*4.5. Co-Culture Assays*

Amoeba and bacterial working solutions were mixed in 25 cm3 flasks by adding the required volume according to Table 1. To provide an example, 10 mL of *W. magna* C2c Maky at 3 × 105 cells/mL was mixed with 0.1 mL of *L. pneumophila* Lens at 3 × 107 CFU / mL. All flasks were left to stand for 2 h at 22 ◦C ± 2 ◦C or at 37 ◦C ± 2 ◦C to allow for amoebae/bacteria contact and the internalization of *L. pneumophila* into amoebae. After the 2-h contact process, each flask was gently shaken 10 times, and the suspension was transferred into a 15 mL Flacon® tube and centrifuged at 3000× *g* for 5 min. This step allowed for the removal of non-internalized (i.e., extracellular) *L. pneumophila* from the co-culture suspensions. The pellet was resuspended in 10 mL of sterile SCYEM, and the suspension was poured into a new 25 cm3 flask and incubated at 22 ◦C ± 2 ◦C or at 37 ◦C ± 2 ◦C. This time point corresponded to the T0 time point of the assay. Each condition was performed for three independent replicates and repeated three times (n = 9), with the exception of the co-culture with strain Philadelphia that was repeated four times at 22 ◦C (n = 15).

#### *4.6. L. pneumophila and Amoeba Quantifications in Co-Culture Assays from T0 to T0* + *96 h*

Occurring at T0, T0 + 24 h, T0 + 48 h, T0 + 72 h, and T0 + 96 h, a washing step was performed. The culture supernatant was removed from each flask and replaced by 10 mL of sterile SCYEM. This step was intended to remove extracellular *L. pneumophila* to allow for the detection of only intracellular bacteria. Each flask was gently shaken 10 times and an aliquot of 1 mL was sampled. Quantification of amoeba populations was performed using 0.1 mL of each aliquot utilizing a haemocytometer cell counting chamber method with Trypan blue. The remaining 0.9 mL were treated with Triton™ X-100 [31] at 0.02% v/v (final concentration) for 2 min to lyse amoebas and to recover the internal *L. pneumophila*. The sample was then serially 10-fold diluted in SCYEM and plated on BCYE plates in triplicate, with the exception of the undiluted conditions that were spread onto five plates when the number of *L. pneumophila* was intended to decrease below the detection limit. BCYE plates were incubated at 36 ◦C, and CFU were counted after 5 days.

#### *4.7. Microscopic Observations in Co-Culture with L. pneumophila Philadelphia at 37* ◦*C*

Co-cultures of *L. pneumophila* Philadelphia using the three amoeba strains at 37 ◦C were sampled from running experiments and stained by the Gimenez technique [50,51] at T0, T0 + 48 h, and T0 + 96 h. Co-cultures (0.1 mL) were deposited onto glass slides by using a Shandon Cytospin 4 cytocentrifuge (Thermo Scientific, Illkirch-France) at 800× *g* for 10 min and then stained using the Gimenez technique. Briefly, each of the glass slides were stained with fuchsin solution for 3 min and washed with water. Then, the glass slides were stained with malachite green for 5–10 s and washed, and this step was repeated twice. Finally, the glass slides were allowed to dry at room temperature.

The observations were performed using a LEICA DM 2500 LED microscope (Leica Microsystemes SAS, Nanterre-France) under an ×100 oil immersion objective.


**2. Materials and Methods**

### *2.1. Sampling*

This is a cross-sectional study carried out from March 2020 until the end of August 2020. The study period coincides with the progression of COVID-19 cases in Saudi Arabia. The number of new cases has been increasing many-fold, ranging from 100 odd cases in March 2020 up to 4919 cases on 17 July 2020. The number of deaths was in single digits during March that went to a peak of 58 deaths on 5 July 2020. However, due to stringent regulations of Saudi Arabian authorities, the spread of the pandemic was well controlled, with the number of new recoveries per day was almost similar to or higher than new reported COVID-19 cases. Additionally, the media of Saudi Arabia was helping the governmental authorities in restricting the spread of rumors. Overall, the situation was alarming but well under control. Ethical approval to conduct this study was obtained from the Research Committee of College of Pharmacy, AlMaarefa University, Riyadh, with approval number (MCST (AU)-COP 2001/RC). Only female adults (aged ≥ 18 years) of Saudi Arabian nationality who provided verbal informed consent and reside in Riyadh (Saudi Arabia) were recruited in the study using convenience and snowball sampling methods. The content validity and reliability check of the questionnaire were done by Cronbach's alpha. The validated questionnaire was translated into the Arabic language, and linguistic validation was done to validate the conceptual translation of the questionnaire with the help of two qualified bilingual (English and Arabic languages) health science researchers. Pretest of the

Arabic version of the questionnaire was conducted to assess the clarity of the questionnaire, suitability to the participants, the time required to complete the questionnaire and to know the possible obstacles.

The sample size was calculated (http://sampsize.sourceforge.net) based on the 28% prevalence of psychological illness in the female population of Saudi Arabia reported in one of the studies [11] with a 5% as a precision percentage and a 95% confidence level. The required sample size was 310 for the infinite population.


*<IP_ADDRESS>.3. Ferruginol*

Ferruginol (abieta-8, 11, 13-triene-12-ol) is the simplest phenolic abietane diterpenoid (3). This abietane occurs in plants belonging to the Podocarpaceae and Lamiaceae families [22].

Biological activity: This diterpene has attracted much attention since it has exhibited important bioactivities, such as antimicrobial [46], miticidal [47], cardioactive [48], and antioxidative [49]. Moreover, it accelerates the gastric ulcer healing process, and such effects have been related with the ability of ferruginol to increase the gastric prostaglandin content in vitro [50–52].