APPLE

FIELD OF THE INVENTION The present invention relates to a method for the production of high amounts of triterpenic acids by an in vitro culture of calluses deriving from the pulp of Red Sentinel (RS) variety apples, among which calluses obtainable in vitro by said method and use thereof.

PRIOR ART In the nutraceutics and cosmeceutics fields there is a growing interest for the production of high amounts of plant-derived natural secondary metabolites, with the aim of avoiding more complex synthetic production methods and conventional growing methods. In the last two decades, the field of nutraceutics, i.e. the discipline studying food components and/or active ingredients contained in foods which entail beneficial health effects, among which disease prevention and treatment, has acquired increasing importance both from a scientific and commercial standpoint. Within the scope of nutraceutics, the functional foods, food supplements, herb-based food products derived from medicinal plants, foods rich in bioactive components, as well as engineered foods, are highly interesting from a scientific standpoint. Most nutraceuticals are of plant origin, among those being polyphenols, glucosinolates or sulfur-based compounds, phytosterols/phytostanols, tocotrienols, etc. Among them, terpenoids and in particular triterpenic acids (triterpene acids) are held in high regard to date in the nutraceutics field owing to their numerous biological activities. In fact, to date compounds such as oleanolic, ursolic, maslinic, corosolic, pomolic, annurcoic and tormentic acids are studied and proposed for their activity in reducing cancer risks, preventing cardio-cerebrovascular disorders, reinforcing the immune system, for their antioxidant activity, the improvement of sportspersons’ muscle performance, etc. Cosmeceutics is that branch of nutraceutics studying the effect of a topical application of substances of natural origin, generally contained in foods, on skin physiology and pathology (with anti-inflammatory and antiaging effects). In this context, raw material quality is key. In fact, it is well-known that the functionality and safety of products obtained from medicinal plant derivatives is strongly related to the quality and features of the raw materials used.

Triterpenic acids are widespread in plants in the form of secondary metabolites. Said triterpenic acids have been used for centuries in popular medicine as anti inflammatory, antidiabetic and hepatoprotective agents, and have recently captured the interest of the scientific community thanks to their anticarcinogenic activity. Triterpenic acids are also highly interesting for their use as functional compounds in cosmetics and pharmaceutical products. For these reasons, singling out new tools for the production of high amounts of metabolites such as triterpenic acids is taking on an ever-increasing importance.

Uvaol is a triterpenic diol found in olive tree leaves and fruits. A percentage of uvaol and erhytrodiol in olive oil equal to 1.4% has been reported in the literature.

Oleanolic acid can be found in the pulp of olives, and an amount of oleanolic acid ranging from 20.26 to 84.14 mg/100 mg in olive pulp (dry weight, or DW) has been reported.

Ursolic acid can be found in many plants, such as Mirabilis Jalapa, as well as in many fruits and herbs used in everyday life (e.g., apples, basil, blueberries, cranberries, elderberry flower, peppermint, rosemary, lavender, wild marjoram, thyme, hawthorn, and plums). Olives and apple peels contain large amounts of ursolic acid and related compounds.

Relatively low concentrations of ursolic acid (about 3.4 mg/100 mg) in air-dried strawberry leaves are reported in the literature.

Maslinic acid can be found in the pulp of olives; an amount of maslinic acid in olive pulp ranging from 38.4 to 131.8 mg/100 mg (DW) has been reported. Corosolic acid is a pentacyclic triterpenic acid, having a structure similar to that of ursolic acid, and it can be found in Lagerstroemia speciosa leaves. The prior art reports a corosolic acid content in L speciosa leaves equal to about 2.2%. The literature moreover reports a corosolic acid content in air-dried strawberry leaves equal to 3.8 mg/100 mg. Tormentic acid is a bioactive triterpenic acid isolated from Luehea divaricata, Agrimonia eupatoria and Rubus chingii. A tormentic acid content in R. chingii fruits equal to about 298.5 mg/5 Kg of dried fruits, corresponding to 5.97 mg/100 mg, is reported in the literature. Again, a tormentic acid content in air-dried strawberry leaves equal to about 3.4 mg/100 mg is reported in the literature (Sallam A. et al. 2016, World J. Pharm. Sci., 4, 287- 290). All of these acids exhibit relevant pharmacological activities; therefore it is of great interest to be able to obtain said substances, in plenty, from material of plant origin. Plant cell culture systems are of particular interest as, not depending on seasonality and not needing agriculture-related complex and costly systems for plant material availability. To date, numerous studies have been conducted by use of cultures of plant cells and organs. The most commonly used plant materials have been mainly developed from plant leaves, nodes, internodes, and petioles.

Saradha et al. (Saradha M. et al. 2014, Schott & Endl. Int. J. Pharm. Sci. Res., 5, 839- 848) have developed a protocol for efficient callogenesis in Hildegardia populifolia with the aim of studying the antioxidant properties of secondary metabolites that might be produced by these cultures. Fruit pulp has rarely been used as starting material in in vitro cultures for the production of secondary metabolites. The literature reports merely a few examples. Nawa and Ohtani (Nawa Y. & Ohtani T. 1992, Biosci. Biotechnol. Biochem., 56, 1732-1736) obtained the yellow pigment crocin from calluses deriving from Gardenia Jasminoides J. Ellis fruits.

Oota et al. (Oota S. et al. 1983, J. Jpn. Soc. Hortic., 52, 117-122) reported the accumulation of anthocyanins in callus cultures deriving from apple pulp. Patent document US 2005/0255569 A1 describes the production of a triterpenes-based composition obtained by calluses deriving from leaves, seeds, stems, buds, roots, or germ cells, yet not from fruit pulp, of banaba, loquat, perilla and guayava, by varying plant hormones inside the culture media (Matsuyama et al., 2005).

Two publications by Verardo G. et al., indicated hereinafter, describe triterpenic acids production in callus cultures deriving from the pulp of two varieties of apples denominated “Mela Rosa Marchigiana” (MRM) and “Golden Delicious” (GD), and from the pulp of a tropical fruit denominated “Acca Seiiowiana” of the Myrtaceae family (Verardo G. et al. 2016, Phytochemical Anal., 28, 5-15; Verardo G. et al. 2019, Food. Res. Int., 119, 596-604). These two articles, for the first time, demonstrated triterpenic acids production by callus cultures deriving from mature fruit pulp.

In said publications it was reported that corosolic acid is produced in an amount equal to 400 and 670 mV/IOO mg (DW) in cultures deriving from pulp of apples of the GD and MRM varieties respectively, whereas A. sellowiana fruit produces only about 90 mg/100 mg DW of corosolic acid. Usually, this compound may be found in Banaba, Lagerstroemia speciosa leaves, containing about 190 mg/100 mg DW of corosolic acid. In vitro and in vivo studies, conducted also in humans, confirmed that corosolic acid possesses antidiabetic activity and is effective in the treatment of renal diseases. Annurcoic acid, an interesting health-promoting ursanic-type triterpene able to increase endothelial nitric oxide synthase (eNOS), is not produced by in vitro culture of A. sellowiana fruit pulp, it being instead produced from GD and MRM cultures (about 840 and 350 mg/100 mg DW, respectively). On the other hand, it has been reported in the literature that the apple of the Annurca variety produces only 0.43-0.58 mg/100 mg (DW) of annurcoic acid.

Therefore, plant cell culture systems for the production of metabolites of interest are available in the literature. The identification of additional sources of plant cells suitable for the production of metabolites of interest, such as, e.g., triterpenic acids, and the setup of systems improving the yield of said metabolites is certainly of great interest due to the above-discussed relevant advantages, that said metabolite sources and methods leading to a production in greater amounts thereof exhibit compared to the extraction of said metabolites directly from parts of the plant.

SUMMARY OF THE INVENTION

To date, numerous studies have been conducted aimed at the production of secondary metabolites from plant cell and organ cultures for use in health promotion. For instance, numerous research groups have studied systems for producing anthocyanins, secondary metabolites of plants having high nutritional value for humans and vast application in the food industry, by in vitro culture of plant cells and tissues. The most commonly used plant materials for the development of said in vitro cultures are mainly the leaves, nodes, internodes, and petioles of the plants. Fruit pulp has rarely been used as a starting material for in vitro culture techniques aimed at the production of secondary metabolites. The present invention is based on the discovery that the culture of calluses deriving from the pulp of an apple variety denominated Red Sentinel (RS), maintained in a culture medium containing appropriate growth regulators and specifically developed for this apple variety, allows to produce surprisingly high amounts of triterpenic acids compared to other plant matrices described in the known art. The triterpenic acids produced according to the method of the present invention can find application in the pharmacology, nutraceutics, cosmeceutics fields, or in the production of active ingredients.

The innovative aspects of the present invention are mainly two: (i) the use of a culture medium appropriate for in vitro culture of RS apple pulp cells which, in the form identified by the Authors, proved to be surprisingly suitable for the production of high amounts of triterpenic acids; (ii) the development of a “callus” cell culture, from apple of the RS variety, a less-known edible variety of apple, mainly used for pectin extraction from the pulp.

Hence, a first aspect of the present invention relates to a method for affording a particularly efficient production of natural triterpenic acids from RS apple pulp, which has been proven to yield said acids in a greater amount compared to alike amounts of material from other plant matrices reported in the known art, as highlighted by the examples reported in the experimental section. These high yields do not derive exclusively from the starting material (i.e. , from explants of RS apple pulp), but are also the result of an appropriate selection of the culture medium and the growth regulators. Notwithstanding the teachings in the literature, the Authors of the present invention have modified the culture medium of the selected plant cells, identifying a precise ratio between the growth regulators auxin/cytokinin used, fostering the optimal development of calluses of pulp of a particular apple type having a high content of triterpenic acids compared to other plant matrices.

Therefore, object of the present invention is a method of cell culture and the related callus from the pulp of RS variety apple and methods for the production of triterpenic acids from said callus. Callus cultures such as that according to the present invention allow to produce greater amounts of triterpenic acids compared to the sole peels or pulp of the present apple variety or of other varieties.

The method described in the present invention affords numerous advantages: (i) a reduction of seasonality-related variability that can strongly influence the production of secondary metabolites in plants grown according to conventional methods; (ii) a reduced environmental impact during the plant cell culture procedure and low water consumptions compared to traditional crops; (iii) a vast availability of raw matter, continually extractable throughout the year; (iv) a higher rate of synthesis of secondary metabolites compared to that of spontaneous plants, or of plants grown according to traditional methods. In fact, through the development of callus subcultures at regular intervals, the method for the production of triterpenic acids described in the present invention envisages the spontaneous regeneration of the cellular material indefinitely. Hence, this allows to produce high amounts of secondary metabolites for nutraceutical and cosmeceutical purposes.

Moreover, the present invention allows to overcome the concept of “balsamic time”. “Balsamic time” is an important parameter referring to the life cycle period in which a medicinal plant possesses the maximum quali-quantitative value of the phytocomplexes. According to Pharmacopoeia, medicinal plants should be harvested during this “balsamic time”. Therefore, it is good practice to identify the plant phenological phases (ripening, growth, blossoming, fructification) with regard to active ingredients content. In fact, in the stages immediately subsequent to harvesting of spontaneous or cultivated plants, it is necessary to abide by the rules related to the degradation processes involving plant tissues (mainly the drying), both spontaneous and saprophyte-induced ones, as those can specifically influence secondary metabolite content.

Since the present invention does not envisage plant tissue treatment in the post harvesting stage, but is based on the use of cellular material, lyophilized “callus”, raw matter quality and functionality remain intact over time. Hence, it is possible to avail oneself of a constant and regular provision of batches of suitable plant material in order to meet the demand of a production line characterized by an uninterrupted productivity, ensuring the availability over time of a standardized product (also of a line of products having a constant formulation). Said product will possess organoleptic characteristics and functionalities constant over time, thereby ensuring an unvaried treatment.

The characteristics of the raw matters used for a nutraceutical or cosmeceutical purpose depend on botanical assessment, chemotypical characterization, genetic characterization, environmental factors, cultivation techniques. A further innovative aspect of the present invention consists in sidestepping said factors, as calluses culture, under appropriate growth conditions, remains stable over time for an undefined (theoretically infinite) period.

In light of the above-described advantages, the invention is particularly useful for the production of active ingredients (in the form of mono-substances or of association of substances having phytocomplex value) in the nutraceutics and cosmeceutics field, as it provides a standardized system for the production of triterpenic acids in large amounts. The present invention therefore provides a system for the production of a large amount of phytochemical substances, without use of synthetic processes, in which the availability of the material for the extraction of said substances does not vary with seasonality and, by the regeneration of calluses described herein, said phytochemical substances can be obtained from the same source of origin.

Therefore, object of the present invention is a method for the production of triterpenic acids from plant material taken from Red Sentinel apples comprising the following steps: preparing Red Sentinel apple pulp discs; subjecting said discs to an in vitro culture with Gamborg B5 medium complemented with from 0.15 a 0.25 mg/L of 6-benzylaminopurine (BA) and from 1.8 to 2.2 mg/L of 1-naphtaleneacetic acid (NAA) until obtaining plant calluses; optionally carrying out subcultures by taking cellular material from said calluses and subjecting said cellular material to in vitro culture under the same conditions used with said apple pulp discs until obtaining further plant calluses; freeze-drying and subsequently pulverizing said plant calluses; carrying out one or more extraction steps in alcoholic or hydroalcoholic solvent of triterpenic acids from said pulverized calluses.

Moreover, object of the present invention is also a callus of RS apple pulp cells. Other advantages and features of the present invention will be apparent from the following detailed description.

BRIEF DESCRIPTION OF THE FIGURES Figure 1. Mature Red Sentinel variety apple.

Figure 2. Culture of calluses deriving from mature Red Sentinel apple pulp.

GLOSSARY

The terms used in the present description are as generally understood by a person skilled in the art, apart from when indicated otherwise.

By the term “callus” (or “calluses”) the present invention refers to an amorphous mass of undifferentiated plant cells exhibiting characters of scarcely specialized parenchymal elements. Said plant cells are able to re-differentiate in response to hormonal stimuli and to originate new plantlets or roots.

The “Red Sentinel” (shortened to RS) denomination in the present invention denotes the Malus variety known under the botanical name of Malus x Robusta, belonging to the Rosaceae family, hybrid, obtained from hybridization of two apple plants of Asian origin: Malus baccata and Malus prunifolia.

The principal UPOV (International Union for the Protection of New Varieties of Plants) botanical name of the Red Sentinel variety described herein is Malus x robusta (Carriere) Rehder, also known as “Malus baccata x Malus prunifolia", or more commonly as “Siberian crab apple”, or “sibirischer Holz-Apfel”. The abovementioned variety is registered in the Community Plant Variety Office (CPVO) database (Number: Malus x /Obusfa_RedSentinel) and in the UPOV database (UPOV Code: MALUS_ROB); the Red Sentinel variety described herein is available in common Garden centers and plant nursery centers, such as, for instance: http://www.igiardinielefronde.it/arbusti/item/221-malus-red- sentinel/6-arbusti, https://www.ashridgetrees.co.uk/malus-red-sentinel-crabapple . The fruits of this plant, apples, are cherry-like, with a diameter of about 2.5 cm, first orange-colored, then shiny red, and characterized by a pectin-rich pulp.

Anywhere in the text of the present description and of the claims, the wording “Red Sentinel” or “RS” can be replaced by any one of the following expressions: “Malus x Robusta Red sentinel”, “Malus x robusta (Carriere) Rehder”, “Malus baccata x Malus prunifolia”, “Siberian crab apple”, or “sibirischer Holz-Apfel”.

By the generic term “triterpenic acids” in the present invention, reference is made to the subclass of terpenic compounds containing six isoprene units, i.e. a base carbon skeleton with 30 Carbon atoms. In nature, triterpenic acids are found in free or esterified form, both in the form of tetracyclic terpenes and of pentacyclic triterpenes. DETAILED DESCRIPTION

The present invention provides a method for the production of triterpenic acids from biological material cultured in vitro, “callus”, deriving from RS apple pulp.

The surprising technical effect observed, clearly seen in Table 2, is the significant increase of the amounts of triterpenic acids produced from said callus compared to the yields obtained by using various plant matrices known in the state of the art. As indicated in Tables 2-4, the mean amount of all triterpenic acids produced according to the method of the present invention is significantly higher not only compared to the amount obtained from calluses deriving from the pulp of other apples, like for instance of GD, MRM apples, or from A. sellowiana pulp, but also compared to the amount of triterpenic acids directly extracted from RS apple peels or pulp.

Such an increase is possible thanks to the in vitro culture of plant material deriving from RS pulp on a culture medium additioned with growth regulators cytokinin/auxin in in a specific ratio. These culture conditions in fact allow to foster the optimal development of calluses with a high content of triterpenic acids. Hence, object of the invention is a method for the production of triterpenic acids from plant material taken from Red Sentinel apples, comprising the following steps: preparing Red Sentinel apple pulp discs; subjecting said discs to an in vitro culture with Gamborg B5 medium complemented with from 0.15 to 0.25 mg/L of 6-benzylaminopurine (BA) and from 1.8 to 2.2 mg/L of 1-naphtaleneacetic acid (NAA) until obtaining plant calluses; optionally carrying out subcultures by taking cellular material from said calluses and subjecting said cellular material to in vitro culture under the same conditions used with said apple pulp discs until obtaining further plant calluses; freeze-drying and subsequently pulverizing said plant calluses; carrying out one or more extraction steps in alcoholic or hydroalcoholic solvent of triterpenic acids from said pulverized calluses. According to the present invention, the apple pulp discs can be isolated by (i) cutting the apple with a sterile blade, and (ii) by carving into the pulp of said apple discs of diameter between 3-5 mm and thickness between 1-3 mm.

The discs as indicated above can be made by using a perforator positioned at a distance of 3 mm from the peel.

According to a preferred embodiment, said pulp discs have a diameter and thickness respectively equal to 5 and 3 mm, and are carved by using a perforator positioned at a distance of about 3 mm from the peel.

Preferably, before the preparing of the pulp discs, the apples are sterilized externally, in order to reduce the possibilities of cell culture contamination and prevent possible bacterial or viral infections of the cultured cells. The sterilization step can be carried out by using any one of the sterilization techniques known to a technician in the field, such as for instance by apple washing with ethanol and/or rapid flaming.

According to the method of the present invention, the apple pulp discs are cultivated in Gamborg B5 (GB5) culture medium. Said culture medium is well-known to a person skilled in the art and was described by Gamborg et al. in 1968 (Gamborg OL et al. 1968 Ex. Cell. Res., 50, 15-158).

The nutrients present in the medium are reported in Table 1 below:

Table 1. Gamborg B5 medium composition Said medium is available from numerous commercial sources, and protocols for its preparation are available.

The medium used for in vitro culture of the calluses may contain other microcomponents selected from vitamins, sugars, solidifying agents, amino acids, phenolic compounds, antioxidants. To stimulate callus formation starting from RS apple pulp, the adding of cyto kin in/auxin plant hormones to the culture medium is particularly relevant, as already mentioned hereto. The ratio between this type of hormones influences the result of plant cell cultures in such a relevant manner as to determine, depending on the relative amounts of cytokinins and auxins added to the culture medium, the development of roots, buds, calluses, etc.

Therefore, the relative amounts and the cytokinin/auxin ratio decisively influence in culture plant cell development and differentiation, also influencing the type and the amount of metabolites synthesized by said cells. Moreover, the concentrations and the cytokinin/auxin ratio can also vary based on the plant type used as source of plant material to be cultured.

Non-limiting examples of auxins include 1-naphtaleneacetic acid (NAA), 2,4- diclorophenoxyacetic acid (2,4-D), indol-3-butyric acid (IBA), indol-3-acetic acid (IAA). Some non-limiting examples of cytokinins include natural cytokinins, like 4-hydroxy-3- methyl-frans-2-butenylaminopurine (zeatin), A/-(2-isopentenyl)adenine (2iP), and synthetic cytokinins, like 6-furfurylaminopurine (kinetin), 6-benzylaminopurine (BA or BAP), thidiazuron (TDZ).

In the method of the present invention, the Authors have found that accurately defined amounts of BA and NAA are fundamental in order to obtain a surprising production of triterpenic acids in calluses derived from RS apple pulp.

As indicated above, BA amounts, in the step of in-medium culture of the pulp discs or of the cells collected from RS pulp calluses according to the invention, are comprised in a very limited range, i.e. of 0.15 to 0.25 mg/L. NAA amounts, in the same steps, range from 1.8 to 2.2 mg/L.

In a preferred embodiment, the Gamborg B5 medium used for the in vitro culture of calluses from RS apple according to the present invention is complemented with NAA in a concentration equal to 2 mg/L and with BA in a concentration equal to 0.2 mg/L.

In the method according to the present invention, the pH of the medium used in the in vitro culture steps is in a range of from 5.5 to 6.5. In one embodiment said pH is of about 5.8.

In the method according to the present invention, pulp discs culture and calluses subculture can be performed at temperatures normally used for in vitro generation of plant calluses. Preferably, the culture in the above-described medium, both as to the use of pulp discs according to any embodiment indicated above and as to cellular material taken from calluses obtained according to the present invention, is performed at temperature ranging from 20-30 °C, but even lower or higher temperatures may be used. In a preferred embodiment, the culture is performed at 25±2 °C. Moreover, aseptic growth conditions, in the dark, are preferred for cell growth. According to a further aspect of the present invention, the cultures of cellular material derived from RS apple pulp according to the present invention can be incubated for a time comprised between 25 and 30 days, before proceeding with triterpenic acids extraction. Said incubation has a time length preferably equal to 28 days.

According to the invention, cellular material collected from plant calluses and obtained with the method described herein, can be subjected to the same above-described culture steps, in all of their embodiments, generating further plant calluses.

Therefore, the triterpenic acids produced according to the method of the present invention can be obtained directly from in vitro cultures of calluses produced by the above-described and claimed steps, or even from subcultures of cellular material taken from said calluses obtained by subjecting said cellular material to in vitro culture according to the same culture conditions and in the same type of medium described for calluses production from RS apple pulp discs provided in the present description and in the claims.

The formation of calluses subcultures provides a stable and continual procedure for the production of triterpenic acids over lengthy periods of time.

Once obtained the plant calluses, the method of the present invention comprises steps for the extraction of triterpenic acids from said calluses.

The calluses are then subjected to freeze-drying and once freeze-dried, are finely fragmented, or even powdered, before being extracted in alcoholic or hydroalcoholic solvent.

The fragmenting/powdering of the freeze-dried (lyophilized) plant material can be carried out according to any technique commonly used by the technician in the field for the extraction of active ingredients from plant cells. Said fragmenting can be for instance carried out by using a pestle mortar.

The step of extracting the triterpenic acids can be carried out by any suitable method known to the technician in the field. For instance, conventional extraction methods in an alcoholic solvent or in a hydroalcoholic mixture can be used. According to a preferred embodiment of the present invention, the alcoholic solvent used is ethanol (EtOH) or methanol (MeOH) or hydroalcoholic mixtures containing 70-96% EtOH or MeOH. The extracts obtained during one or more steps of extraction with an alcoholic or hydroalcoholic solvent can be pooled, and the solvent discarded under vacuum conditions at T < 40 °C.

According to the method of the present invention, said extracts, once the solvent is discarded, can be collected in ethyl acetate, and washed with water.

The extracts obtained according to the method of the present invention comprise oleanolic, ursolic, maslinic, corosolic, pomolic, annurcoic and tormentic acids.

In particular, according to an aspect of the present invention, the total amount of triterpenic acids produced from the plant material callus is equal to at least 7% compared to the weight of said lyophilized callus, preferably equal to at least 8% by weight. Some experimental examples are provided hereinafter to illustrate these results. For instance, from 100 mg of powdered calluses deriving from RS apple pulp, there are obtained on average (see Table 2): 177.1 mg of oleanolic acid, 719.2 mg of ursolic acid, 1083.6 mg of maslinic acid, 1444.2 mg of corosolic acid, 103.2 mg of pomolic acid, 1065.5 mg of annurcoic acid, and 3838.0 mg of tormentic acid, for a total of 8.441 mg/100 mg of triterpenic acids (8.4%).

The method according to the present invention can finally include a further step of analysis of the extracts comprising terpenic acids by gas chromatography coupled to mass spectrometry (GC-MS).

As mentioned above, object of the present invention are also in vitro cultures of calluses deriving from RS apple pulp obtainable by said method.

The present invention also provides the use of said calluses cultures for the production of triterpenic acids comprising the acids enumerated above.

Examples are provided hereinafter in order to show a preferred, yet non-limiting, embodiment of the invention.

EXAMPLES

Some non-limiting examples of the methodologies according to the present invention are reported hereinafter. In vitro culture of the plant material

Mature pulp of Red Sentinel apple (Figure 1) was used as the source of explants used for callus induction. Apples were sterilized by washing the fruit surface with ethanol and subsequently flaming it in the laminar flow cabinet. The apples were then opened by using a sterilized blade.

By means of a perforator positioned at 3 mm from the peel, discs of a diameter and thickness respectively of 5 and 3 mm are carved. The cultures are incubated in the dark, at 25 ± 2 °C, in Gamborg B5 medium additioned with

- 0.2 mg/L of benzylaminopurine (BA, cytokinine)

- 2.0 mg/L of 1-naphtaleneacetic acid (NAA, auxin)

Callus subculture is carried out in the same medium after 28 days. The calluses so obtained (Figure 2) were stored at -20 °C, freeze-dried, pulverized using a pestle mortar and extracted in ethanol prior to chemical analysis.

Extraction with solvent of triterpenic acids

The extraction was carried out as follows: pulverized calluses (DW, 100 mg) were treated with 96% ethanol (EtOH) (25 mL) under magnetic stirring for 1 h at room temperature, and then centrifuged at 5000 rpm for 8 minutes. The supernatant was stored, while pellet was subjected to a second extraction following the same procedure. Obtained supernatants were then pooled and concentrated in vacuo at T < 40 °C. Residual pellet was then resuspended in water (5 mL) and subjected to extraction with ethyl acetate EtOAc (30 mL x 2). Pooled organic phases were washed with water (10 mL) and brine (10 mL). Thereafter, the organic phase was dried by use of anhydrous Na ₂ S0 ₄ , filtered and the solvent removed under vacuum at T < 40 °C. Residual pellets were solubilized in ethyl acetate at the concentration of 1 mg/mL, and stored at 4 °C. Thereafter, an aliquot (1 mL) of the solution of the extract in ethyl acetate was evaporated under Argon atmosphere, and the obtained residue was solubilized in pyridine (500 mί) and treated with Sylon BFT (500 mί).

Quali-quantitative analysis of extracts

A qualitative analysis of the extracts was carried out, sylilating the sample at 80 °C for three hours and injecting 1 m\- of the reaction mixture into the gas chromatography system with mass spectrometer (GC-MS). The quantitative analysis was conducted, by following the same sylilating procedure described above, analyzing the extracts containing cholesterol, added before the extraction procedure, as internal standard.

A first quantitative GC-FID analysis of the extracts obtained from 100 mg (DW) of lyophilized plant material, “callus”, deriving from RS pulp or from the pulp of other plant matrices was carried out.

Table 2 reports the amounts of triterpenic acids present in 100 mg of lyophilized calluses deriving from mature pulp of RS apple, or from the pulp of GD, MRM apple, or of A. sellowiana. Table 2. Quantitative analysis (GC-FID) of lyophilized calluses deriving from fruit pulp.

RS = Red Sentinel; GD = Golden Delicious; MRM = Mela Rosa Marchigiana

Alike results were obtained by using methanol as extraction solvent.

Moreover, a quantitative analysis by GC-FID of the extracts obtained directly from Red Sentinel apple peel or pulp was carried out. The following Table 3 reports the amounts of triterpenic acids present in 100 mg of Red Sentinel apple peel.

Table 3. Quantitative analysis (GC-FID) of Red Sentinel peel.

Finally, Table 4 reports the amounts of triterpenic acids present in 100 g of Red Sentinel apple pulp.

Table 4. Quantitative analysis (GC-FID) of Red Sentinel pulp. From a comparative analysis of the amounts of triterpenic acids obtained from different plant matrices, it is possible to observe that the present invention allows to produce on average (see Table 2): 177.1 mg/100 mg of oleanolic acid, 719.2 mg/100 mg of ursolic acid, 1083.6 mg/100 mg of maslinic acid, 1444.2 mg/100 mg of corosolic acid, 103.2 mV/IOO mg of pomolic acid, 1065.5 mV/IOO g of annurcoic acid and 3838.0 mg/100 mg of tormentic acid, for a total of 8.441 mg/100 mg of triterpenic acids (8.4%).

On the contrary, Red Sentinel apple peel contains for instance only 441.6 mg/100 mg, on average, of ursolic acid (see Table 3). Moreover, the production of triterpenic acids is higher by in vitro culture of the cells deriving from RS apple pulp, compared to the GD and MRM varieties. For instance, the amount of tormentic acid produced by calluses deriving from GD and MRM is equal to 1059.7 and 2905.6 mg/100 mg (DW) respectively, against the 3848.0 mg/100 mg (DW) of tormentic acid produced by calluses deriving from RS pulp. The present invention produces 1.0% of uvaol alone.