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Antinociceptive activity of the volatile oils of Hyptis pectinata L. Poit. (Lamiaceae) genotypes.

Abstract

Hyptis pectinata L. Poit (Lamiaceae) is known popularly in Brazil as "sambacaita" or "canudinho" and is used in the treatment of inflammations, bacterial infections and ache. The antinociceptive activity of the volatile oils of six genotypes, at doses of 100, 200 and 400 mg/kg body wt., were investigated using abdominal writhe models induced by acetic acid and hot plate tests. The volatile oils of all the genotypes are composed mainly of sesquiterpenoids. All the genotypes showed antinociceptive effects in both models used; the SAM002 genotype showed the major inhibitory effect at dose of 100mg/kg body wt. These results suggest that the volatile oil of H. pectinata has peripheral (writhe reduction) and central (time delay of thermal reaction) effects. These observations indicate that H. pectinata may be useful as an analgesic drug.

[c] 2007 Elsevier GmbH. All rights reserved.

Keywords: Hyptis pectinata; Acetic-acid-induced abdominal constrictions; Hot-plate test; Acute toxicity; Native medicinal plant

Introduction

The use of natural products with therapeutic properties is as ancient as human civilization (Esquenazi et al., 2002). Most people living in developing countries are almost completely dependent on traditional medical practices for their primary health care needs and higher plants are known to be the main source for drug therapy in traditional medicine (Calixto, 2005). Volatile oils from a broad spectrum of plant species have shown antinociceptive, antiinflammatory, antimicrobial, antiviral, antitumoral and antioxidant activities (Asekun et al., 1999; Allahverdiyev et al., 2004; Sousa et al., 2004; Al-Burtamani et al., 2005).

The northeast region of Brazil contains a large diversity of native plant species that are known for their medicinal properties and their common use in popular medicine. With increased use of the land for agriculture and exploitation of natural resources, many of these species are in danger of being lost before they have been studied. They include Hyptis pectinata L. Poit. (Lamiaceae), known popularly as "sambacaita" is used as tea, decoction and infusion, mainly as a natural anti-inflammatory.

H. pectinata is an odoriferous plant that grows on wild land or is cultivated near homes in Alagoas and Sergipe states. Because of its popular use as an antiinflammatory, studies have been carried out to ascertain the antinociceptive and antiedematogenic effects of the aqueous leaf extract (Bispo et al., 2001), and antimicrobial activity of the volatile oil of the leaves (Asekun et al., 1999), validating its popular use.

Other studies have shown that genetic variation among the same species can alter the concentration of active principles. Other factors, in addition to genetics, such as climate, soil, seasonality, time and type of culture, and fertilizer, may affect the composition of active principles. Such variations were verified among species of Hyptis suaveolens that were characterized by the evaluation of the yield of the volatile oil and its chemical constituents in different genotypes found in the Brazilian "cerrado" (Azevedo et al., 2002). No studies have been reported to date on Hyptis pectinata concerning the variations in chemical constituents, yield and pharmacological activity of its volatile oil.

The objective of this work was therefore to evaluate the chemical composition and antinociceptive activity of the volatile oils of six genotypes of H. pectinata from the Active Germplasm Bank at "Universidade Federal de Sergipe-UFS".

Material and methods

Plant material

Aerial parts of six genotypes of Hyptis pectinata in different stages of maturation collected from the active germplasm bank in June 2002 were dried in an oven with circulating air at 40 [degree] C. The origin of the genotypes is described in Table 1. Samples of each genotype have been deposited in the Herbarium of the "Universidade Federal de Sergipe", Sao Cristovao-SE, Brazil.
Table 1. Geographical coordinates of the Hyptis pectinata genotypes
harvested in Sergipe State, Brazil, and kept in the active germplasm
bank of the experimental station "Campus Rural da UFS"

Genotypes Origin Geographical coordinates
 (Localization) Latitude (S) Longitude (W)

SAM001 Sao Cristovao 10[degree]55'25.6" 37[degree]11'56.4"
SAM002 Neopolis 10[degree]18'20.7" 36[degree]39'7.2"
SAM003 Santana do Sao 10[degree]16'55.3" 36[degree]38'33.8"
 Francisco
SAM004 Malhada dos Bois 10[degree]21'36.0" 36[degree]54'27.6"
SAM005 Sao Francisco 10[degree]19'1.4" 36[degree]53'36.8"
SAM006 Propria 10[degree]14'26.3" 36[degree]51'11.1"

 Geographical
 coordinates

Genotypes Origin (Localization) Altitude (m) Herbarium
 number

SAM001 Sao Cristovao 24 7455
SAM002 Neopolis 120 7454
SAM003 Santana do Sao 80 7453
 Francisco
SAM004 Malhada dos Bois 130 7452
SAM005 Sao Francisco 55 7451
SAM006 Propria 25 7456

Abbreviations: S--south; W--west; m--meter.


Volatile oil analysis

Volatile oils of dried leaves from six genotypes were obtained via hydrodistillation on a Clevenger-type apparatus (Guenther, 1972) for approximately 3 h Samples of oils were analysed by GC/MS in a Shimadzu QP5050A with a capillary column DB-5 (30 m X 0.25 mm X 0.25 [micro]m) connected to an electron impact detector at 70 eV; helium (flow rate 1 ml/min) was used as carrier gas with the following program: 80 [degree] C (1 min), 3 [degree]C/min, 180[degree]C (0min), 10[degree]C/min, 300 [degree]C (3 min), type of injection split 1:100. The retention index calculations were carried out through co-injections with n-alkanes. The identification of the constituents of the volatile oils was based on the retention time index (Adams, 1995) and comparison with the mass spectral data bank library NIST21 and NIST107. Concentrations of the respective constituents were calculated using the area of the signal in the GC spectra according to the order of elution.

Analgesic activity

Animals

White Swiss mice of both sexes weighing 25-30g each were used distributed in groups of 10 (writhing test) and 8 (hot-plate test) animals for treatment. The animals received Purina ration and water ad libitum throughout the experiment.

Drugs and treatment

The effects of the volatile oils from the six genotypes of H. pectinata were tested. Doses of 100, 200 and 400 mg/kg body wt., in propylene glycol as a vehicle, were administered via subcutaneous injection (s.c.).

Abdominal writhe tests (acetic acid-induced writhing in mice)

Volatile oils were injected 30 min before the intraperitoneal injection (i.p.) of acetic acid 0.6% (10 ml/kg body wt.) (Koster et al., 1959). The control animals received only vehicle and acetic acid. Indomethacin (10 mg/kg body wt., i.p.) was used as a standard drug. Ten minutes after administration of acetic acid, the number of writhes was registered over a period of 20 min.

Hot plate test

The animals were placed on a hot plate (55 [+ or -]0.5 [degree]C) and the thermal stimuli reaction was observed with the maximum incubation time of 30 s. The reaction time (licking of paw, jumping, shaking) was measured at 30, 45, 60 and 90 min after administration of the volatile oils and other drugs. The vehicle (s.c.) and morphine (10 mg/kg body wt., i.p.) were used as control and standard drugs, respectively. The possible participation of the opioid system in the antinociceptive effect was analysed with the volatile oils. The animals were pretreated intraperitoneally with naloxone (5 mg/kg body wt.) 15 min before subcutaneous administration of the volatile oils (100 mg/kg body wt.) or equivalent volumes of the vehicle.

Acute toxicity studies: [LD.sub.50] values

The method described by Lorke (1983) was employed in the determination of the LD(50) values. Swiss mice (n = 10) of both sexes were fasted overnight and the volatile oils were administered at the following doses: 0 (vehicle), 1, 2, 3, 4 and 5g/kg body wt, i.p. Animals were observed for 48 h after treatment and the final [LD.sub.50] value was calculated as the square root of the product of the lowest lethal dose and the highest nonlethal dose, i.e., the geometric mean of the consecutive doses for which 0% and 100% survival rates were recorded.

Statistical analysis

All results were reported as means [+ or -] S.E.M. They were further analyzed using one-way analysis of variance (ANOVA) to calculate the significance of results.

Results and discussion

Analysis and yield of the volatile oils

The volatile oils of the six genotypes of H. pectinata averaged 0.5% on a dry weight basis. Their compounds were identified previously and only the major constituents are listed in Table 2.
Table 2. Main compounds of the essential oil of six H. pectinata
genotypes from the active germplasm bank of the experiment alstation
"Campus Rural da UFS"

Compound (a) KI (b) Percentage in genotypes
 SAM001 SAM002 SAM003

[beta]-Pinene 981 1.24 2.55 5.88
[beta]-Elemene 1393 3.09 4.47 1.40
Cis-[beta]-Guaiene 1494 0.55 2.98 7.58
[gamma]-Cadinene 1516 5.12 1.03 0.56
Cubenol 1617 11.44 0.81 2.01
[beta]-Caryophyllene 1422 12.91 27.10 8.88
Germacrene-D 1483 8.20 9.95 2.37
Caryophyllene oxide 1586 1.98 4.10 2.47
Cubenol 1617 11.44 0.81 2.01
[alpha]-Muurolol 1646 25.45 0.67 0.00
Calamusenone 1688 1.85 21.04 40.70
Monoterpene hydrocarbons 2.81 5.48 11.35
Oxygenated monoterpenes 0.37 0.58 3.14
Sesquiterpene hydrocarbons 49.36 62.57 27.40
Oxygenated sesquiterpenes 43.62 29.44 46.02
Essential oil content (%) 0.50 0.50 0.50

Compound (a) Percentage in genotypes
 SAM004 SAM005 SAM006

[beta]-Pinene 0.46 1.08 0.85
[beta]-Elemene 4.12 2.17 1.53
Cis-[beta]-Guaiene 0.00 4.85 5.30
[gamma]-Cadinene 0.40 1.22 1.38
Cubenol 0.00 1.30 1.22
[beta]-Caryophyllene 45.09 28.15 23.58
Germacrene-D 5.77 5.80 4.77
Caryophyllene oxide 20.92 4.01 9.10
Cubenol 0.00 1.30 1.22
[alpha]-Muurolol 0.00 0.00 0.00
Calamusenone 0.00 26.95 23.00
Monoterpene hydrocarbons 0.46 6.07 2.86
Oxygenated monoterpenes 0.00 0.38 0.00
Sesquiterpene hydrocarbons 66.42 53.27 51.65
Oxygenated sesquiterpenes 25.23 32.26 38.70
Essential oil content (%) 0.45 0.57 0.48

(a).Compounds are listed in order of elution from a DB-5 column.
(b).Kovats indexes.


Analgesic effects of volatile oils of H. pectinata genotypes

The subcutaneous administration of the volatile oils of the six genotypes significantly reduced the number of writhes induced by acetic acid, as compared to animals that received only vehicle (Table 3). The inhibitory effects of the volatile oils ranged from 62.5% to 84.8%. While all the genotypes inhibited the number of abdominal writhes, the genotype SAM002 presented the highest inhibitory effect at a dose of 100 mg/kg body wt (78.4%) followed by the genotype SAM004 (75%). However, these effects were not dose-dependent, because doubling the dose did not significantly alter the response.
Table 3. Analgesic effect of the essential oil of Hyptis pectinata
genotypes on acetic acid-induced abdominal writhes in mice

Group Dose Number of abdominal Inhibition (%)a
 (mg/kg constrictions[+ or -]
 body S.E.M.
 wt.)

Control 33.00 [+ or -] 4.77 -
(vehicle)

Indomethacin 10 8.00[+ or -]1.24 ** 75.7

SAM001 100 11.37 [+ or -] 0.71 ** 65.5

 200 9.00[+ or -]1.94 * 72.7

 400 9.37[+ or -]1.66 * 71.6

SAM002 100 7.25[+ or -]1.33 ** 78.4

 200 7.25 [+ or -] 0.97 ** 78.0

 400 6.75[+ or -]L44 ** 79.5

SAM003 100 12.38[+ or -]1.36 ** 62.5

 200 10.88[+ or -]1.08 * 67.1

 400 7.00[+ or -]1.00 ** 78.8

SAM004 100 8.25[+ or -]1.51 * 75.0

 200 9.87[+ or -]1.38 * 70.1

 400 10.50 [+ or -] 0.94 ** 68.2

SAM005 100 10.25 [+ or -] 2.51 ** 68.9

 200 10.75[+ or -]1.44 ** 67.4

 400 5.75 [+ or -] 0.96 ** 82.6

SAM006 100 10.50 [+ or -] 2.31 ** 68.2

 200 7.87[+ or -]1.22 ** 76.2

 400 5.00 [+ or -] 0.89 ** 84.8

*P[less then]0.05, **P[less then]0.01 vs. control.
(a)Percent inhibition of total writhing test response (n=10).


Using the hot plate test, administration of the volatile oils of all genotypes delayed the time of reaction as compared to the control group (Table 4). In this test, genotype SAM002 showed the best analgesic activity (62.34% at a dose of 100mg/kg body wt).
Table 4. Antinociceptive effect of the essentia! oii from H.
pectinata genotypes in the hot-plate test in mice([albha]=8)
Group Dose Time after administration (min)
 (mg/kg
 body
 wt.)

 30 45

 Latency time (s)(mean [+ or -] S.E.M.)

Control 9.17[+ or -]0.70 9.75[+ or -]0.76

Morphine 10 14.63[+ or -]1.19 ** 13.95[+ or -]0.68 **

SAM001 300 14.99[+ or -]1.15 ** 14.25[+ or -]1.59 *

 200 13.99[+ or -]1.08 ** 15.73[+ or -]1.38 **

 400 15.68[+ or -]0.86 ** 16.51[+ or -]0.94 **

SAM002 100 18.71[+ or -]1.47 ** 15.43[+ or -]1.39 **

 200 14.30[+ or -]1.64 * 17.68[+ or -]1.97 **

 400 15.61[+ or -]1.44 ** 15.81[+ or -]1.49 **

SAM003 100 15.59[+ or -]1.00 ** 14.64[+ or -]0.90 **

 200 13.21[+ or -]0.59 ** 15.09[+ or -]0.71 **

 400 14.90[+ or -]1.14 ** 14.13[+ or -]0.87 **

SAM004 100 12.50[+ or -]1.01 * 14.25[+ or -]1.52 *

 200 12.94[+ or -]0.96 ** 13.11[+ or -]0.75 **

 400 16.41[+ or -]0.83 15.33[+ or -]0.80

SAM005 100 12.79[+ or -]1.23 * 14.23[+ or -]0.86 **

 200 13.61[+ or -]1.06 ** 12.24[+ or -]1.45

 400 13.86[+ or -]0.93 ** 14.31[+ or -]1.01 **

SAM006 100 13.44[+ or -]1.26 * 16.19[+ or -]0.98 **

 200 13.29[+ or -]1.49 * 15.18[+ or -]0.96 **

 400 15.41[+ or -]1.10 * 17.58[+ or -]0.98 **

Group Time after administration (min) Inhibition
 (%) (a)

 60 90

 Latency time (s)(mean [+ or -] S.E.M.)

Control 9.70[+ or -]0.39 10.90 [+ or -]0.89 -

Morphine 13.86[+ or -]1.05 ** 12.94[+ or -]0.68 41.39

SAM001 14.86[+ or -]1.76 ** 14.18[+ or -]2.30 48.85

 16.36[+ or -]1.45 ** 16.18[+ or -]1.97 * 58.97

 15.90[+ or -]1.18 ** 17.46[+ or -]1.30 ** 67.45

SAM002 13.61[+ or -]2.05 15.81[+ or -]1.30 ** 62.34

 15.21[+ or -]1.62 ** 17.24[+ or -]1.49 ** 64.59

 15.78[+ or -]1.24 ** 16.78[+ or -]1.42 ** 63.36

SAM003 15.68[+ or -]0.98 ** 15.40[+ or -]1.23 * 56.62

 14.84[+ or -]0.87 ** 15.80[+ or -]1.11 ** 50.49

 15.65[+ or -]1.23 ** 15.63[+ or -]1.32 * 54.07

SAM004 15.31[+ or -]1.18 ** 12.58[+ or -]0.92 39.56

 13.55[+ or -]1.46 * 15.74[+ or -]1.04 ** 41.29

 15.03[+ or -]0.80 14.31[+ or -]0.46 56.01

SAM005 14.78[+ or -]2.40 13.20[+ or -]1.35 40.48

 12.58[+ or -]0.67 ** 12.61[+ or -]1.15 30.36

 14.13[+ or -]1.30 ** 14.54[+ or -]1.06 * 45.18

SAM006 16.06[+ or -]1.73 ** 15.50[+ or -]2.03 56.31

 13.39[+ or -]0.83 ** 14.60[+ or -]1.20 * 44.16

 16.28[+ or -]0.74 ** 15.99[+ or -]0.61 ** 66.63

*p < 0.05, **p < 0.0l vs. control.
(a)Percent inhibition of total hot-plate response.


Two different models of nociception were employed in the present study with the objective of identifying the possible peripheral and central effects of the volatile oils. The volatile oils from all six genotypes of H. pectinata showed analgesic effects in both models used. This observation indicates that the volatile oil of H. pectinata has peripheral (reduced writhing) and central (delayed reaction time) effects. According to Collier et al. (1968), acetic acid acts indirectly, inducing the liberation of endogenous mediators that stimulate the nociceptive neurons which are sensitive to the antiinflammatory non steroidal drugs (NSAIDs) and to opioids. In general, our results show that the volatile oil of H. pectinata induces variable analgesic effects, depending on the origin of the genotypes. These observations are indicative of the potential for H. pectinata to be used as an analgesic drug.

Bearing in mind that the major constituents of the genotype SAM002 (major inhibitory effect in both models) are [beta]-caryophyllene (27.1%) and pectinone (21.04%) and the major constituents of genotype SAM004 are [beta]-caryophyllene (45.1%) and caryophyllene oxide (20.9%), it appears that the inhibitory effects of the volatile oils are due mainly to the interaction of all constituents and not only to the majority compounds, since previous work has demonstrated that pure [beta]-caryophyllene does not show antinociceptive effects at doses of 400 mg/kg body wt (Santos et al., 1998). The action of the volatile oils is the result of the combined effect of both their active and inactive compounds, and the latter might influence absorption, pharmacokineties, and bioavailability of the active compounds (Svoboda and Deans, 1995).

The administration (i.p.) of the opioid agonist naxolone (5 mg/kg body wt) completely reversed the antinociceptive effect of the volatile oils of all genotypes on hot plate test (Table 5). These results suggest that opioid receptors are involved in the antinociceptive action of the volatile oil of H. pectinata.
Table 5. Effect of naloxone on the antinociceptive effect of the
essential oil from H. pectinata genotypes on the hot-plate test in
mice (n = 8)

Group/dose Time after administration (min)
(mg/kg body
wt.)

 30 45

 Latency time (s)(mean[+ or -]S.E.M.)

Control 10.15[+ or -]0.84 10.30[+ or -]0.74

Morphine 10 14.23[+ or -]1.09 * 14.05[+ or -]0.65 *

Nal 5 8.91[+ or -]0.54 8.89[+ or -]0.97

Morphine 10 9.45[+ or -]0.68 10.22[+ or -]0.98
[+ or -]Nal 5

SAM001 100 12.99[+ or -]0.79 * 14.39[+ or -]1.40 *

SAM001 100 8.11[+ or -]0.87 9.59[+ or -]0.68
[+ or -]Nal 5

SAM002 100 15.46[+ or -]1.30 ** 14.31[+ or -]1.30*

SAM002 100 9.44[+ or -]0.93 10.44[+ or -]1.01
[+ or -]Nal 5

SAM003 100 13.80[+ or -]0.61 ** 14.95[+ or -]1.18 **

SAM003 100 10.35[+ or -]1.18 10.76[+ or -]1.01
[+ or -]Nal 5

SAM004 100 14.19[+ or -]0.41 ** 13.13[+ or -]0.46 **

SAM004 100 11.17[+ or -]1.44 10.76[+ or -]0.99
[+ or -]Nal 5

SAM005 100 14.64[+ or -]1.17 ** 14.95[+ or -]0.91 **

SAM005 100 9.43[+ or -]0.68 10.79[+ or -]1.09
[+ or -]Nal 5

SAM006 100 13.41[+ or -]0.53 * 15.28[+.or.- 0.71 **

SAM006 100 8.59[+ or -]0.84 9.68[+ or -]0.46
[+ or -]Nal 5

Group/dose Time after administration (min) Inhibition
(mg/kg body (%) (a)
wt.)

 60 90

 Latency time (s)(mean[+ or -]S.E.M.)

Control 9.54[+ or -]0.75 9.63[+ or -]0.91

Morphine 10 13.96[+ or -]1.01 * 13.98[+ or -]0.71* 41.50

Nal 5 8.39[+ or -]0.67 9.07[+ or -]0.74 12.65

Morphine 10 9.85[+ or -]0.59 10.12[+ or -]1.01 0
[+ or -]Nal 5

SAM001 100 13.49[+ or -]0.80 ** 14.38[+ or -]1.13 ** 39.45

SAM001 100 9.58[+ or -]1.07 9.50[+ or -]1.02 0
[+ or -]Nal 5

SAM002 100 14.25[+ or -]1.42 * 16.18[+ or -]0.91 ** 53.28

SAM002 100 9.01[+ or -]0.57 10.28[+ or -]1.09 0
[+ or -]Nal 5

SAM003 100 16.05[+ or -]0.96 ** 16.14[+ or -]1.54 ** 53.79

SAM003 100 10.53[+ or -]0.60 9.44[+ or -]0.62 3.70
[+ or -]Nal 5

SAM004 100 15.19[+ or -]1.26 ** 16.25[+ or -]1.23 ** 48.34

SAM004 100 11.49[+ or -]1.21 10.79[+ or -]1.23 14.69
[+ or -]Nal 5

SAM005 100 15.11[+ or -]1.11 ** 16.61[+ or -]0.30 ** 54.80

SAM005 100 11.21[+ or -]1.10 11.45[+ or -]1.26 8.25
[+ or -]Nal 5

SAM006 100 15.58[+ or -]0.93 ** 15.68[+ or -]1.31 ** 51.27

SAM006 100 8.51[+ or -]0.60 10.73[+ or -]0.68 0
[+ or -]Nal 5

*p < 0.05, **p < 0.0l vs. control.
(a) Percent inhibition of total hot-plate response.


The toxicity of the volatile oils was verified; the LD50 value was estimated at 1.51 [+ or -]0.38 g/kg body wt. During the experiment, animals exhibited low motility, respiratory difficulty and shaking when treated with higher doses (4 and 5 g/kg body wt.).

Acknowledgements

The authors wish to thank the 'Tundacao de Amparo a Pesquisa de Sergipe-FAP-SE" for financial support for this research, "Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior-CAPES'1 for the first author's fellowship and "Conselho Nacional de Desenvolvimen-to Cientifico e Tecnologico-CNPq" for the second and fifth author's productivity grants.

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Bispo, M.D., Mourao, R.H.V., Franzotti, E.M., Bomfim, K.B.R., Arrigoni-Blank, M.F., Moreno, M.P.N., March-ioro, M., Antoniolli, A.R., 2001. Antinociceptive and antiedematogenic effects of the aqueous extract of Hyptis pectinata leaves in experimental animals. J. Ethnopharma-col. 76, 81-86.

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*Corresponding author. Tel.: + 55 79 34312410; fax: +55 79 2105 6494.

E-mail address: fatima.blank@terra.com.br (M.F. Arrigoni-Blank).

0944-7113/$-see front matter [c] 2007 Elsevier GmbH. All rights reserved. doi:tO.I016/'j.phymed.2007.09.009

M.F. Arrigoni-Blank (a),*, A.R. Antoniolli (b), L.C. Caetano (c), D.A. Campos (b), A.F. Blank (d), P.B. Alves (e)

(a) Nucleo de Ciencias Biologicas, Campus Prof. Alberto Carvalho, Universidade Federal de Sergipe, Itabaiana, Av. Vereador Olimpio Grande sln, 49500-000 Sergipe, Brazil

(b) Departamento de Fisiologia, Universidade Federal de Sergipe, Sao Cristovao, Sergipe, Brazil

(c) Departamento de Quimica, Universidade Federal de Alagoas, Maceio, Alagoas, Brazil

(d) Departmento de Engenharia Agronomica, Universidade Federal de Sergipe, Sao Cristovao, Sergipe, Brazil

(e) Departamento de Quimica, Universidade Federal de Sergipe, Sao Cristovao, Sergipe, Brazil
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Author:M.F. Arrigoni-Blank; A.R. Antoniolli; L.C. Caetano; D.A. Campos; A.F. Blank; P.B. Alves
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:3BRAZ
Date:May 1, 2008
Words:4122
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