Introduction

In living cells under normal conditions, many physiological and biochemical processes are known to generate reactive oxygen species (ROS) as the by-product. For example, mitochondria consume O2 to make energy and eventually produce H2O. However, in this process, O2 -, H2O2, and OH- are generated [5]. In inflammatory conditions, when phagocytic cells destroy the bacteria or virus, they use an oxidative burst of nitric oxide (NO), O2 -, H2O2, and OCl-. During fatty acid degradation in peroxisome, H2O2 is known to be generated as a by-product [20]. An excessive production of ROS has been implicated in oxidative damage to biomolecules such as carbohydrates, lipids, proteins, nucleic acids, which would lead to the damage of membranes and cellular organelles, and cause cancer [11, 21], cardiovascular diseases [36, 45], inflammatory disorders [4], aging and other diseases in humans [9, 10]. For protection from the oxidative damage, human body is known to take advantage of not only endogenous antioxidant enzymes, such as superoxide dismutase (SOD), catalase, peroxidase, but also exogenous low molecular weight compounds such as tocopherol, ascorbic acid, and polyphenols.

Recently, much attention has been paid to the physiological functionality of foods, due to the increasing interest in human health, and research into the health benefits of foods has been increasing last years. One of the important beneficial functions of foods, beyond the provision of basic nutritional requirements, is mediated by antioxidant action that is supposed to reduce the risk of oxidative stress-related chronic diseases [2, 12]. Since plants have a defense system to come up against the ROS conditions, which is manifested by free radical-scavenging components, such as vitamins, phenolic compounds, and some other intracellular metabolites, plant foods have been considered as an excellent source of antioxidants for human body [6, 27, 44]. While fruits, vegetables, and grains among plants are rich sources of antioxidant substances, the antioxidants found in these plants are mostly phenolic compounds. The grains-derived phenolic compounds are known to be very advantageous in that stability in dry conditions and storage conveniences compared with the fruit- or vegetable-derived compounds.

Agriculture in Korea has traditionally focused on the production of the major grains such as rice, barley and wheat, whereas the cultivation and harvesting of other miscellaneous cereals has remained in a low level. In recent years, however, due to increased demand for well-being foods, the interest in miscellaneous cereal grains as crude fibers and bioactive phytochemical sources that benefit human health and thus the consumption of miscellaneous cereal grains are also increasing in the country. Although several studies have been performed to extend our understanding on nutritional importance, antioxidant, antimicrobial, antimutagenic and anticarcinogenic, and antidiabetic properties of miscellaneous cereal grains harvested in Korea [13, 22, 25, 26, 32], the systematic study on their bioactive components associated with the antioxidant efficacy is still rare. If miscellaneous cereal grains are proven as a proper source of antioxidant phytochemicals, it is likely that these grains become highly effective in preventing oxidative stress-oriented chronic diseases by consuming as diet [28, 38, 40]. Furthermore, it is generally accepted that natural antioxidant components derived from edible plants may be more safely applicable for human health, as compared to synthetic antioxidants.

As an attempt to examine antioxidant activities of eleven selected miscellaneous cereal grains (proso millet, yellow glutinous proso millet, hwanggeumchal sorghum, glutinous sorghum, white glutinous sorghum, yellow glutinous foxtail millet, non-glutinous foxtail millet, green glutinous foxtail millet, golden foxtail millet, barnyard millet, and adlay) harvested in Korea, in the present study, we intended to compare the free radical-scavenging activities of 80% ethanol extracts of the individual grains using 1,-diphenyl-2-picrylhydrazl (DPPH) and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). The 80% ethanol extracts of hwanggeumchal sorghum, glutinous sorghum and barnyard millet grains, which appeared to possess more potent free radical- scavenging activities compared with those of other grains, were sequentially fractionated by n-hexane, methylene chloride, ethyl acetate and n-butanol. Since the DPPH radical-and ABTS radical-scavenging activities were mainly detected in the ethyl acetate fraction followed by the butanol fraction, the antioxidant activity of the ethyl acetate fractions have been examined further by investigating not only their inhibitory effect on lipid peroxidation using ferric thiocyanate (FTC) and thiobarbituric acid (TBA) methods, but also their cytoprotective effect on tertiary-butyl hydroperoxide (TBHP)-induced apoptotic cell death in human HL-60 cells.

Materials and Methods

Chemicals, reagents, cells and culture medium

Dimethyl sulfoxide (DMSO), Folin-ciocaleau reagent, 1,1-diphenyl-2-picryl-hydrazl (DPPH), 2,2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), tertiary-butyl hydroperoxide (TBHP), ferric thiocyanate (FTC), thiobarbituric acid (TBA), α-tocopherol, linoleic acid, ammonium thiocynate, thiobarbituric acid, and tannic acid were purchased from Sigma (St, Louis, MO, USA). Tannic acid was obtained from Avondale Laboratories (Oxon, England). The anti-caspase-3 antibody was purchased from Pharmingen (San Diego, CA, USA). Anti-poly (ADP-ribose) polymerase (PARP), Lamin B and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-caspase-9 was purchased from Cell signaling Technology (Beverly, MA, USA). An ECL Western blotting kit was obtained from Amersham (Arlington Heights, IL, USA) and Immobilon-P membrane was purchased from Millipore (Bedford, MA, USA). Human promyelocytic leukemia cell line HL-60 was obtained from ATCC (Manassas, VA, USA) and maintained in RPMI 1640 (Hyclone, Gaithersburg, MD, USA) containing 10% fetal bovine serum, 20 mM HEPES (pH 7.2), 5×10−5 M β-mercaptoethanol, and 100 μg/ml gentamycin. All other chemicals and organic solvents (ethanol, n-hexane, methylene chloride, ethyl acetate, and n-butanol) were of analytical grade.

Sample extraction

Eleven miscellaneous cereal grains, including proso millet (polished grains), yellow glutinous proso millet (unpolished grains), hwanggeumchal sorghum (unpolished grains), glutinous sorghum (polished grains), white glutinous sorghum (unpolished grains), yellow glutinous foxtail millet (polished grains), non-glutinous foxtail millet (polished grains), green glutinous foxtail millet (polished grains), golden foxtail millet (unpolished grains), barnyard millet (unpolished grains), and adlay (polished grains), harvested in Korea were provided by National Institute of Crop Science, Rural Development Administration, Miryang, Gyeongnam 627-803, Korea. Individual dried grains were milled on a Blender 7012 (Dynamics Corporation, USA), and then extracted with 80% ethanol for 3 h at 80℃. The ethanol extract was evaporated, dissolved in water, and then sequentially extracted with n-hexane, methylene chloride, ethyl acetate, and n-butanol. Each organic solvent fractionation was repeated three times. The organic solvent fraction was concentrated by rotary vacuum evaporator (Heidolph LR 4000, Germany). The dry weights of the 80% ethanol extracts and organic solvent fractionations were described in Supplement 1 [23].

1,1-Diphenyl-2-picryl-hydrazl (DPPH) radicalscavenging activity assay

The 1,1-diphenyl-2-picryl-hydrazl (DPPH) radical-scavenging activity was carried out by modifying the method of Blois [3]. DPPH stock solution (0.2 mM) was prepared in ethanol. To 800 μl of DPPH stock solution, 50 μl of sample dissolved in dimethyl sulfoxide (DMSO) was added. After the mixture was incubated in 37℃ for 30 min, the absorbance was measured at 517 nm. The DPPH-radical-scavenging activity (%) = [(Absorbance of control – Absorbance of sample)/Absorbance of control]×100 [8]. Lower absorbance values of the mixtures indicate higher free radical scavenging activity. The EC50 values denote the effective concentration of sample that is required to scavenge 50% of the DPPH free radicals [17]. All determinations were conducted in triplicates.

2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical-scavenging activity assay

The 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) solution was prepared by reaction of 1 ml of 7 mM ABTS stock solution dissolved in d'H2O and 17.6 μl of 140 mM K2S2O8 dissolved in d'H2O. After incubation in the dark for 16 h, ABTS radical solution was diluted in 50 ml of absolute ethanol. And then 50 μl of sample stock (1 mg/ml) was added to 1 ml of ABTS radical solution. After incubation at room temperature for 20 min, Absorbance value was measured at 734 nm. The ABTS radical-scavenging activity (%) = (Absorbance of control − Absorbance of sample)×100/ (Absorbance of control). The EC50 values denote the effective concentration of sample that is required to scavenge 50% of the ABTS free radicals [30]. All determinations were conducted in triplicates.

Ferric thiocyanate (FTC) assay

The FTC method was performed as previously described [31]. Briefly, a mixture of 1.2 ml of sample (1 mg/ml in DMSO), 1.2 ml of linoleic acid (25 mg/ml in EtOH), 2.4 ml of 0.2 M phosphate buffer (pH 7.0), and 1.2 ml of distilled water was placed at 40℃ in the dark for indicated time periods. Twenty five microliters of the mixture were mixed with 25 μl of ammonium thiocyanate (0.3 μg/ml in H2O), 25 μl of ferrous chloride (2.45 mg/ml in 3.5% hydrochloric acid), and 750 μl of 70% EtOH, and then incubated for 3 min. The optical density (OD) of the reaction of mixture was measured at 500 nm. A mixture without added sample was used as a negative control. The percent of inhibition of lipid peroxidation = 100 − (A1 − A0 )×100, where A0 is the absorbance of the negative control and A1 is the absorbance of the extract samples [8]. The experiment was performed in triplicates.

Thiobarbituric acid (TBA) assay

The TBA assay was performed as described elsewhere [37]. Briefly, 300 μl of 20% trichloroacetic acid (TCA) and 150 μl of 0.67% thiobarbituric acid (TBA) were added to 300 μl of sample solution or standard solution. The mixture was placed in a boiling water bath for 10 min. After cooling, the mixture was centrifuged at 3,000 rpm for 20 min. Absorbance of supernatant was measured at 532 nm. The percentage of inhibition of lipid peroxidation = (Absorbance of control − Absorbance of sample)×100/Absorbance of control. The experiment was performed in triplicates.

Flow cytometric analysis

Flow cytometric analysis for the cell cycle of HL-60 cells was carried out as previously described [46]. Changes in the Δψm were measured after staining with 3,3'-dihexyloxacarbocyanine iodide (DiOC6) [19]. The cells were harvested and incubated with PBS containing 50 nM DiOC6 for 15 min at 37℃ prior to flow cytometric analysis.

The cytoprotective effect of the 80% ethanol extracts and their ethyl acetate fractions obtained from hwanggeumchal sorghum, glutinous sorghum, and barnyard millet grains against tertiary-butyl hydroperoxide (TBHP)-induced cytotoxicity in HL-60 cells was also measured by flow cytometry. Briefly, the cells (0.5×106/ml) were preincubated with a proper dilution of individual samples for 1 h, and then exposed to 100 μM TBHP for indicated time periods. After the cells were harvested, equivalent number of cells were fixed with cold ethanol (67%), stained with propidium iodide, and subjected to flow cytometric analysis to quantify apoptotic sub-G1 cells. Following flow cytometric analysis for apoptotic sub-G1 cells, the protective activity against TBHP-induced apoptosis was determined according to the equation: Cytoprotection ratio (%) = (sub-G1 cell ratio for 100 μM TBHP − sub-G1 cell ratio for 100 μM TBHP plus sample) ×100 / sub-G1 cell ratio for 100 μM TBHP.

Quantification of total phenolic compounds

Total phenolic contents in the 80% ethanol extracts and organic solvent fractions of selected miscellaneous cereal grains were measured by Folin-Ciocalteau method using tannic acid as a standard phenolic compound as previously described [39]. The 80% EtOH extracts and organic solvent fractions were split 100 μl to each test tubes, then 500 μl of Folin-Ciocalteau reagent was added. The mixture was incubated at RT for 5 min and added 400 μl 7.5% Na2CO3. This mixture was incubated at 50℃ for 5 min, and centrifuged at 13,000 rpm for 2 min to remove insoluble precipitation. The absorbance was then measured at 760 nm. Total phenolic content was indicated in μg of tannic acid (TAE) equivalents per milligram of extract (μg TAE/mg extract). All determinations were conducted in triplicates.

Preparation of cell lysates and Western blot analysis

Cellular lysates were prepared by suspending HL-60 cells (5×106 cells) in 250 μl of lysis buffer (137 mM NaCl, 15 mM EGTA, 1 mM sodium orthovanadate, 15 mM MgCl2, 0.1% Triton X-100, 25 mM MOPS, and 5.0 μg/ml proteinase inhibitor E-64, pH 7.2). The cells were disrupted by sonication and extracted at 4℃ for 30 min. An equivalent amount of protein lysate (20∼25 μg) was electrophoresed on a 4-12% NuPAGE gradient gel (Invitrogen/Novex, Carlsbad, CA, USA) with MOPS buffer, and then electrotransferred to Immobilon-P membranes. Detection of individual proteins was performed using an ECL Western blotting kit according to the manufacturer’s instructions.

Statistical analysis

Unless otherwise indicated, each result in this paper is representative of at least three separate experiments. Values represent the mean ± standard deviation (SD) of these experiments. The statistical significance was calculated with Student’s t-test. P values less than 0.05 were considered significant.

Results and Discussion

DPPH radical- and ABTS radical-scavenging activities of the 80% ethanol extracts and their solvent fractions obtained from eleven selected miscellaneous cereal grains

To evaluate antioxidant properties of miscellaneous cereal grains, which were harvested in Korea, including proso millet, yellow glutinous proso millet, hwanggeumchal sorghum, glutinous sorghum, white glutinous sorghum, yellow glutinous foxtail millet, non-glutinous foxtail millet, green glutinous foxtail millet, golden foxtail millet, barnyard millet and adlay, the free radical-scavenging activities of 80% ethanol extracts prepared from the individual grains were measured using DPPH and ABTS methods, both of which have widely been employed to evaluate free radical-scavenging activities of natural compounds in foods and biological systems [34, 43]. As shown in Table 1, the 80% ethanol extract of hwanggeumchal sorghum grains exhibited the highest DPPH radical-scavenging activity followed by those of glutinous sorghum and barnyard millet grains. The EC50 values of the 80% ethanol extracts of hwanggeumchal sorghum, glutinous sorghum, and barnyard millet grains for DPPH radical-scavenging activity appeared to be 33.82, 131.10 and 141.70 μg/ ml, respectively. The ABTS radical-scavenging activity assay also showed that the 80% ethanol extract of hwanggeumchal sorghum grains had the highest ABTS radical-scavenging activity followed by those of barnyard millet and glutinous sorghum grains. The EC50 values of the ethanol extracts of hwanggeumchal sorghum, glutinous sorghum, and barnyard millet grains for ABTS radical-scavenging activity were 8.41, 25.95 and 29.59 μg/ml, respectively.

Table 1.EC50 values of the 80% ethanol extracts of eleven miscellaneous cereal grains for DPPH and ABTS radical-scavenging activity

In order to examine further the antioxidant properties of hwanggeumchal sorghum, glutinous sorghum, and barnyard millet grains, the 80% ethanol extracts of the three miscellaneous cereal grains were sequentially fractionated with n-hexane, methylene chloride, ethyl acetate and n-butanol, and th

en individual organic solvent fractions were tested for the DPPH and ABTS radical scavenging activities. As shown in Table 2, the ethyl acetate and butanol fractions exhibited more potent DPPH radical- and ABTS radical-scavenging activities as compared to other organic solvent fractions of hwanggeumchal sorghum, glutinous sorghum, and barnyard millet. The EC50 values of the ethyl acetate fractions of hwanggeumchal sorghum, glutinous sorghum, and barnyard millet grains were 12.65, 16.03 and 31.41 μg/ml for DPPH radical-scavenging activity, and 4.52, 8.31 and 11.10 μg/ml for ABTS radical-scavenging activity. In addition, the EC50 values of the butanol fractions of hwanggeumchal sorghum, glutinous sorghum, and barnyard millet grains were 14.65, 27.91 and 39.59 μg/ml for DPPH radical-scavenging activities, and 5.48, 9.12 and 16.46 μg/ml for ABTS radical-scavenging activities. On the other hands, the EC50 values of α-tocopherol, which is a well-known natural antioxidant [16], for DPPH radical- and ABTS radical-scavenging activities were 19.84 and 9.81 μg/ml, respectively.

Table 2.EC50 values of the 80% ethanol extracts and their organic solvent fractions of glutinous sorghum, hwanggeumchal sorghum, and barnyard millet for DPPH and ABTS radical-scavenging activity

Consequently, these results showed that the ethyl acetate fraction of glutinous sorghum grains as well as both the ethyl acetate and butanol fractions of hwanggeumchal sorghum grains possessed higher DPPH radical- and ABTS radical- scavenging activities than those of α-tocopherol. In addition, these results raised the possibility that the free-radical scavenging activity-related component(s) in the ethyl acetate fraction of glutinous sorghum grains, and the ethyl acetate and butanol fractions of hwanggeumchal sorghum grains might have more potent antioxidant activity than α-tocopherol.

Inhibitory effect of the 80% ethanol extracts, ethyl acetate fractions, and butanol fractions from hwanggeumchal sorghum, glutinous sorghum and barnyard millet grains on linoleic acid peroxidation

Lipids undergo oxidative degradation (lipid peroxidation) in the presence of reactive oxygen species (ROS) [30]. As a basic mechanism underlying lipid peroxidation, ROS are known to abstract a hydrogen atom from a methylene group of unsaturated fatty acids, such as linoleic acid, linolenic acid and arachidonic acid, and subsequently form free radicals such as peroxyl radicals. Once the free radicals are formed, lipid peroxidation progresses to produce various secondary oxidation products. Ferric thiocyanate (FTC) assay and thiobarbituric acid (TBA) assay, which are frequently used as antioxidant tests for plant and food components, are known to specifically monitor hydroperoxide and malonaldehyde (MA) as the products of lipid peroxidation, respectively. In order to examine whether the antioxidant properties of glutinous sorghum, hwanggeumchal sorghum and barnyard millet grains, which were detectable by DPPH radical- and ABTS radical-scavenging activities, could also prevent lipid peroxidation, we decided to investigate the effects of the 80% ethanol extracts, ethyl acetate fractions, and butanol fractions from hwanggeumchal sorghum, glutinous sorghum and barnyard millet grains on linoleic acid peroxidation by employing both FTC and TBA methods.

In FTC assay, the amount of linoleic acid peroxidation is determined by measuring ferrous oxidation to ferric iron, because the oxidized linoleic acid reacts with ferrous to form ferric ion which then reacts with ammonium thiocyanate to generate ferric thiocyanate possessing red color [30]. As shown in Fig. 1A, the absorbance values of the FTC reaction mixture containing linoleic acid solutions which were obtained after treatment for the indicated time periods appeared to increase in a time-dependent manner and exhibited 3.5-fold elevation after linoleic acid solution was treated at 40℃ for 5 days. This indicated a continuous enhancement in linoleic acid peroxidation during the 5-day treatment. However, the linoleic acid solution treated in the presence of the individual organic solvent fractions of hwanggeumchal sorghum, glutinous sorghum and barnyard millet grains, failed to elevate the absorbance value of the reaction mixtures with the percentage inhibition of 86%-91%. These inhibitory effects of hwanggeumchal sorghum, glutinous sorghum and barnyard millet grains, which lowered the absorbance values of the FTC reaction mixture, were comparable to those of α-tocopherol. These results showed that antioxidant properties of the organic solvent fractions of glutinous sorghum, hwanggeumchal sorghum, and barnyard millet grains could commonly inhibit linoleic acid peroxidation. In TBA assay, the level of malonaldehyde (MA) among lipid peroxidation products is measured to evaluate the antioxidant activity of chemical(s) in linoleic acid peroxidation system, because MA is known to bind TBA to form a red compound MA-TBA adduct. When the TBA reaction mixtures containing the linoleic acid sample prepared in the FTC method was tested, a similar trend was observed with all tested extracts exhibiting low absorbance values corresponding to the percentage inhibition of 38.0-56.1% (Fig. 1B). Under the same conditions, the controls, α-tocopherol showed a slightly lower inhibition of 48.5%, when compared with the inhibition values observed in the FTC assay. These results showed that the data of the TBA assay correlated well with those of the FTC assay. Consequently, current data demonstrated that glutinous sorghum, hwanggeumchal sorghum and barnyard millet grains commonly possessed the antioxidant properties which could inhibit ROS-mediated lipid peroxidation.

Fig. 1.Evaluation of lipid peroxidation in - hibitory activity of the 80% EtOH extracts, and their ethyl acetate and butanol fractions of hwanggeumchal sorghum, glutinous sorghum, and barnyard millet grains by ferric thiocyanate (FTC) method (A) and thiobarbituric acid (TBA) method (B). The experiment was performed in triplicates. Results are expressed as mean ± standard error of the mean (n=3).

Quantification of phenolic compounds in the 80% ethanol extracts and organic solvent fractions

Phenolic compounds are known as very important antioxidant constituents of plants and their radical scavenging ability is due to their redox properties, hydrogen donors, free radical quenchers and metal-chelators [15]. Previously many studies have demonstrated a high correlation between the contents of total phenolic compounds in plants and their antioxidant capacity [7, 18, 33, 35, 47]. To investigate the correlation between antioxidant activities and total phenolic contents, the ethanol extracts and their organic solvent fractions were analyzed for total phenolic contents using Folin-Ciocalteau method.

The concentrations of phenolic compounds in the individual extracts expressed as μg of tannic acid (TAE) equivalents per mg of the extract (μg TAE/mg extract) are shown in Table 3. The 80% ethanol extracts of hwanggeumchal sorghum, glutinous sorghum and barnyard millet grains appeared to contain higher level of total phenolic compounds than other ethanol extracts tested, with exhibiting 121.1 μg TAE/mg, 55.0 μg TAE/mg, and 66.8 μg TAE/mg, respectively. When the phenolic contents in the individual organic solvent fractions of hwanggeumchal sorghum, glutinous sorghum, and barnyard millet grains were compared, most phenolic components of hwanggeumchal sorghum, glutinous sorghum, and barnyard millet grains were distributed in both ethyl acetate and butanol fractions. The contents of phenolic compounds in the ethyl acetate and butanol fractions of hwanggeumchal sorghum grains were 255.2 μg TAE/mg and 244.1 μg TAE/mg, whereas the phenolic contents in the ethyl acetate and butanol fractions of glutinous sorghum grains were 248.4 μg TAE/mg and 159.5 μg TAE/mg, respectively. At the same time, the phenolic contents in the ethyl acetate and butanol fractions of barnyard millet were 198.7 μg TAE/mg and 137.9 μg TAE/mg, respectively. These quantitative levels of total phenolic compounds appeared to be consistent with the antioxidant activity profiles measured in this study. Consequently, current results suggested that the antioxidant activities of the hwanggeumchal sorghum, glutinous sorghum and barnyard millet grains were mainly attributable to the presence of phenolic components.

Table 3.*The concentrations of phenolic compounds in the individual extracts were expressed as μg of tannic acid (TAE) equivalents per mg of the extract (μg TAE/mg extract)

Inhibitory effect of the ethyl acetate fractions of hwanggeumchal sorghum, glutinous sorghum, and barnyard millet grains on TBHP-induced apoptotic signaling and cell death in human HL-60 cells

Since the ethyl acetate fractions of hwanggeumchal sorghum, glutinous sorghum and barnyard millet grains exhibited higher level of DPPH radical- and ABTS radical-scavenging activities and phenolic ingredients than those of the other organic solvent fractions, we decided to examine whether the ethyl acetate fractions could prevent the oxidative stress-mediated apoptotic cell death in human cells. In this context, the effect of the ethyl acetate fractions on TBHP-induced apoptotic sub-G1 peak at concentrations of 12.5 and 25 μg/ml in human HL-60 cells was analyzed by flow cytometry. Previously, it has been reported that TBHP is a potent free radical generating toxin to mimic the oxidative stress and thus can provoke oxidative stress-mediated apoptotic cell death [1, 14].

As shown in Fig. 2, the ethyl acetate fractions of hwanggeumchal sorghum, glutinous sorghum and barnyard millet grains at a concentration of 25 μg/ml could inhibit the TBHP-induced sub-G1 peak to the levels of 51.9%, 65.9%, and 72.2%, respectively. At the same time, the ethyl acetate fractions at a concentration of 12.5 μg/ml could inhibit the TBHP-induced sub-G1 peak to the levels of 25.4%, 48.5%, and 31.1%, respectively. The 80% EtOH extracts (25 μg/ml and 50 μg/ml) of hwanggeumchal sorghum and glutinous sorghum grains appeared to reduce the TBHP-induced sub-G1 peak slightly with the percentage inhibition of < 10%, whereas the 80% EtOH extract of barnyard millet grains at the same dose failed to reduce the level of TBHP-induced apoptotic sub-G1 peak. In addition, the control α-tocopherol (100 μM) was able to inhibit the TBHP-induced sub-G1 peak to the level of 23.7%. It is noteworthy that the presence of α-tocopherol at concentrations of > 100 μM appeared to exert cytotoxicity toward HL-60 cells. Consequently, these results demonstrated that the cytoprotective effect of the ethyl acetate fractions of hwanggeumchal sorghum, glutinous sorghum and barnyard millet grains on TBHP-induced apoptosis was exerted in a dose-dependent manner. These results also suggested that the cytoprotection of the ethyl acetate fractions against oxidative damage might be more potent than that of α-tocopherol.

Fig. 2.Cytoprotective effect of the 80% EtOH extracts and ethyl acetate fractions of hwanggeumchal sorghum, glutinous sorghum, and barnyard millet grains on tertiary-butyl hydroperoxide (TBHP)-induced apoptotic cell death (sub-G1 peak) in human HL-60 cells. The cells were incubated at a density of 5×105 /ml with the indicated concentrations of the 80% EtOH extracts and their ethyl acetate fractions for 5 h. Flow cytometric analysis of the cell cycle distribution was performed as described in Materials and Methods. The experiment was performed in triplicates. Results are expressed as mean ± standard error of the mean (n=3).

To elucidate the biochemical mechanism underlying the cytoprotective effect of the ethyl acetate fractions of hwanggeumchal sorghum, glutinous sorghum, and barnyard millet grains on the TBHP-induced apoptosis, we investigate the effect of the ethyl acetate extracts on the TBHP-induced apoptotic signaling pathway using flow cytometry and western blot analyses. After HL-60 cells were treated with the individual ethyl acetate fractions (25 μg/ml) for 1 h, the cells were exposed to 100 μM TBHP for 5 h. As shown in Fig. 3A, although HL-60 cells following treatment with TBHP exhibited 65.5% of apoptotic sub-G1 cells, the sub-G1 cells declined to the levels of 35.0%, 28.2%, and 30.9% in the presence of the ethyl acetate fractions of hwanggeumchal sorghum, glutinous sorghum, and barnyard millet grains, respectively, indicating a significant reduction in the level of TBHP-induced apoptotic cell death by the ethyl acetate fractions. Under these conditions, the effect of the ethyl acetate fractions on TBHP-induced mitochondrial membrane potential (Δψm) loss was also measured by flow cytometry using DiOC6 staining. Although there was barely detectable Δψm loss in continuously growing HL-60 cells, 58.3% cells exhibited Δψm loss following exposed to TBHP (Fig. 3B). However, the presence of the ethyl acetate fractions of hwanggeumchal sorghum, glutinous sorghum, and barnyard millet grains could reduce the TBHP-induced Δψm loss to the levels of 35.7%, 29.8%, and 36.8%, respectively. This indicated that the oxidative stress-mediated mitochondrial damage in HL-60 cells treated with TBHP could be protected by the ethyl acetate fractions of hwanggeumchal sorghum, glutinous sorghum, and barnyard millet grains. The control α-tocopherol (100 μM) appeared to reduce the TBHP-induced Δψm loss to the level of 42.4%. Because Δψm loss is known to be one of the initial intracellular changes that are accompanied by apoptotic cell death [42], these results suggested that Δψm loss being involved in TBHP-induced apoptosis was prevented by the ethyl acetate fractions of hwanggeumchal sorghum, glutinous sorghum, and barnyard millet grains. These results indicated that the ethyl acetate fractions of hwanggeumchal sorghum, glutinous sorghum, and barnyard millet grains could suppress the oxidative stress-mediated Δψm loss more efficiently than the antioxidant control, α-tocopherol.

Fig. 3.Cytoprotective effect of the ethyl acetate fractions of hwanggeumchal sorghum, glutinous sorghum, and barnyard millet grains on TBHP (100 μM)-induced apoptotic cell death (sub-G1 peak) (A) and mitochondria membrane potential (Δψm) loss (B) in human HL-60 cells. The cells were incubated at a density of 5×105 /ml with the indicated concentrations of the ethyl acetate fractions for 5 h. Flow cytometric analysis of the cell cycle distribution and Δψm loss was performed as described in Materials and Methods. A representative study is shown and two additional experiments yielded similar results.

Since it was reported that Δψm loss precedes the release of cytochrome c into the cytosol, followed by caspase cascade activation [24, 41], it was likely that the suppression of TBHP-induced Δψm loss by the ethyl acetate fractions might be reflected by a reduction in mitochondria-dependent activation of caspase-9 and -3, leading to degradation of PARP and lamin B, in HL-60 cells. To test this prediction, it was investigated in HL-60 cells, by western blot analysis, whether TBHP-induced activation of caspase-9 and -3 could be suppressed by pretreatment with the ethyl acetate fractions. In accordance with the Δψm loss, the cleavage of procaspase-9 (47 kDa) to active forms (37/35 kDa) as well as the cleavage of procaspase-3 (32 kDa) into active form (17 kDa) was detected in HL-60 cells treated with TBHP (Fig. 4). At the same time, the degradation of PARP and lamin B was detected along with the activation of caspase-3 in HL-60 cells treated with TBHP. However, these apoptotic events were significantly reduced in the presence of the ethyl acetate fractions of hwanggeumchal sorghum, glutinous sorghum, and barnyard millet grains. In addition, although these apoptotic events were attenuated by α-tocopherol, the attenuation potency appeared to be much lesser extent as compared to the ethyl acetate fractions. Consequently, these results demonstrated that the antioxidant activities of the ethyl acetate fractions obtained from the grains of hwanggeumchal sorghum, glutinous sorghum, and barnyard millet could inhibit the oxidative stress-mediated mitochondrial damage and thus subsequent activation of mitochondria-dependent caspase cascade in HL-60 cells treated with TBHP.

Fig. 4.Inhibitory effect of the ethyl acetate fractions of hwanggeumchal sorghum, glutinous sorghum, and barnyard millet grains on TBHP (100 μM)-induced activation of caspase-9 and caspase-3, and cleavage of PARP and Lamin B in human HL-60 cells. The cells were incubated at a density of 5×105/ml with the indicated concentrations of the ethyl acetate fractions for 5 h. Western bot analysis was performed as described in Materials and Methods. A representative study is shown and two additional experiments yielded similar results.

In conclusion, these results show that the 80% ethanol extracts of hwanggeumchal sorghum (Sorghum bicolor L. Moench cv. Hwanggeumchalsusu), glutinous sorghum (Sorghum bicolor L. Moench cv. Chalsusu), and barnyard millet (Echinochloa esculenta) grains are rich in phenolic components and can exert potent DPPH radical- and ABTS radical- scavenging activities as well as inhibitory activity on lipid peroxidation. In addition, current results indicate that the ethyl acetate fractions prepared from the 80% ethanol extracts of hwanggeumchal sorghum, glutinous sorghum, and barnyard millet grains can protect human HL-60 cells from oxidative damage-induced apoptotic cell death, and their efficacies are more potent than that of a natural antioxidant α-tocopherol.