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Article

Anti-Obesity Effect of Fresh and Browned Magnolia denudata Flowers in 3T3-L1 Adipocytes

by
Deok Jae Lee
1,
Jae Ho Yeom
1,
Yong Kwon Lee
2,
Yong Hoon Joo
3,* and
Namhyun Chung
1,*
1
Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Republic of Korea
2
Department of Culinary Art & Food Service Management, Yuhan University, Bucheon 14780, Republic of Korea
3
Department of Biosystems and Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Republic of Korea
*
Authors to whom correspondence should be addressed.
Appl. Sci. 2024, 14(20), 9254; https://doi.org/10.3390/app14209254
Submission received: 31 August 2024 / Revised: 27 September 2024 / Accepted: 9 October 2024 / Published: 11 October 2024
(This article belongs to the Section Food Science and Technology)

Abstract

:
The major components of magnolia flower extracts (MFEs) were classified into four substances, such as flavonoids, phenylethanoid glycoside derivatives (PhGs), caffeoylquinic acids (CQAs), and others, in our previous study. The chemical components of MFEs, including the rutin of flavonoid, acteoside and isoacteoside of PhGs, and caffeyolquinic acids, are reported to have physiological effects on anti-obesity effects. The anti-obesity effect of fresh and browned Magnolia denudata flower extracts (FMFE and BMFE, respectively) was investigated in 3T3-L1 adipocytes. The treatment concentrations of FMFE and BMFE were 200 and 400 μg/mL, respectively, as determined with the WST-1 assay. Intracellular lipid accumulation in 3T3-L1 cells was inhibited with the treatment of MFEs, including FMFE and BMFE, as observed with an image of the culture plate, using an optical microscope and Oil red O staining. The expression of the adipogenic target genes involved in adipocyte differentiation, including PPARγ, C/EBPα, perilipin, FABP4, FAS, HSL, and SREBP-1, was suppressed with the treatment of MFEs. Additionally, the phosphorylation of AMPK and ACC in 3T3-L1 cells was significantly increased following treatment with the MFEs. These results suggest that both MFEs have a potential for physiological effects on anti-obesity activity.

1. Introduction

Magnolia denudata flowers have been used as folk medicine in Asian countries. The flowers are often used as a tea material and processed with fermentation to promote aromas, tastes, and flavors [1,2,3]. In addition, the Magnolia denudate flower extract is known to have several physiological effects, including nitrate synthesis inhibitory effect, reduction in lipid accumulation and the expression extent of adipogenic transcription factor, and anti-allergic effects [4,5,6,7]. Although studies of various physiological activities in Magnolia denudata flowers have been reported, extensive studies on the total chemical compounds in non-processed and processed M. denudata flowers are hard to find. Tea has a unique aroma, flavor, and physiological effects. Various fermentation methods are used to achieve unique flavors in tea. For example, to make oolong and black tea, fermentation is often conducted by controlling humidity and temperatures using green tea [8]. In our previous experiment, the browning of Magnolia denudata flowers, like Pu-erh tea, was also fermented by controlling temperature and humidity [1]. In this study, Magnolia denudate flower was fermented similarly to Pu-erh tea.
Our previous study showed that the chemicals’ comparative quantity and composition differed depending on the fermentation degree of Magnolia denudata flowers [1]. Components of fresh, aged, and browned Magnolia denudata flower extract (FMFE, AMFE, and BMFE, respectively; all called MFEs) were analyzed to obtain detailed profiling. The chemical components of magnolia flowers were classified into four categories: flavonoids, phenylethanoid glycoside derivatives (PhGs), caffeoylquinic acids (CQAs), and others. Our previous study showed that the sixteen main compounds of MFE were rutin, quercetin-3-O-glucoside, kaempferol-3-neohesperidoside, keioside, isoverbasoside, yulanoside B, echinacoside, acteoside, isoacteoside, 1-CQA, chlorogenic acid, 4-CQA, galactinol, quinic acid, citric acid, and tyrosol. These main compounds of magnolia flower have been reported to have anti-obesity effects. Rutin, a flavonoid and one of the representative chemical compounds of magnolia flowers, was reported to have the strongest inhibition effect on the differentiation adipocytes such as 3T3-L1 cells [9,10,11,12]. The other chemical components of Magnolia denudata flower extracts (MFEs), such as acteoside and isoacteoside of PhGs, and caffeyolquinic acids, were also reported to have anti-obesity effects [13,14].
Obesity contributes to a significantly increased potential for metabolic disease, such as hyperlipidaemia, type 2 diabetes, cardiovascular disease, fatty liver disease, and several types of cancer [15,16,17,18,19]. The adipose tissue regulates lipid metabolism and functions to secrete and store lipids in glucose metabolism [20,21]. Adipose tissue is composed of adipocytes and a small number of other cells. As a molecular mechanism for adipogenesis, 3T3-L1 cell differentiation efficiency is an important factor for obesity. This process is widely used as an adipocyte differentiation model system [22,23,24,25].
In our previous study, MFEs were shown to have the potential for anti-obesity effects based on the analysis of chemical components [1]. Thus, we have already confirmed the anti-obesity effect of the FMFE and BMFE in a high-fat diet murine model [26]. However, the detailed physiological activity of Magnolia denudata flower extract needs to be explained at the level of intracellular mechanism. Several studies have reported on 3T3-L1 cells with fresh magnolia flowers [9,10,11,12]. However, our study is different because we focused on the anti-obesity effects to observe the detailed molecular mechanism with FMFE (fresh) and BMFE (fermented). While the change in the chemical composition of AMFE was almost the same as FMFE, notable changes were observed between AMFE and BMFE [1,26]. Thus, FMFE and BMFE were chosen to investigate the detailed mechanism of the anti-obesity effects with 3T3-L1 mouse preadipocytes.

2. Materials and Methods

2.1. Chemicals

3-Isobutyl-1-methylxanthine (IBMX), dexamethasone (DEX), insulin, and Oil red O were purchased from Sigma-Aldrich (Saint Louis, MO, USA).

2.2. Preparation of Magnolia denudata Flower

The Magnolia denudata flower used for cell treatment was obtained from Mururang-Daraerang (Damyang, Republic of Korea). Dried magnolia flowers were homogenized and browned for 1 week at 90 °C and under 75% humidity using supersaturated sodium chloride. The fresh and browned Magnolia denudata flowers were extracted in deionized water at 90 °C for 15 min. The infusion was centrifuged at 8000× g for 10 min and filtered using a 0.22 μm aspirator filter (Millipore, Burlington, MA, USA). The filtered infusion was lyophilized to obtain the fresh and browned Magnolia denudata flower extracts (FMFE and BMFE, respectively) powders [26].

2.3. Cell Culture Condition

Mouse preadipocyte (3T3-L1) cell lines were obtained from Korean Cell Line Bank (KCLB, Seoul, Republic of Korea) and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich), containing 10% bovine calf serum (BCS), and 1% penicillin/streptomycin solution in a humidified atmosphere of 5% CO2 at 37 °C.

2.4. Cell Toxicity

3T3-L1 preadipocytes (5 × 104 cells/well) were seeded in 96-well plates overnight and treated with FMFE and BMFE (250 to 6000 µg/mL) for 24 h. According to the manufacturer’s protocol, cell viability was determined using a PreMix water-soluble tetrazolium salts-1 (WST-1) Cell Proliferation Assay System (TaKaTa, Saitama, Japan). The WST-1 reagent with the medium was put into each well and absorbance (450 nm) was measured immediately at 0 min and then every 20 min.

2.5. Cell Differentiation

3T3-L1 preadipocyte cells were seeded in a 24-well plate in DMEM containing 10% BCS with a density of 0.2 × 105 cells per well. Then, 1-week post-confluent 3T3-L1 cells were stimulated and treated for 2 days with differentiation medium (DMEM containing 10% fetal bovine serum (FBS), 0.5 mM IBMX, 1 μM DEX, and 10 μg/mL insulin). After 2 days, the cells were incubated for 2 days with DMEM containing 10% FBS and 10 μg/mL insulin. Subsequently, the cells were incubated with DMEM containing 10% FBS until day 8 when the cells were fully differentiated. During the differentiation, the 3T3-L1 preadipocytes were treated with FMFE and BMFE (200, 400 μg/mL).

2.6. Oil Red O Staining

The differentiated 3T3-L1 cells were stained with Oil red O on day 10 to detect intracellular lipid droplets. The cells were fixed with 4% polyformaldehyde for 60 min and stained with Oil red O in 60% isopropanol for 30 min at room temperature. The stained lipid droplet images were obtained using an optical microscope, and then lipid droplets were dissolved in 100% isopropanol. The supernatant was transferred to 96 wells and quantified by measuring the absorbance at 530 nm.

2.7. Real Time-PCR

The quantification of gene expression of PPARγ, C/EBPα, perilipin, FABP4, FAS, SREBP1, and HSL was measured using a real-time PCR system. The total RNA in the 3T3-L1 cells was isolated using TRIzol reagent (#15596018; Invitrogen, Carlsbad, CA, USA). The total RNA was converted to cDNA using a revertaid first-strand cDNA synthesis kit (#K1622; Thermo Fisher Scientific, Waltham, MA, USA). Real-time quantitative PCR was performed using the RealHelix™ Premier qPCR Kit (#PQL-S200, Nanohelix, Daejeon, Republic of Korea). The cDNA was amplified for 40 cycles of denaturation (95 °C for 5 min), annealing (60 °C for 40 s), and extension (60 °C for 40 s) using a CFX Connect Real-Time PCR machine (Bio-Rad, Hercules, CA, USA). The primer sequences are listed in Table 1. Gene expression was calculated using the 2−ΔΔCt method and normalized to β-actin levels.

2.8. Western Blot

The 3T3-L1 cells were washed twice in cold phosphate-buffered saline. Radioimmunoprecipitation assay buffer (RIPA buffer, Thermo Fisher Scientific) was used for extraction. The cells were vortexed in a 10 min term for 1 h and centrifuged at 13,000 rpm for 20 min at 4 °C. Protein concentration measurements were performed using BCA assay (Thermo Fisher Scientific). An equal amount of protein was loaded in both 6% and 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Burlington, MA, USA). The membranes were blocked with 5% skim milk or bovine serum albumin (BSA) for 90 min in Tris-buffered saline (TBS) containing 0.1% Tween-20. Then, the membranes were incubated with 1/1000 diluted primary antibody solution at 4 °C overnight. Antibodies of AMPK (#2532), p-AMPK (#2535), ACC (#3662), p-ACC (#11818), PPARγ (#2435), C/EBP (#8178), and perilipin (#9349) were purchased from Cell Signaling Technology (Danvers, MA, USA). The membranes were washed three times with TBS containing 0.1% Tween-20. After washing, the membranes were incubated with a secondary antibody (Anti-rabbit IgG, HRP-linked Antibody #7074) for 2 h. Then, the membranes were washed three times with TBS containing 0.1% Tween-20. Finally, the membranes were treated with enhanced chemiluminescence using electrochemiluminescence (ECL). Protein bands were detected using the ChemiDoc XRS + System (Bio-Rad).

2.9. Statistical Analysis

Data were presented as the mean ± standard deviation (SD). Significant differences were analyzed using a one-way ANOVA followed by Tukey’s post hoc multiple comparison test.

3. Results

3.1. Effects of MFEs on Adipogenic Differentiation of 3T3-L1 Cells

Cytotoxicity can adversely affect the anti-adipogenesis properties of FMFE and BMFE. The cell viability of 3T3-L1 cells was determined via WST-1 assay with the treatment of FMFE and BMFE. No significant cell toxicity was detected up to 750 μg/mL of MFEs (Figure 1). Based on these results, we selected much safer concentrations of less than 500 μg/mL and used them in the following studies. We investigated the effect of MFEs on intracellular lipid accumulation in 3T3-L1 cells with a culture plate and optical microscope (Figure 2A,B). The adipogenesis of 3T3-L1 cells was inhibited by treating FMFE and BMFE. The lipid content level decreased compared to the control group and its level was dose dependent. The extent of FMFE groups was significantly reduced to about 55.3% and 29.9% of the differentiation control group in 200 and 400 μg/mL, respectively (Figure 2C). The extent of BMFE groups also significantly reduced to about 55.3% and 28.6% of the differentiation control group in 200 and 400 μg/mL, respectively. The extent of non-differentiation control was about 24.2% (Figure 2C). Thus, when treated with 400 μg/mL of BMFE and FMFE, the extent of lipid contents decreased to a similar level to that of the non-differentiation group.

3.2. Effect of MFEs on the Expression Level of Adipogenic-Specific Genes in 3T3-L1 Cells

The molecular mechanisms by the treatment of FMFE and BMFE to 3T3-L1 cells were investigated to obtain the transcription levels of related genes for preadipocyte differentiation and lipid metabolism. The expression extents of PPARγ were suppressed by about 56.8% and 79.5%, respectively, for 200 and 400 μg/mL of FMFE and by about 22.8% and 57.2%, respectively, for 200 and 400 μg/mL of the BMFE, compared with those of the differentiation control group (Figure 3A). The expression extents of C/EBPα were suppressed by about 65.9% and 92.4%, respectively, for 200 and 400 μg/mL of FMFE, and by about 68.7% and 91.8%, respectively, for 200 and 400 μg/mL of BMFE, compared with those of the differentiation control group (Figure 3B). The expression extent of SREBP-1 was suppressed by about 48.9% and 70.3%, respectively, for 200 and 400 μg/mL of FMFE, and by 52.3% and 65.2%, respectively, for 200 and 400 μg/mL of BMFE, compared with those of the differentiation control group (Figure 3C). The expression extents of FAS were suppressed by about 59.1% and 86.3%, respectively, for 200 and 400 μg/mL of FMFE, and by about 68.0% and 88.5%, respectively, for 200 and 400 μg/mL of BMFE, compared with those of the differentiation control group (Figure 3D). The expression extents of perilipin were suppressed by about 41.4% and 85.8%, respectively, for 200 and 400 μg/mL of FMFE, and by about 42.3% and 91.5%, respectively, for 200 and 400 μg/mL of BMFE, compared with those of the differentiation control group (Figure 3E). The expression extents of FABP4 were suppressed by about 51.9% and 84.6%, respectively, for 200 and 400 μg/mL of FMFE, and by about 30.8% and 59.1%, respectively, for 200 and 400 μg/mL of BMFE, compared with those of the differentiation control group (Figure 3F). The expression extents of HSL were suppressed by about 36.8% and 76.9%, respectively, for 200 and 400 μg/mL of FMFE, and by about 35.3% and 73.0%, respectively, for 200 and 400 μg/mL of BMFE, compared with those of the differentiation control group (Figure 3G).

3.3. Effect of MFEs on the Expression Level of Adipogenic-Specific Markers in 3T3-L1 Cells

The differentiation groups without MFEs had increased expression extents of PPARγ, C/EBPα, and perilipin in 3T3-L1 adipocytes, compared to those of the non-differentiation group. The expression extents of PPARγ, C/EBPα, and perilipin of the differentiation group were increased by about 394.7%, 781.3%, and 3155.9%, respectively, compared to those of the non-differentiation group. The expression extents of PPARγ were suppressed by 28.4% and 45.9%, respectively, for 200 and 400 μg/mL of FMFE, and by 22.0% and 36.6%, respectively, for 200 and 400 μg/mL of BMFE, compared with those of the differentiation control group (Figure 4A). The expression extent of C/EBPα was suppressed by 24.9% and 45.9%, respectively, for 200 and 400 μg/mL of FMFE, and by 22.6% and 41.7%, respectively, for 200 and 400 μg/mL of BMFE, compared with those of the differentiation control group (Figure 4B). The expression extents of perilipin were suppressed by 40.4% and 75.5%, respectively, for 200 and 400 μg/mL of FMFE, and by 27.0% and 51.6%, respectively, for 200 and 400 μg/mL of BMFE, compared with those of the differentiation control group (Figure 4C). In this study, we observed that the treatment with FMFE and BMFE significantly decreased the expression extent of PPARγ, C/EBPα, and perilipin in 3T3-L1 adipocytes. These results suggest that the treatment with the MFEs inhibits the expression extents of PPARγ, C/EBPα, and perilipin.

3.4. Effect of MFEs on AMPK Activation in 3T3-L1 Adipocytes

The groups of FMFE and BMFE had an increased expression ratio of phosphorylated-AMPKα/AMPKα. The 200 and 400 μg/mL of FMFE had a significantly elevated phosphorylated-AMPKα/AMPKα ratio of approximately 150% and 190%, respectively, compared with those of the differentiation control group (Figure 5A). The 200 and 400 μg/mL of BMFE had a significantly elevated phosphorylated-AMPKα/AMPKα ratio of approximately 160% and 180%, respectively, compared with those of the differentiation control group (Figure 5A). The 200 and 400 μg/mL of FMFE had an increased phosphorylated-ACC/ACC expression ratio of approximately 85.6% and 204.5%, respectively, compared with those of the differentiation control group (Figure 5B). The 200 and 400 μg/mL of BMFE had an increased phosphorylated-ACC/ACC expression ratio of approximately 123.6% and 445.7%, respectively, compared with those of the differentiation control group (Figure 5B).

4. Discussion

Processing tea fermentation is a natural post-oxidation process that involves incubating the withered-fresh tea leaves in a closed high or low-humidity storage chamber for a defined period. The chemical components of fermented tea are different from those of green tea. Many studies have shown fermented tea’s antioxidant, anti-mutagenic, anti-atherosclerotic, anti-obesity, and anti-hypercholesterolemic effects [27,28,29,30,31]. In this study, the Magnolia denudata flower was fermented like oolong and black tea. Several studies have reported on 3T3-L1 cells with fresh Magnolia denudate flower [9,10,11,12]. However, our study is different because we focused on the anti-obesity effects to observe the detailed molecular mechanism with fresh and browned magnolia flowers. Thus, study is the first of its kind. We identified the chemical components of fresh and browned Magnolia denudata flower, and the change in the chemical components of Magnolia denudata flower was expected to affect its physiological activities [1,26]. This is because the chemical compounds of MFEs could be a basis for showing the potential for many biological activations. However, it needs to be mentioned that these chemical compounds are cytotoxic if used in a high concentration, as shown in Figure 1, which demonstrates that cell viability decreases with an increasing concentration of more than 1000 µg/mL. Based on this finding, we selected much safer concentrations of less than 500 μg/mL, such as 200 and 400 μg/mL.
PARγ (peroxisome proliferator-activating receptor gamma), C/EBPα (CCAAT/inhancer-binding protein alpha), and SREBP-1 (cholesterol-regulated factor-binding protein 1) are major transcription factors that play an important role in adipogenic and lipogenic control. PPARγ has a role as a major regulator of adipogenesis. It is essential for differentiating adipocyte into mature adipocytes. C/EBPα interacts with PPARγ to enhance the expression of the genes required for adipocyte differentiation. SREBP-1 plays a significant role in fat production. These transcription factors make a complex network that ensures the proper development and functioning of adipose tissue [32]. It has been demonstrated that the expression inhibition of PPARγ, C/EBPα, and SREBP-1 in adipocytes is associated with the prevention of obesity [33,34]. This is because the transcription factors regulate the gene expression of adipogenic targets in adipocytes. The FAS (fatty acid synthase) uses acetyl-CoA and malonyl-CoA as a substrate to generate long-chain fatty acids and promote lipogenesis [35,36,37]. Perilipin participates in the storage and mobilization of lipids [38,39,40,41,42]. FABP4 (Fatty Acid-Binding Protein 4) is a carrier protein for fatty acids in adipocytes and is responsible for intracellular lipid absorption and storage development [43,44,45]. HSL (hormone-sensitive lipase) regulates the triacylglycerol hydrolysis into free fatty acids in lipolysis metabolism [46,47,48]. In the present study, we found that the mRNA expression of all adipogenic target genes significantly decreased by treatment with FMFE and BMFE. We also found that the protein expression of adipogenic target proteins such as PPARγ, C/EBPα, and perilipin significantly decreased by treatment with FMFE and BMFE. These findings suggest that treatment with FMFE and BMFE induces the inhibition of PPARγ, C/EBPα, SREBP-1, FAS, perilipin, FABP4, and HSL in adipocytes.
Cells need to act favorably to available nutrients by amending their metabolism for their energy needs. AMPK (AMP-activated protein kinase) is a highly conserved serine/threonine energy-sensing kinase. AMPK acts as a metabolic master regulator of cellular energy homeostasis. AMPK activity in adipocytes and adipose tissues inhibits lipogenesis by modulating lipogenic enzyme activities, thus suppressing fat accumulation. The phosphorylation of AMPK leads to the inactivation of fatty acid synthesis enzymes such as ACC [49,50,51,52,53]. Therefore, AMPK is suggested as a potential target to treat obesity and diabetes. In this study, when 3T3-L1 adipocytes were treated with FMFE and BMFE, an increase in the phosphorylated AMPKα/AMPKα ratio was induced in 3T3-L1 adipocytes. ACC (acetyl-CoA carboxylase) is involved in fatty acid synthesis. When AMPK is activated, it phosphorylates and inhibits ACC, reducing fatty acid synthesis and promoting fatty acid oxidation. The ACC activation in FMFE- and BMFE-treated groups was inhibited by increasing ACC phosphorylation in a dose-dependent manner. These results suggest that the treatment of 3T3-L1 adipocytes by FMFE and BMFE induces the suppression of adipocyte differentiation and lipid accumulation by AMPK activation and ACC inhibition.
Previous studies demonstrated that fermentation significantly altered the chemical composition of Magnolia denudata flower [1,2,3]. The changed compound compositions following the fermentation process can change the bioactivity of fermented Magnolia denudata flower extracts. Thus, fermented and non-fermented Magnolia denudata flower extracts were expected to exhibit a different extent of the anti-obesity effect. However, the anti-obesity effect was not much different between FMFE and BMFE in a high-fat diet murine model [26]. It is expected that, during fermentation, while the contents of some compounds increase, the contents of the other compounds decrease, compromising the anti-obesity effect. In another case, the anti-obesity effect might be compromised by the interaction of multiple compounds. In the present study, one of the most important changes after the fermentation was isomerization by the induction of the acyl migration of phenylethanoid glycosides. In particular, the levels were dramatically increased during the fermentation process. Rutin, the most common compound of MFEs, was decreased by fermentation, compromising the anti-obesity effect [1]. Due to the increase and decrease in several chemicals, there might be no significant difference in the anti-obesity effects of FMFE and BMFE in the 3T3-L1 cells of this study and a murine model of our previous study [26]. Based on this finding, we suggest that this result is possibly due to the noticeably increased isomerization of phenylethanoid glycosides like isoacteoside, even though rutin was decreased. This finding is also supported by previous studies where the anti-obesity effect of isoacteoside was demonstrated in 3T3-L1 adipocytes and a murine model [13,14].

5. Conclusions

This study aimed to investigate the potential physiological effects of sixteen main compounds in Magnolia denudata flower extracts, and whether fermented and non-fermented Magnolia denudata flower extracts are helpful for the prevention of obesity. Both FMFE and BMFE efficiently reduced adipocyte differentiation in 3T3-L1 cells, irrespective of the changes in the chemical components during the fermentation. No significant cell toxicity was observed up to 750 μg/mL of MFEs. Those results also suggested that both FMFE and BMFE have a suppressive effect on the increase in the lipid contents of the differentiated 3T3-L1 cells. This finding was supported by the expression of the adipogenic target genes involved in adipocyte differentiation, including PPARγ, C/EBPα, perilipin, FABP4, FAS, HSL, and SREBP-1, following treatment with MFEs. Additionally, the phosphorylation of AMPK and ACC in 3T3-L1 cells was significantly elevated with the treatment of MFEs. These results collectively suggest that treatment with MFEs could reduce the accumulation of lipid contents in 3T3-L1 adipocytes (Figure 6). The present and our previous study also demonstrate that both the fresh and browned flower extracts of Magnolia denudata have anti-obesity effects both in vitro and in vivo.

Author Contributions

D.J.L.: writing—original draft, investigation, formal analysis, and data curation. J.H.Y.: formal analysis, data curation, and methodology. Y.K.L.: formal analysis and data curation. Y.H.J.: writing—review and editing, visualization, supervision, and conceptualization. N.C.: writing—review and editing, supervision, project administration, and funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by a Korea University grant in 2014.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article; further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Cell viability of various concentrations of the fresh and browned Magnolia denudata flower extracts (FMFE and BMFE). Different letters indicate significant differences at p < 0.05.
Figure 1. Cell viability of various concentrations of the fresh and browned Magnolia denudata flower extracts (FMFE and BMFE). Different letters indicate significant differences at p < 0.05.
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Figure 2. Effect of the fresh and browned Magnolia denudata flower extracts (FMFE and BMFE) on the lipid accumulation of differentiating 3T3-L1 adipocytes. (A) Images of cell culture plates of 3T3-L1 adipocytes following the Oil red O staining of intracellular lipid droplets. (B) Images from a optical microscope of 3T3-L1 adipocytes following the Oil red O staining of intracellular lipid droplets. (C) Lipid contents of FMFE and BMFE groups. Different letters indicate significant differences at p < 0.05.
Figure 2. Effect of the fresh and browned Magnolia denudata flower extracts (FMFE and BMFE) on the lipid accumulation of differentiating 3T3-L1 adipocytes. (A) Images of cell culture plates of 3T3-L1 adipocytes following the Oil red O staining of intracellular lipid droplets. (B) Images from a optical microscope of 3T3-L1 adipocytes following the Oil red O staining of intracellular lipid droplets. (C) Lipid contents of FMFE and BMFE groups. Different letters indicate significant differences at p < 0.05.
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Figure 3. Effect of FMFE and BMFE on the expression of mRNA related to differentiation in 3T3-L1 adipocytes. (A) PPARγ, (B) C/EBPα, (C) SREBP-1, (D) FAS, (E) perilipin, (F) FABP4, (G) HSL. Different letters indicate significant differences at p < 0.05.
Figure 3. Effect of FMFE and BMFE on the expression of mRNA related to differentiation in 3T3-L1 adipocytes. (A) PPARγ, (B) C/EBPα, (C) SREBP-1, (D) FAS, (E) perilipin, (F) FABP4, (G) HSL. Different letters indicate significant differences at p < 0.05.
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Figure 4. Effect of FMFE and BMFE on the expression genes related to differentiation in 3T3-L1 adipocytes. (A) PPARγ, (B) C/EBPα, (C) perilipin. Different letters indicate significant differences at p < 0.05.
Figure 4. Effect of FMFE and BMFE on the expression genes related to differentiation in 3T3-L1 adipocytes. (A) PPARγ, (B) C/EBPα, (C) perilipin. Different letters indicate significant differences at p < 0.05.
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Figure 5. Effect of FMFE and BMFE on the energy and lipid metabolism in 3T3-L1 adipocytes. (A) p-ACC, (B) p-AMPK. Different letters indicate significant differences at p < 0.05.
Figure 5. Effect of FMFE and BMFE on the energy and lipid metabolism in 3T3-L1 adipocytes. (A) p-ACC, (B) p-AMPK. Different letters indicate significant differences at p < 0.05.
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Figure 6. Anti-adipogenesis effect of FMFE and BMFE in 3T3-L1 adipocytes. Red arrow indicates the increase and decrease in the expression of genes and proteins, and the observed phenomena.
Figure 6. Anti-adipogenesis effect of FMFE and BMFE in 3T3-L1 adipocytes. Red arrow indicates the increase and decrease in the expression of genes and proteins, and the observed phenomena.
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Table 1. Primer sequences used in qRT-PCR analysis *.
Table 1. Primer sequences used in qRT-PCR analysis *.
GenesForward Primer (5′-3′)Reverse Primer (5′-3′)
C/EBP-αTGCTGGAGTTGACCAGTGACAAACCATCCTCTGGGTCTCC
PPAR-γTTTTCAAGGGTGCCAGTTTCAATCCAATCCTTGGCCCTCTGAGAT
PerilipinCTCTGGGAAGCATCGAGAAGGCATGGTGTGTCGAGAAAGA
FABP4AGACGACAGGAAGGTGAAGAGTCATAACACATTCCACCACCAG
FASTGCTGTTGGAAGTCAGCTATGAAGATGCCTCTGAACCACTCACAC
SREBP1CCGAGATGTGCGAACTGGAGAAGTCACTGTCTTGGTTGTTGATG
HSLTCCTGGAACTAAGTGGACGCAAGCAGACACACTCCTGCGCATAGAC
β-actinAGCCATGTACGTAGCCATCCTCCCTCTCAGCTGTGGTGGTGAA
* Primer sequences used in this study are shown in the table.
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Lee, D.J.; Yeom, J.H.; Lee, Y.K.; Joo, Y.H.; Chung, N. Anti-Obesity Effect of Fresh and Browned Magnolia denudata Flowers in 3T3-L1 Adipocytes. Appl. Sci. 2024, 14, 9254. https://doi.org/10.3390/app14209254

AMA Style

Lee DJ, Yeom JH, Lee YK, Joo YH, Chung N. Anti-Obesity Effect of Fresh and Browned Magnolia denudata Flowers in 3T3-L1 Adipocytes. Applied Sciences. 2024; 14(20):9254. https://doi.org/10.3390/app14209254

Chicago/Turabian Style

Lee, Deok Jae, Jae Ho Yeom, Yong Kwon Lee, Yong Hoon Joo, and Namhyun Chung. 2024. "Anti-Obesity Effect of Fresh and Browned Magnolia denudata Flowers in 3T3-L1 Adipocytes" Applied Sciences 14, no. 20: 9254. https://doi.org/10.3390/app14209254

APA Style

Lee, D. J., Yeom, J. H., Lee, Y. K., Joo, Y. H., & Chung, N. (2024). Anti-Obesity Effect of Fresh and Browned Magnolia denudata Flowers in 3T3-L1 Adipocytes. Applied Sciences, 14(20), 9254. https://doi.org/10.3390/app14209254

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