Hepatoprotective Activity of Cordia lutea Lam Flower Extracts Against Paracetamol‐Induced Hepatotoxicity in Rats

In the northern region of Peru, more than 500 plants are traditionally used for medicinal purposes7,8 and, Cordia lutea L. "overo" is an indigenous plant used to treat liver diseases on the northern Peruvian coast.9 Traditionally this plant species is part of an effective and safe treatment to enhance liver function.10 It has also protective activity on the gastric mucosa11, anti-infective activity12 and anticancer13, as well as antiallergic activity.14 Some other species of the genus Cordia L. have demonstrated antibacterial and antioxidant activities.15,16


INTRODUCTION
Although the World Health Organization ranks paracetamol or acetaminophen as one of the essential drugs, the overdose of this drug can cause acute liver failure and even death. 1,2 The metabolic functions of the liver are important for the elimination of waste, the accumulation of which causes complications to the body. 3 The absence of reliable hepatoprotective drugs makes it necessary to search for medicinal plants with this pharmacological property. [4][5][6] In the northern region of Peru, more than 500 plants are traditionally used for medicinal purposes 7,8 and, Cordia lutea L. "overo" is an indigenous plant used to treat liver diseases on the northern Peruvian coast. 9 Traditionally this plant species is part of an effective and safe treatment to enhance liver function. 10 It has also protective activity on the gastric mucosa 11 , anti-infective activity 12 and anticancer 13 , as well as antiallergic activity. 14 Some other species of the genus Cordia L. have demonstrated antibacterial and antioxidant activities. 15,16 Although the hepatoprotective efficacy of Cordia lutea (C. lutea) has been studied, it has only been demonstrated in a mixture of a Peruvian Botanical Remedy, with Curcuma longa rhizome and Annona muricata leaf, so the activity corresponds to the mixture and not to C. lutea alone 11 , likewise, the determination has not been made of the biochemical and histopathological values of the test species.
Preclinical trials of C. lutea are limited 9 and molecularly there are only reports of norcycloartan and rutin glycosides 12,17 , so studies that support this traditional activity is of great importance, a scientific void that this article tries to cover.

Vegetable material
The flowers of Cordia lutea Lam. were collected in the province of Cajamarca, Peru. The deposit of the plant was registered with the Herbarium Truxillense (HUT) of the Universidad Nacional de Trujillo, Peru with the code N° 33425.

Preparation of the extract
The flowers were washed and dried in the environment, they were homogenized to a fine powder and then stored in an amber glass container. Two hundred grams of fine powder and ethyl alcohol 70 °GL were used for the preparation of fluid extract using leaching equipment, macerating for 72 hours. After the maceration period, 75% of the fluid extract was percolated at a constant rate of XX drops/min, storing the extract in an amber colored bottle. Percolation continued until the ferric trichloride test on an aliquot of the extract was negative. The extract was concentrated by rotary evaporation, obtaining 100 mL of fluid extract. Finally, it was filtered through a vacuum pump and dried in a stove at 40 °C. It was stored in an amber colored bottle. 18 Chemicals Sodium chloride and Silymarin were obtained from commercial pharmaceutical products, being Silymarin obtained from Genfar® and sodium chloride from Medifarma®. On the other hand, 37% reactive grade formaldehyde was from Merck® and chemically pure Paracetamol was from Sigma-Aldrich.

Animals
Rattus norvegicus var. albinus of both sexes (220 -250 g) of two and a half months of age were obtained from the Instituto Nacional de Salud (INS) -Sanitary Certificate N° 029-2019. All specimens were kept in standard plastic cages in the animal facility of the Facultad de Farmacia y Bioquímica, Universidad Nacional de Trujillo. The animals were conditioned in a standard environment of photoperiod (12:12 h dark: light cycle) and temperature [(25 ± 2) °C]. They were provided with water ad libitum and food purchased from the INS (6). This study was approved by the ethics committee of the Facultad de Farmacia y Bioquímica of the Universidad Nacional de Trujillo under opinion N° PR003-018 / CEIFYB.

Hepatoprotective evaluation
The rats were divided into six groups of six rats in each group. Group I (normal rat) received only the vehicle (sterile water 10 mL / Kg of body weight p.o.) for 5 days. Group II (Paracetamol) received paracetamol (1000 mg/kg, oral, positive control) for 5 days. Group III (Paracetamol + Silymarin) rats with paracetamol-induced hepatotoxicity treated with silymarin (200 mg/Kg, oral). Group IV (C. lutea 200) rats with paracetamol-induced hepatotoxicity treated with C. lutea 200 mg/Kg. Group V (C. lutea 400) rats with paracetamol-induced hepatotoxicity treated with C. lutea 400 mg/Kg. Group VI (C. lutea 600) rats with paracetamol-induced hepatotoxicity treated with C. lutea 600 mg/ Kg. The animals were sacrificed 48 h after the last administration of paracetamol under mild anesthesia with Ketamine 10%. Blood and liver were collected from each animal. 6,19 Estimation of biochemical parameters The biochemical parameters were determined with the separated serum. Levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), direct bilirubin, indirect bilirubin, total bilirubin, globulin and total proteins were determined. The absorbance of all parameters was measured in a Persee T7 UV-VIS spectrophotometer.

Statistical analysis
The data obtained from the animal experiment are expressed as mean ± SD. The graphs were prepared using Microsoft Excel® and the data were subjected to an analysis of variance (ANOVA) followed by the post-Hoc Tukey test. To evaluate individual variations between the control and treatment groups, p<0.05 was considered statistically significant.

Serum biochemical analysis
The comparison of the biochemical parameters is shown in Figure 1. The levels of ALT, AST, ALP, albumin and total protein in Group II show significant alteration compared to Group I.
The groups that received treatment with C. lutea significantly decreased ALT and AST levels, only Groups V and VI (400 and 600 mg of extract / Kg, respectively) significantly decreased ALP, this difference was also significant with Group III (Silymarin). There was no significant difference with Group I (normal rat).
Although there is a difference in bilirubin levels, they are not statistically significant (p> 0.05). The groups that received C. lutea extract significantly increased the levels of albumin and total proteins compared to Group II (rat paracetamol-induced hepatotoxicity), besides, they did not show significant difference with Group I (normal rat) or Group III (Silymarin).

Histopathological changes
Liver lesions decreased with the administration of C. lutea extract ( Figure 2) compared to the other groups. Group I (Normal rat -Section A) shows that, radially to the central vein (VC), flow cords or hepatocyte plates (inset), including the dilated sinusoids (*) indicative of the activity; the contour and shape of hepatocytes with stained nuclei and cytoplasm (arrows) characteristic of normal tissue are maintained. Group II (Negative control -Section B) shows the presence of several hepatocytes in a degenerative state and cell death or necrosis, some of a powdery and swollen appearance (arrows), the presence of Kupffer cells (ck) and some pyknotic nuclei; change attributed to paracetamolinduced hepatotoxicity. Group III (Treatment with Silymarin -Section C) shows that most hepatocytes show normal architecture (arrows), some swollen (*) some necrotic hepatocytes (n+); the image describes slight regeneration attributable to the hepatoprotective effect of silymarin. In Group IV (Treatment with C. lutea 200 mg/Kg -Section D) many hepatocytes show a normal nucleus, nucleolus and cytoplasm, some in necrosis (arrows) and others with fat droplets that displace the nucleus towards the periphery (*) effects attributable to paracetamol; by the recovery of hepatocytes, it is deduced that C. lutea in low dose has a slight effect in the treatment of toxic conditions. Group V (Treatment with C. lutea 400 mg/Kg -Section E) shows most of the normalappearing hepatocytes arranged in a radial arrangement (inset) to the central vein (VC), few hepatocytes in necrosis (arrows) and some in pyknosis (*), it is generally appreciated that C. lutea at a dose of 400mg/ kg has a hepatoprotective effect. Group VI (Treatment with C. lutea 600 mg/Kg -Section F) shows regeneration of hepatocytes arranged around the central vein (CV), minimal residual hepatotoxic effect by paracetamol (arrows) and dilation of sinusoids (*).

DISCUSSION
C. lutea extract shows hepatoprotective activity against paracetamol. Paracetamol is an antipyretic agent that is safe at therapeutic doses but can produce hepatotoxicity in humans and rats if the dose is increased 20,21 ; therefore, multiple investigations have used paracetamol to search for new agents with hepatoprotective activity. 3,[22][23][24] Paracetamol administered to rats increases the levels of AST, ALT, ALP and all bilirubin (direct, indirect and total), as well as decreases the levels of albumin, globulin and total proteins. 22,25 Analysis of these values, as well as histopathological analysis, are indicative of hepatic necrosis caused by paracetamol. 20,23 The alteration of these values, as well as the damage in the liver tissue, decreased with the pretreatment of the C. lutea extract, being the lowest concentration dose used (200 mg/Kg) comparable in effect to silymarin, a substance that has already shown hepatoprotective activity. 23 The 600 mg/Kg dose shows a better recovery in liver tissue, however, it does not show a significant decrease in ALT, AST and ALP values compared to the 400 mg/Kg dose (p <0.05), likewise, the increase in total proteins was greater in the 400 mg/Kg dose and, unlike the 600 mg/Kg dose, the globulin increase was significant compared to the negative control (p <0.05). Besides, although there are no statistically significant differences, the decrease in bilirubin was greater at the dose of 400 mg/Kg. Although C. lutea is a safe plant, the lower protective effect of 600 mg/ kg might be due to pro-oxidant activity of the extract in the higher dose. That is because the hepatoprotective effect of this plant, at least in part, is due to its antioxidant activity, and antioxidants in some conditions, especially in high doses, might act as pro-oxidants. 10,26 C. lutea is a plant with a great hepatoprotective potential. Although not all its bioactive metabolites have been identified, the presence of rutin has been determined 17 , substance that has been shown to have hepatoprotective activity. 27 However, this activity may be enhanced by the presence of other bioactive metabolites that have probably not yet been identified, since some other compounds such as quercetin and curcumin have already shown this type of pharmacological activity. 23,25 In the most important databases, the information on its activity is very limited, finding only two investigations linked to Peruvian botanical remedies that contain extracts of this plant species 10,11 , however, the activity corresponds to the mixture of plants and not to a specific species; and although there are undergraduate thesis in Peruvian universities that refer to about the activity of this plant species, they are not conclusive. Therefore, the limited evidence on the hepatoprotective activity of this plant makes it necessary to continue research on its hepatoprotective activity and the bioactive metabolites responsible for this activity.