*4.3. Hepatoprotective Effect*

The liver plays a fundamental role in the regulation of various physiological processes, and its activity is related to different vital functions, such as metabolism, secretion and storage. It has the ability to detoxify endogenous and/or exogenous substances, helps in the metabolism of carbohydrates and fats, in the secretion of bile and participates in the supply of nutrients and energy. Unfortunately, "liver disease" (a term that indicates damage to cells, organ structure or function) continues to be one of the main threats to public health around the world. This damage can be induced by biological factors (bacteria, viruses and parasites), autoimmune diseases (immune hepatitis, primary biliary cirrhosis), as well as by the action of different chemicals, such as some drugs [high doses of acetaminophen (APAP) and anti-tuberculosis drugs], toxic compounds [carbon tetrachloride (CCl4), thioacetamide, dimethylnitrosamine (DMN), D-galactosamine/lipopolysaccharide (GalN/LPS)], mycotoxins (aflatoxin B1) and undoubtedly excessive alcohol consumption [12].

Despite the various therapeutic uses attributed to the genus *Opuntia,* scientific research on its hepatoprotective capacity began in 2004, when Wiese et al [69] reported that OFI could reduce the symptoms (nausea, dry mouth and anorexia) characteristic of hangover after consuming excess alcohol. Subsequently, Galati et al. (2005) examined the effects of prickly pear fruit (JPPF) juice against CCl4-induced hepatotoxicity.

After administering 3 mL of JPPF per rat, the liver parenchyma lesion was restored after 72 h. In addition, plasma levels of ALT and AST were reduced. The investigators suggested that hepatoprotection could be related to flavonoids, betalains, and vitamin C, that synergistically, act on the antioxidant activity of JPPF [70].

On the other hand, Ncibi et al [71] demonstrated that a cactus cladode extract (CCE) from OFI could reduce the liver toxicity of the (CPF). Such conclusion resulted when combining the pesticide plus CCE and achieving significant normalization of biochemical parameters: ALT, AST, alkaline phosphatase (ALP), lactate dehydrogenase (LDH), Cho and albumin (Alb), in contrast to animals treated with CPF only where the same parameters were notably affected. Research carried out by Dalel Brahmi's scientific group explored the protective potential of SCC against two hepatocarcinogenic agents in Balb/C mice: Benzo(a)pyrene [B(a)P] [72] and AFB1 [73].

In both studies, the two carcinogens altered EOx markers, such as the level of malondialdehyde(MDA) and catalase activity (CAT), and increased the expression of heat shock proteins (Hsp 70 and Hsp 27). Likewise, the authors demonstrated that pre-treatment with SCC significantly decreased the oxidative damage induced in all the markers tested. In order to confirm the protective capacity of JPPF, in 2012, the team evaluated this property against liver injury induced by chronic consumption of ethanol (EtOH) in Wistar rats. Pretreatment of animals with EtOH plus JPPF (20 and 40 mL/kg body weight, orally) interestingly reduced biochemical markers of liver injury [such as ALT, AST, ALP, LDH, Cho, TG, and gamma glutamyl transferase (GGT)]. An improvement was also observed in lipid oxidation, glutathione (GSH) content, the activity of some antioxidant enzymes [such as SOD, CAT and glutathione peroxidase (GPx)], as well as histopathological lesions induced

by chronic ingestion of EtOH for 90 days. These results again sugges<sup>t</sup> that this property could be attributed to its ability to end free radical chain reactions [74]. Considering that cyclophosphamide (CP) has a high toxicity associated with ROS overproduction, the ability to JPPF to reduce liver damage and cytotoxicity induced by this alkylating agen<sup>t</sup> in mice was evaluated. Again, biochemical markers (AST, ALT, LDH, Alb) and EOx indicators [degree lipid peroxide (LPO) and MDA] were analyzed. Cytotoxicity was also studied by reducing nucleic acids, proteins, and glutathione in liver cells. After a pretreatment with JPPF, all mentioned liver markers were statistically restored [75].

Not only OFI has demonstrated a hepatoprotective potential, since González-Ponce et al. (2016) analyzed the antioxidant activity of OS and OR extracts against APAP-induced acute liver failure (ALF). They also administered both extracts (800 mg/kg/day, orally) to Wistar rats prior to APAP intoxication, which significantly attenuated lesion markers (AST, ALT, and ALP) and improved liver histology.

Furthermore, in a culture of hepatocytes, the extracts reduced LDH leakage and cell necrosis, both prophylactically and therapeutically. Apparently, OR showed higher levels of antioxidants than OS. These results suggested that both extracts could be considered as nutraceuticals to prevent ALF [76]. Finally and considering that OdHw appears to have antioxidant and anti-inflammatory properties, the hepatoprotective effect of a hydroalcoholic extract against lead acetate (Pb) -induced toxicity in an animal and cellular model was analyzed.

In the first case, Wistar rats received the extract (100 and 200 mg/kg/day) plus Pb for ten days. At the end of the period, it was observed that OdHw increased CAT activity and decreased the activity of MDA and serum liver enzymes (ALP, ALT, AST). In the case of the in vitro assay, HepG2 cells were used to measure three concentrations of OdHw (20, 40 and 80 μg/mL) on cell viability, MDA, GSH and TNF-<sup>α</sup>. The measurement confirmed that all concentrations reduced the levels of MDA and TNF-α and increased at GSH [77].

As can be seen, most of the studies analyzed sugges<sup>t</sup> that the main mechanism of hepatoprotective action is to act on the inhibition of EOx. However, because the antioxidant process remains largely unknown, activation of factor 2 related to nuclear erythroid factor (Nrf2) has recently been explored. In this sense, Nakahara et al. [78] demonstrated that an OFI extract showed potent antioxidant activity through the activation of Nrf2. Their conclusion was made by confirming that the antioxidant capacity of OFI was canceled in Nrf2 knockdown keratinocytes.

Cells where Nad (p) h: Quinone oxidoreductase 1 (NQO1) inhibits the generation of ROS induced with B(a)P and TNF-α have been evaluated. The results suggested that OFI upregulated Nrf2-NQO1 through activation of the aryl hydrocarbon receptor (AHR) and AHR-OFI binding regulated the expression of epidermal barrier proteins (such as filaggrin and loricrin) [78].

Recently, INS-1 cells were exposed to Allox with different concentrations of polysaccharides extracted from *O. Milpa Alta* (MAP) to measure the Nrf2 pathway and the activation of apoptosis in response to an increase in EOx. MAP restored cell viability and SOD and GSH activity, while considerably decreased the release of ROS, LDH, MDA and nitric oxide (NO) levels. Possibly, MAP may attenuate the apoptosis induced by Allox by increasing the expression of Bcl-2, decreasing the expression of Bax and the activities of caspase-3 and caspase-9 [79].

### *4.4. Effects on Human Infertility*

Globally, statistics sugges<sup>t</sup> that approximately 15% of couples show concern when they want to conceive and fail after 12 months of regular unprotected sex. For a long time, the female gender was wrongly stigmatized for its inability to conceive, but scientific advances in human reproduction of the last four decades have associated approximately 50% of this infertility with the male gender. Different environmental, physiological, and genetic factors have been identified in "male factor" infertility, especially those related to sperm dysfunction [80].

Oligozoospermia is a disease characterized by low sperm count and quality and responsible of 90% of male infertility. Unfortunately, routine semen studies and analysis have shown that not all men who have normal parameters are fertile. The hidden factor is now known to be EOx and is recognized as a major cause of idiopathic male infertility.

The fact that sperm contain a large amount of unsaturated fatty acids makes them prone to lipid peroxidation, causing DNA damage and activating its apoptotic elimination through a p53-dependent and independent mechanism that can lead to infertility. Some studies have shown that disorders such as poor fertilization, pregnancy loss, birth defects, and poor embryonic development are associated with sperm damaged by excessive EOx [80–83].

In a normal physiological state, seminal plasma contains an antioxidant enzymatic mechanism capable of quenching ROS and protecting sperm. However, a high level of ROS, triggered by factors such as inflammation, DM, AO, alcoholism, smoking, and environmental pollutants can minimize this protective mechanism [80,84–87].

Different studies have suggested that some food supplements such as selenium, zinc, carnitine and arginine increase sperm count and motility; while antioxidants such as vitamin E, C and B12, carotenoids, coenzyme Q and glutathione are beneficial for the treatment of male infertility. In other words, these compounds can help in the balance between ROS generation and the protective enzymatic mechanism of sperm [80,88,89].

In the specific case of *Opuntia* spp., there is little research that has explored its action in human infertility. Meama et al. (2012) conducted the first study and tested the effect of OFI on sperm DNA fragmentation (SDF) exerted by cryopreservation in two sperm populations [PI (brighter) and PI (dimmer)]. Normozoospermic men underwent semen analysis for infertility and their cell samples were subsequently cryopreserved in the presence of OFI extracts.

The process induced an increase in SDF only in the PI sperm population. In contrast, the addition of OFI slightly reduced SDF without affecting cell viability. Their results sugges<sup>t</sup> that OFI probably prevents some damage to sperm during a cryopreservation process [90]. Some studies also sugges<sup>t</sup> that antioxidant treatments administered in high doses can block the oxidative processes essential for the compaction of sperm chromatin.

Consequently, a trial was performed in couples with infertility and at least 2 attempts at assisted reproductive technology (ART) to evaluate a nutritional support (called Condensyl ™) of the cycle of a pure carbon without strong antioxidants. The treatment consisted of a combination of B vitamins, zinc, *Opuntia fig* extract, small amounts of N-acetylcysteine, and vitamin E. A group of 84 patients consumed 2 tablets of Condensyl ™ per day for 12 months.

The final results showed a positive response rate of 64% for the decondensation index and 71% for the DNA fragmentation index. Thus, 18 couples achieved a pregnancy before the planned ART cycle. The rest of the couples underwent a new ART attempt resulting in 22 additional pregnancies and 15 live births. The conclusion with these data was that low doses of Condensyl ™ may have a positive potential on fertility by achieving a pregnancy rate and a live birth rate of 70% and 57%, respectively [91].

Another significant study was the one designed by Hfaiedh et al. (2014) to investigate the protective capacity of CCE (100 mg/kg) on sodium dichromate (SD) -induced testicular damage in male Wistar rats. After a 40 day treatment with SCC, it was possible to restore serum testosterone level, sperm count and motility to levels comparable to the DS control group. A reduction in the elevated level of lipid peroxidation and a significant increase in testicular SOD, CAT, and GPx activities were also registered [92].

Probably, the effect of CCE to minimize the oxidative damage induced by SD motivated the development of another in vivo test; the purpose of which was to analyze the reversible antifertility potential of two doses (300 and 900 mg/kg) of a methanolic extract from the fruit of *O. elatior Mill* (OeM). After administering OeM for 60 days, epididymal sperm count and motility were reduced by 80% without a decrease in serum testosterone levels.

On the other hand, testicular steroidogenesis or libido were not affected, unlike male fertility, which was suppressed when they were mated with virgin female rats. Said suppression of fertility was dose-dependent and reached the 100% at the highest dose. However, withdrawal of treatment for two weeks recovered sperm count, serum testosterone levels, and fertility [93].

Recently, Akacha et al. (2020) determined the role of an OFI ethanolic extract (EEOFI) in methotrexate (MTX) -induced testicular damage in rats. They considered using this chemotherapeutic agen<sup>t</sup> due to its various drawbacks, especially in cells that are constantly dividing and developing. EEOFI (0.4 g/kg) was administered to rats treated with MTX and subsequently the sperm were collected and quantified where their motility was determined.

They also evaluated EOx markers (MDA, CAT, GPx and SOD) and marked serum testosterone levels by radioimmunoassay. The results confirmed that EEOFI had protective effects on rat gonad histology, oxidative stress, and sperm count and motility. Furthermore, serum testosterone levels increased considerably. Their results also sugges<sup>t</sup> that EEOFI improves testicular injury and has a potent stimulating effects on fertility [94]. The contradictory results opens the field of research to confirm the antioxidant action of *Opuntia genus* on fertility.

### *4.5. Chemopreventive and Antigenotoxic Effects*

The chemopreventive and antigenotoxic potential of *Opuntia* spp. is another field of research that has been widely explored by various scientists. The concept of cancer chemoprevention "Use of natural or synthetic biologically active substances that can prevent, inhibit, or reverse tumor progression" was established in the 1970s, and in the specific case of these opuntioid cacti, there have been conducted 21 studies (mainly in vitro and in vivo tests) to date. In general, the results sugges<sup>t</sup> various mechanisms of action to try to prevent the development of this disease.

It is important to remember that the transformation of normal to malignant cells is driven by a multi-step process (conceptually divided into initiation, promotion, progression, invasion, and metastasis) due to genetic alterations that include mutations and/or epigenetic changes caused by genotoxic agents or genotoxins; which by their origin are divided into physical, chemical and biological.

Observations on cancer etiology reveal that as many as 90–95% of carcinoma cases are associated with chemical agents, only 5–10% with physical agents and around 2–5% with biological agents. However, considering that all mutagens are genotoxic, but not all genotoxic substances are mutagenic, the compounds that reduce DNA damage caused by genotoxins are called antigenotoxic and/or antimutagenic agents [3,95].

In general, antimutagens have been classified as desmutagens and bioantimutagens. The first group considers substances that promote the elimination of genotoxic agents from the body, as well as substances that partially or totally inactivate mutagens by enzymatic or chemical interaction before the mutagen attacks DNA.

On the other hand, bioantimutagens (known as true antimutagens) can suppress the mutation process after DNA is damaged and act on the repair and replication processes; resulting in a decrease in the frequency of mutations [3]. The reality is that the mechanisms of action of antigenotoxic and/or chemopreventive agents are varied and complex and it would be very difficult to fully explain them in this document. In summary, observations report that they act in different cellular and molecular events including apoptosis, cell proliferation, cell cycle, EOx regulation, DNA repair, activation/detoxification of carcinogens by xenobiotic metabolizing enzymes, functional inactivation/activation of oncogenes and tumor suppressor genes, angiogenesis and metastasis [3,95].

Practically, the clinical study (the only one so far) developed by Palevitch et al. (1993) where they treated benign prostatic hypertrophy (BPH) with a dried flower preparation of OFI was the one that started in this field of research. Their results showed that patients treated with 2 capsules (250 mg of dried flowers/capsule) orally, three times a day, for 6 and 8 months, significantly improved the discomfort associated with BPH. However,

they were unable to establish the mode of action of this preparation [96]. Table 4 shows the main studies carried out with different plant parts of *Opuntia* spp. that have demonstrated chemopreventive and antigenotoxic potential. In summary, OFI is the most studied species in in vitro models and in different types of extracts [hexane, EtOAc, acetone, methanol (MeOH) and aqueous ones]. Likewise, hexane extracts of its seeds, extracts (aqueous and EtOAc) of its PPFs, juices of its different varieties of PPFs [red-purple (PPRP), whitegreen (PPWG) and yellow-orange (PPYO)] and some of its bioactive compounds such as betanin (betacyanin isolated from its PPFs) and isorhamnetin glycosides. Another species explored is OHF (mainly, in hexane extracts, aqueous and EtOAc); and to a lesser extent, OR extracts, polysaccharides extracted from OdHw and *O. microdasys* at post flowering stage F3 (OMF3). Different cell lines have been used in these studies; the most representative are those extracted from the cervix, bladder, ovarian cancer (OVCA420), immortalized normal ovarian cells (SKOV3), human chronic myeloid leukemia (K562), breast cancer (MCF-7), colon cancer (HT -29), human glioblastoma (U87MG), lung squamous carcinoma (SK-MES-1), human BJ fibroblasts, and Caco-2, SW480, and HeLa cancer cells.

In the case of in vivo tests, only mice (Balb/C and NIH) have been used, causing them genotoxic damage by administering some mycotoxins [zearalenone (ZEN) and AFB1] or a mutagenic agen<sup>t</sup> [such as methyl methanesulfonate (MMS)]. Skin cancer has also been induced by 7,12-dimethyl-benz [a] anthracene (DMBA) and 12- *O*-tetradecanoylphorbol-13- acetate (TPA) and ultraviolet B (UVB) photocarcinogenesis.

In general, the frequency of micronuclei (MN), chromosomal aberrations in bone marrow cells and DNA fragmentation were quantified. Together, the results of these studies sugges<sup>t</sup> that the chemopreventive and/or antigenotoxic effect of OFI, OHF, OR, OdHw and OMF3 is related to their ability to inhibit cell proliferation and induce apoptosis, accumulate ROS (pro-oxidant activity), anticlastogenic potential, modulate lipid peroxidation, induce phase II detoxifying enzyme system and antioxidant capacity. Among the bioactive compounds that exhibited these capacities are flavonoids (such as quercetin, kaempferol, isorhamnetin), betalains (such as betanin and indicaxanthin), carotenoids and phenolic compounds [4,73,97–114].


**Table 4.** Main studies of *Opuntia* spp. on its chemopreventive and antigenotoxic potential.




### **5. Conclusions and Perspectives**

The investigations shown in this review demonstrate the nutritional, medicinal, pharmaceutical and preventive impact of the different species of *Opuntia* spp. However, they also reveal the possibility of expanding and conducting new studies (in vitro, in vivo and clinical) in order to confirm its different mechanisms of action that together favor its beneficial properties. In general, the antiatherogenic, antihyperlipidemic, antihypercholes-

terolemic and antidiabetic effect share two mechanisms of action; the first one related to the soluble fiber content that decreases body weight and slows down the speed of digestion and/or intestinal absorption of glucose and fatty acids. The second, undoubtedly, lies in its antioxidant property, which is directly related to the role that OXs plays in the development of atherosclerosis and cardiovascular diseases that are complications of DM2 and OA. This mechanism is related to the presence of some flavonoids, phenolic compounds and fatty acids, specifically quercetin 3-methyl ether and/or omega-6 linoleic acid from cactus seed oil, both with hypocholesterolemic effects. Likewise, betalains (such as indicaxanthin and betanin) protect the vascular endothelium from inflammation and cytokine-induced oxidative alteration, such as TNFα. Also, the polysaccharide content has shown antiinflammatory activity and the ability to reduce lipoperoxidation and/or sequester ROS. In the control of DM2, the hypothesis that the ingestion of nopal improves the postprandial glucose response and stimulates insulin secretion through a direct action on β pancreatic cells has also emerged.

In addition, the proposal that Cr (III), present in CLD, balances carbohydrate and lipid metabolism, favors a positive effect on insulin signaling and/or function, improves its systemic sensitization and reduces plasma glucose. After studies related to ASCVD, DM2 and OA, another widely explored field of research is its chemopreventive and antigenotoxic potential; where approximately 21 studies (mainly in vitro and in vivo tests) have suggested various mechanisms of action, highlighting the induction of apoptosis, inhibition of cell proliferation and cell cycle, activation of the phase II detoxifying enzyme system and DNA repair.

Furthermore, the antioxidant and/or regulatory effect of EOx has been included. These actions have also been considered as the main hepatoprotective mechanism of *Opuntia* spp. The antioxidant process has been analyzed for many years; yet, it remains largely unknown. So it would be interesting to increase research exploring the activation of Nrf2. In the case of the action of *Opuntia* spp. on human fertility, there is little research and the results are contradictory since some studies sugges<sup>t</sup> that antioxidant treatments administered in high doses can block the oxidative processes essential for the compaction of sperm chromatin; which opens another field of investigation. Taken together, all the studies point to the conclusion that the CLD and PPFs from *Opuntia ficus-indica* (OFI) are the plant parts and the species that have been studied the most. Regarding the form of analysis, hexane, EtOAc, acetone, MeOH and aqueous extracts have been evaluated. Besides OFI, other species studied are OdHw, OHF, OS, OH, OA, OM and OR. It is convenient to remember that the Cactaceae family contains approximately 130 genera and 1500 species, which favors a wide genetic diversity that in conjunction with environmental conditions (climate, humidity), soil type, age of maturity of the cladodes and the harvest season generates differences in the phytochemical composition of their plant parts (PPFs, CLD, roots, flowers, seeds and stems) between wild and domesticated specie, inducing changes in its nutritional values and indisputably in its functional and/or therapeutic properties. Furthermore, although the public and some health care professionals believe that herbal medicines are relatively safe because they are "natural", there are remarkably little data to support this assumption. Therefore, *Opuntia* spp. species are not exempt from possible adverse and toxic effects. In general, OFI has been found to be well tolerated orally, even at high doses, presenting on certain occasions mild diarrhea, increased volume and frequency of stool, nausea, headache and low colonic obstruction [14,114,115]. Both aspects (genetic diversity and toxicology of *Opuntia* spp.) will be analyzed in more detail in part 2 of the manuscript.

In conclusion, by combining all the information in this review favors the field of research in the biotechnology area where new studies could be developed with other species to explore their capacities and pharmacological properties, their doses and administration intervals and analyze their possible toxic effects in the medium and long term. Likewise, the bioactive compounds extracted from the different species could be used in the preparation of drugs and nutraceuticals, as well as to obtain chemopreventive agents directed at cancer and/or chronic degenerative diseases. Future studies on different pure opuntiode

cacti, extracts and isolated bioactive compounds will allow a greater understanding of the properties of *Opuntia* spp., which is a genus of plant consumed by humans for more than 8000 years, with a high frequency throughout the world and which is apparently considered a safe plant.

**Author Contributions:** E.M.-S., E.M.-B., J.A.M.-G., J.P.-R., N.V.-M. designed the concept, wrote the majority of the paper and managed the authors; P.E.M.-G., I.Á.-G., J.A.I.-V., M.S.-G., L.D.-O. conducted the literature search, wrote key sections of the paper; Á.M.-G., L.A.-R. and T.F.-A. wrote sections of the paper and managed the reference list. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors thank Florencia Ana María Talavera Silva for all her academic support. Her comments and observations in reviewing articles are always valuable and we give her immense recognition for her efforts.

**Conflicts of Interest:** The authors declare no conflict of interest.
