Antiobesity Effect of a Novel Herbal Formulation LI85008F in High-Fat Diet-Induced Obese Mice

A variety of natural products have been explored for their antiobesity potential and widely used to develop dietary supplements for the prevention of weight gain from excess body fat. In an attempt to find a natural antiobesity agent, this study was designed to evaluate the antiobesity activity of a novel herbal formulation LI85008F composed of extracts from three medicinal plants in high-fat diet- (HFD-) induced obese mice. After the thirteen-week oral administration of the test materials to mice, the body weight gain, whole-body fat mass, adipose tissue weight, and the expression levels of obesity-related proteins were measured. Our results indicated that LI85008F can suppress body weight gain and lower whole-body fat mass in HFD-induced obese mice. Significant decreases in epididymal and retroperitoneal fat mass were observed in LI85008F-treated groups compared with the HFD-fed control group (p < 0.05). Furthermore, the oral administration of LI85008F caused significant decreases in the expression level of adipogenic (C/EBPα and PPARγ) and lipogenic (ACC) markers and notable increases in the production level of thermogenetic (AMPKα, PGC1α and UCP1) and lipolytic (HSL) proteins. These findings suggest that LI85008F holds great promise for a novel herbal formulation with antiobesity activities, preventing body fat accumulation and altering lipid metabolism.


Introduction
Obesity is a challenging health problem caused by the interaction of various genetic, dietary, lifestyle, and environmental factors [1]. e comorbidities associated with obesity include diabetes, hypertension, and cardiovascular diseases. Excess weight also has an enormous impact on the social, financial, and psychological status of obese individuals, which may contribute to the development of depression [2].
ere are a variety of choices for obesity treatment, including life style changes, exercise, dietary control, weightloss surgery, and prescription weight-loss medications [3]. Medications can facilitate weight loss in obese persons. e first class of medications developed for weight control causes symptoms that mimic the sympathetic nervous system, resulting in the major side effect of this class of medications as high blood pressure. ese medications also lower appetite and increase a sensation of fullness. Another class of antiobesity medications suppresses appetite by increasing the concentration of neurotransmitters at the synaptic cleft, where hunger and fullness are regulated by brain neurotransmitters such as dopamine, serotonin, and norepinephrine [4]. Unfortunately, these weight-loss medications are known to have significant adverse effects including headache, insomnia, irritability, nervousness, abdominal pain, and diarrhea [5,6]. In the face of adverse effects of synthetic drugs, the potential of natural products with antiobesity activity is under exploration [7]. Natural products including crude extracts and isolated pure natural compounds may serve as antiobesity agents via a variety of mechanisms including suppression of adipose tissue growth, inhibition of adipocyte differentiation, stimulation of lipolysis, and induction of apoptosis in existing adipocytes, thereby reducing adipose tissue mass [8].
LI85008F is composed of the extracts of three medicinal plants, Moringa oleifera, Murraya koenigii, and Curcuma longa.
ese medicinal plants were selected by screening hundreds of traditional herbal extracts for their ability to inhibit adipogenesis using a cell-based model [9]. Moringa oleifera, also known as drumstick tree, is indigenous to South Asia and India, and its leaves are highly nutritious and rich in amino acids, vitamins, minerals, and natural antioxidants [10]. e leaves of the plant have been traditionally used in the diet and for the treatment of various inflammatorymediated diseases including cardiovascular diseases and diabetes [11]. Murraya koenigii, known as curry leaf, is used in folk medicine in India and other Asian countries as an analgesic, astringent, antidysenteric, febrifuge, hypolipidemic and hypoglycemic agent [12]. Turmeric (Curcuma longa) has a long tradition of being used as a spice by many cultures across the globe. is commonly consumed spice possesses antioxidant, anti-inflammatory, anticancer, antigrowth, antiarthritic, antiaging, antiatherosclerotic, antidepressant, antidiabetic, antimicrobial, wound healing, and memory-enhancing activities [13]. In traditional Indian medicine, turmeric has also been used to treat different ailments such as gynecological problems, gastric problems, hepatic disorders, infectious diseases, blood disorders, acne, psoriasis, dermatitis, rash, and other chronic ailments [14].
Although these spices have not traditionally been used for weight loss, the combination of M. oleifera, M. koenigii, and C. longa has been clinically shown to support body weight and fat loss in both obese and overweight individuals in a randomized, double-blind, placebo-controlled clinical trial [15]. In addition, Sengupta et al. [9] describe putative mechanisms for activity of this combination in modulating adipogenesis and lipolysis. e present study aimed to further investigate the antiobesity potential of LI85008F in a high-fat diet-(HFD-) induced obese mouse model.

Test
Material. LI85008F, manufactured by Laila Nutraceuticals (Vijayawada, India) and provided by Ju Yeong NS Co., LTD. (Seoul, South Korea), is a novel herbal formulation composed of an aqueous ethanol extract of Moringa oleifera leaves, an aqueous alcohol extract of Murrya koenigii leaves, and Curcuma longa rhizome extract standardized to 95% total curcuminoids, mixed at a ratio of 6 : 3 : 1, respectively. Fresh plant raw materials were collected from local areas in Andhra Pradesh, India. eir voucher specimens are preserved in the Taxonomy Division at Laila Impex R & D Centre (Vijayawada, India). LI85008F is commercially available as Slendacor® or Slimvance®.

Animals and Experimental Diets.
Four-week-old male C57BL/6 mice (Raonbio, Yongin, South Korea) were maintained under a 12-hour light-dark cycle in a temperature-and humidity-controlled room during the experimental period. e mice were given laboratory pellet chow and water ad libitum. Following one week of acclimatization, 20 mice were randomly divided based on their body weight into four groups (n � 5) as follows: the normal group received a chow diet (ENVIGO, Huntingdon, UK); the control group was fed with a 60 kcal% fat high-fat diet (HFD; Research Diet Inc, Burnswick, USA); and the two experimental groups were provided with a HFD supplemented with 250 and 500 mg/kg of LI85008F, respectively. Mice allocated to the experimental groups were orally administered with LI85008F for 13 weeks. e body weight and feed consumption of each mouse were measured once a week and daily throughout the study duration, respectively. All animal care and use protocols following NIH guidelines were approved by the Institutional Animal Care and Use Committee (IACUC) of Daejeon University (approval No. DJUARB2020-003).

Determination of Body Fat Mass.
e body fat mass of mice was measured using dual-energy X-ray absorptiometry (DXA) with an apparatus for small animals (Inalyzer, Medicors Inc., Seongnam, Korea). All scans were performed with the animals positioned prone and spread, with tape attached to each limb on the platform. e fat mass percentage was calculated as fat mass divided by total body weight and multiplied by 100.

Sampling Procedures.
At the end of the 13-wk experimental phase, mice fasted overnight were anesthetized and sacrificed.
e adipose tissues (epididymal and retroperitoneal fat pad) were harvested, weighed immediately, and stored at −80°C until further analysis.

Statistical Analysis.
e results were expressed as mean ± standard deviation (SD). Statistical analyses were conducted using SPSS 21.0 (SPSS, Chicago, USA). e differences among groups were analyzed using one-way 2 Evidence-Based Complementary and Alternative Medicine analysis of variance (ANOVA) with Duncan's test. Significance was set at p < 0.05 for all statistical analyses.

Effect of LI85008F on Body Weight
Gain. e body weight and food intake of each mouse were measured on a weekly and daily basis throughout the study duration, respectively. Distinct separation can be seen from week 3 onwards, whereby the control group which received high-fat diet (HFD) revealed a remarkable increase in body weight when compared with the normal group fed with a regular diet, implying the induction of obesity in animals in the control group. From week 6 onwards, daily administration of test article LI85008F at the dose of each of 250 and 500 mg/kg significantly prevented the body weight gain in both LI85008F-treated groups (control: 17.08 ± 2.53 vs. LI85008F 250 : 10.78 ± 4.39, p < 0.05; LI85008F 500 : 9.37 ± 2.95, p < 0.05) (Figure 1).

Effect of LI85008F on Whole-Body Fat Mass.
To determine whether the observed decrease in body weight by treatment of LI85008F was due to a reduced accumulation of fat, we estimated whole-body fat mass by conducting dualenergy X-ray absorptiometry(DXA) with an apparatus for small animals (Figure 2). e mice in the control group had a significant higher whole-body fat mass at the end of the 13-wk intervention period than in mice fed with a regular diet. In contrast, the daily oral administration of the test material LI85008F at the dose of each of 250 and 500 mg/kg resulted in a significantly reduced whole-body fat mass compared to that of the control group (control: 16.20 ± 3.18 vs. LI85008F 250 : 11.51 ± 2.16, p < 0.05; LI85008F 500 : 11.19 ± 2.63, p < 0.05) (Figure 3(a)). In addition, the fat mass percentage was calculated as fat mass divided by total body weight. In parallel with whole-body fat mass, significant decreases in fat mass percentage were found in mice treated with LI85008F at the dose of 500 mg/kg compared with the control group (control: 36.90 ± 2.95 vs. LI85008F 500 : 31.07 ± 6.20, p < 0.05) (Figure 3(b)).

Effect of LI85008F on Fat Mass of Adipose Tissue.
After daily administration of test article LI85008F for 13 weeks, mice fed with HFD exhibited a significant increase in both the epididymal and retroperitoneal fat pad weights in comparison with the normal group. A significant decrease in epididymal fat pad weight was observed in all animals that received LI85008F at the dose of 250 mg/kg or 500 mg/kg for 13 weeks in a dose-dependent manner in comparison with the control group (control: 2.03 ± 0.16 vs. LI85008F 250 : 1.70 ± 0.37, p < 0.05; LI85008F 500 : 1.20 ± 0.21, p < 0.05) (Figure 4(a)). In retroperitoneal fat pad weight, there was a significant reduction in the group receiving 500 mg/kg of LI85008F compared to the control group (control: 0.68 ± 0.12 vs. LI85008F 500 : 0.45 ± 0.08, p < 0.05) (Figure 4(b)).

Effect of LI85008F on the Expression of Adipogenic and Lipogenic Markers in Adipose Tissue.
To further elucidate the mechanism underlying the blockade of fat accumulation by LI85008F supplementation, the expression of the key markers of adipogenesis C/EBPα and PPARc in retroperitoneal fat pads was examined by western blot analysis. e adipogenesis process is tightly controlled by C/EBPα, which promotes the adipogenic pathway through activation of PPARc [16]. As shown in Figure 5(a), the expression of these two adipogenic markers was suppressed in LI85008F-treated mice compared to HFD-fed control mice. Moreover, the production level of a key lipogenic enzyme, ACC, which is regulated by C/EBPα or PPARc [17], was measured. e intake of LI85008F for 13 weeks significantly decreased the expression level of ACC in a dose-dependent manner ( Figure 5(b)).

Effect of LI85008F on the Expression of ermogenetic and Lipolytic Markers in Adipose
Tissue. It has been reported that phosphorylation of AMPKα triggers the induction of gene expression of thermogenesis markers PGC1α and UCP1 [18]. erefore, we evaluated the effect of LI85008F supplementation on the expression level of AMPKα, PGC1α, and UCP1 in retroperitoneal fat pads of HFD-fed mice. e oral administration of LI85008F for 13 weeks significantly up-regulated the production level of AMPKα, PGC1α, and UCP1 in a dose-dependent manner (Figure 6(a)). In addition, the alterations in the production level of lipase HSL involved in lipolysis by LI85008F intake were also investigated. LI85008F-treated mice showed higher protein expression of HSL than HFD-fed control mice ( Figure 6(b), implying that LI85008F can be acting on the lipolytic pathway in HFD-induced obese mice.
Obesity can be defined as an increase in body weight beyond the limits of physical requirements, which results from an excessive accumulation of fat. Interestingly, in a previous clinical study [15], LI85008F supplementation for 8 weeks resulted in a significant body weight reduction in overweight or obese adults compared to those in the placebo group (p < 0.001), which is parallel with the suppressive effect of LI85008F on HFD-induced body weight gain in mice observed in this study (Figure 1). Furthermore, we have shown that LI85008F treatment significantly reduces wholebody fat mass and fat mass percentage using DXA (Figures 3(a) and 3(b)), most likely through the inhibition of fat accumulation in adipose tissue including epididymal and retroperitoneal fat pads (Figures 4(a) and 4(b)). e current study demonstrated that LI85008F suppressed adipogenesis and decreased lipid accumulation by inhibiting the expression of genes involved in adipogenesis and lipogenesis in adipose tissue (Figures 5(a) and 5(b)), which is consistent with the previous report [9] that LI85008F deactivates the key adipogenic transcription factors C/EBPα and PPARc in 3T3-L1 adipocytes.
As a master energy sensor, AMPK plays a key role in integrating hormones, nutrients, and stress signals to maintain whole-body energy homeostasis [19]. AMPK is activated by allosteric stimulation in response to an Evidence-Based Complementary and Alternative Medicine  Figure 1: e effect of LI85008F on weekly body weight (a) and body weight gain (b) in mice for 13 weeks of treatment. Values are expressed as means ± SD (n � 5). e data were analyzed using ANOVA followed by Duncan's test. # and * indicate the significant difference (p < 0.05) between normal vs. control group and control vs. LI85008F supplemented group, respectively.

Normal
HFD HFD + LI85008F 250 HFD + LI85008F 500  Evidence-Based Complementary and Alternative Medicine increased AMP/ATP ratio or mitochondria activity changes [20]. In skeletal and cardiac muscle, AMPK activation leads to the induction of gene expression of thermogenesis markers PGC1α and UCP1 [18]. In the present study, we demonstrated that LI85008F supplementation elevated AMPKα production level and induced the subsequent activation of PGC1α and UCP1 expression, leading to higher energy expenditure and lower body weight ( Figure 6(a)). HSL, the major enzyme catabolizing triglycerides (TGs), induces lipolysis after phosphorylation of serine residues, leading to translocation of itself to the lipid droplets and markedly enhancing lipolysis [21]. As shown in Figure 6(b), LI85008F treatment to HFD-induced obese mice led to upregulate the expression of HSL involved in lipolysis pathways. Based on these findings, we propose that LI85008F exerts antiobesity activities through a variety of mechanisms including suppression of adipogenesis, inhibition of lipogenesis, stimulation of lipolysis, and upregulation of thermogenesis and energy expenditure in adipose tissue, thereby reducing adipose tissue mass, whole-body fat mass, and body weight gain in HFD-induced obese mice.

Conclusions
e present study demonstrated that L185008F administration for 13 weeks significantly suppressed HFD-induced body weight gain and reduced whole-body fat mass, as well as fat weight of adipose tissue including epididymal and retroperitoneal fat in HFD-induced obese mice. ese findings may be associated with significant decreases in the expression level of adipogenesis and lipogenesis proteins including C/EBPα, PPARc, and ACC, as well as marked upregulation in the production level of thermogenesis and lipolysis markers such as AMPKα, PGC1α, UCP1, and HSL induced by L185008F treatment in HFD-induced obese mice.

Data Availability
All data generated or analyzed during this study are available and included in this article.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.