Finally, we quantified the UA levels in undiluted saliva samples of healthy controls (n = 20) and gout patients (n = 8). The levels were correlated with those measured with conventional salivary UA enzymatic assays as well as serum UA levels. The UOx-paper-based electrochemical UA sensor is a user-friendly and convenient tool to assess salivary UA levels.In this study, we introduce a uricase-immobilized paper (UOx-paper) integrated electrochemical sensor for detection of uric acid (UA) in saliva. The UOx was immobilized on the detection zone in the wax-patterned paper substrate. This UOx-paper was integrated with a Prussian blue-modified, screen-printed carbon electrode after electropolymerization of o-phenylenediamine to construct an electrochemical cell for small-volume (20 μL) of samples. First, we optimized the fabrication conditions of UOx-paper. Next, the amperometric response of the UOx-paper-based electrochemical UA sensor was analyzed using a known concentration of UA standard solution in artificial saliva at an applied potential of − 0.1 V (versus Ag pseudo-reference electrode). The UOx-paper based electrochemical UA sensor showed a sensitivity of 4.9 μA·mM UA is an antioxidant and hypouricemia has been reported to be associated with immune-mediated or degenerative neurological diseases such as multiple sclerosis, Parkinson’s disease, or Alzheimer’s disease3. Recurrent aphthous stomatitis and oral lichen planus were also associated with low levels of salivary UA5. However, hyperuricemia contributes to the development and progression of gout, metabolic syndrome, chronic kidney disease or cardiovascular diseases3. Although hyperuricemia does not always induce gout and the diagnosis of gout is not based on hyperuricemia alone, gout develops in subjects with hyperuricemia leading to the deposition of monosodium urate crystals in tissues. Recent guidelines for the management of gout recommend that urate-lowering therapy should be optimized to achieve and maintain a serum UA level < 5–6 mg/dL, but not < 3 mg/dL7. Therefore, monitoring of serum UA is indispensable for the diagnosis, treatment, and follow-up of hyperuricemia and gout. However, venupunture is invasive and can cause complications including injuries and bleeding. Further, analysis of serum UA requires a laboratory setting using specialized equipment.Uric acid (UA), the final product of purine metabolism in the human body, plays an important role in a variety of physiologic and pathologic conditions, including gout9. UA is mainly produced in the liver and intestines. Most UA is eliminated via kidneys and intestines through urate transporters. However, organic anion and urate transporters are also expressed in the salivary glands10. Additionally, several clinical studies reported a linear relationship between serum and salivary UA levels in most cases, suggesting the potential of salivary UA determination as an alternative for blood tests12. Various analytical methods including high-performance liquid chromatography (HPLC)13, capillary electrophoresis (CE)14, and enzymatic colorimetric assay kits18 have been developed for the detection of salivary UA. However, they are not suitable for daily personal use because they are usually time-consuming and requiring expensive instruments and experts. Although some commercially available enzymatic assay kits can be used for various types of biological matrices such saliva, serum, and urine2, they can be influenced by other interferences such as vitamin C, lipid, or endogenous peroxidase that are present in biological samples, and eventually cause false results22. Besides, they have a short use-by date because peroxidase within reagents is incompatible with preservatives such as sodium azide23. As a result, an overdue reagent may result in a decrease of their sensitivity24.Salivary diagnostics have attracted increased attention in the fields that utilize point-of-care testing (POCT) and in clinical applications for monitoring diseases frequently and easily, in addition to predicting post-treatment outcomes because saliva reflects the physiological and pathological status of the body27. Kim et al. reported a wearable salivary UA mouthguard biosensor using uricase-modified screen-printed electrode (SPE) system with integrated wireless electronics28. Although this mouthguard biosensor might be able to monitor salivary UA level in real-time continuously, its use is limited due to discomfort and biocompatibility issue of electronics. Huang et al. reported a paper-based electroanalytical device for salivary UA analysis29. They fabricated poly –graphene oxide composites on indium tin oxide (ITO) substrates as the working electrodes and covered the prepared electrode with a piece of paper to construct a thin-layer electrochemical cell. Although this device showed high sensitivity, it is inconvenient to use because it requires separate reference and counter electrodes. Therefore, an easy, rapid, cost-effective and sensitive method is needed for the analysis of salivary UA for POCT of UA-associated diseases.Electrochemical sensors received significant attention due to practical advantages, including high sensitivity, rapid response time, portability, low cost, and ease of operation for detecting salivary UAIn this study, we fabricated a facile and effective electrochemical UA sensor based on a uricase (UOx)-immobilized paper (UOx-paper) and a Prussian blue (PrB)-modified, screen-printed carbon electrode (PrB-SPCE). To improve the selectivity and anti-biofouling of the electrode, o-phenylenediamine (o-PD) was electropolymerized on the PrB-SPCE (PPD/PrB-SPCE). The UOx-paper using a wax-patterned lens cleaning tissue with a single circle was integrated with the PPD/PrB-SPCE to construct an electrochemical cell for small-volume samples. First, we optimized the fabrication conditions for UOx immobilization on the paper. Next, we evaluated the analytical performance of the UOx-paper integrated electrochemical UA sensor, based on sensitivity, selectivity, reproducibility, and stability, in artificial saliva. Finally, the UOx-paper integrated electrochemical UA sensor was evaluated by determining the concentration of UA in undiluted saliva samples obtained from non-gout controls (n = 20) and patients with gout (n = 8), comparing serum UA levels and those measured with conventional Salimetrics® salivary UA enzymatic assay kits. The UOx-paper integrated electrochemical sensor for detection of salivary UA is schematically represented in Fig. Candida sp.), o-PD, sodium sulfate (Na2SO4), sodium chloride (NaCl), calcium chloride (CaCl2), potassium chloride (KCl), citric acid, potassium thiocyanate (KSCN), ammonium chloride (NH4Cl), potassium phosphate monobasic (KH2PO4), potassium phosphate dibasic (K2HPO4), L-lactic acid (LA), D-glucose (Glu), L-ascorbic acid (AA), acetaminophen (AP), bovine serum albumin (BSA) and glutaraldehyde (GA) solution were purchased from Sigma Aldrich . Whatman lens cleaning tissue (grade 105) was ordered from GE Healthcare Life Sciences . All reagents were of analytical grade and used without further purification. All aqueous solutions were freshly prepared using deionized water (DW) with 18.2 MΩ· cm resistivity.UA (≥ 99.0%), UOx  with AutoCAD software  for pattern design and a BF-150C drying oven  for wax impregnation. All electrochemical experiments, including cyclic voltammetry and chronoamperometry (CA), were carried out with a Compactstat  at room temperature (RT). PrB-SPCE containing a PrB-modified carbon working electrode (4 mm in diameter), a carbon counter electrode and an Ag pseudo-reference electrode was purchased from DropSens .The lens cleaning tissue with hydrophobic wax barrier measured 11 mm in width and 15 mm in length for one electrode. The hydrophilic zone was 8 mm in diameter  were mixed with 2 mg of BSA and 1 μL of 8% glutaraldehyde solution in 200 μL of potassium phosphate buffer , as shown in Fig. 28. Next, a section of double-sided adhesive tape with a punched hole (8 mm in diameter) was attached to the electrode. Finally, the tape was covered with a UOx-immobilized paper to store the sample solution as well as electrically connect the three-electrode system for electrochemical detection.Prior to fabrication of the UA sensor, we confirmed the electrochemical properties of PrB-SPCE via cyclic voltammetry in KCl solution with a potential range from − 0.1 to 0.4 V (vs. Ag pseudo-reference electrode) and a scan rate of 50 mV/s. Next, poly(o-PD) (PPD) was deposited on the PrB-SPCE by polymerization of o-PD at 0.7 V (vs. Ag pseudo-reference electrode) for 100 s in 0.1 M PB (pH 7.0) solution containing 10 mM o-PD and 5 mM sodium sulfate to minimize biofouling and interference from saliva constituents2, 15 mM KCl, 1 mM citric acid, 1.1 mM KSCN, and 4 mM NH4Cl in DW28. The pH of artificial saliva was adjusted to 6.7. Various concentrations of UA were detected using the CA technique at an applied potential of − 0.1 V (vs. Ag pseudo-reference electrode) for 60 s after 1 min of incubation in the standard solution. The calibration curve was obtained from CA measurements in 20 μL of UA at a concentration range of 50 to 1000 μM in artificial saliva.The CA technique was used to evaluate the UA detection sensitivity of the UOx-paper/PPD/PrB-SPCE. A standard solution of UA was prepared in artificial saliva consisting of 5 mM NaCl, 1 mM CaClg for 15 min at RT) to obtain serum. The whole saliva samples were centrifuged at 4500×g for 10 min at 4 °C. All samples were frozen at − 80 °C until analysis. Serum UA levels were determined using commercially available enzymatic colorimetric assay kits  according to the manufacturer's instruction. For salivary UA analysis, 20 μL of undiluted saliva sample was first dropped on the UOx-paper/PPD/PrB-SPCE. After incubation for 60 s, CA measurement was performed at − 0.1 V (vs. Ag pseudo-reference electrode) for 60 s. The UA concentration in saliva sample was calculated from the slope of the calibration curve. Additionally, salivary UA levels were measured using a salivary UA enzymatic assay kit  according to the manufacturer's instruction.This study and sample collection were approved by the Ethics Committee of Seoul National University Bundang Hospital (IRB No. B-1911-577-303) and written informed consent was obtained from all participants. We enrolled 8 male patients with gout (age (mean ± standard error of mean (SEM)), 39.1 ± 3.0 years) and 20 non-gout males (age 40.8 ± 6.4 years). Serum and saliva samples were simultaneously collected under fasting conditions. Additionally, we obtained 16 paired-samples from patients with gout before and after starting urate-lowering therapy (febuxostat (n = 4), allopurinol (n = 3), or benzbromarone (n = 1)). The duration of treatment was 2.9 ± 1.1 months. Unstimulated whole saliva samples were collected by passive drooling into a plastic tube. Venous blood samples were centrifuged .Continuous variables are presented as mean ± SEM. Independent sample t-test was used for comparison of the two groups. Paired t-test was used to compare UA levels before and after urate lowering therapy. One-way ANOVA was used to compare UA levels across 3 groups and Fisher’s exact test was performed to analyze categorical data. Pearson’s correlation coefficients were calculated to analyze the relationship between serum and salivary UA levels or between salivary UA levels measured by the prepared UA sensor and conventional method. A 2O2) product, it is subject to various electroactive interferences. PrB or ferric hexacyanoferrate has been referred to as an “artificial peroxidase”, because it can enhance electron transport and catalyze the reduction of H2O2 at low overpotential31. Therefore, PrB-SPCE provides selective cathodic detection of H2O2 produced by the enzymatic reaction of UA. However, the PrB may decompose in neutral or weakly alkaline solutions32. Besides, saliva is a complex and difficult matrix to manage due to its high viscosity and protein assembly, as well as other electroactive species33. To improve the stability, selectivity and biocompatibility of PrB-SPCE, we introduced an external protective polymer membrane such as PPD on PrB-SPCE via electropolymerization. PPD membrane is known for its ability to penetrate low-molecular-weight compounds such as H2O2 and reject other electroactive species such as AA and AP, as well as prevent biofouling on the electrode34. To characterize the electrocatalytic property of PPD/ PrB-SPCE toward H2O2, we performed cyclic voltammetry measurements in artificial saliva with and without 1 mM H2O2 in the potential range of − 0.20 to + 0.40 V (vs. Ag pseudo-reference electrode) at a scan rate of 50 mV/sec. As shown in Fig. 2O2 solution. To confirm the electrochemical performance of PPD/PrB-SPCE toward H2O2, the CA technique was used at an applied potential of − 0.1 V (vs. Ag pseudo-reference electrode). The selectivity of PPD/PrB-SPCE was confirmed by the current response of H2O2 in the presence of physiological levels of the relevant electroactive constituents of saliva including UA, AA, and AP. As shown in Fig. 2O2 . In particular, the current responses of AA  and AP (− 0.06 ± 0.002 μA) on PPD/PrB-SPCE were significantly lower than those on PrB-SPCE . However, the cathodic current of H2O2 (− 0.35 μA ± 0.01 μA) on PPD/PrB-SPCE and PrB-SPCE (− 0.38 ± 0.01 μA) was similar. This result indicates that PPD/PrB-SPCE has high selectivity for the detection of H2O2 without any interference effect by possible electroactive species in saliva. In addition, the external PPD membrane did not inhibit the permeability of H2O2 toward PrB-SPCE.Typically, UA biosensors have utilized enzymes such as UOx with high specificity for UA. However, as UOx-based amperometric detection of UA generally requires a relatively high potential (> + 0.65 V) to measure the hydrogen peroxide HO2 produc35. In addition, paper can be easily designed according to the desired pattern by wax printing with a non-toxic reagent. To use paper as a bioanalytical tool for UA detection, we designed a single zone that was integrated to the three-electrode system of SPCE. Next, we immobilized UOx within the hydrophilic zone of wax-patterned lens paper for use as a sample absorption area as well as electrochemical cell to facilitate the reaction between UOx and UA within samples. Therefore, it was necessary to optimize the experimental conditions, including paper type, UOx concentration, and volume of UOx mixture for efficient immobilization of UOx on the paper.Paper-based analytical platforms have several advantages including low cost, simple fabrication, and are easy to use in many applications such as biochemical and analytical sensors36. Therefore, appropriate paper selection appears to be critical to improve the current signal of paper-based biosensors. The effect of paper type was investigated according to the cathodic current response of UOx-paper/PPD/PrB-SPCE on 1000 μM UA at − 0.1 V (vs. Ag pseudo-reference electrode). As shown in Fig. First, we optimized the types of paper including Whatman No. 1, No. 114, and lens cleaning tissues No. 105 for immobilization of UOx. As the pore size and thickness differ with the type of paper Table , the res38. But when enzymes are directly cross-linked to GA, they tend to lose activity. Therefore, mild cross-linking with GA using a feeder rich in amino groups such as BSA can increase the stability of enzyme by reducing the chemical modification of internal amino groups of the enzyme39. Besides, it can reduce the porosity of the film and, thus, increase the responsiveness of the film40. As a result, the UOx-paper prepared using GA and BSA as a crosslinker and stabilizer generated a higher current (− 5.04 ± 0.35 μA) than the paper prepared using only UOx (− 4.03 ± 0.14 μA)  Fig. f. Inset 2 = 0.998), with a detection limit of 13.3 μM according to the standard deviation of the blank and the slope method (3 sbl/slope)42 and a detection sensitivity of 5.0 μA·mM−1 (39.8 μA·mM−1·cm−2). However, the current response of UOx-membrane/PPD/PrB-SPCE, in which UOx was directly immobilized on PPD/PrB-SPCE using GA and BSA, was smaller than that of UOx-paper/PPD/PrB-SPCE. As a result, the slope of the calibration curve of the UOx-paper/PPD/PrB-SPCE was about threefold higher than that of UOx-membrane/PPD/PrB-SPCE (1.7 μA·mM−1). Table We investigated the analytical performance of UOx-paper/PPD/PrB-SPCE for UA detection using the CA technique. The current responses of UOx-paper/PPD/PrB-SPCE in 20 μL of artificial saliva solutions containing various concentrations of UA 01000 μM) were measured at an applied potential of − 0.1 V (vs. Ag pseudo-reference electrode). As shown in Fig. 000 μM we43. PPD is known for having anti-biofouling property in proteinaceous media, possibly due to its greater compactness45. We tested an anti-biofouling ability of UOx-paper/PPD/PrB-SPCE using artificial saliva and real saliva samples. As shown in Fig. To evaluate the selectivity of the UOx-paper/PPD/PrB-SPCE, we compared the current responses of 500 μM UA with that of other potential interfering substances, including 800 μM Glu, 200 μM AA, 100 μM AP and 1000 μM LA. As shown in Fig. In addition, we investigated the reproducibility of UOx-paper/PPD/PrB-SPCE by measuring the current response to UA (500 μM) using 10 sensors on different fabrication dates. As the coefficient of variation (CV) of our UA sensor was 4.8% Fig. c, the UO48. However, its routine use is limited by further dilution of analytes, interference by food, or periodontal or salivary gland health status. In particular, saliva is a difficult and complex matrix for biological analysis because many components in saliva can affect the response of the analyte of interest49. Therefore, a highly sensitive and selective method is required for detection of UA in saliva.Saliva is a biological fluid that is used in clinical diagnosis and management of patients, because of its many advantages, including non-invasive collection, easy-to-use sample, and inexpensive storageTo evaluate the feasibility of our UA sensor for POCT, we measured salivary UA concentrations of non-gout controls (n = 20) and patients with gout (n = 8). In addition, in case of patients with gout, we determined salivary UA levels before and after urate-lowering therapy. The results were compared with those of commercial salivary UA enzymatic assays , together with serum UA levels as reference values.p = 8.77 × 10−10; Fig. p = 1.59 × 10−10). Urate-lowering therapy decreased serum UA in 3/8 (37.5%) gout patients to concentrations lower than 6.0 mg/dL.First, serum UA levels were significantly higher in patients with gout  than in non-gout controls , particularly high in the low concentration range below 2 mg/dL by UA sensor  nm. Although this enzymatic assay is simple and commercially available, the chromogen formation by peroxidase-catalyzed reaction of H2O2 is susceptible to interference from endogenous confounding components in biological matrixes, and eventually may cause false positive or negative results22. The Salimetrics® assay requires centrifugation to remove mucins and other interferents from saliva samples, but it cannot perfectly eliminate multiple compounds that can act as interferers. Second, we evaluated inter-day precision using UA standard (5 mg/dL) and controls (High & Low) in the same kit according to the manufacturer’s instructions. As shown in Table 50. On contrary, the CV of UA sensor was all less than 5% at low concentration range from 1.7 to 5 mg/dL  as shown in Fig. Urate lowering therapy significantly reduced serum UA levels in the gout group from 10.23 ± 0.36 mg/dL to 7.84 ± 0.84 mg/dL p = 0.01 as shownIn summary, we introduced a simple and reliable electrochemical UA sensor using UOx-paper integrated with PPD/PrB-SPCE. The UOx-paper acts as the sample absorption area for reaction between UOx and UA, as well as serving as a filter to prevent interference within saliva. PPD improves the selectivity and anti-biofouling of the electrode. As a result, the prepared UOx-paper/PPD/PrB-SPCE showed robust sensitivity for UA, with a wide linear range, high selectivity, and good reproducibility, and requires low sample volume. Using this UA sensor, we measured UA levels in undiluted, unstimulated saliva, subsequently comparing serum UA levels with those based on conventional UA assays . Although salivary UA levels determined by Salimetrics® assays were significantly higher than those measured with our UA sensor, they were significantly correlated with serum UA levels. Additionally, our UA sensor showed that changes in salivary UA level reflect changes in serum UA levels more closely than the Salimetrics® assay. Therefore, we expect that the prepared UA sensor, UOx-paper/PPD/PrB-SPCE, will be utilized in POCT to evaluate salivary UA levels in subjects with UA-associated diseases.Supplementary Information.


Declining total fertility rates pose a severe challenge to the economy, society, culture, and politics of any region. Low fertility rates among China’s rural floating population with strong fertility are aggravating these challenges. Previous research has confirmed the relationships between health insurance and fertility intention. However, it is still unclear whether the existing association is favorable or not. Moreover, the majority of existing studies in China employ data from either urban or rural populations, whereas evidence from rural floating populations remains scarce. Based on the “China Migrants Dynamic Survey (CMDS)” in 2016, the current study used the logistic regression model to explore the impact of health insurance policy on the fertility intention of the rural floating population in China. Propensity Score Matching (PSM) was used to address potential selection bias. Three important findings were observed: Firstly, participating in the Basic Medical Insurance System (BMISUR) significantly improved rural floating populations’ fertility intentions in China. Secondly, the association between age and the fertility intention of the floating population was “inverted u-shaped” with the highest fertility intention among those aged 25 to 34. There was also a positive correlation between personal income and fertility intention, and it was found between local housing purchase, formal employment, the co-residents scale, and the fertility intention in the rural floating population in China. Interprovincial mobility was positively associated with the fertility intention among rural migrants. Thirdly, the impact of health insurance policies on the fertility intention of the rural migrant population varies by gender, age, and inflow areas. The aforementioned findings can guide the Chinese government in its efforts to improve the fertility intention of the rural floating population, reform the social security system with a focus on “targets”, and implement differentiated welfare policies aimed at promoting the equalization of basic public services, thereby contributing to China’s population structure and long-term development. Since the 1970s, the total fertility rate has been declining year by year and has consistently been significantly lower than the birth rate in China since 1990. The proportion is even lower for the floating population 0.94) than for their counterparts living in urban areas 1.28) and rural residents (1.01) 8 and rur. The rur than forAmong the diversified and multi-level social security types, the health insurance system has the widest coverage and the greatest impact in China. By the end of 2018, the basic medical insurance for urban and rural residents (BMISUR) covered 1.35 billion people . TherefoAt present, there are numerous reports on the fertility intention of Chinese residents, focusing on rural women of childbearing age, the urban population ,18,19, aIn addition to current economic costs, in terms of future-expected benefit, existing studies started from the perspective of social security. Moreover, the impact of social security, represented by the health insurance system, on fertility intention is a controversial topic. One viewpoint is that social security has a positive effect on fertility intention. Some studies have shown that social security relieves medical costs and economic burdens of families , therebyIn summary, current studies have not paid enough attention to the fertility intention of China’s rural floating population, and the research on the relationship between health insurance and the fertility intention of China’s rural floating population has not been explored. In addition, the endogeneity of the relationship between social security and fertility intention has also not been explored. The majority of the rural floating population are in the childbearing age stage, and their fertility intention not only directly affects the implementation effect of China’s fertility policy, but also indirectly affects the fertility intention and behavior of the rural childbearing population of the same age through peer effect, which has an important impact on China’s population structure and long-term sustainable development. In this regard, based on the needs and characteristics of the rural floating population in China, the current study used the data of the China Floating Population Dynamic Monitoring (CMDS) in 2016 to explore the main factors affecting the fertility intention of the rural floating population. It also discusses the relationship between health insurance policies and the fertility intention of the rural floating population. The authors have also used PSM to address the potential selection bias, which will aid in overcoming the possibility of biased findings presented by previous studies on the relationship between fertility intention and social security. In addition, the retrospective design of the current study enhances the reliability and persuasiveness of the research conclusions.The study used the 2016 China Migrants Dynamic Survey (CMDS), which is based on the data of the national floating population in 2015 by the National Health and Family Planning Commission, and obtained via the PPS sampling method of multiple layers and stages. The survey has a total sample size of around 200,600, covers 31 provinces , and Xinjiang Production and Construction Corps. Participants of the current study were the population of childbearing age at 15–49 years old, with rural household registration and non-local residents. A total of 29,437 eligible samples were obtained after excluding individuals who did not meet the criteria. In this study, fertility intention of the rural floating population of childbearing age was taken as the dependent variable. Referring to relevant research on reflecting fertility intention by examining the plans of individuals of childbearing age to have children in a specific period in the future ,44. The Health insurance policy was used as the independent variable in this study. As mentioned above, many local governments in China began to explore merging the NRCMS with the BMISUR into the BMISURR in 2015. The 2016 CMDS was investigated from 31 May 2016, just at the merging stage of NRCMS and BMISUR. Therefore, the current study refers to other scholars’ studies , and comIn addition to health insurance participation, the individual characteristics of the rural floating population of childbearing age include age, age square, sex, and education level. Economic characteristics were also included as control variables, namely personal income, housing property, and employment. Among them, the types of employment were categorized into formal and informal employment. Referring to Maloney’s classification criteria, non-signed labor/service contracts and informal employment relations were regarded as informal employment . In addiiY denotes the probability of the rural floating population’s fertility intention in China, and ix represents the status of the rural floating population participating in health insurance, im indicates a series of control variables, including demographic characteristics, social characteristics, and mobility characteristics, and iε is a random disturbance term. We focus on β1, which reflects the impact of health insurance policy on the fertility intention of the rural floating population. A positive value of β1 indicates that the participation of the rural floating population of childbearing age in a health insurance policy is likely to improve their fertility intention.We used “fertility intention” for the rural floating population as the dependent variable. Since the dependent variable is a binary categorical variable of 0–1, we used a Binary Logistic Regression model to evaluate the impact of health insurance policy on fertility intention of the rural floating population. A Binary Logistic Regression model is always set to analyze the relationship between some variables and fertility intention . The basParticipation in health insurance was included as an independent variable in this study. Due to the moral hazard in the health insurance market, that is, whether or not to participate in the insurance is a choice made based on personal reality and not controlled by human factors, it was impossible to randomly restrict to people who participate in health insurance or not. Therefore, whether the sample in the survey participated in the BMISURR or not has the problem of sample selection ,49, whicPSM analyses are usually divided into three steps. Firstly, propensity scores were estimated using a Binary Logistic Regression model. Based on the above literatures’ review, we set all the controlled variables as independent variables to ensure that the negligibility test is satisfied, and estimated a binary regression model with the fertility intention of the rural migrants as the dependent variable to predict the probability of the rural migrants to participate in health insurance, as shown in Equation (2).iD = 1; otherwise, iD = 0. If the rural floating population participated in the BMISURR, Secondly, propensity score matching was conducted. PSM takes the health insured group as the treatment group and constructs a group of samples with similar characteristics in all aspects, but with differences only in whether to participate in the insurance as the control group. By comparing the differences in fertility intention between the two groups, the size of the effect of health insurance participation and fertility intention of the rural floating population was determined. To ensure the robustness of the results, we adopted four matching methods, namely, nearest neighbor matching, radius matching, kernel matching, and spline matching, and conducted relevant tests .Finally, the average treatment effect (ATT) was calculated. By comparing the difference in fertility intention between the two groups, the effect size of the rural floating population’s participation in BMISURR and fertility intention was determined, as shown in Equation (3).Yi1 and Yi0, respectively, represent the fertility intention of the same floating population in the two different situations of participating in BMISURR and not participating in BMISURR.Among them, p < 0.01). When demographic variables were included in model 2, the coefficient of health insurance became 0.274 (p < 0.01). When socioeconomic variables were included in model 3, the coefficient of health insurance became 0.169 (p < 0.01). When demographic and socioeconomic variables were included in model 4, the coefficient became 0.190 (p < 0.01). In model 5, when the migration characteristic variables were included, the coefficient become 0.201 (p < 0.01). In model 6, when all controlled variables were included, the health insurance coefficient became 0.186 (p < 0.01). It can be appreciated that health insurance has a significant and robust effect on the fertility intention of the rural migrants of childbearing age in China.In order to make the regression results more robust, the method of gradually including control variables was adopted. p < 0.01). Regarding economic features, there is a favorable correlation between personal income and fertility intention among rural migrants. With an increase in income, the fertility intention of rural floating population grew . Housing property has a significant impact on the fertility intention of the rural migrants. Compared with renting, buying a house locally significantly improves the fertility intention of the rural migrants . In terms of employment types, compared with informal employment, formal employment improves the probability of fertility intention of China’s rural floating population . From the perspective of the migration characteristics, the more migration times, the greater the fertility intention of the rural floating population . The larger the co-living scale in the inflow area, the greater the fertility intention of the rural floating population . In terms of mobility range, compared with interprovince migration, the near-distance intercity migration  and intercounty migration  leads to higher fertility intention. At the same time, according to the results of model 6 in B was less than 25, and the R value was between 0.25–2. This indicates that the sample data were balanced after matching. Secondly, before using the propensity score matching method, the synergistic effect of participating in health insurance on fertility intention of the rural floating population was 0.179, which is significantly correlated at the level of 1%. After using different matching methods, the treatment effect increased and remained at 0.220, which is still significantly positive at the 1% level, indicating that the synergistic effect of insurance participation on the fertility intention of the rural floating population is robust.As mentioned above, we used PSM methods to address self-selection bias in participating in health insurance. On this occasion, we deployed radius matching, kernel matching, and spline matching to test the synergistic effect of participating in health insurance on the fertility intention of the rural floating population by reporting the ATT. As shown in In order to test the robustness of the above results, the research processed the dependent variable with different methods, and used a logit regression model and a probit model, respectively, according to different types of dependent variables to test the promoting effect of participating in health insurance on the fertility intention of the rural floating population.p < 0.01). Column 3 still treated fertility intention as a binary variable but used a probit model for regression. Both the logit model and the probit model were suitable for a binary choice model where the independent variable is 0–1, and the occurrence probability of an event depends on the independent variable. The results showed that participation in BMISURR can still significantly improve the fertility intention of the rural migrants of childbearing age . In conclusion, whether the fertility intention variable is treated as a binary or ordered classification variable, and the probit model or logit model was used, health insurance has a significant synergistic effect on the fertility intention of the rural floating population.The above analysis was about the average effect of the whole sample, and did not take into account the heterogeneity of the impact of health insurance on the fertility intention of the rural floating population. Therefore, we further discussed the age, sex, household, and regional heterogeneity of synergies. To facilitate comparison and account for heterogeneity in the regression results, odds ratios were reported in full instead of coefficients. p < 0.05), and has a positive impact on the fertility intention of the rural floating population aged 25–29 . Besides, health insurance has a positive impact on the fertility intention of the rural floating population aged 30–34 , and has a positive impact on the fertility intention of the rural floating population aged 35–39 .In order to more precisely determine the age gap of synergistic impact, this study split the age of rural floating population into seven age groups with a 5-year age gap, based on the perspective of age difference. p < 0.05), but has no significant impact on females’ fertility intention. According to the regression results by region, participating in health insurance has a positive impact on eastern fertility intention , and also has a promoting effect on fertility intention for the rural floating population flowing into the western region . However, health insurance has no effect on the fertility intention of the rural floating population of childbearing age flowing into the central and northeast regions.China’s floating population has a large scale and is dominated by young and middle-aged individuals aged 15–49 with high fertility ability . The ferThe results showed that health insurance has a synergistic effect on the fertility intention of rural migrants in China, rather than a crowding out effect. That is to say, participating in health insurance improves the fertility intention of China’s rural floating population, which is related to the nature of the BMISURR. In 2015, Guangdong, Zhejiang, Shandong, Xinjiang, and other provinces in China began to explore the integration of the NRCMS and BMISUR into the BMISURR . In 2016In addition, the BMISURR continues to follow the principle of voluntary participation, combining individual contributions with government subsidies, and the level of government subsidies is constantly increasing. For example, China’s Yunnan province proposed to increase government subsidies to 420 yuan per capita by 2016. Shanghai proposed that the individual contribution should account for 15% of the total financing, the financial subsidy between the city  and the county and district should be shared 1:1 in 2016. It can be seen that the BMISURR has a high level of subsidies and reimbursement, which relaxes the family budget constraints of the rural floating population and, to some extent, relieves the economic and psychological pressure caused by childbearing so as to reduce the precautionary saving motivation, improve their current consumption ability, and thus enhance their fertility intention. This is consistent with the view of many scholars ,40. ThisAccording to the results in Interestingly, this study found that the nature of housing is significantly correlated with the fertility intention of the rural floating population in China. Compared with renting, local housing can improve their fertility intention, which is inconsistent with the research conclusion of Mulder CH . He arguIn addition, the scale of living together in migrant areas significantly improves the fertility intention of the rural floating population. Family members living together in migrant areas can take care of their children, which greatly reduces the fertility pressure of people of childbearing age and thus improves their fertility intention. This conclusion is related to the recent emergence of the “floating elderly” group in China, who are over 60 years old and continue to follow their children in order to take care of their children and grandchildren . AccordiThis study found that health insurance has different effects on the fertility intention of rural migrants in different ages. The effect of participating in the BMISURR on the fertility intention is mainly concentrated in the population aged 25–39, and has no effect on the population aged below 19 and above 40. However, the population of childbearing age between 25 and 39 years old is not only at the peak of childbearing behavior, but also in the middle age of supporting the elderly and raising children, with great economic, life, and work pressure. The subsidized BMISURR can boost their future income prospects and alleviate their current pressure. The future trajectory of China’s population structure is heavily influenced by the fertility intention and behavior of various groups. This provides a direction for China’s social security reform, and requires that the reform “target” should focus on the rural floating population aged 25–39.Secondly, health insurance has varying effects on the fertility intentions of rural floating populations of both sexes. Participation in the BMISURR had a positive influence on male fertility intention, but no effect on female fertility intention. This is basically in line with the characteristics of the floating population and the family division of labor. Young members of the floating population are mainly migrant workers and businessmen, and males bear the main economic responsibility of the family. Health insurance can alleviate their economic strain on the cost of living in their destination and improve their future aspirations, thus increasing their fertility intention. In this sense, China’s medical insurance reform must include measures that are more sensitive to gender differences.Finally, health insurance has varying effects on the fertility intention of the floating population in various regions. Behind economic restrictions and structural considerations, the fertility goal is reflected from the perspective of the inflow area. The BMISURR has no effect on the fertility intention of the rural floating populations in the central and northeastern areas. On the one hand, although there are more job opportunities and higher living costs in the eastern region, the BMISURR can relieve the pressure of the rural floating population in the eastern region and promote their fertility intention. On the other hand, the cost of living in the western region is low, because the BMISURR is mainly subsidized, it relaxes the family budget and improves the family’s willingness to have children by improving their consumption ability. The results indicated that we must not only consider the fertility intention of the floating population in developed regions, but also the impact of health insurance in developing regions.This study may contribute to the current literature on fertility intention and influencing factors of the rural floating population in China. Firstly, the majority of research on the fertility intention of Chinese residents focuses on urban and rural populations, but there is relatively limited research on the fertility intention of the rural floating population. This study focused on the rural floating population and explored their fertility intention and influencing factors, which could enrich the research scope on the fertility intention of the Chinese population. Secondly, while there are limited studies on the relationship between social security and fertility intention, current research on the relationship between social security and fertility intention remains controversial. This paper selected the BMISURR that covers the largest range of the rural floating population after the implementation of the two insurance policies in China, and firstly discussed the correlation between health insurance policy and the fertility intention of China’s rural floating population. The findings respond to existing research on the controversial relationship between health insurance and fertility intention, and demonstrate that health insurance policy has a significant positive impact on fertility intention for the rural floating population. Therefore, it fills a gap in the lack of research on fertility intention among the rural floating population. We deployed PSM to address the self-selection bias in health insurance participation. To sum up, the findings of this study not only expand the field and content of current studies, but also enrich the research methods of related content and improve the existing research.The study has several limitations. Firstly, due to the fact that the latest data did not involve the fertility intention of the floating population, this paper selects a relatively new 2016 CMDS data. To determine the changes in the fertility intention of the floating population in response to the universal three-child policy, it is still essential to analyze the most recent data. Secondly, only the BMISURR with the largest coverage and the widest range of the rural floating population are considered in this paper, no other types of social security, such as urban residents’ medical insurance, endowment insurance, maternity insurance, etc. are considered. Comprehensive inspection of all insurance types for the influence of the rural floating population fertility intention may become more complicated, hence further research is needed in the future.This current study quantified the impact of health insurance on the fertility intention of rural migrants in China using the 2016 CMDS. After addressing the sample selection bias, the health insurance policy has significantly improved the fertility intention of the rural floating population of childbearing age in China. It was found that participation in the BMISURR significantly increased the fertility intention of the rural floating population, with the probability of increasing the fertility intention of 18.6%. Secondly, the factors that affect the fertility intention of the rural floating population also include age, sex, economic income, housing property, employment type, co-living scale, migration times, and range of migration. Thirdly, the BMISURR has varying effects on the fertility intention of the rural floating population of different ages, genders, and regions. These results provide a new perspective for improving the fertility intention of the rural floating population, and also provide ideas on the reform of social security in China. Future studies on fertility intention should focus on the groups with strong fertility ability but fragile fertility intentions and behaviors in a country or a region, as well as the main factors influencing their fertility intention, so as to provide targeted recommendations for improving the local population structure and achieving sustainable development. For the Chinese government, the future should be in various areas of reform. Firstly, the Chinese government should adjust and improve the BMISURR, so as to increase the health insurance participation rate of the rural floating population, and improve the future expectation guarantee for the fertility intention of the rural floating population. Secondly, the equalization of basic public services should be promoted for the rural floating population, opening urban public resources, especially community medical services to them, and setting up resources and services for the rural floating population in cities where conditions permit, so as to improve the fertility expectation of the rural floating population. Thirdly, government should pay attention to the elderly who follow their children on the move, and provide high-quality elderly care services and medical services for the floating elderly, so as to ensure an adequate supply of family care services. Fourth, the government should implement differentiated welfare policies. The groups with significant improvement effects should become the target groups for future Chinese government reform, including the new generation aged 20–39, men, and the floating population in the eastern and western regions. Specifically, the influences of health insurance policies can be bolstered by continuously increasing the coverage and reimbursement level of the BMISURR, accelerating the policy of reimbursement for the BMISURR in different regions, and enhancing the level of medical insurance coordination, so as to maintain people’s fertility intention. For rural floating females with a low promotion effect of fertility intention, a series of complementary policies to reduce the cost of childbirth should be implemented, focusing on promoting gender equality in the labor market by implementing flexible working hours, and increasing parenting allowances and housing security allowances, so as to reduce the pressure of their fertility costs and enhance their fertility intention.


The Frontiers in Endocrinology Research Topic on fertility preservation in children invited authors from across the globe to participate in the dissemination of knowledge and awareness regarding the best fertility preservation principles in the pediatric population. Although long considered a problem only in adults and post pubertal individuals undergoing cancer therapy, assisted reproductive technologies have rapidly advanced to include ovarian and testicular tissue preservation. This now allows prepubertal patients and families who were previously excluded from fertility conversations, to be included in these profoundly important discussions, which may provide hope for future attempts at parenthood . FertiliDisparities in oncofertility care across the globe are well described, both in high and low resource settings , 3. ManyIn this Research Topic, authors were invited to present their research on optimal methods, timing, and outcomes on fertility preservation in children and adolescents. Data on new populations eligible for fertility preservation is highlighted in this Research Topic.Barrett et al. describe successful oocyte cryopreservation in 19 out of 20 transmen aged 12–20 years (median age 17 years).This is an important study since much of the previously published data is derived from the adult population. Two participants had been on testosterone, which was discontinued during oocyte collection. Around two thirds of patients cryopreserved at least 10 mature oocytes with many patients additionally freezing immature oocytes. There was no difference in outcome in those who attempted oocyte cryopreservation who had been on oral contraception, puberty blockers, or testosterone, compared with those who were naïve to hormonal therapy. Importantly, the use of dysphoria protection protocols in accordance with the World Professional Association for Transgender Health were implemented . The care of patients with DSD requires a multidisciplinary expert approach. Pitfalls in diagnosis after donor transplant are highlighted in this Research Topic by Li et. al.   expression was maintained, preventing non-growing follicle activation, maintaining ovarian reserve, and increasing litter size, providing new evidence for melatonin as a potential adjuvant chemotherapy agent of the future.There is intense interest in innovative fertility preservation techniques, including fertoprotective agents, which may be used as gonadal protectants during chemotherapy. In this Research Topic, 2). This has not previously been well reported. Approximately 30% of the study population had a diagnosis of leukemia, where common consensus is to offer fertility preservation as an interval procedure (prior to bone marrow transplant) when minimum residual disease is negative .Finally, a novel study on the potential to harness the power of social media to disseminate knowledge on the impact of cancer treatment on fertility and fertility preservation options explores questions on how best to communicate with digital savvy adolescents and young adults in ways they may find more familiar. (All authors contributed to conception, critically evaluating the drafts and approving the final draft.


Remyelination is a fundamental repair process in the central nervous system (CNS) that is triggered by demyelinating events. In demyelinating diseases, oligodendrocytes (OLs) are targeted, leading to myelin loss, axonal damage, and severe functional impairment. While spontaneous remyelination often fails in the progression of demyelinating diseases, increased understanding of the mechanisms and identification of targets that regulate myelin regeneration becomes crucial. To date, several signaling pathways have been implicated in the remyelination process, including the Hedgehog (Hh) signaling pathway. This review summarizes the current data concerning the complicated roles of the Hh signaling pathway in the context of remyelination. We will highlight the open issues that have to be clarified prior to bringing molecules targeting the Hh signaling to demyelinating therapy. Remyelination or myelin regeneration is a fundamental repair process in the central nervous system (CNS) that is triggered by demyelinating events. Multiple sclerosis (MS) is the most common demyelinating disease occurring in the CNS . It is gThe bottleneck of remyelination failure is the inability of oligodendrocyte progenitor cells (OPCs) to differentiate into myelinating oligodendrocytes (OLs). In animal models, as well as in humans, myelin regeneration is initiated by both resident OPCs at the site of lesion and neural progenitor cells (NPCs) located in the ventricular-subventricular zone (V-SVZ) of the brain ,9,10,11.Drosophila, where it plays an essential role in the segmentation and body-plan patterning of the larva [Ihh loss-of-function analysis showed that Ihh is also required for OPC specification from NPCs [Several signaling pathways involved in developmental myelination have been implicated in the remyelination program, including the Hh pathway . Moleculhe larva . Three Hhe larva . Among the larva ,21,22,23rom NPCs . In controm NPCs ,26.The activation of Hh signaling can trigger two alternative pathways: canonical and non-canonical . When HhIn addition to the canonical Hh signaling pathway, a number of pathways triggered by Hh ligands, but outside the Hh-Ptch1-Smo-Gli axis and independent of the primary cilium, have been identified and collectively called ‘non-canonical’ Hh signaling pathways ,34,35,36139-151) induces a relapsing–remitting disease course [35-55 peptide induces a chronic course [Since the etiology and histopathology of MS are complex, different types of animal models have been used to study the mechanisms associated with demyelinating diseases and to evaluate the efficacy of novel therapeutic drug candidates. A comparison of histopathological characteristics between different MS animal models is given in ,46,47. Te course , while tc course . Even thc course . Similarc course . MoreoveIn summary, there is no single animal model that can replicate all of the heterogeneous characteristics of human MS. Each single animal model allows us to study distinct aspects of the disease rather than its entire complexity. Consequently, investigators need to carefully select the animal model to accurately address their specific research question and provide outcomes that result in findings applicable to human MS. Nonetheless, animal models have proven to be highly advantageous in developing novel drugs that are directed toward the process of remyelination.There exist sequential waves of OPC generation in vertebrates, firstly localized in the ventral progenitor domains and later in dorsal regions . AlthougThe contribution of the Shh signaling pathway in the context of CNS demyelination and remyelination has been addressed by several groups. In addition to its early role in the induction of embryonic OPCs , Shh sigThe stimulation of Smo activity is compatible with the positive influence that Shh exerts on remyelination. For instance, the microinjection of SAG, a Smo agonist, into the corpus callosum of LPC-induced demyelinated mouse significantly increased OPC proliferation and enhanced remyelination , in accoRecently, another crucial component of the Hh signaling pathway, the type I transmembrane receptor Boc, was identified as a new regulator of myelin formation and repair . During Although the Hh/Smo signaling pathway possesses an important role in promoting remyelination, the development of related therapeutic strategies has been impeded by the lack of U.S. Food and Drug Administration (FDA)-approved Smo agonists. By using high-throughput screening for cells that express the Smo receptor, four FDA-approved drugs, clobetasol, halcinonide, fluticasone, and flucinonide, were identified as agonists of Smo . These dClobetasol is a member of the glucocorticoid family and is commonly used to treat a number of skin disorders . It is aAltogether, the identified Smo agonist Clobetasol might function in multiple cell types and act via a range of targets to promote myelin repair. Importantly, Clobetasol is able to pass through the blood–brain barrier, raising the exciting possibility that Clobetasol could advance to clinical trials for the currently unavailable chronic progressive phase of MS.In the transgenic mice that ectopically expressed Shh in the dorsal neural tube, spinal precursor cells were arrested in an undifferentiated state and exhibited elevated levels of proliferation . RecentlIn fact, Gli1, originally considered to be a reliable readout of Hh signaling activity, has proven to be detrimental during myelination and remyelination. During development, the genetic ablation of Gli1 in NPCs appeared to lead to precocious myelination . SpecifiIn addition, the negative regulation of myelin regeneration by Gli1 was also reported recently. During the demyelination and remyelination processes, the expression of Gli1 appeared to be variable depending on the animal models that were used. When demyelination was induced in the corpus callosum by an injection of LPC, a relatively moderate transcription of Gli1 was seen in OPCs within the lesions . In the As described above, the use of several small molecules able to activate Smo-mediated Hh signaling or to block its key effector Gli1 led to improved remyelination in several animal models , which mAs mentioned above, Smo is able to transduce Hh signaling via both canonical and non-canonical pathways ,36,87,88Under repairing conditions, inflammatory cells in the affected regions, including astrocytes and microglia, are endowed with beneficial or deleterious properties, promoting or impairing the endogenous capacity of OPCs to induce spontaneous remyelination after myelin loss ,93. TherGiven that the process of remyelination is extensive and complicated, boosting remyelination is a current challenge in the field of CNS demyelinated disease. Current approaches for discovering regenerative therapies in MS are mostly based on the assumption of enhancing the differentiation of resident OPCs to OLs. However, a recent single-cell analysis of the white matter in MS patients identified oligodendrogial heterogeneity in MS and proposed that strategies to restore healthy OL heterogeneity should be a major focus in the future treatment of MS ,13. Ther


Prescribing antidepressants in the treatment of bipolar depression remains highly controversial due to the inconsistency between routine clinical practice and the results of controlled trials.To assess the validity of antidepressants use in bipolar depression from the point of view of evidence-based medicine.Database search (Scopus and MEDLINE) followed by analysis of studies concerning the efficacy and safety of antidepressants in the bipolar depression treatment.The search found 23 studies. There was a high degree of inconsistency in the results, apparently related to the methodology. Only two studies compared the effectiveness of antidepressants in monotherapy with placebo. No differences were found in the study with 740 participants but in the study with 70 participants with type 2 bipolar disorder antidepressants were found to be more effective than placebo. Nevertheless, both studies had significant methodological issues. In 6 studies comparing the effectiveness of the combination of antidepressants with mood stabilizers against the combination of mood stabilizers with placebo, only the effectiveness of fluoxetine in combination with olanzapine was confirmed, other antidepressants were ineffective. At the same time, studies where antidepressants were compared with each other in combination with mood stabilizers revealed a significant clinical response to therapy. Risk of the treatment emergency adverse events were relatively low for SSRI.Despite the contradictory literature data, the use of antidepressants in bipolar depression is justified from the point of view of evidence-based medicine for certain groups of patients with taking into account risk factors.


Extracting ursolic acid (UA) from plant resources using organic solvents is incompatible with food applications. To address this, in this study, 15 edible hydrophobic deep eutectic solvents (HDESs) were prepared to extract UA from apple peel, the extraction conditions were optimized, and the optimization strategies were compared. It was found that the solubility of UA in the HDESs can be 9 times higher than the traditional solvent such as ethanol. The response surface optimization concluded that temperature had the greatest effect on the extraction and the optimized test conditions obtained as follows: temperature of 49 °C, time of 32 min, solid–liquid ratio of 1:16.5 g/mL, respectively. Comparing the response surface methodology (RSM) and artificial neural networks (ANN), it was concluded that ANN has more accurate prediction ability than RSM. Overall, the HDESs are more effective and environmentally friendly than conventional organic solvents to extract UA. The results of this study will facilitate the further exploration of HDES in various food and pharmaceutical applications. Pentacyclic triterpenes are based on 30 carbon skeletons, consisting of five six-membered rings or four five-membered rings and one five-membered ring, and are formed by the arrangement of epoxide squalene , which iAs a kind of lipophilic natural pentacyclic triterpenoids, UA has anti-inflammatory , antibaceucalyptus globulus with dichloromethane [ligustrum lucidum fruit with 80% ethanol [Traditional extraction methods of triterpenoids include impregnation, Soxhlet extraction, and hot reflux extraction. The traditional solvents for extracting pentacyclic triterpenes are mainly organic solvents, including methanol, ethanol, and hexane . Kikowskomethane . Xia et  ethanol . HoweverDeep eutectic solvent (DES) is a new type of solvent which is obtained by mixing hydrogen donor (HBD) and hydrogen acceptor (HBA) in a certain molar ratio . DES hasResponse surface methodology (RSM) is an optimization method that uses mathematical and statistical methods to construct an optimal condition for assessing the independent variables’ significance and obtaining predicted response values . Artificadj2) and root mean square errors (RMSE).This experiment aims to extract UA from apple peel using natural Men-based HDESs and systematically compare the test effects of the two optimization methods. Firstly, the type of HDESs with the highest solubility was screened by testing the solubility of UA. Then the molar ratio of HBA and HBD of HDES was further screened to select the HDES with the highest solubility of UA. A single-factor test determined the optimum temperature, time, and solid–liquid ratio of HDES, and then the response surface optimization test was used to obtain the optimum test conditions. This study also used ANN to calculate several sets of data from response surface tests and compares the predictive power of these two methods by comparing their adjust coefficients of determination  were purchased from a local supermarket. UA standard and decanoic acid (purity 99%) purchased from Shanghai Aladdin Biotechnology Company. Menthol (purity 95%) and thymol (purity 99%) were purchased from Wuhan Lanabai Pharmaceutical and Chemical Company. Methanol was purchased from China Ferton Reagent Company (HPLC grade). Pyruvic acid (purity 98%), levulinic acid (purity 99%) and propionic acid (purity 99.5%) were purchased from Shanghai Macklin Biotechnology Co., Ltd. . Formic acid (purity 98%), acetic acid , lactic acid , n-butanol (purity 99.5%), and anhydrous ethanol were purchased from China Pharmaceutical Group Chemical Reagent Co., Ltd. . Isopropanol (purity 99.7%) was purchased from Tianjin Institute of Chemical Reagents. Phosphoric acid (purity 90%) was purchased from Tianjin Comiou Chemical Reagent Co., Ltd. .Based on the method of Peng et al., the HBD and HBA were placed in a reagent bottle and stirred at 80 °C for 30 min at 1 g with a collector thermostatic magnetic stirring bath, then a homogeneous liquid was formed and placed in at dry room temperature and set aside . Cai et Referring to Alhadid et al., the viscosity of all HDESs was measured using a Kinexus advanced rotational rheometer  at room temperature 30 °C and atmospheric pressure with a gap of 0.1 mm and a shear stress of 1 Pa . The tesv/v), and the flow rate was 1 mL/min. UA standard solutions of 20, 40, 80, 160, 320, and 640 μg/mL were prepared, and then, the standard curve was plotted with UA concentration as the horizontal coordinate and peak area  as the vertical coordinate . Then, the regression equation was established (Equation (1)), and the coefficient of determination R2 is 0.9997.The solubility of UA in 17 HDESs was determined according to the method proposed by Silva et al., with slight modifications . The excsol represents the concentration of UA of the tested HDES solubility, D is the dilution multiple of the sample, and 1000 represents the unit conversion.The solubility was calculated by the following equation:The HDES with the highest UA solubility was selected by solubility test, and then, the ratio of HBA and HBD of the best HDES were screened.The HDES with the highest UA solubility among the tested HDESs were further selected to test the UA solubility in HDES under different HBA and HBD molar ratios. The molar ratios of the selected HDESs are shown in g for 10 min, take 0.2 mL of supernatant with 1.8 mL of methanol for dilution, passed through a 0.22 μm membrane and then measured by HPLC to calculate the extraction yield (EY) of UA with the following formula.The apple peels were placed in an oven and baked at 60 °C for 24 h to constant weight. The dried product was crushed and passed through a 60 mesh sieve. After passing through the sieve, the apple powder was baked at 60 °C for 1 h to prevent water reabsorption during the sieving. The 1 g sample was accurately weighed and added into 10 mL HDES solution and extracted with ultrasonic cleaner   at a power of 100 W and temperature of 50 °C for 30 min. The extract was placed in a high-speed centrifuge at 5550× According to the screening of Based on the results obtained from the single-factor test in ANN comprises four parts: input, hidden layer, output layer, and output. The input layer of this experiment was composed of time, temperature, and solid–liquid ratio, and the output layer was the extraction yield of UA. The structure of ANN is shown in In order to obtain better-predicted values and prevent model overfitting, the data is normalized, and the normalized formula is as follows.All the data involved in the training of ANN are derived from 17 sets of data from the RSM optimization experiment, in which the training set accounts for 70%, the validation set, and the test set account for 15%, respectively. The number of hidden layer neurons is calculated as follows:adj2 and RMSE between RSM and ANN. The calculation formula of Radj2 is as follows:The accuracy of performance was evaluated by comparing Rcal is the calculated value of RSM or ANN, YExp is the true value of the experiment, YAvg is the average value of the experiment, and n is the number of experiments for 17.The calculation formula of RMSE is as follows:p < 0.05) between the means was performed using IBM SPSS statistic 26.0  software. All values are expressed as means ± standard deviations (S.D) unless stated otherwise.All experiments were conducted in triplicate. RSM design of experiments and model fit was performed in Design Expert 13 , ANN modeling was performed in MATLAB , and the other statistical analysis was performed using GraphPad Prism 9 . One-way analysis of variance (ANOVA) with Tukey’s test was used to determine the significant differences . In contrast, temperature and time have no significant effect on the extraction yield of UA (p > 0.05). According to the size of the F-value, it can be seen that the solid–liquid ratio has the most significant effect on the extraction yield, then temperature, and finally, time. The R2 in this experiment was 0.9536, and the adjusted R2 was 0.8940 (>0.8), indicating that the experimental data fit well with the second-order polynomial equation. The multiple regression analysis of the data resulted in a second-order polynomial fit regression equation for UA extraction yield, which can be used to calculate UA extraction yield, and the equation is as follows:The correlation between the experimental data and the model was analyzed based on the ANOVA. The ANOVA table from of model . The C.Vp-values of AB (time and temperature) were 0.9171 and 0.3701, respectively. The F and p-values of AC (time and solid–liquid ratio) were 0.3393 and 0.5785, respectively. BC’s F and p-values (temperature and solid–liquid ratio) were 0.3509 and 0.5722, respectively. AB has the most considerable F-value and the smallest p-value. In addition, it can be seen from p-values and large F-values. According to According to the results of ANOVA for the Quadratic model, the F and According to the response surface optimization, the predicted reaction conditions were obtained: time 32.334 min, temperature 49.238 °C, and solid–liquid ratio 1:16.514 g/mL. The calculated UA extraction yield was 1.544%. The optimal reaction conditions were calculated as follows: time 32 min, temperature 49 °C, and solid–liquid ratio 1:16.5 g/mL. The yield obtained by the final verification was 1.566%. Siani et al. extracted UA from dry apple peel with aqueous and hydroethanolic to a content of about 6.12 μg/g , which iANN is a multilayer feedback network model with a feed-forward back propagation algorithm. The ANN model in this study used the Levenberg–Marquardt algorithm in training the data. A two-layer feed-forward network with hidden sigmoid neurons and linear output neurons (fitnet), fits multidimensional mapping problems in extraction prediction arbitrarily well.In this study, a trial-and-error approach was used to train the ANN model one after another, stopping the ANN training when the mean square error (MSE) value is the minimum and R is the maximum. The statement that it is not limited when choosing the number of neurons in the hidden layer is not true . The num−6, the MSE of the validation set is 0.000558, and the MSE of the test set is 0.00256. The accuracy of the experimental model can be demonstrated by the fact that the training and test set errors are close [When the neuron node was determined to be 6, RMSE and R were calculated according to Formulas (6) and (7). The MSE of the training set is 2.22 × 10re close . As seenAs shown in adj2 and RMSE between RSM and ANN. Generally, the higher Radj2, the lower RMSE, and the more accurate the prediction model is [adj2 and RMSE of RSM were 0.9535 and 0.03254, respectively. The Radj2 and RMSE of ANN were 0.9797 and 2.72249 × 10−5, respectively. It can be seen that the Radj2 of ANN is higher than RSM, and its RMSE is far less than RSM. Therefore, it can be proved that the ANN model can better calculate the experimental results than the RSM model, which is related to its general ability to approximate nonlinear systems [The predicted and experimental values of RSM and ANN are shown in model is . The Rad systems . In concadj2 and RMSE, ANN has a better prediction ability than RSM. Therefore, this work has the potential to become the foundation of an efficient technique for the extraction of UA for application in food industry. HDES is a promising extraction solvent, and its significance as a solvent is more than that. More studies need to pay attention to the coexistence of DES in the activity protection and efficacy promotion of plant-active ingredients.This study prepared 17 kinds of menthol-based HDESs composed of different HBA and HBD. This natural hydrophobic solvent was used for the first time to extract UA in apple peels to replace the traditional organic solvent. According to the solubility test, the solubility of UA in HDESs is significantly improved compared to that in the traditional organic solvent. The solubility of UA in ethanol is only 4.16 μg/mL, while the solubility of UA in HDES is up to 36.2 μg/mL. The response surface optimization concluded that temperature had the most significant effect on the extraction, and the optimized test conditions obtained were 49 °C, 32 min, and 1:16.5 g/mL, respectively. In this study, two different prediction methods, RSM and ANN, were used to calculate the values of 17 groups of experiments. According to the analysis of the data parameters in the two models, such as R