(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2018/189672 Al 18 October 2018 (18.10.2018) W !P O PCT
(51) International Patent Classification: Published: A61K 36/752 (2006.01) A61P 3/10 (2006.01) — with international search report (Art. 21(3)) A61K 36/26 (2006.01) A61P 9/12 (2006.01) — before the expiration of the time limit for amending the A61P 3/06 (2006.01) claims and to be republished in the event of receipt of (21) International Application Number: amendments (Rule 48.2(h)) PCT/IB20 18/052498 (22) International Filing Date: 10 April 2018 (10.04.2018) (25) Filing Language: Italian (26) Publication Langi English (30) Priority Data: 102017000040866 12 April 2017 (12.04.2017) IT (71) Applicant: HERBAL E ANTIOXIDANT DERI¬ VATIVES S.R.L. ED IN FORMA ABBREVIATA H&AD S.R.L. [IT/IT]; Zona Industriale Localita Chiusi, Strada Provinciale SNC, 89032 Bianco (RC) (IT). (72) Inventors: BOMBARDELLI, Ezio; Via Gabetta, 13, 27027 Gropello Cairoli (PV) (IT). MOLLACE, Vincenzo; c/o Herbal E Antioxidant Derivatives S.r.l. Ed In Forma Ab- breviata H&ad S.r.l., Zona Industriale Localita Chiusi, Stra da Provinciale SNC, 89032 Bianco (RC) (IT). (74) Agent: MINOJA, Fabrizio; Via Plinio, 63, 20129 MILANO (IT). (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG).
(54) Title: EXTRACTS OF CYNARA CARDUNCULUS AND CITRUS AURANTIUM BERGAMIA, COMBINATIONS THERE OF, AND FORMULATIONS CONTAINING THEM (57) Abstract: Disclosed is a combination of extracts of Cynara cardunculus and Citrus bergamia which are useful for the prevention and treatment of hepatic steatosis and other disorders correlated with dyslipidaemic conditions. EXTRACTS OF CYNARA CARDUNCULUS AND CITRUS AURANTIUM
BERGAMIA. COMBINATIONS THEREOF, AND FORMULATIONS
CONTAINING THEM
T e present invention relates to a combination of extracts of Cynara cardunculus and Citrus bergamia which are useful for the prevention and treatment of hepatic steatosis.
Prior art
The bergamot orange {Citrus bergamia Risso & Poiteau) is a citrus fruit substantially cultivated only in small areas of Calabria, Italy. Three main varieties thereof are known, called Femminello, Fantastico and Castagnaro.
It has long been known that the bergamot orange represents a niche product of particular prestige in the Calabrian citrus fruit industry. Said citrus fruit develops in a limited area of the Province of Reggio Calabria. To date, bergamot orange has been used solely for the properties of its essential oil, which is in particular demand in the perfume industry because it has an exclusive fragrance that is still used as a base to prepare many perfumes by the best-known perfume manufacturers, and for the manufacture of eau de cologne. Said oil is obtained by peeling from the outermost portion (cuticle) of bergamot orange peel.
The bergamot orange is also known to possess significant antiseptic properties, and a derivative of bergamot orange, called Bergamon, has been manufactured and used as a disinfectant in operating theatres for many years.
Moreover, the relaxing properties of bergamot orange extracts, and its important nutraceutical properties, are known from traditional medicine.
Finally, the water-alcohol solution of bergamot orange essence is used to prepare some products (Bergarytal®) manufactured in the form of sprays which have a skin decongestant action, and are particularly effective as a mosquito repellent.
However, the identification of the potentially toxic action of some ingredients of bergamot orange essence, such as bergaptene, has limited its use, since procedures for removing said compound are required to ensure safe use of the essential oil extracts.
Bergamot orange juice, which is rich in polyphenols and flavonoids (brutieridin and melitidin), is also known to possess cholesterol-lowering and blood glucose-lowering activities (Vincenzo Mollace et al, Hypolipemic and hypoglycaemic activity of bergamot polyphenols: From animal models to human studies, Fitoterapia, vol. 82, no. 3, 201 1, pp. 309-316).
US 8,741,362 describes a plant extract obtained from the pith and juice of fresh fruit of Citrus aurantium var. bergamia, and a process for its preparation. Said plant compound, in the form of a dried extract, contains the polyphenols neoeriocitrin, naringin and neohesperidin in the amounts of 29.6±6.0, 32.4±4.0 and 38.0±6% respectively of their total, the flavonoids eriocitrin, neoeriocitrin, melitidin and brutieridin and polymerisation compounds of said flavonoids, and is substantially devoid of furocoumarins and coumarins, allergenic compounds which can have adverse effects on coagulation and the blood count.
The plant compound described in US 8,741,362 normalises the lipid and blood glucose parameters in patients suffering from diet-dependent metabolic disorders or inherited genetic disorders. In pathological hyperlipaemia, a reduction in total cholesterol and LDL cholesterol to concentrations ranging from 20 to 32% has been demonstrated, with a 30% increase in HDL cholesterol. These important variations are associated with a marked reduction in VLDL cholesterol and a dimensional increase in LDLs, leading to a reduction in their oxidation. Bergamot orange extract has also improved the endothelial function, with vascular protection attributable to flavonoids. In double-blind clinical trials, the active dose reported is 650 mg at least twice a day.
The choleretic, cholagogic, hypoglycaemic, antidyspeptic and mildly hypocholesterolaemic action of artichoke (Cynara scolymus) extracts is also known.
However the cholesterol reduction reported in numerous clinical trials is modest, never exceeding 10%. The published clinical trials report modest, erratic increases in HDL cholesterol following administration of high doses of Cynara scolymus extracts, amounting to several grams a day (Naturmed, 13, 17-24,1998; Arzneim Forschung, 50,
260-65, 2000; The Cochrane Library, 2002, Issue 3). Standardised artichoke extracts containing 25% by weight of caffeoylquinic acids, 8% by weight of flavonoids and 7% by weight of cynaropicrin, which possess improved hypolipidaemic and hypoglycaemic activities, have been developed in recent years (Rondanelli et al., Phytotherapy Research
28:33-41 (2014) and Italian Journal of Medicine 2014; Vol 8, suppl 2, p. 113). Said extracts only exhibit a reasonable level of activity if administered twice a day, and in any event have little effect on major hyperlipidaemia.
The choleretic, hypoglycaemic and liver-protecting activity of artichoke extracts is attributed to caffeoylquinic acids, while flavonoids perform a hypolipidaemic action associated with cholesterol synthesis, and cynaropicrin exerts an anti-inflammatory and anti-
STAT 3 action. Cynara scolymus extracts deprived of cynaropicrin, such as those prepared from non-bitter flower heads, have no effect on cholesterol, only on blood glucose.
The cardoon (Cynara cardunculus L.) is a species of plant belonging to the
Asteraceae family. The variety sylvestris Lam. (wild cardoon) grows wild in profusion in the western Mediterranean basin.
WO 2008142460 discloses an anti-diabetic formulation comprising Cynara cardunculus, lemon balm, nettle, chicory and centaury extracts.
WO2014447533 discloses formulations for the treatment of metabolic syndrome comprising extracts of Cynara scolymus and Olea europea. The cynaropicrin content of
Cynara scolymus extract is 10-18% by weight.
WO 2016136441 discloses a plant compound consisting of Citrus bergamia Risso et Poiteau and Cynara scolymus L. extracts, optionally associated with phytosterols and ascorbic acid, which is useful for the treatment of dyslipidaemia. The active ingredients of artichoke (Cynara scolymus) extract include chlorogenic acids and luteolin, but not cynaropicrin, only low percentages of which are contained in artichoke extract.
Description of the invention
It has now been found that by simultaneously atomising aqueous solutions of the plant compound obtained according to US 8,741,362 and a Cynara cardunculus leaf extract obtained from fresh biomass subjected to thermal shock, a composite extract is obtained which exhibits not only a synergic increase in the properties of the single extracts, but also an unexpected preventive and therapeutic activity towards hepatic steatosis.
The step of atomisation of the mixture of the solutions leads to an unexpected increase in the solubility of the ingredients of the extracts.
It has also been found that Cynara cardunculus extract, and in particular Cynara cardunculus var. sylvestris extract, has a much higher cynaropicrin content than Cynara scolymus extracts. The high cynaropicrin content (up to 40% by weight of the Cynara cardunculus var. sylvestris extract, exceeding not only that of C. scolymus but also those of other varieties of cardunculus) gives rise to an increase in therapeutic efficacy against various disorders, as described in more detail below.
It has also surprisingly been found that Cynara cardunculus extract, and especially
Cynara cardunculus var. sylvestris extract, contains catechin polymers which enhance the beneficial effects of the extract.
A first aspect of the invention therefore relates to a composite extract obtained by atomising a mixture of a Cynara cardunculus extract preferably a Cynara cardunculus var. sylvestris extract, and an aqueous extract of Citrus bergami, wherein the Cynara cardunculus extract is obtained by extraction with water of fresh leaves previously subjected to thermal shock, wherein the Citrus bergami extract has at least a 25% content by weight of a polyphenol fraction consisting of neoeriocitrin, naringin and neohesperidin, and a furocoumarin content of less than 400 mg/Kg.
A second aspect of the invention relates to pharmaceutical or nutraceutical formulations comprising the composite extract as defined above, mixed with suitable carriers and excipients.
The invention also relates to said formulations for use in the treatment and prevention of hepatic steatosis and in the treatment of dyslipidaemia, hyperglycaemia, vascular inflammation, hypertension, metabolic syndrome, type 2 diabetes, chemotherapy-induced heart disease, and cardiovascular disease.
The formulations of the invention have also proved useful to stimulate the stem cells of the myocardium, for the treatment of liver cancer and, in general, for the treatment of tumours by inhibiting the STAT-3 pathway.
A further aspect of the invention is an extract of Cynara cardunculus var. sylvestris leaves having a cynaropicnn content ranging from 20 to 40% by weight, a cynaroside content ranging from 5 to 30% by weight, and a catechin polymer content ranging from 5 to 10% by weight.
Finally, the invention relates to a process for the preparation of the composite extract as defined above, which comprises atomisation of a mixture of a Cynara cardunculus extract obtained by extraction with water of the fresh leaves, previously subjected to thermal shock, and an aqueous extract of Citrus bergami.
Detailed description of the invention
The composite extract of the invention is obtained by atomisation or other equivalent techniques of aqueous solutions of:
a) a Cynara cardunculus extract having a cynaropicrin content ranging from 20 to
40%, and preferably amounting to 30% by weight;
b) a Citrus aurantium var. bergamia extract obtained by the steps described in the process according to US 8,741,362, except for the pre-drying step.
The polyphenol ingredients most representative of bergamot orange extract are neoeriocitrin, naringin and neohesperidin, in the following percentages of the total weight of the three compounds:
Neoeriocitrin 29.6% ± 6.0 Naringin 32.4% ± 4.0 Neohesperidin 38.0% ± 6.0 Total 100.0%
The Cynara cardunculus extract is obtained by collecting the leaf mass when it reaches a height of about 40 cm. The biomass is then coarsely chopped in a steam current to inhibit hydrolytic enzymes and oxidants, then pressed after thermal shock at pressures ranging from 100 to 300 bars, preferably 200 bars. After pressing and counterwashes with water in countercurrent, the aqueous extract is centrifuged and filtered, and the clear filtrate is mainly treated with SEPABEADS SP absorption resin or other polystyrene resins; the resin, which retains the polyphenol substances, is washed thoroughly with water to remove inert substances, then eluted with ethanol or an ethanol/water mixture.
The eluate is concentrated by evaporating the ethanol. The concentrate, after optional titration of the active ingredients, is added to the Citrus bergamia extract The mixture is then concentrated until dry in a vacuum.
The resulting extract is formulated in forms suitable for oral administration, for example as conventional or gastroprotected capsules or tablets, sugar-coated pills, soft or hard gelatin capsules, or cellulose capsules.
The compositions of the invention will be formulated according to conventional methods, such as those described in "Remington's Pharmaceutical Handbook", Mack
Publishing Co., N.Y., USA. In particular, the compositions of the invention will be formulated according to conventional plant ingredient formulation techniques, which require particular care to be taken to avoid interactions with the excipients and the capsule matrices.
Particularly suitable carriers are oils rich in o-3 fatty acids, which facilitate the absorption of cynaropicrin and flavonoids.
The formulations can also contain other active ingredients with complementary or otherwise useful activities, in particular one or more extracts of Olea europaea, Berberis aristata, Olea oleracea, Vitis vinifera, Cyclanthera pedata, Gymnema sylvestre, Eugenia jambolana and carotenoids.
The combination with the Olea europaea extract containing 20% oleuropein is particularly preferred. The release of tyrosol and hydroxytyrosol from oleuropein generates a reduction in oxidative processes affecting the cLDLs, which acts synergically with the properties of the combined artichoke and bergamot orange extracts. The combination with the Olea europaea extract therefore maintains its therapeutic efficacy in the treatment of metabolic syndrome, even at low doses of the formulations according to the invention. Said combination, in addition to the primary activity against hepatic steatosis and lipid metabolism, acts on the blood pressure, adipose tissue and vascular inflammation. The growing adipose tissue is infiltrated by macrophages that release proinflammatory cytokines and inflammation mediators such as COX-2 and iNOS; moreover, the circulating fatty acids act as messengers of the adipose cells via the TLR-4 receptors, which induce the expression of inflammatory mediators by activating NF-kB or
JNK. This cascade of events generates a state called "silent inflammation", whose aggressive action against the vascular endothelia causes lesions that act as the site of attack by atheromatous plaque, which is mainly responsible for the atherosclerotic and thrombotic symptoms that form the basis of cardiovascular disease. The Olea europea extracts contain substances useful to counteract the inflammatory process such as triterpenes, ursolic and oleanolic acids, flavonoids such as verbascoside, and the above-mentioned oleuropein.
Hydroxytyrosol, released by oleuropein, has obtained a favourable opinion from the EFSA regarding the claim that it inhibits LDL oxidation, one of the secondary phenomena that induce the formation of atheromatous plaque.
The dose of the composite extract of the invention typically ranges from 400 to
200 mg/dose/day, preferably 300 mg/dose/day, a dose interval much smaller than those reported for the uncombined extracts (1.3 g for bergamot orange extract and 500 mg/day for artichoke extract).
The activity of the composite extract of the invention was evaluated in normal rats, obese Zucker rats and diabetic Zucker rats (ZDF) by comparison with the individual ingredients, extract of Citrus bergamia (BPF) and Cynara cardunculus (CC). The treatment was given for 30 days, and the metabolic state, insulin sensitivity, glucose tolerability and hepatic steatosis were evaluated. All the substances tested improved the parameters examined, but with a different intensity from the controls.
During euglycaemic treatment, hyperinsulinaemic treatment and treatment with BPF, CC and BCS, the Zucker rats and the diabetic Zucker rats required a higher glucose infusion than the controls to maintain a stable glucose concentration (1.6 ± 0.23,
1.5 ± 0.18, 2.01 ± 0.19 and 4.1%, 1 and 6.42 ± 1.03 mg min ) as against values compared with the controls (0.71 ± 0.17 mg min 1 and 2.09 ± 0.71 p<0.05), demonstrating increased insulin sensitivity in both the Zucker rats and the diabetic Zucker rats.
The composite extract of the invention underwent a clinical trial at the dose of 300 mg once a day. The trial involved a total of 86 patients suffering from stable hyperlipaemia (fasting LDL values exceeding 130 mg/dl, triglycerides exceeding 200 mg/dl and HDL lower than 40 mg/dl), with an alcohol intake not exceeding 20 g a day and a fatty liver determined by abdominal ultrasound scan with a hepatorenal index ranging from 2.5 to 3.5 according to the conventional international criteria.
The duration of the clinical trial was 16 weeks, with tests conducted at time zero and at the end of the treatment. The results, set out in tables 1 and 2, demonstrate a synergic effect of the composite extract compared with the two individual extracts on the lipid and glycometabolic parameters, and on the liver parameters correlated with non alcoholic hepatic steatosis.
Table 1
TC mg/dL, total cholesterol; TG mg/dL triglycerides; LDL-c mg/dL low-density lipoprotein; HDL-c mg/dL high-density lipoprotein oxLDL mg/dl
BPF 650 mg twice a day; Cynara cardunculus 100 mg twice a day
Combination 300 mg/single administration
∆ Difference from baseline after 16 week treatment Table 2
ALP alkaline phosphatase; GGT gamma-glutamyltransferase; ALT alanine aminotransferase; AST aspartate aminotransferase; HA hyaluronic acid; PCIII precollagen IV-c type IV collagen. The compositions of the invention exhibited a marked activity in lowering total cholesterol, LDLs and blood glucose, as well as increasing cHDL, reducing blood pressure and modulation of inflammatory parameters, and reducing cardiovascular risk factors. The reduction in serum cholesterol, triglycerides and glucose is 38%, 45% and
28% respectively.
The formulations of the invention have also proved effective on different parameters in a range of patients suffering from metabolic syndrome, in whom normalisation of parameters such as blood glucose, lipid parameters, hypertension and
"silent inflammation" was observed.
The examples set out below further illustrate the invention.
Example 1 - Preparation of Cynara cardunculus var. sylvestris extract
1000 Kg of freshly-picked Cynara cardunculus var. sylvestris leaves are finely ground in a grinder under flowing steam to bring the ground biomass to a temperature of about 85°C in a time compatible with the total enzymatic inhibition of the glycosidases and hydrolases; the biomass is pressed, while still hot, in a screw press, and the aqueous extract is collected, while the squeezed biomass is countercurrent washed with water at
60°C. The combined liquids are clarified by decanter, and the clear solution is absorbed on 20 L of an absorption resin; after washing the resin with water until the non-resin-like substances are completely eliminated, the resin is washed with ethanol and the eluate is concentrated to water.
This solution is cooled and stored until the Citrus bergamia extract, obtained by the procedures reported in US 8,741,362, is available.
Example 2 - Preparation of composite extract of Citrus bergamia and Cynara cardunculus var. sylvestris
5 L of Cynara cardunculus var. sylvestris extract, prepared according to example
1 and concentrated to 40 brix, was mixed with 12 L of Citrus bergamia extract, also concentrated to 40 brix, and the mixture was atomised after filtration.
The resulting extract is a yellow/brown water-soluble powder with a 32% flavonoid content, comprising 4.5% neoeriocitrin, 8.6% naringin, 8.1% neohesperidin and
11.7% cynaropicrin
It presents characteristic IR bands at 3347, 2925, 1712.7, 1634.1, 1514.6, 1269.1,
1171.9 and 1021 cm 1.
Example 3 - Preparation of 300 g of hard gelatin capsules
Unit composition:
Combination of example 2 300 mg
Microcrystalline cellulose 100 mg
Silicon dioxide 5 mg
Magnesium stearate 5 mg
Example 4 - Formulation of the combination of Citrus bergamia extract and
Cynara cardunculus var. sylvestris extract in oily suspension for soft gelatin capsules
Unit composition
Combination of example 2 300 mg
Soya lecithin 200 mg
Beeswax 5 mg
Linseed oil 225 mg CLAIMS
1. A composite extract obtained by atomisation of a mixture of an extract of Cynara scolymus and an aqueous extract of Citrus bergamia, wherein the extract of Cynara scolymus is obtained by waterextraction of fresh leaves previously subjected to thermal shock and wherein the extract of Citrus bergamia has at least a 25% weight content in a polyphenolic fraction consisting of neoeriocitrin, naringin and neohesperidin and a furocoumarin content lower than 400 mg/Kg.
2. An extract according to claim 1 wherein the Cynara cardunculus extract is an extract of Cynara cardunculus var. sylvestris with a cynaropicrin content ranging from 20 to 40%.
3. An extract according to any one of claims 1 - 2 wherein the polyphenolic fraction has the following percentage composition by weight:
Neoeriocitrin 29.6% ± 6.0 Naringin 32.4% ± 4.0 Neohesperidin 38.0% ± 6.0 Total 100.0%
4. An extract of Cynara cardunculus var. sylvestris leaves having a cynaropicrin content ranging from 20 to 40% by weight, a cynaroside content ranging from 5 to 30% by weight, and a catechin polymer content ranging from 5 to 10% by weight.
5. Formulations comprising the extracts according to claims 1-3 in admixture with suitable carriers and excipients.
6. Formulations according to claim 5 further comprising one or more active ingredients selected from extracts of Berberis aristata, Olea europaea, Olea oleracea,
Vitis vinifera, Cyclanthera pedata, Gymnema sylvestre, Eugenia jambolana and carotenoids.
7. Formulations according to claim 5 or 6 in the form of tablets, sugar-coated pills, soft and hard gelatin capsules, and cellulose capsules. 8. Formulations according to claim 7 for use in the treatment or prevention of hepatic steatosis.
9. Formulations according to claim 8 for use in the treatment of dyslipidaemia, hyperglycaemia, vascular inflammation, hypertension, metabolic syndrome, type 2 diabetes and cardiovascular diseases.
10. Formulations for use according to claims 8 and 9 wherein the composite extract is administered at a dose ranging from 400 to 200 mg/dose/day.
11. A process for the preparation of the composite extract according to claims 1-4 which comprises atomisation of a mixture of an extract of Cynara cardunculus obtained by waterextraction of fresh leaves previously subjected to thermal shock and an aqueous extract of Citrus bergamia. A . CLASSIFICATION O F SUBJECT MATTER INV. A61K36/752 A61K36/26 A61P3/06 A61P3/10 A61P9/12 ADD.
According to International Patent Classification (IPC) o r t o both national classification and IPC
B . FIELDS SEARCHED Minimum documentation searched (classification system followed by classification symbols) A61K A61P
Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched
Electronic data base consulted during the international search (name of data base and, where practicable, search terms used)
EPO-Internal , WPI Data, BIOSIS, EMBASE, CHEM ABS Data
C . DOCUMENTS CONSIDERED T O B E RELEVANT
Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.
WO 2015/136441 Al (ESSERRE PHARMA SOCI ET A 1-3 ,5-11 RESPONSABI LIT LI MI TATA SEMPLI FICATA [IT] ) 17 September 2015 (2015-09-17) page 4 , l i ne 20 - l i ne 26; cl aims page 12 , l i ne 24 - page 13 , l i ne 1 page 2 , l i ne 5 - l i ne 10; exampl es
EP 2 719 287 Al (DDF GROUP DI DAM I A NI 1-3 ,5-11 LUCIANO MARCO & C S A S [IT] ) 16 Apri l 2014 (2014-04-16) cl aims ; exampl es
US 8 741 362 B2 (HERBAL & ANTIOXIDANT 1-3 ,5-11 DERIVATIVES S. R. L. ) 3 June 2014 (2014-06-03) ci ted i n the appl i cati on cl aims -/-
X| Further documents are listed in the continuation of Box C . See patent family annex.
* Special categories of cited documents : "T" later document published after the international filing date o r priority date and not in conflict with the application but cited to understand "A" document defining the general state of the art which is not considered the principle o r theory underlying the invention to be of particular relevance "E" earlier application o r patent but published o n o r after the international "X" document of particular relevance; the claimed invention cannot be filing date considered novel o r cannot b e considered to involve a n inventive "L" documentwhich may throw doubts o n priority claim(s) orwhich is step when the document is taken alone cited to establish the publication date of another citation o r other "Y" document of particular relevance; the claimed invention cannot be special reason (as specified) considered to involve a n inventive step when the document is "O" document referring to a n oral disclosure, use, exhibition o r other combined with one o r more other such documents, such combination means being obvious to a person skilled in the art "P" document published prior to the international filing date but later than the priority date claimed "&" document member of the same patent family
Date of the actual completion of the international search Date of mailing of the international search report
12 July 2018 27/09/2018
Name and mailing address of the ISA/ Authorized officer European Patent Office, P.B. 5818 Patentlaan 2 N L - 2280 HV Rijswijk Tel. (+31-70) 340-2040, Fax: (+31-70) 340-3016 Langer, Astri C(Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT
Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.
A VINCENZO MOLLACE ET AL: "Hypol i pemi c and 1-3 ,5-11 hypoglycaemi c acti v i t y of bergamot polyphenol s : From animal model s t o human studi es" , FITOTERAPIA, vol . 82 , no. 3 , 2011 , pages 309-316, XP028365047 , ISSN : 0367-326X, DOI : 10. 1016/J . FITOTE. 2010. 10.014 [retri eved on 2010-11-04] c i ted i n the appl i cati on abstract; f i gures
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-/-- C(Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT
Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.
A, P FERRO A M ET AL: "Hapl otype analysi s of 1-3 ,5-11 the germacrene A synthase gene and associ ati on wi t h cynaropi cri n content and bi ol ogi cal acti v i t i es i n Cynara carduncul us" , MOLECULAR GENETICS AND GENOMICS 20180401 SPRINGER VERLAG DEU , vol . 293 , no. 2 , 16 November 2017 (2017-11-16) , pages 417-433 , XP055491875 , ISSN : 1617-4615 abstract
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WO 2015136441 Al 17-09-2015 EP 3116520 Al 18-01-2017 O 2015136441 Al 17-09-2015
EP 2719287 Al 16-04-2014 NONE
US 8741362 B2 03-06-2014 AU 2009315289 Al 20-05-2010 CN 102215853 A 12-10-2011 EP 2364158 A2 14-09-2011 ES 2393285 T3 20-12-2012 I T 1392535 Bl 09-03-2012 RU 2011124540 A 27-12-2012 SM T201200047 B 14-01-2013 US 2011223271 Al 15-09-2011 W0 2010055490 A2 20-05-2010
W0 2008105023 Al 04-09-2008 EP 2131681 Al 16 -12 -2009 ES 2401203 T3 17 -04 -2013 US 2010055211 Al 04 -03 -2010 US 2013131328 Al 23 -05 -2013 WO 2008105023 Al 04 -09 -2008
WO 2008142460 A2 27-11-2008 HR P20070230 A2 3 -11 -2008 WO 2008142460 A2 27 -11 -2008
US 2015231193 Al 20-08-2015 AU 2013320237 Al 09 -04 -2015 B R 112015005942 A2 04 -07 -2017 CA 2886887 Al 27 -03 -2014 CN 104640555 A 2 -05 -2015 DK 2900251 T3 2 -02 -2017 EP 2900251 Al 05 -08 -2015 ES 2613381 T3 24 -05 -2017 HK 1210597 Al 29 -04 -2016 HU E031901 T2 28 -08 -2017 I L 237803 A 29 -03 -2018 P 6265388 B2 24 -01 -2018 P 2015532917 A 16 -11 -2015 KR 20150058234 A 28 -05 -2015 PL 2900251 T3 3 1-05 -2017 PT 2900251 T 18 -01 -2017 RU 2015109566 A 2 -10 -2016 SG 11201502096V A 29 -04 -2015 US 2015231193 Al 2 -08 -2015 WO 2014044744 Al 27 -03 -2014 INTERNATIONAL SEARCH REPORT
Box No. II Observations where certain claims were found unsearchable (Continuation of item 2 of first sheet)
This international search report has not been established in respect of certain claims under Article 17(2)(a) for the following reasons:
□ Claims Nos.: because they relate to subject matter not required to be searched by this Authority, namely:
□ Claims Nos.: because they relate to parts of the international application that do not comply with the prescribed requirements to such an extent that no meaningful international search can be carried out, specifically:
3 . □I I Claims Nos.: because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4(a).
Box No. Ill Observations where unity of invention is lacking (Continuation of item 3 of first sheet)
This International Searching Authority found multiple inventions in this international application, as follows:
see addi t i onal sheet
□ As all required additional search fees were timely paid by the applicant, this international search report covers all searchable claims.
□ As all searchable claims could be searched without effort justifying an additional fees, this Authority did not invite payment of additional fees.
As only some of the required additional search fees were timely paid by the applicant, this international search report covers ' ' only those claims for which fees were paid, specifically claims Nos. :
No required additional search fees were timely paid by the applicant. Consequently, this international search report is restricted to the invention first mentioned in the claims; it is covered by claims Nos. : 1-3 , 5-11
Remark on Protest The additional search fees were accompanied by the applicant's protest and, where applicable, the ' ' payment of a protest fee. The additional search fees were accompanied by the applicant's protest but the applicable protest ' ' fee was not paid within the time limit specified in the invitation.
I INo protest accompanied the payment of additional search fees.
Form PCT/ISA/21 0 (continuation of first sheet (2)) (April 2005) International Application No. PCT/ I B2018/ 052498
FURTHER INFORMATION CONTINUED FROM PCT/ISA/ 210
Thi s Internati onal Searchi ng Authori t y found mul t i pl e (groups of) i nventi ons i n thi s i nternati onal appl i cati on , as fol l ows :
1. cl aims : 1-3 , 5-11
Composi t e extract obtai ned by atomi zati on of a mi xture of Cynara scolymus/carduncul s and of Ci trus bergami a, formul ati ons contai ni ng thi s composi t e extract, these composi t i ons for use i n the treatment of hepati c steatosi s or dysl i pi demi a, hyperglycaemi a, vascul ar i nfl ammati on , hypertensi on , metabol i c syndrome, type 2 di abetes and cardi ovascul ar di sease as wel l as the process for preparati on of the composi t e extract.
2. cl aim: 4
Extract of Cynara carduncul s var. syl vestri s wi t h a defi ned content i n cynaropi cri n, cynarosi de and catechi n.