Phytochemical characterization and Antibacterial potentials of Mangifera indica L . bark oil

1 Department of Pharmacology, Soniya College of Pharmacy, Dharwad, Karnataka, 580002, India. 2 Department of Pharmacology, College of Medicine, Assiut University, Assiut, Egypt. 3 Department of Family Medicine, Faculty of Medicine, Najran University, Najran, Saudi Arabia 4 Department of Pharmacology, Al-Maarefa Colleges for Science and Technology, Riyadh, Saudi Arabia. 5 Department of Pharmacology, College of Pharmacy, Najran University, Najran, Saudi Arabia.


INTRODUCTION
In the developing countries, infectious diseases account for high proportion of health problems.Resistance to antimicrobial drugs has become an increasingly important and pressing global problem.Of the 2 million people who acquire bacterial infections in US hospitals each year, 70% of cases involve strains that are resistant to at least one drug (Tim-cushnie et al., 2005).This situation forced researchers to discover new antimicrobial substances from various sources, such as medicinal plants (Karaman et al., 2003).Plants as a source of remedies are widely used for the treatment of almost 87% human alignments including fungal, bacterial infections, cancer and different disorders (Abu Mustafa et al.,1961).Mangifera indica L. (family: Anarcardiaceae) is the most economically and culturally important tropical rainforest medicinal plant in Asia and Africa, especially for its edible fruits.The mango tree is an erect perennial tree with a broad rounded canopy which at full maturity may attain up to 10-30 m and 30-35 m in height and width (Shah et al., 2010).Previous studies indicate mango possesses antidiabetic, hypolipidemic, antioxidant, antiviral, cardiotonic, hypotensive, antipyretic and analgesic, anti bone resorption, antihelminthic, antispasmodic, anti-inflammatory, antidiarrheal, antiallergic, gastroprotective, hepatoprotective and immunomodulatory properties (Adeneye et al., 2015).Oil of mango tree bark is an effective home remedy for a sore throat and can be used to cure throat infections (Chetan et al., 2014).
In the present study, we have isolated a fatty fraction of Mangifera indica Linn.bark using petroleum ether and subjected to Gas chromatography-Mass spectroscopy (GC-MS) and physical characterization.The extract was also screened for its antimicrobial activity using Gram positive (S.aureus and B. subtilis) and Gram negative (E. coli and P. aeruginosa) bacteria.

Plant material
Mangifera indica stem barks were collected from the Nigadi region of Dharwad (Karnataka, India) in the month of August 2011.The plant was authenticated by Dr. G. R. Hegde (Voucher specimen number-PB/GRH/KUD-563 deposited in museum Karnataka university, Dharwad-580003).The stem barks were inspected to be healthy, collected and were washed with water thrice in the bucket and dried overnight over a cotton cloth.

Preparation of the extract
The stem barks were grounded to a fine powder using a domestic electric grinder.Powdered stem barks were passed through the sieve no.20.Around 100 g of powder was collected and extracted with Petroleum ether 60 °-80 ° (8 x 300 ml; 8 hrs each) using soxhlet apparatus.Finally, the fatty residue was collected and evaporated to dryness.Spectral characterization of the extract was carried out with the help of TLC and GC-MS.

Thin layer chromatography (TLC)
TLC plate was prepared by using silica gel (GF 254), air dried and kept for activation at 110 ° C for 30 min.n-hexane and ethyl acetate were used as mobile phase in ratio of 9:1.TLC chamber was kept for saturation.MIE (Mangifera indica extract) was dissolved in chloroform and sample spot was put 1 cm from the end of the plate.The plate was further placed in chamber for development.After development, the plate was air dried and kept in iodine chamber for spot visualization.The R f value was calculated.

Gas chromatographymass spectroscopy (GCMS)
The isolated MIE was dissolved in Dimethyl sulfoxide and subjected to GCMS (Shimadzu).

Determination of minimum inhibitory concentration (MIC)
Nine dilutions of each test samples were made with brain heart infusion broth (BHI) for MIC. 10 mg of compound (MIE) was dissolved in 1 ml of DMSO i.e solvent.Then 10 test tubes were arranged serially.In first test tube 300 µl of BHI was added and from 2 nd to 10 th tube 200 µl of BHI was added.After that 100 µl of the drug was added to the 1 st tube (master dilution).Then 200 µl of master dilution was transferred to 2 nd tube and likewise we did till 10 th tube.200 µl was discarded from the 10 th tube.Then 200 µl of the organism was added from 1 st tube to last tube and incubated the tubes in an incubator for 24 hrs at 37 ° C.After 24 hrs tubes were observed for turbidity (Schwalve et al., 2007).

Determination of zone of inhibition
Sterile BHI agar media was used to carry out Disc Diffusion Assay.The agar plates were brought to room temperature prior to use.Selected bacterial colonies were transferred to the plates with help of a loop.Turbidity was adjusted, equal to that of a 0.5 McFarland turbidity standard with broth and standardized the suspension with a photometric device.A hollow tube of 5 mm diameter was heated and pressed it on above inoculated Agar plate and removed immediately by making a well in the plate.Likewise, other four well were made on each plate.75 µl, 50 µl, 25 µl, 10 µl and 5 µl of MIE were added into the respective wells on each plate.Agar plates were incubated for 18-24 h at 37 ºC in the incubator.After incubation, plates were read and zone of inhibition was measured to nearest whole in millimeter by holding the measuring device (Isenberg, 1992).

Analytical profile
The oily fraction was extracted from dried coarse powder of mango tree bark in soxhlet apparatus using petroleum ether.Various analytical profile of extracted oil is mentioned in Table 1.

Thin layer chromatography (TLC)
Chromatographic techniques were adopted to identify purity of the oil.Wherein 5 spots were seen with R f values 0.12, 0.16, 0.23, 0.35, 0.67.Results of TLC are shown in Figure 1.

Gas chromatographymass spectroscopy (GCMS)
GCMS study was undertaken to identify the individual components of the bark oil.Totally there were 13 different peaks observed in the chromatogram.The observed R t values are as follows, 4. 734, 5.115, 5.539, 5.809, 6.609, 6.723, 6.850, 7.142, 7.241, 7.524, 7.726, 7.808, 8.016.The main peak was observed at R t value 6.723, this corresponds to the 26.92% composition of the oil.

Minimum inhibitory concentration (MIC)
The antimicrobial assay results showed that MIE exhibited in-vitro antibacterial activity against Gram-positive and Gram-negative bacteria.The MIC values are depicted in Table 4. MIE showed MIC value for E.coli, P. Aeruginosa and B.subtillis at 0.2 µg/ml respectively.Whereas, MIE showed MIC value for S.aureus at 0.8µg/ml, which was considered as Sensitive (S).

Zone of inhibition
The antimicrobial assay showed that MIE exhibited invitro antibacterial activity against Gram-positive and Gramnegative bacteria.The Concentration of MIE was 10 mg/ml which showed zone of inhibition of 13 mm and 10 mm at 50 µl against P. Aeruginosa and E. coli respectively.Whereas zone of inhibition of 8 mm for B. subtillis at 25 µl and that of 8 mm for S. aureus at 75 µl.The detailed results have been depicted in Table 3.

DISCUSSION
MIE was extracted from locally available mango tree bark which is pale yellow in colour, characteristic odour.Literature survey indicates that MIE has been used for throat infection in home remedies due to the astringent character (Chetan et al., 2014).MIE consists of various volatile oil, fatty acids, which has significant lipophilicity this may be useful for inhibition microbial growth.
In the present study, antimicrobial assays were performed by the agar-well diffusion and MIC method.The antimicrobial activity of the MIE is shown in Table 3 and 4   These bacteria are leading cause of both hospital and community acquired infections and has greater virulence capabilities as well as resistant development than other bacteria.This broad spectrum activity should make it useful for the treatment of infections requiring prompt therapy.Variation in activity among different extracting solvents and also amongst different bacterial strains has been reported.
respectively.All the microbes used in the present study were sensitive to the MIE and showed a potential activity against growth of both gram positive and gram negative bacteria.The activity was concentration dependent against the various micro organisms; which are shown below.B. subtilis, E. coli, P. Aeruginosa,> S. aureus.

Table 1 :
Analytical Properties of MIE.

Table 2 :
Spots and their Rf values.

Table 3 :
Agar well diffusion technique