Experimental immunology
The effect of Echinacea purpurea on the morphology, angiogenic activity and vascular endothelial growth factor (VEGF) concentration of murine L-1 sarcoma tumors

Introduction
Use of Echinacea purpurea (purple coneflower), herb native to the North American prairies, has a long history. North American Indians used this plant internally and topically as antiseptic, anti-inflammatory, and analgesic drug. They used aerial and underground parts of the plant, in the form of infusion, succus or decoction, for curing burns, snake and insect bites, and as general immunoenhancers. Now, this plant is one of the most important world’ medicinal herbs, widely used for the treatment of upper respiratory tract infections [1]. We previously reported that Echinacea purpurea extracts enhance immunity in mice and stimulate various functions of human blood leukocytes in vivo [2-7]. Studies in cancer patients undergoing chemotherapy showed that polysaccharide fraction isolated from Echinacea purpurea may counteract the chemotherapy-induced leukopenia [8].
It was described that Echinacea extracts were able to induce apoptosis of human cancer cell lines by increasing caspase activity and promoting nuclear DNA fragmentation. It was also found that flavonoids and polyphenols, present in Echinacea, inhibit activities of metalloproteinases and some serine proteases important for angiogenesis, and inhibit VEGF receptor phosphorylation [9-12]. We recently reported, that Echinacea purpurea extract diminished neovascular reaction induced in mice skin by human cancer cells [7].
The aim of our present study was to evaluate the effect of feeding mice Echinacea purpurea-containing drugs on 1) neovascular response induced in mice by murine cancer cells 3 days after intracutaneous grafting and, 2) morphology, angiogenic activity and VEGF concentration of murine sarcoma L-1 tumors, 7 days after intracutaneous cells transplantation

Material and Methods
Echinacea purpurea-containing drugs: Immunal forte tablets (LEK, Slovenia) and Echinapur tablets (Herbapol Poznań), 0.6 mg per day.
Animals
Inbred Balb/c mice 8-10 weeks old. Mice have been of local laboratory breed, weighing 20 g each. Animals were handled according to the Polish law on protection of animals and NIH standards. All experiments were accepted by the local Ethical Committee.
Tumor cells
Murine L-1 sarcoma cells from in vitro culture stock were delivered from Warsaw Oncology Center Collection and then passaged through three generations of Balb/c mice as previously described [13].
Cutaneous angiogenesis assay
According to Sidky and Auerbach, with some modifications [13]. Briefly, multiple 0.05 ml samples of 2 × 106 L-1 sarcoma tumor cells were injected intradermally into partly shaved, anesthetized Balb/c mice. In order to facilitate the localization of cell injection sites, the suspension was colored with 0.1% of trypan blue. Next, mice were fed Echinacea preparations in daily dose of 0.6 mg for 3-5 consecutive days. Some mice after 72 hours were sacrificed with lethal dose of Morbital. All newly formed blood vessels were identified and counted in dissection microscope, on the inner skin surface, at magnification of 6×, in 1/3 central area of microscopic field. Identification was based on the fact that new blood vessels, directed to the point of cells injection are thin and/or differ from the background vasculature in their tortuosity and divarications.
The other mice were fed Echinacea drugs for 5 days. At the day 7th mice were sacrificed with lethal dose of Morbital, the tumors were removed and weighted.
Histological evaluation of L-1 sarcoma tumors 7 days after transplantation of cells
Half of excised tumors (6 lesions from each group) was fixed in 10% formalin solution. Next, the specimens were dehydrated in increased concentrations of alcohol and embedded in paraffin. Paraffin tissue block was sectioned on 4 mm thin sections. The specimens were contrasted by hematoxyline and eosine for first screening light microscopic examination.
The second half of removed tumors (6 lesions from each group) was pooled within groups, and frozen at –78°C (suspended in PBS in proportion 100 mg per 1 ml) for later VEGF measuring.
Measurement of VEGF concentration
The tumour samples collected on the day seven after tumor cells grafting were homogenized with an ultrasonic disrupter VirSonic (Virtis) for 2 minutes, at frequency
22.5 KHz. Cytokine levels were determined using standard ELISA R&D kits for mouse VEGF, according to producer instructions. From each material 6 repetitions were established. Optical density was measured at 450 nm using spectrophotometric reader Elx800 (Biotek Instruments, Inc., USA). Cytokine concentration was expressed as pg/ml.
Statistical evaluation of the results
One way analysis of variance ANOVA, Tukey’s Multiple Comparison Test (GraphPad Prism software package).

Results
Echinapur diminished angiogenesis induced by murine L-1 sarcoma cells, as evaluated 3 days after intradermal cells injection. Immunal did not change neovascular reaction (Fig. 1). Both drugs did not reduce tumor mass (Fig. 2) and tumor cells angiogenic activity (Fig. 3), but highly significantly diminished VEGF concentration (Fig. 4) in L1 sarcoma tumor tissue, evaluated 7 days after intradermal cells grafting.
Histologically, there were no changes in diameter of tumors between investigated and control groups. Also, there were no inflammatory infiltrations and areas of necrosis in all groups. In Echinacea-fed groups, however, there were less small blood vessels at a margin of tumors (Figs. 5 and 6).

Discussion
The results of the present study demonstrated that Echinapur, Echinacea purpurea derived drug, diminished neovascular reaction induced in mice skin by syngeneic L-1 sarcoma cells. This effect was not observed when mice were fed for 3 days with other Echinacea purpurea derived drug, Immunal. However, both drugs administered to tumor recipients for 5 days, reduced VEGF concentration in L1 sarcoma tumor tissue, not affecting tumor mass and tumor cells angiogenic activity.
VEGF is one of the most important factors responsible for stimulating angiogenesis during tumor growth. Its production and function may be suppressed by various polyphenolic compounds [14, 15], among them by phenolic acids [16]. It is noteworthy, that Echinacea purpurea extracts contain cichoric, caftaric, chlorogenic and caffeic acids.
7-day old tumors were small and probably we observed the influence of Echinacea on the very early events of their growth - inhibition of VEGF production and lesser number of blood vessels at the tumor margin in Echinacea-fed groups of mice.
Previously we observed angioinhibitory effect of complex herbal remedy (syrup Alchinal), containing Echinacea purpurea, Allium sativum (garlic)and cocoa, as well as its inhibitory effect on sarcoma L-1 growth. However, in that study mice were fed drug for longer time (since –4 to +13 day, in respect to the day of tumor cells grafting). Moreover, the final effect was probably the resultant of action of all remedy compounds, as anti-angiogenic and anti-tumor activities of garlic and cocoa has been described [17].

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