WO2008111918A2

WO2008111918A2 - Combination containing plant extracts and use thereof for the treatment of various types of cancer

Info

Publication number: WO2008111918A2
Application number: PCT/SI2007/000042
Authority: WO
Inventors: Romina Znoj
Original assignee: Romina Znoj
Priority date: 2007-03-14
Filing date: 2007-12-12
Publication date: 2008-09-18
Also published as: SI22466A; WO2008111918A3

Abstract

There is disclosed a combination containing plant extracts, of the following composition: an extract of a plant of the genus Capsicum, an extract of a plant of the genus Allium, and HEPES and elementary sulfur, preferably under the addition of an antibiotic, in various ratios with regard to the purpose, the ingredients being optionally suspended in a saline. The above combination is useful as an agent for triggering apoptosis of cancer cells and at the same time does not trigger a high rate of apoptosis in immortalized cells and does not harm healthy cells.

Description

Combination containing plant extracts and use thereof for the treatment of various types of cancer

Technical field

The invention is from the field of medicine and relates to a combination containing plant extracts, useful for the treatment of various types of cancer. With extracts of plants of the genus Capsicum and genus Allium in various concentrations, various types of cancer, e.g. neuronal, uterine, breast, liver cancers etc. may be treated.

Technical problem

There exists a constant need to treat various types of cancer and to do it by causing the death of cancer cells. One of the most frequently used methods to cause the dying of cancer cells is triggering apoptosis in these cells. By the present invention it was endeavoured to find new products which could trigger apoptosis in cancer cell lines and at the same time would not trigger a high rate of apoptosis in immortalized cells and would not harm healthy cells.

Prior art

It is evident from the history and archaeological data as well as from epidemiological data on cancer incidence in certain geographical regions that various plant extracts have been rather successfully used in the prevention of cancer diseases.

There are not many solutions in the literature. One of them, which seems to be closest to the present invention, is disclosed in EP 92226. It, however, uses plants of other genera; besides, the compound as claimed in EP 92226 does not seem to be selective about cancer/non-cancer cells. The technical solution

There have been efforts to find new products that could trigger apoptosis in cancer cell lines (e.g. cell line of human cancer neurons: SH-SY5Y; cell line of breast cancer: MCF-7; cell line of human cancer cells from uterus: HeLa; cell line of human cancer cells from liver HepG2) and at the same time would not trigger a high rate of apoptosis in immortalized cells (e.g. cell line of human keratinocytes: HaCaT; cell line of mouse endotelial fibroblasts MEF; cell line MEF wt imm) and would not harm healthy cells (e.g. cell line of human fibroblasts: NHDF; cell line HCF 306-05f; cell line RH (R780-30).

Surprisingly, a new product that fulfils the above requirements has been found.

The first object of the invention is a combination containing plant extracts, of the following composition: an extract of a plant of the genus Capsicum, an extract of a plant of the genus Allium, and, as supportive substances on the basis of sulfur, HEPES and elementary sulfur, preferably under the addition of an antibiotic, in various ratios with regard to the purpose, the ingredients being optionally suspended in a saline.

In addition, it has been found that a selective cell death is further enhanced and the percentage of cell necrosis reduced when to the above combination there are added spices, namely oregano, caraway, kitchen salt and black pepper, each spice in an amount of from 0.01 to 1% by weight relative to the combination.

As antibiotics penicillin and streptomycin may be added, suitably in a concentration of 0.5 vol.% each time. An average artisan in the field will easily determine the ratios of the ingredients on the basis of his knowledge and experience.

As an example of plants of the genus Capsicum, e.g. Capsicum chinense should be mentioned.

As an example of plants of the genus Allium, e.g. Allium sativum grown in Egypt and Allium neapolitanum should be mentioned.

Since the quality of plants varies as a result of their origin, an average artisan may, without any special effort, find out which plants are most suitable.

Another object of the invention is a process for preparing the combination according to the invention, characterized in that plants of the genus Capsicum and plants of the genus Allium are dried in the sun and, when completely dry, ground to a powder, the powder is suspended in a saline together with HEPES and elementary sulfur and preferably with antibiotics, left to stand under occasional shaking for a few days so that the solid particles settle, then the suspension is filtered and the filtrate is either stored at 4°C to 10⁰C or dried to a powder.

A further object of the invention is the above-defined combination for use as an agent for triggering the apoptosis of cancer cells. The cancer cells are especially the cell line of human cancer neurons SH-S Y5 Y, the cell line of breast cancer MCF-7, the cell line of human cancer cells from uterus HeLa and the cell line of human cancer cells from liver HepG2.

This has been established in in vitro experiments, which were carried out by standard procedures except that in the culture medium no ingredients of animal origin have been present. The term "combination according to the invention" means all possible combinations which maintain or have a similar specifity and activity as disclosed furtheron.

The invention also relates to the use of said combination for preparing a therapeutic agent for the treatment of cancer, e.g. neuronal, breast and uterine cancer, liver cancer, and this agent also contains a suitable carrier, solvent or other additive together with supportive substances on sulfur basis. A skilled person in the field of cancer therapy will be able to establish the therapeutically effective amount.

Thus, one of the objects of the invention is also the above-defined combination for use as an agent for triggering apoptosis of cancer cells.

Dosage regimens are adjusted to provide the optimum desired response (e.g. a therapeutic response). It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. A dosage unit is to be sensibly used as unitary dosage for treating subjects; each unit shall contain a predetermined amount of the active substance calculated with regard to the desired therapeutical effect together with the required pharmaceutical carrier. The characteristics of the dosage unit according to the invention depend on and are directly determined by a) the unique characteristics of the active ingredient and the particular therapeutic effects to be achieved and b) the limitations inherent to the art of preparing the active substance for the treatment of sensitive individuals (sensitive to the active ingredient or to auxiliary substances or to both).

The terms "parenteral administration" and "administered parenterally" mean the modes of administration other than enteral and topical ones, usually by injection or infusion, and include intravenous, intramuscular, intraarterial, intradermal, intraperitoneal and subcutaneous injections or infusions. The combination according to the invention may be formulated into a pharmaceutical composition together with a suitable carrier or diluent. Examples of suitable aqueous and non-aqueous carriers that may be used for formulating the pharmaceutical composition include water, polysorbate 80, vegetable oils (e.g. olive oil) and injectable esters (e.g. ethyl oleate). These compositions may also contain additives such as preservatives, wetting agents, emulsifying agents and dispersing agents. The absence of microorganisms is ensured by sterilisation techniques (e.g. filtration without the use of heat) and by incorporation of antibacterial and antifungal agents (e.g. penicillin, streptomycin, sorbic acid etc.). It is desirable that also isotonic agents such as sodium chloride etc. are included into the combination according to the invention. In addition, a prolonged absorption of the pharmaceutically active substance for injection may be achieved by adding agents that delay absorption such as gelatine.

When the ingredients of the present invention are administered as pharmaceuticals to animals or humans, they may be given alone or in a pharmaceutical composition in the ratio 0.005 : 0.995 (99.5% of the active ingredient and 0.5% of the pharmaceutically acceptable carrier).

Similarly as in WO 2005/050213 and irrespective of the administration route, the combinations according to the invention, which may be used in a suitable hydrated form and/or in a pharmaceutical composition, are formulated to pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

The actual dosage levels of the active ingredients according to the invention in the pharmaceutical compositions may vary with regard to the preparation of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, as well as with regard to the composition and the mode of administration without being harmful for a patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of each particular composition of the present invention, the mode of administration, the time of administration, the bioavilability of the substances themselves, the duration of the treatment, other drugs (concomitant therapy), the ingredients and/or materials used in the combination with a particular ingredient, the age, sex, body weight, condition, general health and prior medical history of the patient treated and similar factors generally known in medicinal practice.

A physician or a veterinarian may start with doses of ingredients according to the present invention in a pharmaceutical composition and use such an amount of the substance which is the lowest effective therapeutic dose. This effective dose will usually depend upon the factors disclosed above.

Therapeutic doses may be administered by medicinal devices well known in the art. A therapeutic dose according to the invention may be best administered by well-known implants and modules as mentioned in US No 4,447,233 wherein a medication infusion pump for delivering medication at a precise infusion rate is disclosed. The present invention may be administered by means of a device disclosed in US No 4,447,224, wherein a variable flow implantable infusion apparatus for continuous drug delivery is disclosed.

Certain embodiments of the combinations according to the invention may be formulated to ensure proper distribution in vivo. For example the blood-brain barrier (BBB) prevents many highly hydrophilic compounds from crossing. To ensure the crossing (if desired) of the therapeutic active ingredients of the invention, they may be incorporated into liposomes. For methods of manufacturing liposomes see US Nos. 4,552,811; 5,374548 and 5,399,331. Liposomes may contain one or more active ingredients, which are selectively transported into specific cells or organs, thus enhancing the targeted drug delivery rate (V. V. Ranade,1989). Preferably, the combination according to the invention should be incorporated into a liposome. In an improved embodiment, the liposomes are directed to a specific target. In a most preferred embodiment, the therapeutic ingredient contained in the liposome is directly injected to the site where a therapeutic effect is desired (e.g. into the body part with the tumour). The composition should be fluid to the extent that it may be readily squeezed when injected into the organism. It must be stable at conditions of manufacturing and storing and it must be protected against microbial contamination (e.g. contamination with bacteria and fungi).

Effective doses and dosage regimes (as disclosed in WO 2005/050213) for combinations according to the invention depend upon the stage of the disease and health conditions of the patient treated. The dosage regime may be established by a person skilled in the art.

The "therapeutically efective dose" for tumour therapy may be measured by objective responses of a tumour to therapy. The response of the tumour may be complete or partial. A complete response (CR) is defined as the absence of the disease established clinically, radiologically or by some other proof. A partial response (PR) occurs if the reduction in aggregate tumour size is more than 50%. Mean time to progression is a measure that characterizes the duration of tumour response.

The "therapeutically effective dosage" for tumour therapy may also be measured by its ability to stabilize the progression of the disease. The ability of the composition to stop the development of cancer may be evaluated in an animal model and thus the efficiency for treating a human tumour may be predicted. A therapeutically effective amount of a therapeutic composition may reduce the tumour size or in another way reduce the symptoms in a person or animal. By means of one of routine methods in the art, such amount may be determined with regard to the size of a subject, the severity of the subject's symptoms and the specifity of the compositions or the selected administration route. In view of the above-mentioned facts, patients treated with the compositions according to the invention may be additionally treated with therapeutic agents and methods that enhance or strengthen the therapeutic effect of the compositions according to the invention (e.g. photodynamic therapy; Plaetzer et al., 2003). In order to ensure a higher effectiveness of the therapy by derivatives of the above plant extracts, the patients receiving derivatives of the above plant extracts should be on a vegetarian diet throughout the therapy.

In order to prove that a combination according to the invention may trigger apoptosis in cancer cells, suitable cell lines were selected, cell morphology was observed by a light microscope, a cell viability test and a caspase test were performed, the number of apoptotic cells was calculated by FACS Calibur Flow Cytometer and a test of PARP activation was performed.

In order to determine the pathway (intrinsic or extrinsic) used by the combination according to the invention to trigger apoptosis, wide-spectrum inhibitors of caspases and cathepsins (z-VAD-fmk and E-64 d) and inductor of DNA damage (mitomycin C) were used in further investigations. Determination by MitoTracker Red CMXRos and LysoTracker Green DND.

The invention is illustrated by the following non-limiting Examples.

Example 1

Preparation of the combination according to the invention containing a plant extract

Plants Capsicum chinense, Allium sativum and Allium neapolitanum were dried in the sun. After they were completely dry, they were ground into powder. The powder was suspended in a saline together with supportive substances on the basis of sulfur, namely HEPES and elementary sulfur, under the addition of streptomycin (final concentration in the suspension 0.5 vol.%) and of penicillin (final concentration in the suspension 0.5 vol.%). The suspension was left to stand for one week and was occassionally shaken. Then it was filtered. A combination according to the invention was obtained, which may be formulated into pharmaceutical compositions.

Data for one concrete example:

450 mg of the powder of each above-mentioned plant, in a saline up to a concentration of 10 mg/mL;

HEPES 1% with regard to the solution; elementary sulfur 0.01% with regard to the solution.

The solution may be stored as such at 4 to 10⁰C and is effective for 2 months or is dried and processed immediately before use.

Example 2

Process of treating the cells

The solution from Example 1 was incubated with the cells to be investigated (the kind of cells is defined in the Figs, hereinafter) for one week at room temperature and filtered. The solution was then added to a culture medium prepared according to standard methods with the exception that it did not contain any ingredients of animal origin. The cells were grown according to standard methods disclosed in Ausubel et al., Current Protocols in Molecular Biology. Wiley Interscience 1-4 (1998); Bonifacino et al., Current Protocols in Cell Biology. Wiley Interscience 1-3 (2003). The solution was then stored in a refrigerator at 4-10⁰C and under these conditions it was effective for 2 months.

Example 3 In vitro tests

With the solution obtained in Example 2 various tests were carried out in order to determine the rate of apoptosis, namely viability tests, tests for caspase-3 activity, flow cytometry, testing for PARP.

Viability tests and tests for caspase-3 activity were carried out according to standard methods disclosed in Ausubel et al., Current Protocols in Molecular Biology. Wiley Interscience 1-4 (1998); Bonifacino et al., Current Protocols in Cell Biology. Wiley Interscience 1-3 (2003); Lockshin & Zakeri, When Cells Die II. A Comprehensive Evaluation of Apoptosis and Programmed Cell Death. Wiley Interscience 2:37-58 (2004); Holdenrieder & Stieber, Apoptotic markers in cancer. Clin Biochem 37:605- 617 (2004).

Flow cytometry was carried out according to standard methods disclosed in Rahman et al., Introduction to Flow Cytometry. Serotec 2000; Ormerod, Flow Cytometry: A Practical Approach. 3 (2000); Macey, Flow Cytometry: Clinical Applications. 1 (1994); Givan, Flow Cytometry: First Principles. 2 (2002); Haugland, Handbook of Fluorescent Probes and Research Products. 9 (2002); Carleton & Janet, Immunophenotyping. 1 (2000); Watson, Introduction to Flow Cytometry. 1 (2004); Shapiro, Practical Flow Cytometry. 4 (2003).

Testing for PARP was carried out according to standard methods disclosed in Ausubel et al., Current Protocols in Molecular Biology. Wiley Interscience 1-4 (1998); Bonifacino et al., Current Protocols in Cell Biology. Wiley Interscience 1-3 (2003).

The results are shown in Figs. 1 to 38. Each experiment was repeated 2 to 5 times and the obtained results were the same with a relative error ±5-10%.

The concentrations of supportive substances on the basis of sulfur i.e. HEPES and elementary sulfur in the samples ranged from 2 vol.% to 0.5 vol.% for HEPES and from 0.1 vol.% to 1 vol.% for elementary sulfur, and, additionally, penicillin and streptomycin were added up to a final concentration in the suspension of 0.5 vol.% each.

Explanation of Figures:

Fig. 1 shows that healthy fibroblasts from the cell line NHDF were not damaged by the combinations according to the invention with various concentrations of plant extracts. The concentrations increased from sample 1 on, from 1 μg/mL to 10 mg/mL, the last sample being the control (untreated cells). The number of cells per well/unit: 5000; the duration of incubation with the combination according to the invention: 24 hours.

Fig. 2 shows that immortalized cells in the cell line HaCaT managed to maintain their viability over 90% when individual samples of the combination according to the invention with various concentrations of plant extracts were added thereto. The concentrations decreased from sample 1 on: Capsicum from 90 vol.% to 20 vol.% and Allium from 90 vol.% to 20 vol.%. The number of cells per well: 5000; the duration of incubation with the combination according to the invention: 72 hours. Fig. 3 shows caspase activity of immortalized cells from the cell line HaCaT. The first sample represents a buffer, the second sample represents a buffer with substrate, the third sample represents untreated cells (control), the fourth and the fifth samples represent cells treated with the highest concentrations of plant extracts with regard to the results of the viability test (the highest concentrations at which the cells in the viability test showed a viability of more than 90%; these concentrations are: Capsicum and Allium 1 mg/mL, 0.1 mg/mL, 100 μg/mL, 10 μg/mL, 1 μg/mL; HEPES 10 μg/mL and 1 μg/mL; individual concentrations of Capsicum, Allium and HEPES were mutually combined in the ratios Capsicum : Allium 1:1, 1:4, 4:1, HEPES ranged from 2 vol.% to 0.5 vol.% or only Capsicum or only Allium or only HEPES were added). It was found later at treating cancer cells with the above mentioned combinations that the viability of cancer cells was more effectively reduced in combination than when treated only with individual components Capsicum, Allium and HEPES. It is evident that caspase activity was even lower than in untreated cells and this is an indication that the process of apoptosis did not start.

Fig. 4 shows the percentage of apoptotic cells in the cell line HaCaT measured by flow cytometer FACS Calibur and by using Cellquest software (Becton Dickinson, Mountain View, CA, USA). The first sheet shows untreated cells and the second sheet shows cells treated with the same concentrations as in the viability test and the test for determining caspase activity. It is evident that there were no significant variations in the percentage of apoptotic cells between untreated and treated cells when the time of incubation was 24 hours.

Fig. 5 shows that the viability of cancer cells from the cell line SH-SY5Y linearly decreased when the concentrations of the plant extracts increased. The number of cells per well: 10000; the duration of incubation with the combination according to the invention: 24 hours.

Fig. 6 shows the caspase activity of cancer cells from the cell line SH-SY5 Y. The first sample represents a buffer, the second sample represents a buffer with a substrate, the third sample represents untreated cells (control), the fourth, fifth and sixth samples represent cells treated with the highest concentrations of plant extracts with regard to the results of the viability test (highest concentrations which in the viability test showed a viability between 60 and 80%: Capsicum 10 μg/mL + Allium 10 μg/mL in a ratio 1 :1 + HEPES 0.5 vol.%). It is evident that the caspase activity increased proportionally to higher concentrations of plant extracts.

Fig. 7 shows the percentage of apoptotic cells in the cell line SH-SY5Y measured by flow cytometer FACS Calibur and using a Cellquest software (Becton Dickinson, Mountain View, CA, USA). The first sheet shows untreated cells and the second sheet the cells treated with the same concentrations as in the viability test and the test for determining caspase activity. Many tumour cells are apoptotic as shown by determining the percentage of apoptotic cells.

Fig. 8 shows the results of testing for PARP activation. It is evident that PARP was activated, which is expressed and identified by blotting techiques in all three samples of cells SH-SY5Y treated with concentrations of plant extracts mentioned at Figs. 5 and 6.

Fig. 9 shows the viability of cancer cells from the cell line MCF-7, which decreased when the concentrations (see the text at Fig. 3) of plant extracts increased. Since these cells do not contain caspase 3, the test for determining caspase activity was omitted. The number of cells per well: 10000; the duration of incubation with the combination according to the invention: 24 hours.

Fig. 10 shows the percentage of apoptotic cells in the cell line MCF-7 measured by flow cytometer FACS Calibur and by using Cellquest software (Becton Dickinson, Mountain View, CA, USA). The first sheet shows untreated cells and the second sheet shows cells treated with the same concentrations as in the viability test (Fig. 9). {Capsicum 1 μg/mL + Allium 1 μg/mL in a ratio 1 :1 + HEPES 0.5 vol.%). After 72 hours of incubation, the tumour cells were mostly apoptotic and necrotic as shown by determining the percentage of apoptotic and necrotic cells.

Fig. 11 shows the percentage of viability of cells MCF-7 treated with the combination according to the invention as disclosed at Fig. 9, and at the same time with a pan- inhibitor of caspases (z-VAD-fmk), cathepsins (E-64-d) and mitomycin C (Sample 1: Capsicum 1 μg/mL + Allium 1 μg/mL in a ratio 4:1 + HEPES 0.8 vol.%; Sample 2: Capsicum 1 μg/mL + Allium 1 μg/mL in a ratio 4:1 + HEPES 0.8 vol.%, z-VAD-fmk in a concentration of 10 μM; Sample 3: Capsicum 1 μg/mL + Allium 1 μg/mL in a ratio 4:1 + HEPES 0.8 vol.%, E-64-d in a concentration of 15 μM; Sample 4: Capsicum 1 μg/mL + Allium 1 μg/mL in a ratio 4:1 + HEPES 0.8 vol.%, mitomycin C in a concentration of 2 μg/mL). The results show that the present invention induces apoptosis in these cells to some extent through a cascade of caspases and cathepsins since the viability was higher (up to 15%) when the cells were treated with z-VAD- fmk or E-64d. When the cells were treated with the combination according to the invention and mitomycin C, the viability was lower in comparison with the treatment with the combination according to the invention alone and this is an indication that the combination according to the invention does not act in the same way as mitomycin C (mitomycin C induces DNA damages and thus triggers apoptosis). Fig. 12 shows that the number of apoptotic cancer cells in the cell line HeLa was lower than 80% in two samples only. The concentrations that induced the viability of cancer cells lower than 80% (Sample 2: Capsicum 0.1 mg/mL + Allium 0.1 mg/mL in a ratio 1:1 + HEPES 0.8 vol.%, and Sample 4: Capsicum 1 μg/mL + Allium 1 μg/mL in a ratio 4:1 + HEPES 1 vol.%; Sample 1 were untreated cells as control and Sample 3 was Capsicum 40 μg/mL + Allium 1 μg/mL in a ratio 9:1 + HEPES 0.8 vol.%) were used for further tests. The number of cells per well: 10000; the duration of incubation with the combination according to the invention: 48 hours.

Fig. 13 shows the caspase activity of the cancer cells from the cell line HeLa. The first sample represents a buffer, the second sample represents a buffer with a substrate, the third sample represents untreated cells (control), the fourth and the fifth samples represent cells treated with the second and the third highest concentrations of derivatives with respect to the results of the viability test (the second and the third highest concentrations at which the cells in the viability test showed a viability between 50 and 80%: Capsicum 0.1 mg/mL + Allium 0.1 mg/mL in a ratio 1 : 1 + HEPES 0.8 vol.% and Capsicum 1 μg/mL + Allium 1 μg/mL in a ratio 4:1 + HEPES 1 vol.%). It is evident that the caspase activity is present in those cells treated with the above-mentioned concentrations of plant extracts and this further indicates the presence of apoptosis.

Fig. 14 shows the percentage of apoptotic cells in the cell line HeLa measured by flow cytometer FACS Calibur and by using Cellquest software (Becton Dickinson, Mountain View, CA, USA). The first sheet shows untreated cells and the second sheet shows cells treated with the same concentrations as in the viability test and in the test for determining caspase activity. Sample used Capsicum 0.1 mg/mL + Allium 0.1 mg/mL in a ratio 1: 1 + HEPES 0.8 vol.%. After 24 hours of incubation, the tumour cells were mostly apoptotic as shown by determining the percentage of apoptotic cells.

Fig. 15 shows the percentage of viability of cells HeLa treated with the combination according to the invention and at the same time with a pan-inhibitor of caspases (z- VAD-fmk), cathepsins (E-64-d) and mitomycin C (the composition of samples is as in Example 11 except Capsicum 1 μg/mL + Allium 1 μg/mL in a ratio 4:1 + HEPES 1 vol.%). The results show that the present invention induces apoptosis in these cells to some extent through a cascade of caspases and cathepsins since the viability was higher (up to 40%) when the cells were treated with z-VAD-fmk or E-64d. When the cells were treated with the combination according to the invention and mitomycin C, the viability was lower in comparison with the treatment with the combination according to the invention alone and this is an indication that the combination according to the invention does not act in the same way as mitomycin C (mitomycin C induces DNA damages and thus triggers the apoptosis).

Fig. 16 shows that immortalized cells in the cell line MEF (mouse endotelial fibroblasts), namely MEF imm. Bid -/-, maintained the viability on average over 30% when individual samples of the combination according to the invention with various concentrations were added thereto. The concentrations decreased from sample 1 on. The number of cells per well: 5000; the duration of incubation with the combination according to the invention: 24 hours.

Fig. 17 shows that healthy cells in the cell line MEF wt (mouse endotelial fibroblasts wild type) maintained the viability on average over 60% when individual samples of the combination according to the invention with various concentrations were added thereto. This indicates a selectiveness of combinations (see also Fig. 16). The concentrations decreased from sample 1 on. The number of cells per well: 5000; the duration of incubation with the combination according to the invention: 24 hours. Fig. 18 shows that the viability of cancer cells in the cell line HepG2 (human cancer cells from liver) decreased when the concentrations of combinations according to the invention increased. The first sample represents the lowest concentration and the last sample represents the highest concentration of the combinations according to the invention as already mentioned in the above Examples. The number of cells per well: 10000; the duration of incubation with the combination according to the invention: 24 hours.

Fig. 19 shows that healthy fibroblasts from the cell line HCF were not damaged by the combinations according to the invention with various concentrations of plant extracts. The concentrations decreased from sample 1 on, from 10 mg/mL to 1 μg/mL. The number of cells per well/unit: 5000; the duration of incubation with the combination according to the invention: 24 hours. If the duration of incubation was 48 hours, damages occurred already. For comparative reasons also respective plants alone were tested. C means Capsicum extract, AS means Allium extract and E means the inventive combination of plant extracts.

Fig. 20 shows that immortalized cells in the cell line MEF wt imm maintained the viability on average over 70% when individual samples of the combination according to the invention with various concentrations were added thereto. The concentrations decreased from sample 1 on. The number of cells per well: 5000; the duration of incubation with the combination according to the invention: 48 hours. Fig. 21 shows that healthy fibroblasts from the cell line RH were not damaged by the combinations according to the invention with various concentrations of plant extracts. The concentrations decreased from sample 1 on, from 10 mg/mL to 1 μg/mL. The number of cells per well/unit: 5000; the duration of incubation with the combination according to the invention: 24 hours. If the duration of incubation was 48 hours, damages occurred already. For comparative reasons also respective plants alone were tested.

Fig. 22 shows the caspase activity of cancer cells from the cell line HepG2. The first sample represents a buffer, the second sample represents a buffer with a substrate, the third sample represents untreated cells (control), the fourth, fifth, sixth, seventh and eighth samples represent cells treated with the highest to lowest concentrations of the inventive combination with respect to the results of the viability test (the fourth sample represents the highest concentration of Capsicum 10 mg/mL and Allium 10 mg/mL and the eighth sample represent the lowest one of Capsicum 1 μg/mL + Allium 1 μg/mL, all in a ratio 1 :1 + HEPES 0.5 vol.%). The highest caspase activity occured at the highest concentration of 10 mg/mL of the inventive combination. Fig. 23 shows the caspase activity of cancer cells from the cell line MEF imm Bid -/-. The first sample represents a buffer, the second sample represents a buffer with a substrate, the third sample represents untreated cells (control), the fourth, fifth, sixth, seventh and eighth samples represent cells treated with the highest to lowest concentrations of the inventive combination with respect to the results of the viability test (the fourth sample represents the highest concentration of Capsicum 10 mg/mL and Allium 10 mg/mL and the eighth sample represents the lowest one of Capsicum 1 μg/mL + Allium 1 μg/mL, all in a ratio 1:1 + HEPES 0.5 vol.%). The highest caspase activity occured at the highest concentration of 10 mg/mL of the inventive combination.

Fig. 24 shows the caspase activity of cancer cells from the cell line MEF wt imm. The first sample represents a buffer, the second sample represents a buffer with a substrate, the third sample represents untreated cells (control), the fourth, fifth, sixth, seventh and eighth samples represent cells treated with the highest to lowest concentrations of the inventive combination with respect to the results of the viability test (the fourth sample represents the highest concentration of Capsicum 10 mg/mL and Allium 10 mg/mL and the eighth sample represents the lowest one of Capsicum 1 μg/mL + Allium 1 μg/mL, all in a ratio 1:1 + HEPES 0.5 vol.%). The highest caspase activity occured at the concentration of 10 μg/mL of the inventive combination.

Fig. 25 shows the percentage of apoptotic cells in the cell line HaCaT measured by flow cytometer FACS Calibur and using Cellquest software (Becton Dickinson, Mountain View, CA, USA). The cells were incubated for 48 hours with various concentrations of the inventive combination. It is clear that these immortalized cells remained damaged in a very low percentage.

Fig. 26 shows the percentage of apoptotic cells in the healthy cell line HCF measured by flow cytometer FACS Calibur and using Cellquest software (Becton Dickinson, Mountain View, CA, USA). The cells were incubated for 72 hours with various concentrations of the inventive combination. It is clear that these cells show extreme damage only at the highest concentrations, while lower concentrations were much less effective in inducing apoptosis (on average only in 25%).

Fig. 27 shows the percentage of apoptotic cells in the cancer cell line HeLa measured by flow cytometer FACS Calibur and using Cellquest software (Becton Dickinson, Mountain View, CA, USA). The cells were incubated for 48 hours with various concentrations of the inventive combination. It is clear that these cells show a higher percent of damage at lower concentrations.

Fig. 28 shows the percentage of apoptotic cells in the cancer cell line HepG2 measured by flow cytometer FACS Calibur and using Cellquest software (Becton Dickinson, Mountain View, CA, USA). The cells were incubated for 48 hours with various concentrations of the inventive combination. Apoptosis progressed with the increase in concentration.

Fig. 29 shows the percentage of apoptotic cells in the cancer cell line MCF-7 measured by flow cytometer FACS Calibur and using Cellquest software (Becton Dickinson, Mountain View, CA, USA). The cells were incubated for 24 hours with various concentrations of the inventive combination. Apoptosis was the highest when the cells were treated with the highest concentration (10 mg/mL). Fig. 30 shows the percentage of the viability of HeLa cells when they were treated with various concentrations of the inventive combination together with a combination of pan-inhibitors of caspases (z-VAD-fmk), cathepsins (E-64d) and with mitomycin C. The individual samples are of the following constitution: samples 1-3 represent controls (the cells were treated only with the above-mentioned inhibitors or with mitomycin C), sample 4 represents cells treated with a mixture of Capsicum 1 μg/mL + Allium 1 μg/mL in the ratio 4:1 + HEPES 1 vol.%, in sample 5 the cells were treated with a mixture of Capsicum 1 μg/mL + Allium 1 μg/mL in the ratio 4:1 + HEPES 1 vol.% with the addition of z-VAD-fmk in a final concentration of 10 μM, sample 6 represents cells treated with a mixture of Capsicum 1 μg/mL + Allium 1 μg/mL in the ratio 4: 1 + HEPES 1 vol.% with the addition of E-64d in a final concentration of 15 μM, and sample 7 represents cells treated with a mixture of Capsicum 1 μg/mL + Allium 1 μg/mL in the ratio 4:1 + HEPES 1 vol.% with the addition of mitomycin C in a final concentration of 2 μg/mL. The results show that the present invention induces apoptosis through a cascade of caspases and cathepsins since the viability was significantly higher (up to 40%) when the cells were treated with E-64 d. However, the results show that also a non-caspase dependent pathway must be involved since z-VAD-fmk did not prevent the onset of apoptosis in a significant percentage. Concerning mitomycin C, the viability was lower for 60%, when the present invention was combined with mitomycin C in the treatment of the cells, which suggests that the present invention uses a different pathway of apoptosis induction than mitomycin C (mitomycin C damages DNA and in this way induces apoptosis).

Fig. 31 shows the percentage of viability of MCF-7 cells when they were treated with various concentrations of the inventive combination together with a combination of pan-inhibitors of caspases (z-VAD-fmk), cathepsins (E-64d) and with mitomycin C. The individual samples are of the following constitution: samples 1-3 represent controls (the cells were treated only with the above-mentioned inhibitors or with mitomycin C), sample 4 represents cells treated with a mixture of Capsicum 1 μg/mL + Allium 1 μg/mL in the ratio 4:1 + HEPES 0,8 vol.%, in sample 5 the cells were treated with a mixture of Capsicum 1 μg/mL + Allium 1 μg/mL in the ratio 4: 1 + HEPES 0,8 vol.% with the addition of z-VAD-fmk in a final concentration of 10 μM, sample 6 represents cells treated with a mixture of Capsicum 1 μg/mL + Allium 1 μg/mL in the ratio 4: 1 + HEPES 0,8 vol.% with the addition of E-64d in a final concentration of 15 μM, and the sample 7 represents cells treated with a mixture of Capsicum 1 μg/mL + Allium 1 μg/mL in the ration 4:1 + HEPES 0,8 vol.% with the addition of mitomycin C in a final concentration of 2 μg/mL. The results show that the present invention induces apoptosis in these cells to some extent through a cascade of caspases and cathepsins since the viability was higher (up to 15%) when the cells were treated with z-VAD-fmk or E-64 d. Here, however, the results show that a non- caspase dependent pathway prevails since z-VAD-fmk and E-64d did not prevent the onset of apoptosis in a significant percentage. Concerning mitomycin C, the viability was lower for 48% when the present invention was combined with mitomycin C in the treatment of cells, which suggests that the present invention uses a different pathway of apoptosis induction than mitomycin C (mitomycin C damages DNA and in this way induces apoptosis).

Fig. 32 shows the percentage of viability of SH-SY5Y cells when they were treated with various concentrations of the inventive combination together with a combination of pan-inhibitors of caspases (z-VAD-fmk), cathepsins (E-64d) and with mitomycin C. The individual samples are of the following constitution: samples 1-3 represent controls (the cells were treated only with the above-mentioned inhibitors or with mitomycin C), sample 4 represents cells treated with a mixture of Capsicum 10 μg/mL + Allium 10 μg/mL in the ratio 1 :1 + HEPES 0,8 vol.%, in sample 5 the cells were treated with a mixture of Capsicum 10 μg/mL + Allium 10 μg/mL in the ratio 1:1 + HEPES 0,8 vol.% with the addition of z-VAD-fmk in a final concentration of 10 μM, sample 6 represents cells treated with a mixture of Capsicum 10 μg/mL + Allium 10 μg/mL in the ratio 1:1 + HEPES 0,8 vol.% with the addition of E-64d in a final concentration of 15 μM, and the sample 7 represents cells treated with a mixture of Capsicum 10 μg/mL + Allium 10 μg/mL in the ratio 1 :1 + HEPES 0,8 vol.% with the addition of mitomycin C in a final concentration of 2 μg/mL. The results show that the present invention induces apoptosis in these cells to some extent through a cascade of caspases and cathepsins since the viability was higher (up to 15%) when the cells were treated with z-VAD-fmk or E-64 d. Here, however, the results show that a non- caspase dependent pathway prevails since z-VAD-fmk and E-64d did not prevent the onset of apoptosis in a significant percentage. Concerning mitomycin C, the viability is actually higher for 12% when the present invention was combined with mitomycin C in the treatment of the cells, which suggests that the present invention uses different pathway of apoptosis induction than mitomycin C (mitomycin C damages DNA and in this way induces apoptosis).

Fig. 33 shows the lysosomal disruption caused by apoptosis after the incubation of cancer MCF-7 cells for 48 hours with the inventive combination. The lysosomal disruption was measured by LysoTracker Green DND according to standard procedures known to a skilled scientist. It is clear that the lysosomes are disrupted and in this cell line crucial for the onset of apoptosis since a 12% damage of organelles is sufficient for the cell to undergo apoptosis.

Fig. 34 shows the lysosomal disruption caused by apoptosis after the incubation of immortalized cells MEF wt for 48 hours with the inventive combination. The lysosomal disruption was measured by LysoTracker Green DND according to standard procedures known to a skilled scientist. It is clear that the lysosomes are disrupted and in this cell line crucial for the onset of apoptosis.

Fig. 35 shows the lysosomal disruption caused by apoptosis after the incubation of cancer cells SH-SY5Y for 24 hours with the inventive combination. The lysosomal disruption was measured by LysoTracker Green DND according to standard procedures known to a skilled scientist. It is clear that the lysosomes are disrupted and in this cell line crucial for the onset of apoptosis.

Fig. 36 shows the mitochondrial disruption caused by apoptosis after the incubation of cancer cells HepG2 for 48 hours with the inventive combination. The mitochondrial disruption was measured by MitoTracker Red CMXRos according to standard procedures known to a skilled scientist. It is clear that the mitochondria are disrupted and in this cell line crucial for the onset of apoptosis since a 12% damage of organelles is sufficient for the cell to undergo apoptosis.

Fig. 37 shows the mitochondrial disruption caused by apoptosis after incubation of the cancer cells MCF-7 for the 48 hours with the inventive combination. The mitochondrial disruption was measured by MitoTracker Red CMXRos according to standard procedures known to a skilled scientist. It is clear that the mitochondria are disrupted and in this cell line crucial for the onset of apoptosis.

Fig. 38 shows the mitochondrial disruption caused by apoptosis after the incubation of immortalized cells MEF Bid -/- for 24 hours with the inventive combination. The mitochondrial disruption was measured by MitoTracker Red CMXRos according to standard procedures known to a skilled scientist. It is clear that the mitochondria are disrupted and in this cell line crucial for the onset of apoptosis. It is evident from the above explanation that the combinations according to the invention have a selective effect on cancer cells from the cell lines SH-SY5Y, HeLa, MCF-7 and HepG2 where they trigger apoptosis, do not have a significant effect on immortalized cells from the cell line HaCaT and have a very low effect or have no effect on healthy cells from the cell line NHDF.

It is understood that the patent holder has an exclusive right over the present invention and said invention may be only used with the agreement of the patent holder.

Claims

1. A combination containing plant extracts, characterized by the following composition: an extract of a plant of the genus Capsicum, an extract of a plant of the genus Allium, and HEPES and elementary sulfur, preferably under the addition of an antibiotic, in various ratios with regard to the purpose, the ingredients being optionally suspended in a saline.

2. A combination according to claim 1, characterized in that it additionaly contains spices, namely oregano, caraway, kitchen salt and black pepper, each spice in the amount of from 0.01 to 1% by weight relative to the combination.

3. A process for preparing the combination according to claim 1 and 2, characterized in that plants of the genus Capsicum and plants of the genus Allium ared dried in the sun and, when completely dry, ground to a powder, the powder is suspended in a saline together with HEPES and elementary sulfur and optionally spices, namely oregano, caraway, kitchen salt and black pepper, and preferably with antibiotics, left to stand under occasional shaking for a few days so that the solid particles settle, then the suspension is filtered and the filtrate is either stored at 4°C to 10⁰C or dried to a powder.

4. Combination according to claims 1 and 2 for use as an agent for triggering apoptosis of cancer cells.

5. Combination according to claims 1, 2 and 4 characterized in that the cancer cells are a cell line of human cancer neurons SH-SY5Y, a cell line of breast cancer MCF-7, a cell line of human cancer cells from uterus HeLa and a cell line of human cancer cells from liver HepG2.

6. Pharmaceutical formulation, characterized in that, as the active ingredient, contains a combination according to claims 1 and 2, with the proviso that patients are on a vegetarian diet.

7. Formulation according to claim 6, characterized in that it is incorporated into liposomes.