Abstract: Background and Objective: The applications of polyphenols and tocopherols have tempted the interest in the functional foods, nutraceutical and pharmaceutical industries, due to their potential health benefits to humans. The present study was performed to evaluate the yield, phenolic compounds, α-tocopherol contents and antioxidant activity of nano phytochemicals extracted from ground wheat bran by supercritical carbon dioxide (SC-CO2) compared to solvent extraction with methanol. Materials and Methods: Bread wheat variety (Triticum aestivum L.), named Sids 12 and durum wheat variety (Triticum durum L.), named Beni Suef 6 were used. Supercritical carbon dioxide as well as conventional solvent extraction methods was used. Results: Data showed that using SC-CO2 extraction method had the ability to extract the highest yield of nano phenolic compounds and nano α-tocopherol from wheat bran extract compared to methanol extraction. Antioxidant activity of nano phytochemicals extracted by SC-CO2 and methanol extraction methods were highly significantly different (p<0.05) compared to wheat bran extract being 414.39 and 111.58% for SC-CO2 extraction, 255.77 and 66.03% for methanol extraction compared to 42.25 and 33.07% for normal sample extract for Sids 12 and Beni Suef 6, respectively. The SC-CO2 extraction showed a good distribution and produced smaller nanoparticles size compared to those by solvent extraction. The transmission electron microscopy (TEM) results after encapsulation with alginate beads revealed a spherical shape with a narrow size distribution for nanoparticles. Conclusion: It could be concluded that SC-CO2 extraction produced high value nano bioactive compounds with high radical scavenging ability compared to solvent extraction.
INTRODUCTION
The applications of polyphenols and tocopherols have recently attracted great interest in the functional foods, nutraceutical and pharmaceutical industries, due to their potential health benefits to humans. However, the factors affecting polyphenols efficiency include stability, bioactivity and bioavailability of the bioactive compounds. The limited application of most phenolic compounds for its undesirable taste. The use of organic solvent for antioxidant extraction from different plant sources was not environmental friendly. Beside, the production of plant extracts was currently limited by safety and regulatory constraints to the concentration of toxic residues of conventional organic solvents. Supercritical fluid extraction (SFE) has been widely employed as alternative of organic solvent extraction1. Brunner2 and Ge et al.3 found that the application of SFE has grown continuously over classical extraction processes with organic solvents. Carbon dioxide (CO2) was probably the most widely used supercritical fluid because it was nontoxic, nonflammable, noncorrosive, inert to most materials, cheap and readily available in bulk quantity with high purity. Due to low critical temperature, CO2 was applied in SFE processes at near-environmental temperatures thus minimizing heat requirement and thermal damage to bioactive compounds. Supercritical carbon dioxide (SC-CO2) could achieve extraction yields for tocopherols and phenolic acids similar to traditional extraction. Gelmez et al.4 stated that SC-CO2 extraction was effective method to get extracts containing different antioxidant compounds using different extraction conditions i.e. pressure, temperature and time. Supercritical carbon dioxide (SC-CO2) was used as a solvent to extract polyphenols and tocopherols from wheat bran at 60°C and 30 MPa pressure with a carbon dioxide (CO2) flow rate5 of 26.81 g min–1.
Microencapsulation (developed approximately 60 years ago) was defined as a technology of packaging solids, liquids or gaseous materials in miniature sealed capsules that could release their contents at controlled rates under specific conditions. In the food industry, the microencapsulation process could be applied to protect the core material from degradation (by reducing its reactivity to its outside environment), modify their physical characteristics (to allow easier handling). In addition, such process could mask an unwanted flavor or taste of the core material or dilute at small amounts and facilitate the separation of mixture components that would otherwise react with one another. Different encapsulation techniques were used with many food components i.e., flavoring agents, acidulates, sweeteners, colorants, lipids, vitamins, minerals, enzymes and microorganisms6.
Nanoencapsulation involved the formation of active-loaded particles with diameters ranging from 1-100 nm7. For encapsulation of bioactive compounds, a wall around the material to be encapsulated should be formed and ensuring no leakage. The term nanoparticle was a collective name for both nanospheres and nanocapsules. Various techniques such as spray drying, coacervation, liposome entrapment, inclusion complexation, cocrystallization, nanoencapsulation, freeze drying, yeast encapsulation and emulsion were used for encapsulation of polyphenols and tocopherols8.
Therefore, the present study aimed to study the morphological structure and characteristics of nano polyphenols and nanotocopherol extracted from wheat bran.
MATERIALS AND METHODS
Wheat bran preparation: Bread wheat variety (Triticum aestivum L.), named Sids 12 and durum wheat variety (Triticum durum L.), named Beni Suef 6 obtained from the Wheat Research Department, Field Crops Research Institute, Agricultural Research Center, Giza, Egypt were used. The wheat grains were cleaned and conditioned to 14 and 16% moisture content for bread wheat and durum wheat, respectively at ambient temperature for 18-24 h. The grains were milled by Quadrumat Junior flour mill (Brabender OHG, Duisburg, Germany) in Agricultural Research Center, Giza, Egypt. Each meal was fractionated into flour (72% extraction rate), bran and short. This research was conducted from December, 2017 to September, 2018.
Bran samples were dried (at low temperature, 60°C for 24 h), ground for 2 min using mechanical blender, then sieved on wire sieve (0.5 mm) and stored at 2°C. Supercritical carbon dioxide as well as conventional solvent extraction methods was used according to Kwon et al.5.
Extraction methods
Wheat bran extraction by water: Wheat bran (10 g) was transferred into test tube and 40 mL of water were added and vortexed for 30 sec. The supernatant was transferred to a previously weighed clean test tube. The tube containing supernatant was then placed in an oven under vacuum to remove water. The dried extract in the test tube (yield) was weighed then stored at -20°C before further testing.
Extraction by SC-CO2: The set up of a laboratory scale of SFE process (Spe-ed SFE, Model 7030, applied separation, Hamilton, PA, USA) located at National Research Center, Giza, Egypt was used. The extraction was performed according to the method of Kwon et al.5. Twenty grams of wheat bran samples were loaded into the stainless steel extraction vessel (50 mL in volume). Carbon dioxide (CO2) was pumped at constant pressure (digital pressure controller) into the extraction vessel by high pressure pump up to the desired pressure (30 MPa) for 2 h and at 60°C. The flow rate of CO2 was constant at 26.81 g min–1 for all extraction conditions and CO2 volume passing through the apparatus were measured using a gas flow meter. The extracted substances by SC-CO2 were collected by a glass separating vessel. The amount of extract obtained at regular intervals of time was established by weight. The extraction yield (%) was calculated as (g of extract/g of wheat bran)×100. The extract was then stored at -4°C until further analysis. The phenolic compounds and α-tocopherol in the extract were determined.
Extraction by conventional solvent: The conventional solvent extraction was carried out as described by Oufnac et al.9. Wheat bran (10 g) was transferred into test tube and 40 mL of methanol were added and vortexed for 30 sec. The test tube was capped and placed in a water bath at 60°C for 20 min and vortexed twice during the incubation. The solvent layer in tube was then separated by centrifuging at 2.000 rpm for 15 min. The solvent supernatant was transferred to a previously weighed clean test tube. The residue was again mixed with 20 mL of solvent and the supernatant was separated as previously described and combined with the previous one. The tube containing supernatant was then placed in a vacuum to remove solvent. The dried extract in the test tube (yield) was weighed then stored at -20°C before further testing. The extraction yield (%) was calculated as (g of extract/g of wheat bran)×100.
Determination of phenolic compounds: Phenolic compounds of methanol and SC-CO2 wheat bran extracts were determined colorimetry by using Folin-Ciocalteu reagent according to the method of Singleton and Rossi10 as modified by Gao et al.11 and the International Organization for Standardization12 (ISO) 14502-1. The phenolic compounds were expressed as mg g–1 wheat bran extract as gallic acid equivalents (GAE).
Determination of α-tocopherol (Vitamin E): Vitamin E as α-tocopherols was determined by HPLC (Hewlett Packard, series 1050) according to the method of Pyka and Sliwiok13.
Free radical scavenging activity by using DPPH method: The antioxidant activity (AOA) of methanol and SC-CO2 wheat bran extracts was determined based on the radical scavenging ability in reacting with a stable DPPH free radical according to Brand-Williams et al.14 and Oufnac et al.9. Antioxidant activity was calculated as (%) of discoloration (radical scavenging percentage) using the following equation15:
Immobilization of nano polyphenols and nano tocopherols on alginate beads as carrier: Alginate beads were used to immobilize the antioxidant compounds of ground wheat bran extracts through the entrapment technique as described by Erhardt and Jördening16.
Encapsulation of nano polyphenols and nano tocopherols in hard gelatin capsules: Nano phenolic compounds and nano tocopherols extracted from ground wheat bran (by using either SC-CO2 or solvent extraction methods) and entrapped on alginate beads were freeze dried then encapsulated in hard gelatin capsules (obtained from GlaxoSmithKline Company, gsk) under sterilization and kept in dark conditions.
Transmission electron microscopy (TEM): The morphology of the wheat bran extract by SC-CO2 and conventional solvent (enriched in phenolics and tocopherols) and encapsulated nanoparticles were examined by Transmission Electron Microscopy (TEM) using freeze-fraction replica method17.
The sample was fractured in vacuum condition by knocking the upper specimen carrier in a freeze-fracture apparatus (JED 1230, JEOL Ltd., Tokyo, Japan).
Statistical analysis: Statistical analysis was carried out according to Fisher18. LSD (Least significant difference) test was used to compare the significant differences (p<0.05) between means of treatment19.
RESULTS AND DISCUSSION
Physical and chemical properties of nano phenolic compounds and nano tocopherols extracted from wheat bran: The yield, phenolic compounds (PC), α-tocopherol contents and antioxidant activity (AOA) of the prepared nano phytochemicals from bran of the two wheat varieties by using different extraction methods are presented in Table 1.
Results in Table 1 showed that the antioxidant activity (AOA) of nano phytochemicals extracted by SC-CO2 and solvent extraction methods were highly significantly different (p<0.05) compared to wheat bran extract.
| Table 1: | Yield, phenolic compounds (PC), α-tocopherol and antioxidant activity (AOA) of the nano phytochemicals extracted from wheat bran by using different extraction methods |
Values are Means±SD of three measurements, *PC were expressed as mg g–1 wheat bran extract as GAE, means followed by different superscripts within columns are significantly different at 0.05 level | |
They had 414.39 and 111.58% for SC-CO2 extraction, 255.77 and 66.03% for solvent extraction compared to 42.25 and 33.07% for normal sample extract for Sids 12 and Beni Suef 6, respectively. It could be noticed that phyto-nanoparticles exhibited higher antioxidant activity compared to normal particles of the bran extract. This could be attributed to the large surface area of nanoparticles that resulted in greater chemical activity.
Results also showed that using SC-CO2 extraction method had the ability to extract the highest yield of nano PC and nano α-tocopherol from ground wheat bran compared to solvent extraction and wheat bran extract.
Moreover, the yield of nano PC and AOA exhibited the highest values for Sids 12 bran than for Beni Suef 6 bran. This indicated that bran of bread wheat contained the highest amounts of bioactive compounds compared to those in durum wheat.
It could be concluded that SC-CO2 extraction produced high value bioactive compounds with high antioxidant properties and efficiency compared with solvent extraction. The present findings are in agreement with Oufnac et al.9, who found that wheat bran solvent extract (using methanol vs acetone and hexane) produced higher yield (4.86%), PC (241.3 μg of catechin equivalent/g of wheat bran), total tocopherol contents (13.6-15.8 μg g–1 of wheat bran) and free radical scavenging capability (0.042 μmol of trolox equivalent/g of wheat bran). Gelmez et al.4 found that the yield (%), PC, total tocopherol contents (TTC) and antioxidant activity of the SC-CO2 extract for wheat bran were 5.3%, 6, 6.7 mg g–1 and 57.3 mg scavenged DPPH/g, respectively. Also, Kwon et al.5, reported that TPC of wheat bran extract by using SC-CO2 extraction was 3.57 mg g–1.
Morphological structure of the nano polyphenols and tocopherols of wheat bran extract: The morphological structure (particle size) of nano phenolic compounds and tocopherols of ground wheat bran extracts were studied by using transmission electron microscopy (TEM) as shown in Fig. 1, 2.
| Fig. 1(a-b): | Transmission electron microscopy (TEM) of Beni Suef 6, (a) Extracted by methanol and (b) extracted by SC-CO2 |
Results in Fig. 1a, b showed that the prepared nano particles size extracted from ground wheat bran by solvent extraction of Beni Suef 6 were 107, 115, 117 and 223 nm compared to 40, 43, 47 and 48 nm for SC-CO2 extraction method.
| Fig. 2(a-b): | Transmission electron microscopy (TEM) of Sids 12, (a) Extracted by methanol and (b) Extracted by SC-CO2 |
| Fig. 3(a-b): | Transmission electron microscopy (TEM) of Beni Suef 6 (a) Extracted by methanol and (b) Extracted by SC-CO2 after encapsulation |
This indicates that SC-CO2 extraction produced smaller nanoparticles size compared to those by solvent extraction.
Results in Fig. 2a, b also showed that the prepared nano particles size extracted from ground wheat bran by solvent extraction of Sids 12 were 89, 112, 128 and 147 nm compared to 16, 22, 30 and 37 nm for SC-CO2 extraction method. These results indicated that SC-CO2 showed a good distribution and small nanoparticles size as compared to the solvent extraction. In general as the particles size decreased, the specific surface area increased and could enhance drug penetration and systemic activity of drug20.
Encapsulation of nano phenolic compounds and nano tocopherols: The morphological structure of encapsulated nanoparticles with entrapped phenolic compounds and tocopherol was studied by transmission electron microscopy (TEM) as shown in Fig. 3, 4. The TEM results of the encapsulated nano phenolic compounds and tocopherols through alginate beads revealed a spherical shape with a narrow size distribution for nanoparticles.
With the entrapment of phenols and tocopherols in the alginate beads matrix, the encapsulated nanoparticles size of wheat bran solvent extract of Beni Suef 6 bran decreased from 284 and 358 nm to 75 and 135 nm for nanoparticles with SC-CO2 extraction (Fig. 3a, b).
Results also show that the encapsulated nanoparticles size of wheat bran (solvent extract) of Sids 12 bran decreased from 780, 825 nm and 1.32 μm to 186, 194 and 312 nm compared to SC-CO2 extraction (Fig. 4a, b).
It could be concluded that the size of the encapsulated nanoparticles were reduced by using SC-CO2 extraction compared with solvent one.
| Fig. 4(a-b): | Transmission electron microscopy (TEM) of Sids 12 (a) Extracted by methanol and (b) Extracted by SC-CO2 after encapsulation |
CONCLUSION
From the results, it could be concluded that SC-CO2 extraction was an alternative technique to produce high value bioactive compounds with high antioxidant properties and efficiency and a good distribution and produced smaller nanoparticles size compared with solvent extraction.
SIGNIFICANCE STATEMENT
This study discovers the nano bioactive compounds containing high antioxidant characteristics extracted from wheat bran that can be beneficial for functional foods formulation. This study will help the researcher to uncover the critical area of milling added value by-products that many researchers were not able to explore. This is a new theory on nano polyphenols and nano tocopherols extracted from wheat bran may be arrived at.