WO2016157233A1 - Prunus spinosa extracts with anti-tumor activity - Google Patents

Abstract

This invention relates to the use of an extract of Prunus spinosa, alone or in combination with a stone and/or seed extract, in particular Prunus spinosa of the Trigno variety, in combination with amino acids, vitamins and minerals (NAC - Nutraceutical Activator Complex) in the medical and pharmaceutical fields. The composition according to the invention is in particular used for the treatment of human tumors.

Description

"Prunus spinosa extracts with anti-tumor activity"

Technical field of the invention

The present invention relates to a therapeutic composition of extracts of Prunus spinosa, in particular the Trigno variety, with a mixture of amino acids, vitamins and minerals (hereinafter indicated as NAC) and their uses in the medical and pharmaceutical field, in particular for the treatment of human tumors and as a food supplement.

Known art

Prunus spinosa, commonly known as sloe or blackthorn, is a thorny slow-growing deciduous bush of height averaging between 0 and 3 meters, which can reach 4-5 meters depending upon its spreading habit and the environment. It has irregular rather stiff foliage, while the spreading habit is generally bushy and compact. Its name is due to the young branches, which are initially furry and then become hairless, rough, very thorny, with sharp prickly, serrated and snagging spines. Prunus spinosa produces small white flowers which appear from March to May before the foliage.

Prunus fruits between September and October producing small drupes, known as sloes, which are slightly pedunculate and covered by a bluish bloom which becomes bluish-blackish when they are ripe. The drupes have a stone of 8-9 mm, with a rough surface, of a yellow-brown color, of flattened shape, which separates from the tenuous fruit pulp on ripening. The fruit has a sharp very acid and astringent flavor which becomes slightly sweeter after the first frosts.

There are documents in the scientific literature describing the antioxidant activity of Prunus spinosa and its potential use in the foodstuffs field (Fraternale D., Giampieri L., Bucchini A., Ricci D. "Antioxidant Activity of Prunus spinosa L. Fruit Juice" J. Food Sci. 12 (2009) 1665-1670.) The search for natural substances having an anti-tumoral action is becoming increasingly important in the scientific field, as is the need to have new therapeutic strategies to place alongside those already known, and recently the potential anti-tumor activity of Prunus spinosa extract, above all that with an enriched phenolic and/or anthocyanin portion, has been described (R. Guimaraes et al., Plant Foods Hum Nutr (2014) 69:37-42) .

There has been felt a need to obtain compositions based on an extract of Prunus spinosa characterized by greater antiproliferative efficacy for use in anti-tumor therapy.

Summary of the invention

It has now been found that extracts of Prunus spinosa, in particular the Trigno variety, when used in combination with a mixture of amino acids, vitamins and minerals known as NAC (Nutraceutical Activator Complex) show increased antiproliferative activity on tumor lines. This anti-proliferative activity is further increased when the Prunus drupe extract is combined with extracts obtained from the seeds or stones of those drupes.

It has also been demonstrated that the concentrations of such combinations used are not toxic to non-tumor cells, in particular human fibroblast cell lines.

Thus one object of this invention relates to an extract obtained from Prunus spinosa, in particular of Trigno variety, in combination with a mixture of amino acids, vitamins and minerals for use in the pharmaceutical and nutraceutical field as a food supplement.

The composition according to the invention may also be used in combination with other active ingredients known to those skilled in the art. Yet another object of the invention relates to a pharmaceutical composition comprising extracts of Prunus spinosa, alone or in combination with extracts from stones and/or seeds, preferably Prunus of the Trigno variety, in combination with NAC and at least one pharmaceutically acceptable vehicle for the treatment of human tumors in general, in particular colonorectal and uterine cervical adenocarcinomas, carcinoma of the breast and bronchoalveolar adenocarcinoma.

Yet another object of the invention relates to a pharmaceutical composition comprising extracts of Prunus spinosa, alone or in combination with seed or stone extracts, preferably Prunus of the Trigno variety in combination with NAC, and at least one chemotherapeutic agent selected as necessary by a person skilled in the art and a pharmaceutically acceptable vehicle for the treatment of human tumors in general, melanomas and metastatic melanomas, colonorectal and uterine cervical adenocarcinomas, carcinoma of the breast and bronchoalveolar adenocarcinoma in particular.

Yet another object of this invention relates to a method for the pharmaceutical treatment of human tumors in general, melanomas, colonorectal and uterine cervical adenocarcinomas, carcinoma of the breast and bronchoalveolar adenocarcinoma, in particular which comprises the administration of efficacious quantities of Prunus spinosa extract, alone or in combination with extracts of stones and/or seeds, preferably Prunus of the Trigno variety, in combination with a set of amino acids, vitamins and minerals.

Other objects will become evident from the following detailed description of the invention.

Brief description of the figures

Figure 1: Evaluation of the effect of different concentrations of Prunus spinosa var. Trigno (PsT) (86 mg/ml = 86 mg total dry weight, 50 mg/ml = 9.98 mg as total dry weight + PBS, 50 mg/ml = 9.98 mg total dry weight + 84 ul of NAC and 10 mg/ml = 1.98 mg total dry weight + 177 ul of NAC) on HCT116 colon carcinoma cells using the MTT colorimetric test. Staurosporine (1 μΜ), an inducer of apoptosis, is used as a positive control.

Figure 2: Evaluation of the effect of different concentrations of Prunus spinosa var. Trigno (PsT) (86 mg/ml = 86 mg total dry weight, 50 mg/ml = 9.98 mg total dry weight + PBS, 50 mg/ml = 9.98 mg total dry weight + 84 ul of NAC and 10 mg/ml = 1.98 mg total dry weight + 177 ul of NAC) on SW480 colon adenocarcinoma cells using the MTT colorimetric test. Staurosporine (1 μΜ) is used as a positive control.

Figure 3: Evaluation of the effect of different concentrations of Prunus spinosa var. Trigno (PsT) (86 mg/ml = 86 mg total dry weight, 50 mg/ml = 9.98 mg total dry weight + PBS, 50 mg/ml = 9.98 mg total dry weight + 84 ul of NAC and 10 mg/ml = 1.98 mg total dry weight + 177 ul of NAC) on A549 human bronchoalveolar adenocarcinoma cells using the MTT colorimetric test. Staurosporine (1 μΜ) is a positive control.

Figure 4: Evaluation of the effect of different concentrations of Prunus spinosa var. Trigno (PsT) (86 mg/ml = 86 mg total dry weight, 50 mg/ml = 9.98 mg total dry weight + PBS, 50 mg/ml = 9.98 mg total dry weight + 84 ul of NAC and 10 mg/ml = 1.98 mg total dry weight + 177 μΐ of NAC) on HeLa uterine cervical tumor cells, using the MTT colorimetric test, staurosporine (1 μΜ) is used as a positive control.

Figure 5: Growth curve for the HCT116 cell line after 24 hours of treatment with different concentrations of Prunus spinosa var. Trigno (PsT) (10 mg/ml = 1.98 total dry weight + 885 ul of NAC; 5 mg/ml = 1.03 mg total dry weight + 940 ul of NAC; 4.5.mg/ml = 0.86 total dry weight + 950 ul of NAC; 4 mg/ml.= 0.69 mg total dry weight + 960 ul of NAC; 2 mg/ml = 0.34 mg total dry weight + 980 ul of NAC; 1 mg/ml = 0.17 mg total dry weight + 990 ul of NAC; 0.5 mg/ml = 0.086 mg total dry weight + 995 ul of NAC and 0.1 mg/ml = 0.017 mg total dry weight + 999 ul of NAC).

Figure 6: Growth curve for the SW480 cell line after 24 hours of treatment with different concentrations of Prunus spinosa var. Trigno (PsT) (10 mg/ml = 1.98 total dry weight + 885 ul of NAC; 5 mg/ml = 1.03 mg total dry weight + 940 ul of NAC; 4.5 mg/ml = 0.86 total dry weight + 950 ul of NAC; 4 mg/ml = 0.69 mg total dry weight + 960 ul of NAC; 2 mg/ml = 0.34 mg total dry weight + 980 ul of NAC; 1 mg/ml = 0.17 mg total dry weight + 990 ul of NAC; 0.5 mg/ml = 0.086 mg total dry weight + 995 ul of NAC and 0.1 mg/ml = 0.017 mg total dry weight + 999 ul of NAC).

Figure 7 (A, B, C, D, Al, B2, CI, Dl): Effect of different concentrations of Prunus spinosa var. Trigno (PsT) + NAC on HCT116 and SW480 colonorectal tumor cells (series A, B, C, D and series Al, B2, CI, Dl respectively) by means of the clonogenic method. Control (Fig. A and Al); concentrations of 2 mg/ml = 0.34 mg total dry weight + 1960 ul of NAC (Fig. B and B2); 4.5 mg/ml =0.86 mg total dry weight + 1900 ul of NAC (Fig. C and CI); and 10 mg/ml = 1.98 mg total dry weight + 1770 ul of NAC (Fig. D and Dl). Each plate shows colonies of cells which tend to decrease with increasing Prunus spinosa var. Trigno (PsT) + NAC concentration.

Figure 8: Quantitative analysis of the clonogenic test on HCT116 and SW480 colon carcinoma cells (Fig. 8A and 8B), respectively.

Figure 9: Evaluation of the effect of different concentrations of Prunus spinosa var. Trigno stone extract (N) + NAC.

N10 mg/ml = 1.98 mg total dry weight + 177 ul of NAC;

N4 mg/ml = 0.69 mg total dry weight + 192 ul of NAC;

N2 mg/ml = 0.34 mg total dry weight + 196 ul of NAC.

Figure 10: Evaluation of the effect of different concentrations of Prunus spinosa var. Trigno (PsT) extract and stone extract (N) + NAC from the same drupe after 24 hours, 48 hours and 72 hours treatment on HCT116 colon carcinoma cells.

Figure 11: Evaluation of the cytotoxic effect of 5-fluorouracil chemotherapy (concentration 300 uM for 24 hours, 48 hours and 72 hours) in the response to treatment of HCT116 colon carcinoma cells.

Figure 12: Cytotoxic effect of treatment with 10 mg/ml = 1.98 mg total dry weight of Prunus spinosa var. Trigno (PsT ) + NAC, by the MTT test, after 24 hours, 48 hours and 72 hours on MCF-7 WT mammary adenocarcinoma cells.

Detailed description of the invention

It has been found that extracts of Prunus spinosa, in particular the Trigno variety, when used in combination with Nutraceutical Activator Complex (NAC) show strong antiproliferative activity on human colonorectal and uterine cervical adenocarcinoma, breast carcinoma and bronchoalveolar adenocarcinoma tumor lines.

The extract to which the invention relates is preferably obtained from fresh parts of Prunus spinosa, preferably of the Trigno variety. The parts (leaves, sprouts, drupes, stones, seeds and roots) are preferably harvested during their balsamic period.

After harvesting, once the samples have first been cleaned, they can be used for fresh extraction or dried in a ventilated stove until they are completely dehydrated.

Extracts may be obtained according to the methods conventionally used in the field of the preparation of plant extracts known to those skilled in the art, as for example described in "Botanicals, a Phytocosmetic Desk Reference" Frank S. D'Amelio, CRC Press, pg. 39- 48. In the context of this invention, and as described in the official pharmacopeia mentioned above, the term "dry plant tincture" indicates a water/alcohol extract obtained starting from dried plant material, while the term "fresh plant tincture" indicates a water/alcohol extract from fresh plant material. In the context of this invention by "fresh plant tincture" (FPT) is meant a water/alcohol extract (60-70°) obtained from the fresh plant in such a way that 1 g of FPT corresponds to 0.1 g of dehydrated plant. In the context of this invention by "dry plant tincture" (DPT) is meant an extract obtained from the dry plant.

By fresh or dry plant are meant all the parts of the plant, including the stones and seeds, which are just the stones without their ligneous and/or fibrous parts. Prunus stones comprise a seed enclosed in its ligneous and/or fibrous shell. In the context of this invention the stone extract is obtained using the same methods of extraction as for the other parts of the plant, although it is easier and more effective to perform stone extraction procedures on crushed or shredded stones, as well as only coarsely crushed stones, or stones which have been merely pierced or cracked.

The solvents preferably used to perform the extraction are the solvents typically used in the pharmaceutical field, such as for example polar solvents selected from the group comprising water, ethanol, glycerol, propanol, butanol, acetone, glycols such as ethylene-, propylene- and butylene glycol, ethyl acetate, hexane, methylene chloride, methanol, various ethers and their mixtures. A solvent mixture of ethyl alcohol and water in all proportions is particularly preferred.

Extraction is generally performed by macerating the plant material in the solvent, stirring from time to time, for a period of time from a few hours (above all in the case of dry material), for example 24 hours, up to 10 days, 20 days or even 40 days. It is also possible to use other methods of extraction such as percolation or extraction with continuous stirring, or extraction assisted by ultrasound and/or microwaves, as preferred in the industrial field, which yield extracts containing at least 0.5% by weight of total dry residue. The extract may be used as such or subjected to various degrees of concentration up to drying out in order to obtain a fluid, semi-fluid or dry extract. Drying may be performed using the methods conventionally used in this field such as for example evaporation under reduced pressure, spray drying and lyophilization. A quantity of dry extract of between 10 and 900 mg is used in the treatment of human beings.

Indicatively the range of quantities of dry weight used for the treatment of cell cultures is from 86 mg of total dry weight to 0.017 mg of total dry weight.

Prunus spinosa var. Trigno, a plant native to Molise which grows in the Bagnoli del Trigno area, is to be preferred, as will be seen in Table 1.

Table 1

Quality oiPrunus spinosa drupes collected in the following provincial areas

Prunus spinosa var. Trigno extract has proved to be particularly effective in comparison with what has been reported in the known art and in Table 1, and this greater efficacy is attributed to the special nature of the plant complex characterized by the presence of flavones and flavonols, phenolic acids and anthocyanins, and their special distribution. In fact, taking the Prunus extracts reported in Rafaela Guimaraes et al., Plant Foods Hum Nutr (2014) 69:37-42, as a reference, from the analyses performed on extracts of the Prunus spinosa native to Trigno, there is enrichment of flavones/flavonols and phenolic acids. Anthocyanins are present in quantities similar to those reported in the known art, but the distribution of the individual components is different, as explained in Table 2.

What has proved surprising is that the quantity of total phenolic acids varies from 35 to 40 mg/100 g of dry extract and the quantity of total flavones and flavonols varies from 60 to 70 mg/100 g of dry extract.

In particular HPLC analyses have revealed the presence of high quantities of:

quercetin 3-O-rutinoside, quercetin 3-O-glucoside and

kaempferol 3-O-rutinoside, which proved to be more than 50% by weight of the total flavones and flavonols;

3-O-caffeoylquinic and 4-O-caffeoylquinic acids, which proved to be more than 90% by weight of the total phenols.

The 3 and 4-O-caffeoylquinic acids belong to the family of chlorogenic acids, which are esters of caffeic acid and L-quinic acid respectively. 3- O-caffeoylquinic acid has the CAS number 327-97-9 and 4-O- caffeoylquinic acid is an isomer of it.

If Prunus extracts which do not meet these specifications are available it is recommended that the compounds mentioned above should be added in order to fall within the range of compositions indicated. All the compounds mentioned are known to those skilled in the art, who are capable of identifying and preparing them using a normal knowledge of organic chemistry.

Analyses of therapeutic efficacy performed on Prunus extracts have also provided evidence of a special effect, in that when the extract of the whole fruit (or drupe) is combined with the extract obtained from crushed stones or seeds the therapeutic effect is increased and therefore the effective quantity which has to be administered can be reduced, reducing any collateral effects, which are always possible in the context of therapeutic treatment.

The anti-proliferative effect of drupe extracts combined with seed or stone extracts has proved to be synergistic, and the same cytotoxic effect has in fact been observed when the administered dose is reduced by 2.5 times (see an example in Figure 10).

The extract obtained from stones of Prunus spinosa may be prepared in a similar way to the extract starting from the whole fruit, provided that the stones themselves are shredded, breaking up the ligneous shell or using the seeds directly without their ligneous and/or fibrous shell.

The combination of the extract of drupes of Prunus spinosa var. Trigno and the seed or stone extracts can also have NAC added to it.

The NAC mixture of amino acids, vitamins and mineral salts comprises:

at least one amino acid selected from L-arginine, L-asparagine, L-aspartic acid, L-cysteine (sodium salt), L-glutamic acid, L-glutamine, glutathione, glycine, L-histidine, L-proline, L-isoleucine, L-leucine, L-lysine HC1, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine (sodium salt), L-valine and mixtures thereof in quantities from 0.01 to 10% of the whole; at least one vitamin selected from biotin, D-calcium pantothenate, choline chloride, folic acid, I-inositol, nicotinamide, p-aminobenzoic acid, pyridoxal HC1, riboflavin, thiamine HC1, vitamin B12 and mixtures thereof in quantities from 0.0001% to 10% of the whole;

at least one mineral salt selected from Ca (N03)2, KC1, NaCl, NaHC03, Na2HPO₄ and mixtures thereof in quantities from 0.01% to 10% of the whole;

D-glucose may be added to the mixture in a quantity from 0 to 5%, preferably from 0.1 to 5%;

the remainder up to 100 being water.

The relative quantities expressed as % by weight are as follows:

amino acids 30-50%;

vitamins 5-30%;

mineral salts 30-50%;

glucose 0-15%.

NAC can be used in combination with Prunus extracts in the following ratios by weight (Prunus extract)/iVAC varying within the range 0.001-10, preferably 0.01 - 1.

The extract from Prunus spinosa, in particular of the Trigno variety, alone or in combination with the seed and/or stone extract, when used in combination with NAC in accordance with the invention shows higher anti-proliferative activity than that demonstrated by Prunus extract used alone.

It is hypothesized that NAC can encourage metabolization and make the quercetin present in the extract less unstable, thus increasing the antitumoral activity of Prunus extracts.

This result has been observed on different human tumor lines, in particular colonorectal and uterine cervical adenocarcinoma and bronchoalveolar carcinoma. Prunus spinosa in combination with the Nutraceutical Activator Complex according to the invention is capable of reducing cell survival in tumor lines but not in normal cell lines.

In accordance with the invention, Prunus spinosa extract, alone or in combination with the seed or stone extracts, in particular Prunus of the Trigno variety, in combination with NAC, can advantageously be used in the pharmaceutical and nutraceutical fields as a food supplement.

In accordance with the invention the Prunus spinosa extract, alone or in combination with the seed or stone extracts, in particular Prunus of the Trigno variety, in combination with the Nutraceutical Activator Complex may advantageously be used for the treatment of human tumors.

Advantageously the Prunus spinosa extract may be combined with an extract from the stones and/or seeds of Prunus spinosa, preferably in equal quantities.

In a preferred embodiment the Prunus spinosa extract, alone or in combination with the seed and/or stone extract, in particular of the Trigno variety, used in combination with NAC, is used for the treatment of colonorectal and uterine cervical adenocarcinoma, carcinoma of the breast and bronchoalveolar adenocarcinoma.

In accordance with the invention the Prunus spinosa extract, alone or in combination with the seed and/or stone extract, in particular Prunus of the Trigno variety, in combination with NAC, may be administered as an active ingredient in the form of a pharmaceutical composition for the treatment of human tumors. The mean quantity of active principle which has to be administered will vary on the basis of the patient's condition and the recommendations and prescriptions of a qualified doctor.

In addition to the Prunus spinosa extract, alone or in combination with the seed and/or stone extracts , in particular Prunus of the Trigno variety, in combination with NAC, the pharmaceutical composition comprises at least one pharmaceutically acceptable vehicle.

Conveniently the said medicinal product is in the form of a preparation for enteral, preferably oral, parenteral or topical administration, but other forms are also suitable for implementing this invention. Solid and semi-solid forms such as tablets, capsules, granulates, powders, ointments, gels, unguents or emulsions and liquid formulations such as syrups and elixirs are particularly preferred.

The dry weight ratio between Prunus extract and NAC varies within the range 0.001 - 10, preferably 0.01 - 1. The extracts may be those obtained from various components of the plant as mentioned above, in particular they may be extracts from fruits or drupes, seeds or stones or combinations thereof.

In accordance with the invention the Prunus spinosa extract, alone or in combination with the seed and/or stone extract, in particular Prunus of the Trigno variety, in combination with NAC, can advantageously be used in combination with one or more chemotherapeutic agents, according to indications by those skilled in the art, to potentiate activity and increase response to the treatment.

With reference to the human colonorectal and mammary carcinoma lines examined, there is the possibility that Prunus spinosa can be used alone or in combination with the seed and/or stone extract plus NAC in chemosensitizing therapy, combining this natural compound with conventional drugs used in clinical treatment such as 5- fluorouracil and doxorubicin. This combination can increase the efficacy of conventional chemotherapy, with the possibility of reducing doses and thus also undesired collateral effects.

Conveniently the composition according to the invention may be in kit form comprising the Prunus extract alone or in combination with the seed and/or stone extract, and NAC, individually or as mixtures, in predefined dosing units, and instructions for use, possibly in combination with chemotherapy drugs for simultaneous or combined, sequential or delayed administration.

This invention will now be described with reference to the following examples which are not to be regarded as restricting the scope of the invention.

Examples

Characterization of extracts ofPrunus spinosa var. Tris.no

Plant sample

Sloe fruits (Prunus spinosa L., Trigno variety (PsT^®)) were collected manually at the end of October 2014 when the drupes were completely ripe in the Bagnoli del Trigno area, located approximately 35 km to the northeast of Isernia (Molise Region, Italy, latitude 41° 42' N, longitude 14° 27' E, altitude 650 m above sea level.). The Molise Region lies in the eastern part of the Apennines of central/southern Italy and is characterized by a typically Mediterranean climate. The area is characterized by an average annual rainfall of 850 mm and an average temperature of 12.6°C. This area has a low resident population density, very little vehicle traffic and therefore a very low level of pollution, including fine dusts.

The morphological characteristics of the sloes were identified in accordance with what is stated in the volume Flora d'ltalia (Pignatti, 2002). The drupes were collected in coolbags and subsequently transferred to the laboratory where they were stored in a freezer at -20°C awaiting subsequent analyses. Each determination was carried out on three representative samples. The results are expressed as mean ± standard deviation. Process of extraction from the drupes

Determination of phenol and flavonoid compounds

For the HPLC separation of phenol and flavonoid compounds the extracts were prepared as described by Guimaraes et al. (2013). Briefly, starting from the extraction procedure, each sample of dried sloes underwent treatment to encourage extraction of its components in an 80:20 (v/v) mixture of methanol and water at ambient temperature and was subsequently centrifuged at 150 rpm for one hour. The extract obtained was filtered using Whatman No. 4 type filter paper. The residue was re-extracted twice with further additions of 30 ml of the 80:20 (v/v) methanol/water mixture. The extracts so obtained were dried (Buchi R-210 Rotavapor) to remove the methanol. For subsequent purification the aqueous phase was separated on a C- 18 SepPak_Vac 3 cc cartridge column (Phenomenex).

This preliminary stage was followed by analysis of the phenols and flavonoids by HPLC-DAD-ESI/MS. The extracts were analyzed using a Hewlett-Packard 1100 chromatograph (Agilent Technologies) fitted with a diode array detector (DAD) coupled to an HP Chem Station data processing unit (rev. A.05.04). A column of the Waters Spherisorb S3 ODS-2 C18, 3 lm type (4.6-150 mm) thermostatted to 35°C was used. The solvent used was (A) 0.1% formic acid in water and (B) acetonitrile. Double on-line detection was carried out by DAD using two wavelengths at 280 and 370 nm respectively and a mass spectrometer (MS) connected to the HPLC via the DAD outlet. The MS determination was carried out using an API 3200 Qtrap instrument (Applied Biosystems, Darmstadt, Germany) fitted with an ESI source and a mass analyser operated by Analyst 5.1 type software. The MS detector was programmed to acquire the results in two analysis modes: "enhanced MS (EMS)" and "enhanced product ion (EPI)". The EMS mode was used to show the entire spectrum so that an overall view of the ions present in the sample could be obtained. The phenolic compounds present in the sample were characterized by their UV and mass spectra, and retention times comparable to those of standard substances. Quantitative determination of the phenolic compounds was carried out using calibration curves obtained using known standards in different concentrations. The substances used as standards were caffeic acid, chlorogenic acid, gallic acid, isorhamnetin 3-O-glucoside, isorhamnetin 3-O-rutinoside, kaempferol 3-O-glucoside, kaempferol 3- O-rutinoside, quercetin 3-O-glucoside and quercetin 3-O-rutinoside. The results so obtained were expressed as mg per 100 g of dry substance.

Determination of anthocyanins

The anthocyanins present in the sloe sample were separated by HPLC as described by Guimaraes et al. (2013). Briefly each sample was extracted in methanol enriched with 0.5% TFA and subsequently filtered on Whatman No. 4 type filter paper. The residue was extracted twice more using 30 ml of the same solution. Once pooled the sample was dried to remove the methanol. This was followed by a stage of further purification loading the sample onto a C-18 SepPak_Vac 3 cc cartridge column (Phenomenex). Analysis by HPLC-DAD-ESI/MS was then performed. The extract was analysed as already described by Guimaraes et al. (2013). The separation was performed on a reverse phase column thermostatted to 35°C (AQUA, reverse phase C18 column, 5 lm, 150-4.6 mm i.d (Phenomenex)). The solvents used were: (A) 0.1% TFA in water and (B) 100% acetonitrile. A double analysis was performed using DAD at a wavelength of 520 nm and MS with the same arrangements described previously for the determination of phenols. EMS and ESI modes were used to acquire the whole spectrum and to determine the ion fragments found. Anthocyanins present in the sample were characterized by UV and mass spectra and by retention times after comparison with those for standard substances. Calibration curves obtained by injecting variable quantities of known substances such as cyanidin 3-O-glucoside and peonidin 3-O-glucoside into the system were used for quantitative analysis. The results were expressed as \xg per 100 g of dry substance. Analysis of total phenol compounds content

The total content of phenol compounds was determined by comparing results with a standard (gallic acid). The results were expressed as mg of GAE per gram of extract in methanol using the Folin-Ciocalteu method described by Singleton et al. (1999).

Results and discussion

Sloe drupe extract appears to be characterized by the presence of bioactive compounds such as phenols, flavonoids and anthocyanins. In particular, with regard to the anthocyanes, which are present in a total quantity of 38.36 mg/100 g of dry matter, the most representative compounds are 3-O-caffeoylquinic acid and 4-O-caffeoylquinic acid at 26.31 mg/100 g and 8.97 mg/100 g respectively. Among the flavonoids the most representative compounds are: quercetin 3-O-rutinoside (21.98 mg/100 g), quercetin 3-O-glucoside (8.92 mg/100 g) and kaempferol 3-O-rutinoside (6.22 mg/100 g). Anthocyanins are represented by: cyanidin 3-O-rutinoside (42.31 pg/100 g), cyanidin 3-O- glucoside (24.25 pg/100 g), peonidin 3-O-rutinoside (15.53 pg/100 g) and peonidin 3-O-glucoside (13.91 pg/100 g). Finally the total phenols content in a methanol extract was found to be 4.91 ± 0.03 mg GAE/g.

Analysis of the flavone, flavonoid and anthocyanin content of the water-alcohol extract of sloe drupes indicates the presence of many bioactive compounds characterized by significant antioxidant, antiproliferative and antitumoral activity when tested individually. The beneficial properties of the sloe extract tested, and in general all medicinal plants, must be attributed to the phytocomplex, characterized by high molecular diversity which also results from the environment and cultivation technique used (Formisano et al., 2015). From a comparative analysis (see Table 2) with what is reported in the literature (Guimaraes et al., 2013) for sloe drupes harvested in an area in Portugal, those harvested in Bagnoli del Trigno show a larger quantity of compounds obtained from secondary metabolism in the plants. In particular the contents of the following were significantly greater in the drupes from the Molise area: 4-O-caffeoylquinic acid (+163.0%), quercetin 3-O-glucoside (+555.8%) and kaempferol 3-0- rutinoside (+227.3%). These differences are also clearly evident if the phenols and flavonoid contents determined in the Bagnoli del Trigno drupes, (+30.5%) and (+16.8%), respectively, are compared with those from the Portuguese area. In the case of anthocyanins, although greater quantities of cyanidin 3-O-glucoside and cyanidin 3-O- rutinoside are present in the Bagnoli del Trigno drupes, the smaller quantity of peonidin 3-O-rutinoside and other compounds has resulted in the content of these compounds being comparable for the two areas in terms of quantity. In comparison with the Portuguese sample, no apigenin pentoside, quercetin pentosylhexoside, quercetin acetylhexoside and cyanidin 3-O-acetylglucoside were found to be present in the sample from the Bagnoli del Trigno area.

Table 2 - Composition of Prunus spinosa drupes originating from an area of Portugal (according to Guimaraes et al., 2014) and the Bagnoli del Trigno (IS) area.

Data expressed as mg/100 Portugal Difference % Bagnoli del g of dry matter for Bagnoli del Trigno Trigno

"phenolic acids" and vs Portugal

"flavone/ols", and pg/100 g

of dry matter for

"anthocyanins". Compounds

3-O-Caifeoylquinic acid 22.09 ± 0.11 19.10 26.31±0.26

3-p-Coumaroylquinic acid 0.8 ± 0.01 -3.75 0.77±0.02

4-O-Caffeoylquinic acid 3.41 ± 0.03 163.05 8.97±0.08

3-O-Feruloylquinic acid 1.76 ± 0.04 31.25 2.31±0.01

Apigenin pentoside 1.32 ± 0.03 -100.00 0

Total phenolic acids 29.38 ± 0.05 30.57 38.36±0.19

Caffeoyl hexoside 1.07±0.0 -7.48 0.99 ± 0.01

Quercetin pentosylhexoside 1.36 ± 0.04 16.18 1.58 ± 0.02 Quercetin rhamnosylhexoside 2.22 ± 0.03 -9.46 2.01 ± 0.01

Quercetin 3-O-rutinoside 15.6 ± 0.33 40.63 21.98 ± 0.09

Quercetin pentosylhexoside 6.83 ± 0.29 -100.00 0

Quercetin 3-O-glucoside 1.36 ± 0.02 555.88 8.92 ± 0.04

Quercetin hexoside 4.7 ± 0.5 4.68 4.92 ± 0.03

Kaempferol 3-O-rutinoside 1.9 ± 0.05 227.37 6.22 ± 0.02

Quercetin hexosylrhamnoside 7.17 ± 0.42 4.74 7.51 ± 0.05

Quercetin pentoside 8.84 ± 0.26 -9.62 7.99 ± 0.06

Quercetin rhamnoside 1.3 ± 0.02 -3.08 1.26 ± 0.01

Quercetin acetylhexoside 1.9 ± 0.05 -100.00 0

Quercetin acetylrutinoside 1.0 ± 0.01 24.00 1.24 ± 0.02

Total flavone/ols 55.28 ± 0.16 16.90 64.62 ± 0.58

Cyanidin 3-O-glucoside 19.83 ± 0.05 22.29 24.25 ± 0.19

Cyanidin 3-O-rutinoside 31.12 ± 0.11 35.96 42.31 ± 0.35

Peonidin 3-O-glucoside 10.73 ± 0.16 29.64 13.91 ± 0.11

Peonidin 3-O-rutinoside 34.47 ± 0.03 -54.95 15.53 ± 0.09

Cyanidin 3-O-pentoside 1.49 ± 0.12 9.40 1.63 ± 0.01

Cyanidin 3-O-acetylglucoside 1.77 ± 0.01 -100.00 0

Peonidin 3-O-acetylglucoside 0.73 ± 0.05 35.62 0.99 ± 0.01

Total anthocyanins 100.14 ± 0.075 -1.52 98.62 ± 0.63

Process for preparation ofPrunus spinosa (PsT) fresh plant tincture:

Process for preparation of the water/alcohol extracts.

Macerations are performed in 60° alcohol for 40 days (with frequent mixing) using a drug/extract (D/E) ratio of 1/10 calculated on dry weight.

In detail: the moisture content is determined in order to know the dry weight of the sample; this value is multiplied by 10 (D/E ratio 1/10) to know how much solvent to use (60° alcohol). The 60° alcohol is prepared by diluting 96° alcohol, but bearing in mind the quantity of water already present in the Prunus (previously determined by the moisture analysis).

After 40 days maceration the solvent is separated and the Prunus is pressed to recover all the liquid, which is allowed to settle for a day and then filtered. Example: 5 kg of whole drupes (moisture content 55%)→ dry weight: 2.25 kg Preparation of 2.25 x 10 = 22.5 kg of 60° alcohol

Dilution from 96.3° alcohol: 22.5 x 60 / 96.3 = 14 kg of 96.3° alcohol to be used Theoretical water to be used: 22.5 - 14 = 8.5 kg

2.75 kg of water already present in the Prunus (55% moisture content) is subtracted from these 8.5 kg; thus 5.75 kg of water must be used.

In conclusion: the solvent for the maceration of 5 kg of whole drupes must be prepared by mixing 14 kg of 96.3° alcohol and 5.75 kg of water.

The water/alcohol solution is concentrated to remove the alcohol and the concentrate is mixed with NAC (116 ml of water/alcohol Prunus solution + 886 ml of NAC = 1 liter). The solution so obtained is lyophilized or spray-dried to obtain the dry material which, after mixing with suitable excipients, will be packed in capsules.

Process for preparation of the extract + NAC combination:

One aliquot of PsT prepared in accordance with the previous example is mixed with an aqueous solution of NAC having the following composition:

L-arginine 200 mg/litre,

L-asparagine ¾O 56.82 mg/litre,

L-aspartic acid 20.00 mg litre,

L-cysteine (sodium salt) 59.15 mg/litre,

L-glutamic acid 20.00 mg/litre,

L-glutamine 300.0 mg/litre,

Glutathione 1.0 mg/litre,

Glycine 10.0 mg/litre,

L-histidine 15.00 mg/litre,

L-proline 20.00 mg/litre,

L-isoleucine 50.00 mg/litre,

L-leucine 50.00 mg/litre,

L-lysine HC1 40.00 mg/litre, L-methionine 15.00 mg/litre,

L-phenylalanine 15.00 mg litre,

L-proline 20.00 mg/litre,

L-serine 30.00 mg/litre,

L-threonine 20.00 mg/litre,

L-tryptophan 5.00 mg/litre,

L-tyrosine (sodium salt) 24.86 mg/litre,

L-valine 20.00 mg/litre

Biotin 0.20 mg/litre,

D-calcium pantothenate 0.25 mg/litre,

Choline chloride 3.00 mg/litre,

Folic acid 1.00 mg/litre,

I-inositol 35.00 mg/litre,

Nicotinamide 1.00 mg/litre,

p-aminobenzoic acid 1.0 mg/litre,

Pyridoxal HC1 1.0 mg/litre,

Riboflavin 0.20 mg/litre,

Thiamine HC1 1.00 mg/litre

Vitamin B12 0.005 mg/litre,

Ca (N0₃)₂ 69.49 mg/litre,

KC1 400.00 mg/litre,

MgS0₄-7H₂0 100.0 mg/litre,

NaCl 6000 mg/litre,

NaHC0₃ 2000 mg/litre,

Na₂HP0₄ 800.7 mg/litre,

D-glucose 2000 mg/litre,

The NAC solution had a dry residue of 12.406 mg/litre.

Starting from an 86 kg/ml water/alcohol stock solution of Prunus spinosa var. Trigno (PsT) corresponding to 86 mg total dry weight the following dilutions of Prunus spinosa were made in different experiments using singly, NAC, PBS and physiological solution:

• P 50 mg/ml = 9.98 mg of total dry weight

• P 10 mg/ml = 1.98 mg of total dry weight P 5 mg/ml = 1.03 mg of total dry weight P 4.5 mg/ml = 0.86 mg of total dry weight P 4 mg ml = 0.69 mg of total dry weight P 1 mg/ml = 0.17 mg of total dry weight P 0.5 mg/ml = 0.086 mg of total dry weight P 0.1 mg/ml = 0.017 mg of total dry weight P 2 mg/ml = 0.34 mg of total dry weight

In order to confirm the efficacy of the Nutraceutical Activator Complex (NAC), as a means for diluting the PsT, both physiological solution and PBS were used. PsT + PBS (phosphate buffered saline, Sigma) and in a subsequent series of experiments PsT + 0.9% physiological solution in water were used instead of the Prunus spinosa var. Trigno (PsT) + NAC. The results of this study demonstrated that only the combination Prunus spinosa var. (PsT) + NAC induces a significant reduction in cell proliferation in the tumor lines analysed.

To simplify representation of the graphs only the values obtained for the combination Prunus spinosa var. Trigno (PsT) + PBS will be indicated.

• Prunus spinosa (PsT) + NAC

• 0.9% physiological solution: prepared by adding 9 g of NaCl to 1 1 of distilled water.

• PBS (phosphate buffered saline, Sigma): prepared by adding 1 tablet to every 200 ml of distilled water.

Staurosporine (STS) was used as a positive control to induce apoptosis in a concentration of 1 uM. Process for preparing the water I alcohol stone extracts:

Macerations in 60° alcohol are performed for 40 days (with frequent mixing) using a drug/extract (D/E) ratio of 1/10 calculated on dry weight.

In detail: the moisture content is determined in order to know what the dry weight of sample is; this value is multiplied by 10 (D/E ratio 1/10) in order to know how much solvent (60° alcohol) to use. The 60° alcohol must be prepared by diluting 96° alcohol, but bearing in mind the quantity of water already present in the stones (previously determined by moisture analysis).

After 40 days maceration the solvent is separated and the stones are pressed to recover all the liquid, which is allowed to settle for a day and then filtered.

In conclusion: the solvent for the maceration of 5 kg of crushed stones is prepared by mixing 14 kg of 96.3° alcohol and 5.75 kg of water.

The water/alcohol solution is concentrated to remove the alcohol and the concentrate is mixed with RPMI-1640 medium (116 ml of Prunus s.i. + 886 ml of medium = 1 litre). The solution so obtained is lyophilized or spray-dried to obtain the dry material which, after mixing with suitable excipients, will be packed in capsules.

• N 10 mg/ml = 1.98 mg of total dry weight

• N 4 mg ml = 0.69 mg of total dry weight

• N 2 mg/ml = 0.34 mg of total dry weight Cell cultures and cell treatments used

HCT116, ATCC CELL BIOLOGY COLLECTION Number CCL-247. Human colonorectal carcinoma cells; culture medium used for growth: RPMI 1640, Euroclone.

SW480, ATCC Number CCL-228. Human colonorectal carcinoma cells; culture medium used for growth: RPMI 1640, Euroclone.

HeLa, ATCC Number CCL-2. Uterine cervix tumor cells; culture medium used for growth: RPMI 1640, Euroclone.

A549, ATCC Number CCL-185. Human bronchoalveolar adenocarcinoma cells; culture medium used for growth: RPMI 1640, Euroclone.

MCF-7 WT, ATCC Number HTB-22. Mammary adenocarcinoma cells; culture medium used for growth: DMEM, Euroclone.

The cell lines used in the experiments indicated were treated with PsT alone, PsT + NAC and PsT + PBS, N + NAC and PsT + N + NAC when their state of confluence reached 75%. The treatment time was 24 hours in all the experiments. Each point was performed in sextuplicate and each experiment was independent and repeated in triplicate.

Cell proliferative activity assessment test (MTT assay)

"MTT assay", where the acronym indicates the compound 3-(4,5- dimethylthiazol-2-il)-2,5-diphenyltetrazolium bromide, is a standard colorimetric test for measuring cell proliferation. This test measures the activity of the enzymes, above all mitochondrial enzymes, which reduce MTT to formazan, imparting a blue/purple color to the substance. The mitochondrial enzyme succinate dehydrogenase is in fact only active in living cells and severs the tetrazolium ring of the MTT (a substance of a yellow color) with the consequent formation of formazan crystals (a blue salt), which are insoluble in water.

The reaction is assessed and measured by reading the samples using a light meter at a wavelength of 570 nm.

This cell survival test is used in pharmacological research and assesses the mitochrondrial damage caused to cell vitality by drugs or other potentially toxic substances. In the samples with larger cell populations a more intense purple color is observed and can then be evaluated quantitatively.

Figure 1 shows the evaluation of the effect of different concentrations of Prunus spinosa var. Trigno (PsT) (86 mg/ml = 86 mg of total dry weight; 50 mg/ml = 9.98 mg of total dry weight + 84 ul of NAC and 10 mg/ml = 1.98 mg of total dry weight + 177 ul of NAC) on HCT116 colonorectal carcinoma cells. This cell line is positive for TGF beta 1 and TGF beta 2 (transforming growth factor beta 1 and 2). Prunus spinosa var. Trigno (PsT) in absolute amount (concentration 86 mg ml), NAC alone and CTR (control for cells with their growth medium) does not have any adverse effect on tumor cell survival; concentrations of 50 mg/ml + 84 ul of NAC and 10 mg/ml + 177 ul of NAC reduce tumor cell survival. The inhibition of tumor survival is greater if the concentration of Prunus spinosa var. Trigno (PsT) dry extract is reduced. Staurosporine (1 uM), an inducer of apoptosis, is used as a positive control.

Figure 2 shows the evaluation of the effect of treatments on another colon adenocarcinoma line, SW480, deriving from another patient with a mutation of the ras proto-oncogene. Prunus spinosa var. Trigno (PsT) in an absolute amount (concentration 86 mg/ml), NAC alone and CTR (control for cells with their growth medium) do not have any adverse effect on cell survival, but instead diluted concentrations of Prunus spinosa var. Trigno (PsT) 50 mg/ml + 84 ul of NAC and Prunus spinosa var. Trigno (PsT) 10 mg/ml + 177 ul of NAC reduce tumor cell survival. Staurosporine (1 uM) is used as a positive control.

Figure 3 shows the evaluation of the effect of different concentrations of Prunus spinosa var. Trigno (PsT) (86 mg/ml = 86 mg of total dry weight; 50 mg/ml = 9.98 mg of total dry weight + 84 ul of NAC and 10 mg/ml = 1.98 mg of total dry weight + 177 ul of NAC) on A549 human bronchoalveolar adenocarcinoma cells. Prunus spinosa var. Trigno (PsT) in an absolute amount (concentration 86 mg/ml) does not have an adverse effect on cell survival, diluted concentrations of 50 mg/ml + 84 ul of NAC, and 10 mg/ml + 177 ul of NAC reduce cell survival. Staurosporine (1 uM) is used as a positive control.

Figure 4 shows the evaluation of the effect of different concentrations of Prunus spinosa var. Trigno (PsT) (86 mg/ml = 86 mg of total dry weight; 50 mg/ml = 9.98 mg total dry weight + 84 ul of NAC and 10 mg/ml = 1.98 mg total dry weight + 177 ul of NAC) on HeLa uterine cervical tumor cells. Prunus spinosa var. Trigno (PsT) in an absolute amount (concentration 86 mg/ml) does not adversely affect cell survival; the diluted concentrations 50 mg ml + 84 ul of NAC and 10 mg/ml + 177 ul of NAC reduced cell survival. Staurosporine (1 uM) is used as a positive control.

In Figure 12 the cytotoxic effect of treatment with 10 mg/ml = 1.98 mg total dry weight of Prunus spinosa var. Trigno (PsT) + 177 ul of NAC, was evaluated after 24 hours, 48 hours and 72 hours on MCF-7 WT mammary adenocarcinoma cells using the MTT test.

A reduction of 20% in cell survival was observed after 24 hours of treatment, 35% after 48 hours, and at 72 hours vital tumor cells had reduced by 58%.

In the known art it has been found that Prunus spinosa of a variety other than Trigno, in the same concentration used by us for 48 hours treatment, does not inhibit mammary carcinoma tumor cell growth ( Guimaraes et al., 2013). This result confirms the different and greater activity of Prunus spinosa var. Trigno due to the different distribution of chemical components in the phytocomplex.

Cell growth curves

A test was performed to evaluate cell growth inhibition after pharmacological treatment with increasing concentrations of PsT + NAC. In order to evaluate cell vitality an aliquot of cell suspension was combined with 0.4% of Trypan Blu colorimetric solution in a 1:1 ratio after each treatment. The number of cells was counted for each sample using a cell-counting camera. The percentage cell survival was obtained from the ratio between the mean number of negative Trypan Blu cells (vital cells with an intact cell membrane) and the control sample.

Using the total cell count for each treatment compared with the control cells it is also possible to evaluate whether a treatment induces inhibition of tumor growth or is effectively cytotoxic. Figure 5 shows the growth curve for the HCT116 cell line. With a concentration of 4 mg/ml = 0.69 mg total dry weight + 960 ul of NAC rapid change in the gradient of the curve, representing a fall in the number of vital cells, can be observed.

Figure 6 shows the growth curve for the SW480 cell line. At a concentration of 4 mg/ml = 0.69 mg total dry weight + 960 ul of NAC a rapid change in the gradient of the curve representing a fall in the number of vital cells can be observed.

A dose-dependent decrease in cell growth can be observed in the tumor lines examined, from the concentration of 2 mg/ml = 0.34 mg total dry weight + 980 μΐ of NAC. Cell clonosenicitv test

Cytotoxicity was evaluated using a clonal efficiency test, that is by determining the ability of the cells to reproduce and form macroscopic colonies (> 50 cells) following 24 hours of treatment with PsT + NAC in different concentrations. After a certain number of days, depending upon the replicative capacity of the cell line, the colonies were fixed using 95% ethanol, stained with methylene blue and counted by hand. Percentage cell survival was determined from the ratio between the mean number of colonies formed from the treated cells and those formed from the corresponding control cells.

Figure 7 shows the effect of different concentrations οΐ Prunus spinosa var. Trigno (PsT) + NAC (2 mg/ml = 0.34 mg total dry weight, 4.5 mg/ml = 0.86 mg total dry weight, 10 mg/ml = 1.98 mg total dry weight) for 24 hours on colon tumor cells (HCT116 and SW480) using the clonogenic method. Each plate shows the formation of cell colonies comprising at least 50 cells deriving from a single (cloned) tumor cell. As can be seen, the number of colonies formed decreases after treatment with Prunus spinosa var. Trigno (PsT) + NAC. The SW480 cell line is more sensitive to treatment with Prunus spinosa var. Trigno (PsT). In Figure B2 the colonies have a less well defined shape and smaller dimensions than in Figure B. This observation is demonstrated by Figure CI in comparison with Figure C, where no colonies are present at a concentration of 4.5 mg/ml = 0.86 mg total dry weight + 1900 μΐ of NAC in comparison with Figure C, where a few colonies are still visible. These differences in behavior in response to treatment with Prunus spinosa var. Trigno (PsT) + NAC reflect diversity in the genotype of the two lines used. Quantitative analysis of the number of colonies formed confirmed the pharmacological efficacy of the composition Prunus spinosa var. Trigno (PsT) + NAC, in both cell lines (Figure 8).

A greater reduction in cell growth effect is observed with the HCT116 cell line in doses from 0.5 mg/ml to 2 mg/ml, while at concentrations of 4 mg/ml and 4.5 mg/ml the SW480 cell line is more responsive to treatment.

Qualitative analysis of the different sizes and concentrations of the colonies formed is confirmed by the quantitative analysis.

Evaluation of the effect of different concentrations of Prunus spinosa var. Trigno stone extract (N) + NAC (Figure 9)

N10 mg/ml =1.98 mg total dry weight + 177 ul of NAC;

N4 mg/ml = 0.69 mg total dry weight + 192 ul of NAC;

N2 mg/ml = 0.34 mg total dry weight + 196 ul of NAC.

The analysis was performed using the MTT colorimetric test after 24 hours (Figure 9A), 48 hours (Figure 9B) and 72 hours (Figure 9C) of treatment on HCT116 colon carcinoma cells.

These experimental tests were performed to select the sub-cytotoxic concentration to be used in association with the concentration of Prunus spinosa var. Trigno (PsT) + NAC in order to evaluate the additive or synergistic effect.

The concentration of Prunus spinosa var. Trigno (PsT) + NAC to be used in association with the stone extract (N) was selected on the basis of cell vitality experiments (Figure 5). The concentration selected is P2 mg/ml + 196 ul of NAC, which resulted in a cell survival of 80%.

Evaluation of the effect of different concentrations of Prunus spinosa var. Trigno (PsT) extract and stone extract (N) + NAC from the same drupe after 24 hours, 48 hours and 72 hours of treatment on HCT116 colon carcinoma cells (Figure 10).

The concentrations used were P2 mg/ml = 0.34 mg total dry weight + 196 ul of NAC and N2 mg/ml = 0.34 mg total dry weight + 196 ul of NAC. The effect of the P2 + N2 combination gives rise to a fall of 40% in tumor cell survival after 24 hours, 60% after 48 hours and 65% after 72 hours. Thus comparing the results previously obtained (Figure 2) with the concentration of Prunus spinosa var. Trigno (PsT) + NAC alone (P10 mg/ml + 177 ul of NAC), the reduction in tumor cell survival after 24 hours of treatment is around 65%.

The interesting and unexpected result from application of the P2 + N2 + NAC combination is that it is possible to obtain an appreciable reduction in the total concentration used (4 mg/ml), with an efficacy of response in reduction of tumor cell survival that is approximately the same, after 48 hours treatment, and same, after 72 hours treatment, as that observed in treatment with P10 mg/ml + NAC.

Evaluation of the cytotoxic effect of 5-fluorouracil chemotherapy (concentration 300 μΜ over 24 hours, 48 hours and 72 hours) in the response ofHCT116 colon carcinoma cells to treatment (Figure 11).

After 24 hours treatment cell proliferation was observed in comparison with the control cells (135%), indicating a stimulation of cell growth. Conversely a 48% reduction in cell proliferation after 48 hours and 46% after 72 hours in comparison with control cells was observed.

This result indicates that the efficacy of the P2 + N2 + NAC combination on the colon carcinoma cell line after 72 hours of treatment is the same as that observed with the elected chemotherapy. This result demonstrates that it is possible to use the phytotherapeutic combination investigated by ourselves in association with the elected chemotherapy for the purposes of biointegrated medical treatment.

Statistical analysis

The values shown in the graphs are means plus standard deviations (SD) from three independent experiments performed in sextuplicate. The statistical analysis was performed using Student's t-test. Values of p below 0.05 were considered significant. References

- Singleton VL, et al., (1999) Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin- Ciocalteu reagent. Methods Enzymol 299:152-178

- Guimaraes R, et al., Characterization of phenolic compounds in wild fruits from Northeastern Portugal. Food Chem 141:3721- 3730

- Pignatti S., 1982. Flora d'ltalia. Eds. Edagricole, Bologna Prunus spinosa L.—Vol 1 pg 616

Claims

1. Composition for use in the pharmaceutical and nutraceutical fields comprising:

an extract of Prunus spinosa comprising phenolic acids, flavones and flavonols, and

a mixture of amino acids, vitamins and mineral salts comprising: at least one amino acid selected from L-arginine, L-asparagine H2O, L-aspartic acid, L-cysteine (sodium salt), L-glutamic acid, L-glutamine, glutathione, glycine, L-histidine, L-proline, L-isoleucine, L-leucine, L-lysine HC1, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine (sodium salt), L-valine and corresponding mixtures, in quantities from 30 to 50% by weight with respect to the dry weight of the whole;

at least one vitamin selected from Biotin, D-calcium pantothenate, choline chloride, folic acid, I-inositol, nicotinamide, p-aminobenzoic acid, pyridoxal HC1, riboflavin, thiamin HC1, vitamin B12 and corresponding mixtures, in quantities from 5 to 30% by weight with respect to the dry weight of the whole;

at least one mineral salt selected from Ca(N03)2, KC1, NaCl, NaHC03, Na2HP0₄ and corresponding mixtures, in quantities from 30 to 50% by weight with respect to the dry weight of the whole;

D-glucose in a quantity from 0 to 15% by weight with respect to the dry weight of the whole;

the said extract being characterized in that the quantity of total phenolic acids varies from 35 to 40 mg/100 g of dry extract and the quantity of total flavones and flavonols varies from 60 to 70 mg 100 g of dry extract.

2. Composition according to claim 1, wherein the following are present in the extract:

quercetin 3-O-rutinoside, quercetin 3-O-glucoside and

kaempferol 3-O-rutinoside in a quantity greater than 50% by weight with respect to the quantity of total flavones and flavonols; 3-O-caffeoylquinic acid and 4-O-caffeoylquinic acid in a quantity of more than 90% by weight with respect to the quantity of total phenols.

3. Composition according to any one of the previous claims, wherein the ratio between Prunus spinosa extract and NAC varies between 0.001 and 10, preferably between 0.01 and 1.

4. Composition according to any one of claims 1-3, wherein the Prunus spinosa extract is an extract obtained from the whole fruit.

5. Composition according to any one of claims 1-3, wherein the Prunus spinosa extract is an extract obtained from the seed or crushed stone.

6. Composition according to any one of claims 1-3, wherein the Prunus spinosa extract is an extract obtained from the whole fruit in combination with the extract from the seed or crushed stone.

7. Composition according to any one of claims 1-6, wherein the Prunus spinosa is Prunus spinosa of the Trigno variety.

8. Composition according to any one of claims 1-7, formulated in solid or semi-solid form as tablets, capsules, granulates or powders, ointments, gels, unguents or emulsions, or in liquid form such as syrups and elixirs.

9. Composition according to any one of claims 1-8, formulated for oral, enteral, parenteral and topical administration.

10. Composition according to any one of claims 1-9, for use in the treatment of tumors.

11. Composition according to claim 10, wherein the tumors are selected from melanoma, adenocarcinoma of the colon-rectum and uterine cervix, carcinoma of the breast and bronchoalveolar adenocarcinoma.

12. Composition according to any one of claims 1-11 for administration in combination with chemotherapeutic agents.

13. Composition according to claim 12, wherein the chemotherapeutic agent is selected from 5-fluorouracil and doxorubicin.

14. Kit comprising Prunus spinosa extract and the mixture of amino acids, vitamin and mineral salts according to any one of claims 1-8, formulated individually or as mixtures, in predetermined dosage units, together with instructions for use.

15. Kit according to claim 14, wherein the Prunus spinosa extract is an extract obtained from the seed or crushed stone.

16. Kit according to claim 14, wherein the Prunus spinosa extract is an extract obtained from the whole fruit in combination with the extract from the seed or crushed stone.

17. Kit according to any one of claims 14-16, wherein the Prunus spinosa is Prunus spinosa of the Trigno variety.

18. Kit according to any one of claims 14-17, further comprising chemotherapeutic drugs for simultaneous or combined, sequential or delayed administration.

19. Kit according to claim 18, wherein the chemotherapeutic agent is selected from 5-fluorouracil and doxorubicin.

20. Prunus spinosa extract obtained from the seed or crushed stone of that plant for use in the pharmaceutical and nutraceutical fields.

21. Prunus spinosa extract obtained from the whole fruit in combination with extract from the seed or crushed stone of that plant for use in the pharmaceutical and nutraceutical fields.

22. Prunus spinosa extract according to any one of claims 20- 21 for use as an antitumor agent.

23. Extract according to any one of claims 20-22, wherein the Prunus spinosa is Prunus spinosa of the Trigno variety.