Alzheimer s disease (AD) is the most common cause of. Alzheimer s Disease Drug Discovery from Herbs: Neuroprotectivity from -Amyloid (1-42) Insult

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1 THE JOURNAL OF ALTERNATIVE AND COMPLEMENTARY MEDICINE Volume 13, Number 3, 2007, pp Mary Ann Liebert, Inc. DOI: /acm Alzheimer s Disease Drug Discovery from Herbs: Neuroprotectivity from -Amyloid (1-42) Insult DARRICK S.H.L. KIM, Ph.D., 1,2 JIN-YUNG KIM, Ph.D., 1 3 and YE SUN HAN, Ph.D. 4 ABSTRACT Objective: To comparatively evaluate selected herbs for their ability to protect neuronal cells from direct A(1-42) insult. Design: Twenty-seven (27) herbs were selected, extracted with aqueous methanol (90%) and chloroform, and the extracts were evaluated for their ability to protect PC12 rat pheochromocytoma and primary neuronal cells from A(1-42) insult using both 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide reduction assay and lactate dehydrogenase efflux assay. Results: Curcuma aromatia (ul-keum) and Zingiber officinale (ginger) extracts effectively protected cells from A(1-42) insult, followed by Ginkgo biloba (ginkgo), Polygonatum sp. (King Solomon s seal), Cinnamum cassia (Chinese cinnamon), Rheum coreanum (Korean rhubarb), Gastrodia elata (gastrodia), and Scutellaria baicalensis (skullcap). Several extracts showed cytotoxicity at high concentration ( 150 g/ml), whereas other extracts did not at all protect cells from A(1-42) insult. Conclusion: Selective herbs may be potentially important resources to discover drug candidates against the onset of Alzheimer s disease. INTRODUCTION Alzheimer s disease (AD) is the most common cause of progressive cognitive dysfunction that affects approximately 4 million Americans, causing more than 100,000 deaths each year with a total annual cost approaching $100 billion in the United States. 1 One of the principal pathologic characteristics of AD is an extracellular deposition of amyloid ( A) as senile plaques. A(1-42) has been shown to exert direct toxic effects on neurons and inhibit the neurite outgrowth in vitro in a dose-dependent manner. 2 Although A does not appear to cause cell death at low concentration (1 5 g/ml), it was found to cause profound cell damage, such that cell viability is undermined. 3,4 Because A insult to neuronal cells has been hypothesized as one of the major causes of AD pathology, 5,6 neuronal cell protection from A insult has been speculated to be an important therapeutic approach to control the onset of AD. There are large numbers of herbs readily available on the market to treat various ailments. From these herbs, several potential drug candidates to treat AD have been discovered: for example, curcuminoids from Curcuma longa (tumeric), and 3,4 shogaols from Zingiber officinale (ginger), 7,8 huperzine-a from herbal moss Huperzia serrata (toothed clubmoss), 9 and galanthamine from Ungernia sp. (daffodil bulbs). 10 However, these plant extracts represent only a small fraction of herbs used in the world. Thus, it was of interest to identify herbs that could be used as resources for discovering potential drug candidates to treat AD. Because A-induced neuronal cell death is one of the suspected etiologies 1 CurXceL Corporation, The Business and Technology Center, West Lafayette, IN. 2 Program for Collaborative Research in the Pharmaceutical Sciences and Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, IL. 3 Natural Science Research Institute, Seoul Woman s University, Seoul, Korea. 4 Department of Technology Fusion, Konkuk University, Seoul, Korea. 333

2 334 of the onset of AD, it was reasonable to postulate that select herbs may meet the criteria of potential drug candidate leads to treat AD by protecting neuronal cells from A(1-42) insult. To our knowledge, there has been no published comparative evaluation of herbal extracts based on their efficacy toward protecting neuronal cells from A(1-42) insult. Herein, we report results from a pilot study on selected herbs as part of our ongoing effort to discover potential drug candidates to treat AD. Methanolic and chloroform extracts of the following herbs were evaluated for their neuroprotectivity against A(1-42) insult: Aconitum carmichaelis Debs. (Ranunculaceae), Angelica gigas Nakai (Umbelliferae) (Korean angelica), Aralia continentalis Kitagawa (Araliaceae) (Manchurian spikenard), Astragalus membranaceus (Fisch.) Bunge. (Leguminoceae) (astragalus), Atractylodes japonica (Koidz) Kitagawa (Compositae), Bupleurum falcatum L. (Umbelliferae) (bupleurum), Cinnamomum cassia (Nees) Blume (Lauraceae) (Chinese cinnamon), Curcuma aromatia Salisbury (Zingiberaceae) (ul-keum), Dioscorea batatas Decaisne (Dioscoreaceae) (yam), Eleutherococcus senticosus Maxim (Araliaceae), (Siberian ginseng), Gastrodia elata Blume (Orchidaceae) (gastrodia), Ginkgo biloba Linn. (Ginkgoaceae) (ginkgo), Glycyrrhiza glabra Linn. (Leguminoceae) (licorice), Inula heleium Linn. (Compositae) (elecampane inula), Ligusticum officinale Kitagawa (Umbelliferae) (common lavage), Macrocapium officinale Sieb. Et Zucc (Cornaeae), Ophiopogon japonicus Ker-Gagler (Liliaceae) (dwarf lilyturf), Ostericum koreanum Kitagawa (Umbellifereae), Panax ginseng Nees (Araliaceae) (Korean ginseng), Platycodon grandiflorum A. DC. (Campanulaceae) (balloon flower), Polygonatum spp. (Liliaceae) (King Solomon s seal), Poria cocos (Schw.) Wolf. (Polyporaceae) (cocos), Rehmannia glutinosa Liboschtz (Scrophulariaceae) (Chinese foxglove), Rheum coreanum Nakai (Polygonaceae) (Korean rhubarb), Schisandra chinensis Baillon (Schisandraceae) (schisandra), Scutellaria baicalensis Georgi. (Labiatae) (skullcap), and Z. officinale Roscoe (Zingiberaceae). PC12 rat pheochromocytoma and primary neuronal cells were used to evaluate the neuroprotectivity of the herbal extracts by means of 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide (MTT) reduction assay 3,11 and lactate dehydrogenase (LDH) efflux assay. 11,12 MATERIALS AND METHODS Materials PC12 cells were obtained from the American Type Culture Collection (Rockville, MD). Cells were routinely cultured on a poly-l-lysine-coated Corning tissue culture plate (Invitrogen-Gibco, Carlsbad, CA). Culture media and supplements were obtained from Life Technologies (Grand Island, NY). A(1-42) was purchased from Bachem California (Torrance, CA). MTT and other chemicals were purchased from Sigma/Aldrich (St. Louis, MO) and Invitrogen-Gibco (Carlsbad, CA). The herbs were purchased from Korean Kyung- Dong herb market in Seoul, Korea and the voucher samples were labeled and deposited in PCRPS (Program for Collaborative Research in Pharmaceutical Science) storage, Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago. Extraction of herbs Dried herb (100 g) was ground or chopped (average size 0.5 cm), and extracted with 90% aqueous methanol (150 ml 3) by percolation. The methanol extracts were combined, filtered, and the solvent was removed under vacuum at 35 C. The residue was dissolved in 10 ml of methanol, water (100 ml) was added, and partitioned with chloroform (pretreated with NaHCO 3 overnight, 50 ml 4). The chloroform layers were combined and washed twice with H 2 O (100 ml 2), dried (MgSO 4 ), filtered, and evaporated under vacuum to afford a residue. Primary neuronal cell culture KIM ET AL. Dissociated primary neuronal cell cultures were established from embryonic 18-day-old Sprague-Dawley rat fetuses using a previously described method. 13 Pups were delivered by caesarean section while the dam was anesthetized with ether. Hippocampal tissue from embryonic day 18 Spargue-Dawley rat pups was dissected in ice-cold Ca 2 / Mg 2 -free Hank s balanced salt solution (HBSS) containing 100 g/ml each of streptomycin and penicillin. After gentle trituration of the tissue in a conical tube with a sterile pipette in cold buffer, it was centrifuged for 2 minutes at 2000 rpm. The resulting supernatant was discarded and replaced with 3 ml of 0.25% trypsin and 0.05% deoxyribonuclease (DNAased, Typell). Enzymatic digestion was continued for 10 minutes at 37 C with gentle agitation of the tissue. Proteolysis was terminated by adding 3 ml of HBSS containing 0.5% soybean trypsin inhibitor. Remaining tissue fragments were dissociated by repeated trituration using a sterile pipette. The resulting cell suspension was centrifuged at 2000 rpm for 5 minutes. The neurons were seeded into poly-l-lysine-coated 96-well tissue culture plates at a final density of cells/cm 2. The cultures were maintained in Eagle s Minimum Essential Medium supplemented with 10% fetal bovine serum, 10% horse serum, 35 mol/l D-glucose and 2 mol/l L-glutamine, and 100 g/ml each of penicillin and streptomycin in a 5% CO 2 and 95% humidified atmosphere at 37 C. After 3 days of incubation, the cultures were treated with medium containing 35 g/ml uridine and 15 g/ml fluorodeoxyuridine, as well as N 2 supplement to replace the serum and inhibit glial cell proliferation. The medium was changed every 2 3 days. A(1-42) effect on PC12 and primary neuronal cells using an MTT reduction assay PC12 cells were maintained in high glucose Dulbecco s Modified Eagle medium, 10% horse serum, 5% fetal calf

3 HERBS FOR ALZHEIMER S 335 serum, and 1% penicillin/streptomycin. 3,4 Exponentially growing cells (2000 cells/ml) were placed (90 L) in 96- well poly-l-lysine-coated tissue culture plates and incubated for 24 hours prior to the assay. For primary neuronal cells, cells were plated ( cells/ml) in 96-well poly-l-lysine-coated tissue culture plates and incubated for 5 days prior to the assay. A(1-42) stock solution was prepared in dimethyl sulfoxide (DMSO) at 10 mg/ml concentration and aged for 3 days before partition. Cells were incubated with various concentrations of A(1-42) (100, 25, 6.25, 1.56, 0.39, 0.098, and g/ml) for 24 hours at humidified atmosphere containing 5% CO 2 at 37 C. Cell viability was determined using an MTT reduction assay. 3,4 Briefly, after incubation of cells in MTT solution (25 L per well, 1 mg/ml stock solution) for 1 hour at 37 C, 100 L Lysing buffer (50% aqueous dimethylformamide (DMF) and 20% sodium dodecyl sulfate (SDS) at ph 4.7) were added and incubated overnight at 37 C. The optical densities of the resulting solutions were colorimetrically determined at 570 nm using a microplate reader. Dose response curves were obtained and results expressed as EC 50 values in g/ml. for 24 hours. The final DMSO concentration was less than 1%. MTT was dissolved in phosphate-buffered saline (PBS) (ph 7.4) at a concentration of 2.5 mg/ml (6 mmol/l) and stored at 4 C. 1-Methoxyphenazine methosulfate (MPMS) was dissolved in PBS at a concentration of 100 mmol/l and stored at 4 C. LDH substrate mixture (10 ml) was prepared immediately prior to the experiment by dissolving 25 mg of lithium L-lactate and 25 mg of nicotinamide adenine dinucleotide (NAD) in 9.0 ml of 0.2 mol/l Tris-HCl buffer (ph 8.2) containing 0.1% (v/v) Triton X-100, 1.0 ml of MTT stock solution, and 10 L of MPMS stock solution. The culture supernatant (50 L) was transferred to 96-well culture plates and mixed with 50 L of the LDH substrate mixture for 30 minutes. The reaction was stopped by adding 100 L of lysing buffer and was incubated overnight. The optical density of the resulting solutions was colorimetrically determined at 570 nm using a microplate reader. Cell viability assay using MTT The herbal extracts ability to protect PC12 cells and primary cortical neurons from A(1-42) insult was investigated according to the published procedure. 3,4 Cells were plated and prepared as described above and incubated with A(1-42) ([1.0 g/ml for PC12 cells and 3.0 g/ml for primary neuronal cells], prepared from a stock solution [1.0 mg/ml in DMSO]) and herbal extracts at various concentrations (150, 100, 50, 25, 10, and 5 g/ml) for 24 hours. The final DMSO concentration for all experiments was less than 1%. The herbal extracts ability to protect cells from A(1-42) insult was determined by measuring the cell s potential to reduce MTT with respect to the treatment of cells with 1% DMSO only and the treatment of cells with 2.0 g/ml A(1-42) and with or without the presence of herbal extract. MTT reduction assay was performed as described above. Dose response cruves were obtained and results were expressed as EC 50 values in g/ml concentration. Comparative analyses of cell viability using LDH release assay A comparative evaluation of herbal extracts ability to protect PC12 and primary neuronal cells from A(1-42) insult was performed using an LDH release assay. Measurement of LDH released to the extracellular media by nonviable and stressed cells has been a quantitative method for assessing cell viability in cell culture. 11,12 LDH activity in the cell medium was measured using a method described elsewhere. 12 Cells were prepared in 96-well tissue culture plates as described above. Cells were incubated with A(1-42) (5.0 g/ml, prepared from a stock solution [1.0 mg/ml in DMSO]) and the herbal extracts (20 g/ml) FIG. 1. A(1-42) effect on PC12 and primary neuronal cells viability was determined. Cells were incubated with various concentration of A(1-42) for 24 hours and cell viability was determined using an 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide reduction assay. The datapoints were obtained in triplets on three different dates. From the slope EC 50 values were obtained. Data were subject to a one-way analysis of variance. p values of 0.05 were considered to be significant.

4 336 RESULTS A(1-42) effect on neuronal cells Figure 1 shows the effect of A(1-42) on PC12 and primary neuronal cells viability as determined using MTT reduction assay. From the slope of the graphs, EC 50 values were obtained. A(1-42) attenuated the viability of PC12 cells at EC g/ml and the viability of primary neuronal cells at EC g/ml. Efficacy of herbal extracts on neuronal cells protectivity from A insult using MTT reduction assay MTT is a monotetrazolium salt that can be intracellularly reduced to purple-colored formazan and is widely used for measuring cell viability. 3,11 Table 1 shows that chloroform extracts of C. aromatia and Z. officinale extracts effectively KIM ET AL. protected cells from A(1-42) insult at EC g/ml under MTT reduction assay condition, followed by G. biloba extract (EC g/ml). Methanol extracts of C. aromatia and Z. officinale required higher concentration to achieve EC g/ml; and G. biloba, EC g/ml. Chloroform extracts of C. cassia, Polygonatum spp., R. coreanum, I. helenium, G. elata, and S. baicalensis extracts only showed a marginal cell protection from A(1-42) insult (EC g/ml). The methanol extracts of Aconitum carmichaelis, A. continentalis, G. elata, I. helenium, O. koreanum, R. coreanum, and S. bacalensis showed cytotoxicity at 150 g/ml concentration. Chloroform extracts of A. continentalis, B. falcatum, E. senticosus, G. elata, O. japonicus, and R. coreanum showed cell protection against A(1-42) insult, whereas their corresponding methanol extracts did not. The appearance of neuroprotectivity in the chloroform extracts from cytotoxic or nonactive methanol extracts of A. continentalis, B. falcatum, E. senticosus, G. elata, I. helenium, O. japon- TABLE 1. EFFICACY OF CELL PROTECTION FROM A (1-42) INSULT BY HERBAL EXTRACTS EC 50 ( g/ml) Chloroform extract Methanol extract Plant name Common name PC12 Primary neuron PC12 Primary neuron Aconitum carmichaelis Carmichael s aconite CT CT CT Angelica gigas Korean angelica CT Aralia continentalis Manchurian spikenard CT CT Astragalus membranaceus Astragalus CT CT CT Atractylodes japonica Atractylodes CT CT CT Bupleurum falcatum Bupleurum CT CT Cinnamomum cassia Chinese cinnamon CT CT Curcuma aromatia ul-keum CT CT Dioscorea batatas Yam CT CT CT Eleutherococcus senticosus Siberian ginseng CT CT Gastrodia elata Gastrodia CT CT CT CT Ginkgo biloba Ginkgo CT CT Glycyrrhiza glabra Licorice CT CT CT Inula helenium Elecampane inula CT CT 150 CT 150 CT Ligusticum officiale Common lavage Macrocapium officinale 150 CT CT Ophiopogon japonicus Dwarf lilyturf CT CT Ostericum koreanum Kang-hwal CT CT CT Panax ginseng Korean ginseng CT CT CT Platycodon grandiflorum Balloon flower CT CT CT Polygonatum sp. King Solomon s seal CT CT Poria cocos Cocos CT CT CT Rehmannia glutinosa Chinese foxglove CT CT CT Rheum coreanum Korean rhubarb CT CT CT CT Schisandra chinensis Schisandra CT CT CT Scutellaria baicalensis Skullcap CT CT CT CT Zingiber officinale Ginger CT CT EC 50 represents the sample concentration that is required to achieve 50% cell viability, a midpoint between the positive control values and the negative control values. represents no activity given to those extracts that gave 20% cell viability at 150 g/ml conc. CT : cytotoxic at 150 g/ml concentration. The tests were performed in triplets on three different dates. Data were subject to one-way analysis of variance (OriginPro 7.5, Origin- Lab, Northampton, MA). p values of 0.05 were considered significant.

5 HERBS FOR ALZHEIMER S 337 icus, R. coreanum, and S. baicalensis was noteworthy. In general, chloroform extracts showed higher protectivity than methanol extracts did. Comparative analysis of selected herbs ability to protect neuronal cells from A insult using MTT reduction assay Figure 2 shows a comparative analysis of chloroform extracts of selected herb extracts ability to protect PC12 and primary neuronal cells from A(1-42) insult at various concentrations as determined by an MTT reduction assay. The data represent the relative efficacy of the chloroform extracts of selected herbs ability to protect cells from A(1-42) insult with respect to each other. Effective cell protection from A(1-42) insult was clearly demonstrated from C. aromatia and Z. officinale extracts while other herbal extracts cell protectivity from A(1-42) insult lagged behind. Comparative analysis of herbal extracts ability to protect neuronal cells from A insult using the LDH efflux assay In addition to the MTT reduction assay, A(1-42)-induced cell viability reduction can be determined by observing the amount of LDH release in cells whose viability is undermined. 11 Good correlation between the results from the MTT reduction assay and the LDH efflux assay has been reported. 11,12 Figure 3 shows results from a comparative analysis on selected herbal extracts ability to protect PC12 cells and primary neuronal cells in the presence of A(1-42) at 5.0 g/ml concentration using an LDH efflux assay. The results showed that A(1-42) and treated cells (negative control) release the most amount of LDH as expected, whereas 1% DMSO-only treated cells and C. aromatia and Z. officinale extracts-treated cells showed near background level of LDH release. Gastrodia elata, Polygonatum sp. and S. baicalensis showed only a marginal inhibition of LDH release, which is consistent with the result of MTT reduction assay. Herbal extracts that did not show positive results on MTT reduction assay did not inhibit the LDH release. DISCUSSION FIG. 2. Efficacy of herbal extracts on neuronal cells protectivity from A insult using an 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction assay. Chloroform extracts of seven selected herbs were comparatively evaluated for PC12 and primary neuronal cell protectivity from A(1-42) insult. Cells were incubated with various concentrations of herbal extracts in the presence of A(1-42) (1.0 g/ml for PC12 cells and 3.0 g/ml for primary neuronal cells) for 24 hours, and cell viability was determined using an MTT reduction assay. The datapoints were obtained in triplets on three different dates. Data were subject to a one-way analysis of variance. p values of 0.05 were considered to be significant. A. gigas, Angelica gigas (Korean angelica); C. aromatia, Curcuma aromatia (ul-keum); E. senticosus, Eleutherococcus senticosus (Siberian ginseng); R. glutinosa, Rehmannia glutinosa (Chinese foxglove); S. baicalensis, Scotellaria baicalensis (skullcap); S. Chinensis, Schisandra chinensis (schisandra); Z. officinale, Zingiber officinale (ginger). In cancer research, there has been a long history of medicinal plant drug discovery that yielded several clinically effective anticancer agents such as vinca alkaloids, taxanes, and campthothecins. 14 Therefore, it was reasoned that such an approach may be effectively applied in the drug discovery against AD. One of the important criteria for selecting the plant resources was reasoned to be, contrary to anticancer research, their nontoxicity toward cells because protecting neuronal cells from the A(1-42) insult is one of the important keys to discovering AD treatment. One of the reasonable medicinal plant resources for our study was herbs with a long history of safe use, and the herbs were chosen based on their ready availability in the market. Sparked by our effort, we hope scientists will come together to investigate all available herbs to one day discover the treatment against AD. A(1-42) was shown to exert direct toxic effects on neurons to inhibit the neurite outgrowth in vitro in a dose-dependent manner. 2 Although A does not appear to cause cell death at low concentration (1 5 g/ml), it was found to

6 338 FIG. 3. Comparative analysis of selected herbs ability to protect neuronal cells from A insult using a lactate dehydrogenase (LDH) efflux assay. Chloroform extracts of 10 selected herbs were comparatively evaluated for PC12 and primary neuronal cell protectivity from A(1-42) insult. control represents cells without A(1-42) and any herbal extracts, whereas control represents cells with A(1-42) alone. Cells were incubated with A(1-42) (5.0 g/ml) and herbal extracts (20 g/ml) for 24 hours. Cell viability was determined using an LDH release assay. The data points were obtained in triplets on three different dates. Data were subject to a one-way analysis of variance. p values of 0.05 were considered to be significant. A. gigas, Angelica gigas (Korean angelica); C. aromatia, Curcuma aromatia (ul-keum); E. senticosus, Eleutherococcus senticosus (Siberian ginseng); G. elata, Gastrodia elata (gastrodia); P. ginseng, Panax ginseng (Korean gingseng); Polygonum sp. (King Solomon s seal); R. glutinosa, Rehmannia glutinosa (Chinese foxglove); S. chinensis, Schisandra chinensis (schisandra); S. baicalensis, Scutellaria baicalensis (skullcap); Z. officinale, Zingiber officinale (ginger). cause profound cell damage, such that cell viability is undermined. 3,4 Because A insult to neuronal cells has been hypothesized as one of the major causes of AD pathology, 5,6 neuronal cell protection from A insult is a potentially important therapeutic approach to control the onset of AD. As a result of strong interest in finding herbal resources with A-toxicity-modulating property as a chemotherapeutic KIM ET AL. measure to discover potential drug candidates, we comparatively evaluated 27 selected herbs using PC12 cells and primary cortical neurons against A(1-42) insult by means of an MTT reduction assay and LDH release assay. Recently, there have been an increasing number of drug discovery efforts on medicinal herbs to find potential drug candidates to treat neurologic dysfunctions such as AD. A. gigas, Saurus chinensis, and S. chinensis in an 8:1:1 ratio extract was shown to improve scopolamine-induced memory impairment in mice. 15 A. gigas roots have been used traditionally in Korean herbal medicine for the treatment of anemia and as a sedative, anodyne, and tonic agent. Schizandra chinensis fruit has been used as a tonic in traditional medicine and possesses hepatoprotective activity. 16 S. chinensis has been used in traditional medicine for edema, beriberi, jaundice, turbid urine, and gonorrhea in Korea. 17 The ethyl ether fraction of Gastrodia elata, a traditional herb that has been in use as an anticonvulant, and in treating general paralysis and tetanus, 18 was shown to protect A-induced cell death. 19 Shogaols from Z. officinale have both been identified to protect neuronal cells from A insult. 7,8 Baicalein and baicalin from S. baicalensis demonstrated protection of PC12 cells from A insult. 20 Most notably, curcuminods from Curcuma sp. have been recognized for their neuroprotectivity against A(1-42) insult. 3,4 In particular, curcumin, a potential drug candidate to treat AD, has demonstrated inhibition of the formation of A oligomers and fibrils, binds A plaques, and reduces A load in vivo. 21 Thus, it is reasonable to anticipate that rigorous analyses of herbal extracts with neuroprotectivity against A insult may yield potential drug candidates to treat AD. Aqueous extracts from Verbena officinalis, 22 Bambusae concretio Salicea, 23 and Rhizoma acori graminei 24 have shown protection of neuronal cells from A insult. One of the criteria for constituents affecting neuronal cells is their ability to cross the blood brain barrier. Lipophilicity or hydrophobicity is one of the important properties that assist drug molecules to cross the blood brain barrier to affect neuronal cells. 25,26 Consequently, hydrophilic, water-soluble compounds from an aqueous layer could be considered unattractive drug candidates without certain structural modifications or formulations to assist the molecules in crossing the blood brain barrier to influence neuronal cell survival from A(1-42) insult. Interestingly, our results suggest increased neuroprotectivity from chloroform extracts over methanol extracts. Thus, analyses of chloroform extracts may be more important in determining potential drug candidate resources for protection of neuronal cells from A(1-42) insult than those of methanol extracts. The results from investigation using the aqueous portion from chloroform partitioning did not reflect the neuroprotectivity observed from those of methanol or chloroform extracts. Some of the extracts from the aqueous portions were cytotoxic whereas others were nonconclusive and/or noteworthy (unpublished results).

7 HERBS FOR ALZHEIMER S 339 Both methanol and chloroform extracts of G. biloba protected neuronal cells from A(1-42) insult (EC g/ml). The currently marketed ginkgo product, EGb761, contains ginkgo-terpenoids, in particular ginkgolides, along with bioflavonoids such as quercetin as the active constituents. 27,28 However, it is noteworthy that ginkgolides are highly water-soluble polar compounds, and quercetin concentration is nonsignificant in nonprocessed methanol and chloroform extracts. In addition, quercetin is a commonly found flavonoid from various plants without any remarkable report on their neuroprotectivity from A insult. Therefore, our results clearly demonstrate that constituents other than the water-soluble ginkgolides may be responsible for the neuroprotectivity of ginkgo extracts. Efficacy of EGb761 as a preventive measure against the onset of AD has been controversial. 29 As a result, we are currently investigating the source of the neuroprotectivity of G. biloba. CONCLUSION It is reasonable to argue that the window of observation for neuroprotectivity of herbal extracts against A(1-42) insult may have been too restricted to yield unbiased results. Because herbal extracts contain a large number of both active and nonactive compounds, the presence of potent drug candidates in small quantity could have been unaccounted for. Our investigation was designed to provide a reference point in selecting potentially important herbal resources for natural productbased drug discovery effort against AD. Because this investigation only addresses neuroprotectivity from A(1-42) insult, other potentially important drug candidates whose mechanism of neuroprotection are different from those of this study are not detected. Nevertheless, our results provide comparative and comprehensive analyses on extracts of 27 herbs ability to protect cells from A(1-42) insult, which is one of the major recognized causes for the onset of AD. ACKNOWLEDGMENTS The authors gratefully acknowledge the bioassay facility of PCRPS in the College of Pharmacy, UIC, for generous usage of the equipment. The authors are grateful for a partial support by the National Research Laboratory Program (M J ) and the Real Time Molecular Imaging Project of the Korean Ministry of Science and Technology. REFERENCES 1. Garber K. An end to Alzheimer s? Technol Rev 2001;104: Postuma RB, He W, Numan J, et al. Substrate-bound betaamyloid peptides inhibit cell adhesion and neurite outgrowth in primary neuronal cultures. J Neurochem 2000;74: Kim DSHL, Park SY, Kim JY. Curcuminoids from Circuma longa L. (Zingiberaceae) that protect PC12 rat pheochromocytoma and normal human umbilical vein endothelial cells from A(1-42) insult. Neurosci Lett 2001;303: Park SY, Kim DSHL. Discovery of natural products from Curcuma longa that protect cells from -amyloid insult: A drug discovery effort against Alzheimer s disease. J Nat Prod 2002;65: Findeis MA. Approaches to discovery and characterization of inhibitors of amyloid -peptide polymerization. Biochim Biophys Acta 2000;1502: Kawahara M, Kuroda Y. Molecular mechanism of neurodegeneration induced by Alzheimer s -amyloid protein: Channel formation and disruption of calcium homeostasis. Brain Res Bull 2000;53: Kim DSHL, Kim JY. Side-chain length is important in protecting neuronal cells from -amyloid insult. Bioorg Med Chem Lett 2004;14: Kim DSHL, Kim DS, Oppel MN. Shogaols from Zingiber officinalis L. (Zingiberaceae) protect IMR32 human neuroblastoma and normal human umbilical vein endothelial cells from -amyloid(25-35) insult. Planta Med 2002;68: Wang R, Yan H, Tang XC. Progress in studies of huperzine A, a natural cholinesterase inhibitor from Chinese herbal medicine. Acta Pharmacol Sin 2006;27: Kihara T, Sawada H, Nakamizo T, et al. Galantamine modulates nicotinic receptor and blocks A -enhanced glutamate toxicity. Biochem Biophys Res Commun 2004;325: Shearman MS, Ragan CI, Iversen LL. Inhibition of PC12 cell redox activity is a specific, early indicator of the mechanism of -amyloid-mediated cell death. Proc Natl Acad Sci USA 1994;91: Abe K, Matsuki N. Measurement of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction activity and lactate dehydrogenase release using MTT. Neurosci Res 2000;38: Ren RF, Flanders KC. Transforming growth factors- protect primary rat hippocampal neuronal cultures from degeneration induced by -amyloid peptide. Brain Res 1996;732: Balumas MJ, Kinghorn AD. Drug discovery from medicinal plants. Life Sci 2005;78: Kang SY, Lee KY, Koo KA, et al. ESP-102, a standardized combined extract of Angelica gigas, Saururus chinensis and Schizandra chinensis, significantly improved scopolamineinduced memory impairment in mice. Life Sci 2005;76: Kubo S, Ohkura Y, Mizoguchi Y, et al. Effect of Gomisin A (TJN-101) on liver regneration. Planta Med 1992;58: Jung BS, Shin MK. Encyclopedia of Illustrated Korean Natural Drugs. Seoul, Korea: Young Lim Sa, 1998: Yoon KY. Clinical Prescription of Korean Traditional Medical Books. Seoul, Korea: Myung-Bo Publishing Co., Kim HJ, Moon KD, Lee DS, Lee SH. Ethyl ether fraction of Gastrodia elata Blume protects amyloid peptide-induced cell death. J Ethnopharm 2003;84:95 98.

8 Heo HJ, Kim DO, Choi SJ, et al. Potent inhibitory effect of flavonoids in Scutellaria baicalensis on amyloid protein-induced neurotoxicity. J Agric Food Chem 2004;52: Yang F, Lim GP, Begum AN, et al. Curcumin inhibits formation of amyloid oligomers and fibrils, binds plaques, and reduces amyloid in vivo. J Biol Chem 2005;280: Lai SW, Yu MS, Yuen WH, Chang RC. Novel neuroprotective effects of the aqueous extracts from Verbena officinalis Linn. Neuropharmacology 2006;50: Jeong JC, Yoon CH, Lee WH, et al. Effects of Bambusae concretio Salicea (Chunchukhwang) on amyloid beta-induced cell toxicity and antioxidantive enzymes in cultured rat neuronal astrocytes. J Ethnopharmacol 2005;98: Irie Y, Keung WM. Rhizoma acori graminei and its active principles protect PC-1 cells from the toxic effect of amyloidbeta peptide. Brain Res 2003;963: Begley DJ. Delivery of therapeutic agents to the central nervous system: The problems and the possibilities. Pharmacol Therapeut 2004;104: Pardridge WM. The blood-brain barrier: Bottleneck in brain drug development. NeuroRx 2005;2: Bastianetto S, Ramassamy C, Dore S, et al. The Ginkgo biloba extract (EGb 761) protects hippocampal neurons against KIM ET AL. cell death induced by -amyloid. Eur J Neurosci 2000;12: Bastianetto S, Zheng WH, Quirion R. The Ginkgo biloba extract (EGb 761) protects and rescues hippocampal cells against nitric oxide-induced toxicity: Involvement of its flavonoid constituents and protein kinase C. J Neurochem 2000;74: Schneider LS, DeKosky ST, Farlow MR, et al. A randomized, double-blind, placebo-controlled trial of two doses of Ginkgo biloba extract in dementia of the Alzheimer s type. Curr Alzheimer Res 2005;2: Address reprint requests to: Darrick S.H.L. Kim, Ph.D. CurXceL Corporation The Business and Technology Center 1291 Cumberland Avenue West Lafayette, IN curxcel@gmail.com