Preparative TLC was conducted over glass plates coated with silica gel 60 PF254 (0.5 mm thickness), Merck. Gel permeation chromatography was performed using Sephadex LH-20. TLC was also carried out using Merck precoated TLC plates (Silica gel 60, F254), eluting with suitable solvent system. Spots on the TLC plates were visualized under UV light at 254 and 366 nm as well as by spraying with vanillin sulphuric acid followed by heating for 5 min at 110˚C. NMR spectra were recorded in CDCl₃ on a Bruker Advance100 and 400 MHz Ultra shield NMR Spectrophotometer.

The root barks of Zanthoxylum rhetsa were collected from Narsingdi district, Bangladesh in the month of August, 2013. The plant part was identified by a taxonomist (Ms. Nasrin Aktar), Bangladesh National Herbarium where a voucher specimen was deposited for future reference (DACB Accession No. 42528).

The air dried and powdered root barks of Z. rhetsa (3.5 kg) were extracted with methanol over the period of 15 days. The crude methanol extract (40 g) was then fractionated sequentially by petroleum ether (9 g), ethyl acetate (6 g), chloroform (12 g) and methanol (12 g) fractions with continuous stirring. Chloroform (12 g) soluble fraction was subjected to silica gel column and was fractionated with a gradient of petroleum ether-dichloromethane-ethyl acetate-methanol which was given total of 295 fractions each with 20 ml. Similar fractions were mixed together and which showed mixture of several compounds were then subjected to Gel permeation chromatography (GPC) over Sephadex (LH-20) using a mixed solvent system of PE: CF (2:8) followed by mixtures of methanol and chloroform of increasing polarity and finally only with methanol. Depending upon the TLC behavior fractions were mixed and compound 1 (51.7 mg) and 2 (34.6 mg) were isolated followed by eluting with toluene/EtOAc (95:5 and 87:13 respectively) (Figure 1).

8-methoxy-n-methylflindersine(1): Yellow gum; 1H-NMR (400 MHz; CDCl₃): d 7.62 (1H, dd, J = 8, 1.6 Hz, H-5), 7.15 (1H, dd, J = 8, 8 Hz, H-6), 7.07 (1H, d, J = 8 Hz, H-7), 5.55 (1H, d, J = 9.6 Hz, H-3’), 6.77 (1H, d, J = 10 Hz, H-4’), 3.95 (3H, s, OMe-8), 3.91 (3H, s, N-Me), 1.52 (6 H, s, Me-2’ cis, Me-2’ trans); 13C-NMR (100 MHz; CDCl3: 162.2 (C-2), 106.0 (C-3), 154.9 (C-4), 115.6 (C-5), 122.2 (C-6), 114.2 (C-7), 148.5 (C-8), 131.0 (C-9), 18.3 (C-10), 77.2 (C-29), 126.5 (C-39), 118.0 (C-49), 28.2 (2Me-29), 35.1 (N-Me), 56.7 (OMe-8)).

Zanthodioline (2): White crystals; 1H-NMR (500 MHz, CDCl3): δ 7.64 (dd, J = 8.0, 1.2 Hz, H-5), 7.22 (1H, t, J = 8.0 Hz, H-6), 7.12 (1H, dd, J = 8.0, 1.2 Hz, H-7), 4.76 (1H, d, J = 8 Hz, H-4’), 3.97 (3H, s, N-Me), 3.93 (3H, s, OMe-8), 3.84 (1H, d, J = 8 Hz, H-3’), 1.64 (3H s, H-2’ Me), 1.33 (3H, s, H-2’ Me). 13C-NMR (125 MHz, CDCl3): δ 164.6 (C-2), 154.8 (C-4), 148.8 (C-8), 130.8 (C-9), 122.8 (C-6), 118.2 (C-10), 116.2 (C-5), 114.4 (C-7), 106.0 (C-3), 80.9 (C-2’), 75.3 (C-3’), 67.7 (C-4’), 56.7 (OMe-8), 34.8 (N-Me), 26.1 (C-2’ Me), 19.3 (C-2’ Me).

Solvent-solvent fractionation of the crude methanolic extract was conducted by using the protocol intended by Kupchan [8] and altered by Van Wagenen et al. [9] 5 g of the obtained methanolic crude extract. All the four fractions were kept in beakers for analysis (PE 830 mg, CTC 560 mg, CF 675 mg and AQ 400 mg).

The free radical scavenging activity of different soluble extracts of root bark of Z. rhetsa and the pure compound was measured by using DPPH• method of McCune and Johns [12] . Lower absorbance of the reaction mixture indicated

higher free radical scavenging activity [13] . Table 1 shows DPPH radical scavenging activity of the different fractions of the plant and the pure compounds 1 and 2 compared with butylated hydroxyl toluene (BHT). It was observed that the aqueous soluble fraction and 8-methoxy-n-methylflindersine have more proton-donating ability and could serve as free radical inhibitors or scavengers, acting possibly as primary antioxidants. The antioxidant activity of the chloroform and aqueous soluble fractions validated the traditional claims of this plant for the treatment of various ailments (Figure 3) [14] .

Compared to positive control (vincristine sulphate, VS, LC₅₀ 0.42 µg/ml), all the fractions tested showed good brine shrimp larvicidal activity (Table 2). The cytotoxic activity exhibited by the solvent fractions was promising and this clearly indicates the presence of potent bioactive compounds [15] [16] .

In vitro clot lysis activity carried out according to the method of Prasad et al [17] . The fractions of the root bark of Z. rhetsa showed an adequate amount of thrombolytic activity except the pet-ether soluble fractions and the pure compounds. Among all the fractions and pure compounds, the aqueous fraction showed highest clot lysis activity (50.5%), whereas standard streptokinase at 37˚C showed 70.6% lysis of the clot as compared to distilled water showing a negligible lysis of clot (2.4%) (Table 3). This evaluation is previously studied from the leaves of the plant.Further study is required to investigate the in vivo thrombolytic activity and the causative component(s), and mechanism for clot lysis by Z. rhetsa (Figure 4) [18] .

The agar disc diffusion method was employed for the determination of antimicrobial activities [19] . The methanol crude root extracts of Z. rhetsa and its Carbon Tetra Chloride, Chloroform, aqueous soluble fractions and 8-methoxy-n-methylflindersine exhibited significant antibacterial activity against microbial growth which indicated that these extracts contain cheimical substances having antibacterial property [20] . Compound 1 showed significant activity at a concentration of 100 µgm/disc against Sarcina lutea, Staphylococcus aureus and Salmonella paratyphi-A, Shigella dysenteriae, Shigella boydii and Shigella sonnei with high antioxidant activity

has not yet been reported. Pet-ether fraction showed very small zone of inhibition against all other bacteria (Table 4).

The experimental results were expressed as mean ± standard deviation (SD) of three replicates. Statistical significance was determined was considered as significant.

NA = not active; - = absence of growth of microorganism. Diameter of inhibition zone (mm) including well diameter of 8 mm.