Research Article

Received: 24 October 2008 Revised: 11 December 2008 Accepted: 30 December 2008 Published online in Wiley Interscience: 23 February 2009

(www.interscience.wiley.com) DOI 10.1002/jsfa.3532

Chemical composition and antioxidant and radical-scavenging activities of Periploca laevigata root bark extracts
Hajji Mohamed, Masmoudi Ons, Ellouz-Triki Yosra, Siala Rayda, Gharsallah Neji and Nasri Moncef
Abstract
BACKGROUND: The root powder of Periploca laevigata is used for preparing soft drinks and as an aromatic in Tunisia. The infusion or decoction of its root bark has widespread use in folk medicine. The plant is used to treat digestive disorders and hypertensive effects as well as other health problems. RESULTS: The antioxidant activities of extracts of P. laevigata root bark obtained with solvents of different polarity were investigated using assays of 1,1-diphenyl-2-picrylhydrazyl radical-scavenging activity, ferric-reducing capacity, -carotenebleaching ability, hydroxyl radical-scavenging activity and lipid peroxidation inhibition. The methanol extract, with the highest amount of total phenolics and avonoids, showed the highest antioxidant activities in all assays, followed by the water extract. Gas chromatography/mass spectrometry was used to determine the composition of the water and methanol extracts. Thirty-four compounds were identied in the methanol extract, with proavine (516.2 g kg1 dry matter (DM)) and 4-methoxysalicylaldehyde (198.3 g kg1 DM) being the most abundant. Sixteen compounds were identied in the water extract, of which 4-hydroxy-3-methoxyphenethylene glycol (351.2 g kg1 DM) was the main component. CONCLUSION: As far as is known, this is the rst report on the chemical composition and biological activities of phenolic extracts from P. laevigata. The results of the study indicate that the root bark of this plant might be a good candidate for further investigation in developing new antioxidants. c 2009 Society of Chemical Industry Keywords: Periploca laevigata; root bark; phenolic composition; antioxidant activity; gas chromatography/mass spectrometry

INTRODUCTION
Oxidation of polyunsaturated fatty acids, which occurs during storage, processing and heat treatment of raw materials and further storage of nal products, is one of the major factors resulting in losses of fatty food quality by formation of compounds with negative effects on the aroma and nutritional value of foods.1,2 In order to prolong the storage life of foods, synthetic antioxidants are widely used in industrial processing. Moreover, it has been shown that antioxidants and free radical scavengers are crucial in the prevention of pathologies such as cancer, heart diseases, biological damage in living tissues and neurodegenerative diseases, in which reactive oxygen species or free radicals are implicated.3 The two most commonly used synthetic antioxidants are butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), which are added to fatty and oily foods to prevent oxidative deterioration.4 However, use of these chemical compounds has begun to be restricted because of their induction of DNA damage and their toxicity.5,6 Moreover, both BHT and BHA appear to be involved in tumour promotion.7 In recent years there has been great interest in nding new antioxidants from natural sources for use in food and medicinal materials to replace synthetic antioxidants. Vitamins, which are

present naturally in vegetables, fruits and seeds, possess the ability to reduce oxidative damage associated with many diseases, including cancer, cardiovascular diseases, atherosclerosis, etc.8 Recently, the ability of phenolic substances, including avonoids and phenolic acids, to act as antioxidants has been extensively investigated.9 Phenolic compounds are derivatives of the pentose phosphate, shikimate and phenylpropanoid pathways in plants.10 They exhibit a wide range of physiological properties, such as antioxidant, antiallergenic, antiartherogenic, anti-inammatory, antimicrobial, antithrombotic, cardioprotective and vasodilatory effects.3,11,12 The bioactivity of phenolics may be related to their ability to chelate metals, inhibit lipoxygenase and scavenge free radicals.13 Periploca laevigata is an important grazing species in the dry season. In Tunisia it is predominantly found in the south of the

Correspondence to: Hajji Mohamed, Laboratoire de Gnie Enzymatique et de e Microbiologie, Ecole Nationale dIngnieurs de Sfax, BP W 3038 Sfax, Tunisia. e E-mail: hajjimed1989@yahoo.fr Laboratoire de G nie Enzymatique et de Microbiologie, Ecole Nationale e dIngnieurs de Sfax, BP W 3038 Sfax, Tunisia e

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www.soci.org country, especially in the mountains. It is used as a food ingredient (tea) and as a herbal preparation because of its reputed medicinal properties. Many chemical compounds have been isolated and identied from species of the Periploca genus, such as - and -amyrin, lupeol and -sitosterol from P. laevigata.14 Various cardiotonic and sulfated pregnane glycosides have been isolated from Periploca sepium15 and Periploca graeca.16 Moreover, several lupene-type triterpenes and elemane-type sesquiterpenes have been isolated from Periploca aphylla and roots of P. laevigata respectively.14,17 To the best of our knowledge, there are no available reports on the chemical composition and biological activities of phenolic extracts from P. laevigata. Therefore the aim of this study was to evaluate by various methods the antioxidant properties of extracts of P. laevigata root bark obtained with different solvents and to assess whether this species could be a source of natural antioxidants for pharmaceutical and food applications. The chemical composition of volatiles and silylated compounds and the contents of total phenolics and avonoids were also determined.

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Total phenolic content The total phenolic content of PLRB extracts was determined by the FolinCiocalteu method.18 A 0.5 mL aliquot of diluted extract solution was mixed with 0.5 mL of FolinCiocalteus reagent. The mixture was shaken for 5 min, 0.5 mL of 200 g L1 Na2 CO3 solution was added and the mixture was shaken once again for 1 min. Finally, the solution was brought up to 5 mL by adding distilled water. The control reaction contained all reagents except the extract. The reaction mixture was then incubated in the dark at 25 C for 90 min and the absorbance of the resulting colour was measured at 760 nm against a distilled water/sodium carbonate blank. Gallic acid monohydrate was used as standard for the calibration curve. Total phenolic content was expressed as mg gallic acid equivalent (GAE) g1 extract. Values presented are the average of three measurements. Total avonoid content The total avonoid content of PLRB extracts was determined by the method of Zhishen et al.19 Briey, 250 L of each sample was mixed with 1 mL of distilled water and subsequently with 150 L of 150 g L1 NaNO2 solution. After 6 min, 75 L of 100 g L1 AlCl3 solution was added, then the mixture was allowed to stand for a further 5 min before 1 mL of 40 g L1 NaOH solution was added. The mixture was immediately made up to 2.5 mL with distilled water and mixed well. The absorbance of the mixture was then measured at 510 nm. Total avonoid content was expressed as mg quercetin equivalent (QE) g1 extract. Values presented are the average of three measurements. DPPH assay The DPPH radical-scavenging activity of PLRB extracts was determined by the method of Kirby and Schmidt20 with some modications. Briey, 500 L of each extract at different concentrations (550 g mL1 ) was added to 375 l of 99.5% methanol and 125 l of DPPH solution (0.2 mmol L1 in methanol) as free radical source. The mixtures were incubated for 60 min in the dark at room temperature. Scavenging capacity was measured spectrophotometrically (T70 UVvisible spectrometer, PG Instruments Ltd, Wibtoft, UK) by monitoring the decrease in absorbance at 517 nm. In its radical form, DPPH has an absorption band at 517 nm which disappears upon reduction by an antiradical compound. Lower absorbance of the reaction mixture indicated higher free radical-scavenging activity. DPPH radical-scavenging activity was calculated as DPPH radical-scavenging activity (%) =

MATERIALS AND METHODS

Chemicals 1,1-Diphenyl-2-picrylhydrazyl (DPPH), BHA, BHT, thiobarbituric acid (TBA), ethylene diamine tetraacetic acid (EDTA), -carotene, linoleic acid, D-deoxyribose, bis(trimethylsilyl) acetamide, pyridine and L-ascorbic acid were purchased from Sigma Chemical Co. (St Louis, MO, USA). All other chemicals, namely potassium ferricyanide (K3 [Fe(CN)6 ]), trichloroacetic acid (TCA), ferric chloride (FeCl3 ), hydrogen peroxide (H2 O2 ), sodium hydroxide (NaOH), FolinCiocalteus reagent, aluminium chloride (AlCl3 ), sodium nitrite (NaNO2 ), sodium carbonate (Na2 CO3 ), Tween 40 and other solvents, were of analytical grade. All solutions were freshly prepared in distilled water.

Plant material Fresh roots of P. laevigata were collected from Matlegg Mountain (Regueb, Tunisia) on 20 December 2007. The raw material was washed with distilled water and the bark was separated, ground and then dried at 80 C for at least 5 h to obtain P. laevigata root bark (PLRB) powder. The dried preparation was ground further using a mortar and pestle to obtain a ne powder and then stored in glass bottles at room temperature.

Preparation of PLRB extracts The dried powder of PLRB (50 g) was extracted sequentially (by a maceration method) by adding solvents of increasing polarity: hexane, chloroform, ethyl acetate, methanol and water. The powder was rst extracted by stirring with 500 mL of hexane at 30 C for 24 h. The extract was ltered through Whatman No. 1 lter paper in a Buchner funnel. The ltrate was evaporated to dryness under reduced pressure in a rotatory vacuum evaporator (EYELA N1000, Tokyo, Japan) at 40 C. The remaining residues were successively extracted with chloroform, ethyl acetate, methanol and water under the same conditions. The water extract was freezedried. The dried sample of each extract was weighed and the yield of soluble constituents was determined. The dried extracts were kept in the dark at +4 C until further analyses.

[(Acontrol Asample )/Acontrol ] 100 where Acontrol is the absorbance of the control reaction (containing all reagents except the sample) and Asample is the absorbance of the PLRB extract. Ferric-reducing activity The reducing power of PLRB extracts was determined by the method of Yildirim et al.21 Sample solutions (0.5 mL) containing different concentrations (25200 g mL1 ) of dried extracts were mixed with 1.25 mL of 0.2 mol L1 phosphate buffer (pH 6.6) and 1.25 mL of 10 g L1 K3 [Fe(CN)6 ] solution. The mixtures were incubated at 50 C for 30 min. After incubation, 1.25 mL of 100 g L1 TCA was added and the reaction mixtures were

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Composition and antioxidant activity of Periploca laevigata bark centrifuged for 10 min at 3000 g. A 1.25 mL aliquot of the supernatant from each sample mixture was mixed with 1.25 mL of distilled water and 0.25 mL of 1 g L1 FeCl3 solution in a test tube. After a 10 min reaction time the absorbance was measured at 700 nm. Higher absorbance of the reaction mixture indicated higher reducing power. Values presented are the mean of triplicate analyses. -Carotene bleaching by linoleic acid assay The ability of PLRB extracts to prevent bleaching of -carotene was assessed as described by Koleva et al.22 A stock solution of -carotene/linoleic acid was prepared by dissolving 0.5 mg of -carotene, 25 L of linoleic acid and 200 l of Tween 40 in 1 mL of chloroform. The chloroform was completely evaporated under vacuum in a rotatory evaporator at 40 C, then 100 mL of distilled water was added and the resulting mixture was vigorously stirred. The emulsion obtained was freshly prepared before each experiment. Aliquots (2.5 mL) of the -carotene/linoleic acid emulsion were transferred to test tubes containing different extract concentrations. Following incubation for 2 h at 50 C, the absorbance of each sample was measured at 470 nm. Tests were carried out in duplicate. BHA was used as positive standard. The control tube contained no sample. Lipid peroxidation by TBA assay Normal male rats (220 g) obtained from the Science University of Sfax, Tunisia were used for the preparation of liver homogenate. Rat livers were excised, cut into small pieces and homogenised in cold 0.15 mol L1 potassium chloride (KCl) to give a 100 g L1 liver homogenate. The homogenate was centrifuged at 13 000 g for 15 min at 4 C and the cell-free supernatant was used for the study of in vitro lipid peroxidation. Different concentrations (550 g mL1 ) of PLRB extracts dissolved in corresponding solvent were placed in test tubes. A 1 mL aliquot of 0.15 mol L1 KCl and 0.5 mL of rat liver homogenate were added to the test tubes. Peroxidation was initiated by adding 100 L of 0.2 mmol L1 FeCl3 and 100 L of 0.1 mmol L1 ascorbic acid. After incubation at 37 C for 30 min the reaction was stopped by adding 2 mL of icecold 0.125 mmol L1 hydrochloric acid (HCl) containing 10 g L1 BHT, 150 g L1 TCA and 3.8 g L1 TBA (nal concentrations). The reaction mixtures were heated at 80 C for 60 min. The samples were cooled and centrifuged and the absorbance of the supernatants was measured at 532 nm. The inhibition of lipid peroxidation was calculated using a test tube without sample as control. In this test, TBA reacts with malondialdehyde to form a diadduct, a pink chromogen, which can be detected spectrophotometrically at 532 nm.23 Lipid peroxidation inhibition was determined as inhibition (%) = [(Acontrol Asample )/Acontrol ] 100 BHA was used as positive standard. Three replicates were done for each test sample. Hydroxyl radical-scavenging activity This assay was performed as described by Halliwell et al.24 The hydroxyl-radical scavenging activity of PLRB extracts was measured by the competition between deoxyribose and each extract for hydroxyl radicals generated by the Fe3+ /ascorbate/EDTA/H2 O2

www.soci.org system. Sample solutions (0.1 mL) containing different concentrations (5100 g mL1 ) of PLRB extracts were mixed with 0.8 mL of a reaction buffer consisting of 2.8 mmol L1 D-deoxyribose, 0.1 mmol L1 FeCl3 , 0.1 mmol L1 EDTA and 1 mmol L1 H2 O2 in 20 mmol L1 potassium phosphate buffer (pH 7.4). The reaction was started by adding ascorbic acid to a nal concentration of 0.1 mmol L1 and the reaction mixture was incubated for 1 h at 37 C. The extent of deoxyribose degradation was measured by the TBA method. After incubation, 1 mL of 10 g L1 TBA solution in 50 mmol L1 NaOH and 1 mL of 20 g L1 TCA were added and the reaction mixture was heated at 100 C for 20 min. After cooling to room temperature, the amount of chromogen produced was measured at 532 nm against a blank (containing only buffer and deoxyribose). Mannitol, a classical hydroxyl radical scavenger, was used as positive standard. Three replicates were done for each test sample. Hydroxyl radical-scavenging activity was calculated as hydroxyl radical-scavenging activity (%) = [(Acontrol Asample )/Acontrol ] 100 where Acontrol is the absorbance of the control (without extract) and Asample is the absorbance of the extract. Derivatisation step A 100 L aliquot of bis(trimethylsilyl) acetamide was mixed with 100 L of methanol or aqueous extract (suspended in ethanol), then 20 L of pyridine was added. The obtained solution was incubated for 60 min at 80 C and then used for gas chromatography/mass spectrometry (GC/MS) analysis. GC/MS analysis GC/MS analysis of the methanol and water extracts was performed using an Agilent-Technologies 6890N Network GC system equipped with an Agilent-Technologies 5975 Inert XL massselective detector and Agilent-Technologies 7683B Series autoinjector (Agilent-Technologies, Little Falls, CA, USA). For MS detection the electron ionisation mode with an ionisation energy of 70 eV was used, with a mass range of m/z 50550. An HP-5MS capillary column (30 m0.25 mm, lm thickness 0.25 m) (Agilent technologies, Little Falls, USA) was used for GC/MS. The column temperature was programmed from 70 to 220 C at a rate of 4 C min1 , with the lower and upper temperatures being held for 3 and 10 min respectively. The GC injector and MS transfer line temperatures were set at 280 and 290 C respectively. GC was performed in splitless mode. Helium was used as carrier gas at a ow rate of 1 mL min1 . An injection volume of 1 L was used for each diluted PLRB extract. Essential compounds were identied by their retention times and mass fragmentation patterns using data of standards in the Wiley 7.0 library. Statistical analysis Values were expressed as mean standard deviation of three parallel measurements. Analysis of variance (ANOVA) was conducted and differences between variables were tested for signicance by one-way ANOVA using SPSS 11 (Statistical Package for the Social Sciences, The Predictive Analytics Company Chicago, USA). A difference was considered statistically signicant when P 0.05. Correlation and regression analyses were carried out using EXCEL (Microsoft corporation, USA).

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Table 1. Yield and total phenolic and avonoid contents of extracts from Periploca laevigata root bark Yield (g kg1 dried extract) 59.5 1.4 8.3 0.5 5.0 0.1 120 7.0 120 9.0 TPCa (mg GAE g1 extract) ND 272 8.5 399 4.8 623 9.2 570 10.8 TFCb (mg QE g1 extract) ND 136 7.6 188 11.3 410 17.8 248 10.4

Extract Hexane Chloroform Ethyl acetate Methanol Water

Values are expressed as mean standard deviation of three independent determinations; ND, not detected. a Total phenolic content as gallic acid equivalent (GAE). b Total avonoid content as quercitin equivalent (QE).

RESULTS AND DISCUSSION

Extraction yield and phenolic and avonoid contents The PLRB components were fractionated by extraction with solvents of increasing polarity: hexane, chloroform, ethyl acetate, methanol and water. The yield of extractable compounds relative to the weight of dried plant material ranged from 5.0 g kg1 (ethyl acetate extract) to 120 g kg1 (methanol and water extracts) (Table 1). It is well known that phenolic substances and avonoids contribute directly to the antioxidant activity of plant materials.9 Phenolic compounds exhibit considerable free radical-scavenging activities (through their reactivity as hydrogen- or electrondonating agents) and metal ion-chelating properties.9 Flavonoids are a class of secondary plant phenolics with powerful antioxidant activities.25 Therefore the amounts of total phenols and avonoids in the extracts were determined (Table 1). The total phenolics in crude extracts of PLRB were determined by the FolinCiocalteu method. As seen in Table 1, the highest level of total phenolics was found in the methanol extract (623 9.2 mg GAE g1 dried extract), followed by the water, ethyl acetate and chloroform extracts. However, phenolic compounds were not detected in the hexane extract. This result is in agreement with the reports of Hertog et al.26 and Yen et al.,27 which proved that methanol is the most suitable solvent for extraction of phenolic compounds. In addition, water has been reported to be a suitable solvent for extraction of phenolic compounds.28 The content of avonoids varied from 136 to 410 mg QE g1 dried extract. The results in Table 1 show that the chloroform, ethyl acetate, methanol and water extracts contained 136 7.6, 188 11.3, 410 17.8 and 248 10.4 mg QE g1 dried extract respectively. These results are in agreement with the report of Ozsoy et al.29 In addition, avonoids were not detected in the hexane extract. Variations in the yield and phenolic compounds of the different extracts may be attributed to the polarities of compounds present in the raw material. DPPH free radical-scavenging activity It is well known that the antioxidant activity of plant extracts containing polyphenol components is due to their capacity to be donors of hydrogen atoms or electrons and to capture free radicals.30 Free radicals play an important role in autoxidation of unsaturated lipids in foodstuffs. The DPPH radical-scavenging assay is a widely used method for evaluating the ability of plant extracts to scavenge free radicals generated from DPPH reagent.31 DPPH, a stable free radical with a

purple colour, changes into a stable yellow compound on reacting with an antioxidant. In brief, the reduction capacity of DPPH was determined by the decrease in its absorbance at 517 nm, which is reduced by the antioxidant.32 The extent of the reaction depends on the hydrogen-donating ability of the antioxidant.33 As can be seen in Fig. 1, the scavenging activity of all samples tested was concentration-dependent. The methanol extract, which contained the highest amount of total phenolics and avonoids, showed a signicant effect in inhibiting DPPH, reaching 96% at a concentration of 25 g mL1 . It is interesting to note that under the same conditions the methanol extract showed higher free radical-scavenging activity than BHA over the entire concentration range tested. The methanol extract provided a lower IC50 (4 0.5 g mL1 ) than BHA (11 0.5 g mL1 ). The IC50 values of the water and ethyl acetate extracts were 16 0.9 and 50 2.6 g mL1 respectively. It has been reported that free radical-scavenging activity is greatly inuenced by the phenolic composition of the sample.34 The results obtained suggested that some components within the methanol fraction were signicantly strong radical scavengers. Reducing power The reducing power assay is often used to evaluate the ability of an antioxidant to donate an electron,35 which is an important mechanism of phenolic antioxidant action. Many reports have revealed that there is a direct correlation between antioxidant activities and reducing power of certain plant extracts.21,22 In this assay the ability of PLRB extracts to reduce Fe3+ to Fe2+ was determined. The presence of antioxidants in the extracts results in reduction of the Fe3+ /ferric cyanide complex to the ferrous form. Therefore the Fe2+ complex can be monitored by measuring the formation of Perls Prussian blue at 700 nm. Figure 2 shows the reducing power activities (as indicated by the absorbance at 700 nm) of the different extracts compared with BHA as standard. The higher the absorbance of the reaction mixture, the higher is the reducing power. All extracts showed some degree of electron donation capacity, and the reducing power of all extracts increased

100

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40

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0 0 10 20 30 40 50 60 Concentration (g ml-1) BHA Water Methanol Ethyl acetate Chloroform

Figure 1. DPPH free radical-scavenging activity of PLRB extracts ( , , chloroform) at different methanol; , water; , ethyl acetate; concentrations. BHA ( ) was used as reference antioxidant. Values are mean standard deviation (n = 3).

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www.soci.org Antioxidant activity measured by -carotene bleaching The antioxidant assay using the discolouration of -carotene is widely employed to measure the antioxidant activity of bioactive compounds. In this assay, oxidation of linoleic acid produces hydroperoxyl radicals evolving towards lipid hydroperoxides, conjugated dienes and volatile by-products, which simultaneously attack the chromophore of -carotene, resulting in bleaching of the reaction emulsion.38 In this test, -carotene undergoes rapid discolouration in the absence of antioxidant, which results in a reduction in absorbance of the test solution with increasing reaction time. The presence of antioxidant hinders the extent of bleaching by neutralising the linoleic hydroperoxyl radicals formed.39 The antioxidant activity of PLRB extracts as measured by carotene bleaching is presented in Fig. 3. The methanol and water extracts showed the highest ability to prevent bleaching of carotene, followed by the ethyl acetate and chloroform extracts, while the hexane extract showed no detectable preservation of the -carotene emulsion colour. As can be seen in Fig. 3, antioxidant activity increased with increasing extract concentration. At a concentration of 50 g mL1 the chloroform, ethyl acetate,

Absorbance at 700 nm

1.5

0.5

0 0 50 100 150 200

Concentration (g ml-1)
BHA Ethyl acetate Methanol Water Chloroform

Figure 2. Reducing power of PLRB extracts ( , methanol; , water; , ethyl acetate; , chloroform) at different concentrations. BHA ( ) was used as reference antioxidant. Values are mean standard deviation (n = 3).

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0 0 20 40 60 80 100 Concentration (g ml-1) BHA Water Methanol Ethyl acetate Chloroform 120

Concentration (g ml-1) BHA Water Methanol Ethyl acetate Chloroform

Figure 3. Antioxidant activity of PLRB extracts ( , methanol; , water; , ethyl acetate; , chloroform) at different concentrations measured by -carotene-bleaching method. BHA ( ) was used as reference antioxidant. Values are mean standard deviation (n = 3).

Figure 4. Lipid peroxidation inhibition potential of PLRB extracts ( , , chloroform) at different methanol; , water; , ethyl acetate; concentrations using rat liver homogenate. BHA ( ) was used as reference antioxidant. Values are mean standard deviation (n = 3).

100 Scavenging activity (%) 80 60 40 20 0 0 20 40 60 80 100 120

with increasing amount of sample. The methanol extract, which contained the highest amount of total phenolics and avonoids, was the most potent reducing agent, followed by the water extract. However, the ethyl acetate and chloroform extracts, containing the lowest amount of phenolics, showed the weakest activity. The reducing powers of the methanol, water, ethyl acetate and chloroform extracts at a concentration of 200 g mL1 were 1.84, 1.32, 0.96 and 0.36 respectively. The reducing power of the methanol extract was close to that of BHA. For example, at a concentration of 200 g mL1 the reducing powers of the methanol extract and BHA were 1.84 and 1.95 respectively. The ferric-reducing activity correlated well with the content of total phenols and avonoids (R2 = 0.97). A correlation between reducing power and total phenols present in plant extracts has been reported in the literature.36,37

Concentration (g ml-1) BHA Water

Mannitol Ethyl acetate

Methanol Chloroform

Figure 5. Hydroxyl radical-scavenging activity of PLRB extracts ( , methanol; , water; , ethyl acetate; , chloroform) at different concentrations. BHA ( ) and mannitol ( ) were used as reference antioxidants. Values are mean standard deviation (n = 3).

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www.soci.org water and methanol extracts exhibited 22, 33, 78 and 83% antioxidant activity respectively. Interestingly, the inhibition of -carotene bleaching by the methanol extract was close to that by BHA at concentrations up to 25 g mL1 ; however, at higher concentrations, BHA showed higher activity compared with the methanol and water extracts. The antioxidant effectiveness of natural sources has been suggested to be mostly due to phenolic compounds. Some authors have found a correlation between phenolic content and antioxidant activity,40 while others have found no such relationship.41 In this assay the correlation between antioxidant activity and total phenolic content was signicant (R2 = 0.833). This observation is in agreement with a previous report in which high polyphenol content was signicantly associated with antioxidant activity.42 According to the -carotene/linoleic acid assay, the methanol and water extracts have strong effects against the discolouration of -carotene. This suggests that these extracts have potential use as scavengers of free radicals in emulsion-type systems. Inhibition of lipid peroxidation in rat liver homogenate Lipid peroxidation is an oxidative alteration of polyunsaturated fatty acids in cell membranes that generates a number of degradation products. Malondialdehyde is one of the products of lipid peroxidation which has been studied widely as a marker of oxidative stress and an index of lipid peroxidation.43 In the present
(a) Abundance 2800000 2600000 2400000 2200000 2000000 1800000 1600000 1400000 1200000 1000000 800000 600000 400000 200000 0 Time--> 5.00 (b) Abundance 50000 45000 40000 35000 30000 25000 20000 15000 10000 5000 0 Time--> 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00

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study the inhibition of lipid peroxidation by PLRB extracts and BHA as positive control was investigated and the results are shown in Fig. 4. It is clear that the addition of PLRB extracts to rat liver homogenate signicantly inhibited lipid proxidation, indicating a signicant reduction in TBA-reactive substance formation in the homogenate. As can be seen in Fig. 4, the methanol extract exhibited higher inhibition of lipid peroxidation than the water and ethyl acetate extracts. For example, at a concentration of 20 g mL1 the methanol, water and ethyl ether extracts showed 87, 58 and 41% inhibition of lipid peroxidation respectively. Interestingly, under similar conditions the methanol extract showed marginally higher lipid peroxidation inhibition than BHA at concentrations up to 25 g mL1 . These results are in agreement with a previous study by Srivastava et al.,44 who reported that both methanol and water extracts of Decalepis hamiltonii roots were effective in inhibiting lipid peroxidation of liver microsomes. Hydroxyl radical-scavenging activity The hydroxyl radical is an extremely reactive oxygen species that can react with everything in living organisms, especially with proteins, DNA and lipids. Hydroxyl radicals are capable of rapid initiation of the lipid peroxidation process by abstracting hydrogen atoms from unsaturated fatty acids.45 When the hydroxyl radical, generated by the Fenton reaction (containing FeCl3 , EDTA, H2 O2 and ascorbic acid), attacks deoxyribose, it generates fragments

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Figure 6. Chromatograms (GC/MS) of (a) methanol and (b) water extracts of PLRB.

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Composition and antioxidant activity of Periploca laevigata bark that on heating with TBA form a pink chromogen, which can be quantied spectrophotometrically at 532 nm.23,46 Thus one can investigate the inhibition effect of extracts by monitoring changes in absorption. In this study the activities of PLRB extracts were compared with that of mannitol, which has been reported to be an effective hydroxyl radical scavenger.23 The scavenging abilities of PLRB extracts on hydroxyl radical inhibition are shown in Fig. 5. All except the hexane and chloroform extracts showed relatively good hydroxyl radical-scavenging activity (between 24 and 87% at a concentration of 50 g mL1 ). The methanol extract exhibited the highest hydroxyl radicalscavenging activity, greater than that of BHA. At concentrations up to 25 g mL1 , mannitol showed higher activity than the methanol extract, but at 50 and 100 g mL1 the activity of the methanol extract surpassed that of mannitol. For example, at a concentration of 50 g mL1 the methanol extract, mannitol and BHA exhibited 76, 73 and 48.5% hydroxyl-scavenging activity respectively.

www.soci.org GC/MS analysis of methanol and water extracts from PLRB The chemical composition of the methanol and water extracts from PLRB was analysed by GC/MS (Figs 6(a) and 6(b) respectively). The relative retention times and mass spectra of the extract components were compared with those of authentic samples and with mass spectra from a data library. As shown in Table 2, GC/MS analysis of the methanol extract from PLRB resulted in the identication of 34 compounds, with more than 90% similarity with the standard mass spectra in the library, representing 95.71% of the relative area in the methanol extract. The methanol extract was characterised by the presence of phenolics including trimethylsilyl vanillin (0.47%), 4-methoxysalicylaldehyde (19.83%), proavine (51.62%), 4-(hydroxymethyl)-2-methoxyphenol (3.71%) 1,2,3,4-tetrahydroisoquinoline (0.85%), -amyrin (0.44%) and 3O-hydrocinnamoylsucrose (1.29%) and phenolic acids including vanillic acid (2.11%), hexadecanoic acid (3.95) and (Z,Z)-9,12octadecadienoic acid trimethylsilyl ester (2.73%).

Table 2. Chemical composition of methanol extract from Periploca laevigata root bark No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34
a b

RTa (min) 4.310 5.552 7.852 7.932 9.076 14.541 14.838 15.102 15.411 16.269 17.385 17.528 17.608 18.666 19.107 20.761 21.276 22.168 22.775 23.885 25.619 27.123 27.501 31.163 31.289 31.861 34.877 37.886 38.699 38.974 39.946 40.952 41.898 43.249

Compoundb 3H-Pyrazol-3-one 2-Phenyl-5-oxazolecarboxamide Trimethylsilyl ether of glycerol m-Methoxyphenyl trimethylsilyl ester 2-Hydroxy-4-methoxybenzaldehyde Trimethylsilyl vanillin Hexamethylperhydrotetraazacyclopentanouorene 4-Methoxysalicylaldehyde Octanoic acid trimethylsilyl ester Proavine Vanillic acid 4-(Hydroxymethyl)-2-methoxyphenol Bicyclo[2.2.1]heptane-2-acetic acid Tetradecahydrocoronene 1-Vinyl-4-methoxy-9H-pyridoindole 1,2,3,4-Tetrahydroisoquinoline 1,1-Bis[(3-methyl-5-phenyl)-2-furyl]methane 3,6,7,9,11-Pentamethylheptaleno[1,2-c]furan 1-Hydroxy-2-(1-methoxyethyl)-3-methoxyanthraquinone 1H-2-Thiophenylpyridophenothiazine 2-Benzoyl-6,7-dimethoxy-4-methylidene-2H-1,3-benzothiazine Myo-inositol 1,2,3,4,5,6-hexakis-O-trimethylsilyl ester Hexadecanoic acid trimethylsilyl ester (Z,Z)-9,12-Octadecadienoic acid trimethylsilyl ester Oleic acid trimethylsilyl ester Octadecanoic acid trimethylsilyl ester 1-(Diphenylphosphinoylmethylene)-2-(phenylthio)cyclohexene 1,2-Benzenedicarboxylic acid bis-2-ethylhexyl ester 3-O-Hydrocinnamoylsucrose 2,3,4,5-Tetraphenylpyrrole Octadecanoic acid 2,3-bis[(trimethylsilyl)oxy]propyl ester -Amyrin Demecolceine -Taraxasterol acetate Total

% 0.06 0.08 0.32 0.15 0.17 0.47 0.45 19.83 0.13 51.62 2.11 3.71 0.89 0.14 0.11 0.85 0.07 0.56 0.15 0.34 0.26 0.14 3.95 2.73 1.53 1.69 0.11 0.18 1.29 0.42 0.39 0.44 0.20 0.17 95.71

Retention time. Compounds listed in order of retention times.

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Table 3. Chemical composition of water extract from Periploca laevigata root bark No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
a

RTa (min) 4.316 7.857 8.321 20.972 22.237 22.666 23.307 23.398 24.096 24.663 24.892 25.590 25.647 42.103 42.604 43.553

Compoundb 2-Phenyl-5-oxazolecarboxamide 8-Allyl-2-amino-6-methylimidazo[1,5-a]-1,3,5-triazin-4(3H)-one 1-Indanyl methyl ether 2-(3,4-Dihydroxyphenyl)ethanol 1H-Indole-3-carboxaldehyde 4-Hydroxy-3-methoxyphenethylene glycol D-Xylopyranose 1,2,3,4-tetrakis-O-TMS Glucofuranose pentaTMS Benzoic acid 2,3-bis[(trimethylsilyl)oxy]trimethylsilyl ester 6-Deoxy-1,2,3,4-tetrakis-O-TMS Per(trimethylsilyl)-D-xylose 2,2,8,8-Tetramethyl-5-[(trimethylsilyl)oxy]-3,7-dioxa-2,8-disilanonane 1,2,3,4,5,6-Hexakis-O-(trimethylsilyl)-D-glucitol o-Cresol -Amyrin -Sitosterol Total

% 2.41 1.81 0.99 4.55 9.22 35.12 5.06 10.76 1.21 4.55 4.73 5.62 2.03 2.44 0.95 0.47 91.92

Retention time. Compounds listed in order of retention times. TMS: Trimethyl silyl ester

Some of the compounds identied in the methanol extract from PLRB have been reported to exhibit antioxidant activities. Harish et al.47 isolated and puried a natural vanillin from the methanol extract of D. hamiltonii tuberous roots which showed strong antioxidant activities in in vitro assays of lipid peroxidation inhibition and hydroxyl radical, superoxide anion and DPPH radical scavenging. The compound 1,2,3,4-tetrahydroisoquinoline has the quinoline nucleus, and Bickoff et al.48 proved that certain related quinoline derivatives had effective antioxidant activity. Bourgou et al.49 found that vanillic acid and gallic acid contributed signicantly to the antioxidant properties of methanol extracts of Nigella sativa L. shoots and roots. -Amyrin was also identied previously from roots of P. laevigata. Among the compounds identied, proavine, also called diaminoacridine, is well known as a disinfectant bacteriostatic against many Gram-positive bacteria. The water extract from PLRB, showing considerable antioxidant activity in all assays, was also analysed by GC/MS, leading to the identication of 16 chemical compounds representing 91.92% of the relative area in the water extract. As shown in Table 3, the water extract was characterised by the presence of phenolics and glycoside derivatives including 2-(3,4dihydroxyphenyl)ethanol, also called 3-hydroxytyrosol (4.55%), 4hydroxy-3-methoxyphenethylene glycol (35.12%), glucofuranose pentatrimethyl silyl ester (TMS) (10.76%) and o-cresol (2.44%). Chen et al.50 found that 4-hydroxy-3-methoxyphenyl groups exhibited strong peroxidation inhibition activity against free radical-initiated peroxidation of human low-density lipoprotein. In addition, 2-(3,4dihydroxyphenyl)ethanol and o-cresol are known as good natural antioxidant substances.51,52

had the highest phenolic and avonoid contents, followed by the water and ethyl acetate extracts. The results from various antioxidant tests clearly conrmed the antioxidant and free radicalscavenging activities of the extracts. The methanol extract, with the highest amount of total phenolics and avonoids, showed the highest antioxidant activities as measured by all assays, followed by the water extract. The antioxidant activity was correlated with the amount of total phenolics present in the respective extracts in each assay. The results of GC/MS proved the presence of phenolics with antioxidant activity in the methanol and aqueous extracts. These results conrm and contribute to the strong antioxidant capacity of the methanol and aqueous extracts from PLRB. To the best of our knowledge, this is the rst report demonstrating that extracts from P. laevigata have antioxidant activities. The results of the present work indicate that P. laevigata root bark might be a good candidate for further investigation in developing new antioxidants. Further studies should be done on the purication and identication of the bioactive compounds in PLRB.

ACKNOWLEDGEMENTS
This work was funded by the Minist` ere de lEnseignement Superieur, de la Recherche Scientique et de la Technologie, Tunisie. The authors wish to thank Professor Hafedh Belghith (Centre de Biotechnologie de Sfax, Tunisie) for the GC/MS analyses.

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