Leaf samples of mulberry (M. alba) were collected from plantations in the Sangju area of Korea in October 2015, then identified by one of the authors (Professor Young Whan Choi). Voucher specimens (accession number Mul-PDRL-1) were deposited in the herbarium of Pusan National University (Miryang, Korea). C. militaris was kindly provided by Professor Sang Mong Lee in the Department of Life Science and Environmental Biochemistry, Pusan National University and silkworm pupae powder was supplied by Jeongeup Agriculture Cooperative Federations for Silkworm Farming (Jeongeup, Korea). Briefly, the mulberry leaves were dried in a hot-air drying machine (JSR, Seoul, Korea) for 24 h at 60℃, then reduced to powder using an electric blender. Following sterilization at 121℃ for 60 min using an autoclave, the mulberry powder was mixed with silkworm pupae (SWP) powder at four different concentrations (0%-M1, 25%-M2 and 50%-M3) and subsequently inoculated with 10% C. militaris (v/w). The samples were then fermented in a shaking incubator at 25℃ for three different periods (3 weeks; 3M1, 3M2 and 3M3, 4 weeks; 4M1, 4M2 and 4M3, or 6 weeks: 6M1, 6M2 and 6M3). On the final day of fermentation, the mixtures were harvested from the flask and extracted using dH₂O(W), 50% EtOH(50) or 95% EtOH(95) (Table 1). To prepare each extract of fermented mulberry, these powders were mixed with each solvent in a fixed liquor ratio (mulberry powder/solvent ratio, 1:10), then sonicated for 1 h using JAC ultrasonic equipment (KODO, Hwangseong, Korea). The supernatant was collected from this sonicated extract after centrifugation at 3,000 rpm for 10 min. Next, 9 mL of the same solvent was also added into the pellet and then sonicated under the same conditions. Following collection of the supernatant, the above procedure was repeated, after which the samples were filtered through a 0.4 µm filter and concentrated by vacuum evaporation. Finally, 27 EMfCs were lyophilized using circulating extraction equipment (IKA Labortechnik, Staufen, Germany) (Figure 1). Next, these extracts were dissolved in dH₂O or DMSO to 40 mg/mL.

The animal protocol used in this study was reviewed and approved by the Pusan National University-Institutional Animal Care and Use Committee (PNU-IACUC; Approval Number PNU-2017-1461). All SD rats were handled in the Pusan National University-Laboratory Animal Resources Center, which is accredited by the Korea Food and Drug Administration (FDA) (Accredited Unit Number-000231) and AAALAC International (Accredited Unit Number; 001525). Eight-week-old male SD rats were purchased from Samtako Bio-Korea Inc. (Osan, Korea) and provided with ad libitum access to water and a standard irradiated chow diet (Samtako Inc.). During the experiment, rats were maintained in a specific pathogen-free state under a strict light cycle (lights on at 08:00 h and off at 20:00 h) at 23±2℃ and 50±10% relative humidity.

Briefly, the intra-abdominal adipose tissues were dissected from eight-week-old male SD rats after sacrifice using CO₂ gas. These tissues (30 g) were then minced in 5 mL of DMEM supplemented with 1 mg/mL type I collagenase (Worthington Biochemical Co., Freehold, NJ, USA) and 1% BSA (MP Biomedicals, Illkirch, France) and subsequently incubated at 37℃ for 30 min in a shaking incubator (JSR, Gongju-City, Korea). The homogenate of adipose tissue was filtered through a 100 µm nylon mesh and washed three times in KRH (Krebs ringer/HEPES solution: 25 mM NaHCO₃, 125 mM NaCl, 5 mM glucose, 2.5 mM KCl, 1.25 mM NaH₂PO₄, 2 mM CaCl₂, 1 mM MgCl₂, 25 mM HEPES) containing 1% BSA. Finally, the pellets of adipocytes harvested with centrifugation were diluted in KRH containing 3% BSA to generate a cell suspension. Primary adipocytes were grown in a humidified incubator at 37℃ under 5% CO₂ and 95% air in KRH containing 3% BSA. Thereafter, these adipocytes were seeded onto 24-well plates for each experimental purpose and incubated with different concentration of EMfCs to measure the release of free glycerol.

The concentration of cAMP in the culture supernatant of adipocytes was determined using a cAMP ELISA kit (STA-500, Cell Biolabs Inc., Ltd., Wuhan, China) according to the manufacturer's protocol. Following treatment with Vehicle (dH₂O or DMSO), or 10 µM of isoproterenol hydrochloride (Sigma-Aldrich Co., St. Louis, MO, USA), or 100, 200 and 400 µg/mL of EMfCs on adipocytes (4×10⁵ cells/mL) for 24 h, cell culture medium samples were collected by centrifugation at 1,000 rpm for 3 min at 4℃, following which the supernatant was collected for analysis. An acetylation reagent, peroxidase cAMP tracer conjugation and anti-cAMP polyclonal antibody were added and samples were incubated at room temperature for 2 h, followed by the addition of substrate solution for 5 min at room temperature. The reaction was terminated by the addition of stop solution and the plates were read at an absorbance of 450 nm using a VersaMax Plate reader (Molecular Devices, LLC, Sunnyvale, CA, USA).

Free glycerol release was measured using free glycerol reagent (Sigma-Aldrich Co., St. Louis, MO, USA) according to the manufacturer's protocols [1]. To measure the glycerol level, adipocytes were seeded at a density of 2×10⁵ cells/mL in KRH and grown at 37℃ incubator. After 24 h, they were either untreated (No group), treated with Vehicle (dH₂O or DMSO), or pretreated with 10 µM of isoproterenol hydrochloride (Sigma-Aldrich Co.), or 100, 200 and 400 µg/mL of EMfCs. Following incubation for 24 h, the culture medium was collected from primary adipocytes treated with EMfCs and heated at 65℃ for 15 min to inactivate any enzymes released by the adipocytes. The inactivated medium (10 µL) was then mixed with 200 µL of glycerol detection reagent, after which the absorbance was read at 570 nm directly in the wells using a VERSA max plate reader (Molecular Devices, Sunnyvale, CA, USA).

Cell viability was determined using the tetrazolium compound 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) (Sigma-Aldrich Co.). To determine the cell viability, 3T3-L1 cells were seeded at a density of 1×10⁴ cells/0.2 mL and then grown for 24 h in a 37℃ incubator. When the cells attained 70-80% confluence, they were treated with Vehicle (dH₂O or DMSO), 100, 200, or 400 µg/mL of 27 EMfCs. Following incubation for 24 h, the supernatants of the 3T3-L1 cells were discarded, after which 0.2 mL of fresh DMEM media and 50 µL of MTT solution (2 mg/mL in PBS) were added to each well. The cells were then incubated at 37℃ for 4 h, after which the formazan precipitate was dissolved in DMSO and the absorbance at 570 nm was read directly in the wells using a VERSA max Plate reader (Molecular Devices).

The one-dimensional HPLC system (Agilent 1100; Agilent Technologies, Inc., Santa Clara, CA, USA) consisted of a quaternary pump, an auto-sampler, a degasser, an automatic thermostatic column compartment and a diode array detector. Chromatographic conditions for AD, HPC and UD analysis were as follows: A Phenomenex Luna C18 column (150×4.6 mm internal diameter; 5 mm particle size; Phenomenex, Torrance, CA, USA) was used; gradient elution was performed with (A) 0.025% formic acid in water and (B) acetonitrile (0-10 min, 0-5% B; 10-20 min, 5% B; 20-30 min, 5-15% B; 30-40 min, 15% B; 40-50 min, 15-100% B; 50-55 min, 100% B; and 55-60 min, 100-0% B); the flow rate was 0.5 mL/min; and the column temperature was 30℃. For DD and 5-HMF analysis, gradient elution was performed with (A) deionized water and (B) acetonitrile (0-25 min, 30-90% B; and 25-40 min, 90% B). A flow rate of 1.0 mL/min was used. The flow rate and pressure were maintained at 1.53mL/min and 35±2 psi, respectively. The wavelength was set at 254 nm and the output signal of the detector was recorded using Clarity™ Chromatography Software version 6.0 (DataApex, Prague, Czech Republic).

One-way ANOVA was used to identify significant differences between No and other treated groups (SPSS for Windows, Release 10.10, Standard Version, Chicago, IL, USA). Differences in the responses of the Vehicle and EMfCs treated groups, or those treated with 100 µg/mL of EMfCs and other concentrations of EMfCs were evaluated using a post-hoc test (SPSS for Windows, Release 10.10, Standard Version). All values are reported as the mean±standard deviation (SD) and a P value of <0.05 was considered significant.

To identify novel extracts with lipolytic activity among 27 EMfCs, the cAMP levels were analyzed in the culture medium of primary adipocytes derived from SD rats following treatment with 27 EMfCs. Increases in the cAMP level were detected in most groups treated with extracts purified using 4 weeks fermentation product, regardless of the SWP powder concentration, with significant increases observed in primary adipocytes treated with 5 extracts (4M1-W, 4M1-50, 4M1-95, 4M2-W, 4M2-50 and 4M3-95) compared with the Vehicle treated group (Figure 2). Also, several groups treated with extracts purified using 50% EtOH (3M1-50, 3M2-50, 3M3-50 and 6M3-50) showed a significant enhanced level of cAMP (Figure 2). Therefore, these results indicate that some novel extracts derived from fermented mulberry leaves have very strong lipolytic activity.

To evaluate the cytotoxicity of EMfCs against adipocytes, the cell viability was measured in 3T3-L1 adipocytes following treatment with 200 µg/mL of 27 EMfCs. Among all extracts, the most groups treated EMfCs showed some low level of toxicity although their decrease rate was varied. However, eight groups treated with extracts purified using 95% EtOH (3M1-95, 3M2-95, 4M1-95, 4M2-95, 4M3-95, 6M1-95, 6M2-95 and 6M3-95) did not show any significant toxicity in 3T3-L1 cells compared with the Vehicle treated group (Figure 3). These results show that some extracts of 27 EMfCs can't induce any significant toxicity in 3T3L-1 adipocytes at dose of 200 µg/mL.

We finally selected two extracts with high lipolytic activity based on the results of the concentration of cAMP and toxicity (Table 2). To verify the dose dependent effects of these extracts on the lipolytic activity and toxicity, the concentration of cAMP, the levels of free glycerol and cell viability were measured in the culture medium of primary adipocytes and 3T3-L1 cells after treatment with 100, 200 and 400 µg/mL of the extracts. In two groups, the cAMP and glycerol level increased significantly in a dose dependent manner, with a high level being detected in the 400 µg/mL of the extracts treated groups (Figure 4A and B). But, the cell viability decreased in response to treatment with increasing concentrations of 4M3-95 (400 µg/mL), while 4M3-95 treated group were maintained a constant level (Figure 4C). Overall, these findings indicate that 4M3-95 may promote lipolysis of adipocytes derived from SD rats without any significant toxicity.

Cordycepin has been known to exhibit lipid lowering effects as well as multiple-biological effects including modulation of immune response, inhibition of tumor growth, hypotensive and vasorelaxation activities, and promoting secretion of adrenal hormone [22]. Therefore, the presence of the cordycepin in 4M3-95 was confirmed using HPLC analysis. As presented in Figure 5, the peak represented cordycepin were detected in the HPLC chromatogram of 4M3-95 under the optimal conditions, when it compared to standard cordycepin (100 µg/mL). Therefore, these results show that the lipolysis activity of 4M3-95 can be associated with codycepin, one of famous compounds with lipid-lowering effect, in the primary adipocytes.