Phyotochemical Properties of Acalypha indica (L), and its Antimicrobial Potential against Human Pathogens

The present work has been investigated for important medicinal properties of Acalypha indica (L). A. indica crude extract was obtained, significant phytochemicals and antibacterial activity substance for the control of numerous antibiotic resistance bacteria such as Escherichia coli, Salmonella typhi, Staphylococcus epidermidis, Bacillus cereus, Staphylococcus marcescens, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pyogenes and Streptococcus dysgalactiae. Antibacterial activity of A. indica was performed using the crude extract on MullerHinton agar with and without 5% sheep blood by the well diffusion method. We found Ethanol is the most favorable solvent for maximum amount of A. indica extract followed by Acetone, however acetone has more antimicrobial activity. Furthermore, we found Benzene and Diethyl ether are low yielding solvent for A. indica extract. A. indica extract on antimicrobial activity showed remarkable activity in S. typhi, B. cereus followed by S. epidermidis. Eight, phytochemicals tested in A. indica extract showed the existence of tannin, saponin, alkaloid, flavonoid and phenol. Petroleum ether and ethyl acetate solvent (4.2:0.8), was most optimum for the separation of more compounds by TLC analysis. In A. indica four major spots were detected, of these, two major spots were scrabbled from the TLC plates and checked for antimicrobial activity against B. cereus pathogen. B. cereus was selected based on our preliminary results, which showed significant activity among other pathogens.


INTRODUCTION
Medicinal plants have been used for the treatment of human diseases and also used for prophylactic purposes (Nostro et al., 2000). An increasing interest in the herbal based treatment for disease control observed in different countries and herbal remedies integrated into orthodox herbal practice (Wachtel-Galor and Benzieet, 2011). Some of the diseases such as malaria, epilepsy, infantile convulsion, diarrhea, dysentery, fungal and bacterial infections have been treated using medicinal plants (Sofowora, 1996). Medicinal plants are considered as a chemical factory, since it contains alkaloids, glycosides, saponins, resins, oleoresins, sesquiterpene, lactones and oils (Singh, 2005).
A c a l y p h a i n d i c a ( L ) f a m i l y o f Euphorbiaceae, its annual herb and commonly found in gardens, roadsides, waste places, fields throughout the plains of India and commonly known as "Kuppaimeni (Burkill, 1985). In addition, it is found in many parts of Asia, including India, Pakistan, Sri Lanka and throughout Tropical Africa and South America (Davy et al., 2012). A. indica used as emetic, expectorant, laxative, diuretic bronchitis, pneumonia, asthma and pulmonary tuberculosis. In homeopathy, this plant is used for severe cough, bleeding from lungs, haemopytosis and incipient phthisis (Devi et al., 2012).
Synthetic drugs may have side effects and antibiotic resistant microorganisms also increasing every year. Hence, researchers are paying more attention in herbal drug discovery for the treatments of varies diseases (Esakkirajan et al., 2014). The traditional medicinal plantsbased treatment covers the basic health needs in the developing countries (Alavijeh et al., 2012). India is one of the richest and different cultural traditions connected with the use of medicinal plants to cure different diseases. Hence, in this present study, we have taken A. indica, medicinal plants, to check antimicrobial activity against human disease causing bacteria pathogens and to evaluate phytochemical properties to understand the therapeutic value of this important plant.

Plants collection
Healthy, fresh leaves of A. indica were collected in and around the campus of Madurai Kamaraj University, Madurai, Tamil Nadu, India. Collected leaves were washed thoroughly with tap water and then distilled water. The leaves were dried under the shade and grinded into fine powder using an electric grinder.

A. indica extract preparation
A. indica extract was prepared from the dried fine powder, using organic solvents. Five solvents (Polar nature-Ethanol, Acetone, non-polar nature -Benzene, Ethyl acetate, Diethyl ether), were used to extract compounds (Molychem, India). One-gram of powder was added in a 250 ml Erlenmeyer flask with the above said solvents (1:10, w/v) and kept at room temperature for 3 days with frequent mixing. The samples were filtered (Whatman No 3), the extract collected, and solvents were evaporated at room temperature until dry. This process was repeated for three times and dried extracts were dissolved in Dimethyl sulphoxide (DMSO).

Growth and maintenance of bacterial culture
Pathogens such as E. coli, S. typhi, S. marcescens, K. pneumoniae, B. cereus, S. epidermidis, S. aureus were grown LB Agar or LB Broth (Himedia, India), whereas S. agalactiae, S. pyogenes and S. dysgalactiae were obtained from Microbial Technology and Immunology Department respectively, Madurai Kamaraj University, Madurai, Tamil Nadu, India and grown on blood agar or Todd-Hewitt broth with 1% yeast extract. Mueller-Hinton agar with and without sheep blood (5%) was used for antimicrobial activity assay. The cultures were grown at 37°C for 20-24 hrs. All the media were prepared according to manufacturer's instructions (Himedia, India).

Antimicrobial activity assay
Antimicrobial activity with crude extracts was performed using the well diffusion method on Muller-Hinton agar with and without 5% sheep blood (Gupta et al., 2013). Fresh colonies of E. coli, S. typhi, S. marcescens, K. pneumoniae, B. cereus, S. epidermidis, S. aureus were grown one ml of sterilized LB Broth, while S. agalactiae, S. pyogenes and S. dysgalactiae were grown on Todd-Hewittt broth with 1% yeast extract and incubated at 37°C for 18 -20 hrs. A sterile cotton swab was dipped into the culture and was spread uniformly on respective Mueller-Hinton agar plates with and without sheep blood (5%). Crude extract in DMSO at 1, 5, 10 and 15 mg in an each well was added.
Tetracycline (50µg each well) and DMSO were used as positive and negative control respectively for all the experiments. Inoculated plates were incubated at 37°C for 20-24 hrs. Subsequently, a zone of inhibition was measured by a standard ruler scale (Himedia, India).

Tannin assay
A 2 ml A. indica extract was mixed with 2 ml distilled water, then a few drops of Ferric Chloride (FeCl 3 ) solution were added and color formation was observed.

Saponin assay
A 2 ml plant extract was taken in a test tube and shaken vigorously with 5 ml distilled water, warmed in a water bath and then foam formation was observed.

Alkaloid assay
A 3 ml plant extract was mixed with 3 ml HCl (2%) on the steam bath. From the mixture each one ml was added in two test tubes. Few drops of Dragendorff's reagent were added in one tube and Mayer's reagent was added in another tube then color formation was observed.

Flavonoid assay
A 1 ml extract with 1ml of 10% lead acetate solution was added and color formation was observed. Terpenoid assay A 2 ml extract was mixed with 2 ml of CHCl 3 then evaporated to dry. After that 2 ml of concentrated H 2 SO 4 was added and heated for 2 minutes and then color formation was observed.

Glycoside assay
A 2 ml extract with diluted HCl was added then 2 ml of Sodium Nitropruside in Pyridine followed by Sodium hydroxide solution was added and color formation was observed.

Steroid assay Salkowski's test
A 2 ml plant extract was mixed with 2 ml chloroform then 2 ml concentrated sulphuric acid was added then color formation was recorded.

Liebermann Burchard test
A 2 ml plant extract was mixed with 2 ml chloroform then added concentrated sulphuric acid and acetic acid, the development of color formation was observed.

Phenol assay
Ethanol extract (500 mg) was dissolved in 5 ml distilled water. Few drops of neutral 5% ferric chloride solution were added. A dark green color indicated the presence of phenolic compounds.

Thin Layer Chromatography (TLC) analysis
TLC analysis of the crude compound was performed in pre-coated silica gel plates (E-Merck, Germany) to detect, the presence of phytochemicals in A. indica. The crude extract was spotted with capillary tube and solvent front was allowed to run for 80% of the plate. The crude was eluted with Petroleum ether and Ethyl acetate with the ratio 4.2:0.8. Spots with R f values were identified under UV Light (254 nm) absorbance. Antimicrobial activity using partially purified compounds The two major spots in the TLC plates were scrabbled and checked antimicrobial activity against selected pathogen of B. cereus. Briefly, an each spot was collected and dissolved in ethanol, further centrifuged at 10,000 rpm for 15 min. The supernatant was collected air dried until ethanol evaporation after that it was dissolved in 200µl DMSO. Antimicrobial activity was performed using the well diffusion method on Muller-Hinton agar (Gupta et al., 2013). Fresh colonies of B. cereus, were grown in 1 ml sterilized LB broth, incubated at 37°C for 18 -20 hrs. A sterile cotton swab was dipped into the culture and was spread uniformly on Mueller-Hinton agar plates. A 9 mm well was prepared and 100µl of sample (in DMSO), was added to each well. Tetracycline (50µg each well) and DMSO were used as positive and negative control for all experiments. The plates were incubated at 37°C for 20-24 hrs subsequently; zone of inhibition was measured by a standard ruler scale (Himedia, India).

RESULTS AND DISCUSSION Preparation of plant extract
Medicinal plants with different organic solvents were used to extract the active compounds for various applications. In this present study, A. indica leaf extract was used for phytochemical analysis and antimicrobial activity. We used five different solvents including polar and nonpolar solvents to extract compounds. Among five solvents used, the maximum extract was obtained in ethanol (0.56g) followed by acetone (0.26g) (Table 1)

Antimicrobial activity
A. indica crude extract was used to test the antibacterial activity against ten human pathogens which cause different diseases. Among five solvents were used to extract the compounds in A. indica acetone performs well followed by ethyl acetate have good antimicrobial activity compared to other three solvent. In this study, we have shown that Ethanol is optimum for more plant extract; however the antimicrobial activity is low  Zone of inhibition in mm (without disc size, 9 mm) compared to acetone extract. Interesting to note that, each solvent extract inhibited the growth of bacteria, but the zone of inhibition is varied among bacterial pathogens. We used 1 to 15mg of crude extract to check antibacterial activity and acetone extract inhibited S. typhi (3-11mm) followed by B. cereus (3-6 mm) ( Table 2). Further, ethyl acetate extract gave maximum zone of inhibition was observed for K. pneumonia (9-13mm), S. typhi (11mm) and S. epidermidis (9-11), (Table 3, Fig. 1).

Phytochemical assay
The crude acetone extract was used for the analysis of phytochemicals by standard method. We tested for eight phytochemical compounds in A. indica, however, we found tannin (green precipitate), saponin (foam formation), alkaloids (orange color precipitation followed by buff formation), flavonoid (yellow precipitate) and phenol (dark green color) only (Table 4). These phytochemicals detected, might be a active compounds of plants. On the other hand, Bargah et al., (2015) revealed ethanol and aqueous extracts of Moringa pterygosperma contained various phytochemicals such as alkaloid, flavonoid, glycoside, phenol, saponin, steroid and tannin further, these extract used as antioxidants, antifungal, antibacterial, anti abortifacient. Ethanol extract from Bambusa vulgaris, found terpenoid, flavonoid, carbohydrate, protein, tannin, and glycoside with wound healing and anti inflammatory properties (Lodhi et al., 2016). These results suggested different phytochemicals presence depends on the plant nature and their solvent system used.

TLC analysis
TLC is used for identification of compounds in a mixture, when the Rf value of a compound is compared with a known Rf value compound. From the crude extract of A. indica, four major spots were observed with petroleum ether, ethyl acetate (4.

Antibacterial activity using partially purified compounds of A. indica
The two major spots in the TLC plates were scrabbled and checked antimicrobial activity against selected B. cereus pathogen. Among 10 pathogens tested for antimicrobial activity with crude extract, B. cereus also showed maximum and clear zone of inhibition, hence we selected this organism to check inhibition activity for partially purified compounds. The selected spots show significant activity against B. cereus. Of these, two TLC spots, second spot (Rf value 0.70) showed good activity compared to first spot (0.96). These partially purified compounds showed significant activity related to tetracycline antibiotics.

CONCLUSION
In this present study, five solvents were used for A. indica plant extract ethanol followed by acetone is suitable for maximum crude extract. However, acetone extract of A. indica showed good inhibition activity against human pathogens other than ethanol, diethyl ether, ethyl acetate and benzene extracts. Among ten human pathogens tested for antimicrobial activity, S. typhi followed by B. cereus highly inhibited whereas E. coli, and S. epidermidis also pronounced well in A. indica extract. For the pharmacological applications this plant extract would be purified as a single