Antibacterial Activity and Phytochemical Screening of Garcinia pedunculata Roxb. ex Buch.-Ham. fruit extract by HPLC–ESI-MS

Disc and well diffusion methods were used to test the antibacterial activity of methanol extract and ethanol extract of the selected ethnomedicinal plant viz. Garcinia pedunculata Roxb. ex Buch.-Ham. Both the methanol extract and ethanol extract were subjected to antibacterial activity assay against the six clinical isolates. Antibiotic sensitivity test of the test bacteria against standard antibiotics were also determined. All the bacterial pathogens (Staphylococcus aureus, Enterococcus faecalis, Enterobacter cloacae, Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae) were exposed to the plant extract in triplicates. The investigation revealed the potency of G. pedunculata extract as an effective antibacterial agent against both Gram-positive bacteria (GPB) and Gram-negative bacteria (GNB). G. pedunculata are evaluated to be bactericidal against the tested bacteria. The antibacterial activity may be due to an individual compound or synergistic effect of more than one compound present in the medicinal plant extract. The minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of the plant extracts were also investigated by using the broth microdilution method. HPLC-MS of the methanol extract of G. pedunculata fruit revealed the presence of Hydroxy Citric Acid Lactone (MW-190), Garcinone-E (MW-464), a-Mangostin (MW-410), β–Mangostin (MW424), and γ-Mangostin (MW-396).


MATERIALS AND METHoDS Collection and identification of plant
The plant sample ( Fig. 1) was collected from its natural habitat and identified at Botanical Survey of India, Eastern Regional Centre, Shillong. G. pedunculata voucher specimen (Accession No. 96682) has been deposited at Assam Herbarium, BSI, Shillong, India.

Preparation of plant extract
The clean dried pericarp of G. pedunculata were grounded by motor-driven grinder into powder form. Ten (10) gms of powdered plant material was weigh and extracted in methanol and ethanol solvent in Soxhlet Hot Extraction Apparatus. The crude extract was subjected to filtration using Whatmann filter paper no. 1. The collected solution was dried in a rotary shaker (150 rpm; 50° C) and stored at 4°C until further use.
The reconstituted working methanol (MEGP) and ethanol (EEGP) extracts were prepared by dissolving the required amount of dry extract in their respective solvents and are employed directly without any adjustment of pH.

Preparation of standardized inoculum
Each of the bacterial isolates were grown on Mueller-Hinton Agar (MHA, HiMedia) plate for 18-24 hrs. at 37 ± 2°C. Well-isolated colonies were inoculated into sterile Mueller-Hinton Broth (MHB, HiMedia) and the turbidity was adjusted against the 0.5 McFarland standard to yield approximately 1.5 x 10 8 CFU/ml.

Antibacterial activity of methanol and ethanol extracts of G. pedunculata towards UTI clinical bacterial isolates by Disc Diffusion Method
Antibacterial activity of methanol and ethanol extract of G. pedunculata towards different clinical isolates were assessed in terms of diameter of inhibition zone using disc diffusion method as stated by Kirby -Bauer et.al 5 It is the recommended method of European Committee on Antimicrobial Susceptibility Testing (EUCAST) and National Committee for Clinical Laboratory Standard ( NCCLS ),USA .
The isolates to be tested were, firstly, inoculated/spread on the surface of MHA using sterile spreader. Sterile 10-mm diameter blank discs (HiMedia) impregnated with 250 µl of different plant extracts stock solution (200 mg/ ml) were place on the inoculated MHA plate as test disc. Blank disc impregnated with 250 µl methanol and ethanol were used as negative control for methanol and ethanol plant extract respectively. Place the discs in an empty sterile petri dish and then apply the solution to the discs in an installment of 50 µl, 100 µl and 100 µl. Allow 15-30 minutes for the discs to absorb and dry at 40°C in an Oven before applying every installment. Disc of ampicillin (10 mcg) and methicillin (5mcg) antibiotics (HiMedia) were used as reference antibiotics. All test plates were incubated at 37 ± 2°C for 18 to 24 hours and diameter of zones of inhibition produced by the plant extracts were measured. The experiment was repeated three times for each combination of extract and microbe.

Antibacterial activity of methanol and ethanol extracts of G.pedunculata towards UTI clinical bacterial isolates by Well Diffusion Method
The susceptibility of different bacterial clinical isolates to the methanol and ethanol extract of G. pedunculata was determined in terms of diameter of zone of inhibition using agar well diffusion assay. Firstly, the MHA plates were spread with the inoculum and wells (8mm diameter) were cut out using a sterilized stainless steel well borer. The wells were then filled with 250 µl of the plant extracts (200 mg/ml). Wells filled with 250 µl methanol and ethanol were used as negative control for methanol and ethanol plant extract respectively. Disc of ampicillin (10 mcg) and methicillin (5 mcg) antibiotics (HiMedia) were used as reference antibiotics. The bacteriainoculated plates were incubated at 37± 2° C for 18 to 24 hours, and the diameter of any resulting zone of inhibition was measured. The experiment was repeated three times for each combination of extract and bacterial strain.

MIC Determination by Broth Microdilution Method supplemented with Resazurin
The MIC of the methanol extract (MEGP) was determined by using broth microdilution method, as described previously by Wiegand et al 6 supplemented with Resazurin dye. 7 MHB was used as the diluent in a two-fold serial dilution of plant extract. Negative control (inoculum and MHB only) was also maintained. Solvent blanks and positive controls were also included.
The bacterial inoculum was prepared in the same manner as described above for the disc-diffusion method. Fifty (50) μl of the adjusted inoculum were mixed into each Eppendorf tube containing 200 μl of MHB and 250 μl of each plant extract dilution in the dilution series. In an ambient air incubator, the capped microdilution vials were incubated at 37± 2°C for 18 to 24 hours.
After incubation for 18 to 24 h at 37 ± 2°C, resazurin (0.015 %) was added to all the Eppendorf tubes (150 μl per vial), and further incubated for 2-4 h for the observation of color change. On completion of the incubation, the Eppendorf tubes with the lowest concentration of the extract that shows no color change (blue resazurin color remained unchanged) is scored as the MIC value. The experiment was repeated three times.  Footnote : Diameter of zone of inhibition (mm) are given as mean ± SD; (n = 3)

Minimum Bactericidal Concentration (MBC)
The MBC was determined by directly plating the contents of the Eppendorf tube at a concentration higher than the MIC value. The lowest concentration of extract that did not allow any growth was taken to MBC.

LC-MS Instrumentation and Experiment
The methanol extract of G. pedunculata (MEGP) was subjected to LCMS analysis. The LC/ MS was performed on Waters ACQUITY UPLC-TQD Mass spectrometer model ACQ-TQD#QBB1152 with Waters 2424 ELSD detector. The HPLC Column HYDROSPHERE C18, 12nm, 5µm, 250 x 4.6 mm, maintained at temperature of 35° C was used for separation. The solvents used were of LC-MS grade. The sample injection volume was 20.0 µl. A continuous gradient system was followed using mobile phase composed of acetonitrile (ACN) and formic acid (0.1 % FA, v/v in water) for 40 mins. At a flow rate of 1.5 mL/min, the extract was investigated using the following eluent mixtures:

Antibacterial Susceptibility Test of UTI clinical bacterial isolates against standard antibiotics by Disc Diffusion Method
The result of the antibacterial susceptibility test is given in Table 1. The different cultures of the clinical bacterial isolates responded to standard antibiotics in a variable manner resulting in various size of zones of inhibition. The size of zone of inhibition range from 10.3±0.5 (Piperacillin/Tazobactam PIT 100/10mcg against K.pneumoniae) to 59.7±0.5 (Levoflaxacin LE 5mcg against S.aureus).
According to the 31 st edition of CLSI performance standard for antimicrobial susceptibility testing, E.coli was susceptible to all the 12 standard antibiotics tested; E.cloacae was resistant to CAZ, PI, CPM, AT, CPZ and PIT but susceptible to GEN, AK, CIP and LE; K.pneumoniae was susceptible to GEN, AK, CIP, LE, IPM and MRP but resistant to CAZ, PI, CPM, AT, CPZ and PIT; P.aeruginosa was susceptible to CAZ, GEN, AK, CPM, AT, CIP, LE and IPM : S.aureus was susceptible to GEN, CIP and LE: E.faecalis was susceptible to CIP and LE.

Antibacterial activity of methanol and ethanol extracts of G. pedunculata against UTI clinical bacterial isolates -Disc Diffusion Assay
To evaluate the antibacterial activity of G. pedunculata fruit extracts, two different solvents were used to determine which of them      might offer optimal activities against UTI clinical bacterial isolates. The disc diffusion method was used to determine the presence of antibacterial activities. All the methanol and ethanol negative control discs did not produce any zone of inhibition against any of the tested clinical isolates. Table -2 and Fig. -2 & 3 shows the diameter of the zones of inhibition produced by the methanol and ethanol extracts (Disc diffusion method) on the various test bacteria. The data presented in this paper pertains to extracts of crushed plant parts without any pH adjustment. Both the extracts produced very significant antibacterial activities against all the bacterial strains tested. The ethanol extract demonstrated zones of inhibition ranging from 10 mm (E.coli) to 16 mm (E.faecalis). The methanol extract showed zones of inhibition ranging from 10.7 mm (E. cloacae, K.pneumoniae) to 19 mm (E. faecalis).The results were compared with those of ampicillin and methicillin as standard antibiotics. Both the solvent extracts (200mg/ml) of G. pedunculata were not as potent as ampicillin (10 mcg) but are quite effective as methicillin (5 mcg) antibiotics or even better.

Well Diffusion Assay
In order to counter check and corroborate the result of the disc diffusion assay, the antibacterial activity of the methanol extract and ethanol extract of G. pedunculata was also determined by the well diffusion method. It was observe that both the organic solvent extracts of G. pedunculata were inhibitory to all the six bacterial isolates and the diameter of the zones of inhibition were relatively greater than the corresponding zones of inhibition observed in disc diffusion assay. Though the same volume of the extract were used against the six bacteria, the bigger zone of inhibition is expected in the well diffusion assay as the size of the wells (8 mm) are comparatively greater than the size of the disc (6mm). All the methanol and ethanol negative control discs did not produce any zone of inhibition against any of the tested clinical isolates (Table -2

Minimum Inhibitory Concentration (MIC) of G. pedunculata
MIC is used to determine the potency of the antimicrobial compounds towards the susceptible bacteria. An antimicrobial agent with lower MIC value is considered better as less compound is required to inhibit growth of the bacteria. The MEGP showed antibacterial activities against both GPB and GNB. The inhibitory effect of the extract increased with increasing concentration (1.56 -100 mg/ml). The MEGP is approximately equally potent against S.aureus, E.faecalis and P.aeruginosa, with MIC being 12.5 mg/ml. Against E.cloacae, E.coli and K.pneumoniae, the MIC of the   MEGP was observed to be approximately 25 mg/ ml. This result also suggest that the MIC of the MEGP is bacterial genus/species dependent.

Minimum Bactericidal Concentration (MBC) of G. pedunculata
MEGP showed antibacterial activity against all the tested Gram-negative bacteria as well as Gram-positive bacteria. The MBC values was found to be approximately 25 mg/ml against S.aureus, E.faecalis and P.aeruginosa while 50 mg/ ml was the observed MBC against E. cloacae, E.coli and K.pneumoniae.

LC-MS Result
The methanol extract of G. pedunculata (MEGP) was selected for LCMS analysis over ethanol extract (EEGP) since it was observed that it gives larger zone of inhibition against the bacteria tested. It implies that methanol may be a better organic solvent for extraction of antibacterial compounds from G. pedunculata fruit. LC-MS of the methanol extract of G. pedunculata fruit revealed presence of several compounds.

disCussiON
The antimicrobial activity of different species of Garcinia had been investigated by various researchers with an encouraging result till date. Negi et al 11 reported that certain foodborne pathogens and spoilage bacteria, including B.cereus, B.coagulans, B.subtilis, S.aureus, and E.coli, are sensitive to crude hexane and chloroform extracts of Garcinia cowa and Garcinia pedunculata fruit rinds. 11 Torrungruang et al 12 also observed that G.mangostana Pericarp extract exert antibacterial activity against cariogenic Streptococcus mutans. 12 Priya et al 13 study revealed the antibacterial activity of Pericarp extract of G.mangostana against Staphylococcus aureus, Staphylococcus albus and Micrococcus luteus. 13 Mangosteen pericarp extract has long been known for its broad-spectrum antibacterial activity against a number of GPB and GNB, particularly those linked to skin infections, diarrhoea, TB, and acne. a-mangostin, one of the xanthone derivatives derived from mangosteen extract, has been shown to have the strongest antibacterial action. [14][15][16][17][18][19] The present investigation revealed the potency of both methanol and ethanol extracts of G. pedunculata as an antibacterial agent. G. pedunculata is effective as an inhibitory agent against both GPB as well as GNB. The antibacterial activity can be attributed to its phytochemical constituents. 20 The antibacterial activity may be due to an individual compound or synergistic effect of more than one compound present in the medicinal plant extract. Heymsfield et al 8 , Kumar et al 9 and Aravinda et al 10 reported xanthones, biflavonoids, benzophenones, benzoquinones, and triterpenes are bioactive chemicals found in Garcinia species that exhibit antibacterial, antifungal, antioxidant, and cytotoxic properties. [8][9][10] The antibacterial activity of MEGP was quantitatively evaluated by determining its minimum inhibitory concentration (MIC) values and minimum bactericidal concentration (MBC). As the bigger zone of inhibition was observed with methanol extract of G. pedunculata (MEGP) than that of ethanol extract of G. pedunculata (EEGP), MEGP was preferred to EEGP for determination of MIC and MBC. The MEGP is considered to be bactericidal rather than bacteriostatic since its MBC/MIC ratio is ≤ 4. An antimicrobial agent is considered bactericidal if the MBC is not more than fourfold higher than the MIC. 21 Table 3.