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WO2010041290A1 - Natural molecules extracted from bergamot tissues, extraction process and pharmaceutical use - Google Patents

Natural molecules extracted from bergamot tissues, extraction process and pharmaceutical use Download PDF

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Publication number
WO2010041290A1
WO2010041290A1 PCT/IT2009/000456 IT2009000456W WO2010041290A1 WO 2010041290 A1 WO2010041290 A1 WO 2010041290A1 IT 2009000456 W IT2009000456 W IT 2009000456W WO 2010041290 A1 WO2010041290 A1 WO 2010041290A1
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Prior art keywords
flavonoid
natural molecule
fact
fruit
citrus fruit
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French (fr)
Inventor
Giovanni Sindona
Leonardo Dl Donna
Vincenza Dolce
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Universita della Calabria
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Universita della Calabria
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/75—Rutaceae (Rue family)
    • A61K36/752—Citrus, e.g. lime, orange or lemon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
    • C07H17/06—Benzopyran radicals
    • C07H17/065—Benzo[b]pyrans
    • C07H17/07—Benzo[b]pyran-4-ones

Definitions

  • 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.
  • 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
  • CNl 01073610 describes a process for obtaining from Common Hawthorn, organic salts and flavonoids that may be used to lower cholesterol levels.
  • the present invention regards the isolation and the structural determination of new compounds in Citrus species and in particular in Citrus Bergamia (bergamot).
  • HMG 3 -hydroxy-3 -methyl glutaryl
  • 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.
  • Table 1 shows the interpretation of the data coming from ID and 2D NMR experiments performed on molecule 1.
  • 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.
  • 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); 200 ⁇ l of solution of HMGR (catalytic domain) 0,55-0,65 mg/ml, 200 ⁇ l solution of Pravastatin inhibitor.
  • 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 ⁇ mol/L concentration shows inhibition at 80% level after 8 mins (figure 1).
  • Compound 2 at the same conditions shows inhibition at 75% level after 8 mins (figure 2).
  • 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.
  • Flavonoids Chemistry, Biochemistry, and Applications 15. Wattenberg, L. W., Inhibition of carcinogenesis by minor dietary constituents, Cancer Res., 52, 2085, 1992.
  • KeIi 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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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Epidemiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medical Informatics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Cardiology (AREA)
  • Genetics & Genomics (AREA)
  • Vascular Medicine (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Botany (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Mycology (AREA)
  • Urology & Nephrology (AREA)
  • Diabetes (AREA)
  • Hematology (AREA)
  • Obesity (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Saccharide Compounds (AREA)
  • Medicines Containing Plant Substances (AREA)

Abstract

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 and an extraction process 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 in order to separate the flavonoidic portion from the other class of compounds, the flavonoid mixture is separated through a preparative HPLC chromatographic system. Pharmaceutical use of the natural molecule extracted from a citrus fruit used as anticholesterol drug.

Description

Natural molecules extracted from Bergamot tissues, extraction process and pharmaceutical use.
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).
Figure imgf000003_0001
1 R1 = OH1 R2 = OCH3
2 R1 = H1 R2 = OH
O O
H.
VO -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 Ci8 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
Figure imgf000005_0001
Scheme 2
Table 1 shows the interpretation of the data coming from ID and 2D NMR experiments performed on molecule 1.
Table 1. 1H-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-OCH3 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 Na2CO3, 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 1H-NMR data (δ (m)) 13C-NMR data (δ (m))
J-CH3 1.22 (5) 28 fø)
2,4-CH3 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
Figure imgf000007_0001
4 5
Tempo/min
Figure 1
Compound 2 at the same conditions shows inhibition at 75% level after 8 mins (figure 2).
Figure imgf000008_0001
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.
References:
1. Lipkin, M., Uehara, K., Winawer, S., Sanchez, A., Bauer, C , Phillips, R., Lynch, H.T., Blattner, W. A., and Fraumeni, J. F., Seventh-Day Adventist vegetarians have a quiescent proliferative activity in colonic mucosa, Cancer Lett., 26, 139, 1985.
2 Steinmetz, K.A. and Potter, J. D., Vegetables, fruit and cancer. I. Epidemiology, Cancer Causes Control, 5, 325, 1991.
3. Block, G., Patterson, B., and Subar, A., Fruit, vegetables and cancer prevention: a review of the epidemiological evidence, Nutr. Cancer, 18, 1, 1992. 4. Hertog, M.G., Bueno-de-Mesquita, H.B., Fehily, A.M., Sweetnam, P.M., Elwood, P. C, and Kromhout, D., Fruit and vegetable consumption and cancer mortality in the Caerphilly Study, Cancer Epidemiol. Biomarkers Prev., 5, 673, 1996.
5. World Cancer Research Fund, Food, Nutrition and the Prevention of Cancer: A Global Perspective, American Institute for Cancer Research, Washington DC, 1997.
6. Department of Health, Nutritional Aspects of the Development of Cancer, HMSO, London, 1998.
7. Wallstrom, P., Wirfalt, E., Janzon, L., Mattisson, L, Elmstahl, S., Johansson, U., and Berglund, G., Fruit and vegetable consumption in relation to risk factors for cancer: a report from the Malmo Diet and Cancer Study, Public Health Nutr., 3, 263, 2000.
8. Babbs, C. F., Free radicals and the etiology of colon cancer, Free Radical Biol. Med., 8, 191, 1990.
9. Guyton, K.Z. and Kensler, T. W., Oxidative mechanisms in carcinogenesis, Br. Med. Bull., 49, 523, 1993.
10. Goldstein, B. D. and Witz, G., Free radicals in carcinogenesis, Free Radical Res. Commun., 1 1, 3, 1990.
11. Steinmetz, K.A. and Potter, J. D., Vegetables, fruit and cancer. II. Mechanisms, Cancer Causes Control, 5, 427, 1991.
12. Wattenberg, L. W., Chemoprevention of cancer, Cancer Res., 45, 1, 1985.
13. Wattenberg, L. W., Inhibition of carcinogenesis by minor anutrient constituents of the diet., Proc. Nutr. Soc, 49, 173, 1990.
14. 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 15. Wattenberg, L. W., Inhibition of carcinogenesis by minor dietary constituents, Cancer Res., 52, 2085, 1992.
16. 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.
17. KeIi, 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.
18. Yochum, L., Kushi, L. H., Meyer, K., and Folsom, A.R., Dietary flavonoid intake and risk of cardiovascular disease in postmenopausal women, Am. J. Epidemiol., 149, 943, 1999. Andersen and Markham / Flavonoids: Chemistry, Biochemistry, and Applications #2021_c006 Final Proof page 345 8.9.2005 8:53pm Dietary Flavonoids and Health — Broadening the Perspective 345
19. 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.
20. Mermen, L. L, Sapinho, D., de Bree, A., Arnault, N., Bertrais, S., Galan, P., and Hercberg, S., Consumption of foods rich in flavonoids is related to a decreased cardiovascular risk in apparently healthy French women, J. Nutr., 134, 923, 2004.
21. Knekt, P., Jarvinen, R., Seppanen, R., Hellovaara, M., Teppo, L., Pukkala, E., and Aromaa, A., Dietary flavonoids and the risk of lung cancer and other malignant neoplasms, Am. J. Epidemiol., 146, 223, 1997.
22. Garcia-Closas, R., Gonzalez, C. A., Agudo, A., and Riboli, E., Intake of specific carotenoids and flavonoids and the risk of gastric cancer in Spain, Cancer Causes Control, 10, 71, 1999.
23. Birt, D. F., Hendrich, S., and Wang, W., Dietary agents in cancer prevention: flavonoids and isoflavonoids, Pharmacol. Ther., 90, 157, 2001. 24. Hirvonen, T., Virtamo, J., Korhonen, P., Albanes, D., and Pietinen, P., Flavonol and flavone intake and the risk of cancer in male smokers (Finland), Cancer Causes Control, 12, 789, 2001.
25. Arts, I.C., Jacobs, D. R., Gross, M., Harnack, L.J., and Folsom, A.R., Dietary catechins and cancer incidence among postmenopausal women: the Iowa Women's Health Study (United States), Cancer Causes Control, 13, 373, 2002.
26. Sun, C.L., Yuan, J.M., Lee, M.J., Yang, C.S., Gao, Y.T., Ross, R.K., and Yu, M.C., Urinary tea polyphenols in relation to gastric and esophageal cancers: a prospective study of men in Shanghai, China, Carcinogenesis, 23, 1497, 2002.

Claims

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.
PCT/IT2009/000456 2008-10-09 2009-10-08 Natural molecules extracted from bergamot tissues, extraction process and pharmaceutical use Ceased WO2010041290A1 (en)

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CN103304610A (en) * 2012-12-24 2013-09-18 李玉山 Preparation process of methyl hesperidin
US8741362B2 (en) 2008-11-17 2014-06-03 Herbal & Antioxidant Derivatives S.R.L. Phytocomplex from bergamot fruit, process of manufacture and use as dietary supplement and in the pharmaceutical field
CN104297026A (en) * 2014-09-27 2015-01-21 杭州师范大学 Method for extracting effective flavonoid components in traditional Chinese medicine pericarpium citri reticulatae
WO2015186021A1 (en) 2014-06-04 2015-12-10 Dolomiti Fruits S.R.L. Food based food, process for the production and use of the food
CN106565655A (en) * 2016-11-11 2017-04-19 中国科学院兰州化学物理研究所 Method of enriching and extracting flavone compounds in aqueous solution by means of zinc sulfide
CN107857786A (en) * 2017-12-14 2018-03-30 南京中医药大学 A kind of succinyl aurantiin and its application in terms of preparing antibacterial or treating medicine for treating osteoporosis
WO2022107093A1 (en) * 2020-11-20 2022-05-27 Esserre Pharma Srl Composition comprising natural extracts and uses thereof

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EP2033650A4 (en) * 2006-06-22 2009-10-21 Arkray Inc Inhibitor of expression of nuclear transcription factor ap-1, and pharmaceutical product and product using the inhibitor

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