ITCS20080019A1 - NATURAL MOLECULE EXTRACTED FROM A CITRUS, EXTRACTION PROCESS AND PHARMACEUTICAL USE - Google Patents

NATURAL MOLECULE EXTRACTED FROM A CITRUS, EXTRACTION PROCESS AND PHARMACEUTICAL USE Download PDF

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ITCS20080019A1
ITCS20080019A1 IT000019A ITCS20080019A ITCS20080019A1 IT CS20080019 A1 ITCS20080019 A1 IT CS20080019A1 IT 000019 A IT000019 A IT 000019A IT CS20080019 A ITCS20080019 A IT CS20080019A IT CS20080019 A1 ITCS20080019 A1 IT CS20080019A1
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citrus fruit
natural molecule
flavonoid
fruit according
cancer
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Donna Leonardo Di
Vincenza Dolce
Giovanni Sindona
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Univ Calabria
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18Magnoliophyta (angiosperms)
    • A61K36/185Magnoliopsida (dicotyledons)
    • A61K36/75Rutaceae (Rue family)
    • A61K36/752Citrus, e.g. lime, orange or lemon
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00Drugs for disorders of the metabolism
    • A61P3/06Antihyperlipidemics
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00Drugs for disorders of the cardiovascular system
    • A61P9/10Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07HSUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • C07H17/04Heterocyclic radicals containing only oxygen as ring hetero atoms
    • C07H17/06Benzopyran radicals
    • C07H17/065Benzo[b]pyrans
    • C07H17/07Benzo[b]pyran-4-ones

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Description

Molecola naturale estratta da un agrume, processo di estrazione e uso farmaceutico Campo della tecnica dell’invenzione. Natural molecule extracted from a citrus fruit, extraction process and pharmaceutical use Field of the inventive technique.

La presente invenzione riguarda una nuova molecola estratta da un agrume, in particolare dal bergamotto, il processo di estrazione e il suo uso come farmaco anti-colesterolemico. The present invention relates to a new molecule extracted from a citrus fruit, in particular from bergamot, the extraction process and its use as an anti-cholesterol drug.

Stato dell’arte State of the art

I flavonoidi sono presenti in maniera massiccia nelle specie vegetali, e possiedono doti nutrizionali e farmaceutiche notevoli; nel secolo scorso, studi epidemiologici hanno dimostrato che il consumo a lungo termine di vegetali e frutta offre una notevole protezione nei confronti di malattie croniche, come ad esempio il cancro.<1-7>Poichè la produzione incontrollata di radicali liberi è implicata nell’insorgenza del cancro,<8-11>si è focalizzata l’attenzione sul possibile ruolo dei nutrienti di questo tipo di dieta come soppressori e/o spazzini di radicali.<12-15>Flavonoids are massively present in plant species, and possess remarkable nutritional and pharmaceutical qualities; in the last century, epidemiological studies have shown that the long-term consumption of vegetables and fruit offers considerable protection against chronic diseases, such as cancer. <1-7> As the uncontrolled production of free radicals is implicated in onset of cancer, <8-11> attention was focused on the possible role of nutrients in this type of diet as suppressors and / or scavengers of radicals. <12-15>

Studi più recenti suggeriscono che l’assunzione di flavonoli e flavoni, o altri flavonoidi ha un effetto protettivo contro malattie vascolari o alcuni tipi di cancro.<16-26>More recent studies suggest that the intake of flavonols and flavones, or other flavonoids, has a protective effect against vascular diseases or some types of cancer. <16-26>

Nel documento US2008031861 è descritto un integratore che per abbassare il colesterolo in una persona utilizza un integratore contenente un flavonoide ed un enzima. Document US2008031861 describes a supplement that uses a supplement containing a flavonoid and an enzyme to lower cholesterol in a person.

CN101073610 descrive un processo per ottenere dal biancospino sali organici acidi di calcio contenenti flavonoidi che possono essere usati per abbassare il colesterolo. CN101073610 describes a process for obtaining organic acidic calcium salts containing flavonoids from hawthorn which can be used to lower cholesterol.

In US2006182823 viene descritto un metodo per estrarre componenti flavonoidi ed alcaloidi da foglie di loto. In US2006182823 a method for extracting flavonoid and alkaloid components from lotus leaves is described.

Sommario della presente invenzione Summary of the present invention

La presente invenzione riguarda l’isolamento e la determinazione strutturale di alcuni composti presenti negli agrumi e particolarmente nel frutto della specie Bergamia appartenente al genere Citrus (Bergamotto). Essi sono presenti in quantità rilevanti (ca. 300-500 mg/kg) nelle diverse parti del frutto e appartengono alla classe dei flavonoidi. La caratteristica principale di questi nuovi composti è la presenza nella molecola del gruppo 3-idrossi-3-metil glutarile (HMG) sotto forma di estere sulla porzione zuccherina della molecola (schema 1). The present invention relates to the isolation and structural determination of some compounds present in citrus fruits and particularly in the fruit of the Bergamia species belonging to the genus Citrus (Bergamot). They are present in significant quantities (approx. 300-500 mg / kg) in the different parts of the fruit and belong to the class of flavonoids. The main characteristic of these new compounds is the presence in the molecule of the 3-hydroxy-3-methyl glutaryl (HMG) group in the form of ester on the sugar portion of the molecule (scheme 1).

Schema 1 Scheme 1

Il gruppo HMG, presente nelle nuove molecole individuate, ha un ruolo fondamentale nella biosintesi del colesterolo, infatti esso, in vivo, viene biosintetizzato attraverso una serie di reazioni enzimatiche. Uno di questi passaggi chiave prevede la trasformazione (riduzione) dell’acido 3-idrossi-3-metil glutarico coniugato al coenzima-A (HMG-CoA) ad acido mevalonico. Normalmente per bloccare la biosintesi del colesterolo, si utilizzano farmaci denominati “statine”; questi hanno una struttura simile all’HMG-CoA e riescono a bloccare l’enzima HMG reduttasi responsabile della trasformazione (riduzione) descritta sopra. Le nuove molecole, trovate nel succo di bergamotto sono anche esse coniugate dell’HMG (Schema) e possono, quindi, inibire l’enzima reduttasi. Le prove in vitro descritte in fondo a questo abstract danno credito a questa ipotesi. The HMG group, present in the new molecules identified, plays a fundamental role in the biosynthesis of cholesterol, in fact it is biosynthesized in vivo through a series of enzymatic reactions. One of these key steps involves the transformation (reduction) of 3-hydroxy-3-methyl glutaric acid conjugated to coenzyme-A (HMG-CoA) to mevalonic acid. Normally, to block the biosynthesis of cholesterol, drugs called "statins" are used; these have a similar structure to HMG-CoA and are able to block the HMG reductase enzyme responsible for the transformation (reduction) described above. The new molecules found in bergamot juice are also conjugated to HMG (Scheme) and can, therefore, inhibit the reductase enzyme. The in vitro tests described at the bottom of this abstract give credence to this hypothesis.

I composti sono stati isolati attraverso un procedimento estrattivo ed una separazione cromatografia. Il procedimento estrattivo consiste nell’aggiunta di una miscela di solventi (metanolo etanolo e cloroformio in rapporto volumetrico 65:30:5 (v/v/v)),ovvero gli stessi solventi puri od in diverse proporzioni, al frutto precedentemente sminuzzato in tutte le sue parti. Il preparato viene posto sotto agitazione per 2 ore. Alla fine il solvente viene filtrato e ridotto in volume fino a consistenza sciropposa. Il residuo, solubilizzato con acqua, viene sottoposto a estrazione in fase solida attraverso una cartuccia a fase inversa. La cartuccia viene prima attivata con metanolo ed acqua, poi il residuo viene versato sulla cartuccia ed eluito dapprima con acqua per rimuovere gli zuccheri e poi con metanolo o etanolo. Il residuo metanolico che contiene perlopiù flavonoidi viene evaporato a secchezza e sottoposto a separazione cromatografia. Il sistema cromatografico è un HPLC semipreparativo utilizzante una colonna in fase inversa ed un detector UV/MS oppure un sistema MPLC a fase inversa utilizzante gli stessi solventi di eluizione. Per isolare il composto si utilizza una corsa cromatografia con fase mobile acqua e metanolo o etanolo o acetonitrile. Le frazioni corrispondenti agli estratti ionici m/z 755 (composto 1) e m/z 725 (composto 2) vengono raccolte e separate dal solvente per evaporazione per ottenere i composti puri. Il composto 1 è stato caratterizzato attraverso esperimenti di spettrometria di massa ad alta risoluzione (HRMS) e risonanza magnetica ad alta risoluzione (NMR). Lo spettro HRMS in sorgente di 1 genera il picco pseudomolecolare [M+H]<+>con valore 755.2387 che corrisponde alla formula elementare C34H43O19con uno scarto di -0.80 ppm sul valore teorico. Lo spettro HRMSMS della specie [M+H]<+>genera una serie di picchi interpretabili attraverso lo schema 2. The compounds were isolated through an extractive process and a chromatographic separation. The extraction process consists in adding a mixture of solvents (methanol, ethanol and chloroform in a volumetric ratio 65: 30: 5 (v / v / v)), or the same pure solvents or in different proportions, to the fruit previously chopped in all its parts. The preparation is stirred for 2 hours. Finally the solvent is filtered and reduced in volume to a syrupy consistency. The residue, solubilized with water, is subjected to solid phase extraction through a reverse phase cartridge. The cartridge is first activated with methanol and water, then the residue is poured onto the cartridge and eluted first with water to remove the sugars and then with methanol or ethanol. The methanol residue, which mostly contains flavonoids, is evaporated to dryness and subjected to chromatographic separation. The chromatographic system is a semipreparative HPLC using a reversed phase column and a UV / MS detector or a reversed phase MPLC system using the same elution solvents. A water and methanol or ethanol or acetonitrile mobile phase chromatography run is used to isolate the compound. The fractions corresponding to the ionic extracts m / z 755 (compound 1) and m / z 725 (compound 2) are collected and separated from the solvent by evaporation to obtain the pure compounds. Compound 1 was characterized through high resolution mass spectrometry (HRMS) and high resolution magnetic resonance (NMR) experiments. The HRMS spectrum in source of 1 generates the pseudomolecular peak [M + H] <+> with value 755.2387 which corresponds to the elementary formula C34H43O19 with a deviation of -0.80 ppm on the theoretical value. The HRMSMS spectrum of the [M + H] <+> species generates a series of peaks that can be interpreted through scheme 2.

3′′′ 3.59 (dd) 3.59 (dd) 3 ′ ′ ′ 3.59 (dd) 3.59 (dd)

4′′′ 3.62 (dd) 3.66 (dd) 4 ′ ′ ′ 3.62 (dd) 3.66 (dd)

5′′′ 3.90 (m) 3.90 (dd) 5 ′ ′ ′ 3.90 (m) 3.90 (dd)

6′′′ 1.31 (d) 1.31 (d) 6 ′ ′ ′ 1.31 (d) 1.31 (d)

2′′′′ 2.65-2.52 (m) 2.65-2.52 (m) 2 ′ ′ ′ ′ 2.65-2.52 (m) 2.65-2.52 (m)

4′′′′ 2.65-2.52 (m) 2.65-2.52 (m) 4 ′ ′ ′ ′ 2.65-2.52 (m) 2.65-2.52 (m)

6′′′′ 1.26 (s) 1.26 (s) 6 ′ ′ ′ ′ 1.26 (s) 1.26 (s)

La molecola è stata sottoposta a reazione di idrolisi basica per verificare l’esistenza di legami esterei. Dopo 4 ore di reazione con carbonato di sodio, si forma una specie avente ione molecolare a m/z 611. I tempi di ritenzione cromatografici del prodotto di reazione e dello standard neoesperidina, sono uguali per cui si arriva alla conclusione che il prodotto di reazione è proprio neoesperidina. La corsa mostra anche la formazione di un prodotto avente ione molecolare m/z 163, che corrisponde all’acido eliminato dalla reazione di idrolisi. Quest’ultimo prodotto viene separato tramite cromatografia preparativa e sottoposto ad esperimenti HRMS ed NMR (tabella 2), che indicano che esso corrisponde all’HMG. The molecule was subjected to a basic hydrolysis reaction to verify the existence of ester bonds. After 4 hours of reaction with sodium carbonate, a species is formed having a molecular ion at m / z 611. The chromatographic retention times of the reaction product and of the neohesperidin standard are the same so that it is concluded that the reaction product is just neoesperidina. The run also shows the formation of a product having a molecular ion m / z 163, which corresponds to the acid eliminated by the hydrolysis reaction. The latter product is separated by preparative chromatography and subjected to HRMS and NMR experiments (table 2), which indicate that it corresponds to HMG.

Tabella 2. Dati NMR per l’acido 3-metil-3-idrossil glutarico (HMG) ottenuto dopo idrolisi basica. Table 2. NMR data for 3-methyl-3-hydroxyl glutaric acid (HMG) obtained after basic hydrolysis.

<set>dati<1>H-NMR (δ (m)) dati<13>C-NMR (δ (m)) <set> data <1> H-NMR (δ (m)) data <13> C-NMR (δ (m))

3-CH31.22 (s) 28 (q) 3-CH31.22 (s) 28 (q)

2,4-CH32.46 (m) 46.5 (t) 2,4-CH32.46 (m) 46.5 (t)

1,5-COOH 8.21 (s) 173 (s) 1,5-COOH 8.21 (s) 173 (s)

3-OH -- 69 (s) 3-OH - 69 (s)

Il risultato è che la molecola incognita 1 è una molecola di neosperidina coniugata attraverso un legame estereo in posizione 6 del glucosio all’HMG. La posizione del legame estereo è confermata dai dati NMR in tabella 1. The result is that the unknown molecule 1 is a molecule of neosperidin conjugated through an ester bond in position 6 of glucose to HMG. The position of the ester bond is confirmed by the NMR data in Table 1.

L’intera procedura viene ripetuta per il composto 2 che come risultato finale fornisce la struttura della naringina coniugata attraverso un legame estereo in posizione 6 del glucosio all’HMG. The entire procedure is repeated for compound 2 which as a final result provides the structure of the conjugated naringin through an ester bond in position 6 of the glucose to HMG.

Una volta isolate, le molecole 1 e 2 sono state sottoposte ad esperimenti in vitro per verificare il loro potenziale inibitorio del processo biosintetico di produzione del colesterolo. E’ stato utilizzato un kit enzimatico reperibile in commercio; Il kit risulta composto da: 10 ml di tampone 5x; 25 mg di NADPH, 2 ml di substrato (HMG-CoA); 200µl di HMGR (dominio catalitico) 0,55-0,65 mg/ml, 200µl di soluzione dell’inibitore Pravastatina. Il kit è progettato in modo da seguire la diminuzione di assorbanza alla lunghezza d’onda di 340 nm, che rappresenta l'ossidazione del NADPH ad opera della porzione catalitica di HMGR in presenza del substrato HMG-CoA ed eventualmente di un inibitore. Once isolated, molecules 1 and 2 were subjected to in vitro experiments to verify their inhibitory potential of the biosynthetic process of cholesterol production. A commercially available enzymatic kit was used; The kit consists of: 10 ml of 5x buffer; 25 mg of NADPH, 2 ml of substrate (HMG-CoA); 200µl of HMGR (catalytic domain) 0.55-0.65 mg / ml, 200µl of Pravastatin inhibitor solution. The kit is designed to follow the decrease in absorbance at a wavelength of 340 nm, which represents the oxidation of NADPH by the catalytic portion of HMGR in the presence of the HMG-CoA substrate and possibly an inhibitor.

Il composto 1 già a concentrazioni di circa 300 µmol/L mostra un’inibizione dell’80% dopo 8 minuti (grafico 1) Compound 1 already at concentrations of about 300 µmol / L shows an inhibition of 80% after 8 minutes (graph 1)

Grafico 1 Graph 1

Il composto 2 nelle stesse condizioni mostra un inibizione del 75% dopo 8 minuti (grafico 2): Compound 2 under the same conditions shows 75% inhibition after 8 minutes (graph 2):

Grafico 2 Graph 2

I risultati mostrano che tutte e due le molecole derivate dell’HMG hanno una attività inibitoria simile, sebbene a concentrazioni molto maggiori, a quella delle statine utilizzate per combattere il colesterolo. The results show that both HMG-derived molecules have similar inhibitory activity, albeit at much higher concentrations, to that of the statins used to fight cholesterol.

Riferimenti: References:

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Weisburger, J.H., Nutritional approach to cancer prevention with emphasis on vitamins, antioxidants and carotenoids, Am. J. Clin. Nutr., 53, 226S, 1991. Andersen and Markham / Flavonoids: Chemistry, Biochemistry, and Applications #2021_c006 Final Proof page 344 8.9.2005 8:53pm 344 Flavonoids: Chemistry, Biochemistry, and Applications Wattenberg, L.W., Inhibition of carcinogenesis by minor dietary constituents, Cancer Res., 52, 2085, 1992. Weisburger, J.H., Nutritional approach to cancer prevention with emphasis on vitamins, antioxidants and carotenoids, Am. J. Clin. Nutr., 53, 226S, 1991. Andersen and Markham / Flavonoids: Chemistry, Biochemistry, and Applications # 2021_c006 Final Proof page 344 8.9.2005 8:53 pm 344 Flavonoids: Chemistry, Biochemistry, and Applications Wattenberg, L.W., Inhibition of carcinogenesis by minor dietary constituents, Cancer Res., 52, 2085, 1992.

Knekt, P., Jarvinen, R., Reunanen, A., and Maatela, J., Flavonoid intake and coronary mortality in Finland: a cohort study, Br. Med. J., 312, 478, 1996. Knekt, P., Jarvinen, R., Reunanen, A., and Maatela, J., Flavonoid intake and coronary mortality in Finland: a cohort study, Br. Med. J., 312, 478, 1996.

Keli, S.O., Hertog, M.G., Feskens, E.J., and Kromhout, D., Dietary flavonoids, antioxidant vitamins, and incidence of stroke: the Zutphen Study, Arch. Intern. Med., 156, 637, 1996. Keli, S.O., Hertog, M.G., Feskens, E.J., and Kromhout, D., Dietary flavonoids, antioxidant vitamins, and incidence of stroke: the Zutphen Study, Arch. Intern. Med., 156, 637, 1996.

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Andersen and Markham / Flavonoids: Chemistry, Biochemistry, and Applications #2021_c006 Final Proof page 3458.9.2005 8:53pm Dietary Flavonoids and Health Broadening the Perspective 345 Andersen and Markham / Flavonoids: Chemistry, Biochemistry, and Applications # 2021_c006 Final Proof page 3458.9.2005 8:53 pm Dietary Flavonoids and Health Broadening the Perspective 345

Hirvonen, T., Pietinen, P., Virtanen, M., Ovaskainen, M.L., Hakkinen, S., Albanes, D., and Virtamo, J., Intake of flavonols and flavones and risk of coronary heart disease in male smokers, Epidemiology, 12, 62, 2001. Hirvonen, T., Pietinen, P., Virtanen, M., Ovaskainen, M.L., Hakkinen, S., Albanes, D., and Virtamo, J., Intake of flavonols and flavones and risk of coronary heart disease in male smokers, Epidemiology, 12, 62, 2001.

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Claims (1)

Rivendicazioni 1. Molecola naturale estratta da un agrume caratterizzata dal fatto che la sua struttura è un flavonoide coniugato all’acido 3-idrossi-3-metil glutarile 2. Molecola naturale estratta da un agrume secondo la rivendicazione 1 caratterizzata dal fatto che la l’agrume è il bergamotto. 3. Molecola naturale estratta da un agrume secondo la rivendicazione 1 o 2 caratterizzata dal fatto che il flavonoide è coniugato con l’acido 3-idrossi-3-metil glutarile attraverso un legame estereo sulla porzione zuccherina della molecola. 4. Molecola naturale estratta da un agrume secondo la rivendicazione 1 o 2 o 3 caratterizzata dal fatto che il flavonoide è la neoesperidina. 5. Molecola naturale estratta da un agrume secondo la rivendicazione 1 o 2 o 3 caratterizzata dal fatto che il flavonoide è la naringina. 6. Processo di estrazione di una molecola naturale da un agrume secondo una qualsiasi delle rivendicazioni precedenti in cui: - l’estrazione delle parti del frutto triturate viene eseguita in un solvente costituito da metanolo, etanolo e cloroformio puri od in diverse proporzioni per un tempo determinato - il filtrato è evaporato a secchezza - il residuo viene sottoposto ad estrazione in fase solida per purificare i flavonoidi dalle altre classi di composti. - La miscela di flavonoidi viene separata attraverso un sistema cromatografico HPLC preparativo. 7. Processo di estrazione di una molecola naturale da un agrume secondo la rivendicazione 6 in cui il solvente è costituito dalla miscela ternaria metanolo, etanolo e cloroformio in rapporto volumetrico 65/5/30. 8. Processo di estrazione di una molecola naturale da un agrume secondo la rivendicazione 6 in cui la miscela di flavonoidi viene separata attraverso un sistema cromatografico MPLC. 9. Uso farmaceutico di una molecola naturale estratta da un agrume secondo le rivendicazioni 1 o 2 o 3 o 4 o 5 o 6 come farmaco anti-colesterolemico.Claims 1. Natural molecule extracted from a citrus fruit characterized by the fact that its structure is a flavonoid conjugated to 3-hydroxy-3-methyl glutaryl acid 2. Natural molecule extracted from a citrus fruit according to claim 1 characterized by the fact that the citrus fruit is bergamot. 3. Natural molecule extracted from a citrus fruit according to claim 1 or 2 characterized in that the flavonoid is conjugated with 3-hydroxy-3-methyl glutaryl acid through an ester bond on the sugar portion of the molecule. 4. Natural molecule extracted from a citrus fruit according to claim 1 or 2 or 3 characterized in that the flavonoid is neoesperidin. 5. Natural molecule extracted from a citrus fruit according to claim 1 or 2 or 3 characterized in that the flavonoid is naringin. 6. Process of extracting a natural molecule from a citrus fruit according to any one of the preceding claims in which: - the extraction of the shredded fruit parts is performed in a solvent consisting of pure methanol, ethanol and chloroform or in different proportions for a specified time - the filtrate is evaporated to dryness - the residue is subjected to solid phase extraction to purify the flavonoids from the other classes of compounds. - The flavonoid mixture is separated through a preparative HPLC chromatographic system. 7. Process of extracting a natural molecule from a citrus fruit according to claim 6 in which the solvent is constituted by the ternary mixture of methanol, ethanol and chloroform in a volumetric ratio of 65/5/30. 8. A process of extracting a natural molecule from a citrus fruit according to claim 6 wherein the flavonoid mixture is separated through an MPLC chromatographic system. 9. Pharmaceutical use of a natural molecule extracted from a citrus fruit according to claims 1 or 2 or 3 or 4 or 5 or 6 as an anti-cholesterol drug.
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