Chemical characterization
This is the first report of the chemical composition of the essential oil extracted from Eugenia pohliana leaves. The chemical components, their respective retention indices (RI), and relative amounts of EO extracted from E. pohliana leaves are shown in Table 1. The EpEO presented a yellowish color, and the chemical characterization showed 38 compounds, comprising 98.73% of the total. The major components were sesquiterpenes: (E)-β-caryophyllene (15.56%), δ-cadinene (11.24%), and α-cadinol (10.89%).
Table 1
Chemical composition of the essential oil obtained from the leaves of E. Pohliana (EpEO).
Compone | RIa | RIb | % | Compone | RIa | RIb | % |
α-pinene | 931 | 932 | 0.34 | γ-cadinene | 1515 | 1513 | 3.37 |
β-pinene | 973 | 974 | 0.67 | δ-cadinene | 1526 | 1522 | 11.24 |
Limonene | 1027 | 1024 | 0.06 | trans-cadina-1,4-diene | 1534 | 1533 | 0.13 |
δ-elemene | 1337 | 1335 | 3.19 | α-cadinene | 1539 | 1537 | 1.02 |
α-cubebene | 1349 | 1348 | 0.11 | α-Calacorene | 1544 | 1544 | 0.70 |
α-ylangene | 1371 | 1373 | 0.25 | Elemol | 1550 | 1548 | 0.12 |
α-copaene | 1375 | 1374 | 0.62 | Germacrene B | 1558 | 1559 | 0.52 |
β-elemene | 1392 | 1389 | 3.06 | Palustrol | 1568 | 1567 | 0.78 |
α-gurjunene | 1410 | 1409 | 1.73 | Spathulenol | 1578 | 1577 | 1.69 |
β- (E) -caryophyllene | 1421 | 1417 | 12.56 | Globulol | 1585 | 1590 | 2.57 |
α-humulene | 1454 | 1452 | 3.09 | Guaiol | 1593 | 1600 | 2.41 |
Allo-aromadendrene | 1461 | 1458 | 1.50 | Ledol | 1604 | 1602 | 1.48 |
trans-candina-1(6),4-diene | 1474 | 1475 | 1.63 | 1,10-di-epi-cubenol | 1616 | 1618 | 0.74 |
γ-muurolene | 1477 | 1478 | 1.34 | 1-epi-cubenol | 1629 | 1627 | 1.53 |
α-amorphene | 1481 | 1483 | 4.51 | tau.-Muurolol | 1645 | 1644 | 10.81 |
β-selinene | 1487 | 1489 | 1.36 | α-cadinol | 1659 | 1652 | 10.89 |
Bicyclogermacrene | 1498 | 1500 | 8.13 | Shyobunol | 1692 | 1700 | 0.10 |
α-pinene | 931 | 932 | 0.34 | eudesm-7(11)-en-4-α-ol | 1697 | 1700 | 0.10 |
β-pinene | 973 | 974 | 0.67 | γ-cadinene | 1515 | 1513 | 3.37 |
| | | | Total | | | 98.73 |
RIa= Retention rate determined; RIb= Retention index specialized literature; %= area of compost relative to EpEO. |
Table 2
Effect of Eugenia pohliana Essential Oil (EpEO) on leukocyte migration and neutrophil migration in peritoneal exudation in carrageenan-induced.
Treatments | Dose (mg/kg) | Leukocytes (105/ml) | Inhibition (%) | Neutrophils (105/ml) | Inhibition (%) |
Control | - | 8.1 ± 0.9 | - | 5.3 ± 0.5 | - |
Indomethacin | 20 mg/kg | 1.7 ± 0.6* | 79.01 | 1.5 ± 0.3* | 71.69 |
EpEO | 100 mg/kg | 2.4 ± 0.6* | 70,38 | 2.4 ± 0.3* | 54,72 |
| 50 mg/kg | 3.2 ± 0.5* | 60,50 | 2.7 ± 0.3* | 49,06 |
| 25 mg/kg | 3.9 ± 0.5* | 51,86 | 3.3 ± 0.2* | 37,74 |
Values represent the mean ± SEM. * p < 0.001 compared with Control, one-way ANOVA followed by Dunnett’s Test. |
The presence of (E)-β-caryophyllene (BCP), an odorous bicyclic sesquiterpene, common in plants of this family, has been reported as a component in E. brejoensis species (Mendes et al. 2018) E. caryophyllata (Wang et al. 2021), E. uniflora (Mesquita et al. 2017), E. calycina (Silva et al. 2021; Sousa et al. 2015), E. brasiliensis (Silva et al. 2019), E. pyriformis (Durazzini et al. 2019), E. dysenterica (Silva et al. 2018), E. sulcata (Gonzalez et al. 2014)d egensis (Silva et al. 2017).
The presence of (E)-β-caryophyllene (BCP), an odorous bicyclic sesquiterpene, common in plants of this family and has been reported as a component of E. brejoensis species that have antimicrobial activity (Mendes et al. 2018)d calycina, which has larvicidal (Silva et al. 2021), cytotoxic, and antimicrobial activity (Sousa et al. 2015). BCP is also found in E. dysenterica, which has healing activity (Silva et al. 2018); E. sulcata, which has insecticidal activity (Gonzalez et al. 2014)d egensis, which has cytotoxic and antioxidant properties (Silva et al. 2017).
BCP, a phytocannabinoid, acts as a ligand for cannabinoid receptor-2 (CB-2) (Gertsch et al. 2008), a part of the endocannabinoid system that is involved in cell signaling (Meccariello et al. 2020). When activated, this receptor inhibits mediators of inflammation, contributing to the relief of pain and inflammation (Maayah et al. 2020). Compounds that interact with the endocannabinoid system have shown promise for the treatment of various diseases (Shah et al. 2021). Other activities can be attributed to BCP, such as antimicrobial activity against Staphylococcus mutans (Yoo and Jwa. 2018), antiherpetic (Astani et al. 2011), wound healing (Koyama et al. 2019), anti-inflammatory (Brito et al. 2019), and protective effects against ischemic brain injury (Chang et al. 2013).
Another compound present in the composition of EpEO was δ-cadinene, found in the oil of E. caryophyllata (Wang et al. 2021) which showed antinociceptive and anti-inflammatory activities (Taher et al. 2015)d brasiliensis, which showed significant antimicrobial activity (Silva et al. 2019). This compound has demonstrated acaricidal activity against Psoroptes cuniculi (Guo et al. 2017).
The oxygenated sesquiterpene α-cadinol is a termiticide (Morikawa et al. 2014) reported in the composition of E. brejoensis (Mendes et al. 2018)d pyriformis (Durazzini et al. 2019). To date, there are no reports of antinoceptive and anti-inflammatory activities for these species, but other pharmacological activities have been described, such as antimicrobial activity in E. brejoensis (Bezerra Filho et al. 2020)d pyriformis (Souza et al. 2021).
Other species of the Myrtaceae family with biological activity contain α-cadinol, such as Myrcia tomentosa, which has antimicrobial activity (Sa et al. 2017), Eucalyptus occidentalis, which has shown repellent and insecticidal activity (Bande-Borujeni et al. 2018) and Plinia trunciflora, which has antimicrobial activity against yeasts and bacteria (Lago et al. 2011).
Antinociceptive activity
Acetic acid-induced writhing test
The acetic acid-induced writhing model, although not specific, is a simple and sensitive test that is the standard for evaluating candidate drugs with antinociceptive action (Hunskaar and Hole 1987). EpEO reduced writhing in animals by 42.95%, 52.94%, and 70.70% when treated with doses of 25, 50, and 100 mg/kg, respectively. The standard drugs morphine and indomethacin promoted a reduction by 98.83% and 85.88%, respectively, when compared to the control group (Fig. 1).
The genus Eugenia has shown positive results in tests of writhing induced by acetic acid; for example, the methanolic extract of E. uniflora leaves lead to 60% and 74% reduction in writhing at concentrations of 100 and 200 mg/kg, respectively. The EO from E. caryophyllata leaves inhibited 89.6% of abdominal writhing induced by acetic acid in mice in the treatment with a concentration of 100 mg/kg (Taher et al. 2015), which was attributed to the presence of BCP as a major compound in its chemical composition (Wang et al. 2021)
Tail Immersion
The tail-immersion test is a highly sensitive test for opioid drugs and is ideal for the evaluation of drugs that act on the central nervous system (Oliveira et al. 2018; Khatun et al. 2015). EpEO promoted greater analgesic action after 90 min of administration, with an increase in latency time by 86.63–69.12%. Treatment with 100 mg/kg of EpEO or morphine showed an antinociceptive action above 50% at assessment time points. These results indicate that EpEO has a central analgesic effect similar to that of morphine (Kotlinska et al. 2013).
Formalin test
In the formalin-induced nociception test, EpEO showed antinociceptive action at all doses and during both phases of the test. A significant reduction (p < 0.001) in time spent licking the paw was achieved; 61.74%, 65.5%, and 29.54% in the first phase (neurogenic pain) and in 64.87%, 64.3%, and 37.42% in the second phase (inflammatory pain) at doses of 100, 50, and 25 mg/kg, respectively, compared to the control (Fig. 3). Morphine, the standard drug, reduced licking time in both phases (86.5–86.3%), while indomethacin was effective only in the second phase of the test (92%), when compared to the control.
The EO of E. candolleana leaves, with composition dominated by BCP, inhibited the licking time after formalin injection by up to 55.65% in the first phase; however, the results were more significant in the second phase, with inhibition of up to 96.7% at a dose of 100 mg/kg (Guimarães et al. 2009). According to the literature, BCP is among the major compounds of this species (Neves et al. 2017). Santos et al. (2020) reported that acetone extract from Myrciaria floribunda fruits reduced licking time by up to 86.52% in the treatment with 100 mg/kg in the first phase and 82.58% in the second phase. (Silva Barbosa et al. 2020) described the chemical composition of the EO of M. floribunda fruit peels as dominated by BCP and δ-cadinene (Silva Barbosa et al. 2020).
Commercially obtained BCP alone has a significant effect on inflammatory pain in the formalin test; however, it does not have any treatment effect on neurogenic pain (Klauke et al. 2014). Thus, the significant results in the inflammatory pain phase of EpEO treatment may be related to the presence of BCP.
To investigate the mechanism of action and antinociceptive activity using the formalin test, we pretreated the rats with naloxone, a non-selective opioid receptor antagonist, followed by treatment with EpEO 100 mg/kg or morphine. The action of EpEO was almost entirely inhibited, similar to morphine, in the two phases of the formalin test (Fig. 3). These results suggest that the antinociceptive effect of EpEO occurs through the activation of opioid receptors (Lewanowitsch et al. 2006), as suggested by the tail-flick test.
Anti-inflammatory activity
Carrageenan-induced paw edema
As previously mentioned, there are no studies on the biological activities of E. pohliana; however, based on evidence of anti-inflammatory action of the genus Eugenia in addition to the expressive action of the treatment with EO in the second phase (inflammatory phase) of the formalin test, we investigated the anti-inflammatory potential using the paw edema test to screen for this possible effect. Indeed, treatment with EpEO promoted a reduction of 74.93–81.4% in paw edema induced by carrageenan, while indomethacin inhibited it by 81.1% at 3h when compared to control (Fig. 4). The activity of EpEO and indomethacin remained constant until the end of the test.
Similarly, Costa et al. (2020) described that an E. stipitata EO had an anti-inflammatory effect on paw edema, reducing it by up to 96.94% after treatment with 250 mg/kg. According to Sobeh et al. (2019), the paw edema test with the methanolic extract of E. uniflora leaves reduced edema by 32%. In this study, the inhibition potential for Cyclooxygenase-1 (COX-1) and Cyclooxygenas − 2 (COX-2) was determined. The results show significant inhibition, with IC50 values of 5.63 µg/mL for COX-1 and 0.18 µg/mL for COX-2. The inhibition of these enzymes prevents the production of inflammatory substances and the action of anti-inflammatory NSAIDs (Trinh et al. 2021). Mesquita et al. (2017) reported that BCP is a major volatile chemical extracted from E. uniflora.
Peritonitis
After confirming the anti-edematogenic effect of the EpEO treatment, the carrageenan-induced peritonitis test was performed as a model of acute inflammation. Oral administration of EpEO promoted a significant reduction (p < 0.001) of 51.86‒70.38% and 37.74‒54.72% in the influx of leukocytes and neutrophils, respectively, at all doses tested. Indomethacin inhibited 79.01% of leukocyte migration by 71.69% of neutrophils.
Lazarini et al. (2020) showed that oral treatment with ethanolic extract of E. selloi fruits promoted a decrease in neutrophils by 58% and 70% at concentrations of 3 and 10 mg/kg, respectively. A methanolic extract of E. uniflora leaves (100 mg/kg) reduced the number of leukocytes to 4.84 ± 1.97 x 106 mL compared to untreated animals (10.04 ± 2.64 × 106 mL) (Sobeh et al. 2019). BCP, the main compound of EpEO, reduces the peritoneal migration of neutrophils at a dose of 100 µL (Brito et al. 2019). Such anti-inflammatory effects may be related to its ability to activate CB-2 receptors, as the absence of these receptors suppresses the inflammatory response in animal models (Gertsch et al. 2008).
Adverse Effects Assessment
Open field and elevated plus-maze test
In general, analgesic agents have direct side effects on the central nervous system (CNS). To test for these side effects, we used the open field and elevated plus maze tests, which have become a standard for measuring anxiety, sedation, and activity not only in rodents, but also in several other animals (Prut and Belzung, 2003). The absence of the animal's exploratory behavior indicates that the substance tested has sedative and depressant activity (File and Wardill, 1975; Mujumdar et al. 2000).
Thus, to investigate the occurrence of this possible effect in the treatment with EpEO, we tested the animals in the open field and elevated plus maze test after a dose of 1,000 mg/kg, which is 10 times greater than the therapeutic dose used in our tests. Treatment with EpOE in the open field test showed no significant differences in rearing (20 ± 2) and crossing (58 ± 12). However, diazepam resulted in an absence of the animals' exploratory behavior with values of 8 ± 1 for rearing and 37 ± 6 for crossing. The control group showed rearing of 25 ± 3 and crossing of 75 ± 7.
In the LCE test, treatment with EpEO did not promote anxiety symptoms, presenting an anxiety index of 0.54, which was not significant when compared with the control (0.41). Diazepam, on the other hand, had an anxiety index of 0.09, indicating that its neurological activity caused anxiety in the animals.
Thus, our results revealed that EpEO treatment did not affect exploratory activity and anxiety. In contrast, diazepam at 1 mg/kg showed significant values in relation to the control. Based on these results, EpEO can be considered as a possible herbal medicine candidate for the treatment of pain and inflammation.