Evaluation of Î³-oryzanol content and composition from the grains of pigmented rice-germplasms by LC-DAD-ESI/MS

Background Rice is the staple food and one of the worldâ€™s three major grain crops. Rice contains more than 100 bioactive substances including phytic acid, isovitexin, Î³-oryzanol, phytosterols, octacosanol, squalene, Î³-aminobutyric acid (GABA), tocopherol, tocotrienol derivatives, etc. Out of them, Î³-oryzanol is known to have important biological profile such as anti-oxidants, inhibitor of cholesterol oxidation, reduce serum cholesterol levels in animals, effective in the treatment of inflammatory diseases, inhibit tumor growth, reduce blood pressure and promotes food storage stability when used as a food additive, etc. Hence in the present investigation, we aimed to evaluate the content and composition of Î³-oryzanol from pigmented rice germplasms using a liquid chromatography with diode array detection and electrospray ionization-mass spectrometry (LC-DAD-ESI/MS). Findings In the present study, 33 exotic pigmented rice accessions (red, white and purple) have been evaluated. Among them, the contents of Î³-oryzanol varied from 3.5 to 21.0Âmg/100Âg with a mean of 11.2Âmg/100Âg. A total of ten components of Î³-oryzanol including âˆ†7-stigmastenyl ferulate were identified of which, cycloartenyl ferulate, 24-methylenecycloartanyl ferulate, campesteryl ferulate and sitosteryl ferulate were identified as the major components. The mean proportions of steryl ferulates were in the descending order of 24-methylenecycloartanyl ferulate > cycloartenyl ferulate > campesteryl ferulate > sitosteryl ferulate > âˆ†7-campestenyl ferulate > campestanyl ferulate > sitostanyl ferulate > âˆ†7-stigmastenyl ferulate > stigamsteryl ferulate > âˆ†7-sitostenyl ferulate. Almost 11 accessions (33%) showed higher content than the control rice Chucheongbyeo and higher proportions ranged from 10 to 15Âmg/100Âg. Interestingly, the red rice accession Liberian Coll. B11/B-11 (21.0Âmg/100Âg) showed higher content Î³-oryzanol than control rice Jeokjinjubyeo (19.1Âmg/100Âg) and the purple rice accession Padi Adong Dumarat, Mardi No.4376 (20.3Âmg/100Âg) showed a similar content with control rice Heugjinjubyeo (21.4Âmg/100Âg). Conclusions Most of analyzed rice accessions were found to possess higher contents of Î³-oryzanol than the control rice, Chucheongbyeo. In particular, the red accessions showed highest content than the white and purple accessions. The content and composition of Î³-oryzanol in 33 exotic pigmented rice accessions have been evaluated and compared significantly by the present investigation.


Background
Rice is the staple food for more than half the world's population and a valuable food resource as one of the world's three major grain crops. Particularly in Asia, it is a primary food source for most of the countries which can't be replaced by any other crops. However, the rice consumption is showing a considerable downward trend every year as a consequence of the westernization, diversification of dietary patterns and deprived consideration of the nutritional excellence of rice-based diets. Consequently, there is an essential need to upsurge the rice consumption and to solve the economic/social problems of farming communities by develop the rice varieties with more improved nutritional quality and functionality [1][2][3].
At first, γ-Oryzanol was found in rice bran oil in 1954 [33] and it was thought that it consisted of a single component. Later, it was found to be a mixture of different components through a high performance liquid chromatographic analysis (HPLC). However, the number of identified individual components has varied depending on the chromatographic approaches. Diack et al. [34] attempted to separate γ-oryzanol into two fractions using a normal-phase HPLC on a silica column, but their approach failed to isolate and identify the individual components. In contrast, when reverse-phase HPLC was used, Norton et al. [35] and Miller et al. (2003) [36] succeeded in isolating the five different components and Evershed et al. [25] and Rogers et al. [37] succeeded in isolating the six different components. In view of this, recently a reverse-phase HPLC technique has successfully isolated and identified the ten different phytosteryl ferulates of γ-oryzanol including cycloartenyl ferulate, 24-methylenecycloartanyl ferulate and campesteryl ferulate as major components by Xu et al. [38]. Owing to fewer studies on the content and composition of γ-oryzanol in genetic resources and earlier chromatographic analysis involves limitations associated with low peak resolution, long analysis time and large sample size to detect minor components in brown rice, we were tempted to attain an analysis which suggests to irradiate the above limitations and involves a large-scale screening in the evaluation of genetic resources.
Thus, in connection with the above issues and in continuation of our earlier report on the evaluation of anthocyanins in colored potatoes by LC-DAD-ESI-MS [39], the present investigation intends to describe the evaluation of γ-oryzanol content and composition in the seeds of pigmented-rice genetic resources using a liquid chromatography associated with diode array detection and electrospray ionization-mass spectrometry [LC-DAD-ESI/MS]. Figure 1 shows the HPLC chromatograms of standard γ-oryzanol components and extracts from the seeds of control rice varieties, Chucheongbyeo (white rice) and Heugjinjubyeo (red rice). In the present study, a total of 10 components were isolated of which, cycloartenyl ferulate, 24-methylenecycloartanyl ferulate, campesteryl ferulate and sitosteryl ferulate were identified as the major components. Interestingly, herein analysis time was shortened to 40 minutes compared with previous report of 60 minutes and the peak retention time ranged between 15-30 minutes [38,40,41].

Findings
In particular, eight pigmented rice accessions have relatively higher content of γ-oryzanol with more than 15 mg/100 g. Padi Adong Dumarat Mardi No.4376, a purple rice accession showed a similar γ-oryzanol content of 20.3 mg/100 g to the control rice Heugjinjubyeo (21.4 mg/100 g), and γ-Liberian Coll. B11/B-11, a red rice accession showed higher content of 21.0 mg/100 g than the control rice Jeokjinjubyeo (19.1 mg/100 g). The eight pigmented rice accessions showed higher γ-oryzanol content than control-group in the order of 4.5-6 times higher than Chucheongbyeo (3.5 mg/100 g), 2.5-3.5 times higher than Baekjinjubyeo (6.1 mg/100 g), and 1.8-2.5 times higher than Keunnunbyeo (8.8 mg/100 g) ( Table 1). The chemical structure of each isolated component was derived on the basis of mass spectral information from GC-MS and LC-MS analysis [29,39]. When analysis was conducted in negative-ionization mode on a single quadrupole MS equipped with an ESI source, the forehead portion from the whole structure of each isolated component was found to appear as ferulic acid moiety (m/z 177, 178, 193) based on fragment pattern of methyl group (CH 3 ; m/z 15) ( Figure 2).

Materials
In this study, 33 pigmented rice accessions (5 purple and 28 red accessions) and 7 Korean rice varieties (3 white, 3 red and 1 purple rice varieties) obtained from International Rice Research Institute (IRRI) were used for analysis. Hulled rices (whole grain) were used in this study.

Instrumentation and reagents
The instruments were used for the pretreatment process included a refrigerated multi-purpose centrifuge (Hanil Science Industrial Co. Ltd., Korea), and an ultrasonic bath (Daihan Scientific Co. Ltd., Korea). γ-Oryzanol (Wako, Japan) was used as an external standard. The HPLC reagents were methanol, dichloromethane, acetic acid and acetonitrile from Sigma (St. Louis, MO).

Extraction of γ-Oryzanol
A 5 g powdered sample in a conical tube (50 mL) was centrifuged (3000 rpm, 10 min, 4°C) following extraction with 40 ml of dichloromethane-methanol (2:1, v/v) for 30 minutes at 30°C in an ultrasonic shaking water bath. One millilitre of the supernatant solution was collected from the centrifuged sample. A Sep-Pak C 18 cartridge was flushed with 10 mL of dichloromethane followed by the addition of 10 mL of methanol for activation. After loading 1 mL of supernatant (γ-Oryzanol extract) and l mL of standard solution (γ-Oryzanol, 1000 ppm), the cartridge was washed with 10 mL of dichloromethane and eluted with 1 mL of methanol. The γ-Oryzanol filtrate was eluted and concentrated using N 2 gas, and then redissolved in dichloromethane-methanol (2:1, v/v) prior to analysis with LC-DAD-ESI/MS.

Quantitative and qualitative analysis of γ-Oryzanol by LC-DAD-ESI/MS
The quantitative and qualitative analysis γ-Oryzanol in the grains of red and purple rice accessions (whole grain) were analyzed using a Micromass ZQ MS (Waters Co., Milford, MA) and an Alliance e2695 HPLC system (Waters Co.) equipped with a 2998 photodiode array detector (PDA). In addition, YMC PACK ODS-AM reversed-phase column (4.6 x 250 nm I.D., 5 μm; YMC Co. Ltd, Japan) was used. The analysis was conducted at a flow rate of 1.4 ml/min in the detection wavelength of 250-400 nm (a representative wavelength of 325 nm) with the column heater temperature of 30°C. The mobile phases used were methanol: acetonitrile:dichloromethane: acetic acid (50:44:3:3, v/v/v/v) with isocratic elution for 40 minute. The MS was analyzed in negative ionization mode using electrospray ionization (ESI) source. The MS parameters were a cone voltage of 90 V, source temperature of 100°C, desolvation temperature of 500°C, and a desolvation N 2 gas flow of 480 l/h. The range of molecular weights was m/z, 100-800 in full scan mode.

Partial least squares discriminant analysis (PLS-DA)
The SIMCA P+ software (version: 11, Umetrics AB, Umea, Sweden) for multivariate data analysis was used to acquire PLS-DA for discriminate the analyzed rice varieties by arranging and normalizing all quantitative information obtained in this study.