DL DONNA LEONARDO (IT)
DOLCE VINCENZA (IT)
SINDONA GIOVANNI (IT)
DL DONNA LEONARDO (IT)
DOLCE VINCENZA (IT)
KUROYANAGI M ET AL: "Flavonoid glycosides and limonoids from Citrus molasses", NATURAL MEDICINES - SHOYAKUGAKU ZASSHI, JAPANESE SOCIETY OF PHARMACOGNOSY, TOKYO, JP, vol. 62, no. 1, 1 January 2008 (2008-01-01), pages 107 - 111, XP008106395, ISSN: 1340-3443, [retrieved on 20070927]
SAWABE A ET AL: "BIOACTIVE COMPOUNDS IN CITRUS FRUIT PEELS", FOODS AND FOOD INGREDIENTS JOURNAL OF JAPAN, FFI JANARU HENSHU IINKAI, TOYONAKA, JP, vol. 169, 1 January 1996 (1996-01-01), pages 37 - 44, XP008106430, ISSN: 0919-9772
SAWABE AKIYOSHI ET AL: "Studies on physiologically active substances in citrus fruit peel. Part XIV. Structures and hypotensive effect of flavonoid glycosides in young Citrus unshiu peelings", YUKAGAKU, NIHON YUKAGAKU KYOKAI, TOKYO, JP, vol. 38, no. 1, 1 January 1989 (1989-01-01), pages 53 - 59, XP008106407, ISSN: 0513-398X
BERHOW MARK A ET AL: "Acylated flavonoids in callus cultures of Citrus aurantifolia", PHYTOCHEMISTRY, PERGAMON PRESS, GB, vol. 36, no. 5, 10 August 1994 (1994-08-10), pages 1225 - 1227, XP008106358, ISSN: 0031-9422, [retrieved on 20010305]
AVULA BHARATHI ET AL: "Simultaneous quantification of adrenergic amines and flavonoids in C. aurantium, various Citrus species, and dietary supplements by liquid chromatography", JOURNAL OF AOAC INTERNATIONAL, AOAC INTERNATIONAL, ARLINGTON, VA, US, vol. 88, no. 6, 1 November 2005 (2005-11-01), pages 1593 - 1606, XP008106397, ISSN: 1060-3271
HORIE T ET AL: "THREE FLAVONE GLYCOSIDES FROM CITRUS-SUDACHI", PHYTOCHEMISTRY, PERGAMON PRESS, GB, vol. 25, no. 11, 1 January 1986 (1986-01-01), pages 2621 - 2624, XP008106366, ISSN: 0031-9422, [retrieved on 20010301]
MICELI NATALIZIA ET AL: "Hypolipidemic effects of Citrus bergamia Risso et Poiteau juice in rats fed a hypercholesterolemic diet", JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, vol. 55, no. 26, December 2007 (2007-12-01), pages 10671 - 10677, XP002530454, ISSN: 0021-8561
See also references of EP 2424545A1
| Claims: 1. A natural molecule extracted from a citrus fruit characterized by the fact that its structure is a flavonoid linked to 3 -hydroxy-3 -methyl glutaric acid. 2. A natural molecule extracted from a citrus fruit as in claim 1 characterized by the fact that the fruit is bergamot. 3. A natural molecule extracted from a citrus fruit as in claim 1 or 2 characterized by the fact that the flavonoid contains naringin. 4. A natural molecule extracted from a citrus fruit as in claim 1 or 2 characterized by the fact that the flavonoid contains neohesperidin. 5. A natural molecule extracted from a citrus fruit as in claim 1 or 2 characterized by the fact that the flavonoid containing naringin and/or neohesperidin is conjugated to 3-hydroxy-3- methyl glutaric acid as ester on the glycosidic moiety 6. Extraction process of a natural molecule as in above quoted claims in which: the extraction of chopped fruit is conducted in pure or mixed chloroform, ethanol or methanol for a certain time the filtrate is evaporated to dryness the residue is submitted to solid phase extraction to separate the flavonoidic portion from the other class of compounds the flavonoid mixture is separated through a preparative chromatographic system (HPLC) 7. Extraction process as in claim 4 in which it requires a ternary mixture of methanol, ethanol and chloroform 65/30/5 (v/v/v) 8. extraction process as in claim 4 in which the mixture of flavonoid is separated by a MPLC chromatographic system 9. Pharmaceutical use of a molecule as in claims 1 or 2 or 3 or 4 used as anticholesterol drug. |
Field of the invention
The present invention regards new molecules extracted from Bergamot tissues, a fruit of genus citrus, the extraction process and its use as anti-cholesterol pharmaceutical lead.
State of the art
Flavonoids are molecule widespread present in vegetable species; they possess considerable nutraceutical and pharmaceutical properties; in the last century, epidemiological studies have shown that the dietary consumption of vegetables and fruits provides protection against chronic diseases, such as, for example, cancer. 1"7 Since the production of free radicals is implied in the appearance of the cancer, 8" " the attention has been focused on the possible role of these molecules as scavenger of free radicals. 12"13
Recent studies suggest that the consumption of flavonols and flavones, and in general of flavonoids, protects against vascular diseases and some form of cancer. 16"26
In the patent application US2008031861, the consumption of a food additive containing a flavonoid and an enzyme is described to lower cholesterol levels in men.
CNl 01073610 describes a process for obtaining from Common Hawthorn, organic salts and flavonoids that may be used to lower cholesterol levels.
In the patent application US2006182823, an extraction method of flavonoids and alkaloids from Nelumbo leaves is described. Summary of the present invention
The present invention regards the isolation and the structural determination of new compounds in Citrus species and in particular in Citrus Bergamia (bergamot).
These molecules that belong to the class of flavonoids may be found in significant amounts (300- 500 mg/kg) in different tissues of the fruit. The main peculiarity of these compounds is the presence of the 3 -hydroxy-3 -methyl glutaryl (HMG) moiety, esterified on the neohesperidose (sugar) moiety (scheme 1).
1 R 1 = OH 1 R 2 = OCH 3
2 R 1 = H 1 R 2 = OH
O O
H.
V O -R
3; XR= SCoA 4; XR= OR
Scheme 1
The reduction of HMG-CoA conjugate to mevalonic acid by the coenzyme NADPH, in the active site of HMG-CoA reductase, is the key step in cholesterol biosynthesis.
The nucleophilic attack of the coenzyme leads to the reduction of the thioester function of 3 because of the presence of a good leaving group. In the case of ester derivatives such as for molecules of general formula 4 the pKa of the leaving alkoxide prevents the formation of mevalonic acid. It should be considered, however, that the combined effect of recognition and kinetic of ester hydrolysis play a fundamental in in vitro or in vivo testing of substrates of type 4, such the statin family used in conventional therapies.
The new molecules 1 and 2, discovered in bergamot tissues, are neohesperidin and naringin conjugated of the HMG molecule (scheme 1), therefore, it could be expected that they exhibit an inhibitory action against HMG-reductase. This hypothesis has been confirmed by the in vitro tests, described below. The new compounds have been isolated using an extraction procedure followed by a chromatographic step. The extraction procedure consists in the addition of solvents (methanol, ethanol and chloroform) pure or mixed in different proportions, to the fruit previously crushed. The extraction lasts two hours, the solvents are then removed by filtration and under reduced pressure, leaving a residue. The residue is re-dissolved in water and submitted to solid phase extraction using a Ci 8 cartridge. The cartridge is previously activated with methanol and water, then loaded with the residue which is eluted first with water to remove the sugars and then with methanol or ethanol. The alcoholic eluate, that contains flavonoids for the most part, is treated under reduced pressure to remove solvents and submitted to chromatographic separation. The chromatographic hardware consists in a semi-preparative HPLC equipped with a reverse phase column and a UV/MS detector; alternatively, a reverse phase MPLC system may be used. The mobile phase consists of the binary mixture water/methanol or water/ethanol or water/acetonitrile. The fractions corresponding to ionic chromatograms of m/z 755 (compound 1) and m/z 725 (compound 2) are collected; solvent removal from the appropriate fractions affords the pure compounds. Compound 1 has been characterized by high resolution mass spectrometry (HRMS) and nuclear magnetic resonance (HRNMR) experiments. The HRMS spectrum of molecule 1 shows the protonated molecular ion [M+H] + at m/z 755.2387, which corresponds to the elemental formula C34H43O19 with -0.80 ppm error respect to the theoretical mass. The positive HRMSMS spectrum shows a series of peaks whose formation may be described by scheme 2
Scheme 2
Table 1 shows the interpretation of the data coming from ID and 2D NMR experiments performed on molecule 1.
Table 1. 1 H-NMR data (δ) of molecules I e 2
Position Compound 1 Compound 2
2 5.38 (dd) 5.38 (dd)
3 3.09-2.77 {dd) 3.09-3.30 (dd)
6 6.17 [m, ar) 6.17 [m, ar)
8 6.14 (m, ar) 6.14 (m, ar)
2Θ 6.96 (m, ar) 6.96 (m, ar)
40-OCH 3 3.85 (S)
30- H 6.70 (m, ar)
50 6.90 (m, ar) 6.70 (m, ar)
60 6.92 (m, ar) 6.97 (m, ar)
100 SΛQ (d) 5.10 (d) 2ΞΞ 3.62 (dd) 3.66 (dd)
300 3.39 (dd) 3.39 (dd)
4ΞS 3.37 (dd) 3.37 (dd)
5ΘΞ 3.69 (dd) 3.69 (dd)
6ΞΞ 4.20-4.40 (dd) 4.20-4.40 (dd)
1Ξ00 5.26 (d) 5.26 (d)
200Ξ 3.89 (dd) 3.94 (dd)
3ΞΞΞ 3.59 (dd) 3.59 (dd) mm 3.62 (dd) 3.66 (dd)
5H3Ξ 3.90 (m) 3.90 (dd)
6ΞΞΞ 1.31 (d) 1.31 (d)
2ΞΞΞΞ 2.65-2.52 (m) 2.65-2.52 (m)
4ΞΞΞΞ 2.65-2.52 (m) 2.65-2.52 (m) 60.033 1.26 (s) 1.26 (s)
The molecule has been submitted to basic hydrolysis experiments to check the presence of ester moieties. After 4 hours of reaction the molecule 1 dissolved in a solution of saturated Na 2 CO 3 , forms Neohesperidine, as shown by chromatographic experiments. A second reaction product is formed. HRMS and NMR experiments (table 2) demonstrate that the latter molecule is the 3- hydroxy-3-methyl glutaryl acid.
Table 2 NMR data of 3-methyl-3-hydroxyl glutaryl acid (HMG) obtained after basic hydrolysis
~ sei 1 H-NMR data (δ (m)) 13 C-NMR data (δ (m))
J-CH 3 1.22 (5) 28 fø)
2,4-CH 3 2.46 (m) 46.5 (0 7,J-COOH 8.21 (s) 173 (j)
J-OH 69 (5)
The whole structural elucidation is repeated for compound 2.
The isolated molecules 1 and 2 have been submitted to in vitro experiments in order to verify their inhibitory potential against HMG-reductase and, hence, against the cholesterol biosynthesis. A commercially available enzymatic kit, has been used to perform the experiments. The kit includes: 10 ml of 5x buffer; 25 mg of NADPH, 2 ml of substrate (HMG-CoA); 200μl of solution of HMGR (catalytic domain) 0,55-0,65 mg/ml, 200μl solution of Pravastatin inhibitor. To monitor the experiment, the decrease of the absorbance signal at 340 nm wavelength must be followed; this represents the oxidation of NADPH from the catalytic portion of HMGR in presence either of HMG-CoA and an inhibitor. Compound 1 at 300 μmol/L concentration shows inhibition at 80% level after 8 mins (figure 1).
Imbizione dell'HMGR da parte della Brutieridtna 300 μM
4 5
Tempo/min
Figure 1
Compound 2 at the same conditions shows inhibition at 75% level after 8 mins (figure 2).
Figure 2
The experiments performed shows that the two new molecules possess inhibitory properties similar to statins used as anticholesterol drugs.
The anticholesterolemic action of bergamot juice belongs to the tradition of folk medicine experienced by people living in the south ionic cost of Calabria region. The new molecules, never characterized or isolated before, whose anticholesterolemic activity has been unambiguously demonstrated in vitro give a scientific explanation to this empirical observation.
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