Antioxidants are anti-inflammatory which could be used as therapy for certain inflammatory-linked diseases such as autoimmune diseases, asthma, hepatitis, glomerulonephritis and coeliac diseases. Heart disease, stroke and aging are also leading causes of death which can be reversed through antioxidant intake. Several types of antioxidants have differing properties and knowing how each type affects health in specific doses is essential in combating diseases and maintaining a healthy state. Six groups of antioxidants will be reviewed in this experiment: phenolic acids, flavonoids, anthocyanins, proanthocyanidins, tocopherols, tocotrienols, y-oryzanol and phytic acid. In this experiment, iron reduction will be tracked and analysed using ferric reducing antioxidant power assay. In 700 nm, spectrofluorimetry will be utilized to detect absorbance in this spectrum. Data obtained will be calculated using formula for percent reduction. Antioxidant activity will be determined as there is a direct correlation between reduction and antioxidant activity. Researchers found that percent of reduction was greatest in red rice in 1000 ug/mL with a value of 56.181% compared to 500 ug/mL with 30.53%, 250 ug/mL with -1.35% and 125 ug/mL with 4.30%. In black rice, percent reduction was highest with 1000 ug/mL with a value of 66.20%, 500 ug/mL with a value of 55.87%, 250 ug/mL at 38.51% and 125 ug/mL at -15.07%. Thus, black rice has more reduction compared to red rice, according to data.

 

Keywords: Antioxidants, Red rice, Black rice, Stress, Reduction

The nutrition of rice is the focus of this research experiment. Rice has about 345 Cal/100 g. It contains vitamin B1, niacin, vitamin D, calcium, fibre, iron, thiamine and riboflavin. Most importantly, the antioxidants present in rice will be the concentration of this research as it could be used as therapy to leading causes of death such as heart disease, stroke, respiratory diseases and cancer. Antioxidants are compounds that inhibit oxidation. Oxidation is a chemical reaction that transfers electron or hydrogen from substances to an oxidizing agent. Oxidation reactions produce free radicals — unpaired electrons which start a chain reaction that causes cell death. Antioxidants found in fruits and vegetables terminate these chain reactions by removing free radical intermediates and inhibit other oxidative reactions [1].

Antioxidants have 2 modes of action: hydrogen atom transfer measure the capability of antioxidant to quench free radicals peroxyl radicals considered to be biologically more relevant by H-atom donation. In SET-based assays, antioxidant action is simulated with a suitable redox-potential probe, antioxidants react with a fluorescent or colored probe (oxidising agent) instead of peroxyl radicals [1].

These compounds, antioxidants, reverse aging, prevent cancer, heart disease, stroke, and Alzheimer’s disease which are leading causes of death in the world. Antioxidants reduce inflammation thereby also preventing other inflammatory diseases such as allergy, asthma, autoimmune diseases, coeliac disease, glomerulonephritis, hepatitis and inflammatory bowel disease.

Black and red rice both contain antioxidants in differing amounts. Antioxidants compounds in rice were classified into six groups: phenolic acids, flavonoids, anthocyanins, proanthocyanidins, tocopherols and tocotrienols (vitamin E), y-oryzanol  and  phytic  acid  [2].  Phenolic  acids  contain a phenolic ring and an organic carboxylic acid function with absorption maxima at 280 nm for the C6-C1 skeleton of hydroxybenzoic acid derivatives (gallic, protocatechuic, p-hydroxybenzoic, vanillic and syringic acids) and at 320 nm for the C6-C3 skeleton of hydroxycinnamic acid derivatives (p-coumaric, ferrulic, caffeic, sinapic, chlorogenic and cinnamic acids). The phenolic ring can stabilize and delocalize unpaired electrons, conferring an antioxidant property to phenolic acids. Antioxidant property notably depends on the number and the position of hydroxyl groups on the phenolic ring [3]. Phenolic acids are synthesized from the shikimate pathway from L-phenylalanine or L-tyrosine. Phenylalanin and tyrosine are very important amino acids in this pathway since these amino acids are the common precursors of the majority of natural phenolic products [4]. Flavonoids are synthesized by the phenylpropanoid metabolic pathway; most flavonoids have absorption maxima at 370 nm. Flavonoids consist of a 15-carbon skeleton that is organized in two aromatic rings (A and B-rings) interlinked by a three-carbon chain (structure C6-C3-C6). Flavonoids are recognized for both their ability to donate electrons and to stop chain reactions. These activities are attributed to phenolic hydroxyls, particularly in the 3’OH and 4’OH of the three-carbon chain. Flavonoids can be classified into flavones, flavonols, flavanols, flavanonols, isoflavones and flavanones which generally occur as O- or C-glycosides. Of the seven flavonoids that are usually reported in rice, tricin appears to be the major flavonoid in the bran, accounting for 77% of all seven flavonoids. Anthocyanins, another class of antioxidants exhibit maximum absorbance in the green/blue spectrum at 510 nm, are water-soluble glycosides of polyhydroxyl and polymethoxyl derivatives of 2-phenylbenzopyrylium or flavylium (2-phenylchromenylium) salts. They share a common hydroxylation at the C3, C5 and C7 positions on the B-ring. Anthocyanins exist as O-glycosides (mono, di, or tri) and acylglycosides of anthocyanidins in plants. The sugars may be substituted by aliphatic, hydroxybenzoic or hydroxycinnamic acids. The structural characteristics of anthocyanins make them highly reactive toward reactive oxygen species. Tocotrienols and tocopherols share a common structure unit based on amphiphilic 6-chromanol ring and a terpenoid side chain located at position 2 of the ring. The chromanol head group can be joined to a saturated phytyl side chain to form tocopherols or to an unsaturated geranylgeranyl side chain to form tocotrienols. The head group can then be methylated in different configurations, resulting in four alternative forms (α, β, γ and ş). The free hydroxyl group on the chromanol ring is responsible for the antioxidant properties and the hydrogen atom from this group can be donated to free radicals, resulting in a resonance-stabilized vitamin E radical [5]. Gamma-oryzanol is a mixture of steryl ferulate (y-oryzanol) which are formed by esterification of the hydroxyl group of sterols (campersterol, stigmasterol, b-sitosterol) or triterpene alcohols (cycloartanol, cycloartenol, 24-methylenecycloartanol, cyclobranol) with the carboxylic acid group of ferulic acid. Sterols with saturated steroid skeleton are known as stanols, whereas compounds containing a double bond between C5 and C6 or between C7 and C8 are referred to as sterols. Methyl groups at C4 affect the antioxidant properties of steryl ferulates [2].

SCIENTIFIC LITERATURE REVIEW

The history of rice dated back in China. According to archeological evidence, rice was domesticated in the region of the Yangtze River valley in China. Rice is the most important food crop and dietary staple, ahead of wheat, corn and bananas. It is the chief source of food for about 3 billion people, half of the world’s population, and accounts for 20% of all the calories that mankind consumes. In Asia, more than 2 billion people rely on rice for 60-70% of their calories. If consumption trends continue 4.6 billion people will consume rice in 2025 and production must increase 20% to keep up with demand [6].

In Japan, Korea and other countries, farmers now use small diesel-powered rototiller-tractors to plow the rice paddies and refrigerator-size mechanical rice transplanters to plant the rice seedlings. In the old days it took 25 to 30 people to transplant seedlings of one rice paddy [6].

Water depth in the paddy is increased as rice seedlings grow and gradually lowered in increments until field is dry when rice is ready to be harvested. Water is also drained during growing season so the field can be weeded and soil aerated and water is put back in. Rice is harvested when it is golden-yellow color several weeks after water completely drained from the paddy and soil around rice is dry [6].

Phenolic acids undergo absorption in the gastrointestinal tract then these molecules suffer conjugation reactions and several changes in their initial structure and circulate in human plasma as conjugated forma, glucoronide, methylated and sulphated derivatives. These changes in their structures may increase or decrease bioactivity of the initial phenolic acids [4]. Plant antioxidant activity is due to phenolic acid content, especially caffeic and p-coumaric. The greater antioxidant activity of caffeic acid than p-coumaric acid stems from the 3,4-position of dihydroxylation of the phenolic ring. Phenolics behave as antioxidants due to the reactivity of the phenol moiety (hydroxyl substituent) on the aromatic ring. Caffeic acid is expected to have higher antioxidant activity due to additional conjugation in the propanoic side chain, which facilitates electron delocalization, by resonance, between aromatic ring and proanoic group [7]. Detection of phenolic acids at 280nm is the best alternative for the determination of both classes of phenolic compounds. Extensive use of photodiode array detection in the analysis of phenolic acids can be attributed to the ability to collect online spectra without using stopped-flow techniques [7].

MATERIALS AND METHODS

Extraction

Materials were gathered: 4 test tubes, 1 bottle methanol, 1 mortar and pestle, 1 scooper, 4 Erlenmeyer flasks, 4 1/2 kg bags of red rice and black rice measured at 50 g each and 1 graduated cylinder. 50 g of rice underwent cominution using mortar and pestle. Analytical balance was then used to weigh each sample — red and black rice. Each sample was placed inside Erlenmeyer flask. 120 ml of methanol was measured in graduated cylinder. Sample was slightly shaken — rice and methanol and soaked overnight.

Methanol extracts undergone percolation using filter paper and funnel while filtrate was placed in an Erlenmeyer flask. The filtrate was measured at half of the drum vial to be placed in a rotary evaporator and then replenished until mixture is finished. The sample was processed in 37-40C water bath of rotary evaporator with chiller at 4C until liquid was dry. Each dried extract was measured in an analytical balance by weighing.

Amount of reagents prepared

Sodium phosphate buffer Na₂HPO₄=0.85475192 g, NaH₂PO₄=0.47726086 g

1% Potassium Ferricyanide=0.5 g

10% Trichloroacetic acid=5 g

0.1% Ferric chloride (FeCl₃)=0.015 g

50 ml of distilled water was added to each to create a solution except 0.1% ferric chloride (0.015 g) + 15 ml water. pH meter was used for 0.8547 g Na₂HPO₄ + 0.477 g NaH₂PO₄ + 20 ml water.  

Ferric reducing antioxidant power assay (FRAP)

FRAP involves the following steps: a) Preparation of samples; b) Reactions; c) Measuring absorbance of sample and standard at 700 nm using spectrofluorimetry. It is the reduction of ferric to ferrous through an electron transfer process in the presence of antioxidant. High reduction capacity of plant extract would signify that it has an antioxidant activity. 1 mg/mL of dried plant sample of each types of rice — red and black was measured in analytical balance, adding 1000 μl of methanol per sample. This was sonicated for 15 mins. After which, methanol extracts were transferred into 12 microcentrifuge. Each concentration were subdivided into 4 groups — 1000 μg/ml, 500 μg/ml, 250 μg/ml, 125 μg/ml. Per concentration, sample were triplicated. For 1000 μg/ml, concentration was pure. For 500 μg/ml, volume of stock solution would be 250 μl and volume of methanol to be added would be 250 μl, for 250 μg/ml, volume of stock solution was 125 μl and volume of methanol added was 375 μl. For 125 μg/ml, volume of stock solution was 62.5 μl and volume of methanol added was 437.5 μl. This measurement was obtained by using the formula:

C1V1=C2V2

Seventy microliter (70 uL) of standard (butylated hydroxytoluene) and test compounds at different concentrations were mixed with 176.5 uL of 0.2 M sodium phosphate buffer (pH=7.4) and 176.5 uL of 1% potassium ferricyanide. The mixture was incubated at 50°C for 20 min. After incubation, reaction mixtures were acidified with 176.5 uL of trichloroacetic acid (10%). An aliquot of 136.5 uL of the supernatant was added to 136.5 uL of deionized water. Finally, 27.5 uL of FeCl₃ (0.1%) was added to this solution. The absorbance was measured at 700 nm using spectrofluorimeter.

After analysing in the spectrofluorimeter, data was obtained which include absorbance of sample at 700 nm. Percent of reduction was calculated through the formula:

(C1-A1/C1) × 100

Each equation was done in triplicate. Thereafter, results were averaged. Data was bar graphed with concentration in μg/mL in x-axis while percent reduction in y-axis.

RESULTS AND DISCUSSION

The table provided the data comparing black and red rice and BHT (butylated hydroxytoluene) which is a synthetic actioxidant to be able to have a standard to compare the levels of antioxidant activity in different amounts of the extracts from black and red rice — 125, 250, 500 and 1000 ug/mL. Each amount was utilized in triplicate thereafter obtaining the average of the three samples. The mean for each sample was then compared and evaluated.

The Figures 2 and 3 show the shades of green according to antioxidant activity. It is observed that there was greater antioxidant activity upon higher levels of the extract (1000 ug/mL) in both red and black rice. On row A of both Figures 2 and 3, the blank sample was colored yellow which indicated no antioxidant activity. The BHT sample from the next rows showed a parallel result that the shade of green was solid since it is a synthetic antioxidant. The extracts from the next column from Figure 2 showed a darker green comparing to the next rows below it. In Figure 3, there is a parallel relationship with Figure 2.

CONCLUSION

In this research experiment, black rice was found to have more reduction compared to red rice. This signifies that black rice has more antioxidant activity compared to red rice. Percent reduction of black rice was 66.2% for 100 ug/mL and 56.181% for 100 ug/mL.

OPERATIONAL DEFINITION OF TERMS

Antioxidants: Antioxidants is a molecule that inhibits the oxidation of other molecules. Oxidation is a chemical reaction that can produce free radicals, leading to chain reaction that may damage cells.

Anthocyanins: Anthocyanins are water soluble vacuolar pigments that, depending on their pH, may appear red, purple or blue. They belong to a parent class of molecules called flavonoids synthesized via the phenypropanoid pathway [21].

Flavonoid: Flavonoid is a group of natural substances with variable phenolic structures found in fruits, vegetables, grains, bark, roots, stems flowers, tea and wine [22].

Phenolic acids: Phenolic acids are defined chemically as carboxylic acids derived from either benzoic or cinnamic acid skeletons [23].

Proanthocyanidins: Proanthocyanidins are oligomeric flavonoids. They are dimers or oligomers of catechin and epicatechin and their gallic acid esters (phy).

Tocopherols: Tocopherols are a group of lipid-soluble constituents composed of a polar moiety derived from tyrosine, the chromanol ring and a hydrophobic phytyl-derived side chain [24].

Tocotrienols (vitamin E): It contains unsaturated isoprenoid side chains with three carbon-carbon double bonds. It may affect cholesterol biosynthesis by inhibiting HMG-CoA reductase [25].

Y-oryzanol: Y-oryzanol is a combination of ferulate, esters of fatty acid and triterpene alcohol which acts as an antioxidant against free radicals [26].

Phytic acid: Phytic acid is the hexaphosphoric ester of cyclohexane (inositol hexaphosphoric acid). It is usually found as a complex with essential minerals and proteins [27].

Rotary evaporator: Rotary evaporator is an instrument used to evaporate solutions in to extracts.

Methanol extract: Methanol extract is a solvent that can extract both hydrophilic and lipophilic molecules from plant parts.

Reagent: Reagent is a mixture of chemical compounds to test how a reaction occurs.

Cominution: Cominution is a reduction of solid materials from one average particle size to a smaller average particle size.

Percolation: Liquid slowly passing through a filter.

Extract: A preparation containing active ingredient of a substance in concentrated form.

Ferric reducing antioxidant power (FRAP): FRAP an assay utilized to determine the ferric to ferrous reduction of antioxidants.

Metal chelating activity: An assay performed to stimulate lipid peroxidation by Fenton reaction promoting chelation

Butylated hydroxytoluene: A standard utilized in comparing antioxidant activity or reduction.

Fenton type reaction: A chemical reaction that converts hydrogen peroxide a product of mitochondrial oxidative respiration, into a highly toxic hydroxyl free radical [28].

Chelation: Chelation is a type of bonding of ions and molecules to metal ions. It involves formation or presence of two or more separate coordinate bonds between a polydentate ligand and a single central atom (gol).

Lipid peroxidation: A free radical-mediated chain of reactions that, once initiated, results in an oxidative deterioration of polyunsaturated lipids. 

1.       Moharram HA, Youssef MM (2014) Methods for determining the antioxidant activity: A review. Alex J Fd Sci Technol 11: 31-41.

2.       Goufo P, Trindade H (2014) Rice antioxidants: Phenolic acids, flavonoids, anthocyanins, proanthocyanidins, tocopherols, tocotrienols, y-oryzanol and phytic acid. Food Sci Nutr 2: 75-104.

3.       Goffman FD, Bergman CJ (2004) Rice kernel phenolic content and its relationship with antiradical efficiency. Sci Food Agric.

4.       Heleno SA, Martins A, Queiroz MJRP, Ferreira ICFR (2015) Bioactivity of phenolic acids: Metabolites versus parent compounds: A review. 173: 501-513.

5.       Qureshi AA, Mo H, Packer L, Peterson DM (2000) Isolation and identification of novel tocotrienols from rice bran with hypocholesterolemic, antioxidant and antitumor properties. J Agric Food Chem 48: 3130-3140.

6.       Jeffrey H (2009) Rice: Its history, agriculture, production and research. Available at: http://factsanddetails.com/world/cat54/sub343/item1580.html

7.       Goleniowski M, Cusido RM, Bonfill M, Palazon J (2013) Phenolic acids. In: Natural Products, pp: 1951-1973.

8.       Drevon CA (1991) Absorption, transport and metabolism of vitamin E. Free Radic Res Commun 14: 229-246.

9.       Brigelius-Flohé R, Traber MG (1999) Vitamin E: Function and metabolism. FASEB J 13: 1145-1155.

10.    Aggrwal BB, Sundaram C, Prasad S, Kannappan R (2010) Tocotrienols, the vitamin E of the 21^st century: It's potential against cancer and other chronic diseases. Biochem Pharmacol 80: 1613-1631.

11.    Fu JY, Che HL, Tan DM, Teng KT (2014) Bioavailability of tocotrienols: Evidence in human studies. Nutr Metab (Lond) 11: 5.

12.    Fujiwara S, Sakura S, Sugimoto I, Awata N (1983) Absorption and metabolism of gamma-oryzanol in rats. Chem Pharm Bull (Tokyo) 31: 645-652.

13.    Minatel IO, Francisqueti FV, Correa CR, Lima GPP (2016) Antioxidant activity of y-oryzanol: A complex network of interactions. Int J Mol Sci 17: 1-15.

14.    Viskupicova J, Ondrejovic M, Sturdik E (2008) Bioavailability and metabolism of flavonoids. J Food Nutr Res 47: 151-162.

15.    Treml J, Smejkal K (2016) Flavonoids as potent scavengers of hydroxyl radicals. Compr Rev Food Sci Food Saf 15: 724-738.

16.    Lila MA, Burton-Freeman B, Grace M, Kait W (2016) Unraveling anthocyanin bioavailability for human health. Annu Rev Food Sci Technol 7: 1-19.

17.    Ghosh D, Konishi T (2007) Anthocyanins and anthocyanin-rich extracts: Role in diabetes and eye function. Asia Pac J Clin Nutr 16: 200-208.

18.    Turrini E, Ferruzzi L, Fimognari C (2017) Possible effects of dietary anthocyanins on diabetes and insulin resistance. Curr Drug Targets 18: 629-640.

19.    Graf E, Empson KL (1987) Phytic acid. J Biol Chem 262: 11647-11650.

20.    Aparadh VT, Naik VV, Karadge BA (2012) Anti-oxidative properties (TPC, DPPH, FRAP, metal chelating ability, reducing power and TAC) within some cleome species. Annali Di Botanica 2: 49-56.

21.    Andersen OM (2001) Anthocyanins. Encyclopedia of Life Sciences.

22.    Panche AN, Diwan AD, Chandra SR (2016) Flavonoids: An overview. J Nutr Sci 5: e47.

23.    Ferruzzi N (2016) Phenolic acids. Adv Food Nutr Res.

24.    Velasco L, Ruiz-Mendez MV (2015) Sunflower oil minor constituents. In: Sunflower, pp: 297-329.

25.    Stillwell W (2016) An introduction to biological membranes. 2^nd Edn. Elsevier Science.

26.    Agarwal N, Raghav PK, Sharma A (2016) Gamma oryzanol - A therapeutic agent. Int J Adv Res 4: 78-83.

27.    Ahmed A, Randhawa M, Sajid MW (2014) Bioavailability of calcium, iron, and zinc in whole wheat flour. Wheat and Rice in Disease Prevention and Health, pp: 67-80.

28.    Kivrak EG, Yurt KK, Kaplan AA, Alkan I, Altun G (2017) Effects of electromagnetic fields exposure on the antioxidant defense system. J Microsc Ultrastruct 5: 167-176.

29.    Vinayagam R, Xu B (2015) Anti-diabetic properties of dietary flavonoids: A cellular mechanism review. Nutr Metab 12: 1-20.

Download PDF View PDF