Antioxidant Flavonoids from Nicotiana plumbaginifolia Viv . Leaves

Plant derived phenolics and flavonoids exert profound antioxidant effects due to their ability to scavenge free radicals. Nicotiana plubagnifolia Viv. leaves are known to produce large quantities of phenolics and flavonoids. Here we report antioxidant activities of a methanol extract of N. plubagnifolia leaves and its isolates, 3,3’,5,6,7,8-hexamethoxy-4’,5’-methylenedioxyflavone (1), 3,3’,4’,5’,5,6,7,8-octamethoxyflavone (exoticin) (2), 6,7,4’,5’-dimethylenedioxy-3,5,3’-trimethoxyflavone (3) 3,3’,4’,5,5’,8hexamethoxy-6,7-methylenedioxy-flavone (4) and 5-hydroxy-3,3’,6,7,8-pentamethoxy-4’,5’-methylenedioxyflavone (5) against 2,2-diphenyl-1-picrylhydrazyl (DPPH) and NO radicals. This is the first report of 5 from N. plumbaginifolia. The structure of 5 was determined by using high-field 1H and 13C-NMR and by comparison with previously reported values. Amongst the tested materials, the extract and compound 5 produced considerable radical scavenging activity against DPPH and NO radicals, which were comparable to the reference standard, ascorbic acid. All extractives were then evaluated for their toxicity profiles by means of brine shrimp lethality assay. While the methanol extract was lethal against brine shrimp nauplii, the isolated compounds were relatively non-toxic. Thus, our findings suggest that the functionalized flavonoids could be suitable candidates for developing antioxidant drugs with minimal or no cytotoxicity.


Introduction
Reactive free radicals that are frequently generated in cells cause oxidative damages to biological macromolecules and form various toxins.In severe cases, supplementary antioxidants are required to maintain the free radical contents in the body as part of the innate defense mechanism [1].Aberrant regulation of free radicals has been linked to pathogenesis of many diseases including cancer and inflammation [1,2].However, the therapeutic implications of current synthetic antioxidants are somehow limited [3].Natural product derived phenolics and flavonoids can potentially scavenge free radicals via H-or electron-transfer with concomitant production of stable radicals bearing an odd electron [4,5].This newly generated radical is far more stable as the odd electron can delocalize over the aromatic system containing an extended conjugation [6].In addition, there is evidence that the antioxidant properties of phenolics and flavonoids often contribute to anticancer effects [7,8].As part of our continuous impetus in identifying potential antioxidant/ anticancer compounds from natural sources, we investigated Nicotiana plumbaginifolia Viv.(Fam.Solanaceae), a plant that is known to contain phenolics and flavonoids.

NMR Spectroscopy and Melting Point Determination
1 H and 13 C NMR of compound 5 were recorded in CDCl 3 with respect to the residual solvent at 400 and 100 MHz on AVANCE DRX 400 and ASCENDTM 400 (Avance III HD NanoBay), Bruker, Germany, respectively.Melting point of 5 was recorded on a Stuart SMP30 melting point apparatus.The plateau temperature was set at 300 °C and ramp rate was set at 0.5 °C/min.

DPPH Free Radical Scavenging Assay
The scavenging of DPPH free radicals by the crude extract or compounds (1-5) was evaluated as described [24].Briefly, 1.6 mg of standard (ascorbic acid), plant extract or isolates was dissolved in methanol and diluted to obtain the concentrations of 1.5625 -400 µg/ml.An 0.1mM DPPH solution was prepared in methanol.2 ml of DPPH solution was then added to 2 ml of tested materials.The mixture was mixed well and left for 30 min in a dark area at the room temperature.The final absorbance of the mixture was measured at 517 nm using a spectrophotometer (HACH, DR 5000 ™ , Shanghai, China) with respect to DPPH blank solution.Each experiment was carried out in triplicate.The inhibition of DPPH free radicals was measured as percent (%) using the following equation: The concentration required for scavenging 50% of DPPH free radicals (IC 50 ) was determined from the % of inhibition.

NO Scavenging Assay
The assay was conducted as described [25].Briefly, 1.6 mg of plant extract, isolates (1-5) or standard (ascorbic acid) was dissolved in methanol to obtain solutions ranging from 1.5625-400 µg/ml. 1 ml of sodium nitroprusside (5 mM) was then thoroughly mixed with 4 ml of each solution and incubated for 2 h at 30°C.The final absorbance of the mixture was measured at 550 nm followed by the addition of 1.2 ml of Griess reagent (1% sulfanilamide, 0.1% naphthylene diamine dihydrochloride in 2% H 3 PO 4 ).IC 50 values were calculated from % scavenging of NO radicals.The assay was done in triplicates.

Brine Shrimp Lethality (BSL) Assay
The assay was conducted as described [23].Briefly, 1 mg of the methanol extract, its isolates (1-5) or standard (vincristine sulfate) was dissolved in 60 µl of DMSO.For each of standard, control and experimental groups, ten brine shrimp nauplii were taken in a graduated test tube containing 5 mL simulated sea water.30 µL of the test solution was then added to the test tubes leading to final concentrations ranging from 100 -0.195 µg/ml.30 µL DMSO served as a control.Following 24h of the transfer, each test tube was observed for the number of survived brine shrimp nauplii.The concentration of each sample responsible for the 50% lethality of the brine shrimp nauplii (LC 50 ) was determined by Probit analysis.A mean LC 50 value was obtained by performing the experiment in triplicate.were determined using GraphPad Prism 6.05, USA.The multiple comparison between the groups were determined from Tukey's post-hoc test using Statistical Package for Social Sciences (SPSS) software (Version 22, IBM Corporation, USA), where p < 0.05 was considered as statistically significant.

Results and Discussion
Repetitive chromatographic purification led to the isolation of 5 from the leaves of N. plumbaginifolia.Its flavonoid nature was evident from a characteristic dark blue and fluorescent blue colored spot on TLC plate under UV 254nm and UV 366nm light, respectively and a yellow color spot upon vanillin-H 2 SO 4 treatment followed by heating at 11 °C.The 13  five methoxyl groups at δ 3.91 (3H), 3.97 (6H), 4.01 (3H), 4.13 (3H) and a chelated -OH group at 12.36 (1H).The 13 C and DEPT spectra revealed that three out of the five -OCH 3 were downfield between δ C 61.2 -62.1, suggesting their attachment to quaternary carbons.Comparative analysis of 1 H and 13 C-NMR of 5 with similar polymethoxy flavones for the substituted ring A enabled assignment of three -OCH 3 groups [17,20].The 1 H NMR spectrum further showed two aromatic signals at δ 7.54 (1H) and 7.42 (1H) for two meta-coupled protons, which were attributed to H-2' and H-6', respectively.The 1 H NMR signal at δ 6.09 integrated for two protons, revealed the presence of a methylenedioxy (O-CH 2 -O) group which was further supported by the oxygenated methylene carbon at δ C 102.0.The remaining two -OCH 3 at δ 3.91 (δ C 60.2) and δ 3.99 (δ C 56.7) were ascribed to C-3 (δ C 141.6) and C-3' (δ C 143.6) respectively.
The plant extract and the isolated compounds (1-5) were then tested for their ability to scavenge the DPPH and NO radicals.The methanol extract and compound 5 produced statistically significant (p < 0.05) DPPH and NO radical scavenging activity compared to the control.In both assays, they produced concentration dependent effects (Figure 2).In DPPH radical assay, the extract and compound 5 exhibited 84 and 83% scavenging at 400 μg/ ml with the IC 50 values of 14.3 and 90.7 µg/ml, respectively.As compared to 5, compounds 1, 2, 3 and 4 produced 61, 73, 40% and 62% scavenging effect at 400 μg/ml (Figure 2A) with substantially higher IC 50 values of 297.0 (1), 177.6 (2), 212.5 (4) μg/ml (Figure 2B), respectively.IC 50 for 3 could not be measured as it produced <50 % inhibition of DPPH radical at the maximum tested dose.The standard (ascorbic acid) produced >95% scavenging effect at 50-400 µg/ml (Figure 2A) with an IC 50 value of 8.7 μg/ml, which was comparable to that of the methanol extract (Figure 2B).As shown in figure 2C, the extract and compound 5 showed strong NO scavenging activity of 91 and 90%, respectively which was comparable to the standard, ascorbic acid (97% at 400 µg/ml).
Table 1: Cytotoxicity of the methanol extract and its isolated compound (1-5) in brine shrimp lethality assay.

Conclusion
Our results suggest the occurrences of considerable antioxidant principles in the N. plumbaginifolia leaves including the polymethoxoy flavones.In particular, compound 5 demonstrated promising antioxidant properties presumably due to its ability for transferring hydrogen(s) efficiently.The polymethoxoy flavones are unusually rare amongst plants, occurring only in a few genera including Polygonum and Murraya, suggesting a unique biosynthetic pathway.Thus, the observation that N. plumbaginifolia leaves are rich in polymethoxyflaovens warrants further investigations for their potential chemotaxonomic relationships with Polygonum and Murraya.Because these compounds have the potential to scavenge radicals, further research can be directed towards comprehensive Structure-Activity Relationship (SAR) studies aimed at developing polymethoxyflavone-based antioxidants.While we primarily focused on identifying antioxidant flavonoids, the N. plumbaginifolia leaves may also serve as a potential source of cytotoxic compounds that will likely find ways forward to anticancer drug development.