Neuroprotective effect of Portulaca oleracea extracts against 6-hydroxydopamine-induced lesion of dopaminergic neurons

The Portulaca oleracea L. (Portulacaceae) is a cosmopolitan species with a wide range of biological activities, including antioxidant and neuroprotective actions. We investigated the effects of P. oleracea extracts in a 6-hydroxydopamine rat model of Parkinson’s disease, a debilitating disorder without effective treatments. Chemical profiles of aqueous and ethanolic extracts of whole plant were analyzed by thin layer chromatography and the antioxidant activity was assessed by 2,2-diphenyl-1-picrilhidrazila method. Male Wistar rats received intrastriatal 6-hydroxydopamine and were treated with vehicle or extracts (oral, 200 and 400 mg/kg) daily for two weeks. The behavioral open field test was conducted at days 1 and 15. Immunohistochemical analysis was performed 4 weeks after surgery to quantify tyrosine-hydroxylase cell counts in the substantia nigra pars compacta. Extracts presented antioxidant activity in concentrations above 300 μg/kg. The chromatographic analysis revealed the presence of Levodopa, alkaloids, flavonoids, saponins, tannins, terpenoids and polysaccharides. Both extracts improved motor recovery 15 days after lesion and protected from tyrosine-hydroxylase cell loss after 4 weeks, but these effects were more evident for the aqueous extract. Because the dopamine precursor is present, in addition to antioxidant compounds and neuroprotective effects, P. oleracea can be considered as potential strategy for treating Parkinson’s disease. key words: Parkinson Disease, Antioxidant Response Elements, Levodopa, Purslane. Correspondence to: Margarete Zanardo Gomes E-mail: margarete_zanardo@itp.org.br introduCtion Parkinson’s disease (PD) is a neurodegenerative illness characterized by a progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNc). The consequent dopamine depletion in the striatum is clinically related to movement and cognitive disorders (Wichmann and Dostrovsky 2011). Its etiology is not fully understood, however, mechanisms of neuronal degeneration include mitochondrial dysfunction, altered proteasomal and lysosomal proteolysis, glial mediated inflammation and oxidative stress generated by lipid peroxidation, protein and DNA oxidation, and chemical and enzymatic oxidation of dopamine itself (Dexter and Jenner 2013).


iNtroduCtioN
Parkinson's disease (PD) is a neurodegenerative illness characterized by a progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNc).The consequent dopamine depletion in the striatum is clinically related to movement and cognitive disorders (Wichmann and Dostrovsky 2011).Its etiology is not fully understood, however, mechanisms of neuronal degeneration include mitochondrial dysfunction, altered proteasomal and lysosomal proteolysis, glial mediated inflammation and oxidative stress generated by lipid peroxidation, protein and DNA oxidation, and chemical and enzymatic oxidation of dopamine itself (Dexter and Jenner 2013).WALESKA B. MARTINS et al.Currently, there is no available therapy to stop or slow neurodegeneration.
Portulaca oleracea L. (P.oleracea) is a commonly found species and a medicinal food for human consumption.This plant, also called purslane, contains minerals, proteins, carbohydrates, β-carotene, vitamins and fatty acids (Uddin et al. 2012(Uddin et al. , 2014)).Among the bioactive components and biological activities assigned to the P. oleracea, the presence of catecholamines (Chen et al. 2003) and its antioxidant and anti-inflammatory actions (Dkhil et al. 2011, Yue et al. 2015) deserve to be highlighted.
Neuropharmacological actions of the P. oleracea extracts were previously reported in rodent models.Effects included the reduction of the locomotor activity, the increase in the onset time of pentylenetetrazole-induced convulsions in mice, the opioid mediated anti-nociceptive and muscle relaxant activities in rats (Radhakrishnan et al. 2001).Biological activities also include anti-inflammatory actions (Chan et al. 2000, Lee et al. 2012).
It has been shown that P. oleracea protects neurons against hypoxia injury (Wanyin et al. 2012) and D-galactose induced toxicity in vivo (Hongxing et al. 2007).The treatment with betacyanins from P. oleracea improved cognitive deficits and attenuated oxidative damage induced by D-galactose in the brains of senescent mice (Wang and Yang 2010).Moreover, the P. oleracea extracts decreased apoptosis and oxidative-stress-induced neurodegeneration caused by the pesticide rotenone (Al-Quraishy et al. 2012, Abdel Moneim 2013).
These neuroprotective actions and the existence of dopamine and noradrenaline in extracts suggest that P. oleracea may be a potential candidate for the treatment of Parkinson's disease.Thus, the aim of this work was to evaluate the neuroprotective effects of the ethanolic and aqueous P. oleracea extracts, given orally, in animals with 6-hydroxydopamine-induced lesion of the nigrostriatal pathway, an animal model for the study of progressive degeneration in PD.Since ethanolic and aqueous extracts are commonly studied, they were also analyzed regarding chemical profiles and antioxidant activity.

Plant Material
The plant material was collected in August 2012, from the greenhouse for medicinal plants at the campus of the Tiradentes University, located in Aracaju -Sergipe, Northeast of Brazil.The plant was identified by Dr. Ana Paula do Nascimento Prata and a voucher specimen (no.20379) has been deposited at the Herbarium of the Federal University of Sergipe.The whole plant was used for the preparation of the extracts.The fresh vegetal material was manually chopped in pieces about 2 cm.

PreParation of the extracts
The aqueous extract was prepared as previously reported (Hozayen et al. 2011).The vegetal material was boiled in distilled water (80 ºC, 1:1, m: v) for 60 min and then mashed with a blender apparatus (3 cycles of 15 sec).The mixture was coated (2 mm pores strainer) and lyophilized under vacuum.
The plant material was extracted with 100% ethanol (100 mL: 10 g) at room temperature, in a dark and closed container for one week.The extract was centrifuged and filtered through a Whatman number 3 filter and then concentrated using a rotary evaporator.The resultant extract was lyophilized to produce a powder (Wanyin et al. 2012).

PhytocheMical analysis
The thin layer chromatography (TLC) was performed using n-butanol-glacial acetic aciddistilled water 7.0: 2.0: 1.0 (v/v) as mobile phase, through chamber saturation for 5 min and detect by ultraviolet (UV) light.For assessing catecholamine levels, the levodopa (D-9628 Sigma) was used as a positive control and the samples were diluted in hydrochloric acid (Dighe et al. 2008).The phytochemical screening was carried out using standard procedures that have been previously described (Harborne 1998): the frothing test was used for the detection of saponins, the ferric chloride test was used for the detection of tannins, the Dragendorff precipitation assay was used for the detection of alkaloids, Mg-HCl was used for the detection of flavonoids, Liebermann-Burchard and Salkowski reactions was used for the detection of triterpenes and lugol was used for the detection of polysaccharides.
Qualitative anD Quantitative 2,2-DiPhenyl-1-PicrilhiDrazila (DPPH) free raDical scavenging activity assay The extracts were analyzed by TLC in a plate eluted in ethyl acetate and ethanol (50%:50%).After drying, a 0.4 mmol/L solution of DPPH in MeOH was added.The antioxidant activity was determined by the presence of yellow spots on a purple background (Souza et al. 2007).
The quantitative assays were performed according to the method previously described (Djouossi et al. 2015).Samples of the extracts and the positive control (trolox) were prepared in methanol (5 mg/ mL) and diluted in the concentrations of 50, 100, 200, 300, 400 and 550 μg/mL.Measurement of the absorbance in the reactive mixtures (0.3 mL of sample solution with 2.7 mL DPPH solution in a 40 μg/mL concentration) was made on a 515 nm, after 40 minutes incubation at room temperature, protected from light.Absorbance values were converted in percentage of the antioxidant activity and the antioxidant efficacy was stabilized by linear regression analysis (p<0.05 confidence interval).Results were expressed through µmol trolox/g-equivalent antioxidant capacity and the sufficient necessary sample concentration to scavenge 50% of DPPH radical was also estimated.

Animals
Forty-eight adult male Wistar rats weighting 200-250 g were housed in a temperature controlled room (at approximately 25ºC), under 12-h light/ dark cycle, with free access to food and water.The experiments were carried out according to the rules of the local Animal Use and Care Committee (approval number 141110) and the Brazilian National Council for the Control of Animal Experimentation (CONCEA).Every effort was made to minimize animal suffering and to keep a number of animals used to a minimum.

Experimental design
After surgical procedures for the intrastriatal injection of 6-hydroxydopamine (6-OHDA) or saline, the animals were divided into groups (n = 6 per group) that received, via oral administration, vehicle (2% Tween 80 in saline), aqueous extract (AEPO) or ethanolic extract (EEPO) of P. oleracea, at 200 or 400 mg/kg for 15 days, consecutively.Doses were chosen based on previous work (Wanyin et al. 2012).At the first and fifteenth day after surgery, an open field test was performed in order to evaluate the motor abilities of the animals.The animals were sacrificed at 28 days following the beginning of the treatments and their brains were extracted and processed for the histological analysis.

6-OHDA microinjections
The heads of the rats were fixed on a Kopf stereotaxic instrument under general anesthesia (ketamine/xylazine, 90 and 15 mg/kg i.p.).A midline skin incision was made, exposing the skull for subsequent drilling.The neurotoxin, 6-OHDA (20 μg in 4 μl dissolved in 0.9% saline containing 0.02 mg/mL of ascorbic acid; RBI-Sigma) was injected into the right striatum with a 10 μl Hamilton syringe.The stereotaxic coordinates were 1.0 mm WALESKA B. MARTINS et al.
anterior, 3.0 mm lateral (right side) from Bregma, and 5.0 mm ventral to the surface of the skull, with the tooth-bar set at −3.0 mm (Paxinos and Watson 1998).The microinjections were performed in a rate of 1 μl/min with an infusion pump (Insight, BR) and the needle was left in place for an additional 180 s to prevent reflux before being slowly retracted.The movement of an air bubble inside the PE-10 polyethylene tubing connecting the micro-syringe with the needle confirmed drug flow.Sham operated (control) animals were submitted to the same procedure but received ascorbate saline (0.02% ascorbic acid) instead of the neurotoxin.

The open field test
The open-field test was used to assess spontaneous locomotor activity.The following parameters evaluated during 5 mins: locomotion or crossings (number of line crosses) and rearings (the number of times the rat stood on its hind legs) (Whimbey and Denenberg 1967).The open field was made of white colored wood, and it consisted of a quadrilateral with an area of 4,830.25 cm 2 and walls that were 34.5 cm high, with the base subdivided into sixteen quadrants, visibly marked by black lines.

Histological analysis
Animals were euthanized in a CO 2 chamber and their brains extracted, fixed in formalin and paraffin-embedded in preparation of subsequent immunohistochemical procedures.Fifteen micrometer serial, sections were cut within a microtome (Leica).Neuroanatomical sites were identified using a rat brain atlas (Paxinos and Watson 1998).The sections were taken from -5.4 mm from Bregma to -6.0 mm).The brain sections were stained for tyrosine-hydroxylase (TH), a marker for dopaminergic neurons.

Immunohistochemistry of tyrosine-hydroxylase (TH)
For TH immunohistochemistry, the antigen recovery was carried out in citrate buffer (pH 6) by using a microwave (3 cycles of 5 min).The endogenous peroxidase was blocked (0.3 % H 2 O 2 ) and tissue sections were incubated 18 h with the primary antibody (rabbit anti TH: 1/1000, Pel-Freez Biologicals).Sections were processed by the streptavidin-biotin immunoperoxidase method (streptavidin HRP Kit, Dako) and immunopositive cells were visualized by addition of the chromogen 3, 3-diaminobenzidine (DAB; Sigma, 1 m g/mL) and hydrogen peroxide (0.2%).The tissue was always washed in phosphate-buffered saline between procedures.Immunopositive cells were revealed by a brown reaction product.The sections were mounted onto gelatin-coated glass slides, dehydrated in ethanol, cleared in xylene, and cover-slipped for microscopic observations.In all experiments, tissues, from every group were always processed in the same assay (Douhou et al. 2002).

Image analysis
Semi-quantitative analysis of midbrain dopaminergic cells was performed on six sections from 3 different animals.To ensure that neurons were not counted twice, counts were made using every sixth section (i.e., separated by 225 µm).All the histological quantification was performed with experimenters blinded to group identity.Neurons which were positively stained exhibited labeled soma, dendrites and axons.The number of TH positive neurons in the right SNc were counted using a microscope (Nikon), equipped with a 100X objective (numerical aperture 1.4) coupled to a digital photographic camera (Nikon) and connected to a computer system.A grid (200 µm x 200 µm) was overlaid, and positive cells were counted in each square using the cell counting tool of Image J NIH software.Only neurons with the whole cell body included in the square were counted.The cell density (number of positive neurons/0.5mm 2 of the structure) on the side of the lesion in the experimental groups was compared with those on the unlesioned shams (Gomes et al. 2008).

Statistical analysis
Results from antioxidant activity and histological counts were expressed as percentage of controls, while the results from the open field are expressed as the mean ± SEM.Effects of the treatments and time on behavioral tests were analyzed by two way repeated measures (MANOVA).In the case of significant interaction it was followed by one-way analysis of variance (ANOVA) and Tukey post-test.The histological and antioxidant evaluations were submitted to ANOVA followed by Tukey post-test.Values of p<0.05 were considered significant.All statistical analyses were performed using SPSS for Windows (version 19.0).

results
The TLC analysis revealed the presence of three fluorescent bands, observed on the UV light at the same position.Each one corresponded to the AEPO, EEPO and levodopa positive control.In the phytochemical analysis, booth extracts showed positive results for the search of alkaloids, flavonoids, tannins, saponins, polysaccharides and terpenoids.Also, the qualitative evaluation of the extracts by TLC showed the existence of substances with antioxidant activity on both extracts.
Both AEPO and EEPO were able to sequester DPPH radicals.However, the EEPO showed antioxidant activity higher than 70% at the 500 μg/ mL, while it was 52% for AEPO.The amount of extract in the samples necessary to decrease the initial concentration of the DPPH in 50% was 162.62 ± 16.37 μg/mL for EEPO and 463.92 ± 40.88 μg/mL for AEPO (Fig. 1).The statistical analysis (ANOVA) revealed significant differences in both the AEPO [F 5,30 =126.2, p<0.05] and the EEPO [F 5,35 =96.21, p<0.05]:For the AEPO analysis, each increment in concentration (i.e., from 50, 100, 200, 300, 400, and 500 µg/mL) produced a significant increase in its respective antioxidant activity, except that no further increase in antioxidant activity was observed from 400 to 500 µg/mL increase in concentration (Fig. 1a).Similarly, a stepwise increase in antioxidant activity was found for nearly every increase in concentration of EEPO (Fig. 1b).Analysis of the tests conducted 24h after 6-OHDA administration showed significant decrease in locomotion from all 6-OHDA groups compared to saline microinjections, independent of the treatment received orally (crossing: F 9,59 = 45.28,p<0.0001; rearing: F 9,59 = 14.15, p<0.0001, one-way ANOVA followed by Tukey post-test, Fig. 2).When the test was carried out at 15 days after surgery (Fig. 2), a significant reduction of crossing for animals treated with 6-OHDA was still observed when compared with groups which received intracerebral saline, except for 6-OHDA/AEPO 400 mg/kg.Treatments with both extracts at higher doses (6-OHDA/ AEPO and 6-OHDA/EEPO 400 mg/kg) resulted in significantly higher values when compared to those obtained for the group 6-OHDA/vehicle and 6-OHDA/AEPO or 6-OHDA/EEPO 200 mg/kg.Moreover, statistical analysis revealed an increase in crossings for the 6-OHDA/AEPO 400 mg/kg compared with all other 6-OHDA groups (F 9,59 = 44.52,p<0.0001, one-way ANOVA followed by Tukey post-test) reaching values similar to control groups.

disCussioN
In this study, spontaneous motor activity in the open field maze was performed 24 hours and 15 days after the administration of 6-OHDA, in order to access both initial functional symptoms and more stabilized lesion signs (Meredith et al. 2008).The 6-OHDA injections induced a significant decrease in locomotion at 24 hours, corroborating the findings of other authors (Dauer andPrzedborski 2003, Truong et al. 2006).After 15 days, these effects were attenuated in a dose dependent manner in the 6-OHDA-treated rats given AEPO and EEPO.Results of immunohistochemistry are compatible with these observations, since the tyrosine-hydroxylase is the enzyme that catalyzes the rate-limiting step in the synthesis of catecholamines and a marker for dopaminergic reminiscent neurons (Daubner et al. 2011).Here, the administration of both extracts produced a nearly 70% increase in preservation of dopaminergic cells.It is in agreement with the reports that spontaneous locomotion, measured in an open field, is impaired in a manner closely related to striatal dopamine depletion after 6-OHDA in rodents (Kirik et al. 1998, Willard et al. 2015).
Our data indicate that P. oleracea contains bioactive compounds that are able to exert a protective effect against degeneration of dopaminergic neurons.It has already been demonstrated in the rotenone model of PD that aqueous extracts of P. oleracea protects the rat brain from oxidative stress through a decrease in the levels of glutathione (Al-Quraishy et al. 2012).Also, the biochemical changes, including an increase in the intercellular content of calcium and dopamine metabolites and decrease in the complex I activity, as well as apoptosis induced by rotenone in the rat striatum, were effectively counteracted by administration of P. oleracea (Abdel Moneim et al. 2012, Abdel Moneim 2013).
As a catecholaminergic neurotoxin, 6-OHDA, when applied into rat striatum, induces a slow degenerative process in dopaminergic neurons.One of the underling mechanisms of cell death is oxidative stress, which has gained special attention because of its association with the reported H 2 O 2 and free radical formation, decreased superoxide dismutase (SOD) activity, increased monoaminoxidase (MAO) activity and mitochondrial complex toxicity (Martínez-Cerdeño et al. 2010, Decressac et al. 2012).
Both aqueous (Hongxing et al. 2007, Al-Quraishy et al. 2012) and ethanolic (Wanyin et al. 2012) P. oleracea extracts have demonstrated protective effects against oxidative stress-related pathologies.For example, the oral administration of EAPO significantly increased the activities of antioxidant enzymes, such as SOD and decreased the production of MAO (Dkhil et al. 2011).In agreement with previous reports, our results from the DPPH test confirm the antioxidant activity of both types of P. oleracea extracts.The possible neuroprotection mechanism could be related to this antioxidant activity (Molyneux 2004, Chen et al. 2009), since both extracts in concentrations above 300µg/kg dose provided 50% of the antioxidant effects of ascorbic acid.It is interesting to note that P. oleracea ethanolic crude extract did not present toxic effect on normal mice even at 500 mg/kg (Aljeboori et al. 2014).
The antioxidant action of P. oleracea extracts can be attributed to its constituents.It has been reported the presence of gallotannins, omega-3 fatty acids, ascorbic acid, α-tocopherols, kaempferol, quercetin, and apigenin (Yan et al. 2012).The analysis of the extracts showed the presence of alkaloids, flavonoids, tannins, saponins, terpenoids and polysaccharides.The same compounds were found in other studies concerning P. oleracea chemical composition (Lu et al. 2009, Dong et al. 2010, Zhu et al. 2010, Uddin et al. 2012, Tian et al. 2014, Liang et al. 2014).The consumption of foods rich in flavonoids is associated with several beneficial effects for human health and can reduce the risk of developing PD in humans (Gao et al. 2012).Also, the alkaloids compounds betacyanins, soluble pigments found in P. oleracea, had neuroprotective effects in senescent mice, reducing the levels reactive oxygen species damage and lipid peroxidation (Wang and Yang, 2010).
In addition, the tannins demonstrated a strong antioxidant activity (Figueroa-Espinoza et al. 2015), polysaccharides of P. oleracea exhibited free radical scavenging activities and protective effect against oxidative damage (Chen et al. 2009), and terpenoids also exerted a neuroprotective role against neuronal hypoxia (Zhu et al. 2007).Also, previous work showed that AEPO can modulate the activity of acetylcholinesterase (Yang et al. 2012) and increase the monoamine contends due its melatonin, omega-3 fatty acids, flavonoids and phenolic compounds (Abdel Moneim et al. 2012).
The group of flavonoids possesses important biological activities and kaempferol and apigenin have been found in ethanolic extracts of leaves and stems (Xu et al. 2006, Zhu et al. 2010).It may explain the higher antioxidant activity of EEPO.On the other hand, AEPO was more effective against 6-OHDA deleterious effects and it suggests that neuroprotection could have been mediated by a mechanism other than oxidative stress.
The TLC analysis revealed the occurrence of a fluorescent band at the same position of levodopa in both extracts.Catecholamines, such as dopamine, are synthesized from levodopa precursor by tyrosine-hydroxylase and have important roles as neurotransmitters in the central nervous system and in PD, once the dopamine depletion is the responsible for the hallmark symptoms of disease (Missale et al. 1998).The presence of cathecolamines such as dopamine has been reported in P. oleracea (Gandía-Herrero et al. 2009, Weng et al. 2005) as well as the presence of levodopa in P. oleracea and the ability of converting tyrosine to levodopa (Harris et al. 2012).
Considering the importance of levodopa as standard treatment in PD and the fact the antioxidant substances can reduce levodopa-induced WALESKA B. MARTINS et al.
dyskinesia (Del Bel et al. 2014, Padovan-Neto et al. 2009), futures studies are needed to explore the potential of P. olearecea in dopamine replacement therapy.From our results, in addition to the antioxidant effects, the improvement in behavioral performance promoted by P. oleracea could be explained, at least in part, by some dopaminergic-like action.
In conclusion, the present results indicated that aqueous and ethanolic extracts of P. oleracea were able to reverse the behavioral motor deficits and neuronal loss induced by 6-OHDA and that these effects may be related to their antioxidant activity and bioactive compounds.The P. oleracea is, therefore, an important target of study with therapeutic potential for the treatment of neurodegenerative diseases.

Figure 1 -
Figure 1 -Trolox-equivalent antioxidant capacity in different concentrations (50; 100; 200; 300; 400 and 500 µg/mL).Columns indicate the mean % of antioxidant activity and bars indicate the S.E.M.; the left graphic (a) presents results of the aqueous extract of Portulaca oleracea (AEPO), and right graphic (b) represents the ethanolic extract of Portulaca oleracea (EEPO) analysis.The * indicates significant difference from all the other doses under the line and # indicates significant difference between doses compared.

Figure 2 -
Figure 2 -Results from the open field test: Means of crossings (a and c) and rearing (b and d) at 24 h and 15 days after surgery, respectively.# indicates significant decrease from saline groups (p < 0.001), * indicates significant increase from 6-OHDA/vehicle with p < 0.05, *** indicates significant increase from 6-OHDA/vehicle with p < 0.0001 and ( †) indicates significant difference between doses compared (p < 0.05).AEPO: aqueous extract of Portulaca oleracea, EEPO: ethanolic extract of Portulaca oleracea.Columns indicate the mean and bars indicate the S.E.M.

Figure 3 -
Figure 3 -Percentages of tyrosine-hydroxylase positive cell density (TH+) in the substantia nigra pars compacta (SNc), 28 days after surgery, from saline microinjected rats.Different number of asterisks indicates statistical difference between groups (p< 0.05) while equal characters indicate similar results.AEPO: aqueous extract of Portulaca oleracea, EEPO: ethanolic extract of Portulaca oleracea.Columns indicate the mean and bars indicate the S.E.M. (n = 6 per group).