Components and antioxidant, anti-inflammatory, anti-ulcer and antinociceptive activities of the endemic species Stachys mialhesi de Noé

2.1 General procedures

¹H and ¹³CNMR spectra were obtained on a Bruker model 300, AMX-400 and AMX-600 spectrometers with standard pulse sequences operating at 300, 400 and 600 MHz in ¹H and 125, 150 MHz in ¹³C NMR. DMSO-d₆ and CDCl₃ are used as solvents. EIMS and HRE-IMS were taken on a Micromass model Autospec (70 eV) spectrometer. Column chromatography (CC) was carried out on polyamid, and prep. TLC on silica gel 60 PF254 + 366 plates (20 × 20 cm, 1 mm). Indomethacin (Sigma, USA), Acetylsalicylic acid (El Vasr Co.), Carrageenan (BDH, England), Tween 80 (Sigma, Germany), saline (Sigma, Germany). All the chemicals and reagents used were of the highest grade of purity.

2.2 Plant material

Aerial parts of S. mialhesi de Noé were collected on April 2005 at Djebel El-Ouahch Constantine (North Eastern Algerian). The voucher specimen was identified by Professor Gérard De Bélair (University Badji-Mokhtar, Annaba) and was deposited at the Musée botanique de la Ville d’Angers (France) under the reference MBAng2005.10.

2.3 Preparation of the extract

Air-dried and powdered aerial parts (1 kg) of S. mialhesi were extracted with 70% MeOH. The residue was dissolved in water and extracted with petroleum ether, dichloromethane, ethyl acetate and n-BuOH, successively.

Each solvent free extract was dissolved in Tween 80 (7% aqueous solution) as vehicle and biologically tested in different dose levels. The studied biological activities of the tested extract were compared with the reference standard as well as the controls injected with 0.2 ml vehicle (7% Tween 80).

2.4 Isolation of chemical constituents

The n-butanolic extract (10 g) has been chromatographed over polyamid column and eluted with a gradient of toluene/methanol with increasing polarity. Fraction F4, obtained from toluene 92% (50 mg) was subjected to another silica gel column chromatography and eluted with a gradient of dichlormethane/methanol with increasing polarity. Fraction F4.4 (38 mg) was further subjected to preparative silica gel TLC and eluted with dichloromethane/methanol (80:20) leading to compound 1 (7 mg) which was identified as horminone, reported for the first time from Stachys genus and compound 2 (25 mg) which was identified as apigenin-7-β-d-(6″-E-p-coumaroyl)-glucopyranoside, and isoscutellarein-7-O-(6″′-O-acetyl-β-d-allopyranosyl-(1 → 2)-β-d-glucopyranoside) (3). The latter precipitated in all fractions starting from toluene 90% from which 3g of this compound is gathered.

2.5 Animals

Animal studies were conducted in accordance with the internationally accepted principles for laboratory animal use; Animals were obtained from the Animals house colony of the National Research Center, Cairo, Egypt.

Adult albino mice of both sex weighing (20–25 g) and adult male albino rats weighing (120–150 g) were used for determination of the medium lethal dose (LD₅₀), analgesic activity and carrageenan-induced edema, ulcerogenic effect, respectively. The animals were kept under normal laboratory conditions of humidity, temperature (25 ± 1 °C) and light (12 h day:12 h night), and allowed free access to food and water.

2.6 Evaluation of antioxidant activity

2.7 Determination of the medium lethal dose (LD₅₀)

For acute toxicity study, groups of five mice of both sexes (20–25 g) were used, one group injected with the corresponding volume 0.2 ml of the vehicle (Tween 80) and served as control and the tested extract were orally administered in doses ranging from 1000 to 10,000 mg/kg body weight. Animals were observed and the mortality rates were recorded within the first 24 h after extract administration. The LD₅₀ was determined according to Behrens and Kerber method (Behrens and Kerber, 1935).

2.8 Evaluation of analgesic activity

2.9 Evaluation of anti-inflammatory activity

2.10 Ulcerogenic effect

Groups of adult male albino rats of six animals each (110–150 g) were fasted overnight, and orally given the tested extract at a dose level of 5 g/kg body weight, for four consecutive days. Four hours following the last dosing, animals were sacrificed, their stomach were removed, opened along the greater curvature, and the numbers of ulcers were assessed by adopting the method of Correl et al. (1979).

2.11 Data and statistical analysis

Data were expressed as mean ± s.e.m, statistical comparisons between different groups were made by analysis of variance (ANOVA) followed by multiple comparison tests (Post hoc Dunnett’s). Differences with p < 0.05 between experimental groups and control animals were considered statistically significant (Norusis, SPSS INC).

3.1 Characterization of isolated compounds

The n-butanolic extract (BESM), obtained after maceration of the aerial parts of S. mialhesi with MeOH/H₂O (7:3) followed by filtration, concentration and successive liquid–liquid extractions using petroleum ether, dicholoromethane, ethyl acetate and n-butanol, successively, led to the isolation of one diterpenoid, Horminone (1), and two flavonoids glycosides, apigenin-7-O-(6″-E-p-coumaroyl)-β-d-glucopyranoside (2) and isoscutellarein-7-O-(6″′-O-acetyl-β-d-allopyranosyl-(1 → 2)-β-d-glucopyranoside) (3) (Fig. 1). These compounds were identified by the use of ¹H NMR and ¹³C NMR, DEPT, and 2D NMR experiments, COSY, HMQC, HMBC in addition with UV spectroscopy.

Close

Figure 1

Chemical structures of compounds 1–3.

Export to PPT

3.2 Antioxidant activity and phenol composition

The antioxidant and free radical scavenging powers of phenolic compounds have been extensively studied for the past 10 years. The roles of OH groups and π electron delocalization in flavonoids have been largely discussed (Rice-Evans et al., 1996).

Many plants which contain flavonoids are known to possess good antioxidant activities by comparison with reference molecules such as vitamin E.

In this study, it was first speculated that S. mialhesi de Noé might be a source of natural antioxidants. The performed test confirmed the ability of the BESM and compound (3) to scavenge DPPH radical.

The idea that this activity was due to the presence of phenolic compounds led us to investigate the phenolic composition of the BESM.

Fig. 2a,b.shows the concentration of DPPH which gave 50% inhibition (IC₅₀) of BESM and compound (3). By comparison, the value of IC₅₀ of vitamin E was, under the same experimental conditions, 0.025 mg/ml. Quercetin IC₅₀ values were used as a reference (Rice-Evans et al., 1996). In the DPPH test, quercetin IC₅₀ was 0.012 mg/ml. The extract IC₅₀ (0.047 mg/ml) was the result of a mixture of molecules, with or without antioxidant activity. The IC₅₀ value 0.066 mg/ml was the result of a pure molecule (3) (Table 1).

Close

Figure 2

(a) IC₅₀ of BESM. (b) IC₅₀ of compound (3).

Export to PPT

3.3 Acute toxicity

The result of the acute toxicity for BESM showed that the tested extract assayed up to 10 g/kg did not prove to be toxic since it did not induce mortality or toxic manifestations in mice up to 24 h after administration.

3.4 Antinociceptive effect

The results (Table 2) demonstrate that the BESM, when administered orally at different doses (10,000, 5000 mg/kg), caused an inhibition of the writhing response induced by acetic acid (i.p.) (p < 0.05). The significant protective effect was dose-dependent. The percentage inhibitions of writhing produced by the extract were 77.11, 58.22, respectively, for each dose. The standard drug acetylsalicylic acid (200 mg/kg), exhibited about 72.99 inhibitions.

Thus, the results obtained from the antinociceptive effect of the BESM revealed the existence of a peripheral analgesic property (Lee et al., 1994).

The acetic acid induced abdominal constriction method elucidated peripheral activity. The BESM was shown to possess antinociceptive activity at the tested doses. This was evident in all the nociceptive models, which indicates that it possesses both central and peripherally mediated activities (Vongtau et al., 2004).

3.5 Effects of the BESM on carrageenan-induced paw edema

Oral injections of BESM (5000 mg/kg) decreased the carrageenan-induced inflammation significantly (p < 0.05).

The results presented in Table 3 indicated that BESM exhibited a significant anti-inflammatory activity, where it significantly decreased the weight of edema: 52% induced by carrageenan in the rat’s paw (Table 3) using indomethacin (5 mg/kg) as a reference drug.

Carrageenan-induced inflammation is a suitable method for evaluation of the anti-inflammatory effects of the agents (Winter et al., 1962). The inflammation consists of two phases. It appears that the early phase is related to the production of histamine, 5-hydroxytryptamin, bradykinins and cyclooxygenase products, while the delayed phase has been linked to neutrophil infiltration, as well as to the continuing of the production of arachidonic metabolites (Salvemini et al., 1996; Boughton-Smith et al., 1999). BESM was capable of attenuating both early and delayed phases of carrageenan-induced inflammation. The extract in the higher dose failed to inhibit the paw edema associated with the second phase.

Compared to a standard non-steroidal anti-inflammatory drug (NSAID), indometacin, the BESM decreased licking response similar to high doses of indomethacin.

Flavonoids were considered to be the active components responsible for the biological actions of the genus Stachys (El-Ansari et al., 1991).

Anti-ulcer activity. The BESM was found to be devoid of ulcerogenicity at the tested dose level (5000 mg/kg body weight) compared to indomethacin (5 mg/kg body weight, ulcer index 21.6) used as reference drug (Makhlouf et al., 2002).

In the n-butanol-induced stress gastric ulcer test in rats, it was shown that the tested extract produced significant dose-dependent gastroprotective activity.

In conclusion, this study has shown that the BESM exhibited significant antioxidant, antinociceptive and anti-inflammatory effects in laboratory animals. We can also conclude that S. mialhesi de Noé is a natural source of isoscutellarein 7-O-(2″-O-6″′-O-acetyl-β-d-allopyranosyl-β-d-glucopyranoside