Natural matrix mixture for the treatment of osteoporosis

Abstract

The present invention relates to a 100% natural product consisting of a natural matrix that exhibits emerging properties, which product allows to re-establish the correct bone metabolism by correcting the behavior of mesenchymal stem cells and re-establishing the correct balance between the cell populations that allow bone growth or remodeling. As demonstrated by the inventors, the product of the invention is a natural matrix per se, representing a prototypical natural wisdom, which is the only one able to allow a physiologically endogenous interconnection with other entities self-assembled in nature, such as humans. By re-establishing the correct functioning of the bone metabolism, the product achieves beneficial results within the system scale, improving the systemic metabolic and inflammatory status. In fact, the present invention allows a paradigm shift from deterministic properties validation to probabilistic properties validation based on functional redundancy logic, which will provide an industrially viable source of innovation for manufacturers in the field of 100% natural therapeutic or beneficial products.

Description

Technical Field

[0001] This invention relates to a novel composition of substances made of 100% natural materials, which exerts a therapeutic or beneficial effect in the treatment of osteoporosis and has a physiological (rather than pharmacological) mode of action.

[0002] Specifically, this invention relates to the specially selected and used natural matrices, appropriately processed through specific processes and methods, to produce a final product intended for therapeutic or beneficial purposes, to prevent deviations from a balanced physiological state, or to restore the physiological state of vulnerable areas of bone. Each stage of the production process for such products adheres to the One Health principle (which acknowledges the interrelationship between human health, animal health, and environmental health), thus prohibiting the use of artificial forces or substances.

[0003] In fact, a fundamental requirement in the field of this invention is that the final product—that is, a product comprising or composed of one or more natural matrices—must retain the natural intelligence, i.e., the imprint of the biological domain to which each component of the product belongs, thereby maintaining a network capable of interconnecting and recognizing itself with other networks—whether natural or artificial (i.e., networks that were originally natural but acquired a degree of artificiality due to interactions with artificial components). This interconnection is considered the basis for rebalancing any disturbances in the network of events active in each interacting biological system. Each identified matrix will present a biophysical specification that makes it, in itself, an invention.

[0004] Each network is characterized by connections established within the matrix of the final product and within the physiological effects exerted by the product on the receiving organism. Product manufacturing validation can be performed and confirmed using probabilistic models based on the relationship between the retention of physiological activity characteristics and descriptors of the matrix itself generated using various biophysical analysis systems, including spectroscopy (NIR and other techniques), spectrophotometry (targeted and non-targeted metabolomics), and paper or X-ray crystallography (fractal measurements). While useful, traditional molecular chemical definitions of the individual substances contained within a substance cannot be used to validate such products because they do not represent its overall effectiveness and quality.

[0005] The selection of the substrate intended for application must be validated according to the latest and most specific current classification standards of the animal, plant, and mineral kingdoms. When used in conjunction with natural physical phenomena, the relationship between action and utility needs to be verified on a case-by-case basis, considering sound effects (music or other forms) and effects in the wave-particle domain, including effects on quantum properties.

[0006] With current technology, it is not always possible to fully describe the mechanisms of action; however, it is possible to verify the effects and reactions in various network interconnections that have already been verified at the biophysical level.

[0007] The purpose of this invention is to select and provide new entities or products, as well as systems capable of rebalancing, activating, or limiting the physiological functions of an organism under constantly changing specific metabolic states.

[0008] Preparations conceived in this way can rebalance the psycho-neuro-endocrine-immune system, which is viewed as a single system that controls and manages all other systems.

[0009] This invention brings a completely new technology to the medical field, transcending alchemy and originating in the early 16th century, bringing products and processes back to the aforementioned conceptual goal of One Health. This invention proposes a new revolution in artificial technology and naturally self-assembling substances, identifying existing rules or seeking new ones to ensure the construction of verifiable entities, primarily based on the concept of verifying their effects and activity on other organisms. These organisms are constantly changing life forms, requiring assessment of their physiological state at prescribed time intervals—a concept now found in personalized medicine. This invention aligns with the concept of science, which is understood as a set of knowledge capable of demonstrably verifying the effects of theoretical modes of action. Today, these methods are applied to establishing interconnections between all life forms; in this context, technological innovation is progressing so rapidly that it risks undermining the interconnection between human-generated (artificial) intelligence and nature.

[0010] Since the activities of the invention disclosed herein are not currently covered by the prior art, it is necessary to consider the entire product lifecycle from the end user to the relevant social context under the same concept of health.

[0011] The operational paradigm upon which this invention is based is hereby named “Bios Physiological Health”.

[0012] This paradigm aims to introduce an innovative approach to medicine, using natural matrices, alone or in combination, for treatment and self-health management to rebalance the normal physiological state of various organisms, including humans, through product-induced endogenous physiological effects. This involves identifying, selecting, and assembling natural entities with emerging properties that can be validated through the physiological modes of action of the final product and other methods developed in recent decades.

[0013] Interpreting the background from the perspectives of technological science and humanistic paradigms, and integrating them horizontally, forms the basis of this invention. While some properties of each matrix component of the product may already be known, the emerging properties of the new composition are unexpected.

[0014] Of particular interest is the role of identifying the genetic and epigenetic aspects of networks representing the natural matrix and their description at specific isotopic abundance levels.

[0015] To achieve the Bios Physiological Health paradigm, every stage of processing—from the selection of reproductive materials to agricultural and industrial stages, and then to the methods of use—must preserve as much integrity as possible of the original programming heritage, which is embedded in the natural intelligence of each created entity, at least in our known terrestrial dimensions. Validating a matrix derived from an epigenetic reality similar to a reference matrix, considered a benchmark because it possesses specific emerging properties for the metabolism of other organisms, including humans. For example, one factor negatively impacting epigenetic differentiation is varying soil conditions, as well as diurnal rhythms, monthly and annual variations. To protect the properties of natural systems—the only properties that can be claimed to have physiological interconnections with the entire creation—materials derived from alchemical processes (such as distillation), other synthetic or semi-synthetic processes, or products from genetically modified or transgenic organisms cannot be used. A new interpretation of the mysteries of natural programming, the roots of the evolution of life in both organic and inorganic matter, is needed. The consolidation of scientific evolution in recent decades has enabled us to reorient our understanding of the origins of progress based on reductionist determinism, which builds upon the development of alchemical techniques that began in the early 16th century. In the field of medicine, Paracelsus marks the beginning of the current evolutionary process, the Anthropocene.

[0016] The term Anthropocene describes the current stage of human evolution, dating back to various eras. However, in the context of this invention, the key date is 1492, marking the end of the early Renaissance humanist / neoplastic period. Political figures of this era include Cosimo the Elder and Lorenzo de' Medici, as well as artists and scientists such as Piero della Francesca, Luca Pacioli, Leonardo da Vinci, and Albrecht Dürer. In the 16th century, alchemical research was seen as a possibility for human domination of nature, and to this day, it continues to develop with the support of artificial intelligence rather than natural intelligence, inspired by biblical thought that "humans will rule over all creation," with the aim of perfecting divine creation.

[0017] 1492 was a symbolic year: Lorenzo de' Medici and Piero della Francesca died, and Columbus discovered the Americas. Humanity abandoned the Neoplatonist path of the 15th century and embarked on the Judeo-Catholic path; Paracelsus's alchemical practices applied to medicine marked the transition to the 16th-century Renaissance style, which, to this day, has led us toward a total and irreversible sixth extinction.

[0018] This invention demonstrates the feasibility of the resulting industrial discoveries in the medical field, but in principle, it applies to any production sector, aiming to address shifts in evolutionary paradigms. We often talk about protecting biodiversity, yet we never address and deliberately obscure the real problem: billions of tons of exogenous and non-biodegradable artificial matter are being released into planetary systems, inevitably and irreversibly poisoning the source of life, while a "live for the moment" attitude overrides the survival instincts of species.

[0019] This invention is primarily proposed within the context of patents, aiming to open up new research areas to explore and share natural intelligence, rather than artificial intelligence, which is largely incapable of preventing or mitigating the sixth extinction or laying the foundation for alternative developments to current advancements. Inventor Valentino Mercati and his collaborator Jacopo Lucci embarked on a path of studying what is useful to biological systems within nature itself, accumulating knowledge of agricultural and industrial production systems over more than 40 years and filing numerous patent applications based on this implementation strategy. Previous patents related to the method of this invention were primarily based on chemically relevant principles of instruments and diagnostic readings used to link physiological activities to emerging properties of the natural matrix and the innate defenses of each individual organism interconnected with it.

[0020] Because it is technically impossible to read the genetic and epigenetic information recorded in every cell of an organism, and the role of atomic isotope differentiation in molecular self-assembly and the interaction of each individual unit / individual with the "universe," the analysis followed and inspired by the methods disclosed in this paper would have been unimaginable decades ago. The shift from the artificially managed parameters of reliable molecules (at least partially purified and linked by strong thermodynamic forces, allowing strong bonds such as covalent bonds to act on a narrow molecular scale in other organisms) to the concept of a natural matrix (mysterious by definition and still considered partially unreliable for therapeutic purposes) is extremely difficult.

[0021] If, after five centuries of alchemical reductionism, a new and creative explanation is needed for a new medical technique, then this explanation must connect the most distant concepts and processes to a single field of application. As already stated, this is thanks to a philosophical legacy that questions the human condition: was the human species, like all other species, created by primordial life wisdom, and is its very purpose—as far as we can assume—to dominate creation, or has it been endowed through experimentation with abilities distinct from other organisms, and has thus advantageously integrated into creation to constitute a new ecological niche serving the universe?

[0022] For current inventions, the answer to this puzzle does not exist: humanity must return to the Neoplatonist thought of the early Renaissance, liberating the experimental duality of the human species from a dominant mentality in order to share its unique abilities in the universe with all creation. Humanity needs to reconsider Leonardo da Vinci's warning: "Man can only create his own offspring…", and reflect on the melancholic ideas of sophists like Piero della Francesca, Luca Pacioli, and Albrecht Dürer, who believed it impossible to comprehend and represent the beauty of creation and decipher its mysteries.

[0023] The era of establishing new research centers in molecular and cell biology has arrived, with bioinformatics and new physical sciences as indispensable focuses. Today, inventors can base their research strategies on socio-economic applications in new therapeutic areas, particularly those involving complex and / or chronic degenerative diseases, where restoring metabolic balance in organisms, whether naturally or artificially disturbed, will become an integral part of the emerging future.

[0024] This invention represents a new perspective in the field of medicine, re-examining the evolution of science from a viewpoint different from reductionist determinism. This alternative progress, which conflicts with universal or prevailing rules, will have to rely less on artificial intelligence and technological advancements, and more on the evolution of the laws governing our universe and life itself. The shift from artificial treatments targeting specific symptoms (even from a systems perspective of modern systems biology techniques) to a holistic approach encompassing the whole represents the foundation of current progress.

[0025] Specifically, this invention relates to a product composed of a natural matrix derived from the genera *Equisetum*, *Acacia*, *Tetracentron*, coral calcium, eggshell calcium, *Agave*, *Cymbidium*, *Agaricus*, and calcium citrate. This product assists in bone homeostasis by correcting stem cell behavior and re-establishing the correct balance among cell populations that allow for bone growth or remodeling, thus helping the organism rebuild proper bone metabolism. By assisting the proper function of osteocytes, the product achieves beneficial results, readjusting the differentiation process towards a bone lineage rather than an adipocyte lineage, and promoting improvements in systemic metabolism. This invention also relates to the use of said product in the treatment or adjuvant treatment of bone fragility diseases or conditions in subjects in need, and methods for treating or adjuvant treatment of bone fragility diseases or conditions, particularly when such bone fragility occurs postmenopausal or perimenopausal. Background Technology

[0026] Bone fragility is a significant social problem. National scientific associations have developed guidelines for the diagnosis and treatment of this condition, aiming to employ specific tools to assess fracture risk in populations. Osteoporosis is a socially significant disease because its incidence increases with age, affecting the majority of the population over 80 years of age.

[0027] Postmenopausal women are among the groups most affected by this disease. Metabolic changes during this transition lead to increased fat accumulation at the abdominal and bone levels. In particular, an excessively high-energy diet, including overly refined nutrients or an imbalance in the proportions of nutrients, coupled with the physiological trends of the postmenopausal woman's body, results in adipose tissue accumulation characterized by increased inflammation. From a cellular and molecular perspective, dysfunctional and inflamed adipose tissue leads to an imbalance in bone homeostasis, negatively impacting competition for mesenchymal stem cell reserves, which are essential for inducing osteoblast, osteoclast, or adipocyte differentiation, causing this process to unnaturally shift towards osteoclast differentiation.

[0028] This leads to the depletion of mature osteoblasts, which cannot ensure normal mineralization of the cellular matrix, resulting in loss of bone function. The decline in bone mass ultimately leads to osteoporosis, in which the anabolistic effects of osteoblasts on bone metabolism are masked by the catabolic effects of their counterparts, osteoclasts.

[0029] In cases of lipid metabolism disorder and inflammation of adipose tissue, a dysfunctional cycle can form between adipose tissue and bone, leading to the accumulation of fat and osteoclasts, while simultaneously promoting osteoblast components, thereby exacerbating bone fragility.

[0030] Fragility fractures can lead to complex disabilities, severe morbidity, decreased quality of life, and functional limitations. Patients with osteoporosis require comprehensive care, including a multidisciplinary and interdisciplinary approach implemented by a team, and individualized rehabilitation plans that include programs targeting specific intervention areas.

[0031] At the cellular level, osteoporosis involves changes in bone density and strength, leading to an increased risk of fractures. The disease is no longer simply considered a decrease in bone mineral density (BMD), as other factors, such as bone structure, also influence fracture risk. Bone loss begins around age 40 and worsens over time, a condition known as osteopenia, which may remain asymptomatic until it develops into osteoporosis. In osteoporosis, bones become extremely fragile, and even minor trauma or normal weight can cause fractures.

[0032] Bone tissue consists of cellular components and the extracellular matrix. The cellular components, including osteoblasts, osteoclasts, and osteocytes, comprise approximately 2% of bone mass but play a crucial role in bone metabolism. Osteoblasts, differentiated from mesenchymal stem cells (MSCs), are responsible for synthesizing the organic components of bone and mineralizing the matrix. Osteocytes are mature osteoblasts that remain in the matrix and are connected through tubules, forming a network that coordinates cellular activity. Osteoclasts are large, multinucleated cells responsible for bone resorption. Their maturation involves signaling from various molecules, including parathyroid hormone (PTH), estrogen, and interleukins, and is regulated by interactions with receptor activators such as RANKL and osteoprotegerin.

[0033] Adipocytes also play a crucial role in bone metabolism. They originate from the same MSCs that differentiate into osteoblasts and osteoclasts. Adipocytes in bone tissue participate in endocrine functions, secreting adipokines that regulate bone metabolism, such as leptin and adiponectin. While some adipokines stimulate bone formation, others promote bone resorption, which negatively impacts bone density and strength, increases fracture risk, and leads to osteoporosis. Excessive fat deposition in bone, often associated with metabolic syndrome or obesity, reduces bone mineral density, thereby impairing bone health and increasing bone fragility.

[0034] The osteoblast extracellular matrix consists of organic components (65%) and inorganic components (35%). The 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. The inorganic components are primarily composed of calcium phosphate in the form of hydroxyapatite crystals, along with small amounts of magnesium and sodium. Osteoblast mineralization of osteoid is regulated by alkaline phosphatase and osteocalcin, and is crucial for bone strength.

[0035] Bone remodeling is an ongoing process involving the resorption and formation of bone tissue, regulated by various hormones and cytokines. During remodeling, osteoclasts resorb old bone, while osteoblasts form new bone to replace it. This process is crucial 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 within the bone, coordinated by the basic multicellular unit (BMU) composed of osteoclasts and osteoblasts. After osteoclasts remove old bone, they undergo apoptosis, and osteoblasts form new bone; this process is tightly regulated by general and local factors.

[0036] Common factors include hormones such as PTH, calcitriol, and estrogen (which stimulate bone formation), and calcitonin (which inhibits osteoclast activity). Local factors include cytokines such as IL-1, IL-6, and tumor necrosis factor (TNF) (which promote osteoclast differentiation), and osteoprotegerins (which block RANKL-RANK interactions 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.

[0037] In other words, bone fragility is a systemic pathological condition affecting the entire body. As mentioned above, especially during menopause, a significant aspect of bone fragility involves the involvement of adipose tissue, including both intraosseous adipose tissue (osteopenic obesity) and systemic adipose tissue. Osteopenic obesity refers to the accumulation of adipose tissue in the bone marrow, which further damages bone and leads to bone fragility. Increased systemic adipose tissue is often associated with metabolic changes during menopause and can negatively impact bone health through inflammatory mechanisms and altered bone metabolism, leading to bone fragility and an increased risk of fractures. During menopause, women experience a significant drop in estrogen levels, a hormone that plays a crucial role in maintaining bone health. This decrease in estrogen accelerates bone loss, making postmenopausal women particularly susceptible to bone fragility.

[0038] Therefore, it is crucial to use the principles of systems biology and systems medicine to explore the physiological transitions women experience during menopause. This approach should consider the vast network of relationships between cells, tissues, organs, and systems involved in menopausal metabolic changes, explain their functions, and highlight the various contributing factors.

[0039] In contrast to healthy bone, bone fragility may be associated with metabolic disorders in bone tissue, leading to a loss of homeostasis across the entire functional network. This imbalance tends to cause mesenchymal stem cells to differentiate into adipose tissue and osteoclasts, resulting in bone depletion (particularly the osteoblastic component) and impaired bone mineralization. The typical accumulation of bone and abdominal fat in postmenopausal women can lead to an inflammatory state and eventually metabolic syndrome, resulting in a cycle of dysfunction across various bodily systems, including bone tissue.

[0040] In this context, it is important to consider the endocrine function of bone, particularly the secretion of osteocalcin (OCN). Osteocalcin is a hormone that also regulates insulin metabolism, expressed and secreted by mature osteoblasts. It stimulates pancreatic β-cells to secrete insulin, enhances the insulin sensitivity of muscle and white adipose tissue, and lowers blood glucose levels. Regulating this hormone plays a crucial role in reducing adipose tissue volume and inflammatory states, producing anti-adipogenic effects, improving insulin sensitivity, and increasing glucose uptake.

[0041] This framework emphasizes the strategic and functional importance of cell differentiation and bone mineralization processes. Both aspects must be finely modulated and stimulated to ensure good bone health. The most common response to these situations is the use of vitamin D and calcium supplements. While these substances are essential, they meet the body's needs in a limited and incomplete way, interacting with metabolic pathways in a punctual rather than holistic manner, thus failing to achieve satisfactory results. Furthermore, the scientific community has questioned the actual beneficial effects of vitamin D supplements, ultimately leading to the publication of AIFA Note 96 in Italy, which revised the prescribing guidelines for cholecalciferol-based drugs in Category A medicines, stating that vitamin D supplements do not significantly reduce the risk of fractures in uninstitutionalized, independent individuals.

[0042] Therefore, in addition to providing vitamins and calcium, it is necessary to interact with the body's physiological metabolic network through a systematic approach, utilizing the redundancy effect of related products. This method stimulates the differentiation of stem cells in bone tissue.

[0043] In summary, bone fragility is a multifactorial condition characterized by an imbalance between bone resorption and bone formation. Hormonal changes, metabolic disorders, and inflammation exacerbate this imbalance. Understanding the cellular components of bone and the complex processes of bone remodeling is crucial 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 medication and rehabilitation methods to reduce fracture risk and improve quality of life.

[0044] Despite these insights, current treatments fail to adequately address the complex interplay between increased adipose tissue and bone fragility, and no products promote / aid proper restoration of bone and systemic metabolic function. Traditional therapies primarily focus on hormone replacement or bone density maintenance, without adequately addressing the underlying inflammatory processes and metabolic dysfunctions that exacerbate bone fragility. This gap underscores the need for innovative treatments that modulate adipose tissue inflammation and bone metabolism, providing a comprehensive solution for the general prevention and treatment of osteoporosis and bone fragility, with a particular focus on postmenopausal women, promoting proper restoration of bone and systemic metabolic function. Summary of the Invention

[0045] The development of this invention's product (also referred to as "Product C" in the figures and embodiments) is based on the objective of creating a product based on a 100% natural matrix, containing plant and mineral components, that supports physiological bone metabolism, particularly in postmenopausal women. As mentioned above, menopause is a physiological transition process affecting various parts of the body and may be associated with underlying pathological conditions such as osteoporosis and metabolic syndrome. Due to modern epigenetic stimuli that were previously absent or less invasive, adipose tissue plays an increasingly important role in this transition.

[0046] The product of this invention, due to its emerging properties and natural matrix composition, is able to form a network (product) to network (receptor) mechanism and interact with physiological metabolic pathways occurring in women during menopause or in the transitional period (premenopause or perimenopause: premenopause is the time between a woman's first menstruation and the onset of perimenopause. Perimenopause is the transitional phase into menopause, typically lasting about six years). The product of this invention can correctly stimulate the differentiation of mesenchymal stem cells, promote the development of osteoblast lines, and inhibit the formation of osteoclasts and adipocytes, thereby contributing to the establishment of a healthy balance in bone metabolism. Furthermore, it can provide the necessary calcium to ensure the proper deposition of hydroxyapatite crystals within the bone matrix. The results also showed that the product of the present invention can reduce the amount of adipose tissue and inflammatory state by regulating osteocalcin expression, indicating an anti-adipogenic effect, as well as improving glucose tolerance, which suggests further desired effects within the target pathophysiological framework: its local and systemic effects can intervene in the dysfunctional cycle that occurs in systemic inflammation and metabolic disorders, affecting the differentiation of mesenchymal stem cells into white adipocytes and their subsequent accumulation.

[0047] As illustrated in the embodiments and figures, the bone resorption reduction induced by the product of this invention plays a fundamental role in the maintenance and repair of bone itself, promoting a rebalancing of bone density and structure. Furthermore, the product of this invention also exhibits systemic metabolic regulatory effects, inducing the expression level of osteocalcin (OCN), a hormone that also regulates insulin metabolism. Osteocalcin is expressed and secreted by mature osteoblasts and works by stimulating pancreatic β-cells to secrete insulin and enhancing insulin sensitivity in muscle and white adipose tissue, thereby lowering blood glucose levels and regulating total energy expenditure.

[0048] During product development, modern technology and experimental models that represent the physiological transition process of menopausal women from a systemic perspective are used to determine the product's bioactivity.

[0049] The data obtained and disclosed in the examples and figures demonstrate that the dual synergistic and systemic action of the product of the present invention makes it significantly superior to conventional calcium and vitamin D supplements that can only partially support physiological changes in the body. Furthermore, it is evident from the experiments and figures that the beneficial / therapeutic activity of the product of the present invention is delivered through a physiological mechanism of action, meaning that the product interacts with the body according to systems already familiar to the body, rather than according to foreign principles imposed by exogenous entities (e.g., synthetic molecules). This is why the product of the present invention, in addition to containing a vitamin D precursor and a calcium source, is designed to interact with the pool of mesenchymal stem cells in bone and adipose tissue, enabling it to reproduce all the necessary elements to rebalance proper bone turnover, which is crucial for the formation of strong and functional structures. Therefore, overall, the effects of the product have a systemic projection, contributing to the systemic rebalancing of metabolic disorders and underlying inflammation affecting postmenopausal women.

[0050] These characteristics make the product of this invention significantly superior to traditional calcium and vitamin D supplements, which can only partially support the body's physiological changes. It interacts with mesenchymal stem cells in bone and adipose tissue, recalibrating all the necessary elements to restore normal bone turnover, which is crucial for building strong and functional structures, and at a systemic level, rebalancing metabolic imbalances and reducing inflammation.

[0051] Therefore, due to the presence of complex plant substrates and natural calcium from various sources, the products of this invention clearly demonstrate that the complexity of physiological processes can only find the right synergy when accompanied by similar levels of complexity.

[0052] Therefore, the object of the present invention is a product composed of a natural matrix having the following formulation:

[0053]

[0054] And the use of the product in the treatment or improvement of osteoporosis, or the treatment or improvement of osteoporosis, including the administration of the product to patients in need or healthy persons at risk of developing osteoporosis.

[0055] Specifically, the product is used as a beneficial product to help people at risk of bone fragility maintain bone homeostasis. Attached Figure Description

[0056] Figure 1 Lipid metabolism disorders in postmenopausal women. Changes in energy and lipid metabolism leading to weight gain in postmenopausal women: Hormonal changes occur during menopause, including decreased estrogen and increased circulating androgens. These changes make postmenopausal women susceptible to changes in body composition, muscle loss, and abdominal obesity. In particular, the decline in estrogen leads to an increase in bone marrow-derived fat cells, resulting in higher levels of visceral fat in postmenopausal women. Increased lipolysis of visceral fat by adipose tissue lipoprotein lipases produces excess free fatty acids, which can lead to insulin resistance and metabolic diseases. Estrogen loss downregulates genes involved in β-oxidation, preventing the efficient use of free fatty acids as an energy source. Furthermore, older women exhibit increased fat accumulation due to upregulation of genes involved in adipogenesis. As a result, ATP production through β-oxidation decreases, while lipid synthesis increases.

[0057] Figure 2 Bone remodeling. Bone remodeling is a crucial process for maintaining bone strength and health in the human body. This process involves bone resorption controlled by osteoclasts and bone formation controlled by osteoblasts. When this process is imbalanced, bone resorption exceeds bone formation, leading to insufficient mineralization of the cellular matrix, resulting in decreased bone mass and function, and thus osteoporosis. Osteoporosis can cause fractures, pain, disability, decreased quality of life, and in severe cases, even death.

[0058] Figure 3 Bone remodeling in osteoporosis (abnormal bone remodeling). The abnormal bone remodeling process: Key factors influencing bone formation include a) differentiation of mesenchymal stem cells (MSCs) into osteoblasts, b) differentiation of MSCs into osteoclasts and adipocytes, and c) bone matrix deposition. This diagram illustrates abnormal remodeling, where increased bone resorption and decreased osteogenic formation lead to osteoporotic bone. In abnormal remodeling, osteoclasts play a crucial role by excessively breaking down bone tissue, while adipocytes exacerbate the imbalance by promoting adipocyte formation beyond osteocyte formation. As a result, bone becomes brittle and loses its structure and function.

[0059] Figure 4The Influence of Adipocytes on Bone Fragility. Osteopenic obesity is characterized by the infiltration of fat into bone, including adipocytes differentiated from MSCs. These adipocytes are out of balance with the bone cell composition, accompanied by bone loss. Inflammation plays a significant role in the development of osteoopenic obesity and has been extensively studied. Disruption of the connection between bone and adipose tissue is considered a contributing factor. The accumulation of adipose tissue in bone affects bone structure and weakens it.

[0060] Figure 5 ALP activity in osteogenic induction medium (OM). This figure shows the alkaline phosphatase (ALP) activity in hADMSCs cultured in OM under the following different experimental conditions, normalized to DNA content (ALP µU / µg DNA) over time (4 to 35 days): Product C, synthetic calcium with or without DMSO carrier, or vitamin D+ synthetic calcium (with DMSO carrier). ALP enzyme activity data are calculated as the mean ± standard deviation (SD) of four replicates at six different osteoblast induction time points. Significance was calculated using Anova and Bonferroni assays. = p<0.05 vs. OM + DMSO + synthetic calcium; = p<0.01 vs. OM + DMSO + synthetic calcium; $ = p<0.05 vs. OM + synthetic calcium; $$ = p<0.01 vs. OM + synthetic calcium.

[0061] Figure 6 Hydroxyapatite (HA) deposition in bone-inducing medium (OM). Quantitative analysis of calcium mineralization deposits in hADMSCs cultured in OM in the presence of product C, synthetic calcium (with or without DMSO carrier), or vitamin D+ synthetic calcium (with DMSO carrier) for 4 to 35 days. Data are calculated as mean ± standard deviation (SD) of four replicates. Significance was calculated using Anova and Bonferroni tests. $ = p < 0.05 vs. OM + synthetic calcium.

[0062] Figure 7ALP activity in non-osteogenic induction medium (GM). This figure shows the alkaline phosphatase (ALP) activity in hADMSCs cultured in GM under the following different experimental conditions, normalized to DNA content (ALP U / µg DNA) over time (4 to 35 days): Product C, synthetic calcium with or without DMSO carrier, or vitamin D+ synthetic calcium (with DMSO carrier). ALP enzyme activity data are calculated as the mean ± standard deviation (SD) of four replicates at six different osteoblast induction time points. Significance was calculated using Anova and Bonferroni assays. $ = p < 0.05 vs. GM + synthetic calcium.

[0063] Figure 8 Comparison of Product C activity with Vitamin D and Calcium in osteoblast differentiation. The figure shows that while data obtained using Vitamin D do contribute, it does not itself provide differentiation stimulation, nor does it directly provide calcium to promote the formation of mineralized bone matrix. Synthetic calcium alone cannot interfere with the differentiation process; it only provides calcium for bone formation. Product C alone can reproduce the entire physiological process. This product can locally and systematically provide the calcium required for the proper deposition of hydroxyapatite crystals in the bone matrix, and it can also properly stimulate the differentiation of mesenchymal stem cells, which is beneficial to the development of osteoblast lines, and inhibit the formation of osteoclasts and adipocytes.

[0064] Figure 9a and 9b Examples of osteoporosis markers (Column 1), the bioactive network that collectively defines the pathological state (Column 2), and the regulation under the pathological state (Column 3). The regulatory trends of the bioactive network are consistent with those of a healthy physiological state (Column 4). Dark gray: upward regulation; light gray: downward regulation.

[0065] Figure 10a and 10bIn osteoporosis, regulation of selected bioactive networks was assessed in a cell-based assay using human adipocyte-derived mesenchymal stem cell lines (hADMSCs) capable of differentiating into osteoblasts and mineralizing the extracellular matrix (ECM). The column 1 represents the markers, the column 2 the bioactive networks, the column 3 the regulatory trends of the bioactive networks under pathological conditions, the column 4 the regulatory trends of the bioactive networks consistent with healthy physiological states, the column 5 the appropriately induced cells representing the regulation of bioactive networks in unregulated controls, and the column 6 the regulation induced by the test product (Product C). The cell-based assays showed that the product sample regulated the entire selected bioactive network according to trends consistent with characteristics of healthy physiological states. The numbers reported in each box represent the value (z-score) that quantifies this regulation, calculated based on an example representing each observed bioactive regulation.

[0066] Figure 11a and 11b In osteoporosis, regulation of selected bioactive networks was assessed in a cell-based assay using human adipocyte-derived mesenchymal stem cell lines (hADMSCs) capable of differentiating into osteoblasts and mineralizing the extracellular matrix (ECM). The column 1 represents the markers, column 2 the bioactive networks, column 3 the regulatory trends of the bioactive networks under pathological conditions, column 4 the regulatory trends of the bioactive networks consistent with healthy physiological states, column 5 appropriate induction of cells representing regulation of the bioactive networks in unregulated controls, column 6 regulation induced by the test product (Product C), and column 7 regulation induced by the reference drug (DIBASE). The cell-based assay showed that the product sample regulated all selected activities of the network according to regulatory trends consistent with characteristics of healthy physiological states. On the other hand, the reference drug did not effectively regulate the entire bioactive network required to define a healthy physiological state. The numbers reported in each box represent the value (z-score) that quantifies the regulation, calculated based on an example representing each observed bioactive regulation.

[0067] Figure 12a , 12b And 12c. Network analysis of osteoporosis ( Figure 12a (Figure A) and treatment with reference medication ( Figure 12b (small image B) and treatment with product C ( Figure 12c(See Figure C). Gray squares represent basic nodes characterizing pathophysiological or altered physiological states and specific sites of interaction between pathology and the body. Arrows next to the nodes indicate specific regulation for each condition, with intensity expressed as a multiple of the arrow itself. Gray squares further link to a network of bioactivities whose regulation has been experimentally demonstrated. These bioactivities are represented by black (up-regulation) or white (down-regulation) circles, their magnitudes proportional to their experimentally demonstrated regulatory magnitudes. Network analysis shows that product C affects the body in a systemic manner, modulating the desired activity more than reference drug treatment, according to regulatory trends consistent with healthy physiological states.

[0068] Figure 13a and 13b In osteoporosis, regulation of selected bioactive networks was assessed in cell-based assays using human adipocyte-derived mesenchymal stem cell lines (hADMSCs) capable of differentiating into osteoblasts and mineralizing the extracellular matrix (ECM). Column 1 represents the markers, column 2 the bioactive networks, column 3 the regulatory trends of bioactive networks under pathological conditions, column 4 the regulatory trends of bioactive networks consistent with healthy physiological states, column 5 the appropriately induced cells representing the regulation of bioactive networks in unregulated controls, column 6 the regulation induced by batch 1 of product C, column 7 the regulation induced by batch 2 of product C, and column 8 the regulation induced by batch 3 of product C. The cell-based assays showed that all tested product batches regulated all selected active networks according to regulatory trends consistent with characteristics of healthy physiological states (regulation of state and functional resilience). On the other hand, the reference drug failed to effectively regulate all bioactivities required to define a healthy physiological state (it did not act on the networks). The numbers reported in each box represent the value (z-score) that quantifies the regulation, calculated based on examples of regulation of each bioactive network observed.

[0069] Figure 14 FTIR spectrum of product C in batch 1.

[0070] Figure 15 The timetable used for setting up in vitro experiments.

[0071] Glossary

[0072] Unless otherwise defined herein, scientific and technical terms relating to this invention shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include plural terms, and plural terms shall include singular terms.

[0073] In this specification, the term "synthetic calcium" refers to a calcium-containing compound synthesized in a laboratory through a chemical reaction.

[0074] In this specification, the term "calcium citrate" refers to a compound obtained by reacting calcium (e.g., coral calcium) with lemon juice.

[0075] In any point in this specification or claim, the expression "comprising" or "including" may be replaced by "consisting of".

[0076] In this application, "natural matrix" refers to a material composed of a network of numerous components / ingredients, obtained directly from members of nature or their naturally occurring parts (i.e., from natural raw material sources) without significant processing or synthetic alteration. "Without significant processing or synthetic alteration" means that the matrix was obtained from the raw material source without the use of denaturing processes. In other words, the natural raw material source is processed only manually, mechanically, or by gravity, for example by dissolving in water or other naturally occurring solvents, such as water, water-alcohol solutions, etc.; by flotation; by extraction with water or other naturally occurring solvents; by steam distillation or simply by heating to remove water or any other naturally occurring solvent; or by any means of extraction from the air, provided that "natural matrix" does not include the aforementioned members of nature or their naturally occurring parts. Specifically, according to the invention, a natural matrix is ​​a 100% natural and biodegradable material composed of natural components that have not been denatured during the production of the matrix from the starting material, and without the intentional addition of synthetic products throughout the process. In this specification, 100% biodegradability is considered "easily biodegradable" according to OECD biodegradability testing. These characteristics ensure the maintenance of the matrix effect, which is conferred by the structural interactions (material interactions) of the matrix components and the functional interactions (non-material interactions) that become apparent when a biological system is exposed to the natural matrix. In other words, a natural matrix or mixture of natural matrices is a material obtained from self-assembling entities in nature, processed to retain their natural biophysical properties that determine their physiological interactions with other organisms (e.g., human organisms). Their emerging properties can be expressed by contributing to the rebalancing of metabolic processes or states of the receiving organism and / or certain organs or tissues, and the physiological activities that will be activated in each specific condition. According to the invention, a natural matrix can be derived from materials obtained from any source in the living world, namely, prokaryotes, protists, fungi, plants, and animals. Therefore, the term encompasses plant natural matrices, animal natural matrices, fungal natural matrices, protist (archaea or bacteria) natural matrices, and prokaryotic natural matrices. Natural matrices can also include natural inorganic materials, such as minerals obtained from natural raw materials. In this specification, the term "natural matrix" or "one or more natural matrices" is a synonym for "complex natural system" or "natural material" as defined below.

[0077] Examples of naturally occurring parts of an organism can be, for example, the roots, leaves, bark, fruit, flower, or parts, organs, or tissues of a plant.

[0078] In any part of the instruction manual, the general term "natural matrix" may be replaced with:

[0079] Plant-based natural substrates or natural substrates obtained from plants

[0080] Animal-derived natural substrates or natural substrates obtained from animals or animal products (such as eggs or milk),

[0081] Fungal natural substrates or natural substrates obtained from fungi,

[0082] Protozoan natural matrix or natural matrix obtained from protozoa

[0083] Prokaryotic natural matrix or natural matrix obtained from prokaryotes

[0084] Alternatively, plant materials and / or extracts, animal tissue or organ extracts, fungi and / or fungal extracts, or mixtures thereof may be used, wherein the extraction process does not include a denaturing step (e.g., temperature or use of a denaturing solvent).

[0085] The word "plant" is synonymous with "herb".

[0086] The term "natural" matrix emphasizes that, because its acquisition involves no denaturation, it retains the integrity and complexity of the original natural source's component / component network. Therefore, natural matrices do not include compositions of natural sources rich in synthetically produced or isolated specific molecules from natural raw materials. Furthermore, natural matrices can only be obtained through processes that do not involve extensive processing or chemical modification, separation, purification, or molecular extraction.

[0087] Due to the supramolecular self-assembly of natural matrix components and the functional interactions between them, the entire matrix behaves as a complex network that interacts not with individual target molecules, but with the receptor network (also organized as a network) in the receiving organism. Therefore, the interaction between the natural matrix and the receiving organism is not the result of a point-to-point interaction like that of common drug APIs, but rather the result of an interaction between the "interactor" network (i.e., the matrix) and the "receiver" network (i.e., the organism to which the matrix is ​​applied).

[0088] The term "natural matrix" may also be used in any part of the specification and claims to refer to "complex natural system".

[0089] In the specification and claims, the term natural matrix should not be interpreted as a natural product. Rather, a natural matrix is ​​a product obtained from a natural organism as described above and processed (e.g., extracted) by techniques that do not significantly alter the biological structure and the related supramolecular and functional interconnections between the components within the matrix, i.e., without the use of denaturation techniques and without containing additional isolated or synthetic molecules or molecular classes.

[0090] Based on this specification and emerging characteristics in the art, this term defines the properties of a natural matrix or natural material according to this specification, namely, properties that are not represented by the simple sum of the properties of each individual component / part of the matrix / material, but by the functional and structural interactions between all components / parts of the matrix / material, which are also the result of supramolecular self-assembly of the components / parts within the matrix / material itself.

[0091] Therefore, "emerging properties" refer to the technological effects, such as therapeutic or homeostatic auxiliary properties (i.e., beneficial effects), that arise from the interactions and relationships between natural matrix components / components on the receiving biological system. By definition, emerging properties are those that cannot be immediately apparent or even predicted based solely on the individual characteristics of each component / component of the matrix. Instead, they "emerge" when all components / components of the matrix network interact with each other and with the receiving network of the living system in dynamic and complex ways. Emerging properties have been extensively discussed in various scientific and systems-oriented fields, including physics, chemistry, biology, and complex systems theory.

[0092] Therefore, emerging properties are those that cannot be predicted in advance by qualitative and quantitative knowledge of each component in a given composition or matrix, and thus cannot be attributed to one or more specific APIs. Therefore, although multi-drug compositions may exhibit unpredictable synergistic effects, the properties of the composition are still attributed to the specific APIs contained therein and their amounts.

[0093] Regarding emerging properties (specific to natural matrices), the observed emerging properties cannot be redirected to a specific API and are retained in different batches of a given matrix or a mixture of matrices, despite differences in the qualitative and quantitative composition of the batches (functional resilience, see below).

[0094] According to this specification, synthesis has the generally accepted meaning in chemistry. Generally, in chemistry, the term "synthesis" refers to the origin or source of a material or substance. Synthetic substances or materials are produced by humans through artificial synthesis, that is, through laboratory chemical reactions, usually by reacting simpler chemical substances to produce more complex chemical substances. These reactions often use different pathways, temperature conditions, pressure conditions, energy sources, and / or catalysts than those used in living organisms.

[0095] Examples: Synthetic substances or materials include plastics, pharmaceuticals, and many industrial chemicals. For instance, nylon is a synthetic polymer made through chemical synthesis, and aspirin is a synthetic drug produced through specific chemical reactions.

[0096] According to this specification, functional resilience (also known as “redundancy”) means the therapeutic or beneficial (steady-state supportive) resilience of a therapeutic or beneficial product containing or composed of one or more natural matrices; this term describes the maintenance of therapeutic or beneficial properties in different batches of a given product containing or composed of one or more natural matrices, despite differences in qualitative and quantitative composition between batches, which is necessarily (inherent) in a product containing or composed of one or more natural matrices. As those skilled in the art will recognize, each time different batches of starting materials are used, the resulting natural matrices have unique qualitative and quantitative compositions at the molecular level, a characteristic of individual diversity among organisms of the same species.

[0097] A healthy physiological state refers to the state in which the body, organs, apparatus, systems, or body regions and their internal processes of an organism are in optimal condition and operate within the normal parameter range for that individual; that is, a state tending towards homeostasis. In the context of one or more biological activities known to be markers of a specific disease or pathological condition or alteration, a healthy physiological state refers to the state in which said one or more biological activities are in optimal condition and operate within the normal (healthy) parameter range. This state is characterized by the absence of significantly abnormal cellular or molecular processes associated with the specific disease under consideration. When the trend of change in one or more biological activities is known to be consistent with pathology or pre-pathological conditions, a healthy physiological state can be considered to be represented by the opposite trend of change in said activities.

[0098] The term considers markers of a specific disease, that is, unique characteristics or traits typically observed in individuals affected by the disease. These markers may include specific cellular behaviors, molecular pathways, typical pathways, or physiological responses that play a key role in the development or progression of the disease.

[0099] In summary, in the context of a specific disease or pathological / alterative condition, a healthy physiological state refers to a state in which one or more biological activities associated with known markers of that disease or pathological condition are regulated in a direction consistent with the non-pathological / unaltered state (in other words, opposite to the pathological / altered state).

[0100] Therefore, the healthy physiological state according to the invention also indicates the direction of regulation of one or more biological activities known as markers of pathological conditions under homeostasis (i.e., before the onset of a pathological condition), in other words, the direction of homeostatic regulation of one or more biological activities attributable to a specific system, region, device, or organ of a healthy subject.

[0101] Altered physiological state and altered homeostasis are closely related concepts, describing deviations from the normal functioning and balance of the body's internal environment. While they overlap, there are some distinctions between the two terms:

[0102] Altered physiological state: This term encompasses a wide range of changes in the body's normal functions, including disruptions to organ systems, biochemical processes, and cellular function. Altered physiological states can be caused by a variety of factors, such as disease, injury, medications, environmental factors, and psychological stress. Examples include fever, inflammation, hormonal imbalances, and impaired organ function.

[0103] Homeostasis (alteration): Homeostasis refers to the body's ability to maintain a stable internal environment in the face of external or pathological changes. This stability is achieved through regulatory mechanisms that keep variables such as body temperature, blood pressure, pH balance, and blood glucose levels within narrow ranges. When these regulatory mechanisms fail to maintain balance, homeostasis is altered, leading to deviations from the body's normal set point. These deviations may be temporary or chronic and may involve compensatory mechanisms to restore balance.

[0104] In summary, altered physiological state describes observable changes in normal bodily functions, while altered homeostasis refers to the potential disruption of the body's regulatory mechanisms that maintain internal stability.

[0105] Altered homeostasis leads to altered physiological states, as disruption of homeostasis mechanisms results in physiological imbalances and manifestations of disease or dysfunction. Homeostasis-supporting products are those that assist the body in restoring its internal environmental stability when changes occur.

[0106] According to this specification, markers of a disease, pathology, or medical condition have the meaning conventionally used in the art. A marker of a known disease is an indicator that can mark the progression or control of a given disease, pathology, or pre-pathological condition, and often together represent a general pathological state associated with that given pathology. These markers (also called “key indicators”) are typically a set of characteristics or patterns that physicians monitor over time to track the onset, progression, or regression of a particular disease. In summary, a marker of a disease is a defining characteristic or property whose alteration is an indication of a given pre- or medical condition that aids in identification, diagnosis, monitoring, and understanding. For example, for neurodegenerative diseases (NDD), at least eight NDD markers are known in the art: (pathological protein) aggregation, synaptic and neuronal network (dysfunction), (abnormal) protein homeostasis, cytoskeleton (abnormality), (altered) energy homeostasis, DNA and RNA (defects), inflammation (increased), and neuronal cell death (increased). In cancer research, cancer markers are a set of unique features typically found in cancer cells. These features include (continuous) proliferation signaling, (evasion) of growth inhibitors, (resistance) of cell death, (achievement) of replication immortality, (induction) of angiogenesis, and (activation) of invasion and metastasis.

[0107] Disease markers, parameters associated with said markers (e.g., biomarkers), one or more biological activities associated with said characteristics, etc., form a framework for studying diseases, pathologies, or medical conditions using a comprehensive / holistic approach.

[0108] Signs of physiological changes typically include observable changes in various aspects of bodily function, which can be revealed through symptoms, signs, or laboratory test results.

[0109] Changes in physiological state usually reflect a disruption of the body's homeostasis mechanisms, leading to deviations in physiological parameters from normal values. These imbalances may involve changes in thermoregulation, fluid and electrolyte balance, acid-base balance, glucose metabolism, or other regulatory processes.

[0110] Overall, markers of physiological changes provide healthcare providers with valuable clues to identify underlying causes, assess severity, and guide appropriate interventions to restore normal function and promote recovery.

[0111] A reference drug is a standard or preferred treatment that is typically selected or chosen for a particular medical condition or disease. It is usually determined based on factors such as its effectiveness, safety, cost, and clinical experience. The reference drug serves as a benchmark for comparison with other drugs, especially when evaluating generics, new therapies, or alternative treatments. It is often the first-line drug recommended by medical guidelines or healthcare providers for the treatment of a specific disease.

[0112] Primal natural intelligence represents the inherent ability of the natural matrix to preserve and transmit biological and physicochemical information necessary for interaction and integration with other life networks, using the logic inherent to the receiving organism, as this information is known to the organism and therefore endogenous relative to it. This intelligence is a manifestation of natural self-creation, the ability to self-organize and adapt to environmental stimuli without human intervention, which would transmit information according to point-like logic and through a medium unknown to the receiving organism, and therefore exogenous relative to it.

[0113] The term "physiological interconnection" is defined as "endogenous" physiological interconnection, describing the ability of the natural matrix and the recipient's biological system to interact in a harmonious and functional manner, stimulating internal (endogenous) responses to restore a balanced physiological state. This interaction is based on natural dynamics, requires no artificial intervention, and represents a dialogue between the matrix and the organism, promoting self-regulation and physiological recovery.

[0114] Self-assembling entities in nature refer to complex systems composed of multiple components that spontaneously organize into functional structures through chemical and physical interactions occurring under natural environmental conditions. These systems exist in living organisms or natural matrices and exhibit emerging properties arising from their dynamic interactions, which cannot be artificially replicated.

[0115] When referring to subjects who require beneficial or therapeutic treatment, the description refers to humans who are affected by or at risk of developing a pathological condition (e.g., age, weight, sex, etc.). Detailed Implementation

[0116] This invention relates to a novel composition of substances, namely a product composed of 100% natural substances, which exerts a therapeutic or beneficial effect in treating altered bone metabolism and / or bone pathological conditions, said product having a physiological (rather than pharmacological) mode of action.

[0117] In fact, for a product to exert its physiological mechanism of action, it must be 100% natural. Natural materials (e.g., products containing or composed of natural matrices) are entities that at least partially retain the autopoietic properties of their starting materials, which belong to the living domain and exhibit intrinsic properties represented by a network of material and non-material relationships. These properties interact with the network of relationships of the treated subject (interactions between networks), thereby reproducing physiologically similar interactions with physiological characteristics and complexities.

[0118] Therefore, according to this specification, a product containing one or more natural matrices or composed of one or more natural matrices is a 100% natural product, which means that the product does not contain any additional artificial substances, i.e., chemically synthesized substances made by humans through laboratory processes.

[0119] Furthermore, according to this specification, products containing one or more natural matrices do not contain any added isolated molecules, such as excipients or active ingredients, even if they are of natural origin.

[0120] It is important to note that natural materials are fundamentally different from “substances” (including substances of natural origin). Because they are not represented by individual components, specialized models are required. Therefore, describing natural materials necessitates an expanded reductionist approach, leveraging innovations from the last century. Conceptually, this means referencing systems theory. From an experimental perspective, preclinical evidence involves systems biology approaches, such as omics sciences (e.g., transcriptomics) and bioinformatics assessments.

[0121] These allow for proper assessment of the matrix (acting network) and the human body (receiving network), and allow the interaction between the two to be viewed as a “network-to-network” interaction. In each specific case, the mechanisms of coordinated redundancy and resilience accompanying physiological features correspond to “physiological mechanisms of action,” which can be characterized using a network paradigm, unlike the targeted and non-targeted models that describe PhIM and mechano / chemical / physical mechanisms, respectively.

[0122] Specifically, according to the present invention, the natural matrix is ​​a 100% natural and biodegradable material composed of natural components that have not been denatured during the production of the matrix from the starting materials, and no intentionally added synthetic products are present throughout the process.

[0123] As mentioned above, the matrix must be obtained through non-denaturing processes, ensuring that the matrix components are not artificially altered. However, if necessary, additional indicators of maintaining properties present in the initial raw materials can be verified. Furthermore, 100% natural products are expected to be 100% biodegradable. In this specification, 100% biodegradability is considered "easily biodegradable" according to OECD biodegradability testing. These properties ensure the maintenance of the matrix effect, which is conferred by the structural interactions (material interactions) of the matrix components and the functional interactions (non-material interactions) that become apparent when biological systems are exposed to the natural matrix.

[0124] This invention relates to the products defined in the table below.

[0125]

[0126] In one embodiment of the present invention, the product has the following formulation.

[0127]

[0128] In a further embodiment, the product has the following formulation:

[0129] Formula A

[0130]

[0131] Or formula B

[0132]

[0133] Or formula C

[0134]

[0135] In one embodiment, the genus *Equisetum* is *Equisetum Arvense* and / or the genus *Acacia* is *Acacia senegal* and / or the genus *Malpighia* is *Malpighia punctifolia* and / or the coral is *caribbean coral* and / or the bird is *Gallus gallus* and / or the genus *Cetaria* is *Cetaria islandica*.

[0136] Preferably, the *Equisetum* genus is *Hippophae rhamnoides*, the *Acacia* genus is *Acacia senegalis*, the *Gynostemma pentaphyllum* genus is *Gynostemma pentaphyllum*, the coral is Caribbean coral, the bird is red junglefowl, and the *Icelandia* genus is *Icelandia*.

[0137] The calcium citrate in this invention is obtained through a natural reaction between coral skeletons and lemon juice. Specifically, the calcium citrate is prepared by diluting lemon juice with water at a 1:1 (volume) ratio, adding coral powder, stirring for 5 hours, and then freeze-drying.

[0138] According to one embodiment of the invention, it is preferably in a dried form, and the w / w % shown in the table above consists of 6 powders and 3 lyophilized extracts.

[0139] The powder consists of calcium carbonate from coral, calcium carbonate from eggshells, fine powder from agave leaves, and powder from Iceland spore fungi. CetariaFine powder of *Thallus*, from the genus *Agaricus* ( Agaricus ) fine powder of edible fungi, and acacia ( Acacia Gum Arabic ， Mix together at room temperature until the mixture is homogeneous.

[0140] The freeze-dried extracts were derived from the flower tips of the *Equisetum* genus, *Acerola*, and other species of *Acerola*. Malpighia The fruit of ) and calcium citrate (as defined above).

[0141] For preparation, the homogeneous powder mixture was mixed with the freeze-dried extract at room temperature until the mixture was homogeneous.

[0142] When the product is prepared in tablet form, the bulk mixture is compressed by direct compression to obtain the finished tablet.

[0143] In a preferred embodiment, the powder is: calcium carbonate from coral (Caribbean coral), calcium carbonate from eggshells, and calcium carbonate from agave sisal (…). Agave sisalana The fine powder from the leaves, derived from island hyacinth ( Cetaria islandica The fine powder of the fungus comes from shiitake mushrooms (champignon mushrooms) and button mushrooms (Agaricus bisporus). Agaricus bisporus The fine powder of ) and Senegalese acacia gum arabic, and the freeze-dried extract is: horsetail flower top, acerola, red-leaved acerola ( Malpighia punctifolia ) and freeze-dried extracts of calcium citrate.

[0144] The solvent used to prepare the extract of the present invention is water, preferably purified water produced from drinking water by an industrial water treatment plant.

[0145] Aqueous extracts of plant materials are known to those skilled in the art. Non-limiting examples of extract preparation can be applied to *Gynostemma pentaphyllum* with appropriate modifications, as follows:

[0146] Asking for help ( Equisetum arvense Freeze-dried extract of horsetail flower apex: dried horsetail flower apex, freeze-dried at 70°C with 100% ( v / v Extract with water [drug-solvent ratio: 1 / 18] for 2 hours, then filter to remove solid waste. Concentrate the resulting clarified extract under reduced pressure until a concentration factor of 10:1 (v:v, initial extract volume to volume after evaporation) is achieved, then freeze-dry for 72 hours. Store the resulting extract at room temperature away from light and moisture until use.

[0147] According to the present invention, the products of any of the possible embodiments disclosed herein can be formulated into compositions with one or more carriers (e.g., water or other suitable carriers) in the final desired form. In one embodiment, the one or more carriers may be pharmaceutical grade.

[0148] For example only, the composition can be formulated for oral, topical, rectal, vaginal, systemic, and microneedle injection.

[0149] According to non-limiting examples, the products or compositions disclosed or claimed herein can be prepared in the form of lyophilized materials, tablets, soft or hard gelatin capsules, powders, granules, loaded vesicles, loaded liposomes, or loaded nanoparticles.

[0150] According to the present invention, the products or compositions disclosed and / or claimed herein are intended for beneficial or medical use, such as for the treatment or adjunctive treatment of bone fragility in a subject in need.

[0151] In fact, as described in the invention abstract, the applicant’s in vitro studies have enabled a comparison of the efficacy of the mixture of the present invention with that of treatment with synthetic calcium and vitamin D alone, and an assessment of its ability to induce the differentiation (increased alkaline phosphatase ALP activity) of mesenchymal stem cells (hADMSCs) (isolated from the patient’s adipose tissue) into mature osteoblasts, which are capable of mineralizing the cellular matrix (increased deposition of HA crystals) and combating bone fragility.

[0152] Bone induction stimulation treatment with vitamin D combined with osteoinduction medium (OM) showed a statistically significant increase in ALP activity compared to the control group (OM + DMSO + Ca), indicating a possible synergistic effect with OM and confirming its ability to induce stem cell differentiation into osteoblasts. Figure 5 ).

[0153] Although activity increased, vitamin D failed to induce the proper stimulation for functional mineralization. Figure 6 ).

[0154] Even treatment with synthetic calcium in bone-inducing culture medium showed increased ALP activity. Figure 5 It exhibits a typical "bell-shaped" trend, characterized by peak enzyme activity levels on day 28 of treatment, unlike vitamin D treatment, which leads to the proper formation of the extracellular matrix and abundant hydroxyapatite (HA) deposits. Figure 6 ).

[0155] Similar to treatments using vitamin D and synthetic calcium, treatment using the mixture of the present invention in conjunction with bone-inducing stimulation can also promote an increase in ALP activity.Figure 5 The cells exhibited a characteristic bell-shaped trend, with activity peaking on day 21 of treatment, indicating early stimulation of the differentiation process compared to OM medium supplemented only with synthetic calcium.

[0156] In addition to demonstrating the ability to mediate the differentiation of mesenchymal cells into bone lineages, the product of this invention can also induce the mineralization process early and completely, which is beneficial to the deposition of hydroxyapatite (HA) crystals. Figure 6 In fact, after 28 days of treatment, the mixture of the present invention induced a statistically significant increase in hydroxyapatite crystals compared to synthetic calcium.

[0157] The mixture of the present invention not only serves as a support for osteoblast differentiation stimulation in the presence of bone-inducing stimulation, but also as a calcium donor capable of mineralizing the extracellular matrix of bone cells. Furthermore, in GM medium without bone-inducing agents, it results in a statistically significant increase in ALP activity compared to synthetic calcium, indicating that it itself can induce mesenchymal cells to differentiate into mature osteoblasts. Figure 7 Conversely, treatment with calcium and vitamin D did not lead to an increase in ALP activity, confirming that they could not induce cell differentiation in the absence of bone-inducing stimulation.

[0158] Based on current knowledge, while vitamin D and synthetic calcium therapy are fundamental treatments, they only partially explain and meet the organism's actual needs. Although they enter the individual's metabolic pathways precisely, they do not achieve satisfactory results. In fact, they cannot reproduce the physiological differentiation of mesenchymal stem cells into osteoblasts to support bone formation.

[0159] Vitamin D does contribute, but it cannot provide differentiation stimulation on its own, nor can it directly provide the form of calcium to promote the formation of mineralized bone matrix. Figure 8 ).

[0160] Calcium synthesis alone cannot interfere with the differentiation process; it can only provide the material for bone formation. Figure 8 ).

[0161] The mixture of the present invention is itself capable of reproducing the entire physiological process. Figure 8 ).

[0162] By evaluating the regulation of mesenchymal stem cell gene expression after treatment with the mixture of the present invention and differentiation stimulants, the potential molecular mechanisms underlying the decreased activity of the mixture of the present invention at the phenotypic level in inducing appropriate metabolic stimulation with increased ALP and HA were analyzed. The product has a significant effect on stem cells, which serve as precursors to osteoblasts and adipocytes, in vitro, promoting significant differentiation of mesenchymal stem cells into mature osteoblasts capable of mineralizing the extracellular matrix in bone. Therefore, in terms of adipose tissue, the mixture of the present invention promotes fat reduction at both local and systemic levels in bone, reducing the tendency of mesenchymal stem cells to differentiate into mature adipocytes. The synergistic effect of these actions, inducing beneficial effects at both ends of the fat / bone axis, leads to reduced bone fragility and increased bone mass.

[0163] As can be observed in the thermogram (Figure 10), treatment with the mixture of the present invention determines the regulation of the expression profile, which is the basis for reduced bone remodeling, bone resorption and osteopenia, as well as increased osteoblast differentiation and mineralization (in terms of increased bone mineral density).

[0164] Specifically, the product of the present invention promotes osteoblast differentiation by regulating specific proteins associated with osteogenic activity (e.g., inducing RUNX2 and inhibiting sclerostin (SOST) known to inhibit osteoblast activity), which is consistent with what has been observed by analyzing the enzyme activity of ALP.

[0165] This product also induces an increase in bone synthesis markers such as osteocalcin (OCN) and BMP (bone morphogenetic protein), potentially increasing osteoblast activity and forming new bone matrix, consistent with results observed by measuring HA crystal concentration.

[0166] The reduction in bone resorption induced by the mixtures of this invention plays a fundamental role in maintaining and repairing bone itself and promoting the rebalancing of bone density and structure. In particular, the mixtures of this invention promote a decrease in the expression level of sclerosing protein (SOST), which is known to inhibit osteoblast activity, thereby enhancing bone formation.

[0167] Furthermore, the mixture of the present invention also exhibits systemic metabolic regulatory effects, inducing the expression level of osteocalcin (OCN) (Figure 10). Osteocalcin is a hormone that also regulates insulin metabolism. Osteocalcin is expressed and secreted by mature osteoblasts and works by stimulating pancreatic β-cells to secrete insulin and promoting insulin sensitivity in muscle and white adipose tissue, thereby lowering blood glucose levels.

[0168] The results also indicate that the mixture of the present invention has the potential to reduce the amount of adipose tissue and inflammatory processes, demonstrating its anti-lipogenic effects and improved tolerance, thereby identifying further desirable effects within the target physiological and pathological framework.

[0169] Therefore, the local and systemic effects of the mixture of the present invention can enter the dysfunctional cycle established in the context of systemic inflammation and metabolic disorders, interfering with the differentiation of mesenchymal stem cells into white adipocytes and their subsequent accumulation.

[0170] In other words, the mixture of the present invention, due to its novel properties, is able to interact with the physiological metabolic pathways established in postmenopausal women or women facing this transition through network mechanisms. This product not only provides the calcium required for the proper deposition of hydroxyapatite crystals within the bone matrix, but also properly stimulates the differentiation of mesenchymal stem cells, promotes the development of osteoblast lineages, and inhibits the formation of osteoclasts and adipocytes, thereby contributing to a healthy balance in bone metabolism. Furthermore, the improved osteocalcin secretion characteristics indicate that beneficial effects are also induced at the systemic level in organs such as the pancreas, muscle tissue, and adipocytes.

[0171] Compared to commonly used typical calcium and vitamin D supplements, this product's dual synergistic and systemic action makes it highly desirable, as typical calcium and vitamin D supplements can only partially support the physiological changes in an organism. Instead, this support is provided by the mixture of the present invention through physiological mechanisms, enabling it to interact with the body according to its known norms rather than extrinsic norms imposed by exogenous entities (e.g., synthetic molecules). This is because, in addition to containing a precursor of vitamin D and a calcium source, the mixture of the present invention, most importantly, interacts with the pool of mesenchymal stem cells in bone and adipose tissue, allowing for the re-balancing of all elements required for proper bone turnover to form strong and functional structures. Furthermore, the product's effects project systemically, helping to rebalance the metabolic disorders and underlying inflammation that plague perimenopausal women systemically. The mixture of the present invention, with its complex plant matrix and natural calcium from various sources, clearly demonstrates that the complexity of physiological processes can only be properly supported according to their known norms when associated with a 100% natural treatment with the same high degree of complexity.

[0172] Therefore, the present invention also relates to products or compositions defined and / or claimed herein for the treatment or adjunctive treatment of bone fragility conditions, wherein the subject is at risk of developing bone fragility disease.

[0173] According to the present invention, the subjects are humans with osteoporosis or humans at risk of developing osteoporosis.

[0174] Since bone metabolism is also closely related to fat metabolism, as described above, in one specific embodiment of the present invention, bone fragility is associated with increased fat in the subject.

[0175] The increase in fat refers to a significant increase in fat, that is, an increase in fat that exceeds the normal weight fluctuations that occur on average in the subjects.

[0176] For example, common causes of increased fat and brittle bones may be associated with obesity, metabolic syndrome, premenopause, perimenopause or menopause, and male menopause.

[0177] Furthermore, according to the present invention, the condition of bone fragility may be selected from osteoporosis or osteopenia.

[0178] In the course of extensive characterization and research of the products of this invention, the applicant also identified conventional markers and associated biological activities of osteoporosis, the regulation (upregulation or downregulation) of which is the basis of the pathological condition and is defined as the regulatory tendency of said activities toward a healthy physiological state (rather than the regulation observed under altered or pathological conditions) (see Figure 9).

[0179] The applicant further demonstrated that the product of the present invention achieves the desired modulation of all said activities, thereby validating the therapeutic effect of the product (Figure 10). Furthermore, the applicant compared the modulation of said activities by the product of the present invention with that of reference drugs for treating osteoporosis, such as Dibase (vitamin D 10,000 UI / ml), and found that the reference drugs could not: reduce bone remodeling, reduce bone loss, induce osteoblast differentiation, restore mineralization, reduce inflammation, and partially reduce adipose tissue. Therefore, overall, vitamin D administration does not alter the biological activity under the pathological condition of osteoporosis, but rather tends to modulate towards a healthy physiological state (Figure 11).

[0180] Finally, the inventors also unexpectedly discovered that different batches of the present invention (see Examples, Product C, batches 1, 2 and 3) exhibited therapeutic / beneficial functional resilience by modulating the biological activity as described above, despite differences in their chemical composition in both qualitative and quantitative aspects, and shared the same pattern and similar modulation values.

[0181] In fact, although by definition, as described above, different batches of a product containing one or more natural matrices are batches whose qualitative and quantitative compositions are necessarily variable, according to one embodiment, qualitative and / or quantitative analysis is performed on each batch to demonstrate the qualitative and / or quantitative differences present in the molecular composition of each batch (Figure 13). This can be achieved using conventional techniques; non-limiting examples include chromatography, spectrophotometry, atomic absorption spectrometry (AAS), atomic emission spectrometry (AES), inductively coupled plasma (ICP) techniques, chromatography coupled to a detector, and combinations thereof. The analysis can focus on a limited number of selected categories of substances (e.g., Figure 13) or on all components of the product.

[0182] As expected, for products containing or composed of natural matrices, although each batch is prepared according to standardized procedures to pre-obtain a high degree of homogeneity between different batches, detailed qualitative and quantitative analysis of all test batches clearly demonstrates relevant batch-to-batch qualitative and quantitative differences. This leads to the discarding of batches that do indeed have therapeutic effects when using routine validation methods for synthesizing or isolating drugs, and makes it impossible to assume the presence of APIs.

[0183] It should be noted that, due to its inherent nature, even natural substrates obtained from the same source of raw materials can have variable compositions. For example, it is well known to those skilled in the art that, due to the genetic and epigenetic variability of each living organism, even among plants in the same field, a natural substrate obtained from one plant individual will never be exactly the same as another natural substrate obtained from a different individual of the same plant species.

[0184] According to the experiments reported in Figure 13, despite the differences in qualitative and quantitative chemical composition among all the batches analyzed, the authors unexpectedly found that in all the batches tested, the different molecular entities within each matrix appeared to interact redundantly, both functionally and possibly structurally, to provide the same therapeutic or beneficial (homeostatic-adjunctive) effects, despite their differences in qualitative and quantitative molecular composition.

[0185] In fact, despite the qualitative and quantitative differences in molecular composition between batches, they still exhibit functional (therapeutic or beneficial) flexibility.

[0186] In other words, the authors unexpectedly found that, despite batch-to-batch differences in qualitative and quantitative composition, different batches of the same product exhibited a consistent modulation (in terms of trend and magnitude) of all detected biological activities related to the desired therapeutic or beneficial effect, which this paper also defines as the “functional elasticity effect.” The observed maintenance of biological activity may be due to the fact, as mentioned above, that the novel properties of the natural matrix are attributed to the matrix network functioning as a holistic entity with unique properties, rather than to each individual molecule as if it were isolated. The therapeutic effect is achieved through a non-pharmacological mechanism of action, unlike classical therapeutic products based on the structure-activity relationship (SAR) of pharmacological relationships, which is the most relevant relationship considered at the single-molecule level between the active pharmaceutical ingredient (API) and the receptor targeted by the API in classical pharmacological activity.

[0187] This aligns with the inventors' analysis of the possibility that the product may exert its therapeutic or beneficial effects by acting on overall pathophysiology or altered physiological states.

[0188] 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 the restoration of bone homeostasis in the altered physiological state under said bone fragility conditions through a bioactive network, and by exhibiting therapeutic or beneficial functional resilience between different batches of said products or compositions, said functional resilience being intended to maintain the therapeutic or beneficial properties of said products or compositions in different batches, despite them having different qualitative and quantitative compositions between them.

[0189] Specifically, based on all the reasoning provided above regarding natural matrices, and all the experimental data collected by the applicant, it can be said that the products or compositions according to the invention are themselves natural matrices, representing primordial natural intelligence, which is the only one capable of allowing physiological endogenous interconnection with other self-assembling entities in nature (such as humans).

[0190] It is important to note that native natural intelligence represents the inherent ability of the natural matrix to preserve and transmit biological and physicochemical information necessary for interaction and integration with other life networks. This information is acquired using the inherent logic of the receiving organism, as it is known to the organism and is therefore endogenous relative to it. This intelligence is a manifestation of natural autopoiesis, the ability to self-organize and adapt to environmental stimuli without human intervention. Such intervention would transmit information according to point-like logic through a medium unknown to the receiving organism and is therefore exogenous relative to it.

[0191] According to the present invention, the presence of native natural wisdom in a therapeutic or beneficial product or composition ( wherein the product or composition comprises or is composed of a natural matrix) can be determined by verifying that the product or composition itself is a natural matrix with the following emerging properties (therapeutic or beneficial): 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; and the product or composition exhibits batch-to-batch therapeutic or beneficial functional resilience between different batches of the product or composition and when the product or composition modulates an overall altered physiological or pathological condition.

[0192] For the products or compositions of the present invention, this can be achieved by performing the following steps on a sample of the product or composition:

[0193] a. Assess the naturalness of the product or composition by the following methods:

[0194] 1. 14C activity was measured using the ISO-16620-2:2015 (AMS) method.

[0195] 2. Assess the presence of miRNA in the product or composition.

[0196] 3. Assess the presence of exosomes in the product or composition.

[0197] b. Assess the presence of therapeutic or beneficial functional resilience between different batches of the product or composition by comparing the modulation of one or more biological activities under the desired therapeutic or beneficial effects of the product on altered bone metabolism and / or bone pathology in different batches, wherein readings in cell-based tests represent the modulation of the one or more biological activities.

[0198] c. Assessing, from the readings of the cell-based assays, whether the regulation of the bioactivity under the desired therapeutic or beneficial effect results in a regulation of the overall altered physiological or pathological condition; and

[0199] The product or composition is determined to be a natural matrix, representing primordial natural wisdom, when the following are true:

[0200] The measured 14C activity was 99.82 ± 0.22%.

[0201] Detection of miRNAs, exosomes, and therapeutic or functional recovery capabilities, as well as

[0202] The regulation described in c leads to the regulation of physiological or pathological conditions that result in overall changes.

[0203] A product is identified as natural when the following conditions are met: a measured 14C activity of 99.82 ± 0.22%; detection of miRNAs and exosomes; and the ability to exert its activity through a physiological mechanism of action when it alters its state and exhibits therapeutic or beneficial functional resilience (i.e., displays novel properties). The sum of these characteristics allows the product itself to be identified as a natural matrix, thus representing primordial natural wisdom.

[0204] The assessment of functional resilience and state change can be conducted as follows.

[0205] The ability of therapeutic or beneficial products to alter pathophysiological states or altered physiological states (e.g., by assisting homeostatic responses in organisms) is a key characteristic in determining their physiological modes of action. In fact, products with network-to-network interactions are those that are expected to alter states rather than have a single function when applied to a living organism.

[0206] In other words, considering the network-network (product-recipient) interactions that occur through natural matrices, therapeutic or beneficial products containing one or more natural matrices or composed of one or more natural matrices are likely to satisfy this feature. The applicant's patent application PCT / IB2024 / 055892 discloses a method for defining the mode of action of a therapeutic or beneficial product based on a natural matrix.

[0207] Products exhibiting physiological modes of action are also expected to be 100% natural (see above) and demonstrate resilience and self-regulation mechanisms observable in living organisms, where different intracellular and intercellular messages and varying regulation of gene pathways can provide the same results, despite differences in intracellular triggering information. For therapeutic or beneficial products, this equates to the functional resilience of different batches of products (with qualitative and quantitative differences in their chemical composition).

[0208] The inventors determined whether the product of the present invention exerts its therapeutic or beneficial effect by altering a pathological state or an altered physiological (not yet pathological) state, and whether the product exhibits functional resilience (i.e., the maintenance of therapeutic or beneficial properties in different batches of a given product comprising one or more natural matrices, despite differences in qualitative and quantitative composition between batches).

[0209] According to the invention, this can be verified by performing cell-based assays, the readings of which represent the regulation of selected biological activities as defined above, such as those disclosed in the embodiments described in the specification, and can be verified by analyzing and interpreting the data obtained therefrom.

[0210] Assessment of functional resilience (therapeutic or beneficial).

[0211] As indicated in the glossary and specification above, in this specification and claims, functional resilience refers to the ability of different batches of a product to maintain a therapeutic or beneficial measurable effect despite differences in qualitative and quantitative molecular composition.

[0212] Clearly, each batch (e.g., batches 1, 2, and 3 of product C) is intended to be identical in terms of manufacturing process and the type and amount of each ingredient (since the main component of the selected product is a natural matrix, this means that each matrix in the product is produced from the same type of starting material and through the same process, such as a specific type of extract from the same part of the same plant species). Therefore, variations in its qualitative and quantitative molecular composition cannot be attributed to different manufacturing processes or different ingredients, but can only stem from the inherent differences between natural matrices obtained from different organisms of the same species using the same process. The functional resilience of a product can be verified when different batches of the same product containing one or more natural matrices maintain the ultimate regulatory activity behind its therapeutic or beneficial properties in a measurable and verifiable manner, regardless of their qualitative and quantitative composition.

[0213] Therefore, the present invention also relates to a method for determining the presence of primordial natural wisdom in a therapeutic or beneficial product or composition by verifying therapeutic or beneficial emerging properties, the product or composition comprising or consisting of a natural matrix, the method comprising performing the following steps on a sample of the product or composition:

[0214] a. Verify the naturalness of the product through the following methods:

[0215] 1. Measure 14C activity using ISO-16620-2:2015 (AMS) method.

[0216] 2. Assess the presence of miRNA in the product or composition.

[0217] 3. Assess the presence of exosomes in the product or composition.

[0218] b. In a cell-based trial, the presence of therapeutic or beneficial functional resilience between different batches of the product is assessed by comparing the regulation of one or more biological activities under the desired therapeutic or beneficial effect on relevant altered physiological states and / or pathological conditions treated by the product or composition in different batches, wherein the readings of the cell-based trial represent the regulation of the one or more biological activities.

[0219] c. Assessing, from readings of the cell-based assay, whether the modulation of the bioactivity under the desired therapeutic or beneficial effect results in a modulation of the overall altered physiological or pathological condition; and

[0220] The product or composition is determined to be a natural matrix representing native natural wisdom when the following are met: a measured 14C activity of 99.82 ± 0.22%; detection of miRNAs, exosomes, and therapeutic or functional resilience; and regulation of physiological or pathological conditions resulting from the aforementioned regulation in c.

[0221] According to one embodiment of the invention, the method further includes, prior to conducting one or more cell-based tests: (1) providing a list of markers representing the altered metabolic and / or pathological states; (2) identifying one or more bioactivity alterations in the pathological state for each marker, thereby precisely locating a network of bioactivity that regulates in accordance with the pathological state; and (3) identifying one or more parameters whose regulation is consistent with the regulation of the one or more bioactivity under the therapeutic effect of the tested product, and determining an up- or down-regulation trend of the one or more bioactivity in the network consistent with the pathological or healthy state.

[0222] According to a preferred embodiment, the altered physiological state is bone metabolism and / or the pathological condition is osteoporosis, and the marker is selected from: bone remodeling, osteopenia, osteoblast differentiation, bone mineralization, inflammation reduction, adipose tissue reduction, preferably, wherein the bioactivity of (2) for the bone remodeling marker is selected from the bioactivity shown in Figure 9.

[0223] More specifically,

[0224] (1) Provide a list of markers representing pathological states, which are related to pathology or to a physiological state that may originate from the non-pathological changes, i.e., provide a list of markers representing a disease or pathophysiological condition treated by the target product or a physiological state that may originate from a non-pathological change, wherein homeostasis is assisted by the target beneficial product.

[0225] (2) Identifying one or more bioactive modifications under the pathology for each of the stated markers, determining regulation representing a pathophysiological state associated with the pathology, and evaluating the opposite regulation as a regulatory pattern for each of the stated activities representing a healthy physiological state; and

[0226] (3) For each of the one or more biological activities, identify one or more biomarkers and their regulatory patterns under changes detectable in the pathological state, and for each of the parameters, set a regulatory pattern opposite to the identified regulatory pattern as a regulatory pattern consistent with the healthy physiological state.

[0227] In the case of inspecting therapeutic products, according to one embodiment, the method of the present invention may include the following steps:

[0228] (a) Perform at least one of the aforementioned in vitro cell-based assays on the following cell groups,

[0229] (a1) At least one control group and at least two cell test groups with disease phenotypes relevant to the intended use of the therapeutic product; or

[0230] (a2) At least one cell group with a healthy physiological phenotype; and at least one control group and at least two test groups of said cells with healthy physiological phenotypes, wherein a disease phenotype relevant to the intended use of the therapeutic product is induced.

[0231] The test group of each cell was treated with one of the different batches of the treatment product;

[0232] (b) Determine the regulation or regulation mode of each parameter on each of the cell groups in step (a), and calculate the corresponding regulation value for each of the one or more biological activities;

[0233] (c) Compare the modulation values, wherein:

[0234] The therapeutic product demonstrates its therapeutic effect through physiological mechanisms when the following conditions are met:

[0235] For each marker, at least 50% of the said one or more biological activities are regulated by each product batch, wherein the regulatory trend of the network is consistent with the healthy state, and for the test cell group of (a1), the regulation value of each of the at least 50% of the one or more biological activities determined in (b) differs from the regulation value of the control cell group of (a1) by at least 0.15, or

[0236] For each marker, at least 50% of the said one or more biological activities are regulated by each product batch, wherein the regulatory trend of the network is consistent with the healthy state, and for the test cell group of (a2), the regulation value of each of the at least 50% of the said one or more biological activities determined in (b) differs from the regulation value of the control cell group of (a2) by at least 15%, and

[0237] The functional resilience of the product is demonstrated by the fact that the modulated value of each of the one or more biological activities in each of the test cell groups differs from the average value by less than 20%.

[0238] This means that the regulation value of a given biological activity in the test group corresponds to the regulation value of the same biological activity in the control group, and therefore, the difference of at least 0.15 or at least 15% is the difference between the regulation value of a given activity in the treated cell group and the regulation value of the same activity in the cell group representing the control baseline.

[0239] In other words, this method can also be described as a way to assess whether a therapeutic product exerts its therapeutic effect on a pathological state through a physiological mechanism of action, including:

[0240] - We offer different batches of therapeutic products containing one or more natural bases.

[0241] - Provide a list of markers representing the pathological state, identify a set of parameters for each marker, and adjust these parameters to enable the evaluation of the bioactive network under the therapeutic effect of the tested product and to determine the up- or down-regulation trend of the activity in the network under disease and healthy states.

[0242] (a) Perform at least one in vitro cell-based experiment on the following cell groups,

[0243] (1) At least one control cell group and at least two test cell groups having the disease phenotype targeted by the therapeutic product; or

[0244] (2) At least one cell group with a healthy physiological phenotype; and at least one control cell group and at least two test cell groups with healthy physiological phenotypes, wherein the disease phenotype targeted by the therapeutic product is induced.

[0245] Each of the test cell groups was treated with one of the different batches of the treatment product;

[0246] (b) Determine the regulation or regulation mode of each of the parameters for each of the cell groups, and calculate the corresponding regulation value for each of the biological activities;

[0247] (c) Compare the regulatory values ​​of each biological activity in each cell group in step (b), wherein:

[0248] A therapeutic product demonstrates its therapeutic activity through physiological mechanisms when the following conditions are met:

[0249] For each marker, at least 50% of the said bioactivity is regulated by each product batch, wherein the regulation trend is consistent with the health status identified in (b), and for the test cell group of (a)(1), the regulation value of each of the at least 50% of one or more bioactivities identified in (b) differs from the regulation value of the control group of (a)(1) by at least 0.15, or

[0250] At least 50% of the said bioactivity is regulated by each product batch, wherein the regulatory trend is consistent with the health status identified in (b), and for the test cell groups of (a) and (2), the regulation value of each of the at least 50% of one or more of the bioactivities identified in (b) differs from the regulation value of the control group of (a) and (2) by at least 15%.

[0251] Furthermore, the product’s functional flexibility is demonstrated by the fact that the regulation value of each bioactivity in each of the test cell groups differs from the average value by less than 20%.

[0252] The statement "For each mark, at least 50% of the said one or more biological activities are regulated by each product batch, wherein the regulatory trend of the network is consistent with the health status" means that, in the case of a single biological activity of a given mark, to meet the above requirement, 100%, i.e., a single activity, must be regulated by the tested product, and the regulatory trend must be consistent with the health status. The statement "The regulation value of each of the at least 50% of the said one or more biological activities determined in (b)" means that the regulation value of at least 50% of the biological activities determined in (b) satisfies the requirement of regulation according to a regulatory trend consistent with the health status. This applies to all embodiments disclosed herein with appropriate modifications.

[0253] In examining a beneficial product (i.e., a product that exerts a beneficial effect on altered but not yet pathological physiological states by assisting homeostasis), according to one embodiment, the method of the present invention may include the following steps:

[0254] (a) Perform at least one of the aforementioned in vitro cell-based assays on the following cell groups:

[0255] At least one control cell group with a healthy phenotype or at least one control cell group in which the abnormal regulatory phenotype targeted by the beneficial product is appropriately induced, and at least two test cell groups taken from the control group.

[0256] Each of the test cell groups was treated with one of the different batches of the beneficial product;

[0257] (b) Determine the regulation or regulation mode of each parameter in step (a) for each cell group, and calculate the corresponding regulation value for each of the one or more biological activities;

[0258] (c) Compare the adjustment values, wherein:

[0259] When the following holds true, the beneficial product demonstrates that it exerts its homeostatic auxiliary effect through a physiological mechanism of action.

[0260] For each marker, at least 50% of the one or more biological activities are regulated by each product batch, wherein the regulatory trend of the network is consistent with the healthy state, and for the test cell group, the regulation value of each of the at least 50% of the one or more biological activities calculated in (b) differs from the regulation value of the control cell group by at least 0.15, and

[0261] The functional resilience of the product is demonstrated by the fact that the modulated value of each of one or more biological activities in each of the test cell groups differs from the average value by less than 20%.

[0262] This means that the regulation value of a specific biological activity in the test group corresponds to the regulation value of the same biological activity in the control group, and therefore, the difference of at least 0.15 or at least 15% is the difference between the regulation value of a given activity in the treated cell group and the regulation value of the same activity in the cell group representing the control baseline.

[0263] In other words, this method can be defined as a way to assess whether a beneficial product exerts its homeostatic supportive effect by modulating altered physiological states, including:

[0264] - Providing different batches of beneficial products to aid in steady-state conditions; these products contain one or more natural matrices.

[0265] - Provide a list of markers representing pathological states, which may originate from the altered physiological state, identify a set of parameters for each marker, and the regulation of these parameters enables the assessment of the bioactive network under the therapeutic effect of the tested product, and the determination of the up- or down-regulation trend of the activity in the network under disease and health states.

[0266] (a) Perform at least one in vitro cell-based experiment on the following cell groups:

[0267] (1) At least one control cell group with a healthy phenotype or at least one control cell group in which the abnormal regulatory phenotype targeted by the beneficial product is appropriately induced, and at least two test cell groups taken from the control group.

[0268] Each of the test cell groups was treated with one of the different batches of the beneficial product;

[0269] (b) Determine the regulation or regulation mode of each parameter in step (a) for each cell group, and calculate the corresponding regulation value for each biological activity;

[0270] (c) Compare the regulatory values ​​of each biological function in each cell group in step (a), wherein:

[0271] Beneficial products exert their homeostatic supportive effects through physiological mechanisms when the following conditions are met:

[0272] For each marker, at least 50% of the said bioactivity is regulated by each product batch, wherein the regulation trend is consistent with the health status identified in (b), and for the test cell group of (a)(1), the regulation value of each of the at least 50% of one or more bioactivities identified in (b) differs from the regulation value of the control group of (a)(1) by at least 0.15.

[0273] Furthermore, the product’s functional flexibility is demonstrated by the fact that the regulation value of each bioactivity in each of the test cell groups differs from the average value by less than 20%.

[0274] In a preferred embodiment, the control group is regarded as the reference regulatory baseline, and the qualitative and quantitative regulatory value of each of the one or more biological activities is regarded as 0.

[0275] As previously described, the method of the present invention includes: (1) providing a list of markers representing a target pathological state (i.e., a pathological state treated by the analyzed product, or a pathological state that may originate from an altered physiological state, wherein the analyzed beneficial product exerts its homeostatic auxiliary activity on it); (2) identifying one or more bioactivity alterations in the pathological state for each marker, thereby precisely locating a network of bioactivity that regulates in accordance with the pathological state; and (3) identifying one or more parameters whose regulation is in accordance with the regulation of the one or more bioactivity under the therapeutic effect of the tested product, and determining an up- or down-regulation trend of the one or more bioactivity in the network that is in accordance with the pathological state or health state.

[0276] All of the above implementation schemes are able to determine whether a therapeutic or beneficial product exerts its therapeutic or beneficial effect by altering the state of the pathological or abnormal physiological state treated by the product, or by merely a limited number of activities or even a single function, and whether the selected therapeutic or beneficial product maintains functional resilience as defined herein.

[0277] State alteration is a feature that cannot be achieved by a single API drug, thus ruling out traditional drug action modes. However, in principle, state alteration can be achieved by drugs containing a mixture of multiple APIs.

[0278] Physiological modes of action require therapeutic or beneficial products to regulate state by utilizing network-to-network interactions in a manner that involves the overall cellular response, rather than by regulating state in a point-to-network interaction manner based on API modes of action (whether a single API or a mixture thereof).

[0279] This means that, in addition to state regulation, the product also has the ability to provide functional flexibility (therapeutic or beneficial), that is, to provide the same therapeutic / beneficial effect across batches despite differences in qualitative and quantitative composition. In other words, this is the result of adjusting various selected parameters in various ways while still providing a conserved functional outcome.

[0280] Therefore, the present invention also provides a way to express the functional flexibility of therapeutic or beneficial products.

[0281] As explained in the glossary and the above description, functional flexibility (therapeutic or beneficial) refers to the ability of a given product to modulate one or more biological activities in a pathological or altered physiological state, despite differences in the qualitative and quantitative composition of different batches of the same product, thus potentially triggering different signals within the cells to achieve the same end result, i.e., demonstrating bioequivalence in achieving the same end result. As mentioned above, drug (API-based) products known to have different qualitative and quantitative compositions are not considered bioequivalent.

[0282] Physiological mode of action refers to the interaction with the cells of the treated subject as a whole, rather than with individual cellular molecular targets. It is the mode of action exerted by a living organism through network-to-network interactions. Physiological systems in the body often exhibit functional redundancy to maintain homeostasis and adapt to changes or disturbances. Redundancy is a well-known physiological mechanism in organisms used to ensure the achievement of a given goal (e.g., the organism's response to the production of various proteins, the activation of various pathways, etc.). When a therapeutic product interacts with these systems, it may engage in multiple pathways or mechanisms, including redundant pathways or mechanisms, to achieve its intended effect. This redundancy leads to functional flexibility, contributing to the product's physiological mode of action.

[0283] Therefore, the functional resilience of therapeutic / beneficial products (the ability of a therapeutic or beneficial product to maintain its intended function and efficacy despite batch-to-batch differences in qualitative and quantitative composition) is a fundamental characteristic of physiological modes of action.

[0284] The present invention also relates to a method of treating or adjuvant treatment of bone fragility (with necessary modifications), wherein the product or composition of the present invention, alone or in combination with an active ingredient, is administered to a subject in need in a beneficial or therapeutically effective amount.

[0285] The following examples are intended to better illustrate and scientifically support the invention, but are not intended to limit its scope.

[0286] Examples

[0287] 1. Test formulation composition

[0288] The experimental data reported below were generated using the following formulation of the product of the present invention, also referred to hereinafter as "Product C".

[0289] Coral calcium powder 32% w / w

[0290] Eggshell calcium powder 30.2% w / w

[0291] Coral calcium citrate powder 13% w / w

[0292] Button mushroom powder 4.65% w / w

[0293] Horsetail flower apical extract 2% w / w

[0294] Red Leaf Golden Tiger Tail 2% w / w

[0295] Island coating powder 2% w / w

[0296] Agave leaf powder 12% w / w

[0297] Senegalese Acacia Powder 2.15% w / w

[0298] Other product formulations within the scope of this invention were also tested and similar results were obtained (data not shown).

[0299] Three batches of product C were prepared using the same formula, but starting from different batches of raw plant or natural materials, namely batch 1, batch 2 and batch 3.

[0300] Furthermore, arrangements of the above formulations within the scope of claim 1 were prepared, and preliminary data confirmed their correct bioactivity (i.e., bioactivity equivalent to product C) in cell-based experiments, as shown below.

[0301] Calcium carbonate powder from coral (Caribbean coral), calcium carbonate powder from eggshells, fine powder from agave leaves, fine powder from island chrysotile, fine powder from shiitake mushrooms (button mushrooms) and gum arabic (Senegal acacia) were mixed at room temperature until the mixture was homogeneous. Then, freeze-dried extracts of horsetail tops, golden privet, red privet, and calcium citrate were mixed and added at room temperature, in the weight percentages as described above.

[0302] The solvent used to prepare the extract of the present invention is purified water produced from drinking water by an industrial water treatment plant.

[0303] The method for preparing freeze-dried extracts involves freezing each plant material at 70°C with 100% ( v / v The water was extracted for 2 hours, then filtered to remove solid waste. The resulting clarified extract was concentrated under reduced pressure until a concentration factor of 10:1 (v:v, initial extract volume to volume after evaporation) was achieved, and then freeze-dried for 72 hours. The resulting extract was stored at room temperature away from light and moisture until use.

[0304] The product thus prepared is then appropriately dissolved and / or diluted for use in the various tests disclosed below.

[0305] 2. In vitro test

[0306] Human pre-osteoblastic mesenchymal stem cells (hADMSCs) derived from adipose tissue were studied in vitro. These cells represent a convenient and readily available (non-invasive) source of mesenchymal stem cells, capable of differentiating into osteoblast lineages with appropriate induction. In vitro studies demonstrated that hADMSCs can differentiate into mature, viable osteoblasts and produce mineralized bone matrix when appropriately induced in a specific osteogenic induction medium (osteogenic / bone induction medium; OM) containing 10 nM dexamethasone, 0.2 mM ascorbic acid, and 10 mM β-glycerophosphate, as well as calcium and phosphate ester sources. Therefore, these cells are an optimal model for studying osteoblast differentiation and function, mineralization processes, and assessing the effects of various substances on these processes.

[0307] 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 (treatment days 4, 7, 14, 21, 28, and 35).

[0308] The experiment will focus on evaluating the following:

[0309] The ability to provide bioavailable calcium ions for the mineralization of the extracellular bone matrix (measured by hydroxyapatite (HA) crystals).

[0310] Potential to induce and / or enhance osteoblast differentiation (spectrophotometric determination of alkaline phosphatase (ALP) activity),

[0311] Induced transcriptional profiles in cells were analyzed to assess pathways and biological functions that may be affected by treatment (gene expression using a microarray platform).

[0312] The differentiation potential of samples was assessed under two different osteoinduction stimulation conditions (non-osteoinduction medium (GM) and osteoinduction medium (OM)).

[0313] Non-bone induction medium GM:

[0314] (DMEM medium containing antibiotics / calcein / serum + β-glycerophosphate and 2-phosphoascorbic acid, but without dexamethasone): This medium does not contain dexamethasone (an external bone inducer), so the ability of the added formulation to independently induce osteoblast differentiation can be assessed by measuring ALP activity.

[0315] Bone induction culture medium OM:

[0316] (DMEM medium containing antibiotics / calcein / serum + β-glycerophosphate and ascorbic acid 2-phosphate and dexamethasone): This medium contains dexamethasone (an external bone inducer) and can be used to assess whether this formulation has a synergistic (or conversely inhibitory) effect on dexamethasone-induced osteoblast differentiation.

[0317] The experimental model involved adding calcium to the culture medium at a concentration reflecting the physiological amount of calcium in vivo, namely 1.4 mM. To achieve this condition, appropriate amounts of synthetic calcium were added to both culture media (OM and GM) in treatments involving vitamin D (soluble in DMSO, as it is hydrophobic).

[0318] hADMSC cell lines were cultured at 37°C in a humidified atmosphere containing 5% CO2 in 100 mm culture dishes on growth medium (GM) [Ham's F12 Coon's modified medium supplemented with 10% FBS, 100 IU / mL penicillin, and 100 μg / mL streptomycin]. The medium was changed with fresh GM every 3 days. Once confluence was achieved, cells were isolated by trypsin digestion and cultured in GM at a concentration of 1 x 10⁻⁶ cells / mL. 4 cells / cm 2 Cells were seeded at a density of 70% to 80% in 24-well plates until confluence was achieved. The medium was then changed to osteogenic medium (OM) or retained in GM, both supplemented with treatments, and incubated for 7 to 35 days. The treatment medium was changed twice weekly.

[0319] These cells were obtained with informed consent during routine surgery in 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 have been characterized at the Department of Surgery and Translational Medicine, University of Florence, based on the major stem cell markers (CD44, CD105, and STRO1) and by studying their pluripotency in osteogenic phenotypes.

[0320] The timetable used in the experimental setup is as follows: Figure 15 As shown.

[0321] 2.1 ALP test and calcium mineralization sediment test ( Figures 5 - 7 )

[0322] At the end of each incubation time point, cells were washed twice with DPBS, fixed in 4% PFA / DPBS for 15 minutes, washed three times with ultrapure water, dried, and stored at 4°C until the experiment was performed. Each experimental point was repeated four times.

[0323] ALP test

[0324] Each well was incubated with 500 μL of 4-methylumbelliferyl ketone phosphate in 280 mM Tris-HCl buffer (pH 9.0) at 37°C for 15 min. The reaction was terminated by adding 2 mL of 0.1 M NaOH. The fluorescence spectrophotometer was used at 365 nm. Excitation and 445 nm ALP activity was measured under emission conditions, in μU / cm 2 This indicates the standard curve of 4-methylumbelliferone at 50 nM–10 μM using 280 mM Tris-HCl buffer at pH 9.0.

[0325] Calcium mineralization sediment experiment.

[0326] Each well was incubated with 2 mL of 50 mM NaEDTA at 37°C for 30 minutes. The solution was then transferred to a cuvette and analyzed using a spectrophotometer LS55 (PerkinElmer) at 494 nm. Excitation and 517 nm Fluorescence was measured under emission, in μg / cm 2 This indicates that a standard curve was obtained using 25 ng / mL–500 μg / mL calcium mineralized sediments dissolved in 50 mM NaEDTA.

[0327] In vitro studies were able to compare the efficacy characteristics of product C with treatments using synthetic calcium and vitamin D alone. This was achieved by assessing the ability to induce the differentiation (increased ALP activity) of mesenchymal stem cells (hADMSCs) isolated from patient adipose tissue into mature osteoblasts, mineralize the cellular matrix (increased HA crystal deposition), and combat bone fragility.

[0328] The combined treatment of vitamin D and osteoinducible stimulant (OM) showed a statistically significant increase in ALP activity compared to the control group (OM+DMSO+Ca), indicating a possible synergistic effect with OM and confirming its ability to induce stem cell differentiation into osteoblasts. Figure 5 ).

[0329] Treatment with synthetic calcium in bone-inducing culture medium also showed increased ALP activity, exhibiting a typical "bell-shaped" pattern, characterized by peak enzyme activity levels at 28 days of treatment. Figure 5 ).

[0330] Similar to treatments with vitamin D and synthetic calcium, the combination of product C and bone-inducing stimulation also promoted an increase in ALP activity, exhibiting a characteristic bell-shaped pattern, reaching peak activity on day 21 of treatment. This suggests that compared to OM supplementation with synthetic calcium alone, early stimulation of the differentiation process (…) Figure 5 ).

[0331] Bell-shaped curves observed in all groups ( Figure 5 This reflects the dynamic characteristics of ALP activity during osteogenic differentiation, with its increase corresponding to early matrix maturation and its decrease as cells transition to later stages of bone formation. For product C, the early peak of ALP activity highlights its ability to predict key stages of the physiological processes of differentiation and mineralization. This early response indicates its role in promoting the initial stages of osteogenic formation, consistent with the physiological process of bone formation.

[0332] Compared to vitamin D treatment, treatment with synthetic calcium in bone-inducing media showed proper formation of the extracellular matrix and substantial deposition of hydroxyapatite (HA).Figure 6 Product C, in addition to mediating the differentiation of mesenchymal stem cells into the bone lineage, can also induce the mineralization process early and completely, promoting the deposition of hydroxyapatite (HA) crystals. In fact, after 28 days of treatment, the number of hydroxyapatite crystals induced by Product C was statistically significantly increased compared to synthetic calcium. Figure 5 ).

[0333] This result indicates that the early peak of ALP activity observed in product C ( Figure 5 It transforms into effective mineral deposits at a time point of 28 days. Figure 6 In contrast, while OM + vitamin D + synthesized calcium contributed to early ALP activity, it did not translate into significant mineralization by day 28. Figure 6 Compared to other groups, HA content remained significantly lower, highlighting that early differentiation alone is insufficient without effective downstream mineral deposition. This analysis emphasizes that early activation of osteogenic markers (such as vitamin D) does not guarantee successful mineralization unless supported by mechanisms driving the complete differentiation process.

[0334] Both OM + DMSO + synthetic calcium and OM + synthetic calcium served as strong positive controls, demonstrating robust mineralization levels at 28 days. Product C achieved similar results, further demonstrating its effectiveness in supporting the physiological processes of bone differentiation and mineralization. In contrast, Product C effectively combined its early osteogenic activity with sustained mineralization capacity, indicating a more comprehensive role in mimicking physiological bone formation.

[0335] The results showed that product C not only supported osteoblast differentiation in the presence of osteoinductive stimulation, but also served as a calcium donor capable of mineralizing the osteoblast extracellular matrix. Furthermore, in GM medium without osteoinductive agents, it resulted in a statistically greater increase in ALP activity than synthetic calcium, indicating that it itself could induce mesenchymal cells to differentiate into mature osteoblasts. Figure 7 Conversely, calcium and vitamin D treatments did not increase ALP activity, confirming their inability to induce cell differentiation in the absence of bone-inducing stimulation.

[0336] Specifically, the result ( Figure 7 The results showed that in GM medium lacking osteoinducers, product C resulted in a statistically significant increase in ALP activity compared to synthetic calcium, indicating that product C alone could induce mesenchymal cell differentiation into mature osteoblasts. In contrast, treatment with calcium and vitamin D did not increase ALP activity, confirming their inability to induce cell differentiation in the absence of osteoinducible stimulation.

[0337] In osteogenic induction medium, product C also promoted the increase of ALP, exhibiting a typical bell-shaped curve with an activity peak on day 21, indicating that product C has an early stimulatory effect on the differentiation process compared to OM medium containing only synthetic calcium.

[0338] Similarly, ( Figure 6 Treatment with vitamin D showed the same trend in ALP, but induced a statistically larger increase compared to the control (OM+DMSO+Ca). Despite the increased activity, the substance failed to induce the proper stimulation for functional mineralization.

[0339] Product C effectively provided this stimulation, showing a statistically significant increase in hydroxyapatite crystals at 28 days compared to synthetic calcium. Figure ۶ ).

[0340] 3. Gene expression analysis

[0341] At the end of the treatment period, cells were washed with 100 µl PBS, lysed, and collected in RLT buffer (Qiagen, 1053393) supplemented with β-mercaptoethanol (Sigma, M3148) and DX reagent (Qiagen, 19088) for gene expression analysis. Total RNA was extracted from the cell lysates using the QIAsymphony RNA kit (Qiagen) and the QIAsymphony SP instrument (Qiagen).

[0342] RNA quality and quantity were determined using A230, A260, A280, and A320 measurements on a Varioskan™ LUX multimode microplate reader (ThermoScientific™). RNA integrity was checked using a 2100 expert_Eukaryote Total RNA Nano Kit (Agilent). Whole transcriptome expression profiles were evaluated using a Human Clariom™ S PicoAssay HT (Applied Biosystems, ThermoFisher Scientific) on a GeneTitan MC instrument (Applied Biosystems, ThermoFisher Scientific) according to the manufacturer's instructions. Briefly, cDNA was generated using 6 ng of total RNA, and then fragmented and labeled cDNA was hybridized to a HumanClariom S 96 array plate at 45°C for 17 hours. The array was cleaned, stained, and scanned using a GeneTitan MC instrument (Applied Biosystems, ThermoFisher Scientific), and CEL intensity files were generated using the Affymetrix GeneChip command console software (AGCC, ThermoFisher Scientific).

[0343] 3.1 Transcriptomics Data Analysis

[0344] Data analysis was performed using the transcriptome analysis console software (TAC, ThermoFisher Scientific), which provides quality control analysis, standardization and summarization based on the signal spatial transformation-robust multi-chip analysis (SST-RMA) algorithm, and provides a list of differentially expressed genes (Limma Bioconductor package, p-value ≤ 0.05).

[0345] 3.2. Bioinformatics Modeling of Transcriptomics Data Observed in Experiments

[0346] For each study batch, Ingenuity Pathways Analysis (IPA) (QIAGEN Inc., https: / / www.qiagenbioinformatics.com / products / ingenuitypathway-analysis) was used to assess gene expression regulation associated with the target effect.

[0347] IPA is a scientific reference aggregator that allows users to search for information about genes / proteins and build networks that predict the behavior of biological systems based on gene expression status.

[0348] 4. Evaluation of the transcriptional effects of product C treatment on osteoblast differentiation and mineralization in adipose tissue.

[0349] Demonstrated by ALP and HA dosage assessments, gene expression analysis also indicated that product C appears to have a significant effect on both bone and adipose tissue, promoting the clear differentiation of mesenchymal stem cells into mature osteoblasts capable of mineralizing the extracellular matrix, while inducing a reduction in bone adipose tissue, thereby reducing bone fragility and improving bone quality.

[0350] As shown in the heatmap (Figures 10-11), treatment with product C (batch 1) resulted in a modulation of the expression profile, which underpinned bone remodeling, bone resorption and osteopenia, as well as increased osteoblast differentiation and mineralization (in terms of increased bone mineral density).

[0351] Specifically, product C promotes osteoblast differentiation by modulating specific osteogenic proteins (e.g., inducing RUNX2 and inhibiting sclerosing protein (SOST) known to inhibit osteoblast activity), consistent with ALP enzyme activity assays.

[0352] This product can also induce an increase in bone synthesis markers such as osteocalcin (OCN) and BMP (bone morphogenetic protein), highlighting increased osteoblast activity and new bone matrix formation, which is consistent with the results of HA crystal concentration measurements.

[0353] A reduction in bone remodeling processes was also observed, which is crucial for bone maintenance and repair and may rebalance bone density and structure. In particular, product C appears to reduce sclerosing protein (SOST), which is known to inhibit osteoblast activity, thereby enhancing bone formation.

[0354] Finally, product C appears to reduce the amount and inflammatory processes in bone and adipose tissue and at the systemic level, indicating an anti-lipogenic effect, which is confirmed by improving glucose tolerance.

[0355] Therefore, Product C has multiple effects on both bone and adipose tissue, promoting bone formation by increasing osteoblast differentiation and mineralization, and reducing bone remodeling, obesity, and metabolic regulation. All the effects highlighted by the gene expression data are significantly different from those of traditional products consisting only of calcium and vitamin D, and the beneficial effects of Product C are clearly demonstrated.

[0356] The test was repeated for the second and third batches of product C, and the results are as follows: Figure 13a and Figure 13b As shown.

[0357] 5. Definitions of pathophysiological markers used to query IPA diseases.

[0358] Considering the latest changes in the health physiological characteristics of osteoporosis, particular attention is paid to the following areas:

[0359] - Bone remodeling

[0360] - Osteopenia

[0361] - Osteoblast differentiation

[0362] - Mineralization

[0363] - Reduced inflammation

[0364] - Reduction of adipose tissue

[0365] This knowledge is used to query IPA using the "IPA Bioprofiler" tool, using the following keywords: osteoporosis, postmenopausal osteoporosis, bone calcification, osteoblast and osteoclast differentiation, bone mineral density.

[0366] Using the "IPA Bioprofiler," gene clusters causally related to each of one or more identified biological activities and the specific molecular pathways behind them can be identified. Information on gene expression measurements induced for each batch (fold change cutoff values ​​≤ -2 and ≥ +2 and p-value ≤ 0.05) is then overlaid onto the resulting network to determine the affected genes and the regulation of related biological functions.

[0367] Regulation of gene expression is shown in varying intensities of blue (indicating downregulation) or red (indicating upregulation). Based on the literature, the expected calculated impact on relevant biological functions was determined using the "IPA Molecule Activity Predictor" (MAP) tool and displayed in the heatmap visualization.

[0368] Its color and intensity were converted into numerical values. Figure 9 shows the regulatory trends of selected bioactivities under identified osteoporosis markers in altered bone physiological state (metabolic changes) or osteoporotic pathology, as well as the regulatory trends of said bioactivities required to restore healthy physiological state (in contrast to altered / pathological bioactivities).

[0369] The test results compared the performance and mechanism of action of three different batches of product C. Analysis showed that while all batches produced reproducible biological effects, batch-specific fluctuations could be identified in the induced transcriptional patterns. Clearly, induction of slightly different transcriptional patterns still resulted in the same desired regulation of one or more biological activities. This is due to functional elasticity resulting from the redundancy of interactions between the product components and the body; therefore, different batches with different qualitative and quantitative compositions can produce the same effect through a multifocal mechanism of action. Figure 13a and Figure 13b ).

[0370] Because the induction and inhibition patterns are preserved, the analyzed batches are considered to have equivalent biological output.

[0371] Different transcriptional patterns and relative biological effects across different batches should be considered as markers of inherent variability present in formulations composed of biological materials. The results summarized in Figure 13 clearly demonstrate that the observed different transcriptional patterns across batches resulted in highly reproducible biological (functional resilience) effects, leading to an overall modification of the equivalence of pathological processes and overall pathological status across all batches reported in Figure 13.

[0372] 6. Detailed analysis of product C

[0373] Product C was analyzed in detail, and unless otherwise stated, the batch of product used is batch 1.

[0374] The table below summarizes all the methods used.

[0375]

[0376] The results are summarized in the table below:

[0377]

[0378] Note 1: The grey boxes indicate the major chemical classes.

[0379] Note 2. % = Concentration of the compound expressed as a percentage of the whole matrix.

[0380] Note 3. The term "total" refers to the sum of the values of the various compounds making up the corresponding group.

[0381] Note 5. <LdQ = Below the limit of quantification

[0382] In addition, qualitative miRNA characterization was performed on each batch of ultracentrifuged samples, and the results showed that the metabolomics were highly complex and contained typical biomass miRNAs.

[0383]

[0384] 7. Determination of extracellular vesicle and miRNA content in product C

[0385] Flow cytometry analysis of Product C confirmed the presence of particles compatible with extracellular vesicles. This indicates that Product C contains vesicles that may play a role in cell communication. Based on this, small RNA sequencing was performed using the sRNAtoolbox framework to explore the RNA content within these vesicle-sized particles. Among the identified sequences, two mature microRNAs (miRNAs) or their isoforms, miR8175 and miR166, were found; these are well-annotated important miRNAs in the Arabidopsis genome.

[0386] 7.1 Detection of miRNAs and the role of the detected miRNAs in bone metabolism

[0387] The presence of RNA was quantitatively and qualitatively assessed. After homogenization using a QIAshredder column, RNA was extracted using a plant matrix-specific kit (Rneasy PowerPlant kit), followed by the kit's extraction protocol. The size distribution of the obtained RNA was analyzed using a Bioanalyzer 2100 and RNA 6000 Nano, RNA 6000 Pico, and small RNA kits.

[0388] The size distribution of total RNA detected ranged from 4 to 150 nt.

[0389] The role of ath-miR8175 in mediating product C differentiation and mineralization capacity

[0390] The presence of miRNA in the product was characterized by NGS analysis of total RNA isolated from various biological matrices within the product.

[0391] A current and evolving topic in scientific research is the relevance of miRNAs present in food and their role in human nutrition. Many studies have highlighted the importance of these miRNAs in regulating metabolic processes in response to diet.

[0392] RNA analysis of product C identified two mature miRNA sequences (miRNAs) annotated in the Arabidopsis genome (the most annotated plant to date): miR8175 and miR166.

[0393] 7.2 Detection of the supramolecular structure in the natural matrix of Product C

[0394] Dynamic light scattering

[0395] Dynamic light scattering (DLS) is a commonly used technique for analyzing nanoscale particle size. It measures the hydrodynamic size of particles by analyzing the scattering of light through a laser beam passing through a solution. The intensity of the scattered light fluctuates over time, reflecting the Brownian motion of the particles; the smaller the particles, the faster the diffusion rate. DLS is used to measure particle size in colloidal samples, assess formulation stability, and detect aggregation. It is also ideal for analyzing the size distribution of isolated exosomes and microvesicles.

[0396] This analysis was conducted by Alfatest Lab.

[0397] Sample preparation:

[0398] Product C, batch 1 (23D2227), was dispersed at an arbitrary concentration of 1 mg / ml in 0.22 μm filtered demineralized water.

[0399] After dispersion, vortex the sample for 2 minutes to achieve complete dispersion.

[0400] Analysis parameters:

[0401] Measuring unit: Plastic, (DTS0012)

[0402] Detector: Backscatter 173° (NIBS)

[0403] Laser wavelength: 633 nm

[0404] Number of measurements: 3

[0405] Relevant time: Adaptive

[0406] Measurement location: Automatic

[0407] Attenuator: Automatic

[0408] Temperature: 25°C

[0409] Temperature equilibrium time: 120 seconds

[0410] Dispersant: Water

[0411] Dispersant refractive index: 1.33

[0412] Dispersant viscosity: 0.8872 cP (water) at 25°C

[0413] Samples were analyzed under three different conditions:

[0414] - Unfiltered

[0415] - After filtering with a 0.45 μm nylon syringe filter

[0416] - After filtering with a 0.1 μm nylon syringe filter

[0417] The 0.45 μm filtered sample was filtered to 0.1 μm.

[0418] Before each filtration, 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.

[0419] result:

[0420] The table below reports the Z-average and PdI averages obtained from three repeated measurements. The Z-average is the intensity-weighted average diameter, and PdI is the polydispersity index.

[0421]

[0422] The results indicate that the aqueous dispersion contains large particles. In particular, the 0.45 µm filtration exhibits a multimodal distribution, with the particle size signal larger than the filter size (4912.0 nm). This also occurs in the 0.10 µm filtration, where a bimodal system is observed with a signal at 156.8 nm, exceeding the filter size. Therefore, the data suggest the presence of supramolecular aggregates composed of non-covalent intermolecular interactions. The data quality of the filtered samples is good.

[0423] Detection of exosomes in Batch 1 of Product C

[0424] Preparation of samples by ultracentrifugation

[0425] Weigh the starting sample (Product C, Batch 1) for ultracentrifugation and resuspend it in a volume of VIB or vesicle separation buffer (20 mM MES; 2 mM CaCl2; 100 mM NaCl, pH 6.0), maintaining a ratio of 5 mL buffer per 500 mg sample. Incubate the sample at room temperature with stirring for 20–24 hours to promote dissolution. After incubation, centrifuge several times at 4°C with increasing speed to separate particles with sizes between 30–500 nm. A T-1250 rotor (Thermo Fisher Scientific, 11718-5) and a Thermo Scientific™ Sorvall™ WX+ ultracentrifuge (Thermo Fisher Scientific™ 75000080, N°: 15342177) were used for ultracentrifugation. Centrifuge the sample at 700 × g for 20 min; discard the precipitate, and filter the supernatant through a 0.45 µm filter and centrifuge at 10000 × g for 30 min. Then transfer the supernatant to an ultracentrifuge tube and centrifuge at 40000 × g for 70 min. Transfer the supernatant to a new ultracentrifuge tube and centrifuge again at 100000 × g for 70 min. Discard the supernatant, resuspend the 100K precipitate in VIB buffer and centrifuge under the same conditions. Resuspend the precipitate in 600 µL of 25 mM trehalose (Merck, T0167) in PBS and store at 4°C for use within 24 hours, or store long-term at -30°C.

[0426] Extracellular vesicle staining and flow cytometry analysis.

[0427] Extracellular vesicles from samples were stained with CellMask™ Green Plasma Membrane Stain (ThermoFisher Scientific #C37608) according to the manufacturer's instructions and quantified on an Attune NxT flow cytometer. Briefly, 27 μL of exosomes were added to 3 μL of CellMask™ Green Plasma Membrane Stain (10x) for each sample and treated at 37°C for 30 min. Then, 170 μL of PBS (0.22 μm double-filtered) was added to each sample and the results were read. To eliminate any non-specific events in the flow cytometry analysis, PBS stained with CellMask™ Green Plasma Membrane Stain was used as a negative control, and Fluorescent Exosome Standards (Novus Biologicals #NBP3-11691) was used as a positive control. Furthermore, each sample was analyzed unstained to exclude any autofluorescence.

[0428] result

[0429] Samples from Product C, Batch 1, were analyzed using the following flow cytometry: PBS values ​​labeled with CellMask™ Green PlasmaMembrane Stain were excluded from the target region because they were considered blank samples. In Fluorescent Exosome Standards, a large number of elements considered positive were identified in the same region.

[0430] The number of elements present in the same region was then considered when analyzing the samples. The number of extracellular vesicles identified in product C sample was 2.7 × 10⁻⁶. 6 .

[0431] 10. Spectral FTIR characterization

[0432] Fourier transform infrared spectroscopy (FTIR) is an analytical technique used to analyze the absorption or emission spectra of a sample by examining the interaction between infrared radiation and matter. FTIR spectra can be affected by weak interactions in the plant matrix (such as hydrogen bonds, van der Waals forces, and hydrophobic interactions), which can alter the position, intensity, and shape of peaks. These interactions influence the absorption properties of functional groups. The FTIR spectra of each material are unique, making it an effective method for studying the physicochemical properties of plant matrices. Therefore, FTIR is valuable for characterizing plant systems.

[0433] FTIR instruments and setup.

[0434] BRUKER Optics' Alpha Spectrometer. The instrument is equipped with a GLOBAR source emitting in the far-infrared and mid-infrared regions, a ROCKSOLID interferometer (Michelson type), a KBr beam splitter, and an RT-DLATGS detector.

[0435] • Resolution: 2 cm -1

[0436] • Spectral range: 5000-300 cm⁻¹ -1

[0437] • Background scan: 50

[0438] • Sample acquisition and scanning: 50

[0439] Sample preparation

[0440] Transfer the first batch of product C sample into an appropriate sample holder for ATR-FTIR analysis of solids and liquids. Approximately 10 mg of sample was placed and pressed against the diamond crystal of the ATR holder. Before recording the measurement results, confirm that the entire sample holder is properly covered.

[0441] Sample collection

[0442] The sample was measured at least three times to verify the reproducibility of the data. The average of the repeated measurements was then taken to obtain a representative spectrum of the sample used for characterization. Figure 14 ).

[0443] 11. Isotopic abundance

[0444] Isotopic abundance analysis provides an atomic-level description of matter, highlighting the effects of isotopic substitution, such as geometrical isotope effects (GIE) and kinetic isotope effects (KIE). GIE involves changes in molecular geometry due to isotopic substitution, particularly affecting hydrogen bonds. These changes influence molecular geometry and affect physical, chemical, and biological properties. KIE refers to changes in reaction rates caused by isotopic substitution in a molecule. Isotopes have different masses, affecting bond vibrational energies and activation energies. KIE is divided into primary KIE, where substitution directly affects the rate-determining step of the reaction, and secondary KIE, where substitution indirectly affects the reaction rate through changes in molecular geometry or electronic effects. Both effects are crucial for understanding the influence of isotopes on molecular behavior.

[0445] First, isotopic abundance analysis was performed on batch 1 of product C.

[0446] The samples were sent to the Saint-Michel Adige Institute (Edmond Mach Foundation) and stable isotope testing was performed as described below:

[0447] - δ18O: Method PDP 7011:2010 REV. 0 (TC-IRMS), unit ‰ vs V-SMOW.

[0448] - δ13C: Method PDP 7009:2017 REV. 2 (EA-IRMS), unit vs. ‰ V-PDB.

[0449] - δ15N: Method PDP 7009:2017 REV. 2 (EA-IRMS), unit ‰ vs V-AIR.

[0450] - δ34S: Method PDP 7013:2010 REV. 0 (EA-IRMS), unit ‰ vs V-CDT.

[0451] -14C activity was also tested by Chelab (Tentamus).

[0452] -14C activity: Method ISO-16620-2;2015 (AMS), unit % modern carbon (pMC).

[0453] The results are as follows.

[0454]

[0455] Therefore, isotopic characterization was performed on batch 1 of Product C. Furthermore, 14C activity was determined in batch 1 of Product C (excluding the raw material providing calcium carbonate). The measured 14C activity of the sample was 99.82 ± 0.22% modern carbon (pMC), corresponding to measurements from purely bio-based carbon materials. There was no evidence of synthetic origin in the analyzed materials.

[0456] Biodegradability test according to OECD 301F:1992

[0457] Biodegradation refers to the breakdown of organic matter into simple, natural components such as CO2, H2O, and NH3 by microorganisms. Assessing the biodegradability of chemicals is crucial in environmental risk assessment. The Reliable Biodegradability Test (RBT), proposed by the OECD, assesses biodegradability by incubating chemicals in a mineral culture medium containing microorganisms. Metabolic parameters such as oxygen consumption and CO2 production are monitored over 28 days. A chemical is considered readily biodegradable if it passes the RBT. Primary biodegradation refers to structural changes in a substance due to biological processes, which can be measured by chemical analysis. For example, the OECD 301F method assesses biodegradability by measuring oxygen consumption in a respirometer under controlled conditions. The results are expressed as a percentage of oxygen consumption relative to the theoretical oxygen demand (ThOD) or chemical oxygen demand (COD).

[0458] Reagents and substances

[0459] This method involves the use of the following reagents and substances:

[0460] A) Batch 1 of test substance product C was diluted at 100 mg / L in mineral culture medium;

[0461] B) The mineral culture medium used to dissolve the test substance consists of the following four solutions (A, B, C, and D), prepared with ultrapure water to a concentration of 1 L:

[0462] - 10 ml solution A: 8.50 g KH2PO4 + 21.75 g K2HPO4 + 33.40 g Na2HPO4 dihydrate + 0.50 g NH4Cl, diluted with ultrapure water to 1 liter, with a final pH of 7.4;

[0463] - 1 ml solution B: 27.50 g anhydrous CaCl2, diluted with ultrapure water to make 1 liter;

[0464] - 1 ml solution C: 22.50 g MgSO4 heptahydrate, prepared into 1 liter solution with ultrapure water;

[0465] - 1 ml solution D: 0.25 g FeCl3 hexahydrate, prepared into 1 liter solution with ultrapure water.

[0466] C) Bacterial inoculum: The test material is added to an appropriate inoculum and obtained by taking activated sludge from an aliquot of the sample.

[0467] D) The chemical standards used for BOD determination from 5 to 28 days consist of various analytical grade anhydrous sodium acetate solutions.

[0468] Test execution

[0469] Sample preparation

[0470] Samples were processed according to the procedures reported in OECD Standard No. 301F for manometry breath measurement. Sodium acetate was used as a reference material to ensure proper recording using the BOD sensor and with sufficient material for the planned instrumental analysis. Tests were performed at a constant temperature of 22°C.

[0471] Inoculum preparation

[0472] Inoculum was prepared by mixing activated sludge from different sites in equal volumes. The inoculum was aerated, stirred, and then glucose, peptone, and dipotassium hydrogen phosphate were added. Redox potential, oxygen consumption, and total dry matter were monitored daily. Dry matter was measured at 100°C to ensure that the amounts in containers containing the test material were identical (30 mg / mL).

[0473] Inoculum composition

[0474] The inoculum was obtained by mixing activated sludge. The assembled inoculum was aerated, stirred, and then glucose, peptone, and potassium dihydrogen phosphate were added. Oxygen, redox, and total suspended solids values ​​were monitored daily. Total dry matter was measured before use of the inoculum.

[0475] The composition of the microfauna was determined by optical microscopic analysis.

[0476] Reference substance

[0477] The pressure respiration measurement method also requires another test using ultrapure water with added standard (sodium acetate) to assess normal performance and instrument reliability.

[0478] Blank sample

[0479] Blank analysis was performed on the inoculated mineral culture medium to assess the contribution of liquid and inoculum to the final product BOD value.

[0480] Test conditions:

[0481] The container was placed in a constant-temperature refrigerator set at 22±2°C and continuously stirred by the mechanical movement of the anchor; all of this was carried out for 28 days, and the oxygen inhibition value was measured automatically and wirelessly every 6 hours. The oxygen inhibition value after 28 days became the absolute biodegradability value of the sample under study.

[0482] Test Results

[0483] Product C, Batch 1:

[0484]

[0485] Blank sample:

[0486]

[0487] The experiment showed that the BOD contribution between the inoculum and the mineral culture medium was 11.1 mg / l, and this value was applied to the BOD correction of the 100 mg / l mixture in batch 1 of product C.

[0488] The average value fell within the positive range of the test, which is 10 mg / L to 50 mg / L.

[0489] Reference material:

[0490]

[0491] A positive test result is supported by the following evidence:

[0492] A) The value obtained from the chemical standard (30.8 ppm biochemical oxygen demand, BOD) is within the positive range of the experiment, i.e., 31 ± 5 ppm theoretical oxygen demand (ThOD), therefore the chemical control is positive.

[0493] B) After only 7 days, the BOD value reached 89.68% of the total.

[0494] C) After 14 gg, the BOD value was 93.22% of the total.

[0495] D) After 28 gg, the BOD value reaches 99.35% of the total.

[0496] The reference material reached the threshold within 14 days. Therefore, it can be said that the test was performed correctly and the collected data on the biodegradability of the tested material are reliable.

[0497] Biodegradation calculations were performed on the reference material, test sample, and blank sample at each sampling time.

[0498] The total biodegradation of the sample was calculated using the following formula:

[0499] BOD: (Average mg / L of O2 consumed by the test substance) - (Average mg / L of O2 consumed by the blank sample) / mg / L of the test substance in the container.

[0500] There is no evidence that nitrite and nitrate are produced, therefore no nitrification / denitrification correction is performed.

[0501] - O2 consumed by the test substance: 51.25 mg / L.

[0502] - O2 consumed by the blank sample: 11.1 mg / l.

[0503] - O2 mg / l consumed by nitration in the product: /

[0504] - Test chemical in container (mg / l): 100 mg / l

[0505] The average BOD value is calculated as 0.402 mg O2 per milligram of test substance.

[0506] To calculate the percentage of degradation and thus biodegradability, chemical oxygen demand (COD) was assessed by hot acidic dichromate oxidation in ultrapure deionized water; a 50 mg / L Abo11 solution was tested, yielding a COD of 67.1 mg / L.

[0507] COD = (O2 consumed by the test substance, mg / L) / (Test substance in the container, mg / L)

[0508] COD = 0.467 mg of O2 per milligram of test substance.

[0509] Average degradation percentage over 28 days: Average BOD after blank correction / COD (pure substance) = (0.402 / 0.467) 100 = 86.08%

[0510] Interpretation of Results

[0511] Validity criteria

[0512] The test is considered valid under the following conditions:

[0513] - After 14 days of incubation, the average biodegradation rate of the reference material was greater than 60%;

[0514] - The difference between repeated extreme values ​​during the plateau period at the end of the experiment was less than 20%;

[0515] - The oxygen demand of the blank sample should not exceed 60 mg O2 / L.

[0516] Interpretation

[0517] A substance is considered readily biodegradable if it reaches a biodegradation level of more than 60% within 10 days of the onset of degradation (i.e., the time when 10% of the substance has been degraded (10-day window)) or if it reaches a biodegradation level of >60% within 28 days.

[0518] Results

[0519] - Meets the validity criteria of the experiment.

[0520] - Regarding the assessment of the biodegradability of product C, batch 1, at a concentration of 100 mg / L, in the experiments conducted, the product demonstrated readily biodegradable properties under the pressure breath test conditions established according to EC Regulation 440 / 2008 (updated to Regulation 640 / 2012 - Part C: Ecotoxicity determination method - Method c.4. Part V - (Method c.4-d) + OECD 301F:1992); in fact, the product achieved a biodegradability of over 60% (62%) within ten days after reaching a 10% biodegradability rate, meeting the effectiveness criteria of the method.

[0521] - Product C, batch 1, showed no toxicity to microorganisms at a concentration of 100 mg / L, and the tested concentration reached 86.06% of the relevant theoretical COD value, thus demonstrating good biological activity.

[0522] According to the conditions of the pressure breath test (OECD 301F, EC Regulation 440 / 2008 and subsequent updates, Method C.4-D), at a concentration of 100 mg / L, the tested substance was found to be "easily biodegradable" under aerobic conditions. In fact, the substance's biodegradation rate exceeded 60% within ten days after reaching a 10% degradation rate.

[0523] 12. Network Analysis

[0524] Network analysis was conducted on pathological conditions treated or improved by product C, and the data obtained showed how the tested natural matrix-based product affected the body at the system scale.

[0525] Product C

[0526] - Pathological conditions ( Figure 12a(Figure A): Dysfunctional and inflamed adipose tissue leads to an imbalance in bone homeostasis, adversely affecting the competition for mesenchymal stem cell reserves used to induce osteoblast, osteoclast, or adipocyte differentiation, and nonphysiologically shifting this trend towards osteoclast differentiation. This results in a reduced number of mature osteoblasts, failing to ensure proper mineralization of the cellular matrix, leading to loss of bone activity. In cases of lipid metabolism disorders and adipose tissue inflammation, a dysfunctional cycle forms between adipose tissue and bone, leading to the accumulation of adipocytes and osteoclasts, which is detrimental to osteoblast components and exacerbates bone fragility. Bone is also an endocrine organ, and thus can influence events in other tissues at the systemic level, such as by secreting osteocalcin (OCN) to promote insulin secretion from the pancreas, promote insulin sensitivity in peripheral organs such as muscles, and regulate overall energy expenditure (Fukumoto S, Martin TJ. Bone as an endocrine organ. Trends Endocrinol Metab. 2009 Jul;20(5):230-6. Doi: 10.1016 / j.tem.2009.02.001. Epub 2009 Jun 21. PMID: 19546009).

[0527] - Treatment with reference medication ( Figure 12b (Small Figure B): The reference drug can only reduce the number of fat cells.

[0528] - The state of being treated with product C ( Figure 12c (Figure C): Product C, through interaction with pools of mesenchymal stem cells in bone and adipose tissue, is able to reproduce all the necessary elements for restoring proper bone turnover, promoting the formation of strong and functional bone structures, and restoring the balance of metabolic disorders and reducing inflammation at the systemic level.

[0529] 13. Detailed analysis of three different batches of products

[0530] To understand whether the final matrix constituting product C exhibits matrix effect characteristics, a series of analyses were conducted on the three batches 1, 2, and 3 described above to understand the characteristics of the product in different aspects. Figure 13a and 13b The results showed that these three batches had the same expected therapeutic / beneficial effects in in vitro cell-based assays. Targeted metabolomics analyses, capable of identifying most of these molecular components, were performed on the plant matrix components of the different batches (see table below), along with other analyses reported in this paper.

[0531] As mentioned above, the product consists of a plant-based natural matrix and other natural matrices, which are assembled to form the final new natural matrix. Several analytical techniques have been used to identify and quantify the major classes of compounds in plants to assess the composition of the product's plant matrix components. While metabolomics analysis cannot reveal the dynamic changes in matrix components, it can "map" the composition at the moment of analysis.

[0532] Each plant metabolite was specifically studied using "targeted metabolomics." By identifying the compounds present in the material, the analysis captures a qualitative data framework, and by determining the concentration of each compound in the material, it captures a quantitative data framework.

[0533] For product C, an "omics" approach was adopted, using targeted metabolomics analysis based on multiple analytical methods to qualitatively and quantitatively characterize as many primary and secondary metabolites as possible.

[0534] The analytical methods used for chemical characterization of each batch are described below. The most suitable analytical technique was employed based on the chemical properties of the class of compounds present. Chromatographic analysis combined with different detection techniques (e.g., GC and LC, each with appropriate detectors) allows for the proper identification and quantification of organic compounds. Inductively coupled plasma mass spectrometry (ICP-MS) using a single quadrupole mass spectrometer or inductively coupled plasma optical emission spectrometry (ICP-OES) can determine the levels of present elements, while anions are determined by ion chromatography and conductivity detectors.

[0535] Batch 1: 23D2227

[0536] Batch 2: 24F0699

[0537] Batch 3: 23K3123

[0538]

[0539]

[0540] Note 1. The grey boxes indicate the major chemical classes.

[0541] Note 2. % = Concentration of the compound expressed as a percentage of the whole matrix.

[0542] Note 3. (d%) = Percentage deviation: (|Reference 23D2227 (%) - Test (%)| / (Reference 23D2227 (%)) × 100)。

[0543] Note 4. The term "total" refers to the sum of the values of the various compounds making up the corresponding group.

[0544] Note 5. <LdQ = Below the limit of quantification.

[0545] Note 6. / = Percentage deviation not quantifiable

[0546] Qualitative miRNA characterization was performed on each batch of ultracentrifuged samples, and the results showed that the metabolomics were highly complex and contained typical biomass miRNAs.

[0547]

[0548] The results show significant differences in the composition of each batch of plant substrate components, emphasizing that the properties of the substrate cannot be generalized as the sum of its individual components. The work presented and reported in this paper (see cell-based analysis results) and the following data demonstrate that the biological effects induced by the product cannot be summarized by the sum of the effects of individual molecular components, but rather are the result of the interrelationships and interactions between components: the substrate effect. This means that it is impossible to formally define the structure-activity relationship (SAR) according to the API specification principles.

[0549] The results of targeted metabolomics highlighted more or less significant quantitative fluctuations in individual chemical substances and miRNA classes in the plant matrix components of the three batches of Product C. If these fluctuations were taken as a reference parameter, it would lead to the prior expectation that these batches would have different therapeutic or beneficial effects. The summary analysis in Figure 13 shows that, although bioactivity was maintained in all the different batches evaluated, none of the identified individual molecular components met the criteria set for a single API, indicating that the matrix cannot be considered a summary of APIs.

[0550] As shown above, each batch retains the same therapeutic effect (functional flexibility).

[0551] This product can evoke the same response in a biological system as intended for its purpose through a physiological mechanism of action.

[0552] This also highlights the fact that products containing or composed of natural matrices retain structural and functional redundancy mechanisms, exhibiting the functional resilience unique to biomass (achieving the same results despite individual differences among individuals of the same species).

[0553] Isotope abundance of all batches

[0554] To assess the isotopic ratios between different batches of product C, batches made from different starting materials were analyzed. Samples were sent to the Saint-Michel Adige Institute (Edmond Mach Foundation) and stable isotope testing was performed as described below:

[0555] - δ18O: Method PDP 7011:2010 REV. 0 (TC-IRMS), unit ‰ vs V-SMOW.

[0556] - δ13C: Method PDP 7009:2017 REV. 2 (EA-IRMS), unit vs. ‰ V-PDB.

[0557] - δ15N: Method PDP 7009:2017 REV. 2 (EA-IRMS), unit ‰ vs V-AIR.

[0558] - δ34S: Method PDP 7013:2010 REV. 0 (EA-IRMS), unit ‰ vs V-CDT.

[0559] The results are as follows.

[0560] Main isotope δ ratios of product batch C:

[0561]

[0562] Based on the retention of this parameter, the overlap of δ18O, δ15N, δ34S, and δ13C values ​​between batches indicates that the production process has a high degree of repeatability.

[0563] 14. Definition of any unit of product C activity (U - product C):

[0564] Analysis of the reproducibility criteria for natural products led the inventors to focus on the reproducibility of the product's biological effects. Analysis of different batches revealed significant chemical differences. However, despite these differences, each batch exhibited functional resilience, consistent with the concept of maintaining biological activity. This approach is based on the redundancy principle (functional resilience), ensuring that the product retains its biological efficacy despite variations. Therefore, the product must be regulated in units and characterized by non-pharmacological mechanisms. This moves it away from the traditional qSAR concept, highlighting the unique physiological properties of its mechanism of action.

[0565] The product is a 100% natural, biodegradable matrix rich in miRNAs and exosomes from different batches. These characteristics can be better managed by applying the concept of arbitrary activity units (rather than the weight or volume of traditional APIs) to product dosage and batch release strategies.

[0566] Product C unit (U-product C) is defined as one-tenth of the amount of product required to induce a change in the state of hADMSCs, measured at a marker level, using limits and methods defined in Figures 10, 11, and 13, or in a method for assessing whether a therapeutic product exerts its therapeutic effect on a pathological state through the physiological mechanism of action disclosed in the product information.

[0567] In the case of the first batch, based on the experimental parameters described in the Methods and Materials section, 10 U of Product C is equivalent to 50.4 mg of Product C dissolved in 4 ml.

Claims

1. A product for the treatment or adjunctive treatment of bone fragility in a subject in need, said product comprising the following components: Each component is in powder form, and The species mentioned is *Equisetum*, *Acacia*, *Acacia seneca*, *Acacia rubiginosa*, *Agave*, *Agave sisal*, *Agave spp.*, and *Agaricus*.

2. The product according to claim 1, comprising the following components The species mentioned is *Equisetum*, *Acacia*, *Acacia seneca*, *Acacia rubiginosa*, *Agave*, *Agave sisal*, *Agave spp.*, and *Agaricus*.

3. The product according to claim 1, comprising the following components Formula A Or formula B Or formula C The species mentioned is *Equisetum*, *Acacia*, *Acacia seneca*, *Acacia rubiginosa*, *Agave*, *Agave sisal*, *Agave spp.*, and *Agaricus*.

4. The product according to any one of claims 1 to 3, wherein the coral is Caribbean coral and the bird is red junglefowl.

5. A composition for treating or adjuvant treatment of bone fragility in a subject in need, comprising the product according to claim 1 and at least one natural carrier.

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

7. The product according to claim 1 or the composition according to claim 5, wherein it is in the form of lyophilized material, tablet, soft or hard gelatin capsule, powder, granules, loaded vesicles, loaded liposomes, or loaded nanoparticles.

8. The product according to claim 1 or 2 or the composition according to claim 5, wherein the subject is at risk of developing a fragile bone condition.

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

10. The product or composition for the use of claim 9, wherein the increase in fat is associated with obesity, metabolic syndrome, premenopause, perimenopause or menopause, or male menopause.

11. The product or composition for the use of claim 8, wherein the bone fragility condition is osteoporosis or osteopenia.

12. The product according to any one of claims 1 to 3 or the composition according to claim 5, wherein the product or composition is itself a natural matrix.

13. The product or composition according to claim 12, wherein, Whether the product or composition is a natural matrix is ​​determined by verifying its emerging properties in restoring healthy physiological states of bone metabolism when the following conditions are met: its 14C activity is 99.82 ± 0.22% as measured by ISO-16620-2;2015 (AMS) method; miRNA and exosomes are detected in the product or composition; and the product or composition exhibits batch-to-batch therapeutic or beneficial functional resilience between different batches of the product or composition and when the product or composition modulates overall altered physiological or pathological conditions.

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