Bioactive compounds present in plants (more than 2 million identified), also called phytochemicals, are produced by the defense system that is activated by the presence of biotic and abiotic stress conditions; in addition to improving the general health status of plants, they participate in molecular signaling and in plant-environment interaction ^[1][2][1,2]. The biosynthesis of these secondary metabolites in plants has specific localization organs from which they are transported to the whole plant and the main storage parts (vacuoles) [3]. The recovery of these compounds can be developed from any part of the plant (roots, stems, leaves, somatic embryos, callus, flowers) but it should be considered that they can vary in type and concentration depending on the part used, and therefore, in the bioactivity detected. Bioactivities can be developed from various mechanisms of action, both beneficial to the plant and to humans. Due to the great use that can be made of these compounds, it is necessary to know in depth the factors that influence their production, recovery and maintenance techniques, their mechanisms of action, in addition to an emerging factor in recent years, climate change, which without much explanation, could modify the advances already known about these compounds, their natural sources and possible changes of adaptation.

Mexico ranks fifth in the world in biodiversity, offering opportunities for healthy food. Ninety percent of the population of the state of Hidalgo, Mexico, works in the agricultural sector and, what makes the sector a strategic area for said state, as well as for food sovereignty of Mexico. Hidalgo contributes 2.9% of the national volume of agricultural products [4]. Grain corn has a production value for the year 2021 of $2,756,711.47, green forage oats a value of $104,610.10, of a total annual value for the state of $5,081,511.37 (thousands of Mexican pesos); therefore, the importance of these crops (agricultural year) in production and economy is strengthened. The production values of alfalfa and maguey pulquero, the most important production crops (perennial), are greatest, amounting to $1,448,552.37 and $576,812.51, respectively, of a total annual amount of $2,601,538.05 (thousands of Mexican pesos). The third and fourth place in terms of production value (perennial) have an economic value of $74,165.64 and $187,940.07 (thousands of Mexican pesos), which is a lower production value than alfalfa and pulquero maguey (reported as produced honey water) [5]. The criteria considered for the selection of the crops under review at the bioactive level were focused on the annual production and the economic value they represent, as shown in Table 1 and in the description of the previous lines.

On the other hand, Hidalgo stands out for the diversity of its gastronomy characterized by the presence of dishes made with exotic ingredients, traditionally prepared with sophisticated culinary techniques. Within the list of representative foods, the predominant crops in the region are widely present, maguey and corn. Worms are obtained from the maguey; mixiotes (enchilada meat) and ximbo (cooked meat rolled in stalk) are cooked with a film obtained from the stalk; gualumbos or quiotes are obtained from their flowers; pastes, zacahuil (corn), peanut tamale, guajolotes (telera cakes with beans and enchiladas), moles, tecoquitos, bocoles, and molotes are made from corn. Another regional dish is escamoles, which are the larvae or roe of the scale ant [6].

2. Nutrients and Bioactive Compounds in Important Crops

The main crops sown in Hidalgo have a variety of applications in the diet, mainly nutrients and bioactive compounds. Considering and valuing these properties that crops contribute to human health would strengthen the appropriation of regional cultivation, influencing food sovereignty and regional development. By taking advantage of all these crops in an integral way, production would be boosted, generating direct and indirect economic, social, nutritional, and cultural benefits, among others. It is important to remember that the biological activities determined are not exclusive to a molecule per se (unless the solution has been so prepared) considering its physicochemical and structural characteristics (conjugated double bonds, number and position of methyl and hydroxyl groups), since in extracts, it can be attributed to effects of complexes formed with the individual molecules (synergistic, additive or antagonistic) and to the sensitivity of each molecule or complex to different analytical techniques (free radical inhibition, metal reduction). The bioactive properties discussed below may be a response of individual properties or interactions, depending on the sample analyzed.

2.1. Alfalfa (Medicago sativa L.)

According to the Statistical Yearbook of Agricultural Production, SIAP/SADER 2021 [5], the alfalfa production reported for the agricultural year 2020 (perennial cycle) (Table 1) corresponds to the total produced of green alfalfa, not including shrunken alfalfa production. Hidalgo occupies the second national place in alfalfa production (12.95%), only after Chihuahua (7,780,182.40 tons) with a production of 4,477,712 tons, for the same agricultural year (2021). Alfalfa generally is used as fodder, green or dried, in salads and flavored waters. The seed germinates from 2 or 3 °C, with an optimum temperature of 28 to 30 °C, and up to 38 °C, and can survive extreme droughts. It is rich in proteins, minerals, and vitamins [7]. It contains secondary metabolites such as saponins, coumarins, isoflavones, and alkaloids; their content differs with the type of cultivar, tissue, and stage of development. The aerial parts of alfalfa contain mainly glycosides of medicagenic acid substituted at C-3 by glucose or glucuronic acid, zanhic acid, and soyasaponin I tridesmoside ^[8][9][8,9]. It has been reported that the chemical composition of alfalfa sprouts (from the third day) presents water (869.1 g kg⁻¹ DM), crude protein (68.2 g kg⁻¹), ethereal extract (5.2 g kg⁻¹), crude fiber (30.9 g kg⁻¹), and ash (20.4 g kg⁻¹). The composition of phytochemicals and bioactive compounds includes phytoestrogens, sterols, tocols, carotenoids, and saturated and unsaturated fatty acids. The main isoflavones found in alfalfa are secoisolariciresinol diglucoside, daidzein, secoisolariciresinol, coumestrol, isolariciresinol, hydroxymatairesinol, and matairesinol. The main sterols found in alfalfa are stigmasterol (1096.8 mg kg⁻¹ DM), avenasterol (405.9 mg kg⁻¹ DM), β-sitosterol (324.2 mg kg⁻¹ DM), and campesterol (49.5 mg kg⁻¹ DM). Important antioxidant compounds have also been identified: α-tocopherol (314.1 mg kg⁻¹ DM), γ(β)-tocopherol (24.4 mg kg⁻¹ DM), α-tocotrienol (4.1 mg kg⁻¹ DM), δ-tocopherol (2.7 mg kg⁻¹ DM), and γ-tocotrienol (2.1 mg kg⁻¹ DM) [10]. Some reported uses for alfalfa seed flour (composition: 14.71% total starch, 37.59% crude protein, 3.74% ash, 26.22% total dietary fiber, 6.71% soluble dietary fiber, 19.51% insoluble dietary fiber) include improvement of the nutritional value of gluten-free biscuits using different levels of substitution for common rice flour (0, 15, 30, 45% w/w) [11]. In the same context of composition, some studies have reported the factors that may be responsible for variations in the composition and bioactivity of alfalfa (biotic and abiotic) and its biological effect by incorporation in diets. In addition to the inherent factors of plant growth conditions, the effect of light (LED), sound waves, drying, soaking, fermentation, and incorporation of selenium into the crop have been determined in alfalfa, which have diverse effects depending on the stage and potency of incorporation and/or application and the plant’s own metabolism. The incorporation of alfalfa in diets has improved the bioactive composition of the diets including polyunsaturated fatty acids (PUFA), isoflavones, tocopherols, anthocyanins, saponins and total polyphenols, with improvements in the antioxidant capacity of the diet, the consumption of some of these diets has improved the oxidative state of the Longissimus dorsi muscle of rabbits; also, an anti-cholesterol effect has been observed in chickens (see Table 2). On the other hand, it has been reported [19] that alfalfa leaf peptides have a reducer power of 0.69 to 2.00 mg mL⁻¹; they also presented 79.71% (1.60 mg mL⁻¹) and 67.00% (0.90 mg mL⁻¹) scavenging activity of the radicals DPPH (1,1-diphenyl-2-picrylhydrazyl) and superoxide, respectively. In addition, they chelated 65.15% of the ferrous ion at 0.50 mg mL⁻¹. The molecular weight of 67.86% of the peptides was smaller than 1000 Da and was characterized by an amino acid profile with a high nutritional value (glutamic acid, aspartic acid, leucine, arginine, valine, lysine, among others). Alfalfa is a source of isoflavones such as genistein, daidzein, and glycitein [20], and tocopherols (tocols and tocotrienols), compounds with important antioxidant activity. The α-tocopherols are the most abundant of all the tocopherols; their biological activity is double that of the β and γ homologs and 100 times more than the δ homolog [21]. Saponins are a large group of compounds identified in alfalfa, consisting of nonpolar steroidal triterpenoids or aglycones (sapogenins) attached to one or more hydrophilic oligosaccharide moieties via ether or glycosidic ester bond [9]. Saponins function as a chemical protector in the plant’s defense system against harmful agents (e.g., pathogens), but bioactive effects such as antioxidants, antimicrobial, anti-inflammatory, antitumor, antidiabetic, anticholesterol, antiviral, immunomodulatory, antibacterial, antiparasitic, and allelopathic activity have also been identified. Due to their properties, saponins are used as natural surfactants in foods, antimicrobial preservatives, and natural emulsifiers ^[8][9][8,9]. The structural differences between saponins have an impact on the bioactivity demonstrated; for example, due to the lack of sugar, sapogenins have shown better chemical properties (lower molecular weight, higher lipophilicity, or lower molecular flexibility) that improve permeability and bioactivity, in comparison with the precursor saponin [9]. In addition, polysaccharides (e.g., hemicellulose and pectin) with important bioactive functions, such as antioxidant, antitumor, immunomodulatory, anti-inflammatory, and growth-promoting properties, have been obtained from fresh alfalfa ^[22][23][24][22,23,24]; they have also been recognized as natural alternatives to antibiotics when added to animal diets. A pectic polysaccharide from the alfalfa stem was identified as rhamnogalacturonan I (RG-I; pm 2.38 × 10³ kDa) [22]. The polysaccharide (50 µg mL⁻¹) showed a significant anti-inflammatory effect against mRNA expression of the pro-inflammatory genes of the cytokines interleukin (IL)-1β and IL-6, which suggests a potential use in functional foods and supplemented products. Another studied polysaccharide of alfalfa is that formed by galacturonic acid (146.500 μg mg⁻¹), glucose (39.092 μg mg⁻¹), glucuronic acid (29.343 μg mg⁻¹), arabinose (12.282 μg mg⁻¹), galactose (8.649 μg mg⁻¹), mannose (6.791 μg mg⁻¹), xylose (4.811 μg mg⁻¹), and fucose (4.346 μg mg⁻¹). In vitro studies showed that 50 and 100 µg mL⁻¹ of the polysaccharide increased the cell viability of macrophages (RAW 264.7) by improving their immune functions, as well as the secretion and gene expression of inflammatory factors (cytokines, NO/iNOS, IL-6, and tumor necrosis factor (TNF)-α) [23]. The same research group reported a characterization for this same polysaccharide, indicating that the molar ratio of the saccharides is 2.6:8.0:4.7:21.3:3.2:1.0:74.2:14.9 for fucose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid, and glucuronic acid, respectively [24]. Furthermore, the polysaccharide markedly increased the proliferation of B cells and the secretion of IgM in a dose- and time-dependent manner but not the proliferation and expression of cytokines (IL-2, -4, and IFN-γ) of T cells. This represents a biological activity that contributes to the immune system [23]. For alfalfa polysaccharides, in a mouse embryonic fibroblast (MEF) model with oxidative stress induced by hydrogen peroxide (150 µM; H₂O₂), the activation of antioxidant capacity (1.0 mM g⁻¹ (T-AOC)) was detected as a preventive defense mechanism; and 250 µM/12 h was considered as the optimal concentration to stimulate stress in MEF (because it presents the highest expression of the pro-inflammatory gene related to senescence RIG-I). A concentration of 20 µg mL⁻¹ of polysaccharides exhibited the greatest antioxidant effect and the least secretion of inflammatory cytokines [25]. The results demonstrate that alfalfa polysaccharides exert a protective action against oxidative damage induced by hydrogen peroxide. Another study conducted to determine the effect of alfalfa (Medicago sativa L.) polysaccharides considered the growth performance and intestinal health of 200 piglets (35 days old) [26]. Biologically active phytogenic polysaccharides mainly contain carbohydrates comprising β-1,3-D-glycan units. Supplementation with the polysaccharide (0, 300, 500, 800, or 1200 mg polysaccharide kg⁻¹ diet for 42 days) increased average daily gain (ADG) and feed ratio (G/F) in a dose-response manner. The experimental group receiving 500 mg kg⁻¹ of polysaccharide in the diet showed the highest Lactobacillus values in the cecum, colon, and rectum. And the values for Salmonella and Escherichia coli decreased in all sections of the large intestine. The results showed that supplementation of the diet with alfalfa polysaccharides (500 mg kg⁻¹) improved intestinal morphological development and amylase and protease activity in the small intestine and promoted beneficial microbial populations in the large intestine [26]. It determined that alfalfa fiber (12 and 18% in the diet of piglets) decreased diarrhea and increased the composition and diversity of fecal bacteria (Bacteroidetes and Firmicutes were the dominant phyla (98% of the total)), and consequently improved the growth performance of weaning piglets [27]. The supplementation of alfalfa fiber (6–12%) in the diet of 48 crossbred piglets significantly increased growth performance and crude protein digestibility [28], particularly that of albumin, globulins, and total protein; however, it decreased levels of glucose (6% supplemented fiber, from 3.87 to 3.75 mmol L⁻¹), cholesterol (12% supplemented fiber, from 2.3 to 2.06 mmol L⁻¹), triglycerides (12% supplemented fiber, from 0.60 to 0.47 mmol L⁻¹), aspartate aminotransferase (6% supplemented fiber, 48 to 46 µ L⁻¹), and alanine aminotransferase (6% supplemented fiber, 42.5 to 39.5 µ L⁻¹).

2.2. Maguey (Agave spp.)

According to Statistical Yearbook of Agricultural Production, SIAP/SADER 2021 [5], the maguey production reported for the agricultural year 2021 (perennial cycle) (Table 1), corresponds to the total produced of pulquero maguey of aguamiel (58.28% national), not including unclassified pulquero maguey production. Hidalgo occupies the first national place in maguey production, followed by Tlaxcala and Mexico. The same statistical record does not report agave production for the state of Hidalgo, thus recognizing a difference in the use of the terms (maguey and agave) concerning the species and products produced from the plant: the Agave salmiana also named pulquero maguey to produce pulque, and Agave tequilero known to produce tequila. Maguey, also called mixiotero magueys (Agave salmiana) is a plant with rosette leaves, thick and fleshy, with a short stem, and a lower pineapple that does not protrude from the ground. In Mexico there are about 200 species of maguey, a term applied to species of the genus Agave (Asparagaceae). It requires low-humidity soil, intense light, temperatures of 15 to 25 °C, and an approximate altitude of 1700 to 2400 m above sea level [7]. Species of the genus present an important profile of phenolic compounds, such as flavonoids, homoisoflavonoids, and phenolic acids, which have been widely related to important biological, antioxidant, antibacterial, antifungal, antinematode, and immunomodulatory activity [29]. In addition, species of the genus Agave are recognized as an important source of monosaccharides as fructose to produce traditional alcoholic beverages, natural fibers, saponins, high-fructose syrups, and fructans; even the different phytocomponents of the thick leaves act as seasoning or flavor sources during the roasting of meat to prepare a barbecue ^[30][31][30,31]. As a source of saponins, the use of agave is emphasized as having antibacterial, antientomological, antifungal, anticholesterolemic, and anticancer effects) [32]. Agave also contains policosanols and sapogenins; agamenone (5,7-dihydroxy-6,5′-dimethoxy-3′,4′-methylenedioxy flavanone), flavonol, or isoflavones have been identified in concentrated honey water [33]. Mature plants contain low concentrations of saponins, and silage reduces their quantity, improving their characteristics for livestock feed [31]. About 30% of the agave plant is made up of leaves, which have few applications. Saponins are mainly present in the leaves and can be used as precursors for sterols of therapeutic importance. Leaves of A. salmiana and A. tequilana Weber were structurally characterized (light microscopy) and methanolic extracts followed by dichloromethane were recovered, where the presence of saponins was confirmed by hemolytic activity in erythrocytes and a positive reaction with anisaldehyde reagent. A. salmiana presented a higher percentage of protein (7.3%) ^[34][35][34,35]. Multiple in vivo tests have been reported to demonstrate the diverse bioactivity of Agave spp., not having many outstanding investigations on A. salmiana. A. salmiana syrup (honey water) is reported to have antioxidant activity of up to 1096.8 µM TE by DPPH, and a total phenolic content of 904.8 µM GAE [31]. The antidiabetic activity of high-fructose agave syrup from A. salmiana protected against liver steatosis in rats fed 2 and 5 g of serum kg⁻¹ and had a quadratic opposite effect on glycosylated hemoglobin in the blood of diabetic rats (dose 0.5 g kg⁻¹) [36]. Some patents ^[33][37][33,37] reported anticancer activity for the methanolic extract (80%) of concentrated agave syrup (10%) from the species A. atrovirens, A salmiana, and A. lehmanni (15 mg mL⁻¹), with 84.89% inhibition of colon cancer cells (Caco-2) and 67.95% of liver cancer cells (HepG2). In addition, antioxidant activity for the same extract was reported of 61.87 µmol ET g⁻¹ sample. These properties are attributed to the composition that includes phytosterols, polyphenols, flavonoids (agamenone), tannins, policosanols, inulin, and saponins (sapogenins). Another study was carried out on sap concentrated from A. salmiana, evaluating the apoptotic activity in HT-29 cells (IC₅₀, of 3.8 mg mL⁻¹ for the concentrate) of saponins from the acetonic extract. The most bioactive fractions (up to 80% cell inhibition at 75 μg mL⁻¹) presented an IC₅₀ of 108.4, 82.7, and >250 mg mL⁻¹, respectively (partition coefficients (Kd) of 0.23, 0.33 and 0.40); they contained steroidal saponins, mainly magueyoside B (266.4 μg PE mg⁻¹; Kd of 0.33). Flow cytometric analysis has determined that the fraction rich in the glycosides kammogenin and manogenin induces apoptosis and that the presence of gentrogenin and hecogenin is related to a necrotic effect [38]. The phytochemical composition has been proposed as a tool for the classification of different agave syrups (A. tequilana (>60% fructose) and A. salmiana (sucrose 28–32%)), such as infrared spectroscopy coupled to chemometrics (NIR-MIR-SIMCA-PCA) and high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) [39]. The said techniques are reported to identify and classify without significant mistakes. ¹H-NMR-spectroscopy-PCA was used to characterize syrup profiles and chemometrics, what allowed the sweeteners’ classification by origin and kind of Agave. The agave syrups exhibited appreciable amounts of saponins, cardiac glucosides, and terpenoids (excelled in color intensity in the reaction) followed by glucosides, quinones, flavonoids, and coumarins in moderate amounts. In A. tequilana syrup, flavonoids and terpenoids were only detected in a few samples. A. salmiana syrup displayed a positive colorimetric reaction for all the evaluated compounds [40]. In addition to the apoptotic activity, it has been reported [41] that antioxidant activity of agave sap (A. salmiana) is dependent on storage time and is correlated with the browning developed due to heating and storage time, increasing from 18 to 23 eq Trolox µmol g⁻¹ DW in a lot with a high degree of browning (57.7 OD₄₉₀ g⁻¹ fw), after 20 weeks of storage. In addition, they reported that the content of saponins (kammogenin glycosides (magueyosides A and B), manogenins, and hecogenin (agavoside C′)) was different per batch, varying from 224.2 to 434.7 PE g⁻¹ DW in week 2 and varying up to week 20 of storage (207.7 to 462.4 PE g⁻¹ DW). They found no correlation of the browning index and antioxidant capacity (ORAC) with the concentration of free amino acids (serine, phenylalanine, and lysine); the positive correlation found was browning with furosine, an early derivative of the Maillard reaction of lysine, reported as a free radical scavenger [41]. The presence of several compounds in agave syrup (ethanolic extracts), such as saponins, flavonoids, quinones, glycosides, cardiac glycosides, terpenoids, and coumarins, has been identified for several species [40]. The antioxidant activity of agave syrups (A. tequilana, A. salmiana) was in the range of 10–53%, while the content of total phenols was from 24 to 300 GAE 100 g⁻¹ and that of condensed tannins was from 240 to 1900 mg CE g⁻¹. In addition, a relationship between the color and the antioxidant capacity of the syrups is reported, with dark syrups such as those of A. salmiana having the highest antioxidant capacity, about 28.33%, while light syrups show an average capacity of 8.7%. In tests on mice, the consumption of fresh and boiled syrup (A. salmiana sap) promoted weight gain (13%) and increased hemoglobin counts to 4.5 and 9%, respectively; the hematocrit count increased from 2.6 to 5.3%; iron, transferrin, ferritin, and phosphorus increase with the consumption of fresh syrup, while iron increase with boiled syrup [42]. The antioxidant capacity of syrup was determined as 7.1 μmol GAE g⁻¹ DW, that for commercial coffee being 156.1 μmol GAE g⁻¹ DW (commercial coffee). No adverse effects of syrup consumption it is observed. Due to the importance of maguey, A. salmiana, in the production of pulque and functional food industries, processes have been sought to maintain its production and take advantage of its benefits (Table 3). The micropropagation of agave can be an auxiliary process to increase the phytochemicals and bioactivities in the plant. The increase in antioxidant activity in micropropaged plants in vitro has several possible causes: the presence and interaction of cytokines and auxins [43], or the highest concentration of phenols or saponins [44], or bioactive compounds in general, which can be responsive to culture conditions, such as micropropagation [45], which has already been shown to have diverse effects on compositions and properties. Micropropagation needs more research (at different conditions, ages, and physiological states), together with scaling up to primary production levels, so that the effects found in micropropagation can be exploited in the primary production sector and from there to the industrial sector. The study of technologies for the extraction of bioactives from agave bagasse is little to date, the effect of ultrasound assisted with supercritical fluids in Agave salmiana has reported antioxidant activity and saponin content [46], which is a promising area for the recovery of bioactives and the valorization of agave processing residues (Table 3). Antioxidant properties and bioactive compounds of methanolic extracts of A. salmiana leaves were evaluated [50] at different stages of development (I–VI, from 1 to 7 years). The total phenolic content from leaves extracts was found to be between 5 (stage VI) and 13 mg GAE g⁻¹ (stage II) the maximum; the antioxidant capacity presented a negative trend from stages I to VI (from 146 to 52 μmol TE g⁻¹ respectively), the flavonols showing the same behavior (65% reduction from stages I to VI). Five saponins were identified (chlorogenin glycoside 2, chlorogenin glycoside 1, hecogenin glycoside 1, tigogenin glycoside, hecogenin glycoside 2) (also reported [48] in addition to flavonols (maximum concentrations in stage I, kaempferol (0.045 mg g⁻¹ DW) and quercetin (0.07 mg g⁻¹ DW)). Stage III and IV plants presented the highest content of saponins, mainly chlorogenin glycoside, at 3.19 and 2.90 mg PE g⁻¹, respectively. According to Pearson’s correlation, there is a positive relationship between total phenol content and antioxidant activity. Based on these results, it can be said that A. salmiana plants from stages I to IV could be a good source of antioxidants and bioactive agents; in addition to that, the concentration of metabolites could be a marker of the developmental stage. In the same way, the content of saponins was evaluated [51], regarding maturity (before (immature) and at the beginning of the reproductive stage (mature)) of the agaves (8 years old) and syrup. Saponins derived from kammogenin, manogenin, gentrogenin, and hecogenin were found. In syrup form immature A. salmiana, the saponin content was twice as high (478.4 μg PE g⁻¹ DW of syrup) as that of immature A. americana (179.0 μg PE g⁻¹ DW of mead). For both species, the saponin content decreased when the plants reached sexual maturity (up to 325.7 and 60.5 μg g⁻¹ DW of syrup, for A. salmiana and A. americana, respectively). This finding is important to better select the species and maturity stage of agaves used as a source of bioactive.