Color developing test paper card for rapidly detecting polysaccharide content in traditional Chinese medicine extracting solution based on solid phase carrier

Abstract

The invention discloses a color developing test paper card for rapidly detecting the polysaccharide content in a traditional Chinese medicine extracting solution based on a solid-phase carrier, and belongs to the technical field of traditional Chinese medicine content detection. According to the method, a cross-linked polymer is used as a solid-phase carrier, a DNS color developing agent and a stable-form color developing reaction film are adsorbed through an inclusion effect, color developing conditions of DNS and polysaccharide are optimized, a color developing card for rapid detection is prepared, and the polysaccharide content of the traditional Chinese medicine extracting solution is detected through a matched standard color chart and matched detection equipment. The rapid, visual and large-scale detection of the traditional Chinese medicine polysaccharide is realized, the rapid on-site detection analysis is facilitated, and the problems existing in the existing polysaccharide detection technology are solved.

Description

Technical Field

[0001] The present invention belongs to the technical field of content detection of traditional Chinese medicine, and more specifically relates to a colorimetric test strip card for rapidly detecting the polysaccharide content in a traditional Chinese medicine extract based on a solid-phase carrier. Background Art

[0002] The detection and content determination of polysaccharide components in traditional Chinese medicine and its extracts are the most important basis for the quality control of polysaccharide drugs. At present, the detection of the polysaccharide content in traditional Chinese medicine is a difficult point in research. Currently, the methods for quantitative and qualitative detection and analysis of polysaccharides include: spectrophotometry, ion exchange chromatography, colorimetry, thin-layer chromatography, and high-performance liquid chromatography (HPLC), etc. Among them, the most commonly used classical methods in spectrophotometry are the phenol-sulfuric acid and anthrone-sulfuric acid methods. The determination of polysaccharide content by this method is easily affected by monosaccharides in the solution, and turbidity and background value interference occur during the determination process, resulting in poor result stability. At the same time, the redistillation of phenol and the addition of anthrone during the detection have toxic reactions, sulfuric acid has strong corrosiveness, and there are safety risks in the detection operation. The reproducibility of the results is easily affected by the operator and test conditions, which is not conducive to automated and large-scale detection. High-performance liquid chromatography (HPLC) can be used for the precise determination of polysaccharide content, but the operation process is complex, requiring special pretreatment processes such as hydrolysis and enzymatic hydrolysis, and has high requirements for instruments and personnel, which is not conducive to rapid on-site detection.

[0003] The 3,5-dinitrosalicylic acid detection method has the advantages of simple and rapid operation, not being affected by monosaccharides in the sample, and high accuracy of the detection results. However, during the operation process, it is necessary to prepare a variety of reagents, resulting in poor reagent stability, too long color reaction time, and the detection results are measured by a spectrophotometer, and the stability is easily interfered, which is not conducive to rapid on-site detection. Summary of the Invention

[0004] The purpose of the present invention is to provide a colorimetric test strip card for rapidly detecting the polysaccharide content in a traditional Chinese medicine extract based on a solid-phase carrier to solve the problems existing in the above-mentioned prior art. The present invention uses a cross-linked polymer as the solid-phase carrier, adsorbs the DNS color reagent through an inclusion effect to form a stable color reaction film, optimizes the color reaction conditions of DNS and polysaccharides, prepares a colorimetric card for rapid detection, and detects the polysaccharide content of the traditional Chinese medicine extract through a matching standard colorimetric card and supporting detection equipment, realizing rapid, visual, and large-scale detection of traditional Chinese medicine polysaccharides, which is conducive to rapid on-site detection and analysis.

[0005] To achieve the above purpose, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention: provides a preparation method of a colorimetric test strip card for rapidly detecting the polysaccharide content in a traditional Chinese medicine extract based on a solid-phase carrier, including the following steps:

[0007] Mix the activated chitosan and modified β-cyclodextrin to obtain a mixture; adjust the pH value of the mixture to 7.8 - 8.8, and add 3,5-dinitrosalicylic acid, sodium tartrate, potassium hydroxide, polyvinyl alcohol, sodium sulfite, and crystalline phenol thereto. After standing, a color reaction reagent is obtained.

[0008] Dry the prepared color reaction reagent until a semi-wet complex is formed.

[0009] Coat the prepared semi-wet complex on a nitrocellulose membrane to prepare a color reaction membrane; use a hydrophobic membrane to seal the four peripheral edges of the prepared color reaction membrane to prevent water leakage.

[0010] Assemble the color reaction membrane with the water-sealed edges into a color reaction test strip card.

[0011] In the present invention, a cross-linked polymer is used as a solid-phase carrier, and a stable color reaction reagent is formed with 3,5-dinitrosalicylic acid (DNS) reagent by adsorption inclusion. The introduction of Tris buffer system and polyvinyl alcohol (PAV) reagent significantly enhances the stability of the color reaction reagent.

[0012] In the present invention, the four peripheral edges of the prepared color reaction membrane are sealed to prevent water leakage of the color reaction reagent and the entry of moisture in the sample during the color reaction, ensuring the integrity of the color reaction membrane.

[0013] Preferably, the reagent used for activating the chitosan is sodium dodecyl sulfate; the reagent used for modifying the β-cyclodextrin is citric acid.

[0014] Preferably, the reagent used for adjusting the pH value of the mixture is Tris buffer solution.

[0015] Preferably, the molar ratio of the activated chitosan to the modified β-cyclodextrin is 1:3; the addition amount of 3,5-dinitrosalicylic acid is 4.5 - 7 g / 1000 mL; the addition amount of sodium tartrate is 100 - 200 g / 1000 mL; the addition amount of potassium hydroxide is 0.5 - 1 g / 1000 mL; the addition amount of polyvinyl alcohol is 0.4 - 0.8 g / 1000 mL; the addition amount of sodium sulfite is 0.15 - 5 g / 1000 mL; the addition amount of crystalline phenol is 3 - 5 g / 1000 mL.

[0016] Preferably, the standing time is 5 d.

[0017] Preferably, the drying temperature is 40 - 60 °C; the moisture content of the semi-wet complex is 20 - 35%.

[0018] Preferably, the coating thickness is 3 - 5 mm; the preparation raw materials of the hydrophobic membrane include siloxane and chitosan, and the molar ratio of siloxane to chitosan is 1:9.

[0019] Furthermore, the nitrocellulose membrane needs to be activated before coating; the activation includes: first soaking in 2% glutaraldehyde by mass for 15 min, then soaking in absolute ethanol for 10 min, finally ultrasonically cleaning with deionized water for 5 min, and standing at 37 °C until it is completely dry; the purpose of the activation is: the nitrocellulose membrane (NC membrane) is a hydrophilic membrane, and activation can change its hydrophilicity and enhance the binding ability to hydrophobic groups. There are background materials such as surfactants, buffer solutions, and inorganic salts remaining on the surface of the nitrocellulose membrane (NC membrane), which affect the activity of the chromogenic agent and the binding and adsorption effect. Activation can avoid the influence of the above factors on the activity of the chromogenic agent and the binding and adsorption effect.

[0020] Preferably, the assembly includes: cutting the size of the color reaction membrane to 80 mm × 150 mm, connecting a water-absorbing material made of hydrophilic cellulose at the top, maintaining an overlapping area of 3 - 5 mm, cutting a sample loading hole made of glass fiber with a size of 50 mm × 120 mm, making it closely combined with the color reaction membrane, maintaining an overlapping area of 2 - 3 mm, and the assembled combination of the color reaction membrane, sample loading hole, and water-absorbing material is sealed with a polypropylene outer box to ensure complete sealing.

[0021] The second technical solution of the present invention: Provide a chromogenic test strip card prepared by the above preparation method.

[0022] The third technical solution of the present invention: Provide the application of the chromogenic test strip card in the detection of polysaccharide content in traditional Chinese medicine extraction solution.

[0023] Furthermore, when the chromogenic test strip card is used for the detection of polysaccharide content in traditional Chinese medicine extraction solution, in order to achieve quantitative detection and result analysis, the present invention also includes a corresponding standard colorimetric card, and the standard colorimetric card is obtained by the following method:

[0024] Prepare glucose standard solutions with a concentration range of 0.01 - 8.5 mg / mL. Immerse the prepared color reaction membranes into glucose standard solutions with different concentrations respectively, place them in an environment condition of 40 - 60 °C, incubate for 5 min, terminate the reaction with cold water, and after thoroughly drying the water with absorbent paper, fix them on a cardboard with a single-tone background by self-adhesive to prepare a standard colorimetric card with light and dark colors.

[0025] The corresponding concentrations of colorimetric labels 1, 2, 3, 5, 6, 7, 8, 9, and 10 in the standard colorimetric card are 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.5 mg / mL, 4.5 mg / mL, 6.5 mg / mL, and 8.5 mg / mL respectively.

[0026] To meet the need for rapid and on-site detection of traditional Chinese medicine polysaccharides, the present invention also adopts the following technical solutions:

[0027] Equipped with special equipment (the supporting special equipment mainly includes a rotating colorimetric cell with 1 - 8 channels, a 580 nm cold light source, a built-in reading display, and a modular design), just put the completed test strip card into the equipment, and it can display the polysaccharide content in the sample; this special equipment can emit light waves of 580 nm, and by collecting the light wave signals, it can display the polysaccharide content.

[0028] The present invention discloses the following technical effects:

[0029] The present invention provides a colorimetric test strip card for rapid detection of polysaccharide content in traditional Chinese medicine extracts based on a solid-phase multi-microporous membrane as a carrier, which can achieve rapid detection and quantitative determination of polysaccharide content in traditional Chinese medicine extracts. The entire reaction time is completed within 5 minutes, and the color development operation is realized under the conditions of a 40 - 60 °C incubator or water bath. It has the characteristics of simple operation, rapid detection process, quantitative results, suitability for rapid detection of polysaccharide content, and is fast, visual, and highly safe. Brief Description of the Drawings

[0030] Figure 1 The standard colorimetric card prepared for Example 1;

[0031] Figure 2 The standard curve graph of the glucose standard solution during the effect detection process. Detailed Embodiments

[0032] Now, the various exemplary embodiments of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0033] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0034] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0035] Without departing from the scope or spirit of the present invention, various modifications and variations can be made to the specific embodiments of the description of the present invention, which are obvious to those skilled in the art. Other embodiments obtained from the description of the present invention are obvious to those skilled in the art. The description and examples of the present invention are merely exemplary.

[0036] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0037] First, the raw materials used in the present invention are described as follows:

[0038] The preparation steps of activated chitosan are as follows:

[0039] Activate chitosan by the "sodium dodecyl sulfate" method:

[0040] 1. Accurately weigh 10 g of chitosan, add 1.5% glacial acetic acid, and let it stand at room temperature for 2 min;

[0041] 2. Slowly drop 5 mL of 10% sodium dodecyl sulfate, and stir and react for 2.5 h;

[0042] 3. Add 2 mM sodium hydroxide solution to adjust the pH value to neutral (7.0 - 7.4);

[0043] 4. Wash the precipitate with deionized water 5 times, and after drying thoroughly in an oven, it is the activated chitosan product. The preparation steps of modified β-cyclodextrin are as follows:

[0044] Modify β-cyclodextrin by the "citric acid method":

[0045] 1. Accurately weigh 12 g of β-cyclodextrin, 1.8 g of citric acid, 0.2 g of PEG800, and 0.29 g of potassium dihydrogen phosphate, put them into a 500 mL beaker, add 300 mL of deionized water, heat to 80 °C, and stir to dissolve them completely; 2. After complete dissolution, put it into a 135 °C drying oven, react for 3.5 h and take it out, and let it cool naturally at room temperature;

[0046] 3. Wash the cooled product three times with anhydrous ethanol, then wash it five times with deionized water, and place it in a drying oven to dry thoroughly.

[0047] 4. Use a grinder to carefully grind the dried product into powder, which is the prepared modified β-cyclodextrin.

[0048] The preparation steps of the hydrophobic membrane are as follows:

[0049] Prepare the hydrophobic membrane by the "solution method":

[0050] 1. Accurately weigh 1.0 chitosan, put it into a 1000 mL flask, add 400 mL of dimethylformamide solution, stir well and react at 68 °C for 18 - 24 h to form chlorinated chitosan.

[0051] 2. Accurately weigh chlorinated chitosan and dimethyl silicone in a mass molar ratio of 1:9, put them into a 1000 mL beaker, add 200 mL of dimethylformamide solution, stir well and react at 42 °C for 15 - 20 h to form a hydrophobic membrane;

[0052] 3. Wash the hydrophobic membrane three times with anhydrous ethanol and five times with deionized water, and then dry it naturally.

[0053] All raw materials used are commercially available products without special instructions.

[0054] The room temperature involved in the present invention is calculated as 25 ± 5 °C without special instructions.

[0055] Example 1

[0056] (1) Weigh an appropriate amount of activated chitosan and modified β-cyclodextrin according to a molar ratio of 1:3, use Tris buffer solution to adjust the pH value of the system to 7.8, and sequentially add DNS (the addition amount is 6 g / 1000 mL), sodium tartrate (the addition amount is 200 g / 1000 mL), potassium hydroxide (the addition amount is 0.5 g / 1000 mL), PAV (the addition amount is 0.5 g / 1000 mL), sodium sulfite (the addition amount is 4 g / 1000 mL) and crystalline phenol (the addition amount is 5 g / 1000 mL). After heating and dissolving, let it stand at room temperature in the dark for 5 d to obtain a color reaction reagent;

[0057] (2) Place the prepared color reaction reagent in an oven at 60 °C to dry until a semi-wet complex with a moisture content of 35% is formed;

[0058] (3) Coat the prepared semi-wet complex on the NC membrane, with a coating thickness of 4 mm. The semi-wet complex is firmly bound to the NC membrane through electrostatic binding and adsorption to obtain a color reaction membrane;

[0059] (4) Use a hydrophobic membrane to seal the four peripheral edges of the prepared color reaction membrane to prevent leakage of the color reaction reagent and entry of moisture in the sample during the color reaction, ensuring the integrity of the color reaction membrane;

[0060] (5) Assemble the test strip card in the following combination method: Cut the color reaction membrane to a size of 80 mm × 150 mm, connect an absorbent material made of hydrophilic cellulose at the top, maintaining a 4-mm overlap area. Cut a sample loading hole made of glass fiber to a size of 50 mm × 120 mm and make it closely combine with the color reaction membrane, maintaining a 3-mm overlap area. For the assembled combination of the color reaction membrane, sample loading hole, and absorbent material, use a polypropylene outer box for card sealing to ensure complete sealing;

[0061] (6) To achieve quantitative detection and result analysis, the present invention can also prepare a corresponding standard color comparison card, and the preparation steps are as follows:

[0062] Prepare glucose standard solutions with a concentration range of 0.01 - 8.5 mg / mL. Immerse the prepared color reaction membranes into glucose standard solutions with different concentrations respectively, place them in an environment at 60 °C, incubate for 5 min, terminate the reaction with cold water, and after thoroughly blotting the moisture with absorbent paper, fix them on a cardboard with a single-color background by self-adhesive to prepare a standard color comparison card with light and dark colors;

[0063] The corresponding concentrations of color numbers 1, 2, 3, 5, 6, 7, 8, 9, and 10 in the standard color comparison card are 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.5 mg / mL, 4.5 mg / mL, 6.5 mg / mL, and 8.5 mg / mL respectively.

[0064] (7) To meet the requirements for rapid and on-site detection of traditional Chinese medicine polysaccharides, the present invention can also be equipped with special equipment:

[0065] Equipped with special equipment (the supporting special equipment mainly includes a rotary colorimetric cell with 1 - 8 channels, a 580-nm cold light source, a built-in reading display, and a modular design). Just put the completed test strip card into the equipment to display the polysaccharide content in the sample; this special equipment can emit light waves at 580 nm, and by collecting the light wave signals, it can display the polysaccharide content.

[0066] Effect detection:

[0067] 1. Drawing of the standard curve of the glucose standard solution:

[0068] Precisely weigh and prepare glucose standard solutions with a concentration range of 0.01 - 8.5 mg / mL. Immerse the prepared polysaccharide test strips into standard solutions of different concentrations, incubate them for 5 min under the environmental condition of 60 °C, terminate the reaction with cold water, and after thoroughly blotting the water with absorbent paper, use the supporting detection equipment to measure the absorbance at 580 nm. Take the concentration of the standard solution as the abscissa and the absorbance as the ordinate to plot the standard curve. The results show that the linear regression equation of the standard curve is: y = 0.3879x + 0.1737, R 2 = 0.9995, and within the mass concentration range of 0.01 - 8.5 mg / mL, the linear relationship is good.

[0069] 2. Recovery test for the determination of the content of traditional Chinese medicine polysaccharides:

[0070] Precisely weigh 5 portions of traditional Chinese medicine polysaccharides with known contents, respectively and precisely add different volumes of glucose standard solution (concentration 1.0 mg / mL), conduct the determination of the content of polysaccharides according to the determination procedure of the polysaccharide color development test strip, and calculate their recoveries. The results show that the recoveries of the determination of the content of traditional Chinese medicine polysaccharides by the color development test strip are between 99.58 - 101.08%, and the RSDs are between 0.17% - 4.42%. The reliability of the detection results is good, and it can be used for the determination of the content of traditional Chinese medicine polysaccharides.

[0071] Table 1 Recovery test for the determination of polysaccharides

[0072]

[0073] 3. Comparison of the detection results of the traditional Chinese medicine polysaccharide color development test strip and HPLC detection:

[0074] Respectively select 5 different types of polysaccharides extracted from traditional Chinese medicine. Each extracted polysaccharide is divided into 2 portions. One portion is used to detect the content of polysaccharides with the polysaccharide color development test strip, and the other portion is used to detect the content of polysaccharides by high performance liquid chromatography (HPLC method). Analyze the detection results of the two methods and evaluate their performance. The results show that for the detection results of 5 traditional Chinese medicine polysaccharides, both methods have good determination reliability and accuracy. The errors between the determination results of the polysaccharide color development test strip and the HPLC determination results are all controlled below 2.0%, indicating that the polysaccharide color development test strip has very good accuracy for the determination of traditional Chinese medicine polysaccharides.

[0075] Table 2 Comparison of the determination results of the content of traditional Chinese medicine polysaccharides by the color development test strip and HPLC method

[0076]

[0077] In this specification, each embodiment is described in a progressive manner. The key point of each embodiment is to illustrate the differences from other embodiments. For the same or similar parts among the embodiments, reference can be made to each other.

[0078] The foregoing description of the disclosed embodiments enables those skilled in the art to practice or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a color test paper card for rapid detection of polysaccharide content in a traditional Chinese medicine extract based on a solid phase carrier, characterized in that: The steps include: The activated chitosan and the modified β-cyclodextrin are mixed to obtain a mixture; the pH value of the mixture is adjusted to 7.8-8.8, 3,5-dinitrosalicylic acid, sodium tartrate, potassium hydroxide, polyvinyl alcohol, sodium sulfite and crystalline phenol are added thereto, and the mixture is allowed to stand to obtain a color developing reaction agent; Drying the prepared color developing reagent to form a semi-wet complex; The prepared semi-wet complex is coated on a nitrocellulose membrane to prepare a color reaction membrane; The edges of the prepared color reaction membrane are sealed with a hydrophobic membrane; The color test paper card is assembled with a water-proof and sealed color reaction membrane.

2. The preparation method according to claim 1, characterized in that: The reagent used for activating the chitosan is sodium dodecyl sulfate; and the reagent used for modifying the beta-cyclodextrin is citric acid.

3. The preparation method according to claim 1, characterized in that: The reagent used to adjust the pH value of the mixture is Tris buffer.

4. The preparation method according to claim 1, characterized in that: The molar ratio of the activated chitosan and the modified β-cyclodextrin is 1:3; the added amount of the 3,5-dinitrosalicylic acid is 4.5-7 g / 1000 mL; the added amount of the sodium tartrate is 100-200 g / 1000 mL; the added amount of the potassium hydroxide is 0.5-1 g / 1000 mL; the added amount of the polyvinyl alcohol is 0.4-0.8 g / 1000 mL; the added amount of the sodium sulfite is 0.15-5 g / 1000 mL; and the added amount of the crystalline phenol is 3-5 g / 1000 mL.

5. The preparation method according to claim 1, characterized in that: The standing time is 5 days.

6. The preparation method according to claim 1, characterized in that: The drying temperature is 40-60° C.; the moisture content of the semi-wet composite is 20-35%.

7. The preparation method according to claim 1, characterized in that: The coating thickness is 3-5 mm; the raw materials for preparing the hydrophobic film include siloxane and chitosan, and the molar ratio of siloxane to chitosan is 1:

9.

8. The preparation method according to claim 1, characterized in that: The assembly includes: cutting the color reaction membrane to a size of 80 mm×150 mm, connecting the top with a water-absorbing material made of hydrophilic cellulose, maintaining an overlapping area of ​​3 to 5 mm, cutting glass fiber to form a sample injection hole with a size of 50 mm×120 mm, making it tightly combined with the color reaction membrane, maintaining an overlapping area of ​​2 to 3 mm, and the assembled color reaction membrane, sample injection hole and water-absorbing material assembly is sealed with a polypropylene outer box to ensure a complete seal.

9. A color test paper card prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the color developing test paper card according to claim 9 in detecting the polysaccharide content in a traditional Chinese medicine extract.

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