CN105806793B - A kind of simple and effective mulberry leaf polysaccharide detection method - Google Patents
A kind of simple and effective mulberry leaf polysaccharide detection method Download PDFInfo
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Abstract
The invention discloses a kind of simple and effective mulberry leaf polysaccharide detection methods, mulberry leaf water-soluble sugar and mulberry leaf total reducing sugar are specifically first extracted respectively, then hydrolysis is distinguished into reducing sugar, utilize 3, the light absorption value of 5- dinitrosalicylic acid (DNS) colorimetric method for determining mulberry leaf water-soluble sugar extracting solution and mulberry leaf total reducing sugar extracting solution at 540nm, the quality that mulberry leaf water-soluble sugar extracting solution and mulberry leaf total reducing sugar extracting solution in measurement liquid are equivalent to glucose is calculated separately according to glucose standard curve, the content of mulberry leaf water-soluble sugar and mulberry leaf total reducing sugar (in terms of reduced sugar) is calculated again, and mulberry leaf polysaccharide content is calculated by mulberry leaf polysaccharide (%)=[mulberry leaf total reducing sugar (%)-mulberry leaf water-soluble sugar (%)] × 0.9；Present method avoids meeting the problem that water very exothermic keeps color condition uncontrollable because of sulfuric acid, while continuous mode is by the interference of monosaccharide component, and easy to operate, safety, as a result stable, reliable, is suitble to a large amount of mulberry leaf sample polyoses contents of detection.
Description
Technical field
The invention belongs to detection fields, and in particular to a kind of simple and effective mulberry leaf polysaccharide detection method.
Background technique
Mulberry leaf are the leaf of Moraceae (Moraceae) Morus (Morus L.) mulberry kind (Morus alba L.) plant, contain egg
The several functions compositions such as white matter, inorganic salts, vitamin, polysaccharide, polyphenol, flavones, alkaloid, the Ministry of Public Health are classified as medicine food two
With type Chinese herbal medicine.
Modern study pharmacology shows that mulberry leaf polysaccharide as one of mulberry leaf active constituent, has significant hypoglycemic effect.Mesh
Before, mulberry leaf polysaccharide detection method of content is mainly Anthrone-sulfuricacid method and phend-sulphuric acid.Anthrone-sulfuricacid method can almost measure
All carbohydrate, phend-sulphuric acid are mainly used for the measurement of the sugar, pentose and polysaccharide of methylation.With both sides
When method measures mulberry leaf polysaccharide, what is actually get is total sugar content, therefore accurately to detect mulberry leaf polysaccharide content and must first separate
Purifying obtains the higher mulberry leaf polysaccharide of purity, or obtained total reducing sugar result is subtracted reduced sugar result (reduced sugar result need to lead to
It crosses other methods to be measured).But it isolates and purifies and obtains the higher mulberry leaf polysaccharide complex steps of purity, poor reproducibility；And anthracene
Ketone-sulfuric acid process measurement total reducing sugar result can also be influenced by the higher protein of tryptophane, and stability is poor；Phenolsulfuric acid
Method experiment condition requires stringent (requirement of such as Pyrogentisinic Acid is very high), is limited in continuous mode by a variety of conditions, reproducibility is poor.
Therefore, existing detection method cannot simply, accurately and efficiently reflect the content of polysaccharide in mulberry leaf.Therefore, which is badly in need of grinding
Send out a kind of simple, accurate, quickly detection mulberry leaf polysaccharide method.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of detection method of mulberry leaf polysaccharide, easy to operate, safety, knot
Fruit is stable, reliable, is suitble to a large amount of mulberry leaf sample polyoses contents of detection.
For achieving the above object, the present invention utilizes carbohydrate monosaccharide, oligosaccharide and polysaccharide three categories, in certain condition
Under, oligosaccharide and polysaccharide can be hydrolyzed to the monosaccharide with reproducibility.And DNS reagent (3,5- dinitrosalicylic acid solution) with
The amino-compound (3- amino -5-NITROSALICYLIC ACID) of brownish red is reduced into after reduction sugar juice heat together, which exists
There is absorption maximum at 540nm, and in a certain range, the amount and light absorption value of reduced sugar are in a linear relationship.Therefore the present invention first with
The content of total reducing sugar and water-soluble sugar in DNS colorimetric method for determining mulberry leaf recycles formula: mulberry leaf polysaccharide (%)=[mulberry leaf total reducing sugar
(%)-mulberry leaf water-soluble sugar (%)] × 0.9, the content of mulberry leaf polysaccharide is calculated.For this purpose, providing the following technical solutions:
A kind of detection method of simple and effective mulberry leaf polysaccharide extracts mulberry leaf water-soluble sugar and mulberry leaf total reducing sugar, then water respectively
Solution is converted to reducing sugar, obtains mulberry leaf water-soluble sugar extracting solution and mulberry leaf total reducing sugar extracting solution respectively, recycles 3,5- dinitro water
Poplar acid colorimetric method measures the light absorption value of mulberry leaf water-soluble sugar extracting solution and mulberry leaf total reducing sugar extracting solution at 540nm, then according to Portugal
Grape Standard for Sugars curve calculates separately mulberry leaf water-soluble sugar extracting solution and mulberry leaf total reducing sugar extracting solution in measurement liquid and is equivalent to glucose
Quality, then the content of mulberry leaf water-soluble sugar and mulberry leaf total reducing sugar is calculated as follows:
In formula: m2--- it looks into water-soluble sugar in measurement liquid obtained by curve and is equivalent to glucose quality, mg；
m3--- it looks into total reducing sugar in measurement liquid obtained by curve and is equivalent to glucose quality, mg；
V0--- measurement liquid product, mL；
V2--- water-soluble sugar extracting liquid volume, mL；
V3--- total reducing sugar extracting liquid volume, mL；
N2--- water-soluble sugar extracting solution extension rate；
N3--- total reducing sugar extracting solution extension rate；
M --- mulberry leaf sample quality, g.
And mulberry leaf polysaccharide is calculated according to following formula:
Mulberry leaf polysaccharide (%)=[mulberry leaf total reducing sugar (%)-mulberry leaf water-soluble sugar (%)] × 0.9.
In above-mentioned formula, total reducing sugar refers to the summation (except starch, cellulose etc.) of monosaccharide in mulberry leaf, oligosaccharide and polysaccharide,
Water-soluble sugar refers to the summation of monosaccharide in mulberry leaf, oligosaccharide, and total reducing sugar and water-soluble sugar are using hydrolysis as the content of reduced sugar
It is calculated, 0.9 is the coefficient that reduced sugar (with glucose meter) is scaled mulberry leaf polysaccharide.
In the present invention, the preparation method of 3, the 5- dinitrosalicylic acid solution is as follows: preparing contain sodium potassium tartrate tetrahydrate first
Temperature be 40~45 DEG C of aqueous solution, NaOH solution is then added, sequentially adds 3,5- dinitrosalicylic acid, phenol and Asia
Sodium bisulfate, last constant volume make the final concentration of 185g/L of sodium potassium tartrate tetrahydrate, the final concentration of 0.524mol/L of NaOH, 3,5- dinitros
The final concentration of 6.3g/L of salicylic acid, the final concentration of 5.0g/L of phenol and the final concentration of 5.0g/L of sodium hydrogensulfite.
In the present invention, the extracting method of the mulberry leaf water-soluble sugar extracting solution is as follows: taking new fresh mulberry leaf, drying to constant weight, powder
It is broken, distilled water water-bath is then added and extracts, is denoted as extracting solution A, sequentially adds acetic acid zinc solution and potassium ferrocyanide solution, from
The heart collects supernatant, obtains mulberry leaf reduced sugar extracting solution, and hydrochloric acid is then added into mulberry leaf reduced sugar extracting solution and is hydrolyzed, adjusts
PH to 7.5-8.0 obtains mulberry leaf water-soluble sugar extracting solution.Above-mentioned centrifugation is not less than 6000rpm in principle, is centrifuged 10min, twice from
The supernatant collected after the heart is often no less than 60mL, and a part is used for the measurement of reduced sugar, and another part turns for sequential hydrolysis
Change, prepares water-soluble sugar.
The extracting solution A's the preparation method is as follows: take new fresh mulberry leaf temperature be 50 DEG C drying to constant weight, crush, then
60 times of distilled water for being equivalent to mulberry leaf weight is added, 50 DEG C of water-baths extract 20min water-bath, obtain extracting solution A.
The additional amount of hydrochloric acid described in above-mentioned hydrolysis is 3:50 by concentrated hydrochloric acid and mulberry leaf reduced sugar extracting liquid volume ratio；It is described
Hydrolysis is to hydrolyze 15min under 70 DEG C of water-baths.
In the present invention, the extracting method of the mulberry leaf total reducing sugar extracting solution is as follows: taking new fresh mulberry leaf, drying to constant weight, crushes,
Then distilled water is added sufficiently to dissolve, after making total reducing sugar complete hydrolysis with hydrochloric acid, sequentially adds acetic acid zinc solution and potassium ferrocyanide
Solution, adjusts pH to 7.5-8.0, and filtering collects filtrate, obtains mulberry leaf total reducing sugar extracting solution.It is sufficiently molten that distilled water is added in the present invention
When solving mulberry leaf powder, mulberry leaf powder is first tuned into paste with a small amount of water, the distilled water of more amount is added, mixes, the purpose is to make mulberry
Leaf powder is fully dispersed in water to be opened, and the extraction of mulberry leaf total reducing sugar and mulberry leaf water-soluble sugar is conducive to.
The hydrochloric acid additional amount presses mulberry leaf and concentrated hydrochloric acid w/v is 0.5:6 (g/mL), described to be hydrolyzed in boiling water
Bath is lower to hydrolyze 30min, and the most handy single layer nylon cloth of filtering filters, mainly due to containing more glutinous polysaccharide in the extracting solution,
It is extremely complex with filter paper filtering, while excluding interference of the filter paper constituent (carbohydrate) to reducing sugar test.
The condition of the drying is 50 DEG C；The weight that water is added is equivalent to 60 times of mulberry leaf weight；The zinc acetate
Acetic acid zinc concentration is 219g/L in solution；The concentration of potassium ferrocyanide is 106g/L in the potassium ferrocyanide solution.It is added
Acetic acid zinc solution and potassium ferrocyanide solution are added preferably along capacity bottle wall, can reduce the generation of bubble in this way, convenient fixed
Hold.
Most preferably, the regression equation of the glucose standard curve is y=1.5175x-0.0578 or y=1.5802x-
0.063。
During said extracted, the most handy 50mL volumetric flask of reduced sugar extracting solution is measured, and main purpose is that accurate control is gone back
The volume that raw sugar extracting solution is converted reduces experimental error.
Signified concentrated hydrochloric acid refers to that mass fraction is more than 37% hydrochloric acid in the present invention, can will be low under concentrated hydrochloric acid effect
Glycan, polysaccharide are hydrolyzed to the monosaccharide (because total reducing sugar, water-soluble sugar, polysaccharide are calculated with reduced sugar) with reproducibility, and
And the additional amount of hydrochloric acid, bath temperature, water bath time be total reducing sugar, water-soluble sugar hydrolysis be reductive monosaccharide key point,
The condition of hydrolysis is excessively high, too low can all cause last measured value inaccuracy.
The beneficial effects of the present invention are: the present invention containing using total reducing sugar in DNS colorimetric method for determining mulberry leaf and water-soluble sugar
Amount, recycle formula: mulberry is calculated in mulberry leaf polysaccharide (%)=[mulberry leaf total reducing sugar (%)-mulberry leaf water-soluble sugar (%)] × 0.9
The content of leaf polyose；Relative to traditional Anthrone-sulfuricacid method and phend-sulphuric acid, DNS colorimetric method is avoided because sulfuric acid meets water play
The problem that strong heat release keeps color condition uncontrollable, while continuous mode, not by the interference of monosaccharide component, measurement result is closer
True value, and total reducing sugar, water-soluble sugar extraction process are simple, it is easy to operate, therefore the present invention is suitble to a large amount of mulberry leaf sample polyoses contents
Detection, and then filter out the high advantage mulberry leaf kind of polyoses content.
Detailed description of the invention
In order to keep the purpose of the present invention, technical scheme and beneficial effects clearer, the present invention provides following attached drawing:
Fig. 1 is 1 glucose standard curve of embodiment.
Fig. 2 is 2 glucose standard curve of embodiment.
Specific embodiment
Below in conjunction with attached drawing, a preferred embodiment of the present invention will be described in detail.It is not specified in embodiment specific
The experimental method of condition, usually according to conventional conditions or according to the manufacturer's recommendations.
Experimental material is fresh mulberry leaf in the present invention, is taken from Southwest University mulberry field, and drying to constant weight at 50 DEG C, powder
It is broken, it sieves with 100 mesh sieve, is saved backup under -20 DEG C of low temperature environments.
The reagent used is as follows:
Acetic acid zinc solution is prepared: being weighed 21.9g zinc acetate, is added 3mL glacial acetic acid, is dissolved in water and is diluted to 100mL, be made
Acetic acid zinc solution, final concentration of 219g/L.
Potassium ferrocyanide solution is prepared: being weighed 10.6g potassium ferrocyanide, is dissolved in water and is diluted to 100mL, ferrous iron is made
Potassium cyanide solution, final concentration of 106g/L.
Wagner's reagent is prepared: being weighed 5g iodine, 10g potassium iodide, is dissolved in 100mL distilled water, iodine-potassium iodide is made
Solution.
DNS preparation of reagents: the hydrothermal solution of 500mL sodium potassium tartrate tetrahydrate containing 185g is prepared, it is dense to sequentially add 262mL thereto
Degree is the NaOH solution of 2mol/L, 6.3g 3,5- dinitrosalicylic acid, 5.0g phenol and 5.0g sodium hydrogensulfite, stirring and dissolving,
It is transferred to after cooling in 1000mL volumetric flask, is settled to scale with distilled water, be stored in spare in brown bottle, which is
DNS reagent uses after placing 7-10 days under room temperature (18~25 DEG C).
Embodiment 1
A kind of detection method of mulberry leaf polysaccharide, comprising the following steps:
(1) extraction of mulberry leaf reduced sugar and water-soluble sugar
A, mulberry leaf powder 1.00g is weighed in 250mL conical flask, and 60mL distilled water is added (first with a small amount of water by mulberry leaf powder tune
At paste), stirring, 50 DEG C of water-bath 20min are denoted as extracting solution A；
B, extracting solution A obtained by step a (containing residue) is successively transferred in 100mL volumetric flask, with distilled water flushing taper
Bottle 2-3 times, flushing liquor is transferred in volumetric flask together, then the acetic acid that 3mL concentration is 219g/L is sequentially added into volumetric flask
Zinc solution and 3mL concentration are the potassium ferrocyanide solution of 106g/L, and with distilled water constant volume to 100mL, are denoted as extracting solution B；
C, the extracting solution B obtained by single layer nylon cloth filtration step b, filtrate are collected into 50mL centrifuge tube (in view of extracting
Liquid product and subsequent dose, the extracting solution in each volumetric flask need to be collected with 2 50mL centrifuge tubes)；Then in
It is centrifuged 10min under the conditions of 10000rpm, collects supernatant；Supernatant is centrifuged 10min under the conditions of 10000rpm again, in collection
Clear liquid, the supernatant are mulberry leaf reduced sugar extracting solution；
D, mulberry leaf reduced sugar extracting solution obtained by 50mL step c is measured with 50mL volumetric flask, is then transferred to 100mL volumetric flask
In, 3mL concentrated hydrochloric acid is added into volumetric flask, shakes up, is cooled down rapidly after 70 DEG C of water-bath 15min with circulating water, be subsequently poured into 250mL
In conical flask, pH to 7.5-8.0 is adjusted, in the 100mL volumetric flask transferred to, with distilled water flushing conical flask 2-3 times, will be rushed
Washing lotion is transferred to together in corresponding volumetric flask, and constant volume is then transferred in 50mL centrifuge tube to 100mL, is denoted as mulberry leaf water
Dissolubility sugar prepare liquid is stored in spare in 4 DEG C of refrigerators；Remaining mulberry leaf reduced sugar extracting solution is stored in 4 DEG C of refrigerators, for subsequent
Mulberry leaf content of reducing sugar is measured, mulberry leaf reduced sugar prepare liquid is denoted as；
Three parts of each sample parallel extraction according to the method described above, obtains three parts of reduced sugar prepare liquids and three parts of water-soluble sugars wait for
Liquid is surveyed, last measurement result takes its arithmetic mean of instantaneous value.
(2) extraction of mulberry leaf total reducing sugar
A, mulberry leaf powder 0.50g is weighed in 150mL triangular flask, and 20mL distilled water is added (first with a small amount of water by mulberry leaf
Powder is tuned into paste), it stirs evenly；
B, 6mL concentrated hydrochloric acid, boiling water bath 30min hydrolysis are added in Xiang Shangshu triangular flask；
C, 1-2 drop hydrolyzate on white plaque, add 1 drop Wagner's reagent check total reducing sugar whether hydrolyze completely, if not being in
Existing blue then proves to have hydrolyzed completely；
D, 30mL distilled water is added in Xiang Shangshu triangular flask, it is molten that the zinc acetate that 2mL concentration is 219g/L is added after shaking up
Liquid and 2mL concentration are 106g/L potassium ferrocyanide solution, adjust pH to 7.5-8.0 with pH meter；
E, the liquid (containing residue) in triangular flask is transferred in 100mL volumetric flask, with distilled water flushing triangular flask 2-3
It is secondary, flushing liquor is transferred to together in above-mentioned volumetric flask, and constant volume is to 100mL, with single layer nylon cloth by the extraction in volumetric flask
Liquid is filled into 50mL centrifuge tube, is stored in spare in 4 DEG C of refrigerators, which is mulberry leaf total reducing sugar extracting solution, is denoted as mulberry leaf
Total reducing sugar prepare liquid.
Three parts of each sample parallel extraction, obtains three parts of total reducing sugar prepare liquids, and last measurement result takes its arithmetic mean of instantaneous value.
(3) DNS colorimetric method for determining mulberry leaf reduced sugar, water-soluble sugar, total sugar content
A, take respectively mulberry leaf reduced sugar prepare liquid, water-soluble sugar prepare liquid obtained by step (1) and (2), total reducing sugar prepare liquid in
In 10mL centrifuge tube, 10000rpm is centrifuged 10min；
B, it takes each prepare liquid 0.4mL in 10mL colorimetric cylinder respectively, 0.4mL distilled water and 0.6mL DNS reagent is added, shakes
Even, boiling water bath 5min is cooled to room temperature (18~25 DEG C) with cold water immediately after taking-up, then distilled water is added to scale to every pipe
(10mL), shakes up, and replaces prepare liquid to do blank control by same procedure operation with 0.4mL distilled water, measures at 540nm wavelength
Each pipe absorbance value；Each sample prepare liquid is measured in parallel twice, is averaged.
C, glucose standard curve is drawn, first preparation glucose standards solution: it is (pre- accurately weighs glucose 103.6mg
First dry to constant weight at 80 DEG C), after adding a small amount of distilled water to dissolve, it is transferred in 100mL volumetric flask, is settled to 100mL with distilled water,
Spare in 4 DEG C of refrigerators, concentration 1.036mg/mL is stored in after mixing；Then 9 10mL colorimetric cylinders are taken, are separately added into
0.00mL, 0.08mL, 0.16mL, 0.24mL, 0.32mL, 0.40mL, 0.48mL, 0.56mL, 0.64mL glucose solution (are added
0.00mL glucose solution does blank control), it is diluted with water 0.80mL, 0.60mL DNS reagent is added, shakes up, boiling water bath
5min is cooled to room temperature with cold water immediately after taking-up, then distilled water is added to scale (10mL) to every pipe, shakes up, in 540nm wave
Strong point measures each pipe absorbance value.With glucose content (mg) for abscissa, absorbance value is ordinate, draws standard curve,
Obtaining regression equation is y=1.5175x-0.0578, R2=0.9989 (such as Fig. 1).
D, it brings sample absorbance value into standard curve y=1.5175x-0.0578, obtains each measurement liquid and be equivalent to grape
The milligram number of sugar, can calculate containing for mulberry leaf reduced sugar, mulberry leaf water-soluble sugar, mulberry leaf total reducing sugar and mulberry leaf polysaccharide further according to formula
Amount.
Reduced sugar, water-soluble sugar, total reducing sugar and polyoses content are calculated as follows in mulberry leaf:
Mulberry leaf polysaccharide (%)=[mulberry leaf total reducing sugar (%)-mulberry leaf water-soluble sugar (%)] × 0.9
In formula: m1--- it looks into reduced sugar in measurement liquid obtained by curve and is equivalent to glucose in milligrams number, mg；
m2--- it looks into water-soluble sugar in measurement liquid obtained by curve and wants when in glucose in milligrams number, mg；
m3--- it looks into total reducing sugar in measurement liquid obtained by curve and is equivalent to glucose in milligrams number, mg；
V0--- measurement liquid product, V0=0.4mL；
V1--- reduced sugar extracting liquid volume, V1=100mL；
V2--- water-soluble sugar extracting liquid volume, V2=100mL；
V3--- total reducing sugar extracting liquid volume, V3=100mL；
N1--- reduced sugar extracting solution extension rate, N1=1；
N2--- water-soluble sugar extracting solution extension rate, N2=2；
N3--- total reducing sugar extracting solution extension rate, N3=1；
M --- mulberry leaf sample quality, g.
Mulberry leaf reduced sugar, water-soluble sugar, total reducing sugar is calculated, and the results are shown in Table 1:
Mulberry leaf reduced sugar, water-soluble sugar, total reducing sugar measurement result in table 1, embodiment 1
Note: three groups of each sample parallel extraction, twice, the arithmetic mean of instantaneous value measured twice is flat in group for every group of parallel determination
Mean value, the arithmetic mean of instantaneous value of average value average value between group in three groups of groups, similarly hereinafter.
According to the measurement result in table 1, can in the hope of embodiment 1 in mulberry leaf polysaccharide content are as follows:
Mulberry leaf polysaccharide (%)=(24.12%-13.19%) × 0.9=9.84%
Embodiment 2
Related reagent preparation, mulberry leaf reduced sugar, water-soluble sugar, total reducing sugar extracting method with embodiment 1.
Glucose standards solution is prepared: glucose standards solution is prepared according to the method for embodiment 1, it is final concentration of
1.056mg/mL。
Glucose standard curve production: take 11 10mL colorimetric cylinders, be separately added into 0.00mL, 0.08mL, 0.16mL,
(0.00mL is added in 0.24mL, 0.32mL, 0.40mL, 0.48mL, 0.56mL, 0.64mL, 0.72mL, 0.80mL glucose solution
Glucose solution does blank control), it is diluted with water 0.80mL, 0.60mL DNS reagent is added, shakes up, boiling water bath 5min,
It is cooled to room temperature with cold water immediately after taking-up, then distilled water is added to scale (10mL) to every pipe, is shaken up, at 540nm wavelength
Measure each pipe absorbance value.With glucose content (mg) for abscissa, absorbance value is ordinate, draws standard curve, must return
Returning equation is y=1.5802x-0.0631, R2=0.9997 (such as Fig. 2).
The measurement and calculating of sugar content are the same as embodiment 1 in sample.
Mulberry leaf reduced sugar, water-soluble sugar, total reducing sugar measurement result are shown in Table 2 in embodiment 2:
Mulberry leaf reduced sugar, water-soluble sugar, total reducing sugar measurement result in 2 embodiment 2 of table
According to the measurement result in table 2, can in the hope of embodiment 2 in mulberry leaf polysaccharide content are as follows:
Mulberry leaf polysaccharide (%)=(34.56%-17.24%) × 0.9=15.59%
Embodiment 1 and embodiment 2 the result shows that: utilize DNS colorimetric method for determining mulberry leaf reduced sugar, water-soluble sugar and total reducing sugar,
Collimation is good, and the content of mulberry leaf polysaccharide can be found out by calculating.
Finally, it is stated that preferred embodiment above is only used to illustrate the technical scheme of the present invention and not to limit it, although logical
It crosses above preferred embodiment the present invention is described in detail, however, those skilled in the art should understand that, can be
Various changes are made to it in form and in details, without departing from claims of the present invention limited range.
Claims (7)
1. a kind of detection method of simple and effective mulberry leaf polysaccharide, it is characterised in that: prepare respectively mulberry leaf water-soluble sugar extracting solution and
Mulberry leaf total reducing sugar extracting solution recycles 3,5- dinitrosalicylic Acid Colorimetry measurement mulberry leaf water-soluble sugar extracting solution and mulberry leaf total reducing sugar to mention
Light absorption value of the liquid at 540 nm is taken, mulberry leaf water-soluble sugar in measurement liquid is calculated separately then according to glucose standard curve and extracts
Liquid and mulberry leaf total reducing sugar extracting solution are equivalent to the weight of glucose, and containing for mulberry leaf water-soluble sugar and mulberry leaf total reducing sugar is calculated as follows
Amount:
With glucose meter mulberry leaf water-soluble sugar %=
With glucose meter mulberry leaf total reducing sugar %=
In formula: m 2--- it looks into water-soluble sugar in measurement liquid obtained by curve and is equivalent to glucose quality, mg；
m 3--- it looks into total reducing sugar in measurement liquid obtained by curve and is equivalent to glucose quality, mg；
V 0--- measurement liquid product, mL；
V 2--- water-soluble sugar extracting liquid volume, mL；
V 3--- total reducing sugar extracting liquid volume, mL；
N 2--- water-soluble sugar extracting solution extension rate；
N 3--- total reducing sugar extracting solution extension rate；
M --- mulberry leaf sample quality, g；
And mulberry leaf polysaccharide is calculated according to following formula:
Mulberry leaf polysaccharide (%)=[ mulberry leaf total reducing sugar (%)-mulberry leaf water-soluble sugar (%) ] × 0.9；The mulberry leaf water-soluble sugar extracting solution
Extracting method is as follows: taking new fresh mulberry leaf, drying to constant weight, crushes, and distilled water water-bath is then added and extracts, is denoted as extracting solution A, then
Acetic acid zinc solution and potassium ferrocyanide solution are sequentially added, supernatant is collected by centrifugation, mulberry leaf reduced sugar extracting solution is obtained, then to mulberry
Hydrochloric acid is added in leaf reduced sugar extracting solution to be hydrolyzed, adjusts pH to 7.5 ~ 8.0, obtains mulberry leaf water-soluble sugar extracting solution；
The extracting method of the mulberry leaf total reducing sugar extracting solution is as follows: taking new fresh mulberry leaf, drying to constant weight, crushes, distilled water is then added
Sufficiently dissolution, after making total reducing sugar complete hydrolysis with hydrochloric acid, sequentially add acetic acid zinc solution and potassium ferrocyanide solution to zinc acetate and
Potassium ferrocyanide, adjusts pH to 7.5-8.0, and filtering collects filtrate, obtains mulberry leaf total reducing sugar extracting solution.
2. a kind of detection method of simple and effective mulberry leaf polysaccharide according to claim 1, which is characterized in that 3, the 5- dinitro
The salicylic preparation method of base is as follows: temperature of the preparation containing sodium potassium tartrate tetrahydrate is 40 ~ 45 DEG C of aqueous solution first, is then added
NaOH solution, sequentially adds 3,5- dinitrosalicylic acid, phenol and sodium hydrogensulfite, and last constant volume keeps sodium potassium tartrate tetrahydrate dense eventually
Degree is 185 g/L, final concentration of 0.524 mol/L of NaOH, final concentration of 6.3 g/L of 3,5- dinitrosalicylic acids, and phenol is dense eventually
Degree is 5.0 g/L and final concentration of 5.0 g/L of sodium hydrogensulfite.
3. a kind of detection method of simple and effective mulberry leaf polysaccharide according to claim 1, it is characterised in that: the extracting solution A
The preparation method is as follows: take new fresh mulberry leaf temperature be 50 DEG C drying to constant weight, crush, then be added be equivalent to mulberry leaf weight
60 times of distilled water, 50 DEG C of water-baths extract 20 min, obtain extracting solution A.
4. a kind of detection method of simple and effective mulberry leaf polysaccharide according to claim 1, it is characterised in that: the hydrochloric acid adds
Entering amount by concentrated hydrochloric acid and mulberry leaf reduced sugar extracting liquid volume ratio is 3:50；Described be hydrolyzed under 70 DEG C of water-baths hydrolyzes 15 min.
5. a kind of detection method of simple and effective mulberry leaf polysaccharide according to claim 4, it is characterised in that: the hydrochloric acid is added
Amount presses mulberry leaf and concentrated hydrochloric acid w/v as 0.5:6, and unit g:mL, described be hydrolyzed under boiling water bath hydrolyzes 30 min.
6. any one a kind of detection method of simple and effective mulberry leaf polysaccharide according to claim 1 ~ 5, it is characterised in that: described
Acetic acid zinc concentration is 219 g/L in acetic acid zinc solution；The concentration of potassium ferrocyanide is 106 in the potassium ferrocyanide solution
g/L。
7. a kind of detection method of simple and effective mulberry leaf polysaccharide described in claim 1, it is characterised in that: the Glucose standards are bent
The regression equation of line is y=1.5175x-0.0578 or y=1.5802x-0.0631.
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