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Central European Journal of Immunology
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vol. 29

The effect of complex herbal remedy on the angiogenic activity of L-1 sarcoma cells, L-1 sarcoma tumour growth, and on the bacterial infection in mice

Janusz Bany
Ewa Skopińska-Różewska
Joanna Chorostowska-Wynimko
Ewa Rogala
Ewa Sommer
Danuta Zdanowska
Małgorzata Filewska
Henryk Skurzak

Centr Eur J Immunol 2004; 29 (1): 29-34
Online publish date: 2004/12/22
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Correspondence: Janusz Bany, Department of Pharmacology and Toxicology, Military Institute of Hygiene and Epidemiology, 4 Kozielska St., PL-01-163 Warsaw, Poland. Tel. +48 22 685 11 25; fax +48 22 610 84 59, e-mail: tox@future-net.pl; ewaskop@hotmail.com

In recent years an increase has been observed in the interest in drugs of natural origin having immunotropic activity. They may be a valuable complementation to treatment of infection, increasing cellular and humoral immunity of the organism in various clinical situations, among them in cancer patients after chemotherapy.
However, these natural substances should not stimulate tumour growth or vasculature development, since angiogenesis is one of the most important factors connected with the development and spreading of malignant tumours [1, 2].
Vascular endothelial growth factor /VEGF/, potent mitogen for endothelial cells is one of the most important cytokines determining tumour development [3]. So far, we have not been able to find any literature reports on the influence of Echinacea and cocoa, and only a few papers reporting the effect of garlic on angiogenesis and endothelial cells proliferation [4, 5].
Cocoa and Echinacea extracts contain polyphenols. Accumulating evidence demonstrates that diet-derived polyphenols (among them tea catechins) may inhibit tumour invasion and angiogenesis [6-8]. Some of these catechins are also present in cocoa.
In people immunocompromised due to severe burns, or immunosuppressive and cancer therapy, opportunistic infections e.g. with Pseudomonas aeruginosa are frequently observed [9-12].
The immunomodulatory and antiseptic action of extracts of some plants such as Echinacea purpurea and garlic (Allium sativum) is well known [13-15]. The immunomodulatory function and antibacterial properties of cocoa have been confirmed [16]. In recent years attention has been paid to Echinacea purpurea, that was found to augment the activity of NK cells in leukaemic mice [17, 18]. It is also known, that garlic has a broad range of beneficial effects, among them, antimicrobial activity [19]. The active ingredients in
A. sativum are also responsible for the observed antitumour activity and immune stimulation [20].
We believe, that it could be of great importance to find new, non-toxic anti-angiogenic composition of agents among substances of plant origin, which simultaneously will exert antibacterial activity.

In the present study the combination was used consisting of three substances (Echinacea purpurea, Allium sativum, cocoa). The preparation partly suppressed tumour development, diminished tumour angiogenesis, and reduced the development of bacterial infection.
Material and methods

Syrup Alchinal (Gemi, Poland), designated by us herbal remedy (HR), containing in 1 ml 7 mg of Echinacea purpurea extract, 30 mg of Allium sativum extract and 50 mg of cocoa, was administered to mice orally (2x30 µl daily), directly to mouth by Eppendorff pipette, for 10 days or longer, depending on the type of experiment, with the exception of some angiogenesis assays, where mice obtained 3x30 µl daily, for 3 days. For eliminating eventual influence of mice handling, all control mice received water 2x30 ul or 3x30 ul daily, directly to mouth by Eppendorff pipette.
These daily doses, after calculation, taking into account differences in body area to body mass ratios between man and mouse, corresponded to 30 and 45 ml of Alchinal administered to a man weighting about 70 kg.
HPLC analysis of cocoa revealed the presence of catechin (1 mg per 1 g) and epigallocatechin (1.6 mg per 1 g).
Estimation of the tumour growth and angiogenesis assays were performed on F1 hybrids of inbred strains BALB/c and C3H mice, of both sexes, at the age of 10-14 weeks. The tests with P. aeruginosa were performed on inbred C57BL/6 mice, at the age of 10-12 weeks. All experiments were accepted by the local Ethical Committee.
Sarcoma growth
L-1 Sarcoma cells were delivered from Warsaw’s Oncology Center Bank and then passaged through 4 generations of locally bred BALB/c mice. Briefly, tumour cells were grafted (1x106/0,1 ml) into mice subcutaneously, into sub-scapular region. Mice were fed HR or water for 4 days before and 13 days after L-1 sarcoma cells grafting. Starting day 6-th after cell transplantation tumours were measured with electronic calipers. After 14 days tumors were removed, weighted and prepared from them cell suspensions were intradermally grafted to recipient mice (angiogenesis assay), or suspended in PBS (107 cells per ml), homogenized (Virsonic, USA) and frozen at – 750C for later VEGF examination..
Cutaneous angiogenesis test
Tumors were cut for smaller pieces, rubbed through stainless sieve and suspended in 5 ml of phosphate buffered saline (PBS). The suspension was left for 10 min in room temperature. After sedimentation the supernatant was collected and centrifuged for 10 min, 1400 rpm. Obtained tumor cells were washed once in PBS for 10 min, 1500 rpm. Cells were resuspended in Parker medium in concentration of 4x106/ml.
Cutaneous angiogenesis assay was performed according to Sidky and Auerbach [21] method with own modifications Skopińska-Różewska et al. [22]. Multiple samples of two hundred thousands sarcoma cells have been implanted intradermally (while suspended in 0.05 ml of Parker medium) into regionally shaved, anaesthetized (3.6% chloral hydrate) BALB/c mice. In order to facilitate the localization of cells injection sites later on, the suspension was coloured with 0.1% of trypan blue. On the day of cells grafting and on the following two days mice were fed HR, or water (as control, or in experiments where tumor cells were isolated from animals fed HR for 17 days before testing). 72 hours after cells grafting mice were killed with lethal dose of Morbital. All newly formed blood vessels on the inner skin surface were identified and counted in dissection microscope using criteria suggested by the authors of the method (small size, tortuosity and divarications).
Measurement of VEGF concentration
Cytokine levels were determined in homogenates of L-1 sarcoma cell suspensions using ELISA kit (R&D) according to producers instruction. Optical density was measured at 450 nm. VEGF concentrations were expressed as pg/ml.
Bacterial infection
Mice were fed HR (2x30 µl daily) or water (control) for 10 days and on the day 11-th were infected intraperitoneally (i.p.) with Pseudomonas aeruginosa strain ATCC (27853). Four hours after administration of 0.1 ml of bacteria suspension (3x107 CFU) the mice were anaesthetised with barbiturates and killed by spinal dislocation after which the livers were isolated. The livers were homogenised and the number of viable bacteria were estimated by plating.
Statistical methods
Mann-Whitney and Student’s tests were used for statistical analysis and the data with P values less than 0.05 were considered significant.
Tumour growth

The effect of herbal remedy on L-1 sarcoma growth was presented in Table 1.
HR was administered to mice from 4th day before, until 13th day after grafting of tumour cells. Estimation of tumours volumes was accomplished on the days: 6, 7, 8, 10 and 13 after tumour cells grafting. Starting from day 7, mean volumes of tumours from HR – fed group were significantly lower (p<0.05) than in the control, water-fed animals. Angiogenic activity of cells isolated from tumours of HR-fed group, measured by skin test, was significantly lower than that of the controls (Fig. 1). However, VEGF concentration was significantly higher in tumour cell homogenates derived from HR-fed mice than in tumours from untreated mice (Table 2).
Cutaneous angiogenesis assay
When HR was fed to mice for 3 days after sarcoma cells intradermal grafting, diminishion of neovascular response was observed in comparison to the control group (Table 3).
Bacterial infection
A significantly decreased number of bacteria in P. aeruginosa – infected mice after administration of HR as compared with the control group was demonstrated (Table 4).
The studied complex remedy caused inhibition of L1 sarcoma development and tumour angiogenesis in spite of increased VEGF concentration in the tumour cells. The mechanism of this phenomena is not clear. However, it might be connected with the ability of catechins to interfere with VEGF binding to its receptors, such phenomenon was described by Kondo et al [23]. As a result, at the presence of undisturbed VEGF production, accumulation of this cytokine might arise. We observed similar phenomenon in embryos of pregnant mice fed chocolate – lower angiogenic activity and higher VEGF concentration than in controls [24]. In fact, HPLC analysis of cocoa, one of the compounds of our herbal remedy, revealed the presence of substantial amounts of catechin and epigallocatechin.
Khanna et al [25], in different experimental system reported increased VEGF expression in cultures of keratinocytes in the presence of polyphenolic bioflavonoids, proanthocyanidins.
Polyphenols are known to inhibit a wide variety of enzymatic activities associated with cell activation, cell proliferation and tumour progression. Some of these effects are mediated by blocking of VEGF binding to its receptors, inhibition of tyrosine kinase activity and VEGF receptor phosphorylation [26, 27]. It was also reported that tea polyphenols, among them epigallocatechin (also present in cocoa) and flavonoids (present in Echinacea extract) inhibit activities of important for angiogenesis enzymes, metalloproteinases and some serine proteases, in particular, the urokinase-type plasminogen activator -plasmin system [28, 29].
As mentioned above, we have not found in the literature papers about inhibitory action of cocoa or chocolate on angiogenesis, however, there are reports about such effect exerted by catechins present in green tea. Some of these catechins are common for tea and cocoa – for example catechin and epigallocatechin. Demeule et al [8] reported that green tea catechins, in addition to their antioxidative properties, also affect the molecular mechanisms involved in angiogenesis, extracellular matrix degradation, regulation of cell death and multidrug resistance.
Drinking green tea and its polyphenols significantly prevented corneal neovascularization induced by VEGF (30) and also inhibited angiogenesis in other standard animal angiogenesis models [6, 7].
Carnesecchi et al [31] have found, that flavanols and procyanidins of cocoa and chocolate inhibit growth and polyamine biosynthesis of human colonic cancer cells. Kozikowski et al [32] also described inhibition of cancer cell growth by epicatechin-derived procyanidins, through cell cycle arrest.
Regarding the third component of our remedy, garlic (Allium sativum), anti-angiogenic and antitumour activities of its compounds has been described. Shukla et al [4] reported inhibitory effect of diallyl sulfide on angiogenesis in Ehrlich ascites tumour-bearing mice. Thioallyl compounds were described as potent inhibitors of endothelial and tumour cells proliferation [5]. In the study of Tang et al [33] garlic prevented oral precancer induced in rats by chemical carcinogen. The potential application of garlic for the treatment of bladder cancer was discussed by Lamm and Riggs [20]. Diallyl disulfide caused growth retardation of human breast cancer cell lines transplanted to nude mice [34].

The results of the present study also demonstrated that the administered preparation caused inhibition of Pseudomonas aeruginosa growth. Analysing individual components of Alchinal, it can be seen that Echinacea extract contains many compounds with immunomodulatory activity – polysaccharides, alkamides, polyphenols, glycoproteins. Some of these compounds have also anti-inflammatory activity [35, 36]. Other authors [37, 38] reported that polysaccharides from Echinacea purpurea stimulate the activity of phagocytic cells. These authors described stimulatory effect of Echinacea purpurea on non-specific immunity of mice in vivo and in vitro, resulting in protection against systemic infections with Listeria monocytogenes and Candida albicans.
Another component of Alchinal – cocoa, exerted inhibitory effect on the growth of a variety of bacteria (e.g. Shigella, Staphylococci, etc) in various media [16]. Cocoa may cause inhibition of the biosynthesis of polysaccharides by both cell-free and cell – assiociated streptococcal glucosyltransferases [39]. Another study suggested that antioxidant cocoa liquor polyphenols (major component of chocolate) exert immunoregulatory effects [40]. It has been also shown that garlic compounds, diallyl sulphide and diallyl disulphide posses bactericidal effects [19, 41].

In summary it can be said that the preparation used in our study caused modifications of angiogenesis and tumour growth and also reduction of bacterial infection in mice. Therefore, it seems that the substances contained in Alchinal modulate certain parameters of antitumour and antibacterial immunity.
Further studies could help elucidate the mechanism of these phenomena and create an interesting perspective of possible use of Alchinal as tumour angiogenesis inhibitor in the treatment of malignancies (in combination with classic methods) in humans, particularly in patients with impaired immunity. The preparation would also be used for limiting the development of opportunistic infections.

This work was partly supported by scientific program 4/3 of National Institute of Tuberculosis and Lung Diseases and scientific program of Military Institute of Hygiene and Epidemiology in Warsaw.
1. Folkman J (1990): What is the evidence that tumours are angiogenesis-dependent? J Nat Cancer Inst 83: 4-6.
2. Folkman J (1995): Clinical applications of research on angiogenesis. N Engl J Med 333: 1747-1763.
3. Leung DW, Cachianes G, Kuang WJ, Goeddel DV, Ferrara N (1989): Vascular endothelial growth factor is a secreted angiogenic mitogen. Science 246: 1306-1309.
4. Shukla Y, Arora A, Singh A (2002): Antitumorigenic potential of diallyl sulfide in Ehrlich ascites tumour bearing mice. Biomed Environ Sci 15 (1): 41-47.
5. Lee ES, Steiner M, Lin R (1994): Thioallyl compounds: potent inhibitors of cell proliferation. Biochim Biophys Acta 10; 1221 (1): 73-77.
6. CaoY, Cao R, Brakenhielm E (2002): Antiangiogenic mechanisms of diet-derived polyphenols. J Nutr Biochem 13 (7): 380-390.
7. Jung X D, Ellis LM (2001): Inhibition of tumour invasion and angiogenesis by epigallo catechin gallate (EGCG) a major component of green tea. Int J Exp Pathol 82 (6): 309-312.
8. Demeule M, Michaud-Levesque J, Annabi B, et al. (2002): Green tea catechins as novel antitumour and antiangiogenic compounds. Curr Med Chem Anti-Canc Agents 2 (4): 441-463.
9. Spencer RC (1996): Predominant pathogens found in the European prevalence of infection in intensive care study. E J Clin Micr Inf Dis 15: 281-285.
10. Pruitt BA, McManus AT, Kim SH, et al. (1997): Burn wound infections: current status. World J Surg 22: 135-145.
11. Griffith SJ, Nathan RK, Selender RK, et al. (1989): The epidemiology of Pseudomonas aeruginosa in oncology patients in a general hospital. J Inf Dis 160: 1033-1036.
12. Korvick JA Marsh JW, Starzl TE, et al. (1991): Pseudomonas aeruginosa bacteremia in patients undergoing liver transplantation: an emerging problem. Surgery 109: 62-68.
13. Bodinet C, Mentel R, Wegner U, et al. (2002): Effect of Oral Application of an Immunomodulating Plant Extract on Influenza Virus Type A Infection. Planta Medica 68: 896-900.
14. Colic M, Vucevic D, Kilibarda V, et al. (2002): Modulatory effects of garlic extracts on proliferation of T-lymphocytes in vitro stimulated with concanavalin. A. Phytomedicine 9: 117-124.
15. Melchart D, Linde K, Worku F, Bauer R, Wagner H (1994): Immunomodulation with Echinacea-a systematic review of controlled clinical trials. Phytomedicine 1: 245-254.
16. Park CE, Stankiewicz ZK, Rayman MK, et al. (1979): Inhibitory effect of cocoa powder on the growth of a variety of bacteria in different media. Can J Microbiol 25 (2): 233-5.
17. Currier NL, Miller SC (2000): Natural killer cells from aging mice treated with extracts from Echinacea purpurea are quantitatively and functionally rejuvenated. Exp Gerontol 35 (5): 627-639.
18. Currier NL, Miller SC (2001): Echinacea purpurea and melatonin augment natural-killer cells in leukemic mice and prolong life span. J Altern Complement Med 7 (3): 241-251.
19. Chen GW, Chung JG, Ho HC, Lin JG (1999): Effects of the garlic compounds diallyl disulphide and diallyl disulphide on arylamine N-acetyltransferase activity in Klebsiella pneumoniae. J Appl Toxicol 19: 75-81.
20. Lamm DL, Riggs DRY (2000): The potential application of Allium sativum (garlic) for the treatment of bladder cancer. Urol Clin North Am 27 (1): 157-162.
21. Sidky YA, Auerbach R (1975): Lymphocyte-induced angiogenesis a quantitative and sensitive assay of the graft-vs-host reaction. J Exp Med 141: 1084-1100.
22. Skopińska-Różewska E, Piazza GA, Sommer E, et al. (1998): Inhibition of angiogenesis by sulindac and its sulfone metabolite (FGN-1): A potential mechanism for their antineoplastic properties. Int J Tissue React XX. 3: 85-89.
23. Kondo T, Ohta T, Igura K, Hara Y, Kaji K (2002): Tea catechins inhibit angiogenesis in vitro measured by human endothelial cell growth, migration and tube formation, through inhibition of VEGF receptor binding. Cancer Lett 28 (180): 139-144.
24. Skopińska-Różewska E, Borowska A, Skopiński P, et al. (2003): The effect of theobromine or chocolate feeding on pregnant mice and their offspring Immun Letters 87: 286, Greece 15th Europ Immun Cong, June 8-12.
25. Khanna S, Roy S, Bagchi D, Bagchi M, Sen CK (2001): Upregulation of oxidant-induced VEGF expression in cultured keratinocytes by a grape seed proanthocyanidin extract. Free Radic Biol Med 31 (1): 38-42.
26. Liang YC, Lin-shiau SY, Chen CF, Lin JK (1997): Suppression of extracellular signals and cell proliferation through EGF receptor binding by (-) -epigallocatechin gallate in human A431 epidermoid carcinoma cells. J Cell Biochem 67 (1): 55-65.
27. Lamy S, Gingras D, Beliveau R (2002): Green tea catechins inhibit vascular endothelial growth factor reeptor phosphorylation. Cancer Res 15; 62 (2): 381-385.
28. Saito M, Saito K, Kunisaki N, et al. (2002): Green tea polyphenols inhibit metalloproteinase activities in the skin, muscle, and blood of rainbow trout. J Agric Food Chem 20; 50 (24): 7169-7174.
29. Kim MH (2003): Flavonoids inhibit VEGF/bFGF-induced angiogenesis in vitro by inhibibiting the matrix -degrading proteases. J Cell Biochem 89 (3): 529-538.
30. Cao Y, Cao R (1999): Angiogenesis inhibited by drinking tea. Nature 398: 381.
31. Carnasechci S, Schneider Y, Lazarus S, Coehlo D, Gosse F, Raul F (2000): Flavanols and procyanidis of cocoa and chocolate inhibit growth and polyamine biosynthesis of human colonic cancer cells. Cancer Lett 175: 157-156.
32. Kozikowski A, Tuckmantel W, Bottcher G, et al. (2003): Studies in Polyphenol Chemistry and Bioactivity. 4. Synthesis of Trimeric, Tetrameric, Pentameric, and Higher Oligomeric Epicatechin-Derived Procyanidinns Having All -4beta, 8-Interflavan Connectivitty and Their Inhibition of Cancer Cell Growth through Cell Cycle Arrest. J Org Chem 68: 1641-1658.
33. Tang Z, Sheng Z, Liu S, et al. (1997): The preventing function of garlic on experimental oral precancer and its effect on natural killer cells, T-lymphocytes and interleukin-2. Human Yi Ke Da Xue Bao 22 (3): 246-248.
34. Nakagawa H, Tsuta K, Kiuchi K, et al. (2001): Growth inhibitory effects on diallyl disulfide on human breast cancer cell lines Carcinogenesis 22 (6): 891-897.
35. Facino RM, Carini M, Aldini G, Saibene L, et al. (1995): Echinacoside and caffecyl conjugates protect collagen from free radical – induced degradation: A potential use of Echinacea extracts in the prevention of skin photodamage. Planta Med 61: 510-514.
36. Clifford LJ, Nair MG, Rana J, Dewit DL (2002): Bioactivity of alkamides isolated from Echinacea purpurea (L) Moench. Phytomedicine 9: 249-253.
37. Roesler J, Steinmuller C, Kiderlen A, Emmendorffer A, Wagner H, Lohmann Mathes ML (1991): Application of purified polysaccharides from cell cultures of the plant Echinacea purpurea to mice mediates protection against systemic infections with Listeria monocytogenes and Candida albicans. Int J Immunopharmacol 13 (1): 27-37.
38. Steinmuller C, Roesler J, Grottrup E, Franke G, Wagner H, Lohmann-Mates ML (1993): Polysaccharides isolated from plant cell cultures of Echinacea purpurea enhance the resistance of immunosuppressed mice against systemic infections with Candida albicans and Listeria monocytogenes. Int J Immunopharmacol 15 (5): 605-614.
39. Paolino VJ, Kashket S (1985): Inhibition by cocoa extracts of biosynthesis of extracellular polysaccharide by human oral bacteria. Arch Oral Biol 30 (4): 359-63.
40. Sanbongi C, Suzuki N, Sakane T (1997): Polyphenols in chocolate, which have antioxidant activity, modulate immune functions in human in vitro. Cell Immunol 177 (2): 129-36.
41. Chung JG, Chen GW, Wu LT, et al. (1998): Effects of garlic compounds diallyl sulfide and diallyl disulfide on arylamine N-acetyltransferase activity in strains of Helicobacter pylori from peptic ulcer patients. Am J Clin Med 26: 353-364.

Copyright: © 2004 Polish Society of Experimental and Clinical Immunology This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0) License (http://creativecommons.org/licenses/by-nc-sa/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material, provided the original work is properly cited and states its license.
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