INTRODUCTION:

Colibacillosis is an infectious bacterial diseases in birds caused by pathogenic E. coli. The infectious called E. coli or Colisepticaemia is infected worldwide in broilers and breeders of all ages, and other birds like turkeys and ducks. The cause collibacillosis is originated from E. coli enterotoxin. There is certain E. coli strains that can produce Shiga toxin so that these strains are known by various names such as Shiga toxin-producing E. coli (STEC) or verocytotoxigenic E. coli (VTEC). E. coli group that has virulence factors causing diseases, colonization in humans and animals increasing morbidity and mortality¹.

Indonesia is among the top five countries in the world that has high biodiversity of plants and around 55% of them are endemic^2,3,4. The plants also have a variety of pharmacological activities⁵. Therefore, researchers tried to use prevention in handling the problem, which is by giving immunomodulatory and antioxidants that can prevent disease in laying hens by using Phyllanthus niruri L. extract (PNE). PNE has the advantage of being an immunomodulator. PNE is a drug that can restore and repair the immune system where it functions is to impaired or to suppress a function that is excessive. Some of the main substances contained in PNE are lignans, tannins, polyphenols, alkaloids, flavonoids, terpenoids and steroids⁶. The content of flavonoids functions as immunomodulators that are improve immunity function. The specific immune response of P. niruri extract can increase T lymphocyte cells proliferation, increase the secretion of TNFα, IFNγ, IL-4, and reduce IL-2 and IL-10 secretion. For humoral immunity, this PNE can increase the production of immunoglobulin M (IgM) and immunoglobulin G (IgG)^7,8.

Chemically, the PNE are characterized among others by the content of derivatives of lignans, alkaloids, flavonoids, and triterpenoids. Lignans are used as antioxidants⁶. Phyllanthin and hypophyllanthin are derived from hexane extract of P. niruri which shows inhibitory effect on gram-positive bacteria⁹. In addition, the flavonoids action of P. niruri L. is an imunnomodulator whose role is to boost the immune system and improve dysfunctional of the immune system. Therefore, The purpose of this study was to determine the immunomodulatory and antioxidant activities of PNE in laying hens infected by E. coli.

MATERIAL AND METHODS:

Research Methods:

This study used completely randomized design (CRD) with 20 laying hens. Laying hens were divided into five groups consisting of one negative control group given Na-CMC (P0-), one positive control group given E. coli (P0+), and three test groups given the PNE 10% (P1), 20% (P2), and 30% (P3). Each test group was given a sample suspension orally once a day for six consecutive days. At the age of 21 weeks, E. coli infection was 10⁶CFU/mL/body weight. A week after, E. coli infection was given with various dosage of PNE for four weeks. Immunomodulatory effect can be seen from the blood features of leukocyte with differential count and lymphocytes, eosinophils, heterophils, monocytes count. Malondialdehyde (MDA) can be used as an indicator to increase in lipid peroxide formed by free radicals and enzymes. Superoxide dismutase (SOD) is an endogenous enzyme that plays an important role in capturing free radicals.

Calculating the Number of Leukocytes with Differential Count and Lymphocytes, Eosinophils, Heterophils, Monocytes Count:

Whole blood is dropped on a glass object as much as one drop, then it is spread out thinly, and flatten it with another object glass, so that the blood layer becomes homogeneous (smeared blood), then dry it. After drying it, drops with methanol were added till it covers the entire blood smear, then leave it for five minutes. Add one drop of Giemsa (Giemsa staining method) that has been diluted in distilled water (1:20), leave it for 20 minutes. Wash with distilled water. After drying, observe under a microscope. Calculate the number of lymphocytes at 400× magnification.

Measurement of MDA Levels:

The method used for measuring MDA levels in this study was thiobarbituric acid. The principle of this method is based on the ability to form pink complexes between MDA and TBA. One gram of liver tissue was homogenized with 9.0 mL of 1.15% KCl solution with Teflon Potter-Elvehjem homogenizer. 0.2 mL of liver homogenate, added with 0.2 mL of 8.1% SDS and 1.5 mL of 20% acetic acid solution to pH 3.5 and 1.5 mL of 0.8% TBA solution. The mixture was added with water up to 4.0 mL volume, heated at 95 °C for 60 minutes, then cooled using water. After cooling, the mixture is added with 1.0 mL of water and 5.0 mL of the mixture of n-butanol:pyridine (15:1, v/v), and then shaken. Then centrifuged at 4000 rpm for 10 minutes, the organic layer was taken and measured the absorbance at a wavelength of 532 nm using a visible spectrophotometer. The standard solution used is 1,1,3,3-tetraethoxypropane (TEP).

Determination of SOD:

A total of 0.06 mL of liver supernatant was reacted with a mixture consisting of 2.70 mL of 50 mM sodium carbonate buffer containing 0.1 mM EDTA (pH 10), 0.06 mL xantin 10 mM, 0.03 mL of 0.5% BSA, and 0.03 mL NBT 2.5 mM. Next, the addition of xanthine oxidase. Absorbance produced after 30 minutes is measured at a wavelength of 560 nm. As a control solution, the solution used in the preparation of liver samples is PBS containing 11.5 g/L KCl.

Data Analysis:

The data obtained in analyzed by using anova (analysis of variance) and tested by F test. If there are significant differences (p < 0.05), the statistical test is continued with the test smallest significant difference.

RESULTS AND DISCUSSION:

This study shows the results of the study as follows:

Table 1. Mean Table of Leukocytes, Lymphocytes, SOD and MDA Results from Laying Hens given PNE and infected with E. coli after Treatment.

Treatment	Total Leukocytes (X±SD)	Total Lymphocytes (X±SD)	Total Eosinophils (X±SD)	Total Heterophils (X±SD)	Total Monocytes (X±SD)	Average SOD Level (U/g)	Average MDA Level (nmol/gr)
P0-	12.578^a ± 2.751	10203.95^ab ± 1625	457.12^ab ± 148.34	957.97^a ± 325.16	364.56^a ± 76.36	962.125^c± 13.779	7.608^b± 0.105
P0+	19.015^b ± 6.117	14223.25^b ± 25121.65	1217.92^c ± 446.99	1897.89^b ± 784.12	968.75^b ± 225.64	592.765^a ± 12.072	12.953^d± 0.184
P1	16.877^ab ± 3.242	13865.98^b ± 1653.50	455.64^a ± 96.79	1158.69^a ± 167.96	455.72^ab ± 226.32	736.492^b± 10.172	9.802^c± 0.168
P2	16.645^ab ± 3.898	13215.97^ab ± 1315.16	454.75^a ± 99.87	1160.56^a ± 490.76	432.12^ab ± 322.24	1021.618^d± 26.571	3.670^a± 0.158
P3	12.352^a ± 1.821	10468.83^ab ± 1277.71	460.81^ab ± 145.76	978.65^a ± 135.86	485.35^ab ± 210.25	1176.730^e± 8.244	3.428^a± 0.254

Leukocyte statistical analysis uses one way ANOVA with a significance value of p <0.05 which indicates a significant difference in the study. Then proceed with Duncan with a significance level of 5%. P0- (healthy chicken, without treatment), P3 (administration of PNE at a dose of 30%) has not experienced a significant difference in P2 (administration of PNE at a dose of 25%) and P1 (administration of PNE at a dose of 20%). In the results of the count of leukocytes in this study P0- (without infection with E. coli and without PNE) showed the lowest leukocyte count compared to other treatments. P0+ (infected with E. coli but without PNE) showed the highest leukocyte count, which means that the experimental animal was infected with the E. coli. This indicated by the increase in the number of leukocytes due to inflammation caused by bacterial cell infection. The increase in leukocyte count that exceeds normal is called leukocytosis. Leukocytes increase as a physiological response to protect the body from attack by microorganisms. The results showed that P1, P2, and P3 treated with PNE decreased the number of leukocytes, especially treatment P3. Shows that the treatment given by PNE at a dose of 30% was not significantly different from the P0- treatment. The lower number of leukocytes indicates that the inflammation process due to bacterial infection has stopped. This is because the ability of PNE in killing bacteria caused by the content of antibacterial active substances in PNE include tannins, saponins, and alkaloids.

In this study, lymphocytes showed an increase in the treatment of P0+ and decreased in each treatment P1, P2, and P3 although the reduction is not significant. This decrease in lymphocytes is caused by a decrease in inflammation caused by bacteria. The decrease in lymphocyte appointment of cells has begun to recover and the bacteria have died so that phagocytosis is not needed by lymphocytes. In treatments P1, P2, and P3, Eosinophils have a significant decrease, because the saponin in the PNE can increase the permeability of bacterial cell membranes so that it can change the structure and function of the membrane, causing denaturation of membrane proteins so that the cell membrane will be damaged and lysis. In the treatment of P0+, heterophils there was a significant increase compared to treatment P0-. This is because heterophils is an effective defense against microbes, especially bacteria. The heterophilic function as an antibacterial defense through several effective mechanisms, namely chemotaxis (the ability of heterophils to be attracted to the site of infection and inflammation) and as phagocytosis, namely the ability to eat and destroy microbes). So that when infection and inflammation occur heterophils will increase to phagocytosis of microbes. The treatment of P1, P2, and P3 is a decrease in heterophils due to the function of the content in PNE which damages microbial or bacterial cells. The intended content is tannin. Tanin compounds have toxic properties to bacterial cell membranes by inhibiting certain enzymes. Monocytes did not change too significantly at P0+, P1, P2, and P3. This is presumably due to cells damaged due to E. coli infection has improved.

SOD results showed that there was a very significant difference in the SOD level of laying hens given PNE. SOD levels increased at higher doses, the control treatment was very influential with the treatment of PNE at different doses. The P1 treatment increased compared to the control, as did the P2 and P3 treatments compared to the controls. This shows that administration of PNE with different doses can increase SOD levels. Increased SOD levels compared to controls are possible due to active substances in P. niruri. In P. niruri there are triterpenoids as an antioxidant that is able to modulate endogenous SOD enzymes in counteracting free radical compounds in the body due to oxidative stress. Triterpenoids are chemical compounds contained in P. niruri. Triterpenoids are soluble in fat, so that it is categorized in the second antioxidant mechanism, which is secondary antioxidants can slow down the acylation reaction by converting lipid radicals to more stable compounds such as vitamins E and C. The mechanism of free radical capture by triterpenoids as natural antioxidants derived from plants. Triterpenoids are able to modulate endogenous SOD enzymes, so that they can change the compound bonds caused by free radicals to become more stable bonds. In addition, phytochemical compounds found in ethanolic extracts and their fractions of P. niruri consist of flavonoids and polyphenols¹⁰.

The function of flavonoids in general in neutralizing free radicals formed in the body is thought to be through the mechanism of antioxidant capacity and stimulation of genes responsible for the synthesis of antioxidant enzymes. P. niruri has flavonol compounds, namely flavonoids which are most effective at capturing free radicals (hydroxyl radicals, superoxide, and peroxyl) and inhibiting various oxidation reactions, because they can produce phenoxyl radicals which are stabilized by the resonance effect of aromatic rings. P. niruri has tannins, the interaction of tannins with proteins has unique properties and depends on the structure of the tannin. Some tannins have been shown to have antioxidant activity, inhibit tumor growth. The mechanism of action of antioxidants has two functions. The first function is the main function of antioxidants which is as a giver of hydrogen atoms. Antioxidants which have the main function are often referred to as primary antioxidants. These compounds can give hydrogen atoms quickly to lipid radicals or change them to more stable forms, while the radical derivative of antioxidants has a more stable state than lipid radicals, the second function is a secondary function of antioxidants, namely slowing down the rate of autoxidation with various mechanisms outside of the chain termination mechanism autoxidation by converting lipid radicals to a more stable form. The workings of antioxidant compounds are to react with reactive free radicals to form relatively unreactive free radicals. Antioxidants stabilize free radicals by supplementing the lack of electrons possessed by free radicals, and inhibiting the occurrence of chain reactions from the formation of free radicals^9,10,11.

MDA results show that there is a very significant difference in each treatment, except P2 and P3. MDA levels are increasingly decreasing at higher doses. So it can be concluded that the administration of PNE can affect MDA levels in laying hens infected with E. coli. MDA is a toxic compound which is one of the final results of the breakdown of the fatty acid carbon chain in the process of lipid peroxidation. Lipid peroxide formed in the process of lipid peroxide propagation is stable, but if there is a metal transition such as Fe, then the substitution will be catalyzed into peroxy radicals which eventually form the final product, MDA. MDA levels formed are considered identical to the levels of lipid peroxide. MDA levels are low in the body, so usually the antioxidant status in the body is high, as well as low antioxidants in the body, the MDA levels also increase. The active substances involved in decreasing MDA levels are thought to be high antioxidant content and alkaloid elements in P. niruri which play a role in removing free radicals can also reduce MDA levels in the blood. Alkaloid, saponin, and triterpenoid compounds are also thought to have activity in reducing MDA levels. Alkaloid compounds can act as free radical scavengers and can prevent the occurrence of microsomal lipid peroxidation. Indole alkaloids, have the ability to stop the reaction of free radical chains efficiently. These derivative radical compounds from amine compounds have very long termination stages. Other alkaloid compounds that are antioxidants are caffeine which can act as a silencer for hydroxyl radicals¹¹. The strength of the antioxidant activity of flavonoids depends on the number and position of the hydroxyl group (-OH) found in the molecule. The strength of the antioxidant activity of flavonoids depends on the number and position of the -OH group found in the molecule. The more -OH groups in flavonoids, the higher the anti radicic activity¹².

Based on Kim et al. (2008), the phenolic component is a terminator of free radicals and as chelating active redox metal ions. This metal ion allows its role to catalyze lipid peroxidation reactions. These phenolic antioxidants block the oxidation of lipids and other molecules by donating hydrogen atoms to radical compounds to form phenoxyl radical intermediates. The phenoxyl radical intermediate compound is relatively stable so it is no longer able to initiate further radical reactions. The high biological activity in this phenolic compound lies in the position and number of -OH groups¹³.

The active substance of the class of compounds are tannins, alkaloids, flavonoids, saponins which are also useful as antioxidants, namely electron giving compounds. Antioxidants work by donating one electron to an oxidant compound so that the activity of the oxidant compound can be inhibited. Antioxidants stabilize free radicals by complementing the lack of electrons possessed by free radicals, and inhibiting the occurrence of chain reactions of free radical formation^3,4,^6,8.

CONCLUSION:

In sum, we conclude that PNE can increase immune system and antioxidant activity in laying hens infected with E. coli.

ACKNOWLEDGEMENT:

We thank to the Ministry of Research, Technology, and Higher Education of the Republic of Indonesia.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 10.08.2019 Modified on 02.10.2019

Research J. Pharm. and Tech 2020; 13(5): 2246-2250.

DOI: 10.5958/0974-360X.2020.00404.7