TWO NOVEL FLAVONE C-GLYCOSIDES ISOLATED FROM AFROCARPUS GRACILIOR: POM ANALYSES AND IN VITRO CYTOTOXIC ACTIVITY AGANIST HEPATOCELLULAR CARCINOMA

Objective: Cancer is considered as one of the top reasons of death and the number of cases increasing gradually. Cancer is severe clinical difficulty to the health caution system. This study explored two novel polyphenols of Afrocarpus gracilior Pilger growing in Egypt and evaluated their cytotoxic activity. 
Methods: Methanolic (80%) extract of the leaves of A. gracilior was subjected to column chromatography; the chemical structures of the isolated compounds were established by advanced spectral techniques: UV, 1H, 13C NMR, two dimensional NMR (2D NMR) and electron spray ionization mass spectroscopy (ESI-MS). Compounds 1 and 2 were studied for their cytotoxic activity against hepatocellular carcinoma (Hep-G2) using sulforhodamine B (SRB) assay. Furthermore the pharmacokinetics profiles of these molecules were accessed by employing Petra/Osiris/Molinspiration (POM) analyses. 
Results: Two novel C-flavonoid glycosides were isolated [1: Apigenin 8-C-β-D-glucopyranosyl-(1```→4``)-O-β-D-glucopyranoside] and [2: 7-O methyl-luteolin 8-C-β-glucopyranosyl-(1```→4``)-O-β-D-glucopyranoside]. They exhibited significant cytotoxic activity (IC50 = 9.02 and 15.61 µg/ml, respectively) against Hep-G2 cells. The POM analyses revealed that the activity of these two compounds depends on the presence of glucosyl and alkyl groups at the internal and terminal atmosphere of the compounds. 
Conclusion: These findings demonstrated that the leaves of A. gracilior contain a series of bioactive polyphenolic compounds with significant cytotoxic properties against hepatocellular carcinoma and may be used as alternative anticancer agents for doxorubicin. On the basis of POM calculations, it will be interesting to develop some alternative flavones because the deglucosylated derivatives have a better drug score than parent molecules. This preliminary study will be extended to other strains of cancer.


INTRODUCTION
Cancer affects millions of people worldwide despite of the improved molecular diagnostic techniques [1]. Accordingly cancer is a clinically serious problem which possesses significant social and economic changes to the health care system. Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third public reason of cancer linked death throughout world [2]. The synchronous existence of HCC strength may be due to numerous hazard factors such as chronic viral infection diseases with hepatitis B virus (HBV) and hepatitis C virus (HCV), aflatoxin exposure, alcohol drinking, drugs consumption or iron overload [3,4]. In maximum cases, the recovery proportion from HCC is short and existing predictable and adapted therapies are hardly beneficial [5,6]. Thus, there is an urgent need for new therapeutic agents for HCC patients. A collective means of drug finding is the ethno-medical method, in which the choice of a plant is founded on its usage as folkloric system. A large number of anti-cancer drugs have been extracted from plants containing phenolic compounds as flavonoids, tannins, steroid and terpenoids, etc. [7][8][9]. Plants containing polyphenols, flavonoids and/or tannins received considerable attention in recent years for their biological activities [10][11][12][13][14]. For example some species of Afrocarpus genus (family Podocarpaceae) reported to have several biological activities such as antiradical, anti-inflammatory, anti-viral, cytotoxic, anti-microbial properties. These biological activities were revealed for their contents of terpenoid, tannins and flavonoids such as Apigenin 8-C-β-Dglucopyranosyl-(1```→2``)-O-β-D-glucopyranoside (Vitexin 2``-O-β-D-glucopyranoside), Quercetin 3-O-β-D-glucopyranoside and II-4```,I-7-dimethoxyamentoflavone [15][16][17][18][19][20]. Afrocarpus gracilior (syn. Podocarpus gracilior) (Podocarpaceae) (Pg) is an interesting species growing in Egypt which has documented as anti-oxidant and identified to contain taxol [18,[20][21][22]. Therefore, the aim of this study was to take an overview and to continue isolating the potential components responsible for the cytotoxic activity from Afrocarpus gracilior.  HepG2 cells (ATCC source) were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium including 10% heat inactivated fetal bovine serum supplemented by 100 µg/ml penicillin with 100 µg/ml streptomycin at 37 °C under 5% CO2 in air.

Cytotoxic assay
The cytotoxic activity of the isolated pure compounds 1 and 2 ( fig. 1) was carried out according to the method described in literatures [26,27]. This colorimetric assay estimates cell number indirectly by staining total cellular protein with Sulforhodamine-B (SRB) dye. Hep-G2 were seeded in 96-well Fluostar Optima micro-plate at a concentration 5 × 104 cells/well in a fresh medium and placed to attribute to the well-plate for 24 h at 37 °C in a humidified atmosphere of 5% CO2

RESULTS AND DISCUSSION
. The screened samples (compound 1 and 2) were subjected to the wells at diverse concentrations (5, 10, 25, 50 and 100 µg/ml) using doxorubicin® as standard. The results with P<0.05 were observed to be statistically significant.

Investigation of polyphenolic contents (Extraction and isolation)
Powdered, air-dried leaves of A. gracilior (1050 g) were exhaustively extracted with hot 80% MeOH (5×3 l) under reflux. The dry residue obtained (140 g) was extracted with chloroform (3×1 l). The 2D-PC revealed that chloroform soluble portion contains limited polyphenolic contents, while they were concentrated in methanol soluble portion. The aqueous residue (120 g) was fractionated on a polyamide column (300 g, Ø 5.5×120 cm) using a step gradient H2O/MeOH mixture with decreasing polarity from 100% water to MeOH 100% for elution to yield 25 individual fractions, collected into four major collective fractions as illustrated previously by Kamal et al., 2012 [20]. The third collective fraction eluted by 20-50% MeOH/H2O was subjected to chromatographic investigation by PC (using S1, S2 solvent system) and visualization under UV-light. Application of fraction III on cellulose column starting with 20% MeOH/H2O then increase % of MeOH to 60% gave 30 sub-fractions. The collective major sub-fractions eluted at 50-90% MeOH/H2O (using UV light) were collected together. Final purification of these sub-fractions by successive fractionation on Sephedex LH-20 column using 100% MeOH and S3 solvent system (table 1) resulted in chromatographically two pure compounds (1 and 2) identified on the basis of acid hydrolysis, comparative PC, UV, ESI-MS, 1 H-, 13 C-NMR and 2D-NMR spectroscopic analyses ( fig. 2-3).   The dried residue of 80% MeOH extract, which was extracted with chloroform for defatting and aglycones extraction [28] was chromatographed on a polyamide column followed by successive separation on sephadex LH-20 and cellulose columns affording two pure compounds, among which was compound 1, that exhibited chromatographic properties, UV-spectral data of C-glycosylapigenin. The UV spectrum in MeOH exhibited the two characteristic absorption bands at λR maxR (nm) 272 nm (band II) and 335 nm (Band I) of apigenin nucleus. On addition of NaOAc, bathochromic shift of band II ( ≈+7) was diagnostic for free 7 -OH group. The remaining diagnostic shift reagents were in complete accordance with 5, 7, 4`trihydroxy-C-glycosyl flavones structure [24]. Negative ESI-MS spectrum exhibited the molecular ion peak at m/z 593 [M-H]P -P corresponding to M. wt. of 594, molecular formula CR 27RHR30RHR15 Rand fragment ion peak at m/z 431 after loss of a glycosyl moiety indicating apigenin dihexoside structure. P 1 P HNMR spectrum showed an AX coupling system of two ortho douplets, each integrated for two protons at δ ppm 8.12 and 7.06 assigned to H2`/6` and H-3`/5`, respectively of 1`, 4`-disubstituted ring-B, in addition to the two singlet signal resonances at δ ppm 6.64 and 6.46 assignable to H-3 and H-6, respectively characteristic for an apigenin moiety missing H-8 resonance signal. The two anomeric protons appeared as doublets at δ ppm 5.06 with large J value 9.9 Hz and 4.74 with J value 7.4 Hz, gave the suggestion of presence of a C-glucoside and Oglucoside moieties with a β-linkage, respectively. The absence of H-8 gave the expectation of C-glucosidation on C-8. This signal was established from downfield shift of P 13 P C-resonance of C-8 to δ ppm 104.59 (≈+10 ppm) in P 13 P CNMR spectrum. Moreover, the C-glucoside moiety was confirmed as β-glucopyranoside depending on the characteristic upfield location of C-1`` at δ ppm 72.04 and downfield locations of C-5`` and C-3`` at δ ppm 82.69 and 79.59, respectively with respect to those of O-glucoside. The presence of another six carbon resonances with the anomeric carbon at δ ppm 101.08 characteristic for β-O-glucopyranoside structure confirming the presence of a second glucose moiety. In addition to, the downfield shift of C-4`` at δ ppm 77.57 was an evidence for 1```-4`` intra glycosidic linkage. HMBC approved the linkage between the two glucosyl moiety glucopyranosyl-(1```→4``)-O-β-D-glucopyranoside ( fig. 2). All P 1 P H and P 13 P C resonances were assigned by comparison with the corresponding values of structurally related compounds of previously published data [20,[29][30][31][32][33]. In the light of these data compound 1 was identified as Apigenin 8-C-β-D-glucopyranosyl-(1```→4``)-O-β-D-glucopyranoside (Vitexin 4``-O-β-D-glucopyranoside) which is isolated for the first time from nature ( fig. 2).
According to the chromatographic properties, compound 2 was expected to be a glycosyl luteolin [34]. UV-spectrum in MeOH displayed the two distinctive absorption bands I and II of luteolin nucleus at λR maxR 350 and 258 nm, respectively. On addition of NaOAc, no bathochromic shift of band II was observed which is diagnostic for a substituted 7-OH group. The bathochromic shift of band I in AlClR 3R together with hypsochromic shift experimental after adding of HCl confirmed the occurrence of ortho-dihydroxyl groups at C-3` and C-4` in ring B, still the bathochromic shift in band II relative to MeOH continued after adding of HCl designated the occurrence of a free 5-OH group. On explanation of the given above data and the chromatographic properties, compound 2 was expected to be 5,3`,4`trihydroxy glycosyl flavone [34,35]. Negative ESI/MS spectrum exhibited the molecular ion peak at m/z 623 [M-H]P -P , corresponding to molecular weight of 624 and molecular formula CR 28RHR32ROR16R, to support evidence of methyl luteolin-di-hexoside structure. In P 1 P H NMR spectrum a flavone compound was confirmed by the appearance of a singlet at δ 6.62 for H-3. Additionally, the spectrum showed an ABX-  . 3). The presence of carbon resonance at δ 55.98 together with The upfield shift of C-7 at 162.57 ppm were indicative for the presence of methoxy group attached to C-7 which was approved by HMBC spectrum (fig. 3). This structure is confirmed by comparison with previous published reports [34][35][36][37]. Hence, compound 2 was identified as 7-O-methyl-luteolin 8-C-β-glucopyranosyl-(1```→4``)-Oβ-D-glucopyranoside or (4``-glucopyranosyl-7-O-methylorientin), which was isolated for the first time from nature ( fig. 3).

Evaluation of cytotoxic activity of 1, 2
Compounds 1 and 2 showed significant cytotoxic activities against Hep-G2 hepatocellular carcinoma (ICR 50R values 9.02±0.54 and 15.61±1.23 μg/ml, respectively) compared to doxorubicin as a standard drug (ICR 50  The effect of both compounds 1 and 2 were tested against Hep-G2 using the SRB method. SRB dye was used as a stain to estimate cell number indirectly [27]. The National Cancer Institute (NCI) indicated that the cytotoxicity of a plant extract is considered effective with the ICR 50R below 20 µg/ml [38]. Compounds 1 and 2 isolated from P. gracilior have significant cytotoxic activity against Hep-G2 with IC50 values 9.02 and 15.61 µg/ml, respectively ( fig. 4 and 5). Accordingly compounds 1 and 2 could be used as chemopreventive agents since recent studies suggested that using plant derived chemopreventive agents in combination with chemotherapy can increase the usefulness of chemotherapeutic agents and lesser their toxicity to normal tissues [39,40]. POM analyses of the standard drug, molecules 1, 2 and their deglucosylated derivatives (1', 2', 2") revealed that derivatives of 1, 2, contrary to reference drug and the two new flavones 1, 2 are more active (tables 3, 4). They showed better drug scores and can be utilized as therapeutic agents. In fact, structures of the investigated anti-cancer drugs are supposed to present some risks when runned through the mutagenicity, tumorigenicity assessments, and that these two compounds were at low risk comparable with standard drug (SD) as near as irritation and reproductive effects are concerned.
For example, acute side-effects of doxorubicin include vomiting, nausea, and heart arrhythmias. It can also causes neutropenia (reduction in white blood cells) and alopecia (hair loss). An increasing dose of doxorubicin is capable to lead the patient to severe risks of developing cardiac side effects; including congestive heart failure, dilated cardiomyopathy, and death, powerfully rise. Reactive oxygen species (ROS), formed by the contact of doxorubicin with iron, can then harm the myocytes (heart cells), creating myofibrillar loss and cytoplasmic vacuolization [41].
The hydrophilicity character of each constituent had been stated in terms its cLogP value since it had been recognized that the absorption or permeation is importantly affected by this quantity (value of cLogP). Therefore, when the value of cLogP is greater than 5, the absorption or permeation decreases. Our results showed that the new compounds (1 and 2) have similar cLogP of those of the anti-tumor standard drug SD, within the acceptable criteria. As the molecular weight of all parent molecules (1, 2 and SD) is 594-624>500 g, it is necessary to realize more chemical modification (deglucosylation) in goal to make more potentially active analogues. The actual drug-scores of compounds 1 and 2 are very encouraging (positive value of DS) as shown in table 4.

CONCLUSION
The methanolic extract of the leaves of A. gracilior contains a considerable amount of polyphenolic compounds that have significant cytotoxic properties and thus have great potential as a source for natural health products. The POM analyses revealed that the activity of compounds 1, 2 and standard drug (SD) depended on the presence of glucosyl and alkyl groups at the internal and terminal atmosphere of the compounds. The docking analysis revealed that lipophilic and H-bonding interactions were the prominent interactions among flavones and the Cancer-DNA receptor. The POM Analyses of compounds 1, 2 and SD proved to be a useful tool in the prediction of anti-tumor activity of congeneric compounds and some important insights were also originate that will be useful to monitor for the prediction of new cancer inhibitors with enhanced bio-activity.