Antimicrobial Studies on Aqueous and Ethanolic Extract of Root, Stem and Leaves of Phyllanthus niruri (Bhumi Amla): Hepatoprotective Medicinal Plant

 

Reni Nigam, Praveen Garg

Department of Biotechnology, AKS University, Satna, Madhya Pradesh, 485001, India. Department of Science, VITS College, Satna, Madhya Pradesh, India.

 

ABSTRACT:

This study aims to evaluate the antimicrobial potential of ethanolic and aqueous extracts of P. niruri on three bacterial and three fungal pathogens. Agar well diffusion has been used to determine the antimicrobial activities of different parts of P niruri. The extracts showed antibacterial activity against tested bacterial strains while no inhibitory activity against all three tested fungal strains was observed. The ethanol extract of P. niruri (leaves, stem and root) showed potential antimicrobial activity than corresponding aqueous extracts. Furthermore, the least antimicrobial activity of particularly the aqueous stem extract of P. niruri on S. mutans amongst the other bacteria, was observed. This study showed antibacterial activity of P. niruri extracts which depends on the dose, parts of plant and the type of bacterial strain employed. In conclusion, results obtained here suggest that plant extracts are of significant value as natural antimicrobials can be used as antimicrobial agent.

 

 

INTRODUCTION:

People have employed plants as medication of wide variety of health-related applications ranging from treatment of common colds to treatment of cancer.¹ Hepatotoxicity has become serious problem in public health care contributing to more than 50% of acute liver failure cases.² Plant products would be biodegradable and safe to human health therefore, plant products have attracted the attention of microbiologists in several developing countries to search for bioactive constituents for their exploitation as anti-microbial agent in health care services.^3-4There are numerous reports on the phytochemical screening, antimicrobial and antioxidant activity of various herbal extracts in several regions of the world and purified constituents of large number of medicinal plants have shown beneficial therapeutic importance.⁵ Plants are the potential source of antimicrobials which protect them from diverse pathogens.^6-8

In response to microbial infection, plants synthesized plant secondary metabolites; phenols, quinones, phenolic acids, flavonoids, flavones, coumarins and tannins that have demonstrated antimicrobial.^9-10 P. niruri (syn P. amarus, P. fraternus Webster) is a typical kharif weed found in both wastelands and cultivated area belongs to the family Euphorbiaceae. It is a small annual herb, shallow rooted, erect, glabrous that grows 30 to 40 cm in height, generally known as stonebreaker which means seed-under leaf. ¹¹ P. niruri have used as the whole plant as it is a small herb in most of cases.¹²

The powder and fresh juice of P. niruri used in many ayurvedic formulations as well as in other medicinal system such as Chinese and Unani. ^13-14 Different parts of plant like root, fruits, milky juice, leaves and whole plants are used as medication. Fruits are accepted as great solution for many ailments like scabies, wounds, tubercular ulcers, ring worm and sores. The root is valuable for jaundice. The infusion of the root and leaves is used as tonic and diuretic. In India, it is an important component of liver tonics including Liv.-52.¹⁵ Different plant parts of Phyllanthus niruri showed several pharmacological activities viz., hepatoprotective,^16-18 Diuretic activity, ¹⁹ Antidiabetic activity,²⁰ Anti cancerous,^21-22 Antimicrobial activity,^23-24 Antioxidant activity.^25-26 Accordingly, this study conducted screening of different part extracts (root, stem and root) of P. niruri for antimicrobial activity, which had previously raised significant interest of many researchers, in order to identify therapeutically most important parts of plant extracts.

 

MATERIAL AND METHODS:

Collection and authentication of plant material

From different region of chitrakoot, district satna, MP, India, P. niruri were collected. The species was authenticated by Dr. Manoj Tripathi, scientist, DRI, chitrakoot, who is a renowned scientist in the field of herbal medicine.²⁷

 

Preparation of extracts:

Plant sample (root, leaves and stem) were washed with Milli-Q water and shade dried at room temperature for 10 days. The dried plant parts were pulverized and stored in air-tight containers at 4^oC for future use. For defatting of plant samples, all the shade dried powdered plant samples (100g) were defatted with 200 ml petroleum ether by maceration process till the defatting of the plant samples had taken place. The residue obtained were dried and then, subjected to successive extraction by maceration extraction process using two different solvents (ethanol and aqueous) as solvents. Then, filtrates were separately evaporated by distilling the solvents at low temperature using water bath. A brown slurry-like substance was obtained. Finally, all the plant extracts stored at four degree celsius in labelled sterile bottles until further use.²⁷

 

Antimicrobial test :

The microorganisms selected for antimicrobial study were E. coli, S. mutans, B. subtilis, C. albicans, A. niger and A. flavus. The microorganisms were obtained from Microbial Culture collection, National Centre for cell science, Pune, Maharashtra, India. Different bacteria and fungus were used for the study and their name and MTCC No. are listed below.

 

Table 1. Name of pathogenic bacteria/ fungus

 

Antimicrobial sensitivity:

The lawn cultures were prepared with all the microbial strain used under this study and sensitivity of selected microbial strain towards extracts were studied at the concentration of 100mg/ml using agar well diffusion method. Selected bacteria and fungus with inhibition zones were sensitive to the plant extract and without zone of inhibition were considered resistant strain.

Agar well diffusion method:

The agar well diffusion method was adopted to determine the antibacterial activity of the selected plant extracts of P. niruri.²⁸ Firstly, microbial inoculum was swabbed over the surface of culture media. Then, four wells of 6mm in diameter and about 2cm apart were punched in the culture media containing petri plates with pre-sterilized tips. The plant extracts/standard antibiotics of different concentration were placed on the holes. Three different concentration of plant extracts (25, 50 and 100 mg/ml) were selected for antimicrobial test. Ofloxacin and Ciprofloxacin were used as standard. Ofloxacin (10 µg/ml, 20 µg/ml, 30 µg/ml) was used for Streptococcus mutans and Ciprofloxacin (10µg/ml, 20µg/ml, 30 µg/ml) for Escherichia coli and Bacillus subtilis. Subsequently, petri plates were incubated at 37^oC for 24-48hrs. (Bacteria) and after incubation, they were removed and observed to see clear zones of inhibition around the well. The diameter of zone of inhibition formed around the well was measured in millimeter and their average determined.

 

Results and Discussion:

Results of antimicrobial sensitivity are shown in the table no. 2. Phyllanthus niruri extracts did not show any sensitivity against employed fungal strain, which confirmed to those, reported by previous researcher elsewhere.

29-30

 

Table 2. Results of antimicrobial sensitivity of Phyllanthus niruri

 

Table 3. Antimicrobial activity of standard drug on different bacteria

 

Antimicrobial activity of aqueous extract of P. niruri

Table no. 3, 4 and figure no. 1 showed zone of inhibition in culture plate of E. coli, S. mutans and B. subtilis with aqueous extract of leaves, stem and root of P. niruri. It is evident from the results of zone of inhibition that the aqueous extract of P. niruri leaves was highly effective against B. subtilis, moderately effective against S. mutans and least effective against E. coli. Results of the aqueous extract of P. niruri stem at same concentration shows zone of inhibition against E. coli (16±0.11), B. subtilis (13±0.14) and S. mutans (8±0.08). A brief look at table no. 4 figure no. 1, 3, 4  and 5 showed that the lowest zone of inhibition was observed when aqueous root extract of P. niruri was incubated with S. mutans whereas, highest zone of inhibition was observed when extract was incubated with B. subtilis. Leaves (aqueous extract) was more effective against employed bacteria compared to ofloxacin and ciprofloxacin. Aqueous extract of stem and root were less effective against employed bacteria compared to ofloxacin and ciprofloxacin.

 

Table No. 4 Antimicrobial activity of P. niruri aqueous extracts         

Values are expressed as the mean ± SEM of six observations. ^*** P<0.001 vs. control treatment (One-way ANOVA followed by Dunnett's test)

 

Figure 1. Antimicrobial activity of different parts of P. niruri aqueous extracts

 

Antimicrobial activity of ethanol extract of P. niruri

The results of the antibacterial activity for ethanol extract of leaves, stem and root parts of P. niruri against E. coli, S. mutans and B. subtilis are presented in table no. 3, 5 and figure no. 2, 3, 4, 5. The ethanol extract of P. niruri (leaves stem and root) were inhibitory to employed strains with increase in dilutions, zone of inhibition was increased. As indicated in table 3, 5 which ciprofloxacin showed maximum zone of inhibition against B. subtilis (17 ± 0.04) but leaves extract showed maximum zone of inhibition (24 ± 0.16) against B. subtilis. Likewise, ofloxacin showed maximum zone of inhibition (17 ± 0.19) against S. mutans but leaves extract showed maximum zone of inhibition (23 ± 0.18). Ethanol extract of the plant stem was more effective against E. coli, S. mutans and B. subtilis compared to standard ofloxacin and ciprofloxacin. P. niruri root extract had high impact on bacteria such as 18 ± 0.09, 22 ± 0.18, 24 ± 0.08 against E. coli, S. mutans and B. subtilis respectively.

 

Moreover, collective analysis indicated that the leaves, stem and root extracts showed potential antimicrobial activity against bacteria. Earlier workers had also shown that whole plant and leaves of P. niruri exhibit broad antimicrobial activity against bacteria and fungus.^{15, 24} This study showed antibacterial activity of plant extracts which depends on the dose, parts of plant and the type of bacterial strain employed. Previous studies on antimicrobial activities of P. niruri (leaves, stem and root) were in agreement with the present study of strong antimicrobial activity of P. niruri.^{23, 31}

 

Table 5. Antimicrobial activity of P. niruri ethanolic extracts:

Values are expressed as the mean ± SEM of six observations. ^*** P<0.001 vs. control treatment (One-way ANOVA followed by Dunnett's test)

 

 

Figure 2. Antimicrobial activity of different parts of P. niruri ethanolic extracts

Figure 3. Antibacterial activity of Phyllanthus niruri leaves. Frame a, b, c, d, e and f shows zone of inhibition of aqueous and ethanolic leaves extracts against E.coli, S. mutans and B. subtilis respectively.

 

Figure 4. Antibacterial activity of ^{Phyllanthus niruri} stem. Frame a, b, c, d, e and f shows zone of inhibition of aqueous and ethanolic stem extracts against ^{E. coli, S. mutans} and ^{B. subtilis} respectively.

	a		b		c

 

 

Fig- 5 Antibacterial activity of ^{Phyllanthus niruri} root. Frame a, b, c, d, e and f shows zone of inhibition of aqueous and ethanolic root extracts against ^{E. coli, S. mutans} and ^B. subtilis respectively.

 

 

CONCLUSION:

On the basis of the antibacterial assay of this study, bacteria were found (susceptible to the employed plant extracts) than fungi. The standard (ofloxacin and ciprofloxacin) inhibits the growth of all tested bacteria. The results of present study observed variation in in vitro antibacterial activities of the different parts of P. niruri, specially the leaves, stem and root to inhibit bacterial activity. Furthermore, the least antimicrobial activity of particularly the aqueous stem extract of P. niruri on S. mutans amongst the other bacteria, was an indication that antibacterial phytochemicals were not evenly distributed and their absolute concentration may vary within or even absent in some plant organs that constitute the plant system. The ethanol extract of P. niruri (leaves, stem and root) showed potential antimicrobial activity than corresponding aqueous extracts. The present study showed antibacterial activity of plant extracts which depends on the dose and the type of bacterial strain employed. However, a detailed pharmacological study of P. niruri can be done to make new medicines by using bioactive constituent present in leaves, stem and root.

 

CONFLICT OF INTEREST:

The authors have no conflict of interest regarding this investigation.

 

ACKNOWLEDGEMENTS:

The authors are thankful to Director, Scan research laboratory for providing facilities for completion of this work.

 

REFERENCES:

1.       Thalluri J. A review on medicinal plants research & reviews. Journal of Medicinal & Organic Chemistry. 2016; 3(2): 157-63.

2.       Selvanayaki R. Ananthi T. Hepatoprotective activity of aqueous extract of Lawsonia inermis against paracetamol induced Rats. Asian J. Pharm. Res. 2012; 2(2): 75-7.

3.       Ulfatun N. Astana PRW.  Kuncoro H. The effect of antihypertensive herb formula of Indonesian traditional medicines against serum uric acid levels in mild hypertensive patients. Research J. Pharm. and Tech. 2021; 14(1): 254-8. DOI: 10.5958/0974-360X.2021.00045.7

4.       Saha D. Paul S. In vitro screening of antifungal activity of methanol extract of Plumbago indica L. against some pathogenic species of fungi. Asian J. Res. Pharm. Sci. 2012; 2(2): 55-57.

5.       Rao N. Mittal S. Sudhanshu, Menghani E. Assessment of phytochemical screening, antioxidant and antibacterial potential of the methanolic extract of Ricinus communis l. Asian J. Pharm. Tech. 2013; 3(1): 20-25.

6.       Alkofani AS. Abdelaziz A.  Mahmoud I. Aburije M. Huraiti A. Eiogla A. Cytotoxicity, mutagenicity and antimicrobial activity of forty Jordanian medicinal plants. International Journal of Crude Drug Research. 1990; 28(2): 1139-44.

7.       Grayer RJ. Kokubun T. Plant-fungal interactions: The search for phytoalexins and other antifungal compounds from higher plants. Phytochemistry. 2001; 56(3): 253-63.

8.       Olukoya DK. Idugbmi T. Antibacterial activity of some medicinal plants from Nigeria. Journal of Ethnopharmacol. 1993; 39(1): 69-72.

9.       Silva MTG. Simas SM. Batista TGFM. Cardarelli P. Tomassini TCB. Studies on antimicrobial activity, in-vitro, of Physalis angulata L. (Solanaceae) fraction and physalin B bringing out the importance of assay determination. Memorias do Instituto Oswaldo Cruz. 2005; 100(7):779-82.

10.     Cowan MM. Plant products as antimicrobial agents. Clinical Microbiology Reviews. 1999; 12(4): 564- 82.

11.     Ramamurthy V. Abarna T. Evaluation of ethanolic leaf extract of Phyllanthus niruri and its effect on carbon tetrachloride intoxicated hepatotoxicity in albino rats. International Journal of Innovative Research in Science, Engineering and Technology. 2015; 4(7): 6255-261.

12.     Sabdoningrum EK. Sri Hidanah. Yuniarti WM, Sri Chusniati. Warsito SH. Muchtaromah B. Antimicrobial activity of Phyllantus niruri extract on avian pathogenic Escherichia coli Isolated from Chicken with Colibacillosis symptoms. Research J. Pharm. and Tech. 2020; 13(4): 1881-85. doi: 10.5958/0974-360X.2020.00339.X

13.     Barros ME. Schor N. Boim MA. Effects of an aqueous extract from Phyllantus niruri on calcium oxalate crystallization in vitro. Urological research. 2003; 30(6): 374-79.

14.     Bieski IG. Leonti M. Arnason JT. Ferrier J. Rapinski M. Violante IM. Balogun SO. Pereira JF. Figueiredo Rde C. Lopes CR. Da Silva DR. Pacini A. Albuquerque UP. Martins DT. Ethanobotanical study of medicinal plants by population of valley of Juruena region, Legal amazon, Mato grosso, Brazil. Journal of Ethnopharmacology. 2015; 173: 383-423.

15.     Nivetha S. Vetha Roy D. Antimicrobial studies on ethanolic extracts of Phyllanthus niruri. Journal of Chemical and Pharmaceutical Research. 2015; 7(6): 103-6.

16.     Syamasundar KV. Singh B. Thakur RS. Husain A. Kiso Y. Hikino H. Antihepatotoxic principles of

Phyllanthus niruri herbs. Journal of Ethnopharmacol. 1985; 14(1): 41–44.

17.     Sarkar MK. Sarkar K. Bhattacharjee R. Chatterjee M. Sil PC. Curative role of the aqueous extract of the herb, Phyllanthus niruri, against nimesulide induced oxidative stress in murine liver. Biomedical Research. (2005); 16(3): 171-76.

18.     Iqbal Md J. Dewan FZ. Chowdhury SAR. Mamun MIR. Moshiuzzaman M. Begum M. Pre-treatment by n-hexane extract of Phyllanthus niruri can alleviate paracetamol-induced damage of the rat liver. Bangladesh Journal of Pharmacology. 2007; 2(1): 43-48.

19.     Udupa AL. Benegal SA. Prusty V. Kodancha GP. Satish Kumar MC. Bhat V. Ratnakar UP. Diuretic activity of Phyllanthus niruri (Linn.) in rats. Health. 2010; 2(5): 511-12.

20.     Santwana R. Baidyanath K. Glycemic control efficacy of Phyllanthus niruri Linn extract in diabetic mice model. International Journal of Current Microbiology and Applied Science. 2015; 4(4): 599-609.

21.     Sharma P. Parmar J. Verma P. Sharma P. Goyal PK. Anti-tumor activity of Phyllanthus niruri (a medicinal plant) on chemical-induced skin carcinogenesis in mice. Asian Pacific Journal of Cancer Prevention. 2009; 10(6): 1089-94.

22.     Sharma P. Parmar J. Verma P. Sharma P. Goyal PK. Chemo preventive effect of Phyllanthus niruri on DMBA induced skin papillomagenesis in swiss albino mice. International Journal of Biological and Medical Research. 2010; 1(4): 158-64.

23.     Pasha F. Irfan S. A study on antimicrobial properties of Phyllanthus niruri and Ocimum sanctum. Biomedical & Pharmacology Journal. 2011; 4(2): 297-99.

24.     Dangi AS. Sharma M. Yadav A. Arora JP. Chaudhary DRU. Antimicrobial activity of some medicinal plants. International Journal of Environmental Sciences. 2013; 3(2):1308-13.

25.     Anusha JR. Remya RV. Sasi Premila JM. Albin TF. Antioxidant and anticandidal activity studies on phyllanthin compound from Phyllanthus niruri. International Journal of Pharmacy and Pharmaceutical Sciences. 2014; 6(2): 223-26.

26.     Amin ZA. Abdulla MA. Ali HM. Alshawsha MA. Qadirc SW. Assessment of In vitro antioxidant, antibacterial and immune activation potentials of aqueous and ethanol extracts of Phyllanthus niruri. Journal of the Science of Food and Agriculture. 2012; 92(9): 1874-77.

27.     Harborne JB. Phytochemical methods, a guide to modern techniques of plant analysis, 3rd ed. Chapman and Hall, London. 2007.

28.     Bauer AW. Kirby WMM. Sherris JC. & Turck M. Antibiotic susceptibility testing by a standardized single disk method. American Journal of Clinical Pathology. 1966; 36: 493-96.

29.     Jahan N. Sherwani SK. Naqvi GR. Antibacterial potency of plants against clinical samples and toxicity against selected medicinal plant. Int Res J Pharm. 2013; 4(40): 109-12.

30.     Shah RA. Khan S. Sonawane PD. Rehman W. Phytochemical finger printing and antimicrobial activity of Phyllanthus niruri. International Journal of Pharmaceutical Sciences Review and Research. 2017; 44(2): 7-11.

31.     Adebayo OL. Edwin BA. Solvent extracts of different parts of Ghanaian Phyllanthus niruri show potent growth inhibition against six human pathogenic bacteria in vitro. International Journal of Pharmacy and Pharmaceutical Sciences. 2014; 6(7): 272-77.

 

 

Accepted on 27.04.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2022; 15(11):5278-5282.