Cytotoxic activity of the dichloromethane fraction from Vernonia scorpioides ( Lam . ) Pers . ( Asteraceae ) against Ehrlich ’ s tumor cells in mice

Vernonia scorpioides has been widely used in Brazil to treat skin problems and chronic wounds, such as ulcers of the lower limbs and diabetic lesions. In the present study, we investigated the effect of a dichloromethane (DCM) fraction of V. scorpioides leaf extract on Ehrlich ascitic and solid tumor-bearing mice. The animals were treated once a day with the DCM fraction at a concentration of 5 mg/kg, administered ip during and after the development of the tumor. The lifespan, weight, number and type of leukocytes, number of tumor cells, volume of solid and ascitic tumors were measured. The development of the tumor with pre-treated tumor cells in vitro with the DCM fraction (5 mg/kg) was analyzed and the animals were sacrificed after 7 days. The DCM fraction (5 mg/kg) totally inhibited tumor development when in direct contact with tumor cells, and also ascitic tumor development with in vitro treatment or when administered ip, in loco (after 7 days). Animals treated with the DCM fraction increased their lifespan ca. 2 weeks and maintained their body weight for 30 days. When applied immediately after the inoculation of the tumor cells in vivo, it totally abolished tumor development, with tumor development only decreasing when treatment was started 3 days after the tumor challenge. These data suggest an antineoplastic activity of the fraction. Oral or ip administration of DCM fraction (5 mg/kg) for 7 days did not reduce the solid tumor volume. The cytotoxic activity described here differs from the conventional immune suppressing profile of standard chemotherapy because it increases neutrophil influx to the peritoneal cavity. These results show that, besides exhibiting a tumoricidal activity, the DCM fraction also exhibits inflammatory activity. Correspondence


Introduction
Vernonia scorpioides (Lam.)Pers., Asteraceae, popularly known as piracá, enxuga or erva-de-São-Simão in Portuguese, is very common in Brazil, and usually grows in poor and deforested neotropical soils (1).The topical application of the alcoholic extract of its fresh leaves is widely used to treat a variety of skin disorders, including chronic wounds such as ulcers of the lower limbs.Previous studies of the V. scorpioides crude extract and its derived chloroform and hexane fractions have shown fungicidal activity (2), moderate bactericidal activity and mild wound healing effects (3).
In a previous study by our group (3), a pro-inflammatory profile was observed in healthy skin tissue, which prompted an investigation of effect of the crude extract on Ehrlich ascitic and solid tumors in mice.The promising antitumoral result preliminarily obtained with the crude leaf extract led us to partition it in order to obtain fractions of increasing polarity, namely: hexane, dichloromethane (DCM), ethyl acetate and aqueous fractions, tested at 200 mg/kg.Of these, the DCM fraction showed the highest cytotoxicity against tumor cells, especially when applied in loco to the developing tumor.
In the present study, we investigated the effect of the DCM fraction obtained after liquid-liquid partition of the crude hydroalcoholic extract of the V. scorpioides leaf on Ehrlich ascitic and solid tumor-bearing mice.The animals were treated with the DCM fraction at a concentration of 5 mg/kg once a day, administered intraperitoneally (ip) during and after tumor development.The lifes-pan and weight (30 days), number and type of leukocytes, number of tumor cells, volume of the solid and ascitic tumors, and the development of the tumor with tumoral cells pre-treated in vitro with the DCM fraction were analyzed after 7 days of treatment.

Extract, fractions and sample preparation
Flowers and fresh leaves (600 g) of the plant were macerated with 6000 mL ethanol for 7 days, in the absence of light, and the extract obtained was reduced to 1/6 of the initial volume, under vacuum, using a rotary evaporator (Quimis, São Paulo, SP, Brazil).Water (600 mL) was added to the crude extract obtained and the extract was submitted to liquid-liquid fractionation using solvents of increasing polarity.The fractions obtained were denoted: hexane (1.16 g), DCM (420 mg), ethyl acetate (560 mg), and water.After screening with all fractions, DCM showed high activity against the tumor cells (no tumor development was observed) and was selected for further investigation.An aliquot of the dry fractions was dissolved in saline using up to 2% Tween 80 and an ultrasonic bath (10 min) to help dissolution at the following concentrations: 200, 100, 50, 30, 15, and 5 mg/kg in a maximum final volume of 200 µL.
The prepared aliquoted samples were frozen until the day of application.
The DCM fraction is a dark-green semisolid material, its NMR spectra were recorded with a Bruker Avance 400 spectrometer (Rheinstetten, Germany).

Animals
Adult male inbred Swiss mice aged 8 weeks, maintained under standard environmental conditions, were used.The animals were fed a standard diet and received water ad libitum.The animals were used after an acclimatization period of 7 days, and the experiments were conducted in accordance with the Univali Ethics Committee for the treatment of laboratory animals.

Ehrlich tumor
Ehrlich ascitic tumor (EAT) derived from a spontaneous murine mammary adenocarcinoma was maintained in the ascitic form by passages in syngenic Swiss mice by weekly transplantation of 5 x 10 6 tumor cells (ip).The ascitic fluid was removed by opening the belly and collecting all the fluid with a sterile syringe.Ascitic tumor cell counts were carried out in a Neubauer hemocytometer using the Trypan blue dye exclusion method.The animals used for the experiment received 200 µL of a suspension containing 5 x 10 6 tumor cells ip as described in Ref. 21.

Animal treatment
After ip implantation of tumor cells, seven groups of mice (6 mice per group) were treated with different concentrations of the DCM fraction (200, 100, 50, 30, 15, and 5 mg/kg, ip), the positive control group received 5-fluoro-uracil (5-FU) in 0.9% saline solution (20 mg/kg, ip) (22) and the negative control group received saline solution and 2% Tween ip in the same final volume.The body weights of the mice were measured daily until their death.The only animals to survive more than 30 days, those who received 5 mg/kg, were sacrificed 30 days after the beginning of the treatment.

Effect of the DCM fraction (5 mg/kg) on the ascitic tumor
Seven groups of 6 mice each were used.Treatment was started immediately after inoculation of the tumor cells.Group 1 received 5 mg/kg DCM fraction, ip, group 2 received 20 mg/kg 5-FU, ip, group 3 re-ceived 0.9% saline, ip, group 4 received 5 mg/kg DCM fraction by gavage, and group 5 received 0.9% saline by gavage.Group 6 started to be treated with the DCM fraction, ip, 3 days after inoculation of the Ehrlich ascitic tumor cells, and group 7 started to receive 0.9% saline, ip, 3 days after implantation of the tumor cells.After 7 days, the mice were sacrificed and all the ascitic fluid was harvested for volume measurement and ascitic tumor cell count by the Trypan blue dye exclusion method.

Determination of the cytotoxicity of the DCM fraction (5 mg/kg) on Ehrlich ascitic tumor cells in vitro
EAT cells (5 x 10 6 ) were pre-treated in vitro with the DCM fraction for 15 min, and injected into the abdominal cavities of 6 mice (group 1).Group 2 received 5 x 10 6 EAT cells, ip, without pre-treatment.After 7 days, the mice were sacrificed and all the ascitic fluid was harvested for volume measurement and ascitic tumor cell count by the Trypan blue dye exclusion method.

Determination of the effect of the DCM fraction (5 mg/kg) on solid tumor development
Solid tumors were induced by injecting EAT cells, 5 x 10 6 /animal in 50 µL subcutaneously into the hind paw of 5 groups (6 mice/group) of Swiss albino mice.Group 1 was treated with the DCM fraction (5 mg kg -1 animal -1 ) ip and group 2 was treated with the same dose of the DCM fraction by gavage.Group 3 received 5 x 10 6 EAT cells per animal pre-treated with DCM and did not receive any further treatment.Group 4 received 0.9% saline ip, and group 5 received 0.9% saline by gavage.The animals were treated with the DCM fraction daily for 10 days, with the exception of group 3. Footpad volume was measured using a digital pachymeter.All treatments were daily.

Analysis of peritoneal leukocytes
Six tumor-bearing mice per group were treated with 5 mg/kg of the DCM fraction, ip, the positive control group received 20 mg/kg 5-FU, ip, and the negative control group received 0.9% saline, ip.After 30 days, the leukocytes were harvested from the peritoneal cavity with 5 mL saline.The peritoneal fluid was centrifuged, re-suspended in 200 µL saline, distributed (50 µL) on 24-well flat-bottom plates containing round glass coverslips (15 mm) for 1 h at room temperature, processed histologically, and stained with Harry's hematoxylin for microscopic observation.A single blind microscopic evaluation of five fields per coverslip (N = 6) was performed using a reticule eyepiece with 100 quadrants.Based on nuclear shape, neutrophils, monocytes/macrophages and lymphocytes were counted by light microscopy at 100X magnification and the means for the five fields analyzed were calculated.

Morphological analysis of the spleen
The spleens from animals treated ip for 30 days with the DCM fraction (N = 4), from tumor-bearing animals after 7 days of survival, and from normal mice were removed and processed histologically.Sections were stained with hematoxylin-eosin for microscopic observation.

Statistical analysis
Data are reported as means ± SD and were analyzed statistically by the Dunnett test and the F-test.The level of significance was set at P ≤ 0.05.

Evaluation of the lifespan and body weight of the animals after treatment with different concentrations of the DCM fraction
Animals inoculated ip with 5 x 10 6 EAT cells were divided into 5 treated groups (N = 6) receiving different concentrations of the DCM fraction (5, 30, 50, 100, 200 mg/kg), negative control group (saline solution and 2% Tween) and a positive control group treated ip with 20 mg/kg 5-FU (22).As shown in Figure 1 (longevity of the animals), the mice treated with the highest concentration (200 mg/kg) survived for only 12 h, with longevity increasing proportionally with the subsequent decreasing concentrations.The animals that received 5 mg/kg of the DCM fraction survived the treatment and were sacrificed after 30 days.When the animals which had received higher doses were sacrificed, no tumor cells or ascitic fluid could be found in the abdominal cavities.Treatment with a 5 mg/kg dose of the DCM fraction caused a smaller loss of body weight during the 30-day period.A similar profile was observed for treatment with 5-FU.Conversely, the body weight of the mice treated with saline increased, and higher concentrations of the DCM fraction induced loss of body weight.For these groups (control and 15, 30, and 50 mg/kg DCM fraction), the difference between the initial and final body weights was significant (P < 0.05; data not shown).

Antitumor activity of the DCM fraction immediately and after implantation of ascitic tumor cells
Figure 2A shows the effect of ip administration of the DCM fraction against tumor cells, and ascitic volume when treatment was initiated immediately after tumor cell implantation.The result observed was the suppression of 100% of the tumor cells and ascitic volume when the animals were treated immediately after the inoculation of EAT cells, and around 80% tumor cells and 60% (P < 0.001) ascitic volume, when ip treatment was started 3 days after tumor development (Figure 2B).The control groups treated with saline showed a larger volume of tumor and tumoral cells.

In vitro cytotoxicity
When the EAT cells were pre-treated in vitro with 5 mg/kg DCM fraction for 15 min and injected into the abdominal cavity, there was no tumor development.This suggests that this fraction had a direct cytotoxic activity against EAT cells.The relative number of tumor cells and the ascitic volume were determined following daily oral and intraperitoneal (ip) administration once a day for 7 consecutive days of 5 mg/kg of the dichloromethane (DCM) fraction of the Vernonia scorpioides leaf extract, the standard reference drug 5-fluoro-uracil (5-FU, 20 mg/kg), and 0.9% saline (control) immediately after inoculation of 5 x 10 6 Ehrlich ascitic tumor cells.The mean values obtained from one experiment (N = 6 in each group) are presented.No significant difference in cell number was noted between the mice treated by gavage and the control group, as determined by analysis of variance using the F-test (P ≤ 0.05).B, Effect of ip treatment with the DCM fraction after ascitic tumor challenge.Treatment was applied daily starting 3 days after the tumor challenge.The control group received 0.9% saline.The relative number of tumor cells and ascitic volume were calculated.The mean values obtained from one experiment (N = 6 in each group) are presented.A significant difference in number of cells was noted between the mice treated with the DCM fraction and the control group, as determined by analysis of variance using the F-test (P ≤ 0.05).
rect effect of DCM, probably due to the fact that the active compounds did not reach the tumor.Footpad thickness was measured for a period of 10 days and was not reduced when the DCM fraction was inoculated immediately or 3 days after the injection of tumor cells into the animals' paws compared to control.When the tumor cells were pretreated with the DCM fraction before implantation into the animals' footpad there was no solid tumor development.

Analysis of peritoneal leukocytes
Figure 4 shows the number of polymorphonuclear leukocytes present in the peritoneal cavity of EAT-bearing mice treated for 30 days with the DCM fraction and the standard reference drug 5-FU, compared with the number of cells present in the peritoneal cavity of normal mice treated with 0.9% saline.A significant difference was observed between the group treated with 5 mg/kg DCM fraction, the group treated with 5-FU, and normal mice as determined by analysis of variance via the F-test (P ≤ 0.05).This profile is very different from the expected one, as shown by the lymphocyte count.The EAT-bearing mice treated with 0.9% saline died after 10 days.
The data in Figure 4 confirm the increased influx of leukocytes to the peritoneal cavity and illustrate the profile of inflammatory cells in each group.It was possible to compare the different types of leukocytes.Figure 4A shows the peritoneal cells, predominantly macrophages, from tumorbearing mice treated with the DCM fraction for 30 days, Figure 4B shows a reduced number of leukocytes from tumor-bearing animals treated with 5-FU, and Figure 4C shows the results for control animals.

Morphological analysis of the spleen
Significant increases in spleen size were observed in the mice treated with the DCM Figure 3.Effect of the dichloromethane (DCM) fraction of Vernonia scorpioides on solid tumor induced by Ehrlich's ascitic tumor (EAT) cells in mice.Tumor growth on the footpad was determined daily for 7 days following ip administration of the DCM fraction and 0.9% saline (control) immediately and 3 days after the tumor challenge.EAT cells pre-treated in vitro with the DCM fraction were also used for solid tumor induction.The mean values obtained from one experiment (N = 6 in each group) are presented.No significant difference was noted between the treated groups and the control group, as determined by analysis of variance via the F-test (P ≤ 0.05).In vitro pre-treatment of the tumor cells with the DCM fraction inhibited tumor growth and after 3 days the solid tumor volume was significantly lower (P ≤ 0.05, Dunnett test) than that of the saline-treated control.

Effect of DCM fraction on solid Ehrlich ascitic tumor development
Figure 3 shows the development of a solid tumor in the footpad after ip treatment with DCM.This fraction did not reduce the solid tumor volume when systemically inoculated, indicating that there was no indi-fraction compared to normal mice and mice that only received tumor cells.Histological examination of the spleens of mice treated with the DCM fraction demonstrated a significant increase in all white pulp areas, with an increased number of cells in the germinal center and lymphatic vessels.

Discussion
Plant-derived products such as vincristine, vinblastine, taxol, and many other substances in use today are excellent sources for the development of new anticancer chemotherapies.The Vernonia species belongs to the Asteraceae genus, which produces characteristic compounds such as sesquiterpene lactones with several biological properties, including anti-tumoral properties (23).In recent years, a significant number of papers have described the influence of these compounds on the growth of tumors.For example, parthenolide has been considered to be a cytotoxic or cytostatic drug and has been proposed as a complementary therapeutic drug in cancers with constitutively active NF-κB, which mediates the expression of pro-metastatic, pro-angiogenic, antiapoptotic, and multi-drug resistance genes (24).
Several studies focusing on the immunomodulatory effect of plant extracts have been conducted, showing a prominent role of extracts causing prophylactic growth inhibition of tumor cells (25,26).Among the Vernonia species, some cytotoxicity has been described for isolated sesquiterpene lactones from V. cinerea (15), V. lasiopus (16) and V. amygdalina (27).Some active Vernonia extracts have also been found, such as the water-soluble extract of edible V. amygdalina leaves (9), and the water extract from the roots of V. kotschyana (20) and V. anthelmintica (10).
V. scorpioides has been studied in different biological tests and has shown cytotoxic effects in most of them.Previous studies have shown that the DCM and hexane fractions from the extract have fungicidal and bactericidal properties (2).Also, many Vernonia species are used as trypanocidal (28), antiplasmodial (29) and anthelmintic agents (30).
The results presented here confirm the toxic properties of the DCM fraction, a darkgreen semi-solid material rich in pigments that was characterized by infrared spectroscopy, showing strong absorption at ca. 1725 cm -1 , characteristic of the presence of lactones.The NMR spectra showed the presence of acetyl and carbonyl radicals as indicated by intense signals at 168-170 and 20 ppm.Also some hydroxyl/methoxyl derivatives (68 and 55 ppm, intense peaks) and double bonds (124-128 ppm) are probably present.Acetylated sesquiterpene lactones are commonly found in Vernonia species (15).
The DCM fraction totally inhibited tumor development when in direct contact with tumor cells, as also demonstrated by in vitro ip administration, in loco, to the ascitic tumor.Inoculation of the DCM fraction (5 mg/kg) increased the animals' lifespan and maintained their body weight throughout the 30 days of treatment.When applied immediately after inoculation of the tumor cells in vivo, it totally abolished tumor development, and when treatment was started 3 days after the tumor challenge, tumor development was decreased, supporting the existence of a probable antineoplastic activity.
Treatment by the oral or ip routes did not reduce the solid tumor volume, suggesting that some enzymatic routes can deactivate the systemic route.Another property that may exacerbate this cytotoxic activity is the ability of the fraction to increase neutrophil influx into the peritoneal cavity, unlike the conventional immune-suppressing profile of standard chemotherapy.When the tumor developed in the ascitic form, daily inoculation of the DCM fraction led to an increase in peritoneal leukocytes, and the tumor did not grow, with an increase in the animals' lifespan and maintenance of their body weight during the 30 days of treatment.These results show that, in addition to tumoricidal effects, the DCM fraction also has inflammatory activity.Sur et al. (31) showed that the seeds of Trigonella foenum graecum (Fenugreek) inhibited tumor growth by about 70% and also increased the influx of inflammatory cells to the peritoneal cavity.The present results have advanced our understanding of the antitumoral potential of the DCM fraction V. scorpioides.Investigations into the mechanism of action of the tumorreducing activity and also of the compounds responsible for the activity of the DCM fraction are currently in progress.

Figure 2 .
Figure2.A, In vivo tumor cytotoxicity assay.The relative number of tumor cells and the ascitic volume were determined following daily oral and intraperitoneal (ip) administration once a day for 7 consecutive days of 5 mg/kg of the dichloromethane (DCM) fraction of the Vernonia scorpioides leaf extract, the standard reference drug 5-fluoro-uracil (5-FU, 20 mg/kg), and 0.9% saline (control) immediately after inoculation of 5 x 10 6 Ehrlich ascitic tumor cells.The mean values obtained from one experiment (N = 6 in each group) are presented.No significant difference in cell number was noted between the mice treated by gavage and the control group, as determined by analysis of variance using the F-test (P ≤ 0.05).B, Effect of ip treatment with the DCM fraction after ascitic tumor challenge.Treatment was applied daily starting 3 days after the tumor challenge.The control group received 0.9% saline.The relative number of tumor cells and ascitic volume were calculated.The mean values obtained from one experiment (N = 6 in each group) are presented.A significant difference in number of cells was noted between the mice treated with the DCM fraction and the control group, as determined by analysis of variance using the F-test (P ≤ 0.05).