Ultrasonic-Assisted Extraction of Natural Yellow Pigment from Physalis pubescens L . and Its Antioxidant Activities

Physalis pubescens L. is rich in natural pigments but has not yet been fully utilized. Ultrasound-assisted extraction of yellow pigment from Physalis pubescens L. was investigated by response surface methodology in this study. Optimal parameters were ultrasonic power of 29.21%, ultrasonic time of 14.41min, and ultrasonic interval time of 10.55 s. +e yield was 0.193% under optimal parameters. FRAP, ABTS, and superoxide radical scavenging activity of the yellow pigment were 6.11± 0.22mmol/g, 2.80± 0.27mmol/g, and 57281.5± 2749.5U/g, respectively.+e results showed that the yield of yellow pigment could be improved by ultrasonic-assisted extraction and the yellow pigment extracted by ultrasound had antioxidant activity.


Introduction
Physalis pubescens L. is a plant cultivated mainly in Hulunbeier League in Inner Mongolia and Greater Hinggan mountains in Heilongjiang Province, China [1].Physalis pubescens L. is a kind of nutritious and healthy fruit, which contains good amounts of vitamin C, niacin, carotenoids, minerals, antimicrobial molecules, and bioactive withanolides, and it could be a good potential source of essential amino acids [2][3][4].Physalis pubescens L. has been used as a traditional folk medicine to treat sore throat, cough, urethritis, hematuria, and orchitis [5].So Physalis pubescens L. has a nutrient, medicine, and economic importance in many countries.
e fruits of Physalis are consumed either fresh or processed, mainly for jam, canning, or preserves [6], but less attention is paid to the rich natural yellow pigment resources in fruit.Natural yellow pigment is important for food industry, because natural pigment is much safer and healthier [7], so the demand for natural pigments is growing.Ultrasound-assisted extraction could increase the yield of extracted components, achieving reduction in extraction time and higher processing throughput [8].Now ultrasonic-assisted extraction has been successfully applied with extraction of anthocyanins, carotenoids, phenolic compounds, and flavonoids [9][10][11][12].Ultrasonic-assisted extraction has been applied with extraction of Pueraria isoflavonoids, and the ultrasonic-assisted extraction condition was optimized by response surface methodology (RSM), for the maximum recovery of isoflavonoids with high cytoprotective effect [13].Ultrasonic-assisted extraction of phenolic compounds from fresh olives was studied to increase the yield of phenolic compounds [14].e anthocyanins and other phenolic compounds from purple eggplant peels and pulps were extracted by ultrasonicassisted extraction, and these results indicated that suitable ultrasonic pretreatment can reduce the extraction time with high yield of phenolic compounds [15].Ultrasonicassisted extraction has been applied with extraction of phenolics and antioxidant compounds from rhizomes of Rheum moorcroftianum using response surface methodology, and the results indicate that the design to optimize the extraction of polyphenolic compounds is critical for precise quantification of antioxidant phenolics [16].Ultrasonicassisted extraction could be applied to extract the target compounds in complex plant samples [17].
In this paper, Physalis pubescens L. grown in China was chosen as raw material; ultrasound-assisted extraction of yellow pigment of Physalis pubescens L and antioxidant activity of yellow pigment were investigated.e purpose of this research was to study an efficient extraction method of natural yellow pigment of Physalis pubescens L. e result could provide a kind of natural yellow pigment with excellent function to the food industry, which would enhance the economic and nutritional value of Physalis pubescens L.

Materials.
e raw Physalis pubescens L. was purchased in a local market, rutin was provided by National Institutes for Food and Drug Control (Beijing, China), and ethanol was purchased from Tianli Chemical Reagent Co. (Tianjin, China).e superoxide radical scavenging assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Jiangsu, China).Ferric-reducing antioxidant power (FRAP) assay kit and the total antioxidant capacity (ABTS method) assay kit were purchased from Beyotime Institute of Biotechnology (Shanghai, China).

Determination of Yellow Pigment.
Yellow pigment was determined by spectrophotometry based on the modified method by Jia and Wu [18,19].e total amount of reaction system was 25 mL. 5 mL extract was diluted to 10 mL with 75% ethanol and then 0.75 mL NaNO 2 (5% W/V) was added.After 5 minutes, 0.75 mL Al(NO 3 ) 3 (10% W/V) was added into the solution and the mixture was mixed well.After 6 minutes, 5 mL of sodium hydroxide was added into mixture.e mixture was placed at room temperature for 10 minutes.e absorbance was measured by a UV-1800 spectrophotometer (Pgeneral Instrument (Beijing) Co. Ltd., Beijing, China) at 510 nm.Yellow pigment was determined using rutin calibration curves (y � 6.412x + 0.003; R 2 � 0.998, where y is the absorbance and x is the rutin equivalent concentration (mg/ml)).
Yield (%) � m 1 /m 2 × 100%, where m 1 is the weight of the extracted yellow pigment and m 2 is the weight of the sample.

Single-Factor Experiments.
Factors and levels of singlefactor experiment design are shown in Table 1.e three single factors that affected the extraction of yellow pigment were ultrasonic power, ultrasonic time, and ultrasonic interval time.

Response Surface Design.
Response surface design was applied to find the optimal yield of yellow pigment.e optimization of extraction parameters was designed based on the single-factor experiments, and the ranges of the variables investigated are displayed in Table 2: ultrasonic power 25%-35%, ultrasonic time 12-18 min, and ultrasonic interval time (5 s, 10 s, and 15 s).
e prepared ABTS solution was kept at room temperature in dark for 12-16 h.e prepared ABTS solution was then diluted with 10 mM phosphate buffer (PBS, pH 7.0) to obtain an absorbance of 0.70 ± 0.05 at 734 nm [20].
e reaction was initiated with addition of 10 μL sample solution into 200 μL ABTS mixture.e absorbance at 734 nm was recorded after 5 min at room temperature.
e standard curve was constructed using Trolox standard solution [21].Total antioxidant capacity (mmol/g) � C 1 /C 2 , where C 1 is the concentration of the Trolox standard solution when inhibition rate of the sample is equal to the inhibition rate of Trolox standard solution (mmol/L) and C 2 is the concentration of the sample (mg/ml).

Superoxide Radical Scavenging
Activity.Superoxide radical scavenging activity was determined by using an assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions [22].e absorbance of reaction solution was measured at 550 nm.Superoxide radical scavenging activity is expressed as U/g.

FRAP (Ferric-Reducing Antioxidant Power) Assay.
FRAP was measured by commercial kit of Beyotime Institute of Biotechnology (Jiangsu, China).5 μL sample solution was added into 180 μL FRAP working solution at room temperature, and the mixture was incubated for 5 min at 37 °C.
e absorbance of the mixture was measured at 593 nm. e PBS was used as blank control.e standard curve was constructed using FeSO 4 •7H 2 O solution [23].  2 Journal of Chemistry e total antioxidant capacity (mmol/g) � C 1 /C 2 , where C 1 is the concentration of the FeSO 4 solution when the absorbance of the sample is equal to the absorbance of the FeSO 4 solution (mmol/L) and C 2 is the concentration of the sample (mg/mL).
2.9.Statistical Analysis.All the data were analyzed using SPSS 17 software (SPSS Inc., Chicago, IL). e results were expressed as mean ± SD.

Influence of Ultrasonic Power on Yield of Yellow Pigment.
e effect of ultrasonic power on yield of yellow pigment was investigated.Ultrasonic power was taken at 15% (97.5 W), 30% (195 W), 45% (292.5 W), 60% (390 W), and 75% (487.5 W), respectively, while other extraction variables were as follows: the solid-liquid ratio of 1 : 12 and ethanol concentration of 75%.e results are shown in Figure 1.It could be seen that the yield was the highest when the ultrasonic power was 30% (195 W) and the yield value was 0.394 ± 0.033% (Figure 1).e lowest yield among the five points appeared when the ultrasonic power was 45% (270 W) and the yield value was 0.244 ± 0.025%.When the power was increased from 45% to 75%, the yield rose again, but never more than the maximum value.e materials swell and the pores enlarge as a result of the microjet and the violent shock wave produced by the ultrasonic wave, which enhanced the extraction yield.However, the number of cavities might increase sharply above 195 W of ultrasonic power, which negatively influenced the efficiency of the ultrasound energy transmitted into the medium, and reduce the extraction yield [24].

Effect of Ultrasonic Interval Time on Yield of Yellow
Pigment.Ultrasonic interval time (5 s, 10 s, and 15 s) was investigated at the solid-liquid ratio of 1 : 12 and ethanol concentration of 75%.e results are shown in Figure 2.
e results showed that the yield of yellow pigment was the highest when the ultrasonic interval time was 10 s and the yield value was 0.454 ± 0.057%.e yield was lower   Journal of Chemistry at ultrasonic interval time of 5 s and 15 s (Figure 2).It might be that ultrasound facilitated the natural yellow pigment release to the exterior solvent and most of the natural yellow pigment released [25].It could be seen from Figure 3 that the yield increased with increase in ultrasonic time.e highest yield appeared when the ultrasound time was 15 min and the yield value was 0.699 ± 0.018%.ese results could be explained as the increase of extraction time led to the increase of material cell disruption and speeded up both the release and diffusion into water [26].

E ect of Ultrasonic Time on Yield of Yellow
3.4.Response Surface Methodology.Ultrasonic power, ultrasonic time, and ultrasonic interval time were used as the dependent variables, and the yield of yellow pigment was taken as the response value.e response surface analyses were carried out by the design expert software.e results are listed in Table 3.According to a regression analysis of the experimental data, the yield could be explained by the following equation: where Y represents the yield of yellow pigment; A, B, and C are the ultrasonic power, ultrasonic time, and ultrasonic interval time, respectively.P values of the model less than 0.05 indicate that the model terms were significant and lack of fit was not significant (P > 0.05).
e response surface plots shown in Figure 4 demonstrated the changes in the yield of yellow pigment with the interaction effects of the three variables.All the three surfaces were upper convex, with a maximum point in the center of the experimental domain.
is result demonstrated that the ranges of factors were chosen properly [27].
e yield of yellow pigment increased with increasing ultrasonic power from 25% to 29.21% and then decreased slightly above 29.21%.e yield of yellow pigment efficiency increased with the ultrasonic time increasing from about 12 to 14.41 min, but decreased after the optimum value (14.41 min).e possible reason to explain this result was that the ultrasound waves generated cavitation and mechanical forces and improved the mass transfer by disrupting the cell walls.On the other hand, extreme high ultrasonic power and time could decompose target compounds [17,26].

Antioxidant Activity.
e antioxidant activity of yellow pigment was studied by measuring the FRAP assay, superoxide radical scavenging activity (O 2 − ), and ABTS assay.e results are shown in Table 4.In Table 4, the FRAP value of the yellow pigment was 6.11 ± 0.22 mmol/g and this result was higher than the FRAP value of the 50% of ethanolic corn silk extracts (0.44 ± 0.08 mg/g) [28] and the FRAP value of kidney bean (99.36 ± 1.26 μmol/g) [29].
e activity of inhibiting O 2 − free radical was 57281.5 ± 2749.5 U/g, which was much larger than O 2− inhibition activities of Arabidopsis seedlings (<1000 U/g) [22].e ABTS value of the yellow pigment was 2.80 ± 0.27 mmol/g and this result was better than that of the bioactive compounds from palmpressed fiber by ultrasound-assisted extraction, which inhibited the ABTS radical activity of 0.346 mmol/g [30].Yellow pigment exerted significantly higher ABTS•radical scavenging activity as compared to I. galbana (0.086 ± 0.006 mmol/g) and C. calcitrans (0.068 ± 0.003 mmol/g) [31].
e results showed that the yellow pigment extracted from Physalis pubescens L. had a good antioxidant activity, but the relationship between antioxidant activity and yellow pigment concentration and the mechanism of action needed to be further explored.

Conclusions
It could be proved that the method of ultrasonic-assisted extraction of yellow pigment from Physalis pubescens L. was indeed effective and feasible in this study.
e optimal process parameters solved by the regression equation were ultrasonic power of 29.21%, ultrasonic time of 14.41 min, and ultrasonic interval time of 10.55 s. e yield was 0.193% under optimal process parameters.Furthermore, the results showed that the crude yellow pigment from Physalis pubescens L. had antioxidant activity.e results provided evidence for further development and utilization of yellow pigment of Physalis pubescens L.

Figure 1 :Figure 2 :
Figure 1: Influence of ultrasonic power on yield of yellow pigment.

Figure 3 :
Figure 3: Influence of ultrasonic time on yield of yellow pigment.

3
Pigment.Ultrasonic time (3 min, 6 min, 9 min, 12 min, and 15 min) was investigated at the solid-liquid ratio of 1 : 12 and ethanol concentration of 75%.e results are shown in Figure time (s) A : u lt r a s o n ic p o w e r ( % ) B : u lt ra so n ic ti m e (s )

Figure 4 :
Figure 4: Response surface and contour plot of ultrasonic-assisted extraction on yield of yellow pigment.

Table 1 :
Factors and levels of single-factor experiments.

Table 2 :
Response surface design.

Table 3 :
Results of response surface experiment.

Table 4 :
Antioxidant activity of yellow pigment.