Gallic acid (GA) (Sigma-Aldrich, St Quentin Fallavier, France) was used as reference polyphenolic were purchased from Aldrich, Germany. 2,2-diphenyl-1-picrylhydrazyl (DPPH), butylated hydroxyanisole (BHA) were used as free radical donor and antioxidant, Folin-Ciocalteu reagent and Na₂CO₃, methanol (PA) from Sigma Germany. All other organic solvents used for the tests were upgrade. Water used was distilled.

The leaves sample of Lippia rugosa were collected in the month of August 2010 between 7 and 7:30 pm local time in the Guinean type savannah in the neighbourhood of the Ngaoundere University’s campus geographically referenced 13˚22.5E; 07˚25.11N and altitude 1036 m [8] . These data were recorded with a GPS Garmin Geko 301. The plant samples were identified and authenticated by Pr Mapongmetsem JM, botanist and agroforester in the Department of Biological Sciences, University of Ngaoundere. These plants material were cut in smaller pieces, air-dried, pulverized in a traditional mortal and sieved into an average size of 2 mm using kitchen mill.

Five hundred and fifty grams of air-dried powdered of the leaves of Lippia rugosa were extracted exhaustedly at room temperature (25˚C) with 2200 mL of n-hexane by maceration during 4 hours under mechanical agitation at a speed of 3000 rpm using a Prolabo-228 apparatus. The organic layer was then evaporated to dryness at 40˚C under reduced pressure on a 210-Büchii rotary evaporator. This operation was repeated three times to give 13.16 g of hexane extract representing 1.19% of dry material (weight/weight). The residue was then successively extracted in the same manner with organic solvents of increasing polarity respectively with ethyl acetate; ethanol and methanol to give respectively 4.01 g (0.72%), 7.83 g (1.42%) and 6.2 g (1.12%) of ethyl acetate; ethanol and methanol extracts. Theses extracts were kept at 4˚C until used.

Extracts of Lippia rugosa were subjected to preliminary phytochemical screening according to standard procedures. These extracts were tested for the presence of alkaloids, steroids/triterpenoids, phenols, flavonoids, saponins, tannins and carbohydrates [12] [13] .

Total phenolic compounds in the leaves of Lippia rugosa were determined with Folin-Ciocalteu using method described by Singleton and Rossi (1965) amended by Dewanto and calculated using gallic acid as the standard compound [16] . The organic extracts were diluted in methanol. Five point concentrations were prepared with Gallic acid as the positive control. Extracts (100 μl) were added to 50% Folin-Ciocalteu reagent (100 μl). After 3 min, 2 ml of 2% Na₂CO₃ solution was added to the mixture, which was then left to stand for 30 min. Absorbance was measured at 760 nm on a spectrophotometer and compared to gallic acid calibration curves. All analyses were run in three replicates and averaged.

Twenty grams of the ethanol extract of L. rugosa were fractionated by column chromatography (CC) on silica gel 60S (300 g, 230 - 400 mesh) using mixture of n-hexane-ethyl acetate and ethyl acetate-methanol of increasing polarity as eluent. Thin layer chromatography (TLC) was carried out on silica gel precoated plates F-254 Merck and the spots on the TLC plates were visualized by UV₂₅₄, UV₃₆₅ and spraying with 50% H₂SO₄ following by heating at 105˚C. Fractions of 150 ml each were collected and compared by TLC (silica gel) using n-hexane-ethyl acetate and ethyl acetate-methanol of increasing polarity as eluent and the fractions giving similar spots in TLC were combined. In the end seventeen fractions A-Q were obtained. The fractions J (167 - 173), eluted with pure ethyl acetate, were combined on the basis of thin layer chromatography (TLC) analysis, and the presence of a green precipitate was observed, which after recrystallization with n-hexane/ethyl acetate (10/90) (v/v) gives a purified green organic compound identified as compound LRF4 (60 mg) representing respectively 0.41% and 0.0053% of the ethanol extract mass and dry plant powder (w/w) respectively.

Chemical characterization of purified active compound was done through ¹H and ¹³C-NMR spectral analyses. NMR spectra were recorded on a Bruker Avance 500 MHz spectrometer. Chemical shift values (δ) are reported in parts per million (ppm) relative to appropriate internal solvent standard and coupling constants (J values) are given in Hertz. Mass spectra were recorded on a Finnigan MAT 95 high resolution, double focusing magnetic sector mass spectrometer. Accurate mass measurement was achieved using voltage scanning of the accelerating voltage. This was nominally 5 kV and an internal reference of heptacosa was used. Resolution was set between 5000 and 10,000. IR spectra were recorded on a Nicolet 360 FT-IR spectrophotometer and UV spectra on a Thermo Electron Corporation Helios spectrophotometer.

The experimental results were expressed as the mean ± Standard Deviation (SD) using Microsoft Excel 2007. Curves and graphs were obtained by using the software Sigmaplot (version 9.0).

The results of the qualitative phytochemical analysis of the leaves extracts of L. rugosa are giving in Table 1.

−: Absent; +: Present; ++: Abundant.

They indicated that each plant extract contained at least one class of secondary metabolites, the ethanol and the methanol extracts being the richest extracts containing the greatest number of organic compounds which include flavonoids, polyphenols, sterols and triterpenoids whereas hexane and ethyl acetate extracts contained respectively one and two classes of secondary metabolites. Compounds belonging to terpenoids as well as phenolics are well documented for their biological activities. The presence of the flavonoids and polyphenols compounds in the ethanol and methanol extracts corroborate with previous research works carried out on Lippia alba [17] [18] . Their presence in the studied extracts could therefore explain the observed activities and justified the traditional uses.

TLC assay of these extracts spraying with 0.2% DPPH in 96% ethanol showed immediately many bleached bands for the ethanol and methanol extracts while no and few bleached bands were observed after hours respectively on the ethyl acetate and n-hexane extracts indicating their poor and no antioxidative activity of these two last extracts. The best activity seen in the ethanol and methanol extracts prompted us to quantify the antioxidant activity of these extracts. The lower and weakest antioxidant activity of the ethyl acetate and the hexane extracts confirmed the absence of phenolic compounds in both extracts.

The results of the antioxidant activities of the L. rugosa extracts and pure organic compound LRF4, measured by DPPH˚ assay are presented on Figure 1 and Figure 2. It was observed from this curve that inhibition values of L. rugosa ethanol and methanol extracts, compound LRF4 and standard BHA increased with concentration. The ethanol extract of L. rugosa is an antioxidant with a percentage of inhibition reaching 85.668% ± 1.233% at the highest concentration (100 µg/mL) than the methanol extract and ethyl acetate. The ethanol extract of L. rugosa was more effective than the pure isolated compound LRF4 isolated from this extract indicating that the total antioxidant of the extract is due to the synergetic effect of many compounds of the extract including those of compound LRF4. High R² values obtained also showed that there is a good correlation between concentration and radical inhibition. We also note that each concentration, there is a significant difference between the extracted probability threshold p < 5%.

The key role of phenolic compounds as scavengers of free radicals is emphasized in several reports. Amounts of phenolic components of the ethanol extract of Lippia rugosa were found to be 0.0229 ± 0.0003 Gallic acid equivalents (GAEs), similar to those of methanol extract content (Table 2). This result is in agreement with results observed in the antioxidative test showing best result for ethanol and methanol extracts. Although qualitative screening indicated presence of phenolic compounds in ethyl acetate extract, it content in phenolics is very low evaluated at 0.00000 ± 0.00019 GAEs. The phenolic compounds of the ethanol extract may contribute directly to antioxidative action. The high antioxidant activity of the ethanol and methanol extracts could be attributed to their high polyphenolic contents known to be highly rich in antioxidant compounds [16] [19] .

Compound LRF4 was obtained as a green amorphous solid which gives positive reaction to phenolic and flavonoid test reactions. Its ESI-TOF mass spectrum showed a molecular ion peak [M+1]⁺ at m/z 329.5 consistent with the molecular formula C₁₈H₁₆O₆ and eleven insaturations. Its UV spectrum showed two absorption bands at λmax in EtOH at 289 and 332 nm corresponding respectively to the benzoyl and cinnamoyl chromophores of a flavonoid compound. The structure of this compound was totally characterized using ¹H and ¹³C NMR spectrophotometric techniques (Table 3). From the analyses, compound LRF4 was identified as 7-hydroxy-5,6,4'-trime- thoxyflavone or 3-(40-methoxybenzyl)-7,8-methylenedioxy-chroman-4-one (Figure 3). The structure of the isolated flavonoid meet partially the second structural requirement stated by Bors et al.(1990) for a flavonoid molecule to have a maximal radical scavenging activity by bearing a 2,3-double bond in conjunction with a 4- oxo group in the C-ring [20] . Spectroscopic characteristics of 7-hydroxy-5,6,4'-trimethoxyflavone (LRF4) is given below:

ESI-TOF MS m/z: 329.5 [M + 1]⁺; 314.4; 296.5; 268.4; 133.4.

UV λmax (EtOH) nm: 289; 332

¹H and ¹³C NMR see Table 3.

The presence of flavonoids compounds are in agreement with previous results obtained on the genus Lippia. Flavonoid molecules were previously isolated and their antioxidative activity evaluated from L. graveolens [21] . Despite the fact that compound 7-hydroxy-5,6,4'-trimethoxyflavone was previously isolated from the whole plant

−: not measured; BHA: Butylated hydroxyanisole, ARP: antiradical power.

of Eupatorium catarium [22] , this is the first time that it was isolated from the genus Lippia and particularly from L. rugosa from Cameroon and its antioxidative activity evaluated. In recent years, it has been demonstrated that phenolic compounds play a widespread role in the natural plant protection against phytophagous insects and phytopathogenic fungi [23] . The presence of phenolic compounds and 7-hydroxy-5,6,4'-trimethoxyflavone could also explain the traditional uses of L. rugosa to protect maize against pests like Sitophillus zeamais Motsch and Prostephanus truncatus and f against ungi like Fusarium [8] [9] .