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Ocimum gratissimum Linn. Leaf extract inhibits free radical generation and suppressed inflammation in carrageenan-induced inflammation models in rats

  • Abayomi M. Ajayi , Mary O. Ologe , Benneth Ben-Azu , Samuel E. Okhale , Bulus Adzu and Olusegun G. Ademowo EMAIL logo
Published/Copyright: March 22, 2017
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Journal of Basic and Clinical Physiology and Pharmacology
From the journal Journal of Basic and Clinical Physiology and Pharmacology Volume 28 Issue 6

Abstract

Background:

Ocimum gratissimum leaf is used in managing rheumatism and other inflammatory conditions. In this study, we investigated the antioxidant and anti-inflammatory effects of phenolic extract obtained by sequential methanol extraction of O. gratissimum leaves (MEOg).

Methods:

The methanol extract (MEOg) was obtained after sequential maceration (n-hexane, chloroform and methanol) of dried O. gratissimum leaves. The fingerprint of the extract was obtained using a high-performance liquid chromatrographic method. In vitro effects were tested by 1,1-Diphenyl-2-picryl-hydrazyl (DPPH), nitric oxide (NO) free radical scavenging, lipoxygenase, and xanthine oxidase inhibitory assays. MEOg was studied for anti-inflammatory activity in carrageenan-induced paw edema and air pouch inflammation in rats.

Results:

HPLC fingerprint of the extract shows the presence of caffeic acid, rutin, ferulic acid, apigenin, and quercetin. Antioxidant activity of MEOg revealed an IC50 value in DPPH (31.5±0.03 μg/mL) and NO assay (201.6±0.01 μg/mL), respectively. The extract demonstrated strong xanthine oxidase inhibitory and weak antilipoxygenase activities. MEOg (100 mg/kg) significantly inhibited carrageenan-induced paw edema by 43.2%. Furthermore, MEOg (50 and 100 mg/kg) significantly reduced exudate volume, leucocyte count, neutrophil infiltration, TNF-α, nitrites, myeloperoxidase, and malondialdehyde in carrageenan-induced air pouch inflammation. MEOg also elevated the glutathione levels in the inflammatory exudates.

Conclusions:

MEOg shows potential therapeutic benefits in slowing down inflammation and oxidative stress in chronic diseases, such as arthritis.

Keywords: anti-inflammatory; antioxidant; apigenin; carrageenan; phenolic; xanthine oxidase

Introduction

Inflammation is an orchestrated biological process that is induced by microbial infection or tissue injury. Inappropriately triggered or incompletely controlled inflammatory responses are a major cause of ill health, impaired quality of life, loss of time from work or education, and untimely death [1]. Inflammatory diseases affect many people in the world and represent the greatest collective burden of suffering and economic cost. Acute inflammation is a process that is characterized by an increase in vascular permeability and cellular infiltration, leading to edema formation at the site of inflammation [2]. During acute inflammation, recruited mast cells and leukocytes go to the site of damage, causing a “respiratory burst” due to an increased uptake of oxygen, which in turn leads to increased release and accumulation of reactive oxygen species (ROS) at the site of damage. The imbalance between production of ROS and their elimination by protective mechanisms, referred to as antioxidants, is therefore defined as the oxidative stress [3].

The consequences of oxidative stress include oxidative inactivation of antiproteinases, increased sequestration of neutrophils in the microvasculature, and gene expression of proinflammatory mediators [4]. An approach to mitigate continued oxidative stress with herbal extracts having both antioxidative and anti-inflammatory properties has received more attention among researchers working on herbal extracts. The evaluation of antioxidant status during a free-radical challenge is often used as an index of protection against inflammatory processes in experimental conditions for therapeutic measures [5].

Ocimum gratissimum L. is an economically viable multipurpose medicinal herbaceous plant reported in several ethnopharmacological surveys. Popularly known as African basil in English, it is a shrub commonly found around village huts and gardens, is widely used in traditional medicine, and readily accessible to the communities [6, 7]. Ocimum gratissimum, called “efinrin” by the Yoruba people in Nigeria, is widely used as spice and teas, and to treat various ailments associated with inflammation, such as rheumatism, hemorrhoids, bronchitis, and stomatitis [6, 8]. Ocimum gratissimum leaves prepared by maceration, decoction, or infusion in water or alcohol is used in the treatment of different inflammatory disorders [9, 10]. Its leaves are known to be rich in essential oils and phenolic compounds, which are useful ingredients for the treatment of rheumatism [11]. The longstanding and successful use of O. gratissimum in traditional medicine makes it necessary to find a rationale for the pharmacological and therapeutic superiority of its extracts.

Previous reports have shown that aqueous alcohol extracts from the leaves are significantly reducedin carrageenan-induced rat paw edema [12, 13]. The essential oil has also shown anti-nociceptive activity [14]. The ethanol extract of fresh leaves has been reported to suppress collagen-induced arthritis in rats [15]. Secondary metabolites reportedly present in the leaves include rutin, quercetin, caffeic acid, rosmarinic acid, circhoric acid, sitosterol, ursolic acid, salvigenin, and transferulic acid [16, 17]. Therefore, the current study aimed to evaluate the inhibition of free radical generation and the in vivo anti-inflammatory activity by the phenolic-rich extract of O. gratissimum leaves.

Materials and methods

Drugs and chemicals

Carrageenan (type 1), indomethacin, sodium nitroprusside, O-dianisidine, sulfanilamide, N-(1-naphthyl) ethylenediamine dihydrochloride, xanthine, and xanthine oxidase were obtained from Sigma Aldrich (Steinheim, Germany). Sodium nitrite, aluminum chloride, and Folin–Ciocalteu reagent were products of BDH (BDH Chemicals Ltd, Poole, England). The TNF-α ELISA kit came from Biolegend (San Diego, USA). All other solvents and chemicals were analytical-grade reagents.

Plant material collection and extraction

The plant was identified and authenticated by a curator at the herbarium of the Forestry Research Institute of Nigeria (FRIN), Ibadan, by comparing it with existing herbarium specimens deposited at various periods. The voucher specimen of the collected plant sample was deposited at the FRIN herbarium and given the specimen number F.H.I 110191. Fresh leaves were harvested from the fully matured plants and air dried at room temperature. Dried leaves were ground into powder using a mechanical grinder. A sequential extraction of powdered leaves (800 g) of O. gratissimum in solvents of increasing polarity n-hexane, chloroform and 80% methanol was carried out each for 48 h at room temperature (37 °C). The extracts were filtered and concentrated in a Rotary evaporator (Buchi Rotavapor R-124) under reduced pressure. The methanol extract (MEOg) with 5.42% yield was stored in a glass chamber desiccator containing activated silica gel (BDH, England). The extract was prepared fresh daily by dissolving in 1% Tween 80.

High-performance liquid chromatography analysis

The chromatographic system comprised the following: Shimadzu HPLC system consisting of Ultra-Fast LC-20AB prominence equipped with SIL-20AC auto-sampler; DGU-20A3 degasser; SPD-M20A UV-diode array detector; column oven CTO-20AC, system controller CBM-20 Alite and Windows LC solution software (Shimadzu Corporation, Kyoto, Japan); column, VP-ODS 5 μm and dimensions of 150×4.6 mm. The chromatographic conditions included the mobile phase as follows: solvent A: 0.2% v/v formic acid; solvent B: acetonitrile; mode, isocratic; flow rate 0.6 mL/min; injection volume 5 μL of 200 μg/mL solution of MEOg in methanol in the mobile phase; and detection at UV 254 nm wavelength. Reference standards, rutin, quercetin, caffeic acid, ferulic acid, and apigenin (Fluka, Germany) 50 μg/mL in methanol were analyzed under the same condition as the extract. The HPLC operating conditions were programed to give the following: at 0.01 min, solvent B 20%; at 5 min, solvent B 30%; at 15 min, solvent B 60%; and at 40 min, solvent B 20%. The column oven temperature was 40 °C. The total run time was 40 min.

1,1-Diphenyl-2-picryl-hydrazyl (DPPH) free radical scavenging assay

The in vitro antioxidant activity of MEOg was determined using DPPH spectrophotometric assay according to the method described by Aderogba et al. [18] with slight modifications. MEOg and Rutin (12.5, 25, 50, 100 and 200 μg/mL) or control (2 mL) were added to 3 mL of freshly prepared DPPH solution (0.1 mM) in methanol. The mixture was incubated in adark cupboard for 30 min at room temperature, and absorbance was measured at 514 nm on a UV/Vis spectrophotometer (752N INESA, China).

Nitric oxide free radical scavenging assay

Nitric oxide radical scavenging activity assay is based on the method described by Kumaran and Karunakaran [19]. Briefly, sodium nitroprusside (10 mM in 0.1 M sodium phosphate buffer, pH 7.4), was mixed with MEOg and rutin at varying concentrations (50, 100, 200, 400 and 800 μg/mL) and then incubated at room temperature for 150 min. The same reaction mixture without the extract or rutin but with the equivalent amount of water served as control. After the incubation period, 0.5 mL of Griess reagent (equal volume of 1% sulfanilamide in 5% phosphoric acid and 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride) was added. The absorbance was read at 546 nm in a UV/Vis spectrophotometer (752N INESA, China).

Lipoxygenase inhibition assay

The lipoxygenase enzyme inhibitory effect was measured using the Cayman 15-Lipoxygenase inhibitor screening assay kit (Cayman Chemical Company, Ann Arbor, USA). Stock solutions of MEOg and NDGA (positive control) were prepared by dissolving in methanol and diluting in 0.1 M Tris-HCl assay Buffer (pH 7.4). Briefly, 90 μL of 15-LO (in 0.1 M Tris-HCl, pH 7.4) was pre-incubated with 10 μL of MEOg (25–100 μg/mL) or NDGA (100 μM) at room temperature for 5 min. The reaction was initiated by the addition of 10 μL linonelic acid (100 μM), dissolved in potassium hydroxide and incubated for 10 min. Finally, 100 μL of chromogen (equal volume of Kit Item No. 760711 and Item No. 760712) was added to each well to stop enzyme catalysis and develop the reaction. The plate was covered and placed on a shaker for 5 min. The absorbance was measured at 492 nm using a microplate reader.

Xanthine oxidase inhibition assay

The effect of MEOg on the inhibition of xanthine oxidase activity was assayed spectrophotometrically under aerobic condition based on the procedure reported by Noro et al. [20]. The assay mixture consisted of 50 μL of MEOg (25–100 μg/mL) or allopurinol (50 μg/mL), 35 μL of 70 mm phosphate buffer (pH 7.5), and 30 μL of enzyme solution (0.01 units/mL in 70 mm phosphate buffer, pH 7.5). The mixture was incubated for 15 min, after which the reaction was initiated by the addition of 60 μL of substrate solution (150 μM xanthine in the same buffer). The assay mixture was again incubated at room 25 °C for 30 min. The reaction was terminated by the addition of 25 μL of 1 N HCl, after which the absorbance at 290 nm was measured with Microplate reader (Thermo Scientific, USA).

Experimental animals

Female Wistar rats (180–220 g) were obtained from the College of Medicine Central Animal House, University of Ibadan. The animals were acclimatized in the laboratory for 1 week at room temperature of 28±2 °C, relative humidity of 60%–70%, 12:12 h light:dark cycle. All animals were allowed free access to water and fed with standard commercial rat chow pellets (Ladokun Feeds Ltd., Ibadan, Nigeria). All experiments were carried out following the procedure on the ethical conduct of research involving the care and use of laboratory animals outlined by the World Medical Association Declaration of Helsinki, as approved by the University of Ibadan Animal Ethics Committee (UI-ACUREC/app/2015/026).

Carrageenan-induced rat paw edema

Acute inflammation in rats of either sex (weighing 150–200 g) was produced according to a previously described method [21]. Rats were divided into four groups and orally pretreated with vehicle (1% Tween 80; 10 mL/kg), MEOg (50 and 100 mg/kg) and indo (5 mg/kg) for 3 days. At 30 min after last treatment, 0.1 mL of 1% carrageenan (Sigma, type 1) was injected into the left hind foot of each rat under the subplantar aponeurosis. Paw volumes were measured before carrageenan injection and at 1, 3, 5 and 24 h after carrageenan injection with Ugo Basile (7134) digital plethysmometer (Comerio, VA, Italy).

Carrageenan-induced air pouch inflammation model

Air pouch was induced in rats as described in Sedgwick and Lees [22] with slight modification by Martin et al. [23]. Briefly, 20 mL of sterile air was injected subcutaneously on the shaved back of each rat anaesthetized with ketamine (100 mg/kg, i.p.). Four days later, the pouch was re-inflated with another 10 mL of sterile air. Rats were divided into five groups (n=5); saline (1% Tween 80; 10 mL/kg), carrageenan (1% Tween 80; 10 mL/kg), MEOg (50 mg/kg and 100 mg/kg), and indomethacin (5 mg/kg), and orally pretreated for 3 days before the induction of inflammation. On day 6, the saline group was injected with 2 mL sterile normal saline into the air pouch, while groups 2–5 were injected with 2 mL of 1% carrageenan (Sigma, type 1) solution in sterile saline into the pouch. At 24 h after saline or carrageenan injection, rats were anaesthetized with deep ether anesthesia, and each pouch was carefully opened by a small incision. The pouch cavity was flushed with 2 mL phosphate buffered saline (0.01 M, pH 7.4), while the exudates were collected and transferred into a sterile tube; the volume of the recovered exudates was measured. Total leukocytes in exudates were determined after staining with Turk solution, and the number of leukocytes was counted in a Neubauer chamber under a light microscope (Nikkon Eclipse E200, USA). Cell-free exudate supernatant was obtained by centrifugation at 5400 g for 10 min at 4 °C, supernatant aliquoted, and stored at −80 °C for tumor necrosis factor-α (TNF-α), nitrite, reduced gluthathione (GSH), and thiobarbituric reactant substance (TBARS) assays. A portion of the pouch tissue linings was used for the assay of myeloperoxidase activity.

Biochemical assay

TNF-α assay:

The level of TNF-α produced in the carrageenan induced-air pouch exudate was determined using ELISA kit (Biolegend, USA), in accordance with the manufacturer’s instruction.

Nitrite assay:

Nitrite in pouch exudates was determined using spectrophotometric methods with Griess reagent. Griess reagent was freshly prepared from reagents A (1% sulfanilamide in 5% phosphoric acid) and B (0.1% of N-1-naphthyl ethylenediamine dihydrochloride) at a ratio of 1:1. Samples were incubated with Griess reagent and read in a spectrophotometer at 540 nm. The nitrite concentration was estimated from a standard curve obtained from sodium nitrite (0–100 μM).

Myeloperoxidase assay:

The index of neutrophil activation in pouch lining tissue was assessed by following a previously described myeloperoxidase activity assay [24]. Briefly, pouch lining tissue was homogenized in 50 mM potassium phosphate buffer (pH 6.0), and centrifuged for 10,000 rpm at 4 °C for 15 min. The pellet was suspended in extraction buffer (0.5% hexadecyltrimethylammonium bromide in 50 mM potassium phosphate buffer of pH 6.0) and frozen at 20 °C. The process of freeze-thawing and sonication for a 10 s cycle was repeated thrice. The suspension was finally centrifuged at 15,000 rpm at 4 °C for 15 min. MPO activity was assayed by adding 0.2 mL of supernatant to 2.8 mL of mixed solution (containing 0.167 mg/mL O-dianisidine in 50 mM potassium phosphate buffer and 0.15 mM H2O2). The change in absorbance at 450 nm was monitored over a period of 3 min using a UV/Vis spectrophotometer (752N INESA, China). One unit of MPO was defined as that giving a change in absorbance of 0.001/min and the specific activity expressed as a unit of MPO per milligram of protein.

Determination of glutathione concentration in pouch exudates

Ellman’s reagent used in a modified method was employed in determining the amount of reduced glutathione (GSH) in pouch exudates [25]. Briefly, 0.1 mL cell-free exudate supernatant was diluted 10 times and deproteninzed with 1 mL Trichloroacetic acid (20%); the mixture was centrifuged at 10,000 rpm at 4 °C for 10 min. Then, 0.25 mL of the supernatant was mixed with 0.75 mL sodium phosphate buffer (0.1 M, pH 7.4) and 2 mL of 0.0006 M 51, 51-Dithios-nitrobenzoic acid (DTNB). The absorbance was read within 5 min at 412 nm in a UV/Vis spectrophotometer (752N INESA, China). The glutathione concentration, which was determined using a standard curve generated with standard glutathione (0–200 μM), was expressed as a function of the volume of pouch exudates (μM GSH/mL of pouch exudates).

Determination of thiobarbituric acid-reacting substances (TBARS) in pouch exudates

The index of lipid peroxidation in carrageenan-induced pouch was measured using the thiobarbituric reacting substance (TBARS) assay, originally described by Wilbur et al. [26] as modified by Nagababu et al. [27]. Here, 0.1 mL of the sample was diluted 20 times in 0.15 M Tris-KCl buffer, and mixed with 0.5 mL TCA (30%) and 0.5 mL thiobarbituric acid (0.75%). The mixture was incubated in a water bath at 80 °C for 45 min. The reaction was stopped by dipping the test tubes in ice-cold water for 10 min, and was thereafter centrifuged at 4000 rpm for 5 min. The absorbance of the supernatant was measured at 532 nm in a UV/Vis spectrophotometer (752N INESA, China). The majority of TBARS consisted of malondialdyhdes; thus the result was calculated using an index of absorption for MDA (molar extinction coefficient 1.56×105/M/cm). The concentration of TBARS in pouch exudate fluid was expressed as nmol MDA/mg protein. Total protein content in pouch exudates for TBARS and MPO analyses was measured by the biuret method [28]. Protein concentration was determined using a standard curve generated with Bovine serum albumin (0–100 μM).

Histopathological analysis of air pouch lining

A portion of the pouch tissue linings was fixed in 10% neutral buffered formalin; the fixed pouch tissues were dissected longitudinally, placed in embedding cassettes, embedded in paraffin, and then cut into 4-μm sections. Tissue sections were stained with hematoxylin and eosin (H & E) for light microscope examination.

Data analysis

Data were expressed as mean±SEM (standard error of the mean), and statistical significance was taken for p<0.05. Data were analyzed for level of significance using one-way analysis of variance (ANOVA). Significant main effects were further analyzed by Bonferroni’s post hoc test for multiple comparisons of treatment groups. Graphs and statistical analysis were done using Microsoft Excel® (Microsoft Corporation, Redmond, WA, USA) and GraphPad Prism® software version 5.01 (GraphPad Software, Inc. La Jolla, CA 92037 USA).

Results

Chemical characterization

HPLC fingerprinting of MEOg resolved a total of 14 peaks detected at a typical UV absorption wavelength of 254 nm (Figure 1). A comparison of the chromatography results of MEOg with those of pure natural compounds led to the identification of five major peaks. In the chromatogram of MEOg at a wavelength of 254 nm, five peaks appearing at retention times 5.305, 7.510, 8.807, 18.517, and 31.006 were identified as caffeic acid, rutin, ferulic acid, apigenin, and quercetin, respectively.

Figure 1: HPLC chromatogram of the MEOg.Sixteen peaks were detected with retention times of 5.305, 7.510, 8.807, 18.517, and 31.006 min appearing as caffeic acid, rutin, ferulic acid, apigenin, and quercetin, respectively.
Figure 1:

HPLC chromatogram of the MEOg.

Sixteen peaks were detected with retention times of 5.305, 7.510, 8.807, 18.517, and 31.006 min appearing as caffeic acid, rutin, ferulic acid, apigenin, and quercetin, respectively.

Effects of MEOg on DPPH and NO free radical scavenging assay

MEOg showed dose-dependent free radical scavenging activity on both the DPPH and NO free radical scavenging assays (Table 1). There was a corresponding increase in antiradical activity with the increase in concentration, which was comparable to rutin (a flavonoid standard). The antiradical activity was higher in the DPPH assay than in the NO scavenging assay. The performances of the extract, as determined by the 50% inhibitory concentration (IC50), were 31.5±0.03 and 201.6±0.01 on the DPPH and NO assays, respectively. Moreover, rutin performed better than MEOg with IC50 values of 33.7±0.03 and 128.3±0.03, respectively.

Table 1:

In vitro free radical scavenging activities of MEOg and rutin.

DPPH free radical scavenging, %NO free radical scavenging, %
Concentration, μg/mLInhibitory activity, %Concentration, μg/mLInhibitory activity, %
MEOgRutinMEOgRutin
12.559.182.05044.028.1
2572.892.910048.442.8
5088.095.020061.260.9
10092.195.640075.165.9
20095.696.180079.269.9
IC5031.5±0.0333.7±0.03IC50201.6±0.01128.3±0.03

  1. Data represent mean±SEM of triplicate samples.

Inhibitory effects of MEOg on lipoxygenase and xanthine oxidase

MEOg (25, 50 and 100 μg/mL) displayed a weak non-dose-dependent 15-lipoxygenase inhibitory activity (Figure 2A). The percentages of inhibition were 13.8%, 13.8%, and 13.7%, respectively. NDGA (100 μM) demonstrated a significantly superior inhibition of 15-lipoxygenase. MEOg (25, 50 and 100 μg/mL) demonstrated a significant (p<0.001) and dose-dependent xanthine oxidase inhibitory activities at 18.8%, 24.7%, and 92.8%, respectively (2B). MEOg (100 μg/mL) showed superior activity (92.8%) when compared with 80.5% of allopurinol (50 μg/mL).

Figure 2: Inhibitory effects of MEOg (25, 50 and 100 μg/mL) on (A) 15-lipoxygenase, (B) xanthine oxidase.Absorbance rates are mean±SEM (n=3), ***p<0.001 vs. control. Values in percentages are the percent inhibition of enzyme activity.
Figure 2:

Inhibitory effects of MEOg (25, 50 and 100 μg/mL) on (A) 15-lipoxygenase, (B) xanthine oxidase.

Absorbance rates are mean±SEM (n=3), ***p<0.001 vs. control. Values in percentages are the percent inhibition of enzyme activity.

Anti-inflammatory effect of MEOg in carrageenan-induced rat paw edema

Treatment with MEOg (50 and 100 mg/kg) exhibited significant (p<0.001) inhibitory activity by suppressing edema formation after edema induction with carrageenan. The anti-edema activity of MEOg (50 and 100 mg/kg) both showed the same percentage of inhibition (43.2%) at the 5th post-carrageenan injection. Indomethacin (5 mg/kg) showed a more pronounced suppressive effect with an increase in paw volume at different time points (Figure 3).

Figure 3: Effects of MEOg on Carrageenan-induced rat paw edema.Data represent mean±SEM of five rats. *p<0.05, **p<0.01, ***p<0.001 by two-way ANOVA followed by Bonferroni’s post hoc test compared with the carrageenan control group (pretreated orally with 1% tween 80).
Figure 3:

Effects of MEOg on Carrageenan-induced rat paw edema.

Data represent mean±SEM of five rats. *p<0.05, **p<0.01, ***p<0.001 by two-way ANOVA followed by Bonferroni’s post hoc test compared with the carrageenan control group (pretreated orally with 1% tween 80).

MEOg reduced exudate formation and cellular migration in carrageenan-induced air pouch

Carrageenan injection into 6-day old pouches caused significant (p<0.001) increase in fluid exudation into the cavity at 24 h. Exudate formation in the carrageenan-induced air pouch was significantly reduced by MEOg (50 and 100 mg/kg) and indomethacin (5 mg/kg) (Figure 4A). Carrageenan injection into the air pouch caused a significant increase in the migration of leucocytes, particularly of neutrophils (Figure 4B and C). MEOg (50 and 100 mg/kg) demonstrated anti-inflammatory activity by significantly (p<0.001) reducing the total leucocytes and the number of neutrophils migrating to the pouch. The percentages of inhibition of total leucocyte migration by MEOg (50 and 100 mg/kg) were 47.5% and 57.8%, whereas those for neutrophils were 39.2% and 61.8%, respectively. Indomethacin (5 mg/kg) demonstrated more profound inhibition of total leucocytes (79.1%) and neutrophils (77.5%).

Figure 4: Effects of MEOg on carrageenan-induced air pouch.(A) Volume of pouch exudates, (B) total leucocytes, and (C) neutrophils. Each bar represents mean±SEM, n=5; one-way ANOVA followed by Bonferroni’s post hoc test for multiple comparison, **p<0.01, ***p<0.001 vs. carrageenan control, #p<0.001 vs. saline.
Figure 4:

Effects of MEOg on carrageenan-induced air pouch.

(A) Volume of pouch exudates, (B) total leucocytes, and (C) neutrophils. Each bar represents mean±SEM, n=5; one-way ANOVA followed by Bonferroni’s post hoc test for multiple comparison, **p<0.01, ***p<0.001 vs. carrageenan control, #p<0.001 vs. saline.

MEOg suppression of tumor necrosis factor-α, nitrite levels, and the index of neutrophil activation (myeloperoxidase activity)

Carrageenan injection into air pouches induced the release of pro-inflammatory mediators in the exudates compared with animals that were injected only with saline. As shown in Figure 5A, the concentration of TNF-α was markedly lower in the exudates from MEOg-treated animals. Pretreatment with MEOg (50 and 100 mg/kg) showed significant (p<0.05) inhibition of TNF-α by 27.8% and 40.6%, respectively. Similarly, nitrite was reduced by pretreatment with MEOg (Figure 5B). The amount of 50 mg/kg MEOg reduced nitrite by 12.6%, whereas the amount of 100 mg/kg significantly (p<0.001) reduced nitrite by 47.6%. Indomethacin (5 mg/kg) significantly (p<0.05) reduced both TNF-α (34.6%) and nitrite (75.2%) levels in the exudates. In the MEOg-treated animals (50 and 100 mg/kg), the MPO activity was significantly decreased by 39.1% and 54.5%, respectively (Figure 5C). Meanwhile, the control group treated with indomethacin (5 mg/kg) yirled an inhibition rate of 60.9%.

Figure 5: Effects of MEOg on carrageenan-induced air pouch.(A) Tumor necrosis factor-alpha (TNF-α), (B) nitrite concentration in pouch fluid, and (C) myeloperoxidase activity (MPO). Each bar represents mean±SEM, n=5; one-way ANOVA followed by Bonferroni’s post hoc test for multiple comparison, **p<0.01, ***p<0.001 vs. carrageenan control, #p<0.001 vs. saline.
Figure 5:

Effects of MEOg on carrageenan-induced air pouch.

(A) Tumor necrosis factor-alpha (TNF-α), (B) nitrite concentration in pouch fluid, and (C) myeloperoxidase activity (MPO). Each bar represents mean±SEM, n=5; one-way ANOVA followed by Bonferroni’s post hoc test for multiple comparison, **p<0.01, ***p<0.001 vs. carrageenan control, #p<0.001 vs. saline.

MEOg suppressed carrageenan-induced lipid peroxidation and enhances glutathione level

The antioxidative effects of the pretreatment were assessed by looking at the indexes of lipid peroxidation and reduced glutathione in the exudates. The results indicate that carrageenan in the pouches raised the level of TBARS compared with saline. MEOg reduced the index of lipid peroxidation in the carrageenan-injected pouch (Figure 6A). Pretreatment with MEOg (50 and 100 mg/kg) as well as indomethacin (5 mg/kg) prevented the depletion of glutathione (Figure 6B).

Figure 6: Effects of MEOg on carrageenan-induced air pouch.(A) Thiobarbituric acid reacting substances (TBARS) and (B) reduced glutathione (GSH). Each bar represents mean±SEM, n=5; one-way ANOVA followed by Bonferroni’s post hoc test for multiple comparison, ***p<0.001 vs. carrageenan control, #p<0.001 vs. saline.
Figure 6:

Effects of MEOg on carrageenan-induced air pouch.

(A) Thiobarbituric acid reacting substances (TBARS) and (B) reduced glutathione (GSH). Each bar represents mean±SEM, n=5; one-way ANOVA followed by Bonferroni’s post hoc test for multiple comparison, ***p<0.001 vs. carrageenan control, #p<0.001 vs. saline.

Histological changes in the air pouch tissue lining

Histological observation showed that the administration of carrageenan to the air pouch induced tissue edema, which can be characterized by enlargement of the pouch wall and the infiltration of inflammatory cells (large neutrophils and few macrophages) compared with the normal pouch (vehicle group). In the treated groups (Figure 7), a reversal of the inflammatory changes towards normal architecture at varying degrees was observed.

Figure 7: Histology of carrageenan-induced air pouch tissue lining (heamotoxylin-eosin, original magnification×400).(A) Vehicle: air pouch, showing white arrow showing normal dermal layer not infiltrated by inflammatory cells. There is no tissue or cellular degeneration seen. (B) Veh+Carr: air pouch injected with carrageenan; black arrows show areas with severe infiltration of inflammatory cells (black arrow). The adipose tissue layer is moderately infiltrated by inflammatory cells (slender arrow). (C) to (E) air pouch with carrageenan treated with MEOg (50 and 100 mg/kg) and indomethacin (5 mg/kg); blue arrows show areas with mild infiltration of inflammatory cells.
Figure 7:

Histology of carrageenan-induced air pouch tissue lining (heamotoxylin-eosin, original magnification×400).

(A) Vehicle: air pouch, showing white arrow showing normal dermal layer not infiltrated by inflammatory cells. There is no tissue or cellular degeneration seen. (B) Veh+Carr: air pouch injected with carrageenan; black arrows show areas with severe infiltration of inflammatory cells (black arrow). The adipose tissue layer is moderately infiltrated by inflammatory cells (slender arrow). (C) to (E) air pouch with carrageenan treated with MEOg (50 and 100 mg/kg) and indomethacin (5 mg/kg); blue arrows show areas with mild infiltration of inflammatory cells.

Discussion

This study demonstrates the in vitro free radical scavenging and in vivo anti-inflammatory activities of O. gratissimum leaf extracts. Sequential extraction optimizes the separation of the bioactive constituents according to polarity in order to achieve higher concentrations in the target extract. The chromatographic fingerprint of MEOg revealed the presence of rutin, caffeic acid, ferulic acid, quercetin, and apigenin. Rutin and caffeic acid have almost become signatory characteristic constituents in O. gratissimum leaves [16, 29, 30].

MEOg showed potent antiradical properties in both the DPPH and NO free radical scavenging assays. The activity observed paralleled those obtained from rutin in our study. DPPH free radical scavenging assay is a primary assay for investigating the antioxidant properties of extracts. Phenolics are characterized by their potent antioxidant action due to the capacity to neutralize reactive oxygen species [31]. Ocimum gratissimum is a plant known to be rich in phenolic content and has been shown in some studies to have rich antioxidant activities [32, 33].

MEOg showed a moderate inhibition of 15-lipoxygenase in vitro. Non-steroidal anti-inflammatory drugs (NSAIDs) or the selective COX-2 inhibitors re-directs the arachidonic acid metabolism pathway through the lipoxygenase pathway when they inhibit COX-1 and COX-2. This can lead to an overproduction of leukotrienes, which have been implicated in a variety of pathological conditions including asthma, renal insufficiency, gastric ulcerations, and cardiovascular complications [34]. The results suggest that MEOg moderately modulates the formation of leukotrienes from 15-LOX and may prevent an accumulation of these key inflammatory factors, thus contributing to tissue damage through a putative lipoxygenase shunt seen with the NSAIDs [35].

MEOg showed concentration-dependent inhibition of xanthine oxidase. At higher extract concentration (100 µg/mL), xanthine oxidase inhibitory activity proved to be superior to that of allopurinol. Xanthine oxidase produces hydrogen peroxide and superoxide anion during the oxidation of hypoxanthine to xanthine and then to uric acid. Xanthine oxidase contributes to the generation of reactive oxygen species, which increase the oxidative stress levels in gout, arthritis, artherosclerosis, cancer, and aging [36]. Flavonoids have been found to show inhibitory effects on xanthine oxidase [37]; therefore, the presence of flavonoids in MEOg may be responsible for the xanthine oxidase inhibitory effect.

In the carrageenan-induced rat paw edema model, MEOg suppressed edema formation in the paw. The observed effect is consistent with other findings on the ability of the aqueous alcohol extracts of O. gratissimum leaves to suppress edema [12]. Agents that suppressed carrageenan-induced edema are good candidates for further anti-inflammatory screening. MEOg inhibited inflammatory response in the 6-day-old carrageenan-induced air pouch model. Carrageenan injection into the air pouch stimulated fluid exudation and leukocyte migration (80×106 cells per pouch) in the group treated only with vehicle. Meanwhile, pretreatment with MEOg reduced fluid exudation, the number of leukocytic cells, and the inflammatory mediators, particularly TNF-α in the air pouch exudates.

Furthermore, histological analysis of the pouch tissue lining also showed the anti-inflammatory effect of MEOg. It is possible that MEOg targets the migration and activation of neutrophils, as evidenced by the reduction in the myeloperoxidase activity. It has been pointed out that over 96% of the total leucocytes in carrageenan-induced acute inflammation are primarily of the polymorphonuclear cells [25]. Activated neutrophils are present in synovial fluids of patients with rheumatoid arthritis, wherein the progression of inflammation and joint destruction is associated with pronounced released neutrophilic ROS [38]. The anti-inflammatory effect of MEOg is related to the reduced migration of leucocytes, particularly neutrophil recruitment and activation at inflammatory sites.

TNF-α in the carrageenan air pouch had a significantly higher level than in the saline-induced pouch. Pretreatment with MEOg, however, significantly reduced TNF-α level. In the pathogenesis of rheumatoid arthritis, TNF-α is known to be an important mediator that plays a part in regulating leuckocyte migration in the acute inflammatory phase; it also activates neutrophils by promoting extravasation to the lungs, liver, gut and other organs; and has a role in extravasated neutrophil damage tissues by releasing oxygen free radicals and proteases [39, 40]. In this study, MEOg (50 and 100 mg/kg) significantly ameliorated the increase in TNF-α level in carrageenan-induced inflammation in the rat pouch. A similar effect has also been noted by Madhu and Harindran [15], who reported that methanol extract of O. gratissimum reduced TNF-α level in paws of collagen-induced arthritic rats.

The analysis of the lipid peroxidation (LPO) changes induced by carrageenan as a result of released neutrophilic ROS/RNS showed that TBARS (an index of lipid peroxidation) increased in carrageenan-injected pouches as compared with the normal saline-injected pouches. This finding is in agreement with the observation suggesting an increase in exudates and tissue LPO partly mediated via the increase in ROS/RNS radical production in carrageenan-injected animals [41, 42]. Pretreatment with MEOg reversed the TBARs levels significantly. This activity may be connected to the free radical scavenging properties of MEOg, as demonstrated in the in vitro antioxidant activity assays. Glutathione level is known to increase as a response to carrageenan injection, as it is a non-enzymatic antioxidant that reacts with free radicals and yields thiol radicals. This reaction causes depletion of the GSH level with time in animals under inflammatory assault [25, 43]. In this experiment, pretreatment with MEOg prevented the depletion of GSH, which was significantly decreased by carrageenan injection in the air pouch. This effect has also been noted by Mahaptara et al. [44], who showed that methanol extract of O. gratissimum increases glutathione in nicotine-induced peritoneal macrophages.

Conclusions

In conclusion, these experiments showed that carrageenan-induced inflammation and oxidative stress in rats were ameliorated by the sequential methanol extract of O. gratissimum leaves. The anti-inflammatory properties could be related to the free radical scavenging properties of the phenolic compounds.

Corresponding author: Professor Olusegun G. Ademowo, Department of Pharmacology and Therapeutics, College of Medicine, University of Ibadan, Ibadan, Nigeria; and Drug Research Unit, Institute for Advanced Medical Research and Training (IAMRAT), College of Medicine, University of Ibadan, Ibadan, Nigeria, Phone: +2348023342856

Acknowledgments

The authors acknowledge the technical assistance in histological slide preparation and interpretation provided by Mr. Otegbade of the Histopathology Unit of the University College Hospital.

  1. Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

  2. Research funding: None declared.

  3. Employment or leadership: None declared.

  4. Honorarium: None declared.

  5. Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

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Received: 2016-6-21
Accepted: 2017-1-14
Published Online: 2017-3-22
Published in Print: 2017-11-27

©2017 Walter de Gruyter GmbH, Berlin/Boston

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  2. Editorial
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  5. Anti-inflammatory effects and anti-oxidant capacity of Myrathius arboreus (Cecropiaceae) in experimental models
  6. Ocimum gratissimum Linn. Leaf extract inhibits free radical generation and suppressed inflammation in carrageenan-induced inflammation models in rats
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https://doi.org/10.1515/jbcpp-2016-0096
Keywords for this article
anti-inflammatory; antioxidant; apigenin; carrageenan; phenolic; xanthine oxidase

Articles in the same Issue

  1. Frontmatter
  2. Editorial
  3. Inflammation, fever, and body temperature under febrile conditions
  4. Inflammation
  5. Anti-inflammatory effects and anti-oxidant capacity of Myrathius arboreus (Cecropiaceae) in experimental models
  6. Ocimum gratissimum Linn. Leaf extract inhibits free radical generation and suppressed inflammation in carrageenan-induced inflammation models in rats
  7. Intra-articular injections of ketamine and 25% dextrose improve clinical and pathological outcomes in the monosodium iodoacetate model of osteoarthritis
  8. Central mediators of the zymosan-induced febrile response
  9. The use of siRNA as a pharmacological tool to assess a role for the transcription factor NF-IL6 in the brain under in vitro and in vivo conditions during LPS-induced inflammatory stimulation
  10. Effects of single or combined administration of salmon calcitonin and omega-3 fatty acids vs. diclofenac sodium in sodium monoiodoacetate-induced knee osteoarthritis in male Wistar rats
  11. Evaluation of oral multi-herbal preparation of Dashmoolarishta on mice model of osteoarthritis
  12. Behavior and Neuroprotection
  13. Possible modulation of PPAR-γ cascade against depression caused by neuropathic pain in rats
  14. Functional interaction between N-methyl-D-aspartate receptor and ascorbic acid during neuropathic pain induced by chronic constriction injury of the sciatic nerve
  15. Cardiovascular Function
  16. Chronic endurance exercise antagonizes the cardiac UCP2 and UCP3 protein up-regulation induced by nandrolone decanoate
  17. Oxidative Stress
  18. Prevention of renal ischemia/perfusion-induced renal and hepatic injury in adult male Albino rats by oxytocin: role of nitric oxide
  19. Protective effect of salusin-α and salusin-β against ethanol-induced gastric ulcer in rats
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