Natural products have, for decades, been contributing to the development of modern therapeutic drugs. The focus on the therapeutic properties of secondary metabolites have increased due to their great benefits for human being and animals; many phytocompounds are now receiving particular attention from industries because they are important sources of a wide variety of commercially useful base products [1]. Essential oils are important natural products used as raw materials in many fields; one of such is aromatherapy [2]. Aromatherapy is a system in which the healing effects are due to the phytochemicals in essential oils. Thus, essential oils are used to prevent and treat diseases, they are very useful in several healing systems such as treatment of orthopedic (bone, joint, and soft tissue) infections. They also have diaphoretic, disinfectant, antimalarial, antiseptic, analgesic, antipyretic, anti-inflammatory, antimicrobial, expectorant and antioxidant properties. Application of essential oils by either vapor inhalation or oral route provides benefit for both purulent and non-purulent respiratory problems. They are used as remedies for the symptoms of respiratory tract disorders such as cold, flue, pharyngitis, bronchitis, sinusitis, asthma and other respiratory diseases. Essential oils can also be used as quick and effective mood enhancers, for increasing energy, alertness or reducing stress and promoting relaxation [3] [4][5][6] [7]. Essential oils are good natural preservatives and flavoring agent in food and drug because they improve their shelf-life, better than the synthetic preservatives [8][9].

Eucalyptus cloeziana F. Muell, known as Gympie messmate tree, is one of the numerous species of the genus Eucalyptus. Essential oils from Eucalyptus plants are known for their medicinal properties. They are used in medicine to treat pains and respiratory infections [10][11][12]. Eucalyptus oil is included in products used as sealers and solvents for root canal fillings in treating dental problems [13][14]. They are known as potential natural drugs with their potential applications for the treatment of diseases [15] [16]. Eucalyptus essential oils have been classified as non-toxic and safe for human being and animals [17] [18][19][20][21]. Previous studies on E. cloeziana were mainly focused on the chemical composition of the essential oil extracted from the leaves [22][23]. To the best of our knowledge, there is paucity of information on the phytochemical, total phenolic content, free radical scavenging, antioxidant, anti-inflammatory, antinociceptive and antimicrobial potentials of E. cloeziana so far. Therefore, the present study was aimed at looking into the characterization of essential oil from the seed of E. cloeziana and evaluation of its modes of medicinal properties.

The seed essential oil was analyzed using Shimadzu GC-MS-QP2010 Plus (Japan). The separations were carried out using a Restek Rtx-5MS fused silica capillary column (5%-diphenyl-95%-dimethylpolysiloxane) of 30×0.25 mm internal diameter (di) and 0.25 mm in film thickness. The conditions for analysis were set as follows; column oven temperature was programmed from 60–280 °C (temperature at 60°C was held for 1.0 min, raised to 180°C for 3 min and then finally to 280°C held for 2 min); injection mode, Split ratio 41.6; injection temperature, 250°C; flow control mode, linear velocity (36.2 cm/sec); purge flow 3.0 ml/min; pressure, 56.2 kPa; helium was the carrier gas with total flow rate 45.0 ml/min; column flow rate, 0.99 ml/min; ion source temperature, 200°C; interface temperature, 250°C; solvent cut time, 3.0 min; start time 3.5 min; end time, 24.0 min; start m/z, 50 and end m/z, 700. Detector was operated in EI ionization mode of 70 eV. Components were identified by matching their mass spectra with those of the spectrometer data base using the NIST computer data bank, as well as by comparison of the fragmentation pattern with those reported in the literature [25].

Total phenolic content (TPC) in the seed essential oil was determined using Folin-Ciocalteu method according. Gallic acid was used as a standard phenolic compound. 1 ml of Folin-Ciocalteu reagent was added to 1 ml of the sample solution, then the entire solution was diluted with 46 ml distilled water and the content was mixed thoroughly. After 3 min, 3 ml of 2% Na₂CO₃ was added and then the mixture was allowed to stand in dark for 2 h with intermittent shaking. The absorbance was measured at 760 nm. The index of TPC in the essential oil determined as µgmg^-1 of gallic acid equivalent (GAE) using an equation obtained from the calibration curve of gallic acid [26].

In vitro DPPH assay: The free radical scavenging and antioxidant activities of the seed essential oil against the stable free radical DPPH were measured. Briefly, three different concentrations (1000, 100 and 10 µgml^-1) of the essential oil in methanol were incubated with a methanolic solution of DPPH. After 30 min. of incubation at room temperature in the dark, the absorbance at 517 nm was measured with ultraviolet-visible spectrophotometer. Ascorbic acid was used as reference compound. The assay was carried out in triplicate. Scavenging effect was calculated by the percentage (I%) of faded purple DPPH solution color into yellow by the tested sample against the control (DPPH solution only). The IC50 of DPPH assay represents the concentration of the tested sample needed to reduce the DPPH by 50%.

I% = [(Ablank – Aeo)/Ablank] x 100

where: Ablank is the absorbance of blank solution and Aeo is the absorbance of the essential oil. The dose-response curve was plotted and IC50 value for the essential oil and the standard were calculated [25].

The antioxidant activity index (AAI) was calculated as: AAI = [DPPH initial concentration (µgml^-1)]/[IC50 (µgml^-1)]

Antioxidant activity index was classified as weak, when AAI ranged < 0.5, moderate, when AAI ranged between 0.5–1.0. It is strong, when AAI ranged between 1.0–2.0, and very strong, when AAI > 2.0 [27].

In vitro FRAP assay: The sample at different concentrations in distilled water were mixed with phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and potassium ferricyanide [k₃Fe(CN)₆] (2.5 ml, 1%) and incubated at 50°C for 20 min. Then, 2.5 ml trichloroacetic acid (10%) and 0.5 ml of FeCl³ (0.1%) were added to the reaction mixture. The increases in the absorbance were spectrophotometrically measured at 700 nm as an indication of reducing capacity. The activity of ascorbic acid was used as a reference drug over the same concentrations. The assay was carried in triplicate and the results were expressed as mean ± standard deviation. Effective concentration at 50% (EC50) of FRAP value is the sample concentration required to reduce Fe³⁺ to Fe²⁺ [28].

Healthy albino rats (200±30g) were used for this study. All experiments were carried out in strict compliance with the principle of laboratory animal care [29].

Anti-inflammatory activity was assessed on the basis of inhibition of paw edema induced by the injection of 0.1 ml of 1% carrageenan into the subplantar region of the right hind paw of the rat. Three groups of five animals each were used. Carrageenan is known to result in at least neutrophil linked edematous inflammation. The essential oil solution was subjected at a dose of 0.1 ml each of 1000 µgkg^-1, postoperatively were administered orally 30 min before carrageenan injection. Indomethacin 1000 µgml^-1 was used as reference drug. Control group received the vehicle only (10 mlkg^-1). Rat paw volume was measured immediately (0 hour) before the injection of the “irritant” substance and at regular selected time intervals (2 and 4 hours) after injection of the essential oil solution (1000 µgml^-1) or equivalent volume of vehicle, using a digital vernier caliper. Results were expressed as the increase in paw volume (mm) calculated after subtraction of basal paw volume prior to carrageenan irritant injection [30]. The inhibition percentage of the inflammatory reaction was determined for each rat by comparing each group with controls and calculated by the formula below:
I% =1–[dt/dc] ×100

where I% = percentage inhibition and dt is the difference in paw volume in the drug-treated group and dc is the difference in paw volume in control group [31].

Formalin test was used to determine the antinociceptive property of the essential oil [32]. Rats (n = 5 per group) were treated respectively with 1,000 µgkg^-1 of the essential oil solution, 1000 µgkg^-1 of indomethacin. 30 minutes later, the pain was induced by injecting 0.05 ml of 2.5% v/v formalin (formaldehyde) in distilled water into the sub-plantar right hind paw of rat, immediately placed in a transparent plastic cage separately. The licking time and frequency of the injected paw were recorded for 30 min. The amount of time spent licking the injected paw was indicative of pain. The number of lickings from 0–5 min (first phase) and 15–30 min (second phase) of post-injection time was recorded. These phases represented neurogenic and inflammatory pain responses, respectively. The test was performed at room temperature and strict actions were taken to exclude environmental disturbances (high temperature, noise and excessive movement) that might interfere with the animal’s response. The percentage inhibition (I) (analgesic activity %) was calculated by:
I% = [Ao–At/Ao]x100

Where Ao = Average number of stretching of control per group and At = Average number of stretching of test per group [31].

The antibacterial activities of the seed essential oil were evaluated by agar-well diffusion method against multi-drug resistance gram-positive bacteria (Streptococcus agalactiae and Staphylococcus aureus) and gram-negative bacteria (Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa and Salmonella typhimurium). Tested bacteria were cultured on Mueller Hinton Broth media plates at 37oC for 24 h. The turbidity of the bacteria were adjusted to match 0.5 McFarland standard. The bacteria suspensions were used to inundate sterile plates containing nutrient agar. Wells of 6 mm diameter each were bored in the plates with the help of a cork-borer. Different concentration of the essential oil solutions in DMSO were incubated at 37°C for 24 h. The antibacterial activities of the essential oil were compared with synthetic antibiotics i.e., gentamicin (GEN) and cloxicillin (CXC). Antibacterial potentials of different concentrations of the essential oil solutions were evaluated by measuring the clear zones of growth inhibition against the test organisms [33].

The seed essential oil of E. cloeziana analyzed showed that 33 components, representing 99.7% of the seed essential oil were identified (Table 1). The major component of seed essential oil is 1, 8-cineole (23.8%). The other main compounds identified were cis-oleic acid (18.0%), a-pinene (14.5%) and palmitic acid (9.0%).

The essential oil was able to inhibit the formation of DPPH radicals in a concentration dependent manner. The percentage inhibitions of the essential oil at various concentrations (1000, 100 and 10 µgml^-1) were 70.25±0.10, 68.42±0.01 and 62.60±0.00% respectively; while the IC50 value was found to be 2.80 µgml^-1 in comparison to ascorbic acid which gave 96±0.00 69±0.00 and 54±0.00 as the percentage inhibitions with IC50 value of 9.0 µgml^-1. Table 2 the seed essential oil of E. cloeziana showed very strong AAI > 2, while ascorbic acid also showed a strong AAI > 2 [27].

Reduction antioxidant potential of the seed essential oil of E. cloeziana (EC50: 1.5 µgml^-1) was seven times higher than ascorbic acid (11.0 µgml^-1) (Table 3).

The seed essential oil of E. cloeziana investigated has a very high percentage anti-inflammatory value of 68.8% at 1000 µg, this showed that it has comparative properties as indomethacin (93.7%) (Table 4).

The seed essential oil of E. cloeziana showed a very active antinociceptive properties by inhibition in both neurogenic (81.25%) and inflammatory pain (80.34%) induced by intraplantar injection of formalin (Table 5).

The antimicrobial activities of the seed essential oil of E. cloeziana against E. coli, K. pneumoniai, P. aeruginosa, S. typhimurium, S. aureus and S. agalactiae were given in Table 6. The essential oil showed variable activities against tested bacteria. The essential oil was effective on all the bacteria tested. The highest inhibitory effect of the seed essential oil of E. cloeziana was observed against S. aureus (20 mm), E. coli (19 mm), K. pneumoniai (16 mm), P. aeruginosa (15 mm), S. agalactiae (10 mm) but resistant to S. typhimurium. The tested bacteria were found to be resistant to cloxicillin (CXC) while some were sensitive to gentamicin (GEN) synthetic antibiotics.

This study examined the phytochemicals, total phenolic content, antioxidant, anti-inflammatory, antinociceptive and antimicrobial properties of the seed essential oil of E. cloeziana from Nigeria. The seed essential oil of E. cloeziana contained medicinally active phytochemicals (Table 1). Previous studies on the leaf oil of E. cloeziana from Nigeria showed that the composition was dominated by a-pinene (46.6%) and 1,8-cineole (15.4%) [22]. Moreover, the leaf essential oil from Brazil gave a-pinene (74.9%) as their main component [23]. It is worthy to note that the percentage of 1,8-cineole is higher in the seed essential oil while the percentage of a-pinene is low in the seed essential of E. cloeziana compared to the leaf essential oil of this plant [22][23].

The essential oil gave a higher TPC, when compared with the previous study on the related species such as the leaf essential oil of E. globulus from Greece with 10.5±0.3 mgg^-1 gallic acid equivalents which was found to contain a relatively low concentration of phenolic compound compared with the seed essential oil of E. cloeziana investigated in this study [34]. This report indicates that total phenolic content is directly proportional to antioxidant and pharmacological properties of the seeds of the plant. Phenolic compounds have aroused considerable interest recently because of their potential beneficial effects on human health [35].

The DPPH radical scavenging capacity of the seed essential oil of E. cloeziana was higher than that of ascorbic acid [25]. The free radical scavenging and antioxidant properties of the essential oil were found to be three times more active than the synthetic antioxidant as given in Table 2. As the IC50 concentration and the antioxidant capacity have inversely proportional values, E. cloeziana was established to have the high antioxidant capacity. Moreover, the seed essential oil of E. cloeziana inhibited the DPPH free radicals than extracts of other related species such as E. astringens var. obtuse (Iran) with the following IC50: polar (H2O) extract from leaves IC50: 39.8 µgml^-1, non-polar (chloroform) extract from leaves IC50: 217.8 µgml^-1, polar (H2O) extract from flowers IC50: 18.2 µgml^-1 and non-polar (chloroform) extract from flowers IC50: 264.2 µgml^-1 [36]. The antioxidant activity has been related to the number and position of free hydroxyl groups in terpenoids and phenolic compounds, which could be a result of their hydrogen donating ability. The reduction in the number of DPPH molecules can be correlated with the number of available hydroxyl groups [37]. The seed essential oil has the AAI value of 14.3 and could therefore be classified as a very strong antioxidant substance.

The seed essential oil of E. cloeziana had seven times higher metal reducing ion potential than ascorbic acid. The seed essential oil investigated were more effective than the leaf essential oil of E. sideroxylon which FRAP antioxidant potentials as 130.5 µM [38]. The presence of terpenoid and phenolic compounds in the seed essential oil of E. cloeziana are likely to contribute to its higher FRAP value than that of ascorbic acid since these compounds are known to chelate metal ions [39].

The anti-inflammatory activity of the seed essential oil was more effective than the leaf essential oil of E. globulus at concentration of 100 mgkg^-1 which caused inhibition of inflammatory by 76% [40]. This study has shown that the seed essential oil of E. cloeziana investigated possessed a significant antiedematogenic effect on paw edema induced by carrageenan due to the presence of secondary metabolites in the seed essential oil. The nerve damage is caused by inflammation; Inflammation occurs when the body’s own immune cells attack the nervous system [41]. Phenolic compounds have been suggested to be beneficial for the treatment of neurodegenerative diseases [42].

Moreover, the essential oil inhibited the two phases of the formalin response. This indicates the presence of analgesic phytochemical(s) in the seed essential oil. The antinociceptive activities of the seed essential oil investigated were more effective than the leaf essential oil of E. globulus at concentration of 100 mgkg^-1 which caused inhibition of neurogenic pain by 53% [40]. The formalin test has an advantage over other frequently used tests as it involves a biphasic response with an early and a late phase representing respectively neurogenic and inflammatory pain and agents can be screened for activities in these two phases models of pain. This is of interest considering that both phases are sensitive to centrally acting drugs, such as opioids [43]. However, the second phase is also sensitive to NSAIDs (non-steroidal, anti-inflammatory drugs) and corticosteroids [44].

The antibacterial properties of this essential oil were more active than that of leaf essential oils of other Eucalyptus species such as leaves essential oils of eight species of the Eucalyptus from Tunisia (E. bicostata, E. cinerea, E. maidenii, E. odorata, E. sideroxylon, E. astringens, E. lahmannii and E. leucoxylon) showed low inhibitions against H. influenzae, K. pneumoniaie, P. aeruginosa, S. aureus, S. agalactiae, S. pneumoniae and S. pyogenes between 6.0–14.5 mm, which are very low compared to the antibacterial activities of the seed essential oil investigated in this study except for E. maidenii (S. aureus = 22.8 mm and S. pyogenes = 15.5 mm) E. odorata (H. influenzae = 19.2 mm, S. aureus = 27.4 mm, S. agalactiae = 19.4 mm, S. pneumoniae = 17.4 and S. pyogenes = 19.0 mm), E. leucoxylon (S. aureus = 16.4 mm), E. bicostata (S. aureus = 15.6 mm) and E. astringens (S. aureus = 15.5 mm) which showed better inhibitions against few bacteria [45].

The seed essential oil of Eucalyptus cloeziana showed good therapeutic properties. The results of the total phenolic content, free radical scavenging, antioxidant, antiinflammatory, antinociceptive and antimicrobial properties of the part of the plant investigated in this study was thought to be basically due to the synergic effects of the phytochemical constituents in the seed essential oil. The present study suggested that the seed essential oil could be used for the development of naturally occurring antioxidants, anti-inflammatory, analgesic, antibiotics and other forms of drugs. Further studies should be carried out on other medicinal properties of the seed essential oil.

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