WO2021220050A1

WO2021220050A1 - New compositions useful for treating elevated blood lipid levels

Info

Publication number: WO2021220050A1
Application number: PCT/IB2021/000205
Authority: WO
Inventors: Yvonne Beata Silber; Kaylee AZZOPARDI; Kristina GORGIEVSKA; Molly FAIRWEATHER
Original assignee: Health And Happiness (H&H) Hong Kong Limited
Priority date: 2020-05-01
Filing date: 2021-04-20
Publication date: 2021-11-04

Abstract

Provided herein are new compositions that can be employed in the treatment and/or prevention of a disorder or abnormality associated with elevated blood lipid levels such as hypercholesterolemia, wherein the elevated blood lipid levels are the levels of total cholesterol (TC) and/or LDL cholesterol and/or triglycerides. The composition is adapted for oral administration and can be a nutritional or a dietary supplement.

Description

New Compositions useful for Treating Elevated Blood Lipid Levels

Field of the invention

The present invention relates to a novel composition that can be employed in the treatment and/or prevention of a disorder or abnormality associated with elevated blood lipid levels such as hypercholesterolemia, wherein the elevated blood lipid levels are the levels of total cholesterol (TC) and/or LDL cholesterol and/or triglycerides.

The instant composition is adapted for oral administration and can be a nutritional or a dietary supplement.

Background of the invention

Cholesterol is an essential fat that is a component of cell membrane structure as well as being a precursor for the synthesis of steroid hormones, Vitamin D and bile acids (Gandy, J. (2019). Manual of dietetic practice. John Wiley & Sons). Cholesterol is supplied in small amounts in the diet, which is absorbed into the bloodstream via the gastrointestinal tract (Gandy, J. (2019). Manual of dietetic practice. John Wiley & Sons).

Most of the cholesterol (about 85%) is endogenously produced in hepatic cells (Marieb, E. N., & Hoehn, K. (2010). Human anatomy & physiology, ed. B. Cummings 2010, San Francisco, California). This process occurs through the conversion of Acetyl Co-A to cholesterol via the mevalonate pathway. An integral rate-limiting step in this pathway is the conversion of Acetyl- CoA to 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) via the enzyme HMG-CoA reductase.

Cholesterol is transported in the bloodstream via lipoprotein particles, of which there are two broad types (Gandy, J. (2019). Manual of dietetic practice. John Wiley & Sons):

1. Low-density lipoprotein (LDL) cholesterol transports cholesterol that is delivered to cells. LDL cholesterol particles are pathogenic as they can clog the arteries if circulating levels are too high in the blood. l 2. High-density lipoprotein (HDL) cholesterol assists in recycling excess cholesterol from cells to the liver for excretion, which helps to regulate blood cholesterol levels.

High levels of total cholesterol (TC) and low-density lipoproteins (LDL-C) circulating in the bloodstream can damage the arterial endothelium and lead to the formation of plaques which is termed atherosclerosis (Gandy, J. (2019). Manual of dietetic practice. John Wiley & Sons).

High cholesterol is an independent risk factor for both cardiovascular and cerebrovascular disease due to the narrowing of blood vessels which can impede blood circulation to the heart and brain.

To date, cardiovascular disease (hereafter CVD) remains the number one cause of death and disease at a global level. An astounding one in three deaths (18 million; 31%) globally are a result of CVD of which 85% are caused by heart attack or stroke, yet the majority of CVD is preventable (World Heart Federation; WHO, 2019).

In 2017-2018, the Australian Bureau of Statistics found that 6.1% of all Australians (1.5 million people) had hypercholesterolemia (total cholesterol > 5.5mmol/L) (Australian Bureau of Statistics (2019). 4364.0.55.001 - National Health Survey: First Results, 2017-18). According to the World Health Organisation, in 2008 the global prevalence of raised total cholesterol among adults (³ 5.0 mmol/I) was 39%. High blood cholesterol levels double the risk of health disease, with 4.5% of all deaths globally attributed to raised cholesterol levels. Elevated cholesterol is a well-known health issue in Western developed countries and has also emerged as a growing concern in developing countries, with countries such as India and China showing increased prevalence of dyslipidemia- one study found that Indian adults experienced dyslipidemia at rates as high as 30% in urban areas (Gupta, Rao, Misra & Sharma, Indian Heart Journal 69 (2017) 382-392; Zhang et a!., International Journal of Cardiology 260 (2018) 196-203).

The proportion of people with high cholesterol increases to 14.1% among those aged 55-64 years and 21.2% among those aged 65 and over (Australian Bureau of Statistics (2019). 4364.0.55.001 - National Health Survey: First Results, 2017-18). This trend is likely to be observed globally, since the number of people aged 60 or older will rise from 900 million to 2 L billion from 2015 to 2050 and encompass 22% of the global population and age is a major risk factor for dyslipidemia and its related illnesses (World Health Organisation, 2018).

Current treatment for hypercholesterolemia involves dietary and lifestyle advice as a primary intervention, with pharmacological therapies such as HMG-CoA reductase inhibitors (i.e. statin medications) as secondary therapy. These medications function by significantly reducing hepatic cholesterol production, although statin therapy is associated with deranged glucose metabolism, skeletal muscle and neurological side effects (Thompson, P. D., Panza, G., Zaleski, A., & Taylor, B. (2016). Statin-associated side effects. Journal of the American College of Cardiology, 67(20), 2395-2410).

FR2936711 refers to a composition that comprises a combination of hypolipidemic agents including at least one dry extract of artichoke leaves and red yeast rice, for the treatment and/or prevention of hypercholesterolemia.

Given the high prevalence of high cholesterol globally and the side effects of current pharmacological treatments, there is a need for safe, natural alternatives which target cholesterol metabolism via multiple mechanisms in conjunction with a healthy diet and lifestyle.

Brief description of the drawings

Fig. 1 is a graphic comparison between treatment groups on serum lipid outcomes; MCT means medium chain triglycerides.

Fig. 2 is a graphic comparison between treatment groups on serum total cholesterol and non- HDL; MCT means medium chain triglycerides.

Fig. 3 is a graphic comparison between treatment groups on serum oxidative stress markers ; MCT means medium chain triglycerides.

Fig. 4 is a graphic comparison between treatment groups on faecal cholesterol levels; MCT means medium chain triglycerides.

Fig. 5 is a graphic comparison between treatment groups on bile acid excretion; MCT means medium chain triglycerides.

Summary of the invention

It is an object of the present invention to provide a composition that can be employed in the treatment and/or prevention of a disorder or abnormality associated with elevated blood lipid levels such as hypercholesterolemia, wherein the elevated blood lipid levels are the levels of total cholesterol (TC) and/or LDL cholesterol and/or triglycerides. Furthermore, there exists a need in the art for a natural composition which can be used as therapeutic agent for reducing the levels of total cholesterol (TC) and/or LDL cholesterol and/or triglycerides without the side effects of current pharmacological treatments. The present inventors have surprisingly found that this object can be achieved by the composition described hereinafter.

The composition makes use, in general, of four distinct sources of active ingredients: a) Artichoke leaf extract, b) Olive fruits extract, c) Bergamot juice extract, and d) Plant sterols.

Each one of these ingredients are known to have a beneficial effect in the treatment of hypercholesterolemia. Below is reported a summary for the evidences related to the activity of the single ingredients: a) Artichoke leaf extracts (ALE)

ALE is a powder extract of the Cynara cardunculus var. Altilis DC. This cultivar has particularly high concentration of bioactive compounds, notably chlorogenic acids and luteolin- 7-glucoside and derivatives. This extract exerts an effect on HMGR by altering gene expression (Castellino et al., Nutrients, 2019, 11(11), 2580) and reducing cholesterol biosynthesis (Gebhardt, J Pharmacol Exp Ther. 1998, 286(3), 1122-8; Gebhart, Phytotherapy research, 2002, 16(4), 368-372), which is similar to the action of bergamot.

However, much of the preclinical literature on artichoke highlights the ingredient's role in hepatoprotection. This includes beneficial effects on liver enzymes (Castellino et al., Nutrients 2019, 11(11), 2580), protecting against hepatic oxidation (Salem et al., BMC Complementary and Alternative Medicine, 2017, 17(1), 328; Tang et al. Nutrients, 2017, 9(9), 1000; Kucukgergin et al., Biol Trace Elem Res, 2010, 135(1-3), 264-274).

It is important to note that this intracellular antioxidant activity is distinct from the action of hydroxytyrosol (and its derivatives) - see below - which prevents oxidation of LDL molecules in the bloodstream and not the liver. Studies have found that the effects on biomarkers result in histopathological changes in the liver such as reducing lipid droplet and fatty streak formation in the liver (Salem et al., BMC complementary and alternative medicine, 2017, 17(1), 328; Tang et al. Nutrients, 2017, 9(9), 1000). Given the integral role of the liver in cholesterol biosynthesis and processing of dietary cholesterol, these findings are of clinical importance. b) Olive fruits extracts

Hydroxytyrosol (HT) is polyphenol compound, which is found in high concentrations in olive oil. It acts via preventing oxidation of LDL particles which are known to be particularly atherogenic. Adhesion of oxidized LDL molecules to the blood vessel wall contributes to the formation of plaques, which is a key feature of atherosclerosis.

An EFSA claim was released in 2011, stating that a cause-and-effect relationship was established between olive oil polyphenols (standardized to a minimum of 5mg HT and its derivatives e.g., tyrosol) and the prevention of LDL cholesterol molecules from oxidative damage (EFSA Journal 2011, 9(4), 2033). This claim was supported by clinical trials comparing extra virgin olive oil (with high polyphenol content) and refined polyphenol content.

Specific to HT, preclinical research has substantiated the effect of this ingredient on lipid oxidation (Peyrol, Riva & Amiot, Nutrients, 2017, 9(3), 306; Tabernero et al., Food & function, 2014, 5(7), 1556-1563; Fki, Sahnoun & Sayadi., Journal of agricultural and food chemistry, 2007, 55(3), 624-631; Fucelli, Fabiani & Rosignolo, Molecules (Basel, Switzerland), 2018, 23(12),

3212). Furthermore, human clinical research has also demonstrated this benefit. In an acute- dose study Mateos et al. (2016) (Mateos et al., Food chemistry, 2016, 205, 248-256) demonstrated a significant reduction in oxidized LDL in the blood of healthy participants treated with a fortified biscuit containing 5.25mg HT (at certain time points). Another trial conducted by Vazquez-Velasco et al. (2010) (Vazquez-Velasco et al., British journal of nutrition, 2010, 105(10), 1448-1452) demonstrated the HT significantly reduced serum oxidised LDL levels after 3 weeks compared to the control group. c) Bergamot fruit extracts

Citrus bergamia extract, derived from bergamot juice, acts via inhibiting the effect of 3- hydroxy-3-methylglutaryl-CoA reductase (HMGR), which is a HMGR rate-limiting enzyme that catalyzes the synthesis of cholesterol molecules in the liver. Bergamot juice contains various amounts of the flavonoids brutieridin, melitidin, neohesperidin, naringin and neoeriocitrin. Brutieridin and melitidin have been shown to have similar molecular structures to statin molecules via a study which employed functional density theory at the HMGR binding site (Leopoldini, Malaj, Toscano, Sindona & Russo, Journal of agricultural and food chemistry, 2010, 58(19), 10768-10773).

This was further supported by an in vivo study published by Di Donna et al. (2014) (Di Donna et al., Journal of functional foods, 2014, 7, 558-568). Results demonstrated that the simvastatin and bergamot treatment groups produced similar modulatory effects on gene expression of HMGR reductase in a rodent model. The mechanism has been substantiated by Gliozzi et al., (2013) in a clinical trial (Gliozzi et al., International journal of cardiology, 2013, 170(2), 140-145). After treatment with bergamot extract (standardized to neohesperidin, narigin and neoeriocitrin) participants had significantly decreased mevalonic acid excretion compared to placebo. Since mevalonic acid is the product of the reaction that HMGR catalyzes, the results confirm that bergamot contributed to the inhibition of this enzyme in the cholesterol synthesis pathway. d) Plant sterols

Plant sterols are a class of lipid compounds analogous in structure to cholesterol molecules. This ingredient is commonly employed across the supplement and food industry in cholesterol-reducing products. The European Food Safety Authority released a scientific report in 2009 concluding that, based on human clinical trials, plant sterols decrease serum LDL cholesterol levels in doses above 1.5 grams per day (EFSA Journal, 2009, 7(7), 1175).

Preclinical evidence demonstrates that plant sterols act via competitive inhibition of cholesterol through micelle displacement, displacing cholesterol from bile, and decreasing hydrolysis of cholesterol esters in the small intestine (Smet, Mensink & Plat, Molecular nutrition & food research, 2012, 56(7), 1058-1072; Calpe-Bierdel, Escola-Gil & Blanco-Vaca, Atherosclerosis, 2009, 203(1), 18-31). Each of these mechanisms ultimately lead to increased faecal cholesterol excretion. This effect has been further substantiated by clinical research (Racette et al., The American journal of clinical nutrition, 2010, 91(1), 32-38).

Evidence has shown that increased faecal excretion increases endogenous cholesterol production as a compensatory mechanism (Baumgartner, Mensink & Plat, Current pharmaceutical design, 2011, 17(9), 922-932). The composition of the present invention mitigates this effect via the action of bergamot and artichoke on 3-hydroxy-3-methylglutaryl- CoA reductase (HMGR), an enzyme involved in cholesterol synthesis.

In summary, the idea of combining at least three of if not all the above mentioned four ingredients is based on the facts that: a) ALE has been shown to exert strong hepatoprotective effects on the liver through potent antioxidant capacity specific to hepatocytes; b) Olive fruits extract acts via preventing oxidation of the LDL particles in the bloodstream, therefore with the use of the planned formulation any LDL cholesterol that is absorbed into the bloodstream will be less atherogenic and less likely to cause endothelial damage; c) Bergamot fruit extract has been included in the formulation based on its inhibitory effect on HMGR; and d) Plant sterols inhibit cholesterol absorption in the intestinal lumen to increase fecal excretion.

It is well established that negative feedback mechanisms increase hepatic cholesterol biosynthesis in response to the increased excretion induced by plant sterols. However, with the claimed composition, without wishing to be bound by theory, the mechanisms by which bergamot and artichoke work, will offset this compensatory process.

The proposed novel composition targets different pathways of cholesterol metabolism and these pathways, without wishing to be bound by theory, are believed to potentiate each other to product a stronger effect in reducing blood lipid levels, wherein the elevated blood lipid levels are the levels of total cholesterol (TC) and/or LDL cholesterol and/or triglycerides, therefore producing a synergistic effect when these ingredients are administered in combination. The proposed novel composition may also have a synergistically stronger effect on markers of inflammation clinically relevant to cardiovascular disease risk such as high- sensitivity C-reactive protein.

A preclinical study was performed, that investigated the effects of a novel nutraceutical combination i.e., a composition according to the invention (identified as "Nutra+" here below) on low-density lipoprotein-cholesterol (LDL)-cholesterol and other markers of cardiometabolic health in a rodent model of hypercholesterolemia. The latter was aimed to determine the synergistic effects of ingredients the selected nutraceutical combination on serum LDL- cholesterol concentration in a rodent model of hypercholesterolemia.

Outcomes of same are briefly outlined as follows (details are disclosed in Example 3):

- Chronic consumption of Nutra⁺ for 6 weeks lowered serum total cholesterol by 15% and LDL-cholesterol by 20% compared to MCT fed animals.

- Chronic consumption of Nutra⁺ for 6 weeks did not affect negatively the excretion of cholesterol and bile acids or measures of inflammation and liver function or damage.

- Chronic consumption of Nutra⁺ for 6 weeks but not its individual components lowered the concentration of liver MDA compared to MCT fed animals.

Definitions

Within the meaning of the present application the following definitions apply: 'Elevated blood lipid levels' refers to >5mmol/L total cholesterol and/or >2 mmol/L LDL- cholesterol (World Health Organization, 2020; National Stroke Foundation, 2012).

'Altilix*', from the supplier BIONAP:

• Artichoke ( Cynara cardunculus var. Altilis DC) leaf extract that makes makes up 45-60% of the ingredient: o Containing chlorogenic acid and derivates in an amount ranging from 10 to 12% and o Luteolin 7-glucoside and derivates in an amount ranging from 2 to 4%

• Maltodextrin in an amount ranging from 40 to 55% of the ingredient.

'Hydrovas™10', from the supplier BIONAP:

• Olive fruits ( Olea Europea) extract that makes up 60-70% of the ingredient: o Containing hydroxytyrosol and tyrosol in an amount ranging from 10 to 12% and

• Maltodextrin in an amount ranging from 30 to 40% of the ingredient.

'Bergavit^®40', from the supplier BIONAP:

• Bergamot (Citrus bergamia) extract that makes up 75-81% of the ingredient: o Containing citrus flavonoids, such as neohesperidin, naringin and neoeriocitrin, in an amount ranging from 39 to 42% and

• Maltodextrin in an amount ranging from 30 to 40% of the ingredient.

'Natural occurring' compounds are produced by nature without any human intervention.

'Synthetic compounds' are made by humans using methods different than those nature uses, and these Chemical structures may or may not be found in nature.

The patients or subjects in the present invention are typically animals, particularly mammals, more particularly humans.

The preferred definitions given in the 'Definitions' - section apply to all of the embodiments described below unless stated otherwise.

Detailed description of the invention

The composition or combinations of the present invention will be described in the following embodiments. In one embodiment, the present invention relates to a composition comprising at least three, preferably all four of the following ingredients: a) chlorogenic acid, chlorogenic acid derivatives and luteolin-7-glucoside; b) hydroxytyrosol and tyrosol; c) citrus flavonoids, such as neohesperidin, naringin and neoeriocitrin; and d) phytosterol esters.

In a preferred embodiment, the components listed in a) are synthetic or naturally occurring. Preferably they are naturally occurring, more preferably an artichoke leaf ( Cynara cardunculus var. Altilis DC) extract is the natural source. In a further preferred embodiment, the naturally occurring source of Artichoke leaf extract is Altilix™.

In a further preferred embodiment, the components listed in b) are synthetic or naturally occurring. Preferably they are naturally occurring, more preferably an olive fruits ( Olea Europea) extract is the natural source. In a further preferred embodiment, the naturally occurring source of Olive fruits extract is Hydrovas™10.

In a further preferred embodiment, the components listed in c) are synthetic or naturally occurring. Preferably they are naturally occurring, more preferably a bergamot ( Citrus bergamia) extract is the natural source. In a further preferred embodiment, the naturally occurring source of Bergamot juice extract is Bergavit^®.

In a further preferred embodiment, the components listed in d) are synthetic or naturally occurring. Preferably they are naturally occurring, more preferably a vegetable oils extract, nuts or seeds extract, even more preferably sunflower oil extract, is the natural source.

In a further embodiment, the composition according to the invention may include any one of: a) artichoke leaf (Cynara cardunculus var. Altilis DC) extract as the source of chlorogenic acid, chlorogenic acid derivatives and luteolin-7-glucoside; b) olive fruits (Olea Europea) extract as the source of hydroxytyrosol and tyrosol; c) bergamot (Citrus bergamia) extract as the source of citrus flavonoids, such as neohesperidin, naringin and neoeriocitrin; and d) vegetable oils extract, nuts or seeds extract, preferably sunflower oil extract, as the source of phytosterol esters. In a further preferred embodiment, a) the chlorogenic acid, the chlorogenic acid derivatives are in an amount ranging from 0.63 to 0.76% w/w and the luteolin-7-glucoside is in an amount ranging from 0.13 to 0.25% w/w.

In a further preferred embodiment, b) the hydroxytyrosol and tyrosol are in an amount ranging from 0.21 to 0.25% w/w.

In a further preferred embodiment, c) the citrus flavonoids, such as neohesperidin, naringin and neoeriocitrin, are in an amount ranging from 6.16 to 6.64% w/w.

In a further preferred embodiment, d) the phytosterol esters are in an amount of 73.53% w/w.

In a further embodiment, the present invention relates to a composition comprising at least three if not all four of: a) an artichoke leaf (Cynara cardunculus var. Altilis DC) extract as the source of chlorogenic acid, chlorogenic acid derivatives in an amount ranging from 0.63 to 0.76% w/w and luteolin-7- glucoside in an amount ranging from 0.13 to 0.25% w/w; b) an olive fruits ( Olea Europea) extract as the source of hydroxytyrosol and tyrosol in an amount ranging from 0.21 to 0.25% w/w; c) a bergamot (Citrus bergamia) extract as the source of citrus flavonoids, such as neohesperidin, naringin and neoeriocitrin in an amount ranging from 6.16 to 6.64% w/w; and d) a vegetable oils extract, a nuts or seeds extract, preferably a sunflower oil extract, as the source of phytosterol esters in an amount of 73.53% w/w.

The novel composition of the present invention is also reported in the following Table 1.

Table 1 (see following page)

In a further embodiment, the present invention relates to a composition further comprising maltodextrins.

In a further preferred embodiment, the present invention relates to a composition comprising: a) Altilix™ preferably in an amount of 6.3% w/w b) Hydrovas™10 preferably in an amount of 2.1% w/w; c) Bergavit^®40 preferably in an amount of 15.8% w/w; and d) phytosterol esters, preferably in an amount of 75.8% w/w.

The above combination, identified as "Nutra+" from that line on, was selected for the predinical trials reported below in Example 3.

In a further embodiment, the composition according to the invention is suited for oral administration in the form of e.g. tablets, capsules (comprising push-fit capsules made of gelatine as well as soft, sealed capsules made of gelatine and a plasticizer, such as glycerol or sorbitol), pills, dragees, gels, slurries, suspensions, and the like, for oral ingestion by a patient, either as a medicament or a nutritional or dietary supplement. Preferably tablets and capsules.

In a further embodiment, the composition according to the invention is for use in the treatment and/or prevention of a disorder or abnormality associated with elevated blood lipid levels, in particular the levels of total cholesterol (TC) and/or LDL cholesterol and/or triglycerides, more particularly the levels of total cholesterol (TC) and/or LDL cholesterol.

In a further embodiment, the composition according to the invention has a cholesterol lowering activity similar to that of statins without having adverse side effects.

Further embodiments of the invention shall appear either in the lines below or in the claims. Preferred compositions are also illustrated in the examples. Any combination of the embodiments, preferred embodiments and more preferred embodiments disclosed herein is also envisaged in the present invention.

Compositions according to the invention

The compositions according to the invention could normally, but not necessarily, be formulated into pharmaceutical compositions prior to administration to a patient but preferably formulated as a nutritional or dietary supplement.

In a further aspect the invention is directed to a composition for the treatment and/or prevention of a disorder or abnormality associated with elevated blood lipid levels, wherein the elevated blood lipid levels are the levels of total cholesterol (TC) and/or LDL cholesterol and/or triglycerides, comprising the composition of the present invention. Examples of the diseases and conditions which can be treated or prevented include, but are not limited, to hypercholesterolemia which is a risk factor for atherosclerosis, cardiovascular disease, cerebrovascular disease and peripheral vascular disease.

The composition of the present invention will typically be formulated into a dosage form adapted for oral administration such e.g. as tablets, capsules (comprising push-fit capsules made of gelatine as well as soft, sealed capsules made of gelatine and a plasticizer, such as glycerol or sorbitol), pills, dragees, gels, slurries, suspensions, and the like, for oral ingestion by a patient.

When prepared in unit dosage form, the pharmaceutical compositions or preferably the nutritional or dietary supplement according to the invention, typically may contain, for example, from 0.1 g to 5 g, or from 0.5 g to 4 g, or from 1 g to 3 g of the composition of the present invention. The unit dose may be administered, for example, 1 to 6 times per day.

The compositions of the invention are prepared using techniques and methods known to those skilled in the art. Some of the methods commonly used in the art are described in Remington's Pharmaceutical Sciences (Mack Publishing Company).

Within the context of the present invention, the term 'nutritional composition' or 'synthetic nutritional composition' means a composition which may be consumed by a subject and providing such subject with macro (for example lipids, proteins, carbohydrates) and/or micro (for example vitamins, minerals) nutrients. Non-limiting examples of nutritional compositions according to the present invention include: a dietary supplement.

Preferred compositions are illustrated in the examples.

The compositions of the present invention can be prepared by the general method described below. This method is only given for illustrative purposes and should not to be construed as limiting.

• All raw materials containing bioactive components and excipients are received as bulk from supplier.

• Raw materials are combined into a slurry liquid by the contract manufacturer as per standard process, the slurry is assessed by Quality Control and if passed then slurry is released for encapsulation.

• Softgel mass is prepared and checked for correct formulation, colour match and viscosity. • Slurry and softgel mass are brought together and made into capsules.

• Capsules are sent to drying room and dried for the appropriate time

• Capsules are graded and sent to QC for inspection, once passed, capsules are packed as finished goods.

The invention is illustrated by the following examples which, however, should not be construed as limiting.

Examples: All reagents and solvents were obtained from commercial sources and used without further purification.

Example 1: Preparation of the composition

The fbllowing composition has been prepared (Table 2):

Table 2

Example 2: Preparation of the nutritional or dietary supplement

Gelatine Preparation manufacturing process for soft gel manufacturing

• Materials for the gelatine capsule (glycerine, gelatine, binders and emulsifiers) are sourced.

• A mixing vessel is preheated to 75°C using glycerine deionised water then all materials are heated to this temperature to allow for the cooking and mixing process.

• Materials are then added to a gel cooker (also heated to 75°C) and heated/cooked for 45 minutes at a vacuum of -90kPa. The resulting mixture is then stirred manually to ensure homogeneity at this early stage.

• The mixture is then transferred to a gel receiver which is heated to 60°C and held for 6- 8 hours.

• The mixture is then transferred to a gel receiver with a high shear mixture device and mixed. This receiver is heated to 60°C. This resulting processed mixture is then ready for encapsulation once the medicinal ingredients are processed.

Medicine preparation:

The raw material inputs are received from the raw material supplier and added to a medicine mixing vessel to ensure homogeneity when incorporated into the gelatine preparation.

Encapsulation:

• The above two inputs (medicinal mixture and gelatine mixture is then sent to be encapsulated. Encapsulation process is as follows: o Lubricant is added to the gelatine solution. All three inputs are added to a film forming spreader box and heated to 60°C o The mixture is going to capsule filling at a wedge temperature of 39°C, at a rate of 3rpm. It is cooled to 16°C. o The resulting mixture is added to a tumbler at 25-32°C at a rate of 4rpm for 20 minutes o The mixture is then dried at a temperature of 20°C and a humidity of 20%RH. o The resulting soft gel product is then sent for sorting and packing.

Example 3: preclinical trials design

To investigate, as primary objective, the potential synergistic effects of ingredients from Nutra⁺ on serumLDL-cholesterol concentration in a rodent model of hypercholesterolemia and, as secondary objectives among others, to investigate selected bio-markers like : - Serum lipid markers: total cholesterol, HDL-cholesterol, triglycerides, non-HDL- cholesterol(calculated), total cholesterol: HDL-cholesterol (calculated) ;

- Oxidative stress markers: plasma LDL-oxidation, liver malondialdehyde (MDA); and

To investigate further on whether any effects of Nutra⁺ treatment serum LDL-cholesterol were due to changes in:

- Faecal cholesterol and bile acids (measure of cholesterol absorption and excretion) and LDL-receptor gene expression (measure of cholesterol uptake in liver.

The use of animals in this study was approved by the University of Adelaide Animal Experimentation Ethics Committee (Science) under approval number S-2020-084. All experimentalprocedures including the care, handling and maintenance of the experimental animals were performed according to the National Health and Medical Research Council (NHMRC) guidelines forthe use and care of animals for experimental purposes.

The Wistar rat was chosen as it is an outbred albino strain common in laboratory research and is the preferred model for metabolic, physiology and pharmacological research and commonly usedin dietary studies investigating the effects of diets on cholesterol metabolism. In order to minimize variation among rats (experimental unit), and to avoid possible confounding effects of the estrus cycle on LDL-cholesterol levels, male rats were used for the study.

All experimental animals (48 male Wistar rats) were obtained from the Animal Resources Centre (Western Australia) at 6 weeks of age. Rats were housed in groups of 4 in standard open top boxesthat contained environmental enrichment and they were maintained under controlled heating andlighting (23 °C with 12-h light/dark cycle) with free access to food and water. Animals housed together were allocated to the same treatment group.

After arrival the rats were acclimated to the facility for a period of 2 weeks during which they werefed standard AIN93M rat chow (34) that was made in-house. During this period rats were individually ear-notched and familiarized with the staff and the handling procedures. Study treatments were commenced at 8 weeks of age as by this age their growth rate has stabilized, andthey are representative of adult rats.

Rats were killed via exsanguination of the abdominal aorta and blood was collected via X8G needleand syringe from the aorta. Blood was transferred to uncapped vacutainers (serum with clot activator and Ethylenediaminetetraacetic acid (EDTA)) to reduce haemolysis of the samples.

Serum tubes were spun immediately after collection and EDTA tubes were stored on ice for 30minutes prior to 15 minutes centrifugation at lOOOxg at 4°C. The serum and plasma were thenremoved and stored at -80 °C until analysed.

Samples of liver were collected for analysis of LDL-receptor gene expression and malondialdehyde (MDA). A randomized controlled parallel study design was used. After the 2-week acclimatization period, rats were blocked-matched by weight and randomly allocated to one of 6 treatment groups:

Nutra⁺ group: Hypercholesterolaemic diet + 330 mg/kg Swisse Nutra⁺ Cholesterol Balance(including HydrovaslO™ + Bergavit40™ + Altilix™ + Plant sterols)

- MCT group: Hypercholesterolaemic diet + Placebo containing 0.36 mg/kg medium chaintriglycerides (MCT)

- Bergavit 40™ group: Hypercholesterolaemic diet + 30.4 mg/kg Bergavit40™

- Altilix™ group: Hypercholesterolaemic diet + 12.1 mg/kg Altilix™

- Plant Sterol group: Hypercholesterolaemic diet + 145.6 mg/kg Plant sterols

- control group: Normocholesterolaemic diet (AIN93M rat chow) + 0.36 mg/kg Placebo(MCT)

The diets were fed for 6 weeks according to the conventional rules and practice. The Control group was provided with a standard rat diet (AIN-93M) whereas all other animals were fed the hypercholesterolemic diet (standard chow supplemented with 2% cholesterol, 0.2% cholic acid and 20% lard). Food and fluid consumption and urinary and faecal outputs were measured prior to the completion of the study treatment period over 2-days using metabolic cages and after study completion (week 8).

The daily dosages for each treatment were calculated using a human equivalent dose conversionfactor based on the dosages that were determined for the human clinical trial protocol Example 3 : preclinical trial results

All 48 animals used in this study completed the full protocol and their data of interest which were collected were analyzed and are reported below.

Serum lipids : Serum LDL-cholesterol, total cholesterol, HDL-cholesterol, and triglycerides did differ to some extent between treatment groups (Figure 1) and trends for differences between groups were observed (ANOVA p<0.1); rats fed plant sterols tended to have lower serum LDL- cholesterol (ANOVA P=0.08) and total cholesterol (ANOVA P=0.06) concentration compared to rats fed AltilixTM (Figure 1).

There was a trend for total cholesterohHDL-cholesterol ratio to differ between groups (P=0.08).Non-HDL was significant (P=0.043) but differences between groups were less significant. Compared to Altilix™, rats fed plant sterols (P=0.09) and Nutra⁺ (P=0.13) tended to have lowernon-HDL cholesterol (Figure 2).

Oxidative stress : Liver MDA and plasma LDL-oxidation concentrations differed significantly between groups (ANOVA P<0.05 and P O.OOOl respectively). Specifically, liver MDA concentrations were significantly lower in the Nutra+ group compared to the MCT group (P<0.05) (Figure 3). Plasma LDL-oxidation was significantly lower in the plant sterol group compared to MCT, BergavitTM and Nutra+ groups (PO.OOOl) and plasma LDL-oxidation was significantly lower in the AltilixTM group compared to the MCT, BergavitTM and Nutra+ groups (P<0.05) (Figure 3).

Faecal cholesterol and bile acids : Significant differences were seen between treatment groups in faecal cholesterol excretion (ANOVA, P O.Ol). Faecal cholesterol excretion was significantly greater in rats provided with AltilixTM, Bergavit 40TM and plant sterols compared to those receiving the Nutra+ treatment, however no groups were significantly different to the MCT group (Figure 15). When expressed as a percentage of cholesterol consumed, mean excretion of cholesterol was 23% and 20% higher, respectively in the AltilixTM and plant sterol groups compared to Nutra+ (ANOVA, P<0.05, Figure 4).

Claims

1. A composition comprising at least three of the ingredients selected from the group consisting of a), b), c) and d), wherein such ingredients comprise: a) chlorogenic acid, chlorogenic acid derivatives and luteolin-7-glucoside; b) hydroxytyrosol and tyrosol; c) citrus flavonoids, such as neohesperidin, naringin and neoeriocitrin; and d) phytosterol esters.

2. The composition according to claim 1 comprising: a) chlorogenic acid, chlorogenic acid derivatives and luteolin-7-glucoside; b) hydroxytyrosol and tyrosol; c) citrus flavonoids, such as neohesperidin, naringin and neoeriocitrin; and d) phytosterol esters.

3. The composition according to any one of claims 1 or 2 wherein: a) an artichoke leaf ( Cynara cardunculus var. Altilis DC) extract is the source of chlorogenic acid, chlorogenic acid derivatives and luteolin-7-glucoside.

4. The composition according to any one of claims 1 to 3 wherein: b) an olive fruits ( Olea Europea) extract is the source of hydroxytyrosol and tyrosol.

5. The composition according to any one of claims 1 to 4 wherein: c) a bergamot (Citrus bergamia) extract is the source of citrus flavonoids, such as neohesperidin, naringin and neoeriocitrin.

6. The composition according to any one of claims 1 to 5 wherein: d) a vegetable oils extract, a nuts or seeds extract, preferably a sunflower oil extract, is the source of phytosterol esters.

7. The composition according to any one of claims 1 to 6 wherein: a) the artichoke leaf ( Cynara cardunculus var. Altilis DC) extract is the source of chlorogenic acid, chlorogenic acid derivatives and luteolin-7-glucoside; b) the olive fruits (Olea Europea) extract is the source of hydroxytyrosol and tyrosol; c) the bergamot (Citrus bergamia) extract is the source of citrus flavonoids, such as neohesperidin, naringin and neoeriocitrin; and d) the vegetable oils extract, the nuts or seeds extract, preferably the sunflower oil extract, is the source of phytosterol esters.

8. The composition according to any one of claims 1 to 7 wherein: a) the artichoke leaf (Cynara cardunculus var. Altilis DC) extract is the source of chlorogenic acid, chiorogenic acid derivatives in an amount ranging from 0.63 to 0.76% w/w and luteolin-7-glucoside in an amount ranging from 0.13 to 0.25% w/w; b) the olive fruits (Olea Europea) extract is the source of hydroxytyrosol and tyrosol in an amount ranging from 0.21 to 0.25% w/w; c) the bergamot ( Citrus bergamia) extract is the source of citrus flavonoids, such as neohesperidin, naringin and neoeriocitrin in an amount ranging from 6.16 to 6.64% w/w; and d) the vegetable oils extract, the nuts or seeds extract, preferably the sunflower oil extract, is the source of phytosterol esters in an amount of 73.53% w/w.

9. The composition according to any one of claims 1 to 8 wherein: a) the artichoke leaf ( Cynara cardunculus var. Altilis DC) extract, in an amount ranging from 2.84 to 3.78% w/w, is the source of chlorogenic acid, chlorogenic acid derivatives in an amount ranging from 0.63 to 0.76% w/w and luteolin-7-glucoside in an amount ranging from 0.13 to 0.25% w/w.

10. The composition according to any one of claims 1 to 9 wherein: b) the olive fruits (Olea Europea) extract, in an amount ranging from 1.26 to 1.47% w/w, is the source of hydroxytyrosol and tyrosol in an amount ranging from 0.21 to 0.25% w/w.

11. The composition according to any one of claims 1 to 10 wherein: c) the bergamot (Citrus bergamia) extract, ih an amount ranging from 11.85 to 12.80% w/w, is the source of citrus flavonoids, such as neohesperidin, naringin and neoeriocitrin in an amount ranging from 6.16 to 6.64% w/w.

12. The composition according to any one of claims 1 to 11 wherein: a) the artichoke leaf (Cynara cardunculus var. Altilis DC) extract, in an amount ranging from 2.84 to 3.78% w/w, is the source of chlorogenic acid, chlorogenic acid derivatives in an amount ranging from 0.63 to 0.76% w/w and luteolin-7-glucoside in an amount ranging from 0.13 to 0.25% w/w; b) the olive fruits (Olea Europea) extract, in an amount ranging from 1.26 to 1.47% w/w, is the source of hydroxytyrosol and tyrosol in an amount ranging from 0.21 to 0.25% w/w; c) the bergamot (Citrus bergamia) extract, in an amount ranging from 11.85 to 12.80% w/w, is the source of citrus flavonoids, such as neohesperidin, naringin and neoeriocitrin in an amount ranging from 6.16 to 6.64% w/w; and d) the vegetable oils extract, the nuts or seeds extract, preferably the sunflower oil extract, is the source of phytosterol esters in an amount of 73.53% w/w.

13. The composition according to any one of claims 1 to 12 further comprising maltodextrins.

14. The composition according to any one of claims 1 to 13 for oral administration in the form of a tablet, a capsule, a pill, a dragee, a gel, a slurry or a suspension.

15. The composition according to any one of claims 1 to 14 for use as a medicament.

16. The composition according to any one of claims 1 or 15 for use as a nutritional or a dietary supplement.

17. The composition according to any one of claims 1 to 16 for use in the treatment and/or prevention of a disorder or abnormality associated with elevated blood lipid levels.

18. The composition for use according to claim 17 wherein the elevated blood lipid levels are the levels of total cholesterol (TC) and/or LDL cholesterol and/or triglycerides.

19. The composition for use according to any one of claims 17 or 18 wherein the disorder is selected from atherosclerosis, cardiovascular disease, cerebrovascular disease and peripheral vascular disease.