In Vitro Antioxidant and Antibacterial Activity of Boerhaavia diffusa


Jayasri R. and Anuradha R.*

PG and Research Department of Biochemistry, Sengamala Thayaar Educational Trust Women’s College, Sundarakkottai, Mannargudi -614016, Tamilnadu, India.



The present investigation has been carried out to evaluate the in vitro  antioxidant and antimicrobial activity. The in vitro antioxidant activity of leaves of  Boerhaavia diffusa was investigated  by Fe-EDTA method. In this method plant extract (leaves) possess high antioxidant activity when compared with standard ascorbic acid and the antimicrobial activity was visually recorded by measuring zone of inhibition in millimeters. The zone of inhibition varied by plant extract with different concentration. The largest zone of inhibition (19 mm) was found against E. coli using ethanolic extract of leaves of Boerhaavia diffusa. The result obtained indicates that the ethanolic extract of Boerhaavia diffusa showed high antioxidant and antimicrobial activity.


KEYWORDS: Antioxidant activity, Antibacterial activity, Fe-EDTA, Boerhaavia diffusa.




An antioxidant is a molecule capable of inhibiting the oxidation of other molecules. Oxidation is a chemical reaction that transfers electrons from a substance to an oxidizing agent. Oxidation reactions can produce free radicals. In turn, these radicals can start chain reactions that damage cells. Antioxidants terminate these chain reactions by removing free radical intermediates, and inhibit other oxidation reactions. They do this by being oxidized themselves, so antioxidants are often reducing agents such as thiols, ascorbic acid or polyphenols (Sies,1987).


Antimicrobial activity

An anti-microbial is a substance that kills or inhibits the growth of microorganisms. Antimicrobial drugs either kill microbes (microbiocidal) or prevent the growth of microbes (microbiostatic). Disinfectants are antimicrobial substances used on non-living objects or outside the body. Antimicrobial substance inhibits the growth of microorganisms. The plant parts possessing antimicrobial properties can be used as potential source of antimicrobial agents. Infectious diseases caused by number of microorganisms are the world’s major threat to human health and account for almost 50,000 deaths every day (Ahmad and Beg, 2001).


Plants have the major advantage of being effective and cheaper alternative source of drugs (Pretorious and Watt,2001).The use of plant extracts with known antimicrobial properties can be of great significance in therapeutic treatments. In the last few years, studies have been conducted in different countries (Almagboul et al.,1985; Artizzu et al.,1995; Ikram and Inamul, 1984; Izzo et al.,1995; Kubo et al.,1993).



In the present study, in vitro experiment, such as Ferrous EDTA was performed to assess the antioxidant potential of ethanolic extract from the leaf of Boerhaavia diffusa and then the antimicrobial potential of the extract was assessed against E. coli.


Boerhaavia diffusa (Nyctaginaceae) commonly known as Raktapunarnava, Shothaghni, Kathillaka, Kshudra, Varshabhu, Raktapushpa, Varshaketu, Shilatika (Rendle, 1925 and Yelne, 2000) is a herbaceous plant species growing prostrate or ascending upward in habitats like grasslands, agricultural fields, fallow lands, wastelands and residential compounds. The plant was named in honor of Hermann Boerhaave, a famous Dutch physician of the 18th  century (Chopra, 1969).  The plant is mentioned in the Atharvaveda with the name ‘Punarnava’, because the top of the plant dries up during the summer season and regenerates again during the rainy season. Thus the plant generally perennates through the roots in the soil (Singh, 2007). Boerhaavia diffusa (Nyctaginaceae) is one of the most famous medicinal plants in India; it is a perennial herbaceous plant growing in tropical regions such as the Antilles, South America, India and Africa (Correa, 1984).



Collection of plant

The healthy plant leaf of Boerhaavia diffusa leaf was collected from Edaiyarnatham Village, Mannargudi Taluk, Thiruvarur District, Tamil Nadu India. It was collected in early morning and was washed in tap water and shade-dried for 10 days.


Preparation of plant extracts

The shade dried plant material was powdered using kitchen blender and that powder was subjected to soxhlet extraction with Ethanol (60°C) for 24hrs. The solvent extract was distilled and condensed at 40°C .The condensed extract was stored at room temperature in air tight bottles and uses for further studies.


Determination of Antioxidant Property

The antioxidant activity of the ethanolic extract of plant was determined by the invitro method using Fe–EDTA


Anti microbial activity

Collection of microorganisms for Antimicrobial activity

The E.coli isolated from faecal and it was subcultured.


Kirby-Bauer Disc Diffusion Method

Kirby –Bauer disc diffusion method is commonly employed for antibiotic sensitivity test .The test is based on the fact that for given antibiotic, the size of zone of inhibition is related to Minimum Inhibitory Concentration (MIC). MIC is referred as the lowest concentration of antibiotic that exhibit the zone of inhibition of the assay plate.


Determination of concentration of antimicrobial activity

Since agar disc diffusion assay is a qualitative method used in microbial testing, the determination of MIC was done to obtain quantitative results on the antibacterial effects of the plant extracts. The method used to determine the MIC was the agar dilution method of the European Society of Clinical microbiology and infectious diseases. Test microorganisms were spotted on the surface of the solidified extract-agar mixture. Four spots were placed in each plate at an amount of 10 μl (104 cfu for bacteria and yeast or 103 cfu for mold). The plates were inoculated by starting from the lowest concentration upto the highest concentration controls (agar without extract) were also inoculated at the start and at the end of the dilution.



The present study investigate the antioxidant activity of ethanolic extract of  Boerhaavia diffusa by invitro method using Fe–EDTA and  the antimicrobial activity was analysed against E. coli by Kirby Bauer disc diffusion method. The results are described below.


Analysis of Antioxidant Property

The reducing ability of a compound generally depends on the presence of reductants which have been exhibited antioxidative potentially breaking the free radical chain, donating a hydrogen atom. The presence of reductants (i.e. antioxidants) in ethanolic leaf extract of Boerhaavia diffusa causes the reduction of the Fe3+/ferricyanide complex to the ferrous form. Therefore, the Fe2+ can be monitored by measuring at 620 nm. The results were given in the Table 1.


Ebrahimzadeh et al., (2010)  reported that Fe2+ has been shown to cause the production of oxyradicals and lipid peroxidation, minimizing Fe2+ concentration in Fenton reactions affords protection against oxidative damage. Ferrozine can quantitatively form complexes with Fe2+. In the presence of other chelating agents, the complex formation is disrupted with the result that the red colour of the complexes decreases. In this assay, both extract and EDTA interfered with the formation of ferrous and ferrozine complex, suggesting that it has chelating activity and captures ferrous ion before ferrozine.Metal chelating capacity was significant since the extract reduced the concentration of the catalyzing transition metal in lipid peroxidation. It was reported that chelating agents are effective as secondary antioxidants because they reduce the redox potential, thereby stabilizing the oxidized form of the metal ion.


The present study showed the ethanolic leaf extract of Boerhaavia diffusa possessed the antioxidant property compared with standard ascorbic acid. From the observation of results, it had been well noticed that the ethanolic extract of the leaf Boerhaavia diffusa possessed good antioxidant capacity.


Antibacterial activity

The antimicrobial activity was assessed by disc diffusion method. The zone of inhibition was assessed and the plates were kept at room temperature for 24 hours. The plant Boerhaavia diffusa was assessed for the antimicrobial activity against E. coli and the rate of zone of inhibition was 19 mm respectively. The results were given in the Table 2.


Ogundare et al., (2009) reported that the inhibitory activity of the leaf extract of Morinda lucida on E.coli was investigated both in vitro. The in vitro experiment was carried out using the agar well diffusion method and showed that 25 mg/ml of the extract inhibited E. coli with a zone of inhibition measuring 5 mm. The extract has been able to control the establishment of E. coli infection.


From the antibacterial activity, it was confirmed that ethanolic extract of leaf of Boerhaavia diffusa is effective and it may be used for the treatment of bacterial infections.


Table 1: Antioxidant  activity of plant extract as measured by  Fe – EDTA Method


Boerhaavia diffusa(leaf)









S –Standard


E0 - 50% of Ethanolic leaf extract of Boerhaavia diffusa

E -70% of Ethanolic leaf extract of Boerhaavia diffusa


Table 2: Antibacterial activity of  plant extract against E. coli


Zone of Inhibition(mm)



Boerhaavia diffusa (50%)


Boerhaavia diffusa (70%)


Boerhaavia diffusa (80%)






In the present study proved that the ethanolic leaf extract of Boerhaavia diffusa possessed good antioxidant activity by using Fe-EDTA method and also proved that the plant Boerhaavia diffusa was possessed for the antimicrobial activity against E. coli. Further investigation on the isolation and identification of antioxidant and anti microbial component (s) in the plant may lead to chemical entities with potential for clinical use.



The authors are grateful to the management of STET Women’s College, Mannargudi for providing laboratory facilities.



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Received on 02.04.2012

Modified on 28.04.2012

Accepted on 08.05.2012

© A&V Publication all right reserved

Research Journal of Pharmacognosy and Phytochemistry. 4(4): July- August 2012, 223-225