Isolation, Characterization, Complete Structural Assignment, and Anticancer Activities of the Methoxylated Flavonoids from Rhamnus disperma Roots

Different chromatographic methods including reversed-phase HPLC led to the isolation and purification of three O-methylated flavonoids; 5,4’-dihydroxy-3,6,7-tri-O-methyl flavone (penduletin) (1), 5,3’-dihydroxy-3,6,7,4’,5’-penta-O-methyl flavone (2), and 5-hydroxy-3,6,7,3’,4’,5’-hexa-O-methyl flavone (3) from Rhamnus disperma roots. Additionlly, four flavonoid glycosides; kampferol 7-O-α-L-rhamnopyranoside (4), isorhamnetin-3-O-β-D-glucopyranoside (5), quercetin 7-O-α-L-rhamnopyranoside (6), and kampferol 3, 7-di-O-α-L-rhamnopyranoside (7) along with benzyl-O-β-D-glucopyranoside (8) were successfully isolated. Complete structure characterization of these compounds was assigned based on NMR spectroscopic data, MS analyses, and comparison with the literature. The O-methyl protons and carbons of the three O-methylated flavonoids (1–3) were unambiguously assigned based on 2D NMR data. The occurrence of compounds 1, 4, 5, and 8 in Rhamnus disperma is was reported here for the first time. Compound 3 was acetylated at 5-OH position to give 5-O-acetyl-3,6,7,3’,4’,5’-hexa-O-methyl flavone (9). Compound 1 exhibited the highest cytotoxic activity against MCF 7, A2780, and HT29 cancer cell lines with IC50 values at 2.17 µM, 0.53 µM, and 2.16 µM, respectively, and was 2–9 folds more selective against tested cancer cell lines compared to the normal human fetal lung fibroblasts (MRC5). It also doubled MCF 7 apoptotic populations and caused G1 cell cycle arrest. The acetylated compound 9 exhibited cytotoxic activity against MCF 7 and HT29 cancer cell lines with IC50 values at 2.19 µM and 3.18 µM, respectively, and was 6–8 folds more cytotoxic to tested cancer cell lines compared to the MRC5 cells.


Introduction
Rhamnus is a genus of about 110 species of shrubs or small-to medium-sized deciduous or evergreen trees, which are native from temperate to tropical regions and commonly known as buckthorns in the family Rhamnaceae [1,2]. Rhamnus disperma Ehrenb belongs to the family and is a native plant to the northern Middle East and Arabian Peninsula, including Saudi Arabia [3], Syria, Lebanon, Palestine, and considered one of Rhamnus species in the flora of Egypt [1,4]. The plant is reported to contain flavonoids, flavonoid glycosides, and phenolic constituents [5,6]. The Rhamnus species is reported for several biological activities including antioxidant, anti-acetylcholinesterase [7,8], anti-inflammatory, cytotoxic [9,10], and antimicrobial activities [11,12].

Results and Discussion
The ethyl acetate fraction of the alcoholic extract of Rhamnus disperma roots was repeatedly subjected to silica gel column chromatography (Si gel CC) to afford seven fractions (A-G). Fraction A was subjected to Si gel CC followed by HPLC analysis and separation yielding compounds 1-3. Fractions F and G were subjected to SPE-C18 column followed by Si gel CC and sephadex LH-20 to give compounds 4-8 ( Figure 1). The occurrence of compounds 1, 4, 5, and 8 are reported in the current work for the first time from Rhamnus disperma. Although, compounds 2, 3, 6, and 7 were previously isolated from the same plant [5,13]. The current work revised the chemical shifts of the O-methyl

Results and Discussion
The ethyl acetate fraction of the alcoholic extract of Rhamnus disperma roots was repeatedly subjected to silica gel column chromatography (Si gel CC) to afford seven fractions (A-G). Fraction A was subjected to Si gel CC followed by HPLC analysis and separation yielding compounds 1-3. Fractions F and G were subjected to SPE-C 18 column followed by Si gel CC and sephadex LH-20 to give compounds 4-8 ( Figure 1). The occurrence of compounds 1, 4, 5, and 8 are reported in the current work for the first time from Rhamnus disperma. Although, compounds 2, 3, 6, and 7 were previously isolated from the same plant [5,13]. The current work revised the chemical shifts of the O-methyl protons and carbons of the methoxyl groups of compounds 1-3. The structures of the methoxylated flavonoids (1)(2)(3) were also confirmed through the unambiguous assignment based on 2D NMR data (spectra available in the Supplementary Materials). Furthermore, compound 3 was acetylated to give compound 9, which was identified through 1D and 2D NMR analyses.
Compound (4) was obtained as yellow amorphous powder. The 1 H NMR spectrum of compound 4, showed two meta-coupled proton signals at δ 6.41 (1H, J = 1.8 Hz) and 6.82 (1H, J = 1. ion peak at m/z 286 in EIMS, indicated that the aglycon of 4 is kampferol [14]. The anomeric proton signal at δ H 5.54 (brs, H-1"), in addition to the one doublet at δ H 1.11 (J = 6.6 Hz, H-6") observed in the 1 H NMR spectrum of compound 4 established the presence of one rhamnose unit. The anomeric configuration for the rhamnose moiety was established to be in the α-configuration from its chemical shift and 3 J H1,H2 coupling constant [15]. The downfield shifts of H-6 and H-8 of the aglycone compared to those of H-6 and H-8 of kampferol indicates that rhamnose moiety was attached to the C-7 position [16][17][18]. Therefore, compound 4 was established as kampferol 7-O-α-L-rhamnopyranoside and in good agreement with the reported literature [19].
Compound (5) was obtained as yellow amorphous powder and was identified as isorhamnetin-3-O-β-D-glucopyranoside based on comparison of its NMR ( 1 H and 13 C) and EIMS data with those of the literature [17].
Compound (6) was obtained as yellow amorphous powder and its 1 H NMR spectrum was similar to that of compound 4, except for B-ring protons which appear as an ABX system in 6  These data indicated that the aglycone of 6 is a quercetin [14] which confirmed by the observed ion peak at m/z 302 in EIMS spectrum of 6. The 1 H NMR spectrum of 6 exhibited one anomeric proton signal at δ H 5.55 (brs, H-1") and the strong sharp doublet signal at δ H 1.13 ppm (3H, d, J = 6.1 Hz, 6"-CH 3 ) confirmed the rhamnose unit. From the abovementioned data, compound 6 was established as quercetin-7-O-α-L-rhamnopyranoside and in good agreement with the reported literatures [19].
Compound (7) was obtained as yellow amorphous powder. The NMR data of the compound revealed the presence of dirhamnoside moieties attached to the kampferol aglycone. The compound was similar to compound 4 by having kampferol as an aglycone and one rhamnose unit at C-7 position in addition to another rhamnose unit at the C-3 position in compound 7. Therefore, compound 7 was identified as kampferol 3, 7-di-O-α-Lrhamnopyranoside based on the comparison of its 1 H and 13 C NMR EIMS data with those of the literature [17].
Compound (8) was obtained as colorless oil and identified as benzyl-O-β-D-glucopyranoside based on comparison of its 1 H, 13 C NMR, and EIMS data with those of literature [19][20][21].

Cytotoxicity Assay
The result of MTT cytotoxicity assay of compounds 1-3 showed variable IC 50 values against the three tested cancer cells ranging from 0.53 µM to 9.07 µM. However, other isolated compounds 4-7 did not show any inhibition of the cancerous cells proliferation at the tested concentrations. Among the three active compounds, compound 3 showed IC 50 values of 2.76 ± 0.16, 3.73 ± 1.75, and 2.71 ± 1.25 µM against the MCF 7, A2780, and HT 29 cancer cell lines, respectively, and it was 4-5 folds more cytotoxic against cancer cell lines compared to the human fetal lung fibroblasts (MRC5) normal cells. Compound 2 was less active compared to compound 3, as it exhibited IC 50 value ranged from 6.81 µM to 9.07 µM against the three cancerous cell lines, and it was not selective for MRC5 normal cells as it showed IC 50 value of 5.46 ± 1.57 µM against the normal MRC5 cells. Among the three methoxylated flavonoids, compound 1 showed the highest inhibition activity against both MCF 7 and HT 29 cells (IC 50 value of ≤2 µM), and A2780 cells (IC 50 value of 0.53 ± 0.45 µM). Importantly, compound 1 also showed 2-9 folds lower cytotoxicity against MRC5 cells with growth inhibition IC 50 value of 4.40 ± 1.45 µM (Table 1). Following the process of acetylation, compound 9 was tested against MCF7 and HT29 cell lines. It showed IC 50 values in the range of 2-3 µM. It is slightly more active than its precursor, compound 3, against the MCF 7 cell line. Compound 9 was also 6-8 folds less cytotoxic to MRC5 cells compared to tested cancer cell lines ( Table 1). The overall results showed for the higher cytotoxic activity of compounds 1-3 and 9 compared to the other flavonoids (compounds 4-7) are mostly attributed to the presence of the methoxyl moities in the former group of compounds. This claim is completely supported by the literature which proves that poly methoxylated flavonoid derivatives are more potent as cytotoxic agents and have higher ability to inhibit the tumor cells than the flavonoid derivatives with free hydroxylated groups [22][23][24]. The literature also concluded that methoxy-flavonoids showed remarkable chemo-protective properties and are potentially useful as anticancer agents [25]. Apoptosis was quantified by detecting cell surface exposure of phosphatidylserine (PS) in apoptotic cells using annexin V PI/FITC. Living cells stained with neither of the two dyes (PI−/annexin V−), while early apoptotic cells stain only with annexin V (PI−/annexin V+). In late apoptosis, cell membrane integrity is lost allowing penetration of PI (PI+/annexin V+); while in necrosis (death), cells stain with PI only (PI+/annexin V−). In this study, compound 1 induced remarkable apoptosis in a dose dependent manner ( Figure 2).
The cell cycle is a series of changes occurring from the initial phase of cell formation leading to its division as a consequence of a specific mechanism. Cell cycle phases include G 1 (gap 1), S (synthesis), G 2 (gap 2), and M (mitosis), with cell cycle arrest usually taking place in the G 1 /S or the G 2 /M check points. Compound 1 was used to treat the MCF 7 cell line (24 h; 5 µM, 10 µM, and 20 µM) to investigate possible cell cycle effect. Compared to the control, compound 1 caused dose-dependent G 1 arrest (Figure 3).

General Experimental Procedure
UV spectra were collected with a Shimadzu UV-1650PC spectrophotometer; IR spectra were measured on a Shimadzu Infrared-400 spectrophotometer (Shimadzu, Kyoto, Japan). The 1 H-and 13 C-or 13 C-APT NMR measurements were obtained with BrukerAvance III spectrometer operating at 500 or 400 MHz (for 1 H) and 125 or 100 MHz (for 13 C) in DMSO-d 6 solution, and chemical shifts were expressed in δ (ppm) with reference to TMS and coupling constant (J) in Hertz. 13

Plant Material
The roots of Rhamnus disperma Ehrenb were collected from Saint Kathrin Protectorate, South Sinai, Egypt in April 2013, and were kindly identified by Dr. Ibraheem El-Garf, Professor of Plant Taxonomy, Faculty of Science, Cairo University, Egypt. A voucher specimen {RD2013} was deposited in the Pharmacognosy Department, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt. The collected roots were sliced into small pieces and subjected to shade drying at room temperature.

Spectroscopic Analysis of the Isolated Compounds
Spectra are provided in the Supplementary Materials.   5 mg of each compound was separately refluxed with 2M HCl in MeOH (5 mL) at 80 • C for 4 h in a water bath. The reaction mixture was evaporated, and the hydrolysate after dilution with H 2 O (10 mL) was extracted with CHCl 3 (3 × 10 mL). The CHCl 3 extracts were evaporated to afford the aglycones, which were identified as kampferol for 4 and 7, isorhamnetin for 5, quercetin for 6 and benzyl alcohol for 8 by comparison with authentic samples, respectively. The aqueous layer was neutralized with sodium carbonate and concentrated to 1 mL under reduced pressure. The residue was compared with standard sugars by Si gel TLC [(CHCl 3 -MeOH-H 2 O:30:12:4), 9 mL of lower layer and 1 mL of HOAc], which indicated the sugars to be L-rhamnose in compounds 4, 6 and 7 and glucose in compounds 5 and 8.

Cytotoxicity Assay
Three cancerous cell lines, as well as one normal fibroblast cell line, were bought from the ATCC, USA: MCF 7 (human breast adenocarcinoma), A2780 (human ovary adenocarcinoma), HT 29 (human colon adenocarcinoma), and MRC5 (normal human fetal lung fibroblast). The culture procedure was performed as described in the literature [27,28]. Briefly, the cells were sub-cultured at 37 • C, 5% CO 2 , 95% air, and 100% relative humidity. RPMI-1640 media (10% FBS, L-glutamine and 1% antibiotic-antimycotic) was used for all cells except for MRC5, which was maintained in Eagles minimum essential medium (EMEM, 10% FBS and 1% antibiotic-antimycotic). Cytotoxicity of compounds 1-7 and 9 was evaluated by an MTT assay as previously described [29]. Each of the three cell lines and one normal fibroblast were cultured in 96-well plates (3 × 10 3 /well), and incubated at 37 • C overnight. Final concentrations of each compound were: 0, 0.05, 0.5, 5, 25, and 50 µM in media (DMSO 0.1%). Each concentration was tested in triplicates. The plates were incubated for 72 h and then MTT (50 µL) was added to each well. Plates were incubated for 3 h, supernatant was aspirated, and 100 µL of DMSO was added to each well. Absorbance was read on a multi-plate reader. The optical density of the purple formazan A550 is proportional to the number of viable cells. Compound concentrations causing 50% inhibition (IC 50 ) compared to control cell growth (100%) were determined. GraphPad Prism version 5.00 for Windows, GraphPad Software (San Diego, CA, USA) was used for analysis.

Annexin VFITC/PI Apoptosis Assay
The Annexin V FITC/PI assay was devised to quantify apoptosis using MCF 7 cells [30]. MCF 7 cells were cultured in 6 well plates (1 × 10 5 cells/well) in 2 mL medium overnight at 37 • C. Then it was treated by compound 1 (24 h; final concentrations: 0, 5, 10, and 20 µM). Media was collected in tubes and kept on ice, and remaining cells were trypsinized, incubated at 37 • C and added to previous tubes. Cells were centrifuged (350 g) and supernatants were discarded. Cells were washed with PBS, centrifuged and pellets re-suspended in binding buffer (100 µL) and annexin V FITC (10 µL). Tubes were incubated at room temperature in the dark for 20 min. Binding buffer (400 µL) and 10 µL propidium iodide (PI) were added. Samples were analyzed by flow cytometry (BC FC500) within 1 h. Viable cells were differentiated from early and late apoptotic/necrotic cells by annexin V (X axis) and PI staining (Y axis).

Cell Cycle Analysis
Cell cycle analysis was performed using MCF 7 cells, which were cultured in 6 well plates (1 × 10 5 cells/well in 2 mL medium), before treatment with compound 1 (0, 5, 10, and 20 µM; 24 h). Cells were washed with cold PBS and trypsinized. Collected cells were centrifuged at 350 g/5 min, and supernatant discarded. Pellets were washed in cold PBS, centrifuged, and fixed overnight in 70% ice-cold ethanol. Centrifuged cells were re-suspended in cold PBS with the addition of ribonuclease A (15 min), followed by PI (2 µL/mL). Samples were held on ice and analyzed by flow cytometry. Data analysis of the DNA contents (PI bound to DNA) of 20,000 events was carried out using Expo 32 software [31].

Statistical Analysis
Samples were tested in triplicate, and each assay was repeated three times. Data were expressed as mean and standard deviation (mean ± SD). Comparisons were performed using GraphPad Prism version 5.00 for Windows.

Conclusions
In this study, eight compounds 1-8 were isolated from the roots of Rhamnus disperma. Compounds 1, 4, 5, and 8 were isolated for the first time from the plant. Compound 1 exhibited the highest cytotoxic activity against three cancer cell lines: MCF 7, A2780, and HT 29, with IC 50 values of 0.53-2.17 µM and was 2-9 folds more selective against tested cancer cell lines compared to the normal fibroblast cells (MRC5). It also doubled MCF 7 apoptotic populations in a dose dependent manner and caused G 1 cell cycle arrest. It is concluded that compound 1 has cytotoxic, pro-apoptotic, and cell cycle arrest activities, and could have anticancer activity, which should be further tested using in vivo animal models.
Supplementary Materials: The following are available online, Figures S1-S31: NMR spectra of the compounds; Scheme S1: Extraction, fractionation and isolation of chemical constituents of Rhamnus disperma roots.