Bactericidal and antioxidant properties of essential oils from the fruits Dennettia tripetala G. Baker

The UFO as well as the RFO yields of Dennettia. tripetala was 0.62 and 1.10% respectively. Chemical composition of the EOs and identity of constituents are presented in Table 1. The chemical constituents of D. tripetala EOs which were predominantly terpenoids, effectively enhanced the antioxidant and antibacterial properties in the current research. Thirty three constituents were found in the RFO, while the UFO contained 27 compounds representing 95.32 and 94.06% of the total oil content respectively (Table 1). In the UFO, monoterpenoids accounted for 74.60% of the overall oil content and sesquiterpenoids content was 19.45%. The dominant monoterpenoids were 2-methyl phenyl formate (56.05%), α -terpinene (13.93%) and linalool (2.57%). Caryophyllene (6.23%), α –farsenesene (1.50%), trans-cadinol (0.58%), caryophyllene oxide (0.22%) and 1- (+) - ascorbic acid 2,6-dihexadecanoate (0.16%) were important the bioactive sesquiterpenoids identified in UFO. In the RFO, the monoterpenoids and sesquiterpenoids content increased to 75.10%, and 20.10% respectively. The quantity of some of the bioactive compound such as α -terpinene (16.25%), caryophyllene (10.80%), and linalool (3.85%) were significantly higher in ripe fruit. However, the monoterpenoid constituent (2-methyl phenyl formate) found in the RFO decreased to 51.26%.

The presence of phellandrene, (+) -4-carene, terpinen-4-ol, safrol and elemene was significantly higher in the ripe fruit oil, compared to the unripe fruit oil. This may indicate possible physiological effects of ripening which explains pigment change of D.tripetala fruit as reported previously by Adebayo et al. [16]. Linalool, caryophyllene, 4-carene, phenyl ethyl alcohol and 9-octadecenoic acid found in this present study were among the compounds reported by Elekwa et al. [27] as one the major compounds in the seed of D. tripetala. However, some of the reported bioactive compounds found in the RFO such as caryophyllene oxide [28], terpinen-4-ol [29], farnesene [30], and ascorbic acid [31] were not detected in the seed EO report of Elekwa et al. [27]. The study of Kumar et al. [32] revealed palmitic acid, eicosanoic acid, ethyl ester and linoleic acid as major components of D. tripetala solvent extracts. The discrepancy in the composition of D. tripetala essential oil grown in different regions in Nigeria and elsewhere may be due to differences in factors, such as climate, season, geographical conditions, age of the plant, humidity of the harvested plant material, extraction technique and the existence of chemotypes [33]. 1-nitro-2-phenylethane isolated by Oyemitan et al. [14] from dried seed essential oil of D. tripetala was not found in our EOs. However, the yield of 1-nitro-pentane (0.68%) identified in EOs of this study is higher than 1-nitro-2-phenylethane (0.59%) obtained from the seed EO of D. tripetala by Elekwa et al. [27]. This suggests the possibility of chemical transformation of 1-nitro-pentane in the seed EO. To the best of our knowledge the dominant compound 2-methyl phenyl formate found in this present study in D. tripetala EOs has not been reported as component of the plant. Methyl phenyl formate which naturally occurs in capsicum, coffee, pepper and some wine [34], may account for peppery characteristics when the unripe or ripe fruit of D. tripetala is consumed. The two EOs showed some hemolytic activity on Sheep red blood cells. This might be due to the fragile nature of the red blood cells since there has never been any report on human toxicity after consumption of D. tripetala fruits as it is a commonly consumed fruit in West Africa.

Antioxidant activities of UFO and RFO were examined using four (DPPH, ABTS, LP and NO) different radicals. The scavenging effects of the four free radicals by two oils and reference compounds were concentration dependant (Figs. 1, 2, 3 and 4). In the DPPH test (Fig. 1), the inhibitory effect of the two oils were stronger (#) than of reference compound (vitamin C) at all concentrations (0.05 to 0.50 mg/mL), however, when compared with β-carotene, results were similar (* *). The DPPH radical assay illustrates that a donor of electron or hydrogen atom is an antioxidant and its effect is demonstrated as DPPH•, color fades away (purple to yellow) in the test sample due to formation of neutral DPPH-H molecule upon absorption of hydrogen from an antioxidant [35]. The antioxidant strength of tested sample is evaluated by the decrease of UV absorption at 517 nm. However, DPPH test does not differentiate radical species but indicates general radical scavenging potential of an antioxidant [36]. Therefore, to evaluate specific antioxidant efficacy of UFO and RFO, quantitative tests using two different specific radicals species (LP • and NO•) and a cation (ABTS ^{• +}) radical were carried out. A general trend in all four in vitro experiments was observed, where both the RFO and UFO of D. tripetala displayed valuable radicals scavenging effects acting as electron donors in the DPPH, ABTS tests, demonstrated high LP• as well as NO• radical scavenging capacity. Standard curves produced from % inhibitions against concentrations (Tables 2, 3, 4 and 5) of the oils and reference compounds (RC) were all linear in the four assays. Linear regression equation generated (Figs. 5, 6, 7 and 8) from each extract as well for the RC was used to calculate IC₅₀ value. T-Test analysis was applied to test significant differences (Figs. 1, 2, 3 and 4) of % inhibitions against concentrations using SPSS15.0 for windows (IBM SPSS Inc OLRAC SPS registration number 2012/1786646/07). From the standard regression equations generated (Fig. 5) from DPPH data (Table 2), for UFO, y = 10.57× + 40.81; R² = 0.9269: thus x (IC₅₀ for UFO) = 50–40.81/10.57, = 0.87 mg/mL), for RFO, y = 13.49× + 41.59; R² = 0.8214: × = 50–41.59/13.49, (IC₅₀ for RFO) = 0.62 mg/mL, while for vitamin C, y = 14.512× + 0.87; R² = 0.8978, × = 50–0.087/14.512 = 3.39 mg/mL and for β carotene, y = 11.64× + 46.312; R² = 0.9139, × = 50–46.312/11.64, (IC₅₀ for carotene = 0.32 mg/mL). The two oils reduced the DPPH• to a neutral DPPH-H, molecule, attaining 50% decrease with an IC₅₀ value of 0.87 ± 0.23 mg/mL for the UFO, 0.62 ± 0.12 mg/mL for RFO, both lower than the value for vitamin C (3.39 ± 0.12 mg/mL) but significantly higher than that of β-carotene (0.32 ± 0.22 mg/mL) p < 0.05 (Table 2).

In the ABTS test, the scavenging effects by the RFO and β-carotene were similar to that in DPPH model. However, unlike in the DPPH assay, UFO and vitamin C had low activity against ABTS ^{• +} at 0.05–0.2 mg/mL, while RFO and the reference compound (β-carotene) scavenging effects were moderate except at 0.4 and 0.5 mg/mL (Fig. 2). The % inhibitions against concentrations for ABTS data are presented Table 3, while the regression equations generated from the data (Fig. 6) are for UFO, y = 11.546× + 31.608; R² = 0.9732. Thus x (IC₅₀ for UFO) = 50–31.608/11.546 = 1.59 mg/mL; for RFO, y = 12.73× + 38.3, × (IC₅₀ for RFO) = 50–38.3/12.73 = 0.92 mg/mL; for vitamin C, y = 10.638× + 21.072; R² = 0.8935, × (IC₅₀) = 50–21.072/10.638 = 2.70 mg/mL and β – carotene, y = 11.7× + 41.9; R² = 0.9415, × = 50–41.9/11.7 = 0.69. The IC₅₀ values obtained for RFO (0.90 ± 0.02 mg/mL) and β-carotene (0. 69 ± 0.13 mg/mL) from linear regression equations and T-Test analysis using SPSS15.0 for windows were higher compared to DPPH results. The discrepancy observed in the effects of the oils on DPPH and ABTS radicals might be due to factors including the ease at which the oils solvate the radical’s medium as well as complexity, polarity, preferred isomers selection of the radicals which are all factors suggested to influence the activity of volatile constituents in the scavenging of radicals [36].

The scavenging effects of UFO and RFO on lipid peroxide radicals (LP^•) are showed in Fig. 3. The unripe and ripe fruit EOs exhibited more scavenging effect compared to the reference compound (vitamin C) at low concentrations (0.05 mg/mL). The UFO, RFO and β-carotene lipid peroxyl scavenging effects at low (0.05–0.10 mg/mL) and high (0.50 mg/mL) concentrations were similar (* *). At 0.2 and 0.40 mg/mL, β-carotene displayed more scavenging effect than the UFO and RFO (#), however at 0.5 mg/mL the two oils and β-carotene exhibited similar (**) scavenging activity. As in DPPH and ABTS assays, the average percentage inhibitions of lipid peroxyl radicals versus concentrations (Table 4) and the regression equations generated (Fig. 7) were used to calculate the IC₅₀ values for EO and RC. The Interestingly, the IC₅₀ values obtained for UFO and RFO (2.03 ± 0.10 and 1.90 ± 0.00 mg/mL respectively) were comparable to β-carotene (1.67 ± 0.11 mg/mL) and lower than that of vitamin C (3.40 ± 0.10 mg/mL) (Table 2). Fig. 4 shows the scavenging effects of UFO and RFO on nitric oxide radical (NO^•) produced from red-colored complex salt of sodium nitroprusside solution at varying concentrations (0.05–0.5 mg/mL). Ripe fruit oil exhibited more scavenging activity on NO^• compared to UFO as well as the two reference compounds (β-carotene and vitamin C) at 0.05–0.20 mg/mL. At higher concentrations (0.4–0.5 mg/mL) the scavenging effects on NO^• by β-carotene and RFO were similar (**) and higher than that of UFO and vitamin C (Fig. 4). Overall, in the NO^• assay, the IC₅₀ values obtained from standard curves regression equations generated (Fig.8) from the average % inhibitions in nitric oxide experiment (Table 5) for the two EOs and reference compounds indicated that RFO had the highest antioxidant capacity (1.27 ± 0.03 mg/mL), followed by β-carotene (1.85 ± 0.10 mg/mL), then UFO (2.01 ± 0.12 mg/mL) and vitamin C was least (2.33 ± 0.11 mg/mL) (Table 2).

The essential oils isolated from unripe and ripe fruits of D. tripetala displayed strong inhibitory effect against the 5 multi-drug resistant reference strains (S. aureus (NCINB 50080), E. faecium (ATCC19434), L. ivanovii, (ATCC 19119), E. cloacae (ATCC 13047), E. coli O157 (ATCC 700728), as well as 4 multi- drug resistant bacteria E. coli 180, E. coli 179, E. coli 132 and Vibro spp.) from our laboratory stock culture. The unripe fruit oil (UFO) was more active than the ripe fruit oil (RFO) against most of the bacterial strains investigated with MIC values of 0.05–0.20 mg/mL and 0.10–0.20 mg/mL respectively (Table 6). The UFO and RFO were bactericidal against E. faecium at 0.05 and 0.10 mg/mL respectively. The two essential oils were however bacteriostatic against the 8 multi-drug resistant bacterial strains after 24 h (Table 7). The two EOs showed lower activity against E. coli strains which are Gram negative bacteria when compared to Gram positive bacteria (S. aureus, E. faecium, L. ivanovii, E. cloacae) tested. The outer complex membrane of Gram-negative bacteria have been shown in previous studies to contain hydrophilic lipopolysaccharide [37], which create a barrier toward macromolecules and hydrophobic compounds, providing Gram-negative bacteria with higher tolerance toward hydrophobic antibacterial compounds like those found in essential oils [6, 7]. Another probable cause of resistance against phytochemicals has been suggested to be the presence of multi-drug resistant sites that promote the synthesis and secretion of amphipathic toxins [38]. The bioactivity of UFO and RFO tests on the multi-drug resistant bacteria also varied, the variation of components in their chemicals profiles (Table 1) may account for the observed results.

The bioactive compounds in EOs are broadly divided into four groups according to their chemical structure: terpenes, terpenoids, phenylpropenes, and others contain different degradation products originating from terpenes, unsaturated fatty acids, lactones, glycosides, and sulfur- or nitrogen-containing constituents. The constituents of the two EOs of D. tripetala in this current study were predominantly terpenoids. Monoterpenoids as well as sesquiterpenoids were more prominent (95.01%) in the profile of RFO than in UFO (Table 1). Previous essential oils studies have demonstrated bioactivities of some of the individual terpenes and terpenoids including p-cymene [6], α–terpinene, caryophyllene [39], linalool [40], β-ocimene [41] and ascorbic acid found in the EOs of D. tripetala in this present study. Several studies have indicated that p-cymene act as a substitutional impurity in the membrane, which partly perturbs the membrane of microorganisms [6]. Studies on cell and vesicle systems indicate that p-cymene has no effect on the membrane permeability, but acts by decreasing the enthalpy and melting temperature of membranes and also decreasing cell motility [42]. Alpha -terpinene found in both oils, had previously been reported by Takahashi et al. [43] which showed strong potency to inhibit low density lipoprotein oxidation even in the formation phase. Other components such as camphene, α – terpineol, α-pinene and borneol found in the two oils have been previously investigated and found to exhibit a variety of biochemical activities [44‐46], these may have enhanced the activities of the EOs, suggesting possible synergistic interactions between the constituents. In addition, the main component- 2-methyl phenyl formate identified in the two oils for the first time could have reacted with the bacteria and other different radicals through various mechanisms as suggested by Mortein et al. [6] and Foti and Amorati [47]. The results from the current study are in agreement with other reports that have implicated aliphatic terpenes and terpenoids with bioactive properties, while the effect of cyclic monoterpenes and sesquiterpenes with double bonds were similar to the properties of phenolic compounds such as α – tocopherol. The ability of the UFO and RFO to scavenge four varieties of free radicals and exhibit strong activity against some multi-drug resistant bacteria is noteworthy. These observations may suggests that the fruit EO of D. tripetala could possibly be a new active candidate in the search for lead constituents for the management of infectious and oxidative stress-related disorders such as arthritis, cancers, arteriosclerosis, and dementia [48, 49].