3.5.1. Membrane Stabilization Inhibition Assay

The membrane-stabilizing activity of the samples was assessed using hypotonic solution-induced erythrocyte (RBCs) hemolysis [67]. For the preparation of erythrocyte suspension, whole blood was obtained with heparinized syringes from rats through cardiac puncture. The blood was washed three times with isotonic buffered solution (154 mM NaCl) in 10 mM sodium phosphate buffer (pH 7.4) and immediately centrifuged for 10 min at 3000× *g*. The test sample consisted of stock erythrocyte suspension (0.5 mL), 5 mL of hypotonic solution (50 mM NaCl), and the *C. amblyocarpa* EO (7.81–1000 μg mL−<sup>1</sup> in ethanol) or indomethacin (as a standard drug). The control sample consisted of 0.5 mL of stock erythrocyte suspension and hypotonic-buffered saline solution alone. The mixtures were incubated for 10 min at room temperature (25 ± 2 ◦C) and centrifuged for 10 min at 3000× *g*. In 96-well plates, the absorbance of the supernatant was measured at 540 nm. The percentage inhibition of hemolysis or membrane stabilization were calculated according to the modified method described by Shinde et al. [67] as follows:

$$\text{Inhibition }\%=100 \times \text{I(A}\_{\text{control}} - \text{A}\_{\text{treatment}}) \div A\_{\text{control}}\text{ I}\tag{3}$$

where Acontrol is the absorbance control, and Atreatment is the absorbance treatment.

The IC50 value was defined as the concentration of the EO required to inhibit 50% of the RBC hemolysis under the assay conditions. It was calculated graphically by linear regression of the inhibition values of different concentrations using MS-EXCEL 2016.

#### 3.5.2. Lipoxygenase (LOX) Inhibition Assay

The activity of the *C. amblyocarpa* EO on the inhibition of the LOX enzyme (type I-B) was determined according to Granica et al. [68], with slight modifications. Briefly, in 96-well

plates, 100 μL of soybean (*Glycine max*) LOX solution (1000 U/mL in borate buffer solution, pH 9) and 200 μL of borate buffer were mixed together with various concentrations of either EO (to a final concentration range of 0.98–125 μg mL−1) or ibuprofen as a reference drug. Samples were pre-incubated with 100 μL of linoleic acid (substrate) to initiate the reaction and then were incubated at 25 ◦C for 15 min. The absorbance increase was measured at 234 nm using a microplate reader (BioTek Instruments Inc., Winooski, VT, USA). The inhibition percentage and IC50 were calculated as previously mentioned in the membrane stabilization inhibition assay.

#### 3.5.3. Cyclooxygenase (COX 1 and COX 2) Inhibition Assay

The COX activity was monitored as a result of the oxidation reaction of N,N,N,Ntetramethyl-p-phenylenediamine (TMPD) with arachidonic acid according to the protocol of Petrovic and Murray [69], with slight modifications. The activity of the EO or ibuprofen as a reference drug at a concentration range of 0.98–125 μg mL−<sup>1</sup> was determined by monitoring the absorbance of TMPD oxidation reaction with arachidonic acid at 611 nm using a microplate reader (BioTek Instruments Inc., Winooski, VT, USA). The inhibition percentage and IC50 were calculated as previously mentioned in the membrane stabilization inhibition assay.
