Rice (Oryza sativa L., Family Poaceae) is the main staple food crop in almost all Asian countries and is of particular economic value to Thailand. Whereas white and brown rice are the crops that are mostly consumed world-wide, there are a large variety of pigmented wild rice species, and rice cultivars, grown in Asia [1]. These varieties produces rice, with colors ranging from red, purple and black, are now increasingly popular. They demand higher prices in domestic and export markets due to their purported health improving properties. The colors of these pigmented rice varieties are due to a range of anthocyanines in the outer layers [2].

The major by-products during milling of rice are rice husk (20%), rice bran (8%) and rice germ (2%) [3]. Whereas most of these by products have previously been used as animal feeds, or in the brewery industry, their importance and economic value as functional health improving foods has now been recognized, particularly for the pigmented-rice varieties. Therefore, research in the industrial use of pigmented rice by-products, such as rice bran (RB) and rice bran oil (RBO) (and bioactive constituents therein), that might have positive health effects have attracted increased attention in the last 5-10 years.

In addition to fiber and nutritive elements of the whole grain, pigmented rice bran is rich in minerals, antioxidants, ligans and a plethora of phytochemicals. Some of these include water-soluble, fat-soluble and insoluble antioxidants, such as γ-oryzanol, vitamin E, vitamins B1, B2, B3 and B6, tocophenol, tocotrieonols, selenium, phenolic acids, and phytic acid [4-8]. There is some research indicating that consumption of rice bran or rice bran oil of brown rice is beneficial in treating a variety of diseases, including: type 2 diabetes; hypertension; hyperlipidemia; cardiovascular diseases; stomach and colon cancer; improvement of immune system and liver function. Colored rice, broadly known as enriched rice with improved health properties [9], is highly nutritious [10] and it has been used for: reducing blood cholesterol [11]; for its antioxidative properties [12,13] ; to prevent Alzheimer’s disease [14]; as a natural antioxidant to improve the stability of foods [15]; decreasing the incidence of atherosclerosis disease [16]; proposed as a UV-A filter in sunscreen [17]; and natural treatment of inflammatory diseases [18,19].

Relatively fewer data are available for pigmented varieties of rice, and most are in vitro studies that demonstrate that several constituents of such rice species and cultivars exhibit strong antioxidant activities [1,4-8]. There is only one in vitro report of antibacterial activities of pigmented-rice crude extracts from four different types of rice [20]. Recent in vivo studies in animal models has demonstrated the positive effects of black rice bran against chemically-induced inflammation, indicating that pigmented rice bran may contain anti-inflammatory and anti-allergic constituents [19,21,22]. Only two in vitro studies to date have demonstrated the anti-inflammatory effects of purple and black rice [9,18].

Sang Yod rice is the dark red-violet pigmented rice from the south of Thailand (Phatthalung province) that has been supported by Her Majesty Queen Regent Sirikit [23] of Thailand. Sang Yod rice contains various nutritive substances, including vitamin B1, B2, B6, carbohydrate, fibers and protein. It is also rich in various minerals such as iron, calcium and phosphorus [24]. Despite the historic use of Sang Yod rice in southern Thailand as a health improving food, there is a paucity of scientific data supporting the rational for such use. The three main aims of this study were: (a) to develop optimum extraction procedures that afford the highest yield of the crude extract from Sang Yod de-husked whole grain rice, and from Sang Yod rice bran, (b) to use quantitative assays to determine yields of γ-oryzanol and vitamins E, B1, B2 and B3 in the extracts, and finally (c) to study the antiinflammatory activities of each of the extracts using inhibition of nitric oxide (NO) production in lipopolysaccharide (LPS)-induced murine microphage leukemia RAW 264.7 cell line [25,26].

Materials and reagents

Raw 264.7 murine macrophage leukemia cell lines were kindly provided by Assoc Prof Dr. Supinya Tewtrakul, Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical sciences, Prince of Songkla University, Thailand. RPMI Medium 1,640 (RPMI 1,640), L-glutamine, Fetal Bovine Serum (FBS), Penicillin- Streptomycin (P/S), Trypsin-EDTA and trypanblue were purchased from Gibco, NY, USA. Phosphate Buffer Saline (PBS) was from Amresco, Ohio, USA. Sodium bicarbonate was from BHD, Poole, England, lipopolysaccharide (LPS, from Escherichia coli), 3-(4,5-dimethyl-2- thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) were from Sigma, MO, USA. Ninety six well micro plates were also purchased from Costar Corning, NY, USA. Other chemicals were from Sigma, MO, USA and Merck, Darmstadt, Germany. Solvents purchased from Merck (Darmstadt, Germany), standard γ-oryzanol, vitamins E, B1, B2, B3 from Sigma–Aldrich (Seelze, Germany). Supercritical fluid extraction (24L) (Guangzhou, Masson New Separation Technology Co. LTD, China) The HPLC system consisted of an Agilent 1100 series, including an auto sampler, column oven, and a variable wavelength. UV-VIS detector (model G1379A). The column used for γ-oryzanol analyses was Hypersil ODS (4.0 x 250 mm, 5 μm) from Agilent Technologies, Palo Alto, CA, USA. The column used for analyses of vitamins E, B1, B2 and B3 was Phenomenex C18 (4.6 x 100 mm, 3 μm) from Phenomenex, CA, USA.

Plant materials and extraction methods

De-husked whole grain Sang Yod rice and Sang Yod rice bran used in this study were collected from the Phatthalung province, Thailand (organic rice) on October 2011. This rice was identified and confirmed by Rice Department of Phattalung province. The rice bran was placed on a large tray and any extraneous matter removed before use. The whole grain rice was dried (50°C) and stored at the room temperature before extraction. Six different extraction methods utilized were as follows:

Maceration by 95% ethanol

The plant material (100 g) was macerated in 95% ethanol (1 L) for 3 days at room temperature, repeated 2 times, filtered and solvent (3 L) evaporated to dryness using a rotary film at 45 ºC then dried to constant weight by using vacuum desiccator. The extract was stored at -20°C.

Supercritical fluid extraction

The plant material (100 g) was extracted with CO₂ at a critical temperature of 31°C for 21 hours, critical pressure of 74 bars and kept it in the collector tank, and filtered before used. The extract was stored at -20°C.

Boiling in water

The plant material (100 g) was boiled in distilled water (1 L) at 70°C for 30 minutes, filtered and dried by using freeze dryer. The extract was stored at -20°C.

Water from washing rice

The plant material (100 g) was washed with sterile water three times at ratio of rice : sterile water as 1 : 2 and dried by using a freeze dryer. The extract was stored at -20°C.

Expression methods

The plant material (100 g) was put into expression tool, to separate oil and residue. The oil was kept at room temperature for 24 hours and filtered. The extract was stored at -20°C.

Soxhlet extraction method

Using plant material: solvent ratios of 30 g : 1 L, the samples were extracted in a Soxhlet apparatus using solvents of increasing polarity; hexane, chloroform and then methanol at temperatures 40, 50 and 60°C, respectively. The samples were extracted in each solvent for 30 minutes, filtered and evaporated to dryness using a rotary film evaporator at 45°C then dried to constant weight in vacuum desiccator. The extract was stored at -20°C.

Rice bran was extracted by all methods above except the extraction method as washing rice with water. Whole grain rice was extracted only two methods (maceration in 95% ethanol and washing rice with water). The nine different extracts from Sang Yod rice were listed in Table 1.

Table 1: Percentage yields of various crude extracts from whole Sang Yod rice and rice bran using various extraction methods.

Determination of γ-oryzanol content in extracts

γ-oryzanol content in rice bran extract was qualified using reverse phase high performance liquid chromatography (RP-HPLC) according to the method described in the literature [5]. Each extract (10 mg) was dissolved in 1.0 ml of methanol, and filtered through a syringe filter with PTFE (0.2 μm; Ascorbic syringe filter). Aliquots (10 μl) of prepared samples were injected on to the Hypersil ODS column using a rheodyne injector (or autosampler), using a mixture of methanol: acetonitrile: dichloromethane: acetic acid (50:44:3:3, v/v/v/v) as a mobile phase at a flow rate of 1.0 ml/min with UV detection at 330 nm. The content of γ-oryzanol was calculated from calibration graphs obtained by using different concentrations of standard γ-oryzanol. Calibration data was triplicate analyses were performed in three different days. The standard curve was analyzed using the linear least-squares regression equation derived from the peak area and correlation coefficients (r²) of 0.9998. All sample analyses, and cell calibration experiments, were repeated on three separate occasions, with triplicates assays for each sample on any one day. Data for extracts are reported as mg per 100 g of extract.

Determination of Tocopherol (Vitamin E) content in rice bran extract

Tocopherol (Vitamin E) content in each extract was measured by using reverse phase high performance liquid chromatography (RPHPLC) according to the method described in the literature [27] with some modification. Rice extract (10.0 mg) was dissolved in 1.0 ml of methanol and filtered through a syringe filter with PTFE (0.2 μm; ascorbic syringe filter). Aliquots (10 μl) of the samples were injected onto the Phenomenex C18 column using a rheodyne injector (or autosampler), with UV-detection at 292 nm. A mixture of methanol : water (98 : 2 v/v) was used as a mobile phase at a flow rate 1.5 ml/ min in isocratic mode. The content of Tocopherol was calculated from calibration graphs obtained by using different concentrations of standard Tocopherol, calibration data Calibration data was triplicate analyses were performed in three different days. The standard curve was analyzed using the linear least-squares regression equation derived from the peak area and correlation coefficients (r²) of 0.9996. All sample analyses, and cell calibration experiments, were repeated on three separate occasions, with triplicates assays for each sample on any one day. Data for extracts are reported as mg per 100 g of extract.

Determination of Vitamin B1, Vitamin B2, and Vitamin B3, content in rice extracts

Vitamin B1, Vitamin B2 and Vitamin B3 content in extracts were measured by using reverse phase high performance liquid chromatography according to a published method [28], with some modification. Extracts 10.0 mg were dissolved in 1.0 ml of methanol and filtered through a syringe filter with PTFE (0.2 μm; ascorbic syringe filter). Aliquots of the samples (10 μl) were injected onto the Phenomenex C18 column using a rheodyne injector (or autosampler), using a mixture of 0.1 Mol KH₂PO₄ (pH 7) : methanol (90:10 v/v) as a mobile phase at flow rate 0.7 ml/min in isocratic mode. A variable wavelength detector set at 234, 266 and 261, respectively, was used for detection. The standard curve was analyzed using the linear leastsquares regression equation derived from the peak area and correlation coefficients (r²) of standard Vitamin B1, Vitamin B2 and Vitamin B3 (r²=0.997, 0.989 and 0.999 respectively). All sample analyses, and cell calibration experiments, were repeated on three separate occasions, with triplicates assays for each sample on any one day. Data for extracts are reported as mg per 100 g of extract.

Anti-inflammatory activity of extracts using the inhibition of NO production in LPS-induced RAW 264.7 cell line

Inhibitory effects on NO production by murine macrophages like RAW 264.7 cells were evaluated by the following the method described by Tewtrakul and Itharat [29]. The RAW 264.7 cells were washed with phosphate buffer saline (PBS) free of magnesium and calcium. The PBS was decanted and cells were harvested with 0.25% trypsin-EDTA and fresh medium was added. The cell pellet, was obtained by centrifugation (1000 rpm, 6 min), was resuspended in 10 ml of medium to make a single cell suspension. The viable cells were counted by trypan blue exclusion in hematocytometer and diluted with medium to give a final concentration of 1 × 10⁶ cells/ ml for RAW 264.7 cells. One hundred microlitres per well of these cells suspension were seeded in each 96-well microplates with 1 × 10⁵ cells/well and allowed to adhere for 1 hour at 37°C in 5% CO₂. After that the medium was replaced with fresh medium containing 10 μg/ ml of lipopolysaccharide (LPS) together with test samples at various concentrations and then incubated for 48 hours. The extracts were dissolved in dimethyl sulfoxide (DMSO) and the water extracts were dissolved in sterile water, sterilized by filtration (pore size, 0.2 mm) before testing. Stock solutions (10 mg/ml) of the extracts were stored at -20°C until use. The extracts were diluted in medium to produce required concentrations. A hundred microlitres of each concentration was added to each well of plate to obtain final concentrations of 1-100 μg/ml. The final dilution used for treating the cells was contain not more than 1% of the initial solvent; this concentration was used in the solvent control wells. NO production was determined by measuring the accumulation of nitrite in the culture supernatant using the Griess reagent. Cytotoxicity was determined using the 3-(4,5-dimethyl-2- thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) colorimetric method. Briefly, after 48 hours incubation with test samples, MTT solution (10 μl, 5 mg/ml in PBS) was added to the wells. After 2 hours incubation, the medium was removed, and isopropanol containing 0.04 M HCl was then added to dissolve the formazan production in the cells. The optical density of the formazan solution was measured with a microplate reader at 570 nm. The test compounds were considered to be cytotoxic when the optical density of the sample-treated group was less than 70% of that in the control (vehicle-treated) group. Indomethacin was used as positive controls. Inhibition (%) was calculated using the following equation and IC₅₀ values were calculated from the Prism program.

% Inhibition=A-B/A-C × 100

Where, A-C: NO₂^- Concentration (μM) [A: LPS (+), sample (-); B: LPS (+), sample (+); C: LPS (-), sample (-)].

Statistical analysis

The experimental data were expressed as mean ± SEM (n=3) and analyzed using SPSS and Student paired t-test.

From the various extraction methods of rice bran, whole grain and de-husk whole grain of Sung Yod rice were comparison studied on yield, essential chemical content in rice and their anti-inflammatory activity and they were also discussed for useful of Sung Yod rice extraction for health.

The percentage yields of crude extracts from de-husked whole grain Sang Yod rice or rice bran by different methods are shown in Table 1. Boiling rice bran afforded the highest yield (38.2 %w/w) followed by soxhlet extraction using hexane as the solvent (15.23 %w/w). Washing whole grain rice with water, and soxhlet extraction using chloroform as solvent afforded the lowest yields of crude extracts (0.07 and 0.73 %w/w respectively).

The mg %w/w (%w/w) yields of γ-oryzanol and vitamins E, B1, B2 and B3 in all the nine crude extracts are shown in Table 2. The highest yields of γ-oryzanol were in rice bran extracts using soxhlet using chloroform as solvent (344 mg %w/w), followed by expression (309 mg %w/w), and maceration with 95% ethanol (317 mg %w/w). The results are in agreement with previous studies which have reported that pigmented rice bran has the highest content of γ-oryzanol [1,5,10,30,31]. Rice bran extracts, obtained by the maceration (95% ethanol) and soxhlet extraction using chloroform contained the highest levels of vitamin E (211 and 203 mg %w/w). As expected, the water soluble vitamins B1, B2 and B3 were only present in extracts obtained by boiling with water of rice bran or whole de-husked rice grains, there levels being below the detection levels in the organic solvent extracts.

Table 2: Yields of γ-oryzanol and vitamins E, B1, B2, and B3 in various crude extracts of whole Sang Yod rice and rice bran expressed as mg %w/w.

Sang Yod rice and rice bran extracts were investigated for their inhibitory activities against LPS induced NO production in RAW 264.7 cell lines. Measurement of nitrite accumulation in the culture medium, by the Griess reaction, was used to determine NO production. The inhibitory effects of all the nine rice extracts on production of NO are shown in Table 3. Rice bran extract using the 95% ethanolic maceration method (MEB) exhibited the highest inhibitory activity, with an IC₅₀ value of 15.59 ± 1.23 μg/ml (Table 3). The soxhlet extract using chloroform (SXCB) showed the second strongest anti-inflammatory activity with an IC₅₀ value of 16.52 ± 3.36 μg/ml. However pure γ-oryzanol showed the highest inhibitory active of the pure vitamins (IC₅₀ value 1.27 ± 0.71 μg/ml). There is only one previous study on antiinflammatory activities of γ-oryzanol-rich extracts from GAM BOUNG Thai glutinous purple rice bran from northern part of Thailand [18]. Using soxhlet extraction with a mixture of hexane: ethyl acetate (7:3), followed by semi-purification using silica column chromatography they obtained semi-pure extracts which exhibited the high inhibitory effects on NO production (IC₅₀=29.32 ± 2.21 μg/ml). A further purification step employed by Saenjum and coworker’s clearly leads to enrichment of γ-oryzanol, with a consequent high anti-inflammatory effect our crude extract, IC₅₀ value 58.45 μg/ml, Saenjum and coworker’s enriched extracts, IC₅₀ value 29.32 μg/ml. However, Sang Yod rice bran extract obtained by maceration with 95% ethanol (MEB) showed a higher inhibitory effect on NO production than the colored rice from Saenjum and coworker’s report (IC₅₀=15.59 ± 1.23 μg/ml).

Table 3: Anti-inflammatory activities of various crude extracts and isolated pure γ-oryzanol and vitamins E, B1, B2 and B3 from Sang Yod whole rice bran or rice bran.

None of the extracts exhibited any toxic effects against the RAW 264.7 cell line (% Cytotoxicity <20 μg/ml for MTT assay). The extracts obtained by MEB and SXCB need to be further purified to obtain enriched-semi-purified extracts with even a higher anti-inflammatory effect. Already these two crude extracts have a higher activity than indomethacin (IC₅₀ value: MEB=15.59 ± 1.23; SXCB=16.52 ± 3.36; indomethacin=21.01 ± 1.21 μg/ml) which is a positive antiinflammatory compound.

Vitamin E levels in pigmented rice have been reported to be in the range 460-770 mg/100 g of extract [1,5]. The Sang Yod rice and rice bran extracts used in this study contained lower levels of vitamin E (range 93-211 mg %wt), because the type of rice and the extraction methods were different. Our study is the first report on Sang Yod rice and rice bran extracts. The high content of γ-oryzanol and vitamin E in SXCH and MEB extracts account for the strong anti-inflammatory effects of these extracts.

It has been reported that the content of vitamins B1, B2 and B3 in Sang Yod whole rice are in the range of 0.32, 0.01 and 6.40 mg/100 g of Sang Yod whole rice grains, but there are no reports on vitamin content of vitamins in rice extracts. In our study the content of all vitamins B were higher than reported by the Thai Rice Department, Phatthalung province [24]. This is obviously because their data relates to content in Sang Yod whole rice grains, whereas our data is for content in extracts of Sang Yod rice and rice bran. Our study shows that Sang Yod rice bran extracts contain high γ-oryzanol and vitamins E, B1, B2, and B3, suggesting that these extracts would to be a good source of natural antioxidants. The extracts obtained by boiling Sang Yod rice bran showed moderate anti-inflammatory activity compared to pure vitamins B1, B2 and B3. The extract obtained from washing rice grain with water showed high content of vitamins B, especially B2, suggesting that washing Sang Yod rice grain during commercial processing, or prior to cooking will result in the loss of vitamins B1, B2, and B3. This extract also had highest content of vitamin B2, and exhibited higher anti-inflammatory effect (IC₅₀=7.78 ± 0.52 μg/ml) than vitamins B1 and B3. The expression of rice bran yield an extract that had a high content of γ-oryzanol and vitamin E, and this extract had higher anti-inflammatory activity than extract obtained by boiling rice. The expression method can therefore be used as a simple process to obtain an oily product rich in vitamins from Sang Yod rice bran, that can be formulated as oily capsules for sale for the treatment of inflammatory diseases. Supercritical fluid extract had a high content of γ-oryzanol, but did not show any anti-inflammatory activity. This method is complicated and expensive, and therefore not recommended in the production of health products from Sang Yod rice bran.

A few reports have recently been published on the antiinflammatory effects of rice extracts from pigmented rice. Ethanolic extracts of rice bran of colored rice were shown to have an inhibitory effect on edema induced by carragenan in Speague-Dawley rat’s paw [19,22]. Black rice bran extract had inhibitory effected against 12-O-tetradecanolylphorbol-13-acetate (TPA)-induced skin edema and 2,4-dinitrofluorobenzene (DNFB)-induced allergic contact dermatitis (ACD) in inflammatory mouse models [21], and it also showed the inhibition of nitric oxide by lipopolysaccharide (LPS)-induced with RAW 264.7 cells [9]. However, there are no previous reports in animal models of anti-inflammatory activities of extract of Sang Yod rice bran.

MEB and BWB is that maceration of rice bran with 95% ethanol is better than boiling of rice bran with water. However, result from washing with water of whole rice given a very high value (164 mg) for γ-oryzanol. In each case, staring material with rice bran or whole rice were 100 g. γ-oryzanol in whole rice is 164 mg/100 g compared to rice bran (118 mg/100 g) but whole rice only contains about 8% bran which gave γ-oryzanol 9.44 mg in 100 g of whole rice. However, γ-oryzanol obtained from washing with water of whole rice show high yield than rice bran. Thus, washing with water of whole rice which is preparing boiled rice as diary food, should be concern loss on γ-oryzanol.

In summary, Sang Yod rice bran extracted by maceration in 95% ethanol exhibited the highest inhibitory effects on NO production with IC₅₀ value of 15.59 ± 1.23 μg/ml. This extract can be good choice for preparation of commercial products of Sang Yod rice for treatment of inflammatory diseases. However further studies are required to confirm anti-inflammatory activity is mediated by inhibition of COX-1 and COX-2 enzymes. The NO inhibitory activity appears to correlate to content of essential compounds in Sang Yod rice, especially content of γ-oryzanol. Our data provides some rational for the historic use of this rice in southern Thailand as a health enhancing food product, and suggests procedures that might be used for the commercial preparation of marketable functional products of Sang Yod rice.

The authors wish to thank the Nation Research Council of Thailand, National Research University Project of Thailand Office of Higher Education Commission, Center of Excellence on Applied Thai Traditional Medicine Research (CEATMR) and Thammasat University, Thailand, for financial support. In addition, we would like to thank Professor L. A. Damani who is visiting Professor in CEATMR for his critical comments and careful scientific editing of this manuscript.