Novel Senolytic Ingredient, Camellia sinensis Root Extract, Ameliorates Skin Aging-Associated Phenotypes

Novel Senolytic Ingredient, Camellia sinensis Root Extract, Ameliorates Skin Aging-Associated Phenotypes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Novel Senolytic Ingredient, Camellia sinensis Root Extract, Ameliorates Skin Aging-Associated Phenotypes Sunyoung Park, Siyoung Cho, Daejin Min, Hyunjung Choi, Kyeonghwan Hwang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7016169/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Senescent cells can affect neighboring cells via the senescence-associated secretory phenotype (SASP), which involves pro-inflammatory cytokines, chemokines, and proteases. This study aimed to explore the senolytic properties of Camellia sinensis root extract (SENOMUNE), which has therapeutic potential for skin aging-related disorders, with cell viability assays, quantitative reverse transcription polymerase chain reaction, western blotting, and flow cytometry using a stress-induced premature senescence model in normal human dermal fibroblasts (NHDFs). NHDFs were induced to senescence using doxorubicin and insulin-like growth factor-1. The senolytic effect of SENOMUNE was also evaluated through the investigation of senescence-associated β-galactosidase activity, gene and protein expression analysis, and apoptosis assays in NHDFs. The impact of SENOMUNE on the skin barrier function and pigmentation was assessed using conditioned media from senescent fibroblasts and ex vivo skin biopsies. SENOMUNE exhibited a concentration-dependent reduction in senescent cells without affecting young cells and induced apoptosis in senescent cells through a caspase-independent mechanism involving apoptosis-inducing factor and lysosomal membrane permeabilization. SENOMUNE reduced SASP factors and improved skin barrier function and pigmentation by modulating the secretion of inflammatory cytokines from keratinocytes and autophagy. SENOMUNE thus demonstrated novel senolytic properties and therapeutic potential for managing skin-related disorders and is a promising anti-aging phytopharmaceutical ingredient. Biological sciences/Cell biology Health sciences/Diseases Camellia sinensis senescence-associated secretory phenotype senolytics human dermal fibroblasts Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Cellular senescence is the loss of the ability of normal cells to divide and remain in a state of cell cycle arrest. Cellular senescence progresses sequentially: when cell division stops, the non-dividing cells secrete senescence-associated secretory phenotype (SASP) factors, which promotes the spread of senescence causing senescent cells accumulate in tissues (Tchkonia et al., 2013; Kim and Kim, 2019)[ 1 , 2 ]. Normal cells self-destruct through apoptosis, which is difficult to repair due to genetic damage. Instead of apoptosis, senescent cells are eliminated through a process that sends out signaling molecules to inform immune cells to eliminate them (Soto-Gamez and Demaria, 2017)[ 3 ]. A problem occurs when senescent cells increase in number, and the immune cells do not sufficiently remove them, thereby causing an accumulation of senescent cells. Anti-aging treatment to selectively remove the adverse effects of senescent cells is being studied (senotherapy) because an increase in senescent cells causes age-related diseases. Senotherapy includes the use of senolytics and senomorphics. Senolytics are drugs that directly eliminate senescent cells. They focus on apoptosis resistance in senescent cells, and increase suppressing factors in senescent cells and inducing cell death. Meanwhile, senomorphics target the SASP; these are specific cytokines and chemokines secreted by the senescent cells, and certain drugs target the related signaling mechanism (Soto-Gamez and Demaria, 2017; Kim and Kim, 2019; Birch and Gil, 2020; Kirkland and Tchkonia, 2020)[ 2 – 5 ]. Given that senescent cells accumulate in tissues and contribute to aging, their impact is particularly evident in the skin, which is the body's largest organ (Richardson, 2003)[ 6 ]. The skin is the first to exhibit signs of aging visually; therefore, it is a focus of most aging research. Previous research has been conducted on how the interaction of the various cells that make up the skin effects aging. They revealed that senescent melanocytes induce aging in surrounding cells such as fibroblasts, and that skin-aging pigmentation is induced in melanocytes through interaction with senescent fibroblasts (Kim et al., 2022)[ 7 ]. Since this interaction occurs through the SASP produced in senescent skin cells, senolytics and senomorphics are being considered to control skin aging-related disorders caused by aging (Chin et al., 2023)[ 8 ]. Considering the role of senescent cells in skin aging, researchers are exploring natural compounds, such as phytochemicals, for their potential in targeting these cells (Shen et al., 2022)[ 9 ]. Certain phytochemicals are poisons and traditional medicines. Phytochemicals can be classified as terpenes (carotenoids, monoterpenes, and saponins), phenols (polyphenols and flavonoids), organosulfur compounds (indoles, isothiocyanates, and thiosulfonates), organic acids and polysaccharides, and lipids (isoprenoids and omega-3 and omega-6 fatty acids) (Rais et al., 2017)[ 10 ]. Among the phytochemicals, quercetin was the first senolytic, and various cell types and clinical trials have revealed its senolytic activities in combination with dasatinib. Similar to quercetin, other flavonoids (fisetin, curcumin, leteolin, and piperlongumine) are promising senolytic agents (Gurău et al., 2018)[ 11 ]. A previous report confirmed the senolytic activity of flavonoids as well as periplocin and oleandrin, which are saponins (Smer-Barreto et al., 2023)[ 12 ]. Flavonoids, saponins, tannins, phenolic compounds, and triterpenoids are obtained from the leaves, flowers, and roots of Camellia spp. Camellia sinensis leaves are widely used as medicinal herbs because they contain polyphenols such as catechins and epigallocatechin gallate derivatives (Zhao et al., 2022)[ 13 ]. A previous report revealed that epigallocatechin gallate suppresses anti-apoptotic protein Bcl-2 accumulation in senescent cells, thereby promoting apoptosis-mediated cell death (Kumar et al., 2019)[ 14 ]. While tea leaves have long been studied and consumed for their numerous health benefits, there is limited research on the same in the roots of tea plants. Tea plant root extracts contain lower levels of flavonoids than leaf extracts but contain various oleanolic-type triterpenes (Im and Kim, 2022)[ 15 ]. Previous studies have confirmed that tea plant root extracts have high antioxidant and α-glucosidase inhibitory activities owing to the presence of saponins, thereby indicating significant physiological benefits. Additionally, ongoing research explores the anti-cancer, immune-modulating, anti-inflammatory, and antioxidant effects of C. sinensis root extracts (Im and Kim, 2022; Lee et al., 2023)[ 15 , 16 ]. However, the senotherapeutic efficacy of the C. sinensis root extract (SENOMUNE) has not been investigated. Therefore, this study aims to investigate the senotherapeutic potential of SENOMUNE in controlling skin aging-related disorders. We hypothesize that SENOMUNE exhibits senotherapeutic properties by modulating SASP and promoting apoptosis in senescent skin cells. The senolytic effect of SENOMUNE was evaluated using various in vitro assays, and its impact on skin barrier function and pigmentation was assessed with conditioned media from senescent fibroblasts and ex vivo skin biopsies. In this study, we report SENOMUNE as a new senotherapeutic agent for controlling various skin aging-related disorders, including skin aging pigmentation and impaired barrier function. Results Effect of SENOMUNE in human dermal fibroblasts To test senolytic efficacy, we used a stress-induced premature senescence model in normal human dermal fibroblasts (NHDFs). SENOMUNE exhibited a 35% and 59% concentration-dependent reduction in senescent cells at 100 and 150 ppm, respectively; however, it exhibited cell toxicity in young cells at 150 ppm. Contrastingly, the visibility of ABT-737 cells decreased by 49% and 75% at 2.5 and 10 µM, respectively, without affecting the viability of the young cells (Fig. 1 a). The reduction in cell viability induced by ABT-737 and SENOMUNE was observed in the senescence-associated β-galactosidase (SA-β-gal) assay (Fig. 1 b). In ABT-737-treated senescent cells, SA-β-gal reduction was confirmed in a dose-dependent manner. SENOMUNE reduced SA-β-gal at concentrations > 50 ppm (Supplementary Fig. S1 ). Apoptosis induction using SENOMUNE through caspase-independent programmed cell death SENOMUNE did not cause apoptosis in young cells but increased apoptosis by 23% compared with that of the vehicle (16%) in senescent cells (Fig. 2 a). Apoptosis is divided into caspase-dependent programmed cell death (classical apoptosis) or caspase-independent programmed cell death (CI-PCD) (Bhadra 2022)[ 17 ]. ABT-737 increased caspase activity in senescence cells (Fig. 2 b). Protein expression patterns confirmed classical (caspase-dependent) apoptosis of ABT-737 and CI-PCD by SENOMUNE. Cleaved caspase-3 (active form) was unchanged, but cleaved poly(ADP-ribose) polymerase 1 (PARP1) increased in the SENOMUNE-treated senescent cells (Fig. 2 c); PARP-1 is a known caspase cellular substrate. The cleavage of PARP-1 by caspases is considered a hallmark of apoptosis (Elmore, 2007)[ 18 ]. Thus, increased levels of cleaved caspase-3 and PARP-1 indicate that ABT-737 induces classical (caspase-dependent) apoptosis. PARP-1 is a preferred substrate for several “suicidal” proteases, and the proteolytic action of suicidal proteases (caspases, calpains, cathepsins, granzymes, and matrix metalloproteinases) on PARP-1 produces several specific proteolytic cleavage fragments with different molecular weights (Chaitanya et al., 2010)[ 19 ]. A few PARP-1 fragments and their associated binding partners control different forms of cell death (e.g., autophagy, necrosis, and parthanatos). The PARP-1 fragments produced by ABT-737 and SENOMUNE may be derived from the actions of different proteases. The 89-kD PARP-1 fragment, which appears during apoptotic cell death by caspase-3, can be produced by cathepsin-B and D (Elmore, 2007; Chaitanya et al., 2010)[ 18 , 19 ]. The release of cathepsin-B and D from the lysosome to the cytosol was observed following SENOMUNE treatment (Supplementary Fig. S2 ). PARP-1 is thought to be required for apoptosis-inducing factor (AIF) release from the mitochondria during caspase-independent apoptosis (Modjtahedi et al., 2006)[ 20 ]. Bcl-2-associated X protein (BAX) is related to AIF relocation from the mitochondria to the cytosol (Modjtahedi et al., 2006; Bhadra, 2022)[ 17 , 20 ]. PARP1 activation (cleaved PARP1) and an increase in BAX were observed (Fig. 2 c). AIF was observed in the cytosol of cells treated with SENOMUNE but not in the control group (Fig. 2 d). Therefore, CI-PCD induced by SENOMUNE was distinct from the apoptotic signaling pathway triggered by ABT-737. Moreover, AIF promotes caspase-independent cell death following lysosomal membrane permeabilization (LMP) (Boya and Kroemer, 2008)[ 21 ]. The effect of SENOMUNE on lysosomal cell death was evaluated using acridine orange (AO) staining to test the hypothesis that SENOMUNE causes senolysis through LMP (Fig. 2 e). AO is a lysosomotropic metachromatic fluorophore that, when excited with blue light, emits red and green fluorescence at high (when it is observed in lysosomes) and low concentrations (when it is present in the cytosol and the nucleus), respectively (Boya and Kroemer, 2008)[ 21 ]. Lysosomes with high AO concentration were stained and exhibited red dots in young and senescent cells. The red dots disappeared only in senescent cells treated with SENOMUNE. The AO-loaded cells exhibited reduced red fluorescence and increased green fluorescence after LMP treatment, thereby indicating that SENOMUNE exerted a senolytic effect through lysosome-related programmed cell death (Fig. 2 e). Reduction of aging markers and senescence-associated secretory phenotypes by SENOMUNE Lamin B1, phospho-p53, and p21 expression levels were measured in the HDFs to determine the effect of senolytics on aging markers. Lamin B1 was decreased; however, phospho-p53, p53, and p21 levels increased during senescence but were considerably diminished by ABT-737 and SENOMUNE. However, senolytic treatment marginally altered Lamin B1 levels (Fig. 3 a), probably because cell cycle proteins are more unstable than structural proteins. The apoptosis-inducing agents ABT-737 and SENOMUNE may have influenced the stability of the nuclear envelope component Lamin B1 owing to the characteristic feature of nuclear envelope breakdown in apoptotic cell death, which led to a slight reduction. The remaining senescent cells after SENOMUNE treatment were incubated for 1 week, and SASP gene expression was assayed using quantitative PCR (qPCR) to confirm the resistance of senescent cells to the SASP inhibitory effect. Several cytokine and protease genes, such as IL-8, MMP-1, GDF-15, TIMP-1 , and IGFBP-6 , were maintained at low levels. Among these, the IL-8, MMP-1 , and GDF-15 expression levels significantly increased in senescent cells but were markedly reduced following SENOMUNE treatment. TIMP-1 and IGFBP-6 increased in senescent cells; however, their expression levels were lower than those in young cells following SENOMUNE treatment (Fig. 3 b). This suggests that senolytics reduce the number of harmful senescent cells that produce SASP to leave behind less detrimental cells that produce little to no SASP. Since SASP factors promote paracrine senescence in normal cells, reducing these harmful senescent cells may have an anti-aging effect on the skin. Alleviation of the skin barrier function using SENOMUNE Despite the low proportion of senescent keratinocytes, epidermal aging reduces the keratinocyte proliferation and differentiation ability resulting in a thinner stratum corneum (Chin et al., 2023)[ 8 ]. Paracrine signaling between dermal fibroblasts and keratinocytes is essential for tissue homeostasis under certain physiological conditions and dermatological disorders. The interaction between these cell populations is altered during aging (Ho and Dreesen, 2021)[ 22 ]. Therefore, we examined the possibility that aged fibroblasts might affect the function of keratinocytes. Treating keratinocytes with conditioned media from senescent HDFs, which are rich in SASP, reduced differentiation markers such as keratin 10 and loricrin and induced the DNA-damage marker phosphorylated histone H2AX (γ-H2AX). However, conditioned media treated with senolytics (ABT-737 and SENOMUNE) did not damage the skin barrier function or induce DNA double-strand breaks in keratinocytes (Fig. 4 a). Treating keratinocytes with conditioned media from senescent human dermal fibroblasts reduced differentiation markers such as filaggrin and keratin 10 accompanied by an induction of the inflammatory marker IL-6. However, keratinocytes treated with conditioned media from senescent HDFs that were treated with senolytics (ABT-737 and SENOMUNE) exhibited recovery of the skin barrier function and reduced inflammation (Fig. 4 b). In the hanging drop culture model, fibroblasts co-cultured within the collagen gel influenced the epidermis of the MelanoDerm. The proliferation marker Ki67 and differentiation markers, such as keratin-10 and filaggrin, were reduced by senescent fibroblasts. However, senescent fibroblasts treated with SENOMUNE improved the proliferation and differentiation of the epidermis (Fig. 4 c). Alleviation of skin-aged pigmentation using SENOMUNE The association between senescent fibroblasts and melanocytes during aging plays a significant role in the stimulation of melanogenesis and subsequent aging-related pigmentation. SASP from senescent fibroblasts determines the pigmentation phenotype of aging skin (Kim et al., 2020)[ 23 ]. We performed an ex vivo skin biopsy to determine the benefits of the anti-aging effects in native skin. Genoskin is a human skin sample that was derived from the abdominal tissue of a 46-year-old woman who underwent plastic surgery. The ex vivo skin was co-cultured with senescent or non-senescent fibroblasts for 7 days after ABT-737 or SENOMUNE treatment for 1 day. The pigmentation of co-cultured ex vivo skin was significantly increased in the presence of senescent fibroblasts compared with that of non-senescent fibroblasts (Fig. 5 a). ABT-737 or SENOMUNE treatment significantly reduced melanin and tyrosinase activities (Fig. 5 b). A previous study revealed that GDF15 was increased in senescent fibroblasts and that GDF15-overexpressing fibroblasts increased the melanin content and tyrosinase activity levels in melanocytes (Kim et al., 2020)[ 23 ]. The results of the current study confirmed that GDF-15 expression increased in senescent fibroblasts and then decreased with SENOMUNE treatment (Fig. 3 b), thereby suggesting that senolytics may restore aging-related pigmentation by directly reducing melanogenic enzymes, such as tyrosinase and senescent fibroblast-derived pigment-inducing SASP factors, such as GDF15. Inflammatory cytokines produced by keratinocytes are closely associated with melanocyte (MC)-mediated pigmentation (Videira et al., 2013)[ 24 ]. Therefore, SENOMUNE may indirectly affect keratinocytes (KCs), thus, potentially regulating pigmentation. Conditioned media (CM) of senescent HDF was used to treat the keratinocytes and substantiate that SENOMUNE exacerbates anti-pigmentation through autophagy activation in keratinocytes. p62-selective autophagy inhibition by the CM of senescent fibroblasts did not degrade p62, and it accumulated in the cells (Fig. 5 c) to activate downstream nuclear factor-κB and increase the secretion of inflammatory cytokines from keratinocytes (Lee et al., 2011)[ 25 ]. Treatment with SENOMUNE on the CM of senescent fibroblasts did not inhibit p62-selective autophagy, thereby reducing the secretion of inflammatory cytokines from KCs. SENOMUNE exhibited anti-pigmentation effects by selectively activating autophagy and reducing p62 activation (Fig. 5 c). Hence, it can be inferred that the anti-pigmentation effect of SENOMUNE is due to its action on melanocytes and keratinocytes. Discussion C. sinensis leaves have been widely used and extensively studied for their numerous health benefits. 21 However, other parts of the plant, such as the roots, stems, flowers, and seeds, have not been thoroughly studied. Previous studies have focused on the anti-cancer and immune-modulatory effects and anti-inflammatory and antioxidant properties of C. sinensis root extracts (Im and Kim, 2022; Lee et al., 2023)[ 15 , 16 ]. A previous study revealed that SENOMUNE contains 54% of the total pure saponin content, which is higher than that in ginseng extracts. Furthermore, the study showed its protective effects against skin disorders induced by environmental pollutants (Na et al., 2018)[ 26 ]. Saponins are found in many plant species and some marine organisms and have various biological and pharmacological activities, such as immunomodulation, anti-inflammatory properties, blood glucose reduction, antibacterial and antiviral activities, and anti-cancer properties (Shen et al., 2023)[ 27 ]. Saponins play significant roles in cancer therapy through mechanisms involving cell cycle inhibition, antioxidant activities, inhibition of cell invasion, and the induction of apoptosis and autophagy (Elekofehinti et al., 2021)[ 28 ]. Since a key mechanism of senotherapy involves the induction of apoptosis in senescent cells by senolytics, we investigated the potential of saponins for use as effective senolytics. We compared the senolytic potential of plant root extracts (from Codonopsis lanceolata [Deodeok], Platycodon grandiflorus root [balloon flower], and Panax ginseng [ginseng]) known to contain high levels of saponins and anti-inflammatory properties, which are suitable for use as ingredients in cosmetics. These plants, except for Panax ginseng , did not exhibit senolytic efficacy due to variability in the structure and concentration of saponins in the root extracts of each plant (Supplementary Fig. S3 ). A previous study reported that black ginseng, which is derived from processed P. ginseng , can be used as a senolytic agent by reducing cellular senescence (Lee et al., 2022)[ 29 ]. This study revealed that SENOMUNE has potential as a senolytic agent. Saponins, the major constituent of SENOMUNE, induce apoptosis in cancer cells through mechanisms that can be caspase-dependent or -independent (Elekofehinti et al., 2021)[ 28 ]. We have elucidated that SENOMUNE induces the selective eradication of senescent cells via a caspase-independent apoptosis mechanism. We verified that SENOMUNE functions by activating pro-apoptotic factors, such as AIF. Saponin releases AIF into the intermembrane and subsequently translocates to the nucleus, where it binds to DNA, causing chromatin condensation and leading to apoptosis. Unlike conventional senolytics, such as ABT-737, which operate through caspase-dependent apoptosis, caspase apoptosis exhibits a unique mechanism of action for SENOMUNE (Fig. 2 ). Dermal fibroblasts and epidermal cells such as melanocytes and keratinocytes in the skin closely influence each other during aging, and the SASP produced from aged cells acts as a mediator of aging (Birch and Gil, 2020; Chin et al., 2023)[ 4 , 8 ]. Previous studies have suggested that applying senolytics reduces the number of senescent cells, suppresses SASP production, and mitigates aging in neighboring cells (Kim et al., 2022; Chin et al., 2023)[ 7 , 8 ]. We aimed to determine the effects of senescent dermal fibroblasts on the aging of epidermal cells (Videira et al., 2013)[ 24 ]. The impact of the SASP generated from senescent fibroblasts on the skin barrier function and pigmentation has been substantiated through MelanoDerm and ex vivo skin biopsies. Furthermore, we confirmed that SENOMUNE improves aging phenotypes, such as skin barrier dysfunction and hyperpigmentation (Figs. 4 and 5 ). We also established that SENOMUNE improves aging-related pigmentation through various pathways. SASP factors produced by senescent fibroblasts, along with inflammatory cytokines produced by keratinocytes, exacerbate aging-related pigmentation (Lee et al., 2011; Videira et al., 2013)[ 24 , 25 ]. We observed that SENOMUNE treatment reduced the SASP in senescent fibroblasts, which decreased enzymes associated with pigmentation in MCs and activated p62-selective autophagy related to suppressing inflammatory cytokine production in KCs. While most of our experiments were performed using in vitro models, which are valuable for preliminary investigations, they do not fully replicate the complexity of human skin aging in vivo. Future studies should include in vivo studies to more accurately assess the therapeutic potential of SENOMUNE. However, our research has clearly demonstrated multiple senolytic effects of SENOMUNE, which underscored its potential despite the absence of in vivo studies. Moreover, SENOMUNE contains 54% saponins and includes various types of saponins. In this study, we were unable to analyze specific saponins demonstrating senolytic efficacy. However, further research is expected to involve isolating individual saponins comprising SENOMUNE to investigate the efficacy and mechanisms of each component in more detail. Despite these limitations, the current research provides strong preliminary evidence of the bioactivities and therapeutic potential of SENOMUNE as an anti-aging phytomedicine. Therefore, we conclude that SENOMUNE has significant potential as a novel anti-aging phytopharmaceutical ingredient with therapeutic applications. Methods Plant preparation and extraction Root samples were collected from a 30-year-old green tea ( C. sinensis L.) in the Amorepacific Dosun tea garden in Jeju Island, Republic of Korea. The C. Sinensis roots were washed with purified water, dried, and ground into a fine powder. Next, the extract was extracted using ethanol, followed by filtration and vacuum concentration at 40–50 ℃ to prepare the dried plant extract. Cell culture NHDFs were purchased from Clonetics Lonza (Walkersville, MD, USA) and maintained in Dulbecco’s Modified Eagle’s Media (DMEM, Thermo Fisher Scientific, Waltham, MA, USA) with 10% fetal bovine serum (FBS, Thermo Fisher Scientific) and penicillin/streptomycin (100 IU/50 µg/mL). Stress-induced premature senescence DMEM containing doxorubicin hydrochloride (100 ng/mL, Sigma-Aldrich, St. Louis, MI, USA) and insulin growth factor-1 (100 ng/mL, Sigma-Aldrich) containing DMEM were added to the cell culture for 4–7 days to induce senescence. Subsequently, SA-β-Gal activity and the gene and protein expression of senescence-related markers were analyzed (Supplementary Fig. S4 ) (An et al., 2020)[ 30 ]. The viability of young and senescent cells was measured to confirm the senolytic effect of SENOMUNE, and the visibility of the ABT-737 and SENOMUNE cells was compared. Cell viability HDFs were seeded at 1.5 × 10 5 cells/well in a 6-well plate and incubated for 48 h. Young and senescent HDFs were incubated with the test materials for 3 days, followed by treatment with a Cell Counting Kit-8 solution (CCK-8, Dojindo Laboratories, Rockview, MD, USA) for 1 h. The optical density was measured at 450 nm using a plate reader (Spectrostar Nano, BMG Labtech, Ortenberg, Germany). SA-β-Gal activity assay SA-β-gal staining was performed using the Senescence Cells Histochemical Staining Kit (Sigma-Aldrich) according to the manufacturer’s instructions. Briefly, the cells were washed with phosphate-buffered saline (PBS) and fixed for 5 min; thereafter, they were incubated at 37°C for 16 h without CO 2 . Reverse transcription qPCR (RT-qPCR) Total RNA was isolated using a TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and 1 µg of total RNA was used to synthesize cDNA using a reverse transcriptase kit (Invitrogen). Gene expression analyses were performed using TaqMan Universal Master Mix and TaqMan Gene Expression assays (Applied Biosystems, Foster City, CA, USA) in a 7500 Fast Real-time PCR System (Applied Biosystems, Waltham, MA, USA) according to the manufacturer’s instructions. Glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ) was used to normalize variations in cDNA quantities synthesized from different samples. The relative mRNA levels were quantified using the 2 −∆∆Ct method. The primers of CXCL8 (Hs00174103_m1), MMP1 (Hs00899658_m1), GDF15 (Hs00171132_m1), TIMP1 (Hs00171558_m1), IGFBP6 (Hs00181853_m1), FLG (Hs00856927_g1), KRT10 (Hs00166289_m1), IL6 (Hs00985639_m1), CDKN1A (Hs00355782_m1), BCL2L1 (Hs00236329_m1), and COL1A1 (Hs00164004_m1) were used for RT - qPCR. Western blotting The cell pellets were lysed in radioimmunoprecipitation assay buffer (Sigma-Aldrich) containing protease inhibitors (Sigma-Aldrich). The lysate was centrifuged at 15,000 × g for 20 min, and the supernatant was used for western blot analysis. Protein concentrations were determined using the Bradford method, with bovine serum albumin (Sigma-Aldrich) as the standard. Proteins (20 µg per well) were fractionated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Thermo Fisher Scientific). The membranes were blocked with 10% SuperBlock T20 (TBST) blocking buffer (Thermo Fisher Scientific, #37536) for 1 h and were subsequently probed overnight at 4 ℃ with an anti-PARP1 antibody (Cell Signaling Technology, Danvers, MA, USA; Catalog No. 9542), anti-cleaved PARP1 antibody (Cell Signaling Technology, Catalog No. 5625), anti-caspase-3 antibody (Cell Signaling Technology, Catalog No. 9662), anti-cleaved caspase-3 antibody (Cell Signaling Technology, Catalog No. 9661), anti-BAX antibody (Cell Signaling Technology, Catalog No. 5023), anti-Bcl-w antibody (Cell Signaling Technology, Catalog No. 2724), anti-Lamin B1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA; Catalog No. sc-374015), anti-phospho-p53 antibody (Cell Signaling Technology, Catalog No. 9284), anti-p53 antibody (Cell Signaling Technology, Catalog No. 2524), anti-p16 antibody (Santa Cruz Biotechnology, Catalog No. sc-56330), anti-p21 antibody (Cell Signaling Technology, Catalog No. 2947), anti-phospho-p62 antibody (Cell Signaling Technology, Catalog No. 16177), anti-phospho-NF-kB p65 antibody (Cell Signaling Technology, Catalog No. 3033), and anti-β-actin antibody (Santa Cruz Biotechnology, Catalog No. sc-56330). The blots were washed thrice with TBST and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad Laboratories, Hercules, CA, USA) for 1 h. The proteins were detected using the Enhanced Chemiluminescence system (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Flow cytometry Cells were cultured in 60 Petri dishes, and the supernatant was collected and labeled with an allophycocyanin (APC) Annexin V/Dead Cell Apoptosis Kit (Invitrogen, Waltham, MA, USA) with APC Annexin V and SYTOX Green for flow cytometry. The apoptotic cells were detected using flow cytometry. Annexin V-APC-positive apoptotic cells were analyzed using fluorescence-activated cell sorting to determine whether the death of senescent cells by SENOMUNE was caused by apoptosis. Caspase-3/7 activity HDFs were grown in a 96-well plate (Thermo Fisher Scientific) and incubated with test materials for 3 days, followed by treatment with an aminoluciferin-labeled substrate using the Caspase-Glo 3/7 assay kit (Promega, Madison, MI, USA) for 3 h. The luminescence of each sample was measured using a plate-reading luminometer (GloMax Discover GM3000, Promega). Cell fraction assay Cells were cultured in 100 Petri dishes, and the supernatant was collected and fractionated using a Cell Fractionation Kit (Abcam, Cambridge, UK). The cytosolic, mitochondrial, and nuclear fractions were assayed using western blotting. Anti-AIF antibody (Proteintech, Rosemont, IL, USA; Catalog No. 17984-1-AP), anti-COXIV antibody (Cell Signaling Technology, Catalog No. 4844), anti-Lamin A antibody (Santa Cruz Biotechnology, Catalog No. sc-7292), anti-CTSB antibody (Cell Signaling Technology, Catalog No. 31718), anti-CTSD antibody (Cell Signaling Technology, Catalog No. 74089), anti-LAMP1 antibody (Cell Signaling Technology, Catalog No. 9091), and anti-GAPDH antibody (Cell Signaling Technology, Catalog No. 2118) were used for western blotting. AO staining and confocal imaging HDFs were plated on Lab-Tek chambered cover glass (Thermo Fisher Scientific) and incubated with test materials for 3 days, followed by staining with 5 µM AO (Catalog No. A1301, Molecular Probes, Eugene, OR, USA) for 30 min at 37 ℃. After rinsing in PBS (Thermo Fisher Scientific), the fluorescence of AO was imaged using a confocal microscope (LSM980, Carl Zeiss, Oberkochen, Germany) with a corresponding filter and at 470–490 nm excitation and 515 nm emission wavelengths. CM of senescent human dermal fibroblasts The CM of senescent cells were obtained from HDFs that had been passaged > 35-fold. Young or senescent HDFs were seeded on a 100-mm dish, and the test materials were treated for an additional 3 days. The media was changed to 1% FBS containing DMEM and incubated for 2 days. It was then collected, centrifuged, and filtered for further analysis. Hanging drop culture For the hanging drop culture, MelanoDerm (MEL-300-B, MatTek Corp., Ashland, MA, USA) was incubated in an EPI-100-NMM-113-PRF medium (MatTek, Ashland, MA, USA) at 37 ℃ in a 5% CO 2 incubator (Thermo Fisher Scientific). The dermal parts were produced as follows: the dermal mixture was prepared by mixing type I collagen (3 mg/mL, Nitta Gelatin, Tokyo, Japan), reconstruction buffer (Nitta Gelatin), Dulbecco's Eagle concentrated culture solution (Nitta Gelatin), fibrinogen (10 mg/mL, Sigma-Aldrich), aprotinin (0.4 TIU/mL, Sigma-Aldrich), and HDFs. Thrombin (0.625 U/mL, Sigma-Aldrich) was added to initiate the fibrinogen polymerization, and the mixture was loaded onto the bottom of the MelanoDerm (hanging drop, MatTek Corp). The tissues were incubated in EPI-100-NMM-113-PRF medium (MatTek Corp) for 14 days. Co-culture of fibroblasts and ex vivo skin biopsy For the co-culture of fibroblasts and ex vivo skin biopsy, the fibroblasts were seeded into a 6-well plate (Thermo Fisher Scientific) and treated with the test materials for 24 h. Ex vivo skin biopsy samples were purchased from Genoskin, Paris, France. After arrival, the ex vivo skin biopsy was stabilized for 24 h and then co-cultured with fibroblasts for 7 days. Histological examination The tissues were fixed in 10% neutral-buffered formalin (BBC Biochemical, Mount Vernon, WA, USA) and embedded in paraffin. The paraffin-embedded samples were sliced into 5 µm sections, and histological observation was performed following hematoxylin and eosin (Thermo Fisher Scientific) and Fontana-Masson (Sigma-Aldrich) staining. For immunohistochemistry, tissue sections were stained with primary antibodies at 4 ℃ overnight. Primary antibodies for Ki67 (Abcam, Catalog No. ab15580), Keratin 10 (Santa Cruz Biotechnology, Catalog No. sc-23877), Filaggrin (Santa Cruz Biotechnology, Catalog No. sc-66192), Tyrosinase (Cell Signaling Technology, Catalog No. 9319), PMEL (Cell Signaling Technology, Catalog No. 38815), and Claudin-1 (Abcam, Catalog No. ab15098) were used. Then, horseradish peroxidase-conjugated donkey anti-rabbit IgG (Abcam, ab6802) or anti-mouse IgG (H&L) (Abcam, ab6820) was applied as the secondary antibody for 60 min. Immunoreactivity was visualized using 3,30-diaminobenzidine (brown) or 3-amino-9-ethylcarbazoles (red) as a chromogen. The sections were visualized under a light microscope (BX53, Olympus, Japan) using a digital camera (DP72, Olympus). Abbreviations AIF-apoptosis-inducing factor, AO-acridine orange, DI-doxorubicin and insulin-like growth factor 1, GAPDH-glyceraldehyde 3-phosphate dehydrogenase, HDFs-human dermal fibroblasts, SASP-senescence-associated secretory phenotype, SENOMUNE- Camellia sinensis L. root extract Declarations Data availability All data generated or analyzed during this study are included in this published article and its supplementary information files. Additional datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Acknowledgments This work was supported by the Amorepacific R&I Center, Gyeonggi-do, Republic of Korea. Author contributions S. Park and S. Cho conceived and designed the experiments; S. Park, S. Cho, D. Min, H. Choi and K. Hwang performed the experiments; K. Hwang contributed materials; H.-J. Kim, I.S. Kil and W.-S. Park analyzed the data and supervised the project; S. Park and H.-J. Kim wrote the paper. Additional Information Declarations of interests The authors declare no competing interests. References Tchkonia, T., Zhu, Y., van Deursen, J., Campisi, J. & Kirkland, J. L. Cellular senescence and the senescent secretory phenotype: therapeutic opportunities. J. Clin. Invest. 123 , 966-972 (2013). Kim, E. C. & Kim, J. R. Senotherapeutics: emerging strategy for healthy aging and age-related disease. BMB Rep. 52 , 47-55 (2019). Soto-Gamez, A. & Demaria, M. Therapeutic interventions for aging: the case of cellular senescence. Drug Discov. Today 22 , 786-795 (2017). Birch, J. & Gil, J. Senescence and the SASP: many therapeutic avenues. Genes Dev. 34 , 1565-1576 (2020). Kirkland, J. L. & Tchkonia, T. Senolytic drugs: from discovery to translation. J. Intern. Med. 288 , 518-536 (2020). Richardson, M. Understanding the structure and function of the skin. Nurs. Times 99 , 46-48 (2003). Kim, J. C., Park, T. J. & Kang, H. Y. Skin-Aging Pigmentation: Who Is the Real Enemy. Cells 11 , 2541 (2022). Chin, T., Lee, X. E., Ng, P. Y., Lee, Y. & Dreesen, O. The role of cellular senescence in skin aging and age-related skin pathologies. Front. Physiol. 14 , 1297637 (2023). Shen, J. et al. Dietary Phytochemicals that Can Extend Longevity by Regulation of Metabolism. Plant Foods Hum. Nutr. 77 , 12-19 (2022). Rais, J., Jafri, A., Siddiqui, S., Tripathi, M. & Arshad, M. Phytochemicals in the treatment of ovarian cancer. Front. Biosci. (Elite Ed.). 9 , 67-75 (2017). Gurău, F. et al. Anti-senescence compounds: A potential nutraceutical approach to healthy aging. Ageing Res. Rev. 46 , 14-31 (2018). Smer-Barreto, V. et al. Discovery of senolytics using machine learning. Nat. Commun. 14 , 3445 (2023). Zhao, T., Li, C., Wang, S. & Song, X. Green Tea ( Camellia sinensis ): A review of its phytochemistry, pharmacology, and toxicology. Molecules 27 , 3909 (2022). Kumar, R. et al. Epigallocatechin gallate suppresses premature senescence of preadipocytes by inhibition of PI3K/Akt/mTOR pathway and induces senescent cell death by regulation of Bax/Bcl-2 pathway. Biogerontology 20 , 171-189 (2019). Im, S. H. & Kim, J. S. Physicochemical properties of extracts from different parts of Camellia sinensis . Korean J. Med. Crop Sci. 30 , 195-203 (2022). Lee, J. et al. Triterpenoid saponins from Camellia sinensis roots with cytotoxic and immunomodulatory effects. Phytochemistry 212 , 113688 (2023). Bhadra, K. A mini review on molecules inducing caspase-independent cell death: a new route to cancer therapy. Molecules 27 , 6401 (2022). Elmore, S. Apoptosis: a review of programmed cell death. Toxicol. Pathol. 35 , 495-516 (2007). Chaitanya, G. V., Steven, A. J. & Babu, P. P. PARP-1 cleavage fragments: signatures of cell-death proteases in neurodegeneration. Cell Commun. Signal. 8 , 31 (2010). Modjtahedi, N., Giordanetto, F., Madeo, F. & Kroemer, G. Apoptosis-inducing factor: vital and lethal. Trends Cell Biol. 16 , 264-272 (2006). Boya, P. & Kroemer, G. Lysosomal membrane permeabilization in cell death. Oncogene 27 , 6434-6451 (2008). Ho, C. Y. & Dreesen, O. Faces of cellular senescence in skin aging. Mech. Ageing Dev. 198 , 111525 (2021). Kim, Y., Kang, B., Kim J. C., Park, T. J. & Kang, H. Y. Senescent fibroblast-derived GDF15 induces skin pigmentation. J. Invest. Dermatol. 140 , 2478-2486.e4 (2020). Videira, I. F., Moura, D. F. & Magina, S. Mechanisms regulating melanogenesis. An. Bras. Dermatol. 88 , 76-83 (2013). Lee, H. M. et al. Autophagy negatively regulates keratinocyte inflammatory responses via scaffolding protein p62/SQSTM1. J. Immunol. 186 , 1248-1258 (2011). Na, H. W., Lee, Y. R., Park, J. S., Lee, T. R. & Kim, H. J. Green tea root is a potential natural surfactant and is protective against the detrimental stimulant PM2.5 in human normal epidermal keratinocytes. J. Soc. Cosmet. Sci. Korea 44 , 67-72 (2018). Shen, L. et al. Potential immunoregulatory mechanism of plant saponins: A review. Molecules 29 , 113 (2023). Elekofehinti, O. O., Iwaloye, O., Olawale, F. & Ariyo, E. O. Saponins in cancer treatment: Current progress and future prospects. Pathophysiology 28 , 250-272 (2021). Lee, S. J., Lee, D. Y., O’Connell, J. F., Egan, J. M. & Kim, Y. Black ginseng ameliorates cellular senescence via p53-p21/p16 pathway in aged mice. Biology 11 , 1108 (2022). An, S. et al. Inhibition of 3-phosphoinositide-dependent protein kinase 1 (PDK1) can revert cellular senescence in human dermal fibroblasts. Proc. Natl. Acad. Sci. U. S. A. 117 , 31535-31546 (2020). An, S., et al. (2020). "Inhibition of 3-phosphoinositide–dependent protein kinase 1 (PDK1) can revert cellular senescence in human dermal fibroblasts." Proceedings of the National Academy of Sciences 117 (49): 31535-31546. Bhadra, K. (2022). "A mini review on molecules inducing caspase-independent cell death: A new route to cancer therapy." Molecules 27 (19): 6401. Birch, J. and J. Gil (2020). "Senescence and the SASP: many therapeutic avenues." Genes & development 34 (23-24): 1565-1576. Boya, P. and G. Kroemer (2008). "Lysosomal membrane permeabilization in cell death." Oncogene 27 (50): 6434-6451. Chaitanya, G. V., et al. (2010). "PARP-1 cleavage fragments: signatures of cell-death proteases in neurodegeneration." Cell communication and signaling 8 : 1-11. Chin, T., et al. (2023). "The role of cellular senescence in skin aging and age-related skin pathologies." Frontiers in physiology 14 : 1297637. Elekofehinti, O. O., et al. (2021). "Saponins in cancer treatment: Current progress and future prospects." Pathophysiology 28 (2): 250-272. Elmore, S. (2007). "Apoptosis: a review of programmed cell death." Toxicologic pathology 35 (4): 495-516. Gurău, F., et al. (2018). "Anti-senescence compounds: a potential nutraceutical approach to healthy aging." Ageing Research Reviews 46 : 14-31. Ho, C. Y. and O. Dreesen (2021). "Faces of cellular senescence in skin aging." Mechanisms of ageing and development 198 : 111525. Im, S. and J. Kim (2022). "Physicochemical properties of extracts from different parts of Camellia sinensis." Korean Journal of Medicinal Crop Science 30 (3): 195-203. Kim, E.-C. and J.-R. Kim (2019). "Senotherapeutics: emerging strategy for healthy aging and age-related disease." BMB reports 52 (1): 47. Kim, J. C., et al. (2022). "Skin-aging pigmentation: who is the real enemy?" Cells 11 (16): 2541. Kim, Y., et al. (2020). "Senescent fibroblast–derived GDF15 induces skin pigmentation." Journal of Investigative Dermatology 140 (12): 2478-2486. e2474. Kirkland, J. and T. Tchkonia (2020). "Senolytic drugs: from discovery to translation." Journal of internal medicine 288 (5): 518-536. Kumar, R., et al. (2019). "Epigallocatechin gallate suppresses premature senescence of preadipocytes by inhibition of PI3K/Akt/mTOR pathway and induces senescent cell death by regulation of Bax/Bcl-2 pathway." Biogerontology 20 : 171-189. Lee, H.-M., et al. (2011). "Autophagy negatively regulates keratinocyte inflammatory responses via scaffolding protein p62/SQSTM1." The Journal of Immunology 186 (2): 1248-1258. Lee, J., et al. (2023). "Triterpenoid saponins from Camellia sinensis roots with cytotoxic and immunomodulatory effects." Phytochemistry 212 : 113688. Lee, S.-J., et al. (2022). "Black ginseng ameliorates cellular senescence via p53-p21/p16 pathway in aged mice." Biology 11 (8): 1108. Modjtahedi, N., et al. (2006). "Apoptosis-inducing factor: vital and lethal." Trends in cell biology 16 (5): 264-272. Na, H.-W., et al. (2018). "Green tea root is a potential natural surfactant and is protective against the detrimental stimulant PM2. 5 in human normal epidermal keratinocytes." Journal of the Society of Cosmetic Scientists of Korea 44 (1): 67-72. Rais, J., et al. (2017). "Phytochemicals in the treatment of ovarian cancer." Front Biosci (Elite Ed) 9 (1): 67-75. Richardson, M. (2003). "Understanding the structure and function of the skin." Nursing times 99 (31): 46-48. Shen, J., et al. (2022). "Dietary phytochemicals that can extend longevity by regulation of metabolism." Plant Foods for Human Nutrition 77 (1): 12-19. Shen, L., et al. (2023). "Potential Immunoregulatory mechanism of Plant saponins: a review." Molecules 29 (1): 113. Smer-Barreto, V., et al. (2023). "Discovery of senolytics using machine learning." Nature communications 14 (1): 3445. Soto-Gamez, A. and M. Demaria (2017). "Therapeutic interventions for aging: the case of cellular senescence." Drug Discovery Today 22 (5): 786-795. Tchkonia, T., et al. (2013). "Cellular senescence and the senescent secretory phenotype: therapeutic opportunities." The Journal of clinical investigation 123 (3): 966-972. Videira, I. F. d. S., et al. (2013). "Mechanisms regulating melanogenesis." Anais brasileiros de dermatologia 88 : 76-83. Zhao, T., et al. (2022). "Green tea (Camellia sinensis): A review of its phytochemistry, pharmacology, and toxicology." Molecules 27 (12): 3909. Additional Declarations No competing interests reported. Supplementary Files Graphicabstract20250701.docx Suppl.Figure1.tif Suppl.Figure2.tif Suppl.Figure3.tif SupplementaryInfoFile.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7016169","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":487786275,"identity":"0024a2e5-b5fe-4bed-8ac5-8881ba1b5ded","order_by":0,"name":"Sunyoung Park","email":"","orcid":"","institution":"Amorepacific (South Korea)","correspondingAuthor":false,"prefix":"","firstName":"Sunyoung","middleName":"","lastName":"Park","suffix":""},{"id":487786276,"identity":"808b892a-fe03-449d-94d1-6853aa1b9f04","order_by":1,"name":"Siyoung Cho","email":"","orcid":"","institution":"Amorepacific (South Korea)","correspondingAuthor":false,"prefix":"","firstName":"Siyoung","middleName":"","lastName":"Cho","suffix":""},{"id":487786277,"identity":"8074ac7b-5236-4c91-bdf8-8a75bbc984be","order_by":2,"name":"Daejin Min","email":"","orcid":"","institution":"Amorepacific (South Korea)","correspondingAuthor":false,"prefix":"","firstName":"Daejin","middleName":"","lastName":"Min","suffix":""},{"id":487786278,"identity":"e55d1950-2870-409e-930e-966031cb07f5","order_by":3,"name":"Hyunjung Choi","email":"","orcid":"","institution":"Amorepacific (South Korea)","correspondingAuthor":false,"prefix":"","firstName":"Hyunjung","middleName":"","lastName":"Choi","suffix":""},{"id":487786279,"identity":"07f72c1a-3bb0-415e-bd3e-4707b0f84284","order_by":4,"name":"Kyeonghwan Hwang","email":"","orcid":"","institution":"Amorepacific (South Korea)","correspondingAuthor":false,"prefix":"","firstName":"Kyeonghwan","middleName":"","lastName":"Hwang","suffix":""},{"id":487786280,"identity":"f994b75a-52d5-4619-b17e-728fa4489a34","order_by":5,"name":"In Sup Kil","email":"","orcid":"","institution":"Amorepacific (South Korea)","correspondingAuthor":false,"prefix":"","firstName":"In","middleName":"Sup","lastName":"Kil","suffix":""},{"id":487786281,"identity":"1e0b5249-facf-4b5a-9313-0632141af403","order_by":6,"name":"Won-Seok Park","email":"","orcid":"","institution":"Amorepacific (South Korea)","correspondingAuthor":false,"prefix":"","firstName":"Won-Seok","middleName":"","lastName":"Park","suffix":""},{"id":487786282,"identity":"81f5eb3c-cdc3-4484-ae43-2fa1fd468e1e","order_by":7,"name":"Hyoung-June Kim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA90lEQVRIiWNgGAWjYBACPiA2AGI5uAgb88EGvFrYoFqMESJsiYS1gACSMrYE/A5jY28+UMy7wya9f9rhYx8+7rCT52NjbvzAUGMTjVMLz7EEY94zabkzbqclz5x5JtmwjY2xWYLhWFouLuexSeQYGPO2Hc5tuJ1jzMzbxpzAJt/YxsDYcBi3Fvk3IC3/0+VBWv621SewsTES0CLBA9JyIMEApIWx7TARWnjSEgzntiUbbgT6hbH3zHGIXxLw+IWf/fAxg7dtdvJyt5MPM/zcUS0v38b+8MOHGhucWkAWGcCZjDBlCbiVgwDzA0wto2AUjIJRMAqQAACjWVECOt4D+QAAAABJRU5ErkJggg==","orcid":"","institution":"Amorepacific (South Korea)","correspondingAuthor":true,"prefix":"","firstName":"Hyoung-June","middleName":"","lastName":"Kim","suffix":""}],"badges":[],"createdAt":"2025-07-01 05:23:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7016169/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7016169/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87256193,"identity":"9b833820-8b1d-4f92-8faf-86903b0957df","added_by":"auto","created_at":"2025-07-22 05:57:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3300579,"visible":true,"origin":"","legend":"\u003cp\u003eSenolytic activity of SENOMUNE in HDFs. (\u003cstrong\u003ea\u003c/strong\u003e) Viability of young (dark) and senescent cells (light) after cells were treated for 3 days. Dose-dependent senolytic effect of ABT-737 [1–10 µM] and SENOMUNE [5–150 ppm]. * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 compared to young, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 compared to young. (\u003cstrong\u003eb\u003c/strong\u003e) SA-β-gal activity in senescent cells. SENOMUNE: \u003cem\u003eCamellia sinensis \u003c/em\u003eL.\u003cem\u003e \u003c/em\u003eroot extract; HDFs: human dermal fibroblasts; SA-β-gal:senescence-associated β-galactosidase.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7016169/v1/20a7e3fcaed49be083893f42.png"},{"id":87255274,"identity":"dc856631-2a2e-4666-b05d-94b2043456c5","added_by":"auto","created_at":"2025-07-22 05:49:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3400609,"visible":true,"origin":"","legend":"\u003cp\u003eInduction of apoptosis by SENOMUNE via caspase-independent programmed cell death. (\u003cstrong\u003ea\u003c/strong\u003e) Representative flow cytometry plots of the apoptosis assay. Apoptosis was assayed using flow cytometry after staining with Annexin V and SYTOX green. Quantification of the percentage of apoptotic cells (annexin V-APC-positive) 1 day after treatment. (\u003cstrong\u003eb\u003c/strong\u003e) Comparison of caspase-3/7 activity in ABT-737 (left, red line) and SENOMUNE cells (right, green line). Senescence was induced by doxorubicin and insulin-like growth factor-1 for 7 days, and ABT-737 (10 μM) or SENOMUNE (100 μg/mL) cells were treated for 1 or 3 days, respectively. **** \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001 compared to doxorubicin and insulin-like growth factor-1 (DI). (\u003cstrong\u003ec\u003c/strong\u003e) Induction of caspase-dependent programmed cell death by ABT-737. (\u003cstrong\u003ed\u003c/strong\u003e) Translocation of AIF from the mitochondria to the nucleus using SENOMUNE. COXIV, Lamin A, and GAPDH were used as mitochondria, nucleus, and cytosol markers, respectively. (\u003cstrong\u003ee\u003c/strong\u003e) Lysosomal membrane permeabilization using SENOMUNE. Lysosomes with a high concentration of acridine orange (AO) are indicated in red. SENOMUNE: \u003cem\u003eC. sinensis \u003c/em\u003eL.\u003cem\u003e \u003c/em\u003eroot extract; AIF: apoptosis-inducing factor; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; DI: doxorubicin.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7016169/v1/8093194411cf224c16824433.png"},{"id":87254868,"identity":"25d916fc-a35d-4b20-aad9-210e65132a2f","added_by":"auto","created_at":"2025-07-22 05:41:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1052218,"visible":true,"origin":"","legend":"\u003cp\u003eAging marker decrease and SASP inhibitory effect of SENOMUNE on senescent HDFs. (\u003cstrong\u003ea\u003c/strong\u003e) SENOMUNE reduced aging markers. Phospho-p53 and p21 levels increased with aging and decreased with ABT-737 and SENOMUNE treatment, respectively. (\u003cstrong\u003eb\u003c/strong\u003e) SASP inhibitory effect of SENOMUNE. ABT-737 (2.5 μM) and SENOMUNE (100 ppm) were treated for 3 days. Subsequently, cells were transferred to a new culture plate and incubated for an additional 7 days. Gene expression was normalized to that of \u003cem\u003eGAPDH\u003c/em\u003e. * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 compared to DI, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 compared to DI. ## \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 compared to young, ### \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 compared to young. SENOMUNE: \u003cem\u003eC. sinensis \u003c/em\u003eL.\u003cem\u003e \u003c/em\u003eroot extract; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; SASP: senescence-associated secretory phenotype; HDFs: human dermal fibroblasts; DI: doxorubicin.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7016169/v1/1cd373e9cca8c2ea487fb4e6.png"},{"id":87254877,"identity":"d6cc8431-da10-460f-851a-a299ffdedf91","added_by":"auto","created_at":"2025-07-22 05:41:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2476369,"visible":true,"origin":"","legend":"\u003cp\u003eAlleviation of skin barrier function by SENOMUNE. Effect of conditioned medium from fibroblasts on keratinocytes. Conditioned media (CM) from young fibroblasts (YCM) or senescent fibroblasts (OCM) were applied to keratinocytes for 4 days. The protein (A) and gene (B) expression levels were validated. * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 compared to OCM control. (C) Histological examination of epidermal proliferation and differentiation using a hanging drop culture model (C). The scale bar is 500 μm. * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001 compared to young. ## \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ### \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 compared to DI. DI: doxorubicin; SENOMUNE: \u003cem\u003eC. sinensis \u003c/em\u003eL.\u003cem\u003e \u003c/em\u003eroot extract.\u003c/p\u003e","description":"","filename":"Figure4newfiledownload.png","url":"https://assets-eu.researchsquare.com/files/rs-7016169/v1/60bc449c1355d21fd265b247.png"},{"id":87254880,"identity":"4a32fa1a-fefc-4a86-810d-51d45477beb7","added_by":"auto","created_at":"2025-07-22 05:41:21","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2731364,"visible":true,"origin":"","legend":"\u003cp\u003eAlleviation of skin-aging pigmentation. (\u003cstrong\u003ea\u003c/strong\u003e) Ex vivo culture of skin biopsy from young or senescent fibroblasts. Young or senescent fibroblasts were treated with ABT-737 (10 μM) or SENOMUNE (100 μg/mL) for 24 h. The fibroblast medium was replaced with fresh medium and cultured with a skin biopsy for 7 days. The brightness of Genoskin was compared using L-values. ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 compared to the old fibroblast control. (\u003cstrong\u003eb\u003c/strong\u003e) Histological examination of the pigmentation. (\u003cstrong\u003ec\u003c/strong\u003e) Activation of p62-selective autophagy after SENOMUNE treatment. SENOMUNE: \u003cem\u003eC. sinensis \u003c/em\u003eL.\u003cem\u003e \u003c/em\u003eroot extract.\u003c/p\u003e","description":"","filename":"Figure5newfiledownload.png","url":"https://assets-eu.researchsquare.com/files/rs-7016169/v1/ab1c90709bb985a1917fb955.png"},{"id":89070080,"identity":"65ea1a6a-8827-429a-bf62-8a15b4917ed5","added_by":"auto","created_at":"2025-08-14 10:57:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":14319272,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7016169/v1/bc3c4edf-689a-46bb-b3d3-5e95ad76c94b.pdf"},{"id":87255276,"identity":"ddc19c73-ce1d-49a3-ac75-239e9049b1c4","added_by":"auto","created_at":"2025-07-22 05:49:21","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":373687,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicabstract20250701.docx","url":"https://assets-eu.researchsquare.com/files/rs-7016169/v1/9f8504fa1f81b3786b60fa9a.docx"},{"id":87254883,"identity":"e2908fb1-5c3e-470b-b04a-b2958f0bc66c","added_by":"auto","created_at":"2025-07-22 05:41:21","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2268342,"visible":true,"origin":"","legend":"","description":"","filename":"Suppl.Figure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-7016169/v1/bccb570271b6cdcd02a7c22d.tif"},{"id":87254875,"identity":"9b67efa6-76c9-4d41-9b85-4ace78499f99","added_by":"auto","created_at":"2025-07-22 05:41:21","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1032484,"visible":true,"origin":"","legend":"","description":"","filename":"Suppl.Figure2.tif","url":"https://assets-eu.researchsquare.com/files/rs-7016169/v1/8d06f9a60653a4bbc906ae09.tif"},{"id":87255280,"identity":"5fc15752-2e2b-438f-b2c7-4b1c61092f6f","added_by":"auto","created_at":"2025-07-22 05:49:21","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":661772,"visible":true,"origin":"","legend":"","description":"","filename":"Suppl.Figure3.tif","url":"https://assets-eu.researchsquare.com/files/rs-7016169/v1/3a998f0f002a542493f98691.tif"},{"id":87255279,"identity":"ba69da37-1f54-4d3a-ac5f-42d595f6332c","added_by":"auto","created_at":"2025-07-22 05:49:21","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":806914,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInfoFile.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7016169/v1/3e179ebe1a93746e8763f46b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eNovel Senolytic Ingredient, \u003cem\u003eCamellia sinensis\u003c/em\u003e Root Extract, Ameliorates Skin Aging-Associated Phenotypes\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCellular senescence is the loss of the ability of normal cells to divide and remain in a state of cell cycle arrest. Cellular senescence progresses sequentially: when cell division stops, the non-dividing cells secrete senescence-associated secretory phenotype (SASP) factors, which promotes the spread of senescence causing senescent cells accumulate in tissues (Tchkonia et al., 2013; Kim and Kim, 2019)[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Normal cells self-destruct through apoptosis, which is difficult to repair due to genetic damage. Instead of apoptosis, senescent cells are eliminated through a process that sends out signaling molecules to inform immune cells to eliminate them (Soto-Gamez and Demaria, 2017)[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. A problem occurs when senescent cells increase in number, and the immune cells do not sufficiently remove them, thereby causing an accumulation of senescent cells. Anti-aging treatment to selectively remove the adverse effects of senescent cells is being studied (senotherapy) because an increase in senescent cells causes age-related diseases. Senotherapy includes the use of senolytics and senomorphics. Senolytics are drugs that directly eliminate senescent cells. They focus on apoptosis resistance in senescent cells, and increase suppressing factors in senescent cells and inducing cell death. Meanwhile, senomorphics target the SASP; these are specific cytokines and chemokines secreted by the senescent cells, and certain drugs target the related signaling mechanism (Soto-Gamez and Demaria, 2017; Kim and Kim, 2019; Birch and Gil, 2020; Kirkland and Tchkonia, 2020)[\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eGiven that senescent cells accumulate in tissues and contribute to aging, their impact is particularly evident in the skin, which is the body's largest organ (Richardson, 2003)[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The skin is the first to exhibit signs of aging visually; therefore, it is a focus of most aging research. Previous research has been conducted on how the interaction of the various cells that make up the skin effects aging. They revealed that senescent melanocytes induce aging in surrounding cells such as fibroblasts, and that skin-aging pigmentation is induced in melanocytes through interaction with senescent fibroblasts (Kim et al., 2022)[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Since this interaction occurs through the SASP produced in senescent skin cells, senolytics and senomorphics are being considered to control skin aging-related disorders caused by aging (Chin et al., 2023)[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eConsidering the role of senescent cells in skin aging, researchers are exploring natural compounds, such as phytochemicals, for their potential in targeting these cells (Shen et al., 2022)[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Certain phytochemicals are poisons and traditional medicines. Phytochemicals can be classified as terpenes (carotenoids, monoterpenes, and saponins), phenols (polyphenols and flavonoids), organosulfur compounds (indoles, isothiocyanates, and thiosulfonates), organic acids and polysaccharides, and lipids (isoprenoids and omega-3 and omega-6 fatty acids) (Rais et al., 2017)[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Among the phytochemicals, quercetin was the first senolytic, and various cell types and clinical trials have revealed its senolytic activities in combination with dasatinib. Similar to quercetin, other flavonoids (fisetin, curcumin, leteolin, and piperlongumine) are promising senolytic agents (Gurău et al., 2018)[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. A previous report confirmed the senolytic activity of flavonoids as well as periplocin and oleandrin, which are saponins (Smer-Barreto et al., 2023)[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFlavonoids, saponins, tannins, phenolic compounds, and triterpenoids are obtained from the leaves, flowers, and roots of \u003cem\u003eCamellia\u003c/em\u003e spp. \u003cem\u003eCamellia sinensis\u003c/em\u003e leaves are widely used as medicinal herbs because they contain polyphenols such as catechins and epigallocatechin gallate derivatives (Zhao et al., 2022)[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. A previous report revealed that epigallocatechin gallate suppresses anti-apoptotic protein Bcl-2 accumulation in senescent cells, thereby promoting apoptosis-mediated cell death (Kumar et al., 2019)[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. While tea leaves have long been studied and consumed for their numerous health benefits, there is limited research on the same in the roots of tea plants. Tea plant root extracts contain lower levels of flavonoids than leaf extracts but contain various oleanolic-type triterpenes (Im and Kim, 2022)[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Previous studies have confirmed that tea plant root extracts have high antioxidant and α-glucosidase inhibitory activities owing to the presence of saponins, thereby indicating significant physiological benefits. Additionally, ongoing research explores the anti-cancer, immune-modulating, anti-inflammatory, and antioxidant effects of \u003cem\u003eC. sinensis\u003c/em\u003e root extracts (Im and Kim, 2022; Lee et al., 2023)[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, the senotherapeutic efficacy of the \u003cem\u003eC. sinensis\u003c/em\u003e root extract (SENOMUNE) has not been investigated. Therefore, this study aims to investigate the senotherapeutic potential of SENOMUNE in controlling skin aging-related disorders. We hypothesize that SENOMUNE exhibits senotherapeutic properties by modulating SASP and promoting apoptosis in senescent skin cells. The senolytic effect of SENOMUNE was evaluated using various in vitro assays, and its impact on skin barrier function and pigmentation was assessed with conditioned media from senescent fibroblasts and ex vivo skin biopsies. In this study, we report SENOMUNE as a new senotherapeutic agent for controlling various skin aging-related disorders, including skin aging pigmentation and impaired barrier function.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eEffect of SENOMUNE in human dermal fibroblasts\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo test senolytic efficacy, we used a stress-induced premature senescence model in normal human dermal fibroblasts (NHDFs). SENOMUNE exhibited a 35% and 59% concentration-dependent reduction in senescent cells at 100 and 150 ppm, respectively; however, it exhibited cell toxicity in young cells at 150 ppm. Contrastingly, the visibility of ABT-737 cells decreased by 49% and 75% at 2.5 and 10 \u0026micro;M, respectively, without affecting the viability of the young cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The reduction in cell viability induced by ABT-737 and SENOMUNE was observed in the senescence-associated β-galactosidase (SA-β-gal) assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). In ABT-737-treated senescent cells, SA-β-gal reduction was confirmed in a dose-dependent manner. SENOMUNE reduced SA-β-gal at concentrations\u0026thinsp;\u0026gt;\u0026thinsp;50 ppm (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eApoptosis induction using SENOMUNE through caspase-independent programmed cell death\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSENOMUNE did not cause apoptosis in young cells but increased apoptosis by 23% compared with that of the vehicle (16%) in senescent cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Apoptosis is divided into caspase-dependent programmed cell death (classical apoptosis) or caspase-independent programmed cell death (CI-PCD) (Bhadra 2022)[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. ABT-737 increased caspase activity in senescence cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Protein expression patterns confirmed classical (caspase-dependent) apoptosis of ABT-737 and CI-PCD by SENOMUNE. Cleaved caspase-3 (active form) was unchanged, but cleaved poly(ADP-ribose) polymerase 1 (PARP1) increased in the SENOMUNE-treated senescent cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec); PARP-1 is a known caspase cellular substrate. The cleavage of PARP-1 by caspases is considered a hallmark of apoptosis (Elmore, 2007)[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Thus, increased levels of cleaved caspase-3 and PARP-1 indicate that ABT-737 induces classical (caspase-dependent) apoptosis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePARP-1 is a preferred substrate for several \u0026ldquo;suicidal\u0026rdquo; proteases, and the proteolytic action of suicidal proteases (caspases, calpains, cathepsins, granzymes, and matrix metalloproteinases) on PARP-1 produces several specific proteolytic cleavage fragments with different molecular weights (Chaitanya et al., 2010)[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. A few PARP-1 fragments and their associated binding partners control different forms of cell death (e.g., autophagy, necrosis, and parthanatos). The PARP-1 fragments produced by ABT-737 and SENOMUNE may be derived from the actions of different proteases. The 89-kD PARP-1 fragment, which appears during apoptotic cell death by caspase-3, can be produced by cathepsin-B and D (Elmore, 2007; Chaitanya et al., 2010)[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The release of cathepsin-B and D from the lysosome to the cytosol was observed following SENOMUNE treatment (Supplementary Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePARP-1 is thought to be required for apoptosis-inducing factor (AIF) release from the mitochondria during caspase-independent apoptosis (Modjtahedi et al., 2006)[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Bcl-2-associated X protein (BAX) is related to AIF relocation from the mitochondria to the cytosol (Modjtahedi et al., 2006; Bhadra, 2022)[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. PARP1 activation (cleaved PARP1) and an increase in BAX were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). AIF was observed in the cytosol of cells treated with SENOMUNE but not in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Therefore, CI-PCD induced by SENOMUNE was distinct from the apoptotic signaling pathway triggered by ABT-737. Moreover, AIF promotes caspase-independent cell death following lysosomal membrane permeabilization (LMP) (Boya and Kroemer, 2008)[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The effect of SENOMUNE on lysosomal cell death was evaluated using acridine orange (AO) staining to test the hypothesis that SENOMUNE causes senolysis through LMP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). AO is a lysosomotropic metachromatic fluorophore that, when excited with blue light, emits red and green fluorescence at high (when it is observed in lysosomes) and low concentrations (when it is present in the cytosol and the nucleus), respectively (Boya and Kroemer, 2008)[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Lysosomes with high AO concentration were stained and exhibited red dots in young and senescent cells. The red dots disappeared only in senescent cells treated with SENOMUNE. The AO-loaded cells exhibited reduced red fluorescence and increased green fluorescence after LMP treatment, thereby indicating that SENOMUNE exerted a senolytic effect through lysosome-related programmed cell death (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee).\u003c/p\u003e\u003cp\u003e\u003cb\u003eReduction of aging markers and senescence-associated secretory phenotypes by SENOMUNE\u003c/b\u003e\u003c/p\u003e\u003cp\u003eLamin B1, phospho-p53, and p21 expression levels were measured in the HDFs to determine the effect of senolytics on aging markers. Lamin B1 was decreased; however, phospho-p53, p53, and p21 levels increased during senescence but were considerably diminished by ABT-737 and SENOMUNE. However, senolytic treatment marginally altered Lamin B1 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), probably because cell cycle proteins are more unstable than structural proteins. The apoptosis-inducing agents ABT-737 and SENOMUNE may have influenced the stability of the nuclear envelope component Lamin B1 owing to the characteristic feature of nuclear envelope breakdown in apoptotic cell death, which led to a slight reduction.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe remaining senescent cells after SENOMUNE treatment were incubated for 1 week, and SASP gene expression was assayed using quantitative PCR (qPCR) to confirm the resistance of senescent cells to the SASP inhibitory effect. Several cytokine and protease genes, such as \u003cem\u003eIL-8, MMP-1, GDF-15, TIMP-1\u003c/em\u003e, and \u003cem\u003eIGFBP-6\u003c/em\u003e, were maintained at low levels. Among these, the \u003cem\u003eIL-8, MMP-1\u003c/em\u003e, and \u003cem\u003eGDF-15\u003c/em\u003e expression levels significantly increased in senescent cells but were markedly reduced following SENOMUNE treatment. \u003cem\u003eTIMP-1\u003c/em\u003e and \u003cem\u003eIGFBP-6\u003c/em\u003e increased in senescent cells; however, their expression levels were lower than those in young cells following SENOMUNE treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). This suggests that senolytics reduce the number of harmful senescent cells that produce SASP to leave behind less detrimental cells that produce little to no SASP. Since SASP factors promote paracrine senescence in normal cells, reducing these harmful senescent cells may have an anti-aging effect on the skin.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAlleviation of the skin barrier function using SENOMUNE\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDespite the low proportion of senescent keratinocytes, epidermal aging reduces the keratinocyte proliferation and differentiation ability resulting in a thinner stratum corneum (Chin et al., 2023)[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Paracrine signaling between dermal fibroblasts and keratinocytes is essential for tissue homeostasis under certain physiological conditions and dermatological disorders. The interaction between these cell populations is altered during aging (Ho and Dreesen, 2021)[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Therefore, we examined the possibility that aged fibroblasts might affect the function of keratinocytes.\u003c/p\u003e\u003cp\u003eTreating keratinocytes with conditioned media from senescent HDFs, which are rich in SASP, reduced differentiation markers such as keratin 10 and loricrin and induced the DNA-damage marker phosphorylated histone H2AX (γ-H2AX). However, conditioned media treated with senolytics (ABT-737 and SENOMUNE) did not damage the skin barrier function or induce DNA double-strand breaks in keratinocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Treating keratinocytes with conditioned media from senescent human dermal fibroblasts reduced differentiation markers such as filaggrin and keratin 10 accompanied by an induction of the inflammatory marker \u003cem\u003eIL-6.\u003c/em\u003e However, keratinocytes treated with conditioned media from senescent HDFs that were treated with senolytics (ABT-737 and SENOMUNE) exhibited recovery of the skin barrier function and reduced inflammation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn the hanging drop culture model, fibroblasts co-cultured within the collagen gel influenced the epidermis of the MelanoDerm. The proliferation marker Ki67 and differentiation markers, such as keratin-10 and filaggrin, were reduced by senescent fibroblasts. However, senescent fibroblasts treated with SENOMUNE improved the proliferation and differentiation of the epidermis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec).\u003c/p\u003e\u003cp\u003e\u003cb\u003eAlleviation of skin-aged pigmentation using SENOMUNE\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe association between senescent fibroblasts and melanocytes during aging plays a significant role in the stimulation of melanogenesis and subsequent aging-related pigmentation. SASP from senescent fibroblasts determines the pigmentation phenotype of aging skin (Kim et al., 2020)[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. We performed an ex vivo skin biopsy to determine the benefits of the anti-aging effects in native skin. Genoskin is a human skin sample that was derived from the abdominal tissue of a 46-year-old woman who underwent plastic surgery. The ex vivo skin was co-cultured with senescent or non-senescent fibroblasts for 7 days after ABT-737 or SENOMUNE treatment for 1 day. The pigmentation of co-cultured ex vivo skin was significantly increased in the presence of senescent fibroblasts compared with that of non-senescent fibroblasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). ABT-737 or SENOMUNE treatment significantly reduced melanin and tyrosinase activities (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). A previous study revealed that GDF15 was increased in senescent fibroblasts and that GDF15-overexpressing fibroblasts increased the melanin content and tyrosinase activity levels in melanocytes (Kim et al., 2020)[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The results of the current study confirmed that \u003cem\u003eGDF-15\u003c/em\u003e expression increased in senescent fibroblasts and then decreased with SENOMUNE treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), thereby suggesting that senolytics may restore aging-related pigmentation by directly reducing melanogenic enzymes, such as tyrosinase and senescent fibroblast-derived pigment-inducing SASP factors, such as GDF15.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eInflammatory cytokines produced by keratinocytes are closely associated with melanocyte (MC)-mediated pigmentation (Videira et al., 2013)[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Therefore, SENOMUNE may indirectly affect keratinocytes (KCs), thus, potentially regulating pigmentation. Conditioned media (CM) of senescent HDF was used to treat the keratinocytes and substantiate that SENOMUNE exacerbates anti-pigmentation through autophagy activation in keratinocytes. p62-selective autophagy inhibition by the CM of senescent fibroblasts did not degrade p62, and it accumulated in the cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec) to activate downstream nuclear factor-κB and increase the secretion of inflammatory cytokines from keratinocytes (Lee et al., 2011)[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Treatment with SENOMUNE on the CM of senescent fibroblasts did not inhibit p62-selective autophagy, thereby reducing the secretion of inflammatory cytokines from KCs. SENOMUNE exhibited anti-pigmentation effects by selectively activating autophagy and reducing p62 activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Hence, it can be inferred that the anti-pigmentation effect of SENOMUNE is due to its action on melanocytes and keratinocytes.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cem\u003eC. sinensis\u003c/em\u003e leaves have been widely used and extensively studied for their numerous health benefits.\u003csup\u003e21\u003c/sup\u003e However, other parts of the plant, such as the roots, stems, flowers, and seeds, have not been thoroughly studied. Previous studies have focused on the anti-cancer and immune-modulatory effects and anti-inflammatory and antioxidant properties of \u003cem\u003eC. sinensis\u003c/em\u003e root extracts (Im and Kim, 2022; Lee et al., 2023)[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. A previous study revealed that SENOMUNE contains 54% of the total pure saponin content, which is higher than that in ginseng extracts. Furthermore, the study showed its protective effects against skin disorders induced by environmental pollutants (Na et al., 2018)[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSaponins are found in many plant species and some marine organisms and have various biological and pharmacological activities, such as immunomodulation, anti-inflammatory properties, blood glucose reduction, antibacterial and antiviral activities, and anti-cancer properties (Shen et al., 2023)[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Saponins play significant roles in cancer therapy through mechanisms involving cell cycle inhibition, antioxidant activities, inhibition of cell invasion, and the induction of apoptosis and autophagy (Elekofehinti et al., 2021)[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Since a key mechanism of senotherapy involves the induction of apoptosis in senescent cells by senolytics, we investigated the potential of saponins for use as effective senolytics. We compared the senolytic potential of plant root extracts (from \u003cem\u003eCodonopsis lanceolata\u003c/em\u003e [Deodeok], \u003cem\u003ePlatycodon grandiflorus root\u003c/em\u003e [balloon flower], and \u003cem\u003ePanax ginseng\u003c/em\u003e [ginseng]) known to contain high levels of saponins and anti-inflammatory properties, which are suitable for use as ingredients in cosmetics. These plants, except for \u003cem\u003ePanax ginseng\u003c/em\u003e, did not exhibit senolytic efficacy due to variability in the structure and concentration of saponins in the root extracts of each plant (Supplementary Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). A previous study reported that black ginseng, which is derived from processed \u003cem\u003eP. ginseng\u003c/em\u003e, can be used as a senolytic agent by reducing cellular senescence (Lee et al., 2022)[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. This study revealed that SENOMUNE has potential as a senolytic agent.\u003c/p\u003e\u003cp\u003eSaponins, the major constituent of SENOMUNE, induce apoptosis in cancer cells through mechanisms that can be caspase-dependent or -independent (Elekofehinti et al., 2021)[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. We have elucidated that SENOMUNE induces the selective eradication of senescent cells via a caspase-independent apoptosis mechanism. We verified that SENOMUNE functions by activating pro-apoptotic factors, such as AIF. Saponin releases AIF into the intermembrane and subsequently translocates to the nucleus, where it binds to DNA, causing chromatin condensation and leading to apoptosis. Unlike conventional senolytics, such as ABT-737, which operate through caspase-dependent apoptosis, caspase apoptosis exhibits a unique mechanism of action for SENOMUNE (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eDermal fibroblasts and epidermal cells such as melanocytes and keratinocytes in the skin closely influence each other during aging, and the SASP produced from aged cells acts as a mediator of aging (Birch and Gil, 2020; Chin et al., 2023)[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Previous studies have suggested that applying senolytics reduces the number of senescent cells, suppresses SASP production, and mitigates aging in neighboring cells (Kim et al., 2022; Chin et al., 2023)[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. We aimed to determine the effects of senescent dermal fibroblasts on the aging of epidermal cells (Videira et al., 2013)[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The impact of the SASP generated from senescent fibroblasts on the skin barrier function and pigmentation has been substantiated through MelanoDerm and ex vivo skin biopsies. Furthermore, we confirmed that SENOMUNE improves aging phenotypes, such as skin barrier dysfunction and hyperpigmentation (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). We also established that SENOMUNE improves aging-related pigmentation through various pathways. SASP factors produced by senescent fibroblasts, along with inflammatory cytokines produced by keratinocytes, exacerbate aging-related pigmentation (Lee et al., 2011; Videira et al., 2013)[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. We observed that SENOMUNE treatment reduced the SASP in senescent fibroblasts, which decreased enzymes associated with pigmentation in MCs and activated p62-selective autophagy related to suppressing inflammatory cytokine production in KCs.\u003c/p\u003e\u003cp\u003eWhile most of our experiments were performed using in vitro models, which are valuable for preliminary investigations, they do not fully replicate the complexity of human skin aging in vivo. Future studies should include in vivo studies to more accurately assess the therapeutic potential of SENOMUNE. However, our research has clearly demonstrated multiple senolytic effects of SENOMUNE, which underscored its potential despite the absence of in vivo studies. Moreover, SENOMUNE contains 54% saponins and includes various types of saponins. In this study, we were unable to analyze specific saponins demonstrating senolytic efficacy. However, further research is expected to involve isolating individual saponins comprising SENOMUNE to investigate the efficacy and mechanisms of each component in more detail. Despite these limitations, the current research provides strong preliminary evidence of the bioactivities and therapeutic potential of SENOMUNE as an anti-aging phytomedicine. Therefore, we conclude that SENOMUNE has significant potential as a novel anti-aging phytopharmaceutical ingredient with therapeutic applications.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cb\u003ePlant preparation and extraction\u003c/b\u003e\u003c/p\u003e\u003cp\u003eRoot samples were collected from a 30-year-old green tea (\u003cem\u003eC. sinensis\u003c/em\u003e L.) in the Amorepacific Dosun tea garden in Jeju Island, Republic of Korea. The \u003cem\u003eC. Sinensis\u003c/em\u003e roots were washed with purified water, dried, and ground into a fine powder. Next, the extract was extracted using ethanol, followed by filtration and vacuum concentration at 40–50 ℃ to prepare the dried plant extract.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell culture\u003c/b\u003e\u003c/p\u003e\u003cp\u003eNHDFs were purchased from Clonetics Lonza (Walkersville, MD, USA) and maintained in Dulbecco’s Modified Eagle’s Media (DMEM, Thermo Fisher Scientific, Waltham, MA, USA) with 10% fetal bovine serum (FBS, Thermo Fisher Scientific) and penicillin/streptomycin (100 IU/50 µg/mL).\u003c/p\u003e\u003cp\u003e\u003cb\u003eStress-induced premature senescence\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDMEM containing doxorubicin hydrochloride (100 ng/mL, Sigma-Aldrich, St. Louis, MI, USA) and insulin growth factor-1 (100 ng/mL, Sigma-Aldrich) containing DMEM were added to the cell culture for 4–7 days to induce senescence. Subsequently, SA-β-Gal activity and the gene and protein expression of senescence-related markers were analyzed (Supplementary Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e) (An et al., 2020)[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The viability of young and senescent cells was measured to confirm the senolytic effect of SENOMUNE, and the visibility of the ABT-737 and SENOMUNE cells was compared.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell viability\u003c/b\u003e\u003c/p\u003e\u003cp\u003eHDFs were seeded at 1.5 × 10\u003csup\u003e5\u003c/sup\u003e cells/well in a 6-well plate and incubated for 48 h. Young and senescent HDFs were incubated with the test materials for 3 days, followed by treatment with a Cell Counting Kit-8 solution (CCK-8, Dojindo Laboratories, Rockview, MD, USA) for 1 h. The optical density was measured at 450 nm using a plate reader (Spectrostar Nano, BMG Labtech, Ortenberg, Germany).\u003c/p\u003e\u003cp\u003e\u003cb\u003eSA-β-Gal activity assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSA-β-gal staining was performed using the Senescence Cells Histochemical Staining Kit (Sigma-Aldrich) according to the manufacturer’s instructions. Briefly, the cells were washed with phosphate-buffered saline (PBS) and fixed for 5 min; thereafter, they were incubated at 37°C for 16 h without CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eReverse transcription qPCR (RT-qPCR)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTotal RNA was isolated using a TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and 1 µg of total RNA was used to synthesize cDNA using a reverse transcriptase kit (Invitrogen). Gene expression analyses were performed using TaqMan Universal Master Mix and TaqMan Gene Expression assays (Applied Biosystems, Foster City, CA, USA) in a 7500 Fast Real-time PCR System (Applied Biosystems, Waltham, MA, USA) according to the manufacturer’s instructions. Glyceraldehyde 3-phosphate dehydrogenase (\u003cem\u003eGAPDH\u003c/em\u003e) was used to normalize variations in cDNA quantities synthesized from different samples. The relative mRNA levels were quantified using the 2\u003csup\u003e−∆∆Ct\u003c/sup\u003e method. The primers of \u003cem\u003eCXCL8\u003c/em\u003e (Hs00174103_m1), \u003cem\u003eMMP1\u003c/em\u003e (Hs00899658_m1), GDF15 (Hs00171132_m1), \u003cem\u003eTIMP1\u003c/em\u003e (Hs00171558_m1), \u003cem\u003eIGFBP6\u003c/em\u003e (Hs00181853_m1), \u003cem\u003eFLG\u003c/em\u003e (Hs00856927_g1), \u003cem\u003eKRT10\u003c/em\u003e (Hs00166289_m1), \u003cem\u003eIL6\u003c/em\u003e (Hs00985639_m1), \u003cem\u003eCDKN1A\u003c/em\u003e (Hs00355782_m1), \u003cem\u003eBCL2L1\u003c/em\u003e (Hs00236329_m1), and \u003cem\u003eCOL1A1\u003c/em\u003e (Hs00164004_m1) were used for RT\u003cem\u003e-\u003c/em\u003eqPCR.\u003c/p\u003e\u003cp\u003e\u003cb\u003eWestern blotting\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe cell pellets were lysed in radioimmunoprecipitation assay buffer (Sigma-Aldrich) containing protease inhibitors (Sigma-Aldrich). The lysate was centrifuged at 15,000 × \u003cem\u003eg\u003c/em\u003e for 20 min, and the supernatant was used for western blot analysis. Protein concentrations were determined using the Bradford method, with bovine serum albumin (Sigma-Aldrich) as the standard. Proteins (20 µg per well) were fractionated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Thermo Fisher Scientific). The membranes were blocked with 10% SuperBlock T20 (TBST) blocking buffer (Thermo Fisher Scientific, #37536) for 1 h and were subsequently probed overnight at 4 ℃ with an anti-PARP1 antibody (Cell Signaling Technology, Danvers, MA, USA; Catalog No. 9542), anti-cleaved PARP1 antibody (Cell Signaling Technology, Catalog No. 5625), anti-caspase-3 antibody (Cell Signaling Technology, Catalog No. 9662), anti-cleaved caspase-3 antibody (Cell Signaling Technology, Catalog No. 9661), anti-BAX antibody (Cell Signaling Technology, Catalog No. 5023), anti-Bcl-w antibody (Cell Signaling Technology, Catalog No. 2724), anti-Lamin B1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA; Catalog No. sc-374015), anti-phospho-p53 antibody (Cell Signaling Technology, Catalog No. 9284), anti-p53 antibody (Cell Signaling Technology, Catalog No. 2524), anti-p16 antibody (Santa Cruz Biotechnology, Catalog No. sc-56330), anti-p21 antibody (Cell Signaling Technology, Catalog No. 2947), anti-phospho-p62 antibody (Cell Signaling Technology, Catalog No. 16177), anti-phospho-NF-kB p65 antibody (Cell Signaling Technology, Catalog No. 3033), and anti-β-actin antibody (Santa Cruz Biotechnology, Catalog No. sc-56330). The blots were washed thrice with TBST and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad Laboratories, Hercules, CA, USA) for 1 h. The proteins were detected using the Enhanced Chemiluminescence system (Amersham Pharmacia Biotech, Piscataway, NJ, USA).\u003c/p\u003e\u003cp\u003e\u003cb\u003eFlow cytometry\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCells were cultured in 60 Petri dishes, and the supernatant was collected and labeled with an allophycocyanin (APC) Annexin V/Dead Cell Apoptosis Kit (Invitrogen, Waltham, MA, USA) with APC Annexin V and SYTOX Green for flow cytometry. The apoptotic cells were detected using flow cytometry. Annexin V-APC-positive apoptotic cells were analyzed using fluorescence-activated cell sorting to determine whether the death of senescent cells by SENOMUNE was caused by apoptosis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCaspase-3/7 activity\u003c/b\u003e\u003c/p\u003e\u003cp\u003eHDFs were grown in a 96-well plate (Thermo Fisher Scientific) and incubated with test materials for 3 days, followed by treatment with an aminoluciferin-labeled substrate using the Caspase-Glo 3/7 assay kit (Promega, Madison, MI, USA) for 3 h. The luminescence of each sample was measured using a plate-reading luminometer (GloMax Discover GM3000, Promega).\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell fraction assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCells were cultured in 100 Petri dishes, and the supernatant was collected and fractionated using a Cell Fractionation Kit (Abcam, Cambridge, UK). The cytosolic, mitochondrial, and nuclear fractions were assayed using western blotting. Anti-AIF antibody (Proteintech, Rosemont, IL, USA; Catalog No. 17984-1-AP), anti-COXIV antibody (Cell Signaling Technology, Catalog No. 4844), anti-Lamin A antibody (Santa Cruz Biotechnology, Catalog No. sc-7292), anti-CTSB antibody (Cell Signaling Technology, Catalog No. 31718), anti-CTSD antibody (Cell Signaling Technology, Catalog No. 74089), anti-LAMP1 antibody (Cell Signaling Technology, Catalog No. 9091), and anti-GAPDH antibody (Cell Signaling Technology, Catalog No. 2118) were used for western blotting.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAO staining and confocal imaging\u003c/b\u003e\u003c/p\u003e\u003cp\u003eHDFs were plated on Lab-Tek chambered cover glass (Thermo Fisher Scientific) and incubated with test materials for 3 days, followed by staining with 5 µM AO (Catalog No. A1301, Molecular Probes, Eugene, OR, USA) for 30 min at 37 ℃. After rinsing in PBS (Thermo Fisher Scientific), the fluorescence of AO was imaged using a confocal microscope (LSM980, Carl Zeiss, Oberkochen, Germany) with a corresponding filter and at 470–490 nm excitation and 515 nm emission wavelengths.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCM of senescent human dermal fibroblasts\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe CM of senescent cells were obtained from HDFs that had been passaged \u0026gt; 35-fold. Young or senescent HDFs were seeded on a 100-mm dish, and the test materials were treated for an additional 3 days. The media was changed to 1% FBS containing DMEM and incubated for 2 days. It was then collected, centrifuged, and filtered for further analysis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eHanging drop culture\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFor the hanging drop culture, MelanoDerm (MEL-300-B, MatTek Corp., Ashland, MA, USA) was incubated in an EPI-100-NMM-113-PRF medium (MatTek, Ashland, MA, USA) at 37 ℃ in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator (Thermo Fisher Scientific). The dermal parts were produced as follows: the dermal mixture was prepared by mixing type I collagen (3 mg/mL, Nitta Gelatin, Tokyo, Japan), reconstruction buffer (Nitta Gelatin), Dulbecco's Eagle concentrated culture solution (Nitta Gelatin), fibrinogen (10 mg/mL, Sigma-Aldrich), aprotinin (0.4 TIU/mL, Sigma-Aldrich), and HDFs. Thrombin (0.625 U/mL, Sigma-Aldrich) was added to initiate the fibrinogen polymerization, and the mixture was loaded onto the bottom of the MelanoDerm (hanging drop, MatTek Corp). The tissues were incubated in EPI-100-NMM-113-PRF medium (MatTek Corp) for 14 days.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCo-culture of fibroblasts and ex vivo skin biopsy\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFor the co-culture of fibroblasts and ex vivo skin biopsy, the fibroblasts were seeded into a 6-well plate (Thermo Fisher Scientific) and treated with the test materials for 24 h. Ex vivo skin biopsy samples were purchased from Genoskin, Paris, France. After arrival, the ex vivo skin biopsy was stabilized for 24 h and then co-cultured with fibroblasts for 7 days.\u003c/p\u003e\u003cp\u003e\u003cb\u003eHistological examination\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe tissues were fixed in 10% neutral-buffered formalin (BBC Biochemical, Mount Vernon, WA, USA) and embedded in paraffin. The paraffin-embedded samples were sliced into 5 µm sections, and histological observation was performed following hematoxylin and eosin (Thermo Fisher Scientific) and Fontana-Masson (Sigma-Aldrich) staining. For immunohistochemistry, tissue sections were stained with primary antibodies at 4 ℃ overnight. Primary antibodies for Ki67 (Abcam, Catalog No. ab15580), Keratin 10 (Santa Cruz Biotechnology, Catalog No. sc-23877), Filaggrin (Santa Cruz Biotechnology, Catalog No. sc-66192), Tyrosinase (Cell Signaling Technology, Catalog No. 9319), PMEL (Cell Signaling Technology, Catalog No. 38815), and Claudin-1 (Abcam, Catalog No. ab15098) were used. Then, horseradish peroxidase-conjugated donkey anti-rabbit IgG (Abcam, ab6802) or anti-mouse IgG (H\u0026amp;L) (Abcam, ab6820) was applied as the secondary antibody for 60 min. Immunoreactivity was visualized using 3,30-diaminobenzidine (brown) or 3-amino-9-ethylcarbazoles (red) as a chromogen. The sections were visualized under a light microscope (BX53, Olympus, Japan) using a digital camera (DP72, Olympus).\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAIF-apoptosis-inducing factor, AO-acridine orange, DI-doxorubicin and insulin-like growth factor 1, GAPDH-glyceraldehyde 3-phosphate dehydrogenase, HDFs-human dermal fibroblasts, SASP-senescence-associated secretory phenotype, SENOMUNE-\u003cem\u003eCamellia sinensis\u0026nbsp;\u003c/em\u003eL.\u003cem\u003e\u0026nbsp;\u003c/em\u003eroot extract\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll\u0026nbsp;data generated\u0026nbsp;or analyzed during\u0026nbsp;this study are\u0026nbsp;included in this\u0026nbsp;published article\u0026nbsp;and its supplementary\u0026nbsp;information files. Additional datasets\u0026nbsp;used and/or analyzed\u0026nbsp;during the current\u0026nbsp;study are available\u0026nbsp;from the corresponding\u0026nbsp;author on reasonable\u0026nbsp;request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Amorepacific R\u0026amp;I Center,\u0026nbsp;Gyeonggi-do, Republic of Korea.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS. Park and S. Cho conceived and designed the experiments; S. Park, S. Cho, D. Min, H. Choi and K. Hwang performed the experiments; K. Hwang contributed materials; H.-J. Kim, I.S. Kil and W.-S. Park analyzed the data and supervised the project; S. Park and H.-J. Kim wrote the paper.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTchkonia, T., Zhu, Y., van Deursen, J., Campisi, J. \u0026amp; Kirkland, J. L. Cellular senescence and the senescent secretory phenotype: therapeutic opportunities.\u003cem\u003e J. Clin. Invest. \u003c/em\u003e\u003cstrong\u003e123\u003c/strong\u003e, 966-972 (2013).\u003c/li\u003e\n\u003cli\u003eKim, E. C. \u0026amp; Kim, J. R. 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(2022). \"Green tea (Camellia sinensis): A review of its phytochemistry, pharmacology, and toxicology.\" \u003cu\u003eMolecules\u003c/u\u003e \u003cstrong\u003e27\u003c/strong\u003e(12): 3909.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Camellia sinensis, senescence-associated secretory phenotype, senolytics, human dermal fibroblasts","lastPublishedDoi":"10.21203/rs.3.rs-7016169/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7016169/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSenescent cells can affect neighboring cells via the senescence-associated secretory phenotype (SASP), which involves pro-inflammatory cytokines, chemokines, and proteases. This study aimed to explore the senolytic properties of \u003cem\u003eCamellia sinensis\u003c/em\u003e root extract (SENOMUNE), which has therapeutic potential for skin aging-related disorders, with cell viability assays, quantitative reverse transcription polymerase chain reaction, western blotting, and flow cytometry using a stress-induced premature senescence model in normal human dermal fibroblasts (NHDFs). NHDFs were induced to senescence using doxorubicin and insulin-like growth factor-1. The senolytic effect of SENOMUNE was also evaluated through the investigation of senescence-associated β-galactosidase activity, gene and protein expression analysis, and apoptosis assays in NHDFs. The impact of SENOMUNE on the skin barrier function and pigmentation was assessed using conditioned media from senescent fibroblasts and ex vivo skin biopsies. SENOMUNE exhibited a concentration-dependent reduction in senescent cells without affecting young cells and induced apoptosis in senescent cells through a caspase-independent mechanism involving apoptosis-inducing factor and lysosomal membrane permeabilization. SENOMUNE reduced SASP factors and improved skin barrier function and pigmentation by modulating the secretion of inflammatory cytokines from keratinocytes and autophagy. SENOMUNE thus demonstrated novel senolytic properties and therapeutic potential for managing skin-related disorders and is a promising anti-aging phytopharmaceutical ingredient.\u003c/p\u003e","manuscriptTitle":"Novel Senolytic Ingredient, Camellia sinensis Root Extract, Ameliorates Skin Aging-Associated Phenotypes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-22 05:41:16","doi":"10.21203/rs.3.rs-7016169/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"32797635-327b-4515-9e85-89313a87a5fe","owner":[],"postedDate":"July 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":51825183,"name":"Biological sciences/Cell biology"},{"id":51825184,"name":"Health sciences/Diseases"}],"tags":[],"updatedAt":"2025-08-14T10:45:04+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-22 05:41:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7016169","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7016169","identity":"rs-7016169","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}