1. Introduction
Non-steroidal anti-inflammatory drugs (NSAID) are widely used for the alleviation of pain, fever and inflammation. NSAID are the most widely prescribed medications in the world and are used by millions of patients on a daily basis. However, excessive consumption of NSAID has been related to severe side effects caused by oxidative stress, resulting in considerable morbidity and mortality [
1,
2]. Acetaminophen (APAP), a non-prescription drug, is a safe and effective analgesic and antipyretic drug when used at therapeutic doses [
3]. However, an acute or cumulative overdose can cause severe liver injury that may progress to acute liver failure (ALF). In fact, APAP is the most common cause of ALF in developed countries [
4,
5].
The liver is the main organ involved in the metabolism of APAP. At therapeutic doses, APAP is eliminated via glucuronidation and sulfation reactions. However, at high doses, the conjugation pathways are saturated, and part of the drug is converted by cytochrome P450 2E1 (CYP2E1) to the highly reactive metabolite
N-acetyl-
p-benzoquinone imine (NAPQI) that reacts with sulfhydryl groups. Reduced glutathione (GSH) initially traps NAPQI, and the GSH adduct is excreted. However, when GSH is depleted, NAPQI reacts with cellular proteins, including a number of mitochondrial proteins, to form NAPQI adducts. Consequences of this process are the inhibition of mitochondrial respiration and ATP depletion, as well as mitochondrial oxidative stress [
6,
7,
8].
This results in increased susceptibility to liver injury by reactive oxygen species (ROS), including hydrogen peroxide (H
2O
2), superoxide anions (O
2•−) and hydroxyl radicals (·OH). In addition to reducing the GSH level, the APAP overdose also reduces the antioxidant enzyme activities, increases lipid peroxidation and causes hepatic DNA fragmentation, which ultimately leads to cellular necrosis [
9,
10].
Currently, the treatment of choice for APAP overdose is
N-acetyl-
l-cysteine, a precursor of intracellular cysteine and GSH that counteracts the depletion of GSH and allows the excretion of NAPQI as the GSH-adduct. This reduces oxidative stress and, consequently, liver injury [
11]. Unfortunately,
N-acetyl-
l-cysteine is not always effective, and there is an urgent need for more effective interventions.
Medicinal benefits from plants have been recognized for centuries. Vegetables and fruits are very important in human nutrition and as sources of phytochemicals that reduce disease risks, like oxidative stress, inflammation and DNA damage [
12,
13]. The protective effects of diets rich in fruits and vegetables are not only due to fibers, vitamins and minerals, but also to secondary metabolites of plant products [
14]. In recent years, many antioxidant compounds, such as vitamins, pigments and phenolic phytochemicals from fruits, vegetables and herbs, have received special attention due to their protective actions against oxidative damage and genotoxicity [
15,
16].
Cactus (
Opuntia spp.) is used as a common vegetable and medicinal plant on the American continent. There are about 200 recognized species of
Opuntia, and at least 84 are found in México [
17,
18]. Cactus pears are sweet edible fruits from the cactus (
Opuntia spp.) that belong to the Cactaceae family [
19]. These fruits have been used in traditional medicine for the treatment of several diseases [
20] and contain a wide variety of trace elements, sugars and other bioactive compounds, such as betalains, carotenoids, ascorbic acid, flavonoids and other phenolic compounds [
21]. Cactus pear fruits are now recognized as a rich source of nutritional compounds with health-promoting activities, including antioxidant [
22,
23,
24,
25,
26], neuroprotective, anti-inflammatory, cardioprotective, anti-diabetic [
27], anti-clastogenic [
28] and anti-genotoxic actions [
16]. In addition, they have protective effects on erythrocyte membranes [
29] and on acute gastric lesions [
30], and they improve platelet function [
31] and cancer chemoprevention [
18]. Interestingly, APAP-induced liver injury is one of the most widely-used models to evaluate the hepatoprotective potential of natural products [
32].
The aim of this study was to investigate the hepatoprotective effect of Opuntia robusta and Opuntia streptacantha fruits from a semi-arid region of Mexico in a model of APAP-induced liver injury and to perform an initial characterization of the main bioactive compounds in these fruits.
4. Discussion
Natural compounds have a huge structural diversity and many biological activities, thus offering ample opportunities to identify novel compounds for the treatment of different diseases [
49,
50]. The presence or absence of many bioactive compounds in leaves, fruits, roots, seeds and other natural subproducts depends on geographical and environmental factors, such as humidity, temperature, season, pollution, altitude, etc. Therefore, it is very difficult to standardize the composition of natural products, but it is acceptable to link their therapeutic benefits to the presence and concentration of specific compounds in the extracts used [
51]. We studied two
Opuntia species that are widely distributed in the central semi-arid regions of Mexico [
52]. The fruits contained a large quantity of the most important phytochemical compounds with proven therapeutic activity as reported by Vinson et al. [
53] and Coria Cayupán et al. [
24]. A chemical characterization of the main bioactive compounds present in these two species of
Opuntia fruits from different areas has been performed previously by different authors, and the results showed that the main components of them are mostly betalains, specifically betacyanins; flavonoids and phenolic compounds that apparently are responsible for their biologic activity [
20,
23].
Bioflavonoids are widely distributed in fruits and vegetables and have multiple biological effects, including free radical scavenging activity and chelation of metal ions [
54]. It is well known that flavonoid effects are related to their chemical structure. This is especially true for flavonols, such as quercetin, which represents the most abundant dietary flavonoid. Mechanisms of antioxidant action include the suppression of reactive oxygen species (ROS) formation either by inhibition of ROS-generating enzymes; the chelation of trace elements that are involved in free radical generation; or by the induction of antioxidant defenses. These abilities are intimately related to the oxidation/reduction potential and the activation energy for electron transfer of the substance [
55,
56,
57]. Several therapeutic effects of flavonoids have been linked to their antioxidant capacity, e.g., the inhibition of inflammation [
58] and lipoperoxidation [
59], as well as nephroprotective [
60,
61], neuroprotective [
62] and hepatoprotective activities [
63]. In addition, there is increasing evidence that polyphenols protect cellular constituents against oxidative damage and, therefore, limit the risk of chronic diseases associated with oxidative stress [
64].
The fruit extracts of both
Opuntia species investigated in this study contain high concentrations of betalains. The chemical structure of these pigments is derived from betalamic acid, and depending on the structures added to the main structure, they give rise to betacyanins and betaxanthins [
65]. These bioactive compounds are natural antioxidants with a high radical scavenging potential [
66]. The betanin molecule includes phenolic and cyclic amine groups, which are potent electron donors that endow betanin with an exceptionally high free radical scavenging ability [
67]. Studies have investigated the capacity of betalains, mainly betanin, to scavenge free radicals in vitro, and this capacity is even higher than that of vitamin C [
34,
68]. Betanin has also been reported to inhibit cancer cell proliferation in vitro and in vivo [
18], and it protects against acute lung injury and gastric lesions [
30,
69].
At high doses, the metabolism of APAP leads to the generation of the highly-reactive metabolite NAPQI, which leads to GSH depletion and the subsequent reaction of NAPQI with cellular proteins and lipids to form APAP adducts. Mitochondria are one of the most important targets of NAPQI, resulting in ATP depletion, oxidative stress and ultimately hepatocyte necrosis [
7,
8]. It has been reported that the hepatoprotective effect of some natural products is related to their antioxidant capacity to prevent liver cell damage or death, both prophylactically and therapeutically [
70,
71].
Our results suggest that the frequent consumption of
Opuntia robusta and
Opuntia streptacantha provides many bioactive compounds with antioxidant activity to counteract the cellular oxidative damage caused by APAP acute intoxication. Therapeutic treatment more closely resembles the clinical situation of APAP intoxication; however, the primary goal of this study was to demonstrate the protective effect of
Opuntia extracts in APAP intoxication. Although we demonstrate in vitro the value of therapeutic treatment, this needs to be confirmed in vivo, as well. Animal studies have revealed the promising in vivo therapeutic value of antioxidants on liver diseases. Furthermore, APAP is a model of severe oxidative stress, and in many liver diseases, oxidative stress precedes or aggravates existing liver diseases (e.g., in non-alcoholic liver diseases). In fact, oxidative stress is considered as a common mechanism of liver injury in many (chronic) liver diseases, and the application of antioxidants is a rational strategy to prevent or ameliorate liver diseases involving oxidative stress [
72]. Therefore, there is certainly value in the prophylactic use of
Opuntia extracts as anti-oxidants, and we are currently testing
Opuntia extracts in other models of (oxidative) liver damage.
The CYP2E1 enzyme is considered to be the main enzyme responsible for APAP biotransformation, and this enzyme is predominantly expressed in the centrilobular region [
73]. In line with this, we observed the most prominent histological damage in the centrilobular area (Zones II/III of the hepatic acinus). Our histological data are also consistent with necrotic hepatocyte loss. This is confirmed by our in vitro data that clearly show that necrosis (Sytox green, LDH leakage) is the predominant mode of cell death in APAP intoxication. The
Opuntia extracts are still protective when added up to 4 h after APAP intoxication, and in this respect, they are superior to NAC, the currently-used therapy for ALF induced by APAP. The reason for this might be that the
Opuntia extracts contain a multitude of components that counteract oxidative damage via different mechanisms. Future studies should identify the components of
Opuntia extracts that contribute to this protective effect.
In summary, we provide evidence that both Opuntia fruit extracts contain many bioactive compounds with antioxidant activity to counteract the oxidative damage caused by APAP. Our results also suggest that the daily ingestion of Opuntia streptacantha and Opuntia robusta fruit extracts at the indicated doses can increase liver detoxification and could be used as a dietary supplement to prevent APAP-induced acute liver failure. Finally, our results also suggest that the Opuntia extracts can be considered for other oxidative stress-related liver diseases like (non-)alcoholic fatty liver diseases.