In vitro and in vivo delivery of atorvastatin: A comparative study of anti-inflammatory activity of atorvastatin loaded copolymeric micelles

Abstract

Statins have been shown to exert ‘pleiotropic effects’ independent of their cholesterol lowering actions that include anti-inflammatory properties. In this study we synthesized mono methoxy poly (ethylene glycol)–poly (ε-caprolactone) (mPEG-PCL) di block copolymers. The structure of the copolymers was characterized by H nuclear magnetic resonance, Fourier-transform infrared spectroscopy, differential scanning calorimetry and gel permeation chromatography techniques. In this method, atorvastatin was encapsulated within micelles through a single-step nano-precipitation method, leading to the formation of atorvastatin-loaded mPEG-PCL (atorvastatin/mPEG-PCL) micelles. The resulting micelles were characterized further by various techniques such as dynamic light scattering and atomic force microscopy. In this study the anti-inflammatory activity of atorvastatin and atorvastatin/mPEG-PCL micelles on acute models of inflammation are analyzed, to compare the effect of indometacin in rats. Carrageenan induces rat paw edema; six animals of each group (10 groups) received indometacin, atorvastatin, and atorvastatin/mPEG-PCL micelles orally 1, 6, 12 and 24 h before carrageenan injection in paw. The paw edema thickness measured at 1, 2, 3 and 4 h after injection and percentage inhibition of edema in various groups were calculated. The results showed that the zeta potential of micelles was about −16.6 mV and the average size was 81.7 nm. Atorvastatin was encapsulated into mPEG-PCL micelles with loading capacity of 14.60 ± 0.96% and encapsulation efficiency of 62.50 ± 0.84%. Atorvastatin and atorvastatin/mPEG-PCL micelles showed significant anti-inflammatory activity in the present study. The anti-inflammatory activity of atorvastatin and atorvastatin/mPEG-PCL micelles was significant in comparison with indometacin. Atorvastatin/mPEG-PCL micelles showed more anti-inflammatory activity than atorvastatin. This study revealed the anti-inflammatory activity of atorvastatin and atorvastatin/mPEG-PCL micelles and suggested the statins have a potential inflammatory activity along with its lipid lowering properties. Contrary to anti-inflammatory effects, the pro-inflammatory responses are independent of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibition and can be mediated directly by atorvastatin.

Keywords

Copolymer sustained release micelles atorvastatin anti-inflammatory

Introduction

Hyperlipidemia is a major cause of atherosclerosis and atherosclerosis-associated conditions such as coronary heart disease (CHD), ischemic cerebrovascular disease and peripheral vascular disease. Many well-controlled clinical trials established the benefits of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) on fatal and nonfatal CHD events and strokes.^1–5 The observation that statins reduce the risk of cardiovascular events even in the absence of a significant decrease of blood cholesterol levels supports the relevance of the potential ‘pleiotropic’ functions of this drug class.^6,7 A large number of studies reported a prominent role of inflammation and immune response on the development of atherosclerotic plaques and their destabilization.^8–10 Approximately, all in vitro and in vivo studies uniformly support anti-inflammatory roles of statins. Anti-inflammatory properties of statins probably include various mechanisms that may or may not involve the HMG-CoA reductase/mevalonate pathway.¹¹ However, a recent report highlights the pro-inflammatory effects of statins in mitogen-activated peripheral blood mononuclear cells through the activation of caspase-1 and IL-18 secretion in monocytes.¹² Atorvastatin is in a group of drugs called HMG CoA reductase inhibitors, or ‘statins’. Atorvastatin reduces levels of ‘bad’ cholesterol (low-density lipoprotein, or LDL) and triglycerides in the blood, while increasing levels of ‘good’ cholesterol (high-density lipoprotein, or HDL). Atorvastatin is used to treat high cholesterol, and to lower the risk of stroke, heart attack, or other heart complications in people with type 2 diabetes, CHD, or other risk factors. Atorvastatin is used in adults and children who are at least 10 years old. Methoxypoly (ethylene glycol) / poly (ε-caprolactone)/(mPEG/PCL) is a diblock PCL/PEG copolymer that self-assemble into nanoparticles with core–shell structure: a hydrophobic PCL core and a hydrophilic PEG shell. Although encapsulated with the hydrophobic drug, the hydrophobic PCL segment joint with the drug forms the core and the hydrophilic PEG forms the shell of nanoparticles, assembly the drug intravenously injection. An additional entrapment with hydrophobic drugs mPEG/PCL copolymers possibly will improve constancy and systemic distribution of drugs and release drugs at a sustained time in the finest variety of drug concentration. Furthermore, mPEG/PCL copolymers are recyclable, biocompatible and simple to construct, showing hopeful applications in drug-delivery systems.^13–16 New mPEG-PCL micelles were applied to carry hydrophobic drugs with the aim to bring narrative aqueous formulations for these drugs.^17,18 In the previous studies we used magnetic nanoparticles for drug delivery systems.^19–21 In this study, in an effort to support an aqueous formulation for atorvastatin, we organized atorvastatin encapsulated mPEG-PCL micelles. The aim of this study is to compare the anti-inflammatory effects of atorvastatin and atorvastatin loaded micelles in carrageenan-induced rat paw edema. Hence the present study was planned to study and compare the anti-inflammatory activity of atorvastatin and atorvastatin/mPEG-PCL micelles.

Materials and methods

Materials

mPEG (Mn = 5000 Da was from Aldrich (St. Louis, MO, CAS.81323), 2-ethyl-hexanoate (Sn(Oct)2) from Aldrich (CAS. 301100), ε-caprolactone (98% purity) from Acros (NJ, CAS.502443) and carrageenan was from Sigma-Aldrich (Germany). Atorvastatin was a kind gift from Sobhan Pharmaceutical Inc (Sobjan-Daroo, Tehran). Other chemicals and solvent were of chemical lab purity grades, purchased from Emertatchimi.

Synthesis of mPEG–PCL copolymers

mPEG–PCL diblock copolymers were prepared according to our previous report.^22,23 It was synthesized by a ring opening polymerization of ε-caprolactone with mPEG as initial molecule and Sn(Oct)₂ as catalyst. In brief, ε-caprolactone (6 g), mPEG (1 g) and Sn(Oct)₂ (0.01 mmol) were heated to 120°C to initiate polymerization. After 12 h, the resultant copolymers were cooled to room temperature, dissolved in chloroform and precipitated in cold diethyl ether. The copolymer was dried under vacuum at room temperature for 24 h.

Characterization of mPEG-PCL copolymers

The chemical structure of copolymers was known by Fourier transform infrared spectroscopy (FTIR) (Bruker, Tensor 27) and proton nuclear magnetic resonance spectroscopy (1H NMR) in CDCl₃ at 400 MHz (Bruker, Avance 400). Differential scanning calorimetry (DSC) (Mettler Toledo, model Star SW 9.30) was applied for thermal analysis of the synthesized copolymers. Samples were heated at a rate of 10°C min⁻¹ and the results were recorded from 0 to 200°C. The average molecular weight and distribution of the mPEG–PCL copolymers were indomitable by gel permeation chromatography (GPC) (Knaure, Berlin, Germany) set with differential refractometric detector and an Ultrastyra gel column (4.6×30 mm) (Waters, Milford, CT, model HR 4E). The mobile phase was tetrahydrofuran (THF) with a flow-rate of 1 mL/min and the injection volume was 50 µL of stock solutions (0.1–0.5 w/v%). Polymers were characterized by relative elution time to polystyrene monodisperse standards in the range of 1500–35500 Da (Varian Palo Alto, CA) using the calibration obtained before the measurements.

Preparation of atorvastatin-loaded micelles

Atorvastatin-loaded micelles was prepared according to our previous report.^24–26 Nanoprecipitation method was used for preparation of micelles. The acetone was used as the solvent in this method. In brief, mPEG–PCL copolymer (20 mg), and atorvastatin (6 mg) were dissolved in 2 mL of acetone. The solution was, then, added drop-wise (G = 22) into 25 mL of distillated water using a syringe under certain combination rates and stirred magnetically at room temperature awaiting whole disappearance of the organic solvent which caused the amphiphilic copolymers to self-assemble to form the micelles. After removal of acetone by rotary vacuum evaporation at 35°C, the resultant aqueous solution was filtered through a 0.45 µm filter membrane to eliminate the unloaded atorvastatin. The resulting molecules were separated by centrifuging at 20,000g for 20 min and freeze-dried under a pressure of 14 Pa at −78°C until the removal of all the residual solvents and to obtain the final dried form of micelles loaded by atorvastatin.

Characterization of the micelles

Particle morphology

The morphology of micelles was analyzed using atomic force microscopy (AFM) (JPK, Berlin, Germany, model Nano Wizard). For AFM sample preparation, micelles were diluted with water and a droplet of 2 μL was placed onto a freshly cleaved mica substrate (1 cm²) and air-dried. AFM measurements were performed in intermittent contact mode.

Determination of particle size

The particle size distribution of the organized micelles was assigned by dynamic light scattering (DLS) using a nano/zetasizer (Malvern Instruments, Worcestershire, UK, model Nano ZS).

Stability of micelles

For evaluation of the physical stability of micelles the particle size distribution of the micelles was monitored in phosphate-buffered solution (PBS, pH = 7.4) and kept in room temperature for 0, 15, 30, 60, 90, 120, 150 and 180 days after grounding using the method described in the above section.

Determination of loading efficiency

Two parameters including the drug loading ratio and efficiency of entrapment were determined for determining the loading efficiency of the drugs in the micelles. Drug loading ratio was determined as

% D L = \frac{weight of drug in micelles}{weight of micelles} ×100

(1)

where DL% is the drug loading ratio (%), and W_drug and W_micelles signify the weight of the entrapped drug and the total weight of the consequent drug-entrapped micelles, respectively.

For determination of the drug loading ratio, 1 mg of the final freeze-dried nanodispersion was dissolved in 1 mL of acetone, and the drug content was estimated spectrophotometrically (Thermo Fisher Scientific, Madison, model GENESYS™ 10S) at wavelengths of 248 nm.^27,28.

Encapsulation efficiency was determined using the following equation

% E E = \frac{(weight drug in micelles)}{weight of inital drug} ×100

(2)

FTIR analysis

Using FTIR analysis, it is probable to achieve a number of information regarding the incidence of achievable interaction(s) between substances concerned in a nano carrier system. Usually, the interaction between drug and polymer is evaluated during the band shifts exerted by the functional groups as well as through broadening in IR spectra compared to their individual spectra. To verify the attendance of any interactions between drug and polymer, the FTIR spectra of solid micelles were compared with pure drug and individual polymers. Freeze-dried samples were pressed to form the standard disks and the FTIR spectra of the KBr disks were recorded using the aforementioned instrument from 600 to 4000 cm⁻¹.

DSC analysis

DSC analysis was applied to make available further information on the drug–copolymer relationship and possible drug–polymer interaction(s) as well as the physical changes on the drug or polymer can be considered by means of the thermal analysis. DSC analysis was carried out on pure drug and drug-loaded micelles. Samples were heated at a rate of 10°C min⁻¹ and the data were recorded from 0 to 200°C.

Drug release study

This investigation was approved to estimate the release of atorvastatin from micelles. In brief, 5 mg of freeze-dried nanoparticles were dispersed in 2 mL phosphate-buffered saline (PBS) containing 5% (v/v) Tween 80 and the follow-on suspension was sited within a dialysis sac (M_w 12 kDa) and incubated at 37°C while immersed in 15 mL of PBS. Then, at programmed time intervals, 2 mL of the dialysate was taken out and replaced by 2 mL new PBS. The concentration of atorvastatin in the dialysate was assigned by UV-Vis. All the release studies were carried out in triplicate. To facilitate the study of pH-dependency of the drug release, the experiments were also carried out, as specified earlier, using PBS at a pH of 5.5. As controls, the release of free atorvastatin was studied in PBS with pH = 7.4 and 5.5.

In vivo study

Experimental animals

Male Wistar rats (200–250 g; n = 6–8) were used in this study. The animals were given food and water ad libitum. They were housed in the Animal House of Zanjan University of Medical Sciences at a controlled ambient temperature of 25 ± 2°C with 50 ± 10% relative humidity and with a 12-h light/12-h dark cycle (lights on at 7:00 a.m.). This study was performed in accordance with the Guide for the Care and Use of Laboratory Animals of Research affairs of Zanjan University of Medical Sciences, Zanjan, Iran.

Carrageenan-induced paw edema

Carrageenan-induced rat paw edema was used as an acute inflammation model. Male Wistar rats received a sub plantar injection of 100 μL of 1% carrageenan in saline in the right hind-paw. After carrageenan injection the footpad volume was measured by a Vernier caliper and then at hourly intervals from 1–4 h afterwards. Data are expressed as percent increase in paw thickness compared to the pre-injection values. Rats received carboxymethylcellulose (CMC 0.5%; control), indomethacin (10 mg/kg; positive control), or 5, 10, and 20 mg/kg of atorvastatin and nanoparticles in 0.5% CMC orally 24, 12, 6 and 1 h prior to inflammation induction.

Histological examination

Histological studies were carried out to determine leukocytes accumulation and the relationship between microscopic structure of paw tissue and inflammation induced by carrageenan in paw. Biopsies of paws were taken and the tissue slices were fixed in 10% neutral buffered formaldehyde, embedded in paraffin and sectioned. The sections were stained with hematoxylin and eosin and studied by light microscopy.

Cholesterol and triglyceride assay

At the end of experiments rats were anaesthetized by intra peritoneal (i.p) injection of pentobarbital (60 mg/kg) and blood were collected from jugular vein. Serum total cholesterol and triglyceride were assayed by a standard enzymatic method of Watson, Fossati and Principe, respectively.^29,30

Statistics

Data were presented as mean ± SD. Comparisons between groups were made with Student’s t test or ordinary ANOVA as appropriate. If ANOVA analysis indicated significant differences, a Student–Newman–Keuls post test was performed to compare mean values between treatment groups and control. Differences between groups were considered significant at p < 0.05.

Results and discussion

Synthesis and characterization of mPEG–PCL copolymer

mPEG–PCL di-block copolymer was synthesized using the ring-opening polymerization of caprolactone in presence of mPEG, whose hydroxyl end group initiated the ring opening and explained in the previous work (Figure 1). The structure and composition of the synthesized mPEG–PCL di-block copolymer were determined by HNMR spectroscopy in CDCl₃. The presence of methylene (CH₂) in PCL was observed around 1.44, 1.65, 2.33 and 4.09 ppm, the methoxy and methylene protons in methoxy(OCH₃) and methylene (CH₂) groups of PEG were around 3.39 and 3.67 ppm, respectively. Characteristics of the synthesized copolymer are shown in Table 1. FTIR spectrum of mPEG–PCL copolymer is shown in Figure 4(c). In the spectrum shown, the sharp and intense bands at 1728.70 and 1189.25 cm⁻¹ were awardable to the presence of carboxylic ester (C=O) and ether (C–O) groups, thereby indicating that the formation of mPEG–PCL copolymer has been successful. GPC results showed that the weight-based average molecular weight of copolymer is 21,364 Da (Table 1). The endothermic peak (63.58°C) of DSC thermograms of the copolymer included two merged peaks of PEG and PCL.

Table 1.

Molecular characteristics of the synthesized copolymers.

Copolymer	PEG M_w (g/mol)	CL/EG feed	M_w (Da)a	M_n (Da)a	PDIb	T_m (°C)c	DP_PEG	DP_PCL
P1	5000	5	21,364	17,058	1.25	63.58	136.36	143.37

DP: degree of polymerization.

^aDetermined by GPC analysis using narrow molecular weight polystyrene standards.

^bM_w/M_n = polydispersity index (PDI) of the polymers determined by GPC analysis.

^cCalculated from the first run of DSC as half of the extrapolated tangents.

Preparation and characterization of copolymeric micelles

The formation of micellar nanostructures was confirmed by AFM apparently; mPEG–PCL micelles showed a homogeneous spherical morphology, as expected (Figure 2). The size of nanoparticles was measured by DLS technique. As shown in Figure 3, the z-average and zeta potential of atorvastatin loaded micelles were found to be about 81.7 nm with zeta potential −16.6 mV, with their corresponding polydispersity index (PDI) being 0.172 (Figure 3). The micelle size observed by AFM was about 70 nm, a little smaller than that determined by DLS. It can be explained by the fact that the micelle diameter determined by DLS represents the hydrodynamics diameter while that obtained by AFM is related to the collapsed micelles after water evaporation. The loading ratio and encapsulation efficiencies of atorvastatin loaded to mPEG–PCL micelles were determined to be 14.60 ± 0.96% and 62.50 ± 0.84%, respectively.

FTIR analysis

The FTIR spectra of mPEG–PCL copolymer, atorvastatin and atorvastatin/mPEG-PCL micelles were presented in Figure 4. The FTIR spectrum of atorvastatin shows feature bands indicated at 1622.47 cm⁻¹ for C=C bands, 746.25 cm⁻¹ for C–H stretching of aromatic ring, 3412.56 cm⁻¹ (O–H, and N–H stretching) for atorvastatin and 1506.90 cm⁻¹ (C = C band of benzene). By comparing these data with the drug-loaded micelles spectrum indicates the existence of atorvastatin characteristic peaks in the spectrum of micelles which could express the successful loading of atorvastatin in the micelles. The majority remarkable characteristic of the FTIR spectra of micelles was the blue shift of the C=O vibration, from 1720.68 to 1735.88 cm⁻¹ for atorvastatin-loaded micelles compared to the mPEG–PCL spectrum. The shift in the micelles spectrum shows the presence of some form(s) of relationship between atorvastatin and C=O functional group of mPEG–PCL.

DSC analysis

Figure 5 shows the DSC thermograms corresponding to mPEG–PCL copolymer, atorvastatin and micelles loaded by atorvastatin. The thermogram of mPEG–PCL copolymer displayed an endothermic peak at 63.58°C which indicates the melting of crystalline PCL segment of copolymer; the thermogram of atorvastatin displayed an endothermic peak at 168.40°C which was not observed in nanoparticulate atorvastatin. Thus, it could be concluded that the atorvastatin in the NPs was in an amorphous or disordered crystalline phase or in a solid solution state and micelles displayed one endothermic peak at 56.87°C which stand for the melting of copolymer association in the form of micelles. This endothermic peak of micelles presumably confirms a physical interaction between copolymer and atorvastatin upon loading of the drug in micelles, since the melting point of PCL micelles was lower than melting point of copolymer. The peak was observed at 61.37°C for the physical mixture of copolymer and atorvastatin.

Physical stability of micelles

In this work, the stability of particle size was checked over a 150-day course. The variation of the sizes of micelles as a function of incubation time is shown in Figure 6. It can be seen clearly that the size of all micelles was increased slightly. This observation cannot be a sign of aggregation, which usually leads to several fold increases. Probably swelling and/or hydration as a result of the presence of the hydrophilic PEG portions in micelles surfaces can be responsible for this event.

In vitro release of atorvastatin

In order to examine the influence of the chemical and biochemical factors on the release of atorvastatin from micelles, the release study was performed on drug-loaded micelles in neutral (pH = 7.4) and acidified PBS solution (pH = 5.5). For controls, the release of free atorvastatin was studied to verify that the diffusion of drug molecules across the dialysis membrane was not a rate-limiting step during the release process. Free atorvastatin was observed to be rapidly released and reached its peak of 83.3% and 84.14% of the total in the first 10 h at pH 7.4 and pH 5.5, respectively. Figure 7 shows the release profiles of atorvastatin from the drug-loaded micelles, at pH 7.4 and pH 5.5. As expected, no considerable initial burst atorvastatin release was observed from the micelles. As shown in Figure 7, the percentage of atorvastatin released from the micelles increased as the pH value decreased from 7.4 to 5.5. For example, after 72 h incubation, the amounts of atorvastatin released in the media with pH values of 7.4 and pH 5.5 were about 73.32% and 82.21%, respectively. The reason behind this phenomenon lies in the pH sensitivity of the release rate of atorvastatin from the micelles because the copolymer is degradable in acidic condition by hydrolysis. Also, the release is faster in acidic pH than in neutral, as in acidic environment the polymer matrix swells due to protonation of polymer. This behavior is a highly desirable characteristic in many applications especially in anticancer drug delivery where the micro-environments of extracellular spaces of tumors, intracellular lysosomes and endosomes are acidic, which can potentially facilitate the drug release from obtained micelles. The results revealed that the maximum drug releases were 73.32% and 92.21%, respectively, for PBS pH = 7.4, and pH = 5.5 after a period of 72 h. The sustained release of atorvastatin can be attributed to the entrapment of atorvastatin in core of micelles. Therefore, the obtained copolymeric micelles can be regarded as highly attractive nano-carriers for time-controlled drug delivery for hydrophobic drugs to achieve different therapeutic objectives.

Effects of statins on carrageenan-induced paw edema

Sub plantar injection of carrageenan into the paw resulted in swelling of the footpad that can be reproducibly measured after 4 h. Treatment with indomethacin blocked swelling markedly (Figure 8 and Table 2). We found that oral administration of atorvastatin and atorvastatin loaded micelles 24, 12, 6 and 1 h prior to induction of inflammation reduced both the maximal edema response attained during 4 h and neutrophils infiltration into the inflammation zone (Figure 8). Atorvastatin loaded micelles had greater effects than atorvastatin (Figure 8). The strongest inhibitory effect on the total edema response which was comparable to that of indomethacin was seen by atorvastatin loaded micelles (20 and 10 mg/kg). These observations strongly suggest that oral treatment by statins has anti-inflammatory activities. Plasma lipids were measured in samples taken at the end of the experiments for all groups (Figure 9). The statins did not alter plasma cholesterol and triglycerides. Serum C reactive proteins were measured in samples taken at the end of the experiments for all groups (Figure 10). The statins did not alter serum C reactive protein significantly but show a decreasing process. The footpad swelling represents an acute inflammatory response characterized by the influx of polymorphonuclear (PMN) leukocytes. Statins blocked the influx of PMN leukocytes into the paw 4 h after carrageenan injection (Figure 11). As shown in Figure 11, atorvastatin 5 mg/kg did not affect the ability of leukocytes to migrate at the inflammation site, whereas the dose of 10 and 20 mg/kg of atorvastatin reduced the number of total leukocytes recruited by carrageenan (data not shown).

Table 2.

Effects of atorvastatin and atorvastatin loaded nanoparticles (orally) and indomethacin (positive control) on carrageenan-induced paw edema in rats compared to control group (vehicle).^a

Group	Increase in paw thickness (%) ± SD
Group	1 h	2 h	3 h	4 h
Control (carrageenan)	44.12 ± 0.17	46.41 ± 0.19	43.15 ± 0.18	38.6 ± 0.15
mPEG–PCL	42.5 ± 0.03	42.2 ± 0.06	48.1 ± 0.04	45.1 ± 0.03
Indometacin (2.5 mg/kg)	20.09 ± 0.03	15.03 ± 0.02	11.14 ± 0.04	8.4 ± 0.04
Atorvastatin (5 mg/kg)	36.56 ± 0.05	43.67 ± 0.06	42.03 ± 0.06	38.8 ± 0.05
Atorvastatin (10 mg/kg)	26.92 ± 0.08	36.57 ± 0.13	40.77 ± 0.14	37.04 ± 0.13
Atorvastatin (20 mg.kg)	25.32 ± 0.04	37.57 ± 0.07	39.28 ± 0.14	36.59 ± 0.14
Atorvastatin loaded nanoparticles (5 mg/kg)	32.97 ± 0.01	37.72 ± 0.03	37.62 ± 0.05	33.54 ± 0.07
Atorvastatin loaded nanoparticles (10 mg/kg)	33.87 ± 0.04	32.42 ± 0.09	31.98 ± 0.1	29.96 ± 0.08
Atorvastatin loaded nanoparticles (20 g/kg)	21.18 ± 0.06	18.37 ± 0.02	22.65 ± 0.04	19.21 ± 0.05

^aDrugs were given orally 20, 12, 6 and 1 h before carrageenan injection into the paws.

Conclusion

In this work, mPEG–PCL copolymer was synthesized and its structure was confirmed by HNMR and FTIR. Thermal behavior and molecular weight of synthesized copolymer were studied and determined by DSC and GPC, respectively. This copolymer was converted to the micelles by nanoprecipitation method, and applied for loading atorvastatin. The obtained data showed that the drug release from micelles were clearly sustainable for a long time. Therefore, it is to be concluded that the micelles are a suitable carrier for delivery of atorvastatin. Ultimately, the outcome of the present research work provides valuable information about development of novel drug delivery systems, and performance of nonspherical carriers with high strength. Hence, such carriers can lower the systemic side effects of the drugs by reducing the intake doses. To this study we showed that oral administration of atorvastatin and micelles have lipid lowering-independent anti-inflammatory and antileukocyte accumulation activities in carrageenan induced rat paw edema model.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: research deputy of Zanjan University of Medical Sciences, Zanjan, Iran (grant No. A-12-430-5, A-12-430-7).

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