Antioxidant , Cytotoxic , Thrombolytic and Antimicrobial Activity of Zanthoxylum rhetsa Root Bark with Two Isolated Quinolone Alkaloids

The background of this study was to investigate the antioxidant, cytotoxic, thrombolytic and antimicrobial activity of the petroleum ether (PE), carbon tetrachloride (CTC), chloroform (CF) and aqueous (AQ) soluble fractions of crude methanolic Zanthoxylum rhetsa root bark with two isolated quinolone alkaloids, 8-methoxy-n-methylflindersine (1) and zanthodioline (2). Structures were characterized by 1D NMR analyses. Antioxidant activity was assessed by using DPPH assay and antimicrobial activity was screened by disc diffusion method. An in vitro thrombolytic model was used to evaluate the clot lysis effect of different extracts of root bark of Z. rhetsa along with streptokinase as a positive control and distilled water as a negative control and the cytotoxic activity of different extracts of Z. rhetsa root bark was evaluated by Brine Shrimp Lethality Bioassay. AQ fraction exhibited strongest antioxidant, cytotoxic and thrombolytic activity among four fractions. The CTC and CF soluble fractions exhibited significant antioxidant, cytotoxic and thrombolytic activity. CTC and AQ fractions gave highest anti-bacterial activity against Vibrio cholera and Klebsiella pneumonia respectively. 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. The antioxidant, thrombolytic and antimicrobial activity of 8-methoxy-n-methylflindersine and zanthodioline are the first record from root bark of this plant.


Introduction
A great number of medicinal plants contain chemical compounds that exhibit antioxidant properties [1] [2].The brine shrimp lethality assay is considered a useful tool for preliminary assessment of toxicity.It has also been suggested for screening pharmacological activities in plant extracts [3].
Though the currently available thrombolytic [4] are wonderful clot lytics, they have still significant shortcomings, including the need for large doses, limited fibrin specificity, bleeding tendency and allergic reactions [5] and in some cases the thrombi have been proven to be resistant to intravenous t-PA6.In recent years, there has been a growing interest in researching and developing new antimicrobial agents from various sources to combat microbial resistance.The fruits and stem bark of Z. rhetsa are used in the treatment of asthma, bronchitis, heart complaints and rheumatism [6].Chemical analysis of Zanthoxylum rhetsa allows the isolation of two pyranoquinoline alkaloids, 8-methoxy-n-methylflindersine (1) and zanthodioline (2), which has been previously reported from Zanthoxylum rhetsa and Zanthoxylum simulans respectively [7].The present paper also describes antioxidant, thrombolytic, cytotoxicity and antimicrobial activity of the PE, CTC, CF and AQ soluble fractions of Zanthoxylum rhetsa root bark along with two isolated quinolone alkaloids.

General
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 CDCl 3 on a Bruker Advance100 and 400 MHz Ultra shield NMR Spectrophotometer.

Plant Material
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).

Extraction and Isolation
The air dried and powdered root barks of Z. rhetsa (3.5  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).

Preparation of Sample for Biological Investigation
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).

Results and Discussion
Comparison of above 1D NMR data with known physical constants of compound 1 [10] and compound 2 [11] definitely confirmed the structures determination (Figure 2).

Evaluation of Antioxidant Activity
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].

Brine Shrimp Lethality Bioassay
Compared to positive control (vincristine sulphate, VS, LC 50 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].

Evaluation of Thrombolytic Activity
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].

Antibacterial Activity
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).

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

Conclusion
The biological activity found in this investigation justifies the traditional medicinal uses of this plant for treating different dieses.The antioxidant and cytotoxic activity unveiled by the aqueous solvent fraction is promising and this clearly specifies the existence of potent bioactive compounds and will be a good source of herbal medicine.In context of the above discussion it would be interesting to investigate to find out the causative components from the aqueous extract which are responsible for this biological activity.

Figure 1 .
Figure 1.Schematic sequences of the isolation experiment.

Figure 3 .
Figure 3. DPPH scavenging activity of different fractions of root bark of Z. rhetsa and the pure compounds.

Figure 4 .
Figure 4. Comparison of percentage of clot lysis of different fractions and pure compounds of Z. rhetsa.

Table 1 .
Antioxidant activity of different fractions and pure compounds of Z. rhetsa.

Table 2 .
Cytotoxic activity of different fractions and pure compound of Z. rhetsa.

Table 3 .
Thrombolytic activity of different fractions and pure compounds of Z. rhetsa.

Table 4 .
Screening of antimicrobial activity of different fractions and pure compounds of Z. rhetsa.
NA = not active; -= absence of growth of microorganism.Diameter of inhibition zone (mm) including well diameter of 8 mm.