Alzheimer’s disease (AD) is a neurodegenerative disorder, and the most common type of dementia. It is characterized by pronounced loss of neurons, extracellular senile plaque, whose principle component is β-amyloid (Aβ), a 39-43 amino acid peptide derived from the cleavage of the amyloid precursor protein by β and γ-secretase (Ivins et al., 1999). Aβ can directly induce neuronal cell death (Ueda et al., 1997) and cause neurons vulnerable to excitotoxicity (Koh et al., 1990) and oxidative insults (Chen et al., 2007). It is suggested that the mechanisms underlying Aβ neurotoxicity are complex but may involve N-methyl-D-aspartate (NMDA) receptor, a glutamate receptor subtype, modulation induced by glutamate release, sustained elevations of intracellular Ca²⁺ concentration ([Ca²⁺]_i), and oxidative stresses resulting from reactive oxygen species (ROS) generation (Ekinci et al., 2000; Gray and Patel, 1995; Ueda et al., 1997). Aβ has been reported to produce hydrogen peroxide, superoxide, and proinflammatory cytokines in neurons and glial cells (Gitter et al., 1995). Therefore, antioxidants such as α-tocopherol and antiinflammatory agents such as indomethacin reportedly slow the progression of AD (Gasparini et al., 2004; Sano et al., 1997).

Vitis, known as a kind of grapes, has various pharmacological effects including improvement against fatty liver, hypertension, cardiac diseases and arthritis (Wang et al., 2004). Vitis contains large amount of polyphenolic compounds such as resveratrol possessing potent antioxidant properties (Rice-Evans et al., 1995) and anti-inflammatory properties (Wang et al., 2004). Aralia belongs to the families Araliaceae and has long been recognized as therapeutic herbs for antinociceptive, antidiabetic, antioxidant and anti-inflammatory activities in China, Japan and Korea (Bae, 2004). Diterpenes isolated from the root of this plant were known as active constituents for analgesic and anti-inflammatory agents (Han et al., 1985). Recent studies have indicated that extracts from the aerial parts of A. cordata inhibit cyclo-oxygenase (COX)-1, COX-2, and COX-2-dependent prostaglandin E2 (PGE2) generation, as well as hyperalgesia during peripheral inflammation (Dang et al., 2005; Lee et al., 2006; Park et al., 2005). Glycyrrhizae radix, the root of Glycyrrhizae uralensis (Lycophodiaceae), has been widely used in Chinese medicine and food. Current European and Chinese texts list Glycyrrhizae radix as an expectorant in the treatment of bronchitis, catarrh, and coughs, as an anti-inflammatory agent in gastric ulcers, arthritis, and rheumatism, and as an adrenocorticotropic agent in adrenocorticoid insufficiency (Huang, 1999). Moreover, Glycyrrhizae radix has been reported to have antioxidant effects (Tang et al., 2004).

There has been much effort to develop beneficial agents from medicinal plants with free radical scavenging and anti-inflammatory properties to achieve neuroprotection in our laboratory (Jung et al., 2012; Kim et al., 2011). In our previous studies, aerial parts, leaves and stems, of Vitis amurensis and Aralia cordata were demonstrated to show neuroprotective effect against Aβ (25-35)-induced neurotoxicity in in vivo and in vitro (Cho et al., 2009; Jeong et al., 2010). Glycyrrhizae radix also showed neuroprotection in cultured neurons, but not in in vivo (Lee et al., 2012). Therefore, we hypothetized that mixture of three plants might show more effective and synergistic neuroprotective effect with the reduction of possible individual toxicity due to large quantity.

The present study investigated the neuroprotective property of an ethanol extract of mixture of Vitis amurensis, Aralia cordata, and Glycyrrhizae radix, which was named as SSB, against Aβ (25-35)-induced memory deficits in mice and neuronal damage in cultured rat cortical neurons.

Aβ (25-35), which is the core toxic fragment of full length Aβ (1-40), forms a β-sheet structure and induces neuronal cell death, neuritic atrophy, synaptic loss, and memory impairment (Maurice et al., 1996; Pike et al., 1995; Tohda et al., 2004). It has been reported that Aβ (25-35) causes neuronal cell death as shown in the present study, and it was blocked by MK-801, an NMDA receptor antagonist; verapamil, a L-type Ca²⁺ channel blocker; and and N^G-nitro-_L-arginine methyl ester (_LNAME), a nitric oxide synthase inhibitor, in cultured neurons (Cho et al., 2009; Jeong et al., 2010). These results suggest that NMDA glutamate receptor activation, Ca²⁺ influx through L-type voltage dependent Ca²⁺ channel (L-VDCC) and ROS generation are implicated in Aβ- induced neuronal apoptotic death. The present study provides evidence that Aβ (25-35)-induced injury to rat cortical neurons was prevented by SSB, an ethanol extract of a mixture of Vitis amurensis, Aralia cordata, and Glycyrrhizae radix. SSB was able to reduce the [Ca²⁺]_i increase, ROS generation, and apoptotic neuronal death in Aβ (25-35)-induced neurotoxicity. Additionally, SSB prevented memory loss induced by i.c.v. injection of Aβ (25-35) in mice.

Ca²⁺ influx via L-VDCC is considered as a primary insult after Aβ treatment in cultured neurons, because blockade of this channel or Ca²⁺ chelation prevents other consequences (Ueda et al., 1997). In the present study, SSB inhibited a significant increase of [Ca²⁺]_i after the treatment with Aβ (25-35). Many reports have demonstrated the role of ROS formation in Aβ-induced neurotoxicity (Miranda et al., 2000; Morais Cardoso et al., 2002). It has been reported that vitamin-E, an antioxidant, blocked the Aβ-induced generation of ROS, but not Ca²⁺ influx, and reduction of extracellular Ca²⁺ inhibited the Aβ-induced increase in [Ca²⁺]_i as well as generation of ROS, indicating that ROS generation is a consequence of Ca²⁺ accumulation (Ekinci et al., 2000). SSB also decreased the Aβ (25-35)-induced increase of ROS generation. These results indicate that SSB might prevent Aβ (25-35)-induced Ca²⁺ entry through VDCC- and / or NMDA-receptor-coupled channels to inhibit ROS generation and then neuronal death, although the mechanism by which SSB blocks the channels is not clear. Vitis amurensis, Aralia cordata, and Glycyrrhizae radix, the constituents of SSB, have been reported to possess antioxidant principles, resveratrol, oleanolic acid, and isoliquirtigenin (Du and Ko, 2006; Ha et al., 2009a, b; Kumar et al., 2007), respectively, suggesting that inhibition of Aβ (25-35)-induced neuronal death by SSB might be due to their ROS scavenging activity. Further study should be conducted to elucidate the precise mechanism.

Many studies have demonstrated that Aβ is accompanied by multiple events culminating in apoptosis in in vitro neuronal experiments (Ekinci et al., 2000; Yan et al., 1999 ). In the present work, cultured cortical neurons exposed to Aβ (25-35) for 36 h showed increased chromatin condensation, a typical feature of apoptotic cell death, which was reduced by SSB. Activation of caspases after increased [Ca²⁺]_i signaling and ROS generation, or inflammatory responses in Aβ-stimulated neurons have been proposed to play pivotal roles in apoptosis. We have already demonstrated that an increase of caspase-3 activity in Aβ (25-35)-treated cultured cortical neurons is correlated with the increase of [Ca²⁺]_i, ROS generation and neural apoptotic death (Ban et al., 2006; Lee et al., 2005). In the previous studies, Vitis amurensis, Aralia cordata, and Glycyrrhizae radix inhibited caspase activity to reduce Aβ (25-35)-induced neuronal apoptosis (Cho et al., 2009; Jeong et al., 2010; Lee et al., 2012). Therefore, SSB might work in the same way.

I.c.v. administration of Aβ (25-35) into mouse brain induces memory impairment in different behavioral paradigms, including spontaneous alternation, the water maze, and passive avoidance (Maurice et al., 1996; Um et al., 2006). Aβ (25-35) preferentially impairs spatial and non-spatial short-term memory, and these effects remain evident up to 6 months after even a single i.c.v. injection of the peptide (Stepanichev et al., 2003). In the present work, memory impairment in the passive avoidance test was also confirmed in mice 7 days after the i.c.v. injection of Aβ (25-35). Chronic treatment with SSB effectively protected the mice against Aβ (25-35)-induced memory deficit. This result was essentially in agreement with its protective effect on Aβ (25-35)-induced neurotoxicity in vitro. Aβ accumulation associated with cognitive impairment in AD is accompanied by an increase in cholinesterase activity (Atack et al., 1983). Equivalently, the increase of cholinesterase activity by Aβ (25-35) in the brain was inhibited by SSB in the present study.

Many reports have suggested that oxidative stress is responsible for the onset of the cognitive dysfunction as well as the progression of AD (Butterfield et al., 2001; Kontush, 2001). Elevated levels of Aβ induce oxidative stress, increasing the appearance of ROS such as superoxide and NO and subsequently producing ONOO- by a rapid interaction, could mediate the damage seen in AD (Kontush, 2001; Smith et al., 1997). A scavenger of ONOO− protects against Aβ (25-35)-induced memory impairment (Alkam et al., 2007), and anti-oxidants such as α-tocopherol protect against cytotoxicity in vitro as well as against learning and memory deficits induced by Aβ (Sano et al., 1997). In the present study, 15 μM Aβ (25- 35) significantly increased the ROS level in cultured neurons, and this was inhibited by SSB. In addition, the constituents of SSB contain antioxidant components (Du and Ko, 2006; Ha et al., 2009a,b; Kumar et al., 2007). Therefore, it is possible that the favorable effect of SSB on Aβ (25-35)-induced cognitive deficits can be attributed to the inhibition of ROS generation.

In the previous studies (Cho et al., 2009; Jeong et al., 2010; Lee et al., 2012), Vitis amurensis, Aralia cordata, and Glycyrrhizae radix were demonstrated to show neuroprotective effect against Aβ (25-35)-induced neurotoxicity in cultured neurons. Furthermore, Vitis amurensis (Jeong et al., 2010) and Aralia cordata (Cho et al., 2009) but not Glycyrrhizae radix, at the dosage of 50 and 100mg/㎏ revealed protection against Aβ (25-35)-induced memory impairment in mice. Mixture of three plants, SSB, showed a maximal inhibition of Aβ (25-35)-induced memory impairment by a dosage of 100mg/㎏ in the present study. These results indicate that three medicinal plants acted synergistically to produce neuroprotection, since maximal effect of SSB was shown at much lower concentration than the sum of maximal effect concentration of each plant.

In conclusion, it is evident that SSB could provide a marked protection against Aβ (25-35)-induced neuronal cell damage in vitro and Aâ (25-35)-induced memory deficit in vivo. These may explain the inhibitory action of SSB on the progression of AD.