Investigation of Livistona decipiens Leaf Methanol Extract and Evaluation of Antioxidant, Antimicrobial and Cytotoxic Activities

Objectives: This study aimed to isolate the polyphenolic constituents from the methanol extract of Livistona decipiens Becc leaves and evaluate the antioxidant, cytotoxic and antimicrobial activities of the total methanol extract and ethyl acetate fraction. Methods: The ethyl acetate and n-butanol fractions of Livistona decipiens leaf methanol extract were subjected separately to different chromatographic separation techniques. Structures of the isolated compounds were established by different spectroscopic techniques (H / C NMR). Antioxidant activity was evaluated by DPPH assay, while evaluation of cytotoxicity was done according to MTT cell viability assay. Antimicrobial activity was done by agar diffusion method. Results: seven compounds were isolated from the ethyl acetate and n-butanol fractions, five compounds were identified for the first time from this plant as apigenin-8-C-β-D-glucopyranoside (Vitexin) (1), Quercetin-6-C-β-D-glucopyranoside (2),apigenin-6,8-di-C-β-D-glucopyranoside (Vicenin II) (3), 6-O-methyl Kaempferol 3-O-glucopyranoside (4), luteolin-3-C-gentiobiosyl (5), while two compounds vis; luteolin-6-C-β-Dglucopyranoside (Isoorientin) (6) and luteolin-8-C-β-D-glucopyranoside (Orientin) (7), were isolated for the second time from this species. The ethyl acetate fraction has shown moderate activity against Gram positive and Gram negatvie bacteria, also it showed moderate antioxidant activity with IC50 = 23 ± 0.74 μg/ml when compared to ascorbic acid IC50 = 14.2 ± 0.35 μg/ml. Also ethyl acetate extract has shown cytotoxic activity on MCF-7 cells (human breast cancer cell line), HepG-2 (human hepatocellular carcinoma) and HeLa cells (human cervical cancer cell line), whereas, the methanol extract has shown lower activity. Conclusion: Livistona decipiens have potential medicinal value being rich in polyphenolics and being antioxidant, cytotoxic and antimicrobial drug.


INTRODUCTION
Arecaceae, also called Palmae, is among the best known and extensively cultivated plant families 1 .The family has been neglected chemically, probably because of the difficulty of collecting fresh material and getting it authenticated.Most work has been carried out on economically important palms such as Phoenix dactylifera, Cocos nucifera and other palms cultivated for their oils 2 .Among its genera is Livistona R. Br. known as Fountain palm, which is a genus of 34 or possibly more species of hermaphrodite, shrubby or arborescent palms, comprising the most common fanpalms of greenhouses and decorative use, native to East Asia and Australia.Livistona are widely cultivated as ornamentals in the tropics and subtropics regions 3 .L. decipiens Becc leaves are up to 5 ft.long, very deeply divided into about 80 segments.Petioles often toothed only near the base; inflorescence, with glabrous bracts or with hairs only on the margins of smaller bracts, bearing flowers, sepals with solid base as long as erect narrow lobes; fruit Blackish, 1/2 inch in diameter 4 .Reviewing the current literature, it was found that little work was reported on genus Livistona which showed that plants belonging to this genus are rich in flavonoids, phenolic acids, ceramides, glycerides, lipids and acylglucosylsterols 1,[5][6][7][8] .The few phytochemical reports on L. decipiens Becc showed that the lipoidal

Reagents for biological studies
Dimethyl sulfoxide (DMSO), MTT and trypan blue dye was purchased from (Sigma-Aldrich) (St.Louis, Mo., USA).Fetal Bovine serum, DMEM, RPMI-1640, PBS buffer solution, L-glutamine and 0.25% Trypsin-EDTA were purchased from Lonza (Belgium) and 50µg/ml gentamycin was purchased from (Sigma-Aldrich) with 5% CO2 were used for evaluation of cytotoxic activities.Freshly prepared (0.004%w/v) methanol solution of 2,2-diphenyl-1picrylhydrazyl (DPPH) radical was prepared and stored at 10˚C in the dark were used for evaluation of antioxidant activities.

Standard material Authentic flavonoids and sugars
Authentic flavonoids; including Luteolin, Apigenin, Quercetin, Kaempferol, as well as standard sugars; including glucose, rhamnose, xylose, galactose, gentiobiose, were all available in the Department of Pharmacognosy, Faculty of Pharmacy, Helwan University, Cairo, Egypt.

Reference drugs for biological study
Ascorbic acid, Ketoconazole, Gentamycin and Vinblastine Sulfate were supplied by RCMB: Regional Center for Mycology and Biotechnology, Cairo, Egypt.

Apparatus and equipment for chromatographic techniques
Rotary evaporator (Buchi, A.G. Switzerland), glass column for chromatography (Ø 120 x 3.5 cm), Buchner filter funnel, analytical balance (Melter H20, USA), micropipettes for spotting, rectangular glass tank 50 cm x 56 cm x 20 cm was used for PC and 24 cm x 24 cm x 10 cm for TLC, Ultraviolet lamp for localization of spots on paper and thin layer chromatography, (NMR) Nuclear magnetic resonance spectrometer, Bruker a 400, MHz for 1 H NMR and 100.40 MHz for 13 C NMR.The spectra were run in DMSO, and chemical shifts were given in ppm with tetramethylsilane (TMS) as an internal standard.

Apparatus for biological studies
A microplate reader (SunRise, TECAN, Inc, USA), the 96 -well plate used for cytotoxicity evaluation using cell viability assay, a UV-visible spectrophotometer (Milton Roy, Spectronic 1201), used for measuring the absorbance in the antioxidant assay.

Extraction and isolation of phenolic compounds
The air dried leaves of L. decipiens (2 kg) were coarsely ground and extracted three times with 10 L 100% methanol.Then, the leaf extracts were combined and evaporated to dryness under reduced pressure to yield 306 g concentrate.The dried leaf concentrate was reconstituted with 300 ml H2O and then was fractionated with 3 × 300 ml of petroleum ether, ethyl acetate and n-butanol respectively, by liquid-liquid phase separation yielding four fractions weighing (160 g petroleum ether fraction, 10 g ethyl acetate fraction, The ethyl acetate fraction (10 g) was fractionated on polyamide column (Ø 4 × 70 cm), the column was eluted using water then H2O / MeOH mixtures with decreasing polarity to yield 30 individual fractions (150 ml) each, collected into 5 main fractions based on their similarity on PC (I-V), the promising fraction (based on spots detected under UV) was FIII (F24-58), (0.7 g), which was re chromatographed on another polyamide column using 20% MeOH / H2O, giving four subfractions were afford (FIII a -FIII e).FIII c (0.1 g) showed two dark purple spots with minor yellow spot ( under UV on PC (S1) ) then applied on Sephadex LH-20 column for separation using mobile phase n-butanol saturated with water affording compound 1 (11 mg) and compound 2 (20 mg).
Then-butanol fraction (20 g) was fractionated on polyamides column (Ø 5 × 90 cm), the column was eluted using water then H2O / MeOH mixtures with decreasing polarity from 100% H2O to 0% H2O to yield 50 individual fractions, collected into 6 main fractions (FI -FVI) based on their similarity on PC, the most promising fractions were FI and FII.

Hydrolysis of glycosides
Oxidative hydrolysis using ferric chloride (0.2 g in 0.8 ml H2O) refluxed for 6 hours was performed for C-glycosides, while complete acid hydrolysis using 1.5 N HCl in aqueous methanol (50%), refluxed for 2 hours at 100 o C was performed for O-glycosides.Each hydroylsate was then extracted with ethyl acetate.The ethyl acetate extract was subjected to CoPC (comparative paper chromatography) investigation against authentic aglycone.The aqueous layer was then neutralized with sodium carbonate and subjected to CoPC against authentic sugars for identification of the sugar part 9 .

Antioxidant activity
The antioxidant activity of extracts was determined at the Regional Center for Mycology and Biotechnology (RCMB) at Al-Azhar University by the DPPH free radical scavenging assay in triplicate and mean values were considered 10 .

DPPH radical scavenging activity:
Freshly prepared (0.004 %w/v) methanol solution of 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical was prepared and stored at 10˚C in the dark.A methanol solution of the tested extracts were prepared with sample concentrations (0, 10, 20, 40, 80, 160, 320, 640, 1280 µg/mL).A 40 uL aliquot of the methanol solution was added to 3 ml of DPPH solution.Absorbance measurements were recorded immediately with a UV-visible spectrophotometer.The decrease in absorbance at 515 nm was determined continuously, with data being recorded at 1 min intervals until the absorbance stabilized (16 min).The absorbance of the DPPH radical without antioxidant (control) and the reference compound ascorbic acid were also measured.All the determinations were performed in three replicates and averaged.The percentage of DPPH radical scavenging was calculated according to the formula: (scavenging activity) = [{(AC-AT)/ AC} × 100] (1).Where AC = Absorbance of the control at t = 0 min and AT = absorbance of the sample + DPPH at t = 16 min 10 .

Antimicrobial activity
The total methanol extract and ethyl acetate fraction of L. decipiens leaves were assayed for antimicrobial activity using the susceptibility tests.Screening tests regarding the inhibition zone were carried out by the well diffusion method 11 .The inoculums suspension was prepared from colonies grown overnight on an agar plate and inoculated into Mueller-Hinton broth (fungi using malt broth).A sterile swab was immersed in the suspension and used to inoculate Mueller-Hinton agar plates (fungi using malt agar plates).The extracts were dissolved in dimethyl sulfoxide (DMSO) with different concentrations (10, 5, 2.5 mg/ml).The inhibition zone was measured around each well after 24 h at 37°C.Controls using DMSO were adequately done.

Evaluation of cytotoxicity
The total methanol extract and ethyl acetate fraction of L. decipiens leaves were evaluated against (HepG2), (MCF-7) and HeLa cell lines using the MTT cell viability assay.

Cell line propagation
The cells were grown on RPMI-1640 medium supplemented with 10% inactivated fetal calf serum and 50 µg/ml gentamycin + ampicillin.The cells were maintained at 37ºC in a humidified atmosphere with 5% CO2 and were subcultured two to three times a week.

Cytotoxicity evaluation using viability assay
For cytotoxicity assays, the cancer cell lines were suspended in medium at concentration 5 × 10 4 cell/well in Corning® 96 -well tissue culture plates, and then incubated for 24 hr.The tested methanol and ethyl acetate extracts were then added into 96 -well plates (three replicates) to achieve twelve concentrations for each extract.Six vehicle controls with media or 0.5 % DMSO were run for each 96 -well plate as a control.After incubating for 24 h, the numbers of viable cells were determined by the MTT test.Briefly, the media was removed from the 96 -well plates and replaced with 100 µl of fresh culture RPMI 1640 medium without phenol red then 10 µl of the 12 mM MTT stock solution (5 mg of MTT in 1 mL of PBS) to each well including the untreated controls.The 96 -well plates were then incubated at 37°C and 5% CO2 for 4 hours.
An 85 µl aliquot of the media was removed from the wells, and 50 µl of DMSO was added to each well and mixed thoroughly with the pipette and incubated at 37°C for 10 min.Then, the optical density was measured at 590 nm with the micro plate reader (SunRise, TECAN, Inc, USA) to determine the number of viable cells and the percentage of viability was calculated as [(ODt/ODc)] × 100% where ODt is the mean optical density of wells treated with the tested sample and ODc is the mean optical density of untreated cells.The relation between surviving cells and drug plotted to get the survival curve of each tumor cell line.The 50% inhibitory concentration (IC50), required to cause toxic effects in 50% of intact cells, was estimated from graphic plots of the dose response curve for each concentration.using Graph pad Prism software (San Diego, CA.USA) 12,13 .

Statistical Analysis
All experimental results were expressed as means ± SD. Analysis of variance was performed by ANOVA procedures.Correlation coefficient (R 2 ) was used to determine two variables.SPSS software was used for statistical calculations.The results with P<0.05 were regarded to be statistically significant.

Characterization and identification of isolated compounds
Chromatographic separation of ethyl acetate and n-butanol fractions of L. decipiens leaves resulted in seven compounds.Structures of the isolated compounds (Figure 1) were identified by different spectral techniques including 1 H NMR, 13 C NMR, and also by CoPC against standard authentic sugars and aglycones after complete acid hydrolysis.
Compounds 6 and 7: Obtained as yellow amorphous powder each, compound 6 (17 mg) and compound 7 (26 mg) chromatographic properties, Rfvalues (0.43 S1), (0.39 S2) and (0.29 S1), (0.21 S2) respectively, they gave dark purple spot under UVlight, turned to yellow on exposure to NH3 vapors, orange fluorescence on exposure to Naturstoff and green color with FeCl3 spray reagents, Compound 6 and 7 were expected to be a luteolin structure 9 . 1 H NMR spectra (Table 1) showed an ABX-spin coupling system of three proton resonances in each compound at As further confirmation, 13 C NMR spectrum for each compound (Table 1) showed well-resolved typical 15 signals of a luteolin aglycone moiety, including the three key signals of C-3′, C-4′ and C-3 at δ ppm 146.14, 150.07 and 103.11 for compound 6 and at δ ppm 146.51, 150.04 and 102.75 for compound 7. Additionally, the C-glycoside moiety in both structures was confirmed as β-glucopyranoside depending on the characteristic up-field location of C-1′′ at 73.50 and 73.48 ppm for compound 6 and compound 7, respectively, and downfield location of both C-5′′ and C-3′′ to δ 81.92 and 79.18 ppm for compound 6 and to δ 82.37 and 79.03 ppm for compound 7, with respect to those of O-glycosides 19 .The C-glycosidation at C-6 in compound 6 and at C-8 in compound 7 was concluded from the downfield shift of 13 C-signals of C-6 to 109.19 and of C-8 to δ 104.91(~ + 10 ppm) for compounds 6 and 7, respectively.The assignment of all other 13 C NMR resonances of compounds 6 and 7 was achieved by comparison with the corresponding data of structural related compounds 14,19,26,27,29 .Thus according to the above discussed data, compound 6 was confirmed as luteolin-6-C-β-D-glucopyranoside (Isoorientin), while compound 7 was confirmed as luteolin-8-C-β-Dglucopyranoside (Orientin).However this is the second report of their isolation from Livistona decipiens 30 .

Antioxidant activity
The antioxidant activity of both methanol extract and ethyl acetate fraction may be attributed to phenolic contents of this plant; mainly phenolic acids, flavonoids which has been shown in phytochemical screening.The ethyl acetate fraction has moderate activity with SC50 = 23  0.74 µg/ml when compared to ascorbic acid SC50 = 14.2 0.35 µg/ml, while the methanol extract has lower activity with SC50 =55.2  1.9 µg/ml (Table 2 and Figure 2)

Antimicrobial activity
Table 3 showed that the ethyl acetate fraction possess moderate activity against Gram positive and Gram negatvie bacteria,while the methanol extract has shown no action on Gram negatvie bacteria, however both of them possess no anti-fungal activity.

CONCLUSION
Seven compounds were isolated from the ethyl acetate and n-butanol fractions of L. decipiens leaves, for the first time from this species, vies;apigenin

LFigure 2 .
Figure 2. Antioxidant activity (scavenging activity) of methanol extract and ethyl acetate fraction ofLivistona decipiens leaves compared to ascorbic acid

ISSN: 2357-0547 (Print) Research Article / JAPR ISSN: 2357-0539 (Online) Ibrahim et al., 2018, 2 (4), 256-268 http
PCof the fractions revealed the presence of a pronounced number of flavonoid spots in the ethyl acetate and n-butanol fractions, which were detected under UV-light and with specific spray reagents.Thus both ethyl acetate and n-butanol fractions were selected for further phytochemical investigation.

Table 3 . Antimicrobial activity of methanol extract and ethyl acetate fraction of Livistona decipiens leaves Sample Tested microorganisms Total methanolextract of L. decipiens Ethyl acetate fraction of L. decipiens
*NA: No activity.The sample was tested at concentration.10 mg/ml.