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    Leukemia cancer comprises about 8% of all cancers and the fifth most common cause of cancer death in the world. This Cancer is a disease of children, because about 15% of children under 15 who are suffering from this disease. Most of the researches are undergoing herbal drugs to replace them with conventional treatments of various types of cancer and other diseases [1].   Herbal medicines are widely used in all areas of the developing world. Unlike the heavy use, there is too little information about the health and efficacy of treatment by these plants. It has been shown that some medicinal plants have anti-cancer activity. However, Lack of scientific evidence in connection with their mechanism of action reduced their clinical applications [2]. polygonum aviculare is a member of the Polygonaceae family. This plant has spread in Australia, Europe, South Africa, the Mediterranean, North Africa, the Middle East, and India. This plant has astringent properties and is used for the treatment of diarrhea, stop bleeding and healing it. This plant is a diuretic and used for the treatment of urinary retention occurs. In addition, it is used for the treatment of kidney stones [3]. It has been proven that the extract of this plant has large amounts of antioxidants activity. According to this fact that cancer is linked with large amounts of free radicals, we can expect its positive effects on cancer controlling [4]. On the other Research, other types of flavonoids extracted from this plant [5]. Another research was confirmed the anti-tumor effect of Rsvratvl extracted from other species of this family [6]. Some herbal products such as phenolics and flavonoids that are found in Polygonum aviculare have multiple biological effects including antioxidant and anticancer activities with the capacity to scavenge free radicals. These compounds also have shown DNA protective activities [7]. In this paper, the cytotoxic effects of Polygonum aviculare extract have been investigated on all-6 (pre-B-cell leukaemia) cancer cell line.
    2.Materials and methods 
    2.1.Herbal extraction preparation

    polygonum aviculare  was collected from Tehran provinces of Iran in summer of and winter of the year 2012 and was identified and confirmed by the expert. The extraction method was percolation .Aerial parts of this plant were dried at room temperature for 2 weeks and subjected to extraction away from sunlight in the laboratory .methanolic extract of Aerial parts of polygonum aviculare was  prepared separately for two phase (vegetative and generative phase of plant ). Extraction procedure was performed 3 times for 48 hours.
  2.2. Cell culture 
The nalm-6 (pre B-cell leukaemia) cell line was obtained from national cell bank of Iran (NCBI,Pasteur Institute Iran). The cells  were cultured in RPMI-1640 With 10% FBS and 1% Penstrep in 5% CO2 at 37ºC for 2 week and then were treated with herbal extract.

    2.3.Assessment of cell viability 
    The nalm-6 (pre B-cell leukaemia) cells were incubated in presence or absence of the different concentrations ( 50,100,250,500,1000 µg/ml) on two phase (vegetative and generative phase)polygonum aviculare extract for 48 hrs.Following incubation, the cells were harvested and counted with a hemocytometer under phase contrast microscope. The viability of the cells was examined by trypan blue dye staining [6].

    2.4. Evaluation of cytotoxicy using MTT Assay 
    Polygonum aviculare methanolic extract in different concentrations was dissolved in dimethysulfoxide (DMSO) then was used for trating on pre B-cell leukemia cancer cells. At this study extract, effect on cell growth was checked. In addition, cell in culture medium RPMI-1640/DMSO was used as a control. Briefly, 5000 cell/well were cultivated in a 96 well plate. After incubation, cells were treated with different concentrations of methanolic extract of p.aviculare. After incubation, the medium of all wells plate was exchanged with fresh medium and cells were left for 24 hrs in incubator. Then, the medium of all wells was removed carefully and 20 µl of 5µg/ml MTT (Sigma Co, Germany) was added to each well and incubated for three hrs. After removing of well content, 100 µl DSMO was added to wells. Immediately ELISA reader read absorbance of each well in 570 nm using 630 nm as reference wavelength.
    3.Results and Discussion 
    In this study, the effect of polygonum aviculare extract on Nalm_6 cells cancer was demonstrated. The methanolic extracts of the plant selected on cell viability, Nalm_6 cells were incubated with different plant extract concentrations. After 48 hrs, cell viability was determined with MTT. The IC50 was determined for the plant extracts showing cytotoxic activity in vegetative phase (IC50 = 351 µg/ml) and generative phase (IC50 = 253/25 µg/ml) [Figure 1] [Figure 2]. Comparison of concentrations extract  In both of the phase show that the effect of generative phase was more than vegetative phase but There were no significant differences between  them (P>0.05); [Figure 3]. P. aviculare methanolic extracts inhibited growth Nalm_6 cells cell line in a concentration-dependent manner. There were different inhibitions produced by different concentrations of p.aviculare at 48-hrs incubation in every phase separately. Inhibition levels provided by p.aviculare at 1,000 μg/mL were significantly greater than other concentrations after 48-hours incubation (P < 0.01  (for both of the phase. Our results suggest that extract of p. aviculare has cytotoxic effects and may therefore be developed as anticancer agents at 1,000 μg/mL offering the most potential extract (based on IC50 values). Plants used in traditional medicine are often researched, for new anticancer compounds [8]. There is a great need to examine reliable and inexhaustible sources of natural substances. In addition, it is important to find the mechanisms of antitumor agents for future application in cancer therapy [9]. The data presented in this study revealed that Polygonum aviculare extract has strong and important cytotoxic effects on Nalm_6 cells. Some of compounds such as phenolics that are found in Polygonum aviculare at high amounts apply various pharmaceutical effects including antioxidant and anticancer activities [10]. In the present study, total extract used instead of pure components of P. aviculare extract. Because, there is no study about the pure component. According to Chen and Shi report, extraction of pure P.aviculare component is time consumable and needs to HPLC [11]. In the current work, MTT assay showed that methanolic extract of P. aviculare has dose dependent cytotoxicity on the nalm_6 cancer cell line in vegetative and generative phase (IC50 =  351 µg/ml   in vegetative phase and IC50 = 253/25 µg/ml in generative phase ). This finding is in accordance with the results of Rahmati et al in which P.aviculare  on Hela-S cervical cancer cell line and Manoharan et al., in which Polygonum bistorta (Polygonaceae) was evaluated for their cytotoxic activity against P338 (Murine lymphocytic leukemia) cancer cell lins in culture. [12, 13].As regards There were no significant differences in results between vegetative and generative phase , it can be concluded the phases of growth in this plant have no efficacy on cytotoxic effects and  the current study is the first one on Nalm_6 cervical cancer cell line. Organic  compounds such as phenolics that are found in Polygonum aviculare is at high amounts exert various biological effects including antioxidant and anti-tumor activities and have a wide range of medicinal properties [14]. It seems the P.- aviculare might prevent or inhibit nalm_6 cancer  cell line because of its scavenger capability. This finding is in accordance with the results of Habibi RM in which P.- aviculare  on MCF-7 breast cancer [7]. There is no study with P.aviculare about cytotoxicity effects on on nalm_6 cancer cell line and the current study is the first one on it. Therefore, there is a need for further study of P.aviculare extraction constituents in the future. On the other hand, results of another study showed that Polygonum hydropiper L. has cytotoxic compounds soluble in both water and ethanol. Therefore, we consider necessary to further research, both to determine the toxicity of its extracts on genetic material in the event of pro longed administration of infusions or other extracts, and because of it is the- rapeutic potential in antitumor therapy [15]. In summary, the results of the current study showed that methanolic extract of P. aviculare has cytotoxic effect on nalm_6 cancer cell line. The cytotxicity effects on nalm_6 cancer cell line by methanolic extract of P.aviculare are presented for the first time and even any study has not been reported to date. However, similar works on other cell lines or other Polygonum extractions have been performed [16, 17, 18].
    Figure 1.The cytotoxic effects of Different concentrations of polygonum aviculare L. extracts ( 50,100,250,500,1000 μg/mL) on the NALM_6 cell cancer compared to control  in vegetative phase .Maximum inhibition of proliferation was achieved after 48 h at the highest concentration (1000 μg/mL). ( P<0.01**)
    Figure 2.The cytotoxic effects of Different concentrations of polygonum aviculare L. extracts ( 50,100,250,500,1000 μg/mL) on the NALM_6 cell cancer compared to control  in generative phase .Maximum inhibition of proliferation was achieved after 48 h at the highest concentration (1000 μg/mL). (P<0.01**)
    Figure 1.The comparison of cytotoxic effects of  polygonum aviculare L. extracts on the NALM_6 cell line cancer on vegetative and generative phase . There were no significant differences between them (P>0.05).

    The results of these studies have showed that Polygonum extract has cytotoxic effects NALM_6. Therefore, it can have potentially cytotoxic compounds, especially for exploiting in treatment of this cancer. 

    We would appreciate from Cell Bank, Pasteur Institute of Iran for providing cell lines and would like to thank Ali Reza Mousa Mayali for his assistance. 
    [1].Vickers A.Botanical medicines for the treatment of cancer: rationale, overview of current data, and methodological considerations for phase I and II trials. Cancer Invest.2002;20:1069–1079.
    [2].Cassileth BR, Deng G.Alternative and complementary medicine Cancer. Oncologist.1995; 9:80–89.
    [3].Lazarides M, Cowley K, Hohnen, P. Handbook of Australian Weeds. 7th ed. CSIRO publishing. 1997. p. 264.
    [4].HSU CY. Antioxidant activity of extract from Polygonum aviculare L. Biol Res. 2006; 39: 281-288.
    [5].Kruawan K, Kangsadalampai K. Antioxidant activity, phenolic compound contents and antimutagenic activity of some water extract of herbs. Thai J Pharm Sci. 2006; 30:28-35
    [6].by L Feng.Studies on active substance of anticancer effect in Polygonum cuspidatum.Zhong Yao Cai. 2006;29(7):689-91.
    [7].Habibi Roudkenar M. , Mohammadi Roushandeh A. , Delazar A., Halabian R.,  J. Soleimani Rad,  Mehdipour A.,  Bagheri M.,  Jahanian-Najafabadi A.Effects of polygonum aviculare herbal extract on proliferation and apoptotic gene expression of MCF-7, Daru. 2011; 19(5): 326–331.

    [8].Wu W., Bi X.L., Cao J.Q., Zhang K.Q., Zhao Y.Q. Bioorg. Med. Chem. Lett. 2012.  22, 1895.
    [9].Half, E.; Arber, N. Colon cancer: Preventive agents and the present status of chemoprevention. Exp. Opin. Pharmacother.2009;10: 211–219. 

     [11].Chen J, Shi Y . Determination of quercetin and kaempferol in Polygonum aviculare by HPLC. . Biochem. Res .2009; 34(4):423-427.
    [12].Rahmati M, Bannazadeh H, Fazli D, Tahmasebzadeh F,Farrokhi A and Rasmi YThe apoptotic and cytotoxic effects of Polygonum aviculare extract on Hela-S cervical cancer cell line. African Journal of Biochemistry Research. 2011; 5(14): 373-378.

    [13].Manoharan KP, Yang D, Hsu A, Huat BT. Evaluation of Polygonum bistorta for anticancer potential using selected cancer cell lines. Med. Chem. 2007; 3(2): 121-126.

    [14].Hediat M.H. Salama and Najat Marraiki.Antimicrobial Activity and Phytochemical Analysis of Polygonum Aviculare L.(Polygonaceae), Naturally Growing in Egypt. Australian Journal of Basic and Applied Sciences.2008; 3(3): 2008-2015.


    [16].Zhang RC, Liu B, Sun ZX, Xu DY .Effects of extract of Polygonum multiflorum on cell cycle arrest and apoptosis of human liver cell line L02. Zhong Xi Yi Jie He Xue Bao, 2010; 8(6): 554-561.

    [17].Brandao GC, Kroon EG, Duarte MG, Braga FC, de Souza Filho JD, de Oliveira AB. Antimicrobial, antiviral and cytotoxic activity of extracts and constituents from Polygonum spectabile Mart.Phytomedicine, 2010; 17(12): 926-929.

    [18].Rana P, Agarwal  SR.Prostate cancer chemoprevention by silibinin : bench to bedside. Molecular Carcinogenesis. 2006; 45:436-442.