Novel aging reversal method and application thereof

Abstract

The invention discloses a novel method for delaying, decelerating, stopping and reversing cellular senescence of a human subject through a continuous two-stage treatment regimen. In the first stage, an aging clearance therapy with a natural compound is administered to selectively reduce aging cell burden. Standardized extracts rich in flavonols and lignans work cooperatively to target and remove these dysfunctional cells, thereby reducing chronic inflammation and alleviating tissue degradation. In the second stage, a reversal of senescence therapy comprising oleanane triterpenes and beta-alanyl-L-histidine is applied to reprogram any remaining senescent cells back to a non-senescent functionally active state, resulting in tissue repair and recovery of homeostasis. The test results of different human cell types such as fibroblasts, endothelial cells, adipocytes and mesenchymal stem cells show that the continuous method not only eliminates harmful senescent cells, but also enables viable cells to restore vitality, and the method is suitable for large-scale production. A series of age-related diseases including premature senility, metabolic dysfunction and cardiovascular diseases are effectively solved. By integrating both elimination and reversal strategies, this approach overpasses the efficacy of conventional monotherapy in the field, retains beneficial cellular function and promotes tissue regeneration. The layering strategy embodies novelty of treatment intervention of senescence-related conditions, and a promising way is provided for prolonging the health period and improving the overall physiological elasticity.

Description

Summary of the Invention

[0001] This invention proposes a novel strategy for combating age-related disorders, addressing the accumulation of senescent cells and their deleterious effects on health and aging. The core of the invention lies in a two-stage treatment approach:

[0002] Phase 1: senolytic agents

[0003] A synergistic combination of natural flavonols (e.g., fisetin, quercetin, kaempferol, myricetin) with a wider family of lignans (e.g., podocarpin, secoisolariciresinol, pinoresinol, magnolol, purinol). This targeted combination enhances the senolytic potential of these natural compounds, enabling the selective elimination of senescent cells while minimizing off-target effects associated with the use of isolated flavonoids. A prime example of this synergy is the combination of a rich source of flavonols with an oat extract standardized for high lignan content.

[0004] Phase 2: Senoreverting agents

[0005] Utilize the function of combining oleanane-type triterpenes (e.g., oleanolic acid, maslinic acid) with aging-reversing amino acid derivatives or small peptides (e.g., β-alanyl-histidine, taurine, N-acetylcysteine, glutathione, L-carnitine). This combination targets cellular pathways involved in the repair, regeneration, and inhibition of the senescence-associated secretory phenotype (SASP), promoting the regeneration of tissues affected by aging. A powerful example of this preparation is the use of a rich natural triterpene source in combination with β-alanyl-histidine.

[0006] Main advantages of the present invention

[0007] Addresses multiple root causes of aging and its associated decline in health.

[0008] Leverage the efficacy of natural compounds while overcoming the limitations of their individual use through carefully designed synergistic formulations.

[0009] Compared to existing approaches, it provides a novel combination of senolytic and senolytic effects, as demonstrated in various tissue types.

[0010] Offers a promising approach to alleviate various age-related health conditions and potentially extend healthspan. Field of the Invention

[0011] The present invention relates to the fields of nutraceuticals, anti-aging therapies, regenerative medicine, and treatment of age-related diseases. Specifically, a novel approach is introduced to reduce the burden of senescent cells and their deleterious effects on healthspan. The approach utilizes the sequential application of two potent natural agents:

[0012] Senolytic agents: designed to selectively eliminate senescent cells.

[0013] Aging reversal agents: These aim to reverse the cellular characteristics of aging and promote tissue regeneration and repair.

[0014] The present invention has potential applications in the treatment and prevention of a variety of age-related conditions resulting from aging, including but not limited to cardiovascular disease, metabolic disease, neurodegenerative disease, musculoskeletal disorders, skin aging, immune dysfunction and cancer. Background of the Invention

[0015] Aging is a complex process characterized by the progressive accumulation of damage at the molecular, cellular, and tissue levels. This damage leads to a physiological decline known as senescence. Cellular senescence, a key hallmark of aging, is a state in which cells permanently cease dividing but remain metabolically active. Senescence can be triggered by various stressors, including DNA damage, oncogenes, reactive oxygen species (ROS), and chronic inflammation.

[0016] Although cellular senescence initially serves a protective role (e.g., preventing the proliferation of damaged cells), its persistence becomes detrimental with age. The immune system's ability to clear senescent cells declines over time, leading to their accumulation in tissues. These senescent cells secrete proinflammatory cytokines (senescence-associated secretory phenotype, or SASP), creating a chronic inflammatory environment that damages neighboring cells and accelerates age-related tissue dysfunction.

[0017] Senescent cells are more prevalent in disease-prone tissues, where factors such as inflammation, metabolic stress, and toxins promote their formation. This creates a vicious cycle whereby senescent cells exacerbate aging and the development of age-related pathologies.

[0018] Obesity is a prominent example of how aging promotes chronic disease. Adipose tissue acts as a reservoir of senescent cells, triggering local and systemic inflammation that leads to increased insulin resistance, accelerated aging, and a higher risk of mortality.

[0019] Stem and progenitor cells are crucial for tissue maintenance and repair, but they are also susceptible to senescence. Accumulation of DNA damage in these cells leads to replication arrest and the adoption of a proinflammatory phenotype, hampering their regenerative capacity.

[0020] Given the deleterious effects of senescent cell accumulation, strategies to address this issue are crucial. Senescent cell-mediated therapies, which aim to selectively eliminate senescent cells, have emerged as potential interventions to improve healthspan and longevity. Numerous companies have identified protein targets of interest that could be targeted with drugs.

[0021] However, while finding or synthesizing small molecules that target specific proteins is a common drug development strategy, its effectiveness against complex, evolutionarily conserved pathways associated with aging has been shown to be limited. This is supported by the lack of success or adverse reactions observed with existing single-protein senolytic drugs (e.g., those targeting MDM2, BCL-2, HSP90). Notably, the combination of dasatinib plus quercetin (D+Q) stands out and exhibits some clinical efficacy.

[0022] Finally, simply removing senescent cells and replenishing them may have unintended consequences for tissues. This is particularly important for senescent stem cells, which may have to be reprogrammed rather than "killed" to restore their function.

[0023] The present invention addresses these challenges with a novel application approach comprising two treatment phases. The first phase utilizes a naturally derived senolytic agent to selectively eliminate senescent cells with a severe SASP burden, reducing the overall inflammatory burden. Following the senolytic phase, the application introduces another natural agent to induce a "senescence reversal" effect, promoting cellular reprogramming of the remaining senescent cells to exit senescence. This reprogramming aims to restore these cells to a non-senescent state, enhancing their potential for tissue repair and regeneration, without completely eradicating senescent cells that may be beneficial for specific physiological functions.

[0024] This combined senolytic and "senescence reversal" approach addresses the complexity of aging while minimizing the potential negative effects of completely eradicating senescent cells.

[0025] Previous state of the art

[0026] A cornerstone of addressing the aging process is the identification of robust biomarkers that can accurately diagnose the senescent state of a cell. The earliest and most established of these biomarkers is the activity of senescence-associated β-galactosidase (SA-β-gal). Using a direct colorimetric assay, SA-β-gal has helped to demonstrate the accumulation of senescent cells in various mammalian tissues affected by age-related diseases. This biomarker has been a diagnostic tool for aging and has opened the door to targeted interventions, including the development of senolytic drugs designed to selectively eliminate these senescent cells. Depending on the cell type, other supporting biomarkers have been introduced, such as p16 INK4A, lipofuscin, p21, p53, SAHF and various components of the senescence-associated secretory phenotype (SASP), etc.

[0027] As one of the pioneering approaches to targeting senescent cells, researchers used the biomarker p16 INK4A An innovative transgenic model, named INK-ATTAC, was created. This model was specifically designed to conditionally eliminate the expression of p16 following subsequent drug administration. INK4A Specifically, in the context of BubR1 progeria mice, INK-ATTAC was shown to effectively eliminate p16 when a triggering drug was administered. INK4A Senescent cells (Baker et al., 2011). This targeted removal has had a significant impact on tissues susceptible to age-related pathological changes, such as adipose tissue, skeletal muscle, and the eye region. Notably, lifelong elimination of these senescent cells can lead to a delay in the onset of age-related diseases. Furthermore, clearing these cells later in life can mitigate the progression of existing age-related diseases.

[0028] Another macromolecule being added to the senolytic drug pipeline is a peptide called FOXO4-DRI. The peptide in question has been engineered to selectively disrupt the interaction between p53 and FOXO4 (Baar et al., 2017). This interaction facilitates senescent cells' escape from apoptosis, an intrinsic mechanism of programmed cell death. Consequently, disruption subsequently triggers the cells' intrinsic apoptosis. Consequently, selective removal of senescent cells is achieved, preserving the viability of unaffected cells.

[0029] One of the first oral drugs in the field of senolytics came with the discovery of a synergistic effect of quercetin, a plant flavonol, and dasatinib, an anticancer drug (Zhu et al., 2015). Dasatinib is primarily known for its inhibitory effects on certain protein tyrosine kinases, while quercetin is a flavonol that has been associated with PI3K / AKT signaling inhibition. When used in combination, they have been shown to selectively induce apoptosis in senescent cells, thereby extending the healthspan in cell and animal models. Dasatinib + quercetin (D+Q) is currently the only oral drug with clinical efficacy. A short-term treatment regimen of 3 days of dasatinib (100 mg / day) and quercetin (500 mg twice daily) reduced the human senescent cell population (Hickson et al., 2019). The study focused on

[0030] We mark an important milestone by demonstrating the in vivo effectiveness of senolytic clearance in a clinical setting in individuals aged 55 to 79 years with medically controlled diabetes and chronic kidney disease (CKD), urging its use as a reasonable comparison standard for cell-based assay studies.

[0031] Senescent cells are also characterized by elevated expression of proteins that block programmed cell death, particularly members of the BCL-2 family, including BCL-2, BCL-W, and BCL-XL. These proteins confer resistance to stimuli that would normally induce apoptosis. Senolytic drugs such as ABT-737 and ABT-263 (also known as navitoclax) effectively address this resistance by reducing the activity of BCL-2 family members (Chang et al., 2016). This inhibition, therefore, allows senescent cells to undergo apoptosis.

[0032] Recently, a new wave of natural and naturally derived substances with senolytic properties has garnered significant attention. Preliminary validation in cell-based assays and animal studies suggests that these compounds hold considerable promise for translation into clinical applications. For example, fisetin is one such substance that has garnered attention for its senolytic properties both in vitro and in vivo (Yousefzadeh et al., 2018). A clinical study is underway at the Mayo Clinic. Notably, flavonols, including quercetin and fisetin from strawberry extract, have been previously identified as potential senolytic agents.

[0033] According to a study (Wang et al., 2016), piperlongumamide (PL) effectively targets senescence in human WI-38 fibroblasts induced by ionizing radiation, replicative exhaustion, or overexpression of the oncogene Ras. According to these researchers, piperlongumamide induces apoptosis in senescent cells without the need for reactive oxygen species. Furthermore, they revealed that when combined with ABT-263, PL exhibits a synergistic effect in eliminating senescent cells. Preliminary structural modifications have also led to the preparation of PL analogs with enhanced potency and selectivity in inducing the reduction of senescent cells.

[0034] Curcumin has also been examined for its potential senolytic properties. More specifically, researchers explored the senolytic potential of various curcumin analogs, ultimately identifying EF24 as a broad-spectrum agent targeting senescent cells (W. Li et al., 2019). These studies found that EF24 triggers apoptosis in senescent cells independently of the production of reactive oxygen species (ROS). Instead, it works by promoting the proteasomal degradation of the Bcl-2 family of anti-apoptotic proteins, which are thought to be important for protecting senescent cells from apoptosis. Not surprisingly, these researchers also showed that EF24 exhibited a synergistic effect in eliminating senescent cells when combined with ABT-263, a synthetic senolytic agent that inhibits Bcl-2 and Bcl-xl.

[0035] Quercetin alone has been primarily described as a senolytic compound (at doses above 20 μM) rather than a senolytic agent, which was also confirmed by our assay. The proposed mechanism of action is through regulation of NF-κB and SIRT-1 (Zoico et al., 2021).

[0036] Senescence reversals (senoreverters) and senomorphics are recently used terms. Although there is no clear distinction between them, senescence reversals primarily represent drugs that can drive senescent cells to reenter the cell cycle and undergo extensive repair. Senescence modulators are compounds that can reduce the deleterious effects of the SASP or inhibit senescence without inducing SNc death (Zhang et al., 2022).

[0037] Rapamycin can be classified as a senescence-reversing agent (Blagosklonny, 2018). It works by inhibiting the mTOR (mammalian target of rapamycin) pathway, which helps maintain the ability of non-proliferating cells (e.g., senescent or quiescent cells) to re-enter the cell cycle. The ability of rapamycin to inhibit senescence in both in vitro and in vivo studies highlights the potential for designing or discovering similar classes of compounds as senescence-reversing strategies. DETAILED DESCRIPTION

[0038] The present invention relates to a novel method of using potent natural senolytic agents followed by senolytic agents to significantly reduce senescent cells and restore healthy cells, thereby reducing aging-related symptoms.

[0039] In the present invention, aging-related disorders leading to accelerated aging, classical aging are represented by 3 main causes:

[0040] 1. Inducing DNA damage, leading to accelerated aging or pathological conditions derived from DNA damage. DNA damage, in this context, refers to damage caused by increased exposure to DNA-damaging agents, such as reactive oxygen species, reactive nitrogen species, ionizing radiation, cosmic radiation, mutagenic chemicals, toxins, ultraviolet light, and chemotherapeutic agents, which can cause DNA breaks in healthy cells. Diseases associated with a wider range of DNA damage include various hematological malignancies, skin cancers, progeria syndromes, immunodeficiency, infertility, autoimmune diseases, sensory disorders, liver disease, and neurological conditions.

[0041] 2. The natural accumulation of senescent cells, resulting from the accumulation of macromolecular damage and the inability of aging organisms to clear them. Characteristic causes of this damage may be standard exposure to reactive oxygen species, ultraviolet light, the accumulation of dysfunctional macromolecules due to modification of dysfunctional macromolecules (e.g., uncontrolled protein glycosylation), telomere shortening and damage, altered intracellular communication, epigenetic changes in chromatin and cells, and the secretion of senescence messengers from surrounding cells or tissues. Conditions associated with the natural accumulation of senescent cells are also aging-related diseases and conditions: metabolic diseases (type 2 diabetes), chronic kidney disease, hypertension, coronary artery disease, a weakened immune system, joint inflammation, bone density loss, cardiovascular disease, loss of muscle mass, muscle wasting, loss of skin elasticity, slowed recovery from injury, retinal degeneration, fatigue, graying of hair, skin wrinkling, decreased vision, frailty, metabolic syndrome, dementia, cognitive decline, fatigue, sarcopenia, eye-related conditions, and idiopathic pulmonary fibrosis, as well as skin-related conditions such as hyperpigmentation, increased appearance of wrinkles, photosensitivity, or conditions associated with the effects of sunlight.

[0042] 3. Due to existing underlying conditions for various reasons, senescent cells can also accumulate, worsening the overall condition or disease itself. Both young and elderly people can suffer from such diseases, which exacerbate cellular aging and the disease itself through the inflammatory secretome. Such diseases can include fatty liver disease, autoimmune diseases (such as type 1 diabetes, lupus, inflammatory kidney disease, rheumatoid arthritis, psoriasis, and neurodermatitis), monogenic diseases (such as cystic fibrosis, Huntington's disease, muscular dystrophy), chromosomal diseases (such as Down syndrome and Turner syndrome), complex diseases (such as autism spectrum disorder, schizophrenia, and bipolar disorder), mitochondrial diseases (such as mitochondrial myopathy and Leber's hereditary optic neuropathy), mitochondrial-related diseases (such as Alzheimer's disease, Parkinson's disease, phenylketonuria, and Tay-Sachs disease), and skin diseases with various underlying diseases (such as atopic dermatitis, acne, urticaria, rashes, rosacea, and eczema).

[0043] The senolytic formulations representing the first treatment phase according to the present invention hypothesize a synergistic interaction between compounds of natural origin, particularly plant-derived flavonols and plant-derived lignans. In our tests, we used a combination of a standardized strawberry extract comprising 90% flavonols and an oat extract standardized to 90% lignans. However, the lignans referred to in the present invention encompass a broader family of lignans, beyond those found in oat extracts. These include, but are not limited to, podocarpin, secoisolariciresinol, pinoresinol, magnolol, and purinol. Likewise, the flavonols referred to herein refer to a variety of compounds within the flavonol class, not limited to those extracted from strawberry extracts. These include the readily available flavonols fisetin, quercetin, kaempferol, and myricetin, which are derived from edible plants.

[0044] The aging-reversing formulation, representing the second stage of treatment according to the present invention, infers an enhanced regeneration / reprogramming effect derived from a combination of a plant extract rich in oleanane-type triterpenes and one of the aging-reversing amino acid derivatives or small peptides. In our tests, we used a fruit extract standardized to 75% oleanane-type pentacyclic triterpenes in combination with β-alanyl-L-histidine. Regardless, the oleanane-type triterpenes discussed in the present invention encompass the entire family of oleanane-type triterpenes present in plants, including but not limited to oleanolic acid and maslinic acid. Accordingly, the use of β-alanyl-L-histidine in this embodiment infers amino acids, amino acid derivatives, and small peptides that can reduce aging, such as β-alanyl-L-histidine, taurine N-acetylcysteine, glutathione, and L-carnitine, among others.

[0045] Natural compounds such as flavonoids hold compelling therapeutic potential but also present challenges for drug development. Their broad affinity for various protein targets can lead to off-target effects, reduced efficacy, and complex, dose-limiting metabolic profiles. This highlights the need for strategies to enhance the specificity and potency of natural compounds for targeted aging therapies.

[0046] The methods of the present invention address these limitations by using synergistic formulations that combine flavonoids with complementary natural compounds, such as lignans or amino acid derivatives. This strategy minimizes the drawbacks of broad-spectrum binding associated with isolated flavonoids, enabling reduced dosages and enhanced senolytic and aging-reversing effects. These auxiliary ingredients were selected because they can modulate cellular pathways, enhance the effects of flavonols or triterpenes, and avoid competitive interactions. This concept was successfully validated by the combination of dasatinib and quercetin, where neither compound alone exhibited robust senolytic activity at well-tolerated doses, but the combination showed significant efficacy.

[0047] In an exhaustive screen of 98 potential natural compounds, oat extract standardized to 90% lignans showed the highest potency among all tested natural compounds (known natural senolytics) alone at a concentration of 15 μmol, even when compared to the D + Q combination. A preliminary screen was performed on senescent IMR-90 (ATCC-CCL-186) cells pretreated with etoposide as a standardized cellular senescence inducer (Nagano et al., 2016). Briefly, IMR-90 (human lung tissue fibroblasts) were grown in a proprietary culture medium (ATCC-30-2003) according to the manufacturer's instructions. Low-passage (<5 times) cells were treated with 12.5uM etoposide (Cell Signaling Technology, #2200) or an equal amount of DMSO (for non-senescent controls) for 24 hours; the medium was replaced and the cells were allowed to recover for 2 days, and then treated with the above 98 active compounds at a concentration of 15μM, as well as Q+D consisting of 20μM quercetin (Q) and 200nm dasatinib (D). Q+D is an excellent control previously established for validating the effect. After treatment, the percentage of senescent cells was assessed by flow cytometry using a commercially available kit (CellEvent SenescentGreen Flow Cytometry Assay Kit, Invitrogen C10841) according to the manufacturer's instructions. Senescent lung fibroblasts exhibited high activity of senescence-associated β-galactosidase (SA-β-Gal) and accumulated a green fluorescent product that was detected by 488nm laser excitation.

[0048] Initial screening showed that an oat extract standardized to contain 75% lignans (called LIG) significantly reduced the prevalence of senescent cells to less than 40% (as measured by the percentage of SA-β-Gal positive cells in the etoposide fraction), making it a promising compound for further study. These studies were designed to evaluate its performance, either independently or alongside other molecules with senolytic properties as claimed in our initial screen or as indicated in existing scientific publications (suitable for the senolytic phase of the present invention). Figure 1Results are shown in a bar graph showing the effects of selected test compounds on senescent fibroblasts. LIG at 15 μM alone was superior as a senolytic compared to any other single 15 μM compound or combination. Unexpectedly, co-administration of LIG with flavonol-rich strawberry extract (FLA) exhibited significant senolytic effects, effectively eliminating the majority of senescent fibroblasts while exhibiting minimal toxicity. This suggests a synergistic interaction between LIG and these specific, previously undocumented flavonols. In contrast, the known and frequently reported compounds fisetin (FIS) and quercetin (Q), used alone, and their combinations with dasatinib (D+Q and D+FIS), failed to produce similar effects. Fibroblast senescence can lead to various damaging conditions associated with the musculoskeletal system (bone, cartilage, and skeletal muscle) and skin. Therefore, in this embodiment, the synergistic senolytic formulation (LIG+FLA) mentioned above can be used as an effective means to remove cells that cause joint inflammation, bone density loss, muscle mass loss, muscle wasting, osteoarthritis, osteoporosis, periodontal disease, osteoporosis, loss of skin elasticity, slow injury recovery, fatigue, graying hair, skin wrinkling, vision loss, frailty, and idiopathic pulmonary fibrosis.

[0049] In another cellular assay, we tested the senolytic effects of the same compounds to see if they could be replicated in different human tissue types that characterize aging-related diseases, specifically endothelial senescence, a major hallmark of cardiovascular events. As before, a screen was performed to verify a reduction in the percentage of SA-β-Gal after administration, but this time using a different cell line. Human aortic endothelial cells (HAEC, ATCC-PCS-100-011) were grown in appropriate culture medium according to the manufacturer's instructions and pretreated with etoposide. Low-passage (<5) cells were treated with 12.5 μM etoposide (Cell Signaling Technology, #2200) or an equivalent amount of DMSO (for non-senescent controls) for 24 hours; the medium was replaced, and the cells were allowed to recover for 2 days before being treated with 15 μM concentrations of the top four active compounds (LIG, FLA, fisetin, and quercetin), as well as D+Q, consisting of 20 μM Q+200 nM D, and D+F, consisting of 20 μM FIS+200 nM D. After treatment, the proportion of senescent cells was quantified using flow cytometry using a standard commercial kit (CellEvent Senescent Green Flow Cytometry Assay Kit, Invitrogen C10841) according to the manufacturer's instructions. HAECs showed elevated levels of senescence-associated β-galactosidase (SA-β-Gal) activity, likely due to cellular stress caused by etoposide as described in the literature (Honda et al., 2021), and thus accumulated a green fluorescent product that was subsequently detected by excitation with a 488 nm laser. The results are shown in Figure 2. Figure 2 As shown. LIG once again performed well as a single compound, reducing the number of senescent cells to below a control threshold. The combination of LIG and FLA showed a synergistic effect, similar to the situation with fibroblasts. Therefore, this specific mixture of compounds in this embodiment may provide a treatment for conditions associated with endothelial cell senescence, as a dysfunctional endothelium is directly involved in conditions such as peripheral vascular disease, stroke, heart problems, diabetes, insulin insensitivity, chronic renal failure, cancer proliferation, metastatic spread, venous thrombosis, cognitive decline, eye-related conditions, and severe viral infections.

[0050] Cells within white adipose tissue are more susceptible to entering a senescent state, not only due to aging, but also due to factors such as obesity and type 2 diabetes that are unrelated to the individual's actual age. The induction of white adipose tissue senescence is attributed to accelerated lipid accumulation and subsequent release, associated with adipocyte hypertrophy, resulting in impaired insulin responsiveness and lipid homeostasis (Q. Li et al., 2021). In addition, adipocytes have the ability to secrete a variety of inflammatory factors and growth factors, which are characteristics of the senescence-associated secretory phenotype (SASP), leading to a systemic proinflammatory state and accelerating aging-related diseases (Frasca & Blomberg, 2020). To test whether our selected natural compounds can play a role in reducing adipose tissue senescence, we performed a cell assay on senescent human subcutaneous preadipocytes. Since subcutaneous preadipocytes express the SA-β-Gal marker in large quantities fairly early in growth-rich medium (<10 passages), we did not have to apply etoposide to obtain sufficient signal. After only 6 passages, the senescence percentage was 48, which is completely suitable for testing. This is consistent with previous reports on the behavior of subcutaneous preadipocytes and adipocytes (Rouault et al., 2021). As Figure 3 As shown, LIG ​​alone exhibited superior efficacy compared to other treatments. A synergistic effect was observed in effectively eradicating senescence when LIG was co-administered with FLA, confirming previous experimental findings and suggesting a tissue-independent synergistic mechanism that was not obvious or predictable. Therefore, the compound combinations described in this embodiment may confer therapeutic advantages for diseases associated with adipocyte senescence and the senescence-associated secretory phenotype (SASP), including type 2 diabetes, metabolic disorders, inflammatory conditions, chronic liver disease, cardiovascular disease, coronary artery disease, renal dysfunction, hypertension, osteoarthritis, neoplastic conditions, muscle atrophy, dementia, depression, anxiety, and other psychiatric conditions.

[0051] Stem cell aging has been shown to be a major determinant of mammalian aging, as it adversely affects regenerative capacity. In recent years, mesenchymal stem cells (MSCs) have emerged as a therapeutic avenue for the restoration of aged and damaged tissues. The cornerstone approach to tissue regeneration using MSCs involves replacing damaged or missing cells with viable, functional counterparts. MSCs possess inherent plasticity to differentiate into a diverse range of specialized cell lines, including osteoblasts, chondrocytes, myocytes, adipocytes, smooth muscle cells, cardiomyocytes, hepatocytes, and endothelial lineages. The differentiation capacity of aged MSCs changes with aging, which can be attributed to increased sensitivity to reactive oxygen species (ROS) and programmed cell death, or apoptosis, which impairs differentiation capacity (Bruedigam et al., 2010). Upon entering a senescent state, MSCs also exhibit a senescence-associated secretory phenotype (SASP), characterized by the release of proteins that negatively impact neighboring cells, leading to various tissue and organ pathologies (Gnani et al., 2019). This presents a significant challenge given the anti-inflammatory properties of mesenchymal stem cells and their potential therapeutic applications in inflammation-mediated disorders. Pharmacological interventions that can mitigate the accumulation of senescence within mesenchymal stem cell populations are therefore of great importance, particularly given their potential roles in tissue repair and regenerative medicine.

[0052] To evaluate the potential of our selected natural compounds to mitigate senescence in mesenchymal stem cells, we performed a cell-based assay on bone marrow-derived human mesenchymal stem cells, which are known to exhibit elevated SA-β-Gal activity during aging (Bertolo et al., 2020). Similar to previous assays, human mesenchymal stem cells (hMSCs), catalog number ATCC PCS-500-012, were cultured strictly according to the manufacturer's protocol using specialized media (PCS-999-001 for adipose-, umbilical cord-, and bone marrow-derived mesenchymal stem cells, and PCS-500-041 growth kit, customized for bone marrow-derived mesenchymal stem cells). Low-passage number (less than 5) cells were treated with a 24-hour exposure to 12.5 μM etoposide, followed by a 48-hour recovery period before exposure to the previously studied compounds. Figure 4 The results clearly demonstrate that, once again, LIG ​​as an isolated compound is clearly superior in reducing senescence. Interestingly, D+Q treatment resulted in a slight increase in hMSC senescence, which may be due to the negative effect of dasatinib on stemness.

[0053] This phenomenon was observed during its use in breast cancer stem cells (Tian et al., 2018). As evident from the superior senolytic effect, the LIG + FLA combination exhibited a strong synergistic effect. That is, the formulation virtually eliminated senescent cells without any observable toxic effects on healthy cells. This suggests that LIG + FLA exhibits a potent senolytic response throughout tissues due to an unexpected synergistic effect.

[0054] To elucidate the underlying mechanisms of the observed LIG efficacy and its synergistic interaction with the FLAs examined in this study, we combined a comprehensive literature review with a transcriptome pathway review using RNA sequencing data. For transcriptome analysis, cells were processed identically to flow cytometry. After processing, RNA was purified from cells using a commercial kit based on Qiagen RNeasy columns according to the manufacturer's instructions. All experimental points were performed in triplicate. RNA samples were sent to an NGS service provider for quality control (QC), library preparation, and sequencing. QC was performed on an Agilent Bioanalyzer. Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, first-strand cDNA was synthesized using random hexamer primers, followed by second-strand cDNA synthesis, end-repair, A-tailing, adapter ligation, size selection, amplification, and purification. Quantified libraries were pooled and sequenced on the Illumina platform based on the effective library concentration and the required data volume (10 million reads). Libraries were sequenced on the Illumina platform for 150 cycles. In addition, the provider performed thorough quality control on the output data before bioinformatics analysis. After statistical matching of the sequencing short reads (read-mapping), differential gene expression analysis was performed using DESeq2 (Love et al., 2014). The threshold was set to padj (adjusted p-value) < 0.05. Genes below this value were considered to be differentially expressed.

[0055] LIG+FLA demonstrated the ability to influence the expression of multiple genes known to play a role in aging. Notably, downregulation of p16INK, p21, and SERPINE1 (plasminogen activator inhibitor-1 or PAI-1, a known inducer and marker of cellular senescence) was observed relative to controls and etoposide treatment (Safwan-Zaiter et al., 2022; Shtutman et al., 2017; Vaughan et al., 2017). Interestingly, loss-of-function mutations in SERPINE1 have been associated with longevity (in the Berne Amish community) and protection against biological aging in humans (Khan et al., 2017), outlining the importance of its inhibition and / or downregulation in senolytic therapy. To our knowledge, this is the first documented downregulation of SERPINE1 achieved by standardized lignans. Upregulation of Caspase-3 and 9 confirmed all the senolytic actions of the described mixture. Finally, according to Figure 5 , it is clear that LIG+FLA can completely block BRD2 expression while simultaneously downregulating BRD4 expression. This effect of LIG+FLA likely contributes significantly to its more potent senolytic activity compared to its competitors. Recent findings from Wakita and colleagues suggest that drugs that inhibit or attenuate the activity of bromodomain-containing proteins could serve as emerging senolytic therapeutics (Wakita et al., 2020).

[0056] In summary, LIG+FLA demonstrates a mechanism for targeting multiple cellular pathways to mitigate the burden of aging. More importantly, LIG+FLA, as a novel senolytic agent, meets the therapeutic requirements of the first phase of this invention.

[0057] In order to identify potential senescence reversing agents suitable for regenerative cell therapy (Phase 2 of the present invention), several compounds with known senescence cell modulating effects or senescence reversing effects were evaluated in cell-based assays. The starting point was again to examine the ability to reduce the percentage of SA-β-Gal positive cells. We explored the activity of pentacyclic oleanane-type triterpenes (OLEs) alone or in combination with β-alanyl-histidine (BAH) or taurine (TAU) on senescent mesenchymal stem cells (MSCs). D+Q, FIS and Q served as controls. To evaluate the ability of these natural compounds to reverse the replicative senescence of mesenchymal stem cells, we performed assays using bone marrow-derived human mesenchymal stem cells driven into a state of replicative senescence. Following the ATCC protocol (PCS-500-012), human mesenchymal stem cells were cultured using dedicated culture media (PCS-999-001, PCS-500-041) to age over 20 cycles, compared to previous assays using etoposide treatment. Results ( Figure 6) demonstrated that OLE exhibited significant anti-aging activity. Strikingly, the combination of OLE+BAH and OLE+TAU outperformed all individual compounds, indicating a synergistic effect in alleviating senescence in mesenchymal stem cells.

[0058] To validate the senescence-reversing (rather than senolytic) effects of OLE+BAH, we performed RNA sequencing analysis comparing untreated control cells and senescent cells grown for 4 weeks and subsequently treated with 15 μM OLE+BAH, with flow cytometry performed simultaneously. RNA isolation was performed using a Qiagen RNeasy column-based kit, strictly adhering to the manufacturer's protocol. All experimental conditions were analyzed in triplicate. RNA samples underwent rigorous quality control (QC), library preparation, and sequencing by a dedicated NGS service provider. QC was performed using an Agilent bioanalyzer. Using the Illumina platform, after careful library quantification and pooling, 10 million reads were targeted at 150 cycles. A rigorous vendor-led quality control protocol ensured high-quality sequencing data prior to bioinformatic analysis. Differential gene expression analysis was performed using DESeq2, using an adjusted p-value (padj) threshold of <0.05 to identify genes with significant differential expression between control and OLE+BAH-treated samples.

[0059] RNA sequencing analysis results (most obvious expression) are shown in Figure 7 After 4 weeks, increased p21 expression was observed, consistent with stem cell aging. OLE+BAH was able to reduce this. However, there was no significant change in the levels of caspase-3 and caspase-9, indicating the absence of apoptotic activity. OLE+BAH significantly downregulated mTOR (mammalian target of rapamycin). This is valuable for reversing aging, as the mTOR pathway plays a crucial role in aging, as demonstrated in various model organisms. In mice, targeting mTOR with rapamycin significantly extends lifespan and prevents age-related health problems, even in late life (S. Xu, 2014).

[0060] Treatment with OLE+BAH significantly restored telomerase activity, increasing expression by ninefold. This is significant because telomerase deficiency impedes DNA repair and leads to persistent DNA damage signaling, which drives cellular senescence. Telomerase plays a crucial role in maintaining telomere stability, particularly in stem cells. The rapid increase in telomerase expression following OLE+BAH treatment suggests improved efficiency of the cell's repair mechanisms.

[0061] Similarly, OLE+BAH is very good at restoring SIRT1 levels in senescent cells after they have dropped significantly. Due to the role of SIRT1 in metabolism and aging events, the increase of SIRT1 is very important. During aging, the cell's autophagy machinery recognizes nuclear SIRT1 as a target. This leads to its transport to the cytoplasm and ultimate degradation in autophagosomes and lysosomes. This autophagy-lysosomal process plays an important role in the age-related decline of SIRT1 in various tissues (C.Xu et al., 2020). Therefore, the restoration of SIRT1 expression levels indicates the restoration of cellular epigenetic activity, which helps to extend cell lifespan.

[0062] Finally, upregulation of NFKBIA, a gene encoding the IκBα protein and an inhibitor of NF-κB, was observed, which is a major factor in the SASP. Existing evidence suggests that the expression profile of the senescence-associated secretory phenotype (SASP) can be induced both in vivo and in vitro, independent of DNA damage, and entirely through downregulation of the inhibitor of κBα (IκBα) (Kolesnichenko et al., 2021). Therefore, upregulation of NFKBIA / IκBα is one of the key mechanisms for alleviating the SASP and attenuating cellular senescence, as demonstrated by the application of OLE+BAH.

[0063] In summary, the effects of OLE+BAH on cellular repair, reduction of senescence signaling, and decreased inflammation in mesenchymal stem cells support its classification as a potential senescence-reversing compound.

[0064] Usage Examples

[0065] The present invention is directed to a therapeutic strategy designed to address or preemptively combat pathological conditions arising from DNA damage, the spontaneous accumulation of senescent cells or the promotion of their accumulation, and ancillary diseases leading to inflammation in the human body. The strategy involves the sequential administration of two different pharmaceutical agents in various forms.

[0066] In one embodiment, the formulation is characterized by its senolytic properties and is intended for continuous oral administration in capsule form over a 30-day time span. The purpose of the formulation is to target senescent cells, promote their elimination, and significantly reduce their volume in the body.

[0067] Following the completion of the initial phase, the second phase begins immediately. This phase involves the administration of a second medication capsule, a senolytic agent. This agent functions as a cell regeneration agent, and its administration continues for an additional 30 days following the senolytic phase. The primary function of the senolytic agent is to restore and renew previously senescent cells, thereby promoting overall cellular health and vitality.

[0068] This two-step pharmacological interaction results in an effective treatment or preventive measure for conditions induced by DNA damage and the accumulation of senescent cells. This integrated approach facilitates the systemic eradication of senescent cells and their subsequent reversal to a healthier cellular state, promoting overall homeostasis in the body.

[0069] Senolytic Oral Preparations

[0070] a) a therapeutically effective amount of lignans derived from standardized oat extract,

[0071] b) a therapeutically effective amount of flavonols derived from standardized strawberry extract,

[0072] c) optional excipients, fillers or bioavailability enhancing compounds to promote optimal absorption of the active ingredients (a and b).

[0073] The formulation is designed to deliver a minimum effective daily dose of 50 mg lignans and 100 mg flavonols. However, to enhance therapeutic efficacy, a daily dose of 200 mg LIG and 400 mg FLA is recommended.

[0074] Aging-reversing oral preparations

[0075] a) a therapeutically effective amount of OLE from standardized olive fruit extract,

[0076] b) a therapeutically effective amount of taurine

[0077] c) optional excipients, fillers or bioavailability enhancing compounds to promote optimal absorption of the active ingredients (a and b).

[0078] The formulation is designed to deliver a minimum effective daily dose of 100 mg OLE and 1000 mg taurine. However, to enhance therapeutic efficacy, a daily dose of 300 mg LIG and 3000 mg taurine is recommended.

[0079] The present invention further introduces an alternative strategy for the mitigation and preventative treatment of conditions that arise from DNA damage, inherent senescent cell accumulation, or underlying diseases that catalyze senescent cell accumulation and subsequent inflammation in the human body. The technology incorporates a sequential application method involving the use of a senolytic transdermal pharmaceutical formulation for a continuous period of 8 weeks, followed by the use of a senolytic reversal composition in a similar manner for an additional uninterrupted period of 8 weeks.

[0080] The treatment program begins with the use of a transdermal pharmaceutical compound with senolytic properties, administered over an uninterrupted 8-week period. This transdermal formulation is designed to identify and aid in the removal of senescent cells, thereby ameliorating associated inflammation and counteracting the cascade of effects initiated by senescent cell accumulation.

[0081] Following the initial senolytic clearance period, treatment begins with the application of a transdermal senolytic compound for an uninterrupted eight weeks. This compound acts as a regenerative agent, rejuvenating previously senescent cells to promote overall cellular health and vitality.

[0082] This two-phase transdermal intervention serves as an effective therapeutic or preventive strategy to combat the harmful effects of DNA damage or senescent cell accumulation. By promoting the elimination of senescent cells and their subsequent restoration to a healthier state, the process helps to restore overall cellular balance throughout the body.

[0083] Aging-clearing transdermal preparations

[0084] a) a dermatologically effective amount of LIG, derived from lignans of strictly standardized herbal origin,

[0085] b) a dermatologically effective amount of one or more flavonols from plant extracts and

[0086] c) auxiliary ingredients, including but not limited to emulsifiers, wetting agents and other ingredients that enhance the stability, spreadability and bioavailability of the active ingredients, the formulation being intended to achieve a basic effective daily transdermal delivery of at least 10 mg of lignans and 10 mg of selected flavonols.

[0087] Aging-reversing transdermal preparations

[0088] a) a therapeutically effective amount of OLE from standardized olive fruit extract,

[0089] b) A therapeutically effective amount of BAH

[0090] c) optional excipients, fillers or bioavailability enhancing compounds to promote optimal absorption of the active ingredients (a and b).

[0091] The formulation is designed to achieve a basic effective daily transdermal delivery of at least 10 mg OLE and 10 mg BAH.

[0092] The following embodiments encompass a method designed to expand or preserve human mesenchymal stem cells in an in vitro environment, particularly in relation to conditions caused by DNA damage, intrinsic senescent cell accumulation, or causal effects that trigger senescent cell proliferation and subsequent inflammatory responses. The method incorporates a specialized two-stage treatment regimen for the cells.

[0093] The initial phase of the program entails the use of a unique pharmaceutical composition that achieves senolytic capabilities designed to identify and subsequently eradicate senescent stem cells. The formulation functions to reduce the proportion of senescent cells while enhancing overall cellular health and integrity.

[0094] Following Phase 1, Phase 2 utilizes a unique drug formulation designed to restore the stem cells' "stemness" and regenerative capacity. This formulation works synergistically with Phase 1, focusing on cell regeneration and revitalization, thereby enhancing the regenerative potential of the stem cell population.

[0095] This differentiated two-part therapeutic regimen establishes an optimized approach to enhance and maintain mesenchymal stem cells, providing an effective strategy to combat conditions caused by DNA damage, intrinsic aggregation of senescent cells, or causative factors that lead to senescent cell accumulation and subsequent inflammation, particularly in an in vitro setting.

[0096] Senolytic therapeutic agents for mesenchymal stem cells

[0097] a) LIG at a concentration suitable for cell culture, derived from strictly standardized plant extracts,

[0098] b) One or more flavonols at concentrations suitable for cell culture

[0099] c) Other agents, including but not limited to buffers, stabilizers, antioxidants, growth factors and chelating agents, intended to enhance the safety, stability and efficacy of the active ingredients (a and b).

[0100] The formulation is carefully designed to provide a basal effective daily dose to facilitate in vitro treatment of mesenchymal stem cells.

[0101] Aging-reversing therapeutic preparations for mesenchymal stem cells

[0102] a) OLE in a concentration suitable for cell culture, derived from strictly standardized plant extracts,

[0103] b) one or more amino acid derivatives or peptides at concentrations suitable for cell culture, which support senescence reversal and stemness restoration.

[0104] c) Other agents, including but not limited to buffers, stabilizers, antioxidants, growth factors and chelating agents, intended to enhance the safety, stability and efficacy of the active ingredients (a and b).

[0105] The formulation is carefully designed to provide a basal effective daily dose to facilitate in vitro treatment of mesenchymal stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0106] Figure 1Bar graph of IMR-90 cells (ATCC) treated with 12.5 μM etoposide for 24 hours. Etoposide was removed, and cells were allowed to recover for 48 hours before treatment with 15 μM test compound and Q+D, consisting of 20 μM quercetin and 200 nM dasatinib. Ctrl = control, eto = etoposide, Q = quercetin, D = dasatinib, FIS = fisetin, LIG ​​= lignans, FLA = flavonols.

[0107] Figure 2 Bar graph of HAECs, in which human aortic endothelial cells (ATCC) were treated with 12.5 μM etoposide for 24 hours. Etoposide was removed, and cells were allowed to recover for 48 hours before being treated with 15 μM of test compound and Q+D, consisting of 20 μM quercetin and 200 nM dasatinib. Ctrl = control, eto = etoposide, Q = quercetin, D = dasatinib, FIS = fisetin, LIG ​​= lignans, FLA = flavonols.

[0108] Figure 3 Bar graph of human preadipocytes (ATCC) cultured for up to 10 passages. Cells were treated with 15 μM test compound and Q+D consisting of 20 μM quercetin and 200 nM dasatinib. Ctrl = control, eto = etoposide, Q = quercetin, D = dasatinib, FIS = fisetin, LIG ​​= lignans, FLA = flavonols.

[0109] Figure 4 Bar graph of human mesenchymal stem cells (ATCC) treated with 12.5 μM etoposide for 24 hours. Etoposide was removed, and cells were allowed to recover for 48 hours before treatment with 15 μM test compound and Q+D, consisting of 20 μM quercetin and 200 nM dasatinib. Ctrl = control, eto = etoposide, Q = quercetin, D = dasatinib, FIS = fisetin, LIG ​​= lignans, FLA = flavonols.

[0110] Figure 5The results of RNA sequencing of human mesenchymal stem cell-related gene expression (control, treated with etoposide and LIG+FLA after treatment), where human mesenchymal stem cells (ATCC) were treated with 12.5uM etoposide for 24 hours. Etoposide was removed, the cells were allowed to recover for 48 hours, and then treated with 15μM LIG test compound. The RNA samples were sent to the NGS service provider for quality control (QC), library preparation and sequencing. After statistical matching (read-mapping) of the sequencing short sequences, differential gene expression analysis was performed using DESeq2 (Love et al., 2014). The threshold was set to padj (adjusted p value) <0.05. Genes below this value were considered to show expression differences. The X-axis represents the compound, and the Y-axis represents the converted value of RNA expression. P16INK = cyclin-dependent kinase inhibitor 2A, p21 = cyclin-dependent kinase inhibitor 1, BRD2 = bromodomain-containing protein 2, BRD4 = bromodomain-containing protein 4, SERPINE1 = plasminogen activator inhibitor-1 (PAI-1), CAS9 = Caspase-9, CAS3 = Caspase-3.

[0111] Figure 6 Bar graph of human mesenchymal stem cells (ATCC) cultured for 20 cycles over 4 weeks. Treatments were performed with 15 μM test compound, Q+D consisting of 20 μM quercetin + 200 nM dasatinib, and taurine at a concentration of 50 μM. Ctrl = control, Q = quercetin, D = dasatinib, FIS = fisetin, OLE = oleanane-type pentacyclic triterpenoid (75%) from olive fruit extract, BAH = β-alanylhistidine, TAU = taurine.

[0112] Figure 7The results of RNA sequencing of aging-related gene expression are shown. Human mesenchymal stem cells (ATCC) were cultured for 4 weeks or 20 cycles and treated with 15 μM of the test compound formula OLE+BAH. RNA samples of control cells, cells after 4 weeks, and treated cells were sent to an NGS service provider for quality control (QC), library preparation, and sequencing. After statistical matching of sequencing short sequences (read-mapping), differential gene expression analysis was performed using DESeq2 (Love et al., 2014). The threshold was set to padj (adjusted p value) < 0.05. . Genes below this value were considered to show differential expression. The X-axis represents the compound, while the Y-axis represents the converted value of RNA expression. P16INK = cyclin-dependent kinase inhibitor 2A, p21 = cyclin-dependent kinase inhibitor 1, BRD2 = bromodomain-containing protein 2, TERT = telomerase reverse transcriptase, NFKBIA = nuclear factor alpha kappa light polypeptide enhancer of B-cell inhibitor gene (IκBα), SIRT1 = NAD-dependent deacetylase sirtuin-1, CAS9 = Caspase-9, CAS3 = Caspase-3.

[0113] abbreviation:

[0114] Compound:

[0115] D = Dasatinib

[0116] EF24 = 3,5-bis(2-fluorobenzylidene)piperidin-4-one (curcumin analogue)

[0117] ETO = etoposide

[0118] FIS = Fisetin

[0119] LIG = Lignans from oats

[0120] PL = Piperlongum amide

[0121] Q = Quercetin

[0122] FLA = flavonol

[0123] OLE = Oleanane-type pentacyclic triterpenes from olive fruit extract (75%)

[0124] BAH = β-alanylhistidine

[0125] TAU = Taurine

[0126] Gene (or related protein)

[0127] AKT = protein kinase B

[0128] BCL-2 = B-cell lymphoma 2

[0129] BRD2 = bromodomain-containing protein 2

[0130] BRD4 = bromodomain-containing protein 4

[0131] BubR1 = Bub1-related kinase

[0132] BCL-W = Bcl-2-like protein 2

[0133] BCL-XL = B-cell lymphoma-extra large

[0134] FOXO4 = Forkhead box protein O4

[0135] HSP90 = heat shock protein 90

[0136] MDM2 = mouse double minute 2 homolog

[0137] NFKBIA = nuclear factor of B-cell inhibitory kappa-light polypeptide gene enhancer, alpha

[0138] p16 INK =Cyclin-dependent kinase inhibitor 2A

[0139] p21 = cyclin-dependent kinase inhibitor 1

[0140] p53 = tumor protein p53

[0141] PI3K = phosphoinositide 3-kinase

[0142] SA-β-gal = senescence-associated β-galactosidase

[0143] SAHF = Senescence-associated heterochromatin foci

[0144] SERPINE1 = plasminogen activator inhibitor-1

[0145] other:

[0146] CKD = chronic kidney disease

[0147] HAEC = human aortic endothelial cells

[0148] hMSC = human mesenchymal stem cells

[0149] IMR-90 = human lung fibroblasts

[0150] MSC = Mesenchymal Stem Cells

[0151] NGS = Next Generation Sequencing

[0152] PAIN = Pan-analytical interfering compounds

[0153] ROS = Reactive Oxygen Species

[0154] SASP = Senescence-Associated Secretory Phenotype

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Claims

1. A method for delaying, slowing, halting and reversing the signs of aging in a human subject comprising sequentially administering: a) senolytic therapy for the selective removal of senescent cells, and then b) Senescence reversal therapy to revert remaining senescent cells to a non-senescent state.

2. The method of claim 1, wherein the senescence treatment (a) involves administering a combination of active agents that synergistically inhibit, modulate, or interfere with biochemical processes that promote cellular senescence.

3. The method of claim 1, wherein the senescence reversal treatment (b) comprises administering a combination of active agents that stimulate the reversal of cellular senescence and improve tissue repair.

4. The composition for senolytic treatment according to claims 1 and 2, comprising: An herbal extract standardized to: a food plant extract standardized to at least 50% flavonols, and a food plant extract standardized to at least 50% lignans, combined in a 1:1 or 2:1 ratio of flavonols to lignans.

5. The composition for aging reversal therapy according to claims 1 and 3, comprising: Olive fruit extract standardized to at least 50% oleanane-type pentacyclic triterpenes, and an aging reversal agent selected from amino acids, peptides, or amino acid derivatives, combined in a ratio of 1:1 to 1:

10.

6. The composition of claim 4, formulated for oral administration, wherein the minimum effective daily dose comprises at least 100 mg flavonols and 100 mg lignans.

7. The composition according to claim 5, formulated for oral administration, wherein the minimum effective daily dose comprises at least 100 mg of oleanane-type pentacyclic triterpenes and 100 mg of a senescence reversal agent selected from amino acids, peptides or amino acid derivatives.

8. The composition of claim 4, formulated for transdermal application, wherein the minimum effective daily dose comprises at least 10 mg flavonols and 10 mg lignans.

9. The composition according to claim 5, formulated for transdermal application, wherein the minimum effective daily dose comprises at least 100 mg of oleanane-type pentacyclic triterpenoid and 100 mg of an aging reversal agent selected from amino acids, peptides or amino acid derivatives.

10. The composition of claim 4, configured for ex vivo application to human mesenchymal stem cells, wherein the stem cell therapeutically effective minimum dose of each active agent corresponds to a concentration of at least 10 μM.

11. The composition according to claim 5, configured for ex vivo application to human mesenchymal stem cells, wherein the minimum effective dose for stem cell therapy of each active agent selected from amino acids, peptides or amino acid derivatives corresponds to 10 μM.

12. The method of claim 1, wherein subsequent application of the composition of claims 4 and 5 reduces the intensity, alleviates or halts the progression of conditions associated with cellular aging, including but not limited to progeria syndromes, immunodeficiency, infertility, autoimmune diseases, liver diseases, neurological diseases, chronic kidney disease, a weakened immune system, joint inflammation, cardiovascular disease, loss of muscle mass, muscle wasting, loss of skin elasticity, slowed recovery from injury, retinal degeneration, fatigue, skin wrinkling, worsening vision, frailty, metabolic syndrome, dementia, cognitive decline, fatigue, sarcopenia, eye-related disorders and idiopathic pulmonary fibrosis, as well as skin-related conditions such as hyperpigmentation, increased appearance of wrinkles and the aging effects of the sun.

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