## 2 Materials and Methods
### Determination of the Antioxidant Activity
#### 2.4.2 \(\beta\)-Carotene Bleaching Assay  
The antioxidant activity of ZLF extract was evaluated following the beta-carotene bleaching protocol described by [PERSON] and [PERSON] [23]. Indeed, a solution contains 2 mg of  
Figure 1: (**A**) _Z. lotus_ and its habitat; (**B**) Fruits of _Z. lotus_.  
\(\beta\)-carotene, and 10 mL of chloroform were mixed with 20 mg of linoleic acid and 200 mg of Tween-80. After removing the chloroform mixture by rotavapor at 40 \({}^{\circ}\)C, 100 mL of distilled water was added to the flask under vigorous agitation. Then, 0.2 mL of this mixture was added to the individual test tubes containing the sample solution, and then the tubes were incubated at 50 \({}^{\circ}\)C in a water bath under continuous agitation for 2 h. Absorbance was measured at 0 and 2 h of incubation at a wavelength of 470 nm. BHT was used as a standard and distilled water was used as negative control (NC). The measurements were repeated three times to minimize the error. The percentage of oxidized \(\beta\)-carbon was calculated using the following formula:  
\[(\%)\ \text{oxidized}\ \beta-\ \text{carotene}=\frac{Ai(to)-Af(2h)}{\text{Ai}( \text{to})}\ \times\ 100\]  
_Ai_: absorbance in t\({}_{0}\), and _Af_: absorbance after incubation (2 h).  
_2.5. Anti-Hyperlipidemic Activity_  
2.5.1. Animals and Housing  
Twenty-four male Swiss albino mice (19 weeks old) weighing between 20 and 30 g were used in this study. All animals were kept in plastic cages with free access to food and water. The animals were adapted to standard pet shop conditions (23 \({}^{\circ}\)C, 12 h dark/12 h bright) 15 days before treatment. The mice were cared for in compliance according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of the Faculty of Sciences, Oujda, Morocco (01/20-LBBEH-04 and 09/01/2020).  
2.5.2. Preparation of High-Fat Diet  
The high-fat diet was prepared according to the protocol described by [PERSON] et al. (2009) [24]; this diet consisted of the regular chow diet (Society SONABETAIL, Oujda, Morocco), cholesterol 2%, fat 16%, and deoxycholic acid 0.2%.  
2.5.3. Experimental Protocol Design  
After two weeks of acclimatization, the mice were divided into four equal groups (\(n=6\)) and treated as follows:  
* A standard control group (NCG) received only distilled water at 10 mL/kg body weight.
* The Hyperlipidemic Control Group (HCG) freely received the high-fat diet and received distilled water daily (10 mL/kg).
* Two treated groups received 200 or 400 mg/kg of ZLF aqueous extract and then a high-fat diet for 30 days of the treatment.  
At the end of the treatment, all mice were anesthetized by a light ethyl ether inhalation and sacrificed. The blood samples were then taken from their retroorbital sinus into heparinized tubes and centrifuged at 3000 rpm for 10 min, at 4 \({}^{\circ}\)C, to separate the plasma. The separated plasma was stored at \(-20\)\({}^{\circ}\)C for further evaluation.  
2.5.4. Biochemical Analysis  
The biochemical parameters have been measured in plasma. The commercial kits measured high-density lipoprotein cholesterol (HDL-c), triglycerides (TG), total cholesterol (TC), and blood glucose levels. All of the tests were performed with the COBAS INTEGRA(r) 400-Plus analyzer (Roche Diagnostics, Meylan, France).  
2.5.5. The Atherogenic Index Calculation  
The atherogenic index was calculated by the following formula:  
AI = (Total cholesterol) \(-\) (HDL-C)/HDL-C, and in the LDL-C/HDL C, the ratio was calculated as the ratio of plasma LDL-C to HDL-C levels.