In vitro antioxidant capacity and in vivo hepatoprotective effect of Allophylus edulis leaf extract
Antonia K. Galeano 1, Juan R. Centurión 1, María S. Soverina 1, Laura G. Mereles 2, Miguel A. Campuzano-Bublitz 1, María L. Kennedy 1*
1 Departamento de Farmacología, Facultad de Ciencias Químicas, Universidad Nacional de Asunción. Campus UNA, 2169, San Lorenzo, Paraguay; 2 Departamento de Bioquímica de Alimentos, Facultad de Ciencias Químicas, Universidad Nacional de Asunción. Campus UNA, 2169, San Lorenzo, Paraguay
Allophylus edulis is used in folk medicine primarily for liver conditions such as hepatitis, liver cancer and liver cirrhosis. In vitro hepatoprotective activity was previously demonstrated. The aim of this work was to evaluate the antioxidant capacity and hepatoprotective effect of the ethanolic extract of A. edulis in mice. This was done by first determining the acute toxicity of the extract, evaluating the general behavior, and subsequently verifying the effect on paracetamol-induced toxic hepatitis in male mice. Additionally, the phytochemical profile was performed, and the content of total phenols and its total antioxidant capacity were quantified through the 2,2, azinobis- (3-ethylbenzothiazoline)-6 sulfonic acid radical cation (ABTS) method. The extract of A. edulis leaves did not demonstrate adverse effects up to 2000 mg/kg, p.o. Anthraquinones, flavonoids, triterpenoids, and tannins were detected. A high content of total phenolic compounds (TPC) was related to a high antioxidant capacity. Regarding the results of the biological tests, A. edulis did not affect the general behavior of the mice, and all doses tested decreased glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) activity, the main liver enzyme markers of hepatocellular damage. It is concluded that A. edulis has hepatoprotective activity, which could be related to its antioxidant activity.
Keywords: Allophylus edulis; hepatoprotective; acetaminophen; liver enzymes markers; antioxidant capacity
Received: 26/04/2022; Accepted: 06/07/2022
The liver is essential for the metabolism of virtually all foreign substances (1). In addition, it synthesizes plasma proteins, fatty acids and is responsible for the metabolism of carbohydrates. It also stores fat-soluble vitamins, metals, such as iron and copper. This organ detoxifies drugs and other types of chemical products that are not water-soluble by means of liver enzymes that are responsible for oxidization, reduction, hydrolysis, or demethylation. Finally, Kupffer cells are responsible for the immune functions of the liver (2).
Hepatitis is an inflammation of the liver caused by a variety of infectious viruses and noninfectious agents, leading to a range of health problems, some of which can be fatal. Viral hepatitis is the most common cause of liver cirrhosis, liver cancer, and viral hepatitis-related deaths (3). The appearance of a pathological process such as hepatitis results in a public health problem, as it is generally asymptomatic, with symptoms only appearing in an advanced stage of the disease. According to a WHO report, a large percentage of affected persons do not have sufficient resources to pay for the costs of diagnosis and treatment, favoring the advance of this silent disease, which explains its high morbidity and mortality rate in those affected (4).
The infusion of the leaves of Allophylus edulis is popularly used in Paraguay to treat liver conditions (hepatitis, liver cancer, and cirrhosis), as a stimulant of the bile ducts, against inflammation of the throat, for intestinal problems, as an anti-diabetic, and as a cholagogue. The boiled leaves are used to wash wounds and as a treatment against high blood pressure (5,6). Regarding the biological activities, the in vitro hepatoprotective activity of the leaves of A. edulis var gracilis tested in primary culture of hepatocytes with damage induced by carbon tetrachloride (CCl4) and galactosamine has been reported, and it was confirmed that the C-glycosyl flavones present in the leaves have an important role in the hepatoprotective activity of the plant (7). In addition, the inhibitory capacity of the angiotensin-converting enzyme (8), antiulcerogenic activity (9), genotoxic activity (10), antimicrobial activity against Staphylococcus aureus, and insect repellency (11,12) were reported.
This work evaluates the hepatoprotective activity of Allophylus edulis in mice and the antioxidant activity of the extract, in addition to the identification of the main secondary metabolites.
Materials and Methods
Plant material and extraction
Leaves of Allophylus edulis Radlk. (St Hil. Jusset Camb.) syn. Allophylus guaraniticus (StHil.Juss et Camb.) known as kokũ (Sapindaceae) were collected from J.A. Saldívar, Central, Paraguay (25º26´50,4¨S y 57º 27´07,1W). The material was identified by researchers from the Botany Department, and a voucher sample was filed in FCQ Herbarium (G Delmás 284). 200 grams of dried and powdered leaves were extracted with ethanol (1:5), first by ultrasonication (3X/day, 15 minutes each, 3 days, 30ºC). The extracted material was separated, and the residual material was again extracted with ethanol by reflux (15 minutes, 3X, 1L each). The resulting extract (34%) was kept in a desiccator after solvent evaporation. On the day of each experiment, the extract was dissolved in ethanol/propylene glycol/distilled water (0.5:4:5.5) before oral administration in mice.
Reagents and equipment
Absolute ethyl alcohol 99,5% was purchased from CICARELLI Laboratorios (San Lorenzo, Santa Fe, Argentina). TROLOX (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), gallic acid monohydrate, acetaminophen, and silymarin were purchased from Sigma-Aldrich (St. Louis, MO, USA). The 2,2, azinobis-(3-ethylbenzothiazoline)-6 sulfonic acid radical cation (ABTS) was purchased from AppliChem GmbH (Darmstadt, Germany), gentamicin from LASCA (San Lorenzo, Paraguay), and sodium pentobarbital (Nembutal) from Abbott (Toyko, Japan). Ammonium persulfate, propylene glycol and ethanol were purchased locally. Kits for the estimation of alkaline phosphatase (ALP), aspartate aminotransferase (AST or GOT), alanine aminotransferase (ALT or GPT) were purchased from HUMAN Diagnostics Worldwide (Wiesbaden, Germany). Measurements were made in a Biosystem BTS 350 semi-automatic analyzer UV-Vis Spectrophotometer (Shimadzu, Kyoto, Japan).
Preliminary phytochemical analysis
Phytochemical analysis of ethanol extract of A. edulis was performed according to the methodology described (13), based on the color typically developed by major chemical groups. Briefly, anthraquinones were detected with ammonia solution after extraction with an organic solvent. The appearance of a yellow color when the sample was dissolved in a NaOH solution was evidence of flavonoids. The Liebermann-Burchard reaction was performed to identify triterpenoids. The presence of tannin was noted following reaction with ferric chloride.
Total phenol content and ABTS radical inhibition test
Determination of total phenol content in the extract was preceded by ultrasound-assisted extraction of phenols with methanol:water (60:40) and subsequently acetone: water (70:30), following reported methodology (14). Total phenolic compounds (TPC) were measured spectrophotometrically by the Folin–Ciocalteu method (16). The blue-colored complex was quantified at 765 nm. A gallic acid calibration curve was used (5-150 µg/mL), and distilled water was used as the control. The analysis was performed in triplicate and total phenol content was expressed in mg of gallic acid equivalents per 100 g of sample.
Determination of the antioxidant activity of A. edulis extract was carried out according to the methodology described by Re et al 1999 (16). This is based on the discoloration of ABTS•+ cationic radical obtained after the reaction of 7 mM ABTS with 2.45 mM potassium persulfate, incubated 20 hours at room temperature in the dark. On the day of measurement, the solution was diluted with absolute ethanol to reach an absorbance of 0.7 ± 0.02 at 730 nm. The TAC (total antioxidant activity) quantification was performed as described previously using the linear regression method. TROLOX solutions (0-320 μM) were used as standards. The results were expressed as µM Trolox equivalents (TEAC) /g sample, and the antioxidant capacity was expressed in µMTEAC/g per gram of extract (17). In addition, the effective inhibitory concentration (IC50) was determined and subsequently expressed as % inhibition (%I) of sample, which was calculated according to the following: %I = (Abs control-Abs sample/Abs control) x 100 (Abs control: solvent absorbance; Abs sample extract solution absorbance). Measurements were made in triplicate.
Swiss albino male and female mice, weighing 25-35 g, were used. The animals were housed in plastic cages at a constant room temperature (23-25°C), with a 12:12 h light-dark cycle, in a humidity-controlled environment (50-60%). They were fed daily with standard animal pellets (7 g/day each) and water ad libitum. All assays were conducted in accordance with international standards of animal welfare, and the research protocol was approved by the Bioethical Committee of the Facultad de Ciencias Químicas (CEI 469/19). The minimum number of animals and duration of observation required to obtain consistent data were used, and each animal was used once (18).
Acute oral toxicity test and evaluation of general behavior (Irwin test)
The acute toxicity test was conducted following the guidelines of Test No. 420 of the Organization for Economic Cooperation and Development (19). It was conducted in female mice, with oral administration of A. edulis ethanolic extract in fixed doses of 5, 50, 300 and 2000 mg/kg. Male mice were used for the behavioral test (Irwin test), consisting of an observational procedure to evaluate the primary effects of a drug or drug candidate on the behavior and physiological functions of the central nervous system (20, 21). The different groups received the ethanolic extract of A. edulis orally in doses of 50, 100, 200, and 400 mg/kg.
Acetaminophen induced hepatotoxicity and treatments
Six hours fasted Swiss albino male mice were randomly divided into seven groups (n=8). They were treated for 4 days as detailed: Vehicle group (V; 2.5% ethanol: 40% propylene glycol: 57.5% water, p.o.); Acetaminophen group (APAP; water); Silymarin group (SM; 150 mg/kg of silymarin, p.o.); groups Ae50, Ae100, Ae200, Ae 400 (treated with 50; 100, 200 and 400 mg/kg of A. edulis extract, respectively, p.o.). Acute hepatotoxicity was induced on the fourth day using acetaminophen (APAP, 300 mg/kg, i.p.). Two hours after the oral treatment, all animals, except those in the vehicle group, were induced (22). Three hours after APAP administration, a blood sample was collected by cardiac puncture following anesthesia with sodium pentobarbital (50 mg/kg, i.p.). Serum GOT, GPT and ALP were determined.
Glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT) and alkaline phosphatase (ALP) were measured from the blood serum. To obtain the blood serum, the fresh blood samples were incubated in a water bath at 37°C for 20 min and then subjected to centrifugation for 15 minutes at 3000 rpm. The samples were processed immediately after preparation. Control serum (normal and pathological Humatrol) was processed before each measurement as an internal quality control, and the values obtained for the different biochemical parameters were consistently within the expected ranges. The activities of GPT, GOT and ALP were determined through the optimized UV method (IFCC). The absorbance was measured at 340 nm in a spectrophotometer, and the results expressed in U/L (23, 24).
The data were processed using the GraphPad Prism 7.0. (GraphPad Software, Inc., San Diego, CA, USA). The analysis of variance (ANOVA) of one factor followed by Dunnett or the Tukey test was used for comparative analysis. Results were expressed as mean ± standard deviation (SD) and a p-value <0.05 considered statistically significant.
Preliminary Phytochemical Analysis and Total Antioxidant Capacity of A. edulis extract
The TPC content, TAC, and phytochemicals on A. edulis ethanolic extract are presented in Table 1. The IC50 of the extract was observed to be lower than the IC50 of the control, suggesting a high antioxidant capacity of the extract. Preliminary phytochemical analysis of A edulis extract indicated the presence of tannins, anthraquinones, anthraquinones glycosides, flavonoids, and triterpenoids (Table 2).
Acute toxicity and behavioral effect
The acute toxicity assay conducted following Test No. 420 of the Organization for Economic Cooperation and Development determined that the extract does not show acute lethal toxicity, as no mortality was observed. No difference in body weights or evidence of liver, lung, pancreas, heart, or kidney damage was detected after 14 days (19). Water and food consumption were similar to the control group during the observation period. Responses to nociceptive stimuli, behavior, grooming, and postural reflex remained normal and control-like following treatment.
Determination of the hepatoprotective effect of A. edulis against acetaminophen-induced damage
In the hepatotoxicity model used, acute liver damage was induced in the animals on the fourth day of treatment, except for the animals in the control group that were treated with the extract solvent. Measurements were made on the serum of the animals obtained at the end of the experiment (after anesthesia), and the serum activity of GPT (U/L) was verified. When comparing the values obtained in the control and pathological groups (APAP), a statistically significant difference (p<0.0001) between was verified, which indicated that the dose of acetaminophen used effectively induces liver damage in these experimental animals, considering the elevation of this parameter in APAP group. In addition, a statistically significant difference was also verified between the APAP group, and the SM group treated with silymarin, the reference for hepatoprotection effect, and between the APAP group and the Ae50, Ae100, Ae 200 and Ae400 groups, which were treated with different doses of the ethanolic extract of A. edulis (50, 100, 200 and 400 mg/kg, respectively). Serum level of GPT was lower in all extract-treated groups as well as in the SM group. Finally, no statistical difference was observed between the groups treated with the extract or with silymarin and the control group (Figure 1).
The serum GOT activity (U/L) of the different groups was also measured (Figure 2). The results showed that when comparing the values obtained in the control group and the pathological group (APAP), a statistically significant difference was evident (p<0.0001), with activity highly elevated in the APAP group. This again indicated the validity of the method used to evaluate the hepatoprotective activity of the extract. Additionally, when comparing the data of serum levels of GOT of the animals of the APAP group with the data of the groups SM (treated with silymarin, p<0.01), Ae50, Ae100, Ae200 and Ae400 (50, 100, 200 and 400 mg/kg of A. edulis, respectively, p<0.0001), a statistically significant difference was found between them, silymarin reduced GOT values. Furthermore, no difference was observed between the groups that received the extract or silymarin, and the control group.
Serum alkaline phosphatase (ALP) activity in the group treated with silymarin (SM) showed a statistically significant reduction when compared to the group with toxic hepatitis induced by acetaminophen (p<0.05) (Figure 3). The other treated groups did not present a significant difference with respect to the pathological group, presenting similar FA values.
The toxicity test of the ethanolic extract of A. edulis in mice, up to a dose of 2000 mg/kg, showed that the extract did not cause morphoanatomical alterations in the main organs studied or the death of the animal. Therefore, according to the OECD, the extract is safe for oral use. This result is in line with a previous study using similar doses of an aqueous extract of A. edulis in Wistar rats, i.e., no evidence of toxicity was observed; however, an alteration in the weight of the liver was found with very high doses (5000 mg/kg), which suggests signs of toxicity in this organ (12). Following the guidelines of the OECD, doses of up to 2g/kg were used in this study (19). Likewise, the low toxicity of A. edulis has been reported in in vitro models with Artemia salina (25). The results obtained when evaluating the effect of the ethanolic extract of A. edulis on the general behavior of mice also showed the absence of general effects on autonomic or ethological parameters such as lethargy, dysuria, vomiting or regurgitation, diarrhea, spontaneous motility, defecation, piloerection, photophobia, and xerostomia.
It has been reported that A. edulis contains flavonoids, phenolic compounds, triterpenoids, anthraquinones, and that it does not contain saponins in aqueous and ethanolic extracts (10,25), which coincides with our results. Other authors reported the presence of alkaloids, which differs from our results (26)
The phenolic content in the extract was 36.954 ± 0.379 mg GAE/100g, while that reported in another study using A. edulis ethanolic extract was 17.6 ± 0.6 mg GAE/100g and for the aqueous extract was 9.0 ± 0.2 mg GAE/100g (12). This difference could be due to the dilution used by the authors (200 µg/mL), or the time elapsed after the reaction until the absorbance measurement (2 hours), which differs slightly from the conditions used in our study.
The antioxidant activity expressed in IC50 was higher (36.954 ± 0.379 mg GAE/100 g) than that reported in another study using A. edulis ethanolic extract (17.6 ± 0.6 mg GAE/100 g) and other aqueous extract (9.0 ± 0.2 mg GAE/100 g) previously reported (12), this difference could be due to the dilutions used and assay conditions. Our result is similar to data reported from experiments conducted using the ethyl acetate fraction (27). The A. edulis extract was higher antioxidant capacity (lower IC50) than the reference substance TROLOX (Table 1). It has been reported that the antioxidant capacity of A. edulis is related to the extract TPC content (12). On the other hand, a moderate antioxidant activity of the essential oil of A. edulis and its main component viridiflorol has been reported (28). Moreover, aqueous extracts of another member of this genus, A. rubifolius, have exhibited a high antioxidant activity (IC50 7.1±0.1 μg/mL) in the DDPH assay (29).
Regarding the study of the effect of the extract on the model of hepatotoxicity induced by APAP in mice, it was determined that the pathological group exhibited an increase in GPT and GOT activity compared to the control group (p<0.0001) since the metabolism of APAP and hepatic necrosis are the main events during the first hours after the administration of APAP (22). A significant reduction (p<0.0001) of liver enzymes GPT, GOT was also found in animals treated with all doses of A. edulis extract compared to the control group. This result is similar to that observed in the group treated with silymarin (GPT p<0.0001 and GOT p<0.01). In the case of silymarin, this result was what was expected, considering that it is the reference hepatic protective drug (30).
Regarding the activity of ALP, it was found that only the group treated with silymarin presented a statistically significant reduction with respect to the pathological group (p<0.05), in the other groups no difference was observed with the APAP group. This could be because this enzyme is not a specific marker of the liver and therefore the presence of an alteration in the level of ALP is not always indicative of hepatocellular damage (31), but when there is hepatic obstruction of the bile flow, it can be elevated (32). In addition, the time elapsed for sample collection after APAP administration in the hepatotoxicity model used is not sufficient to cause damage at that level.
In general terms, similar values were observed in the GPT and GOT parameters in the animals treated with the different doses of A. edulis extract and those treated with silymarin. This could be due to the antioxidant property of A. edulis that is associated with the presence of phenolic compounds, and it agrees with what was previously found in an in vitro study (7). The liver damage caused in the hepatotoxicity model used involves oxidative stress (30).
At this point it is worth mentioning that the role of phenolic compounds of natural origin in hepato-protection has been reported (33). These compounds can stabilize the free radicals generated by inhibiting the formation and expression of inflammatory cytokines, interleukins, transforming growth factor β (TGF-β), and tumor necrosis factor α (34). Finally, the regulation of the expression of several genes has also been reported according to the hepatotoxicity model studied (33).
In this work we demonstrated that the ethanolic extract of A. edulis is safe to be used orally in the doses tested, it does not affect the general behavior or the general activity of mice. It has been observed that the A. edulis extract has antioxidant capacity, related to presence of total phenolic compounds such as tannins and flavonoids. Finally, according to the evidence obtained in the liver damage test induced by paracetamol in mice, the extract has hepatoprotective activity, which could be associated with its antioxidant activity.
Authors’ Contributions statement
All authors have made substantial contributions to this work, read the final manuscript, and approved the submission. AKG, JRC, MSS, MACB, LK collaborated in the concept and design of biological experiments, data acquisition, data analysis and interpretation, statistical analysis. LGM and AKG conducted the antioxidant experiments. MACB and LK drafted the manuscript. All authors were involved in critical revision and final approval.
The authors acknowledge Professor G. Delmás of the Department of Botany, Facultad de Ciencias Químicas, for plant collection and identification, and Mr. N. Kennedy for the English language revision.
Conflicts of interest
The authors declare that they have no conflicts of interest.
This research was supported by Facultad de Ciencias Químicas, Universidad Nacional de Asunción, Paraguay. AKG conducted the work during her master's program (MCQB) supported by CONACYT, Paraguay (PROCIENCIA POSG16-160).
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