Plant extract

Abstract

A composition comprising a plurality plant derived of peptides obtained by protease digestion of a Brassicae plant extract for use in the enhancement of cellular metabolism in a subject is claimed. Th

Description

Field of the Disclosure The disclosure relates to a composition comprising a plurality of plant derived peptides obtained by protease digestion and its use in the enhancement of cellular metabolism and in the treatment of diseases, for example diseases associated with muscle atrophy such as muscular dystrophy or sarcopenia, that would benefit from enhancement of cellular processes such as the stimulation of transcription and / or translation, and / or bioenergetics. Background to the Disclosure Biological processes are fundamental to the cell and consequently survival of an organism. Biological processes include but are not limited to the control of DNA replication and cell division, transcription of DNA into RNA, stability of transcribed RNA, translation of mRNA into protein, protein stability and function and bioenergetics. Bioenergetics is the energy metabolism of a cell and is a combination of glycolysis and the tricarboxylic acid cycle (TCA cycle) also referred to as the Krebs Cycle. In simple terms glycolysis is the breakdown of glucose to pyruvate via a series of intermediates which is converted to acetyl Coenzyme A which enters the TCA cycle and creates reducing intermediates NADH and FADH2 which are fed into the oxidative phosphorylation pathway to subsequently transduce energy contained in NADH / FADH2 to Adenosine Triphosphate (ATP). ATP is used by the cell in energy requiring reactions such as DNA, RNA and protein synthesis, cell divisional and tissue function such as for example muscle and nerve growth. Muscle tissue can be of different three types. Striated muscle, which primarily is skeletal muscle that allows, amongst other functions, an organism to move its limbs. Smooth muscle is also described as non-straited muscle and can be found in the walls of organs such as the stomach, small intestine and bladder. Cardiac muscle is heart muscle and is a specialised involuntary straited muscle found in the walls of the heart. The growth of muscle tissue fundamentally requires protein synthesis to provide muscle proteins that are incorporated into muscle tissue to enhance tissue growth and function. This is stimulated by, amongst other things, exercise which stimulates and co-ordinates physiological functions to promote muscle growth. There are pathological conditions that do not respond to the normal physiological cues that are associated with muscle growth and can result in loss of muscle mass. One such example is sarcopenia which is associated with aging or loss of mobility. The exact cause of sarcopenia is not well understood although it does appear that there is a failure in normal muscle physiology such as reduced translation and bioenergetics although poor nutrition and a sedentary lifestyle are considered major associated factors. Currently, exercise is recommended to offset the effects of sarcopenia and there are no approved medications to treat the disease. Steroid (e.g. anabolic steroids, testosterone) administration has shown to reverse the effects of sarcopenia but the associated side effects such as potential to induce prostate cancer in males, virilisation in females and effects on cardiovascular function have resulted in cessation of steroid treatment as a treatment of sacropenia. The use of growth hormone to stimulate muscle biogenesis has not been successful. In addition, muscle atrophy includes the wasting of muscle mass. Rare genetic diseases can result in muscle atrophy such as muscular dystrophy which describe a collection of genetic diseases, for example Duchenne muscular dystrophy, Becker muscular dystrophy, and myotonic dystrophy. Current treatments include the use of anabolic steroids, mobility aids and surgery, none of which are effective in reversal of muscular atrophy. There is a need to identify alternative therapeutic interventions to treat muscle wasting diseases such as sarcopenia which is becoming common in an aging population. Protein hydrolysates containing bioactive peptides are typically produced through the enzymatic hydrolysis or gastrointestinal digestion of animal or plant proteins (Chakrabarti et al., 2018). Bioactive peptides are low molecular weight peptides generally with an amino acid chain length of 2-20 amino acids and are thought to exert several physiological functions beneficial to human health, such as immunomodulatory, antihypertensive, antimicrobial or antioxidative properties (Zaky et al., 2022). The bioavailability of bioactive peptides offers a unique avenue for enhancing human health. For instance, peptides can penetrate deeper into certain tissue than the relatively large polypeptides or proteins from which the peptides are derived. The therapeutic potential of bioactive rapeseed peptides noted in recent studies provides increasingly promising results and a mandate for additional research that may promote such entities as active agents in products such as anti-ageing creams or chemotherapeutics. For example, a recent study investigating the antiproliferative effects of rapeseed protein hydrolysates on hormone-dependent breast cancer (MCF-7) cells found that rapeseed proteins undergoing 8.5-9.0% hydrolysis with an average mass of 10 kDa suppresses MCF-7 (83.9%) proliferation whilst not impairing human fibroblast viability (Ferrero et al., 2021). In our co-pending application PCT / GB2024 / 051192, currently unpublished, which we incorporate by reference in its entirety, we disclose a peptide extract prepared from a Brassica napus hydrolysate and its use in wound healing, anti-aging, anti-inflammatory disease and conditions and cosmetic enhancement of skin. This disclosure relates to an alternative use in relation to the enhancement of muscle physiology through the stimulation of protein synthesis, glycolysis and the TCA cycle in relation to improving muscle cell function in normal and diseased muscle cells. Statements of Invention According to an aspect of the invention there is provided the use of a composition comprising a plurality of plant derived peptides obtained by protease digestion of a plant extract prepared from a plant of the genus Brassicae in the enhancement of cellular metabolism in a subject. According to an aspect of the invention there is provided a composition comprising a plurality of plant derived of peptides obtained by protease digestion of a plant extract prepared from a plant of the genus Brassicae for use in the enhancement of cellular metabolism in a subject. According to an aspect of the invention there is provided a composition comprising a plurality of plant derived peptides obtained by protease digestion of a plant extract prepared from a plant of the genus Brassicae for use in the treatment of muscle atrophy in a subject. The composition can be used as a diet supplement to provide the plurality of plant derived peptides to improved and maintain good health and to increase athletic performance or in the treatment of conditions that result in muscle atrophy. “Cellular metabolism” concerns all processes required to maintain a functional cell. In multicellular organisms, cellular metabolism includes all processes required to maintain life of the organism and includes by example and not limitation, DNA / RNA synthesis, protein synthesis and energy metabolism, for example the synthesis of adenosine triphosphate (ATP) via glycolysis and the tricarboxylic acid (TCA) cycle. Altered cellular metabolism can be associated with pathological conditions. For example, pathological muscle wasting conditions such as sarcopenia. Sarcopenia is a type of muscle loss that occurs with ageing and / or immobility (primary sarcopenia). Secondary sarcopenia can be caused by malabsorption, immobility, starvation, hypothyroidism or inflammatory conditions. Sarcopenia is also known to be associated with chronic kidney disease. “Plurality” is construed as a composition comprising at least 2 peptides; preferably at least 3, 4, 5, 6, 7, 8, 9, or 10 peptides. Alternatively, plurality is construed as 10-15, 15 to 20 peptides, 20-25 peptides, 30-35 peptides, 35-40 peptides 40-45 peptides, 45-50 peptides, 50- 55 peptides, 55-60 peptides, 60-65 peptides or 65 to 70 peptides. In a further alternative embodiment of the invention said plurality is construed as a composition comprising at least 70 peptides. In a preferred embodiment of the invention said plurality of peptides comprise peptides that are at least 2 amino acids in length. In a preferred embodiment of the invention said plurality of peptides comprise peptides that vary in length of between 4 to 25 amino acids. In a preferred embodiment of the invention said plurality of peptides comprise peptides that vary in length of between 4 to 20 amino acids. In a preferred embodiment of the invention said plurality of peptides comprise peptides that vary in length of between 4 to 18 amino acids. In a preferred embodiment of the invention said plurality of peptides comprise peptides that vary in length of between 4 to 16 amino acids. In a preferred embodiment of the invention said plurality of peptides comprise peptides that vary in length of between 4 to 14 amino acids. In a preferred embodiment of the invention said plurality of peptides comprise peptides that vary in length of between 4 to 12 amino acids. In a preferred embodiment of the invention said plurality of peptides comprise peptides that vary in length of between 8 to 12 amino acids. In a preferred embodiment of the invention said plurality of peptides comprise peptides that vary in length of between 10 to 12 amino acids. In a preferred embodiment of the invention said plurality of peptides is obtained from a plant of the genus Brassica, for example Brassica napus or Canola. In a preferred embodiment of the invention said plurality of peptides is obtained from Canola. In a preferred embodiment of the invention said plurality of peptides is obtained by digestion with at least one endopeptidase. In a preferred embodiment said at least one endopeptidase is selected from the group consisting of: subtilisin, bacillolysin, Alcalase, Protamex, flavourzyme and actinidin. Preferably, said at least one endopeptidase is subtilisin; alternatively said at least one endopeptidase is bacillolysin; preferably subtilisin and bacillolysin are combined. In a preferred embodiment of the invention said at least one endopeptidase is Flavourzyme® Preferred enzymes are subtilisin and bacillolysin. Alcalase® is a commercial preparation of subtilisin isolated from Bacillus subtilis. Protamex® is a commercial preparation of a complex consisting of subtilisin and a neutral protease called bacillolysin; see https: / / www. brenda--enzymes.org / enzyme. php?ecno-3.4.24.28. Flavourzyme® is a commercial preparation of both endo- and exopeptideases but contains predominantly exoproteases with aminopeptidase activity and is isolated from Aspergillus oryzae. In an alternative embodiment of the invention said at least one endopeptidase is actinidin. In a preferred embodiment of the invention said actinidin is combined with at least one further protease. Preferably, said further protease is subtilisin. In a preferred embodiment of the invention said further protease is bacillolysin In a preferred embodiment said further protease includes both subtilisin and bacillolysin. In a preferred embodiment of the invention, cellular metabolism is enhanced by the stimulation of cytoplasmic translation. In a further preferred embodiment of the invention, cellular metabolism is enhanced by the stimulation of glycolysis. In a further preferred embodiment of the invention, cellular metabolism is enhanced by the stimulation of the tricarboxylic acid (TCA) cycle. In a preferred embodiment of the invention said subject is a mammal; preferably a human. In a preferred embodiment of the invention said cellular metabolism is the enhancement of translation and / or glycolysis and / or the tricarboxylic acid (TCA) cycle in a muscle cell stem cell; preferably a myoblast. In a preferred embodiment of the invention said cellular metabolism is the enhancement of translation and / or glycolysis and / or the tricarboxylic acid (TCA) cycle in a differentiated muscle cell. In a preferred embodiment of the invention the enhancement of cellular metabolism is in the treatment of a muscles atrophy. According to an aspect of the invention there is provided the use of a composition comprising a plurality of plant derived peptides obtained by protease digestion of a plant extract prepared from a plant of the genus Brassicae in the treatment of muscle atrophy in a subject. In a preferred embodiment of the invention said muscular atrophy is associated with muscular dystrophy. In a preferred embodiment of the invention said muscular atrophy is associated with sarcopenia. The medicaments / compositions according to the invention may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, and compatible carriers. Such amounts will depend on the condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size and weight, the duration of the treatment, the nature of concurrent therapy (if any), and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. The medicaments / compositions used contains an effective number of peptides for producing the desired response in a unit of weight or volume suitable for application to a patient. The medicaments / compositions administered to a subject can be chosen in accordance with different parameters, in particular the state of the subject, their body surface area, and their weight. Other factors include the desired period of treatment. If a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. Administration of medicaments / compositions to mammals other than humans, (e.g., for testing purposes or veterinary therapeutic purposes), is carried out under substantially the same conditions as described above although dosages will vary in accordance with the size of the animal treated. A subject, as used herein, is a mammal, preferably a human, and including a non-human primate, cow, horse, pig, sheep, goat, dog, cat or rodent. When administered to a subject the medicaments / compositions are provided in pharmaceutically acceptable amounts and in pharmaceutically acceptable compositions. The term “pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active peptides. Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof and are not excluded from the scope of the invention. Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like. Also, pharmaceutically acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium, or calcium salts. Medicaments / compositions according to the invention may be combined, if desired, with a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” as used herein means one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for application to a human subject and are typically inert. The term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate administration. The components of the medicaments also are capable of being co-mingled with the plurality of peptides, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy. The medicaments / compositions are combined with pharmaceutically acceptable excipients, which may include: (a) fillers such as lactose, manitose, dicalcium phosphate, microcrystalline cellulose, starch, pre-gelatinised starch, (b) binders such as hydroxypropylmethyl cellulose, polyvinyl pyrrolidone, polyvinyl acetate, (c) powder flow enhancers such colloidal silicon dioxide (d) lubricants such as magnesium stearate, sodium stearyl fumarate (e) disintegrants such as sodium starch glycollate and polyvinyl pyrrolidone and (f) anti-sticking agents such as talc (g) taste masking agents such as sucrose, cellulose acetate, cellulose butyrate, polyvinyl acetate, polyvinyl alcohol, polymethacrylates. According to an aspect of the invention there is provided a method to treat a subject suffering from, or having a predisposition to, muscular atrophy comprising administering a composition comprising a plurality plant derived of peptides obtained by protease digestion of a plant extract prepared from a plant of the genus Brassicae to said subject in need of treatment. In a preferred method of the invention said muscular atrophy is associated with muscular dystrophy. In a preferred method of the invention said muscular atrophy is associated with sarcopenia. Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of the words, for example “comprising” and “comprises”, means “including but not limited to”, and is not intended to (and does not) exclude other moieties, additives, components, integers or steps. “Consisting essentially” means having the essential integers but including integers which do not materially affect the function of the essential integers. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise. Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. An embodiment of the invention will now be described by example only and with reference to the following figures: Figure 1. Increased ATP synthesis. L6 myotubes were exposed to 5-5000 pg / mL Valogen material as described under ‘Experimental’. Shown data are averages +1- SEM of 7 independent Seahorse assays in which each condition was probed in quadruplicate. ATP supply rates are plotted in semi-logarithmic space with the respective control rates observed in non-exposed cells represented by dotted horizontal lines. Data were modelled with the ‘simple linear regression’ algorithm of GraphPad Prism v10.2.1 software for Windows. Differences between exposed and non-exposed cells were evaluated for statistical significance by repeated-measures 1-way ANOVA using Dunnett post-hoc analysis (* P <0.05, ** P <0.01). Figure 2. Increased cycloheximide (CHX)-sensitive ATP synthesis. L6 myotubes were exposed to 5-5000 pg / mL Valogen material as described under ‘Experimental’. Shown data are averages +1- SEM of 3 independent Seahorse assays in which each condition was probed in quadruplicate. ATP supply rates are plotted in semi-logarithmic space with the respective control rates observed in non-exposed cells represented by dotted horizontal lines. Data were modelled with the ‘simple linear regression’ algorithm of GraphPad Prism v10.2.1 software for Windows. Differences between exposed and non exposed cells were evaluated for statistical significance by repeated-measures 1-way ANOVA using Dunnett post-hoc analysis (* P <0.05). Figure 3. Hydrolysate material causes a stronger bioenergetic phenotype than leucine. L6 myotubes were exposed to leucine up to 2000 pg / mL (i.e., 15 mM in addition to the 0.45 mM already present in the DMEM growth medium) for 24 h under the same conditions as applied for exposure to the Valogen material. Shown data are averages + / -SEM of 3 independent Seahorse assays in which each condition was probed in quadruplicate. Leucine-stimulated glycolytic ATP supply rates are plotted in linear space and were fitted to a ‘one-phase association’ model using GraphPad Prism (Panel A), while ATP supply data from experiments with cells exposed to Valogen material (Panel B) were analysed as described for Figures 1 and 2. Figure 4. Increased mitochondrial respiratory capacity. L6 myotubes were exposed to 5-5000 pg / mL Valogen material as described under ‘Experimental’ or to leucine as described for Figure 3. Shown data are averages +1- SEM of 6 (panel A) or 3 (panel B) independent Seahorse assays in which each condition was probed in quadruplicate. Oxygen consumption rates (OCR) were measured in the presence of 5 pM BAM 15, a mitochondrial uncoupling agent that allows approximation of the maximum respiratory activity. The obtained respiratory capacities are expressed as uncoupled oxygen consumption rates either normalised to the capacity of non-exposed myotubes (panel A) or to total protein content (panel B). Data were fitted to a ‘one-phase association’ model using GraphPad Prism - the calculated ‘plateau’ parameter was significantly different from the ‘Y0’ value for cells exposed to Valogen material (P = 0.0128) but not for leucine-exposed cells. Differences between exposed and nonexposed cells were further evaluated for statistical significance by repeated-measures 1-way ANOVA using Dunnett post-hoc analysis (* P <0.05). MATERIALSAND METHODS Preparation of Hydrolysate (plurality of plant derived peptides) Rapeseed meal (RSM) or partially defatted rapeseed cake (RSC) is suspended and contacted with water by means of high-shear mixing (Silverston-type mixer), until well incorporated. A solids:liquid ratio of 1:10 is used nominally. The resulting slurry is mixed in a stirred in a vessel for around 40 minutes, with an addition of sodium sulphite 0.025% w / v.. The resulting slurry is transferred to a heated stirring unit where the temperature is increased and maintained between 50 and 60°C. The pH of the slurry is also increased to between 7.5 and 8.5 with the addition of NaOH solution. Once conditions have been met the slurry undergoes enzyme hydrolysis with free enzyme(s), in the stirred reactor vessel. The enzyme(s) are either mixtures of endoproteases and can include optionally exoproteases. Examples include Alcalase® 2.4L (subtilisin) or Protamex®, as endoproteases. A suitable exoprotease includes Flavourzyme®. Typically, addition rates of free enzyme and hydrolysis conditions are given below (Table 1). Table 1 Enzyme example Supplier stated specific activity (U / g) Nominal addition per 100L hydrolysis (II) Temperature (°C) / pH working range Alcalase® 2.4L (subtilisin) >2.4 1.2 30-65°C pH 6.5- 10 Protamex® >1.5 0.75 55-60°C pH 6-9 Flavourzyme® >500 250 30-65°C pH 4.5-7.5 The temperature and pH of the hydrolysis are maintained at, or near, the optima for the enzymes in use, nominally 50-60°C and pH 7.5-8.5 respectively. The hydrolysis is performed for around 3 hours or until the molecular weight profile of the hydrolysis product (for example, determined by HPLC-size exclusion chromatography) reaches a constant or limit. Alternatively immobilised enzymes can be used provided the enzyme hydrolysis takes place after the solid-liquid separation step. In the case of immobilised enzymes, suitable immobilisation onto a beaded polymethacrylate (e.g. Purolite Lifetech™ ECR8415F) support can be achieved using covalent chemistry (e.g. amino-linkages or alternatively via epoxy-linkages). Alternative immobilisation substrates include e.g. Resindion and Sepabeads. This mode of hydrolysis operation can be performed in a plug-in rotating bed reactor, with the immobilised enzyme(s) maintained within the rotating bed basket, or alternatively using a packed or fluidised bed reactor. The extracted and hydrolysed solids are removed by either centrifugation or filtration to leave a clarified supernatant or filtrate containing solubilised protein. The collected solids may be further extracted and hydroylsed, by mixing in the stirred vessel, with a second volume of temperature and pH-adjusted water with enzymes, maintaining a suitable solid:liquid volume ratio (e.g. 1:10). The supernatants or filtrates from the first and subsequent extractions are pooled. The pooled supernatants undergo a filtration stage using an ultrafiltration membrane of either 3 or 1kDa NMWCO polyethersulfone (PES). There is a typical split of 80:20 filtrate to retentate. The filtrate contains the soluble peptides of interest, and the retentate is either sent to waste or recycled to an earlier stage of the process, to allow further hydrolysis of protein to peptides to increase recovery in the product. A subsequent nanofiltration stage is carried out where the Ultrafiltration (UF) filtrate fraction is concentrated further with another 80:20 split of filtrate to retentate using a 500 dalton NMWCO membrane. The nanofiltration (NF) filtrate may be used as the diluent for the original HS mixing extraction step provided the process is topped up with fresh water to prevent the build up of access sodium and other ions. The NF retentate material is freeze-dried or spray dried to yield a solid peptide product, with a nominal protein content by N-analysis (Dumas) of 50%. The supernatants may also be distilled by Rotavap to yield a concentrated solution of material up to 80% solid loading before the solution becomes viscous. Downstream processing of the peptide-containing filtrate An ion exchange (IE) column can be used to further concentrate the peptides (prior to any drying step), possibly separating into positively and negatively charged peptides depending on specific application is found which requires selective separation. An aim of this step is to enrich the protein content of the peptide solution product from the UF stage. The concentrated hydrolysis product is then either passed through a sterile filter (0.2 or 0.45pm) and sent for final storage if a liquid product is preferred or sent to be dried to provide a powder product using either freeze or spray drying. Cell Culture Clonal rat myoblasts were obtained from the European Collection of Cell Culture. L6 myoblasts were cultured at 37 °C under a humidified carbogen atmosphere of 5% CO2 and 95% air in Dulbecco’s modified Eagle medium (DMEM) that contained 5 mM glucose, 4 mM glutamine and 2 mM sodium pyruvate, and that was supplemented with 10% (v / v) fetal bovine serum (FBS). L6 myoblasts were seeded on XFe96 (Agilent Technologies) tissue culture plates at 1.3 x 104 cells / well and grown for 24 h in supplemented DMEM. FBS concentration was then lowered to 2% (v / v) and this ‘lightserum’ medium was refreshed every other day until the cells had visibly turned to myotubes after 7 days. At this point, myotubes were exposed to rapeseed-meal-derived material (Valogen Biosciences Ltd) at 5 - 5000 pg / mL for 24 hours. Roughly half this Valogen material is comprised of peptide hydrolysate. The components of the hydrolysates are listed in Table 2. Following such exposure, cells were washed into a Krebs-Ringer buffer that contained 5 mM glucose and was further comprised of 136 mM NaCI, 3.7 mM KCI, 10 mM Hepes, (pH 7.4), 2 mM NaH2PO4, 1 mM MgCI2 and 1.5 mM CaCI2. After 1h incubation in KRH at 37 °C under air, cells were transferred to a Seahorse XFe96 analyser (Agilent Technologies) to simultaneously measure oxygen uptake and medium acidification. Both glycolytic and oxidative ATP synthesis rates were calculated from these extracellular fluxes as we have described elsewhere (Rosie A. Donnell, 2022). For the calculation it was assumed that cellular energy metabolism was entirely fuelled by glucose under our experimental conditions. Table 2 dmponent Resuit j Energy (KcaiftOOg) 1267 i j Total fat (q / lOOqj (Fatty acids monoun^_______________ Fatty acids polyunsaturated (gCOOq) | 0.1 j ........................................... 10.1 i <0.1 I Fatty acids-tans (u / lOOq) 1 <0.1 j I Carbohydrate total (q / 1®q) I 18.3 j j Carbohydrate available (g / lOOg)____________ Sugars (g / ioog) [ TFO___________ ...............................................................j Fibre (gH 00g)____________________________ Sodium (g / i bbqj 1.7 I ................p .......................... s 8.1 | | Ash at 526 (qHOOq) —---1 6.8 Example 1 Peptide hydrolysates dose-dependently increase the rate of total ATP synthesis in L6 myotubes (Fig. 1A). Increased ATP production is mostly due to stimulated glycolytic ATP supply (Fig. 1B), although a relatively small, statistically non-significant, increase in oxidative ATP supply (Fig. 1C) contributes to the bioenergetic phenotype. Example 2 Peptide hydrolysates dose-dependently increase the rate of total (Fig. 2A) and glycolytic (Fig. 2B) cycloheximide-sensitive ATP synthesis and thus increase ATP supply used to fuel ribosomal RNA translation. This observation indicates that the Valogen material stimulates cellular protein synthesis. Example 3 Increased ATP synthesis (Fig. 1) is likely a response to meet increased ATP demand from stimulated protein synthesis (Fig. 2). Such anabolic stimulation is typically provoked by branched-chain amino acids, most notably by leucine (e.g. Rennie et al., 2006). Exposure to 2000ug / ml leucine for 24h indeed stimulates the glycolytic ATP synthesis rate in L6 myotubes (Fig. 3A). This leucine concentration is higher than the concentration skeletal muscle is expected to face physiologically, but even so, the bioenergetic stimulation is relatively modest (1.4-fold) compared to the 2.2-fold stimulation achieved by equivalent exposure to Valogen material (Fig. 3B). Example 4 Peptide hydrolysates dose-dependently increase mitochondrial respiratory capacity of L6 myotubes (Fig. 4A). The maximum stimulation is roughly 1.2-fold, which is higher than the stimulation achieved by leucine (Fig. 4B). This observation suggests that the oxidative bioenergetic capacity of L6 cells may adapt to increased ATP demand from anabolic stimulation. Chakrabarti, S., Guha, S., and Majumder, K., 2018. Food-Derived Bioactive Peptides in Human Health: Challenges and Opportunities. Nutrients, 10(11), pp.1738. Ferrero, R. L., Soto-Maldonado, C., Weinstein-Oppenheimer, C., Cabrera-Munoz, Z., and Zuniga-Hansen, M. E., 2021. Antiproliferative Rapeseed Defatted Meal Protein and Their Hydrolysates on MCF-7 Breast Cancer Cells and Human Fibroblasts. Foods (Basel, Switzerland), 10(2), pp.309. Zaky, A. A., Simal-Gandara, J., Eun, J. B., Shim, J. H., and Abd El-Aty, A. M., 2022. Bioactivities, Applications, Safety, and Health Benefits of Bioactive Peptides From Food and By-Products: A Review. Frontiers in nutrition, 8, pp.815640. Rosie A. Donnell, Jane E. Carre, Charles Affourtit, Acute bioenergetic insulin sensitivity of skeletal muscle cells: ATP-demand-provoked glycolysis contributes to stimulation of ATP supply, Biochemistry and Biophysics Reports, Volume 30, 2022, 101274. CLAIMS 1. A composition comprising a plurality plant derived of peptides obtained by protease digestion of a plant extract prepared from a plant of the genus Brassicae for use in the enhancement of cellular metabolism in a subject 2. The composition according to claim 1 wherein said plurality of peptides comprise peptides that are at least 2 amino acids in length. 3. The composition according to claim 1 or 2 wherein said plurality of peptides comprise peptides that vary in length of between 4 to 25 amino acids. 4. The composition according to any one of claims 1 to 3 wherein said plurality plant derived of peptides are obtained from a plant of the species Brassica napus or Canola. 5. The composition according to any one of claims 1 to 4 wherein said plurality plant derived of peptides are obtained by digestion with at least one endopeptidase. 6. The composition according to

Claims

1. A composition comprising a plurality plant derived of peptides obtained by protease digestion of a plant extract prepared from a plant of the species Brassicae napus or Canola for use in the in the treatment of muscle atrophy in a subject.

2. The composition for use according to claim 1 wherein said plurality of peptides comprise peptides that are at least 2 amino acids in length.

3. The composition for use according to claims 1 or 2 wherein said plurality of peptides comprise peptides that vary in length of between 4 to 25 amino acids.

4. The composition for use according to any one of claims 1 to 3 wherein said plurality of plant derived peptides are obtained by digestion with at least one endopeptidase.

5. The composition for use according to claim 4 wherein at least one endopeptidase is subtilisin.

6. The composition for use according to claim 4 wherein at least one endopeptidase is bacillolysin.

7. The composition for use according to claim 4 wherein at least one endopeptidase is actinidin.

8. The composition for use according to any one of claims 1 to 7 wherein said subject is a mammal, preferably a human.

9. The composition for use according to claim 8 wherein said muscular atrophy is muscular dystrophy.

10. The composition for use according to claim 8 wherein said muscular atrophy is secondary sarcopenia.

11. Use of a composition comprising a plurality of plant derived peptides obtained by protease digestion of a plant extract prepared from a plant of the species Brassicae napus or Canola in a diet supplement to enhance cytoplasmic translation.5 12. Use of a composition comprising a plurality of plant derived peptides obtained byprotease digestion of a plant extract prepared from a plant of the species Brassicae napus or Canola in a diet supplement to enhance glycolysis.

13. Use of a composition comprising a plurality of plant derived peptides obtained by 10 protease digestion of a plant extract prepared from a plant of the species Brassicae napus or Canola in a diet supplement to enhance the tricarboxylic acid (TCA) cycle.

14. Use of a composition comprising a plurality of plant derived peptides obtained by protease digestion of a plant extract prepared from a plant of the species Brassicae 15 napus or Canola in a diet supplement to enhance muscle function in primary sarcopenia in elderly subjects.s

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