Influence of Some Stachys. Taxa on Carrageenan-Induced Paw Edema in Rats

Abstract

This work investigates the anti-inflammatory activity of methanol extracts of four endemic Stachys. (Labiatae) taxa from the Balkans: S. beckeana. Dörfler & Hayek, S. anisochila. Vis. et Pančić, S. plumosa. Griseb., and S. alpina. L. subsp. dinarica. Murb. As a model of acute inflammation, carrageenan-induced paw edema in rats was used. Extracts, applied at doses of 50, 100, and 200 mg/kg p.o., exhibited dose-dependent activity. S. beckeana. and S. anisochila. extracts were the most active ones (ED₅₀ 154.52 and 162.24 mg/kg, respectively), with the activity comparable with indomethacin at doses of 2 and 4 mg/kg. S. plumosa. extract has shown less-pronounced anti-inflammatory effect (ED₅₀ 220.81 mg/kg). Extract of S. alpina. subsp. dinarica. had the lowest efficiency, attenuating inflammation less than 50%.

Keywords:

• Anti-inflammatory activity
• dinarica
• S. alpina subsp
• S. beckeana
• S. plumosa
• Stachys anisochila

Introduction

The genus Stachys. (Labiatae) comprises about 300 species distributed in Eurasia and America, some of which have a long traditional use as nervine, tonic, and wound healing agents, and as astringent and antidiarrheal drugs (Naghibi et al., 2005; Kukić et al., 2006a). Hence, several pharmacological activities such as antioxidant (Couladis et al., 2003; Kukić et al., 2006a), antimicrobial (Skaltsa et al., 19992003; Petrović et al., 2006), anxiolytic (Rabbani et al., 20032005), antinephritic (Hayashi et al., 1996), and anti-inflammatory (Maleki et al., 2001; Khanavi et al., 2005) have been attributed to genus.

This work describes the anti-inflammatory activity of four endemic Stachys. taxa from the Balkans: S. beckeana. Dörfler & Hayek, S. anisochila. Vis. et Pančić, S. plumosa. Griseb., and S. alpina. L. subsp. dinarica. Murb. S. beckeana., growing on mountains in northern Albania, Montenegro, Herzegovina and southern Bosnia, and S. anisochila. inhabits mountain regions in western Serbia, Bosnia, Herzegovina, and Albania. Both species belong to a very polymorphic S. recta. L. complex. S. plumosa. grows on dry pastures and mountain rocks in southeastern Serbia, Macedonia, western Bulgaria, and northern and central Greece (Ball, 1976). S. alpina. subsp. dinarica. Murb. is Balkan endemic (Croatia, Bosnia and Herzegovina, Montenegro, Serbia, and southwestern Bulgaria) (Diklić, 1974; Ball, 1976; Koeva, 1989). Our ethno-medicinal surveys showed that some of these (e.g., S. alpina. subsp. dinarica.) are used in traditional medicine for bladder inflammation, stomach complaints, and wound healing.

In the above-mentioned taxa, various secondary metabolites have been identified: glycosides of isoscutellareine, hypolaetine, chrysoeriol, apigenin, and luteolin, along with phenylethanoid glycosides, iridoids, phenolic acids, triterpenes, and essential oils (Marin et al., 2004; Meremeti et al., 2004; Kukić et al., 2006b). Previously, essential oil of S. plumosa. was assessed for its antimicrobial activity (Petrović et al., 2006). The antioxidant activity of methanol extracts of investigated species was estimated, too (Kukić et al., 2006a).

As a model of acute inflammation in this experiment, carrageenan-induced paw edema in rats was used.

Materials and Methods

Plant material

The flowering aerial parts of plants were collected from natural populations: S. beckeana. from Mt. Durmitor (Montenegro), August 2003; S. anisochila. in the gorge of Beli Rzav (western Serbia), June 2003; S. plumosa. in Jelašnička klisura gorge (southeastern Serbia), June 2002; and S. alpina. subsp. dinarica. on Mt. Jahorina (Bosnia and Herzegovina), July 2004. Voucher specimens were identified by Mr. Sc. Marjan Niketić (custodian) and deposited in the herbarium collection of the Natural History Museum in Belgrade (BEO ko820033/6, ko620041/4, ko320025/6, and ko720049/83, respectively).

Extraction

Plant material (100 g) was air-dried at room temperature and finely ground. Each sample was bimacerated with chloroform (3 and 2 days; plant material/solvent ratio = 1:7). The marc was further extracted in the same way with methanol and the solvent evaporated under reduced pressure. The obtained dry methanol extracts of S. beckeana., S. anisochila., S. plumosa., and S. alpina. subsp. dinarica. (11.66, 14.88, 12.57, and 13.94 g, respectively) were used for investigations.

Anti-inflammatory activity

The carrageenan-induced rat paw edema test was used as an experimental model for screening the anti-inflammatory activity as reported earlier (Petrović et al., 2003). The investigated Stachys. extracts, dissolved in DMSO, were administered orally (p.o.) in doses of 50, 100, and 200 mg/kg. Indomethacin, dissolved in DMSO, was used as a reference drug in doses of 1, 2, 4, and 8 mg/kg p.o. The control animals were given the vehicle (DMSO) in a dose of 1 ml/kg p.o. One hour after the oral administration of the extracts or indomethacin, 0.1 mL carrageenan-saline solution (0.5%) and saline were injected into the plantar surface of the right and left hind paws, respectively. The left paw served as the control (noninflamed paw). The animals were sacrificed 3 h after the carrageenan and saline injection, and paws were cut off for weighing. The difference in weight between right and left paw, active drug-treated versus vehicle-treated (control) rats, served as an indicator of the anti-inflammatory activity.

Animals

Animal studies were conducted in accordance with the internationally accepted principles for laboratory animal use and care in the European Community (EEC Directive of 1986; 86/609/EEC) adopted by the Ethical Committee of Military Medical Academy, Belgrade. Adult male Wistar rats, weighing 200–250 g, were used in the experiments. Experimental groups consisted of 6–10 animals each. The animals were deprived of food for 18–20 h before the beginning of the experiments but had free access to tap water.

Statistical analysis

Statistical analysis was performed with the Mann–Whitney U.-test and ANOVA. Differences were accepted as statistically significant when p. < 0.05.

The mean effective anti-inflammatory doses (ED₅₀) of extracts and indomethacin were calculated by the method of Litchfield and Wilcoxon (1949).

Results

All investigated Stachys. extracts expressed a dose-dependent anti-inflammatory effect (Table 1). The most prominent reduction of carrageenan-induced inflammation was achieved at the highest dose applied. S. beckeana. and S. anisochila. extracts were the most active ones. These extracts applied at 200 mg/kg p.o. suppressed inflammation by 53.30% and 54.04%, with ED₅₀ of 154.52 and 162.24 mg/kg, respectively. These effects were comparable with those achieved by 2 and 4 mg/kg of indomethacin (50.37% and 58.06%, respectively). S. plumosa. extract was slightly less active reaching the highest activity (46.52%) at maximum dose applied and with ED₅₀ at 220.81 mg/kg. The ED₅₀ for S. alpina. subsp. dinarica. extract could not be determined, because the uppermost dose exhibited an anti-inflammatory effect of 37.17%.

Table 1.. Anti-inflammatory effect of investigated Stachys. extracts.

Treatment	Dose (mg/kg, p.o.)	Anti-inflammatory effect (%)	ED50 (mg/kg)
Control (DMSO)^a.	1 mL/kg	0.00±20.57
Indomethacin^b.	1	27.14±11.83	2.53
	2	50.37±5.96*
	4	58.06±13.87*
	8	74.32±15.70**
S. beckeana. extract	50	20.65±8.97*	154.52
	100	45.43±10.02**
	200	53.30±5.72**
S. anisochila. extract	50	20.33±12.11*	162.24
	100	40.39±6.06**
	200	54.04±11.20**
S. plumosa. extract	50	25.65±7.19*	220.81
	100	40.65±14.23*
	200	46.52±14.70*
S. alpina. subsp. dinarica. extract	50	24.67±9.67*	—
	100	30.43±5.39*
	200	37.17±16.63*

^a.Vehicle.

^b.Reference drug.

*p < 0.05; **p < 0.001 vs. control group.

Discussion

The obtained results are consistent with investigations concerning some other Stachys. species. S. inflata. (Maleki et al., 2001) and S. byzantina. (Khanavi et al., 2005) also attenuated carrageenan-induced rat paw edema similarly to indomethacin (5 mg/kg p.o.). Extract of aerial parts of S. inflata. exhibits a cardioprotective effect, causing a pronounced reduction in myocardial infarct size, and such activity is likely the result of the inhibition of inflammatory components at the site of the reperfused area (Garjani et al., 2004).

As previously reported, secondary metabolites in investigated Stachys. species include various compounds (Marin et al., 2004; Meremeti et al., 2004). Many of these compounds also demonstrated anti-inflammatory activity. In S. plumosa., phenylethanoid glycosides acteoside and forsithoside B were identified (Bankova et al., 1999), which showed selective inhibition toward the COX-2 enzyme (Sahpaz et al., 2002). Acteoside, isolated from S. sieboldii., had a suppressive effect on the accumulation of leukocytes in the nephritic glomeruli (Hayashi et al., 1994) and a modulating activity on NO production, a key mediator in carregeenan-induced rat paw edema (Ohno et al., 2002).

All investigated Stachys. taxa are characterized by the presence of several hypolaetin derivatives. It was previously shown that hypolaetin and its 8-β-d-glucoside are selective inhibitors of lipoxygenase activity in vitro. (Ferrandiz & Alcaraz, 1991). Few luteolin glycosides were identified in S. plumosa. (Marin et al., 2004). Flavonoids resembling luteolin in structure (sharing 3′, 4′, 5, 7-tetrahydroxy substitution) resemble its anti-inflammatory activity, obtained by inhibition of lipopolysaccharide-induced TNF-α production (Ueda et al., 2004). In S. anisochila. and S. beckeana., harpagide and 8-acetylharpagide were identified (Lenherr et al., 1984). These iridoids selectively inhibit thromboxane-synthase enzyme, which may be the primary target of their action and one of the mechanisms through which they exert their anti-inflammatory effects (Bermejo Benito et al., 2000).

It is reasonable to presume that presence of the above-mentioned compounds contributes to the demonstrated anti-inflammatory effects of investigated Stachys. taxa. Our previous experiments revealed the antioxidant and free radical scavenging activities of the investigated extracts (Kukić et al., 2006a). It is well-known that antioxidants directly scavenge reactive oxygen species (ROS), which have been considered to exert their inflammatory effects via a direct toxic action on target cells and also through gene induction and especially through the activation of the redox-sensitive transcription factor NF-κB (Menegazzi et al., 2005). Some antioxidants may also mediate their anti-inflammatory activities by inhibiting different proinflammatory enzymes (COX, LOX, NOS) (Sadik et al., 2003; Sala et al., 2006) and also by preventing induction of the cytokine cascade and upregulation of the expression of adhesion molecules (Menegazzi et al., 2006). It had been shown that the extracts of S. beckeana. and S. anisochila., the most active ones in this study, also exhibited stronger antioxidative activity than extracts of the other two Stachys. species. Such correlation between antioxidant activity and anti-inflammatory effects obtained in the current study suggests that antioxidant activity of investigated Stachys. extracts is at least partly involved in their anti-inflammatory effect. However, for determining the precise mechanisms of the anti-inflammatory action and for recognition of active compounds responsible for such activity, further investigations are needed.

Acknowledgment

This research was supported by the Ministry of Science and Environmental Protection of the Republic of Serbia (grant no. 143012). The authors thank Mr. Sc. Marjan Niketić, custodian of the Natural History Museum in Belgrade, for the collection and the identification of the plant material.