Verbascum nigrum extracts, processes for preparing the same and their uses for treating dysbiosis

Abstract

Verbascum nigrum extracts are disclosed, as well as processes for preparing the same and uses as active ingredients in nutraceuticals. In particular, it is disclosed an enzymatic process wherein a specific enzyme cocktail is used for selectively releasing and extracting compounds from a Verbascum nigrum biomass. It is also disclosed a hydroalcoholic process for releasing and extracting different compounds from a Verbascum nigrum biomass. Said Verbascum nigrum biomass is advantageously obtained from vertical farming, which allows to maximize the stability and properties' reproducibility of the botanical with respect to the corresponding wild-growing plants. The resulting enzymatic and hydroalcoholic extracts can be advantageously used singularly or in combination thereof, thus taking benefit of the overall active compound mixture. Specifically, the extracts can be suitable used in nutraceutical compositions and food supplements.

Description

[0001] VERBASCUM NIGRUM EXTRACTS, PROCESSES FOR PREPARING THE SAME AND THEIR USES FOR TREATING DYSBIOSIS

[0002] DESCRIPTION

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to Verbascum nigrum extracts, processes for preparing the same and uses as active ingredients in nutraceuticals.

[0005] In particular, it is disclosed an enzymatic process wherein a specific enzyme cocktail is used for selectively releasing and extracting compounds from a Verbascum nigrum biomass. It is also disclosed a hydroalcoholic process for releasing and extracting different compounds from a Verbascum nigrum biomass. Said Verbascum nigrum biomass is advantageously obtained from vertical farming, which allows to maximize the stability and properties’ reproducibility of the botanical with respect to the corresponding wild-growing plants.

[0006] The resulting enzymatic and hydroalcoholic extracts can be advantageously used singularly or in combination thereof, thus taking benefit of the overall active compound mixture.

[0007] Specifically, the extracts can be suitable used in nutraceutical compositions and food supplements.

[0008] BACKGROUND OF THE INVENTION

[0009] Herbal medicines are the most ancient form of health remedies known to humankind. Despite the great advances achieved in modern medicine, plants still make an important contribution to health care, and several specific herbal extracts have been demonstrated to be efficacious for specific conditions. Moreover, several chemical substances derived from plants are considered important drugs currently in use, and several other drugs are simple synthetic modifications of natural products. Plants can be regarded as “living factories” producing a variety of chemical compounds, including primary metabolites important for the growth of the plants (amino acids, proteins, carbohydrates) and secondary metabolites (alkaloids, terpenoids, phenylpropanoids, polyketides, flavonoids, saccharides). All these components may work together to deliver a synergistic effect in the finished product.

[0010] Certain herbal medicines, because of the complexity of their chemical content and the variety of bioactivities, can provide the poly-pharmacology that conventional drugs cannot deliver. Natural products are rarely evaluated in the well-controlled clinical trials that are required to receive approval by regulatory bodies; therefore, they tend to have less scientific evidence to support their efficacy. However, all medicinal compounds are chemicals, whether synthesized in plants, animals, or in manufacturing laboratories, therefore, all medicinal chemical compounds should be held accountable to similar standards of quality (identity, purity, and stability), clinical effectiveness and safety. Reliable and consistent quality is the basis of efficacy and safety of herbal medicinal products. Given the nature of products of plant origin, which are highly variable and complex products with numerous biologically active components rarely completely identified, therapeutic results and safety issues vary greatly from product to product, even within a single class. Therefore, the evidence of both benefits and risks is specific to the product tested and cannot necessarily be extrapolated to other products, as is the case for synthetically derived compounds.

[0011] For these reasons, and due to the inherent variability of the constituents of herbal products, it is generally difficult to establish quality control parameter and maintain consistent batch- to-batch quality; variation in the absence of reference standards for identification can start from the collection of raw materials and increase during storage and further processing. The quality of botanicals and herbal medicinal products can be linked to livelihoods of different value chains and sustainability. It is documented that many rural communities and indigenous groups, particularly in Asia, depend on medicinal plant collection for their livelihoods. The downside of this wild collection is as collected plants are depleted in the wild, their scarcity in the marketplace increases along with their economic value. Only an integrated chain using cultivated material may provide a better alternative to these models, however, due to the continuous increase of air and soil pollution, particularly in Asian countries, the level of pesticides, heavy metals and residues are predominantly presence in herbal extract. It is widely known, in fact, that the active ingredients used in food supplement market are not subjected to scientific validation and do not have the same regulatory procedures as instead required for drugs or cosmetics. The vast majority of products on the market contains raw materials that come, as said, from Asian countries, which are not only of very poor quality, but too often rich in toxic substances, heavy metals and with much higher bacterial counts and much lower active ingredients, as compared to those declared.

[0012] Controlled organically cultivated herbs may be a good option, but organically cultivated material is typically more expensive than wild-collected material, especially when it is produced in more economically developed countries.

[0013] Due to the increasing high demand of herbal products globally, there is a pressure to find more material may lead to adulteration of the crop with similar species or adulteration further down the value chain. There are also concerns by some consumers that cultivated material may be in some way inferior to wild-collected material, and subsequently the finished products are less appealing to these consumers.

[0014] In this regard, quality issues of herbal medicines can be classified into two categories: external and internal. External issues are contamination (e.g. toxic metals, pesticides residues and microbes), adulteration and misidentification, whereas complexity and nonuniformity of the ingredients in herbal medicines are the internal factors.

[0015] It is therefore an object of the present invention to provide a process for extracting active ingredients from botanicals, in particular belonging to the Verbascum genus, with the aim to minimize the impact of said external and internal issues, thus improving the overall quality and efficacy of the resulting extracts.

[0016] Among Verbascum genus, Verbascum nigrum, the black mullein or dark mullein, was selected, while being a species used in medicinal products, as expectorant and mucolytic, as well as sudorific, sedative, and diuretic agent. Verbascum species are also used externally for desiccating wounds, and as antiseptic, astringent, demulcent, emollient, expectorant, sedative, narcotic, diuretic and antimalarial agent and for the treatment of tumours, inflammations, migraine, asthma and spasmodic coughs.

[0017] Therefore, is also an object of the present invention to provide Verbascum nigrum extracts that are enriched of suitable active ingredients, thus increasing their yields and efficacy. SUMMARY OF THE INVENTION

[0018] The above object has been achieved by an enzymatic process for preparing an enzymatic Verbascum nigrum extract, as well as a hydroalcoholic process for preparing a hydroalcoholic Verbascum nigrum extract, as set forth by the present claims.

[0019] The present invention thus relates also to the extracts obtained by said processes. In a further aspect, the present invention also relates to a nutraceutical composition comprising both said Verbascum nigrum extracts.

[0020] In an additional aspect, the present invention also relates to a food supplement comprising the enzymatic Verbascum nigrum extract, the hydroalcoholic Verbascum nigrum extract, or the nutraceutical composition, and suitable food excipients.

[0021] BRIEF DRESCTIPTION OF THE DRAWINGS

[0022] The characteristics and advantages of the present invention will be evident from the detailed description given below, from the illustrative non-limiting working examples, as well as from the annexed figures, wherein:

[0023] Figures 1(a) and 1(b) show LC-MS / MS profiles of luteolin in the Verbascum nigrum extracts, as obtained in Example 1, via enzymatic treatment and via hydroalcoholic treatment, respectively;

[0024] Figures 2(a) and 2(b) show LC-MS / MS profiles of verbascoside in the Verbascum nigrum extracts, as obtained in Example 1, via enzymatic treatment and via hydroalcoholic treatment, respectively;

[0025] Figures 3(a) and 3(b) show LC-MS / MS profiles of / ?-coumaric acid in the Verbascum nigrum extracts, as obtained in Example 1, via enzymatic treatment and via hydroalcoholic treatment, respectively;

[0026] Figures 4(a) and 4(b) show LC-MS / MS profiles of caffeic acid in the Verbascum nigrum extracts, as obtained in Example 1, via enzymatic treatment and via hydroalcoholic treatment, respectively;

[0027] Figures 5(a) and 5(b) show LC-DAD profiles at 325 nm in the Verbascum nigrum extracts, as obtained in Example 1, via enzymatic treatment and via hydroalcoholic treatment, respectively;

[0028] Figures 6-9 show the ion abundance levels of specific compounds in the Verbascum nigrum extracts, as obtained in Example 1, via hydroalcoholic treatment, via coupled treatments, and via enzymatic treatment, respectively. Data expressed as mean values with SD depicted by vertical bars.

[0029] DETAILED DESCRIPTION OF THE INVENTION

[0030] In a first aspect, the present invention thus relates to an enzymatic process for preparing an enzymatic Verbascum nigrum extract, the process comprising the steps of: i) providing dry and milled biomass of Verbascum nigrum, where the latter is previously grown and harvested in a vertical farm, ii) adding buffered water at pH 3.5-5.5, to achieve a biomass concentration of 20- 80g / L, iii) adding an enzymatic cocktail consisting of: endo-polygalacturonase (CAS n. 9032-75-1 - EC 3.2.1.15), cellulase (CAS n. 9012-54-8 - EC 3.2.1.4), a-amylase (CAS n. 9000-90-2 - EC 3.2.1.1), and endo-l,4-P-xylanase (CAS n. 9025-57-4 - EC 3.2.1.8), iv) stirring the resulting mixture at a temperature of 30-70°C for 12-36 hours, v) separating the solid residue from the solution, while keeping the solution, which is the Verbascum nigrum extract, wherein said Verbascum nigrum extract comprises -coumaric acid, caffeic acid, caffeic acid-hexoside-rhamnoside, and ferulic acid-hexoside-rhamnoside.

[0031] In step i), Verbascum nigrum is previously grown and harvested in a vertical farm, before being dried and milled.

[0032] For the purposes of the present invention, with the term “vertical farm” or “vertical farming”, it is meant the practice of growing botanicals in vertically stacked layers. It often incorporates controlled-environment agriculture, which aims to optimize plant growth, and soilless farming techniques such as hydroponics, aquaponics, and aeroponics. Some common choices of structures to house vertical farming systems include buildings, shipping containers, or tunnels. The main advantage of utilizing vertical farming technologies is the increased botanical yield that comes with a smaller unit area of land requirement. Another sought-after advantage is the increased plant resistance to weather disruptions because of their placement indoors, meaning fewer plants are lost to extreme or unexpected weather occurrences. Because of its limited land usage, vertical farming is less disruptive to the native plants and animals, leading to further conservation of the local flora and fauna.

[0033] However, it should be also appreciated that the controlled conditions implemented for growing Verbascum nigrum for the purposes of the present invention, allowed to maximize the stability and properties’ reproducibility of the botanical with respect to the corresponding wild-growing plants. In particular, Verbascum nigrum cultivated for the purposes of the present invention, is kept in a controlled and confined environment for its entire life. In details, its germination takes place in a specific area where the plant develops until it is approximately 20 days old, then it is re-potted inside larger pots and inserted inside an industrial phytotron. Here a LED lighting system combined with forced air conditioning are set with the aim to assure controlled and regular growth conditions. At the maximum maturation of the plant, the latter is cut and dried at low temperature for about 24 hours, thus obtaining the Verbascum nigrum biomass to be suitably used for the purposes of the present invention.

[0034] Thus, the biomass of Verbascum nigrum coming from vertical farm, is then milled.

[0035] Preferably, said biomass is dried at 30-50°C for 12-36 hours, and milled down to a particle size lower than 5 mm.

[0036] More preferably, said biomass is dried at 40-50°C for 20-28 hours, and milled down to a particle size lower than 5 mm.

[0037] In step ii), buffered water at pH 3.5-5.5 is added, to achieve a biomass concentration of 20- 80g / L.

[0038] Preferably, in step ii), water is buffered by adding a citrate buffer.

[0039] More preferably, buffered water is at pH 3.8-5.2.

[0040] Preferably, in step ii), a biomass concentration of 30-50g / L is achieved.

[0041] In step iii), an enzymatic cocktail is added, consisting of: endo-polygalacturonase (CAS n. 9032-75-1 - EC 3.2.1.15), cellulase (CAS n. 9012-54-8 - EC 3.2.1.4), a-amylase (CAS n. 9000-90-2 - EC 3.2.1.1), and endo-l,4-P-xylanase (CAS n. 9025-57-4 - EC 3.2.1.8).

[0042] Preferably, said enzymatic cocktail is added so that each enzyme is, independently of one another, at a concentration of 50-200 U / g_DM (i.e. Units per gram of dry biomass).

[0043] More preferably, each enzyme is, independently of one another, at a concentration of 80- 120 U / g_DM.

[0044] In preferred embodiments, the four enzymes are added so as to be at the same concentration.

[0045] In step iv), the mixture resulting from step iii) is stirred at a temperature of 30-70°C for 12-36 hours.

[0046] Preferably, said mixture is stirred at a temperature of 45-65°C for 20-28 hours.

[0047] In step v), the solid residue is separated from the solution, while keeping the solution, which is the enzymatic Verbascum nigrum extract comprising -coumaric acid, caffeic acid, caffeic acid-hexoside-rhamnoside, and ferulic acid-hexoside-rhamnoside.

[0048] In other preferred embodiments, the present invention concerns an enzymatic process consisting essentially of steps i) to v). The term “consisting essentially of’ means that the steps i) to v) are the only steps determining the effective extraction, the possible optional steps being subsidiary and not essential.

[0049] In further preferred embodiments, the present invention concerns an enzymatic process consisting of steps i) to v).

[0050] It is to be understood that all aspects identified as preferred and advantageous for the enzymatic process of the invention, are to be deemed analogously preferred and advantageous also for the enzymatic process of the invention of these preferred embodiments denoted by the terms “consisting essentially of’ and “consisting of’.

[0051] Therefore, in another aspect, the present invention also relates to an enzymatic Verbascum nigrum extract obtained by the enzymatic process above described, said enzymatic extract comprising / ?-coumaric acid, caffeic acid, caffeic acid-hexoside-rhamnoside, and ferulic acid-hexoside-rhamnoside.

[0052] It should be appreciated that the enzymatic process of the invention allows to extract a number of compounds in a significant yield, those compounds being known for a number of advantageous properties. For example, caffeic acid and its derivatives are known for their antibacterial activity, antiviral activity, antioxidant activity, anti-inflammatory activity, anti-atherosclerotic activity, immunostimulatory activity, antidiabetic activity, cardioprotective activity, antiproliferative activity, hepatoprotective activity, anticancer activity, and anti-hepatocellular carcinoma activity. Therefore, it is worthwhile noting that the enzymatic process of the invention leads to an enzymatic Verbascum nigrum extract which features those compounds in an effective amount.

[0053] These results are not achieved when a hydroalcoholic solution is used as extraction medium, thus confirming the purposiveness and specificity of the selected enzyme cocktail. The enzymatic extract above described can therefore be used as an active ingredient or as a co-adjuvant in the treatment of dysbiosis of the intestinal microbiota, and pathologies ascribable thereto.

[0054] Among the pathologies ascribable to dysbiosis of the intestinal microbiota, the following can be mentioned: insulin-resistance, inflammatory bowel disease, irritable bowel syndrome (IBS), and coeliac disease, allergy, asthma, non-insulin-dependent diabetes (NIDDM), syndrome X, obesity, polycystic ovarian disorder (PCOS), hair-AN syndrome, AIDS wasting, intra-uterine growth failure, post-natal growth failure, Prader-Willi syndrome, type 2 diabetes, diabetic complications, hyperglycaemia, dyslipidaemia, metabolic syndrome, hypertension, cardiovascular disease.

[0055] It is well established that p-Hydroxycinnamic acids (HCAs), including ferulic, caffeic, sinapic, and p-coumaric acids, possess a characteristic phenylpropanoid C6-C3 backbone and account for about one-third of the phenolic compounds in our diet. HCAs are typically associated with various plant cell wall components, including mono-, di-, and polysaccharides, sterols, polyamines, glycoproteins, and lignins. HCAs are completely absorbed in their free form upon ingestion and undergo specific reactions upon absorption in the small intestine or liver. The gut epithelium, composed of intestinal epithelial cells (lECs), acts as a physical barrier against harmful bacteria and a site for regulated interactions between bacteria and the gut lumen. Thus, maintaining the integrity of the epithelial barrier is essential for establishing a physiochemical environment conducive to homeostasis. In this regard, in a very recent scientific review, Wang, Z.-Y. et al. (“Biological Activities of p-Hydroxycinnamic Acids in Maintaining Gut Barrier Integrity and Function”, Foods 2023, 12, 2636) have concluded that “HCAs play a protective role in maintaining the integrity of the intestinal barrier. They promote the assembly of epithelial tight junctions and increase the abundance of occludin and ZO-1 proteins, thereby reducing intestinal permeability and mitigating damage to the intestinal mucosa caused by compromised tight junctions. HCAs have also been shown to decrease the levels of pro-inflammatory cytokines IL-6 and IL-8 and the mRNA expression of COX-2 and TNF-a. Furthermore, HCAs can alleviate oxidative stress and inflammatory responses, improve intestinal barrier health, reduce inflammation, and regulate intestinal homeostasis. Animal experiments have demonstrated that supplementing HCAs effectively alleviates DSS-induced colitis in mice by enhancing the intestinal barrier, modulating intestinal microbial homeostasis, and boosting anti-inflammatory and antioxidant capacity. HCAs act as free radical scavengers and antioxidants by increasing the levels of CAT and SOD, reducing the levels of MDA and ROS, and inhibiting free radical formation, lipid peroxidation, and DNA damage. Moreover, HCAs contribute to the restoration of intestinal microbial diversity, leading to a reduction in DSS-induced intestinal damage and protection of the intestinal barrier. HCAs can potentially exert beneficial effects on the intestinal microbiota, and their biotransformation products significantly promote a healthy intestinal flora ecology. In summary, HCAs yield a favourable impact on the protection and restoration of the intestinal barrier through various mechanisms: (1) modulation of intestinal permeability by safeguarding tight junctions, (2) modulation of intestinal immunity, (3) antioxidant properties that benefit the intestinal barrier, and (4) regulation of gut microbiota by HCAs.”

[0056] In view of the above, it is clear the significant advantage achieved by the enzymatic process of the present invention, as well as by the resulting enzymatic extract enriched in said HCAs, within the treatment of dysbiosis of the intestinal microbiota.

[0057] In another aspect, the present invention relates to a hydroalcoholic process for preparing a hydroalcoholic Verbascum nigrum extract, the process comprising the steps of: a) providing dry and milled biomass of Verbascum nigrum, where the latter is previously grown and harvested in a vertical farm, b) adding a hydroalcoholic solution comprising up to 60% ethanol, c) stirring the resulting mixture at room temperature for 1 -6 hours, d) separating the solid residue from the solution, while keeping the solution, which is the Verbascum nigrum extract, wherein said Verbascum nigrum extract comprises luteolin, verbascoside, Forsythoside B, coumaroyl-acetylrhamnosyl-aucubin, and caffeoyl-acetylrhamnosyl-aucubin.

[0058] In step a), Verbascum nigrum is previously grown and harvested in a vertical farm, before being dried and milled. This step a) is the same as step i) for the enzymatic process, so that the same remarks and preferences are applicable in the hydroalcoholic process.

[0059] In step b), a hydroalcoholic solution comprising up to 60% ethanol is added to the biomass. Preferably, said hydroalcoholic solution comprises 50-60% ethanol. In step c), the resulting mixture is stirred at room temperature for 1-6 hours.

[0060] With “room temperature”, it is meant a temperature of 20-25°C.

[0061] Preferably, the resulting mixture is stirred at room temperature for 1-3 hours.

[0062] In step d), the solid residue is separated from the solution, while keeping the solution, which is the hydroalcoholic Verbascum nigrum extract comprising luteolin, verbascoside, Forsythoside B, coumaroyl-acetylrhamnosyl-aucubin, and caffeoyl-acetylrhamnosyl- aucubin.

[0063] In other preferred embodiments, the present invention concerns a hydroalcoholic process consisting essentially of steps a) to d). The term “consisting essentially of’ means that the steps a) to d) are the only steps determining the effective extraction, the possible optional steps being subsidiary and not essential.

[0064] In further preferred embodiments, the present invention concerns a hydroalcoholic process consisting of steps a) to d).

[0065] It is to be understood that all aspects identified as preferred and advantageous for the hydroalcoholic process of the invention, are to be deemed analogously preferred and advantageous also for the hydroalcoholic process of the invention of these preferred embodiments denoted by the terms “consisting essentially of’ and “consisting of’.

[0066] Therefore, in a further aspect, the present invention also relates to a hydroalcoholic Verbascum nigrum extract obtained by the process above described, said hydroalcoholic extract comprising luteolin, verbascoside, Forsythoside B, coumaroyl-acetylrhamnosyl- aucubin, and caffeoyl-acetylrhamnosyl-aucubin.

[0067] It should be appreciated that the hydroalcoholic process of the invention allows to extract a number of compounds in a significant yield, which are different from those extracted via enzymatic process, those compounds being known for a number of advantageous properties. For example, luteolin has pharmacological properties, such as antiinflammatory, antioxidant, neuroprotective, and analgesic activities.

[0068] Verbascoside has various biological and therapeutic activities, such as anti-inflammatory, neuroprotective, and anti -cancer properties.

[0069] Forsythoside B has good antioxidant and anti-bacterial properties in vivo and in vitro, and can reduce cardiovascular disease damage and exert cardiovascular protection.

[0070] Aucubin, as well as its derivatives, has many biological activities, such as antioxidant, anti- aging, anti-inflammatory, antimicrobial, anti-fibrotic, anti-cancer, hepatoprotective, neuroprotective and osteoprotective effects.

[0071] The hydroalcoholic extract above described can therefore be used as an active ingredient or as a co-adjuvant in the treatment of pathologies related to the musculature, skeleton, and circulatory, digestive, and respiratory systems, as well as to treat infectious diseases and organ-sense illnesses.

[0072] In some embodiments, the enzymatic process and the hydroalcoholic process can be performed parallelly and simultaneously, so that the respective extracts are available substantially at the same time. This allows the extracts to be ready to be mixed together in the nutraceutical composition, if desired.

[0073] In view of the advantageously extracted compounds, the present invention thus relates to a nutraceutical composition comprising the enzymatic Verbascum nigrum extract, and the hydroalcoholic Verbascum nigrum extract, as above described.

[0074] Actually, the resulting nutraceutical composition takes benefit of both the extracts, each peculiarly featuring different actives. The mix of said extracts can thus allow to exploit the beneficial properties of all the compounds so provided, in a single and unique nutraceutical product.

[0075] In an additional aspect, the present invention also concerns a food supplement comprising the enzymatic Verbascum nigrum extract, the hydroalcoholic Verbascum nigrum extract, or the nutraceutical composition, and suitable food excipients.

[0076] Suitable food excipients can be, for example, natural starch, partially hydrolysed starch, lactose, glucose, sucrose, mannitol, sorbitol, cellulose and derivatives thereof, microcrystalline cellulose and derivatives thereof, calcium phosphate, calcium carbonate, calcium sulfate, magnesium stearate, maltodextrin, gelatine, gum tragacanth, Arabic gum, xanthan gum, talc, silica, colloidal silica, precipitated silica, magnesium silicates, aluminium silicates, sodium lauryl sulfate, magnesium lauryl sulfate, methacrylate copolymers, and mixtures thereof.

[0077] Said food supplement can be in the form of powder, capsule, tablet, mini-tablet, microtablet, granule, micro-granule, pellet, multi particulate, or micronized particulate. Alternatively, it may be in liquid form, i.e. in solution with suitable solvents of food grade. It is to be understood that all aspects identified as preferred and advantageous for the processes of the invention, are to be deemed analogously preferred and advantageous also for the respective extracts, and both the composition and food supplements.

[0078] It is also to be understood that all the combinations of preferred and advantageous features of the processes, extracts, composition and food supplements are deemed to be hereby described.

[0079] Working examples of the present invention provided for illustrative purposes are reported herein below.

[0080] EXAMPLES

[0081] Example 1

[0082] Process for preparing Verbascum nigrum extracts

[0083] Materials and methods

[0084] The enzymatic cocktail used for the treatment of the plant material consisted of four enzymes: cellulase (EC 3.2.1.4), xylanase (EC 3.2.1.8), pectinase (EC 3.2.1.15), and amylase (EC 3.2.1.1). Said enzymes were purchased from Creative Enzymes and were used in the experimentation:

[0085] • Cellulase (Cat No.: DIS-1017; Lot No.: ECD7011006; Activity: 20340 U / g)

[0086] • Xylanase (Cat No.: DIS-1032; Lot No.: ECD2022310; Activity: 20280 U / g)

[0087] • Pectinase (Cat No.: DIS- 1030 ; Lot No.: ECD0021415; Activity: 30240 U / g)

[0088] • Mid-temperature Refining a- Amylase for beer (Cat No.: BER-1513; Lot No.: ECB3081602; Activity: 4230 U / g)

[0089] The enzymatic treatment was conducted in a citrate buffer. Sodium citrate and citric acid were purchased from Sigma Aldrich. The alcohol used in the hydroalcoholic extraction was ethanol (Carlo Erba).

[0090] Biomass preparation

[0091] The plant material was stored at -20 °C upon arrival from vertical farm.

[0092] Then, the plant material was dried at 45°C for 24 hours. The resulting dried biomass was milled to obtain a particle size lower than 1 mm. The dry matter (DM) of the vegetable biomass was determined to be 40 g / L.

[0093] Biomass enzymatic treatment

[0094] The enzymatic treatment was performed in citrate buffer (0.1 M, pH 4.5). The enzymatic cocktail was added at a loading of 100 U / g_DM (Units per gram of dry vegetable biomass). The treatments were carried out on a rotating wheel, at 55°C, for a duration of 24 hours.

[0095] Hydroalcoholic extraction

[0096] Ethanol (100 vol%) was added to the biomass to reach a concentration of alcohol of 60 vol% in the final water solutions. The hydroalcoholic extractions were performed on a rotating wheel, at room temperature, for a duration of 2 hours.

[0097] Four replicates were conducted for each experiment.

[0098] Characterization of the (poly)phenolic components in Verbascum nigrum extracts

[0099] The extract samples obtained from the enzymatic extraction and the hydroalcoholic extraction were collected and investigated. In particular, an analysis of the (poly)phenolic compounds by liquid chromatography coupled with tandem mass spectrometry and photodiode array detector (UHPLC-DAD-MS / MS) was performed, as described below. Each extract reported was 5-fold diluted with a mixture of water / methanol 1 : 1 (v / v) acidified with 0.1% formic acid. After brief vortexing, each extract was centrifuged at 7,378 g for 5 minutes at 10°C, before UHPLC-DAD-MS / MS analysis.

[0100] For the mobile phase, a binary system of water (phase A) and acetonitrile (phase B) was used, both acidified with 0.01% formic acid. The starting gradient consisted of 99% phase A, maintained for 0.5 min and then decreased to 85% at 3 min. From 3 to 6 minutes phase A was reduced to 50%, then dropped rapidly to 5% at 9 minutes to clean the column. At 11 minutes the starting gradient at 99% of A was restored and maintained until 14 minutes to guarantee a complete equilibration of the column for the next analysis. The mobile phase pumped at 0.4 mL / minute. The column compartment was set at 40°C, while the autosampler temperature was set at 10°C. An Acquity UPLC HSS T3 (2.1 x 100 mm) equipped with a VanGuard Acquity UPLC HSS T3 (2.1 x5 mm) guard column was used as the chromatographic column. The photodiode array detector scanned in a range of 220- 500 nm.

[0101] The injection volume was 2 pL.

[0102] Different spectrometric modalities have been used to characterize the polyphenolic components in the various extracts. Initially, they were analysed in MS Scan mode both in ESI+ and ESI-, scanning from m / z 100 to 1500. Following the MS Scan analyses, both the most abundant ions and the ions inherent in chromatographic peaks with increased / decreased ion abundance between the various samples were fragmented in MS / MS mode. This led to obtain the peak identification or at least to ascribe each chromatographic peak with a specific polyphenolic subclass. In addition, the extracts were analysed in ESI- by monitoring the precursor ions of the fragment ions with m / z 163, 179 and 193, corresponding to coumaric, caffeic and ferulic acids, respectively. This analytical approach has been used since these hydroxycinnamic acids are particularly relevant in Verbascum spp. as glycosides and as esters of iridoids and phenylethanoids (Luca et al. 2019; Iliescu et al. 2020).

[0103] As a result of the comparative analyses mentioned above, a final method in Multiple Reaction Monitoring (MRM) mode was developed, which is the most sensitive and selective method to perform a comparison of the main polyphenolic compounds in the extracts. Each diluted extract was analysed twice.

[0104] Figures 1-4 show LC-MS / MS profiles of the main (poly)phenolic compounds, i.e. luteolin, verbascoside, p-coumaric acid, and caffeic acid, which were different depending on the extraction performed, either enzymatic or hydroalcoholic.

[0105] Actually, the enzymatic treatment alone (sample 65) led to the extraction of p-coumaric acid and caffeic acid, which were barely detected in sample 29 (only traces).

[0106] Differently, the hydroalcoholic treatment alone (sample 29) led to the extraction of luteolin and verbascoside, which were barely detected in sample 65 (only traces).

[0107] Even the LC-DAD profiles recorded at 325 nm (Figure 5) were different among the investigated samples.

[0108] In detail, the enzymatic treatment alone (sample 65) led to the appearance of more chromatographic peaks than the other samples, likely because of the highest release of hydroxycinnamic acids after treatment of the plant material with a mixture of enzymes (cellulase, xylanase, pectinase, and amylase).

[0109] Figures 6-9 showed the ion abundance levels of compounds present in the extracts. For sake of completeness, an additional comparative sample (sample 63) was tested as resulting from a coupled extraction process, i.e. a hydroalcoholic extraction followed by enzymatic treatment, under the same respective conditions as above reported for sample 29 and sample 65.

[0110] Figure 6 shows that luteolin was significantly extracted via hydroalcoholic treatment (sample 29), and a slightly higher amount was recovered when the coupled extraction is performed (sample 63), whereas the enzymatic treatment alone was not suitable for extracting appreciable amount of the same (sample 65).

[0111] Similarly, verbascoside was significantly extracted via hydroalcoholic treatment (sample 29), but differently, when the enzymatic treatment is performed, either alone (sample 65) or coupled with the hydroalcoholic treatment (sample 63), verbascoside was not extracted in appreciable amount.

[0112] Conversely, ferulic acid hexoside was significantly better extracted via enzymatic treatment (sample 65), than via coupled treatment (sample 63), and much more than via hydroalcoholic treatment (sample 29).

[0113] Figure 7 shows that caffeic acid derivatives are better extracted when the enzymatic treatment is performed (sample 65). Conversely, the hydroalcoholic treatment (sample 29) gave unsatisfactory yields of the same, whereas the coupled treatment (sample 63) gave acceptable but lower yields.

[0114] Similar results are shown in Figure 8, where the extraction of other ferulic acid and caffeic acid derivatives is depicted. Again, the enzymatic treatment (sample 65) achieved better extraction profiles.

[0115] Figure 9 shows that coumaroyl-acetylrhamnosyl-aucubin, forsythoside B, and caffeoyl- acetylrhamnosyl-aucubin were significantly extracted via hydroalcoholic treatment (sample 29), whereas the enzymatic treatment alone (sample 65) allowed to significantly extract caffeic acid and p-coumaric acid. The coupled treatment (sample 63) seemed to reduce the overall extraction performance.

[0116] In consideration of the experimental data collected, the best results are obtained by singularly performing the enzymatic treatment and the hydroalcoholic treatment, thus recovering the highest concentrations of compounds of interest, and then joining the resulting two extraction products so obtained, to give a unique and very effective Verbascum nigrum extract composition.

Claims

CLAIMS1. An enzymatic process for preparing an enzymatic Verbascum nigrum extract, the process comprising the steps of: i) providing dry and milled biomass of Verbascum nigrum, where the latter is previously grown and harvested in a vertical farm, ii) adding buffered water at pH 3.5-5.5, to achieve a biomass concentration of 20- 80g / L, iii) adding an enzymatic cocktail consisting of: endo-polygalacturonase (CAS n. 9032-75-1 - EC 3.2.1.15), cellulase (CAS n. 9012-54-8 - EC 3.2.1.4), a-amylase (CAS n. 9000-90-2 - EC 3.2.1.1), and endo-l,4-P-xylanase (CAS n. 9025-57-4 - EC 3.2.1.8), iv) stirring the resulting mixture at a temperature of 30-70°C for 12-36 hours, v) separating the solid residue from the solution, while keeping the solution, which is the Verbascum nigrum extract, wherein said Verbascum nigrum extract comprises -coumaric acid, caffeic acid, caffeic acid-hexoside-rhamnoside, and ferulic acid-hexoside-rhamnoside.

2. The process of claim 1, wherein, in step i), the biomass of Verbascum nigrum is dried at 30-50°C for 12-36 hours, and then milled down to a particle size lower than 5 mm.

3. The process of claim 1 or 2, wherein, in step ii), water is buffered by adding a citrate buffer.

4. The process of any one of claims 1-3, wherein, in step iii), said enzymatic cocktail is added so that each enzyme is, independently of one another, at a concentration of 50-200 U / g_DM (i.e. Units per gram of dry biomass).

5. An enzymatic Verbascum nigrum extract obtained by the process of any one of claims 1-4, said enzymatic extract comprising / ?-coumaric acid, caffeic acid, caffeic acid-hexoside-rhamnoside, and ferulic acid-hexoside-rhamnoside.

6. The enzymatic extract of claim 5 for use as an active ingredient or as a co-adjuvant in the treatment of dysbiosis of the intestinal microbiota, and pathologies ascribable thereto.

7. A hydroalcoholic process for preparing a hydroalcoholic Verbascum nigrum extract, the process comprising the steps of: a) providing dry and milled biomass of Verbascum nigrum, where the latter is previously grown and harvested in a vertical farm, b) adding a hydroalcoholic solution comprising up to 60% ethanol, c) stirring the resulting mixture at room temperature for 1-6 hours, d) separating the solid residue from the solution, while keeping the solution, which is the Verbascum nigrum extract, wherein said Verbascum nigrum extract comprises luteolin, verbascoside, Forsythoside B, coumaroyl-acetylrhamnosyl-aucubin, and caffeoyl-acetylrhamnosyl-aucubin.

8. A hydroalcoholic Verbascum nigrum extract obtained by the process of claim 7, said hydroalcoholic extract comprising luteolin, verbascoside, Forsythoside B, coumaroyl- acetylrhamnosyl-aucubin, and caffeoyl-acetylrhamnosyl-aucubin.

9. A nutraceutical composition comprising the enzymatic Verbascum nigrum extract of claim 5, and the hydroalcoholic Verbascum nigrum extract of claim 8.

10. A food supplement comprising the enzymatic Verbascum nigrum extract of claim 5, the hydroalcoholic Verbascum nigrum extract of claim 8, or the nutraceutical composition of claim 9, and suitable food excipients.

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