Antioxidant Potential of Polyphenol Rich Extract from Hibiscus sabdariffa

This work carried out in collaboration between all Author AOO designed the study, performed the statistical analysis, wrote the protocol and wrote the first draft of the manuscript. Author the analyses of the study the literature sabdariffa is a medicinal plant species that is consumed for its health benefits in Africa, therefore this study investigated the antioxidant properties of Hibiscus polyphenolic rich extract (HPE), Hibiscus sabdariffa Methodology: The antioxidant assays evaluated the scavenging abilities of HPE: Firstly against superoxide ions generated during the xanthine oxidase mediated breakdown of xanthine to uric acid. Secondly against ABTS (2,2-azino-bis-(3-ethylbenzthiazoline 6-sulfonic acid)) radical cation generated by filtering a solution of ABTS through manganese dioxide powder and potassium persulphate. Finally metal chelation ability of HPE against Iron ions (Fe 2+ ) induced oxidative damage in cultured Jurkat-T cells was also assessed. Results: The results showed that 1.0% and 2.5% (v/v) diethyl ether extract of HPE significantly inhibited superoxide ions by 42.35 and 100.00% respectively. The extract also inhibited uric acid production, which suggest that components of HPE inhibit xanthine oxidase activity. In addition, it was found that HPE scavenge ABTS radical cations in dose dependent manner. HPE inhibited Fe 2+ -mediated lipid peroxidation in cultured Jurkat-T cells supplemented with 0.5 mg/ml and 1.0 mg/ml of HPE by 19.67% and 31.69% respectively, metal chelation ability was identified as a potential mechanism behind this observed reduction. Conclusions: HPE is rich in different phenolic compounds; therefore strong antioxidant potential of HPE observed in this study may be related to their polyphenolic constituents. This study demonstrated that Hibiscus sabdariffa is an efficient antioxidant plant species in vitro and may be beneficial in reducing oxidative damage to lipid and thus prevent or reduce the development and progression of free radical mediated diseases.


INTRODUCTION
Oxidative damage to important biomolecules (such as lipids, proteins and DNA), if not repaired will accumulates and leads to physiological attrition and risk of several chronic diseases [1]. For examples, oxidation of low-density lipoprotein (LDL) is an important factor in atherosclerotic plague formation leading to coronary heart disease [2]. Intracellular protein oxidation results in functional changes modulating cellular metabolism [3], and oxidative modification of DNA bases leads to mutation and altered gene function, resulting in cancer formation [4,5]. These oxidative damages are cause by reactive oxygen species (ROS) generated endogenously as by-products of normal metabolic processes. An antioxidant defence system helps to prevent the buildup of ROS and protect living organisms against their deleterious effects [6].
Protective mechanisms against pathogens include the generation of ROS such as superoxide anions, hydrogen peroxide, hydroxyl radicals and hypochlorous acid [7,8]. The generation of ROS, whatever their origin, may play a role in several T cell processes including signalling, proliferation and death [9,10]. Because T cells are present in the inflammatory site they also experience ROS attack. The ability to cope with ROS-induced oxidative stress depend on protective mechanisms the cell can provide [11]. Aging is associated with a loss in protective immunity mainly characterized by poor T cell responses to stimulation, low responses to vaccination and an accumulation of CD8 cells which seem to be in a state of replicative senescence [12,13]. Several hypotheses have been proposed to explain this, with the role of chronic proliferative and oxidative stress featuring as a major factor [14,15]. Epidemiological studies have shown that consumption of high amounts of fruits and vegetables rich in flavonoids/phenolics is associated with a reduced risk of major agerelated diseases such as coronary heart disease and cancer [16,17]. This is because flavonoids and other phenolic compounds have powerful antioxidant activities in vitro and in vivo, such as scavenging of diverse ROS species or inhibiting ROS formation, e.g. by chelating prooxidative metal ions [17,18].
One plant species that is consumed for its health benefits is Hibiscus sabdariffa-Juice/concoction prepared from this plant is taken as a preventive/curative measures against diabetes and hypertension in Nigeria [19,20]. The antihypertensive property of this plant has been demonstrated in previous studies [19,20]. Research demonstrated that constituents of the extract of H. sabdariffa have strong antioxidant properties [21,22]. It has been postulated in several studies that effectiveness of plant extracts against many diseases are due to their antioxidant actions [23,24]. Hibiscus sabdariffa is a rich source of phenolic compounds [25]. Hibiscus polyphenolics rich extract (HPE) prepared from this species has been identified to be rich in numerous phenolic compounds, such as catechin, epigallocatechin, epigallocatechin gallate and caffeic acid [26]. Also, H. sabdariffa is rich in anthocyanins which is responsible for its deep red colouration. However, in spite of this varied phenolic content of H. sabdariffa the contributory role played by these phenolic compounds to antioxidant effects of this plant has not been subjected to many studies.
The aim of this study was to investigate the in vitro antioxidant potential of H. sabdariffa Polyphenolics rich extract (HPE) prepared from H. sabdariffa. Evidence is presented to suggest that H. sabdariffa is an effective antioxidant plant species in vitro and this antioxidant potential may be related to the high concentration of polyphenolic antioxidants in the plant.

Plant Material
The dried calyx of H. sabdariffa (Malvaceae) was bought at a market in Nigeria. The identification and authentication of the plant material was done by a qualified taxonomist, Prof. A.J. Ogunkunle and by comparison with herbarium samples at Department of Pure and Applied Biology, Ladoke Akintola University of Technology, Ogbomoso. The dried calyx were further dried at room temperature and blended to a coarse powder.

Preparation of Hibiscus Polyphenol Rich Extract
Hibiscus Polyphenol Rich Extract (HPE) was prepared according to the method of Lin et al.
[26]. Briefly 100 g of H. sabdariffa were extracted three times with 300 ml of methanol at 50°C for 3 hours. The samples were filtered after each extraction and the solvent was removed from the combined extracts with a vacuum rotary evaporator. The residue was then dissolved in 500 ml of water (50°C) and extracted with 200 ml hexane to remove some of the pigments (i.e. chlorophyll, caratenoids). The aqueous phase was extracted three times with 180 ml ethyl acetate, and the ethyl acetate was evaporated under reduced pressure. The residue was redissolved in 250 ml water and was lyophilized to obtain approximately 1.5 g of HPE and stored at -20°C before use.

Preparation of a Diethyl Ether Extract of HPE
Due to intense colouring of HPE, it was necessary to prepare a less intense extract which did not interfere with some of the spectrophotometric assays. One part of HPE was gently mixed with two parts diethyl ether and centrifuged at 2000 rpm for 10 minutes. The diethyl ether extract was removed and dried under a stream of oxygen-free nitrogen gas. The residue was then re-suspended in phosphate buffered saline (PBS) pH 7.2 to its original volume.

Superoxide and Xanthine Oxidase Activity
This was carried out as reported by Dillion et al. [27]. Superoxide production and xanthine oxidase activity were measured as cytochrome C reduction and uric acid production, respectively. Xanthine oxidase was prepared to a concentration of 107 mU/ml in Phosphate Buffered Saline (PBS), pH 7.2 and Xanthine was prepared as a 1.6 mM solution also in PBS. Superoxide ions were generated in a reaction volume of 1 ml containing 160 µM xanthine and 1.25 mg cytochrome C. The reaction was initiated by the addition of 10.7 mU xanthine oxidase, and superoxide ion production was monitored at 550 nm [28]. In a series of separate experiments, xanthine oxidase activity was monitored as the production of uric acid at 284 nm. Generation of superoxide ions was confirmed by the addition of 50 U superoxide dismutase (SOD), which inhibited the reduction of cytochrome C without affecting xanthine oxidase activity. Extracts were added at 0-10% (v/v). Results for superoxide production are expressed as ∆A550 nm/minute whilst, result for uric acid production are expressed as ∆A284 nm/minute.

Trolox Equivalent Antioxidant Capacity (TEAC) with Manganese Dioxide
The assay was performed as previously described by Schelesier et al. [29]. The ABTS radical cation was prepared by filtering a solution of ABTS (in PBS) through manganese dioxide powder. Excess manganese dioxide was removed from the filtrate by passing it through a 0.2 µm syringe filter. This solution was diluted in 5 mM PBS pH 7.4, adjusted to an absorbance of 0.700±0.020 at 734 nm and preincubated at room temperature prior to use for 2 hours. 1 ml of ABTS •+ solution and various concentrations of the extracts (diluted with water) were vortexed for 45 seconds in reaction tubes, and the absorbance (734 nm) was taken exactly 2 minutes after initiation of mixing. PBS blanks were run in each assay. The antioxidant activity of the extract was calculated by determining the decrease in absorbance at different concentrations by using the following equation: % antioxidant activity = ((A (ABTS•+) -A (Extracts) ) / (A (ABTS•+) ) X 100.

Trolox Equivalent Antioxidant Capacity with Potassium Persulfate
The assay was performed essentially as described by Re et al. [30]. ABTS radical cation was produced by reacting 7 mM ABTS stock solution with 2.45 mM potassium persulphate and allowing the mixture to stand in the dark at room temperature for 12-24 hours before use. The ABTS •+ solution was diluted with water for the hydrophilic assay and with ethanol for the lipophilic assay and adjusted to an absorbance of 0.700±0.020 at 734 nm. For the photometric assay, 1 ml of the ABTS •+ solution and various concentrations of the extracts were mixed for 45 seconds and measured immediately after 1 minute at 734 nm. The antioxidant activity of the extracts was calculated by determining the decrease in absorbance at different concentrations by using the following equation.

Assessment of cell viability
Cells were seeded at a density of 2x10 6 cells/ml and incubated with HPE at various concentrations (0.5, 1.0, 1.5 and 2.0 mg/mL) for 24 hours in 96 well plates. Thereafter the cells were washed twice with filtered PBS before medium (200 µl) was replaced in each well with 100 µl MTT/well. The plates were incubated for 4 hours at 37°C. The MTT was then removed and the dark-blue crystals which remain were dissolved in 100 µl dimethylsulfoxide (DMSO). Complete dissolution of the crystals was achieved by shaking the plates gently for 10 minutes at room temperature. Absorbances were measured using a multi-well plate reader at a wavelength of 550 nm. Plates were always read within 30 minutes of adding the DMSO. The viability of HPE exposed cells was calculated by using the following equation.

TBARS assay
The procedure was based on Mertens et al. [31]. Briefly, 1.0 ml of Jurkat cells suspension was mixed thoroughly with 2 ml 0.25M-HCl containing 15% (w/v) trichloroacetic acid and 0.375% (w/v) TBA. After heating the solution for 15 minutes at 95°C, the samples were cooled and the pink product was extracted with 3 ml butanol. After centrifugation for 10 minutes at 3000 g the absorbance was determined. Hydrolysed tetraethoxypropane was used as the standard. This was prepared as a 10 mM solution in 1% (v/v) H 2 S0 4 ; following hydrolysis at room temperature for 2 hours, the solution was diluted accordingly in PBS and a calibration curve (0-10 µmoles MDA) was constructed using the TBARS assay described. Results were expressed as µmoles of MDA/10 6 cell.

Oxidative treatment
The oxidation of Jurkat cells with iron was performed as described by Erba et al. [32]. Jurkat cells were incubated with 25, 50, 100 and 150 µmol/L FeSO 4 in PBS for 1 hour. The susceptibility of Jurkat cells to Fe 2+ induced oxidative damage was measured in terms of accumulation of TBA -reactive species (TBARS).

Cell supplementation with HPE
HPE was dissolved in RPMI 1640 medium and added to the culture at a concentration range of (0.1 mg/ml-1.0 mg/ml) and maintained for 24 hours at 37°C. The suspension was then washed twice with PBS before the oxidative treatment, which was performed in PBS by adding 100 µmol/L of Fe 2+ (as FeS0 4 ) for 1 hour. Cells were then thoroughly washed twice with PBS, centrifuged and re-suspended in PBS for TBARS analysis.

Determination of iron binding properties of HPE
The iron chelating properties of the HPE was assessed. Cells were pre-incubated with the HPE (0.2-1.0 mg/ml) for 5 minutes, washed thoroughly with PBS twice before being oxidised with 50 µmol/L Fe 2+ for 15 minutes. 50 µM EDTA (ethylenediamine tetraacetic acid) was used as a positive control. Oxidation damage was measured by TBARS assay.

Statistical Analysis
Results are expressed as means ± SEM. Statistical analyses were performed using Student's t test. All analyses were done using Graph Pad Prism software Version 5.00 and p < 0.05 was considered statistically significant.

Superoxide Scavenging Ability of HPE
Superoxide production by xanthine -xanthine oxidase gave a reaction rate of 0.085±0.002 ∆A550 nm/minute while xanthine oxidase activity gave a reaction rate of 0.106±0.001 ∆A284 nm/minute. At 1.0 and 2.5% (v/v) of the reaction volume, diethyl ether extract of HPE significantly inhibited superoxide production i.e the reduction of cytochrome C by 42.35 and 100% respectively ( Table 1). Superoxide production was inversely related to the concentrations of diethyl ether extract of HPE. In addition, uric acid production was significantly affected by diethyl ether extract of HPE.

Effect of HPE on Jurkat Cells Viability
MTT assay showed that HPE up to 1.0 mg/ml concentration exhibited no toxic effect on Jurkat cells (seeded at a density of 2.0x10 6 /ml) incubated with the extract for 24 hours. However the Jurkat cell viability drops when the concentrations of HPE was increased to 2.0 mg/ml (Fig. 1).

Fe 2+ Mediated Oxidation of Jurkat Cells
There was dose dependant increase in Lipid peroxidation observed in Jurkat cells incubated in the presence of Fe 2+ . Lipid peroxidation was significantly increased by about 5 fold in the presence of 100 µM Fe 2+ , when compared with Jurkat cells incubated in PBS only (0.33 µM MDA/10 6 cells vs 1.83 µM MDA/10 6 cells) (Fig.  2). Lipid peroxidation was inhibited in Jurkat cells incubated with 0.5 mg/ml and 1.0 mg/ml of HPE for 24 hours prior to oxidation with 100 µM Fe 2+ by 19.67% and 31.69%, respectively (Fig. 3).

The Fe 2+ Chelation Properties of HPE
Lipid peroxidation measured as TBARS production was 0.33±0.02 and 0.87±0.03 in Jurkat cells incubated for 15 minutes in PBS alone and 50 µM FeSO 4 respectively ( Table 3). The concomitant addition of 50 µM EDTA inhibits the TBARS production by 80%. The addition of 0.2 mg/ml and 0.5 mg/ml HPE in the presence of FeSO 4 inhibit lipid peroxidation (measured as TBARS production) by 63% and 83% respectively (Table 3).    did not significantly affect the cell viability and morphology, at all concentrations used for this study (0 µM-150 µM) and there was significant increase in MDA production (Fig. 2). The increment of MDA concentrations found in Jurkat cells is consistent with the occurrence of freeradical-mediated damage. HPE supplementation at concentrations consistent with viability of cells (0 mg/ml-1 mg/ml) (Fig. 1) decreased the production of MDA in cells subjected to oxidative treatment, indicating the antioxidant role of this extract in preventing lipid peroxidation (Fig. 3).

Values are the means of three experiments ± SEM. Significant difference (p < 0.05) from control (Jurkat cells and PBS only) is indicated by‫٭‬ while significant difference (p < 0.05) from Jurkat cells and Fe 2+ only is indicated by ‫٭٭‬
The anti-lipidperoxidative effect of HPE had been reported in previous study [22].  (Table 3). These Fe 2+ chelating properties of HPE would thus significantly decrease the oxidative modification of lipid in Jurkat cells. Phenolic antioxidants prevent cellular oxidative damage by several mechanisms and chelating metal ions is one of them. It has been identified that chelating ability of polyphenols is dependent upon the localisation of functional hydroxyl groups, while glycosylation of hydroxyl positions reduces the chelating power [40,41].
Hibiscus sabdariffa is a rich source of a plant polyphenols such as flavonoids and phenolics acids [25,26,33]. The presence of flavan-3-ols compounds (catechin, epigallocatechin, epigallocatechin gallate) has been identified in Hibiscus polyphenolics rich extract (HPE) used in this study in previous research [26]. Therefore, it can be inferred that the presence of these antioxidants phenolic compounds (catechin, epigallocatechin, epigallocatechin gallate) in HPE may be associated with alleged medicinal properties of H. sabdariffa. Nevertheless, other phenolic constituents not previously identified could be working synergistically with these flavan-3-ols compounds giving a larger antioxidant effect. Since polyphenols are by far the major antioxidant constituents of H. sabdariffa, this class of compounds appears to be of major relevance for the observed antioxidant effects of this plant species. In addition, ascorbic acid, β-carotene and lycopene previously identified to be present at low concentrations in H. sabdariffa may also contribute to the antioxidants potential of this species [42].

CONCLUSION
It is concluded that H. sabdariffa is an efficient antioxidant plant species as HPE extracted from the plant contains components that actively scavenge ABTS radical cations. It also inhibits both superoxide ion production and Fe 2+mediated lipid peroxidation in cultured Jurkat-T cells. This property may be attributed to the phenolic constituents of the extracts. Therefore the strong antioxidant properties of H. sabdariffa may explain their popular consumption and usage in herbal medicine.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.