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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

  • Sajjad Ahmad1,
  • Farhat Ullah1,
  • Anwar Zeb1,
  • Muhammad Ayaz1,
  • Farman Ullah2 and
  • Abdul Sadiq1Email author
BMC Complementary and Alternative MedicineBMC series – open, inclusive and trusted201616:308

https://doi.org/10.1186/s12906-016-1302-y

Received: 21 May 2016

Accepted: 18 August 2016

Published: 24 August 2016

Abstract

Background

The importance of Rumex genus and the renowned ethnopharmacological and biological potentials of Rumex hastatus is evident from the previous reports. Recently the R. hastatus has been evaluated for anticancer potential against HepG2, MCF7 or LNCaP cell lines with considerable cytotoxicity. We also reported the anti-tumor and anti-angiogenic potentials of R. hastatus. The current study has been arranged to evaluate cytotoxic potential of this plant against HeLa and NIH/3T3 cell lines and sort out the most active fraction of R. hastatus along with the identification of bioactive compounds responsible for cytotoxicity.

Methods

The cytotoxic potential of methanolic extract and sub-fractions of R. hastatus was performed following (3-[4, 5-dimethylthiazole-2-yl]-2, 5-diphenyl-tetrazolium bromide) MTT calorimetric assay. Four concentrations (500, 250, 125 and 62.5 μg/ml) of each sample were used against both cell lines. Two cell lines i.e. HeLa and NIH/3T3 were used in the assay. Furthermore, chemical characterization of chloroform fraction was performed by GC-MS analysis.

Results

The current investigational study demonstrates that all the solvent fractions of R. hastatus were active against HeLa and NIH/3T3 cell lines. Among all the fractions, chloroform fraction was dominant in activity against both cell lines. The observed IC50 values of chloroform fraction were 151.52 and 53.37 μg/ml against HeLa and NIH/3T3 respectively. The GC-MS analysis of chloroform fraction revealed the identification of 78 compounds with the identification of bioactive ones like ar-tumerone, phytol, dihydrojasmone, sitostenone etc.

Conclusion

It can be concluded from our results that Rumex hastatus D. Don possess strong cytotoxic potential. Moreover, the observed IC50 values and GC-MS analysis of chloroform fraction reveal that most of the bioactive compounds are in chloroform fraction. It can be further deduce that the chloroform fraction is a suitable target for the isolation of compounds having potential role in cancer therapy.

Keywords

Rumex hastatus Cytotoxicity Anticancer HeLa NIH/3T3 GC-MS

Background

The leading research teams around the world are in continuous struggle to explore novel aspects to facilitate life. The facilitation of life also encompasses decreased morbidity and mortality [1]. One of leading causes of mortality is cancer worldwide which is considered as the most challenging disease. Several factors have been reported which cause cancer and hyper proliferative conditions [2]. The free radicals induced lesions have been considered as one of the leading causes of cancer [3]. Attention of the advanced clinical investigators has been focused on the therapeutic measures of this disease. Various therapeutic strategies are followed for the treatment of cancer and chemotherapy has been considered as the most acceptable and positive prognostic therapeutic approach [4]. The drugs from natural sources being biodegradable are preferred over the synthetic ones due to their comparative safe and efficacious nature [5]. Several natural anticancer drugs are available in the market like etoposide, docetaxel, irinotecan, pacletaxel, topotecan, vincristine and vinblastine [6]. Various derivatives of natural anticancer drugs are also being synthesized and exploited against cancer [7]. The exploration of anticancer agent is not confined to the laboratory rather their availability is also evidenced in plants, marine animals, bacteria, algae, fungi, reptiles etc [8, 9]. The most feasible and economic source of anticancer agents is plants. Numerous anticancer compounds have been isolated from plants and various investigators have reported plethora of plants’ secondary metabolites with strong anticancer potentials [10]. Several families of plants have been reported to possess anticancer compounds. One of the plants’ families i.e., Polygonaceae is also famous for anticancer activities [11]. Rumex is one of the most important genera of this family and several species of this genus have been reported to possess strong anticancer potentials [12]. Several antitumor compounds have also been isolated from different species of this genus, for example, Rumex hymenosepalus has been reported with the isolation of antitumor compounds, i.e. leucodelphinidin and leucopelargonidin [13]. Several species of Rumex have been employed ethnomedicinally in the treatment of inflammation, swelling, hyper proliferative skin diseases [14].

Rumex hastatus is one of the most important species which has been used traditionally for the treatment of various ailments like rheumatism, tonsillitis, piles etc [1517]. Previously, the R. hastatus has been evaluated for anticancer potential against HepG2, MCF7 or LNCaP cell lines with considerable cytotoxicity [18]. Previously, R. hastatus has been evaluated for anticholinesterase, antioxidant, anti-tumor, anti-angiogenic, phytotoxic and antibacterial potentials [1922]. Based on the ethnomedicinal uses and literature review of R. hastatus, the current study was designed to explore cytotoxic potential of this plant against cell lines and to find out the bioactive phytoconstituents responsible for anticancer activity using GC-MS analysis.

Methods

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;
$$ \%\ \mathrm{Inhibition} = 100-\frac{\mathrm{Mean}\ \mathrm{O}\mathrm{D}\ \mathrm{of}\ \mathrm{test}\ \mathrm{sample}-\mathrm{Mean}\ \mathrm{O}\mathrm{D}\ \mathrm{of}\ \mathrm{negative}\ \mathrm{control}}{\mathrm{Mean}\ \mathrm{O}\mathrm{D}\ \mathrm{of}\ \mathrm{positive}\ \mathrm{control}-\mathrm{Mean}\ \mathrm{O}\mathrm{D}\ \mathrm{of}\ \mathrm{negative}\ \mathrm{control}} \times 100 $$

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.

Results

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.
Table 1

Cytotoxic activity of various samples of Rumex hastatus against HeLa and NIH/3T3 cell lines

Samples

Conc. (μg/ml)

HeLa Cell Line

NIH/3T3 Cell Line

Inhibition (%)

IC50 (μg/ml)

Inhibition (%)

IC50 (μg/ml)

Rh.Cr

500

63.25 ± 0.20***

347.33

74.96 ± 0.21***

174.52

250

41.43 ± 1.15***

59.46 ± 0.54***

125

29.00 ± 1.50***

43.07 ± 1.02***

62.5

20.64 ± 1.60***

35.53 ± 0.61***

Rh.Hex

500

36.33 ± 3.50***

572.61

53.86 ± 0.85***

439.26

250

15.46 ± 2.43***

40.60 ± 0.41***

125

07.33 ± 0.68***

28.33 ± 0.33***

62.5

05.03 ± 0.23***

21.50 ± 0.60***

Rh.Chf

500

81.50 ± 0.86***

151.52

82.13 ± 0.88***

53.37

250

69.00 ± 2.80***

70.66 ± 0.49***

125

43.66 ± 0.89***

64.02 ± 1.11***

62.5

34.22 ± 0.23***

51.43 ± 0.61***

Rh.EtAc

500

79.66 ± 0.89***

166.50

72.76 ± 0.78***

158.73

250

66.32 ± 1.30***

59.00 ± 0.57***

125

40.93 ± 0.49***

46.86 ± 0.85***

62.5

29.83 ± 1.36***

31.43 ± 0.81***

Rh.Aq

500

60.83 ± 1.36***

369.68

65.60 ± 0.41***

237.62

250

42.53 ± 0.46***

51.96 ± 0.21***

125

33.61 ± 1.70***

42.66 ± 0.49***

62.5

21.33 ± 0.33***

36.13 ± 0.88***

Doxorubicin

500

96.63 ± 1.67

<0.1

98.53 ± 1.09

<0.1

250

91.87 ± 0.25

93.76 ± 0.78

125

89.46 ± 2.43

90.33 ± 0.88

62.5

84.50 ± 0.86

87.46 ± 0.54

Data is represented as mean ± S.E.M; n = 3, ***: P < 0.001

Key: Rh.Cr Crude methanolic extract, Rh.Hex n-hexane fraction, Rh.Chf chloroform fraction, Rh.EtAc ethyl acetate fraction, Rh.Aq aqueous fraction

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.
Table 2

Parameters of various components in Chloroform fraction of Rumex hastatus

RT (min)

Height

Height %

Area

Area %

Area Sum %

Base Peak m/z

Width

26.577

536469

7.54

1916591

8.14

2.26

222

0.144

28.475

6E + 06

87.45

22348531

94.9

26.38

88

0.204

31.979

7E + 06

91.91

22675632

96.29

26.77

67.1

0.141

32.106

7E + 06

100

23550533

100

27.8

55.1

0.127

32.173

333815

4.69

496177

2.11

0.59

55.1

0.054

32.525

900308

12.66

2370371

10.07

2.8

88

0.107

34.939

467634

6.58

1286192

5.46

1.52

254

0.1

35.766

331299

4.66

836122

3.55

0.99

88

0.097

37.977

340828

4.79

773168

3.28

0.91

149

0.09

43.667

851097

11.97

2994991

12.72

3.54

43.2

0.134

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.
Table 3

List of compounds in chloroform fraction of Rumex hastatus

S. No

Compound Label

RT

Common Name

Formula

Hits (DB)

1.

Diethyl 2,2-Dihydroxy Sulfide

5.757

Tedegyl

C4H10O2S

3

2.

Benzenemethanol

6.438

Benzyl alcohol

C7H8O

10

3.

2-Pyrrolidinone, 1-methyl

6.567

M-Pyrol

C5H9NO

10

4.

4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-

8.793

NF

C6H8O4

10

5.

Benzoic acid, ammonium salt

9.343

Ammonium benzoate

C7H6O2

10

6.

2-Methoxy-4-vinylphenol

12.609

p-Vinylguaiacol

C9H10O2

10

7.

Trimethylsilyl cyanide

15.284

Trimethyl silyl nitrile

C4H9NSi

10

8.

Bis(2-hydroxyethyl)lauramide

17.708

lauramide

C16H33NO3

10

9.

Dodecanoic acid, ethyl ester

18.281

Ethyl dodecanoate

C14H28O2

10

10.

2-Cyclopenten-1-one, 3-methyl-2-pentyl

18.547

Dihydrojasmone

C11H18O

10

11.

Ethyl.alpha.-d-glucopyranoside

19.004

glucopyranoside

C8H16O6

10

12.

Silane, [(1,1-dimethyl-2 propenyl)oxy] dimethyl-

19.332

NF

C7H16OSi

10

13.

4-[1,5-Dimethyl-1,4-Hexadienyl]-1-Methyl-1-Cyclohexene

19.582

NF

C15H24

10

14.

Ar-tumerone

19.755

Ar-tumerone

C15H20O

10

15.

4-((1E)-3-Hydroxy-1-propenyl)-2-methoxyphenol

21.382

NF

C10H12O3

10

16.

Tetradecanoic acid

21.798

Myristic acid

C14H28O2

10

17.

(-)-Loliolide or Loliolide

22.21

Calendin

C11H16O3

10

18.

Tetradecanoic acid, ethyl ester

22.642

Ethyl myristate

C16H32O2

10

19.

2-Cyclohexen-1-one, 4-hydroxy-3,5,6-trimethyl-4-(3-oxo-1-butenyl)

22.779

NF

C13H18O3

10

20.

p-Hydroxycinnamic acid, ethyl ester

23.832

p-Hydroxycinnamic acid, ethyl ester

C11H12O3

10

21.

7,11,15-Trimethyl,3-Methylene-1-Hexadecene

24.028

Neophytadiene

C20H38

10

22.

2-Pentadecanone, 6,10,14-trimethyl

24.223

Hexahydrofarnesyl acetone

C18H36O

10

23.

Pentadecanoic acid, ethyl ester

25.763

ethyl pentadecanoate

C17H34O2

10

24.

Ethyl (2E)-3-(4-hydroxy-3-methoxyphenyl)-2-propenoate

26.577

NF

C12H14O4

6

25.

Hexadecanoic acid

27.756

Palmitic acid

C16H32O2

10

26.

Ethyl 9-Hexadecenoate

27.899

NF

C18H34O2

10

27.

1,9-Tetradecadiene

28.273

NF

C14H26

10

28.

Hexadecanoic acid, ethyl ester

28.475

Ethyl palmitate

C18H36O2

10

29.

(E)-3-(4-Biphenylyl)-2-propen-1-ol

28.518

NF

C15H14O

8

30.

Peniopholide

29.798

Peniopholide

C15H24O3

10

31.

Heptadecanoic acid, ethyl ester

30.025

Ethyl n-heptadecanoate

C19H38O2

10

32.

Propyl hexadecanoate

30.527

Propyl palmitate

C19H38O2

10

33.

Heptadecanoic acid, ethyl ester

30.607

Ethyl n-eptadecanoate

C19H38O2

10

34.

2-Hexadecen-1-ol, 3,7,11,15-tetramethyl-, [R-[R*,R*-(E)]]-

31.016

Phytol

C20H40O

10

35.

cis-9,cis-12-Octadecadienoic acid

31.507

NF

C18H32O2

10

36.

E-11,13-Tetradecadien-1-ol

31.616

NF

C14H26O

10

37.

Linoleic acid ethyl ester

31.979

Mandenol

C20H36O2

10

38.

Ethyl 9-Octadecanoate

32.104

Ethyl 9-Octadecenoate

C20H38O2

10

39.

exo-4-Methylbicyclo[3.2.1]octan-3-ene

32.121

NF

C9H14

10

40.

16-Methyloxacyclohexadeca-3,5-dien-2-one

33.111

NF

C16H26O2

10

41.

3.beta.-Hydroxydihydroconfertifolin

33.956

NF

C15H24O3

1

42.

Ethyl 9-Hexadecenoate

34.021

NF

C18H34O2

10

43.

Cis-8-methyl-exo-tricyclo[5.2.1.0(2.6)]decane

34.647

NF

C11H18

10

44.

9,10-Anthracenedione, 1,8-dihydroxy-3-methyl

34.942

C.I. Natural Yellow 23

C15H10O4

10

45.

4,8,12-Trimethyltridecan-4-olide

35.181

NF

C16H30O2

10

46.

5-Icosyne

35.305

5-Eicosyne

C20H38

10

47.

Ethyl 9-Hexadecenoate

35.382

NF

C18H34O2

10

48.

Heptadecanoic acid, ethyl ester

35.768

NF

C19H38O2

10

49.

13-Tetradecenal

35.985

NF

C14H26O

10

50.

5-Dodecyne

36.078

5-Dodecyne

C12H22

10

51.

N-Vanillylnonanoamide

37.013

Nonivamide

C17H27NO3

10

52.

1,2-Benzenedicarboxylic acid, bis (2 ethylhexyl) ester

37.978

DNOP

C24H38O4

10

53.

N(4-Hydroxy-3-Methoxybenzyl)-8-Methylnon-6-Enamide

38.186

NF

C18H27NO3

10

54.

delta.13-cis-Docosenoic acid

38.242

Erucic acid

C22H42O2

10

55.

N-(4-Hydroxy-3-Methoxybenzyl)-8-Methyl-Nonanamide

38.489

NF

C18H29NO3

10

56.

Docosanoic acid, ethyl ester

38.566

Ethyl docosanoate

C24H48O2

10

57.

9,10-Anthracenedione, 1,8-dihydroxy-3-methoxy-6-methyl

39.322

Physcion

C16H12O5

10

58.

Methyl palustrate isomer

39.554

Methyl palustrate

C21H32O2

1

59.

1-Bromo-4,8,12-trimethyl-3(E),7(E)-11-tridecatriene

40.642

NF

C16H27Br

5

60.

Oleic acid amide

40.909

Oleamide

C18H35NO

10

61.

Heptadecanoic acid, ethyl ester

41.07

NF

C19H38O2

10

62.

1,1-Di(1,1-dimethylethyl)cyclopropane

41.672

NF

C11H22

3

63.

Arachic alcohol

41.685

n-Eicosanol

C20H42O

10

64.

Aristol-9-en-8-one

42.368

Aristolone

C15H22O

10

65.

2-Bromotetradecane

42.397

NF

C14H29Br

10

66.

Stigmasta-5,22-dien-3-ol, acetate, (3.beta.,22Z)-

42.529

NF

C31H50O2

10

67.

Stigmast-5-en-3-ol, (3.beta.,24S)- (CAS)

42.968

Clionasterol

C29H50O

10

68.

7-methyltocol

43.226

NF

C27H46O2

2

69.

Stigmast-5-en-3-ol, acetate, (3.beta.)-

43.666

β-Sitosterol acetate

C31H52O2

10

70.

alpha.-Tocopherol

44.466

Vitamin E

C29H50O2

7

71.

Cholesta-4,6-dien-3-ol, benzoate, (3.beta.)

45.533

NF

C34H48O2

9

72.

Alpha.-Bisabolol

52.989

.Alpha.-bisabolol

C18H32O

10

73.

Methyl Commate E

53.773

NF

C31H50O5

10

74.

Stigmast-4-en-3-one

55.721

Sitostenone

C29H48O

10

75.

2-Ethylthio-2-ethoxy-3-oxo-N phenylbutanamide

57.414

NF

C14H19NO3S

9

76.

3-(Methoxymethoxy)-5-(phenylmethoxypentanal

58.739

NF

C14H20O4

1

77.

13-Epimanool

62.472

Epimanool-

C20H34O

10

78.

1,2-Dicyclohexyl-1,1,2,2-tetrafluoroethane

70.638

NF

C14H22F4

6

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

Discussion

HeLa is a type of immortal cell line obtained from cervical cancer cells and for the very first time this cell line has been taken from late Henrietta Lacks in 1951 and abbreviated for her name [30]. Similarly, the NIH/3T3 cell line was originated from swiss mice in 1962 which consists of immortal fibroblast cell and widely used for experimental purposes [31]. To figure out the cytotoxicity in these cells, the MTT assay is considered as a rapid and authentic procedure to appraise the cell viability and death by calorimetric analysis [29]. Previously, the MTT assay has been reported by numerous researchers to evaluate the cytotoxicity [32, 33]. Recently, Polygonum hydropiper has been demonstrated with significant cytotoxicity against NIH/3T3 cell line following MTT assay [34]. As this is evidenced from several reports that a specific pharmacological potential within plant species is basically conferred due to specific group of compounds [35]. Similarly, a specific group of phytoconstituents is responsible for the cytotoxic potential of certain plants [36]. The GC-MS is a quick and easy way of finding out various components in a crude mixture of plant extract [37]. In our current research, the GC-MS analysis of chloroform fraction of R. hastatus showed 78 compounds summarized in Table 2. Several compounds identified by GC-MS in the chloroform fraction are reported to have positive role in cell toxicities. For instance, phytol, dihydrojasmone, ethyl α-d-glucopyranoside, anthracenedione, silane, nonivamide, eicosanol, aristolone, ar-tumerone and sitostenone are the compounds with cytotoxic/anticancer potential demonstrated along with their spectra in Figs. 2 and 3.

Phytol present in R. hastatus has been reported to induce programmed cell death in human lymphoid leukemia Molt 4B cells [38]. Dihydrojasmone, one of the member of jasmonate family, which has been implied as a new family of anticancer agents [39]. Ethyl-α-d-glucopyranoside a derivative of glucopyranoside has been reported time and again to possess strong anticancer potential and it is evident from the GC-MS analysis that R. hastatus contain ethyl α-d-glucopyranoside, which may confer the possible anticancer potential to this plant. Anthracenedione has also been reported to possess anticancer properties [40]. Silane has been proven as an efficient agent in a nanoparticle based drug delivery system for anticancer compounds. The chloroform fraction of R. hastatus also possess nonivamide, which is skin permeation enhancer and used in various ointments etc [41]. Similarly, eicosanol is a C20 alcohol present in R. hastatus and C20 aliphatic alcohols has been employed in the treatment of hyperproliferative skin disordersone [42]. Aristolone and Ar-tumerone are sesquiterpenes, and the derivatives of sesquiterpene have been reported to possess the cytotoxic potential [43]. Likewise, vitamin E a phenolic compound with pronounced free radical scavenging and anticancer potential has also been evidenced from Table 2 [44, 45]. Another compound i.e., a natural steroid named sitostenone has also been analyzed in GC-MS spectra and steroids have also been used since long for the treatment of cancer, so this compound may also be involved in cytotoxicity observed in our current studies [46]. The current investigational study demonstrates that the chloroform fraction of R. hastatus was the most active one against two types of cell lines. The regression and correlation analysis shows that this plant has a parallel cytotoxic potential against both the cell lines as depicted in the Fig. 4 with r2 value of 0.881. The current study can also be correlated with the previous cytotoxic activity of R. hastatus against brine shrimps in which the chloroform fraction was the most active fraction [22]. Based on the marked potential of this fraction, it has been chemically characterized and based on the literature survey; the active compounds have been sorted out.
Fig. 4

Regression and correlation of various samples of Rumex hastatus against HeLa cell line Vs NIH/3T3 cell line

Conclusion

Based on our current results, we can conclude that Rumex hastatus is a potential source of cytotoxic compounds. Moreover, the chloroform fraction is the active one among other solvent fractions of R. hastatus. Based on the GC-MS analysis of chloroform fraction, we can conclude that the chloroform fraction of R. hastatus is a rich source of bioactive compounds responsible for cytotoxicity.

Abbreviations

eV: 

Electron volt

FBS: 

Fetal bovine serum

FID: 

Flame ionization detector

GC-MS: 

Gas chromatography-mass spectrometry

HeLa: 

Human cervical carcinoma cell line or Henrietta Lacks cell line

HepG2: 

Human liver cancer cell line/Hepatoblastoma G2 cell line

IC50

Median inhibitory concentration

LNCaP: 

Lymph node carcinoma of the prostate

MTT: 

3-[4, 5-dimethylthiazole-2-yl]-2, 5-diphenyl-tetrazolium bromide

MCF7: 

Breast cancer cell line/Michigan Cancer Foundation-7

NIH/3T3: 

Fibroblast cell line from Swiss mouse embryo/3-day transfer, inoculum 3 x 105 cells

NIST: 

National Institute of Standards and Technology

OD: 

Optical density

Rh.Aq: 

Aqueous fraction

Rh.Chf: 

Chloroform fraction

Rh.Cr: 

Methanolic extract of Rumex hastatus

Rh.EtAc: 

Ethyl acetate fraction

Rh.Hex: 

n-hexane fraction

SEM: 

Standard error mean

Declarations

Acknowledgements

The authors are grateful to Dr. Ali Hazrat, Department of Botany, Shaheed Benazir Bhutto University, Sheringal Dir (U), KPK, Pakistan for the identification of plant.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Availability of data and materials

The data presented in this manuscript belong to the PhD work of Mr. Sajjad Ahmad and has not been deposited in any repository yet. However, the materials are available to the researchers upon request.

Authors’ contributions

SA and AZ carried out experimental work, data collection and literature search. FU designed the project and helped in supervision. MA and FU drafted the manuscript for publication. AS make the final version of publication. All the authors have read and approved the final manuscript for publication.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable for this submission.

Ethics approval and consent to participate

Not applicable for this submission.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Department of Pharmacy, University of Malakand
(2)
Department of Pharmacy, Kohat University of Science & Technology

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Copyright

© The Author(s). 2016

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