Fitoterapia 81 (2010) 1053–1057

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Fitoterapia
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e

A new anxiolytic fatty acid from Aethusa cynapium☆

Richa Shri a, K.K. Bhutani b, Anupam Sharma c,⁎
a
Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala-147002, Panjab, India
b
National Institute of Pharmaceutical Education and Research, S.A.S. Nagar, Panjab, India
c
Pharmacognosy Division, University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh-160014, India

a r t i c l e i n f o a b s t r a c t

Article history: The present investigation was carried out with a view to separate bioactive constituent from
Received 1 May 2010 Aethusa cynapium. Bioactivity guided fractionation of the anxiolytic methanol extract has led to
Accepted in revised form 25 June 2010 the isolation of a novel unsaturated fatty acid. Structure of the acid characterized by UV, IR, 1H
NMR, C13 NMR, MS techniques was found to be trideca-7, 9, 11-trienoic acid. Antianxiety
Keywords: activity was confirmed using the mCPP-induced hypolocomotion test. This new fatty acid—
Aethusa cynapium trideca-7,9,11-trienoic acid, isolated from A. cynapium was found to be responsible for the
Bioactivity guided fractionation antianxiety activity of the plant.
Novel unsaturated fatty acid © 2010 Elsevier B.V. All rights reserved.
Anxiolytic activity

1. Introduction antianxiety in mice [10]. Bioactivity guided fractionation of

Aethusa cynapium L. (Umbelliferae/Apiaceae) or Fool's
Parsley is an annual well-known garden weed native to the
United Kingdom [1]. It has been used in traditional medicine
for gastrointestinal complaints in children, infantile cholera,
summer diarrhea, convulsions, mental tension, sleep dis-
orders, delirium, and as stomachic [2,3]. Phytoconstituents
reported from the plant include a volatile alkaloid cynopine
which resembles coniine in its physical and chemical
characters as well as physiological actions [3–7]; polyacety-
lenes [8] including aethusin, aethusanol A & B; essential oil;
flavone glycosides such as rutoside, narcissine, and ascorbic
acid [2]. Fool's Parsley is poisonous when fresh, but is not
harmful when dried [9]. Toxins like cynopine are destroyed
by drying, and hay containing the plant is not poisonous [2,4].
Methanol extract of A. cynapium (400 mg/kg, p.o.), when
evaluated using elevated plus-maze exhibited significant
☆ None of the authors have any financial interest in any of the products
mentioned in this paper.
⁎ Corresponding author. Tel.: +91 172 2541142.
E-mail address: ans1959@rediffmail.com (A. Sharma).
this extract using solvent partitioning; column chromatogra-
phy and flash chromatography led to the separation of a sub
fraction F-3.1.3.2 which was found to be responsible for the
antianxiety activity of the methanol extract [11]. This sub
fraction comprised two components. Its phytochemical
screening revealed the presence of unsaturated fatty acid in
F-3.1.3.2. The present study was carried out to separate the
two components of F-3.1.3.2, and evaluate their antianxiety
activity.
2. Materials and methods
2.1. Plant material
Aerial parts of A. cynapium, were collected from a
cultivated source (Rati Ram Nursery) at village Khurrammpur
via Kalsia, district Saharanpur (U.P., India) in March 2006. The
identity of the plant was confirmed by Dr. H.B. Singh, Head,
Raw Materials, Herbarium & Museum at the National Institute
of Science Communication and Information Resources, (NIS-
CAIR, CSIR), New Delhi 110067. A voucher specimen no:
NISCAIR/RHM/F-3/3/2005/Consult/698//15 is deposited in
the same herbarium.

0367-326X/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.fitote.2010.06.026
1054 R. Shri et al. / Fitoterapia 81 (2010) 1053–1057

2.2. Test material
Bioactive sub fraction F-3.1.3.2 was obtained from meth-
anol extract of A.cynapium after bioactivity guided separation
and was used for further separation. Subfraction F-3.1.3.2 was
obtained following the scheme depicted below (Scheme 1)
[10,11]. Subfraction F-3.1.3.2 was then subjected to prepar-
ative TLC in order to separate bioactive constituent.
2.3. Animals
Swiss albino mice of either sex, weighing 20–24 g were
procured from the Animal House, Punjabi University, Patiala.
The mice were maintained on standard laboratory feed and
water ad libitum. The animals were fasted 18 h prior to the
biological study. The experiments were conducted in a semi-
sound proof laboratory. The biological studies were carried
out as per the guidelines of the Institutional Ethical
Committee (Reg. no.107/1999/CPCSEA) of Department of
Pharmaceutical Sciences and Drug Research, Punjabi Univer-
sity, Patiala, India.
2.4. Preparative TLC
Preparative thin layer chromatography was performed
using 20 × 20 cm glass plates coated (0.5 mm) with silica gel
G (Loba Chemie).
2.5. Elevated Plus Maze
The anxiolytic activity was evaluated using Elevated Plus
Maze model [12–15]. The experimental animals were divided

Scheme 1. Bioactivity guided separation of anxiolytic fraction from Aethusa cynapium.

 R. Shri et al. / Fitoterapia 81 (2010) 1053–1057 1055

into control group, standard group and test groups each

consisting of 5 animals. Vehicle, diazepam and test material
were administered orally to each mouse, 45 min prior to the
observations. Each mouse was placed at the centre of the
elevated plus-maze with its head facing the open arms. The
behaviour of the animals on the plus-maze apparatus was
recorded for 5 min in terms of: (i) the preference of the
animal for first entry to open or closed arms (ii) number of
entries into open or closed arms (iii) the average time spent
by the mouse in open or closed arms.

2.6. Confirmation of antianxiety activity using mCPP-induced

anxiety model
In this model mCPP which is a metabolite of the
antidepressant drug trazodone was used [16–19]. Mice
were divided into four groups—I to IV. Group I and III animals
were administered vehicle whereas group II and IV animals
were administered test sample (p.o.). Forty minutes after the
above treatment, groups I and II were administered the
vehicle (i.p.) while group III and IV animals received mCPP
(7 mg/kg, i.p.). After 20 min of the treatment with the
vehicle/mCPP, locomotor activity of the animals of all groups
was observed in an actophotometer (Digital Photoactometer,
226001, Techno Electronics, Lucknow, India) for 10 min.
2.7. Characterization of bioactive fraction
Bioactive fraction was subjected to UV, Mass, IR, 1H NMR
and 13C NMR spectroscopy. The following instruments at
National institute of Pharmaceutical Education and Research,
Mohali were used: UV spectrophotometer (Beckmann, DU
7400, Germany); IR spectrophotometer (Multi spoke FT-IR
synthesis monitoring system, Perkin-Elmer, Germany); mass
spectrometer (Finnigan, MAT, LCQ, USA) equipped with a
pneumatically-assisted electrospray ionisation source (ESI).
Spectra were scanned in the range of 0–1400 m/z. 1H NMR
and 13C NMR spectra were obtained on a 400 MHz NMR
spectrometer, (Bruker 400, Ultra Shield, ZH079807, Avane,
Germany) using CDCl3 as solvent. Chemical shifts were
expressed in parts per million (ppm) relative to tetramethyl
silane (TMS) as an internal standard.
Fig. 1. Effect of different treatments on mean number of entries by mice in
open arms of EPM. Values are expressed as mean ± SEM. (n = 5). The data
was analyzed by one way ANOVA and post hoc Tukey's multiple range test.
a = p b 0.05 vs. Control (Vehicle); b = p b 0.05 vs. Diazepam (Standard Drug).
3.1. Preparative TLC of Fraction 3.1.3.2
The TLC profile of F-3.1.3.2 had shown the presence of two
components (Rf 0.53 and 0.67 respectively). These two
components were separated using preparative TLC. From
150 mg of F-3.13.2, two compounds were obtained namely
AC-1 (58 mg) and AC-2 (79 mg). The anxiolytic activity of the
compounds—AC-1 and AC-2 was evaluated using the elevated
plus-maze model (Figs. 1 and 2). AC-1 showed significant
anxiolytic effect.
3.2. Confirmation of antianxiety activiy of AC-1 using mCPP-
induced hypolocomotion model
The antianxiety activity of AC-1 was confirmed using
another experimental model—[1-(3-chlorphenyl)piperazine]
induced hypolocomotion. Results of effect of AC-1 on mCPP-
induced hypolocomotion are presented in Fig. 3.
3.3. Characterization of AC-1
AC-1, a cream coloured, amorphous solid, had a melting
point in the range 31–32 °C. AC-1 was subjected to UV, Mass,
IR, 1H NMR and 13C NMR spectroscopy.

2.8. Statistical analysis

The results of biological studies have been expressed as

mean ± SEM. Further the results of biological evaluations
were analyzed by one way ANOVA. The observations obtained
from the test groups were compared with standard/control
by Tukey's Multiple Range Test. Differences were considered
significant at p b 0.05.

3. Results

Phytochemical screening of the fraction F-3.1.3.2 showed

presence of unsaturated fatty acids. TLC of F-3.1.3.2 using
chloroform:methanol (9:1) as the mobile phase showed two
distinct spots (Rf 0.53, Rf 0.67) in the iodine chamber,
indicating unsaturation.
Fig. 2. Effect of different treatments on mean time spent by mice in open
arms of EPM. Values are expressed as mean ± SEM. (n = 5). The data was
analyzed by one way ANOVA and post hoc Tukey's multiple range test.
a = p b 0.05 vs. Control (Vehicle); b = p b 0.05 vs. Diazepam (Standard Drug).
1056 R. Shri et al. / Fitoterapia 81 (2010) 1053–1057
Fig. 3. Effect of AC-1 on mCPP-induced hypolocomotion. Values are
expressed as mean ± SEM. (n = 5). ANOVA followed by Tukey's multiple
range test. p b 0.05. a = significant with respect to Group I, b = significant
with respect to Group II, c = significant with respect to Group III.
The UV spectrum of AC-1 showed maxima at 271 nm
indicating presence of conjugated unsaturation in the
molecule. The IR spectrum featured bands at 3400.9 (free
OH), 2925.4 (C–H stretch) and 1707.5 cm− 1 (unsaturated
conjugated acid) [20]. In the 1H NMR spectrum, six olefenic
protons were observed in the range between 4.9 and 6.7 ppm.
Signals at 0.91 ppm indicated terminal –CH3; 2.3 ppm
showed presence of –CH2– adjacent to –COOH group;
2.4 ppm indicated presence of allylic protons [21]. The signals
observed in the 13C NMR are presented in Table 1.
The MS data indicated a molecular ion peak [M]+ at m/z
209. Molecular weight is 208.30. Elemental analysis indicated
a molecular formula of C13H20O2. Hence, based on this data,
AC-1 was characterized as trideca-7,9,11-trienoic acid—an
unsaturated fatty acid.
The antianxiety activity of A.cynapium is due to the
presence of a novel unsaturated fatty acid—trideca-7,9,11-
trienoic acid. In the present investigation it was found to be
Table 1
13
C NMR data of AC-1.
Carbon number δC
1 176.2
2 34.3
3 25.1
4 29.1
5 29.4
6 33.5
7 131.3
8 128.0
9 129.3
10 129.4
11 128.7
12 129.5
13 18.8
present to the extent of 0.2% w/w in the aerial parts of the
plant.
4. Discussion
Bioactivity guided fractionation of methanol extract of A.
cynapium ultimately led to the isolation of a novel compound
AC-1, which was found to be responsible for the antianxiety
activity of A. cynapium. The compound AC-1 was character-
ized by UV, Mass, IR, 1H NMR and 13C NMR spectroscopy. The
chemical structure of AC-1 was interpreted as trideca-7,9,11-
trienoic acid—an unsaturated fatty acid. This compound has
been reported for the first time from A. cynapium.
Fatty acids are involved in important structural and
physiological functions. The long chain group of fatty acids
include the polyunsaturated fatty acids (PUFAs), which are
fatty acids containing two or more double bonds. There are
two principal families of PUFAs—the omega-3 and the omega-
6 families. PUFAs have been associated with many health
benefits [22–28]. These play an important role in maintaining
mental health both in experimental animals as well as human
beings. Omega-3 fatty acids may play a role in nervous system
activity, improve cognitive development and reference
memory-related learning, increase neuroplasticity of nerve
membranes, contribute to synaptogenesis, and are involved
in synaptic transmission [29–32]. In primates and humans,
fatty acids play an important role in anxiety, aggression,
depression, attention-deficit/hyperactivity disorder (ADHD)
and schizophrenia [33–40].
The antianxiety activity of AC-1 was confirmed using mCPP-
induced anxiety model. In this model, mCPP [1-(3-chlorphenyl)
piperazine] a metabolite of the antidepressant drug trazodone,
is used. It is shown to have anxiogenic effect both in man and
rats [16–18], and produces hypolocomotion in rats [41,42] and
mice [19]. Anxiety in rodents induced by mCPP is recorded as a
period of hypolocomotion. Agents that have antianxiety activity
reduce this induced hypolocomotion [17,18,43]. In the present
investigation mCPP markedly reduced the number of transits
over 10 min and this was significantly reversed by AC-1 thus,
confirming the anxiolytic activity of AC-1.
To conclude the present investigation bioactivity guided
fractionation of methanol extract of A. cynapium ultimately
led to the isolation of a new fatty acid—trideca-7, 9, 11-
trienoic acid, which was found to be responsible for the
antianxiety activity of A. cynapium.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.fitote.2010.06.026.
References
[1] Bond W, Turner RJ. The biology and non-chemical control of Fool's
Parsley (Aethusa cynapium L); 2004. http://www.organicweeds.org.uk.
[2] Fleming T. PDR for herbal medicines. 2nd edition. Montvale: Medical
economics company inc; 2000. p. 316. NJ07647-1742.
[3] Vikramaditya and Joshi, P., (1997). A guide to important medicinal plants
used in homeopathy, V2, Homeopathic Pharmacopoeial laboratory, India,
4 and 23.
[4] Moreau J. Rudiments of Hahnemann's first material medica. L'
Homdopathie Europ Fenne 1996;5:19–20.
 R. Shri et al. / Fitoterapia 81 (2010) 1053–1057
[5] Clapham AR, Tutin TG, Moore DM. Flora of the British Isles. 3rd edition.
Cambridge, UK: Cambridge University Press; 1987.
[6] Tutin TG. Umbellifers of the British Isles. BSBI Handbook No. 2. London,
UK: Botanical Society of the British Isles; 1980.
[7] Power FB, Tutin F. Chemical examination of Aethusa cynapium. J Am
Chem Soc 1905;27:1461–76.
[8] Andreev GN, Schulz H, Schrader B, Fuchs R, Popov S, Handjieva N. Non-
destructive NIR-Ft-Raman analyses in practice. Part I. Analyses of plants
and historic textiles. Fresenius J Anal Chem 2001;371(7):1009–17.
[9] Salisbury, E. J., (1961). Weeds & Aliens. New Naturalist Series, Collins,
London., Society of the British Isles, London, UK.
[10] Shri R, Singh M, Sharma A. Anxiolytic activity screening studies on
extracts of a few medicinal plants. Comprehensive Bioactive Natural
Products, vol. 2. Efficacy, Safety, and Clinical evaluation I. M/S. USA:
Studium Press LLC; 2010. p. 211–8.
[11] Shri R, Singh M, Sharma A. Bioactivity-directed separation of an
anxiolytic fraction from Aethusa cynapium L. Phcog Res 2009;1(6):
336–41.
[12] Montgomery KC. The relation between fear induced by novel
stimulation and exploratory behaviour. J Comp Physiol Psychol
1958;48:254–60.
[13] Pellow S, Chopin PH, File SE, Briley M. Validation of open:closed arm
entries in an elevated plus maze as a measure of anxiety in the rat. J
Neurosci Meth 1985;14:149–67.
[14] Lister RG. The use of a plus-maze to measure anxiety in the mouse.
Psychopharmacology (Berl) 1987;92(2):180–5.
[15] Kulkarni SK, Reddy DS. Animal behavioral models for testing antianx-
iety agents. Meth Find Exp Clin Pharmacol 1996;18:219.
[16] Curzon G, Gibson EL, Kennedy AJ, Kennett GA, Sarna GS, Whitton P.
Anxiogenic and other effects of mCPP, a5-HT1C agonist. In: Briley M, File
SE, editors. New concepts in anxiety. London: McMillan Press Ltd; 1991.
p. 154–67.
[17] Bilkei-Gorzo A, Gyertyar I, Szabados T. mCPP-induced anxiety—a
potential new method for screening anxiolytic activity. Neurobiology
1996;4:253–5.
[18] Bilkei-Gorzo A, Gyertyar I, Levay G. mCPP-induced anxiety in the light–
dark box in rats—a new method for screening anxiolytic activity.
Psychopharmacology 1998;136:291–8.
[19] Griebel G, Misslin R, Pawloaski M, Vogel E. m-Chloro-phenylpiperazine
enhances aeophobic and anxious behaviour in mice. NeuroRepor
1991;2:627–9.
[20] Rezanka T. Glycosides of polyenoic branched fatty acids from
myxomycetes. Phytochemistry 2002;60:639–46.
[21] Pavia DL, Lampman GM, Kriz GS. Introduction to spectroscopy: a guide
for students of organic chemistry. 3rd edition. New York: Harcourt
College Publishers; 2000. p. 13–166.
[22] Rizzo MT, Regazzi E, Garau D, Akard L, Dugan M, Boswell HS, et al.
Induction of apoptosis by arachidonic acid in chronic myeloid leukemia
cells. Cancer Res 1999;59:5047–53.
[23] WHO. Diet, nutrition and the prevention of chronic diseases. Technical
report series 916. Geneva; 2003. http:// whqlibdoc.who.int/trs/
who_TRS_916.pdf.
[24] Stillwell W, Wassall SR. Docosahexaenoic acid: membrane properties of
a unique fatty acid. Chem Phys Lipids 2003;126:1–27.
[25] Jacobsen C. Developing polyunsaturated fatty acids as functional
ingredients. In: Arnoldi A, editor. Functional Foods, Cardiovascular
Disease and Diabetes. Cambridge, England: CRCPress, Woodhead
Publishers; 2004. p. 307–32.
1057
[26] Timonen M, Horrobin D, Jokelainen J, Laitinen J, Herva A, Rasanen P. Fish
consumption and depression: the Northern Finland 1066 birth cohort
study. J Affect Disord 2004;82:447–52.
[27] Sala-Vila A, Campoy C, Castellote AI, Garrido FJ, Rivero M, Rodriguez-
Palmero M, et al. Influence of dietary source of docosahexaenoic and
arachidonic acids on their incorporation into membrane phospholipids
of red blood cells in term infants. Prostaglandins Leukot Essent Fatty
Acids 2006;74:143–8.
[28] Chavarro JE, Stampfer MJ, Li H, Campos H, Kurth T, Ma J. A prospective
study of polyunsaturated fatty acid levels in blood and prostate cancer
risk. Cancer Epidemiol Biomark Prev 2007;16:1364–70.
[29] Salem Jr N, Kim H-Y, Yergey JA. Docosahexaenoic acid: membrane
function and metabolism. In: Simopoulos AP, Kifer RR, Martin R, editors.
The health effects of polyunsaturates in seafoods. New York: Academic
Press; 1986. p. 263–317.
[30] Salem Jr N. Omega-3 fatty acids: molecular and biochemical aspects. In:
Spiller GA, Scala J, editors. Current topics in nutrition and disease: new
protective roles for selected nutrients. New York: Alan R. Liss; 1989.
p. 109–228.
[31] Lauritzen L, Hansen HS, Jùrgensen MH, Michaelsen KF. The essentiality
of long chain n−3 fatty acids in relation to development and function of
the brain and retina. Prog Lipid Res 2001;40:1–94.
[32] Mazza M, Pomponi M, Janiri L, Bria P, Mazza S. Omega-3 fatty acids and
antioxidants in neurological and psychiatric diseases: an overview.
Progr Neuro-Psychopharmacol Biol Psychiatry 2007;31:12–26.
[33] Mahadik SP, Evans D, Lal H. Oxidative stress and role of antioxidant and
o-3 essential family acid supplementation in schizophrenia. Progr
Neuro-Psychopharmacol Biol Psychiatry 2001;25:463–93.
[34] Simopoulos AP. The importance of the ratio of omega-6/omega-3
essential fatty acids. Biomed Pharmacother 2002;56:365–79.
[35] Morris MC, Evans DA, Tangney C, Bienias J, Wilson RS. Fish consumption
and cognitive decline with age in a large community study. Arch Neurol
2005;62:1046.
[36] McNamaraa RK, Carlson SE. Role of omega-3 fatty acids in brain
development and function: potential implications for the pathogenesis
and prevention of psychopathology. Prostaglandins Leukot Essent Fatty
Acids 2006;75:329–49.
[37] Fedorova I, Salem N. Omega-3 fatty acids and rodent behavior.
Prostaglandins Leukot Essent Fatty Acids 2006;75:271–89.
[38] Ferraz AC, Kiss A´, Arau´ jo RLF, Maria He´, Salles R, Naliwaiko K, et al. The
antidepressant role of dietary long-chain polyunsaturated n−3 fatty
acids in two phases in the developing brain. Prostaglandins Leukot
Essent Fatty Acids 2008;78:183–8.
[39] Benton D. The impact of diet on anti-social, violent and criminal
behaviour. Neurosci Biobehav Rev 2007;31:752–74.
[40] Ferraz AC, Kiss A´, Arau´ jo RLF, Maria He´, Salles R, Naliwaiko K, et al. The
antidepressant role of dietary long-chain polyunsaturated n−3 fatty
acids in two phases in the developing brain. Prostaglandins Leukot
Essent Fatty Acids 2008;78:183–8.
[41] Kennett GA, Wood MD, Bright F, Cilia J, Piper DC, Gager T, et al. In vitro and
in vivo profile of SB 206553, a potent 5-HT2c/5-HT2B receptor antagonist
with anxiolytic like properties. Br J Pharmacol 1996;117:427–34.
[42] Kennett GA, Wood MD, Bright F, Riley G, Holland V, Avenell KY Stean,
et al. Sb 242084, a selective and brain penetrant 5-HT2C receptor
antagonist. Neuropharmacology 1997;36:609–20.
[43] Wallis CJ, Lal H. A discriminative stimulus produced by l-(3-chlor-
ophenyl)-piperazine (mcpp) as a putative animal model of anxiety.
Prog Neuropsychopharmacol 1998;12:647–66.