Dioscorea spp.: Comprehensive Review of Antioxidant Properties and Their Relation to Phytochemicals and Health Benefits

Dioscorea, consisting of over 600 species, is the most important genus in the Dioscoreaceae family; however, the practically used plants, which are commonly called yam, are restricted to a remarkably smaller number of species. Numerous studies have reported the high nutritional value of yam, particularly as an alternative source of starch and some important micronutrients. Several Dioscorea species are widely used for various medicinal purposes as well. In many studies, the bioactivities and health benefits of Dioscorea extracts and other preparations have been related to the presence of phytochemicals, which possess antioxidant properties; they are related mainly to radical-scavenging capacity in chemical assays and positive effects on the endogenous antioxidant system in cell-based and in vivo assays. Considering the increasing number of publications on this topic and the absence of comprehensive and focused review papers on antioxidant potential, this article summarizes the results of studies on the antioxidant properties of Dioscorea spp. and their relation to phytochemicals and health benefits. A comprehensive survey of the published articles has revealed that the majority of studies have been performed with plant tubers (rhizomes, roots), while reports on leaves are rather scarce. In general, leaf extracts demonstrated stronger antioxidant potential than tuber preparations. This may be related to the differences in phytochemical composition: saponins, phenanthrenes and, for some pigment-rich species (purple yams), anthocyanins are important constituents in tubers, while phenolic acids and flavonoids are characteristic phytochemicals in the leaves. The review may assist in explaining ethnopharmacological knowledge on the health benefits of Dioscorea plants and their preparations; moreover, it may foster further studies of poorly investigated species, as well as their wider application in developing new functional foods and nutraceuticals.


Introduction
The role of fruits, vegetables and botanicals, as natural and sustainable sources of valuable nutrients and health-beneficial substances, has been very important for humankind since ancient times. Due to the vast biodiversity in the plant kingdom and the large number of poorly investigated plants, the trend in the search, evaluation and application of natural preparations-as well as the development of new products-remains an important issue in various industries, including foods, pharmaceuticals and cosmetics. Moreover, the importance of this topic is increasing in the era of functional foods, nutraceuticals and personalized nutrition. In addition, the shift in consumer preferences towards 'naturalness' has also played an important role in the popularity of plant origin preparations.
Dioscoreaceae, with nine genera and about 715 species, is considered one of the earliest families of the plant kingdom (Magnoliophyta) [1]. In terms of uses, Dioscorea, consisting of over 600 species, is the most important genus in this family. The plants are perennial herbaceous and dicotyledonous, and bloom with faint flowers and have large roots and/or rhizomes. Dioscorea plants are common in tropical and subtropical regions; however, many of them have successfully adapted to different habitats and, nowadays, are common in other climatic regions as well. Regardless, a large number of existing species-practically used plants, which are commonly called yam-are restricted to a smaller number of species; among them are D. batatas, D. japonica, D. bulbifera, D. opposita, D. tokoro, D. nipponica and D. alata [2]. For instance, D. bulbifera L. (air potato) is native in Asia, North Florida [3], and Georgia (www.gaeppc.org/list/ [4], accessed on 11 November 2021); D. alata L. (water yam, winged/white yam) originates from Southeast Asia [5] but is also found in Africa (FAOSTAT, Database 2021 [6], accessed on 11 November 2021), Georgia (www.gaeppc.org/list/ [4], accessed on 11 November 2021), and America (www.eddmaps.org [7], accessed on 12 November 2021); D. oppositifolia, syn. D. opposita, D. batata, D. polystachya Turcz. (Chinese yam), is native to Asia and is also found in eastern North America.
It should be noted that the occurrence of invasive plant species, albeit beneficial ones, poses a threat to native species and habitats. For example, D. oppositifolia (syn. D. polystachya) is recognized in North America (www.iucngish.org/gisd/, accessed on 22 November 2021, Global Invasive Species Database, 2021 [8]) as a forest or agricultural weed, which can compete with the native species, especially in coastal habitats; while widespread in Africa, D. alata is one of the most valued foods. In general, due to the nutritional and therapeutic benefits and economic income, the contribution of yam to food security is significant in some regions, especially in sub-Saharan Africa [9]. Dioscorea spp. are also known for its association with low-cost food culture, traditional Chinese medicine, modern western medicine, and the pharmaceutical industry. The peculiarities of the diet and traditions of this genus of plant species in many cultures are closely related to the historical and religious aspects of life (FAOSTAT, Database, 2021 [6]), which began much earlier than scientific research. It should be noted that different yam species accumulate a large variety of biologically active compounds and, therefore, various anatomical parts of the plants have been used for different purposes. For instance, D alata, D. japonica Thunb., and the rhizomes of D. schimperiana [10][11][12] are valued as a source of starch in the development of flour, pasta, and desserts.
Traditional medicine mentions such species as D. bulbifera in the treatment of goiter, skin infections, and oncological diseases [13]; D. nipponica in the treatment of arthritis, cough, asthma, and circulatory disorders [14]; D. villosa (wild yam) in the reduction of intestinal disorders [15]; and D. birmanica in the treatment of chronic diseases, whose development is associated with long-term oxidative stress [16]. Food supplements used in modern medicine as natural alternatives to hormone replacement therapy, in the form of powders, capsules, and herbal extracts, are made from the roots of D. villosa; a traditional herbal preparation made from D. nipponica root is used to regulate the immune system and reduce pain and inflammation [14], while the saponins extracted from D. septembola are used in China to treat gout [17].
In addition to the well-known species with high nutritional, economic and medicinal value, it is worth mentioning the other less-known but valuable species that can enrich the diet and improve health, namely: D. birmanica Prain and Burkill [16]; D. hamiltonii Hook. f syn. D. persimilis [18,19]; D. hispida [20]; D. pubera Blume; D. wallichii Hook [21], and others. Some species are poisonous but have healing properties, e.g., D. hispida, which is a promising alternative to conventional chemotherapy [22,23].
Secondary metabolites identified in Dioscorea spp. are often used in physiological and molecular studies. The well-known natural steroid saponin diosgenin has been used in the pharmaceutical industry as a chemical model in the development and/or complete synthesis of hormonal drugs. This metabolite is also reported to reduce oxidative stress damage, may protect the myocardium from ischemia-induced injury [24], and modulates the intestinal microbiota [24,25]. Dioscin and gracillin are also very important metabolites, demonstrating anticancer [26,27] and antioxidant effects. Trillin, found in D. nipponica [28], D. oppositifolia, and D. hamiltonii [29], is a pharmacologically viable steroid saponin with a broad spectrum of physiological effects in the cells, e.g., increasing superoxide dis-mutase (SOD) activity, and lowering lipid peroxidation activity and the oxidative stress response [28].
As may be judged from the records present in various databases (Table 1), Dioscorea plants have been widely studied, and the published results have been reviewed in numerous articles. The topic of Dioscorea antioxidants has also been the focus of many studies; however, the reviews on this topic are less abundant and fragmental.
Obidiegwu et al. [9] published the most comprehensive review on the nutritional and therapeutic potential of the Dioscorea genus. This review covers the nutritional value (starch, fiber, protein, fat, minerals), the profile of bioactive compounds, the therapeutic potential and health benefits (anti-inflammatory, anticancer, anti-diabetic, anti-obesity and anti-hypercholesterolaemic and antimicrobial activities), the management of degenerative disease, menopausal symptoms, and its use as a pharmaceutical excipient. Regarding bioactive compounds, the review includes separate sections on steroidal saponin, dioscorin, alkaloids, flavonoids, phenolic acids and others, while the antioxidant properties of yam are discussed in a very short section. In addition, it refers to seven publications from 2019, while in the period of 2019-2021, fifty-four new articles related to Dioscorea antioxidants have been published.
Padhan and Panda [33], in their review on underutilized and neglected yams, emphasized their potential for improving nutritional security and providing health benefits. The authors focused their article on the ethnobotany of Dioscorea spp., which are consumed by tribal people, and briefly highlighted their nutritional, anti-nutritional, and pharmacological properties. Kumar et al. [34] reviewed the traditional uses and ethnopharmacological potential of wild edible Dioscorea tubers by the local people of the Similipal Biosphere Reserve in India, whereas George et al. [35] highlighted the importance of African yam bean. Salehi et al. [36] reviewed Dioscorea plants as rich in nutrients and pharmacologically valuable constituents; however, their antioxidant activities were summarized in a very short section. Some reviews were focused on individual Dioscorea species and important groups of nutrients and bioactives. For instance, Bhujbal et al. [37] reviewed the pharmacological activity of D. floribunda, and Semwal et al. [38] focused on the traditional uses, bioactive compounds and biological activities of D. deltoidea Wall. ex Griseb. Huang et al. [39] focused their paper on the isolation, structure and bioactivities of yam mucilage polysaccharides and included a short section on antioxidant properties, while Anwar et al. [40] included yam tubers in their review on water-soluble non-starch polysaccharides of root and tuber crops. Zhang et al. [41] focused their review on the development of proteins and peptides from Dioscorea tubers with therapeutic potential. Petropoulos et al. [42] reviewed the antioxidant properties and health benefits of colored root vegetables, which included a section on yam pigments as well. Finally, Yang et al. [43] reviewed the pharmacological activities of dioscin, and Toth et al. [44] reviewed those of phenanthrenes.
The survey of published review articles reveals that a comprehensive review on Dioscorea antioxidant properties has not been published until now, while the pool of data on this topic is quite large. Therefore, the main task of this review was to collect the available results on Dioscorea antioxidant potential, the presence of the main chemical groups of antioxidants, and their relations to health benefits. It should be noted that ethnopharmacological properties and medicinal uses are not covered in this review unless they are closely related to the antioxidant activities.

Evaluation of Crude Extracts and Their Fractions
The antioxidant properties of Dioscorea spp. have been studied using various in vitro chemical methods, cell cultures and in vivo assays, mainly using rat and mouse models. The results of the in vitro chemical assays are summarized in Table 2.
It can be observed that antioxidant properties have been determined using several chemical assays and expressed in various units, e.g., the equivalent amount of the reference antioxidant (trolox, ascorbic acid, catechin, α-tocopherol); the fresh (pfw) and dry weight (pdw) of plant material and/or extract (edw); the effective inhibitory concentration (IC 50 , in some cases abbreviated as EC 50 ) values of the extract or its solution; or the percentage inhibition at the applied extract concentrations. In the latter case, the results are very difficult to compare because it is necessary to know the procedure of the sample preparation for the assays, particularly their concentrations in the solutions. Therefore, if the reader would like to know more details, it is suggested to download the full article. The values expressed in the reference antioxidant equivalents in pdw/pfw and/or edw are the most convenient; the former indicates the antioxidant potential of the whole plant material used in the assay, while the latter is directly related to the antioxidant properties of the isolated extracts and fractions.
Several studies compared the antioxidant potential of different Dioscorea spp. and cultivars of the same species and demonstrated that the differences may be quite remarkable. For instance, Padhan et al. [101] reported the flavonoid, total antioxidant capacity and in vitro antioxidant activity of eight wild (D. oppositifolia, D. hamiltonii, D. bulbifera, D. pubera, D. pentaphylla, D. wallichii, D. glabra, D. hispida) and one cultivated (D. alata) yam tuber from Koraput (India): TPC, TFC, and total antioxidant capacity ranged from 2.19 to 9.62 mg GAE/g pdw, 0.62-0.85 mg QE/g pdw and 1.63-5.59%, respectively; meanwhile, the IC 50 [102]. Bhandari and Kawabata [96] reported that the TPC of D. bulbifera, D. versicolor, D. deltoidei, and D. triphylla from Nepal ranged from 13 ± 1 to 166 ± 10 mg/100 g fpw, while the differences in antioxidant characteristics were less remarkable, particularly in chelating ferrous ion, reducing power, and total antioxidant capacity. Jimenez-Montero and Silvera [103] compared six varieties of D. bulbifera (air potato) from Panama and found that the variations in ascorbic acid, antioxidant capacity, and TPC were not remarkable (6.0-8.1 mg/100 g, IC 50 7.85-8.87, and 175-201 mg GAE/100 g, respectively). However, the cultivars of the same species may differ significantly. For instance, the TPC, TFC, and TAC values of the flour of the Kulonprogo cultivar of D. alata, determined by extraction with acid and with 5% acetic acid water, were higher than those of the Malang cultivar [49]. Alcohol and ester extracts of yellow fresh-cut yam (D. opposita) exhibited higher ORAC and DPPH • scavenging capacity than white fresh-cut yam [69].
The majority of studies used yam; however, some of them were performed with different anatomical parts of Dioscorea as well, including the flesh, peels and leaves. Thus, Alsawalha et al. [104] reported that MeOH extract from D. villosa leaf powder demonstrated strong antioxidant capacity in a FRAP and DPPH • scavenging assay in a dose-dependent manner; at a concentration of 0.1 mg/mL, the inhibition of DPPH • was >90%, while the optical density in the FRAP assay was 1.3 (2 for ascorbic acid). Mondal et al. [105] compared MeOH and EA extracts of D. pentaphylla leaves and found that TPC, DPPH • and • NO scavenging was higher for the former, while the latter had almost 4-fold higher TFC and TAC.
Yams contain various nutrients and phytochemicals, which differ in their chemical structures, molecular weight, polarity and other characteristics. Some studies fractionated yam materials and extracts using different polarity solvents. Duan et al. [60] reported only slight differences between n-BuOH and EA extracts of D. batatas, which were evaluated using different in vitro antioxidant assays. In the case of a thermally treated product, EA extract was more effective in chelating ferrous ion and scavenging • NO, while n-BuOH extract better inhibited linoleic acid peroxidation [59]. Duan et al. [56] compared yam (D. batatas) extracts isolated with 70% MeOH, 70% EtOH and CF-MeOH (2:1, v/v). MeOH extract was most effective at chelating ferrous ion, • NO scavenging and the β-carotene bleaching assay, while CF-MeOH extract better inhibited linoleic acid peroxidation. Ghosh et al. [64] extracted the bulbs of D. bulbifera with cold 70% EtOH, and sequentially with PE, EA and MeOH; the latter extract contained higher TPC, while the EA fraction was richer in TFC (Table 3). The antioxidant properties of EA and alcoholic extracts were stronger compared with the PE extract. Park et al. [89] extracted bulbils of Danma (D. japonica) and Jangma (D. batatas) with MeOH, and the crude extracts were re-extracted with CF, EA, n-BuOH, and water. Xia et al. [106] compared antioxidant properties of PE, EA and n-BuOH fractions of D. nipponica, which were obtained from a dried 60% EtOH extract. The fractions inhibited DPPH • , • O 2 − , and • OH in a concentration range of 2.5-12.5 mg/mL; however, the dependence between radical inhibition percentage and extract concentration was far from linear. Adeniran et al. [107] successively extracted the peel and flesh of D. dumetorum, D. hirtiflora, and D. bulbifera with the increasing polarity solvents HX, DCM, EA, and MeOH. The MeOH extract of D. hirtiflora flesh showed the highest radical-scavenging capacity, while TPC and TFC were higher in the EA extract of D. bulbifera flesh. Among D. quinqueloba extracts isolated with water, MeOH, EtOH, and EA, the EtOH extract was the strongest DPPH • scavenger, whereas EA extract better scavenged ABTS •+ ; both values increased with the addition of water-soluble chitosan [97]. Chen et al. [53] fractionated water solution of dried crude EtOH extract from Taiwan yams (D. alata) via sequential extraction with HX, CF, EA, and n-BuOH. Based on the IC 50 values, the peel extracts better scavenged DPPH • than the flesh products. The EA fraction of the peel of Tainung yam was a particularly strong antioxidant; its EC 50 value was only 14.5 µg/mL, which was lower than that of ascorbic acid (21.4 µg/mL). In addition, the authors applied pressurized solvents and revealed that hot pressurized EtOH was superior to hot pressurized water in extracting the DPPH • scavenging compounds [53].
Only one study was found on the use of biotechnological methods for obtaining antioxidants from yams. Jirakiattikul et al. [108] applied in vitro propagation of D. birmanica shoots; however, the TPC and DPPH • scavenging values were remarkably higher in the mother rhizomes than in the in vitro produced shoots (259.67 ± 7.34 vs. 44.24 ± 8.47 mg GAE/g and 11.42 ± 3.28 vs. 53.67 ± 4.16 EC 50 µg/mL dry extract, respectively).

Evaluation of Polysaccharides and Some Other Compounds
Several studies reported antioxidant properties in yam polysaccharides. The prepurified (dialyzed) polysaccharide of air yam (D. bulbifera) was approx. a 4-fold stronger antioxidant in ABTS •+ , according to a decolorization assay, than the crude (non-dialyzed) polysaccharide; in contrast, in DPPH • and FRAP assays, the latter showed a several-times stronger antioxidant capacity [65]. It can be noted that the reported results are rather strange, because the mechanisms of all the radical-scavenging reactions were based on electron transfer (hydrogen atom transfer may proceed with DPPH • as well). Most likely, extended studies using more specific assays would be required to more precisely clarify the mechanisms of action of yam polysaccharides in the redox systems. Zhang et al. [105] precipitated yam polysaccharide from the centrifugate of D. opposita homogenate with 75% EtOH, and afterwards, degraded it into three low MW derivatives; these were strong antioxidants against • OH (IC 50 = 187, 82 and 55 µg/mL) and • O 2 − (IC 50 was 241, 138 and 125 µg/mL), while the activity of raw polysaccharide was lower. The polysaccharide partially prevented the cyclophosphamide-induced damage, and its effect was probably due to high anti-lipid peroxidation activity and radical-scavenging effects. A previously unknown water-soluble polysaccharide was isolated from the roots of a well-known edible and medicinal plant in China, D. opposite. Its antioxidant capacity IC 50 values against DPPH • and PTIO • were 2.1 ± 0.1 and 1.6 ± 0.1 mg/mL, respectively [109]. Zhao et al. [72] compared ultrasoundassisted, cold, warm, and hot water extraction for the isolation of polysaccharides, and determined that cold water gave the highest uronic acid yield; its DPPH • scavenging, αglucosidase and α-amylase inhibition activities were the best. The MW of polysaccharides recovered using ultrasound-assisted extraction was small, while its reducing power and FRAP were the best. The extracts of tuber pulp from the three D. alata taxa had significant anti-Fenton reaction activity, which was related to the polysaccharide mucilage [110]. Most recently, the antioxidant activity of mucilage isolated from D. opposita was evaluated using DPPH • -scavenging and FRAP assays; at a 5.0 mg/mL concentration, it inhibited 68.57% of radicals, while FRAP was remarkably lower compared to ascorbic acid [111]. The • OHscavenging capacity of diosgenin obtained from D. composita by direct hydrolysis-extraction, under biphasic reaction conditions (1.5% yield), was higher than that of ascorbic acid when the concentration of the scavenger was higher than 2.5 × 10 −3 mmol/L [112]. Various solvents have been used for the extraction and fractionation of antioxidants from Dioscorea. Kwon et al. [45] determined antioxidant, antithrombin, and antimicrobial activities of MeOH extracts prepared from two D. batatas cultivars, two D. alata, one D. bulbifera, and one D. nipponica. Ramos-Escudero et al. [113] reported the TAC, TPC, and antioxidant activity of the pigments extracted from D. trifida.

Evaluation of Proteins and Their Hydrolysis Products
Several studies reported antioxidant and other bioactivities of yam mucilage, proteins and their hydrolysates. Hou et al. [114] purified the storage protein of yam (D. batatas) tuber dioscorin (32 kDa), which scavenged DPPH • (8-46%) at 5.97-47.80 nmol; dioscorin decreased the intensities of the electron spin resonance (EPR) signals used for DPPH • and • OH detection in a dose-dependent manner. Hou et al. [115] extracted and partially purified mucilage from a D. batatas tuber: its IC 50 in DPPH • , • OH and anti-lipid peroxidation and anti-human low-density lipoprotein peroxidation tests were 0.86 mg/mL, 22 µg/mL and 145.46 µg/mL, respectively, using BHT, GSH, or ascorbic acid for comparisons. The intensities of the EPR signals decreased with increasing yam mucilage amounts (IC 50 was 1.62 mg/mL). The mucilage also inhibited the angiotensin-converting enzyme (ACE) [116]. Nagai and co-authors [81,85,[117][118][119][120] studied proteins isolated from D. opposita tuber mucilage tororo. Firstly, they obtained viscous water extract containing 280 mg/mL of protein, which demonstrated the main bands determined by SDS-PAGE at MW~31 kDa (reducing conditions) or~33 kDa (non-reducing conditions), and scavenged • O 2 − (84.1 ± 6.57%), • OH (79.4 ± 6.42%), and DPPH • (38.2 ± 3.14%) [61]. Afterwards they treated the extract with protease or mannanase and noted that the viscosity highly depends on the interactions between certain soluble proteins with mannan [121]. They purified a soluble viscous protein, dioscorin (~200 kDa), from the mucilage using a chromatographic procedure [111] and measured its antioxidant properties. The protein effectively scavenged • OH (IC 50 = 195.1 µg/mL) and • O 2 − (IC 50 = 92.7 µg/mL) [120]. Autolysate and trypsin hydrolysate from yam (D. opposita) ichyoimo tubers exhibited extremely high antioxidant and ACE-inhibitory activities [119]. The lyophilized powder of enzymatic hydrolysates from mucilage tororo effectively scavenged • O 2 − and • OH, particularly when trypsin was used for hydrolysis [118]. The hydrolysates of a yam tsukuneimo tuber mucilage tororo at a 100 mg/mL concentration significantly prolonged the induction period of linoleic acid auto-oxidation (similar to 5 mM ascorbic acid), and scavenged • O 2 − , • OH, and DPPH • [85]. Liu et al. [122] also purified dioscorin from D. alata and D. batatas using DE-52 ion-exchange chromatography and reported antioxidant properties in the protein and its hydrolysates using various assays. Oh and Lim [123] isolated glycoprotein from D. batatas, which has been traditionally used as a folk medicine and health food in Korea; it had an optimal radical-scavenging capacity at an acidic and neutral pH and at temperatures up to 85 • C, while in the presence of Ca 2+ , Mn 2+ , Mg 2+ , and EDTA, its activity reduced.

Effects of Processing and Various Treatments
Processing techniques such as blanching, thermal treatment, soaking, oven-drying, and freeze-drying had different effects on the content of phenolics, DPPH • scavenging and polyphenol oxidase activity in D. esculenta and D. bulbifera tuber flour [124]. Chung et al. [88] reported the effects of blanching, drying and extraction with 50% EtOH, hot water, and water on the antioxidant activities of Taiwanese yam (D. alata). The peel exhibited much higher antioxidant activities. Blanching by immersing the peel in 85 • C water for 30 s caused significant reduction in the antioxidant activities of all peel extracts. Generally speaking, freeze-dried peel maintained higher antioxidant activities than hot air-dried peel. More recently, Santos et al. [90] reported that the freeze-dried purple yam (D. trifida) powder had higher antioxidant capacity and higher TPC and TAC values, compared with the products dried using a refractance window at 70, 80, and 90 • C for 40 min. Compared with the native starch, the antioxidant activity of the starch-palmitic acid complex, prepared using the reaction of different concentrations (0.1-5.0%) of palmitic acid and yam (D. opposita) starch, significantly increased [125]. D. bulbifera tuber extract was used in the synthesis of novel platinum-palladium bimetallic nanoparticles along with individual platinum and palladium nanoparticles. Bimetallic nanoparticles demonstrated enhanced DPPH • , • O 2 − , • NO, and • OH scavenging capacity and more pronounced effects on the HeLa cancer cell death [126]. There was no particle size influence on the content of phenolics (0.27-2.82% dw) or anthocyanin (2.25-15.27 mg/100 g dw) in light purple, purple and dark purple cultivars, or carotene (23.75-132.12 mg/100 g dw) in yellow and orange cultivars of D. alata [99]. Adedayo et al. [47] reported the effects of processing on the TPC, TFC and antioxidant characteristics of white (D. rotundata) and water yam (D. alata) flour (Table 3). Jaleel et al. [127] determined that paclobutrazol (PBZ) treatments had a profound effect on antioxidant metabolism and caused an enhancement in both nonenzymatic and enzymatic antioxidant potentials under treatments in white yam (D. rotundata). Djeukeu et al. [11] used 10-60% of the dried yam (D. schimperiana) flour produced by the traditional and modified process (boiled for polyphenol oxidase inhibition before drying) methods in pasta; a yam ingredient increased TPC, ABTS •+ scavenging, FRAP, and RSA values up to 2.9, 3.80, 14.48, and 2.21-fold; conversely in the case of the modified process, all the values were higher than for the traditional method. The content of total soluble phenolics in the roots of minimally processed yam (commercial Dioscorea spp.) did not change significantly during 14 days of storage at 5 • C (16.9-17.8 mg/g FW) or at 10 • C (16.7-17.5 mg/g FW) [128]. Lu et al. [98] reported an increase in the content of polyphenols, flavonoids, and anthocyanins in the tuber flesh and peel of five commercial yams using arbuscular mycorrhizal fungi treatment. Ratnaningsih et al. [49] compared immersion in water, Na-bisulfite, and ascorbic acid as browning inhibitors of D. alata flour, and found that the treatment provided better TAC, TPC and TFC values.

Application of Dioscorea spp. Ingredients for Increasing Product Antioxidant Properties
Yam flour has been used in the formulations of various foods. Adeloye et al. [10] reported that the addition of water yam (D. alata) flour in the formula of dough at 10-40%, together with plantain and bitter leaves, increased the TPC from 1.26 to 4.44 mg GAE/g, the radical-scavenging capacity from 14.73 to 48.66 mg/g, and the FRAP from 8.57 to 42.52 mg AAE/g, in a dose-dependent manner. The substitution of up to 40% of wheat flour in bread formulation with purple yam (D. alata) flour, containing 220 mg GAE of TPC, 640 mg AAE DPPH scavenging capacity, and 16 mg CGE of TAC in 100 g, showed liking scores in all bread sensory attributes similar to those of the control [129]. The addition of 2-8% D. japonica powder-containing 11.16% protein, 0.73% fat, and 76.39% carbohydrate-into red bean sweet jelly desert 'Yangaeng' increased the TPC from 14.58 to 71.54 mg GAE/100 g, and DPPH • scavenging from 5.84 to 63.98%, in a dose-dependent manner [12]. Extrusion processing increased the TBARS inhibition ability for all yams (D. alata var. Tai-nun and var. Ta-shan, D. doryophora var. Hang-chun and commercial yam flour) and generally had no negative impact on DPPH • , ABTS •+ scavenging, ORAC, and Fe 2+ chelating for most yams in corn-yam extrudates [51]. Extruded yam flour (D. alata) containing 61.20 ± 0.02 mg GAE/g phenolics was used in the formulation of instant drinks [130]. Yams (D. alata) added at 5%, 10%, and 15% to Chinese sausages caused lower thiobarbituric acid (TBA) values during 21 days of storage [131]. Li et al. [132] tested up to 25% of Chinese yam (D. opposita) flour in bread formula. The content of flavonoids increased in the bread from 0.162 to 0.516 mg RE/g, and allantoin from 0.05 to 1.063 mg/g, while the IC 50 value of antioxidant capacity decreased from 124.14 to 101.36 mg/mL. On the other hand, it should be noted that the TPC values increased less significantly, from 0.467 to 0.491 mg GAE/g, which is in some degree of disagreement with the total flavonoids, which belong to the group of phenolics.

In Vitro Assays in Cell Cultures and In Vivo Assays with Animals
Quite a few studies tested Dioscorea preparations in cell cultures and in vivo assays with animals, mainly rats and mice. The results of these studies are summarized in Table 4.   The levels of pro-inflammatory cytokines IL-1, IL-1β, IL-6, IL-10, and TNF-α ↓; suppressed production of oxidant stress makers: NO, MDA; could protect an injured ankle joint from further deterioration. RMD-red mold dioscorea; ROS-reactive oxygen species; SNP-sodium nitroprusside; SOD-superoxide dismutase; STZ-streptozotocin; TBARS-thiobarbituric acid-reacting substances; TGF-β1-transforming growth factor-β1; tGSH-total glutathione; TNBS-trinitrobenzenesulfonic acid; TNF-a-tumor necrosis factor-a; TS-total saponins; TSS-total steroid saponins; YP-yellow powder.
The effects of yam extracts, purified fractions, and individual compounds on various biomarkers were reported, including important factors of endogenous antioxidant defense in the cells, namely superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and reduced glutathione (GSH). This table also includes brief information on chemical assays of enzyme inhibition, which are related to various physiological effects. It should be noted that many authors related the beneficial effects of Dioscorea preparations in cell and animal assays with their antioxidant properties-which were determined using the chemical assays (Table 2)-and the presence of various classes of phytochemicals ( Table 3).
The extracts isolated with different polarity solvents were tested in various cell assays. EtOH extraction of dried, ground D. birmanica (locally called Hua-Khao Yen-Neua) rhizomes yielded 10.56% of the extract, with TPC and TFC values of 170.85 ± 3.02 mg GAE/g and 132.55 ± 3.59 mg CE/g, respectively [16]. The authors suggested that the identified phytochemicals from the extract, which scavenged radicals and moderately chelated metals, may be responsible for various bioactivities in the cells. Water fraction isolated by reflux-extraction of dried D. cirrhosa powder, and afterwards separated by AB-8 macroporous resin with water, 60% and 95% EtOH demonstrated numerous bioactivities in cells as well [148]. The extracts of aerial bulblets of D. japonica inhibited the induction of inflammatory mediators and the expression of iNOS and COX-2 in the cells [149].
Among the five products separated from the purple Chinese yam (D. alata) extracts, the EA fraction of flesh and peel exerted the best antioxidant activity and exhibited excellent antiglycation ability by suppressing different stages of the glycation cascade; they also exhibited cytoprotective effects on methylglyoxal-induced oxidative damage in HepG2 cells [52]. Steroidal glycosides, spirostans, of D. villosa (zingiberensis saponin I, dioscin, deltonin, and progenin III) were found to be cytotoxic, whereas, furostans (huangjiangsu A, pseudoprotodioscin, methyl protobioside, protodioscin, and protodeltonin) were non-cytotoxic. While the treatment of HepG2 cells with compounds prior to H 2 O 2 exposure effectively increased cell viability in a concentration-dependent manner, some of them increased GSH level and decreased intracellular ROS generation [150]. 2,7-Dihydroxy-4,6-dimethoxy phenanthrene, which was isolated from yam (D. batatas) peel extract with 95% EtOH, was found to be a strong antioxidant and anti-inflammatory agent in bio-guided fractionation assays with HepG2-ARE cells [136]. MeOH extract of D. bulbifera leaves had significantly higher antioxidant activity than EA and HX extracts in DPPH • , FRAP, and ABTS •+ assays, and demonstrated a pronounced cytotoxic effect in MDA-MB-231 and MCF-7 cell lines [63]. Ren et al. [151] isolated two new norsesquiterpenoids, dioscopposin A and dioscopposin B, as well as 21 known compounds from the stems and leaves of D. oppositifolia, and evaluated their estrogenic activity using MCF-7 cells; eight of these compounds inhibited cell proliferation. Antioxidant capacity was correlated with phenolic content in the cytoprotective effect on HUVEC viability assay, and D. glabra extract was more active than D. alata against H 2 O 2 -induced oxidative stress [152].
The in vivo studies were focused on the various physiological effects, and some of them reported beneficial effects of Dioscorea preparations in diabetic animals. Potential effects of ethanol extract isolated from a traditional Chinese medicine, D. zingiberensis, against testicular tissue damage via activation of the nuclear factor erythroid 2 (Nrf2) were demonstrated in a diabetic mouse model [153]. A polysaccharide, which was isolated from D. opposita roots with acidified water-extraction following precipitation with EtOH and chromatographic purification, increased SOD activity in alloxan-induced diabetic rats and mouse models at higher doses, and had strong hypoglycemic activity [154]. Fermentable water-soluble polysaccharides of purple and yellow water yam (D. alata) produced shortchain fatty acids, and also demonstrated hypoglycemic effects in rats with alloxan-induced glucose levels [134]. Treatment with crude yam (D. batatas) powder, water extract and allantoin effectively ameliorated antioxidant stress in streptozotocin (STZ)-induced diabetic rats by decreasing MDA formation and increasing SOD/GPx activities [140]. The administration of yam (D. batatas) rhizome aqueous extract and allantoin at high doses had numerous health benefits in the in vivo studies with high-fat-diet (HFD)/STZ-induced dia-betic mice [137]. It is worth mentioning that the traditional Chinese medicine Rehmannia Six Formula for treating diabetes mellitus includes D. opposita, D. alata, and D. batatas [155].
Many in vivo studies reported antioxidant effects and their beneficial contribution to regulating inflammatory events and protecting against damage to important biomolecules through excessive formation of ROS. Several studies reported antioxidant defense-related benefits for gastrointestinal health. Ethanol extracts of D. batatas peel had various effects on human colonic epithelial HCT116 cells, including a reduction in the level of ROS in H 2 O 2 -challenged cells. Oral supplementation inhibited colon inflammation through the upregulation of antioxidant enzymes and subsequent downregulation of inflammatory proteins [156]. D. batatas flesh and peel extracts, obtained with water or 60% or 95% EtOH, were tested in vivo; pre-treatment with 60% EtOH flesh extract before ulcer induction significantly decreased the expression of biomarkers of oxidative stress [138]. Park et al. [18] also reported positive effects of crude mucin and saponins from the Dioscorea rhizome on rats induced with gastric ulcers using alcohol [18]. In the studies with gastric ulcer rats, the administration of crude Dioscorea rhizoma extract activated the antioxidant enzyme CAT and increased the levels of SOD and GP X , while the production of MDA, congestion, and hemorrhage of tissue decreased [157]. An anthocyanin-rich fraction with five identified compounds isolated from D. alata produced potent anti-inflammatory effects in the mouse model of inflammatory bowel disease (IBD) at 80 mg/kg bw; anthocyanins may reduce intestinal permeability, in part, by preserving the ultrastructural integrity of the epithelial mucosa [17]. Yam (D. batatas) increased erythrocyte levels of glutathione, GPx, and CAT in azoxymethane (AOM)-induced colon carcinogenesis in male F344 rats; in addition, the colonic mucosal gene expression of SOD and GPx were up-regulated, and the expression of inflammatory mediators was suppressed [158].
The excessive formation of ROS is also related with the initial phases of carcinogenesis. Yellow pigment from freshly cut yam (D. opposita) exhibited a protective effect against • OHinduced DNA damage, which was attributed to the high • OH-scavenging activity exerted by the pigment [70]. In addition, Liu et al. [75] determined that all the peel extracts of D. opposita had a better effect in the ROS scavenging assay and a stronger antitumor property in the mouse models than in flesh. D. bulbifera polysaccharides attenuated cyclophosphamideheightened oxidative stress in mice; therefore, combination use can potentially enhance the cardiotoxin (CTX) anti-tumor effect and can attenuate CTX-induced immunosuppression and oxidative stress in U14 cervical-tumor-bearing mice [159].
Several studies focused on the evaluation of Dioscorea preparations as cardioprotective agents. Thus, total saponins from D. nipponica, D. panthaica, and D. zingiberensis lowered MDA levels and increased SOD, CAT, GPx, and T-AOC activities in rats; moreover, they demonstrated cardioprotective effects, as was determined by the less severe histological damage [146]. Isoproterenol (ISO)-induced rats pretreated with a flavonoid-rich fraction of D. bulbifera (150 mg/kg) exhibited ameliorated lipid peroxidation and, thereby, enhanced antioxidant status, as evidenced by the increase in the GSH content and the activity of antioxidant enzymes, which also suggest a cardioprotective effect [160]. Feng et al. [24] demonstrated that diosgenins (L, M, H) were the main metabolites produced by rat intestinal microflora from D. nipponica; moreover, when administered orally at up to 80 mg/kg, they protected the myocardium against ischemic insult through increasing enzymatic and nonenzymatic antioxidant (SOD, CAT, GPx, T-AOC) levels in vivo, and by decreasing oxidative stress damage. The intra-peritoneal administration of a steroidal saponin isolated from the rhizomes of D. nipponica, trillin, in rats exerted beneficial effects in improving the levels of lipid peroxidation and SOD activity [28].
Various other health benefits of yam preparations have also been reported. Total steroid saponins (TSS) of a D. zingiberensis rhizome containing 36 quantified compounds [161] alleviated arthritic progression in adjuvant-induced arthritis (AIA) rats in vivo, which correlated with suppression of the overproduction of inflammatory cytokines, oxidant stress makers, eicosanoids, and inflammatory enzymes, while the release of SOD and IL-10 increased [19]. Zhou et al. [145] reported that monosodium-urate-crystals-induced gouty arthritis in rats may be treated with Chinese herbal medicine (D. nipponica) by regulating lysosomal enzymes, antioxidant capacities and the NALP3 inflammasome; the authors demonstrated that the total saponin groups could increase the activities of GPx and SOD, and reduce the activities of several enzymes and the content of several important inflammatory biomarkers. Orally administered TS from D. nipponica remarkably decreased MDA, iNOS and • NO levels in mice, while the levels of GSH, GSH-Px and SOD increased [162]. Water extract from D. opposita significantly increased plasma SOD activity and decreased plasma MDA concentration in hypertensive rats [163]. EtOH (95%) extract from Taiwanese yam (D. japonica, var. pseudojaponica) tubers decreased the LPSinduced • NO production and expressions of iNOS and COX-2 in RAW264.7 cells; moreover, they had effects on the activities of CAT, SOD, and GPx, as well as on the levels of MDA in the edematous-paw mice [142]. The TPC, TFC, and radical-scavenging capacities of the extract were determined (Tables 2 and 3). In the livers of mice treated with the EA fraction of D. bulbifera rhizome, the level of LPO increased remarkably, while the SOD, GPx, GST, GR, and glutamate-cysteine ligase (GCL) of the hepatic tissues all decreased conspicuously [164]. Yam (D. pseudojaponica) was also found to increase the activities of SOD and GPx, and to decrease the MDA level in the brains of D-gal-treated mice [165]. Rats fed with a high-cholesterol diet supplemented with either 0.1% or 0.5% diosgenin, for 6 weeks, exhibited a decrease in TBARS, while SOD in the plasma and liver, GSH-Px in the erythrocytes, and CAT in the erythrocytes and liver were significantly increased in the 0.5% diosgenin group [166]. The activity of SOD and CAT was significantly higher in a probucol and Dioscorea rhizome group of rabbits than in the control group fed chow containing 0.5% cholesterol and 10% corn oil [167]. The pre-intake of Chinese (D. alata) and Japanese (D. japonica) yam containing polyphenols and flavonoids significantly alleviated subsequent LPS-induced oxidative injury in mice by decreasing lipid oxidation levels and fibronectin production, and elevating SOD activity [100]. Strong antioxidant (+)-catechin, which was present in the extract of D. bulbifera bulbils, was suggested as a major bioactive constituent responsible for wound healing by promoting the cell proliferation and cell migration of human fibroblasts [62]. Yam (D. oppositifolia) extract demonstrated antioxidant and protective effects on the liver of rainbow trout [168], and an extract of Chinese yam (D. opposita) activated the antioxidant defense system in oxidatively damaged TM3 cells and KDS-Yang rats [147].

Phytochemicals of Dioscorea
Phytochemicals were studied in various Dioscorea spp., with saponins, phenanthrenes, and anthocyanins being the most widely investigated compounds. Therefore, this section does not discuss the very widely investigated steroidal saponins diosgenin and dioscin: several recently published reviews are available on the health benefits of these compounds (204-210). This section includes only short comments on the most recently-published data, which are summarized in Table 5. The structures of some secondary metabolites important for Dioscorea are given in Figure 1.   Abbreviations: AC-acetone; C-vitamin C; DCM-dichloromethane; DE-diethyl ether; dw-dry weight; EA-ethyl acetate; EtOH-ethanol; FW-fresh weight; HCl-hydrochloric acid; MeOH-methanol; NADES-natural deep eutectic solvent; ND-not detected; α-Toc-α-tocopherol.   Flavonoids and phenolic acids are important and are the most widely studied antioxidants in many plants. The analysis of anthocyanins using LCMS-IT-TOF mass spectrometry in the vines of purple yams (D. alata) revealed that two investigated accessions from 8 months old had cyanidin or peonidin nuclei, while their glycosides were nonacylated, monoacylated, or diacylated with sinapic or ferulic acid. Cyanidin 3-(6-sinapoyl gentiobioside (alatanin C) was the major yam anthocyanin [93]. The compounds identified in the colored tubers of purple sachapapa (D. trifida) were derived from peonidin (Pn), cyanidin (Cy), and pelargonidin (Pg) aglycones, most of them showing the same substitution pattern, anthocyanin 3-acylglucoside-5-glucoside, where the acyl residue consisted of a hydroxycinnamic acid [113].
Some studies applied more modern extraction methods for Dioscorea. Among the 17 phenolic compounds-quantified in D. glabra leaf extracts isolated using microwaveassisted extraction-rosmarinic acid was the major one (22.31 ± 1.33 mg/g edw), while rutin was the dominant flavonoid, followed by quercetin; the latter compound was found in the highest quantity (8.66 ± 0.29 mg/g DW) in D. alata leaves [152]. Ochoa et al. [92] optimized ultrasound-assisted extraction (UAE) conditions for ethanolic anthocyanin-rich extracts from purple yam (D. alata); the best results were obtained at 60 • C for 10 min with 60% amplitude and an EtOH-to-water ratio of 80:20. Using SuperPro Designer ® software (v. 8.5) (Intelligen Inc., Scotch Plains, NJ, USA) the authors determined that the cost of manufacturing decreased from 950.52 to 124.08 USD/kg of extract when the extractor capacity increased from 5 to 500 L; they concluded that UAE would be economically feasible when the selling price is above 170 USD/kg. It is evident that there is a need to expand the studies on using advanced techniques such supercritical and subcritical fluid, pressurized liquid extraction, enzyme assisted, and other methods for the recovery of bioactive constituents. The preservation of heat-sensitive antioxidants, meeting green chemistry principles, and sustainability aspects should be taken into account.
Phenolic acids and flavonoids (in total, 21 compounds) were quantified in 80% ethanol extract isolated from D. alata tubers at ambient temperature. Kaempferol (9.219 mg/100 g pdw) and myricetin (4.613 mg/100 g pdw) were major quantitative constituents, while the content of other identified phenolics was less than 1 mg/100 g pdw [94]. Anthocyanins are the main pigments in the purple yams, while according to the recent report of Zhao et al. [69], the yellow pigments of fresh-cut yam (D. opposita) slices were composed primarily of bisdemethoxycurcumin (73.7%) and two other unknown compounds.
Only a few reports are available on the analysis of Dioscorea extracts using gas chromatography including volatile constituents. 2-Acetyl furan and 2-acetylpyrrole had the highest odor activity (FD ≥ 128) factors in the roasted white yam (D. rotundata), while other compounds among the 29 aroma-active constituents with the FD factors ≥ 32 were 2-methylpyrazine, ethyl furfural, and 5-hydroxy methyl furfural [177]. Dey at al. [133] reported mainly higher fatty acids and their esters in an MeOH extract of freeze-dried tubers of D. alata's various secondary metabolites. Alsawalha et al. [104] performed GC-MS analysis of an MeOH extract of D. villosa leaf powder; however, the identification results of the detected compounds, based on the raw MS software report, are too preliminary.

Conclusions
Numerous studies reported antioxidant properties in extracts and fractions isolated from different anatomical parts of Dioscorea spp. The majority of the published results were obtained in studies on plant tubers/rhizomes, while reports on leaves are rather scarce. Some studies focused on plant bulbils as well. Antioxidant potential studies applied mainly to in vitro chemical and bioassays using cell cultures, while in vivo studies with animal models were also available, albeit in smaller numbers. Some of the reported studies supported radical-scavenging capacity and other antioxidant activities of Dioscorea by investigating the phytochemical composition of the extracts and fractions, isolated using various solvents and methods. In general, leaf extracts were stronger antioxidants than rhizome/root preparations. Moreover, several studies demonstrated that tuber peel accumulate more antioxidants than its flesh. The antioxidant potential of different anatomical parts of Dioscorea may be related to their phytochemical composition. Steroidal saponins, phenanthrenes and anthocyanins (in purple yams) are important constituents in tubers, while phenolic acids and flavonoids are the main antioxidants determined in the leaves. Several studies also reported radical-scavenging properties in the mucilage, purified oligosaccharides and protein hydrolysis products obtained from rhizomes. Several in vivo studies using various animal models demonstrated the anti-inflammatory effects of Dioscorea preparations, which may be related to the increased levels of endogenous cell antioxidants, namely SOD, CAT, GPx, and reduced lipid peroxidation.
The reported data may assist in explaining ethnopharmacological knowledge regarding the health benefits of Dioscorea preparations. Moreover, such knowledge may foster a wider application of Dioscorea plants in developing new functional foods and nutraceuticals. In addition, it may be noted that, until now, published articles have focused mainly on the several most widely used species. A large number of Dioscorea spp. remain poorly investigated, and present a large pool for further studies.