Phenolic Content of Albizia anthelmintica Leaves and Their Antioxidant and Cytotoxic Activity

Objectives: This study aimed to the isolate the polyphenolic constituents from the 80% aqueous methanolic extract of Albizia anthelmintica leaves and evaluates the antioxidant and cytotoxic activities of the pure isolates. Methods: 80% MeOH leaves extracts of the plant subjected separately to different chromatographic separation techniques. Structures of the isolated compounds were established by different spectroscopic techniques (ESI-MS, H/C NMR and 2D NMR experiments). The antioxidant activity was evaluated using DPPH scavenging activity method, the crude extracts and pure isolated compounds form active fractions of A. anthelimintica were tested for cytotoxicity against human prostate normal cell line (PNT 2A), human ovarian carcinoma cell line (A2780) and Human Caucasian breast carcinoma cell line (ZR751). Results: Nine compounds were isolated from A. anthelmintica four were identified for first time from the plant ferulic acid 2, 3`, 7 di-O-methylmyricetin3, Kaempferol 3-O-(6``-E-p-Coumaroyl)-β-D-glucopyranoside 6 and Kaempferol 3-O(6``-E-p-Caffeoyl)-β-D-glucopyranoside7and five were isolated before from the plant methyl gallate 1, gallic acid 4, quercetin 5, Quercetin 3-O-(6``-O-galloyl)-β-D-glucopyranoside 8 and Kaemferol 3-O-(6``-O-galloyl)-β-Dglucopyranoside 9. Compounds 8, 9 and 7showed the most powerful anti-oxidant activity among the compounds tested with IC50 2.75, 3.25 and 5.25 μM, respectively. Compounds 7, 3 and 6 showed activity against human ovarian carcinoma cell line (A2780) with IC50 2.6, 5.23and 6.11μM, respectively. Compound 8 showed strong cytotoxic activity against Human Caucasian breast carcinoma cell line (ZR75-1) with IC50 3.15 μM. Conclusion: A. anthelmintica 80% aqueous methanolic leaf extract contains phenolic compounds, some of which were isolated for first time from this species and some have shown strong antioxidant and cytotoxic activity against the tested cell lines. The three active isolated compounds need to be evaluated in animal models to determine their potential as natural cure products.


INTRODUCTION
The Fabaceae or Leguminosae, commonly known as the legume, pea, or bean family, is a large and economically important family of flowering plants.It includes trees, shrubs, and herbaceous plants perennials or annuals, which are easily recognized by their fruit (legume) and their compound, stipulated leaves.The group is widely distributed and is the third-largest land plant family in terms of number of species, behind only the Orchidaceae and Asteraceae, with 630 genera and over 18,860 species. 1,2The genus Albizia (Fabaceae) comprises about 150 species distributed in Africa, Asia, Central and South America.The Albizia members in Africa are used in folk medicine for the treatment of rheumatism, cough, diarrhea and injuries. 3hytochemical studies carried out on Albizia species led to the isolation of several triterpene glycosides, flavonoids, alkaloids and miscellaneous compounds.Many biological activities were studied for Albizia Authentic reference flavonoid compounds were supplied by Pharmacognosy department, Faculty of Pharmacy, Helwan University.Authentic sugars were purchased from Sigma-Aldrich Co., UK.

Cell line and culture medium
Human prostate normal cell line (PNT 2A), human ovarian carcinoma cell line (A2780) and Human Caucasian breast carcinoma cell line (ZR75-1), All derived from ECACC (Sigma Aldrich, Dorset, UK).All cell lines were cultured in DMEM media supplemented with 10% (v/v) foetal bovine serum, 2 mM glutamine and 50 μg/mL penicillin/streptomycin solution (all from Invitrogen, Paisley, UK).

Extraction and isolation of phenolics
The air dried leaves of A. anthelmintica (1 kg) were coarsely ground and extracted thrice with 5L 80% MeOH.Then, the extracts of A. anthelmintica were combined and evaporated to dryness under reduced pressure to afford 105gm extracts.The dried extract was then reconstituted with 100 mL H2O then fractionated with 3x300 ml of hexane and ethyl acetate by liquidliquid phase separation yielding three subfractions of each extract and the weight of each subfraction was as follow: (17 gm hexane fraction, 32 gm ethyl acetate fraction and aqueous layer).5 mg of each subfraction was dissolved in the appropriate NMR solvent (Hexane fraction in CD3Cl, Ethyl acetate in DMSO and Aqueous layer fraction in Deuterium oxide) to run 1 H NMR experiment, by comparing the 1 H NMR spectrum of each of the three fractions of each plant, it was found that ethyl acetate fraction showed well resolved proton spectrum and characteristic signals in the aromatic region.Therefore, the ethyl acetate fractions were selected for more fractionation work.Purification of compounds from A. anthelmintica leaves extract was performed as shown in the flow chart (Figure 1).

Acid hydrolysis for glycosides
Complete acid hydrolysis was performed by addition of 1.5 N HCl in aqueous methanol (50%) over 4-5 mg of each compound and heating for 2 hours at 100 o C. Each hydrolzate was then extracted with ethyl acetate.The ethyl acetate extract was subjected to CoPC investigation against authentic aglycones.The aqueous layer was then neutralized with sodium carbonate and subjected to CoPC against authentic sugars for identification of the sugar part.

Antioxidant assay
The 1,1-diphenyl-2-picrylhydrazyl radical (DPPH), which possesses an unpaired electron and exhibits a stable violet colour in methanol solution (peak absorbance at 517 nm), is commonly used as a reagent for evaluation of the free radical scavenging activity of antioxidants 8 .The DPPH assay is based on the reduction of DPPH in methanol solution in the presence of a hydrogen-donating antioxidant due to the formation of the non-radical form (DPPH-H) in the reaction.This reduction reaction resulting in color change of DPPH from violet to yellow (reduced form) and as much as the intensity of the yellow color increased as the anti-DPPH activity increased 9 .In this method, a microplate reader and 96 well plates were used to carry out the determination of the spectral absorption values.This assay is based on the classic method developed by Blois, 1958 9 .Various forms of this method are widely used 11,12 .Unlike the commonly used methods, which are labour, time-consuming, and reagent and sample-wasting, this microplate assay method is much more rapid, samplesaving and environmentally-friendly.In this method, methanolic DPPH solutions (100 μg/mL, 50 μL) were added to samples of different concentration (200 μL, 3.125, 6.25, 12.5, 25, 50 and 100 μg/mL for fractions and μM/mL for pure compounds).These solutions were gently mixed and incubated in the dark for 30 min at room temperature.Then the absorbances of the resulting solutions were measured at 517 nm.The scavenging capability of test compounds was calculated using the following equation: DDPH• scavenging activity (%) = 1− (λ517-S / λ517-C) X 100 Where λ517-C is absorbance of a control with no radical scavenger and λ517-S is absorbance of the remaining DPPH in the presence of scavenger.

Cytotoxicity Assay
The crude extracts, fractions, and some pure isolated compounds of A. anthelimintica were tested for cytotoxicity against human prostate normal cell line (PNT 2A), human ovarian carcinoma cell line (A2780) and Human Caucasian breast carcinoma cell line (ZR75-1), All derived from ECACC (Sigma Aldrich, Dorset, UK).The three cell lines were cultured in DMEM media supplemented with 10% (v/v) foetal bovine serum, 2 mM glutamine and 50 μg/mL penicillin/streptomycin solution (all from Invitrogen, Paisley, UK).All kept in a humidified incubator at 37 o C in the presence of 5% CO2.Cells were routinely passaged at 90-95% confluence.The cells were then seeded in transparent 96-well flat bottomed plates and allowed to adhere overnight.Column 1 kept untreated serving as control and column 12 treated with triton X 100 as a positive control.Preliminary screening has been done to evaluate which fractions or compounds have activity at concentration of 30 μM/mL for the tested pure compounds and incubated for 48 hours.Viability was assessed using Alamarblue kit according to the manufacturer's instructions.The luminescence was then measured using a Wallac Victor 2. After performing preliminary screening fractions or compounds showed viability (as % of control) less than 40% considered active (cytotoxic).Active fractions or pure compounds were then added at a concentration range of 0.1 nM/mL to 30 μM/mL (in half log units) for determination of The IC50 values and all results confirmed microscopically.All experiments were done in triplicates.

Statistical analysis
Alamarblue cytotoxicity assay data were analysed using two-factorial analysis of variance (ANOVA), including first order interaction (two-way ANOVA).

Characterization and identification of isolated compounds
Chromatographic separation of 80% aqueous methanol leaves extracts of A. anthelmintica has resulted in nine compounds.They were identified by different spectral techniques including 1 H NMR, 13 C NMR, 2 D NMR and ESI-MS also by CoPC against standard authentic sugars and aglycones after complete acid hydrolysis.
Compound 6 was obtained as pale yellow needles (28 mg).Chromatographic properties: Rf-value 0.60 (S1); dark purple spot under UV-light, turned to intense yellow color after spraying with P-anisaldhyde spray reagent and heating with air gun at 250 C.It gave greenish yellow fluorescence after spraying with Naturstoff spray as well as deep green color with FeCl3.Complete acid hydrolysis resulted in glucose in aqueous layer and kaempferol together with coumaric acid in organic layer (CoPC).Naturstoff reagents and products of acid hydrolysis it was expected to be Kaempferol coumaroyl glucoside 12 .
1 H NMR spectrum of Compound 6 exhibited an A2X2 spin coupling system of two ortho doublet, each integrated to two protons, at 8.00 (H-2`/6`) and 6.87 (H-3`/5`) indicated 4`-hydroxyl B-ring together with the two meta doublets, each integrated for one proton at 6.38 (H-8) and 6.15 (H-6) were intrinsic for kaempferol nucleus.Two ortho doublets each integrated to two protons, at 7.38 (H-2```/6```) and 6.80 (H-3```/5```) together with two doublets with large J value each integrated to one proton at 7.36 and 6.12, characteristic for two olefenic protons7```and 8```, respectively suggesting the presence of coumaryl moiety in the structure.In the aliphatic region the presence of a doublet at  13 The downfield and up field shifts of 13   ).According to the chromatographic properties Compound 7 was expected to be Kaempferol caffoeyl glucoside moities 12 . 1H NMR spectrum of Compound 7 exhibited the characteristic splitting pattern intrinsic for kaempferol nucleus like that of Compound 6, also it showed an ABX spin coupling system of three resonances each integrated for one proton, attributable to H-2```, H-6``` and H-5``` of 3`, 4`dihydroxy benzene ring, two doublets with large J value each integrated to one proton at 7.33 and 6.14 for two olefenic protons which were characteristic for caffoeyl moiety.In the aliphatic region the presence of a doublet at 5.50 integrated for one proton with large J-value of 7.21 Hz.   and products of acid hydrolysis it was expected to be quercetin galloyl glucoside 12 . 1H NMR spectrum of Compound 8 exhibited an ABX spin coupling system of three resonances each integrated for one proton, attributable to H-2', H-6' and H-5' of 3', 4'-dihydroxy Bring and AM spin coupling system of two meta doublets, each integrated for one proton at 6.37 (H-8) and 6.18 (H-6) which are characteristic for 5, 7 dihydroxy A-ring, a singlet signal at 6.89 integrated for two protons characteristic for galloyl moiety.In the aliphatic region the presence of a doublet at 5.45 integrated for one proton with large J-value of 7.29 Hz which was in good agreement with β-glucopyranoside moiety and the down field shift of the two methylene glucose protons (H-6``) at δ ppm 4.25 gave an evidence for galloylation at OH-6``. 13C NMR spectrum exhibited fifteen 13  which was in a good agreement with previously published data 13,17,18 .
Compound 9 was obtained as yellow amorphous powder (39 mg).Chromatographic properties were similar to that Compound 6 and 7: Rfvalue 0.55 (S1); Complete acid hydrolysis resulted in glucose in aqueous layer and kaempferol together with gallic acid in organic layer (CoPC).FeCl3 and Naturstoff reagents) and products of acid hydrolysis it was expected to be Kaempferol galloyl glucoside 12 . 1H NMR spectrum of Compound 9 exhibited an A2X2 spin coupling system of two ortho doublet, each integrated to two protons, at 7.93 (H-2`/6`) and 6.76 (H-3`/5`) indicated 4'-hydroxyl B-ring and AM spin coupling system of two meta doublets, each integrated for one proton at 6.38 (H-8) and 6.18 (H-6) characteristic for 5,7 dihydroxy A-ring.It showed the characteristic signals of (6``-O-galloyl)-β-Dglucopyranoside like that of Compound 8.The attachment of sugar was also deduced to be at the 3position of kaempferol nucleus from the down-fieldshifted carbon signal 157.32 (C-2), together with up-field shift of C-3 at 133.69 indicate O-glycosidation at C-3.The attachment of galloyl moiety to OH-6`` proved by the down and up-field location of C-6`` at 63.31 and C-5`` 74.64 resonances respectively in 13 C NMR spectrum.Compound 9 was identified as Kaempferol 3-O-(6``-Ogalloyl)-β-D-glucopyranoside.A final confirmation of Compound 9 was achieved by comparison with previously published data. 13,17Compound 9 was isolated once before from A. anthelmintica. 19ompound 3 was obtained as a light brown amorphous powder (29 mg).Chromatographic properties: Rf-value 0.75 (S1); dark purple spot under UV-light, turned to intense yellow color after spraying with anisaldhyde and heating with air gun at 250 C.It gave red fluorescence after spraying with Naturstoff spray reagent as well as deep green color with FeCl3.Compound 3 was expected to be myricetin on the basis of its chromatographic properties and complete acid hydrolysis. 12 1H NMR spectrum of Compound 3 showed the characteristic splitting pattern of myricetin nucleus, it exhibited AM spin coupling system of two meta doublets each integrated for one proton at δ ppm 7.04 (H-2`) and 6.67 (H-6`), characteristic for 3`substituted B-ring of myrecetin.Also showed AM spin coupling system of two meta doublets, each integrated for one proton at δ 6.26 (H-8) and 5.48 (H-6) gave an evidence for 5,7-dihydroxy A-ring..Two characteristic methoxy signals displayed at δ 56.63 and 52.51.Cosy spectrum of Compound 3 showed the characteristic correlations of myricetin nucleus between 7.04 (H-2`) and 6.67 (H-6`) and a correlation between 6.26 (H-8) and 5.48 (H-6).Attachment of the two methoxy moieties was assigned to be at C-3` due to up-field shift of C-2` at δ (105.42) and C-7 due to up-field shift of C-8 at δ (91.77).The appearance of C-2 signal at δ (147.15) is indicated that C-3 is not substituted.Accordingly Compound 3 was identified as 3`, 7-di-O-methylmyricetin.A final confirmation of Compound 3 was achieved by comparison with previously published data. 20ompound 5 showed the characteristic chromatographic properties of Quercetin aglycon and also showed the characteristic NMR signals of quercetin.In addition to the isolated flavonoid compounds, 2 phenolic acids ferulic acid 2, gallic acid 4 and it's methyl ester methyl gallate 1.They were identified by CoPC against authentic samples and comparison with previously published data. 17

Antioxidant Activity
All of the main fractions of the plant (Ethyl acetate, hexane and aqueous layer) have been tested for their antioxidant activity and compared to ascorbic acid.The tested concentrations were (3.125-100 μg/mL).(Figure 3) showed the scavenging activities of different concentrations of A. anthelmintica fractions (3.125-100 μg/mL) against ascorbic acid.From the figure presented below it was observed that the ethyl acetate fraction exhibited a good anti-oxidant activity more than that of hexane and aqueous layer, so the ethyl acetate fraction was promising for more fractionation work.

Cytotoxic Activity Evaluating cytotoxicity against normal human prostate cell line (PNT 2A)
After reading plates, the viability as (% of control) has been calculated for each sample at the preliminary screening dose.Samples which showed viability less than 40% are considered to be cytotoxic.(Figure 5) showed cytotoxicity of some of the pure isolated compounds.From the figure presented below it was observed that pure compounds tested are non-toxic to the normal cell line (PNT 2A)

Figure. 5. Cytotoxic activity of some pure compounds isolated against normal human cell line (PNT 2A). Sample concentration 30 µM/ml, n=3
Dose response curve for those active compounds following the concentration range 0.1 nM/ml or ng/ml to 30 μM/ml or μg/ml (in half log units) for determination of The IC50 values.Compound 7 was found to be the most active showing IC50 (2.6 μM/ml) then Compound 3 (5.23 μM/ml) and finally Compound 7 with IC50 (6.11 μM/ml).Figure 6 is showing the dose response curve of these compounds.

Evaluating cytotoxicity against Human Caucasian breast carcinoma cell line (ZR75-1)
All compounds were tested for activity at a preliminary screening dose of 30 µM/mL, only Compound 8 exhibited viability less than 40 %.A dose reponse curve has been done using the concentration range 0.1 nM/mL or ng/mL to 30 μM/mL or μg/mL (in half log units) for determination of The IC50 values.

CONCLUSION
In conclusion the methanolic extract of A. anthelmintica leaves contains phenolic compounds, some of which isolated for first time from this species.Some of the isolated compounds showed strong antioxidant activity and strong cytotoxic activity against both human ovarian carcinoma cell line (A2780) and human Caucasian breast carcinoma cell line (ZR75-1), thus thee active compounds needs to be evaluated in animal models to determine it's potential as natural cure products.

1 H
NMR spectrum of Compound 7 gave evidence that the compound substituted by β-O-glucosyl and caffeoyl moieties. 13C NMR spectrum exhibited thirteen 13 C resonances of the kaempferol 3-Osubstituted moiety with key carbon signals of kaempferol nucleus like that of Compound 6. Six carbon signals are characteristic for O-β-D-glucopyranoside and nine characteristic carbon signals for caffeoyl moiety 166.78 (C-9```), 149.07 (C-4```), 145.39 (C-3```), 145.13 (C-7```-β), 125.49(C-1```), 122.06 (C-6```), 116.74 (C-5```), 115.74 (C-2```), 114.22 (C-8```-α) 14,15,16 .The downfield-shifted carbon signal 156.84 (C-2), together with up-field shift of C-3 at 133.63 indicate O-glycosidation at C-3.The attachment of the caffeoyl group was determined to be adjacent to the OH-6`` position of the glucose moiety by the down field shift of the two dd of the diasteriomeric CH2-6`` protons at 4.27 in its 1 H NMR and down and up-field location of C-6`` at 63.66 and C-5`` at 74.87 resonances respectively in 13 C NMR spectrum.Positive and negative ESI-MS spectrum of Compound 7 showed a molecular ion peak at m/z 609.13 [M-H] -and 611.14 [M+H] + corresponding to the molecular weight of 610.14 which support the evidence that Compound 7 is Kaempferol 3-O-β-D-(6``-E-p-Caffeoyl)-glucopyranoside.A final confirmation of Compound 7 was achieved by comparison with previously published data.

A lb iz ia a n th e lm in tic a m a in fr a c t io n s a n d a s c o r b ic a c id C o n c e n t r a t io n µFigure. 3 . 4 .
Figure. 3. The scavenging activities of different concentrations of A. anthelmintica fractions (3.125-100 μg/mL) against ascorbic acid.After fractionation, purification and Isolation of the polyphenolic constituents of the ethyl acetate extract of the plant, the pure compounds (6, 7, 8 and 9) were tested for their scavenging activity and results are compiled in Fig. 4. From results showed in (Fig. 4.) it is clear that Compound 8 (IC50 =2.75 μM/mL) is the most active one followed by Compound 9 (IC50 = 3.25 μM/mL), Compound 7(IC50 =5.25 μM/mL) then Compound 6(IC50 =25 μM/mL).
14,15onances of C-2 (156.97) and C-3 (133.69)respectively were confirmative evidence for Oglycosidation at C-3.The attachment of the coumaroyl group was proved to be at OH-6`` of the glucose moiety from the downfield shift of the two dd of the diasteriomeric CH2-6`` protons at 4.28 and 4.16 that was shown in 1 H NMR spectrum together with the down and up-field locations of C-6`` (63.31) and C-5`` (74.64) resonances respectively in 13 C NMR spectrum.14,15Negativeand positive ESI-MS spectrum showed a molecular ion peak at m/z 593.13 [M-H] -and 595.