Comparison of Meniran (Phyllanthus niruri Linn.) Extract and Sambiloto (Andrographis paniculata Ness.) Extract on Blood Profile of Animal Model Infected with Salmonella

 

Sri Hidanah1, Emy Koestanti Sabdoningrum1*, Kadek Rachmawati2, Rochmah Kurnijasanti2, Soeharsono3, Sylvia Anggraini4

1Animal Husbandry Division, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia.

2Basic Veterinary Medicine Division, Faculty of Veterinary Medicine,

Universitas Airlangga, Surabaya, Indonesia.

3Veterinary Anatomy Division, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia.

4Undergraduate Program, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia.

*Corresponding Author E-mail: emykoestanti10@gmail.com

 

ABSTRACT:

Salmonella Pullorum is the cause of Pullorum disease. The misuse of antimicrobials has resulted in the evolution of resistant strains. Meniran (Phyllanthus niruri Linn.) and Sambiloto (Andrographis paniculata Ness.) contain compounds that are efficacious as antibacterial and immunomodulatory. This study aims to analyze the comparison of meniran extract and sambiloto extract on the blood profile of broiler chickens infected with Salmonella Pullorum. This study was conducted by making Meniran and Sambiloto extracts. Phytochemicals screening (alkaloids, tannins, saponins, flavonoids, andrographolide, steroids and triterpenoids) of extracts, isolation and identification of Salmonella Pullorum and in vivo study were then conducted. The in vivo study was experimental research with completely randomized design that divided broiler chickens into treatment groups: P0- (chickens were not infected), P0+ (chickens were infected with Salmonella Pullorum), PAB (chickens were infected with Salmonella Pullorum and given Enrofloxacin Antibiotics) then treatment P1 Meniran, P2 Meniran, P3 Meniran (chickens were infected with Salmonella Pullorum and, in order, given meniran extract of 5%, 10% and 20% concentrations) and P1 Sambiloto, P2 Sambiloto, P3 Sambiloto (chickens were infected with Salmonella Pullorum and, in order, given sambiloto extract of 5%, 10% and 20% concentrations).  Data in the form of blood profile was then analyzed statistically by ANOVA test and continued with Duncan's test. This study shows that Meniran and Sambiloto extract has immunomodulator properties that have good impact in restoring blood profile of broiler chickens infected with Salmonella Pullorum. The comparison shows that Meniran extract has better antibacterial activity on blood profile of broiler chickens infected with Salmonella Pullorum compared to Sambiloto extract.

 

KEYWORDS: Meniran (Phyllanthus niruri Linn.), Sambiloto (Andrographis paniculata Ness.), Salmonella, blood profile, food security.

 

 


INTRODUCTION: 

Salmonella Pullorum is a pathogen that causes pullorum disease, a systemic disease that can cause symptoms such as closed eyes (similar to sleepy eyes), bluish comb and decreased appetite.

 

Usually chickens that are affected by this disease have white or greenish-brown diarrhea and a paste-like lump around the cloaca along with leg weakness, dangling wings, dullness, paralysis due to arthritis and shortness of breath. Pullorum can spread vertically and horizontally and cause huge economic losses. Antimicrobials have played an important role in the control of Salmonella bacteria, misuse of antimicrobials has resulted in the evolution of multidrug-resistant strains and made the prevention and treatment more difficult1,2,3,4.

One of the safest ways to prevent bacterial diseases is to use herbs. Indonesia as a tropical country has wealth of herbal potential. Generally herbal plants contain compounds that have potential as natural antibacterial which may serve as an alternative that effective, cheap and safe for treatment of common bacterial infections5,6. Nature serves as the source of rich phytochemical diversity that possess important biological and pharmacological activities7. Phytochemicals are the natural bioactive compounds of plant origin8. Meniran (Phyllanthus niruri Linn.) contains lignans, alkaloids, flavonoids, tripenoids, saponins and tannins, while sambiloto (Andrographis paniculata Ness.) contains andrographolide, alkaloids, saponins, tannins, and flavonoids which are capable of being antibacterial and immunomodulator9. Thus, the administration of meniran (Phyllanthus niruri Linn.) extract and sambiloto (Andrographis paniculata Ness.) extract to chickens can affect the activity of the immune system because they have immunomodulator properties. This is demonstrated, for example, through leukocyte activity and cytokine secretion by immunogenic cells.

 

According to the described background, this study aims to analyze the comparison of meniran (Phyllanthus niruri Linn.) extract and sambiloto (Andrographis paniculata Ness.) extract on the blood profile of broiler chickens infected with Salmonella Pullorum.

 

MATERIALS AND METHOD:

Ethics:

This entire series of study was carried out properly in accordance with ethics in using experimental animals and was approved by the ethics committee of the Faculty of Veterinary Medicine, Airlangga University (Ethical No.: 2.KE.142.08.2018).

 

Manufacture of Meniran (Phyllanthus niruri Linn.) extract and Sambiloto (Andrographis paniculata Ness.) extract:

Meniran (Phyllanthus niruri Linn.) and Sambiloto (Andrographis paniculata Ness.) plants that had previously been dried in an open and shady place were then ground and sieved to obtain the form of powder. Meniran powder and Sambiloto powder were each soaked for three days by maceration using 3L of 96% methanol solution and stirred once a day for three days. Furthermore, the results of the Meniran powder and Sambiloto powder immersions were each squeezed using flannel cloths. The extracts were then evaporated using rotary evaporator at a temperature of 50℃10. The concentrated Meniran extract and Sambiloto extract that obtained were then diluted into 3 concentrations (5%, 10% and 20%).

 

 

Phytochemical screening:

Phytochemical screening is different qualitative tests that were performed for establishing the profile of given extracts to detect various phytoconstituents present in them11. To identify the constituents, standard procedures were followed12.

 

Alkaloid:  Using the Mayer, Wagner and Dragendorff methods. 3Ml of each extract was added 5mL of 2M HCl, stirred and allowed to stand at room temperature then 0.5g NaCl was added to the sample then stirred, filtered and the resulting filtrate was added 3 drops of 2 M HCl. After that, separated into 4 parts (A, B, C, D). Filtrate A was used as blank, Mayer's reagent was added to filtrate B, Wagner's reagent was added to filtrate C and filtrate D was used for confirmation test.

 

Confirmation test was carried out by adding 25% ammonia to filtrate D until the pH changed to 8-9 then chloroform was added and evaporated. Then 2M HCl was added, stirred and filtered. The filtrate then was divided into 3 parts. Filtrate A was used as blank, filtrate B was tested with Mayer's reagent, while filtrate C was tested with Dragendorff's reagent. If identification indicates the presence of alkaloids, precipitate will form

 

Tanin: 2grams of each extract was put into 500mL boiling flask, added 350mL of distilled water and refluxed for 3 hours. The samples were then cooled and transferred into 500ml volumetric flask, filtered and then 2ml of filtrate was put into 100ml volumetric flask. 2ml of Folin Denis reagent and 5ml of concentrated Na2CO3 were added, allowed to stand and the absorbance was measured. 100grams of Na2WO4, 20grams of phosphomolybdic acid and 50ml of 85% phosphoric acid were added to 750ml of distilled water and the mixture was refluxed for 3hours, cooled and 1 liter of distilled water was added. 100ml of distilled water at a temperature of 70-80ºC added 3grams of anhydrous Na2CO3, stirred and cooled overnight.

 

Saponin: Using the Forth method, 2mL of each extract was vigorously shaken with water in test tube, then heated to boil13. The changes that occurred were observed. Stable froth formation indicated the absence of saponins14.

 

Flavonoid: 3mL of each extract was evaporated and washed with hexane. The residue was dissolved in 20 mL of ethanol and filtered. The filtrate from each extract was divided into 4 parts (A, B, C and D). Filtrate A as blank, filtrate B added 0.5mL of concentrated HCl and then heated, a positive result in the Bate Smith-Metchalf method was indicated if there is a dark red color change to purple. The filtrate C was added with 0.5mL of HCl and Mg metal, then the changes were observed (Wilstater method).

 

Andrographolide: Cold maceration of each leaf powder was carried out with a mixture ratio of 1:1 dichloromethane and methanol and continued with recrystallization15.

 

Isolation dan identification of Salmonella Pullorum:

Isolation dan identification of Salmonella Pullorum began with the manufacture of 35g of SSA media dissolved in 1liter of distilled water, then boiled and sterilized using an autoclave for 30 minutes at 121°C, 1 atm16. 10g of sample (which has been mashed) was placed on aluminum foil, then mixed into 90ml of sterile distilled water (10-1 dilution). Shake using cortex until the sample dissolves homogeneously. A total of 1ml of the solution at a dilution of 10-1 was taken and put into a test tube containing 9ml of new sterile distilled water (dilution 10-2). A total of 1 ml of the suspension sample solution at dilutions of 10-1 and 10-2 was taken by pouring dish method on Salmonella-Shigella Agar (SSA) media. Then incubated at 37°C for one day.

 

The Salmonella Pullorum culture was then cultured in liquid media by inserting 4-10 oses of Salmonella Pullorum which had previously been planted on agar media into Erlenmeyer containing 25ml of PBS solution. The turbidity of the bacterial suspension was compared to a Mc Farland tube No. 1 (3 x 108 cell/ml), then diluted by adding 50 ml of PBS solution to obtain 1 x 108cell/ml. The identification was followed by a biochemical test consisting of Triple Sugar Iron Agar (TSIA), Sulfide Indole Motility (SIM), Simmon Citrate Agar (SCA), Urea Agar and a Sugar Test consisting of glucose, mannitol, maltose, sucrose and lactose.

 

In vivo study on broiler chickens:

DOC broiler chickens were reared in brooder cages, fed and watered ad libitum. At the age of 4 days, the chickens were given ND vaccine with eye drops to form the first line of defense. At the age of 14 days, the broiler chickens were adapted to the adaptation period and was carried out for one week by moving the broiler chickens to battery cage before entering the treatment period.

 

Salmonella Pullorum infected at concentration of 108 sel/mL as much as 1mL/head injected using IM method in broiler chickens aged 14 days. After that, clinical symptoms were observed from the age of 14-21 days. Meniran (Phyllanthus niruri Linn.) extract and sambiloto (Andrographis paniculata Ness.) extract were then given to broiler chickens at the age of 21 to 35 days. In vivo study was experimental research with completely randomized design that divided broiler chickens into treatment groups as follows:

 

 

P0-:  Broiler chickens were only given distilled water, without extract and without being infected with Salmonella pullorum.

 

P0+: Broiler chickens were infected with Salmonella Pullorum at concentration of 108 cells/mL as much as 1 mL/head IM.

 

PAB:  Broiler chickens were given Enrofloxacin antibiotics and infected with Salmonella Pullorum at concentration of 108 cells/mL as much as 1mL/head IM.

 

P2 Meniran: Broiler chickens were given 5% concentration of meniran (Phyllanthus niruri Linn.) extract as much as 1mL/head orally and infected with Salmonella Pullorum at concentration of 108 cells/mL as much as 1mL/head IM.

 

P2 Meniran: Broiler chickens were given 10% concentration of meniran (Phyllanthus niruri Linn.) extract as much as 1mL/head orally and infected with Salmonella Pullorum at concentration of 108 cells/mL as much as 1mL/head IM.

 

P3 Meniran: Broiler chickens were given 20% concentration of meniran (Phyllanthus niruri Linn.) extract as much as 1mL/head orally and infected with Salmonella Pullorum at concentration of 108 cells/mL as much as 1mL/head IM.

 

P1 Sambiloto: Broiler chickens were given 5% concentration of sambiloto (Andrographis paniculata Ness.) extract as much as 1mL/head orally and infected with Salmonella Pullorum at concentration of 108 cells/mL as much as 1mL/head IM.

 

P2 Sambiloto: Broiler chickens were given 10% concentration of sambiloto (Andrographis paniculata Ness.) extract as much as 1mL/head orally and infected with Salmonella Pullorum at concentration of 108 cells/mL as much as 1mL/head IM.

 

P3 Sambiloto: Broiler chickens were given 20% concentration of sambiloto (Andrographis paniculata Ness.)  extract as much as 1mL/head orally and infected with Salmonella Pullorum at concentration of 108 cells/mL as much as 1mL/head IM.

 

Sample collection:

Blood samples were taken through the brachial vein as much as 1mL. Next, the anticoagulant EDTA is administered to the blood sample that being examined. The blood sample with a dose of 1mg/1mL was then put into the EDTA tube. Next, the total number of leukocytes and the number of each type of leukocyte were examined17.

 

 

Statistical analysis:

The data was statistically analyzed using the ANOVA test and then continued with the Duncan’s test using SPSS 26 for windows.

 

RESULT:

Phytochemical screening:

The results of phytochemical screening can be seen in Table 1.

 

Table 1. Phytochemical screening results of Meniran (Phyllanthus niruri Linn.) and Sambiloto (Andrographis paniculata Ness.) extract.

S.

No

Phytochemical Screening

Meniran Extract Result

Sambiloto Extract Result

Information

1

Alkaloid

+

+

Presence of chocolate deposits

2

Tannin

+

+

Color change to blackish green

3

Flavonoid

+

+

Color change to more orange

4

Saponin

+

+

Foam formed

5

Steroid/Triterpenoid

+

+

Color change to green

6

Andrographolide

-

+

Andrographolide compounds are crystalline

 

Blood profile:

Data on the total number of leukocytes in broiler chickens infected with Salmonella Pullorum after treatment showed that P0- and P0+ were not significantly different. P0- was significantly different from P2 Meniran, P3 Meniran, P2 Sambiloto and P3 Sambiloto but not significantly different from other treatments. P0+ was significantly different from P1 Meniran, P2 Meniran, P3 Meniran, P1 Sambiloto, P2 Sambiloto and P3 Sambiloto but  not significantly different from PAB.

 

Data on the number of heterophils in broiler chickens infected with Salmonella Pullorum showed that P0- was not significantly different from PAB, P3 Meniran, P1 Sambiloto, P2 Sambiloto and P3 Sambiloto but significantly different from P1 Meniran and P2 Meniran. P0+ was not significantly different from P1 Meniran and P2 Meniran.

 

Data on the number of basophils in broiler chickens infected with Salmonella Pullorum showed that P0- was not significantly different from PAB, P3 Meniran and P3 Sambiloto but significantly different from P0+, P1 Meniran, P2 Meniran, P1Sambiloto and P3 Sambiloto. P0+ differed significantly with all treatments.

 

Data on the number of eosinophils in broiler chickens infected with Salmonella Pullorum showed that P0- was not significantly different from P1 Meniran, P2 Meniran and P3 Meniran but significantly different from P0+, PAB, P1 Sambiloto, P2 Sambiloto and P3 Sambiloto. P0+ was not significantly different from PAB, P1 Sambiloto, P2 Sambiloto and P3 Sambiloto but significantly different from P0, P1 Meniran, P2 Meniran and P3 Meniran.

 

Data on the number of lymphocytes in broiler chickens infected with Salmonella Pullorum showed that P0- was not significantly different from PAB, P3 Meniran and P3 Sambiloto but significantly different from P0+, P1 Meniran, P2 Meniran, P1 Sambiloto and P2 Sambiloto. P0+ was not significantly different from P1 Meniran, P2 Meniran, P1 Sambiloto, P2 Sambiloto but significantly different from PAB, P3 Meniran and P3 Sambiloto.

 

Data on the number of monocytes in broiler chickens infected with Salmonella Pullorum showed that P0- was not significantly different from all treatments. P0+ was not significantly different from P3 Meniran and P3 Sambiloto but significantly different from PAB, P1 Meniran, P2 Meniran, P1 Sambiloto and P2 Sambiloto

 

Data on the total number of leukocytes and each type of leukocyte in broiler chickens infected with Salmonella Pullorum after treatment can be seen on Table 2.


 

 

Table 2. The total number of leukocytes and each type of leukocyte in broiler chickens infected with Salmonella Pullorum after treatment

Treatment

Total leukocyte count

(X±SD)

Heterophile (X±SD)

Basophils (X±SD)

Eosinophils (X±SD)

Lymphocytes (X±SD)

Monocytes (X±SD)

P0-

18233.33ab  ± 635.09

4.33ab  ± 1.53

16.00± 0.87

2.00± 0 .00

79.00± 1.00

3.33ab ± 1.53

P0+

20910.100± 2259.27

10.00de  ± 0.91

35.00± 1.79

7.00± 1.00

87.00± 2.65

7.00 b ± 3.00

PAB

18233.33ab  ± 635.09

5.67ab  ± 0.58

15.33± 4.62

7.33± 1.53

74.67± 2.52

3.00 a ± 1.00

P1 Meniran

15233.67 ac  ± 2622.12

12.00e  ± 2.00

29.33d  ± 2.08

2.00a  ± 1.00

84.67b  ± 4.16

3.00 a ± 1.00

P2 Meniran

13136.67 c   ± 3223.82

8.33cd   ± 1.53

22.33bc   ± 4.04

2.67a   ± 1.15

85.67b   ± 1.53

4.00 a ± 1.00

P3 Meniran

13583.33 c  ± 2217.98

6.33bc  ± 0.58

14.67a  ± 4.51

3.67a  ± 1.53

75.00a  ± 2.00

5.67ab ± 0.58

P1 Sambiloto

15353.33 ac  ± 3865.91

4.00a  ± 0.75

25.00cd  ± 2.64

8.67b  ± 1.53

86.00b  ± 1.00

3.33a ± 0.58

P2 Sambiloto

13436.67 c  ± 1003.11

4.00a  ± 0.00

23.00bc  ± 4.36

8.00b  ± 1.00

84.33b  ± 4.04

4.00 a ± 2.65

P3 Sambiloto

12540.00 c  ± 1338.21

5.00ab  ± 0.86

18.67ab  ± 2.08

8.00b  ± 2.65

79.01a  ± 1.00

7.67 b ±2.08

* Different superscripts in the same column show significant differences (p<0,05).

SD = Standard deviation


 

DISCUSSION:

The results showed that the administration of meniran (Phyllanthus niruri Linn.) extract and sambiloto (Andrographis paniculata Ness.) extract at 5% concentration was able to restore the number of leukocytes in broiler chickens infected with Salmonella Pullorum according to chickens that were not infected. This is different from the total number of leukocytes in chickens infected with Salmonella Pullorum but not given either meniran (Phyllanthus niruri Linn.) extract nor sambiloto (Andrographis paniculata Ness.) extract which increased. Leukocytes are associated with the innate immune system and are one of the first lines of defense against disease and their numbers can increase rapidly when there is infection by bacteria, blood protozoa and larger macroparasites18. A decrease in the total number of leukocytes indicates that the inflammatory process caused by bacterial infection has stopped17. This is because meniran (Phyllanthus niruri Linn.) contains phytochemical compounds that consisted of terpenoids, alkaloids, flavonoids, saponins, and tannins and sambiloto contains major constituent such as andrographolide that function as antibacterial19,20. Phytochemicals are bioactive chemicals of plant origin and have the capacity of exerting a physiologic action on body21,22.

 

The results showed that the administration of meniran (Phyllanthus niruri Linn.) extract at 5%, 10% and 20% concentrations was not able to restore the number of heterophils like healthy chickens (not infected with Salmonella Pullorum) but the administration of sambiloto (Andrographis paniculata Ness.) extract at all concentrations (5%, 10% and 20%) was able to restore the number of heterophils, same as healthy chicken.

 

Heterophiles are important mediators of natural resistance to bacterial infections in poultry. These cells are recruited by bacterial components and host inflammatory mediators to the site of infection where they phagocytize and kill bacteria23. Increased heterophils can be found in cases of infections (bacterial, fungal, and parasitic), inflammation, stress, toxicity, traumatic conditions, and leukemia17,24,25.

 

The results showed that the administration of meniran (Phyllanthus niruri Linn.)  extract and sambiloto (Andrographis paniculata Ness.) extract was able to reduce the number of basophils in broiler chickens infected with Salmonella Pullorum and at 20% concentration, both were able to offset the number of basophils with the group of broiler chickens that were not infected with Salmonella Pullorum and the control group that was given antibiotic. This shows that meniran (Phyllanthus niruri Linn.) extract and sambiloto (Andrographis paniculata Ness.) extract with concentration of 20% were effective in replacing Enrofloxacin antibiotic in broiler chickens infected with Salmonella Pullorum.

 

In poultry, basophils are granulocyte-type leukocytes that function as mediators in the initial inflammatory response26. The increase in the number of basophils in infected chickens may be due to the effect of endotoxin, as a bacterial product, which has chemotactic effect on basophils. In poultry, degranulated basophils release histamine and other vasoactive amines during the inflammatory reaction and contribute to increased vascular permeability and facilitate the infiltration of other types of leukocytes to the site of injury27.

 

The results showed that the administration of meniran (Phyllanthus niruri Linn.) extract in various concentrations (5%, 10% and 20%) was able to balance the number of eosinophils in the group of broiler chickens that were not infected with Salmonella Pullorum but the sambiloto (Andrographis paniculata Ness.) extract at various concentrations (5%, 10% and 20%) was not able to.

 

Eosinophils are rare in chicken and are commonly associated with parasitic disease. The increase in eosinophils, basophils and monocytes is associated with various conditions due to infection and inflammation15. Granule proteins in eosinophils such as ECP, EPO, and MBP have antibacterial properties28.

 

The results showed that the administration of meniran (Phyllanthus niruri Linn.) extract and sambiloto (Andrographis paniculata Ness.) extract at 20% concentration of was able to balance the lymphocyte count from the group of broiler chickens that were not infected with Salmonella Pullorum.

 

Lymphocytes are cells that play an important role in the immune system and control the cellular and humoral immune systems against antigens. An increase in the number of lymphocytes can be associated with the inflammatory effect of infection, while a decrease in the number of lymphocytes can occur due to stress conditions. Chronic antigen-induced stimulation can cause widespread circulation of lymphocytes due to the main function of lymphocytes in the immunologic response, the formation of humoral antibodies mediated by immunocompetent cells. The increased immune system can fight disease. Immunomodulator compounds can enhance the cellular and humoral immune system17,29.

 

The results showed that the number of monocytes in the group of broiler chickens that were not infected with Salmonella Pullorum was not significantly different from all treatments. All treatments were not significantly different. Monocytes are part of the innate immune system that plays an important role for chickens in fighting pathogenic infections30.

 

CONCLUSION:

The administration of Meniran (Phyllanthus niruri Linn.) extract and Sambiloto (Andrographis paniculata Ness.) extract was efficacious in restoring the number of blood profile of broiler chickens infected with Salmonella Pullorum. The comparison shows that Meniran (Phyllanthus niruri Linn.) extract has better antibacterial activity on blood profile of broiler chickens infected with Salmonella Pullorum compared to Sambiloto (Andrographis paniculata Ness.) extract.

 

CONFLICT OF INTEREST:

The authors declare that there is no conflict of interest regarding the publication of this article.

 

ACKNOWLEDGMENTS:

We are grateful to Indonesian Ministry of Education, Culture, Research, and Technology, our research members and all those who participated in this project.

 

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Received on 28.03.2022          Modified on 24.05.2022

Accepted on 06.07.2022        © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(2):581-586.

DOI: 10.52711/0974-360X.2023.00099