WO2016037971A1

WO2016037971A1 - Compositions comprising glucosinolates precursors of sulforaphane in combination with extracts of rosemary

Info

Publication number: WO2016037971A1
Application number: PCT/EP2015/070366
Authority: WO
Inventors: Paolo Tramonti; Giovanni Scapagnini; Cristiano MEGGIATO
Original assignee: Bios Line S.P.A.
Priority date: 2014-09-08
Filing date: 2015-09-07
Publication date: 2016-03-17

Abstract

The present invention relates to a composition comprising a preparation consisting in a mixture of a lyophilized product of Tuscan kale sprouts titrated in glucosinolates precursors of sulforaphane in combination with a rosemary extract titrated in carnosol and carnosic acid phenolic diterpenes. The bioactive phytochemicals characterizing the composition have been shown to exert a synergistic effect on cellular processes controlled by the Nrf2 transcription factor. Accordingly, the composition can be usefully employed in the preparation of food supplements for the prevention and/or the adjuvant treatment of degenerative processes related to aging.

Description

Compositions comprising glucosinolates precursors of sulforaphane in combination with extracts of rosemary

Field of the invention

The invention relates to a composition comprising a preparation consisting in a mixture of a lyophilized product of Tuscan black kale sprouts titrated in glucosinolates precursor of sulforaphane and extracts of rosemary titrated in carnosol and carnosic acid phenolic diterpenes, and its use in the prevention of cellular degenerative processes due to aging and characterized by chronic inflammatory processes.

State of the art

According to the latest theories that attempt to explain biological phenomena of aging, a series of mechanisms, which are partly endogenous and partly exogenous (e.g. environmental), cause many of the cellular changes that occur later in life (e.g. in the membranes of cells, enzymes, proteins, DNA and RNA). These alterations can lead rapidly to aging and death if not countered by the body's defence mechanisms, whose efficiency is genetically controlled. Although the mechanisms that lead to oxidative stress and inflammation are various, many studies suggest an important role in the activation of cellular protection to the route of signal transmission mediated by the nuclear transcription factor "E2-related" 2 (Nrf2). Nrf2, a member of the family of transcription factors Cap'n'Collar that under non-stimulated conditions is sequestered in the cytoplasm by binding to the protein "Kelch ECH associanting" protein 1 (Keapl ). Keapl protein plays a central role in regulating the response of Nrf2. Under normal conditions, Nrf2 is targeted by Keapl , which promotes Nrf2 proteasomal degradation via interactions with an ubiquitin ligase. Keapl further functions as a sensor of stress signals, through stress-induced oxidation of key cysteine residues that lead to conformational changes and the inability to bind Nrf2. Many stimuli, including oxidative stress and electrophilic compounds, such as polyphenols, lead to the disruption of Nrf2/Keap1 complex, freeing Nrf2 for translocation to the nucleus where it interacts with transcription factors "basic leucine zipper", as the members of families Maf and Jun, and binds to a cis-activating element, the antioxidative response element (ARE, also called EpRE or OSRE). The pathway Nrf2/ARE is the major regulatory route of antioxidative responses Phase II, being able to trigger the simultaneous expression of several protective enzymes and "scavengers" substances. The ARE element has been found in many cytoprotective inducible genes that express proteins that constitute cellular responses to stress, such as glutathione-S- transferase, heme oxygenase-1 (HO-1 ), the glutamate-cysteine ligase, ferritin, the gamma-glutamyl cysteine synthetase, NAD(P)H quinone oxidoreductase, thioredoxin, and periredoxine. This implies that the transcription factor Nrf2 is a central hub in the response of cell survival. From the constant activation of this complex genetic system, cellular damage can then be gradually repaired upon their occurrence. On the basis of this evidence, it has been recently suggested that the modulation of Nrf2 and HO-1 in different biological contexts, including the brain, the skin and the vessels, can be a potential pharmacological strategy for the prevention and treatment of degenerative disorders and pathological aging.

Many epidemiological studies in recent years have shown a direct relationship between the type of diet and the incidence and severity of chronic diseases related to aging. This spawned the idea that modulating the activity of genes and proteins, compounds found in food can influence the balance between health and disease in an individual thereby affecting their quality of life, well-being, and the onset of degenerative diseases.

The antioxidant activity of many natural compounds derived from plants has been studied both as regards their ability to act by direct functioning as a "scavenger" of reactive oxygen species (ROS), and as regards their ability to act indirectly activating the transcription factor pathway Nfr2/ARE (Kelsey NA et al., Molecules, 2010, 15, 7792-7814; Scapagnini G et al., Mol. Neurobiol. doi 10.1007/s 12035- 01 1 -8181 -5).

In patent application US2013/0071369, for example, there are described compositions employable as dietary supplements, suited to cell detoxification by expression of genes related to the route Nfr2/ARE, comprising a plurality of components of vegetable origin, and, in particular, at least two selected from a broccoli seed extract, alpha lipoic acid, an extract of red orange, a grape seed extract, an extract from whole grains of grapes, an extract from olive leaves or olives, Qio coenzyme, a pomegranate extract, curcumin, the EGCG catechin, lutein, lycopene, zeaxanthin, resveratrol, an extract of the fruits of Schizandra, an extract of black cherries, a ginseng extract, an extract of rosemary and Cordyceps sinensis. There are described and individually tested the following components: quercetin, Prunus Cerasus extract cv. Balaton, carnosol, broccoli seed extract titrated in 13% sulforaphane/glucosinolate, alpha-lipoic acid, extracted from whole grains and seeds of grapes, aqueous extracts of olive leaf and olive, Schizandra chinensis and Cordyceps sinensis, pomegranate extract, Panax ginseng, and CoQ ubiquinol. The most active components found on the expression of 10 genes representative of the Nrf2/ARE route in liver tissues were the seed extracts of broccoli, red orange and olive and alpha-lipoic acid, while no significant changes were observed in lung tissue with any of the components tested. The alpha-lipoic acid and the pomegranate extract have proved active in the regulation of glutathione synthesis. On the basis of these results four different compositions comprising: (a) red orange extract, grape seed extract, and extract of seeds of broccoli; (b) Cordyceps sinensis, pomegranate extract, and extract of Panax ginseng; (c) red orange extract, grape seed extract, CoQ-io, olive leaf and olive extract; (d) red orange extract, extract of grape seed and extract of broccoli, Cordyceps sinensis, pomegranate extract and extract of Panax ginseng have been prepared and tested. From these were derived two compositions that can be used as dietary supplements consisting of: (1 ) red orange extract, grape seed extract, and extract of seeds of broccoli; (2) olive leaf and olive extract, red orange extract, grape seed extract, and CoQ-io. Finally, the following four compositions have been claimed: (1 ) extract of broccoli seed, red orange extract, and grape seed extract;

(2) olive leaf and olive extract, red orange extract, grape seed extract and CoQ-io;

(3) broccoli seed extract, alpha lipoic acid and grape seed extract; (4) olive leaf and olive extract, alpha-lipoic acid, grape seed extract and CoQ-io.

Among the phytochemicals with antioxidative potent activity, sulforaphane deserves particular attention. It is an isothiocyanate formed by action of the enzyme myrosinase from glucoraphanin, a glucosinolate that is found in some plants belonging to Cruciferae.

Isothiocyanates are basically volatile compounds, which are known for their ability to foster in man the natural antioxidant defences of the organism and to promote the activity of detoxifying enzymes. The transformation of glucosinolates into isothiocyanates in these vegetables is performed by an endogenous enzyme, the myrosinase, which is normally segregated into specific cell districts, but that is activated when the integrity of the plant is injured (Bones A.M. and Rossiter J.T., Physiologia Plantarum 1996, 97, 194-208). In humans, however, the transformation of glucosinolates into isothiocyanates takes place, but only partially, in the intestine by the enzymes of the resident bacterial flora (Fuller Z. et al., Br. J. Nutr. 2007, 98, 364-372). The isothiocyanates exert their beneficial effects by stimulating, through the translocation of the Nrf2 transcription factor from cytoplasm to the cell nucleus, the synthesis of specific enzymes; some facilitate the elimination of toxic substances, others fight oxygen free radicals and promote the repair of cellular damage. This results in an increase of the defences that justify the chemopreventive activity and anti-inflammatory, immunomodulatory and detoxifying effects generally exercised by the isothiocyanates.

However, it is clear that glucosinolates may exert their beneficial effects only when able to render isothiocyanates bioavailable for enzyme transformation by the myrosinase. The bioavailability of isothiocyanates of plant origin derived from the whole plant is strongly influenced by cooking processes, whereby these plants are usually subjected for food purposes, which, by inactivating myrosinase, prevent the enzymatic transformation of the glucosinolates (Rungapamestry V. et al., Br. J. Nutr., 2007, 97, 644-652). In this case, the beneficial effects exerted by these plants depend on the intervention of the intestinal bacterial flora which renders, in part, available in v/Vo the active principles.

The Brassicaceae, and in particular the different varieties of the species Brassica oleracea (commonly known as broccoli), are among the preferred plant sources for the preparation of food supplements enriched in glucosinolates and/or isothiocyanates, the content of which varies depending on the development of the plant. In fact, studies of plant physiology have shown that the levels of glucosinolates vary in time depending on the different stages of plant development and in some cases are higher in the shoots than the adult plant. This was clearly demonstrated in particular for broccoli sprouts, which were found to contain up to 100 times the levels of glucosinolates present in mature plants (Fahey J.W. et al. : Broccoli sprouts: an exceptionally rich source of inducers of enzymes that protect against chemical carcinogens. Proc. Natl. Acad. Sci. 1997, 94, 10367-10372). Among the different varieties belonging to the species Brassica oleracea, lyophilized black Tuscan kale {Brassica oleracea L. var. acephala subvar. Laciniata L.) possesses a high concentration of glucoraphanin especially in the buds. However, it is important in regards to what was mentioned earlier that this plant material is treated in an appropriate way to remedy problems of degradation of phytochemicals present.

There is, therefore, a need to maximize the beneficial effects of sulforaphane bioavailable in vivo for preventive purposes of cellular degenerative processes both by selecting the starting plant material and by making available compositions that, by combining other materials of vegetable origin, are suitable for use as dietary supplements.

Summary

For the aforesaid purpose, the inventors have now developed a composition usable as an oral dietary supplement, which can have a preventive effect in delaying and/or preventing cellular degeneration due to aging.

The composition of this supplement includes compounds which are known for their antioxidant properties, such as glucosinolates / sulforaphane (briefly indicated as Sur) and extracts of rosemary titrated in carnosol and carnosic acid (briefly indicated as RPs). The combination of these phytochemicals, sulforaphane and phenolic diterpenes from rosemary in specific molar ratios, have shown an unexpected ability to activate in a synergic and specific manner the system of the Nrf2 transcription factor and the genes related to the same, and to regulate negatively its activation and the expression of proinflammatory cytokines. These effects contribute to increase the cellular defence reducing both oxidative stress and inflammation. In consequence of these effects, the composition is suitable in promoting the viability and cellular functions, in particular of endothelial cells, of skin and nerve, from deterioration due to aging and to the subsequent degenerative processes.

In one aspect of the invention, it is, therefore, a composition comprising a mixture consisting of a lyophilized product of Tuscan kale sprouts titrated in 5% glucosinolates precursors of sulforaphane in combination with a rosemary extract titrated in 10% carnosol and carnosic acid phenolic diterpenes, wherein in said preparation the sulforaphane precursor glucosinolates and the rosemary phenolic diterpenes phytochemicals are combined in molar ratios comprised from 2:1 to 1 :2.

For the purposes of the present invention, the shoots of Tuscan kale sprouts are collected between 4 and 6 days after sowing and before lyophilization are subjected to a mild treatment with microwaves.

Another aspect of the invention is represented by the use of the aforesaid composition comprising a preparation consisting of a lyophilized product of Tuscan kale sprouts titrated in 5% glucosinolates precursors of sulforaphane in combination with a rosemary extract titrated in 10% carnosol and carnosic acid phenolic diterpenes as a dietary supplement for the prevention and/or treatment of cellular degenerative processes related to aging. In particular, the composition of the invention can be usefully employed for the prevention and/or adjuvant treatment of cellular degenerative processes characterized by chronic inflammatory processes.

Brief description of the figures

Figure 1 . This figure shows the results on human bronchial endothelial cells with sulforaphane (Sur), phenolic diterpenes from rosemary (RPs) and associations of Sur + RPs in different concentrations on: (a) specific primers HO-1 and HO-2 were used to amplify the human RNA; (b) mRNA expression of heme oxygenase 1 (HO- 1 ) at concentrations from 5 to 30 μΜ; (c) - (e) enzymatic activity of heme oxygenase 1 (HO-1 ) at concentrations from 5 to 30 μΜ and at 6 and 24 hours. Figure 2. This figure shows the results obtained with different ratios between the two components sulforaphane (Sur), phenolic diterpenes from rosemary (RPs) in the HO-1 activation measured as mRNA levels.

Figure 3. This figure shows the time-dependent increase in the expression of the Nrf2 nuclear protein after treatment with sulforaphane (Sur), phenolic diterpenes from rosemary (RPs) alone and in combination with each other (Sur + RPs).

Figure 4. This figure shows the time-dependent increase of reduced intracellular GSH glutathione (GSH) after treatment with the association Sur+RPs 15 μΜ.

Figure 5. This figure shows the results on the DNA binding activity of NF-kB p65 in macrophages after stimulation with LPS in the presence or absence of sulforaphane (Sur), phenolic diterpenes from rosemary (RPs) alone and in combination with each other (Sur + RPs).

Figure 6. This figure shows the results obtained on the secretion of inflammatory cytokines in macrophages after stimulation with LPS in the presence or absence of sulforaphane (Sur), phenolic diterpenes from rosemary (RPs) alone and in combination with each other (Sur + RPs).

Detailed Description of the invention

Sulforaphane and phenolic diterpenes from rosemary are undoubtedly among the bioactive compounds of plant origin more attractive for their antioxidant and detoxifying properties.

The inventors have now unexpectedly found that the combination of sulforaphane (Sur) and phenolic diterpenes from rosemary determines a synergistic effect on cellular biological parameters that form the basis of chronic inflammatory and degenerative processes. In particular, it has been found that the combination of the two compounds exerts a synergistic effect with respect to the two compounds taken individually on the inhibition of the route of the pro-inflammatory nuclear factor kappa B (Nf-kb) and on the activation of antioxidant and anti-inflammatory route of the Nrf2 transcription factor.

Before reporting the results, it is necessary to clarify what is meant by "biological synergism" and what is meant by "cellular parameters directly relevant to cellular aging." In the present specification, for "biological synergism" it has been adopted the more scientifically stringent definition derived from studies on drug action. A synergism between the drugs is such when two drugs interact in a way that by their interaction derives an amplification of one or more positive effects or side effects of them. In other words, two drugs that produce similar effects can sometimes produce exaggerated or lesser effects when used together. In this case, it is necessary to make a quantitative assessment to see if it is a simple additive effect or it is a synergistic effect (Tallarida RJ. Drug synergism: its detection and applications. J Pharmacol Exp Ther., 2001 , 298 (3), 865-72). The biological effects, that are the basis of the present invention, consist of a positive modulation of defensive and reparative cellular systems with relevant effects on cell viability, both as regards individual cells and cell populations. Therefore, we must consider that the definition of pharmacological synergism, when translated in terms of molecular events, relates often to the ability of the two bioactive substances producing synergism to enhance cell functions by eliciting pathways, which are originally distinct, but converge at the higher levels in elementary cell activities concurring to the same major cell program. In other words, if there are two ways for a cell to avoid death controlled by two unrelated or minimally related pathways A and B, enhancement of only A or B by two compounds is likely to produce at the highest doses saturation of the pathway, and therefore a combined effect which is less than additive. Full stimulation of A and B, instead, could produce additivity or even synergism (a) because all the activities bringing to that response are engaged, (b) because the cross-talks taking place where the pathways converge can become mutually enhancing, or, finally, (c) because the synchronous activation of both allow the cell to move into discrete but sequential steps of activity.

It is, therefore, evident that for the purposes of preventing degenerative phenomena, usually characterized by chronic inflammatory processes, it is essential to try to obtain effects of synergism, combining among them substances having properties useful for the purpose. The fact of obtaining a synergistic effect is also particularly significant in the development of compositions, suitable for use as a preventive of cell degenerative processes as it allows using limited amounts of these substances thereby remedying possible undesirable side effects, such as the so-called "paradoxical effect" of anti-oxidants that in high doses can become pro-oxidants themselves.

The inventors have identified among the many bioactive natural substances known two phytochemicals that can potentially be used in the preparation of compositions employable as food supplements suitable for use in the prevention of degenerative chronic inflammatory processes related to aging: sulforaphane and the phenolic diterpenes from rosemary.

In particular, the inventors have focused their attention on the ability of the two compounds identified in the prevention of the formation of reactive oxygen species (ROS) mediated by the pathway of the Nrf2 nuclear transcription factor. The Nrf2 Factor interacts with ARE-level as promoter of many inducible genes with critical defensive and reparative properties. The activation of the Nrf2 factor leads to over- expression of these genes by improving the cellular defence mechanisms and inhibiting cell death.

However, the inventors have now found that diterpenes from rosemary (RPs) have the ability to improve very significantly and synergistically the ability of sulforaphane to activate the route of the Nfr2 and, consequently, to favour the expression of genes with inducible defensive and reparative functions, such as heme-oxygenase 1 (HO-1 ), and to increase the amount of intracellular glutathione. Without wishing to be tied to a specific mechanism of action to explain these effects, the same could be due to the fact that the RPs share with sulforaphane the ability to bind sulfhydryl groups that are specific sensors for the activation of Nfr2. In addition, both phytochemicals have been shown to inhibit synergistically the Nf-kb activity and to reduce the expression of cytokines.

As can be seen from the experimental data in detail below and from the figures reported, the combination of sulforaphane and phenolic diterpenes from rosemary induces in vitro a synergistic effect in concentrations of sulforaphane association/ phenolic diterpenes from rosemary comprised between 5 and 30 μΜ. Such synergism is clear already at the concentration of 5 μΜ and is maximum at concentration of the association of 15 μΜ both at 6 and 24 hours. In addition, on the expression of heme-oxygenase 1 , the protein whose potent activity in cellular defence against oxidative stress and the protection of cells against damage caused by free radicals is known, this synergism is occurs in molar ratios between sulforaphane and phenolic diterpenes from rosemary from 2:1 and 1 :2 (Sur:RPs) and is maximum at the molar ratio of 1 to 1 .

Therefore, in the compositions, usable as a nutritional supplement for delaying and/or preventing cellular degeneration consequent to aging and associated chronic inflammatory processes, comprising a preparation consisting of a lyophilized product of Tuscan kale sprouts titrated in 5% glucosinolates precursors of sulforaphane and a rosemary extract titrated in 10% carnosol and carnosic acid phenolic diterpenes, the molar ratios between the aforesaid phytochemicals are essential.

However, in first instance it is necessary in order to develop nutritional supplements containing sulforaphane to pay particular attention both to the sources of this isothiocyanate, or from materials with high content in glucosinolates, and to the treatment to which these materials are subjected in order to preserve the high content in them.

For this purpose, it is preferable to employ as a source of plant materials the Brassica oleracea and, in particular, the black Tuscan kale {Brassica oleracea L. var. acephala subvar. Laciniata L), this variety being particularly rich in glucoraphanin. In particular, there are preferred preparations of Brassica oleracea L. var. acephala subvar. Laciniata L. consisting of lyophilized sprouts so that the high content of glucoraphanin is preserved, and it is maintained also an adequate content of endogenous myrosinase able to generate in vivo the conversion of glucoraphanin in sulforaphane.

In fact, as mentioned previously the Brassicaee, and in particular broccoli, are certainly a vegetable source of primary importance from which extracts with high content of glucosinolates can be obtained, but this high content of glucosinolates is naturally also associated with an appropriate enzyme content, myrosinase, and its co-factor EpithioSpecifier Protein (ESP) capable of degrading them to isothiocyanates, but also to nitriles and epitionitriles. However, it is to be remembered in this respect that the presence of this enzyme and its co-factor is one of the major problems in obtaining extracts with a high content in stable glucosinolates. At the same time, however, it is also necessary to have herbal preparations wherein this enzyme and its cofactor are present and not deactivated, to allow the conversion of glucoraphanin into sulforaphane.

The black Tuscan kale {Brassica oleracea L. var. acephala subvar. Laciniata L.) is compared to other Brassicaceae, such as e.g. broccoli calabrese {Brassica oleracea var. Italic), characterized by seeds containing high levels of both total glucosinolate and glucoraphanin. Such a high content of glucosinolates in the seeds is maintained even in the content of these compounds in the shoots and, thus, the shoots of black cabbage Kale constitute a plant material of choice for the preparation of phytochemicals with a high content of glucosinolates and, in particular, of glucoraphanin from which sulforaphane originates.

From seedlings of black Tuscan kale collected between four and six days from sowing there were obtained, with adequate procedures of single lyophilization preceded by a mild treatment with 600 Watt microwave for 2 minutes to deactivate the ESP cofactor responsible for the conversion of glucoraphanin in sulforafanonitrile but without any post-lyophilization extraction, preparations characterized by a high content of glucosinolates, particularly glucoraphanin, able to release appreciable quantities of sulforaphane in suitable conditions. In this way it is obtained, in fact, an herbal preparation wherein the phytocompounds originally present in the shoots are preserved and are temporarily disabled but available to be reactivated under physiological conditions.

The lyophilizate of shoots harvested at 4-6 days after sowing, deprived of cuticle and treated with microwave contains a quantity of glucoraphanin equal to 3% w/w, compared with a total glucosinolate content of 5% w/w. The treatment with microwaves allows degrading primarily the ESP protein which, if present, directs the transformation of glucoraphanin towards the formation of sulforaphane nitrile instead of sulforaphane. This treatment is therefore essential to block unwanted degradation of glucosinolates present. After the microwave treatment the activity of the enzyme myrosinase, normally present in the shoots of this plant, appears to be only partially reduced, being however always able to transform about 80% of glucoraphanin, present in the lyophilisate, into sulforaphane. A possible alcoholic extraction or hydroalcoholic solution has the disadvantage of completely disabling the endogenous myrosinase thereby blocking an efficient conversion of glucoraphanin present in sulforaphane leaving it only in charge of intestinal microflora.

The title in glucoraphanin/sulforaphane of this preparation of lyophilized sprouts from black Tuscan kale is 5% and is, for the purposes of the invention, associated with a rosemary extract titrated in 10% carnosol and carnosic acid phenolic diterpenes.

To obtain the synergistic effect between these two components, the same are combined in molar ratios considering the phytochemicals sulforaphane (Sur) content in the shoots of black Tuscan kale and the carnosol and carnosic acid phenolic diterpenes from rosemary extract (RPs) between 2:1 (Sur: RPs) and 1 :2 (Sur: RPs) and preferably 1 :1 (Sur: RPs).

In view of what was experimentally found here and briefly discussed, the compositions according to the invention, comprising a mixture consisting of a lyophilized product of Tuscan kale sprouts titrated in 5% glucosinolates precursors of sulforaphane in combination with a rosemary extract titrated in 10% carnosol and carnosic acid phenolic diterpenes, have preferably a content of glucosinolates / sulforaphane between 5.0 mg and 200 mg and a content of phenolic diterpenes from rosemary between 10 mg and 400 mg in relation to the kind of formulation selected for the food supplements developed. Preferably, the total content expressed as a percentage of the association of phytochemicals glucosinolates/sulforaphane and phenolic diterpenes from rosemary in the total weight of the composition is between 5% (1 .6% glucosinolates/sulforaphane + 3.3% phenolic diterpenes) and 30% (10% glucosinolates/sulforaphane + 20% phenolic diterpenes) and preferably is 15% (5% glucosinolates / sulforaphane + 10% phenolic diterpenes).

For the delaying and/or preventing cellular degeneration purposes of the invention, the preparation consisting of the two phytochemicals glucosinolates/sulforaphane and phenolic diterpenes can be included in compositions associating other phytochemicals, as such or in the form of extracts, and/or other bioactive compounds, such as resveratrol, extracts of Andrographis paniculata, extracts of peppermint, extracts of Thyme, extracts of Hedera elix, extracts of Icelandic lichen, N-acetylcysteine, cysteine, Vitamin C.

Such compositions can be in the known forms for oral use in the pharmaceutical and/or parapharmaceutical nutrition field and can therefore be, as known to one skilled in the art, compositions comprising a preparation consisting of a lyophilized product of Tuscan kale sprouts titrated in 5% glucosinolates precursors of sulforaphane in combination with a rosemary extract titrated in 10% carnosol and carnosic acid phenolic diterpenes, wherein these two components are combined with excipients and/or diluents pharmaceutically acceptable or food grade material suitable to obtain such compositions in liquid form, e.g. solutions or suspensions, or in solid form, such as powders in sachets, erodible tablets, tablets, capsules. Preferably, such compositions are selected from tablets, capsules, optionally enteric- or colon-specific, or powders in sachets.

In particular, the gastroprotected compositions are required when one wishes to avoid the release in the stomach of the active ingredients contained in the extracts, and in particular of glucoraphanin, and to obtain a release of sulforaphane in the small intestine, thus promoting a systemic absorption with all positive chemoprotective effects induced at general level by this compound. The gastroprotection both of the capsules and of the tablets is obtainable with pharmaceutical technique methods known to an expert in the art. For example, gastroprotected tablets can be obtained by coating the tablet with an aqueous solution of shellac, ammonium carbonate and tri-ethyl citrate.

In another embodiment, the compositions can be provided in the form of tablets or capsules colon-specific, which allow a release of the active principles of the extracts localized in the colon. Even the colon-specific formulation can be obtained through techniques known to an expert in the art. For example, it can be obtained by coating gelatine capsule with two different protective layers; the inner one protects the capsule from the dissolution at pH 6.8 typical of the small intestine and is obtained by coating the capsule of gelatin with a 15% aqueous solution containing shellac (87.2%), ammonium carbonate (3.3%) and tri-ethyl citrate (9.5%), the outer one instead allows to overcome the acid pH of the stomach and is deposited on the inner one, when dry, using an aqueous solution based on shellac (84.2%), ammonium carbonate (6.3%) and tri-ethyl citrate (9.5%). Double protection thus allows the release of the active compounds only at pH 7.2 typical of the colon.

Pharmaceutical forms or para-pharmaceutical nutritionals, wherein both a rapid dissolution in the stomach and a dissolution in the intestine (small intestine or colon) are combined, cannot be excluded. Examples of such forms can be erodible tablets or micronized powders treated to obtain granules with differentiated coatings depending on the type of release pursued.

Nutritional supplements prepared with the compositions object of the invention, herein described, can be usefully employed for the prevention and/or the adjuvant treatment of degenerative processes related to aging and in particular degenerative processes characterized by chronic inflammatory processes of the respiratory, liver, gastro-intestinal, renal, cutaneous and ophthalmic system and preferably in charge of endothelial cells of the respiratory system.

Considering these relations between the two components below are described by way of example some embodiments of compositions that can be used as nutritional supplements.

Example of composition 1 in capsule mq / capsule

Lyophilized Tuscan kale 20.00-500.00 Rosemary extract 20.00-500.00 Magnesium stearate 10.00

Talc 10.00

Cellulose 50.00

Example of composition 2 in tablets mq / tablet

Lyophilized Tuscan kale 20.00-500.00 Rosemary extract 20.00-500.00 Vegetal magnesium stearate 10.00 Silicon dioxide 10.00

Cellulose 80.0

Calcium phosphate dibasic 100.00

Example 3 composition in slow-release tablets mq / tablet

Tuscan kale lyophilized 15.0-600.0 Rosemary extract 15.00-600.00 Curcuma longa extract 50.00

Magnesium stearate 10.00

Silicon dioxide 10.00

Hydroxypropyl methylcellulose 50.00

Calcium phosphate dibasic 130.00

Example composition 4 in powder mq / sachet Tuscan kale lyophilized 15.00- 1000.00 Rosemary e.s. 15.00- 1000.00 Fructose 1500.00

Aroma 15.00

Silicon dioxide 50.00

Stevia (Rebaudioside A) 2.00

Example composition 5 in buccal dispersible powder mq / sachet Tuscan kale lyophilized 15.00- 750.00 Rosemary e.s. 15.00- 750.00 Fructose 1800.00

Aroma 35.00

Silicon dioxide 50.00

Sucralose 7.00

EXPERIMENTAL PART

Example 1 : effect of Sulforaphane (Sur), and phenolic diterpenes from rosemary (RPs) and their association on the up-regulation of gene for heme oxygenase 1 (HO-1) in human bronchial endothelial cells

Immortalized human bronchial cells (NULL-1 , ATCC CRL-401 1 ) cultured in vitro were incubated for 6 hours with different concentrations (5, 10, 15 and 30 μΜ) of sulforaphane (Sur), diterpenes from rosemary (RPs) or a combination of the two compounds at the total concentrations mentioned (ratio between Sur and RPs = 1 : 1 in moles). The expression of the gene for heme oxygenase 1 (HO-1 ) was measured by means of detection of the levels of mRNA by Real-Time quantitative PCR.

Subsequently, in order to confirm that the over-expression of HO-1 gene also corresponded to an equivalent increase in heme oxygenase enzymatic activity, this was measured after 6 and 24 hours incubation of the cells at the same concentration of Sur, RPs and associations Sur + RPs. The enzymatic activity of heme oxygenase was determined at the end of each treatment as previously described (Scapagnini G. et al. Mol. Pharmacol 2002 vol. 3; 61 : 554-561 ).

Briefly, microsomes prepared from the cells were added to a reaction mixture containing NADPH, glucose-6-phosphate dehydrogenase, cytosol of rat liver as a source of biliverdin reductase, and the substrate hemin. The mixture was incubated in the dark at 37°C for 1 hour and the reaction was stopped by adding chloroform. After a vigorous stirring by means of vortexing and centrifugation, the bilirubin extracted in the chloroform layer was measured by the difference in adsorption between 464 and 530 nm (_ = 40 mM_1 cm_1 ).

In order to determine the best balance between Sur and RPs, different molar ratios between RPs and Sur (Sur: RPS 1 :2, 1 :1 , 2:1 ) to final concentration association Sur + RPs of 15 μΜ were evaluated by measuring mRNA levels through Real- Time PCR.

The results obtained on the expression of mRNA of HO 1 are shown in Figure 1 . In short, it was observed that the treatment resulted in a significant increase (p <0.05) of the expression of mRNA, as measured by Real-Time quantitative PCR, of HO-1 , with a maximum at 15 μΜ. Sulforaphane showed a dose-dependent increase in the mRNA expression of HO-1 , measured with respect to the non- inducible enzyme HO-2 expressed by the paralog gene. This increase reached the maximum (about 6 times) at 15 μΜ, while it was noticed a decrease in the expression of the mRNA to 30 μΜ. The RPs, instead, at the same concentrations have proved much less active in the induction of the expression of the gene HO-1 , but showed again the maximum effect at a concentration of 15 μΜ. Even the combination of the two compounds has demonstrated full activation of gene expression HO-1 to 15 μΜ. It is also noted that the effect of the association Sur + RPs already at 5 μΜ was in the same order of magnitude as that of the only Sur at 15 μΜ (Fig. 1 b).

The trend in increasing the enzymatic activity HO 1 was comparable to that observed on the mRNA levels, confirming the functional significance of data on gene expression (Fig. 1 c). The results shown on this parameter displayed the synergistic effect between Sur and RPs in inducing the activation of gene HO-1 . As shown in Figure 2, the molar ratio of 1 : 1 (Sur: RPs) gave the maximum effect. Example 2: effect of Sulforaphane (Sur), phenolic diterpenes of rosemary (RPs) and their association on the activation of Nrf2/ARE in human bronchial endothelial cells

In immortalized human bronchial cells (Nuli-1 , ATCC CRL-401 1 ) the expression of Nrf2 protein after incubation for 1 , 3, 6 and 12 hours with sulforaphane (Sur), polyphenols of rosemary (RPs) or a combination of the two compounds Sur + RPs (ratio between Sur and RPs = 1 : 1 in moles) at the final concentration of 15 μΜ was evaluated.

The nuclear expression of the Nrf2 protein was determined according to the known method of of Western blotting. The cells were washed twice with PBS and then resuspended in 1 ml PBS and centrifuged at 3000 rpm for 3 minutes at 4° C. The cell pellet obtained was resuspended in 200 μΙ_ of a cold buffer consisting of HEPES 10 mM (pH 7.9), KCI 10 mM, EDTA 0.1 mM, EGTA 0.1 mM, DTT 1 μΜ, and a mixture of protease inhibitors (Roche, Mannheim). The pellet was incubated in ice for 15 minutes to allow the cells to swell, and then 15 μΙ of 10% NP-40 were added. The tube was stirred with a vortex for 10 seconds. The homogenate was centrifuged at 3000 rpm for 3 minutes at 4° C and the nuclear pellet was resuspended in 30 μΙ of a cold buffer consisting of HEPES 20 mM (pH 7.9), NaCI 0.4 M, EDTA 1 mM, EGTA 1 mM, DTT 1 μΜ and protease inhibitors. The pellet was incubated in ice for 15 minutes and stirred with a vortex for 10-15 seconds every 2 minutes. The nuclear extract was centrifuged at 13000 rpm for 5 minutes at 4°C. Nuclear proteins contained in the supernatant were separated by electrophoresis on SDS-polyacrylamide gel and subjected to immunoblotting using anti-Nrf2 antibody (1 :500 dilution) and anti-Sp1 (1 :500 dilution).

As shown in Figure 3, the treatment with sulforaphane (Sur) causes a significant time-dependent increase in the expression of the protein in nuclear extracts. Quantification of three western blots showed that after 1 hour of exposure to sulforaphane 15 μΜ, the expression of Nrf2 increased significantly and remained over-regulated up to 12 hours, while levels of the Sp1 transcription factor were stable. RPs caused a smaller increase in the expression of Nrf2 nuclear. The combination of the two compounds induced a massive expression of the Nrf2.

Example 3: effect of the association of Sulforaphane (Sur) and phenolic diterpenes of rosemary (RPs) on the intracellular induction of reduced glutathione in human bronchial endothelial cells

In immortalized human bronchial cells (NuLi-1 , ATCC CRL-401 1 ) the levels of reduced (GSH) and oxidized (GSSG) glutathione after 1 , 3, 6 and 12 h of incubation with the association Sur + RPs 15 μΜ were measured.

The method used, as described in the literature (Scapagnini G et al. Mol. Pharmacol., 2002 vol. 3; 61 : 554-561 ), is briefly outlined below.

The cells suspended in cold PBS were frozen and thawed three times and an aliquot of the suspension was added to a buffer solution containing 12 mM EDTA and acid 5,5-dithiobis-2-nitro benzoic acid 10 mM. The total glutathione was measured by spectrophotometry at 412 nm optical density using the rate of recycling of glutathione reductase. The oxidized glutathione was measured in an aliquot of cell suspension added to an equal volume of a buffer containing EDTA and /V-ethylmaleilimmide (10 mM). The sample was stirred and centrifuged and passed on a cartridge of C18 Sep-Pak (Waters, Milford, MA) to remove excess of /V-ethylmaleilimmide. The sample was then supplemented with acid 5,5-dithiobis- 2-nitro benzoic acid and glutathione reductase and the detection was performed as for the total glutathione. The intracellular glutathione was calculated by comparison with standard curves obtained with solutions with a known titre of GSH and GSSG and expressed as the number of times of increase compared to the control.

As shown in Figure 4, the GSH levels are increased after 6 h of treatment with Sur+RPs 15 μΜ and persisted for over 12 h with an increase close to 220% compared to starting levels.

Example 4: effect of Sulforaphane (Sur) and phenolic diterpenes of rosemary (RPs) and their association on the binding activity of NF-kB p65 DNA in macrophages cultured in vitro

The NF-kB route plays a key role in the etiopathogenesis of chronic inflammatory diseases. The transcription factors of the NF-kB family, in fact, play a crucial role in the regulation of a variety of genes particularly involved in inflammatory processes and in the expression of cytokines such as IL-1 β, IL-6 and TNF-a.

To evaluate the ability of the association Sur + RPs to inhibit the activity of NF-kb in macrophages, it was used for the test activity of binding capacity of NF-kB p65 DNA using the ELISA kit for the transcription factor p65 TransAM (Active Motif, Carlsbad, CA). A cell line of immortalized cells of macrophages was used. The cells were pretreated for 1 h with Sur 5 μιτι, RPs 5 μιτι or Sur+ RPs 5 μιτι, and then exposed to 500 ng/ml of LPS for an additional 24 h.

Subsequently, the binding activity of the DNA of NF-kB p65 was determined. IL- 1 b, IL-6 and TNF-a were determined with ELISA kit (Abeam, Cambridge, MA).

12 hours after stimulation LPS markedly promoted this binding activity of NF-kB p65 DNA and expression of proinflammatory cytokines IL-1 β, IL-6 and TNF-a. However, a less binding activity of the DNA was observed in macrophages stimulated with LPS in the presence of Sur and RPs, as shown in Figure 5. Surprisingly, the combination of Sur + RPs strongly reduced the binding activity of NF-kB p65 DNA, demonstrating a synergistic effect obtained by the combination of the two compounds. Likewise, similar effects were obtained on the expression of cytokines (Figure 6). Data are expressed as average ± SEM, n=4; ^*p<0.001 vs. LPS, ^**p<0.001 vs. Sur or RPs only.

Claims

1 . Composition comprising a mixture consisting of a lyophilized product of Tuscan kale sprouts titrated in 5% glucosinolates precursors of sulforaphane in combination with a rosemary extract titrated in 10% carnosol and carnosic acid phenolic diterpenes, wherein the sulforaphane precursor glucosinolates and the rosemary phenolic diterpenes phytochemicals are combined in the mixture in molar ratios comprised from 2:1 to 1 :2.

2. The composition according to claim 21 , wherein the sulforaphane precursor glucosinolates and the rosemary phenolic diterpenes phytochemicals are combined in molar ratios of 1 :1 .

3. The composition according to claim 1 , wherein the content in sulforaphane precursor glucosinolates phytochemicals is comprised from 5.0 mg to 200 mg.

4. The composition according to claim 1 , wherein the content in rosemary phenolic diterpenes phytochemicals is comprised from 10 to 400 mg.

5. The composition according to claim 1 , wherein the total content of the mixture consisting in sulforaphane precursor glucosinolates and rosemary phenolic diterpenes phytochemicals is comprised from 5% to 30% by weight on the total weight of the composition.

6. The composition according to claim 5, wherein the total content of the mixture consisting in in sulforaphane precursor glucosinolates and rosemary phenolic diterpenes phytochemicals is 15% by weight on the total weight of the composition.

7. The composition according to claim 1 , wherein further phytochemicals, and/or extracts and/or other bioactive compounds selected from resveratrol, Andrographis paniculata, Peppermint, wild thyme, Hedera elix, Iceland moss, N- acetylcysteine, cysteine, Vitamin C are added.

8. The composition according to one of claims 1 to 6 for use in the preparation of food supplements.

9. The composition according to claim 8, wherein the food supplements are suitable for the prevention and/or adjuvant treatment of cellular degenerative processes related to aging.

10. The composition according to claim 9, wherein the cellular degenerative processes are characterized by chronic inflammatory process of the respiratory, hepatic, gastrointestinal, renal, cutaneous or ophthalmic systems.

1 1 . Use of a preparation consisting of a lyophilized product of Tuscan kale sprouts titrated in 5% glucosinolates precursors of sulforaphane and of a rosemary extract titrated in 10% carnosol and carnosic acid phenolic diterpenes for the preparation of compositions according to one of claims 1 to 6.

12. Use according to claim 1 1 , wherein the compositions are used as food supplements.

13. Use according to claim 12, wherein the food supplements are suitable for the prevention and/or adjuvant treatment of cellular degenerative processes related to aging.

14. Use according to claim 13, wherein the cellular degenerative processes are characterized by chronic inflammatory process of the respiratory, hepatic, gastrointestinal, renal, cutaneous or ophthalmic systems.