In vitro Antibacterial , Antifungal and Phytochemical Analysis of Methanolic Extract of Fruit Cassia fistula

The identification of phytochemical compounds is based on the peak area, retention time molecular weight, molecular formula, MS Fragmentions and Pharmacological actions. GCMS analysis of Cassia fistula revealed the presence of the Oxacyclododecan-2-one, Imidazole ,2-amino-5-[(2-carboxy)vinyl], D-Glucose , 6-O-á-D-galactopyranosyl, 2-Nonanone, Eicosanoic acid , phenylmethyl ester, Phenol , 4-(2-propenyl), Eugenol, Caryophyllene, ß-copaene, Azulene,1,2,3,3a,4,5,6,7-octahydro-1,4-dimethyl-7-(1-methylethenyl), á-acorenol, Spiro[5.5] undec-8-en-1-one, Isoaromadendrene epoxide, Tetraacetyl-d-xylonic nitrile , Benzyl Benzoate, N-Isobutyl-(2E,4Z,8Z,10E)-dodecatetraenamide, Phenethylamine , 3-benzyloxy-2-fluoro-ß-hydroxy, 4a-Hydroxy-4-nitroperhydronaphthalen-1-one, Dasycarpidan -1-methanol, acetate (ester), Propanoic acid , 2-(3-acetoxy-4,4,14-trimethylandrost-8-en-17-yl), Carda-4,20(22)-dienolide,3-[(6-deoxy-3-Omethyl-á-L-mannopyranosyl, Cis-13-Eicosenoic acid , 16-Nitrobicyclo[10.4.0]hexadecane-1-ol-13one, Strychane ,1-acetyl-20á-hydroxy-16-methylene, 2,4,6-Decatrienoic acid , 1a,2,5,5a,6,9,10,10aoctahydro-5,5a-dihydro, Vitamin E and Glycine ,N-[(3á,5ß,12á)-3,12-dihydroxy-24-oxocholan-24yl]. The FTIR analysis of Cassia fistula leaves proved the presence of Alkenes, Aliphatic fluoro compounds, Alcohols, Ethers, Carboxlic acids, Esters, Nitro Compounds, Alkanes, Alcohols and Phenol. Cassia fistula was highly active against Aspergillus terreus (6.99±0.29). Methanolic extract of bioactive compounds of Cassia fistula was assayed for in vitro antibacterial activity against eleven pathogenic bacteria by using the diffusion method in agar. The zone of inhibition were compared with different standard antibiotics. The diameters of inhibition zones ranged from 1.00±0.05 to 6.02±0.23 mm for all treatments.


INTRODUCTION
Cassia fistula plant have naturally occurring bioactive compounds and are mostly secondary metabolites which are now a days being used as medicines, dietary supplements and other useful commercial products 1 .Also, has been reported to contain anthraquinone the principal laxative constituent of many plants used as purgative 2 .Plants have been an important source of medicine with qualities for thousands of years 3,4 .Mainly on traditional remedies such as herbs for their history, they have been used as popular folk medicines 5,6 .Since thousands of years back, plants are used as a major source for medicine as they found to possess a reservoir of bioactive compound [7][8][9] .Cassia fistula contains alkaloids, tannins, flavonoids,terpenes, sugars, and glucosides.Tannins are naturally occurring and water soluble phenolic compounds, which precipitate proteins from aqueous media 10 .Cassia fistula shows the presence of glycoside a natural product, which is used to enhance the cardiac contractile force in patient with congestive heart failure 11 glycoside also plays major role in the cancer therapy 12 .They also play major role in controlling topical disease as eczema etc.Many metabolites have found to possess interesting biological activities such as bactericidal, fungicidal, hepatoprotective and muscle relaxant 13 .A fruit is cylindrical pod and seeds many in black, sweet pulp separated by transverse partitions.The long pods which are green, when unripe, turn black on ripening after flowers shed 14 .Pulp is dark brown in colour, sticky, sweet and mucilaginous, odour characteristic, and somewhat disagreeable 15 .Each compartment contains one seed which is flat, oval, reddish brown with a well-marked raphe.The seed contains a whitish endosperm in which the yellowish embryo is embedded 16 .Fruits are used in the treatment of diabetes, antipyretic, abortifacient, demulcent, lessens inflammation and heat of the body; useful in chest complaints, throat troubles, liver complaints, diseases of eye and gripping 17 .The aim of this research was study the phytochemical composition of Cassia fistula and to evaluate the isolates for possible in vitro antifungal and antibacterial activities.

Solvent extraction
The shade dried powdered of Cassia fistula was extracted with methanol.The extract was filtered with Whatman's filter paper.Filtrate was concentrated under reduced pressure and preserved at 5Cº in dark air tight bottles 18,19 .

Sample preparation for GC-MS analysis
50 µl of sample (Cassia fistula) was dissolved in 2ml of methanol and kept in ultrasonic bath for 25 min and centrifuged for 10 min.at 6000 rpm and supernatant was injected in GC-MS for analysis 20,21 .

Gas chromatography-mass spectrometry (GC-MS) analysis
GC-MS of methanol extract was performed using Agilent 7890A.The run time was 30 minutes.ionization of sample components were performed on EI mode (70 eV).The carrier gas was helium at 1.0ml/min flow rate.0.5 ml of sample was injected in split mode of 20:1.The mass spectrum scan range was set at 29.0 to 500(m/z) 22 .

Identification of compounds
Interpretation of mass spectrum of GC-MS was done using the database of National Institute Standard and Technology (NIST).The mass spectrum of phytochemicals was compared with the spectrum of known compounds stored in the NIST library 23 .

Determination of antibacterial activity of crude bioactive compounds of Cassia fistula
The anti-bacterial activity was evaluated using Mueller-Hinton agar.The bacterial plates were incubated at 37 °C for 24 h.After incubation, the diameter of the inhibition zone was measured to evaluate the antimicrobial activity.Each test was performed twice and the average of the results was calculated.The extraction solvents were used as negative control 24,25 .The test pathogens were swabbed in Muller Hinton agar plates.60ìl of plant extract was loaded on the bored wells.The wells were bored in 0.5 cm in diameter.The plates were incubated at 37C° for 24 h and examined.After the incubation the diameter of inhibition zones around the discs was measured 26 .

Determination of antifungal activity
Five-millimeter diameter wells were cut from the agar using a sterile cork-borer, and 50 ìl of the samples solutions Cassia fistula was delivered into the wells.Antimicrobial activity was evaluated by measuring the zone of inhibition against the test microorganisms.Methanol was used as solvent control.Amphotericin B and fluconazole were used as reference antifungal agent.The tests were carried out in triplicate.The antifungal activity was evaluated by measuring the inhibition-zone diameter observed after 48 h of incubation 27,28 .

Statistical analysis
Results of the study were based on analysis of variance (ANOVA) and differences were considered significant at p < 0.05.

Identification of phytochemical compounds
Plants have been a common source of medicinal property, either in the form of pure active compound or as traditional preparations and it are reasonable to use local plants 29 .Identifying on the plant phytochemistry provides a fundamental use of plants as storage of chemical agents in the field of medicine 13 .The phytochemical analyses of plant from Cassia fistula was studied extensively.Identifying the importance of secondary metabolites in the field of medicine, the presence of tannins, phlobatanins, saponins, flavonoids, terpenoids, glycosides and steroids was detected.Folklore medicine are widely used in our ancient period 13 .Gas chromatography and mass spectroscopy analysis of compounds was carried out in methanolic leaves extract of Cassia fistula, shown in Table 1.The GC-MS chromatogram of the 27 peaks of the compounds detected was shown in Figure 1.Chromatogram GC-MS analysis of the methanol extract of Cassia fistula showed the presence of twenty seven major peaks and the components corresponding to the peaks were determined as follows.The first set up peak were determined to be 1,7-Dioxaspiro [5,5]  undec-2-ene   29).Cassia fistula Linn.(Cassia) family Caesalpiniaceae commonly known as Amulthus has been extensively used in Ayurvedic system of medicine for various ailments 30 .Cassia fistula is widely used in traditional medicinal system of India has been reported to possess hepatoprotective, anti-inflammatory, antitussive, antifungal and used also check wounds healing and antibacterial 15 .Fruits are used as catharatic and in snake bite.Juice of leaves is used in skin diseases [31][32][33] .Flowers and pods are used as purgative, febrifugal, biliousness and astringent.The ethanolic 50% extract of pods show antifertility activity in female albino rats.The heated pods are applied to swellings on the neck due to cold.The fruits are reported to be used for asthma.Padma, (2006) 17 tested the methanolic leaf extract of Cassia fistula for activity against Candida albicans showed highest activity i.e., upto 21 mm which was comparable with the standard antifungal antibiotic, clotrimazole.

Evaluation of antimicrobial activity
In the current study, the anti-microbial activity of the methanolic extract was evaluated by determining the zone of inhibition against five bacteria and fourteen fungi and yeast.Clinical  3. In comparison to the antibiotics used in this study, the plants extracts were far more active against the test bacterial strains.

CONCLUSION
Medicinal property of plant extract is due to presence of secondary metabolites.Twenty seven phytoconstituents were identified from methanol extract of Cassia fistula by gas chromatogram and mass spectrometry (GC-MS) analysis.This plant derived bioactive compounds used as source of antibiotic properties and pharmaceutical industries used for drug formulation.This plant crude extract

Table 3 : Antimicrobial activity of Cassia fistula
ª The values (average of triplicate) are diameter of zone of inhibition at 100 mg/mL crude extract, 30 µg/mL of antibiotics (Streptomycin; Rifambin; Kanamycin; Cefotoxime and chloramphenicol).