Phytochemical screening , gas chromatography-mass spectrometry ( GC-MS ) analysis of phytochemical constituents and anti-bacterial activity of Aerva lanata ( L . ) leaves

The present study is aimed to determine the phytochemical screening and anti-bacterial activity of the extracts (acetone, ethyl acetate and ethanol) of medicinal plant, Aerva lanata leaves against bacterial strains. Acetone, ethyl acetate and ethanol extracts of A. lanata leaves were prepared using Soxhlet apparatus. These extracts of A. lanata leaves were checked for their anti-bacterial activity by well diffusion, minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) techniques against bacterial strains. Preliminary phytochemical screening and gas chromatographymass spectrometry (GC-MS) analysis of phytochemical constituents of A. lanata leaves were performed. Preliminary phytochemical screening of different extracts of A. lanata revealed the presence of alkaloids, proteins, amino acids, flavonoids, tannins, phenolic compounds, saponins, quinone, terpenes and coumarins. Among the three different extracts tested, acetone extract of A. lanata leaves showed maximum anti-bacterial activity. The bioactive components of acetone fraction of A. lanata leaves were evaluated by GC-MS analysis which showed the presence of sixteen chemical compounds. The extracts of A. lanata leaves have a broad spectrum of anti-bacterial activity and support the traditional use of these plants as medicines.


INTRODUCTION
Plants have been widely used to treat various ailments, since ancient times.World Health Organization (WHO) has estimated that nearly 80% of the total population in the developing countries relies on medicinal plants for health care (Muruganantham et al., 2009).Therefore, such plants should be examined to understand their *Corresponding author.E-mail: nbsenthilkumar@gmail.com medicinal properties, safety and efficiency (Ellof, 1998).
Antibiotics are one of our most important weapons in fighting microbial infections and have greatly benefited the health-related quality of human life (Fransworth, 1993).Although, antibiotics have been widely used in last decades, the development of microbial resistance to them is also increased (Cohen, 1992).In order to overcome this problem, scientists deviated their research towards anti-microbial compounds of medicinal plants as an alternative solution.The demand for herbal medicine is due to their wide biological activities, higher safety compared to synthetic drugs and low cost (Grabley and Thiericke, 1999).
Aerva lanata is a medicinal plant and belongs to Amaranthaceae family.The whole plant of A. lanata is used as diuretic, anti-helminthic, anti-diabetic and expectorant and also used in the treatment of lithiasis (Gupta and Neeraj, 2004.).Traditionally, leaves of A. lanata are used as sap for eye-complaints; an infusion is given to cure diarrhea and kidney stone and the root is used in snake bite treatment.A leaf decoction preparation is used as gargle for treating sore throat and is also used in various complex treatments against guinea-worm.They are also used as an antidote for scorpion sting (Vijaya Kumar and Pulliah, 1998), spermatorrhoea and urinary troubles and as an anti-rheumatic (Kakrani and Saluja, 1994).

Plant material collection
The plant A. lanata was collected in and around Salem, Tamil Thangavel et al. 127 Nadu.The collected plant species was identified and confirmed (LOT.NO-68; A/series/F/AMA) by Dr. R. Selvaraj, Professor, Department of Botany, Annamalai University, Annamalai Nagar-608 002, Tamil Nadu, India.The plant was washed with running tap water and finally washed with distilled water to remove the dirt and dried under shade for 7 days.

Preparation of plant extracts
Thoroughly washed and dried leaves of A. lanata were then crushed gently to make it into powder by using mixer grinder.About 25 g of crushed powder was filled into the thimble and was extracted separately with different solvents such as acetone, ethyl acetate and ethanol in Soxhlet apparatus for 24 h.The solvent present in the extracts was allowed to evaporate in open air and the extracts were stored in refrigerator until further use.

Phytochemical screening
The different extracts of plant A. lanata were subjected to preliminary phytochemical screening by using standard procedures (Harborne, 1993;Trease and Evans, 2002) for the detection of alkaloids, proteins, amino acids, anthraquinone glycosides, flavonoids, carbohydrates, saponins, terpenes, coumarin, quinone, tannin and phenolic compounds.

Gas chromatography-mass spectrometry (GC-MS) analysis
GC-MS analysis of acetone extract of A. lanata leaves was performed by using Thermo GC-Trace ultra version 5.0 gas chromatography interfaced to Thermo MS DSQ II mass spectrometer instrument employing the following conditions: DB5-MS capillary standard non polar column (30 × 0.25 mm × 0.25 µm) and helium gas was used as a carrier gas at a constant flow rate of 1 ml/min.The oven temperature was kept at 70°C and was programmed to reach 260°C at a rate of 6°C/min.Mass range was 50 to 650 (m/z).The total running time was completed within 43 min.The chromatogram obtained from gas chromatography was then analyzed in mass spectrometry to get the mass of all fractions.The identification of phytochemical components was achieved through retention time and mass spectrometry by comparing the mass spectra of unknown peaks with those stored in Wiley 9 GC-MS library.

Anti-bacterial activity of plant extracts
Anti-bacterial activity of plant extracts was determined by agar well diffusion method against microbial type culture collection (MTCC) bacterial strains such as Staphylococcus aureus (MTCC 796), Escherichia coli (MTCC 443), Klebsiella pneumoniae (MTCC 109), Salmonella typhi (MTCC 733), Proteus mirabilis (MTCC 422), Pseudomonas aeruginosa (MTCC 6750) and Bacillus subtilis (MTCC 441).All the bacterial strains used in the present study were obtained from MTCC, Chandigarh, India.Muller-Hinton agar (MHA) (HiMedia, India) plates were prepared and 16 h old bacterial culture was swabbed uniformly and was allowed to dry for 5 min.Four wells of 5 mm in diameter were made in each of these plates by using a sterile cork borer.The extracts were prepared at a final concentration of 30 mg/ml by dissolving them in their respective solvents.The different concentrations of the plant extract (300, 600, 900 and 1200 µg) were loaded onto the wells and were allowed to diffuse for 10 min.Then, the plates were incubated at 37°C for 24 h.After the incubation, diameter of inhibition zone around the well was measured and recorded.

Determination of minimal inhibitory concentration (MIC)
Each plant extracts (100 mg) were suspended in 1 ml of their respective solvents and were kept as stock solutions.These were subjected to double fold serial dilution in peptone water to obtain ten different concentrations ranging from 100 to 0.19 mg/ml and added to the respective culture tubes containing 2 ml of peptone water.These tubes were then inoculated with 100 µl of 16 h old broth culture of bacterial strains followed by incubation at 37°C for 24 h.Three tubes containing peptone water, peptone water and extract and peptone water and inoculum were used as control (John et al., 2011).

Determination of minimal bactericidal concentration (MBC)
The MBC of each extract was determined by sub-culturing 10 µl of the test dilutions from MIC tubes on to Muller Hinton agar plates.
Plates were incubated for 24 h.The highest dilution that yielded low or no bacterial colony on the plate was recorded as MBC (Pavithra et al., 2010).

MIC index
The MIC Index (MBC/MIC) was calculated for each extract to determine whether an extract is bactericidal (MBC/MIC < 4) or bacteriostatic (MBC/MIC > 4) on growth of bacterial organisms (Chattopadhyay et al., 2007).The range of MIC Index value which is greater than 4 and less than 32 are considered as bacteriostatic (Cutler et al., 1994).

Statistical analysis
The experiments were repeated thrice and the average data were submitted to analysis of variance using ANOVA (Minitab version 15).A p value < 0.05 was considered statistically significant.
Acetone extract of A. lanata leaves exhibited the highest anti-bacterial activity against P. mirabilis followed by S. aureus, S. typhi, B. subtilis, P. aeruginosa, K. pneumoniae and E. coli.Ethyl acetate leaf extract of this plant showed anti-bacterial activity against S. typhi, S. aureus followed by P. mirabilis, B. subtilis, E. coli, P.aeruginosa and K. pneumoniae.Ethanol extract of leaf showed anti-bacterial activity against E. coli followed by S. aureus, P. mirabilis, P. aeruginosa, S. typhi, K. pneumoniae and B. subtilis (Table 3).
MIC value of acetone extract of A. lanata leaves was found to be 1.53 mg/ml against S. aureus, P. mirabilis, S. typhi and 3.06 mg/ml against E. coli, K. pneumoniae, P. aeruginosa and B. subtilis.MBC value of this extract was 6.12 mg/ml against S. aureus, S. typhi, P. mirabilis and 12.25 mg/ml against B. subtilis (Table 4).MIC index value of acetone extract of plant leaves showed that bactericidal activity against S. aureus, S. typhi, P. mirabilis and B. subtilis and bacteriostatic activity against E. coli, K. pneumoniae and P. aeruginosa (Table 7).MIC value of ethyl acetate extract of A. lanata leaves was found to be 1.53 mg/ml against S. typhi and 3.06 mg/ml against S. aureus, P. mirabilis, B. subtilis and 6.12 mg/ml against E. coli, K. pneumoniae and 12.25 mg/ml against P. aeruginosa.MBC value of this extract was 12.25 mg/ml against S. aureus, S. typhi, B. subtilis and 25 mg/ml against K. pneumoniae, P. mirabilis and 50 mg/ml against E. coli and P. aeruginosa (Table 5).MIC index value of this extract proved that they are found to be bactericidal against S. aureus, K. pneumoniae, P. aeruginosa, B. subtilis and P. mirabilis and bacteriostatic against E. coli and S. typhi (Table 7).
MIC value of ethanol extract of A. lanata leaves was found to be 1.53 mg/ml against S. aureus, E. coli and 3.06 mg/ml against S. typhi, P. mirabilis and 6.12 mg/ml against K. pneumoniae and P. aeruginosa.MBC value of this extract was 6.12 mg/ml against S. aureus and 12.25 mg/ml against E. coli, S. typhi, P. mirabilis, B. subtilis and 25 mg/ml against K. pneumoniae and P. aeruginosa (Table 6).MIC index value of this extract was also found to be bactericidal against S. aureus, E. coli, P. aeruginosa, B. subtilis, S. typhi and P. mirabilis and bacteriostatic against K. pneumoniae (Table 7).All these results proved that the acetone, ethyl acetate and ethanol extract of A. lanata leaves have bactericidal and also bacteriostatic effect on pathogenic microorganisms like against S. aureus, E. coli, P. aeruginosa, B. subtilis, S. typhi and P. mirabilis and K. pneumoniae and these effects were found to be varied from each solvent.

DISCUSSION
The antimicrobial activity of plants have been screened because of their great medicinal relevance with the recent years, infections have increased to a great extent and resistant against antibiotics becomes an ever increasing therapeutic problem.The presence of antifungal and antimicrobial substances in the higher plants are well established as they have provided a source of inspiration for novel drug compounds as plants derived medicines have made significant contribution towards human health.
Knowledge of the phytochemical constituents of plants is desirable, not only for the discovery of therapeutic agents, but also because such information may be of value in disclosing new sources of economic materials such as tannins, oils, gums, flavonoids, saponins, essential oils precursors for the synthesis of complex chemical substances (Akrout et al., 2010).
The results of phytochemical screening of A. lanata leaves clearly imply that the strength of active principle depends upon the use of solvent besides the type of plant species to achieve the positive results.The identified phytochemical compounds have many biological properties.For instance, hexadecanoic acid, methyl ester which is a palmitic acid compound found to be an anti-oxidant, hypocholesterolemic, nematicide, pesticide, lubricant activities and hemolytic 5-alpha is a reductase inhibitors.These results are strengthened by the findings of Sermakkani and Thangapandian (2012) who observed the presence of this compound in methanol extract of Cassia italica leaves.9-Octadecenoic acid is a linoleic acid compound and reported to have an anti-inflammatory, nematicide, insectifuge, hypocholesterolemic, anti-cancer, hepatoprotective, anti-histaminic, anti-acne, anti-arthritic and anti-eczemic properties.Similarly, the presence of 9-octadecenoic acid was observed in the ethanolic root of Plumbago zeylanica by Ajayi et al. (2011).2-Hexadecen-1-ol, 3, 7, 11, 15-tetramethyl-[R-[R*, R*-(E)]]-phytol is a diterpene compound which is reported to possess antimicrobial, anti-cancer, anti-inflammatory and diuretic agent (Praveen Kumar et al., 2010).
Similarly, the presence of phytol was observed in the leaves of Lantana camara (Mariai et al., 2011;Sathish and Manimegalai, 2008) and Mimosa pudica (Sridharan et al., 2011).Phytol was observed to have anti-bacterial activities against S. aureus by damaging the cell membranes which in turn causes leakage of potassium ions from bacterial cells.Phytol is a key acyclic diterpene alcohol that is a precursor for vitamins E and K 1 .It is used along with simple sugar or corn syrup as a hardener in candies (Inoue et al., 2005).Plants are important source

Bacterial strain MIC index Acetone Ethyl acetate Ethanol
of potentially useful compounds for the development of new chemotherapeutic agents.In vitro evaluation of plants for anti-microbial property is the first step towards achieving the goal for developing eco-friendly management of infectious disease of humans by search for new biomolecules of plant origin (Samy et al., 2008;Mohana et al., 2008).
On comparison of three different A. lanata leaves extract, acetone extract showed the highest anti-bacterial activity against the bacterial strains.Senthilkumar et al. (2011) reported that the bark acetone, isopropanol and hexane extract of pomegranate possess anti-bacterial activity against S. typhi and S. paratyphi A isolates and also proved that among three solvents, acetone and hexane extracts showed better efficiency than isopropanol extract.
The increase of antibiotic resistance of microorganisms to conventional drugs has necessitated the search for new, efficient and cost effective ways for the control of infectious diseases.The results of different studies provide evidence that some medicinal plants might indeed be potential sources of new anti-bacterial agents (Kone et al., 2004).The use of medicinal plants is part of the Indian tradition.Many local regions all over India have a great variety of vegetation used by the local population to treat and prevent diseases (Bonyadi et al., 2009).

Conclusion
From this study it is concluded that leaves of A. lanata have a broad spectrum of anti-bacterial activity and supports the traditional use of these plants as medicines.This study demonstrated that herbal medicine can be as effective as modern medicine to combat pathogenic microorganisms and overwhelming the antibiotic resistance.Further studies are needed with this plant on its isolation, structural elucidation of bioactive compounds and about its in vivo toxic effects in experimental animals to formulate a new drug for regular practice.

ACKNOWLEDGEMENT
Authors wish to thank the Muthayammal Educational and charitable Trust, Rasipuram, Tamil Nadu, India, for providing the laboratory facilities.They are also grateful to South Indian Textile Research and Association, Coimbatore, Tamil Nadu for permitting to perform GC-MS studies.

Table 1 .
Preliminary phytochemical screening of different solvent extracts of A. lanata (L.) leaves.

Table 2 .
Phytochemical constituents of acetone extract of leaves of A. lanata (L.) by GC-MS spectra.

Table 3 .
Antibacterial activity of different solvent extracts of A. lanata (L.) leaves against bacterial (MTCC) strains by well diffusion method.

Table 7 .
Antibacterial activity of different solvent extracts of A. lanata (L.) in terms of MIC index (MBC/MIC).