阅读说明：本技术 无患子皂苷标准品的非标定量测定试剂盒及测定方法 (Non-calibration amount determination kit and determination method for soapberry saponin standard substance ) 是由 段志贵 陈爽 黄硕泉 刘炅峰 于 2019-09-30 设计创作，主要内容包括：本发明涉及生物科技检测技术领域,具体公开一种无患子皂苷标准品的非标定量测定试剂盒及测定方法,该方法不需要无患子皂苷标准品,是一种非标定量的无患子皂苷含量测定方法；所述的试剂盒包括溶解剂、水解剂、指示剂、中和剂、显色剂及糖标准品,所述的测定方法包括以下步骤：A糖基含量的测定；B皂苷元含量的测定；C计算无患子皂苷样品中皂苷含量。本发明既可用于无患子皂苷标准品的建立,也可用于任何样品中无患子皂苷含量的测定。该发明针对皂苷中的糖基及皂苷元通过不同工艺分别进行提取、测定,相对于现有技术,本发明成本低、稳定性高。(The invention relates to the technical field of biotechnological detection, and particularly discloses a kit and a method for measuring the non-calibrated amount of a sapindoside standard substance, wherein the method does not need the sapindoside standard substance and is a non-calibrated amount method for measuring the content of sapindoside; the kit comprises a dissolving agent, a hydrolyzing agent, an indicator, a neutralizer, a color developing agent and a sugar standard substance, and the determination method comprises the following steps: measuring the content of A glycosyl; measuring the content of the sapogenin B; and C, calculating the saponin content in the soapberry saponin sample. The method can be used for establishing the soapnut saponin standard product and measuring the soapnut saponin content in any sample. The invention respectively extracts and determines glycosyl and sapogenin in the saponin through different processes, and compared with the prior art, the invention has low cost and high stability.)

1. A non-standard quantitative determination method for a sapindus saponin standard substance is characterized by comprising the following steps:

s1, preparing a standard curve of the optical density value and the corresponding concentration of the anhydrous glucose standard substance by using a DNS colorimetric method;

s2 weighing a W1 sapindoside sample, dissolving with ethanol/acetic acid solution, fixing the volume to v0 with glacial acetic acid, and then carrying out centrifugal treatment;

s3 preparation of blank control: taking supernatant v1 in the step S2, adding distilled water and DNS reagent, adding phenolphthalein after boiling water bath for 5 minutes, cooling to room temperature and fixing the volume to v 2;

s4, preparing a solution to be tested: taking supernatant v1 in the step S2, adding distilled water and 6mol/l hydrochloric acid until the acid concentration is 2.5 +/-0.5 mol/l, shaking and uniformly mixing, adding phenolphthalein after 20-25 minutes of boiling water bath, and dripping to pink by using 6mol/l sodium hydroxide; then adding DNS reagent, boiling water bath for 5 minutes, cooling to room temperature and fixing the volume to v 2;

s5, measuring the optical density value of the solution to be measured in the step S4 by taking the blank control solution of the step S3 as a blank control;

s6, obtaining the content m1 of the reducing monosaccharide in the liquid to be detected according to the standard curve in the step S1 and the optical density value measured in the step S5; and calculating the glycosyl content P1 in the soapberry saponin sample according to the following formula:

s7 hydrolysis: weighing a W1 soapberry saponin sample, adding 2.5 +/-0.5 mol/l hydrochloric acid, fully dissolving, and heating in a boiling water bath for 20-30 minutes;

s8 filtering: vacuum-filtering the hydrolysate with a sand core funnel, and leaching with 1mol/l formic acid solution for 3 times to obtain filtrate and filter residue;

s9, freeze-drying the filter residue obtained in the step S8, and weighing m 2;

s10 neutralizing the filtrate in the step S8 to pH7-8 with sodium hydroxide;

s11, making the neutralized liquid flow through a macroporous non-polar adsorption resin or a C18 reversed phase column with the flow rate of 1ml/min, then washing with 70-80% ethanol solution, and collecting the eluent;

s12, distilling under reduced pressure to remove ethanol, freeze-drying, and weighing m 3;

s13 calculating the sapogenin content P2 in the sapindoside sample according to the following formula:

p2 (%) (m2 × 1.022+ m 3)/W1; p2, sapogenin content; 1.022 is a correction coefficient;

s14 calculating the saponin content of the sapindus saponin sample according to the following formula: P-P1 + P2; p: actual saponin content in the soapberry saponin sample.

2. The method for non-standard quantitative determination of sapindoside standard substance according to claim 1, wherein the step S1 comprises the steps of:

(1) preparing glucose standard mother liquor;

(2) diluting the glucose standard mother liquor with distilled water to prepare a series of glucose standard solutions with concentration gradients, adding an equal amount of DNS reagent into each glucose standard solution, cooling to room temperature after boiling water bath for 5 minutes, fixing the volume, measuring the optical density value of each glucose standard solution, and making a standard curve according to the linear relationship between the concentration and the optical density value of each glucose standard solution.

3. The method for non-standard quantitative determination of sapindoside standard substance according to claim 2, wherein the preparing of the glucose standard mother liquor in step (1) comprises the following steps: the anhydrous glucose standard substance is dissolved in distilled water and diluted to obtain a solution containing 1mg of glucose per 1 ml.

4. The method according to claim 3, wherein 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6ml of the glucose standard mother liquor obtained in step (1) is taken in step (2), 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4ml of distilled water is added to prepare a series of glucose standard solutions with concentration gradients, and the optical density value of each glucose standard solution is determined using distilled water containing 0ml of the glucose standard mother liquor as a blank control.

5. The method for non-standard quantitative determination of sapindoside standard substance according to claim 1, wherein in step S2, W1 sapindoside sample is weighed, dissolved with ethanol/acetic acid solution, and made to volume of 100ml with glacial acetic acid, followed by centrifugation at 12000rpm for 5 min;

the blank control solution prepared in step S3 is prepared by collecting supernatant of step S2 0.1ml, adding distilled water 1ml and DNS reagent 1 ml. After boiling water bath for 5 minutes, adding 5uL of phenolphthalein, cooling to room temperature and fixing the volume to 10 ml;

preparing a solution to be detected in the step S4, namely taking 0.1ml of supernatant in the step S2, adding 1.3ml of distilled water and 1ml of 6mol/l hydrochloric acid, uniformly mixing by shaking, adding 5uL of phenolphthalein after 20-25 minutes of boiling water bath, and dripping to pink by using 6mol/l of sodium hydroxide; DNS reagent was then added, and the mixture was cooled to room temperature in a boiling water bath for 5 minutes and made up to 10 ml.

6. The method according to claim 1, wherein the hydrolysis in step S7 is carried out to obtain W1 sapindoside sample, 2.5 + -0.5 mol/l hydrochloric acid 10W 1ml is added, and the sample is dissolved thoroughly and heated in boiling water bath for 20-30 min.

7. The kit for measuring the non-calibration amount of the sapindoside standard substance is characterized by comprising a dissolving agent, a hydrolyzing agent, an indicator, a neutralizer, a color developing agent and a sugar standard substance, wherein the hydrolyzing agent is 2.5 +/-0.5 mol/l hydrochloric acid, the neutralizer is 6mol/l sodium hydroxide, and the sugar standard substance is 0.1% glucose solution.

8. The kit for non-calibration amount measurement of sapindoside standard according to claim 7, wherein the dissolving agent is 1:1-2 ethanol/acetic acid solution, the indicator is 0.1-0.5% phenolphthalein, and the color developing agent is DNS reagent.

Technical Field

The invention relates to the technical field of biotechnological detection, in particular to a kit and a method for measuring the non-calibrated amount of a sapindoside standard substance.

Background

The patent "a soapberry total saponin rapid detection kit and detection method" (CN201710269527.8) discloses a soapberry total saponin rapid detection kit and detection method, wherein the kit comprises a drying auxiliary agent, an extracting agent, a hydrolyzing agent, an indicator, a color developing agent and a standard substance. The drying auxiliary agent is diatomite or quartz sand, the extracting agent is methanol and acetic acid, the hydrolyzing agent is hydrochloric acid, the indicator is phenolphthalein, the neutralizing agent is sodium hydroxide, the color developing agent is 3, 5-dinitrosalicylic acid, and the standard product is high-purity soapberry total saponin, so that the method is suitable for quality control of soapberry saponin raw materials or products and rapid sampling inspection in an industrial production process, but the technology needs to adopt the high-purity soapberry total saponin as the standard product. The standard substance is used for identification, detection, content measurement and the like of a sample, and in quantitative analysis, the correctness of the standard substance directly influences the measurement result of the sample, so that the purity calibration of the standard substance is very important. In the prior art, although the liquid chromatography-mass spectrometry method and the high performance liquid chromatography can accurately determine and quantify the sapindoside, the standard substance is still needed except for the defects of expensive instrument and equipment, high maintenance cost, high professional level of required operators, expensive standard substance and the like. The vanillin-perchloric acid/concentrated sulfuric acid method adopts a vanillin developer to determine the content of total saponins, glycosyl in a saponin structure can generate color reaction with vanillin, and the saponins with various different glycosyl numbers cause the vanillin to generate color result difference, so that the error is large and the reliability is poor; in addition, the standard is still an irrevocable technical obstacle.

Therefore, the common defects of the conventional methods are that a sapindoside standard product is needed, and no sapindoside standard product is available at present, so that the measured sapindoside content by the method has large deviation. Therefore, in order to solve the above-mentioned contradiction, it is necessary to establish a method for measuring a soapberry standard substance.

Disclosure of Invention

The invention aims to provide a low-cost and high-stability non-calibration quantity measurement kit and a measurement method of a sapindoside standard product, aiming at the existing technical current situation.

In order to achieve the purpose, the invention adopts the following technical scheme:

one object of the present invention is to provide a non-standard quantitative determination method for a sapindoside standard substance, which is characterized by comprising the following steps:

s1, preparing a standard curve of the optical density value and the corresponding concentration of the anhydrous glucose standard substance by using a DNS colorimetric method;

s2 weighing a W1 sapindoside sample, dissolving with ethanol/acetic acid solution, fixing the volume to v0 with glacial acetic acid, and then carrying out centrifugal treatment;

s3 preparation of blank control: taking supernatant v1 in the step S2, adding distilled water and DNS reagent, adding phenolphthalein after boiling water bath for 5 minutes, cooling to room temperature and fixing the volume to v 2;

s4, preparing a solution to be tested: taking supernatant v1 in the step S2, adding distilled water and 6mol/l hydrochloric acid until the acid concentration is 2.5 +/-0.5 mol/l, shaking and uniformly mixing, adding phenolphthalein after 20-25 minutes of boiling water bath, and dripping to pink by using 6mol/l sodium hydroxide; DNS reagent was then added and a boiling water bath was used for 5 minutes. Cooling to room temperature and metering to v 2;

s5, measuring the optical density value of the solution to be measured in the step S4 by taking the blank control solution of the step S3 as a blank control;

s6, obtaining the content m1 of the reducing monosaccharide in the liquid to be detected according to the standard curve in the step S1 and the optical density value measured in the step S5; and calculating the glycosyl content P1 in the soapberry saponin sample according to the following formula:

s7 hydrolysis: weighing a W1 soapberry saponin sample, adding 2.5 +/-0.5 mol/l hydrochloric acid, fully dissolving, and heating in a boiling water bath for 20-30 minutes;

s8 filtering: vacuum-filtering the hydrolysate with a sand core funnel, and leaching with 1mol/l formic acid solution for 3 times to obtain filtrate and filter residue;

s9, freeze-drying the filter residue obtained in the step S8, and weighing m 2;

s10 neutralizing the filtrate in the step S8 to pH7-8 with sodium hydroxide;

s11, making the neutralized liquid flow through a macroporous non-polar adsorption resin or a C18 reversed phase column with the flow rate of 1ml/min, then washing with 70-80% ethanol solution, and collecting the eluent;

s12, distilling under reduced pressure to remove ethanol, freeze-drying, and weighing m 3;

s13 calculating the sapogenin content P2 in the sapindoside sample according to the following formula:

p2 (%) (m2 × 1.022+ m 3)/W1; p2, sapogenin content; 1.022 is a correction coefficient;

s14 calculating the saponin content of the sapindus saponin sample according to the following formula: P-P1 + P2; p: actual saponin content in the soapberry saponin sample.

Preferably, the step S1 includes the steps of:

(1) preparing glucose standard mother liquor;

(2) diluting the glucose standard mother liquor with distilled water to prepare a series of glucose standard solutions with concentration gradients, adding an equal amount of DNS reagent into each glucose standard solution, cooling to room temperature after boiling water bath for 5 minutes, fixing the volume, measuring the optical density value of each glucose standard solution, and making a standard curve according to the linear relationship between the concentration and the optical density value of each glucose standard solution.

Further, the preparing glucose standard mother liquor in the step (1) comprises the following steps: the anhydrous glucose standard substance is dissolved in distilled water and diluted to obtain a solution containing 1mg of glucose per 1 ml.

Further, in the step (2), 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6ml of the glucose standard mother liquor in the step (1) is respectively taken, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4ml of distilled water is respectively added to prepare a series of glucose standard solutions with concentration gradients, and the optical density value of each glucose standard solution is measured by taking the distilled water containing 0ml of the glucose standard mother liquor as a blank control.

Preferably, in the step S2, weighing a W1 sapindoside sample, dissolving with ethanol/acetic acid solution, and making the volume of the solution to be 100ml with glacial acetic acid, and then performing centrifugation, wherein the centrifugation is performed at 12000rpm for 5 min;

the blank control solution prepared in step S3 is prepared by collecting supernatant of step S2 0.1ml, adding distilled water 1ml and DNS reagent 1 ml. After boiling water bath for 5 minutes, adding 5uL of phenolphthalein, cooling to room temperature and fixing the volume to 10 ml;

preparing a to-be-detected solution in the step S4, namely taking 0.1ml of supernatant in the step S2, adding 1.3ml of distilled water and 1ml of 6mol/L hydrochloric acid, uniformly mixing by shaking, adding 5uL of phenolphthalein after 20-25 minutes of boiling water bath, and dripping to pink by using 6mol/L sodium hydroxide; DNS reagent was then added and a boiling water bath was used for 5 minutes. Cooled to room temperature and made up to 10 ml.

Preferably, the hydrolysis in step S7 is to weigh a W1 sample of sapindoside, add 2.5 ± 0.5mol/l hydrochloric acid 10 × W1ml, fully dissolve, and heat in a boiling water bath for 20-30 minutes.

The kit comprises a dissolving agent, a hydrolyzing agent, an indicator, a neutralizer, a color developing agent and a sugar standard substance, wherein the hydrolyzing agent is 2.5 +/-0.5 mol/l hydrochloric acid, the neutralizer is 6mol/l sodium hydroxide, and the sugar standard substance is 0.1% glucose solution.

Further, the dissolving agent is ethanol/acetic acid solution with the ratio of 1:1-2, the indicator is 0.1-0.5% of phenolphthalein, and the color developing agent is a DNS reagent (3.5-dinitrosalicylic acid reagent).

The invention has the beneficial effects that:

the invention overcomes the embarrassment that no soapberry standard substance exists at present, and is an absolute quantitative method without the standard substance. The method can be used for establishing a soapnut saponin standard product and quantitatively detecting a soapnut saponin sample, and is a technical method with strong practicability. The method is used for extracting and measuring glycosyl and sapogenin in the saponin respectively through different processes, and compared with the prior art, the method is low in cost and high in stability.

Detailed Description

The invention is further illustrated by the following examples: