WO2014202803A1 - Procédé d'obtention d'extraits de feuilles de corema album et leur application thérapeutique - Google Patents

Procédé d'obtention d'extraits de feuilles de corema album et leur application thérapeutique Download PDF

Info

Publication number
WO2014202803A1
WO2014202803A1 PCT/ES2014/000099 ES2014000099W WO2014202803A1 WO 2014202803 A1 WO2014202803 A1 WO 2014202803A1 ES 2014000099 W ES2014000099 W ES 2014000099W WO 2014202803 A1 WO2014202803 A1 WO 2014202803A1
Authority
WO
WIPO (PCT)
Prior art keywords
corema
extract
album
leaves
obtaining
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/ES2014/000099
Other languages
English (en)
Spanish (es)
Inventor
Carmen MARTÍN CORDERO
Antonio José LEÓN GONZÁLEZ
María Cruz DIAZ BARRADAS
Inmaculada NAVARRO ZAFRA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universidad de Sevilla
Original Assignee
Universidad de Sevilla
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universidad de Sevilla filed Critical Universidad de Sevilla
Publication of WO2014202803A1 publication Critical patent/WO2014202803A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • 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)
    • A61K36/45—Ericaceae or Vacciniaceae (Heath or Blueberry family), e.g. blueberry, cranberry or bilberry
    • 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
    • A61K31/12—Ketones
    • 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
    • A61K31/12—Ketones
    • A61K31/121—Ketones acyclic
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention aims at an extract of ethyl acetate from the leaves of Corema album (L.) D. Don (Ericaceae), with cytotoxic activity, as well as the isolated active ingredients thereof, which generate reactive oxygen species and induce apoptosis.
  • the present invention belongs to the Pharmaceutical scientific area, since it establishes the methods of obtaining and phytochemical analysis of extracts of the Corema album sheets, as well as isolated active fractions and principles and their cytotoxic activity.
  • the pharmaceutical industry of natural products and the agri-food industry are potential users of the present invention, which may be applied for the development of drugs, nutraceuticals or functional foods in the treatment and prevention of cancer.
  • Chalconas constitute an important group of phenolic compounds that present a great variety of biological activities, such as antioxidant, anti-inflammatory, antibacterial, cytotoxic ... They are naturally in our diet and in different medicinal plants, for example, isoliquiritigenina is abundant in the licorice, obtained from the root of Glycyrrhiza glabra. In recent years, the ability of chalconas and their derivatives to inhibit different steps of the development of carcinogenesis, and their involvement in the mechanisms of apoptosis of transformed cells and the regulation of the cell cycle, have been revealed. as a family of molecules with great potential in chemoprevention and cancer treatment.
  • chalconas can prevent the stage of tumor progression by activating apoptosis, both by extrinsic and intrinsic route, with the Bcl-2 antiapoptotic protein being one of the most frequent targets (Yadav et al., 2011 Int Immunopharmacol, 11 (3), 295-309).
  • Corema album is a species belonging to the family Ericaceae, which also includes species of great agroindustrial interest, such as blueberries (Vaccinium sp.). It grows in the sand and sub-coastal dunes of the Atlantic coast of the Iberian Peninsula. In Spain it appears on the Galician coast of La Coru ⁇ a and Pontevedra and in Andalusia at different points along the coast of Huelva and Cádiz. It is a dioecious shrub, up to 1 m tall, very branched. The lower branches are glabrous and with numerous leaf scars; those of the last years, densely tomentose, with little wavy hairs and grayish color. The leaves are xeromorphic, adapted to the dry climate and measure around 5-6 x 1 -2 mm. There are no previous published data about the chemical composition or pharmacological activity of the leaves of this species.
  • the present invention relates to obtaining and using any extract, fraction or active ingredient isolated from the leaves of Corema album (L.) D. Don, Ericaceae, in the prevention and treatment of neoplastic diseases; preferably the ethyl acetate extract that exhibits marked cytotoxic activity in the HT-29 cell line from colon adenocarcinoma versus the standard 5-fluouracil (Cl 50 : 5.8 ⁇ 2.6 and 16.2 ⁇ 2.6 ⁇ 9 / ⁇ _, respectively).
  • the cytotoxic activity shown by the extract in the HT-29 cell line from colon adenocarcinoma is maintained in the fractions and the isolated compounds, 2 ', 4'-dihydroxydihydrochalcone (Cl 50 : 1.8 ⁇ 0.4 ⁇ ), 2'- Methox ⁇ -4'-hydroxyhydrochalcone (IC 5 or: 8.5 ⁇ 2.1 ⁇ ) and fraction of pinocembrina and 2 ', 4'-dihydrochalcone (Cl 50 : 6.8 ⁇ 1.2 ⁇ Q / r ⁇ ⁇ L) versus the 5-fluorouracil standard ( Cl 50 : 8.7 ⁇ 4.0 ⁇ , 16.2 ⁇ 2.6 ⁇ g / mL). Therefore, the extract, fractions or compounds could be used in antitumor therapy.
  • an extract of the Corema album sheets is provided characterized in that the active principles 2 ', 4'-dihydroxydihydrochalcone, 2'-methoxy-4'-hydroxyhydrochalcone, pinocembrin, 2' are identified , 4'-dihydrochalcone, ursolic acid and oleanolic acid that exhibit cytotoxic activity and this activity is due to the fact that they generate apoptosis and a stop in the cell cycle in the G2 / M phase.
  • a second aspect of the present invention relates to obtaining the extract described above, obtained using an extraction procedure comprising the steps:
  • a third aspect of the present invention relates to the fractionation and analysis of said extract, characterized by chromatography of the silica column extract using n-hexane and ethyl acetate as eluent in different gradients. The fractions obtained were grouped according to their chromatographic behavior (See Table 1).
  • Table 1 Chromatographic fractionation of the ethyl acetate extract from the Corema album sheet.
  • CA1 2 ', 4'-dihydroxydihydrochalcone
  • CA2 2'-methox ⁇ -4'-hydroxyhydrochalcone
  • CA3 pinocembrina
  • CA4 2 ', 4'-dihydrochalcone.
  • composition of the fractions with the highest content in phenolic compounds is determined using different analytical techniques, including spectrometry of masses and nuclear magnetic resonance.
  • the extract of the leaves of Corema album, its fractions or active principles isolated from it may be administered in the form of a medicinal preparation characterized by cytotoxic activity.
  • the formulation of the medicament preparations may be done following any of the conventional procedures, and may be presented in the form of powder, paper, granulate, capsule, seal, tablet, tablet, tablet, solution, syrup, suspension, tincture or any other pharmaceutical form.
  • Pharmaceutical preparations containing leaf extracts of Corema album may be formulated with different vehicles, excipients and diluents in order to optimize the administration thereof by the desired route.
  • Example 1 Obtaining the extract, fractionation and chemical analysis.
  • the sheets of Corema album (L.) D. Don were collected in September in the province of Huelva. After drying them at room temperature, 500 g of leaves were subjected to an ultrasonic bath extraction process. 1000 mL of ethyl acetate was added to the dried leaves without crushing and extracted for 45 minutes at a temperature below 40 ° C in an ultrasound bath. Subsequently, the resulting extract was filtered and concentrated to dryness under reduced pressure on a rotary evaporator.
  • This extract was chromatographed on a silica column using n-hexane and ethyl acetate as eluent in different gradients.
  • the 419 fractions of 15 mL obtained were grouped according to their chromatographic behavior (Figure 1), being revealed in UV light and with oleic and AICI 3 reagents.
  • the cytotoxic activity of the ethyl acetate extract of the Corema album leaves and the isolated and identified compounds was evaluated by the technique of Sulforhodamine B (SRB), presenting a cytotoxic profile similar to that of the 5-fluorouracil standard.
  • SRB Sulforhodamine B
  • the protocols established by the NCI were followed (Monks et al., J. Nati. Cancer Inst. 1991, 83, 757).
  • the HT-29 colon adenocarcinoma cell line was cultured in RPMI 1640 medium (Bio Whittaker) containing 20% fetal bovine serum, 2 mM L-glutamine, 100 U / ml penicillin and 100 ⁇ g / ml streptomycin.
  • the cytotoxicity test was performed using 96-well plates. All cells were maintained at 37 ° C in an atmosphere of 5% C0 2 with 95% humidity. The cells were subcultured weekly, and the culture medium was renewed twice a week.
  • the compounds were initially dissolved in dimethylsulfoxide (DMSO), and 5 serial dilutions were logarithmically prepared in RPMI medium (RosweII Park Memorial Institute medium). The final concentration of DMSO was never greater than 0.6%, to avoid cytotoxicity due to this solvent. The maximum dose tested was 10 "4 M for pure compounds.
  • the compounds were not filtered or sterilized since microbial contamination was controlled by adding gentamicin (50 ⁇ g / mL) to the medium. Then, it was added to each well of the plates incubated above, the different solutions of the extracts or pure compounds (100 ⁇ ) were subsequently incubated for 48 h at 37 ° C in a 5% C0 2 atmosphere.
  • SRB Sulforhodamine B
  • Tumor cells were fixed by the addition of 50 ⁇ of 50% cold trichloroacetic acid, incubating at 4 ° C for one hour. The plates were then washed 5 times with deionized water and allowed to dry. Subsequently, 100 ⁇ of SRB solution (0.4% w / v in 1% acetic acid) was added to each well, incubating 30 minutes at room temperature. The plates were then washed 5 times with 1% glacial acetic acid and allowed to dry. Finally the cells were solubilized in 100 ⁇ of 10 mM Tris buffer, stirred 5-10 minutes and the absorbance at 492 nm was measured in an automatic plate spectrophotometer. Parallel to this test, a control plate was performed, in which this SRB technique was performed after the first incubation.
  • Table 2 Results of the cytotoxicity test ⁇ g / ml) performed with the extract, fraction and isolated compounds of Corema album and the standard drug 5-fluorouracil.
  • the ability of the fraction composed of 2 ', 4'-dihydroxydihydrochalcone and pinocembrin to induce apoptosis was evaluated by an annexin-V kit linked to fluorescein isothiocyanate (Annexin V-FITC). To do this, we planted 50,000 cells per well in 6-well plates with a final volume of 2 mL, and incubated overnight at 37 ° C and 5% C0 2 . The next day we treated with 0, 6 and 12 pg / mL of fraction, and incubated under the same conditions for 48 hours.
  • the analysis of the cell cycle was performed by fluorescence mapping of the nuclei of the cells in suspension with propidium iodide (IP), and the subsequent analysis of the nuclear DNA content of each cell in the population, by flow cytometry. (N ⁇ ez, 2001, Curr issues Mol Biol. 3 (3), 67-70).
  • IP propidium iodide

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Epidemiology (AREA)
  • Botany (AREA)
  • Engineering & Computer Science (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Biotechnology (AREA)
  • Medical Informatics (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicines Containing Plant Substances (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne un extrait cytotoxique provenant des feuilles deCorema album (Ericaceae), ainsi que ses fractions et principes actifs isolés, qui génèrent des espèces réactives de l'oxygène (ERO) et induisent l'apoptose. L'invention concerne aussi les compositions pharmacologiques qui contiennent aussi bien l'extrait que les fractions ou principes actifs isolés de celui-ci, et l'utilisation desdites compositions dans la préparation de médicaments pour la prévention et le traitement du cancer. La présente invention trouve son application dans le secteur pharmaceutique.
PCT/ES2014/000099 2013-06-19 2014-06-17 Procédé d'obtention d'extraits de feuilles de corema album et leur application thérapeutique Ceased WO2014202803A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ES201300600A ES2540457B2 (es) 2013-06-19 2013-06-19 Procedimiento de obtención de extractos de hojas de Corema album y su aplicación terapéutica
ESP201300600 2013-06-19

Publications (1)

Publication Number Publication Date
WO2014202803A1 true WO2014202803A1 (fr) 2014-12-24

Family

ID=52103989

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/ES2014/000099 Ceased WO2014202803A1 (fr) 2013-06-19 2014-06-17 Procédé d'obtention d'extraits de feuilles de corema album et leur application thérapeutique

Country Status (2)

Country Link
ES (1) ES2540457B2 (fr)
WO (1) WO2014202803A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105796548A (zh) * 2016-04-19 2016-07-27 中国科学院西北高原生物研究所 一种黄酮类药物组合物及制备方法和用途

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2181806T3 (es) * 1994-12-20 2003-03-01 Indena Spa Chalconas y sus eteres con actividad antiproliferativa en los tumores de utero, ovario y mama.
ES2244075T3 (es) * 1997-06-19 2005-12-01 Indena S.P.A. Chalconas que tienen actividad antiproliferativa.

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2181806T3 (es) * 1994-12-20 2003-03-01 Indena Spa Chalconas y sus eteres con actividad antiproliferativa en los tumores de utero, ovario y mama.
ES2244075T3 (es) * 1997-06-19 2005-12-01 Indena S.P.A. Chalconas que tienen actividad antiproliferativa.

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
FOREJTNIKOVA, H. ET AL.: "Chemoprotective and toxic potentials of synthetic and natural chalcones and dihydrochalcones in vitro.", TOXICOLOGY, vol. 208, no. 1, 2005, pages 81 - 93, XP004722020, DOI: doi:10.1016/j.tox.2004.11.011 *
LEÓN-GONZÁLEZ, A. J. ET AL.: "Chemo-protective activity and characterization of phenolic extracts from Corema album.", FOOD RESEARCH INTERNATIONAL, vol. 49, no. 2, 2012, pages 728 - 738 *
LEÓN-GONZÁLEZ, A. J. ET AL.: "Phenolic acids, flavonol and anthocyanins in Corema album (L.) D. Don berries.", JOURNAL OF FOOD COMPOSITION AND ANALYSIS, vol. 29, no. 1, February 2013 (2013-02-01), pages 58 - 63 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105796548A (zh) * 2016-04-19 2016-07-27 中国科学院西北高原生物研究所 一种黄酮类药物组合物及制备方法和用途

Also Published As

Publication number Publication date
ES2540457B2 (es) 2015-12-28
ES2540457A1 (es) 2015-07-09

Similar Documents

Publication Publication Date Title
Georgieva et al. Antiproliferative and antitumour activity of saponins from Astragalus glycyphyllos on myeloid Graffi tumour
Kupeli et al. Anti-inflammatory and antinociceptive potential of Maclura pomifera (Rafin.) Schneider fruit extracts and its major isoflavonoids, scandenone and auriculasin
US20170360744A1 (en) Agent containing flavonoid derivatives for treating cancer and inflammation
BRPI0607773B1 (pt) Método para produção de um oligômero de proantocianidina
Zakłos-Szyda et al. The influence of Viburnum opulus polyphenolic compounds on metabolic activity and migration of HeLa and MCF cells
Kaur et al. Isolation of embelin from and evaluation of its anti-cancer potential in Embelia ribes breast cancer
Joseph et al. Identification of anticancer compounds from Linum usitatissimum seed extract and their effect on HeLa cells.
JP6837960B2 (ja) 化学療法に耐性である腫瘍の処置において用いるための、メラトニンおよびフラボノイド類を含む組成物
Lee et al. Benzyl salicylate from the stems and stem barks of Cornus walteri as a nephroprotective agent against cisplatin-induced apoptotic cell death in LLC-PK1 cells
Lalrinzuali et al. Sonapatha (Oroxylum indicum) mediates cytotoxicity in cultured HeLa cells by inducing apoptosis and suppressing NF-κB, COX-2, RASSF7 and NRF2
Alfawal et al. Anticancer Activity of Beetroot (Beta vulgaris L.) Extracts (Human Colon Carcinoma Cell Line)
WO2014202803A1 (fr) Procédé d'obtention d'extraits de feuilles de corema album et leur application thérapeutique
Liu et al. Corilagin induces laryngeal cancer antiproliferation and inhibits growth factor and cytokine signaling pathways in vitro and in vivo
Tanavade et al. In vitro anticancer activity of Ethanolic and Aqueous Extracts of Peristrophe bivalvis Merrill.
ES2906476B2 (es) Extracto etanólico de semillas de Citrullus colocynthis, método para obtenerlo, composición farmacéutica que lo contiene y su uso como agente antitumoral
KR101021975B1 (ko) 암에 대한 방사선 치료 증진용 조성물
He et al. Succinyl rotundic acid inhibits growth and promotes apoptosis in the HeLa cervical cancer cell line
US10188689B2 (en) Method and composition for treating breast cancer
Rahmawati et al. Antioxidant and anticancer activity of Dillenia serrata Thunb ethanol extract against MCF-7 breast cancer cell line
ES2906473B2 (es) Extracto etanólico de semillas de Solanum melongena, método para obtenerlo, composición farmacéutica que lo contiene y su uso como agente antitumoral
Rajkovic¹ et al. An updated pharmacological insight into calotropin as
WINITCHAIKUL et al. Apoptosis induction on colon cancer cells from the Calotropis gigantea stem bark extract
KR102117560B1 (ko) 켈락톤 및 이의 약학적으로 허용가능한 염을 유효성분으로 함유하는 암 예방 및 치료용 조성물
Hamedani et al. The Effect of Glycosylation on Biodistribution and Cytotoxicity of 99mTc-radiolabeled Luteolin
RU2641056C2 (ru) Средство для местного применения в комплексной терапии заболеваний полости рта

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14813810

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 14813810

Country of ref document: EP

Kind code of ref document: A1