CN108107010A - A kind of method for detecting lentinan content - Google Patents
A kind of method for detecting lentinan content Download PDFInfo
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Abstract
The present invention relates to analysis detection field, more particularly to a kind of method for detecting lentinan content.Δ A maximum wavelengths are 544nm, 25 DEG C of reactions 30min, pH7 in the detection method.ΔA544(Y) there is good linear relationship with lentinan concentration (X) in the range of 5~80 μ g/mL, regression equation is Y=0.0049X+0.0043 (R2=0.9985).This method has high sensitivity, specificity and accuracy, precision RSD 1.79%, and stability RSD is 0.85%, and mean sample recovery rate 99.10%, LOD is 1.41 μ g/mL.This method can be used for the measure of the content of structural polysaccharide containing triple helix in lentinan, be a kind of easy, quick, efficiently feasible assay method.
Description
Technical field
The present invention relates to analysis detection field, more particularly to a kind of method for detecting lentinan content.
Background technology
Lentinan (Lentinan, LNT) derives from mushroom (Lentinusedodes (Berk.) Sing.), is mushroom
Effective active composition in entity has the function of the effect of significant balanced immune, can be played by activating body immune system
The multiple pharmacological effects such as antitumor, anti-infective, radioresistance and anticoagulation.
The factors such as the biological activity of LNT and its backbone structure, steric configuration are closely related.Research is found only containing three spiral shells
The LNT for revolving structure just has apparent antitumor activity.β-(1,3)-D- Portugals that LNT triple-helix structures are derived from its structure gather
Glycan molecule.Due to the interaction of polyhydroxy in β-(1,3)-D- dextran molecules, fine and close triple-helix structure is commonly formed.According to report
Road is up to 81% for the LNT of main chain with β-(1,3)-D- glucans to the tumor control rate of S180 tumor-bearing mices.
Mainly there are three classes currently for the detection method of triple-helix structure LNT：1) enzyme process.Enzymatic reaction is a kind of important
Polysaccharide structures research means, but the method complex steps, the factors such as the type and ratio of enzyme have testing result larger impact, and
The single-minded enzyme of high activity is expensive；2) albumen specific recognition method, such as horseshoe crab G-factor method.Horseshoe crab G-factor method has quick, sensitive etc.
Advantage, but easily influenced by dextran molecule amount, degree of branching and polysaccharide three-dimensional structure equally exists complex steps, expensive
The shortcomings of, application is limited；3) inorganic chemistry method, such as phend-sulphuric acid.Inorganic chemistry method is cheap, but time-consuming, and to more
Sugared triple-helix structure is without specificity.Therefore, still lack at present for the convenient of triple-helix structure LNT, high sensitivity, economical and practical
Detection method.
The content of the invention
In view of this, the present invention provides a kind of the quick of Congo red spectrophotometry triple-helix structure lentinan
Quantitative analysis method, the method have height specificity and good sensitivity, accuracy to triple-helix structure lentinan, can use
In the quick measure of triple-helix structure lentinan.
In order to realize foregoing invention purpose, the present invention provides following technical scheme：
The present invention provides a kind of methods for detecting lentinan content, include the following steps：
Step 1：Lentinan standard solution is prepared, takes the lentinan standard items of concentration gradient and Congo red solution anti-
Should, through spectrophotometry, corresponding absorbance is measured, obtains standard curve；
Step 2：Lentinan extract sample solution is prepared, takes sample solution and Congo red solution reaction, through being divided light
Degree method detects, and measures corresponding absorbance, corresponds to the standard curve, obtains the concentration of the sample to be tested；
The wavelength detected described in step 1 or step 2 is 400-600nm.
In some specific embodiments of the present invention, the wavelength detected described in step 1 or step 2 is 544nm.
In some specific embodiments of the present invention, the pH value of mixed solution described in step 1 or step 2 is 5-
10。
Preferably, the pH value of mixed solution described in step 1 or step 2 is 7.
In some specific embodiments of the present invention, the temperature reacted described in step 1 or step 2 is 3-60 DEG C.
Preferably, the temperature reacted described in step 1 or step 2 is 25 DEG C.
In some specific embodiments of the present invention, the time reacted described in step 1 or step 2 is 1-60min.
Preferably, the time reacted described in step 1 or step 2 is 30min.
In some specific embodiments of the present invention, the regression equation of standard curve described in step 1 is Y=
0.0049X+0.0043, linear coefficient R2=0.9985.
It is described in the good linear relation extents of the standard curve in some specific embodiments of the present invention
Lentinan surveys concentration as 5~80 μ g/mL.
In some specific embodiments of the present invention, LOD is 1.41 μ g/mL.
In some specific embodiments of the present invention, precision RSD 1.79%.
In some specific embodiments of the present invention, stability RSD is 0.85%.
In some specific embodiments of the present invention, mean sample recovery rate 99.10%.
In some specific embodiments of the present invention, the sample to be tested is lentinan extract.
The present invention provides a kind of methods for detecting lentinan content, include the following steps：Step 1：It is more to prepare mushroom
Saccharide solution takes the lentinan standard items of concentration gradient to be mixed with Congo red solution, through spectrophotometry, measures
Corresponding absorbance obtains standard curve；Step 2：Prepare lentinan extract sample solution, take sample solution with it is Congo red
Solution mixes, and through spectrophotometry, measures corresponding absorbance, the corresponding standard curve obtains the sample to be tested
Concentration；The wavelength detected described in step 1 or step 2 is 400-600nm.
The condition of the quantitative analysis method of Congo red spectrophotometry lentinan triple helix structure is：Detect body
Δ A maximum wavelengths are 544nm, 25 DEG C of reactions 30min, pH7 in system.ΔA544(Y) with lentinan concentration (X) in 5~80 μ g/
There is good linear relationship, regression equation is Y=0.0049X+0.0043 (R in the range of mL2=0.9985).This method has
There are high sensitivity, specificity and accuracy, precision RSD 1.79%, stability RSD is 0.85%, mean sample recycling
Rate is that 99.10%, LOD is 1.41 μ g/mL.This method can be used for triple helix structural polysaccharide content in lentinan extract
It measures, is a kind of easy, quick, efficiently feasible assay method.
Description of the drawings
It in order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
There is attached drawing needed in technology description to be briefly described.
Fig. 1 shows lentinan Congo red solution absorption spectra variation before and after adding in；
Fig. 2 show lentinan add in after absorbance difference with wavelength variation；
Fig. 3 shows influences of the pH to reaction；
The influence of Fig. 4 temperature displaying functions and time to reaction；
Fig. 5 shows Congo red spectrophotometry lentinan standard curve；
Fig. 6 shows Congo red and various concentration lentinan absorption spectrum；
Fig. 7 shows Congo red and various concentration lentinan Δ A measure；
Fig. 8 shows influence of the chaff interferent to reaction；
Fig. 9 shows that the starch in lentinan standard items excludes experiment；
Figure 10 shows that the starch in lentinan extract excludes experiment.
Specific embodiment
The invention discloses a kind of method for detecting lentinan content, those skilled in the art can be used for reference in this paper
Hold, be suitably modified technological parameter realization.In particular, it should be pointed out that all similar substitutions and modifications are to those skilled in the art
For be it will be apparent that they are considered as being included in the present invention.The method of the present invention and application are by preferably implementing
Example is described, related personnel substantially can not depart from present invention, in spirit and scope to method described herein and
Using being modified or suitably change with combining, to realize and using the technology of the present invention.
In order to realize foregoing invention purpose, the present invention adopts the following technical scheme that：
1. detect the preparation of solution：
1) preparation of lentinan standard solution：Accurately weighed appropriate lentinan standard items are dissolved in 0.1-1mol/L
NaOH or KOH solution, with 0.1-1mol/L HCl solution tune pH to neutrality, with phosphate buffer constant volume, Cord blood；
2) preparation of Congo red solution：It is accurately weighed it is appropriate it is Congo red be dissolved in phosphate buffer, it is firm to be configured to 1mg/mL
Arnotto solution；
3) preparation of lentinan extract sample solution：Accurately weighed appropriate lentinan extract is dissolved in 0.1-
1mol/L NaOH or KOH solution, with 0.1-1mol/L HCl solution tune pH to neutrality, with phosphate buffer constant volume, with 1mg/
ML lentinan extract sample solutions, Cord blood.
2. Congo red spectrophotometry condition：
1) Detection wavelength：Experimental group takes Congo red solution to add in lentinan standard solution, and control group is Congo red molten
Liquid carries out 400~600nm wave bands with ultraviolet specrophotometer and scans, and using wavelength as abscissa, Δ A does figure for ordinate, determines
The corresponding absorbing wavelength of maximum Δ A is best detection wavelength；
2) pH is reacted：Congo red solution and lentinan standard solution are taken, it is 5-10 to adjust solution system pH, certain
At a temperature of react certain time, measure Δ A, screening reaction Optimal pH；
3) reaction temperature and time：Congo red solution and lentinan standard solution are taken, is reacted at a temperature of 3-60 DEG C
After 1-60min, Δ A, screening reaction optimum temperature and time are measured.
3. Congo red spectrophotometry methodological study：
1) precision is investigated：Under the testing conditions optimized, continuous 6 measure lentinan standard solution Δ A, meter
RSD is calculated, investigates instrument precision；
2) study on the stability：Under the testing conditions optimized, continuous 7 measure lentinan standard solution in 12h
Δ A calculates RSD, investigates sample stability；
3) it is loaded recovery test：Lentinan extract sample solution is prepared, is separately added into 0-0.5mL lentinan standards
Product solution and Congo red solution under the testing conditions optimized, measure Δ A, calculate lentinan in Shitake Mushroom P.E and contain
Amount, the rate of recovery and RSD evaluate detection method reliability；
4. the foundation of lentinan standard curve and determining for LOD：
1) lentinan standard curve is established：Various concentration lentinan standard solution is prepared, adds in Congo red solution,
Under the testing conditions optimized, absorbance is measured.Using Δ A as ordinate, lentinan solution standard concentration is abscissa,
Establish lentinan standard curve；
2) LOD is determined：Take Congo red solution and pH7 phosphate buffers, abundant mixing, under the testing conditions optimized,
Parallel multiple measure absorbance, LOD is determined according to 3.3 σ/k.
5. Congo red spectrophotometry specificity is investigated：It is poly- that wood is separately added into Congo red-lentinan solution system
Sugar, glucose, sucrose, malt sugar and starch make its final concentration of 25-1250 μ g/mL, under the testing conditions optimized, survey
Determine Δ A, investigate the detection method specificity.
Congo red molecules can generate complex compound with lentinan triple-helix structure, increase electron delocalization degree, maximum is inhaled
It receives wavelength and red shift occurs, and absorbance enhances, Δ A exists linear with the concentration of triple-helix structure lentinan under certain condition
Relation.The present invention is based on above-mentioned principles, establish determining for Congo red Spectrophotometric Determination triple-helix structure lentinan
Analysis method.The method has many advantages, such as that high sensitivity, specificity are strong, the letter available for triple-helix structure lentinan content
Just, fast and efficiently measure.
Raw materials used and reagent can be bought by market in a kind of method for detecting lentinan content provided by the invention.
With reference to embodiment, the present invention is further explained：
Embodiment 1
1. detect the preparation of solution：
1) lentinan standard solution：Lentinan standard items about 10mg is taken, it is accurately weighed, it is dissolved in 4mL 0.5mol/L
NaOH solution with 0.2mol/L HCl solution tune pH to neutrality, is settled to 100mL with 0.2mol/L pH7 phosphate buffers, obtains
To 0.1mg/mL lentinan standard solutions, 4 DEG C of preservations；
2) Congo red solution：Congo red about 10mg is taken, it is accurately weighed, it is settled to 0.2mol/L pH7 phosphate buffers
10mL obtains the Congo red solution of 1mg/mL；
3) lentinan extract sample solution：Take lentinan extract about 50mg, accurately weighed, addition 4mL
0.5mol/L NaOH solutions are adjusted to neutrality with 0.2mol/L HCl solutions, and pH7 phosphate buffers are settled to 50mL, with 1mg/
ML lentinan extract sample solutions, 4 DEG C of preservations.
2. Congo red spectrophotometry condition：
1) Detection wavelength：
A. experimental group：The Congo red solution of 0.1mL and 1.0mL lentinan standard solutions, and mended with pH7 phosphate buffers
Enough to 4mL；Control group：The Congo red solution of 0.1mL, and complement to 4mL with pH7 phosphate buffers.More than reaction system is 25
DEG C reaction 30min, with ultraviolet specrophotometer carries out 400~600nm wave bands scan, using wavelength as abscissa, Δ A be ordinate
Figure is done, determines that the corresponding absorbing wavelengths of maximum Δ A are best detection wavelength；
Table 1
| Group | Detection wavelength (nm) | pH | Temperature (DEG C) | Time (min) | ΔA |
| Test example 1 | 544 | 7 | 25 | 30 | 0.132 |
| Test example 2 | 400 | 7 | 25 | 30 | -0.018 |
| Test example 3 | 600 | 7 | 25 | 30 | 0.017 |
| Comparative example 1 | 350 | 7 | 25 | 30 | 0.039 |
| Comparative example 2 | 680 | 7 | 25 | 30 | -0.064 |
B. as shown in Figure 1 and Figure 2, Congo red molecules can be with the cavity specific bond of triple helix structure lentinan for result
Form complex compound, it is suppressed that carbon-to-carbon singly-bound rotates, and the coplanarity of molecule increases, and the increase of electron delocalization degree makes characteristic absorption
Peak red shift (Fig. 1).Front and rear Δ A is added in lentinan, figure is done to lentinan concentration, it is known that the corresponding wavelength of maximum Δ A is
544nm (Fig. 2), therefore 544nm is selected as best detection wavelength.
Table 2
2) pH is reacted：
A. into the Congo red solution of 0.1mL add in 1.0mL lentinan standard solutions, and with 0.2mol/L NaOH with
It is respectively 5,6,7,8,9,10 that 0.2mol/L HCl, which adjust pH, and 4mL, 25 DEG C of reactions are complemented to the phosphate buffer of corresponding pH
30min measures Δ A at 544nm；
Table 3
| Group | Detection wavelength (nm) | pH | Temperature (DEG C) | Time (min) | ΔA |
| Test example 4 | 544 | 7 | 25 | 30 | 0.128 |
| Test example 5 | 544 | 5 | 25 | 30 | 0.116 |
| Test example 6 | 544 | 10 | 25 | 30 | 0.107 |
| Comparative example 3 | 544 | 3 | 25 | 30 | 0.104 |
| Comparative example 4 | 544 | 11 | 25 | 30 | 0.105 |
Note：Test example 4 is identical with 1 condition of test example, but is respectively independent experiment twice, there are certain systematic error,
Therefore Δ A data have fine error.
B. the results are shown in Figure 3, and in the phosphate buffer solution of pH5~10, when pH is 7, absorbance difference is maximum, therefore
This experimental selection pH7 phosphate buffers carry out subsequent experimental.
3) reaction temperature and time：
A. 1.0mL lentinan standard solutions are added in the Congo red solution of 0.1mL, and is supplied with pH7 phosphate buffers
To 4mL, respectively in 3,25,40,60 DEG C of reactions 1,10,30,60min, Δ A at 544nm is measured；
Table 4
| Group | Detection wavelength (nm) | pH | Temperature (DEG C) | Time (min) | ΔA |
| Test example 7 | 544 | 7 | 25 | 30 | 0.131 |
| Test example 8 | 544 | 7 | 3 | 1 | 0.093 |
| Test example 9 | 544 | 7 | 3 | 60 | 0.095 |
| Test example 10 | 544 | 7 | 60 | 1 | 0.091 |
| Test example 11 | 544 | 7 | 60 | 60 | 0.122 |
Note：Test example 7 is identical with 1 condition of test example, but is respectively independent experiment twice, there are certain systematic error,
Therefore Δ A data have fine error.
B. the results are shown in Figure 4, and 25 DEG C of whens have little influence on Δ A, and at a temperature of other Δ A occur by a relatively large margin up and down
Fluctuation, therefore 25 DEG C are selected as system reaction temperature；It is Congo red to react very fast with triple helix structure lentinan,
Reaction gradually tends towards stability after 10min, until held stationary after 30min, in order to ensure reaction carries out thorough, selection reaction 30min
After carry out absorbance measurement.
2 Congo red spectrophotometry methodological study of embodiment
1) precision is investigated：
A. 1mL lentinan standard solutions are added in the Congo red solution of 0.1mL, and is complemented to pH7 phosphate buffers
Δ A at 4mL, 25 DEG C of reactions 30min, parallel 6 measure 544nm, calculates RSD；
B. the results are shown in Table 5, RSD 1.79%, shows instrument precision height, favorable reproducibility.
5 precision test of table
2) stability test：
A. 1mL lentinan standard solutions are added in the Congo red solution of 0.1mL, and is complemented to pH7 phosphate buffers
4mL, 25 DEG C of reaction 30min measure Δ A at 544nm respectively at 0,2,4,6,8,10,12h, calculate RSD；
B. the results are shown in Table 6, and RSD 0.85% shows that sample stability is good.
6 stability test of table
3) it is loaded recovery test：
A. lentinan standard solution 0,0.2,0.3,0.4 is added in 1mL lentinan extract sample solutions respectively
And the Congo red solution of 0.5mL and 0.1mL, 4mL is complemented to pH7 phosphate buffers, 25 DEG C of reaction 30min are measured at 544nm
Δ A values are substituted into standard curve, calculate content, the rate of recovery and the RSD of lentinan in Shitake Mushroom P.E by Δ A.
The rate of recovery (%)=(A-B)/C × 100%
Note：A- adds in total lentinan measured concentration (μ g/mL) after lentinan extract；B- lentinan extracts
Measured concentration (μ g/mL)；C- adds in the concentration (μ g/mL) of lentinan standard items；
Content (%)=m of lentinan in Shitake Mushroom P.E1V/1000mV1× 100%
Note：m1The quality (μ g) of lentinan in-Shitake Mushroom P.E substitutes into normal equation and is calculated；M- lentinans
Extract quality 50mg；Sample liquid total volume 50mL made of V- mushroom samples；V1The sample of lentinan extract in-detection architecture
Liquid accumulates 1mL.
B. the results are shown in Table 7, and the rate of recovery is between 96.93~101.00%, average recovery rate 99.10%, and RSD is
1.55%.It understands simultaneously, the quality of lentinan is 35.24 μ g in 1mL Shitake Mushroom P.Es, then lentinan in Shitake Mushroom P.E
Content is 3.52%.Sample recovery rate determination experiment is the result shows that this method accuracy of measurement is high.
Table 7 is loaded recovery test
3 lentinan standard curve of embodiment and foundation and LOD's determines
A. the lentinan standard solution of various concentration is prepared by table 8,25 DEG C of reaction 30min are measured at 544nm and inhaled
Luminosity, using Δ A as ordinate, lentinan concentration (μ g/mL) does figure for abscissa, draws standard curve；
8 congo red method of table measures system
B. the results are shown in Figure 5, and in the range of 5~80 μ g/mL of lentinan final concentration, Δ A has good with concentration
Linear relationship (R2=0.9985), regression equation is Y=0.0049X+0.0043 (Fig. 5).Meanwhile lentinan concentration 5~
In the range of 80 μ g/mL, with the increase of concentration, it is more and more apparent (Fig. 6) to absorb Red Shift Phenomena, by 484nm red shifts to 516nm,
Absorbance also gradually rises simultaneously, and maximum absorption wavelength is obtained at 544nm (Fig. 7) according to Δ A, and selection is corresponding at wavelength herein
Δ A can obtain maximum detection sensitivity.
C. the Congo red solution of 0.1mL and 3.9mL pH7 phosphate buffers are taken, is shaken up, 25 DEG C reaction 30min, parallel 20 times
544nm absorbances are measured, LOD is determined according to 3.3 σ/k；
D.20 the absorbance of secondary parallel determination be respectively 0.333,0.334,0.333,0.332,0.334,0.335,
0.335、0.332、0.334、0.332、0.338、0.333、0.335、0.330、0.330、0.332、0.337、0.335、
0.336th, 0.335, standard deviation 0.002, therefore LOD is 1.41 μ g/mL.
4 Congo red spectrophotometry specificity of embodiment is investigated
A. the Congo red solution of 0.1mL is taken, adds in 1.0mL lentinan standard solutions, addition respectively may interfere with object：Wood
Glycan, glucose, sucrose, maltose, starch, it is respectively 25,250,750,1250 μ g/mL to make its final concentration, is delayed with pH7 phosphoric acid
Fliud flushing complements to 4mL, and 25 DEG C of reaction 30min measure Δ A at 544nm；
B. the results are shown in Figure 8, adds xylan, glucose, sucrose, maltose pair within 50 times of concentration of lentinan
Δ A measure have little to no effect, show these types of polysaccharide to Congo red spectrophotometry triple-helix structure lentinan without
It influences.From Fig. 8 it is also seen that starch can cause certain interference, it can only meet and not disturbed to mushroom under same multiple concentration levels
The measure of polysaccharide, but starch concentration can influence to measure structure, therefore this experiment needs to examine lentinan sample at 1 times or more
Whether contain starch in product；
C. starch excludes experiment：360mg potassium iodide, 130mg iodine are taken, it is accurately weighed, it is placed in mortar, a small amount of water is added to grind
Mill after iodine all dissolving, solution is transferred in brown bottle, is diluted with water to 10mL, is shaken up, obtains Wagner's reagent.
Take 3 tool plug test tubes, label A, B, C.A：3.9mL pH7 phosphate buffers；B：1.0mL1mg/mL lentinan marks
Quasi- product solution+2.9mL pH7 phosphate buffers；C：1.0mL 1mg/mL lentinan standard solution+0.1mL 1mg/mL can
Soluble starch solution+2.8mL pH7 phosphate buffers.0.1mL Wagner's reagents are added in into above-mentioned test tube respectively, are shaken up,
Observe color change；Lentinan standard solution is changed to lentinan extract sample solution, label and solution are prepared same
On, observe color change；
D. result as shown in Figure 9, Figure 10, the lentinan solution containing starch, send out after adding in Wagner's reagent by color
Raw significant change (Fig. 9 C), without amyloid lentinan solution then without color change (Fig. 9 A, 9B), shows that lentinan is molten
Starch in liquid can be excluded by iodine-potassium iodide colour reagent；Lentinan extract sample solution hair only containing starch
Raw apparent color change (Figure 10 C) illustrates not influence without (or containing denier) soluble starch in lentinan extract
The measure of lentinan in extract though also indicating that starch has potential interference to the measure of lentinan, can pass through iodo- iodine
Change potassium solution colour developing to exclude.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
- A kind of 1. method for detecting lentinan content, which is characterized in that include the following steps：Step 1：The lentinan standard items of concentration gradient are taken with after Congo red solution reaction, through spectrophotometry, measuring Corresponding absorbance obtains standard curve；Step 2：Sample to be tested is taken with after Congo red solution reaction, through spectrophotometry, measuring corresponding absorbance, it is corresponding The standard curve obtains the concentration of the sample to be tested；The wavelength detected described in step 1 or step 2 is 400-600nm.
- 2. according to the method described in claim 1, it is characterized in that, the wavelength detected described in step 1 or step 2 is 544nm.
- 3. method according to claim 1 or 2, which is characterized in that mixed solution described in step 1 or step 2 PH value is 5-10.
- 4. method according to any one of claims 1 to 3, which is characterized in that the temperature reacted described in step 1 or step 2 It spends for 3-60 DEG C.
- 5. method according to any one of claims 1 to 4, which is characterized in that reacted described in step 1 or step 2 when Between be 1-60min.
- 6. method according to any one of claims 1 to 5, which is characterized in that the recurrence side of standard curve described in step 1 Journey is Y=0.0049X+0.0043, linear coefficient R2=0.9985.
- 7. according to the method described in claim 6, it is characterized in that, in the good linear relation extents of the standard curve, The lentinan surveys concentration as 5~80 μ g/mL.
- 8. method according to any one of claims 1 to 7, which is characterized in that its LOD is 1.41 μ g/mL；Its precision RSD is 1.79%；Its stability RSD is 0.85%.
- 9. according to claim 1 to 8 any one of them method, which is characterized in that its mean sample recovery rate is 99.10%.
- 10. according to claim 1 to 9 any one of them method, which is characterized in that the sample to be tested extracts for lentinan Object.
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| 梅光明 等: "酸提香菇多糖的分离纯化及结构鉴定", 《现代食品科技》 * |
| 游丽君 等: "超声-高温热水提取香菇多糖及其产物特性研究", 《现代食品科技》 * |
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