Astragalus polysaccharide processed by different processing technologies and application thereof

Abstract

The invention discloses astragalus polysaccharide processed by different processing technologies and application thereof, self-weight polysaccharides of RAPSs, SAPSs, WAPSs and HAPSs are extracted from crude products and processed products obtained by salt processing, wine processing and honey processing, and the influence of different processing methods on the structure and bidirectional immunomodulatory activity of the astragalus polysaccharide is discussed. Results show that different processing methods change the properties and structural characteristics of the astragalus polysaccharide so as to influence the bidirectional immunomodulatory activity of the astragalus polysaccharide; the extraction rate, chemical composition, molecular weight, monosaccharide composition, chain conformation and surface morphology, Zeta potential and particle size, immunity enhancement and anti-inflammatory activity and the like of the four polysaccharides are remarkably different, but the four polysaccharides have the same typical crystal interior, main glycoside residues and O-glucosidic bonds. In a test concentration range, the honey-processed polysaccharide has a relatively strong immunity enhancing capability, and the wine-processed polysaccharide has a relatively strong anti-inflammatory capability.

Description

Technical Field

[0001] The invention relates to an astragalus polysaccharide and a preparation method and application thereof, in particular to an astragalus polysaccharide processed by different processing techniques and an application thereof, belonging to the technical field of polysaccharide extraction and preparation. Background Art

[0002] Astragali Radix (AR) belongs to the Leguminosae family and is a traditional Chinese medicinal material native to northern China. It is known as the "holy medicine for replenishing qi" and has a history of medicinal use of more than 2,000 years. In folk medicine, Astragali Radix is ​​also widely used in food therapy. It is a well-known health tonic and a Chinese medicinal material that can be used as both medicine and food. Modern pharmacological studies have shown that Astragali Radix exhibits significant biological activity in regulating immune system function, anti-tumor, anti-inflammatory and antioxidant aspects. Therefore, it is widely used in functional foods and pharmaceutical fields. The pharmacological activity of Astragali Radix is ​​related to its rich and varied chemical components. So far, a variety of chemical components such as flavonoids, saponins, amino acids, and polysaccharides have been isolated and identified from Astragali Radix. Among them, polysaccharides are one of the main active ingredients of Astragali Radix that exert their medicinal effects. It has a variety of pharmacological activities, such as anti-tumor, anti-aging, hypoglycemic, antibacterial, and immunomodulatory.

[0003] In recent years, astragalus polysaccharides have attracted much attention due to their high efficiency, low toxicity and bidirectional immunomodulatory activity. A large number of studies have shown that astragalus polysaccharides help promote the growth of central (bone marrow, thymus) and peripheral (spleen, lymph nodes and mucosa-associated lymphoid tissue) immune organs, thereby enhancing the body's immunity. On the other hand, when the body's autoimmune level is too high, astragalus polysaccharides (APSs) can regulate the growth, differentiation and cytokine secretion of T lymphocytes, B lymphocytes, natural killer cells and antigen-presenting cells, thereby downregulating the body's immune response level and antagonizing excessive immune responses. APSs can exert bidirectional immunomodulatory effects through multiple targets and multiple pathways to help restore and maintain the body's immune balance.

[0004] Polysaccharide structure is unique and complex, and its biological activity is closely related to its complex chemical structure and different spatial conformations. Therefore, studying and analyzing its unique chemical structure and conformation is the basis for the effective use of polysaccharides. However, many studies have shown that the chemical structure of polysaccharides from the same source will be affected by different processing methods, resulting in changes in their activity. And the processing mechanism has a synergistic effect on the chemistry, pharmacology and pharmacokinetics of the effective ingredients of traditional Chinese medicine. In the prior art, some people have studied the effects of different processing techniques on the structure and antioxidant activity of Cistanche polysaccharides. The results show that the processing technology will affect the physicochemical properties of the polysaccharide and improve its antioxidant activity. Some scholars have also found that the physicochemical properties of processed and unprocessed Polysaccharides from Polygonum multiflorum are significantly different, and the liver protection potential is also significantly enhanced after processing.

[0005] In traditional Chinese medicine theory, the traditional processing of Chinese herbal medicines is called processing. Processing of Chinese herbal medicines can reduce the side effects of herbal medicines, enhance their medicinal properties, and broaden their scope of medicinal use. Reasonable processing can enable Chinese herbal medicines to fully exert their efficacy and comprehensively enhance their clinical efficacy. However, different processing methods may change the changes in polysaccharides and other components in Chinese herbal medicines, ultimately leading to changes in their efficacy. In China, the classic processing methods of Astragalus include salt processing, honey processing, and wine processing. The three different traditional processing methods may affect the physical properties or structural characteristics of APSs, thereby causing changes in their bidirectional immunomodulatory activity. However, the effects of different traditional processing techniques on the structural characterization and bidirectional immunomodulatory activity of Astragalus polysaccharides have not yet been reported. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide an astragalus polysaccharide processed by different processing techniques and its application in view of the deficiencies in the prior art. The present invention processes raw astragalus slices, and obtains three kinds of Chinese medicine slice samples by three classic processing methods: salt-processed AR (SAR), wine-processed AR (WAR) and honey-processed AR (HAR); extracts the obtained raw products and three processed slice samples to obtain four kinds of crude polysaccharides-raw astragalus crude polysaccharides (RAPSs), salt-processed astragalus crude polysaccharides (SAPSs), wine-processed astragalus crude polysaccharides (WAPSs) and honey-processed astragalus crude polysaccharides (HAPSs); the chemical composition, molecular weight, monosaccharide composition, glycosidic bond, molecular conformation, physicochemical properties and bidirectional immunomodulatory activity of these four kinds of polysaccharides are deeply studied. The present invention provides a theoretical basis for the efficient utilization of astragalus polysaccharides and provides a scientific basis for the development of astragalus polysaccharides in the fields of food and medicine.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] The present invention provides a method for processing astragalus polysaccharides with different processing techniques, wherein raw astragalus slices are processed by salt processing, wine processing and honey processing, comprising the following steps:

[0009] (1) Take raw Astragalus slices, add salt water, rice wine and refined honey respectively, mix well and simmer thoroughly, put them in a pot and stir-fry them over low heat until dry, then take them out and let them cool, to obtain salt-processed Astragalus slices, wine-processed Astragalus slices and honey-processed Astragalus slices respectively;

[0010] (2) The salt-processed Astragalus slices, wine-processed Astragalus slices, and honey-processed Astragalus slices obtained in step (1) are respectively crushed using a traditional Chinese medicine crusher, and the crushed samples are respectively weighed and mixed with water, and the crushed samples are heated to dissolve and then centrifuged to remove insoluble residues to obtain supernatants; the supernatants are respectively concentrated to obtain concentrated solutions, and the concentrated solutions are separated by convection through a retention membrane (molecular weight: 5000Da) at a ratio of concentrated solution: water (1:2.5) to obtain separated solutions; the separated solutions are then dialyzed (molecular weight: 3500Da) to obtain dialyzates; the dialyzates are respectively centrifuged, and the supernatants are concentrated and freeze-dried using a freeze dryer to obtain crude Astragalus polysaccharides, crude Astragalus polysaccharides processed with wine, and crude Astragalus polysaccharides processed with honey, respectively.

[0011] Preferably, the present invention first provides a method for preparing astragalus polysaccharide by salt processing, comprising the following steps:

[0012] (1) Take 100 g of raw Astragalus slices (RAR), take 2 g of edible salt and add appropriate amount of water to make brine, add Astragalus polysaccharide brine processed by salt processing method to the raw Astragalus, mix well and stew thoroughly, put it in a hot pot and stir-fry it over low heat until it is dry, then take it out and cool it to obtain salt-processed Astragalus slices (SAR);

[0013] (2) The salt-processed Astragalus membranaceus slices obtained in step (1) are crushed by a Chinese medicine crusher, the crushed sample is weighed and mixed with water, heated to dissolve, and then centrifuged to remove insoluble residues to obtain a supernatant; the supernatant is concentrated to obtain a concentrated solution, and the concentrated solution is separated by convection through a retention membrane (molecular weight: 5000Da) at a ratio of concentrated solution: water (1:2.5) to obtain a separated solution; the separated solution is then dialyzed (molecular weight: 3500Da) to obtain a dialyzate; the dialyzate is centrifuged and filtered, and the supernatant is concentrated and then freeze-dried using a freeze dryer to obtain a crude salt-processed Astragalus membranaceus polysaccharide, which is named SAPSs.

[0014] The present invention also provides salt-processed astragalus crude polysaccharides SAPSs obtained by processing raw astragalus slices through the above-mentioned salt processing method: the average particle size is 115.2nm; the molecular weight is 86.34kDa; the neutral sugar content is 58.70±3.39%, the uronic acid content is 1.80±0.23%, and the total protein content is 9.13±1.54%; the SAPSs includes the following monosaccharides: arabinose 11.52%, glucosamine hydrochloride 0.92%, galactose 11.10%, glucose 74.05%, xylose 1.47%, mannose 0.51%, and galacturonic acid 0.43%.

[0015] The present invention also provides an application of the salt-processed astragalus crude polysaccharide SAPSs in preparing medicines, foods and health foods with bidirectional immunomodulatory effects.

[0016] Preferably, the present invention also provides a method for preparing astragalus polysaccharide by wine processing, comprising the following steps:

[0017] (1) Take 100 g of raw Astragalus slices (RAR), add 20 mL of rice wine, mix well and simmer thoroughly, put in a pot and stir-fry over low heat until slightly yellow, take out and let cool to obtain wine-processed Astragalus slices (WAR);

[0018] (2) The wine-processed Astragalus slices obtained in step (1) are crushed by a Chinese medicine crusher, the crushed sample is weighed and mixed with water, heated to dissolve, and then centrifuged to remove insoluble residues to obtain a supernatant; the supernatant is concentrated to obtain a concentrated solution, and the concentrated solution is separated by convection through a cutoff membrane (molecular weight: 5000Da) at a ratio of concentrated solution: water (1:2.5) to obtain a separated solution; the separated solution is then dialyzed (molecular weight: 3500Da) to obtain a dialysate; the dialysate is centrifuged, and then the supernatant is concentrated and freeze-dried using a freeze dryer to obtain a wine-processed Astragalus crude polysaccharide, which is named WAPSs.

[0019] The present invention also provides a wine-processed astragalus crude polysaccharide WAPSs obtained by processing raw astragalus slices in a wine-processing manner: the average particle size is 378.9 nm; the molecular weight is 112.39 kDa; the neutral sugar content is 69.46±4.40%, the uronic acid content is 2.10±0.002%, and the total protein content is 2.10±0.36%; the WAPSs comprises the following monosaccharides: 8.23% arabinose, 0.70% glucosamine hydrochloride, 8.10% glucose, 80.74% galactose, 1.10% xylose, 0.59% mannose, and 0.54% galacturonic acid.

[0020] The present invention also provides an application of the wine-processed astragalus crude polysaccharide WAPSs in preparing medicines, foods and health foods with anti-inflammatory effects.

[0021] Preferably, the present invention also provides a method for preparing astragalus polysaccharide by using a honey-processing method, comprising the following steps:

[0022] (1) Take 100 g of raw Astragalus slices (RAR), add 25 g of refined honey and an appropriate amount of water to dilute, stir evenly, put into a hot pot after simmering, and stir-fry over low heat until the aroma comes out and the Astragalus is dark yellow and not sticky, take out and cool to obtain honey-processed Astragalus slices (HAR);

[0023] (2) The honey-processed Astragalus slices obtained in step (1) are crushed by a traditional Chinese medicine crusher, the crushed sample is weighed and mixed with water, heated to dissolve, and then centrifuged to remove insoluble residues to obtain a supernatant; the supernatant is concentrated to obtain a concentrated solution, and the concentrated solution is separated by convection through a retention membrane (molecular weight: 5000Da) at a ratio of concentrated solution: water (1:2.5) to obtain a separated solution; the separated solution is then dialyzed (molecular weight: 3500Da) to obtain a dialysate; the dialysate is centrifuged, and then the supernatant is concentrated and freeze-dried using a freeze dryer to obtain a crude honey-processed Astragalus polysaccharide, which is named HAPSs.

[0024] The invention provides a honey-processed astragalus crude polysaccharide HAPSs obtained by processing raw astragalus slices in a honey-processing manner: the HAPSs have an average particle size of 163.5 nm, a molecular weight of 138.70 kDa, a neutral sugar content of 75.69±3.76%, a uronic acid content of 2.23±0.03%, and a total protein content of 9.04±0.91%; the HAPSs comprises the following monosaccharides: 11.32% arabinose, 0.90% glucosamine hydrochloride, 10.55% galactose, 74.65% glucose, 1.37% xylose, 0.58% mannose, and 0.63% galacturonic acid.

[0025] The invention provides an application of the honey-processed astragalus crude polysaccharide in preparing medicines, foods and health foods with immunity enhancement capabilities.

[0026] The present invention simulates the traditional Chinese medicine decoction method to extract four kinds of crude astragalus polysaccharides from raw products and three processed products, which are named RAPSs, SAPSs, WAPSs and HAPSs. The effects of different processing methods on the structure and bidirectional immunomodulatory activity of astragalus polysaccharides were explored. The results showed that different processing methods changed the properties and structural characteristics of astragalus polysaccharides and thus affected their bidirectional immunomodulatory activity. The four polysaccharides obtained had significant differences in extraction rate, chemical composition, molecular weight, monosaccharide composition, chain conformation and surface morphology, Zeta potential and particle size, and immune enhancement and anti-inflammatory activity, but had the same typical crystal interior, main glycoside residues, and O-glycosidic bonds. In addition, within the test concentration range, honey-processed astragalus crude polysaccharide had a stronger immune enhancement ability, while wine-processed astragalus crude polysaccharide had a stronger anti-inflammatory ability. The results of this study provide an important theoretical basis for further revealing the processing mechanism of astragalus. It also provides a scientific basis for the development and efficient utilization of astragalus polysaccharides in the fields of food and medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 : Verify the comparison of the appearance color of the Astragalus slices obtained by different processing methods in Example 1;

[0028] Figure 2 : FT-IR spectra (A), X-ray diffraction patterns (B) and thermal stability patterns (C) of astragalus polysaccharides obtained by different processing methods in Example 3

[0029] Figure 3 : Verification of the microstructures of RAPSs, SAPSs, WAPSs and HAPSs obtained by different processing methods in Example 3. At a magnification of 2000 times, the morphological characteristic diagram (A) and 2D and 3D atomic force microscope (AFM) images (B) of APSs obtained by different treatments.

[0030] Figure 4 :Zeta potential and particle size analysis diagram (A), Congo red analysis diagram (B) and UV scanning diagram of β-elimination reaction (C) of RAPSs, SAPSs, WAPSs and HAPSs obtained by different processing methods in Example 3

[0031] Figure 5 : Verify the NMR analysis spectra of RAPSs, SAPSs, WAPSs and HAPSs obtained by different processing methods in Example 3 (wherein part A represents 1 H NMR spectrum, Part B represents 13 C NMR spectroscopy

[0032] Figure 6 : Bar graph verifying the effect of RAPSs, SAPSs, WAPSs and HAPSs on RAW264.7 cell viability determined by CCK-8 method in the presence or absence of lipopolysaccharide LPS (1 μg / mL) in Example 4 (Note: Values ​​are expressed as mean ± standard deviation (n = 3))

[0033] Figure 7 : Bar graphs verifying the effects of different concentrations of RAPSs, SAPSs, WAPSs and HAPSs on macrophage phagocytic activity (A), NO release (B), TNF-α secretion (C) and IL-6 secretion (D) in Example 4;

[0034] Figure 8 : Bar graphs verifying the effects of RAPSs, SAPSs, WAPSs and HAPSs on the release of NO (A), secretion of TNF-α (B) and secretion of IL-6 (C) by RAW 264.7 macrophages activated by lipopolysaccharide in Example 4 (Note: Values ​​are expressed as mean ± standard deviation (n = 3).

[0035] Fig. 9 : Verify the Pearson correlation analysis between the chemical structures and immune activity of RAPSs, SAPSs, WAPSs and HAPSs in Example 5.

[0036] Fig.10 : Schematic diagram of the bidirectional immunomodulatory activity of RAPSs, SAPSs, WAPSs and HAPSs in the present invention. DETAILED DESCRIPTION

[0037] The specific implementation of the technical solution of the present invention is described in detail below, but the present invention is not limited to the following description:

[0038] Materials and chemical reagents involved in the following embodiments of the present invention: Monosaccharide reference substances arabinose, galactose, glucose, xylose, mannose, galacturonic acid, and glucosamine hydrochloride were purchased from Sigma-Aldrich (purity ≥ 99.9%, Sigma-Aldrich, USA). RAW264.7 cells were purchased from Wuhan Pronosai Life Science Technology Co., Ltd. (Wuhan, China). DMEM culture medium and fetal bovine serum (FBS) were purchased from Bio-Industry Company (Kibbutz Beit Haemek, Israel). Dimethyl sulfoxide (DMSO) was purchased from Solarbio (Beijing, China). Nitric oxide (NO) detection kit Nanjing Jiancheng Bioengineering Institute (Nanjing, China). TNF-α and IL-6 kits were purchased from Nanjing Boyan Biotechnology Co., Ltd. (Nanjing, China). Other chemical reagents were at least analytical grade or higher.

[0039] Embodiment 1:

[0040] A method for preparing astragalus polysaccharide by salt processing, comprising the following steps:

[0041] (1) Take 100 g of raw Astragalus slices RAR, add 2 g of edible salt and appropriate amount of water to make salt water, add the salt water to the raw Astragalus, mix well and simmer thoroughly, put it in a hot pot and stir-fry it over low heat until it is dry, then take it out and let it cool to obtain the salt-processed Astragalus slices SAR.

[0042] (2) The salt-processed Astragalus slices obtained in step (1) were crushed with a Chinese medicine crusher, 55 g of the crushed sample was weighed, mixed with water at a ratio of 1:30 (w / v, dry basis ratio), heated at 90°C for 120 min, and centrifuged to remove insoluble residues. After the supernatant was concentrated to 200 mL at 50°C, it was separated by convection through a cutoff membrane (molecular weight: 5000 Da) at a ratio of concentrate: water (1:2.5) until the separated liquid with a molecular weight greater than 5000 Da was 100 mL. The separated liquid was then placed in a dialysis bag (molecular weight: 3500 Da) and dialyzed with tap water for 3 days. The dialyzate was concentrated to 30 mL by centrifugation and lyophilized with a freeze dryer to obtain salt-processed Astragalus crude polysaccharide, which was named SAPSs.

[0043] Embodiment 2:

[0044] A method for preparing astragalus polysaccharide by wine processing, comprising the following steps:

[0045] (1) Take 100 g of raw Astragalus slices RAR, add 20 mL of rice wine, mix well and simmer thoroughly, put in a pot and stir-fry over low heat until slightly yellow, take out and let cool to obtain wine-processed Astragalus slices WAR.

[0046] (2) The wine-processed Astragalus slices obtained in step (1) were crushed with a Chinese medicine crusher, 55 g of the crushed sample was weighed, mixed with water at a ratio of 1:30 (w / v, dry basis ratio), heated at 90°C for 120 min, and centrifuged to remove insoluble residues. After the supernatant was concentrated to 200 mL at 50°C, it was separated by convection through a cutoff membrane (molecular weight: 5000 Da) at a ratio of concentrate: water (1:2.5) until the separated liquid with a molecular weight greater than 5000 Da was 100 mL. Subsequently, the separated liquid was placed in a dialysis bag (molecular weight: 3500 Da) and dialyzed with tap water for 3 days. The dialyzate was concentrated to 30 mL by centrifugation and freeze-dried with a freeze dryer to obtain the wine-processed Astragalus crude polysaccharide, which was named WAPSs.

[0047] Embodiment 3:

[0048] A method for preparing astragalus polysaccharide by using a honey-processing method, comprising the following steps:

[0049] (1) Take 100 g of raw Astragalus slices RAR, add 25 g of refined honey and an appropriate amount of water to dilute, stir evenly, put into a hot pot after simmering, and stir-fry over low heat until the aroma comes out and the Astragalus is dark yellow and not sticky. Take out and cool to obtain honey-processed Astragalus slices HAR.

[0050] (2) The honey-processed Astragalus slices obtained in step (1) were crushed with a Chinese medicine crusher, 55 g of the crushed sample was weighed, mixed with water at a ratio of 1:30 (w / v, dry basis ratio), heated at 90°C for 120 min, and centrifuged to remove insoluble residues. After the supernatant was concentrated to 200 mL at 50°C, it was separated by convection through a cutoff membrane (molecular weight: 5000 Da) at a ratio of concentrate: water (1:2.5) until the separated liquid with a molecular weight greater than 5000 Da was 100 mL. The separated liquid was then placed in a dialysis bag (molecular weight: 3500 Da) and dialyzed with tap water for 3 days. The dialyzate was concentrated to 30 mL by centrifugation and lyophilized with a freeze dryer to obtain the crude polysaccharide of honey-processed Astragalus, which was named HAPSs.

[0051] Comparative Example 1:

[0052] Take 100g of raw Astragalus slices RAR, crush it with a Chinese medicine crusher, weigh 55g of the crushed sample, mix it with water at a ratio of 1:30 (w / v, dry basis ratio), heat it at 90℃ for 120min, and centrifuge to remove insoluble residues. After the supernatant is concentrated to 200ml at 50℃, it is separated by convection through a cutoff membrane (molecular weight: 5000Da) at a ratio of concentrate: water (1:2.5) until the separated liquid with a molecular weight greater than 5000Da is 100mL. Then, the separated liquid is placed in a dialysis bag (molecular weight: 3500Da) and dialyzed with tap water for 3 days. The dialyzate is concentrated to 30mL by centrifugation and lyophilized with a freeze dryer to obtain raw Astragalus crude polysaccharide, which is named RAPSs.

[0053] Verification Example 1 - Effects of different processing methods on the quality and shape of medicinal pieces:

[0054] 1.1 Method: The salt-processed Astragalus slices, wine-processed Astragalus slices and honey-processed Astragalus slices obtained after processing in Example 1, Example 2 and Example 3 were compared with the raw Astragalus slices to compare the quality and property changes of the samples before and after processing: the appearance morphology changes of the samples before and after processing were observed, and the quality change rate of the samples before and after processing was calculated using the following formula:

[0055] The mass change rate before and after processing (%) = (dry weight after processing (g) - dry weight before processing (g)) / dry weight before processing (g) × 100%. The changes in the mass and shape of the Astragalus slices after different processing are shown in the figure:

[0056] 1.2 Results: Figure 1 It can be seen that compared with RAR, the processed salt-processed Astragalus slices, wine-processed Astragalus slices and honey-processed Astragalus slices have undergone significant changes in quality, appearance color and smell. The surface color of the three samples after processing became darker, among which the color change of honey-processed Astragalus slices was the most obvious, showing a brown-yellow color. In terms of smell, the three samples had different fragrances after processing, and the smell differences were obvious. At the same time, the weight of the three samples after processing also changed significantly. The mass of salt-processed Astragalus slices and honey-processed Astragalus slices increased by 0.38% ± 0.56 and 11.86% ± 1.61, respectively, and their increments can be regarded as the adsorption of salt and refined honey. The mass of wine-processed Astragalus slices decreased by -4.72% ± 0.63, which may be due to the loss of volatile compounds during processing. The Maillard reaction can change the quality of food, such as darkening the color, giving different flavors and aromas, and improving nutritional value. The changes in quality, appearance, color and smell of salt-processed, wine-processed and honey-processed Astragalus slices after processing may be attributed to the Maillard reaction occurring during the thermal processing.

[0057] 1.3 Discussion: Verification Example 1 studied the effects of different processing methods on the quality and shape of medicinal slices. Traditional Chinese medicine processing is a unique pharmaceutical technology that is different from natural medicines. More and more studies have shown that the chemical structure of polysaccharides from the same source will be affected by different processing methods, resulting in changes in their activity. The processing method affects the yield, chemical composition and biological activity of polysaccharides. This study revealed the mass change rate of each sample before and after processing. Its mass change may be caused by a variety of reasons. For example, the increase in the mass of salt-processed Astragalus slices and honey-processed Astragalus slices may be due to the adsorption of processing adjuvants salt and honey. The reduction and difference in the mass of each sample may be attributed to the loss of volatile compounds during processing.

[0058] Verification Example 2 - Determination and analysis of chemical components of crude polysaccharides obtained by different processing methods:

[0059] 2.1 Method: The chemical composition determination and analysis methods of the crude polysaccharides of raw astragalus RAPSs, crude polysaccharides of salt-processed astragalus SAPSs, crude polysaccharides of wine-processed astragalus WAPSs and crude polysaccharides of honey-processed astragalus HAPSs obtained after processing in the comparative example, example 1, example 2 and example 3 are as follows: The polysaccharide extraction rate (%) is calculated by the following formula: Polysaccharide extraction rate (%) = weight of extracted polysaccharide (g) / weight of the crushed sample (g) × 100.

[0060] The content of neutral sugar was determined by phenol-sulfuric acid method with glucose as standard. The content of total protein in each polysaccharide sample was determined by BCA protein quantification kit with bovine serum albumin as standard. The content of uronic acid in the sample was determined by sulfuric acid-carbazole method with galacturonic acid as standard.

[0061] 2.2 Results: The APSs extraction rates of the four different treatment methods and the main chemical compositions of RAPSs, SAPSs, WAPSs and HAPSs are shown in Table 2:

[0062] Table 1: Extraction yield and chemical composition of RAPSs, SAPSs, WAPSs and HAPSs

[0063] sample RAPSs SAPS WAPSs HAPSs Extraction rate (%) <![CDATA[2.21±0.18 a ]]> <![CDATA[1.60±0.12 b ]]> <![CDATA[2.16±0.17 a ]]> <![CDATA[1.65±0.09 b ]]> Neutral sugar content (%) <![CDATA[66.62±5.08 a ]]> <![CDATA[58.70±3.39 b ]]> <![CDATA[69.46±4.40 a ]]> <![CDATA[75.69±3.76 a ]]> Uronic acid content (%) <![CDATA[1.70±0.03 a ]]> <![CDATA[1.80±0.23 b ]]> <![CDATA[2.10±0.002 a ]]> <![CDATA[2.23±0.03 b ]]> Protein content (%) <![CDATA[4.11±0.72 b ]]> <![CDATA[9.13±1.54 a ]]> <![CDATA[2.10±0.36 b ]]> <![CDATA[9.04±0.91 a ]]>

[0064] aData (except monosaccharide composition) are expressed as mean ± standard deviation. Different letters indicate significant differences among different polysaccharides (p<0.05).

[0065] The results in Table 1 show that the processing method has a significant effect on the extraction rate of the four polysaccharides. The yields of SAPSs, WAPSs and HAPSs were 1.6%, 2.16% and 1.65%, respectively, which were all lower than the unprocessed RAPSs (2.21%). The difference in extraction rate may be related to the change in the quality of each sample before and after processing. The total polysaccharide content of the samples obtained by different processing methods was significantly different, in the following order: HAPSs (75.68%) > WAPSs (69.45%) > RAPSs (66.62%) > SAPSs (58.70%). Among the protein contents, HAPSs had the highest protein content, which was 9.04%, while WAPSs had the lowest protein content, which was only 2.10%. The uronic acid content of the three processed polysaccharides SAPSs, WAPSs and HAPSs was higher than that of the unprocessed RAPSs, among which HAPSs had the highest uronic acid content. The results show that different processing methods have a significant effect on the chemical composition of astragalus polysaccharides.

[0066] 2.3 Discussion: Verification Example 2 The chemical composition determination and analysis of crude polysaccharides obtained by different processing methods were studied. In terms of neutral sugar content, the content of processed HAPSs and WAPSs was significantly higher than that of unprocessed RAPSs, while the content of SAPSs was lower than that of RAPSs. First, for HAPSs, its adjuvant honey has the characteristics of natural deep eutectic solvent (NADES), which can promote the solubility and stability of the active ingredients. Secondly, the processing adjuvant of WAPSs, rice wine, contains alcohol, which is a good organic solvent that can dissolve most water-soluble or insoluble substances in traditional Chinese medicine. It also has good permeability and can enter plant tissues to promote the transfer, diffusion and dissolution of plant components, thereby increasing the dissolution and leaching of active ingredients in traditional Chinese medicine. In the above two processing processes, it is also accompanied by a heating process, which can also promote the destruction of the internal cell structure of the medicinal material, making it easier to extract active ingredients such as polysaccharides. In summary, the reasons why the neutral sugar content in the processed samples of honey and rice wine is higher than that of the raw products may include the application of processing adjuvants, heating effects, and changes in the physical properties of polysaccharides. The combined effect of these factors makes the crude polysaccharides extracted after roasting with rice wine and honey show higher advantages in terms of polysaccharide content.

[0067] Verification Example 3—Study on the structural characterization of RAPSs, SAPSs, WAPSs and HAPSs

[0068] 3.1 Structural characterization of RAPSs, SAPSs, WAPSs, and HAPSs

[0069] 3.1.1 Determination of molecular weight of RAPSs, SAPSs, WAPSs and HAPSs

[0070] The molecular weights of RAPSs, SAPSs, WAPSs and HAPSs were determined by high performance size exclusion chromatography (HPSEC). The instrument was an Agilent 1290UPLC Series equipped with a differential detector and a DAWN detector. The four polysaccharide samples were prepared at a concentration of 1.0 mg / mL and analyzed after filtration through a 0.22 μm filter membrane. The molecular weights were then measured using Shodex Ohpak 806HQ (10 μm, 8.0 mm×300 mm) and Shodex Ohpak 804HQ (10 μm, 8.0 mm×300 mm), with a mobile phase of 0.2 M sodium chloride and a flow rate of 1.0 mL / min. The four sets of data obtained were imported into ASTRA8 software for analysis.

[0071] 3.1.2 Determination of monosaccharide composition:

[0072] The monosaccharide composition of RAPSs, SAPSs, WAPSs and HAPSs was analyzed and determined using a pulsed amperometric detector (PDA)-ion chromatograph (ICS500+, Thermo Fisher Scientific, USA). 10.0 mg of RAPSs, SAPSs, WAPSs and HAPSs samples were accurately weighed in turn, placed in ampoules, 10.0 mL of 3M trifluoroacetic acid was added, and the samples were sealed and protected from light at 120°C for 3.0 h for hydrolysis. 2.0 mL of the acid hydrolysis solution was transferred to a 5.0 mL EP tube, and TFA was removed by nitrogen blowing at 80°C. 2.0 mL of anhydrous ethanol was added, and after nitrogen blowing again, 5.0 mL of deionized water was added and vortexed to mix. 100.0 μL was taken and placed in a 1.0 mL volumetric flask, and the volume was adjusted to the mark with deionized water. The sample was filtered through a 0.22 μm microporous filter membrane and analyzed by a pulsed amperometric detector-ion chromatograph. The chromatographic column connected thereto is Dionex CarbopacTM PA20 (3*150); the mobile phase used is 97.5% H2O and 2.5% 100.0 mM sodium hydroxide; the flow rate is 0.5 mL / min; the injection volume is 20 μL; and the column temperature is 30°C.

[0073] 3.1.3 Fourier transform infrared spectroscopy analysis:

[0074] Weigh 2.0 mg of each of the astragalus polysaccharide samples RAPSs, SAPSs, WAPSs, and HAPSs, mix them evenly with potassium bromide powder, press them into tablets, and measure them using a Fourier transform infrared spectrometer to record the wavelengths at 4000-500 cm -1 FT-IR spectrum in the range.

[0075] 3.1.4 Scanning electron microscopy:

[0076] Take a small amount of the four kinds of Astragalus polysaccharide powders that have been thoroughly dried, fix them on the sample stage with conductive glue, vacuum spray gold, and then place them under a field emission scanning electron microscope to capture the three-dimensional morphology of the sample surface. The image is recorded at a magnification of 2000 times at an accelerating voltage of 5.0 kV.

[0077] 3.1.5 Atomic force microscopy:

[0078] 1 mg of RAPSs, SAPSs, WAPSs and HAPSs were dissolved to a concentration of 10 μg / mL, evenly dispersed and centrifuged, and 10 μl of the supernatant was dropped on a clean silicon wafer surface and dried at room temperature. The molecular morphology of the four astragalus polysaccharide samples was observed using atomic force microscopy.

[0079] 3.1.6X-ray diffraction:

[0080] Place the four kinds of astragalus polysaccharide samples in the sample tank, press the powder lightly with a glass slide to fill the tank with the sample, and gently scrape off the excess powder to make the sample surface and the glass rack surface in the same plane. Keep the room temperature constant at 20°C, the relative humidity below 70%, and the vacuum degree ≥10 -5 The data scanning range 2θ was 5-80°, the step length was 0.02°, and the counting time was 0.78 s / step.

[0081] 3.1.7 Thermal stability analysis:

[0082] The thermogravimetric properties of RAPSs, SAPSs, WAPSs and HAPSs were tested using a synchronous thermal analyzer. 2.0 mg of each Astragalus polysaccharide sample was weighed and placed in a sample tray for testing. The specific test conditions are as follows: Test temperature range: room temperature to 800°C; Heating rate: 10°C min -1 ; Scan rate: 1℃·min -1 .

[0083] 3.1.8 Zeta potential and particle size measurement:

[0084] The Zeta potential and particle size of the four astragalus polysaccharide samples were measured using a dynamic light scattering instrument. The specific operation was as follows: 10 mg of each polysaccharide sample was weighed, dissolved in distilled water, and prepared into a 10 mg / mL solution, vortexed, and then centrifuged at 6500 rpm for 5 minutes. 200 μL of the supernatant was diluted 10 times and then measured. Each experiment was repeated 3 times at 25°C.

[0085] 3.1.9 Congo red test:

[0086] The conformations of RAPSs, SAPSs, WAPSs and HAPSs were determined by Congo red assay. Specifically, 2.0 mg of each of the four polysaccharide samples were weighed, prepared into a 1.0 mg / mL solution with distilled water, and then 80 μmol / L of Congo red solution was added, vortexed for 5 min, and mixed thoroughly. 0.5 mol / L NaOH solution was added to the above mixture to make the final concentration of NaOH in the mixture 0.1-0.5 mol / L, and the Congo red solution without polysaccharide sample was used as a control. The maximum absorption wavelength in the range of 200-800 nm was measured at different NaOH concentrations.

[0087] 3.1.10 Nuclear magnetic resonance spectroscopy analysis:

[0088] The structural information of RAPSs, SAPSs, WAPSs and HAPSs was analyzed by nuclear magnetic resonance. Weigh 50 mg of each polysaccharide sample, add 1.0 mL of deuterated water, and shake thoroughly to completely dissolve the sample. Seal with plastic wrap and place in a -80°C refrigerator for pre-freezing overnight. The next day, pierce the plastic wrap and put it in a freeze dryer for 24 hours. Then, dissolve the dried sample again with 1.0 mL of deuterated water, and repeat the above operation 3 times to complete the heavy water exchange. Finally, use 0.6 mL of deuterated water and slowly transfer the sample to the nuclear magnetic tube with a pipette for 1 H NMR, 13 C NMR analysis: Data processing was performed using Bruker NMR data processing software.

[0089] 3.1.11β-elimination reaction:

[0090] Weigh 5 mg of each of RAPSs, SAPSs, WAPSs and HAPSs, add 5 mL of 0.2 mol / L NaOH solution, and place at 45°C for full reaction for 3 h; take another 5 mg sample and use distilled water instead of NaOH solution as a control. Use a UV-visible spectrophotometer to scan the mixture in the wavelength range of 190 to 400 nm.

[0091] 3.2 Structural characterization results of RAPSs, SAPSs, WAPSs and HAPSs

[0092] 3.2.1 Determination of molecular weight of RAPSs, SAPSs, WAPSs and HAPSs:

[0093] The molecular weight of polysaccharides is one of the important factors affecting their activity. Different processing methods, the types and quality of auxiliary materials used in processing, etc. are closely related to the molecular weight of polysaccharides. This study used high performance size exclusion chromatography-light scattering-differential tandem detection (HPSEC-MALLS-RI) to measure and analyze the effects of different processing methods on the molecular weight of astragalus polysaccharides. The results are shown in Table 2:

[0094] Table 2: Molecular weights of RAPSs, SAPSs, WAPSs and HAPSs

[0095] sample RAPSs SAPS WAPSs HAPSs Weight average molecular weight (Mw) kDa 231.30 86.34 112.39 138.70 Number average molecular weight (Mn) kDa 114.99 44.24 53.59 61.32 Polydispersity index (Mw / Mn) 2.01 1.95 2.10 2.26

[0096] According to the data in Table 2, RAPSs, SAPSs, WAPSs and HAPSs all show a peak and there are significant differences in molecular weight and peak area ratio. Specifically, the molecular weight of RAPSs is 231.30 kDa, the molecular weight of SAPSs is 86.34 kDa, the molecular weight of WAPSs is 112.39 kDa, and the molecular weight of HAPSs is 138.70 kDa. From the above polysaccharide molecular weight distribution results, it can be observed that compared with RAPSs, the molecular weights of SAPSs, WAPSs and HAPSs treated by different processing methods are significantly reduced. It may be due to the hydrolysis of polysaccharides under the influence of heat during processing, which leads to a significant reduction in their molecular weights. It is worth noting that the molecular weight of SAPSs decreases most significantly, almost only one-third of the molecular weight of RAPSs. This significant difference may be related not only to its heating, but also to its processing adjuvants. In addition, Mw / Mn (polydispersity index) is used to describe the dispersity of polysaccharide molecular weight. The larger the polydispersity index, the wider the molecular weight distribution. The polydispersity index values ​​of the four kinds of astragalus polysaccharides were all between 1.95 and 2.26, indicating that the molecular weight distribution of the four kinds of astragalus polysaccharides was narrow. The results showed that different processing methods had a significant effect on the molecular weight of astragalus polysaccharides but had little effect on their molecular weight distribution.

[0097] 3.2.2 Determination of monosaccharide composition of RAPSs, SAPSs, WAPSs and HAPSs

[0098] Monosaccharide composition analysis is of great significance for the classification, structural characterization and biological activity research of polysaccharides. Ion chromatography is widely used in the qualitative and quantitative analysis of monosaccharides in polysaccharides due to its high sensitivity, simple and convenient operation, and no need for derivatization pretreatment. The results are shown in Table 3:

[0099] Table 3: Monosaccharide composition of RAPSs, SAPSs, WAPSs and HAPSs

[0100] sample RAPSs SAPS WAPSs HAPSs Arabinose (Ara) 8.15 11.52 8.23 11.32 Glucosamine Hydrochloride(GlcN) 0.64 0.92 0.70 0.90 Galactose (Gal) 8.32 11.10 8.10 10.55 Glucose (Glc) 81.22 74.05 80.74 74.65 Xylose 1.04 1.47 1.13 1.37 Mannose (Man) 0.38 0.51 0.59 0.58 Galacturonic acid (GalA) 0.25 0.43 0.54 0.63

[0101] As can be seen from Table 3, the four polysaccharide samples are quite different, all of which are composed of different molar ratios of arabinose, glucosamine hydrochloride, galactose, glucose, xylose, mannose and galacturonic acid in RAPSs (8.15%, 0.64%, 8.32%, 81.22%, 1.04%, 0.38%, 0.25%), SAPSs (11.52%, 0.92%, 11.10%, 74.05%, 1.47%, 0.51%, 0.43%), WAPSs (8.23%, 0.70%, 8.10%, 80.74%, 1.10%, 0.59%, 0.54%) and HAPSs (11.32%, 0.90%, 10.55%, 74.65%, 1.37%, 0.58%, 0.63%). The types of monosaccharide composition of the four polysaccharides are relatively uniform, all of which are mainly glucose, arabinose and galactose, and contain a small amount of mannose, xylose, glucosamine hydrochloride and galacturonic acid. However, there are differences in the molar ratio of their monosaccharide composition, mainly manifested in the different molar ratios of glucose, arabinose and galactose in the four different polysaccharides. The results show that different processing methods have little effect on the types of monosaccharides, but have a more significant effect on the molar ratio of monosaccharide composition.

[0102] 3.2.3 Fourier transform infrared spectroscopy analysis of RAPSs, SAPSs, WAPSs and HAPSs

[0103] FT-IR spectroscopy is used to distinguish the main functional group structures in RAPSs, SAPSs, WAPSs and HAPSs based on the position and shape of the characteristic peaks. In the infrared spectrum of polysaccharides, it is mainly divided into the following three parts. 1 The broad band in the range represents the stretching vibration of OH; 1200~950cm -1 The range is called the "sugar region", which mainly includes the strong stretching vibrations of CO and CC with high overlap of glycosidic bonds and sugar rings; 950-750cm -1 The range is called the "anomeric carbon region" and mainly contains weaker bands related to the anomeric carbon of the sugar ring. The FT-IR spectra of different Astragalus polysaccharides are shown in Figure 1. Figure 2 As shown in Figure A:

[0104] Depend on Figure 2 From A in the figure, we can see that the infrared spectra of the four polysaccharides all contain characteristic bands that represent the characteristics of polysaccharides, but their absorption intensities are slightly different. The absorption peak intensities of HAPSs are generally higher than those of the other three polysaccharides. -1 The broad absorption peak near 2928cm is the stretching vibration of the hydroxyl group (OH) on the sugar ring; -1 The weak signal appearing nearby is attributed to the stretching vibration of the carbon-hydrogen bond (CH) on the sugar ring; 1721cm-1 There is a weak peak near it due to the stretching vibration of the carbonyl group (C=O), indicating that the four polysaccharides contain uronic acid, which is consistent with the results of monosaccharide composition; the absorption peak near 1654cm-1 is related to the stretching vibration of the carbon-oxygen bond (CO) of the carboxyl group, and the absorption peak near 1410cm-1 is related to the stretching vibration of the carbon-oxygen bond (CO) of the carboxyl group. 1 The weak absorption peaks appearing near are due to the stretching vibration of the asymmetric carbon-hydrogen bond (CH); -1 The absorption peaks near 858 cm indicate that pyranose ring structures may exist in the structures of the four astragalus polysaccharides. In addition, α-configuration glycosidic bonds and β-type glycosidic bonds were found in RAPSs, SAPSs, WAPSs and HAPSs, mainly manifested in the absorption peaks at 858 cm -1 and 762cm -1 There is a weak peak near. Based on the above results, it is inferred that the four polysaccharides are polysaccharides containing α-configuration glycosidic bonds and β-type glycosidic bonds. The results show that RAPSs, SAPSs, WAPSs and HAPSs all contain characteristic absorption signals of polysaccharides, and different processing methods have little effect on the characteristic functional groups of the infrared spectrum of APSs.

[0105] 3.2.4 X-ray diffraction analysis of RAPSs, SAPSs, WAPSs and HAPSs

[0106] X-ray diffraction is a research method that obtains information about material composition, internal atomic structure or morphology by analyzing X-ray diffraction patterns. It is often used to reveal the crystalline and amorphous properties of polysaccharides. Figure 2 Figure B is the X-ray diffraction curves of RAPSs, SAPSs, WAPSs and HAPSs.

[0107] It can be seen from the figure that in the 10-80°2θ region, the four polysaccharides all have an arched diffraction peak at around 20°. This result shows that RAPSs, SAPSs, WAPSs and HAPSs have both crystalline and amorphous components, and different processing methods have little effect on the crystal structure of polysaccharides.

[0108] 3.2.5 Thermal stability analysis of RAPSs, SAPSs, WAPSs and HAPSs

[0109] Thermal stability is an important physicochemical property of polysaccharides. It is mainly determined by measuring the weight change of polysaccharides over time and temperature. The thermal stability of four kinds of astragalus polysaccharides with different processing methods was studied by thermal analysis. The weight changes of the four polysaccharide samples were recorded from room temperature to 800.0℃ by TG, DTG and DSC. Figure 2 Figure C shows the thermal decomposition curves of four polysaccharides.

[0110] Depend on Figure 2As shown in Figure C, it mainly shows a degradation pattern of three steps: the first mass loss occurred at around 120.0℃, and the four samples all lost weight slightly by about 7.00%, which may be due to the volatilization of free water and bound water remaining in each polysaccharide sample. The mass loss occurring at 260.0-340.0℃ is the second stage, and each polysaccharide experienced a serious weight loss process at this stage. Due to the drastic degradation and depolymerization of polysaccharides, RAPSs, SAPSs, WAPSs and HAPSs began to lose weight at 270.7℃, 264.5℃, 264.0℃, and 265.3℃, respectively, and terminated at 335.4℃, 319.8℃, 338.5℃, and 319.9℃. At this stage, the mass changes of each polysaccharide were 61.23%, 37.01%, 59.18%, and 39.05%. Interestingly, the maximum mass loss rate of the unprocessed polysaccharide RAPSs was the highest, while the maximum mass loss rates of the processed SAPSs, WAPSs and HAPSs all decreased to varying degrees, indicating that the mass loss of the processed samples became relatively stable at this stage. As the temperature gradually increased, the drastic degradation and depolymerization of the polysaccharides ended, the thermal decomposition of carbon began, and the third stage of mass loss came. The third stage was similar to the first stage, with a slight weight loss of less than 10.00%. When the temperature rose to 800.0℃, the residual amounts of RAPSs, SAPSs, WAPSs and HAPSs were 20.27%, 21.71%, 18.59% and 23.54%, respectively. The DSC curves of the four polysaccharides had similar trends and two obvious exothermic peaks, but there were slight differences in their peak widths. These differences were attributed to the differences and changes in the molecular weight, monosaccharide composition, structure and water content of the four polysaccharide components. The results of TG, DTG and DSC analysis showed that there was no significant difference in the thermal stability of the raw polysaccharides and the three processed polysaccharides, and different processing methods had little effect on the thermal stability of APSs.

[0111] 3.2.6 SEM analysis of RAPSs, SAPSs, WAPSs and HAPSs

[0112] The surface morphology of polysaccharides RAPSs, SAPSs, WAPSs and HAPSs was characterized by scanning electron microscopy. It was found that the microscopic morphologies of the four types of astragalus polysaccharides were different.

[0113] like Figure 3As shown in Figure A, it can be observed that RAPSs has a dense structure, with a small amount of fine pores and some rice-like particles of different sizes attached to the surface. The surface structure of SAPSs is dense, with irregular corrugations and fine mesh protrusions. The surface of WAPSs is rough, with irregular protrusions and collapses. It can be observed that various holes of different sizes after the water dissipates, with a sponge-like porous structure. The samples processed by wine have a more complex pore structure because the dense tissue of the sample is loosened and the total surface area of ​​the sample is increased during the wine processing. The surface structure of HAPSs is rough, and dense small particles are attached to the surface. Compared with the other three, it can be found that the surface morphology of the processed polysaccharides of RAPSs is damaged to varying degrees compared with the unprocessed polysaccharides. Among them, the surface morphology of WAPSs is most obviously damaged, while the surface pores of SAPSs and HAPSs are significantly reduced. It is speculated that this may be caused by the serious destruction of the structure of its cell wall during the salt and honey processing. The results showed that different processing methods may affect the branching structure and cross-linking network of the four polysaccharides, causing changes in molecular weight, intermolecular distance and interconnection, thereby giving APSs different surface morphology and appearance. The above results are consistent with some reports that the shape and structure of polysaccharides may be affected by pretreatment.

[0114] 3.2.7 Atomic force microscopy analysis of RAPSs, SAPSs, WAPSs and HAPSs

[0115] Atomic force microscopy (AFM) is a scanning probe microscopy technique that has the ability to visualize macromolecular polymers such as polysaccharides down to the level of individual small molecules in a near "native" state. AFM can provide unique insights into the morphology and molecular properties of polysaccharides, including height (diameter), width, contour length, end distance, and polydispersity. It has opened up new horizons for the study of macromolecular polymers such as polysaccharides. AFM microscopy images of RAPSs, SAPSs, WAPSs, and HAPSs are shown in Figure 2. Figure 3 As shown in Figure B:

[0116] The molecular morphology and chain characteristics of the four polysaccharides on the silicon wafer surface were displayed by two-dimensional and three-dimensional AFM images. It can be clearly observed that the four astragalus polysaccharides have molecular morphologies and polysaccharide aggregates of uneven size and shape. RAPSs showed chain structures of varying lengths in the two-dimensional image, mixed with spherical and irregular aggregates. In its three-dimensional image, it can be found that the spherical aggregates appear as white and shiny columnar protrusions, while the irregular aggregates appear as a large number of sharp peak-shaped protrusions. SAPSs appear in a pseudo-snowflake shape in the plane image, accompanied by spherical and irregular aggregates. In its three-dimensional image, it mainly appears as tooth-like protrusions of varying sizes. WAPSs two-dimensional images mainly appear as irregular flakes, accompanied by spherical aggregates that are significantly larger than RAPSs and SAPSs. Its three-dimensional image mainly has irregular block and peak-shaped protrusions, and the spherical aggregates still appear as columnar protrusions. HAPSs also show a large number of irregular spherical aggregates in the two-dimensional image. Spherical and irregular aggregates were observed in the two-dimensional images of the four polysaccharides, but there were significant differences in size. It is speculated that the aggregates may be due to the entanglement of polysaccharide molecules with each other to form various agglomeration structures due to the van der Waals forces and hydrogen bonds within and between molecules. The difference in their size reflects the different molecular weights and aggregation states of the four polysaccharides. The results show that different processing methods can cause Astragalus polysaccharides to form aggregates of different shapes, sizes and numbers, thereby obtaining different molecular forms. The above results are consistent with the results of SEM.

[0117] 3.2.8 Zeta potential and particle size measurement of RAPSs, SAPSs, WAPSs and HAPSs

[0118] The zeta potential of RAPSs, SAPSs, WAPSs and HAPSs can reflect the stability of the four polysaccharide solutions to a certain extent. A higher absolute value of zeta potential will increase the electrostatic repulsion between molecules, reduce the flocculation of the solution, and increase the stability coefficient of the solution.

[0119] Depend on Figure 4As shown in Figure A, the four polysaccharides are always negatively charged, all of which are anionic polysaccharides and have the ability to donate electrons. The zeta potential levels of RAPSs, SAPSs, WAPSs and HAPSs are -4.07±0.1, -6.77±0.3, -7.88±0.4 and -8.14±0.5mV, respectively. It is worth noting that the smaller the absolute value of the zeta potential of the polysaccharide, the easier it is for the polysaccharide to condense or coagulate in the solution. The results show that the absolute value of the zeta potential of the unprocessed raw RAPSs is the smallest, and the polysaccharide will condense rapidly in the solution state. The absolute values ​​of the zeta potential of SAPSs, WAPSs and HAPSs after different processing are greater than that of RAPSs, indicating that after processing, the electrostatic interaction strength between the three polysaccharide particles gradually increases. Compared with the raw polysaccharide solution, the three polysaccharide solutions are more stable. Generally, the negative charge of polysaccharides mainly comes from the uronic acid in their structure. This is consistent with the finding that the proportion of uronic acid content in SAPSs, WAPSs and HAPSs in the monosaccharide composition is higher than that in RAPSs.

[0120] The particle size of polysaccharide molecules is easily affected by many factors, such as particle shape, crystal form, structure and molecular weight. Its size reflects the aggregation level of polysaccharide molecules and is often closely related to the hydrophilicity and biological activity of the molecules. Fig. 9 As shown in the figure, the particle sizes of the polysaccharides obtained after extraction by different processing methods were significantly different. The average particle sizes of RAPSs, SAPSs, WAPSs and HAPSs were 271.7nm, 115.2nm, 378.9nm and 163.5nm, respectively. The results showed that the particle size of polysaccharides processed by rice wine was significantly larger than that of raw RAPSs, while the particle size of polysaccharides processed by salt and honey was smaller than that of raw RAPSs. The three different processing methods would change the particle size and zeta potential of polysaccharides, thereby affecting the pharmacological activity of each polysaccharide and its application in the food and pharmaceutical industries.

[0121] 3.2.9 Congo red assay to analyze the triple helical structures of RAPSs, SAPSs, WAPSs and HAPSs

[0122] The Congo red test is one of the main methods used to identify whether a polysaccharide has a triple helix structure. Generally, in an aqueous solution or a low concentration of NaOH, a polysaccharide with a triple helix conformation will form a complex with the acidic dye Congo red, causing its maximum absorption wavelength to red-shift. However, in a high concentration NaOH solution, the hydrogen bonds between the polysaccharide molecules break, causing the structure of the complex to be destroyed, causing its maximum absorption wavelength to drop rapidly. Therefore, the above phenomenon can be used to determine whether the polysaccharide has a triple helix structure. The Congo red test results of RAPSs, SAPSs, WAPSs and HAPSs are shown below. Figure 4 As shown in Figure B:

[0123] NaOH solution was added to the complex formed by the four polysaccharides and Congo red to make the total concentration of the mixture 0-0.5 mol / L. When the concentration was 0-0.1 mol / L, the maximum absorption wavelength of RAPSs, SAPSs, WAPSs and HAPSs was significantly red-shifted, indicating that the four polysaccharides formed polysaccharide-Congo red complexes in the system. However, when the NaOH concentration in the mixture was 0.2-0.5 mol / L, the maximum absorption wavelength of the four polysaccharides did not have an obvious blue shift, indicating that the complex formed by RAPSs, SAPSs, WAPSs and HAPSs and Congo red did not change due to the depolymerization of the polysaccharide structure. The results show that the four polysaccharides may not have a triple helical structure. However, after the four polysaccharides were mixed with Congo red, a red shift was indeed observed. The reason may be that the four polysaccharides have another ordered conformation, and this spatial structure can also complex with Congo red to produce a red shift, but this complex has greater stability in NaOH solution. Therefore, the spatial conformations of RAPSs, SAPSs, WAPSs and HAPSs need further study.

[0124] 3.2.10 Analysis of β-elimination reactions of RAPSs, SAPSs, WAPSs and HAPSs

[0125] The β-elimination reaction of RAPSs, SAPSs, WAPSs and HAPSs can be used to distinguish glycosidic bonds from peptide bonds. When there is an O-glycosidic bond in the polysaccharide structure, it can be seen that its absorbance at 240nm increases significantly. This is mainly because when the hydroxyl group of serine or threonine is connected to the O-glycosidic bond, an unsaturated amino acid is released, resulting in an increase in absorbance. However, when there is an N-glycosidic bond in the polysaccharide structure, its absorbance does not change much when connected to aspartic acid. The UV scanning results of the β-elimination reaction of RAPSs, SAPSs, WAPSs and HAPSs are shown in Figure 2. Figure 4 As shown in Figure C:

[0126] In each sample, (+) represents the sample treated with NaOH, and (-) represents the polysaccharide solution of the same concentration and volume without NaOH treatment. The results showed that the absorbance at 240nm of the four polysaccharide samples treated with NaOH increased to varying degrees compared with the control, indicating that O-glycosidic bonds exist in RAPSs, SAPSs, WAPSs and HAPSs, and different processing methods have little effect on the glycosidic bonds.

[0127] 3.2.11 NMR spectroscopy analysis of RAPSs, SAPSs, WAPSs and HAPSs

[0128] NMR analysis of RAPSs, SAPSs, WAPSs, and HAPSs can be used to further elucidate the structural characteristics of RAPSs, SAPSs, WAPSs, and HAPSs, including the α- or β-configuration and the glycosyl residues. 1 H NMR spectroscopy and 13 C NMR spectroscopy Figure 5 As shown in A and 5B.

[0129] The NMR spectra of RAPSs, SAPSs, WAPSs and HAPSs are basically similar. The NMR spectra of the four polysaccharides RAPSs, SAPSs, WAPSs and HAPSs are mainly concentrated in the narrow areas of 3.0-5.5ppm and 60-110ppm, which are consistent with the characteristic signal areas of polysaccharides. The signals of the four APSs around 4.7ppm are attributed to the chemical shift of D2O. In general, the chemical shift of α-configuration glycosidic bonds is usually consistent with the signals appearing at 5.0-6.0ppm and 98-103ppm, while the signals at 4.5-5.0ppm and 103-106ppm are consistent with β-configuration glycosidic bonds. Figure 5 Part A ( 1 H NMR spectrum), it can be seen that the four APSs all have chemical shifts in the above two fragments. Therefore, it can be shown that α-configuration glycosidic and β-configuration glycosidic bonds exist simultaneously in the structures of RAPSs, SAPSs, WAPSs and HAPSs. In addition, the signals between 3.0 and 4.2 ppm represent ring protons, indicating the presence of pyranose. The chemical shift at 5.40 to 5.00 ppm characterizes α-pyranose. The results show that α-pyranose also exists in the structures of the four APSs. 13 The signal at δ69.5-77.4 ppm in the C NMR spectrum is the oxygen-oxygen signal peak of sugar. 13 The peaks at about 100.0 ppm and 103.0 ppm were found to be caused by β-Man and β-Glc, respectively. This is consistent with the results of monosaccharide composition. 1 H signal is at 5.28, 3.35, 3.85, 3.74, 3.53, 3.75, 13 The C signals are at 99.62, 71.67, 73.27, 70.98, 71.47, and 60.33 ppm, belonging to H1 / C1, H2 / C2, H3 / C3, H4 / C4, H5 / C5, and H6 / C6, respectively. 1 H and 13In the C spectrum, the C1 / H1, H2 / C2, H3 / C3, H4 / C4, H5 / C5, and H6 / C6 signals of SAPSs, WAPSs, and HAPSs are 5.28 / 99.69, 3.36 / 71.67, 3.85 / 73.28, 3.74 / 70.98, 3.54 / 71.47, 3.75 / 60.39, and 5.2 8 / 99.70, 3.36 / 71.67, 3.85 / 73.28, 3.74 / 70.98, 3.54 / 71.48, 3.75 / 60.39ppm and 5.28 / 99.72, 3.36 / 71.65, 3.85 / 73.29, 3.73 / 70.98, 3.53 / 71.48, 3.75 / 60.33ppm. 1 H and 13 C spectral data showed that RAPSs, SAPSs, WAPSs and HAPSs had similar chemical structures and all contained →4)-α-D-Glc(1→. The δH / δC signals at 5.28 / 99.70, 3.58 / 71.70, 3.90 / 72.80, 3.54 / 76.70, 3.63 / 72.70 and 3.35 / 69.30 ppm were assigned to H1 / C1, H2 / C2, H3 / C3, H4 / C4, H5 / C5 and H6 / C6 of →4,6)-α-D-Glcp-(1→, respectively. The above structures existed in RAPSs, SAPSs, WAPSs and HAPSs. The results showed that different processing methods significantly affected the polysaccharides but did not change their main glycosyl residues, which was consistent with the results of FT-IR analysis.

[0130] 3.3 Discussion: Verify the study of the structural characterization of RAPSs, SAPSs, WAPSs and HAPSs in Example 3. The chemical structure of polysaccharides is the basis of their biological activity. Various factors such as molecular weight, monosaccharide composition, glycosidic bond type and sugar chain conformation will affect their activity. Generally, polysaccharides with larger molecular weights have limited bioavailability due to their low solubility, while too low molecular weight will lead to insufficient polymerization degree of active groups and poor stability, which in turn affects the biological activity of polysaccharides. RAPSs, SAPSs, WAPSs and HAPSs have significant differences in molecular weight, and the molecular weight of the three polysaccharides after processing is significantly lower than that of the raw polysaccharides. The results can be attributed to the hydrolysis caused by heat during processing, which promoted a significant reduction in the molecular weight of SAPSs, WAPSs and HAPSs.

[0131] In terms of structure, RAPSs, SAPSs, WAPSs and HAPSs were all proved to be heteropolysaccharides, including neutral and acidic monosaccharide units. The four polysaccharides showed different monosaccharide compositions, namely, a combination of seven monosaccharides: arabinose, glucosamine hydrochloride, galactose, glucose, xylose, mannose and galacturonic acid. Glucose had the highest content in the monosaccharide composition of the four polysaccharides, reaching 81.22%, 74.05%, 80.74% and 74.65%, respectively. Existing research results have shown that glucose is the main sugar unit of APSs water-soluble polysaccharides, which is consistent with our results. It has been reported that honey processing changes the molar ratio of monosaccharides. The infrared and nuclear magnetic resonance results of the four polysaccharides showed that the four polysaccharides had similar structural characteristics. In addition, both spectra showed that RAPSs, SAPSs, WAPSs and HAPSs had α-configuration glycosidic bonds and β-type glycosidic bonds and the presence of pyranose.

[0132] The structural morphology of polysaccharides is closely related to their solubility, viscosity and various biological activities. In this study, the microstructure of four polysaccharides was analyzed by scanning electron microscopy and atomic force microscopy. The results showed that the four polysaccharides had significant differences in their microstructure. Then the potential and particle size of RAPSs, SAPSs, WAPSs and HAPSs were measured. The results showed that after processing, the electrostatic interaction intensity between the three polysaccharide particles gradually increased, and the particle size also changed significantly. The results of thermal stability measurement showed that the thermal stability of the four polysaccharides was not significantly different.

[0133] In summary, RAPSs, SAPSs, WAPSs, and HAPSs differed in yield, neutral sugar content, uronic acid content, molecular weight monosaccharide composition molar ratio, microstructure, Zeta potential, and particle size. These differences may be due to the application of adjuvants (yellow wine, salt, and honey) and the heating process during processing. These results show that the chemical composition, monosaccharide composition, molecular weight, and surface morphology of APS changed after different processing, but the infrared spectral characteristics, main glycoside residues, and crystal interior were similar, indicating that the three different processing methods had different effects on the degradation of APS without changing its main structure.

[0134] Validation Example 4—Study on the immunostimulatory and anti-inflammatory activities of RAPSs, SAPSs, WAPSs and HAPSs

[0135] 4.1 Methods for studying the immunostimulatory and anti-inflammatory activities of RAPSs, SAPSs, WAPSs and HAPSs:

[0136] 4.1.1 Cultivation and passaging of RAW264.7 macrophages:

[0137] RAW 264.7 macrophages were purchased from Wuhan Pronocell Life Science Co., Ltd. and placed in DMEM medium supplemented with 1% penicillin, streptomycin and 10% fetal bovine serum. They were cultured in an incubator at 37°C and 5% CO2 saturated humidity. When the cell confluence reached 85%, they were passaged with trypsin at a passage ratio of 1:3 to 1:4. Subsequent experiments used cells of passage 12 to 25.

[0138] 4.1.2 Determination of cell viability:

[0139] The CCK-8 method was used to detect the effects of RAPSs, SAPSs, WAPSs, and HAPSs on the viability of RAW264.7 cells. 5 The cells were seeded at a density of 100 μg / well on a 96-well plate. After incubation for 12 h, different concentrations (10, 25, 50 μg / mL) of RAPSs, SAPSs, WAPSs and HAPSs were then incubated for 24 h. Subsequently, 110 μL of rehydration solution (containing 10% CCK-8) was added to each well, and after continued incubation for 1 h, the OD value at a wavelength of 450 nm was measured using an ELISA reader.

[0140] 4.1.3 Phagocytic activity assay

[0141] Neutral red uptake method was used to determine the effects of RAPSs, SAPSs, WAPSs and HAPSs on the phagocytic ability of RAW264.7 cells. RAW264.7 cells were pretreated with different concentrations of RAPSs, SAPSs, WAPSs and HAPSs (10, 25, 50 μg / mL) in the presence or absence of lipopolysaccharide (LPS) (1 μg / mL) for 24 hours, the culture medium was removed, and 200 μL of 0.1% neutral red solution was added to each well. The culture was continued, and after 2 hours, all the culture medium in each well was slowly aspirated, the supernatant was discarded, and the cells were washed 3 times with PBS. Finally, 200 μL of cell lysis solution was added to lyse the macrophages under the action of the lysis solution, and the OD value was measured at 570 nm using an ELISA reader 4 hours later.

[0142] 4.1.4 Determination of nitric oxide (NO) content

[0143] RAW264.7 cells were collected at 1×10 5100 μL of fresh DMEM complete culture medium was used to replace the supernatant, and then 50 μL of Griess reagent I and 50 μL of Griess reagent II were added to each well. At the same time, a standard curve was prepared according to the regulations. Then the absorbance value at 550 nm was measured. The relative release of nitric oxide in each group was calculated according to the standard curve.

[0144] 4.1.5 Cytokine assay analysis

[0145] RAW264.7 cells were seeded in 96-well plates at 1×105 / well and cultured for 12 h. RAW264.7 cells were pretreated with different concentrations of RAPSs, SAPSs, WAPSs, and HAPSs (10, 25, and 50 μg / mL) for 24 h in the presence or absence of lipopolysaccharide (1 μg / mL), and the release of cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the culture supernatant was detected using ELISA kits according to the manufacturer's instructions.

[0146] 4.2 Results of the study on the immunostimulatory and anti-inflammatory activities of RAPSs, SAPSs, WAPSs and HAPSs:

[0147] 4.2.1 Effects of RAPSs, SAPSs, WAPSs, and HAPSs on the activity of RAW264.7 cells

[0148] The most widely used model for studying immunostimulatory and anti-inflammatory activities is RAW264.7 cells. In this study, to test whether RAPSs, SAPSs, WAPSs, and HAPSs have an effect on the survival of RAW264.7 macrophages in the presence or absence of lipopolysaccharide, we used CCK-8 method to detect cell viability after 24 hours of treatment with four polysaccharides at different concentrations (10, 25, 50 μg / mL). Figure 6 As shown in the figure, within the concentration range of 10-50 μg / mL, RAPSs, SAPSs, WAPSs and HAPSs had no significant effect on the viability of RAW264.7 cells in the presence or absence of LPS. Based on the above results, concentrations of 10, 25 and 50 μg / mL were selected for subsequent experiments.

[0149] RAPSs, SAPSs, WAPSs, and HAPSs promote phagocytosis by activating RAW264.7 macrophages

[0150] Phagocytosis is a key step in the immune response and an important parameter for detecting the activation state of macrophages. The enhancement of phagocytic activity is considered to be one of the most significant signs of RAW264.7 cell activation. In this study, the uptake of neutral red dye was evaluated to determine the effects of different concentrations of RAPSs, SAPSs, WAPSs, and HAPSs on the phagocytic activity of RAW264.7 macrophages. Figure 7 As shown in Figure A, compared with the blank control group, different concentrations of RAPSs, SAPSs, WAPSs and HAPSs can increase the phagocytic activity of macrophages, but the degree of stimulation is different. Among them, the HAPSs group significantly promoted the phagocytosis of macrophages, and its phagocytic activity was the highest at 50μg / mL (p<0.01). However, after 24h of action at different concentrations, RAPSs, SAPSs and WAPSs had only a weak positive effect on the phagocytosis of macrophages (p>0.05). The effects of RAPSs, SAPSs, WAPSs and HAPSs on phagocytosis are weaker than their effects on NO and other cytokines.

[0151] RAPSs, SAPSs, WAPSs, and HAPSs promote NO production by activating RAW264.7 macrophages

[0152] Nitric oxide (NO) is an important mediator of immune response, and its release can indirectly reflect the activation state of macrophages. Studies have shown that proper promotion of NO production is necessary for the immune system to attack exogenous substances. In this study, Griess reagent was used to detect the effects of lipopolysaccharide (positive control) and different concentrations of RAPSs, SAPSs, WAPSs and HAPSs on the NO secretion level of macrophages after 24 hours of treatment. Figure 7As shown in Figure B, compared with the control group, the NO secretion level was significantly increased (p < 0.0001) under LPS stimulation, reaching 7.4 μM. Compared with the control group, the RAPSs, SAPSs, WAPSs and HAPSs treatment groups significantly promoted the NO secretion level at 10, 25, and 50 μg / mL concentrations (p < 0.0001). Interestingly, when RAPSs, SAPSs and HAPSs were treated with 50 μg / mL, the NO secretion level of macrophages reached the highest level, 4.60, 3.81 and 4.61 μM, respectively. However, when WAPSs was treated with 10 μg / mL, the highest NO secretion level of macrophages was only 2.88 μM. And at the same concentration (10-50 μg / mL), the order of NO release of the four polysaccharides was HAPSs>RAPSs>SAPSs>WAPSs. Compared with the positive control lipopolysaccharide, the effects of RAPSs, SAPSs, WAPSs, and HAPSs were milder, thus avoiding the apoptosis of macrophages due to excessive secretion of NO.

[0153] 4.2.4RAPSs, SAPSs, WAPSs, and HAPSs promote the secretion of cytokines TNF-α and IL-6 by activating RAW264.7 macrophages

[0154] Activated RAW264.7 cells can directly participate in the defense against pathogen invasion by releasing cytokines such as TNF-α and IL-6. In order to study the differences in immunomodulatory activity among RAPSs, SAPSs, WAPSs and HAPSs, we pretreated RAW264.7 cells with different concentrations of the four polysaccharides for 24 hours, and then used ELISA kits to detect the secretion levels of cytokines such as TNF-α and IL-6 in the cell culture supernatant. Lipopolysaccharide was selected as a positive control. Figure 7 As shown in Figures C and D, RAPSs, SAPSs, WAPSs and HAPSs at concentrations of 10, 25 and 50 μg / mL significantly stimulated RAW264.7 cells to secrete TNF-α and IL-6 (p < 0.0001, p < 0.01). Compared with the control group, when 50 μg / mL of RAPSs, SAPSs, WAPSs and HAPSs were used to treat macrophages, the secretion levels of TNF-α and IL-6 reached the highest level, which were 139.48, 137.85, 133.8, 178.89 pg / mL and 10.61, 10.32, 9.32, 13.67 pg / mL higher than the control group, respectively. Similar to the NO secretion level, among the four polysaccharides, HAPSs had the strongest stimulating effect on the production of cytokines TNF-α and IL-6, thereby better enhancing immune function, while WAPSs had the weakest effect.

[0155] These results indicate that within the tested concentration range, RAPSs, SAPSs, WAPSs, and HAPSs exhibited significant immunostimulatory activity by increasing the secretion of NO, TNF-α, and IL-6 in RAW264.7 cells. In addition, at concentrations of 10, 25, and 50 μg / mL, the order of immunostimulatory activity of the four polysaccharides on RAW264.7 cells was HAPSs>RAPSs>SAPSs>WAPSs. An increasing number of studies have shown that the immunomodulatory activity of polysaccharides is related to their monosaccharide composition, molecular weight, structure, and conformation. Therefore, different processing methods may affect the immunostimulatory activity of APSs by changing their properties and structural characteristics.

[0156] 4.2.5 Effects of RAPSs, SAPSs, WAPSs and HAPSs on LPS-induced NO secretion in RAW264.7 cells

[0157] Excessive activation of macrophages is a key step in the inflammatory process and will lead to the occurrence of inflammatory responses. In order to detect the anti-inflammatory activity of RAPSs, SAPSs, WAPSs and HAPSs, 1 μg / mL LPS was used to stimulate macrophages and an inflammatory model was established. In this study, RAW 264.7 cells were stimulated with LPS for 24 hours, and then different concentrations of RAPSs, SAPSs, WAPSs and HAPSs (10, 25, 50 μg / mL) were used to treat the cells for another 24 hours. Griess reagent was used to detect the effect of NO secretion in the culture supernatant of each group. Figure 8 As shown in Figure A, compared with the blank control, the LPS model group significantly enhanced the secretion of NO by macrophages (p < 0.0001), inferring that the LPS-induced inflammatory model has been successfully established. Compared with the model group, the four polysaccharides at different concentrations can significantly inhibit the LPS-induced NO secretion by RAW264.7 cells (p < 0.0001, p < 0.05). Interestingly, RAPSs, SAPSs, WAPSs and HAPSs all had the strongest inhibitory effect at a low concentration of 10 μg / mL. The inhibition rates were 30.35%, 37.30%, 42.71% and 31.23%, respectively (p < 0.0001). The results showed that within the tested concentration range, WAPSs had the strongest inhibitory effect on the LPS-induced NO secretion by RAW264.7 cells, while RAPSs had a weaker inhibitory effect.

[0158] 4.2.6 Effects of RAPSs, SAPSs, WAPSs, and HAPSs on LPS-induced pro-inflammatory factors in RAW264.7 cells

[0159] It has been reported that pathogenic factors such as LPS can directly or indirectly induce RAW264.7 cells and trigger inflammatory responses. At the same time, a large number of cellular proinflammatory factors such as TNF-α and IL-6 are secreted in the cells. It can cause a series of cascade reactions through humoral and cellular effects, leading to inflammatory diseases and pathological conditions such as fever, asthma, atherosclerosis, tissue damage and rheumatoid arthritis. Therefore, in order to study the therapeutic effects of RAPSs, SAPSs, WAPSs and HAPSs on RAW264.7 macrophage inflammation stimulated by LPS, we further determined the inhibitory effects of the four polysaccharides on the release of cellular proinflammatory factors TNF-α and IL-6. Figure 8 As shown in Figures B and C, compared with the control group, the release of TNF-α and IL-6 in the RAW264.7 cells of the model group activated by LPS alone was significantly increased (p < 0.0001). Figure 8 Figure B shows that the four polysaccharides at different concentrations significantly downregulated the secretion of TNF-α. Compared with the model group, RAPSs, SAPSs, WAPSs and HAPSs had the strongest inhibitory effect at 50μg / mL. At this concentration, their release amounts were 64.89, 107.70, 166.57 and 90.36pg / mL lower than those in the model group, respectively (p<0.0001). Figure 8 Figure C shows that RAPSs, SAPSs, WAPSs and HAPSs all down-regulated the secretion of IL-6, and the inhibition rate reached the maximum at 50 μg / mL, which were 35.40%, 42.67%, 50.66% and 39.03% respectively (compared with the model group, p < 0.0001). The results showed that compared with RAPSs, SAPSs, WAPSs and HAPSs could inhibit the release of TNF-α, IL-6 and other cellular proinflammatory factors. The three processing methods can significantly promote their anti-inflammatory activity, and the promotion degree of different processing methods is different.

[0160] 4.3 Discussion: Validation Example The effects of RAPSs, SAPSs, WAPSs and HAPSs on immunostimulation and anti-inflammatory activity were studied. Macrophages are one of the important innate immune cells and are of great significance to the immune system. It not only induces adaptive immune responses to cancer and immune diseases, but also regulates inflammation. In the process of immunoregulation, macrophages can kill invading pathogens directly through phagocytosis, but also indirectly by secreting pro-inflammatory biological factors, such as TNF-α, IL-6, NO, etc. In the anti-inflammatory process, macrophages are prevented from releasing excessive pro-inflammatory factors. LPS has been shown to activate multiple signaling pathways in macrophages, leading to the production of pro-inflammatory factors. Therefore, LPS-stimulated macrophages were selected as the positive control group and the inflammation model group in this paper.

[0161] In vitro experiments showed that RAPSs, SAPSs, WAPSs and HAPSs promoted the phagocytosis of RAW264.7 cells in the concentration range of 10-50 μg / mL. In addition, the raw and three processed polysaccharides also significantly increased the levels of NO, IL-6 and TNF-α secreted by RAW264.7 cells. Within the tested concentration range, their stimulating activities on RAW264.7 cells were in the order of HAPSs>RAPSs>SAPSs>WAPSs. The immunostimulatory activity of HAPSs was stronger than that of the other three polysaccharides. Among the four polysaccharides, HAPSs did not have the highest galactose content and molecular weight, but it had the strongest stimulating effect on NO, TNF-α and IL-6.

[0162] Excessive inflammation may trigger the apoptosis of immune cells, thereby promoting the occurrence of chronic degenerative diseases or cancer. RAPSs, SAPSs, WAPSs and HAPSs all significantly inhibited the release of NO and pro-inflammatory factors from RAW264.7 cells induced by LPS within the tested concentration range. However, compared with raw RAPSs, the three processed products SAPSs, WAPSs and HAPSs all had stronger abilities to inhibit the secretion of NO and the release of TNF-α, IL-6 and other cellular pro-inflammatory factors from macrophages. And within the concentration range of 10-50 μg / mL, the anti-inflammatory activity of the four polysaccharides was in the order of WAPSs>SAPSs>HAPSs>RAPSs. The three processing methods can significantly promote their anti-inflammatory activity, and the promotion degree of different processing methods is different. Many studies have shown that polysaccharides with relatively large molecular weight have poor solubility, which in turn limits their bioavailability. However, too low molecular weight will lead to low polymerization degree and instability of active groups, affecting the biological activity of polysaccharides. In this study, the molecular weight of the unprocessed raw polysaccharide RAPSs was significantly greater than that of SAPSs, WAPSs, and HAPSs. This is consistent with the result that the anti-inflammatory activity of RAPSs is weaker than that of SAPSs, WAPSs, and HAPSs. WAPSs, which have a larger molecular weight than SAPSs, also showed more significant anti-inflammatory activity. On the other hand, in the correlation analysis, it can be seen that in addition to the molecular weight, the monosaccharide composition of the polysaccharide also significantly affects its anti-inflammatory activity. Studies have shown that the anti-inflammatory activity of polysaccharides is related to the different molar ratios of galactose and glucose in their monosaccharide composition. The monosaccharide composition of RAPSs, SAPSs, WAPSs, and HAPSs is mainly manifested in different molar ratios of glucose, arabinose, and galactose, which may be another important factor leading to the differences in the anti-inflammatory activities of the four polysaccharides. The structural characteristics of polysaccharides, including chemical composition, monosaccharide composition, functional groups, molecular weight, degree of branching, and conformation, will affect the bidirectional immunomodulatory effect of polysaccharides. The complex composition and structural heterogeneity of polysaccharides make it difficult to accurately explain the relationship between their structure and immunomodulatory activity. Therefore, further studies on the structure-activity relationship and immunomodulatory mechanism of RAPSs, SAPSs, WAPSs, and HAPSs are needed.

[0163] In summary, the three classical processing methods of Astragalus membranaceus can affect its bidirectional immunomodulatory activity by changing the physical properties and structural characteristics of polysaccharides, such as Fig. 9As shown in the results, within the tested concentration range, RAPSs, SAPSs, WAPSs and HAPSs all have bidirectional immunomodulatory effects, but different processing methods affect the strength of the bidirectional immunomodulatory activity of the four polysaccharides. In the upregulation of immunostimulatory activity, HAPSs played a better role, while WAPSs played a weaker role. In the downregulation of anti-inflammatory activity, the activities of the three processed polysaccharides SAPSs, WAPSs and HAPSs were greater than those of the unprocessed raw RAPSs. Previous studies have shown that the changes in chemical composition, structure and pharmacological activity during the processing of traditional Chinese medicines are not only caused by hydrolysis reactions under heating to form easily absorbed components, but also by the special formulation characteristics of adjuvants that can establish specific forms with active ingredients. Therefore, different processing methods may change the physical properties and structural characteristics of astragalus polysaccharides through their heating effects and the use of processing adjuvants, thereby causing changes in their pharmacological activities.

[0164] Verification Example 5 - Correlation Analysis between Immune Activity and Physicochemical Properties of Different Raw Sugars

[0165] The chemical composition and bidirectional immunomodulatory activity of astragalus polysaccharides processed by different methods are different. In order to clarify the possible mechanism by which the physicochemical composition of polysaccharides affects the immunoenhancing activity and anti-inflammatory activity of polysaccharides, this study used the Pearson correlation analysis method to study the relationship between the immunological activity and physicochemical properties of astragalus polysaccharides processed by different methods. Fig. 9 In terms of immunopotency, molecular weight and mannose content were correlated with NO release activity (Pearson correlation coefficients were r = 0.51 and r = -0.52, respectively). TNF-α release activity was correlated with galacturonic acid, glucosamine hydrochloride, arabinose, and glucose content (Pearson correlation coefficients were r = 0.60, r = 0.52, r = 0.56, and r = -0.54, respectively). The ability to release IL-6 was correlated with glucose, galactose, glucosamine hydrochloride, and arabinose (Pearson correlation coefficients were r = -0.56, r = 0.53, r = 0.52, and r = 0.59, respectively), and its phagocytic activity was correlated with xylose, galactose, glucosamine hydrochloride, arabinose, and glucose (Pearson correlation coefficients were r = 0.51, r = 0.70, r = 0.57, r = 0.69, and r = -0.66, respectively). In terms of anti-inflammatory activity, molecular weight was significantly negatively correlated with the inhibition rate of NO, TNF-α, and IL-6. The inhibitory capacity of TNF-α and IL-6 was significantly positively correlated with galacturonic acid and mannose. These results indicate that the bidirectional immunomodulatory activity of RAPSs, SAPSs, WAPSs, and HAPSs is significantly correlated with their monosaccharide composition and molecular weight.

[0166] From the above content, it can be seen that the present application has systematically studied the chemical composition, physicochemical properties and bidirectional immunomodulatory activity of polysaccharides in raw and three processed products (processed with salt, processed with rice wine and processed with honey) of Astragalus. The heteropolysaccharides RAPSs, SAPSs, WAPSs and HAPSs are all composed of arabinose, glucosamine hydrochloride, galactose, glucose, xylose, mannose and galacturonic acid, but there are significant differences in polysaccharide yield, chemical composition, molar ratio of monosaccharide composition, molecular weight, Zeta potential and particle size. The four APSs all have typical crystalline interiors, characteristic functional groups of polysaccharides and major glycoside residues. The microscopic morphologies of the four APSs were observed to be different using SEM and AFM. In vitro cell experiments have shown that RAPSs, SAPSs, WAPSs and HAPSs all have bidirectional immunomodulatory effects (such as Fig.10 However, it is worth noting that different processing methods changed the strength of the four polysaccharides in exerting bidirectional immunomodulatory activity. HAPSs had a stronger ability to enhance immunity, while WAPSs polysaccharides had a stronger anti-inflammatory ability. Different processing methods may change the physicochemical properties and structural characteristics of astragalus polysaccharides through their heating effect and the use of processing adjuvants, thereby changing their bidirectional immunomodulatory activity.

[0167] The above examples are only for illustrating the technical concept and technical features of the present invention, and cannot be used to limit the protection scope of the present invention. Any equivalent transformation or modification made according to the essence of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for processing astragalus polysaccharides with different processing techniques, wherein raw astragalus slices are processed by salt processing, wine processing and honey processing, comprising the following steps: (1) Take raw Astragalus slices, add salt water, rice wine and refined honey respectively, mix well and simmer thoroughly, put them in a pot and stir-fry them over low heat until dry, then take them out and let them cool, to obtain salt-processed Astragalus slices, wine-processed Astragalus slices and honey-processed Astragalus slices respectively; (2) respectively crushing the salt-processed Astragalus slices, wine-processed Astragalus slices, and honey-processed Astragalus slices obtained in step (1) with a traditional Chinese medicine crusher, weighing the crushed samples and mixing them with water, heating and dissolving them, and then centrifuging and filtering to remove insoluble residues to obtain supernatants; respectively concentrating the supernatants to obtain concentrated solutions, and separating them through a retention membrane (molecular weight: 5000Da) at a ratio of concentrated solution: water (1:2.5) to obtain separated solutions; Subsequently, the separated liquid was dialyzed (molecular weight: 3500Da) to obtain dialyzate; the dialyzate was centrifuged and filtered, and then the supernatant was concentrated and freeze-dried using a freeze dryer to obtain salt-processed astragalus crude polysaccharide, wine-processed astragalus crude polysaccharide, and honey-processed astragalus crude polysaccharide, respectively.

2. The method according to claim 1, characterized in that The salt-processing method is used to prepare astragalus polysaccharide, which includes the following steps: (1) Take 100 g of raw Astragalus slices (RAR), take 2 g of edible salt and add appropriate amount of water to make brine, add Astragalus polysaccharide brine processed by salt processing method to the raw Astragalus, mix well and stew thoroughly, put it in a hot pot and stir-fry it over low heat until it is dry, then take it out and cool it to obtain salt-processed Astragalus slices (SAR); (2) The salt-processed Astragalus membranaceus slices obtained in step (1) are crushed by a Chinese medicine crusher, the crushed sample is weighed and mixed with water, heated to dissolve, and then centrifuged to remove insoluble residues to obtain a supernatant; the supernatant is concentrated to obtain a concentrated solution, and the concentrated solution is separated by convection through a retention membrane (molecular weight: 5000Da) at a ratio of concentrated solution: water (1:2.5) to obtain a separated solution; the separated solution is then dialyzed (molecular weight: 3500Da) to obtain a dialyzate; the dialyzate is centrifuged and filtered, and the supernatant is concentrated and then freeze-dried using a freeze dryer to obtain a crude salt-processed Astragalus membranaceus polysaccharide, which is named SAPSs.

3. A salt-processed astragalus crude polysaccharide SAPSs obtained by processing raw astragalus slices by the salt processing method as described in claim 2, characterized in that: The salt-processed astragalus crude polysaccharide SAPSs has an average particle size of 115.2 nm, a molecular weight of 86.34 kDa, a neutral sugar content of 58.70±3.39%, a uronic acid content of 1.80±0.23%, and a total protein content of 9.13±1.54%. The SAPSs includes the following monosaccharides: arabinose 11.52%, glucosamine hydrochloride 0.92%, galactose 11.10%, glucose 74.05%, xylose 1.47%, mannose 0.51%, and galacturonic acid 0.43%.

4. Use of the salt-processed astragalus crude polysaccharides SAPSs according to claim 3 in the preparation of medicines, foods and health foods with bidirectional immunomodulatory effects.

5. The method according to claim 1, characterized in that The wine-making method is used to prepare and process astragalus polysaccharide, which includes the following steps: (1) Take 100 g of raw Astragalus slices (RAR), add 20 mL of rice wine, mix well and simmer thoroughly, put in a pot and stir-fry over low heat until slightly yellow, take out and let cool to obtain wine-processed Astragalus slices (WAR); (2) The wine-processed Astragalus slices obtained in step (1) are crushed by a Chinese medicine crusher, the crushed sample is weighed and mixed with water, heated to dissolve, and then centrifuged to remove insoluble residues to obtain a supernatant; the supernatant is concentrated to obtain a concentrated solution, and the concentrated solution is separated by convection through a cutoff membrane (molecular weight: 5000Da) at a ratio of concentrated solution: water (1:2.5) to obtain a separated solution; the separated solution is then dialyzed (molecular weight: 3500Da) to obtain a dialysate; the dialysate is centrifuged, and then the supernatant is concentrated and freeze-dried using a freeze dryer to obtain a wine-processed Astragalus crude polysaccharide, which is named WAPSs.

6. A crude wine-processed astragalus polysaccharide WAPSs obtained by processing raw astragalus slices by the wine-processing method as claimed in claim 5, characterized in that: The wine-processed astragalus crude polysaccharide WAPSs has an average particle size of 378.9 nm, a molecular weight of 112.39 kDa, a neutral sugar content of 69.46±4.40%, a uronic acid content of 2.10±0.002%, and a total protein content of 2.10±0.36%. The WAPSs comprises the following monosaccharides: arabinose 8.23%, glucosamine hydrochloride 0.70%, glucose 8.10%, galactose 80.74%, xylose 1.10%, mannose 0.59%, and galacturonic acid 0.54%.

7. Use of the wine-processed astragalus polysaccharides WAPSs according to claim 6 in the preparation of medicines, foods and health foods with anti-inflammatory effects.

8. The method according to claim 1, characterized in that The astragalus polysaccharide is prepared by using a honey-processing method, comprising the following steps: (1) Take 100 g of raw Astragalus slices (RAR), add 25 g of refined honey and an appropriate amount of water to dilute, stir evenly, put into a hot pot after simmering, and stir-fry over low heat until the aroma comes out and the Astragalus is dark yellow and not sticky, take out and cool to obtain honey-processed Astragalus slices (HAR); (2) The honey-processed Astragalus slices obtained in step (1) are crushed by a traditional Chinese medicine crusher, the crushed sample is weighed and mixed with water, heated to dissolve, and then centrifuged to remove insoluble residues to obtain a supernatant; the supernatant is concentrated to obtain a concentrated solution, and the concentrated solution is separated by convection through a retention membrane (molecular weight: 5000Da) at a ratio of concentrated solution: water (1:2.5) to obtain a separated solution; the separated solution is then dialyzed (molecular weight: 3500Da) to obtain a dialysate; the dialysate is centrifuged, and then the supernatant is concentrated and freeze-dried using a freeze dryer to obtain a crude honey-processed Astragalus polysaccharide, which is named HAPSs.

9. A honey-processed crude astragalus polysaccharide HAPSs obtained by processing raw astragalus slices by the honey-processing method as claimed in claim 8, characterized in that: The honey-processed astragalus crude polysaccharide HAPSs has an average particle size of 163.5 nm, a molecular weight of 138.70 kDa, a neutral sugar content of 75.69±3.76%, a uronic acid content of 2.23±0.03%, and a total protein content of 9.04±0.91%. The HAPSs include the following monosaccharides: arabinose 11.32%, glucosamine hydrochloride 0.90%, galactose 10.55%, glucose 74.65%, xylose 1.37%, mannose 0.58%, and galacturonic acid 0.63%.

10. Use of the honey-processed astragalus crude polysaccharides HAPSs according to claim 9 in preparing medicines, foods and health foods having immunity-enhancing properties.

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