Exploration of  Antibacterial and Antioxidant Potential of Eclipta prostrata Leaf Extract

Eclipta prostrata (L.) commonly known as false daisy, yerba de tago, Guntakalagaraku, Karisalankanni and bhringraj, belongs to Asteraceae family, widespread across the world. It is rich in proteins, vitamins and antioxidants which help to protect the body against certain infections. The plant is known for its ability to support strong health. Bhringraj oil is effective remedy used to promote hair growth as well as various hair concerns for centuries. In fact, all the parts of this plant can be used for therapeutic purposes (Ambu et al., 2020; David et al., 2015).
       
This annual plant grows up to about a foot in height; it grows a solitary, white, winged flower reminiscent of a daisy. The flowers are quite delicate; the slightly curly leaves are strong and thick, with a layer of hairs that can cause the skin to itch when touched. A dark greenish-black liquid obtained up on crushing of flowers can be used to make dyes and inks for tattoos. In ayurvedic medicine, the leaf extract of bhringraj is also considered a powerful liver tonic and detoxifier. It contains several phytochemicals such as alkaloids, polyacetylenes, thiophene derivatives, flavonoids and triterpenes (Timalsina et al., 2021).
       
Eclipta prostrata (L.) is a source of antioxidants (Minh, 2019). Bioactive compounds of Eclipta prostrata are mainly contributed in medicinal value (Akhter et al., 2019). Traditionally E. prostrata was used in the treatment of inflammatory diseases. Methanolic extract of leaves of E. prostrata was proved to be anti-inflammatory activity in albino Wistar rats (Arunachalam et al., 2009). The leaf extract of E. prostrate known for antioxidant and antimicrobial activities (Nahid et al., 2017).  Recent studies also revealed that E. prostrata has curative properties such nephroprotective activity, anti-diabetic, analgesic, antibacterial, anti-breast tumour activity, anticancer activity, anti-hepatotoxic, hair growth promoter activity and memory enhancing activity and it is considered as a potent medicinal plant with tremendous pharmacological significance (Pooja et al., 2020).
       
This present investigation aims at phytochemical analysis of methanol extract of E. prostrate leaves, antioxidant activity and its antibacterial against Klebsiella pneumoniae and Staphylococcus aureus.

Microorganisms
 
Two bacterial isolates used in this study i.e., Klebsiella pneumonia and Staphylococcus aureus culture were provided by Skanda life sciences Pvt. Ltd., Bangalore.
 
Preparation of methanolic extract of Eclipta prostrata
 
Eclipta prostrata leaves were collected from local market of Bangalore. The leaves were shade dried, powdered and subjected to soxhlet extraction using methanol. After the extraction, crude extract was concentrated under reduced pressure using rotary evaporator and evaporated to dryness. The dried material was weighed and stored at 4°C until further use.
 
Phytochemical analysis
 
Phytochemical analysis of methanolic extract of E.prostrata were carried out by reconstituting the semisolid extract in methanol and analyzed  for the presence of different constituents such as alkaloids, carbohydrates, tannins, terpenoids, glycosides, steroids, saponins, flavonoids, glycoprotein, volatile oil, phenols by following standard procedures (Riazunnisa et al., 2013).
 
Estimation of total saponin content
 
Standard saponin solution was prepared by dissolving 10 mg of diosgenin with16 ml of methanol and 4 ml of distilled water. Further, 0.25 ml of 8% vanillin reagent was added to the aliquots for each tube, followed by addition of 2.5 ml of 72% sulphuric acid (v/v,). The solutions were mixed well and the tubes were incubated at 60°C under water bath for 10 mins. After incubation period the tubes were cooled in ice cold water bath. The absorbance was measured at 544 nm against the reagent blank. 0.1 g of freeze dried E. prostrata methanol was dissolved in aqueous methanol (80%, 0.1 ml). Total saponins were determined spectrophotometrically at 544 nm.
 
Ferric reducing antioxidant power (FRAP) assay
 
FRAP assay was carried out calorimetrically using DetectX ® FRAP™ (Ferric Reducing Antioxidant Power) Colorimetric Detection Kit (2 Plate Kit Catalog Number K043-H1). Experiment was carried out as per the manufacturer’s instructions. Briefly, working assay buffer was prepared by diluting concentrated assay buffer (1:10) with distilled water. FRAP colour solution was prepared by mixing  10 ml of assay buffer and  1 ml each of FRAP reagent A and FRAP reagent B reagent respectively  and mixed well. Vitamin C (M.W=176.12 g/mol) was served as control.
       
Different concentrations tested for reference standard i.e., 100, 50, 25, 12.5, 6.25 and 3.125 mg. To this sample 400 ml of assay buffer was added. Then 20 ml of samples/standards was pipette into 96 well plates and for control 20 ml of diluted assay buffer was added. Freshly prepared FRAP colour solution (75 ml) was added to each well and incubated at room temperature for 30 minutes. The absorbance was measured at 560 nm. A stronger absorbance will indicate increased reducing power.
 
Antibacterial activity assay
 
The antibacterial activity of methanol extract was assessed by well diffusion assay (Valgas et al., 2007).  The extract was dissolved in methanol to get 1 and 2 mg/ml concentration. The pre-sterilized, Soya Bean Casein Digested (SBCD) agar plates were poured into 90 mm Petri plates. After solidification, (SBCD) agar plates were inoculated with K. pneumoniae/ S. aureus with pre-adjusted cell density of 2×10⁶ cells/ml. Further, plates werepunched with a sterile cork borer (6 mm diameter) to make open wells at the four edges of the Petri plate. Open wells were filled with E. prostrata extract (1 mg/ml or 2 mg/ml), Standard Ciprofloxacin (0.1 mg/ml). Wells added with 50% methanol serves as control. The treated plates were incubated in at 37°C for 24-36 hrs. After incubation period plates were observed for antibacterial activity and evaluated by measuring the zones of inhibition of microorganism growth surrounding the extract using a millimeter ruler. The antibacterial analysis was carried out under strict aseptic conditions in triplicates and repeated thrice (Eagle and Musselman, 1948; Barry and Sabh, 1974; Bergeron et al., 1973).
 
Determination of minimum inhibitory concentration (MIC)
 
Cell suspension of K. pneumoniae and S. aureus culture were prepared by growing on tryptone broth at 37°C for 24 hrs and cell density was adjusted to 1-2 ×10⁸ cells/ml using 0.5 Mc Farland standards, serves as inoculum. Test concentration was prepared with different concentrations of Ciprofloxacin (8, 4, 2, 1, 0.5, 0.25 and 0.125 mg) in 90 mL tryptone broth. Similarly different concentrations of methanolic extract 1, 0.5, 0.25, 0.125, 0.625, 0.3125 and 0.156 mg in 90 mL tryptone broth. Tryptone broth inoculated only with respective bacterial culture and without any test compounds or standard served as control.
       
Minimum inhibitory concentration was determined by mixing 90 ml test samples/standard of different test concentration with 10 mL inoculum in 96 well plates (100 ml/well). Mix 90 ml tryptone broth with 10 ml inoculum served as control. Treated plates were incubated at 37°C for 24 hours. After incubation optical density (O.D.) at 590 nm was measured using plate reader. Resazurin (0.015 %) was added to all wells (30 ml per well) and further incubated for 2-4 hours for the observation of colour change. On completion of the incubation, columns with no colour change (blue resazurin colour remained unchanged) were scored as above the MIC value. Metabolism of resazurin by active bacterial cells leads to reduction of resazurin (Purpleblue) to resorufin (pink-colourless) pink colour. Determine MIC as minimum concentration of drug giving 50% inhibition of optical density as compared with control (John et al., 2000; Wikler 2006).

Phytochemical analysis
 
The qualitative phytochemical analysis of methanolic extract of E. prostrata exhibited the presence of alkaloids, tannins, glycosides, steroids, saponins, glycoproteins and volatile oil (Table 1).
 

 
Total saponin
 
The total saponin content was determined using vanillin reagent. Diosgenin was used as a standard compound and the total saponin content were expressed as mg/g diosgenin equivalent using the standard curve equation: y = 0.001× + 0.117, R 2 = 0.929, where y is absorbance at 544 nm and × is total saponin content in the methanolic extract expressed in mg/g. The total saponin content in the methanol extract of E. prostrata leaves was found to be 84.175 ± 0.575 in mg/g equivalent of diosgenin.
 
In vitro antioxidant activity; Ferric reducing antioxidant power (FRAP) assay
 
The in vitro antioxidant activity of E. prostrata clearly demonstrated leaves have prominent antioxidant properties. The absorbance increases at 560 nm and can be measured to test the amount of ferric reduced and can be expressed as µmolar Fe²⁺ equivalents or relative to an antioxidant reference standard Ferrous (Fe²⁺) in the methanolic extract of E. prostrata. Higher absorbance value indicates a stronger reducing power of the sample (Table 2).
 

 
Antibacterial activity by well diffusion method
 
Among the varied concentrations of methanolic extract of E. prostrata at 2 mg concentration exhibited potential inhibitory activity against K. pneumonia and S. aureus (Table 3; Fig 1 a and b) however, standard antibiotic, Ciprofloxacin exhibited its inhibitory activity at 2 mg/ml.
 

 

 
Determination of minimum inhibitory concentration (MIC)
 
The minimum inhibitory concentration of the E. prostrate against K. pneumoniae and S. aureus was 0.0625 mg/ml and 0.0312 mg/ml respectively. However, with ciprofloxacin MIC was 1.0 mg/ml and 0.5 mg/ml against K. pneumoniae and S. aureus respectively (Table 4).
 

       
Eclipta prostrata (Bhringraj) is considered to be having medicinal importance because of the presence of various secondary metabolites. This study was made to evaluate the methanol extract of E. prostrata leaves for its phytochemical analysis as well as antibacterial and antioxidant activity. The qualitative phytochemical test of methanolic extract of leaves of E. prostrata (L.) revealed the presence of presence of alkaloids, tannins, glycosides, steroids, saponins, glycoproteins and volatile oil. Recent report by Priya et al., (2018) also in support of current investigation that they reported presence of steroids, tannins, saponins, flavonoids, diterpenes and triterpenes further the GC-MS analysis exposed the compounds propanedinitrile dimethyl, Pentadecane, citronellyl butyrate, Citronellyl propionate, Heptadecanoic acid, methyl ester. Eclipta alba ethanol extract exhibited large zone of inhibition in disc diffusion for antibacterial screening against Gram-negative S.typhi, S. sonnei, E.coli, S.paratyphi, Pseudomonas species (I) and Pseudomonas species (II) and Gram-positive B.subtilis, B.cereus, B. megaterim and S.aureus and antifungal activity was observed against Aspergillus ochraceus (Uddin et al., 2010). In a similar kind of study phyto-chemical screening of the extract revealed the presence of tannins, flavonoids, saponins, alkaloids and the fractionated ethanol extract from Eclipta prostrata could be used against S. typhi pathogen (Karthikumar et al., 2007). The chemical composition of the essential oils from the leaves and stem bark of E. prostrata (L.) led to the identification of 33 and 30 compounds respectively (Ogunbinu et al., 2008).
       
The present study revealed the antimicrobial efficiency of methanol extract of E. prostrata against target pathogens i.e., K. Pneumonia and S.aureus. Shekokar and Nayak (2017) reported the solvent extracts of E.alba exhibited intermediate activity against S. aureus, B. cereus, E. coli, S. typhi, K. pneumoniae, P. aeruginosa, P. mirabilis and S. pyogenes in comparison with standard antibiotics (Ciprofloxacin). Ethanolic extract and active principle compound of E. prostrata was tested for in vitro antimicrobial studies (Sunita and Sudhir, 2010). Zone of inhibition studies and minimum inhibitory concentration exhibited activity against studied strains. The antimicrobial spectrum of E. prostrata was proved against Aspergillus niger, Candida albicans and Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus (Raghuwanshi et al., 2018).     
       
Eclipta prostrata is an important natural source of antioxidants (Sinha and Raghuwanshi 2016). Antioxidant activity of E. prostrata was determined by FRAP, radical scavenging activity, reducing activity and DPPH assay. The antioxidant capacity was increased by increasing the concentration of the extracts similar results were recorded by Jaglan et al., (2013).
       
In the present study from the results of antimicrobial assay it was evident that K. pneumonia 2 mg of E. prostrata methanol extract showed highest inhibitory activity against K. pneumoniae and S. aureus. The minimum inhibitory concentration of the E. prostrata against K. pneumoniae was 0.0625 mg for K. pneumoniae and 0.03125 mg for S. aureus whereas with ciprofloxacin standard it was 15 mg/ml and 0.5 mg/ml respectively. Similar kind of results were observed by Sonia et al., (2013) where ethanolic leaf extract of Catharanthus roseus had recorded the antimicrobial activity against Candida albicans, Pseudomonas aeruginosa and Aspergillus niger with zone of inhibition as 14, 13 and 8 mm respectively.

Eclipta prostrata is an important traditional herb used in the treatment of many diseases, which are usually free from side effects, are economical and also easily accessible to humans. On the basis of our results of the present study it is concluded that the methanolic extract of E. prostrate has significant antimicrobial and antioxidant activity. E. prostrate could be considered for the preparation of nutraceuticals with potent antimicrobial activity suitable for the prevention of plant as well as human diseases too. The extract can be subjected to identification and isolation of the potential antimicrobials and further screening can be used as sources for new drugs or formulations against human or phytopathogens respectively. The results obtained in the current study proved the antimicrobial and antioxidant potential of E. prostrata which is in support of earlier published reports.

The authors declare that they have no potential conflicts of interest  to disclose.