Evaluation of anti-inflammatory activity of derivatives from aerial parts of Baccharis uncinella

Abstract

Context: Species of Baccharis exhibit antibiotic, antiseptic, and wound-healing properties, and have been used in the traditional medicine of South America for the treatment of inflammation, headaches, diabetes, and hepatobiliary disorders.

Objective: To investigate the anti-inflammatory activity of organic phases from EtOH extract of the aerial parts of Baccharis uncinella DC (Asteraceae).

Materials and methods: The crude EtOH extract from the aerial parts of B. uncinella was subjected to partition procedures and the corresponding CH₂Cl₂ and EtOAc phases were subjected to several chromatographic separation procedures. Thus, these phases and their purified compounds were assayed for evaluation of anti-inflammatory activity.

Results: The CH₂Cl₂ phase from EtOH extract from B. uncinella contained two triterpenoids (oleanolic and ursolic acids) and one flavonoid (pectolinaringenin), whereas the respective EtOAc phase showed to be composed mainly by two phenylpropanoid derivatives (caffeic and ferulic acids). The CH₂Cl₂ and EtOAc phases as well as their isolated compounds exhibited anti-inflammatory effects against inflammatory reactions induced by phospholipase A2 (from Crotalus durissus terrificus venom) and by carrageenan.

Discussion and conclusion: The results suggested that the components obtained from partition phases of EtOH extract of B. uncinella could represent lead molecules for the development of anti-inflammatory agents. Additionally, the results confirmed the use of Baccharis genus in the traditional medicine of South America for the treatment of inflammation and other heath disorders. To date, the present work describes for the first time the anti-inflammatory effects of compounds isolated from B. uncinella.

Keywords::

• Secretory phospholipase A2 (sPLA2)
• anti-inflammatory
• triterpenes
• phenolic compounds

Introduction

Genus Baccharis (Asteraceae) comprises some 400 species, 120 of which occur in the southern and south-eastern regions of Brazil (Barroso, 1976; Nesom, 1990; Hind, 1993; Giuliano, 2001). Several species exhibit antibiotic, antiseptic, and wound-healing properties (Feresin et al., 2001) and are widely used in South American folk medicine to treat inflammation, headaches, diabetes, and hepatobiliary disorders (Gené et al., 1996; He et al., 1996; Verdi et al., 2005). Phytochemically, it is reported that Baccharis spp. are composed basically by diterpenoids, triterpenoids, flavonoids, and chromenes (Bohlmann et al., 1979; Labbe et al., 1986; Zdero et al., 1988; Jarvis et al., 1991; Verdi et al., 2005), several of which show biological activity. Based on a systematic study aiming the characterization of bioactive compounds from Brazilian flora (Grecco et al., 2010; Lago et al., 2010), the present work was undertaken to determine the activities of CH₂Cl₂ and EtOAc partition phases as well as their purified derivatives obtained from EtOH extract of aerial parts from Baccharis uncinella DC against inflammation induced by the proinflammatory phospholipase A2 (PLA2) and by carrageenan.

Materials and methods

General

NMR spectra (200 MHz) were recorded on a Bruker (Rheinstetten, Germany) model AC-200 spectrometer with chloroform-d (CDCl₃), dimethyl sulfoxide (DMSO-d₆), or methanol-d₄ (CD₃OD) as solvent and tetramethylsilane (TMS) as internal standard. Chemical shifts (δ) were recorded in parts per million (ppm) relative to TMS. LRESIMS were recorded in a VG Platform II–Micromass spectrometer (positive and negative modes). Silica gel (Merck, Darmstadt, Germany; 230–400 mesh) and Sephadex LH-20 (Amersham Biosciences, Piscataway, NJ) were used for column chromatographic separation, and silica gel 60 PF₂₅₄ (Merck) was used for analytical (0.25 mm) and preparative TLC (1.0 mm). Analytical HPLC were performed using a Dionex chromatograph model P680 with UV–vis detector (model UVD340U) and C₁₈ column (150 × 5 mm; 3 µm) with a flow rate of 0.6 mL/min using MeOH:H₂O 8:2 (pH 3, formic acid) as eluent. Analytical TLC was carried out on Merck silica gel 60 PF₂₅₄ plates (0.25-mm layer thickness).

Plant material

Aerial parts of B. uncinella were collected in the “Campos de Altitude” region, specifically in the city of Campos do Jordão, São Paulo, SP, Brazil in June 2005. The plant was authenticated by Dr. Oriana A. Fávero and voucher specimen deposited at the herbaria of the Prefeitura Municipal de São Paulo (PMSP) with the reference number PMSP8983.

Extraction and fractionation

The dried and powdered aerial parts of B. uncinella (402 g) were deffated with n-hexane (4 × 250 mL) at room temperature. Sequentially, the plant material was exhaustively extracted with EtOH (5 × 1 L) at room temperature, and the resulting solutions were filtered and concentrated under vacuum to afford the EtOH extract (13 g). This extract was dissolved in MeOH:H₂O (1:2 v/v; 500 mL) and organic phases were obtained after partitioning against CH₂Cl₂ (CH₂Cl₂-Bu: 16.4 g) and EtOAc (EtOAc-Bu: 5.2 g), respectively. Part of the CH₂Cl₂-Bu (8.3 g) was subjected to silica gel column chromatography eluted with increasing amount of EtOAc in n-hexane to give 77 fractions, which were pooled together in six groups (D1–D6) after TLC analysis. This procedure afforded 715 mg of a mixture composed by compounds 1 + 2 from group D4 and 134 mg of compound 3 from group D6. Part of the EtOAc-Bu (3.0 g) was subjected to Sephadex LH-20 column chromatography eluted with MeOH to give 45 fractions, which were pooled together in five groups (A1–A5) after TLC analysis. Compounds 4 (12 mg) and 5 (8 mg) were obtained in pure form after preparative TLC (silica gel, CH₂Cl₂:MeOH 8:2, twice) of groups A3 and A4, respectively. Additionally, the EtOAc-Bu phase was dissolved in EtOAc and treated with aqueous NaOH 10% until pH 12. After extraction of aqueous phase, this was acidified with HCl 10% until pH 2 and then extracted with CHCl₃ (3 × 100 mL). After concentration in vacuum, part of this organic phase (350 mg) was separated by preparative TLC (silica gel, CH₂Cl₂:MeOH 8:2, twice), affording compounds 4 (74 mg) and 5 (103 mg).

Animal experiments

Details of this project were submitted to and approved by the Ethical Committee on Animal Experiments of the Biology Institute of State University of Campinas (Certification No. 1320-1, August 22, 2007). All experiments involving animals were conducted in accordance with internationally accepted principles for laboratory animal use and care following the standards set by Centro Multidisciplinar para Investigação Biológica (CEMIB), a scientific member of the International Council for Laboratory Animal Science (ICLAS) and the program ICLAS Network for Promotion of Animal Quality in Research.

Measurement of PLA2 activity

Incubation of PLA2 with CH₂Cl₂-Bu and EtOAc-Bu phases

The activities of CH₂Cl₂-Bu and EtOAc-Bu phases against the induction of inflammation by highly purified PLA2 from rattlesnake Crotalus durissus terrificus (Toyama et al., 2003) were evaluated. Following the procedure described by Iglesias et al. (2005), samples of PLA2 were incubated with fractions CH₂Cl₂-Bu, EtOAc-Bu for 30 min at 37°C and subsequently re-purified to yield samples referred to as PLA2-CH₂Cl₂ (for PLA2 treated with CH₂Cl₂-Bu) and PLA2-EtOAc (for PLA2 treated with EtOAc-Bu).

Incubation of sPLA2 from the C. d. terrificus, bee venom PLA2, and pancreatic sPLA2 with compounds 1–5

The incubation of sPLA2 with fractions composed by compounds 1 + 2 as well as pure compounds 3–5 followed the procedures described by Iglesias et al. (2005). These compounds were dissolved in DMSO, whose concentration never exceeded 1% during incubation. Ten microliters of a solution at 100 nM to each compounds were added to 2000 μL of homogenized solution of purified sPLA2 from the snake or pancreatic (200 μM). The mixed solution was incubated for 60 min in a water bath at 37°C, before the enzymatic or other biological activity.

Measurement of sPLA2 activity

sPLA2 activity was measured following the protocols described by Toyama et al. (2003) for 96-well plate, using as substrate 4-nitro-3-octanoyloxy-benzoic acid (4N3OBA, BIOMOL, Pennsylvania, PA). Enzyme activity, expressed as the initial velocity of the reaction (V_o), was calculated based on the increase in absorbance after 20 min. All assays were performed using n = 12 and absorbances were measured using a SpectraMax 340 multiwell plate reader at 425 nm (Molecular Devices, Sunnyvale, CA). After the addition of native or treated sPLA2 (20 μg), the reaction mixture was incubated for up to 40 min at 37°C, the absorbency being read at 10 min intervals.

Determination of anti-inflammatory activity in vivo

Male Wistar rats (120–150 g) were anesthetized by halothane inhalation. Hind paw edema was induced by a single subplantar injection of treated or non-treated sPLA2 (10 μg per paw). Paw volume was measured immediately before administration of compounds (basal volume). Then the injections of EtOAc-Bu, CH₂Cl₂-Bu, and compounds 1–5 were administered by the intraperitoneal (i.p.) route and at selected time intervals thereafter (30, 60, 120, 180, and 240 min or 15, 30, 60, 90, and 180 min) using a hydroplethysmometer (model 7150; Ugo Basile, Italy) the footpad volume were measured. Results were expressed as the increasing in paw volume (mL) calculated by subtracting the basal volume. Alternatively, groups of rats (n = 4) were injected (i.p.) with 0.1 mL of fractions composed by compounds 1 + 2 and pure compounds 3–5 (3.0 mg/kg) 30 min before the injection of native sPLA2 (10 μg per paw). Dexamethasone and indomethacin were also used as comparing controls for evaluation of the anti-inflammatory effect of compounds 1–5.

Statistical analysis

Results were expressed as the means ± SEM. The significance of differences between means was assessed by an analysis of variance, followed by a Dunnett’s test when several experimental groups were compared with the control group. The confidence limit for significance was 5%.

Results

Phytochemical analysis

TLC, NMR, and HPLC/DAD analysis suggested the presence of non-glycosylated flavonoids and triterpenes from CH₂Cl₂ phase and phenolic derivatives from EtOAc phase, which were purified after several chromatographic steps. NMR and LRESIMS spectroscopic analysis allowed the identification of oleanolic (1) + ursolic (2) acids (Seebacher et al., 2003) and pectolinaringenin (3) (Hase et al., 1995) from CH₂Cl₂ phase as well as caffeic (4) and ferulic acids (5) (Nagatani et al., 2002) from EtOAc phase.

Measurement of PLA2 activity

This measurement was useful to analyze if compounds were able to inhibit the PLA2 activity of purified sPLA2. Figure 1 shows that compounds 1–5 were able to significantly decrease (P < 0.05) the enzymatic activity of bee PLA2 (PLA2 group III), rattlesnake PLA2 (group II PLA2), and pancreatic PLA2 (p PLA2). These results showed that the fraction composed by compounds 1 + 2 presented strong potential to abolish the phospholipase A2 activity and compound 3 presented low inhibition of enzymatic activity of sPLA2. Compounds 4 and 5 showed moderate ability to restrain the enzymatic activity of sPLA2.

Figure 1.  Inhibition of phospholipase A2 activity induced by compounds 1–5. Each of these compounds was previously incubated with each of sPLA2.

Anti-inflammatory activity

The crude EtOH extract from aerial parts of B. uncinella was subjected to partition procedures to afford CH₂Cl₂ and EtOAc phases (CH₂Cl₂-Bu and EtOAc-Bu). Each of these phases was undertaken to determine their activities against inflammation induced by the proinflammatory phospholipase A2 (PLA2) and by carrageenan. The characteristic edema induced in the hind paws of male Wistar rats by native PLA2 was strongly decreased after exposure to CH₂Cl₂-Bu (P < 0.05) (Figure 2A). In contrast, the edema produced by PLA2 that had been pre-treated with EtOAc-Bu was similar to that induced by the native enzyme. In corroboration of these results, the i.p. injection of CH₂Cl₂-Bu prior to the injection with native PLA2 inhibited the edema formation (P < 0.05) (Figure 2B). Otherwise, no significant differences were observed with respect to edemas induced by carrageenan in rats pre-injected with EtOAc-Bu in vivo assay compared with those formed in non-treated animals (Figure 2C).

Figure 2.  Inhibitory effects of the CH₂Cl₂ (CH₂Cl₂-Bu), and the EtOAc phases from B. uncinella (EtOAc-Bu) on edema induction by isolated secretory PLA2 from Crotalus durissus terricus or carrageenan. In panel A, the effects of prior incubation of PLA2 with CH₂Cl₂-Bu (PLA2-CH₂Cl₂; –▴–), or EtOAc-Bu (PLA2-EtOAc; –▾–) on edema induction by PLA2 are displayed (results with native PLA2 are shown by – ▪ –). In panel B, the inhibitory effects of prior intravenous injection of experimental animals with CH₂Cl₂-Bu (– • –), or EtOAc-Bu (–▾–) on edema induction by PLA2 are displayed (results with control animals are shown by – ▪ –). In panel C, the inhibitory effects of prior intravenous injection of experimental animals with CH₂Cl₂-Bu (–▴ –), or EtOAc-Bu (–▾–) on edema induction by 2% carrageenan are displayed (results with control animals are shown by – ▪ –). Each point represents the mean value ± SEM (n = 5); asterisks indicate significant differences in comparison with control (P < 0.05).

Additionally, the fractions composed by compounds 1 + 2 and 5 presented strong inhibitory effects on the edema development induced by native sPLA2 from C. d. terrificus during the experimental time (Figure 3A); moreover, compound 3 presented mild inhibitory effect on the edema evolution. Analyzing the effects against the edema induced by carrageenan (Figure 3B) was possible detect that compound 3 showed weak inhibitory effect while compound 4 did not present significant effect (data no shown). Otherwise, previous injections of the animals with compounds 1 + 2 and 5 strongly inhibited the edematogenic activity induced by carrageenan and these results were very close to that obtained by injection of indomethacin.

Figure 3.  (A) Effect of compounds 1–5 against edema induced by native sPLA2 from the Crotalus durissus terrificus. (B) Effect of previous incubation of compounds 1–5 against the edema induced by carrageenan. *P < 0.05 compared with the corresponding control group.

Discussion

In the present study, the CH₂Cl₂ and EtOAc partition phases from EtOH extract of B. uncinella were assayed with respect to their anti-inflammatory activities against PLA2 purified from rattlesnake venom. As shown in Figure 2, the CH₂Cl₂ phase (CH₂Cl₂-Bu) strongly inhibited the inflammation induced by PLA2 or carrageenan in comparison with EtOAc phase (EtOAc-Bu). After chromatographic steps, compounds 1–3 from CH₂Cl₂-Bu as well as compounds 4 and 5 from EtOAc-Bu were obtained. These compounds showed the potential to inhibit the activity of pharmacologically active sPLA2 isolated from C. d. terrificus by the fact that these compounds were able to inhibit the enzymatic activity of bee venom, pancreatic, and C. d. terrificus sPLA2. However, the results obtained from the previous incubation of C. d. terrificus sPLA2 by different compounds clearly showed that only two compounds effectively inhibited the pharmacologically active of sPLA2 in vivo. Thus, these results suggest an initial dissociation between catalytic activities of PLA2 with pharmacological activity.

Phospholipase A2 from snake venom type (PLA2) (EC 3.1.1.4) are quite fascinating, both biologically and structurally. Although its structure was being conserved, they exhibit a wide range of pharmacological activities, such as myonecrosis, edema, neurotoxicity, and anticoagulant or procoagulant activity. These activities seem to be dependent on the sPLA2 enzymatic activities suggesting that inhibition of enzyme activity could affect the pharmacological activity of these proteins (Toyama et al., 2009). Additionally, carrageenan-induced inflammation in the rat paw represents a classical model of edema formation and hyperanalgesia, and has been employed extensively in the development of nonsteroidal anti-inflammatory drugs and selective COX-2 inhibitors (Guay et al., 2004).

The prior injection of compounds 1 + 2 and 5 in experimental animals before the injection of carrageenan (Figure 3) clearly shows that some of these compounds could not only inhibit the activity of cytosolic PLA2, but also inhibit other pathways of inflammation cascade. Thus, it has been suggested that the fraction composed by compounds 1 + 2 and pure 5 showed a large spectrum of inhibition in enzymatic activity of PLA2 as well as other enzymes of the cascade of inflammation, similar to those detected in several oleanane/ursane triterpene derivatives (Vechia et al., 2009) as well as in caffeic acid (Horiuchi & Seyama, 2008).

Compound 3 did not show a potent inhibitory effect against enzymatic activity of sPLA2 groups II and III, and pancreatic phospholipase A2 (Figure 1), but it showed a significant potency against the edema using a carrageenan-induced inflammation model (data not shown). These results suggested that compound 3 has a preferential action against other enzymes from the inflammation cascade and not specifically against PLA2.

Moreover, the data obtained from the enzymatic activity and edema also suggest that some isolated compounds should interact with sPLA2 by different ways to lead a moderate or strong abolishment of enzymatic activities. A number of polyphenolic compounds have been found to be strong inhibitors of secretory PLA2 (Iglesias et al., 2005), although the flavonoid morin is reportedly able to induce significant changes in the secondary structure of PLA2 from Crotalus durissus cascavella and hence modify the activity of the enzyme. Hydroxycoumarin chemically react with Ser residues of sPLA2, which leads diminishing or abolishment of pharmacological activity of sPLA2 as well as its catalytic power (Toyama et al., 2009), pointed out to the strong effect in the inflammation pathway produced by compounds. The present results indicated that compounds can be important classes of anti-inflammatory molecules and they can be used to develop important medicines. Therefore, the results presented herein corroborated the folk use of this plant in inflammation disorders, showing that traditional knowledge can represent an important target to research and development of new drugs.

Acknowledgements

The authors are grateful to FAPESP, MackPesquisa, LIM-50 HC-FMUSP, and CNPq for financial support. The authors are also grateful to CNPq for fellowships to M.H. Toyama and J.H.G. Lago.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.