In April 2022, the aerial parts of T. aphylla were collected from North Amman, Jordan. The plant material was authenticated by Jamil Salam, serving as a botanist. To ensure future reference, a voucher specimen was submitted to the Hashemite University herbarium in Zarqa, Jordan, with the assigned Herbarium number HU.No. 3884. For the extraction of essential oil (EO), 500 g of dried aerial parts of T. aphylla were subjected to hydrodistillation. A Clevenger-type apparatus was used for the extraction process, with different durations of 1,2,4 hours. The maximum yield of EO (0.4%) was obtained after 4 hours (Siani et al. 2004).

The GC analysis of T. aphylla L essential oil was conducted using a Trace GC ULTRA with flame ionization detector (FID) gas chromatograph. The instrument was equipped with a column (30 m × 0.25 mm × 0.25 μm) type VB-5 (methylpolysiloxane with 5% of phenyl) and a split injection. Mass spectrometry (MS) analysis was carried out using a Polaris Q MS mass spectrometer (with an ion-trap at 70 eV). The column temperature was programmed from 40 °C for 2 minutes and raised to 180 °C at a rate of 4 °C/min. The carrier gas used was helium, with a constant flow rate of 1.4 mL/min. The volatile constituents of the essential oil were identified by automated comparison of their mass spectra with that of the NIST (National Institute of Standards and Technology) library (Stein et al. 1987). Quantitative data were obtained from peak areas using FID, and the areas were normalized using an internal standard (ethyl octanoate). Essential oil samples were analyzed in triplicate.

The study utilized male albino rats (n = 126), 7–8 weeks old and weighing between 120 and 190 grams. These rats were obtained from the animal house at the Hashemite University. They were housed in a controlled environment with constant access to food and water, following a 12-hour light/dark cycle. Before the experiments, the rats were allowed to acclimate to the laboratory conditions for a minimum of 2 hours. Each rat was used only once to ensure data integrity. All in vivo animal studies were conducted in compliance with the Ethics Committee for Animal Experiments at the Hashemite University, Jordan. The care and handling of the animals adhered to the guidelines set by the International Association for the Study of Pain for the ethical use of animals in pain research (Zimmermann 1983).

In order to determine the lethal dose (LD50) of T. aphylla essential oil , rats were randomly divided into seven groups, each containing five rats. The experiment followed the protocol described by Lorke 1983 (Lorke 1983). The rats in six of the groups were orally administered different doses of the essential oil of T. aphylla (EOTA), specifically 10, 100, 500, 1000, 2000, and 3000 mg/kg. The seventh group, serving as the control group, received 1% v/v Tween 80 in sterile saline at a volume of 10 ml/kg. Throughout the experiment, the rats had unrestricted access to food and water. The rats were observed for signs of toxicity and mortality over a period of 72 hours, and these observations were recorded for analysis.

To evaluate the in vivo anti-inflammatory effect of the EOTA, the experiment was conducted on rats. The rats were fasted for 24 hours prior to the experiment but had free access to water during this period (Amanlou 2005). A total of six groups were formed, with each group consisting of six rats.

Group 1 served as the negative control and received the vehicle, which was administered orally as 0.2% Tween 80. Groups 2 to 5 received varying concentrations of the essential oil orally at doses of 10 mg/kg, 31.6 mg/kg, 100 mg/kg, and 316 mg/kg, respectively. doses were calculated to be located at approximately 0.5 log units from each other on a log scale. Group 6, the positive control, received dexamethasone subcutaneously at a concentration of 2 mg/kg. To assess the anti-inflammatory effect of EOTA, a carrageenan-induced rat paw edema model was employed (Morris 2003). One hour after administration of the desired concentration of EOTA or the control substances, acute paw edema was induced by injecting 0.1 ml of a 1% freshly prepared carrageenan suspension in normal saline into the right hind paw of each rat. The paw volume was measured before (0 h) and at intervals of 1, 2, 3, 4, 5, and 6 hours after the carrageenan injection using a water displacement method with a plethysmometer (Choi et al. 2005) (type 7140 Ugo Basile, Italy. The anti-inflammatory action of EOTA was calculated as a percentage using the following formula: (paw final volume – initial volume) / initial volume × 100.

Rats (n = 6/group) were subjected to intraperitoneal injection of carrageenan (1% 250 μL, i.p.) one hour after oral administration of EOTA at concentrations of 10, 31.6, 100, and 316 mg/kg. To assess leukocyte peritoneal infiltration, dexamethasone (2 mg/kg, s.c.) and vehicle (0.2% Tween 80 orally) were used as positive and negative controls, respectively. After four hours of carrageenan injection, the rats were euthanized, and 4 mL of saline containing EDTA (1 mM) were injected into the peritoneal cavity to collect peritoneal lavage. The resulting cell suspension was chilled on ice, centrifuged at 1500 rpm for 10 minutes, and the supernatant was discarded. The cell pellets were then resuspended in 1 mL of saline. Total cell count (number of cells/mL) was determined using a Neubauer chamber, and the cells were stained with Diff-3 stain to differentiate between leukocytes (polymorphonuclear and mononuclear cells).

The ability of EOTA to reduce inflammation was further demonstrated by its ability to inhibit the activity of myeloperoxidase enzyme. At the end of the 4-hour edema measurement, the paw tissue of the rats was homogenized in 50 mM phosphate buffer (pH 6.0) and 0.5% hexadecyl-trimethylammonium bromide. The resulting supernatants were mixed with o-dianisidine dihydrochloride (0.167 mg/mL, in 50 mM phosphate buffer) and 0.005% hydrogen peroxide. The samples’ absorbance was measured at 460 nm using a spectrophotometer, and the results were expressed as units of myeloperoxidase (MPO) (UMPO)/mg of paw tissue. One UMPO represents the amount of enzyme that degrades 1 μmol of H2O2 per minute (Loftus et al. 2011).

To investigate the potential anti-inflammatory mechanisms of EOTA, rats were injected intraperitoneally with carrageenan (1% 250 μL) one hour after oral administration of EOTA (316 mg/kg), subcutaneous administration of Dexamethasone (2 mg/kg) as a positive control, or oral administration of the vehicle (0.2% Tween 80) as a negative control. After six hours of carrageenan injection, the concentration of NOx in the peritoneal lavage was measured spectrophotometrically at 546 nm using the Griess reagent (consisting of 5% H3PO4, 2% sulphanilamide, and 0.1% napthylethylenediamine dihydrochloride) (Bradley et al. 1982). A standard curve was constructed using known concentrations of sodium nitrite.

The antioxidant capacity and free radical scavenging effect of EOTA were evaluated by measuring the inhibition of lipid peroxidation induced by FeSO4 (0.145 mM) or FeSO4 (0.145 mM) in combination with H2O2 (0.4 M). The concentration of oxidized lipids was determined by quantifying the level of thiobarbituric-acid reactive substances (TBARS), as described by Kizil and colleagues. The extent of lipid peroxidation was assessed by measuring the formation of malondialdehyde (MDA), which is a final product generated during lipid peroxidation (Tsikas 2017). Rat’s liver was isolated and homogenized in a phosphate buffer (0.1% w/v in 50 mM phosphate buffer, pH 7.4). The homogenate was then incubated with different concentrations of EOTA (0.001, 0.01, 0.1, 1, 10, 100, 316 μg/ml) along with either 100 μl of 0.145 mM FeSO4 or 100 μl of 0.145 mM FeSO4 combined with 0.4 M H2O2. The mixture was incubated at 37 °C for 1 hour. After incubation, 0.3 mL of the samples was mixed with 0.6 mL of trichloroacetic acid (20% v/v), followed by centrifugation at 10,000 g at 4 °C for 15 minutes. Then, 0.5 mL of thiobarbituric acid (0.67% w/v) was added to the supernatant, and the mixture was heated at 100 °C for 30 minutes. The samples were transferred to a 96-well plate in triplicate, and the absorbance was measured at 532 nm using a spectrophotometer. The experiment included the same range of concentrations of ascorbic acid as a positive control. A standard curve was generated by serial dilution of freshly prepared MDA, which was used to determine the concentrations of TBARS in the samples (Feldman 2019).

The spectrophotometric quantification of nitric oxide (NO) radical scavenging activity was performed. Various concentrations of EOTA (0.001, 0.01, 0.1, 1, 10, 100, 316 μg/ml) were added to separate test tubes before the addition of 0.5 ml of sodium nitroprusside in phosphate buffer saline (10 mM). After incubating all the tubes at 37 °C for 60 minutes, an equal volume of freshly prepared Griess reagent (consisting of 5% H3 PO4, 2% sulphanilamide, and 0.1% napthylethylenediamine dihydrochloride) was added to each tube. A control sample without plant essential oil, prepared in the same manner as the test samples, was used for comparison. As a positive control, the same range of concentrations of ascorbic acid was employed. Subsequently, 150 μL of the reaction mixture from each tube was transferred in triplicate to the wells of a 96-well plate, and the absorbance was measured at 546 nm

Nitric oxide scavenging activity was calculated as: Scavenging activity (%) = (1- [A sample /A control]) ×100 (Rajesh and Natvar 2011):

A sample: The absorbance values obtained from the samples containing plant essential oil.

A control: The absorbance values obtained from the control samples without any plant essential oil.

The collected data was presented as mean ± SEM and subjected to statistical analysis using GraphPad Prism 6. One-way analysis of variance (ANOVA) or two-way ANOVA was performed, followed by a Tukey post hoc test. The significance level was set at P < 0.05.

Table 1 presents the analysis and chemical compositions of the EOTA. A total of twenty-two compounds were detected in the essential oil derived from the plant, constituting 93.2% of the overall oil content. The predominant compounds identified were 6,10,14-Trimethyl-2-pentadecanone (20.2%), β-Ionone (20.1%), Dodecanoic acid (12.2%), and trans-β-Caryophyllene (10%.1).

Rats were given varying doses of EOTA to evaluate their safety and estimate potential toxicity. The doses administered were 10, 100, 500, 1000, 2000, and 3000 mg/kg body weight of the aerial parts essential oil. Results showed that up to 3000 mg/kg was deemed safe to be used for in vivo investigations of the anti-inflammatory effects of EOTA. However, for anti-inflammatory and antioxidant studies, different doses of the plant essential oil were employed (10, 31.6, 100, and 316 mg/kg).

After one hour of administering various doses of EOTA, dexamethasone, and the vehicle, rat paw edema was induced by injecting 0.1 ml of a 1% freshly prepared carrageenan suspension. It was observed that the paw edema increased over time. However, positive control dexamethasone (2 mg/kg) and EOTA significantly reduced rat paw edema at doses of 31.6, 100, and 316 mg/kg between 2 to 6 hours after inducing carrageenan edema, in comparison to the vehicle control. This reduction was statistically significant at all-time points (Fig. 1, n = 6, p < 0.05). The group treated with 316 mg/kg of plant essential oil demonstrated a 64% reduction in edema, while the group treated with 2 mg/kg dexamethasone showed a 75% reduction, both compared to the vehicle control group, 6 hours after inducing carrageenan edema (Fig. 1, n = 6, p < 0.05). However, the group treated with a dose of 10 mg/kg failed to reverse the edema induced by carrageenan in comparison to the vehicle control at all tested time points.

Figure 1. 

The effect of EOTA on rat paw edema was examined to assess its anti-inflammatory properties. Rats were given pre-treatment of either a vehicle, EO at various doses (10, 31.6, 100, 316 mg/kg), or Dexamethasone (2 mg/kg) prior to carrageenan-induced paw injection. The pre-treatment was administered at 6-hour intervals. EOTA demonstrated a significant inhibition of paw swelling induced by carrageenan, particularly at concentrations ranging from 31.6 to 316 mg/kg. The representative blot shown in the study represents a sample size of 6 rats for each experimental group. All values were expressed as the mean ± SEM (standard error of the mean) and analyzed using a two-way analysis of variance (ANOVA), followed by the Tukey Post Hoc test. A p-value less than 0.05 (*) was considered statistically significant when compared to the vehicle control group.

The peritonitis induced by carrageenan was assessed by examining the migration of mononuclear leukocytes (lymphocytes, monocytes, and macrophages) as well as polymorphonuclear leukocytes (neutrophils, eosinophils, and basophils) into the peritoneal cavity (Bradley et al. 1982). Following intraperitoneal injection of carrageenan, there was an influx of leukocytes into the peritoneal cavity over a 4-hour period. Positive control dexamethasone (2 mg/kg) significantly affected the migration of leukocytes and polymorphonuclear (PMN) cells, as indicated by statistical analysis (Fig. 2, n = 6, p < 0.05). Similarly, groups treated with EOTA at doses of 31.6, 100, and 316 mg/kg demonstrated a significant reduction in both total leukocyte and polymorphonuclear (PMN) cell migration (Fig. 2, n = 6, p < 0.05) compared to the vehicle control. However, neither the doses of plant essential oil nor dexamethasone influenced the migration of mononuclear (MN) cells. These findings suggest that EOTA exhibits an anti-inflammatory effect based on the presented results.

Figure 2. 

Anti-inflammatory effect of EOTA treatment on leukocyte migration. Rats were pretreated orally with different doses of EOTA (10, 31.6, 100, 316 mg/kg) or a vehicle containing 0.2% Tween 80, one hour before receiving intraperitoneal injection of carrageenan (1%, 250 μl). The experimental groups consisted of 6 rats each. After the injection, intraperitoneal lavage was performed to collect inflammatory cells. These cells were then assessed to evaluate the anti-inflammatory effect of EOTA using a Neubauer Chamber for cell counting and Diff-3 Stain for leukocyte differentiation. All values were presented as the mean ± SEM and analyzed using a one-way analysis of variance (ANOVA), followed by the Tukey Post Hoc test. Statistical significance was determined as p < 0.05 (*) when comparing the EOTA -treated groups to the vehicle control group.

MPO, also known as myeloperoxidase, is an enzyme released during the degranulation of monocytes and neutrophils. Its primary function is to catalyze the oxidation of various cellular and biological structures. Additionally, MPO plays a role in the development of inflammation, cardiovascular diseases, and immune-mediated conditions. The EOTA has been found to exhibit a significant anti-inflammatory effect by inhibiting MPO activity. This effect was observed after 4 hours of carrageenin induction. Among the different concentrations tested, namely 31.6, 100, and 316 mg/kg of EOTA, there was a significant decrease in MPO activity compared to the control group treated with a vehicle (Table 2, n = 6, p < 0.05). Moreover, these concentrations were comparable to the anti-inflammatory effects of the reference drug dexamethasone (Table 2, n = 6, p < 0.05). However, a dose of 10 mg/kg of EOTA did not show any significant alteration in MPO activity compared to the control group (Table 2, n = 6). Consequently, EOTA demonstrates potential antioxidant and anti-inflammatory effects by inhibiting the activation of inflammatory cells.

After pretreating rats with EOTA at a dose of 316 mg/kg before intraperitoneal injection of carrageenan, there was a significant reduction in the concentration of total NOx (nitrite and nitrate) compared to the control group treated with a vehicle (as indicated in Table 3, n = 6, p < 0.05). Similarly, pretreating rats with dexamethasone also led to a significant decrease in NOx concentration, indicating that EOTA exhibits an anti-inflammatory effect (Table 3, n = 6, p < 0.05).

Antioxidants play a crucial role in preventing oxidative damage caused by reactive oxygen species to cellular DNA, lipids, and proteins. Reactive oxygen species such as hydrogen peroxide and nitric oxide can cause oxidative damage to cells (Tsikas 2017). Furthermore, lipid peroxidation resulting from the action of free radicals can lead to the destruction of cell membranes and eventual cell death (Grisham et al. 1996). Therefore, the use of antioxidants to scavenge free radicals can prevent lipid oxidation. The antioxidant activity of EOTA was assessed by comparing its ability to scavenge nitric oxide with that of ascorbic acid, which was used as a standard. The study revealed that both EOTA and ascorbic acid exhibited dose-dependent increases in scavenging capacity, using the same range of concentrations. EOTA significantly inhibited the generation of nitric oxide (NO) from sodium nitroprusside within the concentration range of 0.01–100 μg/ml, as compared to the vehicle control (Table 4, n = 6, p < 0.05). The highest inhibition was observed with the highest concentration of ascorbic acid, 100 μg/ml (Table 4, n = 6, p < 0.05). To assess lipid peroxidation, the level of thiobarbituric acid reactive substances (TBARS) is measured. This measurement is based on the reaction between thiobarbituric acid (TBA) and malondialdehyde (MDA), a breakdown product of lipid hydroperoxides. In the case of this study, the impact of EOTA on lipid peroxidation induced by FeSO4 (Fe2+) in liver homogenate was evaluated. The results demonstrated that EOAH exhibited an inhibition of lipid peroxidation that was dependent on its concentration. All tested concentrations of EOTA (ranging from 0.01 to 100 μg/ml) as well as ascorbic acid significantly inhibited the generation of TBARS compared to the vehicle control (Table 4, n = 6, p < 0.05). This indicates that EOTA has a concentration-dependent inhibitory effect on lipid peroxidation induced by FeSO4, similar to the effect observed with ascorbic acid.