WO2025168966A1 - Delphinidin-rich maqui berry composition as agent for increasing muscle mass and anti-aging agent - Google Patents

Abstract

The present invention is focused on extending the lifespan and the healthy period of life (healthspan), delaying the development of chronic diseases and dysfunctions, which is a growing need of aging societies. Among the strategies to promote and extend healthspan, nutrition and food ingredients play a central role. The present invention is related to standardized delphinidin-rich maqui berry composition comprising different amounts and combination of delphinidins, which represent a potent class of antioxidant anthocyanins as agent for reducing progressive loss of skeletal muscle, increasing muscle mass and anti-aging agent or geroprotective agent, and thereby, enhancing the healthspan and preserving locomotive functions for longer periods of life. Such effect was measured as climbing ability during life time in an animal model (Drosophila melanogaster), Experimental assays demonstrated that delphinidin-rich standardized maqui berry extracts upregulate the expression of antioxidant enzymes and mitochondrial homeostasis-related proteins, which determine contractile muscle function. Such delphinidin-rich standardized maqui berry composition is useful as antioxidant supplement for adults and elderly.

Description

DELPHINIDIN-RICH MAQUI BERRY COMPOSITION AS AGENT FOR INCREASING MUSCLE MASS AND ANTI-AGING AGENT

BACKGROUND OF THE INVENTION.

FIELD OF THE INVENTION

The present invention is related to nutraceuticals, particularly, nutraceuticals of vegetal origin aimed to improving human or animal health of adults and elderly. Specifically, the present invention is related to standardized delphinidin-rich maqui berry composition comprising different amounts and combination of delphinidins, which represent a potent class of antioxidant anthocyanins as agent for reducing progressive loss of skeletal muscle increasing muscle mass and anti-aging agent or geroprotective agent, and thereby, enhancing the healthspan and preserving locomotive functions for longer periods of life. Such standardized delphinidin-rich maqui berry composition can upregulate the expression of antioxidant enzymes and mitochondrial homeostasis-related proteins, which determine contractile muscle function. Such standardized delphinidin-rich maqui berry composition is useful as antioxidant supplement for adults and elderly either human beings or animals.

BACKGROUND OF THE INVENTION

As life expectancy increases worldwide, there is a general expectation that the age distribution in our societies will move towards the elderly (Lim, W., Wong, S., Leong, I., Choo, P. & Pang, W. Forging a Frailty-Ready Healthcare System to Meet Population Ageing. Int J Environ Res Public Health 14, 1448 (2017)). One of the most pronounced changes in the elderly is the loss of mobility and physical capacity due to a progressive loss of skeletal muscle mass and function (a condition known as sarcopenia), thereby affecting their quality of life and their independency (Larsson, L. et al. Sarcopenia: Aging-related loss of muscle mass and function. Physiol. Rev. 99, 427-511 (2019)). Thus, there is a major challenge for present societies to plan interventions to increase the healthspan of their population, and consequently, to extend the healthy period of life and delay the onset or development of chronic diseases and disabilities until a brief period at the end of life. Sarcopenia is a hallmark of aging and reduced healthspan and begins long before there is any clear physical impact in the senior adult (del Campo, A. et al. Muscle function decline and mitochondria changes in middle age precede sarcopenia in mice. Aging (2018) doi:10.18632/aging.101358; Short, K. R. et al. Decline in skeletal muscle mitochondrial function with aging in humans. Proceedings of the National Academy of Sciences 102, 5618-5623 (2005); y Hughes, V. A. et al. Longitudinal Muscle Strength Changes in Older Adults: Influence of Muscle Mass, Physical Activity, and Health. J Gerontol A Biol Sci Med Sci 56, B209-B217 (2001)). Alterations in skeletal muscle possibly exacerbate other diseases that appear during aging, such as decreased metabolic rate, increased insulin resistance, and a low degree inflammation (Short,

K. R. et al. Decline in skeletal muscle mitochondrial function with aging in humans. Proceedings of the National Academy of Sciences 102, 5618-5623 (2005), McCormick, R. & Vasilaki, A. Age- related changes in skeletal muscle: changes to life-style as a therapy. Biogerontology 19, 519— 536 (2018)). As skeletal muscle frailty is independent of ethnicity, age, morbidity, obesity, income, or health behaviors and is a major public health problem worldwide (Larsson, L. et al. Sarcopenia: Aging-Related Loss of Muscle Mass and Function. Physiol Rev 99, 427-511 (2019); Lim, W., Wong, S., Leong, I., Choo, P. & Pang, W. Forging a Frailty-Ready Healthcare System to Meet Population Ageing. Int J Environ Res Public Health 14, 1448 (2017); y Hughes, V. A. et al. Longitudinal Muscle Strength Changes in Older Adults: Influence of Muscle Mass, Physical Activity, and Health. J Gerontol A Biol Sci Med Sci 56, B209-B217 (2001)). Nutritional interventions have the potential to reach large fraction of the population and have proven effective in extending the healthspan. Examples of these are diets with caloric restriction (Hughes, V. A. et al. Longitudinal Muscle Strength Changes in Older Adults: Influence of Muscle Mass, Physical Activity, and Health. J Gerontol A Biol Sci Med Sci 56, B209-B217 (2001 )) and diets with high protein intake (Muscariello, E. et al. Dietary protein intake in sarcopenic obese older women. Clin Interv Aging 133 (2016) doi:10.2147/CIA.S96017), which although have been linked to reduced oxidative stress and reduction of energy expenditure, there is little adherence in everyday life. Antioxidant dietary compounds offer an alternative to these with proven effects induced muscle atrophy, reducing skeletal muscle mass loss and improving function (Kim, C. & Hwang, J.-K. Flavonoids: nutraceutical potential for counteracting muscle atrophy. Food Sci Biotechnol 29, 1619-1640 (2020)). However, the effects that commercial and standardized antioxidant supplements (which can be scaled to population-level interventions) have over sarcopenia and the healthspan in general still need to be demonstrated.

Growing evidence suggests that muscle loss and malfunctioning begin long before there is any clear physical impact in the senior adult (del Campo, A. et al. Muscle function decline and mitochondria changes in middle age precede sarcopenia in mice. Aging (Albany. NY). (2018). doi:10.18632/aging.101358). After the age of 25, muscle mass begins to decline between 3% to 10% per decade (Short, K. R. et al. Decline in skeletal muscle mitochondrial function with aging in humans. Proc. Natl. Acad. Sci. 102, 5618-5623 (2005)) and reaches a speed of 1% decline per year at older ages (Hughes, V. A. etal. Longitudinal Muscle Strength Changes in Older Adults Influence of Muscle Mass, Physical Activity, and Health. Journals Gerontol. Ser. A 56, B209- B217 (2001)) as estimated. Skeletal muscle frailty is independent of ethnicity, age, morbidity, obesity, income, or health behaviors, and is a major public health problem worldwide (Larsson,

L. et al. Sarcopenia: Aging-related loss of muscle mass and function. Physiol. Rev. 99, 427-511 (2019), Lim, W. S., Wong, S. F., Leong, I., Choo, P. & Pang, W. S. Forging a frailty-ready healthcare system to meet population ageing. Int. J. Environ. Res. Public Health 14, (2017), Hughes, V. A. et al. Longitudinal Muscle Strength Changes in Older Adults Influence of Muscle Mass, Physical Activity, and Health. Journals Gerontol. Ser. A 56, B209-B217 (2001)).

Alterations in skeletal muscle possibly leads to or exacerbate other diseases that appear during aging such as a decrease in metabolic rate, increased insulin resistance and bone loss (Short, K. R. et al. Decline in skeletal muscle mitochondrial function with aging in humans. Proc. Natl. Acad. Sci. 102, 5618-5623 (2005), McCormick Aphrodite Vasilaki, R. Age-related changes in skeletal muscle: changes to life-style as a therapy. Biogerontology 19). Recent evidence has suggested that sarcopenia can occur not only because of aging but also as an outcome of other aging- associated pathologies (Larsson, L. et al. Sarcopenia: Aging-related loss of muscle mass and function. Physiol. Rev. 99, 427-511 (2019), Tieland, M., Trouwborst, I. & Clark, B. C. Skeletal muscle performance and ageing. (2017). doi:10.1002/jcsm.12238). Altogether, these data suggest that interventions to maintain muscle performance and homeostasis for longer periods of life not only should be thought as a health need or an intervention for senior adults but also should be thought to be implemented in younger people, especially those with comorbidities.

In relation to patent documents, KR101982326B1 (Accotech Co., Ltd.) discloses a composition for preventing or treating muscular diseases or improving muscle function comprising fucosterol, mushroom, crushed mushroom, mushroom extract, top, top crush or top extract. The fucosterol, mushroom, mushroom crusher, mushroom extract, top, top crush or top extract are useful for increasing protein expression of major gene p-mTOR involved in muscle protein synthesis, MuRF- 1 and atrogin-1 mRNA expression, and myoD and myogenin mRNA expression, which are involved in muscle differentiation, and thus have an effect of excellently enhancing muscle function. In addition, since the composition is a natural product, it can be safely used without side effects and can be used as medicines, foods, cosmetics, animal feeds or feed additives.

JP2015164967A (Generex Pharmaceuticals Incorporated) is aimed to solve the problem in which longevity still remains a poorly understood area of research although it has long been one of the hottest areas of science and, in particular, aging research involving the search for single-gene mutations with dominant positive effects on lifespan/health span has not been successful, and proposing compounds, extracts and active fractions of the plant Geum japonicum, and methods for increasing longevity and survival potency or for preventing or treating various medical conditions, including diabetes, inflammation, wound healing, bed sores, and ocular disorders. The compounds provided can be formulated into pharmaceutical compositions and medicaments that are useful in the disclosed methods. Also provided are the use of the compounds and extracts in preparing pharmaceutical formulations and medicaments.

Rejuvenation Research, Volume 12, Number 5, 2009, © Mary Ann Liebert, Inc., DOI: 10.1089/rej.2009.0877 is related to a botanical extract (Regrapex-R®) prepared from whole grape (Vitis vinifera) and Polygonum cuspidatum, containing polyphenols, including flavans, anthocyanins, emodin, and resveratrol. The extract has dose-dependent scavenging effects on reactive oxygen species (ROS), and the same demonstrated inhibiting increases of ROS and protein carbonyl in isolated rat liver mitochondria following exposure to 2,2’-azobis (2-amidino propane) dihydrochloride (AAPH), a potent lipid oxidant generator. Also, the extract demonstrated antioxidant effects by protecting enzyme activities of the mitochondrial respiratory electron transport chain (complexes I and II) and pyruvate dehydrogenase in isolated liver mitochondria with AAPH insult. Further, the extract protected pre-treated human neuroblastoma cells (SKN- MC) against AAPH induced oxidation, maintaining cell viability and inhibiting excessive ROS generation. As well, after fed the extract to transgenic Drosophila expressing human a-synuclein to a model for Parkinson disease, male transgenic flies, fed with 0.16-0.64 mg extract/100 g of culture medium, showed a significant improvement in climbing ability compared to controls. Female transgenic flies showed a significant extension in average lifespan. Thus, Regrapex-R would be a potent free radical scavenger, a mitochondrial protector, and a promising candidate to protect against neurodegenerative disease and potentially extend lifespan.

Int. J. Mol. Sci. 2020, 21 , 3893; doi : 10.3390/ijms21113893 is related to the use of olive oil and its biophenolic constituents like hydroxytyrosol (HT) in the health-promoting effects of two hydroxytyrosol preparations, pure HT and Hidrox® (HD, hydroxytyrosol in its “natural” environment) based on an invertebrate model organism Caenorhabditis elegans. HD exposure led to much stronger beneficial locomotion effects in wild type worms compared to HT in the same concentration, and in OW13 worms - a PD-model characterized by a-synuclein expression in muscles, HD exhibited a significant higher effect on a-synuclein accumulation and swim performance than HT, which was also partly confirmed also in swim assays with a UA44 strain, which features a-synuclein expression in DA-neurons. Thus, beneficial effects of HD and HT treatment with similar strength were detected in the lifespan and autofluorescence of wild-type nematodes, in the neuronal health of UA44 worms as well as in the locomotion of rotenone- induced PD-model. Thus, a HD polyphenolic extract treatment could partly prevent or even treat aging-related neurodegenerative diseases and aging itself.

In relation to the use of Drosophila for clinical and nutritional claims and trials, it should be noted that several lines of evidence support that aging-related processes, such as inflammation and muscle loss, begin early in adulthood, many years before age begins (del Campo, A. et al. Muscle function decline and mitochondria changes in middle age precede sarcopenia in mice. Aging (Albany. NY). (2018). doi:10.18632/aging.101358). Therefore, antiaging therapies begin to be administered from adulthood. Costs associated to demonstrating an antiaging compound can be costly methodologically and bioethically, highly costly, and difficult in terms of infrastructure. This is the reason why there are few natural compounds with scientifically proven anti-aging properties in living multicellular organisms. Supplementing the diet of a large number of mice (n=50-100) for a long period of time (18-24 months) presents a methodological, economic and infrastructure problem. Drosophila melanogaster or vinegar fly, shares 90% of its genome with us (Ugur et al, Dis. Model. Meeh, 2016 - Reiter Genome Res, 2001 ), the physiological processes that occur in aging are evolutionarily conserved, it is an economical model and occupies little space, so experiments can be performed with a large number of individuals (Kasai, Y. & Cagan, R. Drosophila as a tool for personalized medicine: a primer. Per. Med. 7, 621-632 (2010)). The use of this organism in preclinical biopharmaceuticals implies a significant decrease in the costs of obtaining the necessary evidence for a compound to enter a clinical trial. The fly has been used successfully in the biopharmaceutical industry to test products with medicinal capacity. Pharmaceutical companies using Drosophila as a preclinical model, for example, Medros used Drosophila for the first time in personalized medicine and discovered the first drug approved by the FDA for a type III clinical trial against cancer (medullary thyroid carcinoma, Das, T. K. & Cagan, R. L. A Drosophila approach to thyroid cancer therapeutics. Drug Discov. Today. Technol. 10, e65-71 (2013)). Further, studies in flies with Parkinson mutations like PINK1 , identified vitamin k2 as an alternative electron carrier molecule that similar to ubiquinone can stimulate the electron transport chain resulting in a rescue of Pinkl -related phenotypes (Vos, M. & Klein, C. The Importance of Drosophila melanogaster Research to UnCover Cellular Pathways Underlying Parkinson’s Disease. Cells 10, 1-22 (2021 )), nominating it to be a promising therapeutic strategy in the treatment of PINK1 -related Parkinson patients. The application of ubiquinone and vitamin k2 as therapy in patients has been initiated in clinical trials (Prasuhn, J. et al. The Use of Vitamin K2 in Patients with Parkinson’s Disease and Mitochondrial Dysfunction (PD-K2): A Theranostic Pilot Study in a Placebo-Controlled Parallel Group Design. Front. Neurol. 1 1 , 592104 (2020); Prasuhn, J. et al. An omics-based strategy using coenzyme Q10 in patients with Parkinson’s disease: concept evaluation in a double-blind randomized placebo-controlled parallel group trial. Neurol. Res. Pract. 1 , (2019); and Prasuhn, J. et al. The Use of Vitamin K2 in Patients with Parkinson’s Disease and Mitochondrial Dysfunction (PD-K2): A Theranostic Pilot Study in a Placebo-Controlled Parallel Group Design. Front. Neurol. 11 , (2021 )) and serves as another excellent example of therapeutic targets identified in flies that are now being tested in patients. Extracts from various sources of the biosphere (e.g., plants, microbes, or marine organisms) have been used for long as food supplements to promote health and/or longevity (Muscariello, E. et al. Clinical Interventions in Aging Dovepress Dietary protein intake in sarcopenic obese older women. Clin. Interv. Aging 11-133 (2016). doi:10.2147/CIA.S96017). Research in “superfoods” or food ingredients using Drosophila has been performed for decades now (Vanacore, D. et al. Effect of restriction vegan diet’s on muscle mass, oxidative status, and myocytes differentiation: A pilot study. J. Cell. Physiol. 233, (2018)). A main issue for this choice is, not only the expression of homologous genes and signaling pathways in equivalent organs but also the fact the fly has an open circulatory system and no brain blood barrier as a membrane, therefore whatever ingredient is being assayed it is bound to hit all organs. In this way different cell types may be assayed at the same time. There are at least 50 different antioxidant food supplements whose properties have been described in Drosophila and are maybe the best-known supplements like resveratrol, apple juice or aloe vera (Yi, Y., Xu, W., Fan, Y. & Wang, H. X. Drosophila as an emerging model organism for studies of food-derived antioxidants. Food Res. Int. 143, 110307 (2021 )). Nonetheless, many more food supplements and ingredients not related to anti oxidative properties or polyphenols have been tried too like, for example, biofortified wheat (Shen, W. et al. Effect of 2-year caloric restriction on organ and tissue size in nonobese 21- to 50-year-old adults in a randomized clinical trial: the CALERIE study. Am. J. Clin. Nutr. 114, 1295-1303 (2021 )). Among the less known are those supplements which come from mushroom (Pan, H. Y. et al. Ergothioneine exhibits longevity-extension effect in Drosophila melanogaster via regulation of cholinergic neurotransmission, tyrosine metabolism, and fatty acid oxidation. Food Funct. 13, 227-241 (2022)) or from ancient oriental traditions. For example, Kamikihito and Unkei-to traditional Japanese supplements were found to be sleep-promoting substances among aged flies and flies manifesting parkinsonian like sleep defects (Mazzoccoli, G., Tevy, M. F., Borghesan, M., Vergini, M. R. D. & Vinciguerra, M. Caloric restriction and aging stem cells: The stick and the carrot? Experimental Gerontology 50, 137-148 (2014)). Drosophila is a well- investigated and highly tractable model organism employed in nutrition research and nutraceuticals discovery since, as mentioned, it shares high homology with several human metabolism and disease-related genes. Consistently, several insights of the molecular mechanisms that affect in vivo aging have been identified by studying the effects of distinct dietary habits and/or components of the fruit fly diet, which have been further translated or verified in mammals.

Among the examples cited on the bibliography by Kim, C. & Hwang, J. K. Flavonoids: nutraceutical potential for counteracting muscle atrophy. Food Sci. Biotechnol. 29, 1619-1640 (2020), it is of particular interest to mention that the only example where an anthocyanin was used (an anthocyanidin), the purpose was to determine the preventive mechanism of delphinidin on disuse muscle atrophy. The current invention presents a way to preserve muscular mass and cellular energy during the natural lifespan, not considering induced muscular disuse nor pathologies.

As exposed at the beginning and as conclusion from the background, it is necessary to have new natural composition or plant extract useful to reduce progressive loss of skeletal muscle mass, increasing muscle mass and providing new anti-aging agents or geroprotective agents, enhancing the healthspan and preserving locomotive functions for longer periods of life and upregulating the expression of antioxidant enzymes and mitochondrial homeostasis-related proteins, which determine contractile muscle function and an antioxidant supplement for adults and elderly, either human beings or animals. BRIEF DESCRIPTION OF THE INVENTION

The present invention is related to a standardized delphinidin-rich maqui berry composition for reducing progressive loss of skeletal muscle mass, increasing muscle mass and providing new anti-aging agents or geroprotective agents, enhancing the healthspan and preserving locomotive functions for longer periods of life and upregulating the expression of antioxidant enzymes and mitochondrial homeostasis-related proteins, which determine contractile muscle function and an antioxidant supplement for living organisms sharing ND-B8, COVIIc and ATPsync mitochondrial genes, having COVIIc a critical role to mitochondrial function of protection, and TM2, MLC1 and TpnEBF muscle genes (see Table 1), including mammals or marine/aquatic animals. Preferably, mammals are selected from human being, pet animals, farm animals, competition animals, emotional support animals, among others. Human beings are not limited to a range of age, gender or ethnicity. More preferably, human beings, pet animal, farm animal, competition animal, emotional support animal, among others, can be selected from adults and elderly, either human beings or animals. Pet animals are selected from dog or cat. Farm animals are selected from cattle, pigs, equines, goats, sheep, poultry, among others, and more preferably, cows, pigs, horses, goats, sheep, chickens, among others. Competition animals are selected from racehorses, dressage animals, beauty pageant animals, among others. Marine/aquatic animals are pisciculture animals or fish of farming. More preferably, such fish of farming is selected from rainbow trout, salmon, tilapia, carp, catfish, among others, and more preferably salmo salar.

Table 1 : ortholog sequences (databases used to ortholog sequences www.uniprot.org, www.orthodb.org, https://flybase.org/) Such standardized delphinidin-rich maqui berry composition comprising 15-40%wt anthocyanins, preferably, 20-35%wt anthocyanins, more preferably, 25%wt or 35%wt anthocyanins, and even more preferably a standardized delphinidin-rich maqui berry composition comprising 15-40%wt anthocyanins having 12-28%wt delphinidin based on the total weight of anthocyanins, preferably, 20-35%wt anthocyanins having 20-25%wt delphinidin based on the total weight of anthocyanins, more preferably, 25%wt anthocyanins having 20%wt delphinidin based on the total weight of anthocyanins, or even more preferably, 35%wt anthocyanins having 25%wt delphinidin based on the total weight of anthocyanins.

Even more preferably, a maqui berry extract composition comprising a first composition comprising a carrier; and about 100 mg to 400 mg of a second composition comprising a plurality of anthocyanins and anthocyanidins, wherein the plurality of anthocyanins and anthocyanidins constitute at least about 25%wt of the second composition, wherein the 25%wt is distributed between delphinidin-3-0-sambubioside-5-0-glucoside, delphinidin-3, 5-O-diglucoside, cyanidin- 3,5-O-diglucoside, delphinidin-3-0-0-sambubioside-5-0-glucoside, cyanidin-3-O-sambubioside, and cyanidin-3-O-glucoside.

Even more preferably, the composition can be selected from the one claimed in US10786522B2 (Maqui New Life), i.e., a maqui berry extract composition comprising a first composition comprising a carrier; and about 100 mg to 400 mg of a second composition comprising a plurality of anthocyanins and anthocyanidins, wherein the plurality of anthocyanins and anthocyanidins constitute at least about 35%wt of the second composition, wherein the 35%wt is distributed between delphinidin-3-0-sambubioside-5-0-glucoside, delphinidin-3, 5-O-diglucoside, cyanidin- 3, 5-O-diglucoside, delphinidin-3-0-0-sambubioside-5-0-glucoside, cyanidin-3-O-sambubioside, and cyanidin-3-O-glucoside; and wherein at least about 15%wt of the plurality of anthocyanins or anthocyanidins or both are sugar-free or sugar-containing delphinidins, wherein the pharmaceutical composition is formulated as a soft cellulose capsule or hard gelatin capsule, and optionally such carrier can be selected from microcrystalline cellulose, lactose, silicon dioxide, glyceryl monostearate, soya lecithin, or Oenothera biennis oil. Further, such optional carrier can be Acacia Gum and maltodextrin.

A first standardized Maqui Berry Extract 1 (35%wt anthocyanins having 25%wt delphinidins based on the total weight of anthocyanins, measured by HPLC, see table 1 ), arbitrarily named SMBE1 , and a second standardized Maqui Berry Extract 2 (25%wt anthocyanins including 20%wt delphinidins based on the total weight of anthocyanins, measured by HPLC, see table 2), arbitrarily named SMBE2, wherein SMBE2 corresponds to an extract obtained by ultrafiltration process, were used in the assays as describe below and both showed improvement in lifespan, healthspan and, geroprotective properties. The above mentioned two standardized polyphenolic extracts were made from Aristotelia chilensis berries (Watson, R. R. & Schonlau, F. Nutraceutical and antioxidant effects of a delphinidin-rich maqui berry extract Delphinol®: a review. Minerva Cardioangiol. 63, 1-12 (2015)). SMBE1 has 1 .4 of the amounts of anthocyanins than SMBE2 and less amounts of disaccharides. Anthocyanins are flavonoids with known antioxidant effect and comprehend a number of molecules (Pandey, K. B. & Rizvi, S. I. Plant polyphenols as dietary antioxidants in human health and disease. Oxid. Med. Cell. Longev. 2, 270 (2009)) such as the delphinidins found in Aristotelia chilensis berries.

Table 1 Profile 1^st standardized Maqui Berry Extract 1 (SMBE1) Table 2 Profile 2^nd standardized Maqui Berry Extract 2 (SMBE2)

To assess the geroprotective properties of SMBE1 and SMBE2 the fruitfly Drosophila melanogaster as a model organism was used. Fruit flies have been used for the past century as model organisms in scientific research and are a robust model to study the mechanisms of aging. Both extracts significantly increase lifespan in males and this effect can also be observed in females. Both extracts ameliorate healthspan significantly in male and female animals as demonstrated. The effects of the extracts on climbing activity of Drosophila as an indirect measure of healthspan as determined. Further, the expression of proteins involved in muscle contraction as a measure of protein synthesis and turnover and the expression of mitochondrial proteins which fuel energy through the electron transport chain were quantified. Altogether the data presented here strongly demonstrates that SMBE1 and SMBE2 can be excellent geroprotector supplementation options, for frequent use, to extend healthspan through the maintenance of muscle homeostasis for longer periods in young adults and during aging.

BRIEF DESCRIPTION OF DRAWINGS

Figures 1A-1 H: Supplementation enhances locomotor behavior and lifespan in aged animals. Locomotor behavior in (Fig. 1 A-Fig. 1C) female and (Fig. 1 D-Fig. F) male aged model animals. The adult animals were fed with control food (black), SMBE1 (S1 , white) supplementation and SMBE2 (S2, oblique lattice) Pure supplementation. Climbing ability and movement was monitored at day 10, 20, 30 and 37 of lifespan (n> 40 animals per group). One way ANOVA followed by Sidak's comparison test was used to derive all p-values. ns: not significant. Survival probability during lifespan. Fig. 1G) Females Fig. 1 H) Males; Control (red), SMBE1 (S1 ) and SMBE2 (S2). Mean lifespan in days is marked with a dotted line: females, control: 32 SMBE1 : 35 SMBE2: 30; males, control: 30 SMBE1 : 32 SMBE2: 29.

Figures 2A-2F: Feeding behavior and body protein composition in aged animals. Feeding behavior was measured as food intake and weight mass (Fig. 2A, Fig. 2B, Fig. 2C) in female and (Fig. 2A', Fig. 2B', Fig. 2C') male aged model animals. The adult animals were fed with control (black) food, SMBE1 supplementation (S1 , white) and SMBE2 supplementation (S2, oblique lattice). Food intake and weight were measured at days 10, 20, 30 and 40 of lifespan (n> 40 animals per group). 2-way ANOVA followed by Tukey's multiple comparisons test was used to derive all p-values. ns: not significant.

Figures 3A-3C’: Flies fed with delphinidins-rich standardized maqui berry extracts have a higher body mass protein composition. Comparative graph between food intake (•) and total protein content (A) of females (Fig. 3A) and males (Fig. 3A’). Thorax to total body protein concentration was measured as a ratio in females (Fig 3B, Fig. 3C) and of males (Fig 3B’, Fig. 3C'). All measurements were taken at days 10, 20, 30 and 40 of lifespan (n> 40 animals per group). Two- way ANOVA followed by Tukey's multiple comparisons test was used to derive all p-values. Control = black; SMBE1 = S1 = white and SMBE2 = S2 = oblique lattice.

Figures 4A-4F’: Antioxidant and longevity gene expression throughout lifespan. Control (black), SMBE1 (S1 , white), SMBE2 (S2, oblique lattice). Catalase: female Fig. 4A, Fig. 4A', males Fig. 4B, Fig. 4B'; SOD: female Fig. 4C, Fig. 4G', males: Fig. 4D, Fig. 4D'; GSTD: female Fig. 4E, Fig. 4E' males Fig. 4F, Fig. 4F'; Hsp70: female Fig. 4G, Fig. 4G', males Fig. 4H, Fig. 4H'. Expression was monitored at day 10, 20, 30 and 40 of lifespan (n> 20 animals per group). One way ANOVA followed by Sidak's comparison test was used to derive all p-values. ns: not significant.

Figures 5A-5F’: Supplementation induces expression changes in mitochondrial genes. Relative expression in whole adult tissues of animals fed with control food (black), SMBE1 (S1 , white) and SMBE2 (S2, oblique lattice) by qPCR of ND-B8 (Fig 5A-5B’), COVIIc (Fig 5C-5D’) and ATPsyn (Fig 5E-5F’). Samples were taken at 10, 20, 30 and 40 days of lifespan (n> 20 animals per group, N=3). One way ANOVA followed by Sidak's comparison test was used to derive all p-values. ns: not significant

Figures 6A-6F’: Supplementation induces expression changes in muscle genes. Relative expression in whole adult tissues of animals fed with control food (black), SMBE1 (S1 , white) and SMBE2 (S2, oblique lattice) by qPCR of TM2 (Fig. 6A-6B’), MLC1 (Fig. 6C-6D’) and TpnC73F (Fig. 6E-6F’). Samples were taken at 10, 20, 30 and 40 days of lifespan (n> 20 animals per group, N=3). One way ANOVA followed by Sidak's comparison test was used to derive all p-values. ns: not significant.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is related to a delphinidin-rich maqui berry composition for reducing progressive loss of skeletal muscle mass, increasing muscle mass and providing new anti-aging agents or geroprotective agents, enhancing the healthspan and preserving locomotive functions for longer periods of life and upregulating the expression of antioxidant enzymes and mitochondrial homeostasis-related proteins, which determine contractile muscle function and an antioxidant supplement for living organisms sharing ND-B8, COVIIc and ATPsync mitochondrial genes and TM2, MLC1 and TpnEBF muscle genes, including mammals or marine/aquatic animals, being such mammals or marine/aquatic animals as mentioned above. Such standardized delphinidin-rich maqui berry composition comprising 15-40%wt anthocyanins, preferably, 20-35%wt anthocyanins, more preferably, 25%wt or 35%wt anthocyanins, and even more preferably a standardized delphinidin-rich maqui berry composition comprising 15-40%wt anthocyanins having 12-28%wt delphinidin based on the total weight of anthocyanins, preferably, 20-35%wt anthocyanins having 20-25%wt delphinidin based on the total weight of anthocyanins, more preferably, 25%wt anthocyanins having 20%wt delphinidin based on the total weight of anthocyanins, or even more preferably, 35%wt anthocyanins having 25%wt delphinidin based on the total weight of anthocyanins and even more preferably, the standardized-rich maqui berry composition comprising a first composition comprising a carrier; and about 100 mg to 400 mg of a second composition comprising a plurality of anthocyanins and anthocyanidins, wherein the plurality of anthocyanins and anthocyanidins constitute at least about 25%wt of the second composition, wherein the 25%wt is distributed between delphinidin-3-0-sambubioside-5-0- glucoside, delphinidin-3, 5-O-diglucoside, cyanidin-3,5-0-diglucoside, dephinidin-3-O-O- sambubioside-5-O-glucoside, cyanidin-3-O-sambubioside, and cyanidin-3-O-glucoside. Even more preferably, the composition can be selected from the one claimed in US10786522B2 (Maqui New Life), i.e., a delphinidin-rich maqui berry extract composition comprising a first composition comprising a carrier; and about 100 mg to 400 mg of a second composition comprising a plurality of anthocyanins and anthocyanidins, wherein the plurality of anthocyanins and anthocyanidins constitute at least about 35%wt of the second composition, wherein the 35%wt is distributed between delphinidin-3-0-sambubioside-5-0-glucoside, delphinidin-3, 5-O-diglucoside, cyanidin- 3, 5-O-diglucoside, dephinidin-3-0-0-sambubioside-5-0-glucoside, cyanidin-3-O-sambubioside, and cyanidin-3-O-glucoside; and wherein at least about 15%wt of the plurality of anthocyanins or anthocyanidins or both are sugar-free or sugar-containing delphinidins, wherein the pharmaceutical composition is formulated as a soft cellulose capsule or hard gelatin capsule, and optionally such carrier can be selected from microcrystalline cellulose, lactose, silicon dioxide, glyceryl monostearate, soya lecithin, or Oenothera biennis oil. Further, such optional carrier can be Acacia Gum and maltodextrin.

The present invention is also related to the use of the composition as described above for reducing progressive loss of skeletal muscle mass, increasing muscle mass and anti-aging agent or geroprotective agent, enhancing the healthspan and preserving locomotive functions for longer periods of life and upregulating the expression of antioxidant enzymes and mitochondrial homeostasis-related proteins, which determine contractile muscle function and an antioxidant supplement for adults and elderly during aging, in mammals as defined above. Specifically, the present invention is related to the use of a standardized delphinidn-rich maqui berry composition for reducing progressive loss of skeletal muscle mass, increasing muscle mass and anti-aging agent or geroprotective agent, enhancing the healthspan and preserving locomotive functions for longer periods of life and upregulating the expression of antioxidant enzymes and mitochondrial homeostasis-related proteins, which determine contractile muscle function and an antioxidant supplement for adults and elderly, in mammals as defined above. Preferably, such composition comprising the use of a standardized delphinidin-rich maqui berry composition comprising 15-40%wt anthocyanins, preferably, 20-35%wt anthocyanins, more preferably, 25%wt or 35%wt anthocyanins, and even more preferably a standardized delphinidinrich maqui berry composition comprising 15-40%wt anthocyanins having 12-28%wt delphinidin based on the total weight of anthocyanins, preferably, 20-35%wt anthocyanins having 20-25%wt delphinidin based on the total weight of anthocyanins, more preferably, 25%wt anthocyanins having 20%wt delphinidin based on the total weight of anthocyanins, or even more preferably, 35%wt anthocyanins having 25%wt delphinidin based on the total weight of anthocyanins.

The composition to be used can be formulated as a capsule, preferably, a soft cellulose capsule or a hard gelatin capsule. Preferably, such carrier can be selected from microcrystalline cellulose, lactose, silicon dioxide, glyceryl monostearate, soja lecithin, or Oenothera biennis oil. Also, the present standardized delphinidin-rich composition can be formulated as gummy chew, strips, powered sachet, powder for food supplements, microencapsulated powder, a masticatory tablet, a masticatory wafer, a swallowing tablet, a stick pack, a tube and scoop, among others.

The present invention is also related to a method for reducing progressive loss of skeletal muscle mass, increasing muscle mass and enhancing the healthspan and preserving locomotive functions for longer periods of life and upregulating the expression of antioxidant enzymes and mitochondrial homeostasis-related proteins, which determine contractile muscle function and an antioxidant supplement for living organisms sharing ND-B8, COVIIc and ATPsync mitochondrial genes and TM2, MLC1 and TpnEBF muscle genes, including mammals or marine/aquatic animals, being such mammals or marine/aquatic animals as mentioned above, comprising administering to such living organism a standardized delphinidin-rich maqui berry composition as described above.

Specifically, but not limited to that, the impact of the use of two antioxidant standardized maqui berry extracts, SMBE1 and SMBE2, in animal physiology, is demonstrated. These supplements have geroprotective properties and are to be useful as healthy aging agents. SMBE1 has already been demonstrated to have anti hyperglycemic properties. Here, geroprotective properties at the molecular level and their correlate in locomotive capacity are demonstrated. Both supplements upregulate the expression of key players of the redox system and effect over the mitochondrial mechanism to fuel energy for the locomotion function.

After, the fruit fly Drosophila melanogaster was used as a model organism to study the effects of two delphinidin-rich standardized maqui berry extracts on aging and the loss of muscle mass. Both maqui berry extracts, SEMB1 and SEMB2, enhance climbing abilities in flies during aging, regardless of sex. This effect is linked to an extension of mean lifespan and life expectancy (Fig. 1A-1 H). Previously, the same effects have been demonstrated in Drosophila melanogaster with other natural extracts rich in anthocyanins, such as those from blueberry (Peng, C. et al. Blueberry extract prolongs lifespan of Drosophila melanogaster. Exp Gerontol 47, 170-178 (2012)), black rice (Zuo, Y. et al. Black rice extract extends the lifespan of fruit flies. Food Funct 3, 1271 (2012)), cranberry (Wang, L. et al. Cranberry anthocyanin extract prolongs lifespan of fruit flies. Exp Gerontol 69, 189-195 (2015)) and aronia (Jo, A. R. & Imm, J.-Y. Effects of aronia extract on lifespan and age-related oxidative stress in Drosophila melanogaster. Food Sci Biotechnol 26, 1399-1406 (2017)). Furthermore, the use of the two delphinidin-rich standardized maqui berry extracts established a clear association between the dietary supplementation of delphinidin-rich standardized maqui berry extracts and the interplay of body mass, protein composition, and climbing abilities since flies receiving these extracts demonstrated a notable increase in their total body protein concentration, even though their food intake remained consistent. This surge in protein content, primarily concentrated in the thorax region, exhibited a direct correlation with improved climbing abilities, particularly among aging flies (Figures 1A-1 H, 2A-2C’, and 3A-3C’).

Oxidative stress has been implicated in aging and other age-related diseases. It contributes to aging process via damage to lipids, proteins, and DNA in various tissues (Staats, S., Luersen, K., Wagner, A. E. & Rimbach, G. Drosophila melanogaster as a Versatile Model Organism in Food and Nutrition Research. J Agric Food Chem 66, 3737-3753 (2018); Beckman, K. B. & Ames, B. N. The Free Radical Theory of Aging Matures. Physiol Rev 78, 547-581 (1998) and Valko, M. et al. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 39, 44-84 (2007)). One of the mechanisms to counteract oxidative damage caused by ROS is through antioxidant enzymes, such as SOD, Cat, and Gstd. It has been demonstrated that the expression of these enzymes decreases during aging in flies (Orr, W. C. & Sohal, R. S. Extension of Life-Span by Overexpression of Superoxide Dismutase and Catalase in Drosophila melanogaster. Science (1979) 263, 1128-1130 (1994) and Sohal, R. S., Arnold, L. & Orr, W. C. Effect of age on superoxide dismutase, catalase, glutathione reductase, inorganic peroxides, TBA-reactive material, GSH/GSSG, NADPH/NADP+ and NADH/NAD+ in Drosophila melanogaster. Meeh Ageing Dev 56, 223-235 (1990)). The antioxidant enzymes SOD, Cat, and Gstd are overexpressed compared to their control (Figure 4A-4H’) in flies fed with SEMB1 and SEMB2 extracts. Interestingly, transgenic flies carrying three genetic copies of SOD and Cat exhibit an extension in lifespan (Orr, W. C. & Sohal, R. S. Extension of Life-Span by Overexpression of Superoxide Dismutase and Catalase in Drosophila melanogaster. Science (1979) 263, 1128-1130 (1994)), a result observed for supplements SEMB1 and SEMB2 (Figure 1 G, 1 H). Not only antioxidant enzymes have been associated with the anti-aging phenomenon, but also the expression of chaperones, such as Hsp70 (Dhanjal, D. S. et al. Plant Fortification of the Diet for Anti-Ageing Effects: A Review. Nutrients 1 , 3008 (2020); Dhalaria, R. et al. Bioactive Compounds of Edible Fruits with Their Anti-Aging Properties: A Comprehensive Review to Prolong Human Life. Antioxidants 9, 1123 (2020); Pandey, M., Bansal, S. & Chawla, G. Evaluation of lifespan promoting effects of biofortified wheat in Drosophila melanogaster. Exp Gerontol 160, 111697 (2022); Salehi, B. et al. Plant-Derived Bioactives and Oxidative Stress- Related Disorders: A Key Trend towards Healthy Aging and Longevity Promotion. Applied Sciences 10, 947 (2020)). In flies supplemented with extracts SEMB1 and SEMB2, thus an increase in the expression of Hsp70 (Figure 4H, 4G’) was demonstrated.

Several studies investigating the impact of aging on mitochondrial H2O2 production have consistently shown a 1 .3-to-2-folds increase in the flight muscles of Drosophila melanogaster (Sohal, R. S., Sohal, B. H. & Orr, W. C. Mitochondrial superoxide and hydrogen peroxide generation, protein oxidative damage, and longevity in different species of flies. Free Radic Biol Med 19, 499-504 (1995) and Sohal, R. S. & Orr, W. C. The redox stress hypothesis of aging. Free Radic Biol Med 52, 539-555 (2012)). This elevation is concomitant with a notable reduction in the activity of electron respiratory chain complex IV (Ferguson, M., Mockett, R. J., Shen, Y., Orr, W. C. & Sohal, R. S. Age-associated decline in mitochondrial respiration and electron transport in Drosophila melanogaster. Biochemical Journal 390, 501-511 (2005)) and a decline in the integrity of cytochrome C oxidase (Ren, J.-C., Rebrin, I., Klichko, V., Orr, W. C. & Sohal, R.

S. Cytochrome c oxidase loses catalytic activity and structural integrity during the aging process in Drosophila melanogaster. Biochem Biophys Res Common 401 , 64-68 (2010)). Moreover, a decrease in the expression of ATP synthase has been identified during the aging process (Landis, G., Shen, J. & Tower, J. Gene expression changes in response to aging compared to heat stress, oxidative stress and ionizing radiation in Drosophila melanogaster. Aging 4, 768-789 (2012)). A supplementation with extracts SEMB1 and SEMB2 leads to a significant increase in the expression of genes associated with oxidative respiration during aging (Figure 5A-5F’). This aligns with findings observed for other natural antioxidants, such as ginger (Zhou, Y., Xue, L., Gao, L., Qin, X. & Du, G. Ginger extract extends the lifespan of Drosophila melanogaster through antioxidation and ameliorating metabolic dysfunction. J. Funct Foods 49, 295-305 (2018)), red ginseng (Liu, Q.-X., Zhang, W., Wang, J., Hou, W. & Wang, Y.-P. A proteomic approach reveals the differential protein expression in Drosophila melanogaster treated with red ginseng extract (Panax ginseng). J Ginseng Res 42, 343-351 (2018)) and ellagic acid (Adedara, A. O., Otenaike,

T. A., Farodoye, O. M. & Abolaji, A. O. Ellagic acid mitigates rotenone-induced damage via modulating mitochondria function in Drosophila melanogaster. J Biochem Mol Toxicol 37, (2023)), which may enhance the synthesis of enzymes, including those involved in mitochondrial function and ATP production.

A characteristic feature observed in aged humans and other mammals is the progressive and debilitating loss of skeletal muscle function and mass, commonly referred to as sarcopenia. In Drosophila melanogaster, the organization and metabolism of skeletal muscle fibers closely resemble those in mammals (Piccirillo, R. & Goldberg, A. L. The p97/VCP ATPase is critical in muscle atrophy and the accelerated degradation of muscle proteins. EMBO J 31 , 3334-3350 (2012) and Taylor, M. V. Comparison of Muscle Development in Drosophila and Vertebrates, in Muscle Development in Drosophila 169-203 (Springer New York). doi:10.1007/0-387-32963- 3_14). During the aging process have also been reported sarcomere-related defects, including alterations in myofibrillar protein composition and function (Miller, M. S. et al. Aging Enhances Indirect Flight Muscle Fiber Performance yet Decreases Flight Ability in Drosophila. Biophys J 95, 2391-2401 (2008)) as well as decreased length and increased disorganization of sarcomeres (Webb, S. & Tribe, M. A. Are there major degenerative changes in the flight muscle of ageing diptera? Exp Gerontol 9, 43-49 (1974) and Takahashi, A., Philpott, D. E. & Miquel, J. Electron Microscope Studies on Aging Drosophila melanogaster III. Flight Muscle. J Gerontol 25, 222-228 (1970)). Through the comparison of gene expression in young and adult flies, a differential gene expression of proteins involved in the structure or function of the sarcomere has been determined, including MLC1 , TM2, and various troponin isoforms (Bordet, G., Lodhi, N., Kossenkov, A. & Tulin, A. Age-Related Changes of Gene Expression Profiles in Drosophila. Genes (Basel) 12, 1982 (2021 )). Additionally, it has been observed that these genes can be upregulated through dietary interventions, such as caloric restriction (Pletcher, S. D., Libert, S. & Skorupa, D. Flies and their Golden Apples: The effect of dietary restriction on Drosophila aging and age-dependent gene expression. Ageing Res Rev 4, 451 -480 (2005)) or resveratrol (Li, Y., Li, S. & Lin, C. Effect of resveratrol and pterostilbene on aging and longevity. BioFactors 44, 69-82 (2018)). In this context, our results demonstrated the overexpression of MLC1 , TM2, and TpnC73F (Figure 6A- 6F’), which would explain the improvement in muscle function during aging.

Thus, the two delphinidin-rich standardized maqui berry extracts (SMBE1 and SMBE2) on animal physiology have geroprotective properties at the molecular level and their correlation in locomotive capacity. In flies fed with both supplements showed an upregulate the expression of key players of the redox system and act over the mitochondrial mechanism to fuel energy for the locomotion function. Further, SMBE1 and SMBE2 act over skeletal muscle contractile proteins as drivers of healthspan, not only at older ages but mainly during midlife. SEMB1 and SEMB2 maintain a contractile muscle function and mitochondrial homeostasis, which are among the critical cellular target mechanisms of these standardized maqui berry extracts. No substantial differences among the effects of these two standardized maqui berry extract supplements even though they contain different amounts of antioxidant agents, indicating a cellular threshold for the effects of antioxidant formulas.

For survival or life span assays, flies were grown at 18^eC and transferred to 29^SC after hatching. Flies were reared at a maximum of 20 individuals per tube and transferred every other day into fresh media and dead flies were counted. Flies were maintained on standard cornmeal (generic Lider Chilean supermarket brand), molasses (Chancaca Deliciosa Chilean brand), yeast (Lefersa Chilean brand), agar media with 60% humidity on a 12:12-h light-dark cycle. SMBE1 and SMBE2 supplements were added (80 pl) to the surface of the tube to cover all the surface so that all flies in the tube have access to supplemented food. Supplements were used at a concentration of 86 mg/ml prepared in distilled water to be used. For each experiment wild type Canton S flies grown permanently in lab were used. Cohorts were built from a collection of flies born with no more than 12 hours of difference. Each experiment was carried out with an initial number of 250 females and 250 males per group, and lifespans experiments were repeated 3 consecutive times for each condition. For each condition only 20 individuals per tube were placed and changed every other day. The data of each cohort was analyzed first separately and then for each sex all experiments were pooled and analyzed together. Kaplan-Meier survival curves, log-rank statistical analysis and pairwise analysis were performed using survival (3-2-13v) package under R statistical environment, version 4.1.2.

For fly climbing, 15 to 20 flies per assay were placed in a clean tube. Tubes were tapped and recorded with HD cell phone camera at 30 frame per second. The trajectory of flies in the tube was traced as a segmented line. Kymograph was used to calculate the speed of each moving object. The slope of each trajectory in the tube was calculated manually using the end point coordinates, multiplied by the length of the tube (in cm) and divided by the speed of the movie (30 frames/s) to obtain an individual speed in cm/s. All procedures were performed on Imaged. Average speeds of individual flies taken randomly from each group from 3 independent crosses. For each group the animals of the extremes of the normal distribution were eliminated and then groups were compared using multiple ANOVA.

For food intake analysis, groups of 8 flies per condition were transferred onto fresh food medium with 2.5% (w/v) blue food dye (F D & C Blue Dye no. 1 ). After a total of 2 hours, 4 flies per condition were transferred to Eppendorf tubes and snap frozen at -20^eC. Subsequently, flies were homogenized in 200 pL of PBS and centrifugate to 13.000 rpm at least one minute. 50 pL of supernatant was transferred to 150 pL of PBS and absorbance of the sample was measured at 630 nm in a TECAN Infinite® 200 PRO Spectrophotometer. Condition-matched flies exposed to non-dyed food were used as the baseline during spectrophotometry. The amount of labelled food in the fly was calculated from a standard curve made by serial dilution in PBS of a sample of F D & C Blue Dye no. 1 . To calculate volume of food consumed, the formula (Ec. 1) was used:

For body weight measurements, adult flies were collected and massed in groups of 10 animals to obtain an average weight of 100 animals per condition in 3 independent experiments.

For protein composition measurements, approximately 30 flies were used per assay. 10 whole flies or 20 dissected thoraxes were homogenized in lysis buffer (140 mM NaCI, 50 mM Tris-HCI, pH 7.4, 0.1% Triton-X, and 1 X protease inhibitor (ThermoFisher, Waltham, MA, USA)). Protein was measured using the Pierce BCA Protein Assay kit (ThermoFisher, Waltham, MA, USA) accordingly to manufacturer’s instructions.

A quantitative PGR technique was used to measure the expression of specific genes. Briefly, RNA was isolated from whole adult flies using a Trizol (Ambion Life Technologies, GA, USA) according to manufacturer instructions. 1 pg of RNA was used to reverse transcription, which was carried out afterwards with Affinity Script Reverse Transcriptase (Stratagene). One microliter of cDNA product was amplified with Brilliant II SYBR Green QRT-PCR Master Mix kit (Stratagene). Reactions were carried out in a LightCycler Nano thermocycler (Roche). A first cycle of 5 min at 95°C was followed by 30 s at 95°C, 30 s at 55.5°C and 30 s at 72°C for 35 cycles with a 5 min, 72°C incubation as the final step.

Table 3

Example 1 A: Preparation of an Aristotelia chilensis extract containing both anthocyanidins (35%) and polyphenols.

Frozen fruits of Aristotelia chilensis (1 kg) containing 15 g anthocyanidins are extracted with 5 x 1 L of ethanol/water, 50% v/v, and each extraction is carried out for 4 hours. In the first liter of solvent for the first extraction, 7.5 g of sodium bisulfite was solubilized. The same quantity of sodium bisulfite is also solubilized in the second liter. The extraction liquids are concentrated under vacuum at a temperature of 30°C up to 2 L, controlling the complete elimination of ethanol. The concentrate is charged on a column containing 2.5 L of a non-polar polystyrenic resin having a particle size of 25-60 mesh. The column is washed with 10 L of water (which promptly eliminates undesired substances) and then washed with 3 L of ethanol (95%). The ethanolic solution is concentrated to 0.5 L (practically to water) under stirring, and the concentrated solution is acidified to pH 1 in a current of nitrogen to remove the SO2, which must be bubbled into the NaOH solution. The deeply red solution is then carefully concentrated under vacuum to dryness. A content of 150 g anthocyanin can be obtained from an extract having 35% anthocyanin. Example 1 B: Preparation of an Aristotelia chilensis extract containing both anthocyanidins (20-35%, solid extract) and polyphenols.

Frozen Aristotelia chilensis (1 kg) fruit containing 15 g total anthocyanidins are extracted with 3 x 750 mL of water, and each extraction is carried out for 3 hours. Each time, the extracted fruit is filtered through a filter press in order to efficiently separate the liquid from the extracted fruit. In the first 750 mL of water for the first extraction, 5.6 g of sodium bisulfite is solubilized. The same quantity of sodium bisulfite is also solubilized in the second and third 750 mL portions of water. The concentrated solution containing anthocyanins and polyphenols is filtered through a nonwoven cloth to remove particulates. The filtered solution is then filtered through a microfiltration membrane with a nominal 0.2 pm cut-off size. The usual required filtering pressures are below 120 bar for 40-60 L/(m²«h) fluxes or more. The filtered solution is then filtered through an ultrafiltration membrane with a nominal 3-10 kDa cut-off size, using typical pressures below 120 bar with permeate fluxes of 40 L/(m²-h) or more. The filtered solution is then filtered through a nanofiltration membrane with a nominal cut-off size of less than 800 Da. Typical cut-off values are 600-800 Da and 300-500 Da, with typical pressures lower than 80 bar and fluxes higher than 18 L/(m²‘h). The filtered solution is subsequently filtered through a low cut-off size nanofiltration membrane (typically 200 Da or less) or a reverse osmosis membrane, to concentrate the anthocyanins and polyphenols and discard the filtrate containing salts and low molecular weight organic molecules. Typical pressures for this filtration step are less than 200 bar for 10 L/(m²«h) fluxes or more. The discarded molecules include mono- and disaccharides, sodium bisulfite, and organic acids. Diafiltration with water can be alternatively used at any stage to accelerate filtration and improve recovery and/or purification. The retentate from the last nanofiltration or reverse osmosis filtration is acidified, using, for instance, HCI, to obtain a concentrated solution containing anthocyanins and polyphenols. This deeply red retentate solution is then carefully concentrated under vacuum to dryness. A content of 15 g anthocyanins can be obtained in a solid extract containing 20-35% anthocyanins. Other filtration cascades can be used by altering the order, number or membrane cut-off and type used in each step, including different membranes with different cut-off sizes, altering the order or number of filtration stages, using diafiltration, or using other current configurations to obtain the desired extract, as can be devised by someone skilled in the art.

Example 2: Standardized maqui berry extracts rich in anthocyanidin (SMBE1 and SMBE2) enhance mobility throughout the lifespan

Based on the experimental conditions as exposed above, to test whether antioxidant standardized maqui berry extracts can be used as complements in strategies to boost healthspan, a model animal Drosophila melanogaster normal food was used (see materials and methods) and such animal was fed with normal food supplemented with one or other extract. This model organism has the advantage of having short life cycles and the physiology and genetic expression of most cell types, including muscle cells, is similar to that in humans. At the tissue level, this organism offers a simplified model from that of mammalian model organisms, however, still has the advantage from in vitro cell culture models of being a fully functional organism.

First the enhance of mobility of flies fed with these supplements was tested as a measure of overall organism health. Separately, the climbing abilities or locomotor activity of males and females at different timepoints during aging were studied. For each group (supplemented with SMBE1 - arbitrarily denominated as supplement 1 -, supplemented with SMBE2 - arbitrarily denominated as supplement 2-, and food without supplementation- arbitrarily denominated as control-) the speed of individual flies across lifespan was quantified. Supplementation enhances the locomotor activity of animals in males and females as showed in Figures 1 A-1 F. Furthermore, these data show the proportion of decay in the climbing ability of the supplemented animals is less than in control animals when the old and young animals among the same cohort or when supplemented old animals are compared to a 10-day younger control group. In other words, old animals maintain the locomotor abilities for longer periods of time throughout lifespan. This is true for males and females and both supplements as the concentration of anthocyanins and delphinidins in both supplements is different, the above suggests the existence of a threshold for the effects of antioxidant activity of the supplements on locomotion.

After checked whether aged-matched flies eat the supplemented food with the same frequency as normal food to rule out dietary restriction or food intake might be affecting the experiment. A higher food intake was observed in older ages females than males, and supplemented food intake is like normal food at the same old age. Complementary, the weight mass throughout lifespan to test whether supplemented food affects this feature was studied and a slightly less mass loss of old flies (D40) reared in supplemented food was observed. Altogether these results suggest that mobility abilities in old flies are not due to differential food intake but rather to the addition of supplements to the same food.

Flies fed with both extracts exhibited minor but significant changes in their mean lifespan (Figure 1 G, Figure 1 H), being SMBE1 and SMBE2 respectively over Control. Nonetheless, the width of the lifespan distribution increased for both supplements. Altogether, these findings suggest that these supplements have a positive effect both on the healthspan and lifespan distribution.

To determine the body composition total proteins in the thorax were measured where most of the muscle proteins which fuel climbing are present. Thoracic total proteins is significantly elevated in supplemented animals with respect to control animals (Figures 2A-2F). To normalize thoracic proteins concentration to body size the ratio of thoracic proteins content to total body protein was measured. This also suggests that the observed tendency of a higher body weight in supplemented animals is localized and can be attributed to a higher thoracic protein concentration. Altogether these results suggest that mobility abilities in old flies are not due to differential food intake but rather to the addition of supplements to the same food and the changes in protein metabolism these ingredients trigger. Thus, taken together, these results eliminate the potential influence of caloric restriction and differential food intake, suggesting that the improved mobility abilities in older flies can be attributed to the inclusion of delphinidins-rich standardized maqui berry extracts in their diet.

Further, to determine whether the increased body mass is due to an enhancement in protein composition, both total body protein and thoracic protein concentration were measured. The thorax is the primary source of muscle proteins that drive climbing and locomotion. The obtained results showed that the total body protein concentration is higher in both female and male flies fed with both extracts, while food intake remains similar (Figure 3A, Figure 3A'). Furthermore, it was demonstrated that the ratio of thoracic to total body protein content in females fed with SEMB1 and SEMB2 is higher, although not statistically significant, possibly due to the bias introduced by yolk proteins when including the abdomen (Figure 3B, Figure 3C). In the case of male flies raised with both extracts, the thoracic protein content is statistically significantly higher compared to control animals (Figure 3B', Figure 3C’). Considering that old, supplemented flies do not consume more food than control flies and experience less weight loss during aging, the maintenance of higher protein content in older flies could be suggested as contributing to improved climbing abilities and, consequently, an extended healthspan.

To confirm the impact of supplements SEMB1 and SEMB2 on the antioxidant response during aging, the genetic expression of the antioxidant enzymes Catalase (Cat), Superoxide dismutase 2 (SOD2), and Glutathione S-transferase D2 (gstD2), and molecular chaperone, Hsp70 were studied. Supplementing flies’ diets with SEMB1 and SEMB2 significantly enhances the expression of Cat (Figure 4A-B') and gstD (Figure 4 E-F') compared to the control group in age- matched animals. The SOD2 gene expression shows sex differential expression in flies with the same genetic background. SOD2 upregulation in female flies occurs after late midlife (day 30) and is significantly higher in supplemented flies with respect to age-matched controls for both supplements (Figure 4C, C'). However, SOD2 is upregulated throughout lifespan in supplemented males with respect to age matched controls and the expression levels are similar (Figure 4 D, D'). With respect to the expression of Hsp70, we observed that both supplements, S1 and S2, result in an increased expression during aging in both sexes when compared to their respective controls (Figure 4G-H’). These results suggest that extracts SEMB1 and SEMB2 may enhance health and lifespan by increasing cellular antioxidant response. To confirm whether the maintenance of mitochondrial protein homeostasis is a cellular mechanism of action to both SEMB1 and SEMB2 extracts, the expression levels of mitochondrial genes involved in the respiratory chain was assessed, including NDB8 (NADH ubiquinone oxidoreductase B8 subunit of mitochondrial complex 1 ), COVIIc (Cytochrome-c oxidase subunit 7C of complex IV), and ATPsyn (mitochondrial membrane ATP synthase component in Complex V). An upregulation of all three enzymes at 20 days of age in supplemented males and females was showed, coinciding with the age at which flies reach their peak metabolic rate. However, at 30 days of age, there is a significant decrease in the expression of these mitochondrial genes, suggesting a shift in metabolism and energy utilization. This phenomenon is more pronounced in supplemented flies, particularly in the expression of NDB8. Nevertheless, towards the end of their lifespan (day 40) another phase of upregulated expression of ETC mitochondrial genes in supplemented flies compared to control flies (Figures 5A-5F’) was determined, possibly to trigger ROS-Reverse Electron Transport (ROS-RET). In summary, these data indicate an 'adaptation to aging,' characterized by a modification in energy production aimed at limiting oxidative damage at older ages.

Further, an increased oxidative stress can lead to alterations in excitation-contraction coupling and disruptions in calcium homeostasis within aging muscles, resulting in dysfunction (Szentesi, P., Csernoch, L., Dux, L. & Keller-Pinter, A. Changes in Redox Signaling in the Skeletal Muscle with Aging. Oxid Med Cell Longev. 2019, 1-12 (2019); Espinosa, A., Henriquez-Olguin, C. & Jaimovich, E. Reactive oxygen species and calcium signals in skeletal muscle: A crosstalk involved in both normal signaling and disease. Cell Calcium 60, 172-179 (2016)), and a decline in the strength of aged muscles (Xu, H., Ahn, B. & Van Remmen, H. Impact of aging and oxidative stress on specific components of excitation contraction coupling in regulating force generation. Sci Adv 8, (2022)). The condition of contractile proteins serves as an indicator of the intrinsic force production potential of myocytes (Xu, H., Ahn, B. & Van Remmen, H. Impact of aging and oxidative stress on specific components of excitation contraction coupling in regulating force generation. Sci Adv 8, (2022)), and may contribute to the observed improvement in climbing and mobility during aging in supplemented animals compared to age-matched controls. To this end, we analyzed the gene expression of contractile proteins, including Tropomyosin (TM2), Myosin Light Chain 1 (MLC1 ) and Troponin-C (TpnC), We observed an upregulation in the gene expression of these contractile proteins at day 20 in supplemented animals, which subsequently declined by day 30. However, TpnC consistently exhibited higher expression in supplemented 40-day-old flies compared to age-matched controls (Figures 6A-6F’). These results suggest a modulatory effect of SEMB1 and SEMB2 extracts on contractile proteins to sustain climbing abilities. SEQUENCE LISTING

<110> Maqui New Life

<120> A delphinidin-rich maqui berry composition as agent for reducing progressive loss of skeletal muscle mass, increasing muscle mass and anti-aging agent or geroprotective agent.

<160> 20

<170> Patentin version 3.5

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<213> Artificial Sequence

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<223> Primer, oligonucleotide sequence

<400> 1 gccaagaagg agaacattgc 20

<210> 2

<211 > 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer, oligonucleotide sequence

<400> 2 gtagtcgaga cgacgcttca 20

<210> 3

<211 > 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer, oligonucleotide sequence

<400> 3 cgtaatcagc agagctgcac 20

<210> 4

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<212> DNA

<213> Artificial Sequence

<220>

<223> Primer, oligonucleotide sequence

<400> 4 gaaaagttgc gtgcaatcac 20

<210> 5

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<213> Artificial Sequence

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<223> Primer, oligonucleotide sequence

<400> 5 cagtttgaca accctgcgta 20

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<213> Artificial Sequence

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<223> Primer, oligonucleotide sequence

<400> 6 caccactttt gcgattttga 20

<210> 7

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<212> DNA

<213> Artificial Sequence

<220>

<223> Primer, oligonucleotide sequence

<400> 7 tgtgtgacag accagatcagc 20

<210> 8

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<213> Artificial Sequence

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<223> Primer, oligonucleotide sequence

<400> 8 tggagaatgg ttgtcgctaa 20

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<213> Artificial Sequence

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<223> Primer, oligonucleotide sequence <400> 9 ttaagaagcg cttgcagtca 20

<210> 10

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<213> Artificial Sequence

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<223> Primer, oligonucleotide sequence

<400> 10 atttagcccc gtctctttcc 20

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<213> Artificial Sequence

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<223> Primer, oligonucleotide sequence

<400> 11 ctggaaaaca ccacccactt 20

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<400> 12 aagtgtgcaa acaggagcaa 20

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<223> Primer, oligonucleotide sequence

<400> 13 accagggcat caagaatctg 20

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<212> DNA <213> Artificial Sequence

<220>

<223> Primer, oligonucleotide sequence

<400> 14 aacttcttgg cctgctcgta 20

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<213> Artificial Sequence

<220>

<223> Primer, oligonucleotide sequence

<400> 15 gacttttact acatgccagg tgg 23

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<212> DNA

<213> Artificial Sequence

<220>

<223> Primer, oligonucleotide sequence

<400> 16 gcaacagata gtcatccttg cc 22

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<223> Primer, oligonucleotide sequence

<400> 17 ggagtcggtg atgttgacct 20

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<223> Primer, oligonucleotide sequence

<400> 18 gttcggtgac aacaccaatg

Claims

1. Use of a standardized delphinidin-rich maqui berry composition comprising 15-40%wt anthocyanins having 12-28%wt delphinidin based on the total weight of anthocyanins for reducing progressive loss of skeletal muscle mass, increasing muscle mass and as an antiaging agent or geroprotective agent to living organisms sharing ND-B8, COVIIc and ATPsync mitochondrial genes and TM2, MLC1 and TpnEBF muscle genes, including mammals, poultry or marine/aquatic animals.

2. The use of the standardized delphinidin-rich maqui berry composition of claim 1 for enhancing the healthspan and preserving locomotive functions for longer periods of life.

3. The use of the standardized delphinidin-rich maqui berry composition of claim 1 for upregulating the expression of antioxidant enzymes and mitochondrial homeostasis-related proteins, which determine contractile muscle function during healthy aging conditions.

4. The use of the standardized delphinidin-rich maqui berry composition of claim 1 wherein such composition comprising 20-35%wt anthocyanins having 20-25%wt delphinidin based on the total weight of anthocyanins.

5. The use of the standardized delphinidin-rich maqui berry composition of claim 5 wherein such composition comprising 25%wt anthocyanins having 20%wt delphinidin based on the total weight of anthocyanins.

6. The use of the standardized delphinidin-rich maqui berry composition of claim 5 wherein such composition comprising 35%wt anthocyanins having 25%wt delphinidin based on the total weight of anthocyanins.

7. The use of the standardized delphinidin-rich maqui berry composition of claim 1 wherein such composition comprising a first composition comprising a carrier; and about 100 mg to 400 mg of a second composition comprising a plurality of anthocyanins and anthocyanidins, wherein the plurality of anthocyanins and anthocyanidins constitute at least about 25%wt of the second composition, wherein the 25%wt is distributed between delphinidin-3-0-sambubioside-5-0- glucoside, delphinidin-3, 5-O-diglucoside, cyanidin-3,5-0-diglucoside, dephinidin-3-O-O- sambubioside-5-O-glucoside, cyanidin-3-O-sambubioside, and cyanidin-3-O-glucoside.

8. The use of the standardized delphinidin-rich maqui berry composition of claim 1 wherein such composition comprising a first composition comprising a carrier; and about 100 mg to 400 mg of a second composition comprising a plurality of anthocyanins and anthocyanidins, wherein the plurality of anthocyanins and anthocyanidins constitute at least about 35%wt of the second composition, wherein the 35%wt is distributed between delphinidin-3-0-sambubioside-5-0- glucoside, delphinidin-3, 5-O-diglucoside, cyanidin-3, 5-O-diglucoside, dephinidin-3-O-O- sambubioside-5-O-glucoside, cyanidin-3-O-sambubioside, and cyanidin-3-O-glucoside.

9. The use of the standardized delphinidin-rich maqui berry composition of claims 7 or 8 wherein such carrier can be selected from microcrystalline cellulose, lactose, silicon dioxide, glyceryl monostearate, soya lecithin, Acacia Gum, maltodextrin or Oenothera biennis oil.

10. The use of the standardized delphinidin-rich maqui berry composition of claim 1 wherein the composition to be used is capsule including a soft cellulose capsule or a hard gelatin capsule, a gummy chew, a powered sachet, a powder for food supplements, a microencapsulated powder, a masticatory tablet, a masticatory wafer, a swallowing tablet, a stick pack, a tube and a scoop.

11 . The use of the standardized delphinidin-rich maqui berry composition of claim 1 wherein such mammal is selected from human being, pet animals, farm animals, competition animals, emotional support animals or marine/aquatic animals.

12. The use of the standardized delphinidin-rich maqui berry composition of claim 11 wherein such human beings, pet animal, farm animal, competition animal, emotional support animal can be selected from adults and elderly.

13. The use of the standardized delphinidin-rich maqui berry composition of claim 12 wherein such pet animal is selected from dog or cat.

14. The use of the standardized delphinidin-rich maqui berry composition of claim 12 wherein such farm animals are selected from cattle, pigs, equines, goats, sheep and chicken.

15. The use of the standardized delphinidin-rich maqui berry composition of claim 12 wherein such competition animals are selected from racehorses, dressage animals, beauty pageant animals.

16. The use of the standardized delphinidin-rich maqui berry composition of claim 12 wherein such marine/aquatic animals are pisciculture animals or fish of farming.

17. The use of the standardized delphinidin-rich maqui berry composition of claim 101 wherein such fish of farming is selected from rainbow trout, salmon, tilapia, carp, catfish.

18. A method for reducing progressive loss of skeletal muscle mass, increasing muscle mass, which determine contractile muscle function, for living organisms sharing ND-B8, COVIIc and ATPsync mitochondrial genes and TM2, MLC1 and TpnEBF muscle genes, including mammals or marine/aquatic animals, comprising administering to such living organism an standardized delphinidin-rich maqui berry composition comprising 15-40%wt anthocyanins having 12-28%wt delphinidin based on the total weight of anthocyanins.

19. A method for enhancing the healthspan and preserving locomotive functions for longer periods of life of living organisms sharing ND-B8, COVIIc and ATPsync mitochondrial genes and TM2, MLC1 and TpnEBF muscle genes, including mammals or marine/aquatic animals, comprising administering to such living organism a standardized delphinidin-rich maqui berry composition comprising 15-40%wt anthocyanins having 12-28%wt delphinidin based on the total weight of anthocyanins.

20. A method for upregulating the expression of antioxidant enzymes and mitochondrial homeostasis-related proteins, which determine contractile muscle function of living organisms sharing ND-B8, COVIIc and ATPsync mitochondrial genes and TM2, MLC1 and TpnEBF muscle genes, including mammals or marine/aquatic animals, comprising administering to such living organism a standardized delphinidin-rich maqui berry composition comprising 15- 40%wt anthocyanins having 12-28%wt delphinidin based on the total weight of anthocyanins.

21. The method of anyone of claims 18 to 20 wherein such composition comprising 20-35%wt anthocyanins having 20-25%wt delphinidin based on the total weight of anthocyanins.

22. The method of claim 21 wherein such composition 25%wt anthocyanins having 20%wt delphinidin based on the total weight of anthocyanins.

23. The method of claim 21 wherein such composition 35%wt anthocyanins having 25%wt delphinidin based on the total weight of anthocyanins.

24. The method of anyone of claims 18-20 wherein such composition comprising a first composition comprising a carrier; and about 100 mg to 400 mg of a second composition comprising a plurality of anthocyanins and anthocyanidins, wherein the plurality of anthocyanins and anthocyanidins constitute at least about 25%wt of the second composition, wherein the 25%wt is distributed between delphinidin-3-0-sambubioside-5-0-glucoside, delphinidin-3, 5-O-diglucoside, cyanidin-3,5-0-diglucoside, dephinidin-3-O-O-sambubioside- 5-O-glucoside, cyanidin-3-O-sambubioside, and cyanidin-3-O-glucoside.

25. The method of anyone of claims 18-20 wherein such composition comprising a first composition comprising a carrier; and about 100 mg to 400 mg of a second composition comprising a plurality of anthocyanins and anthocyanidins, wherein the plurality of anthocyanins and anthocyanidins constitute at least about 35%wt of the second composition, wherein the 35%wt is distributed between delphinidin-3-0-sambubioside-5-0-glucoside, delphinidin-3, 5-O-diglucoside, cyanidin-3, 5-O-diglucoside, dephinidin-3-O-O-sambubioside- 5-O-glucoside, cyanidin-3-O-sambubioside, and cyanidin-3-O-glucoside.

26. The method of claim 24 or 25 wherein such carrier can be selected from microcrystalline cellulose, lactose, silicon dioxide, glyceryl monostearate, soya lecithin, Acacia Gum, maltodextrin or Oenothera biennis oil.

27. The method of claim 18 wherein the composition to be used is capsule including a soft cellulose capsule or a hard gelatin capsule, a gummy chew, a powered sachet, a powder for food supplements, a microencapsulated powder, a masticatory tablet, a masticatory wafer, a swallowing tablet, a stick pack, a tube and a scoop.

28. The method of anyone of claims 18-20 wherein such mammal is selected from human being, pet animals, farm animals, competition animals, emotional support animals or marine/aquatic animals.

29. The method of claim 28 wherein such human beings, pet animal, farm animal, competition animal, emotional support animal can be selected from adults and elderly.

30. The method of claim 28 wherein such pet animal is selected from dog or cat.

31. The method of claim 28 wherein such farm animals are selected from cattle, pigs, equines, goats, sheep and chicken.

32. The method of claim 28 wherein such competition animals are selected from racehorses, dressage animals, beauty pageant animals.

33. The method of claim 28 wherein such marine/aquatic animals are pisciculture animals or fish of farming.

34. The method of claim 33 wherein such fish of farming is selected from rainbow trout, salmon, tilapia, carp, catfish.