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Journal of Food Science and Technology logoLink to Journal of Food Science and Technology
. 2019 Jun 11;56(9):4016–4024. doi: 10.1007/s13197-019-03870-y

Effects of high pressure extraction on the extraction yield, phenolic compounds, antioxidant and anti-tyrosinase activity of Djulis hull

Hsiao-Wen Huang 1, Ming-Ching Cheng 2, Bang-Yuan Chen 3, Chung-Yi Wang 4,✉
PMCID: PMC6706478  PMID: 31477973

Abstract

The hulls of Djulis (Chenopodium formosanum) are a type of agricultural waste. Using 70% ethanol as the extraction solvent, this study compared the extraction yields of high-pressure-assisted extraction (HPE) and conventional oscillation extraction (CE) for Djulis hulls (DH). The total phenolic and flavonoid contents, and antioxidant, anti-inflammatory and anti-tyrosinase activities were also compared. Our findings indicated that 600 MPa/5 min of HPE resulted in higher total phenolic (567–642 mg GAE/g) and flavonoid (47.2–57.2 mg QU/g) concentrations; gallic acid (44.5–53.2 μg/g) and rutin (26.8–34.2 μg/g) were the main phenolic and flavonoid compounds. When the extraction pressure was greater than 450 MPa, HPE extracts showed stronger antioxidant capacity and anti-tyrosinase activity than CE extracts. In a LPS-induced RAW 264.7 cell model of inflammation, HPE extracts had significant inhibitory effects on the cumulative concentrations of nitric oxide and prostaglandin E2. These results indicate that HPE had a better extraction yield, and required a shorter time for the extraction of functional ingredients from DH. Hence, DH could be a potential source for natural antioxidants for the food and biotechnology industries.

Keywords: High pressure extraction, Djulis hull, Antityrosinase, Antioxidant, Anti-inflammatory

Introduction

Djulis (Chenopodium formosanum) is a native cereal plant of Taiwan. This traditional crop has been used as a cereal plant for several centuries, but its identity has only recently been confirmed. One of the species cultivated by the aborigines of Pingtung was recently identified as C. formosanum (Koidz) (Narkprasoma et al. 2012). Djulis is rich in nutritional content, including proteins, essential amino acids, functional dietary fiber, and starch. This plant contains betanin and isobetanin pigments, which have ferric reducing antioxidant power (FRAP) of plasma and DPPH-scavenging capacity (Tsai et al. 2010). Animal experiments have shown that Djulis intake could reduce plasma low-density-lipoprotein (LDL) cholesterol and total cholesterol (Tsai et al. 2011a, b). Djulis extract can also be used as an insecticide and insect repellent against biting midges and apple snails (Chio et al. 2013). Agricultural waste contains a variety of functional ingredients, which is an important source of functional materials for the food and biotechnology industry. Djulis hull (DH) is the outermost layer of Djulis grains separated during the milling process, and is commonly regarded as an agricultural waste. DH accounts for approximately 35% of the total weight, and contains a large amount of nutrients, including polyphenols, flavonoids, resveratrol, γ-aminobutyric acid, and betaine (Chen 2015). With the increase in production, the majority of DH is discarded directly, which could be recovered for extraction of functional ingredients. However, there is currently a lack of research data on DH that can be used by the food or biotechnology industry.

Cereals are the primary sources of energy and nutrients, most of which do not have harmful side effects. Hence, there is increasing interest in natural antioxidants and tyrosinase inhibitors that can be obtained from cereals (Masisi et al. 2016). Polyphenols, flavonoids, anthocyanins, vitamins, fibers, and their derivatives have been isolated from a variety of cereals. Current studies have found that cereals contain a higher level of antioxidant phytochemicals than previously reported. The benefits of cereals to human health have been the topic of extensive research and epidemiological studies. These studies have linked whole grain intake to the prevention of metabolic syndrome, obesity, and related chronic diseases (e.g. cardiovascular diseases and type 2 diabetes) (Craft et al. 2015). The imbalance between pro-oxidants and antioxidants will lead to processes that will generate reactive oxygen species (ROS), resulting in oxidative stress. The oxidative processes in cells are regulated by antioxidants, which will delay or prevent cellular damage. In general, pro-oxidants are balanced with endogenous and/or dietary antioxidants to achieve antioxidant protection (Pisoschi and Pop 2015). Tyrosinase (EC 1.14.18.1) is a copper-containing monooxygenase that is present in many microorganisms, plants, and animals. This enzyme catalyzes the biosynthesis of melanin in human skin, and causes various skin conditions, such as melasma, freckles, and age spots. The application of plant tyrosinase inhibitors in skin health could be a natural solution to maintaining skin whiteness. Such agents have increasingly been applied to cosmetics (Pillaiyar et al. 2017).

High-pressure-assisted extraction (HPE) is an environment-friendly extraction technology that can destroy the cell walls, membranes, and organelles of plant tissues, which also enhances the mass transfer process, increases extraction efficiency, reduces extraction time, and decreases solvent consumption. Currently, the range of pressure in industrial use is between 100 and 600 MPa, which is determined by the processed products (Huang et al. 2013). High-pressure processing is in equilibrium within a sealed container. In other words, pressure transmission is uniform, instantaneous, and adiabatic, which implies it can be applied regardless of food shape or size. Studies have examined the extraction of functional ingredients from food wastes, such as lycopene from tomato paste (Xi 2006), anthocyanins from grape skins (Corrales et al. 2009), and corilagin from longan fruit pericarp (Prasada et al. 2009). However, there are no studies related to the use of high pressure to extract functional ingredients from DH. The aim of this study was to use pressure-assisted extraction of DH for the rapid and effective extraction of functional ingredients, including total phenols and flavonoids. This study also tested the antioxidant capacity, anti-tyrosinase activity, and ability to inhibit inflammatory precursors of the resulting extract. Finally, the extraction efficiency of DH was compared to conventional oscillation extraction of Djulis hull.

Materials and methods

Chemicals

Aluminium nitrate (Al(NO3)3), potassium acetate (CH3CHOOK), quercetin, gallic acid, [2,20-azinobis-(3-ethylbenzothi-azoline-6-sulfonic acid) (ABTS)], potassium ferricyanide, trichloroacetic acid, and LPS from Escherichia coli 0111:B4 were purchased from Sigma-Aldrich (St. Louis, MO). Fetal bovine serum, Dulbecco’s modified Eagle’s medium (DMEM). Antibodies against iNOS (2977) and COX-2 (4842) were purchased from Cell Signaling (Boston, MA).

Preparation of DH extracts

DH was collected from markets in Kaohsiung, Taiwan. DH was extracted according to the method of Wu et al. (2017) with modifications. DH was dried in a hot air oven (FD115, BINDER) at 60 °C for 12 h, milled into a powder, filtered through a 40-mesh filter, placed in a vacuum-sealed bag, and extraction by HPE and CE immediately.

High-pressure extraction

The DH powder extracted by HPE was according to the method of Wu et al. (2017). HPE was conducted by mixing 10 g of DH powder with 200 mL of solvent (water:alcohol = 30:70). The mixture was placed in a sterile polyethylene bag and vacuum sealed, followed by HPE. The high-pressure food processor had a chamber volume of 6.2 L and maximum working pressure of 600 MPa (Baotou Kefa Inc., Baotou City, China). Water was used as the transmission medium. The pressure increased at a rate of about 300 MPa/min. HPE was immediately followed by depressurization. The time taken for the increase and decrease of pressure was not included in the duration of HPE. After the mixture was placed in the pressure chamber, it was treated at 150, 300, 450 and 600 MPa for 5 min at 25 °C (initial temperature), and the water temperature increased by 3 °C for every 100 MPa. After HPE, the extract was vacuum concentrated, freeze-dried (Alpha 1–2/LD-2, vacuum pump RZ-5, Christ, Germany) and the dried extract filtered through Whatman No. 4 paper, and antioxidant activities and content analyses were performed at 4 °C.

Conventional extraction

Oscillation extraction was performed by mixing 10 g of DH powder with 200 mL of solvent (water:ethanol = 30:70) in a 200-mL serum bottle. The mixture was kept in a rotary shaker (150 rpm) at 25 °C for 12 h. After extraction for 8 h, the solution was filtered, freeze dried, and stored at 4 °C for further analysis.

Analysis of contents of DH extracts

The total phenolic content was determined based on the methods described by Baba and Mlink (2015) with modifications. A total of 0.2 mL of the standard (gallic acid) or sample was added to 1 mL of Folin–Ciocalteu’s reagent and 0.8 mL of Na2CO3 (7.5%). The mixture was allowed to stand for 30 min after mixing evenly, and its absorbance was measured at 760 nm using a spectrophotometer. The total phenolic content of the samples was calculated using the standard gallic acid curve as the control. The results were expressed as the gallic acid equivalent (GAE) per gram of dry weight. Flavonoid content was determined using the methods described by Baba and Mlink (2015) with modifications. A total of 1.5 mL of deionized water (or ethanol), 0.1 mL of 10% Al(NO3)3, 0.1 mL of 1 M CH3CHOOK, and 2.8 mL of H2O were added to 0.5 mL of the sample. The mixture was allowed to stand for 40 min after mixing, and the absorbance was measured at 415 nm using a spectrophotometer. The flavonoid content of the samples was calculated using the standard quercetin curve as the control. The results were expressed as milligrams of quercetin per gram of sample (mg QU/g of extract).

Phenolic and flavonoid compounds were identified using high-performance liquid chromatography (HPLC) based on the methods by Özkan and Özcan et al. (2014). In brief, 50 μL of freeze-dried DH extract was re-dissolved in 50% ethanol, and through microporous filter (0.45 μ) and injected to HPLC. The HPLC system was equipped with a system controller, gradient pump, C18 column, column oven and UV detector. The mobile phase was solvent A (acetic acid 2% in water) and solvent B (methanol), and the gradient was adjusted to 72:28 (v/v) in 60 min; the flow rate was 1.0 mL/min, and UV detection was performed at 278 nm. The concentration of each compound was calculated based on the standard curves of gallic acid, chlorogenic acid, coumaric acid, rutin, vitexin and naringin (Sigma).

Antioxidant activity

The total antioxidant capacity of the samples was measured using the methods described by Arnao et al. (2001) with slight modifications. Deionized water (1 mL), peroxidase (4.4 unit/mL, 0.2 mL), H2O2 (50 μM, 0.2 mL) and ABTS (100 μM, 0.2 mL) were mixed evenly and left to stand in a dark room for 1 h to produce stable blue-green ABTS [2,20-azinobis-(3-ethylbenzothi-azoline-6-sulfonic acid)]. Different concentrations of DH extracts were added, the absorbance was measured at 734 nm using a spectrophotometer, and the following formula was used to calculate the total antioxidant capacity.

Totalantioxidantcapacity%=1-A734nm,sample/A734nm,blank×100

Reducing power

Reducing power was measured using the methods described by Duh and Yen (1997). A total of 0.5 mL of 0.2 M phosphate buffer solution (pH 6.6) and 0.5 mL of 1% potassium ferricyanide were added to 1 mL of DH extracts with different concentrations. The mixture was allowed to react in a 50 °C water bath for 20 min and cooled rapidly, and then 0.5 mL of 10% trichloroacetic acid solution was added. After centrifugation at 3000 rpm for 10 min, 1 mL of the supernatant was obtained and added to 1 mL of deionized water and 1 mL of 0.1% ferric chloride solution. The mixture was allowed to stand for 10 min after mixing evenly, and the absorbance was measured at 700 nm using a spectrophotometer. Higher absorbance values indicated greater reducing power.

DPPH radical scavenging activity

The scavenging activity on DPPH radicals by DH extract was determined using the methods by Shimada et al. (1992) with modifications. A total of 5 mL of newly-prepared 0.1 mM DPPH in methanol solution was added to 1 mL of DH extracts with different concentrations. The mixture was allowed to stand for 50 min after mixing evenly. The absorbance was measured at 517 nm using a spectrophotometer, and the DPPH-scavenging capacity was calculated using the following formula.

DPPH - scavengingcapacity%=1-A517nm,sample/A517nm,blank×100.

Intracellular ROS inhibition

RAW 264.7 cells were cultured in triplicate on a 96-well plate at a density of 1 × 105 cells/well for 24 h. Different concentrations of DH extracts were administered, and the cells were incubated at 37 °C for 4 h in 5% CO2 atmosphere. The intracellular ROS level was measured using the fluorescence intensity of the oxidant-sensitive probe 2′,7′-dichlorofluorescein-diacetate (DCFH-DA). This process involves the conversion of DCFH-DA by deacetylase to DCFH, which is then oxidized by various intracellular ROS to produce 2′,7′-dichlorofluorescein (DCF), which is a highly-fluorescent compound. Under the presence or absence of lipopolysaccharides (LPS, 1 μg/mL), the cells were cultured with 50-150 μg/mL of DH extracts for 24 h, after which, the cells were stained with 20 μM DCFH-DA for 15 min at 25 °C, and enzyme-linked immunosorbent assay (ELISA) was performed to detect the production of intracellular ROS.

Measurement of nitrite

Nitrite is the final product of nitric oxide (NO) production by activated macrophages. RAW 264.7 cells were cultured in the presence or absence of LPS (1 μg/mL) with different concentrations of DH extracts for 24 h. Next, 100 μL of Griess reagent (1:1 mixture of 1% sulfanilamide and 0.1% naphthylethylenediamine dihydrocloride in 5% phosphoric acid) was added to 100 μL of cell supernatant in a 96-well plate, and the mixture was stirred automatically at 25 °C for 10 min. An ELISA microplate reader (Molecular Devices Co., USA) was used to measure absorbance at 550 nm. The decrease or increase in nitrite level was estimated as the percentage of absorbance of the samples relative to the controls. The standard curve was plotted based on the serial dilution of nitrite.

Measurement of PGE2

RAW 264.7 cells were cultured in the presence or absence of LPS (1 μg/mL) with different concentrations of DH extracts for 24 h. Then, 100 μL of the culture-medium supernatant was collected, and PGE2 concentration was measured using the PGE2 ELISA kit (Cayman Chemical Company, Ann Arbor, MI, USA). A microplate reader was used to measure absorbance at 405 nm. The standard curve was plotted based on the serial dilution of PGE2.

Enzymatic assay of tyrosinase inhibition

Using the methods described by Kubo and Kinst-Hori (1998), with L-DOPA as the substrate, the anti-tyrosinase activity of the extracts was determined using a spectrophotometer. Firstly, 0.3 mL of 2.25 mM L-DOPA solution was mixed with 0.27 mL of 25 mM phosphate buffer solution (pH 6.8), and was allowed to stand for 10 min at 25 °C. Then, 0.3 mL of each sample solution and 0.03 mL of 400 unit/mL mushroom tyrosinase aqueous solution was added to the mixture sequentially. The linear increase in optical density at 475 nm was measured to monitor the formation of dopachrome in the solution. One unit of enzyme activity (U) was defined as the amount of enzyme needed to increase absorbance by 0.001/min at 475 nm under experimental conditions. The following formula was used to calculate the anti-tyrosinase activity of the samples:

Inhibitionrate%=1-B/A×100

where A = ΔOD475/min of the blank control, and B = ΔOD475/min of test samples.

Statistical analysis

The experiments were performed in triplicate. The data are expressed as mean ± standard deviation (SD), and a statistical analysis system (SAS Inc., NC, USA) was used for analysis. One-way analysis of variance (ANOVA) was performed. Significant differences between the treatments were verified using Duncan’s new multiple range test. A p < 0.05 indicated statistical significance.

Results and discussion

Analysis of extraction yield, phenolic and flavonoid content

Table 1 shows the extraction yield, total phenolic content, and total flavonoid content obtained from DH extract using 70% ethanol as the solvent. The extraction yield calculate as: freeze dried sample (weight) over DH sample (weight). The extraction yield of HPE was highest at 600 MPa (17.1%), followed by 450 MPa (15.9%), 300 MPa (15.2%) and 150 MPa (14.6%), which were all significantly higher (p > 0.05) than the yield of CE (11.2%). Total phenolic content was estimated using gallic acid, and expressed as GAE/g of extract. The total phenolic content was 451.5–642.4 mg GAE/100 g dry weight, and primarily consisted of gallic acid, chlorogenic acid, and coumaric acid. The total flavonoid content of DH 70% ethanol extract was expressed using mg QU equivalent/g of extract, and the range was 40.8–57.2 mg QU/g. Three main flavonoids were identified, which were rutin, vitexin, and naringin. The phenolic and flavonoid contents of the extracts increased with increasing pressure. The gallic acid and rutin contents of DH extracts obtained using HPE-600 were higher than those obtained under other HPE conditions and CE. This indicates that HPE for 5 min at 600 MPa was more effective in extracting antioxidants than 8 h of CE.

Table 1.

Extraction yields and concentrations of phenolics and flavonoids were identified from DH extracts

CE HPE-150 HPE-300 HPE-450 HPE-600
Extraction yield (%) 11.2 ± 1.2c 14.6 ± 0.9b 15.2 ± 1.1b 15.9 ± 1.3ab 17.1 ± 1.2a
Total phenolic (mg GAE/g) 451.5 ± 21.5c 567.2 ± 18.2b 582.4 ± 18.1b 590.2 ± 22.1b 642.4 ± 20.4a
Flavonoid (mg QU/g) 40.8 ± 3.4c 47.2 ± 3.3b 49.3 ± 3.7b 55.6 ± 2.8a 57.2 ± 4.1a
Gallic acid (μg/g) 42.2 ± 1.9b 44.5 ± 2.1b 52.2 ± 1.6a 48.5 ± 1.8a 53.2 ± 2.2a
Chlorogenic acid (μg/g) 18.3 ± 1.4b 21.4 ± 1.8a 23.4 ± 1.8a 20.7 ± 0.9a 22.7 ± 1.4a
Coumaric acid (μg/g) 9.3 ± 0.7a 8.3 ± 0.7a 10.7 ± 0.6a 11.2 ± 1.1a 10.2 ± 0.6a
Rutin (μg/g) 22.3 ± 1.7c 26.8 ± 2.1b 27.4 ± 1.5b 33.2 ± 0.8a 34.2 ± 0.5a
Vitexin (μg/g) 15.8 ± 1.1b 14.9 ± 1.2b 19.8 ± 0.7a 22.1 ± 0.7a 20.7 ± 0.4a
Naringin (μg/g) 8.2 ± 1.3a 9.7 ± 1.2a 9.1 ± 0.2a 8.9 ± 0.6a 10.5 ± 0.9a
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Values are expressed as mean ± SD (n = 3). Means with different superscript letters in every line are significantly different (p < 0.05)

HPE generally involves the application of 100–600-MPa hydrostatic pressure on the extract at room temperature. Under high-pressure conditions, the substantial intracellular and extracellular difference in pressure will cause the solvent to rapidly penetrate the ruptured cell wall. Hence, the mass transfer rate or dissolution rate of the solute is extremely fast. According to the theory of mass transfer, mass transfer rate = mass transfer pressure/resistance. Moreover, according to the theory of phase behavior, dissolution occurs faster under higher pressures (Xi 2013). Longan pericarp has generally been regarded as a by-product of longan fruits. A study applied HPE to obtain extracts of corilagin, lignin and polysaccharides from longan pericarp, and found that HPE had higher extraction yield compared to other extraction methods (Yang et al. 2009). Corrales et al. (2008) used HPE at 600 MPa and 70 °C to extract anthocyanins; compared to hot water extraction, HPE extracts had higher anthocyanidin content (11.21 mg/g DW), and was nearly 50% higher than ultrasound extraction. In addition, the antioxidant capacity of HPE extract was about 1.7 higher compared to ultrasound extraction, and 3 times higher compared to hot water extraction. Singh et al. (2017) also compare ultrasound and conventional method with using different solvents for extraction of polyphenols from whole mung bean, hull and cotyledon. The results showed that acetone was observed the best extraction medium for polyphenols in combination with ultrasound method.

Naghshineh et al. (2013) reported the application of HPE in the enzymatic extraction of pectin. Compared to extraction under ambient pressure, the application of 100- or 200-MPa pressure at 50 °C for 30 min led to significantly higher pectin yield. HPE has also been shown to be an effective method for extracting lycopene from tomato paste. Compared to solvent extraction, HPE procedures provided higher extraction yield and shorter processing time. The extraction yield of lycopene using HPE (41.7 mg/100 g) was far higher than that using solvent extraction (35.6 mg/100 g). The extraction yield of HPE at 500 MPa for 1 min was also higher than that of extraction at room temperature for 30 min (Xi 2006). In this study, extracts obtained by HPE led to higher phenolic and flavonoid contents. HPE samples showed significantly higher antioxidant contents (567–642 mg GAE/g of extract total phenolics and 47.2–57.2 mg QU/g of extract flavonoids) compared to CE (451 mg GAE/g extract total phenolics and 40.8 mg QU/g extract). This may have been due to the higher contribution of antioxidants and anti-tyrosinase activity than CE samples. Exposure to the solvent under high pressure will cause the extracellular matrix to expand, which will increase solvent infiltration into the matrix of the samples, thus increasing the contact of the solvent with the target compounds. This phenomenon can explain the shorter time required for HPE to achieve the same results (Huang et al. 2013).

Antioxidant activity

The total antioxidant capacity of DH extracts from HPE and CE were measured and compared with BHT (Fig. 1a). BHT has significant total antioxidant capacity, which increases with increasing concentration. CE (50 μg/mL) extract showed moderate antioxidant capacity at 43.5%, whereas DH extracts from HPE-150, HPE-300, HPE-450 and H-600 showed higher antioxidant capacity, at 57.1%, 62.7%, 63.8%, and 69.8%, respectively. The total antioxidant capacity was 96.8% when BHT concentration was 100 μg/mL, whereas that of HPE-600 and CE were 76.8% and 48.6%, respectively. When the concentration was 150 μg/mL, the antioxidant capacity of the four HPE extracts increased with increasing concentration and reached approximately 91.2%. Under all tested concentrations, the total oxidant capacity of HPE was higher than that of CE. This indicates that samples prepared using HPE had higher antioxidant capacities than those prepared using CE. The results of reducing power are shown in Fig. 1b. Under each tested concentration, the reducing power of HPE was higher than that of CE. The inhibitory activity of HPE was dose-dependent, and its effects increased with increasing concentration. At a concentration of 50 μg/mL, the reducing power of CE, HPE-600, and BHT were 0.42, 0.68, and 0.71, respectively. Statistical analysis indicated that the antioxidant capacity under different extraction conditions was significantly different (p ≤ 0.05). At 100 μg/mL, the reducing power of HPE extract was within the range of 0.54–0.85. However, at 100 μg/mL, the reducing powers at CE and BHT were 0.48 and 0.92, respectively. When HPE was 150 μg/mL, the reducing power of the DH sample extracted under 600 MPa was the highest at 0.91; when HPE was 50 μg/mL, the reducing power was the lowest, at 0.53. In the DPPH analysis (Fig. 1c), the scavenging activity of 100 μg/mL extract was, in ascending order: CE (68.2%), HPE-150 (69.3%), HPE-300 (72.8%), HPE-450 (75.2%) and HPE-600 (80.5%). At 50-150 μg/mL, the scavenging activity of BHT increased from 7.8 to 96.1%. Similar trends were observed at concentrations of 50 and 150 μg/mL. The scavenging activity of HPE extracts increased with increasing concentration and pressure. Our data indicate that at 50 μg/mL, the DPPH scavenging activity was the lowest for CE (41.8%). More specifically, the DPPH-scavenging activity of BHT was 70.2–91.5%, and that of HPE-150 was 64.5–83.4%. These results show that the DPPH-scavenging activity of HPE was superior to CE, which may have been due to its electron-donating ability.

Fig. 1.

Fig. 1

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a Total antioxidant capacity, b reducing power, c DPPH scavenging, and d ROS inhibition of DH obtained by CE and different HPE conditions (HPE-150, HPE-300, HPE-450, and HPE-600 MPa). Data are expressed as the mean ± SD (n = 3). Different letters above the bars for the same concentration indicate significant differences among means of treatments (p < 0.05)

At 50-1600 μg/mL, the cytotoxicity of CE and HPE to RAW 264.7 cells after exposure for 24 h did not reduce the viability of the cells. Figure 3 indicates that CE and HPE extracts had inhibitory effects on the intracellular ROS production of LPS-induced RAW 264.7 cells. LPS-stimulated cells showed higher intracellular ROS levels, which was estimated using DCFH-DA labelled cells as fluorescent probes. Compared to untreated cells, the large increase in oxygen intake induced by LPS (1 μg/mL) treatment led to a substantial release of intracellular ROS. At 50 μg/mL, the inhibitory activities of CE, HPE-150, HPE-300, HPE-450, and HPE-600 extracts on ROS production were 12.4%, 22.8%, 23.5%, 28.0%, and 32.5%, respectively. However, the ROS inhibitory activities of HPE-150 and HPE-300 did not show significant differences among the various concentrations. When testing LPS-stimulated RAW 264.7 cells, the inhibitory activities of HPE-600 at concentrations of 50, 100, and 150 μg/mL were 32.5%, 30.7%, and 37.4%, respectively. These values were all significantly higher than that of CE at 150 μg/mL (19.2%). In RAW 264.7 cells under LPS stimulation, CE and HPE had inhibitory effects on the expression of inflammatory mediators, NO and PGE2. RAW 264.7 cells stimulated by LPS (1 μg/mL) showed significantly higher cellular NO levels (18.6 μM) compared to unstimulated control cells (3.7 μM). When the concentration of CE, HPE-150, HPE-300, HPE-450, and HPE-600 was 100 μg/mL, the NO concentrations were 17.2, 16.4, 16.5, 16.1, and 12.7 μM, respectively (Fig. 2a). The inhibitory activity of HPE-600 at 150 μg/mL was superior to that of HPEs and CE. As shown in Fig. 2b, compared to the control group, LPS-induced RAW 264.7 macrophages showed increased PGE2 production. HPE-300, HPE-450, and HPE-600 at 100 μg/mL showed better inhibitory activities on PGE2 production compared to CE. The NO and PGE2 concentrations of HPE-150 samples were lower than those of LPS-stimulated cells, but were not significantly different from those of CE. At a concentration of 150 μg/mL, the HPE-600 extract reduced the concentrations of NO and PGE2 released by RAW 264.7 cells by 32.4% and 26.1%, respectively, compared to the LPS group.

Fig. 3.

Fig. 3

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Tyrosinase inhibition of DH obtained by CE and different HPE conditions (HPE-150, HPE-300, HPE-450, and HPE-600 MPa). Data are expressed as the mean ± SD (n = 3). Different letters above the bars for the same concentration indicate significant differences among means of treatments (p < 0.05)

Fig. 2.

Fig. 2

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Effect of CE and HPE on LPS-stimulated a No and b PGE2 production in RAW 264.7 cells. Data are expressed as the mean ± SD (n = 3). Different letters above the bars of same concentration indicate significant differences among treatment means (p < 0.05)

Cereal and its products have become healthy foods, as the antioxidant properties of cereal and its products are related to protection against chronic diseases. Whole-grain foods are a good source of phenolic compounds, which include benzoic and cinnamic acids, anthocyanins, quinines, flavanols, chalcones, flavones, flavanones, and amino phenolic compounds. Several studies have focused on identifying the phenolic and flavonoid compounds, and their antioxidant capacities, in common cereals, such as wheat, corn, rice, barley, sorghum, rye, oats, and millet (Masisi et al. 2016). Djulis contains a variety of phytochemicals, including dietary fiber, pigments, and polyphenols, and hence is beneficial to health. Tsai et al. (2011a, b) found that betanin was the main red pigment in Djulis, and showed that betanin was closely related to antioxidant capacity. HPLC–DAD and HPLC–MS/MS analyses revealed that betanin, rutin, kaempferol, and another 20 compounds are present in the water extract of Djulis, which may be the reason for its protective effects against oxidative stress in human HepG2 cells. (Chyau et al. 2015). Tsai et al. (2011a, b) also reported that a Djulis nanoparticle diet can significantly lower the LDL and total cholesterol levels of hamsters induced by high-fat diets. This may have been due to the large amounts of antioxidants and antioxidases present in nano-processed Djulis. The results of our study showed that DH extracts had antioxidant and anti-tyrosinase activities, and the concentration of gallic acid was the highest among all phenolic acids. HPE-600 showed higher phenolic and flavonoid contents than CE and the other HPE conditions, and had the highest total antioxidant capacity. Compared to CE, HPE-600 showed higher extraction yield, phenolic content, flavonoid content, antioxidant capacity, and anti-tyrosinase activity. Phenolic compounds have redox properties, and can act as antioxidants. Due to the strong radical scavenging capacity of their hydroxyl groups, total phenolic content can serve as the basis for the rapid screening of antioxidant capacity. Flavonoids, which include flavones, flavanols, and condensed tannin, are secondary plant metabolites. Their antioxidant activity is dependent on the presence of free OH groups, especially 3-OH. The literature shows that the phytochemicals measured in different cereals are linearly correlated with DPPH-scavenging activity (Žilić et al. 2011). Deng et al. (2012) found that total antioxidant capacity was positively correlated with total phenolic content (R2 for FRAP and TEAC were 0.8880 and 0.8840, respectively). As the inhibitory pattern is determined by the structures of the substrate and the inhibitor, mushroom tyrosinase has been widely used for the screening and characterization of potential target enzymes for tyrosinase inhibition.

Enzymatic assay of tyrosinase inhibition

At 50 μg/mL, the anti-tyrosinase activity of the extracts obtained by CE, HPE-150, HPE-300, HPE-450, and HPE-600 were 26.5%, 42.1%, 45.2%, 50.6%, and 58.2%, respectively (Fig. 3). At 100 μg/mL, only the extract obtained by CE showed low anti-tyrosinase activity (32.5%), whereas those obtained by HPE-300, HPE-450, and HPE-600 showed moderate anti-tyrosinase activity within the range of 50.4–67.2%. At 150 μg/mL, the anti-tyrosinase activity of HPE extracts obtained by HPE-150, HPE-300, HPE-450, and HPE-600 increased steadily to 73.5%, whereas the inhibitory activity of CE at 150 μg/mL was 47.2%. At 150 μg/mL, kojic acid showed better anti-tyrosinase activity at 90.2%.

Adom and Liu (2002) found that the total phenolic content and antioxidant capacity of real grain extracts were significantly correlated. Similarly, Velioglu et al. (1998) reported that the total phenolic contents of sunflower seeds, flaxseeds, wheat germ, and buckwheat were correlated with their antioxidant capacities. Based on these results, we can conclude that total phenolic and flavonoid contents are significantly correlated with radical scavenging and anti-tyrosinase activity. Therefore, the anti-tyrosinase activity of DH extract increased proportionally with its phenolic and flavonoid content. The processing of plant-derived foods will result in a large amount of by-products. The disposal of these by-products will increase the costs of food processors and have potential negative effects on the environment. Nevertheless, the vast majority of by-products still cannot be used as potentially valuable sources of bioactive compounds. This is partially due to the lack of suitable technology to extract these bioactive compounds. In recent years, a number of novel extraction techniques have been applied to maximize the extraction of bioactive compounds from food by-products. Numerous methods are currently available, including ultrasound extraction, supercritical fluid extraction, microwave-assisted extraction, and HPE (Mustafa and Turner 2011).

Conclusion

The use of HPE can increase antioxidant content and yield. Not only is it economical, it is also environment-friendly, as Djulis by-products can be recycled as ingredients or additives in the food industry. The results of this study indicate that HPE samples had higher phenolic and flavonoid content, and had strong antioxidant and anti-tyrosinase activity than CE. Therefore, HPE is a highly-efficient extraction method, which can enable the use of DH as a potential source of antioxidants in the food and cosmetics industry.

Acknowledgements

This research work was supported by the Ministry of Science and Technology, MOST 107-2221-E-002-110-MY2, Taiwan, Republic of China.

Footnotes

Publisher's Note

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