The Use of Winter Savory ( Satureja Montana )

(19) &

(11) EP 1 793 840 B1

(12) EUROPEAN PATENT SPECIFICATION

(45) Date of publication and mention (51) Int Cl.: of the grant of the patent: A61K 36/53 (2006.01) A61P 15/10 (2006.01) 13.01.2010 Bulletin 2010/02 (86) International application number: (21) Application number: 05788677.2 PCT/EP2005/010481

(22) Date of filing: 28.09.2005 (87) International publication number: WO 2006/037535 (13.04.2006 Gazette 2006/15)

(54) THE USE OF WINTER SAVORY ( SATUREJA MONTANA ) OR THE EXTRACTS THEREOF FOR THE PREPARATION OF MEDICAMENTS FOR THE TREATMENT OF THE PREMATURE EJACULATION VERWENDUNG VON WINTERBOHNENKRAUT (SATUREJA MONTANA) ODER SEINEN EXTRAKTEN ZUR HERSTELLUNG VON MEDIKAMENTEN ZUR BEHANDLUNG VON VORZEITIGER EJAKULATION UTILISATION DE SARRIETTE DES MONTAGNES ( SATUREIRA MONTANA ) OU D’EXTRAITS CORRESPONDANTS POUR PREPARER DES MEDICAMENTS SERVANT A TRAITER L’EJACULATION PRECOCE

(84) Designated Contracting States: • LENA SANTOS: "Satureja montana" [Online] AT BE BG CH CY CZ DE DK EE ES FI FR GB GR 2004, , XP002376438 Retrieved from the Internet: HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI URL:http://home.swipnet.se/~w- SK TR 47128/efile6 .htm> [retrieved on 2006-04-07] the Designated Extension States: whole document AL BA HR MK YU • MICHAEL WEISHAN: "world of gardening" [Online] 1998, , XP002376439 Retrieved from the (30) Priority: 01.10.2004 IT MI20041871 Internet: URL:www.michaleweishan.com/ tradgdnspr98art 3.html> [retrieved on (43) Date of publication of application: 2006-04-07] see esp. paragraph 3 the whole 13.06.2007 Bulletin 2007/24 document • ANGELINI L G ET AL: "Essential Oils from (73) Proprietor: Baraldi, Mario Mediterranean Lamiaceae as Weed Germination 41100 Modena (IT) Inhibitors" JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, AMERICAN CHEMICAL (72) Inventor: Baraldi, Mario SOCIETY. WASHINGTON, US, vol. 51, 9 41100 Modena (IT) September 2003 (2003-09-09), pages 6158-6194, XP002323188 ISSN: 0021-8561 (74) Representative: Minoja, Fabrizio • SEFIDKON F ET AL: "Chemical composition of Bianchetti Bracco Minoja S.r.l. the essential oil of three Iranian Satureja species Via Plinio 63 (S. mutica, S. macrantha and S. intermedia)" 20129 Milano (IT) FOOD CHEMISTRY, ELSEVIER SCIENCE PUBLISHERS LTD, GB, vol. 91, no. 1, June 2005 (56) References cited: (2005-06), pages 1-4, XP004654090 ISSN: • PEPELJNJAK S ET AL: "ANTIMICROBIAL 0308-8146 ACTIVITY OF THE ETHANOLIC EXTRACT OF SATUREJA MONTANA SP. MONTANA" ACTA PHARMACEUTICA, ZAGREB, HR, vol. 49, no. 1, March 1999 (1999-03), pages 65-69, XP009003525 ISSN: 1330-0075

Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 1 793 840 B1

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Description

[0001] The present invention relates to the use of Winter savory or purified extracts thereof rich in rosmarinic acid, and the use of rosmarinic acid or extracts containing it for the preparation of medicaments for the treatment of premature 5 ejaculation.

TECHNOLOGICAL BACKGROUND

[0002] Premature ejaculation, which is ejaculation occurring before the individual wishes, is a dysfunction affecting 10 more than 30% of male population, mainly adolescent, but that may also persist in the adult. Three main causes are apparently involved:

1) organic factors, such as anatomical or physiological alterations of the reproductive system; 2) psychological factors, deriving from wrong sexual education or deep-seated problems related to improper sexual 15 development during growth; 3) external factors, such as use of drugs (amphetamines, hallucinogens), excessive consumption of alcoholics, food and medications.

[0003] Premature ejaculation can ultimately be ascribed to alterations of the neuroconduction and neuromodulation 20 processes which modulate the sexual behaviour both at the central and peripheral levels. Therefore, pharmacological treatment usually consists in the administration of benzodiazepines (alprazolam 0.25 mg), antidepressants acting on the serotoninergic system (paroxetine 20 mg), or in the local application of anaesthetics (prilocaine 25 mg + lidocaine 25 mg). [0004] As this type of therapy does not always prove effective and also involves side effects such as sedation or cardio 25 circulatory problems, there is the need for novel active principles. [0005] Pepeljnjak et al., Acta Pharm. 49 (1999), 65-69, discloses an extract of Satureja montana leaves with 70% ethanol. The antimicrobial activity of said extract has been tested against a variety of bacteria and fungi. [0006] It has now been found that Winter savory (Satureja montana) or the extracts thereof, as well as rosmarinic acid or the extracts containing it, can be used for the preparation of medicaments for the treatment of premature ejac- 30 ulation.

DETAILED DISCLOSURE OF THE INVENTION

[0007] Winter savory (Satureja montana) is a plant belonging to the Lamiaceae family, already known in traditional 35 medicine as a remedy against asthenia or as aphrodisiac. [0008] The object of the invention is the use of Winter savory or the extracts thereof, and of rosmarinic acid or extracts containing rosmarinic acid, for the preparation of medicaments for the treatment of premature ejaculation. [0009] The invention further relates to a Winter savory total extract useful for the preparation of medicaments for the treatment of premature ejaculation. 40 [0010] A further object of the invention is the use of rosmarinic acid or extracts containing it for the preparation of medicaments for treatment of premature ejaculation. [0011] According to the invention, the process for the preparation of the Winter savory purified extract comprises the following steps:

45 a) milling Winter savory dried aerial parts; b) extracting them one or more times with water and recovering the extraction solvent; c) filtering the extraction solvent or the combined extraction solvents and subsequent hot concentration under reduced pressure to obtain a concentrated solution; d) eluting the concentrated solution from step c) on a resin column; 50 e) washing the resin with water; f) eluting with ethanol; g) hot concentrating the organic phase under reduced pressure; h) drying the resulting syrup under vacuum at 60°C for 24 hours; i) milling the resulting solid. 55 [0012] Alternatively to steps d)-f), the solution from step c) is extracted once or repeatedly with butanol; the organic phase (or combined organic phases) is then subjected to steps g), h) and i). [0013] The resulting extract was solubilised in Tween 80 (10%) and water and administered to test animals through

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a gastric probe, in a volume of 5 ml/kg, at different doses, 45 minutes before the in vivo test. [0014] After acute administration per os (25, 50 and 100 mg/kg), a statistically significant increase in ejaculation latency has been observed. [0015] HPLC analysis of Winter savory purified extract evidenced the presence of rosmarinic acid that, when tested 5 under the same experimental conditions at doses of 10 and 20 mg/kg, showed equivalent activity to that of the extract. [0016] Qualitatively similar, although quantitatively different, results were obtained replacing the purified extract with the total aqueous extract of Example I and with the alcoholic extract of Example IV. [0017] For the treatment in humans, the compounds of the invention can be incorporated in pharmaceutical formulations suitable for the oral, intramuscular, transdermal administrations, with conventional excipients and methods. Doses can 10 range from 100 mg to 2000 mg daily, preferably from 200 to 400 mg daily. [0018] The invention is described in more detail by the following examples.

EXAMPLE I: PREPARATION OF TOTAL AQUEOUS EXTRACT

15 [0019] One kg of finely ground Satureja montana dried leaves is extracted by infusion at 70°C with 4 volumes of water for 4 hours. The procedure is repeated for 5 times. The hot extracts are filtered, combined and concentrated under vacuum to give a soft extract, which is subsequently dried under vacuum at 60°C to give 290 g of dry extract.

EXAMPLE II: PURIFICATION OF TOTAL AQUEOUS EXTRACT WITH BUTANOL 20 [0020] 100 g of the extract of Example I is redissolved in 10 volumes of water. The solution is extracted at room temperature with 3x0.5 litres of water-saturated n-butanol. Combined butanolic extracts are concentrated to a soft extract. The butanol residue is replaced with water. The extract is dried under vacuum at 60°C to give 11 g of a dry extract with 9.74% content in rosmarinic acid. 25 EXAMPLE III: PURIFICATION OF TOTAL AQUEOUS EXTRACT WITH ADSORBING RESINS

[0021] 50 g of the extract of Example I is redissolved in 10 volumes of water, absorbed on a column containing 1 litre of duolite XAD761, and washed with 5000 ml of water. Washings are discarded and the column is eluted with 3000 ml 30 of 95% ethanol. The hot ethanol fraction is concentrated under reduced pressure to obtain a soft extract which is subsequently dried under vacuum at 60°C for 24 hours. 5 g of purified extract is obtained, having 10.48% content in rosmarinic acid.

EXAMPLE IV: PREPARATION OF WINTER SAVORY ALCOHOLIC EXTRACT 35 [0022] Dried aerial parts of Winter savory (100 g) are macerated in 500 ml of 90% (v/v) ethanol for 24 hours; the suspension is then filtered and evaporated to dryness.

EXAMPLE V: COPULATORY ACTIVITY 40 [0023] Sprague-Dawley rats were used, both males (weighing approx. 220 g) and females (weighing approx. 160 g), from Harlan Italy (Udine, Italy). The animals were housed under controlled temperature and humidity conditions (221°C, 60% humidity), with 12 hours inverted light-darkness cycle, with lights on at 7 a.m. Females were ovariectomized and subcutaneously injected with estradiol valerate (500 Pg) and progesterone (2 mg) 48 hours after, to induce estrus. Male 45 rats with very short ejaculation latency time were chosen through starting screening consisting of 7 pre-tests [A. Ågmo, Male rat sexual behavior, Brain Research Protocols 1: 203-209, 1997]. [0024] The resulting extract according to Example II or rosmarinic acid were solubilised in Tween 80 (10%) and water and administered to animals, by gastric probe, in a volume of 5 ml/kg, at different doses, 45 minutes before carrying out all the in vivo tests. 50 [0025] Sexual behaviour was evaluated under quiet conditions, with feeble red light, according to the standard procedure [A. Ågmo, Male rat sexual behavior, Brain Research Protocols 1: 203-209, 1997]. The main parameters recorded during the test were:

1) mount latency (ML); 55 2) intromission latency (IL); 3) ejaculation latency (EL); 4) postejaculatory interval (PEI); 5) mount frequency (MF);

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6) intromission frequency (IF).

RESULTS

5 [0026] Table 1 shows that all of the doses of Winter savory extract administered (25, 50, and 100 mg/kg) induced slowing down of the copulatory activity, and a statistically significant prolonging of ejaculation latency. The same results were obtained with rosmarinic acid at doses of 10 and 20 mg/kg (Table 2).

Table 1: Effect of the administration of Winter savory extract on copulatory activity 10 Treatment ML MF IL IF EL PEI (mg/kg) (sec) (n°) (sec) (n°) (sec) (sec) Control 155.041.7 2.50.5 155.141.7 21.03.2 469.280.2 392.220.4 Winter savory 499.7132.8* 1.20.2** 499.8132.7* 23.83.6 1420.0215.9* 686.7101.7* 15 25 Control 247.542.1 3.10.6 283.148.2 27.83.8 496.0107.4 508.667.2 Winter savory 451.1130.0 1.20.2* 451.1130.0 29.64.7 1346.0161.7* 562.0127.0 50

20 Control 37.713.2 8.90.5 73.626.2 12.70.7 470.410.9 301.96.6 Winter savory 160.488.8 6.21.1 189.286.4 9.50.8* 1552.2208.9 458.274.2*** 100 Values are expressed as meanSEM (n= 8-10). Mann-Whitney test: *p<0.05, **p<0.01, ***p<0.001 vs. the respective controls. 25

Table 2: Effect of the administration of rosmarinic acid on copulatory activity Teatment ML MF IL IF EL PEI 30 (mg/kg) (sec) (n°) (sec) (n°) (sec) (sec) Control 135.233.4 2.40.3 147.137.5 22.33.9 483.360.6 383.118.3 Rosmarinic acid 10 382.493.6* 1.60.3 393.989.8* 19.81.2 1282.290.4* 548.545.5* mg/kg 35 Control 184.345.3 2.90.3 199.737.3 24.12.3 470.971.1 444.46.6 Rosmarinic acid 20 425.883.2* 1.10.1* 408.448.6* 27.30.7 1493.181.9** 602.2102.4* mg/kg Values are expressed as meanSEM (n = 8-10). Mann- Whitney test: *p<0.05, **p<0.01, vs. respective controls. 40

CONCLUSIONS

[0027] The results reported above prove that the Winter savory purified extract acts on copulatory activity, significantly prolonging the ejaculation time. The effect is dose-dependent after oral administration and appears after 30-45 minutes. 45

Claims

1. The use of Winter savory or the extracts thereof, and of rosmarinic acid or extracts containing rosmarinic acid, for 50 the preparation of medicaments for the treatment of premature ejaculation.

2. The use as claimed in claim 1 of Winter savory or extracts thereof.

3. The use as claimed in claim 1 of rosmarinic acid. 55

4. The use as claimed in claim 1 of extracts containing rosmarinic acid.

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5. A process for the preparation of a Winter savory extract which comprises the following steps:

a) milling Winter savory dried aerial parts; b) extracting them one or more times with water and recovering the extraction solvent; 5 c) filtering the extraction solvent or the combined extraction solvents and subsequent hot concentration under reduced pressure to obtain a concentrated solution d) eluting the concentrated solution from step c) on a resin column; e) washing the resin with water; f) eluting with ethanol; 10 g) hot concentrating the organic phase under reduced pressure; h) drying the resulting syrup under vacuum at 60°C for 24 hours; i) milling the resulting solid.

6. A process as claimed in claim 5 in which, alternatively to steps d)-f), the solution from step c) is extracted once or 15 repeatedly with butanol and the organic phase (or the combined organic phases) is then subjected to steps g), h) and i).

Patentansprüche

20 1. Die Verwendung von Winterbohnenkraut oder der Extrakte daraus und von Rosmarinsäure oder Rosmarinsäure enthaltenden Extrakten für die Herstellung von Arzneimitteln zur Behandlung von vorzeitigem Samenerguss,

2. Die Verwendung nach Anspruch 1 von Winterbohnenkraut oder Extrakten daraus,

25 3. Die Verwendung nach Anspruch 1 von Rosmarinsäure,

4. Die Verwendung nach Anspruch 1 von Rosmarinsäure enthaltenden Extrakten,

5. Ein Verfahren zur Herstellung eines Winterbohnenkrautextraktes, das die folgenden Schritte umfasst: 30 a) Zermahlen von getrockneten oberirdischen Teilen des Winterbohnenkrauts; b) ein- oder mehrmaliges Extrahieren dieser mit Wasser und Wiedergewinnen der Extraktionslösung; c) Filtrieren der Extraktionslösung oder der kombinierten Extraktionslösungen und anschließendes Wärmekon- zentrieren unter reduziertem Druck, um eine konzentrierte Lösung zu erhalten; 35 d) Eluieren der konzentrierten Lösung aus Schritt c) auf einer Harzsäule; e) Waschen des Harzes mit Wasser; f) Eluieren mit Ethanol; g) Wärmekonzentrieren der organischen Phase unter reduziertem Druck; h) Trocknen des resultierenden Sirups unter Vakuum bei 60 °C für 24 Stunden; 40 i) Zermahlen des resultierenden Feststoffs,

6. Ein Verfahren nach Anspruch 5, bei dem alternativ zu den Schritten d)-f) die lösung aus Schritt c) einmal oder wiederholt mit Butanol extrahiert wird, und die organische Phase (oder die kombinierten organischen Phasen) dann den Schritten g), h) und i) unterworfen wird, 45

Revendications

1. Utilisation de sarriette des montagnes ou des extraits de celle-ci, et d’acide rosmarinique ou d’extraits contenant 50 de l’acide rosmarinique, pour la préparation de médicaments pour le traitement de l’éjaculation précoce.

2. Utilisation selon la revendication 1 de sarriette des montagnes ou d’extraits de celle-ci.

3. Utilisation selon la revendication 1 d’acide rosmarinique. 55 4. Utilisation selon la revendication 1 d’extraits contenant de l’acide rosmarinique.

5. Procédé de préparation d’un extrait de sarriette des montagnes qui comprend les étapes suivantes :

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a) le broyage des parties aériennes séchées de sarriette des montagnes ; b) l’extraction de celles-ci une ou plusieurs fois avec de l’eau et la récupération du solvant d’extraction ; c) la filtration du solvant d’extraction ou des solvants d’extraction combinés et ensuite la concentration à chaud sous pression réduite pour obtenir une solution concentrée ; 5 d) l’élution de la solution concentrée de l’étape c) sur une colonne de résine ; e) le lavage de la résine avec de l’eau ; f) l’élution avec de l’éthanol ; g) la concentration à chaud de la phase organique sous pression réduite ; h) le séchage du sirop résultant sous vide à 60°C pendant 24 heures ; et 10 i) le broyage du solide résultant.

6. Procédé selon la revendication 5 dans lequel, comme alternative aux étapes d) à f), la solution de l’étape c) est extraite une fois ou de façon répétée avec du butanol et la phase organique (ou les phases organiques combinées) est (sont) ensuite soumises aux étapes g), h) et i). 15

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REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Non-patent literature cited in the description

• Pepeljnjak et al. Acta Pharm., 1999, vol. 49, 65-69 •A. Ågmo. Male rat sexual behavior. Brain Research [0005] Protocols, 1997, vol. 1, 203-209 [0023] [0025]