IT202100031673A1 - PROCESS FOR THE PREPARATION OF A STANDARDIZED FERMENTED RED RICE, STANDARDIZED FERMENTED RED RICE, AND ITS USES - Google Patents

Description

DESCRIPTION of the invention entitled:

?PROCESS FOR THE PREPARATION OF A FERMENTED AND STANDARDIZED RED RICE, FERMENTED AND STANDARDIZED RED RICE, AND ITS USES?.

The present invention relates to a process for

preparation of a red yeast rice (RYR), as well as? to

a fermented and standardized red rice. Furthermore, the

This invention refers to a non-use

therapeutic use of said fermented red rice and

standardized as a food or ingredient for

food supplements or nutraceutical products. In the end,

the present invention refers to the said red rice

fermented and standardized for use: (i) in a method

to keep cholesterol levels controlled

plasma levels, or (ii) in a method for reducing the accumulation of cholesterol in the blood and, therefore, for the primary prevention of hypercholesterolemia, or (iii) in a method of treatment, preventive or curative, of a disorder or a disorder or pathology associated with hypercholesterolemia.

Red yeast rice (RYR) is obtained by fermenting rice by adding the yeast Monascus purpureus Went, from which it is obtained? monacolin K was identified, then developed as lovastatin. In particular, red yeast rice is ? the product obtained following the fermentation of common table rice (Oryza sativa L.) with Monascus purpureus Went., a filamentous yeast with a characteristic purple red colour, first isolated in Taiwan in 1930 but whose use in food dates back to to 800 BC. C.

For example, the preparation of fermented red rice involves washing common (white) rice and subsequent steam cooking until a semi-gelled consistency is obtained. After cooling, the rice is polluted with the starter M. purpureus and kept covered with cotton cloths to maintain humidity. at a temperature of 30-35?C for approximately seven days. After this time, the central part of the rice grain takes on the typical red color. For example, the product obtained is left to dry in the sun for approximately 24 hours, or it is dried in an oven at 35-40°C for a shorter time.

The largest fraction plenty of RYR fermented red rice? represented by sugars, in particular starch is the main constituent (73%); followed, in order of quantity, by the protein fraction (15%). RYR is also characterized by the presence of natural orange pigments, monascorubin and rubropunctatin, yellow pigments such as monascin and ankaflavin, and finally red pigments such as monascorubramin and rubropunctamine. Among the other components of RYR we find fatty acids, including oleic, linoleic, linolenic, palmitic and stearic acids; phytosterols, including ?-sitosterol and stigmasterol, flavonoids, such as quercetin and isoflavones. The components of greatest interest, deriving from fermentation, are undoubtedly represented by monacolins, chemical compounds derived from hexahydronaphthalene, with recognized qualities. hypolipidemic. In particular, monacolin K (MK) represents approximately 75% of total monacolins. In addition to this, there are monacolins defined as secondary such as monacolins J, L, M and X, together with their hydroxy- and dehydro-derivatives and compactin. In general, the monacolins present in RYR exist in two forms, namely the lactone form and the acid form: these are in equilibrium with each other and in different concentrations depending on the conditions of the fermentation process.

Therefore, ? felt the need? to develop a process for the preparation of a fermented red rice capable of producing standardized samples in terms of title and concentrations of monacolin K and secondary monacolins, all of which are present in the right proportions between the lactone form and the acid form.

Indeed, ? It is known that freshly fermented red rice mainly contains the acid form of monacolin K (MKA), this is then converted into the lactone form (MKL) during drying and at neutral or basic pH. The RYR on the market contains approximately 85% MKL and 15% MKA. Between the two forms, the acidic one represents the more active in the business of inhibition of the HMGR enzyme (3-hydroxy-3-methylglutaryl reductase enzyme) thanks to the structure similar to the enzyme substrate (HMG-CoA; 3-hydroxy-3-methylglutaryl Coenzyme A enzyme); instead the lactone form requires functionalization through the action of the hydroxylesterase enzyme.

In the RYR? also contains citrinin, a mycotoxin derived from the fermentation of rice. Several in vivo studies have demonstrated that chronic exposure to citrinin can result in a nephrotoxic effect due to hyperplasia of the renal tubular epithelium. For these and other reasons, the European Food Safety Agency (EFSA) has established that the maximum dose of citrinin that can be used? be ingested by humans, without having adverse nephrotoxic effects,? of 20 ?g/kg per day, and for this reason the quantities of citrinin in the RYR have been regulated and must be less than 2 ppm.

Therefore, ? felt the need? to develop a process for the preparation of a red fermented rice capable of producing samples standardized in titre and concentrations of monacolin K and secondary monacolins, as well as? with citrinin quantities lower than 2 ppm.

As reported by EFSA, the bioavailability? of the MK present in the RYR? influenced by the plant ingredients present.

Although the majority of clinical studies produced in the last twenty years take into consideration the safety of RYR use, important side effects, qualitatively similar to those observed during treatment with lovastatin, have rarely been reported.

An Italian studio? was published in 2017 (G, M., Pa, M., E, R., R, D. C. & F, M.-I. Adverse reactions to dietary supplements containing red yeast rice: assessment of cases from the Italian surveillance system. British journal of clinical pharmacology (2017)) with the aim of describing the safety profile of RYR, analyzing spontaneous reports of suspected adverse reactions communicated from April 2002 to September 2015 to the phytovigilance system of the Istituto Superiore di Sanit?. The following disorders or adverse reactions have been reported through these reports: musculoskeletal and connective tissue problems, gastrointestinal disorders, liver damage and skin reactions. Out of 1261 total reports, 55 possible side effects attributable to RYR were recorded; among the reported cases, 14 patients experienced serious adverse reactions, 13 required hospitalization, 28 patients were simultaneously taking other drugs such as ACE inhibitors, diuretics, antibiotics or others. The study cited presented however? some methodological gaps, in fact the possible side effects attributable to the products often used in association with RYR were not analysed; furthermore, the study only evaluated the adverse effects attributable to the consumption of RYR with 3 mg/day of monacolin K, making no reference to rices containing 10 mg/day of MK, even though these are the most common products? consumed from 2011 until today. Despite the obvious shortcomings, the data produced by this work led EFSA to review all safety data on RYR. EFSA therefore necessarily had to provide an opinion regarding the adverse effects relating to RYR; but precisely because of the gaps in the latest Italian data, which only concerned low-dose MK products, EFSA was unable to correlate a dependence of side effects on the dosage of monacolin K taken.

? The impossibility of this was also highlighted. to provide a security definition on the RYR, due to the following reasons:

- The composition, content and relative abundance of monacolins in the different products used? variable;

- are you? variability? of the relationship between monacolina K and KA;

- bioactivity? of components other than monacolin K? unknown;

- impossibility? to evaluate the safety of monacolins in some groups of consumers such as pregnant women, breastfeeding women, breastfed children;

- possible interactions of RYR-based supplements with foods or drugs that inhibit the CYP3A4 isoenzyme, involved in the metabolism of a large number of drugs;

- unknown interactions of monacolins with other ingredients present in the product.

For all these factors, EFSA declares in 2018 that: ?not? Is it possible to establish a safe level of food intake both for the general population and for some specific vulnerable subgroups?

Therefore, ? the need is urgent? to delve deeper into this subject of study and therefore to have more clinical data on the possible interactions of RYR, more data on its use in particular populations, but before that? has the need been established? to define quality parameters? of the products.

The biggest limit linked to the RYR? undoubtedly determined by the considerable variability? of the products on the market and, often, by the relative uncertainty about their quality. Studies conducted on commercial RYR extracts have shown a very variable MK content and MK:MKA weight ratio and high citrinin concentrations (D, H., A, L., Qy, L., S, B. & Vl , G. An analysis of nine proprietary Chinese red yeast rice dietary supplements: implications of variability in chemical profile and contents. Journal of alternative and complementary medicine (New York, N.Y.) (2001);

Marked Variability of Monacolin Levels in Commercial Red Yeast Rice Products: Buyer Beware! Internal Arch. Med. 170, 1722?1727 (2010)).

According to a study published in 2014, a RYR can? be defined as authentic (unadulterated) when it contains 1.20 mg to 1.38 mg of MK per 600 mg of rice (0.20%-0.23%).

It happens that, to satisfy these parameters, the monacolin K titre in samples of red yeast rice is adjusted with synthetic or biosynthetic molecules, such as lovastatin and mevinolin.

It seems clear that the lack of standardization of RYR-based preparations and the findings relating to the low quality of the products on the market, do they? that there is a need? to arrive at an effective evaluation of quality? of the products on the market.

An important confirmation of the low quality? of the RYRs present on the Italian market? was provided by the study conducted by SiFitLab (research laboratory of the Italian Phytotherapy Society) and by the Pharmaceutical Biology laboratory of the University? of Siena. These laboratories, with the collaboration of Federsalus (Italian federation of companies operating in the phytotherapy and nutraceutical sector) have analyzed approximately twenty samples of RYR on the market, using HPLC techniques and sequencing analysis of the genetic material. From the analysis of the results obtained, ? it was highlighted that only less than 25% of the samples analyzed could be defined as authentic; Furthermore ? An increase in the MK titer or the absence of secondary monacolins was often found. From the data obtained, yes? also hypothesized that rice could be fermented with strains other than M. purpureus (BAINI G., CUSI M. G., ISOLDI G., BIAGI M., RED YEAST RICE: FRIEND FOR THE HEART OR FOR THE CHINESE BUSINESS? THE NEED FOR QUALITY CONTROLS , MEDICINAL PLANTS, 2018, 17(1-2)).

At the same time, the Edmund Mach Foundation of San Michele all'Adige published a fine analytical method based on the 12C/13C isotope ratios of the MK isolated from RYR, which allows us to determine the actual origin of the monacolin, and therefore allows us to define whether it comes from from rice, from another cereal or from synthesis. ? Has a parameter of authenticity been therefore established? of the MK, defined by the 12C/13C ratio which should be lower than -28.3%. The data obtained confirmed that in 2017, only a minority of RYR products could be considered authentic (M, P., G, C. & F, C. Stable isotope ratio analysis for authentication of red yeast rice. Talanta (2017)) .

To date, there is no industrial production technique that allows us to obtain quantities of standardized fermented red rice capable of satisfying the needs of a market that demands quality production of fermented red rice. (for example, authentic unadulterated RYR with an MK titer not adjusted by the addition of synthetic or biosynthetic molecules such as lovastatin and mevinolin) and in line with current safety regulations.

? felt, therefore, the need? by operators in the sector and institutions producing red fermented rice with reduced variability? so that, for example, the titer in monacolin K (MK), total monacolins, secondary monacolins, compactin and total polyphenols is precise, repetitive and reliable.

The Applicant, after a long and intense activity? of research and development, has developed a production technique capable of providing an adequate response to the limitations and drawbacks that still exist.

The production technique of the present invention provides - firstly - a process for the preparation of a fermented red rice and - secondly - an analysis method that allows verifying that the productions of fermented red rice obtained with said process are repeatable, uniform and constant and, therefore, are productions of quality standardized red fermented rice? and with reduced variability? industrial so that, for example, the titre in monacolin K (MK), total monacolins, secondary monacolins, compactin and total polyphenols is precise, repetitive and reliable. ? It is important that there is control of the increase in secondary monacolins during fermentation.

The object of the present invention is a process for the preparation of a fermented and standardized red rice, having the characteristics as defined in the attached claims.

Another object of the present invention is a fermented and standardized red rice, preferably prepared through said process, having the characteristics as defined in the attached claims.

Another object of the present invention is a non-therapeutic use of said fermented and standardized red rice as a food or ingredient for food supplements or nutraceutical products, having the characteristics as defined in the attached claims.

It forms another object of the present invention called red yeast rice and standardized for use (i) in a method of maintaining controlled plasma cholesterol levels, or (ii) in a method of reducing the accumulation of cholesterol in the blood and, therefore, for primary prevention of hypercholesterolemia, or (iii) in a method of treatment, preventive or curative, of a disorder or a disorder or pathology associated with hypercholesterolemia, having the characteristics as defined in the attached claims.

Preferred embodiments of the present invention will be described below by way of example, and therefore without limitation, with the aid of the tables, in which:

- Figure 1 illustrates an HPLC-DAD chromatogram of a RYR sample from Example 1, where the main peak at RT (retention time) = 13.13 minutes? monacolin K in lactone form (MKL), while the peak RT = 10.45 minutes identifies monacolin K in acidic form (MKA);

- Figure 2 represents a simplification with graphic identification of the peaks of the chromatogram in Figure 1.

Therefore, the object of the present invention is a process for the preparation of a red fermented rice (RYR). The preparation process of fermented and standardized red rice which is the object of the present invention provides for accurate control with reference to the increase in secondary monacolins during the fermentation phase.

Said preparation process preferably includes? an analysis of at least one monacolin in samples of red yeast rice (RYR). Said analysis preferably includes the following phases:

(I) preparation of first hydroalcoholic solutions of a reference substance, in which said first solutions have known concentration values of said reference substance, and in which said concentration values of said first solutions are different from each other;

(II) analysis of said first solutions obtained from phase (I), via high performance liquid chromatography (HPLC), to give chromatograms with first peak responses associated with each concentration value;

(III) correlation between said first peak responses obtained from phase (II) and said known concentration values of phase (I), to give a correlation (or calibration) tool;

(IV) preparation of second hydroalcoholic solutions, different from said first solutions, of RYR samples containing at least one monacolin, in which said second solutions have unknown concentration values of said at least one monacolin;

(V) analysis of said second solutions obtained from phase (IV), via HPLC, to give chromatograms with second peak responses;

(VI) for each second peak response obtained from phase (V), obtain the concentration values of said at least one monacolin in said second solutions via the correlation tool of phase (III).

It follows that, through the use of an external reference substance and a correlation tool obtained through solutions of said substance, the present method allows to derive the concentration values of said at least one monacolin in said second solutions, and therefore the quantity? of monacolin/s within the RYR sample.

The reference substance of step (I) preferably comprises or, alternatively, consists of lovastatin or mevinolin, preferably mevinolin in acid form and/or in lactone form. As an example, the reference substance of phase (I) ? lovastatin in 20 mg tablets (Lovinacor, Innova Pharma).

Preferably, in phase (I) of preparation of each first hydroalcoholic solution and/or in phase (IV) of preparation of each second hydroalcoholic solution? a quantity? known quantity of said reference substance or a quantity? predefined sample of said RYR sample - is solubilized in a known volume of a hydroalcoholic solvent, via an ultrasonic bath, specifically it is a standard ultrasonic bath, with at least 3 sonication cycles of 10 minutes, temperature between 25 and 35 ?C, at room temperature, for a time ranging from 0.5 hours to 4 hours, preferably ranging from 1 hour to 3 hours, even more? preferably ranging from 1.5 hours to 2.5 hours.

As an example, said quantity? known or said quantity? default? ranging from 1 mg to 25 mg, preferably ranging from 2 mg to 20 mg, more? preferably between 5 mg and 15 mg, even more? preferably ranging from 8 mg to 12 mg, per milliliter of hydroalcoholic solvent.

Preferably, in phase (I) of preparation of the first solutions, said first solutions have concentration values different from each other so as to cover at least an expected range of monacolin concentrations in the second hydroalcoholic solution, and preferably exceeding said expected range by at least 10%. , more? preferably exceeding at least 20%, even more? preferably exceeding at least 50%.

A hydroalcoholic solvent of said first hydroalcoholic solution of phase (I) and/or a hydroalcoholic solvent of said second hydroalcoholic solution of phase (IV) preferably comprises a quantity of of ethanol or methanol (preferably ethanol), in water ranging from 50% to 95% by volume, preferably ranging from 70% to 90% by volume, plus? preferably between 75% and 85% by volume, even more? preferably ranging from 78% to 82% by volume. The water used? preferably ultrapure water, produced with a reverse osmosis system, reprocessed with a Millipore Milli-Q device.

Said analysis of phase (II) following phase (I) of preparation of the first hydroalcoholic solutions and/or said analysis of phase (V) following phase (IV) of preparation of the second hydroalcoholic solutions via HPLC includes or, alternatively, consists of an analysis using high performance liquid chromatography with diode array detector (HPLC-DAD).

As an example, this analysis ? carried out using a Shimadzu Prominence LC 2030 3D instrument equipped with a Bondapak column? RP C18, 10 ?m, 125 ?, 3.9 mm x 300 mm (Waters Corporation, USA).

Preferably, in said analysis of phase (II) and/or in said analysis of phase (V) a chromatographic column is preferably used at a temperature ranging from 25?C to 35?C, preferably ranging from 26?C to 30?C , more? preferably around 28?C. More? preferably, called chromatographic column? a RP-C18 ?Bondapak, 125?, 10 ?m, 3.9 mm

Said analysis of phase (II) and/or said analysis of phase (V) preferably uses an eluent mixture which includes or, alternatively, consists of (A) and (B):

(A) water and formic acid, wherein said formic acid is ? preferably in a quantity? ranging from 0.01% to 1% by volume, more? preferably between 0.05% and 0.5% by volume, even more? preferably ranging from 0.07% to 0.13% by volume; And

(B) acetonitrile and formic acid, wherein said formic acid is ? preferably in a quantity? ranging from 0.01% to 1% by volume, more? preferably between 0.05% and 0.5% by volume, even more? preferably ranging from 0.07% to 0.13% by volume.

The eluent mixture (A) and (B) ? preferably fed with a flow gradient as follows:

- (A) from 65% to 25% by volume and (B) from 35% to 75% by volume in a time ranging from 15 minutes to 25 minutes, preferably ranging from 18 minutes to 22 minutes, even more? preferably in about 20 minutes, with a feed flow of 0.5 ml/min to 2 ml/min, preferably of 0.75 ml/min to 1.25 ml/min; and subsequently

- (A) at 25% by volume and (B) at 75% by volume for a time ranging from 25 minutes to 35 minutes, preferably ranging from 28 minutes to 32 minutes, even more? preferably in 29 minutes, with a feed flow ranging from 0.5 ml/min to 2 ml/min, preferably ranging from 0.75 ml/min to 1.25 ml/min.

Preferably, the chromatograms obtained from phase (II) and/or phase (V) are obtained at 237 nm and 250 nm.

In the correlation phase (III), following the phase (II) of analysis of said first solutions, the first peak responses obtained from phase (II) and the known concentration values of phase (I) are correlated to give a correlation tool (or calibration).

Preferably, said correlation tool obtained from phase (III) ? a linear correlation tool.

Said correlation tool obtained from step (III) preferably comprises or, alternatively, consists of a diagram, a table, a parameter and/or a calculation routine.

Preferably, in the present analysis method, the concentration values obtained from phase (VI) are related to:

(a) Total monacolin K (MKtot) expressed as:

MKtot = MKL MKA

in which:

- MKL = monacolin K in lactone form (lovastatin); - MKA = monacolin K in acidic form; And

(b) one or more? secondary monacolins (Msec) selected from monacolin J, monacolin L, monacolin X, compactin (mevastatin) and their mixtures.

Preferably, said analysis method also includes the following phase:

(VII) absorbance spectrophotometric analysis of third hydroalcoholic solutions, different from said first hydroalcoholic solutions and said second hydroalcoholic solutions, of RYR samples containing at least one monacolin at wavelengths of 405 nm and 510 nm to detect the absorbance maxima of yellow color pigments and red color pigments respectively in said RYR samples.

Ratios of [yellow pigments]:[red pigments] absorbance maxima are generally in the range of 0.75 to 1.40. Approximately 500 ?g/ml of RYR sample should provide absorbance at 405 nm and 510 nm > 0.2. Absorbances ranging from 0.20 to 0.15 suggest samples with little presence of pigments. Absorbances at 405 nm and 510 nm < 0.15 identify samples with a quantity of very poor pigments, indicative of an incomplete or poor quality fermentation process.

Preferably, said analysis method also includes the following phase:

(VIII) evaluation of the polyphenol and phytosterol content Folin-Ciocalteu colorimetric assay.

Preferably, phase (VIII) ? following phase (VI).

The preparation process of fermented and standardized red rice which is the object of the present invention allows to obtain industrial supplies of fermented red rice, having the following characteristics:

- a monacolin K (MK) content of between 1.5% and 5% m/m, with a deviation from the declared value of <10%, compared to the total weight of said fermented red rice; And

- a (MK) content of between 70% and 85%, compared to the weight of the total monacolins; And

- a dehydro-MK content < 50% (min. value 5%), compared to the weight of the monacolins; And

- a total monacolin content of between 1.8% and 6.5% m/m, compared to the total weight of said fermented red rice; And

- a content of secondary monacolins ranging from 15% to 30%, compared to the total monacolins, in which said secondary monacolins are so? divided: monacolin X (MX) monacolin J (MJ) monacolin M (MM) monacolin L (ML) > 20% (max. 90%) of secondary monacolins; And

- a compactin content < 0.15% m/m (min. 0.01%), compared to the total weight of said fermented red rice; And

- a total polyphenol content greater than or equal to 0.75% m/m (max. 2.5%), compared to the total weight of said fermented red rice;

- preferably a 12C/13C isotope ratio of MK isolated from RYR lower than -28.3? ?<13>C; And

- preferably a citrinin content lower than 2 ppm.

The red fermented rice object of the present invention has an in vitro enzymatic test of inhibition of HMG-CoA reductase IC50 that is always better than the MK content alone and in any case has IC50 < 2500 ng/ml when the activated lovastatin, in the same experimental conditions, has IC50 ranging from 60 to 100 ng/ml.

Furthermore, the object of the present invention is a non-therapeutic use of said fermented red rice as a food or ingredient for food supplements or nutraceutical products.

Furthermore, the object of the present invention is called fermented red rice for use:

(i) in a method of keeping plasma cholesterol levels controlled, or

(ii) in a method to reduce the accumulation of cholesterol in the blood and, therefore, for primary prevention of hypercholesterolemia; or

(iii) in a method of treatment, preventive or curative, of a disorder or disorder or pathology associated with hypercholesterolemia.

FRn embodiments of the present invention are reported below.

FR1. A red yeast rice (RYR) having the following characteristics:

- a monacolin K (MK) content of between 1.5% and 5% m/m, compared to the total weight of said fermented red rice; And

- an MK content of between 70% and 85%, compared to the weight of the total monacolins; And

- a dehydro-MK content of between 5% and 50%, compared to the weight of total monacolins; And

- a total monacolin content of between 1% and 10%, preferably between 1.8% and 6.5% m/m, compared to the total weight of said fermented red rice.

FR2. Fermented red rice according to FR1, wherein said red rice also has:

- a secondary monacolin content of between 15% and 30%, compared to the weight of the total monacolins, and in which said secondary monacolins are so? divided into: monacolin

FR3. Red rice fermented according to FR1 or FR2, wherein said red rice also has:

- a compactin content of between 0.01% and 0.15% m/m, compared to the total weight of said fermented red rice.

FR4. Red rice fermented according to any of FR1-3, wherein said red rice further includes:

- a total polyphenol content ranging from 0.75% m/m to 2.5% m/m, compared to the total weight of said fermented red rice.

FR5. Red rice fermented according to any of FR1-4, wherein said red rice further includes:

- a <12>C/<13>C isotope ratio of MK isolated from RYR lower than -28.3? ?13C, e

- a citrinin content of less than 2 ppm.

FR6. Red yeast rice fermented according to any of FR1-5 for use:

(i) in a method of keeping plasma cholesterol levels controlled, or

(ii) in a method to reduce the accumulation of cholesterol in the blood and, therefore, for the primary prevention of hypercholesterolemia; or

(iii) in a method of treatment, preventive or curative, of a disorder or pathology associated with hypercholesterolemia.

FR7. Non-therapeutic use of red yeast rice according to any of FRn FR1 to FR5, as a food or food ingredient, or to prepare food or food supplements or nutraceutical products, or for compositions for medical devices, in accordance with EU Regulation 745/ 2017.

EXPERIMENTAL PART

Samples of RYR fermented red rice obtained with the process for the preparation object of the present invention were subjected to the following chemical analyses.

The chemical analyzes were performed on 6 selected samples: 2 RYR with a declared MKtot titer of 5% m/m, 2 RYR with a titer of 3% MKtot and 2 RYR with a titer of 1.5% MKtot.

All 6 samples comply with the specifications required by the Ministry of Health, as regulated by Legislative Decree 169/2004 which implements EC Dir. 2002/46/EC.

For chemical analyses, solvents and analytical standards (mevinolin) were purchased from Merck, Milan. What about the reference drug? Lovastatin in 20 mg tablets (Lovinacor, Innova Pharma) was used. The water used? ultrapure, produced with a reverse osmosis system, reprocessed with a Millipore MilliQ apparatus. RYR samples were solubilized in 80% V/V ethanol (500 mg in 50 mL) and processed by sonication for 2 hours. In order to obtain the conversion of the lactone form of monacolin K (MKlat) into the active acidic one (MKA),? The solubilization of RYR and lovastatin in 80% V/V ethanol with the addition of 0.1 M NaOH (ethanol:NaOH 0.1M 5:1) was carried out (Scheme 1 below, showing the chemical balance of the two forms) . The hydrolysis reaction? was left to incubate overnight at room temperature.

Scheme 1: Conversion of lactonic monacolin K into acidic form, and vice versa.

The phytochemical analysis of the samples? was conducted using HPLC-DAD following the method described in the "QUALITY CONTROL" work. RED FERMENTED RICE? for the quantification of MK:

1. verification of the total monacolin K titer of the sample expressed as mevinolin;

2. titer in MK, MKL and MKA and titer in secondary monacolins, all expressed as mevinolin;

The HPLC-DAD instrument used? a Shimadzu Prominence LC 2030 3D equipped with a Bondapak column? RP C18, 10 ?m, 125 ?, 3.9 mm x 300 mm (Waters Corporation, USA).

Analyzed samples: solutions in ethanol 80% V/V, 10 mg/ml.

Injected volume: 10 ?l.

Eluent system: A: pure water formic acid 0.1% V/V; B: acetonitrile formic acid 0.1% V/V.

Elution method: B from 35% to 0 minutes, to 75% at 20 minutes, then B 75% up to 29 minutes.

Flow: 1.0ml/min.

Column temperature: 28 ?C

Detection: 250nm

Standard used: mevinolin.

Secondary monacolins were identified and determined using a mass spectrometer coupled to an HPLC-UV. The tool used? an Orbitrap Thermofisher with electrospray source (ESI), used in mode? negative with spray voltage 3.5 kV, sheat gas 20 a.u., auxiliary gas 5 a.u. and capillary temperature 320?C.

The spectrometric analyzes were conducted using high resolution acquisition which allowed the revelation of the exact weight of the analytes and the brute formula.

The identified monacolins were then quantified by HPLC-DAD using mevinolin as a standard for monacolins in lactone form and mevinolin in hydroxyacid form for monacolins in hydroxyacid form.

In this work, RYRs were further analyzed to quantify the different presence of polyphenols and phytosterols. The total polyphenols contained in the extracts were determined by Folin-Ciocalteu colorimetric assay.

To 0.1 ml of each RYR sample, in a solution of 80% V/V ethanol (10 mg/ml) or 80% ethanol and 0.1N NaOH, 2.9 ml of distilled water and 0.5 ml of Folin-Ciocalteu reagent (diluted in water 1:10 V/V). After 30 seconds of shaking, yes? added 1 ml of an aqueous solution of Na2CO3 25% m/m. After incubation at room temperature for approximately 20 minutes, ? absorbance was read at 700 nm using the Victor Nivo 3s instrument (Perkin Elmer, MA, USA). The quantification of polyphenols? was calculated by interpolation of the calibration curve constructed using gallic acid as a standard (Milan, Italy) between 5000 and 78 mg/l.

For the quantification of RYR phytosterols, ? was the acid vanillin method used for total triterpenes, as already? published by

(2018). Briefly, samples are dissolved in 80% V/V ethanol at a concentration of 10 mg/ml. 100 ?l of sample solution is diluted with 100 ?l of glacial acetic acid. 300 liters of a glacial acetic acid solution containing vanillin at 5% m/V are then added; mix for 30 seconds and add 1 ml of perchloric acid (HClO4). The mixture is heated for 45 minutes at 60°C and, after cooling, is brought to a volume of 5 ml with glacial acetic acid. The absorbance is read at 548 nm and the quantification of total triterpenes in the extract is calculated in relation to the calibration curve constructed using ?-sitosterol (6400-100 mg/l) as a standard.

ENZYMATIC 3-HYDROXY-3-METHYLGLUTARYL-COENZYME A REDUCTASE INHIBITION TEST

The enzymatic test? was used to evaluate the capacity? of the different samples of RYR to inhibit the activity? of the enzyme 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMG-CoA reductase), in comparison with the control lovastatin. This enzyme catalyzes the reduction reaction from hydroxymethylglutaryl coenzyme A (HMG-CoA) to Coenzyme A (CoA) and mevalonate, a key step in the biosynthesis of cholesterol, and target of action of statins.

The test is based on a spectrophotometric measurement of the absorbance at 340 nm, a length corresponding to the absorption maximum of NADPH which undergoes oxidation by the subunit? catalytic action of HMG-CoA reductase, in the presence of the substrate HMG-CoA.

The kit used? the CS1090 from (Germany), which is? consisting of:

- 5x buffer

- NADPH

- Substrate solution (HMG-CoA)

- The enzyme HMG-CoA reductase

- A positive control of HMGR inhibition, consisting of pravastatin.

The kit involves setting up the enzymatic reaction inside a 96-well plate. The reagents are added into the wells, according to the instructions described in the protocol provided by the kit and more. precisely in the following order:

1- Samples or pravastatin (control inhibitor) 2- 1X buffer

3- NADPH

4- HMG-CoA

5- HMG-CoA reductase

? A negative control of the reaction was also set up in which the enzyme is not added.

The absorbance kinetics at 340 nm ? was read at an interval of 5 minutes for 20 minutes using the Victor Nivo 3s instrument

The activity of the samples and therefore their capacity? of inhibition of the enzyme HMG-CoA reductase, ? evaluated using the following equation:

Unit? enzym./mgProt. = (?A340t20-t0/20 sample ? ?A340 t20-t0/20 white) x TV / (12.44 x V x 0.6 x LP) where:

12.44 = ? mm ? extinction coefficient for NADPH at 340 nm (6.22 mM?1cm?1) multiplied by the two moles of NADPH consumed for each mole of HMG-CoA;

TV = Total reaction volume in ml (0.2 in the following protocol);

V = volume of enzyme used in the analysis in ml (0.01 in the following protocol);

0.6 = enzyme concentration in mg-protein (mgP) per milliliter (0.50?0.70 (mgP/ml));

LP = optical path in cm (0.55 for the plate).

A?unit? convert? 1.0 micromole NADPH to NADP+ for 1 minute at 37?C. The activity specific ? defined as ?mol/min/mg-protein (Units/mgP). The test ? was first performed on lovastatin in acidic and lactonic form, then on a RYR with MK in acidic and lactonic form, then on all RYR samples under study in acidic form. RYR samples were tested at 6 different concentrations, between 5 ?g/ml and 156 ng/ml (final concentration in the reaction mixture); lovastatin? tested between 1 ?g/ml and 31 ng/ml. Were all experiments conducted in duplicate and for each sample? Was the IC50 calculated, classically understood as the concentration capable of inhibiting 50% of activity? of the enzyme.

CELL CULTURES

In the experiments described in the second part of the thesis work, four different cell lines were used: the murine myoblast line (C2C12), human hepatocytes (HepG2), colorectal adenocarcinoma epithelial cells (Caco-2) and renal embryonic cells. human (Hek-293). All cell lines were grown at a constant temperature of 37?C, 5% CO? and in DMEM medium (Dulbecco's Modified Eagle's Medium) supplemented with 10% inactivated fetal bovine serum (FBS), glutamine (292 ?g/?l), penicillin (100 ?g/?l) and streptomycin (1 U/ml), thereby generating a complete growth medium.

VITALITY TEST? MOBILE PHONE

The Cell counting kit-8 (CCK-8) (Merck, Germany) allows you to determine, via colorimetric assay, the number of viable cells in viability, cell proliferation and cytotoxicity tests. The assay uses the tetrazolium salt, WST-8, which is highly soluble in water and is ? based on the reduction reaction of WST-8 by cellular dehydrogenases and consequent formation of formazan, a yellow-orange product, soluble in the culture medium (DMEM). The quantity? of formazan generated by the activity? of cellular dehydrogenases? directly proportional to the number of living cells.

The test is performed inside a 96-well plate in which 3000 cells per well have been plated. After 24 hours, the cells were treated with the various samples of RYR and lovastatin, solubilized at the appropriate concentration in complete DMEM (100 ?l per well). A 0.5% EtOH solution? was used as a positive control. After 24 hours of stimulation, 100 ?l of the previously prepared 10% v/v CCK-8 solution was added to each well. The cells were then incubated for approximately 1-2 hours until the medium changed from pink to orange, evidence of the formation of formazan salts. Yes ? then proceeded to read at 450 nm using a Multiskan FC spectrophotometer (Thermo Scientific).

STATISTIC ANALYSIS

The statistical analysis of the results obtained from the CCK-8 tests is was performed by applying a One-way ANOVA using GraphPad Prism 6 software, comparing the various groups treated with respect to the control or lovastatin. The values were considered statistically significant with a p-value <0.05.

PHYTOCHEMICAL ANALYSIS OF FERMENTED RED RICE SAMPLES

The HPLC-DAD and HPLC-MS methods used proved to be adequate for the analysis of the samples. In Figures 1 and 2 the DAD chromatogram at 250 nm of a representative sample of RYR titrated at 5% in MK. The chromatogram in Figure 2 represents a simplification with graphical identification of the peaks of the chromatogram in Figure 1. The main peak at retention time (RT) 13.13 minutes? attributable to MK, while the peak that identified MKA was? the one at RT = 10.45 minutes. MKL and MKA were identified and quantified using mevinolin as a reference standard. The main secondary monacolines recognized and quantified are those to which the peaks at RT=9.39 (MX), RT=10.88 min refer. (ML), RT=11.86 (compactin), RT=14.73 min. (MK ethyl ester), RT= 16.46 min. (dihydro MK) and RT= 17.88 min.i (dehydro-MK). As minor monacolins, MLac and MJ are also present in many samples at RT < 9 min. The MM? present in some RYRs, but its quantity? Not ? never higher than 0.01% by weight.

TITLE IN MONACOLINA K OF THE SAMPLES OF FERMENTED RED RICE AND SECONDARY MONACOLIN

The following Table 1 shows, by way of example, the titers of monacolin K (MK; expressed as the sum of the acid (MKA) and lactone (MKL) forms) and of the secondary monacolins (Msec; expressed as MK and calculated as the sum of the three esters, dehydro and dihydroderivatives of MK and monacolins X, J, L and compactin), in six samples of RYR:

Table 1

EVALUATION OF TOTAL POLYPHENOL CONTENT

The evaluation of the polyphenol content? was carried out as these substances could contribute to minimizing the pro-oxidant impact of statins on the biosynthesis of ubiquinone (CoQ10), which in vivo contributes to the onset of side effects induced by these drugs. The data obtained on the initial samples are shown in Table 2. The average polyphenol content? result of 1% and the minimum content of 0.75%, undoubtedly not negligible.

Table 2

The phytochemical results indicate that the samples of red fermented rice produced according to the above method and having the characteristics described have a characteristic phytochemical profile:

- contain between 1.5% and 5% m/m of total MK, with a deviation from the declared < 10%;

- they have MK which represents between 70% and 85% of the total monkshood;

- they therefore have a total monacolin content between 1.8% and 6.5% m/m

- they have secondary monacolins which represent between 15% and 30% of the total monacolins and what are they like? subdivided: MX MJ MM ML > 20% and dehydro-MK < 50% of total secondary monacolins;

- they always have a compactin content <0.15% m/m;

- have a minimum content of 0.75% m/m in total polyphenols.

ASSESSMENT OF THE ACTIVITY OF HMG-COA REDUCTASE INHIBITION OF RED FERMENTED RICE SAMPLES

The evaluation of the activity? of inhibition of HMG-CoA reductase by the different chemically analyzed RYR samples? was carried out using the Merck cell free kit, HMG-CoA Reductase Assay Kit, validated through the internal control (pravastatin). The specificity? of the kit used? was evaluated by the finding that lovastatin in lactone form? was found to be approximately 20 times less active than lovastatin in its acidic form (Table 3). From the analysis of the concentration/activity response? ? It emerged that lovastatin in acid form has an IC50 of approximately 80 ng/ml in agreement with what has already been reported. reported in the literature (85 ng/ml; PERCHELLET, J.-P. H. et al. ?Novel synthetic inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity that inhibit tumor cell proliferation and are structurally unrelated to existing statins?. Int. J. Mol. Med. 24, 633?643 (2009)).

Table 3

Even in the case of RYRs, the activity? ? was found only in samples subjected to conversion of MK to MKA. As regards the different RYRs, an activity is observed in a variable manner. different from that of lovastatin alone (Table 3 and Table 4).

Referring to RYR's IC50s? is it possible to say that the most high content of MK and total monacolins in the different samples? result correlated with greater activity? of enzyme inhibition. The RYR phytocomplex? result, however, increasingly active of lovastatin (=MK) in all samples, on equal terms? of content, demonstrating the fact that the MK is not? the only active element of RYR on HMG-CoA reductase.

Table 4

Therefore, the results obtained, crossed with the phytochemical data, demonstrate that the fermented red rice samples produced according to the above methodology:

- They contain between 1.5% and 5% m/m of total MK, with a deviation from the declared < 10%;

- they have MK which represents between 70% and 85% of the total monkshood;

- they therefore have a total monacolin content between 1.8% and 6.5% m/m;

- they have secondary monacolins which represent between 15% and 30% of the total monacolins and what are they like? subdivided: MX MJ MM ML > 20% and dehydro-MK < 50% of total secondary monacolins;

- they always have a compactin content <0.15% m/m;

- have a minimum content of 0.75% m/m in total polyphenols;

- in the in vitro enzymatic test for inhibition of HMG-CoA reductase they have an IC50 that is always improved compared to the MK content alone and in any case have an IC50 < 2500 ng/ml when the activated lovastatin, in the same experimental conditions, has an IC50 between 60 and 100 ng/ml ml.

SAFETY IN THE USE OF FERMENTED RED RICE

In order to evaluate the possible cytotoxic action of the RYR constituents towards various tissues, we used cell lines of different origins on which concentrations of red rice and lovastatin were tested, hypothetically comparable to those deriving from the metabolism of the same substances in vivo in such districts. Specifically, intestinal (Caco2), hepatic (HepG2), renal (Hek293) and, finally, muscular (C2C12) cell lines were used on which? I know there is an adverse effect from statins. The cells were treated for 24 hours at concentrations of 250, 160, 80, 40, 20, 10, 5 and 1 ?g/ml of the RYR and lovastatin samples in the active form. By means of viability tests? cell (CCK-8) the IC25 and IC50 (Inhibiting Concentration) values were calculated which indicate the concentration of RYR and lovastatin capable of inhibiting the viability of the cell (CCK-8). cell phone by 25% or 50%. The IC25? was analyzed in order to evaluate the concentration that determines a situation of metabolic stress in the cells; The IC50 allows you to determine the concentration that actually causes cytotoxicity. (Table 5).

Table 5: IC25 and IC50 values of RYR and lovastatin on liver cells (HepG2) after 24 hour treatment. SD < 15% of reported means.

Table 6: IC25 and IC50 values of RYR and lovastatin on renal cells (Hek293) after 24-hour treatment. SD < 15% of reported means.

Table 7: IC25 and IC50 values of RYR and lovastatin on C2C12 muscle cells after 24 hours treatment. SD < 15% of reported means.

The results obtained therefore show that RYR samples always have a lower impact on viability. cellular compared to lovastatin; furthermore, in accordance with the literature, they have a greater effect on muscle cells.

Overall, the red yeast rice samples produced as described above and phytochemically standardized have:

- IC50 on human Caco2 (intestinal), HepG2 (liver), Hek293 (renal) cells always greater than lovastatin and in any case > 250 ?g/ml;

- IC50 on C2C12 muscle cells always greater than lovastatin and in any case > 150 ?g/ml;

- IC25 on human Caco2 (intestinal), HepG2 (liver), Hek293 (renal) cells always greater than lovastatin and in any case > 95 ?g/ml and on C2C12 muscle cells always greater than lovastatin and in any case > 50 ?g/ml.

Red yeast rice? processed in the post-fermentation phases to obtain products with variable MK and total monacolin content.

In this work, samples of red fermented rice containing from 1.5% to 5% m/m of total MK or from 1.8% to 6.5% m/m of total monacolins were studied in detail.

Red yeast rice? was analyzed for the first time and in depth to be standardized in the content of MK, various secondary monacolins, compactin and total polyphenols.

In detail, the samples studied:

- contain between 1.5% and 5% m/m of total MK, with a deviation from the declared < 10%;

- they have MK which represents between 70% and 85% of the total monkshood;

- they therefore have a total monacolin content between 1.8% and 6.5% m/m;

- they have secondary monacolins which represent between 15% and 30% of the total monacolins and what are they like? subdivided: MX MJ MM ML > 20% and dehydro-MK < 50% of total secondary monacolins;

- they always have a compactin content <0.15% m/m;

- have a minimum content of 0.75% m/m in total polyphenols.

The samples of red fermented rice produced in an innovative way and chemically characterized all showed high in vitro efficacy towards the biological target HMG-CoA reductase, the key enzyme which leads to the endogenous biosynthesis of cholesterol at the liver level.

For the first time in this job? An in vitro investigation was carried out on several samples of red yeast rice with different contents of MK and total monacolins, compared with lovastatin to evaluate the inhibition of the activity of the HMG-CoA reductase enzyme; for the first time ? The test was validated using a natural form and activated form (hydroxyacid) of the reference statin and monacolins from red yeast rice.

The results obtained showed that the fermented red rice samples produced in an innovative way and chemically characterized:

- in the in vitro enzymatic test for inhibition of HMG-CoA reductase they have an IC50 that is always improved compared to the MK content alone and in any case have an IC50 < 2500 ng/ml when the activated lovastatin, in the same experimental conditions, has an IC50 between 60 and 100 ng/ml ml.

For the first time ? Finally, was the possible effect of metabolic stress and cytotoxicity studied? of fermented red rice on the main areas of accumulation and metabolism of statins: intestine, liver, kidneys, muscle.

The results obtained showed that the innovatively produced and chemically characterized fermented red rice samples have:

- IC50 on human Caco2 (intestinal), HepG2 (liver), Hek293 (renal) cells always greater than lovastatin and in any case > 250 ?g/ml;

- IC50 on C2C12 muscle cells always greater than lovastatin and in any case > 150 ?g/ml;

- IC25 on human Caco2 (intestinal), HepG2 (liver), Hek293 (renal) cells always greater than lovastatin and in any case > 95 ?g/ml and on C2C12 muscle cells always greater than lovastatin and in any case > 50 ?g/ml.

It is concluded that the samples of red fermented rice object of the present invention prepared by the process of the present invention present standardized, precise, repeatable and safe phytochemical characteristics, and in vitro have demonstrated high efficacy on the inhibition of the HMG-CoA reductase enzyme and a low cytotoxicity? on intestinal, hepatic, renal and muscle cell lines.

Each RYR fermentation process can? lead to the presence of secondary monacolins (Msec) more? or less expressed, but the presence of secondary monacolins must be at least 15% of the total monacolins, for which the following ratio applies:

MKtot / Msec > 0.15.

Samples with a MKtot / Msec ratio between 0.15 and 0.05 raise doubts about authenticity; a MKtot / Msec ratio < 0.05 leads to the samples being considered plausibly adulterated due to the addition of monacolin K.

Secondary monacolins (Msec) are preferably identified with a mass spectrometer coupled to HPLC-UV. The usable tool? an Orbitrap Thermofisher with electrospray source (ESI), used in mode? negative with spray voltage 3.5 kV, sheat gas 20 a.u., auxiliary gas 5 a.u. and capillary temperature 320?C. The identified secondary monacolins are preferably quantified in a subsequent step by HPLC-DAD using the reference substance as discussed above. Preferably, the reference substance is ? mevinolin for monacolins in lactone form, and mevinolin in hydroxyacid form for monacolins in hydroxyacid form.

Claims (7)

1. A red yeast rice (RYR) having the following characteristics: - a monacolin K (MK) content of between 1.5% and 5% m/m, compared to the total weight of said fermented red rice; And - an MK content of between 70% and 85%, compared to the weight of the total monacolins; And - a dehydro-MK content of between 5% and 50%, compared to the weight of total monacolins; And - a total monacolin content of between 1% and 10%, preferably between 1.8% and 6.5% m/m, compared to the total weight of said fermented red rice. 2. The fermented red rice according to claim 1, wherein said red rice furthermore has: - a secondary monacolin content of between 15% and 30%, compared to the weight of the total monacolins, and in which said secondary monacolins are so? divided into: monacolin 3. The fermented red rice according to claim 1 or 2, wherein said red rice furthermore has: - a compactin content of between 0.01% and 0.15% m/m, compared to the total weight of said fermented red rice. 4. The fermented red rice according to any of claims 1-3, wherein said red rice further comprises: - a total polyphenol content ranging from 0.75% m/m to 2.5% m/m, compared to the total weight of said fermented red rice. 5. The fermented red rice according to any of claims 1-4, wherein said red rice further comprises: - a <12>C/<13>C isotope ratio of MK isolated from RYR lower than -28.3? ?<13>C, e - a citrinin content of less than 2 ppm. 6. Red yeast rice according to any of claims 1-5 for use: (i) in a method of keeping plasma cholesterol levels controlled, or (ii) in a method to reduce the accumulation of cholesterol in the blood and, therefore, for the primary prevention of hypercholesterolemia; or (iii) in a method of treatment, preventive or curative, of a disorder or pathology associated with hypercholesterolemia. 7. Non-therapeutic use of red yeast rice according to any of claims 1 to 5, as a food or ingredient for foods, or to prepare foods or food supplements or nutraceutical products, or for compositions for medical devices, in accordance with EU Regulation 745/2017.