Cladodes: Chemical and structural properties, biological activity, and polyphenols profile

Abstract The nopal cactus is an essential part of the Mexican diet and culture. The per capita consumption of young cladodes averages annually to 6.4 kg across the nation. In addition to contributing to the country's food culture, the nopal is considered a food with functional characteristics since, in addition to providing fiber, an important group of polyphenolic compounds is present, which has given cladodes to be considered a healthy food, for what they have been incorporated into the diet of Mexican people and many other countries worldwide. Research suggests that polyphenols from cladodes act as antioxidants and antidiabetics. This review studies the main phenolic components in cladodes and summarizes both conventional and novel methods to identify them.


| INTRODUC TI ON
Worldwide, nopal has become a valuable crop due to its health benefits, ease of cultivation, marketing, and climate adaptation (Aruwa et al., 2018). Nopal (Opuntia ficus-indica (L.) Mill) belongs to the Cactaceae family that comprises about 1,500 species (El-Mostafa et al., 2014), some of these species are Opuntia: basilaris, chlorotica, engelmannii, fragilis, humifusa, leucotricha, macrocentra, macrorhiza, dillenii, santa-rita, stricta (Majdoub et al., 2001). Its cultivation represents a major source of income for farmers living in semi-arid regions (Bayar et al., 2016). Nopal can grow in South America and other dry areas such as Africa, Australia, Southern Europe, and Asia (Khouloud et al., 2018;Majdoub et al., 2001). Nevertheless, Mexico accounts for 90% of the world´s production and represent the largest supplier to the United States, Canada, Japan, and European countries. Per capita consumption of nopal in Mexico is 6.4 kg (FAOSTAT, 2016).
Nopal is one of the most consumed species due to its nutritional value (Majdoub et al., 2001); furthermore, recent trends in healthy food consumption aroused scientists' interest to study the effects of nopal polyphenolic compounds in oxidative stress-related diseases (Scalbert et al., 2005). This work describes the nopal as a potential source of polyphenols and the main factors affecting their analytical identification. In addition, we highlight the importance of the relationship structure function in promoting health through cladodes consumption.
The epidermis (Figure 1b) contains numerous stomata (Figure 1c) that control photosynthesis and respiration (Salem-Fnayou et al., 2014). An inner tissue called chlorenchyma (CH) constitutes the second hierarchical level (microstructure), which consists of green plastids and abundant starch. The vascular tissue (V) located at the chlorenchyma tissue and the nucleus tissue (N) junction serves as a water and nutrient transporter into the plant, allowing the tissue to function as water storage for long periods of drought (Ginestra et al., 2009). The colorless central core tissue contains reserves of carbohydrates, proteins, and polyphenols (Feugang et al., 2006).
At the third hierarchical level (nanostructure), the macro cellulose fibers provide structure to the cell wall ( Figure 1e). Alongside the tissues, calcium oxalate crystals are found (decreasing in content as cladodes mature) making calcium more bioavailable in younger cladodes (Contreras-Padilla et al., 2016), which are consumed as vegetables in different stages of maturation ranging from 30 to 90 days (Hernandez-Becerra et al., 2020;Marin-Bustamante et al., 2018).
Finally, on the fourth hierarchical level, we find the cellulose molecular structure (Figure 1f) (Ventura-Aguilar et al., 2017).

| CL ADODE: COMP OS ITI ON AND B IOLOG IC AL AC TIVIT Y
Cladode chemical composition may vary according to soil factors, cultivation season, and plant age ( Table 1). The primary metabolites of cladodes are water, carbohydrates, and proteins. The carbohydrates in cladodes are divided into two types: (a) structural ones that are part of the cell wall, as cellulose (21.6 wt%), hemicelluloses 8.19%, and lignin (3.6 wt%) (López-Palacios et al., 2016;Scaffaro et al., 2019), and (b) the storage carbohydrates constituted by monosaccharides such as arabinose, galacturonic acid, glucuronic acid, galactose, glucose, xylose, rhamnose, mannose, and fructose (Rodríguez-González et al., 2014). Polysaccharides from Opuntia F I G U R E 1 Hierarchical structural organization of nopal ficus-indica (L.) Mill plants build molecular networks with the capacity to retain water, thus they act as mucoprotective agents . Mucilage is the main polysaccharide of cladodes, it contains polymers of βd-galacturonic acid bound in positions (1-4) and traces of R-linked l-rhamnose (1-2) ( Figure 2) (Quinzio et al., 2018).
Mucilage regulates both the cell water content during prolonged drought and the calcium flux in the plant cells (Hernández-Urbiola et al., 2010). In the food industry, mucilage is used as an additive, an emulsifier, and an edible coating to extend the shelf life of food products (Medina-Torres et al., 2013).
Amino acids such as alanine, isoleucine, and asparagine are found in young cladodes, whereas threonine prevails only in mature cladodes (Figueroa-Pérez et al., 2018). Young cladodes have a higher protein content than mature cladodes, which may be related to the increased metabolic activity in the early stages of maturation (Nuñez-López et al., 2013). Furthermore, analyses of plant extracts of the Cactaceae family identified several enzymes (e.g., lipases, proteinases, and glucosidases) (Guevara-Figueroa et al., 2010), and a large content of minerals (23.05%).
Over the years, Mexican people have developed several chronic degenerative diseases such as obesity, diabetes, and cardiovascular diseases (Aparicio-Saguilán et al., 2015). Traditional Mexican medicine recommends consuming cladodes due to their bioactive compounds' effects on health (Table 2); for example, the ability of polyphenols to eliminate free radicals (De Santiago et al. 2019;Filannino et al., 2016;Kim et al., 2016;Petruk et al. 2017   Note: Dissolvent used in the extraction: a NaOH, b water, c methanol, d acidified methanol, e methanol:acetone:water, f ethanol, g methanol: acidified water.

| Extraction techniques for the analysis and characterization of polyphenols
Due to the high fiber content of cladodes (Table 1), other minor compounds (of equal biological importance), such as polyphenols, have not been studied deeply. Therefore, we reviewed the methods to extract and characterize polyphenols. Types of extraction methods include liquid-solid extraction (a procedure that consists of grinding, defatting, solvent extraction, centrifugation, filtration, evaporation, and drying) (Yang et al., 2018) methanol/water/acid, methanol/acetone/water, and methanol/formic acid-based techniques and are optimized by varying methanol concentrations between 50% and 80% (Table 3) Obtaining polyphenol-rich extracts requires sample purification by column chromatography (Nemitz et al., 2015). Chromatography is a physical separation method based on differential migration of the sample components carried by the mobile phase through a stationary phase arranged in a column (Granato et al., 2016). The highest extraction yields were obtained when using C18 reversed-phase HPLC columns (with inner diameter 2-250 mm; particle size 1.8-2 5 μm) and a mobile phase composed of methanol or acetonitrile under isocratic elution or gradient elution (i.e., water and 0.1%-10% acetic or formic acid) conditions (Table 3). However, a factor to consider is the production of raw extracts, in which the management of parameters such as extraction time, temperature, and solvent composition influence the concentration and types of compounds obtained.

| Identification of polyphenols
Several authors have identified polyphenols in cladodes through HPLC and UHPLC because they maximize polyphenol identification accuracy (Tan & Fanaras, 2018). Hence, these techniques lead the separation methods for polyphenols analysis (Table 3).
A study conducted by Petruk et al. (2017) with extracts from    and the characteristic production for each polyphenol.

| CON CLUS I ON S AND FUTURE PER S PEC TIVE S
The present study reviewed the structure function of cladodes, which may provide an nutritional and functional value given the

ACK N OWLED G M ENT
The authors are grateful to the Instituto Politécnico Nacional (IPN-Mexico) for the support provided through the SIP Projects (SIP projects: 20181560, 20196640) and CONACyT projects (241756).

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interest.