Natural matrix composites for the treatment of bone fragility

Abstract

To provide a new composition consisting of 100% natural matter, exerting a therapeutic or beneficial effect in the treatment of bone fragility, having a physiological mode of action.SOLUTION: The present invention provides a 100% natural product consisting of natural matrices exhibiting emerging properties, which enables re-establishing correct bone metabolism by correcting behavior of mesenchymal stem cells and re-establishing a correct balance between cell populations that allow bone growth or remodeling. The natural product consists of, for example, Equisetum flowering tops, Acacia, Malpighia fruit, coral skeleton, avian eggshell, Agave leaves, Cetraria stems, Agaricus, and calcium citrate.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to novel compositions of 100% natural substances that have a physiological (as opposed to pharmacological) mechanism of action and exert a therapeutic or beneficial effect in the treatment of bone fragility.

[0002] The present invention relates to the exclusive selection and use of native matrices, appropriately processed by specific processes and methods, to create final products for therapeutic or beneficial purposes, in particular to prevent deviations from a balanced physiological state or to restore physiological conditions in areas of bone weakness. All stages of the manufacturing process of such products are under the aegis of the One Health principle, which recognizes the interconnectedness of human health, animal health, and environmental health, and therefore does not allow the use of artificial forces or substances.

[0003] Indeed, in the field of the present invention, a fundamental requirement is that the final product, i.e., a product comprising or consisting of one or more natural matrices, must maintain a natural intelligence, i.e., an imprint of the biological domain to which each component of the product belongs, thereby maintaining a network capable of interconnecting and recognizing itself with other networks, whether natural or artificial, i.e., an essentially natural network that has acquired a degree of artificiality through its interaction with artificial components. This interconnection is considered to be the basis for rebalancing any disturbance in the network of events active in each interacting biological system. All the matrices identified present biophysical specifications that themselves represent an invention.

[0004] Each network of each native natural matrix contained in the product that contributes to the formation of the network of the final matrix of the product of the present invention can be defined as a UVCB substance (i.e., a substance of unknown or variable composition, a complex reaction product, or a biological material) according to the REACH (Registration, Evaluation, Authorization and Restriction of Chemicals) definition, since it is a processed product according to its self-assembly properties and therefore cannot be determined or verified based on small molecule chemistry protocols.

[0005] Each network is characterized by the establishment of connections within the matrix of the final product and within the physiological effects exerted by the product on the recipient organism. Validation of product production can be performed and confirmed using probabilistic models based on the association between the preservation of physiological activity profiles and descriptors of the matrix itself, generated using multiple biophysical analytical systems, including spectroscopy (NIR and other techniques), spectrophotometry (targeted and untargeted metabolomics), and paper or X-ray crystallography (fractal measurements). While useful, traditional molecular chemical definitions of the individual substances contained in a substance cannot be used to validate this type of product, as they do not represent its overall efficacy and quality.

[0006] The choice of matrices intended for administration must be verified according to the updated and specific current taxonomic criteria of the animal, plant and mineral kingdoms. When used in combination with natural physical phenomena, the relationship between action and efficacy must be verified on a case-by-case basis, taking into account acoustic effects (musical or other forms) and effects in the field of wave-particles, including those of a quantum nature.

[0007] At the current state of the art, it is not necessarily possible to outline a fully explained mechanism of action, but it is believed possible to examine the actions and reactions of the respective networks in their mutual connections, which have already been verified at the biophysical level.

[0008] The present invention aims to select and provide new entities or products and systems that are able to rebalance, activate or limit physiological functions in specific metabolic states in organisms that are constantly in a state of continuous transformation.

[0009] The preparations thus conceived are capable of rebalancing the psychoneuroendocrine immune system, which is considered as a single system that regulates and controls all other systems.

[0010] This invention contributes to a new cutting-edge technology that goes beyond alchemical techniques in the medical field, dating back to the early 16th century and returning products and processes to the conceptual One Health goal already mentioned. It proposes a new declination of artificial technologies and natural self-assembly of substances, recognizing existing rules or finding new ones to ensure the formation of verifiable entities, primarily based on the concept of verifying their effects and activity on other organisms. The latter are continuously changing living beings that require evaluation of their physiological state within defined intervals, a concept now included in personalized medicine. This invention fits into the concept of science, understood as a set of knowledge that can demonstrably verify the effects of theoretical mechanisms of action. Today, these methods are applied to establishing interconnections between all forms of life, in a context where technological innovation advances at such a pace that it risks undermining the interconnections between human-generated (artificial) intelligence and nature.

[0011] The inventive activities disclosed herein are not currently covered by the state of the art and therefore the entire product cycle from the end user to the relevant societal context needs to be considered under the One Health concept.

[0012] The operational paradigm under which the present invention is implemented is referred to herein as "Bios Physiological Health."

[0013] This paradigm aims to introduce an innovative approach to health treatment and self-management into the field of medical technology using natural matrices alone or in combination to rebalance the normal physiological state of various biological entities, including humans, through endogenous physiological effects induced by the product: by identifying, selecting, and assembling natural entities with emerging properties that can be verified by the physiological mechanism of action of the final product and other methods that have been advanced in recent decades.

[0014] The basis of this invention is a contextual interpretation that incorporates both technoscientific and anthropological norms. While some of the properties of each matrix component of the product may already be known, the emerging properties of the new composition may be unexpected.

[0015] Of particular relevance is its role in determining the genetic and epigenetic aspects that determine the networks representing natural matrices and their interpretation at their specific isotope abundance levels.

[0016] To fulfill the paradigm of biophysical health, each stage of processing, from the selection of regenerative materials to agricultural and industrial stages and methods of use, must preserve as much as possible the inherited integrity of the native programming inserted into the natural intelligence of each creative entity, at least as far as is known on a global scale. It will be essential to verify matrices derived from epigenetic realities similar to those recognized as reference standards for the specific emerging metabolic properties of other organisms, including humans. For example, one of the factors negatively affecting epigenetic differentiation is represented by different soil conditions, along with circadian, monthly, and annual variations. To preserve the properties of natural systems, which are the only ones that can claim physiological interconnections with the whole product, it is impossible to use substances derived from alchemical processes such as distillation or other synthetic or semi-synthetic processes, or products derived from genetically modified or genetically altered organisms. A new interpretation of the mysteries of natural programming responsible for the evolution of organic and inorganic life is needed. The recent establishment of scientific evolution makes it possible to reposition the understanding of the origins of progress based on reductionist determinism on the basis of the development of alchemical processes from the beginning of the 16th century, which, together with Paracelsus in medicine, marked the beginning of the current evolutionary process known as the Anthropocene.

[0017] The term Anthropocene refers to the current stage of human evolution and can be traced back to different eras. When considered in the context of this invention, the key date is 1492, which marks the end of the Early Renaissance / Neoplatonic period. This period was politically represented by Cosimo the Elder and Lorenzo de' Medici, along with artists and scientists such as Piero della Francesca, Luca Pacioli, Leonardo, and Dürer. In the 16th century, the study of alchemy, which was considered the possibility of humans controlling nature, has continued to develop to this day under the aegis of artificial intelligence, as opposed to natural science, with the aim of improving the creation of natural phenomena, so that "man may have dominion over all creation."

[0018] The year 1492 is symbolic: the year Lorenzo de'Medici and Piero della Francesca died, while Columbus discovered America. The human species abandoned its Neoplatonic path in the 15th century to follow the Judeo-Catholic path, the application of Paracelsus' alchemical practices to medicine, and marked the transition to Renaissance Mannerism in the 1500s, leading us to the present day toward a full-scale, irreversible sixth extinction.

[0019] This invention has demonstrated the feasibility of the resulting industrial discovery in the medical field, but is in principle adaptable to any field of production and is intended to address a shift in evolutionary paradigm.While we often speak of protecting biodiversity without addressing the real problem of billions of tons of exogenous, non-biodegradable, man-made materials released into the planetary system, a problem that is clearly obscured, the "carpe diem" approach trumps a sense of species survival.

[0020] This invention, presented primarily in the context of a patent, hopes to open a new field of research exploring and sharing natural intelligence, rather than artificial intelligence, which is unlikely to be able to stop or slow the sixth extinction or to form the basis of alternative advances to the current one. Inventor Valentino Mercati, together with his collaborator Jacopo Lucci, has chosen the path of studying nature in ways that may be useful for living systems, developing knowledge in agricultural and industrial production systems for over 40 years and filing numerous patent applications following this operational strategy. Previously filed patents related to the methods of this invention are essentially based on instrumental and diagnostic readouts based on principles related to the chemistry of physiological effects and the emerging properties of natural matrices and their interconnected relationships with the innate defenses of individual organisms.

[0021] The analysis that inspired the approach disclosed herein was unthinkable just a few decades ago, due to the technical inability to read the genetic and epigenetic information written into the cells of any living organism, as well as the role of atomic isotope differentiation in molecular self-assembly and the interconnectedness of any singular / individual with the "universe." The conceptual difficulty of moving from the reassuringly controlled parameters of molecular artificiality (at least partially purified and linked by powerful thermodynamic forces that allow strong bonds, such as covalent bonds, that act to a reduced extent on the molecules of other organisms) to natural matrices that are by definition mysterious and still considered therapeutically unreliable today is enormous.

[0022] If, after five centuries of alchemical reductionism, a new interpretation of the invention is needed for the new medical status quo, this interpretation must unite the most distant concepts and processes in a single field of application. This, as already mentioned, stems from an ideological legacy that questions the human condition. Was the human species produced by an original vital intelligence, like all other species, for the purpose of life itself, as far as it can be assumed to govern creation, or was it experimentally endowed with capacities different from those of other organisms already advantageously inserted into creation in order to constitute a new ecological niche in the service of the universe?

[0023] The answer to this dilemma does not lie in this invention. Humanity must return to the Neoplatonic thought of the early Renaissance, and the experimental duality of the human species must be liberated from the spirit of domination in order to share its unique capacities within the universe with all of creation. Humanity needs to reconsider Leonardo da Vinci's warning, "Man can only produce his own offspring..." and reflect on the depressing thoughts of sensible figures like Piero della Francesca, Luca Pacioli, and Dürer regarding the impossibility of understanding and expressing the beauty of creation and deciphering its mysteries.

[0024] The time has come to acquire new research centers in molecular and cell biology, with an essential focus on bioinformatics and the new physical sciences. Today, the inventors can base their research strategies and socio-economic applications on new therapeutic fields, especially in the field of complex and / or chronic degeneration, where the restoration of metabolic balance in organisms disturbed naturally or artificially will become an integral part of the already existing future.

[0025] This invention represents a new vision of medical technology that reconsiders scientific evolution from a perspective different from reductionist determinism. This alternative progress requires relying less on artificial intelligence and technological advances, contrary to universal or planetary rules, and more on the development of laws that regulate our universe and life itself. The transition from the artificial treatment of specific symptoms to a holistic approach that embraces the whole, even from a systemic perspective through modern techniques of systems biology, represents the basis of current progress.

[0026] In particular, the present invention relates to a product comprising a natural matrix derived from horsetail, acacia, malafigia, coral calcium, eggshell calcium, agave, settloria, agaric, and calcium citrate that supports bone homeostasis, helping the organism re-establish proper bone metabolism by correcting stem cell behavior and re-establishing the correct balance between cell populations that allows bone growth or remodeling. By supporting the correct function of bone cells, the product achieves beneficial results, resulting in a rewiring of differentiation processes in favor of the bone lineage over the adipocyte lineage, and inducing improvements in whole-body metabolism. The present invention also relates to the use of the product in treating or assisting in the treatment of a bone fragility disease or condition in a subject in need thereof, and to methods of treating or assisting in the treatment of a bone fragility disease or condition, particularly when the fragility is postmenopausal or peri-menopausal.

[0027] Background technology Bone fragility is a significant social problem. Scientific organizations in various countries are developing guidelines for the diagnosis and treatment of this condition, aiming to adopt specific tools for assessing fracture risk in populations. Osteoporosis is a socially important disease because its incidence increases with age, affecting a large proportion of the population beyond their 80s.

[0028] Postmenopausal women are one of the populations most affected by this disease. Metabolic changes that occur during this transition lead to increased adiposity at both the abdominal and bone levels. In particular, an overly energy-rich diet consisting of overly refined nutrients or an imbalanced ratio between them, combined with the physiological tendency of menopausal women's bodies, leads to the accumulation of adipose tissue characterized by an increased inflammatory state. From a cellular and molecular perspective, dysfunctional and inflamed adipose tissue leads to an imbalance in bone homeostasis, negatively impacting competition for mesenchymal stem cell reserves necessary to induce osteoblast, osteoclast, or adipocyte differentiation, unnaturally shifting this process toward osteoclast differentiation.

[0029] This results in a depletion of mature osteoblasts, which are unable to ensure proper mineralization of the cellular matrix, causing a loss of bone functionality. Such a decline in bone quality can lead to the development of osteoporosis, a condition in which the anabolic effects of osteoblasts on bone metabolism are overshadowed by the catabolic effects of their counterparts, osteoclasts.

[0030] In conditions of dysregulated lipid metabolism and adipose tissue inflammation, a dysfunctional loop is formed between adipose tissue and bone, leading to the accumulation of fat and osteoclasts, but promoting osteogenic components and therefore increasing bone fragility.

[0031] Fragility fractures cause multiple disabilities, significant morbidity, reduced quality of life, and functional limitations. Patients with osteoporosis require comprehensive care that includes a multidisciplinary and interdisciplinary approach carried out by a team, with individualized rehabilitation plans that include programs targeting specific areas of intervention.

[0032] At the cellular level, osteoporosis involves changes in bone density and strength, leading to a higher risk of fracture. The disease is no longer considered solely a loss of bone mineral density (BMD), as other factors, such as bone structure, also affect fracture risk. Bone loss, which begins at age 40 and worsens over time, is known as osteopenia and can remain asymptomatic until it progresses to osteoporosis. Osteoporotic bones become very fragile, and even minor trauma or normal body weight can cause fractures.

[0033] Bone tissue consists of both cellular components and extracellular matrix. Cellular components, including osteoblasts, osteoclasts, and osteocytes, account for approximately 2% of bone mass and play important roles in bone metabolism. Osteoblasts, which differentiate from mesenchymal stem cells (MSCs), are responsible for synthesizing the organic components of bone and mineralizing the matrix. Osteocytes are mature osteoblasts that are entrapped in the matrix, maintaining communication through canaliculi and forming a network that coordinates cellular activity. Osteoclasts, large multinucleated cells, are responsible for bone resorption. Their maturation involves signals from various molecules, including parathyroid hormone (PTH), estrogen, and interleukins, and is regulated by the interaction of receptor activators such as RANKL and osteoprotegerin.

[0034] Adipocytes also play an important role in bone metabolism. They arise from the same MSCs that differentiate into osteoblasts and osteoclasts. Adipocytes in bone tissue are involved in endocrine functions and secrete adipokines, such as leptin and adiponectin, which regulate bone metabolism. While some adipokines stimulate bone formation, others promote osteolysis, adversely affecting bone mineral density and strength, potentially increasing fracture risk and contributing to osteoporosis. Excess bone fat, often associated with metabolic syndrome or obesity, can impair bone health and increase fragility by reducing bone mineral density.

[0035] Bone extracellular matrix consists of organic (65%) and inorganic (35%) components. The organic component, or osteoid, includes type I collagen, non-collagenous proteins, proteoglycans, osteonectin, osteocalcin, and other growth factors such as IGF-1 and TGF-β. The inorganic component consists primarily of calcium phosphate in the form of hydroxyapatite crystals and lesser amounts of magnesium and sodium. Mineralization of osteoid by osteoblasts, regulated by alkaline phosphatase and osteocalcin, is important for bone strength.

[0036] Bone remodeling is an ongoing process involving the resorption and formation of bone tissue, regulated by various hormones and cytokines. During remodeling, osteoclasts resorb old bone, while osteoblasts form new bone to replace it. This process is essential for maintaining bone strength, especially in response to mechanical stress, and for regulating calcium levels in the body. Bone resorption and formation occur at different sites in the bone, coordinated by basic multicellular units (BMUs) composed of osteoclasts and osteoblasts. Removal of old bone by osteoclasts is followed by apoptosis, and new bone is formed by osteoblasts, a process carefully regulated by both general and local factors.

[0037] General factors include hormones such as PTH, calcitriol, and estrogen, which stimulate bone formation, and calcitonin, which inhibits osteoclast activity. Local factors include cytokines such as IL-1, IL-6, and tumor necrosis factor (TNF), which promote osteoclast differentiation, and osteoclastic proteins, which block RANKL-RANK interaction, which inhibits osteoclast formation. Furthermore, molecules such as transforming growth factor-β (TGF-β), insulin-like growth factor (IGF), and bone morphogenetic protein (BMP) are released during bone resorption and affect the activity of osteoblasts and osteoclasts.

[0038] In other words, bone fragility is a pathological condition that affects the entire body.As mentioned above, an important aspect of bone fragility, especially during menopause, is the involvement of adipose tissue both in bone (osteopenic adipose tissue) and throughout the body.Osteopenic adipose tissue refers to the accumulation of adipose tissue in bone marrow, which can further reduce bone quality and increase its fragility.Systemically, the increase in adipose tissue, which is often associated with metabolic changes during menopause, can adversely affect bone health through inflammatory mechanisms and changes in bone metabolism, leading to increased bone fragility and fracture risk.During menopause, women experience a significant decrease in estrogen levels, a hormone that plays an important role in maintaining bone health.This decrease in estrogen accelerates bone loss, making postmenopausal women particularly susceptible to bone fragility.

[0039] Therefore, it is essential to approach the physiological transitions that women undergo during menopause using the principles of systems biology and systems medicine. This approach should take into account the vast network of relationships between cells, tissues, organs, and systems involved in the metabolic changes during menopause, understand their functionality, and be acutely aware of the various factors at play.

[0040] In contrast to healthy bones, bone fragility may be related to an imbalance in the physiological metabolism of bone tissue, resulting in a loss of homeostatic response across the entire functional network. This imbalance shifts the differentiation of mesenchymal stem cells toward adipose tissue and osteoclasts, leading to a depletion of bone tissue, especially osteogenic components, and as a result, bone tends to be unable to mineralize properly. The accumulation of fat in both the bones and abdominal region, typical of postmenopausal women, also leads to an inflammatory state, which may eventually develop into metabolic syndrome, creating a dysfunctional loop in various body systems, including bone tissue.

[0041] In this context, it is important to consider the endocrine function of bone, particularly the secretion of osteocalcin (OCN). Osteocalcin, a hormone that also regulates insulin metabolism, is expressed and secreted by mature osteoblasts. It stimulates insulin secretion from pancreatic β cells, enhances insulin sensitivity in muscle and white adipose tissue, and lowers blood glucose levels. Modulating this hormone plays an important role in reducing both adipose tissue mass and inflammatory status, producing an anti-adipogenic effect, improving insulin sensitivity, and increasing glucose uptake.

[0042] This framework emphasizes the strategic and functional importance of cell differentiation and bone mineralization processes. These two aspects must be finely regulated and stimulated to ensure good bone health. The most common response to these conditions is the use of vitamin D and calcium supplements. Although essential, these substances address the body's needs in a limited and incomplete manner and interact with metabolic pathways in a timely and non-comprehensive manner, without achieving satisfactory results. Furthermore, the scientific community has questioned the actual beneficial effects of vitamin D supplementation. This is exemplified by the release of AIFA Note 96 in Italy, which amended the prescription guidelines for cholecalciferol-based drugs in the A-class of drugs, declaring that vitamin D supplementation does not significantly reduce fracture risk in independent, non-institutionalized individuals.

[0043] Therefore, in addition to providing vitamins and calcium, it is necessary to engage the body's physiological metabolic network through a systems-based approach that exploits the redundant effects of the products in question, which stimulates stem cell differentiation in bone tissue.

[0044] In summary, bone fragility is a multifactorial condition characterized by an imbalance between bone resorption and formation. This imbalance can be exacerbated by hormonal changes, metabolic disorders, and inflammation. Understanding the cellular components of bone and the complex process of bone remodeling is essential for developing effective treatment strategies. Interventions aimed at restoring bone health must consider the roles of osteoblasts, osteoclasts, adipocytes, and the extracellular matrix and should include both pharmaceutical and rehabilitation approaches to reduce fracture risk and improve quality of life.

[0045] Despite these insights, current treatments fail to adequately address the complex interplay between adipose tissue expansion and bone fragility, and lack products that can promote / support proper restoration of function in bone and systemic metabolism. Traditional treatment methods primarily focus on either hormone replacement or bone density preservation, and do not adequately target the underlying inflammatory processes and metabolic dysfunction that exacerbate bone fragility. This gap highlights the need for innovative therapeutic approaches that can modulate both adipose tissue inflammation and bone metabolism, thereby promoting proper restoration of function in bone and systemic metabolism, thereby providing a comprehensive solution for the overall prevention and treatment of osteoporosis and bone fragility, with particular focus on the menopausal population.

[0046] Summary of the Invention The development of the product of the present invention (also referred to as "Product C" in the figures and examples) is aimed at creating a 100% natural matrix-based product containing plants and minerals that can support physiological bone metabolism, particularly in women undergoing menopause, a physiological transition that, as explained above, affects various areas of the body and may be associated with underlying pathologies such as osteoporosis and metabolic syndrome. Adipose tissue plays an increasingly important role during this transition due to epigenetic stimuli that have emerged in modern times and were not previously present or widespread.

[0047] Due to its emerging properties and natural matrix composition, the product of the present invention is capable of performing a network (product) over network (recipient) mechanism, interacting with physiological metabolic pathways occurring in the body of menopausal women or women undergoing this transition (premenopause or perimenopause: premenopause is the time between a woman's first period and the onset of menopause; perimenopause is the transition to menopause, typically lasting approximately six years). The product of the present invention precisely stimulates the differentiation of mesenchymal stem cells, promotes the development of osteoblastic cell lines, and inhibits the formation of osteoclasts and adipocytes, thus contributing to the establishment of a virtual balance in bone metabolic processes. Furthermore, it can provide the calcium necessary for the precise deposition of hydroxyapatite crystals within the bone matrix. As a result, the product of the present invention has been shown to be able to reduce both the amount and the inflammatory state of adipose tissue, also by modulating osteocalcin expression, suggesting an anti-adipogenic effect, as well as improving glucose tolerance, revealing further desirable effects in the pathophysiological framework of the target subject: its local and systemic action can intervene in the dysfunctional loop that occurs in systemic inflammation and metabolic dysregulation, which affects the differentiation of mesenchymal stem cells into white adipocytes and their subsequent accumulation.

[0048] The reduction in bone resorption induced by the products of the present invention, as disclosed in the examples and figures herein, plays a fundamental role in the maintenance and repair of bone itself, promoting the rebalance of bone density and structure. Furthermore, the products of the present invention demonstrate a systemic metabolic regulatory effect by inducing the expression levels of osteocalcin (OCN), a hormone also involved in the regulation of insulin metabolism. Osteocalcin is expressed and secreted by mature osteoblasts and acts by stimulating insulin secretion from pancreatic beta cells and enhancing insulin sensitivity in muscle and white adipose tissue, resulting in lower blood glucose levels and regulating overall energy expenditure.

[0049] During the development of this product, the latest technology and experimental models were applied to represent the physiological transition process of menopausal women from a holistic perspective, and the biological activity of the product was defined.

[0050] The data obtained and disclosed in the examples and figures of this application demonstrate that the dual synergistic and systemic effects of the product of the present invention are significantly more desirable than classic calcium and vitamin D supplements, which can only partially support the body's physiological changes. Furthermore, as is evident from the experiments and figures of this application, the beneficial / therapeutic activity of the product of the present invention is provided through a physiological mechanism of action, meaning that the product interacts with the body according to a well-known system, rather than according to an exogenous principle imposed by an exogenous entity such as a synthetic molecule. Therefore, in addition to containing a vitamin D precursor and a calcium source, the product of the present invention is designed to interact with the mesenchymal stem cell pool in bone and adipose tissue, allowing it to reproduce all the elements necessary to rebalance the appropriate bone turnover essential for the formation of solid and functional structures. Therefore, the effect of this product as a whole is expected to be systemic, contributing to the rebalancing of metabolic disorders and subclinical inflammation affecting menopausal women on a systemic scale.

[0051] These features make the product of the present invention significantly superior to conventional calcium and vitamin D supplements. These supplements only partially support the physiological changes in the body. Its interaction with mesenchymal stem cells in both bone and adipose tissue can recalibrate all necessary elements to restore proper bone turnover, which is essential for creating a solid and functional structure. Furthermore, at the systemic level, it can rebalance metabolic dysregulation and reduce inflammation.

[0052] Thus, with its complex plant matrix and the presence of natural calcium from various sources, the product of the present invention clearly demonstrates that the complexity of physiological processes can only find correct synergistic effects when accompanied by a similar level of complexity.

[0053] The object of the present invention is a product consisting of a natural matrix having the following formula: [Table 1]

[0054] Treatment or amelioration of bone fragility or the product for use in the treatment or amelioration of bone fragility is included, as is the administration of the product to a patient in need thereof or to a healthy individual at risk of developing bone fragility.

[0055] In particular, the product is beneficial for use in supporting bone homeostasis in individuals at risk of developing bone fragility. [Brief explanation of the drawings]

[0056] [Figure 1] Figure 1. Imbalances in lipid metabolism in postmenopausal women. Changes in energy and lipid metabolism contribute to weight gain in postmenopausal women. During menopause, women undergo hormonal changes, including a decrease in estrogen and an increase in circulating androgens. These changes predispose postmenopausal women to changes in body composition, muscle loss, and abdominal obesity. In particular, the decrease in estrogen leads to an increase in bone marrow-derived adipocytes, which contributes to higher levels of visceral fat in postmenopausal women. Increased lipolysis of visceral fat by adipose tissue lipoprotein lipase produces excess free fatty acids, which can lead to insulin resistance and metabolic disease. Loss of estrogen downregulates genes involved in β-oxidation, preventing the efficient use of free fatty acids as an energy source. Furthermore, older women show increased fat accumulation due to the upregulation of genes involved in adipogenesis. As a result, ATP production by β-oxidation decreases and lipogenic metabolism increases. [Figure 2] Figure 2. Bone remodeling. Bone remodeling is a critical process that maintains the strength and health of human bones. This process involves bone resorption, controlled by osteoclasts, and bone formation, controlled by osteoblasts. Osteoporosis occurs when there is an imbalance in this process, with bone resorption exceeding bone formation and failing to ensure proper mineralization of the cellular matrix, resulting in decreased bone mass and bone functionality. Osteoporosis can lead to fractures that cause pain, disability, reduced quality of life, and, in severe cases, death. [Figure 3] Figure 3. Bone remodeling in osteoporosis (abnormal bone remodeling). Abnormal bone remodeling process: Key factors affecting bone formation include a) differentiation of mesenchymal stem cells (MSCs) into osteoblasts, b) differentiation of MSCs into osteoclasts and adipocytes, and c) deposition of bone matrix. The figure shows abnormal remodeling with increased bone resorption and decreased bone formation, resulting in osteoporotic bone. In abnormal remodeling, osteoclasts play a key role by excessively destroying bone tissue, while adipocytes contribute imbalance by promoting adipogenesis over osteocytogenesis. As a result, bones become brittle and lose their structure and function. [Figure 4] Figure 4. Effect of adipocytes on bone fragility. Osteopenic steatosis is characterized by bone loss and the infiltration of fat into bone, including adipocytes derived from MSC differentiation that are disproportionate to osteocyte components. Inflammation plays a key role in the development of osteopenic steatosis and has been extensively studied. Disruption of communication between bone and adipose tissue is thought to contribute to this condition. Accumulation of adipose tissue within bone affects bone structure and weakens bones. [Figure 5]Figure 5. ALP activity in non-osteoinductive medium (OM). The graph shows alkaline phosphatase (ALP) activity in hADMSCs cultured in OM, normalized to DNA content (ALP μU / μg DNA) over time (4–35 days) under different experimental conditions: product C, synthetic calcium with or without DMSO vehicle, or vitamin D + synthetic calcium (DMSO vehicle). ALP enzyme activity data were calculated as the mean ± standard deviation (SD) of quadruplicate experiments at six different osteoblast induction time points. Significance was calculated using anova and Bonferroni test. * = p<0.05 vs. OM + DMSO + synthetic calcium; ** = p<0.01 vs. OM + DMSO + synthetic calcium; $ = p<0.05 vs. OM + synthetic calcium; $$ = p<0.01 vs. OM + synthetic calcium. [Figure 6] Figure 6. Hydroxyapatite (HA) deposits in osteoinductive medium (OM). Quantitative analysis of calcium mineralization deposits in hADMSCs cultured in OM in the presence of product C, synthetic calcium with or without DMSO vehicle, or vitamin D + synthetic calcium (DMSO vehicle) for 4 to 35 days. Data were calculated as the mean ± standard deviation (SD) of quadruplicate experiments. Significance was calculated using Anova and Bonferroni test. $=p<0.05 vs. OM + synthetic calcium. [Figure 7] Figure 7. ALP activity in non-osteoinductive medium (GM). The graph shows alkaline phosphatase (ALP) activity in hADMSCs cultured in GM, normalized to DNA content (ALP U / µg DNA) over time (4-35 days) under different experimental conditions: product C, synthetic calcium with or without DMSO vehicle, or vitamin D + synthetic calcium (DMSO vehicle). ALP enzyme activity data were calculated as the mean ± standard deviation (SD) of quadruplicate experiments at six different osteoblast induction time points. Significance was calculated using Anova and Bonferroni test. $=p<0.05 vs. GM + synthetic calcium. [Figure 8]Figure 8. Comparison of Product C activity versus vitamin D and calcium in osteoblast differentiation. This figure shows that while vitamin D contributes to the data obtained in experiments, it does not itself provide a differentiation stimulus or directly provide the calcium needed to promote the formation of mineralized bone matrix. Synthetic calcium alone cannot intervene in the differentiation process; it can only provide calcium for bone formation. Product C alone is able to reproduce the entire physiologically active process. The product is able to provide the calcium necessary for the precise deposition of hydroxyapatite crystals in the bone matrix, and it also has the ability to precisely stimulate the differentiation of mesenchymal stem cells, promote the development of osteoblastic cell lines, and inhibit the formation of osteoclasts and adipocytes both locally and systemically. [Figure 9] Figures 9a and 9b: Illustration of osteoporosis hallmarks (column 1), the network of biological activities consistent with defining the pathological state (column 2), and its degree of regulation in the pathological state (column 3). The regulation trend of the network of biological activities consistent with the healthy physiological state (column 4). Dark grey: degree of upregulation. Light grey: degree of downregulation. [Figure 10] Figures 10a and 10b show modulation of a network of selected bioactivities in a human adipose-derived mesenchymal stem cell line (hADMSC), capable of differentiating into osteoblasts and mineralizing extracellular matrix (ECM) in a cell-based assay for osteoporosis. Column 1 shows the hallmarks; Column 2 shows the network of bioactivities; Column 3 shows the trend of modulation of the network of bioactivities in a pathological state; Column 4 shows the trend of modulation of the network of bioactivities consistent with a healthy physiological state; Column 5 shows the appropriately induced cells representing the network of bioactivities regulated in an unregulated state; and Column 6 shows the modulation induced by the tested product (Product C). The cell-based assay shows that the product sample modulates the entire network of selected activities according to a trend consistent with a healthy physiological state profile. The numbers reported in each box represent values quantifying the degree of modulation (z-score) calculated according to the example, which represents the degree of modulation of each observed bioactivity. [Figure 11]Figures 11a and 11b show modulation of a network of selected bioactivities in a human adipose-derived mesenchymal stem cell line (hADMSC), capable of differentiating into osteoblasts and mineralizing extracellular matrix (ECM) in a cell-based assay for osteoporosis. Column 1 shows hallmarks; Column 2 shows the network of bioactivities; Column 3 shows the trend of modulation of the network of bioactivities in a pathological state; Column 4 shows the trend of modulation of the network of bioactivities consistent with a healthy physiological state; Column 5 shows appropriately induced cells representing modulation of the network of bioactivities in a non-regulated state; Column 6 shows modulation induced by the tested product (Product C); and Column 7 shows modulation induced by the reference drug (DIBASE). The cell-based assay demonstrates that the product sample modulates all selected activities of the network with a single modulation trend consistent with a healthy physiological state profile. Meanwhile, the reference drug fails to effectively modulate all of the entire network of bioactivities required to define a healthy physiological state. The numbers reported in each box represent values quantifying the degree of modulation (z-scores) calculated according to the example, which represent the degree of modulation of each biological activity observed. [Figure 12] Figure 12. Network analysis of osteoporosis (Panel A) and treatment with a reference drug (Panel B) versus treatment with product C (Panel C). Gray boxes represent both the basic nodes characterizing the pathophysiological or altered physiological state and the specific sites where the pathology interacts with the body. Arrows next to the nodes indicate the specific modulation level for each described condition, with strength indicated by a multiplication of the arrow itself. Gray boxes connect to a network of biological activities whose modulation levels have been experimentally verified. These biological activities are represented by black (upregulation level) or white (downregulation level) circles, whose amplitudes are directly proportional to the magnitude of their experimentally verified modulation levels. The network analysis shows that product C affects the body systemically and modulates more desired activities than treatment with the reference drug, in line with the modulation trends associated with a healthy physiological state. [Figure 13]Figures 13a and 13b. Modulation of networks of selected biological activities (biological activities) in human adipose-derived mesenchymal stem cell lines (hADMSCs) capable of differentiating into osteoblasts and mineralizing extracellular matrix (ECM) cell-based assays in osteoporosis: Column 1: Hallmark; Column 2: Network of biological activities; Column 3: Modulation trends of networks of biological activities in pathological conditions; Column 4: Modulation trends of networks of biological activities associated with healthy physiological conditions; Column 5: Appropriately induced cells representing modulation of networks of biological activities regulated by unregulated conditions; Column 6: Modulation induced by Product C Batch 1; Column 7: Modulation induced by Product C Batch 2; Column 8: Modulation induced by Product C Batch 3. The cell-based assays demonstrate that all tested product batches modulate all selected networks of activities according to the modulating trends (modulation of condition and functional resilience) consistent with the healthy physiological condition profile. On the other hand, the reference drug fails to effectively modulate all biological activities necessary to define a healthy physiological state (does not act on the network). The numbers reported in each box represent values quantifying the degree of modulation (in terms of z-scores) calculated according to the examples that represent the modulation of each biological activity of the observed network. [Figure 14] Figure 14. FTIR spectrum of batch 1 of product C. DETAILED DESCRIPTION OF THE INVENTION

[0057] term Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those skilled in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0058] As used herein, the term "synthetic calcium" refers to calcium-containing compounds that are synthetically prepared by chemical reactions in a laboratory.

[0059] As used herein, "calcium citrate" refers to a compound obtained by reacting calcium, such as coral calcium, with lemon juice.

[0060] At any point in this specification or claims, the words "comprising" or "comprise(s)" may be replaced with "consisting of" or "consist(s) of."

[0061] In this application, a "natural matrix" refers to a material consisting of a network represented by a wide range of components / constituents obtained (e.g., extracted) directly from a member of the natural world or its naturally occurring parts (i.e., from a natural source) without significant processing or synthetic alteration. "Without significant processing or synthetic alteration" connotes that no denaturing process is used to obtain the matrix from the source material. In other words, the natural source material is processed only by manual, mechanical, or gravitational means, such as dissolving in water or other naturally occurring solvents, such as water, water-alcohol solutions, flotation, extraction with water or other natural solvents, steam distillation, or heating solely to remove water or any other naturally occurring solvent. Alternatively, the "natural matrix" may be extracted from air by any means, provided that the member of the natural world or its naturally occurring parts itself is excluded. In particular, according to the present invention, a natural matrix is a 100% natural and biodegradable material consisting of natural components that have not been modified by the process for producing the matrix from the starting material, without the intentional addition of synthetic products along the entire process. Herein, 100% biodegradability is considered "readily biodegradable" according to the OECD biodegradability test. These characteristics ensure the maintenance of the matrix effect imparted to the matrix by the structural interactions (material interactions) of its constituent components and the presence of functional interactions (non-material interactions) that become apparent upon exposure of a biological system to the natural matrix. In other words, natural matrices or mixtures of natural matrices are materials obtained from entities that naturally self-assemble and have been processed to preserve their native biophysical properties that determine their physiological interactions with other living organisms, such as human organisms. Their emerging properties may be expressed by contributing to the rebalancing of metabolic processes or states of the recipient organism and / or some organs or tissues, along with physiological effects activated in each specific context. According to the present invention, natural matrices can be derived from materials obtained from any source within the kingdoms of life, namely, Monera, Protesta, Fungi, Plantae, and Animalia.Thus, the term encompasses plant natural matrices, animal natural matrices, fungal natural matrices, protist (archaeal or bacterial) natural matrices, and monera natural matrices. Natural matrices can also include naturally occurring inorganic materials, such as minerals obtained from natural sources. Synonyms for natural matrix or one or more natural matrices herein are "complex natural system" or "natural material," as defined below.

[0062] Examples of naturally occurring parts of an organism may be represented by, for example, roots, leaves, bark, fruits, flowers, plants or sections thereof, organs, tissues.

[0063] In any part of this specification the general term natural matrix may be replaced by: Plant natural matrices or natural matrices obtained from plants, Animal natural matrices or natural matrices obtained from animals or animal products such as eggs or milk, Fungal natural matrices or natural matrices obtained from fungi, Protist natural matrices or natural matrices obtained from protists, Monera natural matrix or natural matrix obtained from Monera, or plant materials and / or extracts, extracts from animal tissues or organs, fungi and / or fungal extracts, or mixtures thereof, where the extraction method does not involve a denaturing step (e.g., temperature or the use of denaturing solvents).

[0064] Plants are synonymous with herbs.

[0065] The term "natural" matrix emphasizes that it retains the integrity and complexity of the component / component network as in the original natural source due to the absence of denaturing treatments to obtain it. Thus, natural matrix does not encompass naturally occurring compositions enriched in specific molecules that have been artificially synthesized or isolated from natural sources. Furthermore, natural matrices can only be obtained by processes that do not involve extensive processing or chemical modification, isolation, purification, or molecular extraction.

[0066] Due to the supramolecular self-assembly of the components / ingredients of the natural matrix and the existence of functional interactions between them, the entire matrix behaves as a complex network that does not interact with a single target molecule, but with a network of recipients (also organized as a network) in the recipient organism. Thus, the interaction of natural matrix recipients is not the result of point-to-point interactions, as with a typical pharmaceutical API, but rather the result of interactions between an "interactor" network (i.e., the matrix) and a "recipient" network (i.e., the organism to which the matrix is administered).

[0067] The term natural matrix may also be substituted with complex natural system in any part of the specification and claims.

[0068] Nowhere in this specification or claims is the term natural matrix construed as a "natural product" per se. A natural matrix is a product obtained from a natural organism and processed therefrom (e.g., extracted) by techniques that do not substantially alter the biological structure and the relevant supramolecular and functional interrelationships between the components within said matrix, i.e., by techniques that do not use denaturing techniques and do not involve additional isolated or synthetic molecules or classes of molecules.

[0069] Emerging properties, as used herein and in the art, are defined as properties of natural matrices or materials, i.e., properties that are not merely represented by the sum of the properties of each isolated component / ingredient of the matrix / material, but are instead represented by both functional and structural interactions between all components / ingredients of the matrix / material, which are also the result of supramolecular self-assembly of the components / ingredients within the matrix / material itself.

[0070] Thus, "emerging properties" refer to the technical effects, such as therapeutic or homeostatic-supportive properties (i.e., beneficial effects), that the interactions and relationships between components of a natural matrix have on a recipient biological system. By definition, emerging properties are properties that are not immediately apparent or even predictable based solely on the individual properties of each component of the matrix. Alternatively, they "emerge" when all components of the matrix network interact in dynamic and complex ways with each other and with the recipient biological system network. Emerging properties have been widely discussed in the art in various scientific and systems-oriented fields, including physics, chemistry, biology, and complex systems theory.

[0071] Thus, emerging properties are those that cannot be predicted a priori by qualitative and quantitative knowledge of each component of a given composition or matrix and, consequently, cannot be attributed to one or more specific APIs. While multi-drug compositions may exhibit unexpected synergistic effects, the properties of such compositions are still attributable to the specific APIs and their amounts contained therein.

[0072] In the case of emerging properties characteristic of the native matrix, the observed emerging properties are not reproducible for a particular API and are maintained in different batches of a given matrix or in a given mixture of matrices, even though the batches are of qualitatively or quantitatively different composition (functional resilience, see below).

[0073] As used herein, synthetic has its conventionally accepted meaning in chemistry. Traditionally, in chemistry, the term "synthetic" refers to the origin or source of a material or substance. Synthetic substances or materials are produced by humans by artificial synthesis, i.e., laboratory chemical reactions that typically react simpler chemicals to produce more complex chemicals through processes that often employ different pathways, temperature conditions, pressure conditions, energy sources, and / or catalysts than those used by living organisms.

[0074] Examples: Synthetic substances or materials include plastics, drugs, and many industrial chemicals. For example, nylon is a synthetic polymer made by chemical synthesis, and aspirin is a synthetic drug made by a specific chemical reaction.

[0075] Functional resilience (also referred to as "redundancy") according to the present invention is intended as the therapeutic or beneficial (homeostatic adjuvant) resilience of a therapeutic or beneficial product comprising or consisting of one or more natural matrices, and this term describes the maintenance of the therapeutic or beneficial properties of different batches of a given product comprising (or consisting of) one or more natural matrices, despite the qualitative and quantitative compositional differences between different batches, which composition is always present (unique) in a product comprising or consisting of one or more natural matrices. As known to those skilled in the art, each time different batches of starting material are used, the resulting natural matrix has a unique qualitative-quantitative composition at the molecular level, which is typical of the individual variations between organisms of the same species.

[0076] A healthy physiological state refers to the state of an organism's body, organ, device, system, or bodily region and its internal processes when they are functioning optimally within the individual's normal parameters, i.e., the state toward which homeostasis tends. In the context of one or more biological activities known to contribute to a given disease or pathology or hallmark of an altered physiological state, a healthy physiological state refers to a state in which the one or more biological activities are operating optimally and within normal (healthy) parameters. This state is characterized by the absence of significant abnormal cellular or molecular processes associated with the particular disease under consideration. When trends in alterations of one or more biological activities consistent with a pathological or pre-pathological state are known, a healthy physiological state can be considered to be represented by the opposite trends in alterations for each of the activities.

[0077] The term considers the hallmarks of a particular disease, which are distinctive features or characteristics typically observed in individuals affected with that disease. These hallmarks may include specific cellular behaviors, molecular pathways, canonical pathways, or physiological responses that play a key role in the development or progression of the disease.

[0078] In summary, a healthy physiological state in the context of a particular disease or pathological / altered condition is one in which one or more biological activities associated with a known hallmark of that disease or condition are modulated in a direction consistent with the non-pathological / unaltered state, in other words, in a direction opposite to the pathological / altered state.

[0079] Therefore, a healthy physiological state according to the present invention also indicates the direction of modulation of one or more biological activities that are known hallmarks of a pathological condition in homeostasis, i.e., before the onset of a pathological condition, in other words, the direction of homeostasis of the degree of modulation of one or more biological activities that are ascribed to a particular system, region, organ or tissue in a healthy subject.

[0080] Altered physiological state and altered homeostasis are closely related concepts that describe deviations from normal function and balance of the body's internal environment. While they overlap, there are some distinctions between the two terms.

[0081] Altered physiological state: This term encompasses a wide range of changes in the normal function of the body, including disruptions to organ systems, biochemical processes, and cellular function. Altered physiological states can result from a variety of factors, such as disease, injury, medications, environmental factors, and psychological stress. Examples include fever, inflammation, hormonal imbalance, and organ dysfunction.

[0082] Homeostasis (altered): Homeostasis refers to the body's ability to maintain a stable internal environment despite external or pathological changes. This stability is achieved by regulatory mechanisms that control variables such as body temperature, blood pressure, pH balance, and blood glucose levels within narrow ranges. Alterations in homeostasis occur when these regulatory mechanisms fail to maintain balance, leading to deviations from the body's normal set points. These deviations can be temporary or chronic and may involve compensatory mechanisms to restore balance.

[0083] In summary, an altered physiological state describes an observable change in the body's normal functioning, while altered homeostasis refers to a fundamental disruption of the body's regulatory mechanisms that maintain internal stability.

[0084] Alterations in homeostasis are the basis for altered physiological states, as disruptions to homeostatic mechanisms can result in physiological imbalances and the development of disease or dysfunction. Homeostatically supporting products are those that condition the body to restore stability to its internal environment when it is altered.

[0085] The term "hallmarks of a disease or pathological or medical condition" as used herein has the meaning conventionally used in the art. Disease hallmarks are known to be indicators that can mark the progression or control of a given disease or pathological or pre-pathological condition, and together they typically represent the general pathological conditions associated with a given pathology. These hallmarks (also called "key indicators") are typically a set of features or patterns that physicians monitor over time to track the onset, progression, or regression of a particular disease. In summary, disease hallmarks are defining features or characteristics whose alterations indicate a given pre-medical or medical condition and aid in its identification, diagnosis, monitoring, and understanding. For example, for neurodegenerative diseases (NDDs), at least eight hallmarks of NDDs are known in the art: (pathological protein) aggregation, synapse and neuronal network (dysfunction), (abnormal) proteostasis, cytoskeleton (abnormal), (altered) energy homeostasis, DNA and RNA (deficiencies), inflammation (increased), and neuronal cell death (increased). In cancer research, the hallmarks of cancer are a set of distinctive properties commonly found in cancer cells, including (sustained) proliferative signaling, (evasion of) growth suppressors, (resistance to) cell death, (enabling) replicative immortalization, (induced) angiogenesis, and (activated) invasion and metastasis.

[0086] Disease hallmarks, parameters (e.g., biomarkers) associated with the hallmarks, one or more biological activities associated with the hallmarks, etc., provide a framework for studying a disease or pathological or medical condition using an integrated / holistic approach.

[0087] Hallmarks of an altered physiological state typically include observable changes in various aspects of bodily function, which may be manifested through symptoms, signs, or laboratory findings.

[0088] Altered physiological conditions typically reflect a disruption of the body's homeostatic mechanisms, resulting in deviations from normal physiological parameters. These imbalances may include changes in temperature regulation, fluid and electrolyte balance, acid-base balance, glucose metabolism, or other regulatory processes.

[0089] Overall, the hallmarks of altered physiological states provide valuable clues for healthcare providers to identify underlying causes, assess severity, and guide appropriate interventions to restore normal function and promote recovery.

[0090] A reference drug is a drug that is commonly selected or chosen as the standard or preferred treatment for a particular medical condition or disease. It is often established based on factors such as its effectiveness, safety profile, cost, and clinical experience. A reference drug serves as a benchmark for comparison with other drugs, especially when evaluating generic versions, new treatments, or alternative therapies. It is typically the first-choice drug recommended by medical guidelines or healthcare providers to treat a particular condition.

[0091] Native natural intelligence represents the inherent ability of natural matrices to store and transmit the biological and physicochemical information necessary to interact with and integrate with other biological networks, using logic that is already known and therefore endogenous to the receiving organism. This intelligence is a manifestation of natural autopoiesis, i.e., the ability to self-organize and adapt to environmental stimuli without artificial intervention, transmitting messages according to a punctate logic and via mediators that are unknown to the receiving organism and therefore exogenous to it.

[0092] The expression physiological interaction is defined as an "endogenous" physiological interaction, which refers to the ability of a natural matrix to harmoniously and functionally interact with the recipient biological system and stimulate internal (endogenous) responses to restore a balanced physiological state. This interaction is based on natural dynamics without artificial intervention and represents a reciprocal dialogue between the matrix and the organism, promoting self-regulation and physiological recovery.

[0093] Self-assembled entities in nature define complex systems composed of multiple components that spontaneously organize into functional structures through chemical-physical interactions that occur in natural environments and conditions. These systems, found in living organisms or natural matrices, exhibit properties that arise from their dynamic interactions and cannot be artificially replicated.

[0094] When referring to a subject in need of beneficial or therapeutic treatment, the description relates to a human suffering from a pathological condition or in a condition (e.g., age, weight, sex, etc.) that puts them at risk for developing a pathological condition.

[0095] Detailed Description of the Invention The present invention relates to novel compositions, or products, consisting of 100% natural products, that exert a therapeutic or beneficial effect in the treatment of altered bone metabolism and / or bone pathologies, which products have a physiological (as opposed to pharmacological) mechanism of action.

[0096] Indeed, to act via a physiological mechanism of action, a product must be 100% natural. Products containing or consisting of natural materials, such as natural matrices, are entities that at least partially maintain the self-generating properties of their starting materials belonging to the biological domain and exhibit unique properties represented by a network of material and non-material relationships (inter-network interactions) that interact with the network of relationships of the treated subject, thereby reproducing physiologically similar features and interactions with complexity.

[0097] Therefore, according to this specification, a product comprising or consisting of one or more natural matrices is a product that is 100% natural, which means that the product does not contain any additional artificial substances, i.e. chemically synthesized substances made by man through laboratory processes.

[0098] Furthermore, according to the present specification, a product comprising one or more natural matrices also does not contain added isolated molecules, such as excipients or active ingredients, even if of natural origin.

[0099] Natural materials are fundamentally different from "materials," including substances of natural origin. Because they are not represented by their individual components, they require dedicated models. Therefore, to describe natural materials, it is necessary to extend reductionist approaches and use innovations from the last century. Conceptually, this refers to systems theory. From an experimental perspective, preclinical evidence includes systems biology approaches such as omics science (e.g., transcriptomics) and bioinformatics evaluation.

[0100] These allow a proper assessment of the matrix (the acting network) and the human body (the receiving network), and allow the interaction between the two to be considered as a "network on a network" interaction. The mechanisms that involve the coordinated redundancy and resilience that characterize physiological functions in each specific situation correspond to "physiological mechanisms of action" and can be characterized by network paradigms that are distinct from targeted and non-targeted models that describe PhIMs and mechanical / chemical / physical mechanisms, respectively.

[0101] In particular, according to the present invention, the natural matrix is a 100% natural and biodegradable material consisting of natural components that are not modified by the process for producing the matrix from the starting materials, without the intentional addition of synthetic products along the entire process.

[0102] As already mentioned, it is essential that the matrix be obtained through a non-denaturing process so that the components of the matrix are not artificially modified; however, if desired, the presence of additional indicators of the maintenance of characteristics present in the original raw materials can be verified. Furthermore, a 100% natural product is one that is expected to be 100% biodegradable. Herein, 100% biodegradability is considered to be "readily biodegradable" according to the OECD biodegradability test. These characteristics ensure the maintenance of the matrix effect imparted to the matrix by the structural interactions (material interactions) of its components and the presence of functional interactions (non-material interactions) that become apparent upon exposure of a biological system to the natural matrix.

[0103] The present invention relates to the products defined in the table below. [Table 2]

[0104] In one embodiment of the invention, the product has the following formulation: [Table 3]

[0105] In a further embodiment, the product has the following formulation: [Table 4] [Table 5] [Table 6]

[0106] In one embodiment, the horsetail is Equisetum Arvense, and / or the acacia is Acacia senegal, and / or the malpighia is Malpighia punctifolia, and / or the coral is Caribbean coral, and / or the bird is Gallus gallus, and / or the cetaria is Cetaria islandica.

[0107] Preferably, the horsetail is Equisetum Arvense, the acacia is Acacia senegal, the malpighia is Malpighia punctifolia, the coral is Caribbean coral, the bird is Gallus gallus, and the cetaria is Cetaria islandica.

[0108] The calcium citrate in the product of the present invention is calcium citrate obtained by the spontaneous reaction that occurs when coral skeletons are exposed to lemon juice. Specifically, the calcium citrate is produced by diluting lemon juice 1:1 (by volume) with water, adding coral powder, mixing for 5 hours, and then freeze-drying.

[0109] According to one aspect of the invention, it consists of six powders and three freeze-dried extracts, preferably in dry form, in w / w % units as shown in the table above.

[0110] The powders are calcium carbonate from coral, calcium carbonate from eggshell, fine powder from Agave leaves, fine powder from Cetaria thallus, fine powder from Agaricus, and gum arabic, acacia, and are mixed together at room temperature until the mixture is homogenous.

[0111] The freeze-dried extracts are from Equisetum flower spikes, acerola, Malpighia fruit and calcium citrate (as defined above).

[0112] For preparation, the homogenous mixture of powders is then mixed together with the freeze-dried extract at room temperature until the mixture is homogenous.

[0113] If the product is prepared in tablet form, the bulk mixture is pressed by direct compression to obtain the final product tablets.

[0114] In a preferred embodiment, the powder is calcium carbonate derived from coral, calcium carbonate derived from Caribbean coral, calcium carbonate derived from eggshell, fine powder derived from Agave sisalana leaves, fine powder derived from Cetaria islandica thallus, fine powder derived from champignon mushrooms, Agaricus bisporus and gum arabic, Acacia senegal, and the freeze-dried extract is freeze-dried extract of Equisetum arvense inflorescence, acerola, Malpighia punctifolia, and calcium citrate.

[0115] The solvent used in the preparation of the extract of the present invention is water, preferably purified water produced from drinking water by industrial water treatment plants.

[0116] Aqueous extracts of plant materials are known to those skilled in the art. Non-limiting examples of extract preparations that can be applied mutatis mutandis to acerola are as follows: Freeze-dried extract of Equisetum arvense flower spikes: Dried Equisetum arvense flower spikes were extracted with 100% water (v / v) [drug-solvent ratio: 1 / 18] at 70°C for 2 hours and filtered to remove solid waste. The resulting clarified extract was concentrated under vacuum to a concentration factor of 10:1 (v:v, initial extract volume compared to the volume after evaporation) and then freeze-dried for 72 hours. The resulting extract was stored at room temperature, away from light and moisture, until use.

[0117] According to the present invention, any possible embodiment of the product disclosed herein can be formulated into a composition comprising one or more carriers, such as water or other suitable carriers, depending on the desired final form. In one embodiment, the one or more carriers can be of pharmaceutical grade.

[0118] By way of example only, compositions may be formulated for oral, topical, rectal, vaginal administration, systemic injection, and microneedle injection.

[0119] By way of non-limiting example, the products or compositions disclosed or claimed herein may be prepared in the form of lyophilisates, tablets, soft or hard gelatin capsules, powders, granules, filled vesicles, filled liposomes, filled nanoparticles.

[0120] In accordance with the present invention, the products or compositions disclosed and / or claimed herein are for beneficial or medical use, such as for use in treating or assisting in the treatment of a bone fragility condition in a subject in need thereof.

[0121] In fact, as mentioned in the summary of the invention, the Applicant's in vitro studies have made it possible to compare the efficacy profile of the mixture of the invention with a treatment with synthetic calcium and vitamin D alone and to evaluate its ability to induce the differentiation of mesenchymal stem cells (hADMSCs) isolated from adipose tissue of patients into mature osteoblasts (increased alkaline phosphatase, ALP, activity) able to mineralize the cellular matrix (increased deposition of HA crystals) and counteract bone fragility.

[0122] Treatment with vitamin D, together with the osteoinductive stimulus provided by osteoinduction medium (OM), showed a statistically greater increase in ALP activity compared to its control (OM+DMSO+Ca), indicating a possible synergistic effect with OM and confirming its ability to induce stem cell differentiation into osteoblasts (Figure 5).

[0123] Despite the increased activity, vitamin D is unable to induce the correct stimulus for functional calcification (Figure 6).

[0124] Even treatment with synthetic calcium in osteoinduction medium showed an increase in ALP activity (Figure 5), with a typical "bell-shaped" trend characterized by a peak in enzyme activity levels at 28 days of treatment, and, unlike treatment with vitamin D, led to the correct formation of extracellular matrix and substantial hydroxyapatite (HA) deposits (Figure 6).

[0125] Similar to treatment with vitamin D and synthetic calcium, treatment with the mixture of the present invention together with osteoinductive stimulation was also able to promote an increase in ALP activity (Figure 5), showing a characteristic bell-shaped trend with a peak in activity at 21 days of treatment, thus indicating an early stimulation of the differentiation process compared to OM medium supplemented with synthetic calcium alone (synthetic calcium).

[0126] In addition to demonstrating the ability to mediate the commitment of mesenchymal cells to the bone lineage, the product of the present invention is able to induce the mineralization process in an early and complete manner, promoting the deposition of hydroxyapatite (HA) crystals (Figure 6). Indeed, after 28 days of treatment, the mixture of the present invention induces a statistically significant increase in hydroxyapatite crystals compared to synthetic calcium.

[0127] The mixture of the present invention not only acts as a support for stimulating differentiation into osteoblasts in the presence of osteoinductive stimuli and as a calcium donor capable of mineralizing bone extracellular matrix, but also produces a statistically greater increase in ALP activity in GM medium without osteoinductive agents compared to synthetic calcium, suggesting that it alone can induce the differentiation of mesenchymal cells into mature osteoblasts (Figure 7). In contrast, treatment with calcium and vitamin D did not determine an increase in ALP activity, confirming that it cannot induce cell differentiation in the absence of osteoinductive stimuli.

[0128] Consistent with what is already known, treatment with vitamin D and synthetic calcium, although basic, interprets the actual needs of the organism in a non-exhaustive way and inserts itself into the metabolic pathways of the individual in a timely manner without achieving satisfactory results. In fact, they are unable to reproduce the physiological differentiation of mesenchymal stem cells into osteoblasts to support bone formation.

[0129] Vitamin D certainly contributes, but alone cannot provide the differentiation stimulus and does not directly provide the material in the form of calcium to promote the formation of mineralized bone matrix (Figure 8).

[0130] Synthetic calcium alone cannot intervene in the differentiation process and can only provide the material for bone formation (Figure 8).

[0131] The mixture of the invention alone is able to reproduce the entire physiologically active process (Figure 8).

[0132] The molecular mechanism underlying the activity of the mixture of the present invention, which induces correct metabolic stimulation at a reduced phenotypic level in increasing ALP and HA, was analyzed by evaluating the regulation of gene expression in mesenchymal stem cells after treatment with the mixture of the present invention together with differentiation stimulation. This product induces significant effects in vitro on stem cells, which are precursors of both osteocytes and adipocytes, and promotes the clear differentiation of mesenchymal stem cells into mature osteoblasts in bone, which can mineralize extracellular matrix. As a result, with respect to adipose tissue, the mixture of the present invention promotes fat loss both locally and systemically in bone, and reduces the tendency of mesenchymal stem cells to differentiate into mature adipocytes. The synergistic effect of these actions leads to a decrease in bone fragility and an increase in bone quality due to the induction of beneficial effects at both ends of the fat / bone axis.

[0133] As can be observed in the heat map (Figure 10), treatment with the mixture of the present invention determines a modulation of the expression profile underlying bone remodeling, bone resorption and bone loss, in addition to an increase in mineralization in terms of increased osteoblast differentiation and increased bone mineral density.

[0134] In particular, the product of the present invention promotes osteoblast differentiation through the modulation of certain proteins related to bone formation, such as the induction of RUNX2 and the inhibition of sclerostin (SOST), which is known to have a function in the inhibition of osteoblast activity, consistent with what was also observed by analyzing the enzymatic activity of ALP.

[0135] The product also induces an increase in bone synthesis markers such as osteocalcin (OCN) and BMP (bone morphogenetic proteins), which underlie increased osteoblast activity and the formation of new bone matrix, consistent with what was observed by measuring the concentration of HA crystals.

[0136] The reduction of bone resorption process induced by the mixture of the present invention plays a fundamental role in the maintenance and repair of bone itself, promoting the rebalance of bone density and structure. In particular, the mixture of the present invention promotes the reduction of the expression level of sclerostin (SOST), which is known to have a function in inhibiting osteoblast activity, thus enhancing bone formation.

[0137] Furthermore, the inventive mixture demonstrated a systemic effect of metabolic regulation, inducing the expression levels of osteocalcin (OCN) (Figure 10), a hormone also involved in regulating insulin metabolic assets. Osteocalcin is expressed and secreted by mature osteoblasts and acts by stimulating insulin secretion from pancreatic beta cells and promoting insulin sensitivity in muscle and white adipose tissue, resulting in lower blood glucose levels.

[0138] The results obtained also demonstrate the potential of the mixture of the invention to reduce both the amount of adipose tissue and the inflammatory process, suggesting an anti-adipogenic effect and improved tolerance, thus identifying further desirable effects in the physiopathological framework of interest.

[0139] Thus, the local and systemic effects of the mixture of the present invention can enter into a dysfunctional loop established in conditions of systemic inflammation and metabolic dysregulation that prevents the differentiation of mesenchymal stem cells into white adipocytes and their consequent accumulation.

[0140] In other words, thanks to its emerging properties, the inventive mixture is able to use network mechanisms to interact with established physiological metabolic pathways in the body of menopausal women or women facing this transition. The product not only provides the calcium necessary for the precise deposition of hydroxyapatite crystals in the bone matrix, but also precisely stimulates the differentiation of mesenchymal stem cells, promotes the development of osteoblastic cell lines, inhibits the formation of osteoclasts and adipocytes, and thus helps establish a favorable balance in bone metabolic processes. Furthermore, the improved secretory profile of osteocalcin suggests beneficial effects at the systemic level in organs such as the pancreas, muscle tissue, and adipocytes.

[0141] This dual synergistic and systemic effect of the product is highly desirable compared to commonly used classic calcium and vitamin D supplements, which can only partially support the physiological changes of an organism. Instead, this support is supported by a mixture of the present invention and physiological mechanisms, i.e., interacting with the body according to a known canon, rather than an external canon imposed by exogenous entities such as synthetic molecules. This is because the present mixture, in addition to containing both a vitamin D precursor and a calcium source, interacts with the mesenchymal stem cell pool in bone and adipose tissue, recreating all the elements necessary to rebalance the correct bone turnover, which is useful for the formation of solid and functional structures. Furthermore, the product's effects reach a systemic projection, helping to rebalance metabolic disorders and the underlying inflammation that afflicts perimenopausal women on a systemic scale. Thanks to the presence of a complex plant matrix and natural calcium from various sources, the present mixture clearly demonstrates how the complexity of physiological processes can only be properly supported according to a known canon when placed in the context of a 100% natural therapeutic solution of a similarly highly complex nature.

[0142] Accordingly, the present invention also relates to a product or composition as defined and / or claimed herein for use in treating or aiding in the treatment of a bone fragility condition, wherein the subject is at risk of developing the bone fragility condition.

[0143] According to the present invention, the subject is a human having a fragile bone condition or at risk of developing a fragile bone condition.

[0144] Since bone metabolism is also strictly interlinked with fat metabolism, as explained above, in certain aspects of the invention, the bone fragility is associated with adiposity in the subject.

[0145] Such fat gain is intended to be significant fat gain, ie, fat gain that is beyond the normal weight fluctuations that occur on average in such subjects.

[0146] Typical triggers of fat gain and bone fragility may be associated with obesity, metabolic syndrome, pre-, perimenopause or menopause, and andropause, by way of example.

[0147] Furthermore, according to the present invention, the bone fragility condition can be selected from osteoporosis and osteopenia.

[0148] In extensive characterization and research activities regarding the products of the present invention, applicants have also identified the traditional hallmark of osteoporosis and related biological activity, where modulation (up and down regulation) underlies the pathology, and defined it as a tendency of modulation of said activity toward a healthy physiological state, as opposed to the modulation observed in an altered or pathological state (see Figure 9).

[0149] The applicant also confirmed that all of the desired modulation of the above activities was achieved using the product of the present invention, thereby verifying the therapeutic efficacy of the product (Figure 10). Furthermore, the applicant compared the modulation of the above activities exerted by the product of the present invention with that exerted by a reference drug used to treat osteoporosis, such as Dibase (vitamin D 10,000 U.I. / ml), and found that the reference drug was unable to reduce bone remodeling, alleviate bone loss, induce osteoblast differentiation, restore mineralization, or reduce inflammation, and could only partially reduce adipose tissue. Therefore, overall, administration of vitamin D does not alter the biological activities underlying the pathology of osteoporosis, with a modulation trend toward a healthy physiological state (Figure 11).

[0150] Finally, the inventors have also surprisingly found that different batches of the product of the present invention (see Product C Batches 1, 2 and 3 in the Examples), despite their qualitatively and quantitatively different chemical compositions, exhibit therapeutic / beneficial functional resilience by modulating the above biological activities in the same pattern and with similar modulation values.

[0151] While different batches of a product containing one or more natural matrices are, by definition, necessarily variable in qualitative and quantitative composition, as discussed fully above, according to one embodiment, qualitative and / or quantitative analysis of each batch was performed to demonstrate the existing qualitative and / or quantitative differences in the molecular composition of each batch (Figure 13). This can be achieved by conventional techniques, non-limiting examples of which include chromatography, spectrophotometry, atomic absorption spectroscopy (AAS), atomic emission spectroscopy (AES), inductively coupled plasma (ICP) techniques, chromatography coupled with a detector, etc., or combinations thereof. The analysis can focus on a limited number of selected classes of substances (e.g., Figure 13) or on all components of the product.

[0152] Although each batch was prepared according to a standardized procedure to obtain a high degree of uniformity between different batches, as would be expected for a product containing or consisting of a natural matrix, a detailed qualitative-quantitative analysis of all tested batches clearly showed relevant batch-to-batch qualitative-quantitative differences that would not allow the traditional validation methods used for synthetic or isolated drugs to assume the presence of API, as well as the discarding of batches that were in fact therapeutically active.

[0153] It is noted again that natural matrices, by their very nature, are variable in composition even when obtained from the same type of raw material: for example, those skilled in the art are well aware that a natural matrix obtained from an individual plant species will never be identical to another natural matrix obtained from a different individual plant species, even from the same field of plants, due to the genetic and epigenetic variability of each organism.

[0154] Following the experiments reported in Figure 13, it was surprisingly found that, although all analyzed batches differed in qualitative and quantitative chemical composition, in all tested batches the different molecular entities within each matrix appeared to interact with each other functionally and possibly structurally in a redundant manner, providing the same therapeutic or beneficial (homeostatic-adjuvant) effect despite the differences in their qualitative-quantitative molecular composition.

[0155] Indeed, the batches exhibited functional resilience (therapeutic or beneficial) despite variability in their qualitative and quantitative molecular composition.

[0156] In other words, Applicant surprisingly found that different batches of the same product exhibited consistent regulation (in terms of trend and magnitude) of all tested biological activities related to the desired therapeutic or beneficial effect, despite batch-to-batch differences in qualitative and quantitative composition, also defined herein as "functional resilience." The observed maintenance of biological activity may be attributed to the fact that, as noted above, the emerging properties of the natural matrix result from the matrix network acting as a whole entity with characteristic properties and cannot be attributed to each single molecule as if it were isolated. The therapeutic effect is due to a non-pharmacological mechanism of action that differs from classical therapeutic products based on a structure-activity pharmacological relationship (SAR), which is most relevant in classical pharmacological activities, considered at the single-molecule level, between the active pharmaceutical ingredient (API) and the receptor targeted by the API.

[0157] This is consistent with the possibility that the products analyzed by the inventors may exert their therapeutic or beneficial effects by acting on an overall pathophysiological or altered physiological state.

[0158] In accordance with the present invention, the defined and / or claimed products or compositions exert their therapeutic or beneficial effects through physiological mechanisms of action by assisting in the restoration of bone homeostasis through a network of biological activities in response to the altered physiological conditions underlying the bone fragility condition, and by exhibiting therapeutic or beneficial functional resilience between different batches of the product or composition, which functional resilience is intended to maintain the therapeutic or beneficial properties of different batches of the product or composition despite the qualitative and quantitative compositional differences between the batches.

[0159] In particular, due to all the inferences regarding natural matrices provided above, together with all the experimental data collected by the applicant, it can be said that the product or composition according to the present invention is itself a natural matrix that represents a native natural intelligence, which only has the ability to allow physiological, endogenous mutual association with other entities that self-assemble in nature, such as the human species.

[0160] We reiterate that native natural intelligence refers to the inherent capacity of natural matrices to store and transmit biological and physicochemical information necessary to interact with and integrate with other biological networks using logic specific to the receiving organism. This logic is already known and is endogenous in relation to it. This intelligence is a manifestation of natural autopoiesis, i.e., the ability to self-organize and adapt to environmental stimuli without artificial intervention. The manner in which it transmits messages follows a punctate logic and transmits through mediators unknown to the receiving organism. It is exogenous in relation to it.

[0161] According to the present invention, the presence of native natural intelligence in a therapeutic or beneficial product or composition, where the product or composition comprises or consists of a natural matrix, can be determined by verifying that the product or composition is a natural matrix with emerging properties (therapeutic or beneficial) if its 14C activity measured using the ISO-16620-2;2015 (AMS) method is 99.82±0.22% percent, if miRNA and exosomes are detected in the product or composition, and if the product or composition exhibits therapeutic or beneficial functional resilience between different batches of the product or composition and from batch to batch when the product or composition as a whole modulates an altered physiological or pathological condition.

[0162] This can be done with the product or composition of the present invention by subjecting a sample of the product or composition to the following steps: a. The natural character of the product or composition; 1. Measure 14C activity using the ISO-16620-2;2015 (AMS) method. 2. Assessing the presence of miRNA in the product or composition; 3. Assessing the presence of exosomes in the product or composition; b. assessing the presence of therapeutic or beneficial functional resilience between different batches of the product or composition by comparing the modulation of one or more biological activities underlying the desired therapeutic or beneficial effect of the product on altered bone metabolism and / or bone pathology from batch to batch in a cell-based assay where the readout represents modulation of one or more biological activities; c. assessing from the cell-based assay readout whether modulation of the biological activity underlying the desired therapeutic or beneficial effect results in modulation of an overall altered physiological state or disease state; below, The measured 14C activity was 99.82±0.22% percent; miRNA, exosomes, therapeutic or functional resilience are detected, and c. If said modulation results in an overall modulation of an altered physiological or pathological condition, then determining that the product or composition itself is a natural matrix representing native natural intelligence.

[0163] A product is determined to be natural if its 14C activity is 99.82±0.22%, miRNAs, exosomes are detected, and it exerts its activity through physiological mechanisms if it exhibits emerging properties that modulate conditions and demonstrate therapeutic or beneficial functional resilience. The sum of these characteristics allows the product to be determined as a natural matrix itself, thereby representing native natural intelligence.

[0164] Assessment of alterations in status and functional recovery can be performed as follows.

[0165] The ability of a therapeutic or beneficial product to modify a pathophysiological or altered physiological state (e.g., by supporting the homeostatic response of an organism) is a key feature for establishing its physiological mechanism of action. Indeed, products that act in network interactions are those that are expected to modify a state rather than a single function when administered to an organism.

[0166] In other words, this feature is likely to be met by a therapeutic or beneficial product that includes or consists of one or more natural matrices, taking into account the network-network (product-recipient) interactions exerted by the natural matrices. Applicant's patent application PCT / IB2024 / 055892 discloses a method for defining the mechanism of action of a therapeutic or beneficial natural matrix-based product.

[0167] Products exerting a physiological mechanism of action are also expected to be 100% natural (see above) and to exhibit the flexibility and self-regulation mechanisms observable in vivo, where different intracellular and intercellular messages and different regulation of gene pathways can provide the same result despite different messages being triggered within the cell. In the case of therapeutic or beneficial products, this corresponds to the functional resilience of different batches of the product (with qualitative-quantitative variability in chemical composition).

[0168] The inventors have determined whether the products of the present invention exert their therapeutic or beneficial effect by modifying a pathological or altered physiological (yet non-pathological) condition, and whether the products exhibit functional resilience (i.e., maintenance of the therapeutic or beneficial properties of different batches of a given product comprising one or more natural matrices despite differences in the qualitative and quantitative composition of the different batches).

[0169] According to the present invention, this can be verified by performing cell-based assays having readouts representative of modulation of a selected biological activity as defined above, for example as disclosed in the examples described herein, and by analyzing and interpreting the data obtained therefrom.

[0170] Assessment of (therapeutic or beneficial) functional recovery. As indicated in the glossary and above specification, in this specification and claims, functional resilience is the maintenance of measurable therapeutic or beneficial efficacy in different batches of product despite variability in their qualitative and quantitative molecular composition.

[0171] It is clear that the batches (e.g., tested batches 1, 2, and 3 of product C) are intended to be identical in terms of the manufacturing process and the type and amount of each ingredient (since the main ingredient of the selected product is a natural matrix, this means that each matrix in the product is manufactured from the same type of starting material in the same procedure, e.g., with a given type of extract from the same plant part of the same plant species), and therefore, variability in their qualitative and quantitative molecular composition cannot be attributed to different manufacturing procedures or different ingredients, but can only result from the inherent differences between natural matrices obtained in the same procedure from different organisms of the same species. The functional resilience of a product can be verified if different batches of the same product containing one or more natural matrices, regardless of their qualitative and quantitative composition, maintain in a measurable and verifiable manner their ultimate regulatory activity underlying their therapeutic or beneficial properties.

[0172] Accordingly, the present invention also relates to a method for determining the presence of a natural intelligence in a therapeutic or beneficial product or composition comprising or consisting of a natural matrix through validation of its therapeutic or beneficial emerging properties, the method comprising, on a sample of said product or composition:

[0173] a. The naturalness of the product shall be verified by measuring the 14C activity according to the ISO-16620-2;2015 (AMS) method. 2. Assessing the presence of miRNA in the product or composition; 3. Evaluating the presence of exosomes in the product or composition; b. assessing the presence of therapeutic or beneficial functional resilience between different batches of the product by comparing the degree of modulation of one or more biological activities underlying the desired therapeutic or beneficial effect of the product on the relevant altered physiological state and / or pathology treated by the product or composition from batch to batch in a cell-based assay whose readout represents the degree of modulation of said one or more biological activities; c. assessing from the cell-based assay readout whether modulation of the biological activity underlying the desired therapeutic or beneficial effect results in modulation of an overall altered physiological state or disease state; below, The measured 14C activity was 99.82±0.22% percent; miRNA, exosomes, therapeutic or functional resilience are detected, and c. If said regulation results in an overall regulation of the altered physiological or pathological condition, determining that the product or composition itself is a natural matrix representing native natural intelligence.

[0174] According to one aspect of the present invention, the method further includes, prior to performing one or more cell-based assays, (1) providing a list of hallmarks representing the altered metabolic and / or pathological state; (2) for each of the hallmarks, identifying alterations in one or more biological activities underlying the pathological state, thereby pinpointing a network of biological activities whose degree of regulation corresponds to the pathological state; and (3) identifying one or more parameters whose degree of regulation corresponds to the degree of regulation of the one or more biological activities underlying the therapeutic effect of the tested product, and determining a trend in regulation in the network for up- or down-regulation of the one or more biological activities that corresponds to the pathological or healthy state.

[0175] In a preferred embodiment, the altered physiological state is bone metabolism and / or the pathological condition is osteoporosis, and the hallmarks are selected from bone remodeling, osteopenia, osteoblast differentiation, bone mineralization, reduced inflammation, and adipose tissue reduction, and preferably, the biological activity of (2) on bone hallmark remodeling is selected from the biological activities shown in Figure 9.

[0176] In more detail, (1) Providing a list of hallmarks representing pathological conditions associated with the pathology that can be attributed to the pathology or the non-pathological altered physiological state, i.e., providing a list of hallmarks representing diseases or pathological conditions treated by the product of interest, or providing a list of characteristics that can be attributed to the non-pathological altered physiological state whose homeostasis is assisted by the beneficial product of interest; (2) for each such hallmark, identifying the alteration of one or more biological activities underlying the pathology, determining the degree of modulation thereof that represents a pathophysiological state associated with the pathology, and assessing the opposite degree of modulation of each such activity as a pattern of modulation that represents a healthy physiological state; and (3) identifying one or more markers and their regulation patterns underlying the detectable alteration in the pathological state for each of the one or more biological activities, and defining, for each of the parameters, the opposite regulation pattern to the identified one as the regulation pattern consistent with the healthy physiological state; is.

[0177] If a therapeutic product is being tested, according to one embodiment, the method of the present invention may comprise: (a) the following cell groups: (a1) at least one control group and at least two test groups of cells having a disease phenotype relevant to the intended use of the therapeutic product; or (a2) performing the at least one in vitro cell-based assay on at least one population of cells having a healthy physiological phenotype, and at least one control group and at least two test groups of the cells having the healthy physiological phenotype in which a disease phenotype associated with the intended use of the therapeutic product has been induced; treating each of the test groups of cells with one of the different batches of therapeutic product; (b) determining the degree or pattern of modulation of each of said parameters for each of said groups of cells of step (a) and calculating a respective modulation value for each of said one or more biological activities; (c) comparing the adjusted values, The therapeutic product: At least 50% of the one or more biological activities for each hallmark are modulated by each product batch such that the modulation trend of the network is consistent with a healthy state, and the modulation value determined in (b) for each of the at least 50% one or more biological activities for the test group of cells in (a1) is different from that of the control group of cells in (a1) by at least 0.15, respectively; or At least 50% of the one or more biological activities for each hallmark are modulated by each product batch such that the modulation trend of the network is consistent with a healthy state, and the modulation value determined in (b) for each of the at least 50% one or more biological activities for the test group of cells in (a2) is different from that of the control group of cells in (a2) by at least 15%, respectively; Comparing the modulation values, where the functional resilience of the product is demonstrated by the modulation value for each of one or more biological activities of each test group of cells differing from the average of the values by less than 20%.

[0178] This means that the modulation value of a given biological activity of the test group is respectively compared to the modulation value of the same biological activity of the control group, and therefore the difference between the modulation value of a given activity in the treated group of cells and the modulation value of the same activity in the group of cells representing the control baseline is at least 0.15 or at least 15%.

[0179] In other words, the method may also be described as a method for evaluating whether a therapeutic product exerts an effect in treating a pathological condition via a physiological mechanism of action, comprising: - providing different batches of a therapeutic product, the product comprising one or more natural matrices; - providing a list of hallmarks representative of said pathological conditions and identifying for each of said hallmarks a set of parameters allowing the assessment of the network of biological activities whose modulation is the basis for the therapeutic effect of the tested product, and determining the modulation trends in terms of up- or down-regulation of activity in the network in diseased and healthy states; (a) the following cell groups: (1) at least one control group and at least two test groups of cells having the disease phenotype targeted by the therapeutic product; or (2) performing at least one in vitro cell-based assay on at least one population of cells having a healthy physiological phenotype and at least one control group and at least two test groups of the cells having the healthy physiological phenotype in which a targeted disease phenotype is induced by the therapeutic product; treating each of the test groups of cells with one of the different batches of therapeutic product; (b) determining the degree or pattern of modulation of each of said parameters for each of said groups of cells and calculating a respective modulation value for each of said biological activities; (c) comparing the modulation values for each biological activity in each cell population of step (b), At least 50% of the biological activity for each hallmark is adjusted for each product batch using the healthy adjustment trend determined in (b) and the adjustment value determined in (b), and each of the at least 50% one or more biological activities for the test group of cells in (a)(1) differs from that of the control group in (a)(1) by at least 0.15, respectively; or comparing the modulation values of at least 50% of the biological activities determined in (b) with the healthy state modulation trend determined in (b) and each of the modulation values of at least 50% of one or more biological activities determined in (b) modulated by each product batch for the test group of cells in (a)(2), which, when each differ by at least 15% from that of the control group in (a)(2), indicate that the therapeutic product exerts its therapeutic activity via a physiological mechanism of action; The functional resilience of the product is demonstrated by the adjusted values for each of the biological activities of each test group of cells differing by less than 20% from the average of the adjusted values.

[0180] The expression "at least 50% of the one or more biological activities for each hallmark" means that the network's modulation trend is modulated by each product batch consistent with a healthy state, and for a single biological activity for a given hallmark, 100%, i.e., the single activity, must be modulated by the tested product to meet the above requirement, with a modulation trend consistent with a healthy state. The expression "modulation value of each of the at least 50% one or more biological activities determined in (b)" refers to the modulation value determined in (b) of at least 50% of the biological activities that meet the requirement of being modulated according to a modulation trend consistent with a healthy state. This applies mutatis mutandis to all aspects disclosed herein.

[0181] If a beneficial product is tested (i.e., a product that has a beneficial effect on an altered, but not yet pathological, physiological state by supporting homeostasis), according to one embodiment the method of the present invention may comprise: (a) the following cell groups: conducting at least one in vitro cell-based assay on at least one control group of cells having a healthy phenotype or at least one control group of cells in which the dysregulated phenotype targeted by the beneficial product has been suitably induced and at least two test groups of cells taken from the control group; treating each of the test groups of cells with one of the different batches of beneficial product; (b) determining the degree or pattern of modulation of each of said parameters for each of said groups of cells of step (a) and calculating a respective modulation value for each of said one or more biological activities; (c) comparing the adjusted values, The beneficial product is demonstrated by at least 50% of the one or more biological activities for each hallmark being modulated with each product batch such that the network's modulation trend is consistent with a healthy state, the modulation value calculated in (b) for each of the at least 50% one or more biological activities for the test group of cells differs from that of the control group of cells by at least 0.15, respectively, and the functional resilience of the product is demonstrated by the modulation value for each of the one or more biological activities for each test group of cells differing from the average of such values by less than 20%. In some cases, it has been shown to exert its homeostatic support effects through physiological mechanisms of action. Comparing the adjustment values.

[0182] This means that the modulation value of a given biological activity of the test group is respectively compared to the modulation value of the same biological activity of the control group, and therefore the difference between the modulation value of a given activity in the treated group of cells and the modulation value of the same activity in the group of cells representing the control baseline is at least 0.15 or at least 15%.

[0183] In other words, the method is a method for assessing whether a beneficial product exerts its pro-homeostatic effect by regulating an altered physiological state, and may be defined as: - Providing different batches of a beneficial homeostasis-supporting product, the product comprising one or more natural matrices. - providing a list of hallmarks representing pathological conditions that may be attributed to said altered physiological state, and for each of said hallmarks, identifying a set of parameters that allow the assessment of a network of biological activities whose modulation underlies the therapeutic effect of the tested product, and determining the modulation trends in terms of up- and down-regulation of said activities in said network in disease and healthy states; (a) the following cell groups: (1) performing at least one in vitro cell-based assay on at least one control group of cells having a healthy phenotype or at least one control group of cells in which the dysregulated phenotype targeted by the beneficial product has been suitably induced and at least two test groups of cells taken from the control group; treating each of the test groups of cells with one of the different batches of beneficial product; (b) determining the degree or pattern of modulation of each of said parameters for each of said groups of cells of step (a) and calculating a respective modulation value for each of said biological activities; (c) comparing the modulation level for each biological function in each cell population of step (a), A useful product comparing at least 50% of the biological activity for each hallmark to the healthy state modulation trend determined in (b) and each modulation value of at least 50% determined in (b) indicates that the test group of cells in (a)(1) exerts an effect in supporting homeostasis via a physiological mechanism when each of the one or more biological activities for the test group of cells in (a)(1) differs from that of the control group in (a)(1), by at least 0.15; The functional resilience of the product is demonstrated by a modulation value for each of the biological activities of each test group of cells that differs from the mean of the values by less than 20%.

[0184] In a preferred embodiment, the control group is considered a reference adjusted baseline and the qualitative-quantitative adjustment value for each of the one or more biological activities is considered to be zero.

[0185] As previously mentioned, the method of the present invention includes: (1) providing a list of prominent hallmarks representative of the pathological condition of interest (i.e., a pathological condition that is treated by the analyzed product or that can result from an altered physiological state for which the analyzed beneficial product exerts its homeostatic supporting activity); (2) for each of the hallmarks, identifying the alteration of one or more biological activities underlying the pathological condition, thereby pinpointing a network of biological activities whose degree of modulation corresponds to the pathological condition; and (3) identifying one or more parameters whose degree of modulation corresponds to the degree of modulation of the one or more biological activities underlying the therapeutic effect of the tested product, and determining a modulation trend in the network, with respect to an up- or down-regulation of the one or more biological activities that corresponds to the pathological or healthy state.

[0186] In all of the above aspects, it is possible to determine whether a therapeutic or beneficial product exerts its therapeutic or beneficial effect by modifying a condition or a limited number of activities, or even a single function, underlying the pathology that is the abnormal physiological condition being treated by the product, and whether the therapeutic or beneficial product, as selected, maintains functional resilience as defined herein.

[0187] Modification of state is a feature not obtainable with a single API pharmaceutical product and therefore excludes the classical pharmaceutical mechanism of action, however, modification of state can in principle also be obtained with pharmaceutical products containing a cocktail of APIs.

[0188] A physiological mechanism of action requires that a therapeutic or beneficial product regulates a condition in a manner that involves the overall cellular response with the network through network interactions, rather than the point of network interaction based on the API mechanism of action (whether a single API or a cocktail thereof).

[0189] This refers to the ability of the product to act in functional recovery (either therapeutic or beneficial) in addition to regulating the condition, i.e., to provide the same therapeutic / beneficial effect from batch to batch despite differences in qualitative-quantitative composition from batch to batch, in other words, to regulate selected different parameters in a variable manner and nevertheless provide a preserved functional result.

[0190] Thus, the present invention also provides an embodiment for demonstrating the functional resilience of a therapeutic or beneficial product.

[0191] As already explained in the terminology section and above, (therapeutic or beneficial) functional resilience is the ability of a given product to modulate one or more biological activities underlying a pathological or altered physiological state, despite variations in the qualitative and quantitative composition of different batches of the same product, and thus eliciting different signals within the cell, despite the possibility of reaching the same end result, i.e., bioequivalence, intended as the same end result. As already mentioned above, it is known that pharmaceutical (API-based) products with different qualitative and quantitative compositions are not considered bioequivalent.

[0192] Physiological mechanisms of action refer to the overall interaction with the cells of a treated subject, not to interactions with specific cellular molecular targets, but to the mode exerted by an organism as a result of network-network interactions. Physiological systems within the body often exhibit functional redundancy to maintain homeostasis and adapt to changes or disruptions. Redundancy is a well-known physiological mechanism in life to ensure a given goal is reached (e.g., the organism's response in producing various proteins, activating various pathways, etc.). When therapeutic products interact with these systems, they may engage multiple pathways or mechanisms, including redundant pathways or mechanisms, to achieve their desired effect. This redundancy, resulting in functional resilience, contributes to the product's physiological mechanism of action.

[0193] Therefore, functional resilience in a therapeutic / beneficial product (defined as the ability of a therapeutic or beneficial product to maintain its intended functionality and effectiveness despite variability in its qualitative and quantitative composition from batch to batch) is an essential feature of a physiological mechanism of action.

[0194] The present invention also relates to a method of treating or assisting in the treatment of a bone fragility condition, mutatis mutandis, by administering to a subject in need thereof a product or composition according to the present invention, alone or in combination with a therapeutically effective amount of a beneficial active ingredient.

[0195] The following examples are intended to better explain and scientifically support the present invention, but are not intended to limit the scope of the present invention.

[0196] example 1. Composition of the test preparation The experimental data reported below was generated using the following formulation of a product of the present invention, also referred to below as "Product C." Coral calcium powder 32% by weight Eggshell calcium powder 30.2% by weight Coral calcium citrate powder 13% by weight Agaricus bisporus powder 4.65% by weight Dry extract of Equisetum arvense flower spikes 2% by weight Malpighia punicifolia 2% by weight Cetraria islandica powder 2% by weight Agave sisalana leaf powder 12% by weight Acacia Senegal powder 2.15% by weight

[0197] Other formulations of the product of the present invention within the claimed range have been tested with similar results (data not shown).

[0198] Three batches of Product C were prepared using the same formula but starting from different lots of raw plant or natural material, namely Batch 1, Batch 2 and Batch 3.

[0199] Additionally, permutations of the above formulations within the scope of claim 1 have also been prepared, and preliminary data have confirmed their correct biological activity in cell-based assays as reported below (i.e., biological activity comparable to that of Product C).

[0200] Coral-derived calcium carbonate powder, Caribbean coral-derived calcium carbonate, eggshell-derived calcium carbonate, Agave sisalana leaf fine powder, Cetaria islandica thallus fine powder, champignon mushroom fine powder, Agaricus bisporus and gum arabic, Acacia senegal were mixed at room temperature until the mixture was uniform, and then freeze-dried extract of Equisetum arvense flower spikes, acerola, Malpighia punctifolia, and calcium citrate were mixed and added at room temperature in the weight-to-weight percentages disclosed above.

[0201] The solvent used for the preparation of the extract of the present invention was purified water from an industrial water treatment plant produced from drinking water.

[0202] Freeze-dried extracts were prepared by subjecting each plant material to extraction with 100% (v / v) water for 2 hours at 70°C and filtering to remove solid waste material. The resulting clarified extract was concentrated under vacuum until a concentration factor of 10:1 (v:v, initial extract volume compared to the volume after the evaporation step) was reached, and then freeze-dried for 72 hours. The resulting extract was stored at room temperature, away from light and moisture, until use.

[0203] The products thus prepared were then appropriately dissolved and / or diluted where appropriate for the various assays disclosed below.

[0204] 2. In Vitro Assay In vitro studies were conducted on adipose tissue-derived human pre-osteoblastic mesenchymal stem cells (hADMSCs). These cells represent a convenient, easily obtainable (non-invasive) source of mesenchymal stem cells that, if properly induced, can differentiate into osteogenic lineages. In vitro studies have shown that when appropriately induced by culture in a specific osteogenic induction medium (osteogenic / osteoinductive medium; OM) containing 10 nM dexamethasone, 0.2 mM ascorbic acid, and 10 mM β-glycerophosphate, along with calcium and phosphate sources, hADMSCs can differentiate into mature, active osteoblasts and produce mineralized bone matrix. Therefore, these cells are an ideal model for studying osteoblast differentiation and functionality, the mineralization process, and evaluating the effects of various substances on these two processes.

[0205] To evaluate the effects of product C, synthetic calcium, and vitamin D on bone metabolism, in vitro experiments were performed by treating hADMSCs for different experimental times (4, 7, 14, 21, 28, and 35 days of treatment).

[0206] The evaluation items focused on in the experiment were as follows: the ability to provide bioavailable calcium ions for mineralization of the extracellular bone matrix (measurement of hydroxyapatite (HA) crystals); the potential to induce and / or enhance osteoblast differentiation (spectrophotometric assay of alkaline phosphatase (ALP) activity); Transcriptional profiling (gene expression using a microarray platform) induced in cells to assess pathways and biological functions potentially affected by the treatment

[0207] The differentiation potential of the samples was evaluated under two different osteoinductive stimulation conditions: non-osteoinductive medium (GM) and osteoinductive medium (OM).

[0208] Non-osteoinductive medium GM: (DMEM medium with antibiotics / calcein / serum plus β-glycerophosphate and 2-phosphoascorbic acid but without dexamethasone): This medium does not contain dexamethasone (an external osteoinductive agent) and therefore allows the evaluation, through measurement of ALP activity, of whether the added formulations are able to independently induce osteogenic differentiation.

[0209] Osteoinductive medium OM: (DMEM medium containing antibiotics / calcein / serum + β-glycerophosphate and 2-phosphoascorbic acid and dexamethasone): This medium contains dexamethasone (an external osteoinductive agent) and allows for the evaluation of whether the formulation may have a synergistic (or conversely, inhibitory) effect on osteoblast differentiation induced by dexamethasone.

[0210] The experimental model involved the addition of calcium to the culture medium at a concentration (1.4 mM) that reflects the physiological amount of calcium in the body. To achieve this condition, in treatments containing vitamin D (solubilized in DMSO due to its hydrophobicity) and calcium, appropriate amounts of synthetic calcium were added to both culture media (OM and GM).

[0211] hADMSC cell lines were cultured in 100 mm Petri dishes at 37 °C in a humidified atmosphere containing 5% CO2 in growth medium (GM) [Ham's F12 Coon's modified medium supplemented with 10% FBS, 100 IU / mL penicillin, and 100 g / mL streptomycin]. The medium was replaced with fresh GM every 3 days. Upon reaching confluence, cells were detached by trypsinization and seeded into 24-well plates at a cell density of 1 x 104 cells / cm2 in GM until they reached 70-80% confluence. The medium was then replaced with osteogenic medium (OM) or maintained in GM supplemented with treatment and incubated for 7-35 days. Treatment medium was refreshed twice a week.

[0212] These cells were obtained with informed consent from three different patients (PA42, PA59, and PA69) during general surgery (Romagnoli et al., “In Vitro Behavior of Human Adipose Tissue-Derived Stem Cells on Poly(ε-caprolactone) Film for Bone Tissue Engineering Applications,” BioMed Research International, vol. 2015, Article ID 323571, 12 pages, 2015. https: / / doi.org / 10.1155 / 2015 / 323571). These cell lines have been characterized for key stemness markers of mesenchymal stem cells (CD44, CD105, and STRO1) and by studying their multipotency towards an osteogenic phenotype at the Department of Surgery and Translational Medicine of the University of Florence.

[0213] The time schedule used in the experimental setup is as follows: [Table 7]

[0214] 2.1 ALP assay and calcium mineralization assay (Figures 5 to 7) At the end of each incubation time point, cells were washed with DPBS (twice), fixed with 4% PFA / DPBS for 15 min, washed with ultrapure water (three times), dried, and stored at 4°C until assayed. Each experimental point was performed in quadruplicate.

[0215] ALP assay Each well was incubated with 500 μL of 4-methylumbelliferyl phosphate in 280 mM Tris-HCl buffer, pH 9.0, for 15 minutes at 37°C. The reaction was stopped by adding 2 mL of 0.1 M NaOH. ALP activity was measured using a spectrofluorometer LS55 (PerkinElmer) at 365 nm λ excitation and 445 nm λ emission, expressed in μU / cm using a standard curve of 50 nM to 10 μM 4-methylumbelliferone in 280 mM Tris-HCl buffer, pH 9.0. 2 It is expressed as:

[0216] Calcium mineralization deposit assay.

[0217] Each well was incubated with 2 mL of 50 mM NaEDTA for 30 min at 37 °C. The solution was then transferred to a cuvette, and fluorescence was measured using a spectrofluorometer LS55 (PerkinElmer) at 494 nm λ excitation and 517 nm λ emission and expressed in μg / cm2 using a standard curve of 25 ng / mL to 500 μg / mL of calcium mineralized deposits solubilized in 50 mM NaEDTA.

[0218] In vitro studies made it possible to compare the efficacy profile of Product C with treatment with synthetic calcium alone and vitamin D alone by evaluating its ability to induce the differentiation of mesenchymal stem cells (hADMSCs) isolated from patients' adipose tissue into mature osteoblasts (increased ALP activity), which can mineralize the cellular matrix (increased deposition of HA crystals) and counteract bone fragility.

[0219] Treatment with vitamin D in combination with osteoinductive stimuli (OM) showed a statistically greater increase in ALP activity than its control (OM + DMSO + Ca), indicating a possible synergistic effect with OM and confirming its ability to induce stem cell differentiation towards osteoblasts (Figure 5).

[0220] Treatment with synthetic calcium also showed an increase in ALP activity in osteoinductive media, with a typical "bell-shaped" pattern characterized by a peak in enzyme activity levels at 28 days of treatment (Figure 5).

[0221] Similar to vitamin D and synthetic calcium treatment, product C treatment in combination with osteoinductive stimulation was also able to promote an increase in ALP activity, showing a characteristic bell-shaped pattern with peak activity at 21 days of treatment, thus indicating an early stimulation of the differentiation process compared to OM supplemented with synthetic calcium alone (Figure 5).

[0222] The bell-shaped curves observed in all groups (Figure 5) reflect the dynamic nature of ALP activity during osteogenic differentiation, with an increase corresponding to early matrix maturation and a decline as cells transition to later stages of osteogenesis. In the case of Product C, the earlier peak in ALP activity highlights its ability to predict key stages in the physiological processes of differentiation and mineralization. This early response promotes the early stages of osteogenesis, suggesting a role consistent with the physiological progression of osteogenesis.

[0223] Treatment with synthetic calcium in osteoinduction medium, in contrast to vitamin D treatment, demonstrated accurate formation of extracellular matrix and substantial deposition of hydroxyapatite (HA) (Figure 6). In addition to its ability to mediate the commitment of mesenchymal stem cells to the bone lineage, Product C is able to induce the mineralization process early and completely and promote the deposition of hydroxyapatite (HA) crystals. Indeed, after 28 days of treatment, Product C induces a statistically significant increase in hydroxyapatite crystals compared to synthetic calcium (Figure 5).

[0224] The results demonstrate that the early peak in ALP activity observed with Product C (Figure 5) translates into effective mineral deposition at 28 days (Figure 6). In contrast, OM + vitamin D + synthetic calcium, despite its contribution to early ALP activity, does not result in significant mineralization at 28 days (Figure 6). HA content remains significantly lower compared to the other groups, highlighting the insufficiency of early differentiation alone without effective downstream mineral deposition. This analysis emphasizes that the early activation of osteogenic markers seen with vitamin D does not guarantee successful mineralization unless supported by mechanisms that drive the full differentiation process.

[0225] Both OM + DMSO + synthetic calcium and OM + synthetic calcium served as strong positive controls, demonstrating robust mineralization levels at day 28. Product C achieved comparable results, further demonstrating its effectiveness in supporting the physiological processes of bone differentiation and mineralization. In contrast, Product C effectively combined its early osteogenic activity with sustained mineralization capacity, suggesting a more comprehensive role in mimicking the physiological bone formation process.

[0226] The results show that Product C not only acts as a support for differentiation stimulation into osteoblasts in the presence of osteoinductive stimuli and as a calcium donor capable of mineralizing the bone extracellular matrix, but also produces a statistically greater increase in ALP activity than synthetic calcium in osteoinductive agent-free GM medium, suggesting that it alone can induce the differentiation of mesenchymal cells into mature osteoblasts (Figure 7). In contrast, calcium and vitamin D treatment did not increase ALP activity, confirming its inability to induce cell differentiation in the absence of osteoinductive stimuli.

[0227] Notably, the results (Figure 7) show that in GM medium lacking osteoinductive agents, Product C results in a statistically significant increase in ALP activity compared to synthetic calcium, suggesting that Product C alone can induce the differentiation of mesenchymal cells into mature osteoblasts. In contrast, treatment with calcium and vitamin D did not induce an increase in ALP activity, confirming the inability to induce cell differentiation in the absence of osteoinductive stimuli.

[0228] In osteoinduction medium, product C also promoted an increase in ALP, showing a typical bell-shaped curve with a peak in activity at day 21, indicating an earlier stimulation of the differentiation process compared to OM medium containing only synthetic calcium (synthetic calcium).

[0229] Similarly, treatment with vitamin D (Figure 6) shows the same trend in ALP, but induces a statistically greater increase compared to its control (OM+DMSO+Ca). Despite the increased activity, the substance fails to induce the correct stimulus for functional mineralization.

[0230] This stimulation was effectively provided by Product C, which showed a statistically significant increase in hydroxyapatite crystals at 28 days compared to synthetic calcium (Figure 6).

[0231] 3. Gene Expression Analysis At the end of the indicated treatment period, cells were washed with 100 μl of PBS, lysed, and collected in RLT buffer (Qiagen, 1053393) supplemented with β-mercaptoethanol (Sigma, M3148) and DX reagent (Qiagen, 19088) for gene expression analysis experiments. Total RNA was extracted from cell lysates using the QIAsymphony RNA kit (Qiagen) with a QIAsymphony SP instrument (Qiagen).

[0232] RNA quality and quantity were determined by A230, A260, A280, and A320 measurements on a Varioskan™ LUX multimode microplate reader (Thermo Scientific™). RNA integrity was confirmed using the 2100 expert_Eukaryote Total RNA Nano Kit (Agilent). Whole-transcriptome expression profiles were assessed using the Human Clariom™ S Pico Assay HT (Applied Biosystems, ThermoFisher Scientific) on a GeneTitan MC Instrument (Applied Biosystems, ThermoFisher Scientific) according to the manufacturer's instructions. Briefly, 6 ng of total RNA was used to generate cDNA, and the fragmented and labeled cDNA was then hybridized to a Human Clariom S 96 array plate at 45°C for 17 hours. Arrays were washed, stained, and then scanned using a GeneTitan MC Instrument (Applied Biosystems, ThermoFisher Scientific), and CEL Intensity files were generated by Affymetrix GeneChip Command Console software (AGCC, ThermoFisher Scientific).

[0233] 3.1 Transcriptome data analysis Data analysis was performed using the Transcriptomic Analysis Console software (TAC, ThermoFisher Scientific), which provided quality control analysis, performed normalization and summarization based on the Signal Space Transformation-Robust Multichip Analysis (SST-RMA) analysis algorithm, and provided a list of differentially expressed genes (Limma Bioconductor package, p-value ≤ 0.05).

[0234] 3.2 Bioinformatic modeling of experimentally observed transcriptome data For each study batch, the degree of modulation of gene expression associated with the desired effect was assessed using Ingenuity Pathways Analysis (IPA) (QIAGEN\Inc., https: / / www.qiagenbioinformatics.com / products / ingenuitypathway-analysis).

[0235] IPA is a scientific literature aggregator that allows for the retrieval of information about genes / proteins and the construction of networks that predict the behavior of biological systems according to their gene expression states.

[0236] 4. Evaluation of the transcriptional effects of Product C treatment on osteogenic differentiation and mineralization of adipose tissue As evidenced by ALP and HA dose assessment, gene expression analysis also indicates that Product C has significant effects on both bone and adipose tissue, promoting distinct differentiation of mesenchymal stem cells into mature osteoblasts capable of mineralizing extracellular matrix, while inducing a decrease in bone adipose tissue, which is believed to result in decreased bone fragility and improved bone quality.

[0237] As shown in the heat maps (Figures 10-11), Product C (Batch 1) treatment resulted in increased osteoblast differentiation and enhanced mineralization with respect to increased bone mineral density, as well as modulation of expression profiles underlying bone remodeling, bone resorption, and bone loss.

[0238] In particular, Product C promotes osteoblast differentiation through the regulation of certain bone formation-related proteins, such as the induction of RUNX2 and the inhibition of sclerostin (SOST), which are known to inhibit osteoblast activity. These results are consistent with the ALP enzyme activity assay.

[0239] This product also induces an increase in bone synthesis markers such as osteocalcin (OCN) and BMP (bone morphogenetic proteins), which is a strong indication of increased osteoblast activity and the formation of new bone matrix, consistent with the measurement of HA crystal concentrations.

[0240] A reduction in bone remodeling processes was also observed, which is important for bone maintenance and repair, potentially rebalancing bone density and structure. In particular, Product C appears to reduce sclerostin (SOST), which is known to inhibit osteoblast activity, thereby enhancing bone formation.

[0241] Finally, product C appears to reduce both the mass and inflammatory processes in bone and adipose tissue as well as at the systemic level, suggesting an anti-adipogenic effect confirmed by an improvement in glucose tolerance.

[0242] Thus, Product C demonstrates multiple effects on both bone and adipose tissue that contribute to bone formation through increased osteoblast differentiation and mineralization, and reduced bone remodeling, adiposity, and metabolic regulation. All effects highlighted by gene expression data clearly distinguish and significantly demonstrate the beneficial effects of Product C compared to traditional products consisting of calcium and vitamin D alone.

[0243] The assay was repeated with batches 2 and 3 of product C and the data obtained are shown in Figures 13a and b.

[0244] 5. Defining the pathophysiological hallmarks of disease for investigating IPA The state of the art "osteoporosis" changes characteristic of a healthy physiological state were examined with particular attention to the following areas of involvement: -Bone remodeling -Osteopenia -Osteoblast differentiation -calcification -Reduces inflammation -Reduction of adipose tissue

[0245] This knowledge was used to interrogate IPAs via the “IPA Bioprofiler” tool using the following keywords: osteoporosis, postmenopausal osteoporosis, bone mineralization, osteoblast and osteoclast differentiation, bone mineral density.

[0246] The use of the "IPA Bioprofiler" enabled the identification of clusters of expressed genes that are causally related to one or more identified biological activities and the specific molecular pathways that support them. Information on the measured gene expression data (fold change cutoffs ≦−2 and ≧+2 and p-value ≦0.05) induced by each batch was then superimposed on the resulting network to define the degree of regulation of affected genes and related biological functions.

[0247] The regulation of expressed genes was shown in different intensities of blue (indicating down-modulation) or red (indicating up-modulation). Based on the literature, the resulting expected calculated impact on the relevant biological function was determined by the "IPA Molecular Activity Predictor" tool (MAP) and presented in a heatmap visualization.

[0248] The colors and intensities were converted to numerical values. Figure 9 shows the modulation trends of selected biological activities underlying the identified hallmarks of osteoporosis in the pathological state of osteoporosis (altered metabolism) or bone physiology, and the modulation trends of the biological activities desired for the restoration of a healthy physiological state (as opposed to altered / pathological).

[0249] The results of the studies conducted resulted in a comparative study of the performance and mechanism of action of three different batches of Product C. Analysis revealed that all batches were able to produce reproducible biological effects, but it was also possible to identify batch-specific variations in the induced transcriptional patterns. Apparently, the induction of slightly different transcriptional patterns still resulted in the same desired regulation of one or more biological activities. This is due to the functional resilience caused by the redundancy of interactions between the product's components and the body, whereby, due to the multifocal mechanism of action, different batches, despite their different qualitative and quantitative compositions, induce the same effect (Figures 13a and 13b).

[0250] Therefore, the analyzed batches are considered to have equivalent biological output since induction and repression patterns are conserved.

[0251] The different transcriptional patterns and relative biological effects of different batches are intended as a hallmark of the inherent variability present in preparations composed of biological materials. From the results summarized in Figure 13, it is clear that the observed transcriptional patterns of different batches induce highly reproducible biological (functional resilience) effects, resulting in a general alteration of the pathological process and equivalence of the overall pathological state for all batches reported in Figure 13.

[0252] 6. Detailed analysis of product C A detailed analysis of Product C was performed and the batch product used was Batch 1 unless otherwise stated.

[0253] The table below summarizes all the methods used. [Table 8]

[0254] The results are summarized in the table below. [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4] [Table 9-5]

[0255] In addition, qualitative miRNA characterization was also performed on ultracentrifuged samples from each batch, and the results show a high metabolomic complexity along with the presence of organism-typical miRNAs. [Table 10]

[0256] 7. Identification of Extracellular Vesicle and miRNA Content in Product C Flow cytometry analysis of product C identified the presence of particles compatible with extracellular vesicles. This suggests that product C contains vesicles that could potentially play a role in cell communication. Based on this, we investigated the RNA content within these vesicle-sized particles using small RNA sequencing with the sRNAtoolbox framework. Among the identified sequences, two mature microRNAs (miRNAs) or their isoforms were found: miR8175 and miR166, prominent miRNAs from the well-annotated Arabidopsis genome.

[0257] 7.1 miRNA detection The presence of RNA was assessed both quantitatively and qualitatively. RNA was extracted using a plant matrix-specific kit (Rneasy PowerPlant kit) after homogenization using QIAshredder columns before proceeding with the kit extraction protocol. Size distribution of the resulting RNA was performed using a Bioanalyzer 2100 equipped with the RNA 6000 Nano, RNA 6000 Pico, and MiniRNA kits.

[0258] A size distribution of total RNA ranging from 4 to 150 nt was detected.

[0259]

[0260] The products are characterized for the presence of miRNAs through NGS analysis of total RNA isolated from various biological matrices within the products.

[0261]

[0262] RNA analysis of product C allowed the identification of two mature miRNA sequences (miRNAs): miR8175 and miR166, annotated in the Arabidopsis thaliana genome (the most annotated plant to date).

[0263] 7.2 Detection of supramolecular structures in product C-based natural matrices Dynamic Light Scattering Dynamic light scattering (DLS) is a common technique for particle size analysis in the nanometer range. It measures the hydrodynamic size of particles by analyzing light scattering from a laser passing through a solution. The intensity of the scattered light fluctuates over time, reflecting the Brownian motion of the particles, with smaller particles diffusing more quickly. DLS is used to measure particle size in colloidal samples, assess formulation stability, and detect aggregation. It is also ideal for analyzing the size distribution of isolated exosomes and microvesicles.

[0264] Analysis was performed by Alfatest Lab.

[0265] Sample preparation: Product C Batch 1 (23D2227) was dispersed in 0.22 μm filtered demineralized water at an arbitrary concentration of 1 mg / ml.

[0266] After dispersion, the sample was vortexed for 2 minutes to ensure complete dispersion.

[0267] Analysis parameters: Measuring cell: Plastic, (DTS0012) Detector: Backscatter 173° (NIBS) Laser wavelength: 633nm Measurement number: 3 Correlation time: adaptation Measurement position: Automatic Attenuator: Automatic Temperature: 25℃ Temperature equilibration time: 120 seconds Dispersant: Water Dispersant refractive index: 1, 33 Dispersant viscosity: 0.8872 cP (water) at 25°C

[0268] Samples were analyzed under three different conditions: -Unfiltered -After filtering through a 0.45 μm nylon syringe filter -After filtering through a 0.1 μm nylon syringe filter

[0269] 0.1 μm filtration was performed on the 0.45 μm filtered samples.

[0270] Before each filtration step, the sample dispersion was vortexed for 30 seconds. The filtered dispersion was allowed to stand at room temperature for approximately 15 minutes and then gently stirred manually before analysis.

[0271] result: The average Z-average and PdI results obtained from three replicate measurements are reported in the table below, where Z-average is the intensity-weighted mean diameter and PdI is the polydispersity index. [Table 11]

[0272] The results indicate that the aqueous dispersion contains large particles. In particular, filtration at 0.45 μm shows a multimodal distribution with particle size signals greater than the filter size (4912.0 nm). This also occurs at 0.10 μm filtration, where a bimodal system is observed with a signal at 156.8 nm, which exceeds the filter size. Therefore, the data suggest the presence of supramolecular aggregates consisting of non-covalent intermolecular interactions. The data quality of the filtered sample is good.

[0273] Exosome detection in Product C Batch 1 Ultracentrifugation sample preparation The starting sample from which the ultracentrifugation was prepared (Product C Batch 1) was weighed and resuspended in a constant volume of VIB or vesicle isolation buffer (20 mM MES; 2 mM CaCl2; 100 mM NaCl, pH 6.0), maintaining a ratio of 5 mL of buffer per 500 mg of sample. The sample was incubated at room temperature for 20-24 hours under agitation to facilitate solubilization. After incubation, several centrifugations were performed at 4°C and increasing speed to isolate particles ranging in size from 30 to 500 nm. Ultracentrifugation was performed using a T-1250 rotor (Thermo Fisher Scientific, 11718-5) and a Thermo Scientific™ Sorvall™ WX+ ultracentrifuge (Thermo Fisher Scientific™ 75000080, No.: 15342177). The samples were centrifuged at 700 x g for 20 minutes, and the supernatant was filtered through a 0.45 μm filter, discarding the pellet, followed by centrifugation at 10,000 x g for 30 minutes. The supernatant was then transferred to an ultracentrifuge tube and centrifuged at 40,000 x g for 70 minutes. The supernatant was transferred to a new ultracentrifuge tube and centrifuged again at 100,000 x g for 70 minutes. The supernatant was discarded, and the 100K pellet was resuspended in VIB buffer and centrifuged under the same conditions. The pellet was resuspended in 600 μL of 25 mM trehalose (Merck, T0167) in PBS and stored at 4°C for use within 24 hours, or at -30°C for long-term storage.

[0274] Extracellular vesicle staining and flow cytometry analysis

[0275] Extracellular vesicles from samples were stained with CellMask™ Green Plasma Membrane Stain (ThermoFisher Scientific #C37608) according to the manufacturer's instructions and quantified using an Attune NxT flow cytometer. Briefly, 27 μL of exosomes were added to 3 μL of CellMask™ Green Plasma Membrane Stain (10x) per sample for 30 minutes at 37°C. Each sample was then read after adding 170 μL of PBS (0.22 μm double-filtered). To eliminate any nonspecific events in the flow cytometry analysis, PBS stained with CellMask™ Green Plasma Membrane Stain was used as a negative control, and a fluorescent exosome standard (Novus Biologicals #NBP3-11691) was used as a positive control. Additionally, each sample was analyzed without staining to rule out autofluorescence.

[0276] result Sample Product C Batch 1 was analyzed using flow cytometry as follows: The PBS value marked by CellMask™ Green Plasma Membrane Stain was considered a blank sample and was therefore excluded from the region of interest. Fluorescent Exosome Standards identified multiple elements in the same area that was identified as positive.

[0277] The samples were then analyzed, taking into account the number of elements present in the same area. The number of extracellular vesicles identified in the Product C sample was 2.7 × 10 6 There were 100 pieces.

[0278] 10. Spectroscopic FTIR Characterization Fourier transform infrared spectroscopy (FTIR) is an analytical technique used to analyze the absorption or emission spectra of a sample by examining the interaction between infrared radiation and a substance. FTIR spectra can be affected by weak interactions in plant matrices, such as hydrogen bonding, van der Waals forces, and hydrophobic interactions, which alter peak position, intensity, and shape. These interactions affect the absorption characteristics of functional groups. FTIR spectra are unique to each material, making them an effective method for studying the physicochemical properties of plant matrices. Therefore, FTIR is valuable for characterizing plant systems.

[0279] FTIR equipment and setup.

[0280] Alpha spectrometer manufactured by BRUKER Optics. The instrument is equipped with a GLOBAR source emitting in the far-infrared and mid-infrared regions, a ROCKSOLID interferometer (Michelson type), a KBr beam splitter, and a RT-DLATGS detector. ·Resolution: 2cm-1 Spectral range: 5000~300cm-1 Background scan: 50 Scans for sample acquisition: 50

[0281] Sample preparation A sample of Product C Batch 1 was transferred to a suitable sample holder for ATR-FTIR analysis of solids and liquids. Approximately 10 mg of sample was deposited and pressed onto the diamond crystal of the ATR support. Before recording measurements, it was ensured that the entire sample holder was properly covered.

[0282] Sample acquisition Each sample was measured at least three times to verify data reproducibility. The replicates were then averaged to obtain a representative spectrum of the sample for characterization (Figure 14).

[0283] 11. Isotopic abundance Isotope abundance analysis provides an atomic-level description of a substance, highlighting the effects of isotope substitution, such as geometric isotope effects (GIEs) and kinetic isotope effects (KIEs). GIEs involve changes in the geometric structure of a molecule due to isotope substitution, particularly affecting hydrogen bonding. These changes affect molecular geometry and can affect physical, chemical, and biological properties. KIEs refer to changes in reaction rates caused by the substitution of an isotope within a molecule. Isotopes have different masses, which affect bond vibrational energy and activation energy. KIEs are classified as first-order KIEs, in which substitutions directly affect the rate-determining step of a reaction, and second-order KIEs, in which substitutions indirectly affect reaction rates through changes in molecular geometry or electronic effects. Both effects are important for understanding the influence of isotopes on molecular behavior. Isotopic abundance analysis was first performed on batch 1 of product C.

[0284] Samples were sent to the Istituto San Michele all'Adige (Fondazione Edmund Mach) and tested for stable isotopes as follows: -δ18O: Method PDP 7011:2010 REV.0 (TC-IRMS), unit ‰ vs V-SMOW. -δ13C: Method PDP 7009:2017 REV.2 (EA-IRMS), units vs. ‰V-PDB δ15N: Method PDP 7009:2017 REV.2 (EA-IRMS), units ‰ vs V-AIR. δ34S: Method PDP 7013:2010 REV.0 (EA-IRMS), units ‰ vs V-CDT. -14C-activity was also tested by Chelab (Tentamus Company): -14C-activity: Method ISO-16620-2;2015 (AMS), unit % modern carbon (pMC).

[0285] The results were as follows: [Table 12]

[0286] Therefore, isotopic characterization was performed on Product C Batch 1. Additionally, 14C activity was performed on the Product C Batch 1 formulation, excluding the calcium carbonate-providing raw material. The 14C activity measurement for the 99.82 ± 0, 22% percent modern carbon (pMC) sample corresponds to that of material from pure bio-based carbon. There is no evidence of synthetic sources in the analyzed material.

[0287] Biodegradability test according to OECD 301F:1992 Biodegradation is the breakdown of organic matter by microorganisms into simple, naturally occurring components such as CO₂, H₂O, and NH₃. Evaluating the biodegradability of chemicals is important in environmental risk assessment. Ready biodegradation tests (RBTs) proposed by the OECD are used to assess biodegradability by incubating chemicals in a mineral medium with microorganisms. Metabolic parameters such as oxygen consumption and CO₂ production are monitored over a 28-day period. A chemical is considered readily biodegradable if it passes the RBT. Primary biodegradation refers to a change in the structure of a substance due to biological processes that can be measured by chemical analysis. For example, the OECD 301F method evaluates biodegradability by measuring oxygen consumption in a respirometer under controlled conditions. Results are expressed as the percentage of oxygen consumed compared to the theoretical oxygen demand (ThOD) or chemical oxygen demand (COD).

[0288] Reagents and Materials The method involves working with the following reagents and materials: A) Test substance Product C batch 1 at 100 mg / l dilution in mineral medium; B) Mineral medium for solubilization of the test substances, consisting of the following four solutions (A, B, C and D) made up to 1 L with ultrapure water: -10 ml of solution A: 8.50 g KH2PO4 + 21.75 g K2HPO4 + 33.40 g Na2HPO4 dihydrate + 0.50 g NH4Cl made up to 1 liter with ultrapure water, final pH 7.4; -1 ml of solution B: 27.50 g of anhydrous CaCl2 made up to 1 liter with ultrapure water; -1 ml of solution C: 22.50 g of MgSO4 heptahydrate made up to 1 liter with ultrapure water; -1 ml of solution D: 0.25 g of FeCl3 hexahydrate was made up to 1 liter with ultrapure water. C) Bacterial inoculum was obtained by adding the test material to an appropriate inoculum and taking equal aliquots of activated sludge. D) Chemical standards for BOD determination consisting of various solutions of sodium acetate anhydrous analytical purity from 5 to 28 days.

[0289] Assay run Sample preparation The samples were treated according to the procedure reported in Respiratory Pressure Measurement Method 301F (OECD). The BOD sensor and sufficient material to perform the planned instrumental analysis were used to ensure proper alignment. Sodium acetate was used as the reference material. The test was performed at a constant temperature of 22°C.

[0290] Inoculum preparation The inoculum was prepared using activated sludge from different locations mixed in equal amounts. The inoculum was oxygenated, stirred, and fed with glucose, peptone, and dibasic potassium phosphate. The redox potential, oxygen consumption, and total dry matter values were monitored daily. The dry matter was determined at 100°C to ensure the same amount (30 mg / mL) in the vessels containing the test substance.

[0291] Inoculum composition The inoculum was obtained by mixing activated sludge. The assembled inoculum was oxygenated, agitated, and fed with glucose, peptone, and monopotassium orthophosphate. Oxygen, redox, and total suspended solids values were monitored daily. Total dry matter was determined before using the inoculum.

[0292] The composition of the microfauna was determined by light microscopic analysis.

[0293] reference material Manometric respirometry also requires the performance of separate tests using ultrapure water enriched with a standard (sodium acetate) to assess proper performance and reliability of the equipment.

[0294] blank A blank analysis was performed on the inoculated mineral medium to assess the contribution of the liquid and inoculum to the BOD value of the final product.

[0295] Assay conditions: The containers are placed in a thermostatic refrigerator set at 22±2°C and kept under constant agitation by the mechanical movement of the anchors. All this is done for 28 days by automatically and wirelessly measuring the oxygen drop value every 6 hours, which becomes the absolute biodegradability value of the sample under investigation over 28 days.

[0296] Test results [Table 13]

[0297] [Table 14]

[0298] This test showed that the contribution to BOD between the inoculum and the mineral medium was 11.1 mg / l, which is the value that should be used for the BOD correction of the 100 mg / l mixture of Product C Batch 1.

[0299] The mean values are within the positive range of the test, which is 10-50 mg / l.

[0300] [Table 15]

[0301] A positive test was obtained from the following evidence: A) The chemical control was positive because the value of the chemical standard obtained (30.8 ppm biochemical oxygen demand, BOD) was within the positive range of the test, which is the theoretical demand for oxygen (ThOD) of 31 ± 5 ppm. B) After just 7 days, the BOD value is 89.68% of the total. C) After 14 gg, the BOD value is 93.22% of the total. D) After 28 gg, the BOD value is 99.35% of the total.

[0302] The reference substance reached the threshold within 14 days. Therefore, it can be stated that the test was performed correctly and the immediate biodegradation data collected for the substance under test are reliable.

[0303] Biodegradation calculations are performed for each sampling time of the reference material, test sample and blank.

[0304] The total biodegradability of the samples was calculated using the following formula: BOD: (average mg / L of O2 consumed of test material) - (average mg / L of O2 consumed of blank) / mg / L of test material in container.

[0305] Based on the lack of nitrification / denitrification correction, there is no evidence of nitrite and nitrate production. -mg / l of O2 consumed by the test substance: 51.25mg / L. -mg / l of O2 consumed by the blank: 11,1mg / L. -mg / l of O2 consumed by nitrification in the product: / - mg / L of test chemical in container: 100mg / L

[0306] Calculated average BOD = 0.402 mg O2 per mg of test material.

[0307] To calculate the degradation rate and therefore the immediate biodegradability, the chemical oxygen demand (COD) was evaluated by hot acid dichromate oxidation in ultrapure deionized water: a test was carried out with a 50 mg / l Abo11 solution, giving a cod of 67.1 mg / l.

[0308] COD = (mg / L of O2 consumed by the test substance) / (mg / L of test substance in the container) COD = 0.467 mg O2 per mg of test material.

[0309] Average percent degradation on day 28: BOD average corrected for blank estimate / COD net material = (0.402 / 0.467) x 100 = 86.08%

[0310] Interpretation of results Efficacy criteria A test is considered valid if: - the average biodegradation rate of the reference substance is more than 60% after 14 days of incubation; - The difference between extreme replicate values at the plateau at the end of the study is less than 20%; - Blank oxygen demand is 60 mg O2 / L or less.

[0311] interpretation A substance is considered readily biodegradable if it reaches a level of biodegradation within 10 days after the onset of degradation, the time when 10% of the substance has been degraded (a 10-day time frame) is considered to be greater than 60%, and furthermore, the level of biodegradation reached at 28 days is >60%.

[0312] result Meet the validity criteria of the study. The assessment of the biodegradability of substance Product C Batch 1 at 100 mg / l shows that during the experiments carried out, the product showed ready biodegradability under the conditions applied in the manometric respirometry test developed in accordance with EC Regulation 440 / 2008 updated to Regulation 640 / 2012 - Part C: Methods for determining ecotoxicity - Method C.4. Part V - (Method C.4-D) + OECD 301F:1992; in fact, the product exceeded 60% (62%) biodegradability within 10 days after reaching 10%, fulfilling the validity criteria of the method. Product C batch 1 at a concentration of 100 mg / L showed no toxic effect on the activity of microorganisms at the tested concentrations, reaching 86,06% of the associated theoretical COD value, thus indicating a well-present biological activity.

[0313] The substance under test, tested at a concentration of 100 mg / L, was found to be "readily biodegradable" under aerobic conditions according to the conditions of the manometric respirometry test (OECD 301F, EC Regulation 440 / 2008 and subsequent updates, method C.4-D). In fact, the substance exceeded 60% biodegradation within 10 days after achieving 10% degradation.

[0314] 12. Network Analysis A network analysis of the pathological conditions treated or ameliorated by Product C was performed, and the data obtained demonstrates how the tested natural matrix-based product can affect the body on a systemic scale.

[0315] Product C - In pathological situations (Figure 12, Panel A): Dysfunctioning and inflamed adipose tissue leads to an imbalance in bone homeostasis, adversely affecting the competition of mesenchymal stem cell reserves to induce osteoblast, osteoclast, or adipocyte differentiation, shifting this phenomenon toward osteoclast differentiation in an unphysiological manner. This leads to a decrease in the number of mature osteoblasts, which are unable to ensure proper mineralization of the cellular matrix with a loss of bone activity. In situations of dysregulated lipid metabolism and adipose tissue inflammation, a dysfunctional loop is established between adipose tissue and bone, leading to the accumulation of adipocytes and osteoclasts, disfavoring osteogenic components and exacerbating bone fragility. Bone is also an organ with endocrine activity and can therefore influence events in other tissues at a systemic level, for example through the secretion of osteocalcin (OCN), which stimulates insulin secretion by the pancreas, insulin sensitivity in peripheral organs such as muscle, and regulation of overall energy expenditure (Fukumoto S, Martin TJ. Bone as an endocrine organ. Trends Endocrinol Metab. 2009 Jul;20(5):230-6. Doi:10.1016 / j.tem.2009.02.001. Epub 2009 Jun 21. PMID:19546009).

[0316] - The situation when treated with the drug reference (Figure 12 Panel B): The drug reference can only reduce the amount of adipocytes.

[0317] - Product C (Figure 12 Panel C): Treatment with Product C is able to recreate all the elements necessary to restore correct bone turnover by interacting with the mesenchymal stem pool in bone and adipose tissue, which is beneficial for the formation of solid functional bone structures, and at the systemic level, restores the balance of metabolic dysregulation and reduces inflammation.

[0318] 13. Detailed analysis of three different batches of product To understand whether the final matrix comprising Product C is characterized by matrix effects, a series of analyses to characterize the product in different ways were performed on the three batches 1, 2, and 3 mentioned above. Figures 13a and 13b show that the three batches have the same desired therapeutic / beneficial effect in in vitro cell-based assays. Targeted metabolomics analyses, which can identify the majority of such molecular components, were performed on the plant-based matrix components (see table below) of the different batches of the product, along with other analyses reported herein.

[0319] Products are composed of plant and other natural matrices that assemble to form a final new natural matrix, as described above. Several analytical techniques have been used to identify and quantify the main classes of compounds present in plants, thereby assessing the composition of the plant matrix components of the product. Metabolomic analysis does not allow for understanding the dynamic changes within the matrix components, but it does provide a "picture" of the composition at the moment the analysis is performed.

[0320] Individual plant metabolites were specifically studied by "targeted metabolomics." This analysis makes it possible to capture a frame on qualitative data by determining the chemical compounds present in the material, and quantitative data by defining the concentration of each compound in the material.

[0321] For product C, a qualitative and quantitative characterization of as many primary and secondary metabolites as possible was performed using an "omic" approach, targeted metabolomics analysis, based on the use of multiple analytical methodologies.

[0322] The analytical methods used for the chemical characterization of each batch are described below. Based on the chemical nature of the classes of compounds present, the most appropriate analytical techniques were adopted. Analysis by chromatographic methods combined with different detection techniques (e.g., GC and LC, each combined with an appropriate detector) made it possible to identify and quantify, where necessary, the organic compounds. Inductively coupled plasma analysis using a single quadrupole mass spectrometer (ICP-MS) or an optical emission spectrometer (ICP-OES) made it possible to establish the levels of elements present, while anions were determined by ion chromatography and a conductivity detector. [Table 16-1] [Table 16-2] [Table 16-3] [Table 16-4] [Table 16-5]

[0323] Qualitative miRNA characterization was also performed on ultracentrifuged samples from each batch, and the results show a high metabolomic complexity, along with the presence of organism-typical miRNAs. [Table 17]

[0324] The results obtained show considerable compositional variability of each plant matrix component in each batch, highlighting the impossibility of reproducing the properties of the matrix as the sum of its single components. The studies performed and reported herein (see cell-based assay results), together with the following data, demonstrate that the biological effects induced by the product cannot be reproduced by the sum of the effects induced by single molecular components, but are the result of mutual associations and interactions between the components: matrix effects. This leads to the impossibility of formally defining the structure-activity relationship (SAR) according to the principles normally applied to APIs.

[0325] The results of targeted metabolomics highlighted more or less significant quantitative variations in individual chemicals and miRNA classes in the plant matrix components of the three batches of Product C. These variations, when considered as reference parameters, would lead to a priori predictions that these batches would have different therapeutic or beneficial effects. The analysis summarized in Figure 13 demonstrates that, although biological activity was maintained across all the different batches evaluated, none of the identified single molecular components adhered to the criteria set for a single API, thus demonstrating that the matrix cannot be considered a collection of APIs.

[0326] As mentioned above, the same therapeutic effect is preserved in all batches (functional recovery). The product is capable of eliciting the same response in a biological system that is relevant to its intended use, through a physiological mechanism of action.

[0327] This also highlights the fact that there are both structural and functional redundant mechanisms of functional resilience typical of living organisms (reaching the same result despite individual differences between individuals of the same species) that are maintained in products containing or consisting of natural matrices.

[0328] Isotopic abundance of all batches To assess the isotope ratio between different batches of product C, analyses were performed on batches prepared from different starting materials. Samples were sent to Istituto San Michele all'Adige (Fondazione Edmund Mach) and tested for stable isotopes as follows: -δ18O: Method PDP 7011:2010 REV 0 (TC-IRMS), units ‰ vs V-SMOW. -δ13C: Method PDP 7009:2017 REV.2 (EA-IRMS), units vs. ‰V-PDB δ15N: Method PDP 7009:2017 REV.2 (EA-IRMS), units ‰ vs V-AIR. δ34S: Method PDP 7013:2010 REV.0 (EA-IRMS), units ‰ vs V-CDT.

[0329] The results were as follows: [Table 18]

[0330] The values δ18O, δ15N, δ34S and δ13C overlap between batches indicating a high reproducibility of the production process due to the conservation of this parameter.

[0331] 14. Definition of the mediation unit of product C activity (U-product C): By analyzing the reproducibility criteria of natural products, the inventors focused on the reproducibility of the product's biological effects. Analysis of different batches revealed significant chemical differences between batches. However, despite these differences, the batches exhibited functional resilience and maintained biological activity, within the concept of redundancy. This approach is based on the principle of redundancy (functional resilience) and ensures that the product retains its biological efficacy despite variability. As a result, the product must be regulated in terms of unity and differentiated by a non-pharmacological mechanism. This deviates from the traditional qSAR concept and emphasizes the unique physiological nature of its mechanism of action.

[0332] The product is a 100% natural biodegradable matrix enriched with miRNA and exosomes from different batches, a feature better controlled by applying the concept of arbitrary activity units to the product positology and batch release policy, rather than traditional API weight or volume.

[0333] A unit of Product C (U-Product C) is defined as 1 / 10 of the amount of product required to induce a change in the state of hADMSCs, which amount is measured using this method, adhering to the limits defined in Figures 10, 11 and 13, or at hallmark levels in a method to assess whether a therapeutic product exerts its effect to treat a pathological condition by the physiological mechanism of action disclosed herein.

[0334] For batch 1, 10U-Product C corresponds to 50.4 mg of Product C dissolved in 4 ml according to the experimental parameters described in the Methods and Materials section.

Claims

1. 1. A product for use in treating or aiding in the treatment of a bone fragility condition in a subject in need thereof, comprising: 【Table 1】 However, each of the above components is in the form of powder, The horsetail is Equisetum Arvense, and / or the acacia is Acacia Senegal, and / or the malpighia is Malpighia punctifolia, the agave is Agave sisalana, and / or the cetaria is Cetaria islandica, and / or the agaricus is Agaricus bisporus.

2. 10. The article of manufacture of claim 1, comprising: 【Table 2】

3. 2. The product of claim 1, comprising the following formulation A, formulation B, or formulation C: 【Table 3】 【Table 4】 【Table 5】

4. 4. The product of any one of claims 1 to 3, wherein the horsetail is Equisetum Arvense, the acacia is Acacia Senegal, the malpighia is Malpighia punctifolia, the coral is Caribbean coral, the bird is Gallus gallus, and the cetaria is Cetaria islandica.

5. 10. A composition for the treatment of a bone fragility condition in a subject in need thereof, comprising the product of claim 1 and at least one natural carrier.

6. 6. The composition of claim 5, formulated for oral, topical, rectal, vaginal, systemic or microneedle injection administration.

7. 10. The product of claim 1 or the composition of claim 5 in the form of a lyophilisate, a tablet, a soft or hard gelatin capsule, a powder, a granule, a filled vesicle, a filled liposome or a filled nanoparticle.

8. 10. A product according to claim 1 or 2, or a composition according to claim 5, for use in the treatment of, or in adjunct to the treatment of, a bone fragility condition in a subject in need of said treatment.

9. 9. The product or composition for use according to claim 8, wherein the subject is at risk of developing a bone fragility condition.

10. 9. The product or composition for use according to claim 8, wherein bone fragility is associated with increased adiposity in the subject.

11. 11. The product or composition for use according to claim 10, wherein the fat gain is associated with obesity, metabolic syndrome, pre-, perimenopause or menopause or andropause.

12. 9. The product or composition for use according to claim 8, wherein the bone fragility condition is osteoporosis or osteopenia.

13. the product exerts its therapeutic or beneficial effect by assisting in the restoration of bone homeostasis through a physiological mechanism of action, a network of biological activities directed against the altered physiological conditions underlying the bone fragility condition, and by demonstrating therapeutic or beneficial functional resilience between different batches of the product; The functional resilience is intended to maintain the therapeutic or beneficial properties of different batches of the product despite differences in qualitative and quantitative composition between the batches. The product according to any one of claims 1 to 3.

14. the composition exerts its therapeutic or beneficial effect by assisting in the restoration of bone homeostasis through a physiological mechanism of action, a network of biological activities directed against the altered physiological conditions underlying the bone fragility condition, and by demonstrating therapeutic or beneficial functional resilience between different batches of the composition; The functional resilience is intended to maintain the therapeutic or beneficial properties of different batches of the composition despite differences in qualitative and quantitative composition between the batches. A composition for use according to claim 8.

15. A product according to any one of claims 1 to 3 or a composition according to claim 5, wherein the product or composition itself is a natural matrix and represents a native natural intelligence that is uniquely capable of enabling physiological, endogenous interconnections with other self-organized entities in nature, such as the human species.

16. 16. The product or composition of claim 15, wherein the presence of native innate intelligence is determined through validation of its emerging properties in restoring a healthy, physiological state of bone metabolism when the following conditions are met: Its 14C activity as measured by the ISO-16620-2;2015 (AMS) method is 99.82±0.22%, miRNA and exosomes are detected in the product or composition, the product or composition exhibits batch-to-batch therapeutic or beneficial functional resilience between different batches of the product or composition, and the product or composition collectively modulates an altered physiological or pathological condition.

17. A method for determining the presence of native natural intelligence in a product according to any one of claims 1 to 3 or in a composition according to claim 5, wherein said product or composition comprises or consists of a natural matrix, said method being through validation of its therapeutic or beneficial emergent properties, comprising the following steps: a. Evaluate the naturalness of a product or composition by:

1. Measuring the 14C activity in the product or composition using the ISO-16620-2;2015 (AMS) method; 2. assessing the presence of miRNA in said product or composition; 3. Assessing the presence of exosomes in the product or composition; b. assessing the presence of a therapeutic effect or beneficial functional restoration between different batches of the product or composition by comparing, from batch to batch, the modulation of one or more biological activities underlying the desired therapeutic or beneficial effect of the product on the relevant altered physiological and / or pathological condition treated by the product or composition in a cell-based assay whose readout is indicative of the modulation of said one or more biological activities; c. assessing from the readouts in the cell-based assay whether modulation of the biological activity underlying a desired therapeutic or beneficial effect results in modulation of an overall physiological or pathological condition; and Determining that the product or composition itself is a natural matrix representing native natural intelligence if: The measured 14C activity was 99.82±0.22%. miRNA, exosomes, therapeutic or functional resilience are detected, and c. Regulation in leads to the overall regulation of an altered physiological or pathological state.

18. (1) providing a list of hallmarks that represent altered metabolism and / or pathological conditions; (2) for each of the hallmarks, defining the alteration of one or more biological activities underlying the pathological condition, thereby pinpointing the network of biological activities whose regulation is consistent with the pathological condition; and (3) identifying one or more parameters having a modulation consistent with modulation of the one or more biological activities underlying the therapeutic effect of the tested product, and determining a modulation trend in terms of up- or down-regulation of the one or more biological activities in the network consistent with the diseased or healthy state; further comprising:

18. The method of claim 17.

19. 19. The method of claim 18, wherein the altered physiological state is bone metabolism and / or the pathology is osteoporosis and the hallmarks are selected from bone remodeling, osteopenia, osteoblast differentiation, bone mineralization, reduced inflammation and adipose tissue reduction.

20. 20. The method of claim 19, wherein the biological activity of (2) for remodeling bone hallmarks is selected from the biological activities shown in Figure 9. Figure 9: Table 1A Table 1B

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