ABTS Assay

The analysis of variance, by one-way ANOVA, showed a significant difference at <0.0005 significance level for the influence of seasonal variation on the antioxidant activity of *A. javanica* flowers' EO using ABTS assay. As shown in Figure 7, the highest antioxidant activities obtained in the spring (3.075 ± 0.263 mg Aq/g) are followed by autumn (1.719 ± 0.025 mg Aq/g), winter (1.685 ± 0.255 mg Aq/g), and summer (0.706 ± 0.248 mg Aq/g), respectively.

**Figure 7.** Effect of seasonal variation on *A. javanica* EO by ABTS assay. The different letters are indicators of the significant variation between groups.

The analysis of multiple comparisons between groups by Tukey HSD test showed significant variations of antioxidant activity recorded in spring with antioxidant results of all seasons at <0.0005 significance level. Furthermore, the variation between the results of summer with spring, autumn, and winter were significant at <0.0005, 0.002, and 0.003 significance levels, respectively. While no significant variation between autumn and winter was observed.

Still, there are many studies that link the influence of exposing the plant to biotic and abiotic stress factors with high production of EO [9,10]. However, in our work there was a negative correlation between the severity of seasonal impacts and the EO yield, which is in agreement with the results obtained by Hussain et al. [5], who reported that during the year the highest antioxidant activity of basil EO was recorded in spring, while the lowest activity was recorded in summer.

Linking the quantitative results of *A. javanica* EO obtained from flowers to our current antioxidant ABTS results shows that the high antioxidant activity recorded in the spring, which then reduced significantly by the arrival of the summer, could be due to the variation in the amounts of the major compounds, which causes the reduction in the availability of the electron-donation group.

It is worth mentioning that the *A. javanica* flowering stage started approximately in November (end of autumn) and continued growing into winter, reaching maturity in the spring, and completing the life cycle by the beginning of summer. This supports the trend of our obtained antioxidant results by ABTS assay.

Our results are similar to other studies found in the literature, which report that the seasonal variation has a significant influence on the qualitative EO yield [5–7].

Correlation between Antioxidant Assays

The correlation analysis using Pearson's correlation test was done to evaluate the strength of the relationship between the results of antioxidant activity obtained by applying the three antioxidant assays: DPPH, FRAP, and ABTS. The Pearson's correlation coefficients were calculated using SPSS statistical software (version 21) at ≤0.01 significance level, in which the significant effect will be illustrated in this section's tables using the sign (\*).

Our correlation analysis of the flowers' EO antioxidant activity measured for the four seasons is shown in Tables 2–5. The illustrated correlation coefficients of spring flowers show strong positive relationship between all tested antioxidant assays. The correlation coefficient of spring flowers was found to be strong (at ≤0.01 significance level) between DPPH and FRAP, DPPH with ABTS, and FRAP with ABTS. While, the correlation coefficient measured for the remaining seasons shows a very strong positive relationship (significant at ≤0.01 significance level) between all the tested antioxidant assays.

**Table 2.** Pearson correlation coefficients of antioxidant activity of spring flowers.


(NA) means Not Applicable.

**Table 3.** Pearson correlation coefficients of antioxidant activity of summer flowers.


(\*) means significant at ≤0.01 significance level. (NA) means not applicable.

**Table 4.** Pearson correlation coefficients of antioxidant activity of autumn flowers.


(\*) means significant at ≤0.01 significance level. (NA) means not applicable.

**Table 5.** Pearson correlation coefficients of antioxidant activity of winter flowers.


(\*) means significant at ≤0.01 significance level. (NA) means not applicable.

#### *3.3. Chemical Composition (by GC-MS)*

Referring to our previous flowers' EO results (yield and antioxidant analysis), it was shown that the highest yield with the best antioxidant activity of the oil was recorded in spring, in which the chemical composition of the oil was investigated in this section.

The chromatogram result of *A. javanica* air-dried flowers' EO extracted during the spring is illustrated in Figure 8. The complete chemical composition of EO, retention time (RT) in minutes, retention indices (RI), and percentages (%) of identified compounds of the flowers' oil are all represented in Table 6.

**Figure 8.** The chromatogram of *A. javanica* air-dried flowers' EO for spring.


**Table 6.** Composition of *A. javanica* EO obtained from air-dried flowers during spring.

The GC-MS analysis of the flowers' EO showed the chemical composition of the oil with 29 identified volatiles (representing 100% of the oil). The major compounds were angustione (20.72%), evodone (14.77%), *methyl*-cresol acetate (12.49%), *γ*-elemene (9.15%), and verbenone (6.06%). Furthermore, 4-hydroxybenzaldehyde (4.56%), (*4Z*)-decen-1-ol (4.07%), and 1-tetradecene (3.98%) were identified as minor components.

Our results show that the *A. javanica* flowers' EO is a rich source of potential hydrocarbons, and supporting the results published by Samejo et al. [12] and Samejo et al. [13]. Our results are justifying the rich ethnomedicinal applications of the flowers [14,15]. Angustione is a ketone reported to have antioxidant, anticancer, and antiviral (anti-HIV) biological activity [16]. Therefore, spring is more favorable to be considered as a collection season of *A. javanica* flowers that provides EO with a higher percentage of angustione, and consequently, better biological activity.

It is worth mentioning that verbenone is a monoterpene (bicyclic ketone terpene) that has a pleasant characteristic aroma, thus it is used in a wide range of industries (e.g., herbal remedies, herbal teas, spices, aromatherapy, and perfumery) [17]. Furthermore, verbenone is an insect pheromone, thus used to control insects [18]. Similarly, *γ*-elemene is a sesquiterpene that has a floral aroma and reported to cause antifungal and antioxidant activity [19].

Truly, the chemical composition of the EO would be significantly varied according to the harvesting season [5–7]. Therefore, the decision of deciding the favorable harvesting season depends upon the proposed application of the extracted oil.; meaning that different applications can consider different harvesting seasons accordingly.
