The Antioxidant Properties of Lavandula multifida Extract Contribute to Its Beneficial Effects in High-Fat Diet-Induced Obesity in Mice

Citation: Molina-Tijeras, J.A.; Ruiz- Malagón, A.J.; Hidalgo-García, L.; Diez-Echave, P.; Rodríguez-Sojo, M.J.; Cádiz-Gurrea, M.d.l.L.; Segura- Carretero, A.; del Palacio, J.P.; González-Tejero, M.R.; Rodríguez- Cabezas, M.E.; et al. The Antioxidant Properties of Lavandula multifida Extract Contribute to Its Beneficial Effects in High-Fat Diet-Induced Obesity in Mice. Antioxidants 2023, 12, 832. https://doi.org/10.3390/ antiox12040832 Academic Editor: Stanley Omaye Received: 2 March 2023 Revised: 23 March 2023 Accepted: 27 March 2023 Published: 29 March 2023 Copyright: © 2023 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/). antioxidants Article The Antioxidant Properties of Lavandula multifida Extract Contribute to Its Beneficial Effects in High-Fat Diet-Induced Obesity in Mice Jose Alberto Molina-Tijeras 1,2,† , Antonio Jesús Ruiz-Malagón1,2,† , Laura Hidalgo-García1,2,*, Patricia Diez-Echave 1,2, María Jesús Rodríguez-Sojo 1,2, María de la Luz Cádiz-Gurrea 3, Antonio Segura-Carretero 3, JoséPérez del Palacio 4, María Reyes González-Tejero 5, María Elena Rodríguez-Cabezas 1,2 , Julio Gálvez 1,2,6 , Alba Rodríguez-Nogales 1,2,* , Teresa Vezza 1,2,7,‡ and Francesca Algieri 1,2,‡ 1Department of Pharmacology, Center for Biomedical Research (CIBM), University of Granada, 18071 Granada, Spain 2Instituto de Investigación Biosanitaria de Granada (ibs. GRANADA), 18012 Granada, Spain 3Department of Analytical Chemistry, Faculty of Science, University of Granada, 18071 Granada, Spain 4Fundación MEDINA, Centro de Excelencia en Investigación de Medicamentos Innovadores en Andalucía, 18016 Granada, Spain 5Department of Botany, University of Granada, 18071 Granada, Spain 6Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Instituto Salud Carlos III, 28029 Madrid, Spain 7Servicio de Digestivo, Hospital Universitario Virgen de las Nieves, 18014 Granada, Spain *Correspondence: [email protected] (L.H.-G.); [email protected] (A.R.-N.); Tel.: +34-958241519 (A.R.-N.) † These authors contributed equally to this work. ‡ These authors contributed equally to this work. Abstract: Obesity is a worldwide public health problem whose prevalence rate has increased steadily over the last few years. Therefore, it is urgent to improve the management of obesity and its comorbidities, and plant-based treatments are receiving increasing attention worldwide. In this regard, the present study aimed to investigate a well-characterized extract of Lavandula multifida (LME) in an experimental model of obesity in mice and explore the underlying mechanisms. Interestingly, the daily administration of LME reduced weight gain as well as improved insulin sensitivity and glucose tolerance. Additionally, LME ameliorated the inflammatory state in both liver and adipose tissue by decreasing the expression of various proinflammatory mediators (Il-6,Tnfα ,Il-1 β ,Jnk-1, Ppar α ,Ppar γ , and Ampk) and prevented increased gut permeability by regulating the expression of mucins (Muc-1,Muc-2, and Muc-3) and proteins implicated in epithelial barrier integrity maintenance (Ocln,Tjp1, and Tff-3). In addition, LME showed the ability to reduce oxidative stress by inhibiting nitrite production on macrophages and lipid peroxidation. These results suggest that LME may represent a promising complementary approach for the management of obesity and its comorbidities. Keywords: obesity; high-fat diet; insulin resistance; inflammation; Lavandula multifida 1. Introduction Obesity is recognized as one of the most prevalent worldwide public health problems of the modern era. According to the World Health Organization, it is an ongoing epidemic that knows no borders and keeps growing, despite the policy interventions implemented in the last few years. It is estimated that 2.7 billion adults will be overweight, and over 1 billion obese by 2025 [ 1 ]. Obesity is a chronic disease with a complex etiopathogenesis involving genetic, biological, behavioral, psychological, cultural, and socioeconomic factors [ 2 ]. It is driven by a continual positive energy balance that gives rise to excessive body fat accumulation. Moreover, obesity increases the risk of chronic diseases such as Antioxidants 2023,12, 832. https://doi.org/10.3390/antiox12040832 https://www.mdpi.com/journal/antioxidants Antioxidants 2023,12, 832 2 of 19 cardiovascular diseases, type 2 diabetes, and different types of cancer that can worsen the quality of life and reduce life expectancy [3–5]. Different anti-obesity drugs are available on the market, but most of them show important side effects and lack long-term efficacy [6]. This limitation has led to the search for alternative and/or complementary approaches for treating and preventing this disorder. Herbal remedies and functional foods of plant origin have been the basis of traditional medicine and are commonly used by obese patients [ 7 ]. One of the plant families most often applied in traditional medicine is Lamiaceae, which is very abundant in the degraded areas of the Mediterranean maquis and rocky, calcareous, or sandy soils. The genus Lavandula (lavender) includes 39 species that are traditionally employed to treat different diseases, including diabetes [ 8 , 9 ], inflammatory conditions, and digestive complaints [ 10 , 11 ]. It has been described that these crude drugs are rich in a wide range of secondary metabolites that are responsible for their beneficial properties, including antimicrobial, antifungal, and antioxidant activities [ 9 ]. Among these Lavandula species, L.multifida has been traditionally used against rheumatism and other inflammatory diseases [ 10 ], as well as for the treatment of diabetes and infectious diseases [ 12 ]. This study intended to assess the effects of a characterized hydroalcoholic extract of L. multifida (LME) on an experimental model of diet-induced obesity in mice, paying special attention to its impact on the altered metabolic and inflammatory status. Furthermore, the antioxidant and anti-adipogenic properties of the extract were also assessed in vitro in RAW 264 macrophages and 3T3-L1 MBX preadipocytes, respectively. 2. Materials and Methods 2.1. Chemicals and Reagents The analytical procedures were carried out by employing water purified by a Milli-Q system from Millipore (Bedford, MA, USA), LC-MS grade acetonitrile, and acetic acid, which were purchased from Fisher Chemicals (Waltham, MA, USA), and Sigma-Aldrich (Steinheim, Germany), respectively. The following reagents were provided from the specified suppliers: sodium carbonate, acetic acid, TPTZ (2,4,6-tris(2-pyridyl)-s-triazine), sodium hydroxide, and hydrochloric acid were acquired from Fluka (Honeywell, NC, USA). Absolute ethanol was purchased from Riedel-de-Haën (Honeywell, NC, USA). Gallic acid, Folin reagent, ABTS (2,2 0 -azinobis (3-ethylbenzothiazoline-6-sulphonate)), potassium persulfate, Trolox (6-hydroxy-2,5,7,8- tetramethylchroman-2-carboxylic acid), sodium acetate, ferric chloride, heptahydrate ferrous sulfate, fluorescein, AAPH (2,2 0 -azobis(2-amidinopropane) dihydrochloride), sodium phosphate monobasic, and dibasic were obtained from Sigma-Aldrich (St. Louis, MO, USA). All chemicals, unless otherwise indicated, were purchased from Sigma-Aldrich (Merck Life Science S.L.U., Madrid, Spain). 2.2. Plant Material and Preparation of the Extract The aerial parts of L. multifida were collected in Ízbor (36 ◦ 53 0 86 00 N 3 ◦ 30.08 0 25 00 W; Granada, Spain) in April 2015. The plant was identified and authenticated by Dr. M. R. González-Tejero and Dr. J.A. Hita from the department of Botany of the University of Granada, Spain. The voucher specimens corresponding to L. multifida L. (GDA 62621) were deposited in the herbarium of the University of Granada (Granada, Spain). The plant extracts were prepared as described before [ 13 ]. Briefly, 5 g of ground plant material was mixed with washed sea sand (Panreac Química S.A.U., Castellar del Vallès (Barcelona, Spain) and extracted with 30 mL of methanol 50% (v/v) at 1500 PSI and 80 ◦ C for 10 min in an ASE200 extraction system (Dionex Corporation, Sunnyvale, CA, USA). After two extraction cycles, liquid extracts were pooled, and the solvent evaporated under a vacuum at 60 ◦ C. The extraction efficiency for L. multifida (expressed as a percentage w/w) was 17%. Antioxidants 2023,12, 832 3 of 19 2.3. Chemical Composition of LME Using UHPLC-MS Conditions The qualitative characterization of LME was carried out using a specifically ACQUITY UPLC H-Class System (Waters, Milford, MA, USA) coupled to an electrospray quadrupoletime of flight mass spectrometer (ESI-qTOF-MS, Synapt G2, Waters Corp., Milford, MA, USA), working in negative-ion mode over a range from 50 to 1200 m/zand following the literature [ 14 ] with minor changes. Briefly, the dried extract was redissolved to a concentration of 5 mg/mL and filtered through a 0.2 µ m filter before analysis. The separation was carried out in an ACQUITY UPLC BEH Shield RP18 Column, 130 Å, 1.7 µ m, 2.1 mm × 150 mm. The injection volume was 10 µ L, and the phytochemical separation was performed at room temperature according to this multistep gradient: 0.0 min 99% A; 2.33 min 99% A; 4.37 min 93% A; 8.11 min 86% A; 12.19 min 76% A; 15.99 min 60% A; 18.31 min 2% A; 21.03 min 2% A; 22.39 min 99% A; and 25.0 min 99% A. The mobile phases were acidified water (0.5% acetic acid, v/v) as solvent A and acetonitrile as solvent B. Finally, the total flow rate was fixed at 0.7 mL/min. MS acquisition was performed using 2 parallel scan functions by rapid switching, in which 1 scan was operated at low collision energy in the gas cell (4 eV) and the other at high collision energy (MSE energy linear ramp: from 20 to 60 eV); desolvation gas flow = 700 L/h, desolvation temperature = 500 ◦ C, cone gas flow = 50 L/h, source temperature = 100 ◦ C, capillary voltage = 2.2 kV, cone voltage = 30 V, and collision energy = 20 eV. The scan duration was 0.1 s, and the resolution was 20,000 FWHM. The MS data were managed with the open-source software MZmine 2.53. 2.4. Total Phenolic Content and Antioxidant Capacity Assays Folin–Ciocalteu (TPC), FRAP, TEAC, and ORAC were performed on a Synergy H1 Monochromator-Based Multi-Mode Microplate reader (Bio-Tek Instruments Inc., Winooski, VT, USA) following the methodology previously reported [ 15 ]. All measurements were made in triplicate. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) test was made to evaluate the free radical scavenging activity of LME [ 15 ]. Briefly, LME, gallic acid, epicatechin, and the positive control ascorbic acid were dissolved in methanol to reach a range of concentrations of 0.1–100 µ g/mL. Then, 10 µ L of each methanolic dilution was mixed with 90 µ L of phosphate buffer at pH = 7 and a 200 µ L of a DPPH solution at 100 µ M. The plate was protected from light and incubated for 30 min at 25 ◦ C. After incubation, the scavenging activity of each compound was measured at 515 nm in a Magellan ® Tecan Infinite F50 spectrophotometer (Tecan Group Ltd., Männedorf, Switzerland). The percentage of radical DPPH scavenging activity (% RSA) was calculated for each concentration employing the following equation: RSA (%) = [(Abs Blank −Abs Sample)/Abs Blank] ×100 where Abs Blank and Abs Sample are the absorbance values at 515 nm of the blank and samples, respectively. The blank absorbance value is the maximum and corresponds with the highest levels of the DPPH radical. Then, the half-maximal inhibitory concentration (IC50) value was calculated for each compound tested. 2.5. In Vitro Studies RAW 264 cells (a mouse macrophage cell line) and 3T3-L1 MBX cells (a mouse preadipocyte cell line) were obtained from the Cell Culture Unit at the University of Granada (Granada, Spain). Cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, ThermoFisher Scientific, Waltham, MA, USA) supplemented with a 10% heat-inactivated fetal bovine serum (FBS) for RAW 264 cells or heat-inactivated fetal calf serum (FCS) for 3T3-L1 MBX cells, L-glutamine (2 mmol/L), penicillin (100 units/mL), and streptomycin (100 units/mL) in a humidified 5% CO2atmosphere at 37 ◦C. RAW 264 cells were seeded into 96-well plates at a density of 5 × 10 5 cells/well and grown until the formation of a monolayer. Then, they were pre-incubated with different concentrations of LME ranging from 0.1 to 100 µ g/mL for 2 h and stimulated with the Antioxidants 2023,12, 832 4 of 19 lipopolysaccharide (LPS) from Escherichia coli 055:B5 (100 ng/mL) for 24 h. For negative and positive controls, untreated unstimulated and LPS-stimulated cells were used, respectively. After the stimulation period, the supernatants were harvested for nitrite determination by the Griess Assay [ 16 ]. An amount of 100 µ L of cell supernatant were mixed with 100 µ L of Griess reagent (0.1% N-(1-naphthy) ethylenediamine solution and 1% sulphanilamide in 5% (v/v) phosphoric acid solution, mixed in a proportion 1:1) and incubated for 10 min. A colored azolic compound was formed and its concentration was measured by a photometric measurement of the absorbance at 550 nm. Cell viability of tested conditions was evaluated by the MTS-based CellTiter 96 ® AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA), being the cellular viability estimated from the absorbance value and by comparing it with the untreated control cells. 3T3-L1 MBX cells were seeded into 6-well plates at 5 × 10 6 cells/well density. 2 days soon after cells reached confluence (day 0), different prodifferentiative agents (0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 5 µ M dexamethasone, and 10 µ g/mL insulin) were added to the medium to initiate cell differentiation. After 48 h (day 2), the culture medium was changed to DMEM and 10% fetal bovine serum (FBS) contained only insulin (10 µ g/mL). On day 4, the medium was replaced with DMEM and 10% FBS, and the cells were incubated for an additional 2 days until fully differentiated adipocyte-like cells were obtained. Treatment of 3T3-L1 cells with LME (10, 25, and 100 µ g/mL) was initiated on day 0 and added whenever the medium was replaced. Images from 3T3-L1 cells were taken to visualize adipocyte differentiation on days 2, 4, and 6. 2.6. Animals and Experimental Design The study was carried out following the “Guide of the Care and Use of Laboratory animals” as promulgated by the National Institute of Health, and all procedures were approved by the Ethics Committee of Laboratory Animals at the University of Granada (Spain) (Ref. No. 28/03/2016/030). A total of 8-week-old male C57BL/6 mice acquired from Janvier labs (St. Berthevin, Cedex, France) were housed in Makrolon cages, kept under controlled light-dark cycles (12 h light/dark), temperature and relative humidity (22 ± 1 ◦ C, 55 ± 10%), and with free access to tap water. They were randomly assigned to 3 groups (n= 8): control diet (CD), high-fat diet (HFD), and HFD-treated group with LME (25 mg/kg) dissolved in water by gavage during all the experimental procedures. Standard chow diet (13% calories from fat, 20% calories from protein, and 67% calories from carbohydrate; Global diet 2014) and high-fat diet (59% calories from fat, 13% calories from protein, and 28% calories from carbohydrate; Purified diet 230 HF) were purchased from Harlan Laboratories (Barcelona, Spain) and Scientific Animal Food and Engineering (Augy, France). Mice were fed the diets and treated with the extract for 30 days. Animal body weight and food and water intake were monitored, and energy efficiency was estimated as the ratio of weight gain (g) to caloric intake (Kcal). Mice were sacrificed by cervical dislocation, and liver and abdominal and epididymal fat were collected, cleaned, and weighed. Fat/weight index was calculated by dividing body weight by tibia length. The samples were frozen in liquid nitrogen and stored at −80 ◦C until additional analysis. 2.7. Glucose Tolerance Test During 1 week before the sacrifice, mice fasted for 8 h and were given a 2 g/kg of body weight glucose solution by intraperitoneal injection. Blood was sampled from the tail vein at 0, 15, 30, 60, and 120 min after injection. A handheld glucometer (Contour XT, Ascensia Diabetes Care, S.L., Barcelona, Spain) was used to determine glucose levels. 2.8. Biochemical Determinations Before sacrifice, mice fasted overnight, and a blood sample was taken by cardiac puncture under isoflurane anesthesia in heparin blood collection tubes. Blood samples were centrifuged for 20 min at 5000 × gat 4 ◦ C, and the resulting plasma was frozen at − 80 ◦ C until further analysis. Plasma glucose, LDL (low-density lipoprotein)-cholesterol, Antioxidants 2023,12, 832 5 of 19 and HDL (high-density lipoprotein)-cholesterol concentrations were determined by colorimetric methods using Spinreact kits (Spinreact, S.A., Girona, Spain). Plasma insulin levels were measured using a mouse insulin ELISA Kit (Alpco Diagnosis, Salem, NH, USA). A homeostatic model assessment of insulin resistance (HOMA-IR) was computed with the formula: fasting glucose (mM) × fasting insulin ( µ U/mL)/22.5. LPS plasma levels were quantified using a Pierce ™ Chromogenic Endotoxin Quant Kit (Thermo Scientific, Inc., Waltham, MA, USA) according to the manufacturer’s recommendations. 2.9. Thiobarbituric Acid Reactive Substance Assay Mouse liver was removed, lysed, and homogenized, and the protein concentration was calculated using the colorimetric method of Bicinchoninic Acid Assay (BCA). Then, the lipid oxidation was determined in the samples by measuring the amount of thiobarbituric acid reactive substances (TBARS), as described before [ 17 ]. Briefly, the malondialdehyde (MDA), resulting from lipid peroxidation, reacts with the thiobarbituric acid (TBA) used in the extraction method. The products of the reaction are TBARS, and its absorbance is assessed at 535 nm. TBARS levels were expressed as µM/mg protein in liver tissue. 2.10. Histological Studies Samples of epididymal adipose tissue and liver were fixed in 4% PFA, dehydrated, and embedded in paraffin. Then, 5 µ m-thick sections were cut and stained with hematoxylin and eosin. Adipocyte size was estimated and evaluated using the Fiji imaging software with the Adiposoft v1.16 plugin. 2.11. Analysis of Gene Expression by RT-qPCR Total RNA was extracted from adipose tissue and liver samples with the RNeasy Mini Kit (Qiagen, Germantown, MD, USA), following the recommended protocol. RNA quantification was obtained using a NanoDrop 200c spectrophotometer (Life Technologies, Thermo Fisher Scientific), and purity was confirmed with the 260 nm/280 nm and 260 nm/230 nm absorbance ratios. Subsequently, RNA was reverse transcribed into cDNA using oligo(dT) primers (Promega, Southampton, UK). Real-time quantitative PCR (qPCR) amplification and detection were carried out on optical-grade 48 well plates in the EcoTM Real-time PCR System (Illumina, San Diego, CA, USA) using 20 ng of cDNA, the MasterMix qPCR SyGreen Kit (PCR Biosystems Ltd., London, UK), and the specific primers (at a final concentration of 100 nM) (Table S1) (see Supplementary Materials). The mRNA relative quantitation was estimated with the ∆∆ Ct method and glyceraldehyde 3-phosphate dehydrogenase (Gapdh) was employed as a housekeeping gene. 2.12. Analysis of Protein Expression by Western Blot Proteins were separated in a 10% SDS-PAGE and then transferred to a PVDF membrane (GE Healthcare Life Sciences, Marlborough, MA, USA). They were blocked and probed at 4 ◦ C overnight with the following anti-mouse antibodies: anti-AMPK (1:2000 dilution), antip-AMPK (1:1000 dilution), anti-AKT (1:1000 dilution), anti-p-AKT (1:1000 dilution), and anti-PPAR γ (1:1000 dilution) (Cell Signaling, Danvers, MA, USA) or anti-SIRT1 (1:1000 dilution). This was followed by 1 h of incubation with peroxidase-conjugated anti-rabbit IgG antibody (1:5000 dilution) and β -actin (1:1000 dilution) (Santa Cruz Biotechnology, Inc., Heidelberg, Germany). The specific proteins were identified by Western Lightning ™ Chemiluminescence Reagent Plus (PerkinElmer Spain SL, Madrid, Spain) and semi-quantified by the ImageJ software (Free Software Foundation Inc., Boston, MA, USA). 2.13. Statistic All results are expressed as the mean ± SEM. Differences between means were assessed for statistical significance using a one-way analysis of variance (ANOVA) and post hoc least significance tests. Differences between proportions were analyzed with the chi-squared test. Antioxidants 2023,12, 832 6 of 19 All statistical analyses were performed with the GraphPad 8 software package (GraphPad Software, Inc., La Jolla, CA, USA), with statistical significance set at p-value < 0.05. 3. Results and Discussion Obesity is a chronic, often progressive condition recognized as an escalating risk in the development of metabolic alterations, including dyslipidemia and glucose intolerance, or cardiovascular diseases, such as hypertension or atherosclerosis [ 18 , 19 ]. At present, the approach to obesity implies the establishment of important modifications in lifestyle, such as caloric restriction or physical exercise, which are frequently difficult to maintain with time. Moreover, the pharmacological treatment of obesity is now plausible, with the administration of lipase inhibitors or anorexigenic drugs; however, they usually show limited efficacy and important side effects [ 6 ]. In this context, and considering the relevance of obesity and its co-morbidities in human health, more efficient and safer treatments are required for its management. This could be the case with plant extracts used traditionally for different purposes. For instance, LME contains different active compounds, including polyphenols, which can contribute to its beneficial effects, given the well-known antioxidant and anti-inflammatory properties ascribed to these compounds [20]. 3.1. Chemical Characterization of LME The present study has characterized the chemical composition of LME by UHPLC-MS for the first time. Figure 1shows the base peak chromatogram (BPC) of LME. A total of 65 compounds were identified, primarily as pentacyclic triterpenes. Antioxidants 2023, 12, x FOR PEER REVIEW 7 of 21 Figure 1. Base peak chromatogram of LME by UHPL-QTOF: (A) full chromatogram, (B) retention time from 8.50 to 16 min, and (C) retention time from 16 to 18.7 min. They are included in Table 1 and numbered in accordance with their elution order. Moreover, this table specifies their retention times (RT), experimental m/z, molecular formula, and proposed compounds. Table 1. Polar profile of LME. Peak RT m/z Molecular Formula Proposed Compounds 1 0.47 343.0356 C13H12O11 Mucic acid lactone gallate 2 0.61 341.1075 C12H22O11 Sucrose 3 4.42 133.0283 C4H6O5 Malic acid 4 6.79 371.0964 C16H20O10 Dihydroferulic acid glucuronide 5 8.87 567.0776 C26H32O14 Phloretin xyloglucoside 6 8.89 301.0705 C16H14O6 Hesperetin 7 9.35 463.0876 C21H20O12 Quercetin glucoside 8 9.43 593.0958 C29H22O14 (Epi)catechin digallate 9 9.79 447.0922 C21H20O11 Luteolin 7-O-glucoside 10 9.96 609.1448 C27H30O16 Rutin 11 10.71 473.0711 C22H18O12 Chicoric acid 12 11.3 489.1024 C23H22O12 Kaempferol acetyl-glucopyranoside 13 11.52 477.0664 C21H18O13 Quercetin glucuronide 14 11.79 461.0717 C21H18O12 Isoscutellarin 8-O-glucoronide 15 12.01 503.3370 C30H48O6 Madecassic acid or its isomer 16 12.27 491.0823 C22H20O13 Isorhamnetin 3-O-glucuronide 17 12.44 307.0446 C14H12O8 Fulvic acid analogue 1 Figure 1. Base peak chromatogram of LME by UHPL-QTOF: ( A ) full chromatogram, ( B ) retention time from 8.50 to 16 min, and (C) retention time from 16 to 18.7 min. They are included in Table 1and numbered in accordance with their elution order. Moreover, this table specifies their retention times (RT), experimental m/z, molecular formula, and proposed compounds. Antioxidants 2023,12, 832 7 of 19 Table 1. Polar profile of LME. Peak RT m/zMolecular Formula Proposed Compounds 1 0.47 343.0356 C13H12O11 Mucic acid lactone gallate 2 0.61 341.1075 C12H22O11 Sucrose 3 4.42 133.0283 C4H6O5Malic acid 4 6.79 371.0964 C16H20O10 Dihydroferulic acid glucuronide 5 8.87 567.0776 C26H32O14 Phloretin xyloglucoside 6 8.89 301.0705 C16H14O6Hesperetin 7 9.35 463.0876 C21H20O12 Quercetin glucoside 8 9.43 593.0958 C29H22O14 (Epi)catechin digallate 9 9.79 447.0922 C21H20O11 Luteolin 7-O-glucoside 10 9.96 609.1448 C27H30O16 Rutin 11 10.71 473.0711 C22H18O12 Chicoric acid 12 11.3 489.1024 C23H22O12 Kaempferol acetyl-glucopyranoside 13 11.52 477.0664 C21H18O13 Quercetin glucuronide 14 11.79 461.0717 C21H18O12 Isoscutellarin 8-O-glucoronide 15 12.01 503.3370 C30H48O6Madecassic acid or its isomer 16 12.27 491.0823 C22H20O13 Isorhamnetin 3-O-glucuronide 17 12.44 307.0446 C14H12O8Fulvic acid analogue 1 18 12.57 839.4089 C42H64O17 Yunganoside G2 or its isomer 19 12.74 519.0928 C27H20O11 Citreaglycon A 20 12.83 533.1661 C26H30O12 Amurensin 21 12.94 839.4052 C42H64O17 Yunganoside G2 or its isomer 22 13.07 545.3464 C32H50O7Hovenidulcigenin B or its isomer 23 13.29 839.4059 C42H64O17 Yunganoside G2 or its isomer 24 13.41 545.3454 C32H50O7Hovenidulcigenin B or its isomer 25 13.86 939.3139 C50H52O18 Unknown 26 13.89 327.2169 C18H32O5Fatty acid 27 14.07 307.0446 C14H12O8Fulvic acid analogue 2 28 14.52 503.3365 C30H48O6Madecassic acid or its isomer 29 14.60 823.4134 C42H64O16 Licoricesaponin J2 or its isomer 30 14.64 329.2487 C30H48O7Fatty acid 31 14.79 823.4134 C42H64O16 Licoricesaponin J2 or its isomer 32 14.82 519.3374 C30H48O7Hydroxyecdysone monoacetonide 33 15.06 287.2228 C16H32O4Fatty acid 34 15.43 501.3208 C30H46O6Medicagenic acid or its isomer 35 15.46 777.2611 C41H46O15 Guaiacylglycerol buddlenol A 36 15.6 501.3208 C30H46O6Medicagenic acid or its isomer 37 15.66 501.3304 C30H46O6Medicagenic acid or its isomer 38 15.84 503.3363 C30H48O6Madecassic acid or its isomer 39 15.93 503.3361 C30H48O6Madecassic acid or its isomer 40 16.07 503.3358 C30H48O6Madecassic acid or its isomer 41 16.16 503.3359 C30H48O6Madecassic acid or its isomer 42 16.56 503.3352 C30H48O6Madecassic acid or its isomer 43 16.63 503.3351 C30H48O6Madecassic acid or its isomer 44 16.73 503.3347 C30H48O6Madecassic acid or its isomer 45 16.76 677.3508 C36H54O12 Bryoamaride or its isomer 46 16.9 677.353 C36H54O12 Bryoamaride or its isomer 47 17.04 485.3261 C30H46O5Quillaic acid or its isomer 48 17.08 485.3249 C30H46O5Quillaic acid or its isomer 49 17.16 441.3369 C29H46O3Camellenodiol 50 17.24 487.3403 C30H48O5Asiatic acid or its isomer Antioxidants 2023,12, 832 8 of 19 Table 1. Cont. Peak RT m/zMolecular Formula Proposed Compounds 51 17.37 677.3522 C36H54O12 Bryoamaride or its isomer 52 17.45 487.3406 C30H48O5Asiatic acid or its isomer 53 17.52 487.3409 C30H48O5Asiatic acid or its isomer 54 17.75 295.2265 C18H32O3Fatty acid 55 17.81 471.3475 C30H48O4Maslinic acid or its isomer 56 17.86 293.2109 C18H30O3Fatty acid 57 17.89 425.3413 C29H46O2Stigmastene dione 58 17.96 469.3408 C30H46O4Glycyrrhetinic acid 59 18.03 471.3473 C30H48O4Maslinic acid or its isomer 60 18.06 471.3464 C30H48O4Maslinic acid or its isomer 61 18.11 471.347 C30H48O4Maslinic acid or its isomer 62 18.14 471.3461 C30H48O4Maslinic acid or its isomer 63 18.27 471.3476 C30H48O4Maslinic acid or its isomer 64 18.38 277.2156 C18H30O2Fatty acid 65 18.4 467.3159 C27H48O6Fatty acid 3.2. Antioxidant Capacity of LME As a previous step to further validate the antioxidant potential of this extract by ORAC, TEAC, and FRAP assays, the total phenolic content (TPC) was estimated in LME by the Folin–Ciocalteu method, which was 179 mg GAE/g of plant extract. Table 2displays the values obtained for every assay. Based on the results obtained, LME showed free radical scavenging activity (Table 2). Table 2. Total phenolic content and antioxidant capacity of LME. Method Value Folin-Ciocalteu (mg GAE/g d.e.) 179 ±1 FRAP (mmol eq. FeSO4/g d.e.) 2.576 ±0.002 TEAC (mmol eq. Trolox/g d.e.) 1.30 ±0.02 ORAC (mmol eq. Trolox/g d.e.) 2.08 ±0.09 GAE—gallic acid equivalents; d.e.—dry extract; eq.—equivalents. The antioxidant activity of LME was also evaluated by carrying out the DPPH assay, a widely used method based on the reduction of alcoholic DPPH solution in the presence of hydrogen-donating antioxidants. LME displayed a dose-dependent neutralizing activity of 10.4%, 21.2%, and 58.6%, at concentrations of 0.1, 1, and 10 µ g/mL of extract, respectively (Figure 2A). The free radical scavenging activity of LME (IC50 = 8.06 µ g/mL) was compared with that of some compounds with a well-known ability to neutralize DPPH radicals: gallic acid (IC50 = 7.94 µ g/mL), epicatechin (IC50 = 6.43 µ g/mL) and ascorbic acid (IC50 = 7.91 µg/mL) [21,22]. Interestingly, numerous studies have reported that the intake of an HFD can cause oxidative stress and increase lipid peroxidation, thus releasing various reactive aldehydes such as 4-hydroxynonenal (HNE) and MDA [ 23 , 24 ]. In addition to being considered biomarkers of lipid peroxidation, these compounds can cause DNA damage, thus inducing pathological processes such as cytotoxicity [ 24 ]. As expected, lipid peroxidation was higher in those mice fed HFD resulting in a significant increase of TBARS compared to CD-fed mice. The antioxidant activity exhibited by LME in vitro was also evidenced in vivo , in which a significant reduction of the amount of TBARS in treated HFD mice (Figure 2B) was observed. Phenolic compounds such as rutin, quercetin glucoside, and epicatechin gallate may be responsible for the restoration of the antioxidant status in mice treated with LME. Antioxidants 2023,12, 832 9 of 19 Antioxidants 2023, 12, x FOR PEER REVIEW 9 of 21 3.2. Antioxidant Capacity of LME As a previous step to further validate the antioxidant potential of this extract by ORAC, TEAC, and FRAP assays, the total phenolic content (TPC) was estimated in LME by the Folin–Ciocalteu method, which was 179 mg GAE/g of plant extract. Table 2 displays the values obtained for every assay. Based on the results obtained, LME showed free radical scavenging activity (Table 2). Table 2. Total phenolic content and antioxidant capacity of LME. Method Value Folin-Ciocalteu (mg GAE/g d.e.) 179 ± 1 FRAP (mmol eq. FeSO4/g d.e.) 2.576 ± 0.002 TEAC (mmol eq. Trolox/g d.e.) 1.30 ± 0.02 ORAC (mmol eq. Trolox/g d.e.) 2.08 ± 0.09 GAE—gallic acid equivalents; d.e.—dry extract; eq.—equivalents. The antioxidant activity of LME was also evaluated by carrying out the DPPH assay, a widely used method based on the reduction of alcoholic DPPH solution in the presence of hydrogen-donating antioxidants. LME displayed a dose-dependent neutralizing activity of 10.4%, 21.2%, and 58.6%, at concentrations of 0.1, 1, and 10 µg/mL of extract, respectively (Figure 2A). The free radical scavenging activity of LME (IC50 = 8.06 µg/mL) was compared with that of some compounds with a well-known ability to neutralize DPPH radicals: gallic acid (IC50 = 7.94 µg/mL), epicatechin (IC50 = 6.43 µg/mL) and ascorbic acid (IC50 = 7.91 µg/mL) [21,22]. Figure 2. (A) DPPH activity scavenging of LME, gallic acid, epicatechin, and ascorbic acid; (B) TBARS production in liver lysates. Data are expressed as means ± SEM (n = 4). Groups with different letters statistically differ (p < 0.05). Interestingly, numerous studies have reported that the intake of an HFD can cause oxidative stress and increase lipid peroxidation, thus releasing various reactive aldehydes such as 4-hydroxynonenal (HNE) and MDA [23,24]. In addition to being considered biomarkers of lipid peroxidation, these compounds can cause DNA damage, thus inducing pathological processes such as cytotoxicity [24]. As expected, lipid peroxidation was higher in those mice fed HFD resulting in a significant increase of TBARS compared to CD-fed mice. The antioxidant activity exhibited by LME in vitro was also evidenced in vivo, in which a significant reduction of the amount of TBARS in treated HFD mice (Figure 2B) was observed. Phenolic compounds such as rutin, quercetin glucoside, and epicatechin gallate may be responsible for the restoration of the antioxidant status in mice treated with LME. Figure 2. ( A ) DPPH activity scavenging of LME, gallic acid, epicatechin, and ascorbic acid; ( B ) TBARS production in liver lysates. Data are expressed as means ± SEM (n= 4). Groups with different letters statistically differ (p< 0.05). 3.3. Effects of LME on Nitrite Production in RAW 264 Cells and Adipogenesis in 3T3-L1 Cells As commented above, oxidative stress is an important pathogenic mechanism of obesity and its associated complications [ 25 ]. Excessive weight gain is characterized by increased adipocyte size and macrophage recruitment, in association with increased free radical and reactive oxygen species production, which contribute to the establishment of a chronic inflammatory state and, consequently, metabolic dysfunctions [ 26 ]. Specifically, pro-inflammatory macrophages contribute to this oxidative stress status by the induction of different enzymes, including inducible NO synthase (iNOS), whose induction favors the release of nitric oxide (NO), a key regulator of body composition and energy metabolism [ 27 ], thus influencing both adipogenesis and insulin resistance. The effects of LME were evaluated in vitro in murine RAW 264 macrophages. The incubation of RAW 264 cells with different concentrations of LME (0.1–100 µ g/mL) during 24 h did not show NO accumulation. Similarly, cell viability was not significantly modified by any of the doses tested (Figure 3). However, LPS (a potent activator of inflammatory signaling pathways) induced a marked NO production, which was dose-dependently decreased by LME pretreatment (Figure 3). Antioxidants 2023, 12, x FOR PEER REVIEW 10 of 21 3.3. Effects of LME on Nitrite Production in RAW 264 Cells and Adipogenesis in 3T3-L1 Cells As commented above, oxidative stress is an important pathogenic mechanism of obesity and its associated complications [25]. Excessive weight gain is characterized by increased adipocyte size and macrophage recruitment, in association with increased free radical and reactive oxygen species production, which contribute to the establishment of a chronic inflammatory state and, consequently, metabolic dysfunctions [26]. Specifically, pro-inflammatory macrophages contribute to this oxidative stress status by the induction of different enzymes, including inducible NO synthase (iNOS), whose induction favors the release of nitric oxide (NO), a key regulator of body composition and energy metabolism [27], thus influencing both adipogenesis and insulin resistance. The effects of LME were evaluated in vitro in murine RAW 264 macrophages. The incubation of RAW 264 cells with different concentrations of LME (0.1–100 µg/mL) during 24 h did not show NO accumulation. Similarly, cell viability was not significantly modified by any of the doses tested (Figure 3). However, LPS (a potent activator of inflammatory signaling pathways) induced a marked NO production, which was dose-dependently decreased by LME pretreatment (Figure 3). Figure 3. Effects of LME (0.1–100 µg/mL) on nitrite accumulation and cell viability in RAW 264 cells. Data are expressed as means ± SEM. The experiments were performed three times. *** p < 0.001 vs. LPS-stimulated cells. In addition, abnormal accumulation of lipids in cells is a characteristic of insulin resistance and obesity. Thus, the reduction of elevated cellular lipid levels could be a potential approach for the management of these pathological conditions. In this sense, the impact of various concentrations of LME on lipid accumulation was assayed in pre-adipo- cyte 3T3-L1 cells, which was checked throughout the experiment. As expected, under appropriate differentiation conditions, 3T3-L1 mature cells showed many lipid droplets in comparison to pre-adipocytes (Figure 4A). Interestingly, LME at the highest dose (100 µg/mL) revealed a lipid-lowering effect, thus highlighting its regulatory impact on adipogenesis. Since adipogenesis is regulated by various transcription factors and adipogenesis-related genes, we continued evaluating LME impact on the protein level of transcriptional factor PPARγ by western blot. PPARγ is a critical component in adipogenesis, where its over-expression aggravates the intracellular triglycerides accumulation in adipocytes as well as cell size. As shown in Figure 4B, the PPARγ level was slightly diminished by LME, especially at the dose of 100 µg/mL. Thus, lower intracellular lipids accumulation observed with LME could be linked to direct changes in the PPARγ protein level. Nevertheless, the exact underlying mechanisms remain to be elucidated. It could be associated with the regulation of other transcriptional factors such as, i.e., CCAAT/enhancerbinding proteins alpha and beta or sterol-regulatory element binding protein-1c, although this has not been explored yet. Figure 3. Effects of LME (0.1–100 µ g/mL) on nitrite accumulation and cell viability in RAW 264 cells. Data are expressed as means ± SEM. The experiments were performed three times. *** p< 0.001 vs. LPS-stimulated cells. In addition, abnormal accumulation of lipids in cells is a characteristic of insulin resistance and obesity. Thus, the reduction of elevated cellular lipid levels could be a potential approach for the management of these pathological conditions. In this sense, the impact of various concentrations of LME on lipid accumulation was assayed in pre-adipocyte 3T3-L1 cells, which was checked throughout the experiment. As expected, under appropriate differentiation conditions, 3T3-L1 mature cells showed many lipid droplets in comparison to pre-adipocytes (Figure 4A). Interestingly, LME at the highest dose (100 µ g/mL) revealed Antioxidants 2023,12, 832 16 of 19 treated mice showed increased hepatic Ampk expression and activation (phosphorylated isoform). Similarly, the treatment with the extract resulted in augmented Sirt1 protein expression in the liver (Figure 8D). This suggests that the high content of maslinic acid in LME could promote the activation and signaling of hepatic Ampk, thus further reducing fat accumulation and steatosis. 3.6. Effects of LME Treatment on Intestinal Barrier Dysfunction It is well documented a that there is a close correlation between obesity and changes in intestinal structure, which can impact gut permeability and result in metabolic complications [ 47 , 48 ]. The present study confirms this since epithelial barrier dysfunction was observed in control HDF-fed mice, evidenced by the reduced expression of several colonic markers of gut integrity, including the peptides Trefoil Factor 3 (Tff-3), occludin and tight junction, as well as the mucins (Figure 9A). Tff-3, which is expressed by goblet cells and typically co-secreted together with epithelial membrane-bound mucins, such as Muc-1, Muc-2, and Muc-3, to protect the mucus layer [ 49 ]. In addition, tight junction protein-1 (Tjp1) works as a tight junction adaptor protein that regulates adherent junctions along with the transmembrane protein occludin, playing a crucial role in maintaining and facilitating epithelial integrity [ 50 ]. This altered epithelial integrity was associated with an increase in plasma LPS levels in untreated obese mice, thus promoting a situation of endotoxemia (Figure 9B). Antioxidants 2023, 12, x FOR PEER REVIEW 17 of 21 have revealed the potential role of maslinic acid, the main component of LME, in regulating lipogenesis in hepatocytes from obese animals. In fact, maslinic acid reduced liver lipid accumulation by modulating the Sirt1/AMPK signaling pathway [46], in accordance with our findings. Indeed, LME-treated mice showed increased hepatic Ampk expression and activation (phosphorylated isoform). Similarly, the treatment with the extract resulted in augmented Sirt1 protein expression in the liver (Figure 8D). This suggests that the high content of maslinic acid in LME could promote the activation and signaling of hepatic Ampk, thus further reducing fat accumulation and steatosis. 3.6. Effects of LME Treatment on Intestinal Barrier Dysfunction It is well documented a that there is a close correlation between obesity and changes in intestinal structure, which can impact gut permeability and result in metabolic complications [47,48]. The present study confirms this since epithelial barrier dysfunction was observed in control HDF-fed mice, evidenced by the reduced expression of several colonic markers of gut integrity, including the peptides Trefoil Factor 3 (Tff-3), occludin and tight junction, as well as the mucins (Figure 9A). Tff-3, which is expressed by goblet cells and typically co-secreted together with epithelial membrane-bound mucins, such as Muc-1, Muc-2, and Muc-3, to protect the mucus layer [49]. In addition, tight junction protein-1 (Tjp1) works as a tight junction adaptor protein that regulates adherent junctions along with the transmembrane protein occludin, playing a crucial role in maintaining and facilitating epithelial integrity [50]. This altered epithelial integrity was associated with an increase in plasma LPS levels in untreated obese mice, thus promoting a situation of endotoxemia (Figure 9B). Figure 9. Effects of LME supplementation on: ( A ) markers of intestinal barrier integrity Muc1,Muc2, Muc3,Occludin,Tjp1, and Tff3, as well as ( B ) plasma LPS levels and gene expression of Tlr4 in the liver. Data are expressed as means ± SEM (n= 8). Groups with different letters statistically differ (p< 0.05). Antioxidants 2023,12, 832 17 of 19 Of note, previous studies have reported the upregulated expression of these markers of epithelial gut integrity in HFD-fed mice after treatment with different polyphenol-enriched plant extracts [ 17 , 28 , 51 ]. Similarly, the administration of LME to HFD-fed mice improved the expression of most of these proteins and reduced plasmatic LPS levels (Figure 9B), thus indicating an improvement in the epithelial barrier function and permeability in comparison with obese non-treated mice. Accordingly, several reports have highlighted the strict association between increased LPS plasma levels and dysregulation of the toll-like receptor (TLR)-4 signaling pathway, including increased Tlr4 expression, which induces pro-inflammatory responses in obesity [ 52 ]. Our research confirmed these findings since the hepatic expression of Tlr4 markedly increased in obese animals compared to the lean ones (Figure 9B). The administration of LME improved the expression of this receptor (Figure 9B), thus confirming an amelioration of the endotoxemia-mediated inflammation in treated-obese mice. 4. Conclusions LME displays a positive impact on decreasing body weight gain and controlling glucose homeostasis in HFD mice. In addition, LME shows antioxidant and adipogenesis inhibitory activities. LME in vivo beneficial effects may be mediated, at least partly, by its antioxidant and anti-inflammatory activities, which are probably ascribed to the synergic properties of its different phytochemical constituents. In sum, our results suggest that LME may be considered a promising complementary approach for the management of obesity and its metabolic complications. Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/antiox12040832/s1, Table S1: RT-qPCR primers sequences. Author Contributions: Conceptualization, T.V., F.A. and J.G.; methodology, J.A.M.-T., A.J.R.-M., P.D.-E., M.J.R.-S., M.E.R.-C., M.d.l.L.C.-G., A.S.-C., J.P.d.P. and M.R.G.-T.; data curation, J.A.M.-T., A.R.-N. and L.H.-G.; validation, T.V., F.A. and J.G.; writing—original draft preparation, J.A.M.-T., A.J.R.-M., T.V. and F.A.; writing—review and editing, M.E.R.-C., A.R.-N. and L.H.-G.; supervision and funding acquisition, T.V., F.A. and J.G. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Junta de Andalucía (CTS 164), by the Instituto de Salud Carlos III (ISCIII) (PI19.01058), and by the Spanish Ministry of Economy and Competitiveness (AGL2015-67995-C3-3-R), with funds from the European Commission (FEDER/ERDF). The CIBEREHD is funded by the Instituto de Salud Carlos III. J.A. Molina-Tijeras (recipient of pFIS (FI20/00159)), Instituto de Salud Carlos III; Programa de Doctorado Nutricion), A.J. Ruiz-Malagón (recipient of Formación de Profesorado Universitario Program, Universidad de Granada; Programa de Doctorado Medicina Clínica y Salud Pública) and M.J. Rodriguez-Sojo (recipient of i-pFI (IFI21/00030)), Instituto de Salud Carlos III; Programa de Doctorado Biomedicina) are predoctoral fellows. T. Vezza is the recipient of Miguel Servet contract (CP22/00153) from the Instituto de Salud Carlos III. Institutional Review Board Statement: This study was carried out in accordance with the “Guide of the Care and Use of Laboratory animals” as promulgated by the National Institute of Health, and the protocols were approved by the Ethic committee of Laboratory Animals of the University of Granada (Spain) (Ref. No. 28/03/2016/030). Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding author. 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