Composition of a plant extract for mitigating the effects of aging by preventing or delaying cellular senescence - Patent Application 20070229933

Abstract

Aging is characterized by a gradual loss of physical and functional integrity, which progressively affects the health of the body. A distinctive feature of aging is the increase in the number of senescent cells that enter a state of irreversible arrest of the cell cycle after exceeding an intrinsic or environmental stress level. Although cellular senescence is an essential process for some physiological processes, it plays a detrimental role in many age-related pathologies. A first aspect of the present invention provides a composition of plant extracts for alleviating the effects of aging, comprising at least 6.0% (by weight) asiaticoside content, preferably from Centella asiatica extract, at least 2.5% (by weight) verbascoside content, preferably from Cistanche extract, at least 12.5% ​​(by weight) hesperidin content, preferably from Sweet Orange extract, and at least 3.0% (by weight) punicalagin content, preferably from Pomegranate extract. Such compositions prevent the adverse effects of aging by reducing the number of cells that reach a senescent state due to the ability of the formulations to prevent or reduce telomere shortening during cell division. The compositions are particularly adapted for preventing the effects of aging on the skin.

Description

[Technical field]

[0001] The present invention falls within the technical field of products for delaying or reducing the effects of aging on cellular tissues, in particular the skin but also internal organs and visceral tissues, by means of an alternative treatment to the usual treatments, based on the delay and / or prevention of cellular senescence. [Background technology]

[0002] Aging is a physiological decline in structural homeostasis as well as functional integrity, which progressively affects the health of organisms (Non-Patent Document 1). A distinctive feature of aging is the increase in the number of senescent cells that enter a state of irreversible arrest of the cell cycle after exceeding an inherent or environmental stress level. Aging is the result of telomere shortening after repeated cell division, and although cellular senescence is an essential process for some physiological processes, it plays a detrimental role in many age-related pathologies. However, current anti-aging products for skin are limited to correcting the visible effects of aging, such as wrinkles, sagging, and lack of brightness, while other invisible effects of aging, namely pathological effects such as reduced protection against pathogens, increased sensitivity to irritants, reduced healing capacity, and increased susceptibility to cancer, remain untreated. References [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Shulin et al,2020:Shuling Song,Tamara Tchkonia,Jing Jiang,James L.Kirkland,Yu Sun;"Targeting Senescent Cells for a Healthier Aging:Challenges and Opportunities";Advanced Science,Dec.2020,Vol 7,Issue 23(Wiley). Summary of the Invention

[0004] A first aspect of the present invention provides a composition of a plant extract for mitigating the effects of ageing comprising an asiaticoside content of at least 6% (by weight), a verbascoside content of at least 2.5% (by weight), a hesperidin content of at least 12.5% ​​(by weight), and a punicalagin content of at least 3% (by weight).

[0005] As shown in the accompanying examples, such compositions prevent or reduce telomere shortening during cell division and under appropriate conditions by stimulating telomerase enzyme activity, and due to the ability of the formulation to extend telomere length, the number of cells that reach a senescent state is reduced, preventing the adverse effects of both visible and pathological aging.If epithelial stem cells have very short telomeres, they will not leave their epithelial niche and will not properly regenerate skin and hair, resulting in premature aging, so by preventing or delaying telomere shortening, the skin will not only be cleaner, but also healthier.The composition also improves the health of all tissues and organs, both external and internal.

[0006] Telomeres are important structures at the ends of eukaryotic chromosomes that consist of copies of guanine-rich repeats. In mammals, the TTAGGG telomeric repeats are repeated several thousand times (Moyzis et al., 1988). Telomeres protect the ends of chromosomes from degradation and from being recognized as breaks in the DNA double helix. Without telomeres, chromosomes would fuse and genetic instability would result (O´Sullivan and Karlseder, 2010). In humans, the average telomere length decreases from 11 kilobases at birth to less than 4 kilobases in old age (Arai et al. 2015). During chromosome replication, the enzymes that replicate DNA are unable to continue their replication to the ends of the chromosomes, and as a result, the ends of the chromosomes are shortened with each replication. Telomeres form the ends or tips of human chromosomes and therefore shorten with each round of cell division. This mechanism limits human cell proliferation to a finite number of cell divisions by inducing replicative senescence, differentiation or apoptosis (Miyata et al. 2004; Smeets et al., 2011).

[0007] Thus, telomere length decreases over time and can predict life expectancy. Telomere shortening has negative health effects and is associated with cellular senescence and many other health problems, including aging and cancer. Accurate and consistent quantification of telomere length is important in many aspects of cell biology, such as chromosomal instability, DNA repair, aging, apoptosis, cell dysfunction and tumorigenesis.

[0008] In this regard, skin is a self-renewing tissue that undergoes extensive proliferation throughout the life of the organism. Telomeres in epithelial cells are particularly susceptible to accelerated telomere shortening due to proliferation and DNA damaging agents, such as solar radiation, environmental pollution or radical oxygen species (Buckingham and Klingelhuts, 2011; D´Errico et al., 2007).

[0009] Preferably, the asiaticoside content is provided as a Centella asiatica extract. Preferably, the composition contains about 36% (by weight) of a Centella asiatica extract. Centella asiatica is a rich source of asiaticoside and is therefore a suitable plant for providing the required asiaticoside content without the need to include large amounts of inactive or inactive compounds in the composition. Preferably, the verbascoside content is provided as a Cistanche extract. Preferably, the composition contains about 32% (by weight) of a Cistanche extract. The Cistanche extract can be of the following Cistanche varieties: Cistanche tubulosa, Cistanche salsa and / or Cistanche deserticola.

[0010] Cistanche is a rich source of verbascoside, and is therefore a suitable plant for providing the required verbascoside content without the need to include a large amount of inactive or inactive compounds in the composition.Optionally, verbascoside content can also be provided in extracts from other plants, such as Lippia citriodora, Budeja species, etc.Preferably, hesperidin content is provided as a citrus extract, namely Citrus sinensis, especially Citrus amara ssp.sinensis cultivar dulcis.Preferably, the composition contains about 23% sweet orange extract.

[0011] Citrus fruits constitute a source rich in hesperidin, and therefore are suitable plants for providing the necessary hesperidin content without the need to include a large amount of inactive or inactive compounds in the composition.Preferably, the punicalagin content is provided as pomegranate extract (Punica granatum).Preferably, the composition contains about 9% pomegranate extract.Pomegranate is a source rich in punicalagin, and therefore are suitable plants for providing the necessary punicalagin content without the need to include a large amount of inactive or inactive compounds in the composition.

[0012] The administration of the composition of the present invention is selected from the formulations containing the composition intended for oral, topical, transdermal or parenteral administration.Preferably, the composition of the present invention can be administered orally in solid or liquid form, containing one or more physiologically compatible carriers or excipients.These formulations can contain conventional ingredients such as physiologically acceptable binders, fillers, lubricants and wetting agents.The formulations can take any suitable form, such as tablets, pills, capsules, lozenges, oily or aqueous solutions, suspensions, emulsions, or dry powder forms suitable for reconstitution with water or other suitable liquid medium before use, for immediate or controlled release.

[0013] Liquid oral forms for administration may also contain certain additives, such as sweeteners, flavorings, preservatives and emulsifiers. Non-aqueous liquid compositions for oral administration may also be formulated, for example, containing edible oils. Such liquid compositions may be conveniently encapsulated, for example, in unit-dose gelatin capsules.

[0014] Preferably, the composition is administered at a dose of 100 mg to 1 g daily, more preferably 100 to 300 mg daily. The composition of the present invention may be administered parenterally in combination with a conventional injectable liquid carrier, such as water or a suitable alcohol. Conventional pharmaceutical adjuvants for injection, such as stabilizers, solubilizers and buffers, may be included in such injectable formulations. The composition of the present invention may be administered intramuscularly, intraperitoneally or intravenously. Similarly, the composition of the present invention may be administered topically in a liquid, aerosol, cream, or the like, a topical application formulation containing one or more physiologically compatible skin carriers or excipients.

[0015] (References) ·Buckingham EM, Klingelhutz AJ.The role of telomeres in the aging of human skin.Exp Dermatol.2011 Apr;20(4):297-302. ·D´Errico M, Lemma T, Calcagnile A, Proietti De Santis L, Dogliotti E.Cell type and DNA damage specific response of human skin cells to environmental agents.Mutat Res.2007 Jan 3;614(1-2):37-47. ·Miyata Y, Okada K, Fujimoto A, Hata K, Kagami H, Tomita Y, Ueda M. The effect of long-term cultivation on telomere length and morphology of cultured epidermis.J Dermatol Sci.2004 May;34(3):221-30. ·Moyzis RK, Buckingham JM, Cram LS, Dani M, Deaven LL, Jones MD, Meyne J, Ratliff RL, Wu JR. A highly conserved repetitive DNA sequence,(TTAGGG)n,present at the telomeres of human chromosomes.Proc Natl Acad Sci US A.1988 Sep;85(18):6622-6. ·O´Sullivan RJ, Karlseder J.Telomeres:protecting chromosomes against genome instability.Nat Rev Mol Cell Biol.2010 Mar;11(3):171-81. ·Smeets SJ, van der Plas M, Schaaij-Visser TB, van Veen EA, van Meerloo J, Braakhuis BJ, Steenbergen RD, Brakenhoff RH. Immortalization of oral keratinocytes by functional inactivation of the p53 and pRb pathways.Int J Cancer.2011 Apr 1;128(7):1596-605. [Brief description of the drawings]

[0016] [Figure 1] 1 shows the effect of senescent cells on epithelial tissue. [Diagram 2] 1 shows an exemplary graph of the effect of a composition according to the present invention on cell proliferation of normal human dermal fibroblasts (MTT assay). [Diagram 3] 1 shows an exemplary graph of the antioxidant effect of a composition according to the present invention on normal human dermal fibroblast cultures. [Figure 4] 1 shows an exemplary graph of the effect of a composition according to the present invention on telomerase activity in normal human dermal fibroblasts. [Diagram 5]1 shows an exemplary graph of the effect of a composition according to the present invention on telomere length in normal human dermal fibroblasts. [Figure 6] 1 shows an exemplary graph of the effect of a composition according to the present invention on telomere shortening of normal human dermal fibroblasts on cell division number. [Figure 7] 1 shows an exemplary graph of the effect of a composition according to the present invention on telomere shortening in normal human dermal fibroblasts on the number of cell divisions per base pair. [Figure 8] 1 shows an exemplary graph of the effect of a composition according to the present invention on melanin production in normal human epidermal melanocytes exposed to ultraviolet radiation. [Figure 9] 1 shows an exemplary graph of the effect of a composition according to the present invention on cell viability in normal human epidermal melanocytes exposed to ultraviolet radiation.

[0017] Detailed Description of the Invention and Examples The following description details, by way of examples, how composition A according to the invention is surprisingly able to mitigate the effects of aging by preventing or reducing the rate at which epithelial cells enter senescence.

[0018] Composition A contains 36.4% (by weight) centella asiatica extract, 31.8% (by weight) cistanche extract, 22.7% (by weight) sweet orange extract, and 9.1% (by weight) pomegranate extract. The centella asiatica extract contains about 20% asiaticoside as determined by high performance liquid chromatography (HPLC).

[0019] Cistanche extract contains about 10% verbascoside as determined by HPLC. Sweet orange extract contains about 60% hesperidin as determined by HPLC. Pomegranate extract contains about 40% punicalagin as determined by HPLC.

[0020] (Epithelial aging) Skin aging is a process in which morphological and functional changes occur progressively in the skin. There is a decline in biological functions and the ability to adapt to metabolic stress, ultimately resulting in the appearance of wrinkles, sagging and changes in pigmentation, among others. The determinants of skin aging can be internal and external. Intrinsic aging is caused by genetic determinants. Extrinsic aging depends on exposure to various environmental factors (UV rays, pollution, etc.) and lifestyle (smoking, food, sleep and stress).

[0021] At the cellular level, fibroblasts, the most important cell type of the dermis, are essential for producing collagen and other proteins that form the extracellular matrix and allow the skin to maintain its proper function.As we age, the number of fibroblasts gradually decreases as they lose their proliferation capacity, and their function gradually deteriorates, for example, the expression of genes that code for collagen isoforms is reduced.In addition, the number of senescent fibroblasts increases with age, especially due to the deterioration of the senescent cell elimination system.

[0022] Furthermore, the number of senescent fibroblasts increases with age, which is due in particular to the deterioration of the senescent cell elimination system. Senescent cells are harmful because they develop into the senescence-associated secretory phenotype (SASP), which is considered one of the main causes of aging. Human fibroblasts adopting the SASP secrete proinflammatory cytokines and matrix metalloproteinases (MMPs) and release reactive oxygen species, all of which lead to the degradation of the extracellular matrix and, consequently, skin aging (see Figure 1 (Freitas et al., 2007)).

[0023] Moreover, reactive oxygen species (ROS) play an important role in both intrinsic and extrinsic aging. With aging, the balance between ROS and antioxidants is lost in favor of the former, leading to the activation of multiple MAP kinase pathways, which induce an increase in matrix metalloproteinase (MMP) synthesis, responsible for collagen degradation in human skin. This imbalance between the enzymes that remodel and repair the dermal matrix promotes the loss of connective tissue and atrophy of the skin, which is associated with the loss of immune function.

[0024] To this end, Examples 1 and 2 evaluate the anti-aging effect of composition A according to the invention after in vitro treatment of normal human dermal fibroblasts (NHDF) subjected to culture conditions simulating the aging process (cellular senescence), by quantifying cell viability in the MTT (cell proliferation) assay and ROS after exposure to a medium containing hydrogen peroxide (H2O2).

[0025] (References) Sandra Freitas-Rodriguez, Alicia R. Folgueras, Carlos Lopez-Otin, The role of matrix metalloproteinases in aging: Tissue remodeling and beyond, Biochimica et Biophysica Acta(BBA)-Molecular Cell Research, (2007)1864, Issue 11, Part A, 2015-2025. EXAMPLES

[0026] Example 1. Formulation A according to the invention prevents the loss of fibroblast proliferative capacity in aging medium by protecting them from cellular senescence.

[0027] (methodology) For cell proliferation assay, normal human dermal fibroblasts (NHDF) were exposed to conditioned aging medium containing H2O2 (500 μM) for 3 hours. After the incubation period, the medium was replaced with fresh medium containing different concentrations of composition A according to the present invention for 96 hours. Epidermal growth factor (EGF, 20 ng / mL) was included as a positive control, and NHDF without pretreatment in H2O2 was included as a non-aging control (control). Cell viability was quantified using MTT assay.

[0028] (result) The results obtained can be observed in the graph of Figure 2. Fibroblasts pretreated with H2O2 (aged control) showed a significantly reduced cell viability (-9.2%) compared to the non-aged control. Cells supplemented with composition A according to the invention restored their proliferation capacity, with a significant increase in cell viability being observed at 0.001% (by weight) (+11.3%) and 0.0005% (by weight) (+10.8%) compared to the aged control, raising the growth capacity to a level similar to that of non-aged fibroblasts and somewhat lower than that of control cells incubated with epidermal growth factor EGF (+22.9%).

[0029] This example demonstrates the strong efficacy of composition A according to the invention in counteracting the effects of cellular aging on the regenerative capacity of tissues.

[0030] Example 2. Formulation A according to the invention inhibits H2O2-induced oxidative stress in human fibroblasts, conditioned aging medium.

[0031] (methodology) After 3 hours of incubation in which normal human dermal fibroblasts (NHDF) were exposed to conditioned aging medium containing H2O2 (500) mM, the medium was replaced with fresh medium containing different concentrations of composition A according to the invention for 24 hours. After this period, the generation of radical oxygen species (ROS) was measured by fluorometry. ROS generation measurements were calculated relative to cells not treated with composition A and aged with H2O2 (aged control).

[0032] (result) The results obtained can be observed in the graph of FIG. Aging-inducing doses of H2O2 showed significantly higher levels of intracellular ROS in cells untreated with composition A (±60.4%), which is characteristic of skin aging. On the other hand, when cells were treated with composition A for 24 hours, ROS levels were reduced. Treatment with 0.005% and 0.01% (by weight) of composition A reduced ROS levels by 32% and 57.7%, respectively. This example demonstrates the strong effectiveness of composition A according to the present invention in reducing the effects of cell stress on the ability to generate oxidative free radicals.

[0033] (Telomeres and Aging) Telomeres are dynamic DNA-protein complexes that protect the ends of chromosomes and promote chromosomal stability. When cells divide, telomeres are not replicated in their entirety, which means their shortening with each replication (Chan and Blackburn, 2004). When telomeres reach a critical length, cells stop dividing and enter senescence (Victorelli and Passos, 2017). Thus, telomere length is considered a marker of cellular aging. The telomere shortening process is reduced with age by the enzyme telomerase, whose function is to add nucleotides to the termini of the ends of DNA molecules (Oeseburg H, 2010).

[0034] Telomerase activity is found in certain cells (e.g., germ cells and stem cells) that divide continuously and must maintain telomeres beyond a certain length to perform their function. By stimulating telomerase activity, it is possible to increase telomere length and, as a result, extend the number of cell divisions that can occur without incurring harmful chromosomal abnormalities or telomere fusions (Sahin E, 2010). This does not immortalize the cell, but it can extend its life cycle. Furthermore, the amount of telomerase in the body decreases with age (Eisenberg, 2011). Thus, by activating telomerase, it is possible to: - Addressing telomere shortening and cellular aging -Helps cells survive longer and continue to function properly. -Making old cells function more like young cells (by changing gene expression to a younger phenotype).

[0035] Example 3A. Composition A according to the present invention exerts anti-aging and anti-aging effects by stimulating telomerase activity.

[0036] (methodology) Measurement of relative telomerase activity by Q-TRAP The DNA polymerase chain reaction (PCR)-based quantitative telomeric repeat amplification protocol is a highly sensitive and accurate PCR-based assay that allows for the measurement of telomerase activity.

[0037] The cell pellet was split using CHAPS splitting buffer ((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate) for protein extraction. Samples were stored at 4 °C and used within 24 h. Protein quantification was performed for each sample using the Bio-Rad protein assay (Bio-Rad Kit 50000002). A minimum protein concentration of 0.3 mg / mL is required to perform sample analysis. The telomerase protein extract was incubated at 27 °C for 30 min to allow telomerase from the protein extract to extend telomeres by the addition of TTAGGG repeat sequences. After the enzymatic reaction, the telomerase extension products were amplified and quantified by real-time qPCR using SYBR green (a fluorescent cyanine dye). PCR was initiated at 95 °C for 10 min, followed by 40 cycles of amplification (95 °C for 15 s, 60 °C for 60 s) and a fusion curve step. The reaction was monitored and analyzed with QuantStudio 5 (Applied Biosystems). Telomerase activity in cell lines or samples was calculated based on the threshold cycle (Ct). All samples were run in triplicate.

[0038] The telomerase positive standard dilution series is plotted against telomerase protein concentration (r2>0.9) as a standard curve of Ct values. The standard curve is created by plotting the threshold cycle (Ct value) of the standard HeLa cell line against the logarithm of 1000, 333, 111, 37.03, 12.34, 4.11, 1.37 and 0.45 ng of protein (whole cell extract). Telomerase activity was measured in human fibroblasts over 24 and 72 hour periods.

[0039] (result) The results of the telomerase activity assay are shown in the graph of FIG. 4. After 6 hours of the assay, the control and samples treated with composition A according to the present invention showed similar levels of telomerase activity. A slight increase in telomerase activity was observed only in samples treated with composition A at a concentration of 0.001% (by weight). After 24 hours, increased telomerase activity was observed in all cell samples treated with composition A compared to the untreated control group. These differences were identified as statistically significant (p<0.01%). After 72 hours, the control and treated samples showed similar levels of telomerase activity.

[0040] (References) ·Chan SR,Blackburn EH.2004.Telomeres and telomerase.Philos Trans R Soc Lond B Biol Sci.359(1441):109-121.doi:10.1098 / rstb.2003.1370 ·Victorelli S, Passos JF.2017.Telomeres and cell senescence-size matters not.EBioMedicine.21:14-20.doi:10.1016 / j.ebiom.2017.03.027. ·Eisenberg DTA.An evolutionary review of human telomere biology:the thrifty telomere hypothesis and notes on potential adaptive paternal effects.American Journal of Human Biology.2011;23:149-167. ·Oeseburg H,et al.Telomere biology in healthy aging and disease.Pflugers Archiv-European Journal of Physiology.2010;459:259-268 ·Sahin E, DePinho RA. Linking functional decline of telomeres, mitochondria and stem cells during aging.Nature.2010;464:520-528. ·Hou M,Xu D,Bjorkholm M,Gruber A.2001.Real-time quantitative telomeric repeat amplification protocol assay for the detection of telomerase activity.Clin Chem.47(3):519-524.

[0041] Example 3B. Composition A according to the present invention exerts anti-aging and anti-aging effects by preventing telomere shortening.

[0042] To test whether Composition A is able to at least partially inhibit telomere shortening, two different experiments were performed using two different techniques and different conditions.

[0043] (Protocol 1) (methodology) For telomere length experiments, NHDF fibroblasts were cultured and grown for 24-29 cell doublings to induce aging and telomere shortening by cell passaging. Throughout this culture, cells were treated with composition A or untreated (untreated control) and the medium was replaced with fresh product every 3 days. After incubation, cells were harvested, gDNA was extracted, and their telomere length was quantified by RT-qPCR. gDNA was also extracted from cells at time 0. The number of cell doublings was calculated by cell counting.

[0044] To calculate telomere length, a reference human genomic DNA sample with known telomere length was included in the qPCR reaction. To perform the analysis of the primary data, the ratio of gene expression to SCR (internal control) was calculated using the Pfaffl method (Pfaffl, 2001). The mathematical expression of relative gene expression in real-time PCR is as follows:

[0045]

number

[0046] (result) The results obtained can be observed in the graph of FIG. Fibroblasts aged 24-29 doublings without treatment showed a significant reduction in telomere length compared to non-aged controls, however, treatment of fibroblasts with concentrations as low as 0.0001% (by weight) of Composition A significantly increased telomere length compared to untreated aged cells.

[0047] To better reflect telomere shortening due to cell division, telomere shortening values ​​were normalized with respect to the estimated number of cell divisions that occurred under the various conditions. When this correction was applied, the results (shown in FIG. 6) show a statistically significant reduction (p<0.05) in telomere shortening caused by Composition A at a concentration of 0.0001% (by weight). Under these conditions, telomeres shortened at a slower rate than untreated cells, by 0.010±0.003 kb / division. This figure is 57.7% slower than the untreated control.

[0048] (Protocol 2) (methodology) For this experiment, normal human fibroblasts were cultured and grown for 8 weeks (14-17 cell doublings) with three different concentrations of Composition A, 0.001%, 0.0005% and 0.0001% (by weight). The cells were cultured and tested under oxidative stress conditions by adding H2O2 (10 μM) as an oxidative stress agent. Changes in telomere length and telomere shortening rate (TSR) were determined for each concentration of Composition A and the results were compared.

[0049] Telomere length measurements were performed using Life Length's proprietary Telomere Analysis Technology (TAT®). TAT® measures telomere length using a previously described HT Q-FISH (high-throughput quantitative fluorescent in situ hybridization) high-throughput technology (de Pedro et al., 2020). The method is based on a modified quantitative fluorescent in situ hybridization method for measuring individual chromosomes in interphase cells. Briefly, telomeres are hybridized with a fluorescent peptide nucleic acid (PNA) that recognizes three telomeric repeats (sequence: Alexa488-OO-CCCTAACCCTAACCCTAA, purchased from Panagene). Images of nuclei and telomeres are captured by a high-content display system. The intensity of the telomere PNA fluorescent signal hybridizing to a given telomere is proportional to the length of that telomere. Fluorescence intensity is converted to base pairs (bp) by a standard regression curve created using a control cell line with known telomere length. TAT® not only measures telomere length in absolute base pairs, but also provides an assessment of telomere length distribution and the percentage of short telomeres, thus allowing a more complete analysis of each sample.

[0050] Therefore, three different variables were measured with the TAT® method: median telomere length, the 20th percentile of telomere length and the percentage of shortest telomeres of 3 Kbp, which is recognized as the amount of very short telomeres that increases the risk of a cell entering senescence.

[0051] All samples were analyzed in quintuplicate. Using normal human dermal fibroblasts, the effect of Composition A on telomeres under conditions of oxidative stress (10 μg / ml H2O2) was observed over a period of 6 and 8 weeks.

[0052] (result) The results obtained can be observed in the graph of FIG. 7. The results show that untreated fibroblasts aged for 6 and 8 weeks under oxidative stress exhibit shorter telomere length and a higher percentage of short telomeres compared to non-aged fibroblasts (time 0 control). In contrast, fibroblasts treated with Composition A exhibited higher median and 20th percentile telomere length and a lower percentage of short telomeres (<3Kb) compared to the untreated control group during both 6 and 8 weeks of treatment.

[0053] Furthermore, after normalizing the data for duplicate populations, a slower rate of telomere shortening was observed in fibroblasts treated with Composition A, suggesting a protective effect of telomeres under conditions of oxidative stress and aging. These experiments demonstrate the potent efficacy of Composition A according to the present invention in slowing cellular progression to a senescent state by reducing the rate of telomere shortening.

[0054] Our skin ages because, individually, all cells in the body age and enter the senescence stage according to a timeline established by the biological cellular clock, namely, telomeres. Telomeres are repeating units of DNA that protect the ends of chromosomes during cell division. As cells divide over time, telomeres shorten and, if the telomere length is very short, the cell will cease replicating by division and enter senescence.

[0055] Senescent cells are more prevalent in older adults due to age-related deterioration of the immune system and senescent cell clearance systems such as the autophagy-lysosome cycle. Senescent cells are harmful because they evolve into the senescence-associated secretory phenotype (SASP), which is suspected to be one of the main causes of aging. SASP cells communicate with neighboring cells using pro-inflammatory cytokines, including catabolic regulators such as matrix metalloproteinases (MMPs) (see Figure 1, right), releasing oxygenated radical species that lead to the degradation of the cellular matrix around them.

[0056] (Environmental and Aging Effects on Skin Pigmentation (UVA Radiation)) The skin pigmentation system is altered by the aging process, and irregular pigmentation is one of the biggest changes associated with aging. Melanin, secreted by melanocytes, is the most important pigment of human skin at the base of the epidermis. Melanin is essential for the photoprotection of human skin from the harmful rays of the sun. However, there are several epithelial hyperpigmentation disorders, such as freckles, age spots, melasma and senile lentigines, that result from the overproduction and subsequent accumulation of melanin, which can be a distressing problem. This overproduction is caused by various factors, the most important of which are exposure to sunlight, age-related hormonal changes or inflammatory skin conditions.

[0057] Hyperpigmentation typically appears in areas of the skin that are regularly exposed to sunlight, such as the hands or face. Ultraviolet radiation (UVA) contributes to melanogenesis by generating cellular oxidative stress and neutralizing antioxidant defenses. Abnormal skin pigmentation is a significant aesthetic condition that can affect a person's quality of life. Furthermore, overproduction of melanin can be associated with melanoma, a type of skin cancer. It is therefore important to research and develop products that can effectively control melanin overproduction, such as Composition A, whose effect on melanocytes is shown in Example 4.

[0058] Example 4. Composition A according to the invention exhibits a bleaching effect by inhibiting the synthesis of melanin and inhibiting cell damage induced by exposure to ultraviolet radiation.

[0059] (methodology) Normal human epidermal melanocytes were cultured with composition A according to the invention for 72 hours. During the incubation period, the cells were exposed twice a day to ultraviolet radiation (total dose = 5 J / cm 2 After the final radiation, samples were incubated for an additional 96 hours before melanin and cell viability were quantified.

[0060] (result) The results obtained can be seen in the graphs of FIG. 8 for melanin production and in FIG. 9 for cell viability. Ultraviolet radiation (UVA) significantly increased melanin levels by 41% compared to the control without UVA exposure. Composition A according to the invention at concentrations of 0.005% and 0.0005% (by weight) significantly reduced melanin levels by 41 and 40%, respectively, compared to untreated irradiated cells. Ultraviolet radiation significantly reduced human melanocyte viability by 25% compared to non-irradiated controls. Composition A according to the invention at concentrations of 0.005% and 0.0005% (by weight) significantly increased cell survival and reduced cell damage caused by UVA radiation by 25% and 20%, respectively, compared to untreated irradiated cells.

[0061] Example 5. Composition A according to the invention exhibits multiple and highly significant beneficial effects on the skin, as demonstrated in this placebo-controlled human clinical study.

[0062] The inventors selected 60 volunteers who showed symptoms of skin aging. The volunteers were divided into two groups, one of which was administered with a daily dose of 225 mg of composition A, and the other was administered with placebo (i.e., capsules not containing composition A). The study lasted for 3 months, and measurements of study parameters were performed after 28 and 56 days, as well as at the end of the study, after 84 days.

[0063] (Results of clinical studies) Overall, composition A has a positive effect on all monitored clinical parameters, both compared to the start of the study (T0) and compared to the placebo group from the first month of treatment (28 days), with the exception of parameter R9, where the results obtained are statistically significant compared to the initial values, but not compared to the group treated with placebo for up to 56 days.

[0064] The following table shows the variation of each parameter relative to the start of the study after 28, 56 and 84 days of treatment. Study implementation: The clinical study was carried out in parallel, randomized, double-blind groups in 60 volunteers with obvious signs of aging. The study lasted for 12 weeks, and the effectiveness of the product was evaluated after 4, 8 and 12 weeks of taking the product. The parameters determined were skin moisture (corneometer®), transepidermal water loss (tewameter®), skin brightness and tone (spectrophotometer), skin elasticity and firmness (cutometer®), wrinkle depth and skin roughness (3D Cousins® image analysis), and epidermal and dermal thickness (ultrasound).

[0065] [Table 1]

[0066] (1) A decrease in the parameter Ra can be expressed in absolute value as an increase in skin smoothness. (2) A decrease in parameter R0 can be expressed in absolute value as an increase in skin firmness. (3) An improvement in parameter R5 indicates an improvement in the elastic recovery of the skin after deformation. (4) The closer this value is to 0, the lower the fatigue effect. The reduction indicates an improvement in the skin. (5) A low ITA angle indicates brown pigmentation, while a high ITA angle value indicates clear pigmentation. S: Statistically significant (p<0.05) relative to the start of the study (T0) | *: Statistically significant vs. placebo (p<0.05) | NS: not statistically significant (p>0.05) relative to the start of the study (T0)

[0067] Considering these results, it can be concluded that Composition A exerts extraordinary and very important benefits on all skin characteristics, such as improving moisture and elasticity, reducing wrinkles and promoting re-densification of the dermis and epidermis, as a result of the synergistic effect of all its components on the telomeres of epithelial cells.Furthermore, it can reduce dark spots on the skin and provide a higher luminosity by making the skin more evenly toned.

Claims

1. A plant extract composition for alleviating the effects of aging, comprising asiaticoside in an amount of at least 6% (by weight), verbascoside in an amount of at least 2.5% (by weight), hesperidin in an amount of at least 12.5% ​​(by weight), and punicalagin in an amount of at least 3% (by weight).

2. 10. The composition of claim 1, wherein the asiaticoside is provided as a Centella asiatica extract.

3. 3. The composition of claim 2, containing about 36% (by weight) of Centella asiatica extract.

4. 10. The composition of claim 1, wherein the verbascoside is provided as a cistanche extract.

5. 5. The composition of claim 4, containing about 32% (by weight) of cistanche extract.

6. 5. The composition of claim 4, wherein the Cistanche extract is an extract of the following Cistanche varieties: Cistanche tubulosa, Cistanche salsa, and / or Cistanche deserticola.

7. 10. The composition of claim 1, wherein the verbascoside comprises a plant extract such as lemon verbena (Lippia citriodora), Budeja species, Rehmania glutinosa, Plantago species, or the like.

8. 10. The composition of claim 1, wherein the hesperidin is provided as a citrus extract.

9. 9. The composition of claim 8, containing about 23% sweet orange extract.

10. 10. The composition of claim 1, wherein the punicalagins are provided as a pomegranate extract (Punica granatum).

11. The composition described in claim 10, wherein the composition contains approximately 9% pomegranate extract.

12. The composition described in claim 1, wherein the composition further reduces melanin production in skin exposed to solar radiation.

13. 10. A formulation for ameliorating the effects of aging, the formulation comprising the composition of claim 1 and further adapted for oral administration in solid or liquid form containing one or more physiologically compatible carriers or excipients.

14. 10. A formulation for ameliorating the effects of aging, the formulation comprising the composition of claim 1 and further adapted for topical administration containing one or more carriers or excipients that are physiologically compatible with human skin.

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