nutrients
Article
The Mechanism of the Anti-Obesity Effects of a Standardized
Brassica juncea Extract in 3T3-L1 Preadipocytes and High-Fat
Diet-Induced Obese C57BL/6J Mice
June-Seok Lim 1 , Ji-Hyun Im 2 , Xionggao Han 2 , Xiao Men 2 , Geon Oh 2 , Xiaolu Fu 2 , Woonsang Hwang 3 ,
Sun-Il Choi 1,2, * and Ok-Hwan Lee 1,2, *
Citation: Lim, J.-S.; Im, J.-H.; Han, X.;
Men, X.; Oh, G.; Fu, X.; Hwang, W.;
Choi, S.-I.; Lee, O.-H. The Mechanism
of the Anti-Obesity Effects of a
Standardized Brassica juncea Extract in
3T3-L1 Preadipocytes and High-Fat
Diet-Induced Obese C57BL/6J Mice.
Nutrients 2024, 16, 846. https://
doi.org/10.3390/nu16060846
Academic Editor: Roberto Iacone
Received: 24 January 2024
Revised: 8 March 2024
Accepted: 12 March 2024
Published: 15 March 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Nutrients 2024, 16, 846. https://doi.org/10.3390/nu16060846
1 Department of Food Science and Biotechnology, Kangwon National University,
Chuncheon 24341, Republic of Korea; dlawnstjr725@naver.com
2 Department of Food Biotechnology and Environmental Science, Kangwon National University,
Chuncheon 24341, Republic of Korea; ijh108020@gmail.com (J.-H.I.); xionggao414@hotmail.com (X.H.);
menxiaodonglei@naver.com (X.M.); dhrjs1@gmail.com (G.O.); fuxiaolu2019@gmail.com (X.F.)
3 STR Biotech Co., Ltd., Chuncheon 24232, Republic of Korea; hwang931@strbiotech.co.kr
* Correspondence: csi23@kangwon.ac.kr (S.-I.C.); loh99@kangwon.ac.kr (O.-H.L.); Tel.: +82-33-250-6456 (S.-I.C);
+82-33-250-6454 (O.-H.L.); Fax: +82-33-259-5561 (S.-I.C. & O.-H.L.)
Abstract: Obesity is a global health concern. Recent research has suggested that the development
of anti-obesity ingredients and functional foods should focus on natural products without side
effects. We examined the effectiveness and underlying mechanisms of Brassica juncea extract (BJE)
in combating obesity via experiments conducted in both in vitro and in vivo obesity models. In
in vitro experiments conducted in a controlled environment, the application of BJE demonstrated
the ability to suppress the accumulation of lipids induced by MDI in 3T3-L1 adipocytes. Addi-
tionally, it downregulated adipogenic-related proteins peroxisome proliferator-activated receptor-γ
(PPAR-γ), CCAAT/enhancer-binding protein-α (C/EBP-α), adipocyte protein 2 (aP2), and lipid
synthesis-related protein acetyl-CoA carboxylase (ACC). It also upregulated the heat generation
protein peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) and fatty acid
oxidation protein carnitine palmitoyltransferase-1 (CPT-1). The oral administration of BJE decreased
body weight, alleviated liver damage, and inhibited the accumulation of lipids in mice with diet-
induced obesity resulting from a high-fat diet. The inhibition of lipid accumulation by BJE in vivo was
associated with a decreased expression of adipogenic and lipid synthesis proteins and an increased
expression of heat generation and fatty acid oxidation proteins. BJE administration improved obesity
by decreasing adipogenesis and activating heat generation and fatty acid oxidation in 3T3-L1 cells
and in HFD-induced obese C57BL/6J mice. These results suggest that BJE shows potential as a
natural method for preventing metabolic diseases associated with obesity.
Keywords: standardized Brassica juncea; 3T3-L1; C57BL/6J; anti-obesity; mechanism
1. Introduction
Obesity is characterized by the abnormal and excessive buildup of fat in adipose
tissue, leading to an unhealthy condition, as outlined by the World Health Organization
(WHO) [1,2]. The body mass index (BMI) of an individual is calculated by dividing their
weight in kilograms by the square of their height in meters. A BMI score that falls within
the range of 25 to 30 is categorized as being overweight or obese [1–3]. Based on the
findings of the WHO’s report on obesity, it has been indicated that there are approximately
1.9 billion adults over the age of 18 who are classified as overweight, with an additional
600 million falling into the category of obesity. These data underscore the significant global
prevalence of obesity as a pressing public health concern [4,5]. Based on data from the
National Health and Nutrition Examination Survey conducted in Korea, it was found that
https://www.mdpi.com/journal/nutrients
Nutrients 2024, 16, 846 2 of 13

the incidence of obesity in individuals aged 19 or older was reported as an estimate of

approximately 26% for women and 40% for men. By 2030, its prevalence is anticipated to
reach approximately 37% in women and 62% in men [6].
The primary cause of obesity is excessive accumulation of body fat resulting from
caloric intake over the body’s energy consumption [7]. Adipocytes function as endocrine
organs by regulating several metabolic processes that contribute to physiological regulation.
These cells release a range of hormones, including leptin and adiponectin, which play essen-
tial roles in the maintenance of metabolic homeostasis [8]. The excessive buildup of lipids
within adipocytes results in impaired functionality, including issues like mitochondrial dys-
function and intracellular endoplasmic reticulum stress. This can lead to insulin resistance,
inflammatory reactions, and an increased susceptibility to complications associated with
obesity, such as cardiovascular disease, hypertension, fatty liver, and cancer [9,10].
Lipid accumulation occurs due to increased adipocyte differentiation and triglyceride
production during adipogenesis [11]. The accumulation of lipids regulates the dimen-
sions of lipid droplets via the augmentation of cell count or proliferation [12]. The 3T3-L1
cell line, isolated from a mouse embryo, is frequently used in obesity-related in vitro
studies [13]. This cell line can differentiate into adipocytes when treated with 3-isobutyl-
1-methylxanthine (IBMX), dexamethasone (Dex), and insulin, which stimulate complex
signaling pathways and various transcription factors [14,15]. The various transcription
factors include CCAAT/enhancer-binding protein α (C/EBP-α), nuclear receptor peroxi-
some proliferator-activated receptor-γ (PPAR-γ), and adipocyte fatty acid-binding protein
(aP2) [16]. In the advanced phase of cellular differentiation, there is a rise in the con-
centrations of C/EBP-α in conjunction with PPAR-γ, resulting in gene stimulation that
promotes adipogenesis, such as aP2 [17,18]. These factors show significantly increased
expression levels in differentiated adipocytes [19]. Controlling the expression of factors
linked to the differentiation of adipocytes is an important approach for preventing and
treating obesity [20]. Therefore, therapeutic agents demonstrating efficacy in promoting
adipocyte differentiation and inhibiting adipogenesis have been developed. Currently, the
drugs targeting obesity include orlistat, topiramate, sibutramine, rimonabant, and phenyl-
propanolamine. However, these drugs come with adverse reactions such as sleeplessness,
loss of appetite, digestive issues, elevated blood pressure, and heart disease [21]. To address
this concern, active research is dedicated to creating treatments for obesity by utilizing
safer natural substances or phytochemicals that have minimal adverse reactions [22].
Leaf mustard (Brassica juncea) is a cruciferous vegatable that is commonly grown in
Korea and Japan and has a history of being utilized in kimchi as an ingredient in Ko-
rea [23]. The nutritional components of Brassica juncea are rich in various free sugars,
fatty acids, amino acids, vitamins, and minerals, and it has been documented to exhibit
exceptional antioxidant activity in cell-based studies [24]. Moreover, glucosinolates and
isothiocyanate compounds found in Brassica juncea, such as allyl isothiocyanate and sini-
grin, are recognized for their diverse health benefits and functional properties, including
their potential anti-cancer effects [25–27]. Sinigrin has been reported to demonstrate anti-
cancer, anti-inflammatory, antibacterial, antifungal, and antioxidant effects. Our previous
study reported that Brassica juncea extract (BJE), extracted under optimized sinigrin ex-
traction conditions, has anti-obesity effects in vitro [28–30]. Furthermore, there have been
reports indicating that BJE exhibits anti-obesity properties in living organisms; however,
research on the specific underlying mechanism remains limited [31]. Based on previous
reports, we hypothesized that BJE induces anti-obesity effects via the regulation of adipo-
genesis, lipid synthesis, heat generation, and fatty acid oxidation. In accordance with our
hypothesis, we investigated the mechanism of how BJE exerts its anti-obesity effect in vivo
and demonstrated its value as a natural product with anti-obesity efficacy.
Nutrients 2024, 16, 846 3 of 13

2. Materials and Methods

2.1. Chemicals and Standards
Bovine serum (BS), penicillin–streptomycin (P/S), trypsin–ethylenediaminetetraacetic
acid (trypsin-EDTA), Dulbecco’s modified Eagle’s medium (DMEM) with high glucose,
phosphate-buffered saline (PBS), and fetal bovine serum (FBS) were purchased from Gibco
(Gaithersburg, MD, USA). The primary and secondary antibodies specific for peroxisome
proliferator-activated receptor gamma coactivator-1α (PGC-1α), PPAR-γ, acetyl-CoA car-
boxylase (ACC), C/EBP-α, phospho-ACC (p-ACC), aP2, and carnitine palmitoyltransferase-
1 (CPT-1) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA), and Cell
Signaling Technology, Inc. (Danvers, MA, USA). Insulin, Dex, IBMX, and sinigrin were
purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).

2.2. Standardized BJE Preparation

Brassica juncea was provided by STR Biotech Co., Ltd. (Chuncheon, Republic of Korea).
In prior research, we established a uniform extraction technique by conducting methodical
experiments that focused on variables such as extraction duration, type of solvent used,
and extraction temperature. Briefly, we adopted a standardized experimental method using
60% ethanol at 70 ◦ C as the extraction solvent, and the extraction time was 3 h [30]. The
materials utilized in the experiment were subjected to a washing procedure to eliminate
any unwanted components, followed by freeze-drying using a freeze dryer (Ilshin, Seoul,
Republic of Korea). The samples were subsequently pulverized and homogenized in
preparation for their utilization in the experiment. Ethanol (60%) was added to 40 kg freeze-
dried Brassica juncea. After subjecting Brassica juncea to reflux extraction at a temperature of
70 ◦ C for a duration of 3 h, the resulting extraction solution underwent filtration using a
0.2 µm filter paper. The extract was evaporated under reduced pressure at 50 ◦ C using a
vacuum evaporator and then freeze-dried to produce BJE powder. The prepared sample
was stored at a temperature of −20 ◦ C and subsequently utilized following suspension
in DMSO.
The concentration of sinigrin in BJE was quantified via High-Performance Liquid
Chromatography (HPLC). The instruments used for analysis were a Shimadzu LC system
(LC-40B XR, Shimadzu Co., Ltd., Kyoto, Japan) and a photodiode array detector (SPD-M40,
Shimadzu Co., Ltd., Kyoto, Japan). Table 1 lists the conditions used in this analysis. The
column used for analysis was CAPCELL PAK C18 UG120 (4.6 × 250 mm, 5.0 µm; Waters
Corporation, Milford, MA, USA).

Table 1. HPLC condition for sinigrin analysis.

Instrument Conditions
Column CAPCELL PAK C18 , UG120 (5.0 µm, 4.6 mm × 250 mm)
Column temp. 30 ◦ C
Mobile phase (isocratic) Isocratic HPLC water containing 0.1 M ammonium sulfate
Detector PDA detector (228 nm)
Flow rate 1.0 mL/min
Injection volume 40 µL
Run time 30 min

2.3. Cell Culture and XTT Assay

3T3-L1 pre-adipocyte cells (CL-173, ATCC, Manassas, VA, USA) were seeded at a
concentration of 2 × 106 cells/mL per plate according to the purpose of the experiment.
The cells were then incubated in DMEM supplemented with 1% P/S and 10% BS until
reaching full confluence. After 2 d of reaching full confluence, adipocyte differentiation was
induced by a medium containing 1% P/S, 10% FBS, and a differentiation-inducing cocktail
consisting of 0.5 mM IBMX, 1 µM Dex, and 1 µg/mL insulin. The medium for differentiation
was replaced every 2 d containing 1 µg/mL insulin. BJE and Garcinia cambogia extracts
(Gar) were added to the cells 2 d after reaching 100% confluence and treated continuously
Nutrients 2024, 16, 846 4 of 13

during differentiation. The total differentiation period was 6 d. 3T3-L1 adipocytes were
cultured in a 96-well plate, and after 6 d of differentiation, a solution combining 1 mL of
2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reagent
with 20 µL of phenazine methosulfate reagent was prepared. This solution was then added
to each well at a concentration of 20% of the medium volume.

2.4. Oil Red O Staining

Following a 6 d differentiation period, the culture medium was removed. 3T3-L1 cells
that had undergone differentiation were rinsed two times with PBS and then fixed at room
temperature for 1 h using 500 µL of a 10% formalin. After removing 10% formalin, the
cells underwent a rinsing process with 60% isopropanol and subsequent air-drying at room
temperature. After drying, the lipids accumulated in the cells were subjected to staining
using a pre-prepared Oil Red O (ORO) working solution, with a ratio of ORO to DW of 6:4,
for a duration of 1 h. The cells were rinsed thrice with DW and subsequently dried. ORO
bound to lipids was separated by elution with 100% isopropanol, subsequently transferred
to a 96-well plate, and absorbance was measured at 490 nm utilizing a microplate reader.

2.5. Animal Experiment

Male C57BL/6J mice, aged four weeks, were procured from DBL Inc. (Incheon,
Republic of Korea), allowed to adjust to a controlled environment with specific conditions
(temperature, 24 ± 5 ◦ C; relative humidity, 55 ± 5%), and provided with ad libitum access
to water and food within a 12/12 h light/dark cycle for a period of 1 week. Via a random
selection process, mice were allocated into five groups: group 1, the control group that
consumed 10% kcal normal-fat diet (CON); group 2, 60% kcal high-fat diet (HFD; D12492;
Research Diet, Inc., New Brunswick, NJ, USA); group 3, HFD + Gar 50 mg/kg/day;
group 4, HFD + BJE 400 mg/kg/day; and group 5, HFD + BJE 800 mg/kg/day. BJE and
Gar dissolved in DW were administered orally once a day for 6 weeks. Food intake was
evaluated by determining the difference between the amount of food given and the amount
left over, and the food efficiency ratio (FER) was determined by dividing the weight gain
(measured in grams) by the amount of food intake (measured in grams). Following a 12 h
fasting period, mice were anesthetized with isoflurane (Hwaseong, Republic of Korea) at
the conclusion of the breeding period. Following this, samples of tissue and blood were
gathered for examination. Subsequently, samples of tissue and blood were collected for
analysis. Blood samples were obtained from the orbital venous plexus of each mouse.
The epididymal adipose tissue (eWAT), liver, spleen, and kidneys were dissected and
rinsed with normal saline, and their weights were recorded. This study was approved by
the Institutional Animal Care and Use Committee of the Kangwon National University
(approval number: KW-221104-2).

2.6. Biochemical Analysis

Blood samples were collected in serum separation tubes (BD Microtainer® tubes,
365967, BD Biosciences, Franklin Lakes, NJ, USA) and left to coagulate at room temperature
for a duration of 30 min. The serum was isolated via centrifugation at a speed of 12,000× g
and a temperature of 4 ◦ C for a duration of 10 min. The levels of alanine aminotransferase
(ALT), aspartate aminotransferase (AST), total cholesterol (TC), and total triglyceride (TG)
in serum were assessed utilizing an automated biochemical analyzer (Hitachi-720, Hitachi
Medical, Tokyo, Japan).

2.7. Histological Analysis

The eWAT was partially excised and fixed in 10% paraformaldehyde. The fixed
eWAT was sequentially dehydrated with a graded series of ethanol solutions using a tissue
processor (TP1020; Leica Biosystems, Nussloch, Germany) and paraffin (Rotary Microtome
RM2255; Leica Biosystems). Paraffin blocks were cut to a thickness of 6 µm; slides were
 2.7. Histological Analysis
The eWAT was partially excised and fixed in 10% paraformaldehyde. The fixed eWAT
was sequentially dehydrated with a graded series of ethanol solutions using a tissue pro-
cessor (TP1020; Leica Biosystems, Nussloch, Germany) and paraffin (Rotary Microtome
Nutrients 2024, 16, 846 RM2255; Leica Biosystems). Paraffin blocks were cut to a thickness of 6 µm; slides 5were
of 13
prepared and stained with hematoxylin and eosin (H&E). The stained adipose tissue was
observed using an optical microscope (ECLIPSE Ni-U; Nikon, Melville, NY, USA).
prepared and stained with hematoxylin and eosin (H&E). The stained adipose tissue was
observed
2.8. using
Western Blot an optical microscope (ECLIPSE Ni-U; Nikon, Melville, NY, USA).
Analysis
eWAT were dissolved in lysis buffer containing 0.1% SDS, 150 mM sodium chloride,
2.8. Western Blot Analysis
1 mM pepstatin, 50 mM Tris-HCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, and 1
eWAT were dissolved influoride,
mM phenylmethanesulfonyl lysis buffer
andcontaining
reacted at 0.1% SDS,
4 °C for 30150 mM
min. Thesodium chloride,
cell lysates un-
1 mM pepstatin, 50 mM Tris-HCl, 1% Nonidet P-40,
derwent centrifugation at 12,000× g and 4 °C for 20 0.25% sodium deoxycholate, and 1
min. The protein concentrations of the mM
phenylmethanesulfonyl
resultant fluoride,
cell lysates were and utilizing
quantified 4 ◦ Bradford
reacted atthe C for 30 min. The assay
protein cell lysates underwent
kit (Bio-Rad La-
centrifugation at 12,000× g and 4 ◦ C for 20 min. The protein concentrations of the resultant
boratories, Inc., Hercules, CA, USA). Equivalent amounts of proteins were separated using
cell lysates were quantified utilizing the Bradford protein assay kit (Bio-Rad Laborato-
a 10% sodium dodecyl sulfate–polyacrylamide gel (SDS–PAGE) and electrotransferred
ries, Inc., Hercules, CA, USA). Equivalent amounts of proteins were separated using a
onto a polyvinylidene difluoride membrane (0.2 µM Immun-Blot PVDF membrane; Bio-
10% sodium dodecyl sulfate–polyacrylamide gel (SDS–PAGE) and electrotransferred onto
Rad Laboratories, Inc., Hercules, CA, USA). Tris-buffered saline containing 5% bovine se-
a polyvinylidene difluoride membrane (0.2 µM Immun-Blot PVDF membrane; Bio-Rad
rum albumin was used to inhibit nonspecific binding. The target proteins, after treatment
Laboratories, Inc., Hercules, CA, USA). Tris-buffered saline containing 5% bovine serum
with primary and secondary antibodies, were detected using Supersignal West Pico chem-
albumin was used to inhibit nonspecific binding. The target proteins, after treatment with
iluminescence (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The Western blot
primary and secondary antibodies, were detected using Supersignal West Pico chemilu-
bands were visualized with the ChemiDoc image software version 5.2.1 (Bio-Rad Labora-
minescence (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The Western blot bands
tories, Inc., Hercules, CA, USA).
were visualized with the ChemiDoc image software version 5.2.1 (Bio-Rad Laboratories,
Inc., Hercules, CA, USA).
2.9. Statistical Analysis
2.9. Statistical
The resultsAnalysis
are expressed as mean ± standard deviation (SD). All statistical analyses
wereTheperformed using
results are SPSS software
expressed as mean(version 24.0;deviation
± standard SPSS Inc., Chicago,
(SD). IL, USA).
All statistical Body
analyses
weight, food efficiency ratio (FER), organ weights, and biochemical parameters
were performed using SPSS software (version 24.0; SPSS Inc., Chicago, IL, USA). Body were ver-
ified using Dunnett’s t-test for statistical significance, and cell viability, lipid
weight, food efficiency ratio (FER), organ weights, and biochemical parameters were veri- accumula-
tion, and Western
fied using Dunnett’s blot results
t-test were verified
for statistical using Duncan’s
significance, and cellmultiple
viability,range test for statis-
lipid accumulation,
tical significance.
and Western blot p-values less than
results were 0.05using
verified were considered staticallyrange
Duncan’s multiple significant.
test for statistical
significance. p-values less than 0.05 were considered statically significant.
3. Results
3. Results
3.1. Sinigrin Content in Standardized BJE
3.1. Sinigrin Content in Standardized BJE
The HPLC chromatogram of BJE is shown in Figure 1. Sinigrin was detected at a re-
The
tention HPLC
time chromatogram
of 7.4 of BJE iscontent
min, and the sinigrin shownininBJE Figure 1. Sinigrinaswas
was quantified 18.0detected
mg/g. at a
retention time of 7.4 min, and the sinigrin content in BJE was quantified as 18.0 mg/g.

HPLC chromatogram
Figure 1. HPLC chromatogram of BJE. The monitoring wavelength for sinigrin was set at 228 nm.

3.2. Effects of BJE on the Viability and Lipid Accumulation of 3T3-L1 Adipocytes
3.2. Effects of BJE on the Viability and Lipid Accumulation of 3T3-L1 Adipocytes
The effect of BJE on cell viability during the induction of 3T3-L1 preadipocyte differ-
The effect of BJE on cell viability during the induction of 3T3-L1 preadipocyte differ-
entiation is shown in Figure 2A. The cells in the BJE treatment group (100–800 µg/mL)
entiation is shown in Figure 2A. The cells in the BJE treatment group (100–800 µg/mL) did
did not exhibit a statistically significant variance in comparison to the MDI group, indicat-
not exhibit a statistically significant variance in comparison to the MDI group, indicating
ing the absence of cytotoxic effects. Hence, this research aimed to assess the anti-obesity
the absence of cytotoxic effects. Hence, this research aimed to assess the anti-obesity
properties linked to the suppression of adipocyte differentiation by administering BJE at
concentrations varying from 100 to 800 µg/mL. The impact of BJE on lipid accumulation
in adipocytes was assessed via the application of Oil Red O staining. The effects of BJE
treatment on lipid accumulation were examined by promoting the differentiation of 3T3-L1
cells over a 6 d duration (Figure 2B). A significant decrease in lipid accumulation was
noted solely at BJE concentrations of 400 and 800 µg/mL, while no notable impact was de-
 properties linked to the suppression of adipocyte differentiation by administering BJE at
concentrations varying from 100 to 800 µg/mL. The impact of BJE on lipid accumulation
in adipocytes was assessed via the application of Oil Red O staining. The effects of BJE
treatment on lipid accumulation were examined by promoting the differentiation of 3T3-
Nutrients 2024, 16, 846 L1 cells over a 6 d duration (Figure 2B). A significant decrease in lipid accumulation 6 of 13 was
noted solely at BJE concentrations of 400 and 800 µg/mL, while no notable impact was
detected at concentrations of 100 or 200 µg/mL. Additionally, lipid accumulation signifi-
tected decreased
cantly at concentrations
as theofBJE
100concentration
or 200 µg/mL. Additionally,
increased fromlipid400
accumulation significantly
to 800 µg/mL.
decreased as the BJE concentration increased from 400 to 800 µg/mL.

120
a 120
ab ab ab
b b
a a

Relative lipid accumulation (%)

100 a
Relative Cell viability (%)

100
c b
80
80
c
60
60

40 40

20 20

0 0
L

I
L

L
L

L
L
L

L
D
m
I

m
m

m
m
D

m
m
m
m
M
g/

g/
g/
M

g/
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g/

g/
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g/
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μ

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(A) (B)
Figure
Figure2. 2. Effect of BJE
Effect of BJEon
oncell
cellviability
viability
(A)(A) and
and lipid
lipid accumulation
accumulation (B)
(B) on on different
different 3T3-L13T3-L1 adipocytes.
adipocytes.
All
All results are presented as the mean ± SD of three independent in triplicate. Bars with differentdifferent
results are presented as the mean ± SD of three independent in triplicate. Bars with
letters
lettersindicate significantdifferences
indicate significant differences at pat<p0.05
< 0.05 using
using Duncan’s
Duncan’s multiple
multiple range range
test. test.

3.3.Effect
3.3. Effect of
of BJE
BJE on
onthe
theRegulation
Regulationof of
Protein Expression
Protein Associated
Expression with Adipogenesis,
Associated Lipid Lipid
with Adipogenesis,
Synthesis, Heat Generation, and Fatty Acid Oxidation in 3T3-L1 Adipocytes
Synthesis, Heat Generation, and Fatty Acid Oxidation in 3T3-L1 Adipocytes
This study evaluated alterations in the protein levels of important genes related to the
Thisofstudy
process evaluated
adipogenesis, alterations
including PPAR-γ,in the proteinand
C/EBP-α, levels
aP2, of important
to clarify genes related to
the mechanism
the process
through of adipogenesis,
which BJE inhibits lipidincluding PPAR-γ,
accumulation C/EBP-α,
(Figure and aP2, to clarify
3A–C). Consequently, within the
cellsmecha-
nism through
exposed to BJE,which BJE of
the levels inhibits
C/EBP-α,lipid accumulation
PPAR-γ, (Figure 3A–C).
and aP2 expression Consequently,
exhibited a significant within
decrease
cells withto
exposed escalating
BJE, theconcentrations.
levels of C/EBP-α, To examine
PPAR-γ, theand
impact
aP2 of BJE on theexhibited
expression protein a sig-
expression
nificant associated
decrease withwith lipid synthesis,
escalating the levels
concentrations. To of p-ACC/ACC
examine expression
the impact of BJEwere
on the pro-
assessed (Figure 3D). In the present study, BJE effectively inhibited lipid synthesis by
tein expression associated with lipid synthesis, the levels of p-ACC/ACC expression were
increasing p-ACC/ACC expression. In order to examine the influence of BJE on the protein
assessed (Figure 3D). In the present study, BJE effectively inhibited lipid synthesis by in-
expression related to the process of lipolysis, the concentrations of CPT-1 and PGC-1α
creasing p-ACC/ACC
were evaluated (Figureexpression.
3E,F). In theIn order to examine
experimental groups the influence
treated of BJE
with BJE, onwas
there theaprotein
expression related
notable increase to levels
in the the process of lipolysis,
of proteins associated the
withconcentrations ofwith
the lipolytic effect CPT-1 and PGC-1α
increasing
were evaluatedin(Figure
concentrations, contrast3E,F).
to the In
MDIthegroup.
experimental groups treated with BJE, there was a
notable increase in the levels of proteins associated with the lipolytic effect with increasing
3.4. Effect of BJE on
concentrations, in Body Weight,
contrast FoodMDI
to the Efficiency,
group.Adipose Tissue Mass, Organ Mass, and Adipose
Tissue Size in HFD-Induced Obese C57BL/6J Mice
To examine the potential anti-obesity properties of BJE, mice with diet-induced obesity
were treated with either 50 mg/kg/day of Gar (HFD + Gar 50 mg/kg/day), 400 mg/kg/day
of BJE (HFD + BJE 400 mg/kg/day), or 800 mg/kg/day of BJE (HFD + 800 mg/kg/day)
over a period of 6 weeks. Body weight assessments were conducted weekly over the course
of the study period (Figure 4A). As anticipated, the consumption of HFD led to obesity in
C57BL/6J mice. The group that consumed HFD showed notable elevations in body weight,
FER, and mass of eWAT in comparison to the CON group. However, the body weight, FER,
and eWAT mass of the BJE group, who received the oral administration of BJE, exhibited a
significant decrease with increasing concentrations of BJE, in contrast to the HFD group.
Furthermore, histological analysis revealed a significant increase in the dimensions of
adipocytes in the HFD group. It was observed that the alterations in adipocyte dimensions
induced by the HFD were effectively reversed by the administration of BJE (Figure 4E).
No significant differences in organ mass were detected across the experimental groups
(Figure 4D).
Nutrients 2024, 16, 846 Nutrients 2024, 16, x FOR PEER REVIEW 7 of 137 of 15

PPAR-γ
C/EBP-α

β-actin β-actin

Relative protein expression of C/EBP-α

120 120
a

Relative protein expression of PPARγ

a
100 100

80 80

b b
60 60
c
bc
40 40
d c

20 20

0 0

L
L
I
I

m
L

m
L

L
D

g/

g/
g/
M

0μ
/

0μ
g/

g/
μg

0μ

0μ

10

80
40
10

ar
40

80

E
E
ar

BJ
E

BJ
G

BJ

(A) BJ (B)
p-ACC
aP2
ACC
β-actin β-actin

Relative protein expression of p-ACC/ACC

120 400
Relative protein expression of aP2

a
100
a

300
80 b b b

60
200
c
40
c
d
100
20

0
L

L
L
I

0
D

m
m
M

g/

g/

L
g/

L
I
0μ

0μ
μ

m
D

m
/
10

g/
M

μg

g/
40

80

0μ
ar

10
E

0
G

40
BJ

BJ

80
ar

E
G

BJ

BJ

(C) (D)

PGC-1α CPT-1

β-actin β-actin
250
Relative protein expression of PGC-1α

140 a
Relative protein expression of CPT-1

ab b
a 120
200 c
b
100
c
150
80

d
100 60

40
50
20

0 0
L
L

L
L
L

L
N

I
m
m

m
m
m

m
D
O

g/
g/

g/
M

g/
g/

g/
C

0μ
μ

0μ
0μ
μ

μ
10

10
40

80
40
80
ar

ar
E

E
E
E
G

BJ

BJ
BJ
BJ

(E) (F)

Figure 3. Effect of BJE on the expression of proteins related to adipogenic, lipid synthesis, fatty acid
oxidation, and heat generation in 3T3-L1. PPAR-γ (A). C/EBP-α (B). aP2 (C). p-ACC/ACC (D). PGC-
1α (E). CPT-1 (F). All results are presented as the mean ± SD of three independent in triplicate. Bars
with different letters indicate significant differences at p < 0.05 using Duncan’s multiple range test.
 in body weight, FER, and mass of eWAT in comparison to the CON group. However, the
body weight, FER, and eWAT mass of the BJE group, who received the oral administration
of BJE, exhibited a significant decrease with increasing concentrations of BJE, in contrast
to the HFD group. Furthermore, histological analysis revealed a significant increase in the
dimensions of adipocytes in the HFD group. It was observed that the alterations in adipo-
Nutrients 2024, 16, 846 cyte dimensions induced by the HFD were effectively reversed by the administration of
8 of 13
BJE (Figure 4E). No significant differences in organ mass were detected across the experi-
mental groups (Figure 4D).

CON
40 HFD
CON
16 HFD + Gar 50 mg/kg/day
38 HFD
### HFD + BJE 400 mg/kg/day

Food efficiency ratio (FER, %)

HFD + Gar 50 mg/kg/day
36 ### 14 HFD + BJE 800 mg/kg/day
HFD + BJE 400 mg/kg/day
34 HFD + BJE 800 mg/kg/day 12

Body weight (g)

32
*
* 10
30
* **
28 8
*** ***
26 6

24
4
22
2
20

0
0 3 6 9 12 15 18 21 24 27 30 33 36 39

Days
(A) (B)
CON
2500 HFD 1200 CON
HFD + Gar 50 mg/kg/day HFD
###
HFD + BJE 400 mg/kg/day HFD + Gar 50 mg/kg/day
1000
2000 HFD + BJE 800 mg/kg/day HFD + BJE 400 mg/kg/day
HFD + BJE 800 mg/kg/day

Organ mass (mg)

eWAT mass (mg)

800
1500 *
** 600

1000
400

500
200

0 0
Nutrients 2024, 16, x FOR PEER REVIEW 9 of 15
Liver Kidney Spleen

(C) (D)

(E)

Figure Figure 4. Effect of BJE on body weight, food efficiency, adipose tissue mass, organ mass, and adipose
4. Effect of BJE on body weight, food efficiency, adipose tissue mass, organ mass, and
tissue size in HFD-induced obese C57BL/6J mice. eWAT were subjected to staining with H&E and
adiposesubsequently
tissue sizeexamined
in HFD-induced obeseChanges
using a microscope. C57BL/6J mice.
in total body eWAT were
weight (A). Foodsubjected to staining with
efficiency ratio
(B). eWAT weight (C). Other tissue weight (D). Histology of the eWAT (E).
H&E and subsequently examined using a microscope. Changes in total body weight (A). Food All values are expressed
as the mean ± SD, and statistical analyses were performed using Dunnett’s t-test. ### p < 0.001 vs.
efficiency ratio (B). eWAT weight (C). Other tissue weight (D). Histology of the eWAT (E). All values
Control group; * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. HFD group.
are expressed as the mean ± SD, and statistical analyses were performed using Dunnett’s t-test.
### p <3.5. Effect
0.001 vs.ofControl
BJE on Serum Biochemical
group; Indexes
* p < 0.05, in 0.01,
** p < HFD-Induced
and *** Obese C57BL/6J
p < 0.001 Mice group.
vs. HFD
Blood samples were obtained from all mice, followed by the separation of serum for
3.5. Effect of BJE on
the purpose Serum Biochemical
of observing biochemical Indexes inwithin
alterations HFD-Induced Obese2).
the serum (Table C57BL/6J Mice
To examine
the impact of BJE on fatty liver disease, changes in serum concentrations of ALT and AST
Blood samples were obtained from all mice, followed by the separation of serum for
were assessed. Elevated concentrations of ALT and AST were noted in the HFD group in
the purpose of observing
comparison to the CONbiochemical
group. However, alterations within the
a notable reduction serum
in the (Table 2).
concentrations To examine the
of both
impactALTof BJE
and AST was observed in all BJE groups compared to the HFD group. To assess and
on fatty liver disease, changes in serum concentrations of ALT al- AST were
terations in serum lipid levels resulting from BJE treatment, TC and TG
assessed. Elevated concentrations of ALT and AST were noted in the HFD group in compar-concentrations
ison towere evaluated.
the CON The HFD
group. group exhibited
However, a notable a significant
reduction increase
in theinconcentrations
TC and TG concentra-
of both ALT and
tions in comparison to the CON group. Conversely, the BJE group demonstrated a signif-
AST was observed in all BJE groups compared to the HFD group. To
icant reduction in TC and TG concentrations relative to the HFD group with increasing
assess alterations in
serumconcentrations.
lipid levels resulting from BJE treatment, TC and TG concentrations were evaluated.
The HFD group exhibited a significant increase in TC and TG concentrations in comparison
to the Table
CON2. group.
Effects of BJE on serum biochemistry in HFD-induced obese C57BL/6J mice.
Conversely, the BJE group demonstrated a significant reduction in TC
and TG concentrations relative to the HFD Groups group with increasing concentrations.
Parameters HFD + Gar 50 HFD + BJE 400 HFD + BJE 800
CON HFD
mg/kg/day mg/kg/day mg/kg/day
ALT (U/L) 32.00 ± 5.24 46.00 ± 12.36 38.00 ± 8.02 30.00 ± 5.82 * 29.00 ± 1.23 *
AST (U/L) 117.00 ± 20.37 243.00 ± 74.80 # 134.00 ± 64.60 126.00 ± 58.65 * 110.00 ± 27.00 *
TC (mg/dL) 106.00 ± 4.53 161.00 ± 9.66 ### 173.00 ± 11.06 163.00 ± 12.16 142.00 ± 5.64 *
TG (mg/dL) 40.00 ± 7.65 86.00 ± 5.81 ### 64.00 ± 8.71 ** 71.00 ± 9.93 57.00 ± 7.86 ***
All values are expressed as the mean ± SD, and statistical analyses were performed using Dunnett’s
t-test. # p < 0.05, ### p < 0.001 vs. Control group; * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. HFD group.
Nutrients 2024, 16, 846 9 of 13

Table 2. Effects of BJE on serum biochemistry in HFD-induced obese C57BL/6J mice.

Groups
Parameters HFD + Gar HFD + BJE HFD + BJE
CON HFD
50 mg/kg/day 400 mg/kg/day 800 mg/kg/day
ALT (U/L) 32.00 ± 5.24 46.00 ± 12.36 38.00 ± 8.02 30.00 ± 5.82 * 29.00 ± 1.23 *
AST (U/L) 117.00 ± 20.37 243.00 ± 74.80 # 134.00 ± 64.60 126.00 ± 58.65 * 110.00 ± 27.00 *
TC (mg/dL) 106.00 ± 4.53 161.00 ± 9.66 ### 173.00 ± 11.06 163.00 ± 12.16 142.00 ± 5.64 *
TG (mg/dL) 40.00 ± 7.65 86.00 ± 5.81 ### 64.00 ± 8.71 ** 71.00 ± 9.93 57.00 ± 7.86 ***
All values are expressed as the mean ± SD, and statistical analyses were performed using Dunnett’s t-test.
# p < 0.05, ### p < 0.001 vs. Control group; * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. HFD group. ALT: alanine
aminotransferase, AST: aspartate aminotransferase, TC: total cholesterol, TG: total triglyceride.

3.6. The Effect of BJE on the Regulation of Protein Expression Associated with Adipogenesis, Lipid
Synthesis, Heat Generation, and Fatty Acid Oxidation in HFD-Induced Obese Mice
Our study demonstrated that BJE effectively inhibited the process of adipogenesis in
differentiated 3T3-L1 adipocytes by reducing the expression levels of PPAR-γ, C/EBP-α,
and ap2 (Figure 3A–C). Consistent with these results, the levels of PPAR-γ, C/EBP-α, and
ap2 expression were notably decreased in the eWAT obtained from BJE groups compared
with those from HFD groups (Figure 5A–C). Furthermore, to assess
Nutrients 2024, 16, x FOR PEER REVIEW 10 of 15
whether BJE could
regulate lipid synthesis-related protein expression in vivo, we measured p-ACC/ACC
expression levels in the eWAT. We confirmed that lipid synthesis was suppressed in the
with those from HFD groups (Figure 5A). Furthermore, to assess whether BJE could
BJE group by significantly
regulate increasing
lipid synthesis-related proteinp-ACC/ACC
expression in vivo, expression compared to the HFD group
we measured p-ACC/ACC
expression levels in the eWAT. We confirmed that lipid
(Figure 5D). Finally, the gene expression related to fatty acid oxidation synthesis was suppressed in the and heat generation
BJE group by significantly increasing p-ACC/ACC expression compared to the HFD
was measured. BJE promoted
group (Figure 5B). Finally, thethe
geneupregulation
expression related tooffatty
PGC-1α andand
acid oxidation CPT-1
heat in vivo, which was
generation was measured. BJE promoted the upregulation of PGC-1α and CPT-1 in vivo,
decreased in the HFD group (Figure 5E,F). In vivo protein expression
which was decreased in the HFD group (Figure 5C). In vivo protein expression results are
results are consistent
with in vitro protein
consistent withexpression results.results.
in vitro protein expression

PPAR-γ C/EBP-α

β-actin
β-actin
Relative protein expression of C/EBP-a

270
180
Relative protein expression of PPARγ

a 240 a
150
210

180
120 b
b b
150
90
c 120 c

60 90
c d
60
30 e
30

0 0
ay

ay
FD

FD
N
ay

ay

da
ay
N

d
O
CO

/d
H

/d

g/
/d

g/

g/
H
C
kg

k
kg

kg

k
g/

g/

g/
g/

g/

g/

m
m

50

0
50

40

80
40

80

ar
ar

JE

JE
JE

JE

+G
+G

+B
+B
+B

+B

FD
FD

FD
FD
FD

FD

H
H

H
H

(A) (B)
p-ACC
aP2
ACC
β-actin
β-actin
Relative protein expression of p-ACC/ACC

180
210
Relative protein expression of aP2

a
150 a
180
b ab
120 c b
bc 150

c
90 120
c
d
90
60

60
30

30
0
y
y
FD

y
N

da
da

da

0
O

/
g/
C

ay
kg

kg

FD

ay

ay
N
k

/d
g/
g/

/d
g/

/d
H
C

kg
m
m

kg

kg
g/
0
50

g/

g/
40

80

m
ar

50
JE

0
+G

40

80
+B

+B

ar
FD

JE

JE
+G
FD

FD
H

+B

+B
FD
H

FD

FD
H

(C) (D)

Figure 5. Cont.
Nutrients 2024, 16, 846 10 of 13
Nutrients 2024, 16, x FOR PEER REVIEW 11 of 15

PGC-1α
CPT-1

β-actin β-actin
120

Relative protein expression of PGC-1α

120

Relative protein expression of CPT-1

a a
a
90 90
ab

b b
60 bc 60 c

c
d
30 30

0
0

ay
N

FD

y
ay

ay
da

ay
FD
N

ay
O

/d
/d

O
H

/d
C

/d
g/

/d
kg

H
g

kg
/k

kg

kg
/k

g/
g
g

g/
g/

g/
m
m
m

m
m

m
0
0
50

80
40

0
50

0
40

80
ar

JE
JE

ar
+G

JE
+G
+B
+B

J
+B

+B
FD

FD
FD

FD

FD

FD
H

H
H

H
(E) (F)
Figure 5. Effect of BJE on the expression of proteins related to adipogenesis, lipid synthesis, fatty
Figure 5. Effect of BJE on
acid oxidation, and the expression
heat generation of proteins
in HFD-induced obese related to adipogenesis,
C57BL/6J mice. PPAR-γ (A). C/EBP-α lipid synthesis, fatty acid
(B). aP2 (C). p-ACC/ACC (D). PGC-1α (E). CPT-1 (F). All results are presented as the mean ± SD of
oxidation, and heat generation in HFD-induced obese C57BL/6J mice. PPAR-γ
3 independent in triplicate. Bars with different letters indicate significant differences at p < 0.05 using
(A). C/EBP-α (B).
Duncan’s multiple
aP2 (C). p-ACC/ACC rangePGC-1α
(D). test. ±
(E). CPT-1 (F). All results are presented as the mean SD of
3 independent in triplicate. Bars with different letters indicate significant differences at p < 0.05 using
4. Discussion
Several
Duncan’s multiple studies
range have focused on both anti-obesity drugs and functional foods [32,33].
test.
Moreover, many studies have aimed to verify the efficacy of natural substances in
functional foods and anti-obesity drugs, with the intention of reducing the various side
4. Discussioneffects associated with commercially available anti-obesity drugs [34–36]. This research
was conducted to confirm the in vivo anti-obesity effect in an animal model, expanding
Severalon studies have
the results of prior focused on both
in vitro investigations on anti-obesity drugs
the inhibition of lipid and functional
accumulation by foods [32,33].
Moreover, BJEmany with anstudies
optimizedhavesinigrin aimed to verify the efficacy of natural substances in func-
content [30].
Sinigrin from Brassica juncea has been documented to exhibit various beneficial
tional foodsproperties
and anti-obesity
such as anticancer,drugs, with the intention
anti-inflammatory, of reducing
antibacterial, antifungal, andthe various side effects
associated with commercially
antioxidant available
effects. In our previous studies,anti-obesity drugs
BJE extracted under [34–36].
optimized This research was con-
sinigrin
extraction conditions was reported to have anti-obesity effects in vitro [28–30]. Therefore,
ducted to confirm the in vivo anti-obesity effect in an animal
we prepared BJE using an optimized extraction method referring to previous studies and model, expanding on the
results of prior
analyzed inthe
vitro
content investigations on the
of sinigrin, the main active inhibition
ingredient of Brassica of lipid
juncea, usingaccumulation
HPLC. by BJE with
The sinigrin content of BJE used in this study was 18.0 mg/g.
an optimized sinigrin contentwere
3T3-L1 preadipocytes [30].treated with MDI to induce their differentiation into
adipocytes.
Sinigrin At the same juncea
from Brassica time, the cytotoxicity
has beenofdocumented
BJE in 3T3-L1 adipocytes was confirmed
to exhibit various beneficial proper-
via XTT analysis, but no cytotoxicity was found at all concentrations investigated (100,
ties such as200,
anticancer, anti-inflammatory,
400, and 800 µg/mL). The inhibitory effect of antibacterial,
BJE (100, 200, 400,antifungal,
and 800 µg/mL) on and antioxidant effects.
adipocyte differentiation was investigated. The morphological features of the cells and
In our previous studies, BJE extracted under optimized sinigrin
stained adipocytes were assessed quantitatively using Oil Red O (ORO) staining. This
extraction conditions was
reported tostudy
have anti-obesity
showed a reduction in effects in vitro
both the quantity [28–30].ofTherefore,
and dimensions weasprepared
adipocytes, as well a BJE using an
optimized decrease
extraction in ORO intensity, in correlation with the concentration of BJE, when compared
method referring to previous studies
to the MDI group. The in vitro anti-obesity mechanism of BJE was investigated via
and analyzed the content of
sinigrin, theWestern
mainblotting.
active ingredient
PPAR-γ, C/EBP-α, and Brassica
of ap2 juncea,
are crucial factors inusing HPLC. The
the transformation of sinigrin content of
BJE used inpreadipocytes
this study into fully developed adipocytes [37]. In a previous study, BJE was reported
was 18.0 mg/g.
to inhibit lipid accumulation by downregulating protein levels of PPAR-γ, C/EBP-α, and
3T3-L1 preadipocytes
ap2, aligning with were
the findings treated
of the current studywith MDI
[30]. ACC to induce
regulates their differentiation into
the condensation

adipocytes. At the same time, the cytotoxicity of BJE in 3T3-L1 adipocytes was confirmed
via XTT analysis, but no cytotoxicity was found at all concentrations investigated (100,
200, 400, and 800 µg/mL). The inhibitory effect of BJE (100, 200, 400, and 800 µg/mL) on
adipocyte differentiation was investigated. The morphological features of the cells and
stained adipocytes were assessed quantitatively using Oil Red O (ORO) staining. This study
showed a reduction in both the quantity and dimensions of adipocytes, as well as a decrease
in ORO intensity, in correlation with the concentration of BJE, when compared to the MDI
group. The in vitro anti-obesity mechanism of BJE was investigated via Western blotting.
PPAR-γ, C/EBP-α, and ap2 are crucial factors in the transformation of preadipocytes into
fully developed adipocytes [37]. In a previous study, BJE was reported to inhibit lipid
accumulation by downregulating protein levels of PPAR-γ, C/EBP-α, and ap2, aligning
with the findings of the current study [30]. ACC regulates the condensation of lipids and
is integral in the overall regulation of energy metabolism [38]. Phosphorylation leads to
the inactivation of this protein, resulting in the inhibition of ACC activity. This process can
effectively suppress lipid production and potentially prevent obesity [39]. BJE inhibited
ACC activation by promoting ACC phosphorylation. CPT-1 serves as a critical modulator
of fatty acid metabolism via its control of malonyl-CoA, which is the initial intermediate
in lipid synthesis [40]. PGC-1α is acknowledged as a significant inducer of mitochondrial
Nutrients 2024, 16, 846 11 of 13

biogenesis in adipocytes [41]. According to our results, BJE exerts its anti-obesity effects by
regulating CPT-1 and PGC-1α to increase fatty acid oxidation and heat generation.
Based on the in vitro results, an animal study was undertaken to evaluate the in vivo
efficacy of BJE in combating obesity. HFD is widely used and acknowledged in nutritional
studies as an effective method for inducing obesity and fat deposition in animal models [42].
Excessive consumption of HFD can lead to weight gain due to white adipose tissue accu-
mulation [43]. As anticipated, mice that were provided with an HFD exhibited obesity and
demonstrated increased eWAT compared to mice that were given a standard control diet.
However, orally administering BJE for 6 weeks to HFD-fed mice significantly decreased
both adipocyte size in eWAT and body weight. Obesity induced by HFD is correlated with
elevated levels of triglycerides and cholesterol in the blood [44]. In this study, BJE decreased
serum TC and TG. Western blotting was conducted to examine the in vivo mechanisms
underlying the anti-obesity properties of BJE. HFD-induced obesity is associated with
high expression of PPAR-γ, C/EBP-α, and ap2 [14]. PPAR-γ, C/EBP-α, and aP2 showed
low expression levels Within the adipose tissue of the epididymis in mice administered
orally with BJE. ACC was activated in HFD-fed mice and promoted adipogenesis [45]. BJE
treatment inhibited ACC activation by restoring ACC phosphorylation in the eWAT of
HFD-fed mice. However, in HFD-fed mice, the expression of PGC-1α, which is involved in
heat generation, and CPT-1, a key enzyme responsible for facilitating the oxidation of fatty
acids [46,47]. In this research endeavor, we investigated the impact of BJE treatment on the
expression of proteins related to heat generation and fatty acid oxidation in vivo. Here, we
showed that BJE administration induced PGC-1α and CPT-1 expression in HFD-fed obese
mice. Therefore, BJE treatment may inhibit lipid accumulation by upregulating the heat
generation and expression of fatty acid oxidation-related proteins.
Overall, these results confirmed that BJE prevented obesity induced by HFD in
C57BL/6J mice. Western blot analysis demonstrated that the anti-obesity properties of
BJE were linked to the suppression of adipogenesis and lipid synthesis, along with the
stimulation of fatty acid oxidation and heat generation. Therefore, BJE may be a promising
source of functional materials that could be utilized in the prevention and management
of obesity.

5. Conclusions
The research demonstrates that BJE can inhibit lipid accumulation in 3T3-L1 and
decrease body weight and eWAT mass in obese mice fed an HFD. These outcomes are
accomplished by inhibiting adipogenic proteins (C/EBP-α, PPAR-γ, and aP2), activating
the phosphorylation of the lipid synthesis protein (ACC), and increasing the levels of fatty
acid oxidation protein (CPT-1) and thermogenesis-related protein (PGC-1α). Therefore,
our data suggest that BJE administration improves HFD-induced obesity by suppressing
the formation of adipocytes and promoting the processes of heat generation and fatty acid
oxidation. However, it is imperative to conduct suitable human trials to investigate its use
as an alternative to anti-obesity drugs for the purpose of controlling obesity.

Author Contributions: Project administration, Visualization, Writing—original draft, J.-S.L.; Method-

ology, Project administration, Visualization, J.-H.I.; Investigation, Validation, X.H.; Investigation,
Software. G.O.; Investigation, Formal analysis, X.M.; Software, Validation, Formal analysis, X.F.;
Resources, Validation, W.H.; Conceptualization, Funding acquisition, Supervision, S.-I.C.; Conceptu-
alization, Funding acquisition, Supervision, Writing—review and editing, O.-H.L. All authors have
read and agreed to the published version of the manuscript.
Funding: The authors declare that this study received funding from the Ministry of Education through
the Basic Science Research Program (NRF-2021R1A6A1A03044242 and 2017R1D1A3B0602846915) and
the BK21 FOUR (Fostering Outstanding Universities for Research) (4299990913942) of the National
Research Foundation of Korea (NRF). The funder was not involved in the study design, collection,
analysis, interpretation of data, the writing of this article or the decision to submit it for publication.
Nutrients 2024, 16, 846 12 of 13

Institutional Review Board Statement: The animal study protocol was approved by the Institu-
tional Animal Care and Use Committee (IACUC) of Kangwon National University. The C57BL/6J
mice were maintained in accordance with Kangwon National University Regulations on Animal
Experimentation (IACUC approval number: KW-221104-2, approved on 21 November 2022).
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available within this article.
Conflicts of Interest: Author Woonsang Hwang was employed by the company STR Biotech Co., Ltd.
The remaining authors declare that the research was conducted in the absence of any commercial or
financial relationships that could be construed as a potential conflict of interest.

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