Total Phenolic Content and Antioxidant Activity of Some Malvaceae Family Species

The antioxidant activity of four species of the Malvaceae family (Sidastrum micranthum (A. St.-Hil.) Fryxell, Wissadula periplocifolia (L.) C. Presl, Sida rhombifolia (L.) E. H. L and Herissantia crispa L. (Brizicky)) were studied using the total phenolic content, DPPH radical scavenging activity and Trolox equivalent antioxidant capacity (TEAC) assays. The antioxidant activity of the crude extract, phases and two isolated flavonoids, kaempferol 3,7-di-O-α-L-rhamnopyranoside (lespedin) and kaempferol 3-O-β-D-(6''-E-p-coumaroil) glucopyranoside (tiliroside) was determined. The results showed that there is a strong correlation between total polyphenol contents and antioxidant activity of the crude extract of Sidastrum micranthum and Wissadula periplocifolia; however, this was not observed between Sida rhombifolia and Herissantia crispa. The ethyl acetate (EaF) phase showed the best antioxidant effect in the total phenolics, DPPH and TEAC assays, followed by the chloroform (CfF) phase, in most species tested. Lespedin, isolated from the EaF phase of W. periplocifolia and H. crispa may not be responsible for the antioxidant activity due to its low antioxidant activity (IC50: DPPH: 1,019.92 ± 68.99 mg/mL; TEAC: 52.70 ± 0.47 mg/mL); whereas tiliroside, isolated from W. periplocifolia, H. crispa and S. micrantum presented a low IC50 value (1.63 ± 0.86 mg/mL) compared to ascorbic acid in the TEAC assay.


Introduction
Reactive oxygen species (ROS), such as superoxide radicals, hydroxyl (OH) radicals and peroxyl radicals, are natural byproducts of the normal metabolism of oxygen in living organisms with important roles in cell signalling [1,2]. However, excessive amounts of ROS may be a primary cause of biomolecular oxidation and may result in significant damage to cell structure, contributing to various diseases, such as cancer, stroke, diabetes and degenerative processes associated with ageing [3,4]. Thus, antioxidants are important inhibitors of lipid peroxidation not only for food protection but also as a defense mechanism of living cells against oxidative damage [5]. Antioxidants have been shown to prevent the destruction of -cells [6,7], and to prevent or inhibit oxidation processes in human body and food products [8].
Plant polyphenols with antioxidant capacity could scavenge reactive chemical species as well as minimise oxidative damage resulting from excessive light exposure. Some plant polyphenols are important components of both human and animal diets and they are safe to be consumed [9].
Food antioxidants such as α-tocopherol, ascorbic acid, carotenoids, amino acids, peptides, proteins, flavonoids and other phenolic compounds might also play a significant role as physiological and dietary antioxidants [10]. Natural antioxidants are known to exhibit a wide range of biological effects including antibacterial, antiviral, antiinflammatory, antiallergic, antithrombotic and vasodilatory activities [11].
Antioxidant capacity is widely used as a parameter for medicinal bioactive components. Various methods are currently used to assess the antioxidant activity of plant phenolic compounds. ABTS + or DPPH  radical scavenging methods are common spectrophotometric procedures for determining the antioxidant capacities of components [12].
Assays based upon the use of DPPH  and ABTS + radicals are among the most popular spectrophotometric methods for determination of the antioxidant capacity of food, beverages and vegetable extracts [13]. DPPH is a stable free radical that reacts with compounds that can donate a hydrogen atom. This method is based on the scavenging of DPPH through the addition of a radical species or an antioxidant that decolourizes the DPPH solution [14].
The Trolox equivalent antioxidant capacity assay (TEAC) is based on the ability of antioxidants to quench the long-lived ABTS radical cation, a blue/green chromophore with characteristic absorption at 734 nm, in comparison to butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), α-tocopherol and Trolox, a water-soluble α-tocopherol analogue [12].
Due to the ethnobotanical importance of the family and the absence of studies that prove their antioxidant activity, the aim of this study was to evaluate the antioxidant activity, using DPPH radical scavenging activity and Trolox equivalent antioxidant capacity, and the total phenolic content of the crude extract (CE) and various phases depending on the species: ethyl acetate (EaF), aqueous (WtF), chloroform (CfF) hexane (HF), n-butanol (n-BF) or dichloromethane (DF) phases of plants of the Malvaceae family: Sidastrum micranthum, Wissadula periplocifolia, Sida rhombifolia and Herissantia crispa. The antioxidant activity of two flavonoids isolated from Wissadula periplocifolia and Herissantia crispa: kaempferol 3,7-di-O-α-L-rhamnopyranoside (lespedin) and kaempferol 3-O-β-D-(6''-E-p-coumaroil) glucopyranoside (tiliroside) was also analyzed. Tiliroside was also isolated from Sidastrum micranthum.

Plant
Leaves of the species under study where dried in an oven, at an average temperature of 40 °C, for 96 h. The dried leaves were macerated with ethanol (95%), for 72 h, at room temperature.

Determination of Total Phenolic Contents
The total phenolic content of the samples was determined using the Folin-Ciocalteau's reagent as described by Gulcin et al. [24]. An aliquot of the samples, dissolved in ethanol, was mixed with Folin-Ciocalteau's reagent (100 µL) and distilled water (3 mL) and mixed for 1 min. Sodium carbonate (300 µL, 15%) was added to the mix. The solution had its volume adjusted to 5 mL with distilled water. After 2 h, absorbance was measured at 760 nm. A standard curve was prepared using gallic acid with a concentration range from 0.5 to 25 µg/mL. Total phenolic content was expressed as mg gallic acid equivalents (GAE)/g of samples.

DPPH Radical Scavenging Activity Assay
The assay was performed according to Silva et al. [25]. An aliquot of the samples was mixed with DPPH solution (5 mL, 23.6 μg/mL in ethanol), followed by incubation of 30 min. The absorbance of each sample was read at 517 nm. Ascorbic acid (0.9, 1.9, 3.9, 4.9, 6.9 μg/mL) was used as positive reference. The percentage of scavenged DPPH was calculated using Equation (1): where Ac is the absorbance of the control and As is the absorbance of the sample. IC 50 values calculated denote the concentration of the sample required to decrease the absorbance at 517 nm by 50%.

Trolox Equivalent Antioxidant Capacity Assay (TEAC)
The ABTS free radical-scavenging activity of each sample was determined according to the method described by RE et al. [26]. The radical cation ABTS + was generated by persulfate oxidation of ABTS. A mixture of ABTS (7.0 mM) and potassium persulfate (2.45 mM) was allowed to stand overnight at room temperature in the dark to form radical cation ABTS + , 12-16 h prior to use. A solution was diluted with ethanol and absorbance measured, at 734 nm. An aliquot of each sample was mixed with the solution of the radical cation ABTS + (5 mL), and the decrease of absorbance was measured at 734 nm after 10 min. Trolox (1.1, 1.7, 2.3, 2.9, 3.5 μg/mL) was used as positive reference. IC 50 values calculated denote the concentration of the sample required to decrease the absorbance at 517 nm by 50%.
All experiments were performed in triplicate. The DPPH and TEAC data were expressed as IC 50 (mg/mL). Total phenolic content was expressed as mg gallic acid equivalents (GAE)/g. Linear regressions were performed to indicate the relationship between the total phenolic contents and data from the antioxidant assays.

Results and Discussion
The results of total phenolic contents and the antioxidant activity of the EEB and phases of Sidastrum micranthum are shown in Table 1. The ethyl acetate (EaF) phase of this species, had the highest content of phenolic compounds (177.44 ± 16.21 mg· GAE/g) and the highest TEAC (IC 50 = 2.267 ± 0.377), showing a better result than the Trolox assay (IC 50 = 3.02 ± 0.014). However, for the DPPH assay, the chloroform (CfF) phase showed better antioxidant activity (IC 50 = 24.7 ± 0. 306).
The results of the antioxidant assays of Wissadula periplocifolia are shown in Table 2. The EaF phase had the highest content of phenolic compounds (260.46 ± 5.74), as well as the best antioxidant activity in the DPPH assay (IC 50 = 20.52 ± 0.16). The TEAC assay, the CfF phase showed better antioxidant activity (IC 50 = 23.98 ± 0.03) compared to the other phases. This discrepancy in total antioxidant activity values depending on the method used indicates that both assays determine different aspects of the antioxidant capacity. Different radicals and mechanisms of reaction are occurring [27].  The EaF of the Sida rhombifolia had the highest content of phenolic compounds (88.311 ± 2.660 mg· GAE/g) and the best antioxidant activity for the DPPH and TEAC assay (IC 50 = 70.503 ±1.629 and 20.580 ± 0.271, respectively), as shown in Table 3. The CfF phase of Herissantia crispa, had the highest content of phenolic compounds (142.397 ± 0.555 mg· EAG/g), and the best antioxidant activity for DPPH assay (IC 50 = 61.52 ± 0.458) and TEAC assay (IC 50 = 27.127 ± 0.567) ( Table 4). Only David et al. [28] have studied the methanolic extract of Herissantia crispa, reporting an IC 50 value for the DPPH assay of 3.9 mg/mL. However, other phases were not studied.
The Folin-Ciocalteu method, generally used to assay the total phenolic compound content also measures the total reducing capacity of a sample. Total phenolics generally correlate with redox and antioxidant capacities as measured by the TEAC or DPPH methods [29,30]. Many studies indicate a linear relationship between total phenolics and antioxidant activity [31][32][33]. A direct correlation between the three methods, in all species, was demonstrated by linear regression analysis. As shown in this study, there is a strong correlation between total polyphenol contents and the antioxidant activity (r 2 = 0.929) of Sidastrum micranthum; as well as Wissadula periplocifolia (r 2 = 0.814) samples.
Liu et al. [34] showed that the correlations of total polyphenol content against the antioxidant activity based on the DPPH assay, TEAC assay, and FRAP (ferric ion reducing antioxidant power) assay of Ilex kudingcha were satisfactory (r > 0.812). The results of that work indicated that polyphenols in kudingcha extracts are largely responsible for the antioxidant activities.
Phenolic compounds are ubiquitous bioactive compounds and a diverse group of secondary metabolites universally present in higher plants [35]. The Folin-Ciocalteu phenol reagent is used to obtain a crude estimate of the amount of phenolic compounds present in an extract. However, the assay has been shown nonspecific not only to polyphenols but to any other substance that could be oxidised by the Folin reagent as reported by various researchers, the poor specificity of the assay [36,37]. This statement may explain the low correlation between total polyphenol contents and the antioxidant activity of Sida rhombifolia and Herissantia crispa (r 2 = 0.618; r 2 = 0.46, respectively). It is suggested, that non-phenolic substances are responsible for antioxidant activity in this species. This lack of relationship is in agreement with Anagnostopoulou et al. [38] who reported a r 2 value of 0.42 between TPC and DPPH for extract obtained from sweet orange peel.
Lespedin showed a high IC 50 value for the DPPH (IC 50 = 1,019.92 ± 68.99) and TEAC assay (IC 50 = 52.70 ± 0.47) (Table 5), which means that this flavonoid is not the main substance responsible for the antioxidant activity of the EaF phase of Wissadula periplocifolia. Tiliroside also showed a high IC 50 value for the DPPH assay (IC 50 = 219.31 ± 9.62), therefore, it cannot be responsible for the antioxidant activity of the EaF phase. However, for the TEAC assay, it exhibited a low IC 50 value (IC 50 = 1.63 ± 0.86), and may contribute to the antioxidant activity of the Eaf phase of Wissadula periplocifolia and Sidastrum micranthum. A study performed by Babbar et al. showed that phenolic compounds alone are not fully responsible for the antioxidant activity of plants. Other constituents such as ascorbates, reducing carbohydrates, tocopherols, carotenoids, terpenes, and pigments as well as the synergistic effect among them could possibly contribute to the total antioxidant activity [48].

Conclusions
In conclusion, species the Malvaceae family have a high content of phenolic compounds and a good antioxidant activity, therefore they can be used to treat several diseases in which there is an increase in free radical production. However, non-phenolic substances can be responsible for the antioxidant activity of Herissantia crispa. Therefore, further studies are needed to identify which phenolic compounds are responsible for the antioxidant activity of the species, and assess the way in which the phenolic substances contribute to this activity. Additional in vivo antioxidant assays are needed to confirm the potential use of these species in disease treatment.