Survey of acetylcholinesterase inhibitory activity in essential oil derived from aromatic plants

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1 , ISSN Vol. 4, No. 1, pp. 1-, March 214 RESEARCH ARTICLE Survey of acetylcholinesterase inhibitory activity in essential oil derived from aromatic plants Watoo PHROMPITTAYARAT* 1, Tapanee HONGRATANAWORAKIT 2, Sarin TADTONG 2, Vipaporn SAREEDENCHAI 2, Kornkanok INGKANINAN 3 1 Faculty of Public Health, Naresuan University, 6 Phitsanulok, Thailand 2 Faculty of Pharmacy, Srinakharinwirot University, 2612 Nakhonayok, Thailand 3 Department of Pharmacognosy and Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Naresuan University, 6 Phitsanulok, Thailand *Corresponding author: Tel , Fax Article History: Received th February 214, Revised 21 st March 214, Accepted 24 th March 214. Abstract: Essential oils have the strong potency to inhibit acetylcholinesterase (AChE) in t heir lipophilicity and small molecular size constituents. Cholinesterase inhibitory activity was determined against AChE using Ellman s colorimetric method of 29 essential oils from aromatic plants. Results revealed of essential oils from Eucalyptus, Cajuput, Sweet marjoram, Camphor tree and Rosemarry showed such activity respectively. The compositions of five essential oils were also analyzed by GC-MS. We also demonstrated that the AChE inhibitory activity of the essential oil could be attributed to 1,8-cineole. Keywords: Acetylcholinesterase inhibitory activity; essential oil; Ellman s colorimetric method. Introduction Alzheimer s disease (AD) is a progressive degenerative neurologic disorder resulting in impaired memory and behavior which caused by a deficit of cholinergic functions in brain. The most effective treatment have been postulated that the deterioration of memory in patients suffering from this disease. Although the cause of AD remains obscure, two main strategies, cholinergic and non-cholinergic, are existed. One of strategy as the cholinergic hypothesis, acetylcholine is recognized as the neurotransmitter involved in the regulation of cognitive function (Enz et al. 1993). For treating the disease is to enhance the acetylcholine level in the brain using acetylcholinesterase (AChE) inhibitors. AChE inhibitors have been studied for reducing the action on the progression of AD. Currently, Galantamine and Physostigmine are the prototypes of the AChE inhibitors from nature (Hostesttman et al. 26). Essential oils have been discovered for AChE inhibitory activity and found strong potency of the inhibitory activity because of the small molecule and the lipophilicity. The essential oils from Salvia species have volatile substances which are likely to cross the blood brain barrier and exert their effect (Perry et al., 2; Perry et al. 1996). In this study, we aimed to screen the essential oils for the AChE inhibitory activity and then investigate their constituents with shown this activity using Ellman s colorimetric method in 96-well microplate (Ellman et al. 1961; Ingkaninan et al. 2). Materials and methods Plant material Cajuput leaves were collected from the south of Thailand. The voucher specimen is kept at Faculty of Pharmacy, Srinakharinwiroj University, Nakhonnayok and Faculty of Pharmacy, Mahidol University, Bangkok (Tadtong1). The plant was identified by Prof.Wongsatit Chuakul, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand. *Corresponding author: ( ) watoop <@> yahoo.com 214 Copyright by the Authors, licensee Open Access Science Research Publisher. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3. Unported (CC BY-NC- ND 3.) License (

2 Essential oils The 29 essential oils were purchased from Thai-China Flavour and Fragrances Industry Co., Ltd. and Perfumesworld, (Bangkok, Tha i- land) They were from Cajuput ( Melaleuca cajuputi Powell) leaves, Eucalyptus (Eucalyptus globulus Labell.) branches and leaves, Sweet majoram ( Majorana hortensis Moench) top, Camphor (Cinnamomum camphora (L.) J.Presl) leaves, Rose marry ( Rosmarinus officinalis L.) top, Lime (Citrus aurantiifolia (Christm.) Swingle) fruit, Lemon grass ( Cymbopogon flexuosus (Nees ex Steud.) Will. Watson) grass, Lemon (Citrus limonum Risso) peel, Lavender (Lavandula angustifolia Mill) top, Thyme (Thymus vulgaris L.) top, Pine ( Pinus palustris Mill) wood, Clove ( Eugenia caryophyllus (Spreng.) Bullock & S.G. Harrison) flower, Spearmint ( Mentha spicata L.) top, Bergamot (Citrus bergamia Risso & Poit.) peel and leaves, Jasmine ( Jasminum sambac (L.) Aiton) flower, Petigan ( Citrus aurantium L.) peel, Patchouli (Pogostemon cablin Benth.) leaves, Ylang Ylang ( Cananga odorata (Lam.) Hook.f. & Thomson) flower, Vetier grass ( Vetiveria zizanioides Nash) root, Peppermint ( Mentha piperita L.) top, Tea Tree oil ( Melaleuca alternifolia Cheel) twig and leaves, Basil (Ocimum basilicum L.) flowering top, Clary sage ( Salvia sclarea L.) flowering top, Cedar wood ( Cedrus atlantica G.Manetti) wood, Geranium ( Pelargonium graveolens L Her) top, Palmarose (Cymbopogon martini (Roxb.) Wats.) grass, Grapefruit (Citrus paradisi Macfad) peel, Ginger (Zingiber officinale Rosco) rhizome, Citronella (Cymbopogon winterianus Jowitt) grass. Extraction Cajuput leaves were dried in the hot air oven at C and blended to coarsely powder. The dried material was extract using the Clevenger apparatus for volatile oil. Chemicals Acetylthiocholine iodide (ATCI), AChE, bovine serum albumin (BSA), 1,8 -cineole and, -dithiobis [2-nitrobenzoic acid (DTNB) were obtained from Sigma (St. Louis, MO.). 2 Fifty millimolar Tris HCl ph 8. was used as a buffer throughout the experiment unless otherwise stated. AChE used in the assay was from electric eel (type VI-S lyophilized powder, 48 U/mg solid, 3 U/mg protein). The lyophilized enzyme was prepared in the buffer to obtain 113 U/ml stock solution. The enzyme stock solution was kept at -8 C.The further enzymedilution was dissolved in.1% BSA in buffer. DTNB was dissolved in the buffer containing.1m NaCl and.2m MgCl2. ATCI was dissolved in deionized water. Microplate assay The assay for measuring AChE activity was modified from the assay described by Ellman et al. (1961) and Ingkaninan et al. (2). Briefly, 12 µl of 3 m MDTNB, 2 µl of 1 mm ATCI, and µl of buffer, 2 µl of sample dissolved in buffer containing not more than 1% methanol were added to the wells followed by 2 µl of.28 U/ml AChE. The microplate was then read at 4 nm every s for 2 min by a CERES UV 9C microplate reader (Bio -Tek Instrument, USA). The velocities of the reactions were measured. Enzyme activity was calculated as a percentage of the velocities compared to that of the assay using buffer without any inhibitor. Inhibitory activity was calculated from 1 subtracted by the percentage of enzyme activity. Every experiment was done in triplicate. Results and discussion Twenty nine essential oils were tested for AChE inhibitory activity using Ellman colorimetric method in 96-well microplate. The results are shown in Table 1. At concentration.12 mg/ml, the essential oil from M. cajuputi, E. globules, O. majoram, C. camphora and R. officinalis showed high AChE inhibitory activity (with percent inhibition of 68.68±4.82, 68.49±1.36, 6.6±, 6.63±7.11 and 1.9±1.81 respectively). At the same concentration, the rest of essential oils were obtained the AChE activity below. Five candidate essential oils (M. cajuputi, E. globules, O. majoram, C. camphora and R. officinalis) were identified for their constituents

3 using GC-MS (analyzed by Central Lab in Chulalongkorn University) in Table 2. The mass Table 1: The inhibitory activity on AChE of essential oils (n=3). 3 spectroscopic data (Figure 1) of the active fractions were compared with the chemical library. Essential oils Scientific name % inhibition±sd 1 Cajuput Melaleuca cajuputi Powell 68.68± Eucalyptus Eucalyptus globulus Labell ± Sweet marjoram Majorana hortensis Moench 6.6± Camphor Cinnamomum camphora (L.) J.Presl 6.63±7.11 Rosemarry Rosmarinus officinalis L. 1.9± Lime Citrus aurantiifolia (Christm.) Swingle 46.86± Lemon grass Cymbopogon flexuosus(nees ex Steud.) Will. Watson 41.76± Lemon Citrus limonum Risso 41.1±4.8 9 Lavender Lavandula angustifolia Mill 38.83±.86 1 Thyme Thymus vulgaris L. 3.88± Pine Pinus palustris Mill 33.39± Clove Eugenia caryophyllus (Spreng.) Bullock & S.G.Harrison 31.94± Spearmint Mentha spicata L. 31.9± Bergamot Citrus bergamia Risso & Poit ± Jasmine Jasminum sambac (L.) Aiton 28.24± Petigran Citrus aurantium L. 28.1± Patchouli Pogostemon cablin Benth. 2.2± Ylang Ylang Cananga odorata (Lam.) Hook.f. & Thomson 24.42± Vetier grass Vetiveria zizanioides Nash 24.16±.94 2 Peppermint Mentha piperita L ±.2 21 Tea tree oil Melaleuca alternifolia Cheel 22.9± Sweet basil Ocimum basilicum L ± Clary sage Salvia sclarea L. 2.7± Cedarwood Cedrus atlantica G.Manetti 14.4± Geranium Pelargonium graveolens L Her 14.7± Palmarose Cymbopogon martini (Roxb.) Wats. 13.4± Grapefruit Citrus paradisi Macfad 13.31±.7 28 ginger Zingiber officinale Rosco 9.2±.2 29 Citronella Cymbopogon winterianus Jowitt 8.14±.6 After the constituents in five essential oils were identified, 1,8-cineole was the greatest active detected constituent. This compound was operated for AChE inhibitory activity at concentration 1 mg/ml. It showed high AChE inhibitory activity and IC were 81.8±.99% and.1±.1µg/ml respectively. Three ( M. cajuputi, E. globules, O. majoram) from five essential oils with high content of 1,8-cineole over % were performed for IC. The IC were.63±.8,.21±.2 and.24±.26µg/ml consequently. The current study showed AChE inhibitory activity efficacy of essential oil of M. cajuputi, E. globules and O. majoram with high content of 1,8-cineole were similar to the previous studies of AChE inhibitory activity of the essential oils with 1,8-cineole (Miyazawa et al., 1998; Perry et al., 2; Dohi et al., 29). Table 2: Active components of essential oils identified by GC-MS Essential oil Constituents % content Cajuput 1,8-Cineole 7.16 α-terpineol 1.8 Limonene.4 α-pinene 1.71 Linalool 1.6 Eucalyptus 1,8-Cineole 83.3 α-pinene 6.83 Limonene 4.9 ortho-cymene 3.83 β-pinene 1.7 Sweet majoram 1,8-Cineole 63.1 α-terpineol Limonene.63 α-pinene 4.67 β-pinene 4.19 Camphor 1,8-Cineole Limonene ortho-cymene 9.6 α-terpineol 4.98 α-pinene 4.91 Rosemarry 1,8-Cineole Camphor ortho-cymene 4.13 Limonene 3.2 Isobornyl acetate 2.62

4 R T : E 7 T IC F : t a p a n e e RT: NL: E7 9 TIC F: MS Tapanee R T : E [A 1,8-cineole [B [C T IC F : t a p a n e e 1 6 RT: R T : [D [E NL:.3E7 TIC F: MS tapanee E 7 T I C F : t a p a n e e Figure 1: GC-MS chromatogram of five essential oils; M. cajuputi [A, E. globules [B, O. majoram [C, C. camphora [D and R. officinalis [E After the constituents in five essential oils were identified, 1,8-cineole was the greatest active detected constituent. This compound was operated for AChE inhibitory activity at concentration 1 mg/ml. It showed high AChE inhibitory activity I and IC were 81.8±.99% and.1±.1µg/ml respectively. Three ( M. cajuputi, E. globules, O. majoram) from five essential oils with high content of 1,8-cineole over % were performed for IC. The IC were.63±.8,.21±.2 and.24±.26µg/ml consequently. The current study showed AChE inhibitory activity efficacy of essential oil of M. cajuputi, E. globules and O. majoram with high content of 1,8-cineole were similar to the previous studies of AChE inhibitory activity of the essential oils with 1,8-cineole (Miyazawa et al., 1998; Perry et al., 2; Dohi et al., 29). Table 3: Active components of essential oils in Cajuput from commercial and in house identified by GC-MS and AChE inhibitory activity Cajuput oil Commercial (%co n- tent) In house(%content) Chemical constituents 1,8-Cineole(7.16) Terpinolene(21.61) γ-terpineol (1.8) g-terpinene(17.8) Limonene(.4) Caryophyllene(12.2) α-pinene(1.71) Platyphyllol(7.) Linalool(1.6) ortho-cymene(7.34) AChE Inhibition* 68.68± ±7.2 * concentration at.12 mg/ml Regarding to prove this hypothesis, M. cajuputi plant material was collected and extracted for essential oil. The essential oil was

5 performed for AChE inhibitory activity and identified for the constituents using GC-MS as following Figure 2 and Table 3. The commercial R T : >7% of 1,8 cineole [A E 7 T I C F : t a p a n e e 1 7 cajuput with high content of 1,8-cineole was shown higher AChE inhibitory activity than the in house cajuput. RT: Figure 2: GC-MS chromatogram of essential oils; commercial M. cajuputi [A and in house M. cajuputi [B <.1% of 1,8-cineole [B E 7 TIC F : ta p a ne e 2 6 Conclusion From the screening of 29 essential oils Eucalyptus, Cajuput, Sweet marjoram, Camphor tree and Rose marry oils showed AChE inhibitory activity and the AChE inhibitory activity could be attributed to 1,8-cineole. Acknowledgement: Financial support by Faculty of Pharmacy, Srinakharinwirot University, Thailand and facility support from Faculty of Pharmaceutical Sciences, Naresuan University are gratefully acknowledged and Ms Kanokwan Changwichit for her helpful References Enz, A., Amstutz, R., Boddeke, H., Gmelin, G., Malonowski, J., Brain selective inhibition of acetylcholinesterase: a novel approach to therapy for Alzheimer s disease. Progress in Brain Research, 98: Dohi, S., Terasaki, M., Makino, M. 29. Acetyl cholinesterase inhibitory activity and chemical composition of commercial essential oils. Journal of Agricultural and food Chemistry, 7: Ellman, G.L., Lourtney, D.K., Andres, V., Gmelin, G., A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology, 7: Hosttettman, k., Borloz, A., Urbain, A., Marston, A. 26. Natural product inhibitors of Acetylcholoinesterase. Current Organic Chemistry, 1: Ingkaninan, K., de Best, C.M., Irth, H., van der Heijden, R., Hofte, A.J.P., Karabatak, B., Tjaden, U.R., van der Greef, J., Verpoorte, R., 2. High performance liquid chromatography with on-line coupled UV-mass spectrophotometric-biochemical detection for identification of acetyl cholinesterase inhibitors from natural products. Journal of Chromatography A, 872: Miyazawa, M., Watanabe, H., Umemoto, K. Kameoka, H Inhibition of acetylcholinesterase activity by essential oil of Mentha spp. Journal of Agricultural and food Chemistry, 46: Miyazawa, M., Tougo, H. Ishihara, M. 21. Inhibition of acetylcholinesterase activity by essential oil of Citrus paradisi. Natural product Letters, 1: Perry, N.S., Houghton, P.J., Theobald, A., Jenner, P., Perry, E.K. 2. In vitro inhibition of human erythrocyte acetylcholinesterase by Salvia lavandulaefolia essential oil and constituents terpenes. Journal of Pharmacy and Pharmacology, 2: Perry, N., Court, G., Bidet, N., Court, J., Perry E European herbs with cholinergic activities: potential in dementia therapy. International Journal of Geriatric Psychiatry, 11:

Purity Specifications

Purity Specifications Purity Specifications Essential oils are pure reflections of the botanicals from which they re derived, but their production is an extremely complicated process with a multitude of