Evaluation of Rumex hastatus D. Don for cytotoxic potential against HeLa and NIH/3T3 cell lines: chemical characterization of chloroform fraction and identification of bioactive compounds

Plant collection, extraction and fractionation

The aerial parts of mature plant of R. hastatus were collected from the surrounding area of University of Malakand, Pakistan. The plant’s name was confirmed by Dr. Ali Hazrat, Plant Taxonomist, Department of Botany, Shaheed Benazir Bhutto University, Sheringal Dir (U), KPK, Pakistan, and deposited with voucher specimen No. 1015SA. The plant’s material was shade dried, powdered and subjected to maceration process. Afterwards, it was filtered and the filtrate was evaporated under reduced pressure using rotary evaporator at 40 °C [23, 24]. Similarly, the crude methanolic extract (Rh.Cr) was obtained weighing 400 g (5.7 %). The suspension of Rh.Cr weighing 300 g was subjected to fractionation process with the order of increasing polarity. In this way, the fractions obtained were 19 (6.3 %), 21 (7 %), 29 (9.6 %) and 120 (40 %) g of n-hexane (Rh.Hex), chloroform (Rh.Chf), ethyl acetate (Rh.EtAc) and aqueous fraction (Rh.Aq) respectively [25, 26].

Gas Chromatography (GC) analysis

Samples were subjected to GC analysis using an Agilent USB-393752 gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) with HHP-5MS 5 % phenylmethylsiloxane capillary column (30 m × 0.25 mm × 0.25 μm film thickness; Restek, Bellefonte, PA) equipped with an FID detector. The initial temperature of the oven was retain at 70 °C for 1 min, followed by increase at the rate of 6 °C/min to 180 °C for 5 min and finally at the rate of 5 °C/min to 280 °C for 20 min. The temperature of injector and detector were set at 220 and 290 °C, correspondingly. Helium was used as carrier gas at a flow rate of 1 ml/min, and diluted samples (1/1000 in n-pentane, v/v) of 1.0 μl were injected manually in the splitless mode.

Gas Chromatography–Mass Spectrometry (GC/MS) analysis

GC/MS analysis of samples were processed using an Agilent USB-393752 gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) with a HHP-5MS 5 % phenylmethylsiloxane capillary column (30 m × 0.25 mm × 0.25 μm film thickness; Restek, Bellefonte, PA) outfitted with an Agilent HP-5973 mass selective detector in the electron impact mode (Ionization energy: 70 eV) working under the same experimental conditions as described for GC.

Identification of components

Compounds were recognized by comparison of their retention times with those of authentic compounds in the literature under the same set of conditions. Further identification were done through the spectral data obtained from the Wiley and NIST libraries and further confirmed by comparisons of the fragmentation pattern of the mass spectra with data published in the literature [27, 28].

MTT assay on HeLa and NIH/3T3 cell lines

Cytotoxic activity of various samples of R. hastatus was assayed in 96-well flat-bottomed micro plates following the standard MTT (3-[4, 5-dimethylthiazole-2-yl]-2, 5-diphenyl-tetrazolium bromide) colorimetric assay [29]. Briefly, HeLa cells (Cervical Cancer) and Mouse embryonic fibroblast NIH/3T3 cell lines were cultured in Minimum Essential Medium Eagle. The media was supplemented with 5 % of fetal bovine serum (FBS), 100 μg/ml of streptomycin and 100 IU/ml of penicillin in 75 cm² flasks and incubated in 5 % CO₂ incubator at 37 °C. Growing cells were harvested exponentially and counted with haemocytometer followed by dilution with a particular medium. Cell culture was prepared having the concentration of 6 x 10⁴ cells/ml and transferred (100 μl/well) into 96-well plates. After overnight incubation, medium was discarded and 200 μl of fresh medium was added with various concentrations of plant samples (1–30 μM). After 48 h, 200 μl MTT (0.5 mg/ml) was added to each well and incubated additionally for 4 h. Afterward, 100 μL of DMSO was added to each well. The extent of MTT reduction to formazan within cells was figured out by measuring the absorbance at 570 nm, employing a micro plate reader (Spectra Max plus, Molecular Devices, CA, USA). The samples causing 50 % growth inhibition for both cell lines were recorded as IC₅₀. The percent inhibition was calculated by the formula given below;\documentclass[12pt]{minimal}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{upgreek}\setlength{\oddsidemargin}{-69pt}\begin{document}$$ \%\ \mathrm{Inhibition} = 100-\frac{\mathrm{Mean}\ \mathrm{O}\mathrm{D}\ \mathrm{of}\ \mathrm{test}\ \mathrm{sample}-\mathrm{Mean}\ \mathrm{O}\mathrm{D}\ \mathrm{of}\ \mathrm{negative}\ \mathrm{control}}{\mathrm{Mean}\ \mathrm{O}\mathrm{D}\ \mathrm{of}\ \mathrm{positive}\ \mathrm{control}-\mathrm{Mean}\ \mathrm{O}\mathrm{D}\ \mathrm{of}\ \mathrm{negative}\ \mathrm{control}} \times 100 $$\end{document}%Inhibition=100−MeanODoftestsample−MeanODofnegativecontrolMeanODofpositivecontrol−MeanODofnegativecontrol×100

The results i.e., Percent inhibition were processed via Soft- Max Pro software (Molecular Device, USA).

Statistical analysis

All the tests were performed in triplicate and values were expressed as means ± S.E.M. Multiple group comparison was performed by Two way ANOVA followed by Bonferroni post test in which the P < 0.05 were considered significant.

MTT assays

The MTT assay was carried out against two types of cell lines, i.e., HeLa and NIH/3T3. The crude methanolic extract and sub-fractions of R. hastatus were assay against both cell lines. All the samples were found active against both cell lines with chloroform fraction more dominant as shown in Table 1. In HeLa cell line cytotoxicity assay, the chloroform fraction revealed significant cytotoxic potential. The observed cytotoxic potential against HeLe cell line were 81.50 ± 0.86, 69.00 ± 2.80, 43.66 ± 0.89 and 34.22 ± 0.23 % at concentrations of 500, 250, 125 and 62.5 μg/ml respectively with IC₅₀ value of 151.52 μg/ml. Similarly, the second highest activity has been demonstrated by ethyl acetate fraction i.e., 79.66 ± 0.89, 66.32 ± 1.30, 40.93 ± 0.49 and 29.83 ± 1.36 % cytotoxic activity at concentrations of 500, 250, 125 and 62.5 μg/ml against HeLa cell line with IC₅₀ value of 166.50 μg/ml. The methanolic extract and aqueous fraction demonstrated moderate cytotoxic potentials with IC₅₀ values of 347.33 and 369.68 μg/ml respectively. Among all the samples of R. hastatus, the least activity was shown by that of n-hexane fraction with IC₅₀ of 572.61 μg/ml.

In NIH/3T3 cell line assay, again the chloroform fraction was found dominant exhibiting 82.13 ± 0.88, 70.66 ± 0.49, 64.02 ± 1.11 and 51.43 ± 0.61 % cytotoxic potential at concentrations of 500, 250, 125 and 62.5 μg/ml with IC₅₀ value of 53.37 μg/ml. Similarly, the ethyl acetate fraction revealed the second highest activity against NIH/3T3 cell line i.e., 72.76 ± 0.78, 59.00 ± 0.57, 46.86 ± 0.85 and 31.43 ± 0.81 % at concentrations of 500, 250, 125 and 62.5 μg/ml with IC₅₀ value of 158.73 μg/ml. The IC₅₀ calculated for the rest of the samples were 174.52, 237.62 and 439.26 μg/ml for methanolic extract, aqueous and n-hexane fractions respectively. The cytotoxic potential of all the test samples of R. hastatus against NIH/3T3 cell line has been summarized in Table 1. The standard drug doxorubicin exhibited IC₅₀ value <0.1 μg/ml against both cell lines.

GC-MS analysis

Based on the high potency in both cell lines assays, the chloroform fraction was subjected to GC-MS analysis. A total of 78 phytoconstituents were identified by the GC-MS analysis. The identified compounds contain important bioactive compounds responsible for the cytotoxic potential of the plant. The parameters of some compounds found in GC-MS analysis have been summarized in the Table 2.

It is evident that area wise the highest percentage has been exhibited by linoleic acid ethyl ester with retention time 31.979 (96.29 %) followed by hexadecanoic acid, ethyl ester with retention time 28.475 (94.9 %). A summary of all identified compounds in the chloroform fraction has been shown in Table 3.

The GC-MS chromatogram of the chloroform fraction is shown in Fig. 1 in which some of the important peaks are clearly visible. Some important bioactive compounds which having a positive role in cytotoxicity are sorted in Fig. 2. Moreover, the integration patterns of some important compounds as elucidated by GC-MS are shown in Fig. 3.

Fig. 1

GC-MS chromatogram of chloroform fraction of Rumex hastatus

Fig. 2

Structures of some anticancer compounds identified in the GC-MS analysis of chloroform fraction of Rumex hastatus.a Phytol b Dihydrojasmone c Ethyl.alpha.-d-glucopyranoside d Anthracenedione e Nonivamide f Silane g Eicosanol h Aristolone i 2-Ethylthio-2-ethoxy-3-oxo-N-phenylbutanamide and j Sitostenone

Fig. 3

GC-MS spectra of some important compounds in chloroform fraction of Rumex hastatus