The antioxidant activity was measured using DPPH and FRAP tests. 0.4 mL of the sample solution (0.025–0.2 mg/mL) was added to 2 mL of the DPPH methanolic solution (0.2 mM) for the DPPH radical scavenging experiment, and the combination was incubated in the dark for 30 min before the absorbance was measured at 517 nm. DPPH radical scavenging activity was measured in milligrams of trolox per gram (33). To conduct the FRAP experiment, 0.5 mL of potassium ferricyanide (1%) was combined with 0.2 mL of NA sample (0.025–0.2 mg/mL). The resultant mixture was then incubated for 20 min at 50°C. After adding 2.5 mL of 10% trichloroacetic acid to the mixture, it was centrifuged at 2200 × g for 20 min. Combining 0.5 mL of the top layer with 0.1 mL ferric chloride and 0.5 mL of distilled water yielded a solution. FRAP was estimated as mg TE/g material after measuring absorbance at 700 nm (33).

According to the literature, acetylcholinesterase (AChE) and butyryl-cholinesterase (BChE) were used to perform in vitro anticholinesterase activities (34). Briefly, 50 μL of NA sample solution (0.025–0.2 mg/mL) was coupled to 5,5-Dithio-Bis(2-Nitrobenzoic) acid plus 25 μL of AChE or BChE solutions in microtiter plates with 96 wells and then incubated at 25°C for 15 min. Following the addition of 25 μL of acetyl-thiocholine iodide or butyryl-thiocholine chloride, the solution was incubated for 10 min at 25°C for AChE and BChE, respectively. At 405 nm, the absorbance was measured. The AchE and BchE in vitro inhibitory activities were determined in milligrams of galantamine equivalents per gram of material (mg GALAE/g).

α-amylase inhibition activity was evaluated according to McCue and Shetty (35). Briefly, 50 μL of the α-amylase solution produced in phosphate buffer was mixed with 25 μL of the sample solution (0.025–0.2 mg/mL) in 96-well microplates. The reaction began by adding 50 μL of 0.5% starch solution to the microplates, which were then incubated for 10 min at 37°C. At 630 nm, the absorbance was measured after adding 25 μL of 1 M HCL and 100 μL of potassium iodine solution (0.25 mM) (35). The α-amylase inhibitory action was reported in mmol ACE/g.

Utilizing L-DOPA as a substrate, the tyrosinase inhibition experiment was carried out in accordance with the technique previously described by Chiari et al. (36). The sample mixture (20 μl, 0.025–0.2 mg/mL), aqueous fungal tyrosinase solution (10 μl, 50 units/mL), and phosphate buffer (pH 6.8, 80 L) were combined and incubated for 5 min at 37°C. Next 90 μl of L-DOPA (2 mg/mL) was added. The mixture was then incubated at 37°C for 20 min. At a wavelength of 475 nm, the concentration of dopachrome was determined. As a control, kojic acid in dimethyl sulfoxide was utilized. The inhibition rate of tyrosinase was computed as follows:

The Barrantes and Guinea (37) method was applied to determine the inhibiting collagenase activity. After adding 800 μL of 0.1 M Tris–HCl (pH 7) and 100 μL of the sample (0.025–0.2 mg/mL) to each tube, a consistent mass of 1 mg of collagen that had been penetrated with Azo dye was measured in the tubes. The mixture was then immediately mixed with 100 μL of collagenase (200 units/mL), and it was let sit for an hour at 43°C. The tubes were then centrifuged for 10 min at 3000 rpm. Each tube’s supernatant was collected on 96-well plates. Negative control was carried out using buffer and substrate but no enzyme. Fluorescence was measured at 495 nm for excitation and 515 nm for emission. All experiments were carried out in duplicate and independently. The inhibition rate of collagenase was determined using the following formula:

Where S is the test sample’s absorbance, Sb is the blank sample’s absorbance, C is the positive control’s absorbance, and Cb is the negative control’s absorbance.

Using the porcine pancreatic elastase enzyme inhibition test, the in vitro elastase-inhibiting activity was assessed (38). This examination was conducted in a buffer containing 0.2 M Tris–HCL (pH 8.0). To create a 100-unit stock solution of porcine pancreatic elastase, the enzyme was dissolved in sterile water. In the buffer, the substrate N-Succinyl- Ala-Ala-p- nitroanilide (AAAPVN) was dissolved. Before adding the substrate to initiate the reaction, the enzyme was incubated for 25 min with the plant extracts. 250 μL of buffer, 10 μg/mL AAAPVN, 0.001 unit of porcine pancreatic elastase, and 25–200 μg/mL of samples comprised the reaction mixture. Maximum velocities (Vmax) were monitored at 410 nm for 25 min at 30-s intervals, and the inhibition rate was computed by employing the equation:

Where Asample is the sample’s absorbance and Acontrol is the assay’s absorbance when the buffer is used in lieu of an inhibitor (sample). Quercetin was the active control drug that was used. The chemical used as a control was tris–HCl.

The inhibition of hyaluronidase was measured as previously reported by Sahasrabudhe and Dedhar (39). 50 μL of the sample (0.025–0.2 mg/mL) was dissolved in 5% dimethyl sulfoxide, followed by the addition of 10 μL of bovine testicular hyaluronidase type 1-S (4200 units/mL) in 0.1 M acetate buffer (pH 3.5). The mixture was then put in a 37°C water bath for 20 min of incubation. Once hyaluronidase activated Ca2+ had been treated with 50 μL of sodium hyaluronate (12 mg/mL) dissolved in 0.1 M acetate buffer (pH 3.5), it was incubated at 37°C for 40 min. The mixture was combined with 10 μL of 0.9 M sodium hydroxide and 20 μl of 0.2 M sodium borate before being incubated in a water bath at boiling temperature for 3 min. After cooling to ambient temperature, the reaction was heated in a 37°C water bath for 10 min. Afterward, 50 μl of a p-dimethylamino benzaldehyde solution was added (0.25 g of p-dimethylamino benzaldehyde dissolved in 21.88 mL of 100% acetic acid and 3.12 mL of 10 N hydrochloric acid). The control group was given 50 μl of 5% dimethyl sulfoxide instead of the sample. At 585 nm, the absorbance was measured. Tannic acid was utilized as a comparative standard. Using the following formula, the enzyme inhibition rate was calculated.

The absorbance of the sample extracts is denoted by “Asample,” and the absorbance of the test conducted in the absence of an inhibitor is denoted by “Acontrol.”

To choose an appropriate DES for NA extraction, six distinct kinds of DES were synthesized based on the various hydrogen bond donors (formic acid, oxalic acid, lactic acid, urea, glycerol, and malic acid). Figure 1A demonstrates how the extraction efficiency changes with various DESs. The highest NA yield was achieved by choline chloride-formic acid (DES-1: 0.97 mg/g). There were significantly lower yields for NA extraction when using choline chloride-citric acid (DES-6: 0.67 mg/g) compared to the other DESs we tested. Because of its greater electrostatic interaction with flavonoids than the other DESs, DES-1 demonstrated better extraction efficiency than the others (40). Moreover, formic acid and choline chloride, which exist naturally in many whole foods, are totally safe to ingest (41). The DES system (DES-1) composed of choline chloride and formic acid was used in the following investigations.

As previously mentioned, the molar ratio of HBD to HBA in the DES regulates the interaction of hydrogen bonding, which in turn influences the viscosity of the DES (42). The physical properties of DES also have an impact on the efficiency with which NA is extracted (41, 42). We did not attempt to increase the choline chloride level since DES crystallizes at a molar ratio greater than 3.5:1. It can be seen in Figure 1B that the selectivity and extraction rate of NA increase with the mole ratio of choline chloride (ChCl) to formic acid (FA), reaching maximum values at a 2:1 mole ratio of ChCl to FA. The increase in ChCl content in DES may increase the interaction of NA with DES, which increases the selectivity and extraction rate. However, when the mole ratio of ChCl to FA is further increased from 2.5:1 to 3.5:1, the selectivity and extraction rate of NA decrease. Due to this, the mole ratio of ChCl-FA in DES-1 used in the subsequent experiments was 2:1.

As discussed earlier, the molar ratio of HBD to HBA in the DES controls the hydrogen bonding interaction, which affects the viscosity and surface tension of DES (43). As a result, optimizing the mass transfer rate and processing efficiency relies on selecting appropriate water content in DES. Figure 1C demonstrates how the amount of water present in the DES-1 significantly affected the extraction yield. Increasing the water ratio in DES-1 from 20 to 50% led to an important improvement in the extraction yield of NA. The high extraction yield may have been achieved because DES-1 viscosity and polarity were optimized by adding water, resulting in more effective interactions between ST and DES-1 (43, 44). Yet, the extraction efficiency dropped dramatically when the water content was brought up to 50%. The low viscosity and great fluidity of DES-1 with a water content of 30–50% made it less expensive to manufacture. Thus, for the next tests, DES-1 with 30–50% water content was chosen.

The successful extraction of bio-compounds from plant material requires optimizing a moderate temperature while keeping economic considerations in mind (45). The optimal temperature for this investigation was between 50 and 70°C. Figure 1D illustrates how yield increased as temperature rose because of enhanced solvation, increased material porosity, and mass transfer. Due to the solvent’s diffusivity in the tissue matrix, the increase in temperature also reduces surface tension and viscosity in extracts, improving extraction yield (46). The quantity of NA that was extracted increased as the temperature rose. The largest quantity of NA that could be extracted was achieved at a temperature of 60°C, after that, when the temperature was raised, the rate of NA extraction fell. This can be mostly since raising the temperature to a certain point enhances NA release. According to this theory, the deterioration of the desirable components occurs at higher temperatures (beyond the optimal temperature) because more cavitation bubbles develop and then collapse, creating shear stress (47). According to a study on the thermal stability of NA (48), the compound is most stable when heated to 60°C. Ultrasonic extraction coupled with thermal hydrolysis significantly improved the extraction of NA from the albedo parts of Citrus paradisi, as reported by Stabrauskiene et al. (8), who also found that 50°C provided a better yield of NA than 70°C. As a result, the concentration of NA in the subsequent studies was highest between 50 and 70°C.

Under the established extraction conditions (temperature, time, solid–liquid ratio, and water content in DES-1 were kept constant at 50°C, 10 min, 1/30 g/mL, and 30%, respectively), the effects of different extraction powers (30, 60, 75, 90, 115, 130, 150, and 180 W) on NA yields are presented in Figure 1E. As the extraction power was increased to 60 W, the NA content was enhanced gradually. The extraction efficiency of natural compounds can be clearly increased by enhancing the speed of molecular motion and solvent penetration because of the mechanical effect, acoustic cavitation, and cavitation intensity characteristics (49, 50). Nonetheless, when the power further increased, the extraction yields of NA declined. During the extraction, the increase in power will cause a lot of impurities, which will lead to a decrease in NA yields. Consequently, the ultrasonic power was set between 60 and 120 W in the following investigations.

The period of ultrasonic exposure has a considerable effect on the extraction of compounds (51). Longer extraction times have been recorded during traditional extraction techniques geared toward the hydrolysis and oxidation of phenolic compounds (52). Ultrasound-assisted extraction is acknowledged as a sophisticated approach that may expedite the mass transfer and consequently improve extraction kinetics (53). Consequently, a helpful aspect of this technology is the shortened extraction time with less solvent usage. The optimal extraction would aid in lowering the method’s energy and cost, as well as making commercialization simpler in pharmaceutical and other relevant industries (53). In the current investigation, the extraction of NA from ST was performed at 5–120 min (Figure 1F). The quantity of NA extracted rises with increasing extraction time, reaching a maximum of 20 min (0.84 mg/g) before beginning to fall. Increased NA extraction by increased release of NA into the solvent from the powder may occur with extended exposure, however, this may also result in NA deterioration, according to (51). To save time and energy and improve the extraction of NA, the following studies used extraction times ranging from 10 to 40 min.

As the biomass-to-solvent ratio has a substantial effect on NA yield, optimizing this variable is crucial for maximizing extraction (54). A solid–liquid ratio between 20 and 70 mL/g was used to extract NA from ST. The ratio of 1–10 g/mL produced the greatest extraction yield of NA, following which equilibrium was restored (Figure 1G). When the solid/liquid ratio is low, more solvent is available to infiltrate the plant matrix, hence enabling the bioactive compounds to dissolve and boosting the extraction yield (55). Hence, the optimal range for testing is between 1/40 and 1/60 g/mL (solid/liquid).

The Design Expert program (version 10.0.7) was used to investigate the key UAE parameters using the RSM according to SFED findings. To maximize the interacting effects of five factors, Supplementary Table S3 displays data from 46 trials with five factors and three levels. The Table 2 presents a summary of the ANOVA findings for Y (NA yield mg/g) as a response. The p-value of the regression model, which is 0.0001, demonstrates the model’s high significance. Because of how well the model suited the data, the p-value for the lack of fit was just 0.1268, indicating that it was not statistically significant. Very high significance (p < 0.0001) was found for the model parameters X1, X2, X5, X1X2, X2², X3², and X5². In other words, X1X4, X2X4, X2X5, and X1² all had statistically significant p-values (p < 0.05). The remaining factors did not reach statistical significance (p > 0.10). Table 2 presents the results of the analysis of variance of the model, which reveals that the most influential factors on NA extraction in ST were ultrasonic temperature (with three significant interaction conditions: X1X2, X2X4, and X2X5), duration of ultrasonication (with two significant interaction conditions: X1X2, X1X4), and DES-1 water content (with one significant interaction condition: X2X5). The connection between ultrasonic power, time, temperature, solid–liquid ratio, and water content in DES-1 is shown as a quadratic equation below:

Where Y is the NA yield in micrograms per gram (μg/g)and X1, X2, X3, X4, and X5 are five numeric variables representing the amount of water in DES-1, the solid–liquid ratio, the time of ultrasonography, and the temperature, respectively. Both the raw and adjusted model correlation coefficients (R²) were used to assess the strength of the relationship between the variables in question and the observed response (Y). With an adjusted R² of 0.9618, a strong model fit is shown, and with an R² of 0.9812, a high prediction for the response model is indicated. Both the R² and the modified R² demonstrate a significant relationship between the theoretical and empirical estimates. Also, the corrected R² of 0.9279 is quite like the expected R² of 0.9279 for Y in this scenario (Table 2). For all 46 experiments, the average output of NA was between 885.68 and 1244.66 μg/g (Supplementary Table S3). Trial 1’s conditions of 10 min of ultrasonication at 50°C, 60 milliliters of solvent per gram of solid, and 75 watts of ultrasonic power produced the highest extraction yield for NA (Supplementary Table S3).

The response surfaces for NA extraction show the interplay of UAE-DES extraction parameters (Figures 2A–D). Figure 2A shows a response surface depicting the temperature’s dependence on the ultrasound’s exposure time. When the combined value of these two parameters is increased, the rate at which NA is extracted lowers. In addition, the figure reveals that the maximal extraction efficiency of total NA rises at low temperatures but falls at high ones, namely those over 70°C. It’s possible that the extremely high temperatures used in extraction damage the flavonoid structures, leading to this outcome (56). Figure 2B demonstrates that at the maximum ultrasonic duration of 45 min, an increase in ultrasonic power results in a greater extraction yield. Although the greatest quantity of NA extraction yield did decrease with increasing microwave ultrasound time, this was not always the case. This is because ultrasonic waves may reduce the sample’s NA if exposed to them for too long (57). The response surface, shown in Figure 2C, demonstrates how ultrasound time and temperature interact to alter the yield of NA extraction. Hence, the longest duration and the coolest ultrasonic temperatures should provide the largest yield. Nevertheless, raising the temperature to 80°C resulted in a decrease in extraction yield as time was increased. There is a trade-off between improving mass transfer and extraction yield and utilizing high ultrasonic temperature, since the latter may disrupt the structure of flavonoids, which are sensitive to damage (58). Figure 2D shows that a higher NA yield is achieved when the ultrasonic temperature is lowered, and the DES-1 sample’s water content is increased. It has also been shown that, at very high temperatures, extraction yield rises in tandem with water content. This evidences a strong interplay between these two parameters on the response surface during NA extraction.