Nutrional composition for treating and / or preventing muscle wasting

Abstract

The invention pertains to a nutritional composition comprising xanthohumol, fucoxanthin and c- phycocyanin. Further, the invention pertains to a nutritional composition comprising xanthohumol, fucoxanthin and c-phycocyanin for use as a medicament, more particularly for therapeutic use in treating and / or preventing muscle wasting.

Description

[0001] NUTRIONAL COMPOSITION FOR TREATING AND / OR PREVENTING MUSCLE WASTING

[0002] Field of the invention

[0003] The invention relates to a nutritional composition for prevention and / or treatment of muscle wasting or muscle decline, particularly for therapeutic use in improving muscle mass, strength and function.

[0004] Background of the invention

[0005] Sufficient muscle energy production and muscle mass is critical to the ageing population. Muscle mass loss becomes pathologic during oncology, hospitalized immobile patients, patients suffering from inflammatory diseases, malnourished elderly or patients suffering from immobilizing diseases like osteoarthritis or rheumatoid arthritis. Muscle functionality and physical ability decline as a result of impaired muscle energy production and muscle mass loss.

[0006] Sarcopenia is a progressive and generalized skeletal muscle disorder characterized by the accelerated loss of muscle mass, strength, and function. It primarily affects older adults and is associated with aging. This condition can significantly impact quality of life by reducing physical capabilities and increasing the risk of falls and fractures. Frailty and sarcopenia are accelerated by acute or chronic diseases. From the age of about 30, humans lose 3-8 % of muscle per decade and this accelerates after 60 years of age.

[0007] Muscle wasting and weakness, driven by severe inflammation is an important medical problem of critically ill patients. Intensive care unit (ICU) acquired weakness, the clinical diagnosis of muscle weakness, occurs in critically ill patients. The ICU patient population is very diverse where different events (e.g. trauma, scheduled operation) can attribute to an admission of a patient to the ICU. The first days in the ICU are considered as acute phase. During the acute phase a lot is happening which can challenge (nutritional) interventions (i.e. inflammatory storm, anxiety, organ failure). Therefore, muscle wasting, high levels of inflammation and oxidative damage are observed in ICU patients during post ICU recovery when the patients are stable.

[0008] Mitochondrial function is crucial to skeletal muscle health and detrimental changes at the level of the mitochondria may contribute to loss of muscle mass and functionality. A main function of mitochondria is the generation of ATP for energy through oxidative phosphorylation (OXPHOS). Mitochondrial dysfunction is associated with impaired immune and inflammatory responses. Acute and chronic inflammatory signalling impairs mitochondrial respiration & energy production via type-2 death receptor signalling, leading up to mitochondrial permeability transition and finally the release of mitochondrial apoptosis inducing factors. Chronic inflammation due to the enhanced production of inflammatory cytokines causes muscle wasting with a reduction in muscle mass and power. Muscle wasting caused by inflammatory cytokines is defined as cytokine-induced muscle wasting. The changes of mitochondrial network influence the production of reactive oxygen species (ROS) that play an important role in muscle function. It is suggested that mitochondrial bioenergetics are altered in aged skeletal muscle, resulting in an increase in ROS production, while conversely genetic / pharmacological approaches that attenuate mitochondrial ROS promote healthy aging and maintenance of muscle mass. Also, exercise improves mitochondrial function by activating mitochondrial biogenesis and mitophagy.

[0009] US2024 / 299472 addresses increasing muscle mass and suppressing a decrease in muscle mass using as an effective component at least one selected from the group consisting of spirulina, phycocyanin, a spirulina enzymatically degraded product, and a phycocyanin enzymatically degraded product. JP2020 / 132576 relates to a sarcopenia ameliorating agent based on fucoxanthin. Yoshikawa et al. "Effects of Fucoxanthin on the Inhibition of Dexamethasone-Induced Skeletal Muscle Loss in Mice", Nutrients, vol. 13, no. 4, (2021-03-26), page 1079 describes how fucoxanthin has preventive effect against muscle atrophy and myotube loss. US2024 / 293441 describes Inhibitor compounds of type 2 iodothyronine deiodinase for use in the therapeutic treatment of muscle wasting and / or in a muscle and / or skin regenerative therapeutic method.

[0010] Outside the field, there is a Whole Body Superfood Dietary Supplement called MegaFlax sold by Designing Health which has 28 essential nutrients to support natural immune function.

[0011] There is a need for a nutritional composition which can support muscle function by stimulating the mitochondria for treating and / or preventing muscle wasting in the ageing population or oncology or hospitalized patients.

[0012] Summary of the invention

[0013] The present invention provides a nutritional composition comprising xanthohumol, fucoxanthin and c- phycocyanin. The inventors have found that the combination of three ingredients xanthohumol, fucoxanthin and c-phycocyanin, improves mitochondrial function in an inflammatory context and hence, helps for treating and / or preventing muscle wasting. Reference is made to the example and Figures 1 and 2 herein. The model used to test synergy is an Agilent Seahorse Cell Mito Stress Test kit which is a widely recognized, well accepted standard assay for assessing mitochondrial function. The assay provided insights into the mechanism of mitochondrial dysfunction.

[0014] Therefore, the present invention provides also a nutritional composition comprising xanthohumol, fucoxanthin and c-phycocyanin for use as a medicament. The invention further provides a nutritional composition comprising the aforementioned combination for use in the treatment and / or prevention of muscle wasting in a human subject. Xanthohumol, fucoxanthin and c-phycocyanin are present in therapeutically effective amounts.

[0015] In an embodiment, bioavailability is further improved by providing at least fucoxanthin, preferably all of xanthohumol, fucoxanthin and c-phycocyanin in encapsulated form, for example in the form of liposomes and nanostructured lipid carriers or spray-dried biopolymer encapsulation. They may be provided in encapsulated form individually or as a combination. List of figures

[0016] Figures 1 and 2 show the effect of xanthohumol, fucoxanthin and c-phycocyanin on mitochondrial function in cytokine-induced muscle wasting. The synergistic effect beyond the expected additive effect based on the individual contributions of the three components is marked in the figures. For figure 2, the numbers: reference (vehicle) 2.523, muscle wasting (trigger) 0.998, treatment with c-phycocyanin (PC) 1.018, xanthohumol (XN) 1.195, fucoxanthin (FX) 0.978, the combination 1.768. For figure 1 , p=0.0068. Figure 3A, 3B and 3C show the individual dose-response curves for FX, PC and XN. There was not so much an effect on ATP production for FX, although there was on maximal and spare respiratory capacity (not shown). Figure 4 shows cell viability (cytotoxicity%) decreasing compared to trigger, which indicates less compromised cells.

[0017] List of preferred embodiments

[0018] 1 . A nutritional composition comprising xanthohumol, fucoxanthin and c-phycocyanin.

[0019] 2. The nutritional composition according to embodiment 1 , wherein the composition comprises a protein.

[0020] 3. The nutritional composition according to embodiment 2, wherein the protein is selected from whey protein, soy protein, pea protein, canola protein or mixtures thereof.

[0021] 4. The nutritional composition according to embodiment 2 or 3, wherein the composition comprises at least 10 g protein per 100 g dry weight, preferably at least 15 g per 100 g dry weight.

[0022] 5. The nutritional composition according to any one of the preceding embodiments, wherein the composition comprises 1 to 40 mg xanthohumol per unit dose, 1 to 20 mg fucoxanthin per unit dose and 5 to 300 mg c-phycocyanin per unit dose.

[0023] 6. The nutritional composition according to any one of the preceding embodiments, wherein the composition comprises xanthohumol, fucoxanthin and c-phycocyanin in a weight ratio of 1 : 0.3 - 1 : 20 - 60.

[0024] 7. The nutritional composition according to any one of the preceding embodiments, being a tube feeding composition, preferably comprising 0.5 to 40 mg / 100 kcal xanthohumol, 0.5 to 20 mg / 100 kcal fucoxanthin and 5 to 200 mg / 100 kcal c-phycocyanin, and wherein the tube feeding composition preferably provides, per 100 ml, 100 - 200 kcal.

[0025] 8. The nutritional composition according to any one of the preceding embodiments for use as a medicament.

[0026] 9. The nutritional composition according to embodiments 1 to 7, for use in the treatment and / or prevention of muscle wasting in a human subject.

[0027] 10. The nutritional composition for use according to embodiment 9, wherein the human subject is an elderly subject of at least 50 years of age, preferably at least 55 years of age; and / or wherein the human subject is suffering from muscle decline; and / or wherein the human subject is suffering from cancer or sarcopenia or Intensive Care Unit (ICU) acquired weakness.

[0028] 11. The nutritional composition for use according to embodiment 9 or 10, wherein the composition provides 1 to 40 mg / day xanthohumol, 1 to 20 mg / day fucoxanthin and 15 mg to 300 mg / day c- phycocyanin. 12. Use of a nutritional composition comprising therapeutically effective amounts of xanthohumol, fucoxanthin and c-phycocyanin in the manufacture of a product for prevention and / or treatment of muscle wasting in a human subject.

[0029] 13. A method of prevention and / or treatment of muscle wasting in a human subject comprises administering a nutritional composition comprising therapeutically effective amounts of xanthohumol, fucoxanthin and c-phycocyanin to the human subject.

[0030] 14. The use according to embodiment 12 or the method according to embodiment 13, wherein the nutritional composition is administered in an amount that provides 1 to 40 mg xanthohumol, 1 to 20 mg fucoxanthin and 15 to 300 mg c-phycocyanin per day.

[0031] 15. The nutritional composition according to any one of embodiments 1 - 7, wherein the composition comprises a protein fraction comprising 32-58 weight % pea protein, 30-56 weight % soy protein and 8-16 weight % free leucine, preferably a protein fraction comprising 36-54 weight % pea protein, 34-52 weight % soy protein and 10 - 14 weight % free leucine, and optionally up to 5 weight % of free branched chain amino acids other than leucine, based on all proteinaceous matter, wherein the composition is essentially free from animal protein; wherein the composition preferably comprises a protein fraction comprising 41-50 weight % pea protein, 39-47 weight % soy protein and 10.5 - 13.5 weight % free leucine, based on all proteinaceous matter; wherein the composition preferably comprises a protein fraction comprising 43-47 weight % pea protein, 41-45 weight % soy protein and 11 - 13 weight % free leucine, based on all proteinaceous matter.

[0032] 16. The nutritional composition according to any one of embodiments 1 - 7, wherein the composition comprises a protein fraction comprising (a) 15 to 35 weight % of casein ; (b) 25 to 50 weight % of whey protein ; (c) 10 to 30 weight % of soy protein ; and (d) 10 to 30 weight % of pea protein, relative to the total protein in the protein fraction, wherein the sum of said proteins preferably equals 100 weight % of the protein fraction; wherein the composition preferably comprises a protein fraction comprising (a) 20 to 30 weight % of casein ; (b) 30 to 40 weight % of whey protein ; (c) 15 to 25 weight % of soy protein; and (d) 15 to 25 weight % of pea protein, relative to the total protein in the protein fraction, wherein the sum of said proteins preferably equals 100 weight % of the protein fraction.

[0033] Detailed description of the invention

[0034] The invention relates to a nutritional composition comprising therapeutically effective amounts of xanthohumol, fucoxanthin and c-phycocyanin. The inventors of the present invention have found that a nutritional composition comprising xanthohumol, fucoxanthin and c-phycocyanin, improves mitochondrial function and hence, helps for treating and / or preventing muscle wasting in a human subject in need thereof. The (prophylactic) treatment of muscle wasting includes therapeutic use in improving muscle mass, strength and function.

[0035] In an embodiment, the nutritional composition according to the invention is for use as a medicament.

[0036] In a preferred embodiment, the nutritional composition is for use in the treatment and / or prevention of muscle wasting in a human subject. The subject is preferably an elderly human. In this respect, it is submitted that in the context of this application, an elderly human is a person of the age of 50 years or more, in particular of the age of 55 or more, more in particular of the age of 60 or more, more in particular of the age of 65 or more. This definition takes into account the fact that the average age varies between different populations, on different continents, etc. Most developed world countries have accepted the chronological age of 65 years as a definition of 'elderly' or older person (associated with the age at which one may begin to receive pension benefits), but like many westernized concepts, this does not adapt well to e.g. the situation in Africa. At the moment, there is no United Nations (UN) standard numerical criterion, but the UN agreed cut-off is 60+ years to refer to the older population in Western world. The more traditional African definitions of an elder or 'elderly' person correlate with the chronological ages of 50 to 65 years, depending on the setting, the region and the country.

[0037] In a preferred embodiment said human subject is suffering from muscle decline In a further preferred embodiment, the human subject is suffering from cancer or sarcopenia or ICU acquired weakness. Sarcopenia, the loss of muscle mass and strength, becomes more common with age. Pathologic muscle mass loss in sarcopenia is typically defined by a reduction in muscle mass that is more than two standard deviations below the mean for young, healthy adults. This significant loss of muscle mass is often accompanied by a decrease in muscle strength and physical performance, which can severely impact daily activities and overall quality of life. Excessive muscle loss is commonly observed in cancer patients. The mechanisms of cancer-related muscle loss are multifactorial, including anorexia, hypogonadism, anaemia, inflammation, malnutrition, and aberrations in skeletal muscle protein turnover and metabolism. Also, low muscle in cancer is associated with an increase in treatment-related toxicities. ICU acquired weakness, the clinical diagnosis of muscle weakness, is observed in approximately half of the ICU patients. Muscle mass decreases rapidly after ICU admission.

[0038] Composition

[0039] The nutritional composition according to the invention comprises xanthohumol, fucoxanthin and c- phycocyanin. These are present in therapeutically effective amounts, meaning that the components are presented in amounts that they altogether result in a reducing (the risk of) muscle wasting beyond the results achieved for corresponding amounts of the individual components.

[0040] Xanthohumol (XN), E)-1-[2,4-Dihydroxy-6-methoxy-3-(3-methyl-2-butenyl)phenyl]-3-(4-hydroxyphenyl)propenone, 2', 4, 4'- Trihydroxy-6'-methoxy-3'-prenylchalcone, is a natural product found in the female inflorescences of Humulus lupulus, also known as hops. This compound is also found in beer and belongs to a class of compounds that contribute to the bitterness and flavour of hops. Xanthohumol is a prenylated chaiconoid, biosynthesized by a type III polyketide synthase (PKS) and subsequent modifying enzymes. Xanthohumol is a prenylated flavonoid that has a variety of biological activities, such as prevention of cancer, the prevention and mitigation of diabetes, and antioxidant, anti-inflammatory, antibacterial and immunomodulatory effects. Such bioactivities can be achieved by the interaction of xanthohumol with different cellular targets and interfering in diverse molecular signalling cascades, including the mitochondria. Xanthohumol could decrease inflammation reactions and prevent osteoarthritis progression by attenuating mitochondria dysfunction / NLRP3 inflammasome axis. This component reveals limited activity in the cytokines-induced muscle mass wasting model; reference is made to Figure 1 .

[0041] If so desired, xanthohumol can be provided in encapsulated form, in order to improve bioavailability and protect against partial degradation in the Gl tract. Encapsulation of fucoxanthin can be achieved using lipid-based systems available in the art (e.g., liposomes, nano-structured lipid carriers), or using spray- dried biopolymer encapsulation.

[0042] Fucoxanthin (FX), is a xanthophyl and is considered to be the most abundant natural carotenoid. Carotenoids are pigments produced by plants and algae and play a role in light harvesting as part of the photosynthesis process. Fucoxanthin is largely found in kelp (brown algae seaweeds) and microalgae (diatoms). Fucoxanthin, one of the main components of marine carotenoids, has strong antioxidant activity. Fucoxanthin has a highly unique structure that contains both an epoxide bond and hydroxyl groups along with an allenic bond (two adjacent carbon-carbon double bonds) and a conjugated carbonyl group (carbon-oxygen double bond) in the polyene chain. All of these features provide fucoxanthin with powerful antioxidant activity. Fucoxanthin has many physiological functions and biological properties, such as antiobesity, antitumor, antidiabetes, antioxidant, anti-inflammatory, and hepatoprotective activities, as well as cardiovascular and cerebrovascular protective effects. That said, it does not show any activity in a muscle mass wasting model; reference is made to Figure 1 . Upon ingestion, fucoxanthin has plasma bioavailability. However, if so desired, fucoxanthin can be provided in encapsulated form, in order to improve bioavailability and protect against partial degradation in the Gl tract. Encapsulation of fucoxanthin can be achieved using lipid-based systems available in the art (e.g., liposomes, nano-structured lipid carriers). Liposomes and nanostructured lipid carriers can increase bioavailability from 25 to over 60%. Spray-dried biopolymer encapsulation improves intestinal absorption and stability. Moreover, after ingestion, a fraction of fucoxanthin is converted into fucoxanthinol in the intestine and part of the latter is turned into amarouciaxanthin A in the liver [8], It can be assumed that fucoxanthin and fucoxanthinol share the same bioactive properties since the only difference between fucoxanthin and fucoxanthinol is the addition of a hydroxyl group to fucoxanthin. The addition of the hydroxyl group increases the polarity of fucoxanthin and renders it more water- soluble so that it can be better distributed in the plasma and excreted.

[0043] C-phycocyanin (PC), is a pigment-protein complex from the light-harvesting phycobiliprotein family, along with allophycocyanin and phycoerythrin. It is an accessory pigment to chlorophyll. C-phycocyanin is found in blue-green algae, such as Spirulina which is a type of cyanobacteria. C-phycocyanin is powerful antioxidant and has anti-inflammatory properties. It has also potential health benefits such as managing inflammation, blood sugar, cholesterol and blood pressure. Additionally, c-phycocyanin has effect on muscle health and mitochondrial function. That said, it does not show much activity in a muscle mass wasting model; reference is made to the figures. PC has significant biological effects due to the potent antioxidant and anti-inflammatory properties of its chromophore phycyanobilin (PCB). The bioactivity of PCB is linked to its ability to induce antioxidant defences to scavenge reactive oxygen species (ROS) and to modulate inflammatory pathways such as blocking cytokine-induced activation of caspase 2 / 8 eliciting mitochondrial demise, or blocking cytokine-induced NF-KB signalling. PCB will be released upon proteolytic digestion of PC and better absorbed than bilirubin which is structurally close to PCB. PCB has greater water solubility and is therefore expected to have better bioavailability than bilirubin. If so desired, PC can be provided in encapsulated form, in order to improve bioavailability and protect against partial degradation in the Gl tract. Encapsulation of PC can be achieved using lipid-based systems available in the art (e.g., liposomes, nano-structured lipid carriers), or using spray-dried biopolymer encapsulation. The study in the attached examples section clearly evidences synergy between the three components. Commercially, individual supplements with either 24 - 180 mg XN, about 8 mg FX or 20 - 200 mg PC, are available, albeit intended for different purposes. The concept of synergy as found in the in vitro study is applied by combining the individual components in amounts in which these are already individually administered to human beings, now making use of the inventors’ findings of synergistic ATP production in the context of skeletal muscle. In one embodiment, the nutritional composition according to the invention comprises 1 to 40 mg xanthohumol per unit dose, 1 to 20 mg fucoxanthin per unit dose and 5 to 300 mg c-phycocyanin per unit dose. More preferably, the nutritional composition according to the invention comprises 5 to 20 mg xanthohumol per unit dose, 2 to 10 mg fucoxanthin per unit dose and 10 to 250 mg c-phycocyanin per unit dose, most preferably 10 to 20 mg xanthohumol per unit dose, 5 to 10 mg fucoxanthin per unit dose and 20 to 200 mg c-phycocyanin per unit dose. The expressions "unit dose" refers to an amount of a components administered to an individual in a single dose. A daily dose is the sum of all single doses in a day. The unit dose is preferably in liquid form, with a volume between 100 and 200 ml.

[0044] For all the individual components a dose response experiment was initially performed on muscle cell viability. This was done to exclude any cytotoxicity of the single component on the muscle cells. Once it was established that a specific dose or range of dose was safe, testing was continued in the Seahorse Mito Stress kit assay, subsequently multiple combinations were tested. Taking from these results and what has been used before in clinical studies for the single components of interest a suggested therapeutic daily dose range is 1 to 40 mg / day xanthohumol, 1 to 20 mg / day fucoxanthin and 15 mg to 300 mg / day c-phycocyanin. More preferably, the daily dose range is 5 to 20 mg / day xanthohumol, 2 to 10 mg / day fucoxanthin and 20 mg to 200 mg / day c-phycocyanin, more preferably the daily dose range is 10 to 20 mg / day xanthohumol, 5 to 10 mg / day fucoxanthin and 20 mg to 200 mg / day c-phycocyanin. It is preferred that the above daily amounts are administered divided over 1 or 2 times per day, although a single intake per day is in any case preferred.

[0045] According to a further embodiment, the nutritional composition according to the invention comprises protein selected from whey protein, soy protein, pea protein, canola protein or mixtures thereof.

[0046] Whey protein is known for its excellent amino acid profile, and for its ability to increase the protein synthesis in a mammal (due to a higher leucine content). Nutritionally speaking, whey protein is known as a naturally complete protein because it contains all of the essential amino acids required in the daily diet. It is also one of the richest sources of branched chain amino acids (BCAAs, in particular leucine) which play an important role in muscle protein synthesis. Whey protein is the preferred choice of proteins to treat persons suffering from sarcopenia.

[0047] Soy protein is categorized as a high-quality, complete protein although the methionine level is slightly below the WHO 2007 recommendation for methionine content. Soy proteins can be divided into different categories according to their production method. Soy protein isolate (SPI) is the most refined form of soy protein. Soy protein isolate contains about 90 % protein. Soy protein concentrate (SPC) is basically soybean without the water soluble carbohydrates. It contains about 70 % of protein.

[0048] Pea protein contributes to forming a good overall mix of amino acids. While whey proteins enter the blood stream very fast, pea proteins are absorbed much slower. Pea protein is quite high in cysteine content and can therefore compensate the inadequate amount of cysteine in casein proteins. Furthermore, pea protein is quite high in arginine compared to casein, soy or whey protein which is required for muscle metabolism and which facilitates the intake of body mass while reducing body fat; and it is quite high in lysine, which is needed to build protein muscle and assist in the maintenance of lean body mass.

[0049] Canola protein is rich in essential amino acids, making it a complete protein. The average protein content of canola is 20 %. The nutritional quality of canola protein, in terms of Protein Digestibility Corrected Amino Acid Score (PDCAAS), is comparable to that of soy protein. As a complete protein, it provides all the essential amino acids necessary for muscle repair and growth.

[0050] In one embodiment, the nutritional composition comprises a protein fraction comprising (a) 15 to 35 weight % of casein ; (b) 25 to 50 weight % of whey protein ; (c) 10 to 30 weight % of soy protein ; and (d) 10 to 30 weight % of pea protein ; relative to the total protein in the protein fraction, wherein the sum of said proteins preferably equals 100 weight of the protein fraction. The protein fraction preferably comprises (a) 20 to 30 weight % of casein ; (b) 30 to 40 weight % of whey protein ; (c) 15 to 25 weight % of soy protein ; and (d) 15 to 25 weight % of pea protein ; relative to the total protein in the protein fraction, wherein the sum of said proteins preferably equals 100 weight % of the protein fraction. According to a preferred embodiment, the proteins including casein, whey protein, soy protein and pea protein are substantially in intact form or non-hydrolysed. In the context of the invention, a "nonhydrolysed" protein is equivalent to an "intact" protein, meaning that the protein has not been subjected to an hydrolysis process. However, minor amounts of hydrolysed proteins may be present in the source of non-hydrolysed proteins, or may be added to the formulation, such as additional amino acids, such as, for example leucine, isoleucine, glutamine, arginine, or dipeptides and the like. In one embodiment of the present invention, the composition may comprise a free amino acid, or a mixture of free amino acids, up to about 1 g / 100 ml, preferably up to about 0.5 gram / 100 ml. The protein fraction preferably essentially consists of vegetable and dairy proteinaceous matter, in particular proteins, preferably casein, whey protein, soy protein and pea protein. Small amounts of free amino acids may be accounted for, preferably less than about 5 %, more preferably less than about 2 %, most preferably less than about 1 % of the protein fraction. In a particularly preferred embodiment, there are no free amino acids, peptides or hydrolysates in the composition. Within the context of this paragraph, with the term "casein" both caseinate and micellar casein is indicated. In one embodiment, the casein is caseinate, preferably Na- caseinate or Ca-caseinate. Preferably, the caseinate is Ca-caseinate.

[0051] In another embodiment, the nutritional composition comprises a protein fraction comprising 32-58 weight % pea protein, 30-56 weight % soy protein and 8-16 weight % free leucine, preferably a protein fraction comprising 36-54 weight % pea protein, 34-52 weight % soy protein and 10 - 14 weight % free leucine, and optionally up to 5 weight % of free branched chain amino acids other than leucine, based on all proteinaceous matter, wherein the composition is essentially free from animal protein. In a preferred embodiment, the composition comprises a protein fraction comprising 41-50 weight % pea protein, 39-47 weight % soy protein and 10.5 - 13.5 weight % free leucine, based on all proteinaceous matter. In a more preferred embodiment, the composition comprises a protein fraction comprising 43- 47 weight % pea protein, 41-45 weight % soy protein and 11 - 13 weight % free leucine, based on all proteinaceous matter. In a preferred embodiment, the sum of said proteins, free leucine and optionally free branched amino acids other than leucine equals 100 weight % of the protein fraction. In a more preferred embodiment, the protein fraction essentially consists of pea protein, soy protein and free leucine. In a preferred embodiment, the composition has no free amino acids, peptides or hydrolysates in the composition, except for free leucine. According to a preferred embodiment, the proteins including soy protein and pea protein are substantially in intact form or non-hydrolysed. In the context of the invention, a "non-hydrolysed" protein is equivalent to an "intact" protein, meaning that the protein has not been subjected to an hydrolysis process. However, minor amounts of hydrolysed proteins may be present in the source of non-hydrolysed proteins, or may be added to the formulation, such as additional amino acids. In a preferred embodiment, the only free amino acids added are branched amino acids leucine, isoleucine and valine. In one embodiment of the present invention, the composition may, in addition to the free leucine, comprise another free amino acid, or a mixture of free amino acids other than leucine, up to about 1 g / 100 ml, preferably up to about 0.5 gram / 100 ml. The protein fraction preferably essentially consists of plant-based proteinaceous matter, in particular proteins, and free leucine. Small amounts of free amino acids other than free leu may be accounted for, preferably less than about 5 %, more preferably less than about 2 %, most preferably less than about 1 % of the protein fraction. In a particularly preferred embodiment, there are no free amino acids (other than leucine), peptides or hydrolysates in the composition.

[0052] In the context of the invention, the term "about" should preferably be interpreted as a deviation of plus or minus 10 % of the given value.

[0053] In a preferred embodiment, the nutritional composition comprises at least 10 g protein per 100 g dry weight, preferably at least 15 g per 100 g dry weight.

[0054] In an embodiment, the nutritional composition according to the invention is an oral nutritional supplement. The oral nutritional supplement according to the invention may take any suitable form, such as a pill, drink or powder. In case the composition is in powder form, it is preferably reconstitutable with an aqueous liquid, such as milk or water. The skilled person is able to determine the most suitable form of the composition according to the invention, for example depending on the target group and the further ingredients that may be present. Preferably, the nutritional composition is packaged, stored and provided in a tube feeding bag. Tube feeding is given to provide nutrition to patients which cannot obtain nutrition by swallowing, using a device such as a nasogastric feeding tube or a naso jejunal feeding tube, or by using a percutaneous endoscopic gastrostomy (PEG) or PEG-jejuno-feeding system. The nutritional composition in tube feeding format according to the invention preferably has the form of a complete food, i.e. it can meet all nutritional needs of the user. As such, the nutritional composition according to the invention preferably contains 1000 to 2500 kcal per daily dosage. Depending on the condition of the patient, a daily dose is about 25 to 35 kcal / kg bodyweight / day. Therefore, a typical daily dose for a 70 kg person contains about 2000 kcal. In the context of this application, the state of being fed by a feeding tube is called enteral feeding, comprising all of the abovementioned tube feeding systems, and the nutrition used in the feeding a feeding tube is called enteral nutrition. The (tube feed) composition preferably provides 1 .0 - 1 .8 g protein / kg body weight per day, more preferably 1 .0 - 1 .8 g protein / kg body weight per day. When the composition is a tube feed composition packaged in a tube feeding bag, the composition preferably comprises 0.5 to 40 mg / 100 kcal xanthohumol, 0.5 to 20 mg / 100 kcal fucoxanthin and 10 to 300 mg / 100 kcal c-phycocyanin. The tube feeding composition preferably provides, per 100 ml, 100 - 200 kcal.

[0055] In one embodiment, the nutritional composition according to the invention is administered to a human subject in an amount that provides 1 to 40 mg xanthohumol, 1 to 20 mg fucoxanthin and 15 to 300 mg c-phycocyanin per day. Preferably, the nutritional composition according to the invention is administered to a human subject in an amount that provides 5 to 20 mg xanthohumol, 2 to 10 mg fucoxanthin and 20 mg to 200 mg c-phycocyanin, more preferably 10 to 20 mg xanthohumol, 5 to 10 mg fucoxanthin and 20 mg to 200 mg per day.

[0056] Alternatively or additionally, the nutritional composition according to the invention comprises xanthohumol, fucoxanthin and c-phycocyanin in a weight ratio of 1 : 0.3 - 1 : 20 - 60; these ratios apply to the unit dose as well as the daily dosage.

[0057] As will be appreciated by the skilled person, the composition may contain further ingredients that are beneficial for the subject. According to a further embodiment, the composition according to the invention optionally comprises one or more of a fat fraction, a carbohydrate fraction, a dietary fibre fraction and micronutrients. Most preferably, the liquid nutritional composition is sterilized or pasteurized. In one embodiment, the xanthohumol, fucoxanthin and c-phycocyanin are added to an already sterilized or pasteurized product through aseptic injection. In another embodiment, the nutritional composition is provided in liquid format, preferably in a ready-to-use liquid form and does not require reconstitution or mixing prior to use. Tube feeding is preferred.

[0058] Application

[0059] According to the present invention, the combination of xanthohumol, fucoxanthin and c-phycocyanin is effective in increasing mitochondrial function and supports muscle function. The nutritional composition according to the invention can advantageously be used for prevention and / or treatment of muscle decline of muscle wasting, particularly for improving muscle protein synthesis (MPS). Muscle decline or wasting involves insufficient MPS, as for example amino acids are spared for fuel utilization or energy is provided for protein synthesis. In the targeted population where muscle mass, strength and function is compromised, promoting MPS is a therapeutic use. Thus, in one embodiment, the nutritional composition is for use in the treatment and / or prevention of muscle wasting in a human subject.

[0060] The invention may also be worded in terms of the use of a nutritional composition comprising therapeutically effective amounts of xanthohumol, fucoxanthin and c-phycocyanin in the manufacture of a product for prevention and / or treatment of muscle wasting in a human subject. Worded differently, the invention also relates to a method of prevention and / or treatment of muscle wasting in a human subject wherein the subject is administered with a nutritional composition comprising therapeutically effective amounts of xanthohumol, fucoxanthin and c-phycocyanin. In prophylactic treatment, the subject in need is preferably at increased risk of muscle wasting as described here above.

[0061] In a preferred embodiment, the invention relates to muscle wasting caused by inflammatory cytokines which in the context of the invention is defined as cytokine-induced muscle wasting.

[0062] Exercise program

[0063] In a preferred embodiment, the human subject receiving the composition of the invention participates in a physical exercise program. The physical exercise regimen or program involves any physical exercise or activity other than or in addition to daily living activities that contributes to a negative energy balance or an activity that costs calories during that activity. Physical activity examples can be, but not limited to, resistance exercise, aerobic exercise or flexibility training or combinations thereof. Endurance training is not recommended, at least not as the sole source of physical exercise, for preserving muscle mass and ameliorating the progression of sarcopenia. Therefore, in one embodiment, the physical exercise regimen does not involve endurance training. It is preferred that the physical exercise regimen involves at least resistance exercise, attributed to its effects on muscle mass maintenance and stimulation of muscle mass increase. Activity patterns could vary from engaging in the above one or more physical activities for a minimum of 1 time per week but preferably 2 times per week and more preferably 3 or more times per week, including at least some form of resistance exercise. There are ACSM / AHA Guidelines for flexibility, endurance and resistance exercise which can be of help to the skilled person to determine a suitable physical exercise regimen, and these are considered incorporated by reference here. The physical exercise regimen involves daily physical exercise activities as described above, preferably comprising resistance exercise. The physical exercise regimen is preferably in accordance with ACSM / AHA guidelines, their contents herein incorporated by reference.

[0064] The invention will now be further elucidated by the examples here below, without being limited thereby. Example 1

[0065] C2C12 mouse myoblasts which are the most commonly used cellular model to mimic skeletal muscle in vitro were cultured in culture medium. Culture medium is Dulbecco's Modified Eagle Medium (DMEM) with high glucose (Sigma) and is supplemented with 2.4 g / L NaHCCh, 10 % Foetal Bovine Serum and 1 % Penicillin-Streptomycin. Cells were cultured twice a week until maximal confluency of about 70%. For differentiation into myotubes, cells were plated in Seahorse XFe 96-well plates at 500,000 cells / mL (80 pL per well). The next day, culture medium was replaced by differentiation medium which is DMEM- high glucose, supplemented with 2.4 g / L NaHCCh, 2 % Horse Serum (heat inactivated) and 1 % Penicillin-Streptomycin. Within 7 days, myoblasts were differentiated into myotubes and exposed to cytokine mixture TNFalpha (10 ng / ml, Bio-Techne 410-TRNC-010 / CF) and Actinomycin D (50 ng / ml, Sigma Aldrich A1410-2MG) for 48 h, and simultaneously incubated with the individual or the combination of the nutritional ingredients.

[0066] To that end, the appropriate amounts of FX, PC and XN to be tested were based on a dose response assay. For all of XN, FX and PC, dose-response curves were prepared. The results of those individual dose-response curves are depicted in Figures 3A, 3B and 3C, respectively.

[0067] Figures 3A - 3C show that:

[0068] A. The mitochondria produced less ATP at concentrations above 1 pM, suggesting a toxic effect. The effect of FX was more driven by increased maximal and spare respiratory capacity (not shown) and not so much by increased ATP production;

[0069] B. PC demonstrated positive effects on mitochondrial function at 25-50 pg / ml;

[0070] C. XN showed increased ATP production starting at a dose of 1 pg / ml, with a significant increase at 2.5 pg / ml.

[0071] As a next step the most optimal, individual doses were tested in combination. However, considering the low bioavailability of the nutrients, the single doses were lowered to levels that are sufficient to assess synergy between the 3 single nutrients: 1.25 pg / mL xanthohumol, 0.494 pg / mL (0.75 pM) fucoxanthin and 50 pg / mL c-phycocyanin. For assessment of the effect of the combination, the amount of xanthohumol had to be reduced since at a concentration of 1 .25 pg / mL XN it overpowered the ATP production results obtained when used in combination with PC and FX. Instead, a combination of 0.62 pg / mL XN, 0.494 pg / mL FX and 50 pg / mL PC was used. The reason to treat cells with a trigger (cytokine mixture) is to induce inflammation driven muscle wasting and mimic the clinical situation.

[0072] In addition to the results reported in the priority, cell viability improvements were assessed. Those results are shown in Figure 4: cell viability decreased compared to trigger, indicating on less compromised muscle cells. Also when combined, cell viability compared to trigger was lowered, suggesting no toxic effects and even improved cell viability in an inflamed context. It indicates that the claimed combination not only improves mitochondrial function but also muscle cell mass in an inflammatory context. Mitochondrial stress test assay

[0073] The Agilent Seahorse Cell Mito Stress Test kit is a widely recognized, well accepted standard assay for assessing mitochondrial function. Reference is made to references (1) - (7) using the same or similar. Multiple parameters are obtained in this one assay including, basal respiration, ATP-linked respiration, maximal and reserve capacities, and non-mitochondrial respiration. The assay provides insights into the mechanism of mitochondrial dysfunction and allows users to investigate functional differences among cell types, drug candidates, and genetic or biochemical interventions. The Cell Mito Stress Test Kit contains the compounds, oligomycin, carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) and rotenone / antimycin A (Rot / A). The Agilent Seahorse Cell Mito Stress Test kit is for use with the Agilent Seahorse XFe96 Analyzer. Using the Agilent Seahorse XFe96 Analyzer, the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of live cells can be measured. This measurement allows for comparing energy engines of mitochondria and glycolysis.

[0074] The hydrated cartridge was placed in a non-C02 incubator for calibration and after 1 hour, the injection ports were loaded with the compounds, oligomycin, FCCP and Rot / A. Oligomycin (1 .5 pM) inhibits ATP synthase and decreases electron flow through the electron transport chain (ETC) which results in reduced mitochondrial respiration / OCR (decreased cellular ATP production). Subsequent FCCP (1.0 pM) injection disrupts the proton gradient and mitochondrial membrane potential and allows uninhibited electron flow across the ETC, and maximum mitochondrial respiration (increase in OCR). Finally, the combined Rot / A solution (0.5 pM each) was added for effectively shutting down mitochondrial respiration (decrease in OCR). This test allows for the calculation of non-mitochondrial respiration.

[0075] After compounds were added and mixed, the measurements were done automatically by the Agilent Seahorse XFe96 analyzer in real-time. Outputs was calculated in terms of basal respiration and ATP production.

[0076] Statistical analyses

[0077] All data are expressed as means±SEM. Statistical analyses were performed using Graphpad Prism 10.0.3. Two-way ANOVA followed by Bonferroni post hoc analysis which was used to compare vehicle and trigger. The result of this analysis should be positive, otherwise the experiment was discarded. Least Significant Difference (LSD) post hoc testing was used to compare trigger and the different nutrient-conditions. Statistical significance is defined as P < 0.05.

[0078] Results and discussion

[0079] The results are shown in terms of ATP production and basal respiration both showing increasing mitochondrial function. As demonstrated in Figures, the individual nutrients were not effective in increasing mitochondrial function. The single nutrients fucoxanthin (FX) and c-phycocyanin (PC) did not show any effect and only slight increase in ATP production and basal respiration was observed with xanthohumol (XN). However, the combination of xanthohumol, fucoxanthin and c-phycocyanin showed synergistic effect and ATP production and basal respiration was increased significantly - a 1 .8 fold increase compared to the muscle wasting condition and almost back to the healthy reference. This was achieved with a combination with XN in an amount which was lower than individually tested, yet still a significant improvement was achieved even though XN already shows improved ATP production and basal respiration in itself. Hence, the synergy is even more profound considering that in figure 1 it is weighed against a higher individual amount of XN.

[0080] From these data, it was concluded that the combination of xanthohumol, fucoxanthin and c-phycocyanin can be used in a nutritional composition to support energy production or cellular nutrition status and prevent / treat muscle wasting, preferably cytokine-induced muscle wasting by stimulating the mitochondrial function.

[0081] List of references

[0082] (1) Jiroutkova et al. “Mitochondrial function in skeletal muscle of patients with protracted critical illness and ICU-acquired weakness" Critical Care (2015) 19:448;

[0083] (2) Tyrrell et al. “Respirometric Profiling of Muscle Mitochondria and Blood Cells Are Associated With Differences in Gait Speed Among Community-Dwelling Older Adults" J. Gerontology series A 70(11) (20150 1394-1399;

[0084] (3) Li et al. “Development of a high-throughput method for real-time assessment of cellular metabolism in intact long skeletal muscle fibre bundles” J. Physiol. 2016; 594(24) 7197-7213;

[0085] (4) Park et al “Oxygen Consumption Rates Between Skeletal Muscle Cells Derived from Young and Old Human Donors Elucidate Mitochondrial Dysfunction” Berkeley Scientific Journal fall 2022

[0086] (5) Fovez et al. “Clinically Relevant Oxygraphic Assay to Assess Mitochondrial Energy Metabolism in Acute Myeloid Leukemia Patients” Cancers 2021 , 13, 6363;

[0087] (6) Simon et al. “An aerobic exercise intervention to improve metabolic health among people living with HIV with at-risk alcohol use: the ALIVE-Ex research study protocof’ AIDS Research and Therapy (2023) 20:35;

[0088] (7) Bharadwaj et al. “Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy” DOI: 10.3791 / 52350-v;

[0089] (8) Hashimoto T et al. British Journal of Nutrition (2012), 107, 1566-1569.

[0090] Example 2 - Composition according to the invention

[0091] A liquid nutritional product for improving muscle mass, strength and function in an elderly person, the product comprising, per serving of 200 ml, about 21 g proteinaceous matter, with 45 weight % pea protein, 43 w weight % soy protein and 12 weight % free leucine, calculated on the weight of the protein fraction. The total amount of leucine is about 19 weight % of all protein. The product has a caloric content of 1 .5 kcal / ml, and comprises about 15 weight % carbohydrates, 5.2 weight % fat and 1 .4 weight % fibre (31 en% fat, 39 en% carbohydrates, 28 en% protein and 2 en% fibre), minerals and vitamins. The composition comprises 10 mg xanthohumol, 5 mg fucoxanthin and 100 mg c-phycocyanin per day.

Claims

Claims1 . A nutritional composition comprising xanthohumol, fucoxanthin and c-phycocyanin.

2. The nutritional composition according to claim 1 , wherein the composition comprises a protein.

3. The nutritional composition according to claim 2, wherein the protein is selected from whey protein, soy protein, pea protein, canola protein or mixtures thereof.

4. The nutritional composition according to claim 2 or 3, wherein the composition comprises at least 10 g protein per 100 g dry weight, preferably at least 15 g per 100 g dry weight.

5. The nutritional composition according to any one of the preceding claims, wherein the composition comprises 1 to 40 mg xanthohumol per unit dose, 1 to 20 mg fucoxanthin per unit dose and 5 to 300 mg c-phycocyanin per unit dose.

6. The nutritional composition according to any one of the preceding claims, wherein the composition comprises xanthohumol, fucoxanthin and c-phycocyanin in a weight ratio of 1 : 0.3 - 1 : 20 - 60.

7. The nutritional composition according to any one of the preceding claims, being a tube feeding composition, preferably comprising 0.5 to 40 mg / 100 kcal xanthohumol, 0.5 to 20 mg / 100 kcal fucoxanthin and 5 to 200 mg / 100 kcal c-phycocyanin, and wherein the tube feeding composition preferably provides, per 100 ml, 100 - 200 kcal.

8. The nutritional composition according to any one of the preceding claims for use as a medicament.

9. The nutritional composition according to claims 1 to 7, for use in the treatment and / or prevention of muscle wasting in a human subject.

10. The nutritional composition for use according to claim 9, wherein the human subject is an elderly subject of at least 50 years of age, preferably at least 55 years of age; and / or wherein the human subject is suffering from muscle decline; and / or wherein the human subject is suffering from cancer or sarcopenia or Intensive Care Unit (ICU) acquired weakness.

11. The nutritional composition for use according to claim 9 or 10, wherein the composition provides 1 to 40 mg / day xanthohumol, 1 to 20 mg / day fucoxanthin and 15 mg to 300 mg / day c-phycocyanin.

12. Use of a nutritional composition comprising therapeutically effective amounts of xanthohumol, fucoxanthin and c-phycocyanin in the manufacture of a product for prevention and / or treatment of muscle wasting in a human subject.

13. Use according to claim 12, wherein the nutritional composition is administered in an amount that provides 1 to 40 mg xanthohumol, 1 to 20 mg fucoxanthin and 15 to 300 mg c-phycocyanin per day.

14. The nutritional composition according to any one of claims 1 - 7, wherein the composition comprises a protein fraction comprising 32-58 weight % pea protein, 30-56 weight % soy protein and 8-16 weight % free leucine, preferably a protein fraction comprising 36-54 weight % pea protein, 34-52 weight % soy protein and 10 - 14 weight % free leucine, and optionally up to 5 weight % of free branched chain amino acids other than leucine, based on all proteinaceous matter, wherein the composition is essentially free from animal protein; wherein the composition preferably comprises a protein fraction comprising 41-50 weight % pea protein, 39-47 weight % soy protein and 10.5 - 13.5 weight % free leucine, based on all proteinaceous matter; wherein the composition preferably comprises a protein fraction comprising 43-47 weight % pea protein, 41-45 weight % soy protein and 11 - 13 weight % free leucine, based on all proteinaceous matter.

15. The nutritional composition according to any one of claims 1 - 7, wherein the composition comprises a protein fraction comprising (a) 15 to 35 weight % of casein ; (b) 25 to 50 weight % of whey protein ; (c) 10 to 30 weight % of soy protein ; and (d) 10 to 30 weight % of pea protein, relative to the total protein in the protein fraction, wherein the sum of said proteins preferably equals 100 weight % of the protein fraction; wherein the composition preferably comprises a protein fraction comprising (a) 20 to 30 weight % of casein ; (b) 30 to 40 weight % of whey protein ; (c) 15 to 25 weight % of soy protein ; and (d) 15 to 25 weight % of pea protein, relative to the total protein in the protein fraction, wherein the sum of said proteins preferably equals 100 weight % of the protein fraction.

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