Phenolic profile and antioxidant capacity of Cnidoscolus chayamansa and Cnidoscolus aconitifolius: A review

Research into ancient cultures has yielded a large body of evidence on the use of medicinal plants for preventive and/or therapeutic purposes. Such plants may have many metabolic activities and functions in the body-antioxidant, anti-inflammatory, platelet aggregation inhibitory and immunological and they can act at different molecular levels. This work offers a comprehensive review of research into the phenolic profile and antioxidant capacity of a plant used since the pre-Columbian era, native to southeast Mexico, commonly known as "chaya". The most prevalent phytochemicals in this plant are its phenolic compounds, and their antioxidant capacity is responsible for many of its health benefits, specifically in controlling chronic diseases. In the chaya leaf, there is a general trend toward the presence of different phenolic groups, such as coumarin, flavonoids, phenols, tannins, anthraquinones and flobotanins in aqueous and alcoholic extracts. Aside from environmental factors, there are differences in the ways samples are treated before the extraction process, such as the treatment type and the drying conditions. There are also differences in the solvents used and in the methods of extraction and concentration of compounds. Finally, a diversity of techniques is used, and even the data are quantified and expressed differently. Chaya has great potential for production as food and as a medicinal plant, but much more research is needed on the composition of its leaf and the biological effects of its components.


INTRODUCTION
The use of plants in medicine goes back to the beginnings of human civilization. Substantial evidence has been found on the use of plants for preventive and/or therapeutic purposes in ancient cultures ( Damme, 2011). According to the World Health Organization (WHO), a medicinal plant is one that contains substances that can be used for therapeutic purposes and/or can serve as active ingredients for the synthesis of new drugs (WHO, 2005). The use of traditional medicines and medicinal plants has been widely observed in most developing countries, where they are seen as therapeutic agents for the maintenance of good health (Soetan and Aiyelaagbe, 2009).
For several decades, various lines of research have been pursued into medicinal plants and their components. One of them focuses on the study of the composition of minority compounds, mainly phenolic compounds, given their various benefits in battling chronic disease, including cardiovascular disease, neurodegeneration, and cancer (Del Rio et al., 2013). They cover a wide range of metabolic activities and have many functions in the body: antioxidant, antiinflammatory, platelet aggregation inhibitory and immunological; and they can act at different molecular levels. Thus, the consumption of phenolic compounds is associated with health benefits (Rangel-Huerta et al., 2015). Also, several studies in plants report on their antioxidant capacity. There are a large number of publications on different plants, applying a variety of methods for extracting and measuring phenolic compounds and antioxidant capacity (Gutiérrez-Grijalva et al., 2016). These publications differ considerably in the types of processing used for the raw material, and also the solvents used (for example, aqueous, alcoholic and non-polar, as well as different mixtures thereof), as well as the times, temperatures, concentration, and other factors. Finally, there are diverse ways of expressing the content of phenolic compounds and antioxidant capacity. This work offers a comprehensive review of the literature on the phenolic profile and antioxidant capacity of a plant that has been in use since the pre-Columbian era, native to southeast Mexico, commonly known as "chaya".

CHAYA (CNIDOSCOLUS SPECIES)
Chaya refers to any group of plants of the genus Cnidoscolus, which is a part of the family Euphorbiaceae (Cifuentes et al., 2010). This genus is composed of 50 species, 20 of which are endemic to Mexico. They are distributed in tropical and subtropical areas, mainly in regions of low deciduous forest and xerophilous scrub of Mexico . Some species of Cnidoscolus are of interest for their nutritional potential, particularly the most commonly used for both consumption and traditional uses such as medicinal and ornamental plants, Cnidoscolus aconitifolius and Cnidoscolus chayamansa . C. aconitifolius has pentalobulated leaves, with lobed, serrated edges, with a long petiole length, without pubescences, with sagittate base, with the presence of glands and with white flowers (Adebiyi et al., 2012). In contrast, C. chayamansa has three-lobed leaves, with smooth lobed edges, with short petiole length, without pubescence, and similarly with sagittate base, with the presence of glands and with white flowers (Standley and Steyerman, 1949) (Figure 1). These two species originated in the Yucatán region of southern Mexico and are fast-growing perennial shrubs (Grubben and Denton, 2004).
The chaya plant is a domesticated shrub, highly valued by people in rural communities of central and southern Mexico as food, as a medicinal plant and as an ornamental. Chaya has been used as food since pre-Columbian times and is currently consumed regularly in some populations (Ross-Ibarra and Molina-Cruz, 2002). In addition, chaya leaves have been found to be an important source of protein, β-carotene, vitamins, ascorbic acid, calcium, potassium, and iron (Jiménez-Arellanes et al., 2014;Kuti and Kuti, 1999).
Chaya is consumed in a manner similar to spinach, which is why it is also called "Mayan spinach" (Ross-Ibarra, 2003). But its nutrient content is far superior to spinach: 78% more proteins, 111% more fiber, 100% more iron and 242% more vitamin C (Kuti and Torres, 1996) (Table 1).
Chaya leaves contain a cyanogenic glycoside called Linamarin. Linamarin is a glucoside conjugate of an acetone and a cyanide (Kuti and Konuru, 2006). It is a secondary metabolite of plants that performs defense functions, since when it is hydrolyzed by enzymes it releases hydrogen cyanide, a process called cyanogenesis. The content of cyanogenic glycosides according to Gonzalez-Laredo et al. (2003) is 2.37 to 4.25 mg/100 g dry matter (DM). These authors tested various thermal treatments to remove this compound from the leaves and reported that 5 min in boiling water is sufficient to remove any residue of cyanide ( Figure 2).
These plants can grow up to 6 m high, with lobed leaves, milky sap and small dichotomous white flowers at the tip of the branches. It is propagated by planting stem cuttings or woody stem cuttings. Within the chaya subspecies there is a considerable morphological and phenological variation. In research carried out by Ross-Ibarra and Molina-Cruz (2002), four cultivated varieties of chaya were identified, with easily separable and quite consistent morphological differences, but their taxonomy is not yet assigned. These are classified as star, beaked, chayamansa and round. Seeds and ripe fruit are rare and unknown (McVaugh, 1994). Given the ease of its  cultivation, its potential productivity, and above all its high nutritional value, chaya has been proposed as a potential crop for regions outside of Mesoamerica (Kuti and Torres, 1996;Molina-Cruz et al., 1997;Ross-Ibarra and Molina-Cruz, 2002).

IMPORTANCE OF PHENOLIC COMPOUNDS AND ANTIOXIDANT CAPACITY
Atoms or molecules containing one or more unpaired electrons are called free radicals. Free radicals are responsible for tissue degeneration through damage to DNA, proteins and lipid peroxidation through oxidative stress, which has been implicated in the pathophysiology of different diseases. Some authors have found that the degree of damage caused by free radicals can be mitigated by supplementation with one or more antioxidants (Marchioli et al., 2001). Several compounds with differential antioxidant properties are found in plants and these plants are considered to have high biological potential in the context of the prevention and treatment of damage caused by free radicals. Several medicinal plants have been examined and evaluated for their properties in antagonism toward free radicals induced by oxidative stress (Esparza-Martínez et al., 2016;Vinson et al., 2001). Some of these plants' medicinal properties are attributed to their phytochemical composition, specifically a variety of minority compounds derived from the secondary metabolism of plants, which have attracted interest recently for their bioactive effects. Phenolic compounds are among these, and are ubiquitous in foods of plant origin. The main functions of phenolic compounds in plants have to do with pigmentation and protection against pathogens and predators. They are chemical compounds having at least one aromatic ring to which one or more hydroxyl groups are attached to aromatic or aliphatic structures (Bravo, 1998). There are over 10,000 different phenolic compounds, ranging from the simplest to the most complex, and their wide diversity in nature is evident upon analysis of their characteristics (Gutiérrez-Grijalva et al., 2016;Neveu et al., 2010;Rothwell et al., 2014;Zare et al., 2014). Many constituents of these plants can contribute to their protective properties, including: vitamins C and E; selenium and other mineral micronutrients; carotenoids; phytoestrogens; glucosinolates and indoles; ditholthiones; isothiocyanates; protease inhibitors; fiber; and folic acid. These compounds may act alone or in combination, as anticarcinogenic or cardioprotective agents, through a variety of mechanisms. One of these protective mechanisms, attributed to vitamins C and E and to carotenoids, is antioxidant activity (radical barrier) (Rice-Evans et al., 1997).
There are several classes of flavonoids, which differ in the level of oxidation and saturation of ring C, and individual compounds within each class differ in the substitution pattern of rings A and B (Wojdyło et al., 2007). Researchers have been looking into the antioxidant properties of many plant species for at least 50 years. There is currently a great deal of interest in the commercial production of plants as sources of antioxidants that can enhance the properties of food, both for nutritional and medicinal purposes. Numerous epidemiological studies have shown an inverse relationship between consumption of fruits, vegetables and cereals and the incidence of coronary heart disease and certain cancers (Gunjan et al., 2011). The plant kingdom is vast, with thousands of species and varieties that demand study. The phenolic composition and antioxidant activity of plants, both wild and cultivated traditionally, are a particularly rich area for future research. The antioxidant capacity of various plants is generally studied with respect to the content of total phenolic compounds using traditional methods, and only one test is used to determine free radical scavenging ability. Although extensive studies of bioactive compounds and their content of total phenolic compounds have been carried out in many species, the phenolic identification data are still insufficient and incomplete. In particular, quantitative data on specific phenolic compounds in plant species remains a pending task. There are also few comparisons of the phenolic constituents identified in several species of different plant families. Further research is required into the structureactivity relationships of phenolic compounds present in plant species (Czapecka et al., 2005;Ivanova et al., 2005). The objective of this work is to review the literature on phenolic composition and the antioxidant capacity of different extracts derived from the leaves of C. aconitifolius and C. chayamansa. A comprehensive search was performed using the terms "Cnidoscolus chayamansa" and "Cnidoscolus aconitifolius" without reducing or limiting the search elements. A total of 57 publications were consulted on the main scientific portals (Scopus, PubMed, Science Direct, Springer-Link, Wiley, Redalyc, Google Scholar, and Web of Science). The information was subsequently analyzed and classified as described subsequently.

PREPARATION FOR PHENOLICS EXTRACTIONS
Plant extracts are a complex mixture, with a multitude of chemical compounds obtained by physical and chemical processes from a natural source and usable in almost any technological field. The WHO estimates that 80% of developing country populations rely on traditional medicines, mostly plant drugs, for their primary health care needs (Soetan and Aiyelaagbe, 2009). Plant extracts have been used since the beginning of civilization because they increase the useful life of the compound. There are few synthetic chemicals that can be used without toxicity or side effects, but nature is a potential source for discovering new structures that may have therapeutic qualities. Various phenolic compounds such as flavonoids can be extracted from fresh or dry material, as long as proper methods and care are used to avoid significant alteration of their contents and composition. Nonpolar or slightly polar solvents are initially used to separate chlorophylls, gums and aglycones from highly methoxylated flavonoids. Flavonoids, which have many unsubstituted hydroxyl groups or sugars, are considered polar, so they are slightly soluble in polar solvents such as methanol, ethanol, acetone, dimethyl sulfoxide (DMSO) or water. The final filtrate is usually concentrated and the solvent is removed (Sarker and Nahar, 2012;Skerget et al., 2005). Most phenolic compounds are found within plant cells in aglyconated or in glycosylated form. This protects them from degradation, diminishes their toxic effects and at the same time aids transport through membranes, increasing their aqueous solubility. These compounds, in any of their forms, are already aglyconated or glycosylated, are in the vacuoles of plant cells and are in a soluble polar fraction.
Therefore, these aglyconated and glycosylated compounds can be extracted relatively easily using polar solvents (Jones and Vogt, 2001). Table 2 shows different forms of extraction of chaya leaf compounds reported in the literature. Awoyinka et al. (2007) report that the aqueous extraction was performed from the dry leaves of C. aconitifolius that had been processed with a mortar and pestle. At the end, the substance was heated in an oven at 45°C until it reached a constant weight, although the proportion of the extraction is not specified. Musa et al. (2008) allowed the leaves of C. aconitifolius to dry at 40°C for 48 h. The reported extraction rate was 20 g of dried ground leaf to 1 L of cold distilled water, mixing for 48 h at a constant    (2015) Only the information available in each of the references is mentioned. NR: Not reported.

Water extraction
temperature. The mixture was then filtered and concentrated in a steam bath until 4.88 g of residue remained. Mordi and Akanji (2012) dried the C. aconitifolius leaves in the sun, and then macerated them. The proportion was 218 g of dry matter to 500 ml of distilled water using a rotoevaporator at 50°C. This residue was then lyophilized. Obichi et al. (2015) mentioned that only C. aconitifolius leaves were harvested, cleaned and air dried at 28°C for 28 days before use. Valenzuela et al. (2015) reported drying the C. chayamansa leaf for 15 days at room temperature in a closed and ventilated area, where the sample was then ground with a mortar and stored at room temperature. The sample was prepared by mixing 5 g of dry matter into 100 ml of distilled water at 90°C for 10 min. This was then filtered with Whatman paper (No. 4, 110 mm) and the extract was stored at 5°C for analysis. Babalola and Alabi (2015) reported four different processes: in the first group, the leaves of C. aconitifolius were bleached at 65°C for 10 min, in the second they were boiled at 100°C for 15 min, in the third the juice was extracted from the leaf, and in the fourth the residue of the juice was collected after extraction. Ramos-Gomez et al.
used a technique gathered from available ethno-botanical information for C. chayamansa, which was to boil 20 g in 1 L of drinking water for 20 min, then to pass this mixture through a 0.5mm pore size filter.  mention that the extraction of C. aconitifolius was 5 g DM in 20 ml of ethanol/acetone/water/acetic acid (40:40:20:0.1 v/v). This is the only study that reports using a microwave oven (1.3 cu ft Panasonic microwave 1000-W), in which the sample was heated for 2 min, to remove the cyanogenic glycoside from the plant. Awoyinka et al. (2007) mention that C. aconitifolius dried leaves were ground in a mortar and that the extraction was carried out with 96% ethanol for 3 h. The resulting solution was placed in a rotoevaporator at 30°C for 25 min, then placed in a drying oven at 45°C until a constant weight was reached. Johnson et al. (2008) reported placing a mixture of 5 g of C. aconitifolius dried leaf in 40 ml of an ethanol/acetone/water/acetic acid solution (40:40:20:0.1 v/v), in a water bath at 60°C for 1 h. Mordi and Akanji (2012) mention that the air-dried powder from C. aconitifolius leaves (1 kg) of fresh matured C. aconitifolius were extracted by percolation at room temperature with 70% ethanol (EtOH). A leaf extract from C. aconitifolius was concentrated under reduced pressure (bath temperature 50°C) and finally defatted with nhexane. The extract was evaporated to dryness. This yielded 69.9 g of dried mass. García-Rodríguez et al. (2014) mentioned that approximately 135 g of C. aconitifolius dried leaves were extracted by maceration using ethanol (9.44 g) at room temperature (25°C). The samples were kept in the dark at room temperature for successive testing during the course of the research reported. The solvent was removed by rotary evaporation to dryness and the resulting material dried completely in an oven at 25°C. Numa et al. (2015) mention that to prepare the soluble extract in ethanol, the C. aconitifolius leaf was dried, ground and macerated for 7 days in a solution of 96% ethanol, changing the solvent daily.

Extraction using methanol and mix polar solvents
The first report was by Kolterman et al. (1984), in which the dry matter of C. aconitifolius was extracted in 70% methanol/30% water. Later, González-Laredo et al. (2003) reported drying the leaves of C. chayamansa at 60°C for 6 h. These authors also performed a duplicate extraction with methanol and rotary evaporation at <40°C. Subsequently a separation was performed in duplicate using hexane, ethyl ether and ethyl acetate. Figueroa-Valverde et al. (2009) performed their extraction by placing 20 g of previously dried leaves of C. chayamansa in 250 ml of 80% methanol for 8 h, then performing a rotary evaporation of the mixture. They then added a chloroform: water solvent mixture (4:1 v/v) to remove the organic phase from the aqueous. The volume of the organic phase was reduced to dryness and the obtained mixture was reconstituted with 70% ethanol to be used as stock solution. Loarca-Piña et al. (2010) reported drying and macerating the C. chayamansa leaves, then performing the extraction by placing 500 g of dry leaf in 1000 ml of solvent (hexane-acetone, 1:1) at room temperature for 5 days, twice daily. Subsequently, the material was extracted with methanol under the same conditions. It was then dried in a rotoevaporator and stored at 4°C. In Adaramoye et al. (2011), approximately 3 kg of dry C. aconitifolius leaves were placed in an extractor at 30°C using methanol for 5 h and the extract was concentrated in a rotary evaporator at 35°C for 30 min. In Ikpefan et al. (2013), C. aconitifolius leaves were air dried for 5 days in a laboratory at room temperature. Oven drying was then carried out at 40°C, followed by milling in powder form, using an electric mill. 1 kg of the dry matter was extracted in 2.5 L of methanol. The extracted liquid obtained was concentrated using a rotoevaporator at a steady temperature of 40°C then kept in refrigeration afterwards. García-Rodríguez et al. (2014) reported extraction from approximately 135 g of dried leaves of C. aconitifolius by maceration, using 5.27 g of ethyl acetate at room temperature (25°C). The solvent was removed by rotary evaporation to dryness and completely dried in an oven at 25°C. Escalante-Erosa et al. (2004) reported that they used 20 freshly cut C. aconitifolius leaves. Subsequently, they added 1 L of methylene chloride for 20 s. Afterwards, the mixture was subjected to rotary evaporation to produce 533.3 mg of wax. García-Rodríguez et al. (2014) reported that approximately 135 g of dried leaves were extracted by maceration using 5.68 g of hexane at room temperature (25°C). The solvent was removed by rotary evaporation to dryness and dried completely in an oven at 25°C. Sarmiento-Franco et al. (2003) mentioned only the use of ground dry matter from C. aconitifolius. Unlike Oyagbemi et al. (2011), they mentioned that the leaves of C. aconitifolius were collected, cleaned and air dried at room  temperature. Aye (2012) reported that the preparation of C. aconitifolius leaves was washed, weighed and cooked in batches of 80 and 90°C for 10 min, and then allowed to dry. Akachukwu et al. (2014) mentioned only that the leaves of C. aconitifolius were dried in an oven at 40°C and subsequently ground. Jiménez-Aguilar and Grusak (2015) reported that C. aconitifolius leaves were dried in an oven at 70°C for 3 days to maintain a constant weight.

Phenolic compounds detected in chaya leaf
In an aqueous extraction, Musa et al. (2008) found different phenolic compounds in different concentrations: 1.86% phenols, 0.93% tannins, 0.30% flavonoids, 0.072% anthraquinones, and 0.065 % flobotannins (Table  3). Mordi and Akanji (2012), also using an aqueous extraction, found a moderate presence of phenols (++), a low presence of tannins (+), and a high presence of flobotannins (+++). In an aqueous extraction of chaya leaf, Obichi et al. (2015) found 5.7% of tannins and 23.7% of flavonoids. Babalola and Alabi (2015) also tested an aqueous extraction of chaya leaf and found 15.17 gallic acid equivalents (GAE)/100 g fresh matter (FM) of total phenolic compounds, and 243.33 mg/100 g FM of flavonoids. Valenzuela et al. (2015) performed a chaya leaf infusion and reported a total phenolic compound concentration of 6.34 mg GAE/ml.      Figure 2 presents the structures of the most reported phenolic compounds in chaya leaves. Valenzuela et al. (2015) reported the presence of quercetin and rutine in an aqueous extraction of C. chayamansa leaf (Table 4).  performed an aqueous extraction of C. chayamansa leaf and analyzed by high performance liquid chromatography with a diodearray detector (HPLC-DAD)/mass spectrometer Table 4. Identification of specific phenolic compounds in chaya leaves.

Antioxidant capacity of chaya leaf
The most commonly used methods for analyzing antioxidant capacity are ABTS+, DPPH, ORAC and FRAP. These are highly reproducible under certain assay conditions, but also show significant differences in their response to antioxidants. The free radical DPPH (DPPH) does not require any special preparation, whereas the radical cation ABTS (ABTS+) must be generated by enzymes or chemical reactions (Arnao, 2000). Another significant difference is that ABTS+ can be dissolved in aqueous and organic media, in which antioxidant activity can be measured, given the hydrophilic and lipophilic nature of the compounds in the samples. In contrast, DPPH can only be dissolved in organic media, especially in ethanol, which is a significant limitation in interpreting the role of hydrophilic antioxidants. In both radicals, however, reductive capacity does not necessarily reflect antioxidant activity, as suggested by Wong et al. (2006), Katalinic et al. (2006) and Wojdyłol et al. (2007). From a scientific standpoint, the best approach is to conduct a variety of tests to evaluate antioxidant capacity, since this yields a more complete and ultimately more accurate analysis.
The content of the phenolic compounds and their antioxidant capacity varies from one extract to another, not only in the environmental factors, but also by the way in which the data are expressed, either in different units or in different states of the sample, for example, lyophilized, dried or fresh matter. The results also vary in that the distinct types of extractions are not usually 100% of a single solvent, but instead use different mixtures and proportions, in addition to various extraction conditions and various determination methodologies.

Future perspectives
The studies presented in this review do not enable us to clearly determine which is the best extraction method for the phenolic compounds of the chaya leaf. This is because of the highly diverse processes mentioned by the different authors, as can be seen in Tables 2 to 4. Apart from the environmental factors, there are differences in the treatment of the sample before the extraction process, such as the type and the drying conditions. There are also differences in the solvents used and in the methods of extraction and concentration of compounds. Finally, a diversity of techniques are used, and even the data themselves are quantified and expressed differently. Even so, it can be said that the greatest amount and variety of phenolic compounds was obtained with different mixtures of hydroalcoholic proportions. Common knowledge tells us that the best drying method is one in which the conditions used to remove the water are not very aggressive with the biological material, for example, temperatures no higher than 40°C and a short drying time to avoid the degradation of the compounds of interest. Specific further study is needed to evaluate different types of solvents and mixtures of them for the extraction of phenolic compounds, where the same methodology is used for sample handling, from the harvesting of chaya leaves, the method of drying, grinding and extraction conditions, through the analysis of the compounds to create a phenolic profile. This would enable researchers to determine the best solvent for extracting certain type of phenolic compounds in chaya leaves. It would also be useful to perform the extractions from both raw and boiled leaves since it is known that the raw leaves have a cyanogenic glycoside that is eliminated by boiling the leaves in water, and this heat treatment could affect the phenolic profile.

CONCLUSIONS
In the chaya leaf, there is a general trend toward the presence of different phenolic groups, such as coumarin, flavonoids, phenols, tannins, anthraquinones, and flobotanins in aqueous and alcoholic extracts. The chaya plant has potential for production as food and as a medicinal plant, but the task of comparing the results obtained from the different research articles is complicated by the different processes used by each of the researchers to report the phenolic compounds and the antioxidant capacity of this plant. Apart from the analysis of different extraction methods, solvents and forms of preparation, as well as the diversity of extracted compounds, further research is also important and necessary through in vitro and in vivo studies of each type of extract in order to evaluate their biological effects on health, for example, in reducing glucose levels, or as a possible chemopreventive or chemoprotector agent against colon cancer.

CONFLICT OF INTERESTS
The authors have not declared any conflict of interests.