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Betacyanins from Portulaca oleracea L ameliorate cognition deficits and attenuate oxidative damage induced by D-galactose in the brains of senescent mice.

ABSTRACT

This experiment was designed to assess the protective effect of betacyanins from Portulaca oleracea L. against the D-galactose (D-gal)-induced neurotoxicity in mice. Betacyanins from Portulaca oleracea markedly reversed the D-gal-induced learning and memory impairments, as measured by behavioral tests. The activities of superoxide dismutases (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) in D-gal-treated mice were enhanced, while the content of the lipid peroxidation product malondialdehyde (MDA) was decreased by betacyanin administration. Furthermore, significant negative correlations were found between mouse latency in finding the platform and the activities of SOD, CAT GR and GPx in the mouse brain, but the level of MDA correlated positively with the latency. These results suggest that the neuroprotective effect of betacyanins against D-gal-induced neurotoxicity might be caused, at least in part, by an increase in the activities of antioxidant enzymes with a reduction in lipid peroxidation. In comparison with vitamin C (VC), the betacyanins had a more pronounced effect on ameliorating cognition deficits in mice.

ARTICLE INFO

Keywords:

Cognition

D-galactose

Mice

Oxidative stress

Portulaca oleracea L

Introduction

The neuron is particularly susceptible to oxidative damage resulted from the production of reactive oxygen species (ROS). It has been proposed that formation of ROS is an important step leading to neuronal death in a variety of age-related neurodegenerative disorders including Alzheimer's disease and Parkinson's disease (Castegna et al. 2002; De Iuliis et al. 2005; Olanow 1993). ROS oxidizes various biological macromolecules, thereby disturbing homeostatics within the neuron and ultimately resulting in cell death. D-galactose (D-gal) can cause the accumulation of ROS, or stimulate free-radical production indirectly through the formation of advanced glycation endproducts in vivo, finally resulting in oxidative stress (Zhang et al. 2005). D-gal could impair neurogenesis in the dentate gyrus, a process similar to natural aging in mice (Cui et al. 2006). Therefore, the D-gal-induced senescent mouse is a good model for studying the aging brain (Wei et al. 2005). Further studies showed that D-gal induced aging-related changes included increased production of free radicals and decreased antioxidant enzyme activities (Wei et al. 2005; Zhang et al. 2005).

Natural products with medicinal value are garnering a lot of attention due to the serious side-effects often caused by medicines of chemical origin (Lu et al. 2007; Zhang et al. 2007, 2008; Zhong et al. 2009). Natural pigments are a class of natural products that have attracted considerable attention as antioxidants. Betalains are water-soluble, nitrogen-containing pigments, which include the red-violet betacyanins and the yellow betax-anthins. Betalains accumulate in most families of the Caryophyllales and some higher fungi. In contrast to other natural pigments such as carotenoids and anthocyanins, the physiological effects of betalains are not well-studied. In recent studies, the antioxidant properties of betalains and incremental improvement of oxidation resistance in humans by betalains have been reported (Gentile et al. 2004; Kanner et al. 2001; Strack et al. 2003; Tesoriere et al. 2003, 2004, 2005; Zakharova and Petrova 1998). Furthermore, the role of betalain pigments in chemoprevention in lung, liver and skin cancers in mice, and inhibition of the cell proliferation of a variety of human tumor cells, have been demonstrated (Kapadia et al. 1996, 2003; Reddy et al. 2005). However, studies of the anti-aging effects of betacyanin in animal models or humans are scarce. Therefore, it is necessary to investigate the effect of betacyanin in an animal model to develop a neuroprotective drug. The aim of the present study was to investigate the effect of betacyanin on the cognition of senescent mice induced by D-gal and on antioxidant parameters in the mouse brain.

Betacyanin was isolated from Portulaca oleracea L, which is used widely not only as an edible plant, but also as a folk medicine in different countries to treat different ailments in humans. Portulaca oleracea has a wide range of pharmacological effects, including antibacterial (Zhang et al. 2002), analgesic, antiinflammatory (Chen 2000), skeletal muscle-relaxant and wound-healing activities (Parry et al. 1987, 1993; Rashed et al. 2003). As is the case with many other members of the Caryophyllales, Portulaca oleracea contains betalains rather than anthocyanins, but little is known about the medicinal values of betacyanin extracted from Portulaca oleracea. This study will therefore strengthen our knowledge about this interesting plant family.

Material and methods

Betacyanin isolation from Portulaca oleracea

Betacyanins were extracted and purified according to a modified version of the method reported by Stintzing et al. (2002). Specifically, 1 g fresh weight of seedlings of Portulaca oleracea was ground with 20 ml MeOH for 30 min at 4[degrees]C. After centrifugation at 10,000 x g for 10 min at 4[degrees]C, the supernatant was discarded and the pellet was re-extracted with distilled water. The sample was then filtered through eight layers of gauze cloth and shaken in a separatory funnel with two-fold volumes of chloroform. The aqueous portion was collected and stored at -70[degrees]C for further use.

The aqueous extracts were purified by passing through a 20-ml capacity [C.sub.18]-reversed phase cartridge previously activated with methanol followed by TFA(pH 3). Betacyanins in the extracts were absorbed on the [C.sub.18] column; sugar, acids and other water-soluble compounds were eluted with two volumes of aqueous TFA. Then betacyanins were subsequently eluted with methanol containing 0.01% HC1 (pH 3). The desalted sample was concentrated and dried in vacuo at 30[degrees]C. The absorbance of betacyanin solution at 538 nm was determined, and the betacyanin content in Portulaca oleracea seedlings was estimated using the molar extinction coefficient for betanin of 65 x [10.sup.6] [cm.sup.2] [mol.sup.-1] (Schwartz and von Elbe 1980).

Animals and drug administration

Ten-week-old male Kunming strain mice (29.86 [+ or -] 5.55 g each) were purchased from the Branch of the National Breeder Center of Rodents (Shanghai). Prior to experiments the mice had free access to food and water and were kept under conditions of constant temperature (25 [+ or -] 2 [degrees]C) and humidity (60-70%). Ten mice were housed per cage on a 12 h light/12 h dark schedule (light on 08:30-20:30). After acclimatization to the laboratory conditions for 1 week, the mice were randomly divided into six groups of ten animals each: the vehicle control group, model group, two betacyanin groups and two vitamin C (VC) groups. The mice of the model group, betacyanin and VC groups were injected subcutaneously with D-galactose (Sigma-Aldrich, MO, USA) at the dose of 50 mg/kg body wt./day, while those of vehicle control group were injected with the same volume of physiological saline (0.9% NaCl) for 8 weeks. From the seventh week, the two betacyanin groups received, respectively, betacyanin at 50 and 100 mg/kg body wt./day in distilled water containing 0.1% Tween 80 by oral gavage for two weeks after injection of D-galactose, and the VC group mice received VC at 50 or 100 mg/kg body wt./day in distilled water containing 0.1% Tween 80 by oral gavage for two weeks after injection of D-galactose. The model control group mice were administered the same volume of distilled water containing 0.1% Tween 80 without betacyanin and VC. Behavioral testing was subsequently conducted for 5 days. Animals were sacrificed after the behavioral test for biochemical assay. All experimental procedures were performed according to the National Institutes of Health Guide for Care and Use of Laboratory Animals.

Behavioral testing

Behavioral testing was performed in the water maze (Morris 1984), which consisted of a black circular tank, 100 cm in diameter and 50 cm in depth. The tank was divided virtually into four equal quadrants and an escape platform was hidden 1.5 cm below the surface of the water in a fixed location in the 3rd quadrant of the pool. After one day's training a trial was started by placing the mice into the pool close to the rim, facing the wall of the tank into one of the four quadrants. Mice were given four trials per session for 5 days, with each trial having a ceiling time of 60s and a trial interval of approximately 30 s. After climbing onto the platform, the animal remained there for 30 s before the next trial. If the mouse failed to reach the escape platform within 60s, it was gently placed on the platform and allowed to remain there for 30s. The time to reach the platform (latency) was measured. The day after the acquisition phase (5 days), a probe test was conducted by removing the platform. The time spent in the target quadrant, which had previously contained the hidden platform, was recorded. The latency to reach the non-exits and the numbers of crossing the non-exits were recorded for each trial.

Preparation of brain tissue and homogenates

According the method of Lu et al. (2007), all mice were deeply anesthetized and sacrificed by decapitation after behavioral testing. Brains were promptly dissected and perfused with 50mM (pH 7.4) ice-cold phosphate buffer saline solution (PBS), then homogenized in 1/10 (w/v) PBS containing a protease inhibitor cocktail (Sigma-Aldrich). The homogenates were divided into two portions and one part was centrifuged immediately at 8000 x g for 10 min to obtain the supernatant for assaying brain catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) activities, MDA level and protein content. The second part of the homogenates was sonicated four times for 30 s with 20 s intervals using a VWR Bronson Scientific sonicator, centrifuged at 5000 x g for 10min at 4[degrees]C, and the supernatant collected for determination of superoxide dismutases (SOD) enzyme activity.

Measurement of antioxidative enzymes and MDA

The assay for total superoxide dismutases (SOD) is based on the ability to inhibit the oxidation of oxymine by the xanthine-xanthine oxidase system (Oyanagui 1984). The hydroxylamine nitrite produced by the oxidation of oxymine had an absorbance peak at 550 nm. One unit (U) of SOD activity was defined as the amount that reduced the absorbance at 550 nm by 50%, and data were expressed as units per microgram of brain protein.

Catalase (CAT) activity was assayed by the method of Aebi (1984). In brief, to a quartz cuvette, 0.65 ml of the phosphate buffer (50 [mmoll.sup.-1]; pH 7.0) and 50 [micro]l sample were added, and the reaction was started by addition of 0.3 ml of 30 mM hydrogen peroxide ([H.sub.2][O.sub.2]). The decomposition of [H.sub.2][O.sub.2] was monitored at 240 nm at 25[degrees]C. CAT activity was calculated as nM [H.sub.2][O.sub.2] consumed in 1 min per milligram of brain protein.

The glutathione peroxidase (GPx) activity assay was based on the method of Mizumo and Ohta (1986). tert-Butylhydroperoxide was used as substrate. The assay measures the enzymatic reduction of [H.sub.2][O.sub.2] by GPx through consumption of reduced glutathione (GSH) that is restored from oxidized glutathione GSSG in a coupled enzymatic reaction by GR. GR reduces GSSG to GSH using NADPH as a reducing agent. The decrease in absorbance at 340 nm due to NADPH consumption was measured in a Molecular Devices [M.sub.2] plate reader (Molecular Devices, Menlo Park, CA). GPx activity was computed using the molar extinction coefficient of 6.22 m[M.sup.-1] [cm.sup.-1]. One unit of GPx was defined as the amount of enzyme that catalyzed the oxidation of 1.0 [micro]mol of NADPH to [NADP.sup.+] per minute at 25[degrees]C.

The glutathione reductase (GR) activity assay was based on the method of Mizumo and Ohta (1986). The enzymatic activity was assayed photometrically by measuring NADPH consumption. In the presence of GSSG and NADPH, GR reduces GSSG and oxidizes NADPH, resulting in a decrease of absorbance at 340 nm, which was measured in a [M.sub.2] plate reader. Quantification was based on the molar extinction coefficient of 6.22m[M.sup.-1] [cm.sup.-1] of NADPH. One unit of GR was defined as the amount of enzyme that reduced 1 [micro]mol of GSSG (corresponding to the consumption of 1 [micro]mol of NADPH) per minute at 25[degrees]C

The level of lipid peroxidation in brain homogenate was indicated by the content of malondialdehyde (MDA) in brain tissue. Thiobarbituric acid reaction (TBAR) method was used to determine the MDA which can be measured at the wavelength of 532 nm by reacting with thiobarbituric acid (TBA) to form a stable chromophoric production (Ohkawa et al. 1979). MDA content was expressed as nmol per milligram of brain protein.

Protein concentration was measured using the method of Bradford (Bradford, 1976). Bovine serum albumin was used as standard.

Statistics

Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Duncan's Multiple Range Test (DMRT). Each value was presented as means [+ or -] standard errors of the mean (S.E.M), with a minimum of five replicates; p < 0.05 was considered as significant.

Results

Effects of betacyanin on the behaviour of D-gal-treated mice

The results of the present study showed that the D-gal-induced model group mice had significant cognitive deficit. As shown in Fig. 1A, the latency to find the platform declined progressively during the training days in all groups. However, the model group mice had longer latency to find the platform throughout the training period than did control mice, showing poorer learning performance due to chronic administration of D-gal. Betacyanin and VC treatment significantly shortened the latency as compared with the D-gal treatment, but at the same dose the effect of betacyanin was more pronounced than that of VC.

[FIGURE 1 OMITTED]

In the probe trial, model group mice failed to remember the precise location of the platform, spending significantly more time first crossing the non-exits than control, betacyanin groups and VC groups. (Fig. 1B). The numbers of target crossing was significantly reduced in the model group mice indicating a spatial navigation deficit, betacyanin and VC treatment significantly reversed these spatial navigation deficits as seen in Fig. 1C. Furthermore, the time spent in the target quadrant was increased by the administration of betacyanin and VC as compared with the model group (Fig. 1D). All these results revealed that both betacyanin and VC could improve spatial learning and memory in D-gal-treated mice, but the betacyanin had a better effect than VC.

Effects of betacyanin on SOD, CAT, GPx, GR activities and MDA content in brain of senescent mice induced by D-galactose

As compared with control group mice, the SOD activity in the brain declined significantly, by 33.2% in the model group mice, betacyanin (50 or 100 mg/kg could increase the activities of SOD nearly to 76.5% and 91.6% of the control group respectively, while VC at 50 or 100 mg/kg body wt.) improved the SOD activities to 70% and 77% of the control (Fig. 2A). Both doses of betacyanin and VC reached significant levels as compared to the model group, and the effect in the betacyanin groups was much better than that of the VC groups. But there were still significant differences between the control and the betacyanin/VC groups (p < 0.01 or p < 0.05).

[FIGURE 2 OMITTED]

The responses of CAT activity to D-gal, betacyanin and VC treatments were similar to but more marked than that of SOD activity (Fig. 2B). For example, the CAT activity in the brain declined by 38.5% in the model group mice as compared with the control group mice, a lower dose of betacyanin (50 mg/kg body wt.) increased the activities of CAT to nearly 83.9% of the control group while CAT activity in the group receiving a higher dose of betacyanin (100 mg/kg body wt.) was almost the same as that of the control group. Thus, only CAT activity in the lower dose (50 mg/kg body wt.) betacyanin group mice reached a significant difference as compared to the control group (p < 0.05). The CAT activities in the two VC groups were lower than corresponding doses of betacyanin groups and both reached significant difference in comparison with the control group (p < 0.01).

The activities of GPx and GR in the brains of the model group mice were significantly lower as compared with the control group (Figs. 2C and D). Betacyanin and VC treatments resulted in a significant elevation in the activities of the two enzymes. The effect of betacyanin was much better than VC on increasing the enzyme activities, and the higher dose of betacyanin at 100 mg/kg body wt. had a more significant effect than at the lower dose of 50 mg/kg body wt. But there were still significant differences between the control and the betacyanin groups (p < 0.01 or p < 0.05).

Model group mice showed a significant increase in MDA level as compared with the control group (Fig. 3). This increase in MDA was also attenuated in the brains of betacyanin- and VC-treated mice. Although the MDA-reducing effect of betacyanin was more pronounced that of VC, there were still significantly differences between the control and the two betacyanin/VC groups, meaning that the betacyanin and VC administration did not reverse the D-gal-induced membrane damage completely.

[FIGURE 3 OMITTED]

The correlation between the antioxidant parameters and cognitive parameters in mouse brain

In order to examine the relationship between latency on the fifth day of the water maze test and the activities of SOD, CAT, GPx and GR and the level of MDA in mouse brain, the data obtained from different D-gal, betacyanin and VC treatments were analyzed statistically as follows:

Y = A+B x X

in which Y indicates latency to find the platform, and X indicates the activities of SOD, CAT, GPx and GR and the level of MDA, R-regression coefficient.

As shown in Table 1, latency to reach the platform on the fifth day was negatively correlated with the activities of SOD (R = -0.795). Such negative correlation between latency and CAT, GPx and GR activities also existed in the mouse brain. But the level of MDA was positively correlated with latency on the fifth day (R-0.877).
Table 1
The correlations between latency and the activities of SOD, CAT, GPx
and GR and the level of MDA in mouse brain.

Relations coefficient             Regression equation  Regression

The latency and the SOD activity  Y = 47.765 -0.454X   R = -0.795
The latency and the CAT activity  Y = 47.543-23.177X   R = -0.815
The latency and the GPx activity  Y = 66.322-2.203X    R = -0.816
The latency and the GR activity   Y = 55.961-3.203X    R = -0.837
The latency and the MDA content   Y = 7.208 + 2.444X   R = 0.877


Discussion

Chronic systemic D-gal exposure induces memory impairments, neurodegeneration, and oxidative damage in mice (Cui et al. 2006; Wei et al. 2005; Zhang et al. 2005). Our results are in agreement with these findings. In our study, chronic administration of D-gal impaired the performance of mice in a water maze task and the groups of mice receiving the two doses of betacyanin showed better cognitive parameters as compared to the model group (Fig. 1), indicating that betacyanin had potential to prevent this kind of learning and memory deficits.

ROS research has become an active field in aging research because of their potential involvement in many neurodegenerative diseases (Castegna et al. 2002; De Iuliis et al. 2005; Harman 1992; Olanow 1993). Recent findings also link the presence of ROS to cell survival or proliferation signals (Ulrich-Merzenich et al 2009). The concentration of ROS is determined by the balance between the rate of production and clearance by various antioxidant enzymes. SOD is the first enzyme of the enzymatic antioxidative pathway to convert superoxide anions into peroxides, which are converted into water by CAT and GPx. In the glutathione peroxide reaction, glutathione is oxidized into glutathione disulfide, which can be converted back to glutathione by GR in an NADPH-consuming process (Deby and Goutier 1990; Halliwell and Gutteridge 1989). In this study, the activities of SOD, CAT, GSH-Px and GR in the mouse brain showed a significant decline in the model group as compared to the control group. Treatment with betacyanin for two weeks clearly improved the activities of these enzymes (Fig. 2). In addition, an obvious enhancement of the MDA level was seen in the model group mice, but it could be significantly reduced after betacyanin administration (Fig. 3). Therefore, betacyanin scavenged ROS mainly via increasing the activity of SOD, CAT, GPx and GR, and consequently, decreased lipid peroxidative damage.

Accumulating evidence (Fukui et al. 2002; Parle and Dhingra 2003) has indicated that treatment with a variety of antioxidants partially reverses oxidative stress and the decline in learning and memory. The task of antioxidants may not be solely the suppression of ROS, but rather a modulatory function on the survival and death signaling of ROS (Ulrich-Merzenich et al 2009). Similarly, there were close correlations between the antioxidant parameters and cognitive parameters in the present study. Significant negative correlations were found between the latency and the activities of SOD, CAT, GSH-Px and GR in mouse brain, while the level of MDA was positively correlated with latency in the mouse brain (Table 1). These results suggest that the oxidative damage may play an important role in the cognitive decline of the senescent mice induced by D-gal, and betacyanin action against oxidative stress to the brain may be involved in its amelioration of the impairments of learning and memory.

The radical scavenging and antioxidant activity of betalain compounds has been shown in several chemical and biological models (Butera et al. 2002; Escribano et al. 1998; Gentile et al. 2004; Kanner et al. 2001; Strack et al. 2003; Zakharova and Petrova 1998). The antioxidant potential of betalains was found to be related to structural features (Cai et al. 2003). Kapadia et al. (1996, 2003) showed a significant inhibitory effect of betalains against skin, liver and lung cancer in mice. In other animal feeding trials, purple cactus pear and garambullo proved to be devoid of toxicity and their pigments did not provoke any allergic reactions (Kuramoto et al. 1996; Stintzing and Carle 2004). Reports of the promising physiological and pharmacological effects of betalains on humans have been published only very recently. Betalains are able to increase the resistance of human low-density lipoprotein submitted to a myeloperoxidase/nitrite-induced oxidation acting as lipoperoxyl radical scavengers (Allegra et al. 2005, 2007; Tesoriere et al. 2003). Furthermore, betalains have been shown to protect vascular endothelium cells, which are a direct target of oxidative stress in inflammation, from cytokine-induced redox state alteration (Gentile et al. 2004). Consumption of cactus pear fruit containing betalains positively affected the body's redox balance, decreased oxidative damage to lipids, and improved antioxidant status in healthy humans (Tesoriere et al. 2004, 2005). The present study showed that betacyanin form Porrulaca oieracea had a better ability than VC to reduce oxidative stress induced by D-gal in the way of activation of many antioxidant enzymes in aging mice, in agreement with a previous study in humans (Tesoriere et al. 2004). The oxidative damage may play a key role in the cognitive decline of the senescent mice induced by D-gal, and betacyanins reversed the impairments of learning and memory via alleviating oxidative stress to the brain.

In conclusion, the present findings indicated that administration of D-gal caused memory impairment, a decrease in antioxidant enzyme activities and an increase in the MDA level in mice. Betacyanin at the dose of 50 or 100 mg/kg body wt. significantly reversed the cognitive impairments, and improved the activities of antioxidant enzymes in the mouse brain. The effect of betacyanins was more pronounced than that of VC in ameliorating cognition deficits in mice. Therefore betacyanin may have potential as an antiaging therapy or in the treatment of neurodegenerative diseases.

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* Corresponding author. Tel.: +86 533 2782141; fax: +86533 2782124.

E-mail address: wangcq@sdut.edu.cn (C.-Q, Wang).

Chang-Quan Wang (a), *, Gui-Qin Yang (b)

(a) College of Life Sciences, Shandong University of Technology, Zibo 255049, Shandong, China

(b) Chinese Medicine Hospital of Zhangdian District. Zibo 255035, Shandong, China

[C]2009 Elsevier GmbH. All rights reserved.

doi: 10.1016/j.phymed.2009.09.006
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Author:Wang, Chang-Quan; Yang, Gui-Qin
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:Jun 1, 2010
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