Natural matrix mixture for 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 a novel composition consisting of 100% natural substances that has a physiological (not pharmacological) mechanism of action and exhibits a therapeutic or beneficial effect in the treatment of bone fragility.

[0002] The present invention particularly relates to the exclusive selection and use of a native matrix appropriately processed by specific processes and methods for the purpose of producing a final product for therapeutic or beneficial purposes, for preventing deviation from a balanced physiological state, or for restoring a physiological state in the area of bone fragility. All stages of the manufacturing process of such a product are under the aegis of the One Health principle, which recognizes the interconnectedness of human health, animal health, and environmental health, and thus does not permit the use of artificial forces or substances.

[0003] In fact, in the field of the present invention, the basic requirement is that the final product, i.e., a product containing or consisting of one or more natural matrices, must maintain the imprint of the nature intelligence, i.e., the domain of the organism to which each component of the product belongs, so that, whether natural or artificial, it can itself be interconnected and recognized with other networks, i.e., maintain its originally natural network that has acquired a certain degree of artificiality through interaction with artificial components. This interconnection is considered fundamental for rebalancing any disturbance in the network of events active in each interacting biological system. All the identified matrices present biophysical specifications such that they represent the invention themselves.

[0004] Each network of the native natural matrices contained in the product that contributes to the formation of the final matrix network 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 definition of REACH (Registration, Evaluation, Authorization and Restriction of Chemicals), but since it is a product processed according to its self-assembly characteristics, it cannot be determined or verified based on small molecule chemical 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 a probabilistic model based on the preservation of the physiological activity profile and the relationship between descriptors of the matrix itself generated using multiple biophysical analysis systems including spectroscopy (NIR and other techniques), spectrophotometry (targeted and non-targeted metabolomics), and paper or X-ray crystallography (fractal measurements). Although useful, the conventional molecular chemical definition of the individual substances contained in the substance does not represent its overall effectiveness and quality, and thus it is impossible to use it to verify this type of product.

[0006] The selection of the matrix intended for administration must be verified according to specific current taxonomic criteria updated for the animal, plant, and mineral kingdoms. When used in combination with natural physical phenomena, it is necessary to verify the relationship between action and effectiveness opportunistically, taking into account the effects in the field of wave-particles including acoustic effects (music or other forms) and 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 considered possible to verify the actions and reactions in the mutual correlation of each network that have already been verified at the biophysical level.

[0008] The present invention aims to select and provide, as a new entity or product, and a system, something that can always continuously transform the physiological functions in a specific metabolic state in a living body in a state of continuous transformation, balance, activate or restrict.

[0009] The preparation thus conceived can balance the psychoneuroendocrine immune system, which is considered as a single system that manages and regulates all other systems.

[0010] The present invention contributes to a new state-of-the-art technology that goes beyond the technology of alchemy in the medical field, whose origin can be traced back to the early 16th century, and returns the products and processes to the already described conceptual One Health goal. The present invention proposes a new declination of artificial techniques and substances that self-assemble in nature, recognizing existing rules or finding new rules in order to guarantee the constitution of entities that can be verified, based mainly on the concept of verifying their effects and activities on other organisms. The latter are living beings that change continuously and require the evaluation of their physiological state within defined intervals, which is a concept included in personalized medicine today. The present invention conforms to the scientific concept understood as a set of knowledge that can demonstrably verify the effects of the theoretical mechanism of action. Today, these methods are applied to the establishment of interconnections between all forms of life in a situation where technological innovation advances at a pace that risks impairing the mutual correlation between human-generated (artificial) intelligence and nature.

[0011] Since the activities of the present invention disclosed herein are not currently covered by the state-of-the-art, it is necessary to consider the entire product cycle from the end-user to the relevant social situation under the concept of One Health.

[0012] The operational paradigm prepared by the present invention is referred to herein as "Bios Physiological Health".

[0013] This paradigm uses natural matrices alone or in combination to introduce an innovative approach for the treatment and self-management of health into the medical technology field, and aims to balance the normal physiological states of various biological entities including humans through the endogenous physiological effects caused by the products. It is to identify, select, and assemble natural entities, which have emerging properties, and it is verifiable by the physiological mechanism of action of the final product and other methods that have advanced in recent decades.

[0014] The context reading by both the integrated technical-scientific and humanistic norms in their transversality constitutes the basis of the present invention. Although it does not deny the possibility that some of the characteristics of each matrix part of the product are already known, the emerging properties of the new composition are unexpected.

[0015] Particularly relevant is the role of determining the genetic and epigenetic aspects that determine the networks representing natural matrices and their descriptions at the level of their specific isotope abundances.

[0016] To meet the paradigm of bios-physiological health, each stage of the process, from the selection of regenerative materials to the agricultural and industrial stages and to the method of use, must preserve as much as possible the integrity of the inheritance of the native programming embedded in the natural intelligence of each creative entity, as far as is known on at least a global scale. It will be essential to verify the matrix derived from an epigenetic reality similar to that of the reference, recognized as a reference standard for the specific emerging properties related to the metabolism of other organisms, including humans. As an example, one of the factors that negatively affect epigenetic differentiation is represented by different soil conditions, along with circadian, monthly and annual variations. In order to maintain the properties of the natural system that can claim a physiological interconnection with the whole product, it is impossible to use substances derived from alchemical processes such as distillation, other synthetic or semi-synthetic processes, or products derived from genetically modified or genetically modified organisms. A new interpretation of the unknown parts (mysteries) in the natural programming responsible for the evolution of organic and inorganic life is needed. The establishment of recent scientific evolution makes it possible to reposition the understanding of the origin of progress based on reductionist determinism, based on 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 represents the current stage of human evolution and can be traced back to different epochs. When considered in the context of the present invention, the key data can be limited to the year 1492, which represents the end of the early Renaissance human / Neoplatonic period. This period was politically represented by artists and scientists such as Piero della Francesca, Luca Pacioli, Leonardo, and Durer, and by Cosimo the Elder and Lorenzo de’ Medici. In the 16th century, the study of alchemy, which was thought to be the potential of humans to dominate nature, has developed to date under the protection of artificial intelligence for the purpose of improving the creation of natural disasters, as opposed to natural things, so that “humans dominate all creations”.

[0018] 1492 is a symbolic date in which Lorenzo de’ Medici and Piero della Francesca died, while Columbus discovered America. The human species abandoned the Neoplatonic path of the 15th century to follow Judeo-Catholicism, and the alchemical practice of Paracelsus was applied to medicine, marking the transition to Renaissance Mannerism in the 1500s and leading us to the current full-fledged and irreversible sixth mass extinction.

[0019] While the present invention has demonstrated the feasibility of industrial discoveries resulting in the medical field, it is in principle adaptable to any production field and is intended to address the changing paradigms of evolution. We often talk about protecting biodiversity without addressing the practical problem of billions of tons of exogenous and non-biodegradable artificial substances released into the galaxy, a problem that has clearly been obfuscated, while the "carpe diem" approach prevails over the sense of species survival.

[0020] The present invention, which is presented mainly in relation to patents, is expected to open up a new field of research that explores and shares natural intelligence rather than artificial intelligence, which can hardly stop or slow down the sixth extinction or build an alternative basis for progress to the current one. Inventor Valentino Mercati, together with his co-researcher Jacopo Lucci, chose the path of research in nature that could be useful in life systems, developed knowledge in agricultural and industrial production systems over 40 years, and presented numerous patent applications following this operational strategy. Patents previously filed in relation to the method of the present invention are essentially based on devices and diagnostic readings based on chemical principles related to the physiological action and the emerging properties of natural matrices and their relation to the innate defenses of individual organisms that are interconnected.

[0021] The analysis inspired by the approach disclosed in this specification would have been unthinkable just a few decades ago. This is due to the fact that it was technically impossible to read the genetic and epigenetic information written in the cells of any living organism, as well as the role of atomic isotope differentiation in molecular self-assembly and the mutual connection of any single / personal nature with the "universe". The conceptual difficulty of transitioning from the governance parameters that give a sense of artificiality to the molecule (linked by strong thermodynamic forces that enable strong bonds such as covalent bonds, which are at least partially purified and act on the reduction of the molecular range of other organisms) to the natural matrix, which is still considered mysterious by definition and therapeutically untrustworthy today, is very high.

[0022] After five centuries of alchemical reductionism, if a new interpretation of the present invention is required for the new medical situation, this interpretation must link the farthest concepts and processes in a single field of application. This is due to the ideological legacy that questions the human condition, as already mentioned. Could the human species have been created by an original vital intelligence, like all other species, for the purpose of life, to the extent that it can be assumed to dominate creation, or could it have been experimentally given different abilities from other organisms already advantageously inserted into creation to form a new ecological niche in the service of the universe?

[0023] The answer to this dilemma does not arise in the present invention. Humanity must return to the neo-Platonic 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 all its unique capabilities within the universe with all creations. Humanity must reconsider Leonardo da Vinci's warning, "Man can only make his own descendants...," and reflect on the morbid thoughts of discriminating individuals such as Piero della Francesca, Luca Pacioli, and Durer regarding the impossibility of understanding and expressing the beauty of creation and deciphering its mystery.

[0024] The time has come to acquire new research centers in molecular biology and cell biology, focusing on what is essential for bioinformatics and the new physical sciences. Today, the inventor can base research strategies and socio-economic applications on new therapeutic fields, particularly those of complex and / or chronic degeneration, and the restoration of the metabolic balance of organisms, whether naturally or artificially disrupted, is already an essential part of the future that exists.

[0025] The present invention represents a new vision of medical technology that reexamines scientific evolution from a perspective different from reductionist determinism. This alternative progress does not need to rely so much on artificial intelligence and technological progress, contrary to universal or planetary rules, but requires relying on the development of the laws that govern our universe and life itself. The shift from artificial treatment of specific symptoms to a holistic approach that encompasses the whole, even when viewed from the modern techniques of systems biology, represents the basis of the current progress.

[0026] In particular, the present invention relates to a product comprising a natural matrix derived from toxsa, acacia, malafigia, coral calcium, eggshell calcium, agave, cetraria, agaricus and calcium citrate, which aids in bone homeostasis by modifying the behavior of stem cells and re - establishing the correct balance between cell populations that enable bone growth or remodeling, thereby helping the organism to re - establish correct bone metabolism. By assisting the correct function of bone cells, the product achieves beneficial results, obtaining a rewiring of the differentiation process in a direction more favorable to the bone lineage than to adipocytes and inducing an improvement in whole - body metabolism. The present invention also relates to the use of said product in assisting the treatment of a bone fragility disorder or condition in a subject in need of treatment or management thereof, and in particular to a method of treating or assisting in the treatment of a bone fragility disorder or condition when this fragility is post - menopausal or peri - menopausal.

[0027] Background Art Bone fragility is a major social problem. Scientific groups in various countries are developing guidelines for the diagnosis and treatment of this condition, aiming to adopt specific tools for assessing the fracture risk in the population. Osteoporosis is a socially important disease because its incidence increases with age and affects the majority of the population over 80 years old.

[0028] Post - menopausal women are one of the groups most affected by this disease. The metabolic changes that occur during this transition lead to an increase in fat accumulation both at the abdominal level and at the bone level. In particular, an overly energy - rich diet consisting of overly refined nutrients or an unbalanced ratio between them, combined with the physiological tendencies of the bodies of peri - menopausal women, accumulates adipose tissue characterized by an increase in inflammatory states. From a cellular and molecular perspective, dysfunctional and inflamed adipose tissue causes an imbalance in bone homeostasis and negatively affects the competition for the reservoir of mesenchymal stem cells necessary to induce the differentiation of osteoblasts, osteoclasts or adipocytes, shifting this process unnaturally towards osteoclast differentiation.

[0029] This results in the depletion of mature osteoblasts, fails to ensure proper mineralization of the cell matrix, and causes loss of bone functionality. Such a decline in bone quality can lead to the onset of osteoporosis, a condition in which the anabolic action of osteoblasts on bone metabolism is masked by the catabolic action of osteoclasts, the counterparts of osteoblasts.

[0030] In states 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, while promoting osteogenic components and thus increasing bone fragility.

[0031] Vulnerable fractures cause complex disorders, significant morbidity, reduced quality of life, and functional limitations. Patients with osteoporosis require comprehensive care, including multiple areas and interdisciplinary approaches implemented by a team in an individualized rehabilitation plan that targets specific intervention areas.

[0032] At the cellular level, osteoporosis is associated with changes in bone density and strength, increasing the risk of fractures. Since other factors such as bone structure also influence fracture risk, this disease is no longer considered to be only about a decrease in bone mineral density (BMD). Osteopenia, which begins at age 40 and worsens over time, is known as bone loss and can remain asymptomatic until it progresses to osteoporosis. Osteoporotic bone becomes very brittle and can fracture even with minor trauma or normal body weight.

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

[0034] Adipocytes also play important roles 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 that regulate bone metabolism. Some adipokines stimulate bone formation, while others promote bone resorption, negatively affecting bone density and strength, increasing the risk of fractures, and potentially contributing to osteoporosis. Excessive bone fat is often associated with metabolic syndrome or obesity, and can impair bone health and increase vulnerability by decreasing bone mineral density.

[0035] The extracellular matrix of bone consists of organic components (65%) and inorganic constituents (35%). Organic components, or osteoid, include type I collagen, non-collagenous proteins, proteoglycans, osteonectin, osteocalcin, and other growth factors such as IGF-1 and TGF-β. Inorganic constituents mainly consist of calcium phosphate in the form of hydroxyapatite crystals and smaller 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 and replace it with new bone. 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 of bone regulated by basic multicellular units (BMUs) composed of osteoclasts and osteoblasts. Apoptosis occurs following the removal of old bone by osteoclasts, and new bone is formed by osteoblasts, and the process is carefully regulated by both general and local factors.

[0037] General factors include hormones such as PTH, calcitriol, and estrogen that stimulate bone formation, and calcitonin that inhibits osteoclast activity. Local factors include cytokines such as IL-1, IL-6, and tumor necrosis factor (TNF) that promote osteoclast differentiation, and osteoclast proteins that block the RANKL-RANK interaction and inhibit 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 whole body. As described above, an important aspect of bone fragility, especially during menopause, is the involvement of adipose tissue both within bone (lipopenia of bone) and systemically. Lipopenia of bone refers to the accumulation of adipose tissue in the 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 have an adverse effect on bone health through inflammatory mechanisms and changes in bone metabolism, leading to an increase in 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. Such a decrease in estrogen accelerates bone loss, making postmenopausal women particularly susceptible to bone fragility.

[0039] Therefore, an approach using the principles of systems biology and systems medicine to the physiological transition that women undergo during menopause is essential. This approach should consider the vast network of relationships between the cells, tissues, organs, and systems involved in the metabolic changes during menopause, understand their functionality, and be highly conscious of the various factors at play.

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

[0041] In this regard, 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 reduces blood glucose levels. Modulating this hormone plays an important role in reducing both the amount of adipose tissue and the inflammatory state, 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 the bone mineralization process. These two aspects must be finely regulated and stimulated to ensure good bone health. The most common response to these situations is the use of vitamin D and calcium supplements. Although essential, these substances address the body's needs in a limited and incomplete way, interacting with metabolic pathways in a non-comprehensive and untimely manner without achieving satisfactory effects. Furthermore, in the scientific community, doubts have been raised about the actual beneficial effects of vitamin D supplementation. The most notable of these is the AIFA Note 96 released in Italy, which revised the prescribing guidelines for colecalciferol-based drugs in Class A drugs and declared that vitamin D supplementation does not significantly reduce the fracture risk in independent individuals not admitted to a facility.

[0043] Therefore, in addition to providing vitamins and calcium, it is necessary to engage the body's physiological metabolic network through a system-based approach that harnesses the redundant effects of the product in question. This approach stimulates the differentiation of stem cells 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 administration and rehabilitation approaches to reduce fracture risk and improve quality of life.

[0045] Despite these insights, current treatments cannot adequately address the complex interactions between increased adipose tissue and bone fragility and cannot utilize products that can promote / support the proper restoration of function in bone and whole-body metabolism. Traditional treatment methods mainly focus on either hormone replacement or bone density preservation and do not sufficiently target the underlying inflammatory processes and metabolic dysfunctions that exacerbate bone fragility. This gap underscores the need for innovative treatment approaches that can regulate both adipose tissue inflammation and bone metabolism and provide comprehensive solutions for preventing and treating osteoporosis and bone fragility in general, particularly in perimenopausal populations, by promoting the proper restoration of function in bone and whole-body metabolism.

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

[0047] Due to its emerging properties and its natural matrix composition, the product of the present invention is capable of performing a network (product)-over-network (recipient) mechanism and interacting with the 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. The perimenopause is the transition period to menopause, which typically lasts about six years). The product of the present invention can accurately stimulate the differentiation of mesenchymal stem cells, promote the generation of osteoblast cell lines, inhibit the formation of osteoclasts and adipocytes, and thus contribute to the establishment of a virtual balance in the bone metabolism process. Furthermore, it can provide the calcium necessary for the accurate deposition of hydroxyapatite crystals into the bone matrix. As a result, the product of the present invention has been shown to be able to reduce both the amount of adipose tissue and the inflammatory state by regulating osteocalcin expression, suggesting an anti-lipogenic effect, and improving glucose tolerance, expressing further desirable effects in the pathophysiological framework of the target subject: Its local and systemic actions can intervene in the dysfunction loops occurring in systemic inflammation and metabolic dysregulation, which affect the differentiation of mesenchymal stem cells into white adipocytes and their subsequent accumulation.

[0048] The decrease in bone resorption induced by the product of the present invention plays a fundamental role in the maintenance and repair of the bone itself and promotes the rebalance of bone density and structure, as disclosed in the examples and figures of the present application. Furthermore, the product of the present invention exhibits a systemic metabolic regulatory effect that induces the expression level 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 β-cells and enhancing insulin sensitivity in muscle and white adipose tissue, resulting in a decrease in blood glucose levels and the regulation of overall energy consumption.

[0049] In the process of developing this product, the latest technologies and experimental models representing the physiological transition process of menopausal women from a holistic perspective were applied to define the biological activity of the product.

[0050] The data obtained and disclosed in the examples and drawings of this application demonstrate that the dual synergistic and systemic effects of the product of the present invention are significantly more desirable compared to classical calcium and vitamin D supplements that can only partially support the physiological changes of the body. Furthermore, as is clear 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 that means the product interacts with the body according to a well-known system, rather than following an exogenous principle imposed by an exogenous entity such as a synthetic molecule. Therefore, in addition to containing precursors of vitamin D and a calcium source, the product of the present invention is designed to interact with the pool of mesenchymal stem cells in bone and adipose tissue, making it possible to reproduce all the elements necessary to balance the appropriate bone metabolic turnover essential for the formation of solid and functional structures. Thus, the effect of this product is, overall, expected to have a systemic effect and contribute to the reversal of metabolic disorders and potential inflammation that affect menopausal women on a systemic scale.

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

[0052] Therefore, due to the 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 the correct synergistic effect 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 formulation. [Table 1]

[0054] In the same way as the product for treating or improving bone fragility or for use in treating or improving bone fragility, it includes the administration of the product to patients in need thereof or to healthy individuals at risk of developing bone fragility.

[0055] In particular, the product is a beneficial product in its use for assisting bone homeostasis in people at risk of developing bone fragility. [Brief Description of the Drawings]

[0056]

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DETAILED DESCRIPTION OF THE INVENTION

[0057] Terms Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular.

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

[0059] In this specification, "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 the claims, the expression "comprising" or "comprise(s)" can be replaced with "consisting of" or "consist(s) of".

[0061] The "natural matrix" in the present application refers to a material composed of a network represented by a wide number of components / components that are directly obtained (e.g., extracted) from members of the natural world or their naturally occurring parts (i.e., from natural raw materials) without significant processing or synthetic changes. "Without significant processing or synthetic changes" is intended to mean that no denaturation process is used to obtain the matrix from the raw materials. In other words, the natural raw material source is treated only by manual, mechanical or gravitational means, for example, by dissolving in water or other naturally occurring solvents such as water, water-alcohol solutions; by flotation; by extraction with water or other natural solvents; by steam distillation, or by heating only to remove water or any other naturally occurring solvent, or the "natural matrix" is extracted from the air by any means, provided that the member of the natural world or its natural part itself is excluded. In particular, according to the present invention, the natural matrix is a 100% natural and biodegradable material consisting of natural components that have not been denatured by a process for manufacturing the matrix from starting materials without intentionally adding synthetic products throughout the process. In this specification, 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) between its components and the functional interactions (non-material interactions) that become apparent upon exposure of the biological system to the natural matrix. In other words, the natural matrix or a mixture of natural matrices is a material obtained from entities that are naturally self-assembled and treated to preserve their native biophysical properties that determine physiological interactions with other living organisms such as the human organism. Their emerging properties can be expressed by contributing to the physiological action activated in each specific situation, as well as to the balance of the metabolic processes or states of the recipient organism and / or some organs or tissues. According to the present invention, the natural matrix can be derived from materials obtained from any source in the living world, i.e., the Monera, Protista, Fungi, Plantae and Animalia kingdoms.Thus, this term encompasses plant natural matrices, animal natural matrices, fungal natural matrices, protist (archaea or bacteria) natural matrices, monera natural matrices. Natural matrices can also include natural inorganic materials such as minerals obtained from natural raw materials. The synonyms for natural matrix or one or more natural matrices herein are the "composite natural system" or "natural material" defined below.

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

[0063] In all parts herein, the general term natural matrix can be replaced by: plant natural matrix or natural matrix obtained from plants, animal natural matrix or natural matrix obtained from animals or from animal products such as eggs or milk, fungal natural matrix or natural matrix obtained from fungi, protist natural matrix or natural matrix 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 include a denaturation step (e.g., use of temperature or denaturing solvents).

[0064] Plant is synonymous with herb.

[0065] The term "natural" matrix emphasizes that, due to no denaturation treatment to obtain it, it retains the integrity and complexity of the network of constituents / components as in the original natural source. Thus, a natural matrix does not include compositions rich in specific synthetic molecules or of natural origin isolated from natural raw materials. Further, a natural matrix can only be obtained by processes that do not act by extensive processing or chemical modification, isolation, purification, or molecular extraction.

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

[0067] The term natural matrix can also be replaced in all parts of the specification and claims by a complex natural system.

[0068] In any part of this specification and the claims, the term natural matrix is not construed as a "natural product" per se. A natural matrix is a product obtained from a natural organism and is processed (e.g., extracted) therefrom by a technique that does not substantially change the biological structure and the associated supramolecular and functional mutual correlations between the constituents within the matrix as described above, i.e., by a technique that does not use denaturing techniques and does not contain additional isolated or synthetic molecules or classes of molecules.

[0069] This specification and the emerging properties according to the art define properties that are represented not by the mere sum of the properties of the individual isolated constituents / components of the matrix / material, i.e., of a natural matrix or natural material according to this specification, but by both the functional and structural interactions between all constituents / components of the matrix / material, which are also the result of the supramolecular self-assembly of the constituents / components within the matrix / material itself.

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

[0071] Thus, emerging properties are not empirically predictable from the qualitative - quantitative knowledge of each constituent of a given composition or matrix, and as a result, they are not attributable to one or more specific APIs. A multi - drug composition may exhibit unpredicted synergistic effects, but the properties of the composition still result from the specific APIs contained therein and their amounts.

[0072] In the case of emerging properties characteristic of a natural matrix, the observed emerging properties are not reproducible with a specific API, and the batches, while qualitatively - quantitatively different in composition, are maintained in different batches of a given matrix or in a given mixture of matrices (functional resilience is referred to below).

[0073] According to the present specification, synthesis has the meaning conventionally accepted in chemistry. Conventionally in chemistry, the term "synthesis" refers to the origin or source of a material or substance. Synthetic substances or materials are manufactured by humans through laboratory chemical reactions that produce more complex chemical substances by artificial synthesis, i.e., usually by reacting simpler chemical substances through a process that often uses 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 manufactured by chemical synthesis, and aspirin is a synthetic drug manufactured by a specific chemical reaction.

[0075] The functional resilience according to the present specification (also referred to as "redundancy") 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. 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 qualitative and quantitative compositional differences between different batches. This composition is necessarily present (inherent) in a product comprising or consisting of one or more natural matrices. As is known to those skilled in the art, each time different batches of starting materials are used, the resulting natural matrices have a unique qualitative-quantitative composition at the molecular level typical of the individual diversity between organisms of the same species.

[0076] A healthy physiological state refers to the state of an organism's body, organs, devices, systems or body regions, and their internal processes when they are functioning optimally within the normal parameters of that individual, i.e., a state showing homeostasis. In the context of one or more bioactivities known to contribute to the hallmarks of a given disease or pathological condition or altered physiological state, a healthy physiological state refers to a state in which the one or more bioactivities 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. If a tendency for modification of one or more bioactivities consistent with a pathological precondition is known, a healthy physiological state can be considered to be represented by an opposite tendency for modification for each of the activities.

[0077] This term takes into account the hallmarks of a particular disease, which are the distinctive features or characteristics typically observed in an individual affected by that disease. These hallmarks can include specific cell behaviors, molecular pathways, canonical pathways, or physiological responses that play important roles in the onset or progression of the disease.

[0078] In summary, in the context of a particular disease or pathological / altered condition, a healthy physiological state is one in which one or more bioactivities related to the known hallmarks of that disease or pathological condition are regulated in a direction consistent with a non-diseased / unaltered state, in other words, in a direction opposite to the diseased / altered state.

[0079] Thus, the healthy physiological state according to the present invention also indicates the direction of regulation of one or more bioactivities that are known hallmarks of a pathological condition in homeostasis, i.e., the direction of homeostasis of the degree of regulation of one or more bioactivities attributable to a particular system, region, organ or organ of a healthy subject prior to the onset of the pathological condition.

[0080] Altered physiological states and altered homeostasis are closely related concepts that explain deviations from the 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 functions. Altered physiological states can be caused by various factors such as disease, injury, medication, environmental factors, and psychological stress. Examples include fever, inflammation, hormonal imbalances, 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 a narrow range. When these regulatory mechanisms are unable to maintain balance and lead to deviations from the body's normal set points, a change in homeostasis occurs. These deviations can be temporary or chronic and may involve compensatory mechanisms to restore balance.

[0083] In summary, an altered physiological state describes observable changes in the normal function of the body, while altered homeostasis refers to a fundamental disruption of the body's regulatory mechanisms for maintaining internal stability.

[0084] As a disruption of the homeostatic mechanisms that can lead to physiological imbalances and the manifestation of disease or dysfunction, a change in homeostasis underlies an altered physiological state. Products that assist homeostasis are those that adjust the body to restore the stability of its internal environment when changed.

[0085] As used herein, a hallmark of a disease or pathological or medical condition has the meaning conventionally used in the art. A disease hallmark is known to be an indicator that can mark the progression or control of a given disease or pathological or pre-pathological condition, and together, typically represent the general pathological state associated with a given pathology. These hallmarks (also referred to as “key indicators”) are typically a set of features or patterns that a physician monitors over time to track the onset, progression, or regression of a particular disease. In summary, a disease hallmark is a defining feature or characteristic whose modification indicates a given pre-medical or medical situation and aids in its identification, diagnosis, monitoring, and understanding. By way of example, for neurodegenerative diseases (NDDs), at least the following eight hallmarks of NDDs are known in the art: (pathological protein) aggregation, synapse and neuronal network (dysfunction), (abnormal) proteostasis, cytoskeleton (abnormalities), (altered) energy homeostasis, DNA and RNA (defects), inflammation (increase), and neuronal cell death (increase). In cancer research, prominent hallmarks of cancer are a set of unique properties commonly seen in cancer cells. These hallmarks include (sustained) proliferative signaling, (evasion of) growth suppressors, (resistance to) cell death, (enablement of) replicative immortality, (induction of) angiogenesis, and (activation of) invasion and metastasis.

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

[0087] Hallmarks of altered physiological states typically include observable changes in various aspects of body function, which can manifest through symptoms, signs, or test findings.

[0088] A changed physiological state typically reflects a disruption of the body's homeostatic mechanisms, leading to a deviation from normal physiological parameters. These imbalances can include changes in temperature regulation, fluid and electrolyte balance, acid-base balance, glucose metabolism, or other regulatory processes.

[0089] Overall, the hallmarks of a changed physiological state provide valuable clues for healthcare providers to identify the root cause, assess the severity, restore normal function, and induce appropriate interventions to facilitate 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 efficacy, safety profile, cost, and clinical experience. The reference drug functions as a benchmark for comparing 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 for treating a particular condition.

[0091] Native natural intelligence represents the inherent ability of natural matrices to interact with and integrate other biological networks, storing and transmitting the biological and physicochemical information necessary to use the logic inherent to the living organisms that receive them, since the natural matrices are already known and thus endogenous in relation to them. This intelligence is the expression of natural autopoiesis, i.e., the ability to self-organize and adapt to environmental stimuli without artificial intervention, transmitting messages according to punctual logic and via mediators that are unknown and thus exogenous in relation to the living organisms that receive them.

[0092] The expression "physiological interrelationship" is defined as "endogenous" physiological interrelationship, representing the ability of the natural matrix to interact harmoniously and functionally with the recipient's biological system, stimulating an internal (endogenous) response to restore a balanced physiological state. This interaction is based on natural dynamics without artificial intervention, representing an interactive 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 chemo-physical interactions occurring in natural environments and conditions. These systems found in living organisms or natural matrices exhibit emergent properties arising from their dynamic interactions and cannot be artificially replicated.

[0094] When referring to a subject in need of a beneficial or therapeutic treatment, the description relates to a human who is suffering from a pathological condition or is in a state (e.g., age, weight, gender, etc.) at risk of developing a pathological condition.

[0095] Detailed Description of the Invention The present invention relates to a novel composition, i.e., a product consisting of 100% natural substances, which exhibits a therapeutic or beneficial effect in the treatment of altered bone metabolism and / or bone pathologies, and the product has a physiological (as opposed to pharmacological) mechanism of action.

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

[0097] Thus, according to the present specification, a product comprising or consisting of one or more natural matrices is a 100% natural product, which means that the product does not contain additional artificial substances, i.e., chemically synthesized substances made by humans via 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 it is of natural origin.

[0099] Note that natural materials are fundamentally different from "substances" that include substances of natural origin. Since they are not represented by their individual components, they require dedicated models. Therefore, in order to explain natural materials, it is necessary to expand the reductionist approach and use the innovations of the previous century. Conceptually, this means referring to systems theory. From an experimental perspective, preclinical evidence includes systems biology approaches such as omics sciences (e.g., transcriptomics) and bioinformatics evaluations.

[0100] These enable an appropriate evaluation of the matrix (action network) and the human body (receiving network), and enable the interaction between the two to be considered as an "etwork - on - network" interaction. The mechanisms accompanied by cooperative redundancy and resilience that characterize physiological functions in each specific situation correspond to the "physiological mechanism of action" and can be characterized by a network paradigm different from the target model and non - target model that describe PhIM and mechanical / chemical / physical mechanisms respectively.

[0101] 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 a process for manufacturing the matrix from starting materials without intentionally adding synthetic products throughout the entire process.

[0102] As already mentioned, it is essential that the matrix be obtained through a non-modifying process so that the components of the matrix are not artificially modified. However, if desired, the presence of additional indicators of the maintenance of the characteristics present in the original raw materials can be verified. Furthermore, a 100% natural product is a product that is expected to be 100% biodegradable. In this specification, 100% biodegradable 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 presence of structural interactions (material interactions) due to its components and functional interactions (non-material interactions) that become apparent upon exposure of the biological system to the natural matrix.

[0103] The present invention relates to a product defined in the following table.

Table 2

[0104] In one aspect of the present invention, the product has the following formulation.

Table 3

[0105] In a further aspect, the product has the following formulation.

Table 4

Table 5

Table 6

[0106] In one aspect, the fern 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 fern 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 a natural reaction that occurs when the coral skeleton is brought into contact with lemon juice. In particular, the calcium citrate is produced by diluting lemon juice with water at a ratio of 1:1 (by volume), adding coral powder, mixing for 5 hours, and then freeze-drying.

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

[0110] The powder is calcium carbonate from coral, calcium carbonate from eggshells, fine powder from Agave leaves, fine powder from Cetaria thallus, fine powder from Agaricus, and gum arabic, Acacia, and is mixed together at room temperature until the mixture is homogeneous.

[0111] The lyophilized extract is from Equisetum flower spikes, Acerola, Malpighia fruits, and calcium citrate (as defined above).

[0112] For preparation, the homogeneous mixture of the powder is then mixed with the lyophilized extract at room temperature until the mixture is homogeneous.

[0113] When 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 from coral, calcium carbonate from Caribbean coral, calcium carbonate from eggshells, fine powder from Agave sisalana leaves, fine powder from Cetaria islandica thallus, fine powder from Champignon mushrooms, Agaricus bisporus, and gum arabic, Acacia senegal, and the lyophilized extract is the freeze dy extract of Equisetum arvense flower spikes, 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 by an industrial water treatment plant manufactured from drinking water.

[0116] The aqueous extracts of plant materials are known to those skilled in the art. Non-limiting examples of extract preparations that can be applied to Acerola with the necessary modifications are as follows; Freeze-dried extract of Equisetum arvense spikes: The dried Equisetum arvense spikes were subjected to extraction with 100% water (v / v) [drug-solvent ratio: 1 / 18] at 70°C for 2 hours, and filtered to remove solid effluent substances. 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, avoiding light and moisture, until use.

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

[0118] By way of mere example, the composition can be formulated for oral, topical, rectal, vaginal, administration, systemic injection, or microneedle injection.

[0119] According to non-limiting examples, the products or compositions disclosed or claimed herein can be prepared in the form of freeze-dried products, tablets, soft or hard gelatin capsules, powders, granules, filled vesicles, filled liposomes, or filled nanoparticles.

[0120] According to the present invention, the products or compositions disclosed and / or claimed herein are for beneficial or medical use, for example, for use in the treatment or adjunct treatment of osteoporosis in a subject in need thereof.

[0121] As a matter of fact, as described in the summary of the present invention, the in vitro studies of the applicant have compared the efficacy profile of the mixture of the present invention with the treatment with synthetic calcium and vitamin D alone, and have been able to evaluate the ability of mesenchymal stem cells (hADMSC) isolated from the adipose tissue of patients to calcify the cell matrix (increase in the deposition of HA crystals) and induce differentiation into mature osteoblasts that can counteract bone fragility (increase in the activity of alkaline phosphatase, ALP).

[0122] Regarding the treatment with vitamin D, it showed a statistically significantly greater increase in ALP activity compared to its control (OM+DMSO+Ca), together with the osteoinductive stimulation provided by the osteoinductive medium (OM), indicating the possibility of a synergistic effect with OM and confirming its ability to induce the differentiation of stem cells into osteoblasts (Figure 5).

[0123] Despite the increase in activity, vitamin D was unable to induce the correct stimulation for functional calcification (Figure 6).

[0124] Even the treatment with synthetic calcium in the osteoinductive medium showed an increase in ALP activity (Figure 5), and the typical "bell-shaped" trend was characterized by a peak in the enzyme activity level after 28 days of treatment. Different from the treatment with vitamin D, it brought about the correct formation of the extracellular matrix and formed substantial hydroxyapatite (HA) deposits (Figure 6).

[0125] Similar to the treatment with vitamin D and synthetic calcium, the treatment with the mixture of the present invention together with the 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 on the 21st day of treatment, and thus indicating an initial stimulation of the differentiation process compared to the OM medium supplemented with synthetic calcium alone (synthetic calcium).

[0126] In addition to showing the ability to mediate the confinement of mesenchymal cells to the bone lineage, the product of the present invention can induce the calcification process in a complete manner at an early stage and promote the deposition of hydroxyapatite (HA) crystals (Figure 6). In fact, 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 acts not only as a support for stimulating the differentiation into osteoblasts in the presence of osteoinductive stimuli and as a calcium donor capable of calcifying the bone extracellular matrix, but also in GM medium without osteoinductive agents, it brings about a statistically greater increase in ALP activity compared to synthetic calcium, suggesting that it can alone induce the differentiation of mesenchymal cells into mature osteoblasts (Figure 7). In contrast, treatment with calcium and vitamin D does not determine an increase in ALP activity and it is confirmed that it cannot induce cell differentiation in the absence of osteoinductive stimuli.

[0128] In line with what is already known, treatment with vitamin D and synthetic calcium, although fundamental, interprets the actual needs of the organism in a non-exhaustive way and inserts into the individual's metabolic pathways in a timely manner without achieving satisfactory effects. In fact, they cannot reproduce the physiological differentiation of mesenchymal stem cells into osteoblasts to support bone formation.

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

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

[0131] The mixture of the present invention alone can reproduce the entire physiologically active process (Figure 8).

[0132] The molecular mechanism underlying the activity of the inventive mixture that induces the correct metabolic stimulation which is decreased at the phenotypic level in the increase of ALP and HA was analyzed by evaluating the regulation of gene expression of mesenchymal stem cells after treatment with the inventive mixture together with a differentiation stimulus. This product induces a significant effect in vitro on stem cells which are precursors of both osteocytes and adipocytes, and promotes in bone the clear differentiation of mesenchymal stem cells into mature osteoblasts capable of calcifying the extracellular matrix. As a result, with respect to adipose tissue, the inventive mixture promotes a decrease in fat both locally in bone and at the systemic level, and decreases the tendency of mesenchymal stem cells to differentiate into mature adipocytes. The synergistic effect of these actions results in 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 inventive mixture determines the regulation of the expression profile underlying bone remodeling, bone resorption and bone loss, in addition to an increase in calcification with respect to the increase in osteoblast differentiation and bone mineral density.

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

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

[0136] The decrease in the 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 balance of bone density and structure. In particular, the mixture of the present invention promotes a decrease in the expression level of sclerostin (SOST), which is known to have a function against the inhibition of osteoblast activity, and thus enhances bone formation.

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

[0138] The results obtained also showed the potential of the mixture of the present invention to reduce both the amount of adipose tissue and the inflammatory process, suggesting an anti-adipogenic effect and an improvement in resistance, and thus identifying further desirable effects in the targeted pathophysiological framework.

[0139] Therefore, the local and systemic actions of the mixture of the present invention can enter into a dysfunction loop established in a state of systemic inflammation and metabolic dysregulation, which prevents the differentiation of mesenchymal stem cells into white adipocytes and their subsequent accumulation.

[0140] In other words, thanks to its emerging properties, the mixture of the present invention can interact, using the network mechanism, with the physiological metabolic pathways established in the body of menopausal women or women facing this transition. The product can not only provide the calcium necessary for the correct deposition of hydroxyapatite crystals into the bone matrix, but also precisely stimulate the differentiation of mesenchymal stem cells, promote the development of osteoblast cell lines, inhibit the formation of osteoclasts and adipocytes, and thus has the ability to help establish an advantageous balance in the bone metabolism process. Furthermore, the improvement of the osteocalcin secretion profile suggests the induction of beneficial effects also at the systemic level of organs such as the pancreas, muscle tissue and adipocytes.

[0141] This dual synergistic and systemic effect of the product is highly desirable compared to classical calcium and vitamin D supplements commonly used, since it can only partially assist the physiological changes of the organism. This assistance is instead provided by the mixture of the present invention with the physiological mechanisms, i.e., it can interact with the body according to the already known canons, rather than external canons imposed by exogenous entities such as synthetic molecules. This is because, in addition to containing both precursors of vitamin D and calcium sources, the mixture of the present invention, in particular, interacts with the pool of mesenchymal stem cells in bone and adipose tissue and makes it possible to reproduce all the elements necessary to rebalance the correct metabolic turnover of bone, useful for the formation of solid and functional structures. Furthermore, the effects of the product reach a systemic projection that helps to balance the metabolic disorders with the potential inflammation that afflicts women in the perimenopausal period on a systemic scale. The mixture of the present invention clearly demonstrates, only when placed in relation to a 100% natural therapeutic solution of a very complex nature thanks to the presence of a complex plant matrix and natural calcium from various sources, the ways in which the complexity of the physiological processes can be correctly assisted according to the known canons.

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

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

[0144] Since bone metabolism is also tightly interconnected with lipid metabolism as described above, in certain embodiments of the present invention, the bone fragility is associated with increased fat in the subject.

[0145] The increased fat is intended as a significant increase in fat, i.e., an increase in fat that exceeds the normal weight fluctuations that typically occur in the subject.

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

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

[0148] In extensive characterization and research activities regarding the products of the present invention, the Applicant has also identified the conventional hallmarks of osteoporosis and related biological activities where dysregulation (up- or down-regulation) underlies the disease state, and defined it as a tendency for regulation of such activities towards a healthy physiological state, as opposed to the regulation observed in the altered state or disease state (see Figure 9).

[0149] The Applicant also confirmed that all desired modulations of the above activities were achieved using the products of the present invention, thereby verifying the therapeutic effectiveness of the products (Figure 10). Furthermore, the Applicant compared the modulation of the activities exerted by the products of the present invention with that exerted by reference drugs used for treating osteoporosis such as Dibase (vitamin D 10,000 U.I. / ml), and found that the reference drugs could not reduce bone remodeling, alleviate bone loss, induce osteoblast differentiation, restore mineralization, reduce inflammation, and could only partially reduce adipose tissue. Thus, overall, administration of vitamin D does not modify the biological activities underlying the pathophysiology of osteoporosis towards a regulatory trend towards a healthy physiological state (Figure 11).

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

[0151] In fact, different batches of products containing one or more natural matrices are, by definition, batches whose qualitative and quantitative compositions are necessarily variable as discussed in detail above. According to one aspect, in order to demonstrate the existing qualitative and / or quantitative differences in the molecular composition of each batch, qualitative and / or quantitative analyses of each batch were performed (Figure 13). This can be achieved by conventional techniques, non-limiting examples of which include chromatography, spectrophotometry, atomic absorption spectrometry (AAS), atomic emission spectrometry (AES), inductively coupled plasma (ICP) techniques, chromatography combined with detectors, etc., or combinations thereof. The analysis can focus on a limited number of selected classes of substances (e.g., Figure 13) or all components of the product.

[0152] As would be expected for products containing or consisting of a natural matrix, each batch was prepared according to standardized procedures in order to obtain a high degree of uniformity between different batches. However, a detailed qualitative-quantitative analysis of all the tested batches revealed qualitative-quantitative differences for each batch that could not assume the presence of an API and that, using the conventional validation methods used for synthetic or isolated drugs, actually led to the rejection of batches that were therapeutically effective.

[0153] Due to its very nature, the natural matrix is again reported to have a variable composition even when obtained from the same type of raw material. As an example, a person skilled in the art is well aware that a natural matrix obtained from an individual of a plant species will never be absolutely identical to another natural matrix obtained from a different individual of the same plant species, even within the same field of plants, due to the genetic and epigenetic variability of each living organism.

[0154] Following the experiments reported in Figure 13, all the batches analyzed had different qualitative and quantitative chemical compositions. Surprisingly, however, in all the batches tested, the different molecular entities within each matrix were found to interact in a functionally and sometimes structurally redundant manner with each other, and to provide the same therapeutic or beneficial (homeostatic-adjuvant) effect despite the differences in their qualitative-quantitative molecular composition. This is surprising.

[0155] In fact, the batches showed a (therapeutically (usable in therapy) or beneficially) functional resilience despite the variability in their qualitative and quantitative molecular composition.

[0156] In other words, the Applicant has surprisingly found that different batches of the same product exhibit a consistent regulation of all tested bioactivities (with respect to trend and magnitude) related to the desired therapeutic or beneficial effect, despite qualitative and quantitative compositional differences between batches, which are also defined herein as "functional resilience". The observed maintenance of bioactivity, as described above, may be due to the emerging properties of the natural matrix giving rise to a matrix network that acts as an entity with characteristic properties and cannot be attributed to each single molecule as if it were isolated, and the therapeutic action is due to a non-pharmacological mechanism of action different from that of classical therapeutic products based on the pharmacological relationship (SAR) between structure and activity, which is the most relevant relationship in classical pharmacological activity considered at the single molecule level between an active pharmaceutical ingredient (API) and the receptor targeted by that 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 the overall pathophysiological state or the altered physiological state.

[0158] According to the present invention, the defined and / or claimed products or compositions exert their therapeutic or beneficial effects through a physiological mechanism of action by assisting in the restoration of bone homeostasis through a network of biological activities against the altered physiological state underlying the osteoporotic condition and by exhibiting therapeutic or beneficial functional resilience between different batches of the product or composition. The functional resilience is intended to maintain the therapeutic or beneficial properties of different batches of the product or composition, despite qualitative and quantitative compositional differences between batches.

[0159] In particular, for all inferences regarding the natural matrix provided above, together with all experimental data collected by the applicant, the product or composition according to the invention is a natural matrix that itself represents native natural intelligence, which can be said to have the sole ability to enable physiological and endogenous mutual binding with other entities self-assembled in nature, such as the human species.

[0160] Native natural intelligence is hereby restated to represent, as an inherent ability of the natural matrix, the preservation and transmission of biological and physicochemical information. It is the biological and physicochemical information necessary to interact with and integrate with other biological networks using the logic inherent to the living body that receives them. This logic is already known and is endogenous in relation to it. This intelligence is the expression of natural autopoiesis, i.e., the ability to self-organize and adapt to environmental stimuli without artificial intervention. The way it transmits messages follows punctual logic and is transmitted via mediators unknown to the living body that receives them. 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, wherein 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 having emerging properties (usable in therapy or beneficial) when its 14C activity measured using the ISO-16620-2;2015 (AMS) method is 99.82 ± 0.22 percent, when miRNA and exosomes are detected in the product or composition, when the product or composition exhibits therapeutic or beneficial functional resilience batch by batch, both between different batches of the product or composition and when the product or composition regulates an overall changed physiological or pathological state.

[0162] This can be done by performing the following steps on a sample of the product or composition of the present invention using the product or composition: a. The naturalness of the product or composition is 1. Measuring the 14C activity by the ISO-16620-2;2015 (AMS) method, 2. Evaluating the presence of miRNA in the product or composition, 3. Evaluating the presence of exosomes in the product or composition, by: b. In a cell-based assay where the readout represents the regulation of one or more biological activities, comparing batch by batch the regulation of one or more biological activities that underlie the desired therapeutic or beneficial effect of the product on altered bone metabolism and / or bone pathology, to evaluate the presence of therapeutic or beneficial functional resilience between different batches of the product or composition, c. From the readout in the cell-based assay, evaluating whether the regulation of the biological activity that underlies the desired therapeutic or beneficial effect results in the regulation of the overall altered physiological or pathological state, Hereinafter, 1. The measured value of 14C activity is 99.82 ± 0.22 percent, miRNA, exosomes, therapeutic or functional resilience are detected, and c. When the regulation in (c) results in the overall regulation of the altered physiological or pathological state, determining that the product or composition itself is a native matrix representing native natural intelligence.

[0163] The product is determined to be natural when the measured value for 14C activity is 99.82 ± 0.22 percent, miRNA and exosomes are detected, it regulates the state and shows therapeutic or beneficial functional resilience, i.e., emerging properties, and exerts its activity via a physiological mechanism of action. The sum of these characteristics enables the determination of the product as a native matrix itself, thereby representing native natural intelligence.

[0164] The evaluation of the modification of the state and functional resilience can be carried out as follows.

[0165] The ability of a therapeutic or beneficial product to modify a pathophysiological state or a changed physiological state (e.g., by assisting the organism's homeostatic response) is an important feature for establishing its physiological mechanism of action. In fact, a product that acts through network - network interactions is expected to modify not a single function but a state when administered to a living body.

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

[0167] A product that exerts a physiological mechanism of action is also expected to be 100% natural (see above) and to exhibit flexibility and self - regulatory mechanisms observable in vivo, where different intracellular and intercellular messages and different regulations of gene pathways can provide the same result despite the different messages induced intracellularly. In the case of a therapeutic or beneficial product, this corresponds to the functional resilience of different batches of the product (with qualitative - quantitative variability in chemical composition).

[0168] The inventors determined whether the product of the present invention exerts its therapeutic or beneficial effect by modifying a pathological state or a changed physiological (not yet pathological) state, and whether the product exhibits functional resilience (i.e., maintenance of the therapeutic or beneficial properties of different batches of a given product containing one or more natural matrices despite different qualitative and quantitative compositions for different batches).

[0169] According to the present invention, this can be verified by performing a cell-based assay having a readout value representing the 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] Evaluation of (therapeutic or beneficial) functional resilience. As shown in the glossary and the above specification, in this specification and the claims, functional resilience is the maintenance of a measurable and verifiable therapeutic or beneficial efficiency in different batches of a product, despite variability in their qualitative and quantitative molecular composition.

[0171] Batches (e.g., tested batches 1, 2, and 3 of product C) are intended to be batches that are identical with respect to the manufacturing process and the type and amount of each component (since the main component 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 by the same procedure, e.g., a given type of extract from the same plant part of the same plant species), and thus, the variability in their qualitative and quantitative molecular composition cannot be attributed to different manufacturing procedures or different components, but can only result from inherent differences between natural matrices obtained from different organisms of the same species by the same procedure. The functional resilience of a product can be verified if different batches of the same product containing one or more natural matrices maintain, in a measurable and verifiable manner, their final regulatory activity underlying their therapeutic or beneficial properties, regardless of their qualitative-quantitative composition.

[0172] Accordingly, the present invention also relates to a method for determining the presence of 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, for a sample of the product or composition:

[0173] a. Measuring the 14C activity of the product according to the 1. ISO-16620-2;2015 (AMS) method to determine the naturalness of the product 2. Evaluating the presence of miRNA in the product or composition 3. Evaluating the presence of exosomes in the product or composition, and an evaluation step by performing these; b. Determining the presence of therapeutic or beneficial functional resilience between different batches of the product by comparing, batch by batch, in a cell-based assay where the readout represents 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 and / or pathological state treated by the product or composition; c. Evaluating whether the modulation of the biological activity underlying the desired therapeutic or beneficial effect results in the modulation of the overall altered physiological or pathological state from the readout in the cell-based assay; Hereinafter, 1. The measured value of 14C activity is 99.82 ± 0.22 percent, miRNA, exosomes, therapeutic or functional resilience are detected, and c. When the modulation in (c) results in the overall modulation of the altered physiological or pathological state, determining that the product or composition itself is a native matrix representing native natural intelligence.

[0174] According to one aspect of the present invention, before performing one or more cell-based assays, the method further includes: (1) providing a list of hallmarks representing the changed metabolic state and / or the pathological state; (2) for each of the hallmarks, identifying one or more modifications of bioactivities underlying the pathological state, thereby accurately identifying a bioactivity network in which the degree of regulation corresponds to the pathological state; and (3) identifying one or more parameters in which the degree of regulation corresponds to the degree of regulation of the one or more bioactivities underlying the therapeutic effect of the tested product, and determining, in the network, a tendency of regulation regarding upregulation or downregulation of the one or more bioactivities corresponding to the pathological state or the healthy state.

[0175] According to a preferred aspect, the changed 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, reduction of inflammation, and reduction of adipose tissue. Preferably, the bioactivity of (2) with respect to bone remodeling of the bone hallmark is selected from the bioactivities shown in FIG. 9.

[0176] More specifically, (1) providing a list of hallmarks representing a pathological state associated with a pathology or a pathological state resulting from the non-pathological changed physiological state, i.e., providing a list of hallmarks representing the disease or pathological state treated by the product of interest, or providing a list of characteristics that may be derived from a non-pathological changed physiological state whose homeostasis is assisted by the beneficial product of interest; (2) for each of the hallmarks, identifying one or more modifications of bioactivities underlying the pathology, determining the degree of regulation thereof representing the pathophysiological state associated with the pathology, and evaluating the opposite degree of regulation as the regulatory pattern of each of the activities representing a healthy physiological state; and (3) Identify the regulatory pattern underlying the detectable modification for each of one or more markers and each of the one or more biological activities in the diseased state, and for each of the parameters, set a regulatory pattern opposite to the one identified as a regulatory pattern conforming to the healthy physiological state. is.

[0177] When a therapeutic product is being tested, according to one aspect, the method of the present invention may include: (a) The following cell groups, (a1) At least one control group and at least two test groups of cells having a disease phenotype related to the intended use of the therapeutic product, or (a2) At least one cell group having a healthy physiological phenotype, and at least one control group and at least two test groups of such cells having a healthy physiological phenotype in which a disease phenotype related to the intended use of the therapeutic product has been induced, perform the at least one in vitro cell-based assay on, The step of treating each of the test groups of cells with one of the different batches of the therapeutic product; (b) Determine the degree of regulation or regulatory pattern of each of the parameters for each of the groups of cells in step (a), and calculate the respective regulatory values for each of the one or more biological activities; (c) A step of comparing the regulatory values, wherein the therapeutic product, for each hallmark, at least 50% of the one or more biological activities is regulated by each product batch such that the regulatory tendency of the network is consistent with the healthy state, and the regulatory values determined in (b) for each of the at least 50% of the one or more biological activities for the test groups of the cells in (a1) are each at least 0.15 different from those of the control group of the cells in (a1), or For each hallmark, at least 50% of the one or more bioactivities are adjusted by each product batch such that the regulatory trend of the network is consistent with a healthy state, and the adjustment values determined in (b) for each of the at least 50% of the one or more bioactivities for the test group of the cells in (a2) are each at least 15% different from those of the control group of the cells in (a2). A step of comparing the adjustment values, wherein the functional resilience of the product is demonstrated by the adjustment values for each of the one or more bioactivities of each test group of the cells being less than 20% different from the average of the values.

[0178] This means that the adjustment value of a given bioactivity of the test group faces the adjustment value of the same bioactivity of the control group respectively. Thus, a difference of at least 0.15 or at least 15% is the difference between the adjustment value of a given activity in the group of treated cells and the adjustment value of the same activity in the group of cells representing the control baseline.

[0179] In other words, the method can also be described as a method for evaluating whether a therapeutic product exerts an effect of treating a pathological condition through a physiological mechanism of action, including the following: - Providing different batches of a therapeutic product, where the product contains one or more natural matrices; - Providing a list of hallmarks representing the pathological condition, and for each of the hallmarks, identifying a set of parameters that enables an evaluation of the network of bioactivities on which the degree of its regulation is based for the therapeutic effect of the tested product, and determining the regulatory trend regarding the degree of upregulation or downregulation of activity in the network in the diseased state and the healthy state; (a) The following cell groups (1) At least one control group and at least two test groups of cells having a disease phenotype targeted by the therapeutic product, or (2) Performing at least one in vitro cell-based assay on at least one cell population having a healthy physiological phenotype, at least one control group and at least two test groups of said cells having a healthy physiological phenotype in which a disease phenotype targeted by a therapeutic product is induced, Treating each of said test groups of cells with one of said different batches of the therapeutic product, (b) Determining the degree or pattern of regulation of each of said parameters for each of said groups of cells and calculating a respective regulation value for each of said bioactivities, (c) Comparing said regulation values for each bioactivity in each cell group of step (b), At least 50% of said bioactivity for each hallmark is regulated by each product batch using the regulation trend of the healthy state determined in (b) and the regulation values determined in (b), and each of said at least 50% of one or more bioactivities for the test group of said cells in (a)(1) is at least 0.15 different from that of the control group in (a)(1), or When at least 50% of said bioactivity is regulated by each product batch for the test group of said cells in (a)(2) using the regulation trend of the healthy state determined in (b) and the respective regulation values of said at least 50% of one or more bioactivities determined in (b), and is at least 15% different from that of the control group in (a)(2) respectively, comparing said regulation values, whereby it is shown that the therapeutic product exerts its therapeutic activity via a physiological mechanism of action, Comparing said regulation values, whereby the functional resilience of the product is demonstrated by the regulation values for each bioactivity of each test group of said cells being less than 20% different from the average of said values.

[0180] The expression "at least 50% of said one or more biological activities for each hallmark" means that the degree of modulation of the network tends to be adjusted by each product batch that conforms to a healthy state. In the case of a single biological activity for a given hallmark, in order to meet the above requirements, it must be 100%, i.e., adjusted by the product in which the single activity was tested, which means that the tendency of modulation conforms to a healthy state. The expression "regulation value of said at least 50% of said one or more biological activities determined in (b)" refers to the regulation value determined in (b) of at least 50% of the biological activities that meet the requirement of being adjusted according to the modulation tendency that conforms to a healthy state. This applies, with the necessary modifications, to all aspects disclosed herein.

[0181] When a beneficial product (i.e., a product that has a beneficial effect on a physiological state that is changing but not yet pathological by assisting homeostasis) is tested, according to one aspect, the method of the present invention may include: (a) The following cell populations, 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 an abnormally regulated phenotype targeted by the beneficial product is appropriately induced and at least two test groups of cells taken from said control group, treating each of said test groups of cells with one of said different batches of the beneficial product, (b) determining the degree or pattern of regulation of each of said parameters for each of said groups of cells in step (a) and calculating a respective regulation value for each of said one or more biological activities, (c) comparing said regulation values, When at least 50% of the one or more bioactivities for each hallmark of the beneficial product is adjusted by each product batch such that the regulatory tendency of the network is consistent with a healthy state, and the regulatory value calculated in each (b) of the at least 50% of the one or more bioactivities for the test group of the cells is at least 0.15 different from that of the control group of the cells, respectively, and the functional resilience of the product is demonstrated by the regulatory value for each of the one or more bioactivities of each test group of the cells being less than 20% different from the average of that value In the case where it is shown to exert its homeostatic auxiliary effect through a physiological mechanism of action The step of comparing the regulatory values

[0182] This means that the regulatory value of a given bioactivity of the test group faces the regulatory value of the same bioactivity of the control group respectively. Thus, the difference of at least 0.15 or at least 15% is the difference between the regulatory value of a given activity in the group of treated cells and the regulatory value of the same activity in the group of cells representing the control baseline

[0183] In other words, the present method is a method for evaluating whether a beneficial product exerts its homeostatic auxiliary effect by regulating a changed physiological state, and can be defined as the following method: - Providing different batches of a beneficial product that aids homeostasis, the product containing one or more natural matrices - Providing a list of hallmarks representing a pathological state that may result from the changed physiological state, and for each of the hallmarks, identifying a set of parameters that enables evaluation of a network of biological activities on which the regulation of the product being tested is based, and determining the regulatory tendency regarding the up - and down - regulation of the activity in the network in the diseased and healthy states (a) The following cell groups (1) At least one control group of cells having a healthy phenotype, or at least one control group of cells in which an abnormally regulated phenotype targeted by the beneficial product is appropriately induced and at least two test groups of cells collected from the control group, perform at least one in vitro cell-based assay on, Treat each of the test groups of cells with one of the different batches of the beneficial product, (b) Determine the degree or pattern of regulation of each of the parameters for each of the groups of cells in step (a) and calculate the respective regulation values for each of the bioactivities, (c) Comparing the regulation values for each biological function in each cell group of step (a), The beneficial product is, When at least 50% of the bioactivity for each hallmark is such that each of one or more bioactivities for the test group of the cells in (a)(1) differs from that of the control group in (a)(1) by at least 0.15 according to the regulation tendency of the healthy state determined in (b) and each regulation value of the at least 50%, demonstrating an effect of assisting its homeostasis through a physiological mechanism by comparing the regulation values, The functional resilience of the product is demonstrated by the regulation values for each bioactivity of each test group of the cells differing from the average of the values by less than 20%.

[0184] In a preferred embodiment, the control group is regarded as a reference regulation baseline and the qualitative - quantitative regulation values for each of one or more bioactivities are regarded as 0.

[0185] As described above, the method of the present invention includes the following: (1) providing a list of prominent hallmarks representing the target pathological state (i.e., the pathological state to be treated by the analyzed product, or the pathological state that may result from the altered physiological state in which the analyzed beneficial product exerts an activity that aids its homeostasis); (2) for each of the hallmarks, identifying one or more modifications of the biological activity(ies) underlying the pathological state, thereby accurately identifying a network of biological activities whose degree of regulation matches the pathological state; and (3) identifying one or more parameters whose degree of regulation matches the degree of regulation of the one or more biological activities underlying the therapeutic effect of the tested product, and determining a regulatory trend regarding the upregulation or downregulation of the one or more biological activities that matches the pathological state or the healthy state in the network.

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

[0187] The modification of the state is a feature that cannot be obtained with a single API pharmaceutical product, and thus this feature excludes classical pharmaceutical mechanisms of action. However, in principle, the modification of the state can also be obtained using pharmaceutical products containing a cocktail of APIs.

[0188] The physiological mechanism of action requires that a therapeutic product or a beneficial product regulates the state in a manner that includes the overall cellular response to the network via network interactions, rather than at the point of network interactions based on the API mechanism of action (whether it is a single API or a cocktail thereof).

[0189] This also means the ability of a product to act with functional resilience (either therapeutic or beneficial), in addition to the regulation of the state, i.e., the ability to provide the same batch-by-batch therapeutic / beneficial effect despite the qualitative-quantitative compositional differences from batch to batch, which is, in other words, the result of regulating various parameters selected in a variable manner and still providing a preserved functional outcome.

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

[0191] As already explained in the terms of the terminology and the above description, (therapeutic or beneficial) functional resilience is the ability of a given product to modulate one or more bioactivities underlying a pathological or altered physiological state, despite the variations in the qualitative and quantitative composition of different batches of the same product, and thus, to induce different signals intracellularly, i.e., to exhibit biological equivalence intended as the same final outcome. As already mentioned above, it is known that pharmaceutical (API-based) products having different qualitative and quantitative compositions are not considered to be biologically equivalent.

[0192] The physiological mechanism of action means the overall interaction with the cells of the treated subject and does not mean the interaction with a specific cellular molecular target, and is the manner exerted by the living body as a result of network-network interactions. Physiological systems in 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 reliably reach a given goal (e.g., the response of an organism in the production of various proteins, the activation of various pathways, etc.). When a therapeutic product interacts with these systems, the therapeutic product can be involved in multiple pathways or mechanisms, including redundant pathways or mechanisms, to achieve its desired effect. This redundancy that brings about functional resilience contributes to the physiological mechanism of action of the product.

[0193] Therefore, functional resilience in therapeutic / beneficial products (intended as the ability of a therapeutic or beneficial product to maintain its intended functionality and effectiveness despite variability in the qualitative and quantitative composition from batch to batch) is an essential feature of the underlying 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, which comprises, if necessary with modifications, administering the product or composition according to the invention, alone or in combination with a therapeutically effective amount of a beneficial active ingredient, to a subject in need thereof.

[0195] The following examples are intended to illustrate the invention better and provide scientific support, and are not intended to limit the scope of the invention.

[0196] Example 1. Composition of the test formulation The experimental data reported below were generated using the following formulation of the product of the invention, also referred to hereinafter as "Product C". Coralline calcium powder 32% by weight Eggshell calcium powder 30.2% by weight Calcium coralline citrate powder 13% by weight Agaricus bisporus powder 4.65% by weight Dry extract of Equisetum arvense 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 invention within the claimed scope have also been tested and similar results have been obtained (data not shown).

[0198] Three batches of Product C were prepared starting from raw plant or natural materials of different lots, i.e., Batch 1, Batch 2, and Batch 3, with the same formulation.

[0199] Furthermore, permutations of the above formulation within the scope recited in Claim 1 were also prepared, and their correct biological activities (i.e., biological activities comparable to Product C) in cell-based assays as reported below were confirmed by preliminary data.

[0200] Powdered calcium carbonate from coral, calcium carbonate from Caribbean coral, calcium carbonate from eggshell, fine powder from Agave sisalana leaves, fine powder from Cetaria islandica thalli, fine powder from Champignon mushroom, Agaricus bisporus, and gum arabic, Acacia senegal were mixed at room temperature until the mixture was homogeneous, and then freeze-dy extract of Equisetum arvense spikes, Acerola, Malpighia punctifolia, and calcium citrate were mixed and added at room temperature in the weight-by-weight percentages disclosed above.

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

[0202] The freeze-dried extract was prepared by subjecting each plant material to extraction with 100% water (v / v) at 70°C for 2 hours, filtering to remove solid discharge materials. The obtained 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 obtained extract was stored at room temperature avoiding light and moisture until use.

[0203] Next, the product thus prepared was appropriately dissolved and / or diluted at a location suitable for the various assays disclosed below.

[0204] 2. In Vitro Assay In vitro studies were conducted on human adipose-derived mesenchymal stem cells (hADMSCs). These cells represent a convenient and readily available (non-invasive) source of mesenchymal stem cells that can differentiate into the osteogenic lineage if appropriately induced. In vitro tests have shown that hADMSCs can differentiate into mature active osteoblasts and produce a mineralized bone matrix when appropriately induced by culturing in a specific osteogenic induction medium (osteogenic / osteoinductive medium; OM) containing 10 nM dexamethasone, 0.2 mM ascorbic acid, and 10 mM β-glycerophosphate as sources of calcium and phosphate. Therefore, these cells are an optimal 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 conducted by treating hADMSCs at 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 biologically available 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), The transcriptional profile induced in the cells to evaluate the pathways and biological functions potentially affected by the treatment (gene expression using a microarray platform)

[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 containing antibiotics / calcein / serum + β-glycerophosphate and 2-phosphoascorbic acid but not dexamethasone): This medium does not contain dexamethasone (an external osteoinductive agent), and thus enables the evaluation of whether the added formulation can independently induce osteogenic differentiation through the measurement of ALP activity.

[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 enables the evaluation of whether the formulation can have a synergistic (or conversely inhibitory) effect on osteoblast differentiation induced by dexamethasone.

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

[0211] The hADMSC cell line was cultured in a humidified atmosphere containing 5% CO2 at 37 °C in a 100 mm Petri dish in growth medium (GM) [modified Ham's F12 Coon's medium supplemented with 10% FBS, 100 IU / mL penicillin, and 100 μg / mL streptomycin]. The medium was replaced with fresh GM every 3 days, and when confluence was reached, the cells were detached by trypsin treatment and seeded into a 24-well plate at a cell density of 1 × 104 cells / cm2 in GM until 70 - 80% confluence was reached. Subsequently, the medium was either replaced with osteogenic medium (OM) or maintained in GM with additional treatments and incubated for 7 - 35 days. The treatment medium was refreshed twice a week.

[0212] These cells were obtained during total surgery with informed consent from three different patients (PA42, PA59, and PA69) (Romagnoli et al., “In Vitro Behaviour 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 were characterized with respect to the main stem cell markers of mesenchymal stem cells (CD44, CD105, and STRO1) and their multipotency for the osteogenic phenotype by studying them in the Surgical and Translational Medicine Department of the University of Florence.

[0213] The time schedule used for the experimental setup is as follows.

Table 7

[0214] 2.1 ALP assay and calcium calcification deposit assay (Figs. 5 - 7) At the end of each incubation time point, the cells were washed with DPBS (twice), fixed with 4% PFA / DPBS for 15 minutes, washed with ultrapure water (thrice), dried, and stored at 4 °C until the assay. 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 at 37 °C for 15 minutes. 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, and expressed as μU / cm using a standard curve of 4-methylumbelliferone from 50 nM to 10 μM in 280 mM Tris-HCl buffer pH 9.0. 2 expressed.

[0216] Calcium calcification deposit assay.

[0217] Each well was incubated with 2 mL of 50 mM NaEDTA at 37 °C for 30 minutes. Then the solution was 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 as μg / cm2 using a standard curve of calcium calcification deposits solubilized in 50 mM NaEDTA from 25 ng / mL to 500 μg / mL.

[0218] The in vitro test made it possible to compare the efficacy profile of Product C with treatments 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 (increase in ALP activity), which calcify the cell matrix (increase the deposition of HA crystals) and can counteract bone fragility.

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

[0220] Treatment with synthetic calcium also showed an increase in ALP activity in the osteoinductive medium, and a typical "Bell-type" pattern was characterized by a peak in enzyme activity levels after 28 days of treatment (Figure 5).

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

[0222] The bell-shaped curves observed in all groups (Figure 5) reflect the dynamic nature of ALP activity during osteogenic differentiation, with the increase corresponding to early matrix maturation and decreasing as the cells transition to the later stages of bone formation. In the case of Product C, the earlier peak in ALP activity emphasizes its ability to predict important stages of the physiological processes of differentiation and mineralization. This early response suggests a role in promoting the initial stages of bone formation and being consistent with the physiological progression of bone formation.

[0223] Treatment with synthetic calcium in the osteoinductive medium showed the precise formation of the extracellular matrix and substantial deposition of hydroxyapatite (HA), in contrast to vitamin D treatment (Figure 6). In addition to its ability to mediate the involvement of mesenchymal stem cells into the bone lineage, Product C was able to induce the mineralization process early and completely, promoting the deposition of hydroxyapatite (HA) crystals. Indeed, after 28 days of treatment, Product C induced a statistically significant increase in hydroxyapatite crystals compared to synthetic calcium (Figure 5).

[0224] This result indicates that the initial peak of ALP activity observed for Product C (Figure 5) is converted into effective mineral deposition at the 28-day time point (Figure 6). In contrast, OM + vitamin D + synthetic calcium, despite its contribution to the initial ALP activity, does not result in significant mineralization on day 28 (Figure 6). The HA content remains significantly lower compared to the other groups, emphasizing that early differentiation alone is insufficient without effective downstream mineral deposition. This analysis emphasizes that the early activation of bone formation markers seen with vitamin D does not guarantee successful mineralization unless supported by a mechanism that drives the complete differentiation process.

[0225] Both OM + DMSO + synthetic calcium and OM + synthetic calcium serve as strong positive controls, showing strong mineralization levels on day 28. Product C achieves comparable results, further demonstrating its effectiveness in assisting the physiological processes of bone differentiation and mineralization. In contrast, Product C effectively combines its initial bone-forming activity with a sustained mineralization ability, suggesting a more comprehensive role in mimicking the physiological bone-forming process.

[0226] The results obtained indicate that Product C acts not only as a support for differentiation stimuli for osteoblasts in the presence of osteoinductive stimuli and as a calcium donor capable of mineralizing the extracellular matrix of bone, but also results in a statistically significantly greater increase in ALP activity compared to synthetic calcium in GM medium without osteoinductive agents, suggesting that it can alone induce the differentiation of mesenchymal cells into mature osteoblasts (Figure 7). In contrast, it is confirmed that calcium and vitamin D treatment does not increase ALP activity and cannot induce cell differentiation in the absence of osteoinductive stimuli.

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

[0228] In the osteoinductive medium, Product C also promotes the increase of ALP, showing a typical bell-shaped curve with a peak of activity on the 21st day, indicating an early stimulation of the differentiation process compared to the OM medium containing only synthetic calcium (synthetic calcium).

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

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

[0231] 3. Gene expression analysis At the end of the described treatment period, the cells were washed with 100 μl of PBS, lysed, and recovered 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 the cell lysates using the QIAsymphony RNA kit (Qiagen) equipped with a QIAsymphony SP device (Qiagen).

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

[0233] 3.1 Transcriptome data analysis Data analysis was performed using Transcriptomic Analysis Console software (TAC, ThermoFisher Scientific), which provides quality control analysis, performs normalization and summarization based on the signal space transformation-robust multi-chip analysis (SST-RMA) analysis algorithm, and provides a list of differentially expressed genes (Limma Bioconductor package, p-value ≤ 0.05).

[0234] 3.2 Bioinformatics modeling of experimentally observed transcriptome data For each investigation batch, the degree of regulation of gene expression related to the target effect was evaluated using Ingenuity Pathways Analysis (IPA) (QIAGEN\Inc., https: / / www.qiagenbioinformatics.com / products / ingenuitypathway-analysis).

[0235] IPA is an aggregator of scientific literature that enables the search for information on genes / proteins and the construction of networks that predict the behavior of biological systems according to their gene expression status.

[0236] 4. Evaluation of the transcriptional effect of product C treatment on osteogenic differentiation and mineralization of adipose tissue As demonstrated by the ALP and HA dosage evaluations, gene expression analysis also indicates that product C has significant effects on both bone and adipose tissue, promotes the clear differentiation into mature osteoblasts capable of mineralizing the extracellular matrix of mesenchymal stem cells, while inducing a decrease in bone adipose tissue, resulting in a reduction in bone fragility and an improvement in bone quality.

[0237] As shown in the heatmaps (Figures 10 - 11), treatment with product C (batch 1) results in the regulation of the expression profiles underlying bone remodeling, bone resorption, and bone loss, in addition to an increase in osteoblast differentiation and enhanced mineralization with respect to the increase in bone mineral density.

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

[0239] This product also induces an increase in bone synthesis markers, such as osteocalcin (OCN) and BMP (bone morphogenetic protein). This strongly indicates that osteoblast activity increases and, furthermore, that new bone matrix is formed, which is consistent with the measurement of HA crystal concentration.

[0240] A decrease in the bone remodeling process is also observed, which is important for bone maintenance and repair and potentially rebalances 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 amount and inflammatory processes in bone and adipose tissue as well as at the systemic level, suggesting an anti-adipogenic effect confirmed by improved glucose tolerance.

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

[0243] The assay was repeated on Batches 2 and 3 of Product C, and the data obtained are shown in Figures 13a and b.

[0244] 5. Definition of Pathophysiological Hallmarks of Diseases for Investigating IPA Changes in the characteristics of the healthy physiological state of "osteoporosis" in the state of the art were examined with particular attention to the following areas involved: - Bone remodeling - Osteopenia - Osteoblast differentiation - Mineralization - Reduction of inflammation - Reduction of adipose tissue

[0245] This knowledge was used to interrogate IPA 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 each of one or more identified bioactivities and the specific molecular pathways that support them. Information regarding the measured gene expression data (fold change value cut-off ≤ -2 and ≥ +2 and p-value ≤ 0.05) induced by each batch was then overlaid onto the resulting network to define the degree of regulation of the affected genes and related biological functions.

[0247] The regulation of the expressed genes was shown in different intensities of blue (indicating downmodulation) or red (indicating upmodulation). Based on the literature, the predicted calculated effects resulting as outcomes on the related biological functions were determined by the "IPA Molecular Activity Predictor" tool (MAP) and resumed with the visualization of the heatmap.

[0248] The color and intensity were converted to numerical values. Figure 9 shows the regulatory trends of selected bioactivities underlying the identified hallmarks of osteoporosis in a changed physiological state of bone (changed metabolism) or the pathological condition of osteoporosis, and the regulatory trends of the said bioactivities desired for the restoration of a healthy physiological state (opposite to the changed / pathological ones).

[0249] The results of the conducted tests led to 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 return reproducible biological effects, but it was also possible to identify batch-specific variations in the induced transcription patterns. Clearly, the induction of slightly different transcription patterns still results in the same desirable regulation of one or more biological activities. This is due to the functional resilience caused by the redundancy of the interaction between the components of the product and the body, whereby, through a multi-focal mechanism of action, different batches have different qualitative-quantitative compositions and induce the same effect (Figures 13a and b).

[0250] Therefore, the analyzed batches are considered to have equivalent biological outputs as the induction and suppression patterns are preserved.

[0251] The different transcription patterns and relative biological effects of different batches are intended as hallmarks of the inherent variability present in preparations composed of biological materials. From the results summarized in Figure 13, it is clear that the observed transcription patterns of different batches induce highly reproducible biological (functional resilience) effects and result in a general modification of the equivalence of the pathological processes and overall diseased states of all batches reported in Figure 13.

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

[0253] The following table summarizes all the methods used.

Table 8

[0254] The results are summarized in the following table.

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 showed high metabolomic complexity along with the presence of miRNAs typical of the organism.

Table 10

[0256] 7. Identification of Extracellular Vesicles 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 can potentially play a role in cell communication. Based on this, small RNA sequencing using the sRNAtoolbox framework was used to investigate the RNA content within these vesicle-sized particles. Among the sequences identified, two mature microRNAs (miRNAs) or their isoforms were found: miR8175 and miR166 are prominent miRNAs from the well-annotated Arabidopsis thaliana genome.

[0257] 7.1 Detection of miRNA and Role of Detected miRNA in Bone Metabolism The presence of RNA has been evaluated both quantitatively and qualitatively. Prior to proceeding with the kit extraction protocol, after homogenization using a QIAshredder column, RNA was extracted using a plant matrix-specific kit (Rneasy PowerPlant kit). The size distribution of the obtained RNA was performed using a Bioanalyzer 2100 equipped with RNA 6000 Nano, RNA 6000 Pico, and small RNA kits.

[0258] The size distribution of total RNA of 4 - 150 nt was detected.

[0259] Role of ath-miR8175 in mediating the differentiation and mineralization ability of Product C

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

[0261] Current and evolving topics in scientific research are the relevance of miRNAs present in food and their role in human nutrition. A number of studies have highlighted the importance of these miRNAs in the regulation of metabolic processes in response to diet.

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

[0263] 7.2 Detection of Supramolecular Structure in Product C Series Natural Matrix 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 the 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 rapidly. DLS is used to measure the particle size in colloidal samples, evaluate formulation stability, and detect aggregation. It is also ideal for analyzing the size distribution of isolated exosomes and microvesicles.

[0264] The analysis was performed by Alfatest Lab.

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

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

[0267] Analysis parameters: Measurement cell: plastic, (DTS0012) Detector: backscattering 173° (NIBS) Laser wavelength: 633 nm Measurement number: 3 Correlation time: adaptive Measurement position: automatic Attenuator: automatic Temperature: 25 °C Temperature equilibration time: 120 seconds Dispersant: water Dispersant refractive index: 1.33 Dispersant viscosity: 0.8872 cP (water) at 25 °C

[0268] The sample was analyzed under three different conditions: - Unfiltered - After filtration with a 0.45 μm nylon syringe filter - After filtration with a 0.1 μm nylon syringe filter

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

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

[0271] Results: The average results of Z-average and PdI obtained from three repeated measurements are reported in the following table. Z-average is the intensity weighted average 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 larger than the filtration size (4912, 0 nm). This also occurs in the 0.10 μm filtration where a bimodal system is observed with a signal of 156.8 nm beyond the filter size. Therefore, the data suggests the presence of supramolecular aggregates consisting of non-covalent intermolecular interactions. The data quality of the filtered samples is good.

[0273] Detection of Exosomes in Product C Batch 1 Preparation of ultracentrifugation samples The starting sample (Product C Batch 1) from which the ultracentrifugation fraction is prepared was weighed and resuspended in a certain amount of VIB or vesicle isolation buffer (20 mM MES; 2 mM CaCl2; 100 mM NaCl, pH 6.0) while maintaining a ratio of 5 mL of buffer per 500 mg of sample. The sample was incubated at room temperature for 20 - 24 hours with stirring to promote solubilization. After incubation, several centrifugations were performed at 4 °C with increasing speeds to isolate particles sized 30 - 500 nm. For ultracentrifugation, a T-1250 rotor (Thermo Fisher Scientific, 11718-5) and a Thermo Scientific™ Sorvall™ WX+ ultracentrifuge (Thermo Fisher Scientific™ 75000080, N°: 15342177) were used. The sample was centrifuged at 700×g for 20 minutes, the supernatant was filtered through a 0.45 μm filter while discarding the pellet, and then centrifuged at 10000×g for 30 minutes. Next, the supernatant was transferred to an ultracentrifuge tube and centrifuged at 40000×g for 70 minutes. The supernatant was transferred to a new ultracentrifuge tube and centrifuged again at 100000×g for 70 minutes. The supernatant was discarded, 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, stored at 4 °C and used within 24 hours, or stored long-term at -30 °C.

[0274] Extracellular vesicle staining and flow cytometry analysis

[0275] Extracellular vesicles from the 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 (10×) per sample and incubated at 37 °C for 30 minutes. Then, 170 μL of PBS (double-filtered at 0.22 μm) was added to each sample and read. To remove any non-specific 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 number NBP3-11691) was used as a positive control. Additionally, each sample was analyzed without staining to exclude autofluorescence.

[0276] Results Sample Product C Batch 1 was analyzed using flow cytometry as follows: Values of PBS marked by CellMask™ Green Plasma Membrane Stain were excluded from the region of interest as they were considered blank samples. In the Fluorescent Exosome Standards, multiple elements were identified in the same area that was considered positive.

[0277] The samples were then analyzed considering the number of elements present in the same region. The number of extracellular vesicles identified in the Product C samples was 2.7×10 6 individuals.

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

[0279] FTIR instrument and setup.

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

[0281] Sample preparation Samples of product C batch 1 were transferred to appropriate sample holders for solid and liquid ATR-FTIR analysis. Approximately 10 mg of the sample was deposited and pressed onto the diamond crystal of the ATR support. Before recording the measurement, it was confirmed that the entire sample holder was properly covered.

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

[0283] 11. Isotope abundance Isotope abundance analysis provides an atomic-level description of substances that emphasizes the effects of isotope substitution, such as geometric isotope effects (GIE) and kinetic isotope effects (KIE). GIE involves changes in the geometric structure of molecules caused by isotope substitution, particularly affecting hydrogen bonding. These changes can influence the molecular geometry and, consequently, the physical, chemical, and biological properties. KIE refers to the change in reaction rate caused by the substitution of isotopes within a molecule. Isotopes have different masses and affect the vibrational binding energy and activation energy. KIE is classified into primary KIE, where the substitution directly affects the rate-determining step of the reaction, and secondary KIE, where the substitution indirectly affects the reaction rate through changes in molecular geometry or electronic effects. Both effects are important for understanding the influence of isotopes on molecular behavior. Isotope abundance analysis was first performed on Batch 1 of Product C.

[0284] The 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. - 14C-activity was also tested by Chelab (Tentamus Company): - 14C-activity: Method ISO-16620-2;2015 (AMS), units % modern carbon (pMC).

[0285] The results were as follows.

Table 12

[0286] Therefore, isotope characterization was performed on Product C Batch 1. Furthermore, 14C activity was carried out on the Product C Batch 1 formulation excluding the raw material providing calcium carbonate. The measured 14C activity of a sample of 99.82 ± 0.22 percent modern carbon (pMC) corresponds to that of a substance from pure biogenic carbon. There is no evidence of a synthetic source in the analyzed substance.

[0287] Biodegradability Test According to OECD 301F:1992 Biodegradation is the decomposition of organic matter by microorganisms into simple natural constituents such as CO2, H2O, NH3, etc. Evaluating the biodegradability of chemical substances is important in environmental risk assessment. The ready biodegradation tests (RBT) proposed by the OECD are used to evaluate biodegradability by incubating chemical substances in mineral media with microorganisms. Over a period of 28 days, metabolic parameters such as oxygen consumption and CO2 production are monitored. A chemical substance is considered readily biodegradable if it passes the RBT. Primary biodegradation refers to the structural changes of a substance resulting from biological processes that can be measured by chemical analysis. For example, the OECD 301F method evaluates biodegradability by measuring the oxygen consumption in a respirometer under controlled conditions. The results are expressed as the percentage of oxygen consumed compared to the theoretical oxygen demand (ThOD) or the chemical oxygen demand (COD).

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

[0289] Assay Execution Sample Preparation The samples were treated according to the procedure reported in Respirometry - Pressure Measurement Method 301F (OECD). By using a BOD sensor and materials sufficient to perform the planned instrumental analysis, correct alignment can be achieved. Sodium acetate was used as a reference substance. The tests were conducted at a constant temperature of 22 °C.

[0290] Preparation of Inoculum Material The inoculum was prepared using activated sludge from different locations mixed in equal amounts. The inoculum was oxygenated, stirred, supplied with glucose, peptone, and monopotassium phosphate, and the values of oxidation - reduction potential, oxygen consumption, and total dry matter were monitored daily. The dry matter was determined at 100 °C to ensure the same amount (30 mg / mL) in the containers containing the test substance.

[0291] Inoculum Material Composition The inoculum was obtained by mixing activated sludge. The assembled inoculum was oxygenated, stirred, and supplied with glucose, peptone, and monopotassium orthophosphate. Oxygen, oxidation - reduction, and total suspended solids values were monitored daily. The total dry matter was determined before using the inoculum.

[0292] The composition of the microfauna was determined by optical microscopy analysis.

[0293] Reference Substance The manometric respirometry also requires the conduct of another test using ultrapure water fortified with a standard (sodium acetate) to evaluate the proper performance and reliability of the equipment.

[0294] Blank To evaluate the contribution of the liquid and inoculum material to the BOD value of the final product, a blank analysis was performed on the inoculated mineral medium.

[0295] Assay Conditions: The vessels are placed in a thermostat refrigerator set at 22 ± 2 °C and kept in a constant stirring state by the mechanical movement of the anchor. All this is carried out for 28 days by automatically measuring the oxygen drop value wirelessly every 6 hours, which becomes the absolute biodegradability value of the sample under investigation in 28 days.

[0296] Test results

Table 13

[0297]

Table 14

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

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

[0300]

Table 15

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

[0302] The reference substance reached the threshold within 14 days. Therefore, it can be stated that the test was conducted correctly and the ready biodegradability data collected for the substance under test is reliable.

[0303] The biodegradation calculations were performed at each sampling time for the reference substance, test sample, and blank.

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

[0305] Based on the fact that no correction for nitrification / denitrification was made, there was no evidence of nitrite and nitrate production. - mg / l of O2 consumed by the test substance: 51.25 mg / L. - mg / l of O2 consumed by the blank: 11.1 mg / L. - mg / l of O2 consumed by nitrification in the product: / - mg / l of test chemical in the container: 100 mg / L

[0306] Average BOD calculated value = 0.402 mg of O2 per mg of test substance.

[0307] To calculate the decomposition rate and thus the ready biodegradability, the chemical oxygen demand (COD) was evaluated by thermal acid dichromate oxidation in ultrapure deionized water: The test was conducted with a 50 mg / l solution of Abo11 and a COD of 67.1 mg / l was obtained.

[0308] COD = (mg of O2 consumed by the test substance / mg of test substance in the vessel) COD = 0.467 mg of O2 per 1 mg of test substance.

[0309] Average percent degradation on day 28: Blank estimate / BOD average corrected with COD pure substance = (0.402 / 0.467) × 100 = 86.08%

[0310] Interpretation of Results Validity Criteria The test is considered valid if: - The average biodegradation rate of the reference substance is greater than 60% after 14 days of incubation; - The difference between the extreme values of the replicate values at the plateau at the end of the test is less than 20%; - The oxygen demand of the blank is 60 mg O2 / L or less.

[0311] Interpretation If the substance reaches the level of biodegradation within 10 days after the start of decomposition, it is considered readily biodegradable, and the time when 10% of the substance is decomposed (10-day time frame) is considered, exceeding 60%, and furthermore, the level of biodegradation reached at 28 days is > 60%.

[0312] Results Meets the validity criteria of the test. The evaluation of the biodegradability of 100 mg / l of substance product C batch 1 was that during the experiment conducted, the product showed ready biodegradability under the conditions applied to the manometric respirometry test developed in accordance with EC Regulation 440 / 2008 updated to Regulation 640 / 2012 - Part c: Methods for the determination of 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% and met 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 test concentration, reached 86.06% of the related theoretical COD value, and thus showed sufficient 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, this substance exceeded 60% biodegradation within 10 days after the achievement of 10% degradation.

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

[0315] Product C - Situation in a diseased state (Figure 12, Panel A): Adipose tissue that has become dysfunctional and inflamed leads to an imbalance in bone homeostasis, adversely affecting the competition of mesenchymal stem cell potentialities that induce the differentiation of osteoblasts, osteoclasts, or adipocytes, and shifting this phenomenon non - physiologically towards osteoclast differentiation. This results in a decrease in the number of mature osteoblasts and an inability to ensure proper mineralization of the cell matrix with a loss of bone activity. In the context of dysregulated lipid metabolism and adipose tissue inflammation, a dysfunction loop is established between adipose tissue and bone, leading to the accumulation of adipocytes and osteoclasts, disadvantaging osteogenic components, and worsening bone fragility. Bone is also an endocrine - active organ and can thus affect events in other tissues at the systemic level, for example, through the secretion of osteocalcin (OCN) that promotes insulin secretion by the pancreas, insulin sensitivity in peripheral organs such as muscle, and the regulation of overall energy consumption (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] - Situation when treated with a drug reference (Figure 12, Panel B): The drug reference can only reduce the amount of adipocytes.

[0317] - Product C (Figure 12, Panel C): The state when treated with Product C can reproduce all the elements necessary to restore the correct bone metabolism turnover by interacting with the mesenchymal stem pool in bone and adipose tissue. This is beneficial for the formation of a solid, functional bone structure and, at the systemic level, restores the balance of metabolic dysregulation and reduces inflammation.

[0318] 13. Detailed analysis of three different batches of the product To understand whether the final matrix that makes up Product C is characterized by a matrix effect, a series of analyses were performed on the aforementioned three batches 1, 2, and 3 to grasp the characteristics of the product in different aspects. Figures 13a and 13b show that the three batches have the same desired therapeutic / beneficial effect on the in vitro cell-based assay. A targeted metabolomics analysis that can identify most of such molecular components, along with other analyses reported herein, was performed on the botanical matrix components of different batches of the product (see the following table).

[0319] As described above, the product consists of botanical and other natural matrices that together result in the final new natural matrix. Several analytical techniques have been used to identify and quantify the compounds belonging to the major classes present in the plants, and as a result, the composition of the botanical matrix components of the product has been evaluated. Metabolomics analysis does not enable an understanding of the dynamic changes within the components of the matrix, but enables a "photo" of the composition at the moment the analysis was performed.

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

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

[0322] The analytical methods used for the chemical characterization of each batch are described below. Based on the chemical properties of the classes of compounds present, the most appropriate analytical techniques have been employed. Analysis by chromatography methods combined with different detection techniques (e.g., GC and LC combined with their respective appropriate detectors) has made it possible to identify and quantify organic compounds as necessary. Inductively coupled plasma analysis using a single quadrupole mass spectrometer (ICP-MS) or an inductively coupled plasma optical emission spectrometer (ICP-OES) has made it possible to establish the levels of the 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 showed a high metabolomic complexity along with the presence of miRNAs typical of the organism.

Table 17

[0324] The results obtained indicate a significant compositional variability of each phytomatrix constituent in each batch, emphasizing that it is impossible to reproduce the properties of the matrix as the sum of its individual constituents. The studies conducted and reported herein (referring to cell-based assay results), together with the following data, demonstrate that the biological effects induced by a product cannot be reproduced by the sum of the effects induced by individual molecular constituents, but rather are the result of the mutual correlations and interactions between constituents: the matrix effect. This leads to the impossibility of formally defining a structure-activity relationship (SAR) according to the principles typically applied to APIs.

[0325] The results of targeted metabolomics highlighted more or less significant quantitative variations in the individual chemicals and miRNA classes within the plant matrix constituents of three batches of Product C. These variations, if considered as reference parameters, would lead to the a priori expectation that these batches have different therapeutic or beneficial effects. The analysis summarized in Figure 13 demonstrates that none of the identified individual molecular constituents comply with the criteria set for a single API, and thus that the matrix cannot be regarded as a set of APIs, despite the bioactivity being maintained across all the different batches evaluated.

[0326] As noted above, the same therapeutic effect is conserved across all batches (functional resilience). The product can induce the same response related to its intended use in a biological system via its physiological mechanism of action.

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

[0328] Isotope abundances of all batches To evaluate the isotope ratios between different batches of Product C, analyses were performed on batches prepared from different starting materials. 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), 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 of δ18O, δ15N, δ34S and δ13C overlap between batches, indicating a high reproducibility of the production process due to the preservation of this parameter.

[0331] 14. Definition of the conciliation unit of Product C activity (U-Product C): By analyzing the reproducibility criteria of natural products, the inventors focused on the reproducibility of the biological effects of the product. Analyses of different batches showed significant chemical differences from batch to batch. However, despite these differences, the batches were within the concept of showing functional resilience and maintaining biological activity. This approach is based on the principle of redundancy (functional resilience) and ensures that the product retains its biological effectiveness despite variations. As a result, the product has to be regulated in terms of units and distinguished by non-pharmacological mechanisms. This deviates from the conventional concept of qSAR and emphasizes the unique physiological properties in its mechanism of action.

[0332] The product is a 100% natural biodegradable matrix enriched with miRNAs and exosomes from different batches. This feature is better managed by applying the concept of any activity unit to the product's posology and batch release policy, rather than the weight or volume of conventional APIs.

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

[0334] For Batch 1, 10 U-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. A product comprising the following: 【Table 1】

2. The product according to claim 1, comprising the following: 【Table 2】

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

4. The product according to any one of claims 1 to 3, wherein the thuya 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.

5. The product according to any one of claims 1 to 4, wherein the thuya 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.

6. A composition comprising the product according to any one of claims 1 to 5 and at least one natural carrier.

7. The composition according to claim 6, formulated for oral, topical, rectal, vaginal, systemic injection or micro-needle injection administration.

8. The product according to any one of claims 1 to 5, or the composition according to claim 6 or 7, in the form of a lyophilizate, tablet, soft or hard gelatin capsule, powder, granule, filled vesicle, filled liposome or filled nanoparticle.

9. The product according to any one of claims 1 to 5 and 8, or the composition according to any one of claims 6 to 8, for use in the treatment of a bone fragility condition or for use in assisting said treatment, in a subject in need of said treatment.

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

11. The product or composition for use according to claim 9 or 10, wherein the bone fragility is associated with an increase in fat in the subject.

12. The product or composition for use according to claim 11, wherein the increase in fat is associated with obesity, metabolic syndrome, premenopause, perimenopause or menopause or andropause.

13. The product or composition for use according to any one of claims 9 to 12, wherein the bone fragility condition is osteoporosis or osteopenia.

14. The product or composition exerts its therapeutic or beneficial effect by assisting in the restoration of bone homeostasis through a biological activity network against the altered physiological state underlying the bone fragility condition by means of a physiological mechanism of action, and by exhibiting a therapeutic or beneficial functional resilience between different batches of the product or composition, The functional resilience is intended to maintain the therapeutic or beneficial properties of different batches of the product or composition, despite the different qualitative and quantitative compositions between batches. The product or composition according to any one of claims 1 to 8, or the product or composition for use according to claims 9 to 13.

15. The product or composition itself is a natural matrix, representing native natural intelligence, which has the sole ability to enable physiological and endogenous mutual binding with other self-organized entities in nature, such as the human species.

16. The product or composition according to claim 15, wherein the presence of native natural intelligence is determined through validation of its emerging property in the restoration of a healthy, physiological state of bone metabolism when the following conditions are met: Its 14C activity 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 among different batches of the product or composition, and the product or composition regulates an overall changed physiological state or pathological state.

17. A method for determining the presence of native natural intelligence in a therapeutic or beneficial product, provided that the product comprises or consists of a natural matrix, and the method is through the validation of its therapeutic or beneficial appearance characteristics and comprises the following steps: a. Evaluating the naturalness of the product or composition by the following:

1. Measuring the 14C activity in the product or composition by the ISO-16620-2;2015 (AMS) method, 2. Evaluating the presence of miRNA in the product or composition, 3. Evaluating the presence of exosomes in the product or composition; b. Evaluating the presence of a therapeutic effect or beneficial functional resilience among different batches of the product by comparing, batch by batch, in a cell-based assay where the read-out represents the modulation of one or more biological activities underlying the desired therapeutic or beneficial effect of the product on the relevant changed physiological and / or pathological states treated by the product or composition; c. Evaluating from the read-out in the cell-based assay whether the modulation of the biological activity underlying the desired therapeutic or beneficial effect results in the modulation of the overall physiological or pathological state; and Determining that the product or composition itself is a natural matrix representing native natural intelligence when 1. The measured value of 14C activity is 99.82 ± 0.22%, 2. miRNA, exosomes, therapeutic or functional resilience are detected, and 3. The modulation in c. results in the overall modulation of the changed physiological or pathological state.

18. (1) Providing a list of hallmarks representing changed metabolism and / or a diseased state; (2) For each of the hallmarks, defining changes in one or more biological activities underlying the pathological state, thereby accurately identifying a network of biological activities having regulation appropriate to the pathological state; and (3) Identifying one or more parameters having regulation consistent with the regulation of the one or more biological activities underlying the therapeutic effect of the tested product, and determining a regulatory trend from the perspective of upregulation or downregulation of the one or more biological activities in the network consistent with the pathological or healthy state, (4) further comprising: (5) The method according to claim 17. (6) Claim 19 (7) The method according to claim 17 or 18, wherein the altered physiological state is bone metabolism and / or the pathological condition is osteoporosis, and the hallmark is selected from bone remodeling, osteopenia, osteoblast differentiation, bone mineralization, reduction of inflammation, and reduction of adipose tissue. (8) Claim 20 (9) The method according to claim 19, wherein the biological activity of (2) for bone remodeling of the bone hallmark is selected from the biological activities shown in FIG. 9.

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