Evaluation In Vivo and In Vitro of the Antioxidant,Antinociceptive,and Anti-Inflammatory Activities of Biflavonoids From Ouratea hexasperma and O. ferruginea

Abstract

Ouratea species are used for the treatment of inflammation-related diseases such as rheumatism and arthritic disorders. The Ouratea genus is a rich source of flavonoids and bioflavonoids and for this reason we evaluated the effects of the biflavonoid fractions from the leaves of O. hexasperma (OHME) and O. ferruginea (OFME) in the in vivo model of complete Freund’s adjuvant (CFA)-induced arthritis and in the in vitro model of oxidative stress and cellular viability. The CFA-induced arthritis model in rats was followed by paw volume, articular incapacitation and Randall-selitto models, as well as quantification of cytokines and serum C-terminal telopeptide of type I collagen levels. OHME and OFME demonstrated antinociceptive and anti-inflammatory activities, as well as improvement in articular incapacity and reduction in levels of interleukin 1β (IL-1β), IL-6, tumor necrosis factor α, and type 1 collagen, and increased cell viability. No adverse effects were observed. The results suggest that OHME and OFME can reduce inflammation and bone resorption besides their antioxidant action.

Rheumatoid arthritis (RA) is a chronic inflammatory disease that leads to a high degree of morbidity and impairment in the quality of life. RA is a predominantly articular disease, but is not confined to the joints and may cause extra-articular manifestations. Administration of complete Freund’s adjuvant (CFA) mimics RA in humans. ¹

Ouratea species (family Ochnaceae) are used in folk medicine for the treatment of inflammation-related diseases such as rheumatism, sprains, and arthritic disorders ² ; they are also the source of biologically active molecules. ³ On the basis of these considerations, the present study aimed to examine the effects of the biflavonoid fractions from the leaves of O. hexasperma (OHME) and O. ferruginea (OFME) in the in vivo model of CFA-induced arthritis and in the in vitro model of oxidative stress and cellular viability. Agatisflavone (17.8 ± 0.08 mg AE/100 mg) and amentoflavone (72.3 ± 0.01 mg AE/100 mg) were identified in the OHME fraction, while amentoflavone (30.5 ± 0.01 mg AE/100 mg) and sequoiaflavone (11.5 ± 0.0 mg AE/100 mg) were identified in the OFME fraction as major constituents. Minor constituents were not identified.

The administration of CFA induces the release of chemical mediators, such as interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), and IL-1β; these mediators stimulate the formation of other inflammatory mediators responsible for increased vascular permeability and consequent formation of edema, such as prostaglandins and adhesion molecules. ⁴ A significant increase in paw volume (PV) was induced by CFA, but dexamethasone, OHME, and OFME (10 mg/kg) maintained the paw volume during 10 days. The OHME group, at a dose of 5 mg/kg, showed an increase in PV only on day 10 (Figure 1a and d). The inflammatory changes induced by the injection of CFA produce bone and cartilaginous destruction in the joint, compromising joint mobility and gait. ⁵ In the current study, CFA produced an increase in the elevation paw time (EPT), but dexamethasone, OHME and OFME (5 and 10 mg/kg) maintained the EPT (Figure 1b and e).

Figure 1

The effects of orally administered OHME and OFME in the paw volume (a and d), articular incapacitation (b and e) and RandallSelitto (c and f) tests. The mice were treated with water, vehicle, dexamethasone (2.25 mg/kg; subcutaneous), or OHME and OFME (5 and 10 mg/kg). In (d), (e), and (f), the graph represents the area under the curve calculated for each day-effect curve. The results are expressed as the mean ± standard error of the mean (n = 7–10). *P < 0.05, **P < 0.01, and ***P < 0.001 when comparing the OHME-, OFME-, vehicle-, and dexamethasone- treated groups with the control group. ^# P < 0.05 when comparing the different days of measurement. OFME: Ouratea ferruginea methanol extract; OHME: Ouratea hexasperma methanol extract.

CFA produces tissue inflammation, which is accompanied by thermal and mechanical hypersensitivity. ⁶ On the fifth and tenth days after the CFA injection, the nociceptive withdrawal threshold was reduced, but dexamethasone, OHME and OFME (10 mg/kg) maintained the withdrawal threshold (Figure 1c and f). During the 10 days of analysis, neither behavioral changes nor significant gastric lesions were observed.

Proinflammatory cytokines stimulate synoviocytes and chondrocytes leading to destruction of cartilage and the synovial membrane. ⁷ For example, IL-6 is able to increase osteoclast activity, leading to increased type 1 collagen (CTX-1) levels in consequence of bone degradation. ⁸ Cross-linked telopeptides of collagen are the most widely used markers of bone resorption. ⁹ They are derived from the carboxyterminal (C-terminal) of type I collagen. ¹⁰ A significant increase in CTX-1 levels demonstrates bone resorption (marker of osteoclastic activity). In the dexamethasone, OHME, and OFME (10 mg/kg) groups, the CTX-I levels were reduced by 52%, 73%, and 80%, respectively (Figure 2a and b). This activity is related to cytokines levels. ¹¹

Figure 2

The effects of orally administered OHME and OFME on CTX-1 levels (a and b) and effect on cellular survival with hydrogen peroxide and preincubation with OHME and OFME (c). The mice were treated with water, vehicle, dexamethasone (2.25 mg/kg; subcutaneous), OHME (a) and OFME (b) (1, 5, and 10 mg/kg). The results are expressed as the mean ± standard error of the mean (n = 7-10). *P < 0.05, **P < 0.01, and ***P < 0.001 when comparing the OHME-, OFME-, vehicle-, and dexamethasone- treated groups with the control group; ^# P < 0.05, when comparing CFA-injected groups with the PBS-injected group. In c, the results are expressed as the mean ± standard deviation. *P < 0.05, **P < 0.01, and ***P < 0.001 when comparing the OHME + H₂O₂-, OFME + H₂O₂-, and H₂O₂- treated groups with the control group. CFA: complete Freund’s adjuvant; OFME: Ouratea ferruginea methanol extract; OHME: Ouratea hexasperma methanol extract; PBS: phosphate-buffered saline.

TNF-α levels were reduced in the dexamethasone, OHME, and OFME (10 mg/kg) groups by 33%, 29%, and 44%, respectively, but IL-10 levels were increased only by dexamethasone (86%) (Figure 3). In the dexamethasone, OHME and OFME (10 mg/kg) groups, the IL-1β levels were reduced by 50%, 40%, and 42%, while IL-6 levels were reduced by 37%, 44%, and 54%, respectively (Figure 2). Studies have shown that products of the reactions of reactive oxygen species (ROS) have been identified in knee-joint tissue/fluid samples collected from patients with RA. The excess of ROS may cause a redox imbalance in the region of the inflammation causing irreversible oxidative damage to tissue cells. ¹² The results show that OHME and OFME fractions increased the viability at the control level (no stress). The viability of the cells under oxidative stress with H₂O₂ was approximately 65% while the OFME and OHME fractions were 97% and 85%, respectively (Figure 2c).

Figure 3

The effects of orally administered OHME and OFME on quantification of TNF-α (a), IL-6 (b), IL-10 (c), and IL-1β (d). The mice were treated with water, vehicle, dexamethasone (2.25 mg/kg; subcutaneous), OHME, or OFME (5 and 10 mg/kg). The results are expressed as the mean ± standard error of the mean (n = 7-10). *P < 0.05, **P < 0.01, and ***P < 0.001 when comparing the OHME-, OFME-, vehicle-, and dexamethasone-treated groups with the control group. IL: interleukin; OFME: Ouratea ferruginea methanol extract; OHME: Ouratea hexasperma methanol extract; TNF: tumor necrosis factor.

The results suggest that OHME and OFME may act as anti-arthritic agents by downregulating cytokines, reducing the bone resorption and inflammatory events induced by CFA. This action produced beneficial responses on the inflammation, nociception, and gait of the evaluated animals. The antioxidant action demonstrated by OHME and OFME could also contribute to the reduction of joint tissue damage induced by RA. The biflavonoids produced neither behavioral alterations nor lesions on the gastric mucosa.

Experimental

Plant Material

The leaves of Ouratea hexasperma (A.St.-Hil.) Baill. were collected in the Tabuleiro Region, João Pessoa-PB, Brazil, and a voucher sample was deposited at Herbarium Lauro Pires Xavier, Joao Pessoa-PB by the botanist Maria de Fatima Agra. The leaves of O. ferruginea Engl. were collected on the campus of Embrapa in Belém, Pará State, Brazil. The species was identified by Master S. T. Rodrigues; a voucher specimen was deposited at the herbarium of Embrapa Amazônia Oriental, Belém, Pará, Brazil.

Preparation of Extracts and Flavonoid-containing Fractions

The dried and powdered leaves (Ouratea hexasperma: 1679.8 g and O. ferruginea: 344.7 g) were extracted initially with CH₂Cl₂ and afterwards with methanol at room temperature. The solvents were removed under vacuum furnishing the residues. The methanol residues were further suspended in MeOH/H₂O (8:2) and extracted with n-hexane, CH₂Cl₂, and EtOAc, obtaining, after removal of the solvents, 3 fractions for each species, P_Hex, P_Cl, and P_Ac. The P_Cl (O. hexasperma: 5.15 g and O. ferruginea: 2.50 g) of both species were individually fractionated on a silica gel column using n-hexane, CH₂Cl₂, EtOAc, and MeOH in increasing polar gradients. The biflavonoid fraction from O. hexasperma methanol extract (OHME: 18.0 mg) was obtained with CH₂Cl₂/EtOAc (1:1), while the biflavonoid fraction from O. ferruginea methanol extract (OFME: 20.0 mg) was obtained with CH₂Cl₂/EtOAc (8:2). After verification of the presence of biflavonoids by silica gel thin-layer chromatography, the fractions were submitted to qualitative and quantitative analysis by high-performance liquid chromatography (HPLC).

HPLC Analysis

HPLC-diode array detection analysis was performed using a Shimadzu Prominence system equipped with an LC-20AT pump module, an SPD-M20A diode array detector, an SIL-10AC autosampler, and a CTO-20A column compartment (Shimadzu, Japan). Data acquisition was achieved using LC Solution software (Shimadzu, Japan). Separation was performed using a Betasil C18 column (250 mm, 4.5 mm, 5 µm; Thermo Fisher Scientific, Runcorn, UK), and the temperature of the column was set at 30°C. An isocratic system with 10% water and 1% acetic acid (solvent A) and 90% methanol (solvent B) was used, and the mobile phase flow rate for the separation was 1 mL/min, as previously described. ¹³ Ouratea fractions were solubilized in methanol and solutions of 2.0 mg/mL were prepared for subsequent injection into the HPLC. The samples were filtered through a 0.45 µm HPLC filter before injection. The injection volume was 20 µL, and the chromatograms were recorded at an absorbance of 320 nm. The identification of biflavonoids present in extracts was based on a comparison of chromatographic data (retention times and UV spectra) with authentic standards (amentoflavone, agatisflavone, sequoiaflavone, putraflavone, podocarpusflavone, 7,7″-O-methyllanaraflavone). Quantitative results were expressed as milligrams of amentoflavone per 100 mg of extracts (mg AE/100 mg). The calibration curve was constructed by dilution of external standard with methanol to achieve the desired concentrations (10, 25, 50, 100, 150 µg/mL). The calibration curve (y = 65 978.157 x – 29 9654.544, R = 0.99992) was plotted relating peak area values obtained from absorbance at 320 nm against concentration (µg/mL), and the data points were well fitted by a linear regression method.

Animals

Male Wistar rats (150-200 g) were obtained from our animal facility. The animals were maintained in a room with a controlled temperature (22 ± 2°C) and a 12-hour light/dark cycle, with free access to food and water. Twelve hours before each experiment, the animals received only water to avoid a possible interference of the food with absorption of the drug. The protocol for this study was approved by the Ethics Committee on the use of animals of the Institute of Biological Sciences and Health of the Federal Rural University of Rio de Janeiro (CEUA-IB – UFRRJ) under number 011/2015.

Treatments

Increasing doses of the OHME and OFME were administered orally (1, 5, and 10 mg/kg) and dexamethasone (2.25 mg/kg subcutaneous administration steroidal anti-inflammatory) ¹⁴ was used as a positive control in the CFA-induced arthritis model. The control group consisted of rats that received only distilled water, and the vehicle group was formed by animals that received distilled water solution in DMSO at a concentration of 1.0% (solubilizing agent).

CFA-induced Chronic Arthritis

Experimental arthritis was induced in rats according to a previously published method. ¹⁵ Each rat was injected with 0.1 mL of CFA into the subplantar region of the left hind paw on day 1. The administration of OHME and OFME started from day 1, once a day, orally, for 10 days, while the dexamethasone was administered intraperitoneally on days 1, 3, 5, 7, and 9. PV, mechanical threshold (MT), and EPT were evaluated on days 1, 5, and 10. On day 10, blood was withdrawn by retro-orbital puncture for assessment of CTX-1 and cytokines levels.

Paw Volume Test

The left hind PVs of all animals were measured before CFA injection (day 1), day 5, and day 10. Edema was measured by a plethysmometer ¹⁶ . The results are expressed as PV and also expressed by calculating the area under the curve (AUC) of responses from day 1 to day 10 after CFA injection, using the following formula based on the trapezoid rule:

$${\displaystyle \mathrm{A}\mathrm{U}\mathrm{C}=5\times \mathrm{P}\mathrm{V}[(\mathrm{d}\mathrm{a}\mathrm{y}1)+\dots +(\mathrm{d}\mathrm{a}\mathrm{y}10)/2]}$$

Articular Incapacitation Test

The articular incapacitation test has been described in detail. ¹⁷ In this test, the animals are placed on a metal cylinder 30 cm in diameter carrying 3 rpm. The cylinder covered by a metallic screen is connected to an electronic device, and this to a computer, which ascertains the total time the injured paw remains in elevation for 5 minutes through identical metal boots engaging the rear feet of the animal. The results are expressed as EPT and also expressed by calculating the AUC of responses from days 1 to 10 after CFA injection, using the following formula based on the trapezoid rule:

$${\displaystyle \mathrm{A}\mathrm{U}\mathrm{C}=5\times \mathrm{E}\mathrm{P}\mathrm{T}[(\mathrm{d}\mathrm{a}\mathrm{y}1)+\dots +(\mathrm{d}\mathrm{a}\mathrm{y}10)/2]}$$

Randall-Selitto Test

The nociceptive withdrawal threshold was assessed by using the Randall-Selitto ¹⁸ electronic algesimeter (IITC 2500 Digital Paw Pressure Meter, IITC Life Science, Woodland Hills, CA). Before the test, each animal was trained for 5 minutes to allow manipulation. The test consisted of the application of an increasing mechanical force, in which the tip of the device was applied onto the medial portion of the plantar or dorsal surfaces of paws injected with CFA until a withdrawal response. The results are expressed as MT and also expressed by calculating the AUC of responses from days 1 to 10 after CFA injection, using the following formula based on the trapezoid rule:

$${\displaystyle \mathrm{A}\mathrm{U}\mathrm{C}=5\times \mathrm{M}\mathrm{T}[(\mathrm{d}\mathrm{a}\mathrm{y}1)+\dots +(\mathrm{d}\mathrm{a}\mathrm{y}10)/2]}$$

Serum C-Terminal Telopeptide of CTX-1 Levels

Serum CTX-1 levels were determined by competitive enzyme immunoassay using a commercial kit (Immunodiagnostic Systems, UK). Inter- and intra-assay coefficients of variation were 10.4% and 6.9 %, respectively.

Cytokines Quantification

The blood was collected and centrifuged at 1500 × g for 10 minutes at room temperature. The supernatants were used to measure IL-1β, IL-6, TNF-α, and IL-10. The cytokines were quantified by an enzyme-linked immunosorbent assay using the protocol supplied by the manufacturer (Peprotech).

Toxicological Evaluation

Toxicological evaluation was determined following the experimental model described previously. ¹⁹ The animals submitted to treatment with OHME and OFME were observed in relation to behavioral parameters (for 10 days), including convulsion, hyperactivity, grooming, loss of righting reflex, either increased or decreased respiration, and sedation. After this period, stomachs were removed and an incision along the greater curvature was made. The number of ulcers (single or multiple erosions, ulcer, or perforation) and hyperemia were measured.

Oxidative Stress and Cellular Viability

BY4741 (MATa; his3; leu2; met15; ura3) was acquired from Euroscarf (Frankfurt, Germany). Stocks of yeast strain were maintained on solid 2% YPD (1% yeast extract, 2% glucose, 2% peptone, and 2% agar). Yeast cells at the mid-log phase of growth (1.0 mg dry weight/mL) were incubated with the compounds at a concentration of 25 µg/mL for 120 minutes at 28°C and 160 rpm. Cells were harvested by centrifugation and washed twice with 50 mM phosphate-buffered saline (PBS), pH 6.0. Thereafter, pellets were resuspended in the same PBS either containing 1.0 mM H₂O₂ and maintained for 60 minutes at 28°C and 160 rpm. Cellular viability was determined by plating in triplicate on solidified YPD medium, after proper dilution, before and after stress conditions, and was expressed as the percentage survival. The plates were incubated at 28°C for 72 hours and the colonies counted. H₂O₂ (30%) solution was obtained from Vetec (Rio de Janeiro, Brazil).

Statistical Analysis

All experimental groups consisted of 7-10 animals. The results are presented as the mean ± standard error of the mean. Statistical significance between the groups was determined using one-way analysis of variance (ANOVA) followed by Tukey’s test for the evaluation of PV, MT, EPT and evaluation of oxidative stress and cellular viability, and two-way ANOVA followed by Tukey’s test was used for the AUC and quantification of CTX-1 and cytokines. P values of less than 0.05, 0.01, and 0.001 were considered to be statistically significant.