Studies on the bioactive natural antioxidants from oilseeds.

Abstract

Oxidation and consequent generation of reactive oxygen species (ROS) is the major cause for deterioration of food lipids. ROS have also been implicated in a variety of degenerative diseases. Epidemiological and experimental evidences suggest close links between ROS and diseases such as cancer, CVD, diabetes, cataract etc. In the human body, ROS is balanced by an array of endogenous and exogenous antioxidants. A disturbance in the pro-oxidant-antioxidant balance in favour of the former, is termed as oxidative stress and can cause potential damage including lipid peroxidation and consequent cytotoxicity.

Antioxidants are substances that when present at low concentrations compared to that of an oxidisable substrate significantly delays or inhibits oxidation of that substrate in food products or in living systems. Antioxidants are either endogenous to the body or derived from the diet. Several types of synthetic antioxidants like BHT, BHA, TBHQ etc. are also used in the food industry. However, findings and subsequent publicity has fostered significant consumer resistance to the use of synthetic food additives as antioxidants, colourants etc. and therefore food industry is in search of potential natural antioxidants from edible sources.

The major dietary sources of antioxidant phytochemicals are cereals, legumes, fruits, vegetables, oilseeds, beverages, spices and herbs. In the present study, we have focused on rice bran and its byproducts. Rice is one of the oldest of food crops and has been a staple food in India from very ancient times. It is also the staple food for about 60% of the world’s population. Rice bran is a byproduct of the rice milling industry and is a potential commercial source of a healthy edible oil viz. rice bran oil and a variety of bio-active phytochemicals. Defatted rice bran (DRB), a byproduct of rice bran oil extraction, is also a good source of insoluble dietary fiber, protein, phytic acid, inositol, vitamin B and a variety of other phytochemicals. Though the antioxidant potential of DRB has been demonstrated, it still remained a relatively unexplored source material, which demanded further investigation especially with regard to its detailed phytochemical profile leading to practical application. The focus of the present investigation therefore has been on DRB primarily to establish its phytochemical status and feasibility of using it as a source of bio-active phytochemicals and natural antioxidants leading to value addition of DRB otherwise used as cattle feed. To gain a better understanding of the value of rice bran as a source of phytochemicals, five popular rice varieties of the region viz. PTB 50, PTB 39, PTB 38, JAYA, and MO 10 and a wild variety (oryza nivara) that is mainly used for medicinal applications in traditional ayurvedic system were characterized along with commercial samples of rice bran. The present study also explains the feasibility of a process for the extraction, enrichment, and isolation of antioxidant compounds from DRB. The antioxidant potential of the extracts were evaluated both in bulk oils and in food relevant model emulsions, using standard in vitro models. Radical scavenging effects, indicative of possible biological effects, were also evaluated. The thesis consists of four chapters. Chapter 1 reviews the literature on oxidation and formation of reactive oxygen species (ROS), oxidative stress and its major consequences, and the role of antioxidants in preventing lipid peroxidation. It also gives comprehensive data on the natural distribution of dietary phytochemicals with special emphasis on oleaginous plant products. This chapter also updates the literature on major rice phytochemicals, their antioxidant and biological effects and highlights the relevance of the present investigation.

Chapter 2 describes in detail about the materials and methods employed for the present investigation. Six rice varieties namely PTB 50, PTB 39, PTB 38, JAYA, MO 10 and oryza nivara obtained from Rice Research Station, Kayamkulam, (Kerala) as well as commercial samples of rice bran and rice bran oil were used for the study. The rice bran and rice bran oil samples were defined in terms of their chemical composition and chemical characteristics respectively. The phytochemical profile of full fat rice bran (FFB), defatted rice bran (DRB), and rice bran oil (RBO) in terms of oryzanols, ferulic acid, and tocols were determined with modified HPLC protocols. The feasibility of a process for the extraction, enrichment, and isolation of antioxidant compounds from DRB is also explained. For enrichment of TPC, oryzanol and ferulic acid content of the crude extracts, sequential extraction technique was employed. The isolation of components present in the extract was carried out by column chromatography. The isolated compounds were identified using IR, UV, NMR and MS data. The antioxidant potentials of the crude extract, enriched fractions, and the isolated compounds were then evaluated using standard in-vitro models. For assessing the antioxidant efficacies of the DRB extracts in bulk oils, Schall Oven Test method (60oC) and differential scanning calorimetry (150oC) were used. To evaluate the activity of the extracts in food relevant emulsions, linoleic acid emulsion method and the β-carotene bleaching test were used. Radical scavenging effects (indicative of possible biological effects) of DRB extracts were studied using the stable DPPH radical and the superoxide radicals generated in-situ by the xanthine-xanthine oxidase system. Synthetic antioxidants viz. BHT and TBHQ were used as reference compounds.

Chapter 3 consists of results and discussions. Rice bran, from major cultivars of the region were analysed for their chemical profile. Significant varietal variations were observed in the levels of different nutrients in rice bran. The mean values for major constituents were dry matter (89.1%), fat (16.8%), protein (10.1%), crude fiber (11.3%), ash (11.4%), and available carbohydrates (50.5%). The mean energy content was 393.5 Kcal/100g. The mean values (ppm) of various minerals followed the order P (13608) > K (9520) > Mg (3844) > Ca (362) > Fe (216) ~ Na (190) > Mn (99) > Zn (39) > Cu (4), with P and Cu being the most and least abundant minerals respectively.

Rice bran oil obtained from the major cultivars were analysed for their chemical characteristics as well as fatty acid composition. The values obtained for FFA, saponification value, iodine value, and unsaponifiable matter were 12.3%, 182.9, 97.3 and 4.8% respectively. RBO had exceptionally high unsaponifiables as compared to that of other edible oils. Major fatty acids of RBO were 16:0, 18:0, 18:1, and 18:2 with mean values of 21.6%, 2.0%, 41.8%, and 32.5% respectively, with saturated to unsaturated ratio of approximately 1:3. The fatty acid profile of RBO is thus close to the ideal ratio of saturated: mono unsaturated: poly unsaturated of 1:1.5:1.

The oryzanol, tocopherol and tocotrienol composition of RBO from the major cultivars were analysed by standardized HPLC protocols. On an average, RBO contained about 2% oryzanols. The various oryzanol components identified in RBO include stigmasteryl ferulate, cycloartanyl ferulate, cycloartenyl ferulate, 24-methylene cycloartanyl ferulate, campesteryl ferulate and β-sitosteryl ferulate with the latter four compounds accounting for more than 95% of the total oryzanols in RBO. RBO was rich in chromanols (tocols) with concentrations ranging from 1042 to 1648 ppm. Seven tocols except β-T3 were separated, identified, and quantitated in all the rice varieties. The major tocopherol isomer in all varieties was α-T, whereas γ-T3 was the major tocotrienol. However, γ-T3 was the predominant vitamin E homolog accounting for about 42-70% of the total vitamin E compounds in the selected varieties.

The defatted meal which remained after the extraction of oil from rice bran was also analysed for oryzanols and tocols. On HPLC analysis, extracts of DRB prepared with various solvents was found to contain significant amounts of oryzanols, tocols as well as ferulic acid. Kinetic studies were designed to select appropriate solvent and to optimise other process parameters like material-solvent ratio, time, temperature etc. for extraction of antioxidants from defatted rice bran. Methanol was found to be the most efficient solvent, with respect to the yield of TPC, oryzanols and ferulic acid from DRB. Other optimized conditions included a material-solvent ratio of 1:15 and a time of extraction of 10 hours using a Soxhlet extractor. The yield of methanol extracts of DRB from the major cultivars ranged from 3.2 to 5.0%. The sugar content of the extracts ranged from 18.8 to 33.8%, protein from 17.9 to 25.0%, TPC from 5.3 to 8.4% and ash from 3.9 to 5.1%. The oryzanol content of the extracts ranged from 2358 to 6602 ppm, ferulic acid from 2541 to 4376 ppm and tocols from 110 to 284 ppm. 24-methylene cycloartanyl ferulate (~45%), and cycloartenyl ferulate (~25%) represented the major oryzanols of the DRB extracts. γ-tocotrienol (~70%), and α-tocopherol (~10%) were the major chromanols.

Enrichment of antioxidants in crude methanol extract was achieved by sequential extraction and fractionation. For this, the CME was re-extracted with less polar organic solvents like ethyl acetate, acetone, ether etc. From this, acetone was found to be the best solvent for ferulic acid and tocols. For further purification of the acetone extract (AE), sequential extraction technique was employed. For this, the dry AE was re-extracted with hexane to give a soluble fraction enriched in lipophilic compounds (AE-LP) and a residue enriched in polar compounds (AE-PP). Considering the bioactive phytochemicals of interest, AE-LP was enriched in oryzanols, and tocols and AE-PP in ferulic acid. Column chromatography was employed to isolate components present in the crude extract. The two pure compounds obtained were identified to be β-sitosterol and tricin based on UV, IR, NMR and MS data. Of these, the flavone tricin is of special phytochemical interest because of its rare occurrence.

The crude extract (CME), the enriched fractions (AE, AE-LP, and AE-PP) and the pure phytochemicals (oryzanols, tocols, ferulic acid, tricin and sterol) were then subjected to a number of antioxidant and antiradical activity assays using standard in-vitro models. To evaluate the antioxidant potential of the extracts and its phytochemical constituents in bulk oils, Schall Oven Test method and differential scanning calorimetry (DSC) were used. The results demonstrated that in bulk oils, some of the DRB extracts (AE-PP) were either equally efficient or better than BHT and that at identical concentrations AE-PP, AE-LP, and AE performed better than the phytochemical constituents ozyzanols, ferulic acid and tocols with respect to PV, DV and DSC data. The increase in activity with fractionation might be due to the enhanced levels of antioxidants in the resultant fractions compared to CME. To evaluate the antioxidant potential in food relevant systems, linoleic acid emulsion method and the β-carotene bleaching test were used. The DRB extracts and its phytochemical constituents proved to have significant activity in these emulsion models as well. Contrary to the bulk oil system (SOT & DSC), where the DRB extracts (AE-PP) were either equally efficient or better than BHT, the latter was more effective in emulsions. None of the pure phytochemicals tested performed better than BHT or TBHQ, both in bulk oils and emulsions indicating that the synergistic effects of phytochemicals in the extracts including that of proteins, sugars and unidentified polyphenols could be contributing to the observed efficacy of DRB extracts. The results further suggest that the DRB extracts could be used both in bulk oils and in food emulsions as natural antioxidants.

The antiradical efficacies of DRB extracts and their phytochemical constituents were studied using the stable DPPH radical and the superoxide radicals generated in-situ by the xanthine-xanthine oxidase system. The DPPH radical scavenging activity of ferulic acid, and Tmix was greater than that of BHT and the activity of AE-PP was equal to that of BHT. It was also found that the DPPH scavenging activity of the fractions AE, AE-LP and AE-PP could be largely attributed to the levels of TPC and ferulic acid in the fractions. H-donating capacity (as evaluated by the DPPH method here) is an important biologically significant property of antioxidants to convert potentially damaging ROS (oxyl and peroxyl radicals) into non-toxic species, and in this context DRB could be a good source of such antioxidants. For evaluating the superoxide scavenging activity of the extracts, the cytochrome C and NBT methods were used. The superoxide scavenging activities of the extracts also followed the order of their TPC and ferulic acid contents. Moreover, the various phytochemical constituents of DRB extracts viz. ferulic acid, tricin and Tmix also exhibited excellent superoxide radical scavenging activity thus directly supporting the superior antiradical efficacies of DRB extracts.

The conclusions arrived from the present investigation is summarized in chapter 4. The study establishes the feasibility of utilization of rice bran, an abundantly available renewable resource, as a commercial source for natural antioxidants. DRB, as the byproduct of rice bran oil extraction, could contain substantial amounts of antioxidant phytochemicals like oryzanols, tocols and ferulic acid that could be harnessed as a source for natural antioxidants. It is also shown that the DRB retained the entire ferulic acid in rice bran as ferulic acid is not amenable to hexane extract (RBO) as practiced in the industry. Detailed information regarding the antioxidant activity of DRB extracts and their phytochemical constituents were provided. The extracts could be used both in bulk oils and in food emulsions as natural antioxidants. They possessed substantial hydrogen donating capacity too and were found to be effective against superoxide radicals. More over, the residue after antioxidant extraction could still be used as cattle feed.