Metabolaid® Combination of Lemon Verbena and Hibiscus Flower Extract Prevents High-Fat Diet-Induced Obesity through AMP-Activated Protein Kinase Activation

Abstract

Lemon verbena (Lippia citriodora) has been used as a food spice, cosmetic, and in traditional medicine formulations to treat asthma and diabetes in South America and Southern Europe. Hibiscus flower (Hibiscus sabdariffa L.) is used in traditional Chinese medicine in the form of a tea to treat hypertension and inflammation. In the present study, we examined the synergistic effects of a formula of Metabolaid^® (MetA), a combination of lemon verbena and hibiscus-flower extracts, on obesity and its complications in high-fat-diet (HFD)-induced obese mice. The results showed that MetA decreased body weight, white adipose tissue (WAT), and liver weight. Additionally, serum and hepatic lipid profiles, glucose levels, glucose tolerance, and cold-induced thermogenesis were significantly improved. Appetite-regulating hormones adiponectin and leptin were significantly increased and decreased, respectively, while the inflammatory-related factors tumor necrosis factor (TNF)-α and interleukin (IL)-6 were downregulated by MetA. Adipogenesis-activating gene expression was decreased, while increased thermogenesis-inducing genes were upregulated in the WAT, correlating with increased phosphorylation of AMPK and fatty-acid oxidation in the liver. Taken together, these results suggest that MetA decreased obesity and its complications in HFD mice. Therefore, this formula may be a candidate for the prevention and treatment of obesity and its complications.

Keywords: AMP-activated kinase; adipogenesis; hibiscus; hyperlipidemia; lemon verbena; obesity; thermogenesis.

Figure 1

Representative high-performance liquid chromatography (HPLC) chromatograms of the Metabolaid^® (MetA). HPLC chromatograms of two extract mixtures at 320 and 520 nm. Delphinidin-3-o-sambubioside, cyanidin-3-o-sambubioside, verbascoside, and isoverbascoside appeared at retention times of 7.1, 8.8, 13.4, and 14.5 min, respectively.

Figure 2

Effect of lemon verbena (LV), Hibiscus flower (HS), and their combination (MetA) on body weight and food intake in high-fat-diet (HFD)-induced obese mice. Changes in body size (A), body weight (B), body weight gain (C), food intake (D), and food efficiency ratio (FER, %, E) of normal diet group (NFD) and high fat diet-induced obese(DIO) mice after 9 weeks of 60% HFD feeding. HFD, 60% high-fat-diet control group; HFD + GC_245, HFD contains Garcinia cambogia extract (245 mg/kg)-fed group; HFD + LV_100, HFD contains lemon verbena extract (100 mg/kg)-fed group; HFD + HS_100, HFD contains Hibiscus flower extract (100 mg/kg)-fed group; HFD + MetA_50, HFD contains the MetA-mixed extract of lemon verbena plus Hibiscus flower (50 mg/kg)-fed group; HFD + MetA_100, HFD contains the MetA-mixed extract of lemon verbena plus Hibiscus flower (100 mg/kg)-fed group. Values are means ± SEM (n = 10). ^a–c Values within arrow with different letters are significantly different from each other at p < 0.05 as determined using Turkey’s multiple-range test.

Figure 3

Effect of LV, HS, and MetA on WAT weight and morphology in HFD-induced obese mice. Representative images (A), final WAT weight (B), hematoxylin and eosin (H and E) staining of WAT (C), and adipocytes area (D) in NFD and DIO mice after 9 weeks of 60% HFD feeding. Images were captured under a light microscope at ×100 magnification. Abdominal subWAT, abdominal subcutaneous fat; EWAT, epididymal white adipose tissue; RWAT, retroperitoneal white adipose tissue; Total, abdominal subWAT + EWAT + RWAT + intestine adipose tissue. HFD, 60% high-fat-diet control group; HFD + GC_245, HFD contains Garcinia cambogia extract (245 mg/kg)-fed group; HFD + LV_100, HFD contains lemon verbena extract (100 mg/kg)-fed group; HFD + HS_100, HFD contains Hibiscus flower extract (100 mg/kg)-fed group; HFD + MetA_50, HFD contains the MetA-mixed extract of lemon verbena plus Hibiscus flower (50 mg/kg)-fed group; HFD + MetA_100, HFD contains the MetA-mixed extract of lemon verbena plus Hibiscus flower (100 mg/kg)-fed group. Values are expressed as means ± SEM (n = 10). ^a–e Values within arrow with different letters are significantly different from each other at p < 0.05 as determined using Turkey’s multiple-range test.

Figure 4

Effect of LV, HS, and MetA on expression of adipogenic regulator genes in HFD-induced obese mice. The mRNA expression levels of adipogenic regulator genes were analyzed using quantitative real-time polymerase chain reaction (qPCR). HFD, 60% high-fat-diet control group; HFD + GC_245, HFD contains Garcinia cambogia extract (245 mg/kg)-fed group; HFD + LV_100, HFD contains lemon verbena extract (100 mg/kg)-fed group; HFD + HS_100, HFD contains Hibiscus flower extract (100 mg/kg)-fed group; HFD + MetA_50, HFD contains the MetA-mixed extract of lemon verbena plus Hibiscus flower (50 mg/kg)-fed group; HFD + MetA_100, HFD contains the MetA-mixed extract of lemon verbena plus Hibiscus flower (100 mg/kg)-fed group. Values are expressed as means ± SEM (n = 10). ^a–d Values within arrow with different letters are significantly different from each other at p < 0.05 as determined using Turkey’s multiple-range test.

Figure 5

Effect of LV, HS, and MetA on energy expenditure and thermogenesis-related gene expression in HFD-induced obese mice. Rectal temperature (A), UCP-1 mRNA expression in subcutaneous fat (B), and UCP-2 mRNA expression in EWAT (C) of NFD and DIO mice after 9 weeks of 60% HFD feeding. Rectal temperature was measured at regular intervals (30, 60, 90, 120, and 180 min) after sample administration when the mice were exposed to 10 °C ambient temperature in a cold room at 8 weeks on HFD (at 18 weeks of age). Gene-expression levels were analyzed using qPCR. HFD, 60% high-fat-diet control group; HFD + GC_245, HFD contains Garcinia cambogia extract (245 mg/kg)-fed group; HFD + LV_100, HFD contains lemon verbena extract (100 mg/kg)-fed group; HFD + HS_100, HFD contains Hibiscus flower extract (100 mg/kg)-fed group; HFD + MetA_50, HFD contains the MetA-mixed extract of lemon verbena plus Hibiscus flower (50 mg/kg)-fed group; HFD + MetA_100, HFD contains the MetA-mixed extract of lemon verbena plus Hibiscus flower (100 mg/kg)-fed group. Values are expressed as means ± SEM (n = 10). ^a–d Values within arrow with different letters are significantly different from each other at p < 0.05 as determined by Turkey’s multiple-range test.

Figure 6

Effect of LV, HS, and MetA on systemic glucose tolerance in HFD-induced obese mice. Serum glucose levels (A), serum IGF-1 levels (B), intraperitoneal glucose tolerance test (C), serum adiponectin levels (D), serum leptin levels (E), adiponectin and leptin mRNA expression in EWAT (F), serum TNF-α levels (G), and serum IL-6 levels (H). Adipokines were quantified using enzyme-linked immunosorbent assay (ELISA) and an intraperitoneal glucose tolerance test (IGTT) was performed at 8 weeks on HFD (at 18 weeks of age). HFD, 60% high fat diet control group; HFD + GC_245, HFD contains Garcinia cambogia extract (245 mg/kg)-fed group; HFD + LV_100, HFD contains lemon verbena extract (100 mg/kg)-fed group; HFD + HS_100, HFD contains Hibiscus flower extract (100 mg/kg)-fed group; HFD + MetA_50, HFD contains the MetA-mixed extract of lemon verbena plus Hibiscus flower (50 mg/kg)-fed group; HFD + MetA_100, HFD contains the MetA-mixed extract of lemon verbena plus Hibiscus flower (100 mg/kg)-fed group. Values are expressed as means ± SEM (n = 10). ^a–d Values within arrow with different letters are significantly different from each other at p < 0.05 as determined using Turkey’s multiple-range test.

Figure 7

Effects of LV, HS, and MetA on liver weight and hepatic lipid accumulation in HFD-induced obese mice. (A) Liver, (B) liver TG levels, and (C) liver H and E and oil-red O staining. HFD, 60% high-fat-diet control group; HFD + GC_245, HFD contains Garcinia cambogia extract (245 mg/kg)-fed group; HFD + LV_100, HFD contains lemon verbena extract (100 mg/kg)-fed group; HFD + HS_100, HFD contains Hibiscus flower extract (100 mg/kg)-fed group; HFD + MetA_50, HFD contains the MetA-mixed extract of lemon verbena plus Hibiscus flower (50 mg/kg)-fed group; HFD + MetA_100, HFD contains the MetA-mixed extract of lemon verbena plus Hibiscus flower (100 mg/kg)-fed group. Values are expressed as means ± SEM (n = 10). ^a–d Values within arrow with different letters are significantly different from each other at p < 0.05 as determined using Turkey’s multiple-range test.

Figure 8

Effects of LV, HS, and MetA on ACC and AMP-activated kinase (AMPK) phosphorylation in liver tissue of obese mice fed HFD. Quantification of proteins in immunoblot (A). A representative blot is shown (A, upper), Densitometric analyses of p-ACC and ACC, and p-AMPK and AMPK (A, lower) in liver. Relative signal strength of AMPK, p-AMPK, ACC, p-ACC, and internal control (β-actin) was quantified for each band and the relative expression levels of quantification of proteins were calculated as a ratio to HFD-HFD gene expression. mRNA expression levels of lipogenesis (B) and fatty acid oxidation (C)-related genes were analyzed using qPCR. HFD, 60% high-fat-diet control group; HFD + GC_245, HFD contains Garcinia cambogia extract (245 mg/kg)-fed group; HFD + LV_100, HFD contains lemon verbena extract (100 mg/kg)-fed group; HFD + HS_100, HFD contains Hibiscus flower extract (100 mg/kg)-fed group; HFD + MetA_50, HFD contains the MetA-mixed extract of lemon verbena plus Hibiscus flower (50 mg/kg)-fed group; HFD + MetA_100, HFD contains the MetA-mixed extract of lemon verbena plus Hibiscus flower (100 mg/kg)-fed group. Values are expressed as means ± SEM (n = 10). ^a–d Values within arrow with different letters are significantly different from each other at p < 0.05 as determined using Turkey’s multiple-range test.