Evaluation of hepatoprotective activity of aqueous extract of Phyllanthus fraternus in Wistar rats.
Plants were used during the age for cure and treatment of diseases since the start of mankind. Phytotherapy is the isolation of pure phytochemicals from the plant and plant extract to cure the diseases. Plants have been used to treat the many diseases such as liver diseases, heart problems, lead and mercury intoxication, and scarlet fever.  Only 1-2% of the global plants have been reasonably well investigated out of 250,000-500, 000 species of plants. Even today, the synthetic drugs are superior in their number than the natural ones, but still many synthetic drugs have been derived from plants and animals. 
The liver is the main organ for biotransformation and elimination. It is always capable with the task of detoxification of xenobiotics, environmental pollutants, and chemotherapeutic agents.  CCl4, paracetamol, D-galactosamine, alcohol, rifampicin, and thioacetamide produce hepatotoxicity through different mechanisms.  Therefore, these drugs are called hepatotoxic agents.
Phyllanthus fraternus belongs to Euphorbiaceae  family. It is frequently called as "Bhumyamlaki" (Nela vusiri). Various biological activities of this plant are hepatoprotective activity, anti-inflammatory activity, antinociceptive activity, antioxidant activity, antimicrobial activity, antifertility activity, antidiabetic activity, antiviral activity, antidote to snake bite, and wound healing activity. [6,7] These properties are speculated to the existence of phyllanthin, hypophyllanthin, nirphyllin, phyllnirurin, phyllanthol, phyllanthenol, rhamnopyranoside, phyllanthenone, lintetralin, astragalin, cymene, niranthin, nirtetralin, niruriside, phyllochrysine, and 4-methoxy-securinine.  Very based on the above medicinal properties, the present study was aimed to examine the hepatoprotective activity of the aqueous extract of P. fraternus (AEPF) against CCl4-induced hepatic damage in rats.
MATERIALS AND METHODS
This was a research article.
The present study was conducted at the Department of Pharmacology, Mamata Medical College, Khammam, Telangana, India.
Five groups of Wistar rats were selected from 30 healthy animals, with six rats in each group. Both male and female, weighing around 200 g, were preferred for the study. All animals were maintained at 27-29[degrees]C. 18 h before the experiment, food was limited to all animals, but free access to water was allowed. All the animals were hydrated orally with 10 ml/kg of 0.9% normal saline for 7 days.
* Group A (n = 6): Normal control (normal saline was administered orally daily for 20 days).
* Group B (n = 6): [CCL.sub.4] control (normal saline and [CCL.sub.4]).
* Group C (n = 6): Standard group (Liv-52 100 mg/kg (p.o) daily for 20 days).
* Group D (n = 6): Test Group-I (P. fraternus extract 250 mg/kg (p.o) daily for 20 days).
* Group E (n = 6): Test Group-II (P. fraternus extract 500 mg/kg (p.o) daily for 20 days).
[CCl.sub.4]: Olive oil was given intraperitonially on 2nd, 5th, and 8th days for all group of rats except Group A (normal control) and Group C.
Carbon tetrachloride (Molychem) and Tab. Liv 52 (The Himalaya Drug Company) were used in the study.
P. fraternus leaves were collected from local places near Khammam city and were authenticated by Professor in the Department of Pharmacology, S.V. Ayurvedic College, Tirupati. Extract was prepared using the Soxhlet extraction.  Phyllanthus leaves were shade dried at a room temperature for almost 1 week. These plant leaves were crushed in the form of fined powder using a mixer grinder. This fine leaf powder was then weighed and used for extraction using distilled water as a solvent. The solvent is evaporated then at the room temperature to get a reddish brownish-colored extract. This obtained extract was stored at a temperature of 2-8[degrees]C until further any other use.
Estimation of Biochemical Parameters
On the 21st day, mild ether was used for anesthetization of animals and cardiac puncture method was used for collection of blood samples. The samples were centrifuged and plasma was separated. Biochemical parameters such as liver functional test, cholesterol, and total proteins were measured using standard methods. 
Livers were isolated from the experimental animals, and they were used for histopathological examination. 
Experimental protocol was approved by the Institutional Animal Ethics Committee of Mamata Medical College.
Data were analyzed using SPSS 20.0 version. One-way ANOVA followed by Tukey's test was applied to observe the significance of difference between the groups. P < 0.05 was considered as statistically significant.
Group B is treated with toxic doses of carbon tetrachloride which had significantly elevated the values of the liver functional test, cholesterol and total proteins levels were reduced when compared to Group A. When treated with hepatotoxic dose of carbon tetrachloride followed by Liv-52 and different doses of P. fraternus (250 and 500 mg/kg po). It showed that significantly lowered values of the liver functional test, cholesterol, bilirubin direct, bilirubin total, and total protein levels were significantly increased (P < 0.05) when compared to CCl4 control group (Group B). As shown in Bardiagram [Figures 1 and 2].
The histopathological examination of Group A livers showed a normal arrangement of the hepatocytes, with clearly visible nuclei, central vein, and portal triad [Figure 3]. In Group B of all rats, hepatocytes show steatosis and cytoplasmic vacuolations with ballooning degeneration, and the midzonal region shows inflammatory infiltration and congestion of central vein [Figure 4]. In Groups C, D, and E, there was a marked reduction in sinusoidal congestion, cloudy swelling, and fatty change. The central veins and sinusoids appeared unremarkable (Figures 5-7).
Normal Control Ccl4 Control LIV-52 AEPF-250 AEPF-500 SGOT 135 314.4 141.6 212.8 172.8 SGPT 60.2 256.4 98 181.2 136 ALP 213.2 430 246.8 310.4 266.8 CHOLESTEROL 120.4 185.8 127.4 161.6 147.2 Figure 1: Effect of Phyllanthus fraternus on serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, alkaline phosphatase, and cholesterol in [CCl.sub.4]-induced hepatotoxicity in Wistar rats Note: Table made from bar graph. Normal Control Ccl4 Control LIV-52 AEPF-250 AEPF-500 BILIRUBIN 0.18 1.2 0.24 0.78 0.54 DIRECT BILIRUBIN 0.94 3.16 1.54 2.66 2.02 TOTAL TOTAL 9.88 6.08 8.58 7.06 7.42 PROTENS Figure 2: Effect of Phyllanthus fraternus on serum bilirubin and total proteins in [CCl.sub.4]-induced hepatotoxicity in Wistar rats. AEPF: Aqueous extract of P. fraternus Note: Table made from bar graph.
The study was aimed to explore the hepatoprotective activity of the AEPF in Wistar rats. Carbon tetrachloride-induced hepatic injury was used for the present study. It was most acceptable method for hepatoprotective activity of plant extracts.  We agree with the previous studies as we have observed in present study significantly reduce the values of the serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, alkaline phosphatase (ALP), cholesterol, bilirubin direct and bilirubin total and A significant increase in total protein levels in Group D and Group E when compared to CCl4 control group (Group B). [8,9] Which is an indication of repair of hepatic tissue and stabilization plasma membrane by plant extract.  ALP is an enzyme and it reflects the pathological amendment in biliary flow.  Increased ALP and bilirubin levels were significantly reduced in Groups D and E; it suggests the possibility of the test drug being able to stabilize biliary dysfunction in rat liver during hepatic injury. In this study, the protein level was reduced in Group B because CCl4 changes the structure of endoplasmic reticulum which leads to reduction in protein synthesis. This might be due to carbon tetrachloride metabolized into trichloromethyl free radical (CCl3) in endoplasmic reticulum, and it is a free radical, which combined with cellular lipids and proteins in the presence of oxygen to induce lipid peroxidation. [15-17] In Groups D and E, marked elevation of total protein levels was observed. Hence, it is evident that plant extracts can restore the normal values of total proteins by increasing the synthesis.  Our results support earlier studies as we have observed that P. fraternus provided significant protection against the toxic effect of CCl4 on the liver. Histological studies reveal the changes, which take place during the damage and recovery. The extract mediated recovery supports the hepatoprotective activity of the same.
The study results support that the P. fraternus has dose-dependent hepatoprotective activity. The study highlights the need of further detailed studies to recommend the implementation of AEPF in the management of liver diseases in general.
Our sincere thanks to Dr. G Sai Sailesh Kumar, Assistant Professor and Head of the Department (I/C), Department of Physiology, Vishnu Dental College, for his support and valuable suggestions.
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Naveen Alasyam (1), Naveen Pokala (2), Premendran John (3)
(1) Department of Pharmacology, Vishnu Dental College, Bhimavaram, Andhra Pradesh, India, (2) Department of Pharmacology, Mamata Medical College, Khammam, Telangana, India, (3) Department of Pharmacology, Mamata Medical College, Khammam, Telangana, India
Correspondence to: Naveen Pokala, E-mail: email@example.com
Received: December 18, 2018; Accepted: January 12, 2019
Source of Support: Nil, Conflict of Interest: None declared.
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|Title Annotation:||RESEARCH ARTICLE|
|Author:||Alasyam, Naveen; Pokala, Naveen; John, Premendran|
|Publication:||National Journal of Physiology, Pharmacy and Pharmacology|
|Date:||Mar 1, 2019|
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