Efficacy of Hot Tea Infusion vs. Ethanolic Extract of Moringa oleifera for the Simultaneous Treatment of Nonalcoholic Fatty Liver, Hyperlipidemia, and Hyperglycemia in a Murine Model Fed with a High-Fat Diet

Moringa oleifera (MO) is a native tree of Asia and is cultivated in some areas of Mexico as part of traditional horticulture. The aim of the present study was to compare the efficacy of MO infusion vs. MO ethanolic extract for the simultaneous treatment of nonalcoholic fatty liver (NAFLD), hyperlipidemia, and hyperglycemia in a murine model fed with a high-fat diet (HFD). BALB/c mice were fed a balanced diet (healthy control) or an HFD for 6 months. With this, the NAFLD model was established before starting a therapeutic intervention with MO for two months. The phytochemical analysis by nuclear magnetic resonance in 1H and 13C experiments showed signals for pyrrole alkaloids and triterpenes as the main constituents of the extract and infusion preparation. A significant reduction of SGPT, SGOT, lipids, urea, and glucose in blood among NAFLD groups treated with MO (infusion or extract) was found, when compared to the NAFLD-placebo group. Steatosis and liver inflammation were found to be decreased in the MO groups, as infusion or ethanolic extract. Infusion produced a better therapeutic effect than the extract in all parameters, except glycemic control, where the extract was better. As an additional finding, it is noteworthy that treatment with MO, particularly through infusion, resulted in improved motor activity. Moreover, a reduction in anxiety-like behavior was observed exclusively with the administration of infusion. These observations provide valuable insights into the potential broader effects of Moringa oleifera beyond the primary aim of the study.

Moringa oleifera (MO) is a native tree of Asia and is cultivated in some areas of Mexico as part of traditional horticulture.Te aim of the present study was to compare the efcacy of MO infusion vs. MO ethanolic extract for the simultaneous treatment of nonalcoholic fatty liver (NAFLD), hyperlipidemia, and hyperglycemia in a murine model fed with a high-fat diet (HFD).BALB/c mice were fed a balanced diet (healthy control) or an HFD for 6 months.With this, the NAFLD model was established before starting a therapeutic intervention with MO for two months.Te phytochemical analysis by nuclear magnetic resonance in 1 H and 13 C experiments showed signals for pyrrole alkaloids and triterpenes as the main constituents of the extract and infusion preparation.A signifcant reduction of SGPT, SGOT, lipids, urea, and glucose in blood among NAFLD groups treated with MO (infusion or extract) was found, when compared to the NAFLD-placebo group.Steatosis and liver infammation were found to be decreased in the MO groups, as infusion or ethanolic extract.Infusion produced a better therapeutic efect than the extract in all parameters, except glycemic control, where the extract was better.As an additional fnding, it is noteworthy that treatment with MO, particularly through infusion, resulted in improved motor activity.Moreover, a reduction in anxiety-like behavior was observed exclusively with the administration of infusion.Tese observations provide valuable insights into the potential broader efects of Moringa oleifera beyond the primary aim of the study.

Introduction
Westernized fast food has resulted in a surge in the consumption and accessibility of lipids, including high levels of saturated fats that can alter the eating habits of the population [1,2].Te associations between the consumption of a high-lipid diet and diabetes, obesity, and liver disease, including nonalcoholic fatty liver disease (NAFLD), have long been recognized [3,4].Te prevalence of NAFLD is up to 30% in developed countries and almost 10% in developing countries, making NAFLD the most common liver condition in the world [5].
NAFLD is the accumulation of hepatic steatosis not caused by excessive alcohol consumption.While it is often regarded as the hepatic manifestation of the metabolic syndrome, closely linked to conditions like obesity, insulin resistance, hyperlipidemia, and hypertension, this characterization has been a topic of ongoing discussion.Contrary to the general assumption, research indicates that around 30% of NAFLD patients do not exhibit symptoms of metabolic syndrome (MetS) [6].Moreover, intriguingly, studies have shown that in many instances, NAFLD precedes the development of MetS [7][8][9].Oxidative stress (OS) is considered an important factor in the pathophysiology and progression of chronic infammatory liver diseases, including NAFLD [5,10].Terefore, the modulation of the antioxidant response emerges as an interesting target to prevent the development and progression of NAFLD.Given the absence of approved treatments by the Federal Drug Administration (FDA) to date, recent guidelines advocate for lifestyle interventions, bariatric surgery, and pharmacotherapy as potential options for managing nonalcoholic fatty liver disease (NAFLD).Tis encompasses the utilization of agents such as glucagon-like peptide-1 receptor agonists (GLP-1RAs), peroxisome proliferator-activated receptor-c (PPAR-c) agonists, and sodium-glucose cotransporter-2 (SGLT-2) inhibitors [11,12].
Recently, there has been a remarkable progress in the feld of herbal therapy due to the growing concerns about the development of drug resistance, drug side efects, and limited advances in the discovery of new drugs [13].In almost all countries, medicinal plants have been widely used throughout history for the treatment of diseases as traditional healing remedies due to their broad therapeutic spectrum and minimal or no side efects [14].Since there are no specifc drugs available for NAFLD, considerable eforts have been focused on the search for new drugs and complementary/alternative medicines from diferent herbal formulations.
Moringa oleifera (MO) Lam. is a plant that belongs to the Moringaceae family, which is widely distributed in the tropics and subtropics and has been reported to possess various medicinal properties [15].In Mexico, the use of Moringa oleifera is widely disseminated in the population, and even the government of the country considers M. oleifera, a plant species of high nutritional value, being used, almost entirely for human consumption [16,17].
Ethnomedical studies refer to the use of M. oleifera tea infusion as follows: regulator of blood glucose levels, enhancer of the immune system, capable of regulating cholesterol levels, and with anti-infammatory activity.Likewise, other ethnomedical precedents refer to the use of MO ethanol macerates to prevent and reduce hypertension [16,17].Phytochemical studies in MO have been shown to be a natural source of antioxidants such as phenolic compounds, vitamins A, C, and E, ascorbic acid oxidase, polyphenol oxidase, and catalase [18,19].Extracts from lyophilized leaves of MO have shown antioxidant and free radical scavenging activity [20][21][22].Terefore, MO could be part of the therapeutic options for NAFLD.
Although MO is a plant currently under study, it has been suggested that the diferent stages of the plant and various extraction methods may infuence its characteristics and potential efects [23], in addition to the fact that its efects are not always considered the same on mobility [24] and anxiety [25].Terefore, the aim of the present study was to compare the therapeutic efcacy of hot tea infusion vs. MO ethanolic extract for the simultaneous treatments of nonalcoholic fatty liver, hyperlipidemia, and hyperglycemia in a murine model fed with a high-lipid diet with NAFLD.

Plant Material.
Te fresh leaves of MO were collected in the city of Colima, Mexico (9.2433 °N latitude and 103.7242 °W longitude).Te plant material was taxonomically identifed and compared with a specimen from the National Herbarium of Mexico (MEXU), with voucher number MEXU: 1115057 serving as the reference.Subsequently, the leaves were conditioned in the laboratory, thoroughly cleaned of extraneous materials, and dried in an oven with air circulation at 37 °C for 60 hours.

Preparation of the Infusion.
Te dried leaves of MO were fnely crushed using a mortar and pestle, achieving a loose tea-like consistency.Daily infusions were prepared, combining 0.7 g of MO leaves with 10 mL of distilled/deionized water.Te water was heated to boiling point (95-100 °C), then poured over the dried MO leaves, and stirred for 15 minutes.Following this, the infusion was fltered through cotton gauze to remove solids.Te resulting fltrate (MO infusion tea) was allowed to cool to body temperature before administration.Tis method adheres to the traditional practices of Colima, Mexico, ensuring the proper preparation of the MO leaf infusion [16,17].

Preparation of the Ethanol Extract.
According to Mousa et al., ethanolic extract was prepared from MO-dried leaves [26].Te cold extraction process was employed to obtain the crude extract.In this process, 1 kg of the powdered material was combined with 2000 mL of ethanol (96%).Te mixture was allowed to macerate for 72 hours at room temperature (22 °C), with intermittent gentle shaking.Following

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Journal of Nutrition and Metabolism maceration, the mixture was fltered sequentially through cotton gauze and Whatman flter paper No. 1, resulting in a green-hued solution.To concentrate the solution, an initial step involved using a vacuum rotary evaporator under specifc conditions (adjustment bath: 40 °C, rotation: 50 rpm, pressure: ∼15 psi, and condenser: 4 °C) to remove ethanol.Te solution was subsequently further concentrated through freeze-drying using a freeze dryer.Te yield of the extract was determined in % (w/w).Te MO ethanol extract was stored within a silicon desiccator until its subsequent utilization.

Phytochemical Screening of the MO Infusion and Ethanol
Extract.A small portion of the dry ethanolic extract or a tea infusion of M. oleifera leaf was utilized for the phytochemical tests, including tannins, favonoids, alkaloids, saponins, and steroids, following established methods with some modifcations [27].
In the preliminary phytochemical analysis of the ethanol extract, a solution was prepared by dissolving 10 mg of the ethanolic extract in the solvent indicated by the specifc test.Regarding the infusion, 1 ml of the infusion fltrate used for the tests (see preparing infusion section) was used.For the tannin test, 1 ml of plant solution extract or infusion fltrate was mixed with 5 ml of distilled water and fltered (using Whatman No. 1 flter paper).Te presence of tannins was indicated by a blue coloration resulting from the addition of ferric chloride reagent to the fltrate.Alkaloid presence was determined by Dragendorf's test; 10 mg of the plant extract or 1 ml of infusion fltrate was dispersed in 5 ml of 1% HCl in a steam bath.A milliliter of the mixture was treated with few drops of Dragendorf's reagent; a positive reaction was considered if a precipitate or turbidity was formed.Te occurrence of a red or orange coloration was indicative of the favonoids in the presence of HCl and magnesium.A freshly prepared 7% blood agar plate was used, and wells were made in it.Te crude extract (10 mg) or infusion fltrate (1 ml) was mixed with 5 ml of a 9 : 1 water/methanol solution and subsequently used to fll the wells bored in the blood agar plates.9 : 1 water/methanol solution was used as a negative control, while commercial saponin solution was used as a positive control.Te plates were incubated at 35 °C for 6 h.A complete hemolysis test of blood around the extract was indicative of saponin.Steroid rings were indicated by the reaction with H 2 SO 4 in chloroform solution.Te reddish-brown color on the interface was taken as positive for steroid rings [27].

Quantitative Determination of Flavonoids, Antioxidant Capacity, and Reducing Power of MO Infusion and Ethanol
Extract.To achieve a comprehensive screening and quantifcation of favonoid compounds, along with assessing the antioxidant capacity and reducing power of the Moringa oleifera (MO) infusion and ethanol extract, additional assays were conducted.For the MO infusion tea, a 1.0 ml sample of the infusion fltrate was pretreated by lyophilization using an ILShinBioBase freeze dryer (model TFD8503, Korea).Tis lyophilization process efectively removed the water content, maintaining the constituents of the infusion in a dry state.Te resulting lyophilized powder was stored in an ultracooler at − 40 °C until analysis.For both the MO extract and the lyophilized infusion, the working concentration was adjusted to 5 mg/ml for subsequent analysis.
Te analysis involved the determination of total favonoid content (TFC), expressed as quercetin equivalent in μg/ mg of extract, using the method by Wakeel et al., 2019 [28].In addition, total antioxidant capacity (TAC) was assessed through the phosphomolybdenum method [29,30], using ascorbic acid as the positive control.Te calculation of antioxidant capacity followed the formula: % antioxidant capacity � [1 − (OD of the sample/OD of the control)] * 100.Furthermore, the ferric reducing power assay (FRPA) was conducted using the potassium ferricyanide-ferrous chloride method [31,32], with modifcations from Wakeel et al., 2019.Te reducing power was calculated as % reducing power � [1 − (OD of the sample/OD of the control)] * 100.All the determinations were carried out using a BioMate THERMO spectrophotometer (USA).

Nuclear Magnetic Resonance General Screening.
In order to obtain a general screening of the compounds in the MO infusion and ethanolic extract that used a nuclear magnetic resonance (NMR) ( 1 H and 13 C), spectra were obtained using a Bruker NMR spectrometer (Bruker, Leipzig, Germany), with an operating frequency of 400 and 100 MHz, respectively.For the Moringa oleifera (MO) infusion tea, a sample pretreatment was conducted by lyophilizing an infusion sample using an ILShinBioBase freeze dryer (model TFD8503, Korea).It is important to note that a larger quantity of the infusion sample was lyophilized for analysis, (30 ml of infusion fltrate) following the conditions described above.In addition, the ethanol extract was directly prepared for analysis using the extract obtained previously.Both the MO-lyophilized infusion and the ethanol extract were analyzed at a concentration of 10 mg/ml for consistency in testing conditions employing CDCl 3 (Chloroformd, Sigma Aldrich, USA) as a solvent.Te chemical shifts are given in δ (ppm), and coupling constants (J) are reported in Hz.Te chemical shifts obtained were compared with some of the isolated compounds from M. oleifera leaves (see S2 Table ).

Experimental Animals.
Te research was conducted as a prospective, single-blind, 4-arm, parallel-group, randomized, preclinical trial, adhering to the guidelines outlined in the "ARRIVE Essential 10" for Animal Research (see Figure 1) [33].Blinding consisted of the evaluators not knowing the group to which the mice or samples analyzed belonged (pathologist, evaluator of blood, and biochemical, motor, or behavioral tests) [33].Te calculation of the sample size in this study was based on the formula derived from previous reports, which takes into account the assessment of incidence [34] and uses the reduction of nonalcoholic fatty liver disease (NAFLD) as the primary outcome measure [35].It was determined that a minimum of nine animals per group was necessary to establish Journal of Nutrition and Metabolism meaningful comparisons.A total of 44 mice were randomly assigned to four groups for the purpose of this study.
Tis experimental study included 44 BALB/c mice (Envigo ® , Mexico) between 4 and 6 weeks old with an initial weight of 22 to 25 g.Te 44 mice were randomly assigned into two groups: a healthy group (n � 11, consisting of 6 males and 5 females) and an NAFLD experimental group (n � 33, comprising 18 males and 15 females).Te mice were kept in cages, with a maximum of 5 mice per group.Conditions were controlled (12 h: 12 h light/dark cycle, 23 °C temperature with 50% humidity), and the animals had access to food and water ad libitum.11 mice were fed with a standard diet made up of 44.2% carbohydrates, 18.6% protein, 3.5% fber, and 6.2% net fat (2018S Teklan and Global 18% Protein Rodent Diet, Envigo ® , USA) and therefore were considered the normality reference (healthy group).All mice were placed with a metal tag with a unique number on the pinna of the ear, which allowed correct identifcation during the experiment.For the induction of the NAFLD model, 33 mice were fed with a high-fat diet (HFD) that was made up of 46.9% carbohydrates, 17.3% protein, 25% net fat, 1.25% cholesterol, and 0.5% cholic acid [35].Te model used to generate nonalcoholic fatty liver (NAFLD) requires 6 months of exposure to a high-fat diet to express pathophysiological alterations similar to the progression of the disease in humans and to facilitate the examination of long-term treatments and their efects [36].After 6 months of administering the aforementioned diets (with the established NAFLD model), HFD-fed mice were randomly divided into three groups, of which two groups were given therapeutic treatment with MO (NAFLD-MO infusion and NAFLD-MO ethanolic extract groups), leaving one group without treatment (the NAFLD-placebo group, with administration of saline solution), which was the point of reference for the pathologic alterations caused by the model.Randomization was performed using computergenerated random allocation cards.Te treatments were administered for 60 days (from the sixth to the eighth month).At the end of the treatment, a glucose tolerance curve and psychomotor analysis were performed.After the completion of the treatment period, euthanasia was performed on all animals twenty-four hours later.Trained research personnel conducted euthanasia in accordance with the American Veterinary Medical Association's (AVMA) Guidelines for Euthanasia of Animals: 2020 Edition.Prior to euthanasia, mice were anesthetized with intraperitoneal administration of sodium pentobarbital at a dose of 50 mg/ kg.Manual cervical dislocation was then carried out, involving the placement of the thumb and forefnger on either side of the neck at the base of the skull, just behind the ears, while keeping the head still.Simultaneously, the other hand pulled on the base of the tail with a frm, steady motion, causing the cervical vertebrae to detach from the skull [37].Subsequently, blood samples were collected by cardiac puncture for further biochemical analyses.Te weight of mice in all groups ranged from 21.2 to 38.3 grams, within the allowable weight for euthanasia by cervical dislocation [37].Te livers were promptly excised, weighed, and subjected to histopathological examination, as illustrated in Figure 1.In addition, the right kidney, left kidney, pancreas, brain, ovaries, and epididymal fat pad were obtained and weighed.Trial protocols were approved by Colima State Cancer Institute's Research Ethics Committee, Mexico (Protocol Number: CEICC-240818-ETAMORI-010), which ensured compliance with national and international legal and ethical requirements.Animals were handled in accordance with institutional guidelines, the Mexican ofcial norm governing laboratory animal use (NOM-062-ZOO-1999) and the National Academy of Science's (2011) Guide for the Care and Use of Laboratory Animals.
Tis experimental study included four groups: (1) healthy group; (2) NAFLD-placebo (saline solution); (3) NAFLD with treatment of ethanolic extract of MO; and (4) NAFLD treatment with infusion of MO.After two months of treatment, blood parameters and the liver tissues were analyzed.Biochemical, histological, and psychomotor analyzes were performed.

Dose Calculation.
Based on the traditional community use, the MO infusion is usually prepared with approximately 3 g of dried leaves in 100 ml of water, which is generally consumed by an adult with a considered average weight of 70 kg.Tis is equivalent to a usual human dose of 42.85 mg/ kg.With these data, the dose for mice was calculated using the US FDA recommendations for dose extrapolation between species [38].Te equivalent mouse dose was 500 mg/ kg/day in 200 μl volume for both extraction systems (infusion and ethanol), determined according to what was reported in the literature as an optimal dose served [39].Administration of all groups of treated or untreated mice was by an orogastric route using saline solution as the vehicle.
2.9.Body Weight and Food Intake.Te body weight (g) was recorded since day one and then weekly consecutively for eight months using an automated electronic scale (A & D Weighing, Limited HR-200, CA, USA).To weigh the mice, a round plastic container was placed on the scale and tarred to zero before placing the mouse inside the container.In addition to this, the daily food intake for each group was measured weekly for eight months.Food intake was estimated as follows: [total food consumed per cage]/[mice per cage] × [days of food consumption].Te data are expressed as the mean ± standard deviation or standard error of the mean.

Hematic Biometry and Biochemical Profle Analysis.
A sample of blood was obtained by cardiac puncture after euthanization.Te samples were collected and deposited in vacutainer tubes without anticoagulant (EDTA) allowing blood to clot.Te blood without EDTA was processed in a Beckman Coulter AC-T instrument (Beckman Coulter, Inc., Brea, CA, USA), and the following blood parameters were estimated: erythrocytes, leukocytes, platelets, hemoglobin, and hematocrit.Te blood without anticoagulant was centrifuged at 3500 rpm for 5 minutes to obtain serum, which was immediately transferred with a pipette to the allocated containers and was determined using an automatic biochemical analyzer (Cobas c111, Roche ® , Mexico).Te serum was analyzed for the estimation of the following biochemical parameters: serum alanine aminotransferase (SGPT), aspartate aminotransferase (SGOT), cholesterol, triglycerides (TG), total lipids, urea, and glucose.

Glucose Tolerance Curve (OGTT).
Fasting glucose was determined at months 0, 3, and 6 (before starting the treatments).An intraperitoneal glucose tolerance test was carried out at the 6 th month (before starting treatment) and 8 th month (after 2 months of treatment), as described by Vinué and González-Navarro [40].Te animals were made to fast for a period of 6 hours.Te blood was collected from the tail vein of each mouse (20 μl), and before initiating the tests, 0-minute blood samples were withdrawn to estimate fasting glucose levels.Te intraperitoneal injection of glucose at a dosage of 2 mg/kg was performed, and the levels of blood glucose were measured at intervals of 0, 30, 60, 90, and 120 minutes following administration.Te area under curve (AUC) was calculated by using the trapezoid rule [41].
2.12.Histopathologic Liver Analysis.Te liver tissues were fxed, processed, embedded in parafn wax, sectioned (5 μm thick), stained (hematoxylin/eosin), and analyzed on digital images of the entire surface of each liver sample (right and left lobules) as previously described [42].A pathologist conducted a blinded evaluation of all histological parameters.Hepatic steatosis was classifed according to the percentage of liver tissue that presented with fat accumulation, which was grade 0 (absent), grade 1 or mild (up to 33%), grade 2 or moderate (between 33 and 66%), and grade 3 or severe (more than 66%) [36].Infammation was evaluated by functioning histologic zones depending on the oxygen supply as previously described, being classifed into four categories according to the percentage of tissue presenting with infammatory infltrate: none (0%), mild (1 to 32%), moderate 33-66%), and severe (>66%) [42,43].

Locomotor Activity Evaluation.
Te psychomotor analysis experiments are crucial for evaluating the potential impact of Moringa oleifera treatment on both anxiety-related behavior and muscular tone in mice.Tese assessments will provide a comprehensive understanding of the treatment's efects, aligning with the goal of exploring the broader implications of Moringa oleifera in the context of nonalcoholic fatty liver disease (NAFLD).
2.14.Open Field.Following the treatment, mice were evaluated for locomotion impairment using an open feld activity monitoring system, which is a useful tool for studying locomotion impairment in animal models [44].Tis test was carried out as previously described by Montes-Galindo et al., 2019 [42], considering an alteration in the locomotor activity in case a signifcant change occurred in the speed of the experimental group, compared to that of the control group [45].

Elevated Plus Maze.
A widely used rodent behavioral assay called the elevated plus maze has been validated for assessing the antianxiety efects of medications [46].Tis evaluation was carried out following previously reported specifcations [47], calculating the anxiety-like behavior index according to Cohen et al. [48].

Rotarod Test.
When the treatments were fnished the mice were trained on 3 consecutive days on a rotarod (LE8300; Letica LSI, Pan-lab Scientifc Instruments, Barcelona, Spain) at a speed of 18 rotations per minute.We recorded rotations and falls.Tese variables were used to identify any alterations in motor coordination and balance [49].
2.17.Statistical Analysis.For descriptive statistics, the data were represented as the mean ± standard deviation, or, in certain cases, utilizing the standard error of the mean (SEM).Te normal distribution of the data was evaluated through the application of the Kolmogorov-Smirnov test.Subsequently, the equality of variances was verifed using Levene's test.To ascertain diferences between groups, oneway ANOVA was performed, followed by Tukey's test post hoc analysis.Te statistical analyses were conducted using IBM SPSS version 20 software (IBM SPSS, Chicago, Illinois, USA).A p value of <0.05 was used to determine statistical signifcance.

Results
Te resultant yield of the ethanolic extract of MO was determined to be 3.5% w/w.Te phytochemical analysis (Table 1) carried out on the ethanolic extract of the MO leaf extract revealed the presence of tannins, alkaloids, steroids, and saponins.On the other hand, in the aqueous infusion, it was possible to detect favonoids, steroids, saponins, and a slight presence of alkaloids.Tese compounds represent some of the prominent metabolite families within the plant species.However, it is important to note that the list provided is not exhaustive, and the presence of other phytochemicals cannot be ruled out.Other compounds not included in the analysis might also contribute to potential pharmacological activities, but further studies are necessary.
Te total favonoid content in M. oleifera samples was quantifed using a standard quercetin calibration curve (y � 0.0027x + 0.0058, R 2 � 0.9986).Te results are expressed in Table 1 as the total favonoid content (TFC) in μg of quercetin equivalents (QE)/mg by extract.Te highest favonoid content was quantifed in the infusion tea, reaching 172.66 ± 7.58 μg/mg extract, while the ethanolic extract showed a concentration of 19.40 ± 10.68 μg/mg.Te reduction of ferricyanide (Fe 3+ ) to ferrous state (Fe 2+ ) by plant phytochemicals is a good indicator of antioxidant activity [50].In this study, the percentage (%) of reducing power of the ethanol extract of M. oleifera was estimated against the standard drug ascorbic acid (Table 1).Te reducing capacity of both Moringa samples (extract and infusion) showed values near that of ascorbic acid.In the case of the antioxidant activity in comparison to the ascorbic acid, both samples show a near 50% activity.In particular, the ethanol extract of Moringa slightly surpasses the infusion by around 10%.
1 H and 13 C NMR spectra as shown in Figures S1-S4 were obtained for the evaluated extracts (MO EtOH and infusion).Signals in the spectra were assigned, and the shifts were compared to isolated compounds from M. oleifera (S1 Table ).Regarding the extract, it could be hypothesized that it is rich in alkaloid-type compounds.In these extracts, some of the signals obtained in 1 H and 13 C are like those reported for niazirin and also similar to niazirinin; alkaloids were identifed from MO leaves [51,52].Both the extract and the infusion presented signals around 7.05 to 9.03 ppm, like what was expected for this type of alkaloids.In respect to the MO infusion, most of the signals assigned are found in the ethanolic extract but in smaller intensity (125 to 140 ppm).Te comparison with the literature allowed us to compare some triterpene molecules, such as β-sitosterol [53], fnding that this possible presence could explain the positive reaction with the sulfuric acid result obtained in the preliminary phytochemical screening.Based on the comparison of the NMR spectra and the phytochemical background of the species, it is suggested that the MO extract and infusion evaluated in these experiments are mostly composed of alkaloids, triterpenes, and favonoids, which are glycosylated evidenced by signals around 3.0-5.0ppm.

Food Intake and Weight
Gain.Regarding food consumption, throughout the period from the fourth to the sixth month (5.32 ± 0.6 in healthy vs. 7.40 ± 0.3 in the NAFLD model for the fourth month and 6.17 ± 1.2 in healthy vs. 8.15 ± 0.2 in the NAFLD model for the sixth month), it was consistently found that the mice in the NAFLD model presented a signifcantly higher consumption than those in the healthy group (p < 0.001 for all periods).
Once the treatments started (sixth month), during 2 months that MO was administered, it was observed that in the last month, the NAFLD-MO infusion group consumed signifcantly less food than the NAFLD-placebo group (5.64 ± 2.0 g vs. 11.0 ± 2.9 g, p � 0.020), and when comparing between the group treated with NAFLD-MO infusion vs. NAFLD-MO ethanolic extract, it was found that the group that received infusion consumed signifcantly less food than the group that received extract (5.64 ± 2.0 g vs. 10.27 ± 1.4 g, p � 0.010) (see Figure 2(a) and SII Table ).
Regarding the weight gain from the third month until the end of the treatments, there was signifcantly greater weight gain in the NAFLD-placebo vs. the healthy group (36.92 ± 2.1 vs. 29.95± 2.2, p ≤ 0.001).In the last month of the treatment, less weight gain was observed only in the NAFLD-MO infusion group compared to the NAFLDplacebo group (25.70 ± 1.2 g vs. 36.92± 2.1 g, respectively, p ≤ 0.001), as well as to the NAFLD-MO ethanolic extract (25.70 ± 1.2 g vs. 35.53± 1.9 g, respectively, p ≤ 0.001), and even with the healthy group (25.70 ± 1.2 g vs. 29.95± 2.2 g, respectively, p ≤ 0.001), see Figure 2(b) and SII Table.

Biochemical and Hematological Parameters.
A signifcant decrease in cholesterol, triglycerides, total lipids, SGOT, SGPT, and glucose was found in the NAFLD groups treated with MO infusion and in ethanolic extract compared to the NAFLD-placebo group (p � 0.001).When doing the comparison between the group treated with infusion vs. ethanolic extract, lower levels of triglycerides SGOT and SGPT 6 Journal of Nutrition and Metabolism were found in the group that received infusion vs. the ethanolic extract (p � 0.020).On the other hand, regarding the hematic parameters, only signifcantly higher hemoglobin levels were found in the group treated with infusion than in the healthy control group (p � 0.001), as shown in Table 2.

Glucose Tolerance Curve.
With the aim of ensuring the comprehensive monitoring of individuals throughout the study, observations were made at months 0, 3, and 6.Basal fasting glucose levels were comparable across all groups at the study's onset (p � 0.153).Te infuence of a high-fat diet on hyperglycemia became evident after three months, as evident when compared with the standard diet (Table 3).Before the commencement of any treatments, a glucose tolerance test was conducted at the 6-month mark.Tis assessment highlighted that the healthy group displayed signifcantly lower glucose levels (across all measurement intervals: basal, 0, 30, 60, 90, and 120 minutes) when contrasted with all NAFLD-aficted groups (p < 0.001).Tis background context sets the stage for understanding the subsequent results.Considering these observations, the MO treatment displayed reductions in glucose levels within the NAFLD-MO ethanolic extract group, establishing its superiority over the NAFLD-placebo group (p � 0.010).Moreover, a direct comparison between the NAFLD-MO infusion group and the NAFLD-MO ethanolic extract group highlighted consistently lower glucose levels in the latter (p � 0.001), as detailed in SIII Table and visually depicted in Figure 3.

Organ's Weight.
Te measurements of organ weights, including the liver, right kidney, left kidney, pancreas, brain, and ovaries, as well as the epididymal fat pad, are detailed in Table 4. Tis comprehensive evaluation was conducted as part of the study's efort to provide insights into the potential  safety implications of MO.Notably, a signifcantly lower liver weight was found in the group treated with infusion compared to the NAFLD-placebo group (p � 0.048), and a corresponding signifcant reduction in epididymal fat pad was also observed (p � 0.001), as indicated in Table 4.

Histopathological Parameters.
Regarding steatosis and infammation, signifcantly lower percentages were found in the two NAFLD groups treated with MO than in the NAFLD-placebo group (p ≤ 0.001).When making the comparison between the NAFLD groups treated with MO, signifcantly lower percentages of steatosis and infammation were observed in the group that received the infusion than in the group treated with the ethanolic extract (p � 0.002), as can be seen in Table 5 and Figure 4.

Locomotor Activity Evaluation.
To evaluate mobility strength, we measured the number of rotations and turns.Regarding the number of rotations, a signifcantly higher number was observed in the NAFLD group treated with infusion than in the SD diet and the NAFLD-placebo group (p � 0.003).On the other hand, regarding the number of falls, a signifcantly lower number of falls were observed in the NAFLD groups treated with MO in relation to NAFLD with placebo (p < 0.001).Finally, the anxiety-like behavior index was calculated in the elevated plus-maze experiment.
In this experiment, the anxiety-like behavior index is referred to as the number of crossings of the mouse between the open and closed arms, as well as, the time that it remained in each of the arms of the maze.In our results, a lower anxiety-like behavior index was found in the NAFLD group treated with MO in the form of infusion than in the NAFLD-placebo group, and even to the healthy group (p < 0.001).When making the comparison between infusion vs. ethanolic extract, the group with infusion showed a lower rate of anxiety-like behavior (p < 0.001), as shown in Table 6.

Discussion
Te objective of the present study was to compare the effcacy of hot tea infusion vs. ethanolic extract of MO for the simultaneous treatment of nonalcoholic fatty liver, hyperlipidemia, and hyperglycemia in a murine model fed with a high-lipid diet and an established NAFLD.An important point to discuss is that the same amount of ethanolic extract was used as the leaf used for the infusion (traditional form) [16,17].Although the concentration of metabolites would be diferent, the objective of the study is to present a comparison of the response to exposure of Moringa oleifera as a refection about what the population consumes.However, future studies are required to clarify the concentration of metabolites consumed by the individual in an infusion and in an ethanolic preparation.
First, the fndings showed that the administration of MO in both infusion and ethanolic extract in individuals with NAFLD reduced food intake, when compared with the NAFLD group with placebo at the seventh month.Tis result coincides with previous studies, which have identifed that the administration of ethanolic extract for 49 days [51] and methanolic extract of MO to mice fed with HFD reduced the food consumption, even, as early as, 21 days after beginning the extract administration [52].In the same way, Rojas-Silva found that the cumulative food intake was lower in the group of mice fed with an HFD +5% MO concentrate from 12 week [53].
It is important to highlight that in the group treated with the infusion of MO, the food intake of mice was signifcantly reduced (from the seventh to the end of the experiment), simultaneously leading to weight loss (see Figure 2) and improvement in NAFLD (see Figure 4).Tese phenomena could be related to the expression of anorexigenic peptides, both of hypothalamic and intestinal origin, which play an important role in the pathology of NAFLD [54,55] and may be modulated by the metabolites present in the infusion (see Table 1).However, further experiments are necessary to identify and isolate the specifc metabolites present, elucidating their mechanisms of action.
Second, the present study identifed a decrease in the levels of total cholesterol, triglycerides, total lipids, SGOT, SGPT, and glucose in the groups treated with MO, in both infusion and ethanolic extracts, compared to the NAFLD group without treatment.Tis fnding supports earlier reports that have suggested that MO at diferent doses lowers total cholesterol [56,57], triglycerides [58], ALT, and AST [59] in rats fed with HFD + MO in diferent doses.
Figure 4 shows that the mice treated with the ethanolic extract of MO presented better glycemic control than the mice treated with the infusion.Tis efect could be due to diferences in the composition and abundance of the secondary metabolites (alkaloids).Some previous phytochemical studies indicate that high polarity extracts (ethanol and methanol) [60] are abundant in pyrrole-type glycosylated alkaloids (niazirin, niazirinin, marumoside A, marumoside B, 4-((4′-0-acetyl-a-L-rhamnosyloxy) benzyl) isothiocyanate, and pyrrolemarumin-4″-O-α-L-rhamnopyranoside) [61,62].Tese high polarity extracts have shown antidiabetic activity, anti-infammatory capacity, and radical scavenging activity [63].It is important to point out that, in the preliminary phytochemical tests, the ethanolic extract showed a more intense response to the alkaloid assay, which could probably explain its better efect on glycemia management.However, more studies are required to identify, isolate, and evaluate these compounds, as well as the molecular mechanisms involved in this phenomenon.
Previous research has revealed that cholesterol decrease is possibly attributable to the presence of bioactive phytoconstituents, known as zand β-sitosterols [60,61] that are known for reducing the level of LDL-c in serum without modifying the high-density lipoproteins or triglycerides [60,63].Another suggested reason why MO exerts hypolipidemic efects is chlorogenic acid (CGA), since it is one of the bioactive components present in sufcient quantities in MO [64].Although the exact phytoconstituent responsible  for the efects observed in this study is not yet known, studies have shown that many of these phytochemicals possess benefcial efects, such as stimulation of glucose transport, inhibition of adipocyte diferentiation, lowering of ALT, AST, total cholesterol, and triglycerides [65], and the attenuation of oxidative stress [57].Tird, one of the indicators used to identify the therapeutic efect of MO on NAFLD has been hepatic steatosis; our results showed a quantitative-qualitative decrease in steatosis after the administration of MO, in infusion or ethanolic extract.Te efect of MO on steatosis has been reported in some studies.For example, Faizi et al., after feeding rats with an HFD for 49 days, observed a development of a high degree of steatosis in the rats; however, the group exposed to the HFD plus daily supplementation of 400 mg/kg of methanolic extract of MO showed a decrease in the development of hepatic steatosis [52].Similarly, another study using MO at a high concentration (15%) showed a lower degree of steatosis in animals fed an atherogenic diet.Finally, MO has been associated with hepatoprotective effects not only in response to the consumption of unbalanced diets but also in mitigating potential liver damage by druginduced toxicity [66,67].Despite the evidence of the positive efect of MO on hepatocytes, the mechanisms involved have not been fully elucidated.Te literature suggests that MO may prevent hepatic steatosis by afecting gene expression related to hepatic lipid synthesis, resulting in lower cholesterol and triglyceride concentrations and reduced infammation in the liver [68].Quercetin and chlorogenic acid have demonstrated the ability to reduce triglyceride by inhibiting diacylglycerol acyltransferase-2 (DGAT2) [68], a key microsomal enzyme involved in the biosynthesis of TG.Given that MO is rich in these compounds [64], it is speculated that their presence in MO may contribute to decrease DGAT2 expression, potentially explaining the observed reduction in triglyceride concentrations in animal models with MO administration [68].
Likewise, it is presumed that the therapeutic efects of MO may be attributable to characteristic phytochemicals, glucosinolates, and isothiocyanates, found in many parts of the MO tree and quite prominently in its leaves, which participate in the elimination of reactive oxygen species and cellular redox balance [20,69].
Fourth, the second and last histological indicator to identify the therapeutic efect of MO on NAFLD was hepatic infammation.Te efect of MO on infammation has been studied to a lesser extent.Among the fndings, there are those that showed that chronic exposure to an HFD + supplementation of methanolic extract of MO prevented the formation of hepatic infammation compared to nonsupplemented animals, who developed a higher degree of infammation [51].
On the other hand, Hamza et al. confrmed the hepatoprotective properties by the marked improvement in the necroinfammatory score in the animals that received MO compared to their counterparts who did not [70,71].Joung et al. demonstrated the suppression of infammation caused by a high-lipid diet with the administration of MO [72].Tese results suggest that MO has hepatoprotective and anti-infammatory properties against liver damage, due to its antioxidant properties and anti-infammatory efects [69].
Te results showed a signifcantly greater efect on hepatic steatosis and infammation and biochemical values of ALT, AST, total cholesterol, and triglycerides when the administration of MO was in infusion.Tea remains one of the most frequently consumed beverages, on account of its antioxidant or pro-oxidant properties.Active compounds in tea, particularly tea polyphenols, can directly or indirectly scavenge ROS to reduce oncogenesis, cancer, and metastasis [73,74].However, it is possible that the combination of polar phenolic compounds like favonoids and tannins with alkaloids is responsible for this efect.Preliminary analysis of the plant's chemical composition showed that both the ethanolic extract and the hot water infusion have similar profles to the tested secondary compounds, except for favonoids (less concentration in MO ethanolic extract than in infusion).Flavonoids were found in the hot water infusion, likely due to higher extractability caused by heat.Tere was also a slight diference in precipitate formation observed during alkaloid selection.Terefore, it can be concluded that the slight diference between the two is due to the variation in phytoconstituent content, which could be caused by diferences in active ingredient concentration or the decomposition of active ingredients during the preparation of the hot tea infusion using gentle heat [75].Tis could explain the better results in the infusion used in our study.
Before addressing behavioral studies, it is important to highlight that these were carried out based on evidence establishing a relationship between nonalcoholic fatty liver disease (NAFLD), anxiety, and depression [76].Our study, although primarily focused on investigating NAFLD under treatment with Moringa, considered the possibility of comparing potential efects on behavioral aspects.Tese aspects serve to explore potential indirect efects associated with the treatment.In the frst place, the detection of alkaloids, for instance, within the extract provides valuable insights into the chemical composition of Moringa oleifera.Alkaloids are known to have diverse physiological efects, including potential impacts on behavior.Our results showed that the group that received MO infusion showed higher mobility than the SD diet group and the ethanolic extract group.Regarding anxiety-like behavior, none of the study groups qualifed for anxious behavior.Tis result may suggest that although the group that consumed the MO infusion increased their mobility, they did not present behaviors related to anxiety-like behavior.Previous studies have suggested that MO has protective efects on behavioral issues.For example, in the study by Islam et al. 2020 [25], efects of MO were found in the central nervous system for the control of anxiety-related disorders, and the administration of MO was found to be potentially an efective stress reliever [77,78].In fact, the direct relevance of these behavioral studies to NAFLD might be limited, and their inclusion contributes to a holistic understanding of the treatment's efects.It is possible that these behavioral changes, although subtle, could indirectly infuence factors such as food consumption or physical activity, which in turn Journal of Nutrition and Metabolism could afect the metabolic parameters under investigation, such as hyperlipidemia and hyperglycemia, but more studies are needed.A strength of the study is the simultaneous analyses of various therapeutic efects of M. oleifera when it is used in individuals who already have established NAFLD.Its preventive efect was not analyzed but rather its ability to reverse various deleterious efects caused by the disease.Likewise, the efect of the ethanolic extract and the hot MO infusion was analyzed, which ofered information on two therapeutic presentations, which can be used in the future, according to the patient's needs or feasibility of use.One of the perspectives for the present study is to evaluate both the M. oleifera infusion and the ethanolic extract in mice on a standard diet or under other diets such as a ketogenic diet to determine the presence or absence of possible protective/ toxic efects.Te authors are aware that this study has several limitations since the molecular mechanisms of the therapeutic efects found were not analyzed.Likewise, the phytochemical study of the species is not ruled out as a future possibility and should be explored to clarify the possible molecular mechanisms responsible for its biological activity.
Furthermore, future studies will aim to investigate the impact of M. oleifera on infammatory cytokines, particularly those afecting insulin signaling.In addition, the efect of M. oleifera treatment on reactive oxygen species (ROS) and certain activities must be explored to better understand its potential role in addressing insulin resistance and hepatic steatosis.As a limitation, it is important to note that the current study did not examine the expression of SGOT and SGPT at the levels of mRNA or protein, nor were any functional assays completed.Understanding the impact of M. oleifera on these specifc markers would provide valuable insights into its comprehensive efects on liver function.Future investigations should consider incorporating these analyses to further enhance the understanding of the mechanisms underlying the observed outcomes.
Despite these limitations, the current study provided evidence for the efects of MO on nonalcoholic fatty liver, hyperlipidemia, and hyperglycemia in an HFD mouse model, which may contribute to knowledge and future studies performed in patients with these diseases.

Conclusion
In conclusion, chronic oral administration of MO infusion leaf extract demonstrates a therapeutic impact, improving liver histology and biochemical markers in a murine NAFLD model.Furthermore, the therapeutic efectiveness of MO extract is notably enhanced when administered as an infusion compared to the ethanolic extract in the same NAFLD murine model.

Supplementary Materials
S1 Table: comparisons of chemical shifts of the MO extract and infusion vs. some of the principal metabolites isolated from the leaves of Moringa oleifera in CDCl 3 .SII Table: food intake and weight of the experimental groups.SIII Table : glucose tolerance curve according to the groups studied.S1 Figure : 1 H NMR spectra for the infusion leaves of Moringa oleifera, 1 H-NMR in CDCl 3 (400 MHz).S2 Figure : 1 H NMR spectra for the ethanolic extract from the leaves of Moringa oleifera, 1 H-NMR in CDCl 3 (400 MHz).S3 Figure: 13 C NMR spectra for the infusion leaves of Moringa oleifera, 13 C-NMR in CDCl 3 (100 MHz).S4 Figure: 13 C NMR spectra for the ethanolic extract from the leaves of Moringa oleifera,

Figure 1 :
Figure 1: General diagram of the research.BALB/c mice were fed a balanced diet (healthy control) or a high-fat diet for 6 months.With this, the NAFLD model was established before starting a therapeutic intervention with MO for 2 months.

Figure 4 :
Figure 4: Histopathological parameters in the groups.It is possible to observe that the liver tissues in the standard diet (SD) group do not show pathological changes.Te NAFLD-placebo group shows a greater presence of signs of steatosis and infammation than the liver tissues of the groups treated with NAFLD-MO.As an example, the black arrow shows an area of steatosis, while the blue arrow shows the presence of infammatory cells.Microphotographs stained with hematoxylin and eosin at xx magnifcation, with the framed area presumably at ×200 magnifcation.
Further investigations are warranted to elucidate the underlying mechanisms driving these observed efects.funded by the University of Colima Faculty of Medicine and the Fundación para la Ética, Educación e Invstigación del Cáncer del Instituto Estatal de Cancerología de Colima, A.C. Mexico.

Table 1 :
Phytochemical screening components of M. oleifera ethanolic extract and infusion.− � completely absent.a TFC: expressed in quercetin equivalents (QE) μg/mg of extract.b FRPA: expressed as % reducing power relative to the ascorbic acid control.c TAC: expressed as % antioxidant capacity relative to the ascorbic acid control.

Table 3 :
Fasting glucose levels (mg/dL) at various times during NAFLD model generation.

Table 4 :
Organ weight of the experimental groups.

Table 6 :
Locomotor activity evaluation according to the groups studied.