- Email: [email protected]
Ethyl ether fraction of Gastrodia elata Blume protects amyloid β peptide-induced cell death
Journal of Ethnopharmacology 84 (2003) 95 /98 www.elsevier.com/locate/jethpharm
Ethyl ether fraction of Gastrodia elata Blume protects amyloid b peptide-induced cell death Hyeon-Ju Kim a, Kwang-Deog Moon a, Dong-Seok Lee b, Sang-Han Lee b, a
Department of Food Science and Technology, Kyungpook National University, Taegu 702-701, South Korea b Korea Research Institute of Bioscience and Biotechnology (KRIBB), Taejon 305-333, South Korea Accepted 26 September 2002
Abstract Alzheimer’s disease is the most common cause of dementia in the elderly. Recently, it has been reported that Alzheimer’s disease is associated with cell death in neuronal cells including the hippocampus. Amyloid b-peptide stimulates neuronal cell death, but the underlying signaling pathways are poorly understood. In order to develop anti-dementia agents with potential therapeutic value, we examined the effect of the herbal compound Gastrodia elata Blume (GEB) on neuronal cell death induced by amyloid b-peptide in IMR-32 neuroblastoma cells. The fractionation of GEB was carried out in various solvents. The hydroxyl radical scavenging effect of the ethyl ether fraction was more potent than any other fractions. In cells treated with amyloid b-peptide, the neuroprotective effect of the ethyl ether, chloroform, and butanol fractions was 92, 44, and 39%, respectively, compared with control. Taken together, these results suggest that the ethyl ether fraction of GEB contains one or more compounds that dramatically reduce amyloid b-peptide induced neuronal cell death in vitro. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Neuroprotection; Amyloid b-peptide; Cell death; Gastrodia elata ; IMR-32 cells
1. Introduction Alzheimer’s disease (AD) is characterized neuropathologically by senile plaques, cerebrovascular amyloidisis, neurofibrillary tangles, and selective neuronal loss. Recent studies suggest that amyloid b-peptide may contribute to the progressive neuronal loss observed in AD. Amyloid b-peptide is an insoluble peptide(s) with 40 /42 amino acid residues (Masters et al., 1985; Kang et al., 1987). Injection of amyloid b-peptide (1 /40) has been reported to result in stable amyloid b-peptide deposits accompanied by localized neurodegeneration (Kowall et al., 1991). The finding is consistent with the observation that insoluble amyloid b-peptide is prevalent within the AD brain (Wisniewski et al., 1989).
Corresponding author. Tel.: /82-42-860-4667; fax: /82-42-8604594 E-mail address: [email protected] (S.-H. Lee).
Gastrodia elata Blume (GEB) is a traditional herb that has been used in oriental countries for centuries (Huh, 1981). GEB has been used as an anticonvulsant, analgesic and sedative against vertigo, hypertension, general paralysis, and tetanus (Yoon, 1992). Vanillyl alcohol and gastrodin derived from GEB are known to have anticonvulsive actions (Wu et al., 1989). Recently, it was reported that compounds found in GEB inhibited glutamate-induced apoptosis in neuronal cells (Lee et al., 1999). Additionally, after pentylenetetrazole-induced seizure activity, the ether fraction of GEB has been shown to attenuate the decrease in g-aminobutyric acid (GABA) and the increase in glutamate content, as well as to have anticonvulsant effects (Ha et al., 2000; Huh et al., 1995). Here we report that compounds in the ethyl ether fraction of GEB dramatically reduce the amount of neuronal cell death in IMR-32 neuroblastoma cells treated with amyloid-b peptide.
0378-8741/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 2 ) 0 0 2 9 0 - 8
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H.-J. Kim et al. / Journal of Ethnopharmacology 84 (2003) 95 /98
2. Materials and methods
wavelength of 570 nm and a reference wavelength of 690 nm.
2.1. Preparation and fractionation of sample 2.4. Assay of hydroxyl radical scavenging activity Three-year-old rhizoma of GEB were obtained from Gastrodia Farm in Kyoungbuk province, Korea. The washed and chopped fresh GEB was deep-frozen ( / 70 8C) until use. Freeze-dried GEB was powdered with a mixer. GEB was extracted with methanol and the methanol extract was resuspended in 10% methanol. The methanol fraction was re-extracted with diethyl ether and divided into two fractions: the ethyl ether fraction and the water fraction, and evaporated at 30 8C under reduced pressure. The water layer was again extracted with chloroform. The remaining water fraction was extracted with ethyl acetate followed by bbutanol. Five fractions were collected: the ethyl ether, chloroform, ethyl acetate, butanol and water fractions (data not shown). The color of each fraction was light to dark brown. All fractions were dried in a rotary evaporator. Ultimately 1.12 g of the ethyl ether fraction was obtained from 100 g of crude GEB powder (Kim et al., 2001).
The hydroxyl radical scavenging activities of several fractions of GEB were assayed by the 2-deoxyribose oxidation method (Chung et al., 1997). 2-Deoxyribose is oxidized by the hydroxyl radical that is formed by the Fenton reaction and degraded to malondialdehyde (Gutteridge, 1987). A solution of 0.1 ml of 10 mM FeSO4 ×/ 7H2O and 0.1 ml of 10 mM ethylenediaminetctraacetic acid (EDTA) was prepared in a test-tube, followed by addition of 0.2 ml of 10 mM 2-deoxyribose, 0.2 ml of the fraction sample and 1.2 ml of 0.1 M phosphate buffer (pH 7.4) to a total volume of 1.8 ml. Finally, 200 ml of 10 mM H2O2 was added to the fraction sample and PBS mixture. After incubation at 37 8 C for 4 h, 1 ml of 2.8% trichloroacetic acid and 1 ml of 1% 2-thiobarbituric acid (TBA) were added to the reaction mixture. The mixture was boiled at 100 8C for 10 min, cooled on ice, and its absorbance measured with a UV-visible spectrophotometer (UV-1601, Shimadzu Co., Kyoto, Japan) at 520 nm.
2.2. Cell culture and stock solutions The IMR-32 cell line was obtained from the National Cancer Institute (NCI), USA, and cultured in the RPMI Medium with 10% calf serum (HyClone). IMR-32 cells were split at the 96-well plates at a concentration of 1 / 105 cells per ml. After 24 h, cells were treated with amyloid-b-peptide (1 /42) (Bachem, Bubendorf, Switzerland) at a final concentration of 10 mM. Cells were then treated with a sample of each of the five fractions diluted in DMSO to a final concentration of 10 mg/ml, and were incubated for 24 h. The final DMSO concentration in each sample was 0.1%, and this concentration did not affect cell growth or death. Stock solutions were diluted 1:1000 using the culture media. 2.3. MTT assay A methylthiazol tetrazolium (3-[4,5-dimethyIthiazol2-yl]-2,5-diphenyltetra-zolmm bromide; MTT) assay was performed to determine the amount of cell death under each treatment condition as described previously (Tada et al., 1986). Briefly, MTT solutions were prepared at 5 mg/ml in phosphate buffered saline (PBS), filtered and stored in the dark at 4 8C. Twenty microliters of MTT at 5 mg/ml was added to each well containing 100 ml of culture medium. After incubation for an additional 7/8 h at 37 8C, formazan crystals were dissolved by addition of 100 ml of 10% sodium dodecyl sulfate (SDS) in 0.01N HCl. Plates were then incubated overnight, and the optical density (OD) of the wells was determined using an ELISA reader at a test
3. Results and discussion Previous work has shown that reactive oxygen species (ROS) can trigger neuronal cell death (Pappolla et al., 1997; Pike et al., 1993) We assumed that some antioxidants may inhibit neuronal cell death. Therefore, we first determined the hydroxy radical scavenging activity of various fractions of methanol extracted GEB (data not shown). The ethyl ether fraction showed a strong hydroxyl radical scavenging activity (80.0%), while BHA and BHT, which are well-known potent antioxidants, showed radical scavenging activity of 80.0 and 89.0%, respectively. Peptides derived from the human amyloid-b sequence have been shown to induce toxicity in neuronal cell lines and primary cultures of neuronal origin as well as in vivo (Pappolla et al., 1997; Pike et al., 1993; Zhang et al., 1994). Moreover, injections of amyloid-b peptide (1 /42) into the nucleus of basalis magnocellularis or the medial septum in rat brain cause neurodegeneration and reduction of the levels of cholinergic markers (Giordano et al., 1994; Pedersen et al., 1996; Giovannelli et al., 1995). In order to examine the neuroprotective effects of GEB, we determined whether cell death induced by amyloid-b peptide in human IMR-32 neuroblastoma cells was reduced by extracts of GEB. In control experiments, amyloid-p peptide (42 /1) was used as a negative control. This peptide did not induce cell death in IMR-32 cells at 20 mM. However, amyloid-b peptide (1 /42) induced cell damage as evidenced by a reduced
H.-J. Kim et al. / Journal of Ethnopharmacology 84 (2003) 95 /98
Fig. 1. Hydroxyl radical scavenging activity of crude G. elata Blume. The hydroxyl radical scavenging activities were assayed by 2-dcoxyribose oxidation method as described in Section 2. M, MeOH-extract; EE, diethyl ether extract. C, control; EA, ethyl acetate. B, butanol. W, water. BHT, butylated hydroxytoluene. BHA, butylated hydroxyanisole.
OD value in the MTT assay (Fig. 1). The ethyl acetate fraction alone or the water fraction alone slightly increased cell growth compared with untreated controls in the absence of amyloid-b peptide (data not shown). Compounds within these fractions may affect cell proliferation via as yet unknown mechanism(s). On the other hand, the ethyl ether, butanol and chloroform fractions reduced cell growth slightly compared with untreated control cultures. Thus the ethyl acetate and water fractions were not studied further. As shown in Fig. 2, we found that ethyl ether fraction of GEB had the predominant effect (92.0%) in reducing amyloid bpeptide induced cell death in IMR-32 cells compared with other fractions. This result suggests that compounds in the ethyl ether fraction of GEB are neuroprotective against cell death induced by amyloid bpeptide. Although the mechanisms underlying amyloidb peptide-induced-cell death are not well understood, it is hypothesized that a death program might be stimu-
97
lated by compromized cellular functions that could result from intraneuronal accumulation of aggregated amyloid-b peptide (Wisniewski et al., 1989; Wu et al., 1996). In AD brain, neurons associated with b-amyloid deposits are predicted to degenerate via the apoptosis pathway (Wisniewski et al., 1989;Wu et.al.;1996). It is reported that amyloid-b peptide can be directly neurotoxic, or can render neurons vulnerable to metabolic or excitotoxic insults (Harkany et al., 1995; Koh et al., 1990). In this study, we demonstrated that ethyl ether soluble compounds in GEB protected against neuronal cell death in IMR-32 neuroblastoma cells. Melatonin, a pineal hormone with recently demonstrated antioxidant properties, has been demonstrated to reduce neuronal cell death (Mattson et al., 1992). Melatonin may have a physiological role in the aging process as Secretion levels of this hormone are decreased in aging and are more severely reduced in AD. In an effort to determine whether agents from natural sources might be neuroprotective, we demonstrate here that the ethyl ether fraction of GEB exhibited a protective activity against b-amyloid as well as a strong hydroxyl radical scavenging activity. The antioxidant effect may be related to the protection against b-amyloid induced neurotoxicity, but the underlying mechanism is unknown (Liu and Mori, 1992). It was previously reported that p -hydroxy benzaldehyde and 4-hydroxy-3-methoxy benzaldehyde from GEB extracts had anticonvulsant activity (Yong et al., 1999). Moreover, GEB was shown to preserve gaminobutyric acid (GABA) content in brain after neurotoxic insult (Huh et al., 1995). We further analyzed the ethyl ether fraction on a gradient using silica gel column chromatography. HPLC analysis demonstrated that this fraction contains b-hydroxy benzaldehyde as well as other minor components (data not shown). Investigating the neuroprotective effects of p -hydroxybenzaldehyde and other benzaldehyde derivatives in amyloid-b peptide-induced cell death in vitro and in vivo may provide insights into novel therapeutic strategies for Alzheimer’s and other neurodegenerative diseases.
Acknowledgements This work was partially supported by a KRIBB Research Fund (S.H. Lee) and a 21st Century Frontier-Plant Diversity Grant (S.H. Lee).
References Fig. 2. Recovery of neuronal cell death by G. elata Blume in IMR-32 cells. Cell death assay was carried out as described in Section 2. Data are mean values of three determinations and bars represent S.D.
Chung, S.K., Osawa, T., Kawakishi, S., 1997. Hydroxyl radical scavenging effects of species and scavengers from brown mustard
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(Brassica nigra ). Bioscience Biotechnology Biochemistry 61, 118 / 123. Giordano, T., Bao Pan, J., Monteggia, L.M., Holzman, T.F., Snyder, S.W., Krafft, G., Ghanbari, H., Kowall, N.W., 1994. Similarities between beta amyloid peptides 1-40 and 40-1: effects on aggregation, toxicity in vitro, and injection in young and aged rats. Experimental Neurology 125, 175 /182. Giovannelli, L., Casamenti, F., Scali, C., Bartolini, L., Pepeu, G., 1995. Differential effects of amyloid peptides beta-(l-40) and beta(25-35) injections into the rat nucleus basalis. Neuroscience 66, 781 /792. Gutteridge, J.M.C., 1987. Ferrous-salt-promoted damage to deoxyribose and benzoate. Biochemical Journal 243, 709 /714. Ha, J.H., Lee, D.U., Lee, J.T., Kim, J.S., Yong, C.S., Kim, J.E., Ha, J.S., Huh, K., 2000. 4-Hydroxybenzaldehyde from Gastrodia elata Bl. is active in the antioxidation and GABAergic neuromodulation of the rat brain. Journal of Ethnopharmacology 73, 329 /333. Harkany, T., Lengyel, Z., Soos, K., Penke, B., Luiten, P.G., Gulya, K., 1995. Cholinotoxic effects of beta-amyloid (1 /42) peptide on cortical projections of the rat nucleus basalis magnocellularis. Brain Research 695, 71 /75. Huh, J., 1981. Young-In Bon. Dong Eu Bo Gam (Korea Traditional Medicine Book). Hang-Sung Book Press, Seoul. Huh, K., Yi, S.J., Shin, U.S., Park, J.M., 1995. Effect of the ether fraction of Gastrodia elata methanol extract on the pentylenetetrazole-induced seizures. Journal of Applied Pharmacology 3, 199 / 204. Kang, J., Lemaire, H.G., Unterbeck, A., Salbaum, J.M., Masters, C.M., Grzeschik, K.H., Multhaup, G., Beyreuther, K., Muller-Hill, B., 1987. The precursor of Alzheimer’s disease amyloid A4 protein resembles a cell-surface receptor. Nature 325, 733 /736. Kim, H.J., Moon, K.D., Oh, S.Y., Kim, S.P., Lee, S.R., 2001. Ether fraction of methanol extracts of Gastrodia elata , a traditional medicinal herb, protects against kainic acid-induced neuronal damage in the mouse hippocampus. Neuroscience Letters 314, 65 /68. Koh, J.Y., Yang, L.L., Cotman, C.W., 1990. Beta-amyloid protein increases the vulnerability of cultured cortical neurons to excitotoxic damage. Brain Research 533, 315 /320. Kowall, N.W., Beal, M.F., Busciglio, J., Duffy, L.K., Yankner, B.A., 1991. An in vivo model for the neurodegenerative effects of beta amyloid and protection by substance P. Proceedings of National Academy Science USA 88, 7247 /7251. Lee, Y.S., Ha, J.H., Yong, C.S., Lee, D.U., Heh, K., Kang, Y.S., Lee, S.H., Jung, M.W., Kim, J.A., 1999. Inhibitory effects of constituents of Gastrodia elata Bl. On glutamate-induced apoptosis in
IMR-32 human neuroblastoma cells. Archives of Pharmaceutial Research 22 (4), 404 /409. Liu, J., Mori, A., 1992. Antioxidant and free radical scavenging activities of Gastrodia elata Bl. and Uncaria rhynchophylla (Miq.) Jacks. Neuropharmacology 31 (12), 1287 /1298. Masters, C.L., Simms, G., Weinman, N.A., Multhaup, G., McDonald, B.L., Beyieuther, K., 1985. Amyloid plaque core protein in Alzheimer disease and down syndrome. Proceedings of National Academy Science USA 82, 4245 /4249. Mattson, M.P., Cheng, B., Davis, D., Bryant, K., Leieberburg, I., Rydel, R., 1992. b-Amyloid peptides destabilize calcium homeostasis and render human cortical neurons vulnerable to excitotoxicity. Journal of Neuroscience 12, 376 /389. Pappolla, M.A., Sos, M., Omar, R.A., Bick, R.J., Hickson-Bick, D.L., Reiter, R.J., Efthimiopoulos, S., Robakis, N.K., 1997. Melatonin prevents death of neuroblastoma cells exposed to the Alzheimer amyloid peptide. Journal of Neuroscience 17, 1683 /1690. Pedersen, W.A., Kloczewiak, M.A., Blusztain, J.K., 1996. Amyloid beta-protein reduces acetylcholine synthesis in a cell line derived from cholinergic neurons of the basal forebrain. Proceedings of National Academy Science USA 93, 8068 /8071. Pike, C.J., Burdick, D., Walencewicz, A.J., Glabe, C.G., Cotman, C.W., 1993. Neurodegeneration induced by beta-amyloid peptides in vitro: the role of peptide assembly state. Journal of Neuroscience 13, 1676 /1687. Tada, H., Shiho, O., Kuroshima, K., Koyama, M., Tsukamoto, K., 1986. An improved calorimetric assay for interleukin 2. Journal of Immunological Methods 93, 157 /165. Wisniewski, H.M., Iqbal, K., Bancher, C., Miller, D., Curie, J., 1989. In vitro assembly and isolation of neurofilaments and microtubules from mammalian CNS. Neurobiology and Aging 10, 409 /412. Wu, H.Q., Xie, L., Jin, X.N., Ge, Q., 1989. The effect of vanillin on the fully amygdala-kindled seizures in the rat. Yao-Hsueh-Hsueh-Yao 24, 482 /486. Wu, C.R., Hsieh, M.T., Huang, S.C., Pens, W.H., Chang, Y.S., Chen, C.F., 1996. Effects of Gastrodia elata and its active constituents on scopolamine-induced amnesia in rats. Planta Medica 62, 317 /321. Yong, C.S., Quan, Q.Z., Kirn, J.A., Ha, J.H., Lee, D.U., Huh, K., 1999. Effect of sodium chloride on the gelation temperature, gel strength and bioadhesive force of poloxamer gels containing diclofenac sodium. Korean Journal of Pharmaceutical Science 29, 47 /53. Yoon, K.Y., 1992. Clinical Prescriptions of Korean traditional medical books. Myung Bo Publishing, Seoul. Zhang, Z., Drzewiecki, G.J., Horn, J.T., May, P.C., Hyslop, P.A., 1994. Human cortical neuronal (HCN) cell lines: a model for amyloid beta neurotoxicity. Neuroscience Letters 177, 162 /164.
Ethyl ether fraction of Gastrodia elata Blume protects amyloid b peptide-induced cell death Hyeon-Ju Kim a, Kwang-Deog Moon a, Dong-Seok Lee b, Sang-Han Lee b, a
Department of Food Science and Technology, Kyungpook National University, Taegu 702-701, South Korea b Korea Research Institute of Bioscience and Biotechnology (KRIBB), Taejon 305-333, South Korea Accepted 26 September 2002
Abstract Alzheimer’s disease is the most common cause of dementia in the elderly. Recently, it has been reported that Alzheimer’s disease is associated with cell death in neuronal cells including the hippocampus. Amyloid b-peptide stimulates neuronal cell death, but the underlying signaling pathways are poorly understood. In order to develop anti-dementia agents with potential therapeutic value, we examined the effect of the herbal compound Gastrodia elata Blume (GEB) on neuronal cell death induced by amyloid b-peptide in IMR-32 neuroblastoma cells. The fractionation of GEB was carried out in various solvents. The hydroxyl radical scavenging effect of the ethyl ether fraction was more potent than any other fractions. In cells treated with amyloid b-peptide, the neuroprotective effect of the ethyl ether, chloroform, and butanol fractions was 92, 44, and 39%, respectively, compared with control. Taken together, these results suggest that the ethyl ether fraction of GEB contains one or more compounds that dramatically reduce amyloid b-peptide induced neuronal cell death in vitro. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Neuroprotection; Amyloid b-peptide; Cell death; Gastrodia elata ; IMR-32 cells
1. Introduction Alzheimer’s disease (AD) is characterized neuropathologically by senile plaques, cerebrovascular amyloidisis, neurofibrillary tangles, and selective neuronal loss. Recent studies suggest that amyloid b-peptide may contribute to the progressive neuronal loss observed in AD. Amyloid b-peptide is an insoluble peptide(s) with 40 /42 amino acid residues (Masters et al., 1985; Kang et al., 1987). Injection of amyloid b-peptide (1 /40) has been reported to result in stable amyloid b-peptide deposits accompanied by localized neurodegeneration (Kowall et al., 1991). The finding is consistent with the observation that insoluble amyloid b-peptide is prevalent within the AD brain (Wisniewski et al., 1989).
Corresponding author. Tel.: /82-42-860-4667; fax: /82-42-8604594 E-mail address: [email protected] (S.-H. Lee).
Gastrodia elata Blume (GEB) is a traditional herb that has been used in oriental countries for centuries (Huh, 1981). GEB has been used as an anticonvulsant, analgesic and sedative against vertigo, hypertension, general paralysis, and tetanus (Yoon, 1992). Vanillyl alcohol and gastrodin derived from GEB are known to have anticonvulsive actions (Wu et al., 1989). Recently, it was reported that compounds found in GEB inhibited glutamate-induced apoptosis in neuronal cells (Lee et al., 1999). Additionally, after pentylenetetrazole-induced seizure activity, the ether fraction of GEB has been shown to attenuate the decrease in g-aminobutyric acid (GABA) and the increase in glutamate content, as well as to have anticonvulsant effects (Ha et al., 2000; Huh et al., 1995). Here we report that compounds in the ethyl ether fraction of GEB dramatically reduce the amount of neuronal cell death in IMR-32 neuroblastoma cells treated with amyloid-b peptide.
0378-8741/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 2 ) 0 0 2 9 0 - 8
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2. Materials and methods
wavelength of 570 nm and a reference wavelength of 690 nm.
2.1. Preparation and fractionation of sample 2.4. Assay of hydroxyl radical scavenging activity Three-year-old rhizoma of GEB were obtained from Gastrodia Farm in Kyoungbuk province, Korea. The washed and chopped fresh GEB was deep-frozen ( / 70 8C) until use. Freeze-dried GEB was powdered with a mixer. GEB was extracted with methanol and the methanol extract was resuspended in 10% methanol. The methanol fraction was re-extracted with diethyl ether and divided into two fractions: the ethyl ether fraction and the water fraction, and evaporated at 30 8C under reduced pressure. The water layer was again extracted with chloroform. The remaining water fraction was extracted with ethyl acetate followed by bbutanol. Five fractions were collected: the ethyl ether, chloroform, ethyl acetate, butanol and water fractions (data not shown). The color of each fraction was light to dark brown. All fractions were dried in a rotary evaporator. Ultimately 1.12 g of the ethyl ether fraction was obtained from 100 g of crude GEB powder (Kim et al., 2001).
The hydroxyl radical scavenging activities of several fractions of GEB were assayed by the 2-deoxyribose oxidation method (Chung et al., 1997). 2-Deoxyribose is oxidized by the hydroxyl radical that is formed by the Fenton reaction and degraded to malondialdehyde (Gutteridge, 1987). A solution of 0.1 ml of 10 mM FeSO4 ×/ 7H2O and 0.1 ml of 10 mM ethylenediaminetctraacetic acid (EDTA) was prepared in a test-tube, followed by addition of 0.2 ml of 10 mM 2-deoxyribose, 0.2 ml of the fraction sample and 1.2 ml of 0.1 M phosphate buffer (pH 7.4) to a total volume of 1.8 ml. Finally, 200 ml of 10 mM H2O2 was added to the fraction sample and PBS mixture. After incubation at 37 8 C for 4 h, 1 ml of 2.8% trichloroacetic acid and 1 ml of 1% 2-thiobarbituric acid (TBA) were added to the reaction mixture. The mixture was boiled at 100 8C for 10 min, cooled on ice, and its absorbance measured with a UV-visible spectrophotometer (UV-1601, Shimadzu Co., Kyoto, Japan) at 520 nm.
2.2. Cell culture and stock solutions The IMR-32 cell line was obtained from the National Cancer Institute (NCI), USA, and cultured in the RPMI Medium with 10% calf serum (HyClone). IMR-32 cells were split at the 96-well plates at a concentration of 1 / 105 cells per ml. After 24 h, cells were treated with amyloid-b-peptide (1 /42) (Bachem, Bubendorf, Switzerland) at a final concentration of 10 mM. Cells were then treated with a sample of each of the five fractions diluted in DMSO to a final concentration of 10 mg/ml, and were incubated for 24 h. The final DMSO concentration in each sample was 0.1%, and this concentration did not affect cell growth or death. Stock solutions were diluted 1:1000 using the culture media. 2.3. MTT assay A methylthiazol tetrazolium (3-[4,5-dimethyIthiazol2-yl]-2,5-diphenyltetra-zolmm bromide; MTT) assay was performed to determine the amount of cell death under each treatment condition as described previously (Tada et al., 1986). Briefly, MTT solutions were prepared at 5 mg/ml in phosphate buffered saline (PBS), filtered and stored in the dark at 4 8C. Twenty microliters of MTT at 5 mg/ml was added to each well containing 100 ml of culture medium. After incubation for an additional 7/8 h at 37 8C, formazan crystals were dissolved by addition of 100 ml of 10% sodium dodecyl sulfate (SDS) in 0.01N HCl. Plates were then incubated overnight, and the optical density (OD) of the wells was determined using an ELISA reader at a test
3. Results and discussion Previous work has shown that reactive oxygen species (ROS) can trigger neuronal cell death (Pappolla et al., 1997; Pike et al., 1993) We assumed that some antioxidants may inhibit neuronal cell death. Therefore, we first determined the hydroxy radical scavenging activity of various fractions of methanol extracted GEB (data not shown). The ethyl ether fraction showed a strong hydroxyl radical scavenging activity (80.0%), while BHA and BHT, which are well-known potent antioxidants, showed radical scavenging activity of 80.0 and 89.0%, respectively. Peptides derived from the human amyloid-b sequence have been shown to induce toxicity in neuronal cell lines and primary cultures of neuronal origin as well as in vivo (Pappolla et al., 1997; Pike et al., 1993; Zhang et al., 1994). Moreover, injections of amyloid-b peptide (1 /42) into the nucleus of basalis magnocellularis or the medial septum in rat brain cause neurodegeneration and reduction of the levels of cholinergic markers (Giordano et al., 1994; Pedersen et al., 1996; Giovannelli et al., 1995). In order to examine the neuroprotective effects of GEB, we determined whether cell death induced by amyloid-b peptide in human IMR-32 neuroblastoma cells was reduced by extracts of GEB. In control experiments, amyloid-p peptide (42 /1) was used as a negative control. This peptide did not induce cell death in IMR-32 cells at 20 mM. However, amyloid-b peptide (1 /42) induced cell damage as evidenced by a reduced
H.-J. Kim et al. / Journal of Ethnopharmacology 84 (2003) 95 /98
Fig. 1. Hydroxyl radical scavenging activity of crude G. elata Blume. The hydroxyl radical scavenging activities were assayed by 2-dcoxyribose oxidation method as described in Section 2. M, MeOH-extract; EE, diethyl ether extract. C, control; EA, ethyl acetate. B, butanol. W, water. BHT, butylated hydroxytoluene. BHA, butylated hydroxyanisole.
OD value in the MTT assay (Fig. 1). The ethyl acetate fraction alone or the water fraction alone slightly increased cell growth compared with untreated controls in the absence of amyloid-b peptide (data not shown). Compounds within these fractions may affect cell proliferation via as yet unknown mechanism(s). On the other hand, the ethyl ether, butanol and chloroform fractions reduced cell growth slightly compared with untreated control cultures. Thus the ethyl acetate and water fractions were not studied further. As shown in Fig. 2, we found that ethyl ether fraction of GEB had the predominant effect (92.0%) in reducing amyloid bpeptide induced cell death in IMR-32 cells compared with other fractions. This result suggests that compounds in the ethyl ether fraction of GEB are neuroprotective against cell death induced by amyloid bpeptide. Although the mechanisms underlying amyloidb peptide-induced-cell death are not well understood, it is hypothesized that a death program might be stimu-
97
lated by compromized cellular functions that could result from intraneuronal accumulation of aggregated amyloid-b peptide (Wisniewski et al., 1989; Wu et al., 1996). In AD brain, neurons associated with b-amyloid deposits are predicted to degenerate via the apoptosis pathway (Wisniewski et al., 1989;Wu et.al.;1996). It is reported that amyloid-b peptide can be directly neurotoxic, or can render neurons vulnerable to metabolic or excitotoxic insults (Harkany et al., 1995; Koh et al., 1990). In this study, we demonstrated that ethyl ether soluble compounds in GEB protected against neuronal cell death in IMR-32 neuroblastoma cells. Melatonin, a pineal hormone with recently demonstrated antioxidant properties, has been demonstrated to reduce neuronal cell death (Mattson et al., 1992). Melatonin may have a physiological role in the aging process as Secretion levels of this hormone are decreased in aging and are more severely reduced in AD. In an effort to determine whether agents from natural sources might be neuroprotective, we demonstrate here that the ethyl ether fraction of GEB exhibited a protective activity against b-amyloid as well as a strong hydroxyl radical scavenging activity. The antioxidant effect may be related to the protection against b-amyloid induced neurotoxicity, but the underlying mechanism is unknown (Liu and Mori, 1992). It was previously reported that p -hydroxy benzaldehyde and 4-hydroxy-3-methoxy benzaldehyde from GEB extracts had anticonvulsant activity (Yong et al., 1999). Moreover, GEB was shown to preserve gaminobutyric acid (GABA) content in brain after neurotoxic insult (Huh et al., 1995). We further analyzed the ethyl ether fraction on a gradient using silica gel column chromatography. HPLC analysis demonstrated that this fraction contains b-hydroxy benzaldehyde as well as other minor components (data not shown). Investigating the neuroprotective effects of p -hydroxybenzaldehyde and other benzaldehyde derivatives in amyloid-b peptide-induced cell death in vitro and in vivo may provide insights into novel therapeutic strategies for Alzheimer’s and other neurodegenerative diseases.
Acknowledgements This work was partially supported by a KRIBB Research Fund (S.H. Lee) and a 21st Century Frontier-Plant Diversity Grant (S.H. Lee).
References Fig. 2. Recovery of neuronal cell death by G. elata Blume in IMR-32 cells. Cell death assay was carried out as described in Section 2. Data are mean values of three determinations and bars represent S.D.
Chung, S.K., Osawa, T., Kawakishi, S., 1997. Hydroxyl radical scavenging effects of species and scavengers from brown mustard
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