Anti-Obesity Activity of Giant Centella asiatica Lava Seawater Extract (GCA-LS-90) Through Regulation of Adipocyte Differentiation and Lipid Metabolism In Vitro

Abstract

Obesity is well-known as a major risk factor for metabolic disorders, and natural compounds are being explored as alternatives to conventional therapies. While Centella asiatica is well known for its medicinal and dietary benefits, the biological activities of Giant Centella asiatica (GCA), especially when extracted with mineral-rich lava seawater, remain poorly characterized. This study aimed to evaluate the anti-adipogenic and lipid-metabolism-regulating effects of a novel GCA extract (GCA-LS-90) and its ability to stimulate GLP-1 secretion in vitro. GCA-LS-90 significantly inhibited lipid accumulation in 3T3-L1 adipocytes by up to 24.3% at 200 µg/mL (p < 0.001). It downregulated adipogenic transcription factors (C/EBPβ, C/EBPα, PPARγ) and lipogenic regulators (SREBP1c, FAS, G6PD, ME), while upregulating KLF2 (all p < 0.001). Western blotting confirmed reduced SREBP1c and SREBP2 protein expression, increased phosphorylation of AMPKα/ACC, and enhanced HSL activity (p < 0.05–0.001). In STC-1 cells, GCA-LS-90 increased GLP-1 secretion (53.5 pmol/L at 90 µg/mL vs. 41.3 pmol/L in control, p < 0.001). The major compounds, 3,5- and 4,5-di-O-caffeoylquinic acids, reproduced these effects. In conclusion, GCA-LS-90 modulated adipogenesis-, lipid-metabolism-, and GLP-1 secretion-related pathways in vitro, suggesting its potential as a functional ingredient for obesity management. Further in vivo studies are needed to confirm efficacy and translational relevance.

1. Introduction

Obesity is a metabolic disorder marked by excess fat accumulation, which increases the risk of diabetes, cardiovascular disease, and other chronic illnesses [1]. With the rapidly increasing population-level prevalence of obesity [2,3,4], there is a growing demand for more effective drugs and therapeutic approaches for sustainable weight management. However, pharmaceutical medications often carry risks such as weight regain and muscle mass loss and have various adverse side effects [5,6], driving interest in the use of naturally occurring compounds to regulate lipid metabolism and reduce associated toxicity [7,8].

Adipogenesis, the process of forming new fat cells within adipose tissue, plays a critical role in obesity development [9]. A network of transcription factors, such as peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), sterol regulatory element-binding protein 1c (SREBP1c), and fatty acid binding protein 4 (FABP4), tightly regulate adipogenesis [10,11,12]. Inhibiting these regulators is a promising strategy for developing anti-obesity drugs and functional foods [13]. Modulating the activity of enzymes, including acetyl-CoA carboxylase (ACC) and hormone-sensitive lipase (HSL), and upstream sensors such as AMP-activated protein kinase (AMPK) has also been reported to impact the balance of lipogenesis and lipolysis [14].

Beyond adipocyte regulation, enteroendocrine hormones such as glucagon-like peptide-1 (GLP-1) also contribute to systemic energy homeostasis. GLP-1 enhances insulin secretion, delays gastric emptying, and reduces appetite, and pharmacological GLP-1 receptor agonists are now widely used as anti-obesity therapeutics [15]. Therefore, natural compounds capable of stimulating GLP-1 secretion may offer an additional mechanism to improve metabolic health.

Centella asiatica (CA), a medical plant, has traditionally been used for its anti-inflammatory, neuroprotective, and wound-healing properties [16]. More recently, CA and its active components, including madecassoside and asiatic acid, have demonstrated anti-obesity effects by inhibiting adipogenic transcription factors and activating AMPK pathways in both cellular and animal models [17,18]. Giant Centella asiatica (GCA), a variety of CA with larger leaves and different phytochemical constituents, has not been studied extensively for its lipid-metabolism- and obesity-related functions. Given its similarity to common CA, GCA is hypothesized to exhibit anti-obesity-related activities comparable to those of common CA. However, scientific evidence validating the anti-adipogenic potential of GCA, particularly when extracted using functional solvents such as mineral-rich lava seawater, remains limited. Lava seawater is known to enhance the extraction of bioactive compounds and may potentiate the functional properties of herbal extracts [19,20].

Lava seawater, obtained from Jeju Island, is seawater that has passed through volcanic rock layers and is enriched with minerals such as calcium, magnesium, and potassium. It has been reported to enhance the extraction of bioactive compounds and improve the antioxidant properties of plant materials [20].

The efficiency of phytochemical extraction is strongly influenced by the physicochemical properties of the solvent system. Recent studies have demonstrated that solvent composition can significantly affect the stability and recoverability of phenolic compounds during extraction [21]. In particular, tailored solvent systems have been shown to enhance polyphenol stability and disrupt complex interactions between phenolic compounds and plant biomass, including hydrogen bonding networks and protein–polyphenol complexes [22]. Such mechanisms facilitate the release of bound phenolics and improve extraction yield. Therefore, the unique mineral composition and ionic characteristics of Lava seawater may enhance the solubilization and stabilization of bioactive compounds, thereby contributing to improved biological activity.

This study was conducted to investigate the anti-adipogenic effects of lava seawater-processed GCA extracts in 3T3-L1 adipocytes by assessing lipid accumulation, gene expression, and protein signaling. The extracts’ ability to stimulate GLP-1 secretion in enteroendocrine STC-1 cell line (STC-1) cells was also examined, and active components contributing to their anti-obesity activity were identified. The results highlight the potential of GCA extracts as a functional food ingredient for obesity prevention.

2. Results

2.1. Inhibition of Lipid Accumulation in 3T3-L1 Cells

The anti-adipogenic potential of five GCA extracts (GCA-EtOH-75, GCA-W-60, GCA-W-90, GCA-LS-60, and GCA-LS-90) was evaluated in 3T3-L1 adipocytes at concentrations of 100 and 200 μg/mL. Lipid accumulation, assessed by Oil Red O staining (Figure 1), was reduced by 11.6% and 23.3% for GCA-EtOH-75, 10.1% and 23.4% for GCA-W-60, 15.3% and 19.9% for GCA-W-90, 11.3% and 22.0% for GCA-LS-60, and 20.7% and 24.3% for GCA-LS-90 at low and high concentrations, respectively. Among the extracts, GCA-LS-90 exhibited the strongest anti-adipogenic activity, showing significant reductions at both concentrations (p < 0.001) and a maximum inhibition of 24.3% at 200 μg/mL. Cell viability after GCA-LS-90 treatment remained at or near 90% at both concentrations (Figure S1). Among the five GCA extracts, GCA-LS-90, which showed significant anti-adipogenic activity at both 100 and 200 μg/mL, was selected for subsequent experiments.

2.2. Modulation of Adipogenic Gene Expression

GCA-LS-90 treatment modulated the mRNA expression of adipogenesis- and lipid-metabolism-related genes (Figure 2 and Figure 3). Early-stage transcription factor CCAAT/enhancer-binding protein β (C/EBPβ) was dose-dependently downregulated (p < 0.05–0.001), while Kruppel-like factor 2 (KLF2), a negative regulator of adipogenesis, was significantly upregulated at all concentrations (p < 0.001). The late-stage regulators C/EBPα and PPARγ, as well as the mature adipocyte marker FABP4, were significantly suppressed at all doses (p < 0.001). The lipogenic transcription factor sterol regulatory element-binding protein 1c (SREBP1c) and its downstream fatty acid synthase (FAS) were also significantly reduced in a dose-dependent manner, whereas sterol regulatory element-binding protein 2 (SREBP2) showed no significant change, although 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) expression decreased at 100 and 200 μg/mL. The nicotinamide adenine dinucleotide phosphate (NADPH)-producing enzymes glucose-6-phosphate dehydrogenase (G6PD) and malic enzyme (ME) were downregulated, indicating a reduction in lipogenic capacity.

2.3. Western Blot Analysis of Lipid-Metabolism-Related Proteins

GCA-LS-90 significantly modulated key proteins involved in lipid metabolism (Figure 4). SREBP1c expression was markedly downregulated (p < 0.001), which is consistent with the suppression of FAS and acetyl-CoA carboxylase (ACC). The p-ACC/ACC ratio increased, indicating functional inhibition via AMPK activation (p < 0.05–0.001). Hormone-sensitive lipase (HSL) phosphorylation was enhanced, supporting increased lipolysis (p < 0.01–0.001). Sterol regulatory element-binding protein 2 (SREBP2) and downstream HMGCR were significantly reduced (p < 0.01–0.001), reflecting inhibition of cholesterol biosynthesis.

Collectively, these findings indicate that GCA-LS-90 exerts multi-target regulatory effects on lipid metabolism. It suppresses lipogenesis by downregulating SREBP1c and functionally inhibiting ACC via AMPK-dependent phosphorylation; reduces cholesterol biosynthesis by downregulating SREBP2; and promotes lipolysis through HSL activation. Together, these data suggest that GCA-LS-90 modulated multiple lipid metabolism-related pathways in 3T3-L1 adipocytes, suggesting its further evaluation as a functional ingredient; however, validation in vivo is required.

2.4. Bioactive Compounds Identified in GCA-LS-90

To identify the major bioactive active compounds potentially responsible for GCA-LS-90’s anti-obesity effects, an Orbitrap-based UHPLC–MS/MS analysis was performed. This comprehensive chromatographic profiling detected a total of 21 compounds, including chlorogenic acid, dicaffeoylquinic acid, miquelianin, cynaroside, and scutellarin (Figure S2).

Based on the UHPLC-MS/MS findings, a total of 21 phenolic and flavonoid compounds were detected in GCA-LS-90 (Figures S2 and S3, Table S2). Among these, five representative phenolic compounds, chlorogenic acid, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, miquelianin, and cymaroside, were selected for subsequent quantitative analysis using HPLC (Figure S3 and

Table 1. Quantification of selected phenolic compounds in GCA-LS-90 by HPLC analysis.

Chemical Name	Contents in GCA-LS-90 (mg/g)
Cynaroside	N.D.
Miquelianin	0.70 ± 0.00
Scutellarin	N.D.
3,5-Di-caffeoylquinic acid	3.50 ± 0.01
4,5-Di-caffeoylquinic acid	1.51 ± 0.01

The contents of five representative phenolic compounds in GCA-LS-90 were analyzed using HPLC. Values are expressed as mg per gram of extract (mean ± SD, n = 3). N.D. = not detected.

). Identification of these compounds was confirmed by comparison of retention times and UV spectra (350 nm) with authenticated reference standards, and quantification was performed using external calibration curves. The results showed that 3,5-di-O-caffeoylquinic acid (3.50 mg/g) and 4,5-di-O-caffeoylquinic acid (1.51 mg/g) were the most abundant phenolic constituents in GCA-LS-90 (Table 1). Other detected compounds such as miquelianin and cynaroside were present in minor amounts or below the limit of detection in HPLC quantification.

These results indicate that di-O-caffeoylquinic acid (DCQA), particularly the 3,5- and 4,5-isomers, is the predominant phenolic constituent of GCA-LS-90. Given their high abundance and the recapitulation of key transcriptional changes, 3,5- and 4,5-di-O-caffeoylquinic acids may contribute, at least in part, to the observed in vitro effects of GCA-LS-90.

2.5. Effects of Identified Bioactive Compounds in GCA-LS-90 on mRNA Expression of Lipid Metabolism Genes

Based on our previous finding that GCA-LS-90 treatment significantly downregulated the expression of SREBP1c and SREBP2 (Figure 3A,C), we selected these two genes for further analysis. To evaluate the specific contributions of the major phenolic compounds in GCA-LS-90, 3T3-L1 adipocytes were treated with 3,5-di-O-caffeoylquinic acid and 4,5-di-O-caffeoylquinic acid. The concentration of each compound was adjusted to match its content in 50, 100, and 200 μg/mL of GCA-LS-90.

As shown in Figure 5A,B, both 3,5-di-O-caffeoylquinic acid and 4,5-di-O-caffeoylquinic acid significantly suppressed SREBP1c and SREBP2 mRNA levels in a dose-dependent manner (p < 0.01 to p < 0.001), mirroring the gene expression and protein suppression patterns observed following GCA-LS-90 administration (Figure 3A,C).

To evaluate the combined effects, low- and high-dose mixtures of both compounds were treated. Both the low-dose mixture (0.35 μg/mL of 3,5-di-O-caffeoylquinic acid and 0.151 μg/mL of 4,5-di-O-caffeoylquinic acid) and high-dose mixture (0.7 μg/mL and 0.302 μg/mL, respectively) significantly inhibited SREBP1c and FAS expression (p < 0.001; Figure 5C,D). Similarly, the expression of SREBP2 and its downstream target HMGCR was significantly reduced in the mixture-treated cells (p < 0.001; Figure 5E,F).

These findings are consistent with our prior Western blot data (Figure 4), which showed that GCA-LS-90 markedly decreased protein levels of SREBP1, SREBP2, FAS, and HMGCR.

2.6. Stimulatory Effects of GCA-LS-90 on GLP-1 Secretion in STC-1 Cells

Non-toxic concentrations of GCA-LS-90 (<100 µg/mL) were determined using a WST assay (Supplementary Figure S1). GLP-1 secretion was significantly reduced in the CON cells (41.29 pmol/L) compared with the NOR cells (62.88 pmol/L, p < 0.001). Treatment with 70 µg/mL and 90 µg/mL of GCA-LS-90 restored GLP-1 secretion to 44.50 pmol/L and 53.54 pmol/L (p < 0.001 vs. CON), respectively, whereas 50 µg/mL showed no significant effect (38.71 pmol/L) (Figure 6). These results demonstrate that GCA-LS-90 enhances GLP-1 secretion in a concentration-dependent manner within the non-toxic range.

3. Discussion

Centella asiatica, commonly known as “Gotu Kola”, has long been utilized in traditional medicine for its wound-healing, anti-inflammatory, and neuroprotective properties and has been applied in cosmetics and nutraceuticals [23]. Beyond its well-established pharmacological effects, emerging studies suggest that C. asiatica extracts may modulate lipid metabolism and adipogenesis, indicating potential anti-obesity benefits [18]. Notably, our study utilized an extract of Giant Centella asiatica, a distinct cultivar bred for its enhanced content of key bioactive compounds [24], in contrast to the traditional variety.

In this study, GCA-LS-90 significantly inhibited lipid accumulation in 3T3-L1 adipocytes (Figure 1). This effect was accompanied by the downregulation of key adipogenic transcription factors, which orchestrate the complex process of adipocyte differentiation. These include early-stage C/EBPβ and late-stage C/EBPα and PPARγ, all of which are recognized as master regulators that drive the transcriptional cascade leading to adipogenesis [25]. Furthermore, the anti-adipogenic factor KLF2 was upregulated (Figure 2). Additionally, markers of mature adipocytes, such as FABP4, which is a crucial protein for intracellular lipid transport, and lipogenic regulators, including SREBP1c, a master regulator of fatty acid synthesis, FAS, G6PD, and ME (which are key enzymes in lipogenesis), were suppressed in a dose-dependent manner (Figure 2 and Figure 3).

In parallel, Western blot analysis confirmed reduced SREBP1c and SREBP2 protein expression (Figure 4) and increased phosphorylation of AMPKα and ACC. Phosphorylation of AMPKα leads to the inactivation of ACC, a rate-limiting enzyme in fatty acid synthesis, thus promoting lipolysis and inhibiting lipid synthesis [26]. Enhanced HSL activity was also observed, which is the primary enzyme responsible for the breakdown of stored triglycerides into free fatty acids and glycerol [27]. These molecular alterations underscore the dual mechanism of GCA-LS-90 in modulating adipocyte differentiation and lipid metabolism, which are central processes in the development of obesity.

GLP-1, an incretin hormone secreted by intestinal L-cells, was also stimulated by GCA in STC-1 cells in a concentration-dependent manner (Figure 6). Beyond its classical role in glucose homeostasis, GLP-1 exerts anti-obesity effects by reducing appetite, slowing gastric emptying, and increasing energy expenditure [15]. GCA’s ability to enhance endogenous GLP-1 secretion may provide an additional indirect mechanism contributing to its anti-adipogenic and lipid-lowering effects in 3T3-L1 cells.

The major bioactive compounds in GCA-LS-90—3,5-di-O-caffeoylquinic acid and 4,5-di-O-caffeoylquinic acid—were present at the highest concentrations (Table 1, Figure S3). These dicaffeoylquinic acids have been reported to modulate adipocyte differentiation and lipid metabolism: 3,5-DCQA inhibits PPARγ-mediated adipogenesis and enhances AMPK activation, reducing lipogenesis and promoting lipolysis [28,29], while 4,5-DCQA exerts similar effects by downregulating the SREBP1c-FAS and SREBP2-HMGCR pathways [30]. Consistently, treatment of 3T3-L1 cells with these compounds individually and in combination reproduced the gene expression patterns observed with GCA-LS-90, confirming their central role in mediating the extract’s anti-obesity effects (Figure 5). This indicates that the high content of 3,5-DCQA and 4,5-DCQA in GCA-LS-90 is likely responsible for the suppression of adipogenic and lipogenic pathways, contributing to reduced lipid accumulation and altered adipocyte differentiation.

The inhibition of C/EBPβ, C/EBPα, PPARγ, and FABP4 observed in our study highlights interference at both early and late stages of adipocyte differentiation, potentially preventing the maturation of preadipocytes into lipid-laden adipocytes. Concurrent suppression of SREBP1c, FAS, G6PD, ME, and SREBP2-HMGCR further indicates reduced lipogenesis and cholesterogenesis, reflecting a multi-targeted modulation of lipid metabolism (Figure 2, Figure 3 and Figure 4). Such coordinated regulation is critical, as excessive adipogenesis and lipid accumulation are hallmarks of obesity and metabolic syndrome.

Taken together, the present findings suggest a coordinated mechanism by which GCA-LS-90 regulated lipid metabolism. Activation of AMPK signaling appeared to play a central role, leading to ACC phosphorylation and subsequent suppression of fatty acid synthesis. In parallel, GCA-LS-90 downregulated key adipogenic and lipogenic transcription factors, including C/EBPβ, C/EBPα, PPARγ, and SREBP1c, while also reducing cholesterol synthesis-related regulators such as SREBP2 and HMGCR. Furthermore, increased phosphorylation of HSL was associated with enhanced lipolysis. In STC-1 cells, GCA-LS-90 stimulated GLP-1 secretion, suggesting an additional metabolic regulatory pathway. The integrated working model derived from these findings is summarized in Figure 7.

Despite these promising in vitro findings, several limitations should be acknowledged. First, our study was restricted to 3T3-L1 and STC-1 cell lines, which may not fully replicate the complex physiology of adipose tissue and enteroendocrine signaling in vivo. Second, although the concentrations used were non-toxic in vitro, they may differ from achievable levels in systemic circulation. Third, potential interactions among other minor bioactive compounds in GCA-LS-90 were not fully explored, and the contribution of triterpenoids relative to DCQA derivatives remains to be clarified.

4. Materials and Methods

4.1. Materials

3T3-L1 preadipocyte cells were obtained from the Korean Cell Line Bank (Seoul, Republic of Korea). Culture media and reagents, including high-glucose DMEM, FBS, penicillin–streptomycin, and IBMX, were purchased from Thermo Fisher Scientific (Gibco, Waltham, MA, USA). Dexamethasone, insulin, Oil Red O, cynaroside, miquelianin, scutellarin, 3,5-DCQA (Isochlorogenic acid A), and 4,5- DCQA (Isochlorogenic acid C) were obtained from Sigma-Aldrich (St. Louis, MO, USA).

4.2. GCA Extract Preparation

GCA leaves were dried and subjected to extraction in various solvents, including distilled water (DW), lava seawater (LS), and ethanol (EtOH). Each extraction was conducted by incubating 30 g of dried plant material in 600 mL of solvent at temperatures of 60 °C, 75 °C, or 90 °C for 4 h. After filtration, the separated supernatant was concentrated under reduced pressure at 50 °C using a rotary evaporator (Eyela N-1200, Tokyo Rikakikai Co., Tokyo, Japan). Maltodextrin (10% w/v) was added, and the mixture was subsequently dried using a spray dryer (Buchi B-290, BÜCHI Labortechnik AG, Flawil, Switzerland) to obtain a powder. The final product was stored at −4 °C until use. The extract samples were supplied by ASK Labs Co. (Daejeon, Republic of Korea). The extract yield of GCA-LS-90 was 41%, with a flavonoid content of 14.3 µg/mg extract and a total polyphenol content of 32.64 µg/mg extract. The extraction yield of GCA-EtOH-05 was 30%, with a total polyphenol content of 34.73 µg/mg extract and a flavonoid content of 3.0 µg/mg extract.

4.3. T3-L1 Cell Culture and Differentiation

3T3-L1 cells were cultured in high-glucose DMEM with 10% FBS and 1% penicillin–streptomycin at 37 °C in a humidified atmosphere of 5% CO₂. Differentiation was induced using 0.5 mM IBMX, 10 µg/mL insulin, and 1 µM dexamethasone at confluence (Day 0), followed by insulin-only treatment (Day 2) and basal medium thereafter. GCA extracts were administered over the entire differentiation period. GCA extracts were administered at final concentrations of 50, 100, and 200 µg/mL over the entire differentiation period. On Day 9, mature adipocytes were identified based on lipid droplet formation and used for subsequent analyses. For compound treatment experiments, differentiated 3T3-L1 cells were treated with 3,5-di-O-caffeoylquinic acid (0.350–1.050 μg) or 4,5-di-O-caffeoylquinic acid (0.151–0.453 μg). For mixture experiments, the two compounds were combined at 0.700 μg + 0.302 μg (total 1.002 μg) or 1.050 μg + 0.453 μg (total 1.501 μg), as indicated in the figures, and administered during the differentiation period.

4.4. Lipid Accumulation Assessment via Oil Red O Staining

Intracellular lipid accumulation was assessed in fully differentiated adipocytes using Oil Red O staining. A stock solution (0.5 g of Oil Red O in 60 mL of 2-propanol) was filtered and diluted with distilled water (3:2, v/v) to prepare the working solution. Differentiated cells were fixed with 10% formalin at 4 °C for 1 h, rinsed, dried at 40 °C, and stained for 20 min in the dark. After removing excess dye, the lipids were eluted with 2-propanol, and absorbance was measured at 520 nm using a microplate reader (Infinite M Nano, Tecan, Switzerland). Absorbance values were normalized to the differentiated control (CON) group and are expressed as a percentage of CON.

4.5. qRT-PCR

Total RNA was extracted from differentiated 3T3-L1 cells using TRIzol reagent (Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA, USA). One microgram of RNA was reverse-transcribed into cDNA. Quantitative real-time PCR was performed using SYBR Green Master Mix (Thermo Fisher Scientific, Inc., Waltham, MA, USA) on an Applied Biosystems StepOnePlus Real-Time PCR System (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Target gene expression related to adipogenesis and lipid metabolism was normalized against β-actin as the reference gene, and relative expression levels were calculated using the 2^−ΔΔCT method. The specific primer sequences used are provided in Table S1.

4.6. Western Blot

Cells were lysed using a homemade lysis buffer consisting of 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.1% SDS, and 0.5% sodium deoxycholate, supplemented with protease and phosphatase inhibitor cocktails (Sigma-Aldrich, St. Louis, MO, USA). Equal amounts (30 µg of protein) were separated by 10% SDS-PAGE, transferred to 0.45 µm PVDF membranes (Immoblilon-P; Merck KGaA, Darmstadt, Germany), blocked with 5% skim milk. Membranes were incubated overnight at 4 °C with primary antibodies (1:1000 dilution; Cell Signaling Technology, Inc., Danvers, MA, USA) After washing, the membranes were treated with HRP-conjugated secondary antibodies and developed using ECL detection reagent (D-Plus™ ECL Pico Alpha Solution A/B, Dongin LS, Seoul, Republic of Korea). Bands were quantified using ImageJ software version 1.50g (National Institutes of Health, Bethesda, MD, USA). Densitometric analysis was performed using ImageJ software. Phosphorylated protein levels were normalized to their corresponding total protein levels, and total protein expression was normalized to β-actin.

4.7. UHPLC–MS/MS Analysis of GCA Extract

The GCA extract was analyzed by UHPLC–MS/MS according to the previously described method [24]. An Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.7 µm; Waters Corp., Milford, MA, USA) with an LTQ XL™ Orbitrap mass spectrometer HESI source (Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used for separation. The mobile phase gradient ranged from 5% to 100% acetonitrile with 0.1% formic acid. The flow rate was 400 µL/min, and the injection volume was 400 µL (15 mg/mL). Data were acquired in positive and negative modes over m/z 150–1500. Key MS parameters included spray voltage of 5.0 kV, capillary voltage of 35 V, capillary temperature of 300 °C, and sheath/auxiliary gas flows of 50/5. Data were processed using Xcalibur software version 4.1 (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Metabolite identification was tentatively performed based on accurate mass measurements and MS/MS fragmentation pattern comparison with previously reported data. Selected compounds were further confirmed by comparison with authenticated reference standards during HPLC-DAD analysis.

4.8. High-Performance Liquid Chromatography (HPLC) Analysis of Dicaffeoylquinic Acids

HPLC analysis was performed using an Agilent 1260 Infinity II system (Agilent Technologies, Inc., Santa Clara, CA, USA) equipped with a diode array detector and a SunFire C18 column (4.6 × 250 mm, 5 µm; Waters Corp., Milford, MA, USA). The mobile phase consisted of 0.1% trifluoroacetic acid in water (solvent A) and acetonitrile (solvent B). The gradient elution profile was as follows: 10% B for 0–8 min, an increase to 20% B from 8 to 15 min, a gradual increase to 23% B between 15 and 40 min, ramping to 100% B from 40 to 45 min, 100% B until 50 min, re-equilibration to 10% B by 54 min, and 10% B until 60 min. The flow rate was 1.0 mL/min, with a 10 µL injection volume of a 30 mg/mL sample, and detection was monitored at 350 nm. Identification of phenolic compounds was confirmed by comparing retention times and UV spectra (350 nm) with authenticated reference standards. Quantification was performed using external calibration curves constructed from the corresponding authentic standards.

4.9. STC-1 Cell Culture

The intestinal secretin tumor cell line (STC-1) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin–streptomycin under a humidified atmosphere of 5% CO₂ at 37 °C.

4.10. Cell Viability and GLP-1 Secretion in STC-1 Cells

Cell viability was assessed using a WST assay (Biomax, Seoul, Republic of Korea). STC-1 cells were seeded in 96-well plates at a density of 1 × 10⁵ cells/mL and incubated for 24 h. The cells were then treated with various concentrations of GCA (25–200 μg/mL) and palmitic acid (PA, 0.1–1.0 mM) for 4 h. Following treatment, the WST reagent was added, and absorbance was measured at 450 nm using a Infinite M Nano microplate reader (Tecan Group Ltd., Männedorf, Switzerland) controlled by Magellan™ software (version 7.2; Tecan Group Ltd.). Non-toxic concentrations determined from this assay were used for subsequent GLP-1 secretion studies.

STC-1 cells were seeded in 12-well plates at 0.5 × 10⁶ cells/mL and incubated for 24 h. After incubation, the cells were washed with Krebs–Ringer buffer containing 11 mM glucose and acclimatized in the buffer for 1 h. To simulate a high-fat environment, the cells were pretreated with PA (0.1–0.5 mM) for 4 h. Subsequently, the cells were exposed to GCA-LS-90 (50–90 µg/mL) or a buffer alone for an additional 4 h. Supernatants were collected after the addition of protease/phosphatase inhibitors, centrifuged at 900× g for 5 min at 4 °C, and stored at −80 °C. Active GLP-1 levels in the supernatants were quantified using a Mouse GLP-1 (Active) ELISA Kit (Crystal Chem, Elk Grove Village, IL, USA) according to the manufacturer’s protocol.

4.11. Statistical Analysis

All data are expressed as means ± standard deviation (SD) and were analyzed using IBM SPSS Statistics software version 26.0 (IBM Corp., Armonk, NY, USA). Differences among groups were evaluated by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. A p-value less than 0.05 was considered statistically significant.

5. Conclusions

This study provides mechanistic insights into the anti-obesity potential of GCA-LS-90, a Giant Centella asiatica preparation. Our findings demonstrate GCA-LS-90’s multi-target activity, through which it inhibits adipocyte differentiation, suppresses lipogenesis, promotes lipolysis, and enhances GLP-1 secretion. These effects appear to be predominantly mediated by 3,5-di-O-caffeoylquinic acid and 4,5-di-O-caffeoylquinic acid. Because these findings were obtained in cell-based models, further studies in appropriate in vivo systems are needed to establish physiological relevance.

Future studies should include in vivo models of diet-induced obesity to evaluate the systemic efficacy of GCA-LS-90 and its impact on GLP-1-mediated metabolic regulation. Further research isolating individual bioactive compounds or their combinations could clarify synergistic mechanisms and optimize formulations for potential nutraceutical applications. This research supports the further development of Giant Centella asiatica as a functional food or nutraceutical for obesity management.