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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

BMC Complementary and Alternative Medicine volume 16, Article number: 308 (2016) Cite this article

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.

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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 [15–17]. 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 [19–22]. 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
Full size table

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
Full size table

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
Full size table

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
figure 1

GC-MS chromatogram of chloroform fraction of Rumex hastatus

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Fig. 2
figure 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

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Fig. 3
figure 3

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

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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
figure 4

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

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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

References

  1. Bird S, Noronha M, Sinnott H. An integrated care facilitation model improves quality of life and reduces use of hospital resources by patients with chronic obstructive pulmonary disease and chronic heart failure. Aust J Prim Health. 2010;16(4):326–33.

    Article  PubMed  Google Scholar 

  2. Borrego-Soto G, Ortiz-Lopez R, Rojas-Martinez A. Ionizing radiation-induced DNA injury and damage detection in patients with breast cancer. Genet Mol Biol. 2015;38(4):420–32.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Valko M, Rhodes C, Moncol J, Izakovic M, Mazur M. Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chem Biol Interact. 2006;160(1):1–40.

    Article  CAS  PubMed  Google Scholar 

  4. Mohamed AF, Gonzalez JM, Fairchild A. Patient Benefit-Risk Tradeoffs for Radioactive Iodine-Refractory Differentiated Thyroid Cancer Treatments. J Thyroid Res. 2015;2015:438235.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Coats JR. Risks from natural versus synthetic insecticides. Ann Rev Entomol. 1994;39(1):489–515.

    Article  CAS  Google Scholar 

  6. Haddadin S, Perry MC. History of small-cell lung cancer. Clin Lung Cancer. 2011;12(2):87–93.

    Article  CAS  PubMed  Google Scholar 

  7. Jordan MA, Wilson L. Microtubules as a target for anticancer drugs. Nat Rev Cancer. 2004;4(4):253–65.

    Article  CAS  PubMed  Google Scholar 

  8. Simmons TL, Andrianasolo E, McPhail K, Flatt P, Gerwick WH. Marine natural products as anticancer drugs. Mol Cancer Ther. 2005;4(2):333–42.

    CAS  PubMed  Google Scholar 

  9. Bermudes D, Zheng L, King I. Live bacteria as anticancer agents and tumor-selective protein delivery vectors. Curr Opin Drug Discov Develop. 2002;5(2):194–9.

    CAS  Google Scholar 

  10. Croteau R, Kutchan TM, Lewis NG. Natural products (secondary metabolites). Biochem Mol Biol Plants. 2000;24:1250–319.

    Google Scholar 

  11. Lajter I, Zupkó I, Molnár J, Jakab G, Balogh L, Vasas A, Hohmann J. Antiproliferative activity of Polygonaceae species from the Carpathian Basin against human cancer cell lines. Phytother Res. 2013;27(1):77–85.

    Article  PubMed  Google Scholar 

  12. Kuete V, Wabo HK, Eyong KO, Feussi MT, Wiench B, Krusche B, Tane P, Folefoc GN, Efferth T. Anticancer activities of six selected natural compounds of some Cameroonian medicinal plants. PLoS One. 2011;6(8):e21762.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Cole JR, Buchalter L. Isolation of a potential antitumor fraction from Rumex hymenosepalus. J Pharm Sci. 1965;54(9):1376–8.

    Article  CAS  PubMed  Google Scholar 

  14. Hussain F, Hameed I, Dastagir G, Khan I, Ahmad B. Cytotoxicity and phytotoxicity of some selected medicinal plants of the family Polygonaceae. Afr J Biotechnol. 2010;9(5):770–74.

  15. Shinwari ZK, Gilani SS. Sustainable harvest of medicinal plants at Bulashbar Nullah, Astore (Northern Pakistan). J Ethnopharmacol. 2003;84(2):289–98.

    Article  PubMed  Google Scholar 

  16. Gorsi MS, Miraj S. Ethnomedicinal survey of plants of Khanabad village and its allied areas, district Gilgit. Asian J Plant Sci. 2002;1(5):604–15.

    Article  Google Scholar 

  17. Manan Z, Razzaq A, Islam M. Diversity of medicinal plants in Wari subdivision district Upper Dir, Pakistan. Pak J Plant Sci. 2007;45:231–4.

    Google Scholar 

  18. Ghufran MA, Qureshi RA, Batool A, Kondratyuk TP, Guilford JM, Marler LE, Chang LC, Pezzuto JM. Evaluation of selected indigenous medicinal plants from the western Himalayas for cytotoxicity and as potential cancer chemopreventive agents. Pharm Biol. 2009;47(6):533–8.

    Article  Google Scholar 

  19. Ahmad S, Ullah F, Sadiq A, Ayaz M, Imran M, Ali I, Zeb A, Ullah F, Raza Shah M. Chemical composition, antioxidant and anticholinesterase potentials of essential oil of Rumex hastatus D. Don collected from the North West of Pakistan. BMC Complement Altern Med. 2016;16:19.

    Google Scholar 

  20. Ahmad S, Ullah F, Ayaz M, Sadiq A, Imran M. Antioxidant and anticholinesterase investigations of Rumex hastatus D. Don: potential effectiveness in oxidative stress and neurological disorders. Biol Res. 2015;48:1–8.

    Article  Google Scholar 

  21. Ahmad S, Ullah F, Ayaz M, Zeb A, Ullah F, Sadiq A. Antitumor and anti-angiogenic potentials of isolated crude saponins and various fractions of Rumex hastatus D. Don. Biol Res. 2016;49(1):1.

    Article  Google Scholar 

  22. Kamal Z, Ullah M, Ahmad S, Ullah F, Sadiq A, Ayaz M, Zeb A, Imran M. Ex-vivo antibacterial, phytotoxic and cytotoxic, potential in the crude natural phytoconstituents of Rumex hastatus D. Don. Pak J Bot. 2015;47(SI):293–9.

    CAS  Google Scholar 

  23. Zeb A, Sadiq A, Ullah F, Ahmad S, Ayaz M. Phytochemical and toxicological investigations of crude methanolic extracts, subsequent fractions and crude saponins of Isodon rugosus. Biol Res. 2014;47(1):57.

    Article  PubMed  PubMed Central  Google Scholar 

  24. Zeb A, Sadiq A, Ullah F, Ahmad S, Ayaz M. Investigations of anticholinestrase and antioxidant potentials of methanolic extract, subsequent fractions, crude saponins and flavonoids isolated from Isodon rugosus. Biol Res. 2014;47(1):1–10.

    Google Scholar 

  25. Ayaz M, Junaid M, Ahmed J, Ullah F, Sadiq A, Ahmad S, Imran M. Phenolic contents, antioxidant and anticholinesterase potentials of crude extract, subsequent fractions and crude saponins from Polygonum hydropiper L. BMC Complement Altern Med. 2014;14(1):145.

    Article  PubMed  PubMed Central  Google Scholar 

  26. Ayaz M, Junaid M, Subhan F, Ullah F, Sadiq A, Ahmad S, Imran M, Kamal Z, Hussain S, Shah SM. Heavy metals analysis, phytochemical, phytotoxic and anthelmintic investigations of crude methanolic extract, subsequent fractions and crude saponins from Polygonum hydropiper L. BMC Complement Altern Med. 2014;14(1):465.

    Article  PubMed  PubMed Central  Google Scholar 

  27. Stein S, Mirokhin D, Tchekhovskoi D, G M: The NIST Mass Spectral Search Program for the NIST/EPA/NIH Mass Spectra Library; Standard Reference Data Program of the National Institute of Standards and Technology: Gaithersburg, MD, USA. 2002.

  28. Adams R. Identification of essential oil components by gas chromatography/mass spectrometry, vol. 804. Carol Stream: Allured Publishing; 2007. p. 804.

    Google Scholar 

  29. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65(1):55–63.

    Article  CAS  PubMed  Google Scholar 

  30. Sharrer T. Opinion-“ HeLa” herself-It’s high time we honored the memory of Henrietta Lacks, the woman who gave us immortal cells. Scientist. 2006;20(7):22–3.

    Google Scholar 

  31. Newbold RF, Overell RW. Fibroblast immortality is a prerequisite for transformation by EJ c-Ha-ras oncogene. Nature. 1983;304(5927):648–51.

    Article  CAS  PubMed  Google Scholar 

  32. Vahedi F, Najafi MF, Bozari K. Evaluation of inhibitory effect and apoptosis induction of Zyzyphus Jujube on tumor cell lines, an in vitro preliminary study. Cytotechnology. 2008;56(2):105–11.

    Article  PubMed  PubMed Central  Google Scholar 

  33. Momtaz S, Hussein AA, Ostad SN, Abdollahi M, Lall N. Growth inhibition and induction of apoptosis in human cancerous HeLa cells by Maytenus procumbens. Food Chem Toxicol. 2013;51:38–45.

    Article  CAS  PubMed  Google Scholar 

  34. Ayaz M, Junaid M, Ullah F, Sadiq A, Subhan F, Khan MA, Ahmad W, Ali G, Imran M, Ahmad S: Molecularly Characterized Solvent Extracts and Saponins from Polygonum hydropiper L. Show High Anti-Angiogenic, Anti-Tumor, Brine Shrimp, and Fibroblast NIH/3T3 Cell Line Cytotoxicity. Front Pharmacol. 2016, 7.

  35. Shen Y, Jin L, Xiao P, Lu Y, Bao J. Total phenolics, flavonoids, antioxidant capacity in rice grain and their relations to grain color, size and weight. J Cereal Sci. 2009;49(1):106–11.

    Article  CAS  Google Scholar 

  36. Palchaudhuri R, Hergenrother PJ. DNA as a target for anticancer compounds: methods to determine the mode of binding and the mechanism of action. Curr Opin Biotechnol. 2007;18(6):497–503.

    Article  CAS  PubMed  Google Scholar 

  37. Zeb A, Ahmad S, Ullah F, Ayaz M, Sadiq A. Anti-nociceptive Activity of Ethnomedicinally Important Analgesic Plant Isodon rugosus Wall. ex Benth: Mechanistic Study and Identifications of Bioactive Compounds. Front Pharmacol. 2016;7:200.

    Article  PubMed  PubMed Central  Google Scholar 

  38. Komiya T, Kyohkon M, Ohwaki S, Eto J, Katsuzaki H, Imai K, Kataoka T, Yoshioka K, Ishii Y, Hibasami H. Phytol induces programmed cell death in human lymphoid leukemia Molt 4B cells. Int J Mol Med. 1999;4(4):377–457.

    CAS  PubMed  Google Scholar 

  39. Flescher E. Jasmonates–a new family of anti-cancer agents. Anti-Cancer Drugs. 2005;16(9):911–6.

    Article  CAS  PubMed  Google Scholar 

  40. Zhang J-Y, Tao L-Y, Liang Y-J, Chen L-m, Mi Y-J, Zheng L-S, Wang F, She Z-G, Lin Y-C, To KK, Fu LW. Anthracenedione derivatives as anticancer agents isolated from secondary metabolites of the mangrove endophytic fungi. Marine Drugs. 2010;8(4):1469–81.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Fang J-Y, Fang C-L, Hong C-T, Chen H-Y, Lin T-Y, Wei H-M. Capsaicin and nonivamide as novel skin permeation enhancers for indomethacin. Eur J Pharm Sci. 2001;12(3):195–203.

    Article  CAS  PubMed  Google Scholar 

  42. Pope LE, Khalil MH, Marcelletti JF, Katz LR, Katz DH. Treatment of hyperproliferative skin disorders with C18 to C20 aliphatic alcohols. In: Google Patents. 2001.

    Google Scholar 

  43. Firestone GL, Sundar SN. Anticancer activities of artemisinin and its bioactive derivatives. Expert Rev Mol Med. 2009;11:e32.

    Article  PubMed  Google Scholar 

  44. Yu W, Jia L, Park SK, Li J, Gopalan A, Simmons‐Menchaca M, Sanders BG, Kline K. Anticancer actions of natural and synthetic vitamin E forms: RRR‐α‐tocopherol blocks the anticancer actions of γ‐tocopherol. Mol Nutr Food Res. 2009;53(12):1573–81.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Baldioli M, Servili M, Perretti G, Montedoro G. Antioxidant activity of tocopherols and phenolic compounds of virgin olive oil. J Am Oil Chem Soc. 1996;73(11):1589–93.

    Article  CAS  Google Scholar 

  46. Salvador JA, Carvalho JF, Neves MA, Silvestre SM, Leitão AJ, Silva MMC, Sá e Melo MLS. Anticancer steroids: linking natural and semi-synthetic compounds. Nat Prod Rep. 2013;30(2):324–74.

    Article  CAS  PubMed  Google Scholar 

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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.

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The authors declare that they have no competing interests.

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Authors and Affiliations

  1. Department of Pharmacy, University of Malakand, Dir (L), Chakdara, 18000, KPK, Pakistan

    Sajjad Ahmad, Farhat Ullah, Anwar Zeb, Muhammad Ayaz & Abdul Sadiq

  2. Department of Pharmacy, Kohat University of Science & Technology, 26000, Kohat, KPK, Pakistan

    Farman Ullah

Authors
  1. Sajjad Ahmad
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  2. Farhat Ullah
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  3. Anwar Zeb
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  4. Muhammad Ayaz
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  5. Farman Ullah
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  6. Abdul Sadiq
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Correspondence to Abdul Sadiq.

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Ahmad, S., Ullah, F., Zeb, A. et al. 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. BMC Complement Altern Med 16, 308 (2016). https://doi.org/10.1186/s12906-016-1302-y

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

Keywords

BMC Complementary Medicine and Therapies

ISSN: 2662-7671

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