Ultrasound-assisted extraction and value of active substances in Muxu

Abstract

This article reviews the latest research progress on ultrasound-assisted extraction of active substances from Muxu, including polysaccharides, polyphenols, leaf proteins, anthocyanins, total flavonoids, and total saponins, in order to provide theoretical references for the extraction of active substances from Muxu. At the same time, its medicinal value, feeding value, ecological value, edible value, and ornamental value were analyzed and summarized. Flavonoids, saponins, and polysaccharides in the bioactive substances of Muxu have good effects on improving animal productivity, enhancing immune function, and improving animal health. Especially when applied to broiler chickens and laying hens, it can improve the quality of meat and eggs and increase the economic benefits of breeding. In addition, there are other beneficial substances in Muxu, such as natural pigments, coumarins, leaf protein, and chlorogenic acid, which also play an important role in livestock and poultry production and health status.

1. Introduction

Muxu (Medicago sativa L.), a perennial herbaceous plant belonging to the legume family, has a planting history of more than 2000 years in China. It is mainly distributed in northern, northeastern, northwestern and other regions of China. Muxu has the advantages of high yield, drought resistance, cold resistance, strong regeneration ability, and good palatability. Due to its tender and delicious stems and leaves, as well as simple planting and strong adaptability, Muxu has been widely planted in countries around the world and is known as the “king of forage”. Numerous studies have shown that Muxu has many biological activities, such as its polysaccharides having pharmacological effects such as anti-infection, anti-tumor, anti-radiation, improving immune regulation ability, and delaying aging [1]. It has minimal toxic side effects on the body and is a highly potential edible material and feed resource with important development and utilization value and broad application prospects [2]. Muxu contains various active substances such as polysaccharides, polyphenols, leaf proteins, anthocyanins, flavonoids, and saponins, which have high value. Herein, the preparation methods and significant values of active substances in Muxu were summarized and discussed (Fig. 1).

Fig. 1.

The active substances and significant values in Muxu.

2. The action mechanism of ultrasound

The basic principle of ultrasonic extraction technology is to use the cavitation, mechanical and thermal effects of ultrasound to accelerate the release, diffusion and dissolution of effective substances inside the cell, thereby improving the extraction efficiency. When high-energy ultrasonic waves act on a medium, the medium is torn into many small cavities, which instantly close, producing an instantaneous pressure of up to thousands of atmospheres, known as cavitation phenomenon. The bursting of tiny bubbles in cavitation generates immense pressure, causing the rupture of plant cell walls and the entire organism to be completed in an instant, shortening the fragmentation time. At the same time, the vibration generated by ultrasound enhances the release, diffusion, and dissolution of intracellular substances, thereby significantly improving extraction efficiency [3], [4] (Fig. 2).

Fig. 2.

Ultrasonic-assisted extraction technology.

There are mainly two types of ultrasound in the field of food research, including ultrasound with different frequencies and vibration intensities. Low intensity detection ultrasound with a frequency greater than 100 kHz is used for food quality control and analysis, which is non-destructive to food. This intensity of ultrasound is mainly used to detect information related to the physical and chemical properties of food. The frequency between 20–100 kHz is low-frequency ultrasound, which has a strong vibration effect. When transmitted to the generator, the vibration force is large, and the mechanical effect is enhanced while generating high energy, which increases the temperature of the system and promotes mass transfer. This type of ultrasound can be used in processes such as sterilization, emulsification, filtration, drying, and peeling, and can improve the quality of food. The use of ultrasound in food processing has many advantages, such as more thorough mixing of different substances, reducing processing temperature, promoting mass transfer, shortening processing time, and increasing extraction efficiency; In addition, the use of ultrasound treatment has high repeatability, low dependence on reagents, and simpler subsequent processing of the product [5].

3. Ultrasound-assisted extraction of active substances in Muxu

3.1. Ultrasound-assisted extraction of Muxu anthocyanins

Anthocyanins are natural compounds extracted from plants, which have fewer side effects compared to artificially synthesized compounds. Currently, anthocyanins are mainly used in the food and pharmaceutical industries, and have great development prospects [6]. The main use of anthocyanins in medicine is to prevent chronic diseases such as cardiovascular disease, metabolic disorders, etc., all of which are due to the antibacterial and antioxidant properties of anthocyanins [7]. In recent years, other physiological activities of anthocyanins have also been discovered, such as reducing DNA damage, DNA breakage, and intracellular reactive oxygen species; Reduce depressive symptoms in adolescents. It has targeted effects on the testicles, etc. [8]. Anthocyanins have numerous and excellent functional properties, as well as the ability to prevent and treat various chronic diseases. Therefore, the research and development of anthocyanins are constantly deepening, and the methods of research and development are also advancing. The method of ultrasound-assisted ethanol acidification was adopted to obtain the best conditions for extracting anthocyanins from Muxu through orthogonal experiments: a solid–liquid ratio of 1:30 (g/mL), extraction at 60 ℃ for 10 min, and the anthocyanin content obtained under this process condition was 93.23 mg/100 g. In further experiments, it was found that the anthocyanins obtained by this method have stronger acid and heat resistance compared to those obtained by ordinary extraction methods; Research has found that food additives used in food processing, such as sucrose and sodium benzoate, do not affect the stability of anthocyanins. However, the presence of sodium nitrite can disrupt the stability of anthocyanins. The effect of metal chloride ions such as Cu²⁺ and K⁺ on the stability of Muxu anthocyanins is relatively small, while Al³⁺ and Zn²⁺ have a certain impact, and Fe³⁺ has the greatest impact on the stability of Muxu anthocyanins [9].

3.2. Ultrasound-assisted extraction of Muxu polysaccharides

Polysaccharides are natural macromolecules with various biological activities. The body itself contains polysaccharides, which are an important substance of the human immune system. Research has found that plant polysaccharides are natural immune modulators with immunomodulatory effects. They promote antibody production and accelerate cytokine release for immune regulation, mainly acting on macrophages, T lymphocytes, natural killer cells, B lymphocytes, etc. They have various effects such as antiviral, anti-inflammatory, antioxidant, anti-adhesion, and anti-sepsis [10], [11], [12]. The ultrasound-assisted method was used to extract polysaccharides from Muxu. Firstly, Muxu was frozen and fused under low temperature regulation, and then subjected to ultrasound extraction. The optimal extraction conditions were obtained as follows: low-temperature freezing for 20 min, repeated freezing and thawing for 7 times, and ultrasound at 70 ℃ for 43 min, resulting in 8.216 mg/g Muxu polysaccharides. The preparation process was optimized using response surface methodology with water as the solvent, and obtained the optimal process as follows: a solid–liquid ratio of 1:37.4, and ultrasound treatment at 74.5 ℃ for 16.69 min. Under these conditions, the polysaccharide yield was 6.19 %, and the stability and efficiency of the polysaccharide content obtained by ultrasound assisted water extraction were better than those of traditional water extraction without ultrasound assistance [13]. A uniform polysaccharide APSU-2a was obtained from Muxu by ultrasonic assisted extraction, DEAE-52 cellulose column chromatography and Sephadex G-75 gel column chromatography. The structural characteristics of APSU-2a were analyzed by high performance gel permeation chromatography, PMP pre column derivatization high performance liquid chromatography, infrared spectroscopy and nuclear magnetic resonance spectroscopy. The antioxidant activity of APSU-2a in vitro was evaluated by DPPH, ABTS and hydroxyl radical scavenging rate. The results showed that the molecular weight of APSU-2a was 311.7 kDa, mainly composed of four monosaccharides: mannose, rhamnose, galactose, and arabinose, as well as a small amount of glucuronic acid and galacturonic acid. The infrared spectroscopy and nuclear magnetic resonance results show that APSU-2 has typical characteristic absorption peaks of polysaccharides, and both α- and β- glycosidic bonds are present. APSU-2a has excellent antioxidant activity, and within a certain concentration range, its in vitro antioxidant activity exhibits a concentration effect relationship. It can be used as a functional ingredient with antioxidant properties for the development of functional foods.

3.3. Ultrasound-assisted extraction of polyphenols from Muxu

Polyphenols are widely present in plants and are secondary metabolites with various biological functions such as antioxidant, antibacterial, anti-inflammatory, and prevention of cardiovascular diseases [14]. Their structure contains multiple phenolic hydroxyl groups, and polyphenols have attracted the attention of many scholars for their numerous benefits. A polyphenol was obtained with the content of 3.642 mg/g in Muxu by adding 4.9 % enzyme and sonicating at 49 ℃ for 74 min. An antioxidant experiment was conducted on Muxu polyphenols, and the IC50 values for DPPH and OH radicals were 10.78 μ g/mL and 19.28 μ g/mL, respectively, indicating good antioxidant activity [15].

3.4. Ultrasound-assisted extraction of total flavonoids from Muxu

Flavonoids, as plant substances, are widely present in nature and have good effects in treating heart disease, hypertension, hyperlipidemia, hyperglycemia, tumors, and other conditions [16]. The total flavonoids in Muxu were extracted using ultrasound assisted biphasic extraction, and the experimental conditions were optimized using response surface methodology. The results showed that the optimal extraction conditions for total flavonoids in Muxu were: ammonium sulfate mass concentration of 0.3 g/mL, ultrasound time of 30 min, liquid to material ratio of 36:1, ultrasound temperature of 55 ℃, and alcohol to water ratio of 1:1. Under these experimental conditions, the total flavonoid extraction amount reached 12.65 mg/g. This method can effectively improve the extraction efficiency compared to ultrasound extraction and ultrasound-assisted semi-biomimetic methods. To fully utilize the flavonoids in Muxu, ultrasound assisted technology was applied to conduct single factor experiments on ethanol concentration, ultrasound treatment temperature, solid–liquid ratio, ultrasound treatment time, and extraction times that affect the total flavonoid extraction of Muxu. Based on this, a 4-factor 3-level orthogonal experiment was conducted to evaluate the interaction efficiency of each factor. The results showed that ethanol concentration, medium temperature, solid–liquid ratio, and ultrasound time all had a significant impact on the extraction of total flavonoids from Muxu. The main and secondary influencing factors are solid–liquid ratio > ethanol concentration > temperature > treatment time. Therefore, the optimal extraction process is determined as follows: 40 % ethanol, 40 ℃, 1:50 solid–liquid ratio, and ultrasonic treatment for 60 min; The actual extraction amount of flavonoids from Muxu using this process is 6.22 mg/g, which is close to the theoretical extraction amount of 6.49 mg/g. The ultrasonic-assisted extraction method is simple, reliable, and efficient. The ultrasound-assisted extraction was used to obtain total flavonoids from Muxu. The evaluation indicators were ABTS free radical scavenging and DPPH free radical scavenging, and the liquid to material ratios were 47.29:1 (mL/g) and 60.30:1 (mL/g), respectively. The extraction temperatures were 62.73 ℃ and 54.56 ℃, the extraction times were 51.62 min and 45.59 min, and the ethanol concentrations were 60 % and 46.67 %, respectively. The corresponding free radical scavenging rates were 87.38 % and 80.72 % [17]. The flavonoids were extracted from Muxu using ultrasound-assisted method, and obtained the optimal process as follows: liquid to material ratio of 1:30 (g/mL), ultrasound with 70 % ethanol at a power of 180 W for 30 min, and obtained a flavonoid content of 4.893 (mg/g) [18]. The preparation conditions were optimized using response surface methodology and obtained the optimal extraction process parameters as ethanol volume fraction of 65 %, ultrasound power of 100 MHz, extraction temperature of 67 ℃, and extraction time of 40 min. Under these process conditions, the yield of total flavonoids in Muxu leaves was 5.29 mg/g [19]. Ultrasound combined with enzymatic method was used to extract total flavonoids from Muxu. The extraction process was optimized using response surface methodology, and the in vitro antioxidant activity of the extract was studied. The results showed that the optimal process for extracting total flavonoids from Muxu by ultrasound combined with enzymatic method was as follows: liquid to material ratio of 40:1, ultrasound time of 55 min, and ultrasound temperature of 60 ℃. Under these conditions, the extraction rate of total flavonoids from Muxu reached 9 % 928 mg/g, with a relative error of 0 40 %, compared to other traditional methods, the extraction amount is greatly increased; In addition, the total flavonoids of Muxu have good ability to scavenge DPPH and • OH free radicals, with IC₅₀ values of 14.31 μ g/mL and 28.23 μg/mL. In order to further optimize the extraction process of flavonoids from Muxu, Qingda No.1 Muxu flowering period green hay powder was selected as the experimental material. The experiment used ultrasound assisted extraction method to extract flavonoids from Muxu. Firstly, a single factor experiment was designed to investigate the effects of four single factors, namely solid–liquid ratio, ultrasound temperature, ultrasound time, and enzymatic hydrolysis time, on the extraction rate of Muxu. The results showed that the four single factor treatments had significant effects on the extraction rate of flavonoids from Muxu (P < 0 05). Based on the results of the single factor experiment, a three-level orthogonal experiment with four factors (material to liquid ratio, ultrasonic temperature, ultrasonic time, enzymatic hydrolysis time) was conducted. The results showed that the factors affecting the extraction rate of flavonoids, in descending order, were the solid–liquid ratio, ultrasound time, enzymatic hydrolysis time, and ultrasound temperature. The optimal extraction conditions obtained through comprehensive analysis are a solid–liquid ratio of 1:30, ultrasound time of 30 min, ultrasound temperature of 50 ℃, and enzymatic hydrolysis time of 1 h. Ultrasound assisted methods can improve the extraction rate of flavonoids from Muxu.

3.5. Ultrasound-assisted extraction of Muxu leaf protein

Leaf protein is a concentrated protein that is widely present in the stems and leaves of plants. It has a balanced nutrition of various amino acids, low total energy, relatively high content of carotenoids and lutein, easy digestion, and contains carbohydrates, fats, crude fibers, and other substances. It has 18 types of amino acids, comprehensive nutrition, anti-aging, lipid-lowering, and immune enhancing functions, effectively preventing various diseases [20]. The ultrasound-assisted extraction was used to extract Muxu leaf protein under alkaline conditions. The orthogonal optimization extraction process was as follows: extraction at pH 8.5, preparation at 40 ℃ for 30 min, and the crude extraction rate of leaf protein was 51.09 %. Compared with the alkaline extraction method without ultrasound, the extraction rate was significantly improved and the extraction time was shortened [21].

3.6. Ultrasound-assisted extraction of total saponins from Muxu

Saponins are a special type of glycoside substance in plants, also known as saponins, which can be added to traditional Chinese medicine for medicinal use. They have the effects of improving anemia, regulating endocrine function, anti-aging, anti-inflammatory, and accelerating metabolism in the body [22]. The ultrasonic method was used to extract Muxu saponins, and the effect of extraction conditions on the total saponin extraction of Muxu was explored through L9 (34) orthogonal experiment. The results showed that the effects of various experimental factors on the extraction of total saponins from Muxu were in the order of ultrasound time, material ratio, ultrasound power, and ultrasound temperature; The optimal process conditions are A2B3C2D1, with a material ratio of 1:20, ultrasonic time of 30 min, ultrasonic power of 80 W, and ultrasonic temperature of 20 ℃. Under the optimal conditions mentioned above, the extraction rate of total saponins can reach 1.975 %. The enzymes were added to extract saponins from Muxu, and the saponin extraction solution was obtained with ultrasound assistance. Then, the extraction solution was extracted with chemical reagents. The best process effect was obtained under the conditions of a material to liquid ratio of 1:30 g/mL, ultrasound temperature of 20 ℃, ultrasound time of 20 min, and power of 360 W [23]. Using ultrasound power, ultrasound time, solid–liquid ratio, and ultrasound temperature as factors and Muxu saponin yield as response values, a regression equation was obtained through response surface analysis to study the main and interactive effects of these four factors on saponin yield, in order to obtain the optimal parameters for improving Muxu saponin yield and maximizing the utilization of raw materials. The optimal extraction process for Muxu saponins was determined through the arrangement and analysis of response surface center combination experiments, which involved ultrasound power of 403w, ultrasound time of 22 min, solid–liquid ratio of 1:51, and ultrasound temperature of 43 ℃. The maximum saponin yield was 4.53 %. The higher yield of saponins in similar studies has certain advantages.

In short, during the ultrasound assisted extraction process, the extraction temperature has a significant impact on the extraction efficiency. Appropriate temperature can improve the permeability and extraction efficiency of solvents, but too high or too low temperature can reduce the extraction effect. In general, ultrasound treatment can achieve good extraction results within 20–45 min. Compared to other influencing factors, ultrasound treatment time has no significant effect on extraction rate. The liquid to material ratio refers to the ratio of solvent to medicinal herbs, and an appropriate liquid to material ratio can improve extraction efficiency [24], [25], [26]. Generally, ethanol, acetone, and other solvents are used. When the liquid to material ratio reaches a certain value, the extraction effect is optimal.

4. Main value

4.1. Medicinal value

4.1.1. Active substance

Muxu saponins: Muxu saponins are unique bioactive substances extracted from Muxu, which are cyclic aldehydes formed by dehydration condensation of sugar hydroxyl groups or hydroxyl groups of non-sugar compounds with aldehyde chains (glycosides). Muxu plants contain nearly 60 species of saponins, all of which are oleanane type pentacyclictriterpenoids. The content of saponins in Muxu varies depending on the variety, location, growth period, and other factors. Although the proportion of total saponins in flowers and seeds is relatively high, their yields are small and low, and the total saponin content in Muxu leaves is not much different from that in seeds. Therefore, leaves are a better material for extracting Muxu saponins. The content of Muxu saponins is highest during the tender shoot stage and lowest during the yellowing stage. With the extension of the growth cycle, there is a gradually decreasing trend [27].

Flavonoids and isoflavones: Muxu contains over 80 types of flavonoids, including flavonoids and their glycosides, flavonols and their glycosides, isoflavones, isoflavones, chalcones, dihydroflavones, dihydroflavones, and rosewood. Chinese scholars isolated seven compounds from the chloroform and n-butanol extracts of Muxu plants, and identified their structures as apigenin 7-O − β − D-glucoside, luteolin-7-O − β − D-glucoside, uridine, β − methyl glucopyranose, apigenin, Muxu extract, and 7,4 '- dihydroxyflavone [28].

Coumarin: Muxu contains coumarin. In addition, Muxu also contains beneficial substances such as growth promoting factors and plant proteins for the human body [29].

4.1.2. Effects on the human body

Reducing cholesterol and blood lipid levels: Muxu diet can prevent the formation of hypercholesterolemia in experimental animals and also reduce their hypercholesterolemia. The effect of Muxu is believed to be mainly caused by its saponins. Research has shown that Muxu saponins can significantly reduce serum cholesterol in hypercholesterolemic rats and promote the clearance of low-density lipoprotein (LDL) by non-receptor pathways (mononuclear macrophage system). This suggests a meaningful approach for finding effective drugs to treat familial hypercholesterolemia (FH). Experimental results have shown that Muxu saponins can significantly convert cholesterol in rat liver into bile acids. The mice fed with Muxu saponins showed a significant increase in the net excretion of total and acidic sterols in their feces compared to the control group, indicating that Muxu saponins have the effect of blocking the enterohepatic circulation of bile acids, thereby promoting the endogenous excretion of cholesterol. Saponins can lower cholesterol levels in animal and human organs and plasma. The saponins in Muxu seeds can be used clinically to reduce cholesterol and triglycerides in human blood, and can also alleviate angina pectoris in coronary heart disease by improving blood circulation in coronary vessels [30].

Regression of atherosclerotic plaque: Muxu saponins can promote atherosclerotic plaque. It is known that NO plays a key role in maintaining vascular homeostasis. It can inhibit the formation of atherosclerosis by inhibiting the activation and adhesion of leukocytes, preventing platelet adhesion and aggregation, inhibiting the proliferation of smooth muscle cells and preventing the formation of foam cells. Muxu saponins can prevent the formation of atherosclerosis by promoting the release of NO from endothelial cells, thus proving the mechanism of Muxu saponins promoting the regression of atherosclerotic plaque to a certain extent [31].

Regulating immune, antioxidant, and anti-aging functions: Flavonoids can significantly improve the carbon clearance function of mouse mononuclear macrophages. Isoflavones extract can inhibit the decrease of thymus index and improve the phagocytic function of mononuclear macrophages. Isoflavones can dose dependently enhance ConA induced lymphocyte proliferation and NK cell activity in vitro, thereby exerting immunomodulatory effects. Flavonoids and isoflavone substances in Muxu can eliminate free radicals in the chain initiation stage during antioxidant reactions, as well as directly capture free radicals in the free radical reaction chain, blocking the free radical chain reaction through phenolic hydroxyl groups. Eating Muxu food year-round can supplement flavonoids, prevent cancer, prevent bone hyperplasia and prostatitis, reduce the incidence of cardiovascular diseases, and alleviate menopausal discomfort in women. Flavonoids and isoflavone substances have strong antioxidant functions, which can clear reactive oxygen species in the human body, protect lipids, proteins, and chromosomes from reactive oxygen species attacks, prevent cell damage, enhance immunity, and delay aging [32].

4.2. Feeding value

Crude protein of Muxu: Muxu is known for its high content of crude protein. The crude protein of Muxu contains a complete range of amino acids with a reasonable composition, including more than 20 types of amino acids, including all essential amino acids for humans and animals, as well as some rare amino acids such as citrulline and coumarin. The protein and amino acid ratio of Muxu is balanced, similar to the protein and amino acid composition in animal bodies, and has a high conversion potency [33].

Carbohydrates in Muxu: The carbohydrates in Muxu are mainly carbohydrates, starch, cellulose, hemicellulose, and lignin, which are important energy nutrients and account for a considerable proportion in animal diets. Muxu carbohydrates not only serve as the main energy source in the diet of ruminant and some non-ruminant animals, but also feeding animals a certain amount of purple Muxu can maintain normal gastrointestinal peristalsis, stimulate the development of the gastrointestinal tract and secretion of digestive fluids, reduce the pH of intestinal contents, change the gastrointestinal environment for the growth and development of pathogenic microorganisms, inhibit the growth and reproduction of pathogenic bacteria such as Escherichia coli, and significantly improve the biological activity of digestive enzymes in the gastrointestinal tract of animals [34].

Vitamins and Mineral Elements of Muxu: Muxu has a wide variety of vitamins and varieties, especially high contents of folate, chlorophyll, VK, biotin, VE, VB2, lutein, and carotene. Muxu also contains multiple mineral elements such as calcium, phosphorus, iron, magnesium, potassium, copper, and manganese. Muxu contains a large amount of carotenoids and lutein, which can improve the color of fish and livestock products and enhance their commercial performance. Adding Muxu extract to the feed of South American white shrimp improves its body color, and Muxu extract is superior to synthetic pigments in coloring effect. Muxu powder in feed can significantly improve the quality of eggs and the coloration of egg yolks. The mineral elements in Muxu can promote animal growth, improve the quality of animal products, and enhance animal immune function [35].

The application effect of Muxu in cattle and sheep: Adding Muxu hay significantly improves milk production, milk protein, and non-fat solids in cows, and significantly improves milk quality, but reduces milk fat percentage. From the perspective of economic benefits in dairy farming, adding Muxu hay to the diet can significantly improve the overall efficiency of dairy farming. Muxu can replace some of the concentrated feed in dairy cows' diets, and can also be used as high-quality roughage to optimize the composition of the diet, and can increase milk production and economic benefits of dairy cows to varying degrees [36].

The application effect of Muxu in pigs: Using Muxu powder instead of soybean meal in the diet of fattening pigs can increase their feed intake and lean meat percentage, reduce feeding costs, and significantly increase breeding efficiency. Adding a certain amount of Muxu powder to the basic diet of pigs can not only reduce feeding costs and increase breeding economic benefits, but also promote the growth and development of fattening pigs and improve the reproductive performance of sows [37].

At different experimental periods, the average feed intake, average body weight, and daily weight gain of the groups treated with 200 mg/kg and 500 mg/kg water-soluble Muxu polysaccharides added to the diet were significantly higher than those of the control group, while the feed to gain ratio was significantly lower than that of the control group. Muxu polysaccharides promote feed conversion efficiency and enhance the digestion and absorption of nutrients. Adding 1 % Muxu polysaccharide to the feed can significantly increase the daily weight gain and body weight of broiler chickens (P < 0.05), and significantly reduce the feed to weight ratio (P < 0.05). An experiment was conducted using Avion broiler chickens to investigate the effect of Muxu polysaccharides on nutrient digestibility by adding 0 %, 0.5 %, 1.0 %, and 2.0 % of Muxu polysaccharides to the basic feed. The results showed that the digestibility of crude protein in the 1 % Muxu polysaccharide group was higher than that in the non-added group, while the 2 % added group was lower than the non-added group, indicating that the 1 % added group had the best effect. However, excessive addition of Muxu polysaccharides can lead to anti-nutritional conditions and hinder the growth of the body. The appropriate addition of Muxu polysaccharides can promote the growth of the body, facilitate the digestion and utilization of nutrients in the diet, improve feed conversion efficiency, and thus promote the growth of broiler chickens [38].

The addition of Muxu flavonoid extract to the diet increased the average daily feed intake, body weight, and daily weight gain of Chongren Ma chicken chicks, and reached a significant level from day 15 to day 21; Reduced feed to weight ratio and improved feed conversion rate; Reduce the utilization rate of crude fat and improve the utilization rate of calcium. Adding an appropriate amount of Muxu flavonoid extract to the diet of chicks can promote their growth, regulate fat metabolism, improve carcass quality, and increase feed utilization efficiency. In addition, adding 300 mg/kg Muxu flavonoids to the diet of Ma chickens can reduce the levels of urea nitrogen, uric acid, triglycerides, cholesterol, and low-density lipoprotein cholesterol in their serum. Increase the content of high-density lipoprotein cholesterol. Reduce the fat content of chest and leg muscles, and lower the abdominal fat percentage. Adding a certain level of Muxu flavonoids to the diet of broiler chickens can increase the metabolic rate of nutrients in the body, reduce cholesterol and abdominal fat, and improve carcass quality [39].

Adding 60 mg/kg Muxu saponins to the diet of laying hens can significantly reduce the cholesterol content in egg yolks and whole eggs (P < 0.05), while adding 90 mg/kg Muxu saponins significantly reduces the cholesterol content in egg yolks and whole eggs (P < 0.01). Compared with the control group, the levels of triglycerides and low-density lipoprotein were significantly reduced at 60 mg/kg and 90 mg/kg (P < 0.05). Moderate Muxu saponins can regulate lipid metabolism in laying hens and reduce cholesterol levels in eggs. Muxu saponins have no significant effect on the growth performance of broiler chickens, but when added at a concentration of 0.12 %, they have an inhibitory effect on the growth of broiler chickens. But Muxu saponins can effectively improve the quality and flavor of broiler chickens, and increase their commercial value. Adding Muxu saponins can enhance the body's immune function, increase the activity of superoxide dismutase in its chest and leg muscles, reduce the content of malondialdehyde, and regulate antioxidant mechanisms. Muxu saponins can effectively regulate the balance of lipid metabolism in broiler chickens, reduce the synthesis of liver cholesterol, and affect the enterohepatic circulation of bile acids; Adding 0.08 % Muxu saponins to the diet of broiler chickens has the best effect. Muxu saponins can improve the quality of broiler chickens from multiple aspects and enhance the utilization value of Muxu saponins in broiler feed. The effects of Muxu total glycosides on the growth performance, blood lipids, and carcass quality of broiler chickens were studied. The results showed that adding Muxu total saponins (0.1 %, 0.2 %) to broiler feed significantly reduced the levels of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) in broiler serum (P < 0.05 or P < 0.01), and significantly increased the level of high-density lipoprotein cholesterol (HDL-C) (P < 0.05 or P < 0.01), while no significant difference was observed between the two levels of addition (P > 0.05); The addition of Muxu total glycosides significantly reduced the abdominal fat percentage of broiler chickens (P < 0.05), decreased lipid deposition, and increased slaughter rate, chest muscle rate, and leg muscle rate of broiler chickens [40], [41].

4.3. Ecological value

Purple Muxu has lush branches and leaves, and can quickly cover the ground when planted in large areas. Especially, purple Muxu has dense, small, and easily wet leaves, which have a high water holding capacity and can effectively intercept precipitation and reduce surface runoff. Not only that, the root system of Zishoukai is also very developed. Root nitrogen fixation can increase the content of soil organic matter. A large number of lateral branches crisscross to form a strong root network and its nitrogen fixation effect, which not only facilitates the formation of soil aggregate structure, but also improves the physical and chemical properties of soil, enhances soil water holding and permeability, and thus plays a role in soil and water conservation. In addition, purple headed Kai has strong adaptability and can be planted in a wide range. Despite multiple harvests or damage during the growth period, it can still grow vigorously and has strong regeneration ability, making it an excellent soil and water conservation plant in mountainous areas [42].

4.4. Edibility

The tender seedlings or stems and leaves were washed, they were blanched in boiling water, removed, rinsed several times with water, drained, chopped into pieces for cold mixing, stirred fry, made filling or steamed with flour [43].

4.5. Ornamental value

As an excellent green manure and feed plant, it has a rich variety of colors and can be used as a ground cover or for slope greening in gardens, as well as for soil improvement [44].

Some important substances and their functions in Muxu were shown in Table 1.

Table 1.

Some important substances and their functions in Muxu.

Substance	Function	References
Protein	Containing high-quality protein, mostly essential amino acids, it is an important source of protein for vegetarians.	[33], [45], [46]
Cellulose	Rich in fiber, it helps promote normal digestive system function.	[34], [47], [48]
Vitamins and minerals	Rich in various vitamins and minerals such as calcium, iron, magnesium, manganese, folic acid, and vitamin K, these substances are very important for maintaining good health.	[35], [49], [50], [51]
Antioxidant	Containing antioxidants such as flavonoids and polyphenols, it can reduce the damage caused by free radicals.	[32], [52], [53]

5. Conclusion and prospect

Traditional mathematical statistical methods generally use orthogonal design to simultaneously consider multiple factors and seek the optimal combination of factors, but cannot find a clear functional expression between factors and response values in the given entire region, thus unable to find the optimal combination of factors and the optimal value of response values in the entire region. The response surface analysis method is a regression analysis method that studies the interaction between several experimental factors through central combination experiments. It has the advantages of fewer experimental times, shorter cycles, and higher accuracy in obtaining regression equations. With the development of computer technology, this method has become a highly accurate, widely used, and practical technology. Response surface methodology has become an effective method for reducing development costs, optimizing processing conditions, improving product quality, and solving practical problems in the production process. It has been widely applied in fields such as agriculture, biology, food, chemistry, and manufacturing.

The roots, stems, and leaves of Muxu are rich in nutrients, mainly including active polysaccharides, peptides, flavonoids, saponins, coumarins, alkaloids, and other compounds. In recent years, there has been an increasing amount of research on Muxu leaf protein. So, Muxu is nutritionally comprehensive. The ultrasound assisted extraction method can significantly improve the extraction efficiency of active substances from Muxu, showing great advantages and providing higher development value for the application of Muxu. As a good natural plant resource, Muxu has attracted much attention. We believe that with the continuous deepening of research, the active substances in Muxu will have broader application prospects in areas such as food, health, and the environment.

CRediT authorship contribution statement

Huilin Wang and Laiqing Deng wrote the manuscript. Gangliang Huang reviewed & edited the manuscript.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.