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Current approaches and challenges for chemical characterization of inhibitory effect against cancer cell line isolated from Gokshur extract Salwa Bouabdallah a,∗ , Rabiaa-M. Sghaier b , Sawssen Selmi c , Daycem Khlifi d , Dhafer Laouini b , Mossadok Ben-Attia a a

Environmental Biomonitoring Laboratory LBE (LR01/ES14), Faculty of Sciences of Bizerta, University of Carthage, Tunisia Laboratory of Transmission, Control and Immunobiology of Infection (11 LR IPT 02), Pasteur Institute, Tunisia c Laboratory of Bioactive Substances, Biotechnology Centre in Borj-Cedria Technopol, BP.901, Hammam-lif, 2050, Tunisia d Laboratory of Molecular Interactions and Chemical Reactivity and Photochemical UMR CNRS 5623, University of Toulouse, University of Paul Sabatier, 118 Route de Narbonne, F-31062 Toulouse, France b

a r t i c l e

i n f o

Article history: Received 30 June 2015 Received in revised form 2 November 2015 Accepted 16 November 2015 Available online xxx Keywords: Anti-cancer ATCM GC–MS Cyclotrisiloxane Gokshur MTTassay RP-HPLC

a b s t r a c t In the present study, the potential effect anti tumor and the chemical composition of different fractions of Gokshur was evaluated. Commonly known as puncture vine, it has been used for a long time in both the Indian and traditional Chinese medicine. It is popularly used as a remedy for fertility disorder in Ayurveda. Samples were collected during June–September 2014 in the Cap Bon (north east of the northern Tunisia). Different organs were separated and extracted by sequential process to compare and investigate their potential anti-tumor effect. For the first time, we report the antiproliferatif effect of leaves n-butannolic fraction against cancer cell lines. The better anti-tumor fraction (94.76 ± 1.52%) has been detected and performed by RP-HPLC has shown a great peak area (5578.21 Mau). Novel designed natural derivatives from Gokshur, a cyclotrisiloxane, major compound identified by GC–MS. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Ovarian cancer is the fifth most important cause of cancerrelated mortality in females and is a lethal disease among all gynecologic malignancies. The main difficulty in arduous ovarian cancer is that the majority of patients are examined at an advanced stage. According to prior studies, as many as 70% of patients have already accessed stages III or IV of the ailment at the time of diagnosis [1]. Due to the latent beginning of ovarian cancer, no effective medical screening process has yet been showed for the mended identification and detection of the disease. In addition, the current approach and clinical therapy for ovarian cancer is optimal primary cytoreductive surgical operation, followed by systemic chemotherapy coupled to paclitaxel and platinum [2]. However, due to the side effects of this medicinal alternative and the high tolerance of ovarian cancer to chemotherapy, the efficiency of chemotherapy is limited. Ayurveda and Traditional Chinese medicine (ATCM) have

∗ Corresponding author at: Environmental Biomonitoring Laboratory (LR01/ES14), Faculty of Sciences of Bizerta, University of Carthage, Jarzouna 7021, Tunisia. Fax: +216 72590566. E-mail address: [email protected] (S. Bouabdallah).

been used in China and India for 1000 years as an antitumor treatment for a number of different malignancies, including lung, liver, and hematopoietic cancers [3–5]. Recently, ATCM are considered to induce few side-effects and little tumor cell resistance and have been known as a key source of novel drugs for targeted therapies. Clinical practices application have also shown that a number of Traditional Chinese medicine (TCMs) exhibit antitumor activity, which supplies a modern therapeutic strategy for cancer treatment. Tribulus terrestris L. (TT) commonly known as Gokshur or Gokharu or puncture vine [6] has been used for a long time in both the Indian and Chinese systems of medicine [7], to treat various kinds of diseases. TT natural products perform various functions and many of them are interesting and useful biological activities [8–14]. TT growing in Bulgaria is a source for the industrial production of the original preparation “TribestanTM” produced by Sopharma AD, Bulgaria. TribestanTM consists of the n-butannolic (n-BuOH) extract of the aerial parts of the same plant and is successfully applied for treatment of sexual deficiency [15]. The active components of TribestanTM are steroid saponins of furostanol type 2–4. The dominating furostanol bis glycosides have been identified as protodioscin and protogracillin. An intensive screening on qualitative and quantitative composition of raw materials from TT and variety of preparations from different origin demonstrated that

http://dx.doi.org/10.1016/j.jchromb.2015.11.023 1570-0232/© 2015 Elsevier B.V. All rights reserved.

Please cite this article in press as: S. Bouabdallah, et al., Current approaches and challenges for chemical characterization of inhibitory effect against cancer cell line isolated from Gokshur extract, J. Chromatogr. B (2015), http://dx.doi.org/10.1016/j.jchromb.2015.11.023

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Bulgarian preparation TribestanTM contains the highest amount of protodioscin and protogracillin. The aphrodisiac property of TT extract was explored in castrated mice [16]. Administration of TT to humans and animals improves spermatogenesis. Most of the chemical investigations described in the literature refer to TT of Chinese, Indian and Bulgarian origin [17]. Despite the great pharmacological profile assigned to this herbaceous, there are limited studies of secondary metabolite in the same plant species from other countries and no study from samples growing in Tunisia flora exept a study developped by Bouabdallah [18]. Molecular identification with precise information on the composition of complex natural extracts (metabolomes) that are derived from medicinal plants is a challenging task that requires sophisticated, advanced analytical methods. In this respect, significant advances in hyphenated chromatographic techniques reversed phase high pressure liquid chromatography (RP-HPLC) and liquid chromatography–mass spectrometry (LC–MS), as well as data mining and processing methods, have occurred together, these tools, in combination with bioassay profiling methods, serve an important role in metabolomics for the purposes of both peak detection, quantification and identification in natural product research. In the present study, a survey of the techniques that are used for generic and comprehensive profiling of secondary metabolites in natural extracts from TT is provided. The various approaches chromatographic methods: RP-HPLC; gas chromatography–mass spectrometry (GC–MS), are discussed with respect to their resolution and sensitivity for extract profiling. In addition in vitro cytotoxic activities of TT on various human ovary tumor cell lines have been examined. So our study report for the first time the best antiproliferatif fraction against tumor’s IGROV (IGROV-1 human ovarian cancer) and OVCAR (OVCAR-3 human ovarian cancer) cell line with respectively (88.19 ± 0.88%; 94.76 ± 1.52%) inhibitory activities. Furthermore, this high antiproliferatif effect derived from leaves n-BuOH fraction has been detected by RP-HPLC process with great peak area (5578.21 Mau) and identified using a GC–MS finger print.

2. Experimental 2.1. Reagents Analytical grade ethanol, chloroform and n-butannol obtained from Merck (Nottingham, UK), all reagents were purchased from Sigma–Aldrich-Fluka (Saint-Quentin, France). Murine macrophage cell line RAW 264.7 (ATCC, TIB-71) was used in this study. For the chemicals, thiazolyl blue tetrazolium bromide [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT), isopropanol, sodium dodecyl sulfate (SDS) and all reagents used for cell culture were purchased from Sigma (St. Louis, MO USA), Fluka Chemie (Buchs, Switzerland) and Merck (Nottingham, UK).

2.2. Collection of plant material Samples of TT were collected from plants grown in the region of Elhawaria (Boukrim) (Fig. 1a–c). Samples collection (June–September 2014) was conducted during the period of young leaves stage, flowering stage, mature fruit stage (when the fruits were ripening) Harvested plants were identified according to Pottier-Alapetite [19]. Furthermore confirmed to previously described methods by our Colleagues Botanists (INRAT, ENAT). Voucher specimens were deposited in the herbarium of our laboratory for future reference. Leaves, fruits, stems and roots were separated and dried at room temperature under dark conditions prior to use.

Fig. 1. Tribulus terrestris Linn. (Gokshura) samples growing spontaneously in the north east of northern Tunisia (El hawaria: Boukrim Ain halloufa).

2.3. Preparation of sample solutions by conventional heat reflux extraction The dried and powdered plant material (powder:30 g) was extracted in a succession by chloroform at room temperature (3 × 270 mL × 1 h) and 70% v/v ethanol (reflux at 80 ◦ C, 3 × 450 mL × 2 h). The combined ethanol solutions were concentrated under vacuum at 70 ◦ C to a small volume ∼150 mL and extracted in the separator funnel with n-butanol (3 times 60, 45,


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VWD1A, Wavelength=205nm(SALWAFB\9A205NMREP2.D)

3

2

5

1000

1

4

500

6 0

7

-500

9

8 -1000

0

5

10

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Fig. 2. Detection of inhibitory activities against human cancer cell lines by RP-HPLC finger print at 205 nm of n-butannolic fraction. Peaks: 1(RT:2.25; PA:1851.85); 2(RT:3.03; PA:5116.82); 3(RT:3.65;PA:5578.23);4(RT:17.4; PA:885.51); 5(RT:17.9; PA:3162.9); 6(RT:18.66; PA:1550.75); 7(RT:19.53; PA:942.22); 8(RT:23.77; PA:710.76); 9(RT:25.98; PA:472.57).

45 mL). The butanol layers were concentrated to dryness giving the crude fraction [20]. 2.4. Preparation of freeze-dried aqueous A 20 g of the powdered aerial parts were suspended in 200 mL distilled boiled water, for 30 min. The decoction obtained was filtered, and the filtrate frozen at −20 ◦ C and then lyophilized. The average yield of the lyophilized material (TT-extract) was approximately 15% it was stored at 4 ◦ C until further use. 2.5. Preparation of gradient program and sample solutions For the present study, we selected two systems composed of the mobile phase consisted of acetonitrile (solvent B) and water with 0.2% formic acid (solvent C). The flow rate was kept at 0.7 mol/min. The gradient programmer was as follows: At 6 min: 35% of B and 65% of C, at 9 min: 60% of B and 40% of C, at 14 min 80% of B and 20% of C, at 25 min 100% B and 0% C, at 30 min 35% of B and 65% of C [21]. The injection volume was 20 ␮L, and peaks were monitored at 205 nm. Samples were filtered through a 0.45 ␮m membrane filter before injection. 2.6. Separation procedure For each separation, the analytical RP-HPLC was carried out using an Agilent Technologies 1100 series liquid chromatography coupled with a UV–vis multi wavelength detector. The separation was carried out on a 250 × 4.6 mm, 4 ␮m Hypersil ODS C18 reversed phase column at ambient temperature. 2.7. GC–MS analysis and identification of phytocompounds peak fraction GC–MS analysis were carried out on a gas chromatograph; an HP 7890 series (II) coupled to an HP 5972 mass spectrometer (Agilent Technologies, Palo Alto, CA, USA) with electron impact ionization (70 eV). Separation was carried out using Phenyl Methyl Silox as capillary column (30m × 0.25 mm, 0.25 ␮m film thickness Agilent Technologies, Hewlett-Packard, CA, USA). Column temperature was programmed as follows 40 ◦ C for 1 min, 8 ◦ C/min to 100 ◦ C for 5 min, 10 ◦ C/min to 200 ◦ C for 3 min, 12 ◦ C/min to 300 ◦ C for 20 min. The carrier gas was helium with a flow rate of 0.9 mL/min and a split ratio of 100:1. Scan time and mass range were 1 s and 50–550 m/z,

respectively. Further identification was made by matching their recorded mass spectra with those stored in the Wiley/NBS mass spectral library of the GC–MS data system and other published mass spectra. Determination of the components percentages was based on peak area normalization.

2.8. Calculation The content of each peak area (PA) was calculated from a calibration curve that was generated using the 1,2,buthylhydroxy-toluene (BHT) as an internal standard.

2.9. Anticancer and cytotoxic activity Cells were grown in complete medium containing endotoxinfree RPMI-1640 medium supplemented with 10% fetal calf-serum (HyClone Laboratories, Logan, UT, USA), air and 5% CO2 at 37 ◦ C. The isolate was added to a medium containing 1 × 106 cells/mL, 2 mM L-glutamine and 50 ␮g/mL gentamycin. Cultures were maintained in a fully humidified incubator for 24 h. RAW 264.7 were dispatched at 3 × 104 cells/well in 96 well tissue culture plates, and kept for one night before treatment.

2.10. Cell viability assay Cytotoxic effects of the extract were evaluated by the conventional MTT assay according to the method described by Mosmann [22]. This assay is based on the capacity of mitochondrial succinyl dehydrogenases of metabolic active viable cells to convert the soluble yellow tetrazolium salt into an insoluble formazan product. After cell incubation, cellular supernatants were removed and replaced with 100 ␮L of 0.2 mg/mL of MTT dissolved in phosphate buffered saline (PBS). Cells were then incubated for 2 h at 37 ◦ C, 5% CO2 . The formazan resulting from the reduction of MTT was solubilized by adding 25 ␮L of isopropanol, followed by another incubation time of 20 min at room temperature in the dark. The absorbance was measured at 540 nm using an ELISA plate reader, and cell viability was estimated as the percentage of samples absorbance relative to non treated cells. Each assay was performed in duplicate and independent experiments were done at least twice.


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T IC: SFB2.D \d a ta .ms 9500000 9000000 8500000 8000000 7500000 7000000 6500000 6000000 5500000 5000000 4500000 4000000 3500000 3.237 3000000 2500000 2000000

37.607 37.551 37.137 37.340 37.011 37.229 35.074 36.306 36.195 36.037 35.885 33.20435.485 35.596 35.243 31.712 34.783 34.839 34.418 34.361 31.642 34.203 31.896 24.361 29.214 30.072 30.687 27.519 26.401 21.909 224.068 3.890 29.017 21.219 25.514 25.008 29.991

1500000

27.108

1000000 3.489 500000 4.022 5.00

15.854 10.476 10.00

15.00

20.00

25.00

30.00

35.00

T ime -->

Fig. 3. Total ion chromatogram for molecular ions from leaves n-butannolic fraction. A GC–MS (EI-SIM) chromatogram profile of secondary metabolites. Good separation and adequate precision for simultaneous quantification of already reported and novel secondary metabolites. Sensitivity of the method was highly dependent on the instrument conditions and therefore ion source cleaning was performed.

Table 1 Majors components detected in the n-butannolic fraction of Gokshur extract. Names of compounds

Retention timea

Area (%)

Molecular formulab

Molecular weight

Heptane Butane, 1,1-dibutoxy1,2-Benzenedicarboxylic acid, dibu Hexadecanoic acid, butyl ester Butyl 9,12,15-octadecatrienoate Pyridine-3-carboxamide, oxime, N 1-Hydroxy-2-o-fluorophenyl-4-nitro Cyclotrisiloxane, hexamethyl-

3.23 4.03 27.08 29.99 31.71 35.07 35.88 36.03

8.05 15.85 6.71 5.89 4.29 4.39 4.18 30.88

C7H16 C7H14 C8H6O4 C20H40O2 C22H38O2 C6H7NO C17H14F4N2O6S C6H18O3Si3

100.21 98.18 166.14 118.17 312.53 334.54 137.14 315.25

a b

RT retention time determined relative to a HP-5MS (5% Phenyl Methyl Silox). Identification method: MS, comparison of mass spectra with those listed in the NIST11 and Wiley 9 libraries.

2.11. Statistical analysis All data were expressed as means ± standard deviations of triplicate measurements. The confidence limits were set at P < 0.05. Correlations were carried out using the correlation and regression in the EXEL program. 3. Results 3.1. Bioanalysis by high-speed counter current chromatography The crude extracts of TT were first analyzed by RP-HPLC, eluting mixture of formic acid/water -acetonitrile was tested at various compositions in an attempt to resolve the polar compounds. The result indicates that the crude sample contains several high polar molecules which have been detected at 205 nm (Fig. 2), the separation of polar compounds were extracted from n-BuOH fraction (peaks 1–9). Retention times (RT) for both polar and less polar samples varied between 2.25 min and 25.98 min. Samples (peaks 1–3)

were eluted first with a mobile phase mixture of formic acid/wateracetonitrile (35% B; 65% C) Secondly, less polar samples (peaks 4–8) were eluted with use of less polar formic acid/water-acetonitrile mixture (80% B; 20% C) and finally sample (peak 9) was eluted using 100% B as the mobile phase. In all samples analyzed, the most abundant compounds were those of peak 2 (RT:3.03 min) and peak 3 (RT:3.65 min). These compounds have a complex hydrocarbure nature and correspond to a saponins derivative [23]. The interpretation on mass spectrum GC–MS was carried out and the spectra of the unknown components were compared to the spectrum of the known components stored in the NIST library. A total of 43 natural compounds were identified from leaves n-BuOH fraction (Fig. 3). RT for both non polar and polar samples varied between 3.23 min and 37.60 min, including major compound cyclotrisiloxane hexamethyl (30.92%; RT:37.01), The majors compounds with theirs RT, molecular formula, molecular weight were ascertained in Table 1. Identification of the edible leaves extract and the proportions (peak area > 4%) of the volatile compounds as part of the total compounds analyzed by GC–MS were shown in Fig. 4.


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Table 3 Cytotoxicity of Gokshur extracts. Samples

IGROV (%inhibitiona )

OVCAR (%inhibitiona )

RAW267.4 (%inhibitiona )

Leaves n-butannolic fraction Leaves aqueous fraction

88.19 ± 0.88 41.58 ± 0.42

94.76 ± 1.52 25.22 ± 1.84

73.1 Non toxic

Values are given as mean ± SD (n = 3). a Cytotoxicity (at 50 ␮g/mL).

4. Discussion

Fig. 4. Identification of the edible leaves and the proportions of the volatile compounds as part of the total compounds analyzed by GC–MS.

Table 2 % Inhibition of Gokshur extracts against tumor cell lines. Samples

IGROV (% of inhibition at 50 ␮g/mL)

Leaves n-butanolic Leaves aqueous lyophilized Fruit ethanolic Leaves ethanolic Fruit n-butannolic

88.19 41.58 58.82 25.44 34.71

± ± ± ± ±

0.88 0.42 12.42 0.75 3.81

OVCAR (% of inhibition at 50 ␮g/mL) 94.76 25.22 12.51 87.17 13.24

± ± ± ± ±

1.52 1.84 2.6 3.2 1.73

Values are given as mean ± SD (n = 3).

3.2. TT stimulates differentiation of ovary tumors in vitro The saponins seem to be strongly cytotoxic against a broad spectrum of malignant tumor cells, such as leukemia HL-60, mouse mastrocarcinoma, human pulmonary adenocarcinoma, large cell carcinoma, and squamous cell carcinoma including adriamycinresistant and camptothecin-resistant cell lines cited in literature [24]. The richness of TT on steroidal saponins [20], plant chosen for our research topic, prompted us to evaluate for the first time the anticancer activity of different extracts performed against IGROV and OVCAR cell line. Our results showed (Table 2) that the leaves n-BuOH fraction exhibited the highest inhibition against Tumor cell proliferation respectively (88.19 ± 0.88% and 94.76 ± 1.52%). 3.3. In vitro cytotoxic effect of solvent on human ovary tumors The antitumor effects of TT were evaluated in the OVCAR, IGROV tumor-bearing human at the dose of 50 ␮g/mL. As shown in Table 2 the inhibition of proliferation tumor’s cell line of leaves n-BuOH was twice as more effective against IGROV cell line and 4 times more active toward OVCAR cell lines than leaves aqueous lyophilized, furthermore the leaves n-BuOH fraction was rather more effective than leaves ethanolic fraction 2 against both cell lines. Leaves n-BuOH fraction with major compound cyclotrisiloxane hexamethyl (30.92%) exhibited the highest antiproliferatif effect against ovary cancer. Cyclotetrasiloxanes, are estrogen-like substances which may produce reproductive effects and may be carcinogenic at high levels of exposure. Hexamethylcyclotrisiloxane, is the simplest member of a series of cyclic oligodimethylsiloxanes primarily used as synthetic equivalents for the reactive intermediate dimethylsilanone. This characteristic is responsible for its extensive application in polymer chemistry.

High performance liquid chromatography on reversed-phase columns remains the best technique for rapid detection of polar molecules and is the most-widely used method for this group of compounds [25,26]. However, the lack of chromophores allowing detection in UV, limits the choice of gradient and detection method. The pre-column derivatisation with benzoyl chloride, coumarin or 4-bromophenacyl bromide has been used successfully in some cases allowing UV detection of separation. Standardization and identification of the peaks in HPLC chromatograms has been based on comparison of the retention times with those observed for authentic standards which needs many replications. The quality of medicinal herbs is defined in terms of the content of its bioactive compounds. Hence, RP-HPLC fingerprint profile of herbal products is such an important and powerful procedure which has often been employed for the determination of bioactive compounds of the herbal medicine [27,28]. RP-HPLC fingerprinting is proved to give a precise liner, an accurate method for herbal identification and can be used further in authentication and standardization of the medicinally important plant. Such finger printing is useful in controlling the quality of herbal products and checking the adulterant. Therefore, it can be useful for the evaluation of different marketed pharmaceutical preparations and plant systematic studies authentic standards. The determination of chemical composition is largely performed by the relatively expensive and often laborious techniques such as gas and liquid chromatography combined with specific detection schemes. In the last few years, GC–MS has become firmly established as a key technological metabolic profiling in plant species. Subtle structural differences in carbohydrates strongly impact their biological activity, and much work remains to be performed to be able to obtain this information by mass spectrometry. Challenging in ion activation will continue to drive progress in the mass spectrometry determination of carbohydrates, with advances in electron-aided methods of activation and photo dissociation is likely to have an important impact in the future. The first antitumor drugs from plants with cancer chemotherapy application were developed 5 decades ago. A great success in this regard is the development of drugs such as: vinblastine and vincristine (Catharanthus roseus), silvestrol (Aglaia foveolata), paclitaxel (Taxus brevifolia), eliptinium (Bleekeria vitensis) artemisinin (Artemisia annua) chrysin (Passiflora incarnate), and others. A large number of the medicinal products nowadays are of natural origin [29]. In this respect, medicinal plants are intensively studied as a potential source of new active components with high antitumor activity. The high saponin levels in TT and the data on their cytotoxic activity toward a number of tumor cell lines presumes a strong antitumor potential of this medicinal plant [30]. So far, most of the studies carried out have been about the cytotoxic effect of the Chinese TT [31–33]. Our study report a variety effects of fractions isolated from TT growing in Tunisia flora, our results showed that nBuOH fraction exhibited the highest antiproliferatif effect against respectively IGROV and OVCAR cell line (88.19%; 94.76%) toward total extract (41.58%; 25.22%) (Table 3) overall in accordance with




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the result developed by [34] indicating decrease in tumor cells viability under treatment of fractionated extract in comparison with the total extract (50% toward 43%), The reason for this quantitative variability is probably due to the different chemical nature and composition of bioactive compounds according to the part used of TT from Tunisia flora. At observation at 50 ␮g/mL OVCAR were rather sensitive to growth-inhibitory activity of fractions from leaves while the IGROV were relatively sensitive of fractions from fruits. So every part of the plant has a different chemical composition for both product qualities than quantities. It is this variation of composition that confers more specific activity of each part of the plant on a specific disease [35]. Furthermore (OVCAR; IGROV) were rather sensitive to growth-inhibitory activity of fractions from leaves n-BuOH than leaves ethannolic fraction (Table 2). The basis for this differential sensitivity of the tumors to the cytotoxic action of TT was not well understood. It could be speculated that the selectivity of TT on different cell lines might depend, at least in part, on the efficacy of the internalization of TT into the cells. As noted above, the murin macrophage was found to be less sensitive to the cytotoxic action of TT. This could be to the fact that TT proprieties interrelate selectively with tumor-cells (OVCAR; IGROV) membranes receptors. 5. Conclusions Despite improvements in analytical techniques for GC–MS, the comprehensive analysis and characterization of metabolites in complex samples remains a challenging task, particularly when they are present at varied concentration levels. The ability to detect a large number of secondary metabolites in low concentrations in a single analysis offers important benefits compared to other analytical methods. We developed for the first time a profiling method which was performed for the quantification of 43 metabolites from leaves n-butannolic fraction, including, siloxanes, alcanes, alcaloids and esters isolated from TT. growing in Tunisia flora. Furthermore we report the antitumor effect evaluated by MTT assay of various extract of TT., so we conclude that leaves n-BuOH fraction with cyclotrisiloxane as major compound exhibits the better inhibitory activities against ovary cancer cell lines. Conflict of interest The authors declare that there are no conflicts of interest. Contributors SB dealt of the choice, the identification, collects and harvested of vegetable matter, conception of ideas, separation and extraction of samples, interpreted the data and wrote all manuscript. SS performed and analytical analysis. R-MS cytotoxic analysis, DK and DL helped SB to perform biological test. MB-A drafted the manuscript and directed all the study. Acknowledgements We think Pr Nedia ben Brahim and Pr Zeineb Ghrab (botanist; INRAT, ENAT), We are thankful to Tawfik Belhaj farmer and owner of an agricultural project at Ain Alloufa (Boukrim, El Hawaria) and finally we are very grateful to our collaborator Pr. Foued Azzouz: owner of Industrial and Agriculture Project (El Hawaria, Tunis, Tunisia) for all supports and encouragement. References [1] J. Ferlay, D.M. Parkin, E. Steliarova-Foucher, Estimates of cancer incidence and mortality in Europe 2008, Eur. J. Cancer 46 (2010) 765–781.

[2] R. Angioli, I. Palaia, M.A. Zullo, et al., Diagnostic open laparoscopy in the management of advanced ovarian cancer, Gynecol. Oncol. 100 (2006) 455–461. [3] H.J. Lee, E.O. Lee, Y.H. Rhee, et al., An oriental herbal cocktail, ka-mi-kae-kyuk-tang, exerts anti-cancer activities by targeting angiogenesis apoptosis and metastasis, Carcinogenesis 27 (2006) 2455–2463. [4] T. Kumagai, C.I. Müller, J.C. Desmond, Y. Imai, D. Heber, H.P. Koeffler, Scutellaria baicalensis, an herbal medicine: anti-proliferative and apoptotic activity against acute lymphocytic leukemia, lymphoma and myeloma cell lines, Leuk. Res. 31 (2007) 523–530. [5] O. Sha, J. Niu, T.B. Ng, E.Y. Cho, X. Fu, W. Jiang, Anti-tumor action of trichosanthin, a type 1 ribosome-inactivating protein, employed in traditional Chinese medicine: a mini review, Cancer Chemother. Pharmacol. 71 (2013) 1387–1393. [6] Ayurvedic Pharmacopia of India, part 2 vol 1, First edition, India, 1989. 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Please cite this article in press as: S. Bouabdallah, et al., Current approaches and challenges for chemical characterization of inhibitory effect against cancer cell line isolated from Gokshur extract, J. Chromatogr. B (2015), http://dx.doi.org/10.1016/j.jchromb.2015.11.023 View publication stats

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