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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 3  |  Issue : 3  |  Page : 63-68

In vitro immunomodulatory, antifungal, and antibacterial screening of Phyllanthus niruri against to human pathogenic microorganisms


1 Department of Pharmaceutics, Sanjo College of Pharmaceutical Studies, Palakkad, Kerala, India
2 Department of Pharmaceutical Biotechnology, Sanjo College of Pharmaceutical Studies, Palakkad, Kerala; Department of Pharmaceutical Technology, Anna University, BIT Campus, Tiruchirappalli, Tamil Nadu, India
3 Department of Pharmacognosy, Sanjo College of Pharmaceutical Studies, Palakkad, Kerala, India
4 Department of Pharmaceutical Chemistry, Sanjo College of Pharmaceutical Studies, Palakkad, Kerala, India
5 Department of Pharmaceutical Technology, Anna University, BIT Campus, Tiruchirappalli, Tamil Nadu, India

Date of Submission29-May-2018
Date of Acceptance19-Aug-2018
Date of Web Publication18-Oct-2018

Correspondence Address:
Mr. K Muddukrishnaiah
Department of Pharmaceutical Technology, Anna University, BIT Campus, Tiruchirappalli - 620 024, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ed.ed_9_18

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  Abstract 

Background: Medicinal plants present a wide range of potentially phytochemical compounds that contain many useful properties including anticancer, enzyme inhibition, anti-inflammatory, antifungal, antibacterial, immunomodulatory, antioxidant, and antiallergic activities. Phyllanthus niruri capsules are extensively recommended to improve the function of the diseased liver. Its leaves root and the whole plant are used as an herbal complement.
Aim: The present study was aimed to focus on the in vitro immunomodulatory activity, antifungal, antibacterial and phytochemical screening of aqueous, methanolic, and ethanolic extract of P. niruri.
Materials and Methods: Immunomodulatory activities were evaluated through nitroblue tetrazolium assay. Antifungal and antibacterial activity were conducted against Candida albicans (NCIM - 3100), Aspergillus niger (NCIM - 1028), Eschericha coli (NCIM - 5346), Bacillus subtilis (NCIM - 2920), and Staphylococcus aureus (NCIM - 5345) by using disc diffusion method.
Results: Medicinal plants contain polyphenolic compounds which have potent anti-cancer and immunomodulator activity. P. niruri has potential immunomodulatory activity. Aqueous, methanolic, and ethanolic extract of P. niruri did not show any significant antifungal activity and 100 mg/ml, 150 mg/ml, and 200 mg/ml. Aqueous, methanolic, and ethanolic extract showed significant antibacterial activity.
Conclusion: From this study, it is concluded that P. niruri does not have antifungal activity but has potent immunomodulatory and antibacterial activity. This immunomodulatory and antibacterial activity of P. niruri may be due to the secondary metabolites such as alkaloid, tannins, terpenoids, flavonoids, and phenol compounds.

Keywords: Antimicrobial activity, immunomodulatory activity, Phyllanthus niruri


How to cite this article:
Shilpa V P, Muddukrishnaiah K, Thavamani B S, Dhanapal V, Arathi K N, Vinod K R, Sreeranjini S R. In vitro immunomodulatory, antifungal, and antibacterial screening of Phyllanthus niruri against to human pathogenic microorganisms. Environ Dis 2018;3:63-8

How to cite this URL:
Shilpa V P, Muddukrishnaiah K, Thavamani B S, Dhanapal V, Arathi K N, Vinod K R, Sreeranjini S R. In vitro immunomodulatory, antifungal, and antibacterial screening of Phyllanthus niruri against to human pathogenic microorganisms. Environ Dis [serial online] 2018 [cited 2021 Nov 27];3:63-8. Available from: http://www.environmentmed.org/text.asp?2018/3/3/63/243633


  Introduction Top


Antibiotic resistance developed by bacteria in healthy individuals is a worldwide problem of the present scenario. Irrational usage of antibiotics is one of the most important factors which leads to antibiotic resistance in living beings. Antibiotics are also commonly used in agriculture and veterinary science to prevent, control, and to treat infectious diseases.[1],[2]

The medicinal plants are extensive reservoirs of potentially secondary metabolic compounds that could be useful as an alternative to allopathic drugs in humans. Medicinal and aromatic plants are important sources of pharmaceutically active compounds, which have a wide range of use in controlling diseases that occur in plants, animals, and humans. The secondary metabolites presented in medicinal plants have been reported to have many useful properties such as anticancer, anti-inflammatory, estrogenic, enzyme inhibition, antimicrobial, anti-allergic, antioxidant, vascular, and immunomodulatory activities.

People from the developing countries, who are exposed to unhygienic conditions in their daily activities, are the worst sufferers of infectious diseases. Their sufferings have been increased many folds due to prolonged illness caused by widespread drug-resistant pathogens (e.g., multidrug-resistant Staphylococcus aureus, vancomycin-resistant Enterococcal strains with 73% mortality rate) and cost of treatment. The usage of novel synthetic compounds because of drug resisting pathogen, for such prolonged illness, makes the treatment costlier and also produces undesirable side effects.[3]

Phyllanthus niruri L. (Phyllanthaceae) a medicinal plant was reported to possess anti-inflammatory, hypoglycemic, antiviral, antioxidant, hepatoprotective, and inhibitory effect on renal stone formation. The plant contains acidic diterpenes, hypophyllanthin, arabinogalactan, alkaloids, flavonoids, lignans, terpenes, tannins, and phyllanthin. In the present study, we evaluated thein vitro immunomodulatory, antifungal, and antibacterial activities.


  Materials and Methods Top


Plant collection

The plants were collected from Pattambi of Palakkad district, South India. The specimens were identified by pharmacognosist. A voucher specimen was prepared in our research lab and maintained with the voucher no. SANJO/DBT/02 for further reference [Figure 1].
Figure 1: Phyllanthus niruri

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Sterilization of plant materials

Disease-free and fresh plants were washed with distilled water for three times. Surface sterilization was performed with 0.1% mercuric chloride for 20 s. The plants were again washed with distilled water for three times.[4]

Solvent extraction

Plants were air dried until the water molecules were evaporated. Dried plants were then grinded using mechanical mixer until they became fine powders, which were transferred into the airtight container with labeling. The methanol, ethanol, chloroform, and aqueous extracts were prepared by 50 g of plant powder material, which were soaked in 100 ml of different solvents in room temperature for 4 days. The extracts were filtered by Whatman filter paper (Grade 1). All the extracts were concentrated by water both evaporator. Methanolic, ethanolic, and aqueous concentrated extracts were preserved for phytochemical screening. Four grams of each extract (methanol, ethanol, chloroform, and aqueous) were dissolved in 10 ml of sterile water to give a concentration of (0.4 g/ml) stock solution. Two-fold serial dilutions were conducted. Four different dilutions were obtained (200, 100, 50, 25, and 12.5 mg/ml) for antifungal and antibacterial activities.[5]

Phytochemical screening

Methanolic, ethanolic, and aqueous extracts of P. niruri were used for phytochemical screening studies using the standard procedure.[6]

Test for alkaloids (Wagner's reagent)

The extract was treated with 4–5 drops of Wagner's reagent (1.27 g of Iodine and 2 g of potassium iodide in 100 ml of water) and observed for the formation colored precipitation.

Test for steroids (Lieberman–Burchardt tests)

A mixture of 2 ml of chloroform and a few drops of acetic anhydride were added to the extract. A few drops of concentrated sulfuric acid from the sides give a reddish ring at the junction of two layers.

Test for phenols (ferric chloride test)

Extract was treated with aqueous 5% ferric chloride and observed for the formation of deep blue or black color.

Tests for flavonoids

Extracts were treated with 10% ferric chloride solution and observed for the formation of green or blue color.

Test for reducing sugars (Fehling's test)

The extract was added to boiling Fehling's solution (A and B) in a test tube. The solution was observed for a color reaction.

Test for cardiac glycosides (Keller–Kelliani's test)

Extract was mixed with 2 ml of glacial acetic acid, a drop of ferric chloride solution and 1 ml concentrated sulfuric acid. A brown ring at the interface indicated the presence of deoxysugar characteristic of cardiac glycosides.

Tests for quinones

Extract was treated with concentrated hydrochloric acid and observed for the formation of yellow precipitate.

Test for saponins (foam test)

Water was added to the extract in the test tube. The mixture was shaken vigorously and observed for foam formation.

Test for terpenoids (Salkowski test)

Chloroform and concentrated sulfuric acid were added to the extract to form a layer and observed reddish brown color.

Test for tannins

Extract was mixed with water in the test tube. It was then stirred well and filtered. To the filtrate, 0.1% of ferric chloride was added and observed for brownish green or blue-black color.

Strain, culture media, and sterile discs

Plant extracts were conducted for bacterial and antifungal activities against Candida albicans (NCIM-3100), Aspergillus niger (NCIM - 1028), Eschericha coli (NCIM - 5346), Bacillus subtilis (NCIM - 2920), and S. aureus (NCIM - 5345) that were procured from the National Collection of Industrial Microorganisms from Pune, Maharashtra. Media used for microbial test was potato dextrose agar (PDA) and Muller Hinton agar (MHA) media of Himedia Pvt. Bombay, India. Sterile discs used for antimicrobial activity were procured from Himedia Pvt. Bombay, India.

Antifungal activity

Antifungal activity of aqueous, methanolic, ethanolic, and chloroform extract was studied by disc diffusion method. Two fungal strains were used for screening the antifungal activity of P. niruri. C. albicans (NCIM - 3100), A. niger (NCIM - 1028) inoculum were prepared by using potato dextrose broth. PDA media were prepared by autoclaving 3.9 g in 100 ml. Preparation of C. albicans (NCIM - 3100) and A. niger (NCIM - 1028) mat on the PDA Petri plates using sterile cotton swabs. Ethanolic, methanolic, aqueous, and chloroform extracts of P. niruri in different concentrations (200, 150, 100, 50% W/V) were placed on sterile discs, which were dried aseptically under laminar air flow to remove solvent. The dried discs were placed on the surface of culture inoculated PDA plates and the plates were incubated at room temperature for 48 h. Antifungal activity was evaluated by HiMedia zone reader.

Antibacterial activity

Antibacterial activity of aqueous, methanolic, ethanolic, and chloroform extract was studied by disc diffusion method. Three bacterial strains were used for screening the antibacterial activity of P. niruri. S. aureus (NCIM - 5345), E. coli (NCIM - 5346), B. subtilis (NCIM - 2920) inoculum were prepared by nutrient broth media. Double strength sterile MHA media were prepared by autoclaving 7.6 g in 100 ml. Preparation of S. aureus (NCIM - 5345), E. coli (NCIM - 5346), B. subtilis (NCIM - 2920) mat on the MHA plates by using sterile cotton swabs. Ethanolic, aqueous, methanolic, and chloroform extracts of P. niruri in different concentrations (200, 150, 100, 50% W/V) were placed on sterile discs, which were dried aseptically under laminar air flow to remove solvents. The dried discs were placed on the surface of culture inoculated MHA plates and the plates were incubated at 37°C for 24 h. Antibacterial activity was evaluated by HiMedia zone reader.[6],[7],[8]

Study of immunomodulatory activity

Leukocyte suspensions were prepared in 0.5 ml of phosphate buffered saline (PBS) solution in different test tubes. One drop of endotoxin activated plasma (standard) and 0.1 ml of PBS solution (control) were added to the first two test tubes. The other tubes were added with 0.1 ml of different concentrations (5, 10, 20, 40, 60, 80, 100, 500, and 1000 μg/ml) of ethanolic extract. Freshly prepared 0.15% nitroblue tetrazolium (NBT) solution was added to each test tube and incubated at 37°C for 20 min. The test tubes were centrifuged at 400 rpm for 5 min, and the supernatant liquids were discarded. The cell pellet were collected and re-suspended in 2 ml of PBS solution. A thin layer made on the slide with drop of from the re-suspended leukocyte solution. Slide was dried, heat fixed, and counterstained with diluted carbolfuchsin. The slide was washed under tap water; air dried and focused under 100 × oil immersion.[9],[10]


  Results Top


Phytochemical screening of Phyllanthus niruri

The present study revealed that the various aqueous, ethanol and methanol extracts of the whole plant of P. niruri contained alkaloids, steroids, quinones, saponins, tannins, cardiac glycosides, phenols, and flavonoids [Table 1]. Aqueous, ethanol, and methanol extracts showed the presence of several of rich secondary metabolites. Terpenoids and reducing sugars are absent in aqueous extract.
Table 1: Phytochemical screening of Phyllanthus niruri

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Antifungal activity of Phyllanthus niruri

[Table 2] shows the Antifungal activity of Aqueous, methanolic and ethanolic extracts of P. niruri. Aqueous, ethanolic, and methanolic extracts of P. niruri does not showing antifungal activity against Candida albicans (NCIM - 3100) and Aspergillus flavus (NCIM - 1028). Antifungal inactivity due to the absence of antifungal compounds in the extracts either C. albicans (NCIM - 3100), A. flavus (NCIM - 1028) showing resistance.[11]
Table 2: Antifungal activity of Phyllanthus niruri

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Antibacterial activity of Phyllanthus niruri

[Table 3] shows the antibacterial activity aqueous, methanolic, and ethanolic extracts of P. niruri. Aqueous, ethanolic, and methanolic extract of P. niruri showing good bacterial activity against S. aureus (NCIM - 5345), E. coli (NCIM - 5346), B. subtilis (NCIM - 2920). Antibacterial activity due to the secondary metabolites (flavonoids, polyphenols, and tannins) present in the extract.[12]
Table 3: Antibacterial activity of Phyllanthus niruri

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Nitroblue tetrazolium test

[Figure 2] shows the immunomodulatory activity of P. niruri. Two hundred and fifty neutrophils were counted from the percentage of NBT positive cells containing blue granules. P. niruri ethanolic extract stimulate the neutrophils provoking phagocytic activity to significant degree (P < 0.05) of 67.91, 61.25, and 6.02, at concentrations of 1000 μg/ml 500 μg/ml and 100 μg/ml.
Figure 2: Immunomodulatory activity

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Statistical analysis

The experiments were conducted in triplicate. The results are given as mean ± standard deviation. To compare between two means, Student's t-test was used. For the comparison of more than two means, one-way analysis of variance was used. A difference was considered statistically significant when P ≤ 0.05.


  Discussion Top


Antibiotic resistance in bacteria and fungi is a natural phenomenon for alteration to antimicrobial agents. Once bacteria become resistant to antibiotic, they pass on this resistance characteristics to their offspring's through horizontal or vertical transfer. The irrational use of antibiotics led to the development of antimicrobial resistance.[13],[14] The evolution of new resistant strains of bacteria and fungi that are somewhat more fatal compared to the parent strains. Screening of antimicrobial agents from source is now being conducted worldwide. Antimicrobial properties in plants are creditable to the presence of active compounds, for example, essential oil quinones, phenols, terpenoids, alkaloids, flavonoids, tannins, lignans, glucosinolates, and other secondary metabolites. Antimicrobial peptides have disulfide bonds and are positively charged. Peptides inhibit bacterial growth by forming ion channels in the bacterial membrane. Thionins peptide compounds are the antimicrobial peptides identified from the plant. Thionins are lipophobic and elicit toxicity to fungi and bacteria through membrane binding with their lipophilic interactions.[15]

P. niruri also called as Chanca Piedra, it means shatter stone. Bhumyamalaki is the Sanskrit name of the magnificent hepatoprotective plant P. niruri. Because of its antihepatotoxic property, P. niruri capsules are extensively recommended to improve the function of the diseased liver. Its leaves root and the whole plant are used as an herbal complement. P. niruri capsules are used for anemia, bronchitis, thirst, leprosy, urinary discharge, diuretic asthma, diuretic, and it also improves immunity. P. niruri containing Ayurvedic formulation.

The phytochemical screening of P. niruri yields secondary metabolites such as alkaloids, flavonoids, phenol, proteins, amino acids tannin, and carbohydrates. Alkaloid, tannins, terpenoids, flavonoids, and phenol are found in the ethanolic and methanolic extracts. Phenol, tannins, terpenoids flavonoids, and tannins components are of importance and interest in pharmaceutical science due to their antibacterial activity.[16]

Aqueous, ethanolic, and methanolic extracts of P. niruri does not showing antifungal activity against C. albicans (NCIM - 3100), A. flavus (NCIM - 1028). Antifungal inactivity due to absence of antifungal compounds in the extracts either C. albicans (NCIM - 3100), A. flavus (NCIM - 1028) showing resistance.

Aqueous, ethanolic, and methanolic extract of P. niruri showing good bacterial activity against S. aureus (NCIM - 5345), E. coli (NCIM - 5346), B. subtilis (NCIM - 2920). Antibacterial activity due to the secondary metabolites (flavonoids, polyphenols, and tannins) present in the extract. These secondary metabolites are naturally synthesized by plants as a mode of protection in opposite to microbial infection.

Medicinal plants contain polyphenolic compounds which have potent anticancer and immunomodulatory activities. These active compounds interfere with the formation of reactive intermediates. Potent radical scavenging properties of polyphenols make the effective weapon against cellular stress. The present work indicates P. niruri has potential immunomodulatory activity.


  Conclusion Top


From this study, it is concluded that P. niruri does not have antifungal activity but has potent immunomodulatory and antibacterial activities. These immunomodulatory and antibacterial activities of P. niruri may be due to secondary metabolites such as alkaloid, tannins, terpenoids, flavonoids, and phenol compounds.

Acknowledgment

The authors wish to acknowledge and thank the support of the Sanjo College of Pharmaceutical Studies and Anna University, BIT campus, Tiruchirappalli for providing all facilities necessary to carry out the work.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

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Purohit MR, Chandran S, Shah H, Diwan V, Tamhankar AJ, Stålsby Lundborg C, et al. Antibiotic resistance in an Indian rural community: A 'one-health' observational study on commensal coliform from humans, animals, and water. Int J Environ Res Public Health 2017;14. pii: E386.  Back to cited text no. 1
    
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Chandra H, Bishnoi P, Yadav A, Patni B, Mishra AP, Nautiyal AR, et al. Antimicrobial resistance and the alternative resources with special emphasis on plant-based antimicrobials-A review. Plants (Basel) 2017;6. pii: E16.  Back to cited text no. 2
    
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Available from: https://www.hannover-re.com/261349/emerging-risks.[Last accessed on 2017 Feb 16].  Back to cited text no. 3
    
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Muddukrishnaiah K and Krishnan S. Study of Bacterial Resistance in Clinical Isolates of Staphylococcus aureus by Comparison of Conventional in-vitro Methods with PCR & RFLP Based Genotypic Methods. British Journal of Pharmaceutical Research 2016;10:1-8.  Back to cited text no. 6
    
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Obiagwu IN, Okechalu OB, Njoku MO. Studies on antibacterial effect of the leaves of Phyllanthus niruri on some enteric pathogens. Niger J Biotech 2011;23:22-7.  Back to cited text no. 7
    
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Muddukrishnaiah K, Singh S. Antimicrobial, synergistic activity and antioxidant studies on multidrug resistance human pathogen using crude extract of Azadirachta indica leaf and Withania somnifera Rhizome. J Plant Pathol Microbiol 2015;S3;1-3.  Back to cited text no. 8
    
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Karri SK, Sheela A. Comparativein vitro antidiabetic and immunomodulatory evaluation of standardized five select medicinal herbs and spectral analysis of Boerhavia erecta L. (Nyctaginaceae). Pharmacogn J 2017;9:336-44.  Back to cited text no. 10
    
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Kotakonda M, Valiyaparambil S, Benson ST, Venkatachalam D, Ravindran VK. Phenotypic characterization of antimicrobial resistance in Kerala Bacillus spp isolated from paddy field. Int J Infect Dis Ther 2017;2:75-8.  Back to cited text no. 12
    
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Available from: http://www.cfsph.iastate.edu/factsheets/pdfs/mrsa-citations.pdf. [Last accessed on 2011 Feb 24].  Back to cited text no. 13
    
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Silva S, Rodrigues CF, Araújo D, Rodrigues ME, Henriques M. Candida species biofilms' antifungal resistance. J Fungi (Basel) 2017;3. pii: E8.  Back to cited text no. 15
    
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Cowen LE, Sanglard D, Howard SJ, David Rogers P, Perlin DS. Mechanisms of antifungal drug resistance. Cold Spring Harb Perspect Med 2015;5:1-22.  Back to cited text no. 16
    


    Figures

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    Tables

  [Table 1], [Table 2], [Table 3]



 

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