A dimeric triterpenoid glycoside and flavonoid glycosides with free ...

Arch Pharm Res Vol 34, No 4, 543-550, 2011 DOI 10.1007/s12272-011-0404-9

A Dimeric Triterpenoid Glycoside and Flavonoid Glycosides with Free Radical-Scavenging Activity Isolated from Rubus rigidus var. camerunensis Télesphore Benoît Nguelefack1, Félicité Hermine Kamga Mbakam1, Léon Azéfack Tapondjou2, Pierre Watcho1, Elvine Pami Nguelefack-Mbuyo1, Beaudelaire Kemvoufo Ponou2, Albert Kamanyi1, and Hee-Juhn Park3 1

Laboratory of Animal Physiology and Phytopharmacology, Faculty of Science, University of Dschang, P.O.Box 67 Dschang, Cameroon, 2Laboratory of Environmental and Applied Chemistry, Faculty of Science, University of Dschang, P.O.Box 183 Dschang, Cameroon, and 3Department of Pharmaceutical Engineering, Sangji University, Wonju 220-702, Korea (Received October 13, 2010/Revised December 28, 2010/Accepted December 29, 2010)

The aerial part of Rubus rigidus var. camerunensis (Rosaceae) is used to treat respiratory and cardiovascular disorders in the Cameroonian traditional medicine. The ethanol extract exhibited more potent antioxidant activity (Emaxs of 119% and 229% activity on DPPH and β-carotene test) than aqueous extract. Bioactivity-guided fractionation of the ethanol extract based on free radical-scavenging assay (DPPH assay) afforded five flavonoid glycosides (four flavonol glycosides and an anthocyanin) and three glucosides of 19α-hydroxyursane-type triterpenoid (two monomeric and one dimeric triterpenoids). The flavonoids were identified as kaempferol 3-O-(2´´-O-E-p-coumaroyl)-β-D-glucopyranoside (1), kaempferol-3-O-β-D-glucopyranoside (astragalin, 2), kaempferol-3-O-α-L-arabinofuranoside (juglanin, 3), quercetin-3-O-β-D-glucopyranoside (isoquercitrin, 4), pelargonidin-3-O-β-D-glucopyranoside (callistephin, 5). The three triterpenoids were 2α, 3β, 19α, 23-tetrahydroxyurs-12-ene-28-O-β-D-glucopyranosyl ester (nigaichigoside F1, 6), 2α, 3β, 19α-trihydroxyurs-12-ene-23-carboxyl-28-O-β-D-glucopyranosyl ester (suavissimoside R1, 7) as monomeric triterpenoids and coreanoside F1 (8) as a dimeric triterpenoid. The flavonoids exhibited potent antioxidant activities (66 to 93.56% against DPPH radical) and they were also active on β-carotene test. Coreanoside F1 exhibited a 63% antioxidant activity, meanwhile the other two triterpenoids showed a weak activity. Three important facts on structure-activity relationship were observed: Compound 8, a dimeric triterpenoid glycoside, strongly enhanced antioxidant activity of its monomers, compound 3 with 3-O-α-L-arabinofuranyl has much more potent activity than compound 2 with 3-O-β-D-glucopyranosyl, and antocyanin (5) is more potent than its corresponding flavonol glycosides. Key words: Rubus rigidus, Antioxidant, Flavonoids, Saponins, Structure-activity-relationship

INTRODUCTION Organisms often produce free radicals from the cell respiratory system. Although some free radicals can serve as cell mediator in the regulation of physiological functions such as vascular tone, cell growth and apoptosis or in defence mechanism, the excessive Correspondence to: Hee-Juhn Park, Department of Pharmaceutical Engineering, Sangji University, Wonju 220-702, Korea Tel: 82-33-730-0564, Fax: 82-33-730-0564 E-mail: [email protected]

generation has harmful effects against many types of cells, because of the imbalance of pro-oxidant and antioxidant homeostasis in the body. Thereby, oxidative stress induces various diseases and further serves as a switch between diseases. Indeed, many diseases such as inflammation, rheumatism, cancer, arterial hypertension, diabetes, neurodegenerative diseases and even genetic mutation are caused or potentiated by the over production of free radicals. These harmful effects can be overcome by antioxidant enzymes, endogenous and exogenous antioxidants.

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There are two main types of antioxidants, namely, primary (chain breaking, free radical scavengers) and secondary or preventive. Secondary antioxidant mechanisms may include deactivation of metals, inhibition of breakdown of lipid hydroperoxides to unwanted volatile products, regeneration of primary antioxidants, singlet oxygen quenching, etc. (Gordon, 1990). Intake of antioxidant chemicals may help to prevent diseases associated with oxidative stress. Therefore, the development of naturally occurring antioxidant compounds is required rather than synthetic ones. Rubus rigidus var. camerunensis is a plant currently used in Cameroonian folk medicine for the treatment of respiratory and cardiovascular ailments which are highly associated to oxidative stress. It has been reported that the Rosaceae family and more precisely the genus Rubus is a reputed source of polyhydroxylated triterpenoids and dimeric triterpene glucosides (Ohtani et al., 1990; Wang et al., 2000). Moreover, they have been found to contain flavonoids and polyphenolic derivatives (Häkkinen et al., 1999; Hussein et al., 2003). Many flavonoids have exhibited a range of biological and pharmacological effects including antioxidant property and inhibition of free radical-mediated processes (Facino et al., 1990; Mora et al., 1990; Yen and Chen, 1995; Cao et al., 1997; Cos et al., 1998). Recently, saponins have also been shown to possess potent antioxidant properties (Fu et al., 2009; Gupta et al., 2009; Kim et al., 2009; Nzowa et al., 2010; Tung et al., 2010). So, plants containing flavonoids and/or saponins may be potential source of new antioxidant with less pro-oxidant effects. Hence, the present work was undertaken to evaluate the antioxidant activities of aqueous and ethanol extracts from the aerial part of Rubus rigidus var. camerunensis and to further isolate through bioactivity-guided fractionation, the active principle responsible for this activity. Since this plant has not been previously subjected to phytochemical studies, its secondary metabolites are herein evaluated for their antioxidant activity for the first time.

MATERIALS AND METHODS Reagents 1,1-diphenyl-2-picrylhydrazyl (DPPH), β-carotène, linoleic acid and tween 80 were all purchased from Sigma-Aldrich Chemie Gmbh. Chloroform, ethanol and methanol were obtained from Ader.

T. B. Nguelefack et al.

Plant material, extraction and bioactivityguided isolation of pure compounds The fresh aerial parts of Rubus rigidus were collected in Dschang (West Cameroon) in March 2008 and identified by Dr Achoundong Gaston at the National Herbarium of Cameroon; Yaoundé, Cameroon by comparison to an existing voucher specimen (N° 23928/SRF/CAM). One hundred grams of the air-dried powdered were macerated in distilled water for 48 h. The mixture was filtered and evaporated in an oven at 40°C to obtain 31 g (yield: 31%) of a brown powder, representing the aqueous extract of the aerial part of Rubus rigidus. The obtained extract was kept at 4°C until use. Concerning the ethanol extraction, 3 kg of the dried and ground aerial parts of this plant were extracted thrice with ethanol (96%) at room temperature for 24 h. Evaporation of solvent from this extract in vacuo gave 190 g of a green syrupy gum which was suspended in water and successively extracted with EtOAc and n-BuOH. The n-BuOH soluble fraction was evaporated to dryness to give a brown gum (55 g) of which 40 g was subjected to sephadex column chromatography using methanol to afford three main fractions A-C. Fraction A and B were essentially consisted of phenolic and polyphenolic compounds respectively, while fraction C constituents were essentially triterpene saponins and residual triterpenes. Fraction A (5.0 g) was rechromatographed on silica gel column (70-200 µm) initially eluted with CHCl3MeOH-H2O (75:25:10) to afford compounds 1 (36 mg), 2 (28 mg), 3 (22 mg) and 4 (32 mg). The sub-fraction eluted with CHCl3-MeOH-H2O (63:37:10) (1.6 g) was submitted to a sephadex column chromatography using methanol to mainly afford compound 5 (22 mg) (Fig. 1). Fraction C (8.0 g) was submitted to silica gel column chromatography (70-200 µm) eluted with CHCl3 containing increasing amounts of MeOH to give four subfractions (I – IV). Compound 6 (350 mg) was precipitated in acetone from sub-fraction II eluted with CHCl3-MeOH (80:20). Sub-fraction III (1.3 g) eluted with CHCl3-MeOH (70:30) was further rechromatographed on silica gel column (70-200 µm) with CHCl3MeOH-H2O (75:25:10) to afford compound 7 (32 mg). Sub-fraction IV (1.0 g) eluted with CHCl3-MeOH (60:40) was purified by silica gel column chromatography with CHCl3-MeOH-H2O (63:37:10) to give compound 8 (18 mg).

Antioxidant Flavonoids and Saponins from Rubus rigidus

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Fig. 1. Chemical structures of flavonoids (1 - 5) and saponins (6 - 8) isolated from the aerial part of Rubus ridigus var. camerunensis.

Kaempferol 3-O-(2″-E-p-coumaroylglucopyranoside) (1) Kaempferol 3-O-(2′′-E-p-coumaroylglucopyranoside) (1) was obtained as a yellow amorphous powder. 1HNMR (500 MHz, CD3OD, δ ppm) 8.06 (2H, d, J = 8.6 Hz, H-2′, H-6′), 7.45 (1H, d, J = 15.2 Hz, H-3′′′), 7.35 (2H, d, J = 8.4 Hz, H-5′′′, H-9′′′), 6.94 (2H, d, J = 8.6 Hz, H-3′, H-5′), 6.92 (2H, d, J = 8.6 Hz, H-6′′′, H-8′′′), 6.90 (1H, d, J = 2.3 Hz, H-8), 6.82 (1H, d, J = 2.3 Hz, H-6), 6.31 (1H, d, J = 15.2 Hz, H-2′′′), 5.27 (1H, d, J = 7.8 Hz, H-1′′); 13C-NMR (125 MHz, CD3OD, δ ppm), 179.5 (C-4), 169.2 (C-1′′′), 166.4 (C-7), 163.5 (C-5), 162.0 (C-4′), 161.5 (C-7′′′), 158.9 (C-9), 159.9 (C-2), 147.0 (C-3′′′), 135.7 (C-3), 132.7 (C-2′ and C-6′), 131.6 (C-5′′′ and C-9′′′), 127.6 (C-4′′′), 123.2 (C-1′), 117.3 (C3′ and C-5′), 116.7 (C-6′′′ and C-8′′′), 106.1 (C-10), 104.4 (C-1′′), 100.5 (C-6), 95.3 (C-8), 78.5 (C-2′′), 76.4 (C-3′′), 76.2 (C-5′′), 72.2 (C-4′′), 64.7 (C-6′′). Kaempferol 3-O-β-D-glucopyranoside (astragalin, 2) Kaempferol 3-O-β-D-glucopyranoside (astragalin, 2)

was obtained as yellow powder (m.p. 174-176°C). 1HNMR (500 MHz, CD3OD, δ ppm) 8.08 (2H, d, J = 8.6 Hz, H-2′, H-6′), 6.94 (2H, d, J = 8.6 Hz, H-3′, H-5′), 6.91 (1H, d, J = 2.2 Hz, H-8), 6.80 (1H, d, J = 2.2 Hz, H-6), 5.26 (1H, d, J = 7.8 Hz, H-1′′); 13C-NMR (125 MHz, CD3OD, δ ppm), 179.0 (C-4), 164.9 (C-7), 162.1 (C-5), 160.5 (C-4′), 157.5 (C-9), 156.2 (C-2), 134.5 (C-3), 131.3 (C-2′ and C-6′), 122.8 (C-1′), 115.1 (C-3′ and C5′), 108.5 (C-10), 104.5 (C-1′′), 98.9 (C-6), 93.8 (C-8), 77.7 (C-3′′), 77.4 (C-5′′), 74.5 (C-2′′), 70.6 (C-4′′), 61.7 (C-6′′).

Kaempferol-3-O-α-L-arabinofuranoside (Juglanin, 3) Kaempferol-3-O-α-L-arabinofuranoside (Juglanin, 3) was obtained as a yellowish white powder. 1H-NMR (500 MHz, CD3OD, δ ppm) 7.88 (2H, d, J = 8.6 Hz, H2′, H-6′), 6.96 (2H, d, J = 8.6 Hz, H-3′, H-5′), 6.91 (1H, d, J = 2.4 Hz, H-8), 6.83 (1H, d, J = 2.4 Hz, H-6), 5.50 (1H, s, H-1′′); 13C-NMR (125 MHz, CD3OD, δ ppm), 178.7 (C-4), 164.9 (C-7), 162.3 (C-5), 160.5 (C-4′), 157.3 (C-9), 155.9 (C-2), 134.5 (C-3), 130.8 (C-2′ and C-6′),

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122.5 (C-1′), 116.1 (C-3′ and C-5′), 108.5 (C-10), 108.2 (C-1′′), 98.6 (C-6), 94.2 (C-8), 87.1 (C-4′′), 82.4 (C-2′′), 74.8 (C-3′′), 63.7 (C-5′′).

Quercetin-3-O-β-D-glucopyranoside (Isoquercitrin, 4) Quercetin-3-O-β-D-glucopyranoside (Isoquercitrin, 4) was obtained as yellow needles crystals (m.p. 210213°C). 1H-NMR (500 MHz, CD3OD, δ ppm) 7.73 (1H, d, J = 2.6 Hz, H-2’), 7.60 (1H, dd, J = 2.6, 8.4 Hz, H6′), 6.90 (1H, d, J = 8.4 Hz, H-5′), 6.42 (1H, d, J = 2.1 Hz, H-8), 6.23 (1H, d, J = 2.1 Hz, H-6), 5.24 (1H, d, J = 7.4 Hz, H-1′′); 13C-NMR (125 MHz, CD3OD, δ ppm), 178.1 (C-4), 164.6 (C-7), 161.6 (C-5), 157.5 (C-9), 157.1 (C-2), 148.4 (C-4′), 144.5 (C-3′), 134.2 (C-3), 121.5 (C1′), 121.8 (C-6′), 116.2 (C-5′), 114.6 (C-2′), 104.3 (C-10), 102.9 (C-1′′), 98.5 (C-6), 93.3 (C-8), 76.9 (C-3′′), 76.7 (C-5′′), 74.3 (C-2′′), 69.8 (C-4′′), 61.2 (C-6′′). Pelargonidin-3-O-β-D-glucopyranoside (Callistephin, 5) Pelargonidin-3-O-β-D-glucopyranoside (Callistephin, 5) was obtained as an amorphous white powder. 1HNMR (500 MHz, CD3OD, δ ppm) 8.11 (2H, d, J = 9.0 Hz, H-2′, H-6′), 6.90 (2H, d, J = 9.0 Hz, H-3′, H-5′), 6.43 (1H, d, J = 2.3 Hz, H-8), 6.23 (1H, d, J = 2.3 Hz H-6), 5.35 (1H, d, J =7.5 Hz, H-1′′), 4.78 (1H, s, H-4); 13 C-NMR (125 MHz, CD3OD, δ ppm) 172.7 (C-7), 165.2 (C-2), 163.5 (C-4′), 161.9 (C-5), 136.1 (C-3), 134.5 (C-3), 132.9 (C-2′ and C-6′), 123.2 (C-4), 116.6 (C-3′ and C5′), 115.7 (C-10), 104.5 (C-1′′), 100.3 (C-6), 95.2 (C-8), 78.5 (C-3′′), 77.7 (C-5′′), 76.1 (C-2′′), 73.9 (C-4′′), 62.5 (C-6′). 2α,3β,19α,23-tetrahydroxyurs-12-ene-28-O-β-Dglucopyranosyl ester (niga-ichigoside F1, 6) 2α,3β,19α,23-tetrahydroxyurs-12-ene-28-O-β-D-glucopyranosyl ester (niga-ichigoside F1, 6) was obtained as a white powder. 1H-NMR (500 MHz, C5D5N, δ ppm) 6.20 (1H, d, J = 7.6 Hz, H-1′), 5.55 (1H, t-like, H-12), 4.75 (1H, d, J = 9.2 Hz, H-3), 4.25 (1H, m, H-2), 4.403.80 (6H, glucose), 2.85 (1H, s, H-18), 1.60 (3H, s, CH327), 1.42 (3H, s, CH3-24), 1.30 (3H, s, CH3-29), 1.25 (3H, s, CH3-26), 1.07 (3H, s, CH3-25), 1.01 (3H, d, J = 6.8 Hz, CH3-30); 13C-NMR (125 MHz, C5D5N, δ ppm) 176.7 (C-28), 139.0 (C-13), 128.1 (C-12), 95.6 (C-1′), 78.9 (C-3), 78.7 (C-5′), 78.1 (C-3′), 73.8 (C-2′), 72.4 (C19), 71.0 (C-4′), 68.6 (C-2), 66.4 (C-23), 62.1 (C-6′), 54.1 (C-18), 48.3 (C-17), 47.8 (C-5), 47.7 (C-1), 47.6 (C-9), 43.3 (C-4), 41.9 (C-14), 41.8 (C-20), 40.4 (C-8), 38.1 (C10), 37.4 (C-22), 32.9 (C-7), 28.9 (C-15), 26.8 (C-29), 26.5 (C-16), 26.3 (C-21), 24.6 (C-11), 24.2 (C-27), 18.5 (C-6), 17.3 (C-25), 17.2 (C-26), 16.4 (C-30), 14.0 (C-24).


2α,3β,19α-trihydroxyurs-12-ene-23-carboxyl-28O-β-D-glucopyranosyl ester (suavissimoside R1, 7) 2α,3β,19α-trihydroxyurs-12-ene-23-carboxyl-28-O-β-Dglucopyranosyl ester (suavissimoside R1, 7) was obtained as a white amorphous powder. 1H-NMR (500 MHz, C5D5N, δ ppm) 6.26 (1H, d, J = 7.8 Hz, H-1′), 5.25 (1H, t-like, H-12), 4.60 (1H, d, J = 9.5 Hz, H-3), 4.30 (1H, m, H-2), 2.87 (1H, s, H-18), 1.75 (3H, s, CH327), 1.60 (3H, s, CH3-24), 1.35 (3H, s, CH3-29), 1.18 (3H, s, CH3-26), 1.15 (3H, s, CH3-25), 1.10 (3H, d, J = 6.2 Hz, CH3-30); 13C-NMR (125 MHz, C5D5N, δ ppm) 180.5 (C-6), 176.6 (C-28), 139.0 (C-13), 127.9 (C-12), 95.6 (C-1′), 80.8 (C-3), 79.0 (C-5′), 78.9 (C-3′), 73.8 (C2′), 72.4 (C-19), 71.1 (C-4′), 68.4 (C-2), 62.2 (C-6′), 54,5 (C-4), 54.1 (C-18), 52.0 (C-5), 48.0 (C-1), 47.9 (C-9), 41.9 (C-14), 41.8 (C-20), 40.5 (C-8), 38.4 (C-10), 37.5 (C-22), 33.4 (C-7), 28.7 (C-15), 26.2 (C-29), 26.4 (C-16), 26.2 (C-21), 24.2(C-11), 24.2 (C-27), 21.5 (C-6), 17.3 (C25), 17.1 (C-26), 16.4 (C-30), 13.1 (C-24). Coreanoside F1 (8) Coreanoside F1 (8) was obtained as a white amorphous powder. 1H-NMR (500 MHz, C5D5N, δ ppm) 6.52 (1H, br s, H-3′), 6.18 (1H, d, J = 7.4 Hz, GlcI-H-1), 6.16 (1H, d, J = 7.6 Hz, GlcII-H-1′), 5.55 (1H, t-like, H-12), 5.54 (1H, t-like, H-12′), 5.10 (1H, br d, J = 10.5 Hz, H-2′), 4.70 (1H, d, J = 10.2 Hz, H-23′a), 4.25 (1H, d, J = 9.5 Hz, H-3), 4.15 (1H, ddd, J = 4.0, 9.5, 10.2 Hz, H-2), 4.00 (1H, d, J = 10.2 Hz, H-23′b), 2.89 (1H, s, H-18), 2.88 (1H, s, H-18′), 1.75-1.11 (9 singlets: CH3-29, CH3-29′, CH3-27, CH3-27′, CH3-26, CH3-26′, CH3-25, CH3-25′, CH3-24), 1.10 (3H, d, J = 6.0 Hz, CH3-30), 1.06 (3H, d, J = 6.6 Hz, CH3-30′); 13CNMR (125MHz, C5D5N, δ ppm) 178.0 (C-23), 176.6 (C28 and C-28′), 139.0 (C-13), 138.9 (C-13′), 128.4 (C-12), 128.0 (C-12′), 81.8 (C-3), 73.8 (C-3′), 72.7 (C-19 and C19′), 67.9 (C-2), 65.8 (C-2′), 65.5 (C-23′), 55.6 (C-4′), 54.5 (C-4), 54.2 (C-18′), 54.0 (C-18), 48.0 (C-1), 44.4 (C1′); Glc-I: 95.5 (C-1), 78.9 (C-5), 78.6 (C-3), 73.7 (C-2), 71.2 (C-4), 62.2 (C-6), Glc-II: 95.6 (C-1′), 78.9 (C-5′), 78.6 (C-3′), 73.6 (C-2′), 71.3 (C-4′), 62.2 (C-6′). Free radical-scavenging activity The free radical scavenging activity of aqueous and ethanolic extracts from the aerial part of Rubus rigidus, EtOAc and n-BuOH fractions from the ethanol extract, phenolic and saponins fractions from the n-BuOH fraction as well as that of the 5 phenolic compounds and the 3 saponins was measured by the method of Blois (1958) using 1,1-diphenyl-2-picrylhydrazyl. Briefly, 1.75 mL of methanol was mixed with 250 µL of methanol (control), ascorbic acid or test


substances prepared in methanol and the absorbance was measured at 517 nm. Then, one milliliter of 0.1 mM solution of DPPH freshly prepared in methanol was added to the mixture for a final concentration of 1, 3, 10, 30, 100 or 300 µg/mL for the tested substance. The mixture was incubated at room temperature for 20 min in dark and absorbance was measured at 517 nm in comparison to the solution made of methanol and DPPH. Lower absorbance of the reaction mixture indicates higher free radical scavenging activity. The test was done in triplicate. The inhibition percentage (%) of radical scavenging was calculated using the following equation: Inhibition (%) = [(A0 − A1) × 100 / A0] Where, A0 is the mean of absorbance in control tubes and A1 is the difference in absorbance of the sample (absorbance in presence of DPPH – absorbance in absence of DPPH) at 517 nm. From the inhibition (%), the concentration of samples (µg/mL) reducing the absorbance by 50% was determined (EC50) using GraphPad Prism 4.0.

β-carotene-linoleic acid assay In this assay, antioxidant capacity is determined by measuring the ability of tested compound to inhibit the linoleic acid oxidation of β-carotene. 3 mg of βcarotene were dissolved in 15 mL of chloroform, 75 µL of linoleic acid and 600 mg of Tween 80 was added. Chloroform was completely evaporated using a vacuum evaporator. Then 150 mL of aerated distilled water was added with vigorous shaking. 1.34 mL of this emulsion was introduced in the test tube with 160 µL of tested substances (ascorbic acid, aqueous and ethanol extracts and isolated compounds) at the final concentration ranging from 1 to 300 µg/mL. The mixture absorbance was measured at 490 nm immediately and after 105 min of incubation at 55°C. The experiment was conducted in triplicate. The antioxidant activity (AOA) was calculated using the following formula: AOA = [(At105 − Ac105) × 1000 / (Ac0 − Ac105)] Where, At105 is the absorbance in test tubes after 105 min of incubation, Ac105 the absorbance in control tubes after 105 min of incubation and Ac0 the absorbance in control tubes before incubation (Koleva et al., 2002).

RESULTS AND DISCUSSION By comparison of spectral data obtained by mainly 500 MHz 1D- and 2D-NMR techniques (1H, 13C, DEPT,

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COSY, HMBC, HMQC and NOESY) with literature data, compounds 1-8 was identified: kaempferol-3-O(2′′-E-p-coumaroylglucopyranoside) (1) (Liu et al., 1999), kaempferol-3-O-β-D-glucopyranoside (astragalin, 2) (Singab et al., 2005), kaempferol-3-O-α-L-arabinofuranoside (juglanin, 3) (Olszewska, 2005), quercetin-3-O-β-D-glucopyranoside (isoquercitrin, 4) (Singab et al., 2005), pelargonidin-3-O-β-D-glucopyranoside (callistephin, 5) (Kuskoski et al., 2003), 2α,3β,19α, 23-tetrahydroxyurs-12-ene-28-O-β-D-glucopyranosyl ester (niga-ichigoside F1, 6) (Seto et al., 1984), 2α,3β,19α -trihydroxyurs-12-ene-23-carboxyl-28-O-β-D-glucopyranosyl ester (suavissimoside R1, 7) (Gao et al., 1985) and coreanoside F1 (8) (Ohtani et al., 1990) (Fig. 1). The aqueous and ethanolic extracts from R. rigidus exhibited potent antioxidant effect on the DPPH assay with a respective EC50 of 9.71 and 0.38 µg/mL and efficiency index (EI) of 0.104 and 0.003. Based on their efficiency index (EC50/Emax), the ethanolic extract was selected as the most active crude extract and was even more potent than ascorbic acid whose EC50 and EI were 1.76 µg/mL and 0.018, respectively. As the ethanolic extract was the most active, it was partitioned to obtain EtOAc, n-BuOH and aqueous fractions. The fractions were assayed and showed free radical scavenging activity with respective EI of 0.09, 0.12 and 0.6. The n-butanol n-BuOH fraction was further separated in phenolic and saponin fractions. The separation of both fraction afforded five flavonoids and three saponins. All of them except compounds 6 and 7 exhibited potent antioxidant activity (Table I). The DPPH test is a non-enzymatic method currently used to provide information on the capacity of compounds to scavenge free radicals (Braca et al., 2003). The compounds which are active in this assay are able to donate hydrogen to the stable DPPH free radical. Therefore, the present results suggest that aqueous and ethanolic extracts as well as flavonoids and compound 8 possess significant hydrogen donating effects. The antioxidant activities of the flavonoids were more potent than the triterpenoid glycoside. Based on the EC50 and the EI data, an anthocyanin callistephin (5) exhibited the highest antioxidant activity. This compound differed from the other phenolic compounds by the absence of ketone group at C-4 in C-ring. This indicates that anthocyanins can be free radical-scavengers than flavonol glycosides. In general, chelating ability of both the ketone group and 5-OH of flavonols enhances the reducing power. In addition, the more the hydroxyl group on the flavonoid skeleton has, the stronger antioxidant activity is (Traykova and Kostova, 2005). By the present experimental results, the anthocyanin (5) with oxonium

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Table I. EC50, Emax and Efficiency Index (EI: EC50/Emax) of the scavenging effects of substances tested on a DPPH stable free radical Substances Ascorbic acid Aqueous extract Ethanolic extract Ethyl acetate fraction n-butanol fraction Aqueous residue Phenolic compounds fraction Saponins fraction 1 2 3 4 5 6 7 8

EC50 (µg/mL)

Emax

EI (EC50/Emax)

1.76 9.71 0.38 8.19 10.94 45.13

95.77 93.65 112.67 91.63 94.03 76.31

0.018 0.104 0.003 0.089 0.116 0.596

2.03

94.42

0.021

15.05 4.84 9.11 0.16 0.69 0.11 68.15 7907.00 0.001

93.97 94.81 88.20 92.22 85.12 86.5 41.02 38.92 63.49

0.160 0.051 0.103 0.002 0.008 0.001 1.661 203.160 0.001

ion made us find the stronger antioxidant ability than the flavonol glycosides (1-4) with ketone group. In addition, there was a big difference in the antioxidant ability between compounds 2 (kaempf. 3O-β-D-glcp) and 3 (kaempf. 3-O-α-L-araf), suggesting that the substitution of the glucosyl moiety by the arabinosyl moiety at C-3 render the compound more antioxidant. Concerning saponins activities, compounds 6 (niga-ichigoside F1) and 7 (suavissimoside R1) showed a very weak antioxidant effect (approximately 40% each). Compound 6 showed a prooxidant activity up to 10 µg/mL concentrations and became antioxidant at the higher concentrations, while compound 7 showed antioxidant effects at all the concentrations

used. Compound 6 is structurally different from 7: the former has 23-carbinol but the latter 23-carboxyl (Fig. 1). Compound 8 (a dimeric triterpenoid), which is a dimer of compound 7 with a supplementary 24hydroxyl, presented an Emax of 63% and was the most active saponin. Based on the EI values (Table I), compound 8 was proven to be as efficient as flavonoid (4, isoquercitrin) and ascorbic acid, thus suggesting that the dimerization of the triterpenoid glycoside may increase its antioxidant activity. It has been shown that the antioxidant activity of diterpenoids is due to the presence of the exocyclic methylene double bond in their skeleton (Dickson et al., 2007). It is therefore possible that the antioxidant activity of triterpenoids or triterpenoids saponins could be attributed to the double bond (C=O, C=C) present in their skeleton. Thus, compound 8 a dimer of triterpenoid saponin containing more double bonds than its monomer (compound 7) should therefore be more active. The β-carotene bleaching test is based on the loss of yellow color of β-carotene due to its reaction with radicals which are formed by linoleic acid oxidation in an emulsion. β-Carotene bleaching, measured by the decrease in the initial absorbance is slowed down in presence of antioxidants. As shown in Table II, almost all the extracts and isolated compounds of the aerial part of R. rigidus protected against the bleaching of βcarotene but at different ranges of concentration. The aqueous extract and compound 4 (quercetin 3-Glc) exhibited the most potent antioxidant activity in this test. The antioxidant activity in this test highly depends on the polarity of the studied molecule (Porter, 1993; Frankel et al., 1994). The less polar antioxidants are then more effective due to their solubility in emulsion and their capacity to concentrate in lipids. The high activity of aqueous extract on this model can indicate

Table II. Concentration-dependent antioxidant activity (%) of tested substances on β-carotene-linoleic acid assay Substances

Concentration (µg/mL) 1

3

10

Ascorbic acid −245.93 986.97 1252.50 Aqueous extract ND 84.74 515.50 −86.15 −93.22 Ethanolic extract ND 1 1.41 46.61 107.34 2 −50.84 11.29 19.77 3 56.50 24.01 128.53 4 −2.82 362.99 504.24 5 36.72 −100.28 −77.68 8 −199.15 −185.03 −183.61 ND, not determined; each value represents the mean of 3 repetitions.

30

100

300

203.58 851.69 46.60 125.71 110.17 257.06 593.20 33.89 −179.35

996.74 1022.60 472.64 525.40 412.43 311.66 733.05 272.60 −56.49

1574.90 1394.00 668.08 608.76 635.59 816.38 1173.70 603.11 73.44


a higher concentration of less polar compounds in this extract. Compound 5, which was the most active flavonoid in the DPPH test, was shown to be less efficient in the β-carotene-linoleic assay, may be due to its higher polarity. However, compound 4 which had an antioxidant activity in DPPH test and structurally quercetin, also exhibited a potent activity in this β-carotene model. This may probably be explained by the supplementary hydroxyl group in the quercetin. Compound 8, a diemric triterpenoid glycoside, was an antioxidant in DPPH test but a prooxidant in β-carotene-linoleic acid test probably due to its very high polarity. In conclusion, these results suggest that Rubus rigidus var. camerunensis extracts may contribute to the preventive effect against the diseases associated with oxidative stress (cardiovascular and respiratory ailments) as a Cameroonian medicinal drug. From this study, two distinct structure-activity relationship was established: even sugar moiety in the flavonoid glycosides like α-L-arabinofuranosyl moiety affects the antioxidant activity, and dimeric triterpenoid glycosides can strengthen the activity of its monomers.

ACKNOWLEDGEMENTS This research work was supported by the IFS (International Foundation for Science, Stockholm, Sweden) programme through grants to Dr T.B. NGUELEFACK (F/4576-1) and Prof. A. Léon TAPONDJOU (RGA No. F/3976-2) and the Korea Research Foundation Grant (MOEHRD) (KRF-2005041-E00487).

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