Chemical changes and antioxidant activities of heated whole barley extracts

Abstract

Chemical profiles of ethanolic (70%) and aqueous extracts of whole barley heated at 150, 190, and 230 °C were analyzed by GC–MS and their antioxidant properties were studied in vitro, in bulk oil, or in an oil-in-water (O/W) emulsion systems. More chemicals were detected in the ethanolic extract than in the aqueous extract from heated barley; heating decreased the contents of detected chemicals. Organic acids, mono- and di-saccharides, sugar alcohols, and glycerol were the major chemicals detected in both the extracts. Ethanolic extracts possessed higher in vitro antioxidant activities than the aqueous extracts. However, this trend was not clearly observed in the bulk oil and O/W emulsion. For O/W emulsions, ethanolic extracts obtained following heating at 150 °C prevented lipid oxidation better than others. Therefore, heat treatment at 150 °C is recommended to enhance the antioxidant activities of whole barley.

Introduction

Barley (Hordeum vulgare L.) is one of the major staples for human beings; it possesses diverse nutrients and phytochemicals, including β-glucan, tocols, and phenolic compounds (Gamel and Abdel-Aal, 2012). Barley hull extracts were used to enforce phytochemical contents in bread (Hao and Beta, 2012) and to prepare brewing tea. Barley extracts have shown beneficial effects, including antiadipogenic and antiobesity properties (Seo et al., 2015), and antioxidant activities (Lahouar et al., 2014; Oh et al., 2015). In vitro antioxidant properties of whole barley have been reported from 80% methanolic extracts (Lahouar et al., 2014) and aqueous extracts (Oh et al., 2014; 2015). Oh et al. (2015) roasted hulled barley at 210 °C for 20 min; the aqueous extracts of these hulled barley samples showed antioxidant properties in O/W emulsion, while some pro-oxidative activities were observed in bulk oils in some environments. These dual properties of roasted barley grains on oxidative stability have been reported using in vitro assays and in vivo studies (Omwamba et al., 2013).

The addition of antioxidants to foods is a practical strategy to control the rates of lipid oxidation, and more consumers tend to purchase products containing antioxidants from natural resources than synthetic antioxidants. Therefore, whole barley could be a possible antioxidant source originating from natural resources. Thermal processing can change the contents of phenolic compounds such as p-coumaric and ferulic acids in barley (Oh et al., 2014; 2015) or generate Maillard reaction products (Duh et al., 2001), which affects the antioxidant efficiency and generation of a unique flavor. In addition, brewing barley tea using roasted whole barley is consumed in several East Asian countries. Via in vitro antioxidant assays and lipid matrices, previous reports on heated barley extracts have shown that the aqueous extracts of these heated barley samples possessed antioxidant properties. Although barley extracts possess diverse nutrients, the chemical profiles of the extracts of whole barley have not been elucidated because identifying or monitoring antioxidative phenolic compounds was the main focus of previous studies.

The objective of this study was to determine the chemical profiles and antioxidant activities of aqueous and ethanolic extracts from heated whole barley. Comprehensive chemical profiles of the extracts were obtained using GC/MS after derivatization, and two matrices, including bulk oil and the oil-in-water emulsion system, were utilized.

Materials and methods

Materials

Husked whole barley, harvested at the year of 2020 in Muan, Republic of Korea, was purchased from Barun F&B (Incheon, Republic of Korea). Methyl hydroxyl chloride amine (MHCA), fluoranthene, pyridine, N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), trimethylchlorosilane (TMCS), DPPH, and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Other chemicals, including ethanol, sodium chloride, n-hexane, acetonitrile, isooctane, and methanol were purchased from Daejung Chemical Co. (Seoul, Korea).

Sample preparation

Husked whole barley (2 kg) was heated at 150, 190, and 230 °C for 1 h in a convection oven (Hysclab, Seoul, Korea). ‘Whole barley’ was used as the same meaning of ‘Husked whole barley’ in this study. To obtain aqueous extracts, heated whole barley was added to distilled water (90 °C) at a ratio of 1:20 (w/w). The mixture was shaken in a shaking water bath (Jeio tech, Seoul, Korea) at 100 rpm for 2 h. To obtain the ethanolic extract, 70% aqueous ethanol solution was added to the heated whole barley samples at a ratio of 20:1 (w/w) and mixed at 250 rpm using a vertical shaker (Taitec Corporation, Koshigaya City, Saitama, Japan) for 2 h at room temperature. Then, the mixture was filtered using a cotton cloth, and the supernatant was obtained by centrifugation (Hanil Science Industrial, Gimpo, Korea) at 6000 g for 10 min. Finally, the solution was concentrated under reduced pressure using Whatman paper # 2 to obtain a clear extract. In the case of the ethanolic extracts, ethanol was removed as much as possible by concentration under reduced pressure at 50 °C, and all extracts were freeze-dried (Ilshinbiobase Co., Ltd., Gyeonggi, Korea).

Chemical profile analysis

Derivatization of the extracts

The freeze-dried extract (5 mg) was added to a glass vial containing 200 μL of 20,000 mg/kg MHCA in pyridine solution, followed by sonication for 10 min. The mixture was incubated at 30 °C for 90 min. The oximated samples (50 µL) were mixed with 50 μL of a mixture of BSTFA and 1% TMCS solution for trimethylsilation. As an internal standard, 10 μL of 500 µmol/g fluoranthene was added, and the mixture was vortex-mixed and heated for 30 min at 60 °C.

GC/MS conditions

The separation and identification of chemical compounds in the derivatized samples was determined by gas chromatography (GC) and analyzed by a mass selective detector (MS) according to the previous report (Park et al., 2021). GC (7890B series, Agilent Technologies, Palo Alto, CA, USA) was connected to a 5977B MS (Agilent Technologies) equipped with a VF-5MS column (60 m length × 0.25 mm i.d. × 0.25 µm film thickness, Agilent Technologies). Helium was used as the carrier gas at a constant column flow rate of 1.5 mL/min. A split injection mode was used at a ratio of 20:1 at 300 °C. The GC oven temperature was maintained at 50 °C for 2 min, raised to 180 °C at a rate of 5 °C /min, and maintained for 8 min. After that, the temperature was increased to 210 °C at a rate of 2.5 °C/min and to 320 °C at a rate of 5 °C/min. The final temperature was maintained for 10 min. Mass spectra were obtained at 70 eV through electron ionization (EI). Data were acquired in the scan mode. To identify the compounds in the extracts, NIST 17 was used as a library. For tentative identification, a compound with a quality of 70% or more was selected, compared to the NIST 17 library. Each compound was quantified using the internal standard ratio.

HPLC analysis for p-coumaric acid

The p-coumaric acid content in the samples was analyzed using a high-performance liquid chromatograph (HPLC) equipped with a photodiode array (PDA) detector (Hitachi, Tokyo, Japan), according to a previous report (Oh et al., 2014). The extracts were diluted in deionized water to a final volume of 10 μL. The stationary phase was a Waters Nova-Pak C₁₈ column (4 µm, 3.9 mm × 150 mm). The mobile phase was a mixture of ultra-pure water, acetonitrile, and acetic acid (88:10:2, v/v), used under isocratic conditions with a flow rate of 0.75 mL/min. The p-coumaric acid levels in the eluent were monitored at 280 nm. The concentration of p-coumaric acid was calculated based on the calibration curves using a standard compound.

In vitro antioxidant assays

The DPPH free radical-scavenging ability of the extracts was determined based on a previous report (Oh et al., 2014). The radical cation-scavenging activity of the extracts was determined using the ABTS method, as described in a previous report (Oh et al., 2014). The ferric reducing antioxidant power (FRAP) assay was conducted as described in a report by Oh et al. (2014). Results are expressed in µg ascorbic acid equivalent/mg extract.

Analysis of antioxidant properties using oil systems

Rancimat analysis

To determine the induction period (IP), a Rancimat assay was conducted. Briefly, triplicate samples of 3 g of corn oil was mixed with 200 mg/kg of aqueous or ethanolic extract solutions (w/w), which were prepared in distilled water and ethanol, respectively. The mixtures were added in a Rancimat tube and placed in the Rancimat instrument (Metrohm, Chennai, India). The temperature was set to 120 °C. All samples were prepared in triplicate.

Oil-in-water (O/W) emulsion system at 50 °C

The O/W emulsion was prepared per the method described by Yi et al. (2018). Briefly, 5% (w/w) corn oil and 0.5% (w/w) Tween 20 were added to distilled water. A coarse emulsion was prepared by homogenizing the mixture for 3 min using an HB873AKR instrument (Tepal, Rumilly, Haute-Savoie, France). This coarse emulsion was passed twice through a high-pressure homogenizer (AVP 1000, SPX flow, NC, USA) at 50 MPa. After homogenization, the extracts were added to the O/W emulsion at a final concentration of 100 mg/kg. TBHQ was added to the corn oil to obtain a final concentration of 100 mg/kg in the O/W emulsion. Two-gram aliquots of each sample were placed in a 10-mL vial with an airtight seal. The emulsion samples were stored at 50 °C in a water bath and analyzed after 14 days. The headspace oxygen content and conjugated diene levels were determined. All samples were prepared in triplicate.

Oxidation parameter analysis

Headspace oxygen analysis

The headspace oxygen level in air-tight sample bottles of corn oil or O/W emulsions was analyzed according to the methods described by Yi et al. (2018). Headspace gas (40 μL) was collected from each sample bottle using an air-tight syringe, and the oxygen content was determined using a Hewlett-Packard 7890 GC (Agilent Technologies, Inc., Santa Clara, CA, USA) with a 60/80 packed column (3.0 m × 2 mm ID, Restek Ltd., USA) and a thermal conductivity detector (TCD). The flow rate of the helium gas was 200 mL/min. The temperatures of the oven, injector, and TCD were 60, 180, and 180 °C, respectively.

Analysis of conjugated dienes in O/W emulsions

The conjugated diene contents in O/W emulsion samples were determined following the method described by Mei et al. (1998). The sample (120 μL) was mixed with 2.7 mL of methanol/1-butanol (2:1, v:v), and the resulting mixture was vortex-mixed thrice for 10 s each. The absorbance of the samples at 233 nm was measured using a UV/VIS-spectrometer (Genesis 10UV, Thermo, Waltham, USA).

Statistical analyses

All data were analyzed statistically by analysis of variance (ANOVA) and Duncan’s multiple range test using SPSS software program version 19 (SPSS Inc., Chicago, IL, USA). Differences with P-values less than 0.05 were considered statistically significant.

Results and discussion

Identification of chemical compounds in the extracts

Compounds from the ethanolic or aqueous extracts of heated whole barley identified tentatively by GC–MS are shown in Table 1. A total of 11 and 5 chemicals were tentatively identified in the ethanolic and aqueous extracts of heated whole barley, respectively. Organic acids, mono- and di-saccharides, sugar alcohols, and glycerol were detected in the extracts by GC/MS analysis. Mannitol and glycerol were found at the highest levels in the ethanolic and aqueous extracts, respectively. Glycolic acid was observed in the aqueous extracts, whereas sugar alcohols including erythritol, arabitol, and mannitol were detected only in the ethanolic extracts. p-Coumaric acid, one of the main phytochemicals in barley, was not detected in the extract, which could be due to the limitation of derivatization for GC/MS analysis. Generally, further heat treatment decreased the contents of these chemicals, while the contents of butanoic acid and hexanedioic acid increased in the ethanolic extracts.

Table 1.

Tentatively identified compounds from aqueous and ethanolic extracts of heated whole barley

Retention time (min)	Chemical compound	Aqueous extract				Ethanolic extract
		NHTb	150 °C	190 °C	230 °C	NHT	150 °C	190 °C	230 °C
Concentration (µmol/g extract)a
15.77	Lactic acid	0.459c	0.386	0.862	0.246	1.232	0.834	1.580	3.701
16.31	Glycolic acid	0.343	0.283	1.226	0.499	NDd	ND	ND	ND
18.44	Hydracrylic acid	ND	ND	ND	ND	0.447	0.449	0.296	0.672
22.12	Glycerol	15.077	7.310	6.526	2.878	16.867	13.755	13.517	9.792
25.90	Butanoic acid	ND	ND	ND	ND	0.672	0.452	1.087	6.598
28.22	meso-Erythritol	ND	ND	ND	ND	1.836	1.318	0.480	0.356
32.90	D-Fructose	0.913	0.726	3.304	0.321	0.663	0.246	1.412	0.820
33.44	Hexanedioic acid	ND	ND	ND	ND	0.434	0.414	0.769	0.809
35.21	D-( +)-Arabitol	ND	ND	ND	ND	20.994	14.412	5.692	5.974
44.91	D-Mannitol	ND	ND	ND	ND	40.182	35.335	20.122	12.087
51.37	Myo-inositol	3.946	3.736	3.542	1.502	2.406	1.788	4.958	2.491
64.82	Lactose	ND	ND	ND	ND	9.372	5.957	4.651	1.251

^aFluoranthene equivalent µmol/g extract, ^b‘NHT’, ‘150 °C’, ‘190 °C’, and ‘230 °C’ are extracts from whole barley of no-heat treatment, at 150, 190, and 230 °C further heat treatment, respectively. ^cmean of data (n = 3), ^dnot detected

Changes in p-coumaric acid levels in the aqueous and ethanolic extracts from heated whole barley are shown in Fig. 1. Due to the analytical limitations of the GC/MS-based post-derivatization of the phytochemicals, as shown in Table 1, HPLC analysis was adapted to determine the contents of p-coumaric acid. Generally, the ethanolic extracts possessed lower p-coumaric acid contents than the aqueous extracts at each heating temperature. The level of p-coumaric acid in the extracts from whole barley samples heated at 150 °C was the highest and heating at higher temperatures decreased the p-coumaric acid contents in both the aqueous and ethanolic extracts (Fig. 1). Therefore, heat treatment at temperatures above 150 °C may not be favorable for the preparation of whole barley extracts with regard to the concentration of p-coumaric acid.

Fig. 1.

Changes of p-coumaric acid in aqueous and ethanolic extracts from heated whole barley. ‘NHT’, ‘150 °C’, ‘190 °C’, and ‘230 °C’ are extracts from whole barley of no-heat treatment, at 150, 190, and 230 °C further heat treatment, respectively. ‘Aqueous’ and ‘Ethanolic’ are types of solvents used for extraction from whole barley. Different letters on the bar are significant at 0.05. Different letters on the bar were significantly different at 0.05

In vitro antioxidant activities of the extracts

The in vitro antioxidant results, i.e., those of the DPPH, ABTS, and FRAP assays of extracts of heated whole barleys are shown in Fig. 2. Generally, ethanolic extracts exhibited higher in vitro antioxidant activities than the aqueous extracts. The DPPH radical-scavenging ability of the ethanolic extracts was the highest in case of the samples heated at 190 °C, followed by that of those heated at 230 °C (p < 0.05); however, the DPPH radical-scavenging ability of the aqueous extracts was not enhanced following heat treatment [Fig. 2(A)]. The results of the ABTS radical-scavenging activities were similar to those of the DPPH assays. The highest ABTS radical-scavenging ability was observed in the ethanolic extracts obtained by heating the barley samples at 190 °C, followed by those obtained by heating the barley samples at 230 °C [Fig. 2(B)]. The free radical-scavenging abilities of the ethanolic extracts obtained from samples heated at 190 °C were 4.5- and 6.1-fold higher than those of the extracts obtained from the un-heated samples, respectively. In case of the FRAP assay, as the heating temperature increased from 0 to 230 °C, significantly higher ferric ion-reducing capacities were observed in the ethanolic extracts (p < 0.05), whereas such enhancing effects were not found in the aqueous extracts, which is consistent with the results of the radical scavenging assays (Fig. 2). Overall, the heating treatment significantly increased the in vitro antioxidant activities in only the ethanolic extracts of whole barley.

Fig. 2.

In vitro antioxidant results including DPPH (A), ABTS (B), and FRAP (C) results from aqueous and ethanolic extracts from heated whole barley. Abreactions in Figures are listed in Fig. 1 legend. Different letters on the bar are significant at 0.05

Antioxidant activities of the extracts in bulk oil and emulsion matrices

A Rancimat assay of corn oil containing aqueous and ethanolic extracts from heated whole barley is shown in Fig. 3. The IP of corn oil containing TBHQ was the highest, followed by IP of corn oils containing extracts of heated whole barley and control corn oil. Further heat treatment did not result in any significant difference in IP of corn oil containing aqueous extracts (p > 0.05). However, ethanolic extracts from whole barley of no-heat treatment and heated at 190 °C had significantly higher IP than those from whole barley heated at 150 and 230 °C (p < 0.05).

Fig. 3.

Rancimat assay of corn oil containing aqueous and ethanolic extracts from aqueous and ethanolic extracts from heated whole barley. ‘NHT’, ‘150 °C’, ‘190 °C’, and ‘230 °C’ are corn oil containing extracts from whole barley of no-heat treatment, at 150, 190, and 230 °C further heat treatment, respectively. ‘Con’ and ‘TBHQ’ are samples without addition of antioxidant and with addition of TBHQ, respectively. ‘Aqueous’ and ‘Ethanolic’ are types of solvents used for extraction from whole barley. Different letters on the bar are significant at 0.05

Changes in headspace oxygen (a) and conjugated diene (b) levels in corn O/W emulsions containing aqueous and ethanolic extracts from heated whole barley are shown in Fig. 4. Generally, ethanolic extracts had higher antioxidant activities than the aqueous extracts, which was differentiated based on the heating treatment. The headspace oxygen content in samples containing TBHQ and aqueous extracts from barley treated at 150 °C were the highest, followed by those from barley heated at 190 °C, 230 °C, and non-heated treatment (in decreasing order), which implies that roasting at higher temperatures decreased the antioxidant properties of the extracts in the O/W emulsions. This effect was also observed in case of the results for the contents of conjugated dienes. Aqueous extracts from whole barley heated at 150 °C had the lowest levels of conjugated dienes, followed by those in samples containing TBHQ and extracts from samples heated at 190 °C.

Fig. 4.

Changes of headspace oxygen (A) and conjugated diene (B) in corn oil-in-water emulsions containing aqueous and ethanolic extracts from heated whole barley. ‘NHT’, ‘150 °C’, ‘190 °C’, and ‘230 °C’ are O/W emulsion containing extracts from whole barley of no-heat treatment, at 150, 190, and 230 °C further heat treatment, respectively. ‘Con’ and ‘TBHQ’ are samples without addition of antioxidant and with addition of TBHQ, respectively. ‘Aqueous’ and ‘Ethanolic’ are types of solvents used for extraction from whole barley. Different letters on the bar are significant at 0.05

In case of ethanolic extracts, all the samples showed higher headspace oxygen contents than the controls, for which the ethanolic extract was not prepared [Fig. 4(A)]. Conjugated diene contents in extracts from samples heated at 150 °C and non-heat treatment were lower than those in the samples containing TBHQ (p > 0.05) [Fig. 4(B)]. Overall, the ethanolic extracts had a greater degree of antioxidant properties than aqueous extracts in O/W emulsions. The heating process decreased the antioxidant activities of the aqueous extracts, whereas such effects were not observed in case of the ethanolic extracts. Interestingly, the antioxidant properties of the extracts were affected by the solvent type and heating process in the O/W emulsion matrix.

Brewing tea made of heated whole barley containing husks or hulls is popular in some Asian countries. Previous reports have focused on the profile changes of phytochemicals possessing antioxidant capacities (Chen et al., 2019; Gallegos et al., 2010; Oh et al., 2014; 2015; Sharma and Gujral, 2011). Heat treatment increased the radical scavenging activity through the generation of Maillard reaction products, such as 5-hydroxymethyl-2-furaldehyde, at high temperatures (Siddhuraju, 2006; Woffenden et al., 2002). Barley flower heated at 230 °C had a greater content of free phenolic compounds than that heated at 170 °C, while glycosidic phenolic compounds were more abundant in samples heated at 170 °C (Chen et al., 2019). Barley flower treated with thermal energy in a microwave oven at 900 W for 120 s had higher antioxidant activities, although heat treatment decreased the contents of phenolic compounds (Baba et al., 2016). Therefore, excessive heat treatment tends to decrease the contents of phenolic compounds, while some heat-induced compounds may contribute to enhancing the antioxidant activities. In the GC/MS data, organic acids, sugar alcohol, lactose, and fructose were observed in both the extracts, implying that substantial nutrients with small molecular weights were present in the husks of whole barley.

Many phenolics, such as p-hydroxyacetophenone, 5,7-dihydroxychromone, naringenin, quercetin, iso-americanol A, p-hydroxybenzaldehyde, 3,4-dihydroxybenzaldehyde, p-hydroxybenzoic acid, vanillic acid, and p-coumaric acid, were already reported in the extracts of barley tea (Etoh et al., 2004). The aqueous extract of heated hulled barley had p-coumaric, ferulic, protocatechuic, chlorogenic, 4-hydroxybenzoic, and vanillic acids (Oh et al., 2015). Specific target compounds reported previously, such as phenolics or flavonoids, other nutrients, including amino acids, fatty acids, and mono- and di-saccharides, were not identified in the whole barley extracts. The major components in the extracts were mannitol, arabitol, glycerol, or fructose (Table 1), which may be related to the specific taste in barley tea.

Since antioxidant properties are the main physiological targets of researches, antioxidants including catechin, α-tocopherol, and lutein were monitored in roasted and unroasted barley (Duh et al., 2001). The aqueous extract of heated whole barley acted as an antioxidant in O/W emulsions under riboflavin photosensitization conditions (Oh et al., 2015), which is consistent with the results of the current study (Fig. 4). Although the pro-oxidant properties of aqueous extracts were observed in bulk oil in case of extracts obtained from samples heated at 60 and 180 °C (Oh et al., 2015), the Rancimat results showed the presence of antioxidant properties in both the extracts (Fig. 3).

In conclusion, heating husked whole barley changed the profiles of chemicals in the barley extracts. The major chemicals in the extracts were mannitol, arabitol, glycerol, or fructose, whose concentrations decreased following the heating treatment. Although phenolic contents were highly correlated with the in vitro antioxidant assays, retardation of oxidation by the extracts was not consistent in the bulk oil and O/W emulsion matrices. Overall, ethanolic extracts prepared from whole barley heated at 150 °C are more suitable for use in an O/W emulsion environment owing to their enhanced oxidative stability in this environment.

Declarations

Conflict of interest

The authors declare no conflict of interest.