http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, Early Online: 1–7 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2014.1001405
ORIGINAL ARTICLE
Chemical composition and acetylcholinesterase inhibitory activity of Artemisia maderaspatana essential oil Jyotshna1, Nidhi Srivastava1, Bhuwanendra Singh1, Debabrata Chanda2, and Karuna Shanker1 Department of Analytical Chemistry and 2Department of Molecular Bioprospection, CSIR – Central Institute of Medicinal and Aromatic Plants, Lucknow, Uttar Pradesh, India
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Abstract
Keywords
Context: To date, there are no reports to validate the Indian traditional and folklore claims of Artemisia maderaspatana L. (syn. Grangea maderaspatana L.) (Asteraceae) for the treatment of Alzheimer’s disease. Objective: The present study characterizes the volatile components (non-polar compounds) of A. maderaspatana and evaluates its acetylcholinesterase inhibition potential. Materials and methods: The essential oils (yield 0.06% v/w) were obtained from fresh aerial part of A. maderaspatana. The characterization of volatile components (non-polar compounds) was performed by GC–MS data and with those of reference compounds compiled in the spectral library of in-house database. The in vitro acetylcholinesterase (AChE) inhibition of the volatile organic constituents (VOC’s) of A. maderaspatana aerial part was evaluated in varying concentration ranges (0.70–44.75 mg/mL) with Ellman’s method. Results: The major components were a-humulene (46.3%), b-caryophyllene (9.3%), a-copaene (8.2%), b-myrcene (4.3%), Z(E)-a-farnesene (3.7%), and calarene (3.5%). Chemical variability among other Artemisia spp. from different climatic regions of India and countries namely Iran and France was observed. The experimental results showed that diverse volatile organic constituents of A. maderaspatana have significant acetylcholinesterase inhibitory activity (an IC50 value of 31.33 ± 1.03 mg/mL). This is the first report on the inhibition of acetylcholinesterase properties of essential oil of A. maderaspatana obtained from fresh aerial part. Conclusions: The present results indicate that essential oil of A. maderaspatana isolated from the northern region of India could inhibit AChE moderately. Therefore, the possibility of novel AChE inhibitors might exist in VOCs of this plant.
a-Humulene, AChE, Alzheimer’s disease, Asteraceae, GC–MS, Grangea maderaspatana, hydro-distillation, medicinal herb
Introduction Alzheimer’s disease (AD) the most common form of dementia, affecting 60–80% of the elderly population. Treatment of AD patients poses many challenges (Alzheimer’s Association, 2012). There is a great interest in the development of new drugs in the field of AD, senile dementia, and age-associated memory impairment. Acetylcholinesterase (AChE) is one of the most essential enzymes in the family of serine hydrolases, catalyzes the hydrolysis of neurotransmitter acetylcholine, which plays a key role in memory and cognition (Dall’Acqua et al., 2010; Lu et al., 2011). Physiological changes due to AChE play critical role in neural transmission. It is still the focus of pharmaceutical research, targeting in treatments of myasthenia gravis, glaucoma, and AD. It has been elucidated that cholinergic deficiency is associated with AD (Silman & Sussman, 2005). The inhibition of biological activity of AChE Correspondence: K. Shanker, Department of Analytical Chemistry, CSIR – Central Institute of Medicinal and Aromatic Plants, Lucknow 226015, Uttar Pradesh, India. Tel: +91 522 2718580. Fax: +91 522 2719072. E-mail: [email protected]
History Received 9 May 2014 Revised 25 November 2014 Accepted 12 December 2014 Published online 17 April 2015
and increased level of acetylcholine in brain are major therapeutic strategies adopted in AD treatment (Dorronsoro et al., 2003). Thus, cholinesterase inhibition is not only the bases of treatment for AD but also considered a promising strategy for the therapy of dementia, myasthenia gravis, and Parkinson’s disease. Several AChE inhibitors such as tacrine, donepezil, rivastigmine, and galanthamine were approved by US Food and Drug Administration (US-FDA) (Sugimoto et al., 2000). Later reports pronounce that these compounds have undesirable effects including gastrointestinal disturbances and problems associated with bioavailability (Mukherjee et al., 2007; Schulz, 2003). The challenge forces researchers to find better AChE inhibitors from natural resources. Keeping all these views in mind, this study was designed involving the chemical characterization and acetylcholinesterase inhibition evaluation of hydrodistillate of fresh plant material (aerial part) of Artemisia maderaspatana L. (syn. Grangea maderaspatana L.) (Asteraceae) collected during flowering phase. About 500 species of Artemisia reported in the world and out of which about 45 species are found in India (Shah, 2014).
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Artemisia maderaspatana is a wide spread herb of semiaquatic areas such as a ponds, lakes, and river-banks. This plant was found throughout India, Pakistan, Bangladesh, Nepal, Sri Lanka, Malaysia, Taiwan, China, Myanmar, and Africa (Guha-Bakshi et al., 2001; Pullaiah, 2006). In India, Artemisia species are mainly used in traditional system of medicine, Unani-tibb and Ayurveda for the management of various ailments. The ethnic communities of Godavari district (Andhra Pradesh, India) are using leaf of the plant for the cure of hysteria and menstrual complaints. The ethnic communities of Rajasthan, India, also use the plant leaf for the treatment of hysteria (Guha-Bakshi et al., 2001). The present in vitro study of acetylcholinesterase inhibition potential of A. maderaspatana is aimed to find a natural product lead for neurodegenerative disorders. The composition of non-polar volatile organic constituents (VOC’s) was also characterized with gas chromatography–mass spectrometry (GC–MS) spectra matching with the laboratory made spectral library. The chemical variability among various other Artemisia spp. was also discussed.
Materials and methods Plant material The aerial part of A. maderaspatana was collected in February 2011 at the young stage of development from three different locations situated at Kursi Road Lucknow, UP, India. The plant identifications (voucher specimens no. 9483) were confirmed by a taxonomist, Dr. Subash C. Singh, Central Institute of Medicinal and Aromatic plants Lucknow, UP, India and deposited in the Botany Department of institute. Chemicals Acetylthiocholine iodide (ATCI), acetylcholinesterase enzyme (AChE) from bovine erythrocytes, physostigmine, and DTNB (5,50 -dithiobis, 2-nitrobenzoic acid) were obtained from Sigma (Poole, UK). Methanol and all other organic solvents (analytical grade) were purchased from Merck (Darmstadt, Germany). Essential oil extraction The aerial parts of fresh plants were subjected to hydrodistillation in a Clevenger apparatus (1 kg each) for 3 h. The distillate was saturated with NaCl and the oil was extracted with n-hexane and dichloromethane. Pooled organic phases were then dried over anhydrous sodium sulfate and then the solvent distilled off at 35 C under vacuum using rotary vacuum evaporator (Buchi, Flawil, Switzerland). The oil yield of plant material was 0.06% (v/w, fresh wt basis). The oil samples were stored at 20 C until analyzed. The quantitative and qualitative analyses of the essential oils were performed by capillary GC, and GC–MS, respectively. Analyses of essential oil Gas chromatography-fast ionization detector A PerkinElmer Autosystem XL gas chromatograph (PerkinElmer Inc., Waltham, MA) with fast ionization detector
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(GC-FID) was used, the system was fitted with an EQUITY–5 [bonded: poly (5% diphenyl/95% dimethylsiloxane), 60 m 0.32 mm, film thickness 0.25 mm, SUPELCO, Bellefonte, PA]. The column temperature ranged from 70 to 250 C, at 3 C/min and 250 to 320 C, at 6 C/min, with a final hold time of 5 min, using H2 as a carrier gas at 10 psi constant pressure, a split ratio of 1:50, an injection size of 0.03 mL and injector and detector (FID) temperatures of 280 C and 300 C, respectively. The percentage compositions were obtained from electronic integration measurements using flame ionization detection without taking into account relative response factors. Gas chromatography-mass spectrometry The sample was analyzed by GC–MS/PerkinElmer turbomass quadrupole mass spectrometer (GC–MS) (PerkinElmer Inc., Waltham, MA) operating at 70 eV with a mass range of m/z 40–450 using bonded phase; poly (5% diphenyl/95% dimethyl siloxane) (EQUITY-5, 60 m 0.32 mm, film thickness 0.25 mm, SUPELCO, Bellefonte, PA) column. The column temperature of 70–300 C was programmed at a rate of 3.0 C/min, with a hold time of 10 min. The oven temperature program was the same as in GC while the injector temperature was 270 C, transfer line and ion source temperatures were 300 C, injection size 0.03 mL neat, split ratio 1:50 using He as a carrier gas at 10 psi constant pressure. The identification of the compounds was achieved on the basis of retention time, Kovats Index, literature reported retention index using a homologous series of n-alkanes (C8–C25 hydrocarbons, Polyscience Corp., Niles, IL), co-injection with standards (Sigma Aldrich, St. Louis, MO), mass spectra library search (NIST, Wiley, and Nbs), and by comparing with the mass spectral literature data (Adams, 1995; Cecchini et al., 2012; Gaviria et al., 2011). Antiacetylcholinesterase assay AChE activity was measured using a 96-well microplate reader based on Ellman’s method (Elgorashi et al., 2004; Ellman et al., 1961; Rhee et al., 2003). The enzyme hydrolyses the substrate acetylthiocholine resulting in the product thiocholine which reacts with Ellman’s reagent (DTNB) to produce 2-nitrobenzoate-5-mercaptothiocholine and 5-thio-2-nitrobenzoate which can be detected at 405 nm. In the 96-well plates, 125 mL of 3 mM DTNB, 25 mL of 15 mM ATCI, and 50 mL of buffer, 25 mL of essential oil sample dissolved in buffer containing not more than 10% methanol (working concentration range 0.70–44.75 mg/mL prepared by dilution of stock 1.0 mg/mL) were added to the wells. The absorbance was measured at 405 nm every 13 s for 65 s. About 25 mL of 0.22 U/mL of AChE enzyme was then added and the absorbance was again read every 13 s for 104 s. The absorbance was read using a SpectraMaxÕ Plus 384 Microplate Spectrophotometer (Molecular Devices, Sunnyvale, CA), at 405 nm. Absorbance was plotted against time and the enzyme activity was calculated from the slope of the line so obtained and expressed as a percentage compared with an assay using a buffer without any inhibitor. Any increase in absorbance due to the spontaneous hydrolysis of substrate was corrected by subtracting the rate of the reaction before adding the enzyme from the rate after adding the enzyme. Inhibition was
Chemical composition and acetylcholinesterase inhibitory
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Table 1. Essential oil composition of A. maderaspatana.
S. no.
Compound namea
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57
(E)-2-hexanal Sabinene b-Myrcene a-Phellandrene b-Ocimene trans-Sabinene hydrate cis-p-Menth-2-en-1-ol trans-p-Menth-2-en-1-ol a-Terpineol trans-Piperitol Geranyl formate Thymoil acetate a-Copaene b-Bourbonene b-Caryophyllene z-Cinnamic acid a-Humulene Germacerene D p-Cresyl isotiglate Z(E)-a-Farnesene 2-Octanoil furan 3-Methyl comounarin a-Muurolene Bicyclogermacrene (E,E)a-Farnesene Cubebol endo-1-Bourbonanol a-Cadinene Germacerene B Germacerene D-4-ol Carryophyllene oxide Spathulenol Guaiol Benzophenone Viridiflorol 1,10-Di-epi-cubenol 10-epi-g-Eudesmol trans-b-Elemenone a-Acorenol Phenyl propyl n-valerate epi-a-Cadinol Geranyl triglate a-Muurolol Calarene Selin-11-en-4a-ol Allylronone-3 n-Heptyl phenyl-acetate 8-a-Acetoxyelemol Phenyl lethyl n-octanate p-Cresyl phenyl acetate Muscone Rimuene Sandaracopimara-8(14),15,diol epi-13-Manoyl-oxide Abietatriene Nezukol trans-Totarol methyl ether
Group component Monoterpenoid hydrocarbons Oxygenated monoterpenes Sesquiterpens hydrocarbons Oxygenated Sesquiterpens Diterpenes Aromatic ketone a
RIb
LRIc
% Content in Aerial part
845 975 987 1026 1043 1096 1120 1138 1194 1206 1251 1359 1382 1391 1426 1431 1467 1479 1482 1486 1487 1490 1498 1504 1519 1524 1527 1536 1560 1578 1584 1587 1595 1602 1609 1615 1621 1624 1630 1636 1645 1649 1659 1667 1670 1689 1717 1790 1816 1826 1834 1889 1953 2005 2045 2133 2239
851 976 989 1026 1043 1093 1119 1140 1193 1205 1302 1357 1383 1393 1429 1430 1463 1480 1482 1484 1487 1490 1499 1505 1517 1524 1525 1538 1559 1578 1584 1589 1596 1604 1606 1614 1622 1623 1630 1635 1643 1650 1658 1629 1669 1689 1717 1789 1819 1827 1831 1894 1961 2010 2054 2126 2234
td 0.1 4.3 0.2 1.1 0.2 0.7 0.3 0.1 0.2 0.4 0.2 8.2 2.3 9.3 0.2 46.3 0.2 0.1 3.7 0.5 0.3 2.4 0.6 2.3 0.5 0.2 0.1 0.2 0.4 0.1 0.4 0.1 0.1 0.6 1.5 0.2 0.5 0.1 0.4 0.2 0.3 0.1 3.5 0.3 0.2 0.2 0.2 0.1 0.4 0.2 0.2 0.1 0.5 0.2 0.1 0.3
Mode of identification RI RI RI, RI, RI, RI RI, RI RI RI RI, RI RI, RI, RI, RI RI, RI RI RI, RI RI RI RI RI, RI, RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI, RI RI RI RI RI RI RI RI RI RI RI RI RI
MS MS MS MS
MS MS MS MS MS MS
MS MS
MS
% 5.7 2.1 76.3 10.9 1.6 0.5
Tentatively identified. RI, retention index on equity-5 columns using a homologous series of n-alkanes C9–C28 hydrocarbons (Polyscience Corp., Niles, IL). c LRI, retention index reported in the literature (based on the same stationary phase). MS, mass spectrum. d t ¼ trace 50.1%. b
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calculated by comparing the rates for the sample to the blank (10% MeOH in buffer). The quantitative results of AChE inhibition were represented as means ± SD of single experiment performed in triplicate. Inhibition concentration (IC50 values) was determined using the binding analysis software of GraphPad Prism 4.0 (GraphPad Software, Inc., San Diego, CA).
Result and discussions
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Essential oil composition of A. maderaspatana The GC and GC/MS analyses of the A. maderaspatana essential oil allowed identification of 79 compounds, accounting for 97% of the total (Table 1). From the data obtained, the essential oils shown to be complex mixtures of several components, predominating sesquiterpenes. It must be emphasized that the crossing data achieved from the retention index (RI) and mass spectrum (MS) allowed the identification of almost all individual compounds detected, even those that presented very similar RI values but different mass spectra could be distinguished. The compounds identification and their abundance, as well as the RI values in order of their elution from Equity-5 column are depicted in Table 1. The essential oils from A. maderaspatana were characterized by high percentages of monoterpene (5.7%) for b-myrcene (4.3%); sesquiterpene hydrocarbon (76.3%) consisted mainly of a-humulene (46.3%), b-caryophyllene (9.3%), a-copaene (8.2%), Z(E)a-farnesene (3.7%), and calarene (3.5%) (Figure 1). The chemical characterization of essential oil of A. maderaspatana is reported first time. However, other Artemisia species such as A. dubia Wall. ex Besser, A. indica Willd., and A. vulgaris L. of Nepal origin have been reported to possess chrysanthenone (29.0%), ascaridole (15.4%), and a-thujone (30.5%), respectively, as major components (Satyal et al., 2012). The putative metabolite identifications of A. maderaspatana indicate more sesquiterpenes than monoterpenes contrary to A. tridentate Nutt. of North American origin (Turi et al., 2014). This may be due to the fact that In Artemisia species, diverse monoterpene and sesquiterpene compounds are produced in the glandular trichomes present on leaf surfaces (Ruhland et al., 2013). Variations in the chemical
Figure 1. Major phytoconstituents of A. maderaspatana.
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composition of the essential oil of the same species from different regions may also be attributed to differences in climatic and geographic parameters such as temperature, altitude, wind direction, rainfall, and soil type (Douglas et al., 2004; Llorens et al., 2014). The chemical constituents other Artemisia species from India and Iran are summarized in Table 2. a-Pinene (41.9%) in Artemisia armeniaca Lam., a-thujone (43.6%) in Artemisia dracunculus L.; methyl chavicol (76.6%) in Artemisia haussknechtii Boiss., and carvanone (47.9%) in Artemisia kermanensis Podl. were observed as the major chemicals (Table 2). A high degree of variability in A. maderaspatana essential oil constituents are mainly due to chemical variability, diverse biochemical pathways, and high polyploidy (Bagchi et al., 2009; Haghi et al., 2010; Juteau et al., 2005; Kazemi et al., 2010; Rustaiyan et al., 2009). Acetylcholinesterase inhibition Acetylcholine is liberated at synaptic gap as a neurotransmitter. The most important change observed in the brain is a decrease in cortical levels of the neurotransmitter acetylcholine. Inhibition of acetylcholinesterase, therefore, can restore the level of acetylcholine in the brain (Howes & Houghton, 2003). Plants are being used traditionally to enhance cognitive function and to alleviate other symptoms of Alzheimer’s disease (Howes & Houghton, 2003). Most of the drugs used for Alzheimer treatment are prepared by an enzyme inhibitor, e.g. galantamine, isolated from the extract of snowdrop (Galanthus nivalis) (Mukherjee et al., 2007). Few reports communicate the acetylcholinesterase inhibitory activity of essential oils (Dohi et al., 2009; Miyazawa et al., 2001; Souza et al., 2010). The AChE inhibitory activity of the essential oils of A. maderaspatana has never been reported before. In order to assess the AChE inhibition potential of A. maderaspatana essential oil, different concentrations of oil were screened using Ellman’s method (1961). The inhibitory activity of different concentration of reference compound (Physostigmine) and essential oil is summarized in Figure 2. Under the experimental condition, the IC50 value of physostigmine and essential oil of A. maderaspatana was 1.03 mg/mL and 31.33 mg/mL, respectively. Although AchE inhibition by essential oil of A. maderaspatana is not effective as a standard drug, i.e. physostigmine, but activity can be considered significant as oil is a mixture of number of components. The volatile organic contents of Artemisia, having several components among b-myrcene a-copaene, b-caryophyllene, a humulene, Z(E)a-farnesene, have reported to possess moderate AChE inhibition (Dohi et al., 2009; Fujiwara et al., 2010; McPartland & Russo, 2001; Paithankar et al., 2010; Savelev et al., 2004). Although a-humulene is major compounds of A. maderaspatana, reported to posses low AChE inhibition (415 mM) (Lee & Ahn, 2013). None of the other Artemisia spp. have been reported to possess AChE inhibition activity except current one i.e. Artemisia maderaspatana. a-Humulene was found to be a major component, while in other species, it was in minor or trace quantity. Essential oils are rich in monoterpenoids (hydrocarbons, alcohols, and ketones). The structure–activity relationship (SAR) studies of monoterpenoid showed that AChE inhibition activity of
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terpene ketones with p-menthane skeleton was higher than the alcohols and hydrocarbons (Miyazawa & Yamafuji, 2005). The terpene hydrocarbon compounds reported similar AChE inhibitory activity to the terpene alcohols (Miyazawa et al., 1997). In bicyclic monoterpenes, the presence of oxygenated functional groups decreased the AChE inhibition. On one hand, the presence of double bond in the mocelular structure of bicyclic monoterpene hydrocarbon has shown a strong inhibition of AChE activity (Aazza et al., 2011). On the other hand, the presence of oxygenated group in sesquiterpenes, inhibitory effect has been improved, especially in ketones (Miyazawa et al., 2001). The sesquiterpene alcohols have also been considered as strong AChE inhibitors. From the data, it can be concluded that out of six major compounds, two monocyclic terpene (a-humulene and b-caryophyllene with three and two double bond, respectively), and two bicyclic
Chemical composition and acetylcholinesterase inhibitory
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hydrocarbons (a-copaene and calarene with one double bond) have contributed for AChE inhibition. However, role of other minor constituent could not be disregarded. Inhibitory activity of A. maderaspatana oil can be expected from the synergistic effect by the combination of a-humulene and b-caryophyllene including minor components. The standardize mixture of a-humulene and b-caryophyllene is already in use in the Brazilian market for topical anti-inflammatory preparation (Bolzani et al., 2012).
Conclusion An effective AChE inhibition potential of A. maderaspatana essential oil makes this plant of attention for further exploration of its chemical and biological potentials. The inhibitory activity of A. maderaspatana oil is not by one
Table 2. Variation in oil composition of different Artemisia species. Major chemical composition Artemisia species
Origin
Artemisia roxbuurghiana
Bhaldana, Uttrakhand, India
Artemisia roxbuurghiana
Bhatwari, Uttrakhand, India
Artemisia roxbuurghiana
Mussoories, Uttrakhand, India
Artemisia kermanensis
Kerman province of Kerman, province of Iran
Artemisia kopetdaghensis
Province of Khorassan, province of Iran
Artemisia haussknechtii
Shahmirzad, province of Semnan, province of Iran
Artemisia dracunculus
Karaj (Tehran), Central Iran
Artemisia verlotiorum
Southern France
Artemisia armeniaca
Kalibar region, province of East Azarbaijan, Iran
Artemisia splendens
Kalibar region, province of East Azarbaijan, Iran
Chemical
% Content
b-Caryophyllene Eugenol Linalyl acetate Linallol b-Caryophyllene a-Thujone Camphore Eugenol 1,8-Cineole Borneol Linalyl acetate a-Humulene Eugenol 1,8-Cineole Carvanone 1,8-Cineole Chrysanthenone Carvacrol acetate Fanchone Carvanone Geranial Ethyl nerolate p-Cymene Neral 1,8-Cineole Camphor Artemisia ketone b-Pinene Methyl chavicol (E)-b-Ocimene (Z)-b-Ocimene A-Thujone 1,8-Cineole B-Caryophyllene Germacrene-D B-Thujone a-Pinene 1,8-Cineole Spathulenol Limonene 1,8-Cineole b-Caryophylene a Pinene Bicyclogermacerene b-Elemene b-Caryophyllene Spathulenol
18.4 16.2 9.6 8.4 16.3 12.0 7.7 5.6 5.1 21.2 7.4 6.7 5.6 5.2 21.4 16.0 14.8 9.3 6.5 47.9 6.5 6.3 5.9 5.4 16.5 14.1 10.5 5.4 76.6 11.0 6.9 43.6 14.6 12.7 10.9 10.8 41.9 20.6 6.7 5.6 14.5 14.3 11.3 11.3 6.8 6.3 6.0
Reference Bagchi et al. (2009)
Bagchi et al. (2009)
Bagchi et al. (2009)
Rustaiyan et al. (2009)
Rustaiyan et al. (2009)
Rustaiyan et al. (2009)
Haghi et al. (2010) Juteau et al. (2005)
Kazemi et al. (2010)
Kazemi et al. (2010)
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Figure 2. Dose-dependent AChE inhibition potential of (a) physostigmine – a standard drug and (b) essential oil of A. maderaspatana.
strong inhibitor but by synergistic effect of several compounds. a-Humulene is the major component of A. maderaspatana unlike other Artemisia spp. The current study not only demonstrated the chemo-taxonomic variation, but also therapeutic/pharmaceutical potential of different Artemisia spp. AChE inhibitory activity suggests that preparations containing A. maderaspatana oil may be of some benefit as part of a coordinated approach for topical anti-inflammation.
Acknowledgement Authors are also thankful to Dr. S. C. Singh, taxonomist of the Institute for the identification of plant herbaria specimen.
Declaration of interest The authors report that they have no conflicts of interest. This research was financially supported by Council of Scientific and Industrial Research (CSIR), New Delhi, as Institutional project [CIMAP/ChemBio/BSC-0203].
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Bolzani VD, Valli M, Pivatto M, Viegas C. (2012). Natural products from Brazilian biodiversity as a source of new models for medicinal chemistry. Pure Appl Chem 84:1837–46. Cecchini C, Silvi S, Cresci A, et al. (2012). Antimicrobial efficacy of Achillea ligustica All. (Asteraceae) essential oils against reference and isolated oral microorganisms. Chem Biodiver 9:12–24. Dall’Acqua S, Maggi F, Minesso P, et al. (2010). Identification of nonalkaloid acetylcholinesterase inhibitors from Ferulago campestris (Besser) Grecescu (Apiaceae). Fitoterapia 81:1208–12. Douglas MH, van Klink JW, Smallfield BM, et al. (2004). Essential oils from New Zealand manuka: Triketone and other chemotypes of Leptospermum scoparium. Phytochemistry 65:1255–64. Dohi S, Terasaki M, Makino M. (2009). Acetylcholinesterase inhibitory activity and chemical composition of commercial essential oils. J Agric Food Chem 57:4313–18. Dorronsoro I, Castro A, Martinez A. (2003). Peripheral and dual binding site inhibitors of acetylcholinesterase as neurodegenerative diseasemodifying agents. Expert Opin Ther Pat 13:1725–32. Elgorashi EE, Stafford GI, Van Staden J. (2004). Acetycholinesterase enzyme inhibitory effects of amaryllidaceae alkaloids. Planta Med 70: 260–2. Ellman GL, Courtney KD, Andres Jr V, Feather-Stone RM. (1961). A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 7:88–95. Fujiwara M, Yagi N, Miyazawa M. (2010). Acetylcholinesterase inhibitory activity of volatile oil from Peltophorum dasyrachis Kurz ex Bakar (Yellow Batai) and bisabolane-type sesquiterpenoids. J Agric Food Chem 58:2824–9. Gaviria M, Quijano C, Pino J, Madrinan S. (2011). Chemical composition and antibacterial activity of the essential oil of Drimys granadensis LF leaves from Colombia. Chem Biodiver 8:532–9. Guha-Bakshi DN, Sensarma P, Pal DC. (2001). A Lexicon of Medicinal Plants in India. Naya Prakash: Calcutta, India. Haghi G, Ghasian F, Safaei-Ghomi J. (2010). Determination of the essential oil from root and aerial parts of Artemisia dracunculus L. cultivated in central Iran. J Essent Oil Res 22:294–6. Howes MJR, Houghton PJ. (2003). Plants used in Chinese and Indian traditional medicine for improvement of memory and cognitive function. Pharmacol Biochem Behav 75:513–27. Juteau F, Masotti V, Viano J, Bessiere JM. (2005). Chemical variation in the oil of Artemisia verlotiorum Lamotte of French origin harvested at a vegetative stage and during flowering. J Essent Oil Res 17:254–6. Kazemi M, Zand MR, Roshanaei K, et al. (2010). Composition of the volatile oils of Artemisia armenica Lan. and Artemisia splendens Willd. from Iran. J Essent Oil Res 22:126–8. Lee DC, Ahn YJ. (2013). Laboratory and simulated field bioassays to evaluate larvicidal activity of Pinus densiflora hydrodistillate, its constituents and structurally related compounds against Aedes albopictus, Aedes aegypti and Culex pipiens pallens in relation to their inhibitory effects on acetylcholinesterase activity. Insects 4: 217–29. Llorens L, Llorens-Molina JA, Agnello S, Boira H. (2014). Geographical and environment-related variations of essential oils in isolated populations of Thymus richardii Pers. in the Mediterranean basin. Biochem Syst Ecol 56:246–54. Lu SH, Wu JW, Liu HL, et al. (2011). The discovery of potential acetylcholinesterase inhibitors: A combination of pharmacophore modeling, virtual screening, and molecular docking studies. J Biomed Sci 18:8. Mcpartland JM, Russo EB. (2001). Cannabis and cannabis extracts. J Cannabis Ther 1:103–32. Miyazawa M, Tougo H, Ishihara M. (2001). Inhibition of acetylcholinesterase activity by essential oil from Citrus paradisi. Nat Prod Lett 15:205–10. Miyazawa M, Yamafuji C. (2005). Inhibition of acetylcholinesterase activity by bicyclic monoterpenoids. J Agric Food Chem 53: 1765–8. Miyazawa M, Watanabe H, Kameoka H. (1997). Inhibition of acetylcholinesterase activity by monoterpenoids with a p-menthane skeleton. J Agric Food Chem 45:677–9. Mukherjee PK, Kumar V, Mal M, Houghton PJ. (2007). Acetylcholinesterase inhibitors from plants. Phytomedicine 14: 289–300. Paithankar VV, Belsare SL, Charde RM, Vyas JV. (2010). Acorus calamus: An overview. Int J Biomed Res 2:518–29.
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Pullaiah T. (2006). Encyclopedia of World Medicinal Plants. New Delhi. India: Regency Publication. Rhee IK, Van Rijn RM, Verpoorte R. (2003). Qualitative determination of false-positive effects in the acetylcholinesterase assay using thin layer chromatography. Phytochem Anal 14:127–31. Ruhland CT, Dyslin MJ, Krenz JD. (2013). Wyoming big sagebrush screens ultraviolet radiation more effectively at higher elevations. J Arid Environ 96:19–22. Rustaiyan A, Tabatabaei-Anaraki M, Kazemi M, et al. (2009). Chemical composition of essential oil of three Artemisia species growing wild in Iran: Artemisia kermanensis Podl., A-kopetdaghensis Krasch., M.Pop et Lincz. ex Poljak., and A-haussknechtii Boiss. J Essent Oil Res 21: 410–13. Satyal P, Paudel P, Kafle A, et al. (2012). Bioactivities of volatile components from Nepalese Artemisia species. Nat Prod Commun 7: 1651–8. Savelev SU, Okello EJ, Perry EK. (2004). Butyryl- and acetylcholinesterase inhibitory activities in essential oils of Salvia species and their constituents. Phytother Res 18:315–24.
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Schulz V. (2003). Ginkgo extract or cholinesterase inhibitors in patients with dementia: What clinical trials and guidelines fail to consider? Phytomedicine 10:74–9. Shah NC. (2014). The economic and medicinal Artemisia species in India. Sci Tech J 1:29–38. Silman I, Sussman JL. (2005). Acetylcholinesterase: Classical and nonclassical functions and pharmacology. Curr Opin Pharmacol 5: 293–302. Souza AD, Lopes EMC, Silva MCD, et al. (2010). Chemical composition and acetylcholinesterase inhibitory activity of essential oils of Myrceugenia myrcioides (Cambess.) O. Berg and Eugenia riedeliana O. Berg, Myrtaceae. Rev Bras Farmacogn 20:175–9. Sugimoto H, Yamanishi Y, Iimura Y, Kawakami Y. (2000). Donepezil hydrochloride (E2020) and other acetylcholinesterase inhibitors. Curr Med Chem 7:303–39. Turi CE, Shipley PR, Murch SJ. (2014). North American Artemisia species from the subgenus Tridentatae (Sagebrush): A phytochemical, botanical and pharmacological review. Phytochemistry 98:9–26.
ORIGINAL ARTICLE
Chemical composition and acetylcholinesterase inhibitory activity of Artemisia maderaspatana essential oil Jyotshna1, Nidhi Srivastava1, Bhuwanendra Singh1, Debabrata Chanda2, and Karuna Shanker1 Department of Analytical Chemistry and 2Department of Molecular Bioprospection, CSIR – Central Institute of Medicinal and Aromatic Plants, Lucknow, Uttar Pradesh, India
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Abstract
Keywords
Context: To date, there are no reports to validate the Indian traditional and folklore claims of Artemisia maderaspatana L. (syn. Grangea maderaspatana L.) (Asteraceae) for the treatment of Alzheimer’s disease. Objective: The present study characterizes the volatile components (non-polar compounds) of A. maderaspatana and evaluates its acetylcholinesterase inhibition potential. Materials and methods: The essential oils (yield 0.06% v/w) were obtained from fresh aerial part of A. maderaspatana. The characterization of volatile components (non-polar compounds) was performed by GC–MS data and with those of reference compounds compiled in the spectral library of in-house database. The in vitro acetylcholinesterase (AChE) inhibition of the volatile organic constituents (VOC’s) of A. maderaspatana aerial part was evaluated in varying concentration ranges (0.70–44.75 mg/mL) with Ellman’s method. Results: The major components were a-humulene (46.3%), b-caryophyllene (9.3%), a-copaene (8.2%), b-myrcene (4.3%), Z(E)-a-farnesene (3.7%), and calarene (3.5%). Chemical variability among other Artemisia spp. from different climatic regions of India and countries namely Iran and France was observed. The experimental results showed that diverse volatile organic constituents of A. maderaspatana have significant acetylcholinesterase inhibitory activity (an IC50 value of 31.33 ± 1.03 mg/mL). This is the first report on the inhibition of acetylcholinesterase properties of essential oil of A. maderaspatana obtained from fresh aerial part. Conclusions: The present results indicate that essential oil of A. maderaspatana isolated from the northern region of India could inhibit AChE moderately. Therefore, the possibility of novel AChE inhibitors might exist in VOCs of this plant.
a-Humulene, AChE, Alzheimer’s disease, Asteraceae, GC–MS, Grangea maderaspatana, hydro-distillation, medicinal herb
Introduction Alzheimer’s disease (AD) the most common form of dementia, affecting 60–80% of the elderly population. Treatment of AD patients poses many challenges (Alzheimer’s Association, 2012). There is a great interest in the development of new drugs in the field of AD, senile dementia, and age-associated memory impairment. Acetylcholinesterase (AChE) is one of the most essential enzymes in the family of serine hydrolases, catalyzes the hydrolysis of neurotransmitter acetylcholine, which plays a key role in memory and cognition (Dall’Acqua et al., 2010; Lu et al., 2011). Physiological changes due to AChE play critical role in neural transmission. It is still the focus of pharmaceutical research, targeting in treatments of myasthenia gravis, glaucoma, and AD. It has been elucidated that cholinergic deficiency is associated with AD (Silman & Sussman, 2005). The inhibition of biological activity of AChE Correspondence: K. Shanker, Department of Analytical Chemistry, CSIR – Central Institute of Medicinal and Aromatic Plants, Lucknow 226015, Uttar Pradesh, India. Tel: +91 522 2718580. Fax: +91 522 2719072. E-mail: [email protected]
History Received 9 May 2014 Revised 25 November 2014 Accepted 12 December 2014 Published online 17 April 2015
and increased level of acetylcholine in brain are major therapeutic strategies adopted in AD treatment (Dorronsoro et al., 2003). Thus, cholinesterase inhibition is not only the bases of treatment for AD but also considered a promising strategy for the therapy of dementia, myasthenia gravis, and Parkinson’s disease. Several AChE inhibitors such as tacrine, donepezil, rivastigmine, and galanthamine were approved by US Food and Drug Administration (US-FDA) (Sugimoto et al., 2000). Later reports pronounce that these compounds have undesirable effects including gastrointestinal disturbances and problems associated with bioavailability (Mukherjee et al., 2007; Schulz, 2003). The challenge forces researchers to find better AChE inhibitors from natural resources. Keeping all these views in mind, this study was designed involving the chemical characterization and acetylcholinesterase inhibition evaluation of hydrodistillate of fresh plant material (aerial part) of Artemisia maderaspatana L. (syn. Grangea maderaspatana L.) (Asteraceae) collected during flowering phase. About 500 species of Artemisia reported in the world and out of which about 45 species are found in India (Shah, 2014).
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Artemisia maderaspatana is a wide spread herb of semiaquatic areas such as a ponds, lakes, and river-banks. This plant was found throughout India, Pakistan, Bangladesh, Nepal, Sri Lanka, Malaysia, Taiwan, China, Myanmar, and Africa (Guha-Bakshi et al., 2001; Pullaiah, 2006). In India, Artemisia species are mainly used in traditional system of medicine, Unani-tibb and Ayurveda for the management of various ailments. The ethnic communities of Godavari district (Andhra Pradesh, India) are using leaf of the plant for the cure of hysteria and menstrual complaints. The ethnic communities of Rajasthan, India, also use the plant leaf for the treatment of hysteria (Guha-Bakshi et al., 2001). The present in vitro study of acetylcholinesterase inhibition potential of A. maderaspatana is aimed to find a natural product lead for neurodegenerative disorders. The composition of non-polar volatile organic constituents (VOC’s) was also characterized with gas chromatography–mass spectrometry (GC–MS) spectra matching with the laboratory made spectral library. The chemical variability among various other Artemisia spp. was also discussed.
Materials and methods Plant material The aerial part of A. maderaspatana was collected in February 2011 at the young stage of development from three different locations situated at Kursi Road Lucknow, UP, India. The plant identifications (voucher specimens no. 9483) were confirmed by a taxonomist, Dr. Subash C. Singh, Central Institute of Medicinal and Aromatic plants Lucknow, UP, India and deposited in the Botany Department of institute. Chemicals Acetylthiocholine iodide (ATCI), acetylcholinesterase enzyme (AChE) from bovine erythrocytes, physostigmine, and DTNB (5,50 -dithiobis, 2-nitrobenzoic acid) were obtained from Sigma (Poole, UK). Methanol and all other organic solvents (analytical grade) were purchased from Merck (Darmstadt, Germany). Essential oil extraction The aerial parts of fresh plants were subjected to hydrodistillation in a Clevenger apparatus (1 kg each) for 3 h. The distillate was saturated with NaCl and the oil was extracted with n-hexane and dichloromethane. Pooled organic phases were then dried over anhydrous sodium sulfate and then the solvent distilled off at 35 C under vacuum using rotary vacuum evaporator (Buchi, Flawil, Switzerland). The oil yield of plant material was 0.06% (v/w, fresh wt basis). The oil samples were stored at 20 C until analyzed. The quantitative and qualitative analyses of the essential oils were performed by capillary GC, and GC–MS, respectively. Analyses of essential oil Gas chromatography-fast ionization detector A PerkinElmer Autosystem XL gas chromatograph (PerkinElmer Inc., Waltham, MA) with fast ionization detector
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(GC-FID) was used, the system was fitted with an EQUITY–5 [bonded: poly (5% diphenyl/95% dimethylsiloxane), 60 m 0.32 mm, film thickness 0.25 mm, SUPELCO, Bellefonte, PA]. The column temperature ranged from 70 to 250 C, at 3 C/min and 250 to 320 C, at 6 C/min, with a final hold time of 5 min, using H2 as a carrier gas at 10 psi constant pressure, a split ratio of 1:50, an injection size of 0.03 mL and injector and detector (FID) temperatures of 280 C and 300 C, respectively. The percentage compositions were obtained from electronic integration measurements using flame ionization detection without taking into account relative response factors. Gas chromatography-mass spectrometry The sample was analyzed by GC–MS/PerkinElmer turbomass quadrupole mass spectrometer (GC–MS) (PerkinElmer Inc., Waltham, MA) operating at 70 eV with a mass range of m/z 40–450 using bonded phase; poly (5% diphenyl/95% dimethyl siloxane) (EQUITY-5, 60 m 0.32 mm, film thickness 0.25 mm, SUPELCO, Bellefonte, PA) column. The column temperature of 70–300 C was programmed at a rate of 3.0 C/min, with a hold time of 10 min. The oven temperature program was the same as in GC while the injector temperature was 270 C, transfer line and ion source temperatures were 300 C, injection size 0.03 mL neat, split ratio 1:50 using He as a carrier gas at 10 psi constant pressure. The identification of the compounds was achieved on the basis of retention time, Kovats Index, literature reported retention index using a homologous series of n-alkanes (C8–C25 hydrocarbons, Polyscience Corp., Niles, IL), co-injection with standards (Sigma Aldrich, St. Louis, MO), mass spectra library search (NIST, Wiley, and Nbs), and by comparing with the mass spectral literature data (Adams, 1995; Cecchini et al., 2012; Gaviria et al., 2011). Antiacetylcholinesterase assay AChE activity was measured using a 96-well microplate reader based on Ellman’s method (Elgorashi et al., 2004; Ellman et al., 1961; Rhee et al., 2003). The enzyme hydrolyses the substrate acetylthiocholine resulting in the product thiocholine which reacts with Ellman’s reagent (DTNB) to produce 2-nitrobenzoate-5-mercaptothiocholine and 5-thio-2-nitrobenzoate which can be detected at 405 nm. In the 96-well plates, 125 mL of 3 mM DTNB, 25 mL of 15 mM ATCI, and 50 mL of buffer, 25 mL of essential oil sample dissolved in buffer containing not more than 10% methanol (working concentration range 0.70–44.75 mg/mL prepared by dilution of stock 1.0 mg/mL) were added to the wells. The absorbance was measured at 405 nm every 13 s for 65 s. About 25 mL of 0.22 U/mL of AChE enzyme was then added and the absorbance was again read every 13 s for 104 s. The absorbance was read using a SpectraMaxÕ Plus 384 Microplate Spectrophotometer (Molecular Devices, Sunnyvale, CA), at 405 nm. Absorbance was plotted against time and the enzyme activity was calculated from the slope of the line so obtained and expressed as a percentage compared with an assay using a buffer without any inhibitor. Any increase in absorbance due to the spontaneous hydrolysis of substrate was corrected by subtracting the rate of the reaction before adding the enzyme from the rate after adding the enzyme. Inhibition was
Chemical composition and acetylcholinesterase inhibitory
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Table 1. Essential oil composition of A. maderaspatana.
S. no.
Compound namea
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57
(E)-2-hexanal Sabinene b-Myrcene a-Phellandrene b-Ocimene trans-Sabinene hydrate cis-p-Menth-2-en-1-ol trans-p-Menth-2-en-1-ol a-Terpineol trans-Piperitol Geranyl formate Thymoil acetate a-Copaene b-Bourbonene b-Caryophyllene z-Cinnamic acid a-Humulene Germacerene D p-Cresyl isotiglate Z(E)-a-Farnesene 2-Octanoil furan 3-Methyl comounarin a-Muurolene Bicyclogermacrene (E,E)a-Farnesene Cubebol endo-1-Bourbonanol a-Cadinene Germacerene B Germacerene D-4-ol Carryophyllene oxide Spathulenol Guaiol Benzophenone Viridiflorol 1,10-Di-epi-cubenol 10-epi-g-Eudesmol trans-b-Elemenone a-Acorenol Phenyl propyl n-valerate epi-a-Cadinol Geranyl triglate a-Muurolol Calarene Selin-11-en-4a-ol Allylronone-3 n-Heptyl phenyl-acetate 8-a-Acetoxyelemol Phenyl lethyl n-octanate p-Cresyl phenyl acetate Muscone Rimuene Sandaracopimara-8(14),15,diol epi-13-Manoyl-oxide Abietatriene Nezukol trans-Totarol methyl ether
Group component Monoterpenoid hydrocarbons Oxygenated monoterpenes Sesquiterpens hydrocarbons Oxygenated Sesquiterpens Diterpenes Aromatic ketone a
RIb
LRIc
% Content in Aerial part
845 975 987 1026 1043 1096 1120 1138 1194 1206 1251 1359 1382 1391 1426 1431 1467 1479 1482 1486 1487 1490 1498 1504 1519 1524 1527 1536 1560 1578 1584 1587 1595 1602 1609 1615 1621 1624 1630 1636 1645 1649 1659 1667 1670 1689 1717 1790 1816 1826 1834 1889 1953 2005 2045 2133 2239
851 976 989 1026 1043 1093 1119 1140 1193 1205 1302 1357 1383 1393 1429 1430 1463 1480 1482 1484 1487 1490 1499 1505 1517 1524 1525 1538 1559 1578 1584 1589 1596 1604 1606 1614 1622 1623 1630 1635 1643 1650 1658 1629 1669 1689 1717 1789 1819 1827 1831 1894 1961 2010 2054 2126 2234
td 0.1 4.3 0.2 1.1 0.2 0.7 0.3 0.1 0.2 0.4 0.2 8.2 2.3 9.3 0.2 46.3 0.2 0.1 3.7 0.5 0.3 2.4 0.6 2.3 0.5 0.2 0.1 0.2 0.4 0.1 0.4 0.1 0.1 0.6 1.5 0.2 0.5 0.1 0.4 0.2 0.3 0.1 3.5 0.3 0.2 0.2 0.2 0.1 0.4 0.2 0.2 0.1 0.5 0.2 0.1 0.3
Mode of identification RI RI RI, RI, RI, RI RI, RI RI RI RI, RI RI, RI, RI, RI RI, RI RI RI, RI RI RI RI RI, RI, RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI, RI RI RI RI RI RI RI RI RI RI RI RI RI
MS MS MS MS
MS MS MS MS MS MS
MS MS
MS
% 5.7 2.1 76.3 10.9 1.6 0.5
Tentatively identified. RI, retention index on equity-5 columns using a homologous series of n-alkanes C9–C28 hydrocarbons (Polyscience Corp., Niles, IL). c LRI, retention index reported in the literature (based on the same stationary phase). MS, mass spectrum. d t ¼ trace 50.1%. b
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calculated by comparing the rates for the sample to the blank (10% MeOH in buffer). The quantitative results of AChE inhibition were represented as means ± SD of single experiment performed in triplicate. Inhibition concentration (IC50 values) was determined using the binding analysis software of GraphPad Prism 4.0 (GraphPad Software, Inc., San Diego, CA).
Result and discussions
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Essential oil composition of A. maderaspatana The GC and GC/MS analyses of the A. maderaspatana essential oil allowed identification of 79 compounds, accounting for 97% of the total (Table 1). From the data obtained, the essential oils shown to be complex mixtures of several components, predominating sesquiterpenes. It must be emphasized that the crossing data achieved from the retention index (RI) and mass spectrum (MS) allowed the identification of almost all individual compounds detected, even those that presented very similar RI values but different mass spectra could be distinguished. The compounds identification and their abundance, as well as the RI values in order of their elution from Equity-5 column are depicted in Table 1. The essential oils from A. maderaspatana were characterized by high percentages of monoterpene (5.7%) for b-myrcene (4.3%); sesquiterpene hydrocarbon (76.3%) consisted mainly of a-humulene (46.3%), b-caryophyllene (9.3%), a-copaene (8.2%), Z(E)a-farnesene (3.7%), and calarene (3.5%) (Figure 1). The chemical characterization of essential oil of A. maderaspatana is reported first time. However, other Artemisia species such as A. dubia Wall. ex Besser, A. indica Willd., and A. vulgaris L. of Nepal origin have been reported to possess chrysanthenone (29.0%), ascaridole (15.4%), and a-thujone (30.5%), respectively, as major components (Satyal et al., 2012). The putative metabolite identifications of A. maderaspatana indicate more sesquiterpenes than monoterpenes contrary to A. tridentate Nutt. of North American origin (Turi et al., 2014). This may be due to the fact that In Artemisia species, diverse monoterpene and sesquiterpene compounds are produced in the glandular trichomes present on leaf surfaces (Ruhland et al., 2013). Variations in the chemical
Figure 1. Major phytoconstituents of A. maderaspatana.
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composition of the essential oil of the same species from different regions may also be attributed to differences in climatic and geographic parameters such as temperature, altitude, wind direction, rainfall, and soil type (Douglas et al., 2004; Llorens et al., 2014). The chemical constituents other Artemisia species from India and Iran are summarized in Table 2. a-Pinene (41.9%) in Artemisia armeniaca Lam., a-thujone (43.6%) in Artemisia dracunculus L.; methyl chavicol (76.6%) in Artemisia haussknechtii Boiss., and carvanone (47.9%) in Artemisia kermanensis Podl. were observed as the major chemicals (Table 2). A high degree of variability in A. maderaspatana essential oil constituents are mainly due to chemical variability, diverse biochemical pathways, and high polyploidy (Bagchi et al., 2009; Haghi et al., 2010; Juteau et al., 2005; Kazemi et al., 2010; Rustaiyan et al., 2009). Acetylcholinesterase inhibition Acetylcholine is liberated at synaptic gap as a neurotransmitter. The most important change observed in the brain is a decrease in cortical levels of the neurotransmitter acetylcholine. Inhibition of acetylcholinesterase, therefore, can restore the level of acetylcholine in the brain (Howes & Houghton, 2003). Plants are being used traditionally to enhance cognitive function and to alleviate other symptoms of Alzheimer’s disease (Howes & Houghton, 2003). Most of the drugs used for Alzheimer treatment are prepared by an enzyme inhibitor, e.g. galantamine, isolated from the extract of snowdrop (Galanthus nivalis) (Mukherjee et al., 2007). Few reports communicate the acetylcholinesterase inhibitory activity of essential oils (Dohi et al., 2009; Miyazawa et al., 2001; Souza et al., 2010). The AChE inhibitory activity of the essential oils of A. maderaspatana has never been reported before. In order to assess the AChE inhibition potential of A. maderaspatana essential oil, different concentrations of oil were screened using Ellman’s method (1961). The inhibitory activity of different concentration of reference compound (Physostigmine) and essential oil is summarized in Figure 2. Under the experimental condition, the IC50 value of physostigmine and essential oil of A. maderaspatana was 1.03 mg/mL and 31.33 mg/mL, respectively. Although AchE inhibition by essential oil of A. maderaspatana is not effective as a standard drug, i.e. physostigmine, but activity can be considered significant as oil is a mixture of number of components. The volatile organic contents of Artemisia, having several components among b-myrcene a-copaene, b-caryophyllene, a humulene, Z(E)a-farnesene, have reported to possess moderate AChE inhibition (Dohi et al., 2009; Fujiwara et al., 2010; McPartland & Russo, 2001; Paithankar et al., 2010; Savelev et al., 2004). Although a-humulene is major compounds of A. maderaspatana, reported to posses low AChE inhibition (415 mM) (Lee & Ahn, 2013). None of the other Artemisia spp. have been reported to possess AChE inhibition activity except current one i.e. Artemisia maderaspatana. a-Humulene was found to be a major component, while in other species, it was in minor or trace quantity. Essential oils are rich in monoterpenoids (hydrocarbons, alcohols, and ketones). The structure–activity relationship (SAR) studies of monoterpenoid showed that AChE inhibition activity of
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terpene ketones with p-menthane skeleton was higher than the alcohols and hydrocarbons (Miyazawa & Yamafuji, 2005). The terpene hydrocarbon compounds reported similar AChE inhibitory activity to the terpene alcohols (Miyazawa et al., 1997). In bicyclic monoterpenes, the presence of oxygenated functional groups decreased the AChE inhibition. On one hand, the presence of double bond in the mocelular structure of bicyclic monoterpene hydrocarbon has shown a strong inhibition of AChE activity (Aazza et al., 2011). On the other hand, the presence of oxygenated group in sesquiterpenes, inhibitory effect has been improved, especially in ketones (Miyazawa et al., 2001). The sesquiterpene alcohols have also been considered as strong AChE inhibitors. From the data, it can be concluded that out of six major compounds, two monocyclic terpene (a-humulene and b-caryophyllene with three and two double bond, respectively), and two bicyclic
Chemical composition and acetylcholinesterase inhibitory
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hydrocarbons (a-copaene and calarene with one double bond) have contributed for AChE inhibition. However, role of other minor constituent could not be disregarded. Inhibitory activity of A. maderaspatana oil can be expected from the synergistic effect by the combination of a-humulene and b-caryophyllene including minor components. The standardize mixture of a-humulene and b-caryophyllene is already in use in the Brazilian market for topical anti-inflammatory preparation (Bolzani et al., 2012).
Conclusion An effective AChE inhibition potential of A. maderaspatana essential oil makes this plant of attention for further exploration of its chemical and biological potentials. The inhibitory activity of A. maderaspatana oil is not by one
Table 2. Variation in oil composition of different Artemisia species. Major chemical composition Artemisia species
Origin
Artemisia roxbuurghiana
Bhaldana, Uttrakhand, India
Artemisia roxbuurghiana
Bhatwari, Uttrakhand, India
Artemisia roxbuurghiana
Mussoories, Uttrakhand, India
Artemisia kermanensis
Kerman province of Kerman, province of Iran
Artemisia kopetdaghensis
Province of Khorassan, province of Iran
Artemisia haussknechtii
Shahmirzad, province of Semnan, province of Iran
Artemisia dracunculus
Karaj (Tehran), Central Iran
Artemisia verlotiorum
Southern France
Artemisia armeniaca
Kalibar region, province of East Azarbaijan, Iran
Artemisia splendens
Kalibar region, province of East Azarbaijan, Iran
Chemical
% Content
b-Caryophyllene Eugenol Linalyl acetate Linallol b-Caryophyllene a-Thujone Camphore Eugenol 1,8-Cineole Borneol Linalyl acetate a-Humulene Eugenol 1,8-Cineole Carvanone 1,8-Cineole Chrysanthenone Carvacrol acetate Fanchone Carvanone Geranial Ethyl nerolate p-Cymene Neral 1,8-Cineole Camphor Artemisia ketone b-Pinene Methyl chavicol (E)-b-Ocimene (Z)-b-Ocimene A-Thujone 1,8-Cineole B-Caryophyllene Germacrene-D B-Thujone a-Pinene 1,8-Cineole Spathulenol Limonene 1,8-Cineole b-Caryophylene a Pinene Bicyclogermacerene b-Elemene b-Caryophyllene Spathulenol
18.4 16.2 9.6 8.4 16.3 12.0 7.7 5.6 5.1 21.2 7.4 6.7 5.6 5.2 21.4 16.0 14.8 9.3 6.5 47.9 6.5 6.3 5.9 5.4 16.5 14.1 10.5 5.4 76.6 11.0 6.9 43.6 14.6 12.7 10.9 10.8 41.9 20.6 6.7 5.6 14.5 14.3 11.3 11.3 6.8 6.3 6.0
Reference Bagchi et al. (2009)
Bagchi et al. (2009)
Bagchi et al. (2009)
Rustaiyan et al. (2009)
Rustaiyan et al. (2009)
Rustaiyan et al. (2009)
Haghi et al. (2010) Juteau et al. (2005)
Kazemi et al. (2010)
Kazemi et al. (2010)
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Jyotshna et al.
Figure 2. Dose-dependent AChE inhibition potential of (a) physostigmine – a standard drug and (b) essential oil of A. maderaspatana.
strong inhibitor but by synergistic effect of several compounds. a-Humulene is the major component of A. maderaspatana unlike other Artemisia spp. The current study not only demonstrated the chemo-taxonomic variation, but also therapeutic/pharmaceutical potential of different Artemisia spp. AChE inhibitory activity suggests that preparations containing A. maderaspatana oil may be of some benefit as part of a coordinated approach for topical anti-inflammation.
Acknowledgement Authors are also thankful to Dr. S. C. Singh, taxonomist of the Institute for the identification of plant herbaria specimen.
Declaration of interest The authors report that they have no conflicts of interest. This research was financially supported by Council of Scientific and Industrial Research (CSIR), New Delhi, as Institutional project [CIMAP/ChemBio/BSC-0203].
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