Flavonolignans and Biological Activity of Nannorrhops ritchiana Leaves

Objectives: This study aimed at isolating the polyphenolic compounds of 70% methanol extract of Nannorrhops ritchiana Griff. leaves and assaying the antioxidant and cytotoxic activities of the extract and main fractions. Methods: The methanol extract of the leaves of N. ritchiana was chromatographically fractionated using a bioactivity-guided approach. The isolated compounds were spectroscopically elucidated by UV, MS, H/C NMR and 2D NMR spectroscopic techniques. The radical scavenging activity of the methanol extract and its fractions was evaluated using DPPH (2,2-Diphenyl-1picrylhydrazyl) radical scavenging assay and their cytotoxic activity was assayed using SRB (3-(4,5-dimethyl thiazol-2yl)-2,5-diphenyltetrazolium bromide) test against human hepatocellular liver carcinoma (HepG2), human alveolar adenocarcinoma (A549) and human prostate carcinoma (PC3) cell lines. Results: Eight compounds were isolated and identified for the first time from the leaves of N. ritchiana, comprising five flavonolignans; Indocalatin A (4) which is a rare compound that has been reported only once in nature, 9′′-O-glucopyranosyl Salcolin A (5), 9′′-O-glucopyranosyl Salcolin B (6) together with their aglycones, Salcolin A (7) and Salcolin B (8) along with three flavone glycosides; Tricin7-O-rutinoside (1), Orientin (2) and Isoorientin (3). The methanol extract of N. ritchiana showed antioxidant activity with SC50 39.4 ± 1.06 μg/ml, while fraction II showed significant effect with SC50 of 6.1 ± 0.24 μg/ml in comparison with ascorbic acid (SC50 1.8 ± 0.35 μg/ml). The methanol extract and the fraction III showed a promising cytotoxic activity against selected cell lines especially the A549 with IC50 9.5 ± 1.98 μg/ml and HepG2 with IC50 8.15 ± 1.3 μg/ml, respectively. Conclusion: N. ritchiana leaf methanol extract is a new source of biologically active compounds, including flavonolignans.


INTRODUCTION
The Arecaceae (Palmae) is a botanical family of perennial climbers, shrubs, a caules and trees commonly known as palm trees.The family with great importance in landscaping and gardening contains 181 genera with around 2600 species which are found throughout equatorial, tropical, and subtropical areas of the world.The abundant presence of C-glycosylflavones, leucoanthocyanins, sulphated flavonoids and tricin derivatives provides interesting chemosystematic marker of the family 1 .The genus Nannorrhops is one of palm trees belonging to this family where Nannorrhops ritchiana Griff. is the sole species of this genus.The species commonly known as Mazari palm is a shrub-like clumping palm, with blue-green to grey-green fan-like leaves and several stems slowly growing and connected to form a single base.It is native to Southwestern Asia, from Southeast of the Arabian Peninsula to east through Iran and Afghanistan to Pakistan 2 .The young leaves of the plant with sweet astringent taste have been used as purgative in livestock 3 .The fruit is edible and used by local communities for the treatment of alimentary tract complaints 4 and other infectious disorders, in Baluchistan, Pakistan 5 .The petroleum ether, butanol, ethyl acetate and methanol extracts of both roots and leaves of N. ritchiana showed good antifungal and weak antibacterial activities against several strains [6][7][8] .

HPLC analysis of flavonoid and phenolic compounds
Flavonoid and phenolic compounds of the samples were detected and determined according to the method described by Goupy et al 9 and Mattila et al 10 using HPLC instrument composed of column C18 hypersil BDS with particle size 5 µm., the solvent system used was a gradient of A (CH3COOH 2.5%), B (CH3COOH 8%) and C (acetonitrile).The best separation was obtained with the following gradient: at 0 min, 5% B; at 20 min, 10% B; at 50 min, 30% B; at 55 min, 50% B; at 60 min, 100% B; at 100 min, 50% B and 50%C; at 110 min, 100% C until 120 min., flow with 1 mL/min where the retention times were compared with those of standards injected in the same run.Quantification was carried out using external standard calibration and expressed in mg/g dry matter of equivalent flavonoid and phenolic compounds.

Extraction and isolation of phenolics from N. ritchiana leaves
The air dried ground leaves (1kg) were exhaustively extracted with 70% methanol by soaking at room temperature for 24 h, thereafter the solvent was evaporated under reduced pressure affording 250 g of methanol concentrate.Sugar content was removed from the methanol extract by dissolving the residual concentrate in least amount of distilled water followed by addition of excess absolute ethanol.Fifty grams of dry concentrate were loaded on 750 g polyamide 6 column (5 cm W. x 120 cm L.) which was then eluted using water then H2O/MeOH mixtures with 20% stepwise decreasing polarity, that yielded 34 fractions of 500 ml each, and similar PC fractions were combined together affording into five major collective fractions; I (7 g) from 100% H2O (1-5), II (3.6 g) from 20% MeOH/H2O (6-12), III (1.2 g) from 40% MeOH/H2O (13-20), IV (1.8 g) from 60-80% MeOH/H2O (21-29) and V (4 g) from pure MeOH (30-34).The interesting biological (antioxidant and cytotoxic) activities of fractions II, III and IV were encouraging for further processing of these fractions mainly by successive column chromatography on Sephadex and preparative paper chromatography using different solvent systems S1 (n-Butanol: Acetic acid: Water 4: 1: 5) and S2 (acetic acid: water 15:85).Fraction II was further applied to subcolumn Sephadex (LH-20) using H2O, then MeOH/H2O mixtures with decreasing polarity to give two major subfractions IIa (eluted with 20% MeOH) and IIb (eluted with 40-60% MeOH).Subfraction IIa was chromatographed by preparative paper chromatography (PPC) using S2 solvent system and finally was purified on column Sephadex using MeOH to give compound 1.Fractionation of subfraction IIb on column Sephadex using 20% MeOH/H2O as an eluting solvent with stepwise decrease in polarity followed by further purification on column Sephadex using MeOH afforded two pure compounds 2 and 3.
Fraction III was subjected to Sephadex column chromatography using 30% MeOH/H2O with increasing amount of MeOH to give major subfraction which was applied to PPC using S2 solvent system to give two bands where the first band was further purified on Sephadex column using methanol resulting pure compound 4. The second band in the same manner afforded isomeric mixture of compounds 5 and 6.Fraction IV was applied to column Sephadex using 40% MeOH/H2O with increasing the ratio of MeOH to give major subfraction which was applied to PPC using S2 and then purified on column Sephadex using MeOH to give epimers of compounds 7 and 8.

Antioxidant assay
DPPH free radical scavenging activity was evaluated by measuring the scavenging activity of the extract on stable 2.2-diphenyl-1-picryl hydrazylradical (DPPH) 11 .A solution of DPPH (0.25 mM) in 70% methanol was prepared.Stock solutions of extract (1.0 mg/ml) and fractions II, III and IV (0.5 mg/ml) in 70% methanol were prepared.Different concentrations of extract (10-100 μg/ml) and fractions (5-25 μg/ml) solutions were added to 0.335 ml (0.25 mM DPPH) and final volume was made to 1 ml with 70% methanol.The mixture was shaken vigorously and kept standing at room temperature for 10 min.Thereafter the absorbance of the mixture was measured at 517 nm on UVspectrophotometer.The decrease in the absorbance indicates an increase in DPPH-radical scavenging activity.The percentage inhibition was calculated by the following equation: DPPH radical scavenging (%) = [(A blank -A sample)/ A blank] ×100 where A blank is the absorbance of control and A sample is absorbance of sample.The experiment was performed in triplicate and Vitamin C (ascorbic acid; 0.5-2.5 μg/ml) was used as a standard drug.The mean values were calculated and SC50 value was calculated as the concentration of sample required to scavenge 50% of DPPH free radicals.

Cytotoxicity Assay
The cytotoxicity against Hep-G2, A549 and PC3 cells were tested according to the SRB (Sulforhodamine B) assay method 12 where doxorubicin was used as the reference drug.Briefly, cells grown in T-75 flasks of stock solution were used when 70% confluence was reached in T-75 flasks.The attached cell line was collected with 0.025% trypsin then plated in 96multiwell plates at densities of 10 4 cells/well in a fresh media and incubated under normal growth condition for approximately 24 h before treatment with the tested sample to allow adherence of cells to the wall of the plate.The N. ritchiana leaves methanol extract and fractions II, III and IV were diluted serially with DMSO-d6 (100%).Then, 200 μl of each aliquot were added in several concentrations (0, 1, 2.5, 5 and 10 μg/ml) and the plates were incubated for 48 h at 37°C in a humidified incubator containing 5% CO2 in air.Control cells were treated with vehicle alone.Each individual concentration was added to three wells.Following 48 h treatment, cells were fixed, washed and stained with Sulforhodamine B stain.Wells were repeatedly washed with 1% (v/v) acetic acid to remove excess dye and treated with Tris EDTA buffer to recover attached stain.The optical density (O.D.) of each well was measured in an ELISA reader spectrophotometer.The amount of dye extracted from the stained cells is directly proportional to the protein content of cells and the survival cell mass.Negative control was treated with the vehicle (0.1% DMSO-d6) used for diluting the tested samples.Doxorubicin (1.0 μg/ml) was used as the positive control.

Statistical analysis
All the aforementioned experiments were conducted in triplicates.Data were expressed as mean ± standard deviation (SD) and at P<0.05.Data were analyzed by using one-way ANOVA followed by Duncan's multiple range tests using SPSS version 12.0 (SPSS Inc., Chicago, IL, USA).

HPLC analysis of flavonoid and phenolic compounds
The experiment revealed the identification of twenty two phenolic compounds in which case, the most abundant one was ellagic acid (7670.04ppm), while twenty one flavonoid glycosides and aglycones were identified, within which Kaempferol 3-O-(2-ρcoumaroyl) glucoside (592.75 ppm) (Table 1) was the major compound.

Characterization and identification of isolated compounds
Promising antioxidant and cytotoxic activities of the aqueous methanol extract of N. ritchiana leaves was the basis for further bioassay-guided fractionations using different chromatographic techniques that resulted in identification of eight compounds which elucidated using various spectroscopic methods including UV, MS, 1 H NMR, 13 C NMR along with 2D NMR and confirmed by comparison of the data with those reported in the literature.

Antioxidant activity
The DPPH radical contains an odd electron, which is responsible for the absorbance at 515-517 nm and also for a visible deep purple color.When DPPH accepts an electron donated by an antioxidant compound, the DPPH is decolorized and this can be quantitatively measured from the changes in absorbance.The methanol extract and its fractions tested for scavenging activity relative to ascorbic acid showed promising activity (Tables 3-5), especially for isolated fractions.The methanol extract showed good antioxidant activity with SC50 39.4 ± 1.06 μg/ml and more free radical scavenging activity appeared interestingly with fractions III and IV that has scavenged 50% of DPPH radicals with values of 9.73 ± 0.67 and 9.77 ± 1.12 μg/ml, respectively.Fraction II showed the most significant antioxidant activity with SC50 6.1 ± 0.24 μg/ml in comparison with ascorbic acid that exhibited SC50 1.8 ± 0.35 μg/ml.This significant activity of fraction II can be attributed to high concentration of flavonoids especially tricin 7-Orutinoside which was the major isolated compound in this fraction.Similarly alike, it is worth mentioning that the flavonolignan present in fractions III and IV can explain the noticeable activity of both fractions.

Cytotoxic activity
Surviving fraction calculated from optical density values was plotted against drug concentration to get the survival curve for each tumor cell line after treatment with tested samples (Figures 2-6), from which IC50 could be calculated and expressed as IC50 ± SD where samples showed mortality more than 50% are considered to be cytotoxic.The methanol extract and fraction III showed promising cytotoxic activity against selected cell lines especially the A549 and HepG2, respectively.The methanol extract exhibited significant cytotoxic activity against A549 with IC50 values of 9.5 ± 1.98 μg/ml and good activity against PC3 and HepG2 with IC50 values of 15 ± 1.77 and 15.6 ± 1.3 μg/ml, respectively.Also, fraction II showed good activity against PC3 with IC50 value of 16.7 ± 1.18 μg/ml despite showing moderate activity against HepG2 and A549 cell lines with IC50 values of 24.3 ± 1.42 and 37 ± 1.59 μg/ml, respectively.Fraction III significantly reduced the growth of HepG2 cell line in a concentration dependent manner with IC50 value of 8.15 ± 1.3 μg/ml, while, it showed moderate cytotoxic activity against A549 and PC3 with IC50 values of 14.7 ± 2.6 and 15.6 ± 0.56 μg/ml, respectively.However, fraction IV showed mild cytotoxic activity against HepG2 and PC3 cancer cells with IC50 values of 42.8 ± 0.63 and 45.7 ± 4.1 μg/ml, respectively while its effect on A549 cell line was lesser compared with doxorubicin (Table 6).

CONCLUSION
According to the obtained results, N. ritchiana leaf methanol extract fractions II, III and IV are rich in phenolic compounds that show significant antioxidant activity and promising cytotoxic activity especially the methanol extract against human alveolar adenocarcinoma (A549) and fraction III against human hepatocellular liver carcinoma (HepG2) cell line.Thus Nannorrhops ritchiana Jriff.leaves could be considered as a new natural source of flavonolignan with valuable biological activity.