Phytochemical compounds from grains are abundant in the human diet, particularly in fruit, vegetables and legumes, which have been consistently associated with a decreased risk of nutritional disease. They constitute one of the most abundant groups of natural metabolites and are now recognized for their important contribution to both health (and diet) effects in humans and animals. Epidemiological studies have consistently shown that regular consumption of fruits, vegetables, whole grains, and other plant foods is associated with reduced risk of developing chronic diseases, such as cancer and cardiovascular disease [1]. Legumes, especially Glycine maxs are widely consumed in the world, and are a staple food in Korea as well as being a major source for protein, energy, vitamins, and minerals. The phytochemicals of Glycine maxs, including phenolic compounds, phytic acid, triterpene glycosides, and phytosterols, may be responsible for their anticancer activity [2]. It was reported that Glycine maxs reduced azoxymethaneinduced colon cancer in a rat model [3]. Legume tannins are 15–30 times more effective at quenching peroxyl radicals than simple phenolics, thus they are potential biological antioxidants [4]. Although biological activity has been discovered from Glycine max, it has not been fully characterized.

Alzheimer’s disease (AD) is an age-related neurodegenerative disease that affects approximately 24 million people worldwide [5]. Neuronal death is an important feature of both acute and chronic neurodegenerative diseases. AD is associated with the accumulation of l-glutamate (Glu) deposits in senile plaques and neurofibrillary tangle lesions in specific areas of brain. Glutamate, a major excitatory amino acid neurotransmitter in the central nervous system (CNS), involved in fast synaptic transmission, neuronal plasticity, outgrowth and survival, memory, learning and behavior. Glutamate also plays an important role in microglial neurotoxicity in AD. Activated microglia produce large amounts of glutamate, which induces excitotoxicity via N-methyl-d-aspartate (NMDA) receptor signaling [6]. Activated microglia release large amounts of glutamate through upregulation of glutaminase expression and induce excitoneurotoxicity through NMDA receptor signaling [7]. Apart from the physiological role of glutamate, excessive activation of its receptors can also evoke neuronal dysfunction and even damage or death. Glutamate-mediated neurotoxicity appears to play a crucial role in several neuropathological disorders, particularly in Alzheimer’s disease, Parkinson’s disease, epilepsy and ischemic stroke [8]. Thus, neuroprotection against glutamate-induced toxicity has been a therapeutic strategy for preventing and/or treating both acute and chronic forms of neurodegeneration [9].

In the context of our natural drugs discovery program dealing with the development of new potential neuroprotection agents, we have examined the isolation of two compounds, 3-O-[α-l-rhamnopyranosyl(1–2)-β-d-glucopyranosyl(1–2)-β-d-glucuronopyranosyl]olean-12-en-3β,22β,24-triol (1) and 3-O-[β-d-glucopyranosyl(1–2)-β-d-galactopyranosyl(1–2)-β-d-glucuronopyranosyl]olean-12-en-3β,22β,24-triol (2) as leads for novel glutamate-induced toxicity inhibitors.

It was found that the methanol extract of Glycine max might inhibit glutamate-induced toxicity in primary cultured rat cortical cells. In order to clarify the neuroprotective components of Glycine max, as a part of continued study of the neuroprotection effects of Glycine max, isolation was performed to seek active fractions and components. After solvent fractionation, the inhibiting effects of various fractions on neuroprotective activity were compared and the ethyl acetate fraction of Glycine max was found to inhibit this activity in a dose-dependent manner in the assay system using glutamate-induced toxicity in primary cultured rat cortical cells. To clarify the active compounds of Glycine max, the effects of the major compounds from Glycine max on neuroprotective activity were examined. Isolation was further performed to yield a single compound. Bioactive triterpene glycosides derivatives were isolated from the ethyl acetate fraction by repeated chromatography and recrystallization. The ethyl acetate fraction (35 g) was further purified by silica gel chromatography (230–400 mesh, 500 × 75 mm) and four subfractions (BB-1–4) whose main active subfraction (BB-3; 5.8 g) was purified by VLC and HPLC, and compound fractions 1 (7.2 mg) and 2 (4.7 mg) were obtained from hexane/ethyl acetate (20:3) elution. Then, the subfraction CH₂Cl₂/MeOH (10:1) was further purified on preparative HPLC using the 250 × 22 mm i.d., 5 μm, Agilent C₁₈ preparative HPLC column, which was eluted isocratically with 20% acetonitrile in water at a flow rate of 8.0 mL/min. Compounds 1 (3.2 mg) and 2 (2.1 mg) were obtained at retention times of 5.3 and 14.7min, respectively. Thin layer chromatography and preparative high-performance liquid chromatography methods of the MeOH extract of harvested Glycine max led to the isolation of two compounds. Compound 1 was identified as 3-O-[α-l-rhamnopyranosyl(1–2)-β-d-glucopyranosyl(1–2)-β-d-glucuronopyranosyl]olean-12-en-3β,22β,24-triol. Compound 2 was identified as 3-O-[β-d-glucopyranosyl(1–2)-β-d-galactopyranosyl(1–2)-β-d-glucuronopyranosyl]olean-12-en-3β, 22β,24-triol (Figure 1). The compounds have previously been reported as antiproliferative and antioxidative activity from the Glycine max seed coats by Dong et al. [10]. In this study, we found that methanol extract of Glycine max might inhibit glutamate-induced toxicity in primary cultured rat cortical cells. In order to clarify the neuroprotective components of Glycine max, as part of a continued study of neuroprotection effects of Glycine max, isolation was performed to seek active fractions and components. After solvent fractionation, we compared the inhibiting effects of various fractions on neurotoxicity activity. The BB-3 fraction of Glycine max was found to inhibit the activity in a dose-dependent manner in glutamate-induced toxicity assay using primary cultured rat cortical cells (Table 1). To clarify the active substances of Glycine max, we examined the effects of the major isolated compounds from BB-3 fraction on neuroprotective activity. Isolation was further performed to yield single compounds.

Two known compounds were isolated from BB-3 fraction by repeated VLC and HPLC. To investigate and compare the neuroprotective activities of these compounds isolated from the methanol extracts of Glycine max, the activities of compounds 1 and 2 were evaluated in glutamate-injured primary cultured rat cortical cells at concentrations ranging from 0.1 to 10 μM (Table 2). It is notable that the neuroprotective activities of compound 2 was comparable to those of MK-801, APV and CNOX, and, only compound 2 displayed an activity which significantly attenuated glutamate-induced toxicity at concentrations ranging from 0.1 to 10 μM and exhibited cell viabilities of 50–70%.

Glutamate-induced neurotoxicity has been implicated in the neuronal cell death of neurological disorders such as ischemia, trauma, seizures, and Alzheimer’s disease [6]. During our search for potential natural products against glutamate-induced neurotoxicity, we have discovered that the ethyl acetate sub-fraction (BB-3 fraction) of Glycine max showed significant protective activity. The neuroprotective activities of the triterpene glycosides glycosides were evaluated by the MTT assay measuring the viability in primary cultured rat cortical cells after glutamate insult. Among these two triterpene glycosides glycosides, compound 2 significantly attenuated neuronal cell death induced by glutamate in cultures. It was found to exhibit the most potent neuroprotective activity at a concentration of 10 μM. Our result supported that compound 2 was isolated from the Glycine max possess neuroprotective activities at low concentration and in a dose-dependent manner in primary cultured rat cortical cells. At present, the cellular and molecular mechanisms that underlie the action of compound 2 are not fully understood. However, future research data will show whether compound 2 acts on glutamate receptors, especially NMDA receptors by the following proposed pathway: (i) a protection of cortical cultures, (ii) selective protection in NMDA-induced neurotoxicity; (iii) blocking Ca²⁺ influx in glutamate insult; (iv) a reduction in NO and cellular peroxide production; (v) a reduction in GSH depletion and membrane lipid peroxidation. On the basis of our present study, the protective effect of compound 2 from Glycine max may provide some ideas for preventing and/or treating the neurodegenerative diseases.