Nutritional compositions and antioxidative capacity of the silk obtained from immature and mature corn

2.1 Sample

Baby corn and sweet corn were purchased from the local wet market in Kota Bharu district, Kelantan state of Peninsular Malaysia. The husks were eliminated and silks were detached from its fruit. Fresh corn silks were chopped and dried in an oven (Memmert, Germany) at 55 °C for 2 days. Dried corn silks were kept in a sealed plastic bag at 4 °C. Prior to analysis, the corn silk was ground into powder form (Waring Commercial, USA).

2.2 Reagents and standards

Folin–Ciocalteu reagent, gallic acid, (+)-catechin, and butylated hydroxytoluene (BHT) were purchased from Sigma–Aldrich (GmbH, Sternheim, Germany). Kalium-hexacyanoferrat (K₃Fe(CN)₆, 2,2′-azinobis (3-ethylbenzthiazoline)-6-sulfonic acid (ABTS⁺), Trolox, sulfuric acid, iron (III) chloride anhydrous (FeCl₃), nitric acid (65%) and other chemicals were purchased from Merck (Darmstadt, Germany).

2.3 Determination of nutritional composition

Nutritional composition (moisture, protein, lipid and ash) was determined according to (AOAC, 1996). Moisture content of fresh and oven dried silks was determined using an oven-dry technique and expressed as fresh and dry basis, respectively. Protein content was determined by using the Kjeldahl method (Gerhardt, Germany) and nitrogen content of samples was multiplied by a factor of 6.25. Lipid content was determined using a Soxhlet apparatus with petroleum ether as the extracting solvent. Total dietary fiber composition of silk was determined by an enzymatic–gravimetric method, using sequential enzymatic digestions by thermostable α-amylase, protease and amyloglucosidase (Sigma–Aldrich (GmbH, Sternheim, Germany)).

2.4 Mineral composition

Mineral content (Ca, Mg, K, Na, Cu, Fe, Mn and Zn) of corn silk was determined using an atomic absorption spectrophotometer (AAS) (Perkin Elmer Analyst, USA). Samples (0.5 g) were ashed and digested in 2 ml concentrated nitric acid. The acid-digests were filtered through ashless filter paper (Whatman 42) and raised to 10 ml with deionized water.

2.5 Extraction

Plant material (20 g) was extracted with three separate solvents (ethyl acetate, ethanol and water) by the using Soxhlet extraction method. Crude extracts obtained were vacuumed evaporated (Heidolph, Germany) to dryness at 50 °C and kept in a screwed cap bottle at −18 °C. The yield of the extract obtained was reported as percentage and was calculated as follows:

2.6 Determination of total polyphenol content

The total phenolic content of extracts was determined using the Folin–Ciocalteau method (Kaur et al., 2008). One milliliter of crude extract (1 mg/ml) was added with 1 ml of Folin–Ciocalteau reagent (1:1) and 1.5 ml of sodium bicarbonate (20% w/v). The mixture was raised to 10 ml with distilled water. After 2 h of reaction at room ambient the absorbance was recorded at 765 nm and used to calculate the phenolic content by comparing with gallic acid standard. The total phenolic content was then expressed as mg gallic acid equivalent (mg GAE/100 g extract).

2.7 Determination of total flavonoid content

Total flavonoid content was determined by a colorimetric assay described by Ozsoy et al. (2008). An aliquot of 0.25 ml of the extract (1000 μg/ml) was added with 75 μl of NaNO₂ (5% w/v) and the mixture was left to stand at room temperature for 6 min. Thereafter, 150 μl of AlCl₃ (10% w/v) was added and allowed to react for another 5 min before adding 500 μl of NaOH (1 M). The volume was then adjusted to 2.5 ml with distilled water and mixed thoroughly. The absorbance was measured at 510 nm against blank which contained the same mixture without sample. Total flavonoid content was expressed as mg CAE/g extract, through the (+)-catechin calibration curve.

2.8 ABTS⁺ free radical scavenging assay

The assay was measured based on the ability of antioxidant from the sample to inhibit the 2,2′-azinobis (3-ethylbenzthiazoline)-6-sulfonic acid or ABTS free radical (ABTS⁺) by comparing with a reference standard (Trolox) (Re et al., 1999). In this assay, ABTS⁺ radical was generated by reacting ABTS solution (7 mM) with 2.45 mM potassium persulfate (K₂S₂O₈) which allowed standing for 15 h in the dark at room temperature. The solution was diluted appropriately with ethanol to obtain the absorbance of 0.7 ± 0.2 units at 734 nm. To perform the ABTS⁺ decolorization assay, 200 μl of plant extract (100 μg/ml) was added with 2000 μl of ABTS free radical solution and reacted for 5 min. After that, the absorbance was measured spectrophotometrically at 734 nm against solvent blanks. The Trolox calibration curve ranging from 0 to 200 μM was prepared according to the sample. The percentage of the ABTS⁺ inhibition was calculated using formula:

Inhibition (%) = (Ac – As)/Ac × 100 where Ac is the absorbance of control; As is the absorbance of sample or standard. Concentration of the extract that inhibit 50% of free radicals (EC₅₀ values) were also evaluated.

2.9 Statistical

All results were presented as mean (±SD) values of three replicates. Analysis of variance (ANOVA) was performed using SPSS V. 19 (SPSS Inc., Chicago, IL, USA) and mean values were statistically different at P ⩽ 0.05. The significantly different results were further separated using the Tukey multiple range test (SPSS Inc., Chicago, IL, USA).

3.1 Nutritional compositions

Nutritional compositions of immature and mature corn silk are shown in Table 1. Overall, the moisture content of immature silks (89.31%) was significantly higher than mature silks (84.42%) (P value <0.05). However, no significant difference of the same component was observed for immature (4.15%) and mature (3.90%) corn of the oven-dried silks. The lipid and protein composition of immature silks were significantly higher than those of mature silks (P value <0.05). Lipid content of immature silks was 1.27% and has dropped to 0.66% in the mature silks. Meanwhile, the protein content of immature silks (12.96%) was also higher than its mature counterpart (8.95%) (P < 0.05). There was no significant difference between the ash and carbohydrate compositions of both immature and mature silks (P > 0.05). The ash (5.28%) and carbohydrate content of immature silks (27.80%) were slightly lower than those of the mature silks. The ash and carbohydrate content of mature silks obtained were 5.51% and 29.74%, respectively. Regarding the TDF composition, both immature and mature silks displayed high content of TDF comprising of 48.50 g/100 g and 51.24 g/100 g, respectively.

3.2 Mineral composition

Mineral contents (Ca, Mg, K, Na, Cu, Fe, Mn and Zn) of the silks from immature and mature corns were shown in Table 2. In general, the content of major elements such as Ca, Mg, K and Na in immature silks was statistically different from the mature silk. Ca and Mg content were much higher in immature silks amounting 1087 and 1219.17 μg/g, respectively compared to the mature silks which contained 707.04 μg/g of Ca and 361.50 μg/g of Mg. However, the content of K and Na was significantly higher in mature silks compared to those of immature silks. K and Na content of mature silks were 35671.67 and 266.67 μg/g, respectively. Immature silks were found to contain lower amount of K (26281.67 μg/g) and Na (190.67 μg/g).

Both immature and mature silks contained appreciable amounts of trace elements (Table 2). The content of Cu, Fe, Mn and Zn varied from 4.12 to 5.60, 2.17 to 4.50, 32.17 to 35.57 and 35.92 to 46.37 μg/g, respectively. Cu and Zn were found significantly higher in immature silks which were 5.60 and 46.37 μg/g, respectively. For mature silks, the content of these minerals was 4.12 and 35.92 μg/g, respectively. In contrast, Fe content was found to be significantly higher in mature silks (4.50 μg/g) compared to the immature silks (2.17 μg/g). Mature silks also had a slightly higher content of Mn (35.57 μg/g) than the immature ones (32.17 μg/g), but the contents were not statistically different (P > 0.05).

3.3 Extract recovery, total polyphenol and flavonoid content

The percentage of recovery, total phenolic and flavonoid contents of immature and mature silks using water, ethanol and ethyl acetate solvent are shown in Table 3. Apparently, the water, ethanol and ethyl acetate extracts of immature silks exhibited significantly higher percentage of recovery (P < 0.05) compared to the mature silks. The highest recovery of immature silks was recorded in ethanol (27.73%) extract followed by water extract (14.63%). Conversely, mature silk displayed the highest percentage of recovery in water extract (13.69%) compared to ethanol extract (3.29%). Both immature and mature silks extracted with ethyl acetate recorded the lowest percentage of recovery that was 3.27% and 1.25%, respectively.

The immature and mature silks had showed significant differences in total polyphenol and flavonoid content (P < 0.05). Total polyphenol content of crude extracts of immature and mature silks varied from 6.70–92.21 mg GAE/g extract to 4.96–64.22 mg GAE/g extract, respectively (Table 4). As for immature silks, the ethanol extract displayed the highest polyphenol (92.21 mg GAE/g extract) content followed by the water (35.35 mg GAE/g extract) and ethyl acetate (6.70 mg GAE/g extract) extract. In contrast, the highest total polyphenol content in mature silk was displayed by water extract (64.22 mg GAE/g extract) followed by the ethanol (49.88 mg GAE/g extract) and ethyl acetate (4.96 mg GAE/g extract) extract.

As for total flavonoid content, the immature silks exhibited higher content compared to the mature silks (Table 5). In addition, the water extract of immature and mature silks gave the highest flavonoid content that was 8.40 mg CAE/g extract and 2.31 mg CAE/g extract, respectively compared to other extracting solvents employed. Meanwhile, the ethanol and ethyl acetate extract of immature and mature silks showed lower content of flavonoid. The flavonoid content of ethanol extract of the immature silk was 7.55 mg CAE/g extract and significantly higher than that of the mature silk (1.96 mg CAE/g extract). For ethyl acetate extract, the flavonoids obtained from mature silks (2.10 mg CAE/g) were higher compared to the immature silk (0.66 mg CAE/g extract) (see Table 6).

3.4 ABTS free radical scavenging capacity

The antioxidant capacity of immature and mature silk extracts as measured by using the ABTS⁺ free radical scavenging method is shown in Fig. 1(a–c). Overall, all extracts had showed a dose-dependent response when exposed to different concentration of extracts (0.1–1.6 mg/ml). Higher percentage of inhibition was observed at higher concentration. Apparently, the water, ethanol and ethyl acetate extract of immature silk demonstrated a higher percentage of inhibition compared to the same extracts of mature silk. Among all three extracts, the ethanol extract of both immature and mature silks exhibited the highest scavenging capacity followed by the water and ethyl acetate extract. These results indicated that antioxidant compounds present in immature silk extracts are prone to donate hydrogen atom to ABTS free radical giving rise to its scavenging capacity. The percentage of inhibition of ethanol extract was ranged from 23.50% to 78.40% in immature silk and 17.40% to 67.40% in mature silk (EC₅₀ value of 0.478 mg/ml and 0.799 mg/ml, respectively) (Table 4). A lower scavenging capacity was observed in water and ethyl acetate extract of both immature and mature silks. The percentage of inhibition of immature silk water extract (19–57%) was however slightly higher than that of mature silk (15.44–53.5%) recording the EC₅₀ value of 0.751 mg/ml and 1.489 mg/ml, respectively. The lowest antioxidant capacity of both silks was exhibited by ethyl acetate extract. The ethyl acetate extract of immature silk however showed lower EC₅₀ value (2.87 mg/ml) compared to the mature silk (6.29 mg/ml). The inhibition of immature and mature silks was ranged from 13.4% to 35% and 15.56% to 44.36%, respectively.

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Figure 1

(a–c): ABTS scavenging capacity of ethanol, water and ethyl acetate extract of immature and mature silks.

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4.1 Nutritional compositions

In moisture content, the oven-dried silks which were dried at 55 °C for 48 h recorded the moisture content of 3.90–4.15%. The differences in moisture content of oven-dried samples in our study with other samples may be due to the differences in the temperature used and duration of drying. The moisture content obtained here was lower than the dried pericarp and seeds of bitter melon (5.0–13.4%) of different maturity stages which had been dried at 40 °C for only 24 h (Horax et al., 2010). The moisture content of both immature and mature silks however was found higher than some dried apple and orange residues (2–4%) (Figuerola et al., 2005), but lower than mango peel powder (10.5%) (Ajila et al., 2008) and its seed (8.5%) (Abdalla et al., 2007).

Among other plant byproducts, the lipid content of maize silks was lower than tomato peel (Elbadrawy and Sello, in press) but comparable with some plant pods (Mateos-Aparicio et al., 2010). Maize silk cuticle was composed of hydrocarbons of silk waxes that was involved in certain hydrocarbon biosynthesis (Perera et al., 2010). Therefore, the accumulation of hydrocarbons in different maturity stage may influence lipid composition.

Immature silk was obtained from unpollinated cob while the mature silk was taken from fully ripen and developed corn fruit. The protein content of immature and mature silks may be influenced by the functions and biosynthesis of amino acids taking place during developmental processes of pollinated and unpollinated silk tissue. It has been reported that amino acids were actively metabolized in immature cob at early stage of silk emergence in order to regulate the seed growth (Seebauer et al., 2004). The protein content of both immature and mature maize silks in the present study was slightly higher than some vegetables (2–4%) (Odhav et al., 2007) and underutilized plant parts such as mango peels (3.6%) and pods (Mateos-Aparicio et al., 2010).

The TDF content of maize silks was higher than some other underutilized plant parts such as seed (Hainida et al., 2008) peel (Elbadrawy and Sello, 2011), and brans (Abdul-Hamid and Luan, 2000). Therefore, maize silks could be considered as a rich source of dietary fiber thus may confer positive health benefits.

4.2 Mineral content

Mineral composition of the silks immature and mature parts was varied. The variations could be attributable to the difference cultivar, plant nutrition, climate and soil conditions (Hamurcu et al., 2010). The distribution of mineral composition is related to certain functions of the plant part during development. Ca is one of essential mineral which is required for structuring of cell wall and membranes (Evans et al., 2001). K ion is very important in biophysical and biochemical functions as it is highly mobile in plants (Szczerba et al., 2009). In stigma, K ion involves in regulation growth of a pollen tube (Holdaway-Clarke and Hepler, 2003; Zienkiewicz et al., 2011). Zn is required in protein synthesis, genetic entities stability and metabolism of carbohydrate and lipid. Mn plays the role as enzymes activator in tricarboxylic acid cycle (Xue et al., 2004), photosynthetic function (Lidon et al., 2004) photosynthesis, fatty acid and carotenoid synthesis (Santandrea et al., 2000). Mg is abundant in plant tissue and is associated with a wide range of cellular functions including photosynthesis (Gardner, 2003), protein synthesis which involves in ribosomal structures and functions (Maathuis, 2009).

4.3 Extract recovery, total polyphenol and flavonoid content

These results demonstrated clearly that the choice of varying polarities solvent had influence on the extractability of bioactive compounds (Trabelsi et al., 2010). The nature of solvent may explain on its capacity to recover as many antioxidants as possible and solvent having higher polarity exerted higher percentage of recovery. In addition, the different affinities of the extraction solvent such as extraction conditions, solvent polarity and temperature toward chemical constituents in plant materials could attribute to the extract recoveries (Moure et al., 2001). In other study, some exotic fruits showed higher yield of polyphenol when combination of ethanol with other solvent was used (Martinez et al., 2012). Successive isolation of flavonoid compounds from tea leaves (Camellia sinensis) was reported by using ethanol and water solvents (Yang et al., 2009). Maize silks extracted with ethyl acetate had little recoveries and found to be lower than the same solvent used to extract tea leaves (Farhoosh et al., 2007).

Maturity stage may influence accumulation of bioactive compounds. In such case, older plant having lower extract recovery than the immature ones had has also been observed in tea leaves extracts (Farhoosh et al., 2007). In apple for instance, the accumulation of total flavonoid and chlorogenic acid was higher in immature fruit but the level had decreased as the fruit reaches the mature and ripening stage (Awad et al., 2001). Some other reasons are due to the physiological and structural changes taking place during the growth period and fertilization requirements (Salvador et al., 2007).

4.4 ABTS free radical scavenging capacity

In this assay, the antioxidant from extract acts as hydrogen donors to ABTS⁺ free radical thus inhibits oxidation process. Polyphenols include many classes of compounds (phenolic acids, flavanols, xanthones, kaempferol etc.) and act as free radical scavengers. Flavonoid which is the major class of polyphenols exhibits strong antioxidant capacities (Fraga and Oteiza, 2011). However, the antioxidant capacity has decreased during maturity stage and this may be associated with apparent decrease in the total polyhenols and flavonoid contents as compared to the immature part. As the plant grew older, new biosynthesis of polyphenols may interrupt or end thus decrease the content of polyphenols. The reduction of polyphenol content has also been attributed by oxidation of polyphenols during the maturity stage (Shwartz et al., 2009).