## 3 Results and Discussion  
### The Impact of Particle Size on the Extractability of the Bioactive Compounds  
This paper presents the effect of sample pre-treatment (grinding) on the extraction of the bioactive compounds from two selected plants. The relation between several bioactive terpenoids and different particle sizes was demonstrated. Such an effect has been studied before, but in the absence of the possibility of a significant release of bioactive during the process of grinding the leaves, which is the main objective of this paper.  
GCMS chromatograms results of un-milled, 500 \(\upmu\)m, 850 \(\upmu\)m, and 1400 \(\upmu\)m Manuka leaves at different times showed no extraction of terpen for the unmilled and the 1400 \(\upmu\)m diameter samples even after 60 min. However, Pinene-alpha-\(>\) was the only monoterenes observed in samples with 850 \(\upmu\)m particle size within the defined time. For the diameter of 500 \(\upmu\)m, the intensity of four compounds, namely Pinene-\(<\) alpha-, RT 5.995, Eucalypt, RT 10.526, Globulol, RT 40.952, and Menthol, 1'-(butyln-3-one-1-yl)-, (1R, 2S, 5R)-, RT 42.84, increased over time, reaching the highest intensity after 60 min. In Figure 4a, the extraction of bioactive terpenoids of Manuka samples with a particle size of d \(\geq\) 250 \(\upmu\)m showed almost no extraction at zero time, and the extraction of all bioactives increased  
Figure 3: Ultrasound-assisted extraction system: without circulating water-bath.  
with time, which is expected since the sample was only mildly grounded. Figure 4b shows that the extraction happens at zero time, indicating that most of the release of bioactives have occurred during grinding, which supports our hypothesis.  
Figure 4: Total of selected bioactive terpenoids in Manuka samples with particle size of: (**a**) d \(\geq\) 250, (**b**) d \(\leq\) 250, (**c**) d \(\geq\) 200 \(\mu\)m.  
Samples with a diameter of d \(\geq\) 200 \(\upmu\)m Figure 4c, a high amount of the terpenes, were again extracted and appeared in the chromatogram at a zero time (in a few seconds). Moreover, the terpen content in the extracts increased little after 3, 5, and 7 min to remain relatively constant. Furthermore, the total terpen content after decreasing the particle size (d \(\leq\) 200 \(\upmu\)m) is represented in Figure 5a. Indeed, the increase of the terpenes content in the extracts with a sample diameter of d \(\leq\) 200 \(\upmu\)m was expected. On a similar approach, [PERSON] et al. studied the effect of particle size on the antioxidant extraction from _Agrimonia euporaria_, Salvia sp., and _Satureja montana_ plants [40]. The particle size of 200 \(\upmu\)m, which was the smallest, obtained by the coffee grinder, gave the highest rate of extraction [40]. We believe that the 50% increase in the extraction shown in Figures 4c and 5a is due to the release of terpenoids during the grinding process, which supports our hypothesis that the maximum terpenoid concentration occurs as soon as the powder is immersed in the solvent. This is also supported by the results of Figure 5b,c for the fine particles d = 100 \(\upmu\)m and 68 \(\upmu\)m, respectively. Interestingly, the extraction of terpenes had slightly decreased after 15 min Figure 5c, which could be the degradation of the terpenes. Data comparison of Manuka extracts with the particle size used in this study at Zero time (in a few seconds) is demonstrated in the GCMS chromatogram Figure S3 in the supplementary file.