Lantana Linn.(from the Latin lento，to bend)probably derives from the ancient Latin name of the genus Viburnum Linn.，which resembles a little foliage and inflorescence(Lu and Atkins， 2004). The plants in Lantana linn. produce a number of metabolites in high quantities and some possess useful biological activities. Lantana Linn. is free from diterpenoids and rich in essential oils. Monoterpenes，triterpenes，flavones，coumarin，steroids，iridoid，glycosides， and caffeic acid derivatives are also reported with triterpenes and flavones being the most abundant secondary metabolites. The plants in Lantana Linn. are used in folk medicine in many parts of the world(Nagao et al，2002; Hayashi et al，2004; Akhtar et al，2006).

From the methanol extract of the dried leaves of L. trifolia L.，a new antimicrobially active and polymethoxylated flavone was isolated and named as umuhengerin(Rwangabo et al，1988). The methanol extract of the aerial parts of L.trifoliahadpreviously been assessed for the anti- inflammatory，antinociceptive， and antipyretic effects in experimental animals. The extract produced an inhibitory effect oncarrageenan-induced edema in the rat paw over a dose range of 10−300 mg/kg. The extract also produced small but significant increase in the response latency of rats subjected to the hot plate， and a thermal pain test only detected analgesia by high efficacy agents. Therefore，the L.trifoliaextract could have therapeutically relevant anti- inflammatory and analgesic properties in human(Uzacátegui et al，2004). In a present study，phytochemical investigation of the ethyl acetate extract of L. trifolia led to the isolation and identification of flavonoids and phenylpropanoids，which were compounds reported to have sedative properties. The sedative effect of L. trifolia extracts on mice cannot be attributed to the direct activation of the central benzodiazepine site but by the action of flavones，phenylpropanoids， and verbascoside(Julião et al，2010). The healing properties of medicinal plants have been typically attributed to their phenolic content，mostly flavonoids and phenolic acids， and their probable role in the prevention of disease associated with oxidative stress(Scalbert et al，2005). The screening of plant extracts using the DPPH free radical method proved to be effective for the selection of those components with anti-oxidativeactivity. These extracts may be rich in radical scavengers，such as flavonoids，known as anti-oxidants. Further more detailed studies on the chemical composition of those extracts，as well as studies with other models such as lipid peroxidation and in vivo assays are essential to characterize them as biological anti-oxidants(Mensor，2001).

There is an increasing interest in anti-oxidants，particularly in those intended to prevent the presumed deleterious effects of free radicals in the human body， and to prevent the deterioration of fats and other constituents of food stuffs. In both cases there is preference for anti-oxidatant from natural rather than from synthetic sources(Abdalla and Roozen， 1999). Lantana Linn. is mostly native to subtropical America，but a few taxa are indigenous to tropical Asia and Africa. In Kenya，it is mainly distributed at Kambi ya moto region of Nakuru county where the leaves are medicinally used in the form of infusions to treat respiratory inflammatory diseases.

The leaves of Lantana trifolia L.(5 kg)，were collected at Kambi ya moto，Nakuru county，Kenya in January 2011. The plant was identified by Thomas Rotich，Botany Department of Egerton University，Kenya. A voucher specimen(PSI-TUTCM)has been retained at the herbarium of Tianjin University of Chinese Traditional Medicine. The dried leaves from L. trifolia were ground into fine powder using an electric hammer mill. The pulverized sample(3.5kg)was exhaustively extracted with 95% and 75% ethanol at room temperature. The extracts were concentrated separately under reduced pressure using rotary evaporator to afford brown syrup. TLC experiment of both 95% and 70% crude ethanolic extracts gave the same result，so they were combined and placed on water bath to evaporate the remaining traces of ethanol. The extract was partitioned between water and organic solvents of increasing polarities to afford the new separate extracts in petroleum ether，chloroform，ethyl acetate， and n-butanol.

DPPH radical-scavenging activity of the extract was determined according to the methods by Blois(1958). An ethanol solution of DPPH radical was prepared by weighing 10 mg of DPPH into 250mL volumetric flask and diluting with absolute ethanol to 250 mL，then stored at 10^oC in the dark. An ethanol solution of the test compound(vitamin C，VC)was prepared by weighing 0.1 g of VC powder into 25mL volumetric flask and diluting with absolute ethanol to 25 mL. Fiveconsecutive serial dilutions were prepared by transferring 12.5 mL from each of the newly prepared 25 mL solution. An ethanol solution of each organic partition from liquid-liquid extraction was prepared by weighing 250 mg to 25 mL volumetric flask and diluting with absolute ethanol to 25 mL. Fiveconsecutive serial dilutions of each sample were prepared by transferring 12.5 mL from each newly prepared 25 mL solution. DPPH solution(1 mL)was diluted with 1mL absolute ethanol and absorbance(A_c)measurements were immediately recorded with a UV-visible spectrophotometer under 517 nm. The decrease in absorbance at 517 nm was continuously determined，with data being recorded at 1 min intervals until the absorbance stabilized. VC sample solution(2 mL)starting from low concentration was added to 2 mL DPPH solution into three different test tubes and set aside for 30 min，then their absorbance(A_i)was detected under 517nm. DPPH solution(2 mL)was added to 2 mL of each sample solution of the serial dilutions. Their absorbance(A_j)was detected under 517 nm. All the determinations were performed in three replicates and averaged.

The total flavonoid content of the ethyl acetate partition was determined according to colorimetric method. Thesample solution(0.5 mL)was mixed with 2mL of distilled water and subsequently with 0.15 mL of 5% NaNO₂ solution. After 6 min of incubation，0.15 mL of 10% AlCl₃ solution was added and then allowed to st and for 6 min，followed by adding 2 mL of 4% NaOH solution to the mixture. Immediately，water was added to the sample to bring the final volume to 5 mL. The mixture was thoroughly mixed and allowed to st and for another 15 min. The absorbance of the mixture was determined at a wavelength of 510 nm. The total flavonoidscontent wascalculated from a calibration curve.

The total phenol content of ethyl acetate partition was analyzed according to the Folin-Ciocalteu method as described by Wu et al(2004). After 2 g of sample was well mixed with 2.5 mL of distilled water，0.5 mL of the Folin-Ciocalteu stock reagent and 1.0 mL of Na₂CO₃ reagent(75 g/L)were added to the mixture. They were then incubated at room temperature for 30 min. The absorbance of the mixture was spectrophotometrically measured at a wavelength of 765 nm. The total phenol content was determined and expressed in gallic acid equivalent(GAE).

Column chromatography technique was used to fractionate the crude extract into enriched fractions and eventually purify the actual active compounds. The ethyl acetate fraction(73 g)was separated using gradient elution approach on silica gel column with a biphasic solvent mixture composed of dichloromethane-ethyl acetate(100:1 to 1:5)at a flow rate of 3 mL/min. Fractions(30 mL)were collected and concentrated using a rotary evaporator. The experiment was monitored by TLC experiment to monitor the change of the polarity of the eluting solvent in relation to elutes which were later pooled according to their retention factor on the TLC plate. Gradient elution on silica gel was not very sufficient for isolation，therefore the collected fractions were further purified using Sephadex LH-20 column with methanol as the eluent at a flow rate of 2 mL/min. HPLC analysis(C₁₈ column，150 mm × 4.6mm，5 µm)using LC solution machine and preparative HPLC(PHPLC)wasemployed to distinctively isolate more compounds. The structures of the isolated compounds were elucidated by Bruker 600 MHz NMR technique(¹H-NMR，¹³C-NMR， and DEPT).

As shown in Table 1，the IC₅₀ values for DPPH radical- scavenging activity of the extracts were 4.23，57.30，17.37，7 9.74， and 4.94 µg/mL for VC(positive control)，samples I−IV(chloroform，n-butanol，petroleum ether， and ethyl acetate extracts)，respectively.The results showed that sample IV(ethyl acetate extract)was the most effective in reducing the stable radical DPPH to the yellow colored diphenyl picryl hydrazine，indicating that it was very active in DPPH radical scavenging. This was attributed to its IC₅₀ value(4.94 µg/mL)that was close to that of the positive control(4.23 µg/mL). We discovered that the radical scavenging effects of partitioned L.trifoliaextract and synthetic anti-oxidants used as positive control on DPPH radical were concentration dependent. The scavenging of DPPH radicals increased with increasing anti-oxidant concentration(Figure 1). Proton radical-scavenging action is one mechanism for oxidation. DPPH has a proton free radical and shows characteristic absorption at 517 nm(purple). When it encounters proton radical scavenger，its purple colour fades rapidly，suggesting that the anti-oxidativeactivity of L. trifolia leaf extract is due to its proton donating ability.

Table 1

Table 1 DPPH essay DPPH absorbance(Ac)= 0.590 Samples Ai Aj Concentration /(µg·mL−1) Scavenging rates / % IC50 /(µg·mL−1) VC(positive control) 0.065 0.037 33.00 95.25 4.23 0.068 0.037 17.00 94.75 0.131 0.036 8.00 83.89 0.384 0.036 4.00 41.02 0.520 0.036 2.00 17.97 0.581 0.036 1.00 7.63 sample I(chloroform extract) 0.311 0.289 1253 96.27 57.30 0.170 0.145 626 95.76 0.103 0.075 313 95.25 0.123 0.040 157 85.93 0.308 0.020 78 51.19 0.426 0.010 39 29.49 sample II (n-butanol extract) 0.082 0.013 157 88.06 17.37 0.080 0.007 78 87.37 0.119 0.004 39 80.10 0.274 0.003 19 53.11 0.409 0.002 9 29.58 0.479 0.002 5 17.47 sample III(petroleum ether extract) 0.107 0.059 401 91.70 79.74 0.148 0.033 201 80.10 0.294 0.006 101 50.17 0.421 0.000 50 27.16 0.483 −0.004 25 15.74 0.507 −0.004 13 11.59 sample IV(ethyl acetate extract) 0.051 −0.005 50 90.31 4.94 0.056 −0.007 25 89.10 0.075 −0.008 13 85.64 0.219 −0.006 6 61.07 0.382 −0.009 3 32.35 0.460 −0.004 2 19.72

Table 1 DPPH essay

Figure 1 Radical scavenging effect of ethyl acetate partition fromL. trifolia leaves and VC by DPPH colouring method

The total phenolic content of ethyl acetate extract from L. trifolia leaves measured by Folin Ciocalteu reagents in terms of GAE was(29.27 ± 1.46)mg/g，while the totalflavonoidscontent was(39.0 ± 1.6)mg/g.

The chemical structures of the compounds separated by column chromatography，HPLC， and PHPLCrespectively were identified. Comparing with reference data，compounds 1−6(Figure 2)were identified as kaempferol-3，7-dimethyl ether(1，Julião et al，2009)，verbascoside(2，Wu et al，2004)，apigenin(3，Julião et al，2009)，umuhengerin(4，Rwangabo et al，1988)，ladanetin(5，Alam et al，1986)， and scutellaein-7-O-β-D-apiofuranoside(6，Julião et al，2010).

Figure 2 Chemical structures of isolated compounds 1−6

In conclusion，ethyl acetate has the highest extract yield and total phenol recovery，as well as the highest anti-oxidative activity when determined by DPPH assay. Therefore our results indicate that the selective extraction from natural materials by an appropriate solvent is important for obtaining the fractions with high anti-oxidativeactivity. The ethyl acetate extract from the leaves of L.trifoliapossesses thepotent anti-oxidative and free radical scavenging activities which aredirectly proportional to its phenol contents. The anti-oxidativeactivities of the extract from the leaves of L. trifolia may be due to its proton donating capability as shown in DPPH radical scavenging results. Acting as an electron donor that can react with free radicals，it converts them to more stable products and terminates radical chain reactions. These mechanisms could have contributed，at least partly，to the therapeutic benefits of L.trifoliafor the treatment of inflammatory and respiratory disorders as a sedative.

We thank Mr. Thomas Rotich，Botany Department of Egerton University for the identification of the plants，Prof. Deng and Dr. Wang for running of NMR spectra. The cooperation of Dr. Mo in anti-oxidant assays is highly acknowledged.