DDR

DRUG DEVELOPMENT RESEARCH 73 : 130137 (2012)

Research Article

Pharmacological and Toxicological Profile of Extract

from Heliopsis longipes and Affinin
Myrna Dciga-Campos,1* Myriam Arriaga-Alba,2 Rosa Ventura-Martnez,3
Berenice Aguilar-Guadarrama,4 and Mara Yolanda Rios4
1
Seccin de Estudios de Posgrado e Investigacin, Escuela Superior de Medicina,
Instituto Politcnico Nacional, Mxico D.F., Mxico
2
Laboratorio de Investigacin en Microbiologa, Direccin de Investigacin y Enseanza,
Hospital Jurez de Mxico, Mxico D.F., Mxico
3
Departamento de Farmacologa, Facultad de Medicina de la, Universidad Nacional Autnoma
de Mxico (UNAM), Mxico D.F., Mxico
4
Centro de Investigaciones Qumicas, Universidad Autnoma del Estado de Morelos,
Cuernavaca, Morelos, Mxico

Strategy, Management and Health Policy

Enabling
Technology,
Genomics,
Proteomics

Preclinical
Research

Preclinical Development
Toxicology, Formulation
Drug Delivery,
Pharmacokinetics

Clinical Development
Postmarketing
Phases I-III
Phase IV
Regulatory, Quality,
Manufacturing

ABSTRACT
Heliopsis longipes is a popular medicinal plant in Mexico. One of the main constituents
that can be extracted from H. longipes is affinin (N-isobutylamide). However, available information
regarding this compound is scarce, and there is only a single report related to the effect of affinin on the
central nervous system. Affinin extracted from H. longipes was evaluated for its psychopharmacological
activity in several models and for its safety. H. longipes extract and affinin demonstrated antinociceptive
effect, modified anxiety behavior and prolonged the time of sodium pentobarbital-induced hypnosis.
Affinin elicited these activities at high doses. Both the extract and affinin decreased the time of clonic and
tonic PTZ-induced seizures. In the Ames test, neither the extract nor affinin induced mutations in the
Salmonella typhimurium strains TA98 and TA100 or TA102 with or without the S9 microsomal fraction.
Lethal dose 50 values in mice suggest that the H. longipes extract may contain sedative principles with
potential anxiolytic activity; however, it may also increase convulsive activity. Drug Dev Res 73 : 130137,
2012.
2012 Wiley Periodicals, Inc.
Key words: Heliopsis longipes; affinin; alkamide; anxiolytic; antinociceptive; mutagenicity

INTRODUCTION

Heliopsis longipes (A. Gray) Blake (Compositae)

is a herbaceous plant found in Guanajuato, San Luis
Potos, and Queretaro states in Mexico. This plant
is known as gold root, and in Mexico, it is known
as chilcuague, chilcuan, chilmecatl, chilicuau,
pelitre, peritre, raz azteca, or raz de oro
[Molina-Torres and Garca, 2001]. The roots of H. longipes have been used as a spice for flavoring and for the
stimulation of salivation. In addition, alcohol extracts
2012 Wiley Periodicals, Inc.

Support information: SIP-IPN 20110653 Grant (MD-C).

*Correspondence to: Myrna Dciga-Campos, Seccin de
Estudios de Posgrado e Investigacin, Instituto Politcnico
Nacional., Plan de San Luis y Daz Mirn s/n Col. Santo Tomas,
Delegacin Miguel Hidalgo. C.P. 11340, Mxico D.F., Mxico.
E-mail: myrnadeciga@hotmail.com; mdeciga@ipn.com.mx
Received 7 December 2011; Accepted 8 December 2011
Published online in Wiley Online Library (wileyonlinelibrary.
com). DOI: 10.1002/ddr.21002

PHARMACOLOGICAL AND TOXICOLOGICAL PROFILE

131

from H. longipes have been used as an anesthetic

for tooth extractions. H. longipes roots are known to
contain bioactive olefinic alkamide compounds. Affinin
(N-isobutyl-2E,6Z,8E-decatrienamide) is the main
lipid component of the plant [Jacobson et al., 1947;
Jacobson, 1954, 1955; Molina-Torres et al., 1995, 1996].
The 2E unsaturation has been proposed to contain
insecticidal activity [Jacobson, 1954]. H. longipes and
affinin have been studied for fungistatic, bacteriostatic,
and analgesic activities [Molina-Torres et al., 2004;
Dciga-Campos et al., 2010]. However, there have
been few reports that pertain to central nervous system
(CNS) activity. We therefore studied the neuropharmacological profile of the plant extract with the present
study being designed to evaluate the CNS activity of
H. longipes and affinin in various rat and mouse experimental models. In addition, the safety profile of
H. longipes extract and affinin were studied.

affinin has been previously reported [Dciga-Campos

et al., 2010]. The total yield of pure affinin was 18.42%
yield with respect to extract weight. The structure was
established on the basis of IR, UV, 1H and 13C NMR,
DEPT, COSY, HSQC, HMBC, and MS (InfraRed,
UltraViolet, Nuclear Magnetic Resonance, Distortionless Enhancement by Polarization Transfer, Correlation
Spectroscopy, Heteronuclear Single Quantum Coherence, Heteronuclear Multiple-Bond Correlation spectroscopy, and Mass Spectrometry) data and compared
with that previously reported [Dciga-Campos et al.,
2010]. The purity of affinin (96.15%) was established
using NMR and comparison of its retention time
(20.57 min) with respect to an authentic sample on gas
chromatographymass spectrometry (GC-MS) analysis
(vide infra) [Dciga-Campos et al., 2010]. The H. longipes extract and affinin were suspended in 1% carboxymethylcellulose in 0.9% saline solution.

MATERIALS AND METHODS

Chemicals and Drugs

Animals

Pentylenetetrazole (PTZ), dimethyl sulfoxide

(DMSO), 2-aminoanthracene (2AA), methyl-Nnitro-N-nitrosoguanidine (MNNG), histidine, biotin,
picrolonic acid (PA), cyclophosphamide (CP), and
mitomycin C (MC) were purchased from SigmaAldrich Co. (St. Louis, MO, USA). Aroclor-1254 was
obtained from Supelco (Bellefonte, PA, USA). Formaldehyde was from J.T. Baker. Sodium pentobarbital
(PB) (SEDAL-VET; Pets Pharma, Estado de Mexico,
Mexico)) and diazepam (Valium) were used in this
study. Salmonella typhimurium strains were kindly provided by Professor Bruce Ames (Berkley, CA, USA). All
treatments were freshly prepared for each use and were
administered in a volume of 0.1 ml/10 g of body weight
i.p. 15 min before the neuropharmacological tests were
performed. For nociception pretreatment, the H. longipes extract, affinin, and vehicle were administered in
a volume of 50 ml in the right (ipsilateral, IL) or left paw
(contralateral, CL) of the rats 15 min before formalin
(50 ml of 1% formaldehyde, IL) injection. The control
animals received the same volume of vehicle (1% carboxymethylcellulose in 0.9% saline solution).

Experiments were performed using male ICR

(Charles River Laboratories, Wilmington, DE) mice
(2530 g) and Wistar rats (180200 g) from Centro
Harlan (Harlan Mxico, SA of CV, Mexico). All experiments were conducted in accordance with the Guidelines on Ethical Standards for Investigation of Mexican
Official Norm for Animal Care and Handing (NOM062-ZOO-1999). Efforts were made to minimize animal
suffering and to reduce the number of animals used.
Animals were housed in groups of seven per standard
cage in climate-controlled rooms (22 2C) with a
12-h light/dark cycle. Each animal was tested once. At
the end of the experiment, animals were sacrificed in a
CO2 chamber.
Plant Material
Fresh roots from H. longipes (Compositae) were
collected on 28 July 2007 at Real de Xichu in Guanajuato and identified by M.C. Ramiro Rios Gmez. A
voucher specimen (number 5904) was deposited at the
FEZA Herbarium at the Facultad de Estudios Superiores Zaragoza (Universidad Nacional Autnoma de
Mxico) in Mxico.
Extract Preparation and Isolation of Affinin
Fresh roots from H. longipes (4.7 kg) were
extracted with acetone (24 l 3) at room temperature.
The extraction solvent was concentrated to dryness
in vacuo to render 106.5 g of extract. The isolation of

Pharmacological Evaluation
Antinociceptive effect
The procedure performed was similar to that previously described by Wheeler-Aceto and Cowan [1991].
The rats were placed in an open Plexiglas observation
chamber for 30 min to accommodate them to their
surroundings. They were then removed for formalin
administration. Formalin (1%, 50 ml) was injected into
the dorsal surface of the right hind paw with a 30-g
Drug Dev. Res.

132

DCIGA-CAMPOS ET AL.

needle. The animal was returned to the Plexiglas

chamber for observation, and nociceptive behavior was
immediately observed after formalin injection. A mirror
was placed behind the chamber to enable unhindered
observation. Nociceptive behavior was quantified as the
numbers of flinches of the injected paw during 1-min
intervals every 5 min up to 60 min after injection.
Flinching was readily identified and was characterized
as rapid and brief withdrawal or flexing of the injected
paw. Formalin-induced flinching behavior is biphasic,
with the initial acute phase (010 min) being followed
by a relatively short quiescent period, which is then
followed by a prolonged tonic response (1560 min).
The rats received an equivalent dose of vehicle (50 ml)
or increasing doses of the H. longipes extract and affinin
15 min before formalin treatment. Curves were constructed by plotting the number of flinches as a function
of time. The area under the number of flinches against
time curve, an expression of the duration and intensity
of the effect, was calculated using the trapezoidal rule.
CNS evaluation
The procedure for CNS activity was performed in
the male mice. All mice were adapted to manipulations
through a daily saline solution injection (10 ml/kg, i.p.)
that began 5 days before evaluation of the H. longipes
extract (130 mg/kg, i.p.), affinin (130 mg/kg, i.p.), or
diazepam (1 mg/kg, i.p.), which served as a positive
control for each experimental procedure. The groups
were composed of seven mice. Ambulatory activity,
antianxiety effects (the elevated plus-maze, hole board
test, and exploratory rearings), sodium PB-induced
hypnosis, and PTZ-induced seizures were evaluated
according to previously published methods [GonzlezTrujano et al., 2006].
Ambulatory activity
Fifty minutes after the administration of vehicle,
diazepam, the H. longipes extract, or affinin, the mice
were placed one by one inside a cage that was divided
into 12 squares (4 cm 4 cm). The number of squares
explored by each mouse in a 2 min interval was
recorded as the ambulatory activity. This mouse continued with the antianxiety test.
Antianxiety Effect
Hole board test
Mice were individually placed in the center of a
perforated board and the number of head dips was
registered during a 3-min trial. The perforated board
test was made by using a table floorboard that was
Drug Dev. Res.

40 cm 40 cm 25 cm with 16 holes (3 cm in diameter) equally spaced in the floor. The holes provided a
measure of the number of head dips.
Exploratory rearings
Each mouse was individually placed on a filter
paper-covered floor in a glass cylinder (16 cm in height
and 11 cm in diameter). The number of spontaneous
rearings on its posterior limbs was counted during the
first 5 min, and a reduced number of exploratory rearings indicated an anxiolytic effect.
The elevated plus-maze
This was composed of two open arms (25 cm
5 cm 15 cm) with an open roof and walls that were
40 cm in height. The number of entries into any of the
arms and the total time spent in each of the two arm types
were observed and recorded by an observed during a
5-min test period after the mice had been placed in the
center of the maze. The total number of entries into the
arms provided a measure of the general activity.
Sodium PB-induced hypnosis
A 40-mg/kg i.p. dose of PB was administered
30 min after administration of the H. longipes extract or
affinin. Each mouse was observed for the onset of uncoordinated motor behavior (sedative phase), loss of righting reflex (hypnosis), and duration of sleep. The criterion
for sleep was defined as the loss and recovery of the
righting reflex, which was recorded as the sleep time.
PTZ-induced seizures
An 80-mg/kg i.p. dose of PTZ was administered
15 min after the injection of the H. longipes extract or
affinin. The latency for onset of the generalized clonic
and tonic seizures was evaluated for 30 min after PTZ
injection. The control animals were administered with
vehicle. If no convulsions or mortality occurred during
the 30-min time limit, the animals were considered to
be protected from PTZ-induced seizures.
Toxicological Evaluation
Acute toxicity study in mice
Experiments were performed using male ICR
mice. Food was withheld for 12 h prior to any experiments with animals having free access to drinking
water. The concentration of the tested compounds was
adjusted for oral administration of 0.2 ml/10 g body
weight. The mice were treated in two phases. In the

PHARMACOLOGICAL AND TOXICOLOGICAL PROFILE

first phase, intragastric doses of 10, 100, and 300 mg/kg

of crude extract were administered (three animals per
dose) according to the Lorke [1983] method. For the
second phase, the doses were administered at 20, 40,
60, and 80 mg/kg. In both phases, the mice were
observed daily for a period of 14 days for mortality, toxic
effects, and/or changes in behavioral patterns.
Ames test
The mutagenicity tests were performed using
the tube incorporation method described by Maron
and Ames [1983] and Dciga-Campos et al. [2007].
S. typhimurium (His) strains TA98, TA100, and TA102
were grown in oxoid-2 nutrient broth for 16 h at 37C
with agitation (90 rpm in an American Scientific-BT23
water bath, Toronto, Canada). After growth, 100 ml of
each strain was transferred to sterile screw-top tubes
with 2 ml of soft agar that contained histidine-biotin
(0.05 mM) and 10 ml of the H. longipes extract or affinin
dissolved in DMSO (1080 mg/Petri dish). The assay was
performed with or without 500 ml of enzymatic liver
fractions (S-9 mix) obtained from male Wistar rats
treated with 20 mg/kg of Aroclor-1254 as previously
described by Maron and Ames [1983]. The total tube
contents were spread onto plates with VogelBonner
media and were incubated for 48 h at 37C. All experiments were performed in triplicate. The number of
histidine reversion colonies was determined using a
Fisher colony counter. The reversion rate was compared
with control plates with and without mutagens. Positive
controls for TA98, TA100, TA102 without the enzymatic
fraction (S-9 mix) added were PA (50 mg/plate), MNNG
(10 mg/plate), and MC (20 ng/plate), respectively. Positive controls employed in the presence of the S-9
mixture were 2AA (20 mg/plate) for the TA98 and TA102
strains and CP (500 mg/plate) for the TA100 strain.
Statistical analysis
All results are presented as a mean of seven
animals SEM except in the toxicological studies
where three animals were used per group [Lorke,
1983]. Statistical differences were analyzed using oneway analysis of variance followed by Dunnetts test, and
a value of P < 0.05 was considered to be significant.
RESULTS

Pharmacological Assays
Administration of 50 ml of formalin (1%) produced a typical flinching behavior. The first phase
began immediately after administration of formalin and
diminished gradually after approximately 10 min. The

133

second phase began after 15 min and lasted for 1 h. IL

administration of the H. longipes extract (0.33 mg/
paw) and affinin (0.1-1.7 mg/paw) produced a dosedependent reduction in the flinching behavior in both
phases (Fig. 1). When the H. longipes extract (3 mg/
paw) and affinin (1.7 mg/paw) were administered in the
CL paw, the antinociceptive effect was similar to IL
administration (Fig. 1).
As shown in Table 1, the H. longipes extract
(130 mg/kg, i.p.) and affinin (130 mg/kg, i.p.) did not
induce significant changes in ambulatory activity at any
of the tested doses. However, changes were observed
with 1 mg/kg of diazepam (27.7 2.3 number of
squares/2 min) with respect to vehicle (60.8 2.1
number of squares/2 min). Anxiety was tested in the
open-side arms, head dipping, and rearing tests. The
H. longipes extract (10 and 30 mg/kg, i.p.) significantly
increased the latency in the open-side arms test. The
antianxiety effect of the extract resulted in approximately 65% of the diazepam-induced antianxiety effect.
The antianxiety effects that were measured by the head
dipping and rearing tests decreased when the mice
were administered 10 and 30 mg/kg of the H. longipes
extract. Affinin administration decreased the number
of head dipping only at the maximum dose evaluated
(30 mg/kg, i.p.). However, in the rearing test, affinin
showed a dose-dependent antianxiety effect (330 mg/
kg, i.p.). Therefore, part of the antianxiety effects of the
H. longipes extract was due to by affinin. Diazepam
(1 mg/kg, i.p.) was used as a positive control, and it
decreased the time in the open-side arms and
decreased head dipping and rearing (see Table 1).
The H. longipes extract modified the latency to
PB-induced sedative-hypnotic effects with respect to
vehicle administration in a dose-dependent manner.
Affinin induce a similar behavior to vehicle administration (Table 2). These results suggest that the
H. longipes extract has sedative effects and that affinin
was not involved in this effect.
The H. longipes extract (130 mg/kg, i.p.) and
affinin (130 mg/kg, i.p.) showed no significant difference with respect to vehicle on latency of the onset to
clonus in the PTZ-induced seizure model. All doses of
the H. longipes extract tested decreased the latency of
the onset to tonus. However, affinin administration did
not modify tonic behavior as the vehicle did, and the
animals died in a dose-dependent manner after tonic
convulsions (Table 3). Diazepam prevented the convulsions and no animals died on its administration.
Toxicological Assays
The lethal dose 50 (LD50) study was performed to
determine the dose of the extract that killed 50% of the
Drug Dev. Res.

134

DCIGA-CAMPOS ET AL.

150

1,000
800

AUC (Fliches/min)

AUC (Fliches/min)

200

*
100

50

600

200

V (IL) V (CL)

0.3

1.0

1.7

3 CL

V (IL) V (CL)

H. longipes extract (mg/paw)

0.3

1.0

1.7

3 CL

H. longipes extract (mg/paw)

C
200

1,000

150
*
*

100

50

AUC (Fliches/min)

AUC (Fliches/min)

400

800
*

600

400
200
0

V (IL) V (CL)

0.1

0.3

1.0

1.7

1.7 CL

V (IL) V (CL)

0.1

0.3

1.0

1.7

1.7 CL

Affinin (mg/paw)

Affinin (mg/paw)

Fig. 1. Local antinociceptive effect of Heliopsis longipes extract and affinin during the formalin test. The rats received H. eliopsis longipes
extract (A, Phase I; B Phase II) and affinin (C, Phase 1; D, Phase II) in either the right (ipsilateral, IL) or left (contralateral, CL) paw and an injection
of 1% formalin (50 ml). Data are expressed as the area under the number of flinches against time curve (AUC). Bars are the mean SEM for six
animals. *Significantly different from the IL vehicle group (P < 0.05), and # significantly different from the CL vehicle (P < 0.05) group, as
determined by analysis of variance followed by Dunnetts test.

TABLE 1. Ambulatory Activity and Antianxiety Effects of Heliopsis longipes, Affinin, and Diazepam in Mice

Treatment
Vehicle
H. longipes extract

Affinin

Diazepam

Dose mg/kg
(i.p.)

Number of squares
(counts/2 min)

Time in open-side
arms (s)

Head dipping
(counts/2 min)

Rearings
(counts/5 min)

1
3
10
30
1
3
10
30
1

60.8 2.1
56.8 2.2
56.9 1.8
57.9 2.3
57.3 1.7
59.2 1.1
58.4 1.7
56.4 1.9
56.8 2.1
27.7 2.3

58.6 2.0
52.1 6.2
63.9 5.6
86.6 8.5*
141.4 10.9*
56.3 3.7
50.2 5.3
56.1 3.6
49.2 6.3
215 9.5*

49.3 2.2
45.9 4.1
42.9 3.4
37.1 2.4*
30.1 3.0*
46.2 1.5
38.5 4.2
41.9 4.6
36.1 2.3*
18.5 1.9*

39.1 1.2
35.9 1.2
34.3 1.9*
28.6 1.9*
25.9 2.0*
36.7 2.5
34.2 1.8*
28.5 1.7*
24.1 1.1*
3.4 1.5*

Data represent the mean SEM for seven trials.

*P 0.05, ANOVA, followed by Dunnetts test compared with the vehicle-treated group.

Drug Dev. Res.

PHARMACOLOGICAL AND TOXICOLOGICAL PROFILE

135

TABLE 2. Effect of Heliopsis longipes Extract and Affinin on Sodium Pentobarbital-Induced Hypnosis Potentiation in Mice

Treatment

Latency to the onset (min)

Dose mg/kg
(i.p.)

Sedation

Hypnosis

Duration of the
hypnosis (min)

1
3
10
30
1
3
10
30

1.13 0.02
0.98 0.06
0.86 0.03*
0.72 0.02*
0.74 0.05*
1.08 0.03
0.96 0.05
0.98 0.12
1.0 0.02

3.21 0.46
2.9 0.31
2.4 0.28*
2.9 0.32
2.5 0.29*
3.1 0.47
2.98 0.56
3.12 0.52
2.8 0.22

19.3 0.74
20.12 0.68
28.44 0.56*
32.21 0.32*
30.35 0.48*
18.31 0.92
19.77 0.62
18.32 0.89
19.18 0.75

Vehicle + pentobarbital
H. longipes extract + pentobarbital

Affinin + pentobarbital

Data represent the mean SEM for seven trials.

*P 0.05, ANOVA, followed by Dunnetts test compared with the vehicle/pentobarbital-treated group.

TABLE 3. Effect of Heliopsis longipes and Affinin on Pentylenetetrazole (PTZ)-Induced Seizures in Mice

Treatment
Vehicle + PTZ
H. longipes extract + PTZ

Affinin + PTZ

Diazepam

Latency to the onset (min)

Dose mg/kg
(i.p.)

Clonus

Tonus

Mortality

1
3
10
30
1
3
10
30
1

0.8 0.2
1.1 0.4
1.1 0.4
0.7 0.3
0.7 0.1
1.1 0.2
1.1 0.3
1.1 0.2
1.4 0.7
30 0***

6.2 1.3
4.5 0.7*
4.2 0.2*
3.6 0.9*
3.8 0.14*
6.0 1.6
6.5 1.4
6.1 0.8
4.3 1.3*
30 0*

7/7
7/7
7/7
7/7
7/7
2/7
2/7
4/7
6/7
0/7*

Data represent the mean SEM for six trials.

*P 0.05, ANOVA, followed by Dunnetts test compared with the vehicle/PTZ-treated group.

animal population by being given i.p. The H. longipes

extract (10, 100, and 300 mg/kg) and affinin (1, 10 and
100 mg/kg) were orally administered in the first stage.
At this step, 2/3 and 3/3 of the animals died when
100 mg/kg of the H. longipes extract and affinin were
administered, respectively. The second dosing experiment was performed with decreased doses of 20, 40, 60,
and 80 mg/kg (3three mice per group). In this study, 2/3
of the animals died with 60 mg/kg of H. longipes
extract, and 2/3 of the animals died with 80 mg/kg of
affinin. Therefore, the LD50 value was calculated with a
geometrical median according to the Lorke [1983]
method, and the H. longipes extract (LD50 = 62.14 mg/
kg) was more toxic than affinin (LD50 = 113.13 mg/kg)
in mice. In addition, H. longipes and affinin were not
mutagenic (Table 4).
DISCUSSION AND CONCLUSIONS

H. longipes has been used for treating pain in folklore medicine, suggesting the presence of constituents
with analgesic and/or anti-inflammatory properties. The

present results show that peripheral application of

H. longipes and affinin into the dorsal surface of the
right hind paw have antinociceptive effects in the two
phases of the rat formalin test. Intraperitoneal administration of the H. longipes extract demonstrated antinociceptive activity in the writhing test [Ogura et al.,
1982; Cario-Corts et al., 2010; Dciga-Campos et al.,
2010], capsaicin-induced nociception [Dciga-Campos
et al., 2010], and hot plate test [Cario-Corts et al.,
2010]. This effect was correlated with antihyperalgesic
effects in carrageenan-induced thermal hyperalgesia
[Acosta-Madrid et al., 2009] and in anti-inflammatory
activity evaluated through the mouse ear edema test
using two irritating agents, arachidonic acid, and
phorbol myristate acetate [Hernndez et al., 2009].
Antinociception may be explained by the fact that the
extract has a complex mixture of oleofinic alkamides
[Molina-Torres et al., 1996] that may synergize the antinociceptive effect. Affinin was identified as the main
alkamide present in the plant extract [Jacobson et al.,
1947; Jacobson, 1954, 1955; Molina-Torres et al., 1995].
Recently, it was observed that affinin-induced nocicepDrug Dev. Res.

136

DCIGA-CAMPOS ET AL.

TABLE 4. Mutagenic Evaluation of the Ethanolic Extract of Heliopsis longipes and Affinin on the Salmonella typhimurium Ames Test
Number of histidine revertants/plate
TA98
S9
0
23.5 6.09
DMSO
25.0 4.78
Positive control 493.67 125.4*
H. longipes extract (mg/Petri)
10
25.58 7.78
20
25.33 5.54
40
25.41 7.08
80
24.75 19.0
Affinin (mg/Petri)
6.25
27.0 9.59
12.5
26.99 6.23
25
31.17 9.26
50
29.5 10.17

TA100
+S9

TA102
+S9

S9

S9

+S9

22.67 7.23
124.00 23.74
124.5 23.68
244.44 72.79
276.67 61.64
23.4 8.20
113.33 16.42
156.33 26.95
298.33 83.94
276.66 61.64
703.6 164.2* 3,709.67 648.9* 2,245.33 316.33* 2,384.88 151.89* 1,322.83 146.76*
32.16 8.38
26.58 7.16
31.03 11.59
28.75 19.00

159.89 27.99
276.83 51.90
169.66 28.0
179.88 26.60

177.01 25.62
165.33 21.49
135.55 33.81
143.44 42.66

244.44 48.50
249.22 60.22
248.22 49.65
250.33 56.33

272.88 35.48
285.44 57.75
295.44 75.05
285.33 46.99

30.83 7.34
27.66 10.65
27.16 4.53
24.01 12.60

123.83 11.09
116.83 12.67
122.0 12.19
128.5 12.96

123.83 11.09
139.83 25.42
141.50 35.12
146.83 25.17

245.88 70.30
240.11 65.53
266.44 76.46
245.33 73.23

262.0 68.03
265.78 70.98
294.56 52.97
319.33 96.30

Positive controls: +S9, 2-AA (10 mg/plate) for TA98 strain; CP (500 mg/plate) for TA100 strain and 2-AA (10 mg/plate) for the TA102 strains. S9,
PA (50 mg/plate) for TA98; MNNG (10 mg/plate) for TA100 and mitomycin C (20 ng/plate) for TA102. The results are reported as the mean SD.
*P 0.05 with respect to vehicle (DMSO). Each value represents the average of nine Petri dishes in three independent studies.

tion involves increasing g-amino butyric acid release in

mice brain slices [Rios et al., 2007] with the latter
resulting in the activation of the opioidergic, and serotoninergic systems as well as nitric oxide-K+ channel
pathways [Dciga-Campos et al., 2010]. The present
results show that peripheral administration of H. longipes extract and affinin, either ipsilaterally or contralaterally, produced a similar antinociceptive effect in
response to formalin-induced nociceptive behavior.
These results demonstrated the systemic antinociceptive activity of this traditional plant and affinin, which
was isolated from the plant. It is known that phase one
of nociceptive behavior is due to direct stimulation of
peripheral nociceptors by formalin resulting in a
barrage of primary afferent activity [Puig and Sorkin,
1996]. It was proposed that the latter leads to the
central sensitization of dorsal horn nociceptive neurons
and that these central changes underlie phase two nociceptive behavior independent of peripheral inputs
[Coderre et al., 1990]. However, the primary afferent
activity persists into phase two, which suggests that this
phase involves both peripheral and central mechanisms
[Puig and Sorkin, 1996; Pitcher and Henry, 2002].
In Mexico, H. longipes is used as a local analgesic
and anesthetic. It produces an intense numbness and
tingling sensation in the lips, tongue, and mouth, and
also stimulates salivation [Correa et al., 1971]. Therefore, we wanted to identify other activities of H. longipes extract and affinin using a neuropharmacological
profile that included antianxiety responses, hypnotic
potentiation, anticonvulsant, and antinociceptive
effects. Administration of high doses of the H. longipes
Drug Dev. Res.

extract generated an antianxiety response in the openside arms, head dipping, and rearing tests. However,
affinin, a natural alkamide, showed low antianxiety
activity. Alkamides are present in some species of Piperaceae [Lee et al., 2008] and Asteraceae [Kraus et al.,
2006] and some have been isolated from Echinacea spp,
a species that is used in traditional medicine for analgesia and also displays anti-inflammatory properties,
modulates the immune response in T-cells [Matthias
et al., 2008], inhibits prostaglandin E(2) production
[LaLone et al., 2007], and inhibits cyclooxygenase-2
activity in human neuroglioma cells [Hinz et al., 2007].
This is the first time that H. longipes extract has
been reported to exert an antianxiety effect, and the
relaxant effect has been correlated with a prolonged
time of sodium PB-induced hypnosis by the extract and
affinin. Cilia-Lpez et al. [2010] showed a controversial
effect of the H. longipes extract using the Irwin [1968]
test, which quantifies a wide variety of drug-related
behavioral changes in experimental animals, including
neurological, autonomic, and toxic. The H. longipes
extract (1 mg/kg) and affinin (10 mg/kg) showed a
stimulant effect by increasing mouse activity as well as a
depressant effect by reducing the reaction to touch and
noise. They proposed that these reactions explained the
traditional use of H. longipes root as a condiment and in
chili sauces given that it has a pungent taste that stimulates salivary secretion while simultaneously reducing
the sensitivity of the tongue. However, a side effect that
was observed with the extract and less so with affinin
was the occurrence of seizures when used with PTZinduced myoclonus and clonus. According to the Lorke

PHARMACOLOGICAL AND TOXICOLOGICAL PROFILE

method, compounds with an LD50 value of greater than

5,000 mg/kg are safe. H. longipes is an extract with a
hazardous toxicity (LD50 = 62.14 mg/kg), and the toxicity for affinin decreased (LD50 = 113.13 mg/kg). Nevertheless, the negative Ames test indicated that when
used at a safe dose, the H. longipes ethanolic extract was
nonmutagenic and may be useful at low therapeutic
doses. However, further evaluation is needed for the
extract to be considered as an alternative drug for its
therapeutic antinociceptive and anticonvulsant activities. The outcome of the present research demonstrates
that the H. longipes extract and affinin produce a significant CNS effects for antinociception and antianxiety
effects. These results suggest that the H. longipes ethanolic extract may represent an alternative medicine.

ACKNOWLEDGMENTS

This work was supported by a SIP-IPN 20110653

grant (MD-C).

REFERENCES
Acosta-Madrid II, Castaeda-Hernndez G, Cilia-Lpez VG,
Cario-Corts R, Prez-Hernndez N, Fernndez-Martnez E,
Ortz MI. 2009. Interaction between Heliopsis longipes and
diclofenac on the thermal hyperalgesia test. Phytochemistry
16:336341.
Cario-Corts R, Gayosso-De-Lucio JA, Ortiz MI, SnchezGutirrez M, Garca-Reyna PB, Cilia-Lpez VG, PrezHernndez N, Moreno E, Ponce-Monter H. 2010.
Antinociceptive, genotoxic and histopathological study of Heliopsis longipes S.F. Blake in mice. J Ethnopharmacol 130:216221.
Cilia-Lpez VG, Jurez-Flores BI, Aguirre-Rivera JR, Reyes-Agero
JA. 2010. Analgesic activity of Heliopsis longipes and its effect on
the nervous system. Pharm Biol 48:195200.
Coderre TJ, Vaccarino AL, Melzack R. 1990. Central nervous system
plasticity in the tonic pain response to subcutaneous formalin
injection. Brain Res 535:155158.
Correa J, Roquet S, Daz E. 1971. Multiple NMR analysis of the
affinin. OMR 3:15.
Dciga-Campos M, Rios MY, Aguilar-Guadarrama AB. 2010. Antinociceptive effect of Heliopsis longipes extract and affinin in mice.
Planta Med 76:665670.
Dciga-Campos M, Rivero-Cruz I, Arriaga-Alba M, CastaedaCorral G, ngeles-Lpez GE, Navarrete A, Mata R. 2007. Acute
toxicity and mutagenic activity of Mexican plants used in traditional medicine. J Ethnopharmacol 110:334342.
Gonzlez-Trujano ME, Carrera D, Ventura-Martnez R, CedilloPortugal E, Navarrete A. 2006. Neuropharmacological profile of
an ethanol extract of Ruta chalepensis L. in mice. J Ethnopharmacol 106:129135.
Hernndez I, Mrquez L, Martnez I, Dieguez R, Delporte C, Prieto
S, Molina-Torres J, Garrido G. 2009. Anti-inflammatory effects of
ethanolic extract and alkamides-derived from Heliopsis longipes
roots. J Ethnopharmacol 124:649652.

137

Hinz B, Woelkart K, Bauer R. 2007. Alkamides from Echinacea

inhibit cyclooxygenase-2 activity in human neuroglioma cells.
Biochem Biophys Res Commun 360:441446.
Irwin S. 1968. Comprehensive observational assessment a systematic
quantitative procedure for assessing the behavioral and physiologic state of the mouse. Psychopharmacology 13:222257.
Jacobson M. 1954. Constituents of Heliopsis longipes species. III.
Cis-trans isomerism in affinin. J Am Chem Soc 76:46064608.
Jacobson M. 1955. Constituents of Heliopsis longipes species. IV.
The total synthesis of trans-affinin. J Am Chem Soc 77:24612463.
Jacobson M, Acree F, Haller HL. 1947. Correction of the source of
affinin (N-isobutyl-2,6,8-decatrienoamide). J Org Chem 12:731
732.
Kraus GA, Bae J, Wu L, Wurtele E. 2006. Synthesis and natural
distribution of anti-inflammatory alkamides from echinacea.
Molecules 11:758767.
LaLone CA, Hammer KD, Wu L, Bae J, Leyva N, Liu Y, Solco AK,
Kraus GA, Murphy PA, Wurtele ES, et al. 2007. Echinacea species
and alkamides inhibit prostaglandin E(2) production in RAW264.7
mouse macrophage cells. J Agric Food Chem 55:73147322.
Lee SW, Kim YK, Kim K, Lee HS, Choi JH, Lee WS, Jun CD, Park
JH, Lee JM, Rho MC. 2008. Alkamides from the fruits of Piper
longum and Piper nigrum displaying potent cell adhesion inhibition. Bioorg Med Chem Lett 18:45444546.
Lorke D. 1983. A new approach to partial acute toxicity testing. Arch
Toxicol 54:275287.
Maron DM, Ames BN. 1983. Revised methods for the Salmonella
mutagenicity test. Mutat Res 113:173215.
Matthias A, Banbury L, Bone KM, Leach DN, Lehmann RP. 2008.
Echinacea alkylamides modulate induced immune responses in
T-cells. Fitoterapia 79:5358.
Molina-Torres J, Garca CA. 2001. Alcamidas en plantas: distribucin e importancia. Avance y Perspectiva 20:377387.
Molina-Torres J, Salazar-Cabrera CJ, Armenta-Salinas C, RamrezChvez E. 2004. Fungistatic and bacteriostatic activities of alkamides from Heliopsis longipes roots: affinin and reduced amidas.
J Agric Food Chem 52:47004704.
Molina-Torres J, Salgado-Garciglia R, Ramrez-Chvez E. 1995.
Presence of the bornyl ester of deca-2E,6Z,8E-trienoic acid in
Heliopsis longipes roots. J Nat Prod 50:15901591.
Molina-Torres J, Salgado-Garciglia R, Ramrez-Chvez E, Del Ro
RE. 1996. Purely olefinic alkamides in Heliopsis longipes and
acmella (Spilanthes) oppositifolia. Biochem Syst Ecol 24:4347.
Ogura M, Cordell GA, Quinn ML, Leon C, Benoit PS, Soejarto DD,
Farnsworth NR. 1982. Ethnopharmacologic studies. I. rapid solution to a problem oral use of Heliopsis longipes by means of a
multidisciplinary approach. J Ethnopharmacol 64:215219.
Pitcher GM, Henry JL. 2002. Second phase of formalin-induced
excitation of spinal dorsal horn neurons in spinalized rats is
reversed by sciatic nerve block. Eur J Neurosci 15:15091515.
Puig S, Sorkin LS. 1996. Formalin-evoked activity in identified
primary afferent fibers: systemic lidocaine suppresses phase-2
activity. Pain 64:345355.
Rios MY, Aguilar-Guadarrama AB, Gutirrez MC. 2007. Analgesic
activity of affinin, an alkamide from Heliopsis longipes (Compositae). J Ethnopharmacol 10:364367.
Wheeler-Aceto H, Cowan A. 1991. Neurogenic and tissue-mediated
components of formalin-induced edema: evidence for supraspinal
regulation. Agents Actions 34:264269.

Drug Dev. Res.