## 4 Discussion  
Management of overweight and obesity with pharmaceutical medicine comes with many unavoidable adverse effects, high costs, and the physical dependency associated with it. Because of these cons, the use of herbal medicine is increasing globally for the treatment of obesity and related metabolic disorders [27]. In order to prepare an effective anti-obesity herbal formulation, we have developed a novel formulation (F2) by homogenizing specific proportions of a trace amount of lemon juice and royal jelly with ethanol extracts of OJ, RV,  
Figure 6: The effect of formulation F2 on the liver and WAT of obese mice. H&E staining was performed for the histological analysis (magnification \(\times\)20). The size of adipocytes in the WAT and deposition of lipid (yellow droplet) in the liver were evaluated. Normal = standard diet; HFD control = high-fat diet control; F2-46 = F2 (46 mg/kg). Figure 7: The effect of formulation F2 on the size of adipocytes in the epididymal WAT. The diameter of the adipocytes was evaluated through ImageJ software. Values are expressed as the mean \(\pm\) SD. A one-way ANOVA was applied to calculate statistical significance. ###\(p\) < 0.001 vs. Normal; ***\(p\) < 0.001 vs. HFD group. Normal = standard diet; HFD = high-fat diet control; F2-46 = F2 (46 mg/kg). and GT [22]. A complete investigation of anti-obesity activity and the standardization of F2 was performed in two consecutive studies. In the first study, the F2 was treated to 3T3-L1 adipocytes to measure its anti-adipogenic efficacy. It was found to exhibit pronounced synergistic anti-adipogenic activity by F2. Furthermore, F2 was co-administered to the HFD fed C57 BL/6] mice to investigate the obesity-preventing activity. HFD was used to induce obesity. In the previous animal study, F2 treatment began from the same day of switching mice to an HFD feed. Interestingly, F2 showed a significant effect in controlling body weight, blood glucose, and protection from HFD-induced liver damage [22]. In the present study, we have further examined the anti-obesity activity of this herbal formulation (F2) in DIO mice model, where male C57 BL/6] mice were first fed with only HFD for five weeks to develop the DIO model, and only after the successful development of obesity, co-administration of F2 were done. This experimental mouse model may closely resemble obesity in humans and may hypothesize if the formulation F2 is effective for obese humans. F2 was further analyzed using the UPLC-DAD method for its major marker compounds (astragalan, ellagic acid, fiestin, fustin, and sulfuretin). A UPLC-DAD method was optimized and validated for the quality control of the formulation. It is well-accepted in many studies that the long-term feeding of an HFD induces obesity in rodents [28; 29; 30; 31]. HFD-induced obesity may be characterized by an increase in body weight gain, body fat content, and FER [28; 32]. Five weeks of the HFD feeding significantly increased body weight and FER in C57 BL/6] mice indicating the development of obesity. The previous studies have also demonstrated that the C57 BL/6] mouse strains were more susceptible to HFD-induced obesity [11; 29; 30; 33]. The obese mice were then treated with vehicle, F2, and GC for 7 weeks, along with continuing the HFD. The fruit extract of _G. cambogia_ is a well-accepted anti-obesity agent and is used as a positive control in anti-obesity studies [34; 35]. Obesity can be categorized as an incurable chronic disease. The restriction in body weight gain without adverse effects is considered an effective therapy in the management of obesity [13; 15]. The body weight gains were found to be significantly reduced in F2 and GC-treated groups during the 7-weeks of drug treatment, with a significant decrement in FER (Table 4). Dietary fat is absorbed only after emulsifying with pancreatic lipase [36]. We have already shown in the previous report that F2 enhanced lipid excretion in feces [22].