Chemical Profiling, Volatile Oil Analysis and Anticholinesterase Activity Of

South African Journal of Botany 115 (2018) 108–112

Contents lists available at ScienceDirect

South African Journal of Botany

journal homepage: www.elsevier.com/locate/sajb

Chemical proﬁling, volatile oil analysis and anticholinesterase activity of Hydrocotyle umbellata L. aerial parts cultivated in Egypt

S.A. Hamdy ⁎, H.M. El Hefnawy, S.M. Azzam, E.A. Aboutabl

Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Kasr El-Aini St, Cairo 11562, Egypt article info abstract

Article history: Hydrocotyle umbellata L. (commonly known as Acaricoba) is an aquatic perennial plant belonging to family Received 14 July 2017 Araliaceae. The plant has been used in folk phytotherapy and in the Ayurvedic medicine to reduce anxiety and Received in revised form 30 November 2017 as a memory enhancer herbal remedy. This study was designated to explore the diversity of the accumulated bio- Accepted 26 January 2018 active metabolites that might account for the potential cholinesterase inhibition activity of H. umbellata L. aerial Available online xxxx parts. UPLC/PDA/ESI-qTOF-MS secondary metabolites profiling revealed similar chemical composition of the leaves fl fi Edited by LJ McGaw and owers. The major identi ed secondary metabolites in both samples were caffeoylquinic acid isomers, quercetin derivatives, and fatty acids. Also, the volatile oil analysis of fresh leaves and flowers using GC/MS showed sim- Keywords: ilar composition. The monoterpenes; α-pinene, β-pinene, and limonene represent the main components of the oil. Hydrocotyle umbellata L. Many reports were found dealing with the cholinesterase inhibition activity of the major identified caffeoylquinic UPLC/PDA/ESI-qTOF-MS acids, quercetin derivatives, as well as the identified volatile monoterpenes. Therefore, the anticholinesterase activ- GC/MS ity of the ethanolic extract of the aerial parts was evaluated using Ellman's spectrophotometric method. The Caffeoylquinic acids ethanolic extract exhibited significant cholinesterase inhibitory activity with IC50 1.88 mg/ml ± 0.19 compared Quercetin to standard cholinesterase inhibitor eserine (IC50 0.27 mg/ml ± 0.15). These findings suggest that the significant Monoterpenes cholinesterase inhibition activity of the ethanolic extract is likely to be mediated by the major identified bioactive Anti-acetylcholinesterase molecules and support the folkloric use of Hydrocotyle umbellata L. as a memory enhancer herbal medicine. © 2017 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction constituents (Rojas et al., 2009), flavonoids (Adams et al., 1989), sterols (Chavasiri et al., 2005), and saponins (Sosa et al., 2011). Genus Hydrocotyle (Family Araliaceae) comprises about 130 species Based on H. umbellata L. presumed therapeutic value and lack of creeping perennial plants (Mabberley, 1997) distributed in tropical of reports concerning the biological and chemical investigation of and temperate regions (Karuppusamy et al., 2014). Plants of the genus H. umbellata L. aerial parts, this study was designed to evaluate the po- have been used in Taiwan folk medicine for treatment of several inflam- tential anticholinesterase activity of the ethanolic extract of Hydrocotyle matory disorders, common cold, dysentery, zoster, eczema and period umbellata L. aerial parts using Ellman's spectrophotometric method, and pain (Lin et al., 2003). to correlate this activity with its secondary metabolites composition. Hydrocotyle umbellata L. is a creeping water-loving specimen, Also, to provide a detailed map for various chemical classes (i.e. phenolic widely grown in the Americas and mainly native to Brazil (Rocha compounds, fatty acids and volatile oil constituents) distribution in et al., 2011). It is a long petiolated plant with peltate-shaped leaves, sim- leaves, as well as flowers using different chromatographic methods. ple umbel inflorescence somewhat exceeding the length of the leaves, UPLC-PDA-ESI-qTOF-MS has been widely used in metabolomic numerous small white flowers and flattened capsule-shaped fruits studies and became a powerful tool for secondary metabolites profiling (Fig. 1)(Godfrey and Wooten, 1981; Florentino et al., 2013). of complicated components in herbal medicines; as it provides rapid Traditionally, H. umbellata L. has been used as anti-inflammatory, metabolites analysis and excellent chromatographic separation with memory stimulant and anxiolytic herbal medicine (Reis et al., 1992; high sensitivity, accuracy, and precision (Farag et al., 2013). Lin et al., 2003). The ethanolic extract of the underground parts of the Alzheimer's disease (AD), the most common cause of dementia, is a plant was reported to possess analgesic, anti-inflammatory (Florentino neurodegenerative disorder that affects the elderly population charac- et al., 2013) and anxiolytic activities (Rocha et al., 2011). The main terized by progressive cognitive disability with declining in daily living secondary metabolites previously identified in the plant were volatile activities and neuropsychiatric symptoms or behavioral changes (Lane et al., 2006). ⁎ Corresponding author. Natural plants are considered a potential source of bioactive second- E-mail address: [email protected] (S.A. Hamdy). ary metabolites that might serve as leads for the development of novel

Entire plant Peltate-shaped leaves

Capsule-shaped fruits

Star-shaped flowers

Fig. 1. Photographs of H.umbellata L. plant. drugs with the ability to inhibit the acetylcholinesterase enzyme, 2.4. Extraction and sample preparation for UHPLC–qTOF– PDA–MS analysis which hydrolyzes the acetylcholine, increasing the acetylcholine available for transmission at the cholinergic synapse (Miyazawa and Leaves and flowers were separately air-dried, reduced to fine pow- Yamafuji, 2005). der (20 mg each) and extracted with 2.5 ml 70% methanol (MeOH) con- Based on the cholinergic theory, these natural cholinesterase in- taining 5 μg/ml umbelliferone (as an internal standard for relative hibitors improve the cholinergic functions in patients suffering from quantification) using a Turrax mixer (11,000 RPM) for five 20 s periods. Alzheimer's disease and mitigate the symptoms of this neurological To prevent heating, a period of 1 min separated each mixing period. disease (Perry and Howes, 2011). Extracts were then vortexed vigorously and centrifuged at 3000g (gravity force) for 30 min to remove plant debris before UPLC-MS analyses, 500 μl

2. Methodology was aliquoted and placed on a C18 cartridge (500 mg) preconditioned with methanol and water. Samples were then eluted using 3 ml 70% 2.1. Plant material MeOH and 3 ml 100% MeOH, the eluent was evaporated under a nitrogen stream and the obtained dry residue was resuspended in 500 μl The plant material was collected in March 2014 at the flowering methanol. Three microliters, aliquots, were used for analysis. Samples stage from El-Orman Botanical Garden, Giza, Egypt and kindly identified were analyzed in negative ESI ionization mode. by Eng. Threase Labib, consultant in Orman Garden and National Gene Bank, Ministry of Agriculture and confirmed by Dr. Mohammed 2.5. High-resolution UPLC-MS analysis El-Gebaly, the senior taxonomist at National Research Center. A voucher specimen (No. 17-8-2016) was kept at the Herbarium of Pharmacog- Qualitative analysis of the secondary metabolites was performed by nosy Department, Faculty of Pharmacy, Cairo University, Egypt. UPLC on an HSS T3 column (100 × 1.0 mm, packed with silica gel RP-C18, particle size 1.8 μm; Waters) by applying the following binary gradient, 2.2. Preparation of ethanolic extract at a flow rate of 150 μlmin−1: 0 to 1 min, isocratic 95% A (water/formic acid, 99.9/0.1 [v/v]), 5% B (acetonitrile/formic acid, 99.9/0.1 [v/v]); The air-dried powdered aerial parts (2.3 kg) were extracted with 1 to 16 min, linear from 5 to 95% B; 16 to 18 min, isocratic 95% B; 18 70% ethanol by cold maceration till exhaustion. The combined ethanolic to 20 min, isocratic 5% B. The injection volume was 3.1 μl (full loop extracts were evaporated under reduced pressure at 55 °C to dryness. injection). Eluted compounds were detected from m/z 100 to 1000 in negative ion mode using the following instrument settings: nebulizer 2.3. Chemicals and reagents gas, nitrogen, 1.6 bar; dry gas, nitrogen, 6 l min−1, 190 °C; capillary, −5500 V (+4000 V); end plate offset, −500 V; funnel 1 RF, 200 Vpp; Acetylcholinesterase (Electric-eel EC 3.1.1.7), Acetylcholine iodide, funnel 2 RF, 200 Vpp; in-source CID energy, 0 V; hexapole RF, 100 Vpp; 5,5-Dithiobis[2-nitrobenzoic acid] (DTNB), Buffers and all other chemicals quadrapole ion energy, 5 eV; collision gas, argon; collision energy, of the highest purity and analytical grade were purchased from Sigma- 10 eV; collision RF 200/400 Vpp (timing 50/50); transfer time, 70 μs; Aldrich (St. Louis, MO, USA). pulser frequency, 10 kHz; spectra rate, 3 Hz. Internal mass calibration 110 S.A. Hamdy et al. / South African Journal of Botany 115 (2018) 108–112 of each analysis was performed by infusion of 20 μl10mMlithiumfor- the measurement of cholinesterase activity. 100 mM sodium phosphate mate in isopropanol: water, 1:1 (v/v), at a gradient time of 18 min buffer (pH 8.0, 140 μl), DTNB (10 μl), and the ethanolic extract of the using a diverter valve. aerial parts (20 μl) at an initial concentration of 10 mg/ml dissolved in a final concentration of 0.001% DMSO which has no effect on the 2.6. GC–MS analysis of volatile constituents enzyme, were added to the first well and serially diluted two-fold down the plate then mixed with acetylcholinesterase (20 μl) and incu- Samples of fresh leaves (210 g) and fresh flowers (180 g), collected bated for 15 min at 25 °C. The reaction was then initiated by the addition in January (winter) were separately subjected to hydro-distillation in of acetylthiocholine (10 μl). The hydrolysis of acetylthiocholine was mon- a Clevenger's apparatus (Egyptian Pharmacopoeia, 2005). The isolated itored by the formation of yellow 5-thio-2-nitrobenzoate anion as a result volatiles in each case were dried over anhydrous sodium sulfate. The of the reaction of DTNB with thiocholine, released by the enzymatic percentage yield was calculated on the fresh weight basis and the hydrolysis of acetylthiocholine at a wavelength of 412 nm (15 min). All samples saved in a refrigerator for further analysis. The hydro-distilled the reactions were performed in triplicate in 96-well micro titre plates volatiles of the two samples under examination were separately sub- and monitored in a SpectraMax 340 (Molecular Devices, Sunnyvale, CA, jected to gas chromatographic/mass spectrometric analysis (GC/MS) USA) spectrometer. The concentration of the ethanolic extract that for investigation of their chemical composition. GC–MS analyses were inhibited the hydrolysis of the substrate (acetylthiocholine) by 50% performed on Shimadzu GC-17A gas chromatograph equipped with (IC50) was determined by monitoring the effect of increasing its concen- SLB-5 column (30 m × 0.25 mm i.d. × 0.25 μm film thickness; Supelco) tration in the assays on the inhibition values. The IC50 value was then and coupled to Shimadzu QP5050A mass chromatograph. The tempera- calculated using a software program (GraphPad Prism, version 5.01, ture of the injector and the interface were kept at 220 °C. The following Inc., 2007, San Diego CA, USA). temperature program was used for analysis: temperature was kept at 40 °C for 3 min, then increased to 180 °C at a rate of 12 °C min−1,kept 3. Results and discussion at 180 °C for 5 min, and finally ramped at a rate of 40 °C for 1 min to 240 °C and kept at that final temperature for 5 min. Helium was used 3.1. UPLC-MS secondary metabolites analysis as a carrier gas with a flow-rate of 0.9 ml/min. 6 μl of oil was diluted in 600 μl dichloromethane. 1 μl of this dilution was injected with split Secondary metabolites of Hydrocotyle umbellata L. leaves and flowers ratio 1:54. The first 5 min of the analysis was considered as solvent were assessed via reversed phase UPLC/PDA/ESI-qTOF-MS, using a gradi- delay and omitted from the final chromatograms. The HP quadruple ent mobile phase consisting of acetonitrile and aqueous formic acid, mass spectrometer was operated in EI mode at 70 eV. The scan range which allow a comprehensive elution of plant analytes. The resulted was set at m/z 40–500. chromatograms (Fig. 2) were similar and characterized by two main The isolated volatile components were identified as described regions; the firstregionwithretentiontime140–290 s with peaks prin- in Farag and Wessjohann (2012), the peaks were de-convoluted using cipally due to caffeoylquinic acid isomers and quercetin derivatives, AMDIS software (www.amdis.net)andidentified by its retention indi- whereas the later elution region starts from 325 to 690 s mainly for ces (RI) relative to n-alkanes (C6–C20), mass matching to NIST, WILEY li- fatty acids. The identities, retention times, UV characteristics, observed brary database. deprotonated molecular ion and fragment ions for individual metabolites are presented in Table 1. The compounds were tentatively identified

2.7. In vitro acetylcholinesterase inhibition assay and determination of IC50 based on the analysis of the data in Table 1 andcomparisonwithprevi- ously published data in the literature (Farag et al., 2013, 2015, 2016). Acetylcholinesterase inhibiting activity was measured according to Three mono-O-caffeic acid and one di-O-caffeic acid conjugates were Ellman's slightly modiﬁed spectrophotometric method (Ellman et al., detected in H. umbellata leaves and ﬂowers, by their predominant frag- 1961). Acetylthiocholine iodide was used as a substrate to assay acetyl- ment of 191 amu for quinic acid in the MS spectrum and characteristic cholinesterase. 5,5′-Dithiobis[2-nitrobenzoic acid] (DTNB) was used for UV max at (325–330) nm. Peaks 1, 3, 4 and 17 exhibited UV spectra

Phenolics Fatty acids

Intens. 6 10+11+12+13+14+15+16 29+30

x10 39 46

18+19+20+21+22+24+25 44

7 43 41 35+36 48 32+33 26

1.00 8 27 40

6 38

3 45 42

17 37 47 0.75 31 34

0.50 1 4

0.25 Flowers

0.00 200 300 400 500 600 700 Time [s]

Intens . 3 6 4 9+11+12+13 38 x10 20+21+22+23+25 44 43 7 16 34 41 5+6 39 29+30 35+36 48 37 46 15 26 0.8 32+33 40 27+28 45 MS response 0.6 42 47 17 31 0.4 1 2

0.2 Leaves

0.0 200 300 400 500 600 Time [s]

rt(sec)

Fig. 2. Representative UPLC/MS traces of H.umbellata L. ﬂowers and leaves methanolic extracts characterized by two main regions: (140–300 s) with peaks mainly due to phenolics and a region from (315–680 s) for fatty acids. S.A. Hamdy et al. / South African Journal of Botany 115 (2018) 108–112 111

Table 1 Metabolites detected in methanolic extracts of leaves (L) and ﬂowers (F) of H.umbellata L. using UPLC/PDA-MS in negative ionization mode.

− Peak no. Rt (sec) U.V (nm) M-H m/z Molecular formula Error of m/z MS-MS Identified compounds L F − 1 142.6 322.8 353.08 C16H17O9 0.9 191 1-O-caffeoylquinic acid + + − 2 162.5 275, 321 shd 577.13 C30H15O12 4.6 559, 451, 425, 407, 289, 245 (Epi)catechin dimer + − − 3 173 293 shd, 325 353 C16H17O9 0.8 191, 127 3-O-caffeoylquinic acid + + − 4 189.8 282.9, 325.3 353 C16H17O9 2.1 191 5-O-caffeoylquinic acid + + − 5 192.5 281, 320 433 C20H33O10 1.9 385, 353, 307, 191 Unidentified + − − 6 195.2 275.9, 344.3 461.16 C20H29O12 1 431, 385, 337, 289, 191 Unidentified + + − 7 202 253, 351 625.14 C27H29O17 1 301 Quercetin-O-di-hexoside + + − 8 203.8 261, 334.8 565.17 C23H33O16 1.7 519, 211, 112 Un-identified − + − 9 207.6 265, 324.6 855.33 C58H47H7 3 711, 625,, 431, 367, 341, 239 Un-identified + − − 10 210 266.9, 308.8 341.12 C16H21O8 0.8 239, 191 Un-identified − + − 11 213 282 shd, 326 595.132 C26H27O16 1 301, 300 Quercetin-O-hexosyl-pentoside + + − 12 220 256 shd, 353 609.15 C27H29O16 2 463, 301, 300 Rutin + + − 13 226 266 shd, 328 463.08 C21H19O12 2.5 301 Quercetin-O-hexoside + + − 14 235 298 shd, 348 593.15 C27H29O15 4 285 Kaempferol-O-rutinoside − + − 15 237.5 2795 shd, 350 447.09 C21H19O12 1 301 Quercetin-O-rhamnoside + + − 16 241 264, 352 433.08 C20H17O11 0.1 301 Quercetin-O-pentoside + + − 17 246.9 Nd 515.12 C25H23O12 0.5 353, 191, 173 1,3-di-O-caffeoylquinic acid + +

18 248.5 277.5, 344 417.21 C20H33O9- 4 377, 317, 248, 161, 112 Un-identified − + − 19 253.8 274, 345.4 957.1 C53H65O16 2.6 665, 517, 473, 393, 273, 187 Un-identified − + − 20 260.5 279, 245 607.29 C28H47O14 2.9 561, 517, 421, 379, 273, 161 Un-identified + + − 21 270 276.9, 344.3 463.25 C22H39O10 1.6 417, 299, 199 Un-identified + + − 22 274 269.9, 343.8 505.22 C23H37O12 4.3 407, 343, 299, 242, 174 Un-identified + + − 23 281.5 270, 345.4 507.24 C23H39O12 2.1 459, 263 Un-identified + − − 24 282.8 265.5, 347.3 591 C28H47013 2.3 507, 447, 343, 263 Un-identified − + − 25 285 268.4, 345.8 493.22 C22H37O12 0.8 447, 299 Un-identified + + − 26 289 255, 372 301 C15H9O7 0.2 179, 151 Quercetin + + − 27 296 268.4, 347.3 445 C22H37O9 0.7 223 Un-identified + + − 28 309.7 275.4, 346.3 553 C34H33O7 3.6 485, 441, 373, 327, 221 Un-identified + − − 29 315.1 274 327.21 C18H31O5 0.1 309, 281 Trihydroxy-octadecadienoic acid + + − 30 330.6 287 329.22 C18H33O5 0.4 311, 283 Trihydroxy-octadecenoic acid + + − 31 405 Nd 309 C18H29O4 6 291,273 Dihydroxy-octadecatrienoic acid + + − 32 420 Nd 721 C34H57O16 3.2 593, 531, 485, 413, 248, 194 Un-identified + + − 33 429 Nd 311 C18H31O4 2 275, 263 Dihydroxy-octadecadienoic acid + + − 34 444 Nd 291.19 C18H27O3 2 273 Hydroxy-octadecatetranoic acid + + − 35 445 Nd 347.19 C24H27O2 8.4 311, 248 Un-identified + + − 36 448 Nd 577 C30H41O11 2.5 520, 467, 415, 325, 265 Un-identified + + − 37 457 Nd 293.2 C18H29O3 0.8 275, 247 Hydroxy-octadecatrienoic acid + + − 38 485 225 295.2 C18H31O3 1 277, 249 Hydroxy-octadecadienoic acid + + − 39 517.7 Nd 487.29 C25H43O9 0.2 441, 381, 321, 293 Un-identified + + − 40 525 Nd 249.18 C16H25O2 0.7 − Un-identified + + − 41 541 Nd 463.29 C23H43O9 1.4 409, 325, 293 Un-identified + + − 42 574.2 Nd 271.2 C16H31O3 0.1 253,225,197 Hydroxy-palmitic acid + + − 43 277.2 Nd 277.2 C18H29O2 0.2 233, 183 Linolenic acid + + − 44 610.5 Nd 279.23 C18H31O2 2 211 Linoleic acid + + − 45 617.5 Nd 253.216 C16H29O2 2.4 183 Palmitoleic acid + + − 46 637.2 Nd 255.23 C16H31O2 0.9 237 Palmitic acid + + − 47 648.7 Nd 281.2744 C18H33O2 3.8 − Oleic acid + + − 48 688 Nd 283.26 C18H35O2 0.2 265 Stearic acid + + Nd; not detected.

comparable to caffeoylquinic acids with high-resolution masses of Table 2 353.0898 for peaks 1, 3 and 4, and 515.1236 for peak 17 (Table 1). Identified components in the hydrodistilled volatile oil of fresh leaves and flowers of fi These peaks were tentatively identi ed as positional isomers of 1-O- H. umbellata L. caffeoylquinic acid (1), 3-O-caffeoylquinic acid (3), 5-O-caffeoylquinic fi + acid (4) and di-O-caffeoylquinic acid (17). Serial Identi ed component RI Percentage M Based on MS and UV/vis spectra, the flavonols of quercetin nucleus Leaves Flowers were the most predominant flavonoidal subclass identified in the 1 α-Thujene 924 0.32 0.47 136 UPLC-PDA-qTOF-MS of H.umbellata L. leaves and flowers. In MS/MS 2 α-Pinene 932 18.5 23.11 136 analysis the nature of the sugar could be revealed by the elimination 3 Sabinene 969 0.81 0.93 136 4 β-Pinene 974 31.2 30.37 136 of the sugar residues, i.e. loss of 162 amu (hexoses), 146 amu 5 Myrcene 988 2.56 2.69 136 (deoxyhexose), 132 amu (pentose) in O-glycosidic linkage (Farag 6 ρ-Cymene 1022 – 0.01 136 et al., 2013). Interpretation of MS spectra allowed for the detection of 7 Limonene 1024 37.19 38.6 136 − 8 β-Phellandrene 1025 0.58 0.50 136 quercetin (m/z 301.0352, C15H9O7 )inpeaks(7, 11, 12, 13, 15, 16, 26). β Negative ionization MS also revealed the presence of hydroxylated 9 Trans- -ocimene 1044 0.11 0.08 136 10 γ-Terpinene 1054 0.05 0.04 136 fatty acids, unsaturated, and saturated fatty acids. There is an increasing 11 Linalool 1095 0.14 0.03 93 interest in hydroxy fatty acids because of their anti-inflammatory, anti- 12 Trans-β-farnesene 1440 0.33 0.07 204 microbial and cytotoxic biological activities (Martin-Arjol et al., 2010). 13 β-Santalene 1457 0.55 0.16 204 However, this is the first report for the presence of oxygenated fatty 14 Germacrene D 1484 0.76 0.04 204 15 α-Chamigrene 1503 0.82 0.22 204 acids in genus Hydrocotyle. 112 S.A. Hamdy et al. / South African Journal of Botany 115 (2018) 108–112

Table 3 Conflict of interests Relative percentages of different classes of constituents identified in the hydrodistilled oils of fresh leaves and flowers of H. umbellata L. All authors have none to declare. Class Leaves (%) Flowers (%)

Hydrocarbons References Monoterpenes 96.5 98.5 fl Sesquiterpenes 2.3 0.5 Adams,A.A.,Norris,J.A.,Mabry,T.J.,1989.A avonoid from Hydrocotyle umbellata L. (Umbelliferae, Hydrocotyllae). Revista Latinoamericana de Química 20, 67–68. Total hydrocarbons 98.8 99 Chavasiri, W., Prukchareon, W., Sawadee, P., Zungsontiporn, S., 2005. Allelochemicals – Oxygenated compounds from Hydrocotyle umbellata Linn. World Congress on Allelopathy. vol. 4, pp. 15 18. Monoterpenes 0.14 0.03 Egyptian Pharmacopeia, 2005. Administration and Pharmaceutical Affairs, Ministry of Health and Population. fourth ed. Cairo, Egypt. Total oxygenated compounds 0.14 0.03 Ellman, G.L., Courtney, K.D., Andres, V., Featherstone, R.M., 1961. A new and rapid color- imetric determination of acetylcholinesterase activity. Biochemical Pharmacology 7, 88–95. Farag, M.A., El-Ahmady, S.H., Elian, F.S., Wessjohann, L.A., 2013. Metabolomics driven 3.2. GC/MS analysis of the hydrodistilled volatile oil analysis of artichoke leaf and its commercial products via UHPLC–q-TOF-MS and chemometrics. Phytochemistry 95, 177–187. The identified volatile components from the leaves and flowers Farag, M.A., Ezzat, S.M., Salama, M.M., Tadros, M.G., 2016. Anti-acetylcholinesterase potential and metabolome classification of 4 Ocimum species as determined via of H. umbellata L. are listed in Table 2 and the relative percentage of dif- UPLC/qTOF/MS and chemometric tools. Journal of Pharmaceutical and Biomedical ferent classes of constituents is recorded in Table 3. The volatile oil iso- Analysis 125, 292–302. lated from fresh leaves and flowers yielded 0.04% v/w and 0.1%v/w, Farag, M.A., Sakna, S.T., El-fiky, N.M., Shabana, M.M., Wessjohann, L.A., 2015. Phytochemical, antioxidant and antidiabetic evaluation of eight Bauhinia L. species from Egypt using respectively (calculated on a fresh weight basis). The two samples ex- UHPLC–PDA–qTOF-MS and chemometrics. Phytochemistry 119, 41–50. hibited nearly the same physical characters being oily, pale yellow Farag, M.A., Wessjohann, L.A., 2012. Volatiles profiling in medicinal licorice roots using in color, with a characteristic odor and readily soluble in ethanol 70%. steam distillation and solid-phase microextraction (SPME) coupled to chemometrics. – fi Journal of Food Science 77, 1179 1184. Hydrocarbons dominated the chromatographic pro les of the two vola- Figueiredo, A.C., Barroso, J.G., Pedro, L.G., Scheffer, J.J., 2008. Factors affecting secondary tiles with prevalent monoterpenoids, among which α-pinene, β-pinene, metabolite production in plants: volatile components and essential oils. Flavour and limonene were the major identified volatile oil constituents in both and Fragrance Journal 23, 213–226. Florentino, F., Nascimento, M.V.M., Galdino, P.M., Brito, A.F., Rocha, F.F., Tonussi, C.R., Lima, samples. In contrast, the published data concerning the volatile oil com- T.C.M., Paula, J.R., Costa, E.A., 2013. Evaluation of analgesic and anti-inflammatory ac- position of Venezuelan H. umbellata L. aerial parts revealed that the tivities of Hydrocotyle umbellata L., Araliaceae (acariçoba) in mice. Anais da Academia sesquiterpenes; germacrene D, β-sesquiphellandrene, and β-bisabolene Brasileira de Ciências 85, 987–997. were the major identified components (Rojas et al., 2009). This qualita- Godfrey, R.K., Wooten, J.W., 1981. Aquatic and Wetland Plants of Southeastern United State: Dicotyledons. University of Georgia Press, Athens, Georgia. tive variation in the chromatographic profiles may be attributed to Karuppusamy, S., Ali, M.A., Rajasekaran, K., Lee, J., Kim, S.Y., Pandey, A.K., Al-Hemaid, F.M., climatic and/or geographical factors (Figueiredo et al., 2008). 2014. A new species of Hydrocotyle L.(Araliaceae) from India. Bangladesh Journal of Plant Taxonomy 21, 167–173. Lane, R.M., Potkin, S.G., Enz, A., 2006. Targeting acetylcholinesterase and butyrylcho- 3.3. In vitro acetylcholinesterase inhibition activity linesterase in dementia. International Journal of Neuropsychopharmacology 9, 101–124. The ethanolic extract of the aerial parts of H. umbellata showed Lin, I.H., Chang, Y.S., Chen, I.S., Hsieh, W.C., 2003. The Catalogue of Medicinal Plant fi Resources in Taiwan. Committee on Chinese Medicine and Pharmacy, Department signi cant cholinesterase inhibition activity compared to standard cho- of Health, Taipei, Taiwan. linesterase inhibitor eserine with IC50 of 1.88 ± 0.19 mg/ml and IC50 Mabberley, D.J., 1997. The Plant Book: A Portable Dictionary of the Vascular Plants. 0.27 ± 0.15, respectively. The obtained results are consistent with bio- Cambridge University Press, p. 353. Martin-Arjol, I., Bassas-Galia, M., Bermudo, E., Garcia, F., Manresa, A., 2010. Identification of logical studies pointing to the anticholinesterase activity of the identi- oxylipins with antifungal activity by LC–MS/MS from the supernatant of Pseudomonas fied phenolic compounds; chlorogenic acid (Farag et al., 2016; Silva 42A2. Chemistry and Physics of Lipids 163, 341–346. et al., 2017) and quercetin (Nugroho et al., 2017; Palle and Neerati, Miyazawa, M., Yamafuji, C., 2005. Inhibition of acetylcholinesterase activity by bicyclic fi monoterpenoids. Journal of Agricultural and Food Chemistry 53, 1765–1768. 2017), as well as the identi ed monoterpenes; limonene (Zarrad et al., Nugroho, A., Choi, J.S., Hong, J.P., Park, H.J., 2017. Anti-acetylcholinesterase activity of 2015), α-pinene and β-pinene (Perry et al., 2003), and suggest that the aglycones of phenolic glycosides isolated from Leonurus japonicus. Asian Pacific H. umbellata L. aerial parts could present a potential source of these bio- Journal of Tropical Biomedicine 7, 849–854. active cholinesterase inhibitor compounds. Palle, S., Neerati, P., 2017. Quercetin nanoparticles attenuates scopolamine induced spatial memory deficits and pathological damages in rats. Bulletin of Faculty of Pharmacy, Cairo University 55, 101–106. 4. Conclusion Perry, N.S., Bollen, C., Perry, E.K., Ballard, C., 2003. Salvia for dementia therapy: review of pharmacological activity and pilot tolerability clinical trial. Pharmacology Biochemistry and Behavior 75, 651–659. The results of metabolomic study and volatile oil analysis of Perry, E., Howes, M.J.R., 2011. Medicinal plants and dementia therapy: herbal hopes for H. umbellata L. leaves and flowers using UPLC/PDA/ESI-qTOF-MS brain aging. CNS Neuroscience and Therapeutics 17, 683–698. and GC/MS, respectively, reveal that both plant parts present similar Reis, H., Gomes, L., Freitas, M., Nogueira, J.O., Silva, E., Maranha, O., Carneiro, D., 1992. fi Como Utilizar Plantas Medicinais. Sistema Único de Saúde- Ministério da Saúde, chemical pro le. Furthermore, this study provides the most detailed Goiânia. map about the chemical composition of the leaves and flowers, Rocha, F.F., Almeida, C.S., Santos, R.T., Santana, S.A., Costa, E.A., Paula, J.R., Vanderlinde, and may explain, in part, the ethnobotanical use of H. umbellata L. as a F.A., 2011. Anxiolytic-like and sedative effects of Hydrocotyle umbellata L., Araliaceae, extract in mice. Revista Brasileira de Farmacognosia 21, 115–120. memory stimulant herbal remedy. Although these results encourage Rojas, L.B., Usubillaga, A., Rondón, J., Vit, P., 2009. Essential oil composition of Hydrocotyle the use of H. umbellata L. as a supplement in treatment of Alzheimer's umbellata L. from Venezuela. Journal of Essential Oil Research 21, 253–254. disease, it should be followed by more detailed preclinical/clinical Silva, L., Rodrigues, A.M., Ciriani, M., Falé, P.L.V., Teixeira, V., Madeira, P., Serralheiro, M.L.M., 2017. Antiacetylcholinesterase activity and docking studies with chlorogenic acid, examination of extracts, preferably the isolates to be more conclusive. cynarin and arzanol from Helichrysum stoechas (Lamiaceae). Medicinal Chemistry Research 26, 2942–2950. Acknowledgment Sosa, A., Rosquete, C., Rojas, L., Pouységu, L., Quideau, S., Paululat, T., Mitaine-Offer, A.C., Lacaille-Dubois, M.A., 2011. New triterpenoid and ergostane glycosides from the leaves of Hydrocotyle umbellata L. Helvetica Chimica Acta 94, 1850–1859. The authors are grateful to Prof. Dr. Mohamed A. Farag for Zarrad, K., Hamouda, A.B., Chaieb, I., Laarif, A., Jemâa, J.M.B., 2015. Chemical composition, metabolomic study and data interpretation, and thankful to Prof. Dr. fumigant and anti-acetylcholinesterase activity of the Tunisian Citrus aurantium L. – Shahira M. Ezzat for acetylcholinesterase inhibition activity, Pharma- essential oils. Industrial Crops and Products 76, 121 127. cognosy Department, Faculty of Pharmacy, Cairo University.