Anti-Obesity Effect of Extract from Nelumbo Nucifera L., Morus Alba L., and Raphanus Sativus Mixture in 3T3-L1 Adipocytes and C57BL/6J Obese Mice

Abstract

The antioxidant and anti-adipogenic activities of a mixture of Nelumbo nucifera L., Morus alba L., and Raphanus sativus were investigated and their anti-obesity activities were established in vitro and in vivo. Among the 26 different mixtures of extraction solvent and mixture ratios, ethanol extract mixture no. 1 (EM01) showed the highest antioxidant (α,α-Diphenyl-β-picrylhydrazyl, total phenolic contents) and anti-adipogenic (Oil-Red O staining) activities. EM01 inhibited lipid accumulation in 3T3-L1 adipocytes compared to quercetin-3-O-glucuronide. Furthermore, body, liver, and adipose tissue weights decreased in the high-fat diet (HFD)-EM01 group compared to in the high-fat diet control group (HFD-CTL). EM01 lowered blood glucose levels elevated by the HFD. Lipid profiles were improved following EM01 treatment. Serum adiponectin significantly increased, while leptin, insulin growth factor-1, non-esterified fatty acid, and glucose significantly decreased in the HFD-EM01 group. Adipogenesis and lipogenesis-related genes were suppressed, while fat oxidation-related genes increased following EM01 administration. Thus, EM01 may be a natural anti-obesity agent.

Keywords: 3T3-L1 adipocyte; C57BL/6J mice; Nelumbo nucifera L., Morus alba L., Raphanus sativus; anti-obesity.

Figure 1

Effects of 26 extracts by mixture ratio of Nelumbo nucifera L., Morus alba L., and Raphanus sativus on antioxidant activity, cell viability, lipid accumulation. (A) DPPH radical scavenging activity. (B) Total phenolic contents. (C) Post-confluent 3T3-L1 preadipocytes were differentiated along with the treatment of each extracts for 6 days. XTT (2,3-bis(2-methoxy-4-nitro-5- sulfophenyl)-2H-tetrazolium-5-carboxanilide) and PMS (N-methyl dibenzopyrazine methyl sulfate reagents) mixture was added to the medium. After 4 h of incubation, cell viability was found out by calculating the absorbance at 450 nm against 690 nm. (D) Stained lipids were extracted and quantified by calculating the absorbance at 490 nm. Data are presented as the mean ± SEM (n = 3). Means with different letters on bars indicate that there is a significant difference at p < 0.05 by Duncan’s multiple range test.

Figure 1

Effects of 26 extracts by mixture ratio of Nelumbo nucifera L., Morus alba L., and Raphanus sativus on antioxidant activity, cell viability, lipid accumulation. (A) DPPH radical scavenging activity. (B) Total phenolic contents. (C) Post-confluent 3T3-L1 preadipocytes were differentiated along with the treatment of each extracts for 6 days. XTT (2,3-bis(2-methoxy-4-nitro-5- sulfophenyl)-2H-tetrazolium-5-carboxanilide) and PMS (N-methyl dibenzopyrazine methyl sulfate reagents) mixture was added to the medium. After 4 h of incubation, cell viability was found out by calculating the absorbance at 450 nm against 690 nm. (D) Stained lipids were extracted and quantified by calculating the absorbance at 490 nm. Data are presented as the mean ± SEM (n = 3). Means with different letters on bars indicate that there is a significant difference at p < 0.05 by Duncan’s multiple range test.

Figure 2

Effect of EM01 and its bioactive compound on lipid accumulation. Post-confluent 3T3-L1 preadipocytes were differentiated along with the treatment of each extracts and its bioactive compound for 6 days. Stained lipids were eluted and quantified by calculating the absorbance at 490 nm. Data are presented as the mean ± SEM (n = 3). Means with different letters on bars indicate that there is a significant difference at p < 0.05 by Duncan’s multiple range test.

Figure 3

Effects of EM01 on (A) body weight, (B) food intake, (C) FER, (D) organ weight, and (E) adipose tissue fat weight in high-fat diet (HFD)-induced obese mice. GC, Garcinia cambogia extract; HFD, mice were fed a high-fat diet (60% kcal fat); ND, mice were fed a normal diet (10% kcal fat); FER, food efficiency ratio (total weight gain/total food intake × 100). Data are presented as the mean ± SEM (n = 12); # p < 0.05, ## p < 0.01, ### p < 0.001 vs. ND; * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. HFD-CTL.

Figure 4

Effects of EM01 on glucose tolerance in HFD-induced obese mice. Data are presented as the mean ± SEM (n = 12); # p < 0.05, ## p < 0.01, ### p < 0.001 vs. ND; * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. HFD-CTL.

Figure 5

Effects of EM01 on serum lipid profile in HFD-induced obese mice. (A) Total cholesterol. (B) HDL cholesterol. (C) LDL cholesterol. (D) Triglyceride. (E) Creatinine. (F) AST & ALT. Data are presented as the mean ± SEM (n = 12); # p < 0.05, ## p < 0.01, ### p < 0.001 vs. ND; * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. HFD-CTL. AST, aspartate aminotransferase; ALT, alanine aminotransferase.

Figure 6

Effects of EM01 on the energy balancing metabolism in HFD-induced obese mice. (A) Adiponectin. (B) Leptin. (C) IGF-1. (D) NEFA. (E) Glucose. Data are presented as the mean ± SEM (n = 12); # p < 0.05, ## p < 0.01, ### p < 0.001 vs. ND; * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. HFD-CTL. IGF-1, insulin-like growth factor-1; NEFA, non-esterified fatty acid.

Figure 7

Effects of EM01 on mRNA expression level of lipid metabolism-related genes in HFD-induced obese mice. (A) Liver. (B) Epididymal adipose tissue. (C) Abdominal subcutaneous fat. Data are presented as the mean ± SEM (n = 12); # p < 0.05, ## p < 0.01, ### p < 0.001 vs. ND; * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. HFD-CTL. FAS, fatty acid synthase; SCD-1, stearoyl-CoA desaturase-1; SREBP-1c, sterol regulatory element binding protein-1c; PPARγ, peroxisome proliferator-activated receptor γ; DGAT1, diglyceride acyltransferase; UCP, mitochondrial uncoupling proteins; ACOX1, peroxisomal acyl-coenzyme A oxidase 1; PPARα, peroxisome proliferator-activated receptor α; ACS1, acetyl CoA synthetase 1; CPT1b, carnitine palmitoyltransferase 1b.

Figure 7

Effects of EM01 on mRNA expression level of lipid metabolism-related genes in HFD-induced obese mice. (A) Liver. (B) Epididymal adipose tissue. (C) Abdominal subcutaneous fat. Data are presented as the mean ± SEM (n = 12); # p < 0.05, ## p < 0.01, ### p < 0.001 vs. ND; * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. HFD-CTL. FAS, fatty acid synthase; SCD-1, stearoyl-CoA desaturase-1; SREBP-1c, sterol regulatory element binding protein-1c; PPARγ, peroxisome proliferator-activated receptor γ; DGAT1, diglyceride acyltransferase; UCP, mitochondrial uncoupling proteins; ACOX1, peroxisomal acyl-coenzyme A oxidase 1; PPARα, peroxisome proliferator-activated receptor α; ACS1, acetyl CoA synthetase 1; CPT1b, carnitine palmitoyltransferase 1b.

Figure 7

Effects of EM01 on mRNA expression level of lipid metabolism-related genes in HFD-induced obese mice. (A) Liver. (B) Epididymal adipose tissue. (C) Abdominal subcutaneous fat. Data are presented as the mean ± SEM (n = 12); # p < 0.05, ## p < 0.01, ### p < 0.001 vs. ND; * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. HFD-CTL. FAS, fatty acid synthase; SCD-1, stearoyl-CoA desaturase-1; SREBP-1c, sterol regulatory element binding protein-1c; PPARγ, peroxisome proliferator-activated receptor γ; DGAT1, diglyceride acyltransferase; UCP, mitochondrial uncoupling proteins; ACOX1, peroxisomal acyl-coenzyme A oxidase 1; PPARα, peroxisome proliferator-activated receptor α; ACS1, acetyl CoA synthetase 1; CPT1b, carnitine palmitoyltransferase 1b.