Extraction Optimization, Preliminary Identification, and Bioactivities in Corn Silk

For thousands of years, corn silk has been widely used as an antidiabetic, antioxidant, and antihyperlipidemic and for other effects, but there is a lack of studies that correlate the extracts of flavonoid composition with their biological activities. Thus, the objectives of this study were to optimize the conditions for extracting flavonoids, identify flavonoids, and correlate the flavonoid composition with the biological activities in corn silk. The response surface experiments showed that the highest flavonoid content was predicted at 45.321 min, 57.349°C, 26.089 mL/g, and 71.269%, respectively. The verification experiment results under these optimized conditions showed an ultrasonic time of 45 min, an ultrasonic temperature of 57°C, a liquid-to-material ratio of 26, and an ethanol volume fraction of 70%. No significant differences (the relative error is 4.378%) were observed between the theoretical and experimental TFC values, indicating that the developed models were accurate. Under these optimum extraction conditions, 20 major compounds were identified and quantified by UPLC-LTQ/Orbitrap MS. Furthermore, these optimum ethanol extracts of corn silk are effective against Bacillus subtilis and hypoglycemic activity compared with the traditional heating reflux extraction method. Six corn silk components seem to be the main contributors to the inhibitory effect against Bacillus subtilis and hyperglycemia activities. These results are useful for the application of corn silk in the food or pharmaceutical industry.

Te choice of an appropriate extraction method is crucial to isolate natural bioactive compounds from plants and materials. Conventional extraction is the most studied and used method to extract compounds. However, it presents a few faws such as long extraction time and bioactive compound degradation [22]. Terefore, it is of great signifcance to employ alternative methods. Many research studies have proved that UAE presents extraction yields in a shorter time [23,24]. Ultrasonic-assisted extraction (UAE) is a simple technique that has the advantages of time-saving, utilisation of a low solvent amount, and high efciency [25]. It has been widely used to design experiments to optimize extraction process conditions [26][27][28]. Te yield and bioactivities of plant extracts are dependent on the extraction conditions such as type of solvent, liquid-to-solid (L : S) ratio, extraction temperature and time, and particle size.
Hence, optimizing an efcient extraction process is very important for a particular variety. Te response surface methodology (RSM) is an efcient mathematical and statistical tool that is used to determine and optimize variable experimental conditions to achieve maximum yields with minimum time and less resource consumption [29][30][31]. Te various parameters and their probable interactions can be evaluated efciently with a mathematical model, reducing the number of experiments.
Ultra-performance liquid chromatography coupled with linear ion trap/Orbitrap mass spectrometry (UPLC-LTQ/ Orbitrap MS), a method with high sensitivity and high accuracy, has been widely used to investigate the constituents of medicinal plants such as Ononis spinosa L. [32]. Terefore, this study used UAE to extract the total favonoid from corn silk and use UPLC-LTQ/Orbitrap MS to analyze the chemical constituents of ethanol extracts of corn silk. Unhealthy habits have led to the frequent occurrence of diseases such as bacterial infammation and hyperglycemia, but drug treatment sometimes may cause adverse reactions [33]. Tus, it is necessary to obtain substances from food extracts to assist or even substitute drug treatments for the treatment of diseases.
To date, ultrasound-assisted extraction with RSM has been used in the extraction of polysaccharides [10], polyphenols [34], and favonoid [35] compounds from corn silk. However, there is a lack of studies that correlate the extracts of favonoid compositions with their biological activities. Te current study was planned to optimize a given set of extraction conditions and reveal the chemical properties and pharmacological activities of ethanol extracts of corn silk, which might provide a theoretical basis for the further development and utilisation of corn silk resources in the future.

Plant Materials.
Corn silk was obtained from Xinxiang, Henan Province, and identifed as authentic by Professor Liu Chunsheng of the Beijing University of Chinese Medicine. It was air-dried to constant weight and pulverized, and the powder was passed through a 65-mesh sieve and stored at 4°C.  [19]. In brief, the ethanol extract was refuxed at 80°C for 3 h, taking 1 : 30 as a solid-liquid ratio with 60% ethanol as an extraction solvent.

Ultrasonic-Assisted Extraction.
In view of the fact that favonoids are the main active components of corn silk, we used the total favonoid content (TFC) as an indicator to optimize the UAE process of corn silk ethanol extracts.

Preliminary Investigations.
To fnd the appropriate range of variables for the extraction process, the ultrasonic power (W), ultrasonic time (min), ultrasonic temperature (°C), liquid-solid ratio (mL/g), and ethanol concentration (%) were tested in a preliminary experiment to investigate the factors and levels of the favonoid extraction process (Table 1). After soaking in solvent for an hour, the constant extraction conditions in each single-factor test were as follows: 70% ethanol solution 30 mL, an ultrasonic power of 500 W, and an ultrasonic temperature of 60°C for 30 minutes. In the selected conditions, a sample was centrifuged at 4°C and 8000 r/min for 10 minutes, and 1 mL sample was diluted 25∼50 times for subsequent color development.

Experimental Design for RSM.
Te three-level fourfactor Box-Behnken design (BBD) was carried out to optimize the UAE condition (Table 2). A total of 29 experimental runs were used to normalize parameters (Table 3).
Te extraction yield of TFC was viewed as the response factor. ANOVA was applied to estimate the infuence of each factor. Te P value of the regression model and the regression coefcient were used to evaluate the scientifc model. Tree-dimensional pictures and two-dimensional pictures were used to demonstrate the infuence of the interaction of the two factors on extraction.

Determination of TFC.
TFC was determined based on the method reported by Sabiu et al. [36] with a slight modifcation. Briefy, 1 mL of extracted samples was dissolved in 14 mL of 70% ethanol and 0.7 mL of 5% NaNO 2 solution. After 5 min, 0.7 mL of AlCl 3 (10% w/v) was added to the solution. Te mixture was allowed to stand for 5 min, and 5 mL of NaOH solution (1 mol/L) was added. Finally, we added 70% ethanol to dilute it to 25 mL, and it was reacted and mixed well and incubated at room temperature for 15 min. Ten, the absorbance was measured at 510 nm. Rutin was used to make the standard calibration curve for TFC. It (10 mg) was dissolved in 50 mL of 70% ethanol, after which

Anti-α-Amylase Assay.
It has been reported that the corn silk ethanol extract has α-amylase inhibitory activity [36]. Herein, the efect of the reaction time and extract concentration on the inhibition rate was investigated by 3,5-dinitrosalicylic acid colorimetry [39]. We added 0.5 mL α-amylase to 0.5 mL of diferent dilutions (0.5 mg/mL, 1.0 mg/mL, and 1.5 mg/mL). After that, at 37°C, water was bathed for 20 min, 30 min, and 40 min, and we added a 1% starch solution of 1 mL. At 37°C, water was bathed for 15 min. Ten, we added 1 mL DNS for color development, and boiling water was bathed for 5 min and cooled to 25°C; fnally, we added 10 mL deionized water. Te absorbance was measured at 540 nm. Each sample was tested in triplicate. Triplicates of the negative control (deionized water) and the positive control (acarbose aqueous solution) were also measured. Te following formula was used to calculate the α-amylase inhibitory activity:    and ACN (B) with linear gradient elution at a fow rate of 0.3 mL/min. Te sample injection volume was 2 μL. Te gradient elution program was set as follows, as shown in Table 4. Te LTQ-Orbitrap XL linear ion trap-tandem electrostatic feld Orbitrap mass spectrometer was equipped with a thermal spray ion source (HESI), and Xcalibur 2.1 ChemStation was operated in a negative ion detection mode. Te ion source temperature was 350°C, the ionization source voltage was 4 kV, the capillary voltage was 35 V, the tube lens voltage was 110 V, and the sheath gas and auxiliary gas were both high-purity nitrogen (purity > 99.99%). Te sheath gas fow rate was 40 arb, and the auxiliary gas fow rate was 20 arb. Te data adopted the Fourier transform high-resolution full scan mode (TF, full scan, resolution 30000) for datadependent acquisition of ddMS 2 and ddMS 3 .

Correlation Analysis of Corn Silk Components with Teir
Biological Activities 2.8.1. Screening of Related Targets. We collected the component targets using the TCMSP (http://lsp.nwu.edu.cn/ tcmsp.php) database. After screening, the UniProt protein database (https://www.uniprot.org) was unifed to standardize the protein targets on which compounds act to standardize protein target information. Mining relevant targets was carried out by setting "Bacillus subtilis," "hyperglycemia," and "hyperglycemia" "as key words" in the DisGeNET (https://www.disgenet.org), TTD database (http://bidd.nus.edu.sg/group/cjttd), GeneCards database (https://www.genecards.org), DRUGBANK database (https://www.drugbank.ca), and OMIM database (http:// www.omim.org). In the GeneCards and DisGeNET databases, the target with a score greater than or equal to the median was set as the potential target. Te results of the fve databases were combined, duplicate values were deleted, and the result was the target related to Bacillus subtilis or hyperglycemia.

Construction of the PPI Network.
Venny2.1 (https:// bioinfogp.cnb.csic.es/tools/venny/) was used to analyze the relationship between the identifed components and Bacillus subtilis or hyperglycemia targets. Te intersection was found, and a Venn diagram was drawn. Te intersection target was submitted to the STRING database (https:// string-db.org) to construct a protein interaction (PPI) network model. 1st and 2nd shells were set as no more than 5 interactors.

TFC of Ethanol Refux Extraction.
We conducted three experiments in parallel, and the average yield was 1.152% ± 0.09%, which was quite diferent from the yield obtained by ultrasonic extraction. It can be seen that in this study, ultrasonic extraction showed obvious advantages in high efciency.

TFC of the UAE Process.
Te rutin standard curve equation was y � 11.565x + 0.0497, R 2 � 0.9998, which illustrated that there was a good linear relationship between the mass concentration and the absorbance value in the range of 0.016 mg/mL-0.08 mg/mL ( Figure 1). Te infuence of diferent single factors on TFC ultrasonic extraction is shown in Figure 2. According to the experimental results, the factors and levels of RSM are determined, as shown in Table 2.
Te regression model analysis of variance is shown in Table 5. R 2 � 0.9656; R 2 Adj � 0.9312; the CV value was 2.85%. It can be seen in Table 5 that the model had a value of P < 0.001, indicating that the regression model was extremely signifcant; the lack of the ft item was not signifcant (P > 0.05), implying that the quadratic regression model ftted well with the actual situation. Te primary and secondary orders of factors afecting the yield of corn silk favonoids were B > D > C > A.

Response Surface Analysis.
Te response surfacecontour map is shown in Figure 3. It can be seen in the fgure that the ultrasonic temperature surface was steepest,        Note. * P < 0.05, the diference is signifcant; * * P < 0.01, the diference is highly signifcant; * * * P < 0.001, the diference is extremely signifcant.    Evidence-Based Complementary and Alternative Medicine indicating that it had the greatest impact on the response value; the ultrasonic time response surface had the gentlest slope, and its impact on the response value was least. Te optimized extraction process parameters obtained by response surface analysis were as follows: the ultrasonic time was 45.321 min, the ultrasonic temperature was 57.349°C, the liquid-to-material ratio was 26.089, and the ethanol volume fraction was 71.269%. Under these conditions, the theoretical prediction value of the favonoid yield was 2.444%.

Verifcation Test Results.
In view of the limitations of the actual operation, the favonoid extraction process conditions were adjusted to an ultrasonic time of 45 min, an ultrasonic temperature of 57°C, a liquid-to-material ratio of 26, and an ethanol volume fraction of 70%. Under these conditions, three experiments were performed to verify the optimized conditions. Te average yield was 2.337% ± 0.118%, and the relative error between the theoretical prediction value was 4.378%, which was less than 5%, indicating that the extraction process parameters obtained by applying RSM optimization were reliable. Previous researchers have designed RSM to determine the optimal extraction conditions of total favonoids in corn silk [40]. Te results are quite diferent from those of this experiment, which may be due to the fact that the ultrasonic temperature or ultrasonic power was not considered for the single-factor test.

In Vitro Activities
3.3.1. Antibacterial Activity. Te corn silk of RSMoptimized extraction exhibited an inhibitory efect against B. subtilis ( Figure 4). However, there were no antibacterial activities against S. aureus and E. coli. As is shown in Figure 4, these data for the inhibition of bacterial activity showed dose-dependent relationships for all of these extracts: the higher the concentration of the applied extract, the higher the inhibition of bacterial activity. Te higher the concentration of the extract, the better the inhibitory efect with MIC values of 25 mg/mL. Moreover, the corn silk obtained by ethanol refux had no inhibitory efect on three bacterial strains. It was speculated that ultrasonic extraction had the ability to extract more antibacterial active substances from corn silk. At present, the application of corn silk along with synthesis exhibited antibacterial activity toward Grampositive and Gram-negative bacteria, as a clean and cheap agricultural waste [41].

Anti-α-Amylase Activity.
In the α-amylase inhibitory activity assay, the results show that the ethanol extract of corn silk has inhibitory efects (Tables 6-8). Te concentration of the sample and the reaction time afected the inhibitory efect. Te results indicated that the corn silk ethanol extract demonstrated the activity of inhibiting α-amylase, and the inhibitory efect increases with  an increase in concentration at 0.5∼1.5 mg/mL. Under the test conditions, the inhibition efect is best when the sample concentration is 1.5 mg/mL and the reaction time is 30 minutes. When the reaction time was 40 minutes, the inhibition rate was very low, and we speculated that this may be due to this inhibition being reversible. However, considerable diferences across these extracts were seen for diferent extraction methods. Te inhibition  Te criteria for antibacterial efects are as follows: diameter >20 mm means extremely efective, 15∼20 mm means highly efective, 10∼15 mm means moderately efective, 7∼9 mm means slightly efective, and absence of inhibition zones means inefective.    Evidence-Based Complementary and Alternative Medicine rate of ethanol refux was signifcantly lower than that of ultrasonic extraction (Tables 6 and 7). It can be seen that the ultrasonic extraction method was more efective in extracting components that inhibit the activity of α-amylase. It can thus be concluded that corn silk is a promising inhibitor of α-amylase and can therefore be considered as antidiabetic potential. Te conclusion that corn silk extracts can be considered as antidiabetic agents is here partly based on previous studies that have shown anti-α-amylase and anti-α-glucosidase activities for polysaccharides [6] and polyphenols [5]. However, the data in the present study demonstrate that these corn silk extracts that contain quercetin have a direct impact on the α-amylase activity and pointed out the fact that other substances present in these extracts may have important impacts on the overall anti-α-amylase activity, which was not shown by other studies. Tese data reinforce the possibility of using corn silk extracts in the human diet and could be further developed as a cheap and plant-derived agent in the management of diabetes.

Qualitative Analysis.
Te determination of total favonoids and the results of in vitro antibacterial tests show that ultrasonic extraction can obtain more active ingredients than refux extraction. Terefore, we conducted a qualitative analysis of the solution obtained by ultrasonic extraction. A total of 20 compounds (Table S1) were identifed, which include two carbohydrates, one organic acid, eight favonoids, seven fatty acids, one ester, and one steroid. We confrmed the components by combining mass spectrometry fragmentation law, database data (https://www. chemspider.com), literature data, MS, MS2, retention time and assumed formula [42][43][44][45][46]. Total ion chromatography (TIC) is shown in Figure 5(a), the chromatograms of 20 compounds are shown in Figure 5(b), and the secondary fragment ion patterns of 20 compounds are shown in Figure 5(c). According to the results, favonoids and fatty acids are the main components. We took rutin as an example to analyze the ionization process of favonoids (Figure 6(a)), and we took linoleic acid as an example to analyze the ionization process of fatty acids (Figure 6(b)).     (Figure 7(a)), and 20 targets were related to hyperglycemia (Figure 7(b)). We submitted the common target to STRING to obtain the PPI network ( Figure 8). In summary, the 6 corn silk components were related to B. subtilis and hyperglycemia-related targets, and their common targets interacted with each other, which provided a theoretical basis for studying the pharmacological efects of corn silk.

Discussion
Conventional extraction techniques (maceration and ethanol refux) require high consumption of available resources and thus are inefective and expensive. In this study, the response surface methodology was used to optimize the ultrasonic extraction of total favonoids from corn silk, which improved yield and had a low processing time compared with ethanol refux extraction. Previous studies reported the successful extraction of favonoids using ultrasonic waves from other plants, such as Ocimum basilicum [49] and immature Citrus unshiu pomace [50]. UAE has a greater efect on the content of extractive substances than other conventional extraction methods [51]. Te UAE extraction procedure has the efect of cavitation and the better degradation of plant tissue so that the extraction of desirable bioactive compounds from the plant material can be easier [52]. Because of these reasons, UAE can be considered the technique of choice for extraction of total favonoid compounds from corn silk. Many natural products contain a variety of bioactive compounds. Natural compounds play an important role in our daily diet and are part of pharmaceutical and cosmetic industries as well. Terefore, it is crucial to fnd antibacterial active ingredients from natural compounds. We have proved the antibacterial ability of corn silk and screened related compounds preliminarily, which can provide reference for the development of antibacterial drugs using corn silk in the future. In addition, we have proved that corn silk can be considered an antihyperglycemia candidate. At present, corn silk has been widely used as tea [1,2] and food ingredients [6,7]. Tat is to say, to relieve the efect of the symptoms of hyperglycemia, the reasonable consumption of corn silk as a functional food is feasible. Nature is regarded as the best source for medicines as a variety of natural compounds exist with promising medicinal values. Here, we identifed 24 components from corn silk extracts and found seven components related to B. subtilis and hyperglycemia activity. Among them, sucrose is related to B. subtilis and hyperglycemia activity because it may contribute to the growth of B. subtilis [47] and induce hyperglycemia [48]. While all the other six components have antibacterial activity and can ameliorate hyperglycemia activity. Studies have shown that quinic acid can inhibit the growth of a variety of bacteria [53,54], and it is a competitive inhibitor of α-amylase [55]. Chitosan/silver (CS/Ag) nanocomposite using rutin has antibacterial activity against B. subtilis [22]; in diabetic rat models, rutin can signifcantly reduce fasting blood sugar [56]. Luteolin and hispidulin have antibacterial activity against B. subtilis as well [57,58], and it is reported that they can ameliorate hyperglycemia [59,60]. Palmitic acid has antibacterial activity [61], and a compressed mixture of insulin and palmitic acid can sustain a reduction in hyperglycemia in rodents [62]. Additionally, research shows that deoxycholic acidtriazole conjugates have good inhibitory efects against B. subtilis [63]; deoxycholic acid is involved in improving glucose metabolism to ameliorate hyperglycemia [64]. In short, we have found 6 possible components of corn silk with antibacterial and hyperglycemia activities. Tis fnding will lay a theoretical foundation for expanding the application of corn silk.
In summary, optimization extraction, preliminary characterization, and antibacterial and anti-α-amylase activities in vitro of corn silk favonoids were carried out. Te optimum extraction conditions of the total favonoid content were determined using the singlefactor experiments and the orthogonal array test. Under optimum reaction conditions, UAE was an efective method to extract favonoids from corn silk compared with ethanol refux extraction. UAE provides a higher extraction yield and can obviously reduce the extraction time. Furthermore, the ethanol extract of corn silk is rich in a variety of chemical ingredients and has an inhibitory efect against B. subtilis and α-amylase. Six corn silk components seem to be the main contributors to the inhibitory efect against B. subtilis and hyperglycemia activities. Tese fndings are important for further research on the chemical composition and biological activity of the corn silk ethanol extract, which is conducive to the rational use of corn silk resources. Our results suggested that corn silk could be a proper candidate for bacteriostatic and hypoglycemic agents due to the presence of bioactive compounds. In the future, we recommend isolating and purifying active compounds and performing more in vitro and in vivo studies for their potential pharmacological applications.

Data Availability
No publicly archived datasets were used to support the fndings of this study.