The anti-obesity effect of mulberry leaf (mori folium) extracts was increased by bioconversion with pectinex

ABSTRACT Mulberry leaf (_Mori Folium_) extract (MLE) is known to have anti-obesity effects. In this study, the enhanced effects of MLE after bioconversion treatment using Pectinex (BMLE) on

obesity were explored, and the underlying mechanisms were investigated using the active components, neochlorogenic acid (5-CQA) and cryptochlorogenic acid (4-CQA), whose amounts were

increased by bioconversion of MLE. Both MLE and BMLE inhibited lipid accumulation in 3T3-L1 adipocytes without cytotoxicity and suppressed the expression of CCAAT/enhancer-binding protein

alpha (C/EBPα). In addition, MLE and BMLE decreased high-fat diet-induced adipose tissue mass expansion. Notably, BMLE significantly increased antiadipogenic and anti-obesity effects

compared to MLE in vitro and in vivo. The active ingredients increased by bioconversion, 5-CQA and 4-CQA, inhibited the protein levels of C/EBPα and the mRNA levels of stearoyl-CoA

desaturase 1 (_Scd1_). These findings provide new insights into the therapeutic possibility of using bioconversion of MLE, by which upregulation of 5-CQA and 4-CQA potently inhibits

adipogenesis. SIMILAR CONTENT BEING VIEWED BY OTHERS 14-DEOXYGARCINOL IMPROVES INSULIN SENSITIVITY IN HIGH-FAT DIET-INDUCED OBESE MICE VIA MITIGATING NF-ΚB/SIRTUIN 2-NLRP3-MEDIATED ADIPOSE

TISSUE REMODELING Article 09 August 2022 SHORT-TERM _CUDRANIA TRICUSPIDATA_ FRUIT VINEGAR ADMINISTRATION ATTENUATES OBESITY IN HIGH-FAT DIET-FED MICE BY IMPROVING FAT ACCUMULATION AND

METABOLIC PARAMETERS Article Open access 03 December 2020 GINSENOSIDE RG3, ENRICHED IN RED GINSENG EXTRACT, IMPROVES LIPOPOLYSACCHARIDES-INDUCED SUPPRESSION OF BROWN AND BEIGE ADIPOSE

THERMOGENESIS WITH MITOCHONDRIAL ACTIVATION Article Open access 22 April 2024 INTRODUCTION Obesity is a disease in which surplus energy caused by an increase in food intake is transformed

into triglycerides and stored in adipose tissue, leading to weight gain1. Maintaining a healthy body weight is important because obesity can cause complications such as type 2 diabetes,

atherosclerosis, and heart attack2,3,4. Since the incidence of obesity has been increasing recently, it is indispensable to discover effective pharmacological drugs that potently inhibit fat

accumulation for treating obesity and metabolic disease. Transcription factors and adipogenic genes function in the molecular mechanism of adipogenesis. Adipogenic transcription factors,

such as CCAAT/enhancer-binding protein (C/EBP) family members and peroxisome proliferator-activated receptor gamma (PPARγ), are essential in the adipogenesis process5. After _Cebpb_ and

_Cebpd_ genes are induced to be expressed quickly in the early stages of adipocyte differentiation, these proteins increase the expression of PPARγ and C/EBPα, which are important for

terminal adipocyte differentiation6,7. In addition, C/EBPα-mediated adipogenic transcription activation, such as stearoyl-CoA desaturase 1 (_SCD1_), has been revealed8,9,10. SCD is an

endoplasmic reticulum (ER) enzyme that catalyzes biosynthesis with monounsaturated fatty acids (MUFAs), which contribute greatly to lipid synthesis11. SCD has isoforms of SCD1, 2, 3, 4, 5,

of which SCD1 and SCD5 are known to be expressed in humans. Furthermore, SCD1 is expressed at higher levels in adult white adipose tissue than SCD512,13,14. Several studies have shown a link

between SCD1 expression and obesity based on the finding that _Scd1_ gene deficiency inhibits lipid accumulation15,16. Therefore, the regulation of SCD1 via C/EBPα can be a treatment

strategy for obesity. Mulberry is a plant that originated from Asia, such as Korea, China, and Japan. Traditionally, all parts of plants, such as leaves, roots, and fruits, have been used

for the purpose of treating obesity or diabetes and for the purpose of eating17,18. Recent studies have reported the effects of mulberry leaves, such as anti-obesity19,20,21, cholesterol

reduction22,23, anti-inflammatory24, antioxidant25, blood pressure improvement26, thrombosis27, and anti-diabetes effects28,29. Bioconversion is a technology that induces the production of

physiologically active ingredients by modifying the chemical structure of natural products using biological methods such as microorganisms and enzyme-mediated fermentation30. Several studies

have reported the effect of potent therapeutic effects through bioconversion. For example, bioconverted Jeju hallabong tangor (_Citrus kiyomi_ × _ponkan_) by cytolase showed antioxidant and

anti-inflammatory effects31, and barley fermented with _Lactobacillus plantarum_ dy-1 showed effects in weight loss, lipid and inflammation improvement compared with natural products32.

Moreover, the bioconversion of _Citrus unshiu_ peel extract with cytolase showed an increased inhibitory effect on adipogenic activity compared with _Citrus unshiu_ peel extract33, and

fermentation of _Panax notoginseng_ by lactic acid bacteria showed an increased anti-obesity effect34. Pectinex used commercially in the food industry is a fungal enzyme complex derived from

_Aspergillus aculeatus_35. Recent studies showed that enzymatic hydrolysis of tea seed extract using Pectinex newly synthesized leucoside36, extracts treated with Pectinex showed a higher

antioxidant effect37,38, and pectins extracted from rapeseed cake using Pectinex showed the effect of inhibiting cancer cell growth39. Therefore, it is expected that mulberry leaves

extracted by grafting bioconversion technology using Pectinex will increase therapeutic activity. We previously reported that mulberry leaf extract (MLE) after bioconversion treatment using

Viscozyme L was superior to unaltered MLE in controlling diabetes both at the cellular and diabetic animal model levels40. In this study, we will investigate whether MLE after bioconversion

treatment using Pectinex (BMLE) are more effective against obesity than MLE in 3T3-L1 adipocytes and high-fat diet-induced mouse models. RESULTS BIOCONVERSION OF MLE ENHANCES THE INHIBITORY

EFFECT OF ADIPOGENESIS WITHOUT INDUCING CYTOTOXICITY IN 3T3-L1 CELLS To investigate the effect of the bioconversion of MLE on adipogenesis, we investigated the effect of lipid droplet

reduction in 3T3-L1 adipocytes (8-day differentiated cells) using Oil red O staining. Compared to differentiated adipocytes, 400 and 800 μg/mL MLE dose-dependently reduced lipid droplet

formation by 11.73% and 15.83%, respectively (Fig. 1a). (−)-epigallocatechin gallate (50 μM, EGCG) was used as a positive control for antiadipogenesis41,42,43. Interestingly, 400 or 800 

μg/mL BMLE reduced lipid droplets by 19.00 and 25.19%, respectively, indicating that bioconversion using Pectinex significantly enhanced the inhibitory effect of lipid accumulation. We

performed an MTT assay to examine whether the inhibitory effect was due to the cytotoxicity of MLE or BMLE in 3T3-L1 adipocytes. Treatment with MLE or BMLE (400 or 800 μg/mL) for 8 days did

not affect cell viability (Fig. 1b). Digitonin (100 μg/mL) was used as a positive control for cytotoxicity44. To investigate which signaling pathways caused BMLE-induced adipogenesis

inhibition, we performed western blots and examined the expression of C/EBPα and PPARγ proteins, major regulators of adipogenesis45. The expression of C/EBPα significantly decreased in the

BMLE-treated groups compared to the equal dose of MLE-treated group (Fig. 1c). In contrast, MLE or BMLE treatment had no significant effect on reducing PPARγ expression. These findings

suggested that bioconversion of MLE reinforced anti-adipogenesis through inhibition of C/EBPα expression. BIOCONVERSION OF MLE ENHANCES SUPPRESSION OF ADIPOSE TISSUE MASS IN HFD-INDUCED

OBESE MICE WITHOUT INDUCING LOSS OF APPETITE To investigate the physiological effect of BMLE on adipose tissue in obesity, mice were fed ND or HFD from 5 weeks of age. After 8 weeks of obese

model establishment, mice were administered MLE (600 mg/kg), BMLE (600 mg/kg) or orlistat (40 mg/kg, used as a positive control for anti-obesity46) for 8 weeks (Fig. 2a). The mice fed HFD

had a significant increase in abdominal adipose mass compared to mice fed ND (Fig. 2b). However, increased abdominal adipose mass by HFD was inhibited by administration of MLE, BMLE or

orlistat. In addition, the abdominal adipose mass of the BMLE-administered mice was severely decreased compared to that of MLE-administered mice. Furthermore, the increased eWAT and rWAT

weights by HFD feeding were significantly inhibited by BMLE compared to MLE administration (Fig. 2c). As shown in Fig. 2d, the fat index increased by feeding HFD was significantly decreased

in the group administered with MLE or BMLE, and the fat index decreased more in the group administered with BMLE compared with MLE. (Fig. 2d). Since weight loss is caused by loss of

appetite47, we examined caloric intake. MLE, BMLE and orlistat did not affect caloric intake, indicating that BMLE-mediated adipose mass inhibition was due to an antiadipogenic effect (Fig. 

2e). Taken together, BMLE significantly increased the attenuation of HFD-induced adipose tissue accumulation compared to MLE without affecting caloric intake. BIOCONVERSION OF MLE WITH

PECTINEX INCREASED NEOCHLOROGENIC ACID AND CRYPTOCHLOROGENIC ACID Bioconversion using enzymes is known to increase or decrease the amount of active components in natural products30,48. We

compared the pattern difference of the main active components with antiadipogenic effects between MLE and BMLE by using HPLC and LC-DAD-ESI-MS (Fig. 3b–e). Among them, three compounds (#1-3)

were distinguished in BMLE compared to MLE. These compounds were identified as neochlorogenic acid (#1, 5-CQA), chlorogenic acid (#2, 3-CQA) and cryptochlorogenic acid (#3, 4-CQA) from MS

and UV spectra and the literature (Fig. 3a)49,50,51. These compounds, as main compounds of MLE and BMLE, were quantified with calibration curves of each compound using HPLC analysis (Fig. 3b

and Supplementary Fig. S2). The compounds 5-CQA (#1, 0.57 ± 0.03 mg/g), 3-CQA (#2, 0.09 ± 0.01 mg/g) and 4-CQA (#3, 0.06 ± 0.01 mg/g) were detected in MLE (Table 1). Additionally, 5-CQA

(0.81 ± 0.02 mg/g) and 4-CQA (0.08 ± 0.01 mg/g) were detected in BMLE, respectively. Therefore, 5-CQA and 4-CQA were significantly increased by 1.42-fold and 1.33-fold, respectively,

different from 3- the CQA (0.07 ± 0.01 mg/g), indicating the role of 5-CQA and 4-CQA in the bioconversion-induced enhancement of the anti-adipogenesis effect. ANTIADIPOGENIC EFFECT OF

NEOCHLOROGENIC ACID AND CRYPTOCHLOROGENIC ACID To investigate the effect of 5-CQA and 4-CQA on lipid accumulation reduction, and cytotoxicity, we performed Oil red O, Nile red assay and MTT

assay in 3T3-L1 cells. The lipid droplets and triglycerides in 3T3-L1 adipocytes were significantly decreased by 40 and 80 μM 5-CQA and 4-CQA treatment without cytotoxicity (Fig. 4a-c).

NEOCHLOROGENIC ACID AND CRYPTOCHLOROGENIC ACID INHIBITS ADIPOGENIC FACTORS TRANSCRIPTION To investigate the effect of 5-CQA and 4-CQA on adipogenic factors, we performed Western blotting and

qPCR in 3T3-L1 cells. 40 and 80 μM 5-CQA and 4-CQA treatment dose-dependently decreased C/EBPα protein levels without affecting PPARγ (Fig. 5a). Since 5-CQA and 4-CQA reduced C/EBPα protein

levels, we examined the mRNA levels of _Scd1_, one of the C/EBPα downstream genes10, and _Cebpa_. The 5-CQA and 4-CQA treatments dose-dependently inhibited the _Cebpa_ and _Scd1_ mRNA

levels, indicating that the C/EBPα protein was regulated by transcriptional regulation and that downregulation of C/EBPα affected adipogenic gene levels (Fig. 5b). Taken together, we showed

that the bioconversion of MLE using Pectinex significantly increased the reduction in lipid droplets in 3T3-L1 adipocytes by downregulating C/EBPα compared to MLE. In addition, BMLE

administration significantly increased the reduction in adipose fat mass in HFD-induced obese mice. These effects were due to the inhibitory effect of _Cebpa_ and _Scd1_ expression by 5-CQA

and 4-CQA, active compounds upregulated via Pectinex-mediated bioconversion. DISCUSSION Mulberry leaf (_Mori Folium_) is a well-known herb traditionally used for metabolic disease

treatment17. In addition, there are several reports that mulberry leaf extract is effective for obesity and diabetes19,20,21,28. Bioconversion is known to increase the production of single

components of natural products30. In the present study, we found that compared with MLE, (1) BMLE increased the reduction in lipid droplet formation and C/EBPα expression, a major regulator

of adipogenesis, in 3T3-L1 adipocytes; (2) BMLE showed an enhanced anti-obesity effect by reducing the tissue mass of eWAT and rWAT in HFD-induced obese mice administered BMLE; (3) 5-CQA and

4-CQA, which are active components of MLE, were increased through bioconversion using Pectinex; and (4) the enhanced anti-adipogenesis effect of BMLE was attributed to the increased levels

of 5-CQA and 4-CQA, and 5-CQA and 4-CQA inhibited adipogenesis by suppressing the _Cebpa_ and _Scd1_ genes. Mulberry extract has been reported to be effective in the management of

obesity19,20,21, diabetes28, cholesterol reduction22,23, anti-inflammatory24, antioxidant25, blood pressure improvement26, and thrombosis improvement27, and clinically, it has been reported

to be effective in improving obesity52 and diabetes53,54. Our results showed that MLE and BMLE inhibited C/EBPα protein expression in 3T3-L1 adipocytes (Fig. 1c), and MLE or BMLE

administration decreased the adipose tissue mass and fat index of eWAT and rWAT in HFD-induced obese mice (Fig. 2b‒d). According to previous results that deficiency of C/EBPα inhibits WAT

fat storage55, modulation of C/EBPα by MLE and BMLE supported the therapeutic effect on fat accumulation and obesity. Furthermore, bioconversion of MLE using Pectinex potently reduced

adipogenesis compared with MLE. Since bioactivity is enhanced by bioprocesses such as enzyme-mediated bioconversion30 and drug dose reduction maintaining efficacy can be possible by

preventing drug safety issues, this pharmacological tool can be useful. Since Pectinex is a complex enzyme with a small amount of hemicellulose and cellulase added to the main enzyme

pectinase and polygalacturonase, it can probably bioconvert plant active ingredients into a variety of compound forms as compared with single enzymes such as glycosylase or cellulase. Thus,

BMLE can be an effective therapeutic method. In the present study, we used HPLC–DAD-ESI–MS/MS to validate the major active components of BMLE. As described above, among the major active

ingredients we measured, only 5-CQA and 4-CQA content increased in MLE after bioconversion treatment using Pectinex. However, it is expected that the reason why Pectinex only increased the

content of 5-CQA and 4-CQA like this can be elucidated through future research. Several studies reported that 5-CQA had an anti-inflammatory effect in brain disease by inhibiting

proinflammatory cytokines56, high potential for the development of human immunodeficiency virus-1 drug by inhibiting its reverse transcriptase activity57, and improved hepatic lipid

accumulation and inflammation by regulating miR-34a58. In addition, 4-CQA was reported to have antioxidant activity59. However, there are not enough studies related to obesity. Our study

first found that 5-CQA and 4-CQA downregulated _Cebpa_ genes in 3T3-L1 adipocytes, thereby decreasing the expression of C/EBPα proteins (Fig. 5b). In addition, we found that these compounds

have an antiadipogenic effect by reducing the _Scd1_ genes known to induce lipid accumulation (Fig. 5b)15. Therefore, the increased inhibitory adipogenesis effect of BMLE was due to

increased 5-CQA and 4-CQA concentrations through bioconversion using Pectinex. CONCLUSION This study demonstrated the enhancement of the antiadipogenic and anti-obesity effects of BMLE and

bioconverted MLE using Pectinex in vitro and in vivo. We showed that the major components of BMLE that were increased compared to MLE were 5-CQA and 4-CQA. In addition, 5-CQA and 4-CQA exert

antiadipogenic effects by suppressing the _Cebpa_ and _Scd1_ genes. These findings provide new insights into the therapeutic possibility using bioconversion of MLE. MATERIALS AND METHODS

REAGENTS Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), bovine serum (BS), phosphate-buffered saline (PBS), penicillin/streptomycin, and

trypsin-ethylenediaminetetraacetic acid (EDTA) were obtained from Gibco, Inc. (Grand Island, NY, USA). 3-Isobutyl-1-methyxanthine (IBMX), dexamethasone, insulin,

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Oil red O, isopropanol, (−)‐epigallocatechin gallate (EGCG), digitonin, neochlorogenic acid (5-CQA), chlorogenic acid

(3-CQA), and cryptochlorogenic acid (4-CQA) were used as reference compounds, and formic acid (HPLC grade) was obtained from Sigma–Aldrich (St. Louis, MO, USA). Anti-PPARγ, anti-C/EBPα and

anti-rabbit antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Anti-β-actin antibody was purchased from AbFrontier (Geumcheon, Seoul, Korea). 5-CQA and 4-CQA

for the in vitro study were purchased from Cayman Chemical (Ann Arbor, MI, USA). Bovine serum albumin (BSAS-AU) was purchased from Bovogen Biologicals (Keilor East, VIC, Australia). Dried

mulberry leaves (_Mori Folium_) were purchased from the Naemome Dah Herb Company (Nam-gu, Ulsan, Korea). Pectinex® Ultra SP-L (3300 PGNU/g) was purchased from Novozymes (Bagsværd, Denmark).

Methanol, ethyl acetate and acetonitrile of HPLC grade were obtained from Honeywell Burdick & Jackson (Muskegon, MI, USA). All other chemicals were of analytical grade. PREPARATION OF

MLE The dried samples were ground using a food mixer (HMF-3080SS, Hanil Electrics, Seoul, Korea), and 2 g of sample powder was weighed, which was extracted with 20 mL of distilled water by

sonication (35 kHz, 118.8 W) for 1 h at room temperature. Then, the extracted solution was centrifuged at 3500 rpm for 5 min. The supernatant was partitioned again with 20 mL of ethyl

acetate for the liquid–liquid extraction (LLE) method. The LLE method was repeated three times. All water layers were evaporated at 40 °C in vacuo, and the residues were dissolved in 2 mL of

methanol, which was filtered with a syringe membrane filter (PVDF, 0.22 μm) for HPLC analysis. Separately, water extracts of MLE for bioassays were filtrated with filter paper (No. 4),

concentrated with a vacuum concentrator and freeze-dried to obtain a powder. BIOCONVERSION OF MLE MLE solution was adjusted to pH 4.5, and Pectinex (Novozymes) was added in an amount to the

1 v/v% weight ratio of MLE solution. The mixture was then fermented at 45 °C for 15 h. Then, the mixture was incubated at 95 °C for 30 min to terminate the enzyme activity. The fermented

solution was filtered with filter paper (No. 4), concentrated with a vacuum concentrator and freeze-dried to obtain a powder. STANDARD PREPARATION AND CALIBRATION CURVE The stock solutions

of each reference standard were prepared to 5-CQA (0.8 mg/mL), 3-CQA (0.05 mg/mL), and 4-CQA (0.6 mg/mL) using methanol and kept at 4 °C until use. To make calibration curves of the main

compounds, each main compound was diluted to five different concentrations with a suitable amount of methanol as follows: 5-CQA of 1.3, 12.5, 25.0, 50.0 and 100.0 µg/mL, 3-CQA of 0.8, 1.6,

3.1, 6.3 and 12.5 µg/mL and 4-CQA acid of 0.1, 1.3, 2.5, 5.0 and 10.0 µg/mL. All standard solutions were filtered by a 0.22-μm syringe filter (PVDF) before injection into the HPLC

instrument. CELL CULTURE Mouse 3T3-L1 preadipocytes were obtained from the American Type Culture Collection (ATCC CL-173™) and cultured as previously described60. 3T3-L1 preadipocytes were

cultured in DMEM containing 10% bovine serum (BS) at 37 °C in a 5% CO2 atmosphere. To induce differentiation, preadipocytes were cultured in MDI [0.5 mM isobutylmethylxanthine (IBMX), 5 μM

dexamethasone, 0.5 μg/mL insulin, and 10% fetal bovine serum (FBS)] and designated day 0 for differentiation. The medium was replaced every 48 h. After 2 days of differentiation, they were

maintained in media containing 1 μg/mL insulin and 10% FBS. The medium containing 10% FBS was replaced every 2 days until 8 days. Cells at passages 5 to 7 were used. MLE and BMLE, 5-CQA, and

4-CQA (dissolved in distilled water) were added during the differentiation period. OIL RED O ASSAY Oil red O staining was performed to observe lipid droplets as previously described60.

Mature 3T3-L1 adipocytes were stained with Oil red O after 8 days of differentiation. Cells were fixed with 4% formaldehyde at room temperature for 1 h and washed with 60% isopropanol. Then,

the fixed cells were stained with filtered Oil red O solution in 60% isopropanol. Subsequently, Oil red O was removed and washed with distilled water. Then, the stained lipid droplets were

dissolved with 100% isopropanol, and the absorbance for quantification was measured at 520 nm using a microplate reader (Tecan Group Ltd., Männedorf, Switzerland). Lipid accumulation was

calculated as a ratio of [(Drug treated each well O.D value)/(Control group each well O.D value)] × 100. MTT ASSAY To examine the cytotoxicity of the substance in 3T3-L1 preadipocytes, an

MTT assay was performed. Cells were seeded in 96-well plates at a density of 2 × 104 cells/mL and incubated at 37 °C under a humidified atmosphere of 5% CO2. Cells were treated with various

concentrations of MLE, BMLE, 5-CQA, 4-CQA or 100 µg/mL digitonin. After 24 h, 5 mg/mL MTT reagent dissolved in serum-free DMEM was added to the wells and incubated for 2 h. After incubation,

dimethyl sulfoxide was added to dissolve the crystals formed. Then, the absorbance at 565 nm was measured using a microplate reader (Tecan Group Ltd.). Cytotoxicity was calculated as a

ratio of [(Drug treated each well O.D value)/(Control group each well O.D value)] × 100. WESTERN BLOT ANALYSIS After termination of the reaction, western blotting was performed to detect

protein levels. Proteins were extracted from cells or tissues using ice-cold RIPA buffer [50 mM Tris–HCl; pH 8.0, 150 mM NaCl; 1.0% NP-40 (Sigma); 2 mM EDTA (Sigma); 5 mM NaF (Sigma); 1 mM

phenylmethylsulfonyl fluoride (PMSF, Sigma); 1 mM sodium orthovanadate (Sigma); 0.5% sodium deoxycholate (Sigma) and 0.1% sodium dodecyl sulfate (SDS, Wako, Osaka, Japan)]. Denatured

proteins were separated by SDS–polyacrylamide gel electrophoresis (SDS–PAGE, 10–12.5%) and transferred to polyvinylidene fluoride membranes (ATTO Corp., Tokyo, Japan). Lysates were

quantified using the BCA protein Assay Kit (Pierce, Rockford, IL, USA). The membranes were blocked for 1 h with 5% BSA dissolved in TBS-T (10 mM Tris, 150 mM NaCl, and 0.1% Tween-20, pH 7.6)

at 4 °C. Then, they were incubated with the appropriate primary antibodies for 16 h at 4 °C and with specific secondary antibodies for 6 h at 4 °C. Protein bands were detected using

enhanced chemiluminescence reagent (ATTO Corp.), and band density was quantified with Image Lab software (Version 5.2.1, Bio–Rad, Hercules, CA, USA). ANIMALS AND DIET Five-week-old C57BL/6

mice (15–23 g) purchased from Orient Bio Inc. (Seongnam, Korea) were used as experimental animals in this study. Mice were acclimatized for one week in an environment controlled by

temperature (22 ± 2 °C), humidity (40 ± 5%), and light (07:00 am–19:00 pm/ h intervals). For the obese mouse model, mice were fed a normal (10% kcal from fat; Research Diets, Inc., New

Brunswick, NJ, USA) or high-fat diet (60% kcal fat, Research Diets, Inc.) for 8 weeks to induce an obese animal model. After 8 weeks, five groups (n = 6/group) were randomly divided into

five groups: normal diet (ND, control), high-fat diet (HFD), HFD + 600 mg/kg MLE (HFD + MLE 600), HFD + 600 mg/kg BMLE (HFD + BMLE 600), HFD + 40 mg/kg orlistat (HFD + Orli 40, positive

control for anti-obesity46). To calculate the caloric intake, the consumed food in grams was examined and multiplied by the calories per gram of the respective type of food (ND: 3.82 kcal/g;

HFD: 5.24 kcal/g). All animal experiments used in this study followed the guidelines of the Committee of Chungnam National University Laboratory Animal Ethics (CNU-00773) and The ARRIVE

(Animal Research: Reporting of in vivo Experiments) guidelines61. DRUG ADMINISTRATION AND DOSAGE INFORMATION Mice were administered MLE, BMLE and orlistat dissolved in solvent orally, with a

daily oral zonde needle. The daily dose of MLE (600 mg/kg) was determined by considering the doses of other studies (200–800 mg/kg)62,63,64,65, and the daily dose of BMLE was the same as

that of MLE. In addition, the daily dose of orlistat (40 mg/kg) was determined by referring to the doses used in other experiments46. TISSUE ISOLATION After feeding and drug administration,

the mice were deeply euthanized by isoflurane, and cervical dislocation was performed. The epididymal white adipose tissue (eWAT) and retroperitoneal white adipose tissue (rWAT) were

separated and weighed with an analytical electronic balance (Shimadzu Analytical Balance; Shimadzu Corporation, Kyoto, Japan). The fat index for each adipose tissue was expressed as the

weight (g) of the adipose tissue per 100 g of the body weight of the individual. IDENTIFICATION OF MAIN COMPOUNDS IN MLE AND BMLE To identify the major components of mulberry leaf (MLE and

BMLE), high-performance liquid chromatography-photo diode array detection-electrospray ionization mass spectrometry (HPLC–DAD-ESI–MS/MS) analysis was performed using the LCMS-8040 system

(Shimadzu, Kyoto, Japan)_._ The LCMS-8040 system, a triple quadruple tandem mass spectrometer, consisted of a CBM-20A communication bus module, an SPD-M20A photodiode array detector, two

LC-20AD pumps, a SIL-20A autosampler, and a CTO-20A column oven in positive and negative electrospray ionization (ESI) mode. Shimadzu Labsolutions software (Ver. 5.60 SP2, Kyoto, Japan) was

used as the operation program. HPLC–DAD-ESI–MS/MS analysis was performed to identify the main components in the samples using an Optimak C18 column (RSTech Co., Daejeon, Korea) with mobile

phases of 0.3% formic acid in water (A) and 0.3% formic acid in methanol (B), followed by a gradient elution of 5–15% B for 0–40 min and 15–19% B for 40–130 min. The main components were

monitored in the UV 190–400 nm (split 1.2 nm) range. LC–MS analysis was performed under the conditions of interface voltages of 4.5 kV and − 3.0 kV of the ESI interface. Another condition

was 15 L/min of a drying gas flow rate, 3 L/min of a nebulizing gas flow rate, 250 °C of a desolation line temperature and 400 °C of a heat block temperature at 0.5 mL/min of a flow rate.

QUANTITATION OF HPLC ANALYSIS To quantify the amounts of the main compounds in mulberry leaves (MLE and BMLE), HPLC analysis was performed using a Shimadzu LC-20A HPLC system (Shimadzu,

Kyoto, Japan) consisting of a CBM-20A communication bus module, an SPD-20A UV/VIS detector, two LC-20AD pumps, a SIL-20A autosampler and a CTO-20A column. It was operated by Shimadzu

Labsolutions software (Ver. 1.25 SP4, Kyoto, Japan). HPLC analysis was performed on an Optimapak C18 column (250 × 4.6 mm (ID), 5 μm, RSTech. Co, Daejeon, Korea) with a mobile phase of 0.3%

formic acid in water (A) and 0.3% formic acid in methanol (B) under the following gradient conditions: 5% ~ 20% B in 0 ~ 80 min and 20% ~ 61% B in 80 ~ 115 min, monitored at a wavelength of

UV 325 nm, column temperature of 25 °C and flow rate of 1.0 mL/min. Each standard and sample solution were injected to 10 µL using an autosampler. NILE RED STAINING Nile red staining was

used to measure intracellular triglycerides levels in 3T3-L1 adipocytes. Mature 3T3-L1 adipocytes were washed in PBS and treated with AdipoRed solution (Lonza, Basel. Switzerland) for 10 

min. Then, the cells were washed with PBS and absorbance was measured with a microplate reader (excitation = 485 nm, emission = 535 nm; Tecan group Ltd.). Images were taken of stained cells

(Olympus IX71 microscope). QUANTITATIVE PCR ASSAY Quantitative polymerase chain reaction (qPCR) was performed to determine mRNA levels. Total RNA was isolated using TRIzol reagent

(Invitrogen, Waltham, MA, USA) according to the manufacturer's instructions. Complementary DNA synthesis was performed with 1 µg of RNA using the AccuPower CycleScript RT premix kit

(Bioneer, Daedeok-gu, Daejeon, Korea). Real-time PCR amplification was performed using TOPreal™ qPCR Premix (Enzynomics, Yuseong-gu, Daejeon, Korea) on a 96-well optical plate. It was

detected with the CFX96 real-time detection system (Bio–Rad). Under the following conditions: 1 cycle at 95 °C for 15 min; 50 cycles of 95 °C for 15 s and 60 °C for 15 s and 72 °C for 30 s,

followed by 1 cycle of 65 °C to 95 °C every 0.5 °C for 1 s. In this study, only primer pairs leading to the synthesis of single fragments of appropriate size were used. The primer sets used

in this study are listed in Table 2. Relative gene expression levels were calculated using the 2 − ∆∆Ct method and normalized to _Actb_ expression levels. STATISTICAL ANALYSIS All data are

shown as the mean ± S.D. of 4–5 independent experiments. Statistical analyses were performed using GraphPad Prism software (Version 9, San Diego, CA, USA). The normality of the data

distributions was tested using the Shapiro–Wilk test. A two-sided, unpaired Student’s t-test was used to analyze the difference between two groups of data with normally distributed

variables. Differences among three or more groups were tested via one-way analysis of variance (ANOVA), followed by a post hoc analysis with Bonferroni’s test the _F_ value was statistically

significant (_P_ < 0.05) and there was no significant variance in homogeneity with the Bartlett’s test. Differences were considered statistically significant at _P_ < 0.05. ETHICS

APPROVAL AND CONSENT TO PARTICIPATE All animal experiments were approved by the Animal Experimentation Ethics Committee of Chungnam National University (Approval Number: CNU-00773), and the

animals were cared for in accordance with the dictates of the National Animal Welfare Law of Korea. DATA AVAILABILITY The datasets used and/or analyzed during the current study are available

from the corresponding author on reasonable request. ABBREVIATIONS * BMLE: Bioconverted mulberry leaf extract * C/EBP: CCAAT/enhancer binding protein * DAD: Diode array detection * EGCG:

(-)-epigallocatechin gallate * ESI-MS: Electrospray ionization mass spectrometry * eWAT: Epididymal white adipose tissue * HFD: High-fat diet * HPLC: High performance liquid chromatography *

MLE: Mulberry leaf extract * LC-MS: Liquid chromatography-mass spectrometry * ND: Normal diet * PPAR: Peroxisome proliferator-activated receptor * rWAT: Retroperitoneal white adipose tissue

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PubMed  Google Scholar  Download references FUNDING This study was supported by National Research Foundation of Korea (NRF) grants funded by the Korean government (No. 2019R1C1C1010698 to

J.H.H and 2021R1F1A1058923 to C.S.M). AUTHOR INFORMATION Author notes * These authors contributed equally: Joo-Hui Han and Hyung-Won Lee. AUTHORS AND AFFILIATIONS * Department of

Pharmacology, College of Pharmacy, Chungnam National University, Daejeon, 34134, Republic of Korea Joo-Hui Han, Hyung-Won Lee, Sang-Hyuk Jung & Chang-Seon Myung * College of Pharmacy,

Chungnam National University, Daejeon, 34134, Republic of Korea Chong Woon Cho, Tae Jeong Kim & Jong Seong Kang Authors * Joo-Hui Han View author publications You can also search for

this author inPubMed Google Scholar * Hyung-Won Lee View author publications You can also search for this author inPubMed Google Scholar * Sang-Hyuk Jung View author publications You can

also search for this author inPubMed Google Scholar * Chong Woon Cho View author publications You can also search for this author inPubMed Google Scholar * Tae Jeong Kim View author

publications You can also search for this author inPubMed Google Scholar * Jong Seong Kang View author publications You can also search for this author inPubMed Google Scholar * Chang-Seon

Myung View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS J.H.H. and H.W.L. designed and performed the experiments. C.W.C. performed the

experiments and analyzed the data. J.S.K analyzed the data. J.H.H., H.W.L., C.W.C. and C.S.M. wrote the manuscript. C.S.M. supervised this project. All authors read and approved the final

manuscript. CORRESPONDING AUTHOR Correspondence to Chang-Seon Myung. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION

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and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Han, JH., Lee, HW., Jung, SH. _et al._ The anti-obesity effect of mulberry leaf (_Mori Folium_) extracts was increased by bioconversion

with Pectinex. _Sci Rep_ 12, 20375 (2022). https://doi.org/10.1038/s41598-022-23856-9 Download citation * Received: 08 April 2022 * Accepted: 07 November 2022 * Published: 27 November 2022 *

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