Journal of Chromatography B, 955–956 (2014) 50–57 Contents lists available at ScienceDirect Journal of Chromatography B jou rn al hom epage: www.elsevier.com/locate/chromb A new approach to develop a standardized method for assessment of acetylcholinesterase inhibitory activity of different extracts using HPTLC and image analysis Amina H. Abou-Donia, Fikria A. Darwish, Soad M. Toaima, Eman Shawky ∗ , Sarah S. Takla Department of Pharmacognosy, Faculty of Pharmacy, Alexandria University, Alkhartoom square, Alexandria 21521, Egypt a r t i c l e i n f o Article history: Received 5 October 2013 Received in revised form 5 February 2014 Accepted 7 February 2014 Available online 19 February 2014 Keywords: HPTLC Autobiography Acetylcholinesterase inhibitory activity Image processing software Quantitative. a b s t r a c t A new, validated, sensitive and cheap method for preliminary quantitative evaluation of acetylcholine esterase inhibitory activity is presented. The proposed method combines HPTLC with data analysis by means of image processing software. An in-situ TLC autobiographic method was employed in which regions of the TLC plate which contain acetylcholinesterase inhibitors show up as white spots against the yellow background. Bleaching of the yellow color, caused by substances with acetylcholinesterase inhibitory activity was observed and recorded using a digital camera. ImageJ, JustTLC and Sorbfil, three image processing programs were evaluated for quantitative measurements. For evaluation of the assay efficiency, acetylcholinesterase inhibitory activity of different Amaryllidaceae plant extracts was expressed as Standard Activity Coefficients (SACs), which are relative measures of the activity to the well known acetylcholinesterase inhibitor eserine. We attempted to validate the method according to the ICH guideline. Different statistical data revealed that all image analysis software are able to detect the acetylcholine esterase inhibitory activity at very low concentration levels with the ImageJ program being the best of all three tested software regarding sensitivity, linearity and precision. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Plants of the Amaryllidaceae family are well-known for their ornamental value but also for the alkaloids they produce. Some of these alkaloids have been found to exhibit interesting phar- macological and/or biological properties. An import example of such alkaloids is galanthamine which is the most interesting for its use in the treatment of Alzheimer’s disease as a cholinesterase inhibitor [1]. Alzheimer’s disease, a slowly progressive neurodegenerative ill- ness, principally characterized by memory deficits, has become the fourth leading cause of death in aged population [1]. The number of people with Alzheimer’s disease has never been greater and is set to increase substantially in the decades ahead as the propor- tion of the population aged 65 years or more rises sharply [2,3]. An important approach to treat Alzheimer’s disease (AD) is directed to the inhibition of acetylcholinesterase (ChE), their clinical efficacy is thought to result from prolonging the half-life of acetylcholine [2]. Until now, no drug of choice for the treatment of this disease ∗ Corresponding author. Tel.: +20 1005294669. E-mail address: shawkyeman@yahoo.com (E. Shawky). has been decided. Therefore, the search for new AChE inhibitors is of great interest [3]. Specific properties of ChE are used for screen- ing of their activity. ChE may provide the hydrolytic decomposition of various esters. Consequently, the determination of ChE activity is usually based on the quantification of the decomposition prod- uct or non-reacted substrate during such reaction [4]. The various methods used for ChE screening include photometric methods [5], flourometric methods [6], manometric methods [4], amperometric methods [7,8], potentiometric methods [9], titrimetric methods [4], radioisotopic assays [10,11], microwave irradiation method [12], enzymatic methods [13,14], chemiluminescence methods [15], mass spectrometry >[4], and biosensors [4]. Thin layer chromatography (TLC) is one of the most widespread analytical methods used in the organic chemistry laboratory. It has some important advantages over the other chromatographic tech- niques like HPLC, LC–MS/MS, capillary electrophoresis and infrared spectroscopy [16], such as low cost of instrumentation, evaluation of the whole sample because of the spatial separation, the abil- ity to make simultaneous separations (even 20 samples) and, of course, shortened time required for analysis [17,18]. Over the last two decades, an explosive growth took place in both the diversity of techniques and the range of applications of image processing. Several pharmacological assays have been modified so that they http://dx.doi.org/10.1016/j.jchromb.2014.02.013 1570-0232/© 2014 Elsevier B.V. All rights reserved.