneously and be placed in 5ml measuring bottle, add Folin-phenol test solution 0.45ml, mix rear standing 2min, add 7.5% sodium carbonate liquor 1.5ml, add water to scale, after 40 ℃ of water-bath 10min, 45 min are secretly put in 25 ℃ of water-baths.Using 50% methyl alcohol as blank, under 760nm wavelength, measure absorbance, take gallic acid content as horizontal ordinate, take its absorbance as ordinate, obtain gallic acid content bioassay standard curve.Obtaining regression equation is y=3.2262x-0.0012, and related coefficient is R
2=0.9999.And try to achieve total phenol content 0.158 mg/ml in solution to be measured.
Get 6 parts of the above-mentioned solution to be measured of 0.1ml, add respectively 30,60,90 μ l reference substance solution, shake up, two parts of operation repetitives, by measuring absorbance, calculate recovery rate after best color condition colour developing completely.The results are shown in Table 11.
the investigation of table 11 application of sample recovery experiment
two, the research of callicarpa nudiflora medicinal material extract method:
Precision takes gallic acid 10.0 mg, puts in 10 ml volumetric flasks, to add 50% methyl alcohol and dissolve and be diluted to scale, shakes up, and obtaining concentration is the reference substance storing solution of 1 mg/ml.Precision pipettes 1 ml reference substance storing solution and is placed in 10 ml volumetric flasks, with 50% methyl alcohol, is diluted to scale, shakes up, and obtains the reference substance solution that concentration is 0.1 mg/ml.Accurate 0,30,60,90,120,150, the 180 μ l reference substance solution of drawing are in 5ml measuring bottle respectively, add Folin-phenol test solution 0.45 ml, mix rear standing 2 min, add 7.5% sodium carbonate liquor 1.5 ml, add water to scale, vibration mixes, and after 40 ℃ of water-bath 10min, 45min is secretly put in 25 ℃ of water-baths.Using 50% methyl alcohol as blank, under 760 nm wavelength, measure absorbance, take gallic acid content as horizontal ordinate, take its absorbance as ordinate, obtain gallic acid content bioassay standard curve, and obtain regression equation.
, Different Extraction Method comparison
Ultrasound wave extracts: take the callicarpa nudiflora raw material of about 0.5g, add appropriate 75% ethanol, ultrasonic processing (300W, 33kHz) 1 h.
Room temperature lixiviate: take the callicarpa nudiflora raw material of about 0.5g, add appropriate 75% ethanol, lixiviate 24 h under room temperature condition.
Heating-condensing refluxing extraction: take the callicarpa nudiflora raw material of about 0.5g, add appropriate 75% ethanol, refluxing extraction 1 h at 95 ℃.
Soxhlet is extracted: take the callicarpa nudiflora raw material of about 0.5g, add appropriate 75% ethanol, refluxing extraction 3 h at 95 ℃.
Above-mentioned each extract 10000r/min, gets supernatant its total phenol content to be measured after centrifugal 1 min.
the comparison of table 12 Different Extraction Method gained total phenol content
By table 12 result, obtained: in four kinds of methods, room temperature lixiviate gained total phenol content is minimum; Soxhlet extracting method gained total phenol content is the highest, but this method solvent used is more, and the time is longer, considers and selects supersonic extracting method to carry out next step research.
, different solvents and variable concentrations solvent comparison
Take respectively the callicarpa nudiflora raw material of about 0.25g, add methyl alcohol, ethanol and the acetone reagent of variable concentrations, ultrasonic processing (300W, 33kHz) 1 h.Above-mentioned each extract 10000r/min, gets supernatant its total phenol content to be measured after centrifugal 1 min.
the comparison of table 13 different solvents and variable concentrations solvent gained total phenol content
From table 13 result: 40% acetone gained total phenol content is the highest, therefore select 40% acetone to carry out next step, extract research.
, different liquid ratios comparison
Take respectively the callicarpa nudiflora raw material of about 0.25g, by liquid ratio, be respectively 40:1; 80:1; 120:1; 160:1; 200:1 adds 40% acetone, ultrasonic processing (300W, 33kHz) 1 h.Above-mentioned each extract 10000r/min, gets supernatant its total phenol content to be measured after centrifugal 1 min.
the comparison of the different liquid ratio gained of table 14 total phenol content
From table 14 result: within the specific limits, total phenol content and liquid ratio correlation.
, different extraction times comparison
Take respectively a certain amount of callicarpa nudiflora raw material, by liquid ratio 200:1, add 40% acetone, ultrasonic processing (300W, 33kHz) (be respectively 30,60,90,120,150, pipette 0.2ml during 180min by extraction time).Above-mentioned each extract 10000r/min, gets supernatant its total phenol content to be measured after centrifugal 1 min.
the comparison of table 15 gained total phenol content of different extraction time
From table 15 result: extract after 60min, total phenol content changes little.
, orthogonal experiment
The concentration of acetone, liquid ratio and extraction time have a certain impact to the equal tool of total phenol content.This experiment adopts orthogonal test to investigate this.By table 16, added after reagent, operation on request, maximum absorption wave strong point is measured absorbance, investigates best color condition.Experimental result is in Table 17.
table 16 factor level table
table 17 extraction conditions is investigated result
From intuitive analysis (in Table 17), take total phenol content as evaluation index, the known preferred plan of intuitive analysis is A
2b
3c
3scheme, therefore this experimental selection option A
2b
3c
3carry out demonstration test.
Take respectively a certain amount of callicarpa nudiflora raw material, by liquid ratio 250:1, add 50% acetone, ultrasonic processing (300W, 33kHz) 60min, above-mentioned each extract 10000r/min, gets supernatant its total phenol content to be measured after centrifugal 1 min.The results are shown in Table 18.
table 18 quadrature demonstration test result (n=3)
As shown in Table 18, A
2b
3c
3the total phenol content that scheme draws is better than other schemes; To sum up, the top condition of determining callicarpa nudiflora medicinal material extract is A
2b
3c
3, that is: take respectively a certain amount of callicarpa nudiflora raw material, by liquid ratio 250:1, add 50% acetone, ultrasonic processing (300W, 33kHz) 60min, above-mentioned each extract 10000r/min, gets supernatant its total phenol content to be measured after centrifugal 1 min.
Accompanying drawing explanation
Fig. 1 represents gallic acid reference substance, the dry extract sample absorption curve under 400nm～800nm wavelength after colour developing.
Embodiment
embodiment mono-
(1). the preparation of need testing solution:
Take the callicarpa nudiflora medicinal material of 0.4156g, be placed in 50ml volumetric flask, add the ultrasonic processing (300W of 60% ethanol 45ml, 33kHz) 0.5h, after letting cool, add 60% ethanol and be settled to scale, shake up, after standing 30min, extract is centrifugal with 10000r/min, after centrifugal 1 min, getting supernatant 5ml is need testing solution.
(2). the making of typical curve: precision takes gallic acid 10.01mg, put and in 10ml volumetric flask, add 50% methyl alcohol and dissolve and be diluted to scale, shake up, obtaining concentration is the reference substance storing solution of 1.001mg/ml.Precision pipettes 1ml reference substance storing solution and is placed in 10ml volumetric flask, with 50% methyl alcohol, is diluted to scale, shakes up, and obtains the reference substance solution that concentration is 0.1001mg/ml.Accurate 0,30,60,90,120,150, the 180 μ l reference substance solution of drawing, in 5ml measuring bottle, add Folin-phenol test solution 0.5ml respectively, mix rear standing 2min, add 5.0% sodium carbonate liquor 2.1ml, add water to scale, shake up, after 40 ℃ of water-bath 10min, 60min is secretly put in 25 ℃ of water-baths.Take 50% methyl alcohol as blank, under 760nm wavelength, measure absorbance, take gallic acid content as horizontal ordinate, take its absorbance as ordinate, obtain gallic acid content bioassay standard curve.
(3). the assay of the total phenol of sample solution: precision measures the described need testing solution 0.1ml of step (1) and is placed in 5ml measuring bottle, add Folin-phenol test solution 0.5ml, mix rear standing 2min, add 5.0% sodium carbonate liquor 2.1ml, add water to scale, shake up, after 40 ℃ of water-bath 10min, 60min is secretly put in 25 ℃ of water-baths, take 60% ethanol as blank, under 760nm wavelength, measure absorbance, according to this absorbance, by typical curve, obtain corresponding concentration, this concentration value is the content of the total phenol of need testing solution, is 0.1652mg/ml.
(4). the calculating of content in medicinal material: according to the content of total phenol in the need testing solution of step (3) gained, conversing the content of total phenol in callicarpa nudiflora medicinal material, is 1.987%.
embodiment bis-
(1). the preparation of need testing solution:
Take the callicarpa nudiflora medicinal material of 0.2015g, be placed in 50ml volumetric flask, add 50% the ultrasonic processing (300W of acetone 45ml, 33kHz) 1h, after letting cool, add 50% acetone and be settled to scale, shake up, after standing 30min, extract is centrifugal with 10000r/min, after centrifugal 1 min, getting supernatant 5ml is need testing solution.
(2). the making of typical curve: precision takes gallic acid 10.00mg, put and in 10ml volumetric flask, add 50% methyl alcohol and dissolve and be diluted to scale, shake up, obtaining concentration is the reference substance storing solution of 1mg/ml.Precision pipettes 1ml reference substance storing solution and is placed in 10ml volumetric flask, with 50% methyl alcohol, is diluted to scale, shakes up, and obtains the reference substance solution that concentration is 0.1mg/ml.Accurate 0,30,60,90,120,150, the 180 μ l reference substance solution of drawing are placed in 5ml measuring bottle respectively, add Folin-phenol test solution 0.45ml, mix rear standing 2min, add 7.5% sodium carbonate liquor 1.5ml, add water to scale, shake up, after 40 ℃ of water-bath 10min, 45min is secretly put in 25 ℃ of water-baths.Take 50% methyl alcohol as blank, under 760nm wavelength, measure absorbance, take gallic acid content as horizontal ordinate, take its absorbance as ordinate, obtain gallic acid content bioassay standard curve.
(3). the assay of the total phenol of sample solution: precision measures the described need testing solution 0.1ml of step (1) and is placed in 5ml measuring bottle, add Folin-phenol test solution 0.45ml, mix rear standing 2min, add 7.5% sodium carbonate liquor 1.5ml, add water to scale, shake up, after 40 ℃ of water-bath 10min, 45min is secretly put in 25 ℃ of water-baths, 50% the acetone of take is blank, under 760nm wavelength, measure absorbance, according to this absorbance, by typical curve, obtain corresponding concentration, this concentration value is the content of the total phenol of need testing solution, is 0.1091mg/ml.
(4). the calculating of content in medicinal material: according to the content of total phenol in the need testing solution of step (3) gained, conversing the content of total phenol in callicarpa nudiflora medicinal material, is 2.707%.
embodiment tri-
(1). the preparation of need testing solution:
Take the callicarpa nudiflora medicinal material of 0.2503g, be placed in 50ml volumetric flask, add the ultrasonic processing (300W of 40% methyl alcohol 45ml, 33kHz) 1.5h, after letting cool, add 40% methanol constant volume to scale, shake up, after standing 30min, extract is centrifugal with 15000r/min, after centrifugal 1 min, getting supernatant 5ml is need testing solution.
(2). the making of typical curve: precision takes gallic acid 10.00mg, put and in 10ml volumetric flask, add 50% methyl alcohol and dissolve and be diluted to scale, shake up, obtaining concentration is the reference substance storing solution of 1mg/ml.Precision pipettes 1ml reference substance storing solution and is placed in 10ml volumetric flask, with 50% methyl alcohol, is diluted to scale, shakes up, and obtains the reference substance solution that concentration is 0.1mg/ml.Accurate 0,30,60,90,120,150, the 180 μ l reference substance solution of drawing are placed in 5ml measuring bottle respectively, add Folin-phenol test solution 0.6ml, mix rear standing 2min, add 7.0% sodium carbonate liquor 1.2ml, add water to scale, shake up, after 45 ℃ of water-bath 15min, 30min is secretly put in 25 ℃ of water-baths.Take 50% methyl alcohol as blank, under 760nm wavelength, measure absorbance, take gallic acid content as horizontal ordinate, take its absorbance as ordinate, obtain gallic acid content bioassay standard curve.
(3). the assay of the total phenol of sample solution: precision measures the described need testing solution 0.1ml of step (1) and is placed in 5ml measuring bottle, add Folin-phenol test solution 0.6ml, mix rear standing 2min, add 7.0% sodium carbonate liquor 1.2ml, add water to scale, shake up, after 45 ℃ of water-bath 15min, 30min is secretly put in 25 ℃ of water-baths, take 40% methyl alcohol as blank, under 760nm wavelength, measure absorbance, according to this absorbance, by typical curve, obtain corresponding concentration, this concentration value is the content of the total phenol of need testing solution, is 0.1098mg/ml.
(4). the calculating of content in medicinal material: according to the content of total phenol in the need testing solution of step (3) gained, conversing the content of total phenol in callicarpa nudiflora medicinal material, is 2.193%.
embodiment tetra-
(1). the preparation of need testing solution:
Precision takes 25.32mg beautyberry extract powder, puts in 25ml measuring bottle, adds the 50% ultrasonic processing of methyl alcohol 10ml (300W, 33kHz) 10min, with 50% methyl alcohol, is diluted to scale, shakes up, and filters, and gained filtrate is made need testing solution.
(2). the making of typical curve: precision takes gallic acid 10.00mg product in contrast, put and in 10 ml volumetric flasks, add 50% methyl alcohol and dissolve and be diluted to scale, shake up, obtaining concentration is the reference substance storing solution of 1 mg/ml; Precision pipettes 1 ml reference substance storing solution and is placed in 10 ml volumetric flasks, with 50% methyl alcohol, is diluted to scale, shakes up, and obtains the reference substance solution that concentration is 0.1 mg/ml; Accurate 0,40,80,120,160,200, the 240 μ l reference substance solution of drawing are placed in 5ml measuring bottle respectively, add Folin-phenol test solution 0.45ml, mix rear standing 2min, add 7.5% sodium carbonate liquor 1.5ml, add water to scale, shake up, after 40 ℃ of water-bath 10min, 45min is secretly put in 25 ℃ of water-baths.Take 50% methyl alcohol as blank, under 760nm wavelength, measure absorbance, take gallic acid content as horizontal ordinate, take its absorbance as ordinate, obtain gallic acid content bioassay standard curve.
(3). the assay of the total phenol of sample solution: precision measures the described need testing solution of 0.1ml step (1) and is placed in 5ml measuring bottle, add Folin-phenol test solution 0.45ml, mix rear standing 2min, add 7.5% sodium carbonate liquor 1.5ml, add water to scale, shake up, after 40 ℃ of water-bath 10min, 45min is secretly put in 25 ℃ of water-baths, take 50% methyl alcohol as blank, according to UV-VIS spectrophotometry, under 760nm wavelength, measure absorbance, according to this absorbance, by typical curve, obtain corresponding concentration, this concentration value is the content of the total phenol of need testing solution, be 0.1433 mg/ml.
(4). the calculating of content in extract: according to the content of total phenol in the need testing solution of step (3) gained, conversing the content of total phenol in beautyberry extract, is 14.149%.
embodiment five
(1). the preparation of need testing solution:
Precision takes 10.26mg beautyberry extract powder, puts in 25ml measuring bottle, adds the 50% ultrasonic processing of methyl alcohol 10ml (300W, 33kHz) 10min, with 50% methyl alcohol, is diluted to scale, shakes up, and filters, and gained filtrate is made need testing solution.
(2). the making of typical curve: precision takes gallic acid 10.00mg product in contrast, put and in 10 ml volumetric flasks, add 50% methyl alcohol and dissolve and be diluted to scale, shake up, obtaining concentration is the reference substance storing solution of 1 mg/ml; Precision pipettes 1 ml reference substance storing solution and is placed in 10 ml volumetric flasks, with 50% methyl alcohol, is diluted to scale, shakes up, and obtains the reference substance solution that concentration is 0.1 mg/ml; Accurate 0,40,80,120,160,200, the 240 μ l reference substance solution of drawing are placed in 5ml measuring bottle respectively, add Folin-phenol test solution 0.55ml, mix rear standing 2min, add 6% sodium carbonate liquor 1.2ml, add water to scale, shake up, after 40 ℃ of water-bath 15min, 60min is secretly put in 25 ℃ of water-baths.Take 50% methyl alcohol as blank, under 760nm wavelength, measure absorbance, take gallic acid content as horizontal ordinate, take its absorbance as ordinate, obtain gallic acid content bioassay standard curve.
(3). the assay of the total phenol of sample solution: precision measures the described need testing solution of 0.1ml step (1) and is placed in 5ml measuring bottle, add Folin-phenol test solution 0.55ml, mix rear standing 2min, add 6% sodium carbonate liquor 1.2ml, add water to scale, shake up, after 40 ℃ of water-bath 15min, 60min is secretly put in 25 ℃ of water-baths.Take 50% methyl alcohol as blank, according to UV-VIS spectrophotometry, under 760nm wavelength, measure absorbance, according to this absorbance, by typical curve, obtain corresponding concentration, this concentration value is the content of the total phenol of need testing solution, is 0.0565 mg/ml.
(4). the calculating of content in extract: according to the content of total phenol in the need testing solution of step (3) gained, conversing the content of total phenol in beautyberry extract, is 13.767%.
embodiment six
(1). the preparation of need testing solution:
Precision takes 49.86mg beautyberry extract powder, puts in 25ml measuring bottle, adds the 50% ultrasonic processing of methyl alcohol 10ml (300W, 33kHz) 10min, with 50% methyl alcohol, is diluted to scale, shakes up, and filters, and gained filtrate is made need testing solution.
(2). the making of typical curve: precision takes gallic acid 10.00mg product in contrast, put and in 10 ml volumetric flasks, add 50% methyl alcohol and dissolve and be diluted to scale, shake up, obtaining concentration is the reference substance storing solution of 1 mg/ml; Precision pipettes 1 ml reference substance storing solution and is placed in 10 ml volumetric flasks, with 50% methyl alcohol, is diluted to scale, shakes up, and obtains the reference substance solution that concentration is 0.1 mg/ml; Accurate 0,40,80,120,160,200, the 240 μ l reference substance solution of drawing are placed in 5ml measuring bottle respectively, add Folin-phenol test solution 0. 5ml, mix rear standing 2min, add 7.5% sodium carbonate liquor 1.5ml, add water to scale, shake up, after 45 ℃ of water-bath 10min, 45min is secretly put in 20 ℃ of water-baths.Take 50% methyl alcohol as blank, under 760nm wavelength, measure absorbance, take gallic acid content as horizontal ordinate, take its absorbance as ordinate, obtain gallic acid content bioassay standard curve.
(3). the assay of the total phenol of sample solution: precision measures the described need testing solution of 0.1ml step (1) and is placed in 5ml measuring bottle, add Folin-phenol test solution 0. 5ml, mix rear standing 2min, add 7.5% sodium carbonate liquor 1.5ml, add water to scale, shake up, after 45 ℃ of water-bath 10min, 45min is secretly put in 20 ℃ of water-baths.Take 50% methyl alcohol as blank, according to UV-VIS spectrophotometry, under 760nm wavelength, measure absorbance, according to this absorbance, by typical curve, obtain corresponding concentration, this concentration value is the content of the total phenol of need testing solution, is 0.2752 mg/ml.
(4). the calculating of content in extract: according to the content of total phenol in the need testing solution of step (3) gained, conversing the content of total phenol in beautyberry extract, is 13.799%.
embodiment seven
(1). the preparation of need testing solution:
Get callicarpa nudiflora capsule, precision takes 10.13mg internal drug, pulverizes, and puts in 25ml measuring bottle, adds 50% methyl alcohol 20ml ultrasonic dissolution, with 50% methyl alcohol, is diluted to scale, shakes up, and filters, and gained filtrate is made need testing solution.
(2). the making of typical curve: precision takes gallic acid 10.01mg product in contrast, put and in 10 ml volumetric flasks, add 50% methyl alcohol and dissolve and be diluted to scale, shake up, obtaining concentration is the reference substance storing solution of 1.001 mg/ml; Precision pipettes 1 ml reference substance storing solution and is placed in 10 ml volumetric flasks, with 50% methyl alcohol, is diluted to scale, shakes up, and obtains the reference substance solution that concentration is 0.1001 mg/ml; Accurate 0,40,80,120,160,200, the 240 μ l reference substance solution of drawing are placed in 5ml measuring bottle respectively, add Folin-phenol test solution 0.5ml, mix rear standing 2min, add 5% sodium carbonate liquor 2ml, add water to scale, shake up, after 45 ℃ of water-bath 15min, 60min is secretly put in 20 ℃ of water-baths.Take 50% methyl alcohol as blank, under 760nm wavelength, measure absorbance, take gallic acid content as horizontal ordinate, take its absorbance as ordinate, obtain gallic acid content bioassay standard curve.
(3). the assay of the total phenol of sample solution: precision measures the described need testing solution of 0.1ml step (1) and is placed in 5ml measuring bottle, add Folin-phenol test solution 0.5ml, mix rear standing 2min, add 5% sodium carbonate liquor 2ml, add water to scale, shake up, after 45 ℃ of water-bath 15min, 60min is secretly put in 20 ℃ of water-baths.Take 50% methyl alcohol as blank, according to UV-VIS spectrophotometry, under 760nm wavelength, measure absorbance, according to this absorbance, by typical curve, obtain corresponding concentration, this concentration value is the content of the total phenol of need testing solution, is 0.0426 mg/ml.
(4). the calculating of content in extract: according to the content of total phenol in the need testing solution of step (3) gained, conversing the content of total phenol in callicarpa nudiflora capsule, is 10.513%.
embodiment eight
(1). the preparation of need testing solution:
Get callicarpa nudiflora tablet, precision takes 49.91mg internal drug, pulverizes, and puts in 25ml measuring bottle, adds 50% methyl alcohol 20ml ultrasonic dissolution, with 50% methyl alcohol, is diluted to scale, shakes up, and filters, and gained filtrate is made need testing solution.
(2). the making of typical curve: precision takes gallic acid 10.01mg product in contrast, put and in 10 ml volumetric flasks, add 50% methyl alcohol and dissolve and be diluted to scale, shake up, obtaining concentration is the reference substance storing solution of 1.001 mg/ml; Precision pipettes 1 ml reference substance storing solution and is placed in 10 ml volumetric flasks, with 50% methyl alcohol, is diluted to scale, shakes up, and obtains the reference substance solution that concentration is 0.1001 mg/ml; Accurate 0,40,80,120,160,200, the 240 μ l reference substance solution of drawing are placed in 5ml measuring bottle respectively, add Folin-phenol test solution 0.45ml, mix rear standing 2min, add 7.5% sodium carbonate liquor 1.5ml, add water to scale, shake up, after 40 ℃ of water-bath 10min, 45min is secretly put in 25 ℃ of water-baths.Take 50% methyl alcohol as blank, under 760nm wavelength, measure absorbance, take gallic acid content as horizontal ordinate, take its absorbance as ordinate, obtain gallic acid content bioassay standard curve.
(3). the assay of the total phenol of sample solution: precision measures the described need testing solution of 0.1ml step (1) and is placed in 5ml measuring bottle, add Folin-phenol test solution 0.45ml, mix rear standing 2min, add 7.5% sodium carbonate liquor 1.5ml, add water to scale, shake up, after 40 ℃ of water-bath 10min, 45min is secretly put in 25 ℃ of water-baths, take 50% methyl alcohol as blank, according to UV-VIS spectrophotometry, under 760nm wavelength, measure absorbance, according to this absorbance, by typical curve, obtain corresponding concentration, this concentration value is the content of the total phenol of need testing solution, be 0.1328 mg/ml.
(4). the calculating of content in extract: according to the content of total phenol in the need testing solution of step (3) gained, conversing the content of total phenol in callicarpa nudiflora tablet, is 6.652%.
embodiment nine
(1). the preparation of need testing solution:
Get callicarpa nudiflora washing lotion, precision measures the callicarpa nudiflora liquid preparation of 1ml, puts in 25ml measuring bottle, with 50% methyl alcohol, is diluted to scale, shakes up, and filters, and gained filtrate is made need testing solution.
(2). the making of typical curve: precision takes gallic acid 10.02mg product in contrast, put and in 10 ml volumetric flasks, add 50% methyl alcohol and dissolve and be diluted to scale, shake up, obtaining concentration is the reference substance storing solution of 1.002 mg/ml; Precision pipettes 1 ml reference substance storing solution and is placed in 10 ml volumetric flasks, with 50% methyl alcohol, is diluted to scale, shakes up, and obtains the reference substance solution that concentration is 0.1002 mg/ml; Accurate 0,40,80,120,160,200, the 240 μ l reference substance solution of drawing are placed in 5ml measuring bottle respectively, add Folin-phenol test solution 0.6ml, mix rear standing 2min, add 6.5% sodium carbonate liquor 2ml, add water to scale, shake up, after 45 ℃ of water-bath 12min, 50min is secretly put in 20 ℃ of water-baths.Take 50% methyl alcohol as blank, under 760nm wavelength, measure absorbance, take gallic acid content as horizontal ordinate, take its absorbance as ordinate, obtain gallic acid content bioassay standard curve.
(3). the assay of the total phenol of sample solution: precision measures the described need testing solution of 0.1ml step (1) and is placed in 5ml measuring bottle, add Folin-phenol test solution 0.6ml, mix rear standing 2min, add 6.5% sodium carbonate liquor 2ml, add water to scale, shake up, after 45 ℃ of water-bath 12min, 50min is secretly put in 20 ℃ of water-baths.Take 50% methyl alcohol as blank, according to UV-VIS spectrophotometry, under 760nm wavelength, measure absorbance, according to this absorbance, by typical curve, obtain corresponding concentration, this concentration value is the content of the total phenol of need testing solution, is 0.1495mg/ml.
(4). the calculating of content in extract: according to the content of total phenol in the need testing solution of step (3) gained, conversing the content of total phenol in callicarpa nudiflora washing lotion, is 0.374%.
embodiment ten
(1). the preparation of need testing solution:
Get callicarpa nudiflora spray, precision measures the callicarpa nudiflora liquid preparation of 5ml, puts in 25ml measuring bottle, with 50% methyl alcohol, is diluted to scale, shakes up, and filters, and gained filtrate is made need testing solution.
(2). the making of typical curve: precision takes gallic acid 10.01mg product in contrast, put and in 10 ml volumetric flasks, add 50% methyl alcohol and dissolve and be diluted to scale, shake up, obtaining concentration is the reference substance storing solution of 1.001 mg/ml; Precision pipettes 1 ml reference substance storing solution and is placed in 10 ml volumetric flasks, with 50% methyl alcohol, is diluted to scale, shakes up, and obtains the reference substance solution that concentration is 0.1001 mg/ml; Accurate 0,40,80,120,160,200, the 240 μ l reference substance solution of drawing are placed in 5ml measuring bottle respectively, add Folin-phenol test solution 0.45ml, mix rear standing 2min, add 7.5% sodium carbonate liquor 1.5ml, add water to scale, shake up, after 40 ℃ of water-bath 10min, 45min is secretly put in 25 ℃ of water-baths.Take 50% methyl alcohol as blank, under 760nm wavelength, measure absorbance, take gallic acid content as horizontal ordinate, take its absorbance as ordinate, obtain gallic acid content bioassay standard curve.
(3). the assay of the total phenol of sample solution: precision measures the described need testing solution of 0.1ml step (1) and is placed in 5ml measuring bottle, add Folin-phenol test solution 0.45ml, mix rear standing 2min, add 7.5% sodium carbonate liquor 1.5ml, add water to scale, shake up, after 40 ℃ of water-bath 10min, 45min is secretly put in 25 ℃ of water-baths, take 50% methyl alcohol as blank, according to UV-VIS spectrophotometry, under 760nm wavelength, measure absorbance, according to this absorbance, by typical curve, obtain corresponding concentration, this concentration value is the content of the total phenol of need testing solution, for 0.3076mg/ml.
(4). the calculating of content in extract: according to the content of total phenol in the need testing solution of step (3) gained, conversing the content of total phenol in callicarpa nudiflora spray, is 0.154 %.
Claims (11)
1. in callicarpa nudiflora, a content assaying method for total phenol, is characterized in that comprising the steps:
(1). the preparation of need testing solution:
Get callicarpa nudiflora medicinal material appropriate, with alcoholic solvent or the ultrasonic extraction of ketone solvent, extract is centrifugal, take supernatant as need testing solution;
(2). the making of typical curve: precision takes the about 10mg of gallic acid product in contrast, put and in 10 ml volumetric flasks, add 50% methyl alcohol and dissolve and be diluted to scale, shake up, obtaining concentration is the reference substance storing solution of 1 mg/ml; Precision pipettes 1 ml reference substance storing solution and is placed in 10 ml volumetric flasks, with 50% methyl alcohol, is diluted to scale, shakes up, and obtains the reference substance solution that concentration is 0.1 mg/ml; The accurate reference substance solution of drawing is mixed with the standard solution of gradient in 5ml measuring bottle respectively again, add chromogenic reagent, take 50% methyl alcohol as blank, according to UV-VIS spectrophotometry, under 760nm wavelength, measure respectively absorbance, take gallic acid content as horizontal ordinate, take its absorbance as ordinate, obtain gallic acid content bioassay standard curve;
(3). the assay of the total phenol of sample solution: precision measures the described need testing solution of 0.1ml step (1), add chromogenic reagent, take corresponding reagent as blank, according to UV-VIS spectrophotometry, under 760nm wavelength, measure absorbance, according to this absorbance, by typical curve, obtain corresponding concentration, this concentration value is the content of the total phenol of need testing solution;
(4). the calculating of content in medicinal material: according to the content of total phenol in the need testing solution of step (3) gained, converse the content of total phenol in callicarpa nudiflora medicinal material.
2. content assaying method according to claim 1, is characterized in that, methyl alcohol or ethanol that in step (1), alcoholic solvent is 40 ~ 60%, and ketone solvent is 40 ~ 60% acetone.
3. content assaying method according to claim 1, is characterized in that, in step (1), the liquid ratio of ultrasonic extraction is 120:1 ~ 250:1, and centrifugation rate is 10000 ~ 15000r/min, extraction time 0.5 ~ 1.5h.
4. content assaying method according to claim 1, is characterized in that, step (1) is:
The preparation of need testing solution:
The ultrasonic extraction of acetone of callicarpa nudiflora use 50%, extraction time is 1 hour, liquid ratio is 250:1, extract is centrifugal with 10000r/min, and after centrifugal 1 min, precision measures supernatant 1-5ml and puts in 25ml measuring bottle, add 50% methyl alcohol and be diluted to scale, shake up, make need testing solution.
5. a content assaying method for total phenol in beautyberry extract, is characterized in that comprising the steps:
(1). the preparation of need testing solution:
Precision takes 0.01 ~ 0.05g beautyberry extract powder, puts in 25ml measuring bottle, adds 50% methyl alcohol 10 ~ 20ml ultrasonic dissolution, with 50% methyl alcohol, is diluted to scale, shakes up, and filters, and gained filtrate is made need testing solution;
(2). the making of typical curve: precision takes the about 10mg of gallic acid product in contrast, put and in 10 ml volumetric flasks, add 50% methyl alcohol and dissolve and be diluted to scale, shake up, obtaining concentration is the reference substance storing solution of 1 mg/ml; Precision pipettes 1 ml reference substance storing solution and is placed in 10 ml volumetric flasks, with 50% methyl alcohol, is diluted to scale, shakes up, and obtains the reference substance solution that concentration is 0.1 mg/ml; The accurate reference substance solution of drawing is placed in the standard solution that 5ml measuring bottle is mixed with gradient respectively again, add chromogenic reagent, take 50% methyl alcohol as blank, according to UV-VIS spectrophotometry, under 760nm wavelength, measure respectively absorbance, take gallic acid content as horizontal ordinate, take its absorbance as ordinate, obtain gallic acid content bioassay standard curve;
(3). the assay of the total phenol of sample solution: precision measures the described need testing solution of 0.1ml step (1), add chromogenic reagent, take corresponding reagent as blank, according to UV-VIS spectrophotometry, under 760nm wavelength, measure absorbance, according to this absorbance, by typical curve, obtain corresponding concentration, this concentration value is the content of the total phenol of need testing solution;
(4). the calculating of content in extract: according to the content of total phenol in the need testing solution of step (3) gained, converse the content of total phenol in beautyberry extract.
6. a content assaying method for total phenol in callicarpa nudiflora solid pharmaceutical preparation, is characterized in that comprising the steps:
(1). the preparation of need testing solution:
Get callicarpa nudiflora solid pharmaceutical preparation, precision takes 0.05 ~ 0.1g internal drug, pulverizes, and puts in 25ml measuring bottle, adds 50% methyl alcohol 10 ~ 20ml ultrasonic dissolution, with 50% methyl alcohol, is diluted to scale, shakes up, and filters, and gained filtrate is made need testing solution;
(2). the making of typical curve: precision takes the about 10mg of gallic acid product in contrast, put and in 10 ml volumetric flasks, add 50% methyl alcohol and dissolve and be diluted to scale, shake up, obtaining concentration is the reference substance storing solution of 1 mg/ml; Precision pipettes 1 ml reference substance storing solution and is placed in 10 ml volumetric flasks, with 50% methyl alcohol, is diluted to scale, shakes up, and obtains the reference substance solution that concentration is 0.1 mg/ml; The accurate reference substance solution of drawing is mixed with the standard solution of gradient in 5ml measuring bottle respectively again, add chromogenic reagent, take 50% methyl alcohol as blank, according to UV-VIS spectrophotometry, under 760nm wavelength, measure respectively absorbance, take gallic acid content as horizontal ordinate, take its absorbance as ordinate, obtain gallic acid content bioassay standard curve;
(3). the assay of the total phenol of sample solution: precision measures the described need testing solution of 0.1ml step (1), add chromogenic reagent, corresponding reagent is blank, according to UV-VIS spectrophotometry, under 760nm wavelength, measure absorbance, according to this absorbance, by typical curve, obtain corresponding concentration, this concentration value is the content of the total phenol of need testing solution;
(4). the calculating of content in preparation: according to the content of total phenol in the need testing solution of step (3) gained, converse the content of total phenol in callicarpa nudiflora preparation.
7. a content assaying method for total phenol in callicarpa nudiflora liquid preparation, is characterized in that comprising the steps:
(1). the preparation of need testing solution:
Precision measures the callicarpa nudiflora liquid preparation of 1-5ml, puts in 25ml measuring bottle, with 50% methyl alcohol, is diluted to scale, shakes up, and filters, and gained filtrate is made need testing solution;
(2). the making of typical curve: precision takes the about 10mg of gallic acid product in contrast, put and in 10 ml volumetric flasks, add 50% methyl alcohol and dissolve and be diluted to scale, shake up, obtaining concentration is the reference substance storing solution of 1 mg/ml; Precision pipettes 1 ml reference substance storing solution and is placed in 10 ml volumetric flasks, with 50% methyl alcohol, is diluted to scale, shakes up, and obtains the reference substance solution that concentration is 0.1 mg/ml; The accurate reference substance solution of drawing is mixed with the standard solution of gradient in 5ml measuring bottle respectively again, add chromogenic reagent, take 50% methyl alcohol as blank, according to UV-VIS spectrophotometry, under 760nm wavelength, measure respectively absorbance, take gallic acid content as horizontal ordinate, take its absorbance as ordinate, obtain gallic acid content bioassay standard curve;
(3). the assay of the total phenol of sample solution: precision measures the described need testing solution of 0.1ml step (1), add chromogenic reagent, corresponding reagent is blank, according to UV-VIS spectrophotometry, under 760nm wavelength, measure absorbance, according to this absorbance, by typical curve, obtain corresponding concentration, this concentration value is the content of the total phenol of need testing solution;
(4). the calculating of content in preparation: according to the content of total phenol in the need testing solution of step (3) gained, converse the content of total phenol in callicarpa nudiflora preparation.
8. according to the arbitrary described content assaying method of claim 1-7, it is characterized in that described developer is Folin-phenol test solution, Na
2cO
3solution and water.
9. according to the arbitrary described content assaying method of claim 1-7, it is characterized in that, temperature of reaction is 40 ~ 45 ℃, and the reaction time is 10 ~ 15min, and developing time is 45 ~ 60min, and colour temp is 20 ~ 30 ℃.
10. according to the arbitrary described content assaying method of claim 1-7, it is characterized in that Folin-phenol test solution and 1.2 ~ 2.1mlNa that described developer is 0.45 ~ 0.6ml
2cO
3solution, described Na
2cO
3concentration of polymer solution is 5.0% ~ 7.5%.
11. according to the arbitrary described content assaying method of claim 1-7, it is characterized in that developer is the Na that the Folin-phenol test solution of 0.45ml and the mass concentration of 1.5ml are 7.5%
2cO
3solution, the reaction time is 10min, and temperature of reaction is 40 ℃, and developing time is 45min, and colour temp is 25 ℃.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111044465A (en) * | 2019-12-20 | 2020-04-21 | 山西省农业科学院农产品加工研究所 | Physiological marking method for evaluating storage capacity of buckwheat rice |
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