GC-MS PROFILE, ANTIBACTERIAL, ANTIFUNGAL, AND ANTICANCER ACTIVITY OF ROOT OF VANDA TESSELLATA AN EPIPHYTIC ORCHID

Background: The presence of phytochemical constituents has been reported from species of the Orchidaceae. Hitherto no reports exist on the GC-MS Profile of Vanda tessellata Hook. Ex G. Don which was an epiphyte from the sacred groove of Penchalikona of Andhra Pradesh. Objective: The current study was to determine the antibacterial, antifungal,anticancer activities, and GC-MS profiles of the root of Vanda tessellata Hook. Ex G. Don. and The root of the Vanda tessellata Hook. Ex G. Don which was an epiphytic The GC-MS profile has shown the presence of different phytochemical compounds in the epiphytic root of tessellata Hook. G. Don. A total of 33 compounds were identified. Gas chromatography-mass spectrometry (GC-MS), analyses were done to find out the active principles in root extracts of V. tessellata in two different solvents. GC-MS analyses revealed a battery of different compounds, many of which are with multi-therapeutic properties. Both methanolic and ethyl acetate root extract a total of 33 analytes are revealed. The data indicate that the ethyl acetate extracts performed better and are a more promising therapeutic agent. The extracts can be used in various pharmaceutical applications.

1556 Disc diffusion method: Media preparation: Dissolve 24 gm of PDB in 1000 ml water to obtain PDB-Potato Dextrose Broth for fungal growth.The broth was sterilized by autoclaving at 121 o C and 15 lb. Pressure for fifteen minutes. The sterilized medium (20 ml) was poured in sterilized Petri dishes under aseptic conditions, allowing them to solidify on a plane table.

Procedure:
Inoculation of fungal strains in autoclaved PDB media and Incubate 3-4 days at 30ºC in a shaker for fungal growth. From that 20 µl of fungal culture was taken and inoculated by inoculation loop on freshly prepared autoclaved agar plates. Filter paper discs (Whatman N0.1 filter paper) of about 6 mm in diameter impregnated withthe test compound at the desired concentration, are placed on the agar surface on the fungal plate. The incubation of the plates was done for 2 to 4 days at 30ºC in the BOD incubator. Zone of inhibition around each disc was measured by measuring scale and recorded. Negative control was prepared with only methanol extract used for extraction.
The inhibition percentage (I %) was calculated using the formula. I%=(C-T)*100/C Where I = Inhibition % of mycelial growth (growth reduction over control), C = Radial growth of the fungus in the control plate (mm), T = Radial growth of fungus on the inoculated plate.

Minimal Inhibition concentration (MIC):
An agar plugs of actively growing fungal culture was placed in the center of the PDA plates, respectively. Plates were incubated for 24h then, wells were made using sterile agar borer 2 cm away from the center where fungal was placed, and different aliquots (100 µg/ml, 75 µg/ml, 50 µg/ml, 25 µg/ml) of root extracts were added in separate wells and incubated at 28 0 C-30 0 C and incubated for 24h-96h.The inhibition percentage (I %) was calculated using the formulaI%=(C-T) *100/C WhereI = Inhibition % of mycelial growth (growth reduction over control), C = Radial growth of the fungus in the control plate (mm), T = Radial growth of fungus on the inoculated plate.
Anticancer activity: SRB assay: Principle: SRB assay is developed in the year 1990. After its development, the SRB assay used to conduct various screening assays to investigate cytotoxicity in cell-based studies [34].This method mainly relies on the property of SRB, which binds stoichiometrically to proteins under mildly acidic conditions and it can be extracted using alkaline conditions, the amount of bound dye considered for proxy for cell mass which can be extrapolated to measure cell proliferation.

Experimental Procedure: Preparation of treatment:
In the present study, the cell lines (Human Hepatoma Cell Line Hep-G2, Murine Skin Melanoma Cell Line B16-F10) were grown in RPMI 1640 medium containing 10% fetal bovine serum and 2 mm L-glutamine. Depending on the doubling time of individual cell lines cells were inoculated into 96 well microtiter plates in 100 µL at plating densities depending on the doubling time of individual cell lines for the present screening experiments.

Incubation of cells:
After the cell inoculation completed, microtiter plates were incubated at conditions 37 0 C,5 per cent CO 2 ,95 per cent air and 100 per cent relative humidity for 24 h before the addition of experimental drugs.
Cell fixation and SRB staining: roots powder was initially solubilized in dimethyl sulfoxide (DMSO) at100 mg/ml and using water diluted to 1mg/ml and stored frozen before use. An aliquote of frozen concentrate (1mg/ml) was melted and a dilute solution of 100 µg/ml, 200 µg/ml, 400 µg/ml and 800 µg/ml prepared with complete medium containing test article before the time of drug addition. Already appropriate microtiter plates containing 90 µl of the medium are present. For 1557 these aliquots of 10 µl of this different drug,dilutions were added resulting in the required final drug concentrations,i.e.,10 µg/ml, 20 µg/ml, 40 µg/ml, 80 µg/ml. After that compound is added to the plates and Plates were incubated at standard conditions for 48 hours, after that by the addition of cold TCA and the assay was terminated. After that gentle addition of 50µl of cold 30 % (w/v) TCA (final concentration, 10 % TCA) the in-situ fixation of the cells took place and incubated for sixty minutes at 4°C. Discarded the supernatant after the experiment, the plates were washed five times with tap water and air-dried. For each ofthe wells Sulforhodamine B (SRB) solution (50 µl) at 0.4 % (w/v) in 1 % acetic acid was and plates were incubated for 20 minutes at room temperature conditions. The unbound dye was recovered after staining, and the residual dye was removed by washing five times with 1 % acetic acid. The plates were air-dried. With a 10 mm trizma base, the bound stain was subsequently eluted. By using a plate reader and the absorbance was read at a 540 nm wavelength using a reference wavelength of 690 nm. On a plate-by-plate basis for test wells, per cent growth was calculated by comparing with control wells. The ratio of average absorbance of the test well to the average absorbance of the control well*100 is called growth percentage.

Absorbance measurement:
Mainly six absorbance measurements (time zero (Tz), control growth (C) and test growth in the presence of drug at four concentration levels (Ti)are taken into account for the calculation of percentage growth at each concentration level. Growth inhibition percentage was calculated as Ti/C*100.

GC-MS Analytical technique: Principle:
GC-MS is a combination of two different analytical techniques, Gas Chromatography (GC) and Mass Spectrometry (MS), which analyse complex biochemical and organic mixtures [28]. The two main components of the GC-MS instrument are the gas chromatography portion and a mobile phase. The gas chromatography portion which splits dissimilar compounds in the sample into pulses of pure substances based on their instability [22] by flowing inert gas (mobile phase), which moves the sample, across a stationary phase fixed in the column [28]. Spectra of compounds are gathered as they are leaving a chromatographic column by the mass spectrometer, which recognizes and counts the chemicals according to the mass-to-charge ratio (m/z). These generated spectra can then be stored on the computer and analysed [22].

Methodology:-
Gas Chromatography-Mass Spectrum Analysis (GC-MS) GC-MS technique was used in this study to identify the phyto components present in the root extracts. The GC-MS analysis of this extract was performed using GC SHIMADZU QP2010 system and gas chromatograph interfaced with a Mass Spectrometer (GC-MS) equipped with Elite-1 fused silica capillary column (Length: 30.0m, Diameter: 0.25 mm, Film thickness: 0.25 µm Composed of 100% Dimethylpolysiloxane). For GC-MS detection, an electron ionization energy system with ionization energy of 70eV was used. For each sample, the lyophilized compounds were dissolved in 50 ll pyridine (Sigma-Aldrich, Steinheim, Germany), sonicated, and then derivatized in 60 ll bis (trimethylsilyl)-trifluoroacetamide (Sigma-Aldrich) at 75 •C for 45 min.
GC-MS analysis was accomplished on a Shimadzu QP 2010 GC-MS system using a 30 m × 0.25 mm (i.d.) × 0.25 lm DB5-MS fused-silica capillary column (J & W Scientific, Folsom, CA).The temperature injector was 280 0 C, and the split ratio was 10: 1. The carrier gas (Helium) is with a linear velocity of 35.0 cm s−1 and an equilibration time of 3 minutes. The temperature of the column keeps at 70 o C at the beginning for 5 min and then increased to 280 0 C at 5 • C min 1, held for 5 min. The MS scan parameters included a mass scan range of m/z 40-600, a scan-interval of 0.5s, a scan speed of 1000 u s−1, and a detector voltage of 0.9 kV. The ion source temperature was fixed to 200 0 C, and the interface temperature was 280 0 C. The solvent cut time was 6 min.
The carrier gas such as Helium was used at a constant flow rate of 1.51ml/minutes, and a volume of injection 2 μl was used. Injector temperature was 200°C, and Ion-source temperature was 200°C. The programming of oven temperature was at 70°C (isothermal for two min.), with an increase of 300°C for 10 min. Mass spectra were noted at 70eV; a scan-interval of 0.5 seconds with a scan range of 40 -1000 m/z. Total GC running time was 35 min. After the comparison of its average peak area to the total areas, the relative percentage amount of each component was calculated Software adapted to handle mass spectra, and chromatograms was a GC MS solution ver. 2.5.

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Results:-Antibacterial activity: Antibacterial activity of different root extracts was conducted in two phases. Preliminary studies were carried out by disk diffusion method to find out the susceptibility of selected bacterial strains to each of the tested root extracts. Based on the primary activity of the tested extracts on the bacterial strains, selected root extracts were tested on the bacterial isolates to determine the minimum inhibitory concentration (MIC) to find out the effectiveness. The lowest concentration of the tested root extracts that inhibits the visible growth of the microorganism tested, recorded as MIC value and is expressed as µg/ml or mg/L. Root extracts of Vanda tessellata, evaluated in three different solvents viz., methanol, n-hexane and ethyl acetate against two gram-positive bacteria Staphylococcus aureus (MTCC -96), Bacillus subtilis and two gram-negative bacteria Escherichia coli (MTCC -443), Pseudomonas aeruginosa (MTCC -424) by disk diffusion method.
Methanolic root extracts of V. tessellata were effective against gram-negative bacteria E. coli and P. aeruginosa. (Table-2    None of the n-hexane root extracts of V. tessellata has shown any inhibitory activity on the bacterial strains tested.

Minimum inhibitory concentrations (MIC's) of the effective root's extracts:
Basing on the results obtained minimum inhibitory concentrations (MIC's) of the effective root extracts were employed to study their bacteriostatic properties.

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Methanolic root extracts of V. tessellata induced inhibitory activity against B. subtilis, E. coli and S. aureus, and exerted the highest inhibition on the gram-negative P. aeruginosa at three lower doses. (Table:3

Antifungal activity:
Antifungal activity of different root extracts was conducted in two phases. Preliminary studies were carried out by disk diffusion method to find out the susceptibility of the selected fungal strains to each of the tested plant extracts. Based on the primary activity of the tested extracts on the fungal strains, minimum inhibitory concentration (MIC) of effective root extracts were employed to evaluate antifungal properties.

Antifungal activity of root extracts:
Root extracts of Vanda tessellata were tested for their antifungal activity against three fungal strains Viz Fusarium oxysporium, Sclerotium rolfsii and Phytophthora infestans by disk diffusion method using three different solvents viz., methanol, n-hexane and ethyl acetate.
Of all the extracts methanolic root extracts V. tessellata was the only one that exerted inhibition on the growth against F. oxysporum. (Table:4, Fig:4&Plate-2) Similarly, ethyl acetate root extracts of V. tessellata exerted an effect on the growth of P. infestans and F. oxysporium. None of the n-hexane extracts of roots of V. tessellata was effective inhibitors against the three fungal strains tested. (Table:4   Results noted with ethyl acetate root extracts of V. tessellata against F. oxysporium and P. infestans were more or less to similar to their methanolic counterparts. (Table:5 Table-6.
An in-vitro study of root extracts of V. tessellata exhibited little anti-proliferative activity on the Hep G2 and B16-F10 cell lines. Based on the calculations from graphs, using GraphPad prism software, it is extrapolated that above 80 μg/ml concentration, of any of the extracts, is necessary to exert 50% growth inhibition of the two cell lines. (Table:6   Hep-G2 B16-F10

GC-MS (Gas Chromatography and Mass Spectroscopy analysis):
Gas chromatography-mass spectrometry (GC-MS), a key technological platform for secondary metabolite profiling in plant species, was carried out on root extracts of Vanda tessellata, using two solvents methanol and ethyl acetate. The total analysis of the extracts of samples revealed about 33 active compounds. Therapeutic values of the compounds are gathered from the PubChem, which is the database of chemical molecules which maintains three types of information namely, substance, compound and bioassays and the web link is https://pubchem.ncbi.nlm.nih.gov/. The list of compounds with their chemical names and therapeutic values are given in Table 8.

GC-MSanalysis of plant extracts:
A maximum number of compounds with antimicrobial property were found in ethyl acetate root extracts of V. tessellata. The methanolic root extract of V. tessellata, revealed four active compounds among which dimethyl ether is used in wart treatment. (Table:8  Arabinitol, pentaacetate Cell immobilization PubChem-Database of chemical molecules which maintains three types of information namely, substance, compound and bioassays.

Discussion and Conclusions:-
One of the key factors influencing the extraction efficiency of bioactive compounds from plant extracts is an extraction solvent. Besides, the concentration of the crude drug, temperature, plant parts used for the extraction of secondary metabolites and rate of diffusion are the other factors that influence the efficacy of the extract [23].
In the overall preliminary screening, ethyl acetate extracts showed more promising activity against tested bacteria compared to its methanolic counterparts [12]. None of the hexane extracts of the root had any inhibitory activity against the bacterial strains tested. Reduced ability of hexane to extract polar solutes could be the reason for non-performance of hexane extracts [30,31].
Bacteriostatic efficacy of V. tessellata against all the four bacterial strains as can be inferred from minimum inhibitory concentrations (MIC), was in accordance with the studies of earlier workers [1,4,5,10,12].Better performance of V.tessellata against the two gram-negative bacteria amply supports the earlier studies [4,5,26].
Antibacterial and the antifungal activity of the root extracts was observed in the present study could be attributable to these metabolites like terpenoids, alkaloids phenolics in general and flavonoids in particular [18].
Absence of anticancer activity by the root extracts, in the present study, could be attributable to the solvent, DMSO, which might have failed to extract the bioactive chemicals that could mediate respective biological activity. Ascertaining the pharmacokinetics of an extract was difficult, if a compound, necessary for anti-prolific activity is insoluble in the solvent [7,31].
The absence of anti-prolific activity by the tested doses of V. tessellata was in line with studies of Chowdary et al., 2014 [9] where in LC 50 value with methanol extracts of V. tessellata was observed at 574.32µg/ml. Similarly,