Research Article

Histopathological Evaluation of Infected Wistar Albino Rat Treated with Phyllanthus niruri L.  

Osho I.B.1 , Oyekanmi B.A.2 , Adetuyi F.C.3 , Oladele A.A.4
1 Department of Animal Production and Health, School of Agriculture and Agricultural Technology, Federal University of Technology, Akure, Nigeria
2 Department of Haematology, College of Health Sciences, Osun State University, Osun State, Nigeria
3 Department of Microbiology, School of sciences, Federal University of Technology, Akure, Nigeria
4 Department of Morbid Anatomy, Obafemi Awolowo University Teaching Hospital Complex, Osun State, Nigeria
Author    Correspondence author
International Journal of Molecular Veterinary Research, 2015, Vol. 5, No. 3   doi: 10.5376/ijmvr.2015.05.0003
Received: 09 Dec., 2015    Accepted: 03 Feb., 2016    Published: 20 Mar., 2016
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This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Osho I.B., Oyekanmi B.A., Adetuyi F.C. and Oladele A.A., 2016, HISTOPATHOLOGICAL EVALUATION OF INFECTED WISTAR ALBINO RAT TREATED WITH Phyllanthus niruri L, International Journal of Molecular Veterinary Research, Vol.5, No.03 1-8 (doi: 10.5376/ijmvr.2015.06.0004)

Abstract

Phyllanthus had been reported to have pharmacological effects such as antibacterial, antiviral activity against hepatitis. P. amarus has been used as chemoprotective agent and antimutagenic. Several active components have been identified in Phyllanthus, some of which are phyllantin, flavonoid, alkaloids, tannins, terpens, and sterols. Phyllanthus niruri L. (Euphorbiaceae) is a well-known hepatoprotective herbal plant. Due to the worldwide usage of Phyllanthus, this study focuses on the toxicology effect of the species in our environment in order to assess the biosafety of the plant. 

 

Apparently healthy Wistar albino rats were given 0.3 ml oral administration of Escherichia coli (ATCC 2522) of standard inoculum. The infected animals experienced reduction in the average body weight gain after three days inoculation, which increased drastically after a week and two weeks treatment, for positive and test animals respectively. At the end of the treatment, negative control and positive control have 6.37% and 15.21% average body weight gain respectively. In the test animals the least percentage weight gain was 10.83% (1200 mg/kgbw) and the highest percentage weight gain was 26.22% (300 mg/kgbw). The hyperchromasia of the liver tissue and vacuolations of the podocytes is suggestive of potential toxic properties of P. niruri L. in chronic administration.

Keywords
Phyllanthus niruri; In vivo; Chemoprotective; Antibacterial; Histopathology

1 Introduction
The increase in resistant strains of micro-organisms encouraged scientists to search for new antimicrobial substances from various sources including medicinal plants. The medicinal plants are widely used by the traditional medical practitioners for treating various diseases in day to day practice. Phyllanthus niruri Linn is one of the medicinal plants traditionally used to treat malaria in India. Phyllanthus niruri L. has been used traditionally to treat various illnesses, including renal stones, gastrointestinal disturbances, cough, hepatitis, gonorrhea, fever and malaria (Tona et al., 2001) In traditional medicine. Phyllanthus niruri L. is used for kidney stones and gallstones (active stones and as a preventive measures); to tone, balance, strengthen, detoxify and protect the kidneys and to reduce uric acid and increase urination; and protect the liver (Thyagarajan et al., 2002). Different species of Phyllanthus are considered to be very effective hepatoprotective agents in the Indian indigenous systems of medicine and are considered bitter, astringent, stomachic, diuretic, febrifuge, deobstruant and antiseptic (Khatoon et al., 2006). Ayurvedic practitioners prescribed fresh juice of 'Bhuiamlki' for jaundice. Various species of Phyllanthus are being sold in India under the trade name 'Bhuiamlki'. During market surveillance of herbal drug, it was observed that almost all the commercial samples, either comprise of Phyllanthus amarus Schum and Thonn. or Phyllanthus maderaspatensis Linn. or mixture of Phyllanthus amarus, Phyllanthus fraternus and Phyllanthus maderaspatensis. (Khatoon et al., 2006).


In Togo and Nigeria Phyllanthus is found easily during the rainy season and is consumed by the population as preventive measures in Togo against all kind of diseases. P. amarus harvested in Togo are rich in tannins and phenols but poor in alkaloids (Polya et al., 1995; Houghton et al., 1996). In Nigeria it is used in home-treatment of illnesses including typhoid fever, malaria fever, stomach troubles, kidney stones and jaundice. In modern medicine P. niruri L. had been shown to cure or treat multiple disorders (Joseph and Raj, 2011) As a result of its wide application in ayurvedic medicine there is the need to assess its effect on internal organs like liver and kidney.


2 Matericls and Methods
2.1 Plant collection and identification
Fresh and apparently healthy whole plants were harvested from Osun state University environment, main campus, Osogbo. The plant was identified and authenticated at the Botany Department, Ife Herbarium, Obafemi Awolowo University, Ile-Ife, Osun state, Nigeria. The plant was adequately washed with sterile water and dried at room temperature. The dry plant was pulverized with a clean and dry grinding machine to obtain smooth powder.


2.2 Preparation of plant extracts
The plant were washed with sterile water, air dried at room temperature and pulverized into fine powder. One hundred grams (100 g) of the pulverized powder was soaked in 1 litre 98% ethanol for 48 h. It was filtered through 3-layered muslin cloth, sterilized using membrane filter and concentrated in vacuo in a rotary evaporator, and stored at 4ºC until use.


2.3 Test organism
Escherichia coli (ATCC 2522).


2.4 Preparation of inoculums
The isolate was obtained on agar slant. The turbidity of the actively growing broth culture was adjusted with sterile distilled water to obtain turbidity optically comparable to that of the 0.5 Mc Farland standards. This resulted in a suspension colony approximately 106 cfu/ml (Barenfangar et al., 1999).


2.5 Animals
Male and female Wistar albino rats weighing 83-105 g were obtained from the animal house of the College of Medicine, Obafemi Awolowo University, Ile-Ife. Animals were housed under standard environmental conditions. Plastic cages were used for their nesting and allowed to acclimate for 5 days. They were fed with standard pellet diet and water under a 12 h light/dark cycle. Feed and water were administered once per day. Certified Wistar albino rats were divided into 6 groups of five: Group 1 was the negative control group that received only feed and clean drinking water while group 6 was the positive control group that was infected and treated with standard drug. Group 2-5 were the test group, which were infected and treated with graded doses of the extract.


2.6 Infectivity dose
Their weights were measured before inoculation with bacterial suspension, three days after inoculation and after weekly treatment. The rats were subjected to oral administration of 0.3 ml of Escherichia coli (ATCC 2522) suspension with the use of canulla. This was administered to each rat in group 2 to 6. The animals were observed for signs of infection and their stool samples cultured to establish infection. The animals were allowed for 3 days for incubation of the bacteria which is in support of signs of infection and the incubation period for Escherichia coli.


2.7 Stool culture of wistar albino rat
Stool samples of animals were collected after 3 days exposures and cultured on Mac Conkey agar and Eosin Methylene Blue to establish Escherichia coli (ATCC 2522) infection in the infected groups. Subsequently the test was repeated weekly.


The stool sample was inoculated into Selenite–F and incubated at 37ºC for 24 h. It was subculture on McConkey agar and Eosin Methylene Blue agar, incubated for 24 h and observed for growth.


2.8 Administration of Antibacterial
The infected rats were treated with the extract according to their body weight, for 2 weeks (Muto et al., 2003). Studies showed that LD50 of ethanolic extract of P. niruri is greater than 5 kg per body weight (Lawson-Evi et al., 2008). Ethanolic extract of Phyllanthus niruri L. graded doses of 300, 600, 1200 and 1500 g per kilogram body weight was administered to group 2-5 respectively (Laboratory manual for pharmacological evaluation of natural products by United States). Ciprofloxacin tablet was purchased from a registered pharmaceutical shop. The concentration was administered according to body weight as it is presented in clinical precision for man of an average body weight. A daily single dose of 1 g per 70 kg body weight contained in 1 ml was administered to each. Each rat in the positive control group was treated with 0.02 mg/ml daily for 7 days.


2.9 Organ harvest
The animals were starved for 6 h and sacrificed by cervical dislocation method. The kidney and the liver were obtained and preserved in 10% formaldehyde buffered saline for histological examination.


3 Results
In vivo testing was conducted on Wistar albino rat. Their weights were taken before and after oral inoculation with E. coli 2522 suspension; after 7 days and 14 days treatment. The average body weight of the negative control increased gradually from day 3 to day 9; weight slightly reduced on day 16 and a sharp increase on day 23 (Table 1). Positive control showed appreciable increase between day 3 to day 6; slight reduction in weight till day 16 and sharp increase on day 23 (Table 1). Group 2 showed gradual increase in the body weight from day 3 till the end of the experiment (Table 1). Group 3 demonstrated increase in the body weight from day 3 till day 23, but a slight drop in the body weight on day 9 (Table 1). Group 4 showed slightly reduced body weight between day 3 and day 6; mild increase on day 9; slightly reduced weight on day 16 and sharp increase in the body weight on day 23 (Table 1). Group 5 demonstrated slight increase in the body weight between day 3 and day 6; mildly reduced body weight on day 9; appreciable increase on day 16 till day 23 (Table 1).

 

 
Table 1 Effect of ethanolic extract of P. niruri L. on the average body weight of E. coli (ATCC 2522) infected wistar albino rat

 
Average weight gain is the difference between the final average weight and initial average weight (after 2 weeks treatment and before treatment was administered, respectively). This was observed and the percentage weight gain determined. The highest average weight gain was observed in group 2 (17.48±1.11b g), followed by group 5 (14.20±6.07ab g), then group 3 (12.86±5.58ab g), followed by the positive control (10.14±3.40ab g), group 4 (7.22±3.65ab), then negative control (4.22±2.01a) in descending order (Table 2). Statistically there was no significant difference in the average weight gain after treatment at p≤0.05. Although the percentage weight gain was considerably high in group 2 and the least percentage was demonstrated in the negative control (Table 2).

 

 
Table 2 Average and percentage weight gain of albino rat after treatment with ethanolic extract of P. niruri Linn

 
The stool culture of negative control rat showed scanty growth throught the experiment while the positive control showed heavy growth on day 9, but scanty growth on day 16 and day 23 (Table 3). Group 2 and 3 showed heavy growth on day 9, moderate growth on day16 and few growths on day 23. Group 4 showed heavy growth on day 9, moderate growth on day16 and scanty growth on day 23 (Table 3). Group 5 showed heavy growth on day 9 and few growth on day16 and day 23 (Table 3).

 

 
Table 3 Effect of Escherichia coli (ATCC 2522) on the stool of Wistar albino rat

 
Photomicrograph of the liver samples from the negative control rats stained with Haematoxilin and Eosin (H&E) showed normal architecture with the basic structural arrangement of the hepatocytes in close proximity with the sinusoids (Plate 1). The positive control liver showed almost normal tissue with scanty polymorphonuclear cells (plate 6). The Phyllanthus niruri Linn treated livers showed hyperchromatic liver tissue and some polymorphonuclear cells around the arterioles (Plate 2); minor leucocyte infiltration (Plate 3); liver tissue with clearly demonstrated central arteriole (Plate 4); hyperchromatic liver tissue infiltrated with polymorphonuclear cells (Plate 5), group 2, 3, 4, and 5 livers respectively (Figure 1).

 

 
Figure 1 Key: P-plate
Plate 1: Normal control liver. Section, showing central arteriole (CA) clearly demonstrated, sinusoids (SS) and hepatocytes (arrows). H&E X40
Plate 2: Liver treated with 300 mg/kg body weight extract of P. niruri L. Section shows hyperchromatic liver tissue and polymorponuclear cells (arrows) seen around the arterioles. H&E X40
Plate 3: Liver treated with 600 mg/kg body weight extract of P. niruri L., arrows show minor leucocyte infiltration
Plate 4: Liver treated with 1200 mg/kg body weight extract of P. niruri L. showing hepatocytes (arrows) and a well demonstrated central arteriole (C)
Plate 5: Photomicrograph of liver treated with 1500 mg/kg body weight extract of P. niruri. Section shows hyperchromatic liver tissue infiltrated with polymorphonuclear cells (arrows). H&E (X40)
Plate 6: Liver treated with ciprofloxacin. Almost normal tissue with arrows showing scanty polymorphonuclear cells infiltration. H&E (X40)

 
Histological sections of the kidney showed negative control kidney tissue with some glomerulus well preserved (Plate 7) and positive control kidney (kidney treated with ciprofloxacin) showed a distorted kidney tissue with numerous polymorphonuclear cells (Plate 12). Sections of kidneys treated with P. niruri L. showed leucocytes infiltration in the kidney tissue (Plate 8) and (Plate 9); almost normal kidney and few polymorphonuclear cells infiltration (Plate 10); vacuolation seen among the podocytes (Plate 11), group 2, 3, 4, and 5 kidneys respectively (Figure 2).

 

 
Figure 2 Key: P-plate
Plate 7: Normal control kidney section shows normal kidney tissue, arrows showing some glomerulus well preserved and convoluted tubules (CT). H&E (X40)
Plate 8: Photomicrograph of kidney treated with 300mg/kg body weight extract of P. nirur L., Section showing leucocyte infiltration (LI) in the kidney tissue. H&E (X40)
Plate 9: Photomicrograph of kidney treated with 600 mg/kg body weight extract of P. niruri L., tissue shows some leucocytes infiltration (LI). H&E (X40)
Plate 10: Photomicrograph of kidney treated with 1200 mg/kg body weight extract of P. niruri L., almost normal kidney tissue, arrows showing few polymorphonuclear cells infiltration. H&E (X40)
Plate 11: Photomicrograph of kidney treated with 1500 mg/kg body weight extract of P. niruri L., arrows showing vacuolation among the podocytes, polymorphonuclear cells infiltration (PI) seen. H&E (X40)
Plate 12: Photomicrograph of kidney treated with ciprofloxacin shows a distorted kidney tissue, arrows showing numerous polymorphonuclear cells. H&E (X40)

 
4 Discussion

The initial reduction in the average body weight was due to the Escherichia coli (E. coli) (ATCC 2522) infection in the rats. A prominent symptom of E. coli infection is frequent passage of stool which often times leads to a drop in the body weight. The improvement in the weight of the Wistar albino rats after treatment indicates improved rate of food consumption and increased absorption rate. This indicates increased feed conversion efficiency (FCE/FCR) which is the ability to turn feed mass to body mass. This development could be the result of recovery from E. coli (ATCC 2522) infection arising from restoration of the physiological activities of the body system. The improvement in the health status of the rats could also be traced to the fact that chemical and biochemical constituents of plants which include carbohydrates, vitamins, glycosides, saponins, protein, alkaloid, fats and oil and mineral elements such as potassium, iron and calcium are able to supply the body tissue with energy and replace worn out tissues. However, the reduction in the weight gain as the concentration intake of P. niruri L. increased is suggestive that chronic administration of the plant extract has potential toxic effect. Adejare et al., (2011) report’s that chronic administration of P. amarus at high doses has potential adverse histological effect in stomach and duodenum digestive organ which agrees with this study.


The liver detoxicates many metabolic products, hormones, drugs and toxins, often prior to their excretion in the kidney. In the present study, histological examination of the liver indicated some morphological variations from normal: polymorphonuclear cells infiltration and hyperchromasia of the liver tissue. Also, the kidneys tissues varied from normal with few polymorphonuclear cells infiltration and some vacuolations. Liver being the primary site of biotransformation and detoxification and the kidneys the principal organ for their excretion are prone to the toxic effect of any toxic substance taken and their metabolites. Polymorphonuclear cells infiltration is indicative of inflammatory reaction which caused the immune response elicited against the invaders in order to eliminate the invading organism and protect the body. The hyperchromasia of the liver tissue and vacuolations of the podocytes is suggestive of potential toxic properties of P. niruri L. in chronic administration.


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