Journal of Nutrition & Food Sciences
Open Access

Antioxidant Properties, Colour Evaluation, Amino acid and Phenolic Profile of Red Sorghum Bran, Roselle calyx and Avocado Leaf Flour Blends

Adedeji Temileye Omotayo^1*, Oluwalana Isaac Babatunde² and Ijarotimi Oluwole Steve^{2 *}Correspondence: Adedeji Temileye Omotayo, Department of Food Science and Technology, Osun State Polytechnic, Nigeria, Tel: +234-8066514332, Email:

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Introduction

Beverages are liquid substances consumed for their thirst quenching properties and calorie value. There is a growing demand for pure natural herbal beverage especially those without the addition of chemicals and sugar. Tea is currently the most widely consumed beverage in the world and therefore ranks as an important world food product [1]. It is generally consumed for its attractive aroma and taste as well as the unique place it holds in the culture of many societies. Traditionally, tea is consumed to improve blood flow, improve resistance to diseases and eliminate toxins [2]. Sorghum (Sorghum bicolor (L.) Moench), the fifth most important cereal grown in the world after maize, rice, wheat and barley which is resistant to semiarid climates, gluten free, and a good source of phytochemical compounds that have been associated with antioxidant, antiinflammatory, and anti-proliferative capacities [3,4]. Pharmacological research has also reported the effects of sorghum and its extracts on cardio vascular disease prevention and glycemic control. Almost all kinds of phenolic are found in sorghum and much more are present in their outer layer (bran) [5]. The leaves of P. americana are reported to possess anti-inflammatory, analgesic, antimicrobial, antiviral, antihypertensive, antihypoglycemic, antiulcer, anticonvulsant, larvicidal antihepatotoxic, vasorelaxant, toxicological activities [5-7]. The genus Hibiscus (family Malvaceae) includes more than 300 species of annual or perennial herbs or shrubs and commonly known as Roselle or red sorrel calyx. Infusions of zobo calyces are traditionally used for their diuretic, choleretic, febrifugal and hypotensive effects, decreasing the viscosity of the blood and stimulating intestinal peristalsis, recommended as a hypotensive. It is also useful in the treatment for cardiac, nerve diseases, cancer and liver toxicity. Besides its importance as a food or traditional medicine in the countries of its geographic origin, it is used worldwide as an important ingredient in industrially produced teas and beverages [8]. The research work therefore deemed it fit to evaluate the antioxidant, phenolic components, amino acids and anti-nutritional constituents of formulated flour blends from underutilized red sorghum bran, zobo calyx and Avocado leaf. The diagrams of red sorghum bran, roselle and avocado leaf are shown in Figures 1-3 [9].

Figure 1: Sorghum stem sheath.

Figure 2: Roselle leaf.

Figure 3: Aocado leaf.

Materials and Methods

Source of materials

Avocado leaves were plucked from Federal College of Agriculture and Research Farm in Akure, Nigeria. All wilting and visibly diseased plant materials were manually removed. Sorghum red bran and Roselle calyx were bought from Oja-Oba market, Akure, Ondo State. The plant materials were authenticated by Crop, Soil and Pest Management Department, Federal University of Technology, Akure.

Methods

All plant materials were carefully inspected and all foreign materials removed. Diseased and damaged leaves were discarded manually just after the collection of fresh leaves. Sorghum red bran and Roselle calyx were picked, sorted and extraneous materials removed. The Avocado leaf was washed under running water to remove extraneous materials and was air dried by spreading on sterile clean green net in a well-ventilated room to prevent loss of nutrients through exposure to high temperature for 4 days. After drying, the leave samples were milled to a coarse form using an attrition mill. The machine was washed before and after milling of each sample. The sieved samples were stored in glass bottles with tight lids and labeled.

Drying of the leaf powders

Sorghum red bran, Avocado leaf and Roselle calyx powders were further dried at 50^°C in a cabinet dryer (model 85, no 64 Shandom, UK) for 30 minutes to reduce moisture content to 10% to enhance increased shelf life. The leaf powders were stored separately in clean air tight containers, protected from light and humidity, and kept in a refrigerator to maintain a temperature below 24^°C (75.2^°F).

Mixing of the blended samples

Each of the leaves sample was weighed into a Kenwood blender for proper mixing in the ratios as shown in Table 1 to obtain seven samples. They were bagged in non-drip tea bags after operating the mixer for three minutes for each blend using a stay material and sealing machine. Each tea bag contained approximately 3 g of the blended samples. The tea bags were stored in glass bottles with tight lids and labeled for analyses.

Analysis

Amino acid profile analysis of red sorghum bran-roselle calyxavocado flour blends: Amino acid composition was determined according to Blackburn, using S433 Amino Acid Analyzer (SYKEM, Germany).

Determination of bioactive compound in red sorghum bran-roselleavocado flour blends: Bioactive compounds examined include: total carotenoid, total flavonoids and anthocyanin [10].

Determination of antioxidant properties of red sorghum bran-roselle calyx-avocado leaf flour blend and drink samples: Antioxidant properties investigated include: the hydroxyl radicalscavenging activity was determined according to a previous method by, ABTS assay, Metal chelating activity, DPPH, total phenol, FRAP.

Determination of antinutritional constituents of red sorghum bran-roselle calyx-avocado flour blends: Red Sorghum bran- Roselle-Avocado leaf flour blends was examined for the following antinutritional components: alkaloids Awah and Verla, phytates content Tannin saponins and oxalate.

Statistical analysis

All determinations were carried out in triplicates. Descriptive statistics, analysis of variance (ANOVA) and Duncan Multiple Range Test were used to interpret the results obtained, and the level of significance was set at p ≤ 0.05.

Results and Discussion

Selection criteria for determining optimal substitution level of avocado leaf and roselle flour and the best outfit samples

Central composite result was used to determine optimal substitution level of Avocado leaf and Roselle calyx according to the modified method of Ijarotimi and Keshinro [11]. In this study, only nutritional criteria (DPPH) were used for the ranking system based on the relative importance of the criteria. The three flour blends of: Red sorghum bran, Avocado leaf and Roselle calyx flour from preliminary study that were very high in DPPH activity were picked to objectively determine the choice of best substitution level along with three controls and a major reference sample. The seven samples were then subjected to further investigation.

Antinutrients content of the enriched red sorghum bran samples

Tannin content of the enriched samples ranged from 1.56 mg/100 g (R50Z40A10) to 1.67 mg/100 g (R50Z45A5) while that of the control (H100) was 0.93 mg/100 g (Table 1). Tannin content of red sorghum bran enriched samples were not significantly different (p ≤ 0.05) from each other but significantly different from that of the control with lower value. Tannin content of the enriched product was higher than 1.03 mg/100 g reported by Fowomola et al. for mango flour, 9.26 mg/100 g reported for raw tigernut flour and (0.25%) obtained for Persea americana (Mill.) leaf flour and (0.97%) obtained for Xylopia aethiopica leaf flour by Gbadamosi et al. [12,13].

Table 1: Anti-nutrient content of Enriched red Sorghum bran samples.

Phytate content of control sample (H100) was 1.56 mg/100 g while phytate content obtained for all the enriched red sorghum bran sample was 1.00 mg/100 g which was higher than (0.52%) obtained for Persea americana (Mill.) leaf flour and (0.09%) obtained for Xylopia aethiopica leaf flour by Gbadamosi et al. [13]. There was significant decrease (p ≤ 0.05) in phytate content of all the enriched samples when compared with the control (1.56 mg/100 g). The result implied that phytate content of enriched product was reduced as a result of processing (such as sieving and drying). Enujuigha (2006) also reported that substitution of Maize with African oil bean seed flour in the production of ogi flour yielded products with lowered phytate content than 100% maize flour. Whether or not high levels of consumption of phytate containing foods will result in mineral deficiency depend on its effect on the bioavailabilty of such minerals which include Ca, Fe, and Zn. The value obtained in this study was higher than 0.21-0.98 mg/100 g reported for ogi flour with different substitution level of African oil bean seed flour, but lower than 15.31-32.9 mg/100 g reported by Ijarotimi et al. for infant flour formula made from germinated popcorn, Bambara groundnut and African locust bean [11]. Phytate markedly decrease Ca, Fe and Zn bioavailability, and Phytate to nutrient molar ratio has been proposed as an indicator of this mineral bioavailability. The critical molar ratio of 0.2, 0.4 and 1.5 has been reported for phytate: Ca, Fe and Zinc, respectively.

Saponin content of control sample (H100) was 1.45 mg/100 g while phytate content obtained for enriched red sorghum bran ranged from 0.99 mg/100 g for (R50Z45A5) to 1.10 mg/100 g for (R50Z40A10). There was significant increase (p ≤ 0.05) in saponin content of all the enriched samples when compared with the control (1.45 mg/100 g). Saponin content of the enriched products (0.99 to 1.10) mg/100 g were lower than the values reported for some important legumes like soybeans flour (5.6%) and chickpea flour (3.6%) by Koratkar and (0.61%) obtained for Persea americana (Mill.) leaf flour and (0.34%) obtained for Xylopia aethiopica leaf flour by Gbadamosi et al. [13].

Bioactive compound of the enriched red sorghum bran samples

Generally all the bioactive components increased with the addition of Avocado leaf and decrease in zobo calyx flour. Total carotenoid content of the enriched samples ranged from 2.84 μg/g for R50Z45A5 to 2.94 μg/g for R50Z40A10 and that of the control (H100) was 3.40 (Table 2). The carotenoid content of the enriched samples (2.84 to 2.94) μg/g were lower than (16.80 to 46) μg/g obtained for Ogi- Moringa seed flour reported by Anwar. The enriched samples are generally low in carotenoid, however can be fortified with carotenoid based fruit flour like mango, guava and pawpaw.

Table 2: Bioactive compounds of Enriched red Sorghum bran samples

Flavonoid contents of the enriched samples are presented in Table 2. Flavonoid content discovered in the control sample (H100) was 0.61% while that of the enriched flour samples ranged from (2.40%- 2.46%). The flavonoid content of the enriched flour (2.40%-2.46%) is higher than (0.0031%-0.0076%) for Ogi-Moringa oleifera seed flour reported by Anwar, (0.03%-0.08%) for Kariya defatted flour obtained by Gbadamosi et al. but lower than (3.443%) for Hibiscus sabdarrifa calyx flour obtained by Chinatu et al. and (776.7%) obtained for Persea americana (Mill.) leaf flour, (431.7%) obtained for Xylopia aethiopica leaf flour by Gbadamosi et al. and (7.36%- 10.24%) obtained for sorrel seed flour by Ayo-Omogie et al. [13-16]. Flavonoid has antioxidants properties. It has been shown to be highly effective scanvengers of most types of oxidizing molecules, including singlet oxygen and various free radicals.

Total anthocyanins content of the enriched samples ranged from 90.88 mg/100 g for R50Z45A5 to 91.28 mg/100 g for R50Z40A10 and that of the control (H100) was 86.32 mg/100 g (Table 2). Total anthocyanins for the enriched samples (90.88 to 91.28) mg/100 g was lower than (493.5-118.2) mg/100 g for roselle-fruit flour blends obtained by Mgaya et al. and (2.8%) obtained for Persea americana (Mill.) leaf flour and (6.2%) obtained for Xylopia aethiopica leaf flour by Gbadamosi et al. [13,17].

Amino acid profile of red Sorghum bran-Roselle calyxavocado leaf flour blends

Table 3 shows the amino acid composition of Sorghum red bran- Roselle Calyx-Avocado leaf flour blend samples. Lysine and leucine generally had the highest amounts in the enriched flour samples as compared to other essential amino acids and compared favourably with the reference, while for the non-essential amino acids, higher values were obtained for arginine, aspartic and glutamic acids in the enriched flour sample. This compared well with previous reports that leucine, lysine, arginine, phenylalanine, valine and glutamic acid are abundant amino acids in sorrel seed flour [18]. The most limiting essential amino acid was Tryptophan. Reverse is the case with Methionine being the most limiting essential amino acid in Sorrel seed flour reported by previous workers [19]. Similar results have been reported in other plant seeds flour also [20]. Also, the lysine content of the enriched flour samples which were higher than the FAO/WHO (1991) human requirement may make this flour useful as a supplement food mixture for poor lysine food sources such as cereals used as weaning foods and a major staple in developing countries. Hence, it may contribute significantly to lysine content of complementary foods when combined with cereals (which are poor in lysine) and thus be useful for combating PEM in young children since values in this flour are higher than FAO/WHO (1991) requirements for infants, pre-school and school age children. The Arg/Lys ratios of the enriched flour (0.73-0.74) g/100 g compared well with (0.73- 0.96) g/100 g reported by Ayo-Omogie et al. but lower than that of soybean (1.40) and higher than that for casein (0.44) [16,19]. Malomo reported that high ratio of Arg/Lys in the diet produces beneficial hypocholesterolemic effects that may improve cardiovascular health and help in regulation of hypertension [21].

Table 3: Amino acid profile of Sorghum red bran-Roselle Calyx -Avocado leaf flour blends (g/100 g).

Total amino acid content of enriched red sorghum bran sample ranged from (73.62 to74.16 g/100 g) was lower than 848.74 to 929.49 g/100 g reported by Oyarekua et al. for Corn-Ogi [22]. TEAA obtained for the enriched samples (28.82-29.10 g/100 g) was lower than 373.2 g/100 g reported by Oyarekua et al. for Corn ogi and 566 g/100 g of egg reference protein. The ArAA (7.83-7.96 g/100 g) falls below the range suggested for infant protein (68-118 g/100 g), however the enriched flour can be fortified with a protein rich source [22]. The ratio of essential amino acids to the total amino acids in the enriched flour was 0.39 which was lower than (0.5) for egg protein but compared well with 0.39 recommended for infant’s food protein, far above 0.26 and 0.11 recommended for children and for adults food protein respectively. Total sulphur amino acid (TSAA) obtained in this study (2.28-2.29 g/100 g) was lower than (58 g/100 g) recommended for infants. Leucine (4.11 to 4.17 g/100 g) and Lysine (6.54 to 6.60 g/100 g) were the most abundant essential amino acids present in the enriched flour samples. Glutamate (10.39- 11.04) g/100 g, Arginine (4.82-4.84) g/100 g, and Aspartate (10.16- 10.20) g/100 g were the most abundant non essential amino acids present in the enriched products.

Antioxidant activity of sorghum red bran-roselle calyx-avocado leaf infused drinks: The DPPH radical scavenging activity of the red sorghum bran-Roselle calyx-Avocado leaf infused drinks is shown in Table 2. The DPPH radical scavenging activity of the enriched samples ranged from 71.57% for R50Z40A10 to 73.44% for R50Z45A5 which was lower than that of the control (H100) was 82.10% (Table 4). The DPPH radical scavenging activity of the enriched samples (71.57%-82.10%) aqueous extract was higher than (16.51%-68.35%) obtained for some local drinks by Oboh et al. (70.18%; 62.55% and 63.90%) reported by Oboh et al. and Al-Hashimi et al. for roselle calyces aqueous extract respectively [23,24]. For all the flour samples and standard (ascorbic acid), the DPPH activity ranged from (32.73%- 90.00%) at different concentrations. The DPPH radical scavenging activity decreased significantly (p<0.5) with increased Avocado leaf and decreased cocentration of Zobo calyx to red sorghum bran flour. This could be attributed to the differential DPPH of the three base materials which are red sorghum bran, Zobo calyx and Avocado leaf flours. The DPPH results showed that red sorghum bran and Zobo calyx contained high phenolic compounds exhibited by high antioxidant activity when determined by DPPH assays. The DPPH radical scavenging activity of the standard (Ascorbic acid) (90.00%) was significantly higher (p<0.05) than that of the control (H100) (82.10).

Table 4: Antioxidant activities of Sorghum red bran-Roselle calyx-Avocado leaf infused drinks.

The Metal Chelating (MC) activity of red sorghum bran-Roselle calyx- Avocado leaf infused drinks and standard (EDTA) are shown in Table 2. The Metal Chelating (MC) activity of the enriched samples ranged from 50.88% for R50Z45A5 to 51.16% for R50Z40A10 which was higher than that of the control (H100) was 44.00% (Table 4). The metal chelating activity of the enriched samples (50.88%-51.16%) aqueous extract was lower than 73.97% reported by Al-Hashimi et al. for roselle calyxes’ aqueous extract [24]. The high metal chelating activity of the enriched samples could enhance ability of tissues to reduce rate of deteriorative metal-catalyzed lipid oxidation. The metal chelating activity of all the samples increased significantly (p<0.5) with increased concentration of Avocado leaf and decreased concentration of Zobo calyx flour. The metal chelating activity of all the extracts revealed their antioxidant potency [25]. The Metal Chelating activity of the standard (EDTA) was significantly higher (87.00%) at (p<0.5) than that of the control (H100) (44.00%).

The Ferric Reducing Antioxidant power (FRAP) of red sorghum bran-Roselle calyx-Avocado leaf infused drinks are shown in Table 2. The FRAP of the enriched samples ranged from 49.11 mgGAE/ml for R50Z45A5 to 51.48 for R50Z40A10 which was lower than that of the control (H100) was 73.06 mg GAE/ml (Table 4). The Ferric Reducing Antioxidant of the enriched samples (49.11 to 51.48) mg GAE/ml aqueous extract was lower than (0.14 to 32.43) mg GAE/ml reported by obtained for some local drinks by Oboh et al. indicated that red sorghum bran is a well-known natural antioxidant and excellent reducing agent [23].

Antioxidant activity of sorghum red bran-roselle calyx-avocado leaf flour blends: The phenolic content of red sorghum bran-Roselle calyx-Avocado leaf infused drinks is shown in Table 2. The phenolic content of the enriched samples ranged from 49.58 mg GAE/g for R50Z45A5 to 52.97 for R50Z40A10 which was lower than that of the control (H100) was 78.42 mg GAE/g (Table 2). The phenolic content of the enriched samples (49.58 to 52.97) mg GAE/g was higher than (7.36 to 10.24) mg GAE/g obtained for sorrel seed flour by Ayo- Omogie et al. [16]. Total phenolic content (TPC) in red sorghum bran (75.60) mg GAE/g and Avocado leaf (84.42) mg GAE/g were high because of their bioactive compositions.

The ABTS radical scavenging activity of the red sorghum bran-Roselle calyx-Avocado flour blends is shown in Table 2. The ABTS radical scavenging activity of the enriched samples ranged from 57.64% for R50Z45A5 to 58.98% for R50Z40A10 which was lower than that of the control (H100) was 60.11 (Table 2). The ABTS radical scavenging activity of the enriched samples (57.64%-58.98%) aqueous extract was higher than (22.3%-37.9%) obtained for four guava genotypes flour by Kriengsak et al. The ABTS radical scavenging activity of the enriched bran decreased insignificantly (p<0.5) with increased Avocado leaf and decreased cocentration of Zobo calyx to red sorghum bran flour.

The OH radical scavenging activity of the red sorghum bran-Roselle calyx-Avocado leaf flour blends is shown in Table 2. The OH radical scavenging activity of the enriched samples ranged from 59.75% for R50Z40A10 to 60.44% for R50Z45A5 which was higher than that of the control (H100) was 54.65% (Table 2). The OH radical scavenging activity of the enriched samples (59.75%-60.44%) was higher than (18.32%-27.28%) obtained for three varieties of sorghum bran flour by Yingying et al. [26]. The OH radical scavenging activity decreased insignificantly (p>0.5) with increased Avocado leaf and decreased concentration of Zobo calyx to red sorghum bran flour (Table 5).

Table 5: Antioxidant activities of Sorghum red bran-Roselle Calyx-Avocado leaf flour blends.

Phenolic profile of Sorghum red bran-Roselle Calyx-Avocado leaf flour blends

The increase in p-coumaric acid ranged from 7.2 mg/100 g for R50Z45A5 to 7.4 mg/100 g for R50Z0A10 compared with the control (H100) 6.39 mg/100 g (Table 3). The p-coumaric acid value (7.2 mg/100 g to 7.4 mg/100 g) of the enriched flour was higher than (0.34 to 0.64) mg/100 g obtained for three varieties of sorghum bran by Yingying (Table 6) [26]. The increase could be a result of the release of bound phenolic compounds under the acidic condition because a significant proportion of phenolic compounds are present in bound form in cereals [27,28].

Table 6: Phenolic profile of Sorghum red bran-Roselle Calyx-Avocado leaf flour blends (mg/100 g).

The decrease in caffeic acid ranged from to 309.96 mg/100 g for R50Z45A5 to 302.38 mg/100 g for R50Z0A10 compared with the control (H100) 620 mg/100 g (Table 3). The caffeic acid value (302.38 mg/100 g to 309.96 mg/100 g) of the enriched flour was however higher than (148 to 172) mg/100 g obtained for red sorghum bran extract by Jeremiah et al. [29,30].

Conclusion

The present study established that the enriched flour blends produced from Red sorghum bran, Roselle calyx and Avocado leaf were very rich in essential nutrients like amino acids, bioactive compounds, phenolic components and antioxidant properties but low in antinutritional factors.

It can be concluded that nutritious health promoting infused beverages can be produced from the enriched flour particularly R50Z40A10, which may be suitable as a functional food ingredient and play protective roles against cardiovascular, neuro-degenerative and age-related diseases compared to commercial sugar laden-fizzy carbonated beverages which are detrimental to human health.

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