4 Methods for Astragalus Root Extraction

Astragalus is mainly divided into two types: Astragalus membranaceus and Astragalus mongholicus. Both are distributed in Heilongjiang, Inner Mongolia, Jiangxi and other places. Astragalus is sweet and neutral in nature, with a slightly warm temperature. It has a medicinal history of more than 2,000 years in China. The first record of Astragalus appeared in the “Fifty-two Disease Prescriptions from the Mawangdui Silk Manuscripts”. Astragalus can be used in combination with peony and licorice to treat gangrene.

Astragalus contains effective ingredients such as astragaloside, polysaccharides, proteins and flavonoids. Polysaccharides are one of the main water-soluble components of astragalus[1]. Astragalus polysaccharides have physiological functions such as anti-oxidation, immune regulation and anti-tumor. Astragalus polysaccharides are composed of arabinose, xylose, mannose, rhamnose, galactose and glucose. Due to its outstanding physiological functions, astragalus polysaccharide has become a research hotspot in medicine, food, breeding and other fields. This paper reviews the extraction, purification and application of astragalus polysaccharide, with a view to providing a reference for the further development and utilization of astragalus polysaccharide.

1 Extraction of astragalus polysaccharides

In the extraction process of astragalus polysaccharides, petroleum ether and ethanol degreasing can be used first, which can make the polysaccharides more soluble. The extraction methods of astragalus polysaccharides include solvent method, enzyme-assisted method, microbial fermentation method, physical strengthening method, etc.

1.1 Solvent method

The solvent method is a commonly used extraction method in industry. It is easy to operate and low in cost, but the extraction time is long, the temperature is high, and it can lead to inactivation of the active components. Table 1 lists the extraction yields of astragalus polysaccharides extracted using three solvent methods.

1.2 Enzyme-assisted extraction

Enzymes can break down the cell wall structure, reduce the resistance of the cell wall and cell interstitial matrix, and improve the extraction rate. Chen et al. [4] compared the extraction rates of astragalus polysaccharides by eight enzymes such as cellulase and pectinase, and found that glucose oxidase had the best effect.

The response surface method was used to optimize the extraction process: enzyme amount 3.0%, treatment time 3.44 d, treatment temperature 56.9 ℃, extraction solvent pH 7.8. Under these conditions, the extraction rate of astragalus polysaccharides reached (29.96 ± 0.14) %, which is 250% higher than that without enzyme assistance. Dong Lingling [5] used a combination of microwave and cellulase extraction, and the polysaccharide extraction rate reached 16.07%. In addition, multiple enzymes are often used together in plant extraction [6]. The use of enzymes for assisted extraction has low pollution, is simple, and low cost, but enzymes have high requirements for environmental conditions and require strict control of environmental conditions.

1.3 Microbial fermentation extraction

Microbial fermentation extraction uses a variety of enzymes produced by microorganisms such as bacteria or fungi during the metabolic process to act on the extraction substrate. These enzymes can destroy or modify the structure of the cell wall, making it easier to release the active ingredients in traditional Chinese medicine, and can also degrade polysaccharides into small molecule polysaccharides and convert them into other types of polysaccharides.

Bian Yabin [7] optimized the process of fermenting astragalus polysaccharides with Lactococcus lactis subsp. lactis (FGM). Under the conditions of an extraction time of 65 min, an extraction temperature of 80 °C, and a liquid-to-solid ratio of 1:9, the content of fermented astragalus polysaccharide was 6.72 mg/mL, and it was confirmed that fermented astragalus polysaccharide could promote the maturation of mouse bone marrow-derived DCs. Su Guilong [8] found that FGM and Bacillus subtilis, and that both the FGM solid and liquid two-step methods could increase the yield of fermented astragalus root, stem and leaf polysaccharides. Fermentation and extraction of the active ingredients of traditional Chinese medicine not only improves the extraction efficiency, but also reduces the toxic side effects of traditional Chinese medicine.

1.4 Physical strengthening method

In recent years, commonly used physical strengthening methods include ultrasonic-assisted extraction, microwave-assisted extraction, and negative pressure cavitation extraction. The physical strengthening method uses physical methods to destroy the cell structure and cause the polysaccharides to flow out of the cells, thereby achieving the goal of improving the extraction rate. It is also a commonly used method in plant extraction in recent years. Compared with ultrasonic and microwave-assisted extraction and negative pressure cavitation extraction, the extraction rate is lower and has been reviewed in detail by previous studies, so it will not be explained in detail in this paper. Table 2 lists the extraction rate of astragalus polysaccharides extracted by physical strengthening methods.

Negative pressure cavitation uses strong cavitation and mechanical vibration to accelerate the entry of active ingredients in plant tissues into the solvent, achieving short-term and low-temperature extraction [12]. It can protect heat-sensitive substances in plants from being destroyed, while also reducing macromolecular impurities such as proteins and starch that are produced by pyrolysis.

Jiao et al. [13] optimized the process of extracting astragaloside polysaccharides using homogenization-assisted negative pressure cavitation. The homogenization time was 70 s, the negative pressure was pressure -0.068 MPa, extraction temperature 64.8 °C, water-to-material ratio 1:13.4, extraction time 53 min, and the extraction rate was 16.74%. At the same time, FTIR results showed that this method does not change the primary structure of astragalus polysaccharides. Compared with the traditional hot water extraction method, negative pressure cavitation extraction has the advantages of high extraction rate and mild conditions. In the extraction of other plants, negative pressure cavitation and other extraction methods are used in combination. Tian Li et al. [14] optimized the process of extracting polyphenols from apple pomace by decompression coupled with ultrasound, and the reducing power of the obtained polyphenol extract was greater than that obtained by ultrasound extraction.

Although the hot water extraction method has a low extraction rate, it is suitable for large-scale industrial extraction. Ultrasonic and microwave-assisted extraction require high-tech equipment and are not suitable for large-scale production. Enzyme-assisted extraction is efficient, but has high environmental requirements. In microbial fermentation, the problem of how to select and breed good strains needs to be solved [15]. Studies have shown that the extraction temperature can affect the physiological activity of astragalus polysaccharides by affecting their structure [16]. Each method has its own advantages and disadvantages. How to make the most of the advantages and avoid the disadvantages, and achieve low-temperature and efficient extraction, should be the focus of future research on astragalus polysaccharide extraction.

2. Purification of astragalus polysaccharides

2.1 Deproteinization

It has been reported that the protein content in crude astragalus polysaccharide is greater than 15%. Protein removal is an important step in obtaining highly active and valuable astragalus polysaccharide products. The commonly used methods for deproteinizing astragalus polysaccharide are shown in Table 3. In order to facilitate the operation and increase the yield of polysaccharides, the enzyme method and Sevage method have been commonly used in recent years. Hu Yuanyuan [20] optimized the deproteinization process using a combination of protease and Sevage methods. The best results were obtained with an enzyme to substrate ratio of 2.0%, pH 5.0, and 50°C water bath enzyme digestion for 24 hours, with a protein removal rate of 89.82% and a astragalus polysaccharide mass fraction of 83.47%. The enzyme method and the Sevage method are used in combination, which has the advantages of good protein removal and low polysaccharide loss, while also simplifying the operation.

2.2 Astragalus polysaccharide purification

2.2.1 Gradient precipitation method

The structure and molecular weight of polysaccharides can lead to differences in polarity, which in turn lead to differences in their solubility in organic solvents. Based on this principle, the solubility of polysaccharides in organic solvents can be determined by successively increasing the concentration of the organic solvent, and polysaccharides with different molecular weights can be precipitated out. In the grading precipitation of polysaccharides,gradual precipitation, ethanol is a commonly used precipitant. Li Hongfa et al. [21] used 30%, 50%, 70%, 75%, 80%, and 90% ethanol solutions to perform graded precipitation of astragalus polysaccharides, obtaining six different fractions. The structure, composition and antioxidant activity of each component were analyzed separately. The results showed that as the ethanol concentration increased, the molecular weight of the polysaccharide gradually decreased, the content of galactose, mannose and rhamnose in the polysaccharide increased sequentially, and the content of glucose decreased sequentially. However, the antioxidant activity increased, and the antioxidant activity was related to the structure of the polysaccharide.

2.2.2 Column chromatography

Ion exchange chromatography and gel chromatography are the most widely used methods for the isolation and purification of astragalus polysaccharides. Ion exchange chromatography is mainly used for the crude separation of single-component polysaccharides in polysaccharide crude extracts. Gel chromatography can be used to separate or further purify polysaccharides according to their structure and molecular weight using different specifications. Qu Jing et al. [22] used a SephadexCL-6B gel column and 0.9% NaCl as the eluent to perform column chromatography on deproteinized astragalus polysaccharide, obtaining a homogeneous polysaccharide with a molecular weight of 5,600 Da and a content of 96.3%. Wang Ruizun [23] first used a DEA-Cellulose ion exchange column to separate Astragalus polysaccharides by chromatography, using 0.5 mol/L NaCl as the eluent, to obtain two components. Then, a Seph CL-6B gel column, with 0.15 mol/L NaCl as the eluent, the polysaccharides were further purified to obtain four molecular weights: AMPSA-a, AMPSA-b, AMPSA-c, and AMPSB-d.

Four molecular weights and each component polysaccharide has significant antioxidant activity.

2.2.3 Membrane separation

The membrane separation process carried out at room temperature has the advantages of not using organic solvents, high separation selectivity, and being easy to use in conjunction with other methods. Ultrafiltration is a membrane separation technology that emerged in the 1960s. It is widely used in the separation and purification of plants because of its high yield and minimal damage to the product. Zhang Qinglei [24] used ultrafiltration with vibration membrane technology and ethanol gradient precipitation for the purification of astragalus polysaccharides, and found that the former method is easy to obtain polysaccharides with relatively uniform molecular weight. Tang Yuwei [25] used hollow fiber membranes with different cut-off values (150, 100, 50, 20, 10, 6 kDa) to separate the deproteinized astragalus polysaccharide into 7 groups. After separation by DEAE-Cellulose and Sephadex G-100 column chromatography, 6 polysaccharides were obtained. Studies have shown that the obtained polysaccharides have microecological regulation functions.

At present, the isolation and purification methods for astragalus polysaccharides are relatively fixed, and the isolation and purification processes of other polysaccharides can be used as a reference to better purify astragalus polysaccharides. For example, membrane integration technology can be used to isolate and purify polysaccharides: microfiltration and ultrafiltration can be used to isolate and purify tea polysaccharides to obtain two fractions. In addition to these, other common membrane separation methods include microfiltration and nanofiltration, which are less commonly used in the purification of astragalus polysaccharides, but have been used in the purification of other plant polysaccharides and can be used as a reference in the separation and purification of astragalus polysaccharides.

3 Applications

3.1 Medical

Astragalus polysaccharides have good immunomodulatory, anti-tumor, anti-inflammatory and other pharmacological effects. At present, astragalus polysaccharides have made great progress in the treatment of diseases. They have been used clinically to treat tumors, asthma, and diabetes. The treatment of some other diseases is still in the animal testing stage.

3.1.1 Tumors

The high mortality rate has made cancer a major threat to human health worldwide. Chemotherapy is currently the main method of treating cancer, but it is often accompanied by toxic side effects and may also lead to drug resistance. Astragalus polysaccharides can enhance the body's immune system, inhibit tumour growth and promote apoptosis while also reducing the toxic side effects of drugs. In recent years, astragalus polysaccharides have developed rapidly in the treatment of cancer. Studies have shown that astragalus polysaccharide has an inhibitory effect on gastric cancer MGC-803 cells, human non-small cell lung cancer A549 cells and human liver cancer HepG2 cells, and can induce apoptosis of gastric cancer MGC-803 cells [26]. Yang Xiaolan [27] used a combination of injectable astragalus polysaccharide and radiotherapy for the treatment of gastric cancer.

The cancer treatment rate and tumor volume reduction rate in the combined treatment group were 63.9% and 59%, respectively, compared to 53% and 51.8% in the control group. At the same time, the functional levels of immunity, hematopoiesis, and the liver and kidney in patients in the combined treatment group were significantly improved compared to the control group. Zhang Ying et al. [28] used a combination of astragalus polysaccharide and cytokine-induced killer cells to treat patients with intermediate and advanced non-small cell lung cancer with a qi deficiency pattern. The disease control rate and the Karasz score were 69.4% and 77.8% in the combined treatment group, and 36.1% and 55.6% in the control group, respectively. In addition, astragalus polysaccharide can also be used in combination with gemcitabine to treat pancreatic cancer, and with doxorubicin liposomes to treat liver cancer.

3.1.2 Asthma

Astragalus polysaccharides may have a beneficial effect on respiratory diseases by regulating the function of immune cells and the expression of cytokines, and enhancing anti-inflammatory activity. Injecting asthma patients with astragalus polysaccharide injection in addition to conventional treatment can significantly reduce the level of inflammatory cells in the patient's BALF or sputum [29], maintain immune balance, improve the body's immune capacity, while restoring lung function and reducing the risk of adverse reactions. Other studies have shown that astragalus polysaccharides can improve the immune stress status of patients and enhance their immunity by exerting an immunomodulatory effect on T lymphocytes, thereby improving the cure rate of patients with bronchial asthma [30].

3.1.3 Diabetes

Astragalus polysaccharides can improve the renal function of patients by regulating their immune function and reducing inflammatory reactions. Deng Haiou et al. [31] used astragalus polysaccharide injection to treat early diabetic nephropathy in the elderly. Lai Yu [32] showed that the combined use of astragalus polysaccharide and Sanyang Huayu Tang can reduce the content of plasminogen activator inhibitor-1, an inflammatory factor in the serum, in patients with early diabetic nephropathy.

3.1.4 Other

In addition, astragalus polysaccharide can also be used to treat cardiovascular and neurological diseases. Chen Tianhua's [33] research shows that astragalus polysaccharide has a good protective effect on Ang II-stimulated myocardial cell hypertrophy and inflammatory response. Wang Aiqing [34] confirmed that astragalus polysaccharide has a protective effect on retinal damage in rats with acute high intraocular pressure, which is related to the dose of the drug.

3.2 Food

The book “Handbook of Health Food Ingredients” states that astragalus polysaccharide can be used as a health food ingredient. Zhou Jianwei et al. used astragalus polysaccharide, ginkgo biloba extract and selenium-rich black tomatoes as the main functional ingredients to develop a type of noodles that has a preventive effect on diabetic complications. Shao Baoping et al. [35] used astragalus polysaccharide as the raw material to develop a functional beverage with a comfortable texture, good color and the ability to enhance immunity and resist fatigue.

3.3 Farming

3.3.1 Immune enhancer

Astragalus polysaccharides can improve the serum environment, stimulate the body's immune response, promote the secretion of cytokines, and enhance the body's antibody levels to enhance the effect of vaccines. Zhu et al. [36] showed by real-time quantitative RT-PCR that in the spleen and head kidney tissues, the mRNA expression of the inflammatory cytokine IL- 1 β mRNA expression increased early in the immune response, stimulating a Th1 immune response. Cytokines IL-2 and IFN-γ2 were elevated throughout the immune period, and serum IgM was significantly enhanced. Astragalus polysaccharide can enhance the efficacy of the Edwardsiella tarda vaccine.

Astragalus polysaccharides, as a natural feed additive, can not only improve the immune system but also enhance the production performance of the body. Chen Yajun [37] found that when the addition of astragalus polysaccharides in the feed was 50-400 mg/kg, it could significantly enhance the non-specific immunity and antioxidant capacity of loach. This may be due to the fact that astragalus polysaccharides can increase the content of NO in white blood cells, red blood cells and serum. Wu et al. [38] confirmed that feeding broilers with 1 g/kg astragalus polysaccharides can promote the growth of young broilers. Compared with the control group, the activity of amylase, lipase and protease in broilers was higher, but when the astragalus polysaccharide content was too high, the activity of digestive enzymes was reduced.

3.3.2 Reproduction

Artificial insemination is widely used in modern pig farms. The lifespan of semen stored in liquid nitrogen is short, and freezing can extend the lifespan of semen. During the freezing and thawing of semen, how to establish a strong antioxidant system for sperm has become an urgent problem that needs to be solved. Astragalus polysaccharides have good antioxidant properties, so they may be a good choice to solve this problem. Fu et al. [39] showed that astragalus polysaccharides can inhibit the dephosphorylation of sperm proteins by affecting the pathway of reactive oxygen species entering cyclic adenosine monophosphate. Song Jian [40] demonstrated that astragalus polysaccharide can improve the antioxidant capacity of sperm by reducing the level of reactive oxygen species in thawed pig semen, thereby improving the efficiency of in vitro fertilization and embryo development.

4 Discussion and outlook

Astragalus polysaccharides have extremely high application value. Although significant progress has been made in the research on astragalus polysaccharides, there are still some problems, such as: (1) Many current studies do not explain the structure-activity relationship, which requires the improvement of the preparation method of astragalus polysaccharides, and the use of techniques such as fingerprinting, similarity analysis and cluster analysis to study the structure of astragalus polysaccharides and formulate a quality control plan, such as determining the molecular weight range of its effective fragments; (2) astragalus polysaccharides are raw materials for health foods, but there are not many health products related to astragalus polysaccharides on the market at present. The mechanism of astragalus polysaccharides can be further improved, and research on research in the food sector; (3) In terms of medical treatment (e.g. neurological and cardiovascular diseases), it has only been used in animal experiments, so further research in this area can be strengthened to further promote clinical development and application; (4) Astragalus polysaccharides have the function of scavenging free radicals [42] and are expected to become active ingredients in anti-aging and anti-oxidant cosmetics.

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