CN108828121A - A kind of white detection method of content for setting the high nojirimycin of α-in medicinal material - Google Patents
A kind of white detection method of content for setting the high nojirimycin of α-in medicinal material Download PDFInfo
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Abstract
The present invention relates to Chinese medicine content detection fields, more particularly to a kind of detection method of content of the high nojirimycin of α-in white tree medicinal material, this method is using the high nojirimycin of α-as reference substance, with acyl chloride derivative reagent column front derivation α-high nojirimycin, and methylene chloride or chloroform acceleration reaction is added, using AgilentExtendC18 chromatographic column as chromatographic column, methanol, the mixture of acetonitrile and water is mobile phase, separation determination and the quantitative analysis of the high nojirimycin of α-are carried out by high performance liquid chromatography, it provides a kind of quick, accurately, the detection method of convenient white tree medicinal material.
Description
Technical field
The invention belongs to Chinese medicine content detection fields, and in particular to the high nojirimycin of α-contains in the white tree medicinal material of one kind
Quantity measuring method.
Background technique
ɑ-glucosidase inhibitor is the oral drugs of a kind of important treatment diabetes clinically used, existing
ɑ-glucosidase inhibitor class representative drugs have acarbose, voglibose, Miglitol.Different from biguanides,
The oral hypoglycemic drugs such as insulin sensitizer and Drugs Promoting Insulin Secretion need to be metabolized by liver kidney and limit liver kidney in vivo
The use of the patient of dysfunction, ɑ-glucosidase inhibitor are able to suppress the work of intestinal epithelial cell surface ɑ-glycosidase
Property, action target spot can delay carbohydrate absorption in small intestine, without inhibiting protein and fatty absorption, almost to liver
Kidney is without side-effects and cumulative effect.Wherein, by the Voglibose of the military field drug development of the Acarbose of Bayer A.G's exploitation and Japan
The first-line drug for the treatment of diabetes is had become, and therefore further expansion indication researches and develops new ɑ-glucosidase inhibitor
It is very necessary, and is of great significance.
Bai Shu, the entitled s μ regadeglomer μ lata (BL.) of Latin belong to Euphorbiaceae Suregada, and arbor is 2-13 meters high；It is small
Branch band grey black or brown.Blade leathery, wide or narrow oblong or how many obovate arrive lanceolar, 5-12 centimetres long, wide
3-6 centimetres, full edge, the anxious point in top is blunt or round, and dilute notch, base portion is gradually narrow, and middle arteries are in two sides protrusion, the every side 5-10 item of lateral vein；
Stipule close to inflorescence wide triangle, it is thicker, be about 1 millimeter, 2 millimeters wide, top is blunt, and the stipule of lower part is caducous, the long 3-8 of petiole
Millimeter (《Hainan flora》, Chen Huanyong chief editor's volume Two first edition nineteen sixty-five, PP177).
Institute of materia medica of chinese medicine science institute is found under study for action：White tree water extract has good ɑ-glucoside
Enzyme inhibition activity, and applied for patent " white tree extract, preparation method and combinations thereof and purposes ", Publication No.
CN1506106A.The content of Chinese medicine directly affects the drug effect and safety of tcm product, therefore establishes the white tree medicinal material of one kind
Content assaying method has great importance for the development and utilization of white tree drug.
In Chinese patent literature CN102462726A, patent name is white tree total alkaloid and polyhydroxylated alkaloid compound
Extracting and developing method and purposes, successively using cation exchange resin extract, anion exchange resin extract and carboxymethyl Portugal
Polysaccharide gel C-25 separation method, is eluted with ammonium hydroxide or distilled water repeatedly respectively, isolated 17 kinds of polyhydroxylated alkaloid classes
Compound recycles carbon-13 nmr spectra and mass spectrum to identify each compound, finally obtains the corresponding content of compound,
Using this kind of method as white tree medicinal material content assaying method, not only process very complicated, time-consuming, is not suitable for conventional analysis,
And extraction separating step is excessive, is easy to appear deviation and measurement result inaccuracy, the problem of precision difference occurs.
Since the UV that feature is not present in separated obtained polyhydroxylated alkaloid class compound in white tree medicinal material absorbs, no
It can be detected with traditional method, be directed to the alkaloid compound without UV absorption at present, generally use evaporative light-scattering inspection
The instruments such as device, differential refraction detector are surveyed to be detected, but the sensitivity of these methods and accuracy are poor, in conjunction with white tree medicine
Similar ingredient is more in material, and structural formula is close, using general extraction separation method, it is difficult to by white tree medicinal material it is various at
Divide and be effectively separated, therefore white tree medicinal material is not suitable for being detected with above-mentioned alkaloid compound conventional detection instrument.
To sum up, the detection method of content for establishing a kind of quick, accurate, convenient white tree medicinal material becomes in the urgent need to address
Technical problem.
Summary of the invention
For this purpose, technical problem to be solved by the present invention lies in white tree medicinal material content assaying method process in the prior art is numerous
It is trivial it is complicated, time-consuming, and to be easy to cause measurement result inaccurate for excessive extraction separating step, so provide it is a kind of quickly,
Accurately, easily it is white tree medicinal material detection method.
In order to solve the above technical problems, the present invention is achieved through the following technical solutions：
A kind of white detection method of content for setting the high nojirimycin of α-in medicinal material, includes the following steps,
(1) prepared by reference substance solution：The accurately weighed high nojirimycin reference substance of α-, is added derivative solvent and acyl chloride is derivative
Reagent carries out derivatization reaction, and the high deoxynojirimycin and derivatives test solution of α-is made, and dilutes constant volume to the high deoxynojirimycin and derivatives test solution of α-
Afterwards to get reference substance solution；
(2) preparation of standard curve：By high performance liquid chromatography, the high nojirimycin of α-in reference substance solution is measured
Peak area, chromatographic condition：Using octadecylsilane chemically bonded silica as the chromatographic column of filler, using mobile phase A as acetonitrile, mobile phase
B is methanol and mobile phase C is water, and using linear gradient elution method, gradient elution program is as follows：0-10min, A：B：C is 31%：
35%：34% → 31%：45%：24%；10-49min, A：B：C is 31%：45%：24%；49-50min, A：B：C is
31%：45%：24% → 100%：0：0；50-60min, A：B：C is 100%：0：0；60-61min, A：B：C is 100%：0：0
→ 31%：35%：34%；61-70min, A：B：C is 31%：35%：34%, establish the high deoxynojirimycin and derivatives concentration-peak α-
Area standard curve obtains standard curve regression equation；
(3) extraction of sample：Smashed after white tree medicinal material drying, be made white tree powder, be added purified water refluxing extraction or
Ultrasonic extraction obtains white tree extracting solution；
(4) prepared by test solution：Derivative solvent is added into the white tree extracting solution and acyl chloride derivative reagent carries out
Derivatization reaction obtains analyte derivative test solution, test solution will be made after the dilution of analyte derivative test solution, constant volume；
(5) measurement of sample：Using high performance liquid chromatography, it is molten that test sample is measured under step (2) described chromatographic condition
The peak area of the high nojirimycin of α-in liquid, then according to the high nojirimycin of α-in regression equation calculation sample in step (2)
Content.
It is further preferred that the derivative solvent of the acyl chloride is for chlorobenzoyl chloride, methyl benzoyl chloride and to methoxy toluene
One of acyl chlorides.
It is further preferred that the derivative solvent is anhydrous pyridine, tetrahydrofuran, one in n,N-Dimethylformamide
Kind.
It is further preferred that further including that methylene chloride or chloroform is added in the step (1) and the step (4)
The step of.
It is further preferred that in step (1), the high nojirimycin reference substance of α-, derivative solvent, acyl chloride derivative reagent and two
The amount ratio of chloromethanes is：(3-12mg)：(1-2ml)：(0.3-1ml)：(0.5-1.5ml).
It is further preferred that in step (4), the use of white tree medicinal material, derivative solvent, acyl chloride derivative reagent and methylene chloride
The ratio between amount is (0.5-2g)：(1-2ml)：(0.3-1ml)：(0.5-1.5ml).
It is further preferred that in step (3) after white tree powder adds purified water to extract, first extracting solution being centrifuged and is cleaned, then
Supernatant is taken to carry out freeze-drying process, the time of the freeze-drying is 6-24 hours.
It is further preferred that the enzyme powder of 0.05-0.5g is added in every 1ml purified water in step (3), the enzyme powder is fibre
Tie up the mixture of plain enzyme, lignin-degrading enzymes or both.
It is further preferred that the enzyme powder be cellulase and lignin-degrading enzymes mixture, the cellulase and
The mass ratio of lignoenzyme is 1:1-1:2.
It is further preferred that reaction condition is 50-60 DEG C of water in the derivatization reaction of the step (1) and the step (4)
Bath heating, reaction time 30-40min.
Technical solution of the present invention has the following advantages that：
1, in white tree medicinal material provided by the invention the high nojirimycin of α-detection method, α-height open country buttocks in this method is mould
Element has stronger ɑ-glucosidase inhibitory active, and the present invention innovatively uses the high nojirimycin of α-to contain as white tree medicinal material
Measure fixed index, using pre-column derivatization by the high nojirimycin of α-be derived as can the product of UV absorption pass through efficient liquid phase again
The content of the chromatograph measurement high nojirimycin of α-.
2, in white tree medicinal material provided by the invention the high nojirimycin of α-detection method, containing there are many give birth in white tree medicinal material
Alkaloids active material at present derives alkaloid compound generally directed to the N atom in compound, using water as molten
Agent, using fluorenylmethyloxycarbonyl chlorine as derivative reagent, but in studying discovery due in the structure of the high nojirimycin of α-in addition to miscellaneous
Outside N atom on ring, it is respectively connected with OH or CH2OH on remaining five carbon atom, causes N atomic space steric hindrance larger, utilized
The white tree medicinal material content results that traditional alkaloid derivatization method effect is poor, detects are relatively low, by the present invention in that with spreading out
Biochemical reagents chlorobenzoyl chloride performs the derivatization reaction to the OH in the high nojirimycin of α-, significantly improves the reaction of the high nojirimycin of α-
Activity shortens the α-high nojirimycin derivative reaction time, improves derivative reaction effect, this kind of derivatization method and efficient liquid
The mutually chromatograph joint used content that can quickly, accurately, easily detect the high nojirimycin of α-in white tree medicinal material, contains as white tree medicinal material
Fixed index is measured, the exploitation of white tree drug is facilitated；
3, the white detection method for setting the high nojirimycin of α-in medicinal material provided by the invention, the present invention pass through in derivative reagent
Middle addition methylene chloride or chloroform can be improved derivatization reaction activity, to accelerate reaction speed；
4, in white tree medicinal material provided by the invention the high nojirimycin of α-detection method, discovery chlorobenzoyl chloride spreads out in research
The high nojirimycin facile hydrolysis of α-after life is unstable, and the impurity generated after hydrolyzing has similar UV absorption with derivative,
Interference measurement is as a result, the present invention is added anhydrous by the way that the extract of white tree medicinal material is freeze-dried to remove moisture in advance
Pyridine performs the derivatization reaction as reaction dissolvent, improves derivatization reaction stability；And it is by controlling sublimation drying
6-24 hours, in the case where guaranteeing that moisture removes, it can improve and be caused because of the too long influence derivative stability of cooling time
The case where assay Lower result, improves the accuracy of measurement result；
5, the white detection method for setting the high nojirimycin of α-in medicinal material provided by the invention, by Extraction solvent purified water
Middle addition enzyme powder, is conducive to the broken wall in extraction process to crude drug cell, and intracellular each constituents are in release conditions, and shortening mentions
The time is taken, and is added in purified water by using the mixture of lignin-degrading enzymes and cellulase as enzyme powder, it can either
It can be degraded the lignin of Bai Shuzhong using lignin-degrading enzymes, and utilize the cellulose of cellulose degraded Bai Shuzhong, the two
Collaboration promotes the release of Bai Shuzhong active constituent, further increases extraction efficiency, and relative to simple water ultrasonic extraction, enzyme is added
It is extracted in 50min after preparation, recovery rate significantly improves；
6, in white tree medicinal material provided by the invention the high nojirimycin of α-detection method, find in derivatization process in research
Although can accelerate derivatization reaction to a certain extent using lasting vortex or electromagnetic agitation, derivatization reaction process is unstable
Fixed, the RSD value for repeatedly measuring content is higher, and therefore, the present invention accelerates reaction speed using 50-60 DEG C of water bath condition, improves
Reaction effect, derivatization reaction process are stablized, and RSD value meets analysis measurement and requires.
Detailed description of the invention
In order to make the content of the present invention more clearly understood, it below according to specific embodiments of the present invention and combines
Attached drawing, the present invention is described in further detail, wherein：
Fig. 1 is the chromatogram of reference substance solution in experimental example 1 2.3, test solution and blank sample solution, wherein A
For reference substance solution；B is test solution；C is blank sample solution；
Fig. 2 is the high deoxynojirimycin and derivatives concentration-peak area standard curve of α-prepared in experimental example 1 2.4；
Fig. 3 is the chromatogram of the corresponding test solution of chromatographic column of different brands in experimental example 1；Wherein, chromatogram A-D
Respectively correspond the chromatographic column 1-4 in table 8.
Specific embodiment
Following embodiments are for further illustrating the present invention but being not limited to the present invention.
Experimental example 1：
One, the methodological study of the pre-column derivatization measurement high nojirimycin content of α-
1 instrument and reagent
Utimate3000 high performance liquid chromatograph (wears peace in the U.S.)；(the limited public affairs of skilful scientific instrument are striven in Shanghai to freeze drier
Department)；Centrifuge (Shanghai Lu Xiang instrument centrifuge Instrument Ltd.)；Ultrasound Instrument (Shanghai High Kudos Science Instrument Co., Ltd.).
The high nojirimycin reference substance of α-(institute of Materia Medica,Chinese Academy of Medical Sciences self-control), acetonitrile and methanol are chromatographically pure,
Water is ultrapure water, and anhydrous pyridine and chlorobenzoyl chloride are super dry grade, remaining reagent is that analysis is pure.
2 methods and result
The preparation of 2.1 reference substance solutions, test solution and blank sample solution
2.1.1 the preparation of reference substance solution
Precision weighs the high nojirimycin reference substance about 3mg of α-, sequentially add anhydrous pyridine 1.5ml, chlorobenzoyl chloride 0.5ml and
Chloroform 1.0ml, closed, 60 DEG C of water-bath 40min place room temperature, and derivative is transferred in 50ml volumetric flask, methanol constant volume,
Precision pipettes 2ml into 25ml capacity again, and methanol constant volume is mixed to obtain the final product.
2.1.2 the preparation of test solution
(1) it after white tree medicinal material drying, smashing to 50 mesh screens are crossed, precision weighs white tree powder 0.5010g respectively,
0.4997g, 0.5003g are placed in three stuffed conical flasks, and accurate respectively that purified water 25ml is added, weighing is 30 minutes ultrasonic,
Extracting solution is obtained, again weighed weight, the weight of reduction, shakes up after supplying ultrasound with purified water, in the item of revolving speed 5000/min
It is centrifuged 10 minutes under part, precision pipettes supernatant 1ml, is placed in 20ml vial, is freeze-dried 6 hours, obtains sample to be tested；
(2) will in resulting sample to be tested be added anhydrous pyridine 1.5ml dissolution, sequentially add chlorobenzoyl chloride 0.5ml and
Solution after reaction is added methanol constant volume to 25ml, shakes up, use by methylene chloride 1.0ml, sealing, 60 DEG C of water-bath 40min
The filtration of 0.45 μm of filter membrane, take subsequent filtrate to get.
2.1.3 prepared by blank sample solution
According to the preparation method of test solution listed by 2.1.2, blank sample solution of the preparation without the white tree medicinal material.
2.2 chromatographic condition
It is filler with octadecylsilane chemically bonded silica；Chromatographic column：AgilentExtendC18 (4.6mm × 250mm, 5
μm), diode array detector (Detection wavelength 230nm)；Mobile phase：Second eyeball-methanol-water；Flow velocity：1.2ml/min, sample volume
10μl.Gradient elution is as follows：
1 condition of gradient elution table of table
| Time/min | Methanol/% | Acetonitrile/% | Water/% |
| 0 | 31 | 35 | 34 |
| 10 | 31 | 45 | 24 |
| 49 | 31 | 45 | 24 |
| 50 | 100 | 0 | 0 |
| 60 | 100 | 0 | 0 |
| 61 | 31 | 35 | 34 |
| 70 | 31 | 35 | 34 |
Number of theoretical plate (pressing the high nojirimycin of α -) should be not less than 3000.
2.3 system suitability
Reference substance solution, test solution and blank sample solution are prepared respectively by test method listed by item 2.1, respectively essence
Close 10 μ l of measurement reference substance solution, 10 μ l of 10 μ l of test solution and blank sample solution inject liquid chromatograph, in item 2.2
It is measured under listed chromatographic condition, records chromatogram, calculate each peak separating degree, theoretical cam curve, judge that blank sample solution is
It is no noiseless to measurement peak.
As shown in Figure 1, blank sample solution is at the noiseless peak in α-high nojirimycin signal peak position under this chromatographic condition
Occur, the high nojirimycin signal peak of α-and adjacent chromatographic peak degree of being directly separated are 1.56 in test solution, illustrate the wild buttocks of α-height
Mycin peak separates preferably with other impurity peaks, and the high nojirimycin signal peak theoretical cam curve of α-is higher than 15000, symmetrical factor and is greater than
0.9, illustrate that the chromatographic condition specificity is strong.2.4 linear relationships are investigated
α-HNJ reference substance about 5.937mg is taken, accurately weighed, anhydrous pyridine 1.5ml, which is added, to be made after being completely dissolved, then successively
Chlorobenzoyl chloride 0.5ml and methylene chloride 1.0ml, sealing is added, solution after reaction is added methanol system by 60 DEG C of water-bath 40min
At every 1ml contain 18.9 μ g high nojirimycin reference substance mother liquors, shake up, with (0.45 μm) of miillpore filter filter, take subsequent filtrate to get
Reference substance mother liquor.Reference substance mother liquor is taken, precision pipettes 0.2ml, 0.5ml, 1.0ml, 2.0ml, 3ml, 5ml to 10ml capacity respectively
In bottle, methanol constant volume dilution is added, a series of reference substance derivative solution of concentration from low to high, the chromatography listed by item 2.2 is made
Under the conditions of measure respectively reference substance derivative solution and 2.1.1 preparation reference substance solution peak area, the results are shown in Table 2.
With reference substance concentration (μ g/ml) for abscissa, (A μm) of integrating peak areas value is ordinate, draws standard curve, knot
Fruit sees Fig. 2.The high nojirimycin regression equation y=1.972x+0.0862 of α-, correlation coefficient r=0.9999 show that the wild buttocks of α-height is mould
Element is in good linear relationship within the scope of 0.378 μ of μ g~18.76 g/ml.
2 α of table-high nojirimycin linear relationship investigation table
| Number | Sample volume (μ g/ml) | The high nojirimycin peak area (A μm) of α- |
| 1 | 0.378 | 0.7622 |
| 2 | 0.945 | 1.9361 |
| 3 | 1.89 | 3.9881 |
| 4 | 3.78 | 7.5802 |
| 5 | 5.67 | 11.1664 |
| 6 | 9.45 | 18.6445 |
| 7 | 18.76 | 37.1257 |
2.5 precision test
Precision measures reference substance solution obtained by item 2.1, continuously repeats 6 needle of sample introduction, surveys by chromatographic condition listed by item 2.1
To determine peak area, records the high nojirimycin peak area of α-, and calculate the RSD% value of peak area, the results showed that instrument precision is good,
RSD is 0.6%, meets the requirement of analysis method, the results are shown in Table 3.
3 α of table-high nojirimycin peak Precision test result
2.6 stability test
Take with a collection of test solution, respectively after preparation 0h, for 24 hours, 36h, 48h, 100h sample introduction, by color listed by item 2.2
Spectral condition is analyzed, and the results are shown in Table 4.
The RSD of the high nojirimycin of α-peak area in 0~100h is 2.0%, the results showed that, sample is placed steady in 100h
It is fixed.
4 α of table-high nojirimycin peak stability test result
2.7 repetitive test
It takes with the white tree medicinal material of a batch, 6 parts of test solutions is prepared in parallel by method listed by item 2.1, by 2.2 lower chromatostrips
Successively sample introduction measures part, calculates the high nojirimycin content of α-, the results are shown in Table 5.
The RSD of the high nojirimycin Content of α-is 2.0%, shows that the repeatability of this method is good.
5 α of table-high nojirimycin repetitive test result
2.8 recovery test
6 parts, every part of about 0.25g of the white tree medicinal material of same a batch of known content are taken, are recycled using sample-adding, according to the form below is accurate respectively
The high nojirimycin reference substance solution of a certain amount of α-, color listed by the preparation method and item 2.2 by item 2.1.2 test solution is added
Spectral condition is measured, and calculates the rate of recovery.Determination of recovery rates the results are shown in Table 6.Show that the rate of recovery of this law meets regulation, method can
Row.
6 α of table-high nojirimycin determination of recovery rates result
The measurement of 2.9 sample sizes
White tree medicinal material is handled to the preparation for pressing test solution listed by item 2.1 with a batch of white tree medicinal material respectively, is pressed
It is measured according to chromatographic condition sample introduction listed by item 2.2, obtains peak area, substituted into the resulting standard curve relational expression of item 2.4, calculated
The content of the high nojirimycin of α-out, each sample measurement three times, the results are shown in Table 7.
The white tree sample size measurement result of table 7
According to existing test result, Bai Shuzhong α-high nojirimycin (C is fixed tentatively at present6H13NO4) weight percent not
It obtains and is less than 0.1%.
Two, the single factor exploration of process conditions
1, the investigation of the chromatographic column of different brands
(1) it after taking white tree medicinal material drying, smashes to 50 mesh screens are crossed, precision weighs white tree powder 0.4998g respectively, is placed in
In stuffed conical flask, purified water 25ml, weighing is added, ultrasound 30 minutes obtains extracting solution, weighed weight, uses purified water again
The weight for supplying reduction after ultrasound, shakes up, is centrifuged 10 minutes under conditions of revolving speed 5000/min, and precision pipettes supernatant 1ml,
It is placed in 20ml vial, is freeze-dried 6 hours, obtains sample to be tested；
(2) preparation of test solution：Anhydrous pyridine 1.5ml dissolution will be added in resulting sample to be tested, then successively add
Enter chlorobenzoyl chloride 0.5ml and methylene chloride 1.0ml, seal, 60 DEG C of water-bath 40min, methanol is added in solution after reaction and is determined
Hold to 25ml, shake up, filtered with 0.45 μm of filter membrane, take subsequent filtrate to get；
(3) measurement of sample：Difference with method listed by 1 middle term 2.3 of embodiment is, respectively using different in the following table 8
Chromatographic column sampling step (2) made from test solution, record each chromatogram, other are consistent, as a result as shown in figure 3,
The separating obtained peak shape of AgilentExtendC18 chromatographic column is symmetrical, good separating effect, therefore selects the model chromatographic column.
The chromatographic column used in the experiment of table 8
| Number | Specification | Filler | Column number |
| Chromatographic column 1 | 250×4.6mm | ThermoDionexBondedSilicaC185μm | 1 |
| Chromatographic column 2 | 250×4.6mm | WatersXBridgeC185μm | 2 |
| Chromatographic column 3 | 250×4.6mm | AgilentExtendC185μm | 3 |
| Chromatographic column 4 | 250×4.6mm | AgilenZORBAXSB-C185μm | 4 |
2, the investigation of extracting liquid volume
A kind of white detection method of content for setting the high nojirimycin of α-in medicinal material, includes the following steps,
(1) preparation of reference substance solution：Anhydrous pyridine is added in the accurately weighed high nojirimycin reference substance 5.937mg of α-
After 1.5ml makes it completely dissolved, chlorobenzoyl chloride 0.5ml and methylene chloride 1.0ml are sequentially added, is sealed, 60 DEG C of water-baths
Methanol constant volume is added to 25ml in solution after reaction by 40min, and precision measures 2ml, adds methanol constant volume to 25ml, every 1ml is made and contains
The high nojirimycin reference substance mother liquor of the α-of 18.9 μ g, shakes up, and is filtered with 0.45 μm of filter membrane, take subsequent filtrate to get；
(2) preparation of standard curve：Take step (1) prepare reference substance solution, respectively precision pipette 0.2ml, 0.5ml,
1.0ml, 2.0ml, 3ml, 5ml are added methanol constant volume dilution, a series of pair of concentration from low to high are made into 10ml volumetric flask
According to product derivative solution, the peak area of reference substance derivative solution and reference substance solution is measured under the chromatographic condition listed by item 2.2 respectively,
With reference substance concentration (μ g/ml) for abscissa, (A μm) of integrating peak areas value is ordinate, draws standard curve；
(3) extraction of sample：After white tree medicinal material drying, smash to 50 mesh screens are crossed, precision weighs white tree powder respectively
0.5003g, 0.4996g, 0.5002g, 0.5004g are respectively placed in four stuffed conical flasks, accurate respectively that purified water is added
15ml, 25ml, 50ml and 75ml, weighing, ultrasound 30 minutes obtain extracting solution, again weighed weight, supply ultrasound with purified water
The weight of reduction afterwards, shakes up, and is centrifuged 10 minutes under conditions of revolving speed 5000/min, precision pipettes supernatant 1ml, is placed in 20ml
In vial, it is freeze-dried 6 hours, obtains sample to be tested；
(4) preparation of test solution：Anhydrous pyridine 1.5ml dissolution will be added in resulting sample to be tested, then successively add
Enter chlorobenzoyl chloride 0.5ml and methylene chloride 1.0ml, seal, 60 DEG C of water-bath 40min, methanol is added in solution after reaction and is determined
Hold to 25ml, shake up, filtered with 0.45 μm of filter membrane, take subsequent filtrate to get；
(5) measurement of sample：Precision pipettes 10 μ l of test solution, injects liquid chromatograph, the chromatography listed by item 2.2
Under the conditions of measure, record peak area, substitute into the high nojirimycin concentration of step (2) resulting α-and the relational expression of peak area, meter
The content for calculating the high nojirimycin of α-in white tree medicinal material, the results are shown in Table shown in 9.
The investigation result table of the Extraction solvent of 9 different volumes of table
A- high nojirimycin is highly polar compound, and selection purified water is Extraction solvent, as shown in table 9, molten with extracting
The increase of agent dosage, the high nojirimycin content of the α-measured increase, and illustrate that the extraction degree of the high nojirimycin of α-improves, work as extraction
When solvent increases to 25ml, continuing the amount for improving Extraction solvent, the content of the high nojirimycin of α-does not obviously increase, therefore, choosing
The amount for selecting Extraction solvent is the white purified water set medicinal powder and correspond to 25ml of every 0.5g.
3, the investigation of ultrasonic time
A kind of white detection method of content for setting the high nojirimycin of α-in medicinal material, includes the following steps,
(1) preparation of reference substance solution：Anhydrous pyridine is added in the accurately weighed high nojirimycin reference substance 5.935mg of α-
After 1.5ml makes it completely dissolved, chlorobenzoyl chloride 0.5ml and chloroform 1.0ml are sequentially added, is sealed, 60 DEG C of water-baths
Methanol constant volume is added to 25ml in solution after reaction by 40min, and precision measures 2ml, adds methanol constant volume to 25ml, every 1ml is made and contains
The high nojirimycin reference substance mother liquor of the α-of 18.9 μ g, shakes up, and is filtered with 0.45 μm of filter membrane, take subsequent filtrate to get；
(2) preparation of standard curve：Take step (1) prepare reference substance solution, respectively precision pipette 0.2ml, 0.5ml,
1.0ml, 2.0ml, 3ml, 5ml are added methanol constant volume dilution, a series of pair of concentration from low to high are made into 10ml volumetric flask
According to product derivative solution, the peak area of reference substance derivative solution and reference substance solution is measured under the chromatographic condition listed by item 2.2 respectively,
With reference substance concentration (μ g/ml) for abscissa, (A μm) of integrating peak areas value is ordinate, draws standard curve；
(3) extraction of sample：After white tree medicinal material drying, smash to 50 mesh screens are crossed, precision weighs white tree powder respectively
0.4996g, 0.4996g, 0.5005g, 0.5002g are respectively placed in four stuffed conical flasks, accurate respectively that purified water is added
25ml, weighing, ultrasound 20 minutes, 30 minutes, 45 minutes and 60 minutes, obtain extracting solution, again weighed weight, with purifying respectively
Water supplies the weight of reduction after ultrasound, shakes up, and is centrifuged 10 minutes under conditions of revolving speed 5000/min, precision pipettes supernatant
1ml is placed in 20ml vial, is freeze-dried 6 hours, is obtained sample to be tested；
(4) preparation of test solution：Anhydrous pyridine 1.5ml dissolution will be added in resulting sample to be tested, then successively add
Enter chlorobenzoyl chloride 0.5ml and chloroform 1.0ml, seal, 60 DEG C of water-bath 40min, methanol constant volume is added extremely in solution after reaction
25ml shakes up, and is filtered with 0.45 μm of filter membrane, take subsequent filtrate to get；
(5) measurement of sample：Precision pipettes 10 μ l of test solution, injects liquid chromatograph, the chromatography listed by item 2.2
Under the conditions of measure, record peak area, substitute into the high nojirimycin concentration of step (2) resulting α-and the relational expression of peak area, meter
The content for calculating the high nojirimycin of α-in white tree medicinal material, the results are shown in Table shown in 10.
The investigation result table of the different ultrasonic times of table 10
As shown in table 10, with the increase of ultrasonic time, α-is high, and nojirimycin content starts to be increased slightly, and illustrates to extract and get over
Completely, when ultrasonic time increases to 30min, ultrasonic time is continued growing, the content of the high nojirimycin of α-does not obviously increase,
Therefore, select ultrasonic time for 30min.
4, the investigation of extraction time
A kind of white detection method of content for setting the high nojirimycin of α-in medicinal material, includes the following steps,
(1) preparation of reference substance solution：Anhydrous pyridine is added in the accurately weighed high nojirimycin reference substance 5.933mg of α-
After 1.5ml makes it completely dissolved, chlorobenzoyl chloride 0.5ml and methylene chloride 1.0ml are sequentially added, is sealed, 60 DEG C of water-baths
Methanol constant volume is added to 25ml in solution after reaction by 40min, and precision measures 2ml, adds methanol constant volume to 25ml, every 1ml is made and contains
The high nojirimycin reference substance mother liquor of the α-of 18.9 μ g, shakes up, and is filtered with 0.45 μm of filter membrane, take subsequent filtrate to get；
(2) preparation of standard curve：Take step (1) prepare reference substance solution, respectively precision pipette 0.2ml, 0.5ml,
1.0ml, 2.0ml, 3ml, 5ml are added methanol constant volume dilution, a series of pair of concentration from low to high are made into 10ml volumetric flask
According to product derivative solution, the peak area of reference substance derivative solution and reference substance solution is measured under the chromatographic condition listed by item 2.2 respectively,
With reference substance concentration (μ g/ml) for abscissa, (A μm) of integrating peak areas value is ordinate, draws standard curve；
(3) extraction of sample：After white tree medicinal material drying, smash to 50 mesh screens are crossed, precision weighs white tree powder respectively
0.5005g is placed in stuffed conical flask, and purified water 25ml, weighing is added in precision, and ultrasound 30 minutes obtains extracting solution, claims again
Determine weight, the weight of reduction, shakes up after supplying ultrasound with purified water, is centrifuged 10 minutes under conditions of revolving speed 5000/min, essence
It is close to pipette supernatant 1ml, it is placed in 20ml vial, is freeze-dried 6 hours, obtains sample to be tested A；Supernatant is poured out and is obtained
Residue, accurate into residue that purified water 25ml is added, weighing is 30 minutes ultrasonic, obtains extracting solution, weighed weight, use are pure again
Change the weight that water supplies reduction after ultrasound, shakes up, be centrifuged 10 minutes under conditions of revolving speed 5000/min, precision pipettes supernatant
1ml is placed in 20ml vial, is freeze-dried 6 hours, is obtained sample to be tested B；Continue to pour out supernatant and obtains residue, to
Accurate in residue that purified water 25ml is added, weighing is 30 minutes ultrasonic, obtains extracting solution, weighed weight, is supplied with purified water again
The weight of reduction, shakes up after ultrasound, is centrifuged 10 minutes under conditions of revolving speed 5000/min, precision pipettes supernatant 1ml, is placed in
In 20ml vial, it is freeze-dried 6 hours, obtains sample to be tested C；
(4) preparation of test solution：Anhydrous pyridine 1.5ml dissolution is separately added into sample to be tested A, B, C, then successively
Chlorobenzoyl chloride 0.5ml and methylene chloride 1.0ml, sealing is added, methanol is added in solution after reaction by 60 DEG C of water-bath 40min
Be settled to 25ml, shake up, filtered with 0.45 μm of filter membrane, take subsequent filtrate to get；
(5) measurement of sample：Precision pipettes above-mentioned three parts of test solutions, 10 μ l respectively, liquid chromatograph is injected, in item
It is measured under chromatographic condition listed by 2.2, records peak area, substitute into the high nojirimycin concentration of step (2) resulting α-and peak area
Relational expression in, calculate it is white tree medicinal material in the high nojirimycin of α-content, the results are shown in Table shown in 11.
The different extraction time contents of table 11 investigate result
As shown in table 11, white tree medicinal powder does not measure a- high nojirimycin content the 2nd, 3 time in extraction, illustrates to extract 1 time
It is i.e. extractable complete, it is thus determined that extraction time is 1 time.
Embodiment 1：(not enzyme in extraction process)
A kind of white detection method of content for setting the high nojirimycin of α-in medicinal material, includes the following steps,
(1) preparation of reference substance solution：Anhydrous pyridine 1ml is added in the accurately weighed high nojirimycin reference substance 5.932mg of α-
After making it completely dissolved, chlorobenzoyl chloride 0.3ml and methylene chloride 1.0ml are sequentially added, is sealed, 60 DEG C of water-bath 40min,
Methanol constant volume is added to 25ml in solution after reacting, and precision measures 2ml, adds methanol constant volume to 25ml, every 1ml is made containing 18.9 μ g
The high nojirimycin reference substance mother liquor of α-, shake up, filtered with 0.45 μm of filter membrane, take subsequent filtrate to get；
(2) preparation of standard curve：Take step (1) prepare reference substance solution, respectively precision pipette 0.2ml, 0.5ml,
1.0ml, 2.0ml, 3ml, 5ml are added methanol constant volume dilution, a series of pair of concentration from low to high are made into 10ml volumetric flask
According to product derivative solution, the peak area of reference substance derivative solution and reference substance solution is measured under the chromatographic condition listed by item 2.2 respectively,
With reference substance concentration (μ g/ml) for abscissa, (A μm) of integrating peak areas value is ordinate, draws standard curve, obtains peak area
With the high nojirimycin concentration relationship formula of α-；
(3) extraction of sample：After white tree medicinal material drying, smash to 60 mesh screens excessively, accurately weighed white tree powder 1.004g,
It is placed in stuffed conical flask, purified water 50ml, weighing is added, ultrasound 30 minutes, obtains extracting solution, again by 45 DEG C of Extracting temperature
Weighed weight, the weight of reduction, shakes up after supplying ultrasound with purified water, is centrifuged 10 minutes under conditions of revolving speed 5000/min,
Precision pipettes supernatant 1ml, is placed in 20ml vial, is freeze-dried 6 hours, obtains sample to be tested；
(4) preparation of test solution：Anhydrous pyridine 1.5ml dissolution will be added in resulting sample to be tested, then successively add
Enter chlorobenzoyl chloride 0.5ml and methylene chloride 1.0ml, seal, 60 DEG C of water-bath 40min, methanol is added in solution after reaction and is determined
Hold to 25ml, shake up, filtered with 0.45 μm of filter membrane, take subsequent filtrate to get；
(5) measurement of sample：Precision pipettes 10 μ l of test solution, injects liquid chromatograph, the chromatography listed by item 2.2
Under the conditions of measure, record peak area, substitute into the high nojirimycin concentration of step (2) resulting α-and the relational expression of peak area, meter
The content for calculating the high nojirimycin of α-in white tree medicinal material, the results are shown in Table shown in 12.
Embodiment 2：(in extraction process plus 0.05g enzyme)
A kind of white detection method of content for setting the high nojirimycin of α-in medicinal material, includes the following steps,
(1) preparation of reference substance solution：Anhydrous pyridine is added in the accurately weighed high nojirimycin reference substance 11.861mg of α-
After 1.5ml makes it completely dissolved, chlorobenzoyl chloride 0.5ml and chloroform 1.5ml are sequentially added, is sealed, 50 DEG C of water-baths
Methanol constant volume is added to 50ml in solution after reaction by 40min, and precision measures 2ml, adds methanol constant volume to 25ml, every 1ml is made and contains
The high nojirimycin reference substance mother liquor of the α-of 18.9 μ g, shakes up, and is filtered with 0.45 μm of filter membrane, take subsequent filtrate to get；
(2) preparation of standard curve：Take step (1) prepare reference substance solution, respectively precision pipette 0.2ml, 0.5ml,
1.0ml, 2.0ml, 3ml, 5ml are added methanol constant volume dilution, a series of pair of concentration from low to high are made into 10ml volumetric flask
According to product derivative solution, the peak area of reference substance derivative solution and reference substance solution is measured under the chromatographic condition listed by item 2.2 respectively,
With reference substance concentration (μ g/ml) for abscissa, (A μm) of integrating peak areas value is ordinate, draws standard curve, obtains peak area
With the high nojirimycin concentration relationship formula of α-；
(3) extraction of sample：Using with embodiment 1 with after dry under the white tree medicinal material the same terms of a batch, smash to 65
Mesh screen, accurately weighed white tree powder 1.9994g, is placed in stuffed conical flask, and purified water 100ml is added in precision, then fills in tool
0.05g enzyme powder (0.02g cellulase+0.03g lignoenzyme) is added in conical flask to be uniformly mixed afterwards, wherein the cellulase
Vigor is 20,000 μ/g, and the lignoenzyme vigor is 50,000 μ/g, and weighing, 35 DEG C of Extracting temperature, ultrasound 50 minutes is extracted
Liquid, weighed weight again, the weight of reduction, shakes up after supplying ultrasound with purified water, is centrifuged under conditions of revolving speed 4500/min
20 minutes, precision pipetted supernatant 1ml, was placed in 20ml vial, is freeze-dried 8 hours, obtains sample to be tested；
(4) preparation of test solution：Anhydrous pyridine 1.5ml dissolution will be added in resulting sample to be tested, then successively add
Enter chlorobenzoyl chloride 0.5ml and chloroform 1.5ml, seal, 50 DEG C of water-bath 40min, methanol constant volume is added extremely in solution after reaction
50ml shakes up, and is filtered with 0.45 μm of filter membrane, take subsequent filtrate to get；
(5) measurement of sample：Precision pipettes 10 μ l of test solution, injects liquid chromatograph, the chromatography listed by item 2.2
Under the conditions of measure, record peak area, substitute into the high nojirimycin concentration of step (2) resulting α-and the relational expression of peak area, meter
The content for calculating the high nojirimycin of α-in white tree medicinal material, the results are shown in Table shown in 12.
Embodiment 3：(in extraction process plus 0.3g enzyme)
A kind of white detection method of content for setting the high nojirimycin of α-in medicinal material, includes the following steps,
(1) preparation of reference substance solution：Tetrahydrofuran 1ml is added in the accurately weighed high nojirimycin reference substance 2.970mg of α-
After making it completely dissolved, sequentially add to methoxy methyl benzoyl chloride 1ml and chloroform 1ml, sealing, 50 DEG C of water-bath 30min,
Methanol constant volume is added to 25ml in solution after reacting, and precision measures 4ml, adds methanol constant volume to 25ml, every 1ml is made containing 18.9 μ g
The high nojirimycin reference substance mother liquor of α-, shake up, filtered with 0.45 μm of filter membrane, take subsequent filtrate to get；
(2) preparation of standard curve：Take step (1) prepare reference substance solution, respectively precision pipette 0.2ml, 0.5ml,
1.0ml, 2.0ml, 3ml, 5ml are added methanol constant volume dilution, a series of pair of concentration from low to high are made into 10ml volumetric flask
According to product derivative solution, the peak area of reference substance derivative solution and reference substance solution is measured under the chromatographic condition listed by item 2.2 respectively,
With reference substance concentration (μ g/ml) for abscissa, (A μm) of integrating peak areas value is ordinate, draws standard curve, obtains peak area
With the high nojirimycin concentration relationship formula of α-；
(3) extraction of sample：Using with embodiment 1 with after dry under the white tree medicinal material the same terms of a batch, smash to 50
Mesh screen, accurately weighed white tree powder 0.9994g, is placed in stuffed conical flask, and purified water 50ml is added in precision, then bores to tool plug
0.3g enzyme powder (0.1g cellulase+0.2g lignoenzyme) is added in shape bottle to be uniformly mixed afterwards, wherein the cellulase activity
For 20,000 μ/g, the lignoenzyme vigor is 50,000 μ/g, and weighing is 30 minutes ultrasonic, 50 DEG C of Extracting temperature, obtains extracting solution, then
Secondary weighed weight, the weight of reduction, shakes up after supplying ultrasound with purified water, and 15 points are centrifuged under conditions of revolving speed 4000/min
Clock, precision pipette supernatant 2ml, are placed in 20ml vial, are freeze-dried 24 hours, obtain sample to be tested；
(4) preparation of test solution：Anhydrous pyridine 1ml dissolution will be added in resulting sample to be tested, sequentially adds
Solution after reaction is added methanol constant volume to 25ml, shaken by chlorobenzoyl chloride 1ml and chloroform 1ml, sealing, 50 DEG C of water-bath 30min
It is even, filtered with 0.45 μm of filter membrane, take subsequent filtrate to get；
(5) measurement of sample：Precision pipettes 10 μ l of test solution, injects liquid chromatograph, the chromatography listed by item 2.2
Under the conditions of measure, record peak area, substitute into the high nojirimycin concentration of step (2) resulting α-and the relational expression of peak area, meter
The content for calculating the high nojirimycin of α-in white tree medicinal material, the results are shown in Table shown in 12.
Embodiment 4：(in extraction process plus 0.5g enzyme powder)
A kind of white detection method of content for setting the high nojirimycin of α-in medicinal material, includes the following steps,
(1) preparation of reference substance solution：Anhydrous pyridine 1ml is added in the accurately weighed high nojirimycin reference substance 5.937mg of α-
After making it completely dissolved, methyl benzoyl chloride 0.5ml and chloroform 0.5ml are sequentially added, is sealed, 55 DEG C of water-bath 35min,
Methanol constant volume is added to 25ml in solution after reacting, and precision measures 2ml, adds methanol constant volume to 25ml, every 1ml is made containing 18.9 μ g
The high nojirimycin reference substance mother liquor of α-, shake up, filtered with 0.45 μm of filter membrane, take subsequent filtrate to get；
(2) preparation of standard curve：Take step (1) prepare reference substance solution, respectively precision pipette 0.2ml, 0.5ml,
1.0ml, 2.0ml, 3ml, 5ml are added methanol constant volume dilution, a series of pair of concentration from low to high are made into 10ml volumetric flask
According to product derivative solution, the peak area of reference substance derivative solution and reference substance solution is measured under the chromatographic condition listed by item 2.2 respectively,
With reference substance concentration (μ g/ml) for abscissa, (A μm) of integrating peak areas value is ordinate, draws standard curve, obtains peak area
With the high nojirimycin concentration relationship formula of α-；
(3) extraction of sample：Using with embodiment 1 with after dry under the white tree medicinal material the same terms of a batch, smash to 65
Mesh screen, accurately weighed white tree powder 0.5004g, is placed in stuffed conical flask, and purified water 25ml is added in precision, then bores to tool plug
0.5g enzyme powder (enzyme powder of 0.2g cellulase+0.3g lignoenzyme) is added in shape bottle to be uniformly mixed afterwards, wherein the cellulose
Enzyme activity is 20,000 μ/g, and the lignoenzyme vigor is 50,000 μ/g, and weighing is 45 minutes ultrasonic, 50 DEG C of Extracting temperature, is extracted
Liquid, weighed weight again, the weight of reduction, shakes up after supplying ultrasound with purified water, is centrifuged under conditions of revolving speed 5000/min
15 minutes, precision pipetted supernatant 1ml, was placed in 20ml vial, is freeze-dried 12 hours, obtains sample to be tested；
(4) preparation of test solution：Anhydrous pyridine 1ml dissolution will be added in resulting sample to be tested, sequentially adds
Methanol constant volume is added extremely in solution after reaction by chlorobenzoyl chloride 0.5ml and chloroform 0.5ml, sealing, 55 DEG C of water-bath 35min
25ml shakes up, and is filtered with 0.45 μm of filter membrane, take subsequent filtrate to get；
(5) measurement of sample：Precision pipettes 10 μ l of test solution, injects liquid chromatograph, the chromatography listed by item 2.2
Under the conditions of measure, record peak area, substitute into the high nojirimycin concentration of step (2) resulting α-and the relational expression of peak area, meter
The content for calculating the high nojirimycin of α-in white tree medicinal material, the results are shown in Table shown in 12.
Comparative example 1：(N atom derivatization).
A kind of white detection method of content for setting the high nojirimycin of α-in medicinal material, includes the following steps,
(1) preparation of reference substance solution：Precision weighs the high nojirimycin reference substance 9.88mg of α-, sets in 50ml measuring bottle, uses
50% methanol dissolves and is diluted to scale.Precision draws above-mentioned solution 1.0ml, sets in 25ml measuring bottle, the boron of 0.4mol/l is added
Acid-potassium chloride buffer (pH=8.5) 1ml, adds the FMOC-Cl acetonitrile solution 2ml of 10mmol/l, is ultrasonically treated at room temperature
30min adds 0.1% acetum to scale, shake up to get；
(2) preparation of standard curve：It is accurate respectively to draw above-mentioned 2,4,8,10,20,30,40 μ l of reference substance solution, injection
Liquid chromatograph, measurement, using peak area as ordinate, sample volume is that abscissa makees linear regression, linear equation Y=
2.361X-0.4658 r=0.995, the range of linearity is 0.0158~0.3162 μ g；
(3) preparation of test solution：Using with after drying under the same white tree medicinal material the same terms of a batch of embodiment 1, smash
To 65 mesh screens are crossed, 50ml0.05mol/l hydrochloric acid solution ultrasound 30min, extracting solution is added in accurately weighed white tree powder 1.002g
It is centrifuged 10min (revolving speed 5000r/min), supernatant is taken to be transferred in 25ml measuring bottle, add 0.05mol/l hydrochloric acid to quarter
Degree.Precision measures above-mentioned solution 1ml, sets in 10ml measuring bottle, boric acid-potassium chloride buffer (pH=8.5) of 0.4mol/l is added
1ml, the FMOC-Cl acetonitrile solution 2ml for adding 10mmol/l are ultrasonically treated (power 250W frequency 53kHz) 30min at room temperature,
Add 0.1% acetum to scale, shake up to get；
(4) measurement of sample：Precision pipettes 10 μ l of test solution, injects liquid chromatograph, and measurement records peak area,
It substitutes into the high nojirimycin concentration of step (2) resulting α-and the relational expression of peak area, it is mould to calculate the wild buttocks of α-height in white tree medicinal material
The content of element, the results are shown in Table shown in 12.
Comparative example 2：(methylene chloride is not added)
A kind of white detection method of content for setting the high nojirimycin of α-in medicinal material, includes the following steps,
(1) preparation of reference substance solution：With the difference of listed method is in the preparation of reference substance solution in embodiment 1,
Methylene chloride is not added during derivatization reaction, other are consistent；
(2) preparation of the high deoxynojirimycin and derivatives concentration-peak area standard curve of α-：With 1 standard curve of embodiment
Listed method in preparation；
(3) extraction of sample：With listed method in the extraction of sample in embodiment 1；
(4) preparation of test solution：With the difference of listed method is in the preparation of test solution in embodiment 1,
Methylene chloride is not added during derivatization reaction, other are consistent；
(5) measurement of sample：With listed method in embodiment 1, the results are shown in Table shown in 12.
Comparative example 3：(anhydrous methanol replaces methylene chloride)
A kind of white detection method of content for setting the high nojirimycin of α-in medicinal material, includes the following steps,
(1) preparation of reference substance solution：Difference with method listed by the preparation of reference substance solution in embodiment 1 is,
1.0ml anhydrous methanol is added during derivatization reaction and replaces methylene chloride, other are consistent；
(2) preparation of the high deoxynojirimycin and derivatives concentration-peak area standard curve of α-：With 1 standard curve of embodiment
Prepare listed method；
(3) extraction of sample：With listed method in the extraction of sample in embodiment 1；
(4) preparation of test solution：With the difference of listed method is in the preparation of test solution in embodiment 1,
1.0ml anhydrous methanol is added during derivatization reaction and replaces methylene chloride, other are consistent；
(5) measurement of sample：With listed method in embodiment 1, as a result as shown in table 12.
Comparative example 4：(dehydrated alcohol replaces methylene chloride)
A kind of white detection method of content for setting the high nojirimycin of α-in medicinal material, includes the following steps,
(1) preparation of reference substance solution：Difference with method listed by the preparation of reference substance solution in embodiment 1 is,
1.0ml dehydrated alcohol is added during derivatization reaction and replaces methylene chloride, other are consistent；
(2) preparation of the high deoxynojirimycin and derivatives concentration-peak area standard curve of α-：With 1 standard curve of embodiment
Prepare listed method；
(3) extraction of sample：With listed method in the extraction of sample in embodiment 1；
(4) preparation of test solution：With the difference of listed method is in the preparation of test solution in embodiment 1,
1.0ml dehydrated alcohol is added during derivatization reaction and replaces methylene chloride, other are consistent；
(5) measurement of sample：With listed method in embodiment 1；As a result as shown in table 12.
Comparative example 5：(anhydrous propanone replaces methylene chloride)
A kind of white detection method of content for setting the high nojirimycin of α-in medicinal material, includes the following steps,
(1) preparation of reference substance solution：Difference with the preparation of reference substance solution in embodiment 1 is, in derivatization reaction
1.0ml anhydrous propanone is added in the process and replaces methylene chloride, other are consistent；
(2) preparation of the high deoxynojirimycin and derivatives concentration-peak area standard curve of α-：With 1 standard curve of embodiment
Listed method in preparation；
(3) extraction of sample：With listed method in the extraction of sample in embodiment 1；
(4) preparation of test solution：With the difference of listed method is in the preparation of test solution in embodiment 1,
1.0ml anhydrous propanone is added during derivatization reaction and replaces methylene chloride, other are consistent；
(5) measurement of sample：With listed method in embodiment 1；As a result as shown in table 12.
Evaluate example
The high nojirimycin content results table of α-measured in table 12 embodiment 1-4 and comparative example 1-4
(1) by embodiment 1 and embodiment 2-4 comparison it is found that for simple water ultrasonic extraction, every 1ml is purified
Again ultrasonic extraction is added after the enzyme powder of 0.05-0.5g in water, and the high nojirimycin assay average value of α-improves, and illustrates enzyme powder
The release that can promote the high nojirimycin of α-is added, significantly improves the recovery rate of the high nojirimycin of α-in white tree medicinal material.
(2) compared with Example 1, the high nojirimycin average content of α-measured with the white tree medicinal material of a batch is aobvious by comparative example 1-4
Writing reduces, and illustrates that the addition of methylene chloride can promote the high nojirimycin derivative reaction of α-, improves derivatization effect；Moreover,
Compared to the anhydrous methanol in comparative example 2, the anhydrous propanone in the dehydrated alcohol and comparative example 4 in comparative example 3, in embodiment 1
Methylene chloride can advantageously promote the high nojirimycin derivative reaction of α-, improve derivatization effect.
Obviously, the above embodiments are merely examples for clarifying the description, and does not limit the embodiments.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or
It changes.There is no necessity and possibility to exhaust all the enbodiments.And thus amplify out it is obvious variation or
It changes still within the protection scope of the invention.
Claims (10)
1. the detection method of content of the high nojirimycin of α-, includes the following steps in a kind of white tree medicinal material,
(1) prepared by reference substance solution：Derivative solvent and acyl chloride derivative reagent is added in the accurately weighed high nojirimycin reference substance of α-
Derivatization reaction is carried out, the high deoxynojirimycin and derivatives test solution of α-is made, after diluting constant volume to the high deoxynojirimycin and derivatives test solution of α-, i.e.,
Obtain reference substance solution；
(2) preparation of standard curve：By high performance liquid chromatography, the peak face of the high nojirimycin of α-in reference substance solution is measured
Product, chromatographic condition：Using octadecylsilane chemically bonded silica as the chromatographic column of filler, it is by acetonitrile, Mobile phase B of mobile phase A
Methanol and mobile phase C are water, and using linear gradient elution method, gradient elution program is as follows：0-10min, A：B：C is 31%：35%：
34% → 31%：45%：24%；10-49min, A：B：C is 31%：45%：24%；49-50min, A：B：C is 31%：
45%：24% → 100%：0：0；50-60min, A：B：C is 100%：0：0；60-61min, A：B：C is 100%：0：0→
31%：35%：34%；61-70min, A：B：C is 31%：35%：34%, establish the high deoxynojirimycin and derivatives concentration-peak face α-
Product standard curve, obtains standard curve regression equation；
(3) extraction of sample：It is smashed after white tree medicinal material drying, white tree powder is made, purified water refluxing extraction or ultrasound is added
It extracts, obtains white tree extracting solution；
(4) prepared by test solution：Derivative solvent is added into the white tree extracting solution and acyl chloride derivative reagent is derived
Reaction, obtains analyte derivative test solution, test solution will be made after the dilution of analyte derivative test solution, constant volume；
(5) measurement of sample：Using high performance liquid chromatography, measured in test solution under step (2) described chromatographic condition
The high nojirimycin of α-peak area, then according in step (2) in regression equation calculation sample the high nojirimycin of α-content.
2. detection method of content according to claim 1, which is characterized in that the acyl chloride derivative reagent is benzoyl
Chlorine, methyl benzoyl chloride and to one of methoxy methyl benzoyl chloride.
3. detection method of content according to claim 1 or 2, which is characterized in that the derivative solvent be anhydrous pyridine,
One of tetrahydrofuran, n,N-Dimethylformamide.
4. detection method of content according to any one of claim 1-3, which is characterized in that in the step (1) and institute
It states in step (4), further includes the steps that methylene chloride or chloroform is added.
5. detection method of content according to claim 4, which is characterized in that in step (1), the high nojirimycin control of α-
Product, the amount ratio for deriving solvent, acyl chloride derivative reagent and methylene chloride are：(3-12mg)：(1-2ml)：(0.3-1ml)：
(0.5-1.5ml)。
6. detection method of content according to claim 4, which is characterized in that in step (4), white tree medicinal material, derivative solvent,
The ratio between dosage of acyl chloride derivative reagent and methylene chloride is (0.5-2g)：(1-2ml)：(0.3-1ml)：(0.5-1.5ml).
7. detection method of content according to claim 1 to 6, which is characterized in that in step (3), white tree powder
After end plus purified water are extracted, first extracting solution is centrifuged and is cleaned, supernatant is then taken to carry out freeze-drying process, the freeze-drying
Time be 6-24 hours.
8. detection method of content described in any one of -7 according to claim 1, which is characterized in that in step (3), every 1ml is pure
Change the enzyme powder that 0.05-0.5g is added in water, the enzyme powder is the mixture of cellulase, lignin-degrading enzymes or both.
9. detection method of content according to claim 8, which is characterized in that the enzyme powder is that cellulase and lignin drop
The mass ratio of the mixture of solution enzyme, the cellulase and lignoenzyme is 1:1-1:2.
10. detection method of content according to claim 1 to 9, which is characterized in that step (1) and step (4)
Derivatization reaction in, reaction condition be 50-60 DEG C of heating water bath, reaction time 30-40min.
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