CN103115887A - Measurement method of total saponins in health food using propolis as raw material - Google Patents
Measurement method of total saponins in health food using propolis as raw material Download PDFInfo
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Abstract
The invention relates to a measurement method of total saponins in health food, and particularly relates to a measurement method of total saponins in health food using propolis as a raw material. The measurement method provided by the invention comprises a standard curve making process, a sample treatment process, a test liquid column chromatography isolation process, a color developing process and a total saponins calculating process. In the standard curve making process, a standard curve is obtained by using an absorbance value and a mass of ginsenoside as curves; in the sample treatment process, liquid to be tested is obtained by taking 0.3g of the health food using the propolis as the raw material; in the test liquid column chromatography isolation process, column chromatography isolation is carried out on the liquid to be tested by using a chromatographic column, so as to obtain an evaporation pan with the liquid to be tested which is evaporated to be dry; in the color developing process, the absorbance value of the liquid to be tested is obtained; and in the total saponins calculating process, the content of the total saponins in the health food using the propolis as the raw material can be obtained. The measurement method provided by the invention has the advantages of simple extraction method, high recovery rate, good accuracy, simplicity, convenience and rapidness.
Description
Technical field
The present invention relates to the assay method of total saponin(e in a kind of health food, especially relate to the assay method of total saponin(e in a kind of health food take propolis as raw material.
Background technology
Propolis is the resin of honeybee herborization, and mixes the tackiness material that forms with self secretion.Propolis can qi-restoratives a little less than, change turbid fat, only quench one's thirst; The external application removing toxicity for detumescence, the convergence myogenic.Propolis has been widely used as healthy food material.
Complicated components in propolis, water insoluble, the propolis after purification is the brown bulk, both has been inconvenient to take, and also is unfavorable for absorbing, and often needs to add auxiliary material to guarantee that propolis can be dispersed in product with the state of fine-powder when therefore being processed into health food.
In health food take propolis as raw material, can be furnished with simultaneously the raw material that American Ginseng, ginseng etc. contain saponins compound, this class health food often is defined as general flavone and total saponin(e the functional component of product.Total saponin(e assay method of bibliographical information has: vanillin assay, thin-layered chromatography, GC method, HPLC method, LC-MS method, CE method etc.; As Shen Xianghong, arbitrary flat, Chen Yi .HPLC method and vanillin assay are measured the panaxoside in health products. Chinese Journal of Health Laboratory Technology, 2004,14 (6): disclose vanillin assay and HPLC method in 681-683.Health food is provided by the assay method of total saponin(e in the health food that provides with technical manual (version in 2003); it is the classical assay method of total saponin(e in present health food; when containing the health food of propolis raw material with this method mensuration; due to the protection that is subjected to the supplies such as propolis, disintegrant, filling agent; in product, total saponin(e is difficult to effectively be extracted; the total saponin content measurement result often is not inconsistent with actual formula ratio, poor accuracy.
the method that is used for measuring total saponin content that other are also arranged now, it was on October 31st, 2012 as open day, publication number is in the Chinese patent of CN102759514A, the assay method of the content of general ginsenoside in a kind of injection qi-tonifying and pulse-restoring preparation is disclosed, the method comprises the following steps: 1) preparation of reference substance respectively precision take the general ginsenoside reference substance, add the methyl alcohol dissolving, make solution, 2) injection Yiqi and vein recovery drug powder is got in the preparation of need testing solution, accurately weighed, the water dissolving, cross resin column, after rinsing with methanol-water, with methanol-eluted fractions, eluent filters, make solution, 3) accurate need testing solution and the reference substance solution drawn of assay method difference, the content of general ginsenoside in the determined by ultraviolet spectrophotometry sample.The method can not be measured the total saponin(e in health food take propolis as raw material.
In sum, also do not have at present a kind of extracting method simple, the recovery is high, and accuracy is good, the assay method of total saponin(e in simple and rapid health food take propolis as raw material.
Summary of the invention
The object of the invention is to overcome above shortcomings in prior art, and provide a kind of extracting method simple, the recovery is high, and accuracy is good, the assay method of total saponin(e in simple and rapid health food take propolis as raw material.
The present invention addresses the above problem the technical scheme that adopts: the assay method of total saponin(e in health food that should be take propolis as raw material is characterized in that: this assay method comprises that standard curve making operation, sample preparation operation, test solution cross post operation, colour developing operation and total saponin(e calculation process;
in described standard curve making operation, draw respectively 0ml, 10ml, 20ml, 40ml, 60ml, 80ml, ginsenoside Re's titer of 100ml and 120ml concentration known is placed in eight evaporating dishes, after ginsenoside Re's titer drying in ware to be evaporated, first adding the 5ml volumetric molar concentration is the sodium hydroxide solution of 0.1M, add again water 20ml, shaking up rear is 6.5-7.0 with the salt acid for adjusting pH value, thereby get eight parts of standard mother liquors, eight parts of standard mother liquors are placed in the ultrasonic 20-40min breakdown of emulsion of water-bath of 45-55 ℃, be cooled to normal temperature, adopt respectively the volumetric flask water constant volume of 50ml to 50ml, shake up rear filtration, discard just filtrate of 5ml, get the 1.0ml subsequent filtrate as standard solution, thereby obtain eight parts of standard solutions, adopting chromatographic column to carry out column operation to every part of standard solution, is first that 70% ethanol is washed post with the 25mL percent by volume, discards eluent, wash post with 25mL again, discard eluent, accurately add the 1.0mL standard solution, cross post, with the washing post of 25mL, wait water-solubility impurity to wash away sugar part, discard eluent, it is 70% ethanol elution ginsenoside with the 25mL percent by volume, collect eluent in evaporating dish, volatilize in the bath that discharges water, thereby obtain eight evaporating dishes after volatilization, accurately adding the 0.2mL percent by volume in the evaporating dish that has volatilized is 5% vanillic aldehyde glacial acetic acid solution, rotate evaporating dish, residue is all dissolved, then add 0.8mL perchloric acid, move into after mixing in 5mL band plug graduated centrifuge tube, be placed in the water-bath below 60 ℃ and heat 10min, take out, after ice bath is cooling, accurately add glacial acetic acid 5.0mL, after shaking up, carry out colorimetric estimation with the 1cm cuvette in 560nm wavelength place together with standard pipe, thereby obtain eight absorbances, the quality of the ginsenoside that contains in absorbance and an evaporating dish is corresponding, adopts the quality of absorbance and ginsenoside to do curve, obtains typical curve,
In described sample preparation operation, the health food 0.3g that gets take propolis as raw material is placed in the 50ml volumetric flask, and adding the 5ml volumetric molar concentration is that the sodium hydroxide solution of 0.1M dissolves health food, after the health food dissolving, add water 20ml, shaking up rear is 6.5-7.0 with the salt acid for adjusting pH value, and ultrasonic 20-40min breakdown of emulsion, be cooled to normal temperature in the water-bath of 45-55 ℃, the water constant volume is to 50ml, shake up rear filtration, discard just filtrate of 5ml, get the 1.0ml subsequent filtrate as test solution to be measured;
Described test solution is crossed in the post operation, and adopt chromatographic column to treat test fluid and carried out column operation, be first that 70% ethanol is washed post with the 25mL percent by volume, discard eluent, then wash post with 25mL, discard eluent, accurately add 1.0mL test solution to be measured, cross post, with the washing post of 25mL, wait water-solubility impurity to wash away sugar part, discarding eluent, is 70% ethanol elution ginsenoside with the 25mL percent by volume, collects eluent in evaporating dish, discharging water volatilizes in bathing, and obtains test solution to be measured and volatilizes evaporating dish;
in described colour developing operation, accurately adding the 0.2mL percent by volume in test solution to be measured volatilizes evaporating dish is 5% vanillic aldehyde glacial acetic acid solution, rotate evaporating dish, residue is all dissolved, add again 0.8mL perchloric acid, move into after mixing in 5mL band plug graduated centrifuge tube, be placed in the water-bath below 60 ℃ and heat 10min, take out, after ice bath is cooling, accurately add glacial acetic acid 5.0mL, after shaking up, carry out colorimetric estimation with the 1cm cuvette in 560nm wavelength place together with standard pipe, thereby obtain the absorbance of test solution to be measured, this absorbance is corresponding with the health food take propolis as raw material,
In described total saponin(e calculation process, in the absorbance substitution typical curve with test solution to be measured, obtain the quality of the ginsenoside that contains in test solution to be measured, thereby obtain the content of total saponin(e in health food take propolis as raw material.
The present invention adopts the health food of sodium hydrate aqueous solution dissolving take propolis as raw material, release is by total saponin constituent of propolis, disintegrant and filling agent embedding, make it soluble in water, then use the salt acid for adjusting pH value to 6.5-7.0, guarantee the stability of total saponin(e in water, and propolis is separated out again, get rid of the interference of propolis, thereby effectively extracted total saponin(e.Measure the total saponin(e in the sample that contains propolis, disintegrant, filling agent with this method, has extracting method simple, the recovery is high, accuracy is good, simple and rapid characteristics, can be used for the quality control of the health food take total saponin(e as functional component take propolis as raw material, also can be used for the mensuration of total saponin(e of general health food.
As preferably, the present invention in the standard curve making operation and test solution cross in the post operation, the long 20cm-25cm of chromatographic column used, internal diameter 0.6cm-0.8cm, the top connects the storage liquid funnel of 30ml; The Amberlite-XAD-2 macroreticular resin that the in-built 6cm of chromatographic column is high, on increase the neutral alumina of 1cm.
As preferably, the present invention when dissolving health food with sodium hydroxide solution, adopts mode that jolting disperses or the mode of ultrasonic aid dispersion to dissolve in the sample preparation operation.
As preferably, the present invention is in the sample preparation operation, and after adding 20ml water in the health food that has dissolved and shaking up, the hydrochloric acid that is used for regulating the pH value is that percent by volume is 10% hydrochloric acid.
As preferably, in standard curve making operation of the present invention, the regression equation of typical curve is y=ax+b, and wherein, y represents absorbance, and x represents the quality of panaxoside, and a is positive number, and b is Arbitrary Digit.
As preferably, in standard curve making operation of the present invention, the regression equation of typical curve is y=0.0036x-0.0043, and the unit of the y of expression absorbance is A, and the unit of the x of the quality of expression panaxoside is mg.
As preferably, the coefficient R of regression equation of the present invention
2=0.9937, illustrate to present good linear relationship in the 20mg-240mg scope.
As preferably, in sample preparation operation of the present invention, the health food take propolis as raw material is the grinds powder.Make thus the dissolving health food that the present invention can be more quickly and effectively.
As preferably, the concentration of ginsenoside Re's titer of the present invention is 2.0mg/mL.
The present invention compared with prior art, have the following advantages and effect: adopt the health food of sodium hydrate aqueous solution dissolving take propolis as raw material, release is by total saponin constituent of propolis, disintegrant and filling agent embedding, make it soluble in water, then use the salt acid for adjusting pH value to 6.5-7.0, guarantee the stability of total saponin(e in water, and propolis is separated out again, get rid of the interference of propolis, thereby effectively extracted total saponin(e.Measure the total saponin(e in the sample that contains propolis, disintegrant, filling agent with this method, has extracting method simple, the recovery is high, accuracy is good, simple and rapid characteristics, can be used for the quality control of the health food take total saponin(e as functional component take propolis as raw material, also can be used for the mensuration of total saponin(e of general health food.
The present invention has made innovation in the selection of extracting reagent, aldehyde radical in phenolic hydroxyl group in the saponin(e structure and vanillic aldehyde structure carries out aldol reaction and generates red condensation product, the phenolic hydroxyl group of glucoside unit is oxidized to carboxyl, with the aldehyde condensation reaction, carbohydrate, flavone compound are because containing the phenolic hydroxyl group interference measurement.The present invention once adopted methyl alcohol to extract total saponin(e, because propolis is soluble in methyl alcohol, thus with methyl alcohol as extraction agent, can extract total saponin(e fully, but sample is when extracting total saponin(e with methyl alcohol, also extracted propolis, for the interference of getting rid of propolis needs methyl alcohol is dried up, more total saponin(e that is dissolved in water, because propolis still is present in methanolic extract, and firmly with total saponin(e embedding, so water is difficult to the total saponin(e of stripping, cause Lower result.The present invention also once adopted the hot water return method to extract, although more better than methyl alcohol method, the recovery increases, but still on the low side, and assay is unstable, and this may be because heating can increase the solubleness of disintegrant, filling agent, but can not improve the solubleness of propolis in water, in addition, unstable result is relevant with factors such as sample thickness, extraction temperature, times.Analyze the character of propolis and total saponin(e, the present invention adopts sodium hydrate aqueous solution to extract total saponin(e, propolis is soluble in alkaline aqueous solution, total saponin(e is able to be released in alkaline aqueous solution from propolis, regulate the pH value to acid, propolis is precipitated again, and total saponin(e is still stayed in water, extract total saponin(e with sodium hydrate aqueous solution, the recovery can reach 90.3%-109.7%.
The present invention has made innovation in the selection of extract concentration, total saponin(e is unstable in alkaline aqueous solution, optimum pH is 6.03, and this is on the books in ginseng liquid body preparation TSPG PHm mensuration by Xu Wenxiang research in " Chinese Journal of Modern Applied Pharmacy magazine " the 15th volume supplementary issue of publishing in August, 1998.Propolis in sample contains a large amount of acid ingredients, the extraction agent sodium hydrate aqueous solution at first with propolis in acidic materials carry out neutralization reaction, thereby the dissolving propolis, the amount of propolis has determined the consumption of extraction agent.If extract concentration is too high, can accelerate the hydrolysis of saponin(e, concentration is too low, and propolis is not dissolved, fully stripping of saponin constituent.The present invention is under the prerequisite of lot of experiments, final high spot reviews volumetric molar concentration be 0.1M, 0.5M and these three kinds of concentration of 1M, and 2mL, 3mL and these three kinds of consumptions of 5mL are to the dissolving situation of sample, find that sample weighting amount is when 0.3g, use the 5ml volumetric molar concentration to be the NaOH of 0.1M dissolution sample well, when regulating the pH value, the consumption of hydrochloric acid is minimum.
The present invention has made innovation in the selection of extracting the pH value, after sample is dissolved by sodium hydroxide solution, need to regulate the pH value, guarantees that total saponin(e is fully extracted and is not hydrolyzed.The present invention under the prerequisite of lot of experiments, final primary study pH value be 5,6,7 o'clock, the recovery of total saponin(e, discovery is that the recovery of 7 o'clock is best in the pH value, possible cause is due under acid condition, when propolis is separated out again embedding due to the part saponin(e.
Embodiment
The present invention is described in further detail below by embodiment, and following examples are explanation of the invention and the present invention is not limited to following examples.
Embodiment.
In health food take propolis as raw material in the present embodiment, the assay method of total saponin(e comprises that standard curve making operation, sample preparation operation, test solution cross post operation, colour developing operation and total saponin(e calculation process.
in the standard curve making operation, draw respectively 0ml, 10ml, 20ml, 40ml, 60ml, 80ml, 100ml and 120ml concentration are that ginsenoside Re's titer of 2.0mg/mL is placed in eight evaporating dishes, after ginsenoside Re's titer drying in ware to be evaporated, first adding the 5ml volumetric molar concentration is the sodium hydroxide solution of 0.1M, add again water 20ml, shaking up rear is 6.5-7.0 with the salt acid for adjusting pH value, thereby get eight parts of standard mother liquors, eight parts of standard mother liquors are placed in the ultrasonic 30min breakdown of emulsion of water-bath of 50 ℃, be cooled to normal temperature, adopt respectively the volumetric flask water constant volume of 50ml to 50ml, shake up rear filtration, discard just filtrate of 5ml, get the 1.0ml subsequent filtrate as standard solution, thereby obtain eight parts of standard solutions.
Adopt chromatographic column to carry out column operation to every part of standard solution, the long 20cm-25cm of chromatographic column used, internal diameter 0.6cm-0.8cm, the top connects the storage liquid funnel of 30ml; The Amberlite-XAD-2 macroreticular resin that the in-built 6cm of chromatographic column is high, on increase the neutral alumina of 1cm.Be first that 70% ethanol is washed post with the 25mL percent by volume, discard eluent, then wash post with 25mL, discard eluent, accurately add the 1.0mL standard solution, cross post, with the washing post of 25mL, wait water-solubility impurity to wash away sugar part, discarding eluent, is 70% ethanol elution ginsenoside with the 25mL percent by volume, collects eluent in evaporating dish, discharging water volatilizes in bathing, thereby obtains eight evaporating dishes after volatilization.
Accurately adding the 0.2mL percent by volume in the evaporating dish that has volatilized is 5% vanillic aldehyde glacial acetic acid solution, rotate evaporating dish, residue is all dissolved, then add 0.8mL perchloric acid, move into after mixing in 5mL band plug graduated centrifuge tube, be placed in the water-bath below 60 ℃ and heat 10min, take out, after ice bath is cooling, accurately add glacial acetic acid 5.0mL, after shaking up, carry out colorimetric estimation with the 1cm cuvette in 560nm wavelength place together with standard pipe, thereby obtain eight absorbances.The quality of the ginsenoside that contains in absorbance and an evaporating dish is corresponding, adopt the quality of absorbance and ginsenoside to do curve, obtain typical curve, adopt EXCEL software, the regression equation of trying to achieve this typical curve is y=0.0036x-0.0043, wherein, y represents absorbance, and the unit of y is A, and x represents the quality of panaxoside, the unit of x is mg, the coefficient R of this regression equation
2=0.9937, illustrate to present good linear relationship in the 20mg-240mg scope.The regression equation of the typical curve in the present invention can be y=ax+b, and wherein, y represents absorbance, and x represents the quality of panaxoside, and a is positive number, and b is Arbitrary Digit.
In the sample preparation operation, the health food 0.3g that gets take propolis as raw material is placed in the 50ml volumetric flask, and adding the 5ml volumetric molar concentration is that the sodium hydroxide solution of 0.1M dissolves health food, after the health food dissolving, add water 20ml, shaking up rear is 6.5-7.0 with the salt acid for adjusting pH value, and ultrasonic 30min breakdown of emulsion, be cooled to normal temperature in the water-bath of 50 ℃, the water constant volume is to 50ml, shake up rear filtration, discard just filtrate of 5ml, get the 1.0ml subsequent filtrate as test solution to be measured.The present invention can adopt the mode of jolting dispersion or adopt the mode of ultrasonic aid dispersion to dissolve when dissolving health food with sodium hydroxide solution; The hydrochloric acid that is used for adjusting pH value can be 10% hydrochloric acid for percent by volume; Health food take propolis as raw material is the grinds powder normally.
Cross in the post operation at test solution, adopt chromatographic column to treat test fluid and carried out column operation, the long 20cm-25cm of chromatographic column used, internal diameter 0.6cm-0.8cm, the top connects the storage liquid funnel of 30ml; The Amberlite-XAD-2 macroreticular resin that the in-built 6cm of chromatographic column is high, on increase the neutral alumina of 1cm.Be first that 70% ethanol is washed post with the 25mL percent by volume, discard eluent, then wash post with 25mL, discard eluent, accurately add 1.0mL test solution to be measured, cross post, with the washing post of 25mL, wait water-solubility impurity to wash away sugar part, discarding eluent, is 70% ethanol elution ginsenoside with the 25mL percent by volume, collects eluent in evaporating dish, discharging water volatilizes in bathing, and obtains test solution to be measured and volatilizes evaporating dish.
in the colour developing operation, accurately adding the 0.2mL percent by volume in test solution to be measured volatilizes evaporating dish is 5% vanillic aldehyde glacial acetic acid solution, rotate evaporating dish, residue is all dissolved, add again 0.8mL perchloric acid, move into after mixing in 5mL band plug graduated centrifuge tube, be placed in the water-bath below 60 ℃ and heat 10min, take out, after ice bath is cooling, accurately add glacial acetic acid 5.0mL, after shaking up, carry out colorimetric estimation with the 1cm cuvette in 560nm wavelength place together with standard pipe, thereby obtain the absorbance of test solution to be measured, this absorbance is corresponding with the health food take propolis as raw material.
In total saponin(e calculation process, in the regression equation with the absorbance substitution typical curve of test solution to be measured, obtain the quality of the ginsenoside that contains in test solution to be measured, thereby obtain the content of total saponin(e in health food take propolis as raw material.
The present invention can adopt the TU-1810SPC ultraviolet-visible pectrophotometer of being made by the Beijing Puxi General Instrument Co., Ltd to measure absorbance, can adopt Sartorius BS224S analytical balance to take sample, can adopt the pH precision test paper to survey the pH value.Provided by Hangzhou Words Bee Apiculture Co.,Ltd take propolis, American ginseng extract as the tablet of main material production in the present invention; Ginsenoside Re's standard items used are Nat'l Pharmaceutical ﹠ Biological Products Control Institute, and lot number is 110754-200822, and the quality percentage composition is 88.8%; It is pure that normal butyl alcohol used, NaOH, hydrochloric acid, methyl alcohol, ethanol, glacial acetic acid, perchloric acid are analysis; Neutral alumina is that chromatography is used; Water can be dual distilled water.
The below analyzes the range of linearity, detectability, the recovery and the precision of the assay method in the present invention.
Linear relationship: the ginsenoside Re standard solution 0ml, 10ml, 20ml, 40ml, 60ml, 80ml, 100ml and the 120ml that are 2.0mg/mL with concentration are placed in evaporating dish, is placed on lower than volatilizing in the water-bath of 60 ℃, or hot blast drying (it is overheated not make).Represent absorbance (A) with y, x represents ginsenoside Re quality (mg), uses EXCEL software, and the regression equation of trying to achieve method is y=0.0036x-0.0043, coefficient R
2=0.9937, illustrate to present good linear relationship in the scope of 20mg-240mg.
Method detectability: to reagent blank METHOD FOR CONTINUOUS DETERMINATION 6 times (seeing Table 1), calculate to detect with 3 times of standard deviations of its absorbance and be limited to 9.9mg.
Precision test: take 6 parts of tablet samples with the said method ungrease treatment and measure, measurement result sees Table 3.Its relative standard deviation is 3.65%.
Recovery test: (lot number is 110801 to take the same batch sample of known content, content 2.51g/100g) 6 parts, every part of about 0.12g, accurately weighed, be placed in the 50ml volumetric flask, the ginsenoside Re reference substance that adds respectively 2.925mg, carry out processing and the mensuration of sample by this assay method, calculate recovery rate the results are shown in Table 4, recovery of standard addition is at 90.3%-109.7%, and relative standard deviation is 6.05%.
In health food take propolis as raw material in the present embodiment the accuracy of the assay method of total saponin(e is high, describes below by the mode of contrast.Adopt respectively assay method (being called for short Ministry of Public Health's method) and this assay method of total saponin(e in health food evaluation and technical manual (version in 2003) health food to process to 3 batches of American ginseng extracts and the 3 batches of products, measurement result sees Table respectively 5 and table 6.As seen from table, American ginseng extract adopts two kinds of methods there is no significant difference, and 3 batches of product differentiation are extremely remarkable, and adopts this assay method measured value consistent with formula ratio, therefore the accuracy of this assay method is high.
The present invention adopts sodium hydrate aqueous solution dissolving propolis, release is by total saponin constituent of propolis, disintegrant, filling agent embedding, make it soluble in water, then use the salt acid for adjusting pH value to 6.5-7.0, guarantee the stability of total saponin(e in water, and propolis is separated out again, get rid of the interference of propolis, thereby effectively extracted total saponin(e.Measure the total saponin(e in the sample that contains propolis, disintegrant, filling agent with this assay method, have that extracting method is simple, the recovery is high, accuracy is good, simple and rapid characteristics, can be used for the quality control of the health food take total saponin(e as functional component take propolis as raw material, also can be used for the mensuration of the total saponin(e in general health food.
Although the present invention with embodiment openly as above; but it is not to limit protection scope of the present invention; any technician who is familiar with this technology not breaking away from change and the retouching of doing in the spirit and scope of the present invention, all should belong to protection scope of the present invention.
Claims (9)
1. the assay method of total saponin(e in the health food take propolis as raw material is characterized in that: this assay method comprises that standard curve making operation, sample preparation operation, test solution cross post operation, colour developing operation and total saponin(e calculation process;
in described standard curve making operation, draw respectively 0ml, 10ml, 20ml, 40ml, 60ml, 80ml, ginsenoside Re's titer of 100ml and 120ml concentration known is placed in eight evaporating dishes, after ginsenoside Re's titer drying in ware to be evaporated, first adding the 5ml volumetric molar concentration is the sodium hydroxide solution of 0.1M, add again water 20ml, shaking up rear is 6.5-7.0 with the salt acid for adjusting pH value, thereby get eight parts of standard mother liquors, eight parts of standard mother liquors are placed in the ultrasonic 20-40min breakdown of emulsion of water-bath of 45-55 ℃, be cooled to normal temperature, adopt respectively the volumetric flask water constant volume of 50ml to 50ml, shake up rear filtration, discard just filtrate of 5ml, get the 1.0ml subsequent filtrate as standard solution, thereby obtain eight parts of standard solutions, adopting chromatographic column to carry out column operation to every part of standard solution, is first that 70% ethanol is washed post with the 25mL percent by volume, discards eluent, wash post with 25mL again, discard eluent, accurately add the 1.0mL standard solution, cross post, with the washing post of 25mL, wait water-solubility impurity to wash away sugar part, discard eluent, it is 70% ethanol elution ginsenoside with the 25mL percent by volume, collect eluent in evaporating dish, volatilize in the bath that discharges water, thereby obtain eight evaporating dishes after volatilization, accurately adding the 0.2mL percent by volume in the evaporating dish that has volatilized is 5% vanillic aldehyde glacial acetic acid solution, rotate evaporating dish, residue is all dissolved, then add 0.8mL perchloric acid, move into after mixing in 5mL band plug graduated centrifuge tube, be placed in the water-bath below 60 ℃ and heat 10min, take out, after ice bath is cooling, accurately add glacial acetic acid 5.0mL, after shaking up, carry out colorimetric estimation with the 1cm cuvette in 560nm wavelength place together with standard pipe, thereby obtain eight absorbances, the quality of the ginsenoside that contains in absorbance and an evaporating dish is corresponding, adopts the quality of absorbance and ginsenoside to do curve, obtains typical curve,
In described sample preparation operation, the health food 0.3g that gets take propolis as raw material is placed in the 50ml volumetric flask, and adding the 5ml volumetric molar concentration is that the sodium hydroxide solution of 0.1M dissolves health food, after the health food dissolving, add water 20ml, shaking up rear is 6.5-7.0 with the salt acid for adjusting pH value, and ultrasonic 20-40min breakdown of emulsion, be cooled to normal temperature in the water-bath of 45-55 ℃, the water constant volume is to 50ml, shake up rear filtration, discard just filtrate of 5ml, get the 1.0ml subsequent filtrate as test solution to be measured;
Described test solution is crossed in the post operation, and adopt chromatographic column to treat test fluid and carried out column operation, be first that 70% ethanol is washed post with the 25mL percent by volume, discard eluent, then wash post with 25mL, discard eluent, accurately add 1.0mL test solution to be measured, cross post, with the washing post of 25mL, wait water-solubility impurity to wash away sugar part, discarding eluent, is 70% ethanol elution ginsenoside with the 25mL percent by volume, collects eluent in evaporating dish, discharging water volatilizes in bathing, and obtains test solution to be measured and volatilizes evaporating dish;
in described colour developing operation, accurately adding the 0.2mL percent by volume in test solution to be measured volatilizes evaporating dish is 5% vanillic aldehyde glacial acetic acid solution, rotate evaporating dish, residue is all dissolved, add again 0.8mL perchloric acid, move into after mixing in 5mL band plug graduated centrifuge tube, be placed in the water-bath below 60 ℃ and heat 10min, take out, after ice bath is cooling, accurately add glacial acetic acid 5.0mL, after shaking up, carry out colorimetric estimation with the 1cm cuvette in 560nm wavelength place together with standard pipe, thereby obtain the absorbance of test solution to be measured, this absorbance is corresponding with the health food take propolis as raw material,
In described total saponin(e calculation process, in the absorbance substitution typical curve with test solution to be measured, obtain the quality of the ginsenoside that contains in test solution to be measured, thereby obtain the content of total saponin(e in health food take propolis as raw material.
2. the assay method of total saponin(e in the health food take propolis as raw material according to claim 1, it is characterized in that: cross in the post operation with test solution in the standard curve making operation, the long 20cm-25cm of chromatographic column used, internal diameter 0.6cm-0.8cm, the top connects the storage liquid funnel of 30ml; The Amberlite-XAD-2 macroreticular resin that the in-built 6cm of chromatographic column is high, on increase the neutral alumina of 1cm.
3. the assay method of total saponin(e in the health food take propolis as raw material according to claim 1, it is characterized in that: in the sample preparation operation, when dissolving health food with sodium hydroxide solution, adopt the mode of jolting dispersion or the mode of ultrasonic aid dispersion to dissolve.
4. the assay method of total saponin(e in the health food take propolis as raw material according to claim 1, it is characterized in that: in the sample preparation operation, after adding 20ml water in the health food that has dissolved and shaking up, the hydrochloric acid that is used for regulating the pH value is that percent by volume is 10% hydrochloric acid.
5. the assay method of total saponin(e in the health food take propolis as raw material according to claim 1, it is characterized in that: in described standard curve making operation, the regression equation of typical curve is y=ax+b, wherein, y represents absorbance, x represents the quality of panaxoside, and a is positive number, and b is Arbitrary Digit.
6. the assay method of total saponin(e in the health food take propolis as raw material according to claim 5, it is characterized in that: in described standard curve making operation, the regression equation of typical curve is y=0.0036x-0.0043, the unit of the y of expression absorbance is A, and the unit of the x of the quality of expression panaxoside is mg.
7. the assay method of total saponin(e in the health food take propolis as raw material according to claim 6, is characterized in that: the coefficient R of described regression equation
2=0.9937, illustrate to present good linear relationship in the 20mg-240mg scope.
8. the assay method of total saponin(e in the health food take propolis as raw material according to claim 1, it is characterized in that: in described sample preparation operation, the health food take propolis as raw material is the grinds powder.
9. the assay method of total saponin(e in the health food take propolis as raw material according to claim 1, it is characterized in that: the concentration of described ginsenoside Re's titer is 2.0mg/mL.
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