Med Chem Res DOI 10.

1007/s00044-011-9582-8

MEDICINAL CHEMISTRY RESEARCH

ORIGINAL RESEARCH

Acetylcholinesterase inhibitory activity of the total alkaloid from traditional Chinese herbal medicine for treating Alzheimers disease
Zhongduo Yang Dongbo Zhang Jin Ren Mingjun Yang Shuo Li

Received: 18 July 2010 / Accepted: 19 January 2011 Springer Science+Business Media, LLC 2011

Abstract Alzheimers disease (AD) is a progressive degenerative neurologic disorder resulting in impaired memory and behavior. A promising treatment strategy for AD has been the use of acetylcholinesterase (AChE) inhibitors. Several potent AChE inhibitors such as huperzine and galanthamine were originally isolated from plants, indicating that herbal medicines are a good source for the search of potential AChE inhibitors. In our study, to search for potential AChE inhibitors, the total alkaloidal extracts of 31 Chinese herbal medicines were tested for their AChE inhibitory activities by Ellmans method and modied TLC bioautographic assay. The results showed that the alkaloidal extracts of Uncaria rhynchophylla (Rubiaceae), Nelumbo nucifera (Nymphaeaceae), Zanthoxylum nitidum (Rutaceae), Portulaca oleracea (Portulacaceae), and Pinellia ternata (Araceae) exhibited remarkable AChE inhibitory activities at the nal concentration of 100 lg/ml. Their IC50 values were 10.8, 12.2, 17.4, 29.4, and 56.2 lg/ ml, respectively. The rest of the alkaloidal extracts showed no or very weak AChE inhibitory activities at the same concentration. The results of this study indicate that the screening of traditional Chinese herbs for AChE inhibitory activity may provide useful lead compounds in the discovery of new drugs for the treatment of AD.

Keywords Alzheimers disease Acetylcholinesterase Alkaloid Ellmans method TLC bioautography Traditional Chinese herbal medicine

Introduction Alzheimers disease (AD) is a progressive degenerative neurologic disorder resulting in impaired memory and behavior (Jann, 1998; Adams et al., 1986). As the aging population increases, AD is more and more prevalent all over the world. A promising treatment strategy for AD has been the use of acetylcholinesterase (AChE) inhibitors, whose basis is the cholinergic hypothesis (Houghton et al., 2006). Up to date, several potent AChE inhibitors such as huperzine and galanthamine have been originally isolated from plants, indicating that herbal medicines are rich sources in the search for potential AChE inhibitors (Ingkaninan et al., 2003; Lin et al., 2008). Therefore, we have chosen frequently prescribed herbs for the screening of their AChE inhibitory activities. So far, various kinds of AChE inhibitors have been isolated from plants and fungi. Types of these inhibitors are mainly divided into alkaloids, terpenoids, shikimatederived compounds, and miscellaneous. Among these AChE inhibitors, alkaloids are regarded as the strongest, and AChE inhibitors marketed to treat AD almost contain N atoms (Houghton et al., 2006). For this reason, in this article, the total alkaloidal extracts from 31 traditional Chinese herbal medicines were tested for their AChE inhibitory activities by Ellmans method (Ellman et al., 1961; Orhan et al., 2004) and modied TLC bioautographic assay (Marston et al., 2002; Yang et al., 2009).

Z. Yang (&) D. Zhang J. Ren M. Yang School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou 730050, Peoples Republic of China e-mail: yangzhongduo@126.com S. Li Laboratory of Chemical Genomics, School of Chemical Biology and Biotechnology, Shenzhen Graduate School of Peking University, Shenzhen 518055, Peoples Republic of China e-mail: lishuobeijing2008@126.com

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Materials and methods Plant materials All herbal medicines were purchased from Huanghe Herb Market in Lanzhou, which were identied by Associate Professor Lin Yang who majored in plant classication, School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou, China. The voucher specimens are deposited at the School of Life Science and Engineering, Lanzhou University of Technology. Chemicals Acetylcholinesterase (EC3.1.1.7, Sigma product No C2888), Fast Blue B salt, acetylthiocholine iodide (ATCI), 5,50 -dithiobis[2-nitrobenzoic acid] (DTNB) and huperzine A were purchased from Sigma (St. Louis, MO, USA). 1-Naphthyl acetate and 1-naphthol were obtained from Sinopharm Chemical Reagent CO., LTD (Shanghai, China). Silica gel GF254 plates were purchased from Qingdao Haiyang Chemical Co., Ltd (Qingdao, China). Extraction of total alkaloids 100 g of each herb was powdered and extracted with 95% ethanol (700 ml 9 3) under reux for 2 h to obtain alcohol extract, which was evaporated to dryness and then suspended in distilled water. The suspension was adjusted to pH 2 by the addition of HCl (2 M). The acid aqueous solution was ltered after one night. The ltrate was basied to pH 11 with NH4OH, and then extracted with chloroform (150 ml 9 2). The chloroform layers were evaporated to dryness under reduced pressure. The alkaloidal extracts obtained were used in the AChE inhibitory activity assays. Microplate assay for AChE inhibitory activity

For the herbal extracts that were shown to exert a signicant inhibition, the inhibitory concentrations (IC50) were further determined by monitoring the effect of increasing concentrations of these samples in assays on the inhibition values. Huperzine A was used as a positive control. All experiments were done in triplicate. TLC bioautographic assay for AChE inhibitory activity The TLC bioautographic assay for AChE inhibitory activity was modied by our group (Marston et al., 2002; Yang et al., 2009). First, 0.1 ll of 10 mg/ml total alkaloidal extract solution was spotted on a silica gel TLC plate and migrated by using 15:1 chloroform:methanol solvent system (v/v). The plate was dried absolutely with a hair dryer. Then AChE (1 U/ml in buffer pH 7.8) and 1-naphthyl acetate (1.5 mg/ml in 40% ethanol solution) were sprayed onto TLC plate subsequently. After each solution was sprayed, TLC plate was blown quickly with cold wind from a hair dryer until no free liquid was found on it. Second, the plate was incubated at 37C for 20 min in a humid atmosphere. Finally, Fast Blue B salt (0.5 mg/ml in distilled water) was sprayed onto the plate. Huperzine A was used as a positive control. White spots on a purple background showed AChE inhibitory activity. False-positive results due to inhibition of 1-naphthol reaction with Fast Blue B salt were eliminated by the method of Yang et al. (2009). Briey, another TLC plate identical to the one in the TLC bioautographic assay was prepared. The developed TLC plate was sprayed with tris HCl (pH 7.8), 1-naphthol (1.5 mg/ml in 40% ethanol solution), and Fast Blue B salt (0.5 mg/ml in distilled water) in sequence. Appearance of white spots on a purple background indicated false-positive results.

Results and discussion The AChE inhibitory activities of the total alkaloidal extracts were assessed by a slightly modied Ellmans method (Ellman et al., 1961; Orhan et al., 2004). Briey, 140 ll of 0.1 M sodium phosphate buffer (pH 8.0), 20 ll of 1 mg/ml sample solution, and 15 ll of 0.28 U/ml AChE were mixed and pre-incubated at 4C for 20 min. The reaction was started by adding 10 ll of 0.01 M DTNB and 10 ll of 0.075 M ATCI, and the total solution was incubated at 37C for 20 min. The optical density was measured at 405 nm. Enzyme activity was calculated by comparing the rate of reaction for the samples relative to that for the blank. The percentage of inhibitory activity was calculated by subtracting the percentage of enzyme activity from 100%. The total alkaloidal extracts of 31 Chinese herbal medicines were tested for their AChE inhibitory activities by Ellmans method and modied TLC bioautographic assay. Microplate assay The AChE inhibitory activities of the total alkaloidal extracts from 31 Chinese herb medicines were evaluated by Ellmans method and the results are shown in Table 1. It was found that the total alkaloidal extracts from Uncaria rhynchophylla, Nelumbo nucifera, Zanthoxylum nitidum, Portulaca oleracea, and Pinellia ternata showed strong

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Med Chem Res Table 1 The acetylcholinesterase inhibitory activities of the total alkaloidal extracts from traditional Chinese herbal medicines at the nal concentration of 100 lg/ml Botanical name Abrus cantoniensis Aconitum kusnezofi Areca catechu Armeniaca mume var. bungo Broussonetia papyrifera Buddleja ofcinalis Corydalis bungeana Curcuma aromatica Desmodium styracifolium Dictamnus dasycarpus Ephedra sinica Glycyrrhiza uralensis Isatis indigotica Lablab purpureus Leonurus artemisia Lobelia chinensis Lycopodium japonicum Nelumbo nucifera Pinellia ternata Piper nigrum Portulaca oleracea Pulsatilla ambigua Sabia japonica Sophora avescens var. kronei Stemona japonica Tripterygium wilfordii Tussilago farfara Uncaria rhynchophylla Vigna umbellata Viscum coloratum Zanthoxylum nitidum
a b

Voucher number Lut 0757 Lut 0741 Lut 0742 Lut 0750 Lut 0749 Lut 0746 Lut 0755 Lut 0748 Lut 0745 Lut 0743 Lut 0752 Lut 0753 Lut 0754 Lut 0747 Lut 0756 Lut 0740 Lut 0758 Lut 0739 Lut 0760 Lut 0759 Lut 0751 Lut 0761 Lut 0764 Lut 0762 Lut 0735 Lut 0738 Lut 0737 Lut 0744 Lut 0736 Lut 0765 Lut 0763

Family Leguminosae Ranunculaceae Palmae Rosaceae Moraceae Loganiaceae Papaveraceae Zingiberaceae Leguminosae Rutaceae Ephedraceae Leguminosae Brassicaceae Leguminosae Labiatae Campanulaceae Lycopodiaceae Nymphaeaceae Araceae Piperaceae Portulacaceae Ranunculaceae Sabiaceae Leguminosae Stemonaceae Celastraceae Compositae Rubiaceae Leguminosae Loranthaceae Rutaceae

Parts used Whole plant Root Fruit Fruit Fruit Flower Whole plant Root Whole plant Root bark Rhizome Rhizome Leaf Seed Whole plant Whole plant Whole plant Leaf Tuber Fruit Upper part Rhizome Stem Root Root Root Bud Stem Seed Stem Root

Yield (%)a 0.082 0.015 0.058 0.098 1.554 0.083 0.106 0.246 0.028 0.208 0.058 0.030 0.139 0.135 0.135 0.190 0.459 0.317 0.035 0.033 0.211 0.156 1.715 0.660 0.112 2.397 0.082 0.358 0.084 0.479 0.101

AChE inhibition (%)b 32.9 1.3 24.4 2.0 13.4 1.9 29.6 2.3 21.7 1.8 36.4 1.3 11.6 1.1 35.8 2.5 49.6 0.9 18.8 2.2 25.7 1.3 41.4 1.1 50.8 1.5 30.4 2.4 15.0 1.7 18.5 1.4 47.8 2.3 88.6 2.1 78.0 0.9 26.5 1.4 68.2 1.5 36.5 0.9 22.6 1.2 15.2 2.3 53.4 1.6 13.1 2.1 46.4 2.6 88.8 1.6 9.0 2.0 18.7 0.8 82.1 1.5

Expressed as percentage weight of air dried plant material Each value represents the mean SD (n = 3)

AChE inhibitory activities at the nal concentration of 100 lg/ml, while the rest of the alkaloidal extracts showed no or very weak AChE inhibitory activities. For these ve alkaloidal extracts mentioned above, the IC50 values of inhibition were further determined and the results are tabulated in Table 2. Among these ve Chinese herbal medicines, the most potent inhibition appeared to be present in the total alkaloidal extracts from U. rhynchophylla, N. nucifera, and Z. nitidum. Their IC50 values were 10.8, 12.2, and 17.4 lg/ml, respectively. Huperinze A was used as a positive control in this study. It showed the concentration that inhibited 50% of AChE activity (IC50) of 74 nmol/l.

Table 2 IC50 values for acetylcholinesterase inhibition of the total alkaloidal extracts from N. nucifera, P. ternata, P. oleracea, U. rhynchophylla, and Z. nitidum Herbs name Nelumbo nucifera Gaertn Pinellia ternata (Thunb.) Breit Portulaca oleracea L Uncaria rhynchophylla (Miq.) Jacks Zanthoxylum nitidum (Roxb.) DC Huperzine A Values are expressed as mean SD (n = 3) IC50 value (lg/ml) 12.2 0.7 56.2 1.8 29.4 1.2 10.8 1.6 17.4 1.5 74.3 2.8 nmol/L

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TLC assay The AChE inhibitory activities of the alkaloidal extracts from 31 Chinese herbal medicines were also evaluated by TLC bioautographic assay and the positive results are shown in Fig. 1a. White spots on a purple background showed AChE inhibitory activities. It was found that the alkaloidal extracts from U. rhynchophylla, N. nucifera, Ephedra sinica (Ephedraceae), and Z. nitidum showed AChE inhibitory activities, while the rest of the alkaloidal extracts show no activities in TLC assays (results not shown). Huperzine A as a positive control showed white spot on a purple background. On the one hand, the results from TLC bioautographic assay conrmed further the antiacetylcholinesterase activities of the total alkaloidal extracts from U. rhynchophylla, N. nucifera, and Z. nitidum. On the other hand, it also provided us further detailed informations as follows: (1) the number of white spots (except for false-positive spots) on a purple background implied that there might be the same number of AChE inhibitors in plant extracts, such as Z. nitidum. (2) After comparing with the false-positive assay (Fig. 1b), it was seen that the white spots on a purple background were seen only in the TLC bioautographic assay, and not in the falsepositive assay. This meant that the false-positive results caused by inhibition of 1-naphthol reaction with Fast Blue B salt barely existed, and the results of TLC assay were reliable. Contrastive analysis of results Comparing the results from microplate assay with those from the TLC assay, it was found that the extracts from P. oleracea and P. ternata identied as active in the microplate assay but inactive in the TLC test were shown to lose activity. The loss of activity might occur in the TLC

assay during the development of the plate or incubation for 20 min at 37C with/without AChE. Another hypothesis to explain the loss of activity is the interaction of either AChE or extracts with the silica of the TLC plates, which might result in an altered afnity of the enzyme for the extract (Di Giouanni et al., 2008). On the contrary, the alkaloidal extract from E. sinica which showed remarkable activity in TLC assay was very weak in activity in the microplate assay (inhibition rate was 25.7% at 100 lg/ml). This contradictory result can be explained as follows: (1) detection limit of the microplate assay was much higher than that of the TLC bioautographic assay, and the content of inhibitors in E. sinica was very low. (2) White spots on a purple background might be a false-positive result. The further isolation of active components is ongoing to conrm whether there are AChE inhibitors in E. sinica.

Conclusions In this study, we rst reported that the total alkaloidal extracts from U. rhynchophylla, N. nucifera, Z. nitidum, P. oleracea, and P. ternata showed signicant AChE inhibitory activities. The results of this study indicate that the screening of traditional Chinese herbs for AChE inhibitory activity may provide useful lead compounds in the discovery of new drugs for the treatment of AD. The further isolation of active components is ongoing.
Acknowledgments This study is supported by the National Natural Science Foundation of China (No. 20802031) and the Excellent Young Teachers Program of Lanzhou University of Technology (No. Q200904). Conict of interest The authors declare no conict of interest.

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Fig. 1 a Huperzine A (positive control) (1 9 10-4 lg) and 1 lg total alkaloidal extracts from U. rhynchophylla, N. nucifera, E. sinica, and Z. nitidum were applied from left to right in turn to a silica gel G plate, which was eluted by chloroformmethanol (15:1 v/v) and the TLC bioautographic assay was carried out. b Another TLC plate identical to the one in the TLC bioautographic assay was prepared and the false-positive assay was carried out

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