Materials and Methods

Plant material. Eriobotrya japonica leaves (500 g, fresh weight) were obtained from the Botanical Garden “Francisco Javier Clavijero” of the Instituto de Ecología (INECOL), A.C., in Xalapa, Veracruz, Mexico, using three adult individuals of this species, the leaves were collected in the spring of 2021. A specimen was identified by curators of the herbarium XAL, and a voucher was deposited in the herbarium XAL (number XAL0106251). Leaves were washed with distilled water and were dried at 25 °C for one day. The dried leaves were then stored at -20 °C before being lyophilized for six days in a freeze dryer (FreeZone 1, Labconco, Kansas, MO, USA). After lyophilization, the leaves were ground in a mortar and stored in a plastic bag at 4 °C prior to further processing.

Total leaf content of carbon, nitrogen, and macro elements. Total C and N content from 1 g of freeze-dried leaves (n = 3) was determined by dry combustion using an auto-analyzer (TruSpec CN, LECO, Corporation, St. Joseph, MI). Quantification of phosphorus (P), sodium (Na), potassium (K), calcium (Ca), and magnesium (Mg) was performed according to ^{Etchevers (1988)}. Macro elements of leaves were quantified by atomic absorption (Ca and Mg) using a fast-sequential atomic absorption spectrometer (AA240FS, Varian) and flame spectrophotometry for K and Na were analyzed using a flame photometer (410 corning). For the quantification of total P, the colorimetric method of vanadomolybdophosphoric acid was used (^{Etchevers 1988}). The absorbance of the standard dilutions was measured with a spectrophotometer (Genesys 20, Thermo Scientific, Waltham, Massachusetts, USA). A triplicate was performed for each measurement.

Identification and quantification of phenolic compounds by UPLC-QqQ-MS analysis. The leaf methanolic extract (LME) was obtained using an accelerated solvent extraction system (ASE 350, Dionex Corporation, Sunnyvale, CA, USA) following the protocol previously described by ^{Infante-Rodríguez et al. (2020)}. For this purpose, 3 g of plant material were mixed with 1 g of diatomaceous earth (Thermo Scientific, Waltham, MA, USA) and placed in a 34 mL cell. The method consisted of a single static cycle of 15 min at 60 °C. The methanolic extract was concentrated by rotary evaporation under reduced pressure at 40 °C (RII, Büchi, Flawil, Switzerland). The identification and quantification of phenolic compounds was performed in an ultra-performance liquid chromatograph coupled to a triple quadrupole mass spectrometer (UPLC-QqQ-MS, 1290-6460 Agilent Technologies, Santa Clara, CA, USA) using a dynamic multiple reaction monitoring (dMRM) method following the protocol previously described by Infante-Rodríguez et al. (2020). For this, chromatographic separations were carried out on a ZORBAX SBC18 column (1.8 µm, 2.1 × 50 mm) (Agilent Technologies) with the column temperature at 40 °C. Mobile phase consisted of (A) water containing 0.1 % formic acid and (B) acetonitrile containing 0.1 % formic acid. The gradient conditions of the mobile phase were: 0 min 1 % B, 0.1-40 min linear gradient 1-40 % B, 40.1-42 min linear gradient 40-90 % B, 42.1-44 min isocratic 90 % B isocratic, 44.1-46 min linear gradient 90-1 % B, 46.1-47 min 1 % B isocratic (total run time 47 min). The flow rate was 0.1 mL/min, and 5 µL of sample injection volume. dMRM were obtained on an Agilent 6460 Triplequadropole (QqQ) mass spectrometer. The ESI source was operated in positive and negative ionization modes, desolvation temperature of 300 °C, Cone gas (N2) flow of 5 L/min, nebulizer 45 psi, sheath gas temperature 250 °C, sheath gas flow of 11 L/min, capillary voltage (positive and negative) 3,500 V, nozzle voltage (positive and negative) 500 V. For quantitation of each phenolic compound, a calibration curve in a concentration range of 0.25 to 19 µM was constructed (R ² values = 0.99 were considered for the linearity range) (Table 1) and quantities were established by using MassHunter Workstation Software version B.06.00 (Agilent Technologies). Results are expressed as µg/g of sample (dry weight).

Identification of volatile compounds by GC-MS analysis. The leaf petroleum ether extract (LPE) for gas chromatography coupled to mass spectrometer (GC-MS) analysis was obtained using the method of Ferguson (^{Borgonetti et al. 2020}). An amount of 10 g of lyophilized vegetable powder was taken and soaked in methanol for 24 h. Subsequently, the filtrate was extracted with petroleum ether; the LPE was analyzed using a GC coupled to a single quadrupole MS (2010 Plus-QP2010 Ultra, Shimadzu, Tokyo, Japan) equipped with a ZB-5MSi column (30 m × 0.25 mm ID × 0.25 μm). Electron impact (70 eV) spectra were obtained. Helium was the carrier gas (0.8 cm³/min, constant flow), and a split-less injector (temperature of 250 °C, split valve delay of 3 min) was used to inject the sample. The oven temperature was held at 50 °C for 2 min, then programmed to increase at a rate of 15 °C/min to 280 °C, which was held for 10 min. The ion source temperature was 250 °C. Tentative identifications were made by comparison of fragmentation patterns with those patterns available in the NIST/EPA/NIH Mass Spectral Library, NIST 11, Software version 2.0 (National Institute of Standards and Technology, www.nist.gov) using a range of 84-100 % similarity values, with the Lab solutions GC-MS solutions 2.72 software (Shimadzu, Tokyo, Japan). Some identifications were confirmed by comparison of retention times and mass spectra with commercially available standards analyzed in the same instrument and analytical conditions. The relative area was calculated integrating each compound peak area and determining the contribution of each compound to the total area (sum of the individual areas).

α-Glucosidase enzyme inhibition assay. The α-glucosidase enzyme inhibition by the LME of E. japonica was determined by in vitro enzymatic inhibitory assays according to ^{Infante-Rodríguez et al. (2022)}. The α-glucosidase enzyme (≥ 100 U/mg protein) from the yeast Saccharomyces cerevisiae (Desm.) Meyen (Sigma Aldrich St. Louis, MO, USA) was diluted to 0.005 mg/mL in phosphate buffer (PB, 100 mM, pH 7.2, Sigma Aldrich, St. Louis, MO, USA). Then, 20 μL of LME dissolved at 1 mg/mL in PB was mixed with 100 μL of 4-nitrophenyl-α-D-glucopyranoside (Sigma Aldrich, St. Louis, MO, USA) at 1 mM in PB. Acarbose (Sigma Aldrich, St. Louis, MO, USA) was used as a positive control (30 mM). The reaction mixture was incubated for 5 min at 30 °C. After the incubation time, the enzyme was added to each well, and the microplate was incubated for 30 min at 30 °C. Absorbance was measured at 405 nm at the beginning and after 30 min in a microwell spectrophotometer (Multiskan FC, Thermo Scientific, Waltham, MA, USA). The inhibition percentages (PI) were calculated with the equation:

Where ABS_control, ABS_extract, and ABS_blank correspond to the absorbance of the negative control, inhibitor (LME or acarbose) and PB, respectively.

Statistical analysis. The total C and N, and the quantification of P, Na, K, Ca, and Mg were expressed in percentage or cmol/Kg. Quantification of individual phenolic compounds are expressed in μg/g of dried sample. The α-glucosidase enzymatic inhibition results were analyzed with Wilcox rank sum test for group comparisons. All statistical analyses were performed with the Agricolae library (^{De Mendiburu 2010}) in R software v. 4.1.2 (^{R Core Team 2020}).

Results

Total leaf content of carbon, nitrogen, and macro elements. The total C and N content in E. japonica leaves were 49.9 ± 2.95 % and 1.77 ± 0.7 %, respectively; resulting in a C:N ratio of 28.19 ± 10.94 (Table 2). In addition, the leaves content of Na, K, Ca, and Mg were 97.96 ± 64.9 cmol/Kg, 36.73 ± 15.87 cmol/Kg, 37.29 ± 16.97 cmol/Kg, and 27.85 ± 16.41 cmol/Kg, respectively. Leaves exhibited a lower P content of 3.73 ± 0.36 cmol/Kg (Table 2).

Identification and quantification of phenolic compounds by UPLC-QqQ-MS analysis. Twenty-two phenolic compounds plus a precursor (shikimic acid) were identified and quantified in LME, mainly phenolic acids and flavonoids (Table 3). The most abundant compounds are the flavonoids rutin, kaempferol-3-O-glucoside and quercetin-3-glucoside (Table 3). In addition, the phenolic acids that exhibited the highest content are caffeic acid, 4-hydroxyphenylacetic acid, and chlorogenic acid. Also, the proanthocyanidin procyanidin B2 and the dihydrochalcone glucoside phloridzin were quantified (Table 3).

Identification of volatile compounds by GC-MS analysis. Nineteen compounds, including fatty acids (7), terpenes (9), and aromatic (2) and aliphatic (1) compounds were identified through GC-MS analyses in the LPE. The identified compounds with the highest relative peak area (%) were phytol and the palmitic, linoleic, and stearic acids, as well as phytol acetate (Table 4).

α-glucosidase enzyme inhibition assay. LME derived from E. japonica demonstrated an 86.05 ± 0.73 % inhibition of α-glucosidase enzyme in vitro, showing similar effectiveness to the control substance of acarbose, with an α-glucosidase inhibition of 83.43 ± 0.44 %. No significant differences in α-glucosidase inhibition were observed between the two treatments (P = 0.1) (Figure 1).

Figure 1 Inhibitory activity of the enzymes by acarbose, and leaf methanolic extract (LME) of E. japonica (1 mg/mL). Bars represent mean ± SD of inhibitory activity (3 mM for α-glucosidase), Wilcox rank sum test (P > 0.05). 

Discussion

The main aims of the present study were to identify and quantify the presence of different minerals, and secondary metabolites extracted from the leaves of this plant species. In addition, to test the α-glucosidase inhibition in the LME.

Only a few studies have reported that E. japonica leaves contain minerals, fiber, and several vitamins. For example, ^{Hwang et al. (2010)} found that the mineral content of loquat leaf from a Korean population were greater in order of Ca > K > Mg > Na > Fe > Mn > Zn. An analysis of a loquat leaves from a Moroccan population made by ^{Khouya et al. (2022)} revealed high levels of Ca, K, and Mg with values of 267.50, 953.80, and 279.60 mg/100 g, respectively. Also, they reported a moderate content of Na (40 mg/100 g), and a relatively low amount of Fe (0.5 mg/100 g). The results of the present study showed that E. japonica leaves were a rich source of carbon-based compounds. Conversely, the leaves were poor in N, suggesting that the leaves have a relatively low concentration of nitrogen-containing compounds. The observed C:N ratio (28.19 cmol/Kg) indicates that C (49.9 cmol/Kg) is present in a significantly higher proportion compared to N (1.77 cmol/Kg) in our samples of E. japonica leaves. Furthermore, the leaves are also found to have high levels of the minerals Na, K, Ca, and Mg (Table 2). These minerals contribute to the overall nutritional profile of the leaves. However, the content of nutrients and mobile elements (such as N, P, K, and Mg) could vary significantly among seasons and loquat populations (^{Quiñones et al. 2013}); these variations could be related to nutrient mobility along with individuals, high-intensity shading, soil type and soil bioavailability of nutrients, and the annual physiological cycle of each loquat cultivar; in some cases, long-term and high-intensity of shading can increase foliar mineral nutrients in this species (^{Shan et al. 2020}).

Certain micronutrients play a critical role in human metabolism and are essential for maintaining optimal health and preventing some diseases (^{Shergill-Bonner 2013}). Many diseases, including diabetes mellitus, have been experimentally shown to be controlled by medicinal plant extracts. The elements Mg, K, Ca, Mn, Fe, Zn, Br, Rb, Cr, Ti, Cu, V, Cl and Pb were identified in antidiabetic medicinal plants traditionally used in the management of diabetes mellitus (^{Arika et al. 2016}, ^{Gholamhoseinian et al. 2020}).

Mg (that was one of major mineral found in loquat leaves) is a trace mineral of importance to human biology and health and increasing evidence suggest that play an important role in glucose metabolism (^{Carneiro et al. 2013}). Also, Mg is an important ion in all living cells being a cofactor of many enzymes, especially those utilizing high energy phosphate bounds. The relationship between insulin and Mg has been recently studied and chronic Mg supplementation (3g/day for 3 weeks) can contribute to an improvement in both islet Beta-cell response and insulin action in non-insulin-dependent diabetic subjects (^{Paolisso et al. 1990}). People with high blood sugar are prone to develop deficiency in some minerals like K, Zn and Mg. Low levels of insulin causes decreased utilization of glucose by body cells, increased mobilization of fats from fat storage cells and depletion of proteins in the tissues of the body, keeping the body in crisis (^{Pathak 2014}). The functional foods of plant origin can help achieving optimal physiological metabolism and cellular functions helping the body to come out of these crises (Pathak 2014). High Mg in raw fruit peel and leaves of P. guajava was observed providing antidiabetic benefits in alloxan induced diabetic rats (^{Rai et al. 2007}). In people at high risk of diabetes Mg supplementation significantly improved plasma glucose levels after a 2 h oral glucose tolerance test, Mg supplementation appears to have a beneficial role and improves glucose parameters in people with diabetes and improves insulin-sensitivity parameters in those at high risk of diabetes (^{Veronese et al. 2016}). Minerals such as Na, K, Ca, and Mg are crucial for human health, as they play vital roles in functions such as fluid balance, muscular function, bone health, and regulation of blood pressure (^{Schiefermeier-Mach et al. 2020}). A normal concentration of K is required for optimal insulin secretion, and a deficiency of K leads to diabetic acidosis (^{Narendhirakannan et al. 2005}). K depletion can result in impaired glucose tolerance. In diseases associated with kidney disease (^{Narendhirakannan et al. 2005}), Na and K ions play an important role. Obtaining these minerals from plant sources in the diet is critical to prevent deficiencies and maintain a balanced intake, thus reducing the risk of cardiovascular diseases, bone issues, and other nutrition-related disorders (^{Kear 2017}). These suggest possibilities for the use of loquat leaves as a source of minerals for human health benefits.

Several studies have been carried out on the phytochemistry of E. japonica leaves, ^{Uysal et al. (2016)} identified and quantified 11 phenolic compounds in aqueous and methanolic extracts of loquat leaves collected in the Mediterranean region of Turkey including gallic acid, catechin, p-hydroxybenzoic acid, chlorogenic acid, p-coumaric acid, ferulic acid, and kaempferol, being the most abundant chlorogenic acid (5.79 mg/g of methanolic extract). ^{Chen et al. (2017a)} identified 20 compounds including phenolic acids, flavonoids, and triterpene acids in an ethanol/water (50:50) extract obtained from loquat leaves collected from China. Among the phenolic compounds identified were kaempferol xylose, chlorogenic acid, rutin, procyanidin C1, procyanidin B2, among others. ^{Chen et al. (2017b)} quantified gallic acid, protocatechuic acid, (+)-catechin, vanillic acid, caffeic acid, syringic acid, epicatechin, p-coumaric acid, ferulic acid, rutin, isoquercitrin, quercitrin, and quercetin in E. japonica leaves collected from the south of China. The most abundant compounds were (+)-catechin (883.23 µg/g of dried leaves) and epicatechin (266.49 µg/g of dried leaves). ^{Wu et al. (2018)} performed an HPLC-MS analysis using growing and fallen loquat leaves and report 12 compounds, including the presence of chlorogenic acid, vomifoliol-9-O-β-D-xylopyranosyl-(1→6)-β-D-glucopyranside, quercetin-3-O-galactosyl-(1→6)-glucoside, quercetin-3-O-sophoroside, quercetin-3-O-rutinoside, kaempferol-3-O-sophoroside, kaempferol-3-O-rutinoside, hyperoside, quercetin-3-O-glucoside, kaempferol-3-O-galactoside, quercetin-3-O-rhamnoside, and kaempferol-3-O-glucoside. ^{Park et al. (2019)} isolated and identified eight phenolic compounds from the methanolic extract of loquat leaves including chlorogenic acid, 5-O-caffeoylshikimic acid, 4-O-caffeoylshikimic acid, epicatechin, quercetin-3-O-galactoside, quercetin-3-O-glucoside, naringenin-6-C-(2′′-O-acetyl)-glucoside, and naringenin-6-C-(2′′,4′′,6′′-O-triacetyl)-glucoside. ^{Silva et al. (2020)} performed a chemical profiling of E. japonica leaves by paper spray mass spectrometry using hydroalcoholic (ethanolic and methanolic) extracts and different extraction methods. They report 49 compounds including organic acids, phenolic acids, flavonoids, sugars, quinones and terpenes. In addition, ^{Silva et al. (2020)} quantified the content of chlorogenic acid (159.81 - 511.04 µg/g extract), caffeic acid (25.05 - 82.01 µg/g extract), ellagic acid (0.92 - 2.73 µg/g extract), and quercetin (0.13 - 0.26 µg/g extract) in loquat levels. The best results were obtained using methanol/water (50:50) as extraction solvent and ultrasound as extraction method. The phytochemical analysis made by ^{Khouya et al. (2022)} revealed ten compounds in loquat leaves, consisting of protocatechuic acid, chlorogenic acid, rutin, quercetin, naringenin, epicatechin, epigallocatechin-3-gallate, kaempferol-rhamnose, and kaempferol, and a condensed tannin, procyanidin C1. The most abundant compounds were naringenin (10.93 ± 0.19 mg/g), procyanidin C1 (9.33 ± 0.16 mg/g), epicatechin (8.43 ± 0.04 mg/g) and rutin (7.55 ± 0.12 mg/g). In our study, the most abundant phenolic compounds found in MLE were flavonoids, and their glycoside derivatives included rutin, kaempferol-3-O-glucoside, quercetin-3-glucoside, (+)-catechin, quercitrin, quercetin 3,4-di-O-glucoside, and procyanidin B2. Despite the high number of chemical compounds reported in E. japonica leaves, few studies had performed quantitative determinations and as far as we know our study reports for first time shikimic acid, quercetin 3,4-di-O-glucoside, luteolin-7-O-glucoside, and phloridzin, contributing with the phytochemical knowledge of this species. Moreover, nineteen compounds were identified using the GC-MS approach, including seven fatty acids, nine terpenes, two aromatic compounds, and one aliphatic compound. ^{Zhou et al. (2019)} previously reported the presence of sesquiterpenes, triterpenes, monoterpenes, and fatty acids in the low-polarity fractions of E. japonica. ^{Tai et al. (2008)} identified several main compounds in the essential oils of E. japonica leaves, including n-hexadecanoic acid, (E)-nerolidol, (Z,Z,Z)-9,12,15- octadecatrien -1-ol, (+)-carvone, 2-hexanoylfuran, elemicin, dihydroactinidiolide, farnesyl acetate, farnesol, and α-bisabolol. ^{Hwang et al. (2010)} reported that loquat leaves contain fatty acids such as lauric acid, myristic acid, pentadecanoic acid, stearic acid, and oleic acid. ^{Hong et al. (2011)} found 109 constituents in loquat leaves extracted with petroleum ether using capillary GC-MS, with the highest concentration of constituents being phytol. In our study, we also identified compounds such as linoleic acid, phytol isomer, and phytol in the LPE. Besides, we have reported the presence of 16 additional compounds including palmitic acid, stearic acid, linolenic acid, ethyl ester, hexadecanoic acid, ethyl ester, tetradecanoic acid, dodecanoic acid, phytol acetate, hexahydrofarnesyl acetone, (2E,6E)-7,11-dimethyl-2,6,10-dodecatrien-1-ol, β-famesene, α-bergamotene, β-bisabolene, β-eudesmene, acetophenone, methyl methanthranilate, and 3-eicosyne.

Diabetes mellitus is a metabolic disorder caused by hyperglycemia resulting from a defect in insulin secretion or action (^{Pandhi et al. 2020}). Recent research suggests that certain compounds found in the leaves of E. japonica may have antidiabetic properties (^{Shih et al. 2010}, ^{Dhiman et al. 2021}). These compounds include flavonoids, tannins, and triterpenoids from the leaves or seeds, which may be useful in the prevention and control of type 2 diabetes (^{Liu et al. 2016}). The primary approach to mitigate the metabolic changes associated with type 2 diabetes is to inhibit the activity of the enzymes α-glucosidase and α-amylase (^{Proença et al. 2017}, ^{Infante-Rodríguez et al. 2022}). However, the side effects of some synthetic drugs have led to great interest in nontoxic and safe natural products for the treatment of these conditions (^{Fu et al. 2019}).

The LME showed in vitro inhibition of α-glucosidase enzyme. This enzyme plays a key role in the intestinal digestion of carbohydrates and its activity is one of the main factors affecting postprandial blood glucose (^{Yang et al. 2021}). Inhibition of α-glucosidase allows the production of hydrolyzed glucose from carbohydrates to be delayed, thus controlling postprandial blood glucose levels (^{Zabidi et al. 2021}). This finding could validate the use of this plant species in medicine for the treatment of diabetic patients (^{Dhiman et al. 2021}).

Compounds such as cinchonain-Ib, thymosaponin, chlorogenic acid, and epicatechin, which have been identified in the leaves and seeds of E. japonica, can reduce blood glucose, total cholesterol, and triglycerides, improve insulin secretion and sensitivity (^{Ansari et al. 2020}), and improve glucose tolerance (^{Ansari et al. 2022}). Also, chlorogenic acid exhibited an in vitro IC₅₀ of 9.2 µg/mL, while epicatechin exhibited an IC₅₀ of 290 µg/mL with a competitive mechanism by the pocket of enzyme (^{Oboh et al. 2015b}, ^{Abioye et al. 2023}).

Rutin, also identified in the LME, is a flavonoid found in medicinal plants with antidiabetic properties (^{Ansari et al. 2022}) and has been shown to have a wide range of activities, including antihyperglycemic effects (^{Ghorbani 2017}). Rutin exhibited an IC₅₀ of 0.037 µM/mL in vitro against α-glucosidase enzyme (^{Oboh et al. 2015a}), also, it has shown to reduce glucose absorption in the small intestine in vivo models by inhibiting the enzymes α-glucosidase and α-amylase involved in carbohydrate digestion (^{Hunyadi et al. 2012}, ^{Gao et al. 2015}). Kaempferol-3-O-glucoside is another compound that has been reported to have significant antidiabetic and antioxidant effects in extracts of Moringa oleifera Lam. (^{Irfan et al. 2017}) and Annona squamosa L. (^{Panda & Kar 2007}). Extracts of the Erica multiflora L. plant is rich in kaempferol-3-O-glucoside and have been used to ameliorate fatty liver disease induced by a high-fat, high-fructose diet by modulating metabolic and inflammatory pathways in rats (^{Khlifi et al. 2020}). In addition, this flavonoid inhibits the enzyme α-glucosidase with IC₅₀ values greater than 200 µmol/L (^{Li et al. 2022}). Catechin and quercitrin have also been reported to have potent antihyperglycemic activity (^{Dai et al. 2013}, ^{Mechchate et al. 2021}).

Apart from phenolic compounds, plants can contain saturated fatty acids that exhibit strong inhibitory activity against α-glucosidase and protein tyrosine phosphatase 1B (PTP1B), which is involved in insulin receptor desensitization and has become a drug target for the treatment of type II diabetes (^{Rocha et al. 2021}). By blocking the action of PTP1B, insulin sensitivity can be improved, promoting a more effective insulin response in bodily tissues; therefore, PTP1B inhibitors have the potential to be used in the treatment of diabetes and other related metabolic disorders (^{Genovese et al. 2021}). Several PTP1B inhibitors have already been found that interact with the binding site of the enzyme, surrounding the catalytic amino acid Cys215 and the adjacent region, or with the allosteric site of the enzyme (^{Rocha et al. 2021}). Fatty acids, phenols, flavonoids, flavonols, flavanones, isoflavones, lignans, and phenolic acids found in plants have been reported as PTP1B inhibitors (^{Zhao et al. 2017}).

According to the chemical composition of LPE, palmitic, linoleic, and stearic acids, and phytol and phytol acetate stand out compared to the other classes of compounds. Palmitic and linoleic acids are also abundant in E. japonica seeds (^{Henmi et al. 2019}) and are the main components of the saturated fats we consume in the human diet (^{Trinick & Duly 2013}). Palmitic acid, a saturated fatty acid, seemed the most potent inhibitor of PTP1B (IC₅₀= 45.5 µM), showing an affinity for this enzyme like some unsaturated fatty acids. Also, it has inhibitory activity towards α-glucosidase (IC₅₀= 111.51 µM) (^{Genovese et al. 2021}). Palmitic acid has been reported as a compound present in leaf extracts of Psychotria malayana Jack, a plant used in the treatment of diabetes. In vitro experiments have shown an IC₅₀ value of 8.04 µg/mL of palmitic acid inhibiting the enzyme α-glucosidase (^{Nipun et al. 2021}).

Linoleic acid plays an important role in plant defense against abiotic stress because it can induce the transcription of genes involved in plant defense (^{Kessler & Baldwin 2001}). It is an essential fatty acid for humans (^{Uysal et al. 2015}) and its consumption has been associated with a reduced risk of type 2 diabetes, better glycemic control, and insulin sensitivity (^{Belury et al. 2018}). It has also been reported that oleic and linoleic acids may contribute to high inhibition of α-glucosidase enzyme exhibiting IC₅₀ values of 0.022 and 0.032 µg /mL, respectively (^{Su et al. 2013}).

Stearic acid, according to ^{Marset et al. (2009)}, is the second most consumed saturated fatty acid. When a diet is rich in this fatty acid, plasma cholesterol levels reduction is observed (^{Bonanome & Grundy 1988}, ^{Mensink 2005}). Additionally, ^{Habib et al. (1987)} discovered that stearic acid inhibits tumor cells in both mice and humans. Also, it inhibits PTP1B activity, which could potentially boost insulin receptor signaling and trigger glucose uptake. In a PTP1B cell-free assay, stearic acid suppressed PTP1B activity in a dose-dependent manner ranging from 1-30 µM. At 30 µM concentration, stearic acid promoted glucose uptake into adipocytes by itself and significantly increased insulin-stimulated phosphorylation of insulin receptors at Tyr1185; however, insulin-induced phosphorylation of Akt remained unchanged (^{Tsuchiya et al. 2013}).

Phytol and phytol acetate are compounds that are commonly found in the leaves of plants (^{Hong et al. 2010}, ^{Syeda & Riazunnisa 2020}). Phytol has a wide range of biological activities, including anti-inflammatory, anti-allergic, immunostimulant, antinociceptive, antimicrobial, antioxidant, and antitumor effects (^{Pejin et al. 2014}). Taken together, our results suggest that the flavonoids, some glycoside derivatives, and fatty acids present in LME and LPE of E. japonica are good candidates that could explain the antidiabetic activity observed in the leaf extracts. These compounds could exert different action mechanisms of enzyme as competitive or non-competitive inhibitors of enzymes (^{Zhu et al. 2019}, ^{Su et al. 2013}). For example, it has been described that some flavonoids and fatty acids bind to the pocket site of the enzyme, which is considered a competitive inhibition (^{Zhu et al. 2019}, ^{Su et al. 2013}). Also, some phenolics such as chlorogenic acid can change their action mechanism to “mixed type" in synergic to other drugs such as acarbose (^{Abioye et al. 2023}). Moreover, some flavonoids may close the channel to the active center, that is usually associated with non-competitive inhibition (^{Zhu et al. 2019}). Due to the high complexity of extracts and this work's objective, it is impossible to define a mechanism action at this level over the enzyme. For that reason, it is recommended for future studies isolated the active metabolites to describe their IC₅₀ values, as well to study the specific actions mechanisms, and synergic effects.

In conclusion, the analysis of Eriobotrya japonica leaves performed on individuals collected in Veracruz, Mexico has revealed a composition of nutrients and minerals such as C, Na, K, Ca, Mg and P. Some of these minerals, such as Mg, may be important for human health, and there is increasing evidence of their importance in glucose metabolism (^{Carneiro et al. 2013}. Mg supplementation may contribute to the improvement of both beta cell response and insulin action in non-insulin-dependent diabetics (^{Paolisso et al. 1990}). E. japonica leaf extracts are a rather complex mixture of secondary metabolites and miscellaneous compounds, among which phenolic compounds and phytol, palmitic acid, linoleic acid, stearic acid, and phytol acetate stand out. The LME exhibited potent α-glucosidase enzyme inhibition. The presence of various phenolic compounds, including flavonoids and their glycoside derivatives, may explain the inhibitory activity against α-glucosidase enzyme observed in vitro and further supports the traditional use of E. japonica as an herbal medicine with antidiabetic properties. Estimation of IC₅₀ of LME and evaluation of α-glucosidase inhibitory potential of LPE are recommended in future studies.