CN102778437B - Method for identifying propolis and poplar glue by detecting chlorophyll content - Google Patents
Method for identifying propolis and poplar glue by detecting chlorophyll content Download PDFInfo
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- CN102778437B CN102778437B CN201210175857.8A CN201210175857A CN102778437B CN 102778437 B CN102778437 B CN 102778437B CN 201210175857 A CN201210175857 A CN 201210175857A CN 102778437 B CN102778437 B CN 102778437B
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Abstract
The invention provides a method for identifying propolis and poplar glue by detecting chlorophyll content. Chlorophyll in a sample is extracted by using acetone, layered extraction is performed by using ether, the extract is cleaned and purified by using sodium sulfate solution, and colorimetry is performed under certain wavelength at two places to identify. The chlorophyll in the propolis or the poplar glue which is manufactured from poplar bark or poplar buds by using an organic solvent with very low polarity to performing extraction and separation, the chlorophyll content is quantitatively detected by spectrophotometry, the peak value and the average value of the chlorophyll content in the propolis and the poplar glue are calculated respectively, the safety limit value of the chlorophyll content in the propolis is set to be 15mg/100g, and the propolis and the poplar glue can be accurately identified by compared detection. The detection method is simple and convenient to perform, the detected sample and equipment are simple and available, and the detection accuracy is high.
Description
Technical field
The present invention relates to propolis and yang gum authentication technique field, particularly differentiated a kind of method of propolis and yang gum by the content detection of chlorophyll content.
Background technology
Propolis have reducing blood lipid, hypoglycemic, improve body immunity, strengthen Abwehrkraft des Koepers, anti-oxidant, antitumor, various biological characteristic such as prevention infection etc., being widely used among body-care and disease treatment, is the focus of recent domestic bee product research and development.
Propolis is the resin of apis mellifera herborization bud, leaf or bark rent secretion and is mixed into the fragrant solids of honeybee mandibular gland secretion and beeswax, the place such as gap, frame ear of beehive is coated on, through the bee products with natural attribute artificially collected by honeybee.China's natural propolis annual production about 400 tons, per kilogram procurement price is at 400 yuan.Yang gum is the colloid manually utilizing the spray of Salicaceae poplar, young shoot, bark etc. to carry out boiling extraction, is the main Types of domestic false propolis, its cost price per kilogram about 150 yuan.Due to yang gum low price, drive by interests, current China's propolis market has a considerable amount of product to be the propolis product pretended to be with yang gum, the concern of extremely domestic apiculture circle of this phenomenon and relevant department.
Propolis and yang gum are quite similar in sensory properties, and total flavonoids substance chemical composition is very nearly the same, and the foundation domestic relevant criterion about propolis at present, is difficult to both differentiations.Such as: 1, The Ministry of Agriculture of the People's Republic of China, MOA issues in NY5136-2002 pollution-free food propolis, with the general flavone content in high effective liquid chromatography for measuring propolis, expensive equipment, process is numerous and diverse, and eight kinds of flavones Galangins, rutin, Quercetin, myricetin, the luxuriant and rich with fragrance alcohol of camphane, apiolin, pinocembrin, chrysin standard specimen are expensive, domestic purchase is incomplete.And too containing the ratio difference that aforementioned eight kinds of flavones are respective in yang gum, cannot fundamentally distinguish propolis and yang gum.
2, General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China issues in GB/T 24283-2009 propolis, by ultraviolet spectrophotometry, take rutin as reference substance, measure the general flavone content in propolis, detecting instrument and and reference substance cost higher, and in yang gum with rutin be contrast general flavone identical with general flavone in propolis, propolis and yang gum cannot be distinguished equally.
3, in NY5136-2002 and GB/T 24283-2009, all have and measure ethanol extract content and oxidization time, yang gum can meet this two Testing index equally under the same conditions, also cannot distinguish propolis and yang gum.
Summary of the invention
Technical matters to be solved by this invention is to provide a kind of method of effective discriminating propolis and yang gum.
Technical matters of the present invention is solved by following method:
Detect the method that chlorophyll content differentiates propolis and yang gum, with the chlorophyll in acetone extraction sample, with ether layering extraction, metabisulfite solution washing, purifying, colorimetric assay under the wavelength that two places are certain, differentiates; Concrete technology step is as follows:
A, analytical procedure
A.1, the process of preparing Chinese medicine of propolis and extraction: get a mao propolis sample, first below-5 ~-10 DEG C freezing 24 ~ 48 hours, then smash to pieces accurately weighed; Be placed in glass mortar, add and the calcium carbonate of sample equivalent and silica sand
,grinding, the ethanol adding suitable double sample amount continues to grind to form pasty state; Abrasive material is all transferred in tool plug conical flask, with ethanol washing mortar to colourless, makes amount of alcohol reach 20 times of sample size, close plug, cold soaking, jolting mixing, leave standstill 24 ~ 48 hours, use G
3glass funnel suction filtration, drains with aqueous acetone solution washing; Refunded in mortar by residue in funnel and add aqueous acetone solution grinding, transfer in funnel, suction filtration, the residue of continuation in aqueous acetone solution washing mortar and funnel, colourless to filtrate, use aqueous acetone solution amount reaches 20 times of sample size; 1/4th of the total suction filtration liquid of accurate absorption, be placed in the evaporating dish of dry constant weight, in water-bath after evaporate to dryness, drying 3 hours at 90 DEG C ~ 110 DEG C temperature is also cooled to room temperature, rapid accurately weighed weight, calculates the content (%) of extract in mao propolis with dry product;
A.2, the dissolving of sample: get above-mentioned dry propolis extract, accurately weighed, put in beaker, add aqueous acetone solution and test sample is all dissolved, for subsequent use;
A.3, isolation of purified: said sample lysate is all proceeded in the separating funnel of interior Sheng metabisulfite solution, with ether gradation washing beaker, be incorporated in separating funnel, jolting, makes layering, and water layer puts into another separating funnel, add the extracted with diethyl ether of sample lysate 3/5ths, discard water layer; Organic layer is incorporated in first separating funnel, washes with water, discards water layer; Ether layer is filtered in brown volumetric flask by the little funnel that is equipped with anhydrous sodium sulfate, with the washed with diethylether separating funnel of sample lysate 1/5th, and washes away the pigment in anhydrous sodium sulfate layer, constant volume, shake up, stand-by;
A.4, measure: get above-mentioned solution in brown volumetric flask, use ether constant volume, shake up, on spectrophotometer, measure the light absorption value at 642nm and 660nm wavelength place immediately, do with ether blank.
A.5 the content of sample Determination of Chlorophyll is gone out by certain formulae discovery.
Described reagent is chemical pure or analyzes pure, and water is distilled water.
Described reagent comprises: ethanol, calcium carbonate, silica sand: the one in SILVER REAGENT, acetone, anhydrous sodium sulfate, ether, aqueous acetone solution and aqueous sodium persulfate solution.
Described reagent anhydrous sodium sulfate is the anhydrous sodium sulfate at 650 DEG C of calcination 4h.
The mass volume ratio of described reagent acetone aqueous solution is 85+15, V/V.
Described reagent of sulfuric acid sodium water solution is 2%, m/V.
Be measurement unit with g when said sample and admixture are solid, be measurement unit with ml when sample and admixture are liquid, when multiple converts, 1g is equivalent to 1ml.
The organic solvent extraction that the present invention adopts polarity lower and the chlorophyll in the yang gum being separated propolis or make for material with poplar bark or bud, with spectrophotometric method, its content is quantitatively detected, obtain propolis and the yang gum mxm. of chlorophyll content and mean value separately respectively, setting propolis chlorophyll content safety limit is 15mg/100g, detects chlorophyll content can accurately differentiate propolis and yang gum by contrast.If detected sample chlorophyll content value < 15mg/100g, can judge that sample is true propolis; If detected sample chlorophyll content value > 15mg/100g, can judge that sample is false propolis, or be mixed with yang gum in propolis, detect sample chlorophyll content value higher explanation yang gum incorporation larger.Detection method of the present invention is easy, detect sample and equipment simple and easy to get, Detection accuracy is high.
specific implementation method
Embodiment 1
Method And Principle: with the chlorophyll in acetone extraction sample, with ether layering extraction, metabisulfite solution washing, purifying, colorimetric assay under 642nm and 660nm wavelength.
Reagent: ethanol.
Instrument and equipment:
Porous electronic thermostatic water-bath, spectrophotometer, tool plug conical flask, 250ml, porcelain evaporating dishes, glass mortar, 150ml, G
3glass sand core funnel, separating funnel, 250ml, brown volumetric flask, 50ml, transfer pipet, 10ml, 20ml.
1, analytical procedure
1.1, the process of preparing Chinese medicine of propolis and extraction: get a mao propolis sample 5g, first freezing 24h at-5 DEG C, then smashs to pieces accurately weighed; Be placed in glass mortar, add calcium carbonate 5g and silica sand 5g, slowly grind to form impalpable powder, add ethanol 10ml and continue to grind to form pasty state; Abrasive material is all transferred in 250ml tool plug conical flask, closely colourless with ethanol washing mortar, make the cumulative volume of ethanol to 100ml, close plug, cold soaking, jolting constantly in front 6h, then leave standstill 18h, all use G
3glass funnel suction filtration, drains with in aqueous acetone solution (85%) cyclic washing conical flask to funnel; Refunded in mortar by residue in funnel and grind 5min with aqueous acetone solution, transfer in funnel, suction filtration, continue with the residue in aqueous acetone solution cyclic washing mortar and funnel, closely colourless to filtrate, the cumulative volume using aqueous acetone solution is 100ml.Accurate absorption suction filtration liquid 50ml, is placed in the evaporating dish of dry constant weight, in water-bath after evaporate to dryness, at 110 DEG C of dry 3h, puts in exsiccator and cool 30min to room temperature, rapid accurately weighed weight, calculate the content (%) of extract in mao propolis with dry product.
1.2, the dissolving of sample: get above-mentioned dry propolis extract and be about 0.2g, accurately weighed, put in 50ml beaker, add aqueous acetone solution (85%) 50ml and test sample is all dissolved, obtain 50ml sample solution, for subsequent use.
1.3, isolation of purified: said sample lysate is all proceeded in the 250ml separating funnel of interior Sheng 50mL 2% metabisulfite solution, with 20ml ether gradation washing beaker, be incorporated in separating funnel, jolting 1min, make layering, water layer puts into another separating funnel, adds 20ml extracted with diethyl ether, discards water layer; Organic layer is incorporated in first separating funnel, washes twice with water, and each 20ml, discards water layer.Ether layer is filtered in the brown volumetric flask of 50ml by the little funnel that is equipped with 5g anhydrous sodium sulfate, uses washed with diethylether separating funnel, and wash away the pigment in anhydrous sodium sulfate layer, constant volume, shake up, stand-by.
1.4, measure: get in the brown volumetric flask of above-mentioned solution 10ml to 50ml, use ether constant volume, shake up, on spectrophotometer, measure the light absorption value at 642nm and 660nm place immediately, 1 ㎝ cuvette, do with ether blank.
1.5 results calculate:
The Chlorophyll massfraction content in sample is calculated by following formula (1), (2).
X
1=7.12A
660+16.8A
642…………………………………（1）
X
2=【（X
1×V×F）÷（m×1000）】×100 ……………（2）
In formula: X
1the content of Chlorophyll in-sample liquid, unit is milligrams per liter (mg/L);
A-respective wavelength place (660nm, 642nm) records light absorption value;
X
2the massfraction of Chlorophyll in-sample, unit is milligram every 100 grams (mg/100g)
The cumulative volume of V-extract, unit is milliliter (ml);
The extension rate of F-sample;
The quality of m-sample, unit is gram (g);
Measurement result is made even the arithmetical mean of row test findings, to retain after radix point first.
Parallel experiment permissible error (relatively) is not more than 3%.
A. the testing result of willow (bud) glue sees the following form 1: table
1
Note: willow (bud) glue is purification glue, all dissolves in ethanol and acetone solution, without residue thing, so need not measure and calculate the content of extract.
As can be seen from above testing result, in the Chlorophyll content of 6 yang gums, mxm. 163.5 mg/100g, minimum 74.7 mg/100g, average content is 111.2mg/100g, and parallel experiment metrical error is all less than 1.6%.
B. the testing result of yang gum preparation (capsule) sees the following form 2: table
2
Note: contain pure yang gum content in yang gum preparation between 40 ~ 60%.
As can be seen from above testing result, in the Chlorophyll content of 3 willow (bud) glue preparations, mxm. 77.05 mg/100g, minimum 64.02 mg/100g, average content is 68.9mg/100g, and parallel experiment metrical error is all less than 1.5%.
C. the testing result of propolis sees the following form 3: table
3
Note: extract content is the ethanol soluble extraction content in mao propolis, chlorophyll content is the Chlorophyll content in mao propolis.
As can be seen from above testing result, in the content of 12 propolis raw material Chlorophylls, mxm. 11.47mg/100g, minimum 3.61 mg/100g, average content is 6.73mg/100g, and parallel experiment metrical error is all less than 1.62%.
D. being chosen at chlorophyll content is mix willow (bud) glue that the different proportion chlorophyll content such as 5%, 10% is 163.5mg/100g in the bee glue purifying sample of 17.35 mg/100g, find that chlorophyll content obviously increases along with increasing of yang gum incorporation, direct ratio linear relationship is there is between incorporation and chlorophyll recruitment, its regression equation is: Y=17.385+9.3004X, the coefficient of variation, close to 1, illustrates that the method has higher detection sensitivity; Average recovery mean value reaches 93.8%, and the recovery is higher, illustrates that the method has certain accuracy.
E. the mean value of propolis and false propolis chlorophyll content has separately been solved out in test, and according to propolis Determination of Chlorophyll content mxm. 11.47mg/100g, average content is 6.73mg/100g; Yang gum chlorophyll content minimum 74.7 mg/100g, average content is 111.2mg/100g and yang gum preparation chlorophyll content minimum 64.02 mg/100g, average content is the test result of 68.9mg/100g, on the comprehensive basis analyzed, setting propolis chlorophyll content safety limit is 15mg/100g, detects chlorophyll content can accurately differentiate propolis and yang gum by contrast.
F. find out from testing result, true propolis detects the whole < 15mg/100g of sample chlorophyll content value; False propolis and preparation thereof detect the whole > 15mg/100g of sample chlorophyll content value.The result of application of sample recovery test, confirms the deduction that chlorophyll content value higher yang gum incorporation is larger.
Embodiment 2
Method And Principle, reagent, instrument and equipment lead to embodiment 1.
1, analytical procedure
1.1, the process of preparing Chinese medicine of propolis and extraction: get a mao propolis sample 5g, first freezing 48h at-10 DEG C, then smashs to pieces accurately weighed; Be placed in glass mortar, add calcium carbonate 5g and silica sand 5g, slowly grind to form impalpable powder, add ethanol 10ml and continue to grind to form pasty state; Abrasive material is all transferred in 250ml tool plug conical flask, closely colourless with ethanol washing mortar, make the cumulative volume of ethanol to 100ml, close plug, cold soaking, jolting constantly in front 10h, then leave standstill 48h, all use G
3glass funnel suction filtration, drains with in aqueous acetone solution (85%) cyclic washing conical flask to funnel; Refunded in mortar by residue in funnel and grind 5min with aqueous acetone solution, transfer in funnel, suction filtration, continue with the residue in aqueous acetone solution cyclic washing mortar and funnel, closely colourless to filtrate, the cumulative volume using aqueous acetone solution is 100ml.Accurate absorption suction filtration liquid 50ml, is placed in the evaporating dish of dry constant weight, in water-bath after evaporate to dryness, at 9 DEG C of dry 3h, puts in exsiccator and cool 30min to room temperature, rapid accurately weighed weight, calculate the content (%) of extract in mao propolis with dry product.
1.2, the dissolving of sample: get above-mentioned dry propolis extract and be about 0.2g, accurately weighed, put in 50ml beaker, add aqueous acetone solution (85%) 50ml and test sample is all dissolved, obtain 50ml sample regulator solution, for subsequent use.
1.3, isolation of purified: said sample lysate is all proceeded in the 250ml separating funnel of interior Sheng 50mL 2% metabisulfite solution, with 20ml ether gradation washing beaker, be incorporated in separating funnel, jolting 1min, make layering, water layer puts into another separating funnel, adds 20ml extracted with diethyl ether, discards water layer; Organic layer is incorporated in first separating funnel, washes twice with water, and each 20ml, discards water layer.Ether layer is filtered in the brown volumetric flask of 50ml by the little funnel that is equipped with 5g anhydrous sodium sulfate, uses washed with diethylether separating funnel, and wash away the pigment in anhydrous sodium sulfate layer, constant volume, shake up, stand-by.
1.4, measure: get in the brown volumetric flask of above-mentioned solution 20ml to 50ml, use ether constant volume, shake up, on spectrophotometer, measure the light absorption value at 642nm and 660nm place immediately, 1 ㎝ cuvette, do with ether blank.
1.5 results calculate and contrast with embodiment 1.
Claims (6)
1. detect the method that chlorophyll content differentiates propolis and yang gum, with the chlorophyll in acetone extraction sample, with ether layering extraction, metabisulfite solution washing, purifying, colorimetric assay under two place's wavelength, differentiates; Concrete technology step is as follows:
A, analytical procedure
A.1, the process of preparing Chinese medicine of propolis and extraction: get a mao propolis sample, first at-5 DEG C ~-10 DEG C freezing 24 ~ 48 hours, then smash to pieces accurately weighed; Be placed in glass mortar, add and the calcium carbonate of sample equivalent and silica sand
,grinding, the ethanol adding suitable double sample amount continues to grind to form pasty state; Abrasive material is all transferred in tool plug conical flask, with ethanol washing mortar to colourless, makes amount of alcohol reach 20 times of sample size, close plug, cold soaking, jolting mixing, leave standstill 24 ~ 48 hours, use G
3glass funnel suction filtration, drains with aqueous acetone solution washing; Refunded in mortar by residue in funnel and add aqueous acetone solution grinding, transfer in funnel, suction filtration, the residue of continuation in aqueous acetone solution washing mortar and funnel, colourless to filtrate, use aqueous acetone solution amount reaches 20 times of sample size; 1/4th of the total suction filtration liquid of accurate absorption, be placed in the evaporating dish of dry constant weight, in water-bath after evaporate to dryness, drying 3 hours at 90 DEG C ~ 110 DEG C temperature is also cooled to room temperature, rapid accurately weighed weight, calculates the content (%) of extract in mao propolis with dry product;
A.2, the dissolving of sample: get above-mentioned dry propolis extract, accurately weighed, put in beaker, add aqueous acetone solution and test sample is all dissolved, for subsequent use;
A.3, isolation of purified: said sample lysate is all proceeded in the separating funnel of interior Sheng metabisulfite solution, with ether gradation washing beaker, be incorporated in separating funnel, jolting, makes layering, and water layer puts into another separating funnel, add the extracted with diethyl ether of sample lysate 2/5ths, discard water layer; Organic layer is incorporated in first separating funnel, washes with water, discards water layer; Ether layer is filtered in brown volumetric flask by the little funnel of the anhydrous sodium sulfate that 1/4th ether amounts are housed, with the washed with diethylether separating funnel of sample lysate 3/5ths, and washes away the pigment in anhydrous sodium sulfate layer, constant volume, shake up, stand-by;
A.4, measure: get above-mentioned solution in brown volumetric flask, use ether constant volume, shake up, on spectrophotometer, measure the light absorption value at 642nm and 660nm wavelength place immediately, do with ether blank;
A.5 the content of sample Determination of Chlorophyll is calculated.
2. the discrimination method of a kind of propolis according to claim 1 and yang gum, is characterized in that: described reagent is chemical pure or analyzes pure, and water is distilled water.
3. the discrimination method of a kind of propolis according to claim 1 and 2 and yang gum, is characterized in that described reagent comprises: ethanol, calcium carbonate, silica sand: the one in SILVER REAGENT, acetone, anhydrous sodium sulfate, ether, aqueous acetone solution and aqueous sodium persulfate solution.
4. the discrimination method of a kind of propolis according to claim 3 and yang gum, is characterized in that described reagent anhydrous sodium sulfate is the anhydrous sodium sulfate at 650 DEG C of calcination 4h.
5. the discrimination method of a kind of propolis according to claim 3 and yang gum, is characterized in that the mass volume ratio of described reagent acetone aqueous solution is 85+15, V/V.
6. the discrimination method of a kind of propolis according to claim 3 and yang gum, is characterized in that described reagent of sulfuric acid sodium water solution is 2%, m/V.
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| CN1765214A (en) * | 2005-11-04 | 2006-05-03 | 余内逊 | Method for preparing blood sugar-decreasing, stomach-benefiting, health-preserving soybean yoghurt with micron holoside, ginseng, solomonseal, chlorophyll, propolis and chromium powder |
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| CN1765214A (en) * | 2005-11-04 | 2006-05-03 | 余内逊 | Method for preparing blood sugar-decreasing, stomach-benefiting, health-preserving soybean yoghurt with micron holoside, ginseng, solomonseal, chlorophyll, propolis and chromium powder |
Non-Patent Citations (3)
| Title |
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| 《蜂胶》;中华人民共和国国家质量监督检验检疫总局;《中华人民共和国国家标准》;20091231;正文第1-7页 * |
| 《蜂胶与杨树叶和杨树芽中叶绿素含量的比较研究》;周立东等;《中国蜂业》;20071231;第58卷(第6期);第5-7页 * |
| 《进出口螺旋藻粉中藻蓝蛋白、叶绿素含量的测定方法》;中华人民共和国国家质量监督检验检疫总局;《检验检疫行业标准》;20020520;正文第2-3页 * |
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