3.2.1. Quantitative Analysis (by Yield)

The influence of seasonal variations on the EO yield (%, *v*/*w* of dry weight) of *A. javanica* air-dried flowers is illustrated in Figure 4. The mean results are represented with their standard deviations (SDs) and standard deviation error bars.

The analysis of variance, by one-way ANOVA, showed significant effect for the seasonal variation on flowers' EO yield (quantitatively) at 0.042 significance level (*p* ≤ 0.05). The analysis of multiple comparisons between groups by Tukey HSD test showed significant variations of EO yield obtained in spring at 0.036 significance level with results obtained in autumn. Our results are similar to other studies found in the literature, which report that a seasonal variation has significant influence on EO yield [5–7]. According to Hussain et al. [5], the highest EO yield was obtained in spring, which was reduced afterward. Therefore, our findings are in agreement with their obtained results.

Annually, *A. javanica*'s flowering stage started approximately in November (end of autumn season), continuing to grow in the winter, reaching maturity in the spring, and completing the life cycle by the beginning of summer. This corresponds with the trend of our obtained EO yields, which shows the lowest results in the autumn, followed by an increased EO yield in winter, reaching the maximum yield in spring, which then starts decreasing by the beginning of summer.

**Figure 4.** Effect of seasonal variation on flowers of *A. javanica* EO yield. The different letters are indicators of the significant variation between groups.

> Our results are in agreement with the results obtained by Hussain et al. [5], Omer et al. [6], and Villa-Ruano et al. [7], which report that the seasonal variation has a significant influence on the quantitative EO yield. According to Hussain et al. [5], high EO yield was recorded in spring and reduced afterward, which is similar to our results.

> During spring, the flowers reach the maturity stage, thus the EO yield from the glandular cells will reach maximum levels, which will play a major role in the pollination process. However, with the arrival of the summer, which is characterized by high temperature and high variations between daily minimum and maximum temperatures, the proline content of the leaves will reach maximum levels, while flowers will end their life cycle, and accordingly the glandular cells will collapse, thus flowers' EO content will be reduced.

> Based on our results, the best season to extract the highest quantitative EO yield (0.011 ± 0.002%) obtained from *A. javanica* flowers is spring, followed by early summer (0.009 ± 0.001%) and winter (0.007 ± 0.000%). While extracting the oil from flowers collected during autumn provides the lowest yield (0.006 ± 0.001%), thus not recommended.

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

3.2.2. Qualitative Analysis (by Antioxidant Activity) DPPH Assay

The DPPH molecule (2,2-diphenyl-1-picryl hydrazyl) is one of the few stable organic nitrogen radicals. It is characterized by deep purple color, with maximum absorbance at 517 nm wavelength. The antioxidants cause the reduction reactions against the free radical DPPH by pairing; it has an odd electron with the free radical scavenging antioxidant. This reaction causes color loss of the purple DPPH due to the formation of the reduced DPPH-H. The higher the color loss, the higher the antioxidant concentration would be, thus higher DPPH scavenging activity [8].

In this work, the antioxidant concentration was calculated as Trolox equivalent from a calibration curve and the antioxidant activity was expressed as mg Trolox eq/g of extract.

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 DPPH assay. As shown in Figure 5, the highest antioxidant activities are obtained in the spring (12.20 ± 1.44 mg Aq/g), followed by winter (11.5 ± 0.15 mg Aq/g), autumn (7.77 ± 0.21 mg Aq/g), and summer(7.08 ± 0.21 mg Aq/g), respectively.

**Figure 5.** Effect of seasonal variation on *A. javanica* EO by DPPH 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 summer and autumn both at <0.0005 significance level, while no significant variation between spring and winter was observed. The antioxidant activity recorded in winter was significant with summer and autumn also at <0.0005 and 0.001 significance levels, respectively.

Although, there are many studies that link the influence of exposing the plant to biotic and abiotic stress factors with the high productivity of EO [9–11]. 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 and qualitative results of *A. javanica* EO obtained from flowers to our current antioxidant DPPH results shows that the high antioxidant activity recorded in the spring, which was then reduced significantly by the arrival of summer, could be due to the variation in amounts of major compounds, which causes the reduction in the availability of the electron-donation group.

It is worth mentioning that the *A. javanica* flowering stage starts approximately in November (end of autumn) and continues growing into the winter reaching maturity in spring, and completing the life cycle by the beginning of summer. This supported the trend of our obtained antioxidant results by DPPH 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]. According to Hussain et al. [5], basil EO extracted in spring was rich in oxygenated monoterpenes, while the oil obtained in summer was rich in sesquiterpene hydrocarbons. Meaning that, seasonal variation has a significant influence on the EO qualitative yield, which is similar to our obtained antioxidant results.
