Life Sciences 73 (2003) 1667 1681

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Investigation of Bolivian plant extracts for their radical scavenging activity and antioxidant activity
Irene Parejo a, Francesc Viladomat a, Jaume Bastida a, Alfredo Rosas-Romero b, nez d, Carles Codina a,* Gloria Saavedra c, M. Antonia Murcia d, Antonia M. Jime
` cia, Universitat de Barcelona, Departament de Productes Naturals, Biologia Vegetal i Edafologia, Facultat de Farma Avda. Joan XXIII s/n, 08028 Barcelona, Spain b mica, Universidad Simo n Bol var, Apartado 89000, Caracas 1080A, Venezuela Departamento de Qu c a Agroindustrial, Facultad de Ciencias y Tecnolog a, Universidad Mayor de San Simo n, Centro de Tecnolog Cochabamba, Bolivia d n y Bromatolog a, Facultad de Veterinaria y Ciencia y Tecnolog a de Alimentos, Campus de Espinardo, Area de Nutricio 30100 Murcia, Spain Received 6 January 2003; accepted 11 March 2003
a

Abstract Fifty-four different extracts of nine Bolivian plants belonging to the family Asteraceae were evaluated for their radical scavenging activity by the DPPH, NBT/hypoxanthine superoxide, and OH/luminol chemiluminescence methods, and for their antioxidant activity by the h-carotene bleaching test. The total phenolic content was also determined by the Folin-Ciocalteu method, and the oxidative stability by the Rancimat test. Both remarkably high phenolic content and radical scavenging and antioxidant activities were found mainly in the ethyl acetate fractions among the different plant extracts. Some ethyl acetate and even some defatted crude extracts exhibited activities comparable to those of commercial extracts/compounds, thus making it possible to consider some of the studied plants as a potential source of antioxidants of natural origin. D 2003 Elsevier Inc. All rights reserved.
Keywords: Antioxidant activity; Asteraceae; Bolivia; Oxidative stability; Plant extracts; Radical scavenging activity; Rancimat; Total phenolic content

* Corresponding author. Tel.: +34-934024493; fax: +34-934029043. E-mail address: ccodina@farmacia.far.ub.es (C. Codina). 0024-3205/03/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0024-3205(03)00488-0

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Introduction It is commonly accepted that in a situation of oxidative stress, reactive oxygen molecules (ROS) such as superoxide (O 2 , OOH ), hydroxyl (OH ) and peroxyl (ROO ) radicals are generated. The ROS play an important role related to the pathogenesis of various serious diseases, such as neurodegenerative disorders, cancer, cardiovascular diseases, atherosclerosis, cataracts, and inflammation (Aruoma, 1998). The use of traditional medicine is widespread and plants still present a large source of natural antioxidants that might serve as leads for the development of novel drugs. Several antiinflammatory, digestive, antinecrotic, neuroprotective, and hepatoprotective drugs have recently been shown to have an antioxidant and/or antiradical scavenging mechanism as part of their activity (Perry et al., 1999; Lin and Huang, 2002; Repetto and Llesuy, 2002). In the search for sources of natural antioxidants, in the last few years some medicinal plants have been extensively studied for their antioxidant activity and radical scavenging activity (De las Heras et al., 1998; Desmarchelier et al., 2000; Schinella et al., 2002; VanderJagt et al., 2002). The present paper deals with a preliminary screening of some Bolivian plants from the Asteraceae family for their radical scavenging and antioxidant activities: Baccharis pentlandii, B. platypoda, Chromolaena tunariense, Erechtites hieraciifolius, Mikania psilostachya, Pluchea sagittalis, Tagetes maxima, Tessaria fastigiata and Wulffia baccata. Plants of this family have been used in traditional medicine in South America in the treatment of different pathologies (Yan et al., 1999; Kadarian et al., 2002). Although some asteraceous species belonging to the genus Baccharis (Mongelli et al., 1997), Chromolaena (Thang et al., 2001), Mikania (Suyenaga et al., 2002), Pluchea (Sen et al., 2002), Tagetes (De las Heras et al., 1998), and Tessaria (Desmarchelier et al., 2000), have been reported for their antiinflammatory and/or antioxidant properties, to our knowledge there is no information on the plant species here studied, with the exception of Pluchea sagittalis, which has been found to posses rez-Garc a et al., 1996). antiinflammatory activity (Pe In this work, 54 plant extracts/fractions of different polarity obtained from the nine Bolivian plants listed before were studied in order to assess their radical scavenging and antioxidant activity. The three assays used to evaluate the radical scavenging activity were those of the DPPH (2,2-diphenyl-1picrylhydrazyl), superoxide-nitro-blue tetrazolium (NBT) hypoxanthine/xanthine oxidase, and OH/ luminol chemiluminescence. The h-carotene bleaching test and the Rancimat test were used to evaluate the antioxidant activity and the oxidative stability of the extracts and fractions, respectively. Furthermore, the total phenolic content was determined by the Folin-Ciocalteu method. The results were compared with those obtained with different reference products: quercetin, an antioxidant of natural origin; BHA (butylated hydroxyanisole), one of the most widely used synthetic antioxidants employed in the food industry, and three commercially available extracts of natural origin with high antioxidant activity: rosemary, green tea and grape seeds.

Methods Plant material The different botanical taxa studied in this work are shown in Table 1, together with some information concerning their vernacular names, date and site of collection. The plants were authenticated by Lic.

I. Parejo et al. / Life Sciences 73 (2003) 16671681 Table 1 Botanical and geographical data of the plants studied Scientific name Baccharis pentlandii DC Baccharis platypoda DC Chromolaena tunariense (Hieron) B.L. Robins Erechtites hieracifolius (L.) Raf. ex DC Mikania psilostachya DC Pluchea sagittalis (Lam.) Cabrera Tagetes maxima Kuntze Tessaria fastigiata Griseb. Wulffia baccata (L.f.) Kuntze Voucher specimen MM 1825 MM 1838 MM 1830 MZ 340 MZ 394 MZ 337 MM 1837 MM 1845 MZ 339 Clavelin Cana Canita Suico Alto, Alko Suico Uri Uri Kunamisilli Popular name Chilca Kellu Tola Date of collection April, 1998 April, 1998 April, 1998 October, 1997 December, 1997 October, 1997 April, 1998 May, 1998 December, 1997 Site of collection Icoya, Independencia Machaca, Independencia Vega, Independencia Chimore Chimore Bulo bulo Sivingani Tiquipaya Chimore

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Province Ayopaya Ayopaya Ayopaya Chapare Chapare Carrasco Ayopaya Quillacollo Chapare

rate and Lic. Magaly Mercado, from the Unidad de Biodiversidad y Gene tica, of the Modesto Za n Universidad Mayor de San Simon, and the plants were deposited at the Herbario Nacional Mart rdenas, in Cochabamba (Bolivia). Voucher numbers are also indicated in Table 1. The plant material Ca consisted in all cases of aerial parts (stems, leaves and flowers). Chemicals All the chemicals used in this work were purchased from Sigma Aldrich (USA), with the exception of the Folin-Ciocalteus reagent, which was purchased from Panreac (Spain). All the chemicals and reagents were of analytical grade. Sample preparation and extraction The plant material was air dried until dryness at room temperature and in darkness, and then powdered with a mill. Two extracts and four fractions of each plant species were obtained using an extraction and fractionation procedure previously described (Parejo et al., 2002). In summary, the dried and powdered plant material (30 g) was extracted with MeOH (600 mL) by maceration at room temperature for 48 hours stirring several times throughout the process. After filtering through folded paper, the methanol was evaporated and the extract redissolved in water (f 1 g of extract was first dissolved in the minimum amount of methanol and then 200 mL of water were added), kept at 4 jC for 12 hours, and filtered again thus obtaining the crude extract (CE1). This CE1 was then partitioned with hexane, thus obtaining both the hexane fraction (HxF) and the clean or defatted crude extract (CE2). The CE2 was then successively partitioned with dichloromethane and ethyl acetate, thus obtaining the dichloromethane (DCF), the ethyl acetate (EAF) and the final aqueous (WF) fractions. For test dilutions, every extract or fraction was dried and redissolved in methanol (Folin Ciocalteu, DPPH, and chemiluninescence assays), water (superoxide assay) or dimethyl sulfoxide (h-carotene bleaching test). In the Rancimat test, the dry extract/fraction was directly mixed with the oil.

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Determination of total phenolics The total phenolic content (TPC) was determined according to the Folin-Ciocalteu method (Singleton and Rossi, 1965). The reaction mixture was composed of 0.1 mL of extract, 7.9 mL of distilled water, 0.5 mL of the Folin-Ciocalteus reagent, and 1.5 mL of 20% sodium carbonate. The opaque flasks were mixed and allowed to stand for 2 hours. The absorbance was measured at 765 nm in a HITACHI U-2000 Spectrophotometer (the same equipment was used in all the assays, except for that of chemiluminescence). The total phenolic content was determined as gallic acid equivalents (GAE)/mg of extract. Free radical scavenging activity The different extracts were measured in terms of hydrogen donating or radical scavenging ability using the stable radical DPPH (Brand-Williams et al., 1995). 0.75 mL of a methanolic solution of the extract at different concentrations were mixed with 1.5 mL of a DPPH methanolic solution (20 mg/L). The absorbance was measured at 517 nm after 20 minutes of reaction. The percent of DPPH decolourization of the sample was calculated according to the equation % Decolourization = [1 (ABSSAMPLE/ABSCONTROL)] 100. The decolourization was plotted against the sample extract concentration, and a logarithmic regression curve was established in order to calculate the IC50 (inhibitory concentration 50, Ag/mL) which is the amount of sample necessary to decrease by 50% the absorbance of DPPH. Hydroxyl radical scavenging activity The radical scavenging activity was determined through the Co(II)/EDTA/OH/H2O2-luminol system according to the method established by Parejo et al., 2000. The intensity of chemiluminescence (CL) was measured as relative light units (RLU) in a Turner Designs TD-20/20 luminometer. The highest CL intensity of the reaction (control light) is decreased by hydroxyl radical scavenging substances (Merfort et al., 1996; Parejo et al., 2000). The reaction mixture was composed of buffer pH 9 Co(II)/EDTA, buffer pH 9 luminol and methanolic extract (methanol for the control). Finally, H2O2 was added to start the reaction in dark conditions. CL intensity (RLU) was measured 20 minutes after the reaction started. The percent of inhibition of the CL was calculated for each concentration according to the equation % Inhibition = [1 (RLUSAMPLE/RLUCONTROL)] 100. The RLU was plotted against the sample extract concentration, and a linear regression was established in order to calculate the IC50 (Ag/mL), which is the amount of sample necessary to decrease by 50% the CL intensity. Superoxide anion scavenging activity The superoxide radicals were generated in vitro by the hypoxanthine/xanthine oxidase system. The scavenging activity of the extract is determined by the nitro-blue tetrazolium (NBT) reduction method. 2+ In this method O 2 reduces the yellow dye (NBT ) to produce the blue formazan, which is measured spectrophotometrically at 560 nm. Antioxidants are able to inhibit the purple NBT formation (Cos et al., 1998; Chu et al., 2000). The capacity of the extracts to scavenge the superoxide radical was assayed as follows. A reaction mixture was prepared with 50 mM phosphate buffer (pH 7.5) containing EDTA (0.05 mM),

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hypoxanthine (0.2 mM), NBT (1 mM), aqueous or ethanolic extract (distilled water for the control), and xanthine oxidase. The xanthine oxidase (1.2 U/AL) was added last. For each sample a blank was carried out. The solutions were prepared daily, and kept from light. The subsequent rate of NBT reduction of the control was determined on the basis of sequential spectrophotometric determinations of absorbance at 560 nm. The absorbance of the samples was measured at the time of the maximum absorbance of the control. The results are expressed as the percentage inhibition of the NBT reduction with respect to the reaction mixture without sample (buffer only), and were calculated by the equation % Inhibition = {[(CABS CBABS) (SABS SBABS)] / (CABS CBABS)} 100 where SABS, SBABS, CABS, and CBABS were the absorbances of the sample, the blank sample, the control, and the blank control, respectively. Determination of the antioxidant activity In this method, antioxidant activity is measured by the ability of a compound to minimize the coupled oxidation of linoleic acid and h-carotene in an emulsified aqueous system, which loses its orange color when reacting with the radicals (Rosas-Romero et al., 1999). In this work AAPH (2,2V -azobis (2methylpropionamide) dihydrochloride) was used as a water-soluble radical azo-initiator, which decomposes itself at a temperature-controlled rate, 32 jC, yielding molecular nitrogen and two carbon radicals (R). Then the R. can react rapidly with molecular oxygen to produce ROO. The reaction was carried out in situ in the cuvette: 1990 AL of the aqueous emulsion (h-carotene/linoleic acid and saturated with oxygen) were equilibrated at 32 jC for 6 minutes. The oxidising reaction was started by adding 10 AL of AAPH (0.9 M). Ten minutes after vortexing the mixture, 100 AL of plant extract dissolved in DMSO at a concentration of 250 Ag/mL (DMSO for the control) were added and the mixture was vortexed again. The absorbance was measured at 470 nm in a spectrophotometer until the plateau (90 minutes). BHA (250 Ag/mL) was used as a reference synthetic antioxidant and a Tween 20 solution was used as a blank. All the solutions and emulsions were prepared daily. The antioxidant activity (AOA) was given by the equation % AOA = {1 [(SABS at 0 min SABS at 90 min) / (CABS at 0 min CABS at 90 min)]} 100 where SABS is the absorbance of the sample and CABS is the absorbance of the control. Rancimat test for oxidative stability Sample preparation for the Rancimat test consisted of maceration of the dry extract (problem or standard)/corn oil mixture at the concentration of 10 mg/ml, or the reference antioxidant/corn oil mixture at the concentration of 100 ppm (w/v), which is that permitted as food additive. The Rancimat method (Metrohm model 743, Herisan, Switzerland) determines the induction periods by measuring the increase in the volatile acidic by-products released from the oxidizing oil at 110 jC with an air flow rate of 20 L/h. The concentration of the degradation products, which are transferred into distilled water, is assessed by measuring the conductivity. Longer induction periods suggest stronger activity for the added antioxidants or extracts. The relative activity is expressed by the protection factor (PF), which is calculated by dividing the induction period of the oil with added antioxidants by that of the control (corn oil alone) (Schwarz and Ernst, 1996). Although this technique has been questioned (Frankel, 1993), we decided to use it, as it is a procedure commonly used in the food industry and governmental analytical laboratories (Murcia et al., 2001).

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Statistical analysis All the analyses were carried out in triplicate, and they were repeated if some coefficient of variation was higher than 10%. A one-way ANOVA analysis (Newman-Keul test) was carried out to compare the results of the extracts and fractions, as well as the nine studied plants. The calculations belonging to the free radical and hydroxyl radical scavenging activity were made using the antiradical efficiency (AE) values (AE = 1000/IC50). The statistical and regression analyses were performed using the computer software Statistica for Windows. Regression analyses were carried out using the Statistica programme. Differences at P < 0.05 were considered to be significant.

Results Total phenolic content The TPC values of the different extracts/fractions ranged from 409.5 to 2.9 GAE/mg of extract (Table 2). Considering all the fractions, the ethyl acetate ones were found to contain the highest phenolic content (especially those of Tessaria fastigiata, Mikania psilostachya and Tagetes maxima), with the exception of Chromolaena tunariense, where the clean crude extract (CE2) exhibited a TPC value higher than the ethyl acetate fraction. The total phenolic content of the ethyl acetate fractions of both T. fastigiata and M. psilostachya was significantly different from that of the other extracts/fractions. The CE2 extracts was the second group to exhibit high TPC values in five of the nine plants studied. Surprisingly, the dichloromethane fractions were found not to give good results in comparison to a similar work on Mediterranean herbs (Parejo et al., 2002). In all cases, the lowest amounts of phenolics were found in the hexane fractions, with the exception of Wulffia baccata, where the dichloromethane fraction showed the lowest TPC value. The higher phenolic content in the ethyl acetate fractions than in the crude extracts was probably due to the purification and concentration of phenolics throughout the fractionation procedure. Radical scavenging activity The radical scavenging activity of the different extracts/fractions are also shown in Table 2. The best free radical (DPPH) scavenging activity was shown by the clean crude extract (CE2) of Tagetes maxima (IC50 = 5.6), followed by the ethyl acetate fractions of this species and that of Mikania psilostachya (IC50 = 9.4 and 8.3, respectively). With the exception of T. maxima, all the other ethyl acetate fractions showed the highest free radical scavenging activity compared to the different extracts/fractions. The activity exhibited by the EC2 of T. maxima and the EAF of M. psilostachya was significantly higher than that of the other extracts/fractions of these two species. Some relatively high activity was also exhibited by the water fractions, especially that of Tessaria fastigiata (IC50 = 14.6), being the second most active fractions in four of the nine plants. All the hexane fractions were found to be inactive at the concentration used in this work, and no free radical scavenging activity was detected in the dichloromethane fractions in six of the nine plants species analysed. It is noteworthy that no activity was detected in any of the extracts and fractions of Wulffia baccata.

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Concerning the hydroxyl radical scavenging activity, the best results were exhibited by the clean crude extract (CE2) of Mikania psilostachya and Tagetes maxima, with IC50 values of 12.1 and 12.6, respectively (Table 2), which were found to be significantly higher than those of any other extract or fraction. The third best result was that of the crude extract (EC1) of T. maxima (IC50 = 19.5). Apart from these two plant species, the most active fractions were generally found to be those of dichloromethane (in four of the nine fractions) and ethyl acetate (in three of nine), although their IC50 values were higher than 20. Thus, some relatively good results were exhibited by the ethyl acetate fractions of T. maxima and Tessaria fastigiata (IC50 = 20.3 and 29.3, respectively) and by the dichloromethane fraction of Chromolaena tunariense (IC50 = 24.8). The lowest activity was always found in the hexane fractions. The values of superoxide scavenging activity ranged from 96.6 % in the crude extract (EC1) of Tagetes maxima to 3% in the hexane fraction (HxF) of Chromolaena tunariense, although some extracts did not show activity (Table 2). As for the DPPH and CL assays, in general, the highest inhibition of the superoxide anion was found mainly in the ethyl acetate fractions (in six of nine), although in Baccharis pentlandii, Erechtites hieraciifolius and Tagetes maxima the highest value was observed in the crude extracts (EC1). As in the other scavenging activity assays, weak superoxide inhibitory activities were found in the hexane fractions. Five of the nine hexane fractions did not exhibit superoxide scavenging activity, as well as the two extracts (EC1 and EC2) of Wulffia baccata. This assay revealed Tagetes maxima to be the most active plant as three of its six extract/fractions were found to exhibit values of superoxide scavenging activity higher than 95%. This high activity was in agreement with a relatively high scavenging activity of superoxide anion also shown by Tagetes pusilla (De las Heras et al., 1998). Apart from this species, only the crude extract of Baccharis pentlandii exhibited a value higher than 90% of inhibition (93%). Antioxidant activity The antioxidant activity determined by the h-carotene bleaching method was different according to the plant material analysed, but in general it was also higher in the ethyl acetate fractions (in five of nine plants), thus coinciding with the results of other scavenging activity assays (Table 2). The highest values were shown by the ethyl acetate fractions of Tagetes maxima and Tessaria fastigiata (with 60.7% and 55.5% of inhibition, respectively), and the dichloromethane fraction of Chromolaena tunariense (56.5% of inhibition), which were found to be significantly different from those of the respective ethyl acetate and dichloromethane fractions. No other results higher than 50% of inhibition were found, except for the activity of the clean crude extract (EC2) of T. maxima (54.3% of inhibition). This result is in good agreement with the activities shown by this plant species in the other assays. The lowest antioxidant activity was always exhibited by the hexane fractions, with the exception of Wulffia baccata where it was found to be similar to that of the dichloromethane fraction. Rancimat results The highest protection factor was exhibited by the ethyl acetate fraction of Tessaria fastigiata (PF = 1.35), which was significantly higher than that of the other extracts or fractions analysed. The second fraction showing high oxidative stability was the ethyl acetate fraction of Pluchea sagittalis (PF = 1.26), a plant species that exhibited little activity in the other assays, although it is used as an antiinflammatory

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Table 2 Antioxidant and radical scavenging activities, and protection factor of the different extracts and fractions of the nine Bolivian plants Extract/ fraction Total phenolic contenta DPPH radical scavenging activityb 56.7 63.8 nd nd 15.0 50.3 F 1.2 F 1.7 Hydroxyl radical scavenging activityb 125.9 110.3 nd 223.2 53.1 81.5 F 5.6 F 6.3 F 4.1 F 0.9 F 6.8 Superoxide scavenging activityc 93.0 76.7 8.7 41.6 86.4 70.5 F F F F F F 7.0 2.7 0.6 2.5 2.5 2.3 Antioxidant activityc Oxidative stabilityd

Baccharis pentlandii DC. CE1 114.9 F 7.1 CE2 101.7 F 5.9 HxF 33.1 F 1.4 DCF 71.3 F 2.8 EAF 207.5 F 8.8 WF 110.2 F 10.4 Baccharis platipoda CE1 121.5 F CE2 143.3 F HxF 31.3 F DCF 106.0 F EAF 292.3 F WF 126.1 F

F 0.3 F 1.2

15.0 16.1 3.5 13.5 12.8 14.6

F F F F F F

1.4 1.2 0.5 0.9 1.3 1.5

1.08 1.09 0.98 1.00 1.22 1.03

F F F F F F

0.03 0.03 0.01 0.01 0.01 0.02

7.7 4.2 0.9 5.6 14.5 0.9

49.5 80.9 nd nd 20.5 73.3

F 0.8 F 1.8

F 1.3 F 8.6

61.2 135.6 nd 222.8 56.7 96.9

F 2.8 F 4.5 F 10.3 F 1.6 F 0.5

36.7 43.2 nd 20.7 82.9 42.1

F 1.5 F 2.0 F 1.0 F 2.2 F 1.7

9.8 16.3 nd 3.7 21.0 8.7

F 0.4 F 0.9 F 2.5 F 1.8 F 1.0

0.92 1.08 nd 0.91 1.14 0.97

F 0.03 F 0.00 F 0.03 F 0.00 F 0.01

Chromolaena tunariense CE1 147.1 F 6.8 CE2 270.2 F 8.7 HxF 47.0 F 4.4 DCF 173.8 F 10.7 EAF 229.9 F 25.2 WF 187.0 F 5.6

nd 124.1 F 3.9 nd nd 32.7 F 0.3 190.0 F 2.3

115.6 103.7 309.0 24.8 46.8 116.9

F F F F F F

4.6 6.1 10.3 2.6 0.5 4.1

35.1 54.1 3.0 63.0 86.1 75.2

F F F F F F

0.5 3.4 0.0 3.6 2.8 1.7

nd 15.1 nd 56.5 11.5 2.0

F 1.8 F 0.3 F 2.9 F 1.1

1.24 1.08 1.06 0.97 1.00 0.98

F F F F F F

0.02 0.05 0.03 0.01 0.00 0.03

Erechtites hieraciifolius (L.) Raf. Ex DC. CE1 177.1 F 6.1 25.8 F 0.6 CE2 190.4 F 7.7 21.5 F 0.9 HxF 18.0 F 0.9 nd DCF 96.6 F 6.9 nd EAF 380.9 F 12.4 14.9 F 0.4 WF 180.3 F 6.8 25.9 F 0.4 Mikania psilostachya DC. CE1 126.9 F 4.9 CE2 229.8 F 10.0 HxF 15.2 F 0.1 DCF 154.2 F 7.8 EAF 404.3 F 27.3 WF 171.3 F 6.7 Pluchea sagittalis (Lam.) CE1 76.3 F CE2 134.4 F HxF 2.9 F DCF 85.9 F Cabrera 3.6 8.8 0.1 4.2

58.0 55.1 739.4 36.4 45.1 74.7

F F F F F F

1.7 0.8 27.2 0.8 3.2 0.3

56.3 49.7 nd 21.9 48.2 47.2

F 3.0 F 1.0 F 1.8 F 1.3 F 3.8

32.5 24.8 9.9 13.0 29.8 23.8

F F F F F F

0.7 2.1 1.5 1.3 1.0 2.9

1.10 1.01 0.83 0.96 1.02 1.02

F F F F F F

0.03 0.02 0.04 0.02 0.01 0.01

68.5 33.2 nd 37.6 8.3 30.6

F 2.8 F 0.3 F 1.7 F 0.1 F 0.9

78.1 12.1 nd 42.3 34.9 71.9

F 1.7 F 6.2 F 1.5 F 1.7 F 6.9

57.3 80.2 35.5 80.8 88.2 75.2

F F F F F F

4.5 1.0 2.1 2.1 3.4 4.7

14.1 26.5 nd 16.6 40.6 30.5

F 1.0 F 0.7 F 2.7 F 0.7 F 0.5

0.88 0.93 0.88 1.06 1.20 na

F F F F F

0.01 0.02 0.03 0.02 0.05

124.0 F 1.3 119.4 F 1.4 nd 156.5 F 6.7

161.9 F 4.3 154.4 F 0.9 nd 44.6 F 0.9

42.3 F 1.6 37.0 F 4.1 nd 46.1 F 5.2

10.7 F 1.6 10.8 F 1.3 nd 13.1 F 1.2

1.07 F 0.01 1.00 F 0.01 nd 1.06 F 0.03

I. Parejo et al. / Life Sciences 73 (2003) 16671681 Table 2 (continued) Extract/ fraction Total phenolic contenta DPPH radical scavenging activityb Hydroxyl radical scavenging activityb 56.2 F 3.0 139.1 F 1.4 Superoxide scavenging activityc 62.3 F 3.5 41.5 F 4.4 Antioxidant activityc

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Oxidative stabilityd

Pluchea sagittalis (Lam.) Cabrera EAF 237.9 F 28.0 17.4 F 0.4 WF 71.0 F 0.8 103.8 F 1.4 Tagetes maxima Kuntze CE1 269.5 F 6.2 CE2 315.8 F 5.1 HxF 28.8 F 0.7 DCF 79.5 F 3.6 EAF 369.2 F 18.6 WF 128.0 F 4.3

37.8 F 0.7 12.4 F 2.6

1.26 F 0.01 1.05 F 0.04

15.0 5.6 nd 135.7 9.4 123.8

F 1.3 F 0.4 F 19.1 F 0.4 F 8.3

19.5 12.6 219.8 70.2 20.3 76.9

F F F F F F

1.7 0.1 0.6 2.6 1.5 4.8

96.6 95.6 13.6 27.9 95.8 38.2

F F F F F F

2.9 3.2 0.9 3.7 5.4 2.6

25.2 54.3 nd 13.5 60.7 nd

F 3.1 F 0.4 F 0.9 F 0.2

0.96 1.02 0.96 1.10 1.22 0.93

F F F F F F

0.02 0.00 0.02 0.04 0.01 0.03

Tessaria fastigiata (Griseb.) Cabrera CE1 126.3 F 4.1 53.2 CE2 191.9 F 7.4 20.8 HxF 36.3 F 1.6 nd DCF 62.4 F 5.9 nd EAF 409.5 F 25.3 10.7 WF 211.5 F 18.0 14.6 Wulffia baccata (L.) Kuntze 32.2 F 1.8 CE1 CEe 62.5 F 1.9 HxF 24.9 F 0.5 DCF 24.5 F 0.5 EAF 102.4 F 10.7 WF 75.6 F 3.6 Standards Quercetin BHA Grape seed Rosemary Green tea

F 3.5 F 2.2

F 0.5 F 0.2

64.6 40.8 262.5 41.1 29.3 44.6

F F F F F F

4.0 1.0 7.3 1.3 1.1 0.9

62.7 68.7 nd 7.1 81.5 66.4

F 3.6 F 0.9 F 0.3 F 1.4 F 2.1

28.9 28.3 nd 20.4 55.5 44.3

F 2.5 F 1.1 F 0.9 F 0.5 F 0.3

0.99 0.95 0.87 1.07 1.35 1.00

F F F F F F

0.00 0.02 0.03 0.03 0.01 0.01

nd nd nd nd nd nd

235.3 100.2 715.4 58.5 65.7 135.0

F F F F F F

12.7 2.9 42.5 3.1 1.3 8.5

nd nd nd 22.0 F 0.4 30.8 F 1.6 19.9 F 2.4

20.7 14.8 11.8 3.4 5.1 10.9

F F F F F F

1.8 2.4 2.0 1.1 2.7 1.6

1.02 0.99 0.95 0.98 1.06 1.01

F F F F F F

0.02 0.01 0.03 0.01 0.04 0.02

851.3 F 12.8 132.0 F 5.6 387.2 F 7.7

6.1 34.1 6.9 35.0 11.8

F F F F F

0.5 0.6 0.2 0.2 0.6

5.1 2.1 29.5 46.8 13.3

F F F F F

0.1 0.0 1.4 1.3 0.4

97.7 67.5 88.3 38.5 92.1

F F F F F

4.3 0.3 0.5 1.0 4.9

83.3 89.2 65.6 55.7 72.3

F F F F F

6.3 5.4 2.4 1.2 1.2

1.46 1.01 0.67 2.04 0.97

F F F F F

0.05 0.01 0.01 0.04 0.03

nd = not detected, na = not available (see Methods for the identification of extracts and fractions). a Values expressed as GAE/mg extract. b Values expressed as IC50 (Ag/mL). c Values expressed as percentage of inhibition. d Values expressed as protection factor.

rez-Garc a et al., 1996). Similar results were found in the crude extract of in South America (Pe Chromolaena tunariense (PF = 1.24), and the ethyl acetate fractions of Baccharis pentlandii (PF = 1.22), Tagetes maxima (PF = 1.22), and Mikania psilostachya (PF = 1.20). In general, the best oxidative stability was exhibited by the ethyl acetate fractions, with the exception of C. tunariense and Erechtites hieraciifolius, where the highest protection factor was shown by the crude extracts (EC1). The oxidative stability exhibited by the other extracts and fractions was not remarkable.

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Total phenolic content versus radical scavenging activity and antioxidant activity Among all the extracts analysed, a significant total phenolic content (values higher than 200 GAE/mg, with the exception of Wulffia baccata) and radical scavenging and antioxidant activities were found mainly for the ethyl acetate fractions (Table 2). In general, extracts or fractions with higher radical scavenging and antioxidant activities showed a higher phenolic content, and some good correlations were found among these parameters. When subjecting the results (radical scavenging and antioxidant activity and phenolics) of all the extracts and fractions of each plant species to the regression analysis (Table 3), one can observe that the highest correlation coefficients were exhibited between the TPC and the DPPH scavenging activity (with four of the nine plants showing correlation coefficients higher than 0.94), and between the TPC and the antioxidant activity/h-carotene bleaching method (with five of the nine plants exhibiting correlation coefficients higher than 0.90). The lowest correlation coefficients were found between the total phenolic content and the hydroxyl radical scavenging activity. Considering simultaneously the four correlation coefficients between the total phenolic content and the four methods of radical scavenging and antioxidant activity used, the two plants species exhibiting the best results were found to be Tagetes maxima and Baccharis platipoda, with average values of 0.91 and 0.89, respectively. Although they generally did not reach the highest values (only in the case of the superoxide scavenging activity), they exhibited quite good correlation coefficients in the four kinds of analyses (Table 3). In general, the ethyl acetate fractions exhibited the highest antioxidant activity (h-carotene bleaching method) and free radical and superoxide scavenging activities and TPC values, whereas the dichloromethane fractions showed better results in the hydroxyl radical scavenging test, thus confirming earlier results on other plant species (Parejo et al., 2002). Within the nine ethyl acetate fractions, only those of Baccharis platipoda and Tessaria fastigiata exhibited the best results in both the antioxidant and radical
Table 3 Correlation coefficients (R) between the radical scavenging and antioxidant activity and the total phenolic content Plant material Baccharis pentlandii Baccharis platipoda Chromolaena tunariense Erechtites hieraciifolius Mikania psilostachya Pluchea sagittalis Tagetes maxima Tessaria fastigiata Wulffia baccata All the plants mixed
a b c d e f

N 18 18 18 18 18 18 18 18 18 162

R DPPH/TPCa 0.96e 0.94f 0.53f 0.92e 0.94e 0.89e 0.86e 0.95e 0.81e CLb/TPC 0.92f 0.73e 0.40 0.51f 0.36 0.57f 0.89e 0.76e 0.39 0.59e SOc/TPC 0.78 0.97e 0.78e 0.68f 0.81e 0.82e 0.96e 0.82e 0.58f 0.64e BCd/TPC 0.48f 0.90e 0.23 0.76e 0.92e 0.92e 0.92e 0.90e 0.23 0.67e

TPC = total phenolic content. CL = Chemiluminescence. SO = Superoxide anion. BC = h-carotene bleaching. p < 0.001. p < 0.05.

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scavening activities and the highest TPC values (Table 2). Other results should be pointed out. For instance, only Tagetes maxima exhibited the best radical scavenging activity (DPPH and CL), as well as the highest total phenolic content among the nine clean extracts (CE2). Likewise, within the nine dichloromethane fractions, only that of Chromolaena tunariense showed the best hydroxyl scavenging and antioxidant activities. In all cases the hexane fractions were found to contain the lowest amounts of phenolics and to exhibit the worst antioxidant and radical scavenging activities, with the exception of Wulffia baccata, where the dichloromethane and ethyl acetate fractions (DCF and EAF) showed an antioxidant activity slightly lower than that of the hexane fraction (Table 2). Comparative study among the different extracts and fractions The behaviour of the different extracts and fractions in relation to the radical scavenging and antioxidant activities was checked independently of the plant species. Thus, significant differences between the extracts and fractions were found, with the ethyl acetate fractions exhibiting the highest antioxidant activity and free radical and superoxide radical scavenging activities (Fig. 1), in good agreement with earlier results (Mensor et al., 2001; Parejo et al., 2002). The highest hydroxyl radical scavenging activity was exhibited by the clean crude extracts (CE2), although no statistically significant differences were observed between them and both ethyl acetate (P = 0.0661) and dichloromethane (P = 0.3557) fractions. The Rancimat test also revealed significant differences in the oxidative stability of the ethyl acetate fractions with respect to the other extracts and fractions analysed. The lowest antioxidant and radical scavenging activity was always exhibited by the hexane fractions, which were significantly different (P = 0.5665) from the other extracts/fractions, although no significant differences were observed between the free radical scavenging activity of both hexane and dichloromethane fractions. A comparison of the different radical scavenging activity and antioxidant activity of each kind of extract or fraction among the different plants was also carried out through a one-way ANOVA. For every kind of activity measured, significant differences were found among the results (AE values or percentages of inhibition) of each of the nine different extracts/fractions. Thus, the free radical scavenging activity and oxidative stability of the ethyl acetate fractions of Mikania psilostachya and

Fig. 1. Distribution of the radical scavenging and antioxidant activities among the different extracts and fractions, separately for methods (DPPH = free radical; CL = chemiluminescence; SO = superoxide anion, BCB = h-carotene bleaching). Values are expressed as antiradical efficiency (AE = 1000/IC50) and percentages of inhibition (see Methods for identification of extracts and fractions).

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Tessaria fastigiata, respectively, were significantly higher than that of the other ethyl acetate fractions. Likewise, the hydroxyl radical scavenging activity of the CE2 of this plant species was also significantly higher than that of the rest of the clean crude extracts. For both the superoxide radical activity and antioxidant activity, the ethyl acetate fraction of Tagetes maxima was significantly higher than the other ethyl acetate fractions. Regarding the total phenolic content, in general there were statistically significant differences among extracts and fractions, although not between the crude extracts and water fractions. Comparative study with reference antioxidants The results of both the antioxidant and scavenging activities of the reference substances and extracts studied in this work, as well as those of the oxidative stability, are also shown in Table 2. Quercetin was found to exhibit the highest free radical scavenging activity (IC50 = 6.1), followed by that of the grape seed extract (IC50 = 6.9), whereas BHA showed the highest antioxidant (89.2% inhibition) and hydroxyl radical scavenging (IC50 = 2.1) activities. It has already been reported that the DPPH radical savenging nchezactivity is higher in quercetin than BHA (Brand-Williams et al., 1995; Chen and Ho, 1997; Sa Moreno et al., 1998), and this coincides with quercetin displaying a slower kinetic behaviour than BHA (Parejo et al., 2000). Green tea and grape seed extracts, as well as quercetin, were found to exhibit quite similar superoxide radical activities, higher than BHA and rosemary extracts. The rosemary extract showed the lowest antioxidant and radical scavenging activities, but the highest protection factor (PF = nez-Tome et al., 2001), followed by quercetin 2.04), in complete agreement with earlier results (Mart (PF = 1.46). Grape seed and green tea extracts, however, did not exhibit a good oxidative stability in the Rancimat test, in contrast with their high phenolic content (Table 2). The different polyphenolic composition of these reference extracts might explain this difference in such an activity. Some of the obtained extracts and fractions exhibited quite strong antioxidant and radical scavenging activities, which were found to be similar, and in some cases even higher, than those of the reference compounds or extracts. Thus, the clean crude extract of Tagetes maxima showed a free radical scavenging activity higher than quercetin and grape seed extract, the best reference compound and extract here tested, respectively (Table 2). All the ethyl acetate fractions, with the exception of Wulffia baccata, were found to be more active than BHA and rosemary extract, but only those of Mikania psilostachya, Tagetes maxima and Tessaria fastigiata exhibited a free radical scavenging activity higher than that of green tea. No extracts or fractions were found to exhibit a higher hydroxyl radical scavenging activity than that of BHA or quercetin, but the clean crude extracts of both Mikania psilostachya and Tagetes maxima showed a higher activity than that of the three reference extracts. The hydroxyl radical scavenging activity of both the crude extract and ethyl acetate fraction of T. maxima was lower than that of the green tea extract but higher than that of the extracts of grape seeds and rosemary. Other fractions, such as the dichloromethane fraction of Chromolaena tunariense and the ethyl acetate fraction of Tessaria fastigiata also exhibited a higher hydroxyl radical scavenging activity than that of grape seeds and rosemary extracts. The two extracts (CE1 and CE2) and the ethyl acetate fraction of Tagetes maxima, and also the crude extract of Baccharis pentlandii, exhibited a higher superoxide radical scavenging activity than that of the different reference substances and extracts studied in this work. No other extracts or fractions showed a higher activity than that of grape seeds or quercetin, with the exception of the ethyl acetate fraction of

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Mikania psilostachya which was found to be more active than grape seeds extract but less active than quercetin. None of the extracts or fractions obtained exhibited a higher antioxidant activity than that of the reference substances or extracts, with the exception of the ethyl acetate fraction of Tagetes maxima and the dichloromethane fraction of Chromolaena tunariense, which were found to be more active than that of the rosemary extract (Table 2). BHA and quercetin showed a high antioxidant activity, which was moderate in the reference commercial extracts, probably as a consequence of the interactions between their individual constituents and both linoleic acid and Tween. The extracts and fractions from the studied plants, which are highly complex mixtures, could act similarly to these commercial extracts. Finally, none of the extracts and fractions obtained from the nine studied plants exhibited a higher oxidative stability than that of the rosemary extract or quercetin. However, the ethyl acetate fraction of Tessaria fastigiata, which was found to show the highest phenolic content, exhibited an oxidative stability comparable to that of quercetin.

Discussion Often it is difficult to decide in a screening for antioxidants from natural sources which of the plant species studied can be considered the best one, as each of them exhibits different antioxidant and/or scavenging activities. During the screening of nine plants in this work, Tagetes maxima and Mikania psilostachya, in this order, were found to be the most promising ones. T. maxima exhibited the best free radical and superoxide radical scavenging activities, and also antioxidant activity (by the bleaching method), whereas M. psilostachya showed the best hydroxyl radical scavenging activity, followed by T. maxima. On the other hand, although the total phenolic content of the ethyl acetate fraction of M. psilostachya was higher than that of the same fraction of T. maxima, considering the sum of the TPC values determined in all the extracts and fractions, the total phenolic content of T. maxima (1191 GAE/ mg) was higher than that of M. psilostachya (1102 GAE/mg). Moreover, Tagetes maxima showed the second highest PF value in the Rancimat test, and was the plant species exhibiting the best average correlation coefficient among the different methods used (R = 0.91), thus suggesting to possess good antioxidant properties. Of the six different extracts/fractions of T. maxima analysed, the clean or defatted extract (CE2) was found to show very good results in all the tests, in most assays even better than those of the ethyl acetate fraction. This CE2 extract also exhibited better antioxidant properties (free radical and superoxide scavenging activity) better than those of the reference chemicals and extracts used for comparison. Furthermore, this extract is especially interesting for its possible use in the food industry or in any other application because it would not be necessary to subject it to a further process of fractionation or purification. The good results exhibited by Mikania psilostachya are in agreement with the antiinflammatory activity shown by other species of this genus, like M. laevigata and M. involucrata (Suyenaga et al., 2002). Apart from Tagetes maxima and Mikania psilostachya, which are considered the two best plants of those studied here, Tessaria fastigiata and Baccharis pentlandii also exhibited relatively good radical scavenging and antioxidant activities. This is similar to other species belonging to these genera, in particular T. integrifolia (Desmarchelier et al., 2000), B. coridifolia (Mongelli et al., 1997) and B. trinervis (De las Heras et al., 1998), which are commonly used in traditional medicine in South America. No extracts/fractions of Pluchea sagittalis were found to show significant antioxidant activity, in

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contrast to the activity observed by an aqueous extract of this plant and a methanolic extract of P. indica. rez-Garc a et Both plant species exhibit also antiinflammatory activity (Sen and Nag Chaudhuri, 1991; Pe al., 1996; Sen et al., 2002). This work reveals that the Bolivian flora can be an interesting source of new antioxidative plant extracts, such as those of Tagetes maxima and Mikania psilostachya, with a potential use in different fields (food, cosmetics, pharmaceutical). A more detailed investigation of T. maxima to identify the compounds responsible for the antioxidant activity is in progress. Acknowledgements This work was financially supported by the Generalitat de Catalunya (2001SGR-00124), and n Bol var, Venezuela, and was carried Decanato de Investigaciones y Desarrollo de la Universidad Simo a para el Desarrollo out within the frame of the Programa Iberoamericano de Ciencia y Tecnolog n Cient fica con Iberoame rica. I.P. is grateful to (CYTED, project IV.11) and the Programa de Cooperacio Generalitat de Catalunya for the provision of a research fellowship. The authors are grateful to Ferran nchez and Luca Lavelli for their technical support. Antonio Man es (EUROMED, Spain) and Bernd Sa Weinreich (RAPS, Germany) are acknowledged for supplying us with extracts of grape seeds, and of green tea and rosemary, respectively. References
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