Lab Anim Res.  2017 Sep;33(3):270-279. 10.5625/lar.2017.33.3.270.

Lipolytic effect of novel extracts from mulberry (Morus alba) leaves fermented with Cordyceps militaris in the primary adipocytes derived from SD rats

• Lee MR
• Kim JE
• Yun WB
• Choi JY
• Park JJ
• Kim HR
• Song BR
• Choi YW
• Kim KM
• Hwang DY

• KMID: 2391102
• DOI: http://doi.org/10.5625/lar.2017.33.3.270

Abstract

Mulberry (Morus alba) leaves are known to have therapeutic effects on lipid metabolism including lipogenesis, lipolysis and hyperlipidemia. However, novel compounds with strong lipolytic ability among 27 extracts of the mulberry leaves fermented with Cordyceps militaris (EMfCs) have not yet been identified. Therefore, the cAMP concentration and cell viability were measured in the primary adipocytes of SD (Sprague Dawley) rats and 3T3-L1 cells after treatment of 27 EMfCs. Briefly, mulberry leaves powders amended with three different concentrations (0, 25 and 50%) of silkworm pupae (SWP) powder were fermented with 10% C. militaris (v/w) during three different periods (3, 4 and 6 weeks). A total of 27 extracts were obtained from the fermented mulberry leaves powders using three different solvents (dH2O, 50% EtOH and 95% EtOH). Among the 27 EMfCs treated groups, a significant increase in the concentration of cAMP was detected in primary adipocytes treated with 10 extracts when compared with the Vehicle treated group. However, their cAMP concentration did not agree completely with the non-toxicity, although most extracts showed non-toxicity. Furthermore, the concentration of cAMP and level of free glycerol gradually increased in a dose dependent manner (100, 200 and 400 µg/mL) of 4M3-95 contained cordycepin without any significant toxicity. Overall, the results of this study provide strong evidence that 4M3-95 extract derived from EMfCs can stimulate the lipolysis of primary adipocytes at an appropriate concentration and therefore have the potential for use as lipolytic agents to treat obesity.

Keyword

Mulberry leaves; Lipolysis; Primary adipocytes; Glycerol; cAMP

MeSH Terms

3T3-L1 Cells
Adipocytes*
Animals
Bombyx
Cell Survival
Cordyceps*
Glycerol
Hyperlipidemias
Lipid Metabolism
Lipogenesis
Lipolysis
Morus*
Obesity
Powders
Pupa
Rats*
Solvents
Therapeutic Uses
Glycerol
Powders
Solvents
Therapeutic Uses

Figure

• 

  Figure 1 Schematic procedure of EMfCs preparation. After preparation of mulberry leaves powder and silkworm pupae powder, 27 EMfCs were extracted from the fermented mulberry leaves powder using dH2O, 50% EtOH and 90% EtOH as described in the Materials and Methods.

• 

  Figure 2 Analysis of cAMP concentration. After 27 EMfCs treatment, the concentration of cAMP was measured in the supernatant of primary adipocytes treated with 27 EMfCs (200 µg/mL) that were fermented for 3 weeks (A), 4 weeks (B) and 6 weeks (C). The cell images of the three groups were observed with an inverted microscope at 100× magnification. The data shown represent the means±SD of three replicates. *P<0.05 relative to the Vehicle treated group. ISOP; isoproterenol.

• 

  Figure 3 Cytotoxicity of 27 EMfCs. The cell viability was measured in 3T3-L1 adipocytes treated with 27 EMfCs (200 µg/mL) that were fermented for 3 weeks (A), 4 weeks (B) and 6 weeks (C). The cell images of the three groups were observed with an inverted microscope at 100× magnification. The data shown represent the means±SD of three replicates. *P<0.05 relative to the Vehicle treated group.

• 

  Figure 4 Dose dependent response of two candidates with strong lipolytic activity. The concentration of cAMP (A), free glycerol release (B) and cell toxicity (C) were measured in primary adipocytes and 3T3-L1 cells treated with 100, 200 and 400 µg/mL of two candidates that were fermented for 4 weeks. The data shown represent the means±SD of three replicates. *P<0.05 relative to the Vehicle treated group. ISOP; isoproterenol.

• 

  Figure 5 Chromatograms of 4M3-95 containing cordycepin. High performance liquid chromatography were performed the specific condition described in Materials and Methods section. The peak height/area reflected the concentration of the cordycepin in 4M3-95.

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