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Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Effect of Drying Methods on the Microstructure, Bioactivity Substances, and Antityrosinase Activity of Asparagus Stems Qun Yu, Jinwei Li, and Liuping Fan*

J. Agric. Food Chem. Downloaded from pubs.acs.org by MIDWESTERN UNIV on 01/29/19. For personal use only.

State Key Laboratory of Food Science & Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu 214122, People’s Republic of China ABSTRACT: The impacts of vacuum drying (VD), far-infrared drying (FIRD), hot air drying (HAD), and freeze drying (FD), as representative food drying methods, on structural characterization, bioactive substances, and antityrosinase activity of Asparagus have been assessed. The microstructure characterization by scanning electron microscopy indicated that VD treatment led to serious breaking of the vascular bundle and epithelial cells and provided higher free polyphenol (FP) and bound polyphenol (BP) contents. Besides, the smaller individual molecule (weight and hydroxy and phenolic rings) polyphenols bound to cellulose to a lesser extent than larger molecules, i.e., rutin and quercetin. In contrast, FD extracts possessed lower polyphenol contents but higher saponin and chlorophyll contents. The antityrosinase activity inhibition rates of FD and VD extracts were higher than those of FIRD and HAD for both mono- and diphenolase. The FP extract of VD, which possessed more polyphenolic compounds, had greater antityrosinase activity than BP. KEYWORDS: Asparagus, drying methods, free polyphenols, bound polyphenols, antityrosinase activity suitable for different active substances in the same fruit.19 For the flavanone or flavone extraction, freeze drying (FD) was more helpful. For the flavanol extraction, hot air drying (HAD) was more applicable. Sun et al. also found that FD was suitable for retaining polyphenol compounds and HAD was more efficient for the preservation of flavonoids.20 Thus, it is necessary for us to discuss the influences of different drying methods on Asparagus. Asparagus contains higher levels of polyphenol, saponin, flavonoid, and chlorophyll compounds, which represent positive effects on the human body. There are literature are about the total polyphenols, saponins, and flavonoids and antioxidant activity of the Asparagus extract, but little report is related to the effects of various drying methods on the free phenolic and bound phenolic compounds and their antityrosinase activity. Therefore, the aims of the present research were to compare the application of VD, FIRD, HAD, and FD in the vegetable processing of dehydrated Asparagus in terms of microstructure, color, total polyphenols, individual polyphenols, saponins, flavonoids, and chlorophyll. Moreover, this study also aimed at evaluating the FP and BP and their antityrosinase activity of Asparagus.

1. INTRODUCTION Asparagus (Asparagus officinalis L.) belongs to the Liliaceae family, which is a high-nutritional-value vegetable.1,2 Besides being a delicious food, it also possesses herbal medicine efficacy in many European and Asian countries. Plant-derived foods are widely consumed as a result of the nutritional components, including cellulose, polysaccharides, aspartic acid, polyphenols, saponins, glutamic acid, etc.3−5 Asparagus extract shows ameliorating scopolamine-induced memory impairments, antitumor activities, ameliorating sleep disturbances, and antioxidant and antityrosinase activities,2,3,6−8 which indicate that it is necessary for researchers to study the relationship between nutrition and function. Among them, polyphenols and saponins possess many biological activities; however, the best characteristic is their capacity to act as antityrosinase inhibitors.9−11 Both the chemical structure and food processing conditions can affect the biological activities of vegetables. As a representative processing method, a lack of knowledge on different drying methods affects their biological effects [especially free polyphenol (FP) and bound polyphenol (BP)] during vegetable processing. Dried products related to Asparagus have also been popular in recent years. Drying methods regulate the polyphenol, saponin, and chlorophyll contents and their biological activities in our consumption.12,13 Different drying methods have their own features.14 Vacuum drying (VD) is suitable for the substances that can react with oxygen easily because of the low vacuum degree. Nevertheless, VD needs a longer drying time as a result of the absence of air and the difficulty of heat convection.15 In comparison to VD, far-infrared drying (FIRD) accelerates the drying rate obviously. The electromagnetic wave energy can be absorbed directly by the materials, so that different parts of the material possess the same temperature.16−18 In addition, LedesmaEscobar et al. examined that different drying methods are © XXXX American Chemical Society

2. MATERIALS AND METHODS 2.1. Reagents. L-Tyrosine, Folin−Ciocalteu reagent, and gallic acid were purchased from the China National Pharmaceutical Group (Sinopharm) in Shanghai, China. Rutin, cinnamic acid, ferulic acid, quercetin, kaempferol, and sarsaparin were purchased from the Yuan Ye Biological Technology Company, Limited in Shanghai, China. 3,4Dihydroxy-L-phenyl-alanine (L-DOPA) was purchased from the B-mei Company. Received: November 20, 2018 Revised: January 10, 2019 Accepted: January 14, 2019

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DOI: 10.1021/acs.jafc.8b05993 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry 2.2. Plant Materials. Fresh asparagus (A. officinalis L.) stems were collected from Jiangsu Province, China, on August 2018. Fresh Asparagus were gently washed with pure water to clean the soil and dirt. Afterward, the fresh Asparagus were packaged and stored at 4 °C until dry. It is noted that the fresh Asparagus needs to be cut into cubes (3−5 mm) before drying. 2.3. Drying Methods. 2.3.1. Vacuum Drying (VD). A VD oven (DP33C, Yamao, Japan) was employed. The maximum load was 200 ± 5 g. The Asparagus cubes were put on wire netting in a drying oven. At the same time, the vacuum degree and temperature were set at −0.098 MPa and 70 °C, respectively. The final moisture content (FMC) of Asparagus was 0.116 ± 0.05 kg of water/kg of dry weight (dw). 2.3.2. Far-Infrared Drying (FIRD). A lab-scale far-infrared dryer was used for FIRD. The major parameter of this lab-scale far-infrared dryer is the drying temperature. Before the experiment, the farinfrared dryer was turned on under the setting temperature for 30 min. The temperature was set at 80 °C for 5 h. At last, the FMC of Asparagus was 0.093 ± 0.05 kg of water/kg of dw. 2.3.3. Hot Air Drying (HAD). HAD was accomplished in the drying and heating oven (Binder, Germany), and the setting temperature was 60 °C for 4.5 h. The FMC of Asparagus was 0.116 ± 0.05 kg of water/ kg of dw (243 ± 8 min) after the drying process. 2.3.4. Freeze Drying (FD). Fresh Asparagus stems were immediately frozen under −80 °C in a freezer. The FD of the Asparagus was carried out by the freeze dryer (Labconco, Kansas City, MO, U.S.A.), and the setting temperature was −64 °C. During the drying process, the pressure can maintain 0.035 mbar for 48 h. At the end of the drying process, the FMC of Asparagus was 0.134 ± 0.05 kg of water/ kg of dw. 2.4. Scanning Electron Microscopy (SEM). SEM was employed to study the microstructure of Asparagus cubes on the basis of the steps described by Liu et al., with minor modifications.21 Asparagus cubes were dried following the steps described in section 2.3. Then, they were dipped into 5% glutaraldehyde in methanol solution for 1 day, followed by another 24 h in glutaraldehyde. Then, they were introduced to 0.1 M phosphate buffer to rinse. Asparagus stem were fixed by 1% osmium tetroxide and then introduced into 0.1 M phosphate buffer to rinse again. Finally, the Asparagus cubes were dehydrated under the critical point temperature. Plasma sputtering was conducted for 30 s. Asparagus cubes were observed in SEM, Quanta 200 (FEI Company, Eindhoven, Netherlands), accelerated at 5 kV, with 10 mm working distance, and under a vacuum pressure of 40 Pa. 2.5. Color Measurement. The color of Asparagus stem powder was investigated by an UltraScan Pro1166 spectrophotometer (HunterLab, Reston, VA, U.S.A.). L, a*, and b* were selected to show the color. Among them, L is lightness (0 for black and 100 for white), a* is from red to green, and b* is from yellow to blue. Before the test, the color meter should be adjusted by a standard white. Data should be shown as an average value of five measurements [±standard deviation (SD)].14 The hue angle (h°) was calculated by the following equation:

2.6.2. Extraction of Bound Phenolics. Alkaline hydrolysis was employed according to the steps of Monente et al., with slightly alteration.23 A total of 40 mL of 3 mol/L NaOH solution was added to the residues. Then, the solutions were flushed with nitrogen gas. The mixture was incubated for 4 h at 40 °C. After the hydrolysis, samples were neutralized with 2 mol/L HCl to pH 2, and the mixtures were centrifuged for 15 min at 5000g. The supernatant was obtained and extracted with 20 mL of ethyl acetate mixture 3 times. The ethyl acetate layer was then evaporated to dryness at 50 °C and redissolved in 10 mL of methanol. All extracts were preserved at −20 °C until analysis. 2.6.3. Extraction of Saponins. A total of 20 mL of 90% ethanol was added to the beaker to extract 1 g of Asparagus powder. During extraction, the mixture was stirred (150 rpm) at 75 ± 2 °C and then passed through filter paper. The same steps described above were repeated 3 times. After that, the extracts were also evaporated to dryness at 50 °C. Ethanolic extracts should be stored at −20 °C until analysis.24 2.7. Quantification of Total Polyphenol Content (TPC) and Total Flavonoid (TF) Contents. The TPC was determined as gallic acid equivalents and measured by the Folin−Ciocalteu method.25 Briefly, 1 mL of Asparagus powder extract at an applicable concentration was mixed with 0.5 mL of Folin−Ciocalteu. After reaction for 6 min, 2 mL of 7.5% sodium carbonate was added to the mixture and put in a dark place for another 30 min at 70 °C. Finally, the absorbance of the solution was measured at 750 nm by an ultraviolet−visible (UV−vis) spectrophotometer (L8, INESA Co., Ltd., Shanghai, China). All of the experiments were repeated 3 times. Results were expressed as milligrams per gram of dw of Asparagus stem powder. The TF content can be determined according to the aluminum chloride colorimetric method described by Samad et al., with modification.26 Briefly, 1 mL of extract (applicable concentration) or rutin standard solution was mixed with 0.3 mL of 5% sodium nitrite and then reacted for 5 min. Then, 0.3 mL of 10% aluminum chloride was added to the test tube and reacted for 6 min. Then, 4 mL of 1 mol/L sodium hydroxide reagent was mixed with it. Distilled water was added to the solution to make the final volume of 10 mL. After the mixture reacted at room temperature for 12 min, the absorbance was measured against a blank at 510 nm with an UV−vis spectrophotometer. Results can be expressed as micrograms per gram of dw of Asparagus stem powder. 2.8. Analysis of Polyphenol by High-Performance Liquid Chromatography−Ultraviolet (HPLC−UV). The liquid chromatograph (HPLC) system equipped with a ultraviolet detector (UV) was employed to analyze the individual polyphenols.27 Polyphenol compounds can be separated by a Waters Atlantis C18 reversephase analytical column (4.6 mm inner diameter × 15 cm length, 3 μm particle size). The gradient profile for the separation of polyphenols was formed using solvent A (water with 0.1% acetic acid) and solvent B (acetonitrile with 0.1% acetic acid) in the following procedure: from 0 to 5 min, 30% B; from 5 to 25 min, linear gradient to 60% B; from 25 to 30 min, linear gradient to 100% B; and from 30 to 35 min, linear gradient to 7% B. 2.9. Quantification of Saponin. Saponin quantification was investigated using the perchloric acid method, with minor modifications.28 Perchloric acid was emplyed to quantificate the saponin content because it has the ability to produce a light brown stain as long as the solution contains saponin compounds. At first, 0.2 mL of extract of Asparagus powder was added to the test tube and had a water bath under 65 °C until it became dry. Then, the mixing sample with 5 mL of perchloric acid, with absorbance at 310 nm, was measured in all solutions after 15 min. A calibration curve based on sarsaparilla saponin was employed to investigate the final saponin contents (milligrams per gram of dw) in Asparagus powder. 2.10. Determination of the Antityrosinase Activity. The antityrosinase activity is referred to as the inhibition rate of bioactive substances on the ability of tyrosinase to catalyze L-tyrosine and LDOPA. Mono- and diphenolase inhibitory activities can be measured as a result of the melanine accumulation, and melanine has the

h° = arctan(b* /a*) 0° = red, 90° = yellow, 180° = green, and 270° = blue 2.6. Preparation of the Asparagus Stem Extract. 2.6.1. Extraction of Free Phenolics and Flavonoids. The dried Asparagus stems were smashed into powder. The extraction should be completed in lucifugal containers. The dried powder of Asparagus stems (1 g) was extracted with 30 mL of 70% (v/v) ethanol for 40 min. The speed of the magnetic stirring apparatus was set as 150 revolutions per minute (rpm), and the temperature was 26 ± 2 °C. The residue was subjected to the above extraction 3 times. Afterward, extracts were centrifuged for 15 min at 5000g. Then, the supernatants were combined and evaporated with a rotary evaporator at 60 °C under vacuum. Meanwhile, the residues were further extracted to obtain the bound extracts.22 B


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Journal of Agricultural and Food Chemistry Table 1. Consistency of the Reaction Mixture reaction component

group 1 (mL)

group 2 (mL)

group 3 (mL)

1.9 1 0.1 3.0

0.1 1.8 1 0.1 3.0

0.1 1.9 1

sample sodium phosphate buffer L-tyrosine/L-DOPA tyrosinase total volume

3.0

maximum absorption wavelength at 475 nm.9 All of the reactions were proceeded in 25 mmol/L sodium phosphate buffer (pH 6.8). The consistency of the reaction mixture can be seen in Table 1. The reaction was performed at 26 °C. The inhibition of tyrosinase activity can be calculated according to the following equation: inhibition (%) = [1 − (A2 − A3)/A1] × 100%. 2.11. Quantification of Chlorophyll. During the whole experiment, all steps were carried out in a dark place. A total of 2− 5 g of each sample was extracted with 10 mL of ethanol/acetone (1:1, v/v) and left to stand for 5 h in the dark, ensuring complete contact of plant material. The maximum absorption wavelengths were 645 and 663 nm.29 The spectrophotometer was adjusted to zero by the control (1:1 ethanol/acetone). According to the following equations, the chlorophyll a, chlorophyll b, and chlorophyll total contents can be estimated. chlorophyll a (mg/g) = (12.72A 663) − (2.59A 645) chlorophyll b (mg/g) = (22.88A 645) − (4.67A 663)

chlorophyll total (mg/g) = (8.05A 663) + (20.29A 645) 2.12. Statistical Analysis. An analysis of variance (ANOVA) was employed to estimate the significant effects. The difference was considered significant at the 95% confidence level (p < 0.05). All determinations were repeated 3 times, and the data are represented as means ± SD. Standard analysis were calculated by SPSS software (SPSS 20.0, IBM, Chicago, IL, U.S.A.).

3. RESULTS AND DISCUSSION 3.1. Morphology of Fresh and Dried Asparagus Stems. SEM characterization of the fresh and dried Asparagus stems was carried out to explore some structural changes induced by the drying treatments. Figure 1 exhibits 10 representative images of different Asparagus stems. The fresh Asparagus stems presented a integrated structure about its vascular bundle and epithelial cells regularly oriented, with an average diameter of around 100 and 50 μm, respectively (panels A and B of Figure 1). However, the average diameter of the FIRD, HAD, and FD samples significantly shrinked (around 16, 20, and 45 μm for the FIRD, HAD, and FD Asparagus stems, respectively). From these images, FD samples seemed to maintain the pore sizes (panels I and J of Figure 1). Related research has explained that FD is key for these samples to keep an integrated structure about its vascular bundle and epithelial cells similar to those of the fresh Asparagus stems.30 It could be because the strong inhibition of polyphenol binding in the FD Asparagus stems, in agreement with Liu et al. as a result of the drying process, should have fewer −OH to contribute to cellulose−polyphenol interactions.21,31 As clearly seen in panels C and D of Figure 1, the VD treatment led to serious breaking of the vascular bundle and epithelial cells, which could be explained by the higher temperature and vacuum degree. Such breaking was not observed in the FIRD-, HAD-, and FD-treated samples, suggesting that bioactivity substances may be expelled from the epithelial cells at a higher rate. Probably, the food drying process might increase the contents of polyphenol compounds that are released because

Figure 1. Scanning electron micrographs of (A) fresh, 300×, (B) fresh, 2400×, (C) VD, 300×, (D) VD, 2400×, (E) FIRD, 300×, (F) FIRD, 2400×, (G) HAD, 300×, (H) HAD, 2400×, (I) FD, 300×, and (J) FD, 2400× at two different scales (200 and 20 μm), showing the structural characterization of the Asparagus stems after different drying methods.

C


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Journal of Agricultural and Food Chemistry

Figure 2. Changes in color (L, h°, a*, and b*) and photos for the color difference of Asparagus powders obtained by selected drying methods. Different letters (a−d) denote statistically significant differences among samples by Duncan’s multiple range test at p < 0.05. VD, vacuum drying; FIRD, far-infrared drying; HAD, hot air drying; and FD, freeze drying.

of the breakdown of cellular constituents.32 A previous study also mentioned that there are more polyphenol substances in the outer textures of vegetables than in the vacuoles.33 This may be a good explanation for the later results. 3.2. Color. The Asparagus powder pictures and color parameter changes by four drying methods are represented in Figure 2. Different drying methods caused significant darkening in lightness (L) scores (Figure 2I). The highest and lowest values of parameter L were established for FD and HAD, respectively. This means that the HAD samples presented the darkest color, which is not a welcome surface. We can know that HAD may trigger a more severe browning reaction. The lowest value of parameter h° was established for HAD (Figure 2II). The h° parameter indicated a red pigment in the Asparagus, and therefore, the results expected that FD possessed a more violet pigment. The differences in the color tone among various drying powders could be the result of diverse hydroxyl groups attached to the anthocyanin and polyphenol molecules, consistent with the studies of Samad et al.26 Also, the h° parameter of the fresh green Asparagus ranged from 88.90° to 103.22°, which showed that the color circle represented green pigments and FD could maintain a similar color of fresh Asparagus (Figure 2II). At the same time, no significance differences in the h° parameter were detected

between VD and FIRD. From Figure 2III, we can note that a* values were all below zero, suggesting that Asparagus powders tended much more to green and a lower value represented better sensory quality. FD showed significantly lower a* than others, indicating a greener color. This might be due to the fact that the higher temperature in the dry chamber is a disadvantage for a greener color. A significant difference was observed in b*, among all drying methods. However, b* of HAD was up to the highest value, indicating that the HAD Asparagus powder will become yellow during drying. This phenomenon may be related with the degradation of carotene during the drying process. Our results confirm that the color tone of the samples is influenced by red pigments but also by green (chlorophyll) and yellow (carotenoid) pigments. It is also in agreement with previous literature.26,34 3.3. Total Polyphenol Content (TPC) and Individual Polyphenol Content (IPC). The TPC in the FP and BP extracts from Asparagus powder ranged from 605.62 to 791.79 and from 527.44 to 631.31 μg/g of dw, respectively (Table 2). Considerably variable amounts of TPC in Asparagus powders were previously reported, indicating the significant impact of processing on the stability of polyphenols in the Asparagus powders.26,35,36 The data suggested that the highest TPC was obtained by VD regardless of FP or BP extracts, which was D


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10.294 ± 0.05

11.332 ± 0.03

11.764 ± 0.02

16.297 ± 0.05

19.755 ± 0.02

B, rutin

C, cinnamic acid D, ferulic acid

E, quercetin

F, kaempferol

For each analytical method and sample, different letters indicate significant differences (p < 0.05) between different drying methods. FP, free polyphenol; BP, bound polyphenol; and ND, not detected.

4.580 ± 0.02 A, gallic acid

approximately 791.79 and 631.31 μg/g of dw higher when compared to powders using FIRD, HAD, and FD. That is to say, the highest retention of TPC was noted after VD, whereas their content was lower when FIRD and HAD were employed. The oxidation reaction of polyphenol is affected by the high vacuum degree, which can cause lower pressure and oxygen content.21,37 According to the microstructure mentioned above, the disruption of cell walls leads to the release of oxidative and hydrolytic enzymes, which, at the same time, could break the polyphenol compounds in Asparagus. However, 70 °C of the VD process would deactive these enzymes. Therefore, it is possible for VD to maintain the highest FP and BP contents compared to others. Drying methods influenced the IPC in Asparagus powders, and this was strictly connected with the structure of cellulose. Asparagus is rich in polyphenols and cellulose. From Table 2, the highest quantity of IPC in powders made from FP extracts was noted after VD (151.40, 117.90, 159.41, 244.46, and 118.67 μg/g of dw for gallic acid, rutin, cinnamic acid, ferulic acid, and quercetin, respectively). VD might be successfully used in terms of the polyphenols released during powder production. A previous study has confirmed that the interaction between polyphenol and cellulose can happen during the drying process.21 At the same time, the polyphenols can also bind to cell walls in the small intestine conditions.38 As observed in Table 2, it is interesting to find that gallic acid (A), cinnamic acid (C), and ferulic acid (D) of FD in FP extracts are 46.42, 44.32, and 160.90 μg/g of dw, respectively, and in comparison to VD, a significant decrease occurred. This may explain that FD can significantly reduce the binding capacity.21 During the drying process, cellulose becomes more tight, which may change the availability of the hydroxy group. According to Figure 3, gallic acid belongs to the smaller molecules because of its smaller weight and less hydroxy and phenolic rings compared to rutin. At the same time, the rutin content of VD is 16.54 μg/g of dw more than that of FD, but the gallic acid content of VD is 104.98 μg/g of dw more than that of FD (Table 2). This phenomenon was probably related to the binding capacity of polyphenols and cellulose. The smaller molecule (weight and hydroxy and phenolic rings) polyphenols binds to cellulose to a smaller extent than larger molecules, i.e., rutin (B) and quercetin (E). In fact, Liu et al. also found a similar phenomenon.21 According to a previous study, the polyphenol molecules can move freely in cellulose.39 In addition, the presence of changeable hydroxyl groups and conformational flexibility of phenolic rings favor the binding of polyphenols with cellulose.39−42 Cellulose can selectively absorb IPC, which has distinct binding affinities. These results indicate that different drying methods change the interaction of polyphenols onto cellulose, thereby affecting the TPC and IPC. 3.4. Saponin, TF, and Chlorophyll. To evaluate the effect of drying methods, saponin, TF, and chlorophyll contents were also measured. In the drying Asparagus powders, the saponin contents ranged from 1.73 to 2.69 mg/g of dw (Table 3) and were in the rank order of FD > FIRD > VD > HAD during different treatments, with statistically significant difference (ANOVA; p < 0.05) for each drying method. However, previous data on the Asparagus byproducts (between 2.14 and 3.64 mg/g) are in agreement with our results.28 As we know, onion and garlic are also widely used in folk medicine because of these pharmacological activities (antimicrobial, antityrosinase, anticancer, diuretic, hypoglycaemic, antithrombotic, etc.),

a

23.08 ± 3.57 a 22.59 ± 1.17 a 21.10 ± 1.08 a 22.24 ± 0.43 a ND ND ND ND 0.99908

34.04 ± 1.69 a 34.33 ± 1.01 a ND ND 75.45 ± 0.81 b 106.63 ± 3.04 a 81.52 ± 2.18 b 118.67 ± 9.10 a 0.99941

40.23 ± 2.00 c 66.26 ± 3.16 a 20.58 ± 2.25 d 51.84 ± 2.27 b 160.90 ± 2.82 b 244.80 ± 7.57 a 240.71 ± 9.86 a 244.46 ± 9.65 a 0.99973

52.87 ± 2.76 c 154.92 ± 8.18 a 151.76 ± 6.80 a 117.38 ± 4.28 b 44.32 ± 0.96 d 142.39 ± 6.75 b 125.24 ± 6.77 c 159.41 ± 8.54 a 0.99977

31.91 ± 1.15 b 34.03 ± 0.96 b 23.11 ± 0.17 c 39.33 ± 1.06 a 101.36 ± 2.38 a 106.77 ± 8.70 a 111.42 ± 2.00 a 117.90 ± 8.77 a 0.99752

19.50 ± 1.83 a 19.91 ± 0.83 a 18.19 ± 1.01 a 19.11 ± 0.68 a 46.42 ± 1.51 d 92.25 ± 2.00 c 108.78 ± 8.67 b 151.40 ± 6.88 a 0.99918

555.95 ± 5.06 b

FD HAD

553.53 ± 2.26 b 527.44 ± 5.65 c

FIRD VD

631.31 ± 4.39 a 605.62 ± 6.36 d

FD HAD

620.85 ± 6.32 c 685.68 ± 5.36 b

FIRD VD

791.79 ± 8.53 a 0.99554

y = −0.0013 + 0.0087x y = −49535.8 + 33793.81x y = −15744.2 + 11766.585x y = −83289.4 + 67445.8x y = −44969.8 + 35422.235x y = −46030.2 + 19066.13x y = −57209.4 + 30276.35x

r2 regression equation tR ± SD (min) polyphenolic compound

TP

BP (μg/g of dw) FP (μg/g of dw)

Table 2. Bioactive Substances of the Chromatographic Parameters for Individual Polyphenols in Asparagus FP and BP Extracts Obtained by VD, FIRD, HAD, and FDa


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Figure 3. Separation of (A) gallic acid, (B) rutin, (C) cinnamic acid, (D) ferulic acid, (E) quercetin, and (F) kaempferol using reversed-phase (RP)-HPLC−UV (detection at 280 nm).

which are because of the existing steroidal saponin. Its content is 2−3 mg/g of dw in onion and garlic, similar to Asparagus powders.43 Flavonoid also existed in the drying powders from Asparagus stems. Plant flavonoid bioactivity substances are the most widespread in secondary metabolism.26 Asparagus flavonoids play an important part in the antityrosinase characteristics. Hence, before studying the antityrosinase abilities of the FP and BP extracts, it is necessary to measure their TF contents. Both VD and FD samples (833.80 and 837.62 μg/g of dw) had higher contents of flavonoids than FIRD and HAD samples (742.65 and 592.71 μg/g of dw), indicating that FIRD and HAD led to a decrease of the TF content. As observed, the temperature and vacuum degree play an important role in the stability of flavonoids during drying. In addition, another condition should be taken into account during the extracting process, because continuous stirring was in an open beaker. Some flavonol compounds may react with oxygen. The lower content of saponins and TF in Asparagus powders led to a lower antityrosinase activity, which will be discussed below. In this case, VD and FD, which are considered as ideal drying methods, showed 833.80 and 837.62 μg/g of dw TF, respectively. Therefore, saponin and TF contents of the extracts indicate that Asparagus may be a potential antityrosinase inhibitor. The chlorophyll content is also an crucial indicator to evaluate different drying methods. The chlorophyll a, chlorophyll b, and total chlorophyll contents of dried Asparagus powders are shown in Table 3. FD samples possessed the highest total chlorophyll content compared to

VD, FIRD, and HAD, which was 0.401 mg/g of dw. Thus, thermal dehydration had greater degradation effects than nonthermal drying, and the result was similar to a previous study.3 Chlorophyll is closely related with human nutrition. It can be observed that, although samples of FD maintained a better green, it was a high energy consumption drying method as a result of its longer drying time. 3.5. Antityrosinase Activity. To estimate the antityrosinase activity of the dehydrated Asparagus, L-tyrosine and LDOPA were employed as substrates, separately. The percentages of mono- and diphenolase inhibition rates of FP and BP extracts are described in Table 3. Among the 16 treatments, all extracts exhibited a significant effect on the activity of tyrosinase. Regardless of the processing method employed, the FP extract of VD, which possesses more polyphenolic compounds, had greater antityrosinase activity than BP. These data suggested that the drying methods could affect the inhibition of tyrosinase. The inhibition rates of VD, FIRD, HAD, and FD about monophenolase in FP were 46.53, 23.30, 21.10, and 55.24%, respectively, indicating that the rates of FD and VD were higher than those of FIRD and HAD. FDprocessed samples produced higher antityrosinase activity, which accounted for the important role of saponins in the antityrosinase activity. According to previous papers, Asparagus possesses abundant steroidal saponins (shatavarins I−IV). Besides, gallic acid, rutin, quercetin, and kaempferol were also found in the roots and stems of Asparagus.9 On the one hand, tyrosinase inhibitors could bind with the copper active site; on the other hand, it also inhibits oxidation by the electrochemical process.6 Different bioactive substances have distinct inhibition F


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2.03 0.69 2.01 2.03 46.53 41.38 43.68 62.26 a c d b 0.89 0.56 0.62 0.69 57.98 11.84 8.65 44.44 b c c a 1.03 1.36 1.02 0.96 ± ± ± ± 46.74 23.30 21.10 55.24 d b c a 0.02 0.01 0.02 0.12 ± ± ± ± 0.229 0.350 0.262 0.401 c b c a 0.02 0.12 0.02 0.01 ± ± ± ± 0.045 0.106 0.060 0.152 c a b a 0.05 0.04 0.10 0.01 ± ± ± ± 0.184 0.244 0.202 0.249 1.98 2.25 1.73 2.69

0.39 0.36 0.66 0.43

c b d a

833.80 742.65 592.71 837.62

7.23 5.03 5.20 4.02

a b c a ± ± ± ± ± ± ± ± VD FIRD HAD FD

mechanisms. Kojic acid is widely used as a tyrosinase inhibitor, which can bind with the copper atoms in the active sites of the enzyme and the same inhibition mechanism with flavonoids. In addition, quercetin inhibited the reaction of L-DOPA by an electrochemical process. Although the inhibition mechanism of some crude extracts was still unclear, it is possible that hydroxyl groups and the furan ring can help increase its antityrosinase activity. In short, the structure similarity (3hydroxy-4-keto moiety) is an crucial substructure to interact with the copper ions in the active site.6 In summary, VD is shown to be effective in preserving the polyphenol compounds and also disrupts both the vascular bundle and epithelial cells. However, FD can result in better color and a marked increase in sapanins and chlorophyll. The drying time of FIRD is shortened obviously. Besides, Asparagus extracts exerted significant antityrosinase activity in vitro. Further experiments about the nutrition of Asparagus and the effects of food processing conditions on it are still needed.

a For each analytical method and sample, different letters indicate significant differences (p < 0.05) between different drying methods. TF, total flavonoid; FP, free polyphenol; BP, bound polyphenol; MPA, monophenolase; and DPA, diphenolase.

c b d a 0.68 0.30 0.95 1.02 ± ± ± ± 26.11 29.03 19.70 41.76 ± ± ± ± ± ± ± ±

b d c a

MPA inhibition rate (%) DPA inhibition rate (%) MPA inhibition rate (%) total chlorophyll (mg/g of dw) chlorophyll b (mg/g of dw) chlorophyll a (mg/g of dw) TF (μg/g of dw) saponin (mg/g of dw) drying method

FP

Table 3. Saponin, Flavonoid, and Chlorophyll Contents and Antityrosinase Activity (Mono- and Diphenolase) of Different Drying Method Samplesa

BP

DPA inhibition rate (%)


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AUTHOR INFORMATION

Corresponding Author

*Telephone: 0086-0-510-85876799. E-mail: fanliuping@ jiangnan.edu.cn. ORCID

Liuping Fan: 0000-0003-1312-8057 Funding

This research was financially supported by the Jiangsu Agriculture Science and Technology Innovation Fund [CX(18)3070], the China National Natural Science Foundation (31871840), the Six-Talent Peaks Project in Jiangsu Province, and the QingLan Project, which have enabled us to carry out this study. Notes

The authors declare no competing financial interest.

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