Bidens pilosa L. (Asteraceae) cultivated in Brazil on acute liver disease in dogs

[Bidens pilosa L. (Asteraceae) cultivada no Brasil na doença hepática aguda em cães]

ABSTRACT

Bidens pilosa L. is a medicinal plant popularly used for treatment of liver diseases. In this study, the dry extract of aerial parts of Bidens pilosa and Silymarin, a phytocomplex obtained from the Silybum marianum fruits and marketed as hepatoprotective, were tested in dogs experimentally acutely intoxicated with carbon tetrachloride. The liver activity was evaluated by hematological and biochemical profiles, and histological and ultrasound analyzes. It was observed that the lowest serum activities of ALT and serum concentrations of total bilirubin occurred in the groups treated with the dry extract of Bidens pilosa, while only decreased serum concentrations of total bilirubin occurred in the group treated with Silymarin. Best liver recovery was also observed for the dry extract of B. pilosa at a 400mg/Kg dose by ultrasonography. This study showed that the dry extract of Bidens pilosa acted more efficiently in the treatment of acute toxic hepatitis induced in dogs than Silymarin.

Keywords:
carbon tetrachloride; liver diseases; Bidens Pilosa

RESUMO

Bidens pilosa L. é uma planta medicinal utilizada popularmente para tratamento de doenças hepáticas. Neste trabalho, o extrato seco das partes aéreas da Bidens pilosa e a silimarina, um fitocomplexo obtido dos frutos da Silybum marianum e comercializado como hepatoprotetor, foram testados em cães intoxicados experimentalmente de forma aguda com tetracloreto de carbono. A atividade hepática foi avaliada por meio dos perfis hematológico e bioquímico, análises histológica e ultrassonográfica. Observou-se que, nos grupos tratados com o extrato seco da Bidens pilosa, ocorreram as menores atividades séricas da ALT e de concentrações séricas de bilirrubina total, enquanto no grupo tratado com silimarina, ocorreu apenas diminuição de concentrações séricas de bilirrubina total. Melhor recuperação hepática também foi verificada para o extrato seco de B. pilosa na dose de 400mg/kg por ultrassonografia. Este estudo evidenciou que o extrato seco da Bidens pilosa atuou de forma mais eficiente no tratamento da hepatite aguda tóxica induzida em cães do que a silimarina.

Palavras-chave:
tetracloreto de carbono; doença hepática; picão

INTRODUCTION

Bidens pilosa L. belongs to the Asteraceae family originated from South America, is popularly known in Brazil as picão, picão preto, marcela-do-campo and carrapicho de agulha and in the USA as black-jack, beggar-ticks, cobbler's pegs and Spanish needle, and grows spontaneously in agricultural crops in all regions of Brazil. This is a cosmopolitan species and occurs in tropical and subtropical regions of the Americas and Asia (Lorenzi and Matos, 2008; Alonso, 2016). Flavonoids and polyacetylene compounds outstand among the chemical components of the aerial parts of B. pilosa. Phenylheptatriine, glycopyranosyl-OH-tetradecene and glycopyranosyloxy-OH-tridecene, quercetin 3-O-beta-D-glycoside, nicotinic acid, tannic acid, hydrocarbons with 28-30 carbons, among others have already been identified in this plant (Alonso, 2016; Silva et al., 2011). Some of the therapeutic activities associated with Bidens pilosa are antioxidative, immunomodulatory, anti-inflammatory and anti-ulcerogenic (Bartolome et al., 2013; Santos Filho et al., 2018). In Brazil, this species is included in the National List of Medicinal Plants of Interest to the Unified Health System - RENISUS, which aims to guide research and studies. There is even a specific monograph on Bidens pilosa prepared by the Ministry of Health (Brasil, 2015).

The aqueous extract of B. pilosa has been used in case of liver injuries produced by acetaminophen and carbon tetrachloride in rats and decreased the injury areas significantly (Chin et al., 1996), also acting in cases of liver fibrosis in mice (Yuan et al., 2008). It has also showed antioxidant activity (Yang et al., 2006; Krishnaiah et al., 2011), what could contribute to the treatment of liver diseases.

Another plant used in liver diseases of different etiologies is Silybum marianum (L.) Gaertn (Asteraceae). A phytocomplex commercially known as Silymarin is extracted from the ripe and dry fruit of S. marianum. This phytocomplex features seven flavonolignans and polyphenols in its composition, and silybin is its main component (Alonso, 2016). In addition to its action in liver diseases, studies report that Silymarin shows antioxidant cytoprotective activity (Asghar and Masood, 2008) mediated by the transcription factor NFkB (Tsai et al., 2010), and anti-inflammatory and anticancer activity for suppressing the tumor necrosis factor (TNF) (Shulze-Osthoff and Haussinger, 2007; El-Kamary et al., 2009). It is assumed that this phytocomplex is safe and effective in the improvement of symptoms in cases of acute hepatitis and in patients with alcoholic cirrhosis (Basu, 2003).

On the experimental evaluation of liver activity, carbon tetrachloride (CCl₄) has been used to induce liver injury in different experimental models, because this compound produces oxidative stress, with fatty acid and membrane phospholipid peroxidation, causing complete destruction of cell and intracellular membranes (Basu, 2003; Chen et al., 2005). The activity of a substance in the presence of liver injury induced by CCI₄ may be evaluated by determining the serum activity of alanine aminotransferase (ALT), alkaline phosphatase (ALP) and gamma-glutamyltransferase (GGT). In addition to these enzymes, other substances are correlated with the structural and functional integrity of the organ and may be analyzed, as the serum concentrations of albumin and urea (Chen et al., 2005). Thus, in this study, the liver activity of standardized dry extract of aerial parts of Bidens pilosa and Silymarin phytocomplex have been evaluated in dogs acutely intoxicated by oral administration of carbon tetrachloride.

MATERIAL AND METHODS

The experimental protocol was approved by the Animal Research Ethics Committee of the Universidade Federal de Goiás (process 164/2009). Aerial parts of Bidens pilosa L., in the form of plant drug, were acquired from Empresa Santa Efigênia (Goiânia, GO, Brazil). The morphological characteristics of this sample were analyzed considering the descriptions reported in the literature (Alonso, 2016; Lorenzi and Matos, 2008) and by comparison with a standard sample of Bidens pilosa L. deposited in the Herbarium of the UFG, under nr. 43835.

The aerial parts of B. pilosa were grounded in a knife mill (Wiley type), and the powder was submitted to percolation extraction using hydroalcoholic solution. The liquid extract was concentrated in a rotary evaporator at a temperature of 40°C, up to about 40% of total solids, becoming the ethanolic extract of Bidens pilosa (EEB) and characterized regarding pH, relative density, viscosity, alcohol content, solids content and total flavonoid content. The total flavonoid content was quantified by spectrophotometry and calculated as rutin, according to Rollim et al. (2005). The EEB was dried by nebulization/atomization, in spray drying, and was deposited in the Bank of Patents at the National Institute of Intellectual Property (BR10-2012 0076675).

The Silymarin phytocomplex, in the form of dry extract, was provided by a magisterial pharmacy of Goiânia, GO, Brazil. This pharmacy also provided the fractionation and encapsulation of the Silymarin phytocomplex and EEB, according to the drug dose and weight of the dogs. Twenty-four adult mongrel male dogs from the Zoonosis Control Center of the city of Goiânia, with an average weight of 15 ± 6.2Kg were used. At the end of the study, after confirming that the dogs were healthy, they were sent to foster homes. The animals were housed in individual pens and received balanced ration for adult dogs (Finotrato-VB rations) and water at ease, being monitored for a period of three months before the experiment.

For the administration of CCl₄, the animals were anesthetized with propophol (6mg/Kg/EV) followed by placement of tracheal tube for orogastric probing. Carbon tetrachloride PA (99%) was administered in a single dose of 2.5mL/Kg. The dogs were distributed randomly into four groups (n = 6), which had the following treatments by oral administration, after the induction of liver injury: group I - Control (400mg/Kg starch), group II treated with EEB (400mg/Kg), group III - treated with EEB (100mg/Kg) and group IV treated with Silymarin (30mg/Kg). Group I functioned as negative control and group IV as positive control. The treatments were performed once a day, at the same time, for a period of 21 days.

The animals were observed daily, throughout the experimental period, aiming at detecting episodes of vomiting, diarrhea, anorexia and any other change in the health conditions of the animals. For evaluating the enzymatic activity and other analytes, blood collections were conducted at the following times: before the intoxication, 24 hours after the intoxication induction and on days 2, 3, 5, 8, 11, 14, 19 and 21 after intoxication. The blood was collected by jugular venipuncture in a vacuum collection system (Vacutainer® - Becton Dickinson Ind. Cirúrgicas Ltda., Brazil).

The samples were processed in less than six hours as of the collection time. The counting of blood cells was conducted by an automatic method, using the BC-2800 vet device (Auto Hematology Analyzer, Mindray® Bio-Medical Electronics Co. Ltd., Shenzhen-Guangdong), adapted with a reading card proper to the canine species. For the differential counting of leukocyte, blood smears prepared with fresh blood and stained by the Rosenfeld technique were used.

For biochemical analyzes, standardized commercial reagents (Labtest® - Labtest Diagnóstica S.A., Lagoa Santa, MG, Brazil) were used, with kinetic, enzymatic and colorimetric methodologies, in a temperature of 37°C, and the reading was conducted in a semi-automatic spectrophotometer (Analisador Bioquímico Bio-Plus®). The reactions were performed according to the guidelines of the manufacturer. The evaluations included the determination of the serum activity of enzymes: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamyltransferase (GGT); quantification of total and direct bilirubin, total cholesterol, total protein, albumin, globulins, urea and creatinine.

Ultrasound monitoring was conducted in four evaluations throughout the experimental period, being: one before the intoxication, 48 hours after the intoxication, on the 7^th day and on the 21^st day. Abdominal images in real time were obtained with ultrasound equipment (My Lab™ 30 Vet, The Esaote Group, Genoa, Italy) coupled to a multifrequency microconvex transducer (5.0 - 7.5 MHz). The longitudinal, dorsal and transverse section plans were used to the liver parenchyma scan. The liver was evaluated regarding size, echogenicity, echotexture and presence of biliary sludge, according to the Nyland et al. (2004) definitions.

The liver biopsy guided by ultrasonography was conducted when the animal was properly anaesthetized with isoflurane after induction with propophol (6mg/Kg, EV), using Tru-Cut® needle (CardinalHealth). Two fragments were removed (by examination) from the left medial liver lobe, positioning the needle at the right of the linea alba, below the last rib, with an average inclination of 35° directed cranially. The liver fragments, collected by ultrasound-guided biopsy, were processed according to the histological routine and stained by hematoxylin and eosin (HE) in the Department of Pathology of the Escola de Veterinária e Zootecnia of the Universidade Federal de Goiás. The results were submitted to statistical treatment using the GraphPad InStat software (Version 3.05 for Windows). ANOVA and Tukey post-test were used for the analysis of parametric variables. The significance level was P<0.05 (Sampaio, 2002).

RESULTS

The total flavonoids content of the EEB, calculated as rutin, was 366.8mg/100g of extract. This value is in accordance with literature data for this plant species (Brazil, 2015). In the first two days after the acute intoxication with CCl₄ the animals of all groups showed clinical signs of vomiting and diarrhea. In the 100mg and 400mg EEB groups, vomiting ceased in the 3^rd and 4^th days after intoxication, respectively, while in the Silymarin and control groups vomiting remained until the 8^th day. Diarrhea episodes ceased in the 100mg and 400mg EEB groups on the 5^th day after intoxication and in the Silymarin and control groups it remained until the 18^th day. Anorexia was not observed in any of the groups evaluated. There was no death during the experimental stage.

No significant differences were observed (P>0.05) concerning the hematological parameters between the experimental groups. The evaluation of the liver disease marker enzymes indicated that, 24 hours after intoxication of the dogs, there was significant increase in the serum activity of ALT, AST and ALP enzymes compared to the basal level. However, in the 72-hour time, there was significant decrease (P=0.001) of the serum ALT activity only in the groups treated with EEB (100mg/Kg) and EEB (400mg/Kg), compared to the negative control group (Table 1). Still regarding the ALT, significant increase was observed in the group treated with Silymarin (P<0.001), in the 24-hour time, compared to the negative control group (Table 1).

During the 11-day period after the intoxication, decrease (P=0.008) of the plasma concentration of total bilirubin was observed in the EEB groups (400mg/Kg and 100mg/Kg) and Silymarin compared to the group treated with starch, negative control (Table 2). In the same period, considering the direct bilirubin, a significant difference (P=0.02) was observed in the EEB 400mg/Kg group compared to the negative control group (Table 2). Serum concentrations of urea and creatinine did not vary and remained within the physiological limits of the species. Regarding the size of the liver, hepatomegaly was observed in animals of all groups, 48 hours after intoxication. On the 7^th day, the animals treated with 400mg EEB already showed normal liver size. The animals of the other groups showed reduced liver size only on the 21^st day.

Forty-eight hours after intoxication, one of the animals of the EEB 100mg, 400mg EEB and control group showed diffuse hyperechogenicity of the liver parenchyma. On the 7^th day, this result was found in 50% of the 100mg EEB group and in 33.33% of the 400mg EEB and Silymarin groups. On the 21^st day, one of the animals of the 100mg EEB and 400mg EEB groups still showed diffuse hyperechogenicity. Regarding the echotexture, changes were seen on the 7^th day in 50% of the animals of the control group and in 33.33% of the other groups. On the 21^st day, this change was seen only in one dog of the Silymarin group.

In 48 hours, presence of biliary sludge was observed in 33.33% of the animals of the control and 400mg EEB groups, while in the 100mg EEB group this result was observed in 16.66%. On the 7^th day, there was presence of biliary sludge in 33.33% in the control and 100mg EEB groups and 66.66% in the 400mg EEB group. On the 21^st day, this change was observed in 50% of the animals in the 400mg EEB group and in 16.66% in the control group. In the animals of the Silymarin group, there was presence of sludge in 16.66% on the 7^th day and in 66.66% on the 21^st day.

Table 1
Mean values of ALT, AST, ALP and GGT of dogs (n=6) treated for 21 days with starch (400mg), dry extract of Bidens pilosa L. (Asteraceae) (EEB) (100 and 400mg/Kg) and Silymarin (30mg/Kg) after oral administration of CCL₄ (2.5mL/Kg), Goiânia, Goiás, Brazil

	ALT	BL*	24h	48h	72h	5d	8d	11d	14d	17d	21d
Control	Mean	38.33a	266.83b	419.50a	334.67a	236.67a	161.40a	130.10a	64.86a	64.20a	61.60a
	Std. Dev.	6.77	75.68	133.05	83.11	109.73	53.05	102.50	27.70	18.71	12.24
	CV	17.67	28.36	31.72	24.83	46.36	32.87	78.78	42.70	29.15	19.87
EEB 400mg/Kg	Mean	44.08a	267.33b	253.67a	138.33b	150.83 a	118.33a	74.17a	65.28a	60.50a	58.33a
	Std. Dev.	16.79	96.64	119.46	32.98	51.09	30.49	12.86	27.80	13.34	25.86
	CV	38.08	36.15	47.09	23.84	33.87	25.76	17.33	42.58	22.05	44.33
EEB 100mg/Kg	Mean	42.92a	276.58b	306.83a	167.33b	142.33 a	144.67a	144.92a	99.33a	86.33a	79.67a
	Std. Dev.	10.95	83.32	75.34	79.38	49.01	48.23	68.42	61.58	36.14	40.06
	CV	25.52	30.13	24.55	47.44	34.44	33.34	47.21	61.99	41.86	50.28
Silymarin	Mean	39.00a	434.17b	422.33b	224.33a	182.83a	159.17a	98.67a	71.50a	67.50a	61.50a
	Std. Dev.	10.88	142.67	70.66	89.76	41.83	52.93	40.44	34.41	20.95	18.60
	CV	27.89	32.86	16.73	40.01	22.88	33.25	40.98	48.13	31.04	30.24
	AST	BL	24h	48h	72h	5d	8d	11d	14d	17d	21d
Control	Mean	34.17a	253.50a	327.00a	207.92a	196.67a	132.00a	107.40a	53.00a	65.20a	47.60a
	Std. Dev.	13.35	106.53	89.92	65.03	122.53	53.70	26.93	13.75	35.80	7.09
	CV	39.08	42.02	27.50	31.28	62.30	40.68	25.08	25.94	54.91	14.90
EEB 400mg/Kg	Mean	36.75a	145.50a	202.17a	163.33a	111.07a	83.72a	72.00a	59.00a	53.58a	53.83a
	Std. Dev.	12.11	55.50	59.52	48.36	22.86	29.78	20.78	22.10	12.94	14.69
	CV	32.94	38.14	29.44	29.61	20.58	35.57	28.87	37.46	24.15	27.29
EEB 100mg/Kg	Mean	35.00a	148.67a	252.67a	155.00a	114.17a	77.83a	91.17a	78.00a	74.05a	49.00a
	Std. Dev.	15.95	66.43	77.15	47.08	32.52	27.68	37.71	24.86	25.79	18.09
	CV	45.57	44.68	30.54	30.38	28.49	35.56	41.37	31.87	34.83	36.92
Silymarin	Mean	31.92a	224.17a	254.50a	181.37a	114.67a	94.33a	83.92a	75.83a	58.25a	57.67a
	Std. Dev.	7.41	63.81	32.20	27.21	43.48	36.27	28.40	19.65	37.41	31.36
	CV	23.20	28.47	12.65	15.00	37.92	38.45	33.84	25.91	64.22	54.38
	ALP	BL	24h	48h	72h	5d	8d	11d	14d	17d	21d
Control	Mean	24.67a	216.17a	246.17a	172.00a	167.00a	99.00a	79.60a	66.80a	41.80a	37.80a
	Std. Dev.	6.98	118.67	151.16	123.91	115.31	56.86	64.46	44.21	25.16	30.58
	CV	28.28	54.90	61.41	72.04	69.05	57.43	80.98	66.19	60.20	80.90
EEB 400mg/Kg	Mean	27.17a	185.00a	174.83a	125.00a	99.00a	60.83a	60.17a	47.83a	36.33a	32.00a
	Std. Dev.	8.13	79.80	76.94	93.23	93.29	44.67	43.74	28.73	13.75	13.19
	CV	29.92	43.14	44.01	74.58	94.23	73.44	72.70	60.06	37.84	41.22
EEB 100mg/Kg	Mean	21.33a	174.17a	235.67a	134.00a	114.50a	67.17a	52.50a	36.67a	41.17a	28.83a
	Std. Dev.	4.14	46.47	102.21	39.92	49.07	38.50	17.95	7.99	16.93	8.54
	CV	19.42	26.68	43.37	29.79	42.85	57.32	34.20	21.80	41.12	29.63
Silymarin	Mean	28.92a	237.00a	176.17a	143.33a	95.50a	92.17a	62.67a	49.33a	42.00a	30.00a
	Std. Dev.	8.05	86.21	74.04	74.93	56.79	71.45	19.22	16.08	14.32	9.59
	CV	27.85	36.38	42.03	52.28	59.47	77.52	30.67	32.60	34.11	31.97
	GGT	BL	24h	48h	72h	5d	8d	11d	14d	17d	21d
Control	Mean	6.38a	11.90a	12.75a	8.50a	9.35a	6.12a	9.24a	6.12a	6.12b	8.16a
	Std. Dev.	0.00	4.16	2.79	4.16	3.84	2.28	4.29	2.28	2.28	2.79
	CV	0.00	34.99	21.91	48.99	41.06	37.27	46.40	37.27	37.27	34.23
EEB 400mg/Kg	Mean	5.95a	11.05a	11.90a	8.50a	7.65a	9.35a	6.80a	6.80a	6.80b	6.80a
	Std. Dev.	0.66	3.84	2.63	2.63	2.79	3.84	2.63	2.63	2.63	2.63
	CV	11.07	34.74	22.13	30.98	36.51	41.06	38.73	38.73	38.73	38.73
EEB 100mg/Kg	Mean	5.95’a	9.35a	11.05a	11.05a	9.35a	8.50a	7.65a	5.95a	5.95b	5.95a
	Std. Dev.	0.66	2.08	3.84	3.84	2.08	4.16	4.27	2.08	2.08	2.08
	CV	11.07	22.27	34.74	34.74	22.27	48.99	55.78	34.99	34.99	34.99
Silymarin	Mean	6.38a	8.50a	12.75a	11.90a	7.65a	9.35a	8.50a	9.35a	6.20a	8.50a
	Std. Dev.	1.40	4.16	2.79	2.63	2.79	3.84	2.63	2.08	0.00	4.16
	CV	21.91	48.99	21.91	22.13	36.51	41.06	30.98	22.27	0.00	48.99

Table 2
Mean values of direct bilirubin (directBB), total bilirubin (totalBB), total proteins (TP), globulin albumins and total cholesterol of dogs (n=6) treated for 21 days with starch (negative control), dry extract of Bidens pilosa L. (Asteraceae) (EEB) (100 and 400mg/Kg) and Silymarin (30mg/Kg) after oral administration of CCL₄ (2.5mL/Kg), Goiânia, Goiás, Brazil

	totalBB	BL*	24h	48h	72h	5d	8d	11d	14d	17d	21d
Control	Mean	0.72a	1.06a	1.08a	1.20a	1.27a	1.06a	1.18b	0.97a	1.02a	1.00a
	Std. Dev.	0.15	0.17	0.12	0.42	0.46	0.17	0.15	0.24	0.19	0.20
	CV	20.47	16.43	10.85	34.96	36.58	15.79	12.57	24.76	18.22	20.29
EEB 400mg/Kg	Mean	0.62a	1.04a	0.97a	0.94a	0.94a	0.86a	0.88a	0.83a	0.82a	0.87a
	Std. Dev.	0.04	0.10	0.16	0.20	0.12	0.12	0.23	0.20	0.21	0.24
	CV	6.58	9.48	16.91	21.53	12.84	14.31	26.23	24.17	25.44	28.26
EEB 100mg/Kg	Mean	0.73a	0.88a	0.89a	0.98a	0.80a	0.91a	0.90b	0.89a	0.87a	0.79a
	Std. Dev.	0.15	0.16	0.19	0.12	0.14	0.10	0.10	0.09	0.12	0.24
	CV	20.99	17.90	20.87	11.89	17.68	11.10	11.58	10.29	13.58	29.97
Silymarin	Mean	0.68a	1.07a	1.00a	0.99a	0.82a	0.94a	0.90b	0.82a	0.82a	0.80a
	Std. Dev.	0.17	0.18	0.15	0.08	0.28	0.18	0.17	0.18	0.16	0.14
	CV	25.44	16.53	14.52	8.28	34.12	18.81	18.59	22.54	19.62	16.93
	directBB	BL	24h	48h	72h	5d	8d	11d	14d	17d	21d
Control	Mean	0.19a	0.60a	0.36a	0.40a	0.30a	0.32a	0.38a	0.35a	0.39a	0.36a
	Std. Dev.	0.07	0.25	0.08	0.12	0.31	0.10	0.05	0.12	0.11	0.03
	CV	35.92	41.49	23.40	30.89	0.30	30.91	12.46	34.39	27.96	9.02
EEB 400mg/Kg	Mean	0.20a	0.37a	0.31a	0.35a	0.30a	0.27a	0.25b	0.32a	0.29a	0.31a
	Std. Dev.	0.07	0.09	0.03	0.07	0.05	0.06	0.07	0.10	0.11	0.08
	CV	37.52	24.33	8.71	21.25	17.79	23.66	29.75	32.27	36.69	25.88
EEB 100mg/Kg	Mean	0.17a	0.47a	0.33a	0.33a	0.32a	0.31a	0.32a	0.38a	0.39a	0.36a
	Std. Dev.	0.08	0.08	0.06	0.07	0.05	0.05	0.04	0.08	0.10	0.07
	CV	45.52	16.55	18.30	21.83	16.55	17.81	12.81	21.96	25.07	18.79
Silymarin	Mean	0.21a	0.43a	0.35a	0.34a	0.48a	0.33a	0.33a	0.33a	0.28a	0.28a
	Std. Dev.	0.07	0.19	0.05	0.09	0.24	0.29	0.08	0.06	0.05	0.05
	CV	35.21	44.59	15.55	27.02	50.74	88.63	23.39	17.60	19.16	16.75
	TP	BL	24h	48h	72h	5d	8d	11d	14d	17d	21d
Control	Mean	6.48a	7.22a	7.03a	7.43a	7.53a	7.74a	7.44a	7.38a	7.90a	7.50a
	Std. Dev.	0.48	0.92	0.68	0.85	0.86	0.53	0.44	1.00	0.62	0.72
	CV	7.46	12.71	9.63	11.47	11.47	6.87	5.90	13.50	7.85	9.66
EEB 400mg/Kg	Mean	6.75a	6.72a	6.42a	6.85a	7.22a	7.00a	7.08a	7.07a	7.23a	7.30a
	Std. Dev.	0.38	0.81	0.50	0.69	0.97	0.50	0.60	0.73	0.48	0.51
	CV	5.68	12.00	7.85	10.15	13.39	7.11	8.54	10.31	6.58	6.93
EEB 100mg/Kg	Mean	6.77a	7.03a	7.02a	7.18a	7.32a	7.38a	7.55a	7.52a	7.63a	8.03a
	Std. Dev.	0.49	0.94	1.02	0.66	0.81	1.02	0.55	0.89	1.06	0.46
	CV	7.28	13.33	14.55	9.13	11.08	13.80	7.24	11.82	13.93	5.71
Silymarin	Mean	6.15a	6.35a	6.37a	6.95a	7.37a	6.88a	6.90a	6.82a	6.97a	6.83a
	Std. Dev.	0.53	0.48	0.94	0.46	0.94	0.60	0.27	0.44	0.84	0.75
	CV	8.65	7.50	14.82	6.61	12.78	8.74	3.89	6.39	12.07	10.98
	Albumin	BL	24h	48h	72h	5d	8d	11d	14d	17d	21d
Control	Mean	2.88a	2.75a	3.23a	3.05a	2.68a	2.81a	2.99a	3.04a	2.98a	2.51a
	Std. Dev.	0.26	0.54	0.76	0.27	0.34	0.47	0.58	0.38	0.37	0.31
	CV	9.05	19.58	23.51	8.94	12.77	16.60	19.25	12.41	12.52	12.37
EEB 400mg/Kg	Mean	3.09a	2.82a	2.92a	3.36a	2.79a	3.05a	2.69a	2.77a	2.99a	2.98a
	Std. Dev.	0.30	0.46	0.71	0.56	0.18	0.32	0.39	0.51	0.36	0.25
	CV	9.73	16.26	24.44	16.55	6.45	10.66	14.52	18.41	12.12	8.56
EEB 100mg/Kg	Mean	3.00a	3.18a	3.46a	3.04a	3.16a	3.07a	2.68a	2.99a	2.97a	3.05a
	Std. Dev.	0.23	0.31	1.10	0.33	0.50	0.51	0.26	0.32	0.37	0.33
	CV	7.52	9.72	31.84	10.69	15.77	16.70	9.87	10.59	12.61	10.84
Silymarin	Mean	2.77a	2.65a	3.16a	2.90a	2.97a	2.94a	3.05a	3.22a	3.02a	2.86a
	Std. Dev.	0.25	0.40	1.44	0.34	0.38	0.23	0.49	0.35	0.48	0.23
	CV	8.88	15.28	45.64	11.60	12.76	7.66	16.09	10.84	16.04	8.20
	Globulins	BL	24h	48h	72h	5d	8d	11d	14d	17d	21d
Control	Mean	3.60a	4.46a	3.81a	4.39a	4.85a	4.11a	3.71a	3.62a	4.10a	4.16a
	Std. Dev.	0.52	1.41	0.90	0.86	1.08	2.05	1.94	1.87	2.18	2.22
	CV	14.39	31.56	23.61	19.62	22.27	49.94	52.30	51.82	53.19	53.39
CV	5.95	25.31	23.76	29.68	19.12	17.21	22.03	15.22	11.38	10.06
EEB 100mg/Kg	Mean	3.77a	3.86a	3.56a	4.14a	4.16a	4.31a	4.88a	4.53a	4.67a	4.98a
	Std. Dev.	0.39	0.71	1.72	0.71	1.09	0.72	0.57	1.06	1.23	0.66
	CV	10.39	18.47	48.35	17.15	26.28	16.65	11.63	23.33	26.38	13.18
Silymarin	Mean	3.38a	3.71a	3.21a	4.05a	4.40a	3.94a	3.85a	3.60a	3.95a	3.97a
	Std. Dev.	0.65	0.80	2.00	0.65	0.79	0.52	0.52	0.75	0.93	0.81
	CV	19.26	21.72	62.21	15.99	17.87	13.12	13.58	20.74	23.56	20.46
	Cholesterol	BL	24h	48h	72h	5d	8d	11d	14d	17d	21d
Control	Mean	170.25a	200.17a	147.17a	183.50a	208.67a	185.20a	223.80a	244.00a	248.00a	211.20a
	Std. Dev.	25.03	52.83	27.92	49.71	66.96	24.17	65.49	32.76	53.61	32.81
	CV	14.70	26.39	18.97	27.09	32.09	13.05	29.26	13.42	21.62	15.54
EEB 400mg/Kg	Mean	175.00a	176.83a	161.33a	195.00a	210.00a	206.00a	233.17a	217.67a	220.83a	196.00a
	Std. Dev.	32.78	59.17	31.95	13.51	84.10	56.59	46.01	46.31	64.66	39.31
	CV	18.73	33.46	19.80	6.93	40.05	27.47	19.73	21.27	29.28	20.06
EEB 100mg/Kg	Mean	186.67a	196.17a	175.67a	207.17a	244.17a	253.67a	221.33a	192.83a	213.00a	210.33a
	Std. Dev.	24.71	71.41	37.56	59.23	88.60	142.97	36.19	46.15	74.73	53.85
	CV	13.24	36.40	21.38	28.59	36.29	56.36	16.35	23.93	35.09	25.60
Silymarin	Mean	186.58a	168.17a	199.17a	196.67a	193.17a	157.50a	208.17a	257.00a	236.83a	203.00a
	Std. Dev.	36.15	49.29	69.68	56.82	68.10	62.45	52.88	54.71	45.76	71.62
	CV	19.37	29.31	34.99	28.89	35.26	39.65	25.40	21.29	19.32	35.28

• BL - Base Line. Equal letters in the same column do not differ between themselves (ANOVA and t. Test, P<0.05).

The histological analysis of samples collected 48 hours after intoxication revealed the presence of microvacuolar hydropic degeneration of hepatocytes and bile ducts and necrosis (Figure 1). Generally, necrosis was observed in isolated hepatocytes or in small groups, especially in areas close to the central vein. Other changes observed were: presence of lymphoplasmocytic and eosinophilic inflammatory infiltrate, congestion of blood vessels and atherosclerosis (Figure 1). On the 7^th day after the intoxication, the persistence of histological changes was observed. However, in the animals treated with EEB at a dose of 400mg/Kg, lower incidence of changes was observed, when compared to the other groups, however, significant differences (P>0.05) were not detected. On the 21^st day of analysis, the samples collected for histopathological analysis showed no significant changes, with adequate recovery of the liver tissue in all experimental groups.

Figure 1
Photomicrograph of a hydropic degeneration area of hepatocytes and necrosis in a liver of control group dog (A); microvacuolar degeneration and hepatocyte and blood vessel necrosis in the liver of a dog of the group treated with ethanolic extract of Bidens pilosa L. (Asteraceae) at a dose of 100 mg/kg (B); focal degeneration of the bile duct and inflammatory infiltrate near a bile duct in a dog of the group treated with silymarin (C); cholangitis in a dog treated with ethanolic extract of Bidens pilosa L. (Asteraceae) at a dose of 400 mg/kg (D), 48 hours after intoxication by CCl₄ in a single oral dose of 2.5mL/Kg. HE, 20X. Goiânia, Goiás, Brazil.

DISCUSSION

Overall, the clinical, biochemical, ultrasound and histological changes observed after the intoxication with carbon tetrachloride in the different experimental groups were similar and reversible, corroborating the results reported in the literature (Aguilera-Tejelo, 1988; Assy and Minuk, 1997) which used a similar methodology for the induction of toxic liver injuries in dogs. The use of CCl₄ as intermediate in chemical reactions was severely restricted due to its high toxicity. However, once it induces hepatotoxicity tissue, enzymatic and clinic changes, the CCl₄ has been used in experimental protocols to elucidate the mechanisms of acute and chronic hepatitis and their treatments (Weber et al., 2003).

Clinical signs observed in animals after the oral administration of CCL₄, although nonspecific, are consistent with the clinical condition of acute hepatotoxicity. According to Rogers (1986), anorexia, depression, vomiting and diarrhea, may be observed in dogs with hepatotoxicity by CCl₄. According to Center (1999), in acute liver failure, especially in those caused by xenobiotics, diarrhea could be observed, as noticed in some dogs of this study. Despite no significant difference was generally observed among the groups treated, a lower incidence of clinical signs was noticed in the groups treated with EEB at a dose of 100mg/Kg, followed by the animals that received EEB at a dose of 400mg/Kg and those treated with Silymarin. The animals of the control group showed a higher number of episodes of vomiting and diarrhea.

Specifically, the increased plasma concentration of ALT, an enzyme present in the cytosol, results from the membrane permeability, and consequently, indicates cell death (Center, 1999; Giannini et al., 2005; Grigorescu, 2006). The hepatotoxic effect of CCL₄ is related to cell death by necrosis and/or induction of apoptosis, which could justify the increased serum activity of ALT (Golstein and Kroemer, 2012). In this study, the lowest serum activities of ALT after intoxication were observed in the groups treated with Bidens pilosa extract, indicating that the extract showed hepatoprotective effect against lipid peroxidation, an important mechanism of protection against acute liver injury. This finding corroborates the study by Chin et al. (1996) conducted in rats, where the ethanolic extract of this plant species showed hepatoprotective effect in mice intoxicated acutely by CCl₄.

Silymarin acts in the prevention of liver injuries induced by different chemicals and toxins (Lieber et al., 2003; Shaker et al., 2011; Li et al., 2012). In this study, the Silymarin showed significant increased serum activity of ALT after acute intoxication in the group treated, indicating that this compound did not show hepatoprotective action in the first 24 hours after liver injury. On the other hand, a hepatoprotective effect of the Silymarin administered for six days after the hepatic injury was observed in other studies (Lieber et al., 2003; Shaker et al., 2011).

The dogs treated with Bidens pilosa extract and Silymarin showed the lowest serum concentrations of total bilirubin and, therefore, acted positively on the hepatocytes. A similar result for Silymarin was reported by Yadav et al. (2008) in a study developed in rats. Whereas direct bilirubin reflects the conjugation ability of the hepatocytes (Giannini et al., 2005), it could be observed that the Bidens pilosa extract reestablished the liver conjugation process more quickly, when compared to the Silymarin and control groups, at all times evaluated, after injury with carbon tetrachloride. No other studies were found on the effect of Bidens pilosa on serum concentration of bilirubin in dogs.

The ultrasound aspect of the liver of dogs before the intoxication was consistent with that of a healthy organ, whereas the description by Nyland et al. (2005), because it showed regular contour and size, hyperechoic or isoecogenic parenchyma in relation to the renal cortex, homogeneous echotexture and no signs of biliary sludge. Liver ultrasound changes were observed with higher frequency on the 7^th day after the intoxication, as well as histological changes, in all experimental groups. Sen et al. (2005) obtained similar results and reported that the dogs experimentally intoxicated by CCL₄ showed significant ultrasound changes on the 5^th day. At the end of the evaluation period, the liver size of most (70.83%) dogs returned to normal in all experimental groups, as described by other authors who induced the acute hepatotoxicity in dogs experimentally after a single dose of carbon tetrachloride (Sen et al., 2005).

On histological evaluation, necrosis in isolated hepatocytes or in small groups was observed, especially in areas close to the central vein. Other changes observed were: presence of lymphoplasmocytic and eosinophilic inflammatory infiltrate, congestion of blood vessels and atherosclerosis. These changes were observed uniformly when among the groups and are in accordance with those reported by Sen et al. (2005) in dogs intoxicated with CCL₄. On the 7^th day after the intoxication, the persistence of the histological changes described could be observed, with no significant statistical difference among the groups. Already on the 21^st day of analysis, the samples collected for the histopathological analysis showed no significant changes, with adequate recovery of liver tissue in all experimental groups. The result is also in accordance with those found by Sen et al. (2005).

CONCLUSION

The dry extract of Bidens pilosa proved to be more efficient than Silymarin in the treatment of acute toxic hepatitis induced in dogs.

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Publication Dates

• Publication in this collection
  14 Aug 2020
• Date of issue
  Jul-Aug 2020

History

• Received
  20 Mar 2019
• Accepted
  16 Mar 2020