WO2013088225A1

WO2013088225A1 - Waterborne extract derived from a plant bulb rich in sulphur substances containing selenium and glucose and supplemented with copper salts, having properties against tumors, fungi and dermatitis, including autoimmune dermatitis

Info

Publication number: WO2013088225A1
Application number: PCT/IB2012/002697
Authority: WO
Inventors: Guido MARCONE; Angelo DE MARTINO
Original assignee: Marcone Guido
Priority date: 2011-12-14
Filing date: 2012-12-13
Publication date: 2013-06-20
Also published as: CH705879A2

Abstract

Compound comprising garlic, preferably Allium sativum, and supplemented with selenium, copper salts and sugars, preferably glucose, said compound having an apoptotic, cytotoxic or cytostatic action against tumor cells.

Description

WATERBORNE EXTRACT DERIVED FROM A PLANT BULB RICH IN SULPHUR SUBSTANCES CONTAINING SELENIUM AND GLUCOSE AND SUPPLEMENTED WITH COPPER SALTS, HAVING PROPERTIES AGAINST TUMORS, FUNGI AND DERMATITIS, INCLUDING AUTOIMMUNE DERMATITIS .

The invention relates to a galenic preparation, i.e. a water- or hydroalcohol- borne extract derived from a plant rich in sulphur compounds and bio-active molecules (based on glucose-, selenium- and sulphur-compounds) and supplemented with a metal at concentrations well below the toxicity threshold, for medical and pharmacological uses.

The transition metal which is added to the present extract binds to molecules in the extract to create Cu-coordination (observable by EPR) and Se- coordination sugar-based complexes (glycated molecules) which allow the so- formed complexes to be more easily transported into cells in an increased amount, said complexes having specific cytostatic and cytotoxic functionalities against tumor cells, specific anti-fungal activities, and being pharmacologically active against different types of dermatitis, including autoimmune dermatitis (e.g. psoriasis). These molecules have the specific ability of being active in inducing programmed cell death (apoptosis) in tumor cells of various origins, with the ultimate advantage of having no cytotoxic and cytostatic effects on healthy cells and differentiated cells, as shown by the herein attached experiments.

In summary, the discovery relates to molecules which are present in our extract and capable of specific action against tumor cells while leaving healthy cells unaffected and showing no effect on tumor cells which are differentiated with retinoic acid (cells induced to have features similar to those of the original non- tumor cells), thereby exhibiting a selective efficacy against tumor cells and fungi.

From numerous studies and publications since the 2000's, it is clear that liquid extracts derived from Allium sativum show a cytostatic-cytotoxic action against liver tumor cells HepG2 without exhibiting any adverse cytotoxic and cytostatic effect.

It has also been shown that a copper-allicin complex synergically enhances the toxic action of copper against various strains of fungi and yeast. The copper- allicin complex has been shown to lower the toxic dose of copper required against these micro-organisms as well as to increase toxicity of copper against several strains of fungi, including certain phytopathogenic fungi, at a decreased concentration.

In the context of our discovery, the choice of using a water-born extract derived from a plant extract is based on a consideration with reference to certain publications (J. Nutr. <2001 Mar; 131 (3s): 955S-62S. Intake of garlic and its bioactive components. Amagase H., Petesch B.L., Matsuura H., Kasuga S., Itakura Y.) which show that liposoluble compounds derived from extracts of Allium sativum, including allicin, have a very high toxic effect and are detoxified as ketone bodies by liver tissue (measurement of acetone in breath following the intake of liposoluble compounds). On the contrary, compounds extracted via a water- soluble route show no toxicity while having beneficial effects on health and no toxic activity on healthy cells.

Now, the authors of the present invention have developed a method for the preparation of a special extract derived from a plant bulb rich in sulphur - selenium - glucose - compounds and free of allicin which has an effective and selective action against tumor cells after being supplemented with copper salts. The particular effectiveness of the combination according to the invention is greater than that obtained with an extract derived from a plant bulb rich in selenium without the addition of the metal. This is due to the synergistic activity occurring between the metal, the selenium compounds and the sulphur compounds existing in the aqueous extract. When the bulbs of the plants used are grown in a soil rich in selenium, the latter is able to form a series of selenium compounds by substituting for the sulphur. All these selenium- and sulphur- compounds are water-soluble and are those that bind copper in our extract to give rise to the specific cytotoxic synergy.

It was also surprisingly found that the combination according to the present invention is also effective in the treatment of mycoses and dermatitis, including autoimmune dermatitis (psoriasis).

Therefore, the specific object of the present invention is a combination of extracts derived from plant bulbs grown in a soil rich or enriched in selenium without the presence of allicin and with the addition of a copper salt such as, for example, copper sulphate, copper chloride, copper acetate, etc. Adding the copper salt to the aqueous or hydroalcoholic plant bulb extract results in the formation of Cu/molecule complexes which act in a selective manner. According to data available in the scientific literature, several water-soluble sulphur-, selenium- and glucose-based compounds identified in polymers may bind and/or coordinate to copper (see Table 1 and Figure 1 a).

Table 1

γ-glutamyl-Se-methyl-selenocysteine Y-Glu-SeMeCys

methylselenocysteine MeSeCys

selenomethionine SeMet

Selenate

Se-allyl-selenocysteine AllSeCys

Se-propyl-selenocysteine

γ-glutamyl-Se-methyl-selenomethionine

PSeCys

SeEt

SeCy

PrSeCys

SeCys₂

Y-GIu-SeMet

y-glutamyl-methyl-Se-selenocysteine Y-Glu-MeSeCys

Se-methyl-selenocysteine

selenate

dimethyl selenide

Methaneseleninic acid

selenomethionine

selenocysteine

Se-propyl-selenocysteine Se-cystathionine

γ-glutamyl-selenomethionine

selenite

dimethyl diselenide

allylmethyl selenide

selenocystathione

Se-1-propenyl selenocysteine selenoxide

Se-methyl-selenocysteine selenoxide

S-allyl-cysteine SAC

Trans-S-1-propenyl-L-cysteine SIPC

S-methyl-L-cysteine SMC

S-propyl-cysteine

S-ethyl-cysteine

γ-glutamyl-S-methylcysteine

γ-glutamyl-S-allylcysteine

γ-glutamyl-S-propylcysteine

S-allyl-N-acetylcysteine

S-allylsulphonyl alanine

S-alk(en)yl cysteine

S-allyl mercaptocysteine

alliin (S-allyl-L-cysteine sulphoxide)

allicin (diallyl thiosulphinate)

allyl methyl thiosulphinates

allyl trans-1-propenyl thiosulphinates

allyl thiosulphinates

allyl sulphide γ-glutamyl-S-allylcysteine

Y-glutamyl-S-trans-1 -propenylcysteine

Y-glutamyl-S-cis-1-propenylcysteine

S-allyl-cysteine

γ-glutamyl phenylalanine

dialk(en)yl thiosulphinates

S-alk(en)yl sulphenic acid

Therefore, all the molecules reported in Table 1 which form Se-metal- and SH-metal-complexes after being additioned with copper and show a cytotoxic and cytostatic activity against tumor cells of different origins as well as an antifungal activity and an activity against various forms of dermatitis, including autoimmune dermatitis, are also an integral part of this invention.

The spectrograms illustrated in Figures 1 and 2 for the subject extract of this invention are described below under the Drawings section.

The spectrograms of Figures 1 and 2 provide basic information on the complexes and organic molecules existing in our preparation, and they also show the glucose molecules present in the extract. Furthermore, this finding allows us to extend the use of the above-listed molecules containing a glucide linkage as well. This glucide linkage makes available glycated or glycosylated molecules (molecules having one or more glucose molecules attached thereto) which allow the compound to be transported across either the cell membrane or the fungal wall with an enhanced effectiveness. These complexes, i.e. metal-activated sugar- based molecules, could also explain the selectivity for membrane receptors existing only on tumor cells and not on healthy cells. As it is known from data available in the literature, the cell membrane of a tumor cell differs in a whole range of molecules, proteins and receptors which are present on both external and internal layers of the plasma membrane. This complex, i.e. the molecule-metal- sugar complex, recognizes only receptors existing on the membrane of tumor cells and not existing - for some reason - on non-tumor cells. These membrane receptors are the key to the selectivity of our compounds.

Another important fact to be emphasized is that the metal added to our extract give rise to molecules which do not only bind a sulphur or selenium atom, but also coordinate to more atoms, thereby showing a very high molecular stability.

This stability is also increased by the undoubted presence of glucose molecules which are linked in a stable manner. In fact, in experiments performed by heating our compound, its toxic, cytostatic and cytotoxic responsiveness and effectiveness did not change. As can be seen from the EPR (electron spin resonance) spectrogram illustrated in Panel C of Figure 6, the subject molecule or molecules show a metal-SH atom multiple coordination in which one of the stabilized bonds is undoubtedly the selenium bond. This spectrogram, together with the above-reported and mentioned spectrogram, provides a characteristic print (fingerprint) of the subject compound of the present invention. Explanation of the subject experiment and of the various spectra occurring in culture. As shown in Figure 3 of the Drawings section. The extract composition according to the present invention does not contain allicin (which is toxic and pungent in odor), is almost completely odorless, did not show toxicity problems, acts selectively against several tumor cell lines (liver hepatoma HepG2, neuroblastoma SHSY-5Y, gastric adenocarcinoma AGS, melanoma, colon cancer CaCo2, leukaemia U937), and shows no effect on either the same tumor cells which are differentiated or primary cells (fibroblasts, keratinocytes). The therapeutically effective molecules according to the present invention have a cell-specific action which is effective against tumor cells and ineffective towards healthy cells. They also have a specific action against several strains of fungi (Candida albicans strains of various origins and selected drug-resistant strains), thus showing a very selective and specific efficacy also in this case.

According to the present invention, it is claimed that the composition can be administered in different prescription forms, for example as an ointment or cream, as a vaginal suppository, a spray solution, a drinkable beverage or a dry extract (tablet or capsule), in an oral or intravenous form, as an aerosol for inhalation, or in a parenteral or intravenous administration form as highlighted in the practical experimentation illustrated in Figure 4 of the Drawings section, in which Panel A shows certain apoptotic markers, whereas Panel B shows that Apoptosis Inducing Factor, AIF, is increased in a time-dependent manner. Figure 5 shows that the extract-metal complex can reduce the number of lung metastases in treated mice. The experiment was carried out with 20 nude mice per test by injecting 1x10x6 million melanoma cells and, one day later, starting the treatment with intraperitoneal injections of the subject drug. Note: melanoma cells were inoculated intravenously into the tail. The disproportionately high amount of inoculated melanoma cells did not allow for a complete healing within the very short time span, however, the dramatic reduction of side effects is clear as a result of the intraperitoneal therapy for a period of 20 days.

Report on the effectiveness of the compound against tumor cells in total absence of toxicity or apoptotic action against healthy, differentiated and nonneoplastic cells.

The extract derived from a plant rich in sulphur compounds and bioactive molecules and supplemented with a metal at concentrations well below the toxicity threshold shows a selective anti-tumor activity. Such a selectively has been evaluated by testing the compound on several types of tumor cells and various types of differentiated and primary cells, in which the compound shows a cytotoxic and cytostatic activity which is only selective against tumor cells, while leaving the functionality of differentiated and primary cells unaffected. The used tumor cells are from different types of cancer formations, such as hepatoma cells, neuroblastoma cells, intestine cancer cells and circulating tumor cells (and other types of tumor cells). The product showed a high cytotoxic activity at concentrations of 1 % v/v of plant extract supplemented with the metal at a concentration of 100 μΜ. From Figure 6 of the Drawings section it can be seen that, at a concentration of 0.1 % of the extract with the addition of the metal at the concentration of 100 μΜ, a progressive time-dependent decrease of active cells occurs on hepatoma cells ΗβρΘ2.μ With reference to Figure 7 of the Drawings section, Panel A shows that the chosen metal exhibit no cytotoxic and cytostatic effects at a concentration of 100 μΜ. Panel B shows that increasing the concentration of the extract without changing the concentration of the metal results in an increased cytotoxicity. From Panel C it can be seen that increasing the concentration of the metal while leaving the concentration of the extract unchanged will result in cytotoxic effects. In Figure 8 of the Drawings section there are shown (A) the same hepatoma cells HepG2 treated with different concentrations of compound (0.1 % - 0.5% and 1 %) while keeping the metal at a steady concentration of 100 μΜ. The compound shows a very high cytotoxic activity^ The same differentiated cells (B) treated with the same concentrations show no cytotoxic and cytostatic activities, thus emphasizing the specificity of the compound only against tumor cells, while leaving the cellular functionality unaffected. The same compound was tested on differentiated cells of various types of tumor (in vitro), and it did not provide noteworthy results. The same result is obtained with primary cells (fibroblast cells and keratinocyte cells). This demonstrates that the metal-molecule complex is selective against tumor cells without showing any cytotoxic and cytostatic effect towards "normal" cells. Figure 9 shows that the metal crosses the cell membrane and it is concentrated within the nucleus. The metal was dosed in both the cytosol and the nucleus of the treated cells. All the metal is concentrated within the nucleus with no trace remaining in the cytosol. The extract-metal complex crosses the tumor cell membrane, passes into the cytosol and enters the nuclear membrane, where it is deposited at equimolar concentrations with respect to the concentration of treatment, as shown in Figure 10 under the Drawings section. Other metals were also tested, but they showed no effect on cell viability.

Claims

1. Compound comprising garlic, preferably Allium sativum, and supplemented with selenium, copper salts and sugars, preferably glucose, said compound having an apoptotic, cytotoxic or cytostatic action against tumor cells.

2. Compound comprising garlic, preferably Allium sativum, and supplemented with selenium, copper salts and sugars, preferably glucose, said compound having an action against fungi and autoimmune dermatitis as well as an action against psoriasis.

3. Compound according to claim 1 or 2, wherein the concentration of selenium in the garlic extract is at least 5 nmol/ml.

4. Compound according to any one of the preceding claims, wherein the copper salt is at a concentration from 20 M to 0.9 mM.

5. Compound according to any one of the preceding claims, wherein the copper salt is selected from the group consisting of copper sulphate, copper chloride, copper acetate or other copper salt capable of releasing Cu ions which are available to bind to Sh groups, Se groups, Glucose groups or any sugar.

6. Compound according to any one of the preceding claims, for the simultaneous, separate or sequential use of the garlic extract, the copper salt and the optional sugar against the above mentioned diseases.

7. Compound consisting of the combination as defined in any one of the preceding claims and supplemented with any active substance in combination with one or more pharmaceutically acceptable excipients and/or adjuvants.

8. Compound according to claim 7, wherein the supplemental active substance belongs to the group of anti-tumor substances.

9. Compound according to claim 7, wherein the supplemental active ingredient is selected from the group of antifungal substances.

10. Use of the compound as defined in any one of the preceding claims for the preparation of a medicament for the treatment of tumors.

1 1. Use of the compound as defined in any one of the preceding claims for the preparation of a medicament for the treatment of mycoses.

12. Use of the compound as defined in any one of the preceding claims for the preparation of a medicament for the treatment of psoriasis.

13. Compound according to the preceding claims, which administered topically, enterally, intramuscularly or intravenously.