Activity of some Mexican medicinal plant extracts on carrageenan-induced rat paw edema.

Abstract

The extracts obtained from 14 plants of the Mexican medicinal flora were assessed for anti-inflammatory activity by carrageenan-induced rat paw edema model. The i.p. administration of the extracts at a dose of 400 mg/kg produced a high reduction of edema with 70% of the plant extracts. Oenothera rosea methanol extract, Sphaeralcea angustifolia chloroform extract, Acacia farnesiana, Larrea tridentata and Rubus coriifolius methanol extracts as well as the aqueous extract of Chamaedora tepejilote were demonstrated to be particularly active against the induced hind-paw edema. Moderate inhibition of edema formation was also demonstrated with the methanol extracts of Astianthus viminalis, Brickellia paniculata, C. tepejilote and Justicia spicigera.

[c] 2004 Elsevier GmbH. All rights reserved.

Keywords: Mexican medicinal plants; Anti-inflammatory; Edema inhibition; Nor-dihydroguaiaretic acid

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Introduction

Herbal remedies are an important resource in Mexican traditional medicine, the inhabitants of the urban and rural zones still use medicinal plants as an alternative to resolve their primary health problems. Ethnobotanical information gives a number of species widely used in the country to treat ailments related to the inflammatory process and which could represent an important source to find compounds with anti-inflammatory properties. In the course of our search for new pharmacologically active compounds from Mexican medicinal plants, the present study deals with a preliminary screening of the effects produced by crude extracts obtained from 14 species in the carrageenan-induced rat paw edema test.

Materials and methods

Plant material and extracts preparation

The plants were collected in different regions of Mexico and the species were botanically identified by MSc Abigail Aguilar, voucher specimens were deposited at IMSSM Herbarium (Table 1). Acacia farnesiana, Astianthus viminalis, Justicia spicigera and Piper auritum were collected in Morelos State, Brickellia paniculata, Helianthemum glomeratum and Rubus coriifolius in the highland of Chiapas, Chamaedora tepejilote in Veracruz, Lantana hispida in Oaxaca, Larrea tridentata in San Luis Potosi and Teloxys graveolens, Oenothera rosea, Eucalyptus globulus and Sphaeralcea angustifolia in Mexico City. The species were air dried and 100 g ground plant material was extracted for seven days by maceration at room temperature with 11 of the organic solvent, the procedure was repeated 2-3 times. When more than one solvent was used, subsequent extraction was performed with solvents of increasing polarity (Table 1). Extracts were concentrated to dryness under low pressure at 40[degrees]C. Aqueous extracts from C. tepejilote and L. hispida were prepared by digestion of the plant material at 60[degrees]C during 45 min and the extracts obtained were lyophilized. Just prior to the biological testing, all the extracts were dissolved in the appropriate vehicles.

Rat paw edema assay

The carrageenan-induced hind paw edema test was performed according to Winter (Winter et al., 1962). Briefly, male Sprague-Dawley rats of 150-200 g body weight, divided into groups of 5-8 animals, were injected subcutaneously into the plantar side of the hind paw with 0.1 ml of a freshly prepared 1% carrageenan suspension ([lambda] type IV Sigma). The extracts (400 mg/kg b.w.) dissolved in DMSO-[H.sub.2]O (2:3), Tween 20-[H.sub.2]O (2:3) or [H.sub.2]O were injected intraperitoneally (i.p.) 60 min before carrageenan in a volume not exceeding 0.5 ml. The same volume of the vehicle was administered to the control groups. Rat paw edema was assessed by volume displacement method (plethysmometer (Ugo Basile 7150) before administration of the proinflammatory agent (initial volume) and, after carrageenan injection each 10 min during the first hour and at 2, 3, 5, 7 and 24 h afterwards. The time course of the increase in paw swelling was determined by comparing it to the initial volume and the percent inhibition of edema was calculated for each group with the respective vehicle-treated control (Chattopadhyay et al., 1994). Phenylbutazone and nor-dihydroguaiaretic acid (NDGA) were used as references (Sigma, USA).

Statistical analysis

Results were expressed as the mean value [+ or -] standard error of the mean. Treated groups were compared with the controls for statistical significant differences (p < 0.05) using paired Student's t-test.

Results

The subplantar injection of carrageenan produced a local edema that increased progressively to reach its maximum in 3-5 h. At this time, the volume of the injected paw was almost 100% greater than the initial measured before carrageenan, afterwards the edema decreased but remained obvious 24 h after. As already reported, a biphasic development of edema was registered during the 1-5 h of the process, the first phase reached a maximum between 1 and 1.5 h followed by a second phase with increased edema formation (Fig. 1).

By evaluating the selected plant species it was found that P. auritum at the tested dose of 400 mg/kg was the only inactive extract. On the other hand, mortality was registered with the hexane extracts of A. farnesiana, C. tepejilote, J. spicigera, S. angustifolia, the chloroform extracts of A. farnesiana, J. spicigera, the methanol extract of E. globulus and the acetone-water extract of H. glomeratum. The animals died within 24 h of administering these extracts and they were not included in the analysis of the obtained results. The rats tolerated i.p. administration of the rest of the extracts well.

The early phase in edema formation (1 h) was not affected by phenylbutazone but it was suppressed by pretreatment with most of the polar extracts and NDGA (Table 2). The magnitude of the effect of S. angustifolia chloroform extract was found to be higher in the early phase (78.8 [+ or -] 8.8% at 60 min) than the registered effect at 3 h (57.5 [+ or -] 6.4%) (Table 3). On the contrary, inhibition of edema formation with the methanol extract of A. farnesiana was 54.0 [+ or -] 2.3% in the early phase but increased significantly at 3 h (72.8 [+ or -] 2.3%). It was determined that C[H.sub.2][Cl.sub.2]-MeOH (1:1) extract of R. coriifolius exhibited potent edema inhibition at 60 min after carrageenan administration (85.8 [+ or -] 0.7%), close to the effect obtained at 3 h (89.7 [+ or -] 4.4%). Methanol extract of O. rosea showed a similar profile with a reduction of the paw edema by 79.8 [+ or -] 4.1% and 86.2 [+ or -] 1.8% after 60 min and 3 h injection of carrageenan, respectively. A significant anti-edematogenic action was demonstrated also with the aqueous extract of C. tepejilote (71.4 [+ or -] 5.8% at 60 min and 77.9 [+ or -] 1.3% at 3 h). Another group of extracts were found to be less active: methanol extracts of A. viminalis, B. paniculata, C. tepejilote and J. spicigera produced inhibition with values between 33% and 45% and the effect was similar at both phases of edema. Feeble activity was registered with L. hispida extracts.

[FIGURE 1 OMITTED]

All the extracts of L. tridentata were active, the effect of the methanol one, being slightly more potent. In Fig. 2 the time course of edema formation with the methanol extract of the plant and a commercial sample of NDGA is shown; at the dose of 400 mg/kg of the extract, inhibition of paw swelling at 3 h (64.1 [+ or -] 4.8%) was near to that obtained with 12 mg/kg NDGA (53.2 [+ or -] 5.3%).

Discussion

Anti-phlogistic drugs are needed to control inflammatory processes in several health problems including different types of rheumatic diseases. Although anti-inflammatory effects of medicinal plants have been extensively studied, evaluation of these properties in Mexican medicinal species are scarcely reported, this prompted us to screen 14 plants with presumable anti-inflammatory activity. As far as the anti-edematogenic effect is concerned, except for P. auritum root methanol extract, all the evaluated species were effective in reducing the carrageenan-induced edema. This, may support the popular use the plants have in the treatment of inflammation or diseases related with an inflammatory response.

As reported previously, a biphasic response in the time course of edema development is observed after injection of carrageenan in rat hind-limb, a nonphagocytic edema is measured at 20-60 min (first phase) and neutrophils in this phase are not detected in histological sections. Histamine, 5-hydroxytryptamine, bradykinin and platelet activating factor have been reported to be the mediators released in this first phase of carrageenan-induced rat foot edema (Hwang et al., 1986). After 3 h an intense phagocytic inflammation with large numbers of neutrophils and tissue edema is observed (Vinegar et al., 1987). Thus, from the results here obtained, it may be concluded that dichloromethane-methanol extract of R. coriifolius, aqueous extract from C. tepejilote and the methanol extract of L. tridentata possess a potent anti-inflammatory activity, both in the first and second phases of edema formation. The aerial parts of R. coriifolius are used in southeastern Mexico to treat bloody diarrhea; (-)-epicatechin, (+) catechin and ellagic acid were isolated from the dichloromethane-methanol extract (Alanis et al., 2003). Anti-inflammatory properties of polyphenols have already been reported, although hyperin, which is also present in the extract (Alanis et al., 2003), could account for the effect.

[FIGURE 2 OMITTED]

C. tepejilote is an ornamental plant used in Mexico to treat pneumonia and cough. In our study, intraperitoneal administration of the hexane extract showed to be toxic even at doses of 25 mg/kg and, at lower doses, anti-edematogenic effect was not detected. On the contrary, a potent inhibitory effect on both phases of carrageenan induced edema was observed with the nontoxic aqueous extract of C. tepejilote and chemical studies are in progress to identify the active principles of this plant. In Mexico, L. tridentata is widely employed to treat urinary and renal disorders as well as edematous processes. The lignane nor-dihydroguaieretic acid in the plant has been described as a potent anti-oxidant agent which inhibits lipooxigenase and cyclooxigenase exerting a potent and long-lasting anti-inflammatory effect (Della Logia et al., 1988). In all the extracts here assayed, NDGA was detected by thin layer chromatography. The activity profile of NDGA differs from that of phenylbutazone, for NDGA induces an inhibitory effect close to the maximal activity already 30 min following the injection of the phlogistic compound, the effect remained steady during several hours.

O. rosea is a well known plant used in cases of bruises and wound healing, stomach pain, and diarrhoea. No chemical and pharmacological studies that could support the anti-edematous effect have been reported on this species, nevertheless, the evaluated methanol extract found of O. rosea with its sustained high effect during 7 h, was to be a good candidate to be submitted for further investigation.

At the same time, another group of the evaluated species showed a moderate activity, among them, A. viminalis that was included in this assay because its content in ursolic acid, a triterpene, which has already been reported as very active on carrageenan induced edema test (Deepak and Swami, 2000). In the present evaluation, inhibition produced by the methanol extract of A. viminalis was close to the effect registered with the methanol extract of B. paniculata and J. spicigera (40-45% inhibition). B. paniculata extract contains xanthomicrol in high concentrations (Meckes et al., 2002), this methoxiflavone has proved to be a LTC4 and thromboxane B2 inhibitor (Skaltsa et al., 2000). Anti-inflammatory activity of J. spicigera has not been reported before, although a potent activity was described for lignan glycosides isolated from the species Justicia ciliata (Day et al., 2000).

The methanol extract of A. farnesiana inhibited both phases of the edema process being highly active at 3 h with an inhibition of 72%. The flavonoids apigenin-6,8-di-C-[beta]-D-glucopyranoside (vicenin-2) and linamarin which have been isolated from the leaves (Thieme and Khogali, 1974; Secor et al., 1976; Martinez-Vazquez et al., 1998) were reported for its anti-oxidant and anti-edematous properties (Gil-Izquierdo et al., 2002; Martinez-Vazquez et al., 1998) and could account for the here described effect.

Finally, by comparison with S. angustifolia methanol extract, the chloroform extract of the plant showed the best activity, specially on the first phase of edema formation (78%). This finding is interesting in view of the fact that in Mexico, the plant is more frequently used by topical application.

The present screening permits to support, in part, the attributed properties of the evaluated species, often reported to treat ailments in which inflammatory processes could be involved. Current chemical and pharmacological research is being carried out on the active extracts of O. rosea, C. tepejilote and S. angustifolia, three of the species which may be a potential source of natural products with anti-inflammatory activity.

Table 1. Plant extracts tested on carrageenan paw edema test

Botanical species          Voucher (IMSSM)  Medicinal uses

Acacia farnesiana          14293            Diarrhoea, inflammation
  (aerial parts)
Astianthus viminalis       LA24 (a)         Diabetes, hemorrhage
  (aerial parts)
Brickellia paniculata       7471            Stomach pain, colics,
  (leaves)                                    diarrhoea
Chamaedora tepejilote      13080            Pneumonia, cough
  (leaves)
Eucalyptus globulus        14291            Bronchitis, throat
  (leaves)                                    inflammation
Helianthemum glomeratum    14142            Bloody diarrhoea
  (aerial parts)
Justicia spicigera         14150            Colics, inflammation
  (leaves)
Lantana hispida (aerial     8815            Stomach pain, inflammation
  parts)
Larrea tridentata (aerial  14411            Lithiasis, diarrhoea
  parts)
Oenothera rosea (aerial    14412            Bruise, swelling
  parts)
Piper auritum (roots)      14292            Analgesic, diuretic
Rubus coriifolius (aerial  11874            Bloody diarrhoea, cough
  parts)
Sphaeralcea angustifolia   14294            Bruise, swelling
  (aerial parts)
Teloxys graveolens         11133            Diarrhoea, stomach pain
  (aerial parts)

Botanical species          Extracts

Acacia farnesiana          Hex/CH[Cl.sub.3]/MeOH
  (aerial parts)
Astianthus viminalis       MeOH
  (aerial parts)
Brickellia paniculata      MeOH
  (leaves)
Chamaedora tepejilote      Hex/MeOH/[H.sub.2]O
  (leaves)
Eucalyptus globulus        Hex/MeOH
  (leaves)
Helianthemum glomeratum    [Me.sub.2]CO-[H.sub.2]O
  (aerial parts)
Justicia spicigera         Hex/CH[Cl.sub.3]/MeOH
  (leaves)
Lantana hispida (aerial    Hex/MeOH/[H.sub.2]O
  parts)
Larrea tridentata (aerial  Et pt/C[H.sub.2][Cl.sub.2]/MeOH
  parts)
Oenothera rosea (aerial    Hex/CH[Cl.sub.3]/MeOH
  parts)
Piper auritum (roots)      MeOH
Rubus coriifolius (aerial  C[H.sub.2][Cl.sub.2]-MeOH
  parts)
Sphaeralcea angustifolia   Hex/CH[Cl.sub.3]/MeOH
  (aerial parts)
Teloxys graveolens         MeOH
  (aerial parts)

(a) Herbarium at Morelos University.

Table 2. Inhibition of the first phase (at 1 h after carrageenan
injection) by 400 mg/kg i.p. administration of the active extracts
of low (hexane, petroleum ether); medium (chloroform, dichloromethane)
and high (methanol, acetone-aqueous) polarities

Botanical species  % Inhibition of edema
                   Polarity of the organic extracts
                          Low                       Medium

A. farnesiana
A. viminalis
B. paniculata
Ch. tepejilote
E. globulus        23.3 [+ or -] 6.7
J. spicigera
L. hispida         25.5 [+ or -] 4.0
L. tridentata      57.5 [+ or -] 2.9           52.0 [+ or -] 2.7
O. rosea           41.3 [+ or -] 1.2           46.7 [+ or -] 2.6
R. coriifolius
S. angustifolia                                78.8 [+ or -] 8.8
T. graveolens

Botanical species  % Inhibition of edema
                   Polarity of the organic extracts       Aqueous
                          High

A. farnesiana      54.0 [+ or -] 2.3
A. viminalis       39.0 [+ or -] 2.6
B. paniculata      44.1 [+ or -] 8.5
Ch. tepejilote     34.6 [+ or -] 4.4                 71.4 [+ or -] 5.8
E. globulus
J. spicigera       39.0 [+ or -] 2.6
L. hispida         31.9 [+ or -] 1.1                 11.5 [+ or -] 1.3
L. tridentata      62.9 [+ or -] 4.2
O. rosea           79.8 [+ or -] 4.1
R. coriifolius     85.8 [+ or -] 0.7
S. angustifolia    36.9 [+ or -] 3.4
T. graveolens      32.3 [+ or -] 1.5

NDGA was used as a reference at a dose of 12 mg/kg.
NDGA (12 mg/kg) 55.0 [+ or -] 4.3%.

Table 3. Inhibition produced by the administration of the active
extracts (400 mg/kg i.p) on the 2nd phase of carrageenan induced edema
(3 h)

Botanical species  % Inhibition of edema
                   Polarity of the organic extracts
                          Low                       Medium

A. farnesiana
A. viminalis
B. paniculata
Ch. tepejilote
E. globulus        43.0 [+ or -] 6.4
J. spicigera
L. hispida         26.7 [+ or -] 2.4
L. tridentata      52.3 [+ or -] 7.5           55.6 [+ or -] 3.9
O. rosea           50.0 [+ or -] 1.1           37.2 [+ or -] 4.4
R. coriifolius
S. angustifolia                                57.5 [+ or -] 6.4
T. graveolens
NDGA (12 mg/kg)                                53.2 [+ or -] 5.3%
Phenylbutazone
  (20 mg/kg)                                   26.4 [+ or -] 5.8%

Botanical species  % Inhibition of edema
                   Polarity of the organic extracts       Aqueous
                          High

A. farnesiana      72.8 [+ or -] 2.3
A. viminalis       32.8 [+ or -] 1.6
B. paniculata      43.1 [+ or -] 1.2
Ch. tepejilote     40.8 [+ or -] 6.5                 77.9 [+ or -] 1.3
E. globulus
J. spicigera       40.3 [+ or -] 4.8
L. hispida         24.3 [+ or -] 2.7                 11.4 [+ or -] 1.1
L. tridentata      64.1 [+ or -] 4.8
O. rosea           86.2 [+ or -] 1.8
R. coriifolius     89.7 [+ or -] 4.4
S. angustifolia    20.3 [+ or -] 0.0
T. graveolens      12.6 [+ or -] 2.6
NDGA (12 mg/kg)
Phenylbutazone
  (20 mg/kg)

Phenybutazone, and NDGA were used at a dose of 20 and 12 mg/kg,
respectively.

Acknowledgements

We thank the National Council of Science and Technology for the financial support of this work (34761-M). The authors are grateful to the IMSSM Herbarium for their valuable assistance in the botanical identification of the plants.

Received 11 November 2002; accepted 16 June 2003

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M. Meckes*, A.D. David-Rivera, V. Nava-Aguilar, A. Jimenez

Unidad de Investigacion Medica en Farmacologia de Productos Naturales, Centro Medico Nacional Siglo XXI, IMSS., Av Cuauhtemoc 330, Col. Doctores, Mexico D.F., Mexico

*Corresponding author. Tel.: [phone]x3235; fax: [phone].

E-mail address: [e-mail] (M. Meckes).