CN103869026A - Pretreatment method for compound folium mori preparation and method for detecting 1-deoxynojirimycin - Google Patents
Pretreatment method for compound folium mori preparation and method for detecting 1-deoxynojirimycin Download PDFInfo
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Abstract
The invention discloses a pretreatment method for a compound folium mori preparation and a method for detecting 1-deoxynojirimycin. The pretreatment method comprises the steps of uniformly mixing a solution of the compound folium mori preparation, a buffering solution and a solution of fmoc chloride to obtain a mixture, then uniformly mixing the mixture with glycine, and filtering to obtain the pretreated compound folium mori preparation, wherein a solvent in the solution of the compound folium mori preparation consists of 80 percent of a methyl alcohol solution and/or 80-100 percent of an ethyl alcohol solution by volume; the buffering solution is a borate buffering solution. The detection method comprises the pretreatment method for the compound folium mori preparation. The pretreatment method is quick, convenient and high in separation degree; a target can be precisely obtained; furthermore, the stability is high, the sensitivity is high, and the error is small; according to the detection method, a detection result is precise, and the quality of the compound preparation can be objectively evaluated.
Description
Technical field
The present invention relates to the detection method of preprocess method and the 1-DNJ of mulberry leaf compound preparation.
Background technology
1-DNJ, Chinese another name: (2R, 3R, 4R, 5S)-2-hydroxymethyl piperidine-3,4,5-triol, 1-deoxidation nojirimycin, polyhydroxylated alkaloid or 1-deoxidation azepine-D-Glucose; English name: 1-Deoxynojirimycin, is called for short 1-DNJ or DNJ; Molecular formula: C
6h
13nO
4; Molecular weight: 163.17; No. CAS: 19130-96-2.
1-DNJ is white powder, can from branch, leaf and the root of moraceae plants mulberry leaf (Mulberry Leaf), extract and obtain, and be a kind of α-raisins enzyme inhibitor, there is significant effect to reducing blood sugar for human body value.
In prior art, mainly concentrate in the chemical composition of 1-DNJ about the research of 1-DNJ.And relatively less for the research of the qualitative or quantitative detection method of 1-DNJ, because the preprocessing process of 1-DNJ is comparatively complicated in preparation, make detection method complicated operation and error larger.
Yang Haixia measures the content (research of DNJ separation and purification and assay in mulberry leaf, middle traditional Chinese medicines section, 2003:1) of 1-DNJ in mulberry leaf single preparations of ephedrine with derivatization-spectrophotometer.The preprocessing process of the method causes the loss of 1-DNJ, and testing result is inaccurate; The mulberry-leaf extract and the purified that obtain are colored solutions, larger to the determination influences of absorbance, and measurement result accuracy is low.
In prior art, the pre-service of 1-DNJ, is also only confined to the pretreated research of 1-DNJ in single preparations of ephedrine.Research for compound preparation is less.Compound preparation is compared with single preparations of ephedrine, and composition and content thereof are more complicated, and the interaction between each composition makes the pre-service of 1-DNJ in compound preparation more complicated, has directly affected the detection to 1-DNJ in compound preparation.
Summary of the invention
Technical matters to be solved by this invention is, overcome in prior art still less than the defect for the preprocess method of 1-DNJ in compound preparation and the research of detection method thereof, the detection method of 1-DNJ in a kind of preprocess method of mulberry leaf compound preparation and mulberry leaf compound preparation is provided, this preprocess method is quick and convenient, degree of separation is good, can accurately obtain object, and stability is high, highly sensitive, error is little, this detection method testing result exact method, product quality that can objective appraisal compound preparation.
The present inventor, for precision detects the 1-DNJ in compound preparation, preprocess method for mulberry leaf compound preparation has carried out a large amount of and deep research, in research process, inventor finds: compound preparation is compared with single preparations of ephedrine, composition and content thereof are more complicated, only apply mechanically the preprocess method of 1-DNJ in single preparations of ephedrine, can not extract accurately and effectively and detect the 1-DNJ in compound preparation.For example, with hydrochloric acid extraction 1-DNJ, after derivatization, HPLC analyzes, and sample separation degree is bad, and hangover or acromion appear in peak shape, and assay is inaccurate, and measurement result is unstable.
Through after permanent research, inventor finds: compound preparation is dissolved in specific solvent, with specific derivatization reagent, in specific damping fluid, carry out derivative reaction, can generate the complex compound with uv absorption, carry out separating treatment, the material obtaining can be detected and be obtained by high performance liquid chromatography-ultraviolet, and the accuracy, precision, the repeated texts excellence that detect again.
One of object of the present invention is, a kind of preprocess method of mulberry leaf compound preparation is provided, and described preprocess method comprises the following steps:
The solution of the solution of mulberry leaf compound preparation, buffer solution and fluorenes methoxy dicarbonyl chloride is mixed, then mixes with glycocoll, filter; Wherein, the solvent in the solution of described mulberry leaf compound preparation is the methanol solution of percent by volume 80% and/or the ethanolic solution of percent by volume 80%-100%; Described buffer solution is borate buffer solution.
Below, for the further preferred condition of above-mentioned preprocess method, do following explanation:
Those skilled in the art all understand: compound preparation refers to two or more medicine mix preparation.Preprocess method of the present invention is applicable to the pre-service of 1-DNJ in the mulberry leaf compound preparation of this area routine.Described mulberry leaf compound preparation preferably comprises the mulberry-leaf extract of 40%-50%, Bitter Melon P.E, the Astragalus Root P.E of 12%-17% and the ophiopogon japonicus extract of 5%-9% of 25%-35%, and described number percent is mass percent.Described mulberry leaf compound preparation is more preferably Xi Er board compound mulberry leaf capsule.
In the present invention, the preparation method of the solution of described mulberry leaf compound preparation is preferably: described mulberry leaf compound preparation is dissolved in described solvent, shakes up and/or ultrasonic processing.Described ultrasonic processing preferably for to process 20min under power 100W and frequency 40kHz condition.
In the solution of described mulberry leaf compound preparation, the addition of described solvent is preferably the solvent that every 0.2-0.6g mulberry leaf compound preparation adds 25mL, is more preferably the solvent that every 0.3g mulberry leaf compound preparation adds 25mL.
Described borate buffer solution is preferably boric acid-potassium chloride damping fluid.The concentration of described boric acid-potassium chloride damping fluid is preferably 0.2mol/L; The pH value of described boric acid-potassium chloride damping fluid is preferably pH8.5; The addition of described boric acid-potassium chloride damping fluid preferably adds boric acid-potassium chloride damping fluid of 1.67mL for every Ke Sangye compound preparation.
The addition of the solution of described fluorenes methoxy dicarbonyl chloride is in fluorenes methoxy dicarbonyl chloride, preferably for preferably adding the fluorenes methoxy dicarbonyl chloride of 10mmol in every 1-3 gram testing sample, more preferably for preferably adding the fluorenes methoxy dicarbonyl chloride of 10mmol in every 1.5 grams of mulberry leaf compound preparations.
The concentration of the fluorenes methoxy dicarbonyl chloride in the solution of described fluorenes methoxy dicarbonyl chloride is 20mmol/L.
The preferred solvents ground of the solution of described fluorenes methoxy dicarbonyl chloride is acetonitrile.
The volume ratio of the solution of solution, buffer solution and the fluorenes methoxy dicarbonyl chloride of described mulberry leaf compound preparation is preferably 1:1:4.
Method and condition that the described solution by the solution of mulberry leaf compound preparation, buffer solution and fluorenes methoxy dicarbonyl chloride mixes are method and the condition of the reagent mix of this area routine.The described solution by the solution of mulberry leaf compound preparation, buffer solution and fluorenes methoxy dicarbonyl chloride mixes preferably for carrying out ultrasonic processing after shaking up.Described ultrasonic processing preferably for to process 20min under power 100W and frequency 40kHz condition.
The addition of described glycocoll is preferably the glycocoll that every Ke Sangye compound preparation adds 0.05-0.1mol, is more preferably the glycocoll that every Ke Sangye compound preparation adds 0.08mol.
Described filtration was preferably the organic system miillpore filter of 0.45 μ m.
Two of object of the present invention is, the detection method of 1-DNJ in a kind of mulberry leaf compound preparation is provided, described detection method comprises the following steps: (1) carries out mulberry leaf compound preparation the preprocess method processing of mulberry leaf compound preparation as above, obtains testing sample solution; (2) described testing sample solution is detected.
In step (2), described detection is preferably that high performance liquid chromatography-ultraviolet detects.
The chromatographic column of the detection of described high performance liquid chromatography is preferably C
18chromatographic column is more preferably Agilent20RBAX SB-C18; The specification of described Agilent20RBAX SB-C18 is preferably 4.6*150mm*5 μ m or 4.6*250mm*5 μ m.
The mobile phase of the detection of described high performance liquid chromatography preferably comprises: mobile phase A: acetonitrile; The vinegar aqueous acid of Mobile phase B: 0.1%-0.2%; Described mobile phase A and the volume ratio of Mobile phase B are preferably (28-32): (72-68), be more preferably 32:68; Described number percent is mass percent.The concentration of described vinegar aqueous acid is preferably 0.2%, and described number percent is mass percent.
The column temperature of the detection of described high performance liquid chromatography is preferably 30 DEG C.
The flow velocity of the detection of described high performance liquid chromatography is preferably 1.0mL/min.
The wavelength of the detection of described high performance liquid chromatography is preferably 265nm.
The sample size that described high performance liquid chromatography-ultraviolet detects is preferably 10-20 μ L.
After described detection, preferably calculate the content of 1-DNJ in mulberry leaf compound preparation according to the calibration curve method of this area routine.The operation steps of described calibration curve method is preferably: be mixed with standard model with standard model, and under the chromatographic condition identical with testing sample, equal-volume sample introduction; Measure the peak area at each peak of each standard model solution, with facing sample concentration drawing standard curve in peak.According to the peak area of the testing sample recording, calculate the concentration of testing sample again.
Wherein, the preprocess method of described standard model is identical with the preprocess method of the mulberry leaf compound preparation in above-mentioned steps (1).
In the time quantitatively detecting, testing sample solution, according to this area routine, refilters after first constant volume.Described constant volume is preferably for using acetic acid constant volume.The concentration of described acetic acid is preferably 0.1%-0.2%, is more preferably 0.1%, and described number percent is mass percent.
Meeting on the basis of this area general knowledge, above-mentioned each optimum condition, can combination in any, obtains the preferred embodiments of the invention.
Agents useful for same of the present invention and raw material be commercially available obtaining all.
Positive progressive effect of the present invention is: preprocess method of the present invention is quick and convenient, and degree of separation is good, can accurately obtain object, and stability is high, highly sensitive, error is little, this detection method testing result exact method, product quality that can objective appraisal compound preparation.
Brief description of the drawings
Fig. 1 is the typical curve that embodiment 1 makes.
Fig. 2 is specificity experimental result picture; Wherein, A: the chromatogram of negative sample (having removed the Xi Er board compound mulberry leaf capsule of element of mulberry leaf) solution; B: the chromatogram of testing sample (Xi Er board compound mulberry leaf capsule) solution; The chromatogram of C:1-DNJ standard model solution.
Embodiment
Mode below by embodiment further illustrates the present invention, but does not therefore limit the present invention among described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example, according to conventional method and condition, or selects according to catalogue.
In following examples, the instrument of use, material and reagent are as follows:
Liquid chromatograph: Agilent company of the U.S. produces, model: 1260, this liquid chromatograph comprises binary pump, automatic sampler, column oven, detecting device, workstation;
KQ-250DB numerical control supersonic cleaning apparatus: Kunshan Ultrasonic Instruments Co., Ltd.;
PH accommodometer: Shanghai Yi electricity scientific instrument company limited, model: PHS-3C;
Balance: METTLER200 type electronic balance, BP211D electronic analytical balance (SartoriusCO.).
Standard model: 1-DNJ, upper Hiroad standing grain Pharmaceutical Technology Co., Ltd;
Testing sample: Xi Er board compound mulberry leaf capsule, the natural medicine company of Hunan Xi Er.
Except acetonitrile is chromatographically pure, it is pure that all the other reagent are analysis.
Embodiment 1
The preprocess method of mulberry leaf compound preparation, comprises the following steps:
Get the measuring bottle that Xi Er board compound mulberry leaf capsule 0.2907g is placed in 25mL, add percent by volume 100% ethanol 2mL, ultrasonic processing 10min, ethanol is settled to scale, is storing solution; Get the measuring bottle that 1mL storing solution is placed in 25mL, add boric acid-potassium chloride damping fluid (pH8.5) 1mL of 0.2mol/L, add again the acetonitrile solution 4mL of the fluorene methyl dicarbonyl chloride of 20mmol/L, shake up, ultrasonic processing (power 100w, frequency 40kHz) 20min, then add the glycocoll 2mL of 0.05mol/L, shake up; Be settled to scale with 0.1% acetum, shake up, cross 0.45 μ m filter membrane.
The detection method of 1-DNJ in mulberry leaf compound preparation
(1) preparation of typical curve
Getting the measuring bottle that 1-DNJ standard items are placed in 25mL, add 80% dissolve with methanol solution, be made into the solution of the 1-DNJ of 0.2mg/mL, is storing solution;
1mL storing solution is placed in to the measuring bottle of 25mL, add boric acid-potassium chloride damping fluid (pH8.5) 1mL of 0.2mol/L, add again the acetonitrile solution 4mL of the fluorene methyl dicarbonyl chloride of 20mmol/L, shake up, ultrasonic processing (power 100w, frequency 40kHz) 20min, then add the glycocoll 2mL of 0.05mol/L, shake up; Be settled to scale with 0.1% acetum, shake up, obtain standard model solution;
Standard model solution, according to volume 4 μ L, 8 μ L, 10 μ L, 12 μ L, 14 μ L, 18 μ L, 20 μ L sample introduction respectively, is carried out to high performance liquid chromatography-ultraviolet and detects;
The testing conditions that described high performance liquid chromatography-ultraviolet detects is:
Chromatographic column: 20RBAX SB-C18(Agilent) (specification 4.6*150mm*5 μ is m)
Mobile phase: mobile phase A: acetonitrile; The vinegar aqueous acid of Mobile phase B: mass percent 0.1%-0.2%; The volume ratio of mobile phase A and Mobile phase B is 32:68;
Column temperature: 30 DEG C;
Flow velocity: 1.0mL/min;
Wavelength: 265nm;
The measurement result of peak area is as shown in table 1;
Table 1:1-DNJ typical curve data
| Sampling volume μ L | 4 | 8 | 10 | 12 | 14 | 18 | 20 |
| DNJ quality μ g | 0.032384 | 0.064768 | 0.08096 | 0.097152 | 0.11334 | 0.145728 | 0.16192 |
| Peak area | 191.893 | 387.586 | 484.807 | 581.579 | 678.240 | 873.446 | 971.617 |
Obtain thus typical curve equation: Y=6012.7X-2.4587, R=1; Wherein: X represents the quality of DNJ, μ g; Y represents peak area.
Concentration drawing standard curve with peak area to standard model solution, the typical curve making as shown in Figure 1.
(2) detect
Get the testing sample solution that the pre-service of embodiment 1 obtains, carry out high performance liquid chromatography-ultraviolet and detect, the testing conditions when testing conditions that high performance liquid chromatography-ultraviolet detects is prepared with typical curve, sample size is 10 μ L.
The preprocess method of testing result: embodiment 1, the complex compound of generation obtains good separation with other chemical compositions.It is 272.30 that the solution of mulberry leaf compound preparation detects the peak area obtaining; According to above-mentioned typical curve equation, the quality that calculates DNJ in 10 μ L samples is 0.0457 μ g again, and in sample, the quality of DNJ is 2856 μ g; The content of DNJ is 0.983%.
Embodiment 3
The preprocess method of mulberry leaf compound preparation, comprises the following steps:
Get the measuring bottle that Xi Er board compound mulberry leaf capsule 0.06350g is placed in 25mL, add 80% methyl alcohol 2mL, shake up; Add boric acid-potassium chloride damping fluid (pH8.5) 1mL of 0.2mol/L, then add the acetonitrile solution 4mL of the fluorene methyl dicarbonyl chloride of 20mmol/L, shake up, ultrasonic processing (power 100w, frequency 40kHz) 20min, then add the glycocoll 2mL of 0.05mol/L, shake up; Be settled to scale with 0.1% acetum, cross 0.45 μ m filter membrane;
Embodiment 4
Carry out the preparation of typical curve, preparation method is with embodiment 2.
The testing sample solution that embodiment 3 preprocess methods are obtained carries out high performance liquid chromatography-ultraviolet and detects, and the testing conditions that high performance liquid chromatography-ultraviolet detects is with embodiment 2, and sample size is 20 μ L.
The preprocess method of testing result: embodiment 3, the complex compound of generation obtains good separation with other chemical compositions.It is 299.64 that testing sample detects the peak area obtaining; Press again the regression equation of standard specimen: Y=6012.7X-2.4587(R=1 according to DNJ-FMOC chromatographic peak area; Wherein: X represents the quality of DNJ, μ g; Y represents peak area);
The quality that the quality that calculates DNJ in 20 μ L samples is DNJ is 0.0502 μ g, and in sample, the quality of DNJ is 628 μ g; The content of DNJ is 0.989%.
Embodiment 5
(1) preparation of typical curve, preparation method is with embodiment 2;
(2) pre-service of mulberry leaf compound preparation:
Get the measuring bottle that Xi Er board compound mulberry leaf capsule 0.3755g is placed in 25mL, add percent by volume 80% methyl alcohol 2mL, ultrasonic processing 10min, 80% methanol constant volume, to scale, is storing solution; Get the measuring bottle that 1mL storing solution is placed in 25mL, add boric acid-potassium chloride damping fluid (pH8.5) 1mL of 0.2mol/L, add again the acetonitrile solution 4mL of the fluorene methyl dicarbonyl chloride of 20mmol/L, shake up, ultrasonic processing (power 100w, frequency 40kHz) 20min, then add the glycocoll 2mL of 0.05mol/L, shake up; Be settled to scale with 0.1% acetum, shake up, cross 0.45 μ m filter membrane;
(3) carry out high performance liquid chromatography-ultraviolet and detect, the testing conditions that high performance liquid chromatography-ultraviolet detects is with embodiment 2, and sample size is 10 μ L.
Testing result: peak area 375.8; The quality that calculates DNJ in 10 μ L samples is 0.0629 μ g; In sample, the quality of DNJ is 3942.8 μ g; The content of DNJ is 1.05%.
(1) preparation of typical curve, preparation method is with embodiment 2;
(2) pre-service of mulberry leaf compound preparation:
Get the measuring bottle that Xi Er board compound mulberry leaf capsule 0.3621g is placed in 25mL, add percent by volume 80% ethanol 2mL, ultrasonic processing 10min, 80% ethanol is settled to scale, is storing solution; Get the measuring bottle that 1mL storing solution is placed in 25mL, add boric acid-potassium chloride damping fluid (pH8.5) 1mL of 0.2mol/L, add again the acetonitrile solution 4mL of the fluorene methyl dicarbonyl chloride of 20mmol/L, shake up, ultrasonic processing (power 100w, frequency 40kHz) 20min, then add the glycocoll 2mL of 0.05mol/L, shake up; Be settled to scale with 0.1% acetum, shake up, cross 0.45 μ m filter membrane;
(3) carry out high performance liquid chromatography-ultraviolet and detect, the testing conditions that high performance liquid chromatography-ultraviolet detects is with embodiment 2, and sample size is 10 μ L.
Testing result: peak area 329.4; The quality that calculates DNJ in 10 μ L samples is 0.0552 μ g; In sample, the quality of DNJ is 3476.2 μ g; The content of DNJ is 0.96%.
Embodiment 7
(1) preparation of typical curve, preparation method is with embodiment 2;
(2) pre-service of mulberry leaf compound preparation:
Get the measuring bottle that Xi Er board compound mulberry leaf capsule 0.04011 is placed in 25mL, add 100% ethanol 2mL, shake up; Add boric acid-potassium chloride damping fluid (pH8.5) 1mL of 0.2mol/L, then add the acetonitrile solution 4mL of the fluorene methyl dicarbonyl chloride of 20mmol/L, shake up, ultrasonic processing (power 100w, frequency 40kHz) 20min, then add the glycocoll 2mL of 0.05mol/L, shake up; Be settled to scale with 0.1% acetum, cross 0.45 μ m filter membrane;
(3) carry out high performance liquid chromatography-ultraviolet and detect, the testing conditions that high performance liquid chromatography-ultraviolet detects is with embodiment 2, and sample size is 10 μ L.
Testing result: peak area 408.6; The quality that calculates DNJ in 10 μ L samples is 0.0684 μ g; In sample, the quality of DNJ is 429.2 μ g; The content of DNJ is 1.07%.
Comparative example 1
Preprocess method and detection method are any in following methods:
1-1：
Pre-service: the Xi Er board compound mulberry leaf capsule of getting 0.2999g is dissolved in water, crosses 732 type resin cations (3*20cm), with 10% the ammonia scrubbing of 250mL, collect eluent, concentrate drying, is transferred in the volumetric flask of 25mL, add methanol constant volume to scale, shake up;
Detect: high performance liquid chromatography-ultraviolet detects; Testing conditions is with embodiment 2;
Testing result: without peak area.
1-2：
Pre-service: the Xi Er board compound mulberry leaf capsule of getting 0.2999g is dissolved in water, crosses 732 type resin cations (3*20cm), and with 80% the alcohol washing of 250mL, concentrate drying, is transferred in the volumetric flask of 25mL, adds methanol constant volume to scale, shakes up;
Detect: carry out high performance liquid chromatography-ultraviolet and detect; Testing conditions is with embodiment 2;
Testing result: without peak area.
Comparative example 2
In preprocessing process, different kinds of liquid solvents is on detecting the investigation of termination impact really
Investigation method: Xi Er board compound mulberry leaf capsule is put in the measuring bottle of 25mL, and the kind of the quality of sample and the solvent adding is as follows; The addition of solvent is 2mL, and sample size is 10 μ L; Wherein group 2-7 refilters after representing to add the water of 10mL; All the other testing conditions are with the testing conditions in embodiment 2, and testing result is as shown in table 2;
Table 2: the impact of different kinds of liquid solvents on testing result
| Group | Solvent | Sample heavy (g) | Peak area | Have or not acromion or hangover |
| 2-1 | Ethanol | 0.02907 | 685.87 | Without acromion |
| 2-2 | The methyl alcohol of percent by |
0.03230 | 702.98 | Without acromion |
| 2-3 | Water | 0.03002 | 314.83 | There is acromion |
| 2-4 | Mass percent 0.1% vinegar aqueous acid | 0.02930 | 302.32 | There is acromion |
| 2-5 | The methyl alcohol of percent by volume 50% | 0.03539 | 533.36 | Hangover |
| 2-6 | Methyl alcohol | 0.02930 | 1379.90 | Hangover |
| 2-7 | Water (10mL) filters | 0.03209 | 90.75 | Without acromion |
As shown in Table 2: in group 2-1 and 2-2, added the methyl alcohol of ethanol and 80%, in the chromatogram obtaining, without acromion or conditions of streaking, and measurement result is accurate.
In group 2-3 to 2-6, due to the kind difference of solvent, testing result is in chromatogram, to have acromion or hangover to occur, show that degree of separation is bad, accuracy is low.
In group 2-7, peak area is very little, may be due in the process of filtering, and after DNJ is adsorbed, removes, and has reduced the content of the DNJ in testing sample solution, and therefore, measurement result is inaccurate.
Effect embodiment 1
Precision Experiment
Get standard model, repeat to test 6 times, preprocess method is with the preparation of the typical curve of embodiment 2, and testing conditions is with the testing conditions of embodiment 2, and sample size is 10 μ L.With measurement result to recently determining the precision of detection method of the present invention.Its result is as shown in table 3;
Table 3: precision measurement result (n=6)
As shown in Table 3: detection method RSD of the present invention is 0.09976%, and withinday precision is good.
Stability experiment
Get standard model, in different time sections, repeat 9 experiments, preprocess method is with the preparation of the typical curve of embodiment 2, and testing conditions is with the testing conditions of embodiment 2, sample size is 10 μ L, with the measurement result of different time sections to recently determining the stability of detection method of the present invention.Its result is as shown in table 4;
Table 4: Stability Determination result
As shown in Table 4: detection method of the present invention is good at 72h internal stability.
Effect embodiment 3
Repeated experiment
The Xi Er board compound mulberry leaf capsule of getting same batch of different quality, repeats to test 5 times according to the method for embodiment 2, with measurement result to recently determining the repeatability of detection method of the present invention.Its result is as shown in table 5;
Table 5: repeated measurement result
As shown in Table 6: repeat after 5 times, detection method RSD of the present invention is low, illustrates that detection method of the present invention is reproducible.
Effect embodiment 4
Average recovery experiment
Get the solution (being respectively 2 1.5mL and 2 2mL according to group 4-2 to 4-5 volume) that difference is got the testing sample of testing sample content, the solvent in the solution of testing sample is 80% methyl alcohol; Get 1-DNJ (DNJ) standard items 2.93mg, the methanol constant volume with 80%, in 10ml measuring bottle to scale, is designated as group 4-1.The 1-DNJ standard solution that adds respectively specified quantitative in above-mentioned solution, in this solution, the addition of 1-DNJ is as shown in table 6; Detect according to the detection method shown in embodiment 4, obtain the amount of recording.And calculate recovery data according to the computing formula of average recovery.The computing formula of average recovery is:
The average recovery %=(amount of recording-sample size)/actual addition × 100%;
Result of calculation is as shown in table 6;
Table 6: recovery testu result
As shown in Table 6: detection method average recovery of the present invention is high.
Effect embodiment 5
Specificity experiment
Standard items: 1-DNJ standard model; The preparation method of standard solution is with embodiment 1;
Testing sample: Xi Er board compound mulberry leaf capsule; The preparation method of testing sample solution is with embodiment 2;
Negative control: the Xi Er board compound mulberry leaf capsule (its composition mainly comprises in 0.3g: the ophiopogon japonicus extract of the Bitter Melon P.E of 0.1016g, the Astragalus Root P.E of 0.0508g and 0.0200g) of having removed element of mulberry leaf; The preprocess method of negative control solution is with embodiment 1.
Specificity experimental technique: measuring under identical condition with testing sample, carry out the detection of negative control solution, testing conditions is with embodiment 2, and sampling volume is 10 μ L.
The chromatogram that detection obtains as shown in Figure 2, in Fig. 2: standard model and testing sample solution all go out peak, the peak that this peak is DNJ in about 6min.In negative control solution, the appearance time of all the other materials and the appearance time of DNJ do not have overlapping.During high efficiency liquid phase-ultraviolet detects in the retention time of 1-DNJ, only there is 1-DNJ compound composition, other compositions do not disturb the detection of the 1-DNJ of this time period, and therefore the assay of 1-DNJ is not subject to the impact of all the other materials; Testing result can be got rid of false positive; Detection method specificity of the present invention is good.
Claims (10)
1. the preprocess method of mulberry leaf compound preparation, is characterized in that: described preprocess method comprises the following steps:
The solution of the solution of mulberry leaf compound preparation, buffer solution and fluorenes methoxy dicarbonyl chloride is mixed, then mixes with glycocoll, filter; Wherein, the solvent in the solution of described mulberry leaf compound preparation is the methanol solution of percent by volume 80% and/or the ethanolic solution of percent by volume 80%-100%; Described buffer solution is borate buffer solution.
2. preprocess method as claimed in claim 1, is characterized in that: described borate buffer solution is boric acid-potassium chloride damping fluid; The concentration of described boric acid-potassium chloride damping fluid is preferably 0.2mol/L; The pH value of described boric acid-potassium chloride damping fluid is preferably pH8.5; The addition of described boric acid-potassium chloride damping fluid preferably adds boric acid-potassium chloride damping fluid of 1.67mL for every Ke Sangye compound preparation;
The solvent of the solution of described fluorenes methoxy dicarbonyl chloride is acetonitrile.
3. preprocess method as claimed in claim 1, it is characterized in that: the addition of the solution of described fluorenes methoxy dicarbonyl chloride is counted the fluorenes methoxy dicarbonyl chloride that adds 10mmol in every 1-3 gram testing sample with fluorenes methoxy dicarbonyl chloride, preferably for preferably adding the fluorenes methoxy dicarbonyl chloride of 10mmol in every 1.5 grams of mulberry leaf compound preparations; The concentration of the fluorenes methoxy dicarbonyl chloride in the solution of described fluorenes methoxy dicarbonyl chloride is preferably 20mmol/L.
4. preprocess method as claimed in claim 1, is characterized in that: the volume ratio of the solution of solution, buffer solution and the fluorenes methoxy dicarbonyl chloride of described mulberry leaf compound preparation is 1:1:4.
5. preprocess method as claimed in claim 1, it is characterized in that: described mulberry leaf compound preparation comprises the mulberry-leaf extract of 40%-50%, Bitter Melon P.E, the Astragalus Root P.E of 12%-17% and the ophiopogon japonicus extract of 5%-9% of 25%-35%, and described number percent is mass percent; Described mulberry leaf compound preparation is preferably Xi Er board compound mulberry leaf capsule;
The preparation method of the solution of described mulberry leaf compound preparation is preferably: described mulberry leaf compound preparation is dissolved in described solvent, shakes up and/or ultrasonic processing; Described ultrasonic processing preferably for processing 20min under power 100W and frequency 40kHz condition;
In the solution of described mulberry leaf compound preparation, the addition of described solvent is preferably the solvent that every 0.2-0.6g mulberry leaf compound preparation adds 25mL, is more preferably the solvent that every 0.3g mulberry leaf compound preparation adds 25mL;
The method that the described solution by the solution of mulberry leaf compound preparation, buffer solution and fluorenes methoxy dicarbonyl chloride mixes is for carrying out ultrasonic processing after shaking up; Described ultrasonic processing preferably for to process 20min under power 100W and frequency 40kHz condition.
6. the preprocess method as described in any one in claim 1-5, is characterized in that: the addition of described glycocoll is the glycocoll that every Ke Sangye compound preparation adds 0.05-0.1mol, is preferably the glycocoll that every Ke Sangye compound preparation adds 0.08mol;
Described filtration was preferably the organic system miillpore filter of 0.45 μ m.
7. the detection method of 1-DNJ in a mulberry leaf compound preparation, it is characterized in that: described detection method comprises the following steps: (1) carries out mulberry leaf compound preparation the preprocess method processing of the mulberry leaf compound preparation as described in any one in claim 1-6, obtains testing sample solution; (2) described testing sample solution is detected.
8. detection method as claimed in claim 7, is characterized in that: in step (2), described detection is that high performance liquid chromatography-ultraviolet detects.
9. detection method as claimed in claim 7, is characterized in that: the chromatographic column of the detection of described high performance liquid chromatography is C
18chromatographic column is preferably Agilent20RBAX SB-C18; The specification of described Agilent20RBAX SB-C18 is preferably 4.6*150mm*5 μ m or 4.6*250mm*5 μ m;
The mobile phase of the detection of described high performance liquid chromatography comprises: mobile phase A: acetonitrile; The vinegar aqueous acid of Mobile phase B: 0.1%-0.2%; Described mobile phase A and the volume ratio of Mobile phase B are preferably (28-32): (72-68), be more preferably 32:68; Described number percent is mass percent; The concentration of described vinegar aqueous acid is preferably 0.2%, and described number percent is mass percent;
The column temperature of the detection of described high performance liquid chromatography is 30 DEG C; The flow velocity of the detection of described high performance liquid chromatography is 1.0mL/min; The wavelength of the detection of described high performance liquid chromatography is 265nm; The sample size that described high performance liquid chromatography-ultraviolet detects is 10-20 μ L.
10. detection method as claimed in claim 7, is characterized in that: the content that calculates 1-DNJ in mulberry leaf compound preparation after described detection by calibration curve method; The preprocess method of described calibration curve method Plays sample is identical with the preprocess method of the mulberry leaf compound preparation in described step (1).
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| CN107192777A (en) * | 2017-05-24 | 2017-09-22 | 江苏耐雀生物工程技术有限公司 | The detection method of 1 DNJ content in a kind of mulberry-leaf extract |
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Non-Patent Citations (3)
| Title |
|---|
| KAZUHISA YATSUNAMI，ET AL: "1-Deoxynojirimycin Content and Alfa-Glucosidase Inhibitory Activity and Heat Stability of 1-Deoxynojirimycin in Silkworm Powder", 《FOOD AND NUTRITION SCIENCES》, vol. 2, 30 April 2011 (2011-04-30), pages 87 - 89 * |
| 朱见: "桑叶、蚕体中1-脱氧野尻霉素的含量测定与分析", 《西南大学硕士学位论文》, 31 December 2008 (2008-12-31) * |
| 花俊丽等: "超声波法提取1-脱氧野尻霉素", 《食品与生物技术学报》, vol. 28, no. 6, 23 November 2009 (2009-11-23) * |
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| CN107192777A (en) * | 2017-05-24 | 2017-09-22 | 江苏耐雀生物工程技术有限公司 | The detection method of 1 DNJ content in a kind of mulberry-leaf extract |
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