Corn stigma, as a kind of traditional Chinese medicine, is the dry style and stigma of the gramineous plant maize, and it is widely cultivated in China, the United States, France, Turkey, and other countries (Oyabambi et al., 2021). Corn stigma is rich in flavonoids, terpenes, polysaccharides, and other active ingredients (Zhou, Song, & Wang, 2013; Zhou, Zhang, Chen, & Hou, 2012). In addition, corn stigma can be used as tea beverage and diuretic additive. Accumulating evidences have confirmed that corn stigma polysaccharides (CSPs) showed various biological activities including immunomodulatory, anti-inflammatory, antibacterial, antihypertensive, hypoglycemic, and other activities (Guo et al., 2019; Yuan, Xue, & Yang, 2019). Hence, CSPs have been widely used in fields such as food, medicine, cosmetics, etc. Notably, the extraction and purification of CSPs are the main prerequisite for their development and utilization. Therefore, it is particularly important to find efficient method to extract and purify CSPs.

At present, traditional and advanced extraction methods have applied to extract natural polysaccharides. Hot water extraction is one of the most commonly used methods for extracting polysaccharides from natural materials due to its some merits including no special equipment and simple operation (Yun, Li, Yang, & Zhang, 2019). Nevertheless, this method has the disadvantages including large solvent consumption, long time-consuming, and low extraction efficiency (Romdhane et al., 2017). Enzyme assisted extraction is a method suitable for small-scale extraction of high-purity polysaccharides. This method needs to strictly control the extraction temperature to avoid enzyme inactivation caused by high temperature (Park & Lee, 2021). Microwave-assisted extraction, as a potential extraction method, shows some merits including of high extraction efficiency and environmental protection (Golbargi, Gharibzahedi, Zoghi, Mohammadi, & Hashemifesharaki, 2021). However, microwave radiation causes local high temperature of the extract, which limits the application of microwave in the field of extraction (Baltacoglu, Baltacoglu, Okur, Tanrvermi, & Yal, 2021). Ultrasound assisted extraction (UAE) is a potential extraction method that has lower solvent consumption and extraction cost, higher extraction efficiency and recovery compared with the traditional solvent extraction method (Wang et al., 2021). Ultrasound can produce cavitation and mechanical thermal effects in the extraction solvent, accelerate the rupture of plant cell wall, make the target component in the cell diffuse into the solvent faster, and then improve the yield of the target component (Chen, Wang, & Kan, 2021). Hence, UAE is widely used to extract biologically active ingredients from natural plant resources. However, our analysis found that the above all extraction methods used a single extraction solvent. The yield of the active ingredients obtained by single-phase solvent extraction is relatively low. Moreover, the active components obtained through the above extraction method contain a large amount of impurities, which increases the cost of subsequent separation and purification. With the continuous development of extraction technology, aqueous two-phase extraction (ATPE) has attracted more and more attention in the field of extraction, which is attributed to its high extraction efficiency, low cost, energy conservation, and little damage to the activity of target components (Jiang, Wang, Zhu, Wu, & Tian, 2021; Mokgehle, Madala, Gitari, & Tavengwa, 2022). Aqueous two-phase extraction system (ATPS), as a new extraction solvent, can extract and separate different kinds of natural compounds, and the extraction and separation process are completed in one step (Leong, Yang, & Chang, 2020). In particular, short chain alkanol solution is widely used in the extraction of active components. Ultrasound assisted aqueous two-phase extraction (UATPE) integrating UAE with ATPE is a novel combination for simultaneous extraction of different polysaccharides from two phases, showing the some merits of UAE and ATPE (Ayoub Khan, Fuzail Siddiqui, & Alam Khan, 2019; Mahindrakar & Rathod, 2020; Zhou et al., 2021). In addition, UATPE can also complete the extraction and purification of target active components in one step. To data, there are few reports on the study of UATPE polysaccharides from corn stigma.

Some experimental parameters will directly affect the yield of the target compound. Hence, optimizing the extraction process is necessary to achieve the maximum yield of CSPs. A large number of tests need to be carried out to obtain the desired results, which is time-consuming and cumbersome. This problem can be overcome by appropriate mathematical models, which can predict the relationship between various input variables and output variables. RSM and ANN are the most commonly used methods for model construction (Abdullah, Pradhan, Pradhan, & Mishra, 2020). RSM presents more accurate results than orthogonal optimization method when the delta function is a quadratic function (Mza et al., 2020). ANN has strong prediction ability owing to its high learning ability and can be applied to various types of non-linear problems. By comparing the R2, MSE, RMSE, and ADD between ANN and RSM model, it is found that the trained ANN showed stronger advantages of nonlinear fitting than RSM model (Dash & Das, 2021). Genetic algorithm (GA) is a computational model, which mainly searches for the optimal solution by simulating Darwin's biological evolution theory. The GA combined with ANN (GA-ANN) method is an advanced optimization method. Hence, the extraction process of different kinds of bioactive components was optimized by GA-ANN method (Desta, Sinha, Ramulu, & Singh, 2021; Ghosh, Mandal, & Mondal, 2017; Kalathingal, Basak, & Mitra, 2019). Nevertheless, the optimization process of UATPE of polysaccharides from corn stigma by GA-ANN is rarely reported. Moreover, there are limited research reports on the CSPs purification and immunomodulatory activity of the purified CSPs fraction.

In this study, the objective is to: 1) establish RSM and ANN models for predicting CSPs yield; 2) optimize the extraction parameters of UATPE polysaccharides from corn stigma by GA-ANN; 3) purify the crude CSPs by column chromatography and characterize its structure through UV–vis, fourier-transform infrared spectroscopy (FT–IR), gas chromatography–mass spectrometry (GC–MS), SEM, AFM, etc.; 4) evaluate its immunomodulatory activity by RAW264.7 cells.