Antibacterial and Antioxidant Activities of 3O-methyl Ellagic Acid 4 ’-rhamnoside from Stem Bark of Polyalthia longifolia Thw .

The plant Polyalthia longifolia (Annonaceae) is an ornamental tree that finds its reference in Indian medicinal literature owing to its popular Hindi name Ashoka i.e, Saraca indica. However, P. longifolia is equated with the name Asoka and often used as an adulterant or substitute of the genuine Asoka bark. The present investigation was carried out with an object to separate and isolate active phytomolecule(s) from stem bark of P. longifolia and to screen their antibacterial and antioxidant potential. Column chromatography of the butanol fraction of the hydroalcoholic extract (methanol:water, 1:1) has led to the isolation of a phenolic compound. Structural elucidation was done by IR, 1 H NMR, 13 C NMR, DEPT, COSY, HSQC, HMBC and mass spectroscopy techniques, and purity was checked by HPTLC and HPLC. Butanol fraction and the isolated compound were screened for antibacterial activity (against facultative aerobic and fastidious aerobic bacterial strains) and antioxidant potential (DPPH method). The compound was revealed to be 3-O-methyl ellagic acid 4’-rhamnoside (1), and the purity of the compound was 99.2%. The isolated compound comprises promising antibacterial and antioxidant activities.


Introduction
The plant Polyalthia longifolia (Annonaceae) is an ornamental tree, that finds its reference in Indian medicinal literature owing to its popular Hindi name Ashoka.Ashoka, a Sanskrit name in Ayurveda stands for the plant Saraca indica.However, Polyalthia longifolia is equated with the name Asoka and due to its easy availability, often used as an adulterant or substitute of the genuine Asoka bark [1].As such, no medicinal attributes are accorded to P. longifolia [2].P. longifolia is indigenous to the southernmost part of the India and to Ceylon; it has been cultivated in Bombay and other parts of India.It is useful in fever, skin diseases, ulcer, diabetes, hypertension, helminthiasis and vitiated conditions of vata and pitta [3][4][5][6].It is also used in the treatment of burning sensation, thirst, worm infestations, wound, diarrhoea, scrofulous gland tumors and uterine disorders.The plant contains diterpenoids, alkaloids, tannins and mucilage.The chief components among others are aporphine and azafluorene alkaloids, clerodane and ent-halimane diterpenoids and sesquiterpenes [7][8][9][10][11][12].The present study was designed to isolate phytoconstituent(s) from the hydroalcoholic extract of the bark of the plant and to study their antibacterial and antioxidant activities.

Determination of minimum inhibitory concentration
The minimum inhibitory concentration of compound 1 and butanol fraction was found to be in the range of 80-160 µg/ml and 160-320 µg/ml respectively.Compound 1 exhibited higher antibacterial potential against all most all tested bacterial strains than butanol fraction, but the potency is less as compared to standard drugs (Table 2 and 3).

Antioxidant activity
The highest antioxidant activity of the compound was 57.95% at 40 µg/ml and butanol fraction was 66.05% at 40 µg/ml.Isolated compound exhibited better antioxidant property than the standard drug, vitamin C (Table 4).Literature surveys indicated that plant phenolics constitute one of the major groups of compounds found in both edible and inedible plants and reported to have multiple biological effects, including antioxidant activity [13][14][15][16][17]. Hence, the antioxidant and antimicrobial activities of P. longifolia may be due to presence of the isolated compound.

Plant material
The stem bark of Polyalthia longifolia (Sonn) Thw. was obtained from Banasthali University campus, Rajasthan, India and identified by Dr. Vinod Kumar Sharma, Department of Botany, Rajasthan University, Jaipur, India (Voucher No.: RUBL 211351).A voucher specimen was preserved in department of pharmacy, Banasthali University, Rajasthan for future references.

Preparation of extract
The stem bark of P. longifolia was first air dried for few days and then dried under controlled temperature (45⁰ C).It was then crushed to smaller pieces followed by coarsely powdered in a grinding mill and stored in an air tight labeled container in a cool place till further use.Air dried and coarse powdered stem bark (2kg) was extracted by cold maceration technique with hydroalcohol (methanol:water, 1:1) at room temperature for twenty four hours, three times successively.Filtered the extracts and pooled, concentrated in rota vapor (Buchi, Switzerland) under reduced pressure, a dark brown viscous mass (178g) was obtained.

Characterization of compound 1
FTIR studies were conducted on the IR ARD/1402 (FTIR Spectrophotometer, Perkin Elmer, USA); 1 H and 13 C NMR was recorded on AVA-NCE (Bruker, Switzerland) at 400 and 100 MHz respectively.The two-dimensional experiments (HSQC, HMBC, COSY) were also performed.Samples were dissolved in DMSO based on the solubility of the sample.Mass spectra were recorded on Direct MS (Waters, USA).Purity of the isolated compound was done by HPTLC [Sample applicator: Linomat 5, stationary phase: precoated silica gel G 60 F 254 , mobile phase: chloroform:methanol:water (7:3:0.5),detection: under UV at 254 nm] and waters HPLC system equipped with waters 2996 PDA detector in combination with Empower software [column: C18(ODS), 250*4.6 mm, 5 micron, Kromasil; sample cabinet temperature: 20 0 C; sample prepared in methanol; injection volume of 20µl; mobile phase: acetonitrile:buffer; flow rate: 1ml/min; run time: 30min; detection: 254 nm; and purity determination by area normalization].Erythromycin (for S. pnemoniae, S. pyogens and S. viridens), vancomycin for S. aureus, oxacillin for MRSA and ciprofloxacin (for P. aeruginosa, E. coli and A. aumannii) were used as standard drugs.

Bacterial inoculum preparation
Picked up 3-4 isolated bacterial colonies from the respective medium plates individually with the help of inoculation needle one day prior to incubation of the plate.Picked up colonies were added in 2ml of pre sterilized saline solution (0.85% NaCl), mixed it properly with the help of vortex mixer (Remi, New Delhi) to make a homogeneous suspension.Bacterial density was adjusted to 1.0-1.1 Mac Farland for fastidious bacterial strains and 0.5-0.8Mac Farland for facultative aerobic bacterial strains using densitometer (Biomerieux, France).Both adjusted Mac Farland absorbance were represented as 0.5-1.5 x 10 8 cfu/ml.

Preparation of drug samples
The stock solution of 2 mg/ml of butanol extract and isolated compound was prepared in DMSO.From the stock solution different concentrations as 320, 160, 80, 40, 20, 10, 5, 2.5, 1.25, 0.625 µg/ml were prepared.The different concentrations of standard drugs used were 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.062, 0.031, 0.015 µg/ml.Assay method Micro titter 96 well plate was used to determine MIC value of each drug.For the good growth of fastidious organisms one of the growth supplement i.e., sheep blood (5%) was added in Mullere Hinton broth.On the other side, Muller Hinton broth was used for facultative aerobic bacteria.The prepared inoculum 0.5-1.5 x10 8 cfu/ml was serially diluted to make a final concentration of 1 x 10 5 cfu/ml.The final concentrations of drugs and culture inoculum (1 x 10 5 cfu/ml) were added in micro titer well as per protocol.The final volume was 200µl per micro titter well.Simultaneously DMSO control and positive control as media with inoculum were prepared.Micro titter plates were incubated at 35-37 0 C for 24 hrs and then minimum inhibitory concentration was determined [18,19].

Evaluation of antioxidant activity of butanol fraction and isolated compound
Antioxidant activity was measured on the basis of the scavenging activity of the stable 1,1diphenyl-2-picrylhydrazyl (DPPH) free radical [20,21] and compared with Vitamin C (CAS No.: 50-81-7; purity: 99%; HIMEDIA).Various concentrations of the compound and standard drug were added to 0.004% methanolic solution of DPPH.After 30 min the absorbance at 517 nm was determined, and the percentage inhibition activity was calculated using the following formula.% inhibition = [(Ac − At) / Ac] × 100 Where, Ac = absorbance of control sample and At = the absorbance of test sample.