Method for rapid preparation of type ii arabinogalactan (AG-ii), method for quantification and quality control, and use thereof

Abstract

Disclosed in the present invention is a method for preparing type II arabinogalactan (AG-II). The method comprises the following steps: (i) weighing a proper amount of a polysaccharide sample, dissolving same in a salt solution, centrifuging same to obtain a supernatant, adding to the supernatant a Yariv reagent solution the volume of which is equal to that of the supernatant, uniformly mixing same, reacting same for a period of time, and performing centrifugation to obtain a precipitate; (ii) adding a salt solution to the precipitate, vortexing same, centrifuging same, removing a supernatant, and repeating the operations 2-4 times; and (iii) adding a sodium dithionite solution having a certain concentration to the washed precipitate, and after the solution becomes faint yellow or colorless, dialyzing same with cellophane in flowing water for a period of time, and drying same to obtain the type II arabinogalactan (AG-II). The polysaccharide sample contains the type II arabinogalactan (AG-II). The present invention uses the Yariv reagent as a chemical method for rapidly preparing the polysaccharide AG-II and uses same as a means for controlling the quality of traditional Chinese medicinal materials rich in polysaccharide AG-II.

Description

A rapid method for the preparation, quantification, and quality control of type II arabinogalactan AG-II and its application. Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a method for the rapid preparation, quantification, and quality control of type II arabinogalactan AG-II, and its application. Background Technology

[0002] The main structural types of polysaccharides derived from traditional Chinese medicine include dextran, mannan, galactan, and HG-type (galacturonic acid as the main chain), RG-type pectin polysaccharides (galacturonic acid and rhamnose disaccharide repeating units as the main chain), and AG-type polysaccharides (galactose as the main chain). Polysaccharides possess a variety of unique biological activities, including immunomodulation, anti-inflammation, anti-tumor, hypoglycemic, hypolipidemic, and antioxidant effects, all of which are related to their complex structures. The field of polysaccharides still faces numerous scientific and technological challenges. For example, issues in the production of sugar-based drugs include how to achieve specific homogenization of sugar chains; low-cost, large-scale preparation of polysaccharide components; the structure of active polysaccharides; rapid acquisition and characterization; quality control; and the development of high-precision analytical methods. Only by solving these problems can solutions be provided for the large-scale production and quality control of drugs.

[0003] The Chinese Pharmacopoeia records that the main method for quality control of polysaccharides in medicinal materials is the determination of total sugar content, which mainly uses the classic phenol-sulfuric acid method or ketone-sulfuric acid method. However, this method can only reflect the quality of polysaccharides to a certain extent. It has limitations such as poor selectivity and specificity, which can easily lead to large deviations between the measured values ​​and the true values. The literature reports three solutions: (1) Strengthen the specificity of detection methods. The existing total sugar content only reflects the quality of polysaccharides and cannot specifically reflect the structural characteristics of polysaccharides. There may be large differences between different batches of medicinal materials and finished products, so it is necessary to strengthen the detection methods for the specific structure of polysaccharides; (2) Develop standards suitable for the quality control of polysaccharides in traditional Chinese medicine. Most experiments use dextran with different relative molecular mass labels as controls. There is no standard suitable for the quality control of polysaccharides in traditional Chinese medicine; (3) Study detection methods related to functional activity. At present, the research on the quality standards of polysaccharides in traditional Chinese medicine focuses on the analysis of their structural characteristics and rarely involves the correlation between polysaccharide activity and function. Establishing detection indicators related to their active functions is also a major trend in the quality evaluation of polysaccharide drugs.

[0004] To better understand the pharmacodynamic basis of polysaccharides, they generally need to be separated and purified to obtain homogeneous polysaccharides, typically using ion exchange chromatography and gel permeation chromatography. However, polysaccharide purification is limited by the ion exchange medium, suffering from slow flow rates and poor stability. Although faster-flowing and more stable ion exchangers have been developed, their cumbersome and time-consuming operation and slow enrichment rate limit their use to laboratory preparations, failing to meet the requirements of industrial production. Furthermore, the yield of polysaccharides is affected by extraction, separation, and purification steps. The unknown higher-order structures of polysaccharides, difficulties in characterization, and high costs severely restrict subsequent efficacy evaluation and quality control, becoming a major technical bottleneck in the development of glycoside drugs that urgently needs to be addressed. Summary of the Invention

[0005] Based on this, the present invention provides a method for preparing type II arabinogalactan AG-II, the method comprising the following steps:

[0006] (i) Weigh an appropriate amount of polysaccharide sample, dissolve it in a salt solution, centrifuge to obtain a supernatant, add an equal volume of Yariv reaction reagent solution to the supernatant, mix well and react for a period of time, centrifuge to obtain a precipitate;

[0007] (ii) Add salt solution to the precipitate, vortex, centrifuge, remove the supernatant, and repeat 2–4 times; and

[0008] (iii) Add a certain concentration of sodium dithionite solution to the precipitate after washing. After the solution turns pale yellow or colorless, dialyze it with cellophane and running water for a period of time, and then dry it to obtain the type II arabinogalactan AG-II.

[0009] The polysaccharide sample contained type II arabinogalactan AG-II.

[0010] According to another aspect of the present invention, a method for quantitatively determining the content of type II arabinogalactan AG-II in a polysaccharide test sample is provided, the method comprising the following steps:

[0011] (1) Preparation of type II arabinogalactan AG-II reference solution: (a) Weigh an appropriate amount of type II arabinogalactan AG-II reference standard, dissolve it in salt solution, make up to volume and mix well; (b) Take an appropriate amount of reference solution into a container, add salt solution to dilute, then add an equal volume of Yariv reaction reagent solution, mix well and react for a period of time, centrifuge and remove the supernatant; (c) Add salt solution to the precipitate, vortex, centrifuge, remove the supernatant, repeat 2 to 4 times; and (d) After washing, blow the precipitate with nitrogen, add alkaline solution to it, vortex to dissolve, and obtain the type II arabinogalactan AG-II reference solution;

[0012] (2) Preparation of polysaccharide test solution: (a) Weigh an appropriate amount of polysaccharide test sample, dissolve it in salt solution, make up to volume and mix well; (b) Take an appropriate amount of polysaccharide test solution into a container, add salt solution to dilute it, then add an equal volume of Yariv reaction reagent solution, mix well and react for a period of time, centrifuge and remove the supernatant; (c) Add salt solution to the precipitate, vortex, centrifuge, remove the supernatant, repeat 2 to 4 times; and (d) After washing, blow the precipitate with nitrogen, add alkaline solution to it, vortex to dissolve it, and obtain the polysaccharide test solution;

[0013] (3) Take appropriate amounts of the type II arabinogalactan AG-II reference solution and the polysaccharide test solution, and measure the absorbance value using an enzyme-linked immunosorbent assay (ELISA) reader:

[0014] (4) Prepare the standard curve for type II arabinogalactan AG-II reference standard; and

[0015] (5) Substitute the absorbance of the polysaccharide test solution into the standard curve to obtain the concentration of type II arabinogalactan AG-II in the polysaccharide test solution, and take the average value of the parallel determinations.

[0016] Furthermore, the salt solution is a sodium chloride solution.

[0017] Furthermore, the volume concentration percentage of the sodium chloride solution is 0.5% to 3%, for example, about 1%.

[0018] Furthermore, the concentration of the Yariv reagent solution is 0.5% to 2%, for example, about 1%.

[0019] Furthermore, the reaction is carried out under static conditions.

[0020] Furthermore, this resting period should be a light-protected resting period.

[0021] Furthermore, the reaction time is 1–25 hours, or even 3–5 hours, for example, about 4 hours.

[0022] Furthermore, the reaction temperature is 1°C to 10°C, and even more specifically, 3°C to 5°C, for example, about 4°C.

[0023] Furthermore, the centrifuge speed is 8000–12000 r / min, for example, about 10000 r / min.

[0024] Furthermore, the centrifugation time is 5 to 15 minutes, for example, about 10 minutes.

[0025] Furthermore, this is repeated 3 times.

[0026] Furthermore, the polysaccharide sample or the polysaccharide test specimen was derived from the medicinal herb Notopterygium incisum.

[0027] Furthermore, the polysaccharide sample or the polysaccharide test specimen was obtained from the dried rhizomes and roots of *Notopterygium incisum* Ting ex HTChang or *Notopterygium franchetii* H. de Boiss., which were extracted with water and precipitated with alcohol. The crude polysaccharide was then separated using DEAE Sephrose™ FastFlow anion exchange resin to obtain the elution fraction of 0.2M sodium chloride solution.

[0028] Furthermore, the polysaccharide sample or the polysaccharide test specimen is gum arabic.

[0029] Furthermore, the concentration of the polysaccharide sample is 1.5–2.5 mg / mL, for example, about 2 mg / mL.

[0030] Furthermore, the concentration of the sodium dithionite solution is 8% to 12%, for example, about 10%.

[0031] Furthermore, the dialysis time is 40–60 hours, for example, about 48 hours.

[0032] Furthermore, the drying process is freeze drying.

[0033] Further, in step (1) or step (2), the mass ratio of the type II arabinogalactan AG-II reference standard or the polysaccharide test sample to the mass of the Yariv reaction reagent is 0.5 to 2, for example, about 1.

[0034] Further, in step (1) or step (2), the concentration of the reference solution or the polysaccharide test solution is 0.5 to 2 mg / mL, for example, about 1 mg / mL.

[0035] Further, in step (1) or step (2), the alkaline solution is a sodium hydroxide solution.

[0036] Further, in step (1) or step (2), the molar concentration of the sodium hydroxide solution is 0.005 to 0.02 mol / L, for example, about 0.01 mol / L.

[0037] Furthermore, the volume of the sodium hydroxide solution is 0.5 to 1.5 ml, for example, about 1 ml.

[0038] Further, in step (3), the amount of the reference solution taken is 100 to 250 μl, for example 200 μl.

[0039] Further, in step (3), the volume of the test solution is 100 to 250 μl, for example 200 μl.

[0040] Furthermore, in step (3), the detection wavelength of the ELISA reader is 450 nm.

[0041] Furthermore, in step (5), the number of parallel determinations is 2 to 5, for example, 3 times.

[0042] Furthermore, for polysaccharide samples or test specimens derived from Notopterygium incisum, the standard curve for type II arabinogalactan AG-II reference standard was y = 25.396x + 0.0393, R0 2 =0.9998.

[0043] Furthermore, for polysaccharide samples or test articles derived from Notopterygium incisum, the limit of quantitation standard curve for type II arabinogalactan AG-II reference standard was y = 0.0237x + 0.0385, R0 2 =0.9998.

[0044] Furthermore, for polysaccharide samples or test articles derived from Notopterygium incisum, the detection limit for type II arabinogalactan AG-II reference standard is 0.72 μg.

[0045] Furthermore, for polysaccharide samples or test samples derived from Notopterygium incisum, the limit of quantification for type II arabinogalactan AG-II reference standard is 2.18 μg.

[0046] Furthermore, for gum arabic, the standard curve for type II arabinogalactan AG-II reference standard is y = 4.3187x + 0.0263, R0 2 =0.9999.

[0047] Furthermore, for gum arabic, the limit of quantitation standard curve for type II arabinogalactan AG-II reference standard is y = 0.0476x + 0.0353, R0 2 =0.9995.

[0048] Furthermore, for gum arabic, the detection limit for type II arabinogalactan AG-II reference standard is 0.89 μg.

[0049] Furthermore, for gum arabic, the limit of quantification for type II arabinogalactan AG-II reference standard is 2.69 μg.

[0050] According to another aspect of the present invention, the above-described method is provided for use in the quality detection and / or quality evaluation and / or quality control of medicinal materials containing type II arabinogalactan AG-II or their exudates.

[0051] The beneficial effects of this invention are:

[0052] This invention develops a novel method for preparing type II arabinogalactan, which has the advantages of being rapid and efficient. This method improves the enrichment efficiency and material utilization of arabinogalactan by optimizing reaction conditions, including reaction time and raw material ratios. Furthermore, this invention also quantitatively analyzes the prepared type II arabinogalactan, establishing a rapid and reliable detection method to determine its content variations under different conditions.

[0053] This invention not only provides an efficient method for preparing type II arabinogalactan, but also conducts in-depth research on its quantitative analysis. This method is of positive significance for deepening the understanding of the synthesis process of type II arabinogalactan and promoting its application in food, medicine and other fields. Attached Figure Description

[0054] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without exceeding the scope of protection claimed by the present invention.

[0055] Figure 1 is a schematic diagram of the screening results of AG content in the Chinese herbal medicine database.

[0056] Figure 2 shows the HPGPC spectrum of QH-2-AG, with the horizontal axis representing retention time and the vertical axis representing response value.

[0057] Figure 3 shows the sugar composition spectrum of QH-2-AG, with the horizontal axis representing retention time and the vertical axis representing peak signal intensity. In the figure, 1 represents rhamnose; 2 represents fucose; 3 represents arabinose; 4 represents xylose; 5 represents mannose; 6 represents glucose; and 7 represents galactose.

[0058] Figure 4 shows the methylation spectrum of QG-2-AG, with the horizontal axis representing retention time and the vertical axis representing peak signal intensity. (a) GC spectrum of QH-2-AG after methylation; (b) GC spectrum of QH-2-AG-on after methylation; (c) GC spectrum of QH-2-AG-Re after methylation; where T-Araf: terminal furanyl arabinose; 1,3-Araf: 1,3-linked furanyl arabinose; T-Glcp: terminal pyranyl glucose; T-Galp: terminal pyranyl galactose; 1,4-Galp: 1,4-linked pyranyl galactose; 1,3-Galp: 1,3-linked pyranyl galactose; 1,6-Galp: 1,6-linked pyranyl galactose; 1,3,6-Galp: 1,3,6-linked pyranyl galactose.

[0059] Figure 5 is a schematic diagram of the results of optimizing reaction time and temperature (at room temperature).

[0060] Figure 6 is a schematic diagram of the results of the optimized reaction time and temperature (4℃).

[0061] Figure 7 is a schematic diagram of the results of ingredient ratio optimization (1).

[0062] Figure 8 is a schematic diagram of the results of ingredient ratio optimization (2).

[0063] Figure 9 shows the standard curve of GAG.

[0064] Figure 10 shows the standard curve of QH-2-AG.

[0065] Figure 11 shows the standard curve of the limit of quantitation of GAG reference standard.

[0066] Figure 12 shows the standard curve of the quantitation limit of QH-2-AG. Detailed Implementation

[0067] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0068] Unless otherwise stated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by one of ordinary skill in the field of this invention or the field of application of such terms. While any methods, conditions, substances, or materials similar to or equivalent to those disclosed herein may be used in the practice of this invention, preferred methods, conditions, substances, or materials are described herein.

[0069] This invention is intended to cover all options, variations, and equivalents that may be included in the field of prior art as defined in the claims. Those skilled in the art will recognize many similar or equivalent methods and substances described herein that can be applied in the practice of this invention. This invention is by no means limited to the description of methods and substances.

[0070] The singular forms “a,” “an,” and “the” used in the specification and appended claims include plural indicators unless the context clearly specifies otherwise.

[0071] In this invention, the term "comprising" and "including" are synonymous. The terms "comprising," "including," "having," "containing," or any other variations thereof as used herein are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such a composition, step, method, article, or apparatus.

[0072] As described in the background section, existing polysaccharide isolation and purification methods are cumbersome and time-consuming, have slow enrichment rates, and can only be used for laboratory preparations, failing to meet the requirements of industrial preparations. To address these issues, this invention provides a method for preparing type II arabinogalactan AG-II, comprising the following steps:

[0073] (i) Weigh an appropriate amount of polysaccharide sample, dissolve it in a salt solution, centrifuge to obtain a supernatant, add an equal volume of Yariv reaction reagent solution to the supernatant, mix well and react for a period of time, centrifuge to obtain a precipitate;

[0074] (ii) Add salt solution to the precipitate, vortex, centrifuge, remove the supernatant, and repeat 2–4 times; and

[0075] (iii) Add a certain concentration of sodium dithionite solution to the precipitate after washing. After the solution turns pale yellow or colorless, dialyze it with cellophane and running water for a period of time, and then dry it to obtain the type II arabinogalactan AG-II.

[0076] The polysaccharide sample contained type II arabinogalactan AG-II.

[0077] According to another aspect of the present invention, a method for quantitatively determining the content of type II arabinogalactan AG-II in a polysaccharide test sample is provided, the method comprising the following steps:

[0078] (1) Preparation of type II arabinogalactan AG-II reference solution: (a) Weigh an appropriate amount of type II arabinogalactan AG-II reference standard, dissolve it in salt solution, make up to volume and mix well; (b) Take an appropriate amount of reference solution into a container, add salt solution to dilute, then add an equal volume of Yariv reaction reagent solution, mix well and react for a period of time, centrifuge and remove the supernatant; (c) Add salt solution to the precipitate, vortex, centrifuge, remove the supernatant, repeat 2 to 4 times; and (d) After washing, blow the precipitate with nitrogen, add alkaline solution to it, vortex to dissolve, and obtain the type II arabinogalactan AG-II reference solution;

[0079] (2) Preparation of polysaccharide test solution: (a) Weigh an appropriate amount of polysaccharide test sample, dissolve it in salt solution, make up to volume and mix well; (b) Take an appropriate amount of polysaccharide test solution into a container, add salt solution to dilute it, then add an equal volume of Yariv reaction reagent solution, mix well and react for a period of time, centrifuge and remove the supernatant; (c) Add salt solution to the precipitate, vortex, centrifuge, remove the supernatant, repeat 2 to 4 times; and (d) After washing, blow the precipitate with nitrogen, add alkaline solution to it, vortex to dissolve it, and obtain the polysaccharide test solution;

[0080] (3) Take appropriate amounts of the type II arabinogalactan AG-II reference solution and the polysaccharide test solution, and measure the absorbance value using an enzyme-linked immunosorbent assay (ELISA) reader:

[0081] (4) Prepare the standard curve for type II arabinogalactan AG-II reference standard; and

[0082] (5) Substitute the absorbance of the polysaccharide test solution into the standard curve to obtain the concentration of type II arabinogalactan AG-II in the polysaccharide test solution, and take the average value of the parallel determinations.

[0083] In this invention, when times, concentration, time, temperature, rotation speed, ratio, volume, absorption amount, wavelength, or other values ​​or parameters are expressed as ranges, preferred ranges, or a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range "2 to 4" is disclosed, the described range should be interpreted as including ranges "2 to 4", "2 to 3", "3 to 4", etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

[0084] In a preferred embodiment, the salt solution is a sodium chloride solution.

[0085] In a preferred embodiment, the sodium chloride solution has a volume concentration percentage of 0.5% to 3%, for example, about 1%.

[0086] In a preferred embodiment, the concentration of the Yariv reagent solution is 0.5% to 2%, for example, about 1%.

[0087] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1%" includes 1% ± 5%, or from 0.95% to 1.05%.

[0088] In a preferred embodiment, the reaction is carried out under static conditions.

[0089] In a preferred embodiment, the resting period is a light-protected resting period.

[0090] In a preferred embodiment, the reaction time is 1 to 25 hours, more specifically 3 to 5 hours, for example, about 4 hours.

[0091] In a preferred embodiment, the reaction temperature is 1°C to 10°C, more specifically 3°C to 5°C, for example, about 4°C.

[0092] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 4" includes ±5% of 4, or from 3.8 to 4.2.

[0093] In a preferred embodiment, the centrifugation speed is 8000 to 12000 r / min, for example, about 10000 r / min.

[0094] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10000" includes ±5% of 10000, or from 9500 to 10500.

[0095] In a preferred embodiment, the centrifugation time is 5 to 15 minutes, for example, about 10 minutes.

[0096] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0097] In a preferred embodiment, the repetition is performed three times.

[0098] In a preferred embodiment, the polysaccharide sample or the polysaccharide test material is derived from the medicinal herb Notopterygium incisum.

[0099] In a preferred embodiment, the polysaccharide sample or the polysaccharide test specimen is derived from the dried rhizomes and roots of *Notopterygium incisum* Ting ex HTChang or *Notopterygium franchetii* H. de Boiss., a plant belonging to the Apiaceae family. The crude polysaccharide obtained by water extraction and alcohol precipitation is then separated by DEAE Sephrose™ Fast Flow anion exchange resin to obtain the elution fraction of 0.2M sodium chloride solution.

[0100] In a preferred embodiment, the polysaccharide sample or the polysaccharide test specimen is gum arabic.

[0101] In a preferred embodiment, the concentration of the polysaccharide sample is 1.5 to 2.5 mg / mL, for example, about 2 mg / mL.

[0102] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 2" includes 2 ± 5%, or from 1.9 to 2.1.

[0103] In a preferred embodiment, the concentration of the sodium dithionite solution is 8% to 12%, for example, about 10%.

[0104] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10%" includes 10% ± 5%, or from 9.5% to 10.5%.

[0105] In a preferred embodiment, the dialysis time is 40 to 60 hours, for example, about 48 hours.

[0106] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 48" includes ±5% of 48, or from 45.6 to 50.4.

[0107] In a preferred embodiment, the drying is freeze drying.

[0108] In a preferred embodiment, in step (1) or step (2), the mass ratio of the type II arabinogalactan AG-II reference standard or the polysaccharide test sample to the mass of the Yariv reaction reagent is 0.5 to 2, for example, about 1.

[0109] In a preferred embodiment, in step (1) or step (2), the concentration of the reference solution or the polysaccharide test solution is 0.5 to 2 mg / mL, for example, about 1 mg / mL.

[0110] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0111] In a preferred embodiment, in step (1) or step (2), the alkaline solution is a sodium hydroxide solution.

[0112] In a preferred embodiment, in step (1) or step (2), the molar concentration of the sodium hydroxide solution is 0.005 to 0.02 mol / L, for example, about 0.01 mol / L.

[0113] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.01" includes ±5% of 0.01, or from 0.0095 to 0.0105.

[0114] In a preferred embodiment, the volume of the sodium hydroxide solution is 0.5 to 1.5 ml, for example, about 1 ml.

[0115] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0116] In a preferred embodiment, in step (3), the amount of the reference solution taken is 100 to 250 μl, for example 200 μl.

[0117] In a preferred embodiment, in step (3), the volume of the test solution is 100 to 250 μl, for example 200 μl.

[0118] In a preferred embodiment, in step (3), the detection wavelength of the ELISA reader is 450 nm.

[0119] In a preferred embodiment, in step (5), the number of parallel measurements is 2 to 5, for example, 3.

[0120] In a preferred embodiment, for polysaccharide samples or test articles derived from Notopterygium incisum, the standard curve for type II arabinogalactan AG-II reference standard is y = 25.396x +

[0121] 0.0393, R 2 =0.9998.

[0122] In a preferred embodiment, the limit of quantitation standard curve for polysaccharide samples or test articles derived from Notopterygium incisum is y = 0.0237x + 0.0385, R0 2 =0.9998.

[0123] In a preferred embodiment, the detection limit for type II arabinogalactan AG-II reference standard is 0.72 μg for polysaccharide samples or test articles derived from Notopterygium incisum.

[0124] In a preferred embodiment, the limit of quantification for type II arabinogalactan AG-II reference standard is 2.18 μg for polysaccharide samples or test articles derived from Notopterygium incisum.

[0125] In a preferred embodiment, the standard curve for gum arabic, type II arabinogalactan AG-II reference standard, is y = 4.3187x + 0.0263, R0 2 =0.9999.

[0126] In a preferred embodiment, the limit of quantitation standard curve for gum arabic, type II arabinogalactan AG-II reference standard, is y = 0.0476x + 0.0353, R0 2=0.9995.

[0127] In a preferred embodiment, the detection limit for gum arabic type II arabinogalactan AG-II reference standard is 0.89 μg.

[0128] In a preferred embodiment, the limit of quantification for gum arabic type II arabinogalactan AG-II reference standard is 2.69 μg.

[0129] According to another aspect of the present invention, the above-described method is provided for use in the quality detection and / or quality evaluation and / or quality control of medicinal materials containing type II arabinogalactan AG-II or their exudates.

[0130] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or conditions recommended by the manufacturer.

[0131] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0132] The features mentioned above in this invention, or the features mentioned in the embodiments, can be combined arbitrarily. All features disclosed in this patent specification can be used in any compositional form, and each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0133] Example

[0134] 1. Materials and Methods

[0135] 1.1 Experimental Materials

[0136] Sample to be tested: QH-2 (crude polysaccharide obtained by water extraction and alcohol precipitation of dried rhizomes and roots of *Notopterygium incisum* Ting ex HTChang or *Notopterygium franchetii* H. de Boiss., a plant of the Apiaceae family, followed by DEAE Sephrose analysis). TMFast Flow anion exchange resin was used to separate the elution fraction of 0.2M sodium chloride solution, namely the polysaccharide QH-2 and gum arabic (General-reagent, purchased from the Exploration Platform, product number 011135934, original product number 88873B, brand Adamas).

[0137] Standards: QH-2-AG (prepared by precipitating QH-2 with Yariv reagent, followed by washing, centrifugation, dissociation, dialysis, and lyophilization), GAG (prepared by precipitating gum arabic with Yariv reagent, followed by washing, centrifugation, dissociation, dialysis, and lyophilization);

[0138] Other reagents: sodium dithionite (General-reagent), gum arabic (General-reagent), NaCl (Sinopharm Group Pharmaceutical Co., Ltd.), and Yariv reagent (self-made).

[0139] Preparation of Yariv reagent: Anhydrous methanol is added to p-nitroglucose, and after mixing and stirring, palladium catalyst on carbon is added. Hydrogen gas is introduced and stirring is continued. The reaction is carried out at room temperature for 4 hours. After complete conversion, the mixture is cooled to room temperature, filtered through diatomaceous earth, and the filtrates are combined and concentrated under reduced pressure to obtain p-aminophenyl glucosinolate. The obtained p-aminophenyl glucosinolate is dissolved in pure water, and then hydrochloric acid and sodium nitrite solution are added dropwise. The reaction is carried out at 0-5℃ for 2.5 hours for diazotization. Then, pyrogallol solution is added dropwise, and after stirring evenly, the pH is adjusted to 9.0 with sodium hydroxide solution. The coupling reaction is carried out at 0-5℃ for 2.5 hours. After the reaction is completed, the mixture is diluted with an equal volume of anhydrous methanol and left to stand overnight at 4℃. The precipitate is filtered and dried to obtain β-glucosyl-Yariv reagent.

[0140] 1.2 Instruments and Equipment

[0141] Fischer centrifuge (Shanghai Fischer Analytical Instruments Co., Ltd., SF-TGL-16M), microplate reader (Gene Company Limited), analytical balance (Sartorius Scientific Instruments (Beijing) Co., Ltd., SQP)

[0142] 1.3 Experimental Methods

[0143] 1.3.1 Preparation of Yariv reagent solution

[0144] Accurately weigh 50.00 mg of Yariv reagent, add 1% NaCl solution to it, and make up to 50 mL, with a concentration of 1.00 mg / mL.

[0145] 1.3.2 Preparation of AG-II reference standard

[0146] Gum arabic is commonly used as a standard in the literature, but experiments have shown that its linearity is insufficient. Therefore, this invention proposes to purify it, name it GAG, and then use it as a standard for quantitative analysis.

[0147] This invention involves screening a large number of polysaccharides from various parts of a traditional Chinese medicine (TCM) library. 4 mg of each polysaccharide was weighed and dissolved in 2 mL of 1% NaCl solution. The solution was centrifuged, and the supernatant was collected. An appropriate volume of the supernatant was placed in a 2 mL centrifuge tube and diluted to 1.00 mL with 1% NaCl solution. An equal volume of Yariv reagent solution was then added, mixed, and allowed to stand in the dark for 4 hours. The mixture was then centrifuged at 10000 rpm for 10 minutes. The supernatant was carefully removed, and 1.00 mL of 1% NaCl solution was added. The mixture was vortexed thoroughly, centrifuged again, and the supernatant was carefully removed. This process was repeated three times. The resulting precipitate was then purged with nitrogen, and 1.00 mL of 0.01 M NaOH solution was added and vortexed thoroughly to dissolve it. The herb with the highest absorbance, i.e., the highest AG-II content—Notopterygium incisum—was selected and purified to obtain the QH-2-AG reference standard. The screening results of AG content in the TCM library are shown in Figure 1.

[0148] 1.3.2.1 Preparation of GAG reference standard

[0149] Weigh 500 mg of gum arabic and dissolve it in 250 mL of 1% NaCl solution. Centrifuge and collect the supernatant. Add an equal volume of Yariv reagent, mix well, and incubate at 4°C in the dark for 4 hours. Then centrifuge at 10000 rpm for 10 minutes. Carefully remove the supernatant, add 1.00 mL of 1% NaCl solution, vortex thoroughly, centrifuge again, and carefully remove the supernatant. Repeat this process three times. Add sodium dithionite to bring the final concentration to 10%. After the solution turns pale yellow or colorless, dialyze through cellophane and running water for 48 hours, then freeze-dry to obtain the product, named GAG.

[0150] 1.3.2.2 Preparation of QH-2-AG reference standard

[0151] Weigh 500 mg of the polysaccharide from the QH-2 fraction and dissolve it in 250 mL of 1% NaCl solution. Centrifuge and collect the supernatant. Add an equal volume of Yariv reagent, mix well, and incubate at 4°C in the dark for 4 hours. Then centrifuge at 10000 rpm for 10 minutes. Carefully remove the supernatant, add 1.00 mL of 1% NaCl solution, vortex thoroughly, centrifuge again, and carefully remove the supernatant. Repeat this process three times. Add sodium dithionite to bring the final concentration to 10%. After the solution turns pale yellow or colorless, dialyze through cellophane and running water for 48 hours, then freeze-dry to obtain the product, named QH-2-AG.

[0152] This invention used HPGPC, sugar composition analysis, and nuclear magnetic resonance (NMR) to detect its structure. HPGPC results showed a homogeneous structure; the sugar composition showed that it mainly consisted of galactose and arabinose in a 95:5 ratio, satisfying the composition of AG-II type polysaccharides; methylation results showed its linkage mode as (to be supplemented), proving that QH-2-AG is indeed an AG-II type polysaccharide. The results are shown in Figures 2, 3, and 4.

[0153] 1.3.3 Preparation of AG-II reference solution

[0154] 1.3.3.1 Preparation of GAG reference solution

[0155] Accurately weigh 5.00 mg of GAG sample, add 1% NaCl solution, and dilute to 50 mL. Mix well; the concentration is 0.10 mg / mL. This solution will be used as a reference solution.

[0156] Take an appropriate volume of GAG reference solution and place it in a 2 mL centrifuge tube. Dilute to 1.00 mL with 1% NaCl solution. Add an equal volume of Yariv reagent solution, mix well, and let stand in the dark for 4 hours. Then centrifuge at 10000 rpm for 10 minutes. Carefully remove the supernatant, add 1.00 mL of 1% NaCl solution, vortex thoroughly, centrifuge again, and carefully remove the supernatant. Repeat this process three times. After nitrogen blowing, add 1.00 mL of 0.01 M NaOH solution and vortex thoroughly to dissolve the precipitate.

[0157] 1.3.3.2 Preparation of QH-2-AG reference solution

[0158] Accurately weigh 5.00 mg of QH-2-AG sample, add 1% NaCl solution, and dilute to 50 mL. Mix well; the concentration is 0.10 mg / mL. This solution will be used as a reference solution.

[0159] Take an appropriate volume of QH-2-AG reference solution and place it in a 2 mL centrifuge tube. Dilute to 1.00 mL with 1% NaCl solution. Add an equal volume of Yariv reagent solution, mix well, and let stand in the dark for 4 hours. Then centrifuge at 10000 rpm for 10 minutes. Carefully remove the supernatant, add 1.00 mL of 1% NaCl solution, vortex thoroughly, centrifuge again, and carefully remove the supernatant. Repeat this process three times. After nitrogen blowing, add 1.00 mL of 0.01 M NaOH solution and vortex thoroughly to dissolve the precipitate.

[0160] 1.3.4 Preparation of AG-II test solution

[0161] 1.3.4.1 Preparation of gum arabic test solution

[0162] Accurately weigh five portions of gum arabic sample, each 5.00 mg, add 1% NaCl solution to each portion, and dilute to 50 mL. Mix well; the concentration is 0.10 mg / mL. Prepare the solution according to the method described in section "1.3.3" and use it as the test solution.

[0163] 1.3.4.2 Preparation of QH-2 test solution

[0164] Accurately weigh five portions of QH-2 sample, each 5.00 mg, add 1% NaCl solution to each portion, and dilute to 50 mL. Mix well; the concentration is 0.10 mg / mL. Prepare the solution according to the method described in section "1.3.3" and use it as the test solution.

[0165] 2. Methodological Examination

[0166] 2.1 Condition Optimization

[0167] 2.1.1 Optimization of reaction time and temperature

[0168] Accurately weigh 1 mg of GAG sample into a centrifuge tube and add NaCl solution to a final volume of 1.00 mL. Follow the procedure outlined in section "1.3.3". Take 200 μL of the sample and measure the absorbance at 450 nm using a microplate reader. The results are shown in Figures 5 and 6. The highest absorbance value was observed after precipitation at 4°C for 4 hours, resulting in the complete dissolution of the precipitate.

[0169] 2.1.2 Optimization of ingredient ratio

[0170] Accurately weigh 1 mg of GAG sample into a centrifuge tube and add NaCl solution to 1.00 mL. Perform the procedure according to section "1.3.3" (note: the concentration of Yariv reagent used may vary). Take 200 μL and measure the absorbance at 450 nm using a microplate reader. The results are shown in Figures 7 and 8. The absorbance value of the dissolved precipitate was the highest when the material ratio was 1:1.

[0171] 2.1.3 Optimization of washing cycles

[0172] Accurately weigh 1 mg of GAG sample into a centrifuge tube and add NaCl solution to 1.00 mL. Perform the procedure according to section "1.3.3", repeating steps 2, 3, and 4. Take 200 μL of each sample and measure the absorbance at 450 nm using a microplate reader. The results are shown in Table 1. Some Yariv reagent remained after two washes, and the absorbance change was not significant in the 3rd and 4th washes. The 3rd wash was chosen for its best cost-effectiveness.

[0173] Table 1 Optimization of Washing Frequency

[0174] 2.1.4 Optimization of salt concentration in solution preparation

[0175] Accurately weigh 1 mg of GAG sample into a centrifuge tube, and add 0.5%, 1%, 2%, and 3% NaCl solution to a final volume of 1.00 mL. Perform the procedure as described in section "1.3.3", and take 200 μL of the solution. Measure the absorbance at 450 nm using a microplate reader. The results are shown in Table 2. The highest absorbance was observed when the salt concentration was 1%.

[0176] Table 2 Optimization of Salt Concentration Conditions

[0177] 2.1.5 Summary

[0178] Experiments revealed that the optimal concentration of the Yariv reagent in the AG-II solution (1% salt concentration, 4℃ temperature, 1:1 material ratio) reached its peak. Three washes of the precipitate were sufficient; washing it more frequently resulted in precipitate loss and negatively impacted the experimental results.

[0179] 2.2 Linear Examination

[0180] 2.2.1 Linearity Study of GAG Reference Standard

[0181] Take 0.00, 0.10, 0.20, 0.30, 0.40, and 0.50 mL of GAG reference solution respectively and place them in centrifuge tubes. Add NaCl solution to a final volume of 1.00 mL. Perform the procedure according to section "1.3.3". Take 200 μL of each solution and measure the absorbance at 450 nm using a microplate reader. The linear regression equation is y = 4.3187x + 0.0263. The linear fit is R0. 2 =0.9999, indicating good linearity, as shown in Figure 9.

[0182] 2.2.2 Linearity Study of QH-2-AG Reference Standard

[0183] Take 0.00, 0.10, 0.20, 0.30, 0.40, and 0.50 mL of QH-2-AG reference solution and place them in centrifuge tubes, respectively, and add NaCl solution to a final volume of 1.00 mL. Follow the procedure in section "1.3.3". Take 200 μL of the standard solution and measure the absorbance at 450 nm using a microplate reader. The linear regression equation is y = 25.396x + 0.0393. The linear fit R0 is... 2 =0.9998, indicating good linearity, as shown in Figure 10.

[0184] 2.3 Stability

[0185] 2.3.1 Stability of GAG Reference Standard

[0186] Take 0.30 mL of GAG reference solution, add NaCl solution to a final volume of 1.00 mL, and then add an equal volume of Yariv reagent solution. Perform the procedure according to section "1.3.3". Take 200 μL of the same concentration of reference solution at 0.5, 1, 2, 4, 8, 10, 12, and 24 hours, and measure the absorbance at 450 nm. Perform three parallel tests. The relative standard deviation was 0.37%, indicating good solution stability. The results are shown in Table 3.

[0187] Table 3 Stability of GAG reference solution

[0188] 2.3.2 Stability of QH-2-AG Reference Standard

[0189] Take 0.30 mL of QH-2-AG reference solution, add NaCl solution to a final volume of 1.00 mL, and then add an equal volume of Yariv reagent solution. Perform the procedure according to section "1.3.3". Take 200 μL of the same concentration of reference solution at 0.5, 1, 2, 4, 8, 10, 12, and 24 hours, and measure the absorbance at 450 nm. Perform three parallel tests. The relative standard deviation was 0.55%, indicating good solution stability. The results are shown in Table 4.

[0190] Table 4 Stability of QH-2-AG reference solution

[0191] 2.4 Instrument Precision

[0192] Take 0.20 mL of QH-2-AG reference solution, add NaCl solution to 1.00 mL, and add an equal volume of Yariv reagent solution. Perform the operation according to the method in section "1.3.3". Take 200 μL of the solution and measure it 6 times at 450 nm. The relative standard deviation is 0.07%, and the instrument precision is good. The results are shown in Table 5.

[0193] Table 5 Instrument Precision

[0194] 2.5 Repeatability

[0195] 2.5.1 Repeatability of GAG reference standard

[0196] Take 0.30 mL of GAG reference solution, add NaCl solution to a final volume of 1.00 mL, and then add an equal volume of Yariv reagent solution. Perform the procedure according to section "1.3.3", repeating 6 times. Take 200 μL of solution for each sample and measure the absorbance at 450 nm. Perform three parallel groups. The relative standard deviation was 0.42%, indicating good repeatability. The results are shown in Table 6.

[0197] Table 6 Repeatability test of GAG reference standard

[0198] 2.5.2 Repeatability of QH-2-AG Reference Standard

[0199] Take 0.20 mL of QH-2-AG reference solution, add NaCl solution to a final volume of 1.00 mL, and then add an equal volume of Yariv reagent solution. Perform the procedure according to section "1.3.3", repeating 6 times. Take 200 μL of solution for each sample and measure the absorbance at 450 nm. Perform three parallel groups. The relative standard deviation was 0.5%, indicating good repeatability. The results are shown in Table 7.

[0200] Table 7 Repeatability test of QH-2-AG reference standard

[0201] 2.6 Recovery Test

[0202] 2.6.1 GAG reference standard spiking recovery test

[0203] Take 0.7 mL of the same concentration of gum arabic test solution, add 0.15 mL of GAG reference solution, add NaCl solution to a final volume of 1.00 mL, and then add an equal volume of Yariv reagent solution. Perform the procedure according to section "1.3.3", and measure the absorbance at 450 nm. Perform six parallel tests. The average recovery rate was 99.95%, with a relative standard deviation of 0.56%, which meets the pharmacopoeia requirements. The results are shown in Table 8.

[0204] Table 8. Recovery rate of GAG reference standard after spiking

[0205] 2.6.2 Recovery test of QH-2-AG reference standard

[0206] Take 0.35 mL of the same concentration of QH-2 test solution, add 0.15 mL of QH-2-AG reference solution, add NaCl solution to a final volume of 1.00 mL, and then add an equal volume of Yariv reagent solution. Perform the procedure according to section "1.3.3", and measure the absorbance at 450 nm. Perform six parallel tests. The average recovery rate was 99.61%, with a relative standard deviation of 0.77%, which meets the pharmacopoeia requirements. The results are shown in Table 9.

[0207] Table 9. Recovery rate of QH-2-AG reference standard.

[0208] 2.6 Limit of Detection and Limit of Quantification

[0209] 2.6.1 Limits of Detection and Limits of Quantification of GAG Reference Standards

[0210] Accurately measure an appropriate amount of QH-2-AG reference solution, dilute the reference solution, and perform the determination according to the method in section "1.3.3". The detection limit and quantitation limit were set to LOD = 3.3δ / S and LOQ = 10δ / S, respectively. The detection limit of QH-2-AG reference solution was 0.89 μg, and the quantitation limit was 2.69 μg. The results are shown in Figure 11 and Table 10.

[0211] Table 10 Limit of Quantification and Limit of Detection

[0212] 2.6.2 Limits of Detection and Limits of Quantification for QH-2-AG Reference Standard

[0213] Accurately measure an appropriate amount of QH-2-AG reference solution, dilute the reference solution, and perform the determination according to the method in section "1.3.3". The detection limit and quantitation limit were set to LOD = 3.3δ / S and LOQ = 10δ / S, respectively. The detection limit of QH-2-AG reference solution was 0.72 μg, and the quantitation limit was 2.18 μg. The results are shown in Figure 12 and Table 11.

[0214] Table 11 Limit of Quantification and Limit of Detection

[0215] 2.7 Summary

[0216] The method for precipitating AG-II with Yariv reagent was optimized and the methodology was examined. The linear regression equation was y = 25.396x + 0.0393. The linear fit R0 was [value missing]. 2=0.9998, indicating good linearity; the test solution showed good stability within 24 hours, with an RSD of 0.55%; after three replicates, the RSD was 0.5%, indicating good method repeatability; the recovery test met the limits specified in the Chinese Pharmacopoeia; the limits of quantitation and detection were calculated according to the requirements of the Chinese Pharmacopoeia.

[0217] 3. Application of the method

[0218] Using the above methodology, a batch of samples were measured according to the procedures in section "1.3.3", as shown in Table 12.

[0219] Table 12 Methodological Examples

[0220] The embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The descriptions of the embodiments above are only for the purpose of helping to understand the method and core ideas of the present invention. Furthermore, any changes or modifications made by those skilled in the art based on the ideas of the present invention, its specific implementation methods, and its application scope, are all within the scope of protection of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A method for preparing type II arabinogalactan AG-II, characterized in that, The method includes the following steps: (i) Weigh an appropriate amount of polysaccharide sample, dissolve it in a salt solution, centrifuge to obtain a supernatant, add an equal volume of Yariv reaction reagent solution to the supernatant, mix well and react for a period of time, centrifuge to obtain a precipitate; (ii) Add salt solution to the precipitate, vortex, centrifuge, remove the supernatant, and repeat 2–4 times; and (iii) Add a certain concentration of sodium dithionite solution to the precipitate after washing. After the solution turns pale yellow or colorless, dialyze it with cellophane and running water for a period of time, and then dry it to obtain the type II arabinogalactan AG-II. The polysaccharide sample contains type II arabinogalactan AG-II.

2. A method for quantitatively determining the content of type II arabinogalactan AG-II in a polysaccharide test sample, characterized in that, The method includes the following steps: (1) Preparation of type II arabinogalactan AG-II reference solution: (a) Weigh an appropriate amount of type II arabinogalactan AG-II reference standard, dissolve it in the salt solution, make up to volume and mix well; (b) Take an appropriate amount of the reference solution into a container, add salt solution to dilute it, then add an equal volume of Yariv reaction reagent solution, mix well and react for a period of time, centrifuge and remove the supernatant; (c) Add salt solution to the precipitate, vortex, centrifuge, remove the supernatant, repeat 2 to 4 times; and (d) After washing, blow the precipitate with nitrogen, add alkaline solution to it, vortex to dissolve it, and obtain the type II arabinogalactan AG-II reference solution; (2) Preparation of polysaccharide test solution: (a) Weigh an appropriate amount of polysaccharide test sample, dissolve it in salt solution, make up to volume and mix well; (b) Take an appropriate amount of polysaccharide test solution into a container, add salt solution to dilute, then add an equal volume of Yariv reaction reagent solution, mix well and react for a period of time, centrifuge and remove the supernatant; (c) Add salt solution to the precipitate, vortex, centrifuge, remove the supernatant, repeat 2 to 4 times; and (d) After washing, blow the precipitate with nitrogen, add alkaline solution to it, vortex to dissolve, and obtain the polysaccharide test solution; (3) Take appropriate amounts of the type II arabinogalactan AG-II reference solution and the polysaccharide test solution, and measure the absorbance value using an enzyme-linked immunosorbent assay (ELISA) reader: (4) Prepare the standard curve of type II arabinogalactan AG-II reference standard; as well as (5) Substitute the absorbance value of the polysaccharide test solution into the standard curve to obtain the concentration of type II arabinogalactan AG-II in the polysaccharide test solution, and take the average value of the parallel determinations.

3. The method according to claim 1 or 2, characterized in that, The salt solution is a sodium chloride solution; Preferably, the volume concentration percentage of the sodium chloride solution is 0.5% to 3%, for example, about 1%. Preferably, the concentration of the Yariv reaction reagent solution is 0.5% to 2%, for example, about 1%; Preferably, the reaction is carried out under static conditions; Preferably, the resting period is a light-protected resting period; Preferably, the reaction time is 1 to 25 hours, more preferably 3 to 5 hours, for example, about 4 hours; Preferably, the reaction temperature is 1°C to 10°C, more preferably 3°C to 5°C, for example, about 4°C; Preferably, the centrifugation speed is 8000-12000 r / min, for example, about 10000 r / min; Preferably, the centrifugation time is 5 to 15 minutes, for example, about 10 minutes; Preferably, the repetition is performed 3 times.

4. The method according to claim 1 or 2, characterized in that, The polysaccharide sample or the polysaccharide test sample is derived from the medicinal herb Notopterygium incisum. Preferably, the polysaccharide sample or the polysaccharide test sample is derived from the dried rhizomes and roots of *Notopterygium incisum* Ting ex HTChang or *Notopterygium franchetii* H. de Boiss., which are obtained by water extraction and alcohol precipitation. The crude polysaccharide is then separated by DEAE Sephrose™ Fast Flow anion exchange resin to obtain the elution fraction of 0.2M sodium chloride solution.

5. The method according to claim 1 or 2, wherein the polysaccharide sample or the polysaccharide test sample is gum arabic.

6. The method according to claim 1, characterized in that, The concentration of the polysaccharide sample is 1.5–2.5 mg / mL, for example, about 2 mg / mL; Preferably, the concentration of the sodium dithionite solution is 8% to 12%, for example, about 10%; Preferably, the dialysis time is 40-60 hours, for example, about 48 hours; Preferably, the drying is freeze drying.

7. The method according to claim 2, characterized in that, In step (1) or step (2), the mass ratio of the type II arabinogalactan AG-II reference standard or the polysaccharide test sample to the mass of the Yariv reaction reagent is 0.5 to 2, for example, about 1; Preferably, in step (1) or step (2), the concentration of the reference solution or the polysaccharide test solution is 0.5 to 2 mg / mL, for example, about 1 mg / mL; Preferably, in step (1) or step (2), the alkaline solution is a sodium hydroxide solution; Preferably, in step (1) or step (2), the molar concentration of the sodium hydroxide solution is 0.005 to 0.02 mol / L, for example, about 0.01 mol / L; Preferably, the volume of the sodium hydroxide solution is 0.5 to 1.5 ml, for example, about 1 ml; Preferably, in step (3), the amount of the reference solution taken is 100-250 μl, for example 200 μl; Preferably, in step (3), the volume of the test solution is 100-250 μl, for example 200 μl; Preferably, in step (3), the detection wavelength of the ELISA reader is 450 nm; Preferably, in step (5), the number of parallel measurements is 2 to 5, for example, 3.

8. The method according to claim 4, characterized in that, For polysaccharide samples or test samples derived from Notopterygium incisum, the standard curve for type II arabinogalactan AG-II reference standard is y = 25.396x + 0.0393, R0 2 =0.9998; Preferably, for polysaccharide samples or test samples derived from Notopterygium incisum, the limit of quantitation standard curve for type II arabinogalactan AG-II reference standard is y = 0.0237x + 0.0385, R0 2 =0.9998; Preferably, the detection limit for type II arabinogalactan AG-II reference standard is 0.72 μg for polysaccharide samples or test samples derived from Notopterygium incisum. Preferably, the limit of quantification for type II arabinogalactan AG-II reference standard is 2.18 μg for polysaccharide samples or test samples derived from Notopterygium incisum.

9. The method according to claim 5, characterized in that, For gum arabic, the standard curve for type II arabinogalactan AG-II reference standard is y = 4.3187x + 0.0263, R0 2 =0.9999; Preferably, for gum arabic, the limit of quantitation standard curve for type II arabinogalactan AG-II reference standard is y = 0.0476x + 0.0353, R 2 =0.9995; Preferably, the detection limit for gum arabic type II arabinogalactan AG-II reference standard is 0.89 μg; Preferably, the limit of quantification for gum arabic type II arabinogalactan AG-II reference standard is 2.69 μg.

10. Use of the method according to any one of claims 1 to 9 in the quality detection and / or quality evaluation and / or quality control of medicinal materials containing type II arabinogalactan AG-II or their exudates.

Images