Anti-inflammatory and immunomodulatory effects of Critonia aromatisans leaves: Downregulation of pro-inflammatory cytokines

Author’s Accepted Manuscript Anti-inflammatory and immunomodulatory effects of Critonia aromatisans leaves: Downregulation of pro-inflammatory cytokines Villa-De la Torre Fabiola, Kinscherf Ralf, Bonaterra Gabriel, Arana-Argaez Victor Ermilo, Méndez-González Martha, Cáceres-Farfán Mirbella, Borges-Argáez Rocio

PII: DOI: Reference:

www.elsevier.com/locate/jep

S0378-8741(16)30364-6 http://dx.doi.org/10.1016/j.jep.2016.06.006 JEP10208

To appear in: Journal of Ethnopharmacology Received date: 9 February 2016 Revised date: 30 May 2016 Accepted date: 3 June 2016 Cite this article as: Villa-De la Torre Fabiola, Kinscherf Ralf, Bonaterra Gabriel, Arana-Argaez Victor Ermilo, Méndez-González Martha, Cáceres-Farfán Mirbella and Borges-Argáez Rocio, Anti-inflammatory and immunomodulatory effects of Critonia aromatisans leaves: Downregulation of pro-inflammatory cytokines, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2016.06.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Anti-inflammatory and immunomodulatory effects of Critonia aromatisans leaves: Downregulation of pro-inflammatory cytokines Villa-De la Torre Fabiolaa*, Kinscherf Ralfc1, Bonaterra Gabrielc1, Arana-Argaez Victor Ermilob2, Méndez-González Marthaa3, Cáceres-Farfán Mirbellaa4, Borges-Argáez Rocioa4 a

Unidad de Biotecnología, Centro de Investigación Científica de Yucatán, Mérida,

Yucatán, México b

Laboratorio de Farmacología, Facultad de Química, Universidad Autónoma de Yucatán,

Mérida, Yucatán, México c

Institute of Anatomy, Philipps Marburg University, Marburg, Germany

[email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] *

Corresponding autor. Unidad de Biotecnología, Centro de Investigación Científica de

Yucatán, Mérida, Yucatán, México. Calle 43 No. 130, Colonia Chuburná de Hidalgo. 97200, Mérida, Yucatán, México, Tel:+52 999 942 83 Ext 157. Current address: Laboratorio de parasitología, CIR-Biomédicas, Unidad Inalámbrica, Universidad Autónoma de Yucatán. Calle 43 No. 613 x Calle 90 Col. Inalámbrica. C.P. 97069. Mérida, Yucatán, México. Tel: +52 999 924 5809. Abstract Ethnopharmacological relevance Critonia aromatisans (Asteraceae), commonly known as “Chiople”, is a cultivated species that is used in Mayan traditional medicine to treat inflammation, joint pain and rheumatism. 1

Robert Koch Strasse 8, 35032 Marburg, Germany. Tel: +49 64212866245. Calle 43 No. 613 x Calle 90 Col. Inalámbrica. C.P. 97069. Mérida, Yucatán, México. Tel: + 52 999 922-57-11 Ext 119. 3 Calle 43 No. 130, Colonia Chuburná de Hidalgo. 97200, Mérida, Yucatán, México, Tel:+52 999 942 83. 4 Calle 43 No. 130, Colonia Chuburná de Hidalgo. 97200, Mérida, Yucatán, México, Tel:+52 999 942 83 Ext 157. 2

Aim of the study To evaluate the in vivo and in vitro anti-inflammatory and immunomodulatory properties of aqueous and organic extracts prepared from Critonia aromatisans leaves. Materials and methods Methanol, ethyl acetate, methylene chloride, hexanic, and aqueous extracts were obtained from the leaves of C. aromatisans. The anti-inflammatory properties of the extracts were tested in vivo to evaluate their ability to reduce the inflammatory response in the carrageenan-induced hind paw edema model in NIH mice. In addition, to explore the immunomodulatory effects of C. aromatisans, in vitro testing was performed to determine whether C. aromatisans leaf extracts are capable of decreasing macrophage production of nitric oxide (NO), tumour necrosis factor alpha (TNF-, and cytokines IL-1, IL-6, and cyclooxygenase 2 (COX-2) without affecting macrophage viability. Results Single orally administered doses (100 mg/kg or 200 mg/kg) of a hexanic extract of C. aromatisans leaves significantly reduced carrageenan-induced paw edema in mice (P<0.001) by 76% and 84%, respectively. The effect of the extract in this model was generally comparable to those of the standard drugs used. In the in vitro determination, the extracts reduced the amount of NO mainly at 500 and 1000g/mL. Hexanic extract and subfractions C, D, E, and F at 50 and 100g/mL produced the lowest concentration of mediators in culture supernatants (protein) and at the mRNA/gene level by the significant down-regulation of cytokines. These findings explain some of the anti-inflammatory activity of this species. Purification of fractions C and D allowed the complete identification of cyclocolorenone, stigmasterol and stigmasterol derivatives as some of their main components.

Conclusion A hexanic extract of C. aromatisans displayed anti-inflammatory effects, validating the traditional practice of Mayan communities wherein an ointment with a petrolatum base, a non-polar substance, is used to treat inflammation. Additionally, C. aromatisans showed strong in vivo and in vitro activity, and one of the mechanisms of its anti-inflammatory

response was shown to be inhibition of the production of NO and pro-inflammatory cytokines. The results of this study provide a pharmacological basis for the use of C. aromatisans leaves in the treatment of inflammatory disorders. The presence of stigmasterol and cyclocolorenone could be the responsibles of the anti-inflammatory activity of this specie. Further studies should be done on the antioxidant and antiinflammatory properties of cyclocolorenone. The results of this study provide a pharmacological basis for the use of C. aromatisans leaves in the treatment of inflammatory disorders. Keywords

Critonia

aromatisans,

Eupatorium

hemipteropodum,

Inflammation,

Cyclocolorenone, Stigmasterol, TNF-

1. INTRODUCTION Inflammation is a response induced by pathogens, damaged tissue or autoimmune and rheumatic diseases. Physiologically, inflammation operates in a restricted environment to eradicate infected or devitalized tissue. Although usually beneficial to the host, inflammation is intrinsically destructive to the immediate surroundings (Ahmed et al., 1995). During the complex process of inflammation, which produces characteristic signs (redness, heat, tumour, pain and loss of function), regulatory cytokines are released in response to infection, inflammation and trauma as part of the immune response. A variety of molecules participate in the inflammatory response, including prostaglandins (PG), nitric oxide (NO), tumour necrosis factor alpha (TNF-), interleukins (ILs), prostanoids and leukotrienes (Hewett and Roth, 1993; Gallin and Snyderman, 1999; Kubes and McCafferty, 2000). Leukocytes release inflammatory mediators such as neuropeptides, histamine, serotonin, potassium ions, bradykinin and metabolites of arachidonic acid (Rang et al., 1991). The macrophages contribute to acute and chronic inflammation, locally and systemically (Wynn et al., 2013). Some important roles of these cells are contributing to tissue remodeling and wound healing .The huge capacity of these cells for phagocytosis clearance of apoptotic and necrotic cells, secretion of proinflammatory cytokines, such as IL-1β, IFN-γ, TNF-α and tumor necrosis factor β (TNF-β) (Rabinovitch and Suarez-Pinzon, 1998).When swelling increases during inflammation, the massive production of TNF-, IL1 and IL-6 by macrophages can cause severe tissue damage and dysfunction in various organs (Delgado et al., 2003; Miyake, 2004; Ahmed et al., 2005). TNF- and IL-1 are

particularly important mediators in that they contribute to chronic inflammatory disorders such as rheumatoid arthritis (RA) (Bingham III, 2002). TNF- is also involved in inducing cell damage in mitochondria under conditions of oxidative stress (Sies, 1997).

NO

production by activated macrophages has been shown to mediate immune functions, including antimicrobial and antitumor activities. However, excess NO production has been implicated in inflammatory processes, septic shock and autoimmune diseases such rheumatoid arthritis (MacMicking et al., 1995). Therefore, the inhibition of NO production has become a therapeutic target for the treatment of inflammatory diseases (Azadmehr, 2009). To treat inflammatory disorders and rheumatic pain, the Mayan population has used plants such as Critonia aromatisans (DC.) R. M. King & H. Rob (synonym Eupatorium hemipteropodum), commonly called “Chiople” (Asteraceae) by the Mayan population of the Yucatan peninsula and in Cuba as "Trébol de olor”. C. aromatisans is commonly used alone, or in different plant mixtures to prepare anti-inflammatory and antiarthritis remedies. Mayan people traditionally apply the leaves directly to the painful area; for example, the leaves are applied to the forehead to treat migraines. To treat edema or an inflamed area, the leaves can be used in a decoction for water baths, as a poultice, or in ointments enhanced with petrolatum alone or mixed with other species. The production of such ointments involves pulverizing the leaves and boiling them with petrolatum. The mixture is filtered and placed in containers. Then, it is ready to use topically on the affected area of the body. The leaves have also been used to aromatize tobacco (Roig et al., 1974, Méndez et al., 2012). Based on this ethnobotanical information, the aim of the present work was to evaluate the in vivo and in vitro anti-inflammatory and immunomodulatory effects of extracts prepared from the leaves of C. aromatisans.

2. MATERIALS AND METHODS

2.1 Plant material Leaves of C. aromatisans were collected in October 2009 and July 2010 from the botanical garden of the Traditional Medical Centre of Yaxcabá, Yucatán, México. The species were identified by Dr. Martha Mendez-González, and a voucher specimen was deposited at the

herbarium "U najil tikin xiw" of the Centro de Investigación Científica de Yucatán (CICY) under the collection number “M. Méndez 2455”. 2.2 Preparation of the extracts 2.2.1 Preliminar in vivo screening Powdered leaves (872 g) were extracted during 3 days with methanol (MeOH) at room temperature. This process was repeated 3 times to obtain the C. aromatisans MeOH extract (“total extract”, 243 g). A portion of the methanolic extract (20.8 g) was dissolved in MeOH, and acetonitrile (ACN) was added at a ratio of 1:3. Water was added to the mixture, and the mixture was extracted 3 times with n-hexane to obtain the Hx fraction (6.5 g). The process was repeated with dichloromethane (DCM) to obtain the DCM extract (11 g) and with ethyl acetate (AcOEt) to obtain the AcOEt fraction (0.15 g); finally, the aqueous remainder was collected and named as aqueous fraction (1.2 g). 2.2.2 Purification of the n-hexane (Hx) extract Powdered leaves from C. aromatisans (3 kg) were extracted during 3 days with methanol (MeOH) at room temperature. This process was repeated 3 times to obtain the C. aromatisans MeOH extract (“total extract”, 410 g). A portion of the methanolic extract was dissolved in MeOH, and acetonitrile (ACN) was added at a ratio of 1:3. The mixture was extracted 3 times with hexane Hx to obtain the Hx fraction. The Hx fraction (34 g) was subjected to a vacuum liquid chromatography (VLC) column on silica gel (7  10 cm, 200400 mesh) eluted with Hx and EtOAc mixtures of increasing polarity (100 % Hx , Hx/EtOAc 98: 2 to 50:50, 100 % EtOAc, EtOAc/MeOH 50:50 and 100 % MeOH) to produce seven final fractions (A-G). Fraction C (1.1 g) was further purified on a second VLC column (7  5 cm, 200-400 mesh) and eluted with n-hexane (Hx) and dicloromethane (DCM) mixtures of increasing polarity (99:1 to 4:96 and 0:100) followed for 50:50 DCM/ EtOAc, 100% AcOEt, 100% MeOH, to yield eight final fractions (C1-C8). From fraction C6 (29 mg) cyclocolorenone (12.7 mg) was obtained by preparative TLC (DCM/acetone 97:3). Flash chromatography (35  5 cm) of fraction D (8 g) on n-hexane and DCM mixtures of different proportion (99:1, 98:2, 97:3, 95:5, 90:10, 85:15, 50:50, 100% DCM). After a recrystallization with ether, subfractions D1, D2 and D3 yielded a white precipitate that was identified as a mixture of Stigmasterol and stigmasterol derivatives. 2.3 In vivo assays

Male and female NIH mice (25-30 g) were provided by the Animal House of “Centro de Investigaciones Regionales”, UADY. The animals were maintained on a standard pellet diet and water ad libitum. Five to ten animals were used in each group. The animals were handled according to the ethical guidelines for Mexican procedures (NOM-062-ZOO1999). 2.3.2 Preparation of test samples Extracts were administered per os in a unique dose dissolved in a solution of 5% Tween 80. The animals in the control group received a solution of 5% Tween 80 suspension in distilled water and the same experimental handling as those in the test groups. A dose of 10 mg/kg indomethacin (anti-inflammatory drug) was used as a positive control. 2.3.3 Carrageenan-induced hind paw edema in NIH mice The in vivo anti-inflammatory activity of the extracts was evaluated using the carrageenaninduced hind paw edema model as previously described by Winter et al. (1962) with a few modifications. Briefly, two control groups and ten experimental groups were formed; the mice were weighed, and either an extract (10, 100, or 200 mg/kg), indomethacin (10 mg/mL) or vehicle was administered per os. Thirty minutes later, the subplantar tissue of the right hind paw of each mouse was injected with a suspension of carrageenan (Sigma, St. Louis, MO, USA) at 1% (0.25 L/ paw) in sterile physiological saline solution (0.9%). The hind paw was measured prior to oral administration and 3 h after carrageenan injection. The difference in footpad thickness was measured using a precision micrometre. The mean values of the animals in the treated groups were compared with the mean values of the animals in a control group, and the percentage inhibition of inflammation was calculated.

2.4 In vitro assays of nitrite production 2.4.1. Isolation of peritoneal macrophages Macrophages were isolated from the peritoneal cavities of male NIH mice. Briefly, macrophages were harvested from the peritoneal cavity with two washes (10 mL each) of cold sterile PBS solution. The cells were resuspended and adjusted to 1x106 cells/mL in Dulbecco`s Modified Eagle`s Medium (DMEM) containing 10% inactivated foetal bovine serum, 100 U/mL penicillin, and 100 µL streptomycin (this combination is referred to as complete DMEM).

2.4.2 Treatment of peritoneal macrophages Peritoneal macrophages (5×104 cells/well) were incubated in 96-well plates with 200 µL of complete DMEM for 24 h at 37º C in a humidified 5% CO2 incubator. Non-adherent cells were removed by gentle washing with DMEM. After washing, medium containing C. aromatisans extracts at various concentrations (1, 10, 100, 500, 1000 µg/mL) was added, and the cells were cultured for 24 h at 37º C in a humidified 5% CO2 incubator. After incubation, the supernatants were removed, and LPS (1 µg/mL) was added. The culture supernatants were collected after 72 h of LPS treatment for nitrite determination.

2.4.3 Measurement of nitric oxide production Nitrite production, an indicator of NO synthesis, was determined by the Griess reaction (Green et al., 1982). Briefly, 50 µL of supernatant from the 96-well plates was incubated with an equal volume of Griess reagent (1% sulphanilamide/0.1% naphthalene diamine dihydrochloride/2.5% H3PO4) for 10 min at room temperature to quantify the accumulation of nitrite (Ding et al., 1988). The absorbance of the mixture at 550 nm was determined using a microplate reader. Conversion of absorbance to µM of NO was determined using dilutions of sodium nitrite at known concentrations (0-100 µM) in culture medium.

2.4.4. Determination of cell viability of peritoneal macrophages Cell viability was assessed by the mitochondria-dependent reduction of 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to purple formazan. Cells were obtained as described previously and plated in a 96-well culture plate at a concentration of 5×104 cells/well in complete DMEM. After an overnight culture period, the cells were washed with DMEM, C. aromatisans leaf extracts at varying concentrations (1, 10, 100, 500, 1000 µg/mL in culture medium) were added in a total volume of 200 µL, and the cells were cultured for 24 h at 37ºC in a humidified 5% CO2 incubator. The supernatants were then removed, and the cells were incubated with MTT (5 mg/mL in PBS) for 4 h at 37 ºC and 5% CO2. The supernatants were removed by aspiration, and the residual formazan crystals were dissolved in DMSO. The reduction of MTT was measured at 550 nm using a microplate reader. The percentage of proliferation was calculated using the following equation: % proliferation = (sample OD - control OD / control OD) x 100

2.5 Measurement of in vitro inflammatory mediators 2.5.1 Cell culture A human acute monocytic leukaemia cell line, THP-1 (Cell Lines Service, Eppelheim, Germany) was cultured at a concentration of 1x106 cells/mL in 12-well plates in RPMI 1640 medium (PAA Laboratories Gmb, Pasching, Austria) containing 10% inactivated foetal bovine serum, 100 U/mL penicillin and 100 g/mL streptomycin. To differentiate monocytes into macrophages, we added 100 ng of PMA (phorbol myristate acetate, Sigma-Aldrich) for 48 h at 37ºC in a humidified 5% CO2 incubator. Non-adherent cells were removed by suction, and adherent cells were washed with fresh medium.

2.5.2 Determination of TNF- by the ELISA method Differentiated THP-1 cells were treated with extracts of C. aromatisans at concentrations of 200, 100, 50, and 25 g/mL). The extracts were pre-incubated for 1 h with THP-1 cells; LPS (1 g/mL) was then added, and the cells were incubated for an additional 4 h. The supernatants were collected, and the amount of TNF- released was evaluated using an ELISA immunoassay kit (Thermo Fisher Scientific Inc., Rockford, IL, USA). As controls, we used untreated cells (negative control), cells treated with LPS (positive control), cells treated with dexamethasone (anti-inflammatory control) and cells exposed to diluent (negative control). 2.5.3 Viability of THP-1 cells Determination of cell viability was conducted in cultures of THP-1 cells differentiated into macrophages and exposed to similar concentrations of extracts and fractions as cultures used to evaluate the induction of inflammatory mediators. Briefly, we used 12-well plates containing 1x106 cells/well; after differentiation with 100 ng PMA for 48 hours, pretreatment with test extracts and fractions and incubation for 1 h, we added LPS (1 g/mL) and incubated the plates for an additional 4 hours. The culture was washed 2-fold with medium (RPMI 1640). Finally, 1 ml of medium was set and the reagent PrestoBlue® Cell Viability reagent (Life Technologies Corporation, California, USA) was added to a final concentration of 10%. Incubation was continued for 45 minutes at 37 ° C, after which the absorbance was read on a microplate spectrophotometer (SUNRISE ELISA reader, Tecan, Salzburg, Austria) at a wavelength of 570 nm using absorbance at 600 nm as a reference. We used two colour controls: medium without cells and medium containing PrestoBlue®. The absorbance value of the PrestoBlue + medium control was considered

to represent 100% cytotoxicity. High absorbance values are correlated with increased overall metabolic activity and increased numbers of cells. 2.5.4 Extraction of mRNA After collection of the cell supernatants, the cells were homogenized in 1 ml of peqGOLD Tri-Fast (PEQLAB, Erlangen, Germany), and the RNA was purified according to the supplier's instructions. Briefly, peqGOLD/chloroform partitioning was performed, the mixture was centrifuged, and the aqueous phase was treated with isopropanol to precipitate total RNA. After 10 minutes of centrifugation at 12,000 xg and 4 °C, the pellet was washed twice with 75% ethanol; after each wash, insoluble material was recovered by centrifugation for 10 min at 12,000 xg at 4 °C. After removal of the supernatant, the pellet was allowed to dry at room temperature and resuspended in an appropriate amount of DNase/RNase-free

water

(UltraPure™

DNase/RNase-Free

Distilled

Water,

Life

Technologies Corporation, California, USA). The concentration and purity of the RNA was assessed by determination of its A260/A280 ratio (A260/A280 = 1.7-2.0) using a NanoDrop 2000c spectrophotometer (Thermo Scientific, Schwerte, Germany). RNA integrity was determined using a "lab-on-a-chip" NanoChip 6000 kit and the Agilent 2100 Bioanalyser (Agilent Technologies, Waldbronn, Germany).

2.5.5 Synthesis of cDNA An aliquot (1 g) of RNA was treated with 1 unit of DNase (Fermentas, St. Leon-Rot, Germany) for 30 min at 37 °C. Reverse transcription of RNA was carried out with the first oligo (dT) 12-18, reverse transcriptase RT AffinityScript (Agilent Technologies, Inc. La Jolla, USA) and 24 units of RNase inhibitor LockTM Ribo (Fermentas, St-Leon-Rot, Germany). Reverse transcription was performed for 45 min at 42 °C following by heating for 5 min at 95 °C to inactivate the enzyme and terminate the cDNA synthesis reaction.

2.5.6 Determination of TNF-, IL-1, IL-6 and COX-2 expression by qRT- PCR The cDNA obtained from the cultured cells was used to quantify the expression of various genes

using

real

time

qRT-PCR.

The

primers

were

purchased

from

QuantiTect/PrimerAssays (QIAGEN GmbH, Hilden, Germany). The genes corresponding to prostaglandin-endoperoxide synthase 2 (COX-2, NM_000963) 68-bp amplicon, tumour necrosis factor alpha (TNF-, NM_000594), interleukin 6 (IL-6, NM_000600), and interleukin-1 beta (IL-1, NM_000576) were analysed; actin (ACTB, NM_001101) was

used as a reference gene for normalization of the expression of the genes of interest. The cDNA was amplified using Brilliant® II SYBR® Green qRT-PCR Master Mix (Stratagene, Agilent Technologies, Waldbronn, Germany). The thermal profile consisted of 1 cycle at 95 °C for 3 minutes followed by 40-45 cycles at 95 °C (10 sec) and 60 °C (20 sec). To confirm the specificity of the obtained products, a cleavage protocol (55 °C - 92 °C) was carried out after the PCR analysis. Amplification and data analyses were performed using the Mx3005P™ qPCR system and Mx3005P analysis software (Stratagene, Agilent Technologies). For relative quantification, a standard curve was generated using serial dilution of a pool of cDNA. The relative amount of specific DNA was calculated using linear regression analysis of the respective standard curves.

2.6 Statistical analysis For in vivo pharmacological evaluations, experiments were performed with 5-10 animals per group. The experimental values obtained in in vivo and in vitro tests are expressed as the mean ± standard error of the mean (S.E.M.). Statistical analysis was performed by one-way analysis of variance (ANOVA) with Dunnett’s post hoc multiple comparisons test using PRISMA software (GraphPad Software, Inc., San Diego, CA, version 5.01). In both cases, p ≤ 0.05 was used as the criterion of statistical significance (sample vs. control).

2.7. Gas chromatography-mass spectrometry (GC-MS) analysis of the Hx fraction, cyclocolorenone and subfraction D. The CG-MS analyses of Hx and fractions C and D were performed using an Agilent Technologies 6890N gas chromatograph (Santa Clara, CA, USA) connected to an Agilent 5975B (MSD; electron impact ionization, 70 eV). Preliminary analyses of the Hx fraction were performed with an HP-5 MS capillary column (30 m × 25 mm  0.25 µm). Helium was used as the carrier gas; the injection volume was one microliter, and the temperature program used was as follows: 50°C (3 min); an initial temperature of 80°C (5 min.); an increase to 215°C at 4°C/min; and a hold at 215°C for 5 min. Cyclocolorenone was run using the following chromatographic conditions: split injection of 1 µL of a 1% concentration sample; HP 5MS column (30  0.25 mm i.d), flow rate 0.8 mL/min (Helium as carrier gas); oven temperature program T1= 50oC (10 min), T2=310oC(10 min), gradient 15oC/min. Injector and detector temperature (FID) at 310oC. Stigmasterol Sigma-Aldrich reference and sterol fraction were run on split injection of 1 µL of a 1% concentration

sample; HP 5MS column (30  0.25 mm i.d), flow rate 1 mL/min (Helium as carrier gas); oven temperature program T1= 100oC (3 min), T2=280oC (30 min), gradient 10oC/min. Injector and detector temperature (FID) at 280oC. Co-injection of the mixture with the standard of stigmasterol also was performed. 2.8 NMR structure elucidation of cyclocolorenone 1

H,

13

C, HMQC and HMBC spectra for Cyclocolorenone were recorded on a Brucker

Avance III 500 Mhz spectrometer in CDCl3, with TMS as an internal standard.

3. RESULTS 3.1 Carrageenan hind paw edema assay In a previous plant screening, we found that an ACN/MeOH extract from C. aromatisans displayed some anti-inflammatory effect (data not shown). In the present work, fractions obtained by partitioning the MeOH extract with ACN/MeOH, Hx, DCM, and EtOAc and the aqueous residue of this partitioning were evaluated in the common albino Swiss mouse (NIH) strain at doses of 10 and 100 mg/kg. In this mouse strain, the ACN/MeOH fractions had no significant inhibitory effect on the development of plantar edema; however, the hexane fraction showed greater than 80% inhibition of swelling at a dose of 100 mg/kg. Based on this result, the Hx fraction and the total ACN/MeOH extract were tested at a higher concentration (200 mg/kg); at this concentration, inhibition of inflammation was not increased (Table 1). Because the DCM extract, the EtOAc extract and the aqueous residue did not cause more than 50% inhibition of plantar swelling, the hexane fraction was selected to continue further investigations.

3.2 Nitric oxide (NO) determination in peritoneal macrophages from NIH mice The NO production by peritoneal macrophages in presence of the extract and fractions was evaluated. As seen in Table 2, the highest activity was found in the Hx fraction. This fraction almost completely inhibited the production of NO. The Tween 80 diluent employed in the preparation of the dilutions for the evaluation of the extracts had

no effect on the NO content of the cells (Saline C (-) = 1.11 ± 0.06 M, Saline C + Tween 80 = 1 ± 0.29 M, Saline C + LPS 1 g/mL = 97.49 ± 2.25 M, LPS + Tween 80 = 91.02 ± 3.13 M). In addition, no effects on the viability of peritoneal macrophages exposed to different concentrations of the extracts (10-1,000 g/mL) were observed (data not shown), indicating the specificity of these extracts against NO production in NIH mouse macrophages. The results show that treatment with fraction D at 100 g/mL reduced the nitric oxide content of the cells by 89.3 %. Furthermore, the Hx extract, as well as fractions B and C, caused a reduction of greater than 50% in cellular nitric oxide levels at concentrations of 500 and 1,000 g/mL, and fraction A showed an inhibition of NO of 29.6%. In the case of E and F, inhibition was greater than 50%. The G fraction showed no significant activity. With those results, we decided that subfractions with higher effect (C and D) were re-evaluated to confirm these findings. In this second evaluation we used the ACN/MeOH and Hx extracts. In summary, the results showed that the Hx fraction, as well as fractions C and D, inhibit NO production by more than 80 % and that the ACN/MeOH fraction has a lesser effect. We observed that at higher concentrations of the Hx fraction and subfraction D, NO production decreased, suggesting that metabolites present in fraction D are capable of reducing the production of this mediator. 3.3 Cell viability To demonstrate that the extracts and fractions tested in this study exert an inhibitory effect on NO production in macrophages and not a cytotoxic effect, a cell viability assay was performed (data not shown). The fact that all of the cells produced high similar amounts of formazan as the control (close to 100%) in the MTT assay using Tween 80-diluted extracts suggests that neither Tween 80 nor C. aromatisans extract fractions have a cytotoxic effect.

3.4 Determination of TNF- levels by ELISA This method was used for initial evaluation of the extracts. The results illustrated in Figure 1 show the decrease in the production of TNF- by THP-1 cells. Treatment of cells with the hexane extract at a concentration of 100 g/mL produced the highest level of inhibition of TNF- production, whereas the total extract, ACN/MeOH and hexane fractions displayed inhibitory effects of greater than 80%. Furthermore, nearly 90% inhibition of

TNF- production by the hexane fraction was obtained at a concentration of 50 g/mL. These results indicate that the hexane fraction has a good inhibitory effect on the production of TNF- in the culture medium. Fractions A, C, E and F all have a good inhibitory effect on this mediator; however, fractions B, D and G reach approximately 50% of inhibition, so these fractions may also contain some active metabolites. Overall, these results show that the hexane extract and the fractions derived from it produce similar inhibition of TNF- secretion when used at a concentration of 50 g/mL. Therefore, with respect to inhibition of TNF-, it is possible to employ the hexane fraction as an inhibitor of this cytokine without the need for further fractionation.

3.5 Determination of TNF-, IL-1, IL-6 and COX-2 levels by qRT-PCR Figure 2 shows the relativity index of TNF-, IL-1, IL-6 and COX-2 expression. This mRNA expression of these mediators was lower after treatment of cell with the Hx fraction at 100 g/mL in the presence of the LPS stimulus. For this reason, an assessment at a lower dose of extract (50 g/mL) was performed; at this dosage, only IL-1 levels were affected. The Hx fraction was further subfractionated, and the seven fractions obtained were tested at a dose of 50 g/mL. Fractions C, E and F were the most active, and fraction C was the single most active fraction in all cases. Although ELISA determination showed that the Hx fraction produced a large decrease in the release of TNF- to the medium, PCR analysis revealed that fraction C showed a significant difference from the Hx extract at the mRNA expression level, with C causing a greater decrease than the Hx extract. Therefore, the C fraction should be considered for further analysis and for the identification of chemical components.

3.6 Viability of THP-1 cells THP-1 cells were evaluated by the Presto Blue staining method, which measures cellular metabolic activity. The results indicated that cellular metabolic activity remained close to 100% in the treated cells (data not shown). 3.7. Determination of cyclocolorenone, stigmasterol and stigmasterol derivatives on fractions C and D by CG-MS and NMR structure elucidation of cyclocolorenone

Gas chromatography-mass spectrometry analysis of the Hx extract of C. aromatisans showed that it is primarily composed of fatty acids and terpenoids, which included 2H-1-benzopyran-2-one, isolongifolen-5-one (misidentified as isolongifolenone by NITS library), methyl palmitate and Lup-20 (29)-en-3-ol, acetate, respectively. Other minor components were identified as ledol, 2-pentadecanone, stigmasterol, lupeol and sitosterol. Purification of fractions C and D, allowed the complete identification of their main metabolites present in those fractions. Compound 1 (m/z 218) was identified as cyclocolorenone, isolated from fraction C as a yellow oil with an Rf 0.7 in the solvent system DCM:acetone 97:3. The UV spectrum ( 265 nm) together with a

13

C signal

resonating at  208.3 and quaternary signals at  140.3 and 176.4, indicated the presence of an unsaturated carbonyl group. The LREIMS and

1

H and

13

C NMR

established the molecular formula to be C15H22O.The 1H NMR spectrum accounted for all 22 protons, which were recognized as four methyls: δ 0.79 (d, J=6.95 Hz, 3H), 1.02 (s, 3H), 1.24 (s, 3H) and 1.72 (d, J=1.85 Hz, 3H), a doublet of doublet signal at δ2.48 (J=6.60, 18.45 Hz, 1H), a doublet at δ2.06 (J=18.45 Hz, 1H), a singlet at δ 2.95 (1H) and multiplets at δ1.25 (1H), δ1.42 (1H), δ2.07 (1H), δ1.47 (1H), δ1.59 (1H),. δ1.96 (1H) and δ1.99 (1H). The presence of a cyclopropane ring was suggested by the 1H peak resonating at δ 1.47 (d, J= 8.5 Hz, H6) and 1.99 (m, H7) and signals at δ 1.02 (s, 3H) and 1.24 (s, 3H) which were consistent with a gem-dimethyl substitution. HMBC and HMQC correlations were similar to the aromadendrane type sesquiterpene identified as cyclocolorenone. On the other hand, GC/MS of subfraction D suggested the presence of a sterol mixture. The identification of the mixture components was performed using National Institute Standard and Technology Version 5.0 Library (NITS library). Stigmasterol (RT=24.39 min with peak area 64%) was the major compound identified in fraction D. Additionally, two minor components were present that include Stigmasta-7,25-dien-3-ol (RT= 26.63 min) and Stigmast-7-en-3-ol (RT= 26.63 min). Co-injection of sterol mixture and Sigma-Aldrich Stigmasterol standard grade confirmed the presence of this main metabolite on the mixture with the same retention time and same pattern fragmentation. (GC-MS profiles and all spectroscopic data are available as Supporting information).

4. DISCUSSION

C. aromatisans is used in traditional medicine as an ointment that is prepared by placing crushed leaves of the plant into a fire along with melted petrolatum. Due to its nonpolar nature, petrolatum maintains a semi-solid structure at room temperature, allowing it to be applied to the skin and to form a waxy layer before it melts with body heat. The composition of the compounds in the traditional ointment is not known, but we believe the active compounds remain in the petrolatum because the heated leaves liberate some essential oils that are captured by the petrolatum due to its consistency and polarity. The petrolatum removes the less polar compounds contained in the leaves, and these compounds are responsible for the pharmacological activity of the plant. The fact that the active Hx fraction contains major compounds of low polarity supports this theory. To gain a better perspective, it is important to evaluate in further studies the essential oils in the antiinflammatory models used and compare with our results. Preliminary in vivo anti-inflammatory plant screening conducted by our group in BALB/c mice indicated that both ACN/MeOH and Hx extracts of C. aromatisans have strong anti-inflammatory activity (Villa de la Torre, 2014). For this reason, we repeated the test in Swiss albino NIH mice, a less sensitive strain than BALB/c. We found that the ACN/MeOH extract had no significant effect on paw edema, whereas the Hx extract showed significant activity at 100 and 200 mg/kg. Therefore, the inhibitory activity of the Hx extract on NO production in activated macrophages was determined. It was found that treatment of cells with the extract at concentrations of 10 to 1,000 g/mL decreased NO production to below baseline. Macrophages are actively involved in the inflammatory response, releasing cytokines and factors that recruit other cells to sites of infection and to sites at which tissue alteration or damage has occurred. In addition, the expression of genes responsible for the synthesis of bioactive lipids derived from arachidonic acid and reactive oxygen and nitrogen species such as NO is rapidly increased in activated macrophages. These molecules contribute to the regulation of the inflammatory response (Boscá, 2005). The use of macrophages contributes to the determination of the mechanism of action of the anti-inflammatory substances tested in this study. Our results suggest that part of the mechanism of action of the Hx extract of C. aromatisans is inhibition of the production of some of the pro-inflammatory mediators produced by macrophages, including NO. To confirm this, it was necessary to perform a cell viability test to be sure that the reduction of NO was not due to cell death but to the inhibition of NO production. The results of this test suggested that the Hx extract was active and that it decreases the production of this mediator.

The importance of the cellular and molecular analysis performed in this study is that it provides information on the mechanism of action of the extracts and fractions obtained from C. aromatisans. It was found that both the Hx extract and fraction C reduce the amount of nitric oxide present in cells and that treatment with these fractions inhibits the release of some important pro-inflammatory mediators, such as TNF-α, into the culture medium. Based on this result, we evaluated the extracts and fractions using quantitative RT-PCR to measure the production of the mediators at the mRNA level and thereby to determine at precisely what level the Hx fraction or subfractions stop the expression of mediators. Because the production of TNF-, IL-1, IL-6 and COX-2 is inhibited by the Hx fraction and by subfraction C, we conclude that the inhibition occurs within the cell nucleus. These mechanisms could explain the results obtained in the in vivo carrageenan assay (carrageenan activates the release of inflammatory mediators), in which the decrease in inflammation was greater than 80 % due to inhibition by C. aromatisans of the release of various mediators. The fractions responsible for these effects may be C and D; gas chromatography-mass spectrometry analysis of the Hx extract of C. aromatisans showed that it is primarily composed of fatty acids and terpenoids. Purification of fractions C and D, allowed the complete identification of their main metabolites present in those fractions. Compound 1 isolated in fraction C was identified as cyclocolorenone and fraction D resulted on a sterols mixture based on stigmasterol, stigmasta-7,25-dien-3-ol and stigmast-7-en-3-ol. Cyclocolorenone has been previously reported on the leaf extract of Magnolia grandiflora L. To the best of our knowledge, this compound showed antimicrobial activity against B. subtilis, B. cereus, M. thermosphactum, E. coli, E. cloacae, C. freundii, C. lunata, and C. cochliodes spinusum. Furthermore, cyclocolorenone showed good growthinhibitory activity in an etiolated wheat coleoptile assay and also was phytotoxic to greenhouse corn, bean and tobacco plants (Jacyno et al., 1991). To date, no records have been found on its probable anti-inflammatory activity and other biological effects of cyclocolorenone, however the presence of an ,β unsaturated cyclopentenone ring, a reactive structure element similar to sesquiterpen lactones, it can contribute to the interaction with sulfhydryl reagents such as cysteine and glutathione thus showing pharmacological effect (Schmidt, 1997). For example, cysteine functions as a free radical scavenger via two pathways, either by binding with the radicals or by promoting the synthesis of and endogenous antioxidant; glutathione (GSH) (Zhang and Forman, 2012).

GSH is a major intracellular redox regulator peptide involved in inflammation (Nadeem et al., 2014) and it is ubiquitously found in all cell types as a major non-protein sulfhydryl compound. GSH plays a significant role in some respiratory disorders and constitutes the first line of defence against oxidative inflammation along with other enzymatic/nonenzymatic antioxidants. GSH can also affect cellular signalling through redox sensitive regulation of kinases, phosphatases and transcription factors (Sahiner et al., 2011; Ghezzi 2011). We hypothesized that cyclocolorenone might interact by a Michel-type addition with thiol groups, such as cysteine and glutathione residues modulating the inflammatory process. On the other hand, different species of the Asteraceae family, including this, contain sterols such as stigmasterol, some examples includes Eupatorium glotinosum and E. ripartum which also showed an anti-inflammatory effect (El-Seedi, 2002; Perez-Garcia et al., 2005). It has been shown that some extracts of species of the genus Eupatorium decrease the production of NO, as in the case of E. ripartum; the methanolic extract inhibits NO production in LPS-stimulated macrophages with an IC50 of 42 g/mL without causing a cytotoxic effect. From this species have been isolated compounds such as stigmasterol, epi-fidelinol acetate taraxasteril palmitate; stigmasterol and taraxasteril acetate having anti-inflammatory effect (Patra et al., 1981; Perez-Garcia et al., 2005). Plant sterols are found in seeds, roots, stems, branches, leaves and blossoms of various plants, including medicinal herbs, edible plants, shrubs and trees. The three major phytosterols in plants include sitosterol, campesterol and stigmasterol. Phytosterols have been reported to have anti-inflmmatory properties through the inhibition of proinflammatory cytokines including IL-6 and TNF-α (Jeong et al., 2001; Othman and Moghadasian, 2011). In other study, a sterol fraction composed of campesterol (7.6%), stigmasterol (28.4%) and -sitosterol (61.1%) showed anti-inflammatory activity in “in vivo” murine models of inflammation. It decreased carrageenan paw edema in mice after oral administration and inhibited mouse ear edema induced by 12-Otetradecanoylphorbol acetate (TPA) after topical application (Navarro et al., 2001). Stigmasterol, specifically, inhibits some pro-inflammatory and pro-degradative mediators involved in osteoarthritis; is able to bind to chondrocyte membrane and possesses potential anti-inflammatory and anti-catabolic properties. Stigmasterol counteracts the expression of the MatrixMetalloproteinases involved in cartilage degradation along with an inhibitory effect on the pro-inflammatory mediator PGE2, at least in part via the inhibition of the NF-kappa B

pathway (Gabay et al., 2010). The nuclear factor NF-kB pathway is considered a prototypical pro-inflammatory signaling pathway, largely based on the role of NF-kB in the expression of pro-inflammatory genes including cytokines, chemokines, and adhesion molecules (Lawrence, 2009). For these reasons, sterols and other components, either individually or synergistically, may contribute to or produce the anti-inflammatory effect observed. By the other hand, Glucocorticoids are a widely used class of anti-inflammatory and immunosuppressive drugs. According to Crinelli (2000), dexamethasone is a potent glucocorticoid analogue, which can be encapsulated in erythrocytes and selectively delivered to macrophages. Lipopolysaccharide (LPS) stimulation of dexamethasonetargeted macrophages results in the suppression of TNF-alpha secretion. The administration of dexamethasone to macrophages by means of opsonized red blood cells allows efficient interference with NF-kB activation. Furthermore, NF-kB inactivation correlated with downregulation of TNF-alpha mRNA expression in dexamethasonetargeted cells occurs at the transcriptional level. Our results showed that Hx fraction and subfractions of C. aromatisans produced a stronger anti-inflammatory effect, similar to that obtained with dexamethasone, which is promising for finding a new and better remedies with anti-inflammatory, anti-rheumatic and analgesic effect.

C. aromatisans is a potential source for the development of anti-inflammatory drugs and perhaps also for other applications in which decreased levels of TNF-α are desired (e.g., in the treatment of cancer or/and to produce an immunomodulatory effect). By the other hand, the fractions showed to be acting at the level of the enzyme COX-2, which could provide an analgesic effect. Furthermore, to attribute the observed pharmacological effect to the major components obtained, it is necessary to obtain a higher amount of the compounds and carry out several experiments. Even our results suggest that one of the mechanisms involved in the anti-inflammatory effect of C. aromatisans is inhibition of cellular mediators such NO, TNF-, IL-1, IL-6 and COX-2 enzyme, this may not be the only mechanism responsible. Therefore, exploration of the involvement of other pathways and mediators that may enhance the anti-inflammatory effect of this plant species is recommended. C. aromatisans is a plant used in traditional medicine for the treatment of inflammation and some of the symptoms caused by rheumatoid arthritis. It has been used

for many years by the Mayan communities in the Yucatan Peninsula. This research validates the medicinal effects that have traditionally been attributed to this plant.

5. CONCLUSION In this study, we report for the first time the effects that have the leaves of Critonia aromatisans. Hexanic extract of C. aromatisans displayed anti-inflammatory and immunomodulatory effects, validating the traditional practice of Mayan communities wherein an ointment with a petrolatum base, a non-polar substance, is used to treat inflammation. Additionally, C. aromatisans showed strong in vivo and in vitro activity, and one of the mechanisms of its anti-inflammatory response was shown to be inhibition of the production of NO, pro-inflammatory cytokines such like IL-1, IL-6 TNF-, and COX-2 enzyme. The presence of stigmasterol and cyclocolorenone could be the responsibles of the anti-inflammatory activity of this specie. Further studies should be done on the antioxidant and anti-inflammatory properties of these compounds. The results of this study provide a pharmacological basis for the use of C. aromatisans leaves in the treatment of inflammatory disorders

Acknowledgements The authors thank to CONACyT [Grant number 201275] for providing us the funding support to perform this study. We would also like to thank the technicians from the Institute of Anatomy, Phillips Marburg University for the assistance and support. We would also like to acknowledge Alfredo Dorantes for his support during sample collection and processing of the plant material. Finally, we thank for the technical assistance of Fundación Medina in allowing to run the NMR spectra of cyclocolorenone.

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TABLES Table 1. Evaluation of different extracts and fractions of C. aromatisans in Carrageenan (1%) -induced hind paw edema model in NIH mice. Extracts

Dose p.o. (mg/kg)

-2

Edema at 3 h (x 10 mm) ±

S.D.

Inhibition of Inflammation (%)

Control: Saline and Tween 80 at 5%

Indomethacin C. aromatisans ACN/MeOH C. aromatisans Hx C. aromatisans Hx C. aromatisans Hx

_____

75 ± 12

0

10

18 ± 10

c

76.5

200

45 ± 16

a

40.0

200

20.6 ±18

10

60 ± 9

100

12.1 ± 7

c

84.0

10

54.6 ± 10

27.1

100

57.5 ± 10

23.4

10

48 ± 22

a

35.4

100

40 ± 18

b

46.8

10

48 ± 22

a

35.4

100

43 ± 21

a

43.0

c

76.0

24.6

C. aromatisans

DCM C. aromatisans

DCM C. aromatisans EtOAc C. aromatisans EtOAc C. aromatisans Aqueous C. aromatisans Aqueous

*Values are expressed as mean ± S.D., a = p<0.05; b = p<0.01; c = p<0.001.

Table 2. Inhibition of NO production with Hx and A-G fractions obtained from C. aromatisans

in

LPS-activated

peritoneal

mice

macrophages

employing

immunoassay.

HX

A

B

C

D

E

F

G

%

%

%

%

%

%

%

%

1000

69.5

24.1

49.7

57.6

92.2

51.9

52.6

27.7

500

44.2

21.2

48.2

48.2

90.0

49.3

45.4

20.2

g/mL

ELISA

100

43.4

13.3

29.8

38.9

89.3

32.8

15.1

22.0

10

1.8

5.04

0

21.2

74.9

27.0

18.7

1.4

*Values are expressed as percentage inhibition of inflammation. For positive control cells (0% of inhibition) were only treated with LPS (Control + LPS), for negative control, cells were not treated with any substance (Control).

FIGURES Fig. 1. Evaluation of different concentrations of extracts and fractions of C. aromatisans in the inhibition of TNF- production in LPS-activated THP-1 cells, employing ELISA immunoassay. A one way ANOVA with a Dunnett’s “post hoc” test was done with a p≤ 0.05. Each extract (Tot, Hx and MA) were tested in a concentration of 50 and 100g/mL, Hexanic subfractions (A-G) were evaluated at 50g/mL. For positive control cells were only treated with LPS (Control + LPS), for negative control, cells were not treated with any substance (Control). Simultaneously, dexamethasone (Dexa), an anti-inflammatory steroid, was used also as a control. *Tot = Total extract, Hx = Hexanic fraction, MA = ACN/MeOH fraction. Fig. 2. Relative quantification of inflammatory mediators in LPS-activated THP-1 cells treated with extracts and fractions at different concentrations, employing qRT-PCR technique. A one way ANOVA with a Dunnett’s “post hoc” test was done with a p≤ 0.05. Each extract (Tot, Hx and MA) were tested in a concentration of 50 and 100g/mL, Hexanic subfractions (A-G) were evaluated at 50g/mL. For positive control cells were only treated with LPS (Control + LPS), for negative control, cells were not treated with any substance (Control). Simultaneously, dexamethasone (Dexa), an anti-inflammatory steroid, was used also as a control. *Tot = Total extract, Hx = Hexanic fraction, MA = ACN/MeOH fraction. A) Relative quantification of TNF- B) IL-1; C) IL-6; D) COX-2 enzyme.

C

C on on t tr ol rol + LP D ex S a to 0.2 ta l1 0 H 0 x 10 M 0 A 10 To 0 t5 0 H x 50 M A 50 A 50 B 50 C 50 D 50 E5 0 F5 0 G 50

TNF- (pg/mL)

Figure 1.

10000

7500

5000

2500

* *

* *

* * * *

* *

Extract concentration (g/mL)

*

* *

*

0

10

100

50

*

* * * * *

*

* *

*

*

Extract concentration (g/mL)

*

5

* *

*

* *

Relative quantification (arbitrary units)

15

0

200

C

150

*

* *

*

0

*

Extract concentration (g/mL)

*

*

IL-6

10

*

*

A5 0 B5 0 C5 0 D5 0 E5 0 F5 0 G 50

* * * *

Co Co nt nt ro ro l+ l L De PS xa To 0. ta 2 M l 10 eO 0 H 1 He 0 0 x 1 To 00 ta M l 50 eO H 5 He 0 x 50

Relative quantification (arbitrary units)

*

A

Relative quantification (arbitrary units)

on Co tr nt ol ro + l D LP ex S To a 0 t . M al 2 eO 10 H 0 H 10 ex 0 To 10 0 M tal eO 50 H H 50 ex 50 A 50 B 50 C 50 D 50 E5 0 F5 0 G 50

C

TNF-

on Co tr nt ol ro + l D LP ex S To a 0 . M tal 2 eO 10 H 0 H 10 ex 0 To 10 0 M tal eO 50 H H 50 ex 50 A 50 B 50 C 50 D 50 E5 0 F5 0 G 50

Relative quantification (arbitrary units) 20

C

Co Co nt nt ro ro l+ l De LPS x To a 0. ta 2 M l1 eO 00 H He 1 0 0 x To 100 ta M l5 eO 0 H He 5 0 x 50 A5 0 B5 0 C5 0 D5 0 E5 0 F5 0 G 50

Figure 2.

40

IL-1

B

30

20

* *

10

*

* * *

Extract concentration (g/mL)

*

*

0

Extract concentration (g/mL)

50

COX-2

D

40

30

20

* * *

0

Graphical abstract