2.5.1. Sample Preparation

The flowers collected throughout the year in each of the four seasons (as described in Table 1) were washed thoroughly with distilled water three times, and left in a well ventilate shaded area (27 ◦C) for two weeks (until completely dried). The flowers were then weighed (dry weight), labelled, and stored in clean and closed paper bags until use for further analysis.

On the distillation day, the flowers were cut into homogenous sizes (2 mm), measured by U.S. metric sieves (SOILTEST, ASTM), and directly subjected to hydrodistillation. It is worth mentioning that the size of *A. javanica* flowers, which is 2 mm, was obtained by separating the flowers from the mother plant by hand. The dry matter contents were measured using the following formula:

$$\text{DM}(\%) = \frac{\text{DW}}{\text{FW}} \times 100$$

where DM is the dry matter, FW is the fresh weight, and DW is the dry weight.

2.5.2. Extraction Conditions and Yield Determination

The hydrodistillation process was performed for around 100 g of plant matrix (dry weight) at 85 ◦C, for a period of 4 h, until no further EO was extracted. At the end of the distillation, the EO was accumulated as a waxy extract collected and easily separated from water. Thus, drying with anhydrous sodium sulfate (anhydrous Na2SO4) was not needed. The pure collected EO was weighed and stored in a sealed glass amber vial at 4 ◦C until

analyzed. After the distillation, the Clevenger apparatus was subjected to a process using water, soap, methanol, and ethanol.

The EO yield was calculated based on the plant dry weight and the herbal extract weight using the following equation:

$$\text{EO yield } (\%) = \frac{\text{W}\_{\text{EO}}}{\text{W}\_{\text{plant}}} \times 100\%$$

where, Wplant is the dry weight of the plant (in grams) and WEO is the weight of the extracted EO (in grams).

#### *2.6. Antioxidant Activity*

The antioxidant activity was determined in microplate to evaluate the antioxidant activity (in vitro) by spectrophotometer (Microplate reader, BioTek, EPOCH 2) using a 96-well plate. The tested antioxidant assays were: DPPH (2,2-diphenyl-1-picryl hydrazyl), FRAP (ferric reducing antioxidant power), and ABTS (2, 2-azinobis (3-ethylbenzothiazoline-6-sulfonate)).

#### 2.6.1. Sample Preparation

EO waxy samples of *A. javanica* (flowers) for different seasons (in triplicate) were dissolved in 500 μL of dimethyl sulfoxide (DMSO) and vortexed very well.
