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Title:
PHARMACEUTICAL AND NUTRACEUTICAL COMPOSITIONS AND COMBINATIONS COMPRISING ACTIVE INGREDIENTS USEFUL IN THE TREATMENT AND PREVENTION OF HEPATIC PATHOLOGIES AND KITS COMPRISING THEM
Document Type and Number:
WIPO Patent Application WO/2019/244023
Kind Code:
A1
Abstract:
The present invention relates to pharmaceutical and/or nutraceutical combinations and compositions of active ingredients useful in the treatment and prevention of hepatic diseases, and kits comprising said combinations and compositions.

Inventors:
PIZZONI ANGELO (IT)
PIZZONI PAOLO (IT)
Application Number:
PCT/IB2019/055087
Publication Date:
December 26, 2019
Filing Date:
June 18, 2019
Export Citation:
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Assignee:
APHARM SRL (IT)
International Classes:
A61K36/9066; A61K36/28; A61K36/74; A61P1/16
Domestic Patent References:
WO2012027603A22012-03-01
Foreign References:
FR3058319A12018-05-11
US20160158305A12016-06-09
US20170368135A12017-12-28
EP2740473A12014-06-11
Other References:
LABRECQUE, D. ET AL.: "World Gastroenterology Organisation Global Guidelines: Nonalcoholic Fatty Liver Disease and Nonalcoholic Steatohepatitis", JOURNAL OF CLINICAL GASTROENTEROLOGY, vol. 48, no. 6, July 2014 (2014-07-01), pages 467 - 473
KIM ET AL., J AGRIC FOOD CHEM, vol. 63, 2015, pages 9729 - 39
CHUNG MY ET AL., NUTRITIONAL RESEARC, vol. 4, 2017, pages 1 - 10
VIDYASHANKAR ET AL., TOXICOLOGY IN VITRO, vol. 27, 2013, pages 945 - 953
"The ethical policy of the University of Florence complies with the Guide for the Care and Use of Laboratory Animals", 1996, NATIONAL INSTITUTES OF HEALTH
ROSSMEISL ET AL., DIABETES, vol. 52, 2003, pages 1958 - 1966
FELLMAN ET AL., PHARMACOL THER, vol. 137, 2013, pages 331 - 340
Attorney, Agent or Firm:
SANTORO, Tiziana et al. (IT)
Download PDF:
Claims:
Claims

1. A combination of pharmaceutical and/or nutraceutical compositions for the use in the treatment and/or prevention of the hepatic diseases, said combination comprising:

a pharmaceutical and/or nutraceutical composition, Form (A), comprising:

- milk thistle: 50-800 mg;

- Coffea arabica : 40-400 mg;

- feverfew: 150-1000 mg; and

- choline or a physiologically acceptable salt thereof: 50-500 mg (as the weight of the non-salified choline);

and possibly conventional excipients and carriers;

and

a pharmaceutical and/or nutraceutical composition, Form (B), comprising:

- turmeric: 100-600 mg;

DHA: 150-1000 mg;

- choline or a physiologically acceptable salt thereof: 50-500 mg (as the weight of the non-salified choline); and

- Vitamin E: 10-100 mg;

and possibly conventional excipients and carriers;

said use comprising administering at least one composition Form (A) and at least one composition Form (B) per day.

2. The combination for the use according to claim 1, characterized in that it comprises:

a composition Form (A) comprising:

- milk thistle: 400 mg;

- Coffea arabica. 300 mg;

- feverfew: 400 mg; and

- choline or a physiologically acceptable salt thereof: 200 mg (as the weight of the non-salified choline);

and possibly conventional excipients and carriers; and

a composition Form (B) comprising:

- turmeric: 200 mg;

DHA: 400 mg;

- choline or a physiologically acceptable salt thereof: 200 mg (as the weight of the non-salified choline); and

- Vitamin E: 60 mg;

and possibly conventional excipients and vehicles.

3. The combination for the use according to claim 1 or 2, characterized in that said hepatic diseases are NAFLD and/or NASH.

4. The combination for the use according to any one of claims 1 to 3, characterized in that the composition Form (A) is administered in the morning and the composition Form (B) at another time of the day.

5. The combination for the use according to claim 4, characterized in that the composition Form (B) is administered together with a meal, preferably in the evening.

6. The combination for the use according to any one of claims 1 to 5, characterized in that said compositions Form (A) and Form (B) are in the form of solid, semi-solid, gel or liquid oral compositions.

7. The combination for the use according to claim 6, characterized in that said compositions Form (A) and Form (B) are in the solid form.

8. A kit comprising at least one composition Form (A) and one composition Form (B) as defined according to any one of claims 1, 2, 6 and 7, the number of two different compositions being the same, possibly together with a leaflet.

9. The kit according to claim 8, comprising 1 to 100 compositions Form (A) and 1 to 100 compositions Form (B).

10. The kit according to claim 9, comprising 14-30 compositions Form (A) and 14-30 compositions Form (B).

11. A pharmaceutical and/or nutraceutical composition comprising:

milk thistle: 400 mg;

Coffea arabica : 300 mg; - feverfew: 400 mg; and

- choline or a physiologically acceptable salt thereof: 200 mg (as the weight of the non-salified choline);

and possibly conventional excipients and vehicles.

12. A pharmaceutical and/or nutraceutical composition comprising:

- turmeric: 200 mg;

DHA: 400 mg;

- choline or a physiologically acceptable salt thereof: 200 mg (as the weight of the non-salified choline); and

- Vitamin E: 60 mg;

and possibly conventional excipients and vehicles.

13. A pharmaceutical and/or nutraceutical mixture consisting of 400 mg milk thistle, 400 mg feverfew, 300 mg Coffea arabica and 200 mg choline.

14. A pharmaceutical and/or nutraceutical mixture consisting of 200 mg turmeric, 400 mg DHA, 200 mg choline or a physiologically acceptable salt thereof and 60 mg

Vitamin E.

15. The compositions of claims 11 and 12 for the use in the treatment of the hepatic diseases, such as NAFLD and NASH.

16. The mixtures of claims 13 and 14 for the use in the treatment of the hepatic diseases, such as NAFLD and NASH.

Description:
“Pharmaceutical and nutraceutical compositions and combinations comprising active ingredients useful in the treatment and prevention of hepatic pathologies and kits comprising them”

Description

Summary of the Invention

The present invention relates to pharmaceutical and/or nutraceutical compositions and combinations of active ingredients useful in the treatment and prevention of hepatic diseases, and kits comprising said compositions.

Technical Field

Non-alcoholic steatosis (NAFLD) is a disease linked to the intracellular buildup of triglycerides which can evolve up to necrosis of the hepatic cell. The first step of the disease provides for an excessive cellular storage of lipids which generates hepatocellular lipotoxicity in a second step. This condition is characterized by cellular stress starting signaling processes able to induce the monocyte recruitment in the liver and their differentiation in inflammatory macrophages. The hepatocellular lipotoxic damage induced by lipids is generally insufficient to cause cell death, but is sufficient to trigger pro-inflammatory signaling cascades (NF-kB pathway) intended to alter tissue homeostasis. The clinical picture is characterized by oxidative stress, mitochondrial dysfunction, cytokine release and insulin resistance with consequent hyperglycemia. If the pro-inflammatory stimulus persists, the inflammatory response from acute becomes chronic and causes tissue remodeling and fibrosis. For this reason, some patients affected by NAFLD develop hepatic cell injuries and inflammation, that is the etiological picture of the non-alcoholic steatohepatitis (NASH).

10-25% of the Italian population between 18 and 75 years of age suffers from NAFLD and it represents the cause of severe hepatic disorders all over the world, not only in the Western countries. This is what is stated by the new published guidelines of the World Gastroenterology Organisation (WGO) (see LaBrecque, D. et al. World Gastroenterology Organisation Global Guidelines: Nonalcoholic Fatty Liver Disease and Nonalcoholic Steatohepatitis. Journal of Clinical Gastroenterology: July 2014 - Volume 48 - Issue 6 - p 467-473. WGO Guidelines).

In the next years an increase is expected, hand in hand with the increasing prevalence of obesity, diabetes and metabolic syndrome. In Italy it is estimated that there are 2 new cases on 100,000 inhabitants in one year.

The epidemiological incidence has been evaluated as follows:

Middle East 32%

South America 31%

Asia 27%

Europe and North America 20/30%.

The presence of NAFLD/NASH increases the mortality risk both for hepatic causes and cardiovascular diseases.

The metabolic risk factors associated with NAFLD/NASH are mainly:

- Overweight (BODY MASS INDEX or BMI >25 Kg/m2);

- Visceral adiposity increased (waist circumference M >94 cm, F>80 cm);

- Arterial hypertension (A.H. >l35/85mmHg);

- Hypertriglyceridemia;

- Total hypercholesterolemia increased and HDL decreased;

- Hyperferritinemia (>350 mg/L);

- First degree familiarity for obesity and/or diabetes.

Thus, NAFLD/NASH can be defined as“dysmetabolic hepatopathy” and at the moment an effective therapy is lacking.

WGO experts underline that the fat liver not always indicates the need of an aggressive therapy, and that the doctors should reserve the liver biopsy to the patients with risk factors for NASH and/or other hepatic diseases.

The first approach should consist in an adequate diet and physical exercise but as it is easily understandable, such remedies are almost never resolutive.

Therefore, there is a need to provide novel therapeutic approaches directed to the treatment of subjects affected with hepatic diseases such as NAFLD and/or NASH, and to the prevention of such diseases in subjects with predisposition to their development.

Brief Description of the Figures Figure 1. Cell viability and cell proliferation. **P<0.0l vs. BSA + vehicle; L R<0.05 vs. OA/BSA + vehicle.

Figure 2. SOD activity. *P<0.05 and **P<0.0l vs. BSA + vehicle; L R<0.05 and LL R<0.01 vs. OA/BSA + vehicle.

Figure 3. Lipid peroxidation. **P<0.0l vs. BSA + vehicle; L R<0.05 and LL R<0.01 vs. OA/BSA + vehicle.

Figure 4. Triglycerides concentration. **P<0.0l vs. BSA + vehicle; L R<0.05 and LL R<0.01 vs.

OA/BSA + vehicle.

Figure 5. Total cholesterol concentration. *P<0.05 and **P<0.0l vs. BSA + vehicle; L R<0.05 and LL R<0.01 vs. OA/BSA + vehicle.

Figure 6. Steatosis grade. **P<0.0l vs. BSA + vehicle; LL R<0.01 vs. OA/BSA + vehicle.

Figure 7. Body weight. **P<0.0l vs. normal diet + vehicle; LL R<0.01 vs. HFD + vehicle

Figure 8. Food intake. **P<0.0l vs. normal diet + vehicle

Figure 9. Liver weight **P<0.0l vs. normal diet + vehicle; L R<0.05 and LL R<0.01 vs. HFD + vehicle.

Figure 10. Heart weight. **P<0.0l vs. normal diet + vehicle; L R<0.05 vs. HFD + vehicle.

Figure 11. Kidney weight.

Figure 12. Perigonadal fat pad weight normalized to body weight. **P<0.0l vs. normal diet + vehicle; L R<0.05 vs. HFD + vehicle.

Figure 13. Histology of adipose tissue and area of adipocytes. **P<0.0l vs. normal diet + vehicle; L R<0.05 vs. HFD + vehicle.

Figure 14. Histology of liver and steatosis index. **P<0.0l vs. normal diet + vehicle; L R<0.05 and LL R<0.01 vs. HFD + vehicle.

Figure 15. Oxidative damage of liver: lipid peroxidation. **P<0.0l vs. normal diet + vehicle; L R<0.05 vs. HFD + vehicle.

Figure 16. Oxidative damage of liver: protein carbonylation. **P<0.0l vs. normal diet + vehicle; L R<0.05 and LL R<0.01 vs. HFD + vehicle Figure 17. Oxidative damage of heart: lipid peroxidation. **P<0.0l vs. normal diet + vehicle; L R<0.05 vs. HFD + vehicle.

Figure 18. Oxidative damage of heart: protein carbonylation. **P<0.0l vs. normal diet + vehicle; L R<0.05 vs. HFD + vehicle.

Objects of the Invention

It is an object of the invention to provide a combination of active ingredient compositions for the use in the treatment and/or prevention of hepatic diseases such as NAFLD and/or NASH.

It is another object of the invention to provide novel pharmaceutical and/or nutraceutical compositions comprising said active ingredients, and their use in the treatment and/or prevention of hepatic diseases such as NAFLD and/or NASH.

It is a further object of the invention to provide novel pharmaceutical and/or nutraceutical compositions comprising said active ingredients together with probiotic bacteria and their use in the treatment and/or prevention of hepatic diseases such as NAFLD and/or NASH.

It is a further object of the invention to provide kits comprising the combinations and compositions of the invention.

Description of the Invention

Thus, according to one of its aspects, the invention describes pharmaceutical and/or nutraceutical compositions comprising at least three components selected from milk thistle, turmeric, feverfew, Coffea arabica, DHA, choline or a physiologically acceptable salt thereof and Vitamin E, for their use in the treatment and/or prevention of hepatic diseases, in particular NAFLD and/or NASH, possibly together with conventional excipients and carriers, preferably at least four of the components listed above.

“Milk thistle” herein means a Silybum Marianum extract, preferably a seed extract of said plant. According to a preferred embodiment, said extract contains at least 60%, preferably at least 70%, advantageously at least 75%, for example about 80% or more, by weight of sylimarin. According to a preferred embodiment, said extract is in the powder form.

“Curcuma” herein means an extract of Curcuma , preferably of Curcuma Longa , preferably an extract of dry rhizome of said plant. According to a preferred embodiment, said extract contains at least 10%, preferably at least 15%, advantageously at least 18%, for example about 20% or more, by weight of curcumin. According to a preferred embodiment, said extract is in the powder form. According to a particularly preferred embodiment, said extract is the extract currently sold with the trading name of CurcuWIN ® (by the OmniActive company) having a particularly high bioavailability.

“Feverfew” herein means a Tanacetum parthenium extract, preferably a flower extract of said plant. According to a preferred embodiment, said extract contains at least 0.4%, preferably at least 0.6%, advantageously about 0.7-0.9%, for example 0.8 by weight of parthenolides. According to a preferred embodiment, said extract is in the powder form.

“ Coffea arabica” preferably means a seed extract of said plant. According to a preferred embodiment, said extract contains at least 30%, preferably at least 35%, advantageously at least 40%, for example about 45% or more, by weight of chlorogenic acid. According to a preferred embodiment, said extract is in the powder form.

“DHA” herein means the powdered docosahexaenoic acid, preferably having a purity of at least 60%, advantageously at least 70%, for example about 75%, wherein by purity is meant that the aforementioned powder comprises said percentage amounts of DHA together with excipients, inert substances and other Omega 3 fatty acids, for example EPA (eicosapentaenoic acid).

“Choline or a physiologically acceptable salt thereof’ herein means 2-hydroxy- N,N,N-trimethylethan-ammonium, or a physiologically acceptable salt thereof, wherein by “physiologically acceptable salt” is meant a salt non-toxic for the organism of a mammal, preferably a human, if taken orally. According to a preferred embodiment, choline is in the salified form, advantageously is the choline hydrochloride and is in the powder form.

Vitamin E is well known to the person skilled in the art and herein means any tocopherol and/or tocotrienol and/or derivatives thereof, advantageously DL-alpha- tocopheryl acetate. According to a preferred embodiment, DL-alpha-tocopheryl acetate is in the powder form, advantageously in partially water-soluble powder, for example 50% CWS (“Cold Water Soluble”, i.e. soluble in water at 50%).

According to one of its aspects, subject-matter of the invention is a combination of pharmaceutical and/or nutraceutical compositions for the use in the treatment and/or prevention of hepatic diseases, said combination comprising:

a pharmaceutical and/or nutraceutical composition, herein called Form (A), comprising:

- milk thistle: 50-800 mg, preferably 100-500 mg, more preferably about 400 mg;

- Coffea arabica : 40-400 mg, preferably 100-350 mg, more preferably about 300 mg;

- feverfew: 150-1000 mg, preferably 200-800 mg, more preferably about 400 mg; and

- choline or a physiologically acceptable salt thereof: 50-500 mg (as the weight of the non-salified choline), preferably 100-300 mg, more preferably about 200 mg; and possibly conventional excipients and carriers;

and

a pharmaceutical and/or nutraceutical composition, herein called Form (B), comprising:

- turmeric: 100-600 mg, preferably 50-300 mg, more preferably about 200 mg;

- DHA: 150-1000 mg, preferably 150-600 mg, more preferably about 400 mg;

- choline or a physiologically acceptable salt thereof: 50-500 mg (as the weight of the non-salified choline), preferably 100-300 mg, more preferably about 200 mg; and

- Vitamin E: 10-100 mg, preferably 20-80 mg, more preferably about 60 mg; and possibly conventional excipients and carriers;

said use comprising the administration of at least one composition (A) and at least one composition (B) per day.

According to an embodiment, said hepatic diseases are selected from NAFLD and NASH.

According to a preferred embodiment, the compositions of the invention are solid, semi-solid, gel or liquid oral compositions, possibly, and preferably, with conventional excipients and carriers.

The compositions of the invention are preferably in the form of tablets, possibly coated, granules, fine granules, powders, hard capsules, soft capsules, syrups, emulsions, suspensions and solutions suitable for the oral administration, preferably in the form of tablets. However, other pharmaceutical forms can be used.

Such compositions can be taken alone or with water or else, mainly when they are in the form of powders or granules, contained in multi-dose containers or preferably in single dose sachets, can be blended with other food, for example with yogurt or the like.

Alternatively, the compositions of the invention can be in ready-to-use drinkable form, such as for example in the form of drinkable sachets, of the“stick pack” type, preferably in the form of gel.

The types of pharmaceutical additives used in the preparation of the compositions of the invention, the content ratios of the additives with respect to the active ingredients and the methods for preparing the pharmaceutical composition can be suitably selected by the person skilled in the art.

As carriers and conventional excipients, organic or inorganic substances, or solid or liquid substances such as excipients and carriers, provided that they are edible, physiologically acceptable and compatible with all the other components of the composition, can be used.

When a solid composition is prepared in the form of tablets, for example, excipients conventionally used in the pharmaceutical art, for example diluents such as for example lactose, microcrystalline cellulose, starch, dicalcium phosphate; ligands such as for example polyvinyl pyrrolidone, hydroxypropyl methyl cellulose; disrupting agents such as cross-linked polyvinyl pyrrolidone, cross-linked carboxymethyl cellulose; sliding agents such as silica, talc; lubricants such as magnesium stearate, stearic acid, glyceryl tribehenate, sodium stearyl fumarate can be added to the aforementioned active ingredients. Also wetting agents or surfactants such as sodium laurylsulphate, polysorbate 80, poloxamer 188 can be added.

As mentioned, the person skilled in the art is perfectly able to select the most suitable carriers and excipients.

The tablets can be made with different techniques, direct compression, dry granulation, wet granulation, hot-melt. The tablets can be non-coated or coated (for example with sucrose) or covered with polymers or other suitable materials. The tablets can have an immediate, delayed or extended release by making polymer matrices or by using specific polymers at the filming level.

Other preferred compositions according to the invention are the hard or soft capsules containing fine powders or granules of the active ingredients, possibly in combination with suitable conventional excipients.

Other preferred compositions according to the invention are the fine powders or granules of the active ingredients, possibly in combination with suitable conventional excipients contained in mono-dose sachets.

However, other dosages of the compositions of the invention can be formulated and administered to a subject depending on the need and/or according to the doctor's opinion.

Form A and Form B are preferably oral forms, advantageously solid oral forms, such as tablets, powders, capsules and granules.

The compositions comprising milk thistle, feverfew, Coffea arabica and choline, as defined herein, and the compositions comprising turmeric, DHA, choline or a physiologically acceptable salt thereof and Vitamin E, as defined herein, as well as the specific compositions herein called Form A and Form B, are further subject- matters of the invention.

According to another of its aspects, subject-matter of the invention is also a mixture of active ingredients consisting of milk thistle, feverfew, Coffea arabica and choline, as defined herein, preferably in the amounts and weight ratios indicated above for the composition of Form A.

Advantageously, subject-matter of the invention is also a mixture consisting of 400 mg milk thistle, 400 mg feverfew, 300 mg Coffea arabica and 200 mg choline.

According to another of its aspects, subject-matter of the invention is also a mixture of active ingredients consisting of turmeric, DHA, choline or a physiologically acceptable salt thereof and Vitamin E, as defined herein, preferably in the amounts and weight ratios indicated above for the composition of Form B.

Advantageously, subject-matter of the invention is also a mixture of active ingredients consisting of 200 mg turmeric, 400 mg DHA, 200 mg choline or a physiologically acceptable salt thereof and 60 mg Vitamin E.

The expression“as defined herein” means that the components can be present in the compositions and combinations in any form described herein and advantageously, but not only, in the preferred forms.

According to the present invention (and as it is apparent to the person skilled in the art), “composition comprising” means a pharmaceutical and/or nutraceutical composition comprising the listed active ingredients and which can comprise also other possible active ingredients, together with possible conventional excipients and carriers.

Still according to the invention (and as it is apparent to the person skilled in the art) “mixture consisting of’ means a mixture the active ingredients of which constitute 100%. Said mixtures can be administered in pharmaceutical and/or nutraceutical compositions together with conventional excipients and carriers and/or other possible active ingredients.

Within the scope of the present invention the compositions and mixtures comprising not necessarily the components herein described and extracted from the plants containing them are also included, but for example also substances such as sylimarin instead of milk thistle as defined herein, parthenolides instead of feverfew as defined herein, chlorogenic acid instead of Coffea arabica as defined herein and curcumin instead of turmeric as defined herein, said substances also being able to come from other natural sources or being of synthetic origin.

It is also a further subject-matter of the invention the use of the compositions Form A and Form B and mixtures of the invention, which are described above, in the treatment and/or prevention of hepatic disorders, in particular but not only NAFLD and/or NASH.

The subject to be treated with the composition of the invention is preferably mammal, in particular the human being.

According to another of its aspects, subject-matter of the invention is the use of Form A and Form B for oral administration, each Form once per day, for the treatment and/or prevention of hepatic disorders, in particular NAFLD and/or NASH. Preferably, the two different compositions, Form A and Form B, are administered in two different times of the day.

According to a preferred embodiment, for the use according to the invention, Form A is administered in the morning and Form B is administered at another time of day, preferably together with a meal, for example at noon or in the evening, preferably in the evening.

According to a preferred embodiment, Form A and Form B are solid oral compositions, for example tablets, hard or soft capsules, and powders or granules contained in mono-dose sachets.

According to another of its aspects, subject-matter of the invention is also a kit comprising at least two compositions as defined in any embodiment of the present invention, said at least two compositions being different from each other.

According to an embodiment, subject-matter of the invention is a kit comprising at least one composition according to Form A and a composition according to Form B, the number of the two different compositions being the same.

According to an embodiment, the kit comprises 1 to 100 compositions according to Form A and 1 to 100 compositions according to Form B, for example 7 to 50, preferably 10 to 30 of each composition, for example 14, 20 or 30 of each composition, the number of the compositions of Form A and Form B being the same. Preferably, the kit of the invention also comprises a leaflet also providing indications concerning posology of the two compositions A and B.

According to another of its aspects, subject-matter of the invention is the use of a kit comprising at least one composition according to Form A (preferably 1 to 100, for example 14, 20 or 30) and at least one composition according to Form B (preferably 1 to 100, for example 14, 20 or 30), for the treatment and/or prevention of the hepatic diseases, in particular but not only NAFLD and/or NASH, wherein said use comprises administering a composition according to Form A in the morning and a composition according to Form B at another time of day, preferably together with a meal, for example at noon or in the evening, preferably in the evening. The treatment according to the invention is preferably carried out in cycles for example of 2-3 months and repeated along the year.

According to another of its aspects, subject-matter of the invention is a method for the treatment and/or prevention of the hepatic diseases, in particular NAFLD and/or NASH, comprising administering the combination, compositions or mixtures as described above, in any one of the dosages and according to the posologies indicated above, to a subject in the need thereof. According to a preferred embodiment, the method of the invention uses the kit of the invention as defined herein.

According to an alternative embodiment, in the method of the invention the compositions of Form A and Form B are not contained in a single kit.

According to a preferred embodiment, subject-matter of the invention is a method for the treatment and/or prevention of the hepatic diseases, in particular NAFLD and/or NASH, comprising administering a composition Form A and a composition Form B at different times of the day, preferably Form A in the morning and Form B at another time of day, preferably together with a meal, for example at noon or in the evening, to a subject in the need thereof.

In vitro assays have been carried out on a human hepatic cell line by using the active ingredients herein described, thus obtaining optimal results in terms of protection from the fat absorption, inflammation, oxidative stress and glucose alteration.

The mixtures of the invention have also been tested in a screening for evaluating the in vivo protective effects on the mouse. In this assay the mixtures of the invention provided extremely interesting results demonstrating their actual effectiveness in the treatment and also in the prevention of hepatic disorders and in particular NASH and NAFLD.

Said assays are described in detail in the following Experimental Section.

The Experimental Section illustrates representative forms of the invention for non limiting purpose.

Experimental Section

Example 1

Representative composition of Form A

A single-dose sachet is prepared, each dose comprising - milk thistle (sylimarin titre at least 80%) : 200 mg;

- Coffea arabica (chlorogenic acid titre at least 2%): 70 mg;

- feverfew (parthenolides titre about 0.8%): 500 mg;

- choline hydrochloride: 200 mg,

possibly and preferably together with pharmaceutically acceptable conventional carriers and excipients.

Example 2

Representative composition of Form B

A tablet is prepared, each comprising

- turmeric (curcumin titre at least 20%) : 150 mg;

- DHA (75% purity): 300 mg;

- choline hydrochloride: 200 mg,

- DL-alpha-tocopheryl acetate: 60 mg;

possibly and preferably together with pharmaceutically acceptable conventional carriers and excipients.

Example 3

Representative composition of Form A

A single-dose sachet is prepared, each dose comprising

- milk thistle (sylimarin titre at least 80%): 150 mg;

- Coffea arabica (chlorogenic acid titre at least 2%): 80 mg;

- feverfew (parthenolides titre about 0.8%): 400 mg;

- choline hydrochloride: 150 mg,

possibly and preferably together with pharmaceutically acceptable conventional carriers and excipients.

Example 4

Representative composition of Form B

A tablet is prepared, each comprising

- turmeric (curcumin titre at least 20%) : 200 mg;

- DHA (75% purity): 400 mg;

- choline hydrochloride: 150 mg, - DL-alpha-tocopheryl acetate: 50 mg;

possibly and preferably together with pharmaceutically acceptable conventional carriers and excipients.

Example 5

Representative composition of Form A

A single-dose sachet is prepared, each dose comprising

- milk thistle (sylimarin titre at least 80%): 250 mg;

- Coffea arabica (chlorogenic acid titre at least 2%): 60 mg;

- feverfew (parthenolides titre about 0.8%): 600 mg;

- choline hydrochloride: 250 mg,

possibly and preferably together with pharmaceutically acceptable conventional carriers and excipients.

Example 6

Representative composition of Form B

A tablet is prepared, each comprising

- turmeric (curcumin titre at least 20%) : 100 mg;

- DHA (75% purity): 350 mg;

- choline hydrochloride: 250 mg,

- DL-alpha-tocopheryl acetate: 60 mg;

possibly and preferably together with pharmaceutically acceptable conventional carriers and excipients.

Example 7

Representative composition of Form A

A single-dose sachet is prepared, each dose comprising

- milk thistle (sylimarin titre at least 80%): 400 mg;

- Coffea arabica (chlorogenic acid titre at least 2%): 300 mg;

- feverfew (parthenolides titre about 0.8%): 400 mg;

- choline hydrochloride: 200 mg,

possibly and preferably together with pharmaceutically acceptable conventional carriers and excipients. Example 8

Representative composition of Form B

A capsule is prepared, each comprising

- turmeric (curcumin titre at least 20%) : 200 mg;

- DHA (75% purity): 400 mg;

- choline hydrochloride: 400 mg,

- DL-alpha-tocopheryl acetate: 60 mg;

possibly and preferably together with pharmaceutically acceptable conventional carriers and excipients.

Example 9

Combination of compositions

A combination of the compositions of Example 7 and Example 8 is prepared, for their daily administration, the composition of Example 7 in the morning and the composition of Example 8 in the evening.

Example 10

A kit is prepared by packing in the same container the combination of Example 9, preferably together with a leaflet illustrating the method of administration.

Example 11

A kit is prepared by packing in the same container 14 compositions of Example 7 and 14 compositions of Example 8, preferably together with a leaflet illustrating the method of administration.

Example 12

A kit is prepared by packing in the same container 1 or 5 or 7 or 10 or 15 or 30 compositions Form A and at least 1 or 5 or 7 or 10 or 15 or 30 compositions Form B, as defined herein, preferably together with a leaflet illustrating the method of administration.

Example 13

Kits are prepared by packing in the same container 1 or 5 or 7 or 10 or 15 or 30 compositions Form A of Example 7 and 1 or 5 or 7 or 10 or 15 or 30 compositions Form B of Example 8, preferably together with a leaflet illustrating the method of administration. Example 14

Kits are prepared by packing in the same container 30 sachets containing the composition Form A of Example 7 and 30 capsules containing the composition Form B of Example 8, preferably together with a leaflet illustrating the method of administration.

Example 15

Kits are prepared by packing in the same container 20 sachets containing the composition Form A of Example 7 and 20 capsules containing the composition Form B of Example 8, preferably together with a leaflet illustrating the method of administration.

Example 16

Evaluation of the in vitro effect in a hepato-steatosis model

Evaluation of the protective effects in vitro of a multicomponent mixture planned for intervening on the different aspects of the disorder. In particular, the mixture contains products able to prevent lipid load, reduce inflammatory processes, oxidative unbalance and the consequent glucose metabolism alteration.

The steatosis model was reproduced in vitro using the human hepatocellular carcinoma cell line HepG2. Cells were treated with 0.5 mM oleic acid (in l%BSA; OA/BSA) for 24 h (Kim et ak, J Agric Food Chem 2015;63:9729-39; Chung MY et al, Nutritional Researc, 4: 1-10, 2017; Vidyashankar et al, Toxicology in Vitro 27, 2013, 945-953).

The effect of 3 differently composed mixtures co-incubated with OA/BSA (24 h; preventive protocol) or incubated for 48 h suddenly after 24h treatment with OA/BSA (intervention protocol) was evaluated.

The components of the analyzed mixtures were:

- omega 3 fatty acid (DHA), choline chloride, Tanaceum parthenium extract (0.8% parthenolides), Sylibum marianum (80% sylimarin), Coffea arabica (green) extract (45% chlorogenic acid), Curcuma longa extract (20% curcumin); dl a-tocopheryl acetate: 7.5 pg/ml. Mixtures 1, 2, 3 present the same components and the same dose ratio among components but they are different in concentrations as depicted in Table 1 Table 1

The cell incubation for 24 h with OA/BSA did not significantly modify cell viability (Figure 1, preventive). On the other hand, after treatment, cells showed lesser proliferative activity than control condition. Mixtures 2 and 3 were able to restore the proliferative capabilities (Figure 1, intervention).

In Figure 2 is shown that OA/BSA treatment reduced the antioxidant properties of cells and in particular the mitochondrial one. Activity of superoxide dismutase (SOD) was reduced both after 24h treatment as well as when additional 48h of recovery were allowed. In the preventive protocol all mixtures were significantly protective, in the intervention similar results were obtained with mixtures 2 and 3.

The redox unbalance evoked a cell damage highlighted as measure of lipid peroxidation. As shown in Figure 3 OA/BSA induced a progressive increase of TBARS (thiobarbituric acid reactive substances) 24h after treatment and 48h after the recovery indicating that lipid damage is a persistent hallmark of pathology. When co-incubated with OA/BSA, the mixture evoked a protective, concentration- dependent effect (Figure 3, preventive). When incubated after damage, during the recovery period, the analyzed mixture was lesser effective. Mixture 3 only, was able to significantly improve the damage (Figure 3, intervention).

The inflammatory cytokines TNF-a and interleukin 8 were measured in the culture medium after treatments. Table 2 shows a significant increase of both after OA/BSA treatments, values were higher after 24h incubation and slightly decreased after the 48h of recovery. The presence of the mixtures as preventive as well as intervention was able to concentration-dependently reduced cytokines level. Table 2

Preventive, 24 hours co-incubation

Intervention, 24 h OA + 48 h mixture

TNF-a and IL-8 concentrations in cell medium. **P<0.0l vs. BSA + vehicle; L R<0.05 and LL R<0.01 vs. OA/BSA + vehicle.

Aimed to evaluate if the here described protective features of the mixture could be able to interfere also with the most relevant pathological signatures of metabolic syndrome, we measured alterations of glucides, cholesterol and triglycerides. As regards carbohydrates metabolism (Table 3), we evaluated the cell glucose uptake stimulated by insulin treatment. After OA/BSA incubation, the sensitivity to insulin was significantly reduced, mixtures (all concentrations) protected by this alteration in the prevention protocol. When the treatment started 24 after damage (intervention protocol) a concentration-dependent effects was observed reaching an almost complete rescue with mixture 3.

Table 3

Preventive, 24 hours co-incubation

Intervention, 24 h OA + 48 h mixture

Glucose uptake. **R<0.01 vs. BSA ± vehicle; L R<0.05 and LL R<0.01 vs. OA/BSA ± vehicle.

As regards the intracellular concentration of triglycerides, it was increased by OA/BSA. As shown in Figure 4, mixture 3 was the only able to significantly reduce the alteration when co-incubated with OA/BSA (preventive). On the contrary, the post-treatment of cells for 48h after the OA/BSA damage allowed to highlight a more effective role of mixtures (intervention)

OA/BSA increased the total cholesterol intracellular concentration (Figure 5), both protocols of treatment induced a significant protection.

The infarction of cells of lipidic substances was measured by staining cells with Oil red. As shown in the upper panels of Figure 6 and quantified in the lower graphs, OA/BSA strongly increased lipid accumulation (steatosis grade) immediately after 24h incubation. This effect appeared unchanged after 48h recovery. Both protocol treatments were efficiently protective, in particular mixture 3 in the intervention treatment was able to evoke a strong improvement of the steatosis grade (Figure 6). Methods

Cell culture

The cell line HepG2 (hepatocellular carcinoma cell line) was obtained by American Type Culture Collection (Manassas, VA, USA). Cells were cultured in Dulbecco modified Eagle medium (DMEM; GIBCO Life Technologies, Grand Island, NY, USA) with 10% (vol/vol) bovine fetal serum in the presence of 1% antibiotic- antimycotic solution at 37°C with 5% C02.

Steatosis induced by oleic acid

Confluent cells were treated with 0.5 mM oleic acid in the presence of l%albumin for 24h. Control condition was performed allowing incubation with albumin.

Cell viability (MTT)

Cell viability was evaluated by the reduction of 3-(4,5-dimethylthiozol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) as an index of mitochondrial compartment functionality. Post treatments, after three extensive washing, 1 mg/ml MTT was added into each well and incubated for 2 hours at 37 °C. After washing, the formazan crystals were dissolved in 100 mΐ dimethyl sulfoxide. The absorbance was measured at 580 nm. Experiments were performed in quadruplicate on at least 3 different cell batches.

Measurements of superoxide dismutase (SOD) activity

Total SOD activity was determined by monitoring the inhibition of the reduction of ferricytochrome C at 550 nm, using the xanthine-xanthine oxidase system as the source of superoxide. One unit of the SOD is defined as the amount of the enzyme required to inhibit 50% of the rate of cytochrome-C reduction (Flohe and Otting, 1984). Catalase activity was measured by following the rate of H202 consumption spectrophotometrically at 240 nm.

Evaluation of lipid peroxidation (Thiobarbituric acid reactive substances TBARS) After treatments, cells were scraped and cell suspension underwent a freeze/thaw cycle. The suspension was added to 4 ml reaction mixture consisting of 36 mM thiobarbituric acid (Sigma-Aldrich, Italy) solubilized in 10% CH3COOH, 0.2 % SDS, pH was adjusted to 4.0 with NaOH. The mixture was heated for 60 minutes at l00°C and the reaction was stopped by placing the vials in ice bath for 10 minutes. After centrifugation (at 1,600 x g at 4°C for 10 minutes) the absorbance of the supernatant was measured at 532 nm (Perkin-Elmer spectrometer) and TBARS were quantified in pmoles/milligram of total protein using 1, 1,3, 3 -tetramethoxy propane as standard.

Evaluation of pro-inflammatory cytokines

TNF-alfa and IL-8 were measured by using a commercially available enzyme-linked immunosorbent assays (Biosource, Italy).

Evaluation of glucose uptake

The cell uptake of 6-hydroxy glucose was measured a colorimetric reaction (Abeam, Italy).

Evaluation of steatosis grade by Oil red staining

After treatments cells were fixed in 4% formaldehyde for 15 min, then washed in PBS and incubated for 5 min with 60% isopropanol. Cells were treated with 0.1% oil red solution for 1 h. After an additional wash images were captured under a light microscope.

Evaluation of total cholesterol and triglycerides

After treatment, lipids of cells were extracted with chloroform: isopropanol: tween- 20 (7: 11 :0.1). Enzymatic methods applied to commercial kit were used to perform specific quantitative measurements (Biosource, Italy).

Example 18

Effect of the mixture in vivo in a mouse model of non-alcoholic steatohepatitis induced by the prolonged feeding of animals with a high fat diet (HFD. 60% fat).

The mixtures have been tested for their protective activities against metabolic and tissutal damages following a preventive (co-treatment) or a therapeutic (post-injury) protocol of administration.

The hepato-steatosis model was induced in C57BL/6 mice fed by a high fat diet (60% fat, 20% protein and 20% carbohydrate, as a percentage of total Kcal; Watanabe et al, 2012) in comparison to control mice fed with a standard diet (24% protein, 58% carbohydrate, 18% fat;).

Animals were fed for 12 weeks ad libitum (6-7 weeks of treatment need for developing metabolic alteration). The studied mixture was composed as follows:

- omega 3 fatty acid (DHA), choline chloride, Tanaceum parthenium extract (0.8% parthenolides), Sylibum marianum (80% sylimarin), Coffea arabica (green) extract (45% chlorogenic acid), Curcuma longa extract (20% curcumin); dl a-tocopheryl acetate. Two different dosages were tested, the final composition (as daily intake) of mixtures 1 and 2 (equal in type of components and dose ratio but different in quantities) is depicted in Table 4.

Table 4. Mixtures composition as daily intake

To obtain a better distribution of supplements during the day, the administration was divided in two parts, a morning treatment (lh after the light phase of the circadian light/dark cycle in the animal facility) and another in the evening lh before the dark phase. Components were split as follows:

Morning treatment

Evening treatment

Mixtures were suspended in 1% carboxymethylcellulose sodium salt and per os administered. Two protocols of treatment were scheduled:

Co-treatment: from week 1 for 12 weeks (days 1 - 84), concomitantly with HFD feeding

Post-injury: from week 7 (day 49, when metabolic alterations are established) to week 12 (day 84).

Groups:

1 ) N ormal di et + vehi cl e

2) HFD + vehicle

3) HFD + mixture 1 co-treatment

4) HFD + mixture 2 co-treatment

5) HFD + mixture 1 post-injury 6) HFD + mixture 2 post-injury

Body weight was measured every week during the treatment. HFD-fed animals showed a significantly increased body weight starting from day 30. The HFD-group that received the mixture 2 following the co-treatment protocol of administration presented a body weight lower in comparison to HFD + vehicle from day 41 to day 84. The mixture 2 administered following the post-injury protocol induced significant decrease from day 66 to day 78. Other treatments were ineffective on this parameter (Figure 7).

The intake of food was lower in HFD-fed animals in comparison to animals that received normal diet. No effects were shown by mixtures (Figure 8).

To evaluate the metabolic alterations induced by HFD, total cholesterol, triglycerides and glucose were monitored every week by blood analysis. Starting from day 42, all these parameters were enhanced with respect to normal diet-fed group. (Table 5). For this reason the post-injury treatment started on day 49 when hepatosteatosis syndrome was established. In Table 6, the effect of both mixtures, in the co-treatment and post-injury protocols, were reported till day 84. Group 4 (HFD + mixture 2 co- treatment) was fully active in normalize total cholesterol over time, as well as LDL measured on day 84. Group 3 (HFD + mixture 1 co-treatment) was active on days 70 and 77, Group 6 (HFD + mixture 2 post-injury) was active starting from day 70, the efficacy was maintained also on day 84 for total, LDL and HDL cholesterol. Similar consideration can be desumed by the analysis of triglyceride values in Table 7.

The modulation of glucose levels seems to be lower in comparison to lipids. As shown in Table 8, only group 4 was able to significantly reduced hyperglycemia from day 42 to day 84 without reaching the values of control. Group 6 was active from day 70 to day 84. Similarly, the ability to respond to a glucose load was preserved in group 4 and, at a lesser extent, in group 6 (Table 9).

Table 5

Blood dosages

Table 5. Blood dosages of cholesterol, triglyceride and glucose on day 42 (week 6). **P<0.0l vs. normal diet + vehicle

Table 6

Blood dosages

Table 6. Cholesterol blood levels over time (day 49 - day 84). **P<0.0l vs. group 1; LR<0.05 and LL R<0.01 vs. group 2. Groups of treatment: Group 1 : normal diet + vehicle; Group 2: HFD + vehicle; Group 3: HFD + mixture 1 co-treatment; Group 4: HFD + mixture 2 co-treatment; Group 5: HFD + mixture 1 post-injury; Group 6: HFD + mixture 2 post-injury.

Table 7

Blood dosages

Table 7. Triglyceride blood levels over time (day 49 - day 84). **P<0.0l vs. group 1; L R<0.05 and LL R<0.01 vs. group 2. Groups of treatment: Group 1 : normal diet + vehicle; Group 2: HFD + vehicle; Group 3: HFD + mixture 1 co-treatment; Group 4: HFD + mixture 2 co-treatment; Group 5: HFD + mixture 1 post-injury; Group 6: HFD + mixture 2 post-injury.

Table 8

L R<0.05 vs. group 2. Groups of treatment: Group 1 : normal diet + vehicle; Group 2: HFD + vehicle; Group 3: HFD + mixture 1 co-treatment; Group 4: HFD + mixture 2 co-treatment; Group 5: HFD + mixture 1 post-injury; Group 6: HFD + mixture 2 post-injury.

Table 9

Glucose tolerance test (Day 84)

HFD + vehicle.

On day 84, animals were sacrificed and ex vivo analysis were performed. The weight of organs involved in metabolic disorders were recorded. In Figure 9, a dramatic increase of liver weight after HFD diet is shown. It was dose-dependently prevented by mixture treatment when administration started from day 1. Mixture 2 was also able to significantly decrease liver weight also in the post-injury regimen. As regards heart weight, the HFD-dependent increase was prevented by mixture 2 co-treatment protocol (Figure 10). In Figure 11, the lack of alterations in the kidney weight. On the contrary, HFD increased the weight of adipose tissue measured as perigonadal fat pad, again mixture 2 in the co-treatment regimen was significantly protective (Figure 12). The histological evaluation of this peculiar adipose tissue revealed a HFD- dependent enlargement of adipocyte (cell area) reduced by mixture 2 in both protocols (Figure 13).

Focusing on liver, the histologic analysis showed a strong steatotic appearance of liver parenchima of HFD group. Damage was quantified in numbers by the steatosis index. The degree of steatosis was graded‘0’ to‘ 10’ based on the average percent of fat-accumulated hepatocyte per field at 20 c magnification under H&E staining (grading 0, <5% of fat accumulation and grading 10, >75% of fat accumulation) (Figure 14). Mixtures, both dosages in the co-treatment protocol, and the high dosage (mixture 2) in the post-injury one, were able to improve this condition.

To deep inside the effects of mixtures on liver protection, the concentrations of lipids in the parenchyma homogenate were measured (Table 10). HFD induced a 5 fold increase of LDL and HDL and a 6 fold increase of triglyceride. The co-treatment protocol allowed to observe a significant normalizing effect: mixture 2 was effective on all parameters, mixture 1 on HDL and triglycerides. The post-injury treatment was also active when dosed as mixture 2.

Moreover, mixture 2 (co-treatment protocol) was able to prevent the HFD-induced oxidative damage in the liver evaluated as lipidic oxidation (Figure 9); the alteration of proteins (carbonyl ation) was, dose-dependently, reduced by both treatments (Figure 10).

As regards the protection by oxidative damage, ex vivo analysis were performed also on heart. As shown in figures 11 and 12, mixture 2 significantly reduced lipid peroxidation and protein carbonylation after the co-treatment and the post-injury treatments.

Finally, the pro-inflammatory cytokines IL-8 and TNF-a were dosed in blood. Both protocols of treatment were able to decrease these general inflammation parameters (Table 11).

Table 10

Liver dosages (Day 84)

vs. normal diet + vehicle; L R<0.05 and LL R<0.01 vs. HFD + vehicle. Groups of treatment: Group 1 : normal diet + vehicle; Group 2: HFD + vehicle; Group 3: HFD + mixture 1 co-treatment; Group 4: HFD + mixture 2 co-treatment; Group 5: HFD + mixture 1 post-injury; Group 6: HFD + mixture 2 post-injury.

Table 11

Table 11. Cytokines blood concentration. **P<0.0l vs. group 1; L R<0.05 vs. group 2. Groups of treatment: Group 1 : normal diet + vehicle; Group 2: HFD + vehicle; Group 3: HFD + mixture 1 co-treatment; Group 4: HFD + mixture 2 co-treatment; Group 5: HFD + mixture 1 post-injury; Group 6: HFD + mixture 2 post-injury.

Conclusion

The multicomponent product tested in these experiments is effective in improving the different alterations induced in mice by HFD. A prolonged feeding with a high fat provision evokes a steatohepatitis state characterized by body weight increase, alteration of lipids and glucose levels, histopathological damage of liver and adipose tissue as well as a general oxidative and inflammatory condition. The tested product induces protective effects dependent by the dosage. An almost complete prevention is obtained when the mixture 2 is daily administered from day 1 (co-treatment treatment). Nevertheless, it is important to note that a significant recovery is also obtained when treatments start after the establishment of the metabolic alterations. Methods

Animals

Male C57BL/6 mice (Envigo, Varese, Italy) weighing approximately 20 g at the beginning the experimental procedure were used. Animals were used in Ce.S.A.L (Centro Stabulazione Animali da Laboratorio, University of Florence) and used at least one week after their arrival. Twelve mice were housed per cage (size 26 X 41 cm) kept at 23.0 ± 1.0 °C with a 12 h light/dark cycle, with lights on at 7 a.m.; during acclimatization they were fed a standard laboratory diet and tap water ad libitum.

All animals manipulations were carried out according to the Directive 2010/63/EU of the European parliament and of the European Union council (22 September 2010) on the protection of animals used for scientific purposes. The ethical policy of the University of Florence complies with the Guide for the Care and Use of Laboratory Animals of the US National Institutes of Health (NIH Publication no. 85-23, revised 1996; University of Florence assurance number: A5278-01). Formal approval to conduct the experiments described was obtained from the Animal Subjects Review Board of the University of Florence. Experiments involving animals have been reported according to ARRIVE guidelines (McGrath and Lilley, 2015). All efforts were made to minimize animal suffering and to reduce the number of animals used. Induction of metabolic syndrome

Metabolic syndrome was induced feeding the animals with a high fat diet (HFD; Research Diets, New Brunswick, NJ) for 12 weeks ad libitum. The model is consistent, with minor modification, to what was previously published (Rossmeisl et al, Diabetes 2003;52: 1958-1966; Fellman et al, Pharmacol Ther, 2013; 137:331- 340). HFD contains: 60% fat, 20% protein and 20% carbohydrate, as a percentage of total Real (Watanabe et al, 2012). Control animals were fed ad libitum for 12 weeks with a standard diet (24% protein, 58% carbohydrate, 18% fat; Envigo, Varese, Italy).

Body weight and food intake

During the experiment, the body weight of mice was measured every 3 days while the food intake was measured weekly.

Collection of blood and analytical methods

Blood was collected from the facial vein of the mice under light ether anaesthesia into Eppendorf tubes containing heparin (20 microl, 25000 IU/5 ml) weekly starting from week 1. Samples were analyzed for glucose, triglycerides and total cholesterol levels using a Reflotron reflectance photometric analyser (Reflotron, Roche Diagnostics and Vitros, Johnson & Johnson). LDL, HDL cholesterol were measured using the same method only on week 12 (day 84).

Glucose tolerance tests

On week 12, the glucose tolerance test were performed by intraperitoneally injections of glucose (1 mg kg-l). Blood glucose was measured by Accu-Check Aviva at 0 min, 15 min and 30 min after glucose injections.

Tissues explant

On week 12 (day 84), after the behavioural measurements and the biochemical analysis, mice were sacrificed by cervical dislocation and tissues explant was performed. In particular, perigonadal fat pad, liver, heart, and kidney were collected. Liver histopathology

Liver tissue samples were fixed in 4% formalin and processed for embedding and sectioning. Liver sections (5 microns) were stained with hematoxylin and eosin (H&E). The quantitative scoring of H&E-stain based liver tissue sections was conducted according to the previous methods with slight modifications, regarding steatosis (Brunt, 2001; Kleiner et al, 2005). Briefly, each section was examined by a specialist who was blinded to the sample information and hepatic steatosis and inflammation scores were evaluated. The degree of steatosis was graded‘O’ to‘10’ based on the average percent of fat-accumulated hepatocyte per field at 4 or 20 c magnification under H&E staining (grading 0, <5% of fat accumulation and grading 10, >75% of fat accumulation).

Lipid levels in liver.

Liver homogenate was dosed for triglycerides, LDL and HDL cholesterol using a Reflotron reflectance photometric analyser (Reflotron, Roche Diagnostics and Vitros, Johnson & Johnson).

Adipose tissue histopathology

The perigonadal fat pad was collected for histological and biochemical analyses. For histology, the tissues were drop-fixed in 10% neutral buffered formalin for 2-3 days, after which they were processed for paraffin embedding. Sections (5 pm) were then cut, collected, stained with hematoxylin and eosin, and digitized. Adipocyte size was measured by an investigator blinded to the experimental grouping in x40 microscope fields by counting the total number of adipocytes within predefined area grids and then dividing the area by the total number of adipocytes within the grids to calculate average adipocyte size. For each sample, three replicate tissue sections were analyzed, with three fields counted in each section, for a total of nine fields averaged per sample.

Lipid peroxidation (thiobarbituric acid-reactive substances assay)

Thiobarbituric acid-reactive substances (TBARS) assay was assessed as an index of lipid peroxidation according to Cinci et al. (2015). The TBARS determination was carried out in mice liver and heart homogenate obtained from tissue homogenized in PBS at the final concentration of 10% w/v. Then were added FeCl3 (20 mM, Sigma- Aldrich, St. Louis, MO, USA) and ascorbic acid (100 mM, Sigma-Aldrich) to obtain the Fenton reaction. At the end of incubation, the mixture was added to 4 ml reaction mixture consisting of 36 mM thiobarbituric acid (Sigma-Aldrich) solubilized in 10% CH3COOH, 0.2% SDS, and pH was adjusted to 4.0 with NaOH. The mixture was heated for 60 min at l00°C and the reaction was stopped by placing the vials in an ice bath for 10 min. After centrifugation (at 1600 g at 4°C for 10 min) the absorbance of the supernatant was measured at 532 nm (PerkinElmer spectrometer) at 550 nm (PerkinElmer spectrometer) and TBARS were quantified in pmol/mg of total proteins using l,l,3,3-tetramethoxypropane as the standard.

Carbonylated proteins

Carbonylated proteins were evaluated in liver and heart tissue homogenate. Tissue proteins extract was quantified by BCA. Twenty pg of each sample were denatured by 6% SDS and derivatized by 15-minute incubation with 2-4 dinitrophenyl hydrazine (DNPH; Sigma-Aldrich, Italy) at room temperature. Samples were separated on a 4-12% sodium dodecyl sulfate (SDS)-polyacrylamide gel by electrophoresis and transferred onto nitrocellulose membranes (Biorad, Italy). Membranes were blocked with 5% non fat dry milk in phosphate-buffered saline (PBS) containing 0.1% Tween 20 (PBST) and then probed overnight with primary antibody specific versus DNPH (Sigma-Aldrich, Italy) 1 : 5000 in PBST/5% non fat dry milk. After washing with PBST, the membranes were incubated for 1 hour in PBST containing the appropriate horseradish peroxidase-conjugated secondary antibody (1 : 5000; Cell Signalling, USA) and again washed. ECL (Pierce, USA) was used to visualize the peroxidase-coated bands. Densitometric analysis was performed using the“ImageJ” software (NIH Bethesda, Maryland, USA). For each experiment the density of all bands showed in a lane was reported as mean. The total protein amount quantified by Ponceau staining was used to normalize all samples.

Evaluation of pro-inflammatory cytokines

TNF-alfa and IL-8 were measured in plasma by using a commercially available enzyme-linked immunosorbent assays (Biosource, Italy).

Statistical analysis Measurements were performed on 12 animals for each group analyzed in 2 different experimental sets by researchers blinded to the treatment procedure. Results were expressed as means ± S.E.M. and the analysis of variance was performed by ANOVA test. A Bonferroni's significant difference procedure was used as a post hoc comparison. P values less than 0.05 were considered significant. Data were analyzed using the“Origin 8.1" software.