Extracts of <i>Cynara cardunculus </i>and <i>Citrus aurantium bergamia</i>, combinations thereof, and formulations containing them

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.

Claims

1. A composition of matter comprising neoeriocitrin, naringin, neohesperidin and cynaropicrin in relative weight proportions of about 13.5:25.8:24.3:35.1.

2. The composition of matter of claim 1, said composition of matter having a furocoumarin content lower than 400 mg/Kg.

3. The composition of matter of claim 1, said composition of matter substantially free of bergaptene.

4. The composition of matter of claim 1, comprising about 13.5 mg neoeriocitrin, about 25.8 mg naringin, about 24.3 mg neohesperidin, about 35.1 mg cynaropicrin and about 100 mg of pharmaceutically-acceptable excipient, said composition formed into an oral dosage form.

5. The invention of claim 4, wherein said pharmaceutically-acceptable excipient comprises oil rich in omega-3 fatty acid in an amount sufficient to facilitate the absorption of said neoeriocitrin, naringin, neohesperidin or cyanopicrin by the human gastrointestinal system.

6. The composition of matter of claim 1, said composition substantially free of hydrolytic enzymes and oxidants.

7. The composition of matter of claim 1, wherein said neoeriocitrin, naringin, neohesperidin and cynaropicrin have been subject to atomization, wherein said atomization increases the solubility of said neoeriocitrin, naringin, neohesperidin and cynaropicrin.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) The composite extract of the invention is obtained by atomisation or other equivalent techniques of aqueous solutions of:

(2) a) a Cynara cardunculus extract having a cynaropicrin content ranging from 20 to 40%, and preferably amounting to 30% by weight;

(3) b) a Citrus aurantium var. bergamia extract obtained by the steps described in the process according to U.S. Pat. No. 8,741,362, except for the pre-drying step.

(4) 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%

(5) 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.

(6) 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.

(7) 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.

(8) Particularly suitable carriers are oils rich in co-3 fatty acids, which facilitate the absorption of cynaropicrin and flavonoids.

(9) 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.

(10) 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.

(11) 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.

(12) 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).

(13) 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.

(14) 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.sup.−1) as against values compared with the controls (0.71±0.17 mg min.sup.−1 and 2.09±0.71 p<0.05), demonstrating increased insulin sensitivity in both the Zucker rats and the diabetic Zucker rats.

(15) 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.

(16) 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.

(17) TABLE-US-00001 TABLE 1 Substances Dose CT cLDL cHDL TG oxLDL BPF 1300 241 ± 11 169 ± 18 35 ± 4  251 ± 8  131 ± 14 Δ 16 − 50 ± 8 Δ 16 − 40 ± 4 Δ 16 13 ± 2  .sup.  Δ 16 − 46 ± 6 Δ −18 ± 6  CYNARA C. 200 246 ± 9  165 ± 12 39 ± 2    239 ± 7.5 127 ± 4 Δ −28 ± 6   Δ −20 ± 8   Δ 12 ± 4  Δ −31 ± 6 Δ −11 ± 3  COMB. ES. 3 300 252 ± 10 172 ± 16 38 ± 3  210 ± 6 128 ± 4 Δ −76 ± 9   Δ −39 ± 5   Δ 21 ± 4  Δ −34 ± 2 Δ −26 ± 2  PLACEBO 251 ± 12 155 ± 8  39 ± 3   136 ± 14 129 ± 6 Δ −6 ± 2  Δ −6 ± 15  Δ 5 ± 2  Δ −10 ± 4 Δ 4 ± 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 Δ Difference from baseline after 16 week treatment

(18) TABLE-US-00002 TABLE 2 Dose ALP GGT ALT AST HA PCIII IVC Hep/ Substances mg (u/L) (U/L) (u/L) (u/L) (ng/ml) (ng/ml) (ng/ml) Index PLACEBO Baseline 65.26 ± 0.7 67.44 ± 3.9   52 ± 4.1 44.5 ± 3.9  85.7 ± 10 85.7 ± 10   85.7 ± 10  85.7 ± 10 16 weeks  −1.3 ± 0.7 −2.69 ± 1.1 −0.35 ± 05  −046 ± 0.6   −2.8 ± 3.1 −1.83 ± 1.5  −1.57 ± 0.6  −0.3 ± 01 BPF Baseline 1300 64.61 ± 5.1 66.31 ± 4.2 59.71 ± 4.1 46.58 ± 3.9  80.53 ± 8.5 71.81 ± 7.5  57.62 ± 5.8  3.7 ± 0.4 16 weeks   14 ± 1.6 .sup. −16 ± 7.2 −13.4 ± 2.sup.  11.30 ± 0.6    21 ± 5.2 −16.4 ± 3.5   15.3 ± 3.2 −12.2 ± 0.3 CYNARA c. Baseline 200  62.5 ± 4.9  65.6 ± 10 59.80 ± 3.9 41.6 ± 2.7 76.6 ± 9  74.9 ± 6.2  54.6 ± 6.2 127 ± 4 16 weeks .sup. −10 ± 1.1 .sup. −12 ± 6.4 .sup. −15 ± 3.1 12.3 ± 0.8   18 ± 3.2  −12 ± 2.8 10.2 ± 2  −10.2 ± 0.1 COMB. Baseline 300  64.2 ± 5.1 67.38 ± 1.7 57.69 ± 2.6 45.62 ± 3.1  83.67 ± 7.4 72.91 ± 6.5  58.11 ± 3.9  3.9 ± 0.5 16 weeks  −18 ± 47 .sup. −18 ± 5.4 .sup. −14 ± 3.6 13.2 ± 4.5 24.58 ± 6.5 18.3 ± 5.8 17.6 ± 6  −12.1 ± 0.2 ALP alkaline phosphatase; GGT gamma-glutamyltransferase; ALT alanine aminotransferase; AST aspartate aminotransferase; HA hyaluronic acid; PCIII precollagen IV-c type IV collagen.

(19) 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.

(20) 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.

(21) The examples set out below further illustrate the invention.

Example 1—Preparation of Cynara cardunculus Var. Sylvestris Extract

(22) 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.

(23) This solution is cooled and stored until the Citrus bergamia extract, obtained by the procedures reported in U.S. Pat. No. 8,741,362, is available.

Example 2—Preparation of Composite Extract of Citrus bergamia and Cynara cardunculus var. sylvestris

(24) 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.

(25) 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

(26) It presents characteristic IR bands at 3347, 2925, 1712.7, 1634.1, 1514.6, 1269.1, 1171.9 and 1021 cm.sup.−1.

Example 3—Preparation of 300 mg of Hard Gelatin Capsules

(27) Unit Composition:

(28) TABLE-US-00003 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

(29) Unit Composition

(30) TABLE-US-00004 Combination of example 2 300 mg Soya lecithin 200 mg Beeswax 5 mg Linseed oil 225 mg