1. Introduction
Obesity is considered a chronic metabolic disorder that enables the advent of multiple other metabolic disorders such as cardiovascular diseases, type 2 diabetes, fatty liver disease, and certain cancers [
1]. Obesity is further associated with dyslipidemia characterized by elevation of serum total cholesterol, triglyceride (TG), and low-density lipoprotein (LDL)-cholesterol and demotion of high-density lipoprotein (HDL)-cholesterol [
2]. Obesity develops as a result of hypertrophy and/or hyperplasia of adipocytes which occurred due to the differentiation of pre-adipocytes through adipogenesis [
3]. Adipogenesis is a complex process involving the synergistic action of numerous adipogenic factors. In particular, the essential transcription factors such as peroxisome proliferator-activated receptor-gamma (PPARγ), CAAT/enhancer-binding protein alpha (C/EBPα), adipocyte protein 2 (aP2), and sterol response element-binding protein-1c (SREBP-1c) regulates the whole process of adipogenesis [
4,
5]. At the maturity stage, C/EBPα and PPARγ combinedly regulate adipogenesis [
6]. Therefore, inhibition of these essential adipogenic factors is a potential target to inhibit adipogenesis, and so on obesity [
7]. Deletion of PPARγ in adipose tissue ameliorated high fat-diet (HFD) induced obesity and insulin resistance [
8]. C/EBPα deficient adipocytes accumulated less lipid in vitro [
6].
The complex pathophysiology of obesity requires a multi-dimensional approach to deal with it. While studying anti-obesity activity, a mouse obesity model is a well-characterized and reliable strategy. More than 200 genetic mouse models are practicing obesity-related research. The ob/ob model, lacking leptin production, is an important monogenic model for obesity and diabetes [
9]. As most human obesity is developed as a result of high energy consumption, the diet-induced obesity (DIO) model is considered as the most relevant to correlate with human obesity [
10]. The pathogenesis of the DIO model mimics that of human obesity in order to increase body weight, dysregulation of serum lipids, dysregulation of glucose metabolism, and insulin resistance. Therefore, DIO is understood as a human-like obesity model [
9,
10]. The C57BL/6J strain mice are popular for the DIO model as they develop obesity, hyperinsulinemia, hyperglycemia, hepatic steatosis, and insulin resistance on HFD feeding for more than 4 weeks [
11].
Obesity can be treated or prevented by assimilating certain interventions such as lifestyle change (diet, exercise, and behavior therapy) and medical or surgical approaches (pharmacotherapy or bariatric surgery). Surgical interventions are utilized to individuals who failed to lose weight based on lifestyle changes [
12]. Unfortunately, due to the appearance of numerous mild to life-threatening adverse effects, there are controversies in using pharmaceutical therapies. Though several pharmaceutical anti-obesity drugs have received approval from regulating bodies, many drugs have been withdrawn from the market due to the appearance of psychiatric and cardiac-related adverse effects [
13]. Therefore, it is necessary to find a safe, effective, and economic therapy for obesity. Compared to synthetic medicines, therapies based on natural resources are considered safe and more acceptable for obesity [
14]. A variety of herbal medicines or natural products including crude extracts, isolated compounds, or herbal formulations have been widely used for weight loss therapy [
15]. Due to the presence of a variety of phytoconstituents in polyherbal formulations, they showed multidimensional action in a vast number of ailments. Most of the herbal formulations exhibit a wide therapeutic range showing activity even in low concentrations and are safe at high doses [
16]. In addition, herbal formulations have low-cost, are eco-friendly, and are easily available. Therefore, the demands for polyherbal formulations are increasing globally [
16,
17].
According to the World Health Organization (WHO), more than 80% of the global population still uses traditional and complementary medicines (TCM) for healing. Most TCMs consists of herbal medicine [
18]. However, various factors such as geographical location, climate condition, environmental hazards, harvesting methods, and collecting protocols affects the phytochemical variation in plants, and so the quality of herbal medicines [
16]. The chemical profile reflects the quality of any herbal medicine, which determines the safety and efficacy of the medicine [
19]. Chromatography is the most used analytical technique for exploring the chemical profiles of herbal medicines. Thin-layer chromatography (TLC) is a cheap and rapid technique but high-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography (UPLC) are even more reliable tools to analyze a complex mixture of plant constituents accurately [
20,
21].
Recently, we have reported the anti-adipogenic activity of herbal formulation F2 in 3T3-L1 adipocytes in vitro and preliminary anti-obesity activity in HFD-fed C57BL/6J mice in vivo [
22]. F2 was prepared by mixing specific proportions of a trace amount of royal jelly and lemon juice with ethanol extracts of
Orostachys japonica (OJ),
Rhus verniciflua (RV), and
Geranium thunbergii (GT). Our observations suggested that the ingredients of F2 worked synergistically as in the formulation and inhibited adipocyte differentiation, restricted dietary fat absorption, and reduced fat accumulation in tissues. In addition, F2 showed potential anti-oxidant and blood-glucose-lowering action [
22]. The interesting anti-obesity observations encouraged us to evaluate the therapeutic efficacy of F2 for obesity in the DIO mice model. The objectives of this study are to confirm the anti-obesity activity of F2 in the DIO mice model and validate the previously developed analytical method for the standardization of F2. Furthermore, the effect of F2 on HFD-induced insulin resistance was also determined.
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, 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 C57BL/6J 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 C57BL/6J 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 (astragalin, ellagic acid, fisetin, 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 C57BL/6J mice indicating the development of obesity. The previous studies have also demonstrated that the C57BL/6J 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]. Therefore, lower FER in the F2-treated group might be due to an interfering with the pancreatic lipase causing reduced dietary fat absorption. The extent of weight gain in 7-weeks of drug treatment was significantly lower than that in 5-weeks of the obesity induction period (
Figure 5). These findings reinforced the FER and weight gain reducing effect of F2 observed in the previous study [
22].
Obesity is associated with hyperlipidemia characterized by the elevation of serum total cholesterol and triglyceride (TG) levels [
37]. Hyperlipidemia contributes a major pathophysiological role in cardiovascular diseases, stroke, and diabetes [
38]. Formulation F2 was found to be effective in reducing serum TG and total cholesterol compared to the HFD control group (
Figure 4), indicating that F2 may have a potential in the management of dyslipidemia and associated comorbidities. Previous studies have revealed that an 80% ethanol extract and ethyl acetate fraction of OJ and 70% ethanol extract of GT showed the greatest activity in lowering serum lipids and abdominal fat in rodents [
39,
40,
41]. Long-term consumption of a high-fat diet promotes a high level of circulating lipid species and free fatty acids in rodents [
42]. Excess accumulation of such lipids results in adipocyte hypertrophy and, therefore, obesity. The accumulation of lipids into adipocytes during adipogenesis requires a sequential role of various adipogenic factors such as PPARγ, C/EBPα, SREBP-1c, aP2, Leptin, LPL, STATs, FAS, etc. [
5,
43,
44]. The evaluation of WAT for the expression of such adipogenic transcription factors may offer a general idea that the drug samples affect the regulation of adipogenesis. The current study revealed that the gene expression level of PPARγ, SREBP-1c, aP2 were significantly down-regulated in the WAT of F2-treated mice. Whereas C/EBPα, LPL, and leptin expression were perceived as slightly reduced but were not significantly different with the HFD control group (
Figure 5). The results indicate that the formulation F2 inhibited adipogenesis in WAT. In addition, an inflammatory cytokine, IL-6, was found to be under-expressed in WAT, indicating the formulation F2 has a positive response in ameliorating obesity-associated inflammatory responses. The secretion of IL-6 and TNFα by adipose tissue in obesity leads to chronic inflammation, and so promotes insulin resistance [
28,
44]. In the previous report, we showed that the gene expression level of the aforementioned adipogenic transcription factors is down-regulated in F2 treated 3T3-L1 adipocytes [
22]. From all the observations, it may be suggested that the anti-obesity action of F2 might be due to the restriction of adipogenesis in adipose tissue.
It is well known that the β cells of pancreatic islets secret insulin to maintain the basal level of blood glucose. In normal conditions, the β cell function and population dynamics are influenced by the blood glucose concentration [
45]. Insulin travels through the body and induces muscles and fat cells to absorb glucose. In addition, insulin selectively induces glycogenesis when postprandial blood glucose levels become elevated. Once the blood glucose level returns to its basal level, insulin secretion is also adapted [
46]. When the body systems are exposed to high fat for a long time, it may cause an impairment on insulin binding to its receptors and/or glucose transporter in muscle and fat cells, resulting in decreased insulin sensitivity [
47]. As a result, pancreatic β cells secret more insulin to cope with peripheral insulin resistance in muscle and adipose tissue to maintain the normal level of blood glucose. This condition has been termed as compensatory hyperinsulinemia [
45]. The previous literature has also revealed that dietary high-fat leads to promote insulin resistance and hyperinsulinemia before the development of type 2 diabetes in rodents [
48,
49,
50]. High-fat diet-induced hyperinsulinemia or insulin resistance may occur due to a decrease in the glucose transporter, insulin receptors, and glucose metabolism [
10,
28]. The systemic insulin resistance was quantified in the term of the HOMA-IR index [
25]. In the current study, the serum insulin level was found to be drastically increased in the HFD control group. However, in F2 and GC-treated groups, the insulin level was significantly diminished (
Table 5). However, the blood glucose level was not altered among the groups. These observations indicated that the mice were developing compensatory hyperinsulinemia in the HFD control group as a result of partial insulin resistance. F2 and GC were found to ameliorate HFD-induced insulin resistance. In contrast with the observations of the current experiment, the blood glucose level was significantly reduced by F2 compared to the HFD control in the previous study [
22]. These findings indicated that the formulation F2 could have preventive effect in controlling the blood glucose level. In other words, seven weeks treatment of F2 in obese mice was effective enough for protection against insulin resistance. Therefore, it may be suggested that the F2 might be effective in reducing the blood glucose level in obese mice if the treatment period would be longer.
The effect of F2 on the weight of visceral organs of obese mice and their histological evaluations were analyzed in the current study following a similar procedure as in the previous study [
22]. Similar results were found in both studies that the weights of the WAT in the HFD control group were significantly increased due to the over deposition of lipids into them. Whereas F2 WAT mass was found to be significantly reduced. The fat deposition into WAT was observed by histological analysis and the results showed that the size of adipocytes of the F2-treated mice was reduced compared to the HFD control mice (
Figure 6 and
Figure 7). In addition, supporting the results of the previous study [
22], F2 was found to be effective in reducing the fat deposition into the liver in this study (
Figure 6). The massive fat globules into the liver of the HFD control mice in the current study indicated that the long-term consumption of HFD induced severe liver steatosis in obese mice [
51]. Though, F2 was found to be rationally effective in the management of liver steatosis. There are some limitations to this study. A complete molecular mechanism of the anti-obesity activity of F2 remains to be investigated. A further study on genetically challenged obesity models (in vitro and/or in vivo) can be used for this approach. The effect of F2 on the protein expression level of adipogenic markers is yet to be investigated.