Hepatoprotective Effect of Opuntia robusta and Opuntia streptacantha Fruits against Acetaminophen-Induced Acute Liver Damage

Acetaminophen (APAP)-induced acute liver failure (ALF) is a serious health problem in developed countries. N-acetyl-l-cysteine (NAC), the current therapy for APAP-induced ALF, is not always effective, and liver transplantation is often needed. Opuntia spp. fruits are an important source of nutrients and contain high levels of bioactive compounds, including antioxidants. The aim of this study was to evaluate the hepatoprotective effect of Opuntia robusta and Opuntia streptacantha extracts against APAP-induced ALF. In addition, we analyzed the antioxidant activities of these extracts. Fruit extracts (800 mg/kg/day, orally) were given prophylactically to male Wistar rats before intoxication with APAP (500 mg/kg, intraperitoneally). Rat hepatocyte cultures were exposed to 20 mmol/L APAP, and necrosis was assessed by LDH leakage. Opuntia robusta had significantly higher levels of antioxidants than Opuntia streptacantha. Both extracts significantly attenuated APAP-induced injury markers AST, ALT and ALP and improved liver histology. The Opuntia extracts reversed APAP-induced depletion of liver GSH and glycogen stores. In cultured hepatocytes, Opuntia extracts significantly reduced leakage of LDH and cell necrosis, both prophylactically and therapeutically. Both extracts appeared to be superior to NAC when used therapeutically. We conclude that Opuntia extracts are hepatoprotective and can be used as a nutraceutical to prevent ALF.


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 The reconstituted extract of each Opuntia species was clarified (12,000× g for 15 min at 15 • C) and used to determine the bioactive compounds. Flavonoids were quantified by the colorimetric method of Zhishen et al. [33], and values were expressed as µg quercetin equivalents/mL. The total content of betalains (betacyanins and betaxanthins) was determined according to the methods described by Stintzing et al. [34] and Sumaya-Martínez et al. [26], and results were expressed as mg of betacyanin or betaxanthin equivalents/L. Ascorbic acid content in the extracts was determined by the method of Dürüst et al. [35], and the results were expressed as mg ascorbic acid equivalents/L. The total phenolic content was determined by the Folin-Ciocalteu method described by Georgé et al. [36] and was expressed as mg gallic acid equivalents/L. All measurements were performed in triplicate on a Biotek PowerWave XS microplate reader.

Determination of Free Radical Scavenging and Chelating Activities
The antioxidant activity of the Opuntia fruit extracts was determined by different methods. The DPPH (2,2-diphenyl-1-picrylhydrazyl) method was performed according to Morales and Jimenez [37], and the antioxidant activity was expressed as mmol Trolox equivalents/L. The ABTS (2,2 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) method was performed according to Re et al. [38] and Kuskoski et al. [39], and the results were expressed as mg ascorbic acid (AA)/100 mL. FRAP (ferric reducing antioxidant power) was determined as described by Hinneburg et al. [40], and the results were expressed as mg ascorbic acid (AA)/100 mL. The assay to determine the chelating activity was performed as described by Gulcin et al. [41] and was expressed as mol EDTA equivalent/L. All measurements were performed in triplicate on a Biotek PowerWave XS microplate reader. The ORAC (oxygen radical absorbance capacity) was determined as described by Huang [42], and the results were expressed as mmol Trolox equivalents/L. This assay was performed in triplicate on a Biotek Synergy HT microplate reader.

Rat Hepatocyte Isolation
Hepatocytes were isolated from male Wistar rats by two-step collagenase perfusion as described by Conde de la Rosa et al. [43] and Vrenken et al. [44]. The cell viability was determined by trypan blue exclusion assay and was always higher than 85%. After isolation, the hepatocytes were plated on 6-well coated plates in William's E medium supplemented with 50 µg/mL gentamycin, 1% penicillin-streptomycin-fungizone and 10% fetal calf serum. Cells were cultured for 4 h to attach in a humidified incubator at 37 • C and 5% CO 2 .
Liver damage was assessed by measurement of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) serum activities using a commercial spectrophotometrical method (SPINREACT, Girona, Spain). The values represent the mean of three measurements (±standard deviation) and are expressed as IU/L.
The GSH content in liver tissue homogenates was determined according to Hissin and Hilf [46], using o-phtaldehyde (OPT) as the fluorescent reagent. The fluorescence intensity was measured at 420 nm using 350 nm as the excitation wavelength, using a Luminescence Spectrophotometer (Model LS-50B, PerkinElmer Inc., Waltham, MA, USA).
For histological studies, animals were anesthetized with sodium pentobarbital and perfused via the portal vein with saline solution (sodium chloride 0.9%), containing 0.5% heparin and 0.1% procaine and fixed in situ with neutral formalin (10%). The hepatic tissue was embedded in paraffin blocks, and sections of 5 µm were prepared with a microtome (Leica RM2125RT). The sections were stained with periodic acid Schiff (PAS) reagent. Liver tissue images were captured using a Carl Zeiss microscope (Axioskope 40) and processed using Image-Pro Plus software.

In Vitro Study
After the attachment period of 4 h, the medium was changed for medium without fetal calf serum. Monolayer cultures were exposed to a toxic dose of acetaminophen (20 mmol/L). A single dose of 125 µL (8% v/v) of Opuntia streptacantha or Opuntia robusta reconstituted extract or 5 mmol/L N-acetyl-L-cysteine (NAC) was added 30 min before (prophylactic regimen) or 30 min, 1, 2, 4 and 8 h after the addition of acetaminophen (therapeutic regimen).
To quantitatively assess hepatocyte injury, LDH leakage into medium was evaluated. LDH activity was measured spectrophotometrically at 340 nm by determining NADH oxidation in the presence of sodium pyruvate in a microplate reader (BioTek EL808) for 30 min at 37 • C and expressed as fold induction versus control values [47]. Hepatocyte necrosis was also assessed by Sytox Green staining to confirm the LDH leakage results. Sytox green binds to nuclear DNA, but can only enter cells with ruptured plasma membranes, as occurs in necrotic, but not in apoptotic cell death. Necrosis of hepatocytes was determined by incubation for 15 min with Sytox green (1:40,000) nucleic acid stain as described by Woudenberg-Vrenken et al. [48]. Necrosis was quantified by counting fluorescent nuclei and the total number of cells in three randomly chosen high power fields. Fluorescent nuclei were visualized using a Leica microscope DMI 6000B at 450-490 nm.

Statistical Analysis of Data
For the analysis of the components and antioxidant activities of cactus fruit extracts, the unpaired t-test was carried out with a confidence level of 99%. The analysis of biochemical results was done using the one-way ANOVA test, and the means of the different experimental groups were compared using the Tukey test with a confidence level of 95%. GraphPad Prism 5 software was used for the statistical analysis (La Jolla, CA, USA).

Yields of the Juice Extraction Method
Depending on the size and ripeness stage, approximately three fruits of Opuntia robusta and six fruits of Opuntia streptacantha were used to obtain 50 mL of juice extract. After lyophilization of 50 mL of each fruit juice, 6.89 ± 0.80 g of Opuntia robusta and 6.68 ± 0.63 g of Opuntia streptacantha powder were obtained (Figure 1). The difference in yield between the two cactus species was not statistically significant (p > 0.05). For the in vivo experiments, a dose of 800 mg/kg in rats (200-250 g b.w.) corresponded to approximately 1.5 mL of the reconstituted extract. For the in vitro experiments, a dose of 8% v/v in primary rat hepatocytes corresponded to approximately 125 µL of the reconstituted extract. of hepatocytes was determined by incubation for 15 min with Sytox green (1:40,000) nucleic acid stain as described by Woudenberg-Vrenken et al. [48]. Necrosis was quantified by counting fluorescent nuclei and the total number of cells in three randomly chosen high power fields. Fluorescent nuclei were visualized using a Leica microscope DMI 6000B at 450-490 nm.

Statistical Analysis of Data
For the analysis of the components and antioxidant activities of cactus fruit extracts, the unpaired t-test was carried out with a confidence level of 99%. The analysis of biochemical results was done using the one-way ANOVA test, and the means of the different experimental groups were compared using the Tukey test with a confidence level of 95%. GraphPad Prism 5 software was used for the statistical analysis (La Jolla, CA, USA).

Yields of the Juice Extraction Method
Depending on the size and ripeness stage, approximately three fruits of Opuntia robusta and six fruits of Opuntia streptacantha were used to obtain 50 mL of juice extract. After lyophilization of 50 mL of each fruit juice, 6.89 ± 0.80 g of Opuntia robusta and 6.68 ± 0.63 g of Opuntia streptacantha powder were obtained ( Figure 1). The difference in yield between the two cactus species was not statistically significant (p > 0.05). For the in vivo experiments, a dose of 800 mg/kg in rats (200-250 g b.w.) corresponded to approximately 1.5 mL of the reconstituted extract. For the in vitro experiments, a dose of 8% v/v in primary rat hepatocytes corresponded to approximately 125 μL of the reconstituted extract.

Bioactive Compounds of Cactus Pear Fruit Extracts
The extracts of Opuntia robusta and Opuntia streptacantha ripe fruits contain a high quantity of bioactive compounds. Opuntia robusta had a significantly higher amount of flavonoids, ascorbic acid and total phenolic compounds than Opuntia streptacantha (Table 1). Opuntia robusta contained a significantly higher amount of betacyanin than Opuntia streptacantha. In addition, both Opuntia fruit extracts had more betacyanin than betaxanthin, causing the red-purple color of the fruits ( Table 2). Similar values of these compounds have been reported in other Opuntia species [26,34].

Bioactive Compounds of Cactus Pear Fruit Extracts
The extracts of Opuntia robusta and Opuntia streptacantha ripe fruits contain a high quantity of bioactive compounds. Opuntia robusta had a significantly higher amount of flavonoids, ascorbic acid and total phenolic compounds than Opuntia streptacantha (Table 1). Opuntia robusta contained a significantly higher amount of betacyanin than Opuntia streptacantha. In addition, both Opuntia fruit extracts had more betacyanin than betaxanthin, causing the red-purple color of the fruits ( Table 2). Similar values of these compounds have been reported in other Opuntia species [26,34]. Values are the mean of three measurements ± SD. Opuntia robusta vs. Opuntia streptacantha * p < 0.01. eq.: equivalent. Values are the mean of three measurements ± SD. Opuntia robusta vs. Opuntia streptacantha * p < 0.01.

Free Radical Scavenging and Chelating Activities of Opuntia Extracts
Both Opuntia species demonstrated free radical scavenging capacity, but to different extents ( Table 3). Opuntia robusta had superior antioxidant activity compared to Opuntia streptacantha, which might be due to its chemical composition since Opuntia robusta contains higher amounts of bioactive compounds (Tables 1 and 2). On the other hand, extracts of Opuntia streptacantha contained more chelating activity than Opuntia robusta (Table 4).

In Vivo Experiments
The plasma levels of transaminases and alkaline phosphatase, as well as the GSH content in liver homogenates of experimental animals 4 h after APAP intoxication are shown in Table 5. APAP treatment significantly increased plasma levels of transaminases and ALP compared to control animals (p < 0.05). The prophylactic administration of both Opuntia fruit extracts (Or + APAP and Os + APAP) in APAP-treated animals significantly decreased all markers of liver cell injury compared to APAP-intoxicated animals (p < 0.05). The extracts alone did not cause any changes in these plasma markers (Table 5). GSH appeared to be less effective in attenuating liver damage compared to the Opuntia extracts (Table 5).
GSH was almost completely depleted after APAP overdose. Opuntia robusta extract completely and Opuntia streptacantha extract partially prevented the depletion of GSH induced by APAP treatment. Opuntia extracts alone did not change hepatic GSH levels. GSH administration partially prevented the depletion of hepatic GSH levels, comparable to Opuntia streptacantha. Thus, Opuntia robusta had superior protective effects than Opuntia streptacantha in acute APAP intoxication.
In the APAP group, an extensive hydropic vacuolation was observed ( Figure 2B), as well as glycogen depletion ( Figure 2C) and focal necrosis of cells with pyknotic nuclei ( Figure 2D) of the hepatocytes nearest to the central vein compared to the control group (Figure 2A). Opuntia extracts alone did not cause morphological changes, glycogen depletion or necrosis ( Figure 3A,B). Both Opuntia extracts attenuated the histological changes induced by APAP alone and maintained cytoplasmic glycogen stores, without signs of vacuolation or necrosis in the hepatic acinus ( Figure 3C,D). GSH supplementation also prevented vacuolation and necrosis, although some hepatocytes lost glycogen stores ( Figure 3E). prevented the depletion of hepatic GSH levels, comparable to Opuntia streptacantha. Thus, Opuntia robusta had superior protective effects than Opuntia streptacantha in acute APAP intoxication. In the APAP group, an extensive hydropic vacuolation was observed ( Figure 2B), as well as glycogen depletion ( Figure 2C) and focal necrosis of cells with pyknotic nuclei (Figure 2D) of the hepatocytes nearest to the central vein compared to the control group (Figure 2A). Opuntia extracts alone did not cause morphological changes, glycogen depletion or necrosis ( Figure 3A,B). Both Opuntia extracts attenuated the histological changes induced by APAP alone and maintained cytoplasmic glycogen stores, without signs of vacuolation or necrosis in the hepatic acinus ( Figure 3C,D). GSH supplementation also prevented vacuolation and necrosis, although some hepatocytes lost glycogen stores ( Figure 3E).

In Vitro Experiments
Both Opuntia extracts protected the hepatocytes against APAP-induced cell necrosis when added prior to APAP (prophylactic regimen), as shown in Figure 4. NAC was also protective, but tended to be less potent than the Opuntia extracts. In the therapeutic regimen, both Opuntia extracts protected against APAP-induced toxicity when added up to 4 h after APAP, whereas NAC was only protective when added up to 1-2 h after APAP (Figure 4).

In Vitro Experiments
Both Opuntia extracts protected the hepatocytes against APAP-induced cell necrosis when added prior to APAP (prophylactic regimen), as shown in Figure 4. NAC was also protective, but tended to be less potent than the Opuntia extracts. In the therapeutic regimen, both Opuntia extracts protected against APAP-induced toxicity when added up to 4 h after APAP, whereas NAC was only protective when added up to 1-2 h after APAP (Figure 4). To confirm the results obtained by determining the LDH release, we also used Sytox green as a fluorescent dye for necrotic cells. APAP (20 mmol/L) induced significant cell necrosis in primary cultures of rat hepatocytes ( Figure 5B) in comparison to the Control ( Figure 5A), which was almost completely abolished by the Opuntia extracts ( Figure 5F,G). NAC ( Figure 5H) showed a similar protective effect as the Opuntia extracts. These results confirm the data obtained with the LDH determinations. Results are expressed as fold-increase relative to control group; * p < 0.05, ** p < 0.01 compared to the APAP group.
To confirm the results obtained by determining the LDH release, we also used Sytox green as a fluorescent dye for necrotic cells. APAP (20 mmol/L) induced significant cell necrosis in primary cultures of rat hepatocytes ( Figure 5B) in comparison to the Control ( Figure 5A), which was almost completely abolished by the Opuntia extracts ( Figure 5F,G). NAC ( Figure 5H) showed a similar protective effect as the Opuntia extracts. These results confirm the data obtained with the LDH determinations. To confirm the results obtained by determining the LDH release, we also used Sytox green as a fluorescent dye for necrotic cells. APAP (20 mmol/L) induced significant cell necrosis in primary cultures of rat hepatocytes ( Figure 5B) in comparison to the Control ( Figure 5A), which was almost completely abolished by the Opuntia extracts ( Figure 5F,G). NAC ( Figure 5H) showed a similar protective effect as the Opuntia extracts. These results confirm the data obtained with the LDH determinations.

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 ROSgenerating 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

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.