Celastrus paniculatus seed water soluble extracts protect against glutamate toxicity in neuronal cultures from rat forebrain☆
Introduction
Indian medicinal plants have been used in the treatment of cognitive dysfunction, insomnia, and epilepsy (Chopra et al., 1958). One popular plant is Celastrus paniculatus (CP) Willd (Celastraceae), which is known for its ability to improve memory (Nadkarni, 1976). Pharmacological studies suggest that the oil obtained from the seeds of CP possess sedative and anticonvulsant properties (Gaitonde et al., 1957). Analgesic and anti-inflammatory effects of a CP seed methanolic extract has been reported in mice and rats by Ahmad et al. (1994). CP seed oil has a long history and has been reported to improve memory processes in rats (Karanth et al., 1981), be beneficial to psychiatric patients, (Hakim, 1964), and increased the I.Q. of mentally retarded children (Nalini et al., 1986). More recently, rats treated with CP seed oil for 15 days exhibited a significant decrease in the levels of norepinephrine, dopamine, serotonin and their respective metabolites in both brain and urine (Nalini et al., 1995). Chronic treatment with CP seed oil reversed scopolamine-induced deficits in navigational memory performance of rats (Gattu et al., 1997). A methanolic extract of CP seed oil exhibited free radical scavenging effects (Russo et al., 2001).
In marked contrast to organic extracts, aqueous extracts of CP have not been well characterized. Only recently was there a report (Kumar and Gupta, 2002) demonstrating that rats treated with aqueous extracts of CP seeds showed improvement in learning and memory tasks. They concluded that aqueous extracts of CP seed reduced oxidative stress in the brain by increasing endogenous antioxidant enzymes. However, a biochemical rational for these observations is still required. The reduction in neurotoxicity by antioxidants and a variety of other compounds has been attributed to several biochemical processes, including modulation of glutamate pathways. For example, ischemia or oxygen/energy deprivation trigger glutamate release leading to the primary pathological events associated with the initiation of neuronal injury. When uncontrolled in animals, glutamate causes long-lasting maintenance of seizure activity and subsequent neuropathology due to over stimulation of glutamate receptors. Various potential therapeutic strategies are currently used to prevent glutamate-induced neurotoxicity. These include the use of calcium channel blockers, N-methyl-d-aspartate (NMDA) or α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor blockers, intracellular calcium chelators, excitatory amino acid antagonists, GABA-inverse agonists, and the reduction of reactive oxygen species (ROS) (Zivin and Choi, 1991, Choi, 1994, Hall and McCall, 1994).
To further define the molecular mechanism of action and protection afforded by the aqueous CP-WSE extracts, we studied the effects of CP-WSEs on glutamate-induced toxicity in primary neuronal cell cultures and the effects of WSE on NMDA activated whole-cell currents in forebrain neurons using patch–clamp technique (Hamill et al., 1981). We show that these water soluble plant extracts exert their neuroprotective effects by inhibiting NMDA receptor channels. Thus, the CP-WSE have been identified as new neuroprotective drugs from plant extracts, which can be readily obtained, easily prepared, and exhibit low side-effects.
Section snippets
Chemicals
All chemicals were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).
Plant material
The Celastrus paniculatus seeds were purchased from the local market of Mumbai, India, and were identified by the Agharkar Research Institute, Pune, India.
CP seed water soluble extracts (WSE)
To generate WF (the fraction soluble at room temperature, 25 ± 5 °C), 25 g of finely powdered CP seeds were vortexed with 400 ml of deionized water for four h. The resulting fluid was centrifuged at 10 000 × g for 30 min. WF was obtained from the clear supernatant by vacuum
Data analysis
Glutamate-induced toxicity results are presented as mean ± S.D. and evaluated for statistical significance with an analysis of variance (one-way ANOVA) test. Dunnet’s test was used for comparisons of all treatments with the control. Statistical differences between treatments with glutamate alone versus glutamate and WSE pre-treatment were determined using the F-test.
For electrophysiological studies, an NMDA concentration-response curve was fitted using Origin (Microcal Software Inc.,
Effects of CP on the neuronal cells and on glutamate-induced toxicity
Fig. 1, Fig. 2, Fig. 3 show the effects of a 90-min pre-treatment of neuronal cells with the CP-WSE fractions AF, WF, and HF, respectively. For the control cells receiving only the CP-WSE fractions, while AF and WF did not show any adverse effects on cell viability at concentrations ranging from 0.01 to 10 μg/ml, at 10 μg/ml HF, nearly 51% of the neuronal cells died. However, pre-treatment of the primary neuronal cells with a 10-fold lower concentration of HF resulted in no neurotoxicity (Fig. 3,
Discussion
Glutamate-induced neurotoxicity is one of the contributing mechanisms of neuronal cell death in neurodegenerative diseases such as Alzheimer’s, Parkinson’s, Huntington’s, stroke, and epilepsy (Choi, 1994). Elevation of intra-cellular calcium ions mediates a delayed neuronal degeneration that is caused by activation of glutamate receptors, and eventually energy depletion, cell exhaustion, and death (Coyle and Puttfarken, 1993). Glutamate channel blockers can exert neuroprotective effects by
Acknowledgements
This work was supported by the National Research Council (PFG). This research was supported in part by the Office of Naval Research work unit no. 601153N.4114.114.AO109 (AR).
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Research was conducted in compliance with the Animal Welfare Act, and other Federal statutes and regulations relating to animals and experiments involving animals, NIH publication 85-23. The views of the authors do not purport to reflect the position of the Department of the Army, the Department of the Navy, or the Department of Defense, (para 4-3), AR 360-5.