INTRODUCTION

Several studies have showed that free radicals and related reactive oxygen species (ROS) mediate much of the neurological damage that occurs after an ischemic brain insult. Brain ischemia and particularly reperfusion induce generation of powerful free radicals such as superoxide and hydroxyl radicals, which overwhelm endogenous antioxidative mechanisms (Love, 1999; Allen and Bayraktutan, 2009; Manzanero et al., 2013; Zhao et al., 2016). This condition of oxidative stress in the highly vulnerable brain tissue leads to lipid peroxidation, protein oxidation, different types of DNA damage, and eventually to neuronal cell death. In this sense, antioxidants of varying chemical structures have been investigated as therapeutic agents in the treatment of this acute injury (Cuzzocrea et al., 2001; Wang et al., 2006; Alonso-Alconada et al., 2013; Ren et al., 2015). Flavonoids, one of the most potent naturally occurring antioxidants, represent the most common group of polyphenolic compounds in the human diet and are widely distributed in plants. Recently, there has been great interest in the potential of flavonoids to protect the brain from ischemic damage (Dajas et al., 2003; 2013; Lin, 2011; Panickar and Jang, 2013; Pérez-Hernández et al., 2016). Antioxidation, but also modulation of signalling cascades and gene expression as well as anti-inflammation appears as the main protective mechanisms (Dajas et al., 2013).

Gossypitrin (Gos) is a flavon-3-ol glycosylated at the 7-position in ring A. It contains two free OH groups at C₅ and C₈ in ring A, a catechol moiety at C₃´-C₄´ in ring B and a free OH group at C₃ in the pyrone ring C (Fig. 1). It can be found with high prevalence in the flowers from the genus Hibiscus, where it joins the red anthocyanins producing a co-pigmentation effect found in their extracts (Cuéllar-Cuéllar et al., 2010; 2011). It is also the main bioactive substance of the flowers of Talipariti elatum Sw. (Majagua azul), traditionally used in Cuban island as expectorant and antiasthmatic (Roig, 1974). Only few reports have documented the biological properties of this flavonol, despite its optimized structural arrangement for the antioxidant action, encompassing five hydroxi-aromatic groups. Therefore, it could be hypothesized that the antioxidant capacity of gossypitrin could contribute to its cytoprotective capacity in vitro, which would be important to define a putative neuroprotective action against ischemic brain damage. Thus, the present study was designed to address the antioxidant and cytoprotective effects of gossypitrin against cyanide-induced oxidative stress and cell death in PC12 cells.

MATERIAL AND METHODS

Plant material

Flowers were collected in January 2016 in the gardens of the Faculty of Pharmacy and Foods at Havana University, and identified at the herbarium of National Botany Garden of Havana, where the voucher specimen no. HAJB 82587 was deposited and registered as Talipariti elatum S.w.

Extract and samples preparation

Dark red flowering types were collected daily. The petals used were dried in an oven with controlled temperature at 40°C, during 5 days. The extracts were prepared with the ground material (60 g), using a Soxhlet apparatus and 95% ethanol (675 mL) for 20 hours. The ethanolic extracts were concentrated and roto-evaporated under vacuum to 200 mL at 120 rpm, 70°C, and 500 mBar. For to the purification, 1 g of solid was dissolved in 25 mL of diethyl ether and the volume was completed to 100 mL with ethanol. The sample was refrigerated until the appearance of abundant yellowish-green solid, which was recovered by filtration. This process was done twice, and was monitored by thin layer chromatography (TLC) on pre-coated 60 F254 silica gel aluminum backed plates (Merck), using a mixture of n-butanol, acetic acid and water (4:1:5 v/v/v) as eluent (Cuéllar-Cuéllar et al., 2010). Solid obtained were characterized using 1D- and 2D NMR, and GC/MS experiments. 1D-NMR (1H and 13C) and 2D–NMR (1H-1H COSY, HSQC and HMBC) spectra were acquired on a Bruker Advance 500 spectrometer (500 MHz, DMSO-d6) (Bruker Co., Karlsruhe, Germany). Peak positions are relative to TMS for 1H NMR and 13C NMR spectra. GC–MS was recorded using an Agilent Gas Chromatograph (series 6890 N) (Agilent Technologies, Inc., Wilmington, DE, USA) coupled to a mass detector (series 5953 N) (Agilent Technologies, Inc., Wilmington, DE, USA). Injection of the sample was performed in the split mode with a 1:10 ratio. Temperatures in the injector, interphase and detector were 280, 280 and 150°C, respectively. Chromatographic separation was accomplished in a SPB-5 (15 m × 0.25 mm × 0.10 μm) column, using He flow (1 mL/min). The mass spectrum was recorded by electron impact as the ionization technique (70 eV). Gossypitrin was detected as [M−H] ions at m/z 481.1 (positive mode ion) and m/z 479.2 (negative mode ion). The MS spectra of the compound are dominated by the neutral loss of the O-linked hexosyl-group (-162 u), but differ in the ratio of the resulting Y₀⁻ and [Y₀⁻H]•− ions at m/z 318.1 (Márquez et al., 1999). NMR 1D (¹H) and (¹³C) revealed the presence of 21 identified carbons: (11 C sp2, 4 CH sp2, 5 CH sp3 and 1 CH₂ sp3) utilizing the ¹³C, DEPT 135 and DEPT 90 experiments. The results by 1D and 2D (proton, COSY, HSQC) allowed the confirmation of 4 aromatic protons (δ = 8.08 ppm (d, 1H, J4 = 2.21 Hz, 2’-H), (δ = 7.92 ppm (dd, 1H, J3 = 8.72 Hz - J4 = 2.21 Hz, 6’-H), (δ = 6.99 ppm (d, 1H J3 = 8.71 Hz, 5’-H). The observed multiplicity (ABX system) is characteristic of a catechol moiety. Observed chemical shift value of proton 6-H (δ= 6.25 ppm) and carbon 6-C (δ = 97.98 ppm) confirmed the presence of hydroxyquinol (ring A). The pres- ence of a glucoside was confirmed by the presence of 6 carbons sp3 (5CH and 1 CH₂) where the protons at (δ = 4.83 ppm (d, 1H, J = 7.32 Hz, 1’’-H), ), δ = 3.76 ppm (d, 1H, J = 11.96 Hz, 6’’-H), δ = 3.59 ppm (dd, 1H, J = 11.96 Hz, J = 4.10 Hz, 6’’-H), δ = 3.44-3.34 ppm (m, 4H, 2’’, 3’’, 4’’, 5’’-H) resonating in the characteristic zone of polyhydroxylated compounds. Signal at 10.39 ppm (C-7) disappeared in gossypitrin ¹H NMR spectra (Márquez et al., 1999).

PC12 cell culture

Pheochromocytoma (PC12) cells were kindly supplied by Beatriz Caputto, Ph.D., from Cordoba National University, and Sandra Verstraeten, Ph.D., from Buenos Aires University, Argentina. Cells at passage seven were routinely maintained in Dulbecco's Modified Essential Medium (DMEM) containing L-glutamine (Sigma, St. Louis, MO) and supplemented with 10% heat-inactivated equine serum (Gibco, Life Technologies, USA), 5% inactivated fetal bovine serum, 50 U/mL of penicillin and 10 mg/mL of streptomycin (all of which were obtained from Sigma, St. Louis, MO) in a humidified atmosphere containing 95% air/5% CO₂ at 37°C. The growth medium was replaced every three days. At day seven, the cells were trypsinized (Sigma, St. Louis, MO) and seeded at a density of 1.5×10⁵ cells/mL in 96 multiwell flat bottom plates (Corning Costar, Sigma) in 200 μL of culture medium at 37°C and were flushed with 5% CO₂ in air for 24 h (Satpute et al., 2008; Figueredo et al., 2011).

Induction of chemical hypoxia in PC12 cells

For the cyanide-induced injury, the cells were exposed to 0.6 mM KCN in DMEM without glucose in the absence (control) or presence of Gos (0.001–10 mM), or Rut (0.01-0.15 mM) over the course of 1 h in a humidified atmosphere containing 95% air and 5% CO₂. Gos or Rut were added 30 min before KCN. Hypoxia was terminated by aspirating the poisoned medium and washing the cells with fresh media in all of the experimental groups, including the control and reference cultures. The reperfusion state was simulated by the addition of DMEM without KCN and with high glucose content for 24 h (Satpute et al., 2008).

In vitro antioxidant activity of Gos on cell-free system

Scavenging activity of Gos against 100 µM 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) was monitored by the change in absorbance at 517 nm (U-2910 Hitachi spectrophotometer, Japan), 5 min after incubation in 40 mM sodium acetate ethanolic solution, pH 5.5 (Blois, 1958). As a positive control, the synthetic antioxidant Trolox was used. All determinations were performed in triplicate.

The antioxidant capacity was also estimated in terms of the ABTS^●+ radical [2,2-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid)] scavenging activity following the procedure described by Long and Halliwell (2001). After the addition of 10 µL of Gos or Trolox to 4 mL of diluted ABTS^●+ solution, the absorbance (U-2910 Hitachi spectrophotometer) was measured at 30 min.

Ferric ion reducing antioxidant power (FRAP) was assayed through the reduction of Fe³⁺ to Fe²⁺ by Gos or ascorbic acid as reference. The Fe²⁺-2,4,6-tripiridils-triazine complex was detected spectrophotometrically (U-2910 Hitachi spectrophotometer) at 593 nm and ascorbic acid was used as antioxidant control (Benzie and Strain, 1996).

Lipid peroxidation was assessed by malondialdehyde (MDA) production in rat’s brain homogenate, as previously described (Genet et al., 2002). MDA concentration was calculated from ε = 1.56 × 10⁵ M⁻¹·cm⁻¹ (Buege and Aust, 1978).

PC12 cell viability assays

Cell viability was measured by the levels of blue formazan products formed from the colorless 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazo-liumbromide (MTT) by mitochondrial dehydrogenases, which are only active in viable cells. The absorbance was measured at 540 nm using a microplate reader (POLARstar Omega fluorescence spectrophotometer, Germany). The results are expressed as the percentage of absorbance relative to the undamaged control cells.

Measurement of oxidative stress parameters in PC12 cells

For the antioxidant enzymes and lipid peroxidation assays, the cultures were washed with ice cold PBS and then pooled in 0.5% Triton X-100 and 0.1 M PBS - 0.05 mM ethylenediamine tetra acetic acid (EDTA) buffered solution and homogenized. The homogenate was centrifuged for 20 min at 3000 xg at 4°C and the supernatants were used in the assays. For the absorbance readings, a U-2910 Hitachi spectrophotometer (Japan) was used.

The extent of lipid peroxidation was determined quantitatively as previously described (Buege and Aust, 1978). The amount of MDA was measured by its reaction with thiobarbituric acid, monitored at 532 nm. MDA concentration was calculated from its extinction coefficient (ε) mentioned before.

The assay of catalase was based on the consumable rate of H₂O₂ measured at λ = 240 nm (Aebi, 1984).

The superoxide dismutase activity was determined by the quantification of the superoxide anion radical (O₂^•−) generated by the xanthine/xanthine oxidase system. The assay is based on the spectrophotometric determination of nitroblue tetrazolium reduction products at 560 nm (Nagai et al., 2001).

For GSH measurement aliquots (100 µL) of the supernatant were mixed with 2 mL of 100 mM NaH₂PO₄ buffer, pH 8.0, containing 5 mM EGTA. One hundred microliters of o-phthalaldehyde (1 mg/mL) was added, and the fluorescence was measured 15 min later using the 350/420 nm excitation/emission wavelength pair and an F-4500 fluorescence spectrophotometer (Hitachi) (Hissin and Hilf, 1976; Pardo-Andreu et al., 2007). As in the hypoxia/re-oxygenation assay, Rut (0.01-0.10 mM) was used as reference compound.

Quantitative estimation of cellular protein was made by the standard method of Lowry et al. (1951).

Statistical analysis

GraphPad Prism 6.0 software (GraphPad Software Inc., USA) was used for statistical analyses. The data are expressed as the mean ± S.E.M. Comparisons among different groups were performed using a one-way analysis of variance (ANOVA), followed by the Newman–Keuls multiple comparison post hoc test. Differences were considered statistically significant at p < 0.05. The IC₅₀ and EC₅₀ values were estimated using a non-linear regression algorithm.

RESULTS

Free radicals scavenging and anti-lipoperoxidative activities of Gos in a free cell system

In vitro antioxidant methods used for the evaluation of antioxidant activities of Gos included DPPH, ABTS, FRAP and lipid peroxidation assays. The results were expressed as the IC₅₀ values for DPPH, ABTS and lipid peroxidation i.e., the quantity of Gos needed to scavenge 50% of the radicals or lipoperoxides produced in the reaction mixture, or EC₅₀ for FRAP i.e., the quantity of Gos required to reduce 50% of total ferric iron in the assay medium. The Gos IC₅₀ values for the DPPH and ABTS radicals scavenging were lower than those for Trolox, a hydrosoluble derivative of α-tocopherol, suggesting a potent intrinsic antioxidant activity (Table 1). The EC₅₀ value for FRAP of this polyphenol was also lower than that of ascorbic acid, evidencing a strong reducing power that probably could explain its ability to reduce the above mentioned artificial radicals. Finally, Gos also showed a concentration dependent inhibition of lipid peroxidation, with a low IC₅₀ value of 24.26 µM (Table 1).

Effects of Gos on the viability of PC12 cells submitted to KCN-mediated hypoxia

Incubation of PC12 cells with KCN (600 µM) in glucose-free medium for 1 h, followed by 24 h reperfusion (fresh growth medium without KCN and with a high glucose content), resulted in approximately 46% cell death. Fig. 2 shows a strong cytoprotective effect of Gos when it was pre-incubated with cells (the lower cytoprotective concentration was 0.001 mM), with a very low IC₅₀ value (4.82 ± 0.84 µM). Rutin, the well-known glycoside derivative of quercetin showed a weaker cytoprotective effects, with an IC₅₀ value around 12 times higher (57.3 ± 1.52 µM).

Effects of Gos on the redox status of PC12 cells submitted to KCN-mediated hypoxia

To determine whether the protective effects of Gos on hypoxia-induced injury were mediated by their antioxidant functions, the activities of antioxidant enzymes, and lipids peroxidation in PC12 cell under hypoxia with or without Gos were examined. As shown in Table 2, the exposure of PC12 cells to low glucose and hypoxia for 1 h followed by 24 h reperfusion diminished the activities of SOD and CAT by 55 and 70%, respectively; being catalase more sensitive to cyanide than SOD as reported elsewhere (Kanthasamy et al., 1997). The levels of the endogenous antioxidant GSH was also decreased by 34% in close association to a marked increase (3.7 folds) of lipid peroxidation, evidencing the instauration of an oxidative stress condition on the hypoxic-damaged cells. Gos significantly attenuated the oxidative damage on PC12 under hypoxia in a concentration-dependent manner, as reflected by the recovery of the antioxidant enzymes activities and GSH, and by a significant diminution of lipid peroxidation. The EC₅₀ values for the antioxidant effects of Gos were at the very low micromolar concentrations, ranging from 1-5 µM (Table 2). Rutin, here used as a reference compound, also counteracted the oxidative damage to PC12 cells, but its antioxidant effects were attained at higher concentrations (EC₅₀ values > 100 µM for CAT and SOD restoration, and 45.2 µM for MDA inhibition).

DISCUSSION

Hypoxia is a component of physiological events and multiple pathophysiological conditions, including neonatal hypoxia, and cerebral ischemia (Lendahl et al., 2009), which treatment options are still empirical due to poor mechanistic understanding. Thus, it is important to explore the mechanisms underlying the potential protective effects of compounds through hypoxia models. In this sense, the chemical hypoxia model induced by KCN exposure in PC12 cells is a useful in vitro model to elucidate the mechanisms associated to the hypoxic damage and to test novel neuroprotective compounds because it reproduces many hypoxic conditions, including reactive oxygen species generation, mitochondrial impairment, cytosolic calcium and pH deregulations, and apoptosis induction (Johnson et al., 1987; Maduh et al., 1990; Mills et al., 1996; Kanthasamy et al., 1997; Gao et al., 2003; Satpute et al., 2008). Using PC12 as in vitro model, Mills et al. (1996) demonstrated that cyanide induced apoptotic oxidative stress. Accordingly, it has been observed that treatment with the antioxidants N-acetyl cysteine (Satpute et al., 2008), ascorbate (Kanthasamy et al., 1997) and inhibitors of lipid peroxidation (Müller and Krieglstein, 1995) significantly reduced the incidence of cyanide-induced apoptosis. The antioxidant-optimized structure of Gos together with the poor documentation of its antioxidant-mediated biological effects prompted us to determine the effects of this flavonoid on PC12 cells subjected to chemical hypoxia. Our data showed that treatment of PC12 cells with 600 mM of KCN resulted in 46% decrease in the cell viability. Nevertheless, cells pre-incubation with Gos exerted a protective effect against KCN-induced cell viability decrease, reaching full protection (no difference from the Control-unpoisoned cells) between 5-10 µM. Interesting, the neuroprotective effects followed the pattern of the antioxidant actions on both the cells free and the cells lysates systems. The former data evidenced a potent intrinsic antioxidant potential (free radical scavenging and reducing power) even superior than those from the classical antioxidant Trolox and ascorbic acid used as positive controls. Gos has a privileged chemical structure that justifies is antioxidant actions.

On the basis of available experimental studies (Bors et al., 1990; van Acker et al., 1996; Arora and Nair, 1998), several key features of the structure–antioxidant activity relationship in flavonoids include the catechol group in ring B, the carbonyl group in the 4 position in conjugation with the double bond at C₂ and C₃, an OH group at C₅, and a free OH group at C₃. Since gossypitrin contains all these key features, it is likely to be a powerful antioxidant. In this regard it has been shown that quercetin, a well-known antioxidant flavonoid that fulfil the above mentioned structural arrangement, showed marked cytoprotective capacities in experimental conditions resembling molecular feature of ischemic brain damage (Dajas et al., 2003; 2013). It is worth to notice that the in vitro antioxidant effects of Gos on PC12 cells were higher than those of Rut, here used as reference compound. Rut possesses two carbohydrate structures as its side-chain, but Gos has only one carbohydrate side-chain and an extra hydroxyl group. The enhanced antioxidant effects of Gos could be correlated with higher liposolubility and stability and improved biological properties when compared to the Rut.

The protective effects of Gos in cells are probably the biological consequence of its antioxidant actions, evidenced by the Gos capacity to restore the antioxidant enzyme activities (SOD and CAT) and to recover the endogenous GSH levels that could be used for the restorative activities of the cells. In this regard, antioxidants are known to inhibit the cell injury, scavenged ROS formation, increased antioxidative enzyme activity, and attenuated the lipoperoxidative damage in oxidative-stressed neuronal cells (Hou et al., 2003; Marín-Prida et al., 2012; 2013). Excessive ROS production in the brain are believed to contribute to neurodegenerative processes and compounds inhibiting this neurotoxic process, i.e. antioxidants have been considered as promising therapeutic candidates against ischemic stroke (Broussalis et al., 2012).

Not only antioxidants, but also protein kinase C (PKC) modulators play a role in hypoxic damage. PKC down-regulation or inhibition (with chelerythrine or staurosporine) conferred protection against KCN-induced cytotoxicity (Pavlaković et al., 1995). Ca²⁺ channel blocker also protected against chemical hypoxia (Pavlaković et al., 1995). The hypoxia-inducible factor-1 (HIF-1), is also involved in the hypoxic mediated cells death since it has been shown that the protective role of ginkgolides, garlic derivative and fisetin were due to the modulation of HIF-1α protein expression (Li et al., 2008; Chen et al., 2015; Orozco-Ibarra et al., 2016). MAPK and mitochondrial mediated signaling pathways have also been involved in the neuroprotective effects of carnosic acid and resveratrol, respectively on neuronal cells under hypoxic stress (Hou et al., 2012; Liu et al., 2016). Since most of the above mentioned neuroprotective effects were elicited by antioxidants, we do not discard the contribution of some of them to the neuroprotection elicited by Gos. In this regard, additional in vitro and in vivo studies are necessary to fully understand the neuroprotective mechanism of action of Gos against neuronal hypoxic damage.

CONCLUSIONS

These results showed for the first time that Gos was able to protect PC12 cells against hypoxia induced injury through direct free radical scavenging and modulation of endogenous antioxidants and antioxidant enzymes. The strong neuroprotective activity of Gos in vitro, as evidenced in this study, prompted us to extend our research to in vivo experimental models of brain damage, which are now underway.

Acknowledgements

Authors confirm that this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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Additional information

CONFLICT OF INTEREST: The authors declare no conflict of interest.

Citation Format: Bécquer-Viart MA, González-Yaque J, Fonseca-Fonseca LA, Núñez-Figueredo Y, Pardo Andreu GL (2018) Antioxidant and neuroprotective effects of gossypitrin, a flavonoid from Talipariti elatum, against chemical hypoxia-induced PC12 cell death. J Pharm Pharmacogn Res 6(2): 72–80.

Alternative link

http://jppres.com/jppres/pdf/vol6/jppres17.314_6.2.72.pdf (pdf)

http://jppres.com/jppres/antioxidant-and-neuroprotective-effects-of-gossypitrin/ (html)