Antinociceptive effect of extracts and compounds from Hofmeisteria schaffneri

Article abstract of DOI:10.1016/j.jep.2010.07.009

Ethnopharmacological relevance: Hofmeisteria schaffneri (Asteraceae) is a medicinal plant widely commercialized in the most important Markets of Mexico City for the treatment of gastro-intestinal complaints and skin afflictions. Aim of the study: The main goals of this study were to establish the potential acute toxicity and the antinociceptive activity in animal models of several preparations and compounds from Hofmeisteria schaffneri. Materials and methods: The aqueous and organic extracts as well as the essential oil of Hofmeisteria schaffneri were prepared by infusion, maceration and hydrodistillation, respectively. Investigation of the acute toxicity was accomplished by the Lorke method. The antinociceptive effect was assessed using the writhing and the hot plate tests. Natural compounds were isolated by standard phytochemical procedures. In addition, a few thymol esters were prepared by chemical synthesis. The stability of natural and synthetic esters was qualitatively analyzed by measuring their susceptibility to hydrolysis by pig liver estearase and mouse plasma at 37°C. Results: The LD₅₀ for each preparation tested was higher than 5000mg/kg revealing that they were not toxic to mice after exposure for short space of time. On the other hand, the extracts showed significant antinociceptive effect when tested in the hot plate model. The most active natural product as antinociceptive agent was hofmeisterin III (1) which also was the most stable in the stability study. Its pharmacological effect seems to be partially mediated by an opioid mechanism since naloxone inhibits its action. Using compound 1 as a lead molecule, several synthetic thymol esters were prepared and only compounds 13, 15 and 17 were antinoceptive at the dose of 1mg/kg. Conclusions: The present investigation provided evidence of the efficacy of several preparations of Hofmeisteria schaffneri as antinociceptive agents. The most active preparation was the essential oil which contained large amount of hofmeisterin III (1) and other thymol derivatives. Some novel synthetic analogs of hofmeisterin III with antinociceptive properties were discovered. The nature of the ester chain of these analogs did not have a clear impact on the antinociceptive activity. The phyto-preparations analyzed in this study were not toxic to mice according to the Lorke's test; therefore considering their long term use of the plant they might be secure for human consumption.

Full text of DOI:10.1016/j.jep.2010.07.009

Journal of Ethnopharmacology 131 (2010) 425–432
Contents lists available at ScienceDirect
Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm
Antinociceptive effect of extracts and compounds from Hofmeisteria schaffneriଝ
Guadalupe Angeles-Lópeza, Araceli Pérez-Vásqueza, Francisco Hernández-Luisa,
Myrna Déciga-Camposb, Robert Byec, Edelmira Linaresc, Rachel Mataa,∗
a Departamento de Farmacia, Facultad de Química, Universidad Nacional Autónoma de México D.F. 04510, Mexico
b Sección de Estudios de Posgrado e Investigación, Escuela Superior de Medicina del Instituto Politéctico Nacional, México, D.F. 11340, Mexico
c Instituto de Biología, Universidad Nacional Autónoma de México D.F. 04510, Mexico
a r t i c l e i n f o
a b s t r a c t
Article history:
Ethnopharmacological relevance: Hofmeisteria schaffneri (Asteraceae) is a medicinal plant widely commer-
cialized in the most important Markets of Mexico City for the treatment of gastro-intestinal complaints
and skin afflictions.
Aim of the study: The main goals of this study were to establish the potential acute toxicity and the
antinociceptive activity in animal models of several preparations and compounds from Hofmeisteria
schaffneri.
Received 1 March 2010
Received in revised form 15 June 2010
Accepted 6 July 2010
Available online 13 July 2010
Keywords:
Materials and methods: The aqueous and organic extracts as well as the essential oil of Hofmeisteria
schaffneri were prepared by infusion, maceration and hydrodistillation, respectively. Investigation of
the acute toxicity was accomplished by the Lorke method. The antinociceptive effect was assessed using
the writhing and the hot plate tests. Natural compounds were isolated by standard phytochemical proce-
dures. In addition, a few thymol esters were prepared by chemical synthesis. The stability of natural and
synthetic esters was qualitatively analyzed by measuring their susceptibility to hydrolysis by pig liver
estearase and mouse plasma at 37 ◦C.
Hofmeisteria schaffneri
Asteraceae
Acute toxicity
Antinociceptive effect
Stability
Thymol derivatives
Results: The LD50 for each preparation tested was higher than 5000 mg/kg revealing that they were not
toxic to mice after exposure for short space of time. On the other hand, the extracts showed signif-
icant antinociceptive effect when tested in the hot plate model. The most active natural product as
antinociceptive agent was hofmeisterin III (1) which also was the most stable in the stability study. Its
pharmacological effect seems to be partially mediated by an opioid mechanism since naloxone inhibits
its action. Using compound 1 as a lead molecule, several synthetic thymol esters were prepared and only
compounds 13, 15 and 17 were antinoceptive at the dose of 1 mg/kg.
Conclusions: The present investigation provided evidence of the efficacy of several preparations of
Hofmeisteria schaffneri as antinociceptive agents. The most active preparation was the essential oil which
contained large amount of hofmeisterin III (1) and other thymol derivatives. Some novel synthetic analogs
of hofmeisterin III with antinociceptive properties were discovered. The nature of the ester chain of these
analogs did not have a clear impact on the antinociceptive activity. The phyto-preparations analyzed in
this study were not toxic to mice according to the Lorke’s test; therefore considering their long term use
of the plant they might be secure for human consumption.
© 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
promote their rational use. Consequently, the present investiga-
tion was undertaken to initiate the preclinical pharmacological and
In Mexico an important segment of the population relies on
botanical raw materials for primary health care, therefore it is
very important to investigate these herbs from the pharmacologi-
cal and toxicological points of view to establish their real efficacy
and safety. The results of such investigations will be also useful
to integrate the scientific monographs of these plants in order to
toxicological analyses of Hofmeisteria schaffneri (A. Gray) R.M. King
& H. Robinson (Asteraceae), a widely commercialized medicinal
species in Central Mexico. The infusion prepared from the fresh or
dried aerial parts of the plant is highly valued for treating gastro-
intestinal complaints, including stomach aches, related or not with
irritable bowel syndrome and dyspepsia, and bleeding diarrhea
(King, 1967; Mendoza-Castelán et al., 1997; Pérez-Vásquez et al.,
2005, 2008). In addition, the plant is highly valued as a topic anti-
septic agent.
ଝ
This work was taken in part from the PhD thesis of Guadalupe Angeles-López.
Previous chemical work of the plant allowed isolation of sev-
eral thymol and northymol derivatives (Pérez-Vásquez et al., 2005,
∗
Corresponding author. Tel.: +52 5 55 622 5289; fax: +52 5 55 622 5329.
E-mail address: rachel@servidor.unam.mx (R. Mata).
0378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2010.07.009

426
G. Angeles-López et al. / Journal of Ethnopharmacology 131 (2010) 425–432
Fig. 1. Structures of thymyl esters.
2008) including hofmeisterin III (1), thymol itself (2) and 8,9-
epoxy-10-acetoxythymyl angelate (3) (Figs. 1 and 2). Furthermore,
the essential oil and infusion of the plant revealed significant
antimicrobial properties against Gram+ bacteria. The antimicrobial
essential oil of the plant harvested at different seasons during a year
period was also chemically analyzed by GC and capillary GC–MS.
Forty four compounds representing ∼90% of the total constituents
were identified. Compounds 1–3, thymyl isovalerate (4), thymyl
isobutyrate (5) were the major components of the oils but only 3
and 5 were active against Staphyloccocus aureus and Bacillus subtilis
(Pérez-Vásquez et al., submitted for publication).
2. Materials and methods
2.1. Plant material
The aerial parts of Hofmeisteria schaffneri (4 kg) were collected
Fig. 2. Structures of thymol (2) and 8,9-epoxy-10-acetoxythymyl angelate (3).
in San Luis Potosi, Mexico on November 2002. An authenticated

G. Angeles-López et al. / Journal of Ethnopharmacology 131 (2010) 425–432
427
voucher (Bye & Linares 31018) specimen is deposited in the
National Herbarium (MEXU), Instituto de Biología, UNAM.
products were used to prepare esters 4, 6, 7, 8, 10, 11, 15 and 16,
respectively.
2.2. General chemistry procedures
2.7.2. Thymyl esters 4–16
To a stirred mixture of thymol (0.5 g, 0.003 mol) and 10% aque-
ous NaOH (4.5 ml) the acid chloride previously obtained was slowly
added. In each case, the reaction mixture was stirred for 30 min
at room temperature. Every reaction mixture was extracted with
CH2Cl2 (3× 10 ml). The organic layer was dried over anhydrous
Na2SO4 and concentrated under reduced pressure; the crude prod-
ucts were purified by open column chromatography [silica gel,
hexane–EtOAc (98:2)] to yield pure 4–16 (Fig. 1). Compound 14
was obtained as colorless oil in a yield of 70%. The remaining prod-
ucts 4–13 and 15–16 were yellow oils and the yields were 46, 61,
50, 41, 48, 65, 41, 35, 62, 71, 45 and 52%, respectively. The spec-
tral properties of compounds 4–8, 12, 15, and 16 were identical
to those previously described (Mathela et al., 2008; Martinez et
al., 1988; Paolini et al., 2005, 2007; Schmitz et al., 1979; Rice and
Coats, 1994; Viana et al., 1981; Ateeque et al., 2002; Sheng et al.,
1984; Kumar et al., 2008, respectively). Compounds 9, 10, 11 and
13 were new chemical entities therefore their NMR (Table 1) and
other spectral data are presented in this paper.
IR (films) spectra were recorded in a Perkin Elmer 59913 spec-
trophotometer. NMR spectra were registered on a Varian Mercury
300 spectrometer in CDCl3, at 400 MHz (1H) and 100 MHz (13C) with
tetramethylsilane (TMS) as internal standard. Electron impact (EI)
mass spectra (MS) were obtained on a JEOL SX 102 mass spectrome-
ter. Open column chromatography: silica gel 60 (0.063–0.200 mm),
70–230 Mesh (Merck). TLC analyses were carried out on silica gel 60
F254 plates (Merck) using a ceric sulphate (10%) solution in H2SO4
as color reagent.
2.3. Preparation of organic extract
The organic extract of Hofmeisteria schaffneri was obtained
from dried and shredded aerial parts (750 g) macerating with
CH2Cl2–MeOH (1:1) (2 l × 3) during 14 days at room tempera-
ture. After filtration, the extract was concentrated in vacuo to yield
172 g of a greenish residue. Hofmeisterin III (1) (100 mg), thymol
(2) (79 mg), and 8,9-epoxy-10-acetoxythymyl angelate (3) (20 mg)
were isolated as previously described (Pérez-Vásquez et al., 2008).
Thymyl 2-methylvalerate (9): UV (MeOH) ꢀmax nm (log ε): 222
(4.71), 263 (4.36), 271 (4.34), IR (film) ꢁmax cm−1: 2962, 2934,
2873, 1756, 1621, 1506, 1458, 1149, 1127, 815, 730, EI-MS m/z (rel.
intensity): 248 [M+ (12)], 150 (34), 135 (45), 115 (10), 91 (25), 71
(100).
2.4. Preparation of the essential oil
Dried aerial parts (500 g) cut in small pieces were hydrodistilled
during 3 h using a Clevenger-type apparatus. When the condensed
material cooled down, the essential oils were separated from
the water by liquid–liquid extraction with CH2Cl2 (2 l × 3). After
removal of the solvent, 0.4 g of the oil was obtained. The oil was
stored at 4 ◦C until analysis (WHO, 1998).
Thymyl 3-methylvalerate (10): UV (MeOH) ꢀmax nm (log ε): 222
(4.81), 263 (4.46), 271 (4.44), IR (film) ꢁmax cm−1: 2963, 2930, 2874,
1759, 1621, 1505, 1461, 1148, 954, 815, 577, EI-MS m/z (rel. inten-
sity): 248 [M+ (11)], 150 (49), 135 (87), 115 (15), 91 (60), 71 (100).
Thymyl 4-methylvalerate (11): UV (MeOH) ꢀmax nm (log ε): 222
(4.71), 263 (4.36), 271 (4.23), IR (film) ꢁmax cm−1: 2960, 2930, 2871,
1760, 1621, 1505, 1467, 1150, 950, 815, EI-MS m/z (rel. intensity):
248 [M+ (20)], 150 (70), 135 (100), 115 (10), 105 (20), 91 (40).
Thymyl hexanoate (13): UV (MeOH) ꢀmax nm (log ε): 224 (4.75),
263 (4.40), 271 (4.38), IR (film) ꢁmax cm−1: 2960, 2931, 2871, 1760,
1621, 1505, 1458, 1151, 815, 728, 577, EI-MS m/z (rel. intensity):
248 [M+ (15)], 150 (75), 135 (100), 115 (10), 105 (15) 91 (35).
2.5. Preparation of the aqueous extract
The dried aerial parts (900 g) were extracted with boiling water
(25 l) during 30 min. The resulting aqueous extract was partitioned
with CH2Cl2 (25 ml × 3). The organic phases were dried over anhy-
drous Na2SO4 and concentrated in vacuo to yield a brown residue
(3.43 g).
2.7.3. Thymyl acetate (17)
In a 50 ml round-bottomed flask, thymol (0.5 g, 0.003 mol),
acetic anhydride (1.5 ml, 0.015 mol) and sulphuric acid (0.1 ml,
0.001 mol) were mixed and stirred for 45 min. The reaction mix-
ture was then extracted with CH2Cl2 (3× 10 ml). The organic layer
was dried over anhydrous sodium sulphate and concentrated in
vacuo. The residue was purified by open column chromatography
on silica gel, eluting with hexane–EtOAc (98:2) to yield a yellow
oil (72%). Its spectral properties were identical to those previously
described by Viana et al. (1981) and Ateeque et al. (2002).
2.6. Chemicals and drugs
Morphine (MOR) was a donation from Alfredo Covarrubias
Gómez, MD, and Luis Antonio Reyes Vallejo, MD. Dipirone (DIP)
was purchased from Laboratorios Pisa (Mexico City); the vehi-
cle (VEH) was prepared with isotonic solution of NaCl and
tween 80 (0.05%); pig liver esterase, 4-nitrophenyl acetate (4-
NPA), glibenclamide (GLIB), N(G)-nitro-l-arginine methyl ester
(l-NAME), naloxone (NLX), acetic anhydride, valeroic, tiglic, isova-
leric, propionic, 3-methylvaleric, 4-methylvaleric, 2-methylbutiric
and 3,3-dimethylacrylic acids as well as 2-methylvaleric, butyric,
hexanoic, benzoic and isobutyric acid chlorides were purchased
from Sigma–Aldrich (St. Louis. MO, USA). Compounds 4–17 were
prepared by synthesis as describe below.
2.8. Qualitative stability test of thymol esters
The stability of esters 1, 4 and 5 toward pig liver esterase
(2 mg suspended in 10 ml of distilled water) was estimated using
the procedure described by Schlacher et al. (1998). The enzyme
activity was qualitatively corroborated by monitoring the produc-
tion of 4-nitrophenol, yellow, from the colorless 4-nitrophenyl
acetate (4-NPA) (Innocenti et al., 2008). The stock solution, in
water–acetonitrile (99:1), of 4-NPA (0.1 mM) was freshly prepared
before use. For the analysis, 10 ␮l solution acetate buffer (pH 5.0)
or phosphate buffer (pH 7.4), 100 ␮l (0.1 mM) of test compound (1,
4 and 5) or positive control (4-NPA, 1 mM) and enzyme prepara-
tion (10 ␮l) were added to Eppendorf tubes (Jewell et al., 2007).
The resulting reaction mixtures were shaken for 1 h at 37 ◦C. Suit-
able blanks using compounds 1, 4, 5 and 2 were also prepared in
2.7. Synthesis of thymol derivates
2.7.1. Preparation of acyl chlorides of isovaleric, tiglic,
2-methylbutiric, propionic, 3-methylvaleric, 4-methylvaleric,
valeric and 3,3-dimethylacrylic acids
Thyonyl chloride (2.5 ml; 0.034 mol) was added drop wise to a
stirred solution of appropriate carboxylic acid (0.002 mol) in CH2Cl2
(1 ml). The resulting mixtures were refluxed gently for 1 h. In all
cases, the excess of thyonyl chloride was distilled and the resulting

428
G. Angeles-López et al. / Journal of Ethnopharmacology 131 (2010) 425–432
Table 1
1H and 13C (400 MHz and 100 MHz, respectively) NMR spectroscopic data of compounds 9, 10, 11 and 13.
Nuclei
Compounds
9
10
11
13
ıH
ıC
ıH
ıC
ıH
ıC
ıH
ıC
1
2
–
175.6
39.8
36.0
20.7
–
–
171.7
41.1
–
31.7
19
–
172.4
32.2
–
33.5
–
–
172.7
34.6
–
24.9
–
2.73 m
1.32 d (7.2)
1.52 m
–
2␣ 2.35 dd (15.2, 8), 2␤ 2.6 dd (14.8, 6)
–
1.38 m
1.17 d
2.59 m
2.5 t (7.6)
–
1.78 m
–
2-CH3
3
3-CH3
1.68 m
–
4
1.45 m
–
0.98 t (7.2)
–
–
–
7.19 d (8)
7.01 br d (8)
–
6.77 d (0.8)
2.30 s
2.97 h (6.8)
1.19 d (6.8)
1.19 d (6.8)
17.3
–
14.2
–
4␣ 1.45 m 4␤ 1.35 m
–
0.97 t (7.4)
–
–
29.1
–
10.9
–
1.71 m
0.98 d (7.2)
0.98 d (7.2)
–
–
–
7.19 d (8)
7.0 br d (8)
–
6.79 d (0.8)
2.31 s
2.95 h (6.8)
1.18 d (6.8)
1.18 d (6.8)
27.4
21.9
21.9
–
1.38 m
–
1.38 m
0.92 t (7.2)
–
31.5
–
4-CH3
5
22.5
14.1
148.1
137.2
126.5
127.2
136.7
122.9
21.0
27.2
23.2
23.2
6
1ꢀ
148.2
137.2
126.4
127.1
136.6
122.8
21.0
27.1
23.1
23.1
147.6
136.7
126.0
126.7
136.2
122.4
20.5
26.7
22.7
22.7
147.6
136.7
126.0
126.7
136.18
122.4
20.5
26.7
22.7
22.7
2ꢀ
–
–
3ꢀ
7.18 d (8)
7.0 br d (8)
–
6.78 d (0.8)
2.30 s
2.97 h (6.8)
1.20 d (6.8)
1.20 d (6.8)
7.17 d (8)
7.0 br d (8)
4ꢀ
5ꢀ
6ꢀ
6.78 d (0.8)
2.30 s
2.95 h (6.8)
1.17 d (6.8)
1.17 d (6.8)
5ꢀ-CH3
1ꢀꢀ
2ꢀꢀ
3ꢀꢀ
J values are in parentheses in Hz.
the same conditions but in the absence of the enzyme. In all cases,
the test was carried out by triplicate. After incubation, the mixture
of each tube was partitioned with 100 ␮l CH2Cl2; organic phase
aliquots were withdrawn and immediately analyzed by TLC on sil-
ica gel (hexane–AcOEt 8:2) to detect the presence of 2. For each
compound, the procedure was repeated using incubation periods
of 4.5 and 24 h. Finally, the same set of experiments was repeated
using mouse plasma instead pig liver esterase.
2.9.3. Writhing test
The acetic acid-induced writhing test was performed in mice as
previously described (Zimmerman, 1983; Williamson et al., 1996).
The organic extract (10–316 mg/kg) suspended in the VEH was
orally administered 30 min before intraperitoneal (i.p) injection
of 0.6% acetic acid. Control animals received a similar volume of
VEH or dipirone (DIP, 100 mg/kg) p.o. Animals were placed in an
observation box, and the abdominal constrictions were counted
cumulatively over a period of 30 min. Antinociceptive activity was
expressed as the reduction in the number of abdominal constric-
tions. The results corresponded to the area under the curve.
2.9. Pharmacological and toxicity studies
2.9.1. Animals
2.9.4. Hot plate test
ICR male mice weighing 20–25 g were used in the experiments.
Mice were purchased from Centro UNAM-Harlan (Harlan Méx-
ico, S.A. de C.V.). They were housed under standard laboratory
conditions and maintained on standard pellet diet and water ad
libitum. Procedures involving animals and their care were con-
ducted in conformity with the Mexican Official Norm for Animal
Care and Handing (NOM-062-ZOO-1999) and in compliance with
international rules on care and use of laboratory animals. After the
experiments, all animals were sacrificed in a CO2 chamber. Fur-
thermore, clearance for conducting the studies was taken from the
Ethics Committee for the Use of Animals in Pharmacological and
Toxicological Testing, Facultad de Química, UNAM, which in turn is
coordinated by UNAM Central Committee of Ethics for Animal Care
and Handing. For the pharmacological studies groups of six animals
were used, whereas for the toxicological tests the groups were of
three mice. All doses are in mg/kg of body weight.
The hot plate apparatus (Ugo Basile, Italy) was used to mea-
sure the antinociceptive effect (Zimmerman, 1983; Williamson et
al., 1996). Mice were placed into an acrylic cylinder over a heated
surface (55.5 0.2 ◦C), the time between placement and shaking
or licking paws or/and jumping was recorded as latency response.
The organic extract (10–562 mg/kg), essential oil (1–100 mg/kg),
aqueous extract (10–316 mg/kg), thymol (2) (10–100 mg/kg) or
thymol derivates (1, 3 and 4–17) (0.1–17.7 mg/kg) were orally
administered 30 min before beginning each experiment. Mice were
observed before and 30, 60, 90, 120 and 150 min after each drug
administration. A cut off of 30 seconds was used to avoid injury.
MOR (3.16 and 5 mg/kg, i.p) was used as positive control. The cumu-
lative antinociceptive effect was determined measuring the area
under the curve of the course time (AUC) analysis. Curves were
plotting with AUC (trapezoidal method) and doses.
2.9.5. Preliminary studies on the mode of action of hofmeisterin
III (1)
2.9.2. Acute toxicity study in mice
The treatments were orally administrated in two phases, in
both phases four groups with three animals were used; in the first
phase, mice were treated with doses of 10, 100 and 1000 mg/kg of
the organic extract, essential oil or aqueous extracts; these treat-
ments were suspended in the VEH. In the second phase, the animals
received 1600, 2900 and 5000 mg/kg of the treatment according to
the Lorke procedure (Lorke, 1983). In both stages, the animals were
observed during 14 days. After this period of time, the lungs, heart,
stomach and intestines were removed under dissection to detect
any macroscopic injury.
Naloxone (NLX, 1 mg/kg, in VEH), glibenclamide (GLIB,
10 mg/kg, in DMSO 2% and VEH) or l-NAME (l-NAME, 30 mg/kg, in
VEH) were i.p administrated 15 min before the oral treatment with
compound 1 (1 mg/kg). In each case, the antinociceptive effect was
recorded as describe above (Déciga-Campos et al., 2007).
2.9.6. Statistical analysis
Data are expressed as the mean SEM for the number (n = 6) of
animals in all groups. In all cases the results of treated-animal (com-

G. Angeles-López et al. / Journal of Ethnopharmacology 131 (2010) 425–432
429
Table 2
pounds, extracts and reference drugs) groups were compared with
the VEH groups. Statistical analysis was performed with one-way
ANOVA followed by Dunnett post hoc to determine the source of
significant differences where appropriate; *P < 0.05, **P < 0.01 and
***P < 0.001 were considered statistically significant. Prisma Graph-
Pad (version 4.0) software was used for statistics and plotting.
Effect of the organic extract of Hofmeisteria schaffneri in the contortions induced by
acetic acid.
Treatment
VEH
Doses (mg/kg)
AUC E.E
3725 100
316
100
31.6
10
3440 289.1
3202.5 1033.8
4162.5 855.8
3842.5 595
Organic extract
3. Results and discussion
As part of our investigations dealing with the preclinical efficacy,
quality control and safety of selected Mexican medicinal plants, the
present study was undertaken to determine the acute toxicity and
the antinociceptive action of Hofmeisteria schaffneri. The antinoci-
ceptive activity was selected on the basis of its extensive use for
treating painful complaints. In all cases the primary target for test-
ing was the plant crude extracts considering their similarity with
the preparations used in traditional medicine, although some major
pure compounds isolated from the plant were also evaluated in
order to find out the active principles.
DIP
770 269.9*
Data are expressed as the mean SEM for the number (n = 6) of animals in every
group. ANOVA followed by Dunnett post hoc *P < 0.05.
terin III (1), thymol (2), 8,9-epoxy-10-acetoxythymyl angelate (3),
thymyl isovalerate (4), thymyl isobutyrate (5) were tested at differ-
ent doses. For this endeavor, compounds 4 and 5 were synthesized
by condensing thymol with isovaleryl and isobutyryl chlorides,
respectively. The spectral properties of 4 and 5 were identical to
those described by Mathela (Mathela et al., 2008). Compounds
1–3 were isolated from the plant material as previously described
(Pérez-Vásquez et al., 2008).
3.1. Acute toxicity
Compound 1 (0.1–17 mg/kg) was the most active increasing
significantly the latency to thermal stimulis in a dose dependent
manner up to the dose of 1 mg/kg. At higher doses (3.16 and
17.7 mg/kg) the effect remains constant (Table 4). The pharma-
cological effect of 1 was also assessed after pretreatment (15 min
before) of mice with NLX (1 mg/kg), a non-selective opiate recep-
tor antagonist, GLIB (10 mg/kg), a Na+ channel blocking, or l-NAME
(30 mg/kg), an inhibitor of nitric oxide synthase. According to the
results in Table 5, l-NAME and GLIB were not able to decrease
the antinociceptive activity of 1. However, the effect of 1 was sig-
nificantly reversed by the treatment with NLX suggesting that its
mode of action involves opioids receptors and/or an increment of
endogenus opioids.
Investigation of the acute toxicity is the first step in the toxico-
logical investigations of herbal drugs. Lorke’s method is perhaps the
most widely used approach since the experiments are carried out
with a minimum number of experimental animals (Lorke, 1983).
Thus, to asses any potential toxic effects of the plant mice were
treated orally with Hofmeisteria schaffneri essential oil as well as
with the organic and aqueous extracts using doses in the range
of 10–5000 mg/kg. After 14 days, treated mice did not present
any visible toxic effect. Furthermore, no lesions or bleedings were
observed in internal organs such as lungs, kidneys, liver, heart and
stomach. Since no death or damage was observed throughout the
experiments, the LD50 for each preparation tested was higher than
5000 mg/kg revealing that they were not toxic to mice after expo-
sure for short space of time.
Compounds 4 and 5 (0.1–17 mg/kg) showed similar activity than
hofmeisterin III (1) while 2 (10–100 mg/kg) was less active; finally,
compound 3 (0.316–17.7 mg/kg) did not show any activity sug-
gesting that the esterification of the free phenolic group of thymol
improved the activity (Table 4). Altogether this information reveals
3.2. Antinociceptive effect using writing and hot plate tests
The potential antinociceptive effect produced by extracts and
essential oil was assessed using two well known models of noci-
ception, the writhing and the hot plate tests. The former is an
inflammatory visceral pain model and is very useful to detect
painful complaints due to inflammatory disorders of internal
organs such as the stomach or intestines (Al-Chaer and Traub,
2002). This method shows good sensitivity for weak analgesics, but
shows poor specificity because abdominal constrictions could be
suppressed by smooth muscle relaxants so results could be mis-
understood. This problem can be overcome using other models
of nociception such as the hot plate test. Human preclinical pain
tests have frequently employed heat stimulation for physiological,
pathological and pharmacological assessments (Arendt-Nielsen
and Chen, 2003). In addition, this model measures animal behavior
and has good sensitivity and specificity.
Graded doses of the organic extract (10–562 mg/kg, p.o.) did
not decrease acetic acid induced contortions in mice (Table 2).
Therefore, the essential oil and aqueous extracts were not further
evaluated.
On the other hand, the organic extract significantly increased
(P < 0.001) the latency to thermal stimuli when tested at the doses
of 177 and 562 mg/kg; the effect was dose-dependent. The essential
oil and aqueous extract were also very active in the hot plate test
at doses ranging from 1 to 316 mg/kg (Table 3), however, the effect
was not dose dependent (Table 3).
Table 3
Antinociceptive effect of extracts and essential oil from Hofmeisteria schaffneri as
detected in the hot plate test.
Treatment
Doses (mg/kg)
AUC E.E
VEH
Organic extract
983.1 39.6
31.6
56.2
100
177
562
1136.4 62.3*
1268.1 65.6*
1381.1 79.9*
1661.6 100.8***
1914.6 117.8***
2638.1 200.1***
MOR
3.16
VEH
Aqueous extract
983.1 39.6
10
56.2
100
177
316
3.16
1788.7 172.9***
1974.4 116.3***
2212.5 61.2***
2107.6 89.3***
2071.9 124***
2638.1 200.1***
MOR
VEH
Essential oil
1494.5 97.6
2280 158**
1
3.16
17.7
31.6
100
3.16
2328.7 152.5***
2353.1 124.7***
1755.9 138.7
1618.3 126.8
3078.7 177.8***
MOR
Since the essential oil was the most active its major components
(Pérez-Vásquez et al., submitted for publication), namely hofmeis-
Data are expressed as the mean SEM for the number (n = 6) of animals in every
group. ANOVA followed by Dunnett post hoc *P < 0.05, **P < 0.01 and ***P < 0.001.

430
G. Angeles-López et al. / Journal of Ethnopharmacology 131 (2010) 425–432
voltage-operated Na+ (Haeseler et al., 2002) and K+ (Elliot and
Elliot, 1997) channels and directly activates ␥-aminobutyric acid
GABAA receptors (Mohammadi et al., 2001). It was also suggested
that the demonstrated effects accounted for its pain-reliever prop-
erties and contributed to its sedative–hypnotic actions (Haeseler
et al., 2002). On the other hand, thymol inhibited prostaglandin
synthesis by microsomes of bovine tooth pulp. This inhibition was
reversible and probably related with the analgesic effect of thymol
in endodontic therapy (Anamura et al., 1988). Furthermore, Beer et
al. (2007) speculated that thymol could have analgesic effect due
to its agonistic effect on ␣1-, ␣2- and ␤-adrenergic receptors.
Table 4
Antinociceptive effect of compounds 1–5, 13, 15 and 17 as detected in the hot plate
test.
Treatment
Doses (mg/kg)
AUC E.E
VEH
1494.5 97.6
1775.6 85.7
1976.2 78*
2460 83.7***
2465.6 61.3***
2546.2 114.4**
3078.7 177.8***
0.1
0.316
1
3.16
17.7
3.16
Hofmeisterin III (1)
MOR
VEH
1726.9 84.5
1
10
56.2
100
3.16
1427.4 169.5
2116.9 68.1
2096.2 133.5
2420.6 102.6**
2783.1 148.3***
Thymol (2)
3.3. Antinociceptive effect of thymol derivates (6–17) in hot plate
test
MOR
Structural modification of a lead molecule could improve its
efficacy since the pharmacokinetics parameters as well as the inter-
action with the molecular targets could be improved. Therefore,
12 analogs of compound 1, namely 6–17, were prepared condens-
ing thymol with the corresponding acid chlorides in a single step
reaction. The resulting esters were then evaluated in the same phar-
macological model. Compounds 9, 10, 11 and 13 are new chemicals
entities but compounds 6 (Paolini et al., 2005), 7 (Schmitz et al.,
1979; Paolini et al., 2007), 8 (Rice and Coats, 1994; Ateeque et
al., 2002), 12 and 14 (Viana et al., 1981; Ateeque et al., 2002),
15 (Sheng et al., 1984), 16 (Kumar et al., 2008) and 17 (Viana et
al., 1981; Ateeque et al., 2002) have been reported as natural or
synthetic products and their spectral data are in agreement with
those previously described. Compounds 9–11 and 13 were iden-
tified by spectral means. In all cases their IR exhibited the typical
phenolic ester carbonyl at ∼1760 cm−1. The NMR spectra (Table 1)
were very similar to those of the other thymyl esters under study
with the aromatic region displaying the characteristic ABX system
of the trisubstituted benzene ring of thymol (Table 1). The spec-
tra differed only in the resonances due to the acid residues which
were easily assigned to a tiglic, 2-methylvaleric, 3-methylvaleric, 4-
methylvaleric and hexanic acids, respectively by comparison with
the information previously reported for similar structures (Mathela
et al., 2008; Martinez et al., 1988; Paolini et al., 2005, 2007; Schmitz
et al., 1979; Rice and Coats, 1994; Viana et al., 1981; Ateeque et al.,
2002; Sheng et al., 1984; Kumar et al., 2008).
VEH
1547.3 116.7
1778.3 108.7
1804 107.7
1662.7 166.5
1790.9 189.9
2371.6 122.1***
0.316
1
3.16
17.7
3.16
8,9-Epoxy-10-acetoxythymyl
angelate (3)
MOR
VEH
4
1597.6 52.2
1889.2 271.4
2196.2 141*
2245.4 178.1*
2114.1 113.1*
3372.8 100***
0.1
0.316
1
17.7
5
MOR
VEH
5
1483.3 91.6
0.316
1
3.16
17.7
5
1474.9 260.8
1998.8 86.1*
1964.1 207.6*
1948.6 125*
3221.4 133.4***
MOR
VEH
13
1483.3 91.6
1642.7 281.8
1986 55.3*
1709.8 86.9
3221.4 133.4***
0.316
1
17.7
5
MOR
VEH
15
1371.4 120
0.316
1773.4 122.6
2253.3 133.7**
1759.6 209.2
2900.9 178***
1
17.7
5
MOR
Testing all analogs (6–17) at three different doses in the range
of 1–17.7 mg/kg revealed that only compounds 13, 15 and 17 were
active. These results indicated that the nature of the ester chain did
not have a clear impact on the antinociceptive activity (Table 4).
Considering that the stability of drugs in biological systems plays
an important role in their activity (Di et al., 2005) and that degra-
dation of drug esters in biological matrices is mainly attributed to
their hydrolysis by esterases, the stability in vitro of the most active
(1, 4 and 5), and one inactive (6) thymyl esters was assessed during
24 h. The stability study was qualitatively performed measuring the
susceptibility to hydrolysis of esters 1 and 4–6 in the presence of
a commercial pig liver estearase solutions in two buffers (pH 7.4
and 5.0) as well as in mouse plasma at 37 ◦C (Di et al., 2005). The
hydrolysis of the esters was monitored on a period of 24 h by TLC.
According to the results summarized Table 6, in general, the
ester compounds were unstable in blood plasma; only compound
1 was stable during the first hour of the experiment. In the pres-
ence of the enzyme the degradation time was different for each
compound. Thus, compound 1 was stable in the control solutions
(pH 5.0 and 7.4) throughout the experiment; however, it was totally
hydrolyzed in the enzymatic solution (pH 7.4) and mouse plasma
after 4.5 h. On the other hand, at pH 5.0 this compound was par-
tially hydrolyzed after the same period of time and completely
hydrolyzed after 24 h. These results suggested that compound 1
could have an acceptable stability in the gastro-intestinal tract.
VEH
17
1371.4 120
1670.7 62.1
2101 223.9**
1775.1 74.1
2900.9 180***
0.316
1
17.7
5
MOR
Data are expressed as the mean SEM for the number (n = 6) of animals in every
group. ANOVA followed by Dunnett post hoc *P < 0.05, **P < 0.01 and ***P < 0.001.
that the thymyl esters of Hofmeisteria schaffneri are responsible of
the antinociceptive activity of the different preparations tested in
this work.
Thymol (2) was only active at the dose of 100 mg/kg, however
it has been previously shown that this compound partially block
Table 5
Antinociceptive effect of hofmeisterin (1) from Hofmeisteria schaffneri in the pres-
ence of GLIB (10 mg/kg), NLX (1 mg/kg) or l-NAME (30 mg/kg).
Treatment
AUC E.E
VEH
1 (1 mg/kg)
1 (1 mg/kg) + GLIB (10 mg/kg)
1 (1 mg/kg) + NLX (1 mg/kg)
1 (1 mg/kg) + l-NAME (30 mg/kg)
MOR (3.16 mg/kg)
1373.7 78.2
1950.2 123.4**
1716.7 433.7*
1380 96.2
1817.9 314.6**
3070 96.2***
Data are expressed as the mean SEM for the number (n = 6) of animals in every
group. ANOVA followed by Dunnett post hoc *P < 0.05, **P < 0.01 and ***P < 0.001.

G. Angeles-López et al. / Journal of Ethnopharmacology 131 (2010) 425–432
431
Table 6
In vitro stability of thymol esters derivatives toward pig liver estearase.
Compounds
pH 7.4
1 h
pH 5.0
1 h
Mouse plasma
1 h
4.5 h
A
24 h
A
4.5 h
A
24 h
A
4.5 h
A
24 h
A
A
B
B
B
A
B
B
B
A
B
B
B
1
4
5
6
−
−
++
−
−
−
++
++
++
++
++
−
++
+
++
++
++
++
++
−
−
−
++
−
−
−
+
++
+
++
++
−
++
−
−
−
++
++
++
++
++
−
++
−
−
−
+
−
++
−
−
−
++
++
++
++
++
−
++
−
−
−
++
++
++
++
++
−
++
+
++
+
++
+
++
−
++
++
++
++
++
++
−
++
−
++
++
−
4NPA
−
−
A: esterase or mouse plasma; B: buffer or distilled water; h: hours.
−: not hydrolyzed; +: partially hydrolyzed; ++: totally hydrolyzed.
Compound 5 was stable in buffer solution at pH 5.0 but partially
hydrolyzed at pH 7.4 after 4.5 h. With regard to mouse plasma and
enzymatic solutions, this compound was totally hydrolyzed at 1
and4.5 h, respectively. Therefore one would expect a similar gastro-
intestinal stability than for compound 1. Finally, esters 4 and 6 were
completely hydrolyzed in all test conditions; this result raised the
possibility that these compounds might hydrolyze before intestinal
absorption.
On the whole, the results indicated that the better antinoci-
ceptive activity of compounds 1 and 5 could be partially due to
a better stability, however in the case of 4 other factors might be
involved such as a better absorption in vivo, although substrate
specificity differences in vitro could not be ruled out. Further work
is in progress to determine the bioavailability of these esters in vivo.
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