Abstract: The hydroxyl and nitric oxide radical scavenging activities and total phenolic content of artemether were investigated in vitro. The concentrations of artemether (200-1000 μg mL^-1) did not exhibit hydroxyl radical scavenging activity except at 200 μg mL^-1 concentration. The mean values for the hydroxyl radical scavenging for the different concentration (200, 400, 600, 800 and 1000 μg mL^-1) of artemether were 47.55±11.28, -3.49±13.16, -30.09±6.09, -34.74±4.29 and -49.00±7.75%, respectively. The total phenolic content of artemether in ethylacetate ranged from 208.00±48.33 (SD) to 340.00±14.14 μg mL^-1, while the total phenolic content of artemether in methanol ranged from 90.00±6.3 (SD) to 122.00±35.44 μg mL^-1. Total phenolic content of artemether was significantly higher in ethyl acetate than in methanol at all concentration investigated (p<0.05). The total phenolic content arthemeter was influenced by the organic solvent used as a medium for preparing artemether.

INTRODUCTION

Artemisinin or Qinghaosu is an extract of medicinal plant Qinhao (Artemisia annua) (Novartis, 2005), a herb which has been used in traditional Chinese medicine for over 2000 years (Winstanley et al., 2004). Artemisinin is a sesquiterpene lactone with a peroxide bridge and its antimalarial activity has been attributed to its peroxide moiety (Klayman, 1985) that generates free radicals which damages the parasite membrane (Meshnick et al., 1989). Artemisinin compounds include artemether, artesunate and dihydroartemisinin (Mueller et al., 2004). Artemisinin has no nitrogenous heterocyle (Schmid and Hofheinz, 1983).

Artemether is an oil-soluble derivative of artemisinin of sesquiterpene lactone peroxide linkages (White and Olliaro, 1998; Hien and White, 1993). Its chemical structure is decahydro-10-methoxy-3, 6, 9-trimethyl-3, 12-epoxy-12H-pyrano-1, 2-benzodioxepin and pharmacological name is artemetherum (Utzinger et al., 2001). Artemisnins which do not possess peoxidic oxygen are devoid of antimalarial activity (Avery et al., 1992). Amorpha -4, 11-diene synthase is a key enzyme for artemisinin synthesis (Bouwmeester et al., 1999). Artemisinins possess antimalarial (Woodrow et al., 2005), antitumor (Efferth et al., 2002), antibacterial, antiviral (Efferth et al., 2007), immunosuppressive (Zhang et al., 2002; Tam et al., 2000) activities. Hitherto, there is no scientific study on in vitro hydroxyl and nitric oxide radical scavenging activities of artemether. This study was therefore carried out to assess the in vitro hydroxyl and nitric oxide radical scavenging activities of artemether and assess the relationships between hydroxyl radical scavenging ability, nitric oxide scavenging activity and total phenolic concentration of artemether.

MATERIALS AND METHODS

Chemical reagents: Folin-Ciocalteau reagent, FeSO₄.7H₂O and 1,10-Phenanthroline used were products of British Drug House (BDH), UK.

Sample: The brand of Artemether used was Lariether brand with a stock concentration of 40 mg mL^-1. The drug was purchased from Taki Medicine Store, Ogbomoso, Oyo State, Nigeria.

Preparation of the different concentrations of artemether from the stock conc. of 40 mg mL^-1: About 200-1000 ug mL^-1 concentrations of artemether were prepared in ethylacetate and methanol from the stock concentration of artemether (40 mg mL^-1).

Biochemical assays
Total phenolics content estimation: Total phenolic content of artemether was estimated according to the method of Hung et al. (2002). This is based on the principle that the phenolic group present in the sample reacts with Folin-Ciocalteau reagent in an alkaline medium using Sodium Carbonate (Na₂CO₃) solution thus giving a blue colour. About 0.5 mL of Folin-Ciocalteau (10%, w/v aqueous) was added to 0.1 mL of each concentrations of artemether, followed by the addition of 0.4 mL of aqueous Na₂CO₃ (7.5% w/v). The mixture was allowed to stand in the dark for 30 min. The absorbance of the blue color solution was read at 765 nm on a UV visible spectrophotometer (Genesy 10 vis, Thermoelectric corporation, USA) against blank (distilled water). Total phenolic concentration (mg mL^-1) of the artemether was extrapolated from a standard curve using tannic acid as a standard.

Nitric oxide scavenging activity: The nitric oxide radical scavenging activity of artemether was assayed according to the method of Marcocci et al. (1994). Nitric oxide was generated from sodium nitroprusside and the nitrite formed was measured by the Greiss reaction. Sodium nitroprusside in aqueous solution at physiological pH spontaneously generates nitric oxide (Green et al., 1982; Marcocci et al., 1994) which interacts with oxygen to produce nitrite ions that can be estimated by use of Greiss reagent. Scavengers of nitric oxide compete with oxygen leading to reduced production of nitric oxide (Ruch et al., 1989). About 0.1 mL aqueos sodium nitroprusside and 0.2 mL of 0.2M Na₂HPO₄ (pH 7.8) were added to 0.4 mL of different concentrations of artemether and the mixture incubated at room temperature for 2 h and 30 min. About 0.12 Griess reagent was added and the absorbance of the pink colour was read at 540 nm against blank (distilled water).

Where:

A_control=Absorbance of the control in the absence of sample

A_sample=Absorbance of sample

Hydroxyl radical scavenging activity assay: The scavenging capacity for hydroxyl radical of artemether was assayed according to the method of Yu et al. (2004). Hydroxyl radical is generated in vitro by mixing iron (II) sulphate which generates ferrous ion (Fe²⁺) with hydrogen peroxide and 1, 10-phenanthroline. The 1, 10-phenanthroline was used since phenanthroline-Fe²⁺ is a commonly used indicator of redox reaction. The H₂O₂/Fe²⁺ system produces hydroxyl radical through the Fenton reaction and phenanthroline-Fe²⁺ complex oxidizes to Fe³⁺. The hydroxyl radical produced can then be determined due to a change in absorbance at 560 nm. About 60 uL of FeSO4.7H₂O (1 mM) was added to 90 uL of aqueous 1, 10-phenanthroline (1 mM). About 2.4 mL of 0.2M Na₂HPO₄ (pH 7.8) was added to the mixture, followed by the addition of 150 uL of H₂O₂ (0.17 M) and 1.5 mL of different concentrations of artemether (250-1000) in sequence. The mixture was incubated for 5 min at room temperature. The absorbance of the mixture was read at 560 nm after 5 mm on the UV/visible spectrophotometer (Genesy 10 vis, Thermoelectric corporation, USA) against blank (distilled water).

Where:

A_control=Absorbance of the control

A_sample=Absorbance of the sample in the presence of other reagents in the reaction mixture

RESULTS AND DISCUSSION

Artemether was observed to exhibit moderate in vitro hydroxyl radical scavenging activity (47.55%) at the lowest concentrations of arthemeter (200 μg mL^-1). At other concentrations (400-1000 μg mL^-1) of artemether, the drug failed to scavenge hydroxyl radical in vitro. The drug at those concentrations (400-1000 μg mL^-1) accelerated hydroxyl production as evidenced by their negative values. At all concentrations investigated (200-1000 μg mL^-1), artemether did not exhibit nitric oxide radical activity in vitro as evidenced by their negative values (Table 1). The drug accelerated the generation of nitric oxide radical in vitro. The solvent used in preparing artemether before total phenolic determination influenced the total phenolic content of artemether. The total phenolic content artemether in ethyl acetate was significantly higher than the total phenolic content (p<0.05) in methanol at all concentrations investigated in vitro (200-1000 μg mL^-1). The total phenolic content of artemther in ethylacetate ranged from 208.00±48.33 to 340.00±14.14 (SD) mg mL^-1, while the total phenolic content of artemether in methanol ranged from 90.00±6.33 to 122.00±35.44 (SD) mg mL^-1. Hydroxyl radical scavenging activity of artemether in ethyl acetate showed a strong non-significant negative correlation with total phenolics concentration of artemether (r = -0.972; p = 0.05) at 200 μg mL^-1 concentration (Table 2).

Table 1:	Changes in levels of hydroxyl and nitric oxide radical scavenging activities and total phenolic concentration of artemether in vitro (in ethylacetate and methanol)
Values are mean±S.D of 5 analyses per concentration

Table 2:	Pearson correlation of hydroxyl radical scavenging ability and total phenolics of artemether in ethyl acetate

Table 3:	Pearson correlation of total phenolic content in ethyl acetate and nitric oxide radical scavenging activity of artemether

At 600 μg mL^-1 of artemether, a strong positive non-significant was observed between hydroxyl radical scavenging activity and total phenolics concentration of artemether in ethylacetate (r = 0.932; p = 0.05).

Nitric oxide radical activity of artemether in ethylacetate showed weak non-significant negative correlations with total phenolics at 200, 400 and 1000 μg mL^-1 of artemether (r = -0.382; p = 0.05 at 200 μg mL; r = -0.119; p = 0.05 at 400 μg mL^-1). A high positive non-significant correlation was observed between nitric oxide radical scavenging activity and total phenolics of artemether (r = 0.637; p = 0.05) (Table 3).

Hydroxy radicals can be generated from H₂O₂ in the presence of transition metals such as Fe²⁺ by Fenton reaction (Halliwell and Gutteridge, 1992). The short-lived hydroxyl radicals are assumed to be more unspecially destructive to biomolecules because they are located less than a few nanometers from the site of generation (Hipeli and Elstner, 1997).

Hydroxyl radical is generated in vitro by mixing iron (II) sulphate which generate ferrous ion (Fe²⁺) with hydrogen peroxide and 1-10-phenanthroline (Liu, 2003). The 1,10-phenanthroline was used since phenanthroline-Fe²⁺ is a commonly used indicator of redox reaction. The H₂O₂/Fe²⁺ system produces hydroxyl radical through the Fenton reation with phenanthroline-Fe²⁺ complex oxidies to Fe³⁺ (Liu, 2003).

CONCLUSION

Artemether failed to scavenge nitric oxide radical at all the concentrations investigated in vitro. These findings suggested that artemether could be a potentiator of inflammation and it corroborated its traditional role as a free radical generator.