Leonardo Gobbo-Neto1 Michel D. Santos1 Alexandre Kanashiro1 Maria C. Almeida2 Yara M. Lucisano-Valim1 Joo L. C. Lopes1 Glria E. P.

Souza1 Norberto P. Lopes1

Evaluation of the Anti-Inflammatory and Antioxidant Activities of Di-C-glucosylflavones from Lychnophora ericoides (Asteraceae)
Original Paper

Abstract The Brazilian medicinal plant Lychnophora ericoides is commercially available as an analgesic and anti-inflammatory agent. The phytochemical investigation of the leaf polar extract yielded 6,8-di-C-b-glucosylapigenin (1) and the new compound 6,8-diC-b-glucosylchrysin (2). 6,8-Di-C-b-glucosylapigenin (1) showed significant anti-inflammatory activity in the carrageenan-induced rat paw edema. We did not observe any statistical differ-

ence between the two compounds (1 and 2) in inhibiting chemiluminescence in opsonized zymosan-stimulated polymorphonuclear leukocytes, suggesting that the anti-inflammatory property of 6,8-di-C-b-glucosylapigenin (1) is not related to its antioxidant activity. Key words Lychnophora ericoides Asteraceae anti-edematogenic activity C-glycosylflavones 6,8-di-C-b-glucosylchrysin vicenin-2

Introduction Species of Lychnophora (Asteraceae) are popularly known in Brazil as arnica, falsa arnica or arnica da serra. These endemic plants are used in folk medicine as analgesic and anti-inflammatory agents [1]. Among Lychnophora species, Lychnophora ericoides Mart (Asteraceae) is the most widely used and its intact leaves, root powder and hydroalcoholic extracts are commercially available as phytotherapeutic products. The apolar extracts of L. ericoides leaves and roots yielded sesquiterpene lactones (SLs), flavonoids and lignans [2], [3], [4]. Although some SLs isolated from Lychnophora and Eremanthus can modulate the inflammatory process in vitro by inhibiting the transcription factor NF-kB [5], [6], there is no in vivo evidence for this activity. Furthermore, the lignans showed analgesic but not anti-inflammatory or antipyretic in vivo activities [3]. Until now, there is insufficient information about the chemical composition of the polar extracts or their biological activity. The folk use of this plant and the lack

of chemical and biological data about the polar compounds from L. ericoides motivated us to perform a phytochemical investigation followed by an anti-inflammatory assay. Furthermore, we evaluated the radical scavenging activity in vitro by quantifying the inhibition of chemiluminescence in opsonized zymosan-stimulated polymorphonuclear leukocytes (PMNs) in order to relate (or not) the anti-inflammatory to the antioxidant activity.

Material and Methods General experimental procedures H-, 2D-NMR and 1D selective TOCSY spectra were recorded on a Bruker AMX-500 and 13C-NMR on a Bruker DRX-400, and chemical shifts (d) were referenced to the solvent signal. Accurate mass ESI-MS analyses were performed on a Bruker Daltonics Apex IV 7.5 Tesla FT-MS. Elemental analysis were recorded on a Perkin El1

Affiliation Department of Physics and Chemistry, Faculty of Pharmaceutical Sciences of Ribeiro Preto, USP, Ribeiro Preto, So Paulo, Brazil 2 Department of Physiology, Faculty of Medicine of Ribeiro Preto, USP, Ribeiro Preto, So Paulo, Brazil
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Correspondence Norberto P. Lopes Department of Physics and Chemistry Faculty of Pharmaceutical Sciences of Ribeiro Preto USP Av. do caf s/n CEP 14040903 Ribeiro Preto So Paulo Brazil Phone: +55-16-602-4168 Fax: +55-16-633-2960 E-mail: npelopes@fcfrp.usp.br. Received April 6, 2004 Accepted August 31, 2004 Bibliography Planta Med 2005; 71: 36  Georg Thieme Verlag KG Stuttgart New York DOI 10.1055/s-2005-837742 ISSN 0032-0943

mer CNH-2400 instrument. Preparative HPLC was performed on a Shimadzu LC-6A apparatus with a UV detector SPD-6AV. Edema was measured with a plethysmometer (Ugo Basile, Italy). Chemiluminescence was measured in an automatic luminometer (Autolumat LB953, EG&G Berthold, Wildbad, Germany). Plant material Leaves from L. ericoides were collected in Ibiraci, Minas Gerais, Brazil and were identified by Prof. Dr. Joo Semir, University of Campinas, where a voucher specimen was deposited (NPL-123; herbarium UEC). Isolation of constituents Fresh leaves were washed with CH2Cl2 for 10 min to remove the constituents in glandular trichomes (mostly SLs). The washed leaves were air-dried, powdered (370 g) and extracted with MeOH for 24 h at room temperature. The extract (30 g) was suspended in 600 mL of MeOH-H2O (8 : 2) and extracted with hexane (300 mL 3) followed by CH2Cl2 (300 mL 3) and yielded 4.5 g of hexane fraction, 7.0 g of CH2Cl2 fraction and 18.0 g of MeOH-H2O fraction. 4.0 g of the MeOH-H2O fraction were suspended in H2O (200 mL) and extracted with n-BuOH (60 mL 3), yielding 2.1 g of n-BuOH fraction and 1.9 g of aqueous fraction. The aqueous fraction was submitted to CC with Amberlite XAD2 (100 g, 4.5 cm 35 cm) consecutively eluted with H2O (1.5 L), MeOH-H2O (1.5 L) and MeOH (2.0 L), affording 0.93 g of H2O fraction (0 1.5 L), 0.35 g of MeOH-H2O fraction (1.5 3.0 L) and 0.31 g of MeOH fraction (3.0 5.0 L). The first sub-fraction (H2O) consisted basically of inorganic salts, sugars and free amino acids and after comparative analysis fractions 2 (MeOH-H2O) and 3 (MeOH) were pooled. Preparative HPLC (ShimPack ODS 5 mm Shimadzu, 250 20.0 mm column; MeOH/H2O linear elution gradient starting from 30 % MeOH up to 55 % MeOH in 25 min; l = 280 nm, flow 9.8 mL/min) of this new sub-fraction afforded 1 (60 mg) and 2 (33 mg). 6,8-Di-C-b-glucosylchrysin (6,8-di-b-glucopyranosyl-5,7-dihydroxy-2-phenyl-4H-1-benzopyran-4-one) (2) was obtained as an amorphous powder: 1H-NMR (500 MHz, DMSO-d6): d = 13.36 (1H, s, 5-OH), 8.18 (2H, br d, J = 8.0 Hz, H-2 and H6), 7.60 (3H, m, H-3, H-4 and H-5), 7.01 (1H, s, H-3), 4.83 (1H, d, J = 9.8 Hz, H-1), 4.78 (1H, d, J = 9.8 Hz, H-1), 3.85 (2H, t, J = 9.5 Hz, H-2 and H-2), 3.70 (2H, d, J = 12.0 Hz, H-6b and H-6b), 3.55 (1H, dd, J = 12.0, 8.0 Hz, H-6a), 3.48 (1H, dd, J = 12.0, 8.0 Hz, H-6a), 3.42 (1H, t, J = 9.0 Hz, H-4), 3.39 (1H, t, J = 9.0 Hz, H-4), 3.37 (1H, m, H-5), 3.32 (1H, t, J = 9.0 Hz, H3), 3.30 (1H, t, J = 9.0 Hz, H-3), 3.27 (1H, m, H-5); 13C-NMR (100 MHz, DMSO-d6): d = 182.8 (C, C-4), 163.8 (C, C-2), 161.7 (C, C-7), 158.9 (C, C-9), 155.6 (C, C-5), 132.5 (CH, C-4), 131.3 (C, C-1), 129.4 (CH, C-3 and CH, C-5), 127.3 (CH, C-2 and CH, C-6), 108.1 (C, C-6), 105.8 (C, C-8), 105.2 (CH, C-3), 104.4 (C, C-10), 82.2 (CH, C5), 81.3 (CH, C-5), 79.1 (CH, C-3), 78.2 (CH, C-3), 74.4 (CH, C1), 73.7 (CH, C-1), 72.3 (CH, C-2), 71.2 (CH, C-2), 70.8 (CH, C4), 69.5 (CH, C-4), 61.4 (CH2, C-6), 60.3 (CH2, C-6); HR-ESI-MS: m/z = 579.1711 [M H]+ (calcd. for C27H31O14+: 579.1708); anal. found: C 56.08 %, H 5.20 %; calcd for C27H30O14: C 56.06 %, H 5.23 %. Animals Male Wistar rats weighing 180 200 g were housed at 24  1 8C on a 12 : 12 h light dark cycle (lights on at 06 : 00 am), and with
Gobbo-Neto L et al. Evaluation of the Planta Med 2005; 71: 3 6

free access to food and tap water. Eight hours before the experiment only tap water was available to the animals. All experiments were conducted between 10 : 00 and 17: 00 h and were in accordance with the ethical guidelines of the International Association for the Study of Pain [7]. Induction and measurement of rat paw edema Rat paw edema was induced by intraplantar injection of 100 mL of carrageenan (Marine Colloids), 1 % in sterile saline, into the right hind paw. The contralateral paw received the same volume of sterile saline and was used as the control. Edema was measured at 1 h intervals up to 4 h after carrageenan injection. The results are expressed in milliliters as the difference between the right and left paws. Treatments and statistical analysis Animals were orally treated (p. o., 0.5 mL) with the methanolic extract, MeOH-H2O (100 mg/kg) or aqueous fraction from L. ericoides leaves (10, 100 and 200 mg/kg), 6,8-di-C-b-glucosylapigenin (1) or 6,8-di-C-b-glucosylchrysin (2) (15 and 90 mg/kg), all diluted in the vehicle (saline plus Cremophor RH40, BASF, 10 %). Control animals received indomethacin (5 mg/kg, Merck, Sharp & Dohme) diluted in Tris-HCl buffer, pH 8.2, or vehicle. All treatments were performed 60 min before sub-plantar injection of carrageenan. Values are reported as mean  SEM and were analyzed for statistical significance by one-way ANOVA followed by Dunnett's test using the SPSS statistical software. The level of significance was set at P < 0.05. Chemiluminescence assay PMNs were isolated from venous blood of New Zealand rabbits as described by Lucisano and Mantovani [8]. The radical scavenging activity of 1, 2 and rutin was evaluated in vitro by quantifying the inhibition of luminol (5-amino-2,3-dihydro1,4-phthalazinedione; Sigma, USA) enhanced chemiluminescence in opsonized zymosan-stimulated PMNs [9]. The control experiment was performed without adding the compounds to the chemiluminescence reaction. The integrated area of CLlum, i. e., the area under the time-course curve of CLlum (AUC) is reported as mean  SEM of 3 determinations and represents the total amount of ROS produced by the stimulated PMNs in 15 min at 37 8C. The flavonoid glycone rutin (PVP S.A.), a known natural anti-inflammatory compound was used as a reference drug [10]. The data were analyzed for statistical significance by one-way ANOVA followed by Tukey's test using the GraphPad Prism software version 3.0 for Windows. The level of significance was set at P < 0.05.

Original Paper
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Results and Discussion In a preliminary evaluation, the methanolic extract of L. ericoides leaves (previously washed with dichloromethane) showed a significant anti-edematogenic activity on the carrageenan-induced edema in the rat at a dose of 100 mg/kg (37 and 30 % of inhibition, at 3 and 4 h, respectively, when compared with the vehicle). The active extract was resuspended in MeOH-H2O and partitioned with hexane and CH2Cl2 to remove the apolar constituents. The MeOH-H2O fraction also showed an anti-edematogenic effect at the dose of 100 mg/kg, which inhibited the edema by 35 and 41 %

at 3 and 4 h, respectively. This fraction was partitioned between n-BuOH and H2O, and these two polar fractions were also assayed in the rat paw edema, but only the aqueous (aq) fraction showed anti-edematogenic activity at 100 and 200 mg/kg doses (Fig. 1). This active fraction was consecutively submitted to column chromatography with Amberlite XAD-2 and preparative HPLC, yielding 6,8-di-C-b-glucosylapigenin (1), commonly known as vicenin-2, and the new compound 6,8-di-C-b-glucosylchrysin (2) (Fig. 2). Compound 1 was identified by comparison with spectroscopic data published in the literature [11], [12], [13], [14], [15], [16], [17]. Compound 2 showed essentially the same spectroscopic data as 1, except for the signals concerning the flavone B ring which, in this compound, are characteristic of a mono-substituted ring. The HMQC experiment carried out on 2 showed correlations between the two anomeric protons (d = 4.83 and 4.78) and two carbon signals at d = 74.4 and 73.7, respectively. From these data the C-bonds between sugars and the aglycone could be deduced. The HMBC spectrum of 2 showed a long-range coupling between the anomeric proton at d = 4.83 and the C-5 at d = 155.6 and C-7 at d = 161.7 while the signal at d = 4.78 was long-range coupled with the C-7 and C-9 at d = 158.9. Accurate ESI-MS analysis and the elemental analysis are in agreement with chrysin di-C-hexosyl structure. In 1D selective TOCSY experiments, the sugar proton signals showed the characteristic all-trans coupling pattern (between 9.8 and 8.0 Hz) of glucose, providing definitive proof for the relative stereochemistry of the sugars.

Compounds 1 and 2 were then assessed in the carrageenan-induced edema in the rat paw. As can be seen in Fig. 3A, pre-treatment of rats with 1 at doses of 15 and 90 mg/kg significantly reduced the paw edema induced by carrageenan. At doses of 15 mg/kg compound 1 inhibited the paw edema in both the 3rd and 4th hour while at the dose of 90 mg/kg, like indomethacin, the inhibition started at the second hour and remained for all the experimental period. In contrast, compound 2 at the same doses was ineffective in promoting any change in this experimental model of edema (Fig. 3B). Among the several mediators involved in the edematogenic response to carrageenan, the prostaglandins are the target for the anti-edematogenic activity of the non-steroidal and steroidal anti-inflammatory drugs in rats [18]. The involvement of prostaglandins in rat paw edema is reinforced here by the robust inhibition of the paw edema by indomethacin. Since prostaglandin synthesis depends on the breakdown of arachidonic acid by cyclooxygenases-1 and -2, both involved in the mechanism of the anti-inflammatory effect of indomethacin, we cannot rule out the possibility that 1 also acts by inhibiting the synthesis of these eicosanoids. On the other hand, according to Abeysekera et al. [19], compounds that can effectively inhibit the oxidative burst (high consumption of oxygen) of activated leukocytes may contribute to the anti-inflammatory activity. In order to assess the in vitro antioxidant properties of 1 and 2, we measured the radical scavenging activity by quantifying the inhibition of chemiluminescence in opsonized zymosan-stimulated PMNs [9]. The production of reactive oxygen species (ROS) by PMNs plays a key role in the defense system [20]. PMNs are stimulated through the activation of a membrane-associated NADPH oxidase which converts molecular oxygen into the superoxide anion (O2), resulting in the formation of other ROS. The ROS generation can be measured with

Original Paper

Fig. 1 Anti-inflammatory effects of the aqueous fraction from Lychnophora ericoides leaves on carrageenan-induced rat paw edema. Animals received the fraction at 10 (~), 100 (!) or 200 (l) mg/kg; control animals received indomethacin (*, 5 mg/ kg) or vehicle (n, saline plus Cremophor RH40 10 %) p. o., 0.5 mL, 60 minutes before sub-plantar injection of 100 l of carrageenan 1 %. The values represent the mean  SEM of the variation in the paw volume of 6 10 animals for each group. * P < 0.05, indomethacin 5 mg/kg and fraction 200 mg/kg versus vehicle; # P < 0.05, fraction 100 mg/kg versus vehicle. Fig. 2 Chemical structures of 6,8-diC-b-glucosylapigenin (1) and 6,8-di-C-bglucosylchrysin (2).

Fig. 3 Anti-inflammatory effects of 6,8-di-C-b-glucosylapigenin (1, A) and 6,8-di-C-b-glucosylchrysin (2, B) on carrageenan-induced rat paw edema. Animals received either 1 or 2 at 15 (~) and 90 (l) mg/kg; control animals received indomethacin (*, 5 mg/kg) or vehicle (n, saline plus Cremophor RH40 10 %) p. o., 0.5 mL, 60 minutes before subplantar injection of 100 l of carrageenan 1 %. The values represent the mean  SEM of the variation in the paw volume of 6 10 animals for each group. * P < 0.05, indomethacin 5 mg/kg versus vehicle; # P < 0.05, 1 at 15 and 90 mg/kg versus vehicle.
Gobbo-Neto L et al. Evaluation of the Planta Med 2005; 71: 3 6

luminol, a chemiluminescent probe used to assess the production of total ROS, in particular those generated by the myeloperoxidase system [9]. In the present study, we found that 1 and 2, in comparison with the negative control (no drug added), inhibited the luminol-enhanced chemiluminescence (CLlum) in opsonized zymosan-stimulated PMNs in a concentration-dependent manner, and these activities are very similar to that shown by rutin. Furthermore, the data obtained show that there is no statistical difference between the activities of the two compounds (1 and 2, Fig. 4). Similarly, the literature reports that the flavonoids apigenin and chrysin present similar activities in the Trolox equivalent antioxidant activity [21]. Structurally, apigenin and chrysin represent the aglycone moiety of 1 and 2, respectively.

References
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Original Paper
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In conclusion, the phytochemical investigation afforded two Cglucosylflavones as the major constituents of the L. ericoides polar leaves fraction. The 6,8-di-C-b-glucosylapigenin (1) showed anti-inflammatory activity in inhibiting the edema. On the other hand, the new flavonoid 6,8-di-C-b-glucosylchrysin (2) was inactive in the same assay. When it comes to the anti-oxidant assays, both compounds markedly inhibited the CLlum when compared to the reference compound rutin and no significant statistical differences between the two flavonoids were observed in this assay. This indicates that the anti-inflammatory action shown by 1 cannot be related to its anti-oxidant activity, since 2 has no effect in reducing the edema but has similar antioxidant activity compared to 1. Finally, the similar anti-inflammatory activity observed for the extract and 1 indicate the possible presence of other anti-inflammatory compounds in lower concentration and helps to explain the popular use of the plant.
Fig. 4 Concentration-dependent inhibitory effects of 6,8di-C-b-glucosylapigenin (1, crosshatched bars), 6,8-di-C-b-glucosylchrysin (2, open bars) and reference drug rutin (dotted bars) in opsonized zymosan-induced chemiluminescence in isolated rabbit PMNs. AUC, integrated area of CLlum reported as mean  SEM of 3 determinations in 15 min at 37 8C; C, control value (closed bar); * P < 0.05 versus control.

Acknowledgements This work was supported by FAPESP and CNPq. The authors acknowledge Prof. Dr. Joo Semir (Departamento de Morfologia e Sistemtica Vegetal, Instituto de Biologia da Universidade Estadual de Campinas) for the plant identification. Thanks are also due to Mrs. M.C.C. Melo, Mrs. J.A. Vercesi and Mr. R.C. Penatti for technical assistance and to Dr. Paul J. Gates (School of Chemistry, Bristol University) for performing accurate mass analysis and for the English language revision. Thanks are also given to BASF (Brazil) for Cremophor RH40 and to Merck, Sharp & Dohme (Brazil) for indomethacin. The authors also thank the University of Bristol, proteomics facility for access to the FT-MS.
Gobbo-Neto L et al. Evaluation of the Planta Med 2005; 71: 3 6