CN103076325B - Quality control method for blood sugar-lowering preparation - Google Patents
Quality control method for blood sugar-lowering preparation Download PDFInfo
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- CN103076325B CN103076325B CN201210592158.3A CN201210592158A CN103076325B CN 103076325 B CN103076325 B CN 103076325B CN 201210592158 A CN201210592158 A CN 201210592158A CN 103076325 B CN103076325 B CN 103076325B
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Abstract
A detection method for a blood sugar-lowering preparation is extracted from main raw materials of L-arabinose and oligochitosan, and is used for quantitatively testing the L-arabinose and qualitatively identifying the oligochitosan respectively, wherein a method for quantitatively testing the L-arabinose comprises the following steps: adding a sodium hydroxide solution, and a methanol solution of 1-phenyl- 3-methyl-5-pyrazolone into a test sample to be tested for derivatization reaction, after completion of the derivatization reaction, adding a proper amount of hydrochloric acid for neutralization, filtering by using a filtration membrane and performing highly-efficient liquid chromatography; and a method for qualitatively identifying the oligochitosan comprises chemical chromogenic identification or thin-layer chromatographic identification. By the detection method for the blood sugar-lowering preparation, good quality control of the blood sugar-lowering preparation can be achieved.
Description
Technical field
The present invention relates to a kind of detection method of tool function of blood sugar reduction preparation, refer to a kind of compound preparation detection method made for primary raw material with Arabinose and chitosan oligosaccharide especially, belong to the technical field of food or health food being carried out to quality control.
Background technology
It is a kind of that to have function of blood sugar reduction preparation be a kind of compound preparation by making for primary raw material with Arabinose and chitosan oligosaccharide that the applicant develops, be that 201210333553X name is called a kind ofly have the composition of auxiliary hyperglycemic function and the patented claim of goods thereof see the applicant in the application number that 2012-09-10 applies for, chitosan oligosaccharide is by β-1 by 2-20 D-Glucosamine, the water soluble oligosaccharide of 4 glycosidic bond compositions, the product of shitosan after chemistry or enzyme degraded, Arabinose is valeral monose, usually be combined with other monose, be present in widely in plant with heteroglycan form.Clinical trial certificate, both all have hypoglycemic activity, chitosan oligosaccharide and Arabinose are carried out combining to take to have synergy, secreted by the promotion human insulin in conjunction with chitosan oligosaccharide, increase the effect of islet receptor susceptibility, suppress sucrose degradation with Arabinose, reduce glucose absorption effect and combine, play dual regulation, can effectively blood glucose value be controlled within the scope of human normal.
Quality standard has great significance to product safety is stable, quality controllable, and this product is compound preparation, qualitatively or quantitatively determines functional component in product, by the degree of controllability improved the quality of products, ensures product curative effect.In view of there is no chitosan oligosaccharide discrimination method in preparation, Arabinose content assaying method at present, the present invention will set up chitosan oligosaccharide discrimination method and Arabinose content assaying method in preparation first.
Summary of the invention
A kind of detection method containing Arabinose and chitosan oligosaccharide, tool function of blood sugar reduction preparation of the present invention, comprise proterties, inspection, discriminating and assay, wherein differentiate employing chemical staining method and thin-layered chromatography to differentiate chitosan oligosaccharide in preparation, assay adopts the Arabinose of high performance liquid chromatography to derivatization to detect.
Through literature query and experimental studies have found that, this product raw material chitosan oligosaccharide is polysaccharide composition, standard items cannot be provided, therefore cannot assay be carried out, but there is distinctive free amine group in its molecular structure, thus can carry out the discriminating of Elson-Morgan method and TLC distinguish to the peculiar Glucosamine produced after hydrolysis, specificity is strong.
Arabinose can adopt high-efficient liquid phase technique to carry out assay, but adopt efficient liquid phase-Composition distribution, efficient liquid phase-evaporative light detecting device method is when measuring, there is severe jamming in the mensuration of polysaccharide chitosan oligosaccharide to Arabinose, and when adopting column front derivation-efficient liquid phase-ultraviolet spectrophotometer to measure, the interference of assay can be effectively reduced.
Have a detection method for function of blood sugar reduction preparation, said preparation is made for primary raw material extracts with Arabinose and chitosan oligosaccharide, carries out quantitative measurement and Qualitive test respectively to Arabinose and chitosan oligosaccharide, wherein,
As follows to the method for quantitatively determining of Arabinose: the methanol solution adding sodium hydroxide solution and 1-phenyl-3-methyl-5-pyrazolones ketone in sample to be tested carries out derivative reaction, add appropriate hydrochloric acid neutralization after reaction terminates, after membrane filtration, carry out high-performance liquid chromatogram determination;
Chemical colour reaction is adopted to differentiate or indentification by TLC to the Qualitive test of chitosan oligosaccharide.
The chromatographic condition of described high-performance liquid chromatogram determination is take octadecylsilane chemically bonded silica as filling agent, 0.1-0.2mol/L pH5.0-6.0 ammonium acetate buffer solution-acetonitrile is mobile phase, the volume ratio of ammonium acetate buffer solution-acetonitrile is 80-70:20:30, and determined wavelength is 244-252nm.
When in described response sample solution, Arabinose concentration is 0.0133mol/L, the concentration of sodium hydroxide solution is 0.07-0.15mol/L, the concentration of methanol solution of 1-phenyl-3-methyl-5-pyrazolones ketone is 0.2-0.4mol/L, namely controls NaOH, the mol ratio of 1-phenyl-3-methyl-5-pyrazolones ketone and Arabinose three is 5.22-11.18:11.21-22.43:1.
Described derivative reaction condition is: react at least 30min in 60-80 DEG C of water-bath, and every 10-20min jolting once.
Described chemical colour reaction discrimination method is according to forming the former 2-methyl-3-diacetyl azole derivatives that adds lustre under chitosan oligosaccharide acid condition with diacetone condensation, add lustre to and formerly in concentrated hydrochloric acid methanol solution, complex reaction occurs with p-dimethylbenzaldehyde again, generate purple solution, thus differentiate.
Described TLC Identification be with hydrochloric acid hydrolysis after chitosan oligosaccharide be test sample, aminoglucose hydrochloride is reference substance, the methyl alcohol with volume ratio 7-9:3-1: ammoniacal liquor is developping agent, and ninhydrin solution is the TLC Identification of developer.
A kind of detection method with function of blood sugar reduction preparation that the present invention sets up, chemical staining method discriminating is carried out according to feature amino group in chitosan oligosaccharide molecule, or be that reference substance carries out TLC distinguish to the chitosan oligosaccharide after hydrolysis with aminoglucose hydrochloride, after derivatization is adopted to Arabinose in preparation, adopt high performance liquid chromatography-ultraviolet spectroscopy to measure, the good quality that can realize having function of blood sugar reduction preparation controls.
Accompanying drawing explanation
Accompanying drawing 1 is chemical colour reaction discrimination test collection of illustrative plates of the present invention, wherein 1 is blank solvent, 2 is negative blank, 3 is reference substance solution, 4 is need testing solution (lot number: 20120501), 5 be need testing solution (lot number: 20120502), 6 is need testing solution (lot number: 20120503);
Accompanying drawing 2 is indentification by TLC collection of illustrative plates of the present invention, and wherein 1 is reference substance solution, and 2 is formulation samples solution, and 3 is chitosan oligosaccharide material solution, and 4 is the non-hydrating solution preparation of chitosan oligosaccharide, and 5 is negative control solution preparation;
Accompanying drawing 3 is assay of the present invention system suitability test-reference substance solution collection of illustrative plates, and wherein 1 is derivative reagent peak, and 2 is derivatization Arabinose peak;
Accompanying drawing 4 is assay of the present invention system suitability test-need testing solution collection of illustrative plates, and wherein 1 is derivative reagent peak, and 2 is derivatization Arabinose peak;
Accompanying drawing 5 is assay of the present invention system suitability test-negative control solution collection of illustrative plates, and wherein 1 is derivative reagent peak;
Fig. 6 is Arabinose content range of linearity figure in 4.4 joints of embodiment.
Embodiment
For the ease of understanding the present invention, spy enumerates following examples.Its effect is understood to be explaination of the present invention but not to any type of restriction of the present invention.
The chemical colour reaction differential method that embodiment one one kinds has function of blood sugar reduction preparation is investigated
The identification experiment of chitosan oligosaccharide carries out with reference to Elson-Morgan method, and is improved.Its principle is that chitosan oligosaccharide can generate Glucosamine monose through hydrochloric acid hydrolysis, free amine group in Glucosamine structure forms the former 2-methyl 3-diacetyl azole derivatives that adds lustre in the basic conditions with diacetone condensation, add lustre to and formerly in concentrated hydrochloric acid methanol solution, complex reaction occurs with P-DABA (p-dimethylaminobenzaldehyde) again, generate purple solution.
Test method
This product content 0.1g is got in need testing solution preparation, puts in tool plug test tube, adds hydrochloric acid 3ml, close plug, heating water bath 3 hours, after being cooled to room temperature, in the 10ml gradation that adds water washing evaporating dish, evaporate to dryness, the 30ml gradation that adds water is dissolved, and filter, filtrate is incorporated in 50ml measuring bottle, be diluted with water to scale, obtain final product.
It is appropriate that aminoglucose hydrochloride reference substance is got in reference substance solution preparation, adds water and make the solution of every 1ml containing 0.5mg, product solution in contrast.
Scarce chitosan oligosaccharide sample (i.e. Arabinose) 0.05g is got in negative control solution preparation, puts in tool plug test tube, adds hydrochloric acid 3ml, close plug, heating water bath 3 hours, after being cooled to room temperature, in the 10ml gradation that adds water washing evaporating dish, evaporate to dryness, the 30ml gradation that adds water is dissolved, and filter, filtrate is incorporated in 50ml measuring bottle, be diluted with water to scale, obtain final product.
Getting each 5ml of reference substance solution, need testing solution and negative control solution respectively puts in tool plug test tube respectively, separately get 1, tool plug test tube, adding distil water 5ml is as solvent blank, respectively add diacetone test solution and (get diacetone 2ml, add 0.5mol/L sodium carbonate liquor to 50ml, configure before use) 1.0ml, shake up, put in boiling water bath, heat 25 minutes, take out, after cooling rapidly with frozen water, (paradime thylaminobenzaldehyde 0.8g, adds ethanol,aldehyde free 15ml and hydrochloric acid 15ml to add paradime thylaminobenzaldehyde test solution, shake up) 1.0ml.
Experimentally result, need testing solution and reference substance are composed, equal displaing amaranth, and negative control solution and blank reagent all do not develop the color, therefore this method can be used for the discriminating of chitosan oligosaccharide.The results are shown in Figure 1.
Investigate result according to above-mentioned chemical colour reaction differential method, it is as follows that formulation one has chitosan oligosaccharide chemical colour reaction discrimination method in function of blood sugar reduction preparation:
Get this product content 0.1g, put in tool plug test tube, add hydrochloric acid 3ml, close plug, heating water bath 3 hours, after being cooled to room temperature, in the 10ml gradation that adds water washing evaporating dish, evaporate to dryness, the 30ml gradation that adds water is dissolved, filter, filtrate is incorporated in 50ml measuring bottle, be diluted with water to scale, getting 5ml puts in tool plug test tube, add diacetone test solution and (get diacetone 2ml, add 0.5mol/L sodium carbonate liquor to 50ml, configure before use) 1.0ml, shake up, put in boiling water bath, heat 25 minutes, take out, after cooling rapidly with frozen water, add paradime thylaminobenzaldehyde test solution (paradime thylaminobenzaldehyde 0.8g, add ethanol,aldehyde free 15ml and hydrochloric acid 15ml, shake up) 1.0ml, i.e. displaing amaranth.
The tlc identification method that embodiment 21 kinds has function of blood sugar reduction preparation is investigated
Thin-layered chromatography discriminating is carried out to chitosan oligosaccharide hydrolysate Glucosamine, with aminoglucose hydrochloride reference substance, because in structure, 2 hydroxyls are replaced by amino, and present alkalescence, thus methyl alcohol/strong aqua is adopted, the developping agent systems such as chloroform/methanol/strong aqua are investigated, result with methyl alcohol/strong aqua for development system degree of separation is good, clear spot, launch effective, in formulation samples chromatogram, in chitosan oligosaccharide raw material chromatography space on the position corresponding to reference substance chromatogram, the spot of aobvious same color, and lack the negative control chromatogram of chitosan oligosaccharide, be not hydrolyzed in chitosan oligosaccharide chromatogram noiseless, accompanying drawing 2 is shown in by collection of illustrative plates.
Experimental technique is as follows:
Aminoglucose hydrochloride reference substance is got in reference substance solution preparation, adds water and makes the solution of every 1ml containing 5mg, product solution in contrast.
This product content 0.1g is got in the preparation of formulation samples solution, puts in tool plug test tube, adds hydrochloric acid 3ml, close plug, heating water bath 3 hours, after being cooled to room temperature, in the 10ml gradation that adds water washing evaporating dish, evaporate to dryness, the 30ml gradation that adds water is dissolved, and filter, filtrate is incorporated in 50ml measuring bottle, get 20ml and be concentrated into 5ml, to obtain final product.
Chitosan oligosaccharide 0.05g is got in the preparation of chitosan oligosaccharide material solution, puts in 25ml volumetric flask, is dissolved in water and is diluted to scale, to obtain final product.
Chitosan oligosaccharide 0.05g is got in the non-hydrating solution preparation of chitosan oligosaccharide, puts in tool plug test tube, by the preparation of formulation samples solution manufacturing method, to obtain final product.
Negative control sample (i.e. Arabinose) 0.05g of scarce chitosan oligosaccharide is got in negative control solution preparation, by the preparation of formulation samples solution manufacturing method, to obtain final product.
The each 2 μ l of point sample reference substance solution, formulation samples solution, chitosan oligosaccharide material solution, the non-hydrating solution of chitosan oligosaccharide and negative control solution
Thin layer plate silica G plate
Developping agent methyl alcohol: strong aqua=9:1
Expansion mode developping agent pre-equilibration 30 minutes, ascending development
Location is taken out, and dries, and 105 DEG C are dried 3 minutes, spray with ninhydrin solution, dry, inspect under putting daylight at 105 DEG C.
Investigate result according to above-mentioned tlc identification method, it is as follows that formulation one has chitosan oligosaccharide thin-layer identification method in function of blood sugar reduction preparation:
Get this product content 0.1g, put in tool plug test tube, add hydrochloric acid 3ml, close plug, heating water bath 3 hours, after being cooled to room temperature, in the 10ml gradation that adds water washing evaporating dish, evaporate to dryness, the 30ml gradation that adds water is dissolved, filter, filtrate is incorporated in 50ml measuring bottle, gets 20ml and is concentrated into 5ml, as need testing solution; Get aminoglucose hydrochloride reference substance simultaneously, add water and make the solution of every 1ml containing 5mg, product solution in contrast.According to thin-layered chromatography (Chinese Pharmacopoeia version in 2010 annex VI B) test, draw each 2 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, methyl alcohol-ammoniacal liquor (9:1) is developping agent, launch, take out, dry, dry 3 minutes at 105 DEG C, spray, with ninhydrin solution, is dried at 105 DEG C, is inspected under putting daylight, on the position corresponding to reference substance chromatogram, the purple dot of aobvious same color respectively.
Embodiment 31 kinds has the content assaying method Selecting research of function of blood sugar reduction preparation
At present not for method for quantitatively determining and the standard of Arabinose in preparation, consider that carbohydrate can be transformed into the material with uv absorption after derivatization, originally the composition being difficult to detect can be detected by conventional UV-detector, this patent adopts 1-phenyl-3-methyl-5-pyrazolones ketone (PMP) to carry out derivatization treatment for Arabinose in preparation first, to derivative reagent consumption in sample pretreatment process, temperature of reaction, reaction time, alkali consumption, the derivatization conditions such as organic reagent extraction times are investigated, in conjunction with high performance liquid chromatography, draw Arabinose assay condition.
The selection of 3.1 liquid-phase conditions and determined wavelength
Take octadecylsilane chemically bonded silica as filling agent, respectively to 1. water-acetonitrile (75:25) 2. 0.15mol/L (pH5.5) ammonium acetate buffer solution-acetonitrile (75:25) 3. 0.1mol/L (pH6.5) phosphate buffered solution three kinds of flow visualizing investigate, adopt DAD detecting device.
Sample-pretreating method: precision takes 0.2g Arabinose and puts in 100ml volumetric flask, be dissolved in water and be diluted to scale, getting 2ml puts in tool plug test tube, add 0.1mol/L NaOH solution 2ml, add 0.2mol/LPMP methanol solution 1.5ml again, controlling temperature of reaction is 70 DEG C, reaction 60min, period every 20min jolting once, put into cold water after reaction terminates and be cooled to room temperature, and add 0.1mol/L HCL solution 2.5ml and neutralize, wash with water in 100ml volumetric flask, and be settled to scale with water, after filtering with microporous membrane, get 10 μ l injecting chromatographs, record chromatogram, be simultaneously that blank is measured in the same method with reagent.
Experimental result shows, under ammonium acetate buffer solution-acetonitrile mobile system condition, it is best that Arabinose derives peak system flexibility, theoretical cam curve is greater than 4000, be greater than 1.5 with derivative reagent peak degree of separation, thus select 0.15mol/L (pH5.5) ammonium acetate buffer solution-acetonitrile (75:25) for liquid phase assay method.Simultaneously according to DAD full wavelength scanner result, Arabinose chromatographic peak has absorption maximum at 248nm place, and experimental result shows that this liquid-phase condition is applicable to the assay that Arabinose derives peak, and determined wavelength is 248nm.
3.2 derivative reagents are removed and are investigated
Usually unnecessary derivative reagent need be with an organic solvent extracted, to eliminate the interference of derivative reagent to sample determination chromatographic peak after derivatization reaction completes.This experiment is investigated the chloroform extraction process that derivatization reaction terminates rear test solution.Getting 0.2g Arabinose is dissolved in 100ml water, getting 2ml puts in tool plug test tube, add 0.1mol/L NaOH solution 2ml, add 0.2mol/L PMP methanol solution 1.5ml again, controlling temperature of reaction is 70 DEG C, reaction 60min, period every 20min jolting once, add appropriate HCL after reaction terminates to neutralize, wash with water in 50ml volumetric flask, and be settled to scale with water, getting 10ml puts in separating funnel, respectively with after chloroform 10ml extraction once with twice, aqueous solution evaporate to dryness, by water-soluble solution and dilution be settled in 25ml volumetric flask, HPLC is adopted to analyze, get non-extraction solution 10ml, dilute with water is settled in 25ml volumetric flask, HPLC is adopted to analyze, the results are shown in Table 1.
Table 1 extraction times-clearance/recovery is investigated
According to table 1 result, the PMP derivative reagent percentage extraction that employing chloroform extraction is twice is about 70%, but the recovery of Arabinose is less than 90%, affect the detection of Arabinose, and complex operation, poor repeatability, PMP chromatographic peak does not affect the assay of Arabinose simultaneously, and degree of separation is greater than 1.5, PMP derivative reagent peak does not disturb the mensuration of Arabinose chromatographic peak, thus for ensureing assay accuracy, without the need to the derivative reagent adopting chloroform extraction unnecessary.
3.3 NaOH and Arabinose react mol ratio and determine
Getting 0.2g Arabinose is dissolved in 100ml water, get solution 2ml to put in tool plug test tube, add the NaOH solution 2ml of variable concentrations by table 2, then add 0.2mol/L PMP methanol solution 1.5ml, controlling temperature of reaction is 70 DEG C, reaction 60min, period every 20min jolting once, adds appropriate HCL after reaction terminates and neutralizes, wash with water in 100ml volumetric flask, and be settled to scale with water, adopt HPLC to analyze, the results are shown in Table 2.
Table 2 NaOH addition is investigated
According to table 2 result, when add 2ml 0.07mol/L NaOH or add the amount of NaOH and Arabinose ratio be 5.22 time, peak area has maximal value, and continue to strengthen concentration to 0.15mol/L, peak area remains unchanged, find when concentration adds to 0.20mol/L and is above simultaneously, Arabinose is degraded, and be decomposed into two chromatographic peaks, thus reaction conditions is the NaON adding 2ml 0.07-0.15mol/L, and top condition is select to add 2ml 0.07mol/L NaOH or add the amount of NaOH and Arabinose ratio is 5.22.
3.4 derivatization reaction times were investigated
Getting 0.2g Arabinose is dissolved in 100ml water, get 2ml to put in tool plug test tube, add 0.07mol/LNaOH solution 2ml, 0.2mol/L PMP methanol solution 1.5ml, controlling temperature of reaction is 70 DEG C, react 30min, 45min, 60min respectively, period jolting 2 times, add appropriate HCL after reaction terminates and neutralize, wash with water in 100ml volumetric flask, and be settled to scale with water, adopt HPLC to analyze, the results are shown in Table 3.
Table 3 reaction time is investigated
According to table 3 result, react when 45min, peak area reaches maximal value, and compared with during 60min no significant difference, thus the reaction time is decided to be 45min, every 15min jolting once.
3.5 PMP and Arabinose react mol ratio and determine
Getting 0.2g Arabinose is dissolved in 100ml water, get 2ml to put in tool plug test tube, add 0.07mol/LNaOH solution 2ml, according to the form below adds the PMP methanol solution 1.5ml of variable concentrations, control temperature of reaction 70 DEG C, reaction 45min, period every 15min jolting once, adds appropriate HCL after reaction terminates and neutralizes, wash with water in 100ml volumetric flask, and be settled to scale with water, adopt HPLC to analyze, the results are shown in Table 4.
Table 4 PMP addition is investigated
As can be known from Table 4, when add 1.5ml 0.3mol/L PMP methanol solution or add the amount of PMP and Arabinose ratio be about 17 time, Arabinose derives peak-to-peak area maximal value, and concentration increases peak area does not have significant change.Due to the participation reagent that PMP is Arabinose derivatization reaction, consideration method need investigate linearly (sample concentration 70-130%) and the recovery (sample concentration 80-120%), thus PMP addition suitably relaxes, and namely adds 1.5ml 0.4mol/L PMP methanol solution.
3.6 derivatization reaction temperature are investigated
Getting 0.2g Arabinose is dissolved in 100ml water, get 2ml to put in tool plug test tube, add 0.07mol/LNaOH solution 2ml, 0.2mol/L PMP methanol solution 1.5ml, control temperature of reaction 60,70,80 DEG C respectively, reaction 45min, period every 15min jolting once, adds appropriate HCL after reaction terminates and neutralizes, wash with water in 100ml volumetric flask, and be settled to scale with water, adopt HPLC to analyze, the results are shown in Table 5.
Table 5 temperature of reaction is investigated
According to table 5 result, temperature of reaction is 70 DEG C time, and peak area reaches maximal value, and compared with during 80 DEG C of maximal values no significant difference, simultaneous reactions temperature 80 DEG C time between parallel sample checkout discrepancy comparatively large, thus temperature of reaction is decided to be 70 DEG C.
Investigate result according to above-mentioned sample preparation and liquid phase process, it is as follows that formulation one has Arabinose content assaying method in function of blood sugar reduction preparation:
Chromatographic condition and system suitability take octadecylsilane chemically bonded silica as filling agent, 0.15mol/L ammonium acetate buffer solution (gets ammonium acetate 11.6g, be dissolved in water and be diluted to 1000ml, pH to 5.5 is regulated with acetum)-acetonitrile (75:25) is mobile phase, determined wavelength is 248nm.Theoretical cam curve calculates should be not less than 4000 by derivatization Arabinose peak, and derivatization Arabinose peak and derivative reagent peak degree of separation are not less than 1.5.
Determination method is got this product content and is about 0.37g, accurately weighed, put in 100ml volumetric flask, add water appropriate, jolting is dissolved and is diluted to scale, shake up, precision measures 2ml and puts in tool plug test tube, add 2ml 0.07mol/L sodium hydroxide solution and 1.5ml 0.4mol/L PMP (1-phenyl-3-methyl-5-pyrazolones ketone) methanol solution, shake up, be placed in 70 DEG C of water-baths and react 45min, period every 15min jolting once, cold water is put in taking-up, add 2.5ml0.07mol/L hydrochloric acid solution simultaneously, be cooled to room temperature, move in 100ml volumetric flask, the gradation that adds water is washed, washing lotion is incorporated in volumetric flask, thin up is settled to scale, shake up, 0.45 μm of membrane filtration, as need testing solution, precision measures 10 μ l injection liquid chromatographies, record chromatogram, another precision takes Arabinose reference substance and is about 0.2g, puts in 100ml volumetric flask, is measured in the same method.By external standard method with derivatization Arabinose peak-to-peak areal calculation, to obtain final product.
The content assaying method that embodiment 41 kinds has function of blood sugar reduction preparation is investigated
4.1 system suitability test
Prepare reference substance solution and need testing solution in accordance with the law; Separately get scarce Arabinose sample appropriate, prepare negative control solution by the preparation method of need testing solution.Get above-mentioned three kinds of solution, the chromatographic condition formulated by embodiment three is sample introduction analysis respectively, and chromatogram is shown in accompanying drawing 3-5.
Result shows: under this chromatographic condition, and in reference substance solution, in derivatization Arabinose peak and need testing solution, the retention time at derivatization Arabinose peak is basically identical; Negative control solution chromatographic peak is noiseless on derivatization Arabinose position.
4.2 tolerance test
Prepare need testing solution in accordance with the law, respectively tolerance test carried out, record chromatogram to the aqueous-phase concentration of the column temperature of liquid-phase condition, flow velocity, determined wavelength, mobile phase, pH value, aqueous phase and organic phase ratio, method with the results are shown in Table 6.
Table 6 durability experimental result
Test findings shows, under this product chromatographic condition, through different column temperatures, flow velocity, determined wavelength, mobile phase ratio, derivatization Arabinose peak and derivative reagent peak degree of separation are all greater than 1.5, theoretical cam curve is all greater than 4000, shows that this product chromatographic condition durability is good.
4.3 precision test
The method formulated according to embodiment three prepares reference substance solution, and according to the chromatographic condition that embodiment three is formulated, continuous sample introduction 6 times, record derivatization Arabinose peak area, calculates relative standard deviation, the results are shown in Table 7.
Table 7 Precision test result
Experiment shows, under this liquid-phase condition, derivatization Arabinose peak area precision is good.
4.4 linear relationships are investigated
Get Arabinose appropriate, accurately weighed, add water be mixed with concentration be respectively 1.0,1.4,1.7,2.0,2.3,2.6, the Arabinose solution of 3.0mg/ml.The chromatographic condition formulated according to the embodiment three respectively accurate 2ml that draws is prepared into need testing solution, injection liquid chromatography, record chromatogram, and with working sample concentration for horizontal ordinate, absorption value is ordinate mapping, calculates linear relationship and related coefficient.The results are shown in Table 8, Fig. 6.
Table 8 Arabinose content range of linearity measurement result
Result shows that Arabinose is good in 20.06-59.54 μ g/ml concentration range internal linear relation under this liquid-phase condition.
4.5 stability test
Get one there is the method that function of blood sugar reduction preparation (lot number 20120526) sample formulates by embodiment three to prepare need testing solution, within 0,1,3,8 hour, measure respectively at after preparation, record derivatization Arabinose peak area, calculates relative standard deviation, the results are shown in Table 9.
Table 9 stability test result
Experiment shows, it is stable in 8 hours that one has function of blood sugar reduction preparation need testing solution.
4.6 repeatability and Intermediate precision test
Get one and there is 12 parts, function of blood sugar reduction preparation (lot number 20120526) sample, respectively on not same date, Agilent and Shimadzu chromatograph, the method formulated by embodiment three prepares test sample and reference substance solution, and measure content, calculate relative standard deviation, the results are shown in Table 10.
Table 10 replica test result
Experiment shows, in disaccharide capsule Arabinose content assaying method repeatability and Intermediate precision good.
4.7 recovery test
By 80%, 100% of recipe quantity, 120% precision takes each three parts of Arabinose reference substance, add blank auxiliary and other raw materials, sample is prepared into the solution totally 9 parts of basic, normal, high 3 kinds of concentration by the method that embodiment three is formulated, and sample introduction measures respectively, by external standard method with calculated by peak area.The average recovery rate of the basic, normal, high 3 kinds of strength solution of Arabinose is respectively 100.18,100.47,101.79%, the results are shown in Table 11.
Table 11 recovery is tested
Result shows, this content assaying method recovery is good.
Embodiment kind on May Day has the assay quality standard of function of blood sugar reduction preparation
Proterties: this product is hard shell capsules, content is white particle and brown ceramic powder potpourri, puckery, acid.
Differentiate: (1) gets this product content 0.1g, put in tool plug test tube, add hydrochloric acid 3ml, close plug, heating water bath 3 hours, after being cooled to room temperature, in the 10ml gradation that adds water washing evaporating dish, evaporate to dryness, the 30ml gradation that adds water is dissolved, filter, filtrate is incorporated in 50ml measuring bottle, be diluted with water to scale, getting 5ml puts in tool plug test tube, add diacetone test solution and (get diacetone 2ml, add 0.5mol/L sodium carbonate liquor to 50ml, configure before use) 1.0ml, shake up, put in boiling water bath, heat 25 minutes, take out, after cooling rapidly with frozen water, add paradime thylaminobenzaldehyde test solution (paradime thylaminobenzaldehyde 0.8g, add ethanol,aldehyde free 15ml and hydrochloric acid 15ml, shake up) 1.0ml, i.e. displaing amaranth.
(2) get this product content 0.1g, put in tool plug test tube, add hydrochloric acid 3ml, close plug, heating water bath 3 hours, after being cooled to room temperature, in the 10ml gradation that adds water washing evaporating dish, evaporate to dryness, the 30ml gradation that adds water is dissolved, and filter, filtrate is incorporated in 50ml measuring bottle, get 20ml and be concentrated into 5ml, as need testing solution; Get aminoglucose hydrochloride reference substance simultaneously, add water and make the solution of every 1ml containing 5mg, product solution in contrast.According to thin-layered chromatography (Chinese Pharmacopoeia version in 2010 annex VI B) test, draw each 2 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, methyl alcohol-ammoniacal liquor (9:1) is developping agent, launch, take out, dry, dry 3 minutes at 105 DEG C, spray, with ninhydrin solution, is dried at 105 DEG C, is inspected under putting daylight, on the position corresponding to reference substance chromatogram, the purple dot of aobvious same color respectively.
3) check: moisture gets this product content, measures according to aquametry (Chinese Pharmacopoeia version in 2010 annex Ⅸ H). must not 9.0% be crossed.
Content uniformity gets this product 10, and accurately weighed weight, inclines and content (must not lose softgel shell) respectively, and hard capsule softgel shell little brush or other suitable apparatus are wiped only, then distinguish accurately weighed softgel shell weight, obtain the loading amount that every intragranular is tolerant.Every loading amount is compared with sign loading amount, and content uniformity limit should within ± 10.0%, and what exceed content uniformity limit more than 2, and must not must not have 1 overrun one times.
Heavy metal and harmful element measure according to lead, cadmium, arsenic, sclera remodeling method (Chinese Pharmacopoeia version in 2010 annex Ⅸ B atomic absorption spectrophotometry or annex Ⅸ D inductively coupled plasma mass spectrometry), and lead must not cross 5/1000000ths; Cadmium must not cross 3/10000000ths; Arsenic must not cross 1,000,000/; Mercury must not cross 3/10000000ths.
Microbial limit measures according to microbial decolorization (Chinese Pharmacopoeia version in 2010 attached XIII C), and the every 1g of total bacteria must not cross 10000cfu, and mould and yeast count must not cross 100cfu, and Escherichia coli must not detect.
Assay:
Chromatographic condition and system suitability take octadecylsilane chemically bonded silica as filling agent, 0.1mol/L ammonium acetate buffer solution (gets ammonium acetate 7.7g, be dissolved in water and be diluted to 1000ml, pH to 5.5 is regulated with acetum)-acetonitrile (75:25) is mobile phase, determined wavelength is 248nm.Theoretical cam curve calculates should be not less than 4000 by derivatization Arabinose peak, and derivatization Arabinose peak and derivative reagent peak degree of separation are not less than 1.5.
Determination method is got this product content and is about 0.37g, accurately weighed, put in 100ml volumetric flask, add water appropriate, jolting is dissolved and is diluted to scale, shake up, precision measures 2ml and puts in tool plug test tube, add 2ml 0.07mol/L sodium hydroxide solution and 1.5ml0.4mol/L PMP (1-phenyl-3-methyl-5-pyrazolones ketone) methanol solution, shake up, be placed in 70 DEG C of water-baths and react 45min, period every 15min jolting once, cold water is put in taking-up, add 2.5ml0.07mol/L hydrochloric acid solution simultaneously, be cooled to room temperature, move in 100ml volumetric flask, the gradation that adds water is washed, washing lotion is incorporated in volumetric flask, thin up is settled to scale, shake up, 0.45 μm of membrane filtration, as need testing solution, precision measures 10 μ l injection liquid chromatographies, record chromatogram, another precision takes Arabinose reference substance and is about 0.2g, puts in 100ml volumetric flask, is measured in the same method.By external standard method with derivatization Arabinose peak-to-peak areal calculation, to obtain final product.
This product every must not be less than 0.25g containing Arabinose.
[usage and consumption] is oral, one time 2,3 times on the one.
The heavy 0.5g of [specification] every.
[storage] seals.
Claims (6)
1. have a detection method for function of blood sugar reduction preparation, said preparation is made for primary raw material extracts with Arabinose and chitosan oligosaccharide, it is characterized in that: carry out quantitative measurement and Qualitive test to Arabinose and chitosan oligosaccharide respectively, wherein,
As follows to the method for quantitatively determining of Arabinose: the methanol solution adding sodium hydroxide solution and 1-phenyl-3-methyl-5-pyrazolones ketone in sample to be tested carries out derivative reaction, add appropriate hydrochloric acid neutralization after reaction terminates, after membrane filtration, carry out high-performance liquid chromatogram determination;
Chemical colour reaction is adopted to differentiate or indentification by TLC to the Qualitive test of chitosan oligosaccharide.
2. a kind of detection method with function of blood sugar reduction preparation as claimed in claim 1, it is characterized in that: the chromatographic condition of described high-performance liquid chromatogram determination is for being filling agent with octadecylsilane chemically bonded silica, 0.1-0.2mol/L pH5.0-6.0 ammonium acetate buffer solution-acetonitrile is mobile phase, the volume ratio of ammonium acetate buffer solution-acetonitrile is 80-70:20:30, and determined wavelength is 244-252nm.
3. a kind of detection method with function of blood sugar reduction preparation as claimed in claim 1, it is characterized in that: in described derivative reaction process, control NaOH, the mol ratio of 1-phenyl-3-methyl-5-pyrazolones ketone and Arabinose three is 5.22-11.18:11.21-22.43:1.
4. a kind of detection method with function of blood sugar reduction preparation as claimed in claim 1, it is characterized in that: described derivative reaction condition is: react at least 30min in 60-80 DEG C of water-bath, every 10-20min jolting once.
5. a kind of detection method with function of blood sugar reduction preparation as claimed in claim 1, it is characterized in that: described chemical colour reaction discrimination method is according to forming the former 2-methyl-3-diacetyl azole derivatives that adds lustre under chitosan oligosaccharide acid condition with diacetone condensation, add lustre to and formerly in concentrated hydrochloric acid methanol solution, complex reaction occurs with p-dimethylbenzaldehyde again, generate purple solution, thus differentiate.
6. a kind of detection method with function of blood sugar reduction preparation as claimed in claim 1, it is characterized in that: described TLC Identification be with hydrochloric acid hydrolysis after chitosan oligosaccharide be test sample, aminoglucose hydrochloride is reference substance, methyl alcohol with volume ratio 7-9:3-1: ammoniacal liquor is developping agent, ninhydrin solution is the TLC Identification of developer.
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| L-阿拉伯糖对2型糖尿病大鼠糖脂代射的影响;王鑫 等;《卫生研究》;20120331;第41卷(第2期);全文 * |
| 低分子量壳寡糖改善II型糖尿病大鼠症状及其作用机制研究;王鑫 等;《食品科学》;20071130(第11期);全文 * |
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Address after: Tongan District Xiamen city of Fujian Province Jin RI Lu 361100 gold industry village (production and business address - Xiamen City Industrial Zone Tongan Yung source road No. 88) Applicant after: Golden day Pharmaceutical (China) Co., Ltd. Address before: Tongan District Xiamen city of Fujian Province Jin RI Lu 361100 gold industry village Applicant before: Xiamen Jinri Pharmaceutical Co., Ltd. |
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