HPTLC Profiling and Antibacterial Efficacy of Melia Azedarach Linn. Leaf Extracts Against Secondary Bacterial Pathogens of Dermatophytosis

Medicinal plants are potential source of antimicrobial agents, used traditionally to treat various human microbial infections worldwide. The present study was aimed to determine the antibacterial efficacy of Melia azedarach Linn. leaf extracts against secondary bacterial pathogens of dermatophytosis such as Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus and to investigate the presence of phytocompounds through High Performance Thin Layer Chromatographic (HPTLC) method of the potential extract. The leaf extracts of the selected plants were tested for antibacterial potentiality using disc diffusion method and gentamicin (10μg/disc) was used as positive control. The results revealed that the positive control had more inhibitory effect than the solvent extracts used. Comparatively, acetone extract of M. azedarach leaves was more effective against two test pathogens, S. aureus (12.93±0.65 mm) at 150 μl/disc and P. aeruginosa (11.5±0.10 mm) at 100 μl/disc concentration with significant difference of p=0.05 using one-way analysis of variance (ANOVA). The varying degree of extract concentrations has a greater influence in the inhibitory effect against test pathogens. The different Rf values, maximum percentage concentration, area percentage of polyvalent chemical constituents was recorded in HPTLC profiling of acetone leaf extract, where the maximum percentage concentration was found to be 14.07% at 0.09 Rf. The HPTLC studies has confirmed that the compounds present in the acetone extract might be responsible for the inhibitory effect against the bacterial pathogens and are more soluble in semi-polar solvent. Therefore, the present investigation forms the basis as preliminary study of antibacterial efficacy of M. azedarach leaf extracts and phytocompound HPTLC profiling of potential extract, which could be used for quality evaluation of compound and standardisation of drug in future work.

Dermatophytic infections are considered as global health problem. Dermatophytosis is an infectious condition caused by keratinophilic pathogenic fungi which belong to three major genera Trichophyton, Microsporum and Epidermophyton sps. It has a tendency of reoccurrence due to several reasons such as poor hygiene, over population, humid environmental conditions and invasion of opportunistic microbes on the infected part. Skin infection occurs when pathogenic micro-organisms (bacterial, fungal, viral and parasitic) penetrate the skin, spread and cause swelling, colour change, pain and discomfort. A rash is an area of swollen or irritated skin, primarily remain as symptom and then paves way for opportunistic microbes causing secondary bacterial infections 1 .
The major cause of skin infections is the occurrence of secondary bacterial skin infections which are common complications of primary dermatoses or dermatophtosis, primary non-bacterial skin infections, traumatic lesions, ulcers, cutaneous infestations. Aerobic and anaerobic, gram-negative and gram-positive organisms present in such secondary infections, include Staphylococcus aureus, S. epidermidis, Streptococcus sps., Escherichia coli, Enterobacter sps., Pseudomonas aeruginosa, Proteus sps, Peptostreptococcus sps., Clostridium sps., Eubacterium sps., Bacteroides sps., Porphyromonas sps., Fusobacterium sps., Candida sps. Local application of antibacterial agents remains as an important component of treatment whereas, infection may also persist as a result of resistance to antibiotic drugs. Thus, treatment of serious skin infections should include systemic antimicrobial therapy 2 .
Medicinal herbs with high therapeutic value are used to treat multitude of ailments and diseases. Plants synthesize abundant chemical compounds (phytochemicals) that possess pharmacological actions with medicinal properties widely used in traditional medicine, since prehistoric times. Phytochemicals are rich in secondary metabolites such as alkaloids, flavonoids, tannins and terpenoids, which are known to possess antimicrobial properties 3 in phyto-research field. Several innovative therapeutic approaches revealed that phtyochemicals exhibit potent activity against bacterial resistance 3,4,5,6,7 .
The complex mixtures of phytocompounds commonly known an 'active constituents' are standardised, analysed and purified for its therapeutic potentiality against various diseases using several chromatographic techniques. High Performance Thin Layer Chromatography (HPTLC) has been well known for its advanced technology that provide qualitative and quantitative data of an active ingredient or phytoconstituent 8 . It is an effective analytical technique extensively used to identify phytocompounds, standardize and provide quality control of herbal formulations in the development of potential drug 9 . The significant degradation of active compounds due to exposure of heat, light and air can be corrected and minimized by increasing the volume of analyte in HPTLC plate, which serves as a major advantage when compared to other analytical methods 10 .
In south Asia, Melia azedarach, Linn. is well known for its tremendous medicinal properties. It is a tree belonging to the family meliaceae. In traditional system of medicine, the plant was recognized to possess significant therapeutic properties such as blood detoxifier, anti-inflammatory, antipyretic, anthelmintic and antimicrobial agent especially used in the treatment of skin diseases 11 . With the knowledge of traditional medicinal system and the medicinal properties of plants, the present investigation was aimed to analyze the antibacterial efficacy of traditional medicinal plant Melia azedarach Linn. leaf extracts against three bacteria Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, which are responsible for secondary bacterial infections of dermatophytosis, also to study High Performance Thin Layer Chromatographical (HPTLC)profiling of the plant extract.

Collection and preparation of plant extracts
The fresh leaves of Melia azedarach Linn. was collected from Tiruchirappalli district, Tamil Nadu, washed several times in tap water and shade dried at room temperature for 10 -15 days. The dried leaves were powdered using an electric grinder.
Soxhlet extraction (hot continuous extraction) procedure was undertaken to extract the phytocompounds of the plant sample. 15g of coarsely ground leaf powder was mixed with 100ml solvents such as petroleum ether, chloroform, ethanol, acetone, and aqueous in the Soxhlet apparatus. The extracts were sequentially collected in separate containers and was evaporated at low pressure using Buchi Rotavapor at 10R"C. The crude extracts were stored at 4R"C for further use.

Antimicrobial assay Selection of microbes
The microorganisms selected for the present study were obtained from the Department of Microbiology, K.A.P Viswanathan Govt. Medical College, Tiruchirappalli, Tamil Nadu. The three bacterial strains employed as test organisms were listed in appendix-1.

Preparation of microbial inoculums Nutrient broth selected for the growth of the bacteria
The peptone broth (nutrient broth) was procured from Himedia laboratory Pvt. Ltd., Bombay, India. Microbial inoculums were prepared by sub culturing the commercially available strains procured from clinics. A loop full of organisms were taken and inoculated into 5ml of nutrient broth and incubated at 37ºC for 24 hours till a moderate turbidity was developed. This was used as a source of bacterial inoculum.

Preparation nutrient medium Nutrient medium preparation for the growth of the bacteria
The Muller -Hinton agar medium (nutrient media) was procured from Himedia laboratory Pvt. Ltd., Bombay, India. 28g of nutrient agar medium was taken in a conical flask and dissolved in 1000ml of distilled water. The contents were mixed thoroughly. Then, the conical flask with the medium was tightly plugged with cotton and subjected to sterilization.

Sterilization of nutrient medium
The steam sterilization process was carried out using an autoclave. Along with the nutrient medium, necessary glass wares such as petridishes, forceps and inoculation needle were also sterilized at 15lb psi pressure at 121ºC for 15 minutes.

Antibacterial Sensitivity test -Disc Diffusion method
Nutrient agar plates were prepared for each bacterium in sterilized petriplates. 20ml of the sterile Muller-Hinton agar medium was poured carefully under aseptic conditions and allowed to remain undisturbed for the medium to solidify. Each petriplate was labeled according to the bacterial strains to be used for streaking. Each bacterial pure culture was swabbed on the surface of the nutrient medium. On the petriplates, antibiotic discs with plant extracts along with the positive control antibiotic disc (gentamicin, 10µg/ disc) were placed at equidistant on the surface. The inhibition zone formed against each bacterial strain by the plant extracts was compared with the standard positive control antibiotic disc. The diameter of the inhibition zone was denoted in millimeters(mm) using a measuring scale 12 .

Statistical analysis
The results of the antibacterial activity were expressed as mean ± SD of three experiments. All the data were analysed statistically using oneway analysis of variance (ANOVA).

High performance thin layer chromatography
The acetone leaf extract of M. azedarach was analyzed for their qualitative phytoconstituent fingerprinting by HPTLC method 13,14 .

Sample Preparation
The acetone leaf extract was evaporated under reduced pressure using rota-evaporator and the extract residue was re-dissolved in 1ml of chromatographic grade solvent methanol, which was used as sample.

Developing Solvent System
A n u m b e r o f s o l v e n t s y s t e m s were tried and a satisfactory resolution was obtained in the solvent system of Toluene:Ethylacetate:Methanol:Formicacid (6:2:1.5:0.5) for the extract used.

Sample Application
Application of bands of each extract was carried out (6mm in length and 100µl in concentration for leaf) using spray technique. The sample was applied in duplicate on pre-coated silica gel 60F254 aluminium sheets (4 x 10 cm) with the help of Linomat 5 applicator attached to CAMAG TLC Scanner system, which was programmed through winCATS Planar chromatography manager software.

Development of Chromatogram
After the application of sample, the chromatogram was developed in Twin trough glass chamber 10 x 10 cm saturated with solvent Toluene:Ethylacetate:Methanol:Formicacid (6:2:1.5:0.5) with the total volume of 10ml for 20 minutes between 60 to 120°C.

Detection of Spots
The air-dried plates were viewed in ultra-violet radiation to mid-day light. The chromatograms were scanned by densitometer at 420nm after spraying with specific reagent. The plates were kept in photo-documentation chamber and images were captured at white light, UV-254nm and UV-366nm wavelengths. After derivatization, the plates were scanned for peak table, display and densitogram were recorded. The R f values and % area were calculated using winCATS software

RESULTS
The antibacterial efficacy of Melia azedarach leaf extract was determined against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. The different solvent extracts used were ethanol, methanol, acetone, at 100, 150, 200 and 250µl/disc concentrations whereas, chloroform and petroleum ether at 200, 300, 400 and 500µl/disc concentration. The values were expressed in mean ± standard deviation of three replicates indicating a significant difference of pdH0.05, according to One-Way Analysis of Variance (ANOVA). The positive control gentamicin antibiotic disc (10µg/disc), revealed maximum inhibitory effect against all the test organisms when compared to all the leaf extracts used. The results were tabulated in Table -1. Acetone leaf extract was more effective against two test pathogens, S. aureus and P. aeruginosa, whereas, the maximum zone of inhibition was registered to be 12.93±0.65 mm at 150 µl/disc and 11.5±0.10 mm at 100 µl/disc concentration respectively (Fig.3). The maximum zone of inhibition (12.70±0.20 mm) by ethanolic leaf extract against P. aeruginosa was recorded at 100 µl/disc concentration (Fig.1). Methanolic leaf extract exhibited inhibition (11.56±0.35mm) against S. aureus at 250 µl/disc concentration (Fig.2). Chloroform leaf extract  No. Bacteria Strain 1.
Pseudomonas aeruginosa Gram -negative   inhibited Escherichia coli (11.45±0.63) at 500 µl/ disc concentration (Fig.4). Also, petroleum ether extract registered inhibitory effect (10.26±0.24) against S. aureus at 200 µl/disc concentration (Fig.5). Correspondingly, S. aureus was inhibited by methanol, acetone and petroleum ether leaf extracts. There was no inhibition by ethanol and chloroform extracts. Inversely, E. coli was inhibited only in chloroform extract and did not show any inhibition against all other leaf extracts. Whereas, P aeruginosa was inhibited in ethanol and acetone leaf extracts. Also, methanol, chloroform and petroleum ether did not exhibit any inhibition against the organism. Therefore, difference in the concentrations of leaf extracts might be responsible for the wide range of variations in the inhibitory effect.
Based on the preliminary invitro examination on antibacterial efficacy, acetone and ethanol extract was considered to be more   Table  2 and shown in Fig. 6. Also, the 3-dimensional chromatogram and spectra of the extract was depicted in Fig. 7 and 8 respectively. Thus, it has been found that acetone extract of the M. azedarach leaf contains mixture of compounds and the pharmacological activity revealed by them are due to the cumulative effect of all the composite compounds.

DISCUSSIONS
The present study finds supportive evidence of several scientific research reports. The influence of ethanolic and methanolic extract on inhibition against selected micro-organisms were found to be some extent, depending upon the varying solvent concentrations. The ethanolic extract of Limonia acidissima leaves 15 and   coli, Pseudomonas aeruginosa and Aspergillus niger. They reported that the chloroform extract showed good inhibitory properties against all pathogens and even at very low concentrations 23 . Moringa oleifera leaf extracts of different solvents showed varying degree of inhibition according to the type of solvent extract and its concentration against Bacillus subtilis and Klebsiella pneumoniae which revealed susceptibility as well resistant to all the extracts 24 . A significant antibacterial activity has been reported in M. azedarach leaves 25,26,27,28 and fruit extracts 29 against certain gram-positive and gram-negative strains. Therefore, it can be concluded that M. azedarach may contains certain antimicrobial components that could be very useful in the treatment for various infectious diseases, especially against secondary bacterial pathogens of skin infection.
The quality of plant extracts depends on the presence of active phytoconstituents which can be identified and determined by an analytical technique High Performance Thin Layer Chromatography (HPTLC). Densitometry provides data with peak area, peak height for the quantitative determination of bioactive constituents 30 32 . Similar studies on the crude extract of different parts of Vernonia cinerea L indicated the chemical profile of potential compounds that possess biological activity 33 . A HPTLC densitogram reported major phytoconstiutents with several peaks scanned at 254 nm and 366nm from the methanolic extract of Fumaria parviflora (whole plant) 34 . The methanolic extracts of Verbesina sphaerocephala leaves and flowers were reported to possess high phenolic and flavonoid compounds through HPTLC analysis with relevant antibacterial and antioxidant activity 35 . Two phytocompounds rutin and kaempferol-3-O-rutinoside were identified in Bauhinia rufescens by HPTLC with antioxidant and antidiabetic potential 36 . The methanolic leaf and root extracts of Hypochaeris radicata has confirmed the existence of major phytocompounds through HPTLC method responsible for bioactivity against pathogenic organisms of communicable and non-communicable aliments 37 . Thus, HPTLC fingerprinting helps to determine the major active biocompounds especially secondary metabolites, present in medicinal plants with reliable scanning profiling of qualitative and quantitative measurements.

CONCLUSION
According to World Health Organisation (WHO), every year, millions of fatalities occur due to microbial infections all around the world. This remains a biggest challenge in health society. In this regard, researchers focus on natural products as an alternative to existing less effective antibacterial drugs. The present research findings may provide an authentic conclusion that, the phytochemical compounds present in the leaves of M. azedarach may have a promising role in the antibacterial activity against the tested microbes. These phytocompounds may serve as selective agents for the maintenance of human health and a potent remedy for secondary bacterial pathogens of dermatophytosis. Also, the HPTLC profiling has proved the presence of major phytocompounds in the acetone leaf extract. Based on the separation of bands along with obtained R f values, percent area of the potent extract and its correlation with literature study, it can be stated that the chemical constituents present in the extract may include phenols, flavonoids, alkaloids, terpenoids and other secondary metabolites which can be authenticated and purified as maker compounds for drug delivery in future work.

ACKNOWLEDGEMENT
We are grateful to thank DST-FIST for providing the infrastructural facilities. We are extremely thankful to Department of Microbiology, K.A.P Viswanathan Govt. Medical College, Tiruchirappalli, for providing the microbial strains to undergo antibacterial study. Also, we extend our sincere gratitude to SRM university, Chennai, for providing facilities to undertake HPTLC analysis.