!"#$%&! '()" * --- "!"#$%&!. '()" * (/ +,, Full Length Research Paper Isolation and anti-inflammatory effects of maturin acetate from the roots of Psacalium peltatum (Asteraceae) Nadia A. Rojano-Vilchis1*, Manuel Jimenez-Estrada1, A. Nieto Camacho1, Armando Torres Avilez1, Robert. A. Bye2, Victor M. Chavez Avila2, M. Canales-Martinez3, K. S. MartinezElizalde3 and M. A. Rodriguez-Monroy3 1 Instituto de Química, Universidad Nacional Autónoma de México, UNAM, Coyoacán 04510, México D.F., México. Jardín Botánico, Instituto de Biología, Universidad Nacional Autónoma de México, UNAM, Coyoacán 04510, México D.F., México. 3 UBIPRO, F.E.S. IZTACALA, Universidad Nacional Autónoma de México, UNAM., Coyoacán 04510, México D.F. México. 2 Accepted 26 March, 2013 Anti-inflammatory effects of maturin acetate (MA) isolated from the roots of Psacalium peltatum were investigated using in vitro and in vivo models. Inhibitory effects of increasing concentrations of MA on the production of pro-inflammatory cytokines (TNF-α and IL-1β) by lypopolysaccharide (LPS)-activated peritoneal macrophages were measured in vitro. MA at 75 µg/ml concentration inhibited TNF-α production by 80% and IL-1β by more than 85% (p < 0.01). MA inhibited inflammation by 75% when applied topically to 12-O-tetradecanoylpholbol-13-acetate (TPA) induced, dose-dependent acute ear edema. Myeloperoxidase enzyme levels were also reduced in the inflamed tissue. MA showed a significant inhibition level of 58.95% at the onset of carrageenan-induced inflammation (1 h) and the effect persisted up to 5 h. Thus, MA shows high cytokine production and marked anti-inflammatory effects. Key words: Psacalium peltatum, maturin acetate, pro-inflammatory cytokines, 12-O-tetradecanoylpholbol-13acetate (TPA)-induced acute ear edema, myeloperoxidase enzyme, carrageenan-induced inflammation. INTRODUCTION The biological activity of natural plant products has motivated extensive worldwide research to determine the pharmaceutical applications of their active compounds. Psacalium peltatum (Kunth) Cass. (synonyms: Cacalia peltata Kunth, Senecio peltiferus Hemsley) is an endemic medicinal plant, member of the matarique complex, which grows in central Mexico. The matarique medicinal plant complex consists of different plant species that are 01 (($* )#&)/ "',+ ( 2 %"& )"#&"( .") 34"+ ! % %5 grouped under the same common name, among them are Acourtia thurberi, Psacalium decompositum, Psacalium sinuatum and Psacalium palmeri (Linares and Bye, 1987). These plants share certain features, such as morphological characteristics of the employed plant and the same medical application. In Mexico, P. peltatum roots have traditionally been macerated in alcohol to treat conditions that cause inflammation such as wounds, skin Rojano-Vilchis et al. 1601 Figure 1. Structure of maturin acetate, cacalol and cacalone. ulcers and rheumatism (Bye et al., 1995). The antidiabetic effects of P. peltatum roots have also been studied (Contreras-Weber et al., 2002, 2005). The furanoeremophilane-type sesquiterpene named maturin acetate (MA) isolated from P. peltatum is similar to cacalol and cacalone isolated from P. decompositum (Figure 1). These two latter compounds are known to inhibit edema, showing a dose-dependent antiinflammatory effect when tested in in vivo models (Jimenez-Estrada et al., 2006). Further investigations have shown that cacalone in a natural mixture with epicacalone exhibits higher anti-inflammatory effect than cacalol using an in vivo 12-O-tetradecanoylpholbol-13acetate (TPA) model (Acevedo-Quiroz et al., 2008). The present work reports the anti-inflammatory effects of the bioactive compound MA isolated from P. peltatum using in vitro and in vivo models. MATERIALS AND METHODS Sample preparation and phytochemical analysis Roots of P. peltatum were collected from the pine-oak forest at Mineral del Chico, Hidalgo, Mexico, [20° 09´ 55” N and 98° 45´ 08” W]. A voucher specimen was deposited at the National Herbarium (MEXU 1138692) of the Institute of Biology, UNAM, México. Airdried and powdered roots (4.381 kg) of P. peltatum were sequentially extracted with n-hexane by thorough maceration (3 times × 2 l) at room temperature. The extract was filtered and concentrated. After the removal of solvent in vacuum, it afforded an oil residue. The n-hexane P. peltatum root extract (6.0 g) was fractionated by column chromatography over silica gel and eluted with n-hexane, ethyl acetate, and mixtures of these solvents. The collected fractions and compounds were monitored by thin layer chromatography (TLC) on 0.2 mm pre-coated silica gel 60 F254 plates (E. Merck), with the hexane/ethyl acetate (8:2) solvent system. Compounds were observed under ultra violet (UV) light and by spraying with 1.0% ceric sulfate/H2SO4 solution. The isolated compound was recrystallized in acetone-hexane to yield a pure compound. The structure was examined using infrared spectra (IR), 1 H, 13C nuclear magnetic resonance (NMR) spectra and mass spectra (MS). Uncorrected melting points (m.p.) were determined on a Fisher Jones apparatus. Infrared radiation (IR) was recorded with a Nicolet fourier transform infrared spectroscopy (FT-IR) SX spectrometer using chloroform as solvent. 1H, 13C NMR spectra were measured with a Varian Gemini-2000 and a Varian VXR-300 (200 MHz) spectrometer. Chemical shifts are reported in ppm relative to tetramethylsilane (Me4 Si, δ = 0) in CDCl3 as solvent and J values in Hz. Electron impact mass spectra were measured with a Jeol JMSAX 505 HA spectrometer. MA was detected and quantified by highperformance liquid chromatography (HPLC). Sample solutions were prepared by dissolving the MA and P. peltatum root extract in methanol. A Synergi Polar-RP column (150 × 2.00 mm, 4 µm particle size; Phenomenex) was used. The mobile phase consisted of deionized water (A) and acetonitrile HPLC grade (B). Gradient elution was performed starting with 70A/30B and changing the composition to 100B after 25 min. The detection wavelength, flow rate and column temperature were set at 254 nm, 0.2 ml/min, and 40°C, respectively. For all solutions (samples and standard), 10 µl were injected. A sample of 1.00 mg of the compound was dissolved in 5.00 ml methanol and serial dilutions of this stock solution with methanol were done to obtain calibration curves. Within the concentration range injected (200.0 to 2 µg/ml), the detector response was linear (R2 ≥ 0.9948), with a detection limit of less than 0.002 µg/ml (data not shown). Experimental animals Male ICR-CD1, BALB/c mice weighing 25 to 30 g and Wistar rats weighing 180 to 200 g were provided by the Instituto de Fisiología 1602 J. Med. Plants Res. Celular, UNAM, and approved by the Animal Care and Use Committee (NOM-062-ZOO-1999). All animals were housed under a 12 h light-dark cycle at 24 ± 2°C, humidity 50 ± 5% with ad libitum access to food and water. All experiments were carried out using six animals per group. In vitro determination of cytokine production in peritoneal macrophages To obtain peritoneal macrophages, 8-week-old male BALB/c mice were killed by cervical dislocation. Peritoneal cells were obtained by lifting the peritoneal wall and injecting 4 ml of sterile phosphate buffered saline (PBS). The peritoneum was then massaged, and the fluid (approximately 4 ml) was drawn into a syringe. Cells were washed twice and re-suspended in Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma) containing 10% fetal bovine serum (FBS) (GIBCO) and 100 g/ml gentamycin (GIBCO); later the concentration was adjusted to 3 × 106 cell/ml. Cell viability was determined as described in Badisa et al. (2003). Absorbance was measured on a microplate reader at 515 nm. Cytokines IL-1β and TNF-α were measured by enzyme-linked immunosorbent assay (ELISA) in the supernatant of peritoneal macrophages. These cell supernatants used for cytokine determination were prepared as follows: macrophages were washed twice, adjusted to a density of 106 cells/ml, and cultured at 37°C in 5% CO2 for 24 h in RPMI complete medium either alone or with 2 µg/ml LPS in the absence or the presence of increasing doses of maturin acetate (MA). After 24 h, samples were centrifuged at 2,500 rpm for 20 min at 18°C and the supernatant collected and assayed for IL-1β (range of detection 63 to 4000 pg/ml) and TNF-α (range of detection 16 to 2000 pg/ml) using an ELISA kit (Peprotech) in a format of sandwich. In vivo TPA-induced acute myeloperoxidase (MPO) activity ear edema assay and The assay involving TPA-induced acute ear edema in mice was based on the method described by Merlos et al. (1991) modified by Jimenez-Estrada et al. (2006). Increasing concentrations of MA and indomethacin as positive control were dissolved in ethanol:acetone (1:1). The degree of neutrophil infiltration was indirectly quantified by determination of ear myeloperoxidase (MPO) activity (Bradley et al., 1982; Suzuki et al., 1983). Enzyme activity was determined by colorimetry at a wavelength of 450 nm, using a BIOTEK micro plate reader ELx808. Activity was expressed as absorbance increase and enzyme inhibition percentages. In vivo carrageenan-induced rat paw edema assay The assay involving carrageenan-induced rat paw edema was based on the method described by Winter et al. (1962), with certain modifications. Rats were administered sodium naproxen (Sigma) and MA at 100 mg/kg i.p. Sodium naproxen was used as positive control because it is widely accepted as a non-steroidal antiinflammatory drug (NSAID) and is dose-related (Delporte et al., 2005). The 100 mg/kg MA dose was chosen as it is high enough to evidence anti-inflammatory effects; this has been shown in ibuprofen (In-Tae et al., 2004) and diclofenac (Ojewole, 2005). When there is no prior evidence of activity, natural extracts are commonly used at this concentration (Bolanle et al., 2012; Arawwawala et al., 2012; Prakash Babu et al., 2011). Before treatment, the mean volume (from 3 or 4 measurements) of the right paw of each animal was determined (Vo, basal volume and Vt, edema at different time) using a Plethysmometer 7159, Ugo Basile. These individual records were used to calculate the variation of edema (Vt - Vo) in each group. Percentages of inhibition (I%) were determined using the following formula: I% = 100 - [B × 100] / A, where A is the mean variation of edema (Vt - Vo) in the control group and B is the (Vt - Vo) in the groups treated with MA and sodium naproxen. Statistical analysis Data obtained from animal experiments were expressed as mean values ± standard error (± SEM). Statistical differences between the treated and control groups were evaluated by one-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparisons. Values of P ≤ 0.05 (*) and P ≤ 0.01 (**) were considered to be statistically significant (Tallarida and Murria, 1981). RESULTS AND DISCUSSION Medicinal plants and their products have been used for many centuries to treat different kinds of acute and chronic inflammatory diseases such as wound-healing, edema and rheumatoid arthritis (Gayathri et al., 2007). MA has been isolated from the roots of Mexican species such as P. beamanii (Perez et al., 2004), P. radulifolium (Garduño and Delgado, 2003), Roldana angulifolia (Arciniega et al., 2006), Trichilia cuneata (Doe et al., 2004) and from the South African species Senecio digitalifolius (Bohlmann and Zdero, 1978), Senecio lydenburgensis (Bohlmann et al., 1979) and Senecio affinis (Bohlmann and Bapuji, 1982). In this work, phytochemical analysis of dried n-hexane P. peltatum root extract (6 g) by silica gel column chromatography revealed MA as the root’s main constituent. Fractions eluted with hexane afforded a mixture of β-sitosterol/stigmasterol (15 mg) (Voutquenne et al., 1999). Fractions eluted with hexane/ethyl acetate (99:1) yielded maturinin (12 mg) (Correa and Romo, 1966) and MA (2.34 g) in the form of yellow needles (Figure 1). MA recrystallized from acetone-hexane. Crystallographic analysis of MA allowed the unambiguous assignment of the structure (RojanoVilchis, 2012). The 13C NMR data from MA have not been reported before and were unequivocally assigned as: 13C NMR (75.4 MHz, CDCl3) δ 121.03 (C-1), 130.24 (C-2), 124.77 (C-3), 130.36 (C-4), 133.44 (C-5), 122.05 (C-6), 129.38 (C-7), 142.41 (C-8), 141.78 (C-9), 125.42 (C-10), 116.44 (C-11), 148.94 (C-12), 58.48 (C-13), 193.45 (C14), 26.62 (C-15), 61.10 (O-CH3-9), 170.56 (C-1´) 21.00 (C-2´) ppm. In the present work, MA was directly obtained from the n-hexane root extract without undergoing the procedure of preparing an acetylated product with acetic anhydride in pyridine from maturin, as has been the case in previous studies (Correa and Romo, 1966). Chromatographic conditions of isolated MA were 1603 Absorbance (mAU) Rojano-Vilchis et al. Time (min) Figure 2. HPLC profile of maturin acetate. optimized by HPLC to evaluate compound purity in the shortest period possible. MA showed one peak, with retention time of 29.29 min and with concentration of 228.75 mg/g in the n-hexane P. peltatum root extract. (Figure 2). Cytokine synthesis is a primary mode of response of macrophages to inflammatory stimuli by bacterial products including LPS (Cheng et al., 2008). TNF-α is a cytokine with very diverse biological activities playing a role in various physiological and pathological phenomena, such as infection, inflammation, immunomodulation, cancer, cachexia and lethal septic shock. IL1β is among the most potent and multifunctional cell activators. IL-1β is a key mediator in the series of host responses to infection and inflammation known as acutephase response, and plays a particularly important role in the induction of acute-phase proteins whose synthesis is increased during inflammation. IL-1β has been studied regarding its pathogenetic role in rheumatoid arthritis, vasculitis, Alzheimer disease, diabetes and autoimmune diseases in general (Mire-Sluis et al., 1998). As shown in Figure 3, LPS-activated macrophages produced considerable levels of the two cytokines TNF-α and IL-1β. The treatment of cells with MA induced a concentration-dependent inhibition of the production of the two pro-inflammatory cytokines by LPS-stimulated macrophages. 75 µg/ml of MA resulted in 80% inhibition of TNF-α and in more than 85% inhibition of IL-1β (p < 0.01). These results confirm previously reported data, indicating that many medicinal plant extracts contain antiinflammatory substances which act as inhibitors of proinflammatory responses (Calixto et al., 2004; Grabley et al., 1999; Tan et al., 2004). Inflammation is a rapid local response to tissue damage with edema formation. Edema is characterized by reddening and swelling of the skin at the damage site (Ahamed et al., 2007). In this study, topical administration of MA and indomethacin significantly decreased acute ear edema induced by TPA at all administered doses compared with control groups (p < 0.01). Edema inhibition percentage (EI%) by indomethacin was 89.19% at a dose of 0.46 mg/ear, and MA showed its maximum inhibition level (75.50%) at a dose of 0.56 mg/ear. MA and indomethacin showed a dose-dependent anti2 inflammatory effect and ID50 values of 0.40 mg/ear (r = 2 0.99) and 0.097 mg/ear (r = 0.97), respectively (Table 1). MA showed a greater anti-inflammatory effect (75.50% edema inhibition) than cacalol (45.5% edema inhibition) in the same experimental model (Jimenez-Estrada et al., 2006). The TPA-induced acute ear edema response is 1604 J. Med. Plants Res. Figure 3. Effect of maturin acetate on LPS-induced cytokine secretion. Peritoneal macrophages (1 × 106/ml) were plated in 24-well plates and stimulated with LPS (2 µg/ml) in the absence or presence of the indicated concentrations of compound for 24 h. At the end of the culture period, culture medium was collected for the determination of IL-1β, TNF-α by ELISA kits. Results are expressed as mean ± SEM of cytokine concentration (µg/ml). (**p < 0.01) versus LPS-stimulated cells. associated with an increase in myeloperoxidase (MPO) concentration resulting from chemotaxis of neutrophils in the inflamed tissues (Smith, 1994). MA at doses of 0.31, 0.42 and 0.56 mg/ear reduced MPO activity (Figure 4). Topical application of MA significantly reduced MPO levels in ear homogenates, indicating that neutrophil migration control participates in the observed topical antiinflammatory activity. Anti-inflammatory effect observed Rojano-Vilchis et al. 1605 Table 1. Anti-inflammatory effects of increasing concentrations of indomethacin and maturin acetate on TPAinduced mouse ear edema. Substance Control Indomethacin Control Maturin acetate Dose (mg/ear) 0.05 0.15 0.46 Weight of ears (mg) 16.24±0.86 10.53±1.04** 7.10±1.34** 1.57±0.33** Edema inhibition (%) 35.15 56.29 89.19 0.31 0.42 0.56 15.35±0.42 11.04±0.23** 6.78±1.55** 3.64±0.39** 25.71 54.41 75.50 ID50 (mg/ear) 2 0.097, r =0.97 2 0.40, r =0.99 The weight of ears was represented as the mean ± SEM (standard error of mean) of six animals. **p < 0.01. Figure 4. Measurement of myeloperoxidase (MPO) activity using neutrophil infiltration within the dermis, treated with TPA alone and TPA + MA. Values are expressed as mean ± SEM of absorbance at 450 nm. Each bar represents the mean of six replicates. The asterisk denotes the significance levels when compared with controls (** p < 0.01). in TPA ear model was confirmed using the carrageenaninduced rat paw edema assay. Before injection of carrageenan in the paw edema assay, the basal paw volume values (Vo) ranged from 0.69 to 1.00 ml (0.87 ± 0.04 ml) and the mean variations of edema (Vt - Vo) ± standard error of mean (SEM) in the control group (N = 6) were 1 h (0.42 ± 0.10 ml), 3 h (0.98 ± 0.12 ml) and 5 h (1.06 ± 0.06 ml). MA showed significant (p < 0.05) inhibition of 58.95% of the inflammation induced by carrageenan at the beginning of the inflammatory process (1 h), and the effect persisted up to 5 h (Table 2). Administration of MA inhibited edema starting from the first hour along all phases of inflammation, which was probably due to inhibition of different aspects and chemical mediators of inflammation (Ahamed et al., 2005). The development of edema in the rat paw after the 1606 J. Med. Plants Res. Table 2. Anti-inflammatory effects of sodium naproxen and maturin acetate (100 mg/kg) on carrageenan-induced paw edema. Substance Sodium naproxen Maturin acetate Edema inhibition (%) 1h 3h 4.70±24.9 49.91±12.7 * 58.95±12.4 * 35.08±14.6 * 5h 41.6±10.9 * 35.22±6.1 Each value is represented as the mean ± SEM (standard error of mean) of six animals. *p < 0.05. injection of carrageenan has been described as a biphasic event. The early phase (2.3 to 3 h) of inflammation is due to the release of vasoactive amines such as histamine and serotonin. The later phase (4.5 to 6 h) is due to the activation of kinin-like substances such as prostaglandins induced by cyclooxygenase, proteases and lysosomes (Olajide et al., 2000), producing an edema dependent on the mobilization of neutrophils (Vinegar et al., 1987; Hwang et al., 1996). Present results indicate that MA reduces the exudation of the acute inflammation process and the early phase inflammatory response related to the release of proinflammatory mediators such as histamine and serotonin. This edematous response was reduced significantly in rats pretreated with indomethacin, a compound known to be a cyclooxygenase inhibitor. Conclusions Maturin acetate (MA) was isolated and quantified as the main constituent of the n-hexane P. peltatum root extract and exerted an important anti-inflammatory effect due to the modulation of macrophage responses and cytokine production. In addition, MA suppressed LPS-induced proinflammatory IL-1β and TNF-α production by mouse peritoneal macrophages acting as in vitro antiinflammatory agent. Furthermore, MA showed antiinflammatory effects in experimental animal models of acute inflammation generated with TPA and carrageenan. MA displayed an effective anti-inflammatory response similar to the indomethacin, which has been found to be effective in the treatment of rheumatic conditions as a cyclooxygenase inhibitor limiting prostaglandin biosynthesis. In this study, MA significantly reduced MPO levels by a mechanism related to the inhibition of neutrophil migration induced by carrageenan. MA showed a significant percentage of inhibition (58.95%) at the beginning of the inflammatory process (1 h) and the effect persisted up to 5 h, in comparison to sodium naproxen (4.70%). The present analysis confirms some of the beneficial effects ascribed in traditional medicine to the roots of P. peltatum. The anti-inflammatory effects produced by MA may be therapeutically used for diverse inflammatory diseases. Further experiments are required to determine to what extent this sesquiterpenoid compound and the P. peltatum root extract can be used as therapeutic agents with anti-inflammatory activity. ACKNOWLEDGMENTS We are grateful to N. Zavala-Zegovia, Ma. A. PeñaGonzález, R. Patiño-Maya, E. Huerta-Salazar, L. Velasco-Ibarra and F. J. Perez-Flores of the Institute of Chemistry for the determination of all spectra. We also thank E. 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